key: cord- - ic twhy authors: ding, dan; du, yimei; qiu, zhihua; yan, sen; chen, fen; wang, min; yang, shijun; zhou, yanzhao; hu, xiajun; deng, yihuan; wang, shijia; wang, liangping; zhang, hongrong; wu, hailang; yu, xian; zhou, zihua; liao, yuhua; chen, xiao title: vaccination against type angiotensin receptor prevents streptozotocin-induced diabetic nephropathy date: - - journal: j mol med (berl) doi: . /s - - - sha: doc_id: cord_uid: ic twhy abstract: recently, our group has developed a therapeutic hypertensive vaccine against angiotensin (ang) ii type receptor (at r) named atrqβ- . to explore its potential effectiveness on streptozotocin-induced diabetic nephropathy, male sprague dawley rats were randomly divided into two groups: a control and a diabetic model. after week, the diabetic rats were divided into four subgroups (each with rats) for -week treatments with saline, olmesartan, atrqβ- , and qβ virus-like particle (vlp), respectively. in addition to lower blood pressure, atrqβ- vaccination ameliorated biochemical parameter changes of renal dysfunction, mesangial expansion, and fibrosis through inhibiting oxidative stress, macrophage infiltration, and proinflammatory factor expression. furthermore, atrqβ- vaccination suppressed renal ang ii-at r activation and abrogated the downregulation of angiotensin-converting enzyme -ang ( – ), similar to olmesartan treatment, while no obvious feedback activation of circulating or local renin-angiotensin system (ras) was only observed in vaccine group. in rat mesangial cells, the anti-atr- antibody inhibited high glucose-induced transforming growth factor-β (tgf)-β /smad signal pathway. additionally, no significant immune-mediated damage was detected in vaccinated animals. in conclusion, the atrqβ- vaccine ameliorated streptozotocin-induced diabetic renal injury via modulating two ras axes and inhibiting tgf-β /smad signal pathway, providing a novel, safe, and promising method to treat diabetic nephropathy. key messages: overactivation of ras plays a crucial role in the development of the dn. our aim was to verify the effectiveness of atrqβ- vaccine in stz-induced dn. the atrqβ- modulated two ras axes and inhibited tgf-β /smad signal pathway. the vaccine therapy may provide a novel, safe, and promising method to treat dn. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. diabetic nephropathy (dn) is the most common cause of endstage renal diseases, responsible for over % of all cases in the usa, and this number is likely to increase unabated [ ] . overactivation of the renin-angiotensin system (ras) plays a crucial role in the development of the disease [ ] . current treatment, aimed at slowing progression, concentrates on two inter-related therapeutic strategies: blood pressure reduction and blockade of the ras [ ] [ ] [ ] . such treatments have been shown to reduce the functional changes seen in dn dan ding, yimei du and zhihua qiu contributed equally to this work. electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. and also to attenuate the structural abnormalities that characterize this disease [ ] [ ] [ ] . at present, blockade of the ras is achieved by two major drug classes: angiotensin-converting enzyme (ace) inhibitors and angiotensin ii (ang ii) receptor blockers (arbs), agents that also lower systemic blood pressure [ ] . however, they are not completely effective in preventing or reversing the progress of dn, in part due to a compensatory increase in plasma renin activity (pra) or ang ii [ ] . current therapies are insufficient, necessitating the search for new therapeutic strategies in dn. recently, we developed a therapeutic hypertensive vaccine atrqβ- , a peptide (atr- ) derived from human ang ii receptor type (at r) conjugated with qβ bacteriophage virus-like particles, which decreased the blood pressure of hypertensive animals effectively through diminishing the pressure response and inhibiting signal transduction initiated by ang ii with no obvious feedback activation of circulating or local ras [ ] . in our previous study, we demonstrated that an epitope from the rat at r, designated as atr , could decrease sbp of spontaneously hypertensive rats (shrs) and provide excellent protections in target organs [ ] . following our work, hiroshi itoh and his colleagues emphasized vaccination against at r for the prevention of l-name-induced nephropathy in shrs, not only for the attenuation of hypertension [ ] . ang ii and other components of ras also have a central role in the pathogenesis and progression of diabetic renal injuries. accordingly, this study was designed to explore the possibility of the atrqβ- vaccine in ameliorating of experimental dn and the potential mechanisms. the study was undertaken in sprague dawley rats treated with streptozotocin that developed renal injury similarities to human dn [ ] . detailed methods are available in the online supplement. male sprague dawley rats weighing - g were purchased from the experimental animal research center (hubei province, china). all animals were kept in the pathogen-free room in the experimental animal center (tongji medical college of huazhong university of science and technology, wuhan, china), and all experiments were carried out in accordance with guidelines for the care and use of laboratory animals (science and technology department of hubei province, china, ). experimental diabetes was induced by intraperitoneal injection of the β cell toxin streptozocin ( mg/kg) dissolved in fresh sodium citrate buffer (ph . ) following an overnight fast. animals with plasma glucose concentrations in excess of . mmol/l, week postinduction of diabetes, were included in the study. sham-injected control animals (sodium citrate buffer, ph . ) were followed concurrently. diabetes rats were then randomized into four groups (n= ), receiving one of the following treatments: ( ) dn group: equal volume saline injection subcutaneously (s.c); ( ) om group: olmesartan, mg/kg/day via oral gavage; ( ) atrqβ- group: the atrqβ- vaccine, which was immunized s.c μg on days , , and , formulated in aluminum hydroxide gel; ( ) virus-like particle (vlp) group: μg qβ vlp injected as group . each week, rats were weighed and their blood glucose levels were measured. atr- -specific antibody titers were detected in every weeks. every weeks, systolic blood pressure (sbp) was determined in preheated conscious rats via tail-cuff plethysmography using a non-invasive blood pressure controller and powerlab system. the rats were decapitated between am and am. blood samples were collected and divided into two parts: one was mixed with the enzyme inhibitor mixture ( ml blood in μl inhibitor mixture including μl . mol/l edta, μl . mol/l dimercaprol, and μl . mol/l -ohquinoline sulfate) for pra and ang ii concentration measurement by radioimmunoassay (ria) according to the assay kit instruction (nibt, beijing); the other was used for biochemical experiments. for tissue ang ii and ang ( - ) measurement, immediately after harvesting and weighing, the kidney cortex were immersed in ice-cold methanol, minced, and homogenized with tissue homogenizers. the homogenates were centrifuged ( rpm, °c, min), and the supernatants were dried overnight in a vacuum centrifuge. the dried residue was reconstituted in ml ria buffer and then was subjected to hplc to separate ang ii from other substances. the tissue ang ii concentration was detected according to the assay kit instruction (nibt, beijing), and ang ( - ) was measured by hplc. the plasma samples were used for the measurement of glucose, lipid level, creatinine (cr), and blood urea nitrogen (bun). urine samples were collected using metabolic cages, and the supernatant was used for examination of the -h urinary protein. parts of fresh left renal cortex were immediately fixed in . % glutaraldehyde for transmission electron microscopy ( t e m ) . t h e o t h e r p a r t s w e r e f i x e d w i t h % paraformaldehyde overnight, embedded in paraffin for histopathology. sections were stained with hematoxylin-eosin (h&e), masson's trichrome, and periodic acid-schiff (pas). frozen sections were stained with dihydroethidium (dhe). immumohistochemical staining was performed to detect the expression of tgf-β ( : , everest biotech) and macrophages (cd , : , abd serotec) in glomeruli. immunofluorescence staining of nephrin and podocin expression was performed on the paraffin sections of kidneys using monoclonal anti-nephrin ( : , abcam) and anti-podocin antibodies ( : , abcam). rat mesangial cells (rmcs) were cultured in dulbecco's modified eagle's medium supplemented with % fetal bovine serum, u/ml penicillin, and mg/ml streptomycin at °c in % air and % co . cells plated on -mm dishes were cultured to % confluence and divided into five groups: control group, in which cells were incubated in mmol/l d-glucose dmem, and mmol/l d-mannitol was added to the medium in order to take into the account of the effect of high osmolarity in other cell groups; hg group, in which cells were stimulated with a high concentration of glucose ( mmol/l) only for h; los group, in which cells were pretreated with − mol/l losartan for h; anti-atr- group, in which cells were pretreated with anti-atr- for h; anti-natr- group, in which cells were pretreated with anti-natr- for h. all cells except control group received stimulation with a high concentration of glucose ( mmol/l) for h after treatment. cell protein and messenger rna (mrna) were extracted for western blot and qrt-pcr analysis. data were shown as the mean±sem. statistical analyses of the data were performed with one-way anova using spss . . p< . was considered statistically significant. in comparison with control animals, diabetic rats had significantly reduced body weight, which was unaffected by treatment. the ratio of kidney weight/body weight was increased, while atrqβ- vaccine and olmesartan treatments decreased the level. blood glucose and lipid levels were elevated to a similar extent in all diabetic rat groups, irrespective of treatment (table ) . no evidence of skin damages at the site of subcutaneous injection was noted in vaccine-treated animals. less activities and poorer hair were shown in diabetic rats, but the reactivity was the same as normal rats. atrqβ- vaccination effectively reduced blood pressure and reversed biochemical parameters of renal dysfunction in stz-induced diabetic rats to examine antibody production, we measured the amounts of anti-atr- antibodies produced in response to vaccination with atrqβ- . after the second injection of the vaccine, the atr- -specific antibody titer was : , to : , , and it rose after the third injection, then peaked on day ( : , ) and gradually decreased thereafter (fig. a) . to investigate the efficacy in blood pressure, systolic blood pressure (sbp) levels were measured by the tail-cuff method. over the course of the study, sbp was elevated in dn compared to normal rats, while rats immunized with the atrqβ- vaccine were decreased compared to the vlp group, with a maximum decrease of . mmhg ( . + . vs. . + . mmhg, p< . , fig. b) . to evaluate the biochemical parameters of renal function, h urine volume, and total protein excretion, blood urea nitrogen and creatinine were measured. diabetic rats exhibited higher levels in all these parameters. atrqβ- vaccine and olmesartan-treated rats significantly reduced these parameters compared to dn and vlp groups, and no significant differences were between these two groups ( fig. c-f ). to confirm the effect of vaccination, we evaluated renal pathological changes. podocytes form the filtration slit diaphragms that prevents the escape of plasma protein from the glomerular circulation. the disruption of podocytes contributes to proteinuria and further development of glomerular sclerosis [ ] . reduced nephrin and podocin immunostaining in glomeruli were showed in diabetic rats, and atrqβ- vaccination prevented the reduction (fig. ) . in addition to podocyte injury, glomerular mesangial expansion is also a hallmark of dn. extracellular matrix deposition was detected by periodic acid-schiff staining, and glomerular sclerosis index (gsi) was calculated. compared with dn and vlp groups, gsi was significantly decreased in atrqβ- -vaccinated group, similar with olmesartantreated group, which was also confirmed by transmission electron microscopy (tem) (fig. ) . to further investigate the mechanisms, renal fibrosis and inflammation parameters were measured. the extent of interstitial expansion was quantified by masson's trichrome-stained sections. the interstitial expansive index was higher in dn and vlp groups, and atrqβ- vaccination blocked the increase (fig. ) . glomerular and interstitial inflammation was quantified by the cell numbers of macrophages stained positively with cd antibody, and atrqβ- vaccination successfully prevented the increased inflammation (fig. ) . besides, we detected transforming growth factor-β (tgf-β ) expression and reactive oxygen species, which was also decreased by atrqβ- vaccine treatment. further, the mrna expressions of profibrotic and proinflammatory factors in kidney cortex were measured. as shown in fig. f , g, the increased mrna levels in these factors were significantly suppressed by atrqβ- vaccination. there were no significant differences between atrqβ- -and olmesartan-treated groups. atrqβ- vaccination suppressed renal ang ii-at r activation and abrogated the downregulation of ace -ang ( - ) with no feedback of ras to determine whether blockade of at r lead to feedback activation of circulating ras, we detected the pra and ang ii concentration. the pra in atrqβ- vaccinated group was ± pmol/h/l, which had no significant difference with vlp group ( , ± pmol/h/l, p = . ). however, a distinct increase was observed in olmesartan group ( ± pmol/h/l, p= . ; fig. a) . similarly, the plasma concentration of ang ii in olmesartan group was higher than vlp group ( . ± . vs. . ± . pmol/l, p = . ), whereas no significant difference was observed between vlp and vaccine groups ( . ± . vs. . ± . , p= . ; fig. b ). to further examine the local ras, kidney ang ii concentration was measured. the concentration of ang ii in kidneys was lower in olmsartan-and vaccine-treated groups compared to dn and vlp groups (fig. d) . to investigate whether the protection effect was a result of increase ang ( - ) activation with simultaneous ang ii suppression, we subsequently measured plasma and kidney ang ( - ). the decline of ang ( - ) in diabetic rats was improved in atrqβ- vaccine and olmesartantreated groups (fig. c, e) . activation of ras in streptozotocin (stz)-induced diabetic kidneys was further confirmed by western blot and quantitative real-time pcr (fig. ) . the mrna expression of kidney renin in olmesartan group was obviously higher than vlp group (p< . ), while no difference was found between atrqβ- vaccine and vlp groups (fig. g) . ace and at r expressions were increased in diabetic rats, and both atrqβ- vaccine and olmesartan groups attenuated the increasing expressions. ace -ang ( - )-mas axis counteract ang ii-mediated effects in various organs, including the kidneys, and we observed reduced expression of ace -ang ( - )-mas in the diabetic kidneys. atrqβ- vaccine and olmesartan displayed upregulation of ace -ang ( - )-mas expression compared with vlp group. further, we also detected the downstream signal transduction and the activation of erk / , and p mapk phosphorylation was inhibited by atrqβ- vaccination, similar to olmesartan treatment (fig. e-f ). to further explore the mechanisms, cell experiment was undertaken by anti-atr- stimulation. glomerular mesangial cells are believed to be responsible for overproduction of ecm proteins in various pathologic conditions, and tgf-β has been proposed to play an important role. the tgf-β / smad signal pathway regulates the hypertrophic and prosclerotic changes in diabetic renal injury [ ] . anti-atr- and losartan treatment significantly decreased the expression of tgf-β and the phosphorylation levels of smad in rmcs stimulated by high glucose. additionally, we examined the expression of collagen iv and fibronectin, which showed similar change as tgf-β (fig. ) . for safety consideration, normal sd rats were immunized with the atrqβ- vaccine, and the histological changes of kidney were observed by light microscopy and tem. no evidence of skin damages at the site of subcutaneous injection was noted in vaccine-treated animals. compared with control group, no obvious cell proliferation and pathological changes in the mesangial area were shown in vaccine group. also, tem demonstrated that no immune complexes were observed in the basement membrane, and the structure of the glomerulus was intact (fig. ). in this study, we demonstrated for the first time that in a rat model of dn, a vaccine targeting at r named atrqβ- the rat dn model was induced by an intraperitoneal injection of stz that has been widely used to create diabetes models in rodents with pathological features similar to human diabetic nephropathy [ ] . the nephropathy subcommittee of the animal models of diabetic complications consortium (amdcc) has published the following validation criteria for rodent models of dn based on the clinical and pathological features of human dn: ( ) > % decrease in renal function, ( ) > -fold increase in albuminuria, and ( ) pathological features including advanced mesangial matrix expansion (±nodules), thickening of the glomerular basement membrane, arteriolar hyalinosis, and tubulointerstitial fibrosis [ ] . an ideal model of dn would display all of these criteria; however, no current model entirely satisfies them. that is the limitation in animal experiments. as the rodent models share many similarities with human disease, we cannot ignore the potential therapeutic value of the vaccine in the treatment of human diseases. our results showed that rats presented severe hyperglycemia and renal dysfunction manifested as increased plasma creatinine level, bun, h urinary volume, and protein excretion, indicating that a dn animal model was successfully established. the ratio of kidney weight/body weight in diabetic groups was significantly increased compared with that in control group, substantial to the presence of renal hypertrophy in diabetic rats. during the course of the study, systolic blood pressure was elevated in diabetic rats, notwithstanding the limitations of tail-cuff sphygmomanometry, atrqβ- vaccination lowered blood pressure (bp) smoothly, and olmesartan decreased to a greater extend. it is worth noting that our animal experiments showed many times that the antihypertensive effect of atrqβ- vaccine was dependent on the baseline of the bp level and exhibited no decreasing effect in normotensive rats. oppositely, arbs decreased bp irrespective of the basic level and had an increase risk of hypotension. therefore, atrqβ- vaccine may be more practical in diseases where the bp was not very high, such as dn. glomerular mesangial matrix expansion, renal fibrosis, and inflammation are typical pathological features of dn. atrqβ- vaccination ameliorated these morphologic changes of renal injury as well as decreased expression of profibrotic and proinflammatory factors. to further confirm the effects, rmcs were used to investigate the mechanism in vitro. the anti-atr- antibody treatment effectively inhibited high glucose-induced extracellular signal-regulated kinase phosphorylation and tgf-β /smad signal pathway. these results strengthened the renoprotection of atrqβ- vaccination in addition to the antihypertensive effect. the inhibition of oxidative stress and macrophage infiltration as well as proinflammatory factor expression may contribute to the amelioration of renal pathological changes. furthermore, compared with the obvious ras feedback of arbs, circulating or local ras were not elevated in diabetes animals immunized with atrqβ- vaccine. nevertheless, kidney ang ii concentration and at r expression were decreased in both groups. the recent development of ace -ang ( - )-mas concept has provided new insights into the effects of ang ii in animal experimental models. this novel concept states that ras has two axes: the ace-ang ii-at r axis and ace -ang ( - )-mas axis. the former axis induces vasoconstriction, proliferation, and proinflammatory functions through ang ii, and the latter axis counteract the effects of the former axis through the major heptapeptide effector ang ( - ) [ , ] . ace cleaves ang ii to produce abundant levels of ang ( - ) in the proximal tubule, which produces vasodilation and antiproliferative, natriretic, and diuretic effects [ ] . despite its significant role, the exact role of ace and ang ( - ) in kidney disease is not clearly understood. it has been reported that inhibition of ace function accelerates diabetic injury and human recombinant ace reduces the progression [ , ] . moreover, ang ( - ) attenuates the progression of diabetic nephropathy in animal models [ , ] . our study showed a downregulation of ace -ang ( - )-mas axis in diabetic rats, and the beneficial effects of atrqβ- vaccine and olmesartan treatment may partly due to the upregulation of this axis. although the exact mechanism of how these two axes counteract with each other remains unknown, we can conclude that the vaccine regulates the two ras axes, not only inhibiting at r overactivation. safety considerations are paramount when developing any vaccine but are particularly important when targeting a condition for which many safe and effective alternative therapies are available. four key factors define the safety of therapeutic vaccine: ( ) the targeted molecule, ( ) the reversible of antibody response, ( ) antibody-dependent cell-mediated cytotoxicity (adcc) and complement-mediated cytotoxicity, and ( ) fig. no imumune-mediated injury was observed in vaccinated animals. a representative staining of hematoxylin-eosin (h&e), masson's trichrome, and periodic acid-schiff (pas) and transmission electron microscopy (tem). b normal sd rats were immunized on days and , and the atr- -specific antibody titers were screened on days , , , , , , , and activation of t cells against self-molecules [ ] . combined with our previous studies, vaccination-targeted at r showed no immune-mediated injuries [ , ] and was also confirmed in this study. the antibody response was reversible as the possibility of halting antibody production. for adcc and complement-mediated cytotoxicity, there are still several controversies in terms of its onset and regulation. nevertheless, the potential effects caused by the vaccine needed further investigation. as the target peptide of the vaccine was only eight amino acids in length, then it was smaller than the minimal size of a t cell epitope and therefore should not be able to induce a t cell response [ ] . several studies had estimated that vaccines comprising self-molecules coupled to vlps present optical candidates capable of achieving efficacious antibody levels while fulfilling the necessary safety criteria [ , ] . examples like the ang ii vaccine cyt- -angqb, which showed effectively reduced bp without any uncontrolled immune stimulation in both animals and patients [ , ] . similar to the composition, the kidney damage caused by immune complexes was not detected and no visible pathological changes were observed by light microscopy and tem in atrqβ- -vaccinated animals. from the results above, the atrqβ- vaccine was found to be basically safe, although further assessments are needed to confirm this conclusion. compared with chemical drugs, the vaccine therapy has potentially superior advantages. the half-life of the anti-atr- antibody was . days [ ] , longer than any other chemical drugs presently used. no obvious feedback activation of circulating or local ras was observed. additionally, long-term treatment of chronic diseases is costly, tedious, and at the population level rather unsuccessful. monoclonal antibodies specific for host proteins have proven to be highly effective, and the movement from the passive administration of monoclonal antibodies to active vaccination against selfmolecular could provide affordable medicines and broader patient acceptance and compliance. the question today in many patient populations is not whether to block the ras but rather how best to inhibit its activity. from a theoretical perspective, vaccination has much to recommend it as a strategy. atrqβ- vaccine modulates two major axes of ras and ameliorates stz-induced diabetic injury with no obvious immune-mediated damages, providing a 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glomerular injury in association with increased ace expression in streptozotocin-induced diabetic mice human recombinant ace reduces the progression of diabetic nephropathy angiotensin( - ) attenuates the progression of streptozotocin-induced diabetic renal injury better than angiotensin receptor blockade angiotensin - mediates renoprotection against diabetic nephropathy by reducing oxidative stress, inflammation, and lipotoxicity therapeutic vaccination for chronic diseases: a new class of drugs in sight syfpeithi: database for mhc ligands and peptide motifs virus-like particles-universal molecular toolboxes the influence of antigen organization on b cell responsiveness a vaccine for hypertension based on virus-like particles: preclinical efficacy and phase i safety and immunogenicity effect of immunisation against angiotensin ii with cyt -angqb on ambulatory blood pressure: a double-blind, randomised, placebocontrolled phase iia study open access this article is distributed under the terms of the creative commons attribution . international license (http:// creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord- - rmuvb i authors: lew, rebecca a.; warner, fiona j.; hanchapola, iresha; smith, a. ian title: characterization of angiotensin converting enzyme- (ace ) in human urine date: - - journal: int j pept res ther doi: . /s - - - sha: doc_id: cord_uid: rmuvb i angiotensin converting enzyme- (ace ) is a recently described membrane-bound carboxypeptidase identified by its homology to ace, the enzyme responsible for the formation of the potent vasoconstrictor angiotensin ii (ang ii). ace inactivates ang ii and is thus thought to act in a counter-regulatory fashion to ace. ace is highly expressed in epithelial cells of distal renal tubules, and recent evidence indicates that expression is increased in a range of renal diseases. a soluble form of ace, generated by proteolytic cleavage of the membrane-bound form, has been shown to be present in urine; although evidence for a similar release of ace has been reported in cell culture, it is not yet known whether this occurs in vivo. the present study has identified ace in human urine, both by a sensitive fluorescence-based activity assay and by western immunoblot. levels of ace were surprisingly higher than ace, which may reflect preferential targeting of the enzyme to the luminal surface of the renal epithelium. future studies will determine whether increased expression of ace in renal diseases are reflected in higher urinary levels of this novel enzyme. the renin-angiotensin system is well-documented as a key regulator of cardiovascular and renal function, with the peptide angiotensin ii (ang ii) considered the primary bioactive component (figure ). ang ii is generated by two successive enzymatic steps: first, an inactive decapeptide (ang i) is liberated from a liver-derived protein angiotensinogen by the aspartic protease renin in the circulation; the active eightresidue ang ii peptide is then formed by the action of the membrane-bound metallopeptidase, angiotensin converting enzyme (ace). inhibition of ace has proven to be a powerful therapeutic in the treatment of certain forms of hypertension characterized by overactivity of the renin-angiotensin system. recently, a homolog of ace, termed ace , has been described which appears to inactivate ang ii by removal of the c-terminal phe residue (donoghue et al., ; tipnis et al., ) . the resulting peptide, ang - , has no affinity for the angiotensin type receptor, the main mediator of ang ii effects. furthermore, increasing evidence suggests that, far from being an inactive degradation product, ang - activates its own distinct receptor, with consequent biological effects, which tend to oppose those of ang ii (santos et al., ) . thus the balance between ace and ace activity may determine the local and circulating levels of ang ii, and any imbalance may reflect or result in cardiovascular and/or renal disease. both ace and ace are type i integral membrane proteins, comprising a large extracellular catalytic domain anchored to the plasma membrane via a single hydrophobic span, followed by a short c-terminal cytoplasmic tail. like a number of membrane proteins, ace has been shown to be proteolytically released from the cell surface (parkin et al., ) , resulting in the presence of a catalytically active form circulating through the bloodstream, as well as in other biological fluids, including urine and seminal plasma (hooper, ) . we have recently shown that such cleavage-secretion also occurs for ace , at least in vitro, and that the metalloprotease tumor necrosis factor-a converting enzyme (tace) is responsible for the enhanced shedding of ace elicited by phorbol esters (lambert et al., ; unpub. obs.) . evidence for shedding of ace in vivo is limited, with anecdotal reports of ace in urine and plasma. in the present study, we have undertaken a more thorough examination of soluble ace in human urine, and critically assess its potential role in angiotensin metabolism within the urinary tract. for ace and ace activity assays, first morning urine was collected from normal individuals ( male, female, ages - ), without any signs of renal complications or associated disorders, kept at °c and used within h. in some cases, urine was subjected to an ultracentrifugation step ( , g, min, °c), and the supernatant and pellet (resuspended in assay buffer at the original volume) were assayed for ace activity or used for western analysis (see below). for all other experiments, first morning urine from one or two female subjects was used. urine ( ll) was incubated with an ace -specific quenched fluorescent substrate (qfs), ( -methoxycoumarin- -yl)-acetyl-ala-pro-lys( , -dinitrophenyl); auspep, parkville, victoria, australia), as previously described in duplicate. assays were performed in black -well microtiter plates with lm qfs in a final volume of ll per well ace assay buffer ( mm tris, m nacl, ph . ). reactions proceeded at °c for min with continuous monitoring of liberated fluorescence (k ex = nm, k em = nm) using a fluostar optima plate reader (bmg labtechnologies, offenburg, germany). cleavage of the qfs was attributed to ace by the use of the specific inhibitor (s,s) -{ carboxy- -[ -( , -dichloro-benzyl)- h-imidazol- -yl]-ethylamino}- -methyl-pentanoic acid (mln- ; a generous gift of dr. natalie dales, millenium pharmaceuticals, cambridge, ma, usa.) at nm final concentration (dales et al., ) . the rate of substrate cleavage was determined by comparison to a standard curve of the free fluorophore, -amino-methoxycoumarin (mca; sigma). urinary ace activity was determined using a modification of a standard ace assay (barton et al., ) . briefly, ll urine was incubated at °c with the ace substrate hippuryl-his-leu ( mm) in a total volume of ll buffer ( mm tris-hcl, mm nacl, ph . ), in the presence or absence of the acespecific inhibitor ramipril ( lm), for h. following incubation, ll . n naoh and ll o-phthaldialdehyde ( mg/ml in methanol) were added. after min at room temperature, ll n hcl was added, the tubes were centrifuged at , rpm in a tabletop microfuge for min, and the supernatants transferred to a round-bottomed black -well microtiter plate. fluorescence angiotensinogen angiotensinogen (k ex = nm, k em = nm) was measured using a fluostar optima plate reader (bmg labtechnologies, durham, north carolina, usa). the rate of substrate cleavage was determined by comparison to a standard curve of the product his-leu. urine ( ml) was concentrated -fold (final volume = . ml) using a kda molecular weight cut-off nanosep centrifugal concentrator (pall sciences), and ll ( - lg protein) were loaded onto a - % gradient polyacrylamide gel (nupage, invitrogen) for separation by sds-page under reducing conditions. in some cases, concentrated urine was first separated by ultracentrifugation prior to sds-page of the supernatant and pelleted (resuspended in same volume as supernatant) fractions. proteins were transferred onto an immobilon p poly(vinylidene difluoride) membrane in a tris-glycine buffer ( . m glycine, . m tris-base) with % (v/v) methanol. the membrane was saturated with tris-buffered saline with tween- (tst) ( . m tris-base, . m nacl, . % tween- , ph . ) containing % (w/v) skim milk powder for min at room temperature, then incubated overnight at °c with a polyclonal anti-human ace antiserum raised in goat (r&d systems, catalog # af , : ) diluted in % (w/v) bovine serum albumin in tst buffer, or with an antiserum raised in rabbit against a peptide from the ectodomain of human ace ( : dilution; described in douglas et al., ) . following washing, membranes were incubated for min at room temperature with hrp-conjugated secondary antibody (donkey anti-goat/sheep, : , or goat anti-rabbit, : ; silenus laboratories) diluted in % skim milk in tst, and bound antibodies were detected by using the enhanced chemiluminescence system (catalog # nel , perkin-elmer life science). following concentration as described above, urinary proteins were deglycosylated using peptide:n-glycosidase f (pngase f; new england biolabs), according to the manufacturer's instructions. in brief, urinary proteins were denatured in . % sds, % b-mercaptoethanol at °c for min. samples were then cooled to room temperature and % np- (v/v) was added prior to incubation with pngase f at room temperature for or h. following deglycosylation, urinary proteins were separated by sds-page and ace immunoreactive bands detected by western analysis as described above. synthetic angiotensin i or ii ( lg; auspep) was incubated at °c with -ll urine in a total volume of ll tris-buffered saline ( mm tris-hcl, mm nacl, ph . ) either in the presence or absence of mln- (ace inhibitor, nm). aliquots ( ll) were taken at and h time points, precipitated with ll of % trifluoroacetic acid (tfa) in methanol, and dried on a centrifugal vacuum evaporator (speed-vac, savant, farmingdale, ny, usa) prior to hplc analysis using a agilent series lc (agilent technologies, palo alto, ca, usa). samples were loaded onto a zorbax eclipse c column (maintained at °c) in . % acetonitrile/ . % tfa/ . % acetic acid at . ml/ min, and eluted with a min linear gradient to % acetonitrile/ . % tfa. ace activity was detected in human urine, averaging ± pmol qfs/min/ml (figure ). in contrast, ace activity was much lower; our initial assay using -ll urine incubated for h resulted in very low activity, which was in fact undetectable in of samples. the assay was repeated with a lower volume of urine ( ll) to reduce background fluorescence, and extended incubation time ( h). ace activity under these conditions averaged . ± . nmol hip-his-leu/min/ml (n = ), with no detectable activity in of samples. the ratio of ace :ace was surprisingly high, as in our previous report , suggesting that ace is actively shed into the urine. this is consistent with the observation in the same publication that ace expressed in polarized renal proximal tubule epithelial cells (mdckii) is preferentially localized on the apical (luminal) side of the cell; in contrast, ace is equally distributed on both apical and basolateral cell surfaces . it is not yet known which part of the renal tubular system is the source of urinary ace activity. ace is localized to renal proximal tubules in human kidney (donoghue et al., ; lely et al., ) , whilst semiquantitative rt-pcr and immunohistochemistry of micro-dissected rat kidney suggest that the enzyme is expressed throughout the nephron, with highest expression in the proximal straight tubules and inner medullary collecting ducts (li et al., ) . in contrast, ace expression is confined to the glomeruli, and proximal straight and convoluted tubules in the human (schulz et al., ) and the rat (li et al., ) . thus it is possible that either the release of these ectoenzymes is restricted to the lower nephron, or that enzymes released within the proximal tubule (e.g., ace) are degraded along the nephron. interestingly, neprilysin (nep), another membrane-bound zinc metallopeptidase expressed in the renal brush border, is preferentially trafficked to the apical surface of renal epithelial cells (lanctoˆt et al., ) , like ace , and is present in relatively high amounts in urine. thus, as expected, secretion likely depends on the subcellular localization of the enzyme. immunoblot analysis of urinary ace revealed an immunoreactive band at approximately kda in each of two individual urine samples (figure ). in one of these samples ( figure b ), a second band was observed at $ kda, corresponding to the molecular mass of full-length recombinant ace ; a faint band at this position was present in the other urine sample as well ( figure a ). in contrast, immunoreactive ace could not be detected (data not shown), consistent with the much lower activity levels of this enzyme. following deglycosylation with pngase, the apparent molecular mass of the kda ace band in each sample was reduced to $ kda, while an additional band at $ kda was generated in the first urine sample, likely to have arisen from deglycosylation of the kda band. a smaller immunoreactive band at $ kda was also observed in both samples, but was not altered by pngase treatment; this may represent non-specific immunoreactivity of an unrelated protein, or possibly an unglycosylated degradation fragment, as which of the potential n-linked glycosylation sites in ace are post-translationally modified is not currently known. it is also not yet known whether urinary ace is a truncated form derived via proteolytic shedding, as demonstrated in cell culture (donoghue et al., ; lambert et al., ) , or a full-length, membranebound form. membrane-bound enzymes such as ace, nep, and aminopeptidase p have been shown to be present within urinary exosomes, small vesicles arising from intracellular multivesicular bodies, which are released from cells (pisitkun et al., ) . our observation of more than one immunoreactive band in urine suggests that urinary ace may constitute both a cleaved form and a full-length form, and that the relative amounts of these forms may vary between individuals or in different circumstances. the mass difference between these forms ($ kda) is similar to that seen in cell culture (lambert et al., ) , suggesting that ectodomain shedding of ace in vivo resembles that in vitro. in addition, significant ace activity ( . ± . % of total activity, n = ) was observed in the pellet following ultracentrifugation of urine, further suggesting that a sizeable portion of urinary ace exists in a full-length form. western blot analysis of these urine fractions revealed that while the majority of immunoreactive ace (still in two bands) was in the supernatant, a single ace -positive band was also seen in the pellet ( figure c) . furthermore, the apparent molecular mass of this insoluble ace was identical to that of the full-length, membrane-bound recombinant control, whereas the two bands in the soluble fraction were clearly of smaller size. these results suggest that the majority of urinary ace is derived from proteolytic shedding, although a portion apparently remains membrane-bound and may result from cell death or exosome release. further studies involving mass spectrometric analysis of affinity-purified urinary ace from both membrane and soluble urine fractions will be required to determine the precise nature of the ace species present. despite the clear presence of ace in urine by both qfs cleavage and immunoblotting, we were unable to detect any significant metabolism of angiotensin peptides that could be attributable to this enzyme. extended incubation of synthetic ang i and ii with dilute urine resulted in limited, but detectable degradation ( figure ). the addition of the ace inhibitor mln- had no discernible effect, nor did inhibition of ace (ramipril) and nep (thiorphan), both enzymes previously reported to be present and active in urine (data not shown). the lack of effect of the various peptidase inhibitors may be attributable to the relatively high concentration of angiotensin used ($ lm), well above its k m for these enzymes, which was necessary for identification of degradation fragments by uv absorbance. thus the more sensitive fluorescence-based assay is perhaps more appropriate to detect the relatively low levels of ace in urine. indeed, at concentrations near its k m for ace ( - lm), ang ii could readily inhibit qfs cleavage by urine at levels comparable to the mln inhibitor, both in soluble and pelleted fractions of urine (table i) . interestingly, individual urine samples displayed differences in the extent of inhibition by mln and ang ii (ranging from to %), suggesting the presence of other qfs-cleaving enzymes in some samples; however, the near-identical degree of inhibition by mln and ang ii in each sample supports the hypothesis that urinary ace cleaves ang ii. the absence of significant processing of synthetic angiotensin peptides by urine in our lc experiments does not necessarily discount a role for ace or other metallopeptidases in the metabolism of these peptides within the nephron. for example, li et al. ( ) describe the generation of ang - from ang i by microdissected rat proximal straight tubules, which was blocked by inhibition of ace by the peptide dx . surprisingly, proximal tubule ace did not cleave ang ii, its preferred substrate in vitro (rice et al., ) . this discrepancy may result from differences in experimental conditions, such as the ph or chloride concentration of buffers, both known to affect ace activity (guy et al., ) , or the concentration of angiotensin substrate used. as urinary levels of angiotensin peptides are quite low (variously reported between ) and ) m, depending on species and peptide), and require sensitive and specific techniques such as radioimmunoassays and mass spectrometry for accurate quantitation, it remains to be verified whether ace participates in angiotensin metabolism within the renal tubule. in this paper, we have shown, using both a sensitive catalytic assay and immunoblotting, that ace is present in normal human urine, at levels that are in excess of its homolog ace. although a role for ace in the metabolism of angiotensin peptides in urine or within the nephron has yet to be established, renal ace expression is greatly increased in a range of kidney diseases in man (lely et al., ) , as well as in a mouse model of type diabetes (ye et al., ) ; if these changes are reflected in urinary ace levels, measurement of this peptidase may have potential in the diagnosis or prognosis of renal diseases. proc. natl. acad. sci. usa key: cord- - qkzd w authors: ferreira, anderson j.; murça, tatiane m.; fraga-silva, rodrigo a.; castro, carlos henrique; raizada, mohan k.; santos, robson a. s. title: new cardiovascular and pulmonary therapeutic strategies based on the angiotensin-converting enzyme /angiotensin-( – )/mas receptor axis date: - - journal: int j hypertens doi: . / / sha: doc_id: cord_uid: qkzd w angiotensin (ang)-( – ) is now recognized as a biologically active component of the renin-angiotensin system (ras). the discovery of the angiotensin-converting enzyme homologue ace revealed important metabolic pathways involved in the ang-( – ) synthesis. this enzyme can form ang-( – ) from ang ii or less efficiently through hydrolysis of ang i to ang-( – ) with subsequent ang-( – ) formation. additionally, it is well established that the g protein-coupled receptor mas is a functional ligand site for ang-( – ). the axis formed by ace /ang-( – )/mas represents an endogenous counter regulatory pathway within the ras whose actions are opposite to the vasoconstrictor/proliferative arm of the ras constituted by ace/ang ii/at( ) receptor. in this review we will discuss recent findings concerning the biological role of the ace /ang-( – )/mas arm in the cardiovascular and pulmonary system. also, we will highlight the initiatives to develop potential therapeutic strategies based on this axis. the renin-angiotensin system (ras) plays a key role in several target organs, such as heart, blood vessels, and lungs, exerting a powerful control in the maintenance of the homeostasis [ ] [ ] [ ] [ ] . this system is activated by the conversion of the angiotensinogen to the inactive peptide angiotensin (ang) i through the renin action [ ] . subsequently, ang i is cleaved by the angiotensin-converting enzyme (ace) generating ang-( - ) by ace is important to regulate the ras activity since ang-( - ) induces opposite effects to those elicited by ang ii [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . additionally, ace can form ang-( - ) less efficiently through hydrolysis of ang i to ang-( - ) with subsequent ang-( - ) formation [ ] . the relevance of the ras is highlighted by the success obtained in therapeutic strategies based on the pharmacological inhibition of this system in cardiovascular and respiratory diseases [ ] [ ] [ ] [ ] [ ] [ ] . blockade of the ras with ace inhibitors (acei) or at receptor antagonists (arbs) improves the outcomes of patients with hypertension, acute myocardial infarction, and chronic systolic heart failure [ ] [ ] [ ] . furthermore, based on the involvement of the ace/ang ii/at axis in respiratory diseases and the crucial role of the lungs in the ras metabolism, several studies have reported the contribution of the ras in lung pathophysiology [ , , , [ ] [ ] [ ] [ ] [ ] . importantly, it has been shown that administration of acei and arbs causes substantial increases in plasma ang-( - ) levels, leading to the assumption that part of their clinical effects might be mediated by this heptapeptide [ ] [ ] [ ] . indeed, some effects of acei and arbs can be blocked or attenuated by a- , a mas antagonist, confirming the role of ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) in the actions of these compounds [ ] . the beneficial effects of ang-( - ), as well as its likely participation in the effects of the acei and arbs, represent evidences for the potential of the ace /ang-( - )/mas axis as a therapeutic target. in this review, we will focus on the recent findings related to the pathophysiology actions of the ace /ang-( - )/mas axis in the cardiovascular and respiratory system. also, we will discuss the promising initiatives to develop new therapeutic strategies based on this axis to treat pathological conditions. the heart is one of the most important targets for the actions of the ace /ang-( - )/mas axis. in the heart, ace international journal of hypertension is expressed in the endothelium [ ] , myofibroblasts [ ] , cardiomyocytes, and fibroblasts [ , ] . classical pharmacotherapeutic agents used to treat heart failure, including acei, arbs, and aldosterone receptor blockers, increase ace activity and/or expression, indicating its importance in the cardiac diseases establishment and progression [ ] [ ] [ ] . additionally, pharmacological and genetic (transgenic animals and gene transfer) approaches have evidenced the significance of ace in cardiac pathologies. despite some controversies concerning the consequences of the ace deficiency, in general, evidences indicate a protective role of ace in the heart [ , [ ] [ ] [ ] [ ] [ ] [ ] . crackower and colleagues [ ] were the first to demonstrate that genetic ablation of ace results in severe blood-pressure-independent systolic impairment. also, disruption of ace was able to accelerate cardiac hypertrophy and shortened the transition period to heart failure in response to pressure overload by increasing local ang ii [ ] . recently, it has been demonstrated that loss of ace enhances the susceptibility to myocardial infarction, with increased mortality, infarct expansion and adverse ventricular remodeling [ ] . in keeping with these genetic findings, pharmacological inhibition of ace exacerbated cardiac hypertrophy and fibrosis in ren- hypertensive rats [ ] . on the other hand, cardiac overexpression of ace prevented hypertension-induced cardiac hypertrophy and fibrosis in spontaneously hypertensive rats (shr) and in ang-ii-infused rats [ , ] . indeed, transfection of lenti-ace (lentivirus containing ace cdna) or ad-ace (recombinant adenovirus carrying the murine ace ) into the surrounding area of the infarcted myocardium was protective against pathological remodeling and cardiac systolic dysfunction in a rat model of myocardial infarction [ , ] . this effect was associated with decreased expression of ace and ang ii and increased expression of ang-( - ) [ ] . collectively, these observations reveal that ace effectively plays a protective role in the cardiac structure and function. since the discovery of ang-( - ) in the late s [ , ] , several studies have demonstrated important effects of this peptide in hearts. the presence of ang-( - ) and its receptor mas in the heart [ , ] and the ability of this organ to produce ang-( - ) [ , ] are evidences of the role of this peptide in cardiac tissues. functionally, ang-( - ) induces an antiarrhythmogenic effect against ischemia/reperfusion injuries in rats [ , ] as well as prevents atrial tachycardia and fibrillation in rats and dogs [ , ] . treatment with ang-( - ) improved the coronary perfusion and cardiac function in rats after myocardial infarction [ ] and after ischemia/reperfusion injury [ ] . increases in circulating ang-( - ) levels in transgenic rats reduced the cardiac hypertrophy [ ] and fibrosis [ , ] induced by isoproterenol administration. these effects are apparently independent of changes in blood pressure since grobe and colleagues [ ] have demonstrated that the antifibrotic and antihypertrophic actions of ang-( - ) are still observed in ang-ii-infused hypertensive rats. local overexpression of ang-( - ) in hearts of mice and rats improved the myocardial contractility and prevented the isoproterenol-and hypertension-induced cardiac remodeling [ , ] . altogether, these findings support a direct effect of ang-( - ) in the heart. further evidence for the role of ang-( - )/mas in the pathophysiology of the heart came from experimental protocols utilizing mice with genetic deficiency of mas. they revealed that the cardiac function is impaired in mas knockout mice likely due to the increased extracellular matrix proteins deposition in the heart [ , ] . this profibrotic phenotype may be related to changes in matrix metalloproteinases (mmps) and tissue inhibitors of metalloproteinases (timps) levels and/or activities [ , ] . although further elucidations regarding the signaling pathways involved in mas activation are necessary, some mechanisms have been proposed. overexpression of ang-( - ) in hearts of rats causes an improvement in the [ca + ] handling in cardiomyocytes and increases the expression of serca a [ ] . in keeping with these results, cardiomyocytes from mas-deficient mice present slower [ca + ] i transients accompanied by a lower ca + atpase expression in the sarcoplasmic reticulum [ , ] . although acute ang-( - ) treatment failed to alter ca + handling in ventricular myocytes of rats [ ] , these findings suggest an important role of the ang-( - )/mas in the long-term maintenance of the ca + homeostasis in the heart. one of the mechanisms by which ang-( - ) plays its effects in the heart is stimulating the nitric oxide (no) production. indeed, it has been demonstrated that ang-( - ) via mas increases the synthesis of no through a mechanism involving the activation of the endothelial no synthase (enos). these effects were abolished by a- and are absent in cardiomyocytes from mas-deficient mice [ ] . recently, gomes et al. [ ] found that the treatment of isolated cardiomyocytes of rats with ang-( - ) efficiently prevents the ang-ii-induced hypertrophy by modulating the calcineurin/nfat signaling cascade. these effects were blocked by no synthase inhibition and by guanylyl cyclase inhibitors, indicating that these effects are mediated by the no/cgmp pathway. also, ang-( - ) inhibits serum-stimulated mitogen-activated protein kinase (mapk) activation in cardiac myocytes [ ] and prevents the ang-ii-mediated phosphorylation of erk / and rho kinase in hearts in a dosedependent manner [ ] . in line with these data, activation of endogenous ace significantly reduced the phosphorylation of erk / in hearts of hypertensive rats (shrs) [ ] . however, mercure et al. [ ] reported that overexpression of ang-( - ) in hearts of rats decreases the ang-ii-induced phosphorylation of c-src and p kinase, whereas the increase in erk / phosphorylation was unaffected by the expression of the transgene, thereby suggesting a selective effect of ang-( - ) on intracellular signaling pathways related to cardiac remodeling. overall, these data reveal a key role of the ace /ang-( - )/mas axis in the pathophysiology of the cardiac structure and function. activation of this axis might be an important strategy to develop a new generation of cardiovascular therapeutic agents against cardiac dysfunction and pathological remodeling of the heart. early studies have reported the endothelium as the major site for generation [ ] and metabolism [ ] of ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in addition to ang-( - ), endothelial cells also express ace and mas [ , ] . thus, now it is recognized that the ace /ang-( - )/mas axis is present in vascular endothelial cells and modulates its function promoting vasorelaxation [ ] , reduction of the oxidative stress [ , ] , and antiproliferative effects [ , ] . the vasodilatory actions of ang-( - ) have been reported in many studies in several vascular beds and preparations, including mouse [ , ] and rat [ ] aortic rings, canine [ ] and porcine [ ] coronary arteries, canine middle cerebral artery [ ] , porcine piglet pial arterioles [ ] , feline mesenteric vascular bed [ ] , rabbit renal afferent arterioles [ ] , and mesenteric microvessels of normotensive [ ] and hypertensive [ ] rats. vascular ang-( - ) actions are still controversial in human. for example, it has been shown that ang-( - ) causes vasodilation in forearm circulation of normotensive subjects and patients with essential hypertension [ ] while other studies were unable to report any significant effect of ang-( - ) in the same vascular territory in acei-treated patients [ ] . the mas receptor is critically involved in the vascular effects of ang-( - ). in fact, many of these actions are completely abolished by a- or partially blocked by this antagonist [ , , ] . importantly, the endothelium-dependent relaxation induced by ang-( - ) in mouse aortic rings is absent in vessels derived from mas-knockout mice [ ] . however, other studies have shown that ang-( - ) also interacts with ace, at , and at -like receptors, suggesting the existence of additional sites of interaction for ang-( - ) [ , , ] . indeed, silva et al. [ ] reported evidence for the presence of a distinct subtype of ang-( - ) receptor sensible to d-pro -ang-( - ), a second mas antagonist, but not to a- in aortas of sprague-dawley rats. the vascular effects of ang-( - ) are endothelium dependent and involve the production of vasodilator products, such as prostanoids, no, and endothelium-derived hyperpolarizing factor (edhf) [ , , ] . pinheiro and coworkers [ ] found that ang-( - ) promotes an increase in no release in mas-transfected chinese hamster ovary (cho) cells [ ] . furthermore, short-term infusion of ang-( - ) improved the endothelial function by a mechanism involving no release in rats [ ] . mas deletion resulted in endothelial dysfunction associated with an unbalance between no and oxidative stress [ ] . also, mas activation by ang-( - ) in human endothelial cells stimulated enos phosphorylation/activation via the aktdependent pathway [ ] . other mechanisms appear to be involved in the ang-( - ) vascular actions. roks et al. [ ] have shown that ang-( - ) inhibits the vasoconstriction induced by ang ii in human internal mammary arteries, thereby suggesting that ang-( - ) can regulate the ang ii effects [ ] . in fact, ang-( - ) negatively modulates the ang ii type receptor-mediated activation of c-src, and its downstream targets erk / and nad(p)h oxidase [ ] . the counterregulatory action of ang-( - ) on ang ii signaling has been also observed in cardiomyocytes [ ] , vascular smooth muscle cells [ ] , and fibroblasts [ ] . additionally, an interaction between mas and bradykinin (bk) type (b ) receptors may modulate some of the ang-( - ) effects in blood vessels [ ] . indeed, it has been demonstrated that ang-( - ) potentiates the vasodilator and hypotensive effects of bk in several vascular beds [ , [ ] [ ] [ ] . as the major enzyme involved in ang-( - ) formation, ace has also a crucial role in vessels. lovren et al. [ ] have demonstrated that ace ameliorates the endothelial homeostasis via a mechanism involving reduction of the reactive oxygen species production [ ] . of note, this effect was attenuated by a- [ ] . moreover, overexpression of ace in vessels of hypertensive rats resulted in reduction in the arterial blood pressure and improvement of the endothelial function associated with increased circulating ang-( - ) levels [ ] . overall, these data indicate that the beneficial effects of ace are, at least in part, mediated by ang- ( - ) . recently, we have demonstrated that activation of endogenous ace causes a dose-dependent hypotensive effect in normotensive and hypertensive rats [ ] . also, the response to bk administration was augmented in rats chronically treated with xnt, an ace activator [ ] . however, we were unable to demonstrate any significant effect of xnt on blood pressure in response to the administration of ang ii or losartan in normotensive and hypertensive rats ( figure ). in the past few years, the participation of the ace /ang-( - )/mas axis in the establishment and progression of pulmonary diseases has become evident. indeed, the important role of the ras in the lung pathophysiology and the side effects and pulmonary toxicity induced by the acei raised the interest to evaluate the activation of the ace /ang-( - )/mas axis as an alternative target to treat pulmonary pathologies. thus, it has been reported beneficial outcomes induced by the activation of this axis in animal models of acute respiratory distress syndrome (ards), pulmonary hypertension (ph), fibrosis, and lung cancer [ , , [ ] [ ] [ ] [ ] . these studies pointed out that the imbalance between the ace/ang ii/at and the ace /ang-( - )/mas axes of the ras might be relevant in lung diseases. taking into account that systemic hypotension is an important limitation to the use of acei and arbs in pulmonary patients, therapies based on the ace /ang-( - )/mas axis emerge as a safe and efficient approach since studies using the ace activator xnt or ace gene transfer have shown that these strategies induce beneficial pulmonary outcome without changes in systemic blood pressure in rats and mice [ , , ] . imai and colleagues [ ] demonstrated the role of ace in ards pathogenesis. they found that a more severe ards was reached in ace knockout mice, and this phenotype was reversed by double genetic deletion of the ace and ace genes or by the treatment with recombinant human ace (rhace ). furthermore, ang ii levels were related international journal of hypertension to the severity of the lung injury. of note, ace is widely expressed in the pulmonary endothelium, vasculature, and pneumocytes [ , ] . also, rhace inhibited the increase of ang ii and tnf-α levels, attenuated the arterial hypoxemia and ph, and ameliorated the distribution of the pulmonary blood flow in lipopolysaccharide-induced lung injury in piglets [ ] . therefore, these studies suggest that ace is a suitable target to arrest the development of ards in patients at risk. the stimulation of the ace /ang-( - )/mas axis has been successful used to prevent and reverse ph and fibrosis in animals. ace activation using the compound xnt or induction of ace overexpression by gene transfer efficiently prevented and, more importantly, reversed the increase of the right systolic ventricular pressure (rsvp), pulmonary fibrosis, imbalance of the ras, and inflammation in animals (rats and mice) with ph induced by monocrotaline (mct) or in rats with pulmonary fibrosis caused by bleomycin treatment [ , , ] . in keeping with these findings, ang-( - ) gene transfer into the lungs triggered similar protective actions in mct-treated rats [ ] . in addition, ang-( - ) via mas prevented the apoptosis of alveolar epithelial cells and the jun n-terminal kinase (jnk) activation induced by bleomycin [ ] . the involvement of the ang-( - )/mas in ph was further evidenced by the observation that the xnt effects are blocked by a- [ ] . furthermore, in both lung specimens from patients with idiopathic pulmonary fibrosis and from animals with bleomycin-induced pulmonary fibrosis were reported a reduction in mrna, protein, and activity of ace with a reciprocal increase in ang ii level [ ] . a growing body of studies has focused on the relevance of the ace /ang-( - )/mas axis in the pulmonary cancer pathophysiology. the protein expression of ace is reduced in non-small-cell lung carcinoma (nsclc) along with an increase in ang ii levels. moreover, overexpression of ace in cultured a lung cancer cells and in human lung cancer xenografs inhibited the cell growth and the vascular endothelial growth factor-a (vegfa) expression induced by ang ii [ , ] . gallagher and tallant [ ] evaluated the effects of several angiotensin peptides [ang i, ang ii, ang-( - ), ang- ( ) ( ) ( ) ( ) ( ) ( ) , and ang-( - )] in sk-lu- cancer cells growth, and only ang-( - ) showed significant attenuation of the dna synthesis and proliferation. the antiproliferative effect of ang-( - ) was mediated by its receptor mas and inhibition of the erk / pathway. neither the blockage of at nor at succeeded in inhibiting the action of ang-( - ). in keeping with these data, the antiproliferative effect of ang-( - ) was observed in human a lung tumor xenograft growth along with a marked decrease in the vessel density in mice through a mechanism involving cyclooxygenase- (cox- ) [ , ] . of note, in a nonrandomized phase i clinical trial conducted by petty and colleagues [ ] , subcutaneous injections of ang-( - ) were administered in patients with advanced solid tumors refractory to standard therapy. despite the mild adverse effects observed with the ang-( - ) treatment, generally it was well tolerated. there were no treatment-related deaths. clinical benefits were observed in % of the patients. altogether, these studies provide insights into the involvement of the ace /ang-( - )/mas axis in lung cancer. many advances have been achieved regarding the therapeutic regulation of the ras. current therapies based on the modulation of the ras include the acei, arbs, and renin inhibitors. in general, these drugs prevent or reverse endothelial dysfunction and atherosclerosis, reduce cardiovascular mortality and morbidity of patients with coronary artery disease, and hold antihypertensive effects [ ] . classically, the mechanisms of action of the acei and arbs involve the blockade of the synthesis and actions of ang ii, respectively. however, the ras is a complex hormonal system and, consequently, other mechanisms are likely implicated in the actions of these drugs [ , , ] . they cause substantial increase in plasma levels of ang-( - ), leading to the assumption that their clinical effects might be partly mediated by this heptapeptide [ , ] . indeed, a variety of effects of the acei and arbs can be abolished or attenuated by mas antagonism, confirming the role of ang-( - ) in the actions of these compounds [ , ] . the beneficial effects of ang-( - ) as well as its likely involvement in the effects of the acei and arbs represent a strong evidence for the therapeutic potential of the activation of the ace /ang-( - )/mas axis (figure ). ang-( - ) formulations. the beneficial effects of ang-( - ) are well known; however, the therapeutic utilization of this peptide is limited due to its unfavorable pharmacokinetic properties. ang-( - ) has a short half-life (approximately seconds) since it is rapidly cleaved by peptidases [ ] . furthermore, ang-( - ) is degraded during its passage through the gastrointestinal tract when orally administrated. thus, new strategies are crucial to make feasible the clinical application of ang-( - ) . recently, a formulation based on the ang-( - ) included into hydroxypropyl β-cyclodextrin [hpβcd/ang-( - )] was developed by lula and colleagues [ ] . cyclodextrins are pharmaceutical tools used for design and evaluation of drug formulations, and they enhance the drug stability and absorption across biological barriers and offer gastric protection [ ] . the amphiphilic character of cyclodextrins allows the possibility of formation of supramolecular inclusion complexes stabilized by noncovalent interactions with a variety of guest molecules [ , ] . in this regard, the formulation hpβcd/ang-( - ) allowed the oral administration of ang-( - ). pharmacokinetic and functional studies showed that oral hpβcd/ang-( - ) administration significantly increases plasma ang-( - ) levels and promotes an antithrombotic effect that was blunted in mas deficient mice [ ] . marques and colleagues [ ] have found that chronic oral administration of hpβcd/ang-( - ) significantly attenuates the heart function impairment and cardiac remodeling induced by isoproterenol treatment and myocardial infarction in rats [ ] . in addition, liposomal delivery systems represent an alternative method to administer ang-( - ) [ ] . administration of liposomes containing ang-( - ) in rats led to prolonged hypotensive effect for several days in contrast to the response observed when the free peptide was used [ , ] . a strategy used to protect the ang-( - ) against proteolytic degradation was proposed by kluskens and coworkers [ ] . using the ability of prokaryotes to cyclize peptides, they synthesized a cyclic ang-( - ) derivative [thioetherbridged ang-( - )] which presented an increased stability in homogenates of different organs and plasma and enhanced the ang-( - ) bioavailability in rats [ ] . furthermore, cyclized ang-( - ) induced a relaxation in precontracted aorta rings of rats which was blocked by the ang-( - ) receptor antagonist d-pro -ang-( - ), providing evidence that cyclized ang-( - ) also interacts with mas [ ] . agonists. ave was the first nonpeptide synthetic compound developed with the intention of stimulating the mas receptor. this compound mimics the ang-( - ) effects in several organs such as vessels [ , ] , kidney [ ] , and heart [ , ] . similar to ang-( - ), ave induced a vasodilation effect which was absent in aortic rings of mas-deficient mice [ ] . moreover, its effects in aortic rings were blocked by the two ang-( - ) receptor antagonists, a- and d-pro -ang-( - ) [ ] . ave potentiated the acetylcholine-induced vasodilation in conscious normotensive rats, and this effect was abolished by a- and l-name [ ] . similarly, it was able to increase the hypotensive effect of bk in normotensive rats, and a- also blocked this effect [ ] . ferreira et al. [ , ] reported that ave protects the heart against cardiac dysfunction and remodeling caused by isoproterenol treatment or by myocardial infarction in rats [ , ] . in mas-transfected cells, ave induced no release which was blunted by a- and not by at or at antagonists [ ] . all these data support the concept that ave is an ang-( - ) mimetic and that its actions are mediated by the interaction with mas. using a computational discovery platform for predicting novel naturally occurring peptides that may activate gpcr, two novel peptides, designated as cgen- and cgen- , with amino acid sequence unrelated to angiotensin peptides, were found to display high specificity for mas [ ] . these peptides elicited ca + influx in cho cells overexpressing mas without any activity in at or at receptors [ ] . cgen- s, a derivative of the cgen- peptide, induced beneficial cardiovascular effects similar to those caused by ang-( - ) [ ] . this compound competes with ang-( - ) for the same bind site in mas-transfected cells. furthermore, similar to ang-( - ), cgen- s produced a vasodilation effect which was absence in mas-deficient mice, indicating that this compound also acts via mas [ ] . this was confirmed by the inhibition of the cgen- s effects by the mas antagonist a- . importantly, savergnini et al. [ ] showed that cgen- s promotes antiarrhythmogenic effects and produces a small dose-dependent decrease in arterial pressure of conscious shr [ ] . a new approach addressing the therapeutic potential of the activation of the ace /ang-( - )/mas axis was proposed by hernández prada et al. [ ] . based on the crystal structure of ace and using a virtual screening strategy, it was identified small molecules that may interact with this enzyme leading to changes in its conformation and, consequently, enhancing its activity [ ] . thus, the ace activator, namely xnt, was identified and its administration in shr decreased blood pressure, induced an improvement in cardiac function, and reversed the myocardial and perivascular fibrosis observed in these animals [ , ] . the beneficial effects of xnt were also observed in rats with ph induced by mct [ ] . furthermore, this compound attenuated the thrombus formation and reduced the platelet attachment to vessels in hypertensive rats [ ] . it appears that the pharmacological activation of ace promotes its beneficial effects due to an increased ang-( - ) production with concomitant degradation of ang ii. in fact, coadministration of a- abolished the protective effects of xnt on ph [ ] . in addition, the antifibrotic effect of xnt observed in hearts of shr was associated with increases in cardiac ang-( - ) expression [ ] . however, it is also pertinent to point out that off-target effects of xnt on these beneficial outcomes cannot be ruled out at the present time. the complexity of the ras is far beyond we could suspect few years ago. there is growing evidence that changes in the novel components of the ras [ang-( - ), ace , and mas] may take part of the establishment and progression of cardiovascular and respiratory diseases. importantly, these new components of the ras, due to their counter regulatory actions, are candidates to serve as a concept to develop new cardiovascular and respiratory drugs. role of the reninangiotensin system in control of sodium excretion and arterial pressure kidneys and fluids in pressure regulation: small volume but large pressure changes angiotensin-( - ): an update the pulmonary renin-angiotensin system role of the renin-angiotensin-aldosterone system and proinflammatory mediators in cardiovascular disease purification and properties of angiotensin i-converting enzyme in human lung and its role on the metabolism of vasoactive peptides in pulmonary circulation a memorial to robert tiegerstedt: the centennial of renin discovery recent advances in angiotensin ii signaling biological functions of angiotensin and its receptors localization and function of angiotensin at receptors molecular and cellular mechanisms of angiotensin ii-mediated cardiovascular and renal diseases angiotensin ii cell signaling: physiological and pathological effects in the cardiovascular system angiotensin type receptor dephosphorylates bcl- by activating mitogen-activated protein kinase phosphatase- and induces apoptosis role of at receptors in angiotensin ii-stimulated contraction of small mesenteric arteries in young shr angiotensin-( - ) and the rat aorta: modulation by the endothelium angiotensin-( - ) is an endogenous ligand for the g proteincoupled receptor mas expression of an angiotensin-( - )-producing fusion protein produces cardioprotective effects in rats prevention of angiotensin ii-induced cardiac remodeling by angiotensin angiotensin( - ) blunts hypertensive cardiac remodeling by a direct effect on the heart reduced isoproterenol-induced reninangiotensin changes and extracellular matrix deposition in hearts of tgr(a - ) rats attenuation of isoproterenol-induced cardiac fibrosis in transgenic rats harboring an angiotensin-( - )-producing fusion protein in the heart lifetime overproduction of circulating angiotensin-( - ) attenuates deoxycorticosterone acetate-salt hypertensioninduced cardiac dysfunction and remodeling vascular relaxation, antihypertensive effect, and cardioprotection of a novel peptide agonist of the mas receptor hydrolysis of biological peptides by human angiotensin-converting enzymerelated carboxypeptidase a human homolog of angiotensinconverting enzyme: cloning and functional expression as a captopril-insensitive carboxypeptidase a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - the renin-angiotensin system: importance in physiology and pathology lisinopril attenuates acute hypoxic pulmonary vasoconstriction in humans the importance of the renin-angiotensin system in cardiovascular disease angiotensin ii is mitogenic for human lung fibroblasts via activation of the type receptor pulmonary capillary endothelium-bound angiotensin-converting enzyme activity in acute lung injury the tissue reninangiotensin system and intracellular signalling vascular and cardiac benefits of angiotensin receptor blockers reninangiotensin-aldosterone system blockade for cardiovascular diseases: current status renin-angiotensinaldosterone blockade for cardiovascular disease prevention compared evolution of plasma fibronectin and angiotensin-converting enzyme levels in septic ards angiotensin-converting enzyme protects from severe acute lung failure phase i and pharmacokinetic study of angiotensin-( - ), an endogenous antiangiogenic hormone the angiotensin-converting enzyme /angiogenesis-( - )/mas axis confers cardiopulmonary protection against lung fibrosis and pulmonary hypertension regulation of alveolar epithelial cell survival by the ace- /angiotensin - /mas axis metabolism of angiotensin-( - ) by angiotensin-converting enzyme angiotensin-( - ) contributes to the antihypertensive effects of blockade of the renin-angiotensin system effect of angiotensin-( - ) and bradykinin in patients with heart failure treated with an ace inhibitor role of angiotensin-( - ) in the modulation of the baroreflex in renovascular hypertensive rats the role of ace in cardiovascular physiology functional angiotensin-converting enzyme is expressed in human cardiac myofibroblasts regulation of ace in cardiac myocytes and fibroblasts angiotensin-converting enzyme activation protects against hypertension-induced cardiac fibrosis involving extracellular signal-regulated kinases effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme mineralocorticoid receptor blocker increases angiotensinconverting enzyme activity in congestive heart failure patients olmesartan improves left ventricular function in pressure-overload hypertrophied rat heart by blocking angiotensin ii receptor with synergic effects of upregulation of angiotensin converting enzyme angiotensinconverting enzyme is an essential regulator of heart function altered blood pressure responses and normal cardiac phenotype in ace -null mice deletion of angiotensin-converting enzyme accelerates pressure overload-induced cardiac dysfunction by increasing local angiotensin ii primary role of angiotensin-converting enzyme- in cardiac production of angiotensin-( - ) in transgenic ren- hypertensive rats loss of angiotensinconverting enzyme accelerates maladaptive left ventricular remodeling in response to myocardial infarction prevention of angiotensin ii-mediated renal oxidative stress, inflammation, and fibrosis by angiotensin-converting enzyme inhibition of angiotensin-converting enzyme exacerbates cardiac hypertrophy and fibrosis in ren- hypertensive rats protection from angiotensin ii-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ace in rats ace gene transfer attenuates hypertension-linked pathophysiological changes in the shr cardiac overexpression of angiotensin converting enzyme protects the heart from ischemia-induced pathophysiology ace overexpression ameliorates left ventricular remodeling and dysfunction in a rat model of myocardial infarction converting enzyme activity and angiotensin metabolism in the dog brainstem release of vasopressin from the rat hypothalamo-neurohypophysial system by angiotensin-( - ) heptapeptide cardiac angiotensin-( - ) in ischemic cardiomyopathy impairment of in vitro and in vivo heart function in angiotensin-( - ) receptor mas knockout mice production of angiotensin-( - ) by human vascular endothelium angiotensin-( - ): cardioprotective effect in myocardial ischemia/ reperfusion mas receptor axis is expressed in sinoatrial node cells of rats enalapril, irbesartan, and angiotensin-( - ) prevent atrial tachycardiainduced ionic remodeling angiotensin-( - ) attenuates the development of heart failure after myocardial infarction in rats angiotensin-( - ) improves the post-ischemic function in isolated perfused rat hearts effects of genetic deletion of angiotensin-( - ) receptor mas on cardiac function during ischemia/reperfusion in the isolated perfused mouse heart interplay of angiotensin ii and angiotensin( - ) in the regulation of matrix metalloproteinases of human cardiocytes angiotensin-( - ) ameliorates myocardial remodeling and interstitial fibrosis in spontaneous hypertension: role of mmps/timps molecular mechanisms involved in the angiotensin mas signaling pathway in cardiomyocytes angiotensin-( - ) prevents cardiomyocyte pathological remodeling through a nitric oxide/guanosine , -cyclic monophosphate-dependent pathway angiotensin-( - ) inhibits growth of cardiac myocytes through activation of the mas receptor angiotensin-( - ) has a dual role on growth-promoting signalling pathways in rat heart in vivo by stimulating stat and stat a/b phosphorylation and inhibiting angiotensin ii-stimulated erk / and rho kinase activity ace , a new regulator of the renin-angiotensin system angiotensin-( - ) through receptor mas mediates endothelial nitric oxide synthase activation via akt-dependent pathways angiotensin-( - ) and its receptor as a potential targets for new cardiovascular drugs endothelial dysfunction and elevated blood pressure in mas gene-deleted mice ace -angiotensin-( - )-mas axis and oxidative stress in cardiovascular disease molecular mechanisms of inhibition of vascular growth by angiotensin therapeutic implications of the vasoprotective axis of the reninangiotensin system in cardiovascular diseases angiotensin-( - ) dilates canine coronary arteries through kinins and nitric oxide release of nitric oxide by angiotensin-( - ) from porcine coronary endothelium: implications for a novel angiotensin receptor angiotensin-( - ) causes endothelium-dependent relaxation in canine middle cerebral artery comparative effects of angiotensin-( - ) and angiotensin ii on piglet pial arterioles differential responses to angiotensin-( - ) in the feline mesenteric and hindquarters vascular beds vasodilator action of angiotensin-( - ) on isolated rabbit afferent arterioles synergistic effect of angiotensin-( - ) on bradykinin arteriolar dilation in vivo potentiation of bradykinin by angiotensin-( - ) on arterioles of spontaneously hypertensive rats studied in vivo effects of angiotensin-( - ) on forearm circulation in normotensive subjects and patients with essential hypertension a- attenuates angiotensin-( - ) depressor response in salt-induced hypertensive rats angiotensin-( - ) augments bradykinin-induced vasodilation by competing with ace and releasing nitric oxide angiotensin-( - ) acts as a vasodepressor agent via angiotensin ii type receptors in conscious rats evidence for a new angiotensin-( - ) receptor subtype in the aorta of sprague-dawley rats signal transduction mechanisms involved in angiotensin-( - )-stimulated arachidonic acid release and prostanoid synthesis in rabbit aortic smooth muscle cells nonpeptide ave is an angiotensin-( - ) receptor mas agonist in the mouse kidney shortterm angiotensin( - ) receptor mas stimulation improves endothelial function in normotensive rats angiotensin-( - ) is a modulator of the human renin-angiotensin system inhibition of pkc and erk / in cultured rat vascular smooth muscle cells by angiotensin the counterregulating role of ace and ace -mediated angiotensin - signaling against angiotensin ii stimulation in vascular cells infusion of angiotensin-( - ) reduces glomerulosclerosis through counteracting angiotensin ii in experimental glomerulonephritis evidence for mas-mediated bradykinin potentiation by the angiotensin-( - ) nonpeptide mimic ave in normotensive rats angiotensin( - ) potentiates bradykinin-induced vasodilatation in man potentiation of the hypotensive effect of bradykinin by short-term infusion of angiotensin-( - ) in normotensive and hypertensive rats angiotensin-( - ) potentiates the coronary vasodilatatory effect of bradykinin in the isolated rat heart angiotensin converting enzyme- confers endothelial protection and attenuates atherosclerosis transgenic angiotensin-converting enzyme overexpression in vessels of shrsp rats reduces blood pressure and improves endothelial function structure-based identification of small-molecule angiotensin-converting enzyme activators as novel antihypertensive agents haemodynamic and hormonal effects of captopril in primary pulmonary hypertension antifibrotic effect of captopril and enalapril on paraquat-induced lung fibrosis in rats angiotensin converting enzyme- is protective but downregulated in human and experimental lung fibrosis evidence for angiotensin-converting enzyme as a therapeutic target for the prevention of pulmonary hypertension prevention of pulmonary hypertension by angiotensin-converting enzyme gene transfer tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis reverse transcription polymerase chain reaction (rt-pcr) analysis of proteolytic enzymes in cultures of human respiratory epithelial cells recombinant angiotensin-converting enzyme improves pulmonary blood flow and oxygenation in lipopolysaccharide-induced lung injury in piglets regulation of alveolar epithelial cell survival by the ace- /angiotensin - / mas axis the angiotensin-converting enzyme in tumor growth and tumor-associated angiogenesis in non-small cell lung cancer overexpression of ace produces antitumor effects via inhibition of angiogenesis and tumor cell invasion in vivo and in vitro inhibition of human lung cancer cell growth by angiotensin angiotensin-( - ) inhibits growth of human lung adenocarcinoma xenografts in nude mice through a reduction in cyclooxygenase- angiotensin-( - ) inhibits tumor angiogenesis in human lung cancer xenografts with a reduction in vascular endothelial growth factor the role of the renin-angiotensin system in the development of cardiovascular disease antithrombotic effect of captopril and losartan is mediated by angiotensin vasodepressor actions of angiotensin-( - ) unmasked during combined treatment with lisinopril and losartan the role of ang ( - ) in mediating the chronic hypotensive effects of losartan in normal rats converting enzyme determines plasma clearance of angiotensin study of angiotensin-( - ) vasoactive peptide and its β-cyclodextrin inclusion complexes: complete sequence-specific nmr assignments and structural studies design and evaluation of cyclodextrin-based drug formulation an orally active formulation of angiotensin-( - ) produces an antithrombotic effect an oral formulation of angiotensin-( - ) produces cardioprotective effects in infarcted and isoproterenol-treated rats site-specific microinjection of liposomes into the brain for local infusion of a short-lived peptide long-lasting cardiovascular effects of liposomeentrapped angiotensin-( - ) at the rostral ventrolateral medulla angiotensin-( - ) with thioether bridge: an angiotensin-converting enzyme-resistant, potent angiotensin-( - ) analog the endothelium-dependent vasodilator effect of the nonpeptide ang( - ) mimic ave is abolished in the aorta of mas-knockout mice ave , a nonpeptide mimic of the effects of angiotensin-( - ) on the endothelium the nonpeptide angiotensin-( - ) receptor mas agonist ave- attenuates heart failure induced by myocardial infarction isoproterenol-induced impairment of heart function and remodeling are attenuated by the nonpeptide angiotensin discovery and validation of novel peptide agonists for g-protein-coupled receptors ace activation promotes antithrombotic activity key: cord- -x kankxd authors: romero, cesar a.; orias, marcelo; weir, matthew r. title: novel raas agonists and antagonists: clinical applications and controversies date: - - journal: nat rev endocrinol doi: . /nrendo. . sha: doc_id: cord_uid: x kankxd the renin–angiotensin–aldosterone system (raas) regulates blood pressure homeostasis and vascular injury and repair responses. the raas was originally thought to be an endocrine system critically important in regulating blood pressure homeostasis. yet, important local forms of the raas have been described in many tissues, which are mostly independent of the systemic raas. these systems have been associated with diverse physiological functions, but also with inflammation, fibrosis and target-organ damage. pharmacological modulation of the raas has brought about important advances in preventing morbidity and mortality associated with cardiovascular disease. yet, traditional raas blockers such as angiotensin converting enzyme (ace) inhibitors and angiotensin receptor blockers (arbs) only reduce the risk of disease progression in patients with established cardiovascular or renal disease by ∼ % compared with other therapies. as more components of the raas are described, other potential therapeutic targets emerge, which could provide improved cardiovascular and renal protection beyond that provided by an ace inhibitor or arb. this review summarizes the present and future pharmacological manipulation of this important system. supplementary information: the online version of this article (doi: . /nrendo. . ) contains supplementary material, which is available to authorized users. more than seven decades ago, angiotensin, a poly peptide that is the product of the enzymatic activity of renin, was found in venous samples of ischaemic dog kidneys. in the s and s, two forms of angiotensin were reported: angiotensin- (ang i), which is amino acids long, and angiotensin- (ang ii), with amino acids. the latter form results from the metabolism of ang i by a dipeptidyl carboxypeptidase named angiotensinconverting enzyme (ace). the substrate of renin was found to be angiotensinogen, a serum globulin produced by the liver. also at this time, the role of ang ii in the regulation of aldosterone production by the adrenal cortex emerged. in the s, the main catalytic cascade of the reninangiotensin-aldosterone system (raas) was described ( figure ). plasma angiotensinogen is cleaved by renal renin, producing ang i, which is then converted to ang ii by endothelial ace, a process that occurs most extensively in lung tissue. ang ii was considered the most important raas mediator, with increased levels of ang ii being associated with vasoconstriction and increased blood pressure. ang ii binds to the type- ang ii receptor (at ) in a variety of tissues, including vascular smooth muscle and the adrenal grand, to mediate many mechanisms that lead to raised blood pressure. the stimulation of aldosterone production via the at receptor in the adrenal gland facilitates sodium retention by the kidney when aldosterone binds to the mineralocorticoid receptor. on the basis of the first experimental studies of ang ii, in which supraphysiological doses of this peptide were tested in dogs, the raas was related to the pathophysiology of hypertension mainly through the effects of this system on vascular constriction. however, more recent studies have helped clarify that blood pressure control by ang ii at physiological concentrations is more related to sodium and water handling than to arterial constriction. ang ii shifts the natriuresis-blood pressure curve, with increased levels of ang ii requiring increased blood pressure to eliminate the same quantity of sodium. part of this effect occurs via direct mediation by ang ii, which activates sodium transporters in the proximal tubules of the kidney. [ ] [ ] [ ] furthermore, ang ii, through binding to the at receptors, is the most potent regulator of aldosterone secretion in the adrenal cortex. , aldosterone binding to the mineralocorticoid receptor induces nongenomic (rapid) and genomic effects in many tissues, but mainly in the kidney, where it increases the epithelial expression of epithelial sodium channel (enac, also known as amiloride-sensitive sodium channel) at the level of the distal tubules, which favours the retention of sodium and water. blockade of the raas was a major breakthrough in the treatment of cardiovascular disease, lowering mortality and improving quality of life in patients with hyper tension, chronic kidney disease (ckd), myocardial infarction and heart failure. [ ] [ ] [ ] [ ] [ ] the classic raas antagonists target the angiotensinogen-angiotensin-at -aldosterone axis. however, as knowledge about the raas expands, the number of potential therapeutic targets in this system is increasing. in this review we the components of the raas are schematically represented in figure . renin is the rate-limiting step in ang ii production and is released by the juxta glomerular apparatus in the kidney in response to decreased renal perfusion pressure, low tubular salt load or sympathetic activation. renin is synthesized from prorenin, a pre-p roenzyme that is constitutively released, with plasma levels -fold higher than those of active renin. prorenin can be proteolytically activated in the kidney by neuroendocrine convertase (also known as prohormone convertase and proprotein convertase ) or cathepsin b and non-proteolytically (reversibly) in many tissues by the renin receptor (also known as renin/p rorenin receptor). these two types of activation require the propeptide to expose its active site, a cleft between two similar domains. the renin receptor also binds to mature renin, increasing its catalytic activity by - fold. the renin receptor can initiate ang ii-independent signal ling pathways, such as the mapk/erk pathway and pathways involving the zinc finger protein plzf. the effects of these signalling pathways are not clear and the renin receptor has not been shown to have a role in cardiovascular disease when the effects of ang ii are blocked. the secretion of renin is controlled by short-term and long-term negative feedback loops. short-term regulation is mediated directly by ang ii through binding to at , which induces inhibition of renin gene expression. the long-term negative feedback loop is an indirect consequence of the effects of increased ang ii levels: increased blood pressure, sympathetic inhibition of baroreceptor mechanisms and increased intratubular sodium levels. the classic effects of ang ii are mediated by the at receptor, which is present in practically all tissues (box ). blood pressure effects are modulated by vascular at receptors, whose activity produces vasoconstriction (rapid pressure effects) and by renal at receptors that mediate sodium and water reabsorption. many fibrotic and inflammatory effects of ang ii are also a consequence of at receptor activation. however, ang ii can also activate the type- ang ii receptor (at ), which is more sparsely expressed in body tissues than the at receptor, but which is upregulated in many cases of injury (for example, to the brain, heart and kidney). at is part of the protective arm of the raas, and, when activated, has antifibrotic and anti-inflammatory effects (box ). activation of the at receptor does not seem to have blood pressure effects. other non-classic raas components have been described in the past few years. a novel enzyme similar to ace was identified and called angiotensin-converting enzyme (encoded by ace and hereby referred to as ace ). this carboxypeptidase has great ang ii affinity and can transform this peptide to angiotensin - (hereby referred to as ang - ). ace can also convert ang i to angiotensin - (ang - ), which is then converted to ang - by ace. similarly, plasmatic endopeptidases can convert ang i to ang - . ang - binds to a g-protein-coupled receptor called proto-oncogene mas (encoded by mas and hereby referred to as mas ). this ace -ang - -mas axis has been reported to function as a counterbalance to the classic ace-ang ii-at axis, with opposite effects than those mediated by the classic axis (box ). another non-classic axis in the raas is the pathway mediated by angiotensin- (ang iv) and its receptor, leucyl-cystinyl aminopeptidase (also known as insulin-regulated membrane aminopeptidase, insulinresponsive aminopeptidase or irap). , this receptor, which was originally reported to colocalize with the glucose transporter glut- , has a role in the degradation of vaso pressin and oxytocin. ang iv inhibits the enzymatic activity of irap and knockout models and studies of non-peptide inhibitors suggest a role of irap in c ardiovascular disease, mainly in target-organ damage. [ ] [ ] [ ] the effects of irap inhibition are listed in box . beyond the original description of the endocrine roles of the raas, experimental studies have demonstrated that the system also has paracrine, autocrine and intracrine functions. different tissues (reproductive and digestive tissues, heart and brain) and cells (adipocytes and white blood cells) express raas components, which not only perform the classic raas function (fluid volume homeostasis), but are also involved in other physiological functions, such as ovulation, secretion of insulin by the pancreas, cognitive function, inflammation and cancer. , in addition, activation of local raass is strongly related to target-organ damage, mostly in the kidney and heart. four clinically established groups of drugs inhibit the raas: ace inhibitors, angiotensin receptor blockers (arbs, which block the at receptor), renin inhibitors and mineralocorticoid receptor blockers (box ). despite the important improvements achieved with these agents in slowing the progression of established cardiorenal disease, the ace inhibitors and the arbs only provide a % reduction in the relative risk of key points ■ renin-angiotensin-aldosterone system (raas) blockade with an angiotensin converting enzyme inhibitor or an angiotensin receptor blocker provides a % relative risk reduction for the progression of established cardiorenal disease compared with other non-raas blocking therapies ■ the raas is an endocrine, paracrine and autocrine system that regulates blood pressure homeostasis through effects on a variety of target organs, as well as having a role in the responses to vascular injury and repair ■ the raas is a complex system with a variety of sites suitable for pharmacological intervention ■ novel molecules that alter the production of various raas peptides or that alter receptor density, function or responsiveness to these peptides could have an important influence on haemodynamics and vascular structure and function www.nature.com/nrendo progression of cardiovascular disease when compared with non-raas blocking therapy. , , , moreover, no data have demonstrated beneficial effects of ace inhibitors or arbs in the primary prevention of either cardiac or kidney disease. therefore, much consideration has been given to how to improve upon raas inhibition with the classic, established drugs, by investigating new agonists and antagonists of the complex raas cascade. many theories about why traditional raas inhibition has not provided more clinical benefits have been proposed, but none of these theories has been validated clinically. in the early s, early clinical studies of the first oral ace inhibitor, captopril, demonstrated this agent has antihypertensive properties and improves the clinical status of patients with heart failure. ace inhibitors do not affect the levels of ace . even though the main mechanism of action of ace inhibitors is to limit the formation of ang ii, other activities, such as reducing the catabolism of other vasodilator oligopeptides (such as bradykinin) by ace, might have a role in the pharmacological effects of ace inhibitors. another tetrapeptide, n-acetyl-seryl-aspartyl-lysil-proline (ac-sdkp), whose levels are elevated in patients receiving ace inhibitors, could also be responsible for many of the antifibrotic and immune-beneficial effects of these agents. furthermore, ace inhibitors increase the levels of ang i, which shifts raas activity to the axis producing ang - . , some of the favourable pharmacological effects of ace inhibitors might be mediated though increased activity of the ace -ang - -mas axis. , currently, ten ace inhibitors are available in the usa and several more are available globally. the clinical effects of the various ace inhibitors are not markedly different, except for trandolapril and quinapril having a longer half-life and broader tissue distribution than the other agents owing to their liposolubility. many years of use have confirmed very good safety and tolerability profiles for these drugs, with cough ( - % of patients) and angioedema ( % of patients) being the most common adverse effects. this drug class is the first-line therapy for patients with hypertension, ckd and heart failure, [ ] [ ] [ ] with ace inhibitors successfully controlling blood pressure and reducing left-ventricular hypertrophy and all-cause mortality. the first oral arb, losartan, was made available in the s, and currently nine arbs exist for clinical use (box ). despite different pharmacokinetic characteristics, most arbs are used as once-daily drugs and have fewer adverse effects than ace inhibitors. in general, the indications for using arbs are the same as for using ace inhibitors; arbs are efficacious in hypertension - and heart failure, figure | components of the renin-angiotensin-aldosterone system and main classes of pharmacological activators and inhibitors of the system. prorenin can be activated proteolytically in the kidneys (by neuroendocrine convertase or cathepsin b) or nonproteolitically by the renin receptor in many tissues. circulating renin can also bind to the renin receptor, which increases its enzymatic activity. renin converts angiotensinogen to ang i, which can then enter three main pathways. these three axes, ace-ang ii-at -aldosterone, ace -ang - -mas and ang iv-irap, are highlighted. activation of the at receptor in the adrenal gland results in production of aldosterone, which can then bind to the mineralocorticoid receptor. abbreviations: ace, angiotensin-converting enzyme; ace , angiotensin-converting enzyme ; ang, angiotensin; arb, angiotensin receptor blocker; arn, angiotensin receptor-neprilysin; at , type- ang ii receptor; at , type- ang ii receptor; irap, leucyl-cystinyl aminopeptidase (also known as insulin-regulated membrane aminopeptidase or insulin-responsive aminopeptidase); mas , proto-oncogene mas; rh, recombinant human. protection. as with ace inhibitors, use of arbs increases the levels of ang ii as a substrate of ace and could have clinical benefits resulting from stimulation of the ace -ang - -mas axis. , in theory, combining ace inhibitors with arbs or with a renin inhibitor could have pharmacological benefits. however, no evidence of clinical benefits beyond those achieved with single-agent approaches was seen when combination therapy was used in patients with hypertension, coronary artery disease or ckd, and c ombination therapy could potentially be harmful. , mineralocorticoid receptor antagonists the mineralocorticoid receptor antagonist spironolactone was first used in the s as a potassium-sparing diuretic. since then, the systemic effects of aldosterone in hypertension, heart failure and target-organ damage became better known. randomized clinical trials confirmed the effectiveness of spironolactone to treat hypertension and showed that the drug has antifibrotic and anti-inflammatory properties. spironolactone binds to other receptors in addition to the mineralocorticoid receptor, such as androgen and progesterone receptors, which can have adverse effects such as gynaecomastia and decreased libido. in , a second mineralocorticoid receptor antagonist, eplerenone, was introduced. eplerenone has less affinity for sex hormone receptors than spironolactone; the drug has a nearly similar antihypertensive effectiveness to that of spironolactone, yet is less potent and has fewer adverse effects. , a third antagonist, canrenone, is available in some countries but scant clinical data on this drug exist. in addition to their use in primary hyperaldosteronism, mineralocorticoid receptor blockers are commonly used to treat ascites, heart failure and resistant hypertension. , direct renin inhibitors in , and after many attempts, the first oral direct renin inhibitor (dri), aliskiren, became available, replacing first-generation inhibitors of low bioavailability. aliskiren is an effective antihypertensive agent and dual therapy with aliskiren and an arb was tested in the altitude trial. however, in this trial, the combination of aliskiren and losartan did not show any benefits on cardiovascular or renal outcomes versus losartan alone, and dual therapy was associated with an increased rate of adverse events. improving the benefits of raas blockade beyond the effects of ace inhibitors or arbs will be challenging, but research in this area is abundant. several new nonpeptide drugs have been developed that modify raas activity (box ); however, most of them are at preclinical or early clinical stages. angiotensin-receptor-neprilysin inhibitors natriuretic peptides have an important role in sodium and water homeostasis, inducing natriuresis, inhibiting the hypothalamic-pituitary-adrenal system at all regulatory levels , and promoting vasodilatation; they also possess antiproliferative properties. , neprilysin is a vasopeptidase that metabolizes natriuretic peptides and other peptides, such as bradykinin. neprilysin inhibition increases the plasma levels of natriuretic peptides, but has modest effects in reducing blood pressure. however, combining neprilysin with a raas blocker is a powerful tool to increase natriuresis and vasodilation. the first therapeutic agent in this class was omapatrilat, a vasopeptidase that inhibits both neprilysin and ace. the cardiovascular effects of omapatrilat were encouraging, but a high rate of angioedema stopped clinical trials of this drug. the next generation of drugs in this group combines a natriuretic peptide inhibitor with an arb. lcz , the first agent of the angiotensinreceptor-neprilysin (arn) inhibitor class, combines a neprilysin inhibitor moiety with a valsartan moiety. this dual therapy decreases blood pressure in animals and healthy humans with low rates of adverse effects. this type of drug intervention is promising in the fields of h ypertension and heart failure. abbreviations: at , type- angiotensin ii receptor; at , type- angiotensin ii receptor; irap, leucyl-cystinyl aminopeptidase (also known as insulin-regulated membrane aminopeptidase or insulin-responsive aminopeptidase); mas , proto-oncogene mas; raas, renin-angiotensin-aldosterone system. a randomized clinical trial (rct) evaluated the effect of lcz in the treatment of hypertension. patients with mild to moderate hypertension (n = , ) received a single dose of , or mg of lcz , or , or mg of valsartan in a -week placebo-controlled trial. lcz showed a good safety profile and was s uperior to placebo and valsartan in reducing office blood pressure and pulse pressure. more than % of patients had their blood pressure controlled with the mg dose of lcz , whereas this proportion was only % in the group receiving mg of valsartan. in approximately patients per arm, ambulatory blood pressure monitoring was utilized. mean diastolic h blood pressure was not different between the groups receiving the two therapies, but mean systolic blood pressure was significantly reduced in the lcz group because of larger nocturnal decreases in blood pressure than those observed in the valsartan or placebo groups. the greater blood pressure reduction seen in patients receiving lcz when compared with patients in the other groups was not related to increased levels of the second messenger (cgmp) that mediates the actions of natriuretic peptides. a second rct in an asian population also confirmed the efficacy of lcz in treating hypertension. the parameter study is an ongoing study in elderly patients with wide pulse pressure, which evaluates the vascular effects of arn inhibition, including changes in arterial stiffness and central haemodynamics, and efficacy in reducing blood pressure. the trial will be finished in . the paramount study evaluated the effects of mg of lcz compared with mg of valsartan in almost patients with heart failure and preserved ejection fraction. even though the levels of n-terminal probnp (also known as b-type natriuretic peptide) were decreased and functional new york class of heart failure was improved at weeks of follow-up, no differences in echocardiographic parameters were observed. no changes were observed in the levels of plasma inflammation markers. surprisingly, proteinuria increased in the lcz group, an increase that was not observed in patients treated with lcz in the hypertension trials. this increase could be related to increased intraglomerular pressure related to increased levels of natriuretic peptides. the most important observations in this study were a mmhg decrease in systolic blood pressure and a mmhg decrease in diastolic blood pressure, which both persisted at weeks of follow-up in the lcz group, whereas the decreases in blood pressure seen in the valsartan group were . mmhg and . mmhg, respectively, over the same period. the paradigm-hf trial, in which therapy with mg of lcz was compared with therapy with mg of enalapril for the treatment of heart failure (with decreased ejection fraction), was stopped according to prespecified rules because lcz reduced the frequency of the primary outcome, a composite of death from cardiovascular causes or hospitalization for heart failure (hr . , p < . ). , blood pressure was signifi cantly lower in the lcz group than in the enalapril group. the lcz group had higher proportions of patients with hypertension and nonserious angioedema, but fewer patients with renal impairment and hyperkalaemia than the enalapril group. urinary levels of cgmp in the lcz group did not change during the trial. some post-hoc analyses have noted that the benefits of lcz in the treatment of heart failure do not correlate with the antihypertensive effects of the drug. arn inhibition could be a breakthrough in the hypertension field. the observed reductions in systolic nocturnal blood pressure and in pulse pressure might be an indication of a substantial effect of lcz on vascular function or on the central nervous system rather than an effect on natriuresis. nonsteroidal mineralocorticoid receptor antagonists (third-generation and fourth-generation compounds) have been developed with the aim of achieving cardiovascular benefits with fewer renal adverse effects than those of classic mineralocorticoid receptor antagonists (spironolactone, eplerenone and canrenone). abbreviations: ace, angiotensin-converting enzyme; ace , angiotensin-converting enzyme ; ang, angiotensin; arb, angiotensin receptor blocker; arn, angiotensin receptorneprilysin; at , type- angiotensin ii receptor; at , type- angiotensin ii receptor; irap, leucyl-cystinyl aminopeptidase (also known as insulin-regulated membrane aminopeptidase or insulin-responsive aminopeptidase); mas , proto-oncogene mas; raas, renin-angiotensin-aldosterone system. finerenone, previously called bay - , is a potent and promising mineralocorticoid receptor antag onist that conferred extensive cardiovascular and renal protection in rat models of hypertension and heart failure when compared with eplerenone. the arts trial assessed the efficacy and safety of this drug in patients with systolic heart failure and mild ckd. this phase ii study consisted of two parts. part a, which involved patients, evaluated the safety and tolerability of . , or mg daily doses of finerenone versus placebo. part b assessed the safety and efficacy of the drug in patients randomized in a : : : : : design to receive finerenone orally at doses of . , , or mg daily, or mg twice daily; placebo; or open-label oral spironolactone, which was given at an initial dose of mg daily and uptitra ted to mg daily if serum potassium concentrations allowed. finerenone decreased the levels of bnp and n-terminal probnp. the drug decreased albuminuria by a similar degree to spironolactone and was associated with lower incidence of hyperkalaemia and acute renal failure. more data are needed to assess the clinical effects of finerenone. the arts hf trial, which has been completed but has not been published yet, assessed the efficacy and safety of this drug when compared with eplerenone in patients with diabetes mellitus but without ckd or chronic left-ventricular dysfunction. another nonsteroidal drug, br- , has high potency and selectivity for the mineralocorticoid receptor but also blocks the l-type calcium channel, which might help lowering blood pressure. after binding to the mineralo corticoid receptor, br- triggers degradation of this receptor. no clinical trials of br- have been reported. pf- is a potent and selective nonsteroidal inhibitor of the mineralocorticoid receptor that has been tested in healthy humans , and patients with diabetic nephropathy, but was discontinued owing to adverse effects. sm- is another agent of this class that has potent activity as an antagonist of the mineralocorticoid receptor and, in hypertensive rats, diminishes blood pressure and reduces the urinary levels of inflammatory markers. sm- has not been tested in humans. aldosterone synthase inhibitors a different approach to avoid the deleterious effects of aldosterone is by inhibition of the expression of cyp b , the gene than encodes aldosterone synthase. inhibition at this level of the raas would block the effects that are mediated through the mineralocorticoid receptor and also those that are independent of this receptor. an additional potential benefit of this approach is the prevention of a reactive increase in aldosterone levels, which occurs when the receptor is blocked. the potential downside of this strategy is that aldosterone synthase inhibitors might also suppress cortisol release by inhibiting cytochrome p b , mitochondrial (also known as steroid β-hydroxylase, encoded by the cyp b gene), because the two enzymes have a high sequence homology. fad a is an isomer of fadrozole, an aromatase inhibitor. this drug decreased plasma and urinary aldosterone concentrations and improved cardiac and renal target-organ damage in several animal models. [ ] [ ] [ ] fad a has only a mild antihypertensive effect and, therefore, might be more clinically important in the treatment of heart failure than other approaches that have a more potent effect in lowering blood pressure. lci is an orally active and nonselective aldosterone synthase inhibitor that has been evaluated in human studies. in phase i studies, a . mg daily dose of lci was well tolerated and lowered plasma and urinary aldosterone levels without affecting cortisol secretion. at higher doses, however, cortisol secretion was diminished. phase ii studies demon strated that plasma and urinary aldosterone levels are decreased with lci therapy, but the raas is upregulated and slight hyponatraemia and increased serum potassium levels were reported. in a large study, patients were randomized to receive either lci . mg once daily (n = ), . mg once daily (n = ), . mg twice daily (n = ) or . mg once daily (n = ); eplerenone mg twice daily (n = ); or placebo (n = ) for weeks. lci intake was associated with a modest reduction in blood pressure that was lower than that achieved with eplerenone.when aldosterone synthase is inhibited by lci , cortisol levels remain normal, but -deoxycorticosterone levels increase, which means that the adrenocorticotropichormone-cortisol axis is stimulated by the inhibition of expression of the cyp b gene. the stimulation of this axis might be one of the reasons why the decrease in blood pressure is only modest even at high lci doses. lack of aldosterone synthase selectivity, a short halflife and lack of adequate antihypertensive efficacy in primary aldosteronism and resistant hypertension hinder the usefulness of lci in these areas. this agent, however, might be of value in the treatment of cushing disease. therefore, research is now set to develop second-generation aldosterone synthase inhibitors with better selectivity than lci , which would be associated with improved effects on reduction of blood pressure. n-(pyridin- -yl) benzamides are potential agents for future development in this drug class. , post-transcriptional regulation of cyp b and cyp b expression by dicer-dependent micrornas (mirnas) can affect secretion of aldosterone and cortisol in adrenocortical cells. therefore, adrenal mirnas might also be potential therapeutic targets. the rationale behind complete raas inhibition through blockade of the rate-limiting enzyme renin, and the expectation that this approach would more effectively prevent ang ii production than approaches targeted at other components of the raas is theoretically attractive. however, aliskiren is the only dri available. as add-on therapy to arbs, aliskiren did not improve renal or cardio vascular outcomes and was associated with more adverse effects than arb therapy alone. aliskiren has not been evaluated as initial or solo therapy, because other drugs have proven benefits when administered in this way. in the usa, the package insert of aliskiren contains a formal recommendation by the fda that the drug should not be used as dual therapy in association with arbs or ace inhibitors in patients with diabetes mellitus and renal disease. in , a new dri, act- , was shown to have an adequate safety profile in humans when administered once daily. act- inhibits plasma renin activity, while the levels of immunoreactive renin increase, a pattern typical of dris. however, the decrease in plasma renin activity is transient and is not dose-dependent at days. the mechanism under lying these observations is not clear, because the plasma levels of act- remain constant. the effects of this dri on blood pressure are controversial, as only one study showed a decrease in blood pressure when act- was used, and this effect was seen in the supine position, whereas other studies showed no differ ences between the effects of act- and placebo on blood pressure in healthy individuals and patients with hypertension. , more studies are necessary to clarify this inconsistency. the same pharmaceutical company that developed act- is developing another dri, act- . in early phase i and phase ii studies, act- has shown a good safety profile. not much information is yet a vailable about the antihypertensive efficacy of act- . for years, different agonist and antagonist peptides were used to study the function of the at receptor. the reported protective effects of this receptor were related to its antifibrotic and anti-inflammatory properties (box ). in , a selective non-peptide at agonist, compound , was introduced for research. most of the known effects of this agent have been described in different animal models. in rats with spontaneous hypertension, the drug reduces the amount of collagen in the extra cellular matrix and in vascular tissue in a bloodpressure-independent manner. compound has a similar effect in animal models of myocardial infarction, reducing scar formation and improving cardiac function. compound reduced proteinuria in the kidney of rat models of hypertension and kidney disease, and also decreased the levels of inflammatory markers in these models. , in the central nervous system, at receptor activation has neuroprotective effects and induces neuro regeneration. compound reproduces these effects, but has to be administered centrally to bypass the blood-brain barrier. however, systemic administration of compound might still have effects on neuroregeneration. compound does not have a net effect on blood pressure, which is similar to the situation seen with other at agonists. however, when the effects of the at receptor on vasoconstriction are blocked, the vasodilator properties of compound are revealed, and blood pressure decreases. , an at antagonist can reverse this effect. a study published in demonstrated that the increased urinary sodium excretion that is induced by compound is mainly due to effects on proximal tubular cells of the kidney, namely internalization of sodium/ hydrogen exchanger (nhe- ) and na + /k + atpase. continuous compound infusion recruited more at receptors to the proximal tubule brush border than an acute infusion. acute compound infusion did not change blood pressure, but in a -day experiment in which concomitant intrarenal ang ii infusion was administered, compound blocked the blood pressure effects of ang ii, increasing natriuresis. more studies are needed to clarify whether infusion time, time to achieve at receptor upregulation, or administration route can influence blood pressure responses to compound . even though no clinical trials are published yet, available evidence suggests that compound could modulate fluid retention, h ypertension and target-organ damage. the benefits of ace in vascular disease might be brought about by two different mechanisms. first, by participating in ang ii degradation, ace decreases the interaction of ang ii with at receptors, and second, by increasing ang - synthesis, ace induces vasodilation. the main function of ace has been proposed to be the counter balancing of the effects of the ace-ang ii-at -aldosterone axis. decreases in ace levels or increases in ace and ang ii levels induced damage in animal models of diabetic nephropathy or hyper tension. , in animal models, the human recombinant form of ace (rhace ) reduced progression of diabetic nephro pathy, decreased blood pressure (in models of hyper tension), and decreased cardiac fibrosis. , in , the pharmaco kinetic and pharmacodynamic characteristics of rhace were described in healthy humans. daily rhace intake decreased the levels of ang ii in a dose-dependent manner over h, with a transient increase in ang - levels, and more sustained increases in ang - degradation products, such as ang - . the authors of this study suggest that rhace is likely to reach the extravascular space, a hypothesis that is based on the large spread of rhace distribution they observed. this enzyme has a good safety profile and did not induce immunogenicity, even when repeated doses were administered. similarly to results reported in healthy animals, rhace did not affect blood pressure or heart rate in humans. thus, rhace is a promising drug for treatment of patients with intolerance to other drugs that target raas components, and also perhaps in acute illnesses, such as myocardial infarction or decompensated heart failure, in which the balance between ace and ace is disturbed. this drug is attractive in the renal field, because rhace might quickly reduce proteinuria, even in associ ation with an ace inhibitor. these two classes of drugs used together might have important protective effects mediated through substantial increases in ang - levels. we look forward to more human clinical trials in this area. aside from cardio vascular effects, some evidence exists that ace might facilitate recovery from acute lung injury, as rhace has been reported to ameliorate virus-mediated lung injury in mouse models. thus, rhace therapy in virus-mediated lung injury, especially influenza, in which ang ii levels are correlated with the degree of lung injury and with mortali ty, is potentially promising. similarly to ace administration, ang - binding to the mas receptor has shown beneficial effects in animal models; these effects are related to decreased fibrosis and inflammation and to blood pressure reduction by vaso dilation and enhancement of baroreflex sensitivity. more than years ago, the non-peptide compound ave showed similar effects to those of ang - in endo thelial cells. subsequent data demonstrated that ave and ang - compete for the same mas receptor in the kidney. this non-peptide mas receptor agonist has been studied in animals, in which it demon strated a capa city to decrease blood pressure, alone or in combination with renin inhibitors. ave was also demonstrated to attenuate acute and chronic renal injury in animal models. , in , ave was shown to improve penile erection through nitric oxide release in rats. ave has also been shown to ameliorate kidney inflammation, and, more interestingly, improve cell infiltration, cytokine release and histology in two rodent models of arthritis. unfortunately, no clinical trials have yet been performed to test this promising therapeutic approach in humans. the development of compounds that protect ang - from enzymatic degradation has also been the focus of interest. the combination of ang - with hydroxylpropyl-β-cyclodextrin protects ang - from degradation and acts as a long-lasting release system, enabling uptake of ang - in the colon. this drug combination has been tested in animal models of hypertension, in which it showed antihypertensive effects, and also had beneficial effects in models of diabetes mellitus and atherosclerotic inflammation. , phase i clinical trials are currently testing the safety profile of this combination therapy in humans (r. a. santos, personal communication). many oligopeptides result from ang ii degradation by endopeptidases. the hexapeptide ang - , known as ang iv, has been studied because it might have a role in protecting cognitive function. irap, the ang iv receptor, which is inhibited by ang iv, was identified in . irap colocalizes with the glucose transporter glut- and was originally thought to contribute to basal intracellular retention of the transporter. irap is linked to vasopressin and oxytocin degradation. a group of nonpeptide inhibitors of irap has been developed. hfi- is an irap-selective pyridinyl compound that enhances memory in rats. an australian group is working on the cardiovascular-protective effects of irap inhibition with hfi- in mice. preliminary data demonstrate that hfi- prevents cardiac and endothelial damage induced by ang ii, independent of blood pressure. hfi- also has anti-inflammatory properties. the association of irap with glut- is intriguing, and whether irap inhibitors could be used to improve insulin sensitivity remains an interesting opportunity. more studies are n ecessary to explore therapeutic uses for irap inhibitors. vaccines against the raas the high prevalence of hypertension in the world and the related cost of this burden have made the development of vaccines against the raas an attractive strategy, with the aim of achieving increased treatment compliance and public access to therapy, and a decrease in public-health costs. many attempts were made to generate vaccines against renin, ang i, ang ii and at . however, most of these attempts have been harmful or have not had clinical efficacy. an rct in which an anti-ang ii vaccine (cyt -angqb) was tested passed phase iia without safety problems, and showed a promising h reduction in blood pressure of . mmhg (systolic) and . mmhg (diastolic). however, subsequent studies could not confirm these results. novel strategies are currently in development, such as the association of ang ii as an epitope to a hepatitis a virus-like particle to generate an improved immune response. future studies are needed to explore the efficacy and safety of an i mmunological, chronic and irreversible blockade of the raas. although to improve upon the cardiovascular and renal protection offered by ace inhibitors and arbs will be diffi cult, novel drugs are being developed to shift the activity of the raas towards the protective cardiovascular arm (at and mas receptors). a role for raas blockade in other, non-cardiovascular pathologies (such as lung injury and cognitive disorders) is emerging. the combination of raas inhibition with other autacoid systems (endothelin blockade or natriuretic peptide stimulation) could improve the efficacy of traditional raas blockade and be the basis of specific tissue-targeted drug development. the substance causing renal hypertension the existence of two forms of hypertensin studies on angiotensinogen formation in a liver perfusion system stimulation of aldosterone secretion by angiotensin ii. a preliminary report mechanism of pulmonary conversion of angiotensin i to angiotensin ii in the dog 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hypertension in rats renoprotective effects of ave , a nonpeptide mas receptor agonist, in experimental acute renal injury beneficial effects of the activation of the angiotensin-( - ) mas receptor in a murine model of adriamycininduced nepopathy ave , a non-peptide mas-receptor agonist, facilitates penile erection anti-inflammatory effects of the activation of the angiotensin-( - ) receptor, mas, in experimental models of arthritis an orally active angiotensin-( - ) inclusion compound and exercise training produce similar cardiovascular effects in spontaneously hypertensive rats treatment with angiotensin-( - ) reduces inflammation in carotid atherosclerotic plaques oral administration of angiotensin-( - ) ameliorates type diabetes in rats evidence that the angiotensin iv (at( )) receptor is the enzyme insulin-regulated aminopeptidase insulin-regulated aminopeptidase is a key regulator of glut trafficking by controlling the sorting of glut from endosomes to specialized insulin-regulated vesicles identification and characterization of a new cognitive enhancer based on inhibition of insulin-regulated aminopeptidase vaccine for hypertension: modulating the renin-angiotensin system effect of immunisation against angiotensin ii with cyt -angqb on ambulatory blood pressure: a double-blind, randomised, placebo-controlled phase iia study construction, expression and immunogenicity of a novel anti-hypertension angiotensin ii vaccine based on hepatitis a virus-like particle author contributions c.a.r. researched data for the article. c.a.r. and m.r.w. wrote the article. all authors contributed substantially to discussion of content and reviewing and/or editing the manuscript before submission. key: cord- - he sjpf authors: goldstein, benjamin; trivedi, malav; speth, robert c. title: alterations in gene expression of components of the renin-angiotensin system and its related enzymes in lung cancer date: - - journal: lung cancer int doi: . / / sha: doc_id: cord_uid: he sjpf objectives: the study assessed the existence and significance of associations between the expression of fifteen renin-angiotensin system component genes and lung adenocarcinoma. materials and methods: ncbi's built-in statistical tool, geo r, was used to calculate student's t-tests for the associations found in a dna expression study of adenocarcinoma and matched healthy lung tissue samples. the raw data was processed with genespring™ and then used to generate figures with and without sidak's multiple comparison correction. results: ten genes were found to be significantly associated with adenocarcinoma. seven of these associations remained statistically significant after correction for multiple comparisons. notably, agtr , which encodes the at( ) angiotensin ii receptor subtype, was significantly underexpressed in adenocarcinoma tissue (p < . ). agtr , ace, enpep, mme, and prcp, which encode the at( ) angiotensin ii receptor, angiotensin-converting enzyme, aminopeptidase n, neprilysin, and prolylcarboxypeptidase, respectively, were also underexpressed. agt, which encodes angiotensinogen, the angiotensin peptide precursor, was overexpressed in adenocarcinoma tissue. conclusion: the results suggest an association between the expression of the genes for renin-angiotensin system-related proteins and adenocarcinoma. while further research is necessary to conclusively demonstrate a link between the renin-angiotensin system and lung cancers, the results suggest that the renin-angiotensin system plays a role in the pathology of adenocarcinoma. lung cancer continues to be the leading cause of cancer deaths, and lung adenocarcinomas account for approximately % of all lung cancers (https://www.cancer.org/cancer/ lungcancer-non-smallcell/index (accessed / / )). to assess factors associated with lung adenocarcinomas, a gene expression study comparing normal lung tissue and lung tumor was undertaken using the hg-u a affymetrix gene chip [ ] . while such associations do not establish causation, they characterize the underlying molecular marks or tumors and identify potential target sites for treatments. to ascertain if the renin-angiotensin system (ras) is altered in lung adenocarcinomas we evaluated the expression of genes of the classical ras ( figure ) and extended components of the ras as reported in a previous study [ ] . the ras (table ) functions primarily to maintain fluid and electrolyte homeostasis and regulate the cardiovascular system; however, the ras is pleiotropic and is now recognized to have effects that extend beyond its primary functions. for example, angiotensin ii (ang ii), the primary hormone of the ras, promotes the proliferation of mouse fibroblast t cells (but not sv transformed t cells in culture) [ ] , dna synthesis and proliferation of adrenal cortical cells in culture [ ] , and the hypertrophic and hyperplastic growth of vascular smooth muscle cells [ ] . both myointimal hyperplasia and vascular smooth muscle dna synthesis are increased in rats infused with ang ii [ ] . all at receptor figure : pathways of the renin-angiotensin system (ras) cascade. the classical ras cascade is shown with light shading and includes the two major angiotensin ii receptor subtypes, at and at . nonclassical components of the ras cascade would include the ability of the (pro)renin receptor to activate prorenin, as well as enhance the activity of renin to metabolize angiotensinogen to ang i, the ability of chymase to convert ang i to ang ii, metabolism of ang ii to iii and iii to iv by aminopeptidases a and n, respectively, and the novel at receptor for ang iv which is also known as insulin-regulated aminopeptidase. in addition, there is the ace /ang - /mas axis of the ras which is considered to be a counterregulator of the actions of the classical ras. this includes the enzyme angiotensin-converting enzyme (ace ) which primarily forms ang - from ang ii, but can also form ang - from ang i, prolylcarboxypeptidase which also forms ang - from ang ii, and the enzymes neprilysin and prolylendopeptidase which forms ang - directly from ang i, and mas, the putative receptor for ang - . the names of the genes that encode the various proteins of the ras and related proteins are shown in italics after the protein names. these proliferation-promoting effects of ang ii suggest that the ras might play a critical role in cancer [ ] [ ] [ ] [ ] . canonically, most studies suggest that ang ii acting via the at receptor can promote cancer by a variety of mechanisms. ang ii is angiogenic [ , ] and can promote blood flow to tumors by stimulating vascular endothelial growth factor (vegf) release; see reviews [ , ] . moreover, the tumor vessels are less responsive to ang ii such that the systemic vasoconstrictor actions of ang ii shunt blood to the tumor vasculature [ , ] . stimulation of the at receptor by ang ii transactivates the egf receptor [ , ] , a receptor that is overexpressed in many cancers [ , ] . activation of the at receptor promotes the release of a host of growth factors that also promote cell proliferation [ ] . additionally, in several animal models of cancer, both angiotensinconverting enzyme (ace) inhibitors and at angiotensin receptor subtype blockers (arbs) inhibit tumor growth [ ] . since both ace inhibitors and arbs are widely used antihypertensive agents, meta-analyses have been conducted to identify any correlation for cancer incidence in patients taking these drugs and/or the effects of these drugs on cancer risk. surprisingly, there is not universal agreement on the effects of ace inhibitors and arbs on cancer incidence. for example, a meta-analysis reported in [ ] found a slight increase in cancer risk in individuals that were being treated with arbs. however, a subsequent meta-analysis published the following year [ ] found no association between the use of arbs and cancer risk. however, they did observe an increase in cancer risk in patients taking both an arb and an ace inhibitor. of note the previous study [ ] compared arb + ace inhibitor treated groups compared to ace inhibitor only groups to show the association of arb use with increased cancer risk. an epidemiological study of ace inhibitor and/or arb usage reported an increased risk of cancer deaths [ ] ; however, a study from the united states food and drug administration [ ] found no evidence for an increased cancer risk from arbs. a recent meta-analysis of ras blockade with ace inhibitors and/or arbs suggested that ras blockade reduces cancer risk [ ] . another recent meta-analysis focusing on lung cancer risk in patients taking arbs indicated that arbs reduce lung cancer risk [ ] with all but one [ ] of studies showing a reduced risk of cancer with of the studies showing a significant reduction in lung cancer risk on their own [ , ] . thus the preponderance of clinical data suggests that arbs reduce lung cancer risk but that there may be individual or population differences in lung cancer risks associated with ras blockers. to further explore this putative relationship this study investigates changes in gene expression of ras and related protein in a population of lung adenocarcinoma patients [ ] . this could help to identify specific genes that could be investigated further to determine the exact role of ras and extended ras pathway genes in lung adenocarcinomas. the study is an in silico analysis of the expression of reninangiotensin system genes in connection with lung cancer. a total of renin-angiotensin system related genes were studied (see table and figure ). the gene expression analysis was conducted on data sourced from ncbi's geo database as log values. the specific dataset used, gse , profiled the gene expression of lung adenocarcinoma tissue samples and paired noncancerous tissue samples [ ] . the dataset was analyzed through the geo database's integrated statistics utility, geo r, in addition to prism (v. ), genespring (v. . ), and microsoft excel. more specifically, geo r calculated values in order to identify significant differences in gene expression while genespring extracted and normalized the raw expression data using provided algorithms to allow for further statistical calculations using prism (graphpad software), which was also used to generate graphs and other figures from tables of raw data created in excel. one value for anpep in the tumor group was excluded based upon the criterion of being more than standard deviations from the mean. since the number of samples in this specific data set differed between the tumor ( = ) and the normal tissue ( = ) groups, comparisons were made using an unpaired student's -test with welch's correction for heterogeneity of variance. data is presented with and without sidak's correction for multiple comparisons: = . , = − ( − . ) ( / ) ( genes were studied) to provide a relative estimate of significance ( table ) there were a number of profound differences in the expression of the genes encoding the proteins comprising and closely associated with the renin-angiotensin system (ras), between normal lung tissue and the lung tumor tissue (figures and and table ). additionally, there were substantial differences in the level of expression of the mrna for the different proteins. among the proteins that comprise the classical ras, the gene for the at subtype of the ang ii receptor was the most highly expressed in normal lung tissue (figures and s , and table ). the mrna for the other major ang ii receptor subtype at was expressed at less than half the intensity as the at receptor subtype. the gene expression for both ang ii receptor subtypes was dramatically reduced, and %, respectively, ( figure s ), for at and at in the lung tumor tissue ( < . , corrected for multiple comparisons, table ). the agt gene which encodes angiotensinogen was also highly expressed in the normal lung tissue, and its mrna was nearly doubled in the lung tumor tissue ( < . , corrected for multiple comparisons table ). ace, the gene that encodes the enzyme that converts the inactive precursor angiotensin i (ang i) to the active hormone, ang ii, was expressed at a lower level and its expression was reduced by about half in the lung tumor tissue ( < . corrected for multiple comparisons table ). ren, the gene for renin, the enzyme which cleaves angiotensinogen to make ang i, was the least highly expressed gene of the classical ras proteins and its expression level was unchanged in the lung tumor tissue. genes of the proteins that have ancillary interactions with the ras also showed a considerable variation in expression level in normal lung tissue, and the expression of several of these genes were altered in the lung tumor tissue (figures and s and table ). atp ap which encodes the (pro)renin receptor, but which may have a more important cellular function in promoting hydrogen ion transport and wnt signaling via the frizzled receptor [ ] was the most highly expressed gene in the normal figure : expression of genes of proteins that directly interact with renin-angiotensin system. these genes encode proteins that can alter the function of ras but have many other functions. atp ap encodes the prorenin receptor (atpase h(+)-transporting accessory protein ), cma encodes chymase, lnpep encodes the ang iv (at ) receptor (insulin-regulated aminopeptidase), enpep encodes aminopeptidase a, and anpep encodes aminopeptidase n. one value for anpep was excluded from the data analysis because it was > standard deviations apart from the rest of the dataset. * < . by unpaired test with welch's correction for heterogeneity of variance and sidak's correction for multiple comparisons. lung tissue. there was a small, %, increase in atp ap expression ( figure s ) that was not statistically significant after correction for multiple comparisons. ren expression in lung tumor tissue was also % higher than in normal lung lung cancer international tissue ( figure s ) and also was not statistically significant after correction for multiple comparisons (table ) . cma , the gene for chymase, which can facilitate the ras by converting ang i to ang ii, was expressed at a low level and its expression did not differ between normal lung tissue and lung tumor tissue. lnpep, the gene which encodes the at receptor, but which may have a more important cellular function as insulin-regulated aminopeptidase [ ] , was also expressed at a low level and its expression did not differ between normal lung tissue and lung tumor tissue. expression of enpep, the gene which encodes aminopeptidase a, a major ang ii metabolizing enzyme, was expressed at a moderate level in normal lung tissue with a statistically significant ( < . , corrected for multiple comparisons, table ) % reduction in its expression in lung tumor tissue ( figure s ). anpep, the gene which encodes aminopeptidase n, a major ang iii metabolizing aminopeptidase, was also expressed at a moderate level in normal lung tissue and at a slightly lower level in lung tumor tissue, but did not retain statistical significance ( = . ) after correction for multiple comparisons (table ). the genes for the primary component proteins of the ace /ang - /mas axis of the ras, ace , and mas were expressed at low levels in normal lung tissue and expression levels did not differ between normal lung tissue and lung tumor tissue (figures and s and table ). however, the genes for two enzymes that have ancillary functions associated with the ace /ang - / mas axis; mme and prcp, which encode neprilysin and prolyl carboxypeptidase, respectively, were highly expressed in normal lung tissue. their expression was significantly decreased in lung tumor tissue ( < . , corrected for multiple comparisons, table ) by % and %, respectively (figure s ) . prep, which encodes prolyl endopeptidase, another enzyme that can promote the functionality of the ace / ang - /mas axis, was expressed at a moderate level in normal lung tissue, but its expression was unaltered in lung tumor tissue. in view of the potential involvement of the ras in promoting lung as well as other cancers (see reviews [ , ] ) the extent and changes in the expression of genes encoding proteins of the ras, as well as proteins whose actions include alteration of classical ras activity, are of considerable interest. the high level of expression of the gene encoding the at receptor in the lung suggests that ang ii acting upon the at receptor plays an important role in lung function and cell growth. consistent with an oncogenic activity of the ras in lung, at receptor inhibition decreases the metastasis of lung cancer cells [ ] . see also reviews - . an additional mechanism whereby the ras could increase tumor growth by activating at receptors is by selectively increasing blood flow in newly formed vessels of tumors which, in contrast to the systemic vasculature, do not constrict in response to ang ii [ ] . corollary to such a mechanism, inhibitors of ang ii formation, agents that increase metabolism of ang ii, and at receptor blockers that lower bp may decrease tumor growth by disproportionately reducing blood flow to the tumor. also consistent with the ability of the at receptor to promote cancer cell growth, the at receptor can transactivate the egf receptor, a known oncogenic stimulus [ ] . however, the reduced expression of the at receptor gene in lung tumor cells, suggests that at receptor stimulation of the tumor cells may not be a driving force for their oncogenic activity. conversely, the slightly greater reduction in at receptor gene expression could contribute to the oncogenic activity of lung tumor cells by reducing the inhibitory actions on cell growth that ang ii causes by activating the at receptor. indeed, when at receptors were overexpressed in lung adenocarcinoma cells, their growth was inhibited [ ] . the increased expression of the precursor protein of the ras, angiotensinogen, in lung tumor tissue suggests that there may be an increased activation of the ras in lung tumor cells. angiotensinogen can be rate limiting in the production of ang ii, although the concentrations of renin, prorenin, and the (pro)renin receptor are the primary determinants of the rate of formation of ang i and ang ii. of note, however, there is a non-ras functionality of angiotensinogen to interact with dna, in particular to reverse formation of carcinogenic dna adducts generated by the contents of tobacco smoke [ ] . in this case the increase in angiotensinogen gene expression in lung tumor cells could be a compensatory response of the cells to normalize their dna by removing the adducts formed by tobacco smoke carcinogens. additionally, angiotensinogen, as well as des-angiotensin i angiotensinogen, has antiangiogenic effects lung cancer international and reduces metastasis of b -f melanoma cells to the lungs of mice [ ] . consistent with the reduced expression of ace in the lung tumor tissue, serum levels of ace are decreased in lung cancer patients to a greater extent than in other cancers [ ] . while ace inhibitors are heavily used as blockers of the ras actions on the at receptor, ace is not the rate limiting step in ang ii production. moreover, ang ii can also be generated by chymase, and the mrna for chymase was not altered in the lung tumor cells. another consideration is that the mrna transcribe from the gene enpep which encodes aminopeptidase a, also known as angiotensinase a, a major metabolic enzyme of ang ii, is reduced and this could indicate a prolongation of the stimulation of at and at receptors by ang ii. while ang iii the product of aminopeptidase a metabolism of ang ii is a full agonist at at and at receptors, it is known to be rapidly metabolized following its formation, such that any slowing of the conversion of ang ii to ang iii will increase the stimulation of at and at receptors. moreover, early studies of the proliferative effects of ang ii suggested that ang iii lacked the ability to stimulate cell proliferation [ ] . the low level and apparent lack of differential expression of the genes encoding components of the ace /ang - /mas axis in normal lung tissue and lung tumor tissue are surprising in view of the large body of literature suggesting that the ace /ang - /mas axis plays an important role in lung function [ ] , susceptibility to the sars virus [ ] , and inhibition of angiogenesis in lung cancer [ ] . the reductions in the high level of expression of the genes for two enzymes capable of cleaving ang i and ang ii to form ang - ; neprilysin and prolyl carboxypeptidase, respectively, may cause a reduction in the shunting of ang i and ang ii away from their roles in the classical ras and reduce the antagonistic action of the ace /ang - /mas axis. this would promote the actions of ang ii at the at receptor while simultaneously reducing the stimulation of the mas receptor by ang - . it is well established that not all cancers are the same and it is not surprising that alterations in the ras in different cancers are not consistent. for example, a large-scale study of estrogen receptor-positive breast cancer tumors revealed an increase in agtr mrna expression [ ] , which is the opposite of what we observed in the lung cancer study from which this data is derived. equally contradictory, agtr as well as ace mrna expression is upregulated in hormoneindependent prostate cancer [ ] . while the changes observed in mrna expression in some of the components of the ras in lung adenoma tumor cells are substantial, there are three caveats that must be acknowledged. first, it is uncertain whether these changes in mrna are contributing to the phenotypic changes in lung cells to become cancer cells, or if they are a result of the phenotypic changes associated with the transformation of normal lung cells to cancer cells. second, alterations in mrna expression do not always reflect alterations in protein expression. to assess this second caveat it will be necessary to determine the level of expression of the proteins encoded by the mrnas that showed significant changes. the third caveat relates to possible changes in signal transduction by the at , at , and mas receptors. it is possible that signal transduction in response to activation of these receptors by ang ii, ang iii, and ang - could be increased or decreased. it is known that the at receptor transduces responses to ang ii via g protein-coupled and g protein independent pathways [ ] and that peptide antagonists of the at receptor can signal exclusively through the g protein independent pathway [ ] , so it is not unreasonable for at receptor activation to have differential effects on cell proliferation due to differences in the efficiency of these two signaling pathways in cancer cells. while it would be of interest to assess the mrna expression of these signal transduction proteins, it is not possible to specify that their changes are specifically linked to these angiotensin receptors since they mediate transduction by a variety of hormones, cytokines, and neurotransmitters. the current study emphasizes the involvement of components of the ras (both direct and extended) in the development and progression of lung cancer. in spite of the several limitations pertaining to the dataset, this study provides a novel therapeutic targeting modality for treatment of lung cancer. moreover, the current study also provides a platform to mimic our approach of data mining previously conducted genome-wide expression studies to identify novel therapeutic targets for a variety of cancers and other diseases. this will decrease the cost burden of investigating new drug entities and possibly allow for the repurposing of already approved drugs. the authors declare that there are no conflicts of interest regarding the publication of this paper. gene expression signature of cigarette smoking and its role in lung adenocarcinoma development 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proteinmediated erk activation by the angiotensin ii receptor the authors thank homood as sobeai for assistance in using genespring software. key: cord- - ygb sc authors: van meurs, matijs; kümpers, philipp; ligtenberg, jack jm; meertens, john hjm; molema, grietje; zijlstra, jan g title: bench-to-bedside review: angiopoietin signalling in critical illness – a future target? date: - - journal: crit care doi: . /cc sha: doc_id: cord_uid: ygb sc multiple organ dysfunction syndrome (mods) occurs in response to major insults such as sepsis, severe haemorrhage, trauma, major surgery and pancreatitis. the mortality rate is high despite intensive supportive care. the pathophysiological mechanism underlying mods are not entirely clear, although several have been proposed. overwhelming inflammation, immunoparesis, occult oxygen debt and other mechanisms have been investigated, and – despite many unanswered questions – therapies targeting these mechanisms have been developed. unfortunately, only a few interventions, usually those targeting multiple mechanisms at the same time, have appeared to be beneficial. we clearly need to understand better the mechanisms that underlie mods. the endothelium certainly plays an active role in mods. it functions at the intersection of several systems, including inflammation, coagulation, haemodynamics, fluid and electrolyte balance, and cell migration. an important regulator of these systems is the angiopoietin/tie signalling system. in this review we describe this signalling system, giving special attention to what is known about it in critically ill patients and its potential as a target for therapy. critical illness is a life-threatening disease by definition. patients treated for critical illness in the intensive care unit have underlying causes such as infection, trauma, major surgery, hemorrhagic shock, pancreatitis and other major insults. despite maximal supportive care, severely ill patients treated in intensive care units are still likely to die, usually after an episode of increasing failure of multiple organs [ ] . the mechanisms that underlie multiple organ dysfunction syndrome (mods) are not known [ ] , although several have been proposed, including overwhelming infection or immune response, immune paralysis, occult oxygen debt and mitochondrial dysfunction [ ] [ ] [ ] . although these potential mechanisms have features in common, it is not clear whether mods is a final common pathway or when it is engaged. the innate and adaptive immune systems, coagulation, and hormonal and neuronal signalling are undoubtedly involved and are all connected. for example, the hypoxic response is linked to innate immunity and inflammation by the transcription factor nuclear factor-κb (nf-κb) [ ] . it is no coincidence that the few interventions that appear to be of benefit, although this is still under debate, have pleiotropic mechanisms of action [ ] [ ] [ ] . thus, it seems reasonable to study the intersections between and within cellular and molecular systems to elucidate the interactions and to develop therapeutic options. one of the central cellular players in this system is the endothelial cell (ec). once thought to serve as an inert vascular lining, ecs are highly heterogeneous and constitute an active disseminated organ throughout the circulatory system. ecs form the border between every organ and the bloodstream and thus with the rest of the body. the ec receives and gives signals, stores active substances of multiple systems, and regulates the passage of fluids, electrolytes, proteins and cells. the ec has a time and place dependent phenotype that is dynamically controlled, and its reactions to stimuli are specific to organ and vascular bed [ ] [ ] [ ] [ ] . the ec merits robust investigation in critical illness, as in vascular medicine [ ] . genesis and organogenesis in normal physiology and in wound repair, but it is considered pathologic in tumour growth and diabetes [ ] . second, in the adult organism, ecs help to maintain homeostasis, including fluid, electrolyte and protein transport, and cell migration into and out of the vessel, and to regulate blood flow. third, ecs react and respond to disturbances of homeostasis (for example, in inflammation, coagulation and hypoxia/reperfusion). all three functions are involved in mods, in which ecs are shed, blood flow regulation is hampered, vessels become leaky, cells migrate out of the vessel and into the surrounding tissue, and coagulation and inflammation pathways are activated [ ] . the machinery involved -receptors, signalling pathways and effectors -is largely the same in each function, but the net effect is determined by the balance between the parts of the machinery and the context [ ] . the angiopoietin/tie signalling system (ang/tie system) appears to be crucial in all three functions [ , ] . the ang/tie system, which was discovered after vascular endothelial growth factor (vegf) and its receptors, is mainly restricted to ec regulation and is the focus of this review. accumulating evidence suggests that this system is nonredundant and is involved in multiple mods-related pathways. all components of potential pathophysiological mechanisms in mods should be viewed within their own context, because all systems are mutually dependent. thus, examination of the ang/tie system might offer insight into the mechanisms underlying mods and provide opportunities for therapeutic intervention. the notion that the ang/tie system contributes to disease pathogenesis is supported by clinical studies and studies in animal models, and by the relation between symptoms of critical illness and disturbances in this system. in mice, ang- over-expression in glomeruli causes proteinuria and apoptosis of glomerular ecs [ ] . in a rat model of glomerulonephritis, tie is over-expressed by ecs, and ang- and ang- are over-expressed by podocytes in a time-dependent manner during the repair phase [ ] . therefore, ang/tie might be involved in renal failure and repair. lung dysfunction is common in critical illness, and evidence of ang/tie involvement has been found in animal models. in a rat model of acute respiratory distress syndrome, ang- reduces permeability and inflammation, whereas tie deficiency increases damage [ ] . in an experimental model of asthma, ang- mrna was decreased, and ang- supplementation decreased alveolar leakage and nf-κb-dependent inflammation [ ] . in hypoxia-induced pulmonary hypertension in rats, decreased activity of the tie pathway contributed to right ventricular load, and this effect was antagonized by ang- [ ]. on the other hand, a causative role for ang- in pulmonary hypertension has also been suggested [ ] . in hyperoxic lung injury, ang- is involved in lung permeability and inflammation [ ] . ang/tie also may contribute to critical illness in patients with pulmonary conditions. ang- and ang- concentrations in sputum from asthma patients correlated with airway microvascular permeability [ ] . in patients with exudative pleural effusion, the ang- level was increased whereas ang- was unchanged [ ] . ang- levels are associated with pulmonary vascular leakage and the severity of acute lung injury. plasma from patients with acute lung injury and high ang- concentrations disrupts junctional architecture in vitro in human microvascular ecs [ , ] . patients with cardiovascular disorders also exhibit changes in the ang/tie system. circulating ang- concentrations are stable in patients with atrial fibrillation, but ang- concentrations are increased, along with markers of platelet activation, angiogenesis and inflammation [ ] . patients with hypertension resulting in end-organ damage have increased levels of circulating ang- , ang- , tie and vegf [ ] . congestive heart failure is associated with elevated plasma levels of ang- , tie and vegf, but normal levels of ang- [ ]. a similar pattern is seen in acute coronary syndrome [ ] . circulating levels of components of the ang/tie system have been measured in patients admitted to the critical care unit. in trauma patients plasma ang- , but not plasma ang- or vegf, was increased early after trauma, and the level correlated with disease severity and outcome [ ] . in children with sepsis and septic shock, ang- levels in plasma were increased and once again correlated with disease severity, whereas ang- levels were decreased [ ] . the same ang- / ang- pattern is seen in adults with sepsis [ , , [ ] [ ] [ ] [ ] . the results of studies of the ang/tie system in humans are summarized in table . in sepsis, vegf and its soluble receptor sflt- (soluble vegfr- ) are also increased in a disease severity-dependent manner [ ] [ ] [ ] .the picture that emerges from these studies is that the ang/tie signalling system appears to play a crucial role in the symptoms of mods. findings in animal models and in patients suggest that ang- stabilizes ecs and ang- prepares them for action. the close relation with vegf is also apparent. four (ang- ) and two (ang- ) subunits [ , ] . the dissimilarity between ang- and ang- signalling lies in subtle differences in the receptor binding domain that lead to distinct intracellular actions of the receptor; differential cellular handling of both receptor and ligands after binding and signalling initiation may also play a role [ , ] . the receptors are tie and tie [ ] . tie is a -kda tyrosine kinase receptor with homology to immunoglobulin and epidermal growth factor [ , ] . tie receptors have an amino-terminal ligand binding domain, a single transmembrane domain and an intracellular tyrosine kinase domain [ ] . ligand binding to the extracellular domain of tie results in receptor dimerization, autophosphorylation and docking of adaptors, and coupling to intracellular signalling pathways [ , [ ] [ ] [ ] . tie is shed from the ec and can be detected in soluble form in normal human serum and plasma; soluble tie may be involved in ligand scavenging without signalling [ ] . tie shedding is both constitutive and induced; the latter can be controlled by vegf via a pathway that is dependent on phosphoinositide- kinase (pi k) and akt [ ] . shed soluble tie can scavenge ang- and ang- [ ] . tie does not act as a transmembrane kinase; rather, it regulates the binding of ligands to tie and modulates its signalling [ ] [ ] [ ] . ang- is produced by pericytes and smooth muscle cells ( figure ). in the glomerulus, which lacks pericytes, ang- is produced by podocytes [ ] . ang- has a high affinity for the extracellular matrix, and so circulating levels do not reflect tissue levels, which in part is probably responsible for the constitutive phosphorylation of tie in quiescent endothelium [ ] [ ] [ ] [ ] . ang- is produced in ecs and stored in weibel-palade bodies (wpbs) [ , ] . the release of ang- from wpbs by exocytosis can be regulated independently of the release of other stored proteins [ ] . tie is expressed predominantly by ecs, although some subsets of macrophages and multiple other cell types express tie at low levels [ , ] . in ecs, tie is most abundant in the endothelial caveolae [ ] . the ang- and ang- genes are located on chromosome . functional polymorphisms have not been identified in the ang- gene, but three have been identified in the coding region of ang- [ ] . in ecs under stress, ang- mrna expression is induced by vegf, fibroblast growth factor and hypoxia [ , ] . upregulation of ang- induced by vegf and hypoxia can be abolished by inhibiting tyrosine kinase or mitogen-activated protein kinase [ ] . ang- mrna expression can be downregulated by ang- , ang- , or transforming growth factor [ ] . after inhibition of pi k by wortmannin, ang- mrna production is induced by the transcription factor foxo (forkhead box o ) [ ] . ec-specific ang- promoter activity is regulated by ets- and the ets family member elf- [ , ] . because tie signalling is required under circumstances that usually hamper cell metabolism, its promoter contains repeats that ensure transcription under difficult circumstances, including hypoxia [ ] . tie is present in phosphorylated form in quiescent and activated ecs throughout the body [ ] . signalling is initiated by autophosphorylation of tie after ang- binding and is conducted by several distinct pathways [ , , , ] . tie can also be activated at cell-cell contacts when ang- induces tie /tie homotypic intercellular bridges [ ] . in human umbilical vein endothelial cells (huvecs), ang/tie signalling resulted in upregulated genes and downregulated genes [ , ] . akt phosphorylation by pi k with interaction of nitric oxide is the most important intracellular pathway [ , [ ] [ ] [ ] [ ] ; however, erk / , p mapk, and sapk/jnk can also participate in ang/tie downstream signalling [ , , , [ ] [ ] [ ] [ ] . endothelial barrier control by ang- requires p rhogap, a gtpase regulator that can modify the cytoskeleton [ ] . the transcription factors foxo , activator protein- , and nf-κ b are involved in ang/tie-regulated gene transcription [ , [ ] [ ] [ ] . ang- induced signalling is has also been implicated in cell migration induced by reactive oxygen species [ ] . abin- (a -binding inhibitor of nf-κb ), an inhibitor of nf-κb, is involved in ang- -regulated inhibition of endothelial apoptosis and inflammation in huvecs [ ] . however, the downstream signalling of tie varies depending on cell type and localization and whether a cell-cell or cell-matrix interaction in involved, which results in spatiotemporally different patterns of gene expression. for example, ang- /tie signalling leads to akt activation within the context of cell-cell interaction, but it leads to erk activation in the context of cell-matrix interaction. the microenvironment of the receptor in the cell membrane plays a central role in this signal differentiation. adaptor molecules such as dok and shp and the availability of substrate determine which protein is phosphorylated [ ] . after binding of ang- , and to a lesser extent ang- , tie is internalized and degraded, and ang- is shed in a reusable form [ ] . vegf is an important co-factor that can exert different effects on ang- and ang- signalling [ ] . ang- is antiapoptotic in the presence of vegf but induces ec apoptosis in its absence [ ] . autophosphorylation and subsequent signalling are inhibited by heteropolymerization of tie and tie [ ] . although the ang/tie system appears to play its role mainly in paracrine and autocrine processes, its circulating components have been found in plasma. the significance of this finding in health and disease has yet to be determined. the ang/tie system is an integrated, highly complex system of checks and balances ( figure ) [ , ]. the response of ecs to ang- and ang- depends on the location of the cells and the biological and biomechanical context [ , ] . it is believed that pi k/akt is among the most important downstream signalling pathways and that vegf is one of the most important modulators of effects. below we describe in more detail how this system responds to changes in homeostatic balances under various conditions of damage and repair. angiogenesis, inflammation and homeostasis are highly related, and the ang/tie system lies at the intersection of all three processes [ , ] . the ang/tie system is critically important for angiogenesis during embryogenesis, but in healthy adults its function shifts toward maintenance of homeostasis and reaction to insults. except for follicle formation, menstruation and pregnancy, angiogenesis in adults is disease related. neoplasia-associated neoangiogenesis and neovascularization in diabetes and rheumatoid arthritis are unfavourable events, and improper angiogenesis is the subject of research in ischaemic disorders and atherosclerosis. finally, failure to maintain homeostasis and an inappropriate reaction to injury are detrimental features in critical illness. a schematic model of the angiopoietin-tie ligand-receptor system. quiescent endothelial cells are attached to pericytes that constitutively produce ang- . as a vascular maintenance factor, ang- reacts with the endothelial tyrosine kinase receptor tie . ligand binding to the extracellular domain of tie results in receptor dimerization, autophosphorylation, docking of adaptors and coupling to intracellular signalling pathways. signal transduction by tie activates the pi k/akt cell survival signalling pathway, thereby leading to vascular stabilization. tie activation also inhibits the nf-κb-dependent expression of inflammatory genes, such as those encoding luminal adhesion molecules (for example, intercellular adhesion molecule- , vascular cell adhesion molecule- and e-selectin). ang- is stored and rapidly released from wpbs in an autocrine and paracrine fashion upon stimulation by various inflammatory agents. ang- acts as an antagonist of ang- , stops tie signalling, and sensitizes endothelium to inflammatory mediators (for example, tumour necrosis factor-α) or facilitates vascular endothelial growth factor-induced angiogenesis. ang- -mediated disruption of protective ang- /tie signalling causes disassembly of cell-cell junctions via the rho kinase pathway. in inflammation, this process causes capillary leakage and facilitates transmigration of leucocytes. in angiogenesis, loss of cell-cell contacts is a prerequisite for endothelial cell migration and new vessel formation. ang, angiopoietin; nf-κb, nuclear factor-κb; pi k, phosphoinositide- kinase; wpb, weibel-palade body. angiogenesis is dependent on multiple growth factors and receptors and their signalling systems and transcriptional regulators [ ] . the process is complex and encompasses the recruitment of mobile ecs and endothelial progenitor cells, the proliferation and apoptosis of these cells, and reorganization of the surroundings [ ] . to form stable new blood vessels, the response must be coordinated in time and space, and the ang/tie system is involved from beginning to end. to prepare for angiogenesis, ang- destabilizes quiescent endothelium through an internal autocrine loop mechanism [ , ] . before vascular sprouting starts, focal adhesion kinase and proteinases such as plasmin and metalloproteinases are excreted [ ] . often, this stage is preceded by activation of innate immunity and inflammation [ ] . apparently, the machinery to clean up after the work has been finished is installed before the work is commenced, again illustrating the close relations among the different processes [ ] . ang- maintains and, when required, restores the higher order architecture of growing blood vessels [ , , , ] . this is achieved by inhibiting apoptosis of ecs by tie mediated activation of pi k/akt signalling [ ] [ ] [ ] . ang- / tie signalling is involved in angiogenesis induced by cyclic strain and hypoxia [ , ] . although its role is less clear, tie might be involved in ec reactions to shear stress [ ] . ang- is a chemoattractant for ecs [ ] [ ] [ ] , and both ang- and ang- have proliferative effects on those cells [ , ] . at the end of a vascular remodelling phase, ang- induces apoptosis of ecs for vessel regression in competition with the survival signal of ang- [ ] . this apoptotic process requires macrophages, which are recruited by ang- [ , ] . ecs require support from surrounding cells such as pericytes, podocytes, and smooth muscle cells [ ] . these cells actively control vascular behaviour by producing signalling compounds (for instance, ang- and vegf) that govern the activity and response of ecs [ ] . to attract ecs, ang- secreted by support cells binds to the extracellular matrix. in quiescent ecs, this binding results in tie movement to the site of cell-cell interaction. in mobile ecs, ang- polarizes the cell with tie movement abluminal site [ ] . in tumour angiogenesis and in inflammation, ang- recruits tie positive monocytes and causes them to release cytokines and adopt a pro-angiogenic phenotype [ ] . the ang/tie system provides vascular wall stability by inducing ec survival and vascular integrity. however, this stability can be disrupted by ang- injection, which in healthy mice causes oedema [ , , , ] that can be blocked by systemic administration of soluble tie [ ] . ang- can impair homeostatic capacity by disrupting cell-cell adhesion through e-cadherin discharge and ec contraction [ , ] . in contrast, through effects on intracellular signalling, the cytoskeleton and junction-related molecules, ang- reduces leakage from inflamed venules by restricting the number and size of gaps that form at endothelial cell junctions [ , , ] . ang- also suppresses expression of tissue factor induced by vegf and tumour necrosis factor (tnf)-α, as well as expression of vascular cell adhesion molecule- , intercellular adhesion molecule- and e-selectin. as a result, endothelial inflammation is suppressed [ ] [ ] [ ] [ ] . in primary human glomerular ecs in vitro, ang- stabilizes the endothelium by inhibiting angiogenesis, and vegf increases water permeability [ ] . similar observations were made in bovine lung ecs and immortalized huvecs, in which ang- decreased permeability, adherence of polymorphonuclear leucocytes and interleukin- production [ ] . reaction to injury can be seen as an attempt to maintain homeostasis under exceptional conditions. ecs can be affected by several noxious mechanisms. the ang/tie system is considered crucial in fine-tuning their reaction to injury and in containing that reaction. ang- -deficient mice cannot mount an inflammatory response to peritonitis induced chemically or with staphylococcus aureus [ ] , but they can mount a response to pneumonia, suggesting the existence of inflammatory reactions for which ang- is not mandatory. ang- sensitizes ecs to activation by inflammatory cytokines. in ang- -deficient mice, leucocytes do roll on activated endothelium but they are not firmly attached, owing to the lack of ang- -dependent upregulation of adhesion molecules and the dominance of ang- -regulated suppression of adhesion molecules [ ] [ ] [ ] [ ] ] . in bovine retinal pericytes, hypoxia and vegf induce ang- and tie gene expression acutely without altering ang- mrna levels. the opposite occurs in bovine aortic ecs and microvascular ecs, underscoring the heterogeneity of ecs from different microvascular beds [ , , ] . lipopolysaccharide (lps) and pro-inflammatory cytokines can shift the ang/tie balance, rouse ecs from quiescence and provoke an inflammatory response. in rodents lps injection induces expression of ang- mrna and protein and reduces the levels of ang- , tie and tie phosphorylation in lung, liver and diaphragm within hours, which may promote or maintain vascular leakage. the initial increase in permeability is probably due to release of ang- stored in wpbs [ , ] . in a mouse model of lps-induced lung injury, pulmonary oedema was found to be related to the balance between vegf, ang- and ang- [ ] . in a comparable model, ang- -producing transfected cells reduced alveolar inflammation and leakage [ ] . in choroidal ecs, tnf induces ang- mrna and protein before affecting ang- and vegf levels [ ] . in huvecs, tnf-induced upregulation of ang- is mediated by the nf-κb pathway [ ] , and tnf-induced tie expression can be attenuated by both ang- and ang- . without tnf stimulation, only ang- can reduce tie expression [ ] . ang- sensitizes ecs to tnf, resulting in enhanced expression of intercellular adhesion molecule- , vascular cell adhesion molecule- and e-selectin [ , , ] . by inhibiting those endothelial adhesion molecules, ang- decreases leucocyte adhesion [ ] . angiopoietins can mediate the synthesis of platelet-activating factor by ecs to stimulate inflammation [ ] . moreover, both ang- and ang- can translocate p-selectin from wpbs to the surface of the ec [ ] , and both can also increase neutrophil adhesion and chemotaxis and enhance those processes when they are induced by interleukin- [ , , ] . in a rat model of haemorrhagic shock, ang- reduced vascular leakage, and it inhibited microvascular endothelial cell apoptosis in vitro and in vivo [ , ] . in this model, ang- promoted cell survival was partly controlled through integrin adhesion [ ] . it has been suggested that ec apoptosis in haemorrhagic shock contributes to endothelial hyperpermeability [ ] [ ] [ ] . apoptosis is one of the reactions to modsrelated injury as demonstrated in hypoxia/reperfusion [ ] . ang- and ang- are involved in cell-cell and cell-matrix binding [ , [ ] [ ] [ ] . endothelial permeability is greatly dependent on cell-cell adhesion. the major adherens junction is largely composed of vascular-endothelial cadherin. this complex can be disrupted by vegf, leading to increased vascular permeability [ , ] , which can be antagonized by ang- [ , ] . ecs can also bind to the matrix through the binding of ang- to integrins, which can mediate some of the effects of ang- without tie phosphorylation [ , ] . at low ang- concentrations, integrin and tie can cooperate to stabilize ecs [ ] . ang- might play a role in inflammatory diseases such as vasculitis by disrupting the cell-cell junction and inducing denudation of the basal membrane [ ] . ang- can mediate the translocation of tie to endothelial cell-cell contacts and induce tie -tie bridges with signal pathway activation, leading to diminished paracellular permeability [ ] . in the mature vessel, ang- acts as a paracrine signal to maintain a quiescent status quo, whereas ang- induces or facilitates an autocrine ec response [ , ] . in general, ang- can be viewed as a stabilizing messenger, causing continuous tie phosphorylation, and ang- as a destabilizing messenger preparing for action [ ] . attempts to unravel the exact molecular mechanisms that control the system are complicated by microenvironment-dependent endothelial phenotypes and reactivity and by flow typedependent reactions to dynamic changes [ , , ] . hence, the ec must be viewed in the context of its surroundings -the pericyte at the abluminal site, and the blood and its constituents on the luminal site [ ] . the ang/tie system certainly functions as one of the junctions in signal transduction and plays a key role in multiple cellular processes, many of which have been linked to mods. a therapy should intervene in the right place and at the right time, with the proper duration of action and without collateral damage [ , ] . the ang/tie system is involved in many processes and lies at the intersection of molecular mechanisms of disease. thus, interventions targeting this system might have benefits. as in other pleiotropic systems, however, unexpected and unwanted side effects are a serious risk. the absence of redundant systems to take over the function of ang/tie has the advantage that the effect of therapeutic intervention cannot easily be bypassed by the cell. on the other hand, because the cell has no escape, the effect may become uncontrolled and irreversible. moreover, the exact function of the ang/tie system in the pathological cascade is not fully established. what we see in animal models and in patients is most probably the systemic reflection of a local process. we do not know whether this systemic reflection is just a marker of organ injury or even a mediator of distant organ involvement. of the three main functions of the ang/tie system, it is mainly angiogenesis that has been evaluated as a therapeutic target. so far, the focus of ang/tie modulation has been on inhibiting angiogenesis related to malignant and ophthalmological diseases and to complications of diabetes [ , ] . in peripheral arterial occlusive disease, stimulation of angiogenesis seems a logical strategy to attenuate the consequences of ongoing tissue ischaemia. in a rat model of hind limb ischaemia, combined delivery of ang- and vegf genes stimulated collateral vessel development to the greatest extent [ , ] . thus far, therapy directed at vegf has reached the clinic, but not therapy directed at ang/tie [ ] . targeting homeostasis and repair/inflammation in critically ill patients is an attractive option and has already led to the development of new drugs [ , , ] . from current knowledge, one can speculate about the best options for therapy aimed at the ang/tie system. in critical illness, ang- is considered to be the 'good guy' because it can create vascular stability and thus its activity should be supported. in contrast, ang- appears to be a 'bad guy' that induces vascular leakage, so its activity should be inhibited [ ] . production of recombinant ang- is technically challenging as ang- is 'sticky' because of its high affinity for the extracellular matrix [ ] . however, stable ang- variants with improved receptor affinity have been engineered. a stable soluble ang- variant has anti-permeability activity [ ] . when injected intraperitoneally in mice, human recombinant ang- can prevent lps-induced lung hyperpermeability [ ] . in diabetic mice, a stable ang- derivative attenuated proteinuria and delayed renal failure [ ] , and manipulating the ang- /ang- ratio changed infarct size [ ] . a more profound ang- effect can be achieved by locally stimulating ang- production. in experimental acute respiratory distress syndrome, transfected cells expressing ang- reduced alveolar inflammation and leakage [ ] . an adenovirus construct encoding ang- protected mice from death in an lps model, and ang- gene therapy reduced acute lung injury in a rat model [ , , ] . in hypertensive rats, a plasmid expressing a stable ang- protein reduced blood pressure and end-organ damage [ ] . if used in a disease with a limited duration, as critical illness should be, virus/plasmid-driven production of ang- could easily be shut down when it is no longer needed. manipulating ang- activity is also difficult. ang- stored in wpbs is rapidly released and must be captured immediately to prevent autocrine/paracrine disruption of protective ang- / tie signalling. soluble tie or ang- inhibitors should be effective [ , ] . neutralizing antibodies against ang- might also be an option. replenishment of ang- stores could be abolished by small interfering rna techniques or spiegelmer/aptamer approaches [ , , ] . however, no bad guy is all bad, and no good guy is all good. for example, ang- has been linked to the development of pulmonary hypertension [ ] . also, under certain circumstances ang- can act as a tie agonist and exert effects similar to those of ang- -an unexplained finding that illustrates our limited understanding of the ang/tie system [ ] . complete blockade of ang- might also hamper innate immunity and revascularization. finding the right balance and timing will be the major challenge when developing therapies to target the ang/tie system. in the meantime, we might have already used ang/tie-directed therapy with the most pleiotropic of all drugs -corticosteroids. in the airways, steroids suppressed ang- and increased ang- expression [ , , ] . interventions further downstream targeting specific adaptor molecules, signalling pathways, or transcription factors have yet to be explored. in patients with malignant disease, the ang/tie system might serve as a tumour or response marker. in patients with multiple myeloma, normalization of the ang- /ang- ratio reflects a response to treatment with anti-angiogenesis medication [ ] . in patients with non-small-cell lung cancer, ang- is increased in serum and indicates tumour progression [ ] . after allogeneic stem cell transplantation in patients with high-risk myeloid malignancies, the serum ang- concentration predicts disease-free survival [ ] , possibly reflecting a relation between cancer-driven angiogenesis and ang- serum level. in nonmalignant disease, the levels of ang/tie system components correlate with disease severity [ , , [ ] [ ] [ ] [ ] ]. however, current data are insufficient to justify the use of serum soluble tie /ang levels for diagnostic and prognostic purposes. in critical illness, assessment of the ang/tie system in patients with different severities of disease and with involvement of different organ systems might help to define our patient population and allow us to rethink our concepts of mods. in this way, such work may lead to enhanced diagnosis and prognostication in the future [ ] . accumulating evidence from animal and human studies points to the involvement of the ang/tie system in vascular barrier dysfunction during critical illness. many processes in injury and in repair act through this nonredundant system. thus far, only preliminary studies in critically ill patients have been reported. methods to manipulate this system are available but have not been tested in such patients. the response to treatment is difficult to predict because of the pleiotropic functions of the ang/tie system, because the balance among its components appears to be more important than the absolute levels, and because the sensitivity of the endothelium to disease-related stimuli varies, depending on the environment and the organ involved. to avoid disappointment, further experimental and translational research must be carried out, and ang/tie modulation must not be introduced into the clinic prematurely. implementing the results of this research in critical care represents an opportunity to show what we have learned [ ] . ang/tie signalling is a very promising target and must not be allowed to become lost in translation [ ] . treating sepsis sepsis: rethinking the approach to clinical research multiorgan failure is an adaptive, endocrine-mediated, metabolic response to overwhelming systemic inflammation sir isaac newton, sepsis, sirs, and cars apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction nf-kappab links innate immunity to the hypoxic response through transcriptional regulation of hif- alpha efficacy and safety of recombinant human activated protein c for severe sepsis effect of treatment with low doses of hydrocortisone and fludrocortisone on mortality in patients angiopoietins have distinct modular domains essential for receptor binding, dimerization and superclustering activation of tie by angiopoietin- and angiopoietin- results in their release and receptor internalization functional significance of tie signaling in the adult vasculature structure of the extracellular domain of tie receptor tyrosine kinases and localization of the angiopoietinbinding epitope protein tyrosine kinase structure and function tie receptors and their angiopoietin ligands are context-dependent regulators of vascular remodeling identification of tek/tie binding partners. binding to a multifunctional docking site mediates cell survival and migration identification of a soluble form of the angiopoietin receptor tie- released from endothelial cells and present in human blood vegf induces tie shedding via a phosphoinositide -kinase/akt dependent pathway to modulate tie signaling regulated proteolytic processing of tie modulates ligand responsiveness of the receptor-tyrosine kinase tie activation of the orphan endothelial receptor tie modifies tie -mediated intracellular signaling and cell survival interaction between tie receptors modulates angiogenic activity of angiopoietin in endothelial progenitor cells. cardiovasc res functional symbiosis between endothelium and epithelial cells in glomeruli tie expression and phosphorylation in angiogenic and quiescent adult tissues endothelial/pericyte interactions the role of pericytes in blood-vessel formation and maintenance angiopoietins assemble distinct tie signalling complexes in endothelial cell-cell and cell-matrix contacts formation and function of weibel-palade bodies the tie- ligand angiopoietin- is stored in and rapidly released upon stimulation from endothelial cell weibel-palade bodies dynamics and plasticity of weibel-palade bodies in endothelial cells tie -expressing monocytes and tumor angiogenesis: regulation by hypoxia and angiopoietin- tie -expressing monocytes: regulation of tumor angiogenesis and therapeutic implications localization of tie and phospholipase d in endothelial caveolae is involved in angiopoietin- -induced mek/erk phosphorylation and migration in endothelial cells genomic structures of the human angiopoietins show polymorphism in angiopoietin- hypoxia and vascular endothelial growth factor selectively upregulate angiopoietin- in bovine microvascular endothelial cells regulation of angiopoietin- mrna levels in bovine microvascular endothelial cells by cytokines and hypoxia angiopoietin- functions as an autocrine protective factor in stressed endothelial cells expression of angiopoietin- in endothelial cells is controlled by positive and negative regulatory promoter elements transcriptional regulation of human angiopoietin- by transcription factor ets- internal translation initiation mediated by the angiogenic factor tie signaling mechanisms regulating endothelial permeability angiopoietin- requires p rhogap to protect against vascular leakage in vivo transcriptome of angiopoietin -activated human umbilical vein endothelial cells gene expression analysis of tek/tie signaling angiogenic actions of angiopoietin- require endothelium-derived nitric oxide mechanisms which mediate the antiapoptotic effects of angiopoietin- on endothelial cells angiopoietin- induces endothelial cell sprouting through the activation of focal adhesion kinase and plasmin secretion angiopoietin chemotactic activities on neutrophils are regulated by pi- k activation angiopoietin- activates both anti-and proapoptotic mitogen-activated protein kinases signaling and regulation of endothelial cell survival by angiopoietin- angiopoietin- at high concentration can enhance endothelial cell survival through the phosphatidylinositol '-kinase/akt signal transduction pathway angiopoietins- and - are both capable of mediating endothelial paf synthesis: intracellular signalling pathways angiopoietin- promotes endothelial cell proliferation and migration through ap- -dependent autocrine production of interleukin- angiopoietin- modulates endothelial cell function and gene expression via the transcription factor fkhr (foxo ) abin- protects endothelial cells from death and has a role in the antiapoptotic effect of angiopoietin- roles of reactive oxygen species in angiopoietin- /tie- receptor signaling differential function of tie at cell-cell contacts and cell-substratum contacts regulated by angiopoietin- angiopoietin- displays vegfdependent modulation of capillary structure and endothelial cell survival in vivo differential response of lymphatic, venous and arterial endothelial cells to angiopoietin- and angiopoietin- biological action of angiopoietin- in a fibrin matrix model of angiogenesis is associated with activation of tie angiogenesis and inflammation face off innate immunity and angiogenesis transcriptional regulators of angiogenesis extracellular matrix mediates a molecular balance between vascular morphogenesis and regression the tie- ligand angiopoietin- destabilizes quiescent endothelium through an internal autocrine loop mechanism angiopoietin- and vascular endothelial growth factor induce expression of inflammatory cytokines before angiogenesis recombinant angiopoietin- restores higher-order architecture of growing blood vessels in mice in the absence of mural cells contextual role for angiopoietins and tgfbeta in blood vessel stabilization angiopoietin- is an apoptosis survival factor for endothelial cells direct actions of angiopoietin- on human endothelium: evidence for network stabilization, cell survival, and interaction with other angiogenic growth factors angiopoietin- inhibits endothelial cell apoptosis via the akt/survivin pathway cyclic strain regulates the notch/cbf- signaling pathway in endothelial cells: role in angiogenic activity expression of tie- by human monocytes and their responses to angiopoietin- transcriptional and post-translation regulation of the tie receptor by fluid shear stress changes in vascular endothelial cells chemotactic properties of angiopoietin- and - , ligands for the endothelial-specific receptor tyrosine kinase tie obligatory participation of macrophages in an angiopoietin -mediated cell death switch angiopoietin- causes inflammation in vivo by promoting vascular leakage expressional regulation of the angiopoietin- and - and the endothelial-specific receptor tyrosine kinase tie in adrenal atrophy: a study of adrenocorticotropin-induced repair angiopoietins: a link between angiogenesis and inflammation angiopoietin- decreases plasma leakage by reducing number and size of endothelial gaps in venules angiopoietin- protects the adult vasculature against plasma leakage angiopoietin- negatively regulates expression and activity of tissue factor in endothelial cells angiopoietin- is an antipermeability and anti-inflammatory agent in vitro and targets cell junctions angiopoietin- reduces vegf-stimulated leukocyte adhesion to endothelial cells by reducing icam- , vcam- , and e-selectin expression angiopoietin- inhibits endothelial permeability, neutrophil adherence and il- production angiopoietin and vascular endothelial growth factor modulate human glomerular endothelial cell barrier properties angiopoietin- sensitizes endothelial cells to tnf-alpha and has a crucial role in the induction of inflammation hypoxia and vascular endothelial growth factor acutely up-regulate angiopoietin- and tie mrna in bovine retinal pericytes hypoxic regulation of angiopoietin- expression in endothelial cells regulation of angiopoietin expression by bacterial lipopolysaccharide angiogenic growth factors in the pathophysiology of a murine model of acute lung injury prevention of lps-induced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin sequential induction of angiogenic growth factors by tnfalpha in choroidal endothelial cells tumor necrosis factoralpha upregulates angiopoietin- in human umbilical vein endothelial cells regulation of tie expression by angiopoietin-potential feedback system osteoprotegerin upregulates endothelial cell adhesion molecule response to tumor necrosis factor-alpha associated with induction of angiopoietin- angiopoietin-mediated endothelial p-selectin translocation: cell signaling mechanisms ang- and pdgf-bb cooperatively stimulate human peripheral blood monocyte fibrinolysis angiopoietins can directly activate endothelial cells and neutrophils to promote proinflammatory responses angiopoietin- inhibits intrinsic apoptotic signaling and vascular hyperpermeability following hemorrhagic shock angiopoietin- promotes cardiac and skeletal myocyte survival through integrins apoptotic signaling induces hyperpermeability following hemorrhagic shock alpha lipoic acid attenuates microvascular endothelial cell hyperpermeability by inhibiting the intrinsic apoptotic signaling trauma-hemorrhagic shock mesenteric lymph induces endothelial apoptosis that involves both caspase-dependent and caspase-independent mechanisms rho-kinase-dependent factin rearrangement is involved in the inhibition of pi -kinase/akt during ischemia-reperfusion-induced endothelial cell apoptosis besides adhesion: new perspectives of integrin functions in angiogenesis. cardiovasc res direct cell adhesion to the angiopoietins mediated by integrins effects of protein and gene transfer of the angiopoietin- fibrinogen-like receptor-binding domain on endothelial and vessel organization the role of adherens junctions and ve-cadherin in the control of vascular permeability vegf receptor and the adherens junction as a mechanical transducer in vascular endothelial cells opposing effect of angiopoietin- on vegf-mediated disruption of endothelial cell-cell interactions requires activation of pkc beta angiopoietin- prevents vegfinduced endothelial permeability by sequestering src through mdia stable interaction between alpha beta integrin and tie tyrosine kinase receptor regulates endothelial cell response to ang- elevated serum angiopoietin- correlates with degree of arteriosclerosis in ckd v patients emerging roles of the angiopoietin-tie and the ephrin-eph systems as regulators of cell trafficking endothelial immunogenicity: a matter of matrix microarchitecture vascular bed origin dictates flow pattern regulation of endothelial adhesion molecule expression angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation angiogenesis treatment, new concepts on the horizon inhibition of tie- signaling induces endothelial cell apoptosis, decreases akt signaling, and induces endothelial cell expression of the endogenous anti-angiogenic molecule, thrombospondin- kerbel rs: tumor angiogenesis coadministration of angiopoietin- and vascular endothelial growth factor enhances collateral vascularization combined angiopoietin- and vascular endothelial growth factor gene transfer restores cavernous angiogenesis and erectile function in a rat model of hypercholesterolemia inhibitors of growth factor receptors, signaling pathways and angiogenesis as therapeutic molecular agents biomedical significance of endothelial cell specific growth factor, angiopoietin angiopoietin- : modulator of vascular permeability in acute lung injury comp-ang : a designed angiopoietin- variant with nonleaky angiogenic activity renoprotective effect of comp-angiopoietin- in db/db mice with type diabetes critical role of angiopoietins/tie- in hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis vascular endothelial growth factor gene therapy increases survival, promotes lung angiogenesis, and prevents alveolar damage in hyperoxia-induced lung injury: evidence that angiogenesis participates in alveolarization protective role of angiopoietin- in endotoxic shock angiopoietin- prevents hypertension and target organ damage through its interaction with endothelial tie receptor angiopoietin concentrations in infants developing bronchopulmonary dysplasia: attenuation by dexamethasone inhibition of in vivo tumor angiogenesis and growth via systemic delivery of an angiopoietin -specific rna aptamer therapeutic application of rnai: is mrna targeting finally ready for prime time? angiopoietin/tie pathway influences smooth muscle hyperplasia in idiopathic pulmonary hypertension relationship between vascular endothelial growth factor and angiopoietin- in asthmatics before and after inhaled beclomethasone therapy normalization of the serum angiopoietin- to angiopoietin- ratio reflects response in refractory/resistant multiple myeloma patients treated with bortezomib serum angiopoietin- as a clinical marker for lung cancer angiopoietin- predicts disease-free survival after allogeneic stem cell transplantation in patients with high-risk myeloid malignancies the full cycle angiopoietin is a potential mediator of high-dose interleukin -induced vascular leak angiopoietin- serum levels are elevated in patients with liver cirrhosis and hepatocellular carcinoma the author(s) declare that they have no competing interests. key: cord- -gk fnb authors: kehoe, patrick gavin; wong, steffenny; al mulhim, noura; palmer, laura elyse; miners, j. scott title: angiotensin-converting enzyme is reduced in alzheimer’s disease in association with increasing amyloid-β and tau pathology date: - - journal: alzheimers res ther doi: . /s - - - sha: doc_id: cord_uid: gk fnb background: hyperactivity of the classical axis of the renin-angiotensin system (ras), mediated by angiotensin ii (ang ii) activation of the angiotensin ii type receptor (at r), is implicated in the pathogenesis of alzheimer’s disease (ad). angiotensin-converting enzyme- (ace- ) degrades ang ii to angiotensin – (ang ( - )) and counter-regulates the classical axis of ras. we have investigated the expression and distribution of ace- in post-mortem human brain tissue in relation to ad pathology and classical ras axis activity. methods: we measured ace- activity by fluorogenic peptide substrate assay in mid-frontal cortex (brodmann area ) in a cohort of ad (n = ) and age-matched non-demented controls (n = ) for which we have previous data on ace- activity, amyloid β (aβ) level and tau pathology, as well as known ace (rs ) indel polymorphism, apolipoprotein e (apoe) genotype, and cerebral amyloid angiopathy severity scores. results: ace- activity was significantly reduced in ad compared with age-matched controls (p < . ) and correlated inversely with levels of aβ (r = − . , p < . ) and phosphorylated tau (p-tau) pathology (r = − . , p < . ). ace- was reduced in individuals possessing an apoe ε allele (p < . ) and was associated with ace indel polymorphism (p < . ), with lower ace- activity in individuals homozygous for the ace insertion ad risk allele. ace- activity correlated inversely with ace- activity (r = − . , p < . ), and the ratio of ace- to ace- was significantly elevated in ad (p < . ). finally, we show that the ratio of ang ii to ang ( – ) (a proxy measure of ace- activity indicating conversion of ang ii to ang ( – )) is reduced in ad. conclusions: together, our findings indicate that ace- activity is reduced in ad and is an important regulator of the central classical ace- /ang ii/at r axis of ras, and also that dysregulation of this pathway likely plays a significant role in the pathogenesis of ad. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. ad in human brain tissue [ , ] . angiotensin ii type receptor blockers (arbs) and angiotensin-converting enzyme inhibitors (aceis) reduce the amount of ad-like pathology and improve cognitive performance in most but not all mouse models of ad [ ] [ ] [ ] [ ] [ ] [ ] . translation of these treatments in ad is also supported in secondary outcomes of clinical trials of various arbs and aceis, as well as in epidemiological studies where the prevalence of ad was reduced [ ] [ ] [ ] [ ] [ ] . last, the ace- indel polymorphism (rs ) is a genetic risk factor for sporadic ad [ ] . this finding has previously been supported by several meta-analyses [ ] [ ] [ ] [ ] [ ] but not by recent genome-wide association studies. ace- is a zinc metallopeptidase which shares % sequence homology within the ace- catalytic region [ , ] . the ace- metalloprotease is expressed mostly as a transmembrane protein, but it also exists in an active soluble truncated form [ ] . it is expressed predominantly in endothelial and arterial smooth muscle cells throughout the body [ ] , but it is also expressed in non-vascular cells within the brain, including neuronal cell bodies [ ] and astroglial cells [ ] . upon its discovery, ace- was shown to generate angiotensin - (ang ( - )) from ang ii, and, to a lesser extent, angiotensin - (ang ( - )) from ang i [ , , ] . emerging data suggest that ace- -mediated conversion of ang ii to ang ( - ) and subsequent activation of the mas receptor by ang ( - ) (comprising the ace- /ang ( - ) /mas axis) oppose the local actions of the classical ras pathway in both the periphery (reviewed in [ ] ) and brain (reviewed in [ ] [ ] [ ] [ ] ). in experimental animal studies, ace- regulates blood pressure by counteracting the effects of the classical axis. a reduction in ace- expression has been implicated in cardiac and renal pathologies (reviewed in [ ] ) associated with chronic hypertension. activation of brain ace- has been shown to be neuroprotective in animal models of ischaemic stroke [ , ] . previous studies have suggested a link between reduced activity of the ace- /ang ( - )/mas axis and neurodegenerative conditions, including multiple sclerosis [ ] . a recent study provided the first clues of an association with ad and reported reduced serum ace- activity in patients with ad compared with control subjects [ ] . notably, this study also identified that ace- converts aβ (an early deposited and highly amyloidogenic form of aβ that seeds plaque formation [ ] ) to aβ , which in turn is cleaved by ace- to less toxic aβ and aβ species [ ] . ang ( - ) levels were also reduced in a mouse model of sporadic ad in association with hyperphosphorylation of tau [ ] . in the present study, we investigated the expression and distribution of ace- in relation to ad pathology and the classical ras axis in human post-mortem brain tissue. we show, for the first time to our knowledge, that ace- activity is reduced in human post-mortem brain tissue in ad in relation to aβ and tau pathology, and also that ace- correlates inversely with ace- activity. we also show that the ratio of ang ii to ang ( - ) (a proxy measure of ace- activity) was increased in ad, indicating reduced conversion of ang ii to ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) . together, these data indicate that the ace- / ang ( - )/mas axis is dysregulated in ad and that loss of function of this regulatory arm of ras may contribute, at least in part, to overactivation of the classical ras axis associated with ad pathogenesis. brain tissue was obtained from the south west dementia brain bank, university of bristol, uk, with local research ethics committee approval (national research ethics service /h / + ). tissue was dissected from the mid-frontal cortex (brodmann area ) in cases of ad and age-matched controls. brains had been subjected to detailed neuropathological assessment according to the national institute on aging-alzheimer's association guidelines [ ] , and ad pathology was a sufficient explanation for the dementia in these cases. control brains were from people who had no history of dementia, had been extensively assessed neuropathologically, and had few or absent neuritic plaques, braak tangle stage iii or less, and no other neuropathological abnormalities. the demographic data for these cases are presented in table , and the medical research council uk brain banks network (mrc uk-bbn) database identifiers are shown in additional file : table s . previous measurements of ace- activity, measured by fluorogenic activity assay, were available for all cases [ , ] . total soluble (nonidet p- -extracted) and insoluble ( m guanidine hydrochloride-extracted) aβ levels were measured previously by sandwich enzyme-linked immunosorbent assay (elisa) [ ] , and cerebral amyloid angiopathy (caa) severity, which was graded semi-quantitatively on a -point scale by a method adapted from that of olichney et al. [ ] , had previously been reported [ ] . phosphorylated tau (p-tau) load (area fraction of cerebral cortex immunopositive for p-tau) had been measured for all cases, as previously reported [ , ] . ace genotype data for the alu -bp insertion(i)/deletion(d) (indel) polymorphism (rs ) in intron of the ace gene were previously reported [ , ] . last, all cases had previously been apolipoprotein e (apoe)-genotyped [ , ] by a polymerase chain reaction method [ ] . the right cerebral cortex had been fixed in % formalin for a minimum of weeks before the tissue was processed and paraffin blocks were taken for pathological assessment. the left cerebral hemisphere had been sliced and frozen at − °c until used for biochemical assessment. for each case, mg of dissected frozen brain tissue was homogenised in a precellys homogeniser (stretton scientific, stretton, uk) as previously described [ , ] . the samples were centrifuged at , rpm, and the clarified supernatants were aliquoted and stored at − °c until required. total protein was measured using the total protein kit (sigma-aldrich, poole, uk) following the manufacturer's guidelines. all brain tissue was obtained within h after death. ace- activity was measured in brain tissue using the sensolyte® ace activity assay kit (catalogue number as- ; anaspec, fremont, ca, usa). the assay was performed in black, flat-bottomed, non-binding, well nunc fluoronunc plates (fisher scientific, loughborough, uk) following the manufacturer's guidelines with minor modifications. brain tissue homogenates were prepared in assay buffer provided in the kit, to which . % triton x- was added. samples were centrifuged at , rpm for minutes at °c, and supernatants were removed and stored at − °c until used. supernatants were diluted : in the proprietary ace- assay buffer and incubated for minutes at room temperature prior to addition of the ace- -specific fluorescence resonance energy transfer (fret) peptide and then incubated for minutes in the dark. cleavage of the ace- fret peptide was measured using a bmg fluostar op-tima microplate reader (bmg labtech, aylesbury, uk) at an excitation/emission wavelength of / nm. ace- activity was interpolated from a serial dilution of -methoxycoumarin- -yl-acetyl (mca) fluorescence reference standard, and measurements for each case were repeated in duplicate. to confirm the specificity of the commercial ace- assay kit, we measured ace- activity in a subset of samples (ten controls and ten ad) for which we had previously measured ace- activity as outlined above. the assay was performed in black, flat-bottomed, nonbinding, -well nunc fluoronunc plates. recombinant human ace- ( - ng/ml) (r&d systems, cambridge, uk) and brain tissue supernatants (diluted : ) were diluted in assay buffer ( mm tris, m nacl, ph . ) and pre-incubated with an ace- specific inhibitor, mln ( μm) (calbiochem, nottingham, uk) or assay buffer alone for minutes at °c. an ace- fluorogenic peptide mca-apk(dnp) (enzo life sciences, exeter, uk) was then added, and the reaction was incubated at °c for minutes in the dark. fluorescence was read at an excitation/emission wavelength of / nm using a bmg fluostar optima microplate reader. ace- -specific activity was calculated after subtracting fluorescence in the presence of mln- from the uninhibited sample. we observed a very strong correlation between the independent measurements of ace- in the presence of mln ( μm) and with the kit, indicating the specificity of the ace- assay kit (additional file : figure s ). ang ii levels were measured in brain tissue homogenates extracted in % sds lysis buffer ( μm nacl, mm tris, ph , μm phenylmethylsulphonylfluoride, μg/ml aprotinin [sigma-aldrich] and % sds in distilled water) using a commercially available sandwich elisa kit (abcam, cambridge, uk) following the manufacturer's guidelines. in brief, recombinant human ang ii or brain tissue supernatants (diluted : in pbs) were added in duplicate to wells that had been pre-coated with an ang ii-specific capture antibody and incubated for h at room temperature. after a wash step, the wells were incubated for h with biotinylated anti-ang ii antibody at room temperature. the plate was again washed, followed by a -minute incubation with streptavidin/hrp. after a final wash, , ′, , ′-tetramethylbenzidine (tmb) substrate was added for minutes, and the absorbance at nm was read using a fluostar optima plate reader. the concentration of ang ii was interpolated from a serial dilution of recombinant ang ii ( - . pg/ml) and measured in duplicate for each case. ang ( - ) levels were measured in human brain tissue homogenates in % sds lysis buffer (see above) using an in-house direct elisa kit. recombinant human ang ( - ) (enzo life sciences) or human brain tissue homogenates (diluted : in pbs) were incubated for h in a clear, high binding capacity nunc maxisorp plate (thermo fisher scientific, waltham, ma, usa) at °c with shaking. the wells were washed five times in pbs with . % tween- and blocked for h in pbs: % bovine serum albumin (sigma-aldrich). after another five washes, the wells were incubated with biotinylated antihuman ang - ( μg/ml in pbs) (cloud-clone, wuhan, china) for h at °c with shaking, followed by a further wash step. streptavidin/hrp ( : ) in pbs/ . % tween- was added to each well, which was incubated at room temperature for minutes in the dark. tmb substrate (r&d systems) was added after a further wash and left to develop in the dark for minutes. absorbance at nm was read following the addition of n sulphuric acid ('stop' solution) using a fluostar optima plate reader. ang ( - ) concentration was interpolated from a standard curve generated by serially diluting recombinant human ang ( - ) ( - . pg/ml). the assay showed minimal cross-reactivity with a number of closely related peptides, including ang i, ang ii and ang iii. formalin-fixed, paraffin-embedded tissue sections ( μm) were cut and de-waxed prior to immunohistochemistry. sections were pre-treated in trisodium citrate buffer ( mm), ph , and microwaved for minutes, left to stand for minutes, and boiled for a further minutes before being left to stand for minutes at room temperature. sections were then rinsed thoroughly and covered in horse serum blocking solution, rinsed again, and incubated overnight at room temperature with anti-ace- antibody ( . μg/ml, ab ; abcam). bound antibody was visualised using a biotinylated universal antibody followed by vectastain elite abc avidin-biotin complex kit (vector laboratories, peterborough, uk) and a reaction with . % h o . specificity of the antibody was assessed by preadsorption of the ace- antibody with a -fold molar excess of recombinant human ace- protein (r&d systems). unpaired two-tailed t tests or analysis of variance (anova) with bonferroni's post hoc analysis was used for comparisons between groups, and pearson's test was used to assess linear correlation with spss version (spss, chicago, il, usa) and graphpad prism version (graphpad software, la jolla, ca, usa) software. p values < . were considered statistically significant. ace- enzyme activity is reduced in alzheimer's disease in association with increasing aβ load and tau pathology ace- activity was significantly reduced by approximately % in the mid-frontal cortex in ad compared with age-matched controls (p < . ) (fig. a) . ace- varied according to disease severity when the controls and ad cases were grouped and stratified into the following braak tangle stage groups: -ii, iii-iv, and v-vi (p < . by anova). post hoc analysis using the bonferroni correction for multiple comparisons revealed that ace- activity was significantly reduced in braak tangle stages v-vi compared with stages -ii (p < . ) and stages iii-iv (p < . ) (fig. b) . no difference was observed between braak stages -ii and stages iii-iv. in a combined ad and control cohort, ace- activity correlated inversely with total insoluble aβ levels (r = − . , p < . ) (fig. c) but not with soluble aβ (data not shown). ace- correlated inversely with βsecretase activity (r = − . p < . ) (additional file : figure s ). ace- correlated inversely with p-tau load (r = . , p < . ) (fig. d) . ace- activity is reduced in relation to apoe and ace polymorphisms and caa severity ace- activity was significantly lower in individuals possessing an apoe ε allele, an established genetic risk factor for sporadic ad [ ] , than in those without (p < . ) (fig. a) . ace- activity also differed significantly post hoc analysis revealed that ace- activity was reduced in braak tangle stages v-vi compared with stages -ii and iii-iv (p < . and p < . respectively) and in braak tangle stages iii-iv compared with stages -ii, but the difference was not statistically different. the bars indicate the mean value and sem. c and d scatterplots showing that ace- activity was inversely correlated with insoluble amyloid-β (aβ) load (measured by enzyme-linked immunosorbent assay) (r = − . , p < . ) and phosphorylated tau (p-tau) load (measured by field fraction analysis) (r = − . , p < . ). the solid inner line indicates the best-fit linear regression and the outer lines the % confidence intervals. *p < . , **p < . , ***p < . , ****p < . . rfu relative fluorescence units between ace (rs ) indel genotypes (p < . ), with individuals who were homozygous ii for ace- (previously associated with increased risk for ad [ ] ) having the lowest ace- activity, although post hoc analysis revealed that this did not reach statistical significance (fig. b) . we assessed ace- activity in relation to caa severity and found, as for ace- activity [ ] , a tendency, although not significant, towards increased ace- activity in cases with moderate to severe caa compared with absent to mild caa (p = . ) (fig. c) . ace- is inversely correlated with ace- , and the ratio of ace- to ace- is increased in alzheimer's disease ace- activity correlated inversely with ace- activity in a combined ad and control cohort (r = − . , p > . ) (fig. a) . the same pattern was observed and remained statistically significant when the control (r = − . , p < . ) and ad (r = − . , p < . ) groups were analysed separately. previous reports have suggested the ratio of ace- to ace- is a good proxy measure for the activation status of classical and regulatory ras pathways [ ] . with this in mind, we calculated the ace- /ace- ratio for all cases and found that it was significantly increased in ad compared with controls (p > . ) (fig. b) . the ace- /ace- ratio also correlated positively with insoluble aβ level, approaching significance (r = . , p = . ) (fig. c) , and significantly with p-tau (r = . , p < . ) (fig. d) . the ace- /ace- ratio was increased in individuals possessing an apoe ε allele, approaching significance (p = . ) (fig. e) , and differed significantly according to ace (rs ) indel polymorphism (p < . ). post hoc analysis revealed that the ratio was significantly higher in individuals with ace ii (ad risk factor) than in dd (p < . ) and in id than in dd (p < . ) (fig. f) . ang ii levels were significantly increased in mid-frontal cortex in ad compared with age-matched controls (p < . ) (fig. a) , whereas ang ( - ) levels were unchanged (fig. b) . we calculated the ang ii/ang ( - ) ratio (as a proxy indicator of ace- activity) and found that the ang ii/ang ( - ) ratio was significantly increased in ad (p > . ) (fig. c) . these data indicate that the conversion of ang ii to ang ( - ) is likely to be reduced in ad because of lower ace- activity. ace- expression in human brain tissue ace- was localised primarily to capillaries but also had a perivascular distribution around larger arterioles (fig. a) . ace- labelled non-vascular cells that strongly resembled astrocytes ( fig. b and c) . labelling was not observed with pre-adsorption of the ace- antibody with recombinant human ace- , demonstrating specificity of the antibody (fig. d ). in the present study, we show that ace- activity is reduced in post-mortem brain tissue in ad in association with increased aβ and tau pathology. the reduction in ace- was more pronounced in individuals carrying an apoe ε allele and in those who were homozygous ii for the ace (rs ) indel polymorphism (both of which are suggested genetic risk factors for ad [ ] ). ace- activity correlated inversely with ace- activity (which we have previously shown to be increased in ad [ , ] ), and the ace- /ace- ratio was higher in ad. together, these data strongly suggest that reduced ace- fig. angiotensin-converting enzyme (ace- ) activity is reduced in association with apolipoprotein e (apoe) ε and ace (rs ) indel polymorphism and increased in cerebral amyloid angiopathy (caa). a bar chart showing reduced ace- activity in individuals with an apoe ε allele (p < . ). b bar chart showing that ace- activity varied according to ace indel polymorphism (p < . ), with a trend towards reduced ace- activity in ace- ii homozygotes. c bar chart showing elevated ace- activity in moderate to severe caa compared with absent to mild caa, approaching significance (p = . ). the bars indicate the mean value and sem. *p < . . rfu relative fluorescence units fig. angiotensin-converting enzyme (ace- ) activity is inversely correlated with ace- activity, and the ace- /ace- ratio is increased, in alzheimer's disease (ad). a scatterplot showing a strong inverse relationship between ace- and ace- activity in mid-frontal cortex (r = −. , p < . ). the inner solid line indicates the best-fit linear regression and the outer lines the % confidence intervals. each dot represents an individual brain. b bar chart showing elevated ace- /ace- ratio in ad (p < . ). c and d scatterplots showing positive correlation between the ace- /ace- ratio and insoluble amyloid-β (aβ) load (r = . , p = . ) and p-tau load (r = . , p < . ). e bar chart showing a trend towards increased ace- /ace- ratio in individuals who possessed an apolipoprotein e (apoe) ε allele. f bar chart showing lower ace:ace- ratio in individuals who were homozygous dd for the ace (rs ) indel polymorphism compared with ii (p < . ) and id (p < . ). the bars indicate the mean value and sem. *p < . , **p < . , ****p < . . rfu relative fluorescence units fig. the ratio of angiotensin ii (ang ii) to angiotensin ( - ) (ang ( - ) ) (a proxy measure of ace- activity) is increased, indicating reduced conversion of ang ii to ang ( - ) in alzheimer's disease (ad). bar charts showing a elevated ang ii levels in ad and b unchanged ang ( - ) levels in ad compared with age-matched controls in mid-fontal cortex. c bar chart showing the ang ii/ang ( - ) ratio was significantly increased in ad (p < . ). the bars indicate the mean value and sem. ***p < . , ****p < . activity within the brain contributes to ad pathogenesis and is associated with increased activation of the central classical ras axis. the brain has its own intrinsic ras [ ] [ ] [ ] , and we have shown in our previous studies that ace- , the rate-limiting enzyme in the production of ang ii, is overactive in ad [ , ] . it is widely accepted that ang ii-mediated signalling via at r (commonly termed the classical axis) is overactive in ad and is associated with ad pathogenesis (reviewed in [ ] ). this view has been supported in various animal studies in which infusion of ang ii resulted in elevated plaque and tau pathology and significant cognitive impairment [ , ] . secondary observations in clinical trials and epidemiological studies have provided further evidence that ras-targeting drugs that either block the production of ang ii or prevent at r-mediated signalling reduce the prevalence of ad [ ] [ ] [ ] [ ] [ ] , while cognitive performance is improved and pathology reduced, in animal models of ad [ ] [ ] [ ] [ ] [ ] [ ] . until recently, the prevailing view of the ras in ad has been oversimplified because it has failed to consider the contribution of the other downstream ras regulatory pathways within the brain. in this study, we found reduced brain ace- activity in ad, which supports a recent study showing lower peripheral serum ace- levels in ad [ ] . ace- activity correlated inversely with parenchymal aβ load and increased p-tau levels. we also observed a strong inverse relationship between ace- and β-secretase activity, suggesting that ace- may contribute in some way to regulating the amyloidogenic processing of app. there are several possible mechanisms that link reduced ace- activity to the pathogenesis of ad. firstly, lower ace- activity will, via a lower conversion of ang ii to ang ( - ), result in elevated ang ii levels (as we have shown in this study). an increase in ang ii/ang ( - ) ratio has commonly been reported in other chronic conditions associated with overactivation of the central axis [ ] . secondly, ace- is primarily responsible for generating ang ( - ) from ang ii [ , , ] , and subsequent ang ( - ) activation of the mas receptor counter-regulates the detrimental effects of the classical (ace- /ang ii/at r) axis [ ] [ ] [ ] and has been linked with enhancing learning and memory processing [ , ] . lastly, ace- has recently been shown to convert aβ , a highly amyloidogenic form of aβ that seeds plaque formation [ ] , to aβ , which in turn is cleaved by ace- to aβ or, to a lesser extent, aβ , which have reduced toxicity [ ] . lower ace- activity in ad may therefore promote the early deposition of aβ and prevent downstream cleavage of aβ by ace- .together, these data suggest a putative protective role of the ace- /ang ( - )/mas pathway, not only against the development of pathology but also against the decline in cognitive function, that is lost in ad. our findings indicate that the balance between the classical (ace- /ang ii/at r) axis and regulatory (ace- / ang ( - )/mas) axis of ras is disturbed in ad, as previously shown in various mouse models of cardiovascular disease [ ] and diabetic nephropathy [ ] . ace- activity is reduced in ad and is inversely correlated with increasing ace- activity, and the ace- /ace- ratio is increased in ad in association with disease pathology. these findings support commonly observed traits in cardiac and renal pathologies showing that dysregulation of the ace- /ang ( - )/mas pathway, including reduced ace- activity, is associated with sustained hypertension mediated by overactivation of the classical axis (reviewed in [ , ] ). despite the ratio of ang ii to ang ( - ) (a proxy measure of ace- activity) being increased in ad (i.e., reduced conversion of ang ii to ang ( - )), we did not observe an overall reduction in total ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) in ad. this is inconsistent with a recent report showing reduced serum ang ( - ) levels, rather than reduced ace- activity, in senescence-accelerated mouse prone , a mouse model of sporadic ad (involving overexpression of app). the authors observed that ang ( - ) levels correlated inversely with ang ii and p-tau levels [ ] . the reason for the discrepant findings between human and mouse brain tissue is unclear; however, both studies indicate that the ace- /ang ( - )/mas pathway is dysregulated in ad and that further work is required to determine the exact contribution of each component of the pathway in ad. activation of the ace- /ang ( - )/mas pathway, by inducing ace- activity, or infusion of ang ( - ) or a mas receptor agonist, is protective in various experimental animal models of cardiovascular disease and is associated with a reduction of the classical ras pathway (reviewed in [ , ] ). neuronal overexpression of brain ace- is also neuroprotective in a chronic hypertension mouse model (transgenic for renin and angiotensinogen that overproduces ang ii) following experimental induction of ischaemic stroke [ , , ] . these protective effects were partially reversed in the presence of a mas receptor antagonist, demonstrating the specificity of the ace- /ang ( - )/mas pathway, and they have been shown to be mediated by counter-regulating the effects of ang ii-mediated reactive oxygen species production [ ] . in ad, there is growing recognition that re-positioning of brain-penetrating arbs and aceis may have clinical benefits in ad [ ] . in addition to reducing the central pool of ang ii, arbs and aceis might also exert their protective effects by preventing at r-mediated reduction in ace- activity [ ] that can be reversed by arbs [ , [ ] [ ] [ ] [ ] . ace- activation is also associated with reduced ace- activity [ ] and with down-regulation of ang ii levels and at r expression [ , , [ ] [ ] [ ] . these studies suggest that activation of ace- may exert protective effects in ad above and beyond dampening ras activation that the use of aceis and arbs currently allow. lastly, we explored the distribution of ace- within the mid-frontal and temporal cortices and found it to be localised predominantly within endothelial cells and smooth muscle cells of cerebral arteries, as previously reported [ ] . interestingly, as for ace- , we also observed extensive perivascular ace- expression and found that ace- activity was increased in individuals with moderate to severe caa, as has previously been shown for ace- [ ] . we speculate that the sequential cleavage of aβ , first by ace- , and the subsequent cleavage of aβ to aβ (the predominant species in caa [ ] ) by ace- , provides a potential mechanistic link with caa. further studies are required to determine the relationship between ace- and caa severity. these data indicate that reduced activity of the ace- / ang ( - )/mas axis is strongly linked to overactivity of the classical ras pathway and with ad-related pathology. additional file : table s . mrc identifiers for all cases. (doc kb) additional file : figure s . scatterplot showing a strong positive correlation between two independent measures of ace- activity in brain tissue samples. ace- was measured using either a commercially available ace- activity assay kit (sensolyte® ) or an ace- fluorogenic peptide substrate (mca-apk[dnp]) in the presence of a selective ace- inhibitor, mln ( μm). the solid inner line indicates the best-fit linear regression, and the outer lines the % confidence intervals. each point represents a separate brain. ****p < . . (tif kb) additional file : figure s . scatterplot showing an inverse relationship between ace- activity and bace- activity in a combined alzheimer's disease and age-matched control cohort. ace- activity was measured using the sensolyte® ace- activity assay kit, and bace- activity was measured using the β-secretase specific fluorogenic substrate angiotensins in alzheimer's disease -friend or foe? central angiotensin ii stimulation promotes β amyloid production in sprague dawley rats central angiotensin ii-induced alzheimer-like tau phosphorylation in normal rat brains angiotensin-converting enzyme (ace) levels and activity in alzheimer's disease, and relationship of perivascular ace- to cerebral amyloid angiopathy angiotensinconverting enzyme levels and activity in alzheimer's disease: differences in brain and csf ace and association with ace genotypes protective effects of intranasal losartan in the app/ps transgenic mouse model of alzheimer disease perindopril, a centrally active angiotensin-converting enzyme inhibitor, prevents cognitive impairment in mouse models of alzheimer's disease angiotensin ii type receptor blocker losartan prevents and rescues cerebrovascular, neuropathological and cognitive deficits in an alzheimer's disease model cognitive deficit in amyloid-β-injected mice was improved by pretreatment with a low dose of telmisartan partly because of peroxisome proliferator-activated receptor-γ activation valsartan lowers brain β-amyloid protein levels and improves spatial learning in a mouse model of alzheimer disease effect of a centrally active angiotensin-converting enzyme inhibitor, perindopril, on cognitive performance in a mouse model of alzheimer's disease associations of antihypertensive treatments with alzheimer's disease, vascular dementia, and other dementias prevention of dementia in randomised double-blind placebo-controlled systolic hypertension in europe (syst-eur) trial use of angiotensin receptor blockers and risk of dementia in a predominantly male population: prospective cohort analysis effects of blood pressure lowering with perindopril and indapamide therapy on dementia and cognitive decline in patients with cerebrovascular disease effects of telmisartan on cognition and regional cerebral blood flow in hypertensive patients with alzheimer's disease variation in dcp , encoding ace, is associated with susceptibility to alzheimer disease systematic metaanalyses of alzheimer disease genetic association studies: the alzgene database alzheimer disease risk and genetic variation in ace: a meta-analysis haplotypes extending across ace are associated with alzheimer's disease large meta-analysis establishes the ace insertion-deletion polymorphism as a marker of alzheimer's disease candidate singlenucleotide polymorphisms from a genomewide association study of alzheimer disease a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - a human homolog of 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ischaemic stroke suppressing inflammation by inhibiting the nf-κb pathway contributes to the neuroprotective effect of angiotensin-( - ) in rats with permanent cerebral ischaemia angiotensin-converting enzyme (ace) and ace levels in the cerebrospinal fluid of patients with multiple sclerosis conversion of aβ to aβ by the successive action of angiotensin-converting enzyme and angiotensin-converting enzyme aβ is the earliest-depositing aβ species in app transgenic mouse brain and is converted to aβ by two active domains of ace angiotensin-( - ) is reduced and inversely correlates with tau hyperphosphorylation in animal models of alzheimer's disease national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of alzheimer's disease: a practical approach ace variants and association with brain aβ levels in alzheimer's disease higher soluble amyloid β concentration in frontal cortex of young adults than in normal elderly or alzheimer's disease the apolipoprotein e ε allele is associated with increased neuritic plaques and cerebral amyloid angiopathy in alzheimer's disease and lewy body variant apoe ε influences the pathological phenotype of alzheimer's disease by favouring cerebrovascular over parenchymal accumulation of a β protein cholinesterase inhibitors reduce cortical aβ in dementia with lewy bodies cholinesterase inhibitors may increase phosphorylated tau in alzheimer's disease apoe and cerebral amyloid angiopathy in the elderly apolipoprotein e genotyping by one-stage pcr apolipoprotein e ε and cerebral hemorrhage associated with amyloid angiopathy brain renin-angiotensin-a new look at an old system the brain renin-angiotensin system: a diversity of functions and implications for cns diseases brain renin angiotensin in disease angiotensin-( - ): a novel peptide to treat hypertension and nephropathy in diabetes? brain angiotensin-converting enzymes: role of angiotensin-converting enzyme in processing angiotensin ii in mice hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase advances in biochemical and functional roles of angiotensin-converting enzyme and angiotensin-( - ) in regulation of cardiovascular function angiotensin-( - ): an update angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas angiotensin-( - ) enhances ltp in the hippocampus through the g-protein-coupled receptor mas angiotensin-( - )/mas axis integrity is required for the expression of object recognition memory angiotensin-converting enzyme : a new target for neurogenic hypertension neuronal overexpression of ace protects brain from ischemia-induced damage activation of the ace / ang-( - )/mas pathway reduces oxygen-glucose deprivation-induced tissue swelling, ros production, and cell death in mouse brain with angiotensin ii overproduction current status of renin-aldosterone angiotensin system-targeting anti-hypertensive drugs as therapeutic options for alzheimer's disease angiotensin ii type receptor-mediated reduction of angiotensin-converting enzyme activity in the brain impairs baroreflex function in hypertensive mice ace and ace : their role to balance the expression of angiotensin ii and angiotensin the ang-( - )/ace /mas axis in the regulation of nephron function increased expression of angiotensin converting enzyme in conjunction with reduction of neointima by angiotensin ii type receptor blockade role of angiotensin-converting enzyme /angiotensin-( - )/mas axis in the hypotensive effect of azilsartan ace overexpression in the paraventricular nucleus attenuates angiotensin ii-induced hypertension exercise training normalizes ace and ace in the brain of rabbits with pacing-induced heart failure brain-selective overexpression of angiotensin-converting enzyme attenuates sympathetic nerve activity and enhances baroreflex function in chronic heart failure angiotensin-converting enzyme overexpression in the subfornical organ prevents the angiotensin ii-mediated pressor and drinking responses and is associated with angiotensin ii type receptor downregulation amyloid β protein (aβ) in alzheimer's disease brain: biochemical and immunocytochemical analysis with antibodies specific for forms ending at aβ or aβ ( ) we acknowledge professor seth love, university of bristol, for his academic input and neuropathological assessment. all data within the article is linked to the mrc uk-bbn by a unique numeric mrc uk-bbn identifier (additional file : figure s ). there is no risk of disclosure of personal information, because all of the information held within the database has been anonymised.authors' contributions jsm carried out the angiotensin-ii measurements and validated the ace- activity measurements, performed the statistical analysis and was primarily responsible for drafting and finalizing the manuscript. sw carried out the ace- activity measurements, performed statistical analysis and helped to draft the manuscript. nam carried out the angiotensin ( - ) measurements, performed statistical analysis and helped draft the manuscript. lep carried out the ace- immunolabelling and analysis and revised the manuscript. pgk conceived and was responsible for overall planning and design of the study, and helped to revise and finalize the manuscript. all authors read and approved the final manuscript. all authors are members of the dementia research group, clinical neurosciences, school of clinical sciences, university of bristol, bristol, uk. the authors declare that they have no competing interests. the use of human brain tissue for this study was approved by the management committee of the south west dementia brain bank (human tissue authority licence number ) under the terms of bristol research ethics committee approval of the brain bank (reference /h / + ). all participants provided consent to post-mortem removal of whole brain and csf and the retention of these for use in research. consent included access to the donor's medical records to collect information on past medical history relevant to the donation, but that in all publications this information would be anonymised. key: cord- -hynkb a authors: acharya, k. ravi; sturrock, edward d.; riordan, james f.; ehlers, mario r. w. title: ace revisited: a new target for structure-based drug design date: journal: nat rev drug discov doi: . /nrd sha: doc_id: cord_uid: hynkb a current-generation angiotensin-converting enzyme (ace) inhibitors are widely used for cardiovascular diseases, including high blood pressure, heart failure, heart attack and kidney failure, and have combined annual sales in excess of us $ billion. however, the use of these ace inhibitors, which were developed in the late s and early s, is hampered by common side effects. moreover, we now know that ace actually consists of two parts (called the n- and c-domains) that have different functions. therefore, the design of specific domain-selective ace inhibitors is expected to produce next-generation drugs that might be safer and more effective. here we discuss the structural features of current inhibitors and outline how next-generation ace inhibitors could be designed by using the three-dimensional molecular structure of human testis ace. the ace structure provides a unique opportunity for rational drug design, based on a combination of in silico modelling using existing inhibitors as scaffolds and iterative lead optimization to drive the synthetic chemistry. the neural activity of the sympathetic nervous system, regulating (through adrenergic receptors) cardiac and vascular function. the resistance to blood flow, which is directly proportional to the extent of vasoconstriction, is one of the primary determinants of blood pressure. angiotensin i (ang i) and an octapeptide called angiotensin ii (ang ii) . two years later a chloride-dependent, metalloenzyme that could convert ang i to ang ii was purified from horse plasma , and was later referred to as ace. ace, also known as peptidyl-dipeptidase a (ec . . . ), belongs to the m family of the ma clan (a protein clan contains all the modern-day polypeptides that have arisen from a single evolutionary progenitor) of zinc metallopeptidases . it is a dipeptidyl carboxypeptidase that catalyses the hydrolytic cleavage of dipeptides from the carboxyl terminus of a wide variety of oligopeptides in vitro. its best known function is the in vivo conversion of ang i (drvyihpfhl), which circulates in plasma, into the potent vasopressor ang ii by removal of the c-terminal his-leu (fig. ) . ang i is generated primarily by the renin-catalysed hydrolysis of the leu -val peptide bond of angiotensinogen, a liver-derived -kda plasma protein. ace also affects blood pressure by cleaving bradykinin (bk, rppgfspfr), thereby abolishing its vasodilating activity (because of this, ace is sometimes referred to as kininase ii (kininase i is carboxypeptidase n)). bk is generated from a kininogen precursor by the action of plasma kallikrein (a serine proteinase) in a process analogous to that of ang i formation (fig. ) . human ace is a monomeric zinc metalloenzyme that is synthesized as a , -amino-acid polypeptide, is processed to a , -residue mature form, is heavily glycosylated ( % by weight), and is localized to the plasma membrane of endothelial and absorptive epithelial and neuroepithelial cells . a sequence of hydrophobic amino acids located near the carboxyl terminus of the protein serves as a transmembrane domain that anchors ace to the cell surface. this creates a -residue cytosolic domain and a , -residue glycosylated extracellular domain. its cellular orientation defines ace as a type i transmembrane ectoprotein and, in the case of endothelial cells, positions it optimally for interaction with its circulating substrates. the amino acid sequence of human endothelial ace provides clear evidence for a gene duplication event in its evolutionary history. it consists of an n-terminal domain of about amino acids, a -residue interdomain sequence and a -residue c-terminal domain. a -aminoacid segment of the n-domain has more than % sequence identity to the corresponding segment of the c-domain (fig. ) . each domain contains a five-residue sequence of amino acids, hemgh, which is characteristic of catalytic zinc sites found in a large family of neutral endoproteinases. detailed kinetic and mutational analyses have established that both of the zinc sites have catalytic activity , . the physiological consequence of such tandem active sites in an enzyme is unknown. some differences in catalytic properties have been observed for these two sites: the n-domain site is notably -times more active toward the haemoregulatory peptide n-acetyl-ser-asp-lys-pro (acsdkp) , -times here, we provide an overview of ace and the ras, current ace inhibitors and their clinical utility, insights from the tace crystal structure, and the rationale and prospects for developing second-generation, domainselective inhibitors by structure-guided design. the recognition that an elevated basal blood pressure can lead to a shortened life expectancy and higher morbidity (due to cardiovascular complications such as kidney failure, heart failure and stroke) only evolved during the first half of the twentieth century, when numerous clinical surveys demonstrated a normal distribution of blood pressures and a - % prevalence of hypertension in an apparently healthy population . hypertension occurs in millions of people worldwide, the large majority of whom are unaware of their condition; a recent report indicated that prevalence rates are even higher than previously suspected, at % in north america and % in europe . many long-term studies were required before the increased morbidity and mortality associated with hypertension became accepted and it was classified as a disease in need of treatment . the underlying cause of hypertension in most cases was, and still is, unknown. therapy was initially directed at reducing blood volume by the use of diuretics, sympathetic tone by the use of adrenergic-blocking agents, or vascular resistance by the use of vasodilators. a major breakthrough in our understanding of blood pressure regulation came with the discovery of the ras and ace (reviewed in ref. ). on the basis of a suspected relationship between kidney and blood pressure, goldblatt, houssay, page and others identified the renal enzyme renin and the pressor substance angiotensin in the s and s [ ] [ ] [ ] . in the mid- s, skeggs and co-workers purified angiotensin and found that it existed in two forms: a decapeptide called is much less so , . however, given the relative constancy of plasma chloride concentration, the significance of this chloride dependency is not obvious. human endothelial ace, also known as somatic ace, is encoded by a single gene that consists of exons, more sensitive to inhibition by the phosphinic peptide rxp (ref. ) , and more than -times less sensitive to inhibition by rxpa (ref. ace is released from the endothelial cell surface by the action of a so-called ace-secretase, or sheddase, that cleaves the arg -ser peptide bond near the transmembrane region , . soluble ace is a minor component of total ace activity, and its physiological function is unknown. activators of protein kinase c stimulate the release of ace from endothelial and other cells in culture, and markedly elevated concentrations of plasma ace are observed in certain diseases, particularly sarcoidosis. besides its role in blood pressure regulation, ace also has been postulated to participate in 'local' or 'tissue' rass. the ang ii arising from such systems is thought to act as a paracrine growth factor . this activity has been implicated in the development of left ventricular dysfunction that can occur after a major heart attack, and seems to account for the beneficial effects of ace inhibitor therapy in such situations. the structural and functional conservation of the gene encoding ace are indications of its widespread evolutionary importance as a key metallopeptidase involved in the metabolism of regulatory peptides . the drosophila melanogaster genome contains six genes (acer, ance, ance- , ance- , ance- and ance- ) that encode ace-like proteins. two of these, angiotensinconverting enzyme (ance) and angiotensin-converting enzyme-related (acer), are enzymatically active and share % sequence identity with human ace . ance displays properties very similar to those of the human c-domain, whereas acer is inhibited by the n-domainselective inhibitor rxp (ref. ). ace (also known as aceh) is an ace homologue found in humans and rodents that functions as a carboxypeptidase with a preference for c-terminal hydrophobic or basic residues (reviewed in refs , ) . it is expressed mainly in the heart, kidney and testis and is important for the regulation of blood pressure and cardiac function. interestingly, ace is not inhibited by ace inhibitors such as lisinopril, captopril and enalaprilat. one of the main impediments to determining the structure of ace by x-ray crystallography was the production of diffraction-quality crystals. the c-domain of ace has seven potential n-linked glycosylation sites, six of which are located on the surface of the protein and the seventh in the juxtamembrane region. two concurrent approaches succeeded in paving the way for the crystallization and successful x-ray structure determination of tace . five of the n-linked sites were disrupted by substituting glutamines for each of the asparagine residues in the glycosylation sequences and a truncated form was expressed in the presence of a glucosidase inhibitor yielding crystals suitable for x-ray diffraction. the latter protein was used for the subsequent threedimensional structure determination of tace. highresolution crystal structures of the human tace and its complex with the widely used inhibitor lisinopril at . Å resolution were recently reported . all but one of which, exon , is transcribed into the corresponding messenger rna . a form of ace found only in adult testis (tace) is encoded by the same gene but its mrna begins before exon and continues through exon . it is transcribed by the interaction of a promoter with a site present in intron that is active only in adult male germinal cells . translation of this mrna results in a -amino-acid version of ace that, except for the first residues, is identical to the c-terminal domain of somatic ace . testis or germinal ace is found in developing sperm cells and mature sperm. sperm lacking ace are deficient in transport within the oviduct and in binding to the zonae pellucidae, and male ace -/mice have markedly diminished fertility . a polymorphism involving a -base-pair insertion corresponding to an alu repetitive sequence has been found in intron of the human ace gene . the insertion is all or none: in one study the (i)/(d) frequency ratio was : . the deletion (d) allele of the ace gene lacks the sequence that is present in the insertion (i) allele. the d allele is associated with a higher plasma and tissue ace activity. the dd genotype is positively correlated with plasma ace activity and numerous attempts have been made to search for associations between the dd genotype and specific cardiovascular diseases, but the conclusions remain controversial . the association of allele i with athletic performance is equally controversial. some studies have shown that this polymorphism is associated with endurance performance, whereas others have shown the d allele to be associated with that of elite shortdistance athletes , . notable differences in hydrophobicity and charge are observed in the lid-like structure comprising helices α , α and α (using tace nomenclature; fig. ). this part of the structure seems to affect the substrate specificity of the n-or c-domain, as tace mutants which had this region replaced by the corresponding n-domain sequence showed a preference for n-domain substrates (z. l. woodman et al., unpublished data). second, the zn + -binding site with the canonical hexxh motif is conserved. third, it has been shown that the c-domain has greater chloride dependence than the n-domain, both in terms of substrate hydrolysis and inhibitor binding , . from the model we can predict that arg (a key residue for binding one of the two chloride ions in tace) is replaced by his in the n-domain. so, in the n-domain only one chloride-ion-binding pocket is plausible, involving arg and tyr . fourth, positioning of the lisinopril molecule in the active site of the n-domain model revealed that the full complement of structurally conserved residues was found as observed in the tace structure, confirming that the n-domain of somatic ace could also bind lisinopril with similar affinity, as previously reported , . fifth, the s sub-site is formed by asn and thr , which replace a serine and a valine, respectively, in the c-domain. furthermore, the asparagine occurs in an n-glycosylation sequon (nvt) that is unique to the n-domain; the attachment of any glycan to this residue would occlude the s pocket. this, in part, might explain how the bulky aromatic ring of the benzamido group in keto-ace (an ace inhibitor; see fig. a) is accommodated by the s site in the c-domain (tace), making keto-ace more c-domain selective. sixth, the ace inhibitor rxp has an n-acetyl group that confers a certain degree of n-domain specificity . the model of rxp and the n-domain reveals two residues, arg and tyr , that form van der waals interactions with the n-acetyl carbonyl/methyl groups (fig. d) . however, a glutamate and phenylalanine are substituted for these two residues in the c-domain and might exert repulsive forces in the binding of the inhibitor to the c-domain active site. homology modelling of ace using the atomic coordinates of the tace structure revealed differences in the ligand-binding pockets of the two homologues that account for their substrate and inhibitor selectivity . first, the accommodation of the s ′ sub-site in tace increases the substrate-binding cavity and permits the binding of an additional amino acid to the obligatory binding site; second, gln , which interacts with the carboxyl terminus of lisinopril in tace, is replaced by an arginine in ace . this represents an elongation of the side chain of residue , which causes steric conflict with the p ′ residue of lisinopril when it is docked in the active site of the ace model. in addition, the substitution of the leucine and phenylalanine for the hydrogen-bonding lys and tyr , respectively, and replacement of thr in tace by the more bulky phe residue, probably account for the changes in substrate specificity. there are no striking differences in the large s ′ sub-sites of tace and ace except for a proline in ace which corresponds to ala in tace. drosophila ace (ance) contains a single domain similar to tace. however, it has only three potential n-linked glycosylation sites, which are not required for secretion and enzymatic activity . recently, the crystal structures of this homologue (which has considerable similarity to the tace structure), bound to captopril and lisinopril, were reported . the wild-type protein was expressed in a baculovirus expression system and the oligosaccharides did not hamper crystallization. however, it is unlikely that this will be the case with the ace homologue, the ace n-domain or the full-length somatic ace structures -ace structural milestones that are still eagerly awaited. prominent features of the tace structure include an abundance of α-helices and a deep central cavity that divides the molecule into two halves, which pack together into an overall ellipsoid shape (fig. ) . the active site, identified by the catalytic zn + ion bound to the hexxh sequence (and to lisinopril in its complex with the enzyme), is located in the deep cavity, some Å from its entrance. the amino-terminal helices (α - ) form a lid-like extension that partially covers the activesite channel, which limits the access of substrates and inhibitors. in fact, the aperture of the channel opening is approximately Å in diameter, which is too small for most peptide substrates, indicating that tace must undergo some conformational change, possibly associated with its unique chloride ion activation, for the substrate to enter the channel. from the structure of the lisinopril-tace complex (fig. ) it is evident that the phenyl ring of the inhibitor interacts with the s sub-site in the active site, the lysine with the s ′ sub-site, and the proline occupies the s ′ sub-site. the carboxyl group, located between the phenylpropyl group and the lysine, binds to the zn + in the active site and also forms a hydrogen bond with the side-chain carboxylate of glu . other key interactions occur between the sidechain amino group of the inhibitor lysine and glu of tace, and between the c-terminal proline carboxyl group with lys and tyr . the binding of the inhibitor to the s , s ′ and s ′ pockets and its zinc coordination form the basis for the structure-guided design of improved domain-selective ace inhibitors. in addition, two buried chloride ions that are important for the activation of the enzyme were identified in the crystal structure (outside the active site), both distant ( . Å and . Å, respectively) from the catalytic zn + ion. the first is bound to arg , arg and trp , whereas the second is bound to arg and tyr . the structure is indicative of an indirect mechanism for chloride activation, possibly through effects on active-site structure. this study also revealed that the structure of tace (ma sub-clan of m clan of peptidases (m clan peptidases are metalloenzymes and the metal is involved in catalysis)) resembles that of rat neurolysin and a newly identified carboxypeptidase from the hyperthermophilic archaeon pyrococcus furiosus -both of which are members of the ma clan -despite low sequence similarity. structure-based modelling of the n-domain of somatic ace (using the three-dimensional structure of tace , which has ~ % amino-acid sequence identity to somatic ace) reveals some interesting features. first, glycosylation sequon consensus sequence asn-x-ser/thr whose core glycosylation generally occurs at the asn residue. nevertheless, the use of cpa as a model led to key conceptual insights in inhibitor design, because cpa was much better understood and its crystal structure was known. cushman and ondetti reasoned that ace was an exopeptidase like cpa, with the difference that ace cleaved the penultimate peptide bond to release a dipeptide product. the second major breakthrough in inhibitor design derived from an earlier observation that an extract from the south american pit viper bothrops jararaca, known as bradykinin potentiating factor (bpf), could inhibit ace [ ] [ ] [ ] . bpf was a mixture of peptides , which were shown to be potent and specific inhibitors of ace (table ), and structure-activity studies indicated that the optimal c-terminal inhibitory sequence was phe-ala-pro . this work led to the proposal that the venom peptides were substrate analogues that bound competitively to the obligatory substrate-binding sites in the ace active site (fig. a) . what was needed were orally active, non-peptide analogues of bpf. the third key insight derived from work by byers and wolfenden describing a new design concept for inhibitors of cpa based on benzylsuccinic acid, referred to as by-product analogues. cushman and ondetti recognized that part of the binding affinity of benzylsuccinic acid derived from coordination of the active-site zinc by the carboxyl group, and predicted that a similar succinylamino acid derivative would inhibit ace if its structure was analogous to the dipeptide product of ace activity. on the basis of the phe-ala-pro sequence derived from the bpf peptides, they synthesized methylsuccinyl-pro (analogous to carboxy-ala-pro) and indeed found it to be a specific inhibitor, with an ic of µm . cushman and ondetti then searched for a superior zinc-binding group and the potency breakthrough was achieved by replacing the carboxyl with a sulphydryl group, yielding captopril with an ic of nm , (fig. a) . captopril became the first ace inhibitor in clinical use (first approved in ) and rapidly established itself as a powerful new therapeutic agent in the treatment of hypertension and heart failure. reports of captoprilrelated side effects, such as loss of taste and skin rash, prompted patchett and colleagues to focus on the design of non-sulphydryl ace inhibitors, by reverting to carboxyl compounds and introducing additional functionalities that would complete the by-product design. captopril did not make use of at least two potential binding sites: the s binding site and a hydrogen-bonding site for the amide nitrogen of the (substrate) scissile bond (fig. a) . so, structure-activity studies were performed on a series of n-carboxyalkyl dipeptides of the general formula r-chco h-a -a . the r group, occupying the s pocket, was best served by benzylmethylene, whereas a -a was either ala-pro (as in the bpf peptides) or, surprisingly, lys-pro, leading to enalaprilat and lisinopril, respectively (fig. a) , which show nanomolar inhibition constants. enalaprilat and lisinopril are essentially tripeptide analogues with a zn + -coordinating carboxyl group substituting for the substrate scissile amide carbonyl. enalaprilat closely resembles the phe-ala-pro sequence the story of the design and synthesis of the first orally active, potent inhibitors of ace is one of the great success stories of modern medicinal chemistry. it has been described as one of the first examples of true 'rational drug design' , and although this might not be true in the sense that we understand that term today, the design of the ace inhibitors in the late s and early s was certainly based on a series of brilliant insights that, together with a sprinkling of serendipity, constituted what might now be called 'rational intuition' . it is not our intention to provide a comprehensive review of the events leading to the design and synthesis of captopril, enalaprilat and lisinopril, the original group of potent ace inhibitors that formed the basis for all subsequent compounds of what can now be termed first-generation ace inhibitors. several excellent reviews of this history have been published during the past two decades, especially by the inventors themselves [ ] [ ] [ ] [ ] [ ] [ ] . however, it is instructive to consider some of the key insights that led to these drugs, especially in the context of the crystal structure that is now available and the structure-guided drug design of second-generation ace inhibitors that can now be undertaken. a role for a metal in the catalytic mechanism had been suspected since the discovery of the enzyme and was confirmed in the s , , leading to the proposal that ace was mechanistically similar to carboxypeptidase a (cpa) , . we now know that ace is unrelated to the cpa class of enzymes, but instead falls into the group of metallo-endopeptidases characterized by the hexxh zinc-binding motif (cpa possesses hxxe) . has been attributed to a direct vascular protective and anti-atherogenic effect of ace inhibitors, because it has been observed even in normotensive individuals . a major debate concerns the mechanism by which cardiovascular benefits are conferred by ace inhibitors. it is generally accepted that ang ii not only has direct pressor effects and stimulates salt and water retention (via aldosterone release), but also stimulates myocyte proliferation and exerts pro-atherogenic effects via the induction of oxidative stress, endothelial dysfunction and vascular inflammation , . however, there is also considerable evidence that some of the benefits of ace inhibition derive from potentiation of bk signalling, which stimulates release of the vasodilator nitric oxide and of the fibrinolytic protein tissue plasminogen activator , . indeed, co-administration of the specific bk-receptor antagonist icatibant significantly attenuated the hypotensive effect of captopril in both normotensive and hypertensive subjects . further complicating this debate is the recent appreciation that the ras is more complex than originally thought. there are multiple ang peptides and at least three or four ang receptors, some of which have opposing activities (fig. ) . for instance, the principal ang ii receptor, the at receptor, mediates the well-known effects of ang ii described above, but the at receptor, which has a more limited tissue distribution, mediates largely opposing effects. similarly, ang - , which is derived from both ang i and ang ii by the action of various peptidases including ace and ace , opposes the actions of ang ii , . moreover, as already discussed, ace also acts on non-ang peptides, including bk, substance p, luteinizing hormone-releasing hormone (lh-rh) and the haemoregulator acsdkp; with the exception of bk, the importance of these peptides for the cardiovascular effects of ace inhibitors is unknown. some insights might derive from comparisons of the efficacy and side-effect profiles of ace inhibitors and at -receptor blockers (arbs). ace inhibitors are generally well tolerated, but certain class-specific side effects have emerged, in particular cough and angioedema . the incidence of cough has been estimated at - % of patients and might result in the discontinuation of treatment , . angioedema affects . - . % of patients and can be life-threatening . both cough and angioedema have been attributed to alterations in levels of non-ang peptides, especially raised bk concentration. recently, an association has been found between aceinhibitor-related angioedema and low plasma levels of aminopeptidase p, an enzyme that is also involved in the metabolism of bk, indicating that these individuals are at risk for developing angioedema when treated with ace inhibitors . the potentiation of bk signalling by ace inhibitors (fig. ) seems to result not only from reduced bk degradation but also from inhibition of desensitization of the b bk receptor, possibly by inducing crosstalk between ace and the b receptor [ ] [ ] [ ] . therefore, both the benefits and side effects arising from increased bk signalling by ace inhibitors seem to be mechanistically complex and have implications for the design of n-or c-selective inhibitors (see below). that was found to be the optimal c-terminal sequence among the venom peptides. the structure of the ace-lisinopril complex confirms that lisinopril makes extensive contacts with the active site, including occupation of the s , s ′ and s ′pockets, binding of a lysine by the c-terminal carboxylate, a hydrogen bond involving the (substrate) scissile amide nitrogen, and coordination of the active-site zn + by the carboxyalkyl carboxylate (fig. ) . lisinopril binds with twofold greater affinity than enalaprilat , probably because the lysine side chain makes better contacts with the deep s ′ pocket, including a weak hydrogen bond between the lysyl ε-amine and glu (ref. ). the design of captopril, enalaprilat and lisinopril was later extended by others, and a total of ace inhibitors have been approved for clinical use . the later compounds are all variations on the original theme, with most of the differences residing in the functionalities that bind the active-site zinc and the s ′ pocket . the different types of zinc-coordinating groups are of interest, because these have also found application in the design of inhibitors for other metalloproteases, such as the matrix metallopeptidases. on the basis of work first described by holmquist and vallee , phosphonates have proven useful , , as have hydroxamates , ketones and silanediols . since their introduction in , ace inhibitors have been studied extensively (for recent reviews, see refs , , ) . ace inhibitors are first-line therapy for hypertension, congestive heart failure, left ventricular systolic dysfunction and myocardial infarction, and are recommended for slowing the progression of diabetic and non-diabetic nephropathy , . more recently, ace inhibitors have also been shown to slow the progression of atherosclerotic vascular disease. this vascular benefit conditions , , , . this difference might be due to the additional effect of ace inhibitors on bk, although this comes at the cost of increased side effects , . interestingly, and contrary to earlier assumptions, angioedema has also been reported with arb therapy , , which might be related to unopposed activation of the at receptor leading to increased bk concentrations . in light of the enormous importance of the ras in cardiovascular pathophysiology, there is continued interest in novel compounds that target this system . eplerenone, the first selective aldosterone antagonist, the arbs were introduced more recently but have also been studied extensively , . as is the case for ace inhibitors, arbs have been shown to be effective in the treatment of hypertension and heart failure, in reducing cardiovascular morbidity and mortality, and in slowing the progression of nephropathy. studies in myocardial infarction and heart failure have indicated a trend towards lower mortality in patients treated with ace inhibitors versus arbs, leading to the recommendation that ace inhibitors remain the first-line agents in these c-domain site will allow some level of bk degradation to continue, catalysed by the n-domain. this could be sufficient to prevent the excessive bk accumulation that has been observed during attacks of angioedema . second, bk potentiation by b receptor resensitization is maximal when both the n-and c-domains are inhibited , indicating that a pure c-selective inhibitor will have a lower propensity for excessive bk stimulation. third, the multiple ang and non-ang peptides known to be vasoactive are not hydrolysed equally by the two domains , , making it likely that the ratio of vasopressor to vasodilator peptides will differ between c-selective and mixed inhibitors. so, a highly selective c-domain inhibitor has the potential for effective blood pressure control with reduced vasodilator-related side effects. in contrast to a c-selective inhibitor, an n-selective inhibitor might open up novel therapeutic areas. as discussed, the n-domain seems to play a minor role in blood pressure control in vivo. at least three physiologically important peptides are hydrolysed preferentially or exclusively by the n-domain: lh-rh, ang - and acsdkp , , . the contribution of ace to the metabolism of lh-rh and ang - in vivo is unclear, but there is increasing evidence that ace is the principal metabolizing enzyme for acsdkp, a natural haemoregulatory hormone . acsdkp has antiproliferative and antifibrotic activities, and might have utility in protecting haematopoietic stem cells against chemotherapyinduced injury and in limiting cardiac fibrosis . administration of ace inhibitors results in a four to sixfold elevation of acsdkp plasma levels , . this might be the basis for the observed association between ace inhibitors and anaemia, and the effective treatment of altitude polycythaemia by the ace inhibitor enalaprilat . current-generation ace inhibitors in clinical use are essentially mixed n-and c-domain inhibitors . although a modest degree of domain selectivity can be observed in some cases, this is not likely to be clinically significant. nevertheless, these differences might be instructive and can guide future attempts to develop highly domain-selective inhibitors. captopril has been noted to be modestly n-selective, depending on clconcentration, whereas lisinopril and enalaprilat are more c-selective , . more recently, keto-ace, originally described in (ref. ), was found to exhibit a - -fold c-selectivity ; one of the bpp peptides, bppb, was shown to be -fold more c-selective ; and the phosphinic tetrapeptide rxpa is , -fold more c-selective . by contrast, bpp- b is -fold, and the phosphinic tetrapeptide rxp , -fold, more n-selective , (table ) . examination of these compounds reveals a number of features (table and fig. a) . captopril is the smallest inhibitor and can be viewed as an n-thioalkyl derivative of the dipeptide ala-pro, whereas enalaprilat, lisinopril and keto-ace are tripeptide analogues of phe-ala-pro, phe-lys-pro and phe-gly-pro, respectively. this might indicate that a bulky p group -present in enalaprilat, lisinopril and keto-ace, but absent in was approved for the treatment of hypertension in september and will probably also find use in the treatment of severe heart failure . an orally active renin inhibitor, aliskiren, is now in clinical development for hypertension . of particular interest are the vasopeptidase inhibitors, which are dual ace-neutral endopeptidase (nep) inhibitors, of which omapatrilat is the most advanced in clinical development. nep, also a zinc-metallopeptidase, is the principal enzyme responsible for the degradation of natriuretic peptides, which are vasodilatory and diuretic peptides that reduce volume loading and are therefore beneficial in both hypertension and heart failure . as expected, omapatrilat was equivalent or superior to ace inhibitors in clinical trials, but was found to be associated with a significantly higher incidence of angioedema, which has delayed its approval . the higher incidence of angioedema is probably related to the fact that nep also inactivates bk (fig. ) , and its inhibition might result in even higher plasma bk levels, which together with raised concentrations of natriuretic peptides (and potentially other vasodilatory peptides) might promote vasodilation-related adverse events , . nep might also be required for the removal of the neurotoxic amyloid β-peptide, and so chronic nep inhibition could lead to neurodegeneration . the clinical results with omapatrilat have had a sobering effect on the field and have indicated that broad inhibition of vasoregulatory peptides should be approached with caution . indeed, overly vigorous inhibition of neuro-hormonal activation during heart failure, by simultaneous treatment with an ace inhibitor, an arb and a β-blocker was associated with excess mortality in the valsartan heart failure trial (val-heft) . so, efforts to develop triple inhibitors of ace, nep and endothelin-converting enzyme- (ece- ) might need to be re-evaluated. ece- , which is structurally and functionally related to nep, is the principal activating enzyme for the potent vasopressor peptides endothelin- and - (ref. ), and so triple inhibitors might show even greater efficacy than single ace or dual ace/nep inhibitors. however, there is clearly an even greater potential for adverse events such as angioedema. in light of these developments, highly specific, singledomain inhibitors of ace offer an attractive alternative. as we have attempted to show in this review, our understanding of the ras (and related vasoregulatory systems) has come a long way since the introduction of the first ace inhibitors. the n-and c-domain sites of ace hydrolyse ang i and bk at comparable rates in vitro, but in vivo it seems that the c-domain is primarily responsible for regulating blood pressure , . this might indicate that a c-selective inhibitor would have a profile comparable to current mixed inhibitors, but this is not necessarily the case. first, whereas ang i is hydrolysed predominantly by the c-domain in vivo , bk is hydrolysed by both domains and therefore selective inhibition of the be predicted that this sub-site is significantly different in the n-domain active site. modelling of the s sub-site in the n-domain has also revealed differences, as expected (see earlier), confirming its potential utility for conferring domain selectivity (fig. c,d) . these considerations can form the starting point for the structureguided design of domain-selective inhibitors, which will be refined further once the n-domain structure becomes available. an important caveat in considering the design and pharmacological utility of domain-selective ace inhibitors is the potential for conformational effects that have not yet been observed in the tace crystal structure. for instance, it is unknown whether chloride binding and dissociation result in significant movement of the n-terminal 'lid' formed by helices α , α and α , and thereby restrict substrate access . even more importantly, the physical orientation of the n-and c-domains in somatic ace is unknown, as is whether there is any significant degree of domain interaction or cooperativity. inhibitor titrations in vitro and studies with domainselective inhibitors in vivo have provided indirect evidence for some form of domain interaction, which could have significant effects on the pharmacological profile of domain-selective inhibitors. the past decade has seen major advances in structurebased drug design approaches, including technologies such as mass spectrometry, x-ray crystallography and nuclear magnetic resonance. these are important tools in structural proteomics and to some extent have eliminated the scepticism about the feasibility and value of the structure-based approach. in particular, high-throughput structure-based drug design using protein crystallography has become a very attractive proposition for the pharmaceutical industry. many examples exist today in which a combination of the three-dimensional structure of the target protein, computer-aided drug design (in silico or virtual screening), and a rational approach using highthroughput screening have produced important lead compounds that are now being evaluated in clinical trials (for recent reviews see refs , , , ) . in addition to mining the untapped riches of the human proteome, the application of modern structure-based drug design methods to existing drug targets will generate more selective compounds for known disease targets, such as ace. we expect that next-generation, domain-selective ace inhibitors will be a result of such endeavours. captopril -confers c-selectivity, and that a larger p ′ side chain also promotes c-selectivity (fig. b) , because lisinopril is more c-selective than enalaprilat. interestingly, trandolaprilat, although a potent inhibitor for both domains, was tenfold more c-selective (table ) . trandolaprilat contains a c-terminal hexahydroindoline group, which also indicates that a bulky p ′ group confers c-selectivity. this is confirmed by results from radioligand-binding studies, which indicated that both perindoprilat and quinaprilat, which contain hexahydroindoline and tetrahydroisoquinoline groups, respectively, in the p ′ position, were - -fold more c-selective . similarly, the highly c-selective tetrapeptide rxpa also contains a bulky methylindole group in the p ′ position. these studies also indicated that lisinopril and a (a hydroxybenzamidine analogue of lisinopril) were - -fold more c-selective, reinforcing the importance of a bulky p ′ group . on the other hand, structure-activity studies performed with a series of phosphinic tetrapeptides indicated that a phenylalanine in the p position did not confer c-selectivity. instead, the single most important determinant for n-selectivity was an amidated c-terminal carboxyl in the p ′ position, followed by an acidic group in the p position (fig. d) . the bradykinin potentiating peptides (bpps) confirm the conclusion regarding the p group: bppa, bppb, bppc and bpp all end with the sequence ile-pro-pro, yet only bppc is unselective. bppc has a proline in the p position versus a lysine in the most c-selective peptide, bppb . similarly, the c-selectivity of keto-ace can probably be ascribed to a bulky p benzyl group (fig. c) ; the selectivity of rxpa is also consistent with the proposed importance of a phenyl p group (fig. a) . taken together, these data indicate that c-selectivity is conferred by bulky p ′ and p ′ groups and a large, neutral or basic p group, whereas n-selectivity is conferred by a blocked c-terminal carboxyl and an acidic p group. the ace c-domain crystal structure revealed that the s ′ pocket was surprisingly deep, easily accommodating the lysyl group of lisinopril, with a hydrogen bond between glu and the ε-amine (figs and b) . additional modifications to the p ′ group will potentially further enhance c-selectivity. moreover, the c-terminal carboxylate of lisinopril was found to bind to lys , explaining the importance of a free c-terminal (p ′) carboxyl for binding to the c-domain active site . binding to lys , instead of to an arginine (as originally predicted), might prompt investigation of functionalities other than carboxylates in this position. since n-selectivity is conferred by a blocked p ′ carboxylate, it can also high-throughput crystallography and lead discovery in drug design the genesis of high-throughput structure-based drug discovery the angiotensin-converting enzyme gene family: genomics and pharmacology outstanding review of the ras and of the landmark studies that established the clinical utility of ace inhibitors crystal structure of the human angiotensinconverting enzyme-lisinopril complex first report on the three-dimensional structure of human testis ace and its interactions with the potent ace inhibitor lisinopril at the molecular level the development of cox inhibitors comparative pharmacology of h antihistamines: clinical relevance hypertension: pathophysiology, diagnosis, and management hypertension prevalence and blood pressure levels in european countries, canada, and the united states hypertension: pathophysiology, diagnosis, and management studies on experimental hypertension. i. the production of persistent elevation of systolic blood pressure by means of renal ischemia secretion hipertensora del rinon isquemaido a crystalline pressor substance (angiotonin) resulting from the interaction between renin and renin activator the purification of hypertensin i references - are a classic series of papers describing the isolation and characterization of ang i handbook of proteolytic enzymes first report of the molecular cloning of somatic ace and the surprising finding of a two-domain structure the two homologous domains of human angiotensin i-converting enzyme are both catalytically active differences in properties and enzymatic specificities between the two active sites of angiotensin -converting enzyme the hemoregulatory peptide n-acetyl-ser-asp-lys-pro is a natural and specific substrate of the n-terminal active site of human angiotensin-converting enzyme rxp , a phosphinic peptide, is a potent inhibitor of angiotensin i converting enzyme able to differentiate between its two active sites roles of the two active sites of somatic angiotensin-converting enzyme in the cleavage of angiotensin i and bradykinin the two homologous domains of human angiotensin i-converting enzyme interact differently with competitive inhibitors structure of the angiotensin i-converting enzyme gene. two alternate promoters correspond to evolutionary steps of a duplicated gene molecular cloning of human testicular angiotensinconverting enzyme: the testis isozyme is identical to the c-terminal half of endothelial angiotensin-converting enzyme angiotensin-converting enzyme and male fertility an insertion/deletion polymorphism in the angiotensin -converting enzyme gene accounting for half of the variance of serum enzyme levels angiotensin converting enzyme gene insertion/deletion polymorphism and cardiovascular disease: therapeutic implications human performance: a role for the ace genotype? endurance and the ace i/d polymorphism proteolytic release of membranebound angiotensin-converting enzyme: role of the juxtamembrane stalk sequence shedding of somatic angiotensinconverting enzyme (ace) is inefficient compared with testis ace despite cleavage at identical stalk sites insect angiotensin-converting enzyme. a processing enzyme with broad substrate specificity and a role in reproduction the acer gene of drosophila codes for an angiotensin-converting enzyme homologue functional conservation of the active sites of human and drosophila angiotensin i-converting enzyme just the beginning: novel functions for angiotensin-converting enzymes deglycosylation, processing and crystallization of human testis angiotensin converting enzyme a major breakthrough using protein-engineering tools on testis ace, making it amenable for structural study drosophila melanogaster angiotensin iconverting enzyme expressed in pichia pastoris resembles the c domain of the mammalian homologue and does not require glycosylation for secretion and enzymic activity crystal structure of drosophila angiotensin i-converting enzyme bound to captopril and lisinopril structure of neurolysin reveals a deep channel that limits substrate access crystal structure of a novel carboxypeptidase from the hyperthermophilic archaeon pyrococcus furiosus effects of the n-terminal sequence of ace on the properties of its c-domain first biophysical demonstration that somatic ace indeed contains two zinc-binding active sites and that lh-rh enzymes of the renin-angiotensin system and their inhibitors the design and properties of n-carboxyalkyldipeptide inhibitors of angiotensin-converting enzyme design of angiotensin converting enzyme inhibitors angiotensin-converting enzyme inhibitors natural products and design: interrelated approaches in drug discovery the discovery of captopril spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung pulmonary angiotensin-converting enzyme. structural and catalytic properties design of potent competitive inhibitors of angiotensinconverting enzyme. carboxyalkanoyl and mercaptoalkanoyl amino acids design of specific inhibitors of angiotensin-converting enzyme: new class of orally active antihypertensive agents landmark paper describing the brilliant series of insights leading to the first clinically useful ace inhibitor, captopril zinc coordination, function, and structure of zinc enzymes and other proteins conversion of angiotensin i to angiotensin ii fate of angiotensin i in the circulation conversion of angiotensin i to angiotensin ii by cell-free extracts of dog lung activity of various fractions of bradykinin potentiating factor against angiotensin i converting enzyme angiotensin-converting enzyme inhibitors from the venom of bothrops jararaca. isolation, elucidation of structure, and synthesis binding of the by-product analog benzylsuccinic acid by carboxypeptidase a important extension of the cushman-ondetti model, laying the groundwork for a series of additional ace inhibitors inhibition of rabbit lung angiotensinconverting enzyme by n α (s)- -carboxy- -phenylpropyl]llysyl-l-proline metal-coordinating substrate analogs as inhibitors of metalloenzymes dipeptidehydroxamates are good inhibitors of the angiotensin iconverting enzyme synthesis and biological activity of a ketomethylene analogue of a tripeptide inhibitor of angiotensin converting enzyme siliconbased metalloprotease inhibitors: synthesis and evaluation of silanol and silanediol peptide analogues as inhibitors of angiotensin-converting enzyme the renin-angiotensin system: a review of trials with angiotensin-converting enzyme inhibitors and angiotensin receptor blocking agents mechanisms of cardiovascular risk reductions with ramipril: insights from hope and hope substudies using ace inhibitors appropriately the at -type angiotensin receptor in oxidative stress and atherogenesis. part i: oxidative stress and atherogenesis inflammation in early atherogenesis: impact of ace inhibition contribution of bradykinin to the cardioprotective effects of ace inhibitors effect of bradykinin-receptor blockade on the response to angiotensin-converting-enzyme inhibitor in normotensive and hypertensive subjects effects of losartan and captopril on mortality and morbidity in high-risk patients after acute myocardial infarction: the optimaal randomized trial aminopeptidase p in individuals with a history of angio-oedema on ace inhibitors potentiation of the actions of bradykinin by angiotensin i-converting enzyme inhibitors. the role of expressed human bradykinin b receptors and angiotensin-converting enzyme in cho cells enhancement of bradykinin and resensitization of its b receptor angiotensin-converting enzyme inhibitor ramiprilat interferes with the sequestration of the b kinin receptor within the plasma membrane of native endothelial cells angiotensin blockade for hypertension: a promise fulfilled effect of losartan compared with captopril on mortality in patients with symptomatic heart failure: randomized trial -the losartan heart failure survival study elite ii comparison of candesartan, enalapril, and their combination in congestive heart failure. randomized evaluation of strategies for left ventricular dysfunction (resolvd) pilot study can angiotensin receptor antagonists be used safely in patients with previous ace inhibitor-induced angioedema? angiotensin ii suppression in humans by the orally active renin inhibitor aliskiren (spp ): comparison with enalapril vasopeptidase inhibition and angio-oedema β-amyloid catabolism: roles for neprilysin (nep) and other metallopeptidases? omapatrilat -the story of overture and octave vasopeptidase inhibition: a double-edged sword? a randomized trial of the angiotensinreceptor blocker valsartan in chronic heart failure n-[ -(indan- -yl)- -mercapto-propionyl] amino acids as highly potent inhibitors of the three vasopeptidases (nep, ace, ece): in vitro and in vivo activities hydrolysis of peptide hormones by endothelin-converting enzyme- . a comparison with neprilysin the critical role of tissue angiotensinconverting enzyme as revealed by gene targeting in mice rxp , a selective inhibitor of the n-domain of angiotensin i-converting enzyme, blocks in vivo the degradation of hemoregulatory peptide acetyl-ser-asp-lys-pro with no effect on angiotensin i hydrolysis bradykinin potentiation by angiotensin-( - ) and ace inhibitors correlates with ace c-and n-domain blockade n-domain-specific substrate and c-domain inhibitors of angiotensin-converting enzyme. angiotensin-( - ) and keto-ace angiotensin i-converting enzyme and metabolism of the haematological peptide n-acetyl-seryl-aspartyl-lysyl-proline effect of n-acetyl-seryl-aspartyl-lysylproline on dna and collagen synthesis in rat cardiac fibroblasts angiotensin-converting-enzyme inhibition therapy in altitude polycythaemia: a prospective randomised trial selective inhibition of the c-domain of angiotensin i converting enzyme by bradykinin potentiating peptides the c-type natriuretic peptide precursor of snake brain contains highly specific inhibitors of the angiotensin-converting enzyme structural constraints of inhibitors for binding at two active sites on somatic angiotensin converting enzyme integration of virtual and high-throughput screening structure-based screening and design in drug discovery angiotensin-converting enzyme- (ace- ): comparative modelling of the active site, specificity requirements and chloride dependence the ace research in k.r.a.'s and e.d.s.'s laboratories is supported by the wellcome trust, uk. we would like to acknowledge the help of r. natesh and s. iyer in the preparation of this manuscript. the following terms in this article are linked online to: locuslink: http://www.ncbi.nlm.nih.gov/locuslink/ ace | ace | acer | angiotensinogen | at receptor | at receptor | bk | ece- | endothelin- | endothelin- | lh-rh | renin access to this interactive links box is free online. key: cord- -z movens authors: ferrario, carlos m.; averill, david b.; brosnihan, k. bridget; chappell, mark c.; diz, debra i.; gallagher, patricia e.; neves, liomar; tallant, e. ann title: regulation of cardiovascular control mechanisms by angiotensin-( – ) and angiotensin-converting enzyme date: - - journal: hypertension and hormone mechanisms doi: . / - - - - _ sha: doc_id: cord_uid: z movens among the molecular forms of angiotensin peptides generated by the action of renin on angiotensinogen (aogen), both angiotensin ii (ang ii) and the amino terminal heptapeptide angiotensin-( – ) [ang-( – )] are critically involved in the long-term control of tissue perfusion, cell-cell communication, development, and growth. whereas an impressive body of literature continues to uncover pleiotropic effects of ang ii in the regulation of cell function, research on ang-( – ) has a shorter history as it was only yr ago that a biological function for this heptapeptide was first demonstrated in the isolated rat neuro-hypophysial explant preparation ( ). on the contrary, the synthesis of angiotonin/ hypertensin (now ang ii) was first obtained in ( ), three decades ahead of the discovery of ang-( – ) biological properties. carboxy-terminal hydrophobic or basic residues, whereas ace cleaves two amino acids from its substrate. the ace sequence is similar to those of the testis-specific or germinal form of ace (tace) and the drosophila homolog of ace (ance; sequence identities of % and %, and similarities of % and %, respectively) ( ) . conformational differences between the somatic form of ace and germinal ace account for the demonstration that ace activity is not blocked by ace inhibitors ( , ) . the potential importance of ace , not only in the regulation of cardiac function and blood pressure but also in other disease states, has been realized with the discovery that ace also serves as the cellular entry point for the severe acute respiratory syndrome (sars) virus ( ) ( ) ( ) ( ) . prabakaran et al. ( ) built a homology model of the ace structure with a root-mean-square deviation less than . Å from the aligned crystal structures of tace and ance. according to the authors a prominent feature of the model is a deep channel on the top of the molecule that contains the catalytic site ( ) . negatively charged ridges surrounding the channel may provide a possible binding site for the positively charged receptor-binding domain (rbd) of the s-glycoprotein, which they recently identified ( ) . several distinct patches of hydrophobic residues at the ace surface were noted at close proximity to the charged ridges that could contribute to binding. these results may help explain the structure and function of ace . we have employed immunocytochemistry to identify the tissues expressing ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) focusing primarily on the heart, kidney, brain, vascular system, and the utero-placenta complex. early studies have demonstrated the ang-( - ) immunoreactivity in the cell bodies and in the axons of magnocellular neurons of the paraventricular nucleus (pvn) and supra-optic nucleus (son) of the hypothalamus as well as in the neurons of the nucleus circularis ( ) . in fact, ang-( - ) immunoreactivity in neurons of the pvn and son was co-localized with vasopressin-like immunoreactivity whereas ang-( - ) was not co-localized in paraventricular neurons immunoreactive for oxytocin ( , ) . the same pattern of ang-( - ) immunoreactivity, seen in sprague-dawley rats, was also observed in the brains of (mren ) transgenic rats ( ) in which a subpopulation of nitric oxide synthase-containing neurons also contained ang-( - )-like immunoreactivity. collectively, the co-localization of ang-( - ) and vasopressin immunoreactivity in neurons of the magnocellular division of the pvn and the son are congruent with functional studies demonstrating a role of ang-( - ) in the regulation of hydro-mineral balance involving neurons of the hypothalami-neurohypophysial pathway. in addition, the colocalization of ang-( - ) immunoreactivity in nitric oxide synthase-containing neurons of the pvn is especially interesting because a number of studies now show that the angiotensin peptides may modulate the disposition of reactive oxygen species in the pvn of animals with heart failure ( ) . our interest in the role of ang-( - ) in cardiovascular regulation during heart failure led to the investigation of expression of ang-( - ) in the heart ( ) . ang-( - ) immunoreactivity in lewis rats was restricted to myocytes of both the right and left ventricles. fig. shows that ang-( - ) staining in myocytes had a granular appearance throughout the cytoplasm. in contrast, there was a distinct absence of staining for the peptide in vascular smooth muscle as well as in interstitial cells of the heart. when ang-( - ) was examined in the hearts of rats subjected to ligation of the left main coronary artery, we observed a significant increase in ang-( - ) staining in ventricular myocytes that had undergone hypertrophic remodeling. in the region of ischemic damage there was a marked absence of ang-( - ) staining in fibroblasts and connective tissue. the increase in ang-( - ) staining in rats with congestive heart failure was positively correlated with an increase in left ventricular end-diastolic pressure and negatively correlated with part i / ferrario et al. (fig. ). fig. b shows a granular reaction product for ang-( - ) staining in the juxtaglomerular cells of the afferent arteriole ( ) . it is interesting that blood vessels (arterioles) of the kidney exhibit ang-( - ) staining whereas this was not the observation for blood vessels in the heart or in other organs. vasodilation produced by ang-( - ) was first described in by benter et al. ( ) in rats that were made areflexic by spinal cord destruction. the vasodilator properties of the peptide were confirmed in isolated canine ( , ) and porcine ( ) coronary artery vessels, rabbit pial arterioles ( ) , the rat mesenteric circulation ( ) , the spontaneously hypertensive rats (shr) ( ), [mren ] hypertensive transgenic rats ( ) , two-kidney one-clip hypertensive dogs ( ) , and in the human forearm circulation ( ) . as reviewed elsewhere ( , ) , the vasodilator response is mediated by release of vascular endothelium nitric oxide (no), prostacyclin, and a receptor-mediated potentiation of bradykinin. the biological action of ang-( - ) satisfies the concept that the peptide acts to oppose the vasoconstrictor and hypertensive effects of ang ii by raising the activity of intrinsic vasodilator autocoids. ang-( - ) is present in hypothalamus, amygdala, and medulla oblongata at concentrations equivalent to or greater than those of ang ii ( ) and evidence of a functional role for ang-( - ) exists in brain regions known to respond to other components of the ras. ang-( - ) excites neurons in the pvn and in the nucleus of the solitary tract, dorsal motor nucleus of the vagus, and rostral and caudal ventrolateral medulla. the population of cells excited by ang-( - ) and ang ii shows some overlap, but the majority of cells respond to one or the other peptide. in addition to direct cellular actions, ang-( - ) releases other neurotransmitters including monoamines, substance p, vasopressin, glutamate, prostaglandins, and no as reviewed recently ( ) . like ang ii, the ang-( - )mediated release of vasopressin may involve tachykinin peptides ( ) ( ) ( ) . in contrast, intracerebroventricular injections of ang-( - ) do not increase blood pressure or promote drinking, which are thought to be mediated by ang ii acting on monoaminergic pathways ( , ) . an opposing action of ang-( - ) on central mechanisms regulating blood pressure is demonstrated by the finding that cerebroventricular injections of antibodies to ang-( - ) produce opposite effects to those of ang ii ( ) . evidence from studies using c-fos reveals that ang-( - ) activates pathways in the organum vasculosum of the lamina terminalis and median preoptic nucleus, whereas ang ii activates these pathways as well as those involving the pvn and subfornical organ ( , ) . pathways in the medulla oblongata appear separate for the two peptides as well, since sino-aortic denervation potentiated the responses to ang-( - ) but not those to ang ii in the nucleus of the solitary tract ( ) . important indicators of a role for the endogenous peptide in cardiovascular regulation come from studies using the ang-( - ) antagonist d-ala -ang-( - ) ( ) . blockade of endogenous ang-( - ) within the solitary tract nucleus augments baroreceptor reflex control of heart rate, providing an independent and opposite effect from those of ang ii ( , ) . a loss of tonic input by ang-( - ) may accompany the age-related decrease in reflex gain ( ) . in asraogen animals deficient of glial angiotensinogen, a role for endogenous ang-( - ) to facilitate baroreflex sensitivity persists, whereas the attenuating effect of ang ii is lost ( ) . these data suggest different sources of the two peptides consistent with their divergent functional effects. in the rostral and caudal ventrolateral medulla, ang-( - ) exerts excitatory actions mimicking those of ang ii ( , ) , although clear differences exist in terms of regulation of the responses to the two peptides ( ) ( ) ( ) . in brain, receptor subtypes exhibiting varying degrees of selectivity for either the at or at antagonists and d-ala -ang-( - ) appear to mediate the actions of ang-( - ) ( , , ) . the pharmacology of the receptor involved may be dependent on the specific transmitter studied, but blockade by d-ala -ang-( - ) is a common feature as reviewed in detail by ferrario et al. ( ) . as an explanation for the fact that either d-ala -ang-( - ), at or at receptor antagonists, or some combination of the three antagonists is effective in blocking actions of ang-( - ) in brain, there is evidence that distinct subpopulations of classical at sites in the nucleus tractus solitarius (nts) and dorsal motor nucleus of the vagus (dmnx) exist showing differential sensitivity/selectivity to ang-( - ) and the at antagonist ( ) . moreover, at receptor antagonists (either losartan or candesartan) and d-ala -ang-( - ) block the actions of the peptide in the nts ( ) and ventrolateral medulla ( ) . it is now recognized that the mas gene codes for a receptor responsible for vascular and renal effects of ang-( - ) ( ) . mice deficient in the mas receptor show impairments in the baroreceptor reflex and alterations in responses to ang ii ( , ) , providing additional evidence of a role for endogenous ang-( - ) in these functions. there are few studies about the direct actions of ang-( - ) on heart function, although the peptide is highly expressed in rat myocardium ( ) and can be detected in larger concentrations in the cardiac interstitium or the coronary sinus blood after acute coronary artery ligation ( ) ( ) ( ) . in the isolated perfused heart of a rat, ang-( - ) appears to act as an anti-arrhythmogenic factor during reperfusion injury ( , ) , although a study from the same group showed that ang-( - ) increased reperfusion arrhythmias ( ) . functional data in whole animals suggest that ang-( - ) may have cardioprotective functions because an wk infusion of ang-( - ) in sprague-dawley rats, started -wk postmyocardial infarction, attenuated heart failure progression together with restoration of vascular endothelial function ( ) . that the heart may be an important site for ang-( - ) actions is highlighted by the demonstration that ace activity in plasma and atrial tissue is inhibited by ang-( - ) ( ) whereas the peptide enhances tritiated norepinephrine release from isolated atrial tissue at doses comparable to those for ang ii ( ) . the studies by us ( , ) and others on the relation between ang-( - ) function and cardiac ace expression are shedding light on an intracardiac role for ang-( - ) as counterbalancing the hypertrophic and inotropic actions of ang-( - ) following myocardial injury. ang-( - ) was formed in the intact human myocardial circulation in patients with postcardiac transplantation and its levels were decreased when ang ii formation was suppressed by enalaprilat ( ) . the evolution of myocardial infarction in the rat d after coronary artery ligation was accompanied by large increases in plasma ang-( - ) concentrations; further plasma ang-( - ) augmentation accompanied by reversal of cardiac remodeling because of continuous blockade of ang ii receptors with either losartan or olmesartan was accompanied by a threefold increase in cardiac ace mrna ( ) . in addition, emerging data suggest that ang-( - ) may oppose the atherogenic actions of ang ii through inhibition of smooth muscle proliferation and blockade of inflammatory cytokines. this interpretation is supported by the finding that the chronic effects of losartan in the prevention of fatty streak formation and monocyte activation in monkeys was associated with large increases in plasma ang-( - ) concentrations ( - ). although ang-( - ) is processed from either ang i or ang ii in the circulation and in many tissues, it is important to emphasize that the processing pathways for ang-( - ) in the circulation and kidney are distinct. the endopeptidase neprilysin is the primary enzyme forming ang-( - ) from ang i or ang-( - ) in the circulation ( , ) . although levels of neprilysin are low to undetectable, the enzyme is appropriately localized to the exocellular surface of endothelial and smooth muscle cells to contribute to the formation of ang-( - ). in the kidney, ang-( - ) is the primary product formed in preparations of isolated proximal tubules and exists in urine at significantly higher levels than ang ii ( ) . neprilysin may contribute to both the formation as well as the degradation of the peptide. neprilysin cleaves ang i to ang-( - ), but continues to metabolize ang-( - ) at the tyr -ile bond to form ang-( - ) and ang-( - ) ( , ) . indeed, neprilysin inhibitors increase the urinary levels of ang-( - ) in both human and rat ( , ) . moreover, the combined ace/neprilysin inhibitor omapatrilat augmented renal and urinary levels of ang-( - ), but produced a blunted ang-( - ) response in plasma in comparison to ace inhibition alone ( , ) . omapatrilat was also associated with increased expression of ace and ang-( - ) in the proximal tubule ( ) . these data suggest that upregulation of ace may contribute to the renal protective effects of ace or combined vaso-peptidase inhibitors through increased conversion of ang ii to ang-( - ), as well as the reduced metabolism of ang-( - ). indeed, ace (-/-) mice exhibit enhanced intrarenal levels of ang ii and pronounced glomerulosclerosis ( ) . we found that ang-( - ) and ace are present within the proximal tubular regions of the mouse and the rat kidney ( , ) . these data clearly support the concept of a complete ras within the proximal tubule including expression of renin, ace, angiotensinogen, the at and at receptors ( , ) . the presence of ang ii and ang-( - ) in kidney supports the concept of important and divergent actions for the two peptides. for ang ii, renal actions include potent vasoconstriction, retention of sodium and water, as well as a stimulus for inflammation, and oxidative stress. in contrast, ang-( - ) stimulates diuresis and natriuresis that are associated with modest increases in the glomerular filtration rate ( ) . ang-( - ) induces vasodilation of afferent arterioles through a no-dependent pathway ( ) . indeed, immunocytochemical staining for the ang-( - ) receptor mas is evident in the afferent arteriole, as well as throughout the proximal tubules of the renal cortex providing biochemical support for the functional actions of the peptide ( ) . ang-( - ) and its metabolite ang-( - ) are potent inhibitors of na + , k + -atpase activity in the renal epithelium ( ) ( ) ( ) . ang-( - ) also inhibits the transcellular flux of sodium, which was associated with activation of phospholipase a (pla ) ( ). ang-( - ) dependent inhibition of sodium transport is potentiated by ace inhibition suggesting a shift toward the formation and protection of ang-( - ) within the proximal tubules. the chronic and pronounced diuresis following omapatrilat treatment was associated with large increases in urinary excretion of ang-( - ) and enhanced immunocytochemical staining of the peptide in the kidney ( ) . recent studies have revealed that ang-( - ) abolished the ang ii-dependent stimulation of the na + -atpase activity in ovine kidney ( , ) . furthermore, intrarenal administration of ang-( - ) blocked the antinatriuretic actions of ang ii ( ) . these studies emphasize that the actions of ang-( - ) within the kidney may be particularly relevant in the setting of an activated ras. moreover, the discrete localization of ang- ( - ) , ace , and the mas receptor provides evidence for an alternative ras within the proximal tubule epithelium that may antagonize the actions of ang ii in this renal compartment. pregnancy is a physiological condition characterized by increased ras activity ( ) that does not manifest in increased blood pressure ( ) . merrill et al. ( ) evaluated the effect of pregnancy on ang-( - ) in nulliparous preeclamptic patients and in third trimester normotensive pregnant controls ( rd t; matched for parity, race, and gestational age). plasma ang-( - ) was increased by % (p < . ), whereas plasma ang ii was increased by % (p < . ). in preeclampsia subjects plasma ang-( - ) was reduced ( ± pg/ml, p < . vs third t); plasma ang ii was also reduced ( ± pg/ml, preeclamptic vs rd trimester normal pregnant, p < . ), but remained elevated as compared to nonpregnant subjects and % higher than plasma ang- ( - ) . other components of the ras, with the exception of ace, were reduced in preeclamptic subjects. assessment of the relationship between ang-( - ) and blood pressure revealed a negative correlation of ang-( - ) with systolic (r = - . , p < . ) and diastolic (r = - . , p < . ) blood pressures. these data suggested a potential role for reduced production of ang-( - ) contributing to the elevated blood pressure. in preeclampsia, the decreased levels of plasma ang-( - ) in the presence of persistent elevated plasma ang ii are consistent with the development of hypertension. additionally, a -h urinary excretion of ang-( - ) and ang ii was evaluated during the ovulatory menstrual cycle, single normotensive pregnancies, and their subsequent lactation ( ) . no significant differences in urinary ang-( - ) were observed between the follicular and luteal phase of the normal menstrual cycle. there was a progressive rise of urinary ang-( - ) throughout normal human gestation, attaining levels that are -fold greater than that of the normal menstrual cycle. urinary ang ii showed a similar pattern reaching levels that were -fold higher than the values at the menstrual cycle. at wk of gestation, ang-( - ) was the predominant angiotensin peptide in the urine, reaching levels that were sixfold higher than ang ii. the urinary excretion levels may reflect local kidney production of peptides. thus, increases in renal ang-( - ) levels may play a role in the vasodilatory adaptations of mid and late human pregnancies. to understand the contribution of the ras during pregnancy, studies were conducted in pregnant rats at late gestation ( th day) and compared to virgin female rats at diestrus phase of the estrous cycle. twenty-four hour urinary excretion of the angiotensin peptides was significantly increased in pregnant animals by % (ang i), % (ang ii), and % [ang-( - )] of values found in virgin rats. kidney ang i and ang-( - ) concentrations were significantly increased by seven-and fivefold, respectively (p < . ) in pregnant animals as compared to virgin females. in contrast, there was no significant change in renal ang ii concentrations of pregnant and virgin females. these studies provide evidence that urinary excretion of angiotensin peptides reflect local kidney content of angiotensins during pregnancy. the potential contribution of ang-( - ) to vascular control in pregnancy was also documented from increased vasodilator responses to the local application of the peptide in isolated small mesenteric arteries obtained from pregnant rats ( ) . in alignment with this interpretation, ang-( - ) and ace staining in the kidney of d pregnant sprague-dawley rats showed higher intensity when compared with virgin rats ( ). ang-( - ) is a poor competitor at pharmacologically defined at or at receptors ( - ) , did not compete for binding at rat adrenal at or at receptors, and did not attenuate pressor or contractile responses to ang ii, demonstrating selectivity for ang-( - ). we identified ang-( - ) binding sites on bovine aortic endothelial cells (baec), canine coronary artery rings, and rat blood vessels that are sensitive to [d-ala ]-ang-( - ) ( ) ( ) ( ) . in addition, a multitude of physiological responses to ang-( - ) are selectively blocked by [d-ala ]-ang-( - ) or the sarcosine analogs of ang ii, but not by at or at receptor antagonists, including the depressor response to ang-( - ) in the pithed rat and the lowering of blood pressure in hypertensive rats ( , , ) . collectively, these results demonstrate that the hypotensive response to ang-( - ) is mediated by a non-at , non-at , [d-ala ]-ang-( - )-sensitive receptor. we refer to this receptor as the at ( ) ( ) ( ) ( ) ( ) ( ) ( ) receptor, as defined by its sensitivity to ang-( - ), its antagonism by [sar -thr ]-ang ii and [d-ala ]-ang-( - ), and its lack of response to at or at receptor antagonists, either functionally or in competition for binding. identification of an at ( ) ( ) ( ) ( ) ( ) ( ) ( ) receptor is confounded by reports of responses to ang-( - ) that are sensitive to at or at receptor antagonists or both. some of the renal and central effects of ang-( - ) are mediated by a losartan-sensitive receptor ( , , ) . arachidonic acid release from rabbit vsmcs and hypothalamic norepinephrine release were blocked by both [d-ala ]-ang-( - ) and the at antagonist pd ( , ) . additional responses to ang-( - ) in brain and heart are blocked by both at and at receptor antagonists ( , ) . these results provide evidence for additional subtypes of the at ( ) ( ) ( ) ( ) ( ) ( ) ( ) receptor that are sensitive to losartan and/or pd or suggest an interaction with the at and/or the at receptor. many of the physiological and cellular responses that are mediated by the at ( ) ( ) ( ) ( ) ( ) ( ) ( ) receptor are linked to the production of prostaglandins. ang-( - ) stimulates prostaglandin production in endothelial cells, vsmcs, astrocytes, and renal tubular epithelial cells ( , , ( ) ( ) ( ) ( ) ) . in addition, physiological responses to ang-( - ) are dependent on prostanoid production, based on the studies using the cyclooxygenase inhibitor indomethacin (fig. ) . recently, santos et al. ( ) reported that the orphan g protein-coupled receptor mas is an ang-( - ) receptor. ang-( - ) bound with high affinity to cells transfected with the mas receptor, which was blocked by [d-ala ]-ang-( - ), and renal or depressor responses to ang-( - ) were lost in mas-depleted mice. we recently showed that antisense oligonucleotides or sirnas to mas prevent the ang-( - )-mediated inhibition of growth in vsmcs, which is also blocked by [d-ala ]-ang-( - ) ( ). these results suggest that the mas receptor serves as a selective ang-( - ) binding site. fig. shows the signal transduction pathway by which ang-( - ) activates the g protein-coupled mas receptor to increase the production of no and prostacyclin (pgi ) via increases in cgmp and camp, respectively. ang-( - ) also reduces the mitogen-activated protein kinases (mapks) by either increasing mapk phosphatases or reducing the mapk kinase mek. the increase in camp and cgmp and the decrease in mapk activity cause vasodilation and inhibit cell growth. the mas receptor is predominantly expressed in the testis and the hippocampus and amygdala of the mammalian forebrain with minimal levels in the rodent heart and kidney. this tissue distribution differs from previous reports of ang-( - ) binding and functional responses, suggesting the existence of other at ( ) ( ) ( ) ( ) ( ) ( ) ( ) receptors. ang-( - ), a product of both ang i and ang ii metabolisms, functions to antagonize the actions of ang ii by acting primarily through binding to a non-at /at receptor and also the mas receptor. ang-( - ) stimulates vasodilation through increased production of vasodilator prostaglandins and no, as well as amplifying the intrinsic actions of bradykinin. in the kidney, ang-( - ) promotes natriuresis and diuresis through an effect on transport mechanisms involved in water and electrolyte absorption within the renal tubules and collecting ducts. in addition, ang-( - ) modulates the actions of tubular vasopressin via an effect on its v receptor. in the heart, ang-( - ) counteracts the hypertrophic, pro-fibrotic, and pro-thrombotic effects of ang ii as well as increases myocardial blood flow. ang-( - ) contributes to the antihypertensive effects of ace inhibitors and ang ii antagonists by several mechanisms ( , , , ) . inhibition of ace increases blood and tissue concentrations of ang-( - ) by preventing ace-mediated ang-( - ) degradation and increasing substrate (ang i) availability. in contrast, blockade of ang ii receptors increases blood and tissue levels of ang-( - ) by: (i) increasing ang i substrate availability through the dis-inhibition of ang ii-mediated renin release, and (ii) augmenting the rate of ang ii conversion into ang-( - ) via increased ace expression and activity. both ace and ace represent critical steps at which modulation of angiotensin peptide functions are precisely regulated, the specific mechanisms of which need further investigation. genomic studies are also urgently needed to determine whether polymorphisms in the ace gene or the genes for ang-( - )-forming enzymes exist. release of vasopressin from the rat hypothalamo-neurohypophysial system by angiotensin-( - ) heptapeptide synthesis and pharmacology of the octapeptide angiotonin angiotensin-( - ): a new hormone of the angiotensin system angiotensin-( - ). a member of circulating angiotensin peptides evidence that prolyl endopeptidase participates in the processing of brain angiotensin production of angiotensin-( - ) by human vascular endothelium inhibition of angiotensin converting enzyme by the metalloendopeptidase . . . inhibitor c-phenylpropylalanyl-alanyl-phenylalanyl-p-aminobenzoate pathways for angiotensin-( - ) metabolism in pulmonary and renal tissues pathways of angiotensin-( - ) metabolism in the kidney metabolism of angiotensin-( - ) by angiotensin converting enzyme release of angiotensin-( - ) from the rat hindlimb: influence of angiotensin-converting enzyme inhibition converting enzyme determines plasma clearance of angiotensin-( - ) vasodepressor actions of angiotensin-( - ) unmasked during combined treatment with lisinopril and losartan angiotensin-( - ) contributes to the antihypertensive effects of blockade of the renin-angiotensin system a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - aceh/ace is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ace inhibitors. can the angiotensin-converting enzyme gene family: genomics and pharmacology hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase heart block, ventricular tachycardia, and sudden death in ace transgenic mice with downregulated connexins a model of the ace structure and function as a sars-cov receptor the secret life of ace as a receptor for the sars virus angiotensin-converting enzyme is a functional receptor for the sars coronavirus ace : from vasopeptidase to sars virus receptor the sars-cov s glycoprotein: expression and functional characterization immunocytochemical localization of angiotensin-( - ) in the rat forebrain angiotensin-( - ) immunoreactivity in the hypothalamus of the (mren- d) transgenic rat angiotensin-( - ) and nitric oxide synthase in the hypothalamoneurohypophysial system brain angiotensin ii: new insights into its role in sympathetic regulation cardiac angiotensin-( - ) in ischemic cardiomyopathy angiotensin-( - ) attenuates the development of heart failure after myocardial infarction in rats vasopeptidase inhibition and ang-( - ) in the spontaneously hypertensive rat cardiovascular actions of angiotensin angiotensin-( - ) dilates canine coronary arteries through kinins and nitric oxide effect of the angiotensin-( - ) peptide on nitric oxide release release of nitric oxide by angiotensin-( - ) from porcine coronary endothelium: implications for a novel angiotensin receptor comparative effects of angiotensin-( - ) and angiotensin ii on piglet pial arterioles characterization of angiotensin-( - ) receptor subtype in mesenteric arteries angiotensin-( - ) in the spontaneously hypertensive rat contribution of angiotensin-( - ) to blood pressure regulation in salt-depleted hypertensive rats angiotensin-( - ) and nitric oxide interaction in renovascular hypertension angiotensin-( - ) potentiates bradykinin-induced vasodilatation in man angiotensin-( - ): its contributions to arterial pressure control mechanisms angiotensin-( - ): an update identification of angiotensin-( - ) in rat brain. evidence for differential processing of angiotensin peptides the hypothalamic paraventricular nucleus is a site of action for the central effect of tachykinins on plasma vasopressin opposing actions of angiotensin-( - ) and angiotensin ii in the brain of transgenic hypertensive rats vasopressin release induced by intracranial injection of eledoisin is mediated by central angiotensin ii acute depressor actions of angiotensin ii in the nucleus of the solitary tract are mediated by substance p characterization of receptors for angiotensin-induced drinking and blood pressure responses in conscious rats using angiotensin analogs extended at the n-terminal angiotensin-( - ) is an antagonist at the type angiotensin ii receptor the association of thirst, sodium appetite and vasopressin release with c-fos expression in the forebrain of the rat after intracerebroventricular injection of angiotensin ii, angiotensin-( - ) or carbachol actions of angiotensin peptides after partial denervation of the solitary tract nucleus angiotensin peptides and baroreflex control of sympathetic outflow: pathways and mechanisms of the medulla oblongata angiotensin peptides as neurotransmitters/ neuromodulators in the dorsomedial medulla reduced baroreflex sensitivity and diminished role for endogenous angiotensin-( - ) at the nucleus tractus solitarii in older rats may reflect reduced formation of the peptide glial versus non-glial angiotensin ii and angiotensin-( - ) in the nucleus tractus solitarii independently modulate control of the circulation alterations of the reninangiotensin system at the rvlm of transgenic rats with low brain angiotensinogen angiotensin peptides acting at rostral ventrolateral medulla contribute to hypertension of tgr(mren ) rats potentiation of the hypotensive effect of bradykinin by short-term infusion of angiotensin-( - ) in normotensive and hypertensive rats central and peripheral actions of angiotensin-( - ). braz characterization of a new angiotensin antagonist selective for angiotensin-( - ): evidence that the actions of angiotensin-( - ) are mediated by specific angiotensin receptors characterization of the receptors mediating the actions of angiotensin-( - ) in the nucleus tractus solitarri the cardiovascular effects of angiotensin-( - ) in the rostral and caudal ventrolateral medulla of the rabbit angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas interaction between mas and the angiotensin at receptor in the amygdala metabolism of angiotensin i in isolated rat hearts. effect of angiotensin converting enzyme inhibitors the renin-angiotensin system during acute myocardial ischemia in dogs angiotensin peptides modulate bradykinin levels in the interstitium of the dog heart in vivo angiotensin-( - ): cardioprotective effect in myocardial ischemia/reperfusion angiotensin-( - ) improves the postischemic function in isolated perfused rat hearts. braz effect of angiotensin-( - ) on reperfusion arrhythmias in isolated rat hearts. braz angiotensin-( - ) is a modulator of the human renin-angiotensin system effects of angiotensin ii and angiotensin-( - ) on the release of [ h]norepinephrine from rat atria upregulation of angiotensin-converting enzyme after myocardial infarction by blockade of angiotensin ii receptors angiotensin-( - ) formation in the intact human heart: in vivo dependence on angiotensin ii as substrate renin-angiotensin system as a therapeutic target in managing atherosclerosis angiotensin-( - ) reduces smooth muscle growth after vascular injury inhibition of early atherogenesis by losartan in monkeys with diet-induced hypercholesterolemia mechanisms linking angiotensin ii and atherogenesis renin-angiotensin system expression in rat bone marrow haematopoietic and stromal cells effect of reduced angiotensin-converting enzyme gene expression and angiotensin-converting enzyme inhibition on angiotensin and bradykinin peptide levels in mice angiotensin-converting enzyme is an essential regulator of heart function effects of omapatrilat on the renin-angiotensin system in salt-sensitive hypertension evidence for the formation of angiotensin-( - ) and the inhibitory actions of the peptide on na + , k + -atpase activity in rat proximal tubules omapatrilat treatment is associated with increased ace- and angiotensin-( - ) in spontaneously hypertensive rats novel aspects of the renal renin-angiotensin system: angiotensin-( - ), ace and blood pressure regulation regulation of intrarenal angiotensin ii in hypertension why are angiotensin concentrations so high in the kidney? vasodilator action of angiotensin-( - ) on isolated rabbit afferent arterioles renal actions of angiotensin-( - ): in vivo and in vitro studies angiotensin-( - ) can interact with the rat proximal tubule at( ) receptor system autoradiographic analysis and regulation of angiotensin receptor subtypes at( ) modulation of phospholipase a activity and sodium transport by angiotensin angiotensin-( - ) modulates the ouabain-insensitive na + -atpase activity from basolateral membrane of the proximal tubule angiotensin-( - ) reverts the stimulatory effect of angiotensin ii on the proximal tubule na(+)-atpase activity via a a -sensitive receptor intrarenal infusion of angiotensin-( - ) modulates renal functional responses to exogenous angiotensin ii in the rat the renin-angiotensin system in pregnancy and parturition chesley's hypertensive disorders in pregnancy angiotensin-( - ) in normal and preeclamptic pregnancy urinary vasodilator and vasoconstrictor angiotensins during menstrual cycle, pregnancy, and lactation pregnancy enhances the angiotensin (ang)-( - ) vasodilator response in mesenteric arteries and increases the renal concentration and urinary excretion of ang enhanced renal immunocytochemical expression of ang-( - ) and ace during pregnancy characterization of angiotensin receptors mediating prostaglandin synthesis in c glioma cells alterations in prostaglandin production in spontaneously hypertensive rat smooth muscle cells differential regulation of prostaglandin synthesis by angiotensin peptides in porcine aortic smooth muscle cells: subtypes of angiotensin receptors involved human astrocytes contain two distinct angiotensin receptor subtypes counterregulatory actions of angiotensin-( - ) evidence that prostaglandins mediate the antihypertensive actions of angiotensin-( - ) during chronic blockade of the renin-angiotensin system bovine aortic endothelial cells contain an angiotensin-( - ) receptor cardiovascular effects of angiotensin-( - ) in conscious spontaneously hypertensive rats angiotensin-( - ) has a biphasic effect on fluid absorption in the proximal straight tubule angiotensin-( - ) reduces norepinephrine release through a nitric oxide mechanism in rat hypothalamus signal transduction mechanisms involved in angiotensin-( - )-stimulated arachidonic acid release and prostanoid synthesis in rabbit aortic smooth muscle cells effects of angiotensin analogues and angiotensin receptor antagonists on paraventricular neurones effects of angiotensin ii and angiotensin-( - ) on the release of [ h] norepinephrine from rat atria inhibition of map kinase activity by angiotensin-( - ) in vascular smooth muscle cells is mediated by the mas receptor characterization of angiotensin-( - ) in the urine of normal and essential hypertensive subjects effects of captopril related to increased levels of prostacyclin and angiotensin-( - ) in essential hypertension the study described in this review was supported in part by grants from the national heart, lung and blood institute of the national institutes of health (hl- , hl- , and hl- , hl- , hl- ), and the american heart association (aha ). an unrestricted grant from the unifi corporation (greensboro, nc) is also acknowledged. key: cord- -arczqdza authors: khajah, maitham a.; fateel, maryam m.; ananthalakshmi, kethireddy v.; luqmani, yunus a. title: anti-inflammatory action of angiotensin - in experimental colitis date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: arczqdza background: there is evidence to support a role for angiotensin (ang) – in reducing the activity of inflammatory signaling molecules such as mapk, pkc and src. enhanced angiotensin converting enzyme (ace ) expression has been observed in patients with inflammatory bowel disease (ibd) suggesting a role in its pathogenesis, prompting this study. methods: the colonic expression/activity profile of ace , ang – , mas -receptor (mas -r), mapk family and akt were determined by western blot and immunofluorescence. the effect of either exogenous administration of ang – or pharmacological inhibition of its function (by a treatment) was determined using the mouse dextran sulfate sodium model. results: enhanced colonic expression of ace , ang - and mas -r was observed post-colitis induction. daily ang – treatment ( . – . mg/kg) resulted in significant amelioration of dss-induced colitis. in contrast, daily administration of a significantly worsened features of colitis. colitis-associated phosphorylation of p , erk / and akt was reduced by ang – treatment. conclusion: our results indicate important anti-inflammatory actions of ang – in the pathogenesis of ibd, which may provide a future therapeutic strategy to control the disease progression. the renin-angiotensin-aldosterone system (raas) plays an important role in the homeostatic control of cardiovascular, renal and adrenal functions. this peptide-based hormonal system is comprised of two main pathways; angiotensin converting enzyme/angiotensin ii/angiotensin type / receptor (ace/ang ii/at / r; which is the classical raas), and ace /ang - / mas- receptor (mas -r) (a recently described version of raas) [ , ] . the proteolytic enzyme renin is released by the juxtaglomerular cells in the kidneys to cleave the n-terminal region of angiotensinogen to form ang i (or decapeptide). ang i is further degraded to ang ii by the removal of two c-terminal amino acids by proteases such as ace and chymas- . ang ii binds to two main receptors; at r and at r. whereas at r plays an important role in blood pressure control and cardiac cell remodeling, at r antagonizes the signaling associated with at r stimulation [ ] . furthermore, ang ii can be converted to smaller peptide products with various biological activities. one of these, ang - , can be synthesized not only from ang ii (via postproline carboxypeptidase) but also from ang i (via tissue endopeptidases; neprilysin, prolyl endopeptidase, and thimet oligopeptidase), or directly from ang - following its formation from ang - by ace , bypassing the synthesis of ang ii [ ] . ang - induces its biological effects through the only known receptor for this peptide; the mas- r oncogene gprotein coupled receptor [ ] . the physiological roles of ang - on renal function, fluid homeostasis, vascular tone and cardiac contractility and remodeling are well documented, and most of these actions are directly opposed to ang ii-mediated effects [ ] [ ] [ ] [ ] . it is thought that the beneficial effects of ace-inhibitors (aceis) and angiotensin receptor blockers (arbs) on blood pressure control and in delaying/inhibiting the cardiac remodeling process is through increasing serum levels of ang - [ ] [ ] [ ] [ ] . several studies have suggested an important role for ang ii in the pathogenesis of inflammatory bowel disease (ibd), a chronic disorder of the gastrointestinal tract which comprises two conditions: crohn's disease and ulcerative colitis [ , ] . when the production and/or the activity of ang ii is reduced, colitis severity is ameliorated in various animal models of ibd through various mechanisms. treatment with aceis or arbs resulted in significant reduction in colitis severity in , , -trinitrobenzene sulphonic acid (tnbs) and dextran sulfate sodium (dss) induced models of experimental colitis [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, colitis severity can also be ameliorated by making mice homozygous for targeted disruption of the angiotensin gene [ ] or atr a deficiency [ ] . these protective effects are considered to be due in part to inhibition of ang ii mediated enhancement of nfκb phosphorylation, adhesion molecule expression in the gut (the mucosal addressin; madcam- ) and serum pro-inflammatory cytokine release (tnfα, ifnγ, il- β). furthermore, acei or arb treatment in mice results in reduced colonic epithelial cell apoptosis and enhanced expression of the anti-inflammatory cytokine il- . interestingly, ace gene polymorphism was detected in ibd patients and is associated with reduced ace serum levels; this might be associated with the disease pathogenesis and its extraintestinal side effects [ ] [ ] [ ] . it is thought that chronic treatment with raas blockers (acei or arbs) may lead to increased ang - , which could then oppose the harmful effects of ang ii in the cardiac and renal system, and possibly in the gut as well. anti-inflammatory effects of ang - have been reported in atherosclerosis plaque [ ] and in an arthritic model [ ] which was mediated through inhibition of nfκb activity and reduction of cytokines and chemokines such as tnfα, il- β, mcp- and cxcl . several reports [ ] [ ] [ ] [ ] have suggested that ang - treatment reduces the activity of intracellular signaling molecules such as mapk family (p , erk / and jnk), protein kinase c (pkc) and c-src kinase, which play an important role in intensifying the inflammatory response. ang - has been found to reduce the severity of allergic inflammation in mice by suppressing the activity of erk / and nfκb pathways [ ] . of particular interest, ace (the main enzyme responsible for ang - generation) expression has been observed in epithelial and sub-mucosal cells throughout the gut, with significant expression in the ileum and the colon [ ] [ ] [ ] . in the present study, we used the mouse dss colitis model to examine the ability of ang - to influence colitis severity. we show for the first time that the expression of ace /ang - / mas- r are modulated post colitis induction. blockade of the endogenous function of ang - (by the mas- r antagonist a ) exacerbated colitis severity, while daily administration of ang - ameliorated it. the anti-inflammatory effects of ang - were associated with reduction in the expression level of ang ii and the phosphorylation of p mapk, erk / and akt in the colon. male and female balb/c mice ( : ratio, - weeks old, mean weight g.) were supplied by the animal resource center of the health sciences center at kuwait university. all animals were kept under standard conditions including controlled temperature ( °c), a -h lightdark cycle and had free access to food and drinking water ad libitum. all experimentations were approved by the animal care committee at kuwait university health sciences center and conformed to their rules and regulations. during this study, the general health and wellbeing of the mice was continuously monitored and overseen by a veterinary physician in the animal house. all mice were monitored for their eating/drinking habits, activity, or other severe signs of hunched or lateral recumbency or starry fur or lethargy. there were no signs of illness or mortality during the treatment time points that required sacrificing the animals. the number of animals in each control and treated group for each set of experiments is indicated in the respective figure/table legends. colitis was induced in mice by delivering dss polymers ( . % w/v, m.wt kd; mp biomedicals, france) in autoclaved drinking water and provided ad libitum for - days [ ] . control (untreated; ut) mice received autoclaved tap water only. the dss solution was replaced every days for the duration of the experiment. there was no difference in the amount of water consumption by mice between the groups. decrease in body weight provides indirect indication of colitis severity. mice were sacrificed by cervical dislocation at - days post-colitis induction. blood samples were collected by cardiac puncture using a heparinized syringe (for total and differential wbc count). colons were resected and fixed in % formalin solution. treatment protocols. angiotensin fragment - acetate salt hydrate (ang - ; m.wt ; sigma-aldrich, st louis, usa) was dissolved in . % saline (vehicle) at mg/ml and stored at - °c. various doses ( . , . , . , . and mg/kg) were freshly prepared from the stock each day of the experiment, and administered to mice by daily intra-peritoneal (i.p) injections in a volume of μl per injection, either before (prophylactic approach) or after (treatment approach) dss treatment. a (mas- r antagonist; m.wt ; genscript, usa) was similarly dissolved in distilled water at mg/ml and stored at - °c. a freshly prepared dose of mg/kg was administered to a second group of mice by daily i.p injections in a volume of μl daily (for days) along with colitis induction (prophylactic approach). a third group of mice received dss containing water and daily i.p injections of . % saline (vehicle). the fourth group received dss containing water along with daily i.p injections with dexamethasone (dex) at doses of . - . mg/kg or its vehicle ( . % saline) (prophylactic approach). gross (macroscopic) assessment of colitis severity. using sterile forceps and scissors, the entire colon of each mouse was removed by a ventral midline incision and opened longitudinally. its length and maximal bowel thickness was measured (in mm) with calipers. several other macroscopical parameters were used to assess the colitis severity including stool consistency, blood in stool, adhesion, erythema, edema and ano-rectal bleeding [ ] . the data is presented as the percentage of mice in each group showing these features. histological (microscopic) assessment of colitis severity. the colon was cleaned from stools and blood with a few drops of sterile . % saline and 'swiss-rolled' from the descending to the ascending part. samples were fixed in % neutral buffered formalin and placed in tissue processing and embedding cassettes in pbs for a few minutes and then overnight in a leica asp tissue processing machine. tissues were rinsed in two changes of formalin, then dehydrated in several changes of graded alcohol ( %, % and %). three changes of xylene were used for tissue condensation and clearing. using an slee-mps embedding machine, processed tissues were embedded in paraffin wax ( h at room temperature) and stored at °c before trimming and sectioning using an leica rm microtome. sections ( μm thick) were floated in a water bath and then placed on uncoated slides at °c overnight. after de-paraffinisation in three changes of xylene ( min each) and rehydration by serial immersion for - min in each of absolute, % and % alcohol, sections were washed briefly with distilled water and stained in meyer's alum haematoxylin solution for min followed by thorough rinsing with running tap water. before counter-staining sections in eosin solution for min, slides were dehydrated in graded alcohol. a clearing step was performed by rinsing in three changes of xylene ( min each) followed by mounting with dpx. the stained sections were (blindly) scored by observers using a standard semi-quantitative histology scoring system [ ] [ ] [ ] which graded the following features: extent of destruction of normal mucosal architecture ( , normal; , , and , mild, moderate, and extensive damage respectively), presence and degree of cellular infiltration ( , normal; , , and , mild, moderate, and transmural infiltration respectively), extent of muscle thickening ( , normal; , , and , mild, moderate, and extensive thickening respectively), presence or absence of crypt abscesses ( , absent; , present) and the presence or absence of goblet cell depletion ( , absent; , present). the scores for each feature were summed with a maximum possible score of . the extent of ulceration was determined on each section along the muscularis mucosa and expressed as percentage ulcerated mucosa [ ] . differential white blood cell counts. during dissection, blood was collected by cardiac puncture using a ml heparinized syringe. to determine differential white blood cell count (wbc), a blood smear was made on a glass slide by placing a drop of blood on one slide surface and using a second glass slide to push the blood forward from the edge of the drop, and left to dry for h. siemens, diff-quik™ stain set was used for rapid differential staining. slides were dipped times in a methanolic fixative to stabilize cellular components, then dipped times in a buffered solution of eosin y and times in a buffered solution of thiazine dye (methylene blue and azure a). finally, slides were rinsed in distilled water, dried and stored at room temperature. wbcs were identified as monocytes, neutrophils, basophils, eosinophils and lymphocytes. a total of cells were randomly counted in each slide and the data are presented as % of the presence of each cell type. colon tissue samples (descending part) were cut and homogenized (with teflon glass homogenizer) in ml of buffer composed of . g of mm hepes, . g of mm nacl, ml of . m edta, ml of % triton x- and ml of deionized water. a protease inhibitor cocktail ( μg/ml aprotinin, μg/ml leupeptin and μm pmsf) was added separately. homogenates were centrifuged at , rpm for min at °c, and the supernatant collected. protein concentration was determined by the bradford assay (bio-rad, hercules, ca). samples containing μg protein were dissolved in an equal volume of x lammeli sample buffer and β-mercaptoethanol, heated at °c for min and loaded onto a . % sds-polyacrylamide gel and electrophoresed at v for h. proteins were transferred (at v for h) onto a pvdf membrane (millipore, ireland) and then blocked with % bsa for min before overnight incubation at °c with primary antibodies for actin (cell signaling technology, boston, ma, usa; / dilution), mas- r (m , santa cruz, tx, usa; / dilution), ang ii (novus biological; / dilution), and ace (santa cruz, tx, usa; / dilution). membranes were washed times for h with x tbs-t buffer and incubated with appropriate horseradish peroxidase (hrp)-labeled secondary antibodies [anti-rabbit igg, hrp linked antibody (cell signaling technology, boston, ma, usa; / dilution), and donkey anti-goat igg-hrp linked antibody (santa cruz, tx, usa; / dilution)]. bands were visualized using super signal ecl substrate (thermo scientific, rockford, usa) and kodak x-ray film. colon sections ( μm) were deparaffinized, rehydrated through a series of washes in graded ethanol and water, followed by an antigen retrieval step (by boiling the sections in mm sodium citrate buffer, ph . for min). sections were then incubated in blocking solution ( % bovine serum albumin (bsa) + . % triton x- in pbs) for h, followed by incubation overnight at °c with primary antibodies [p-erk / , p-akt ( : dilution), p-p mapk and mas- r ( : dilution); cell signaling, usa, and ang ii ( / dilution); novus biological] diluted in % blocking solution. on the following day, sections were washed and incubated with secondary antibody conjugated to alexa fluor (goat anti rabbit sfx kit; life technologies,usa, : dilution) for h at room temperature) in the dark. after washes in pbs sections were stained with ', diamidino- -phenylindole and mounted. images were captured on a ziess lsm confocal microscope and fluorescence intensity estimated in defined fields using image j software package. colon tissue ( mg/ml) was homogenized as described above and sonicated for min for further disruption of the cell membrane. homogenates were centrifuged for min at , rpm and the supernatants collected for subsequent analysis. blood was collected by cardiac puncture, centrifuged at , rpm for min and the plasma stored at- °c. ang - was measured using mouse angiotensin - elisa kit (mybiosource # mbs ). following the manufacturer's protocol: μl of standards, samples and blank (pbs) were pipetted into the pre-coated wells and mixed with μl of the balance solution. enzyme conjugate ( μl) was added to samples (except blank) and kept for h at °c in the dark. after removal of suspension, wells were washed five times with x wash solution prior to addition of hrp substrates a and b and min later, of stop solution. intensity of the yellow complex was measured at nm and concentration of ang - interpolated from a standard curve. data were analyzed using graphpad prism version . for windows (graphpad software, calfornia, usa). differences between groups were assessed using unpaired students' t test or oneway anova, with p . being regarded as significant. a seven fold decrease in the plasma level of ang - was demonstrated in dss treated mice compared to untreated (ut) group at day post colitis induction (fig a) . on the other hand, a significant increase in ang - was observed in colon homogenates of dss treated mice at day ( . ng/ml) compared to ut mice ( . ng/ml) as shown in fig b . ang ii expression determined by immunoblotting was increased at days and post-colitis induction compared to ut mice (fig a) , as was ace . an elevation was seen at day in the mas- r (fig b) . immunofluorescent staining showed presence of ace and mas- r in the mucosal layers of colon sections (fig c) observed at days and post-colitis induction, whereas very little reaction was detectable in colon tissue of untreated control mice. the effect of ang - on modulating colitis severity in mice was tested by daily i.p injections of various doses of the peptide. dss administration resulted in approximately % drop in body weight from day onwards (in contrast with mice receiving tap water only). ang - at doses of . and . mg/kg (but not at higher doses of . - mg/kg; data not shown) reduced this drop in body weight by - % compared with the dss/saline i.p treated group (fig a) . dss treatment in the dss/saline i.p group resulted in increased circulating neutrophils; this was prevented by ang - treatment at doses of . - mg/kg (fig b) . no significant changes in circulating lymphocytes were observed between control and treatment groups. the decrease in colon length and thickness seen after dss treatment was prevented by ang - treatment at doses of . - . mg/kg but not at higher doses (fig c and d ). as indicated in table , at the gross level, dss administration for days with daily i.p injections of saline (vehicle) resulted in diarrhea, blood in stool, adhesion, erythema, edema and ano-rectal bleeding, none of which was seen in the ut group. daily administration of ang - at doses of . - . mg/kg significantly reduced most of these effects. the presence of erythema was reduced by - %, incidence of diarrhea by - %, ano-rectal bleeding by - %, blood in stool by %, and edema by - % relative to dss/saline i.p group. ang - at . mg/kg dose reduced ano-rectal bleeding, blood in stool and erythema by - % but at the groups indicated. panel g represent plasma levels (ng/ml) of ang - in mice treated with dss (for days) plus either daily i.p saline or ang - ( . mg/kg). histobars represent means ± sem for - mice in each group ( for ut, for dss/i.p saline, for ang . mg/kg, for ang . mg/kg, for ang . mg/kg, for ang . mg/kg and for ang . mg/kg). asterisks denote significant difference from ut mice with p< . (*) and p< . (**). # denotes significant difference from dss/i.p saline treated mice with p< . . . panels e and f represent quantitatively the histological assessment of colitis severity and the % of ulceration in the whole colon section for each group as indicated. histobars represent means ± sem for - mice . - . mg/kg, this protective effect was lost. the histological score of colitis correlated well with the macroscopic score (as shown in fig e) ; a significant reduction in colitis severity was seen with ang - treatment only at the lower doses of . - . mg/kg. similarly, the % of ulceration (fig f) was significantly reduced ( - %) but again only with the lower dose of . - . mg/kg ang - . furthermore, daily i.p injections of ang - at . mg/kg dose significantly increased the plasma levels of ang - compared to mice receiving daily i.p saline at day post-colitis induction (fig g) . the histological features of the resected tissues are illustrated in fig . compared to the normal architecture in untreated (ut) controls, dss/i.p saline treatment for days resulted in significant mucosal destruction and infiltration by immune cells (fig b) . injection of dss treated animals with . - . mg/kg doses of ang - improved mucosal integrity and reduced the degree of immune cell recruitment to the colon (fig c and d ). this anti-inflammatory effect was lost with higher ang - doses (fig e, f and g). to determine whether ang - can reduce the severity of colitis once it is established, mice were given daily i.p injections of ang - (for days) days after dss administration (when effects are less severe). by days, at both doses of . or . mg/kg, ang - reversed the loss in body weight induced by dss (fig a) . in addition, the elevation in % circulating neutrophils seen with dss treatment was also reversed by ang - back to levels seen in the ut group ( fig b) . the small increase in colon thickness observed in the dss group was reversed by ang - though this effect did not reach statistical significance (fig d) . histological examination ( fig g- i and quantified in fig e and f ) showed that ang - could reverse some of the dss induced mucosal damage and ulceration, though in the case of the latter it did not reach statistical significance. at the macroscopic level, ang - was seen to reduce the dss induced increases in edema and erythema but not in adhesion ( table ) . we compared the effects of ang - with the classic anti-inflammatory agent dexamethasone (dex) using the same treatment approach. the dss induced loss in body weight was partly in each group ( for ut, for dss/i.p saline and for ang . mg/kg and ang . mg/kg). in panels a asterisks denote significant difference from ut mice with p< . (*) and p< . (**). # denotes significant difference from dss/ i.p saline treated mice with p< . . panel g shows colon section taken from a mouse treated with dss for days followed by days of i.p saline (vehicle) indicating the extent of mucosal destruction and immune cells recruitment. panels h and i show colon sections taken from dss mice subsequently treated with ang - . significant improvement in the mucosal integrity and reduction in immune cell recruitment can be seen. bars represent μm. reversed after days of dex treatment at . mg/kg dose ( fig a) . as observed with ang - , this effect was lost at higher doses. dex did not reverse the increase in neutrophils (data not shown). whereas there was no difference in the colon thickness, colon length was significantly increased by dex treatment at . - . mg/kg doses (but not higher) compared to dss/i.p saline group (fig b) . both histological score of colitis and extent of ulceration (fig d and e ) were reduced at low (but not higher) doses of dex. with respect to the macroscopic parameters, dex treatment specifically at . mg/kg dose significantly reduced diarrhea, blood in stool, ano-rectal bleeding and erythema, but did not have any effect on either the edema or adhesion ( table ) . to determine the effect of inhibiting the action of endogenous ang - , the mas- r antagonist a was administered (for days) with daily i.p injections at mg/kg dose to dss treated mice. dss/i.p saline group did not show weight reduction since dss treatment was given for days only ( fig a) . a treatment exacerbated the weight loss but did not affect the changes in lymphocytes or neutrophils induced by dss (fig a and b ). there was also no effect on colon length or thickness but histologically, there was significant additional deterioration of the mucosal structures and increased ulceration as compared to the dss/i.p saline group as shown by the examples in fig panels g and h and quantitatively in panels e and f. at the macroscopic level, a treatment resulted in reduction of the dss induced edema but worsened the erythema, with no effect on the other parameters (table ). prolonged administration of dss plus a to mice (for - days) resulted in death (data not shown), highlighting the intensified colitis severity as a result of prolonged inhibition of the endogenous levels of ang - . the level of phosphorylated forms of three key signaling intermediates, erk / (fig ) , p mapk (fig ) and akt (fig ) , were measured by immunofluorescence in sections from resected colon tissue of untreated mice or mice treated with dss (for days) plus daily ang - or saline (vehicle) treatment. in each case, expression was enhanced by dss and reduced by ang - back to the basal levels observed in the ut group. in the case of erk / , ang - at . mg/kg dose suppressed phosphorylation even below basal levels ( fig e) . . panels e and f represent histological assessment of colitis severity and the % of ulceration in the whole colon section respectively in the groups indicated. histobars represent means ± sem for - mice in each group ( for ut and for dss/i.p saline and dss/a ). asterisks denote significant difference from ut mice with p< . and # denotes significant difference from dss/ i.p saline treated group with p< . . panels g and h show representative colon sections from dss/i.p saline or dss/a treated mice. the expression level of ang ii was examined by immunofluorescence in sections from resected colon tissue of untreated mice (ut) or mice treated with dss (for days) plus daily ang - or saline (vehicle) treatment. ang ii expression was enhanced by dss administration (fig b) compared to ut mice ( fig a) . ang ii expression was reduced by daily administration of ang - at doses of . - . mg/kg (fig c- e ). in fact, daily administration of ang - at . mg/kg dose ( fig d) reduced ang ii to the basal levels observed in the ut group. daily administration of a high dose of ang - ( . mg/kg, fig f) resulted in increased ang ii levels similar to levels seen in dss/i.p saline group. in this study, we have presented data indicating a novel role of ang - in reducing colitis severity in the murine dss model. daily i.p injections (at doses of . - . mg/kg) ameliorated colitis severity when given either prophylactically or after colitis induction. blockade of the mas- r (by a ) aggravated colitis severity, highlighting the involvement of the ang - /mas- r axis in the pathogenesis of ibd. this protective effect was in part modulated through the expression/activity of several signaling molecules such as ang ii, p mapk, erk / , and akt. it has been demonstrated that ace is expressed in the epithelial, sub-mucosal and muscular layers of the colon in both humans and mice [ ] [ ] [ ] , and its expression levels are increased in the plasma of ibd patients [ ] . we observed increased expression of ace and mas- r proteins in the colonic tissues of mice after dss challenge (fig ) , and higher ang - levels in the inflamed colon homogenates (fig b) . plasma levels of ang - were significantly reduced at days post dss treatment ( . ± . ng/ml) when compared to healthy controls (ut; . ± . ng/ml). partial restoration was achieved with daily i.p injections of ang - at . mg/kg dose ( . ± . ng/ml; fig g) . the enhanced expression of the ace /ang - /mas- r axis post dss challenge might reflect a compensatory protective mechanism to reduce the effect of the inflammatory response. daily ang - administration significantly reduced colitis severity observable at both gross (table ) and histological levels (fig e and f ). in addition, it also opposed the dss-induced drop in body weight (fig a) , and restored the changes in colon length and thickness post colitis induction (fig c and d) . the protective effect of ang - was lost with higher doses in vivo (fig ) . one explanation is that higher concentrations of ang- - attenuate the mas- r responsiveness through receptor desensitization [ ] [ ] [ ] . another report also showed that ang - treated hek t cells at high concentrations redistributed the mas- r to intracellular vesicles and co-localized ang - with rab and the adaptor protein complex , suggesting regulation by a clathrin-mediated pathway [ ] . enhanced activity of members of the mapk family, p mapk and erk / as well as akt (the downstream target of pi k), which we observed in colonic sections post dss treatment (figs - ) has previously been reported in colonic biopsies from ibd patients [ ] [ ] [ ] [ ] . treatment with inhibitors of these signaling molecules ameliorated colitis severity in mice and reduced the disease activity index in patients, underlining their role in ibd pathogenesis [ ] [ ] [ ] [ ] . in this study, we showed that the anti-inflammatory effects of ang - treatment were associated with a reduction in the phosphorylated forms of p mapk, erk / and akt post dss induction (figs - ) , consistent with previous reports showing reduced activity of these molecules post ang - treatment in the anterior pituitary [ ] , proximal tubular cells [ ] , and lung tissues in an animal model of lung inflammation [ ] . using immunofluorescence analysis, we observed enhanced colonic expression of ang ii at day post-dss treatment (fig ) , confirming western blotting data presented in fig a. ang ii expression was reduced by daily ang - treatment (at doses of . - . mg/kg, fig c- e ). this suggest that the antiinflammatory properties of ang - are in part mediated through reduction of ang ii levels. regarding the influence of the ace /ang - /mas- r axis in modulating colitis severity, one report demonstrated that administration to mice of gl (as a chemical inhibitor of ace ) reduced colitis severity [ ] . however, it should be noted that the specificity of this inhibitor is questionable, since ace inhibition may increase the levels of other substrates such as ang i/ii, ghrelin, dynorphin, bradykinin, neurotensin and apelin [ ] . other studies (in the murine tnbs model) have demonstrated that ace deficiency results in increased likelihood of developing intestinal inflammation due to the disruption of dietary amino acid homeostasis, innate immunity and gut microbial ecology [ ] , as well as increased leukocyte recruitment and pro-inflammatory cytokine release in the colonic tissues. these data are consistent with a protective role of the ace /ang - /mas- r axis observed in this study. in regard to the effect of dex on modulating colitis severity, targeted delivery of dex to the colon was shown to reduce colitis severity in rodents [ ] [ ] [ ] . however, daily i.p administration of dex was shown to either increase [ ] , or decrease colitis severity [ , ] . in our study, we showed that daily i.p injection of dex at doses . - . mg/kg significantly reduced colitis severity at macroscopic (table ) and histological level (fig d- e) . in conclusion, this study is the first demonstration (to our knowledge) that the ang - / mas- r axis plays a role in modulating colitis severity, in part through down-regulation of the levels of ang ii and the phosphorylation of key signaling molecules involved in the inflammatory process such as erk / , p mapk and akt. blockade of the mas- r inhibited the protective effects of endogenous ang - and increased the severity of colitis. renin-angiotensin system revisited the renin-angiotensin system: an old, newly discovered player in immunoregulation the renin-angiotensin system: peptides and enzymes beyond angiotensin ii the mas oncogene encodes an angiotensin receptor angiotensin-( - 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) in the rat renal cortex signal transduction mechanisms involved in angiotensin-( - )-stimulated arachidonic acid release and prostanoid synthesis in rabbit aortic smooth muscle cells circulating rather than cardiac angiotensin-( - ) stimulates cardioprotection after myocardial infarction angiotensin ( - ) induces mas receptor internalization inhibition of stressactivated map kinases induces clinical improvement in moderate to severe crohn's disease. gastroenterology anti-inflammatory and anti-oxidative activities of paeonol and its metabolites through blocking mapk/erk/p signaling pathway. inflammation inhibition of phosphoinositide -kinase ameliorates dextran sodium sulfate-induced colitis in mice pi k/akt signaling pathway is involved in the pathogenesis of ulcerative colitis effects of the ace inhibitor gl on acute dextran sodium sulfate-induced colitis in mice hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase ace links amino acid malnutrition to microbial ecology and intestinal inflammation investigation of the colon-targeting, improvement on the side-effects and therapy on the experimental colitis in mouse of a resin microcapsule loading dexamethasone sodium phosphate immunomodulation of rheum tanguticum polysaccharide (rtp) on the immunosuppressive effects of dexamethasone (dex) on the treatment of colitis in rats induced by , , -trinitrobenzene sulfonic acid dexamethasone -sulfate improves the therapeutic properties of dexamethasone against experimental rat colitis by specifically delivering the steroid to the large intestine the effect of dexamethasone treatment on murine colitis bioluminescence imaging for il- beta expression in experimental colitis expression of na-h exchanger- isoform is suppressed in experimental colitis in adult rat: lack of reversibility by dexamethasone this work was supported by kuwait university research sector grant pt / . parts of this work were supported by grant srul / to the research unit for genomics, proteomics and cellomics studies (omics), kuwait university. key: cord- - llgr authors: chappell, mark c. title: role of ace, ace and neprilysin in the kidney date: journal: frontiers in research of the renin-angiotensin system on human disease doi: . / - - - - _ sha: doc_id: cord_uid: llgr nan from the initial description of renin activity over a century ago, ongoing study of the renin-angiotensin-aldosterone system (raas) continues to yield novel findings that redefine the functional nature of this system, as well as reveal the complexity of the interplay among the various raas components. indeed, the recent discoveries of angiotensin converting enzyme (ace ) donoghue et al ; tipnis et al ) ; the renin receptor (nguyen et al ) and the angiotensin-( - ) [ang-( - )] receptor (santos et al ) represent important examples of our evolving concepts of the raas and cardiovascular regulation. coupled with the emerging view that the raas is not defined as simply an endocrine system, these local or tissue systems may exhibit distinct functional and processing pathways (chappell et al ; chappell et al ; paul et al ) . the kidney is clearly an important target organ of the circulating raas, particularly the actions of ang ii and aldosterone to promote sodium and water reabsorption, as well as their influence on the progression of tissue injury and fibrosis (harris ) . the kidney also exhibits a local raas that expresses ang ii, ang-( - ), and multiple ang receptor subtypes that mediate the distinct actions of these two peptides in both normal and pathophysiolgocial conditions such as hypertension or diabetes (burns ; carey & siragy ; navar et al ) . figure illustrates one current view of the renal raas network that emphasizes the distinct synthetic pathways of ang ii and ang-( - ), as well as functional actions mediated by the at , at and at − receptors. although the emergence of receptor subtypes distinguishes the distinct signaling pathways of ang ii and ang-( - ), the post-renin enzymes that form and degrade these peptides must be considered in lieu of the overall regulation of the functional raas within the kidney. the inclusion of ang-( - ) as a potential intermediate in ang ii formation via a renin-independent pathway reflects the recent figure . scheme that depicts the processing pathways involved in the formation and degradation of angiotensin ii (ang ii) and ang-( - ) within the kidney. ang ii binds to either at or at receptor (r) subtypes, while ang-( - ) recognizes an at − r. ace, angiotensin converting enzyme; eps, endopeptidases; nep, neprilysin demonstration of endogenous levels of the peptide in the kidney, circulation and other tissues (nagata et al ) . in this chapter, the roles of ace , ace and the endopeptidase neprilysin in the functional expression of the intrarenal hormones of the raas are reviewed. ace may be considered the activation step in the catalytic cascade for the formation of ang ii from ang i (fig. ) . although evidence of non-ace pathways for biosynthesis of ang ii is evident (sadjadi et al a; tokuyama et al ) , ace likely represents the major, if not sole enzyme responsible for ang ii formation under normal physiological conditions in humans and other species. this is not to imply that ace has no other substrates than ang i (see below), but that a primary role for ace is the generation of ang ii. indeed, the identification of ace and the characterization of the enzymatic properties must be considered a pivotal achievement in our understanding of the raas and cardiovascular disease, as well as leading to the successful development of ace inhibitors in the treatment for hypertension and renal disease. ace is a metallopeptidase composed of a single monomeric protein. somatic ace contains two catalytic regions designated as the amino (n) and carboxy (c) domains. selective inhibitors against both catalytic domains of somatic ace are now available, however, the functional significance of the two domains is presently unknown (dive et al ; georgiadis et al ) . the enzyme cleaves two residues from the carboxy end of various peptides and, hence, its description as a dipeptidyl-carboxypepitdase. within the kidney, somatic ace is primarily a glycosylphosphatidylinositol-anchored membrane protein and the majority of the enzyme including both catalytic regions faces the extracellular space. ace is localized throughout the kidney with high concentrations in vascular endothelial cells, proximal tubules and interstitial cells. ace is also released from the apical surface of epithelial cells into the proximal tubular fluid and likely contributes to the urinary levels of the enzyme (hattori et al ) . indeed, the tubular fluid should be considered a distinct intrarenal compartment that contains raas processing enzymes and the peptide products may interact with ang receptors along the entire tubular area of the kidney. the release of ace from the cell membrane is a specific process as releasing enzymes or "sheddases" have been identified that recognize a unique motif on the stalk region of the enzyme (beldent et al ) . the conversion of membrane-bound ace to a soluble form does not appear to substantially alter the substrate preference or the catalytic properties of the enzyme. although the significance of this event is not currently understood, enzyme shedding may underlie an endocrine process to transport ace to more distal areas of the nephron that are deficient in this peptidase activity for the discrete production of ang ii. in this regard, casarini and colleagues have presented intriguing data that the urinary excretion of the n-terminal domain of ace may serve as a urinary marker in both humans and experimental hypertensive models (marques et al ) . extensive evidence suggests that intrarenal ace participates in the direct formation of ang ii from ang i. the renal administration of ace inhibitors reduces interstitial levels of ang ii and attenuates blood pressure. moreover, in an animal model of tissue-depleted ace that preserves circulating levels of the enzyme, renal ang ii is significantly reduced (modrall et al ) . interestingly, intrarenal levels of ang i were also markedly reduced in the tissue ace null mouse while renal ang-( - ) concentrations were maintained (modrall et al ) . these data serve to emphasize that ace participates in the metabolism of other peptide hormones (skidgel & erdos ) . in the case of ang-( - ), ace efficiently metabolizes the peptide to ang-( - ), a product which is presently not known to exhibit functional activity deddish et al ; rice et al ) . we have postulated that the formation of ang-( - ), particularly under prolonged activation of the raas, is considered to balance or attenuate the constrictor and proliferative actions of ang ii ferrario et al c) . indeed, ang-( - ) exhibits vasodilatory, natriuretic and anti-proliferative actions through the stimulation of nitric oxide and arachidonic acid metabolites (sampaio et al ) . ang-( - ) abrogates the ang ii-dependent activation of map kinase in primary cultures of proximal tubule epithelial cells (su et al ) . moreover, the inhibitory actions of ang-( - ) were blocked by the ang-( - ) antagonist [d-ala ]-ang-( - ) suggesting a receptor mediated pathway distinct from either at or at receptor subtypes (su et al ) . similar effects of ang-( - ) were originally demonstrated in non-renal cells (tallant et al a) . in the circulation, ace inhibitors increase circulating levels of ang-( - ) and augment the in vivo half life of the peptide by almost fold yamada et al ) . the urinary excretion of ang-( - ) increases in both human and experimental hypertensive models following acute administration of ace inhibitors yamada et al ) . the increased excretion of ang-( - ) most likely reflects the reduced intrarenal metabolism of the peptide and the efficient shunting of the ang i pathway to formation of ang-( - ). our recent studies in isolated sheep proximal tubules reveal that without prior inhibition of ace, ang-( - ) derived from either ang i or ang ii was rapidly converted to ang-( - ) (shaltout et al ) . blockade of ang-( - ) partially reverses the beneficial actions of ace inhibitors on blood pressure in hypertensive rats as an ang-( - ) monoclonal antibody or the [d-ala ]-ang-( - ) antagonist increase blood pressure (iyer et al ; iyer et al ) . moreover, studies by benter and colleagues find that the renoprotective effects of exogenous ang-( - ) in lname-treated shr were not further improved with the ace inhibitor captopril (benter et al a) . apart from ang ii and ang-( - ), renal ace may also participate in the metabolism of other peptides including kinins, substance p and the hematopoietic fragment acetyl-ser-asp-lys-pro (ac-sdkp). bradykinin-( - ) is very rapidly metabolized by ace in a two -step process to the inactive fragments bradykinin-( - ) and bradykinin-( - ). ace inhibition is associated with increased circulating and tissue levels of bradykinin-( - ) and the renal content of kinin is higher in the tissue ace null mouse (campbell et al ) . in general, bradykinin is a potent vasodilator and inhibitor of cell growth through stimulation of nitric oxide, as well as exhibiting natriuretic actions within the kidney (scicli & carretero ) . interestingly, santos and colleagues have reported that the functional activity of ang-( - ), under certain conditions, is dependent on the increased release of bradykinin (fernandes et al ) . moreover, the kinin b receptor antagonist hoe blocked nitric oxide release by the non-peptide ang-( - ) agonist ave (wiemer et al ) . similar to ang-( - ), circulating levels of the ac-sdkp are markedly increased with ace inhibition and the enzyme cleaves the lys-pro bond of the tetrapeptide (azizi et al ; raousseau et al ) . although current evidence does not support a role for ac-sdkp in the regulation of blood pressure, the peptide does exhibit potent anti-fibrotic and anti-inflammatory actions (peng et al ) . indeed, exogenous administration of ac-sdkp attenuates proteinuria and improves renal function in several models of renal injury and hypertension (omata et al ) . interestingly, ang-( - ) and ac-sdkp may be the only known endogenous substrates that are exclusively cleaved by the n-terminal catalytic domain of human ace (raousseau et al ; deddish et al ) . moreover, prolyl (oligo)endopeptidase, an enzyme that processes ang i or ang ii to ang-( - ) in endothelial and neural cells (chappell et al ; santos et al ) , may also convert thymosin- to ac-sdkp in plasma and tissue (cavasin et al ) . the unusual specificity of the n-domain of ace for ang-( - ) and ac-sdkp suggests an overlap of the activities of these two peptide systems within the kidney as well. although elucidation of the signaling mechanisms and receptors for ang-( - ) and ac-sdkp is at an early stage, future studies should consider whether there is a basis for the functional similarities between these peptides. the role of raas enzymes including ace and renin has been primarily emphasized for their catalytic properties; however, compelling evidence now reveals receptor-like properties for these two enzymes. indeed, a renin receptor was recently cloned by nyguen and colleagues with significant concentrations of the protein in the glomerulus and vascular smooth muscle cells. (diez-freire et al ; nguyen et al ) . receptor-bound renin exhibits increase proteolyitc activity for ang i formation, but both pro-renin and renin also induce distinct signaling pathways following binding. in isolated mesangial cells, exogenous renin increased tgf-expression and other matrix proteins including plasminogen activator inhibitor (pai- ) and fibronectin that was apparently independent of ang ii synthesis (huang et al ) . ace inhibitors may also induce cell-specific signaling by inducing conformational changes in membrane-bound ace without alterations in ang ii or other peptides (benzing et al ) . two kinases, c-jun kinase and map kinase kinase associate with the intracellular portion of ace. moreover, ace inhibitors increase the phosphorylation and nuclear trafficking of phosphorylated cjun kinase (kohlstedt et al ) . this aspect of ace-dependent activation of various kinases has been demonstrated in human endothelial cells and the question remains as to what extent this occurs in other cells or tissues. in addition, ace inhibitors or the angiotensin peptides ang-( - ) and ang-( - ) induce the association of ace and the bradykinin b receptor that prevents the rapid down-regulation of the ligand-receptor complex, thus potentiating the actions of bradykinin (burckle et al ; chen et al ) . almost years following the discovery of ace, a new homolog of the enzyme termed ace was identified by two separate groups (donoghue et al ; tipnis et al ) . ace activity is not attenuated by ace inhibitors nor does the enzyme share the same catalytic properties. in this regard, ace contains a single zincdependent catalytic site that corresponds to the c-terminal domain of somatic ace. ace exhibits carboxypeptidase activity cleaving a single amino acid residue at the carboxyl terminus of various peptides. the original studies assessed ang i as the peptide substrate for ace , given the similar homology to ace and the existing evidence for ace-independent pathways; however, ace converted ang i to the nonapeptide ang-( - ) (donoghue et al ) . this product is currently not known to exhibit functional activity, but may serve as a substrate for the further processing to ang ii or ang-( - ) . the subsequent kinetic studies of over peptides found that the conversion of ang ii to ang-( - ) was much preferred over that for ang i (vickers et al ) . indeed, ace exhibits an approximate -fold greater kcat/km for ang ii versus ang i and has the highest efficiency among the known ang-( - )-forming enzymes (fig. ) . these studies also revealed that other peptides including apelin and dynorphin are cleaved by ace at a similar or slightly greater efficiency than ang ii (vickers et al ) . at this time, the majority of studies have focused on the role of ace in the metabolism of angiotensins (see below), principally ang ii to ang-( - ), and the role of ace in the processing of apelin or other peptides has not been sufficiently addressed. similar to ace, ace exists in both soluble and membrane-associated forms with high expression in the kidney, heart, brain, lung and testes (harmer et al ) . although there is significant circulating ace activity in various species, plasma levels of ace are quite low, but may vary among species (elased et al ; rice et al ) . recent studies in the sheep reveal appreciable ace in the plasma, albeit the activity was significantly lower than that for ace (fig. , inset) (shaltout et al ) . for this assessment, we compared the enzyme activities using the endogenous substrates for both ace and ace (ang i and ang ii, respectively) data are n = - , mean ± sem;* p < . vs. control at equimolar concentrations under identical incubation conditions. interestingly, as shown in fig. , male sheep exhibited higher ace activity than females that likely contributes to the lower half-life (t / ) of serum ang ii in males . addition of the specific ace inhibitor abolished the conversion of ang ii to ang-( - ) as measured by a hplc- i-detector and markedly increased the ang ii-[t / ] in both male ( fold) and female ( fold) sheep (fig. ) . these ex vivo data in sheep serum demonstrate that circulating ace constitutes a major pathway in the metabolism of ang ii and support the increase in circulating ang ii levels in the ace null mouse . furthermore, we did not find that soluble ace in the serum contributed to the direct conversion of ang i to ang-( - ) even in the presence of complete ace inhibition (shaltout et al ) . within the kidney, ace is primarily localized to the apical aspect of the proximal tubule epithelium. indeed, expression of ace in the renal mdck cell line revealed exclusive trafficking of the enzyme to the apical side, while the distribution of expressed ace was different, trafficking to the basolateral and luminal aspects of the cell (guy et al ) . consistent with the apical expression of ace in the renal epithelium, we found significant urinary ace activity that converted ang ii to ang-( - ), but did not process ang i to ang-( - ) (shaltout et al ) . the glycosylated form of ace is approximately , daltons and the filtration of the enzyme into the tubular fluid is highly unlikely (shaltout et al ) . in this regard, lambert and colleagues report that the metallopeptidase adam may function as a secretase to release ace from extracellular side of the cell membrane . interestingly, adam does not release ace suggesting that the regulation for the secretion for ace and ace is distinct. the localization of ace in the proximal tubule epithelium along with other elements of the ras (ace, angiotensinogen, ang receptors) supports a role for the enzyme in the processing of angiotensin peptides. in the rat kidney, burns and colleagues found no evidence that ace or other peptidases metabolize ang ii in proximal tubule preparations or in perfused proximal tubule segments isolated from male sprague dawley rats . however, ace activity was clearly evident in the rat tubules as the conversion of exogenous ang i to ang-( - ) was sensitive to the ace peptide inhibitor dx- . ang-( - ) was subsequently converted to ang-( - ) by ace, a pathway similar to that reported for ang i metabolism in isolated cardiomyocytes (donoghue et al ) . in contrast to the rat, we found that ace was the predominant activity to convert ang ii to ang-( - ) in sheep proximal tubules (shaltout et al ) . the addition of the nonpeptide ace inhibitor mln- significantly attenuated the metabolism of ang ii at early time points. however, as shown in fig. , the significant ace and neprilysin activities required prior inhibition to protect ang-( - ) from rapid degradation in the proximal tubules. we could not demonstrate that ace participated in the direct metabolism of ang i, particularly under conditions where other enzymatic pathways were blocked (shaltout et al ) . indeed, ang i was directly converted to ang ii and ang-( - ) via ace and neprilysin, respectively. the preferred conversion of ang ii to ang-( - ) by ace in the sheep kidney is entirely consistent with kinetic studies on various peptide substrates by the human enzyme (rice et al ; vickers et al ) , as well studies in membrane fractions of mouse kidney and rat renal cortex that demonstrated ace -dependent conversion of ang ii to ang-( - ) ( elased et al ; ferrario et al b) . an explanation for the discrepancy in the metabolism studies for angiotensin metabolism is not readily apparent; however, if the rat exhibits different kinetic properties for ang i and ang ii than sheep or human, then the role of ace is likely to be quite different among species. additionally, these studies have important figure . ace inhibition blocks the conversion of ang ii to ang-( - ) in isolated proximal tubules from female sheep. sequential addition of peptidase inhibitors on the formation of ang ii metabolites include: ap, amniopeptidase (amastatin, bestatin); chy, chymase, carboxypeptidase a (chymostatin, benzyl succinate); cy, cysteine protease (pcmb); nep, neprilysin (sch ), ace, angiotensin converting enzyme (lisinopril); ace (mln- ). data are n = , mean ± sem implications on the role of ace as well, particularly whether ace is involved in the formation or degradation of ang-( - ) chappell et al ; yamada et al ) . although campbell and colleague demonstrate significant quantities of endogenous ang-( - ) in the rat kidney (campbell et al ) , chronic ace inhibition or combined ace/at blockade (chappell, unpublished observations) did not attenuate renal ang-( - ) levels in the rat. in addition, ang-( - ) levels within the kidney were maintained in tissue ace knockout mice (modrall et al ) . thus, these in vivo studies do not strongly support an ace -ace cascade leading to the formation of ang-( - ) and ang-( - ) in the kidney. the molecular studies utilizing ace knockout mice provide additional evidence for the enzyme's role to balance the expression of ang ii and ang-( - ). we originally showed that ace null mice exhibit higher circulating and tissue levels of ang ii . indeed, the increased ratio of renal ang ii to ang-( - ) may contribute to the renal pathologies observed in older ace null mice (oudit et al ) . furthermore, the incidence of glomerulosclerosis and proteinuria in the male mice was markedly attenuated by at receptor blockade. several hypertensive models including the spontaneously hypertensive rat (shr), stroke-prone shr and sabra salt sensitive rat exhibit lower mrna levels and protein expression for ace in the kidney than normotensive controls zhong et al ) , as well as human prehypertensives (keidar et al ) . tikellis and colleagues find that renal ace expression is actually higher in the shr than wky normotensive controls at day one following birth, similar at days and then dramatically declines in adult shr by days (tikellis et al ) . ace activity, however, was markedly lower in the shr kidney at all time points measured and declined in both strains at days. apart from the interesting pattern of development for ace in the kidney, these data emphasize the need to at least consider alterations in both ace and ace in characterizing the functional output of the raas. moreover, parallel studies to document the changes in renal ang ii and ang-( - ) during this developmental period are critical to establish the relevance to altered ace and ace . it is clear that not all hypertensive models exhibit reduced ace in the kidney. our studies in the male mren .lewis rat, a model of tissue renin expression with increased renal ang ii, found no difference in renal cortical ace activity as compared to the normotensive lewis strain, although cardiac activity was indeed lower in the hypertensive rats ferrario et al b; pendergrass et al ) . chronic blockade with either an ace inhibitor or at antagonist increased ace activity in the kidneys of both the mren .lewis and lewis rats, but enzyme activity was significantly higher in the normotensive strain following treatment . this may reflect the situation where raas blockade does not completely reverse the extent of renal injury in the male mren .lewis model. in this regard, the reduced ace and elevated renal ang ii in the injured kidney of albumin-loaded rats was associated with increased nf-b expression (takase et al ) . in contrast, ace and its product ang-( - ) increase in the kidney of the rat during pregnancy . it is well known that the raas is activated during pregnancy, yet blood pressure is not altered in normal pregnancy, and it will be of interest to determine whether ace expression within the kidney is altered with pre-eclampsia. diabetic nephropathy is clearly dependent on an activated raas and both ace inhibitors and at receptor antagonists are effective in attenuating the progression of injury. indeed, the renal expression of ace is reduced in the proximal tubules of the streptozotocin-induced model of type i diabetes (tikellis et al ; wysocki et al ) . moreover, the attenuation of renal injury in this model by ace inhibition is associated with increased ace expression. a protective role for renal ace is also evident from the findings that chronic ace inhibition in the diabetic db/db mice exacerbates the extent of albuminuria almost -fold . although angiotensin content was not measured, the db/db mice exhibited increased glomerular expression of ace and reduced ace as compared to the control db/dm mice. interestingly, the localization studies revealed distinct patterns of staining for ace and ace within the glomerulus -ace in podocytes and ace in the endothelial cells (liebau et al ) . ang-( - ) or the nonpeptide agonist ave attenuates proteinuria and improves renal vascular activity in the streptozoticin type diabetic rat, but did not reverse the urinary excretion of lysozyme, a marker of tubulointerstitial damage (benter et al ) . moreover, the ratio of ang-( - ) to ang ii formed from exogenous ang i was lower in glomeruli isolated from the kidneys of diabetic rats, however, the identity of the ang-( - )-forming activity was not determined in this study (singh et al ) . thus, in addition to the proximal tubule epithelium, the glomerulus may be a second key site within the kidney where ace may influence the local expression of angiotensin peptides and renal function. in the kidney, the endopeptidase neprilysin constitutes significant peptidase activity, particularly within the brush border region of the proximal tubules. similar to ace and ace , neprilysin is a zinc-dependent metallopeptidase that is anchored to the apical or extracellular region of the membrane, but is apparently resistant to enzymatic shedding. although neprilysin was initially recognized for its enkephalindegrading activity and frequently referred to as "enkephalinase", studies now reveal that this enzyme contributes to the metabolism of various peptides with cardiovascular actions including adrenomedullin, angiotensins, kinins, endothelins, substance p and the natriuretic peptides (skidgel & erdos ) . indeed, the development of neprilysin inhibitors, and more recently, dual or mixed inhibitors that target ace as well remain potential therapies in cardiovascular disease (veelken & schmieder ) . in general, these dual inhibitors were either equally or more effective in lowering blood pressure and reducing renal injury as compared to monotherapy with an ace or neprilysin inhibitor (kubota et al ; tikkanen et al ) . however, two large clinical trials (octave, overture) with the mixed inhibitor omapatrilat revealed an increased incidence of angioedema. moreover, the drug was no more effective than an ace inhibitor alone (kostis et al ; packer et al ) . a subsequent experimental study has shown that omapatrilat inhibits amniopeptidase p and, although less potent than its actions against ace and neprilysin, this may further augment the local concentrations of kinins or substance p to exacerbate vascular permeability (sulpizio et al ) . in this aspect, the development of more selective inhibitors against ace and neprilysin may be of clinical benefit. the rationale for neprilysin inhibition primarily resides in preserving the "cardioprotective" peptides bradykinin and anp or bnp. however, neprilysin readily metabolizes ang ii to the inactive fragment ang-( - ) which undergoes further hydrolysis to the dipeptides asp-arg and val-tyr. neprilysin also cleaves endothelin, although it is not clear to what extent reduced intrarenal levels of endothelin are beneficial given the functional diversity of the endothelin a and b receptor subtypes within the kidney (schiffrin ) . the additional ace inhibition would prevent the accumulation of ang ii and further contribute to the protection of kinins, as well as possibly reduce endothelin release. one possible caveat to this approach is that neprilysin is the major ang-( - )-forming activity from ang i or ang-( - ) in the circulation (campbell et al ; yamamoto et al ) . indeed, acute administration of the potent neprilysin inhibitor sch reduced circulating levels of ang-( - ) and increased blood pressure in the shr chronically treated with the ace inhibitor lisinoropril (iyer et al ) . although plasma levels of neprilysin are low to non-detectable, the enzyme is appropriately localized to the ectocellular surface of endothelial and smooth muscle cells to contribute to the formation of ang-( - ) within the vasculature (llorens-cortes et al ) . in the kidney, neprilysin may contribute to both the formation as well as the degradation of the ang-( - ) (allred et al ) . neprilysin cleaves the pro -phe bond of ang i to ang-( - ), but the very high levels of the enzyme in the kidney may continue to metabolize ang-( - ) at the tyr -ile bond to form ang-( - ) and ang-( - ) (allred et al ; chappell et al ) . indeed, the mixed inhibitor omapatrilat augmented the urinary levels of ang-( - ) in both human hypertensives and the shr model (ferrario et al a; ferrario et al b) . the clinical study revealed a strong correlation between the reduction in blood pressure and increased excretion of ang-( - ) with the dual peptidase inhibitor (ferrario et al a) . interestingly, chronic treatment of male shr with omapatrilat ( weeks, mg/kg daily) was also associated with the increased renal expression of ace . as shown in fig. , immunocytochemical studies demonstrate enhanced expression figure . increased expression of ang-( - ) and ace in the renal cortex of shr following treatment with omapatrilat. immunocytochemical staining for ang-( - ) in control (a) and treated (b) shr; ace staining in control (c) and treated (d), group. ace staining in renal artery of treated shr; arrow indicates intimal layer (e). renal cortical ace mrna levels are significantly increased -fold following omapatrilat treatment (f); inset: ace and ef- bands in the presence of the specific rt primers (rt+). data are n = - , mean ± sem of both ace and ang-( - ) within the renal cortex of the treated-shr . omapatrilat treatment also revealed the renal vascular expression of ace with staining evident in the intimal, medial and adventitial regions of the renal artery (fig. e ); vascular staining for the enzyme was undetectable in the untreated shr group (fig. c ). cortical mrna of ace expressed as a ratio to ef- increased -fold suggesting that transcriptional regulation contributes to the enhanced expression of ace within the kidney (fig. f ). these studies are of interest as they reveal an additional mechanism of the vasopeptidase inhibitor that may result in the enhanced conversion of ang ii to ang-( - ) by ace , as well as protecting ang-( - ) from both neprilysin-and ace-dependent degradation within in the kidney. furthermore, these data suggest an important ability of the dual peptidase inhibitor (as well as the administration of other raas inhibitors alone) to restore ace levels in the hypertensive kidney which may mitigate against the ang ii-at receptor axis of the raas. indeed, raizada and colleagues show that lenti-viral expression of ace has amelioratory effects on blood pressure and cardiac fibrosis in the shr, although the renal effects of enhanced enzyme activity were not ascertained (diez-freire et al ) . their data clearly demonstrate that ace can markedly alter the balance of an activated raas pathway towards a normotensive phenotype. further study is required to determine the extent that the beneficial actions of increased ace reflect the greater inhibition of ang ii or the increased accumulation of ang-( - ) in the kidney or other tissue. the positive influence of ace in the shr kidney following blockade of ace and neprilysin emphasizes the complex regulation of raas components within the kidney (see fig. ). we have also shown that ace inhibition alone or at receptor antagonism increases either renal ace mrna or activity (igase et al ; ferrario et al b) . consistent with these data in the intact animal, gallagher figure . a potential regulatory scheme for the stimulatory and inhibitory pathways of the reninangiotensin-aldosterone system within the kidney. ace, angiotensin converting enzymes; aogen, angiotensinogen; aldo, aldosterone; at r, angiotensin type receptor; jg, juxtaglomerular and colleagues demonstrate that ang ii directly down regulates ace through activation of the at receptor . although ang-( - ) alone did not influence the basal expression of ace in these cells, the peptide attenuated the inhibitory effects of ang ii on ace via a receptor-dependent mechanism . in contrast to the negative influence on ace , ang ii increases ace expression within the kidney sadjadi et al b) . ang ii also positively influences the expression of its precursor protein angiotensinogen (kobori et al ; zhang et al ) , and in selective areas of the kidney, either maintains or up regulates the at receptor as well . this effect on the at receptor may also lead to increased renal levels of ang ii via receptor mediated uptake and stable sequestration of the circulating peptide (ingert et al ) . in contrast, ang-( - ) can down regulate the at receptor through stimulation of a cylocoxygenase pathway (clark et al ; clark et al ) . there are few studies on the regulation of the ang-( - ) receptor, although chronic ace or at blockade reduced mas mrna expression in the renal cortex of the ren lewis congenic rat . consistent with the positive feedback concept, the current evidence suggests that aldosterone down regulates ace (keidar et al ; tallant et al b) while the mineralocorticoid increases expression of ace, at receptor, renin and intrarenal ang ii (bayorh et al ; klar et al ; schiffrin ) . thus, the renal raas appears to function in a positive regulatory manner on these components to promote or maintain ang ii content. in this regard, ace may serve as an important mechanism to break or reduce the positive gain of the system for ang ii production or enhanced signaling in the kidney. although ang ii potently reduces juxtaglomerular (jg)derived release and expression of renin, renin is not suppressed but increases in the collecting duct and distal tubules (prieto-carrasquero et al ) . the negative feedback by ang ii on jg renin may also be balanced by renin-independent pathways that contribute to the formation of ang i. alternatively, nagata and colleagues (nagata et al ) find significant concentrations of the novel peptide ang-( - ) in the kidney and other tissues that may not require renin for the peptide's synthesis (see fig. ). the infusion of ang-( - ) produced an immediate increase in blood pressure that was abolished by either an ace inhibitor or an at receptor antagonist (nagata et al ) . these data suggest that following formation of ang-( - ), the peptide or its intermediate is converted to ang ii by ace. the elucidation of the enzyme(s) responsible for the formation of ang-( - ) and the factors that influence its expression may greatly contribute to our understanding the regulation of the intrarenal raas, particularly under pathophysiological conditions. the majority of experimental studies on the raas and the regulation of blood pressure have utilized male animals. as there is overwhelming evidence for sex differences in the extent of hypertension and cardiovascular injury, the consideration of gender in the regulation of the renal raas enzyme cascade should be carefully considered (bachmann et al ; brosnihan et al ; reckelhoff et al ) . for example, the presence of renal damage in the male ace knockout mice was not evident in the estrogen replete female littermates and possibly there is greater expression of raas components in the males with the loss of ace (oudit et al ) . we and others have shown that estrogen depletion is associated with altered expression of renin, at receptors, ace and nos isoforms, as well as exacerbates hypertension and salt-sensitive renal injury (bayorh et al ; brosnihan et al ; chappell et al ; chappell et al ; harrison-bernard et al ; roesch et al ; yamaleyeva et al ) . moreover, in lieu of the negative outcomes for estrogen or combined hormone replacement in older women (hers, whi), the influence of aging on the response of the intrarenal raas and other systems may be of equal importance. clearly, understanding the regulation and interplay of ace, ace and neprilysin within the kidney, as well as other areas including the heart, brain and vascular beds are critical to treating the burgeoning problem of cardiovascular disease. - ) metabolism in pulmonary and renal tissues high plasma level of n-acetyl-seryl-aspartyl-lysyl-proline: a new marker of chronic angiotensin-converting enzyme inhibition sexual dimorphism of blood pressure: possible role of the renin-angiotensin system effects of enalapril, tempol, and eplerenone on salt-induced hypertension in dahl salt-sensitive rats the role of gender in salt-induced hypertension proteolytic release of human angiotensinconverting enzyme: localization of the cleavage site 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responses to renin n-acetyl-seryl-aspartyl-lysyl-proline ameliorates the progression of renal dysfunction and fibrosis and wky rats with established anti-glomerular basement membrane nephritis loss of angiotensin-converting enzyme- leads to the late development of angiotensin ii-dependent glomerulosclerosis comparison of omapatrilat and enalapril in patients with chronic heart failure. the omapatrilat versus enalapril randomized trial of utility in reducing events (overture) physiology of local renin-angiotensin systems differential expression of nuclear at receptors and angiotensin ii within the kidney of the male congenic mren .lewis rat ac-sdkp reverses cardiac fibrosis in rats with renovascular hypertension enhancement of collecting duct renin in angiotensin ii-dependent hypertensive rats the hemoregulatory peptide n-acetyl-ser-asp-lys-pro is a natural and specific substrate of the n-terminal active site of human angiotensin-converting enzyme gender differences in development of hypertension in spontaneously hypertensive rats: role of the renin-angiotensin system circulating activities of angiotensin-converting enzyme, its homolog, angiotensin-converting enzyme , and neprilysin in a family study evaluation of angiotensin-converting enzyme (ace), its homologue ace and neprilysin in angiotensin peptide metabolism estradiol attenuates angiotensininduced aldosterone secretion in ovariectomized rats angiotensin converting enzyme-independent angiotensin ii production by chymase is up-regulated in the ischemic kidney in renovascular hypertension angiotensin ii exerts positive feedback on the intrarenal renin-angiotensin system by an angiotensin converting enzyme-dependent mechanism angiotensin-( - ) through receptor mas mediates endothelial nitric oxide synthase activation via akt-dependent pathways angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas production of angiotensin-( - ) by human vascular endothelium role of endothelin- in hypertension effects of aldosterone on the vasculature renal kallikrein-kinin system angiotensin metabolism in renal proximal tubules, urine and serum of sheep: evidence for ace -dependent processing of angiotensin ii a novel mechanism for angiotensin ii formation in streptozoticindiabetic rat glomeruli angiotensin converting enzyme (ace) and neprilysin hydrolyze neuropeptides: a brief history, the beginning and follow-ups to early studies angiotensin-( - ) inhibits angiotensin ii-stimulated phosphorylation of map kinases in proximal tubular cells mechanism of vasopeptidase inhibitor-induced plasma extravasation: comparison of omapatrilat and the novel neutral endopeptidase . /angiotensin-converting enzyme inhibitor gw nf-kappab-dependent increase in intrarenal angiotensin ii induced by proteinuria angiotensin-( - ) inhibits growth of cardiac myocytes through activation of the mas receptor differential regulation of cardiac angiotensin converting enzyme (ace ) and ace by aldosterone characterization of renal angiotensin-converting enzyme in diabetic nephropathy developmental expression of ace in the shr kidney: a role in hypertension? combined inhibition of neutral endopeptidase with angiotensin converting enzyme or endothelin converting enzyme in experimental diabetes a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase differential regulation of elevated renal angiotensin ii in chronic renal ischemia neutral endopeptidase inhibition: the potential of the new therapeutic approach in cardiovascular disease evolves hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase a comparison of the properties, and enzymatic activity of three angiotensin processing enzymes: angiotensin converting enzyme, prolyl endopeptidase and neutral endopeptidase . distinct gender differences in the ace -dependent metabolism of angiotensin ii in the serum of sheep ave , a nonpeptide mimic of the effects of angiotensin-( - ) on the endothelium ace and ace activity in diabetic mice converting enzyme determines the plasma clearance of angiotensin-( - ) differential response of angiotensin peptides in the urine of hypertensive animals discoordinate regulation of renal nitric oxide synthase isoforms in ovariectomized mren .lewis rats in vivo metabolism of angiotensin i by neutral endopeptidase (ec . . . ) in spontaneously hypertensive rats glomerular localization and expression of angiotensin-converting enzyme and angiotensin-converting enzyme: implications for albuminemia in diabetes essential role(s) of the intrarenal renin-angiotensin system in transforming growth factor-beta gene expression and induction of hypertrophy of rat kidney proximal tubular cells in high glucose upregulation of angiotensinconverting enzyme by all-trans retinoic acid in spontaneously hypertensive rats these studies were supported in part by grants from the national institute of health grants (hl- and hl- ) and the american heart association (aha- and aha- ). an unrestricted grant from the unifi corporation (greensboro, nc) and the farley-hudson foundation (jacksonville, nc) is also acknowledged. key: cord- -q m srvp authors: nakagawa, pablo; gomez, javier; grobe, justin l.; sigmund, curt d. title: the renin-angiotensin system in the central nervous system and its role in blood pressure regulation date: - - journal: curr hypertens rep doi: . /s - - - sha: doc_id: cord_uid: q m srvp purpose of the review: the main goal of this article is to discuss how the development of state-of-the-art technology has made it possible to address fundamental questions related to how the renin-angiotensin system (ras) operates within the brain from the neurophysiological and molecular perspective. recent findings: the existence of the brain ras remains surprisingly controversial. new sensitive in situ hybridization techniques and novel transgenic animals expressing reporter genes have provided pivotal information of the expression of ras genes within the brain. we discuss studies using genetically engineered animals combined with targeted viral microinjections to study molecular mechanisms implicated in the regulation of the brain ras. we also discuss novel drugs targeting the brain ras that have shown promising results in clinical studies and trials. summary: over the last years, several new physiological roles of the brain ras have been identified. in the coming years, efforts to incorporate cutting-edge technologies such as optogenetics, chemogenetics, and single-cell rna sequencing will lead to dramatic advances in our full understanding of how the brain ras operates at molecular and neurophysiological levels. the physiological relevance of the renin angiotensin system (ras) in blood pressure regulation and electrolyte homeostasis is well established and undisputable. the ras is traditionally described as a hormone system, which promotes arterial blood pressure elevation primarily by inducing vasoconstriction, sodium retention, and aldosterone release. the sustained overactivation of the ras could lead to hypertension, a disease affecting almost half of us american adults [ ] . the activation of the endocrine ras is initiated upon the release of renin from juxtaglomerular cell granules into the circulation. by catalyzing the cleavage of angiotensinogen to release angiotensin i peptide, renin acts as the rate limiting enzyme of the ras, at least in humans. thus, it is not surprising that there are a number of complex mechanisms regulating renin expression and secretion [ ] . the subsequent conversion of angiotensin (ang)-i to ang-ii is catalyzed by angiotensin converting enzyme (ace) which is localized to endothelial cells and is abundant in the lungs. most of the functions inducing blood pressure elevation are mediated through binding of ang-ii to angiotensin type receptor (at r), whereas, binding of ang-ii to angiotensin type ii receptors (at r) has been reported to generally oppose the actions of at r. other peptides of the ras, such as ang-( - ) and alamandine, also act to counter regulate the action of ang-ii at at r [ , ] . drugs targeting the ras are effective as treatments for hypertension and other diseases including heart failure, chronic kidney disease, diabetic nephropathy, marfan's syndrome, and some autoimmune diseases [ ] [ ] [ ] [ ] [ ] [ ] . however, it is unclear why these drugs are effective even in patients exhibiting low or normal circulating renin activity [ , ] . the answer to this observation may lie in the existence of an independent autocrine/paracrine ras acting locally within several tissues, including the brain. the existence of the brain ras, which was initially proposed by bickerton and buckley in , has changed the traditional view of the ras [ ] . since the discovery that central ang-ii induces a potent pressor response, several new functions of the brain ras have been identified. central administration of ang-ii elicits potent dipsogenic responses, induces sodium intake, triggers sympathetic outflow to the kidney and other organs, and recently, evidence has established that the brain ras modulates metabolic function primarily through distinct nuclei within the hypothalamus [ ] [ ] [ ] [ ] . most of these effects can be attenuated by administration of ras blockers or by genetically ablating at r in specific brain regions or cell types [ ] [ ] [ ] . resistant hypertension, in which high blood pressure remains above / mmhg despite use of or more antihypertensive drugs (including a diuretic), accounts for approximately % of patients with essential hypertension [ ] . resistant hypertension and sympathetic overactivity have been linked to brain ras overactivation [ ] . thus, novel drugs targeting the brain ras might be useful to treat resistant hypertension and/or diseases associated with elevated sympathetic outflow such as heart failure [ ] . this article aims to bring the reader up-to-date on the important new findings and the currently controversial topics in the field. then, novel translatable strategies to attenuate the upregulation of brain ras activity in human resistant hypertension will be also discussed. although more than years of research supports the important role of the brain ras in modulating several physiological functions, it is not completely clear how angiotensin peptides are generated within the central nervous system (cns). there is extensive evidence indicating that angiotensinogen is highly expressed in astrocytes and in some specific populations of neurons, which suggests that the extracellular space of the cns has abundant renin substrate [ , [ ] [ ] [ ] [ ] [ ] . the distribution of the two main ang-ii receptors, at r and at r, was mapped initially by autoradiography and subsequently confirmed by either in situ hybridization or utilizing transgenic mice expressing reporter genes under the control of either at r or at r promoters [ , , •] . at r is highly expressed in most of the circumventricular organs such as the subfornical organ, the organum vasculosum laminae terminalis (ovlt), and the area postrema. however, the elevated expression of at r in some regions behind the bloodbrain barrier such as the paraventricular nucleus of the hypothalamus (pvn), the nucleus tractus solitarius (nts), the rostral and caudal ventrolateral medulla (rvlm and cvlm, respectively), the medial preoptic nucleus (mnpo), and some neurons within the arcuate nucleus (arc) suggests a role for ang-ii as a neuromodulator. expression of at r in the brain predominates over at r expression during fetal development [ ] . however, recent studies from de kloet et al. utilizing bacterial artificial chromosome transgenic at r-enhanced green fluorescent protein (egfp) reporter mouse confirmed the presence of at r in adult brains particularly in neurons and/or fiber terminals in circumventricular organs, hypothalamic nuclei, and the hindbrain [ ] . although most of the components of the ras have been identified in the brain, the lack of a reliable method to detect renin in small neuroanatomical structures had led some to question whether the central generation of ang-ii requires renin. indeed, renin-independent ang-ii generating biochemical pathways involving tonin and cathepsin d have been proposed [ ] [ ] [ ] [ ] . others have performed experiments which they interpret as a refutation of the brain ras [ ] . the difficulties in measuring renin stem from several factors. first, considering that renin is the rate-limiting enzyme for the generation of ang-i, and given the elevated bioavailability of angiotensinogen in the extracellular space of the brain, it is expected renin must be tightly controlled and secreted from specific cells within selected neuroanatomical nuclei. supporting this, minimal elevation of ang-ii levels in discrete neuroanatomical regions is expected to elicit extremely profound effects. indeed, evidence indicates that ablation or stimulation of angiotensinergic signaling within a few cells in the brain leads to extremely prominent cardiovascular and metabolic phenotypes [ ] [ ] [ ] . second, we and others have described the existence of an alternative renin isoform termed renin-b, which is the predominant transcribed renin isoform in the brain, but is absent in other tissues [ , ] . renin-b lacks the signal peptide and the first third of the pro-segment which implies that ( ) renin-b protein is catalytically active and ( ) is predicted to remain in an intracellular compartment [ ] . this observation was particularly compelling as an intracrine ras had been previously proposed [ , ] . notably, evidence that ang-ii can be generated intracellularly in presynaptic neurons and subsequently released to the synaptic terminal upon presynaptic depolarization would provide a strong argument to define ang-ii as a neurotransmitter. to test the hypothesis that renin-b is involved in the generation of intracellular ang-ii in the brain, we generated transgenic mice lacking the alternative renin-b, while renin-a was preserved [ ] . paradoxically, mice lacking renin-b exhibited a mild increase in blood pressure during the light cycle, which was attenuated by intracerebroventricular infusions of at r blockers or ace inhibitors [ •] . these data indicate that intracellular renin-b might play a regulatory role on the brain ras rather than generating intracellular ang-ii as initially proposed [ ] . even though our data do not support the existence of an intracellular ang-ii generating mechanism, they neither disprove it. the brain is one of the most vascularized systems because it receives . - . % of total cardiac output; thus, distinguishing renin levels generated within brain tissues (autocrine/paracrine) from the circulating renin (endocrine) is extremely challenging [ ] . despite evidence that renin activity and ang-ii have been identified in the cerebrospinal fluid (csf) and brain tissues from nephrectomized animals, those who resist the existence of the brain ras postulated that samples must be contaminated with traces of trapped blood [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . following this rationale, van thiel and colleagues attempted to provide evidence that the brain lacks the capacity to generate ang-ii by measuring renin activity (angiotensin-generating activity) in brain structures before and after organ buffer perfusion [ ] . it is not surprising that there was a significant decrease in renin activity within several brain structures after buffer perfusion because ( ) the brain has blood, and ( ) neurons unlikely store renin within granules as do specialized renal juxtaglomerular cells. indeed, the most direct and accurate conclusion from this study was that the brain indeed contains blood. it is surprising the authors decided to ignore their own data that renin activity remained at detectable levels in some buffer-perfused tissues including the brainstem. this is particularly interesting because in a double-transgenic mouse in which enhanced green fluorescence protein (gfp) is expressed under the control of the renin promoter and β-galactosidase is controlled by the angiotensinogen promoter, we identified unique reninexpressing cells in close proximity to angiotensinogenexpressing cells specifically within the rvlm, a brainstem nucleus [ , ] . it is of utmost importance that one contrasts a classical endocrine system and views the brain ras as a neuroendocrine, autacoid, or neurotransmitter system where single neurons or small collections of neurons mediate output from the cns. thus, cellular, neuroanatomical, and molecular specificities are the key aspects to be considered when neuroendocrine systems, such as the brain ras, are studied. it remains unfortunate that equivocal interpretations from experiments lacking sensitivity and neuroanatomical selectivity, those which are not required when studying the classical ras, re-emerge from time to time to question the existence of the brain ras [ ] . in the final section of this article, we will discuss how the development of state-of-the-art technology to study precise molecular signaling and neuronal circuits within the cns are appropriate to elucidate the key mechanisms regulating generation and action of angiotensin peptides within different neuroanatomical regions. the proteolytic activation of prorenin that normally occurs in secretory granules in renal jg cells is unlikely to occur in extrarenal tissues, including the brain [ ] . thus, it has been proposed that activation of renin in extrarenal tissues requires its binding to atpase h(+)-transporting lysosomal accessory protein , also known as the prorenin receptor (prr), encoded by the atp ap gene. prr has the unique capability to bind prorenin and induce its activation without prosegment removal [ ] . prr is highly expressed in neurons and some microglia cells and has been detected in several brain regions implicated in cardiovascular and autonomic control including the subfornical organ (sfo), paraventricular nucleus (pvn), nucleus of the solitary tract (nts), and the rostral ventrolateral medulla (rvlm) [ •, ] . despite initial reports indicating that prr might be involved in hypertension through local generation of ang-ii, other roles that are independent of ras activation have been described (reviewed in [ ] ). for instance, the transmembrane domain of the prr interacts with the vacuolar h(+)-atpase and plays an important role in lysosomal function and neuronal development [ ] . in humans, a unique mutation (c. c>t, p.d d) in the prr locus is associated with x-linked mental retardation and epilepsy [ ] . although many ras-independent functions of prr are well defined in kidney, the ras-independent functions of prr in the brain are not completely understood [ , , [ ] [ ] [ ] . in mice where neuronal prr was ablated at embryonic daỹ . (neuron filament promoter-cre crossed with prrfloxed mice; nefh-prrko), central generation of ang-ii in response to intracerebroventricular administration of recombinant prorenin was attenuated. to study the role of neuronal prr in the pathogenesis of hypertension, feng's lab utilized the model of low-renin hypertension induced by deoxycorticosterone acetate (doca) infusion and high dietary sodium. intracerebroventricular infusion of ace inhibitor prevents and reverses high blood pressure, demonstrating that production of ang-ii in the brain is required for doca-salt hypertension even though circulating ras activity is suppressed [ ] . moreover, two separate studies have shown that central infusion of an at r blocker mimics the effects of central delivery of ace inhibitors [ , ] . thus, it is well accepted that the blood pressure elevation in doca-salt hypertension is strongly driven by a neurogenic mechanism involving activation of the brain ras [ ] . li et al. reported that the selective ablation of prr in neurons attenuated the elevation of blood pressure in doca-salt hypertension when . m nacl was the only fluid offered [ ] . in contrast, experiments where doca-treated nefh-prrko mice were exposed to two-bottle choice paradigm for the assessment of sodium preference revealed that the ablation of prr in neurons is insufficient to decrease blood pressure but suppressed doca-induced saline intake [ ] . these data suggest expression of prr in specific brain regions controlling drinking behavior, such as the sfo is of physiological relevance. recently, souza and colleagues examined the role of prr in the pvn, a key integratory nucleus involved in blood pressure control [ ] . in this study, pvntargeted ablation of prr was induced in prr-floxed mice by bilateral stereotactic microinjection of adeno-associated virus (aav)-cre-gfp. the reduction of prr expression in the pvn by less than % was sufficient to attenuate doca-salt induced blood pressure elevations, cardiac and vasomotor sympathetic over-activity, and improved cardiac parasympathetic function [ • ]. in addition, live-cell calcium recordings utilizing a novel calcium biosensor (gcamp ) revealed pvntargeted ablation of prr attenuates calcium influx in response to ang-ii in doca-salt hypertension. despite the growing evidence indicating the importance of prr in the cns to control cardiovascular function, there is no generalized consensus whether the underlying mechanisms require local ang-ii generation. it is likely that both ang-ii-dependent and ang-ii-independent mechanisms might occur simultaneously at different degrees depending on the neuroanatomical localization and cell types, as well as different physiological and pathological circumstances [ ] . there is a growing interest in the soluble prr fragment (sprr) which arises from the proteolytic cleavage of prr by furin or site- protease to generate a -kd transmembrane/ cytoplasmic fragment and a -kd soluble prr form [ , ] . growing evidence supports that elevation of circulating sprr levels are associated with high blood pressure, chronic kidney disease, preeclampsia, and obstructive sleep apnea [ ] [ ] [ ] [ ] . however, the biological function and the physiological relevance of sprr were completely unknown until recently. many functions of sprr controlling renal function have been reported. for instance, sprr exerts antidiuretic actions in part by inducing frizzled -dependent stimulation of aquaporin expression in the collecting duct [ ] . studies specifically aiming to elucidate the role of sprr in the cns have not been reported. recently, gatineau et al. demonstrated that the selective deletion of adipose tissue prr elevates systolic blood pressure concomitant with increased circulating sprr levels in high fat diet-fed mice [ •] . in males, systemic infusion of recombinant mouse epitope tagged sprr resulted in blood pressure elevation and this increase was attenuated by ganglionic blockade, but not administration of at r blockers, indicating that autonomic dysfunction, but not circulating ras overactivation, is a key mechanism underlying sprr-mediated blood pressure elevation in obese male mice. in contrast, infusion of sprr in females failed to induce autonomic dysfunction but it induced elevated vasopressin levels and plasma renin indicating the existence of sex differences in sprr-mediated responses [ ] . given that the source of vasopressin is exclusively from the pvn and supraoptic nucleus (son), these observations support that sprr is biologically active in the cns and elevations of sprr in the brain might be implicated in certain forms of neurogenic hypertension linked to obesity. it has been proposed that many of the effects of ang-( - ) in the cns oppose many of the at r-mediated actions of ang-ii in the brain [ ] . ang-( - ) induces its effects mainly through mas receptor activation, although it has been reported to also act through at r [ ] . other studies proposed that mas receptors form heterodimers with at r in astrocytes [ ] . teixeira et al. recently reported that ang-( - ) binds to at r but fails to engage its canonical g protein signaling [ ] . instead, ang-( - ) triggers β-arrestin recruitment and intracellular signaling, suggesting it may have the properties of a biased agonist for at r. several synthetic at r biased agonists have been designed, but the existence of an endogenous functional biased ligand for at r has not been reported. ang-( - ) can be generated from the direct cleavage of ang-ii by angiotensin converting enzyme (ace ), but ace can also catalyze the conversion of angiotensin i to angiotensin-( - ), which subsequently is converted to ang-( - ) by ace or neutral endopeptidase [ ] . downregulation of ace and suppression of central ang-( - ) levels are thought to be one of the underlying mechanisms causing low renin hypertension [ , ] . in recent years, significant progress has been achieved particularly on the molecular mechanisms controlling brain ace activity. lambert et al. described a process termed "ace shedding" in which a disintegrin and metalloprotease (adam ) catalyze the cleavage of membrane anchored ace [ ] . however, advances on the physiological and pathophysiological role of adam -mediated ace shedding in the brain have only been recently demonstrated. xia and sriramula et al. demonstrated that neuron-targeted overexpression of ace is sufficient to ameliorate elevated blood pressure, autonomic dysfunction, and vasopressin release in response to doca-salt hypertension [ ] . moreover, braintargeted ablation of adam utilizing central infusions of sirna suppressed doca-salt induced hypertension concomitant with blunted reduction of ace activity in the hypothalamus and cerebrospinal fluid, indicating that ace shedding by adam in the brain is a relevant mechanism contributing to neurogenic hypertension. new evidence indicates that activation of at r is required for adam -mediated ace shedding possibly via reactive oxygen species and phosphorylation of extracellular signal-regulated kinase in neurons [ ] . it has been previously shown that a reduction of ace expression in the rvlm is a contributing factor in the development of hypertension in spontaneously hypertensive rats [ ] . mukerjee et al. provided evidence of the importance of ace /adam pathway in pre-sympathetic neurons within the pvn [ •] . interestingly, ace is expressed in gabaergic inhibitory neurons projecting onto the hypothalamus. thus, ace is thought to maintain a normal gabaergic inhibitory tone to the presympathetic neurons in the pvn in normal physiological conditions, while disinhibition of this pathway might lead to hypertension. in contrast, adam is expressed in single-minded family basic helixloop helix transcription factor (sim )-positive excitatory neurons within the pvn and promotes excitatory activity. ablation of adam in the pvn blunts pressor responses to acute pvn-targeted microinjection of ang-ii. this accumulating evidence suggests the importance of the opposing roles of ace and adam in modulating of sympathetic activity and central control of blood pressure. finally, it has been recognized that adam has a role in processing the proinflammatory cytokine, tumor necrosis factor alpha (tnf-α). indeed, upregulation of the brain adam was associated with elevated tnf-α implicating that attenuation of tnf-α-related mechanisms could be mediating part of the phenotype observed in mice lacking hypothalamic adam . numerous reports suggest elevation of tnf-α and activation of inflammatory cells (microglia) within certain brain regions triggers hypertensive responses [ ] [ ] [ ] . therefore, future studies are expected to clarify whether tnf-α plays a regulatory role in ace activity and other ras components. a quarter of hypertensive patients exhibit low-renin hypertension [ ] . it has been suggested that low-renin hypertension is in part driven by elevated angiotensinergic signaling in the brain [ , ] . therefore, the development of novel drugs modulating the brain ras might represent an effective solution to treat resistant hypertension coincident with elevated sympathetic activity and suppressed circulating renin activity. two decades ago, llorens-cortes's laboratory demonstrated that the conversion of ang-ii into angiotensin iii (ang-iii) in the brain is catalyzed by aminopeptidase a (apa), a zinc metalloprotease [ ] . importantly, ang-iii has been hypothesized to be the major biologically active peptide of the brain ras. this is based on the observation that inhibition of the brain apa completely prevents elevated blood pressure in models of neurogenic hypertension with elevated brain ras [ ] . it took years to translate the observation that pharmacological inhibition of apa can be used as a therapeutic tool to treat resistant hypertension in humans. recently, a new multicenter, open-label, phase ii study was released evidencing the efficacy of a brain penetrating inhibitor of apa, firibastat (previously named rb ), in reducing blood pressure in overweight patients of multiple ethnic origins without angioedema [ •] . moreover, an additional pilot double-blinded randomized placebo-controlled study in hypertensive patients demonstrated that blood pressure in firibastat-treated patients trended to be decreased compared to placebo controls without affecting systemic ras activity [ ] . firibastat is the first oral medication that may target the brain ras with promising clinical application. current efforts in designing new brainpenetrating apa inhibitors led to a -fold more potent new prodrug, ni /qgc , which has been shown to exert powerful antihypertensive effects in rats treated with doca-salt [ ] . in recent years, multiple pleiotropic roles of the brain ras, namely neuroinflammation, autophagy, er stress, and mitochondrial dysfunction, have emerged. these findings resulted in considerable advances in utilizing brain ras blockade or ras modulation as a therapeutic strategy to treat diseases beyond neurogenic hypertension. this is specifically relevant in stroke and cerebrovascular diseases, alzheimer's disease, cognitive dysfunction, parkinson disease, aging, and others [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . numerous other new molecules or administration routes to target the brain ras are currently under investigation. for instance, new classes of at r biased agonists, which can selectively activate β-arrestin without activating the classical g protein-coupled signaling, might represent potential tools to modulate angiotensin signaling within the brain. for example, carvalho-galvão et al. demonstrated that intracerebroventricular infusion of trv , a β-arrestin-biased at r-agonist effectively attenuated autonomic dysfunction and decreased arterial pressure in spontaneously hypertensive rats (shr) [ ] . thus, the development of brain-penetrating at r-biased agonists would be a promising strategy to treat resistant hypertension. finally, torika et al. demonstrated that intranasal administration of telmisartan is effective at reducing brain inflammation and ameliorating the progression of alzheimer's disease in mice indicating that novel routes of administration can also be employed to inhibit the brain ras without systemic off targets effects [ ] . the development of several cutting-edge technologies to study the cns predicts that we might witness a profound advance in this field in the near future. several laboratories are currently utilizing novel in situ hybridization techniques with significantly higher specificity and sensitivity. these are powerful tools to identify the anatomical and cellular localization of cells expressing components of the ras within the cns and to query the molecular and/or neural significance of these cells by multiplexing with different probes. we used rnascope® technology to confirm the abundance of agt and the presence of at r and prr in discrete cells within the brain. using this same technique, we and others have identified the distribution of ras genes in specific neun+ neurons, gfap+ astrocytes, and iba- + microglia cells in the sfo, pvn, arc, and the rvlm (unpublished). in addition, the development of an extensive array of transgenic mice carrying conditional alleles of ras genes as well as mice expressing tamoxifen-inducible and/or cell-specific cre recombinase expression facilitates further exploration of novel molecular and physiological functions of the ras within specific cell types within the brain. novel and previously generated animals expressing cre recombinase under the control of specific promotors including at r-cre or ren -cre mice, which can be crossed with mice expressing cre-dependent tdtomato (or other reporter genes) as well as mice expressing a fluorescent reporter gene under the control of ras genes such as at r-egfp or at a r-egfp (nz ), allow fluorescent labelling of cells expressing ras genes [ , •, , ] . significant progress has been made in developing techniques to identify the distinct roles of specific neurons in the brain. these techniques include optogenetics and designer receptors exclusively activated by designer drugs (dreadd), a chemogenetic technique, in which neuronal activity can be stimulated or suppressed in specific brain nuclei utilizing light or designer drugs, respectively. using these techniques, several laboratories recently showed the distinct roles of specific cells within selected nuclei controlling cardiovascular, metabolic, and autonomic function. for instance, de kloet el al. recently reported that optogenetic stimulation of at r expressing neurons in the pvn promotes blood pressure elevation and activation of the hypothalamic-pituitary-adrenal axis [ •] . similarly, nation et al. utilized dreadds to study the role of sfo neurons in thirst and salt appetite [ •] . stimulation of neuronal firing and activation of gq signaling in mice receiving sfo-targeted microinjection of a virus (aav -camkii-ha-hm d(gq)-ires-mcitrine) to induce selective neuronal expression of gαq via a designer receptor (hm d) that is exclusively activated by clozapine n-oxide, resulted in strong dipsogenic responses and preference to . m saline. the combination of these novel techniques with the array of transgenic mice described above are powerful tools to inquire the responses to stimulating or inactivating different neuronal circuits in the cns. finally, emerging "omics" techniques are becoming more accessible and reliable to study the transcriptome profile in different neuronal populations. there is particular interest in single-cell and single-nuclear rna sequencing technology to identify different clusters expressing ras genes to evaluate the molecular signature of cells. although studies utilizing these techniques to specifically evaluate the brain ras are not yet available, sapouckey recently reported an in silico re-analysis of hypothalamic single-cell rna sequencing datasets revealing that at r is expressed in a specific cluster of neurons expressing both agouti-related peptide (agrp) and leptin receptors [ • ]. this seminal discovery may illuminate the underlying mechanisms by which the brain ras controls resting metabolic rate and sympathetic activity in obesity-related hypertension. although the ras in the brain has been studied for decades, interesting and seminal discoveries continue to be made to this day. these include assessing the functional significance of newly identified components of the ras (such as prorenin receptor), the action of unconventional ras peptides (such as ang iii), enzymes which modify ras components (such as adam ), and new therapeutic tools to combat neurogenic hypertension. new technologies are making it easier to answer old questions-what is the localization of angiotensin receptors-and investigate new ones-what are function of specific subsets of at r-containng neurons? new genomic technologies such as single cell sequencing will provide novel platforms to understand the diversity of neuronal types which respond to ras activation or mediate downstream ras signaling. conflict of interest the authors declare no conflicts of interest relevant to this manuscript. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. papers of particular interest, published recently, have been highlighted as: • of importance •• of major importance heart disease and stroke statistics- update: a report from the classical renin-angiotensin system in kidney physiology angiotensin-( - ) counterregulates angiotensin ii signaling in human endothelial cells discovery and characterization of alamandine: a novel component of the renin-angiotensin system the use of renin-angiotensin system inhibitors in patients with chronic kidney disease heart failure drug treatment treatment of diabetic nephropathy with angiotensin ii 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agouti-related peptide (agrp) and leptin receptors publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -r gfr gk authors: kondo, masateru; imanishi, masaki; fukushima, keijo; ikuto, raiki; murai, yoichi; horinouchi, yuya; izawa-ishizawa, yuki; goda, mitsuhiro; zamami, yoshito; takechi, kenshi; chuma, masayuki; ikeda, yasumasa; fujino, hiromichi; tsuchiya, koichiro; ishizawa, keisuke title: xanthine oxidase inhibition by febuxostat in macrophages suppresses angiotensin ii-induced aortic fibrosis date: - - journal: am j hypertens doi: . /ajh/hpy sha: doc_id: cord_uid: r gfr gk background: several reports from basic researches and clinical studies have suggested that xanthine oxidase (xo) inhibitors have suppressive effects on cardiovascular diseases. however, the roles of a xo inhibitor, febuxostat (feb), in the pathogenesis of vascular remodeling and hypertension independent of the serum uric acid level remain unclear. methods: to induce vascular remodeling in mice, angiotensin ii (ang ii) was infused for weeks with a subcutaneously implanted osmotic minipump. feb was administered every day during ang ii infusion. aortic fibrosis was assessed by elastica van gieson staining. mouse macrophage raw . cells (raw) and mouse embryonic fibroblasts were used for in vitro studies. results: feb suppressed ang ii-induced blood pressure elevation and aortic fibrosis. immunostaining showed that ang ii-induced macrophage infiltration in the aorta tended to be suppressed by feb, and xo was mainly colocalized in macrophages, not in fibroblasts. transforming growth factor-β (tgf-β ) mrna expression was induced in the aorta in the ang ii alone group, but not in the ang ii + feb group. ang ii induced α-smooth muscle actin-positive fibroblasts in the aortic wall, but feb suppressed them. xo expression and activity were induced by ang ii stimulation alone but not by ang ii + feb in raw. feb suppressed ang ii-induced tgf-β mrna expression in raw. conclusions: our results suggested that feb ameliorates ang ii-induced aortic fibrosis via suppressing macrophage-derived tgf-β expression. many reports have suggested that patients with hyperuricemia are at risk for cardiovascular diseases, hypertension, and chronic kidney disease. [ ] [ ] [ ] indeed, some reports have demonstrated the contribution of uric acid to endothelial cell dysfunction. [ ] [ ] [ ] however, because uric acid itself has anti-oxidative activity and because hypouricemia decreases renal hemodynamics, a low serum uric acid level can negatively affect the cardiovascular system. on the other hand, several recent reports have suggested that xo acts as an inflammation activator in endothelial cells or macrophages. xo was shown to induce interleukin (il)- β secretion via nlrp inflammasome activation in macrophages, and angiotensin ii (ang ii) was shown to induce xo expression in endothelial cells or macrophages. , thus, based on these reported beneficial effects of xo inhibitors or feb on cardiovascular events, xo inhibitors may have beneficial effects independent of the level of uric acid. in this study, we used a mouse model of hypertension induced by ang ii characterized by aortic fibrosis and macrophage accumulation to investigate the effects of a xo inhibitor, feb, on the pathogenesis of vascular remodeling independent of the level of uric acid. we found that feb has beneficial effects on vascular fibrosis and hypertension. because accumulated macrophages in aortae following ang ii administration expressed high levels of xo, the major target cells of feb may be macrophages. our results suggested that feb inhibited ang ii-induced transforming growth factor (tgf)-β expression in macrophages and suppressed fibrotic processes in aortae. feb may be potentially effective against hypertension accompanied by aortic stiffness in clinical practice via its additive effects outside of ameliorating hyperuricemia. details for materials and methods section are in the online-only supplementary data. this study was carried out in accordance with the recommendations of "the guidelines for animal experimentation administered by tokushima university, the animal care and use committee for tokushima university. " the protocol was approved by the "the animal care and use committee for tokushima university. " c bl/ j male mice ( weeks old) were purchased from clea japan (tokyo, japan). the animals were housed in a temperature-controlled room at °c under a -hour light/dark cycle, with free access to food and water. ang ii (a ; sigma-aldrich) dissolved in normal saline was continuously infused at . mg/kg/d for weeks with a subcutaneously implanted osmotic minipump (alzet model ; alza, mountain view, ca); the dose used in this study was based on the results of another study and can induce vascular remodeling with blood pressure elevation. mice without ang ii administration were shamoperated (skin incision). feb (f ; tokyo chemical industry, tokyo, japan) ( mg/kg/day, p.o.) suspended in % carboxymethylcellulose-normal saline was administered every day during ang ii infusion. the mice were divided into groups: control, ang ii, feb, and ang ii + feb (n = - ). systolic blood pressure was measured noninvasively with a computerized tail-cuff system (bp- a; softron, tokyo, japan) (n = - ). two weeks after ang ii infusion, the mice were anesthetized with an intraperitoneal injection of both pentobarbital sodium (somnopentyl; kyoritsu seiyaku, tokyo, japan) (dosage: mg/kg) and xylazine (selactar; bayer, osaka, japan) (dosage: mg/kg), and % lidocaine hydrochloride (xylocaine; astrazeneca, london, uk) was injected subcutaneously for local analgesia. the absence of a pedal withdrawal reflex was checked frequently to ensure the adequacy of anesthesia, and more anesthetic was injected if the mice exhibited any signs of pain during the surgery. aortae were isolated for further analysis at the end of the experiments (qrt-pcr, histomorphology, and immunofluorescence). blood samples were incubated at °c for minutes and centrifuged at , rpm for minutes. the supernatant serum was used to measure the xo activity and uric acid. rat aortic smooth muscle cells were isolated as previously described. mouse embryonic fibroblasts were isolated as previously described, and we checked that almost of the isolated cells were fibroblasts by immunofluorescence with fibroblast-specific protein (fsp) antibody. rat aortic smooth muscle cells and mouse embryonic fibroblasts were cultured in dmem ( - ; nacalai tesque, kyoto, japan). raw . mouse macrophage cells were purchased from atcc (manassas, va) and were cultured in rpmi ( - ; nacalai tesque). all cells were maintained at °c in a humidified atmosphere of % co and % air. feb ( nm) was added hour prior to ang ii stimulation. for western blotting and mtt assays, the culture medium was changed to serum-free medium days before the stimulation. we tested for normality and equal variance before parametric data analysis. data are presented as the mean ± sem. statistical significance was assessed by -way analysis of variance followed by bonferroni post-hoc testing for multiple comparisons; values of p < . or p < . were considered significant. the number of individual experiments is indicated by n. for the gene expression analysis with genevestigator, mann-whitney's u-test was used for statistical analysis. ang ii administration did not affect serum xo activity or serum uric acid level. feb administration suppressed serum xo activity and serum uric acid level in sham-operated as well as ang ii-infused mice (figure a and supplementary figure ), confirming that the feb treatment produced its pharmacological effects in mice irrespective of ang ii infusion. feb did not affect the ang ii-induced heart weight increase ( figure b ) but suppressed ang ii-induced systolic blood pressure elevation (figure c ). elastica van gieson staining showed that feb suppressed ang ii-induced aortic perivascular fibrosis (red-stained area) but not medial thickening in mice (figure d and e). these results suggested that the suppressive effects of feb on ang ii-induced aortic fibrosis and blood pressure elevation were independent of the circulating uric acid level. since smooth muscle cell proliferation is linked to medial thickening in aortae, we also assessed the effects of feb on aortic smooth muscle cells in vitro (supplementary figure a) . feb did not affect ang ii-induced vsmc proliferation. in figure . the effects of feb on ang ii-induced aortic fibrosis and medial thickening. (a) feb administration ( mg/kg/day, weeks) significantly suppressed serum xo activity in mice. statistical significance was assessed by -way anova, and multiple comparisons were made by bonferroni's t-test. means ± sem, *p < . vs. vehicle administration. (n = - ) (b) feb did not affect ang ii-induced heart weight elevation adjusted for body weight. statistical significance was assessed by -way anova, and multiple comparisons were made by bonferroni's t-test. means ± sem, **p < . vs. without ang ii. (n = - ) (c) feb suppressed ang ii-induced systolic blood pressure elevation. statistical significance was assessed by -way anova, and multiple comparisons were made by bonferroni's t-test. means ± sem, **p < . vs. without ang ii, ## p < . vs. with ang ii alone. (n = - ) (d) evg staining showed that feb suppressed ang ii-induced aortic fibrosis (stained red), but not ang ii-induced medial thickening. scale bar = . mm. (e) four graphs indicate medial layer (µm), fibrotic layer (µm), medial layer to the diameter, and fibrotic layer to the diameter. statistical significance was assessed by -way anova, and multiple comparisons were made by bonferroni's t-test. means ± sem, *p < . , **p < . . (n = - ). abbreviations: ang ii, angiotensin ii; anova, analysis of variance; evg, elastica van gieson; feb, febuxostat; xo, xanthine oxidase. addition, feb did not affect ang ii-induced erk / and akt phosphorylation, in rat aortic smooth muscle cells (supplementary figure b,c) . these results agreed well with the effect of feb on ang ii-induced medial thickening. to determine how feb suppressed ang ii-induced aortic fibrosis, we examined inflammation in aortae. f / immunofluorescence staining showed that ang ii induced macrophage accumulation in aortae, whereas it was attenuated by the feb treatment (figure a) . f / mrna expression was also induced in aortae in mice treated with ang ii alone but not in mice treated with ang ii and feb (figure b) . the mrna expression levels of monocyte chemotactic protein (mcp)- , il- β and lysozyme m (lysm) were increased in aortae in the ang ii alone group but not in the ang ii and feb group (figure c) . these results suggest that under feb administration, ang ii does not induce inflammation in aortae. to determine the target cells of feb, we used xo immunofluorescence staining of the aorta sections from mice treated with ang ii. the results indicated that cells highly expressing xo were scattered about the adventitia, but the medial area and endothelium had very low levels of xo expression (figure a and supplementary figure ) . some of the xo-expressing cells colocalized with f / (figure a ), but not with fibroblast-specific protein (fsp) (supplementary figure ) . these results suggest that xo is highly expressed in macrophages and that feb mainly modulates the function of macrophages accumulating in the aortae upon ang ii infusion. because our results suggested that feb mainly affected the accumulated macrophages, we used raw . macrophage cells in an in vitro system. feb reduced ang ii-induced xo activity in the cells (figure b ). we further examined the effect of ang ii and feb on xo expression. similar to previous results, , we found that ang ii induced xo expression (figure c ). however, in the presence of feb, ang ii did not induce xo expression (figure d) . during fibrotic processes, macrophage-derived tgf-β plays key roles in fibroblast differentiation into myofibroblasts and extracellular matrix production by fibroblasts and myofibroblasts. feb significantly suppressed ang ii-induced tgf-β mrna expression in macrophages (figure a) . moreover, in aortic tissue, we found that ang ii administration induced tgf-β mrna expression, but not under feb administration (figure b ). fibroblast differentiation into myofibroblasts expressing α-smooth muscle actin (α-sma) is a key factor in fibrotic processes. macrophage-derived tgf-β is the primary factor that induces their differentiation. to assess differentiation, we used double immunofluorescence staining of aortae with α-sma antibody and a fibroblast marker, fsp antibody. α-sma is usually expressed in the medial area; however, in the ang ii alone group, α-sma-positive cells were also scattered around the outer side of the medial area (figure c ). fsp -expressing cells were found in the outer side of the medial area in the ang ii group and ang ii + feb group (figure c ). in the ang ii alone group, several fsp expressing cells, fibroblasts, were α-sma positive, but almost of the fsp -expressing cells were α-sma negative in the ang ii + feb group (figure c) . thus, feb reduced the number of α-sma-positive fibroblasts induced by ang ii in the outer side of the medial area. these results agreed well with , hours) induced tgf-β mrna expression, but not in the presence of feb ( nm) in raw . . statistical significance was assessed by -way anova, and multiple comparisons were made by bonferroni's t-test. means ± sem, *p < . . (n = - ) (b) tgf-β mrna expression was induced by ang ii, but not under the feb administration in the aortae. statistical significance was assessed by -way anova, and multiple comparisons were made by bonferroni's t-test. means ± sem, *p < . . (n = - ) (c) by ang ii administration, fsp -expressing cells, fibroblasts, were located in the fibrotic area. in ang ii alone group, several fibroblasts were α-sma positive (white arrowheads), but almost of fibroblasts were α-sma negative in ang ii + feb group. scale bar = µm. abbreviations: α-sma, α-smooth muscle actin; adv, adventitia; ang ii, angiotensin ii; anova, analysis of variance; feb, febuxostat; m, media; tgf, transforming growth factor. the results indicating the suppressive effects of feb on ang ii-induced macrophage-derived tgf-β mrna expression. we also examined the effects of feb on fibroblasts using mouse embryonic fibroblast in an in vitro system. we found that ang ii did not induce tgf-β mrna expression in the fibroblasts and that feb did not alter this effect (supplementary figure a) . we also found that ang ii slightly induced fibroblast proliferation but that feb did not affect it (supplementary figure b) . these results suggest that macrophage-derived tgf-β expression is a key factor in fibrotic processes and that feb suppresses ang ii-induced macrophage-derived tgf-β expression. the major finding of this study was that a xo inhibitor, feb, suppressed ang ii-induced vascular fibrosis by affecting mainly the accumulated macrophages in the adventitia. we also found that inhibition of xo by feb in macrophages suppressed ang ii-induced tgf-β expression, which is linked to promoting fibroblast differentiation into myofibroblasts during the fibrotic process. because we did not find serum uric acid changes in the mouse model of ang ii-induced vascular remodeling we used in this study, these effects of feb are likely independent of uric acid. in the present study, feb suppressed ang ii-induced hypertension, although it did not affect ang ii-induced heart weight elevation. a previous study reported that lysmpositive monocytes mediate ang ii-induced hypertension. our lysm mrna expression results in aortae agreed well with this report. several studies have suggested the beneficial effects of feb or xo inhibitors on endothelial function and hypertension. landmesser et al. have demonstrated that ang ii induces reactive oxygen species (ros) production in part via xo activation in endothelial cells. therefore, feb might have protective effects in part on endothelial cells in addition to macrophages, which appeared to be the major target of feb in the present study. the discrepancy between the effects of feb on hypertension and heart weight suggests that feb does not affect ang ii signal transduction leading to cardiomyocyte hypertrophy. our data suggest that feb had little effect on smcs, which is in line with low expression of xo in the aortic media regardless of ang ii infusion. to clarify the mechanisms by which feb suppressed ang ii-induced aortic fibrosis, we evaluated macrophage accumulation. both f / immunofluorescence staining and mrna expression in aortae showed that under feb administration, ang ii did not induce much macrophage accumulation and the mrna expression levels of inflammatory cytokines, mcp- and il- β. these results may partly explain the suppressive effects of feb on fibrosis. during the tissue fibrotic processes, macrophage-derived tgf-β production plays key roles in extracellular matrix production by fibroblasts or myofibroblasts and fibroblast differentiation into myofibroblasts, which produce more extracellular matrix than normal fibroblasts. myofibroblasts are active fibroblasts and express α-sma, whereas normal ones do not. we elucidated that the target of feb, xo, was highly expressed in accumulated macrophages in aortae and that feb significantly suppressed ang ii-induced tgf-β expression in macrophages. some reports have suggested that ros induces tgf-β expression, and our preliminary data suggest that feb can inhibit ang ii-induced ros production in aortae (supplementary figure ) . thus, feb might suppress ang ii-induced tgf-β expression in part via suppressing ros production. in addition to reduce xo activity, feb suppressed xo expression induced by ang ii in macrophage cells. landmesser et al. have demonstrated that ang ii induces xo expression via redox-sensitive pathway involving the nicotinamide adenine dinucleotide phosphate (nadph) oxidase. yisireyili et al. have also demonstrated that feb suppressed the expression levels of nadph oxidase subunits. thus, feb might also suppress expression of xo in part via affecting nadph oxidase. in the present study, we concluded that feb suppressed ang ii-induced vascular fibrosis, via mainly inhibiting the tgf-β expression in the accumulated macrophages in the adventitia, as its additive effects outside of ameliorating hyperuricemia. our results suggest the possibilities that ang ii/xo/tgf-β signaling axis existed in macrophages. in addition, ives et al. have demonstrated that xo induces il- β production via mitochondrial ros generation in macrophages. we showed that under the feb administration, ang ii did not induce il- β expression in aortae. thus, ros might be involved in mediating xo effects. feb is frequently used in patients with hyperuricemia accompanied with metabolic syndrome. these additive effects of feb can be useful for suppressing the cardiovascular events in patients with hyperuricemia. supplementary data are available at american journal of hypertension online. xanthine oxidase inhibition by febuxostat attenuates experimental atherosclerosis in mice use of xanthine oxidase inhibitor febuxostat inhibits renal interstitial inflammation and fibrosis in unilateral ureteral obstructive nephropathy febuxostat ameliorates doxorubicin-induced cardiotoxicity in rats xanthine oxidase inhibitors for prevention of cardiovascular events: a systematic review and meta-analysis of randomized controlled trials renoprotective effects of febuxostat compared with allopurinol in patients with hyperuricemia: a systematic review and meta-analysis the impact of serum uric acid reduction on renal 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dysfunction in patients with coronary disease nicorandil, a k(atp) channel opener, alleviates chronic renal injury by targeting podocytes and macrophages smooth muscle cellspecific hif- α deficiency suppresses angiotensin ii-induced vascular remodelling in mice endothelial nitric oxide synthase-independent protective action of statin against angiotensin ii-induced atrial remodeling via reduced oxidant injury nitrosonifedipine ameliorates angiotensin ii-induced vascular remodeling via antioxidative effects induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors essential role of vascular endothelial growth factor in angiotensin ii-induced vascular inflammation and remodeling macrophages in tissue repair, regeneration, and fibrosis lysozyme m-positive monocytes mediate angiotensin ii-induced arterial hypertension and vascular dysfunction xor inhibition with febuxostat accelerates pulmonary endothelial barrier recovery and improves survival in lipopolysaccharide-induced murine sepsis sars coronavirus papain-like protease induces egr- -dependent up-regulation of tgf-β via ros/p mapk/stat pathway xanthine oxidase inhibition by febuxostat attenuates stress-induced hyperuricemia, glucose dysmetabolism, and prothrombotic state in mice the authors declared no conflict of interest. key: cord- -voyzq n authors: de mello, walmor c. title: regulation of cell volume and water transport – an old fundamental role of the renin angiotensin aldosterone system components at the cellular level date: - - journal: peptides doi: . /j.peptides. . . sha: doc_id: cord_uid: voyzq n the expression and the role of renin angiotensin aldosterone system (raas) components on regulation of cell volume and water transport on vertebrates and invertebrates were reviewed. the presence of these components even in simple organisms like leeches and their relevance for the control of cellular volume and water transport supports the view that the expression of these components, at cellular level, is an acquisition which was preserved throughout evolution. the preservation of cell volume is fundamental for cell survival requiring the presence of regulatory mechanisms such as the regulatory volume decrease (rvd) or the regulatory volume increase (rvi). the inhibition of rvd has serious consequences for cellular function leading to activation of membrane ionic channels and of several kinases resulting in abnormalities of cellular function and is commonly associated with cell shrinkage through activation of akt [ ] . furthermore, changes in cell volume can represent signals triggering cell migration, proliferation and release * tel.: + ; fax: + . e-mail address: walmor.de-mello@upr.edu of hormones and transmitters [ ] . cell swelling occurs during hypothermia and hypoxia/ischemia, increase in the extracellular k+ concentration [k+] as well as intracellular acidosis/diabetic ketoacidosis. variations in cell volume are not necessarily associated with extracellular hypotonicity or hypertonicity because increase of intracellular osmolarity due to synthesis or elimination of osmolytes, is responsible for cell swelling [ ] . evidence is available that in mammals, there are local renin angiotensin systems in different organs including the heart, kidney and possibly in the brain in which raas components have been identified intracellularly (see , , , , volume and water transport is an old event preserved throughout evolution. evidence is available, for instance, that components of the raas are present in invertebrates including annelids [ ] , crustaceans [ , ] , molluscs [ ] , insects [ , , , ] . in leech (theromyzon tessulatum), brain neurons immunoreaction to angiotensin ii has been described [ ] and immunocytes showed immunoreactivity to antihuman-ang ii and antihuman at receptors [ , ] . also, an asparyl protease of kda exhibiting % of sequence identity with mammalian renin, has been found in the leech [ , ] . biochemically, renin, ace-and at -like receptor were identified in the leech immune cells [ ] and ace has been found in bacteria [ ] . so far, only soluble, single domain aces from invertebrates have been cloned, and these have been implicated in reproduction in insects [ ] . interestingly, bacterial dna sequences could encode putative ace-like proteins, strikingly similar to vertebrates enzymes [ ] . aces from invertebrates have been isolated from insects like drosophila melanogaster [ ] showing kinetics very similar to mammalian ace [ ] . immunochemical studies indicated ace intracellularly co-localized with myotropine peptides suggesting a convertase activity [ ] . immunostaining of leech sections, revealed labeling in neurons and glial cells of the central nervous system (cns), immunocytes, the nephridial canal canaliculi and the periphery of the ciliated funnel, as well as the epithelium lining nephridia. renin is localized in the excretory system and in the nervous system of leech [ , ] and its first appearance has been detected in bony fish [ ] . the first genomic characterization of non-mammalian vertebrates renin genes found in zebrafish was recently described [ ] . according to fournier et al. [ ] , practically all components of the renin angiotensin aldosterone system appear in bony fish with exception of mas receptor of ang ( - ) which appeared in amphibians. an important question is the physiological meaning of the expression of raas components. for many invertebrates, an important factor for survival when the medium changes, is the capability of the sodium pump to regulate the increased intracellular sodium concentration caused by an incremented osmolarity of the medium [ ] . evidence is, available that ang ii regulates water flow through aquaporins in clam worm [ ] and experiments performed on leeches in vivo, indicated that ang ii amide is involved on the control of water balance [ ] . measurements of transepithelial short-circuit current in leeches, showed that ang ii amide reduced this current-an effect mediated by cl− secretion [ , ] and in consequence, water follows the peptide eliciting water loss across the epithelium. cell swelling is known to activate ionic channels like the swelling-dependent chloride channel (i clswell ) which plays an important role on the regulation of cell volume [ , [ ] [ ] [ ] ] . eggs of the ascidian boltenia villosa have an inwardly rectifying cl− current whose amplitude varies by more than -fold during each cell cycle and osmotically produced swelling also increased cl− current amplitude in unfertilized eggs [ ] . these findings indicate that raas components play an important role on regulation of cell volume and water transport. the appearance of a sophisticated kidney function in mammals made it possible the efficient regulation of blood volume and blood pressure through the activation of the systemic renin angiotensin aldosterone system (raas). independently of a circulatory raas, the expression of raas components at cellular level in vertebrates because immunodetection of renin-angiotensin system (ras) components were found in anterior pituitary cells, particularly in lactotropes [ ] and prorenin, renin and angiotensinogen were identified not only in lactotropes and gonadotropes but also in somatotropes, corticotropes, and thyrotropes. the highest levels of renin were detected in lactotropes and gonadotropes [ ] . the detection of angiotensinogen and both its specific cleavage enzyme and its proenzyme within the same granule, suggests intragranular processing of this component [ ] indicating an important intracrine mechanism involved in the secretory process. in mammalians, components of the renin angiotensin system have been detected in several tissues including the heart, adrenal gland, kidney and in the brain [ , , , , , ] and many of the old properties of raas components as regulators of cell volume and water transport seen in invertebrates, are present in the mammalians. the raas is involved in the regulation of cell volume in normal mammalian heart as well as in the failing heart [ ] . indeed, in myocytes isolated from the failing ventricle and exposed to renin plus angiotensinogen or to ang ii, an increase of cell volume was seen concurrently with the inhibition of the sodium pump [ ] while the intracellular administration of ang ii had an opposite effect [ ] . the activation of the na-k- cl cotransporter is involved in the effect of ang ii because bumetanide abolished the swelling induced by the peptide [ ] . the regulation of cell volume, involves other components like ion channels and their voltage dependence. cell swelling induced by ang ii in cardiac cells, causes the activation of ionic channels like the swelling-activate chloride current (i cl,swell ) [ ] leading to membrane depolarization, reduction of action potential duration [ ] and consequent changes in cardiac excitability. the increase of (i clswell ) in the failing and in the normal mammalian heart [ , ] elicited by changes in cell metabolism or by ang ii, is particularly relevant because the activation of this current seems involved in generation of cardiac arrhythmias including early after depolarizations [ ] . in mammals, cell swelling also activates potassium channels (k(v) . / . ) which are the primary subunits of i (to,fast) , through phosphorylation/dephosphorylation of serine/threonine phosphatases [ ] in the mammalian heart [ , ] . it is known, for instance, that ang ii causes cell swelling and increases icl swell in the failing as well as in normal heart [ , ] and that the inability of the heart cell to regulate its volume through a lack of activation of the regulatory volume decrease (rvd) leads to serious impairment of heart cell function [ ] . components of the renin angiotensin system in the myocardium [ ] play an important role on regulation of cell volume and water transport because extracellular ang ii or renin inhibit the sodium pump, counteracts the decline in cell volume. on the other hand, when the cell is exposed to hypotonic medium, the increment of cell volume can be reduced by intracellular ang ii or renin [ ] . in this way, raas components contribute to the regulation of cell volume. this physiological property of ang ii is quite old because evidence is available that the peptide suppressed body weight loss of the clam worm perinereis sp. under a hyper-osmotic condition, and enhanced body weight gain under a hypo-osmotic condition [ ] . the earliest corticoid receptor have been found in lamprey and hagfish. the mineralocorticoid (mr) and the glucocorticoid (gr) first appear in skates and sharks while aldosterone first appears in lobe-finned fishes [ , , ] . a mutation corresponding to his- in human mr may have been important in the physiological changes associated with emergence of old world monkeys from prosimians [ , ] . bridgham et al. [ ] provide strong evidence that a gr with a preference for cortisol over aldosterone, arose in an elasmobranch descendent that was the common ancestor of ray finned fish and land vertebrates. it is well known that aldosterone is a steroid hormone involved in the regulation of blood volume, salt and water homeostasis. although aldosterone has a genomic effect by activating the mineralocorticoid receptor, a rapid action of aldosterone has been described. the mechanism involved in this rapid effect of the hormone, probably involves interaction with ang ii. evidence is available that the mineralocorticoid receptor is involved in the regulation of cell volume. the increase in cell volume caused by extracellular ang ii in cardiac cells of the mammalian heart, for instance, was enhanced when the cells were incubated with aldosterone for h [ ] -an effect abolished by spironolactone. the mineralocorticoid receptor is an important component of the intracrine action of ang ii because the decline in cell volume elicited by intracellular administration of ang ii, was increased by aldosterone and inhibited by spironolactone [ ] . the effects of aldosterone and spironolactone were related, in part, to a change in expression of at receptors at surface cell membrane [ ] . ace [ ] belongs to the angiotensin-converting enzyme family of dipeptidyl carboxydipeptidases and has considerable homology to human angiotensin converting enzyme [ ] . in addition to regulation of cardiovascular and kidney function, the encoded protein is a functional receptor for the spike glycoprotein of the human coronaviruses sars. angiotensin ii is hydrolyzed to angiotensin ( - ) by ace [ ] with consequent generation of angiotensin ( - ), which counteracts some harmful effects of ang ii [ ] including the block of impulse propagation seen during ischemia/reperfusion [ ] . according to fournier et al. [ ] the first appearance of ang ( - ) mas receptor was in amphibians suggesting that ace also appears in amphibians. mutation of angiotensin-converting enzyme-related (acer) gene, a drosophila ace homolog, was proven to result in a severe defect during heart morphogenesis and regulates heart physiology in adult flies [ ] . acer also plays an important role in regulating sleeping behavior [ ] . ang ( - ) generated from angiotensin ii [ ] in vertebrate animal models and humans [ , ] , has been found to counteract many effects of ang ii [ ] , reduces the heart cell volume and the swelling-activate chloride current (i cl,swell ) enhanced by ang ii [ ] . since cell swelling induced by myocardial ischemia or heart failure, represents an important arrhythmogenic mechanism, the formation of ang ( - ) through the activation of the ace /ang ( - )/mas receptor axis might be of benefit to the ischemic heart [ ] . the beneficial effect of angiotensin ( - ) on impulse propagation seen under these conditions, is related to the activation of the sodium pump and hyperpolarization of cell membrane [ ] . ace is over-expressed during heart failure leading to a higher formation of ang ( - ) [ ] and recent findings indicate that angiotensin ( - ) increments the sodium pump and reduces the cell volume by % within min in the failing heart [ , ] . it is then conceivable, that the improvement of cardiac function and decreased incidence of cardiac arrhythmias during ischemia/reperfusion elicited by ang ( - ), be related, at least in part, to the decrease of heart cell volume. according to these observations, the balance between ace and ace expression seems to be a determinant factor on the regulation of heart cell function and volume. although evidence is available that the mas receptor for ang ( - ) first appear in amphibians [ ] , no information is available on the role of the hexapeptide on cell volume and water transport on invertebrates. ( ) the presence of raas components in cells of invertebrates and their role on the regulation of cell volume and water transport, represents an important acquisition which was maintained throughout evolution; ( ) the organization of a renin angiotensin aldosterone system only appeared later on the evolutionary scale; and ( ) although the intracellular and extracellular expressions and functions of ace, ace , angiotensinogen, renin and ang ii preceded the appearance of a sophisticated circulating renin angiotensin system, its acquisition represents an important regulatory factor of cellular homeostasis in mammals. evolution of hormone selectivity in glucocorticoid and mineralocorticoid receptors structural analysis of the evolution of steroid specificity in the mineralocorticoid and glucocorticoid receptors swelling-activated chloride channels in cardiac physiology and pathophysiology evolution of hormone-receptor complexity by molecular exploitation cloning and expression of an evolutionary conserved single domain angiotensin converting enzyme from drosophila melanogaster memory enhancement by the angiotensinergic system in the crab chasmagnatus is mediated by endogenous angiotensin ii novel aspects of angiotensin ii action in the heart. implications to myocardial ischemia and heart failure cell swelling, impulse conduction and cardiac arrhythmias in the failing heart. opposite effects of angiotensin ii and angiotensin ( - 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) forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin converting enzyme homolog ace key: cord- -pnnkfid authors: ioakeimidou, a.; pagalou, e.; kontogiorgi, m.; antoniadou, e.; kaziani, k.; psaroulis, k.; giamarellos-bourboulis, e. j.; prekates, a.; antonakos, n.; lassale, p.; gogos, c. title: increase of circulating endocan over sepsis follow-up is associated with progression into organ dysfunction date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: pnnkfid how circulating inflammatory mediators change upon sepsis progression has not been studied. we studied the follow-up changes of circulating vasoactive peptides and cytokines until the improvement or the worsening of a patient and progression into specific organ dysfunctions. in a prospective study, concentrations of tumor necrosis factor-alpha (tnfα), interleukin (il)- , il- , il- , interferon-gamma (ifnγ), endocan and angiopoietin- (ang- ) were measured in serum by an enzyme immunoassay in patients at baseline; this was repeated within h upon progression into new organ dysfunction (n = ) or improvement (n = ). endocan and ang- were the only parameters that were significantly increased among patients who worsened. any increase of endocan was associated with worsening with odds ratio . (p < . ). this increase was independently associated with progression into acute respiratory distress syndrome (ards) as shown after logistic regression analysis (odds ratio . , p: . ). changes of circulating cytokines do not mediate worsening of the critically ill patients. instead endocan and ang are increased and this may be interpreted as a key-playing role in the pathogenesis of ards and septic shock. any increase of endocan is a surrogate of worsening of the clinical course. despite progress made in early recognition and management, sepsis remains the leading cause of death accounting for more than , deaths every year in the united states [ ] . sepsis is nowadays defined as a life-threatening organ dysfunction induced from the dysregulated response of the host to an infection [ ] . this definition is coming from our better understanding of the complex pathophysiology of sepsis acquired in recent years. we have managed to understand that when sepsis develops not all patients react in a similar way; in some hosts a aikaterini ioakeimidou and evgenia pagalou contributed equally to this submission. pro-inflammatory response prevails whereas in others an antiinflammatory response emerges [ ] . however, this distinction is not clear-cut in all patients. everyday practice teaches that unfavourable outcome is due to the deterioration of the initial host response to progressive organ dysfunction. it is highly probable that the mechanisms underlying this deterioration are individualized from patient to patient. these mechanisms are poorly studied and their knowledge is mandatory for future immunotherapy studies. deterioration of the host from an initial septic state to multiple organ dysfunctions most probably results from the dynamic interaction of circulating cytokines and peptides secreted from endothelial cells. vascular endothelium is s t i m u l a t e d b y c y t o k i n e s f o r t h e p r o d u c t i o n o f angiopoietin- (ang- ) and endocan. ang- increases vascular permeability and mediates progression into shock [ ] whereas endocan inhibits leukocyte migration in the lungs and kidneys [ ] . our aims were to monitor the changes of circulating levels of pro-inflammatory and antiinflammatory cytokines and of vasoactive peptides of critically ill patients at well-defined time-points of the clinical course and to understand how these changes mediate progression to organ dysfunction in an individualized way. this is a prospective study that was conducted in ten intensive care units (icus) participating in the hellenic sepsis study group from january until december . the study protocol was approved by the ethics committees of the participating hospitals. a patient was enrolled after written informed consent provided by his first-degree relatives. inclusion criteria were: (a) written informed consent, (b) age equal to or above years, (c) both genders, (d) at least two signs of the systemic inflammatory response syndrome (sirs) as defined elsewhere [ ] , (e) one of the following infections: acute pyelonephritis, community-acquired pneumonia, acute intraabdominal infection, primary bacteremia or ventilatorassociated pneumonia. the diagnosis of infections was done by international definitions [ ] , (f) first blood sampling within the first h from the presentation of the first two signs of sirs, and (g) repeat blood sampling at a follow-up time point. the follow-up time point could be h from the first blood sampling or within the first h from the onset of any new organ dysfunction provided that this was at least h apart from the first blood sampling. this -h time difference between the two time-points of sampling was selected in order to increase the likelihood that the changes described over-time of the studied mediators were associated with the new organ dysfunction and not with the existing baseline inflammatory process. exclusion criteria were: (a) infection by the human immunodeficiency virus − , (b) neutropenia defined as an absolute neutrophil count less than /mm unless due to sirs per se, and (c) intake of corticosteroids at a daily dose of more than . mg/kg of equivalent prednisone for at least consecutive days. five ml of blood was sampled after venipuncture of one forearm vein under sterile conditions. blood was poured into pyrogen-free tubes (vacutainer, becton dickinson, cockeysville, md) and centrifuged at room temperature. the supernatant serum was immediately aliquoted and aliquots were shipped in dry-ice on the same day into the central lab at the laboratory of immunology of infectious diseases as the th department of internal medicine of attikon university hospital. aliquots were stored there at − °c until measurement. patients were then followed-up daily for days. patients who progressed into new organ dysfunctions less than h from the first blood sampling were excluded from the study. a second blood sample was collected from the remaining patients h after the first blood sampling. patients were then followed daily for days and sequential organ failure assessment (sofa) score was measured daily. if the patient was presenting with a new organ dysfunction, blood sampling was done within h and the blood sample drawn at h was discarded. if not, the blood sample originally collected at h was processed for analysis to be used as comparator. organ dysfunctions were defined as follows: (a) acute respiratory distress syndrome (ards) as diffuse shadows in chest x-ray and ratio of partial oxygen tension to fraction of inspired oxygen below [ ] , (b) acute kidney injury (aki) as less than . mg/kg/min of urine output for at least two consecutive hours provided that the negative fluid balance was restored [ ] , (c) disseminated intravascular coagulation (dic) as absolute platelet count below , /mm accompanied by an increase of the concentrations of d-dimers and decrease of circulating fibrinogen [ ] , and (d) septic shock as decrease of systolic arterial pressure below mmhg not responding to fluid resuscitation and requiring the administration of vasopressors [ ] . for all patients retrospective evaluation was done using the recently published sepsis- definitions [ ] . those who did not meet the new definitions at study enrolment were excluded from analysis. concentrations of tumor necrosis factor-alpha (tnfα), of interleukin (il)- , il- , il- , interferon-gamma (ifnγ), angiopoietin- (ang- ) and of endocan were measured by an enzyme immunoassay. reagents for tnfα, il- , il- , il- and ifnγ were purchased from ebiosciences (san diego, usa); those for ang- by r&d (minneapolis, usa); those for endocan were a kind donation by lunginnov s.a.s (campus de l'institut pasteur de lille, , lille, france). the lower limits of detection were pg/ml for tnfα, il- , il- , il- and ifnγ; pg/ml for ang- ; and . ng/ml for endocan. the primary study endpoint was the correlation between the follow-up changes of each measured parameter from baseline until the improvement or the worsening of a patient. a patient was considered worsened if his total sofa score was increased on the follow-up time point by at least two points from the baseline sofa score on study enrolment. a patient was considered improved if his total sofa score was decreased on the follow-up time point by at least two points from the baseline sofa score on study enrolment. the secondary endpoint was the comparative changes of the measured parameters for specific organ dysfunctions. results of measured parameters were expressed as median and % confidence intervals. for the primary endpoint, pair-wise comparisons were done between the two time points of sampling separately for patients worsening and for patients improving by the wilcoxon test. the percent of change of parameters from baseline was analyzed by receiver operating characteristic curve (roc) analysis. using co-ordinate points of the curve, the cut-off with specificity greater than % for worsening was chosen. the sensitivity, specificity, positive and negative predictive values of that cut-off were calculated. the odds ratio (or) and % confidence interval (ci) for worsening at the depicted cut-off point were calculated by mantel and haenzel's statistics. for the secondary endpoint, pair-wise comparisons between baseline and follow-up measurements were done within the subgroups of patients developing specific new organ dysfunctions. the association of changes of circulating peptides with the development of a specific organ failure was further analyzed by logistic regression analysis; the presence of at least one comorbidity and baseline severity entered the equation as co-variates. any value of p below . after correction for multiple comparisons was considered significant. the study flow-chart is shown in fig. . a total of patients were screened. the average number of beds per participating icu was ten. taking into consideration that the median patient pair-wise comparisons showed that the only parameters that were significantly increased upon worsening of the patients were endocan and ang- (fig. ) . endocan significantly decreased among patients who improved. roc curves of the percent changes of endocan and ang- from the baseline were designed to explore the use of endocan and of ang- as surrogate markers of worsening. only the roc curve of the change of endocan provided a significant area under the curve (fig. a) . using the coordinate points of roc curve, it was found that any increase of endocan from baseline could be associated with sepsis worsening with . % sensitivity and . % positive predictive value (fig. b) . any baseline increase of endocan was associated with or . ( % cis: . - . , p: × − ) for worsening. when pair-wise comparisons between baseline and follow-up measurements were done within the subgroups of patients developing new organ dysfunctions, it was found that the only parameters significantly changing were endocan and ang- . more precisely, both were increased on development of ards and septic shock; ang- was also increased on development of aki (fig. ) . no changes were found upon development of dic (data not shown). logistic regression analysis showed that any increase of endocan from baseline was independently associated with the progression into ards. a similar association was shown for the progression into septic shock with a trend towards statistical significance (table ). the present study measured the concentrations of a range of circulating protein molecules in the sera of patients at icu admission and upon progression into a new organ dysfunction. results showed that only vasoactive and endothelialderived molecules like endocan and ang- are increased upon deterioration of the patient into a more severe stage as this is defined by any increase of the total sofa score by at least two points. increase of endocan and ang- were mainly found on progression into ards and septic shock whereas ang- was also increased among patients who progressed into aki. any increase of endocan was associated with increased likelihood for the deterioration of a patient. in a recently published single-center prospective study, patients with sirs, nine patients with sepsis, patients with severe sepsis and patients with septic shock were enrolled. serum levels of endocan were measured on days , and . results showed that concentrations of endocan were greater among patients at septic shock, and that day levels of endocan could prognosticate both -day and six-month outcome [ ] . although at first glance our study appears similar in design, it presents with some clearly distinctive characteristics in the design: (a) serial sampling at clinically indicative time points of progression into a new organ dysfunction or improvement, (b) analysis based on the new sepsis- definitions, and (c) correlation of changes of baseline endocan with progression into specific organ dysfunctions. endocan is a proteoclycan located on endothelial cells of the lungs and kidneys. at the event of a systemic inflammation, the molecule is cleaved through the activity of cathepsin g of neutrophils and a kda polypeptide is generated at greater concentrations in patients with sepsis than in healthy volunteers [ ] and also in patients with severe sepsis and septic shock than in patients with sirs [ , ] . former studies have shown that this glycoprotein may be involved in the pathophysiology of an infection without its contribution been clearly defined. in a prospective study of patients, serum endocan greater than . ng/ml was associated with the presence of bacteremia [ ] . our overall findings are in general agreement with those of recent studies enrolling a lower number of patients. these studies have explored the probable associations of admission endocan levels with the development of organ failures within two to five days. in a study of patients with multiple injuries, developed acute lung injury (ali) and did not. admission levels of endocan in the icu were lower among patients who developed ali compared to those who did not [ ] . a similar finding was described in critically ill patients; admission serum endocan was lower among those who developed respiratory failure on day [ ] . their findings may at first appear contradictory to ours. however follow-up measurements were missing so it could be hypothesized that serum endocan might increase on the day of respiratory failure. the first study on critically ill patients showed that endocan was greater among patients who developed organ failures and mods within the first h post admission [ ] . the second study on patients with ards showed that admission endocan levels were greater in nonsurvivors than in survivors [ ] . our study is not using the traditional design that compares the concentrations of circulating peptides between patients with infection and patients with sepsis. instead, all enrolled patients had sepsis at baseline and the changes of circulating peptides between those improving and those worsening were compared. our findings suggest that changes of circulating cytokines do not mediate progression of the clinical course of sepsis. probably this is explained by the fact that upon deterioration of sepsis the patient is already at the state of immunosuppression where further cytokine production does not take place [ , ] . instead endocan and ang are increased and this may also be interpreted as a key-playing role in the pathogenesis of ards and septic shock. from the practical aspect, it becomes evident that any increase of endocan is a surrogate of prediction for the overall deterioration of the patient. our findings clearly show that endocan and angiopoietin- are mediators increased upon deterioration of the clinical course of sepsis. any baseline increase of endocan is a specific surrogate marker to indicate worsening of the patient. increases of endocan are independently associated with progression into adult respiratory distress syndrome. the findings require validation by an independent cohort where changes of endocan are correlated with respective changes of the sofa score. compliance with ethical standards the study protocol was approved by the ethics committees of the participating hospitals. funding the study was funded by the hellenic institute for the study of sepsis. immunoassay reagents for endocan were a kind donation by lunginnov s.a.s (campus de l'institut pasteur de lille, , lille, france). conflict of interest none of the authors has any conflict of interest related to this submission. ethical approval the study protocol was approved by the ethics committees of korinthos general hospital, korgialeneion benakeion general hospital, attikon university hospital, bg.gennimatast hessaloniki general hospital, sotiria general hospital,baghios pavlos^thessaloniki general hospital, and tzaneion general hospital. informed consent a patient was enrolled after written informed consent provided by his first-degree relatives. community-and healthcareassociated infections in critically ill patients: a multicenter cohort study the third international consensus definitions for sepsis and septic shock (sepsis- ) sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy angiopoietins in angiogenesis and beyond endocan is a novel chondroitin sulfate/dermatan sulfate proteoglycan that promotes hepatocyte growth factor/scatter factor mitogenic activity sis international sepsis definitions conference the international sepsis forum consensus definitions of infections in the intensive care unit diagnosis and treatment of disseminated intravascular coagulation (dic) according to four dic guidelines endothelial cell-specific molecule- /endocan: diagnostic and prognostic value in patients suffering from severe sepsis and septic shock identification of a kda fragment generated by cathepsin g, a novel circulating biomarker in sepsis endocan, a new endothelial marker in human sepsis characteristics of serum endocan levels in infection lower serum endocan levels are associated with the development of acute lung injury after major trauma evaluation of endothelial biomarkers as predictors of organ failures in septic shock patients endocan is a useful biomarker of survival and severity in sepsis endocan levels in peripheral blood predict outcomes of acute respiratory distress syndrome inhibition of caspase- activation in gram-negative sepsis and experimental endotoxemia reversal of immunoparalysis in humans in vivo: a double-blind, placebo-controlled, randomized pilot study key: cord- -s wf pix authors: prehn, jochen h m; jirström, elisabeth title: angiogenin and trna fragments in parkinson’s disease and neurodegeneration date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: s wf pix in this review, we summarise the evidence for a role of the ribonuclease angiogenin in the pathophysiology of neurodegenerative disorders, with a specific focus on parkinson’s disease (pd). angiogenin is a stress-induced, secreted ribonuclease with both nuclear and cytosolic activities. loss-of-function mutations in the angiogenin gene (ang) have been initially discovered in familial cases of amyotrophic lateral sclerosis (als), however, variants in ang have subsequently been identified in pd and alzheimer’s disease. delivery of angiogenin protein reduces neurodegeneration and delays disease progression in in vitro and in vivo models of als and in vitro models of pd. in the nucleus, angiogenin promotes ribosomal rna transcription. under stress conditions, angiogenin also translocates to the cytosol where it cleaves non-coding rna into rna fragments, in particular transfer rnas (trnas). stress-induced trna fragments have been proposed to have multiple cellular functions, including inhibition of ribosome biogenesis, inhibition of protein translation and inhibition of apoptosis. we will discuss recent evidence of trna fragment accumulation in pd, as well as their potential neuroprotective activities. acta pharmacologica sinica ( ) : - ; https://doi.org/ . /s - - - parkinson's disease (pd) is characterized by the progressive degeneration and loss of dopaminergic neurons in the substantia nigra pars compacta and catecholaminergic neurons in the locus coeruleus. loss of neurotrophic or neuroprotective support may therefore increase the likelihood of developing pd. angiogenin is a . kda protein that belongs to the superfamily of vertebrate secreted ribonucleases. other members of this family include rnase (pancreatic ribonuclease), rnase (eosinophil-derived neurotoxin), rnase (eosinophil cationic protein), rnase , rnase (k ), rnase and rnase [ ] . in contrast to most members of this family, angiogenin exhibits relatively low ribonuclease activity [ ] . angiogenin was initially discovered as a tumour-derived angiogenic factor in human colon adenocarcinoma cells [ ] . subsequent studies focused on its role in angiogenesis, cancer, ischaemia and infection (reviewed in [ ] ). as the name suggests, angiogenin stimulates endothelial cell proliferation and has been shown to be required for the angiogenic activity of vascular endothelial growth factor (vegf) and fibroblast growth factor- (fgf- ) [ ] . while vegf and fgf- signal through tyrosine kinase receptors to activate protein synthesis (via stimulation of mtor and s kinase pathways), angiogenin increases ribosomal rna (rrna) transcription in the nucleus [ ] . it thereby acts synergistically with vegf and fgf- to increase protein synthesis in endothelial cells and is required for their proliferation [ ] . angiogenin-induced rna transcription also requires the ribonuclease activity of angiogenin and occurs via an epigenetic activation of the ang promoter [ , ] . angiogenin has also been proposed to indirectly stimulate the pi -kinase and akt kinase pathways in endothelial cells and bladder cancer cells [ ] [ ] [ ] . additional cytosolic activities of angiogenin have recently emerged. under conditions of cellular stress such as oxidative stress, disruption of proteostasis or withdrawal of trophic factors, angiogenin accumulates in cytosolic stress granules [ , ] . stress granules are cytoplasmic foci containing untranslated mrna and rna-binding proteins that are formed in response to cellular stress and function to arrest protein translation [ ] [ ] [ ] . angiogenin is also a secreted factor that can be taken up by endocytosis into the cytoplasm of target cells [ ] [ ] [ ] where it may function in paracrine [ ] . as angiogenin is secreted from cells, several studies investigated potential angiogenin receptors and cellular uptake mechanisms. angiogenin has been shown to be a ligand for surface receptors of the plexin family. plexin-b was identified by yu et al. as receptor for angiogenin in endothelial, cancer, neuronal, and normal hematopoietic and leukaemic stem and progenitor cells [ ] . plexin-b is expressed in the postnatal and adult nervous system particularly in subventricular zone (szv)-derived neural stem cells [ ] . angiogenin may, however, also been taken up through additional mechanisms into other cell types. studies from our group demonstrated that neuronally-secreted or exogenous angiogenin protein is effectively taken up into astroglia [ ] and alters their secretome [ ] . indeed, astroglia are now considered an important contributor to neurodegenerative disorders including pd and amyotrophic lateral sclerosis (als) [ , ] . uptake and subsequent rna cleavage in astrocytes have been shown to require clathrin-mediated endocytosis (cme) and dependent on heparan sulfate proteoglycans [ ] . it is possible that other angiogenin-binding proteins responsible for angiogenin uptake and signalling will be identified going forward. the activity of angiogenin is critically regulated by an endogenous inhibitor, ribonuclease/angiogenin inhibitor (rnh ) [ ] . like angiogenin, rnh is expressed in endothelial cells and many other cell types, including neurons and glial cells [ ] [ ] [ ] . rnh inhibits the activity of angiogenin by binding to its catalytic triad residues lys- , his- and his- [ ] . under stress and anabolic conditions, rnh accumulates in the nucleus where it binds angiogenin and inhibits rrna transcription to save energy [ ] . angiogenin activity is therefore a process that is determined by the relative abundance and co-localisation of both proteins in cells. similar to other pro-angiogenic factors such as vegf, transcription of the ang gene is regulated by the transcription factor hypoxia-inducible factor- α (hif- α). through this mechanisms, increased ang expression stimulates angiogenesis in tissues that have insufficient oxygen supply [ ] . the hypoxia responsive element within the ang gene has been mapped to the consensus hif- α binding site ′-rcgtg- ′ [ , ] . hif- α has also been shown to be required for ang expression in neural cells in response to hypoxia [ ] . ang expression is also positively regulated by the transcription factor hepatic nuclear factor- α (hnf- α) [ ] . this transcription factor is involved in glucose and lipid metabolism in liver and pancreatic beta-cells. angiogenin became of interest to neuroscience with the initial discovery that ang gene variants were associated with familial and apparently sporadic cases of als in scottish, irish, english, swedish and northern american families [ ] . as part of this study, our laboratory identified that angiogenin is expressed and enriched in motor neurons. subsequent studies have identified ang variants in italian, french, german, dutch, belgian, hungarian, chinese and indian als patients ( table ) . several of the ang variants identified were predicted and subsequently validated to affect angiogenin's ribonuclease activity due to their proximity to the catalytic site of the protein (table ) . other angiogenin variants have been shown to inhibit the shuttling of angiogenin between nucleus and cytoplasm [ ] , or to reduce the stability of the protein [ ] . subsequent studies showed that angiogenin exerts neuroprotective activities in vitro in models of excitotoxic, hypoxic and trophic factor-withdrawal-induced injury to motor neurons and other neural cells, including dopaminergic sh-sy y neuroblastoma cells [ , , ] . many of the ang variants identified were shown to have reduced neuroprotective activity compared with wild-type ang when overexpressed at similar levels in neurons [ , ] . however, it is currently unknown which cell types in the nervous system (including endothelial cells) are susceptible to ang mutations. of note, subsequent studies also identified ang variants in familial forms of pd [ , ] . the two studies identified several non-synonymous ang variants in northern american, german, dutch and italian pd patients ( table ). the frequency of pd ang variants were highly similar in both studies ( . %/ . %) compared with controls ( . %/ %). furthermore, van es et al. reported similar frequency of als patients and pd patients carrying ang variants ( . %/ . % compared with . % in controls) [ ] . many of the reported pd ang variants were predicted to impair angiogenin protein function [ ] . in a more recent study, several of these variants were validated to have reduced levels of ribonuclease activity in comparison with wildtype angiogenin [ ] . interestingly, a recent study demonstrated ang mutations in familial cases of alzheimer's disease (ad) [ ] . collectively, these findings point to a general role of angiogenin as a protective factor for central nervous system neurons. of note, ang knock-out mice do not appear to develop reported by [ ] neurodegeneration and als-, pd-, or ad-like symptoms or neuropathology in their life span [ ] , highlighting that aging and/or additional disease processes are required to trigger neurodegeneration. ang can therefore be added to list of genes that regulate stress responses in neurons and are mutated and contribute to neurodegeneration in various neurological disorders (including pd), as seen in the case of tardbp mutations, fus mutations, c orf repeat expansions, and dj- mutations [ ] [ ] [ ] [ ] . the ang mutations that have been reported in als and pd patients suggested a direct involvement of angiogenin in pathways leading to motoneuron degeneration or degeneration of dopaminergic neurons. we demonstrated that angiogenin protects cultured primary mouse motor neurons against alsassociated, stress-induced cell death including excitotoxic injury by promoting and sustaining cell survival signalling through pi kinase/akt kinases [ ] . in further preclinical work, we demonstrated that daily, systemic (intraperitoneal) delivery of recombinant human angiogenin protein significantly increased life span and improved motor function in sod g a mice, an established mouse model of als [ ] . cell survival signalling in motoneurons was preserved in angiogenin-treated mice. importantly, the effect of angiogenin, when delivered post-symptom onset (from day onward) on life span and disease progression, was comparable to the effect of a pre-symptom angiogenin treatment (from day onward). these results suggested that angiogenin protein delivery may be beneficial in treating patients with newly diagnosed als. to further validate these findings, our group performed a sod g a mouse model study according to the preclinical guidelines for als animal studies set by the european als/ mnd group [ ] . in this study, we demonstrated that systemic delivery of human angiogenin three times per week postsymptom onset (from day onward) delayed motor dysfunction, significantly enhanced survival and protected against motoneuron loss and vascular network regression in the lumbar spinal cord [ ] . angiogenin may also be neuroprotective in pd. steidinger et al. demonstrated significantly decreased levels of endogenous angiogenin in an alpha-synuclein transgenic mouse model of pd and showed that recombinant human angiogenin protected against dopaminergic neuronal cell death and inhibited caspase- activation in neurotoxin-induced in vitro models of pd [ ] . a subsequent study by the same group found that virally-mediated overexpression of human angiogenin in the substantia nigra did not protect against dopaminergic cell loss in a neurotoxin-based mouse model of pd [ ] . these findings suggest that further in vivo studies are required to explore potential neuroprotective functions in animal models of pd, in particular in genetic models. during stress conditions, angiogenin accumulates in the cytosol where it cleaves non-coding rnas, including transfer rnas (trnas). cleavage of trnas by angiogenin generates fragments termed 'stress-induced trna fragments' or 'tirnas' [ , , ] . cleavage of trnas by angiogenin occurs in their anticodon loop, a process that is highly regulated by trna modifications [ ] [ ] [ ] [ ] [ ] , so that only specific subsets of trna fragments are generated [ ] . tirnas have been shown to inhibit ribosome assembly [ ] and to inhibit cap-dependent protein translation via interaction with the initiation factor eif f [ ] . both processes may facilitate the recovery of cells during stress conditions, so that resourceconsuming or error-sensitive cell functions are stalled during periods of stress. tirna generation has also been linked to the process of stem cell maintenance and inhibits the proliferation of hematopoietic stem/progenitor cells [ ] . due to their multiple mechanisms of action, lack of tirna production could be involved in the pathogenic effects of ang variants in human disease. trfs in general are now considered a new class on small noncoding rnas with multiple cellular functions, of which tirnas are only a subclass. trfs have been detected in various biological systems suggesting that trna cleavage by angiogenin and other ribonucleases is an evolutionary conserved process. other new functions of trfs beyond the regulation of protein translation have also been reported. 'shot-rnas', which are analogous to angiogenin-produced tirnas, were identified in hormone responsive cancer cells where they stimulate their proliferation [ ] . two studies in showed that trfs fragments are enriched in sperm cells and delivered to the zygote at fertilization where they modified gene expression by binding to the elements in the zygote's genome [ , ] . multiple ribonucleases other than angiogenin are able to generate such fingerprints, including the ribonucleases z and dicer [ ] [ ] [ ] [ ] [ ] . the identification of trf 'fingerprints' in different biological systems and disease conditions is still at its infancy, but interesting observations are now beginning to emerge. a study from our laboratory showed that specific trfs are associated with epilepsy [ ] . small rna sequencing analysis of plasma samples collected during video eeg monitoring of patients with focal epilepsy identified significant differences in three specific trfs fragments compared with healthy controls. interestingly, these trfs are different from angiogenin-generated fragments as cleavage did not occur in the anticodon loop. these fragments were elevated in the pre-seizure period, but lower in post-seizure samples, and may represent a novel class of biomarkers indicating seizure risk in epilepsy patients. a recent study performed in pd patients identified diseasespecific trfs in brain and biofluids [ ] . reanalyses of rnaseq data from three previous studies identified multiple differentially abundant trfs between pd patients and healthy controls in prefrontal cortex, cerebrospinal fluid and serum. of note, a subset of the identified trfs successfully distinguished pd patients from controls with high sensitivity and specificity in each sample collection. further research is required as to whether these fragments are generated by angiogenin or other ribonucleases. collectively, these findings suggested that trf signatures are promising candidates as non-invasive pd biomarkers. there is a significant body of evidence suggesting that angiogenin is a stress-induced survival factor for central nervous system neurons. while it has been shown that angiogenin is able to protect against dopaminergic neuron loss in vitro, further research is required to explore its role in animal models of pd. the arrival of new animal models of pd will likely accelerate this translation, as seen in als models. due to its pleiotropic mechanism of action, angiogenin may indeed be an interesting candidate for the treatment of neurodegenerative disorders. it stimulates angiogenesis in endothelial cells and promotes neuronal survival through akt signalling and possibly through the formation of tirnas, thereby facilitating the recovery of stressed neurons. moreover, trfs generated by angiogenin and other ribonucleases may deliver novel diagnostic or prognostic tools for the management of neurodegenerative disorders. studies are now required to explore the biological functions of these fragments in vitro and in vivo. rnase , a novel rnase a superfamily ribonuclease expressed uniquely in placenta characteristic ribonucleolytic activity of human angiogenin isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells three decades of research on angiogenin: a review and perspective endogenous angiogenin in endothelial cells is a general requirement for cell proliferation and angiogenesis the nuclear function of angiogenin in endothelial cells is related to rrna production angiogenin stimulates ribosomal rna transcription by epigenetic activation of the ribosomal dna promoter angiogenin-induced protein kinase b/akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in huve cells angiogenin induces nitric oxide synthesis in endothelial cells through pi- and akt kinases angiogenin interacts with ribonuclease inhibitor regulating pi k/akt/mtor signaling pathway in bladder cancer cells angiogenin-induced trna-derived stress-induced rnas promote stress-induced stress granule assembly angiogenin-induced trna fragments inhibit translation initiation rna-binding proteins tia- and tiar link the phosphorylation of eif- alpha to the assembly of mammalian stress granules dynamic shuttling of tia- accompanies the recruitment of mrna to mammalian stress granules evidence that ternary complex (eif -gtp-trna(i)(met))-deficient preinitiation complexes are core constituents of mammalian stress granules motoneurons secrete angiogenin to induce rna cleavage in astroglia the cellular uptake of angiogenin, an angiogenic and neurotrophic factor is through multiple pathways and largely dynamin independent nuclear translocation of angiogenin in proliferating endothelial cells is essential to its angiogenic activity plexin-b mediates physiologic and pathologic functions of angiogenin plexin-b regulates the proliferation and migration of neuroblasts in the postnatal and adult subventricular zone angiogenin induces modifications in the astrocyte secretome: relevance to amyotrophic lateral sclerosis non-cell autonomous toxicity in neurodegenerative disorders: als and beyond the a astrocyte paradigm: new avenues for pharmacological intervention in neurodegeneration human placental ribonuclease inhibitor abolishes both angiogenic and ribonucleolytic activities of angiogenin ribonuclease inhibitor: structure and function proteomics. tissue-based map of the human proteome human protein atlas. version . rnh binding of placental ribonuclease inhibitor to the active site of angiogenin ribonuclease/ angiogenin inhibitor regulates stress-induced subcellular localization of angiogenin to control growth and survival regulation of angiogenin expression and epithelial-mesenchymal transition by hif- alpha signaling in hypoxic retinal pigment epithelial cells angiogenin protects motoneurons against hypoxic injury the mouse rnase and rnase /ang locus utilizes dual promoters for tissue-specific expression ang mutations segregate with familial and 'sporadic' amyotrophic lateral sclerosis angiogenin lossof-function mutations in amyotrophic lateral sclerosis characterization of human angiogenin variants implicated in amyotrophic lateral sclerosis control of motoneuron survival by angiogenin human angiogenin is a neuroprotective factor and amyotrophic lateral sclerosis associated angiogenin variants affect neurite extension/pathfinding and survival of motor neurons angiogenin variants in parkinson disease and amyotrophic lateral sclerosis angiogenin variation and parkinson disease structural insights into human angiogenin variants implicated in parkinson's disease and amyotrophic lateral sclerosis a novel nonsense angiogenin mutation is associated with alzheimer disease angiogenin promotes hematopoietic regeneration by dichotomously regulating quiescence of stem and progenitor cells decoding als: from genes to mechanism the p.a t tardbp gene mutation in sardinian patients affected by parkinson's disease and other degenerative parkinsonisms dj- mutations and parkinsonism-dementia-amyotrophic lateral sclerosis complex c orf repeat expansions are a rare genetic cause of parkinsonism guidelines for preclinical animal research in als/mnd: a consensus meeting pleiotropic activity of systemically delivered angiogenin in the sod (g a) mouse model a neuroprotective role for angiogenin in models of parkinson's disease angiogenin in parkinson disease models: role of akt phosphorylation and evaluation of aav-mediated angiogenin expression in mptp treated mice stress induces trna cleavage by angiogenin in mammalian cells angiogenin cleaves trna and promotes stress-induced translational repression rna methylation by dnmt protects transfer rnas against stress-induced cleavage queuosine modification protects cognate trnas against ribonuclease cleavage transfer rna demethylase alkbh promotes cancer progression via induction of trna-derived small rnas dnmt mediates intergenerational transmission of paternally acquired metabolic disorders through sperm small non-coding rnas aberrant methylation of trnas links cellular stress to neuro-developmental disorders angiogenin generates specific stressinduced trna halves and is not involved in trf- -mediated gene silencing shot-rnas: a novel class of trna-derived functional rnas expressed in hormone-dependent cancers sperm tsrnas contribute to intergenerational inheritance of an acquired metabolic disorder biogenesis and function of trna fragments during sperm maturation and fertilization in mammals filtering of deep sequencing data reveals the existence of abundant dicer-dependent small rnas derived from trnas mouse es cells express endogenous shrnas, sirnas, and other microprocessor-independent, dicerdependent small rnas extensive terminal and asymmetric processing of small rnas from rrnas, snornas, snrnas, and trnas a novel class of small rnas: trna-derived rna fragments (trfs) human trnaderived small rnas in the global regulation of rna silencing elevation in plasma trna fragments precede seizures in human epilepsy trna-derived fragments as sex-dependent circulating candidate biomarkers for parkinson's disease angiogenin mutations in hungarian patients with amyotrophic lateral sclerosis: clinical, genetic, computational, and functional analyses identification of new ang gene mutations in a large cohort of italian patients with amyotrophic lateral sclerosis identification of novel angiogenin (ang) gene missense variants in german patients with amyotrophic lateral sclerosis a case of als-ftd in a large fals pedigree with a k i ang mutation evidence for an oligogenic basis of amyotrophic lateral sclerosis accumulation of tdp- and alpha-actin in an amyotrophic lateral sclerosis patient with the k i ang mutation fus mutations in familial amyotrophic lateral sclerosis: genotype-phenotype correlations mutations of the ang gene in french patients with sporadic amyotrophic lateral sclerosis a novel angiogenin gene mutation in a sporadic patient with amyotrophic lateral sclerosis from southern italy identification of a novel missense mutation in angiogenin in a chinese amyotrophic lateral sclerosis cohort sod g d sporadic amyotrophic lateral sclerosis (sals) patient with rapid progression and concomitant novel ang variant identification and characterization of novel and rare susceptible variants in indian amyotrophic lateral sclerosis patients structural and molecular insights into the mechanism of action of human angiogenin-als variants in neurons jhmp and ej wrote the paper. competing interests: jhmp is a beneficiary of a patent relating to the use of angiogenin as a diagnostic and therapeutic for als and other neurodegenerative disorders. ej declares no competing interest. key: cord- -iggkxbbf authors: phillips, m. ian; de oliveira, edilamar menezes title: brain renin angiotensin in disease date: - - journal: j mol med (berl) doi: . /s - - - sha: doc_id: cord_uid: iggkxbbf a brain renin angiotensin system (ras) and its role in cardiovascular control and fluid homeostasis was at first controversial. this was because a circulating kidney-derived renin angiotensin system was so similar and well established. but, the pursuit of brain ras has proven to be correct. in the course of accepting brain ras, high standards of proof attracted state of the art techniques in all the new developments of biolo gy. consequently, brain ras is a robust concept that has enlightened neuroscience as well as cardiovascular physiology and is a model neuropeptide system. molecular biology confirmed the components of brain ras and their location in the brain. transgenic mice and rats bearing renin and extra copies of angiotensinogen genes revealed the importance of brain ras. cre-lox delivery in vectors has enabled pinpoint gene deletion of brain ras in discrete brain nuclei. the new concept of brain ras includes ace- , ang – , and prorenin and mas receptors. angiotensin ii (ang ii) generated in the brain by brain renin has many neural effects. it activates behavioral effects by selective activation of ang ii receptor subtypes in different locations. it regulates sympathetic activity and baroreflexes and contributes to neurogenic hypertension. new findings implicate brain ras in a much wider range of neural effects. we review brain ras involvement in alzheimer’s disease, stroke memory, and learning alcoholism stress depression. there is growing evidence to consider developing treatment strategies for a variety of neurological disease states based on brain ras. of drinking obviously requires neural circuits in the brain. it was reasoned that the effects of ang ii in the brain were isolated from circulating ras by the blood-brain barrier (bbb). however, circumventricular organs (cvos) lack a bbb and the circulating ras and the endogenous brain ras appear to have a complex interface in the cvos [ , ] . to prove a brain ras existed, neurophysiological techniques were required. felix and phillips [ ] showed that ang ii activated neurons in the brain and in the subformical organ (sfo), and a peptide antagonist of ang ii, saralasin, inhibited ang ii-induced activity in those neurons. this suggested that ang ii is a nonclassical neurotransmitter. taking the concept a step further, it was reasoned that if ang ii, formed by brain renin, was involved in hypertension, then inhibiting brain ras would lower blood pressure. simply injecting saralasin alone into the brain of stroke prone hypertensive rats (spshr) temporarily reduced high blood pressure [ ] . this occurred even in the absence of kidney renin when the spshr rats were bilaterally nephrectomized [ ] . the finding was later confirmed by molecular inhibition of ang ii synthesis in the brain [ ] . it meant that brain ras plays a role in neurogenic hypertension. immunocytochemistry with renin antibodies showed staining in neurons [ , ] . renin-like activity was evident in the hypothalamus, pituitary, and pineal glands. antibodies to ang ii revealed a clear distribution of ang iilike staining in the hypothalamus and cvos and in higher concentration in shr [ ] . what was needed were actual measurements of the renin and ang ii proteins. this was the achieved with high-performance liquid chromatography [ , ] and direct protein assay [ ] . brain levels of ang ii were higher than circulating levels of ang ii, suggesting independence of the two systems. having shown that ang ii was in the brain, the next level of proof required demonstrating that it had been synthesized in the brain. molecular biology was used to measure and locate mrna for agt and renin in the brain [ , ] . stornetta et al. [ ] identified glial cells as the site of synthesis of agt using in situ hybridization mrna. mendelsohn et al. [ ] , using autoradiography with pseudocolor imaging, were able to map the entire distribution ang ii receptor binding sites in the brain. this proved that there were sites within the brain that had evolved to be activated by ang ii made in the brain. minute injections of ang ii into key brain nuclei showed that ang ii could differentially produce pressor effects, elicit drinking, release vasopressin, inhibit the baroreflex, and increase sympathetic nervous system activity [ , ] . the confirmation that there was a separate brain ras, independent of the circulating ras, gave rise to the more generalized concept of local or tissue angiotensins in most other tissues including heart, blood vessels, and, ironically, the kidney [ ] whitebread et al. [ ] and chiu et al. [ ] reported that there were two subtypes of the ang ii receptor, at and at . from this has sprung not only insight into genes involved in brain function but also the highly successful angiotensin receptor blockade class of antihypertensive drugs. the development of the transgenic mice [ ] was relevant to brain ras with transgenic mice overexpressing agt by a metallothionein promoter [ ] . transgenic mice, overexpressing agt in brain and liver, led to high blood pressure [ , ] . but, what about renin? renin exists in only one form in humans, ren- , but in two forms in mice, ren- and ren- . after inserting a mouse ren- gene into a transgenic rat [tgr(mren- ) ], mullins et al. [ ] reported fulminant hypertension in transgenic rats, even though circulating ang ii levels were low. the homogenous offspring developed malignant hypertension and had high mortality. the heterozygous offspring had end organ damage in the heart and kidney with extreme hypertension. hypertension disappeared when the brains of these mice was anesthetized. curt sigmund's group [ ] developed transgenic mice that overexpressed human renin and human agt. with either a glial fibrillary acidic protein promoter or a synapsin- promoter, to stimulate gene expression in neurons or glial cells, hypertension and increased drinking resulted. transgenic animals must undergo embryonic development. if the inserted gene or the deleted gene of the transgenic animal is not embryonically lethal, it may be compensated for by other genes duplicating its role. this may account for the surprisingly benign effects of at knockout transgenic mouse [ , ] . to overcome this possibility, we developed gene suppression methods in adult animals using in vivo rna-based inhibition. antisense inhibition of agt mrna or at r mrna oligonucleotides delivered to the brain reduces hypertension in shr [ ] . antisense inhibition of at r specifically diminishes at receptors in the paraventricular nucleus and organum vasculosum of the lamina terminalis [ ] . functionally, at antisense inhibited the effects of ang ii in the brain including the pressor response, vasopressin release, and drinking [ ] . in the double transgenic mice developed by sigmund and colleagues [ ] , to overexpress both human renin and human agt so that they constantly have high ang ii levels, antisense to at r mrna dramatically reduced the blood pressure. [phillips et al., unpublished] . in an elegant study, davisson et al. [ ] , using a combination of at a -and at b -deficient mice, showed that at a receptors are mainly involved in central blood pressure (bp) control, while at b receptors mediate the drinking response. the question of which genes and exactly where in the brain cardiovascular effects are regulated may now be answered with greater precision. davisson and colleagues, who previously demonstrated uptake of vectors into specific location of the brain, reported an approach to gene deletion in mice with the cre-lox system [ ] . cre, a bacteriophage p -derived dna recombinase, serves to recombine precisely defined dna sequences that have been "floxed" by loxp sequences prior to embryonic development. because cre recombines the loxp sequences, the floxed segment is deleted and rendered nonfunctional. sinnayah et al. [ ] microinjected cre with a promoter into selective targets, such as the sfo and supraoptic nucleus, delivered by adenovirus vectors or feline immunodeficiency virus. with this technique, the sigmund group have demonstrated the presence of brain ras and its physiological and blood pressure effects [ , ] . the classical brain ras components must now include ang iii, ang iv, ang ( - ), ace , prorenin, and mas receptors (fig. ) . ang iii is a metabolite of ang ii found in brain and cerebrospinal fluid. it appears to act on the at receptor. ang iv is an endogenous agonist that has been identified in the brain in areas distinct from those where at and at are located and probably acts on an at receptor. an action of brain ras is implied in memory and learning by the finding that stimulation of at receptors potentiates the release of acetylcholine from hippocampal brain slices [ ] . ace generates ang ( - ) from ang i [ , ] ; ace is a human ace homolog that differs greatly from ace in substrate specificity and its activity is not modified by ace inhibitors [ ] . ace is widely expressed in a variety of tissues including brain [ ] [ ] [ ] [ ] . mice lacking the ace gene showed elevation in plasma ang ii level [ ] and enhanced pressor response to ang i. ang ( - ) is formed from ang ii via the action of several tissue-specific endopeptidases or from ang i via ace . ang ( - ) can be metabolized by ace to ang ( ) ( ) ( ) ( ) ( ) ; it is a peptide with no known biological activity. ang ( - ) produces its biological effects such as vasodilatation, diuresis, natriuresis, and antitrophic via the mas receptor [ ] . mas receptor stimulation also induces to production of nitric oxide (no) and prostacyclin. this new [ , ] . this points to a complex interface between the brain ras and the systemic ras in controlling vital cardiovascular functions. prorenin receptors were first cloned by nguyen et al. [ ] . they bind prorenin with higher affinity than renin [ ] . tissue prorenin [ , ] is active and renin per se has cellular effects independent of ang ii generation [ , ] . prorenin receptors may help to explain the presence of tissues ras, where the generation of ang ii is independent of systemic ras. there are two forms of renin expressed in the brain of rodents and humans: secreted prorenin and nonsecreted renin. this suggests that renin is constitutively active [ ] . using transgenic mice that express nonsecreted active renin or secreted prorenin in the brain, bp and drinking volume were increase similarly in both models. bp was normalized by an intracerebral ventricular injection of losartan in both models, whereas the same dose given systemically had no effect. these data support the concept of an intracellular form of renin in the brain, which may promote functional changes in fluid homeostasis and bp regulation [ ] . ace , ang ( - ), and mas receptors ace is a carboxypeptidase that counterregulates the vasoconstrictors effects of ang ii to degrade it to the vasodilator peptide ang ( - ) [ ] . the first functional evidence of the ang ( - ) in the brain showed that the peptide stimulates the release of vasopressin, equivalent to that obtained with ang ii [ ] . in the central nervous system, ang ( - ) acts as neuromodulator, especially in areas related to tonic and reflex control of arterial pressure. its effects are blocked by a mas receptor antagonist a- [ ] . becker et al. [ ] showed mas receptor in cardiovascular and hydroelectrolytic control areas of the rat brain. ang-( - ) appears to act through the mas receptor. the distribution of ace is widespread in the mouse brain, predominantly in neurons. ace mrna is found in rat medulla oblongata, and ace overlaps with compo-nents of the ras in the same areas [ , ] . ace- , ang ( - ), and mas receptors are all localized in brain areas related to the control of cardiovascular function. the physiological effects of central ang ii are mainly mediated by at r, including vasoconstriction effects, vasopressin release, retention of salt and water, cell growth, and stimulation of aldosterone from the adrenal gland [ ] . however, via at , the ang ii mediates a wide variety of actions, including vasodilatation, inhibition of cellular growth, cell differentiation, and apoptosis. in addition to the brain ras role in neurogenic hypertension discussed above, evidence is accumulating that brain ras is involved in alzheimer's disease, stroke, memory and learning, alcoholism, depression and, emotional stress. alzheimer's disease (ad) is an age-related, neurodegenerative disease with a prevalence of - % in individuals over years [ ] . the dementia presents as primary indicator with memory loss following progressive cognitive impairment and personality changes [ ] . two hypotheses have been proposed to explain the pathogenesis of ad [ ] . the amyloid plaque (ap) hypothesis postulates that ap is generated by proteolytic activity of the βand γ-secretase processing the amyloid precursor protein (app). however, although ap was observed in transgenic mice with mutations of app and presenilin- (ps ) genes, the mice did not exhibit neurodegeneration or memory loss observed in ad patients. supporting the presenilin hypothesis, it postulates the neurodegenerative and behavioral effects independent of ap. an increase in the ace activity and alteration in others components of brain ras has been demonstrated in ad [ ] [ ] [ ] [ ] . the beneficial effects of ace inhibitors and at blockers have been shown in animals and humans suggesting reducing brain ras activity is important. there are many effects of decreased ang ii activity including reduced blood pressure, less acetylcholine release, and increase of substance p-a substrate of ace which is reported to increase neprilysin activity, a recognized amyloid β degrading enzyme [ ] . treatment with ace inhibitors and at antagonists has been shown to prevent stroke in spshr and in salt-loaded dahl salt-sensitive rats [ ] . some clinical trails (syst-eur, systolic hypertension in europe; progress, peridopril protection against recurrent stroke) and randomized studies using antihypertensive treatments have been performed using different combinations of ace inhibitors: at receptor antagonists, β-blockers, and diuretics treatments [ ] [ ] [ ] [ ] . at receptor antagonists normalized the expression of cerebrovascular nitric oxide synthase, cerebrovascular compliance, and protected cerebrovascular flow [ ] . similarly, cerebral blood flow was increased by ang ( - ) and reduced by a selective ang ( - ) antagonist a- [ ] . these data suggest that ang ( - ) could have protective effects in ischemia. in general, the results of treatment show a reduction of bp, stroke, vascular dementia, and cognitive decline [ ] . zhou et al. [ ] showed evidence of an active local ras in brain microvessels influenced by circulating prorenin and ang ii with a predominant localization to the microvessel endothelium. kagiyama et al. [ ] induced stroke with middle cerebral artery occlusion in rats and found that local angii levels and at receptor concentrations increased. they concluded that brain ras and at receptors increase apoptosis in cerebral ischemia. several studies have indicated brain ras in memory function. at antagonist treatment reduced anxiety and improve learning, spatial working memory, and motor performance in the aged rat [ ] [ ] [ ] [ ] . denobel et al. [ ] reported that intracerebroventricular infusion of renin disrupts passive avoidance learning. coadministration of ace inhibitor (captopril) or at antagonist attenuates this deficit induced by renin. at receptor antagonist was not effective. ace inhibitors have been shown to enhance memory learned by fear or habituation [ ] . recently, bonini et al. [ ] showed that the intrahippocampal infusion of ang ii in rats induces amnesia. the effect was completely reversible and did not alter locomotion activity, exploratory behavior, or anxiety state. this effect of ang ii was blocked by the at antagonist but not by losartan. long-term potentiation (ltp) is a correlation of learning and memory processes. ang iv and ang ( - ) increase the excitability and ltp of neurons of the hippocampus [ ] . furthermore, mas receptor knockout mice do not show alteration of hippocampal ltp [ ] . a picture emerges of inhibitory influence by ang ii acting at the at receptor, and a facilitatory role by ang iv acting at the at to increase excitability, ltp, associative and spatial learning, and memory. although, the ang ii participation in memory consolidation is controversial, results suggest that ang iv and ang - via mas receptor in the brain are involved in cognitive behavior. alcohol intake ang ii induces drinking, but does it induce alcohol drinking? losartan, the at receptor blocker, has been reported to block toxic effects of low doses of ethanol [ ] . maul et al. [ ] showed that in at knockout mice, ang ii affected alcohol consumption, but at receptor and bradykinin receptors are not involved [ ] . furthermore, ethanol-induced inhibition of hippocampal ltp is mediated by at receptors [ ] . recently, sommer et al. [ ] , using rats treated with spirapril-a blood-brain-penetrating inhibitor of ace-or transgenic rats [tgr(asraogen) ] with reduced central agt expression, showed that increased ethanol consumption was associated with an upregulation of agt transcript levels and a downregulation of local at mrna. taken together, these data suggest that reducing brain ras could be a strategy for treating alcoholism. the etiology of depression involves several neurotransmitter systems. the first evidence that brain ras may be important in depression was observed in hypertensive patients undergoing captopril treatment. depressed patients in the group noted reduced depression [ ] . in this context, ras polymorphisms have been investigated for their association with depression, including the agt t allele, ace d allele, and at r c allele, which are associated with markedly higher plasma agt and ace activity and with greater responsiveness to ang ii at lower concentrations. the ace d allele was more prevalent in depressed patients of japanese population [ ] . agt m allele was associated with increased susceptibility for bipolar affective disorder in brazilian patients [ ] . recently, saab et al. [ ] observed that at c allele was prevalent in a depressed lebanese population. ras polymorphisms associated with depression are, in some cases, associated with suicidal behavior [ ] . among males, the risk for suicide in i/i homozygotes was about % higher than in subjects with other genotypes. ace i/d polymorphism may also increase the vulnerability to suicide [ ] . however, not all studies have found associations of these polymorphisms with depression, so the jury is still out. brain ras distribution of at receptors follows the hypothalamic-pituitary-adrenal axis. during stress, both the peripheral and the central ang ii systems are stimulated, with increases in at receptors expression and ang ii levels. different types of stress have been associated with peripheral sympathetic nerve stimulation, increased plasma renin activity, and higher circulating ang ii [ ] . at r are increased in brain by isolation stress induced by short periods of isolation and to -h isolation stress. at receptor blocker prevented this response and the ulcerations of the gastric mucosa produced by isolation stress [ ] . peng and phillips [ , ] demonstrated that in cold-induced hypertension, agt and ang ii in the brain predominate and in different brain areas, at r are upregulated and at r downregulated. at antisense treatment reduced the cold-stress-induced high blood pressure [ ] . experimental studies also showed that pretreatment with losartan provide protection from stress induced by immobility or forced swimming [ ] . ang ii is an important stress hormone, and blockade of at receptors might be considered for stress-induced disorders. to inhibit central at receptors may be impractical, but the hypothesis of paton suggests inhibition of at r in brain capillaries could be an alternative if no is involved [ , ] . detlev ganten's bold hypothesis in has proven correct. the brain ras is a model for neuropeptide processing and action in the brain. brain ras has neuronal effects far beyond cardiovascular and fluid homeostasis and may be developed for therapies for neurological dysfunctions the discovery of renin years ago angiotensin-forming enzyme in brain tissue a micromethod for the measurement of renin in brain nuclei: its application in spontaneously hypertensive rats the cerebral ventricles as the avenue for the dipsogenic action of intracranial angiotensin subfornical organ: a dipsogenic site of action of angiotensin ii angiotensin ii in central nervous system physiology specific angiotensin ii receptive neurons in the cat subfornical organ lowering of hypertension by central saralasin in the absence of plasma renin antisense inhibition of at receptor mrna and angiotensinogen mrna in the brain of spontaneously hypertensive rats reduces hypertension of neurogenic origin wide distribution of immunoreactive renin in nerve cells of human brain renin-like immunocytochemical activity in the rat and mouse brain angiotensin-like immunoreactivity in the brain of the spontaneously hypertensive rat angiotensin synthesis in the brain and increased turnover in hypertensive rats angiotensin ii in rat brain comigrates with authentic angiotensin ii in blood pressure liquid chromatography presence of renin in primary neuronal and glial cells from rat brain localization of angiotensinogen messenger rna sequences in the rat brain identification of renin and angiotensinogen messenger rna sequences in rat brain astrocytes synthesize angiotensinogen in brain autoradiographic localization of angiotensin ii receptors in rat brain visualization of specific angiotensin ii binding sites in the brain by fluorescent microscopy functions of angiotensin in the central nervous system preliminary biochemical characterization of two angiotensin ii receptor subtypes identification of angiotensin ii receptor subtypes germ-line transformation of mice generation of transgenic mice with elevated blood pressure by introduction of the rat renin and angiotensinogen genes studies on the regulation of renin genes using transgenic mice high blood pressure in transgenic mice carrying the rat angiotensinogen gene fulminant hypertension in transgenic rats harbouring the mouse ren- gene the brain reninangiotensin system in transgenic mice carrying a highly regulated human renin transgene behavioural and cardiovascular effects of disrupting the angiotensin ii type- receptor in mice effects on blood pressure and exploratory behaviour of mice lacking angiotensin ii type- receptor antisense inhibition of hypertension: a new strategy for renin-angiotensin candidate genes a decrease in angiotensin receptor binding in rat brain nuclei by antisense oligonucleotides to the angiotensin at receptor antisense oligonucleotide to at receptor mrna inhibits central angiotensin induced thirst and vasopressin the brain renin-angiotensin system contributes to the hypertension in mice containing both the human renin and human angiotensinogen transgenes divergent functions of angiotensin ii receptor isoforms in the brain targeted viral delivery of the cre recombinase induces conditional gene deletion in cardiovascular circuits of the mouse brain localization of renin expressing cells in the brain using a ren-egfp transgenic model efficient liver-specific deletion of the floxed human angiotensinogen transgene by adenoviral delivery of the cre recombinase in vivo potentiation of cholinergic transmission in the rat hippocampus by angiotensin iv and lvv-hemorphin- a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - distinct roles for ang ii and ang-( - ) in the regulation of angiotensin-converting enzyme in rat astrocytes new mass spectrometric assay for angiotensin-converting enzyme activity angiotensin-converting enzyme is an essential regulator of heart function angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas detection of angiotensin ii mediated nitric oxide release within the nucleus of the solitary tract using electron-paramagnetic resonance (epr) spectroscopy pivotal role of the renin/prorenin receptor in angiotensin ii production and cellular responses to renin binding properties of rat prorenin and renin to the recombinant rat renin/prorenin receptor prepared by a baculovirus expression system tissue activity of circulating prorenin in vivo enzymatic assay reveals catalytic activity of the human renin precursor in tissues specific receptor binding of renin on human mesangial cells in culture increases plasminogen activator inhibitor- antigen evidence supporting a functional role for intracellular renin in the brain effects of reninangiotensin system blockade on renal angiotensin-( - ) forming enzymes and receptors release of vasopressin from the rat hypothalamoneurohypophysial system by angiotensin-( - ) heptapeptide cardiovascular effects of angiotensin-( - ) injected into the dorsal medulla of rats immunofluorescence localization of the receptor mas in cardiovascular-related areas of the rat brain differential expression of neuronal ace in transgenic mice with overexpression of the brain reninangiotensin system is inhibition of the reninangiotensin system a new treatment option for alzheimer's disease transcriptional regulation of the presenilin- gene: implication in alzheimer's disease effects of brain-penetrating ace inhibitors on alzheimer disease progression angiotensin-converting enzyme inhibitors and incidence of alzheimer's disease in japan prevention of dementia: lessons from syst-eur and progress prevention of dementia in randomised double-blind placebo-controlled systolic hypertension in europe (syst-eur) trial stroke prevention by losartan in stroke-prone spontaneously hypertensive rats brain angiotensin ii: new developments, unanswered questions and therapeutic opportunities systemic and regional. hemodynamic effects of angiotensin-( - ) in rats at receptor blockade regulates the local angiotensin ii system in cerebral microvessels from spontaneously hypertensive rats expression of angiotensin type and type receptors in brain after transient middle cerebral artery occlusion in rats non-peptide angiotensin ii receptor antagonist and angiotensin-converting enzyme inhibitor: effect on a renin-induced deficit of a passive avoidance response in rats angiotensin ii blocks memory consolidation through an at receptor-dependent mechanism angiotensin ii disrupts inhibitory avoidance memory retrieval angiotensin-( - ) enhances ltp in the hippocampus through the gprotein-coupled receptor mas losartan reduces ethanol intoxication in the rat central angiotensin ii controls alcohol consumption via its at receptor ethanol and diazepam inhibition of hippocampal ltp is mediated by angiotensin ii and at receptors plasticity and impact of the central renin-angiotensin system during development of ethanol dependence captopril treatment of major depression with serial measurements of blood cortisol concentrations frequency of the fragile x syndrome in japanese mentally retarded males angiotensinogen and angiotensin converting enzyme gene polymorphisms and the risk of bipolar affective disorder in humans renin-angiotensin-system gene polymorphisms and depression association between a functional polymorphism in the renin-angiotensin system and completed suicide angiotensin ii-an important stress hormone reduction of cold-induced hypertension by antisense oligodeoxynucleotides to angiotensinogen mrna and at -receptor mrna in brain and blood the predominant role of brain angiotensinogen and angiotensin in environmentally induced hypertension brain and peripheral angiotensin ii play a major role in stress differential baroreflex control of sympathetic drive by angiotensin ii in the nucleus tractus solitarii acknowledgements em oliveira is recipient of a brazil-cnpq fellowship. mip was supported by nih merit award r -hl and nih r , r -hl . key: cord- -z ddajd authors: shenoy, vinayak; ferreira, anderson j.; katovich, michael; raizada, mohan k. title: angiotensin-converting enzyme /angiotensin-( - )/mas receptor axis: emerging pharmacological target for pulmonary diseases date: - - journal: the protective arm of the renin angiotensin system (ras) doi: . /b - - - - . - sha: doc_id: cord_uid: z ddajd experimental and clinical evidence supports an active role of the renin–angiotensin system (ras) in the pathogenesis and progression of lung diseases. angiotensin ii (ang ii), a key vasoactive peptide of the ras, has been implicated in pulmonary disorders such as pulmonary arterial hypertension, lung fibrosis, chronic obstructive pulmonary disease, and acute respiratory distress syndrome. over the past few years, the classical concept of the ras has undergone substantial changes to include several new active components. among them, the identification of angiotensin-converting enzyme (ace ), its metabolic product angiotensin-( - ) (ang-( - )), and the mas receptor has been of biological significance since these components form a counterregulatory axis (ace /ang-( - )/mas) that opposes the detrimental actions of ang ii. in this chapter, we will discuss the role of the ace /ang-( - )/mas axis in lung diseases and describe novel therapeutic approaches to activate this axis for the treatment of pulmonary disorders. the renin-angiotensin system (ras) has long been recognized as an important player in the pathogenesis of several lung diseases, including pulmonary arterial hypertension (pah), pulmonary fibrosis (pf), chronic obstructive pulmonary disease (copd), and acute respiratory distress syndrome (ards). evidence for this stems from the following observations: (a) pah and pf are associated with higher circulating levels of angiotensin ii (ang ii) , ; (b) increased concentrations of angiotensinogen (the precursor for ang ii peptide) and angiotensin-converting enzyme (ace), the major generator of ang ii, have been observed in the lungs of fibrotic and pulmonary hypertensive subjects , ; (c) patients carrying the ace id/dd genotype, which confers increased ace levels, are susceptible to copd and ards , ; (d) human lung fibroblasts obtained from patients with pf, but not from normal lungs, generate ang ii, suggesting a causative role for this peptide in disease pathogenesis; (e) ang ii induces apoptosis of the alveolar epithelial cells, a key initiating factor for lung fibrogenesis ; (f) ang ii is a potent pulmonary vasoconstrictor with mitogenic properties, that produces migratory, hypertrophic, and proliferative effects on the lung smooth muscles to cause pah ; (g) ang ii mediates oxidative stress and cytokine signaling, factors that contribute to the pathophysiology of lung diseases; and (h) pharmacological blockade of the ras using ace inhibitors (acei) or angiotensin receptor blockers (arb) offers protection against animal models of copd, pah, and lung fibrosis. , , collectively, these findings indicate an active involvement of the ras in lung diseases. however, clinical studies with acei or arbs have yielded mixed results, failing to reach a consensus opinion on the use of these agents for treating pulmonary disorders. discovery of several new members has led to the emergence of a new arm of the ras, which has forced a reevaluation of the role of this hormonal system in pulmonary physiology and pathobiology. these new components include the monocarboxypeptidase angiotensin-converting enzyme (ace ), the vasoactive peptide angiotensin-( - ) (ang-( - )), and its g protein-coupled receptor mas, which together form a protective arm, the ace /ang-( - )/mas axis of the ras. this axis counteracts the vasoconstrictive, proliferative, inflammatory, and fibrotic actions of ang ii in several target organs, including the lungs. our objective in this chapter will be to (i) summarize the importance of the ace /ang-( - )/mas axis in lung diseases and (ii) explore novel strategies to upregulate this axis that hold great potential for clinical development in the treatment of pulmonary disorders. the classical ras comprises a cascade of proteolytic enzymes and peptides located primarily within the circulatory system. however, in addition to the circulating ras, a tissue-specific ras has been identified in several organs, including the heart, kidneys, and lungs. biochemical and molecular studies have revealed that all the necessary components required for the active biosynthesis of ang ii, namely, renin, angiotensinogen, and ace, are present within the lung tissue. furthermore, expression of at r and at r at both mrna and protein levels has been detected in the pulmonary system. since at r gene expression is more abundant than at r in the adult lungs, this receptor mediates most of the biological effects of ang ii. the intrapulmonary ace-ang ii-at r axis has been shown to promote vasoconstriction, smooth muscle cell proliferation, and fibrosis, along with induction of oxidative stress and inflammation. thus, this axis forms the vasodeleterious arm of the ras. on the other hand, identification of ace and mas receptor within the lung parenchymal cells has lent credence to the existence of a vasoprotective arm of the ras. we believe that an imbalance between vasodeleterious and vasoprotective axes of the ras plays a crucial role in the pathophysiology of lung diseases. ace is a zinc-dependent metalloproteinase that is widely expressed on the pulmonary endothelium, type ii alveolar epithelial and smooth muscle cells of the lungs. its primary physiological function is to metabolize ang ii to ang-( - ), a biologically active heptapeptide that binds to the mas receptor to exert vasodilatory, antiproliferative, and antifibrotic actions in the pulmonary system. it is becoming increasingly evident that downregulation of the ace /ang-( - )/mas axis contributes to the development and progression of lung diseases. the importance of ace in pulmonary physiology was appreciated when this enzyme was identified as a functional receptor for the sars coronavirus (sars-cov), the causative agent of severe acquired respiratory syndrome (sars). in vivo experimental studies revealed that lung ace expression is markedly decreased upon sars-cov infection, which consequently leads to respiratory failure and death. the protective role of ace against lung injury was further underscored by studies carried out on ace -deficient animals. knockout mice for ace displayed enhanced vascular permeability, increased lung edema and neutrophil accumulation, which worsened pulmonary function in experimental models of ards. with respect to fibrotic lung diseases, li et al. were the first to demonstrate severe downregulation of ace and its catalytic activity in the lungs of animals, as well as patients with pf. also, inhibition/silencing of pulmonary ace in rodents was associated with excessive lung collagen accumulation, suggesting that decreased ace levels play a causal role in lung fibrogenesis. consistent with this interpretation, mice deficient in ace gene were more susceptible to bleomycin-induced injury, exhibiting excessive deposition of extracellular matrix proteins in the lungs as compared with wild-type animals. a growing body of studies has focused on the relevance of the ace /ang-( - )/mas axis in pah. pah is a fatal lung disease that is associated with increased blood pressure in the pulmonary arteries. decreased expression of ace and enzymatic activity has been observed in animal models of pah and pulmonary hypertensive patients, along with a substantial increase in circulating ang ii levels. , thus, it is evident that restoring the balance between the vasodeleterious and the vasoprotective axes of the ras could be therapeutically beneficial in attenuating disease pathogenesis. ards is the most severe form of acute lung injury that is triggered by sepsis, gastric juice aspiration, or pathogenic infections such as sars-cov and h n avian influenza virus. in experimental models of ards, treatment of wild-type mice and ace knockout mutants with catalytically active recombinant ace protein rescued them from lung failure. likewise, administration of ang-( - ) conferred beneficial effects against acute lung injury, an effect that was abolished by blocking the mas receptor. with regard to pah, the enzymatic activity of ace offers a dual beneficial effect. on one hand, it decreases the levels of ang ii, which are significantly elevated during pah, and on the other, it increases the concentration of the vasoprotective heptapeptide, ang-( - ). we were the first to demonstrate that pulmonary overexpression of ace attenuates monocrotaline (mct)-induced pah and associated pathophysiology. similarly, genetic overexpression of ang-( - ) rendered beneficial effects against this disease. interestingly, coadministration of a- , a mas antagonist, abolished the cardiopulmonary protection offered by ang- ( - ) , suggesting the involvement of mas-mediated signaling pathway. in a separate set of experiments, kleinsasser et al. demonstrated that administration of recombinant human ace protein (rhace ) decreased hypoxia-induced pulmonary vasoconstriction. on the clinical front, it was observed that the serum ace activity was significantly reduced in patients with pah. apparently, elevating circulating levels of ace in these pulmonary hypertensive patients could yield beneficial results. pf is a debilitating disease marked by scarring in the lungs. treatment of ace knockout animals with rhace attenuated bleomycin-induced pf and improved lung function. furthermore, studies from our group have shown that pulmonary overexpression of either ace or ang-( - ) by lentiviral-mediated gene transfer prevents lung fibrosis in the bleomycin model. one of the hallmarks of pf is the loss of alveolar epithelial cells (aecs). mechanistic studies carried out in cultured aecs revealed that ace mediates cell survival by balancing the proapoptotic ang ii and its antiapoptotic metabolic product ang-( - ). this is evidenced by the fact that silencing of the ace gene in aecs or its pharmacological inhibition resulted in increased ang ii and decreased ang-( - ) levels, which was associated with apoptotic cell death. on the contrary, treatment of aecs with ang-( - ) attenuated ang ii and bleomycin-induced apoptosis. this cell survival effect of ang-( - ) was mediated through inhibition of caspases (caspases and ) and downregulation of the jun n-terminal kinase (jnk) pathways. also, these ang-( - )-mediated effects were reversed by blocking the mas receptor or with antisense nucleotides against the mas mrna. taken together, these findings implicate a potent antifibrotic role for the ace / ang-( - )/mas axis against pf. administration of rhace has been shown to exert promising effects in a variety of disease models with pathologically elevated ang ii levels or a dysregulated ras. these experimental findings have provided compelling evidence for initiating clinical trials with rhace . in this regard, glaxosmithkline is currently evaluating the effects of gsk (formerly apn ), a soluble form of rhace , in a multicenter phase ii clinical trial for the treatment of acute lung injury. previous phase i trials have established that administration of single and multiple doses of rhace is well tolerated by healthy humans, with no serious adverse events or dose-limiting toxicities. furthermore, in a separate clinical study, the efficacy of rhace is being assessed for the treatment of pah (clinicaltrials.gov; nct ). although clinical trials are currently under way, the use of protein therapy faces several challenges. among these, the cost of manufacturing the recombinant protein, its stability, repetitive intravenous dosing, and patient compliance pose major obstacles in realizing the full therapeutic potential of ace therapy. an ideal approach to overcome the aforementioned limitations of recombinant protein therapy would be to identify small synthetic molecules that can favorably activate endogenous ace . using a structure-based drug design, we have identified synthetic enhancers of ace such as resorcinolnaphthalein, -[[ -(dimethylamino)ethyl]amino]- -(hydroxymethyl)- -[[( -methylphenyl)sulfonyl]oxy]- h-xanthone (xnt), and diminazene aceturate (dize). [ ] [ ] [ ] in silico docking studies revealed that binding of these small molecules to a novel pocket in the hinge-bending region of the ace protein stabilizes the enzyme in an open conformation. this is important because only the open conformation favors/accelerates enzyme activity. among the three compounds, dize was found to be the most potent, followed by xnt, and finally resorcinolnaphthalein. pharmacologically, all these ace activators were found to be effective in attenuating lung injury. in addition, xnt exhibited strong antithrombotic actions along with decreased platelet attachment, whereas dize therapy was associated with enhanced endothelial progenitor cell (epc) function in pulmonary hypertensive animals, factors that could potentiate the cardiopulmonary beneficial effects of these agents. however, there are some critical issues that impede the successful translation of these ace activators into the clinic. xnt has an undesirable pharmacokinetic profile. it is poorly soluble in water and requires acidic ph for solubilization, making it an unsuitable drug candidate. with regard to dize, it is associated with toxic side effects. chronic treatment with dize has been shown to harm the liver, kidneys, and brain, which could potentially result in life-threatening situations. in fact, a preslaughter withdrawal period of - days has been recommended for dize when treated animals are intended for human consumption. besides, dize was found to be mutagenic, but not teratogenic. nonetheless, xnt and dize could serve as lead compounds that require structural modification to make them safe and druggable. it is evident that ang-( - ) holds promise for the treatment of lung diseases. however, the use of ang-( - ) as a therapeutic agent is limited because of its short half-life in vivo. also, being of peptide nature, ang-( - ) will be rapidly degraded in the gastrointestinal tract when given orally. to overcome these issues, formulations of ang-( - ) with extended half-life and improved oral bioavailability are currently being developed. ang-( - ) complexed with beta-cyclodextrin (hpβcd/ ang-( - )) enabled effective oral administration, with significant increases in plasma ang-( - ) levels. in addition, kluskens and coworkers synthesized a cyclic analog of ang-( - ) (thioether-bridged ang-( - )) that exhibited better in vivo pharmacokinetic profile. alternatively, liposomal delivery system represents an effective method to administer ang-( - ). administration of liposomes containing ang-( - ) was also found to increase the half-life of this heptapeptide. importantly, all these modified forms of ang-( - ) were pharmacologically active when tested in experimental models. it is worth noting that txa , a synthetic analog of ang-( - ) from tarix pharmaceuticals, is currently in clinical trials for the treatment of pah. txa could also be a promising candidate for pf therapy. given that stimulation of the mas receptor is protective, it would be reasonable to evaluate the effects of mas agonists for pulmonary therapeutics. ave is the first nonpeptide agonist of the mas receptor that was shown to promote beneficial effects against several cardiovascular diseases. an important aspect of this compound is that it is orally active and mimics the effects of ang-( - ) in several organs by binding to the mas receptor. recently, two novel peptides (cgen and cgen ) with high specificity for the mas receptor have been discovered. all these mas agonists have the potential to treat pulmonary diseases. the oral route is the most widely accepted means of drug administration. but oral delivery of therapeutic proteins like ace and ang-( - ) is not feasible since they will be easily degraded in the gastrointestinal tract. however, we have developed a low-cost oral delivery system for administering ace and ang-( - ) using transplastomic technology. the use of transplastomic technology allows chloroplasts to generate high levels of therapeutic proteins within plant leaves. since chloroplast expression of ace or ang-( - ) enables the bioencapsulation of these therapeutic proteins within plant cells, it protects them against gastric enzymatic degradation upon oral delivery. we observed that oral feeding of rats with bioencapsulated ace or ang-( - ) prevented the development of mct-induced ph and was associated with improved right heart function. it is possible to produce bulk quantities of therapeutically active ace and ang-( - ) using transplastomic technology, which holds great potential for clinical development as an oral delivery system. reduced number and impaired function of circulating/resident stem cells have been associated with poor cardiopulmonary outcomes in patients and experimental models of lung diseases. in this regard, exogenous administration of stem cells such as endothelial progenitor cells, bone marrow-or adipose-derived mesenchymal stromal cells (mscs), and umbilical cord blood cells could offer protection. however, such cell-based therapy presents a number of unique challenges before they can be effectively employed in the clinical setting. the hostile environment characterized by inflammation and high oxidative stress within the diseased lungs could threaten survival and impair homing of the injected stem/progenitor cells. genetic modification of these cells to overexpress ace or ang-( - ) could improve survival and enhance their potential to effectively repair lung injury. in fact, we have shown that ace priming of endothelial progenitor cells not only enhances their function but also increases their therapeutic efficacy to render protection against stroke. conclusions a dysregulated pulmonary angiotensin system contributes to the pathogenesis and progression of lung diseases. discovery of the protective ace /ang-( - )/mas axis has provided a novel and more promising target for disease treatment than the classical approach of using acei or arbs. innovative pharmaceutical strategies that can effectively activate this beneficial ace /ang-( - )/mas axis hold great promise for treating pulmonary pathologies. dysregulated renin-angiotensin-aldosterone system contributes to pulmonary arterial hypertension extravascular sources of lung angiotensin peptide synthesis in idiopathic pulmonary fibrosis angiotensin signalling in pulmonary fibrosis expression of pulmonary vascular angiotensin-converting enzyme in primary and secondary plexiform pulmonary hypertension renin-angiotensin system blockade: a novel therapeutic approach in chronic obstructive pulmonary disease angiotensin converting enzyme insertion/deletion polymorphism is associated with susceptibility and outcome in acute respiratory distress syndrome angiotensin ii induces apoptosis in human and rat alveolar epithelial cells vasoconstrictor effects of angiotensin ii on the pulmonary vascular bed role of the renin-angiotensin system in vascular diseases: expanding the field role of angiotensin-converting enzyme and angiotensin ii in development of hypoxic pulmonary hypertension losartan attenuates bleomycin induced lung fibrosis by increasing prostaglandin e synthesis targeting the vasoprotective axis of the renin-angiotensin system: a novel strategic approach to pulmonary hypertensive therapy the pulmonary renin-angiotensin system regulation of alveolar epithelial cell survival by the ace- /angiotensin - /mas axis tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis angiotensin-converting enzyme is a functional receptor for the sars coronavirus a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury angiotensin-converting enzyme protects from severe acute lung failure angiotensin converting enzyme- is protective but downregulated in human and experimental lung fibrosis angiotensin converting enzyme abrogates bleomycin-induced lung injury angiotensin-( - ) protects from experimental acute lung injury prevention of pulmonary hypertension by angiotensin-converting enzyme- gene transfer the angiotensin converting enzyme /angiotensin-( - )/mas axis confers cardiopulmonary protection against lung fibrosis and pulmonary hypertension recombinant angiotensin-converting enzyme suppresses pulmonary vasoconstriction in acute hypoxia the changes of serum angiotensin-converting enzyme in patients with pulmonary arterial hypertension due to congenital heart disease pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme in healthy human subjects structure-based identification of small-molecule angiotensinconverting enzyme activators as novel antihypertensive agents diminazene attenuates pulmonary hypertension and improves angiogenic progenitor cell functions in experimental models evidence for angiotensin-converting enzyme as a therapeutic target for the prevention of pulmonary hypertension ace activation promotes antithrombotic activity an orally active formulation of angiotensin-( - ) produces an antithrombotic effect angiotensin-( - ) with thioether bridge: an angiotensinconverting enzyme-resistant, potent angiotensin-( - ) analog long-lasting cardiovascular effects of liposome entrapped angiotensin-( - ) at the rostral ventrolateral medulla the nonpeptide angiotensin-( - ) receptor mas agonist ave- attenuates heart failure induced by myocardial infarction vascular relaxation, antihypertensive effect, and cardioprotection of a novel peptide agonist of the mas receptor oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells oral delivery of angiotensin-converting enzyme and angiotensin-( - ) bioencapsulated in plant cells attenuates pulmonary hypertension angiotensin-converting enzyme priming enhances the function of endothelial progenitor cells and their therapeutic efficacy key: cord- -ksacxsdk authors: shoieb, sherif m.; el-kadi, ayman o. s. title: resveratrol attenuates angiotensin ii-induced cellular hypertrophy through the inhibition of cyp b and the cardiotoxic mid-chain hete metabolites date: - - journal: mol cell biochem doi: . /s - - - sha: doc_id: cord_uid: ksacxsdk several reports demonstrated the direct contribution of cytochrome p b (cyp b ) enzyme and its associated cardiotoxic mid-chain, hydroxyeicosatetraenoic acid (hetes) metabolites in the development of cardiac hypertrophy. resveratrol is commercially available polyphenol that exerts beneficial effects in wide array of cardiovascular diseases including cardiac hypertrophy, myocardial infarction and heart failure. nevertheless, the underlying mechanisms responsible for these effects are not fully elucidated. since resveratrol is a well-known cyp b inhibitor, the purpose of this study is to test whether resveratrol attenuates angiotensin ii (ang ii)-induced cellular hypertrophy through inhibition of cyp b /mid-chain hetes mechanism. rl- and h c cells were treated with vehicle or μm ang ii in the absence and presence of , or μm resveratrol for h. thereafter, the level of mid-chain hetes was determined using liquid chromatography–mass spectrometry (lc/ms). hypertrophic markers and cyp b gene expression and protein levels were measured using real-time pcr and western blot analysis, respectively. our results demonstrated that resveratrol, at concentrations of and μm, was able to attenuate ang-ii-induced cellular hypertrophy as evidenced by substantial inhibition of hypertrophic markers, β-myosin heavy chain (mhc)/α-mhc and atrial natriuretic peptide. ang ii significantly induced the protein expression of cyp b and increased the metabolite formation rate of its associated mid-chain hetes. interestingly, the protective effect of resveratrol was associated with a significant decrease of cyp b protein expression and mid-chain hetes. our results provided the first evidence that resveratrol protects against ang ii-induced cellular hypertrophy, at least in part, through cyp b /mid-chain hetes-dependent mechanism. pathological cardiac hypertrophy is a complex condition that is characterized by increased thickness of the cardiac muscle and a reduction of the size of the chambers of the heart, representing a major risk factor for developing most phenotypes of heart failure [ , ] . heart failure (hf) remains a global pandemic that represents a leading cause of morbidity and mortality worldwide. in spite of considerable gains in the treatment options, there are yet cases where hf patients continue to be symptomatic, posing a major burden on the health care system [ , ] . according to heart and stroke foundation of canada, hf is affecting more than , canadians with , new cases being diagnosed with the disease annually and it is responsible for an estimated health expenditure of around $ . billion per year as a direct cost [ ] . searching for possible treatments that halt the development and progression of cardiac hypertrophy is regarded as an important step in the prevention of a major risk factor of heart failure and will allow for designing novel therapeutic modalities. a plethora of molecular mechanisms are involved in the pathogenesis of cardiac hypertrophy and the consequent cardiac remodeling. one of these mechanisms is cytochrome p (cyp) enzymes and their associated arachidonic acid (aa) metabolites [ , ] . in the heart, cyp enzymes metabolize aa into either cardioprotective metabolites, such as epoxyeicosatrienoic acids (eets) and -hydroxyeicosatetraenoic acid , or cardiotoxic metabolites exemplified as mid-chain hetes and -hete [ , ] . recently, cardiac cyp b and its associated mid-chain hetes, typified by -, -, -, -, -and -hete, have received a particular interest as a potential druggable target. mid-chain hetes are group of biologically active eicosanoids that are produced from the metabolism of aa by both lipoxygenaseand cyp-catalyzed bis-allylic oxidation reaction [ , ] . a growing body of evidence demonstrated that mid-chain hetes possess cardiotoxic effects and they are involved in the pathogenesis of cardiovascular diseases (cvd) including atherosclerosis, hypertension, cardiac hypertrophy and hf [ ] [ ] [ ] . several reports showed that overexpression of cyp b in the cardiac muscle caused an increase in the level of cardiotoxic mid-chain hetes leading to deterioration of the cardiac histo-architecture and function [ , , ] . on the contrary, strategies to mitigate cyp b activity either through pharmacological inhibition or genetic deletion have been employed to protect against cvd. , ′, , ′-tetramethoxystilbene (tms), a well-known cyp b inhibitor, was able to significantly decrease the level of mid-chain hetes and protect against angiotensin ii (ang ii)-induced cardiac hypertrophy [ ] . in addition, cyp b genetic disruption resulted in a reduction of cardiac hypertrophy in ang ii-induced model of hypertension in mice [ ] . resveratrol is a naturally occurring polyphenol that is found in many food sources and beverages including black grapes, peanuts, blueberries and red wine and as a commercially available dietary supplement [ ] . resveratrol possesses anti-oxidant, anti-inflammatory, anti-apoptotic and anti-fibrotic effects. of interest, resveratrol has been shown to improve the myocardial energetics and is experimentally used for prevention and treatment of multiple cvd [ , ] . resveratrol has been shown to exert an inhibitory effect on recombinant cyp b supersomes with ic = . µm [ ] . in addition, it inhibited tcdd-mediated induction of cyp b in cultured human mammary epithelial cells and human breast cancer cell line mcf- [ , ] . several reports have demonstrated that resveratrol protects against cardiac hypertrophy through modulation of various pathways [ ] [ ] [ ] . however, none of these studies investigated the effects of resveratrol on cyp b . therefore, the purpose of this study is to investigate whether resveratrol protects against ang ii-induced cellular hypertrophy through inhibition of cyp b /mid-chain hetes mechanism. human cardiomyocyte (rl- ) cells and rat cardiomyoblast (h c ) cell lines were obtained from american type culture collection (atcc) (manassas, va, usa). rl- cells were grown in dmem/f- , with phenol red supplemented with . % fetal bovine serum, μm l-glutamine, % penicillin-streptomycin. h c cells were grown in dmem containing % heat-inactivated fetal bovine serum, and % penicillin-streptomycin. cells were grown in cm cell culture flasks at °c in a % co humidified environment. cells were treated with vehicle (serum-free dmem/f- containing . % dmso for rl- cells and serum-free dmem containing . % dimethyl sulfoxide (dmso) for h c cells), μm ang ii, μm ang ii in combination with ( , or μm resveratrol) or ( , or μm resveratrol alone) in serum-free medium (sfm) for the time indicated for each experiment as described in the figure legends. resveratrol was prepared as a stock solution in dmso and maintained in dmso at − °c until use. the treatment of cells was performed in the corresponding culture media in -well cell culture plates for rna assay and -well cell culture plates for protein and determination of aa metabolites assays. ang ii stock solution was prepared in saline and maintained at − °c until use. diluted solutions of ang ii and resveratrol were freshly prepared in sfm before each experiment. in all experiments, the dmso concentration did not exceed . % (v/v). mtt assay was used to determine the viability of rl- and h c cells. the main principle of this assay is the ability of viable cells to reduce tetrazolium dye mtt -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide to its insoluble colored formazan crystals. briefly, cells were seeded in -well plates, treated with varying concentrations of resveratrol, incubated for h at °c in a % co humidified incubator, incubated with μl/well mtt ( . mm) dissolved in phosphate-buffered saline (pbs; ph . ) for h at °c, and then μl/well isopropyl alcohol to dissolve the formazan for min at room temperature. the optical density was assessed at a wavelength of nm using the bio-tek synergy h hybrid multi-mode microplate reader (bio-tek instruments, winooski, vt, usa). the cell viability was presented as a percentage of the control mean absorbance value. rl- and h c cells were seeded in -well plates and treated with the test compounds for h. thereafter, cells were collected and total rna was isolated using trizol reagent (invitrogen®) according to the manufacturer's instructions, and quantified by measuring the absorbance at nm while the purity was determined by measuring the / ratio (> . ). afterward, first-strand cdna synthesis was carried out using the high-capacity cdna reverse transcription kit (applied biosystems) according to the manufacturer's instructions. in brief, . μg of total rna from each sample was added to a mix of . μl × rt buffer, . μl × dntp mix ( mm), . μl × reverse transcriptase random primers, . μl multiscribe™ reverse transcriptase enzyme and . μl nuclease-free water. the final reaction mixture was kept at °c for min, heated to °c for min, heated for °c for min and finally cooled to °c. real-time pcr was utilized to quantitatively determine specific mrna expression of different targets by subjecting the resultant cdna to pcr amplification using -well optical reaction plates in the abi prism system (applied biosystems). the reaction mixture ( μl) contained . μl of μm forward primer and . μl of μm reverse primers ( nm final concentration of each primer), . μl of sybr green universal master mix, . μl of rnase/ dnase-free water, and . μl of cdna sample. human primer sequences for cyp b , atrial natriuretic peptide (anp), α-myosin heavy chain (α-mhc), β-myosin heavy chain (β-mhc), and β-actin as well as rat primer sequences for cyp b , anp, α-mhc, β-mhc, and β-actin are listed in table . primers were purchased from integrated dna technologies idt (coralville, ia, usa). analysis of the real-time pcr data was carried out using the relative gene expression (i.e., ΔΔct) method. in brief, the fold change in the level of target genes between treated and untreated cells, corrected for the level of housekeeping gene (β-actin), was determined using the following equation: fold change = −Δ(Δct) , where Δct = ct(target) − ct(β-actin) and Δ(Δct) = Δct(treated) − Δct(untreated). the thermal cycle parameters were as follow: initiation of the reaction at °c rl- and h c cells were grown in -well plates and incubated with the test compounds for h. thereafter, lysis buffer containing mm hepes, . m sodium chloride, . mm magnesium chloride, mm edta, % (v/v) glycerol, % triton x- , and μl/ml protease inhibitor cocktail was added to collect the cells. cell lysates were incubated on ice for h to prepare the cell homogenates while they are sporadically vortexed every min, followed by centrifugation at , ×g for min at °c. supernatant of total cellular lysate was collected and maintained at − °c. subsequently, lowry assay was carried out to determine the concentration of protein using bovine serum albumin as a standard [ ] . western blot analysis was performed according to previously detailed assay [ ] . briefly, total cell lysates ( μg) were separated by % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page), samples were undergone electrophoresis at v for h and separated proteins were transferred onto immun-blot® pvdf membrane. afterward, protein membranes were blocked overnight at °c using blocking solution containing . m sodium chloride, mm potassium chloride, mm tris-base, % skim milk, % bovine serum albumin, and . % tween- . following blocking, the blots were subjected to washing cycles times for min with tris-buffered saline (tbs)-tween- . the blots were subsequently incubated for h at °c with primary antibodies in tbs solution ( . % (v/v) tween- , . % sodium azide). incubation with secondary antibodies (peroxidase-conjugated igg) in blocking solution was performed for min at room temperature. visualization of the bands was carried out using the enhanced chemiluminescence method according to the manufacturer's guide (ge healthcare life sciences). imagej software (national institutes of health, bethesda, md, usa; https ://rsb.info. nih.gov/ij) was used to quantify the intensity of the protein bands in relation to the signals acquired from gapdh loading control. data, given in the figures, are represented as relative protein intensity (%) + sem, as compared to control group. to investigate the effect of resveratrol on mid-chain hete metabolites, rl- and h c cells were treated, as previously described, for h and then the cells were incubated with μm aa for h. extraction of aa metabolites was carried out using ethyl acetate and dried using speed vacuum (savant, farmingdale, ny, usa). the resultant metabolites were analyzed using liquid chromatography-electrospray ionization mass spectrometry (lc-esi-ms) (waters micromass zq spectrometer) method. the analysis of mid-chain hete metabolites was performed using lc-esi-ms as previously described [ ] . briefly, negative ionization mode was the mode of the mass spectrometer with single ion monitoring: m/z = for midchain hete metabolites, and m/z = for internal standard, -hete-d . the nebulizer gas was supplied from an in house nitrogen source with high purity. all results are presented as the mean ± sem. multiple group comparisons was performed using one-way analysis of variance (anova) followed by the student-newman-keuls as a post hoc test. differences between means were considered significant at p < . . all analyses were performed using sigmaplot® for windows (systat software, san jose, ca, usa). mtt assay was used to assess the effect of different concentrations of resveratrol on cell viability. rl- and h c cells were grown to - % confluency in -well culture plates and treated for h with increasing concentrations of resveratrol ( , , , , or µm). cells in the control group were treated with sfm without resveratrol. after incubation of cells with mtt for h, mtt is reduced by viable cells to produce formazan dye which is then solubilized using isopropyl alcohol. data in fig. a , b showed that resveratrol, at all concentrations used, did not significantly alter the cell viability of both cell lines (measured cell viability was more than %). therefore, , and µm resveratrol concentrations were selected to perform all consequent experiments in the current study based on mtt assay results. to examine the potential protective effect of resveratrol against the development of cellular hypertrophy, rl- and h c cells were treated with vehicle, μm ang ii, μm ang ii in combination with resveratrol ( , or μm) or resveratrol alone ( , or μm) in sfm. the results showed that incubation of cells with μm ang ii for h resulted in cellular hypertrophy as demonstrated by significant increase in β/α-mhc ratio and anp by approximately % and %, respectively in rl- cells, compared to control group (fig. a) . also, in h c cells, ang ii caused significant elevation of β/α-mhc ratio and anp by approximately % and %, respectively, compared to control group (fig. b) . while low concentration of resveratrol ( μm) was not able to revert ang ii-induced effects, cotreatment with higher concentration of and μm was able to significantly protect against ang ii-mediated elevation of β/α-mhc ratio and anp to approximately control levels in both cell lines. notably, treatment of cells with low and high concentrations of resveratrol alone did not have any significant effects on hypertrophic markers. these results indicate that resveratrol protects against ang ii-induced cellular hypertrophy as evidenced by a reduction in hypertrophic markers and prompted us to further investigate the effects of resveratrol on cyp b and its cardiotoxic midchain hete metabolites. we investigated the effect of low and high concentrations of resveratrol on mrna and protein expression levels of cyp b in rl- and h c cells and the results showed that while ang ii ( μm) had no significant effect on cyp b mrna level in comparison to control group in both cell lines, co-treatment with ang ii and resveratrol ( , or μm) or resveratrol alone ( , or μm) significantly decreased the transcription level of cyp b , compared to ang ii group, in both cell lines (figs. a, a) . in rl- cells, resveratrol ( , or μm) in combination with ang ii decreased cyp b mrna expression by approximately %, % or %, respectively. while , or μm resveratrol alone decreased the expression of cyp b by approximately %, % or %, respectively. in h c cells, resveratrol ( , or μm) in combination with ang ii decreased cyp b mrna expression by approximately %, % or %, respectively. however, , or μm resveratrol alone decreased the expression by approximately %, % or %, respectively. we examined whether different concentrations of resveratrol have a protective effect against ang ii-induced cellular hypertrophy through inhibiting the protein expression of cyp b . for this reason, western blot analysis was performed and the results showed that the protein expression of cyp b was significantly increased by ang ii treatment in comparison to control group in rl- and h c cells, by data are presented as the percentage of control (set at %) ± sem (n = ). data were analyzed using one-way anova followed by student-newman-keuls as post hoc test approximately % and %, respectively (figs. b, b) . in rl- cells, low and high concentrations of resveratrol ( , or μm) in combination with ang ii significantly decreased cyp b protein expression by approximately %, % or %, respectively, compared to ang ii group. interestingly, , or μm resveratrol alone induced cyp b protein expression by approximately . -, . -or -folds, respectively. in h c cells, low concentration of resveratrol ( μm) did not significantly alter cyp b protein level compared to ang ii group. however, high concentrations of resveratrol ( or μm) in combination with ang ii significantly decreased cyp b protein expression by approximately % or %, respectively, compared to ang ii group. intriguingly, , or μm resveratrol alone increased cyp b protein expression by approximately . -, . -or -folds, respectively. to investigate the effect of low and high concentrations of resveratrol on the formation of mid-chain hetes fig. effect of resveratrol on ang ii-mediated induction of hypertrophic markers in rl- and h c cells. rl- (a) and h c (b) cells were treated for h with vehicle, μm ang ii, μm ang ii in combination with ( , or μm resveratrol) or ( , or μm resveratrol alone) in sfm. total rna was isolated using trizol, the mrna expression levels of β-mhc/α-mhc and anp were assessed using real-time pcr and their levels were normalized to β-actin housekeeping gene. the results are presented as the mean and sem based on at least individual experiments. data were analyzed using one-way anova followed by student-newman-keuls as post hoc test. + p < . significantly different from control group. *p < . significantly different from ang ii-treated group metabolites, rl- and h c cells were treated for h with vehicle, μm ang ii or μm ang ii in combination with ( , or μm resveratrol). thereafter, the cells were incubated with μm aa for h and mid-chain hete metabolites were analyzed using lc-esi-ms. the results showed that treatment of rl- cells with ang ii significantly increased the metabolite formation rate of -, -, -, -and -hete by approximately %, %, %, % and %, respectively, while it had no significant effect on -hete (fig. a, b) . resveratrol at concentration of μm was able to decrease ang ii-mediated induction of -and -hete by approximately % and %, respectively, compared to ang ii-treated group. while it was not able to reverse this increase in case of -, -, and -hete (fig. a, b) . on the other hand, higher doses of resveratrol significantly protected against the increase in cardiotoxic mid-chain hete metabolites. resveratrol at concentration of μm significantly inhibited fig. effect of resveratrol on mrna expression and protein expression levels of cyp b in rl- cells. rl- cells were treated for h with vehicle, μm ang ii, μm ang ii in combination with ( , or μm resveratrol) or ( , or μm resveratrol alone) in sfm. cyp b mrna expression (a) and protein expression levels (b) were determined using real-time pcr and western blot analysis, respectively. for real-time pcr, total rna was isolated using trizol reagent, the mrna level was quantified and its level was normalized to β-actin housekeeping gene. for western blot analysis, protein levels were detected using the enhanced chemiluminescence method. the intensity of protein band was normalized to the signals obtained for β-actin or gapdh protein and quantified using imagej®. the results are presented as the mean and sem based on at least individual experiments. data were analyzed using one-way anova followed by student-newman-keuls as post hoc test. + p < . significantly different from control group. *p < . significantly different from ang ii-treated group ang ii-mediated increase of the metabolite formation rate of -, -, -, -and -hete by approximately %, %, %, % and %, respectively, compared to ang ii-treated group (fig. a, b) . also, the highest concentration of resveratrol ( μm) was able to decrease ang ii-mediated increase of the metabolite formation rate of -, -, -, -and -hete by approximately %, %, %, % and %, respectively, compared to ang iitreated group (fig. a, b) . to further confirm the results in another species, we have performed the metabolic analysis study in rat h c cells. the results showed that treatment of h c cells with μm ang ii significantly increased the metabolite formation rate of -, -, -and -hete by approximately %, %, % and %, respectively, while it had no significant effect on -or -hete (fig. a, b) . resveratrol at concentration of μm was only able to decrease ang ii-mediated increase of -hete by approximately %, compared to ang ii-treated group. while it was not able to reverse, this increase in case of -, -and -hete (fig. a, b) . interestingly, higher concentrations of resveratrol also significantly inhibited ang iimediated increase in cardiotoxic mid-chain hete metabolites. resveratrol at concentration of μm significantly inhibited ang ii-mediated increase of the metabolite formation rate of fig. effect of resveratrol on mrna expression and protein expression levels of cyp b in h c cells. h c cells were treated for h with vehicle, μm ang ii, μm ang ii in combination with ( , or μm resveratrol) or ( , or μm resveratrol alone) in sfm. cyp b mrna expression (a) and protein expression levels (b) were determined using real-time pcr and western blot analysis, respectively. for real-time pcr, total rna was isolated using trizol reagent, the mrna level was quantified and its level was normalized to β-actin housekeeping gene. for western blot analysis, protein levels were detected using the enhanced chemiluminescence method. the intensity of protein band was normalized to the signals obtained for β-actin or gapdh protein and quantified using imagej®. the results are presented as the mean and sem based on at least individual experiments. data were analyzed using one-way anova followed by student-newman-keuls as post hoc test. + p < . significantly different from control group. *p < . significantly different from ang ii-treated group the results are presented as the mean and sem (n = ). data were analyzed using oneway anova followed by student-newman-keuls as post hoc test. + p < . significantly different from control group. *p < . significantly different from ang ii-treated group -, -, -and -hete by approximately %, %, % and %, respectively, compared to ang ii-treated group (fig. a, b) . moreover, the highest concentration of resveratrol ( μm) was able to decrease ang ii-mediated increase of the metabolite formation rate of -, -, -and -hete by approximately %, %, % and %, respectively, compared to ang ii-treated group (fig. a, b) . according to world health organization (who), cvd are the leading cause of death worldwide, they are responsible for the death of approximately . million people each year representing % of all deaths globally [ ] . accumulating studies have demonstrated that cyp enzymes and their associated aa metabolites play a central role in the maintenance of cardiovascular health and also in the pathogenesis of cvd [ , ] . interestingly, cyp b and its associated cardiotoxic mid-chain hete metabolites have been given a considerable attention due to their evident role in the pathogenesis of cvd such as pathological cardiac hypertrophy, atherosclerosis, hypertension and hf [ , ] . moreover, several studies have established an inverse relationship between developing cvd and the dietary consumption of vegetables and fruits. these protective effects have been attributed to polyphenols including flavonoids (such as resveratrol) and stilbenoids that are naturally present in high concentration in heart-healthy diets. nevertheless, the molecular cardioprotective mechanisms for some of these natural compounds have not yet been elucidated [ , ] . resveratrol has been widely studied as a protective agent against many cvd. also, resveratrol has been demonstrated to possess an inhibitory effect on cyp b [ ] . however, the possible beneficial effects of resveratrol as an inhibitor of cyp b in case of cardiac hypertrophy have never been investigated. therefore, in the current study, we evaluated the effects of three different concentrations of resveratrol ( , and μm) on cardiac cyp b and its associated cardiotoxic mid-chain hete metabolites. using two different cell lines, we investigated whether resveratrol is able to protect against ang ii-induced cellular hypertrophy. moreover, we characterized the effect of resveratrol on both transcriptional and translational levels of cyp b and its effect on the metabolite formation of cardiotoxic mid-chain hetes including -, prior to investigating the effect of resveratrol on ang iiinduced cellular hypertrophy, we tested the effect of increasing concentrations of resveratrol on cell viability. based on the mtt assay results, resveratrol did not have any toxic effect on cell viability following treatment for h at concentrations of - µm, suggesting that the concentrations used in the current study have no cytotoxic effects. this comes in agreement with previous studies that reported the safe use of resveratrol at concentration up to µm without observing toxic effects [ , ] . therefore, we tested the potential protective effect of resveratrol concentrations ( , and µm) against ang ii-induced cellular hypertrophy. treatment of both cell lines with ang ii resulted in cellular hypertrophy as evidenced by the elevation in β-mhc/α-mhc ratio and anp. the concentration of ang ii has been chosen based on previous work done in our lab. rl- cells were treated for h with various concentrations of ang ii ( , , . , , and μm) and cellular hypertrophy markers were measured using real-time pcr. ang ii increased the expression of hypertrophic markers in a concentrationdependent manner. the maximum induction was observed . data were analyzed using oneway anova followed by student-newman-keuls as post hoc test. + p < . significantly different from control group. *p < . significantly different from ang ii-treated group at the highest concentration tested, μm, and it was associated with increase in the cell surface area without affecting cell viability [ ] . in another study, ang ii has been shown to induce cyp b protein expression at μm concentration without negatively affecting cell viability in two different cell lines [ ] . the ratio of β-mhc to α-mhc is regarded as a sensitive marker of cardiac hypertrophy in several models [ ] [ ] [ ] . the mechanical properties of the cardiac muscle are largely dependent on the expressed isoform of mhc [ ] . several studies have reported that α-mhc isoform has greater atpase activity than β-mhc, and shifting from α-mhc to β-mhc isoform during cardiac hypertrophy is considered as an adaptive response for improved energy economy. however, it also leads to disruption in the contractile machinery of the heart resulting in impaired cardiac function and promoting diseases progression [ ] . furthermore, treatment of cells with ang ii caused a significant increase in anp, a hypertrophic marker that is found to be increased at the mrna and protein levels in human hypertrophic hearts supporting the correlation between higher anp expression and cellular hypertrophy [ ] . in the current study, low concentration of resveratrol ( µm) was not able to significantly protect against ang ii-induced increase in β-mhc/α-mhc ratio and anp. on the other hand, higher concentrations ( and µm) were able to reverse ang ii-mediated increase of hypertrophic markers to nearly control level, with no significant difference between the concentrations. notably, resveratrol alone groups did not have any significant effects on the mrna level of hypertrophic markers in both cell lines, rl- and h c . cyp b is a heme-thiolate monooxygenase enzyme that is constitutively expressed in non-hepatic tissues including cardiovascular system, and is responsible for nadphdependent metabolism of endobiotics and xenobiotics [ ] . moreover, cyp b expression was found to be induced in several experimental models of cardiac hypertrophy such as isoproterenol-induced, pressure overload-induced and ang ii-induced cardiac hypertrophy, pointing out to the role of ang ii in the progression of cardiac hypertrophy [ , , ] . in the current study, while treatment of cells with ang ii did not show significant effects on mrna expression level of cyp b , it significantly induced the protein expression of cyp b . this comes in agreement with previous study that used ang ii as an inducer of cardiac hypertrophy [ ] . several cardioprotective effects of resveratrol have been previously reported in various experimental and clinical settings. experimentally, it attenuated pressure overloadinduced cardiac fibrosis and diastolic dysfunction, it protected against chronic intermittent hypoxia-induced cardiac hypertrophy and it also restored the cardiac function and structure in case of pressure overload-induced cardiac hypertrophy [ , , ] . clinically, multiple reports provided evidences that resveratrol exerts protective effects on the cardiovascular system in patients with coronary artery diseases in the secondary prevention against myocardial infarction. resveratrol prevented platelet aggregation, reduced ldl cholesterol level and enhanced left ventricular diastolic function [ , ] . while several molecular mechanisms have been proposed to be responsible for the cardioprotective effects of resveratrol, this study is focusing on the effect of resveratrol on cyp b and its associated cardiotoxic midchain hetes. in the current study, resveratrol at concentrations of and µm was able to significantly decrease protein expression of cyp b compared to ang ii group, in both cell lines. this comes in agreement with previous reports demonstrating the inhibitory effect of resveratrol on protein expression of cyp b in several tissues [ , ] . although resveratrol inhibits cyp b on the protein expression and activity levels, it has been found to inhibit other cytochrome p enzymes such as cyp a and cyp a with higher affinity to cyp a [ ] . moreover, our lab has shown that resveratrol also inhibits the activity of human recombinant cyp a , cyp f and cyp f b enzymes with higher potency on cyp f [ ] . several lines of evidence previously demonstrated the role of cardiotoxic mid-chain hetes in the pathogenesis of cvd such cardiac hypertrophy and heart failure. -hete has been reported to elevate rat neonatal cardiomyocytes sensitivity to isoproterenol-induced β-adrenergic response. moreover, -, -, and -hete showed a direct inducing effect of cellular hypertrophy in rl- cells via mapk-and nf-κb-dependent pathways [ , ] . inhibiting the formation of -hete metabolite reduced ang ii-induced vascular remodeling and cardiac hypertrophy in mice proposing that inhibition of the -hete formation might be considered as a new therapeutic target in the treatment of cardiac hypertrophy [ ] . in the present study, treatment of cells with ang ii significantly induced the metabolite formation rate of the majority of mid-chain hete metabolites including -, -, -and -hete. interestingly, treatment of cells with resveratrol at concentrations of and µm was able to significantly decrease the metabolite formation rate of midchain hetes compared to ang ii-treated group, in both cell lines. these results are in line with previous study showing that resveratrol improved cardiac function and exercise performance in mi-induced heart failure through the inhibition of cyp b and cardiotoxic hete metabolites [ ] . moreover, our laboratory has previously shown that resveratrol was able to inhibit the formation of -hete generated by incubation of aa with human liver microsomes. resveratrol has shown concentration-dependent inhibition of the -hete formation with maximum inhibition i max of % and ic of μm. these data suggest that inhibition of the pro-fibrotic -hete may contribute to the beneficial and protective effects of resveratrol in case of cardiac hypertrophy [ ] . in conclusion, the results of the current study showed that resveratrol, at concentrations of and μm, was able to protect against ang-ii-induced cellular hypertrophy as evidenced by significant inhibition of β-mhc/α-mhc ratio and anp. while ang ii significantly induced the protein expression of cyp b and increased the metabolite formation rate of its associated mid-chain hetes, resveratrol was able to attenuate ang ii-mediated effects and caused a significant decrease of cyp b protein expression 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resveratrol ameliorates cardiac dysfunction induced by pressure overload in rats via structural protection and modulation of ca + cycling proteins cardioprotection by resveratrol: a human clinical trial in patients with stable coronary artery disease cigarette smoke toxins-induced mitochondrial dysfunction and pancreatitis involves aryl hydrocarbon receptor mediated cyp gene expression: protective effects of resveratrol alteration of benzo(a)pyrene biotransformation by resveratrol in apc min/+ mouse model of colon carcinogenesis resveratrol is a selective human cytochrome p a inhibitor repurposing resveratrol and fluconazole to modulate human cytochrome p -mediated arachidonic acid metabolism -, -and -hydroxyeicosatetraenoic acids induce cellular hypertrophy in the human ventricular cardiomyocyte, rl- cell line, through mapk-and nf-κb-dependent mechanism -lo/ -hete mediated vascular adventitia fibrosis via p mapk-dependent tgf-β a pirinixic acid derivative (lp ) inhibits murine -lipoxygenase activity and attenuates vascular remodelling in a murine model of aortic aneurysm conflict of interest the authors declare that they have no conflict of interest. key: cord- - kt ose authors: esch, joep h.m. van; danser, a.h. jan title: local angiotensin generation and at( ) receptor activation date: journal: frontiers in research of the renin-angiotensin system on human disease doi: . / - - - - _ sha: doc_id: cord_uid: kt ose nan the renin-angiotensin system (ras) plays an important role in the regulation of blood pressure and body fluid homeostasis. traditionally, the ras has been viewed as a circulating system ("circulating" ras). however, it is now well-established that angiotensin (ang) generation also occurs at tissue sites ("tissue" ras). the complexity of the system has increased even further now that we know that ang ii activates more than one receptor, that ang ii has metabolites which activate their own receptors, and that there may even be receptors for renin and prorenin. this review summarizes the latest insights on tissue angiotensin generation and focuses in particular on the activation of the ang ii type (at ) receptor by locally generated ang ii. renin belongs to the family of aspartyl proteases and has only one known substrate, angiotensinogen, the precursor of all angiotensin peptides. structure analysis revealed that renin consists of homologous lobes which form a cleft containing the active site. renin has an inactive precursor, prorenin, in which the active site is covered by the prosegment. the renin gene was cloned in the s in human, rat and mouse. most species have one renin gene (ren- c ), although some mouse strains have two renin genes, ren- d and a submandibular variant, designated as ren- . the ren- gene is encoding for a nonglycosylated prorenin, as opposed to the ren- gene which can be glycosylated at three asparagine residues. the renin gene is located on chromosome in human and mouse, whereas it is localized on chromosome in rat. the renin gene encodes for pre-prorenin consisting of a presegment of amino acids, a prosegment of amino acids and the actual renin protein of amino acids (morris ) . the presegment functions as a signal peptide directing prorenin to the secretory pathway. recently, a splice-variant of the renin gene was discovered which lacks the signal peptide and part of the prosegment. this truncated prorenin displays enzymatic activity because the truncated prosegment only partially covers the enzymatic cleft. it is thought to remain intracellular (clausmeyer et al ) , although truncated prorenin has also been demonstrated extracellularly (shinagawa et al ) . mice lacking the ren- d gene are characterized by sexually dimorphic hypotension (leading to a significant reduction of blood pressure in female mice), absence of dense secretory/storage granule formation in juxta-glomerular cells, altered morphology of the kidney, and a significant increase of plasma prorenin levels (clark et al ) . deletion of the ren- gene resulted in increased renin and decreased prorenin levels (sharp et al ) , but no changes in blood pressure, nor morphological changes occurred. transgenic mice overexpressing human renin did not develop hypertension whereas transgenic mice expressing both human renin and human angiotensinogen showed a significantly increased blood pressure (fukamizu et al ) . the plasma concentrations of ang i and ang ii were - -fold increased in double transgenic mice as compared to either control mice or transgenic mice overexpressing human renin. these results demonstrate that human renin does not crossreact with mouse angiotensinogen, thereby illustrating the unique species specifity of the ras. prorenin can be activated through cleavage of the prosegment (proteolytic activation) or via a conformational change induced by low ph or low temperature (non-proteolytic activation) (danser and deinum ) (fig. ) . proteolytic activation is an irreversible process in which the prosegment is cleaved, e.g., by kallikrein, trypsin or plasmin. in vivo, proteolytic activation is probably mediated by a proconvertase in the renin-producing cells of the juxta-glomerular apparatus of the kidney. non-proteolytic activation of prorenin is a reversible process in which prorenin is converted from the 'closed ' (inactive) to the 'open' (active) conformation by unfolding of the prosegment from the enzymatic cleft . acid activation leads to complete activation of prorenin whereas exposure to cold ('cryoactivation') only leads to partial activation (∼ %). kinetic studies have shown that an equilibrium exists between the closed and open conformations of prorenin, and that under physiological conditions (ph . , o c) < % of prorenin is in the open conformation (danser and deinum ) . the kidneys are the main source of circulating (pro)renin. however, following a bilateral nephrectomy, prorenin, in contrast with renin, remains detectable. this suggests that prorenin is also produced outside the kidney. potential extrarenal prorenin-producing tissues are the eye, adrenal, ovary and testis (sealey et al ; danser et al ; itskovitz et al ; clausmeyer et al ) . normally, the concentration of prorenin in human plasma is times higher than that of renin. the reasons for this excess are unknown, as prorenin does not seem to be activated (lenz et al ) . one possibility is that prorenin has functions unrelated to angiotensin generation. in this regard, it is of interest to note that it has recently been suggested that prorenin binds to a '(pro)renin receptor', thereby activating second messenger pathways in a manner that is independent of ang ii (nguyen et al ; saris et al ) . (pro)renin receptors may also mediate the uptake of renin and/or prorenin into tissues that do not synthesize renin and prorenin themselves, like the heart and the vessel wall. to date, two (pro)renin-binding receptors have been identified: the mannose- phosphate (m p) receptor (saris et al ) and the above-mentioned (pro)renin receptor. the m p receptor is identical to the insulin-like growth factor ii (igfii) receptor and binds igfii, m p-containing proteins such as prorenin and renin, and retinoic acid at distinct sites (kornfeld ; kang et al ) . prorenin and renin are both rapidly internalized after binding to this receptor, and internalized prorenin is proteolytically converted to renin. however, binding to this receptor did not result in angiotensin generation, either intra-or extracellularly. this, in combination with the fact that intracellularly generated renin was found to be degraded within a few hours, suggests that m p/igfii receptors function as clearance receptors for (pro)renin. alternatively, since binding of m p-containing proteins to m p/igfii receptors results in the activation of second messenger pathways involving g-proteins (di bacco and gill ) , (pro)renin may act as an m p/igfii receptor agonist. the (pro)renin receptor was cloned by nguyen and co-workers (nguyen et al ) . prorenin and renin bind equally well to this receptor, without being internalized or degraded. interestingly, the catalytic activity of bound renin was increased -fold, and receptor-bound prorenin became fully active in a non-proteolytic manner. thus, apparently, this receptor allows prorenin to generate angiotensins at tissue sites. importantly, binding of (pro)renin to the (pro)renin receptor in human mesangial cells also induced ang ii-independent effects, such as an increase in dna synthesis, activation of the mitogen-activated protein kinases (mapks) extracellular signal-regulated kinase (erk) (p )/erk (p ), and plasminogenactivator inhibitor- release. furthermore, in cardiomyocytes, prorenin activated the p mapk/heat shock protein pathway, resulting in changes of actin filament dynamics (saris et al ) . these non-angiotensin-mediated effects may underlie the blood pressure-independent cardiac hypertrophy in rats with a hepatic prorenin overexpression (véniant et al ) . finally, peters and co-workers demonstrated ren- prorenin internalization in cardiomyocytes of transgenic rats expressing the mouse ren- gene in the liver (peters et al ) . since ren- prorenin is nonglycosylated, this phenomenon cannot be mediated by m p/igfii receptors. the internalization contrasts with the observations on the recently cloned (pro)renin receptor. thus, there may be a third (pro)renin receptor, the identity of which is currently unclear. angiotensinogen, the precursor of all angiotensin metabolites, is the only known substrate for renin. the angiotensinogen gene encodes for a glycoprotein of amino acids with a molecular weight of ∼ kda. the gene is located as a single copy on, respectively, chromosome in rats, chromosome in mice and chromosome in humans. in , doolittle reported a significant sequence homology of angiotensinogen to -antitrypsin ( %), ovalbumin ( %) and antithrombin iii ( %) (doolittle ). these proteins are members of the serine proteinase inhibitor family and are closely associated with acute inflammation reactions. acute inflammation induces gene expression via acute-response which increases the angiotensinogen concentration in plasma (kageyama et al ) . the similarity between the structural organization of the angiotensinogen and antitrypsin genes suggests that both genes have evolved from a common ancestor (kitamura et al ) . although angiotensinogen mrna has been detected in brain, adipocytes, heart and the reproductive system, its main source is the liver (paul et al ) . hepatocytes constitutively secrete angiotensinogen into the extracellular fluid, without intracellular storage. blood plasma/extracellular fluid functions as the major reservoir for angiotensinogen. angiotensinogen plasma concentrations (∼ μm) approximate the michaelis-menten constant of the renin reaction, which makes ras activity sensitive to small changes in angiotensinogen concentration. deletion of the angiotensinogen gene in mice leads to hypotension, low body weight gain after birth, and an abnormal morphology of kidney and heart . in turn, overexpression of angiotensinogen led to the development of hypertension (kimura et al ) . two isoforms of ace exist: somatic ace and testis (germinal) ace. somatic ace is abundantly expressed throughout the body, whereas testis ace is exclusively expressed in the testis. cloning of the ace gene provided a better understanding of the relationship between somatic and testis ace. both forms are transcribed from the same gene by using different promoters (hubert et al ) . in humans the ace gene is located on chromosome . somatic ace has homologous domains which share % sequence homology. both domains contain a catalytically active site (wei et al a) and are situated at the n-and c-terminal side of ace. according to their position they are designated as n-and c-domain. the majority of somatic ace is membrane-bound on endothelial cells. circulating ace is derived from ace-expressing cells by proteolytic cleavage at the juxta-membrane stalk region (wei et al b) . testis ace possesses only one catalytic domain which is identical to the c-domain of somatic ace. studies selectively blocking the cand n-domain of somatic ace revealed that conversion of ang i to ang ii by membrane-bound ace depends on the c-domain, whereas both domains contribute to this conversion in soluble ace (van esch et al ) . degradation of bradykinin at tissue sites also required both domains (tom et al ) . deletion of both somatic and testis ace (ace −/− ) in mice led to hypotension, male infertility and changes in kidney morphology (esther et al ) . vascular expression of germinal ace in ace null mice restored renal morphology but did not normalize blood pressure, thus demonstrating that germinal ace cannot functionally substitute for somatic ace (kessler et al ) . recently, a homologue of somatic ace called ace was discovered (donoghue et al ) . ace shares % homology with the c-and n-terminal domains of somatic ace. the gene encoding ace is located on the x chromosome and ace is mainly expressed in endothelial cells of heart, kidney and testis. like somatic ace, ace can be released into the circulation after proteolytic cleavage (turner and hooper ) . unlike somatic ace, ace has only one catalytically active site which can convert ang i and ang ii to ang ( - ) and ang ( - ), respectively (donoghue et al ; vickers et al ) . these data suggest a potential role of ace in the counterregulation of high blood pressure by inactivation of ang ii. indeed, in a model of ang ii-dependent hypertension, blood pressures were substantially higher in ace -deficient mice than in wildtype controls (gurley et al ) . mice lacking the ace gene were originally described to develop an abnormal heart function with severely impaired contractility (crackower et al ) , but this was not confirmed in a follow-up study (gurley et al ) . remarkably, ace also functions as a receptor for the virus causing severe acute respiratory syndrome, thereby stressing the importance of ace in a manner unrelated to the ras (li et al ) . initially, it was thought that the responses to ang ii were mediated by a single ang ii receptor. at the end of the s, the discovery of specific ang ii receptor ligands such as losartan, pd , pd and cgp made it possible to identify several ang ii receptor subtypes. we now know that the biological actions of ang ii in man are mediated by at least two types of ang ii receptors: ang ii type (at ) and at receptors (fig. ). at receptors mediate virtually all the known physiological actions of ang ii, such as vasoconstriction, inotropy, chronotropy, aldosterone release, noradrenaline release and growth stimulation. the at receptor gene encodes for a protein of amino acids with a molecular weight of kda. the gene was first cloned in from rat vascular smooth muscle cells (murphy et al ) and bovine adrenal gland (sasaki et al ) . cloning and genetic analysis of the human at receptor gene revealed that the human at receptor gene is located on chromosome and can produce two isoforms by alternative splicing. both isoforms have similar binding -and functional properties. in rodents two subtypes of the at receptor have been identified: at a and at b (elton et al ) . the origin of these subtypes lies in a gene duplication which occurred after the divergence of rodents from the human/artiodactyls group about million years ago. at a and at b share % sequence homology and are located on chromosome and in rat and chromosome and in mice, respectively. not surprisingly, both subtypes have similar ligand binding affinities and signal transduction properties but varying expression levels in different tissues. the at a receptor predominates in heart, kidney, lung, liver and vascular smooth muscle, whereas the at b receptor is mainly expressed in the adrenal and pituitary gland (burson et al ) . to date, there are no pharmacological antagonists which clearly discriminate at a and at b receptors. studies in mice using targeted gene manipulation provided more insight in the functional role of both subtypes in vivo. deletion of the at a receptor gene significantly decreased resting blood pressure in both heterozygous at a +/− and homozygous at a −/− receptor mice (ito et al ) . ang ii infusions resulted in a diminished pressor response in at a +/− receptor mutants whereas this response was virtually abolished in at a −/− mutants. additionally, both the expression levels of renin mrna and plasma renin activity were markedly increased in at a receptor knockout mice (sugaya et al ) . deletion of the at b receptor gene did not affect resting blood pressure, nor altered the pressure response to ang ii (chen et al ) . taken together, these findings indicate the important role of the at a receptor in mediating the pressure response in mice. at a or at b receptor deficiency is not associated with an impaired development or survival, but double knockout mice lacking both receptors display a phenotype similar to that observed in angiotensinogen knockout mice (tsuchida et al ) . these observations, together with the fact that ang ii does cause a pressor response in at a knockout mice after enalapril pretreatment (oliverio et al ) , suggest a compensatory role for the at b receptor. additionally, in vitro studies demonstrated that the at b receptor is the most important regulator of ang ii contractile responses in the mouse aorta and femoral artery (zhou et al ) . the at receptor belongs to the seven-transmembrane g-protein-coupled receptor superfamily, and couples to a wide variety of second messenger systems, including the phospholipase c/inositol- , , -triphosphate/diacylglycerol/protein kinase c pathway, the phospholipase a /arachidonic acid pathway, the phospholipase d/phosphatidylcholine/phosphatidic acid pathway, and tyrosine kinases such as the map kinases erk / , p and c-jun n-terminal kinase (mehta and griendling ) . at receptor stimulation results in a rapid internalization of the ang ii-at receptor complex, followed by either receptor degradation in lysosomes or receptor recycling to the cell surface (mehta and griendling ) . internalized ang ii has been proposed to activate cytoplasmic or nuclear receptors prior to its intracellular degradation (thomas et al ) . furthermore, zou and co-workers recently demonstrated that mechanical stretch resulted in at receptor activation in a ligandindependent manner. interestingly, the consequences of such activation could be prevented by an at receptor blocker (zou et al ) . several reports have described crosstalk between at receptor and other receptors, e.g. the bradykinin type (b ) receptor, the at receptor, and the adrenoceptor. at and b receptors form stable heterodimers with an enhanced g-protein activation and altered receptor sequestration (abdalla et al ) . at receptor- -adrenoceptor crosstalk enhances the response to -adrenoceptor agonists (purdy and weber ) . interestingly, although the postjunctional at receptor interacting with the -adrenoceptor is of the at a subtype, the prejunctional at receptor which facilitates noradrenaline release from sympathetic nerve endings is of the at b subtype (guimaraes and pinheiro ) . in contrast to the well-characterized at receptor, the function of the at receptor is much less understood. in general, it is assumed that at receptors counteract the responses mediated by the at receptor (hein et al ; ichiki et al ; abdalla et al ; schuijt et al ; batenburg et al ) . at receptors are involved in physiological processes like development and tissue remodeling (by inhibiting cell growth and by stimulating apoptosis), regulation of blood pressure (vasodilatation), natriuresis and neuronal activity. evidence for at receptor mediated vasodilatation is largely based on two approaches: an indirect approach, showing an enhanced response to ang ii in the presence of at receptor blockade or gene disruption (hein et al ; ichiki et al ; batenburg et al ; van esch et al ) , and a direct approach showing at receptor-induced responses by applying either the (partial) at receptor agonist cgp a or ang ii in the presence of an at receptor blocker (widdop et al ; li and widdop ) . the at receptor gene was first cloned in (mukoyama et al ) . the at receptor gene shares % sequence homology with its at receptor counterpart and encodes for a protein of amino acids with a molecular mass of kda. it is located on the x chromosome in both humans and rodents. in fetal tissues the at receptor is the predominant subtype. this situation changes rapidly after birth, resulting in the at receptor becoming the dominant subtype in most adult tissues (widdop et al ) . yet, in adults, at receptors can still be detected in a variety of tissues, including uterus, ovary, adrenal medulla, heart, blood vessels and brain (bottari et al ) . here it is important to consider that the distribution of the at receptor depends on age and species, but is also subject to changes in expression during pregnancy and pathological conditions such as hypertension, heart failure and vascular injury (see below) (bottari et al ; de gasparo et al ) . in , two groups reported that deletion of the at receptor in mice led to an increased pressor response to ang ii (hein et al ; ichiki et al ) . additionally, ichiki et al reported a significant increased blood pressure in hemizygous at −/y receptor mice whereas blood pressure was not significantly increased in a similar model described by hein and co-workers. mutants lacking the at receptor gene showed a lower body temperature and impaired exploratory behavior. remarkably, despite its wide expression in the fetus, the at receptor does not seem to be required for embryonic development, as no morphological and developmental differences were found between homozygous at −/− or hemizygous at −/y receptor mice and their wildtype controls. possibly, at receptor knockout mice display a delayed expression of calponin and h-caldesmon after birth (yamada et al ) . during pregnancy, ang ii levels are elevated. because the fetus is also exposed to these high ang ii levels, it has been postulated that the at receptor plays a role in the regulation of ang ii responsiveness in order to prevent fetal hypertension (perlegas et al ) . like at receptors, at receptors belong to the g protein-coupled receptor superfamily. however, in contrast to the at receptor, the at receptor is not internalized upon binding of ang ii (widdop et al ) . two major pathways have been described for at receptor signaling (nouet and nahmias ) : (a) activation of protein phosphatases causing protein dephosphorylation and (b) activation of the nitric oxide (no)/guanosine cyclic ', '-monophosphate (cgmp) pathway. up to now, three specific phosphatases have been linked to at receptor activation: mapk phosphatase , sh -domain-containing phosphatase and protein phosphatase a. growth factors, including ang ii via the at receptor, mediate their growth promoting actions through tyrosine kinase receptors and several kinase-driven phosphorylation steps. activation of the at receptor counteracts these growth-promoting actions by dephosphorylation through subsequent activation of phosphatases. in addition to the inhibitory effect on growth, dephosphorylation (e.g., of erk / ) also seems to play an important role in the stimulation of apoptosis (horiuchi et al ) . several studies have shown that at receptor-mediated vasodilation is an endothelium-dependent phenomenon involving b receptors, no and cgmp (wiemer et al ; siragy and carey ) (fig. ) . initially, in vitro studies using endothelial cells showed that the stimulatory effect of ang ii on cgmp production, figure . at receptor-mediated relaxation involves either intracellular activation of kininogenase and subsequent bradykinin type (b ) receptor activation, or a direct activation of no synthase (nos) a downstream signaling product of no production, was abolished by blocking both b receptors and nitric oxide synthase (nos) (wiemer et al ) . subsequent in vivo studies confirmed that the at receptor-induced rise in cgmp involves bradykinin and no (siragy and carey ) . in vitro studies in endothelial cells reported that intracellular acidosis, as a result of at receptor activation, stimulates bradykinin formation by activating kininogenases (tsutsumi et al ) . katada and majima were able to show production of bradykinin after at activation in rat mesenteric arteries, suggesting that the b receptor mediates vasodilatation by endogenous bradykinin released upon at receptor activation (katada and majima ) . in agreement with this concept, deletion of the b receptor enhanced the ang ii-induced hypertensive response in vivo (cervenka et al ) . additional studies concluded that no production following at receptor stimulation may also occur independently of b receptors, through direct nos activation (abadir et al ) , possibly involving the calcineurin/nuclear factor of activated t cells pathway (ritter et al ) . as both at and b receptors are co-expressed in various tissues, the hypothesis was raised that both receptors form heterodimers which can interact through receptor crosstalk. recent studies in rat pheochromocytoma cells, applying fluorescence resonance energy transfer, confirmed this hypothesis (abadir et al ) . heterodimer formation appeared to be dependent on the receptor number that was expressed, but also required at receptor stimulation. as a consequence of heterodimer formation, it is possible that at receptor activation results in b receptor activation without intermediate bradykinin synthesis (batenburg et al ) . in addition to its interaction with the b receptor, at receptors are also known to interact with their at counterpart. transfection studies in fetal fibroblasts showed that at and at receptors form heterodimers in which the at receptor functions as a specific at receptor antagonist (abdalla et al ) . possibly, at receptorinduced vasodilatation depends on simultaneous at receptor activation, as no at receptor-mediated responses were noted in the absence of at receptors (van esch et al ) . furthermore, it is important to consider that data obtained in absence of the at receptor are complex because at receptors downregulate at receptors in a ligand-independent manner (jin et al ) and at receptor knockout mice display an increased at receptor expression . in addition to its interaction with at receptors, the at receptor also downregulates renin biosynthesis, thereby inhibiting the formation of ang ii (siragy et al ) . ang i and ii are metabolized by a whole range of peptidases ('angiotensinases'). although initially it was thought that all metabolites other than ang ii were inactive, it is now clear that at least several of these metabolites have functions of their own, which are sometimes mediated via non-at /at receptors. the most important of these peptides are ang ( - ), ang ( - ) (ang iii) and ang ( - ) (ang iv) (fig. ) . ang ( - ) can be formed from ang i by the action of neutral endopeptidase or prolyl endopeptidase but also from the ang i degradation products ang ( - ) and ang ii (vickers et al ) . ang ( - ) is generally believed to counteract the response of ang ii although there are reports of similar or distinct actions from ang ii (santos et al ) . ang ( - ) induces relaxation in several vascular beds. the fact that this relaxation could be blocked by the selective ang ( - ) antagonist a- [d-ala -ang ( - )] suggested the involvement of a specific ang ( - ) receptor (santos et al ) . indeed, in the mas proto-oncogene, a g protein-coupled receptor, was proposed to be the receptor for ang ( - ) (santos et al ) . ang ( - ) potentiates bradykinin-induced responses (tom et al ) and releases no (brosnihan et al ) via mas receptor stimulation. mas receptor mrna expression has been detected in heart, testis, kidney and brain (metzger et al ) . mice deficient for the mas-receptor lack the antidiuretic action of ang ( - ) after an acute water load, and their aortas no longer relax in response to ang ( - ) (santos et al ) . mas −/− mice are also characterized by an impaired heart function, indicating an important role of the mas receptor in the maintenance of the structure and function of the heart (santos et al ) . although the mas-receptor is now held responsible for most of the responses to ang ( - ) , there are several other pharmacological mechanisms and receptors that are affected by ang ( - ) . as a slow substrate for ace, ang ( - ) may also function as an ace inhibitor, resulting in decreased ang ii formation and potentiation of bradykinin-induced vasodilatation (tom et al ) . furthermore, ang ( - ) acts as an at receptor antagonist at low concentrations (stegbauer et al ) , and exerts at receptor agonistic effects at high concentrations (van rodijnen et al ) . a link between ang ( - ) and the at receptor has recently been proposed, because infusion of ang ( - ) during at receptor blockade unmasked a vasodepressor response in conscious shr rats that could be attenuated by blockade of at receptors, b receptors and nos . possibly, mas-at and/or mas-at receptor heterodimers exist (castro et al ; lemos et al ) . through the action of aminopeptidase a, ang ii is converted to ang iii, which in turn can be converted to ang iv by aminopeptidase n (ardaillou and chansel ) . ang iii mediates some of the classical responses of ang ii (such as stimulation of aldosterone secretion and vasoconstriction) and this most likely involves binding to at and at receptors. the affinity of ang iii for these receptors is somewhat lower than that of ang ii (wright and harding ) . the responses to ang iii are less efficacious than those of ang ii, possibly due to its accelerated metabolism in the circulation. the latter relates to the wide distribution of aminopeptidase n that initiates the hydrolysis of ang iii but not ang ii. it is thought that ang iii might be the final mediator of some of the actions of ang ii. for example, the central action of ang ii on vasopressin secretion in rats is dependent on ang iii, as this effect was absent after specific blockade of aminopeptidase a (zini et al ) . additionally, ang iii, and not ang ii, mediates the excretion of na + excretion through at receptors in the presence of at receptor blockade (padia et al ) . ang iv was initially believed to have no biological activity. this was based on two important findings: both at and at receptors display a poor affinity for ang iv, and ang iv does not elicit the characteristic ang ii responses like ang iii. the discovery of a specific ang iv binding site, designated as the at receptor, changed this view (swanson et al ) . after purification, the receptor was identified as insulin-regulated aminopeptidase (albiston et al ) , a protein which is abundantly found in vesicles containing the insulin-sensitive glucose transporter (glut ) (keller et al ) . at receptor expression occurs in brain, spinal cord, heart, kidney, colon, prostate, adrenal gland, bladder and vascular smooth muscle cells (wright and harding ; de gasparo et al ) . ang iv and the at receptor appear to be involved in the facilitation of memory and learning (wright et al ) . ang iv infusions cause vasorelaxation in cerebral and renal vascular beds, possibly by increasing nos activity (patel et al ) . on the other hand, there are also studies showing that ang iv, because of its weak agonistic activity towards the at receptor, induces vasoconstriction (van rodijnen et al ) . the close association of the at receptor with glut suggests that ang iv might modulate glucose uptake. as soon as it was realized that angiotensin production at tissue sites is of greater importance than angiotensin generation in the circulation, many investigators started to unravel how and where such local angiotensin production might occur. initially, it was thought that all components required for local ang ii production (i.e., renin, angiotensinogen and ace) would be produced at tissue sites. infusions of radiolabeled angiotensins, allowing the quantification of uptake of blood-derived angiotensin in tissues, confirmed that the majority of tissue ang i and ii is produced at tissue sites, and not derived from blood (schuijt and danser ) . ace is well-known to be abundantly expressed in virtually every tissue of the body, its main site being the surface of endothelial cells. thus, its local synthesis is beyond doubt. although angiotensinogen mrna has been detected outside the liver, direct proof for actual angiotensinogen synthesis at important sites of local angiotensin production (e.g., heart and vessel wall) is lacking. for instance, the isolated perfused heart does not release angiotensinogen . therefore, the majority of tissue angiotensinogen is probably of hepatic origin. the fact that angiotensinogen is neither internalized, nor binds to membranes, combined with the observation that angiotensinogen-synthesizing cells release angiotensinogen to the extracellular space (klett et al ) , rather than storing it intracellularly, indicates that angiotensin generation must occurs extracellularly. thus, tissue angiotensin generation is restricted to the interstitial space and/or the cell surface (fig. ) . following a bilateral nephrectomy, tissue renin and angiotensin levels drop to levels at or below the detection limit (campbell et al ; danser et al ; katz et al ) . this suggests that the majority of tissue renin is not locally produced, but kidney-derived, and that without renin, there is no angiotensin production. the presence of renin in cardiac membrane fractions (danser et al ) suggested that circulating renin, in addition to its diffusion into the interstitial space (katz et al ; van den eijnden et al ) , may bind to renin-binding proteins or receptors at tissue sites. the recent discovery of several of such receptors, as discussed above, supports this concept. an interesting additional observation is that these receptors also bind prorenin, and that prorenin, upon binding, becomes catalytically active. in view of the much higher prorenin than renin levels, an attractive concept is that prorenin rather than renin contributes to tissue angiotensin generation. studies with (pro)renin receptor blockers in diabetic rats confirmed this concept (ichihara et al ) . unexpectedly however, these blockers did not affect tissue angiotensin levels in control rats, although the prorenin levels of the latter rats were only ≈ -fold lower than those of the diabetic rats. moreover, despite the fact that prorenin is still present in circulating blood after a nephrectomy (danser et al ) , tissue angiotensin levels are close to zero. this suggests that, if prorenin contributes to tissue angiotensin production, this involves prorenin of renal rather than extrarenal origin. currently, the only known difference between renal and extrarenal prorenin relates to their degree of glycosylation. in vitro studies using the isolated perfused rat langendorff heart fully confirmed the idea of renin and angiotensinogen uptake underlying tissue angiotensin production. during buffer perfusion, no release of ras components could be demonstrated in the coronary effluent or interstitial fluid . after adding renin to the perfusion fluid, renin started to accumulate in the interstitial fluid, reaching steady-state levels in this compartment that were identical to its levels in the coronary circulation. findings on angiotensinogen were similar. stopping the exposure to renin revealed a biphasic washout curve, in agreement with the concept that renin is not only present in extracellular fluid but also binds to receptors. angiotensinogen washout was mono-phasic. angiotensin synthesis only occurred during simultaneous perfusion with renin and angiotensinogen. interestingly, in hearts of transgenic rats overexpressing angiotensinogen, angiotensin release continued after stopping the renin perfusion, i.e., when renin was no longer present in the coronary circulation (müller et al ) . this was due to the fact that receptor-bound renin continued to generate ang i. at steady state, the cardiac tissue levels of ang i were as high as expected assuming that ang i is restricted to the extracellular fluid (de lannoy et al ; schuijt et al ) . in contrast, the tissue ang ii levels were much higher. pretreatment with an at receptor antagonist greatly reduced the cardiac tissue ang ii levels during renin + angiotensinogen perfusion. this suggests that locally generated ang ii accumulates at tissue sites through binding to at receptors. subsequent subcellular fractionation studies confirmed that tissue ang ii, but not ang i, is located intracellularly (schuijt et al ; van kats et al ) . this is due to the fact that at receptor-bound ang ii is rapidly internalized, after which intracellular degradation occurs. based on these observations, it is not surprising that the tissue ang ii content correlates directly with tissue at receptor density . a wide range of in vitro studies has provided evidence for the existence of enzymes other than renin and ace generating ang i and ii, including cathepsin d, kallikrein, tonin and chymase (hackenthal et al ; urata et al ) . the in vivo importance of these alternative pathways is questionable. the fact that ang i and ii are virtually absent in plasma and tissue of nephrectomized animals (including humans) argue against a role of non-renin angiotensinogen-converting enzymes in vivo. a similar situation exists for chymase which is present in the cardiac interstitium, mast cells and endothelial cells. in vitro studies have provided evidence for an important role of chymase in the conversion of ang i to ang ii (urata et al ; tom et al ) , but in vivo evidence for chymase-dependent ang ii generation could not be obtained (saris et al ) . moreover, angiotensinogen and ace knockout mice have similar phenotypes (tanimoto et al ; krege et al ) , and ace deletion reduced the ang ii levels in both tissue and circulation by up to % (campbell et al ) . thus, at least in mice, ace is the main, if not only ang ii-generating enzyme in vivo. as discussed above, at receptor expression is low or undetectable in adult tissues, in contrast with its high expression in fetal tissues. however, at receptors reappear under pathophysiological conditions. for instance, in the kidney, at receptor expression increases when inflammation, apoptosis, and proteinuria occur (ruiz-ortega et al ) . interestingly, transgenic at receptor-overexpressing mice displayed less glomerular injury, proteinuria and transforming growth factor expression in a subtotal nephrectomy model (hashimoto et al ) . this suggests that the re-appearance of at receptors under pathological conditions is part of a protective mechanism, for instance related to enhanced no production (hiyoshi et al ) . however, not all studies confirm the counterregulatory, protective actions of at receptors in the kidney. duke and co-workers report that at receptors mediate vasoconstriction in the renal medulla of -kidney, -clip rats, as opposed to the vasodilator effects mediated by at receptors in this model (duke et al ) . in the heart, a wide range of animal studies revealed increased at receptor expression under pathological conditions, e.g. during pressure overload, hypertension and ischemia, and post-myocardial infarction (wiemer et al ; wu et al ; schuijt et al ; yayama et al ) . studies in failing human hearts confirmed the animal data, and simultaneously showed a downregulation of at receptors (asano et al ; wharton et al ) . from studies with at receptor antagonists it is widely accepted that at receptors play a major role in the post-myocardial remodeling process, mediating both fibrosis and hypertrophy (schieffer et al ) . since the beneficial effects of at receptor blockade following myocardial infarction were diminished in at −/y receptor mice , it is reasonable to assume that the increased ang ii levels that will occur during at receptor blockade (see below) exert beneficial effects via at receptor stimulation. indeed, transgenic mice overexpressing at receptors in the heart displayed improved cardiac hemodynamics post-myocardial infarction in an no-dependent manner bove et al ) . furthermore, treatment with either an at receptor antagonist or a b receptor antagonist reduced the beneficial effects of at receptor blockade in wildtype mice following myocardial infarction . therefore, the beneficial effects of at receptors in the heart involve the b receptor/no/cgmp pathway. in contrast with these observations, a few studies have shown that at receptors, like at receptors, induce cardiac hypertrophy and fibrosis (senbonmatsu et al ; ichihara et al ) . to explain these discrepant data, it has been hypothesized that at receptor upregulation is beneficial in the early pathological phase, by counteracting hypertrophy and fibrosis, but that chronic stimulation of the at receptor (for instance by the high ang ii levels that will occur during at receptor blockade) has deleterious effects on cardiac recovery (schneider and lorell ) . knowledge on the effects of at receptors in the human heart comes from polymorphism studies, although the data are often contradictory. at receptor gene variants have been linked to both cardiac hypertrophy and coronary ischemia (schmieder et al ; herrmann et al ; alfakih et al ) , without knowing however whether this is based on inceased or decreased at receptor density. at receptor-mediated vasodilation in isolated human coronary microarteries increases with age (batenburg et al ) . since endothelial function decreases with age, this could point to increased at receptor expression in the face of decreased endothelial function, again in agreement with the concept that at receptor density increases under pathological conditions. at receptor expression also increased in peripheral resistance arteries of hypertensive diabetic patients during treatment with an at receptor blocker, and this resulted in enhanced ang ii-induced vasodilation (savoia et al ) . recent studies have shown that at receptors are also expressed in various carcinomas (deshayes and nahmias ) . assuming that at receptors contribute to tumor growth and vascularization (fujita et al ) , one may predict that, here too, at receptors will counteract the effects of the at receptor stimulation, thus inhibiting growth and vascularization (silvestre et al ) . however, proangiogenic effects of at receptors have also been described, occurring in conjunction with at receptor activation . blocking the ras is possible at three levels: renin, ace and the at receptors. beta-adrenoceptor blockers, by antagonizing the renin-releasing -adrenoceptors in the juxta-glomerular cells, were the first drugs to suppress the ras. these drugs will lower renin (campbell et al ) , ang i and ang ii, thereby reducing the degree of at and at receptor stimulation (table ) . subsequently, the ace inhibitors were introduced. these drugs will lower ang ii. given the wide variety of available angiotensinases, this will not lead to substantial ang i accumulation, but rather result in metabolism of ang i through different (compensatory) pathways, e.g. by neutral endopeptidase. as a consequence, ang-( - ) levels will rise during ace inhibition, thereby allowing ang-( - ) to contribute to the beneficial effects of ace inhibitors (tom et al ) . simultaneously, due to the interference with ang ii generation, the negative feedback loop system regulating renin release is affected, and thus, the kidneys will release more renin. therefore, depending on the degree of ace inhibition, ang ii levels may rise again, sometimes to levels above baseline (campbell et al ; van kats et al ) . for instance, at % ace inhibition, a -fold rise in renin is sufficient to fully restore ang ii levels, whereas a -fold rise in renin would increase ang ii twofold above its baseline levels. in addition, prolonged ace inhibition is known to upregulate ace. given these compensatory mechanisms, it is not surprising that it has proven difficult to show that blood plasma and tissue ang ii levels remain suppressed during continuous ace inhibition (van kats et al ) . indeed, in pigs treated with captopril for weeks post-myocardial infarction, cardiac ang ii levels were increased as compared to untreated control pigs (fig. ) . although this ang ii may theoretically stimulate at and at receptors, it must be kept in mind that such receptor stimulation may occur less efficiently than normal. without ace inhibitor treatment, ace generates ang ii in a highly efficient manner, in close proximity of at receptors. during chronic ace inhibition, the increase in ang i generation will still allow ang ii generation, either by noninhibited ace or by non-ace converting enzymes like chymase (van kats et al ) . however, this type of ang ii generation is less efficient, because it does not result in a high level of regional at receptor stimulation. in particular, ang ii generated by chymase (which is localized in the adventitia) will be subject to rapid metabolism in the interstitial space on its way to at receptors (schuijt et al ; de lannoy et al ) and thus is less likely to result in a high regional at receptor occupancy. therefore, a low overall at receptor occupancy will occur, below the minimum per cell required to induce an effect. at receptor blockers, available since the early s, will also cause a rise in renin. ang i and ii in blood and tissues (as well as their metabolites) will increase in parallel with renin, and although this will not result in at receptor stimulation, non-at receptors (including at receptors and mas) may now be stimulated excessively. as discussed above, it is feasible that, at least part of the beneficial effect of at receptor blockers is due to such at receptor stimulation (widdop et al ) . finally, renin inhibitors will soon be clinically available. these drugs lower both ang i and ii, and evidence for this, at least in blood plasma, is already available (nussberger et al ; azizi et al ) . whether renin inhibitors also decrease tissue ang i and ii levels is not yet known. this relates to the fact figure . plasma and cardiac tissue angiotensin levels in pigs that were either untreated or treated with the ace inhibitor captopril or the at receptor antagonist eprosartan for weeks after a myocardial infarction. *p< . vs. untreated. data are derived from (van kats et al ) that renin inhibitors primarily block human renin, and not (or to a much lesser degree) rat, mouse or porcine renin. thus, renin inhibitors cannot be tested easily in well-established animal models. theoretically, the decreased ang i and ii levels during renin inhibition will prevent at and at receptor stimulation, as well as the stimulation of any other receptor by angiotensin metabolites. although renin will rise during renin inhibitor treatment (like it does during any ras blocker treatment), this renin cannot be enzymatically active due to the presence of the renin inhibitor. thus, renin inhibitors may offer a more complete suppression of the ras, although this also implies that the putative beneficial effects mediated by at or mas receptors will now no longer occur. so far, this does not appear to diminish the effects of renin inhibitors, at least on blood pressure (gradman et al ) . ang ii generated at tissue sites stimulates both at and at receptors. this local generation depends largely on angiotensinogen and renin and/or prorenin taken up from blood, the latter uptake possibly involving the recently discovered (pro)renin receptor. ace is generated locally, and appears to be the main, if not the only, ang ii-generating enzyme. ang ii has a whole range of metabolites, the most important of which are ang ( - ), ang iii and ang iv. the enzymes generating these metabolites, including ace , have recently been characterized, as well as their putative (non-at /at ) receptors, like the mas and at receptor. stimulation of at receptors most likely contributes to the beneficial effect of ras blockers, in particular during at receptor antagonism. these receptors are upregulated under pathophysiological conditions, and are generally believed to counteract the effects of at receptor stimulation. however, not all studies agree on this aspect, and thus it remains to be seen how the effect of drugs that completely suppress the ras, i.e., renin inhibitors, compare to 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release mechanical stress activates angiotensin ii type receptor without the involvement of angiotensin ii key: cord- -c fu jv authors: lu, chen-chen; wang, gui-hua; lu, jian; chen, pei-pei; zhang, yang; hu, ze-bo; ma, kun-ling title: role of podocyte injury in glomerulosclerosis date: - - journal: renal fibrosis: mechanisms and therapies doi: . / - - - - _ sha: doc_id: cord_uid: c fu jv finding new therapeutic targets of glomerulosclerosis treatment is an ongoing quest. due to a living environment of various stresses and pathological stimuli, podocytes are prone to injuries; moreover, as a cell without proliferative potential, loss of podocytes is vital in the pathogenesis of glomerulosclerosis. thus, sufficient understanding of factors and underlying mechanisms of podocyte injury facilitates the advancement of treating and prevention of glomerulosclerosis. the clinical symptom of podocyte injury is proteinuria, sometimes with loss of kidney functions progressing to glomerulosclerosis. injury-induced changes in podocyte physiology and function are actually not a simple passive process, but a complex interaction of proteins that comprise the anatomical structure of podocytes at molecular levels. this chapter lists several aspects of podocyte injuries along with potential mechanisms, including glucose and lipid metabolism disorder, hypertension, ras activation, micro-inflammation, immune disorder, and other factors. these aspects are not technically separated items, but intertwined with each other in the pathogenesis of podocyte injuries. the glomerular filtration membrane constituted by three components, porous endothelial cells, glomerular basement membrane (gbm), and epithelial cells in the gbm, which also called podocytes. podocytes are highly differentiated epithelial cells consist of three distinct parts: cell body, major processes, and foot processes (fps). podocytes have a voluminous cell body, which is at the central position of the cell and lies in the urinary space. the cell body contains a nucleus, abundant endoplasmic reticulum, a well-developed golgi system, lysosomes, and mitochondria. the densely distributed organelles in the cell body suggest a high level of anabolic and catabolic activity. microtubules and intermediate filaments, such as vimentin and desmin, are the dominated cytoskeleton components in cell body to accounts for the unique shape of podocytes (pavenstadt et al. ) . from the cell body, podocytes give rise to primary processes that reach to glomerular capillary, forming an affixation by fps. podocytes are polarized epithelial cells which contain a apical/luminal and a basal cell membrane. the apical surface domain forms a few fingerlike protrusions which bulge into bowman's space. the apical domain is negatively charged, which limits the passage of albumin (also negatively charged) and maintain the separation of adjacent podocytes by anion charge. the basal cell membrane mediates the affixation to the gbm by α β integrin and αand β-dystroglycans, which play the function by connecting to certain matrix proteins within the gbm (kreidberg et al. ; raats et al. ) . both of apical and basal membranes contain numerously distributed cholesterol-rich domains, and it was found that specific membrane proteins of podocytes are obviously arranged in rafts simons et al. ) . fps functionally consist of a luminal or apical membrane domain and a basal cell membrane domain. fps are characterized by a podosome-like cortical network of short, branched actin filaments and by the presence of highly ordered, parallel contractile actin filament bundles, which are thought to modulate the permeability of the filtration barrier through changes in fp morphology (greka and mundel ) . the foot processes of neighboring podocytes are bridged by slit diaphragm (sd), which is the site of convective fluid flow through the visceral epithelium and the final barrier to urinary protein loss. similar to the apical membrane domain of podocytes, the sd is also covered by a thick surface coat mainly constituted by sialoglycoproteins, including podoendin, podocalyxin, and others, which are responsible for the high negative surface charge of the podocytes. in addition, several molecules, including zo- (zonula occludens protein), nephrin, cd ap (cd -associated protein), fat, and p-cadherin, have all been shown to be expressed within the sd, and some of those molecules play a major role for its integrity (pavenstadt et al. ) . the unique shape of podocyte and the maintenance of its processes are owing to a well-developed cytoskeleton, which serves as the podocyte's "backbone." and also, the actin-rich cytoskeleton makes podocytes to be able to alter shape continually and dynamically. the cytoskeleton is comprised by microfilaments ( - nm diameter), intermediate filaments ( nm diameter), and microtubules ( nm diameter), which are mainly defined by their diameter. microtubules and intermediate filaments are predominant cytoskeletal constituents in the cell body and the primary processes. in the fps, microfilaments are the main cytoskeletal component, which consist of a network with densely accumulated f-actin and myosin. fp actin cytoskeleton is extensively distributed in all three domains of fps, resulting to an important role of actin for the function and dysfunction of podocytes. fp effacement requires the activation of actin filaments reorganization, a process which is regulated by multiple signaling events involving integrin activation, g protein-coupled receptor (gpcr) and growth factor receptor, and calcium (ca + ) influx pathways as upstream modulators of the actin cytoskeleton (takeda et al. ) . the complex architecture of podocytes, in particular on the maintenance of highly ordered, parallel, contractile actin filament bundles in fps, is required for the highly specialized functions of podocytes, which include (i) a size barrier to protein; (ii) charge barrier to protein; (iii) maintenance of the capillary loop shape; (iv) counteracting the intraglomerular pressure; (v) synthesis and maintenance of the gbm; (vi) production and secretion of vascular endothelial growth factor (vegf) required for gen integrity (shankland ) . podocyte is the most differentiated cell type in the glomerulus, which plays a crucial role in the glomerular filtration barrier. podocyte foot processes with the interposed sd represent the last filtration barrier of gbm. the sd is a subtle signal transduction unit characterized by a modified adherens junction that bridges the - -nm-wide filtration slits (reiser et al. ) . transmembrane proteins such as nephrin and fat constitute the rod-like units of sd which are connected by numerous linear bar, forming a network with pores the same size as or smaller than albumin (mundel and kriz ) . meanwhile, negatively charged apical domain of sd works as a charge barrier to prevent the albumin loss. thus, the podocyte is the important size and charge barrier of gbm, and podocytes' damage leads to the disruption of gbm integrity and proteinuria. podocytes stabilize glomerular architecture owing to fps counteract distensions of the glomerular basement membrane, which is regulated by vasoactive hormones. in this regard, they are responsible for % of the hydraulic resistance of the filtration barrier (pavenstadt ) . ang ii regulates the contractile state of their foot processes by activating a cl − conductance and increasing [ca + ] i, camp in podocytes, thereby modulating the ultrafiltration coefficient kf. other agonists such as avp, oxytocin, norepinephrine, and parathormone have also been reported to modulate [ca + ] i in podocytes. vasoactive hormones may also alter charge properties of the podocyte and thereby enhance urinary protein excretion (pavenstadt ) . vegf family consists of five secreted homodimeric glycoproteins: vegf-a, vegf-b, vegf-c, vegf-d, and placental growth factor. in human and murine kidneys, vegf-a isoform is constitutively expressed in podocytes, while playing its role mainly by contact with vegfr- and vegfr- predominately localized on the glomerular endothelial cells. it was assumed that vegf-a is critical for the regulation of endothelial cell survival, proliferation, differentiation, and migration as well as endothelium-dependent vasodilatation and vascular permeability (advani ) . the complex paracrine signaling pathway between podocytes and glomerular endothelial cells plays a central role in maintaining the structure and integrity of the kidney filtration barrier. podocyte injury is the common pathological process in many glomerular diseases such as minimal change disease, membranous glomerulopathy, focal segmental glomerulosclerosis (fsgs), diabetic nephropathy (dn), and lupus nephritis. physiological stresses or pathological stimuli like mechanical stress, oxidative stress, and immunologic stress disrupt the homeostasis of glomerular filtration barrier. transcapillary pressure increment is induced by glomerular hypertension/hyperfiltration, and podocyte processes' elongation is induced by capillary expansion contribute to cytoskeletal dysregulation and intrinsic stress (neal et al. ) . pathological factors, such as ischemia-reperfusion, chemical/toxic substances from the primary urine, usually cause reactive oxygen species (ros) production in podocytes . it was also reported that aldosterone and angiotensin ii promoted receptormediated ros generation in podocytes (liu et al. ) . immunologic stress is induced by cytokine/complement, such as cc chemokine receptor , tumor necrosis factor, and sublytic c b- -mediated intracellular stress in podocytes (nagata ) . typical electron microscopy manifestations of podocyte injury include microcystic, pseudocystic changes, vacuolization, the presence of cytoplasmic inclusion bodies, and detachment from the gbm. besides those changes, foot process effacement is the most characteristic change in podocyte injury. the damage of sd proteins contributes to cytoskeleton disorganization, leading to podocyte effacement and proteinuria (shankland ) . sd between adjacent podocytes is constituted predominantly by sd proteins including nephrin, podocin, cd ap, neph , and fat . mutations/abnormalities of those proteins result proteinuria and kidney disease. studies have shown that sd proteins regulate cytoskeleton organization and podocyte shape by interacting with proteins associated with actin cytoskeleton. fat- is an organizer of actin polymerization. cd ap connects the nephrin complex with the actin-modifying proteins wasp, capz, cortactin, and the arp / complex. reduced podocyte number causes proteinuria and glomerulosclerosis. podocyte detachment, podocyte apoptosis, and the lack of adequate podocyte proliferation are three main reasons leading to the decrease in podocyte number also called "podocytopenia." the lack of charge-and size-selective barriers induced by podocyte loss leads to proteinuria. studies have demonstrated the correlation of podocytes reduction with the onset and progression of glomerulosclerosis. because podocytes counteract the outward forces of glomerular pressures and maintain capillary loop shape, podocyte loss results to local bulging of the gbm when glomerular pressures increase in many renal diseases. the denuded gbm tends to form a synechia attachment by contacting with the parietal epithelial cells and bowman's capsule, which is thought to be the first "committed step" of focal segmental glomerular sclerosis (fsgs) (kriz et al. , a . podocytes maintain a healthy intraglomerular environment by cross talk with glomerular endothelial cells. endothelial cell swelling and attenuation of fenestrae are observed in podocyte injury models by ultrastructural study (kriz et al. ) . it was illustrated that podocyte injury disrupts intracapillary homeostasis, causing thrombotic micro-angiopathy and mesangial abnormalities by reducing vegf signaling (eremina et al. ; kobayashi et al. ) . glomerulosclerosis is a terminal consequence of podocyte injury. the classic type of glomerulosclerosis, as defined by segmental obliteration of glomerular capillaries by the extracellular matrix, has been believed to progress to complete sclerosis without regression (nagata ) . in early stage of fsgs, cellular lesions including transformed podocytes were accompanied by segmental sclerosis, supporting the fact that podocyte damage might be an early event of glomerulosclerosis. in a recent elegant review by kim js and his colleagues, the essential steps of glomerulosclerosis were suggested as follows: ( ) increased glomerular capillary pressure and filtration flow through podocyte slits, ( ) foot process effacement as an adaptive response, ( ) podocyte hypertrophy and glomerulomegaly, ( ) mismatch between glomerular tuft growth and podocyte hypertrophy, ( ) stretching and attenuation of podocyte cell body, ( ) pseudocysts formation by hindered flow of filtrates beneath the podocyte that is partially detached on bare areas of gbm, ( ) complete podocyte detachment by enlarged pseudocysts and adhesion to bowman's capsule, ( ) glomerular tuft's adhesion to bowman's capsule, ( ) spreading of filtrates to interstitium out of nephron through adhesion structure, and ( ) interstitial proliferation and nephron degeneration (kim et al. ). podocytes' injury and depletion was a crucial step in the development of albuminuria in dn. in dn, the number of podocyte-specific markers and podocytes number is decreased, which leads to the occurrence of albuminuria and further develops into glomerulosclerosis. hyperglycemia is the main pathological change of diabetes and plays an important role in promoting the occurrence and development of dn. increased intracellular glucose could induce multiple cell and molecular events in podocyte: ( ) generation of reactive oxygen species (ros) and advanced glycation end products (ages), ( ) increased flux of polyols and hexosamines, ( ) activation of protein kinase c (pkc), ( ) increased cytokines and growth factors, ( ) aberrant notch signaling, and ( ) activate the renal ras. these abnormal molecular pathological changes mediate the functional and morphological changes of podocytes in a direct or indirect way, including podocyte hypertrophy, epithelial mesenchymal transition (emt), podocyte detachment, and podocyte apoptosis. podocyte injury is a key factor in the development of dn. recent studies in both type and type diabetes have proposed that a reduction in the number of podocytes may lead to the development of proteinuria. it is reported that the structure and function of podocytes are abnormal under high glucose conditions, such as podocyte fusion, septal injury, and podocyte loss. there has been evidence that podocytes pos-sess a completely functional system for glucose uptake (lewko et al. ). coward et al. have revealed that the cultured human podocytes express glucose transporter (gluts) in two forms: glut and glut , which participate in insulin-dependent glucose transport to the cell (coward et al. (coward et al. , . in addition, the podocyte split protein, such as nefin, is also involved in glucose transport. schiffer et al. have demonstrated that podocytes also express another insulin-sensitive glucose transporter, glut (schiffer et al. ) . glut is the primary glucose transporter in most cells as well as in podocytes (coward et al. (coward et al. , . in diabetes, hyperglycemia and alteration of glucose transporter cause increased intracellular glucose concentration in podocyte and lead to severe impairment of the glomerular filtration barrier. conversely, zhang et al. found that enhancement of glut expression in diabetic podocyte significantly reduced the mesangial expansion and fibronectin accumulation by inhibiting the expression of vascular endothelial growth factor (vegf) (zhang et al. ) . similarly to other cells, under high glucose condition, podocyte can undergo many pathological changes induced by aberrations in various cellular and molecular events. high glucose induces generation of advanced glycation end products (ages) and reactive oxygen species (ros), increased flux of polyols and hexosamines, increased activity of protein kinase c (pkc), upregulated expression of cytokines and growth factors including vascular endothelial growth factor (vegf), and transforming growth factor-beta (tgf-β), induces aberrant notch signaling, and activates the renal ras (anil kumar et al. ) . pathomechanism of podocyte injury in dn mainly includes podocyte hypertrophy, emt, podocyte detachment, and podocyte apoptosis. podocyte hypertrophy is the initial stage of podocyte injury in early dn. hyperglycemia upregulated the expression of cyclin-dependent kinase p kip , which leads to further cell cycle arrest and hypertrophy. it was found that p kip -/-mice had significantly improved renal damage in dn (wolf et al. ) . several studies suggested high glucoseinduced podocyte hypertrophy by activating mtorc pathway (fantus et al. ). in addition, hyperglycemia increased expression of nuclear stat via the activation of the upstream signal transduction element gp , which eventually leads to podocyte hypertrophy. excessive hypertrophy could result in degenerative changes in podocyte structure and functions, leading to its detachment from glomerular basement membrane (gbm). previous studies have shown that phenotype conversion of podocyte was involved in the early stage of podocyte deletion in dn by inducing podocyte detachment or podocyte apoptosis. podocyte emt is a manifestation of podocyte phenotype conversion and one of the initiating factors leading to a variety of glomerular diseases. when emt occurs, the cells lose their original characteristics, resulting in disappearance of intercellular contact, impaired cell polarity, and expression of mesenchymal markers such as alpha smooth muscle actin (alpha-sma) and fibroblast-specific protein (fsp ). emt is also an explanation for podocyte depletion in dn (yamaguchi et al. ). emerging evidence suggested that podocytes could undergo emt in dn, characterized by loss of epithelial features such as nephrin and p-cadherin, while expressing mesenchymal markers such as fsp- , type i collagen, and fibronectin (reidy and susztak ). xing et al. ( ) demonstrated that stimulation with high glucose for h could activate the pi k/akt pathway in podocyte, and thereby induce the protein expressions of α-sma and desmin. dai et al. ( ) suggested that connective tissue growth factor (ctgf) and integrinlinked kinase (ilk) were involved in high glucose-induced phenotypic alterations of podocytes. lv et al. ( ) findings elaborated that rac /pak signaling contributed to high glucose-induced podocyte emt via promoting β-catenin and snail transcriptional activities, which could be a potential mechanism involved in podocytes injury in response to stimuli under diabetic conditions. guo et al. indicated high glucose can also activate β-catenin and snail expressions by upregulating gsk- β. in addition, hyperglycemia-induced podocyte detachment by decreasing the expression of key proteins involved in the foot process actin cytoskeleton, split diaphragm (sd) integrity, and podocyte-gbm interactions. a b integrins are the important transmembrane protein involved in anchoring foot processes in the gbm. high glucose regulates the expression of integrin subunits and inhibits the synthesis of agrin. therefore, high glucose affects not only the structure of podocytes, but also their ability to adhere to gbm (chen et al. ; han et al. ; yard et al. ) . it is found that high glucose can alter podocyte adhesion by decreasing expression of integrin α β v which was an important receptor that could tightly connect podocyte with the gbm and participated in the adhesion function of podocyte. in addition, α-actinin, an actin filament for protein crosslink, is also an important factor required for podocyte adhesions (dandapani et al. ) . high glucose and age treatment resulted in α-actinin- expression changes and induces cytoplasmatic translocation in podocyte (ha ) . there are some evidences that podocyte apoptosis played a role in reduction in density and number of glomerular in dn. high glucose led to podocyte apoptosis by increased production of ros, activation of poly(adp-ribose) polymerase, nf-kb, and p map kinase szabo et al. ) . in diabetes, the surface receptors of the ages are upregulated in the podocytes (tanji et al. ) . binding ages to receptors activates activated transcription factor foxo , which also induced podocyte apoptosis via p protein kinase signaling pathways (cohen et al. ). in addition, high glucose increased the protein expression of nestin, which is a vi intermediate filament protein-related cell cytoskeleton, thereby increased podocyte apoptosis rate . high glucose increased the expression of tgf-β in podocyte. tgf-β could induce podocyte apoptosis by directly activate smad , p map kinase, and notch pathway (li et al. ) . in addition to its direct effects, elevated glucose may act indirectly, via the proinflammatory response, ang ii-dependent pathways, and lipid accumulation. under high glucose conditions, secretion of the mcp- protein by cultured podocytes was increased rapidly (han et al. ) , and similar effect was observed in podocyte stimulated with ages (gu et al. ) . podocyte can also express tnf-α, a cytokine produced by various immune cells, in response to high glucose stimulation and in diabetic conditions (ikezumi et al. ; ruster et al. ). high glucose could stimulate activity and expression of the local ras components in podocyte, including ang ii and its at receptors (yoo et al. ). following that, it was recently demonstrated that local ras activation would lead to podocyte injury through a variety of pathways. ang ii could induce podocyte apoptosis through activation of nadph oxidase and production of ros, and upregulate the expression of glut transporters (gill and wilcox ; nose et al. ). in addition, ma et al. found that lipid accumulation in podocytes was increased under the high glucose stimulation, which is mediated through the disruption of low-density lipoprotein receptor (ldlr) pathway (zhang et al. a ). interestingly, reducing lipid accumulation in podocytes decreased the protein expression of sma and increased the expression of nephrin in podocyte. these studies reveal that high glucose-induced lipid accumulation is involved in the podocyte injury in dn. therefore, the above shows that high glucose could induce various other metabolic disorders and indirectly lead to podocyte injury. lipid metabolism disorder is commonly observed in patients with chronic kidney disease (ckd), accompanied by increased fasting triglyceride levels and decreased high-density lipoprotein cholesterol (hdl-c) (bianchi et al. ) . it is increasingly recognized that dysregulation of lipid metabolism is involved in the development and progression of ckd, such as obesity-related renal disease and dn (de vries et al. ) . podocytes, as specialized cells of glomerulus, play an important role in the pathologist of ckd when they are injured (fiorina et al. ) . and excessive lipid accumulation in podocytes can lead to cellular dysfunction and death, which is called lipotoxicity. between neighboring podocytes, there is a unique interdigitating structure bridged by sd, maintaining the proper glomerular filtration (ruotsalainen et al. ) . researches have revealed that sd is a lipid raft structure containing multiple podocyte-specific proteins, such as podocin and nephrin (schermer and benzing ) . in particular, podocin can recruit and bind to cholesterol to form sd, and this binding can influence the composition of lipid membrane, allowing cholesterol to contact with the ion-channel transient receptor potential canonical (trpc ) (huber et al. ). this suggests that cholesterol homeostasis is essential for glomerular functions. however, excessive cholesterol can also negatively disrupt the mutual binding of podocyte sd proteins, or interfere with the binding between podocyte sd proteins and caveolin- , a lipid raft-associated protein, binding nephrin, and cluster of differentiation (cd )-associated protein (sorensson et al. ) it is regulated by some functional proteins such as atp-binding cassette transporter a (abca ) involving cholesterol efflux, -hydroxy- -methyl-glutaryl coa reductase (hmg-coa reductase, hmgcr) regulating cholesterol synthesis and lowdensity lipoprotein receptor (ldlr) mediating cholesterol influx. the expression of hmgcr and ldlr is regulated by some transcription factors, such as the sterol regulatory element-binding protein (srebp), under negative feedback loops. when cells are rich in cholesterol or its derivatives, the transcription of ldlr gene or other genes necessary to lipid synthesis are suppressed. as a result, the cells are not able to generate and uptake cholesterol, and then establish cholesterol homeostasis. in contrast, when intracellular sterols are exhausted, the transcriptions of srebp target genes will be activated, increasing intracellular cholesterol (zhang et al. ). this enables cellular cholesterol homeostasis despite physiological fluctuations in cholesterol requirements and exogenous supply. however, it is demonstrated that the cellular cholesterol imbalance of podocytes can induce proteinuric glomerular diseases (merscher et al. ) . it is demonstrated that human glomerular podocytes express abca , hmgcr, and ldlr (merscher-gomez et al. ). ma et al. found that some pathogenic factors such as inflammation can disrupt ldlr pathway (zhang et al. b ). thus, excessive lipid accumulates in podocytes, resulting in effacement of the foot processes and epithelial mesenchymal transition of podocytes (zhang et al. b) . it is recently demonstrated that human podocytes treated with the sera from diabetic kidney disease (dkd) patients had increased cholesterol accumulation compared with human podocytes exposed to the sera of patients with diabetes, but no dkd. this was associated with a reduction of abca and an impairment of cholesterol efflux (merscher-gomez et al. ) . besides, it is showed that c-x-c motif ligand (cxcl ) is the main scavenger receptor for oxidized ldl (oxldl) in human podocyte (gutwein et al. ). the expression of glomerular cxcl was increased in patients with membranous nephropathy, accompanied with higher levels of oxldl (gutwein et al. ). and in diabetic db/db mice, cxcl pathway was activated, in parallel with increased cholesterol accumulation in kidney (hu et al. ) . in vitro, oxldl can induce loss of nephrin expression from cultured podocytes (bussolati et al. ) . in summary, cholesterol metabolism disorder can destroy the structure and function of podocytes, leading to the progression of ckd. in addition to hypercholesterolemia, free fatty acids (ffas) can also affect podocyte function in kidney disease. the essential role of fatty acids is to form the phospholipid bilayers of the cell membranes and act as phospholipid messengers, transmitting vital intracellular signals (lee ) . normal cellular fatty acid homeostasis reflects a balance between generation or delivery and utilization. srebp- c is involved in fatty acid and tg synthesis, targeting lipogenic enzymes including acetyl-coa carboxylase (acc) and fatty acid synthase (fas) (horton et al. ) . ffas can be transported into cells by the scavenger receptor platelet glycoprotein (also called as cd ) or via the assistance of vascular endothelial growth factor b (vegf-b) (hagberg et al. ; masuda et al. ). cellular ffas are esterified or transported into the mitochondria for oxidation and subsequent energy production (lee ) . palmitic and stearic acids, belonging to saturated ffas (sfas), and oleic acid, belonging to monounsaturated ffas (mufas), account for - % of plasma ffas (raclot et al. ) . sfas can induce insulin resistance and cell death, involving the pathogenesis of diabetes mellitus type (t dm) (lennon et al. ; sieber et al. ) . in contrast, mufas can prevent sfa-induced lipotoxicity (sieber et al. ) . in human podocytes, insulin resistance can be induced by palmitic acid (lennon et al. ). it is observed that insulin sensitivity in glomeruli of obese and diabetic rats is reduced (mima et al. ) . podocyte-specific insulin receptor knockout mice develop albuminuria and glomerulosclerosis, indicating that normal insulin signaling is critical for podocyte function and survival (welsh et al. ) . these findings imply that ffas play potential roles in insulin resistance, promoting the development and progression of obesity-related renal disease and dn. in the tubulointerstitial and glomerular segment of renal biopsies obtained from patients with dn, endoplasmic reticulum (er) stress is observed (sieber et al. ) . importantly, in a t d mouse model, the progression of dn can be attenuated by ameliorating er stress (qi et al. ) . er dyshomeostasis can decrease the er folding capacity, thereby leading to accumulation of unfolded and misfolded proteins. this in turn initiates the unfolded protein response (upr), adaptively maintaining proper er function (ma and hendershot ) . but if er stress persists, apoptosis will be induced by the proapoptotic transcription factor c/ebp homologous protein (chop) (rasheva and domingos ) . in podocytes, er stress induced by palmitic acid results in the upregulation of several upr markers/effectors, such as the er chaperone heavy chain-binding protein (bip), and chop, while monounsaturated palmitoleic and oleic acids only upregulated bip but not chop (sieber et al. ) . as bip can attenuate palmitic acid-induced apoptosis (laybutt et al. ) , the beneficial effect of mufas may own to the upregulation of bip. in addition to the unfolded proteins, alterations in er membrane lipid composition can also sensitively affect the expression of the er stress sensor inositol requiring enzyme (ire- ) (promlek et al. ) . it is shown that small molecule compound m c, specific ire- inhibition, can attenuate palmitic acid-induced podocyte death (sieber and jehle ) . enhanced ffa uptake by podocytes is induced by increased expression of cd and a decrease in fatty acid β-oxidation, leading to excessive intracellular lipid accumulation (soetikno et al. ). in animal model of type diabetes (t d), increased expression of srebp- in renal results in upregulation of enzymes responsible for ffa synthesis and as a consequence of a high level of triglyceride (tg) in renal (hashizume and mihara ) . accumulated lipids in podocytes limited mitochondrial fatty acids β-oxidation. it induced mitochondrial damage and inhibition of amp kinase (ampk) activity, leading to endoplasmic reticulum (er) stress, autophagy, and apoptosis in podocytes. as a result, mitochondrial dysfunction caused decreased podocyte density and increased in foot process width, together with inflammation (szeto et al. ) . renal accumulation of tg is associated with reduced expression of the ultrasensitive energy sensor ampk strongly. this suggests that the imbalance between energy-generating and energy-consuming pathways might be related to podocyte dysfunction in dkd and other disorders in ckd, due to lipid accumulation (wahl et al. ) . and hypertriglyceridemia can also increase podocytic de novo expression of desmin, which represents podocyte injury (joles et al. ) . since the first description that glycosphingolipid accumulation in the renal results in glomerular hypertrophy in streptozotocin (stz)-induced diabetic mice, several studies have highlighted the role of sphingolipids and gangliosides in podocyte biology (merscher-gomez et al. ) . analysis of kidney biopsy compartments from patients with fabry disease using unbiased quantitative stereology indicated age-dependent accumulation of globotriaosylceramide (gb ) in podocytes (najafian et al. ) . in vitro, globotriaosylsphingosine (known as lysoglobotriaosylceramide) acts as a profibrotic metabolite in cultured human podocytes (sanchez-nino et al. ) . ganglioside gm (gm ) is a receptor for soluble flt , locating in lipid raft domains in the sd of podocytes. binding of soluble flt to gm plays essential roles in autocrine preservation of the podocyte actin cytoskeleton and in prevention of proteinuria (jin et al. ) . o-acetylated disialosyllactosylceramide (gd ), a sialic-acid-containing lipid, was identified as a podocyte-specific ganglioside in rat (reivinen et al. ) . treating mice with an antibody against gd caused nephrin phosphorylation and dislocation from the podocyte sd . it is an emerging concept that sphingolipids act as modulators of podocyte function in fsgs and other glomerular diseases. patients with fsgs are more likely to have recurrence of proteinuria after kidney transplantation. and the number of acid sphingomyelinase-like phosphodiesterase b (smpdl b) positive podocytes is decreased in patients with recurrent proteinuria (fornoni et al. ) . to sum up, lipid metabolism disorder is involved in the pathogenesis of podocyte injury. cholesterol helps form sd between podocytes, maintaining the proper glomerular filtration. ldl-cholesterol uptake is mediated via the ldlr or cxcl and may cause er stress. cholesterol metabolism is regulated by several nuclear receptors and transcription factors, including srebp. excessive cholesterol accumulation in podocytes may contribute to kidney disease. free fatty acids are primarily transported via cd , causing oxidative and er stress based on the degree of saturation. sphingolipids and gangliosides also play a role in podocyte biology. binding of soluble flt to gm plays essential roles in autocrine preservation of the podocyte actin cytoskeleton and in prevention of proteinuria ( fig. . ). hypertension has become the second leading cause of end-stage renal disease (esrd) after diabetes mellitus (udani et al. ) . high blood pressure can affect renal vessels, glomeruli, and tubulointerstitium. recently, more and more studies have indicated that podocyte damage play an important role in hypertensive nephrosclerosis. decreased intrarenal podocyte and increased urinary podocyte were observed in hypertensive nephrosclerosis ). as terminally differentiated cells, podocyte loss leads to denudation of the glomerular basement membrane (gbm) and focal adhesion of the tufts to bowman's capsule, which finally results in glomerulosclerosis and reduced filtration (cellesi et al. ) . podocyte loss in hypertension includes detachment of viable cells and apoptosis (kriz et al. ) . the major factor for podocyte loss in hypertension is the capillary hypertension, which cause glomerular hypertrophy and hyperfiltration (kriz and lemley ) . glomerular hypertrophy results in relatively decreased podocyte density. puelles et al. ( ) examined the effect of hypertension on podocyte depletion using kidneys obtained from autopsy, and they did not observe a difference in total podocyte number solely driven by hypertension, while the relative podocyte depletion is associated with glomerular hypertrophy which resulted in the reductions in podocyte density. hyperfiltration gives rise to increased shear stress by elevating driving force and augmenting gbm area. podocytes cultured in vitro are sensitive to shear stress, which induces reorganization of cytoskeleton (friedrich et al. ) , and this helps them to cover an expanding gbm which further leads to foot process effacement. in desoxycorticosterone-trimethylacetate (doca) hypertensive mice, chloride intracellular channel a, which is highly enriched in podocytes foot process, protects against hypertension-induced podocyte injury through weakening the tensile strength of the actin cytoskeleton in rac -dependent manner (tavasoli et al. ). this has been considered to be the protective response for podocyte to escape detachment. however, this strategy is not always successful and finally results in podocyte detachment from gbm, as seen in progressive stage of fawnhooded hypertensive (fhh) (kriz et al. c) and doca hypertensive rat model . apoptosis is another cause for podocyte loss under shear stress in hypertension, which is before or in conjunction with cell detachment (kriz et al. ; ying et al. ) . besides mechanical stress, renin-angiotensin-aldosterone system (raas) plays central role in the pathogenesis of hypertensive nephrosclerosis, mainly through its actions on the subtype receptor. mechanical strain increased angiotensin ii production and upregulation of angiotensin receptor (at ) in cultured podocytes, while the increased apoptosis induced by mechanical strain was also in an angiotensin ii-dependent manner (durvasula et al. ) . increased angiotensin ii results in decreased expression of podocin and integrin β , which are both vital in viable podocytes adhesion to the gbm and interaction of podocytes with other gbm components. this might elucidate that the elevated intraglomerular pressure is translated into a maladaptive response in podocyte probably due to the activation of local tissue angiotensin system. furthermore, angiotensin ii is also considered to be associated with the rearrangement of the actin cytoskeleton (macconi et al. ) . aldosterone, an important mediator of the effect of angiotensin, has become a hot spot concern in hypertensive nephropathy. using only the inhibition of aldosterone by eplerenone dramatically alleviated podocyte injury in dahl salt-hypertensive rats, an animal model inclined to hypertensive glomerulosclerosis (nagase et al. ) . in a doubleblind, randomized, placebo-controlled trial, additional use of low-dose eplerenone to renin-angiotensin system inhibitors has renoprotective effects in hypertensive patients with non-diabetic chronic kidney disease (ando et al. ). these findings suggested that aldosterone plays an important role in hypertension-induced podocyte injury. the underlying mechanism is primarily due to aldosterone-induced mitochondrial dysfunction, which increased oxidative stress. in uninephrectomized rats infused with aldosterone and fed with high-salt diet, podocyte-associated proteins nephrin and podocin were dramatically decreased, along with reduced nicotinamideadenine dinucleotide phosphate oxidase activation, increased oxidative stress, and enhanced aldosterone effector kinase sgk . thus, podocyte is the prominent target for aldosterone by inducing oxidative stress and sgk ). selective mineralocorticoid receptor (mr) antagonist eplerenone also ameliorated the saltinduced proteinuria and podocyte injury in hypertensive rat model ). after detachment from gbm, podocyte moves through meshes of bowman's capsule to the urine and might keep alive. unfortunately, the detection of viable podocytes in the urine is a complex procedure, which is still unavailable in all laboratories. however, elevated mrna levels of podocin and nephrin can be examined in urine of hypertensive patients (kelder et al. ) . recent studies suggested that increased podocyte-derived extracellular vesicles may predict podocyte stress and subsequent podocyte loss in hypertensive patients, which might provide a novel non-invasive detective method (kwon et al. ) . podocytes, as the gatekeepers of protein in glomerular filtration barrier, are major targets of high blood pressure. in all, hypertension could cause mechanical stress and the activation of raas (mentioned below). mechanical stress further induces capillary hypertension, promoting glomerular hypertrophy and hyperfiltration. these changes would lead to reduced podocyte density and the reorganization of cytoskeleton in podocytes, resulting in detachment of viable podocyte and podocyte apoptosis, progressing to final glomerulosclerosis. more studies are needed to prove that podocytes can be the detective marker for hypertensive nephrosclerosis and find the more specific method for early diagnosis and treatment. hemodynamic changes and ras of the glomeruli are key factors of ckd patients' persistent proteinuria and disease progression. many investigations suggest that local intrarenal ras activation contributes to kidney tissue injury (gurley et al. ) , and ras activation accelerates renal injury by various mechanisms. angiotensinogen (agt), the original of ras, transforms into ang ii through the conversion of ang i as a result of the enzymatic cleavage process by renin and ace. as the most active peptide of ras, ang ii was demonstrated to induce tgf-β expression and provoke oxidative stress and inflammation, which are main factors in the initiation, development, and progression of ckd (ruggenenti et al. ) . under a condition of continuous glomerular hypertension in ckd, podocytes may undergo actin cytoskeletal reorganization, compensatory hypertrophy, weakened local adhesion ability due to downregulation of adhesion molecules of basement membrane cells, and apoptosis of podocytes induced by local ang ii activation. the continuous increase of ang ii caused by mechanical stress further affects the capillary intraglomerular pressure, resulting in a vicious circle and contributing to the pathogenesis of glomerulosclerosis (ruster and wolf ) . in addition to causing podocyte lesions by altering glomerular hemodynamics, ang ii also has a direct effect on the structure and functions of podocytes, which is mentioned later in this section. podocytes, in possession of a complete ras (marquez et al. ) , can produce functional ras elements themselves and participate in local ras systems as well, playing an important role in not only its own physiological process but pathological status ( fig. . ) . it has been reported that mechanical stress and high glucose could increase the production of local ang ii and at receptor (at r) in podocytes (durvasula et al. ; durvasula and shankland ) , with inducing the expression of other ras elements (sakoda et al. ) . there are also important elements of the ras system expressing in human differentiated podocytes, including angiotensin, renin, ace, at r, and at r subtype mrna, but the related proteins were not detected (liebau et al. ) . therefore, podocytes could not only be a target of the damage caused by ang ii, but a source of localized ang ii as well. however, it has been found that ang ii secreted by podocytes was not blocked by renin inhibitors, acei, and chymase inhibitors (liebau et al. ) , suggesting that there might be an unknown pathway for ang ii formation in podocytes. velez et al. ( ) used matrix-assisted laser desorption/ionization time of flight mass spectrometry (maldi-tof-ms) to quantify the presence of rasrelated peptide chains in rat podocytes, in order to further explore the role of podocytes in the metabolism of ras elements. as a result, after co-incubated with ang i, mesangial cells mainly produced ang ii while the main product of podocytes was ang ( - ) with almost no ang ii. furthermore, it was confirmed that podocyteproducing ang ( - ) is mainly through the neprilysin pathway, as ace-mediated ang ii production did not result in an increase of ang ii concentration in podocytes, which might be related to podocytes' degradation of ang ii through ace and aminopeptidase a pathways. as a new member of ras, ace might have a negatively regulatory effect on ace-produced ang ii of traditional ras, mainly by accelerating the degradation of ang ii to attenuate its effect, and through the generation of ang ( - ), which has the most expression in podocyte ras. there has been no evidence that podocytes express the receptor of ang ( - ), i.e., mas, yet ang ( - ) and its receptor seem to be involved in the renal protection for dn, such as regulating inflammation, oxidative stress, and retaining the progression of renal fibrosis. therefore, podocytes probably play an essential role in maintaining the balance of local ras system in the kidney, similar to that between systemic ang ii and intrarenal ras system, by degrading the systemic ang ii filtered from the glomeruli, and/or promoting the conversion of glomerular-filtrated ang i and agt to ang ( - ), thereby regulating the damage caused by the whole systemic ang ii to the kidney. the pathways of ang ii signaling mediating podocyte injury can be generally divided into the following aspect: ( ) to damage the function of the pore membrane and structure of the cytoskeleton the sd is an essential structure of the glomerular filtration barrier, which is connected to the foot processes adjacent to podocytes. nephrin and zonula occludens (zo)- are main proteins of sd, preventing macromolecules from entering the urine. it has been found that sd is susceptible to damage, leading to decreased expression of nephrin and zo- , and cytoskeletal reorganization of podocytes. it has been found that the expression of nephrin in renal biopsy specimens from patients with t dm-induced dkd was significantly reduced compared with healthy volunteers, and the patient's urinary concentration of nephrin was significantly positively correlated with their urinary protein level (jim et al. ) . ren et al. ( ) have found in vitro that ang ii could directly cause the downregulation and dephosphorylation of nephrin, which mediates podocyte injury. besides, application of acei and arb has been reported to inhibit the rearrangement of cytoplasmic zo- and reduced the degree of proteinuria (macconi et al. ) . ( ) to induce podocyte apoptosis one of the main causes of podocyte loss in ckd patients is podocyte apoptosis, and the occurrence of urinary podocyte plays an important role in glomerular sclerosis. ang ii reportedly could induce the apoptosis of rat podocytes cultured in vitro in a dose-and time-dependent manner, and this process required cells to be exposed to tgf-β and tgf-β antibody could inhibit apoptosis of podocytes (ding et al. ) . after activation of tgf-β in diabetic glomeruli, the nuclear factor κb might be inhibited via the gene smad , resulting in podocyte apoptosis. the advanced glycation end products (ages) were also found to activate the ras system of podocytes, upregulate ang ii levels, and induce podocyte apoptosis via a ages receptor-pik /protein kinase b (akt)-dependent signaling pathway; arb could attenuate ang ii-induced podocyte apoptosis. ( ) to cause cell phenotypic transformation and hypertrophy p kip encodes a protein which belongs to cyclin-dependent kinase (cdk) inhibitor proteins, which could control the cell cycle progression at g phrase, thereby inhibiting cell proliferation. it was found that ang ii could directly increase the levels of p kip mrna and protein in podocytes cultured in vitro and in vivo in dn, which was inhibited by arb. ang ii-induced upregulation of p kip expression might lead to podocyte hypertrophy (xu et al. ) . it was also observed that ang ii can upregulate the expression of p kip protein, causing pathological podocyte hypertrophy similar to that in a dn text (romero et al. ) . as an essential factor promoting the progression to renal fibrosis, emt in podocytes will result in loss of epithelial markers with de novo expression of emt markers; in more severe cases, it may lead to podocyte detachment from the glomerular basement membrane, thereby aggravating proteinuria and glomerulosclerosis loeffler and wolf ) . a recent study reported that a high concentration of glucose and ang ii promoted emt in podocytes, which could be reversed by silencing tcf (bai et al. ). ( ) to induce podocyte membrane depolarization and damage the charge barrier studies by using patch clamp recording technique in isolated glomeruli in vitro have demonstrated that ang ii could cause sustained and irreversible depolarization of podocyte membranes. stimulation of ang ii resulted in an immediate calcium influx of cultured podocytes (greka and mundel ) . studies have confirmed that trpc colocalized with podocyte nephrin and podocin, and its functional mutation could disrupt the integrity of the pore membrane, leading to proteinuria and fsgs (reiser et al. ; winn et al. ) . numerous studies have found that abnormal calcium signaling may be the main cause of related podocyte diseases. for example, calcium increases evoked by ang ii are primarily mediated via trpc channels and this pathway could be pharmacologically targeted to abate the development of dkd (nijenhuis et al. ; sonneveld et al. ). ( ) to induce podocyte autophagy as terminally differentiated cells, podocytes mainly reduce intracellular accumulation of damaged dna and macromolecular substances through autophagy rather than cell division (pan et al. ) . in vitro experiments, animal experiments, and human kidney biopsy indicate that podocytes have a high-level basis of autophagy, which plays an important role in maintaining the stability of podocytes. recent research using a ckd animal model has demonstrated that autophagy is an essential intracellular process to encourage the survival of renal cells (huber et al. ) , while excessive and dysfunctional autophagy might result in podocyte injury (de rechter et al. ) . it has been found that ang ii could enhance the ros production and increase oxidative stress in the renal system by enhancing the activity of systematic nadph, leading to detrimental podocyte autophagy (de rechter et al. ; yadav et al. ) , the underlying pathways of which is dependent or independent on mtor (mao et al. ) . a recent study has found that autophagy could enhance the cell viability of ang ii-treated podocytes, suggesting improving autophagy may become a new targeted therapy to relieve ang ii-induced podocyte injury . traditionally concerned solely as an inactive precursor of renin, prorenin actually participates in the functional regulation of body through the hydrolysis of agt to produce ang i and can also bind to prorenin/renin receptor (prr) (non-proteolytic pathway) to activate, like mitogen-activated protein kinases (mapks), initiating intracellular signal transductions. the plasma prorenin/renin ratio in diabetic patients was significantly higher, and the prorenin levels began to increase before the appearance of micro-albuminuria without changes in renin levels (sakoda et al. ) , suggesting that prorenin itself exerts somewhat important effects on dn. immunofluorescence double-labeling studies have showed that prorenin activated by non-proteolytic pathway coexisted with the podocyte marker nephrin, and electron microscopy also displayed that prr was distributed on podocyte foot processes (ichihara et al. ) . handle region peptide (hrp) is a polypeptide blocker of prorenin receptor. ichihara et al. ( ) have found that gene deletion of at r or using acei inhibitor could to some extent delay the occurrence of proteinuria and glomerular sclerosis in streptomycin-induced dn rats, while continuous instillation of hrp could almost completely block the progression of dn. it is noteworthy that the mapk signaling pathway was activated in at r-deficient mice, and hrp could significantly inhibit mapk, indicative of an equally important role of prorenin coupling with prr-induced angiotensin-independent pathway in diabetic kidney injuries. besides, sakoda et al. ( ) have confirmed that adding prorenin to human podocytes cultured in vitro could increase the intracellular level of ang ii and activate the mapk intracellular signal transduction pathway, resulting in podocyte damage. in mammalians, the acute-phase reaction is beneficial for eliminating acute insults for protection against microorganisms, limiting tissue damage, and maintaining homeostasis. this reaction would become disadvantageous under a chronic condition called micro-inflammation. micro-inflammation is a state with low-intensity, chronic persistent and dominant inflammation caused by the infection of non-pathogenic microorganisms, which is characterized by mild persistent elevation of inflammatory cytokines in the systemic circulation (kaysen ; schomig et al. ) . micro-inflammation is a continuous and relatively secretive action, the essence of which is immune inflammation. micro-inflammation state has no obvious clinical symptoms, there is no specific diagnostic criteria, and the diagnosis of micro-inflammation relies mainly on the examination of circulating inflammatory biomarkers such as c-reactive protein (crp) and serum amyloid a (saa), tumor necrosis factor alpha (tnf-α), and interleukin- (il- ). the acute-phase reactants including the above proteins are mainly synthesized by hepatocytes, such as complement components, coagulation proteins, and metal-binding proteins. it is important to note that when we are in the diagnosis of micro-inflammatory state, other causes and diseases of increased inflammatory markers must first be ruled out, such as connective tissue disease and recent microbial infection. during acute-phase reaction, the concentration of crp may increase over fold compared with normal levels (kaysen ). in addition, crp follows the course of a disease with little delay due to its short half-life. crp is supposed to bind multiple other binding specificities such as opsonin of bacteria, immune complexes, and chromatin. crp reflects not only the activity of inflammation, is also a sign of cytokine activation, its levels was positively associated with the degree of infection. the diagnosis of state of micro-inflammation based on crp is the level of crp > mg/l but not more than - mg/l. saa is a sensitive acute-phase reactant in micro-inflammatory state. the level of saa obviously rises before other acute-phase reaction proteins. a variety of inflammatory cytokines have emerged as being closely involved in the micro-inflammation state. immune cells and intrinsic renal cells such as podocytes secrete proinflammatory cytokines including interleukin- (il- ), il- , tnf-α, and monocyte chemoattractant protein- (mcp- ), which may contribute to the inflammatory process and aggravate diseases progression. for dn as an example, a strong induction of mcp- and keratinocyte chemoattractant (kc) by fetuin-a (feta) or lipopolysaccharide (lps) is associated with exacerbated palmitic acid-induced podocyte death. moreover, the prevention of mcp- and kc secretion and inhibition of il- attenuates the inflammatory and ultimate cell death response elicited by feta alone or combined with palmitic acid. the study offers evidence that inflammation aggravates palmitic acid-induced podocyte death and the il- β signaling might be novel potential therapeutic targets for prevention and treatment of dn (orellana et al. ) . infiltrating macrophages/monocytes are associated with chronic, low-grade inflammation. the macrophages can interact with resident renal cells to generate a proinflammatory micro-environment that amplifies tissue injury and promotes scarring. macrophage-derived tnf-α had a direct role in the progression of dn. conditional deletion of tnf-α from macrophages markedly reduced albuminuria, lessening the increase of plasma creatinine and histopathologic lesions (awad et al. ) . likewise, tonicity-responsive enhancer-binding protein (tonebp) in macrophages promotes hyperglycemia-mediated proinflammatory activation and chronic renal inflammation leading to dn and ckd (choi et al. ). lipids such as triglycerides and cholesterol may accumulate ectopically in the kidney, which contributes to a lipotoxicity process. palmitic acid-treated podocytes had intracellular lipid accumulation and abnormal lipid metabolism, accompanied by the process of inflammation, insulin resistance, and rearrangements of the sd and actin cytoskeleton of podocyte. thus, lipotoxicity accelerated podocyte damage through lipid accumulation related inflammation (martinez-garcia et al. ) . lipoproteins including ldl, vldl, and idl might act as proinflammatory mediators, which promote the production of inflammatory cytokines, such as tgf-β, platelet-derived growth factor (pdgf), and il- secreted from human mesangial cells. lipoprotein-mediated cytokine production may cause recruitment of monocytes, lipid-mediated cell proliferation and apoptosis, and extracellular matrix production, thus contributing to podocyte injuries and glomerulosclerosis. chronic inflammation can reduce podocyte insulin sensitivity. nucleotide-binding oligomerization domain-containing (nod ) is a subtype of intracellular pattern recognition receptor (prr), playing functions in innate immunity. of particular interest, increased levels of nod were observed in dn patients and high fat diet (hfd)/stz-induced mice models. furthermore, hfd/stz-induced diabetes mice models with nod knock-out showed reduced podocyte injury and proteinuria compared with wild-type diabetic mice (du et al. ) . in vitro, nod which was activated by bacterial component muramyl dipeptide in podocytes reduced insulininduced glucose uptake and inhibited serine phosphorylation of irs- . another study has explored the role of other prr toll-like receptors (tlrs) in the db/db mice model of dn. administration of a selective tlr / / inhibitor git improved insulin sensitivity, reduced albuminuria and urinary nephrin levels, indicative of reduced podocyte damage. tlr expression in podocytes was found to be highest expressed (cha et al. ) . given the links between some specific prrs activation and insulin stimulation in podocytes, how podocyte insulin responses are altered following prrs activation and inhibition may need specifically investigated. ikb/nf-κb is another important pathway of insulin resistance in podocyte, and nf-κb expression was increased in kidney tissues of patients with type diabetes. nf-κb can increase the level of irs serine phosphorylation and the expression of inflammatory mcp- , il- , and tnf-α. moreover, the increased expressed inflammatory factors can further activate the nf-κb. the inflammatory cytokines and the activation of nf-κb pathway form positive feedback to induce insulin resistance. xu et al. reported that chronic systemic inflammation exacerbates lipid accumulation in the kidney of apoe knockout mice by diverting lipid from the plasma to the kidney via the scap-srebp -ldlr pathway and causing renal injury (xu et al. ) . consisted with this, il- β stimulation in vitro increased the lipid accumulation in the podocytes by increasing the expression of lipid metabolism related proteins, for instance, ldlr, sterol regulatory element-binding protein- (srebp- ) and srebp cleavage-activating protein (scap), and through promoting translocation of the scap/srebp- complex from the endoplasmic reticulum to the golgi in the podocytes (zhang et al. b) . compared with db/db mice, podocyte injury was more severe in db/db mice with subcutaneous casein injections, which are supposed to induce inflammatory stress in vivo. altogether, inflammation may be associated with high risk for chronic renal fibrosis. activation of intrinsic proinflammatory signaling in podocytes such as nf-κb signal pathway aggravates podocyte injury and proteinuria. in stz-induced diabetic mice models with ccr knock-out, transgenic ccr overexpression in the podocytes resulted in significantly increased albuminuria and podocyte loss, without concurrent increase in kidney macrophage infiltration or inflammatory cytokine production. these findings support that activation of ccr signaling cascade in podocytes mediates diabetic renal injury, which is independent of macrophage recruitment (you et al. ). il- , a proinflammatory cytokine which is upregulated by high glucose and tgf-β , can increase mcp- and tgf-β expression in podocytes and induce apoptosis in podocytes through activating caspase- . in stz-induced early dn mice models, anti-il- monoclonal antibody ( e) treatment or il- r -deficiency led to lower blood glucose and improved renal functions, and il- is proved to be expressed in podocytes. collectively, intrinsic proinflammatory signaling in podocytes contributes to podocyte damage ( fig. . ). immune injuries are common causes of podocyte damage. processes interfering with podocyte's structural or functional integrity lead to disruption of the glomerular filtration barrier. primary-cultured human podocytes synthesize and secrete complement c physiologically, and the stimulation of inflammatory factor inf-γ could increase the production of c . under physiological conditions, c produced by glomerular podocytes can resist the invasion of foreign pathogens and protect local tissues. c activation can lead to decreased immune complex formation and increased disintegration. on the other hand, c activation leads to increased production of vasoactive molecules and chemokines, which in turn recruits more inflammatory mediators into the glomerulus. the activation of complement would produce proinflammatory components of complement, i.e., c a. in immune complex diseases and ischemia-reperfusion injury, c a is an important mediator that triggers an inflammatory cascade (heller et al. ) . the kidney is one of the organs that are most susceptible to abnormally activated complement, which can be seen in various glomerulonephritis. the main pathogenesis of idiopathic membranous nephropathy (imn) is caused by the binding of igg to the intrinsic antigen on the basement membrane side of glomerular podocytes, which combine to form an antigen-antibody complex, thereby activating the complementforming membrane attack complex (takano et al. ) . in imn, the concentrations of complement cleavage products such as c a, c a and c b- are significantly increased. c b- is the final product of complement activation in three pathways of complement activation, causing podocyte injury not through conventional lysis, but probably via the mechanism related to the activation of corresponding intracellular signaling pathways in a subdissolved form. ronco and debiec have confirmed that the podocyte surface antigen megalin binded to the corresponding antibody underwent an immune complex reaction, activated the complement system, and promoted the formation of the membrane attack complex c b- (ronco and debiec ) . as a stimulant of podocytes, c b- could destroy podocyte cytoskeletal proteins, inserting in the membrane to increase cell permeability, and activating a series of transduction pathways, resulting in the diffuse thickening of gbm and defects in glomerular filtration barrier, clinically leading to significant proteinuria. in addition, podocytes begin to express complement receptor (cr , or c br, or cd ) during the capillary synthesis stage of renal development and are evenly distributed on the cell membrane and the membrane of foot processes. cr is expressed as a cofactor of the complement factor i and expressed in most circulating cells. cr is the only physiological blocker of complement synthesis in podocytes and inactivates the lysate of complement to promote the clearance of immune complexes, protecting podocytes from complement-mediated damage (alexander et al. ). it has been reported that the production of cr was reduced in several glomerular diseases, making podocytes vulnerable to complement attacks. complement regulatory proteins include crry, cd , and decay acceleration factors (daf or cd ), which are vital to limiting the activation of podocyte complement (cheng et al. ) . podocyte expression of crry and cd could inhibit c invertase and the synthesis of c b- , thus to protect podocytes from injuries induced by antibody-complement activation. in addition, podocytes both in vitro and in vivo could be detected of daf. in a mouse model of nephritis, deficiency of daf resulted in serious podocyte foot fusion, indicating that daf might protect podocytes from complement-mediated injury (bao et al. ). in both physiological and pathological texts, podocytes of humans, rats, and mice all express the receptors of cytokines interleukin (il- ), il- , and il- . after stimulating podocytes cultured in vitro with il- and il- , the skeletal structure and intercellular-link protein of podocytes were damaged and the permeability increased (ha et al. ; kim et al. ) , suggesting that il- and il- could damage podocytes by binding to its receptors. in early minimal change disease (mcd), fsgs, and mn, podocytes increasingly express inflammatory mediators il- α/β along with il- type receptor (il- ri), and il- ri is decreasingly expressed at late stage of the disease when glomerular cell hyperplasia and sclerosis appear (brahler et al. ) , indicating that these molecules participate in podocyte damage and repair, glomerular local inflammation. in addition, both podocytes cultured in vitro and renal tissue express receptors of functional cc chemokine receptor (ccr) and cxc chemokine receptors (cxcr), which could couple with corresponding chemokines to promote the production of cytoplasm ca + and ros and be involved in podocyte injuries (huber et al. ) . moreover, it has been found that podocytes themselves could produce il- (ligand of cxcr /cxcr ), thus podocytes could be activated via autocrine. cxcl might play an important role in the inflammatory response of kidney diseases. podocytes overexpress cxcl under the stimulation of proinflammatory factors. soluble cxcl plays a chemotactic role in inflammation and immune response, while transmembrane cxcl removes oxldl (gutwein et al. ), which is harmful to the kidney. therefore, abnormal expression of cxcl in podocytes might cause renal damage due to excessive immune-inflammatory reaction or an accumulation of oxldl. it has been found that the expression of cxcl and oxldl in the glomeruli of mn patients increased not only significantly but consistently as well (gutwein et al. ). the inflammatory factor ifn-γ is the strongest stimulator of cxcl , which upregulates several forms and overall cell expression levels of cxcl , consequently promoting podocyte damage (wang et al. ) . under physiological conditions, podocytes of humans and mice could express tlr . stimulating cultured murine podocytes in vitro with the ligand of tlr -like lps, lipid a, and fibrins (endogenous ligand), resulted in an increasing expression of ccl and cxcl. in the mouse model of cryoglobulinemia membrane proliferative glomerulonephritis, podocytes expressed more tlr , promoting the synthesis and secretion of chemokines and further leukocyte recruitment and glomerular injury (banas et al. ) . it has been shown that under the stimulation of endogenous tlr ligand, podocytes upregulate tlr , promote the production of proinflammatory chemokines, and actively participate in the recruitment of inflammatory cells, all leading to glomerular injuries (banas et al. ) . apart from tlr , other members of the tlr family have also been proved to participate in podocyte injury. a recent study has pointed out that the overexpression of tlr- correlates with the progression of podocyte injury in glomerulonephritis, suggesting that altered levels of urinary tlr mrna might reflect the degree of podocyte injury in murine autoimmune gn (kimura et al. ) . tlr- and tlr- expressed by b cells and dendritic cells have been considered as important molecules involved in the pathogenesis of systemic lupus. recent study demonstrated that active ln onset in childhood expressed more tlr- , accompanied by weakened expression of podocyte sd protein nephrin, podocin, and synaptopodin; in the meantime, patients showed proteinuria and high ds-dna antibody and low complement (machida et al. ) . therefore, under pathological conditions, tlrs link the innate immune system with podocyte and glomerular injuries. b - (cd ) belongs to the immunoglobulin superfamily, mainly expressed in antigen-presenting cells, and provides a costimulatory signal by coupling with corresponding molecular receptors expressed on t cells, i.e., cd and ctla , regulating the immune responses induced by activated t cells. it has been found that b - was expressed on podocytes of lupus nephritis (ln) (reiser et al. ) , and the expression of podocyte b - in ln patients and ln mouse models is positively correlated with the degree of proteinuria. however, new evidence has stricken up a discordant tune (baye et al. ), leading to further mandatory studies of the application of b - blockers in treating proteinuric patients (novelli et al. a) . studies have shown that under the induction of hypoxia, high glucose, or bacteriocin lipopolysaccharide (lps), the expression of b - would be induced in podocytes which does not occur under physiological conditions, and participate in podocyte cytoskeletal reorganization and the pathogenesis of proteinuria (chang et al. ; fiorina et al. ; shimada et al. ) . in the glomerulus of nephritis, podocyte-expressed b - may also recruit t cells to where gbm is damaged and promote further inflammation. podocytes from necrotic crescentic nephritis rat model and cultured rat podocytes in vitro could express both mhc i/ii molecules and intercellular adhesion molecule (icam- ) after stimulation of ifn-γ, suggesting that cytokines could present the antigen to infiltrating t cells (goldwich et al. ) . recently, it has been pointed that compared to normal people, mcd patients but not fsgs patients excreted more urinary b - , while podocytes of relapsed mcd patients and fsgs patients did not express b - , thus b - might be used to identify mcd and fsgs (novelli et al. b ). the glomerulus is a well-recognized target of miscellaneous immune-mediated injuries, and the pathogenesis of immune-mediated glomerular disease is multifactorial ( fig. . ) . in mn, the surface molecules of glomerular podocyte act as antigens and trigger systematic immune responses, resulting in the formation of in situ immune complexes. the classic animal model of mn, heymann nephritis, reproduces typical mesangial lesions by eliciting auto-antibodies against the podocyte membrane protein megalin in rats (ronco and debiec ) . it has been reported in vivo that the occurrence of human newborns mn was due to the production of auto-antibodies against glomerular podocyte membrane proteins. neutral endopeptidase (nep) is a membrane protein expressed on the surface of human podocytes. studies have shown that neonatal mn occurs due to the presence of anti-nep auto-antibodies in children (herrmann et al. ). its origin is due to the mother's carrying the relevant mutation gene and lacking nep. if the mother bred a normal healthy fetus, the mother will produce an anti-nep antibody against the fetus during pregnancy and the antibody enters the fetus through the placenta. anti-nep antibodies react with nep antigens on fetal podocytes, forming an immune complex on the epithelial side, leading to neonatal mn. although the incidence of this type of patients is very low, its pathogenesis confirms the role of anti-podocyte antigen antibodies in the development of human mn (pozdzik et al. ) . t cell dysfunction and the release of cytokines (circulatory factors) causing podocyte injury are associated with the formation of proteinuria in mcd patients. it is currently believed that the cytokines produced by th and th cells in t cells are involved in the occurrence of mcd, but the cytokines produced by th cells (il- , il- , il- ) might be more important (mack ). animal experiments have found that the injection of il- to rats can reduce the content of heparin sulfate on the surface of podocytes, weaken the membrane filtration barrier of charge, and trigger proteinuria. there are also receptors for il- and il- on the podocyte, and the increase of circulating or local il- and il- can directly damage the podocyte through the receptors on the podocyte and increasing the permeability of the filtration membrane. shimada et al. ( ) have proposed that mcd is the result of a "two-hit" attack from podocyte immune dysfunction: the first hit is the effects of bacterial products, viruses, and various cytokines on podocytes, resulting in an abnormal expression of cd in podocytes, and further cytoskeleton reorganization and morphological changes of podocytes, increasing the permeability of gbm which might bring about proteinuria. however, due to the self-regulation of the body, podocytes can downregulate the expression of cd . if the auto-regulatory function of podocytes and the body is defective, the sustained expression of cd would lead to proteinuria and even mcd. moreover, ishimoto et al. ( ) also observed increasing expression of cd in the urine of mcd patients; in view of the fact that the expression of cd in podocytes can be induced by il- and bacterial products through the tlr pathway and regulated by ctla , suggesting that defective immune functions of podocytes is an essential cause of mcd. certain exogenous antigens (small molecular weight, positively charged) are implanted on the epithelial side and can also lead to the formation of in situ immune complexes. hepatitis b virus (hbv)-associated nephropathy is often manifested as mesangial lesions (especially in children), and hbeag plays an important role in its occurrence. the hbeag molecule is of small mass and negatively charged and can be implanted across the glomerular basement membrane (gbm) on the epithelial side, triggering the formation of in situ immune complexes (gupta and quigg ) . under inflammatory conditions, podocytes would inhibit the expression of mhc class ii molecules, promoting the remove of immune complexes from the gbm. in some cases, podocytes might act as antigen-presenting cells themselves, taking up and processing antigens to initiate specific t cell responses. there has been evidence that transgenic mice with a loss of mhc class ii exclusively in podocytes developed only a very moderate degree of nephrosclerosis and glomerular crescent formation compared to the control animals, indicative of their defective capacity to activate cd + t cells (goldwich et al. ). viral infection, such as human immunodeficiency virus (hiv)- , parvovirus b , cytomegalovirus (cmv), hepatitis b virus (hbv), and hepatitis c virus (hcv), is associated with podocyte injury. hiv-associated nephropathy (hivan) mostly manifests collapsing glomerulopathy or classic fsgs (chandra and kopp ) . podocyte infection is associated with podocyte injury and dedifferentiation and rapid loss of renal function. studies have reported that hiv virus can be internalized by podocytes in vitro, which might be associated with receptors, such as viral coat protein gp , and subsequent endocytosis, phagocytosis, or pinocytosis (bruggeman ) . although the transmission of virus in vitro has been well documented, further studies are needed to demonstrate the definite mechanism by which the virus enters podocyte in vivo. structural viral proteins, gag and pol, and non-structural proteins, vpr, nef, and tat, have been considered to be associated with hivan (conaldi et al. ; reid et al. ; zuo et al. ). hbv is a major cause for membranous nephropathy and fsgs scarcely, which can be diagnosed by evidence of hbv antigen or antibodies on kidney biopsy. the possible mechanisms of hbv-induced podocyte injury might be as follows: detective infection of the cells by hbv, deposition of circulating immune complex in renal cells, effects of hbv-induced immunological mediators (bhimma and coovadia ; sakai et al. ) . podocytes are also targets of some toxicity drugs, which may further progress to glomerulosclerosis. for example, gold, bucillamine, and d-penicillamine, which are used for the treatment of rheumatoid arthritis, are confirmed to cause mn. the possible mechanism might be closely related to stress, energy metabolism, and inflammation (fujiwara et al. ; seguin et al. ) . other drugs, like non-steroid anti-inflammatory drugs and interferon, also can be inductor of podocyte injury. organic solvents, like gasoline, dimethylbenzene, and formaldehyde, can induce podocyte injury including foot process fusion and decreased expression of nephrin and podocin (qin et al. ) . hypoxia can be induced by various pathogenic conditions including hypertension and diabetes. chronic hypoxia can trigger endoplasmic reticulum (er) stress, which result in increased ros. nephrin and alpha-actin- , the structural components of sd, are subject to mutations, which cause defective protein folding in the er of podocytes. the underling mechanism might include transient receptor protein and complement complex and increased expression of mcp- cybulsky ; maekawa and inagi ) . targeting hypoxia and er stress and the possible signal networks might be the novel target for intervention of podocyte injury in ckd. living in an environment of a variety of pathological stresses and stimuli, podocytes adapt to maintain the integrity and stability of the glomerular basement membrane, depending on their highly differentiated characteristics which also reflect the vulnerability of this barrier. the different responses of podocytes to injury are associated with the pathology and prognosis of glomerular diseases. as a vital type of renal intrinsic cells, podocyte damage is an important cause of nephrotic proteinuria and glomerular sclerosis. however, as a highly differentiated terminal cell, podocyte has no proliferative potential, and loss of podocyte is associated with poor renal outcomes such as increased proteinuria, glomerulosclerosis, and renal disease progression. podocytes have different responses to injuries, including endoplasmic reticulum stress and autophagy reactions caused by abnormal energy metabolism. this chapter lists several aspects of podocyte injuries along with potential underlying mechanisms, including glucose and lipid metabolism disorder, hypertension, ras activation, micro-inflammation, immune disorder, and other factors. these aspects are not technically separated items, but intertwined with each other in the pathogenesis of podocyte injuries. injured podocytes would undergo a series of morphological changes: fp disappearance, cellular shrink, pseudocysts form, cell hypertrophy, cytoplasmic lysosomal enrichment, etc. these changes eventually cause podocytes to detach from the gbm. moreover, due to the lack of proliferative capacity, the number of glomerular podocytes would become less and less, until reduced by more than % when glomerulosclerosis occurs. glomerulosclerosis is not a specific disease but a state representing podocyte injury which is mediated by diverse causes. podocytes interact with gbm and capillary loops tightly, dysfunction of which is an early event leading to glomerulosclerosis. glomerulosclerosis seems like a station to stay in just before arriving to destination. unanswered questions in the pathogenesis of podocyte injury and glomerulosclerosis are still ill-defined, and the causing list will continue to grow. uncovering the selective targeting to pathogenesis and underlying mechanism of podocyte injury and glomerulosclerosis is bound to provide clues to answer for treatment and prevention of the disease in the future. vascular endothelial growth factor and the kidney: something of the marvellous mouse podocyte complement factor h: the functional analog to human complement receptor anti-albuminuric effect of the aldosterone blocker eplerenone in non-diabetic hypertensive patients with albuminuria: a double-blind, randomised, placebo-controlled trial molecular and cellular events mediating glomerular podocyte dysfunction and depletion in diabetes mellitus macrophage-derived tumor necrosis factor-alpha mediates diabetic renal injury knocking down tcf inhibits high glucose-and angiotensin ii-induced epithelial to mesenchymal transition in podocytes tlr links podocytes with the innate immune system to mediate glomerular injury focal and segmental glomerulosclerosis induced in mice lacking decay-accelerating factor in t cells the costimulatory receptor b - is not induced in injured podocytes hepatitis b virus-associated nephropathy lipid metabolism abnormalities in chronic kidney disease intrinsic proinflammatory signaling in podocytes contributes to podocyte damage and prolonged proteinuria hiv- infection of renal cells in hiv-associated nephropathy statins prevent oxidized ldl-induced injury of glomerular podocytes by activating the phosphatidylinositol -kinase/akt-signaling pathway podocyte injury and repair mechanisms renal protective effects of toll-like receptor signaling blockade in type diabetic mice viruses and collapsing glomerulopathy: a brief critical review b - expression regulates the hypoxia-driven cytoskeleton rearrangement in glomerular podocytes altering expression of alpha beta integrin on podocytes of human and rats with diabetes calcium entry via trpc mediates albumin overload-induced endoplasmic reticulum stress and apoptosis in podocytes role of nadph oxidase-mediated reactive oxygen species in podocyte injury complement regulatory proteins in kidneys of patients with anti-neutrophil cytoplasmic antibody (anca)-associated vasculitis tonicity-responsive enhancer-binding protein mediates hyperglycemia-induced inflammation and vascular and renal injury evidence linking glycated albumin to altered glomerular nephrin and vegf expression, proteinuria, and diabetic nephropathy human immunodeficiency virus- tat induces hyperproliferation and dysregulation of renal glomerular epithelial cells the human glomerular podocyte is a novel 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and transplantation role of podocyte b - in diabetic nephropathy rituximab targets podocytes in recurrent focal segmental glomerulosclerosis podocytes are sensitive to fluid shear stress in vitro proximal tubules and podocytes are toxicity targets of bucillamine in a mouse model of drug-induced kidney injury angiotensin ii induces calcium-mediated autophagy in podocytes through enhancing reactive oxygen species levels nadph oxidases in the kidney podocytes are nonhematopoietic professional antigen-presenting cells balancing calcium signals through trpc and trpc in podocytes cell biology and pathology of podocytes role of receptor for advanced glycation end-products and signalling events in advanced glycation end-productinduced monocyte chemoattractant protein- expression in differentiated mouse podocytes glomerular diseases associated with hepatitis b and c at a angiotensin receptors in the renal proximal tubule regulate blood pressure cxcl is expressed in podocytes and acts as a scavenger receptor for oxidized low-density lipoprotein high glucose and advanced glycosylated end-products affect the expression of alphaactinin- in glomerular epithelial cells montelukast improves the changes of cytoskeletal and adaptor proteins of human podocytes by interleukin- vascular endothelial growth factor b controls endothelial fatty acid uptake effect of retinoic acid in experimental diabetic nephropathy high glucose and angiotensin ii increase beta integrin and integrin-linked kinase synthesis in cultured mouse podocytes atherogenic effects of tnf-alpha and il- via up-regulation of scavenger receptors selection of a c a receptor antagonist from phage libraries attenuating the inflammatory response in immune complex disease and ischemia/reperfusion injury membranous nephropathy: the start of a paradigm shift srebps: activators of the complete program of cholesterol and fatty acid synthesis in the liver inflammation-activated cxcl pathway contributes to tubulointerstitial injury in mouse diabetic nephropathy expression of functional ccr and cxcr chemokine receptors in podocytes podocin and mec- bind cholesterol to regulate the activity of associated ion channels emerging role of autophagy in kidney function, diseases and aging prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin ii type a receptor-deficient mice activated macrophages down-regulate podocyte nephrin and podocin expression via stress-activated protein kinases minimal change disease: a cd podocytopathy dysregulated nephrin in diabetic nephropathy of type diabetes: a cross sectional study soluble flt binds lipid microdomains in podocytes to control cell morphology and glomerular barrier function early mechanisms of renal injury in hypercholesterolemic or hypertriglyceridemic rats the microinflammatory state in uremia: causes and potential consequences quantitative polymerase chain reaction-based analysis of podocyturia is a feasible diagnostic tool in preeclampsia secondary focal segmental glomerulosclerosis: from podocyte injury to glomerulosclerosis b cell-derived il- acts on podocytes to induce proteinuria and foot process effacement overexpression of toll-like receptor correlates with the progression of podocyte injury in murine autoimmune glomerulonephritis podocyte injury-driven intracapillary plasminogen activator inhibitor type accelerates podocyte loss via upar-mediated beta -integrin endocytosis alpha beta integrin has a crucial role in kidney and lung organogenesis podocyte damage is a critical step in the development of glomerulosclerosis in the uninephrectomised-desoxycorticosterone hypertensive rat a potential role for mechanical forces in the detachment of podocytes and the progression of ckd the role of podocytes in the development of glomerular sclerosis progression of glomerular diseases: is the podocyte the culprit? from segmental glomerulosclerosis to total nephron degeneration and interstitial fibrosis: a histopathological study in rat models and human glomerulopathies development of vascular poleassociated glomerulosclerosis in the fawn-hooded rat the podocyte's response to stress: the enigma of foot process effacement elevated urinary podocyte-derived extracellular microvesicles in renovascular hypertensive patients endoplasmic reticulum stress contributes to beta cell apoptosis in type diabetes mechanisms and consequences of hypertriglyceridemia and cellular lipid accumulation in chronic kidney disease and metabolic syndrome saturated fatty acids induce insulin resistance in human podocytes: implications for diabetic nephropathy characterization of glucose uptake by cultured rat podocytes advanced glycation end products activate smad signaling via tgf-beta-dependent and independent mechanisms: implications for diabetic renal and vascular disease enhanced epithelial-to-mesenchymal transition associated with lysosome dysfunction in podocytes: role of p /sequestosome as a signaling hub functional expression of the renin-angiotensin system in human podocytes nestin protects mouse podocytes against high glucose-induced apoptosis by a cdk -dependent mechanism roles of na(+)/h(+) exchanger type and intracellular ph in angiotensin ii-induced reactive oxygen species generation and podocyte apoptosis epithelial-to-mesenchymal transition in diabetic nephropathy: fact or fiction? rac /pak signaling promotes epithelialmesenchymal transition of podocytes in vitro via triggering beta-catenin transcriptional activity under high glucose conditions the unfolding tale of the unfolded protein response effect of angiotensin-converting enzyme inhibition on glomerular basement membrane permeability and distribution of zonula occludens- in mwf rats expression of toll-like receptor in renal podocytes in childhood-onset active and inactive lupus nephritis podocyte antigens, dendritic cells and t cells contribute to renal injury in newly developed mouse models of glomerulonephritis stress signal network between hypoxia and er stress in chronic kidney disease ginsenoside rg inhibits angiotensin ii-induced podocyte autophagy via ampk/mtor/pi k pathway renin-angiotensin system within the diabetic podocyte renal lipotoxicity-associated inflammation and insulin resistance affects actin cytoskeleton organization in podocytes chylomicron remnants are increased in the postprandial state in cd deficiency metabolism, energetics, and lipid biology in the podocyte-cellular cholesterol-mediated glomerular injury cyclodextrin protects podocytes in diabetic kidney disease glomerular-specific protein kinase c-beta-induced insulin receptor substrate- dysfunction and insulin resistance in rat models of diabetes and obesity structure and function of podocytes: an update podocyte injury underlies the glomerulopathy of dahl salt-hypertensive rats and is reversed by aldosterone blocker salt-induced nephropathy in obese spontaneously hypertensive rats via paradoxical activation of the mineralocorticoid receptor: role of oxidative stress podocyte injury and its consequences progressive podocyte injury and globotriaosylceramide (gl- ) accumulation in young patients with fabry disease glomerular filtration into the subpodocyte space is highly restricted under physiological perfusion conditions angiotensin ii contributes to podocyte injury by increasing trpc expression via an nfat-mediated positive feedback signaling pathway regulation of glucose transporter (glut ) gene expression by angiotensin ii in mesangial cells: involvement of hb-egf and egf receptor transactivation another piece of the puzzle of podocyte b - expression: lupus nephritis any value of podocyte b - as a biomarker in human mcd and fsgs? fetuin-a aggravates lipotoxicity in podocytes via interleukin- signaling presecretory oxidation, aggregation, and autophagic destruction of apoprotein-b: a pathway for late-stage quality control roles of the podocyte in glomerular function cell biology of the glomerular podocyte anti-nep and anti-pla r antibodies in membranous nephropathy: an update membrane aberrancy and unfolded proteins activate the endoplasmic reticulum stress sensor ire in different ways human podocyte depletion in association with older age and hypertension attenuation of diabetic nephropathy in diabetes rats induced by streptozotocin by regulating the endoplasmic reticulum stress inflammatory response mixed organic solvents induce renal injury in rats expression of agrin, dystroglycan, and utrophin in normal renal tissue and in experimental glomerulopathies selective release of human adipocyte fatty acids according to molecular structure cellular responses to endoplasmic reticulum stress and apoptosis an hiv- transgenic rat that develops hiv-related pathology and immunologic dysfunction epithelial-mesenchymal transition and podocyte loss in diabetic kidney disease the glomerular slit diaphragm is a modified adherens junction induction of b - in podocytes is associated with nephrotic syndrome trpc is a glomerular slit diaphragm-associated channel required for normal renal function a cell-type specific ganglioside of glomerular podocytes in rat kidney: an o-acetylated gd angiotensin ii induces nephrin dephosphorylation and podocyte injury: role of caveolin- parathyroid hormone-related protein induces hypertrophy in podocytes via tgf-beta( ) and p (kip ): implications for diabetic nephropathy molecular pathomechanisms of membranous nephropathy: from heymann nephritis to alloimmunization target antigens and nephritogenic antibodies in membranous nephropathy: of rats and men mechanisms and treatment of ckd nephrin is specifically located at the slit diaphragm of glomerular podocytes angiotensin ii as a morphogenic cytokine stimulating renal fibrogenesis angiotensin ii upregulates rage expression on podocytes: role of at receptors focal segmental glomerulosclerosis as a complication of hepatitis b virus infection aliskiren inhibits intracellular angiotensin ii levels without affecting (pro)renin receptor signals in human podocytes podocytes as a target of prorenin in diabetes globotriaosylsphingosine actions on human glomerular podocytes: implications for fabry nephropathy lipid-protein interactions along the slit diaphragm of podocytes localization of the glut glucose transporter in murine kidney and regulation in vivo in nondiabetic and diabetic conditions the microinflammatory state of uremia podocin, a raft-associated component of the glomerular slit diaphragm, interacts with cd ap and nephrin gene expression profiling in a model of d-penicillamine-induced autoimmunity in the brown norway rat: predictive value of early signs of danger the podocyte's response to injury: role in proteinuria and glomerulosclerosis podocyte as the target for aldosterone: roles of oxidative stress and sgk minimal change disease: a "two-hit" podocyte immune disorder? toll-like receptor ligands induce cd expression in human podocytes via an nf-kappab-dependent pathway free fatty acids and their metabolism affect function and survival of podocytes regulation of podocyte survival and endoplasmic reticulum stress by fatty acids involvement of lipid rafts in nephrin phosphorylation and organization of the glomerular slit diaphragm curcumin decreases renal triglyceride accumulation through ampk-srebp signaling pathway in streptozotocin-induced type diabetic rats glucose specifically regulates trpc expression in the podocyte in an angii-dependent manner glomerular endothelial fenestrae in vivo are not formed from caveolae glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy poly(adp-ribose) polymerase inhibitors ameliorate nephropathy of type diabetic leprdb/db mice protection of mitochondria prevents high-fat diet-induced glomerulopathy and proximal tubular injury complement-mediated cellular injury loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton expression of advanced glycation end products and their cellular receptor rage in diabetic nephropathy and nondiabetic renal disease the chloride intracellular channel a stimulates podocyte rac , protecting against hypertension-induced glomerular injury epidemiology of hypertensive kidney disease characterization of reninangiotensin system enzyme activities in cultured mouse podocytes systemic and renal lipids in kidney disease development and progression podocyte loss in human hypertensive nephrosclerosis oxldl-induced lipid accumulation in glomerular podocytes: role of ifn-gamma, cxcl , and adam insulin signaling to the glomerular podocyte is critical for normal kidney function a mutation in the trpc cation channel causes familial focal segmental glomerulosclerosis (kip ) knockout mice are protected from diabetic nephropathy: evidence for p (kip ) haplotype insufficiency pten inhibits high glucose-induced phenotypic transition in podocytes angiotensin ii receptor blocker inhibits p kip expression in glucose-stimulated podocytes and in diabetic glomeruli inflammatory stress exacerbates lipid-mediated renal injury in apoe/cd /sra triple knockout mice ang ii promotes autophagy in podocytes epithelialmesenchymal transition as a potential explanation for podocyte depletion in diabetic nephropathy decreased glomerular expression of agrin in diabetic nephropathy and podocytes, cultured in high glucose medium induction of apoptosis during development of hypertensive nephrosclerosis activation of the renin-angiotensin system within podocytes in diabetes podocytespecific chemokine (c-c motif) receptor overexpression mediates diabetic renal injury in mice podocyte-specific overexpression of glut surprisingly reduces mesangial matrix expansion in diabetic nephropathy in mice dysregulation of low-density lipoprotein receptor contributes to podocyte injuries in diabetic nephropathy inflammatory stress exacerbates lipid accumulation and podocyte injuries in diabetic nephropathy dysregulation of the low-density lipoprotein receptor pathway is involved in lipid disorder-mediated organ injury hiv- genes vpr and nef synergistically damage podocytes, leading to glomerulosclerosis acknowledgements this study was supported by grants from the national key research and development program of china ( yfc ). key: cord- -kxrmzyo authors: wei, wen-ying; zhao, qing; zhang, wen-zhong; wang, mao-jing; li, yan; wang, shi-zhong; zhang, ning title: secreted frizzled-related protein prevents pressure-overload-induced cardiac hypertrophy by targeting the wnt/β-catenin pathway date: - - journal: mol cell biochem doi: . /s - - -x sha: doc_id: cord_uid: kxrmzyo background and aim: secreted frizzled-related protein (sfrp ) has been reported to be involved in cardiovascular diseases. however, its role in cardiac hypertrophy induced by pressure overload is still elusive. we aimed to examine the role of sfrp in the development of cardiac hypertrophy in vivo and in vitro. methods and results: following cardiac hypertrophy stimulated by aortic banding (ab), the expression of sfrp was downregulated in the hypertrophic ventricle. adeno-associated virus (aav ) was injected through the tail vein to overexpress sfrp in the mouse myocardium. overexpression of sfrp alleviated cardiomyocyte hypertrophy and interstitial fibrosis, as identified by the reduced cardiomyocyte cross-sectional area, heart weight/body weight ratio, and left ventricular (lv) collagen ratio. additionally, sfrp decreased cardiomyocyte apoptosis induced by pressure overload. western blot showed that sfrp prevented the expression of active β-catenin. the wnt/β-catenin agonist licl ( mmol/kg) abolished the inhibitory effects of sfrp on cardiac hypertrophy and apoptosis, as evidenced by the increased cross-sectional area and lv collagen ratio and the deterioration of echocardiographic data. conclusion: our study indicated that decreased sfrp levels were observed in failing mouse hearts. overexpression of sfrp attenuated myocyte hypertrophy and interstitial fibrosis induced by hypertrophic stimuli by inhibiting the wnt/β-catenin pathway. we revealed that sfrp may be a promising therapeutic target for the development of cardiac remodeling. electronic supplementary material: the online version of this article ( . /s - - -x) contains supplementary material, which is available to authorized users. heart failure and its underlying cardiac hypertrophy processes are spreading worldwide [ ] . cardiac hypertrophy is identified as compensatory pathological changes in response to chronic arterial hypertension and/or aortic valve stenosis. characterized by the deposition of cardiac interstitial collagen and cardiomyocyte enlargement, cardiac hypertrophy is regarded as a major predictor for the development of heart failure. hence, inhibition of pathological cardiac hypertrophy can serve as an effective therapeutic strategy for curing heart failure. the wnt/β-catenin pathway is evolutionarily conserved throughout metazoans and participates in various developmental processes including early cardiomyogenesis [ ] . wnt/ β-catenin signaling is usually silent in adult organs, but reactivates after acute injury. previous studies have found that wnt/β-catenin signaling is closely related to the mediation of cardiomyocyte enlargement and apoptosis. it has been demonstrated that increased wnt a expression activated caspases and aggravated myocyte apoptosis induced by hypoxia reoxygenation [ ] . furthermore, activation of wnt/ β-catenin is observed in cardiac and renal tissues following pressure overload stimulation, and blockage of the signaling attenuates chronic organ remodeling in the heart and kidney [ ] . secreted frizzled-related protein (sfrp ), as a soluble protein, directly interacts with wnt extracellularly and prevents its interaction with frizzled; this is involved in the regulation of wnt/β-catenin signaling. lin et al. found that sfrp was downregulated after cardiac infarction in mice and elaborated that sfrp may be an antifibrotic target by regulating cardiac fibroblast (cf) growth and extracellular matrix remodeling [ ] . nevertheless, whether sfrp can affect pressure-overload-induced cardiac hypertrophy through wnt/β-catenin signaling remains elusive. in the present study, we explored the role of sfrp in the pathogenesis of cardiac hypertrophy induced by aortic banding (ab), as well as the underlying mechanism. c bl/ male mice ( weeks) were obtained from the institute of laboratory animal science, cams & pumc (beijing, china). antibodies against β-catenin (ab ), total-gsk- β (ab ), phospho-gsk- β (ab ), bax (ab ), bcl- (ab ), cleaved caspase- (ab ), and gapdh (ab ) were purchased from abcam. active β-catenin ( , ) was obtained from cell signaling technology and angiotensin (ang ii, a ) and lithium chloride (licl, , ) were purchased from sigma-aldrich. recombinant adeno-associated viruses of serotype (aav ) were produced and purchased from hanbio biotechnology co. (shanghai, china). aav vectors were packaged with single-stranded dna containing enhanced green fluorescent protein (gfp) gene or sfrp , which was driven by the human cytomegalovirus (cmv) promoter (aav -cmv-gfp or aav -cmv-gfp-sfrp ). vector titers were determined by using qpcr with primers against the cmv promoter region. recombinant sfrp ( -fr) was obtained from r&d systems. all animal procedures were by approved the guidelines for the care and use of laboratory animals published by the united states national institutes of health (nih publication, revised ) and the institutional guidelines of the animal care and use committee of affiliated hospital of qingdao university (qingdao, china). all eighty male c bl/ mice were housed and drank freely under specific pathogen-free conditions under a -h light/ -h dark cycle and fed standard chow diet ( . kcal% fat, . kcal% protein, . kcal% carbohydrate). aav -cmv-sfrp or aav -cmv-gfp ( × drp) was injected through the tail vein to overexpress sfrp in the mouse myocardium. four weeks later, c bl/ mice were anesthetized by intraperitoneal injection of % sodium pentobarbital and placed on a ventilator. the ab and sham operation were performed in - g male c bl/ j mice ( weeks old) according to a previous study [ ] . additionally, adequate aortic banding was confirmed by doppler echocardiogram. the sham mice underwent a similar operation without aorta constriction. licl, a wnt/β-catenin signaling agonist, was administered by intragastric administration via a mmol/kg dose once a day and maintained for weeks. mouse echocardiography was performed under sedation by . % isoflurane inhalation. short-axis parasternal views of the left ventricular systolic and diastolic parameters were obtained immediately at the mid-papillary level using a vivid echocardiography apparatus with a -mhz probe (esaote spa, genoa, italy). m-mode measurements of the magnitude of the lv walls and chamber were achieved by two-dimensional guidance from the left ventricular shortaxis view in a blinded manner as described previously [ , ] . lv end-diastolic diameter (lvedd), lv end-systolic diameter (lvesd), and end-diastolic interventricular septal thickness (ivsd) were determined. the lv fractional shortening (fs) was computed using the following formula: fs (%) = (lvedd-lvesd)/ lvedd × . for invasive hemodynamic analysis, mice were primarily anesthetized with . % isoflurane inhalation. an incision was subsequently made in the right carotid artery and pressure-volume (pv) loop measurements were performed using a . fr micromanometer conductance catheter (millar, houston, usa). hemodynamic data were collected at steady state; pv loop parameters including the maximum rate of pressure development (dp/dt max) and minimum rate of pressure decay (dp/dt min) were calculated using the pvan data analysis software (millar, houston, usa). the harvested heart was fixed in % paraformaldehyde for h, subsequently dehydrated in ethanol and xylene series, and finally embedded in paraffin. to evaluate the cross-sectional sizes of cardiomyocytes and cardiac interstitial fibrosis, -μm cardiac tissue slides were stained with hematoxylin and eosin (h&e) and picrosirius red (psr). subsequently, images of the slides were obtained by optical microscopy with a nikon photo-imaging system (h l, tokyo, japan). for cardiomyocytes sizes and interstitial fibrosis quantification, images were analyzed with the image-pro plus . software (maryland, usa). for immunohistochemistry of myocardial sfrp expression, the cardiac tissue sections were deparaffinized, blocked with % goat serum, and then incubated with : diluted anti-sfrp antibody overnight at °c. after washing, the slides were incubated for h with anti-rabbit hrp reagent (gene tech, shanghai, china) at °c, rinsed, and developed with a peroxide-based substrate dab kit for min (gene tech, shanghai, china). finally, images of the slides were obtained by light microscopy with a nikon photo-imaging system (h l, tokyo, japan). apoptosis was detected in the cardiac tissues using the apoptosis assay kit (millipore, temecula, ca) according to the manufacturer's instructions. the myocardial tissue sections were deparaffinized and preconditioned with proteinase k ( μg/ml), after which they were incubated with fluorescein-labeled dutp. quantitation of apoptotic cells, as shown by nuclear fragmentation and chromatin condensation, was conducted with the image-pro plus . software. nrcms were isolated from -to -day-old sprague-dawley (sd) rats as shown previously [ ] . briefly, twenty neonatal rat ventricles were minced into mm small fragments and digested with . g/l trypsin and g/l collagenase at °c six times for min. the resultant suspension was collected by centrifugation at g for min and strained through a -μm filter. then, the cell suspension was reseeded into fresh dmem/f medium containing % (v/v) fetal bovine serum (fbs) (ausbian, australia) for . h. afterwards, the cardiac fibroblasts were abandoned and the extracted cardiomyocytes were transferred to a new plate and cultured accordingly. for in vitro experiments, cardiomyocytes were prepared as quiescent by serum starvation for h, after which the cells were incubated with licl ( . mm, h) followed by ang ii ( μm) stimulation for the indicated amount of time. for evaluation of the myocyte surface area, cardiomyocytes were seeded on coverslips and incubated with nm sfrp and/or not . mm licl following stimulation with ang ii for h. to block the wnt/β-catenin signaling, nrcms were pretreated with licl for h followed by ang ii stimulation. the coverslips were washed twice with icecold pbs and fixed with pbs plus % paraformaldehyde for min. the myocytes were permeabilized with pbs containing . % triton x for min and then blocked with % bsa in pbs for h. cardiomyocytes were incubated with primary antibody against α-actin (abcam, cambridge, ma) overnight at °c and reacted with a fluorescent secondary antibody. for determination of the myocyte surface area, fluorescent images were captured by an inverted fluorescence microscope and calculated using the image-pro plus . software. lv myocardial tissues were immediately dissected and total rna was extracted from mouse cardiac tissues and cultured cardiomyocytes using the trizol reagent (invitrogen, carlsbad, ca, usa). first strand cdna was made using the cdna reverse transcription kit (roche, mannheim, germany). quantification of specific gene expression was performed using the lightcycler sybr green master mix (roche, mannheim, germany) and mrna sequencespecific pcr primers were designed using primer express software; the primer sequences are shown in supplementary table . gene expression of the housekeeping gene gapdh was utilized as a relative quantitative reference. cardiac tissues and cultured nrcms were homogenized in ripa lysis buffer, and the concentration of the protein lysates was determined by a bca assay kit as indicated previously [ ] . protein extracts were separated by % sds polyacrylamide gels under denaturing conditions and blotted onto polyvinylidene difluoride (pvdf) membranes. pvdf membranes were blocked with % bovine serum albumin/ tbst and then incubated with the following primary antibodies: β-catenin, active β-catenin, phospho-gsk- β, total-gsk- β, bax, bcl- , and cleaved-caspase diluted in % bovine serum albumin, followed by incubation with the appropriate conjugated anti-rabbit or anti-mouse secondary antibodies (cell signaling technology). signals were obtained using a two-color infrared imaging system (odyssey; li-cor biosciences, lincoln, ne, usa). blot quantification was performed with the imagej software (nih, bethesda, md, usa) and normalized to gapdh protein expression. data were presented as the mean ± sd. differences between groups were analyzed with one-way analysis of variance (anova) using graphpad prism (graphpad software inc, la jolla, ca). comparisons of two unpaired groups utilized the two-sided student's t tests. p ≤ . was considered statistically significant. we detected the expression of sfrp in c bl/ mice for , , and weeks after ab operation. our data showed that sfrp mrna and protein expression was increased in the st week after ab operation and subsequently decreased during the th week ( fig. a-b) . nrcms were cultured in vitro and stimulated with μm ang ii for h. western blotting results indicated that sfrp expression was significantly decreased in ang ii-treated nrcms compared with the pbs-treated group (fig. c) . these results suggested that the changeable expression of sfrp may have possible roles in ab-induced cardiac hypertrophy. to examine the effects of sfrp on ab-induced cardiac hypertrophy and accompanying dysfunction, we specifically overexpressed sfrp in the myocardium by injecting aav encoding sfrp into the tail vein of mice [ ] . we observed that compared with baseline levels, cardiac sfrp expression was increased by approximately . fold in aav -sfrp -infected mice after weeks (fig. a-c) . to investigate the potential protective role of sfrp in the development of pressure-overload-induced cardiac hypertrophy, ab and sham operations were performed weeks after aav -sfrp or aav -gfp injection. we detected mrna expression of fetal gene bnp in hypertrophic hearts. our results demonstrated that sfrp overexpression retarded the development of cardiac hypertrophy and improved the deteriorative cardiac functions in ab-treated mice, as indicated by their reduced heart weight/body weight (hw/bw) ratio and decreased bnp mrna expression (fig. e-f ). four weeks after ab operation, h&e staining was used to evaluate the cross-sectional area of cardiomyocytes. the results showed that cardiomyocyte cross-sectional areas were decreased in aav -sfrp -treated mice after ab insult compared with aav -gfp-treated mice (fig. g) . the echocardiographic and hemodynamic measurements reflected the anti-hypertrophy and cardiac function protective effects of overexpressed sfrp , which presented as a reduction in lvedd, lvedd, and ivsd and increase in dp/ dt max, dp/dt min, and fs in mice subjected to ab surgery (fig. h) . these data suggested that the protective role of sfrp was achieved in response to hypertrophic insults. to evaluate the role of sfrp in cardiac fibrosis, psr staining was performed. as identified by the decreased interstitial fibrosis ratio (fig. i, k) , overexpression of sfrp markedly mitigated myocardial fibrosis induced by hypertrophic stimuli. meanwhile, the induction of fibrotic markers including collagen iα, collagen iiiα, and periostin by ab were markedly increased in hypertrophic mice; sfrp significantly blunted collagen iα, collagen iiiα, and periostin mrna expression (fig. k) in hypertrophic hearts, whereas there were no statistically significant differences between the sham-sfrp -vs. sham-gfp-treated mice. these results implied that sfrp plays a role in mitigating cardiac fibrosis under pressure overload. we further investigated the effects of sfrp on cardiac apoptosis. tunel staining was performed in cardiac data are presented as the mean ± sd. *p < . tissue sections. our results indicated that a large number of tunel positive nucleus were present in the hypertrophic cardiac slices, whereas positive nucleus signal was significantly reduced in the sfrp -overexpressing hearts (fig. a, b) . protein levels of cleaved-caspase , bax, and bcl- were assessed by western blotting and indicated that overexpressed sfrp markedly decreased the expression of the pro-apoptosis proteins cleaved-caspase and bax and increased the expression of anti-apoptosis proteins bcl- (fig. c) . a previous study revealed that sfrp can potently blunt canonical wnt/β-catenin signaling in epithelial ovarian cancer [ ] , while in our present study, pressure overload induced by ab could also induce the activation of this canonical wnt/β-catenin signaling (fig. d) . more importantly, overexpression of sfrp dramatically inhibited the wnt/β-catenin pathway, indicated by the decreased expression of β-catenin and active β-catenin (fig. d) . collectively, these results suggested a cardioprotective role for sfrp that reduces cardiomyocyte apoptosis via inhibition of the wnt/β-catenin pathway. since ang ii stimulation resulted in a remarkable downregulation of sfrp expression (fig. c) , a subsequent study was performed to evaluate the specific molecular mechanisms of sfrp in ang ii-induced myocyte hypertrophy. nrcms were isolated and stimulated by ang ii ( μΜ) for h to establish cardiomyocyte hypertrophy model in vitro. as shown in fig. e -g, sfrp mitigated ang ii-induced cardiomyocyte hypertrophy, which was indicated by a smaller cell surface area and decreased bnp mrna expression compared with the positive control group. additionally, sfrp alleviated the vast apoptosis of cardiomyocytes induced by ang ii (fig. h) . to examine whether sfrp regulates ang ii-induced cardiac hypertrophy and apoptosis in a wnt/β-catenin signaling-mediated way, nrcms were pretreated with . mm licl (a wnt/β-catenin signaling agonist) for h followed by ang ii for h. thereafter, the role of licl in sfrp coincubated with angii stimulation was evaluated. our data showed that preincubation with licl reversed the downregulation of β-catenin expression and increased levels of cleaved-caspase (fig. h) . moreover, licl abolished the protective role of sfrp followed by ang ii stimulation, as evidenced by the larger cell surface area (fig. e) and increased bnp mrna expression (fig. g) . the results demonstrated that sfrp mitigated ang ii-induced cardiac hypertrophy and apoptosis, mainly by inhibiting the wnt/βcatenin pathway. to validate the critical effects of sfrp overexpression on wnt/β-catenin signaling in mouse cardiac hypertrophy, licl ( mmol/kg per day) was intraperitoneally injected into cardiac hypertrophy mice two weeks before sacrifice. our results showed that licl abolished the anti-hypertrophic role of sfrp , as identified by the increased cardiomyocyte sectional areas (fig. a-b) , hw/bw ratios, and bnp mrna expression (fig. d-e) . additionally, compared with the ab + aav -sfrp group, mice in the ab + aav -sfrp + licl group presented more collagen deposition in the myocardium (fig. a, c) . echocardiographic data indicated that licl abrogated the alleviative development of cardiac hypertrophy induced by ab following sfrp overexpression, which manifested as similar lvef (%), lvedd, and lvesd levels between the ab + aav -gfp and ab + aav -sfrp + licl groups (fig. f-g) . taken together, these results revealed the protection of sfrp against cardiac hypertrophy and fibrosis depending on the blockage of the wnt/β-catenin signaling pathway. overarching conclusions from previous studies indicated a critical role for the dysregulated wnt/β-catenin pathway in the pathogenesis of a hypertrophic heart [ , ] . inhibition of the wnt/β-catenin pathway exerted a beneficial role and resulted in improved cardiac function following ischemic insults [ ] . our study was focused on sfrp , which is similar to frizzled, but lacks transmembrane domains [ ] . sfrp , a novel secreted factor, is considered a multifaceted regulator of the wnt/β-catenin pathway. a previous study demonstrated that sfrp expression in the failing heart was increased in a δ-sarcoglycan-null to strain hamster [ ] ; however, we found that sfrp expression in the hypertrophic heart of c bl/ mice was increased after week and then decreased after weeks. meanwhile, reduced sfrp expression was also detected in nrcms stimulated by ang ii; this discrepancy might be attributable to the different stages of cardiac remodeling. in the early phase of a failing heart, sfrp expression in cardiac tissues could be increased, which is consistent with our results that cardiac sfrp levels increased week after ab surgery. therefore, the mechanism to mediate the expression and functional relevance of sfrp requires further investigation. wnt/β-catenin signaling was activated in cardiac tissues during the progression of cardiac hypertrophy. blankesteijn et al. observed increased frizzled- expression during the development of cardiac hypertrophy induced by aortic constriction in rat [ ] . the levels of disheveled (dvl), a downstream molecule of frizzled in the wnt/β-catenin pathway, was shown to be upregulated after pressure overload stimulation in mice [ , ] . moreover, overexpression of β-catenin resulted in hypertrophic growth of cardiomyocytes in a rat myocardial infarction model [ ] . moreover, cardiac-specific deletion of β-catenin alleviated the development of cardiac hypertrophy stimulated by thoracic aortic constriction [ ] . overall, studies have indicated that wnt/β-catenin mediates hypertrophic responses upon various stimuli. notably, sfrp mediated the differentiation of mouse embryonic carcinoma stem cells by blocking wnt a and exerted a beneficial role to alleviate apoptosis during hypoxia reoxygenation in h c cells [ , ] . sfrp also plays a vital role in regulating the survival signal of mesenchymal stem cells in the ischemic myocardium [ ] . in the present study, we utilized aav -sfrp to transfect myocardial tissue and then performed an ab operation to establish a cardiac hypertrophy model. our results showed that overexpression of sfrp significantly alleviated pressure-overload-induced myocardial hypertrophy via inhibition of the wnt/β-catenin pathway. even though cardiac hypertrophy is considered as the adaptability to multiple stimuli, increased apoptosis aggravates cardiac dysfunction when the compensatory hypertrophic response exceeds its ability. therefore, inhibition of apoptosis is a critical strategy to prevent cardiac remodeling. many studies have revealed that increased apoptosis is associated with aberrant activation of wnt signaling [ ] , and activation of wnt/β-catenin could mediate apoptosis following intestinal ischemia/reperfusion (ir) injury [ ] . as depicted in fig. , we proposed a cascade of pathological change in ab-mediated hypertrophic response that triggers wnt/β-catenin activation in the myocardium. sfrp prevented apoptosis following ir injury by blocking canonical wnt a [ ] . in our study, sfrp expression was decreased after hypertrophic stimuli both in vitro and in vivo, and overexpression of sfrp attenuated ab-induced apoptosis by inhibiting wnt/β-catenin signaling. moreover, our in vitro study delineated that licl abolished the anti-apoptotic role of sfrp in nrcms with ang ii stimulation. cardiac fibrosis is another important feature in the development of cardiac remodeling and is closely associated with cardiac diastolic dysfunction. previous studies have delineated that wnt expression is a major downstream molecular pathway of tgf-β-induced cardiac fibrogenesis [ ] . meanwhile, sfrp scavenges extracellular wnts and blocked tgf-β-mediated myofibroblast formation in autoimmune myocarditis, resulting in the alleviation of cardiac fibrosis [ ] . an in vitro study showed that sfrp promoted c c myoblast proliferation, but did not reveal a change in α-sma expression after sfrp treatment [ ] . in our study, sfrp mitigated ang ii-induced nrcms hypertrophy. excessive activation of wnt/β-catenin signaling is associated with abnormal cardiac fibrosis, while sfrp is traditionally considered a wnt inhibitor in that it completely interacts with frizzled receptors [ ] . however, sfrp possessed fig. sfrp overexpression prevents the establishment of cardiac hypertrophy. a schematic timeline to investigate sfrp overexpression effects in ab-induced left ventricular (lv) hypertrophy; ab and sham operations were performed weeks after aav -cmv-sfrp or aav -cmv-gfp injection. b and c quantification of sfrp expression in cardiac tissues following aav -sfrp infection at the indicated times (n = ; *p < . ). d four weeks after ab operation, h&e staining was used to evaluate the cross-sectional area of cardiomyocytes hematoxylin and eosin (h&e) staining; characteristic images of heart cross-sections showing hypertrophic growth in the indicated mice (n = mice per group, at least cells were measured per group). bottom scale bars, μm. e the heart weight to body weight ratio (hw/bw) in sfrp -or gfp-overexpressing hearts weeks after the sham or ab operation (n = mice per group). f real-time polymerase chain reaction (rt-pcr) analysis of brain natriuretic peptide (bnp) expression in sfrp -or gfp-overexpressing hearts weeks after the sham or ab operation (n = mice per group), sfrp overexpression retarded bnp mrna expression in ab-treated mice. g cardiomyocyte cross-sectional area (csa) quantified in the sham or ab mice from the indicated groups (n = mice per group, at least cells were measured per group). h echocardiographic parameters including the heart rate (hr), end-diastolic interventricular septal thickness (ivsd), left ventricle end-diastolic diameter (lvedd), left ventricle end-systolic diameter (lvesd), left ventricle fractional shortening (fs), and hemodynamic parameters, such as the maximal rate of pressure development (dp/dt max), maximal rate of pressure decay (dp/dt min), of each group weeks after ab or sham surgery (n = mice per group). i characteristic images of picrosirius red (psr) staining of left ventricular sections in sham and ab animals. j quantification of interstitial fibrosis in mice from the sham and hypertrophy groups (n = mice per group, at least different areas were measured per group). k mrna expression of collagen iα , collagen iiiα , and periostin detected by rt-pcr analysis in each indicated group (n = mice per group), sfrp significantly blunted collagen iα, collagen iiiα ,and periostin mrna expression in hypertrophic hearts. data are presented as the mean ± sd. *p < . vs. the sham group, # p < . vs. the ab + gfp group, and §p < . vs. the sham group. ◂ wnt-activating activity in different organ systems under multiple physiological conditions. in our opinion, there is a cardioprotective effect of sfrp under specific pathological circumstances. there were some limitations and pitfalls in our study. one issue is the uncertainty of sfrp expression in human heart failure tissues. future studies are warranted to evaluate human samples from patients with end-stage heart failure. another issue is that different cell type-specific conditional overexpression of sfrp mice are needed to further elucidate its role in the pathogenesis of cardiac hypertrophy. obviously, more studies are needed for future investigations. in conclusion, our present study showed that sfrp expression was reduced during the progression of heart failure. moreover, overexpression of sfrp by aav -sfrp infection retarded cardiac remodeling induced by pressure overload by blocking wnt/β-catenin signaling. consistent with our in vivo results, licl markedly abrogated the inhibitory effects of sfrp on cardiomyocyte hypertrophy following ang ii stimulation. our studies implied that inhibition fig. sfrp overexpression prevents apoptosis in the development of cardiac hypertrophy. a representative terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) images of myocardium overexpressing sfrp or gfp, a large number of tunel positive nucleus were present in the hypertrophic cardiac slices, whereas positive nucleus signal was significantly reduced in the sfrp -overexpressing hearts. b quantification of positive tunel nuclei (n = mice per group). c protein expression of cleaved-caspase , bax, and bcl- was analyzed by western blot (left); quantitative results of cleaved-caspase and the bax/bcl- ratio (right). d western blot and quantitative results of active β-catenin and total β-catenin protein in the heart of mice weeks after ab (n = repetitions). e nrcms were co-incubated with licl ( . mm, h) followed by stimulation with ang ii ( μm, h). representative images of cardiomyocyte hypertrophy. (blue: nuclear; green: α-actinin; scale bars: μm). f quantitation of the nrcm cell surface area (at least cells per group). g bnp mrna expression was detected by rt-pcr analysis (n = repetitions). h protein expression of cleaved-caspase and β-catenin in nrcms after ang ii stimulation for h. representative western blots (left) and quantitative data (right) are presented (n = repetitions). data are presented as the mean ± sd. *p < . vs. the sham group, #p < . vs. the ab + gfp group, and *p < . , §p < . vs. the sham group. ◂ fig. sfrp exerted protective effects in the development of cardiac hypertrophy via inhibition of wnt/β-catenin signaling. a representative images of the morphological analysis of cardiac hypertrophy as reflected by h&e staining (top) and psr staining (bottom) (n = mice per group, at least different areas were measured per group). b cardiomyocyte cross-sectional area (csa) quantified from the sham or ab mice in the indicated groups (n = mice per group, at least cells were measured per group). c quantification of interstitial fibrosis from mice with sham or ab surgery (n = mice per group, at least different areas were measured per group). d the heart weight to body weight ratio (hw/bw, n = mice per group). e rt-pcr analysis of bnp mrna expression (n = mice per group). f representative echocardiographic images of each group. g echocardiographic parameters including the left ventricle end-diastolic diameter (lvedd), left ventricle end-systolic diameter (lvesd), and left ventricle ejection fraction (lvef) in the indicated groups (n = mice per group). data are presented as the mean ± sd. *p < . of β-catenin by carrying sfrp with aav virus may be a novel strategy for protecting against cardiac remodeling in the therapy of patients with heart failure. pathophysiology of heart failure wnt- activation of a non-canonical wnt signalling pathway is required for cardiogenesis secreted frizzled related protein protects cells from apoptosis by blocking the effect of canonical wnt a wnt/beta-catenin signaling mediates both heart and kidney injury in type cardiorenal syndrome sfrp activates wnt/beta-catenin signaling in cardiac fibroblasts: differential roles in cell growth, energy metabolism, and extracellular matrix remodeling overexpression of ctrp protects against sepsis-induced myocardial dysfunction in mice pellino -mediated tgf-beta synthesis contributes to mechanical stress induced cardiac fibroblast activation critical role for stromal interaction molecule in cardiac hypertrophy tet inhibits emt of ovarian cancer cells through activating wnt/beta-catenin signaling inhibitors dkk and sfrp the wnt/frizzled pathway as a therapeutic target for cardiac hypertrophy: where do we stand? beyond wnt inhibition: new functions of secreted frizzled-related proteins in development and disease ) sfrp suppression of bone morphogenic protein (bmp) and wnt signaling mediates mesenchymal stem cell (msc) self-renewal promoting engraftment and myocardial repair secreted frizzled-related protein as a target in antifibrotic therapeutic intervention increased expression of a homologue of drosophila tissue polarity gene "frizzled" in left ventricular hypertrophy in the rat, as identified by subtractive hybridization interruption of wnt signaling attenuates the onset of pressure overload-induced cardiac hypertrophy wnt signaling is critical for maladaptive cardiac hypertrophy and accelerates myocardial remodeling beta-catenin overexpression reduces myocardial infarct size through differential effects on cardiomyocytes and cardiac fibroblasts sfrp regulates cardiomyogenic differentiation by inhibiting a positive transcriptional autofeedback loop of wnt a secreted frizzled related protein (sfrp ) is the key akt-mesenchymal stem cell-released paracrine factor mediating myocardial survival and repair activation of wnt/beta-catenin signaling increases apoptosis in melanoma cells treated with trail protective effects of ginsenoside rg on intestinal ischemia/reperfusion injury-induced oxidative stress and apoptosis via activation of the wnt/beta-catenin pathway entanglement of gsk- β, β-catenin and tgf-β signaling network to regulate myocardial fibrosis transforming growth factor-beta-dependent wnt secretion controls myofibroblast formation and myocardial fibrosis progression in experimental autoimmune myocarditis canonical wnt signaling is involved in switching from cell proliferation to myogenic differentiation of mouse myoblast cells exogenously administered secreted frizzled related protein (sfrp ) reduces fibrosis and improves cardiac function in a rat model of myocardial infarction key: cord- - pjpe authors: mahmudpour, mehdi; roozbeh, jamshid; keshavarz, mohsen; farrokhi, shokrollah; nabipour, iraj title: covid- cytokine storm: the anger of inflammation date: - - journal: cytokine doi: . /j.cyto. . sha: doc_id: cord_uid: pjpe patients with covid- who require icu admission might have the cytokine storm. it is a state of out-of-control release of a variety of inflammatory cytokines. the molecular mechanism of the cytokine storm has not been explored extensively yet. the attachment of sars-cov- spike glycoprotein with angiotensin-converting enzyme (ace ), as its cellular receptor, triggers complex molecular events that leads to hyperinflammation. four molecular axes that may be involved in sars-cov- driven inflammatory cytokine overproduction are addressed in this work. the virus-mediated down-regulation of ace causes a burst of inflammatory cytokine release through dysregulation of the renin-angiotensin-aldosterone system (ace/angiotensin ii/at r axis), attenuation of mas receptor (ace /masr axis), increased activation of [des-arg ]-bradykinin (ace /bradykinin b r/dabk axis), and activation of the complement system including c a and c b- components. the molecular clarification of these axes will elucidate an array of therapeutic strategies to confront the cytokine storm in order to prevent and treat covid- associated acute respiratory distress syndrome. the coronavirus infectious disease that is spreading at the global scale is caused by a severe acute respiratory syndrome coronavirus (sars-cov- ), an enveloped singlestranded rna virus belonging to the coronaviridae family, genus beta-coronavirus [ ] . nonstructural proteins of this virus play a crucial role in virus replication while structural and auxiliary proteins are involved in morphogenesis and interfere with the host immunity response, respectively [ ] . accumulating evidence suggests that the host immunity response is contributing in severe forms of mers-cov, sars-cov and sars-cov- infections [ ] [ ] [ ] . this immune response has been associated with a higher intensive care unit (icu) admissions and mortality in covid- . in fact, higher concentrations of granulocyte-colony stimulating factor (g-csf), interferon gammainduced protein (ip ), monocyte chemoattractant protein (mcp ), macrophage inflammatory protein alpha (mip a), and tumor necrosis factor alpha (tnfα) in comparison to non-icu patients were reported in patients with covid- [ ] . in another study, higher levels of interleukin- (il- ) receptor, interleukin- (il- ), interleukin- (il- ), interleukin- (il- ), and tnfα were found in deceased patients with covid- compared to patients who had recovered from the disease [ ] . these immunologic reactions in severe covid- may characterize the cytokine storm that is associated with untoward clinicopathological consequences. the cytokine storm is an out-ofcontrol cytokine release that has been observed in some infectious and noninfectious diseases, leading to a hyperinflammation condition in the host ( fig. ) [ ] . this uncontrolled cytokine response might be accompanied with more immune cells activation including t helper cell (th ) differentiation from cd + lymphocytes. in fact, increased th responses were reported in mers-cov, sars-cov and sars-cov- [ ] [ ] [ ] [ ] . at least % of the patients with severe covid- will eventually present lung injury, acute respiratory distress syndrome (ards) and involvement of multiple organs within - days of the onset of their illness [ ] . these severe cases that develop respiratory failure show a series of pathological findings such as hyaline membrane formation, inflammatory infiltration with multinucleated syncytial cells in their lung pathology and a burst of cytokine release leading to morbidity and mortality [ , ] . the initial cellular entry phase of the sara-cov- requires binding of its envelope homotrimeric spike glycoprotein to the membrane-bound form of angiotensin-converting enzyme (ace ) on the target cell [ , ] . the attachment of the virus with ace , as its cellular receptor, triggers internalization of the complex into the target cell, leading to the down-regulation of the ace [ ] . ace internalization and its subsequent down-regulation would potentially result in unopposed function of angiotensin ii (angii) and decreased levels of angiotensin-( - ) [ ] . since angiotensin-( - ) has a key counter-regulatory role in many of the angiotensin type receptor (at r)-related physiopathological functions, the sara-cov- -mediated downregulation of ace and the resulting increased overall ratio of ang ii to angiotensin-( - ) leads to the deterioration of the pulmonary function and lung injury [ , ] . therefore, the imbalance of ace /ace levels in covid- and the dysregulated angiotensin-ii /at r axis of the renin-angiotensin-aldosterone system (raas) may partially be responsible for the cytokine storm and the resulting pulmonary damage [ , ] . the loss of the modulatory effect of angiotensin-( - ) via its binding to the mas receptor (masr) that attenuates inflammatory response may be a further contributing factor to the hyper-inflammation status of severe cases of covid- . beyond ace catalytic activity in raas and masr-mediated actions, it has interesting effects on multiple molecular pathways which are involved in inflammatory response and cytokine release. however, potential cellular and molecular mechanisms of the cytokine storm in covid- have not yet been explored extensively. in this review, we specifically discuss the complex inflammatory molecular consequents of downregulation of ace in the context of sars-cov- , with a particular emphasis on the complement system and [des-arg ]-bk or (dabk) in addition to ace/ angiotensin-ii/at r and ace /masr axes. we propose a unifying molecular model to better understand the complex molecular events behind out-of-control cytokine response in severe covid- patients. undoubtedly, this insight will be pivotal to obtain a harmonized therapeutic strategy to confront this deadly viral infection and to protect the lungs during the cytokine storm. the renin-angiotensin-aldosterone system (raas), through its vasoactive peptides, regulates blood pressure, fluid volume, sodium and potassium balance. this elegant system also plays a significant role in the promotion and maintenance of inflammation [ ] . it appears that activation of the raas system can induce inflammation in an independent mechanism of blood pressure through the at receptor (at r) in the kidney and vasculature [ ] . the secretion of profibrotic cytokines such as transforming growth factor beta (tfg-β) is stimulated during raas activation [ ] [ ] [ ] . furthermore, increased production of ang ii and activation of at r are accompanied with a pro-inflammatory response via activation of the complement cascade including c a, c b- [ ] . this implies a cross-talk between raas and the complement system. ang ii can activate the nuclear factor kappa b (nf-κb) pathway [ , ] via stimulation of the phosphorylation of the p subunit of nf-κb [ ] . this will lead to increased production of il- [ ] , tnfα, il- b and il- [ ] . after at r activation, ang ii regulates mitogen-activated protein kinases (mapk) (erk / , jnk, p mapk), which have important functions on cellular processes including the release of cytokines such as il- , il- , il- and tnfα (fig. ) [ ] [ ] [ ] . ace enzyme is expressed in the heart, kidneys, testes, gastrointestinal tract and lungs [ , ] . it cleaves the angiotensin i (ang i) to generate the inactive ang-( - ) peptide, which can be changed to the vasodilatory peptide ang-( - ) by ace or other peptidases. ace can also directly metabolize ang ii to generate ang-( - ) [ ] . sars-cov- uses ace in type ii pneumocytes of lung alveoli and club cells in bronchioles as the cellular entry receptor. very recently, tmprss (as a major host protease), and ace coexpression was reported among a subset of type ii pneumocytes in the lung [ ] . after the attachment of sars-cov- spike (s)-protein to ace , its intracellular binding site down-regulates ace . consequently, following this down-regulation of ace , ang ii level increases in the serum leading to augmentation of the ang ii/at r axis activation, which is followed by trans-signaling of il- -sil- ra complex, in which the gp -mediated activation of stat occurs in the lungs' epithelial cells. although sars-cov- itself activates nf-κb through pattern recognition receptors, it is the simultaneous activation of nf-κb and stat that enhances nf-κb activation machinery (the il- amplifier). this hyper-activation of nf-κb via the il- amp in the lungs induces a cytokine storm with subsequent ards that had been observed in severe covid- patients [ , ] . indeed, down-regulation of ace which was accompanied with enhancement of ang ii levels in different types of lung injury triggered those pulmonary pathological changes that are commonly observed in ards [ ] . given the above premises, it seems reasonable to speculate that depletion of ace and activation of ace/angiotensin ii/at r axis might have a pivotal role in the clinical presentations of covid- . in fact, higher circulatory levels of ang ii were reported in covid- patients than the control subjects and these plasma levels of ang ii were linearly associated with lung injury [ ] . therefore, in contrast to earlier clinical experts' opinions, raas inhibitors should not be discontinued in stable cases of covid- because the discontinuation of ace inhibitors and arbs may potentially have detrimental effects on these patients [ , , ] . in a recent study, the first clinical evidence has shown that ace inhibitors or arb therapy in covid- patients with hypertension were associated with a lower rate of disease severity, a trend toward lower il- levels, and higher circulatory cd + and cd + t cells counts [ ] . the protective effect of ace in severe acute lung failure has been shown in animal models [ ] . the accumulating clinical-epidemiological evidence about covid- implies that sars-cov- associated ace depletion is accompanied with a severe clinical course of disease in those clinical and epidemiological conditions that jeopardize the levels of ace expression including older age, male sex, and medical conditions (diabetes mellitus, hypertension and cardiovascular diseases, and obesity) [ , ] . under these medical conditions, the covid- infection-induced ace depletion could not overcome already exaggerated ace/angiotensin ii/at r axis activity. hence, administration of recombinant soluble ace to patients with severe covid- infection may be a therapeutic modality. however, it is worthwhile to consider targeting downstream of ace/angiotensin ii/at r axis, such as il- -stat axis [ ] to combat the observed cytokine storm in covid- in order to prevent lung inflammation and end organ damage. ace , ang-( - ) and ang-( - ) receptor mas are the constituents of the other arm of the ras system which counteracts and attenuates the effects of ace-ang ii-at r axis [ , ] . ace derives vasodilatory peptide ang-( - ) from ang ii following a cleavage activity. this vasodilatory peptide has anti-proliferative, anti-thrombotic and anti-inflammatory activities [ ] [ ] [ ] . ang-( - ) reduces the expression of p mapk and nf-κb and inflammatory factors such as il- , tnfα and il- [ ] [ ] [ ] [ ] . thus, ang-( - ) per se has an anti-inflammatory effect and ameliorates inflammatory damages, as revealed in several animal studies [ , ] . it has been shown that ang-( - ) reduces inflammatory cardiac injury in diabetic hypertensive rats [ ] and glomerular involvement in mesangial proliferative glomerulonephritis (mpgn) rat models [ ] . the protection of vascular endothelium and renal tubular cells, diuresis and vasodilationdependent ang-( - ) effects occur via masr [ , ] . mas receptors express in the epithelium and bronchial smooth muscle; therefore, ang ( - ) could modulate acute and chronic inflammatory processes in the lung via activation of masr [ ] . a range of physiological effects of ang- ( - ) is present in different tissues such as heart, brain and kidney by its action on masr [ ] . ang-( - ) also attenuates ang ii induced intercellular adhesion molecule- (icam- ), vascular cell adhesion molecule- (vcam- ) and mcp expression by masr activation leading to inhibition of the p mapk and nf-κb pathways [ ] . erk / pathway modulates production of il- [ ] that induces differentiation of t-helper toward th -type. th- regulates immune responses by producing anti-inflammatory cytokines like il- , il- , il- and il- [ ] . additionally, il- is an anti-inflammatory cytokine that may be involved in the prevention of tissue damage [ , ] . ang-( - ) modulates the activity of erk / therefore, ang-( - ) has an anti-inflammatory function via modulation of nf-κb, mapk and erk / pathways. hence, it may be proposed that sars-cov- associated suppression of ace , which would be accompanied with reduction of all mas receptor-mediated functions, leads to accentuation of the cytokines release and frank inflammatory responses. because ang-( - ) exerts a critical role in counteracting the pro-inflammatory effect of raas, protecting from endothelial cell activation and resulting lung damage from inflammatory mediators in the cytokine storm, the administration of ang-( - ) or one of its similar agents to patients with covid- pneumonitis has been suggested [ , ] . the kinin-kallikrein system includes kininogen, kallikrein enzyme, bradykinin (bk- - or bk), and [des-arg ]-bk or dabk [ , ] . the active products of this system interact with two distinct g-protein coupled receptors, the bradykinin b receptor (bkb r) and bradykinin b receptor (bkb r); the main ligand of bkb r is dabk and the ligand of bkb r is bk [ , ] . bkb r could hardly be detected in peripheral tissues in normal states; however, it is expressed on the cell types involved in inflammation. therefore, it is an inducible pro-inflammatory receptor [ ] . the expression of bkb r, as a specific receptor of bradykinin pathway, is highly sensitive to inflammatory mediators such as lipopolysaccharide (lps) and interleukins [ , [ ] [ ] [ ] . it is also up-regulated by cytokines such as il- b and tnfα [ ] [ ] [ ] . il- , ifnɣ, epidermal growth factor (egf), and oncostatin increase the rate of bkb r receptor mediated response [ ] [ ] [ ] [ ] . it should be mentioned that il- b and tnfα-induced bkb r expression is related to nf-κb activity [ ] . sodhi et al. [ ] showed that activation of bkb r enhances the neutrophil attraction to tissue by release of chemokine c-x-c motif chemokine (cxcl ). activity of this receptor leads to expression of fgf- [ ] , and to increased levels of il- b [ ] and mcp (fig. ) [ ] . dabk is a known pulmonary inflammatory factor [ ] [ ] [ ] [ ] . it is interesting that ace also cleaves terminal residue of dabk [ , ] . this reaction results in deactivation of dabk. therefore, it could be postulated that covid- -induced reduction of ace activity would be accompanied with increased activity of dabk and the resulting accentuation of the aforementioned inflammatory cascade, leading to increased cytokine release. hence, targeting the ace /dabk/ bradykinin b receptor axis has been suggested by some authors [ , ] . to prevent or control ards in patients with severe covid- . although several bkb antagonists have passed phase ii clinical trials, none have been approved yet for clinical use [ ] . the complement system is an ancient system that contributes to innate immune response. this system includes many proteins and cleavage products that plays a key functional role in defense against microorganisms including viruses. the viral inactivation by the complement cascade involves virus uptake and clearance by phagocytic cells, coating virions resulting in prevention of attachment with their receptors, virus lysis by pore formation, and destruction of its membrane by membrane attack complex formation (c b- ) [ ] . following viral-induced complement cascade activation, inflammatory processes are promoted. complement factor a (c a) is the strongest inflammatory peptide in the complement cascade that induces release of pro-inflammatory cytokines [ ] [ ] [ ] [ ] . ]. c a can also induce secretion of tnf-α [ , ] . terminal products of the complement cascade can induce the production of cytokines such as tnf-α and il- [ , [ ] [ ] [ ] [ ] . terminal complement component c b- induces release of il- via activation of redox-sensitive transcription factor nf-κb and activator protein- (ap- ) [ ] and monocyte chemoattractant protein- (mcp ) from vascular smooth muscle cells [ ] . also, the increased production of c a leads to production of pro-inflammatory cytokines such as il- , il- and tnf-α [ ] . the involvement of the complement system in the pathogenesis of syncytial virus infection, [ ] [ ] [ ] . the hyper activation of complement components including c a in sera and c b- in lungs was observed in mers-cov-infected hdpp -transgenic mice. the lung and spleen-induced pathological damages and inflammatory responses were alleviated through blockade of the c a-c ar axis in those transgenic mice [ ] . gralinski et al. [ ] showed that mice models deficient in c (c -/-) loads in the lung had milder sars-cov induced pathologic features such as better respiratory function, lower weight loss, reduced pathologic findings in respiratory system, and lower circulatory and tissue cytokines and chemokines, despite equal lung viral loads compared to the controls. these results showed that although the complement system had no role in virus replication, activation of complement system in the lungs of sars-cov infected mice might lead to immune mediated damages in the lungs [ ] . the complement cascade can be activated by the lectin pathway (fig. ) . by priming the immune system and enhancing clearances of viruses and virus-infected cells [ ] ], the lectin pathway may also be involved in covid- pathogenesis. recently, gao et al. [ ] reported in a very stimulated macrophages [ , ] . however, the c a-c r axis inhibition by the available pharmaceutical agents would be partial, and the activity of residual terminal complement components might remain. therefore, targeting the upstream activators of the complement cascade such as c a-c ar axis may be more effective in restraining the uncontrolled complement pathway activation in severe covid- [ ] . very recently, the compstatin-based complement c inhibitor amy- was administered to a patient with ards due to covid- , in which very good clinical responses with a high level of safety were reported [ ] . the overall clinical benefits of targeting the c a-c ar/c a-c ar axis in order to control maladaptive immune-inflammatory consequences of the complement pathways in severe covid- the covid- cytokine storm, like the other cytokine storms in infectious and non-infectious conditions, may be considered as a complex network. the complex network of the cytokine response was described by tisoncik et al. [ ] as "a series of overlapping networks, each with a degree of redundancy and with alternative pathways". our aforementioned pathogenesis of the so-called covid- cytokine storm through the four described distinct axes clarified that this cytokine storm complex network has many components which might cross-talk with each other in multiple known and unknown interfaces. these interactions in a network state imply the complex nature of the covid- cytokine storm. the dynamic equilibrium of the network components could be disturbed at multiple sites to emerge an untoward behavior. in covid- cytokine storm, this perturbation is initiated via attachment of the sars-cov- spike protein to its receptor, ace , followed by the ace/ang ii/at r axis activation leading to hyper-activation of nf-κb by il- stats axis [ ] . in the normal dynamic equilibrium state, the ace/ang ii/at r axis activation is counterbalanced by ace /masr axis and production of ang-( - ) that reduces the expression of p mapk and nf-κb and inflammatory factors such as il- , tnfα and il- [ ] [ ] [ ] . however, sars-cov- associated down-regulation of ace suppresses these immunomodulatory effects, leading to accentuation of the cytokine release response. we currently know that dabk is a pulmonary inflammatory factor whose deactivation by ace is deranged by covid- -induced reduction of ace activity. this derangement is followed by ace /dabk/ bradykinin b receptor axis activation that creates a pro-inflammatory synergistic effect for sars-cov- associated ace/ang ii/at r axis activation. the resulting effect would be a more inflammatory state, neutrophil recruitment and enhancement of pathological pulmonary changes that are observed in ards of severe covid- . we have already discussed the involvement of the complement system in the pathogenesis of sars-cov- via its nucleocapsid (n) protein-mediated masp- auto-activation and binding to mlb in the lectin arm of c a-c ar/c a-c ar axis [ ] . however, the increased production of ang ii and activation of at r may be accompanied with pro-inflammatory response via activation of the complement cascade including c a, c b- [ ] . this implies a cross-talk between ace/angiotensin ii/at r axis and the complement system. similar to other complex networks, elements from the complex network of the covid- cytokine storm may experience much cross-talk with elements from known and unknown pathways and networks. for example, ang ii as an element from ace/ang ii/at r induces tgfβ expression [ , ] . tgf-β has a role in the differentiation of t helper cells from naive cd + t-cells. th- cells generate il- , gm-csf, il- and il- . il- itself promotes the production of a vast amount of pro-inflammatory cytokines and chemokines [ , ] . il- was among the cytokines that were significantly correlated with the lung injury murray score and disease severity in covid- [ ] . an increment of the highly pro-inflammatory ccr + th in cd t cells was reported in covid- associated with ards [ ] . therefore, targeting il- and t helper responses in the cytokine storm of covid- have been suggested [ , ] . therapeutic plasma exchange is a well-known therapeutic option that can be considered in the treatment of autoimmune diseases. the beneficial effect of this modality works through elimination of autoantibodies, complement components, immune complexes and cytokines. therefore, this option may be valuable in the treatment of severe covid- [ ] . however, application of this therapeutic modality is a general approach to confront the cytokine storm. hence, other approaches may be considered in the treatment of the covid- driven cytokine storm in order to protect lungs from injury (table- ). according to the aforementioned axes which may be involved in the cytokine storm of severe cases of covid- , some potential targets could be considered as therapeutic options. the first one is recombinant human ace . there are some trials regarding the efficacy and safety of this agent in clinicopathological settings related to ace decrement such as congestive heart failure (chf) [ ] , ards [ , ] , and lung injury due to viral illness such as respiratory syncytial virus (rsv) [ ] . the reported findings about safety and efficacy were promising. very recently, monteil et al. [ ] reported that human recombinant soluble ace (hrsace ) can prevent entry of sars-c v- to the human blood vessel organoid and human kidney organoid; this finding may suggest a highly compelling therapeutic intervention to protect lung injury in covid- . we suggested ace /bradykinin/dabk may be involved in the inflammatory response of sars cov- ; therefore, blockade of this axis by inhibiting bkb r may ameliorate a part of the cytokine storm which occurs in covid- infection. lf - (safotibant) is a bkb r antagonist with promising anti-inflammatory results [ , ] . several clinical trials have been conducted to evaluate the effect of this drug in multiple medical settings; they have had promising results (fig. ) [ ]. regarding the pivotal role of the complement system in the cytokine storm and activation of th- , every effort should be made to suppress the activation of this elegant cascade. c a, as a potent component of this system, is a good target for alleviation of pro-inflammatory responses to severe covid- . eculizumab is a monoclonal antibody with high affinity to c near its cleavage site [ ] . this agent prevents the formation of c a and c b- . it has been reported that this monoclonal antibody has beneficial effects in the treatment of diseases where their pathogenesis are based on complement activation [ ] . in a preliminary study conducted by gao (fig. ) [ ] . in conclusion, accumulating evidence suggests that sars-cov- driven ace down-regulation leads to an array of complex and intertwined molecular interactions via at least four axes consisting of dysregulation of the ace/angiotensin ii/at r axis, attenuation of ace /masr axis, increased activation of ace /bradykinin b r/dabk axis, and activation of the complement cascades, resulting in the observed cytokine storm in severe covid- . the elucidation of molecular events of the aforementioned axes which might be involved in the pathogenesis of ards and lung injury in fulminant infections with sars-co-v- will promise novel therapeutic options for prevention or attenuation of the inflammatory cytokine release response that are observed nowadays in patients with severe covid- . mm, in, jr, sf and mk interpreted and wrote the manuscript. in and mm supervised the study. all authors read and approved the final manuscript. an award was given to iraj nabipour by "novo nordisk pars" (the best innovator of diabetes in iran ). for the remaining authors none were declared. the technical assistance of mr. dara joukar in drawing of figures is gratefully acknowledged. the authors also thank ms. melissa t. yang, phd for her kind language editing of the manuscript. captions for figures: a doubt of multiple introduction of sars-cov- in italy: a preliminary overview emerging coronaviruses: genome structure, replication, and pathogenesis clinical features of patients infected with novel coronavirus in plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome mers-cov infection in 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map kinases in proximal tubular cells - ) attenuates airway remodelling and hyperresponsiveness in a model of chronic allergic lung inflammation the ace /angiotensin-( - )/mas axis of the renin-angiotensin system: focus on angiotensin-( - ) angiotensin-( - ) attenuates angiotensin ii-induced icam- , vcam- , and mcp- expression via the mas receptor through suppression of p and nf-κb pathways in huvecs polar opposites: erk direction of cd t cell subsets t helper cytokine patterns: defined subsets, random expression, and external modulation il- dampens tnf/inducible nitric oxide synthaseproducing dendritic cell-mediated pathogenicity during parasitic infection interleukin- and the regulation of mitogenactivated protein kinases: are these signalling modules targets for the anti-inflammatory action of this cytokine? molecular mechanisms of inhibition of vascular growth by angiotensin inhibition of human lung cancer cell growth by angiotensin angiotensin-( - ) inhibits growth of cardiac 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selective bradykinin b receptor antagonists discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria many drugs for many targets: novel treatments for complement-mediated glomerular disease soluble cr therapy improves complement regulation in c glomerulopathy increased activity of the raas system occurs in the covid- induced cytokine storm.the covid- induced cytokine storm is accompanied with attenuation of masr activity.the sars-cov- associated ace induces dabk and bkb r hyper-activation.the covid- induced cytokine storm leads to hyperactivity of the complement system. an award was given to iraj nabipour by "novo nordisk pars" (the best innovator of diabetes in iran ). for the remaining authors none were declared. key: cord- -p msabl authors: obukhov, alexander g.; stevens, bruce r.; prasad, ram; li calzi, sergio; boulton, michael e.; raizada, mohan k.; oudit, gavin y.; grant, maria b. title: sars-cov- infections and ace : clinical outcomes linked with increased morbidity and mortality in individuals with diabetes date: - - journal: diabetes doi: . /dbi - sha: doc_id: cord_uid: p msabl individuals with diabetes suffering from coronavirus disease (covid- ) exhibit increased morbidity and mortality compared with individuals without diabetes. in this perspective, we critically evaluate and argue that this is due to a dysregulated renin-angiotensin system (ras). previously, we have shown that loss of angiotensin-i converting enzyme (ace ) promotes the ace/angiotensin-ii (ang-ii)/angiotensin type receptor (at r) axis, a deleterious arm of ras, unleashing its detrimental effects in diabetes. as suggested by the recent reports regarding the pathogenesis of severe acute respiratory syndrome coronavirus (sars-cov- ), upon entry into the host, this virus binds to the extracellular domain of ace in nasal, lung, and gut epithelial cells through its spike glycoprotein subunit s . we put forth the hypothesis that during this process, reduced ace could result in clinical deterioration in covid- patients with diabetes via aggravating ang-ii–dependent pathways and partly driving not only lung but also bone marrow and gastrointestinal pathology. in addition to systemic ras, the pathophysiological response of the local ras within the intestinal epithelium involves mechanisms distinct from that of ras in the lung; however, both lung and gut are impacted by diabetes-induced bone marrow dysfunction. careful targeting of the systemic and tissue ras may optimize clinical outcomes in subjects with diabetes infected with sars-cov- . infection. studies indicate that - % of individuals with severe infections have diabetes ( , ) . however, a recent meta-analysis of six clinical studies involving , covid- patients provided evidence that individuals with diabetes exhibited a similar prevalence of being infected with sars-cov- as the overall population, but presence of diabetes was a critical comorbidity that increased the risk of a poor outcome ( ) . certain racial groups such as african americans and native americans are highly prone to developing diabetes and experience disparities in health care making them particularly vulnerable to covid- ( ) . however, to date there is a paucity of data regarding comorbidities, covid- outcomes, and mechanisms that modulate viral pathogenesis. in this perspective, we bring attention to specific factors that may complicate covid- in individuals with diabetes including ) the presence of bone marrow changes (myeloidosis) that predispose those with diabetes to an excessive proinflammatory response (cytokine storm) and contribute to insulin resistance and reduced vascular repair, and worsening function of the heart, kidney, and systemic vasculature as a whole; ) increased circulating furin levels that could cleave the spike protein and increase infectivity of sars-cov- ; ) dysregulated autophagy that may promote replication and/or reduce viral clearance; and ) gut dysbiosis that leads to widespread systemic inflammation, increased gut glucose and sodium absorption, and reduced tryptophan and other key amino acid absorption needed for incretin secretion and glucose homeostasis. central to each of these dysfunctions is the dysregulated ras, in particular, the global loss of ace , which we propose is a unifying mechanism that could lead to the increased risk of morbidity and mortality in individuals with diabetes presenting with covid- . the ras is a key hormonal circuit tasked in regulating extracellular fluid volume and blood pressure in mammals. if blood pressure falls, the juxtaglomerular cells in the kidneys produce and secrete renin, which cleaves serum angiotensinogen to produce angiotensin i (ang-i). then, angiotensin-i converting enzyme (ace) further converts ang-i into angiotensin ii (ang-ii) in the lungs by removing the c-terminal dipeptide from ang-i (fig. ) . ang-ii in turn activates the g-protein-coupled angiotensin type receptor (at r) on adrenal zona glomerulosa cells to produce aldosterone, which causes sodium retention, an increase in blood volume, and blood pressure stabilization. besides its beneficial function in regulating extracellular fluid volume, a dysregulated ras, as seen in diabetes, could lead to an increase in serum levels of ang-ii that could cause a plethora of potentially harmful effects including vasoconstriction, inflammation, and increased oxidative stress. ace converts ang-ii into ang- - , and ang- - acts through the mas receptor (masr) to oppose the effects of ang-ii. this ability of ace to convert the serum vasopressor ang-ii into the vasodilating ang- - identifies it as a "negative" regulator of ras. notably, covid- patients exhibit increased serum levels of ang-ii ( ) , which would support less cleavage by ace and thus potentially less ace activity. in addition to systemic ras, "local" ras exists within each tissue including a lung ras, intestinal ras, and bone marrow ras. interestingly, the ras system in the intestinal mucosa significantly contributes to the regulation of glucose, salt, and water uptake. emerging covid- gastrointestinal disturbances implicate a central role of intestinal pathophysiology in exacerbation of hyperglycemia and blood pressure in individuals with diabetes infected with sars-cov- . ace protein: function, interaction between ace and adam , and ace as the receptor for sars-cov and sars-cov- ace was discovered in ( ), just - years before the first wave of sars-cov coronavirus pandemic, when ace was identified as the major sars-cov "receptor" on host cells. ace functions as a metallocarboxypeptidase, a plasma membrane-bound proteolytic enzyme ( fig. a ) that removes a single carboxy-terminal amino acid from specific bioactive oligopeptides, such as ang-i and ang-ii to form ang- - and ang- - , respectively ( fig. ). unlike ace, which is a peptidyl dipeptidase removing two carboxy-terminal amino acids from ang-i and ang- - , ace is not inhibited by typical ace inhibitors, such as captopril. the ectodomain of ace can be shed into the systemic circulation as a soluble protein, preserving the catalytic activity of ace (soluble ace ) and its ability to generate in the circulation the vasoprotective peptide, ang- - . shedding of the ace ectodomain occurs after proteolytic cleavage by plasma membrane-anchored endopeptidases, enzymes capable of breaking nonterminal peptide bonds, such as a disintegrin and metallopeptidase domain protein (adam ) ( ) or the type ii transmembrane serine proteases (tmprss ) ( ) . human ace (hace ) is predominantly expressed in the nasal epithelium, airways, lungs, heart, adipose tissue, kidneys, small intestine, and colon ( , ( ) ( ) ( ) . the high density of hace is found in human nasal epithelium goblet cells, human ciliated cells of the airways, the type alveolar (at- ) epithelial cells, and bronchial transient secretory cells ( , ) . high hace expression in the nasal epithelium is consistent with clinical observations that symptomatic individuals with covid- present with a higher viral load in the nasal cavity compared with the throat ( ) and that some covid- patients complain of inability to smell ( ) . the hace -expressing nasal epithelium may provide an "intermediate site" for viral replication before its invasion of the lungs to cause symptomatic covid- and may serve as a sanctuary niche for sars-cov- survival without spreading to the lungs in human subjects, thus perhaps permitting asymptomatic human-to-human transmission. notably, the sars-cov- -positive individuals may shed the virus for up to days ( ). the ace protein contains two cofactors: zn and cl ions ( fig. a) . the zinc binding site, coordinating zn , is critical for the catalytic activity of ace and consists of h -e -x-x-h in hace . the cl binding site regulates the efficacy of ace to cleave its substrates (ang-i and ang-ii) in an extracellular cl -dependent manner ( ) . the spike proteins of sars-cov and sars-cov- bind the membrane-bound ace to enter the host cells ( ) ( ) ( ) . the interface of the sars-cov- receptor binding domain located on the s subunit of the spike protein and the n-terminal segments of hace was mapped using cryo-em and x-ray crystallography (fig. b ) ( ) ( ) ( ) , and the structure data shed light on the underlying mechanisms. there are several virus binding hotspots on the surface of hace that are critical for virus infectivity; these include hotspot (lysine ), hotspot (lysine ), and the hydrophobic interaction site (tyrosine y ) ( ) . compared with sars-cov, sars-cov- forms additional hydrogen bonds, dipole-dipole interactions, and salt bridges ( ) , suggesting stronger interaction. the affinity binding data indicate that the receptor binding domain of sars-cov- has a greater affinity to hace compared with that of the sars-cov virus ( ) , potentially explaining the enhanced ability of sars-cov- to quickly spread and infect a great number of hosts. while membrane-bound hace is the major cellular receptor for sars-cov- binding and internalization, the soluble form of hace is efficient at preventing the coronaviruses attachment to the membrane-bound hace ( ) . proteolytic cleavage of the homotrimeric spike protein ectodomains at the s /s subunit junction is critical for entry of coronaviruses into host cells. the ectopeptidase tmprss and endosomal peptidases cathepsin b/l are the major cellular enzymes that mediate coronavirus "priming" in sars-cov- ( ) by cleaving the spike protein at the s / s cleavage site (iay↓tms) (fig. ) . the sars-cov- spike protein has an additional canonical furin cleavage site (prrar↓sv) located upstream of the conserved iay↓tms cleavage site ( , ) . this is a unique property of sars-cov- because the furin site is not present in sars-cov ( , ) . the presence of a furin-like cleavage site in viral spike-shaped hemagglutinin proteins has been associated with an increased virulence and pathogenicity in avian and human influenza viruses. consistently, the current pandemic epidemiological data confirm the increased transmissibility and pathogenicity of sars-cov- as compared with sars-cov ( ) . renin is produced and secreted in the juxtaglomerular cells of the kidney when plasma nacl decreases or blood pressure falls. renin cleaves angiotensinogen to produce ang-i, which is further converted into ang-ii by ace in the lungs. ang-ii induces aldosterone secretion from adrenal zona glomerulosa cells, which in turn promotes sodium and water retention in the kidneys, increasing blood pressure. thus, initial serum ang-ii levels are set by renin. however, the steady-state serum ang-ii level is also markedly affected by the rate of its conversion to ang- - by ace . therefore, ace activity contributes to regulating the steady-state levels of ang-ii. if we consider an example of a rainwater barrel and assume that renin is the actual rainfall amount, ang-ii is the rainwater, and ace activity is the barrel's outlet spigot, then the rainfall amount (renin) would always determine the rainwater (ang-ii) inflow rate and level into the barrel. but if we would keep the barrel outlet spigot always open (ace is active) during and after rainstorms, the final level of rainwater would not be as high as in the case if the barrel's outlet spigot were closed (ace is inactive). aldo, aldosterone; zg, zona glomerulosa. the structure of the sars-cov- spike protein provides insight on why the addition of the furin cleavage site may increase transmissibility of the virus. according to the structure, the tmprss cleavage site (iay↓tms) is located in a shallow pocket on the lateral surface of the sars-cov- spike protein ( fig. ) , whereas the short solvent-exposed protein loop harboring the furin-cleavage site (not solved in the structure and shown as the dotted lines in fig. ) appears to hang over the tmprss cleavage site, obstructing access. the newly biosynthesized sars-cov- viral particles are likely released by budding in a golgi compartment-dependent manner. since furin is a ca -dependent endopeptidase, which is present and active only in the golgi compartment (fig. ) , the complete cleavage of the furin site is expected in golgi compartment-processed sars-cov- spike proteins and is experimentally confirmed ( ). furin-cleaved s /s subunits remain noncovalently bound in the homotrimeric spike protein assembly. it is possible that in the furin precleaved sars-cov- spike protein, the tmprss cleavage site is no longer obstructed and is more accessible for tmprss and/or cathepsins. however, experimental confirmation will be needed for this hypothesis. the sars-cov- spike protein can be in the closed (folded) or open conformation when the viral receptor binding domain unfolds and extends above the trimeric spike protein structure (fig. ). whether furin cleavage affects the equilibrium between the two spike protein conformations also remains unclear and awaits experimental evidence. as covid- progresses, sars-cov- may also involve the lytic release pathway for newly produced viral particles, bypassing the budding process utilizing the furin-containing golgi compartments (fig. ). in such cases, the spike protein of sars-cov- may remain at least partially uncleaved by intracellular furin. at this stage of covid- , extracellular furin may be utilized to complete the cleavage of spike protein's furin cleavage sites, facilitating the virus spread in the infected host. notably, circulating levels of furin are elevated in patients with diabetes ( ), and patients with diabetes infected with sars-cov- present with increased mortality ( ) and delayed recovery from sars-cov- infection. also, individuals with high plasma furin concentration typically have a pronounced dysmetabolic phenotype and elevated risk of diabetes. there is increasing evidence that dysregulated autophagy contributes to the pathogenesis of diabetes and its complications. autophagy is primarily recognized for its essential role in cellular housekeeping and homeostasis through the sequestration and transfer of intracellular components to lysosomes for degradation. however, the endocytic pathway and autophagy are key processes affecting virus infection and replication, including the coronavirus family ( ) . viral rna replication in coronavirusinfected cells occurs in double membrane vesicles that resemble autophagosomes (fig. ) . additionally, nonstructural protein (nsp ) of sars-cov- can generate autophagosomes, and an associated mutation in nsp is identified in covid- patients ( , ) . interestingly, inhibition of the canonical autophagy pathway, using in vitro approaches, does not appear to have an effect on sars-cov replication, suggesting a noncanonical process. however, a key autophagy protein, lc , colocalizes with viral replication-transcription complexes, and an s-phase kinase-associated protein (skp ) reduces autophagy protein beclin levels in coronavirus infections ( , ) . in both cases, fusion between autophagosomes and lysosomes is blocked, leading to an accumulation of autophagosomes favoring replication of the virus. inhibiting skp or enhancing autophagy flux has been shown to reduce the replication of coronaviruses ( , ) . ras can be an important regulator of autophagy. ang-ii activation of angiotensin type receptor (at r) attenuates autophagy, whereas ang-ii activation of at r induces autophagy through ampk/mtor signaling. ang- - induces autophagy via the cofilin receptor ( ) . activation of intestinal ras promotes paneth cell autophagy leading to bowel inflammation and arrested release of antimicrobial factors including defensin , which inhibits sars-cov- infection by cloaking ace ( ) . given the strong association between the ras and autophagy, both may serve as therapeutic targets to ablate sars-cov- infection and replication, and this may further explain the possible beneficial effects of ace inhibitors/atr blockers in the treatment of covid- , discussed further below. several mechanisms may contribute to increased severity of covid- progression in subjects with diabetes. individuals with diabetes are more vulnerable to most infections and may exhibit decreased viral clearance due to reduced neutrophil chemotaxis, phagocytosis, and intracellular killing of microbes. under noninfectious conditions, chronic diabetes in both human and rodent models was associated with myeloidosis ( ), with monocytes expressing higher levels of proinflammatory cytokines that may, in patients with acute respiratory distress syndrome (ards), contribute to cytokine storm. once bound to ace , sars-cov was shown to downregulate cellular expression of ace , and the unopposed action of ang-ii was deemed responsible for worsening lung injury ( ) . whether this is the case with sars-cov- is not known. ang-ii receptor blockers, ace inhibitors, thiazolidinediones, incretin glp- agonists, and statins figure -block diagram of the homotrimeric sars-cov- spike protein assembly. "rbd" stands for receptor binding domain, "fp" stands for fusion peptide, and the ipf block depicts the location of internal fusion peptide. s and s are two segments of sars-cov- ectodomain that can be cleaved with the indicated endopeptidases. the cryo-em structure ( ) of the spike protein is shown in the center of the figure. the proteolytic sites are shown in green. in the structure, the residues preceding and following the furin cleavage site are colored in brown. the inset shows a magnifying view of the tmprss /cathepsin cleavage site. the structure of the homotrimeric sars-cov- spike protein complex was replotted from pdb id: vyb ( ) . each spike protein in the homotrimer is color coded for better identification. this is a positive-sense single-stranded rna virus. sars-cov- viral rna serves to code the viral genome and as mrna for direct protein translation by the host cell ribosomes. indeed, viral rna contains a poly-a tail at the end and a typical mrna cap structure at the end. sars-cov- viral rna is nonsegmented. viral rna genome translation starts with the production of two replicase polyproteins, pp a and pp ab, which consist of or covalently linked nonstructural proteins (nsp), respectively. these two large polyproteins are subject to proteolytic cleavage by proteases resulting in the formation of individual nsp -nsp . viral nsp functions as a papain-like protease and is important for cleaving the interdomain junctions between nsp and nsp , whereas nsp is a chymotrypsin-like protease, which is also named "main protease" because it is responsible for cleaving interdomain junctions between nsp and nsp . nsp can induce small-diameter autophagosome formation in infected cells. nsp (rdrp) is an rna-dependent rna polymerase, which is critical for a largescale replication of viral rna. nsp requires several cofactors, such as nsp and nsp . the rna helicase nsp (hel) is important for are typical medications for diabetes that are known to increase ace expression. lack of evidence regarding the risk or benefit of ace inhibitors and angiotensin receptor blockers (arbs) has resulted in the american college of cardiology, american heart association, american society of hypertension, and european heart association recommendations that patients should continue treatment with their usual antihypertensive therapy ( ) . however, we would propose that drug-induced increases in ace expression would potentially be beneficial in subjects with diabetes by increasing ang- - and shifting the ras axis away from the profibrotic, proinflammatory arm of ras. thus, in subjects with diabetes, infection with sars-cov- would potentially result in additional loss of ace expression in blood vessels and could exacerbate the already compromised vasculature ( ) . the existence of specific ras systems in organs including the bone marrow has been well established. local ras is active in primitive embryonic hematopoiesis ( ) and continues to regulate each stage of physiological and pathological blood cell production in the adult via autocrine, paracrine, and intracrine pathways. local ras peptides directly regulate myelopoiesis, erythropoiesis, thrombopoiesis, and the development of other cellular lineages ( ) . the bone marrow plays a critical role in the pathogenesis of diabetic complications. individuals with diabetes with vascular complications typically have reduced numbers and migratory function of bone marrow-derived vascular reparative cells, called circulating angiogenic cells (cacs or cd cells). ang- - improved migration, restored bioavailable nitric oxide, and reduced reactive oxygen species in diabetic cacs. ang- - gene modification of cacs restored the cells in vivo vasoreparative function ( ) . a unique set of individuals with diabetes that remained free of microvascular complications, despite . years of poor glycemic control, had higher mrna levels for ace and masr in their cacs compared with age-, sex-, and glycemia-matched individuals with diabetes with microvascular complications ( ) . in akita mice, global loss of ace (ace /y -akita mice) was associated with a reduction of hematopoietic stem/progenitor cells (hs/ pc), a shift of hematopoiesis toward myelopoiesis in bone marrow, and an impairment of hs/pc migration and proliferation. migratory and proliferative dysfunction of these cells was corrected by exposure to ang- - ( ) . these data support that activation of the protective ras is beneficial for the dysfunctional diabetic bone marrow. diabetes-associated bone marrow dysfunction and loss of vascular reparative cells, such as cacs, may contribute to vascular dysfunction in covid- patients that can be manifested as cardiac disease including arrhythmias, viral myocarditis, heart failure, and cardiac arrest ( ) ( ) ( ) . the impact of global loss of ace in cardiac dysfunction is supported by preclinical studies showing that hearts from akita mice exhibit marked systolic dysfunction and that ace /y -akita mice show impaired flow-mediated dilation of the femoral artery in response to ischemia/reperfusion injury, indicative of endothelial dysfunction. in contrast, gain-of-function studies using ace overexpression, via adenoviral gene delivery, in type diabetic rats decreased collagen accumulation and improved left ventricular remodeling and function ( ). the impact of dysregulated ras is seen in obesity and type diabetes models. heart failure with preserved ejection fraction (hfpef) is a proinflammatory state closely linked to obesity-related cardiovascular dysfunction. loss of ace increases epicardial adipose tissue macrophage polarization to proinflammatory m -like phenotype and worsens hfpef in response to diet-induced obesity. ang- - has potent anti-inflammatory effects in adipose tissue of obese type diabetic mice and protects against diabetic cardiomyopathy and nephropathy. importantly, ang- - decreased macrophage m polarization and preserved cardiac function in diet-induced obese ace knockout mice ( ) . in covid- patients, the prevalence of kidney disease on admission and the development of acute kidney injury during hospitalization is high and associated with in-hospital mortality ( ) . patients with diabetes with nephropathy have reduced ace . global loss of ace replication. nsp is a viral n -methyltransferase ensuring the fidelity of replication. the viral rna also encodes four structural proteins: the spike protein (s), envelop protein (e), membrane protein (m), and nucleocapsid protein (n). in sars-cov- virions, viral rna is enveloped with a membrane that is stabilized by the imbedded structural proteins, including s, e, m, and n proteins. the s or spike protein is a homotrimer that gives the viral particles a characteristic appearance of spiky corona. the s protein is critical for the viral entry into the host cells. the s subunit of the sars-cov- spike protein utilizes the hace protein as its cellular receptor. the tmprss protein is the key endopeptidase that is important for priming the spike protein of sars-cov- , allowing viral entry into host cells. cathepsin b/l is an endosomal protease that can substitute tmprss activity during spike protein priming before viral rna gains access into the cellular cytosolic compartments. sars-cov- replication takes place in double membrane vesicles (dmv) that are associated with the specific areas of the rough endoplasmic reticulum or other intracellular membranes, including autophagosomal membranes. the m, e, and n structural proteins together with the s protein are important for formation and stabilization of the sars-cov- viral particles. viral structural protein modification takes place in the golgi compartment before viral particles are ready for budding. furin is a ca -dependent endopeptidase enriched in the golgi compartments that precleaves the spike protein at a specific cleavage site in the golgi compartments, with s and s subunits remaining noncovalently bound in budding virions. adam proteolytic activity generates soluble hace . exacerbates diabetic kidney injury while potentiating ang-ii-mediated cardiorenal fibrosis and oxidative stress in the heart and kidney ( ) . in akita mice, recombinant hace (rhace ) treatment for weeks resulted in decreased glomerular mesangial matrix expansion, which was associated with increased ang- - levels and lowered ang-ii levels, along with reduced nadph oxidase activity. the loss of ace via adam proteolytic cleavage, which is strongly activated in covid- patients, will likely promote further injury to the cardiovascular system and kidneys in patients with diabetes ( , ) . importantly, ace overexpression increases the antihypertensive components of the ras and pretreatment with rhace prevents ang-ii-induced hypertension in preclinical experimental models. however, these results have yet to be validated in human hypertension. while the lung is not considered a target tissue for diabetic complications, covid- patients with diabetes experience worse pulmonary disease than those without diabetes. ace knockout mice exhibit ards pathology. ards triggers multiple pulmonary diseases and is observed in covid- patients. importantly, ace deficiency or treatment with at r blockers of ace /y mice rescues them from ards ( ) . taken together, these studies support that in individuals with diabetes with vascular complications, the loss of the protective ras would serve to intensify sars-cov- -induced pathology. the recent demonstration of sars-cov- actively infecting human enterocytes and the mounting gastrointestinal symptomology implicate gastrointestinal tract pathophysiology in covid- infection ( , ). the digestive system possesses the body's site of greatest relative expression of ace , which in the gut exists as a tetramer with b at (fig. ) . while b at is not expressed in lung pneumocytes, ace :b at complex in the gut acts as a central player in local gut ras and regulates uptake of glucose, sodium, water, and amino acids ( ) ( ) ( ) . however, ace : b at complex internalization by sars-cov- (fig. ) destabilizes the gastrointestinal tract's role in diabetes and blood pressure regulation (fig. ) . b at (slc a ) is the intestine's primary epithelial apical membrane transporter serving na -coupled uptake of neutral amino acids, such as tryptophan. b at was originally discovered and functionally characterized by stevens et al. ( ) , and the transporter was initially named nbb, b, b , or b( ) in the literature but was subsequently called b at . ace chaperones the trafficking of b at to form the stabilized dimer of ace :b at ( ) in the apical membrane (fig. ) . importantly, b at substrates, notably tryptophan and glutamine, signal downregulation of lymphoid proinflammatory cytokines, promote tight junction formation, activate the release of antimicrobial peptides, and modulate mucosal cell autophagy as defense mechanisms. in the models shown in figs. and , binding of sars-cov- s to ace ( ) (fig. ) results in downregulating both intestinal ace and b at , with consequences of disrupting sodium and glucose transport, promoting leaky gut syndrome, elevating plasma bacterial lipopolysaccharide, and enhancing inflammation (figs. and ) . intestine, lumen-facing ace, and ace participate in the food digestion process but are also intertwined in cross talk with gut microbiome metametabolomics of bioactive peptides. such peptides include a balance of agonists and antagonists of enterocyte apical membrane masr and at r, which are physiologically tasked with regulating uptake of dietary na via nhe and glucose absorption via sglt and glut (fig. ). intestinal ace :b at dimer of heterodimers promotes enterocyte na -coupled uptake of phenylalanine, glutamine, tryptophan and its microbiome-generated metabolites, and other neutral amino acid agonists of nutrient-sensing receptors. these stimulate release of glp- and gip into the blood from gut mucosal enteroendocrine l cells (fig. ) ( ) . these incretins circulate to activate pancreatic b-cells, suppress a-cells, and afford brain satiety. sars-cov- infection of gut mucosa results in endocytosis of apical ace , thereby downregulating its activity ( ) , resulting in gut luminal accumulation of at r agonist peptides and disrupting all functions of b at . the dysregulated ras in the bone marrow with its accompanying myeloidosis promotes chronic inflammation that can contribute to both lung and gut pathology (fig. ). an extensive literature supports the concept of communication between the gut and bone marrow. the gut microbiota is a critical extrinsic regulator of hematopoiesis ( ) , as very low concentrations of microbial antigens set the size of the bone marrow myeloid cell pool, and the size of this pool correlates strongly with the complexity of the intestinal microbiota. in turn, bone marrow cells migrate to the gut and impact gut function via changes in blood flow, gut immunity, and epithelial and endothelial tight junction integrity. recruitment of bone marrow-derived immune cell to the gut is necessary for host defense and contributes to inflammation resolution and tissue healing. loss of ace in diabetes results in phylogenetic differences in the gut bacterial community composition with increases in bacteria that have been associated with peptidoglycan generation, which promotes systemic inflammation ( ) . overactivation of bone marrow-derived immune cells including proinflammatory monocytes results in secretion of a large number of harmful cytokines into the circulation that promotes insulin resistance. in the patient with diabetes infected with covid- , developing pneumonia can be devastating, as preexisting systemic inflammation can rapidly lead to multiple organ failure. inflammatory cytokine storm is a notable cause of death in critically ill covid- patients and may be driven as much by gutinduced inflammation as lung injury. thus, imbalance in the bone marrow ras system (fig. ) may represent a central mechanism to not only initiate but also propagate lung and gut injury. from the perspective of gut enterocyte local ras, orally delivered ace inhibitors upregulate expression of both intestinal ace and b at with their attending nutrientsignaled release of glp- , gip, and mucosal antimicrobial peptides ( ) (fig. ) . in a preclinical colitis model, the arb irbesartan restored intestinal b at and ace expression and tryptophan homeostasis with concurrent reduction of intestinal inflammasome activity through an mtor s kinase pathway ( ) . irbesartan further shifted the gut microbiota composition toward favorable taxa and away from stress-related dysbiosis ( ) . activation of enterocyte at r signaled apoptosis with reduced mucosal villus height, while losartan-mediated blockage of gut at r resulted in increased mucosal cell proliferation and reduced apoptosis. increasing gut ace by engineering probiotic species such as lactobacillus paracasei (lp) to express this recombinant protein was a strategy used to prevent microvascular complications in diabetic mice. lp expressing the secretable ace fused with the nontoxic subunit b of cholera toxin (which acts as a carrier to facilitate transmucosal transport), showed increased ace activities in serum and tissues, and reduced diabetic complications ( ) . these results provide proof of concept for feasibility of using engineered probiotic species as a live vector for delivery of decoy hace for possible treatment of enteric covid- infection. rhace given as intravenous medication may be explored as beneficial to covid- patients with pulmonary complication, as it increases pulmonary blood flow and oxygenation in a pig model of lipopolysaccharide-induced ards. supplementation with ace or inhibition of ang-ii improves outcomes in acute lung injury. a pilot trial demonstrated that rhace is well-tolerated in ards patients and showed the anticipated changes in ras peptides. taken together, evidence unequivocally supports the concept that ace is critical in pulmonary function and its imbalance in covid- infection contributes to the devastating lung consequences. an ace activator, diminazene aceturate (dize) is a known antiprotozoal drug used in humans, but it has additional benefits including potent anti-inflammatory the complex comprises a dimer of heterodimers formed by two b at subunits (red) contacting with two ace subunits (green), with each ace interfacing with a single sars-cov- spike (brown). the complex was stabilized using na cotransporter b at transport substrate leucine within the center membrane-spanning domain, known to serve tryptophan, glutamine, and other neutral amino acids in addition to leucine. intestinal apical membranes express the b at :ace complex, which does not occur in lung pneumocytes ( ) . b: side view showing charged moiety interactions in the extracellular region of the gut lumen (top inset) and also hydrophobic interactions of b at tm and tm interacting with the single long transmembrane domain of ace within the apical membrane (bottom inset). data coordinates were obtained from pdb id: m ( ) . ( ) . luminal agonist and antagonist bioactive peptides are derived from interactions of gut digestive enzymes intertwined with microbiome metabolism. oral arbs and ace inhibitor drugs impact gut ras. gut ras governs sodium and glucose uptake via nhe , sglt , and glut . the ace :b at complex dimer of heterodimers ( ) serves the na -coupled transport of neutral amino acids, including tryptophan. in enteroendocrine l cells, basolateral tryptophan stimulates glp- and gip secretion. these incretins maintain gut tight junctions, preventing dysbiosis, stimulate pancreatic b-cells, and blunt a-cells, thereby modulating plasma glucose levels. sars-cov- binding to ace disrupts this homeostasis. and antifibrotic activity. dize has been used in type diabetes to prevent nephropathy and gastric inflammation. dize modulated the ras by reducing serum ang-ii and the expression of at r, but it increased ang- - ( ). dize not only increased ace activity but also increased the expression of ace in select cell types where dize inhibited the expression of il- , il- , and mcp- at both mrna and protein levels following stimulation with lipopolysaccharide. collectively, these results show that dize downregulates proinflammatory cytokine production by many distinct cell types and suggest that this drug may provide benefit to covid- patients by reducing pulmonary inflammation and fibrosis, gut inflammation, and cytokine storm. as the global pandemic unfolds and rapidly spreads, there is an urgent need for basic and clinical studies to address the many unanswered questions posed by covid- . this perspective has directed attention to the disruption of ras in the lung, gastrointestinal tract, and bone marrow as possible mechanisms of sars-cov- disease pathogenesis. the dysregulated ras can potentially impact clinical outcomes in individuals with diabetes resulting in increased morbidity and mortality. ace has emerged as the pleiotropic regulator of the ras, by metabolizing ang-ii into the beneficial peptide ang- - , while being harmful as the sars-cov- receptor. loss of ace indirectly via proteolytic processing, autophagy, and shedding partly could not only drive lung pathology but also gut disease in individuals with diabetes infected with covid- . sars-cov- , by downregulating intestinal ace -b at , could promote leaky gut syndrome with elevated plasma bacterial lipopolysaccharides and/or peptidoglycans enhancing systemic inflammation. careful targeting of the ras axis may represent a strategy for improving clinical outcomes in subjects with diabetes infected with covid- . ace , a pleiotropic regulator of the ras, is hijacked as a receptor for sars-cov- to promote viral infection. loss of ace indirectly via proteolytic processing, autophagy, and adam -mediated shedding (not shown) partly drives not only lung but also gut disease in individuals with diabetes with covid- . sars-cov- s binding to ace initiates internalization of ace :b at complex (gut) or ace (outside of gut). thus, sars-cov- by downregulating intestinal ace -b at would promote leaky gut syndrome with elevated plasma bacterial lipopolysaccharides and/or peptidoglycans enhancing systemic inflammation. in the lung, virus internalization also promotes a reduction in ace that results in pulmonary pathology. careful targeting of the ras axis will likely optimize clinical outcomes in subjects with diabetes infected with sars-cov- . wbc, white blood cell. china medical treatment expert group for covid- . clinical characteristics of coronavirus disease in china clinical characteristics of patients infected with sars-cov- in wuhan clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study prevalence and impact of diabetes among people infected with sars-cov- covid- testing, hospital admission, and intensive care among , , united states veterans aged - years clinical and biochemical indexes from -ncov infected patients linked to viral loads and lung injury sars-cov- receptor and regulator of the renin-angiotensin system: celebrating the th anniversary of the discovery of ace tumor necrosis factor-alpha convertase (adam ) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme- (ace ) tmprss and adam cleave ace differentially and only proteolysis by tmprss augments entry driven by the severe acute respiratory syndrome coronavirus spike protein hca lung biological network. sars-cov- entry factors are highly expressed in nasal epithelial cells together with innate immune genes sars-cov- receptor ace and tmprss are primarily expressed in bronchial transient secretory cells digestive system is a potential route of covid- : an analysis of single-cell coexpression pattern of key proteins in viral entry process sars-cov- : olfaction, brain infection, and the urgent need for clinical samples allowing earlier virus detection sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor structure, function, and antigenicity of the sars-cov- spike glycoprotein structural and functional basis of sars-cov- entry by using human ace structural basis of receptor recognition by sars-cov- structural basis for the recognition of sars-cov- by full-length human ace structure of the sars-cov- spike receptor-binding domain bound to the ace receptor characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov the spike glycoprotein of the new coronavirus -ncov contains a furin-like cleavage site absent in cov of the same clade quantifying sars-cov- transmission suggests epidemic control with digital contact tracing plasma levels of the proprotein convertase furin and incidence of diabetes and mortality targeting the endocytic pathway and autophagy process as a novel therapeutic strategy in covid- skp attenuates autophagy through beclin -ubiquitination and its inhibition reduces mers-coronavirus infection autophagy in metabolic syndrome: breaking the wheel by targeting the renin-angiotensin system human intestinal defensin inhibits sars-cov- invasion by cloaking ace a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury renin-angiotensin system blockers and the covid- pandemic: at present there is no evidence to abandon renin-angiotensin system blockers angiotensin-converting enzyme (cd ) marks hematopoietic stem cells in human embryonic, fetal, and adult hematopoietic tissues a role for the renin-angiotensin system in hematopoiesis activation of the ace / angiotensin-( - )/mas receptor axis enhances the reparative function of dysfunctional diabetic endothelial progenitors loss of angiotensin-converting enzyme exacerbates diabetic retinopathy by promoting bone marrow dysfunction cardiac involvement in a patient with coronavirus disease (covid- ) association of cardiac injury with mortality in hospitalized patients with covid- in wuhan, china cardiovascular implications of fatal outcomes of patients with coronavirus disease (covid- ) kidney disease is associated with in-hospital death of patients with covid- angiotensin converting enzyme : a doubleedged sword sars-cov- productively infects human gut enterocytes human intestine luminal ace and amino acid transporter expression increased by ace-inhibitors imbalance of the renin-angiotensin system may contribute to inflammation and fibrosis in ibd: a novel therapeutic target? the meaning of mas multiple transport pathways for neutral amino acids in rabbit jejunal brush border vesicles glutamine triggers and potentiates glucagon-like peptide- secretion by raising cytosolic ca and camp renin-angiotensin-aldosterone system inhibitors in patients with covid- microbiota-derived compounds drive steady-state granulopoiesis via myd /ticam signaling bone marrow-derived cells restore functional integrity of the gut epithelial and vascular barriers in a model of diabetes and ace deficiency angiotensin receptor blocker irbesartan reduces stress-induced intestinal inflammation via at a signaling and ace -dependent mechanism in mice expression of human ace in lactobacillus and beneficial effects in diabetic retinopathy in mice ace x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis key: cord- -fsumpj n authors: kintscher, ulrich; slagman, anna; domenig, oliver; röhle, robert; konietschke, frank; poglitsch, marko; möckel, martin title: plasma angiotensin peptide profiling and ace (angiotensin-converting enzyme)- activity in covid- patients treated with pharmacological blockers of the renin-angiotensin system date: - - journal: hypertension doi: . /hypertensionaha. . sha: doc_id: cord_uid: fsumpj n nan p harmacological blockade of the renin-angiotensin system (ras) with ace (angiotensin-converting enzyme) inhibitors or angiotensin type receptor blockers (arb) reduces morbidity and mortality in various cardiovascular diseases. one of the key ras-modulating enzymes, ace , has recently gained increasing attention because it converts not only angiotensin (ang) ii to the alternative ras metabolite ang-( - ) but also functions as the cellular entry receptor for sars-cov- . at the beginning of the sars-cov- pandemic, some investigators suggested that because ace inhibitor or arb may lead to upregulation of ace expression/activity, use of these agents in coronavirus disease (covid- ) patients might be associated with worsened outcomes. meanwhile, several observational studies have shown that neither the risk of covid- nor its severity is negatively affected by ace inhibitor or arb. , however, it remains unclear how ras activity, particularly ace , is regulated in covid- and how this is altered by ace inhibitor/arb therapy. in this study, we analyzed distinct ras components in plasma from covid- patients±ace inhibitor/arb therapy using lc-ms/ms. the study was approved by the charité-universitätsmedizin, berlin, germany, institutional ethics committee (ea / / , amendment ) and registered in the german registry for clinical studies (drks ). surplus plasma samples were collected at the time of admission to the emergency room from different patient groups (total, n= [women, ]): sars-cov- negative control group (ctrl, n= [ ] ), sars-cov- negative with ace inhibitor (ctrl-ace inhibitor, n= [ ] ), sars-cov- negative with arbs (ctrl-arb, n= [ ] ), covid- without ace inhibitor/arb (covid, n= [ ] ), covid- with ace inhibitor (covid-ace inhibitor, n= [ ] ), and covid- with arbs (covid-arb, n= [ ] ). equilibrium levels of ang-peptides (ang i, ang ii, ang- [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and ang- [ ] [ ] [ ] [ ] [ ] ) were measured using lc-ms/ms technology (attoquant diagnostics). ang-based markers for ace (ang ii/ang i) and plasma renin activity (ang i+ang ii) were calculated from ang-peptide levels. ace activity was assayed by a classical kinetic approach applying its natural substrate (ex vivo spiked ang ii) and measuring the turnover to ang-( - )±ace inhibitor mln- . the inhibitor-sensitive ace -specific turnover was converted to an ace concentration using a calibration curve of recombinant human ace . ang-peptide concentrations/ratios, ace activity, and age between groups were compared using the kruskal-wallis test. in case of a significant result, the dunn-test for pairwise comparisons using bonferroni correction was applied. a p of < . was considered statistically significant, although results have to be considered exploratory. ang-peptide equilibrium concentrations did not significantly differ between the ctrl and covid groups without ace inhibitor/arb treatment (figure [b] , left). more importantly, ang i+ii, ang ii/ang i, and ace activity were not significantly different between both groups (figure [c] ). these data suggest that covid- patients are not those with increased ras activity levels and that particularly covid- -induced alternative ras activation, potentially mediated through circulating ace , is not a typical feature in our patient cohort. november comparison of all groups, including ace inhibitor/arb treatment groups, revealed no significant differences of ang i+ii levels between the groups (figure [c] , upper left). ang i+ii is a reliable marker for plasma renin activity and did not change significantly, despite the use of ace inhibitor/ arb, while median values were clearly increased in patients on ace inhibitor/arb. this is consistent with previous observations demonstrating a broad spectrum of intensity in compensatory renin secretion in patients treated with ace inhibitor or arb. as expected, patients in the ctrl-ace inhibitor and covid-ace inhibitor group showed increased ang i and markedly suppressed ang ii levels (figure [b] ), resulting in a significant reduction of the ang ii/ang i ratio (figure [c] , lower left). ang-( - ) levels did not significantly differ between groups, whereas ang-( - ) was significantly increased in the covid-ace inhibitor group versus covid without ace inhibitor/arb (p= . ) and versus covid-arb (p= . ). ace activity was significantly higher in covid- patients treated with ace inhibitor compared with covid- patients without ace inhibitor/ arb (figure [c] , right). ace activity was also increased in the ctrl-ace inhibitor and ctrl-arb group but did not reach statistical significance (figure [c] , right). arb treatment in covid- did not significantly affect ace activity (figure [c] , right). the main findings of this study are as follows: ( ) covid- patients are not characterized by major changes in ras activity in plasma including ace activity, ( ) ace inhibitor therapy significantly suppressed ang ii/ang i ratios, the angbased marker for ace, in covid- and in non-covid- patients, and ( ) plasma ace activity is increased in covid- patients treated with ace inhibitor. these data are consistent with previously published results in sars-cov- -negative patients treated with ace inhibitor or arb demonstrating an ang ii/ang i suppression and a more profound increase of ang-( - ) under ace inhibitor compared with arbs. the data published so far on plasma ace activity and ang-( - ) levels in patients without covid treated with ace inhibitor or arbs are controversial. some studies showed an increase in circulating ace activity and ang-( - ) levels that cannot be proven by other studies. in addition, increased ace activity has been identified in multiple cardiovascular diseases such as hypertension, cad, and chf, which are usually treated with ace inhibitor. whether the ace inhibitor treatment in our study plays a role in ace upregulation or whether these changes are mediated by the increased presence of cardiovascular disease in this group requires further investigation. furthermore, the clinical significance of the elevated ace activity in covid- patients treated with ace inhibitor is currently not completely understood. whether plasma ace level may be a reliable marker of the full-length membrane bound form and whether ace serves as a marker for disease severity or endothelial regeneration in the lung need to be clarified in future studies. some of the major limitations of this study include small sample sizes, lack of a power analysis, lack of any data on blood pressure when the plasma samples were obtained, and lack of any data on duration of illness. finally, it should be emphasized that the majority of the study patients were not experiencing severe covid- . however, we provide for the first time a snapshot of distinct systemic ras components in covid- patients under ace inhibitor/ arb therapy that helps to understand the clinical data on a molecular pharmacological level. renin-angiotensin-aldosterone system inhibitors in patients with covid- reninangiotensin-aldosterone system blockers and the risk of covid- renin-angiotensin-aldosterone system inhibitors and risk of covid- angs (angiotensins) of the alternative renin-angiotensin system predict outcome in patients with heart failure and preserved ejection fraction pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid- key words: angiotensin ii type receptor blockers ◼ angiotensinconverting enzyme ◼ cardiovascular diseases ◼ peptide fragments ◼ renin-angiotensin system we thank fabian holert, jana eberst, and beata hoeft for the support with sample preparation/handling and clinical data collection. key: cord- -fkk ry authors: jing, yan; run-qian, li; hao-ran, wang; hao-ran, chen; ya-bin, liu; yang, gao; fei, chen title: potential influence of covid- /ace on the female reproductive system date: - - journal: mol hum reprod doi: . /molehr/gaaa sha: doc_id: cord_uid: fkk ry the novel coronavirus ( -ncov) appeared in december and then spread throughout the world rapidly. the virus invades the target cell by binding to angiotensin-converting enzyme (ace) and modulates the expression of ace in host cells. ace , a pivotal component of the renin-angiotensin system, exerts its physiological functions by modulating the levels of angiotensin ii (ang ii) and ang-( - ). we reviewed the literature that reported the distribution and function of ace in the female reproductive system, hoping to clarify the potential harm of -ncov to female fertility. the available evidence suggests that ace is widely expressed in the ovary, uterus, vagina and placenta. therefore, we believe that apart from droplets and contact transmission, the possibility of mother-to-child and sexual transmission also exists. ang ii, ace and ang-( - ) regulate follicle development and ovulation, modulate luteal angiogenesis and degeneration, and also influence the regular changes in endometrial tissue and embryo development. taking these functions into account, -ncov may disturb the female reproductive functions through regulating ace . corona virus disease is an emerging acute communicable disease that was identified in patients with pneumonia in december , which was declared a pandemic by the world health organization on th march, . a total of , , laboratory-confirmed cases and , deaths have been reported as of th april, (world health organization, . epidemiologically, the genome of the novel coronavirus ( -ncov) is composed of , nucleotides, with an % identity to bat sars-like-covzxc and % to human sars-cov (chan et al., ) . -ncov infects the target cell by binding to angiotensin-converting enzyme (ace) through its surface spike protein zhou et al., ) , modulates the expression of ace and causes severe injuries, similar to sars-cov (kuba et al., ; wang and cheng, ) . ace is a zinc metalloprotease which shares homology with ace in its catalytic domain (donoghue et al., ) , and is composed of amino acids including a -amino acid n-terminal signal sequence and a c-terminal membrane binding domain (tipnis et al., ) . ace contains a single hexxh zinc-binding motif and is able to hydrolyze angiotensin i (ang i) to produce angiotensin-( - ) and also has a high affinity for angiotensin ii (ang ii) to generate ang-( - ) (vickers et al., ) . ang ii, the major component of the ace/ang ii/at (angiotensin ii type ) axis, facilitates vasoconstriction, promotes cell proliferation (bataller et al., ; campbell-boswell and robertson, ; hiruma et al., ; ray et al., ) , and maintains the hydro-salinity balance (hall et al., ; johnson and malvin, ) . as an important modulator of the human renin-angiotensin system, ang-( - ) is an endogenous ligand for the g protein-coupled receptor mas and specifically inhibits ang ii by the antagonism of at receptors (roks et al., ) . moreover, ang-( - ) enhances vasodilation (brosnihan et al., ; oliveira et al., ) , protects the heart (ferreira et al., ; iwata et al., ; santos et al., ) and alleviates metabolic syndrome (giani et al., ; liu et al., ) . evidence has been accumulating that besides lung injury, -ncov also damages the human heart zheng et al., ) , liver (chen et al., c; zhang et al., b) , kidney (chen et al., c; huang et al., ; and nervous system (li et al., c; mao et al., ) . recently, cases of covid- during pregnancy have been reported liu et al., ; zhu et al., ) , but the influence of -ncov on the female reproductive system needs further investigation. in this review, we analyzed the distribution and function of ace , trying to predict the possible targets and transmission routes, as well as the influence on the female reproductive system, of -ncov. ace presents in stroma and granulosa cells as well as oocytes in immature rat ovaries, the expression of which is enhanced in antral and preovulatory follicles subjected to equine cg treatment (pereira et al., ) . in bovine theca cells and granulosa cells, ace also exists (barreta et al., ; tonellotto dos santos et al., ) . notably, ace mrna transcripts were detected in ovaries from reproductive-age women and postmenopausal women . we analyzed ace data from the genecards (https://www.genecards.org/cgi-bin/carddisp.pl?gene=ace #protein_expression) database, and found that ace is most abundantly expressed in the ovary. in the meantime, data obtained from bgee (https://bgee.org/?page=gene&gene_id=ensg ) showed that the expression level of ace in oocytes is relatively high. therefore, the ovary and oocyte might be potential targets of -ncov. ace is the key enzyme in the axis that plays a synergistic role in balancing the levels of ang ii and ang-( - ). ang ii induces steroid secretion (hayashi et al., ; shuttleworth et al., ) , facilitates follicle development (ferreira et al., ; shuttleworth et al., ) and oocyte maturation (giometti et al., ; stefanello et al., ; yoshimura et al., ) , contributes to follicular atresia (kotani et al., ; obermuller et al., ; tanaka et al., ) , influences ovulation (acosta et al., ; ferreira et al., ; guo et al., ; kuji et al., ; kuo et al., ; miyabayashi et al., ; pellicer et al., ; yoshimura et al., ; yoshimura et al., ) and maintains corpus luteum progression (sugino et al., ) . ang-( - ) promotes the production of estradiol and progesterone (costa et al., ) and enhances ovulation (muthalif et al., ; tonellotto dos santos et al., ; viana et al., ) and the resumption of meiosis in the oocyte . a recent study showed that the level of ang-( - ) is also associated with the maturation of human oocytes (cavallo et al., ) . ace mrna has been identified in the uterus of human (vaz-silva et al., ) and rat (brosnihan et al., ) . vaz-silva et al. ( ) claimed that ace mrna is more abundant in epithelial cells than in stromal cells, and higher in the secretory phase than in the proliferative phase (vaz-silva et al., ) . moreover, we confirmed the presence of ace in uterus and vagina after analyzing the data from the human protein atlas (https://www.proteinatlas.org/ensg -ace /tissue) and genecards. noteworthy is the report of a high infection rate among sexual partners of -ncov-positive females (cui et al., ) , suggesting the possibility of sexual transmission. however, the confirmation of sexual transmission still needs extensive investigation. ang ii plays a dual role in vascular bed and endometrium regeneration, and initiates menstruation through spiral artery vasoconstriction (ahmed et al., ; ahmed, a, ) . the balance between ang ii and ang-( - ) could regulate the regeneration of endometrium (vaz-silva et al., ) and myometrium activity (deliu et al., ; vaz-silva et al., ) . moreover, ang ii increases the proliferation of uterus epithelial and stroma cells and enhances endometrial fibrosis, an effect that can be inhibited by ang-( - ) (hering et al., ; shan et al., ; shan et al., ) . notably, the normal function of ang ii in endometrium is necessary for regular menstrual cycles, and alterations in its distribution and the level of the receptors may be related to dysfunctional uterine bleeding associated with hyperplastic endometria (li and ahmed, b) . furthermore, many authors have described in the literature that intense ace and ang ii expression correlates with the metastasis and prognosis of endometrial carcinoma (delforce et al., ; shibata et al., ; watanabe et al., ) , and highlighted that the increased ace /ang-( - )/mas/at r pathway activity in endometrial cancer can be an important mechanism to counteract the actions of ang ii/at r (abdalla et al., ; kostenis et al., ) . ace is widely expressed in human placenta (valdes et al., ) . in placental villi, ace is mainly expressed in the syncytiotrophoblast, cytotrophoblast, endothelium and vascular smooth muscle of primary and secondary villi. in the maternal stroma, ace is expressed in the invading and intravascular trophoblast and in decidual cells. ace is also found in arterial and venous endothelium and smooth muscle of the umbilical cord (valdes et al., ) . of note, ace reaches the highest level in early gestation (pringle et al., ) . during early gestation, ace is expressed in the primary and secondary decidual zone, and in luminal and glandular epithelial cells. during late gestation, ace staining is visualized in the labyrinth placenta, and amniotic and yolk sac epithelium (ghadhanfar et al., ; neves et al., ) . moreover, ace in placenta of rat begins to increase from mid-gestation (vaswani et al., ) . according to the genecards, the expression of ace in the placenta is greater than that detected in the lung, suggesting a possibility of viral infection of the placenta. recently, early-onset -ncov infection was identified in infants whose nasopharyngeal and anal swabs were positive on day and of life (zeng et al., ) , and a neonate born to a mother with covid- had elevated igm antibodies at hours after birth (dong et al., ) . given that the identification of -ncov in human airway epithelial cells requires at least hours of culture (national health commission of the people's republic of china, ), we speculate that intrauterine infection with -ncov may appear and the fetuses may be infected during gestation. additionally, the human protein atlas and genecards database showed the presence of ace in female breasts. wu et al. ( ) claimed that of samples of breast milk was positive for -ncov in nucleic acid testing (wu, ) , indicating the chance of transmission through breastfeeding. even if there is no virus in milk, contact transmission during breastfeeding should be taken into account. given the weaker immune system of newborns, we advise that pregnant patients who are confirmed as positive for -ncov should carry out artificial feeding instead, or start breastfeeding after a -day isolation following recovery and discharge. concurrently, considering its benefits in decreasing respiratory tract and gastrointestinal tract infections, sudden infant death syndrome and diabetes of the infants (section on breastfeeding, ), breastfeeding might not be completely forbidden. nevertheless, ferrazzi et al. ( ) reported that when breastfed by two postpartum women diagnosed with covid- and wearing no masks, the newborns tested positive (ferrazzi et al., ) . we strongly support that all possible precautions should be taken to avoid spreading the virus to the infant, including washing hands before touching the infant and wearing a face mask during breastfeeding (baud et al., ) . however, these precautions may not be strictly adhered to, hence increasing the risk of infection in the infants. therefore, mothers who intend to breastfeed are encouraged to use a dedicated breast pump, and after each pumping session, the breast pump should be appropriately disinfected. during pregnancy, ang ii, ace and ang-( - ) function mainly through regulating blood pressure and fetus development. meanwhile, they also interact to maintain normal uterine physiology. ang ii stimulates trophoblast invasion in rat and human cells (hering et al., ) . ang-( - ) and ace may act as a local autocrine/paracrine regulator in the early (angiogenesis, apoptosis, and growth) and late (uteroplacental blood flow) events of pregnancy . ace hydrolyzes ang ii into ang-( - ), and ang i into ang-( - ), which is quickly converted to ang-( - ) and thereby controlling the blood pressure and hydro-salinity balance of pregnant women (pringle et al., ) . the aberrant expression of ang ii, ace and ang-( - ) may be involved in hypertension of pregnancy, pre-eclampsia and eclampsia (anton et al., ; anton et al., ; brosnihan et al., ; merrill et al., ; sykes et al., ; yamaleyeva et al., ) . brosnihan et al. ( ) described that pre-eclamptic women presented with suppressed plasma ang-( - ) levels when compared with normal pregnancy subjects (brosnihan et al., ) . high expression of ang ii in the placental villus during pre-eclampsia causes a decreased blood flow and nutrition supply in fetuses (anton et al., ; anton et al., ; shibata et al., ) . meanwhile, low levels of ace and ang-( - ) in placenta are associated with intrauterine growth restriction (ghadhanfar et al., ) . in gestational ace -/mice, plasma ang-( - ) decreases and placental ang ii increases, accompanied by abnormal placental functions (including placental hypoxia and uterine artery dysfunction) and ultimately fetal growth retardation (bharadwaj et al., ; yamaleyeva et al., ) . moreover, chen et al. ( ) found that the maternal ang-( - )/ang ii ratio is independently associated with gestational hypertension or pre-eclampsia, factors causing preterm birth . additionally, it has been shown that the up-regulation of ace /ang-( - )/mas prevents premature birth (lumbers, ) . it is noteworthy that premature birth and intrauterine growth restriction may predict the cardiovascular disorders that appear in adulthood (irving et al., ) . bessa et al. ( ) reported that activation of the ace /ang-( - )/mas axis in hypertensive pregnant rats could attenuate the cardiovascular dysfunction in adult offspring (bessa et al., ) , confirming the engagement of the ace axis in pregnancy. -ncov infection poses a great threat to pregnant women and fetuses, causing premature birth ( . %, / ), fetal distress ( . %, / ), premature rupture of fetal membranes ( . %, / ) and cesarean section ( . %, / ) chen et al., b; ferrazzi et al., ; li et al., a; liu et al., ; zeng et al., ; zhu et al., ) . the considerable cesarean section rate is mainly due to the concern about -ncov and obstetrical indications (chen et al., b) . it is worth mentioning that current data are still insufficient and some reports lack concrete details. therefore, whether it is -ncov/ace that causes the placental dysfunction remains elusive and needs further evaluation. moreover, just like sars-cov patients, patients infected with -ncov also demonstrate complicated acute renal impairment, renal dysfunction and renal failure (chu et al., ; fan et al., ; li et al., b; li et al., d; zhang et al., a) . pacciarini et al. ( ) found that sars-cov infects human tubular kidney cells (pacciarini et al., ) . of note is that ace level in the renal tubules of pregnant mice increases by % compared to non-pregnant mice, which may contribute to the maintenance of blood pressure (brosnihan et al., ) . we suppose that pregnant women with covid- may be susceptible to renal injury. -ncov may infect the ovary, uterus, vagina and placenta through the ubiquitous expression of ace . moreover, -ncov/ace may disturb the female reproductive functions, resulting in infertility, menstrual disorder and fetal distress. we suggest a following-up and evaluation of fertility after recovery from -ncov infection, and delaying becoming pregnant, if possible, especially for young female patients. moreover, we should persistently pay close attention to the situation of pregnant patients as well as fetuses, and take timely measures. what is more, to decrease the incidence of -ncov infection, special nursing should be conducted for 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hospitalized patients with covid- in wuhan, china: a retrospective case series study angiotensin-( - ) in normal and preeclamptic pregnancy changes of mrna expression of vascular endothelial growth factor, angiopoietins and their receptors during the periovulatory period in ecg/hcg-treated immature female rats signal transduction mechanisms involved in angiotensin-( - )-stimulated arachidonic acid release and prostanoid synthesis in rabbit aortic smooth muscle cells national health commission of the people's republic of china. the notice of launching guideline on diagnosis and treatment of the novel coronavirus pneumonia ace and ang-( - ) in the rat uterus during early and late gestation immunohistochemical and mrna localization of the angiotensin ii receptor subtype (at ) in follicular granulosa cells of the rat ovary synergistic effect of angiotensin-( - ) on bradykinin arteriolar dilation in vivo persistent replication of severe acute respiratory syndrome coronavirus in human tubular kidney cells selects for adaptive mutations in the membrane protein blockage of ovulation by an angiotensin antagonist gonadotropin stimulation increases the expression of angiotensin-( -- ) and mas receptor in the rat ovary the expression and localization of the human placental prorenin/renin-angiotensin system throughout pregnancy: roles in trophoblast invasion and angiogenesis? angiotensin ii receptor-mediated proliferation of cultured human fetal mesangial cells angiotensin-( - ), its receptor mas, and the angiotensin-converting enzyme type are expressed in the human ovary angiotensin-( - ) is a modulator of the human renin-angiotensin system expression of an angiotensin-( - )-producing fusion protein produces cardioprotective effects in rats angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas section on breastfeeding. breastfeeding and the use of human milk effect of angiotensin-( - ) and angiotensin ii on the proliferation and activation of human endometrial stromal cells in vitro angiotensin-( - ) and angiotensin induce the transdifferentiation of human endometrial epithelial cells in vitro angiotensin ii decreases system a amino acid transporter activity in human placental villous fragments through at receptor activation possible involvement of adipocyte-derived leucine aminopeptidase via angiotensin ii in endometrial carcinoma in vitro development of pig preantral follicles cultured in a serum-free medium and the effect of angiotensin ii effect of angiotensin ii with follicle cells and insulin-like growth factor-i or insulin on bovine oocyte maturation and embryo development angiogenesis in the human corpus luteum: changes in expression of angiopoietins in the corpus luteum throughout the menstrual cycle and in early pregnancy fetal sex and the circulating renin-angiotensin system during early gestation in women who later develop preeclampsia or gestational hypertension characterization of angiotensin ii receptor type during differentiation and apoptosis of rat ovarian cultured granulosa cells a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase molecular characterization and regulation of the angiotensin-converting enzyme type /angiotensin-( - )/mas receptor axis during the ovulation process in cattle distribution of angiotensin-( - ) and ace in human placentas of normal and pathological pregnancies the rat placental renin-angiotensin system -a gestational gene expression study the vasoactive peptide angiotensin-( - ), its receptor mas and the angiotensin-converting enzyme type are expressed in the human endometrium tissue specific localization of angiotensin-( - ) and its receptor mas in the uterus of ovariectomized rats angiotensin-( - ) induces ovulation and steroidogenesis in perfused rabbit ovaries hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in increasing host cellular receptor-angiotensin-converting enzyme (ace ) expression by coronavirus may facilitate -ncov infection adipocyte-derived leucine aminopeptidase suppresses angiogenesis in human endometrial carcinoma via renin-angiotensin system world health organization. coronavirus disease (covid- ) situation reports viral shedding of covid- in pregnant women intraovarian actions of anti-angiogenic agents disrupt periovulatory events during the menstrual cycle in monkeys local delivery of angiopoietin- into the preovulatory follicle terminates the menstrual cycle in rhesus monkeys hemodynamic responses to angiotensin-( - ) in women in their third trimester of pregnancy uterine artery dysfunction in pregnant ace knockout mice is associated with placental hypoxia and reduced umbilical blood flow velocity angiotensin ii directly induces follicle rupture and oocyte maturation in the rabbit locally produced angiotensin ii induces ovulation by stimulating prostaglandin production in in vitro perfused rabbit ovaries neonatal early-onset infection with sars-cov- in neonates born to mothers with covid- in wuhan, china clinical characteristics of death cases with covid- liver injury in covid- : management and challenges covid- and the cardiovascular system a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical analysis of neonates born to mothers with -ncov pneumonia this work was supported by national natural science foundation of china (no. ). none declared. key: cord- - j m authors: campbell, duncan j. john title: ace inhibition in heart failure and ischaemic heart disease date: journal: frontiers in research of the renin-angiotensin system on human disease doi: . / - - - - _ sha: doc_id: cord_uid: j m nan angiotensin converting enzyme (dipeptidyl carboxypeptidase i, kininase ii, ec . . . , ace) plays a major role in the metabolism of many different peptides, including angiotensin (ang) i, bradykinin, kallidin, and n-acetyl-seryl-aspartyllysyl-proline (acsdkp). ace inhibitors are established therapy for heart failure and ischaemic heart disease, and alterations of ang ii, bradykinin, kallidin, and acsdkp peptide levels are implicated in the mechanisms of this therapy. this chapter briefly describes the renin angiotensin, kallikrein kinin, and acsdkp systems, and their role in cardiovascular physiology and disease. the role of ace inhibition in treatment and prevention of heart failure and ischaemic heart disease is summarised, and the possible mechanisms of the therapeutic benefits of ace inhibitors are described. this is not an exhaustive review, but focuses on those aspects most relevant to the clinical application of ace inhibitors. figure shows an outline of the pathways of ang peptide formation and metabolism. in addition to the classical pathway involving renin and ace, alternative pathways have been proposed (campbell ) . there remain many questions concerning the mechanisms of ang peptide formation in discrete tissue compartments such as the heart. serine proteases, for example, may form ang ii by processes independent of renin at sites of inflammation or coagulation, where kallikrein and/or cathepsin g may be active. figure . pathways of ang peptide formation and metabolism. adapted from (campbell ) studies of nephrectomised animals show the main mechanism of ang peptide formation in the heart involves kidney-derived renin (campbell et al ; danser et al ) . renin messenger rna (mrna) levels in the heart are very low or undetectable (de mello et al ) . cardiac renin expression may, however, be induced by myocardial infarction and macrophages and myofibroblasts may express renin at the site of repair (sun et al ) . all ang peptides are derived from angiotensinogen. although angiotensinogen may be produced in low levels in the heart (dostal et al ; paul et al ) , plasma is the main source of angiotensinogen for ang peptide formation in the heart. ace is a membrane-bound zinc-containing metallopeptidase, some of which is cleaved from membranes and released as soluble ace found in plasma and other fluids (erdos ). ace has two catalytic domains with differential substrate specificities and susceptibility to ace inhibitors (wei et al ; wei et al ; jaspard et al ) . table lists the many substrates of ace. those ace substrates most related to cardiac function are ang i, the bradykinin and kallidin peptides, and acsdkp. both catalytic domains of ace posses dipeptidyl carboxypeptidase and endopeptidase activities and can cleave ang i, bradykinin-( - ), bradykinin-( - ), and substance p. however, the n-terminal catalytic domain cleaves of lutein- bradykinin-( - ), bradykinin-( - ), and bradykinin-( - ) lys -bradykinin-( - ) (kallidin), lys -bradykinin-( - ), and lys -bradykinin-( - ) substance p n-acetyl-seryl-aspartyl-lysyl-proline (acsdkp) chemotactic peptide neurotensin luteinising hormone-releasing hormone (lh-rh) enkephalins cholecystokinin gastrin adapted from (ehlers et al ; erdos ; hooper ; rieger et al ) ising hormone-releasing hormone (lh-rh) and acsdkp more efficiently than the c-terminal domain (jaspard et al ; rousseau et al ) . the two catalytic domains of ace interact differently with ace inhibitors. captopril, enalapril, lisinopril, and trandolapril are all highly potent inhibitors of both domains. whereas trandolapril, lisinopril and enalapril show preference for the c-terminal catalytic domain, captopril shows preference for the n-terminal catalytic domain (wei et al ) . ace has a widespread tissue distribution, including vascular endothelium and smooth muscle cells, the brush border of proximal tubule cells of the kidney, and the brain (erdos ) . ace is expressed by the endothelium of the coronary vasculature, and by the endocardium and epicardium, but not by the valves in the human heart (dostal et al ) . ace is also expressed by cardiac fibroblasts, and fibroblast expression of ace is increased in the border zone of myocardial infarction (dostal et al ; burrell et al ) . cardiac ace expression is up-regulated in heart failure (hirsch et al ; studer et al ) . many different cell types express ang receptors in the heart. the type ang (at ) receptor is expressed by coronary smooth muscle and endothelial cells, cardiomyocytes, fibroblasts, nerves, and conduction tissue (regitz-zagrosek et al ) . at receptors are expressed by fibroblasts and endothelial cells (regitz-zagrosek et al ) . in heart failure, cardiomyocyte at receptor expression may be down-regulated, whereas fibroblast expression of both at and at receptors is increased (ohkubo et al ) . the at receptor mediates most of the known actions of ang ii. there is continuing uncertainty about the role of the at receptor, which may mediate actions of ang ii in the vasculature and heart that differ from those of the at receptor (carey et al ; voros et al ) . the at receptor is described further by danser in chapter of this volume. human heart chymase was initially discovered in homogenates of human heart and proposed to be the major pathway of conversion of ang i to ang ii in the heart (urata et al ) . given that chymase is not inhibited by ace inhibitors, it represented a potential pathway of continued ang ii formation in patients taking ace inhibitor therapy (dell'italia et al ) , and thereby provided a rationale for a possible superiority of at receptor blocker (arb) therapy over ace inhibitor therapy. however, studies of the effects of ace inhibition in rats, mice, and humans, and of ace gene knockout in mice, show ace is the dominant pathway of ang ii formation in the heart (campbell et al ; campbell et al ; zeitz et al ; campbell et al a) . ace-related carboxypeptidase (ace ), like ace, is a membrane-associated and secreted metalloprotease expressed predominantly on endothelium (donoghue et al ; tipnis et al ; hamming et al ) . ace is expressed in all human tissues, with relatively high levels in renal and cardiovascular tissues, and also in the gut (harmer et al ) . in contrast to the dipeptidyl carboxypeptidase activity of ace, ace cleaves ang i to ang-( - ) and also cleaves ang ii to ang-( - ). ace is not inhibited by ace inhibitors. kinetic considerations make it unlikely that ace contributes to ang i metabolism in vivo (jaspard et al ; vickers et al ) . ace and ace have similar k m for ang i ( and . μmol/l, respectively) but the k cat for ace ( s − ) is approximately -fold higher than that for ace ( . s − ), such that the k cat /k m ratio is approximately -fold higher for ace ( . x l/mol per s) than for ace ( . x l/mol per s). by contrast, the k m ( μmol/l), k cat ( . s − ), and k cat /k m ratio ( . x l/mol per s) of ace for ang ii (vickers et al ) make it more likely to participate in ang ii metabolism. initial genetic studies suggested an important role for ace in ang peptide metabolism in the heart. the ace gene knockout mouse was reported to have a cardiomyopathic phenotype associated with increased ang ii levels in plasma, heart, and kidney. additionally, the cardiomyopathic phenotype was ameliorated by concomitant ace gene knockout, suggesting that altered ang peptide metabolism contributed to the phenotype (crackower et al ) . in subsequent studies the ace gene knockout mouse had a normal cardiac phenotype, although it had an enhanced pressor response to ang ii administration (gurley et al ) . ace activity is reported to be increased in the hearts of patients with heart failure (zisman et al ) . however, measurement of ang peptides in coronary venous blood of patients with heart failure or ischaemic heart disease does not support an important role for ace in either ang i or ang ii metabolism in the human heart (campbell et al b) . elucidation of the role of ace in ang ii metabolism must await the development of specific ace inhibitors. effects of the ras on the heart and vasculature both systemic and local actions of ang ii impact on the heart. systemic actions of ang ii include its vasoconstrictor action to increase blood pressure and the stimulation of aldosterone secretion. increased aldosterone levels may produce hypokalaemia and contribute to cardiac fibrosis (brilla et al ) . local cardiac actions of ang ii include inotropic and hypertrophic effects, and cardiac remodelling (paul et al ) . at receptor stimulation induces both myocyte hypertrophy and collagen synthesis (regitz-zagrosek et al ) . moreover, ang ii may contribute to oxidative stress, inflammation, and thrombosis (dzau ; duprez ) . at -mediated nadph oxidase activation leads to generation of reactive oxygen species, widely implicated in vascular inflammation and fibrosis (li et al ; mehta et al ) . ang ii also activates gene transcription factors involved in vascular inflammation and remodelling (oettgen ) . ang ii and its metabolite ang iv may promote thrombosis by stimulating plasminogen activator inhibitor type (pai- ) and pai- production by the vasculature (van leeuwen et al ; feener et al ; kerins et al ) . additionally, ang ii may promote thrombosis by activation of nuclear factor b-dependent proinflammatory genes and accelerating vascular expression of tissue factor (dielis et al ) . ang ii stimulates endothelin release (kohno et al ; moreau et al ) and endothelin blockade prevents some of the cardiovascular actions of ang ii (webb et al ; rajagopalan et al ; herizi et al ) . ang-( - ) is a biologically active peptide (ferrario et al ) . the main pathway of ang-( - ) formation is by cleavage of ang i by neutral endopeptidase (nep, endopeptidase . ) (yamamoto et al ; duncan et al ) (fig. ). ang-( - ) may also be formed by ace cleavage of ang ii, but the significance of this pathway remains to be established. many actions of ang-( - ) are contrary to those of ang ii, and ang-( - ) is proposed to function as a counter-regulatory hormone in blood pressure control, and in other cardiovascular actions of ang ii. ang-( - ) reduces blood pressure and produces endothelium-dependent vasodilatation (benter et al ; pörsti et al ; benter et al ; nakamoto et al ; brosnihan et al ; le tran et al ) , actions that may be due in part to potentiation by ang-( - ) of the hypotensive effects of kinins (paula et al ; lima et al ) and/or to stimulation of vascular prostaglandin production (benter et al ; paula et al ) . in support of a role for kinin-mediated nitric oxide production in its vasodilator effects, ang-( - ) induced vasodilatation and hypotension were attenuated by nitric oxide synthase (nos) inhibition (pörsti et al ; gorelik et al ) , by the type bradykinin (b ) receptor antagonist icatibant (pörsti et al ; abbas et al ; lima et al ; gorelik et al ) , and also by at receptor antagonism (lima et al ) . moreover, ang-( - ) stimulation of nitric oxide release from coronary vessels was blocked by icatibant (brosnihan et al ) . high concentrations of ang-( - ) inhibit ace, leading to the suggestion that ang-( - ) potentiates the effects of bradykinin through ace inhibition (li et al ) . however, the ic for ang-( - ) inhibition of ace was nmol/l and it is unlikely endogenous ang-( - ) levels would be sufficient to produce this effect. ang-( - ), like other ace inhibitors, may potentiate the actions of a b receptor agonist by an indirect mechanism that is independent of bradykinin hydrolysis (deddish et al ) , possibly by sensitisation of the b receptor (marcic et al ) . this mechanism of potentiation of kinin-induced hypotension by ang-( - ) is unlikely to operate in vivo, however, because micromolar concentrations of ang-( - ) were required to produce this effect (deddish et al ) . plasma ang-( - ) levels are less than ang ii levels, except during ace inhibition when ang-( - ) levels increase several-fold, in parallel with the increase in ang i levels (lawrence et al ; menard et al ) . tissue levels of ang-( - ) are very low or undetectable, even with ace inhibition (campbell et al ; ) . there is, therefore, uncertainty whether ang-( - ) levels are sufficient to play a role in cardiovascular physiology and disease states in humans. . . figure shows an outline of the pathways of kinin peptide formation. a proportion of kininogens is hydroxylated on pro of the bradykinin sequence, leading to the formation of hydroxylated kinin peptides. the kininogens are the sole precursors of the kinin peptides and are coded by a single gene. differential splicing of the initial mrna transcript produces two different mrna coding for either high or low molecular weight kininogen. each is a glycoprotein that contains the kinin sequence in its mid portion. tissue kallikrein and plasma kallikrein are both serine proteases. whereas a single gene codes for plasma kallikrein there is a large family of tissue kallikrein genes, although klk is the only tissue kallikrein known to generate kinin peptides (yousef et al ) . kininogens and tissue kallikrein are expressed in many different tissues. plasma kallikrein is predominantly expressed in liver, although recent studies suggest expression of plasma kallikrein in the brain (takano et al ) . in humans, plasma kallikrein forms bradykinin from high molecular weight kininogen, whereas tissue kallikrein forms kallidin from high or low molecular weight kininogens (fig. ) . by contrast, both plasma and tissue kallikrein generate . an outline of the formation of kallidin and bradykinin peptides in humans. a proportion of high molecular weight kininogen is hydroxylated on pro of the bradykinin sequence, giving rise to both hydroxylated and non-hydroxylated peptides. adapted from (campbell ) bradykinin in rodents (bhoola et al ) . bradykinin may also be generated by aminopeptidase-mediated cleavage of kallidin. alternative pathways of kinin formation involving enzymes other than kallikreins may operate in disease states. although low molecular weight kininogen is a poor substrate for plasma kallikrein, it will form bradykinin in the presence of neutrophil elastase which, by cleaving a fragment from low molecular weight kininogen, renders it much more susceptible to cleavage by plasma kallikrein (sato et al ) . moreover, the combination of mast cell tryptase and neutrophil elastase releases bradykinin from oxidized kininogens that are resistant to cleavage by kallikreins (kozik et al ) . kinin production in vivo is controlled in part by endogenous inhibitors of the kallikrein enzymes. the main inhibitors of plasma kallikrein are c inhibitor, -macroglobulin and antithrombin iii (bhoola et al ) . an important inhibitor of tissue kallikrein is kallistatin, although the function of kallistatin in vivo is uncertain (chao et al ) . all components of a functional kks are expressed in the heart . the heart and vasculature express tissue kallikrein (oza et al ; xiong et al ; nolly et al ; nolly et al ) . in addition, plasma kallikrein, a member of the contact system, generates bradykinin at the endothelial surface of blood vessels (campbell ) . . dose related effects of the ace inhibitor perindopril on ang ii, ang i, and bradykinin levels in the cardiac ventricles of control rats and rats with myocardial infarction. *, p < . compared to mg/kg per day perindopril. data adapted from (campbell et al ; duncan et al ) kinins act via two types of kinin receptor, the b and the b receptors. the b receptor normally predominates, whereas the b receptor is induced by tissue injury. the kks generates bioactive kinin peptides: bradykinin, hyp bradykinin, kallidin, and hyp -kallidin act on the b receptor, whereas their carboxypeptidase metabolites des-arg -bradykinin, des-arg -hyp -bradykinin, des-arg -kallidin, and des-arg -hyp -kallidin act on the b receptor. hydroxylated kinins have similar biological activity to non-hydroxylated kinins. of particular interest is the recent report that the human b receptor is activated by both plasma and tissue kallikrein (hecquet et al ) . cathepsin g and trypsin similarly activate the b receptor and activation is blocked by icatibant. thus, the b receptor may belong to a new group of serine-protease-activated receptors (hecquet et al ) . ace is one of many enzymes that metabolise kinin peptides (campbell ) and the efficiency of metabolism is an important determinant of their levels in blood and tissues. consequently, inhibition of any single enzyme that contributes to kinin metabolism causes only a modest increase in kinin levels. kinin peptides have a broad spectrum of activities and both systemic and local cardiac actions impact on the heart (bhoola et al ). kinin peptides act through many different second messenger systems, in particular nitric oxide and prostaglandins (bhoola et al ) . the b receptor participates in an inhibitory interaction with endothelial nos (enos) that is reversed by bradykinin (ju et al ) . this interaction may recruit enos to the b receptor and allow for effective coupling of bradykinin signalling to the nitric oxide pathway. kinins are potent vasodilators and promote diuresis and natriuresis. kinins in high concentration also participate in the cardinal features of inflammation, producing vascular permeability, neutrophil chemotaxis and pain (bhoola et al ) . cardiac bradykinin levels are increased during the acute phase of myocardial infarction in rats (duncan et al ) . by contrast, we found decreased kallidin levels in coronary sinus blood of subjects with heart failure, suggesting downregulation of the cardiac kks in heart failure (duncan et al ) . there is a large body of evidence demonstrating anti-hypertrophic and cardioprotective actions of the kks (griol-charhbili et al ; koch et al ; park et al ; spillmann et al ) . the cardioprotective effects of bradykinin included the reduction of arrhythmias, reduction of lactate, lactate dehydrogenase, and creatine kinase release, and increase in myocardial contractility and myocardial levels of glycogen, adenosine triphosphate and creatine phosphate during postischaemic reperfusion of the isolated working rat heart (linz et al ) . moreover, bradykinin suppressed endothelin release from the post-ischaemic rat heart (brunner et al ) . kinins protect against ischaemia-reperfusion injury by decreasing endothelial adherence of leukocytes, leading to attenuation of post-ischaemic leukocyte adherence, attenuation of disruption of the microvascular barrier and reduced tissue injury (shigematsu et al ) . many of the actions of kinins counteract those of ang ii, by causing endothelium-dependent vasodilatation through endothelial release of nitric oxide and prostacyclin (pelc et al ; lamontagne et al ; gallagher et al ) . kinins also counteract the hypertrophic actions of ang ii and reduce collagen formation (gallagher et al ; ritchie et al ) . administration of kinin receptor antagonists indicates a role for endogenous kinins in the regulation of the coronary vasculature and in the myocardial response to myocardial infarction. icatibant reduced flow-dependent vasodilatation of human coronary arteries, indicating a role for kinins in the regulation of coronary vasculature (groves et al ) . icatibant enhanced myocardial interstitial deposition of collagen following myocardial infarction in the rat, indicating a role for endogenous kinins in the modulation of collagen deposition; however, icatibant did not modify morphological and molecular markers of cardiomyocyte hypertrophy (wollert et al ) . kinins participate in the process of ischaemic preconditioning, and have also been shown to limit reperfusion injury (baxter et al ) . kinins may also protect against thrombosis by stimulating endothelial release of nitric oxide, prostacyclin, and tissue plasminogen activator (dielis et al ) . new properties of kinin peptides are being discovered. for example, b receptors may have an important role in angiogenesis (emanueli et al ) . acsdkp is an inhibitor of pluripotent haemopoietic stem cell proliferation (lenfant et al ; bonnet et al ) , and is normally present in human plasma and mononuclear cells (pradelles et al ) . acsdkp is released from its precursor thymosin-ß by prolyl oligopeptidase (cavasin et al ) and it is cleaved to an inactive form by the dipeptidyl carboxypeptidase activity of the n-terminal catalytic domain of ace (rousseau et al ) . acsdkp has a . min half-life in the circulation and is probably released continuously . the importance of ace in acsdkp metabolism is shown by the -fold increase in acsdkp plasma levels that accompany ace inhibition . acsdkp inhibits dna and collagen synthesis by cardiac fibroblasts (rhaleb et al ) , and both prevents and reverses myocardial inflammation and fibrosis in rats with heart failure after myocardial infarction . acsdkp and thymosin-ß stimulate coronary vasculogenesis and angiogenesis (wang et al ; smart et al ) , and acsdkp increases myocardial capillary density in rats with myocardial infarction (wang et al ) . many clinical trials demonstrate the therapeutic benefit of ace inhibition in heart failure and ischaemic heart disease. it is of note, however, that the effects of ace inhibitors are dose related. large clinical trials, by necessity, use only one dose of any drug. the results of such trials are just as much a measure of the effect of the dose as they are a measure of the effect of the drug. use of a less than optimal dose may fail to reveal a drug's true therapeutic potential. this is of particular concern in a head-to-head comparison of two active drugs, where the result may be more due to choice of dose than to choice of drug. clinicians should strive to achieve drug doses that have proven to be of benefit in clinical trials. at present, a large proportion of patients receiving ace inhibitor therapy are receiving less than optimal doses (lenzen et al ) . measurement of plasma ang peptide levels is not feasible for the monitoring of ace inhibitor therapy, but measurement of plasma acsdkp levels may assist in this regard (struthers et al ) . heart failure is associated with neurohormonal activation that includes increased renin, ang ii, and aldosterone levels, and activation of the sympathetic nervous system (francis et al ) . increased ang ii, aldosterone, noradrenaline, and adrenaline levels predict increased mortality in heart failure patients (swedberg et al ) . therapies that counteract the effects of ras and sympathetic nervous system activation are the cornerstone of heart failure therapy (hunt et al ; swedberg et al ) . acute ace inhibition in heart failure patients promotes arterio-and venodilatation, with reduction in both afterload and preload, and an associated increase in cardiac output, stroke volume, and stroke work index, along with a decrease in pulmonary capillary wedge pressure, indicating improved left ventricular (lv) function (gavras et al ; ader et al ) . the cooperative north scandinavian enalapril survival study (consensus) demonstrated reduced mortality and improved symptoms with enalapril therapy in patients with severe heart failure (the consensus trial study group ). moreover, mortality was lower with enalapril therapy than with hydralazine-isosorbide dinitrate therapy in the second veterans administration cooperative vasodilator-heart failure trial (v-heft ii) (cohn et al ) . the studies of left ventricular dysfunction (solvd) confirmed the survival benefits of enalapril therapy in patients with reduced lv ejection fraction and heat failure (the solvd investigators ) and also demonstrated the prevention of heart failure in asymptomatic subjects with reduced lv ejection fraction (the solvd investigators ). ace inhibition improves survival, symptoms, and functional capacity, and reduces hospitalisation in patients with moderate and severe heart failure and lv systolic dysfunction (flather et al ; abdulla et al ) . ace inhibition is recommended as first-line therapy in patients with a reduced lv ejection fraction with or without symptoms, and should be up-titrated to the doses shown to be effective in clinical trials (hunt et al ; swedberg et al ) . although the patients recruited to the consensus, v-heft ii, and sovd studies had reduced lv ejection fraction due most often to ischaemic heart disease, they were enrolled several months or more after a myocardial infarction. studies in rats demonstrated survival advantage of ace inhibitor therapy commenced days after myocardial infarction (pfeffer et al b) . additionally, ace inhibition reduced arterial pressure and total peripheral resistance, attenuated lv remodelling, prevented deterioration in cardiac output and stroke volume index, and prevented the increase in lv volume, lv chamber stiffness and lv end diastolic pressure in rats with myocardial infarction (pfeffer et al a) . these benefits of ace inhibition in rats with myocardial infarction were confirmed in patients. the survival and ventricular enlargement (save) trial showed reduced mortality with ace inhibitor therapy when commenced - days after myocardial infarction in patients with asymptomatic lv dysfunction (pfeffer et al ) . in addition, ace inhibitor therapy reduced the incidence of both fatal and nonfatal major cardiovascular events, including the development of severe heart failure and recurrent myocardial infarction. the benefits of ace inhibitor therapy after myocardial infarction were confirmed in the acute infarction ramipril efficacy (aire) and the trandolapril cardiac evaluation (trace) studies (the acute infarction ramipril efficacy (aire) study investigators ; kober et al ) . the aire study recruited patients - days after myocardial infarction who had shown clinical evidence of heart failure at any time. the trace study recruited patients - days after myocardial infarction who had a lv ejection fraction ≤ %. both the aire and trace studies showed survival advantage with ace inhibitor therapy and the trace study showed less development of severe heart failure. other large clinical trails confirmed the benefits of ace inhibition after myocardial infarction (gissi- gruppo ; isis- collaborative group ) . in addition to mortality benefit and reduction of severe heart failure, ace inhibition after myocardial infarction attenuates lv remodelling, lv enlargement and increase in lv mass, and improves lv ejection fraction after myocardial infarction (pfeffer et al ; sharpe et al ; sogaard et al ; johnson et al ) . by contrast, the consensus ii trial found the commencement of ace inhibitor therapy within hours of myocardial infarction did not improve survival (swedberg et al ) . the failure of ace inhibition to improve outcomes in the consensus ii trial may have been due to its protocol. ace inhibitor treatment was started with intravenous infusion of mg enalaprilat within hours after the onset of chest pain, followed by administration of oral enalapril. intravascular administration of ace inhibitor had a negative inotropic effect in several human studies (foult et al ; haber et al ; zeitz et al ) , although not in another (friedrich et al ) . thus, the failure of ace inhibitor therapy to produce benefit in the consensus ii trial may have been due to the negative inotropic effect of intravenously administered enalaprilat, in addition to its administration within hours of chest pain. current european society of cardiology guidelines recommend the initiation of ace inhibitors after the acute phase of myocardial infarction in patients with signs or symptoms of heart failure, even if transient, to improve survival and to reduce re-infarctions and hospitalisations for heart failure . the hope study was based on emerging evidence that ace inhibition reduced the risk of myocardial infarction in patients with low ejection fraction (pfeffer et al ; yusuf et al ; lonn et al ) . it examined the effects of addition of mg ramipril to standard therapy in patients aged at least years with a history of coronary artery disease, stroke, peripheral vascular disease, or diabetes, plus at least one other cardiovascular risk factor (hypertension, elevated total cholesterol level, low high-density lipoprotein cholesterol level, cigarette smoking, or microalbuminuria). patients were excluded if they had heart failure, were known to have a low ejection fraction, were taking an ace inhibitor or vitamin e, had uncontrolled hypertension or overt nephropathy, or had had a myocardial infarction or stroke within weeks before the study began. during a mean follow-up of years ramipril reduced the primary outcome (composite of myocardial infarction, stroke, or death from cardiovascular causes) from . % to . % (relative risk . , % confidence interval . to . ; p < . ). treatment with ramipril reduced the rates of death from cardiovascular causes and all-cause mortality, myocardial infarction, revascularisation procedures, cardiac arrest, heart failure, and complications related to diabetes. the europa study examined the effects of addition of mg perindopril to standard therapy in patients with previous myocardial infarction, angiographic evidence of coronary heart disease, coronary revascularization, or a positive stress test. past history of heart failure was recorded in . % of subjects, but none had clinical signs of heart failure, with % in new york heart association class i and none in class ii or higher. during a mean follow-up of . years, perindopril reduced the primary outcome (composite of cardiovascular death, non-fatal myocardial infarction, cardiac arrest with successful resuscitation) from . % to . % (relative risk . , % confidence interval . to . ; p< . ). the main contributor to this reduction in the primary outcome was the reduction in non-fatal myocardial infarction. perindopril also reduced the incidence of heart failure requiring hospitalisation. by contrast, the prevention of events with angiotensin converting enzyme inhibition (peace) study failed to show an effect of ace inhibition on its primary endpoint (the peace trial investigators ). the peace study examined the effects of addition of mg trandolapril to standard therapy on cardiovascular events in patients with stable coronary heart disease and preserved lv function. during a median follow-up of . years, trandolapril produced non-statistically significant reductions in the primary endpoint (composite of cardiovascular death, myocardial infarction, and coronary revascularization) from . % to . %, and in cardiovascular death and non-fatal myocardial infarction from . % to . %, although trandolapril reduced hospitalisation or death due to heart failure from . % to . %. participants in the peace study were at lower risk of cardiovascular events than those in the hope and europa studies. the baseline blood pressure of peace participants was less than that of patients in the hope and europa studies, and was similar to the level achieved with active therapy in the hope and europa studies. in addition, peace participants received more intensive management of risk factors than did those in the hope and europa studies, with % of peace participants receiving lipid lowering therapy ( % in hope, % in europa), and % had undergone coronary revascularization before enrollment ( % in hope, % in europa). thus, peace participants had an event rate similar to that of the general population ( . % annualised rate of death), and the more aggressive management of their risk factors may have negated any potential benefit from ace inhibitor therapy. there has been debate about the reasons for the failure of the peace study to show an effect of trandolapril on the primary endpoint (pitt ; fox et al a) . although the dose and type of ace inhibitor may be implicated, the most likely explanation is the low event rate in its relatively low risk population (necessitating the inclusion of revascularisation as part of the primary endpoint), such that the study did not have sufficient statistical power to achieve its aim. the ischemia management with accupril post bypass graft via inhibition of angiotensin converting enzyme (imagine) study similarly showed a lack of benefit from mg quinapril in optimally treated low-risk patients after coronary artery bypass grafting (keuper et al ) . pooled analysis of the hope, europa, and peace trials showed ace inhibition reduced all cause and cardiovascular mortality, non-fatal myocardial infarction, stroke, heart failure, and coronary artery bypass surgery, leading to the recommendation that ace inhibitors be considered in all patients with atherosclerosis (dagenais et al ) . a meta-analysis of the hope, europa, peace, and other studies came to a similar conclusion (al-mallah et al ) . however, the number needed to treat for . years to prevent either one death, one nonfatal myocardial infarction, or one coronary revascularisation procedure was (al-mallah et al ) . current european society of cardiology guidelines state: "ace inhibition is well established in the treatment of heart failure or lv dysfunction and in the treatment of diabetic patients. thus, it is appropriate to consider ace inhibitors for the treatment of patients with stable angina pectoris and co-existing hypertension, diabetes, heart failure, asymptomatic lv dysfunction and post-myocardial infarction. in angina patients without co-existing indications for ace inhibitor treatment the anticipated benefit of treatment (possible absolute risk reduction) should be weighed against costs and risks for side-effects, and the dose and agent used of proven efficacy for this indication" (fox et al b) . et al ) . the systemic effects include the reduction of circulating ang ii and aldosterone levels and the increase in kinin and acsdkp levels. decreased ang ii and increased kinin levels contribute to the reduction of blood pressure by ace inhibition. there is ongoing debate about the extent to which the benefits of ace inhibition are related to blood reduction, as opposed to intrinsic benefits of ace inhibition (sever et al ) . a major contributor to the benefits of ace inhibition in heart failure and ischaemic heart disease may be the reduction in systemic blood pressure, and consequent reduction in heart work. ace inhibition may improve cardiac function by reducing coronary vascular resistance in patients with heart failure, thereby augmenting cardiac blood flow (dietz et al ) . ace inhibition reduces circulating and tissue levels of ang ii in both animals and humans (campbell et al ; duncan et al ; campbell et al ; zeitz et al ) . ace inhibition produced a modest reduction in ang ii levels in europa participants (ceconi et al ) . however, the effects of ace inhibition on ang ii levels can be variable, and depend on the responsiveness of renin secretion . in situations where renin shows little increase in response to ace inhibition, the levels of ang ii and its metabolites show a marked fall, with little change in the levels of ang i and its metabolites. by contrast, a large increase in renin levels in response to ace inhibition also increases the levels of ang i and its metabolites. the increased ang i levels promote ang ii formation by residual uninhibited ace and by serine protease pathways of ang i conversion, thereby buffering any fall in ang ii levels during ace inhibition . improved survival of heart failure patients with ace inhibitor therapy is associated with reduction in ang ii and aldosterone levels (swedberg et al ) . the role of renin in determining the response of ang ii levels to ace inhibition is most evident in heart failure, where many patients continue to have elevated ang ii levels despite ace inhibitor therapy (roig et al ; campbell et al ) . it is of note that maximally recommended doses of ace inhibitor do not completely prevent ace mediated formation of ang ii in heart failure (jorde et al ) . the beneficial therapeutic effects of concomitant ß-blocker therapy in heart failure may be due in part to the associated reduction in renin and ang ii levels (campbell et al ) . the effects of ace inhibitors on ang ii levels are dose dependent (fig. ) . studies in rats showed tissue-specific differences in the dose-related effects of ace inhibition on ang ii levels (campbell ) . renal ang ii levels were reduced by lower doses of ace inhibitor than were required to reduce ang ii levels in other tissues such as the heart (fig. ) . ang-( - ) . this is due in part to the increase in ang i levels, with subsequent conversion to ang-( - ). another mechanism for the increase in ang-( - ) levels during ace inhibition is the inhibition of ang-( - ) metabolism, given that ace is an important pathway of ang-( - ) metabolism yamada et al ) . studies in rats led to the proposal that increased ang-( - ) levels mediate in part the hypotensive effects of ace inhibition (iyer et al a; iyer et al b) . however, there is as yet no evidence that these mechanisms operate in patients receiving ace inhibitor therapy. there is ample evidence that kinin peptides contribute to the therapeutic effects of ace inhibitors (linz et al ; bönner ) . ace inhibitors increase circulating and tissue levels of bradykinin in animals (fig. ) and humans (campbell et al ; duncan et al ; zeitz et al ) . the effect of ace inhibition on kinin peptide levels in any tissue compartment depends on the contribution of ace, relative to other kininases, to kinin peptide metabolism in that compartment. ace inhibitor therapy did not increase either bradykinin or kallidin peptide levels in cardiac atria of patients with ischaemic heart disease, despite the reduction in ang ii levels . the maintenance of low levels of kinin peptides by their efficient metabolism is relevant to the success of ace inhibitor therapy. ace inhibition has only a modest effect on kinin peptide levels because of the many other kininases that contribute to kinin metabolism. it is for this reason that ace inhibitors are generally free of the side effects, such as angioneurotic oedema, that one might expect from increased kinin peptide levels (nussberger et al ; nussberger et al ) . studies with kinin receptor antagonists indicate a role for kinins in the cardiovascular actions of ace inhibitors in animals and humans (linz et al ) . studies in humans indicate a role for the b receptor in flow-dependent vasodilatation in normal volunteers (hornig et al ) and in the hypotensive effects in patients with hypertension (gainer et al ; squire et al ) . a role for the b receptor is indicated in the systemic haemodynamic effects of ace inhibition in patients with heart failure (witherow et al ; cruden et al ) . cardioprotective effects of ace inhibition that were attenuated by icatibant included the reduction of arrhythmias, reduction of lactate, lactate dehydrogenase, and creatine kinase release, and increase in myocardial contractility and myocardial levels of glycogen, adenosine triphosphate and creatine phosphate during reperfusion of the ischaemic isolated working rat heart (linz et al ) . icatibant attenuated the ace inhibitor-induced increase in coronary flow and nitric oxide levels in dogs with myocardial ischaemia (kitakaze et al ) . icatibant also prevented the potentiation of ischaemic preconditioning by ace inhibition in human atria (morris et al ) . the post-ischaemic anti-arrhythmic effect of ace inhibition may be mediated by kinin-induced suppression of endothelin release (brunner et al ) . icatibant prevented the reduction in myocardial infarct size and the reduction in post-infarct remodelling by ace inhibition in animal models (linz et al ; hartman et al ; stauss et al ; mcdonald et al ; hu et al ) . however, a subsequent study in an in vivo canine model of myocardial ischaemic injury did not show an effect of ace inhibition on infarct size (black et al ) . moreover, icatibant did not modify the antihypertrophic effect of ace inhibition in rats with myocardial infarction, although it partially reversed the reduction in myocardial collagen deposition by ace inhibitor therapy in one study (wollert et al ) . possible mechanisms by which kinin peptides mediate the therapeutic benefits of ace inhibition include the promotion of endothelial production of nitric oxide and prostacylin, thereby contributing to the correction of endothelial dysfunction and reduced oxidative stress (linz et al ; bönner ; münzel et al ) . ace inhibition induced endothelial nos (enos) in vasculature of control rats, and attenuated the induction of inducible nos (inos) in rats administered bacterial lipopolysaccharide (bachetti et al ) . icatibant prevented the increase in nitric oxide formation in the heart and reduction in myocardial oxygen consumption that accompany ace inhibition in dogs (zhang et al ) . icatibant also prevented the antiproliferative effect of ace inhibition in neointima formation following endothelial injury to the rat carotid artery (linz et al ) , and the increase in capillary density induced by chronic ace inhibitor treatment in stroke-prone spontaneously hypertensive rats (gohlke et al ) . part of the benefits of ace inhibition may be due to the enhancement of insulin-mediated muscle glucose uptake, that is also attenuated by icatibant (henriksen et al ; henriksen et al ) . ace inhibition also affects the kks by mechanisms separate from prevention of kinin degradation. for example, chronic ace inhibition in mice and rats induced both renal and vascular b receptor expression without modification of b receptor expression (marin-castano et al ) . moreover, enalaprilat and other ace inhibitors in nanomolar concentrations were shown to directly activate the human b receptor, in the absence of ace and b receptor ligands (ignjatovic et al ) . several studies show ace inhibitors may potentiate the effects of bradykinin by a mechanism independent of prevention of kinin metabolism, that involves direct interaction between ace and the b receptor (fleming ) and attenuation of the sequestration of the b receptor (benzing et al ; chen et al ) . additionally, membrane ace appears to have its own signalling cascade that is activated by binding of ace inhibitors (fleming ) . one approach to differentiation of the respective roles of the ras and kks in mediating the therapeutic benefits of ace inhibition is the comparison of ace and arb therapy. comparison of ace inhibitor and arb therapy after myocardial infarction, or in patients with heart failure, did not show any difference in outcomes (pitt et al ; dickstein et al ; pfeffer et al ; mcmurray et al ) . these studies suggest ace inhibitor and arb therapy act through blockade of the ras, but a role for bradykinin cannot be excluded because losartan was shown to increase bradykinin levels in hypertensive humans (campbell et al ) . maximally recommended doses of ace inhibitors do not completely prevent ace mediated formation of ang ii in heart failure (jorde et al ) . combination of ace inhibitor and arb therapy produces more complete blockade of the ras that is dependent on the dose regimens of the individual therapies (menard et al ; azizi et al ) . this combination therapy improves outcomes in heart failure patients (cohn et al ; mcmurray et al ) , but not following myocardial infarction mcmurray et al ) . ace inhibition causes a several-fold increase in acsdkp levels that may contribute to decreased cardiac inflammation and fibrosis, and to increased myocardial capillary density after myocardial infarction (wang et al ; yang et al ) . elevated acsdkp levels during ace inhibitor therapy may also contribute to the anaemia experienced by heart failure patients receiving ace inhibitor therapy (van der meer et al ). heart failure patients have increased plasma aldosterone levels consequent to stimulation of aldosterone secretion by increased ang ii levels (weber ) . evidence that reduced aldosterone levels may contribute to the therapeutic benefits of ace inhibition is the reduced hypokalaemia in patients receiving ramipril therapy in the hope study (mann et al ) . in addition to promotion of sodium retention and oedema formation, aldosterone may promote cardiac fibrosis and deterioration in cardiac function (brilla et al ) . the possible clinical importance of this mechanism is shown by the benefits of aldosterone receptor antagonists in patients with heart failure, and in patients with lv dysfunction after myocardial infarction (pitt et al ; pitt et al ) . many authors have suggested the reduction in sympathetic activity that may accompany ace inhibition is due to a reduction in the stimulation of sympathetic activity by ang ii. however, although ace inhibitor therapy leads to reduction in sympathetic nervous system activity in heart failure, this is thought to be mainly secondary to the improvement of cardiovascular haemodynamics, rather than the specific consequence of reduced stimulation of the sympathetic nervous system by ang ii (esler et al ) . cardiac hypertrophy is well recognised as a risk factor for death and cardiovascular events (levy et al ) . ace inhibitors reduce cardiac hypertrophy in hypertensive patients (dahlof et al ) and also reduce progressive lv remodelling after myocardial infarction (ferrari ) . ventricular remodelling has a dominant role in the pathogenesis of heart failure, and the prevention of remodelling is considered to be an important mechanism of the benefit of ace inhibitor therapy in heart failure and after myocardial infarction (cohn ; abdulla et al ) . reduction of myocardial infarction and other ischaemic events by ace inhibition raises the possibility that these drugs inhibit atherosclerosis. ace inhibitors correct endothelial dysfunction in patients with heart failure and ischaemic heart disease mancini et al ; ceconi et al ) . these effects of ace inhibition may be due to the reduction of oxidative stress, vascular remodelling and inflammation by reduced ang ii levels and increased kinin levels. however, current evidence does not allow these data to be extrapolated to a reduction in atherogenesis by ace inhibition in humans. despite the prevention of atherosclerosis in animal models, ace inhibitor therapy was not able to reduce atherogenesis in patients. ace inhibition with cilazapril did not prevent restenosis after angioplasty (mercator), (mercator study group ; faxon ) . similarly, quinapril did not reduce restenosis after coronary stenting; in fact, late loss in minimum lumen diameter was significantly higher in the quinapril group than in controls (meurice et al ) . additionally, ace inhibition with enalapril failed to reduce progression of coronary atherosclerosis, as assessed by intravascular ultrasound, in patients with coronary artery disease (nissen et al ) . a meta-analysis of randomised controlled studies of the effect of antihypertensive therapies in progression of carotid intima-media thickness showed only a weak, non-significant reduction in progression of carotid intima-media thickness by ace inhibitor therapy, with significant heterogeneity between studies (wang et al ) . some studies showed a reduction in progression of intima-media thickness by ace inhibition and some did not. of note, calcium channel blockers were significantly more effective than ace inhibitors in their reduction of progression of intima-media thickness (wang et al ) . reduced rates of myocardial infarction with ace inhibitor therapy may also be due to an effect of this therapy on the mechanisms of thrombosis and fibrinolysis. ace inhibition reduced plasma levels of pai- antigen and activity in normal subjects on low salt diet and in subjects following myocardial infarction (wright et al ; moriyama et al ; oshima et al ; vaughan et al ; brown et al ; brown et al ) , although this effect of ace inhibition was not confirmed in other studies of patients with previous myocardial infarction (zehetgruber et al ; pedersen et al ) . ace inhibition also reduced pai- antigen, but not pai- activity, in subjects with congestive cardiac failure (goodfield et al ) . diabetes is well recognised to accelerate the processes of cardiovascular disease, and reduction of diabetes incidence may contribute to the therapeutic benefits of ace inhibition. many large clinical trials, including the hope, peace, and solvd studies, showed a reduced incidence of type diabetes with ace inhibitor therapy (abuissa et al ) . however, the diabetes reduction assessment with ramipril and rosiglitazone medication (dream) study found ramipril did not reduce diabetes incidence among persons with impaired fasting glucose levels or impaired glucose tolerance, although it significantly increased regression to normoglycaemia (the dream trial investigators ) . this improvement in insulin resistance may be due in part to the enhancement of insulin-mediated muscle glucose uptake by ace inhibition (henriksen et al ; henriksen et al ) . aortic compliance is an important determinant of coronary blood flow (o'rourke et al ) . a recent meta-analysis showed ace inhibitors decrease arterial stiffness (mallareddy et al ) . ace inhibitors, by increasing aortic compliance, may reduce central systolic blood pressure and maintain diastolic blood pressure, thereby reducing heart work without compromising myocardial perfusion. decrease in arterial stiffness by ace inhibition may be due to reduced collagen deposition, as suggested by studies in spontaneously hypertensive rats (benetos et al ) . reduction of aortic collagen deposition by ace inhibition was not affected by icatibant, suggesting that this effect of ace inhibition was not mediated by kinins (benetos et al ) . atrial fibrillation is an important contributor to poor prognosis in heart failure (wang et al ) , and prevention of atrial fibrillation by ace inhibition may contribute to the therapeutic benefits of this therapy (vermes et al ). given that kinin peptides mediate in part the therapeutic benefits of ace inhibition, and that some of the actions of kinins are mediated by prostaglandins, the question arises whether a drug that inhibits prostaglandin synthesis may attenuate the effects of ace inhibition. this question was addressed in a systematic review of the interaction between aspirin and ace inhibitor therapy (teo et al ) . the solvd study found aspirin prevented the reduction of death by ace inhibition, but this interaction between aspirin and ace inhibitor therapy was not significant in the other trials examined. however, both solvd and the other trials showed aspirin attenuated the prevention of myocardial infarction or reinfarction by ace inhibition. by contrast, there was no evidence that aspirin attenuated the prevention of stroke, hospital admission for heart failure, or revascularisation by ace inhibitor therapy. when the composite of major vascular events including death, myocardial infarction or reinfarction, hospital admission for heart failure, 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captopril therapy on endogenous fibrinolysis in men with recent, uncomplicated myocardial infarction identification, purification, and localization of tissue kallikrein in rat heart converting enzyme determines plasma clearance of angiotensin-( - ) in vivo metabolism of angiotensin i by neutral endopeptidase (ec . . . ) in spontaneously hypertensive rats ac-sdkp reverses inflammation and fibrosis in rats with heart failure after myocardial infarction the new human tissue kallikrein gene family: structure, function, and association to disease effect of enalapril on myocardial infarction and unstable angina in patients with low ejection fractions the aceinhibitor lisinopril affects plasma insulin levels but not fibrinolytic parameters myocardial uptake and biochemical and hemodynamic effects of ace inhibitors in humans ace inhibitors promote nitric oxide accumulation to modulate myocardial oxygen consumption - )-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme homologue ace this work was supported by a senior research fellowship from the national health and medical research council of australia (id ), and by the national heart foundation of australia (id g m ). key: cord- -qq h vc authors: fyhrquist, f.; saijonmaa, o. title: renin‐angiotensin system revisited date: - - journal: j intern med doi: . /j. - . . .x sha: doc_id: cord_uid: qq h vc new components and functions of the renin‐angiotensin system (ras) are still being unravelled. the classical ras as it looked in the middle s consisted of circulating renin, acting on angiotensinogen to produce angiotensin i, which in turn was converted into angiotensin ii (ang ii) by angiotensin‐converting enzyme (ace). ang ii, still considered the main effector of ras was believed to act only as a circulating hormone via angiotensin receptors, at and at . since then, an expanded view of ras has gradually emerged. local tissue ras systems have been identified in most organs. recently, evidence for an intracellular ras has been reported. the new expanded view of ras therefore covers both endocrine, paracrine and intracrine functions. other peptides of ras have been shown to have biological actions; angiotensin – heptapeptide (ang iii) has actions similar to those of ang ii. further, the angiotensin – hexapeptide (ang iv) exerts its actions via insulin‐regulated amino peptidase receptors. finally, angiotensin – (ang – ) acts via mas receptors. the discovery of another ace was an important complement to this picture. the recent discovery of renin receptors has made our view of ras unexpectedly complex and multilayered. the importance of ras in cardiovascular disease has been demonstrated by the clinical benefits of ace inhibitors and at receptor blockers. great expectations are now generated by the introduction of renin inhibitors. indeed, ras regulates much more and diverse physiological functions than previously believed. have been shown to have biological actions; angiotensin - heptapeptide (ang iii) has actions similar to those of ang ii. further, the angiotensin - hexapeptide (ang iv) exerts its actions via insulin-regulated amino peptidase receptors. finally, angiotensin - (ang - ) acts via mas receptors. the discovery of another ace was an important complement to this picture. the recent discovery of renin receptors has made our view of ras unexpectedly complex and multilayered. the importance of ras in cardiovascular disease has been demonstrated by the clinical benefits of ace inhibitors and at receptor blockers. great expectations are now generated by the introduction of renin inhibitors. indeed, ras regulates much more and diverse physiological functions than previously believed. keywords: angiotensin, angiotensin-converting enzyme, angiotensin receptor, renin. the classical paper on the discovery of renin by the finnish physiologist robert tigerstedt and his swedish student per bergman in [ ] was based on experiments performed - at the karolinska institute. saline extracts of rabbit kidney were shown to slowly raise the blood pressure (bp) when injected into rabbits. the principle causing this was present in kidney cortex but not in medulla and was destroyed by heating. the authors concluded that the substance was a protein and they named it renin. they speculated that 'renin might in some direct or indirect way be associated with hypertrophy of the heart found in renal disease and high bp'. however, these early results could not be repeated in other laboratories, and it was not until late s when renin was 'rediscovered'. an immense amount of early research on the reninangiotensin system (ras) paved the way for improved understanding of its physiology and pathophysiology. in the early s, the major components of 'classical' circulating ras (fig. ) were identified and there was compelling evidence to indicate important roles for ras in the regulation of fluid balance and bp. at that time, however, there was widespread skepticism review | regarding the role of ras in cardiovascular disease. it was not until the discovery of orally effective angiotensin-converting enzyme (ace) inhibitors, the first of which was captopril [ ] , that the paramount importance of ras in cardiovascular homeostasis and disease was being appreciated. the introduction of losartan, the first orally active and effective angiotensin receptor type blocker [ ] further strengthened this concept. fig. the present view of the expanded renin-angiotensin system. rpr, renin ⁄ prorenin receptor; mas, mas oncogene, receptor for ang - ; at r, angiotensin type receptor; at r, angiotensin type receptor, irap, insulin-regulated aminopeptidase; ang iv receptor ampa, aminopeptidase a; ampm, aminopeptidase m; ace, angiotensin-converting enzyme; ace , angiotensin-converting enzyme ; nep, neutral endopeptidase. angiotensin ii exerts its actions via at and at receptors which in principle, but not invariably, mediate opposite functions. at receptors mediate actions with potentially harmful consequences, if not properly counterbalanced. at receptors are thought to mediate protective actions, the clinical relevance of which has not yet been clearly established. angiotensin ii is a major regulator of fluid and sodium balance and haemodynamics, but also of cellular growth and cardiovascular remodelling. thus, at receptors mediate vasoconstriction, thirst and release of vasopressin and aldosterone, fibrosis, cellular growth and migration. more recently, ang ii has been shown to cause generation of oxidative radicals via at receptors and to be involved in inflammatory processes including atherosclerosis and vascular ageing. at receptors mediate vasodilation, release of nitric oxide (no) and usually inhibition of growth. angiotensin ii infusion caused decrease of plasma adiponectin, an insulin sensitizer, apparently via at receptors in the rat [ ] . suppression of adiponectin may represent a mechanism whereby ang ii causes impaired glucose tolerance. other metabolic actions of ang ii include pro-inflammatory modulation [ ] , increased insulin secretion [ ] , b-cell apoptosis [ ] , reduction of gluconeogenesis and hepatic glucose output [ ] and increased plasma triglycerides [ ] . we decided not to enter the complex field of intracellular at and at receptor signalling and therefore refer the reader to recent reviews [ , ] . angiotensin receptor type is generally reported to mediate effects opposing and counterbalancing those mediated by at receptors (fig. ), e.g. vasodilatation, no release and inhibition of proliferation and growth. however, at receptors may also mediate neurotrophic effects in the central nervous system [ ] . moreover, upregulated at receptors in the peri-ischaemic brain may exert protection against ischaemic damage [ ] . the authors speculate that such a protective effect mediated by at receptors might in part explain superior protection against stroke in patients treated with losartan vs. treatment with atenolol in the life study [ ] . another explanation may be that losartan lowers central bp more effectively than atenolol [ ] . in rat kidney, ang iii but not ang ii was recently reported to induce natriuresis mediated by at receptors [ ] . this natriuresis was augmented by blockade of aminopeptidase n, an enzyme metabolizing ang iii to ang iv ( fig. ) [ ] . the authors speculated that blockers of aminopeptidase n might be developed to treat diseases characterized by sodium and fluid retention such as hypertension and heart failure. in theory, such inhibitors may also exert beneficial actions via reduced tissue levels of ang iv (see below). interestingly, renal interstitial fluid has been shown to contain roughly -fold higher concentrations of ang ii and ang iii than found in the plasma [ ] . effects of unopposed stimulation of at receptors are slightly controversial [ ] . thus, beneficial effects include bradykinin-no vasodilatory effects, natriuretic and antifibrotic effects. potentially detrimental effects are apoptosis, nuclear factor-kappa b (nf-jb) signal transduction and induction of chemokines. despite a plethora of promising experimental results strongly suggesting beneficial actions of at stimulation [ ] the final clinical proof is lacking. though treatment with at receptor blockers (arbs) substantially increase plasma ang ii levels and presumably cause increased stimulation of at receptors, there is no conclusive evidence to prove the clinical relevance of increased at receptor activity. angiotensin ii may be enzymatically generated from ang i by chymase (fig. ) in certain pathological conditions. chymase is stored in macromolecular complex with heparin proteoglycan in secretory granules of mast cells [ ] . to become enzymatically active, complexed chymase must be released from f. fyhrquist & o. saijonmaa | review: renin-angiotensin system mast cell granules, e.g. by vascular damage caused by ballooning or other damage. therefore, chymase is enzymatically inactive in normal vascular tissue and may produce ang ii only in damaged or atherosclerotic arterial walls. it is of note that endogenous serine protease inhibitors present in interstitial fluid [ ] are potent inhibitors of chymase. chymase inhibitors reportedly prevent neointimal lesions following vein grafting or arterial ballooning in dogs, whereas ace inhibitors are ineffective [ ] . however, the effects of chymase inhibitors may depend on other effects of these compounds such as decrease in transforming growth factor-b (tgf-b) generation and stabilization of mast cell granules and not on decreased ang ii formation. in addition, arbs which block ang ii actions irrespective of generating enzyme(s) have not proven superior to ace inhibitors in large clinical trials [ , ] . though animal experimental results with chymase inhibitors are promising [ ] , the possible importance of ang ii generation by chymase is unclear and chymase inhibitors that are safe and useful for human trials have not yet been developed. angiotensin iii has been known since the s to cause vasoconstriction and release of aldosterone. it is generated from ang ii by aminopeptidase a (fig. ). ang iii exerts its actions, in principle similar to those of ang ii, via at and at receptors. while ang ii is considered the main effector of ras, ang iii may be equally or even more important in some actions mediated by at receptors, e.g. release of vasopressin [ ] . systemic infusion of ang ii or ang iii to conscious dogs was recently shown to result in equipotent effects at the same plasma concentration on bp, aldosterone secretion, sodium excretion and plasma renin activity; all effects inhibited by candesartan. however, the metabolic clearance rate of ang iii was five times that of ang ii [ ] . this study indicated that ang ii plays a dominant role as an effector of the 'classical circulating ras'. angiotensin iv may be generated from ang iii by aminopeptidase m (fig. ). this biologically active peptide has caught increasing interest following the discovery and cloning of insulin-regulated amino peptidase receptors (irap), [ , ] , a binding site and a probable receptor (at ) of ang iv. actions of ang iv mediated by irap (fig. ) include renal vasodilation, hypertrophy and activation of nf-jb leading to increased expression of platelet activator inhibitor-i (pai- ), monocyte chemoattractant protein (mcp- ) interleukin- and tumour necrosis factor-a [ , ] . several studies suggest that ang iv has important regulatory functions in cognition, renal metabolism and cardiovascular damage [ , ] . ang iv regulates cell growth in cardiac fibroblasts, endothelial cells and vascular smooth muscle cells. it appears that ang iv is involved in the vascular inflammatory response and could therefore play a role in cardiovascular pathophysiology. angiotensin - heptapeptide was thought for a long time to be devoid of biological actions, in spite of early reports on biological effects [ ] . the importance of ang - was emphasized by the relatively recent discovery of a 'new' ace . this enzyme generates ang - from ang ii. ang - may also be generated from ang i or ang ii by other peptidases. already back in , ang ( - ) was shown to release vasopressin as effectively as ang ii from neurohypophyseal explants [ ] . ang ( - ) was found to have actions opposing those of ang ii (fig. ) , namely vasodilation and antitrophic effects and amplification of vasodilation caused by bradykinin [ ] [ ] [ ] . numerous experiments suggest an important interaction between ang ( - ) and prostaglandin-bradykinin-no systems. ang ( - ) appears to counterbalance several actions of ang ii. ang ( - ) binds to the mas receptor (figs and ) which mediates vasodilating and antiproliferative actions of the heptapeptide. angiotensin-converting enzyme was discovered and cloned rather recently [ , ] . this discovery brought both ace and its main product, ang - into the focus of intense research. ace is a carboxypeptidase which cleaves one residue from ang i to generate angiotensin - and a single residue from ang ii to generate ang - (fig. ) . ace is most abundant in vascular endothelium of kidney, heart, hypothalamus and aortic wall. ace is also found in testis [ ] . regulation of ace expression has not yet been fully clarified [ ] . neither ace inhibitors nor arbs do inhibit ace activity, but they both appear to upregulate ace expression in rat myocardium and renal cortex [ ] . expression of ace in the heart is increased by myocardial infarction [ ] . disruption of the ace gene in mice was reported to result in a severe cardiac contractility defect, increased ang ii plasma levels and upregulation of cardiac hypoxia-induced genes [ ] . the authors concluded that ace is an 'essential regulator of heart function'. genetic ablation of ace in ace null mice completely normalized the cardiac phenotype, which fits the proposed mutually counterbalancing roles for the ace ⁄ ang ii and ace ⁄ ang - arms of ras. the authors interpreted these findings as evidence for ace being an essential regulator of heart function. this interpretation [ ] has recently been challenged by a study also on ace null mice, showing 'no detectable effect on cardiac dimensions or ejection fraction in conscious mice under basal conditions' [ ] . these investigators reported increased pressor sensitivity to ang ii infusion and higher plasma levels and renal concentrations of ang ii during infusion in ace null mice. this study suggested an important role for ace in degrading ang ii and regulation of vascular responses to ang ii. the abundance of ace in kidneys notably in the proximal tubule is of particular note. ace may be critical in regulating the balance between renal effects of ang ii and ang ( - ) and may therefore become a target for future therapeutic approaches [ ] . ace seems to have a protective role in the kidney [ ] . angiotensin-converting enzyme and ang - may play an important role in cardiovascular physiology and pathophysiology, e.g. by modulating or counterbalancing excess activity of the 'classical' ras [ , ] . the expression and activity of ace in heart [ ] and kidney is differently increased by treatment with ace inhibitors. this leads to organ-wise regulated increase of local production of ang - , as demonstrated in rats [ ] [ ] [ ] . this would offer an additional beneficial effect of ace inhibitors, possibly explaining in part why ace inhibitors and arbs review: renin-angiotensin system remain effective despite increased plasma renin activity and angiotensin peptide concentrations [ ] [ ] [ ] . renin receptors were identified and cloned rather recently and shown to be functional [ ] . renin receptors bind both prorenin and renin [ ] . two receptors have been characterized; the m p ⁄ insulin-like growth factor ii receptor, a clearance receptor and the specific renin receptor, which activates intracellular signalling and enhances receptor-bound renin catalytic activity on the cell surface (fig. ) . renin receptors are abundant in heart, brain and placenta, lower levels being found in kidney and liver [ ] . visceral adipose tissue also expresses renin receptor, whereas subcutaneous adipose tissue expressed less renin receptors [ ] . it appears that renin receptors participate in local production of ang ii and may contribute to systemic ang ii levels as well. binding of prorenin to the renin receptor (fig. ) leads to activation of prorenin to active renin, activation of mitogen-activated protein kinases p ⁄ and tgf-b, ultimately increasing contractility, hypertrophy and fibrosis [ , ] . it has been suggested that blocking renin receptors may be a new target for tissue protection. the 'handle and gate' hypothesis of activation of receptor-bound renin offers exciting perspectives [ ] . a pentapeptide reproducing the 'handle' region of the prosegment of prorenin (fig. ) that covers the active site has provided promising results in the treatment of diabetic nephropathy [ ] and in the prevention of hypertensive glomerulopathy in mice [ ] . if local ras systems have been identified in most organs and tissues investigated as recently reviewed [ , ] . they contain all components necessary for the production of ang ii and other angiotensin peptides and their respective receptors, and in addition, renin ⁄ prorenin receptors. the majority of studies indicate that most if not all renin found in local ras systems is derived from renal renin. tissue ras systems exert diverse actions in many organs. in some organs, they operate independently of the 'circulating ras', e.g. the adrenal glands and brain. other local ras systems, e.g. heart and kidney operate in close interaction with the 'circulating' ras. thus, circulating components of ras like renin and agt may be taken up by tissues. circulating ras and local tissue ras are thought to operate in a complementary fashion [ ] , not opposing each other. a proper balance between regulating and counter-regulating factors of tissue ras appears important in maintaining normal physiological functions of many organs. the circulating ras is seen as a regulator of systemic volume and electrolyte balance and of bp homeostasis, whilst 'local' ras systems have local tissue effects involving proliferation, growth, protein synthesis and organ functions, e.g. in kidney, heart, brain, reproductive organs and pancreas [ , ] . some recent discoveries concerning local ras systems may deserve particular interest, namely those of the heart, brain and adipose tissue. thus, ace expression is increased in the heart following myocardial infarction [ ] , in heart failure [ ] and during treatment with ace inhibitors or arbs [ ] . ace is the main generator of ang - from ang ii in the heart, and the amount of ang - is increased in the peri-ischaemic area following myocardial infarction [ ] . brain ras components mediate a large variety of neurobiological activities [ ] which are gradually being understood. for instance, neuronal at receptors mediate ang ii effects on bp, salt and water intake and secretion of vasopressin whereas at receptors mediate, e.g. apoptosis and possibly neural regeneration after neural injury [ ] . in addition to ang ii and ang iii, ang iv and ang - appear to be involved in modulation of brain functions, including learning and memory responses. adipose tissue also contains all components of ras including functional renin receptors co-localizing with renin and may be involved in the regulation of visceral adipose tissue accumulation [ ] . thus, visceral ras may play a role in the pathophysiology of the metabolic syndrome [ ] . adipose tissue was shown to be an important source of both local and circulating agt [ ] and might thereby participate in systemic bp regulation. however, this has not been shown in humans. testicular ace (ca kda), a smaller isoform of ace ( - kda) was recently shown to play a crucial role in fertilization by releasing a glycosylphosphatidylinositol (gpi)-anchored protein from sperm cells. ace knock-out sperm cells showed deficient binding of egg cells [ ] . the impact of this observation awaits further clarification. however, treatment with ace inhibitors is not reported to interfere with male fertility. evidence suggesting the existence a complete, functional intracellular ras within cells has been provided recently [ , ] . intracellular ras is reported to mediate changes in ca + fluxes and activation of genes [ ] . intracellular ang ii reportedly caused cardiac hypertrophy in vivo in mice [ ] . in these experiments, a plasmid construct under the control of an a-myosin promoter caused increased intracellular ang ii and % increase in relative heart weight. the mechanisms by which intracellular ang ii exerts its actions are not fully understood. thus, intracellularly applied arbs can only partly block intracellular ang ii. the role of intracellular ras is presently unclear. surprisingly, ace has been shown to function as a receptor of severe acute respiratory syndrome (sars) coronavirus [ , ] . ace is thought to contribute to pulmonary tissue damage and oedema by generating ang ii. ace is believed to normally counteract these harmful effects, but following attachment of sars virus to ace and replication, ace expression is diminished, less ang - is formed from ang ii and at receptor activation is intensified. in support of this contention, injection of recombinant ace into mice protected these mice from acute lung injury caused by sepsis or acid aspiration [ ] . thus, functioning ace may protect against the potentially lethal lung injury associated with sars [ ] . ageing is associated with alterations of several structural and functional properties of large arteries. increased wall thickness and stiffness, pulse wave velocity and pulse wave augmentation and deterioration of endothelial function are hallmarks of arterial ageing [ ] . these alterations form fertile soil for age-associated cardiovascular disease. conversely, cardiovascular disease causes acceleration of these detrimental changes. several lines of evidence support an important role of ras in arterial ageing as well as in cardiovascular disease. arterial components of the ang ii-signalling cascade increase with ageing [ ] . ang ii signalling via at receptors increases collagen production within the arterial wall, promotes reduced forms of nicotinamideadenine dinucleotide phosphate oxidase activity and enhances migration of vascular smooth muscle cells. increased formation of reactive oxygen species (ros) leads to activation of metalloprotease, less no bioavailability and endothelial dysfunction. formation of ros induced by ang ii may contribute to tissue ageing and age-related cardiovascular disease [ ] . ang ii also causes activation of nf-jb pathway and proinflammatory cytokines [ , ] . thus, judging by mechanistic criteria, ang ii appears to play a central role in many stimuli that govern arterial ageing and its functional responses. a recent study [ ] showed that treatment of male wistar rats with an ace inhibitor (enalapril mg · kg · day) or an arb (losartan mg · kg · day) for months or life-long resulted in prolongation of life span by % (enalapril) or % (losartan) compared with untreated control rats. the difference in life span could not be explained by cardiovascular protection. the authors speculate that prolongation of life span could be explained by reduction by ras inhibitors of ros formation and reduction of oxidative burden. in accordance with the generally acknowledged importance of ras in pathophysiology of cardiovascular disease, many mutations in ras component genes are associated with hypertension and cardiovascular diseases [ ] . the agt gene has been associated with hypertension [ ] , but attempts to predict responses to antihypertensive drugs based on agt polymorphisms have yielded inconsistent results [ ] . variants of the at and the at receptor genes are reportedly associated with hypertension, but they are also inconsistently associated with response to antihypertensive therapy [ ] [ ] [ ] [ ] . in a chinese cohort of hypertensive patients, the association with combined agt and at receptor single nucleotide polymorphisms (snp) haplotypes was modest ( % for systolic, % for diastolic bp reduction with ace inhibitor [ ] . in a prospective study comprising uk men, the at receptor genotype cc was associated with increased cardiovascular risk irrespective of bp [ ] whereas the at ii receptor a allele was associated with increased risk only at high systolic bp (> mm hg). in general, the magnitude of predictive power of ras gene, snps has been rather modest. a possible role for the at receptor in the central nervous system was first suggested by attenuated exploratory behaviour in at receptor-deficient (knockout) mice [ ] [ ] [ ] . in humans, the absence of or mutations in the at receptor gene was shown to be associated with severe x-chromosome linked mental retardation [ ] , showing a link between a ras component and development of cognitive functions. interestingly, a unique mutation of the renin receptor gene was later shown to be present in patients with x-linked mental retardation and epilepsy [ ] . functional analysis revealed that the mutated renin receptor could bind renin and increase renin catalytic activity. 'this finding confirmed the importance of the ras in cognitive processes and indicated a novel specific role for the renin receptor in cognitive functions and brain development' [ ] . considerable widening and deepening of our understanding of ras' physiology and pathopysiology has been achieved by genetical manipulation of experimental animals, e.g. rats [ ] and in mice [ ] . for instance, 'ace ⁄ mice', compound heterozygotes for the ace genes [ ] have no endothelial ace, but are nevertheless capable of maintaining normal physiology. the explanation for this appeared to be a compensatory increase in renal renin production followed by increased ang ii generation by nonendothelial ace, showing the plasticity of ras. this is just one example of fascinating gene manipulation revealing secrets of ras physiology that would otherwise have remained enigmatic. angiotensin-converting enzyme inhibitors and arbs (fig. ) are well established corner stones in the prevention and treatment of hypertension and cardiovascular disease, as demonstrated by numerous clinical trials and world wide clinical practice. according to a recent meta-analysis [ ] , ace inhibitors and arbs have similar bp-dependent effects for the risks of stroke, coronary heart disease and heart failure. for ace inhibitors, but not for arbs, there is evidence for bp-independent effects on the risk of coronary disease events [ ] . the benefits of ace inhibitors or arbs on renal outcomes probably result from bplowering effects [ ] whereas renoprotective benefits in diabetic patients may partly depend on factors beyond bp lowering [ ] . in fact, several studies suggest that both ace inhibitors and arbs offer benefits in addition to those mediated by bp lowering only. several trials have demonstrated a - % reduction of new onset diabetes during treatment with ace inhibitors or arbs [ ] . the mechanism behind this protective effect of ras inhibition is not clear, but it offers a significant advantage for ras inhibitors as we are experiencing a global epidemy of increasing incidence of diabetes. combined blockade of the ras by ace inhibitors and arbs has been shown to provide additional benefits compared with either drug class [ ] [ ] [ ] . however, these expectations were not confirmed by the recently published ontarget study [ ] which compared treatment with telmisartan, ramipril or both drugs combined in a megatrial comprising patients with high cardiovascular risk profile. of particular interest is the use in ontarget of telmisartan, by far, the most prominent activator of peroxisome proliferator activated receptor-c, a mediator of a host of favourable metabolic actions [ , ] . in the ontarget study, mean bp was lower in both the telmisartan group ( . ⁄ . mm hg greater reduction) and the combination therapy group ( . ⁄ . mm hg greater reduction) than in the ramipril group. telmisartan was equivalent to ramipril in terms of primary outcomes in patients with vascular disease or diabetes. the combination of telmisartan and ramipril was associated with more adverse events (hypotension, renal dysfunction) without an increase in benefit. the recent introduction of the first orally effective renin inhibitor, aliskiren, has raised additional interest in new possibilities of almost complete blockade of ras as a tool (fig. ) , perhaps, more effective than earlier, in the prevention and treatment of cardiovascular disease [ , , , ] . early reports on the use of aliskiren are promising, showing at least, an antihypertensive effect of aliskiren potent as those of other antihypertensive drugs [ ] . in particular, the combination of renin inhibitors with ace inhibitors and arbs may offer a solution to the 'renin escape' phenomenon [ ] , which implies that ace inhibitors or arbs may lose part of their effect during long time treatment. many questions are also raised, for instance, what would the consequences be if the 'beneficial' angiotensin peptide, ang - was not generated at all or 'benefical' at receptor mediated effects disappeared completely? only well conducted experiments and trials may answer such questions. we may look forward to interesting years ahead whilst waiting for results and answers. having witnessed an amazing plethora of ras discoveries from renin, in , to mutations in the at receptor and of renin receptor genes associated with x-chromosome linked mental retardation, we cannot avoid concluding that the physiology of ras is by far more complex and multilayered than one would have thought of. it appears quite unlikely that we have seen the whole picture yet. this challenging complexity and the central position of ras in the pathophysiology of cardiovascular disease will continue to inspire research and drug trials aiming at creating optimal pharmacological tools for ras modulation or blockade. design of specific inhibitors of angiotensin-converting enzyme: new class of orally active antihypertensive agents angiotensin ii receptors and 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antidiabetic mechanisms of angiotensin-converting enzyme inhibitors and angiotensin ii receptor antagonists: beyond the renin-angiotensin system renin inhibition with aliskiren: where are we now and where are we going? oral renin inhibitors fax: + ; e-mail: frej.fyhrquist@helsinki.fi) no conflict of interest was declared. key: cord- -egbl ljp authors: allen, a.m.; o’callaghan, e.l.; mendelsohn, f.a.o.; chai, s.-y. title: neuronal angiotensin date: - - journal: encyclopedia of neuroscience doi: . /b - - . - sha: doc_id: cord_uid: egbl ljp many tissues, including the brain, contain all components of the renin–angiotensin system and generate angiotensin peptides independent of the systemic, circulating system. within the brain renin, some questions remain as to how the precursor, angiotensinogen, and its processing enzymes interact to produce the active compounds, angiotensin ii/iii, because they are rarely localized to the same brain nucleus let alone the same cell. these questions aside, there is clear evidence for actions of angiotensin peptides in regions behind the blood–brain barrier. receptors for angiotensin peptides, including at( ) and at( ) receptors, are distributed in a characteristic pattern throughout the brain, with many of these sites behind the blood–brain barrier. stimulation of these receptors affects multiple physiological functions – actions which often complement the physiological roles established for the systemic renin–angiotensin system. these include effects on fluid and electrolyte homeostasis, autonomic and neuroendocrine regulation, and modulation of sensory function. moreover, administration of selective receptor antagonists attenuates several of these functions when they are activated in response to physiological stimuli, such as dehydration. together, these observations point to important roles for brain-derived angiotensin peptides in a wide range of physiological functions. a m allen, e l o'callaghan, f a o mendelsohn, and s-y chai, university of melbourne, melbourne, vic, australia elsevier ltd. all rights reserved. our understanding of the renin-angiotensin system began in the late s with the discovery of a pressor agent, called renin, derived from the kidney. further work throughout the s defined a humoral system in which the active agent, angiotensin (ang ii), was derived from a protein precursor, angiotensinogen (aogen), via the sequential action of two enzymes, renin and angiotensin converting enzyme (ace) (figure ). in this humoral system, aogen is produced by the liver and secreted into the circulation. renin, an aspartyl protease, is secreted into the plasma from cells of the juxtaglomerular apparatus in the kidney. renin's only known substrate is aogen, and the activity of this enzymatic reaction is considered to be the rate-limiting step in the pathway. cleavage of aogen yields a large glycoprotein of no known function and an inactive decapeptide, ang i. cleavage of ang i by the zincdependent carboxypeptidase, ace, produces the active octapeptide, ang ii. ace is located on endothelial cells throughout the vascular system, with a particularly high concentration in the lung. subsequent n-terminal cleavage of ang ii produces the heptapeptide ang iii, which is equipotent at ang ii receptors and may be an important ligand in some tissues. receptors for ang ii were cloned in the s and shown to be g-protein-coupled receptors with seven transmembrane spanning domains. two receptors, with approximately % homology, were cloned and termed at and at receptors. the at receptor, which mediates most of the classical actions of ang ii, was shown to occur in two isoforms in rodents, at a and at b . although the number of intracellular signaling pathways employed by the at receptor continue to expand, many of the principal actions involve coupling to the a q/ g-protein subunit. activation results in cleavage of phosphatidyl-inositol ( , ) bisphosphate, by phospholipase c b , to yield diacylglycerol and inositol trisphosphate with subsequent increases in intracellular calcium concentrations and activation of protein kinase c. the angiotensin at receptor signals via activation of the protein phosphatases pp a, sh domain-containing phosphatase, and mitogen-activated protein kinase phosphatase , resulting in the dephosphorylation and inactivation of kinases including growth factor tyrosine kinase receptors and extracellular signal-regulated kinase (erk- ) and (erk- ). antiproliferative and proapoptotic actions mediated by the at receptor appear to involve the inactivation of erk- and - by dephosphorylation. the binding of ang ii to the at receptor has also been shown to activate nitric oxide and phospholipase a in certain tissues. investigation into the signaling pathways associated with at receptor activation is further complicated by the complex physiological roles of the receptor, often in pathological states. studies have indicated the involvement of other enzymes and receptors in the activity of the reninangiotensin system ( figure ). angiotensin converting enzyme (ace ) was cloned and found to exhibit % homology with ace but with one catalytic domain. in contrast to ace, ace cleaves single amino acid residues from the c-terminus of peptides. ace is thought to have a counterregulatory role in the renin-angiotensin system, being involved in the degradation of ang ii and the generation of ang - , an effector peptide that often shows opposing actions to ang ii. the receptor for ang - was found to be the 'orphan' g-protein-coupled receptor that is encoded by the proto-oncogene mas. mice with a deletion of the mas proto-oncogene are not responsive to ang - and lose the binding site for ang - in the kidney. ace is also the receptor for some coronaviruses, including the virus that causes severe acute respiratory syndrome (sars-cov) and another virus associated with respiratory tract infections, hcov-nl . ace facilitates viral entry into and infection of target cells by binding specifically and with high affinity to the s protein of the viruses. angiotensin iv is generated from ang iii by the cleavage of the n-terminal arginine by aminopeptidase n. angiotensin iv was initially thought to be biologically inactive because it exhibited very low affinity for the at and at receptors. however, injection of ang iv into the cerebral ventricles enhances performance in memory tasks, and it was shown to bind to a specific, high-affinity site termed the angiotensin at receptor. subsequent work identified the at receptor as an enzyme, insulin-regulated amino peptidase. in the early s, evidence of local formation of angiotensin peptides in specific tissues began to emerge. it has since been demonstrated that many tissues, including kidney, heart, blood vessels, adrenal gland, uterus, testes, and brain, have the potential to produce ang independently of the circulating renin-angiotensin system. although the exact mechanism of formation remains controversial for many of these tissues, the existence of independently regulated tissue renin-angiotensin systems is widely accepted. as discussed in the following sections, there is also considerable evidence for the local generation of ang peptides in the brain which act on receptors within the blood-brain barrier to regulate a variety of functions. angiotensinogen is expressed in relatively high levels by the brain, with mrna levels reported to be approximately one-third those produced by the liver. measurements of aogen concentrations in cerebrospinal fluid by radioimmunoassay indicate that it represents % or % of the total protein content. this aogen is identical in amino acid structure to that produced by liver but may be differentially glycosylated. most aogen mrna expression in the brain occurs in ependymal and astroglial cells. interestingly, there is regional heterogeneity of expression, with highest levels in hypothalamic, midbrain, and medullary regions that are associated with the known actions of ang peptides. transgenic mouse lines with reporter protein expression under the control of the aogen promoter demonstrate neuronal expression of aogen but only in a very small number of cell groups. most evidence indicates that aogen synthesized by glia is constitutively secreted into the extracellular space; however, studies have demonstrated that some aogen might be directed to the nucleus. the relative proportion of intracellular versus secreted aogen existing in vivo and the role of the intracellular protein have not been examined. transgenic rat and mouse models demonstrate the functional importance of brain-derived aogen. transgenic rats which produce an aogen antisense under the regulation of a glial-specific promoter show multiple functional deficits, including decreased blood pressure and impaired fluid and electrolyte balance. evidence for the converse is available in mice, in which global overexpression of renin and aogen leads to increased blood pressure that can be reduced by glial-specific ablation of aogen production. together, these observations indicate that brain aogen plays an important role in the regulation of blood pressure, but they do not shed light on how the aogen is processed to yield ang ii and other active components of the system. the presence of high concentrations of aogen in astrocytes may also be suggestive of wider roles for the brain renin-angiotensin system. investigation of functional alterations in the aogen knockout mouse revealed impairment of blood-brain barrier function which can be restored by exogenous administration of ang ii or ang iv. this blood-brain barrier dysfunction is thought to be due to the decreased expression of glial fibrillary acidic protein and laminin in the astrocytes of these animals. efforts to demonstrate the existence and localization of renin in brain have proven extremely difficult. initial biochemical studies, which made every effort to remove contamination from systemic renin, demonstrated renin-like enzymatic activity in extracts of brain but did not establish the identity of renin. several other enzymes, including cathepsin d and g, tonin, and kallikrein, have the capacity to form ang peptides from renin substrate, and it remains a distinct possibility that enzymes other than renin might contribute to the generation of ang peptides in brain. the use of renin antibodies in immunohistochemical and western blot analysis to identify brain renin was also fraught with issues pertaining to antibody specificity. initial attempts to demonstrate the existence of renin mrna, using in situ hybridization or northern blot analysis, also produced variable results, most likely due to the use of whole brain, which thus diluted any renin present in discrete brain regions to below the level of detectability. using northern blot hybridization methods, dzau and colleagues found evidence for the existence of renin mrna in brain with levels approximately % of those found in kidney. in the kidney, renin is produced as preprorenin that is sequentially cleaved to form active renin. the renin gene has nine exons, the first of which encodes a signal peptide which targets the nascent protein to the endoplasmic reticulum for glycosylation and subsequent secretion from the cell. exon has three different transcriptional initiation points, which form different renin isoforms. experiments have demonstrated two distinct renin isoforms formed by tissue-specific transcriptional initiation in brain. initiation from exon a forms the classic 'renin a' found in the kidneys and systemic circulation, whereas the exon b transcription start point forms 'renin b.' this latter isoform lacks the coding for the signal peptide and is likely to be a constitutively active intracellular enzyme. these data raise the possibility of two distinct mechanisms of ang peptide formation in brainextracellular and intracellular. however, these studies did not provide information on the distribution of renin or evidence of the cell type responsible for production. other efforts have focused on the use of long lengths of renin promoter to drive expression of marker proteins in transgenic mice. using a . kbp of the flanking sequence of the renin gene to drive expression of green fluorescent protein, gross and colleagues demonstrated tissue-specific expression and regulation of the marker protein. using this mouse model, sigmund and colleagues localized renin expression in different regions of the mouse central nervous system. this expression occurs in many brain regions known to be involved in the regulation of fluid and electrolyte balance and autonomic control by ang ii and is predominantly localized to neurons. thus, current evidence supports the view that renin is expressed in the brain predominantly by neurons. the presence of renin isoforms indicates the potential for both intracellular and extracellular enzymatic activity. questions remain regarding the localization of renin in relation to the precursor, aogen, and how these interact to produce ang peptides in brain. the highest levels of renin expression occur in the cerebral and cerebellar cortices in regions where aogen expression is low or absent. observations in double transgenic mice, in which long sequences of the renin and aogen promoters are used to drive expression of different reporter proteins, indicate that coexpression of renin and aogen is rare; this is in line with observations that they are expressed in different cell types. however, adjacent cellular expression was observed in several nuclei, providing evidence to support the view that secreted aogen might be cleaved locally in the extracellular space or taken into neighboring neurons for further processing to ang i. within the circulation, ang i is rapidly converted to ang ii by the action of ace, a zinc-dependent metallopeptidase that cleaves a dipeptide from the c-terminus of ang i to form the effector peptide ang ii. specific inhibitors of ace are effective in inhibiting the formation of ang ii in vivo and attenuating the physiological effects of the peptide. hence, these inhibitors have been developed very successfully into therapeutic drugs that are widely used to treat hypertension, heart failure, vascular disease, and renal disease. in addition to enzymatic cleavage of ang i, ace cleaves a range of other small peptide substrates, many of which are present in brain, including bradykinin, substance p, met-enkephalin, beta-endorphin, dynorphin - , neurotensin, and luteinizing hormone releasing hormone. two isoforms of ace exist in mammals and both are membrane bound, although a secreted form of ace that is catalytically active is found in the circulation. the more abundant kda somatic enzyme occurs on the plasma membrane of endothelial and epithelial cells and has a large extracellular domain that contains two homologous regions, each with an active site. the testicular isoenzyme contains only one active site, which shares sequence homology with the c-terminal active site of the somatic enzyme and is the site that has been shown to selectively cleave ang i. in the many brain regions, ace occurs in high concentrations. many of these sites are also enriched with ang ii-containing cell bodies and fibers and ang at receptors, including the circumventricular organs, several hypothalamic nuclei, and the dorsal vagal complex in the medulla. it is proposed that at these sites, ace participates in the local production of ang ii, either from circulating ang i (as is the case for the circumventricular organs) or from brain-derived ang i. however, ace is much more abundantly distributed and high concentrations of the enzyme are detected in the basal ganglia, choroid plexus, hippocampus, cerebral cortex, and cerebellum, where its roles remain to be elucidated and may involve cleavage of other neuropeptide substrates. although the predominant form of ace in the brain appears to be membrane bound, a low level of the enzyme has been detected in cerebrospinal fluid, at a concentration -fold lower than that detected in serum. because they are cleavage products of a precursor that is secreted into the extracellular space, the cellular localization of ang peptides in the brain has proven difficult. immunohistochemistry has been problematic because of uncertain antibody specificity. detailed maps of the distribution of ang ii-like immunoreactive cells and fibers have been produced but have mostly relied on results obtained with one antibody. due to this scarcity of reliable antibodies, our knowledge of the neuronal circuitry that may utilize ang ii as a neurotransmitter is limited. however, in support of the veracity of the maps of ang ii distribution, there is a very high concordance in the distributions of ang ii-like immunoreactive nerve terminals and ang ii at receptors throughout the brain. there are some notable exceptions; for example, the magnocellular nuclei of the hypothalamus and central nucleus of the amygdala contain high levels of ang ii-like immunoreactive nerve terminals but low to undetectable concentrations of receptor. as outlined previously, several controversies exist in relation to the production of ang ii in the brain. some of the most convincing evidence for the existence of a brain renin-angiotensin system is derived from examination of the distribution of the ang receptors and the physiological actions exerted through them. unequivocal evidence points to the existence of ang at and at receptor mrna and binding sites (figure ) present in a characteristic distribution throughout the brain. these receptors show identical binding profiles to the peripheral receptor, including antagonism by the nonpeptide at and at receptor antagonists. in some cases, brain at and at receptors are located outside the blood-brain barrier, where they are accessible to systemic ang peptides. however, the vast majority of sites are behind the blood-brain barrier, where they are presumably accessed by brain-derived ang peptides. administration of ang peptides into these regions generally results in neuronal excitation and a characteristic range of site-dependent physiological responses. more important, in the context of establishing a role for brain-derived ang peptides, pharmacological blockade of these receptors with specific and selective antagonists attenuates many physiological responses, such as drinking following dehydration or autonomic activation in response to mental stress. together, these observations provide strong support for the existence of brain-derived ang peptides that regulate physiological functions in the central nervous system. there is an extensive distribution of ang ii at receptors in the brain. moderate to high levels of at receptors occur in the sensory circumventricular organs (subfornical organ and vascular organ of the lamina terminalis and area postrema); piriform cortex; lateral septum; bed nucleus of the stria terminalis; median preoptic nucleus; periventricular, parvocellular paraventricular, and dorsomedial nuclei of the hypothalamus; locus coeruleus; lateral parabrachial nucleus; nucleus of the solitary tract; spinal nucleus of the trigeminal tract; and ventrolateral medulla. most of these sites also possess high levels of aogen-expressing astrocytes and ace. electrophysiological studies have demonstrated that neurons in many of the sites listed previously are responsive to local application of ang ii. the predominant response is excitation via an at receptor-mediated inhibition of outward potassium channels. microinjection studies with exogenous ang ii and/or at receptor antagonists have identified several actions of ang ii in the brain. in brief, these include the following: . alterations in autonomic activity to the cardiovascular system, leading to increases in blood pressure and heart rate, through direct stimulation of efferent pathways and regulation of sensory afferent (e.g., the baroreceptor reflex) information . increases in fluid and salt intake . stimulation of the secretion of several neuroendocrine hormones, including vasopressin, adrenocortictropic hormone, and luteinizing hormone releasing hormone . effects on learning and memory angiotensin at receptors occur in high levels with a wide distribution in the brain during development. in the adult, this distribution becomes restricted to a few sites, including some thalamic nuclei, the cerebellum, and the inferior olivary nucleus. this distribution indicates a role in sensory information processing. the phenotype of the at receptor knockout mouse also points to some effects of ang ii on these receptors in the brain. these mice show reduced spontaneous movement and exploratory behavior, and there is evidence for effects of ang ii on cognitive function and memory. however, these are often contradictory in regard to potentiation or inhibition of memory formation. it has been proposed that the detrimental effects of ang ii on memory consolidation and retrieval are mediated by the at receptor. further support for the angiotensin at receptor altering cognitive function comes from the detection of mutations in the coding region of this receptor in patients with mental retardation. schematic diagram representing the distribution of angiotensin at and at receptors in the brain. the distribution of receptors, determined by either in vitro autoradiography or in situ hybridization histochemistry, is depicted on midsagittal sections of the rat brain. ang - and its binding site, the mas oncogene angiotensin - may play a role in the central regulation of blood pressure because microinjection of ang - into various sites alters arterial pressure and stimulates release of vasopressin. ang - has also been reported to enhance long-term potentiation in the lateral nucleus of the amygdala and in the ca region of the hippocampus, suggesting a role in memory formation. ang iv and its binding site, irap intracerebroventricular injection of ang iv in rats enhances performance in several different memory tasks. a binding site that recognizes ang iv with high affinity, but not ang i, ang ii, at , or at antagonists, was named the at receptor based on its distinct pharmacology. a surprising finding was the discovery that this receptor is the previously known insulin-regulated aminopeptidase (irap), a transmembrane zinc metallopeptidase known to traffic with the glut- glucose transporter in response to insulin. ang iv, lvv-hemorphin- , and other at ligands were all found to be competitive inhibitors of irap but were not cleaved to any appreciable extent by the enzyme. the mechanism by which ang iv and other at ligands mediate their memory effects is yet to be resolved. during the past several decades, it has been established that ang peptides are generated in the brain. however, despite considerable effort, many questions regarding the biochemical pathways involved in the generation of this peptide system remain incompletely resolved. this system appears to have unique properties with, for example, precursor and processing enzymes existing in different cellular compartments and the potential for neurotransmitter, paracrine, and even intracrine effects. yet despite these deficiencies in our knowledge, it is clear that ang peptides play important physiological and pathological roles in the central nervous system regulation of a wide variety of functions, including those that complement its peripheral actions (e.g., autonomic regulation and fluid and electrolyte balance) and other more disparate functions, such as modulation of memory and blood-brain barrier formation. see also: angiotensin ii; angiotensin actions on and within brain. central neural regulation of cardiovascular function by angiotensin -a focus on the rostral ventrolateral medulla brain renin-angiotensin system: lessons from functional genomics the angiotensin iv/at receptor physiology of local renin-angiotensin systems molecular evidence of tissue renin-angiotensin systems: a focus on the brain angiotensin-( - ) and the renin-angiotensin system key: cord- -f e ynhu authors: sarangarajan, rangaprasad; winn, robert; kiebish, michael a.; bountra, chas; granger, elder; narain, niven r. title: ethnic prevalence of angiotensin-converting enzyme deletion (d) polymorphism and covid- risk: rationale for use of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers date: - - journal: j racial ethn health disparities doi: . /s - - - sha: doc_id: cord_uid: f e ynhu rationale: hypertension, obesity and diabetes are major risk factors associated with morbidities underlying covid- infections. regression analysis correlated presence of ace insertion/deletion (i/d) polymorphism to covid- incidence and mortality. furthermore, covid- prevalence correlated to allele frequency of angiotensin-converting enzyme (ace) deletion (d) polymorphism within the european population. objective: homozygous ace deletion polymorphism is associated with increase in ace and angiotensin ii (ang-ii), sustained levels can result in inflammation, fibrosis and organ damage. the ace dd polymorphism is also associated with hypertension, acute respiratory distress and diabetic nephropathy, all considered high risk for covid- infection and outcomes. the study objective was to describe a biological framework associating ethnic prevalence of ace deletion polymorphism to covid- comorbidities providing rationale for therapeutic utility of ace-i/arbs to improve outcomes. method and results: the allele frequency database (alfred) was queried for frequency of rs representing ace i/d polymorphism. in a total of worldwide population samples, frequency of ace d allele was higher in european, asian, and africans cohorts. in the usa, the frequency of ace d allele was higher in non-hispanic black compared with non-hispanic white and mexican americans. conclusion: covid- binding mediated reduction/inactivation of ace-ii can increase ace/ang-ii signalling pathway and related pathologies. the presence of ace dd polymorphism with covid- infection likely augments ace/ang-ii activities, increasing severity of covid- morbidities and impacts outcomes. thus, ethnic prevalence of ace dd polymorphism can explain in part the severity of covid- morbidity providing rationale for the use of ace-i/arbs to improve outcomes. the sars-cov- (covid- ) infection has infected in excess of seventeen million individuals around the globe and is designated as a pandemic by the world health organization. the global efforts are focused on understanding the disease onset, progression and to identify causal linkage for differences in observed outcomes among the affected population and within specific demographics. despite worldwide spread of the covid- infections, european countries and the usa appear to have experienced higher incidence and mortality rates [ ] [ ] [ ] . hypertension, obesity, and diabetes were identified as the most common comorbidities associated with covid- infection; higher severity of disease and mortality was generally reported in the elderly (> years) population. angiotensin-converting enzyme (ace ) is the predominant receptor for sars-cov viral entry and infection, resulting in the reduction of expression of ace [ , ] . ace is an enzyme component of the renin-angiotensin system (ras), a complex integrated network of peptides-enzyme combination, generating catalytically active peptides with prominent influence on the vascular, renal, cardiac, and immune system [ ] . in this report, we describe a framework of the pathophysiological consequence of covid- -induced reduction in ace , i.e., overactivation of the ras pathway with the potential to have deleterious effect on organ functions including the lungs, kidneys, heart, and immune system. the deleterious activities of ras within the covid- -infected cohorts can be further amplified by the presence of genetic polymorphism in the angiotensin-converting enzyme (ace). increased prevalence in frequency of the ace polymorphism within ethnic groups, in part, is likely responsible for the observed severity of covid- comorbidities and mortality in this population. this is substantiated by recent regression analysis linking presence of ace- i/d (insertion/deletion) polymorphism with incidence and mortality with covid- infection [ ] . renin-angiotensin system: ace, ang-ii, and inflammation the ras system has a prominent role in the regulation of vascular dynamics; its components directly or indirectly influence functions of the lung, heart, kidney, brain and the immune system [ ] . in addition to central ras components, i.e., renin (kidney), ace (lungs), and angiotensinogen (liver), tissue-specific localized systems including the kidney, heart, and lungs have been identified [ , ] . within ras, the canonical angiotensin-converting enzyme (ace) is responsible for conversion of angiotensin- (ang-i) to angiotensin- (ang-ii) (fig. a) . subsequently, ang-ii mediates its effects through activation of at- and at- receptors, resulting in distinct intracellular signalling pathways [ ] [ ] [ ] . activation of at- receptors is associated with the well-characterized physiological actions of ang-ii in various organs including the lung, heart, kidney, and the vascular system [ ] . in addition to its hemodynamic effect, ang-ii has significant pro-inflammatory effects, promoting generation of reactive oxygen species (ros), cell proliferation, extracellular matrix remodelling, and regulation of gene expression via signalling pathways leading to tissue injury [ , ] . ang-ii promotes expression of proinflammatory chemokines in the kidneys, heart, and vasculature to induce inflammation [ ] . several studies have characterized key inflammatory processes influenced by ang-ii on macrophages, dendritic cells, and mesangial cells resulting in mobilization and activation of cytokines, chemokines, and pro-inflammatory factors resulting in tissue damage and progressive organ failure [ ] . due to profound influence of ang-ii signalling pathways that are predominantly adverse when unmitigated, the potency of ang-ii is tightly regulated via proteolytic activities of enzymes to generate various angiotensin peptide fragments with physiological activities different from ang-ii [ ] (fig. a) . ace is an enzyme component of ras, with proteolytic activities different from the canonical ace. ace is responsible for cleaving angiotensin i to ang ( - ) and angiotensin- to ang ( - ) peptides respectively (fig. ) , of which the latter is a potent vasodilator [ , ] . several studies support a major role for ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) in providing the counterbalance to the physiological effects of ang-ii [ ] [ ] [ ] . thus, the pro-inflammatory effects of ace/ang-ii axis are balanced by activation of anti-inflammatory pathways by ace and other systems. two recent publications reported that ace insertion/ deletion polymorphism correlated to infectivity and mortality associated with covid- infections [ , ] . in humans, the gene encoding ace is located on chromosome and exhibits an insertion/deletion polymorphism that is characterized by an insertion (allele i) or deletion (allele d) of a base pair marker in intron that results in three different genotypes, i.e. dd or ii homozygotes or id heterozygotes. it is reported that the deletion (d) allele occurs in % of the population and associated with increased ace activity, implicating the presence of d allele with disease pathologies associated with ras activity [ ] . the allele frequency database (alfred; https://alfred. med.yale.edu/alfred/index.asp; rrid:scr_ ) was queried for frequency of rs representing ace i/d polymorphism, one of the best studies of all ace polymorphisms. the allelic frequencies of the insertion (i, +) and deletion (d, −) genotypes within various geographic regions from population samples were obtained from alfred and are summarized in table . inclusion of data from all european studies demonstrated almost equal distribution of the ace (i) or ace (d) allele, with italians, ashkenazi jews and canarians demonstrating slightly higher prevalence compared with the population averages. in contrast to europe, among the african population, the frequency of d allele was almost twice compared with the i allele among population samples with highest levels observed in pygmies, ethiopian jews, moroccan, nigerian and tunisian populations. these are consistent with other studies reporting significant increase in the frequency of deletion polymorphism of ace observed in individuals of african descent and associated with disease pathology [ ] . specifically, a prevalence of the d allele of % has been reported in individuals of african descent [ ] . in the usa, the non-hispanic black population has higher frequency of the d allele (table ) compared with non-hispanic white and mexican american population [ ] . the frequency of the d allele was increased compared with the i allele within the middle eastern population with higher values observed in both arab and saudi arabia sample populations. in contrast to africa and middle east, increased frequency of the i allele was observed in sample populations from asia (india, pakistan nepalese, tajik regions and sri lanka), oceania (new zealand, papua new guinea and micronesia), east asia (china, japan, korea, taiwan, cambodia, vietnam, philippines and malaysia) and south american countries. fig. a overview of the reninangiotensin system. the figure describes the basic components of the renin angiotensin system with focus on the impact of ace and ace in the generation of angiotensin peptides, the respective cognate receptor(s) and corresponding physiological consequence of receptor activation. b influence of ace deletion (dd) polymorphism on reninangiotensin system. the figure describes the consequence of the ace deletion polymorphism, the increase in levels of ace and angiotensin ii resulting in activation of at- receptor and downstream pathophysiological effects. c consequence of covid- infection and ace deletion (dd) polymorphism on reninangiotensin system. the figure describes the increased activation of ace and generation of ang-ii as a consequence of covid- -mediated reduction in ace in the presence of ace deletion polymorphism. the result is disruption of physiological balance of the ace/ace axis resulting in overactivation of at -r signalling and associated pathological consequence ace deletion (d) polymorphism and disease-increased susceptibility and severity to co-morbidities associated with covid- although the ace i/d polymorphism is located in a noncoding region, its presence is directly linked to regulation of renin-angiotensin system and associated pathological conditions. a positive association between d allele and high blood pressure, atherosclerosis, coronary artery disease, stroke, diabetic nephropathy and alzheimer's disease has been extensively reviewed [ ] . the molecular underpinning of these diseases is multi-factorial and complex, and the presence of the ace deletion polymorphism may contribute to influence disease pathology. indeed, to date, there is distinct lack of consensus studies linking the presence of ace deletion polymorphism to disease causality. nevertheless, the increase in levels of ace in individuals with the id and dd genotypes and potential augmentation of the ras system and associated signalling cascades can influence pathways to influence disease pathology [ ] (fig. b) . indeed, increased levels of ace and ang-ii have been implicated in the pathophysiology of [ , ] ) and kidney disease (chronic kidney disease, diabetic nephropathy [ , ] ). in the african american population, the deletion polymorphism is associated with increase in systolic blood pressure, hypertension and altered vascular reactivity with potential impact on cardiovascular disease [ ] [ ] [ ] . a subset of individuals with a positive diagnosis of covid- infection have rapid progression of lung dysfunction leading to acute respiratory distress with potential need for ventilatory support [ , ] . presence of ace insertion/ deletion (i/d) polymorphism is associated with susceptibility and is an independent risk factor for mortality in patients with acute respiratory distress syndrome (ards) [ , ] . of the three ace polymorphisms, there is positive association with frequency of the dd allele and incidence of ards, increased fatality and a prognostic factor of outcomes [ ] [ ] [ ] . further, the dd genotype is usually associated with higher ace levels relative to other genotypes and with increased mortality in acute lung injury (ali)/ards patients [ , ] . elevated levels of ace have been observed in the bronchoalveolar fluid of individuals with ards [ ] . although decreases in circulating ace have been reported in ards patients [ ] , this might be a consequence of the progressive damage to lung tissue as increased levels of ace are evident in the bronchoalveolar fluids of individual with ards [ ] . the positive relationship between dd genotype and ali/ards and the corresponding increase in ace levels suggest the potential involvement of increased ang-ii in the etiopathology of ards. during the avian (h n ) flu infections, approximately % of patients developed ards [ ] . in a subset of infected patients, increase in plasma ang-ii levels was linked to severity and fatal outcomes [ ] . within the covid- -infected population, there is increased incidence of kidney injury associated with higher mortality rates [ , ] . chronic kidney disease (ckd) is associated with severity of covid- infection [ ] . interestingly, both ace and ace expressions in the kidneys are predominant in the proximal tubules with minor expression in the glomerular apparatus [ ] . the balance between ang-ii and ang ( - ) affects renal ras to maintain balance of kidney functions; imbalance of the ratio results in kidney disease [ ] [ ] [ ] . chronic kidney disease is characterized by decreases in cardiac and renal ace in human [ ] . diabetic nephropathy (a ckd) is characterized by decrease in ace , increased ace and ang-ii-mediated tubular and glomerular damage as a result of renal ras activation [ , ] . based on these studies, the ability of covid- to bind and decrease ace in target tissues is most likely responsible for the observed increase in blood urea nitrogen, proteinuria and hematuria associated with kidney damage [ ] . thus, covid- -associated decrease in ace most likely results in disruption of the ace/ace balance in the kidney leading to sustained activation of ace and ang-ii activities and kidney damage. ace insertion/deletion polymorphism is also associated with diabetic kidney disease, the frequency of dd and id genotype distribution being higher compared with non-diabetic kidney disease cohorts, leading to functional decline [ , ] . the above observations suggest that presence of the dd genotype of ace in patients with covid- infection may be associated with severe respiratory distress compared with the other genotypes. multiple studies have reported on the prevalence of ace i/ d polymorphism, specifically the id and dd polymorphism in increasing levels of ace and ang-ii, which could in part influence susceptibility to underlying pathologies considered high risk for covid- infections, progressive organ dysfunction and poor outcomes. thus, presence of id and dd polymorphism by itself is a potential underlying risk factor associated with severity and outcomes in individuals with positive diagnosis of covid- infection [ , ] . the proteolytic cleavage of ang-ii by ace to generate ang ( - ) represents a major event leading to the physiological inactivation of ang-ii function. thus, in patients with active covid- infections, decrease in ace expression/activity should most likely lead to sustained ace-mediated generation of ang-ii and downstream signalling deleterious to organ functions including that of lung, kidney and heart [ ] . although the status of circulating and lung ace levels in covid- patients is unclear, the ability of sars-cov- binding specifically to ace decreases its expression and activity suggesting upregulation of ace/ang-ii-mediated activities. this is consistent with the observation that knockdown of ace is associated with severe ards in multiple rodent models compared with corresponding wild-type controls [ ] . loss of ace expression in mutant mice is associated with worse lung function and characterized by increases in vascular permeability, lung oedema and neutrophil accumulation [ ] . interestingly, reduced plasma levels of ace are also observed within populations of african descent including african americans, specifically in individuals with pre-hypertensive status, diabetes and renal disease [ , ] . administration of a catalytically active recombinant ace protein improved symptoms of acute lung injury in ace knockout and wild-type mice [ ] . in a pilot clinical investigation, administration of recombinant human ace (apn ) in patients with acute respiratory distress was associated with rapid decrease in ang-ii level and did not significantly influence oxygenation indices in the treated population compared with placebo-controlled group [ ] . the recombinant human ace is undergoing renewed clinical testing in the covid- patient population to investigate clinical outcomes [ ] . sars-cov- binding to ace results in reduction of protein expression, activity and ability to generate anti-inflammatory signalling, all of which contribute to a pro-inflammatory phenotype due to presence of ace activity and ang-ii signalling (fig. c) . presence of ace d polymorphism increases ace levels and ang-ii leading to pro-inflammatory phenotype and is associated with disease susceptibilities considered high risk for covid- infections. recently, it was proposed that reduced plasma levels of ace in individuals of african descent most likely lowers potential for covid- infection [ ] ; the overall outcomes in individuals with presence of ace deletion polymorphism after infection with covid- most likely leads to exacerbation of comorbidities and overall deleterious outcomes. based on the described biological consequence of covid- infections on the ras system, treatment with ace-i and arbs should be associated with improved outcomes within the overall covid- patient cohorts. indeed, several meta-analyses provide preliminary support for the potential benefits of the use of ace-i/arbs in management of covid- infections. in a multicenter study of adult patients with hypertension with positive covid- diagnosis, in-patient use of ace-i/arb was associated with reduced risk of mortality from all causes when compared with patients not treated with the medications [ ] . recent publications further highlight the use of ace-i and arbs in providing cardiovascular and renal benefits to patients with covid- diagnosis [ , ] . in a metaanalysis, patients treated with ace-i/arbs had % reduction in odds of developing severe disease and death compared with patients not treated with ace-i/arbs [ ] . these studies provide rationale for investigation into the utility of ace-i/arbs in the ethnic population with known prevalence of ace deletion polymorphisms in an effort to mitigate severity and improve outcomes in response to covid- infections. ace is a multi-functional, relatively non-specific peptidase enzyme with a wide range of substrate specificities that impact physiological pathways in influencing blood pressure, haematopoiesis, hormone regulation, renal function and immune responses. the specificity of hypertension and cardiovascular disease as underlying causes for severity of covid- infection, the inherent role of ace-mediated generation of ang-ii and downstream signalling to potentially exacerbate inflammation and organ damage along with genotypic impact on ace status provide compelling support of the use of ace-i and arbs in the clinical management of patient with positive diagnosis of covid- . the biological impact of the presence of deletion polymorphism of ace in individuals with covid- infection provides a significant rationale for serious consideration of shortterm use of ace-i and/or arbs in patients without underlying issues with blood pressure or cardiovascular disorder. the guidance statement issued by the heart failure society of america (hfsa), the american college of cardiology (acc) and american heart associated (aha) states that in the absence of favourable or detrimental effects of ace-i and arbs in the covid- setting, the recommendation is to not arbitrarily or pre-emptively discontinue these agents in patients currently on the medication as standard of care (acc. org). indeed, both ace-i and arbs have been extensively used in conditions ranging from hypertension, congestive heart failure, prevention of kidney failure and other indications. both classes of drugs have extensive use history, understanding of safety, tolerability, efficacy, adverse events profile and drug interactions. the significant genetic, scientific and clinical data supporting a potential role for increased ace levels and associated ang-ii effect in target organs provides compelling argument for use of ace-i and arbs in the clinical management of patients with covid- infections to improve outcomes. high salt sensitivity-associated low plasma renin activities are responsible for the attenuated blood pressure-lowering response of ace-i in the african american population [ ] . however, this particular phenomenon might be of potential advantage in dosing and management of severity of covid- -associated morbidities in african american and other ethnic populations with ace deletion polymorphism. in summary, this study describes the biological relevance of genetic polymorphism of ace deletion with higher prevalence in certain ethnic populations including african americans in context of covid- infection and rationale for the use of ace-i/arbs for therapeutic management of severity of morbidity and improving outcomes associated with covid- . author contributions rs is responsible for concept; mak, cb, eg, nrn contributed to the structure and content of the manuscript. funding mr. carl e. berg supported this work. data availability all data obtained from public resources and referenced in the manuscript. conflict of interest the authors declare that they have no conflict of interest. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. covid- in europe: the italian lesson coronavirus disease (covid ) in the eu/eea and the uk -ninth update hospitalization rates and characteristics of patients hospitalized with laboratoryconfirmed coronavirus disease -covid-net, states sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor tissue distribution of ace protein, the functional receptor for sars coronavirus: a first step in understanding sars pathogenesis working outside the system: an update on the unconventional behaviour of the renin-angiotensin 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syndrome compared evolution of plasma fibronectin and angiotensin-converting enzyme levels in septic ards angiotensin ii plasma levels are linked to disease severity and predict fatal outcomes in h n -infected patients kidney impairment is associated with in-hospital death of covid- patients caution on kidney dysfunctions of covid- patients chronic kidney disease is associated with severe coronavirus disease (covid- ) infection expression of ace and ace in individuals with diabetic kidney disease and healthy controls high-salt diet increases glomerular ace/ace ratio leading to oxidative stress and kidney damage ace alterations in kidney disease synergistic expression of angiotensin-converting enzyme (ace) and ace in human renal tissue and confounding effects of hypertension on the ace to ace ratio angiotensin converting enzyme activity in patients with chronic kidney disease the association of ace gene polymorphism with diabetic kidney disease and renoprotective efficacy of valsartan 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converting enzyme inhibitors) in coronavirus disease (covid- ) the effect of angiotensin converting enzyme inhibitors and angiotensin receptor blockers on death and severity of disease in patients with coronavirus disease [covid- ]: a meta analysis. medrxiv. ; his version posted a review of ace inhibitors and arbs in black patients with hypertension publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -sgts l authors: simões e silva, ana cristina; lanza, katharina; palmeira, vitória andrade; costa, larissa braga; flynn, joseph t. title: update on the renin–angiotensin–aldosterone system in pediatric kidney disease and its interactions with coronavirus date: - - journal: pediatr nephrol doi: . /s - - - sha: doc_id: cord_uid: sgts l the last decade was crucial for our understanding of the renin–angiotensin–aldosterone system (raas) as a two-axis, counter-regulatory system, divided into the classical axis, formed by angiotensin-converting enzyme (ace), angiotensin ii (ang ii), and the angiotensin type receptor (at r), and the alternative axis comprising angiotensin-converting enzyme (ace ), angiotensin-( - ) (ang-( - )), and the mas receptor. breakthrough discoveries also took place, with other raas endopeptides being described, including alamandine and angiotensin a. in this review, we characterize the two raas axes and the role of their components in pediatric kidney diseases, including childhood hypertension (htn), pediatric glomerular diseases, congenital abnormalities of the kidney and urinary tract (cakut), and chronic kidney disease (ckd). we also present recent findings on potential interactions between the novel coronavirus, sars-cov- , and components of the raas, as well as potential implications of coronavirus disease (covid- ) for pediatric kidney diseases. the renin-angiotensin-aldosterone system (raas) was first conceived as a single-arm hormonal axis centered on the biological functions of angiotensin ii (ang ii). the raas has subsequently grown in scientific relevance due to its multiplicity of physiological interactions. nowadays, the raas is considered a two-arm, counter-regulatory system, with the most important peptides being ang ii and angiotensin-( - ) (ang-( - )) [ ] . figure shows the raas cascade. the present knowledge about raas, however, has gradually developed over the years. the concept of the raas classical axis started with the discovery of vasoactive substances related to left ventricular hypertrophy [ ] . the primary raas fragments were described after several experimental studies using arterial hypertension models as a result of renal ischemia [ ] . the first fragment identified by tigerstedt and bergman was the aspartyl-type acid protease renin. the formation of ang ii by means of the removal of two amino acids from angiotensin i (ang i) by the angiotensin-converting enzyme (ace), however, was not described until by groups in argentina and the usa [ ] . ang ii, hence, was determined as a potent vasoconstrictor that elevates arterial blood pressure (bp) by increasing vascular resistance. further characterization occurred in the s, as the binding mechanism of ang ii to the membrane-binding sites, later named as angiotensin ii receptor types and (at r and at r), was clarified after studies based on raas nonspecific antagonism with saralasin [ ] . hence, it was established that ang ii contributes to sodium/potassium balance, body fluid volume and arterial pressure homeostasis by stimulating vasoconstriction, sodium reabsorption, and aldosterone release [ ] . the complementary biological functions of the ace/ang ii/at r axis included the discovery of new roles for ang ii, including its proinflammatory properties, by several research groups [ ] . the first evidence of the alternative raas axis was established in , when santos et al. [ ] described the formation of ang-( - ) by an ace-independent pathway. in the same year, schiavone et al. [ ] reported that ang-( - ) is equipotent to ang ii in releasing vasopressin (avp) from the rat hypothalamo-neurohypophysial system (hns) in vitro. a year later, campagnole-santos et al. [ ] first described the biological actions of ang-( - ) in vivo, including several vasodilatory effects related to the release of nitric oxide and prostaglandins, therefore antagonizing the well-established classical raas arm. in , tipnis et al. [ ] and donoghue et al. [ ] simultaneously discovered the production of ang-( - ) by means of the removal of a phenylalanine from the ang ii, catalyzed by the carboxypeptidase angiotensin-converting enzyme (ace ). more importantly, in , the g proteincoupled mas receptor (masr) was identified as the specific binding-site for ang-( - ) [ ] . similar to the classical axis, the ace /ang-( - )/masr axis also has functions beyond the kidney and cardiovascular systems, including inhibition of cell growth and anti-inflammatory actions [ ] . when the molecular basis for the physiological function of the alternative raas axis was clearly established, the system was conceived as composed of two main axes with opposite local and systemic effects [ ] . figure shows the opposite actions of both raas axes. the diversity of the raas, however, goes far beyond these two axes. new hormone fragments and functions continue to be discovered, including pro-renin, alamandine, angiotensin-( - ) and angiotensin iv [ ] . given the expanded understanding of the raas, this review aims to summarize the biochemical and physiological roles of the raas, as well as to discuss the involvement of this system in pediatric kidney diseases and related conditions. furthermore, we also present recent findings on the potential interactions between the novel coronavirus, sars-cov- , and raas components and some potential implications of coronavirus disease (covid- ) for pediatric kidney diseases. the raas is well-known for its major role in bp control and fluid balance [ ] . its endocrine (tissue-to-tissue), paracrine (cell-to-cell), and intracrine (intracellular/nuclear) actions are crucial for cardiovascular, kidney, immune, and neural homeostasis [ ] . metabolic pathways of the classical raas axis start with the conversion of angiotensinogen (agt) into angiotensin i (ang i) by renin. the subsequent cascade of events depends on the activity of the two angiotensinconverting enzymes to either form ang ii or ang-( - ), the effector peptides of the raas [ ] (fig. ) . the agt gene is located on chromosome and leads to the expression of a amino acid peptide (asp-arg-val-tyr-ile-his-pro-phe-his-leu-leu-val-tyr-ser) that is fig. then renin-angiotensin system cascade. legend: the liver produces angiotensinogen (agt), a -amino-acid peptide, which is secreted in sinusoidal capillaries. the -amino-acid ( kda) human agt glycoprotein contains a signal peptide that is removed co-translationally to yield the -amino-acid substrate of renin resulting in amino acids plus amino acids. renin, an enzyme produced by the juxtaglomerular apparatus of the kidney, is also secreted in the circulation. in the blood stream, angiotensinogen is cleaved by renin into ang i, which can also be cleaved by ace , producing ang- - ; by nep or pep, producing ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ; and by ace, producing ang ii. from that point, ang ii can also be cleaved by ace and produce ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) or it can be cleaved by apa, producing ang iii. ang ii can still have its first amino acid replaced, forming ang a, which can be cleaved by ace into alamandine. this heptapeptide alamandine can also be formed by ang-( - ) due to replacement of one amino acid. ang iii can be cleaved by apn and form ang iv. ang i, angiotensin i; ang ii, angiotensin ii; ang iii, angiotensin iii; ang iv, angiotensin iv; ang - , angiotensin ( - ); ang - , angiotensin ( - ); ang a, angiotensin a; ace , angiotensin-converting enzyme; nep, neutral-endopeptidase; ace, angiotensin-converting enzyme; ad, aspartate decarboxylase; apa, aminopeptidase a; apn, aminopeptidase n; apb, aminopeptidase b primarily synthesized by hepatocytes. however, adipose tissue, brain, spinal cord, heart, kidney, lung, adrenal gland, small and large intestine, stomach, spleen, ovaries, and vascular endothelium are also capable of producing agt [ ] . once synthesized, agt is released into the bloodstream and has a -h half-life [ ] . agt is the only substrate of renin. although renin is the main enzyme that converts agt into ang i, this conversion may also occur by the enzymes chymase; tonin; cathepsins a, d, and g; proteinase- ; and neutrophil elastase [ ] . the physiological roles of these enzymes, however, require further characterization [ ] . renin is an aspartyl protease with two isoforms. the first isoform is expressed in the juxtaglomerular (jg) cells and the collecting duct (cd) and has extracellular functions [ ] . renin expressed in jg cells is secreted as pro-renin (inactive) or renin (active) itself and is capable of catalyzing the production of ang ii, which negatively regulates jg production of the enzyme. on the other hand, renin secreted by the cd into the tubular fluid also produces ang ii, and in turn the octapeptide upregulates cd production of renin. both are key rate-limiting steps in determining ang ii levels [ ] . hence, regulation of renin expression and secretion significantly impact overall activity of the raas. the main stimuli for renin release include the kidney baroreceptor reflex [ ] , sodium chloride (nacl) concentration in the macula densa, and renin microrna expression and stimulation by other molecular pathways, including nitric oxide, prostaglandins, adenosine, and β -adrenergic receptors [ ] . the second isoform of renin is related to the intracellular environment. the pathways and location of this second isoform are as yet not well established, but findings suggest an intra-mitochondrial location and association with increased bp [ ] . ang i, the product of agt, is an inactive peptide with a amino acid chain (asp-arg-val-tyr-ile-his-pro-phe-his-leu). cleavage of angiotensinogen also generates a amino acid chain named des-(ang i)-agt. this remaining fragment appears to have function in plasma, but its actions on bp lack characterization [ ] . subsequently, in the classical raas axis pathway, ang i is converted into ang ii by ace, which is a dicarboxypeptidase with four isoforms, generated by alternative splicing of the ace gene [ ] . regarding ace isoforms, isoforms , , and are expressed somatically, while isoform is expressed in the testicles [ ] . somatic ace is an ectoenzyme located on the surface of endothelial cells throughout the body, particularly abundant in lungs, intestine, choroid plexus, placenta, and on brush border membranes of kidney tubular cells. a soluble circulating form of ace can be produced after the enzymatic cleavage of tissue-bound ace at its transmembrane domain [ ] . ace has two main substrates, both related to bp homeostasis: ang i and bradykinin, a peptide with vasodilator and natriuretic properties [ ] . ang ii is the central octapeptide (asp-arg-val-tyr-ile-his-pro-phe) of the classical raas axis. the biological functions of ang ii take place both in the intra-and extracellular space and are mediated by two cell surface receptors, at r and at r [ ] . the receptors belong to a -transmembrane receptor family divided by nonpeptide antagonists' affinity difference; however, at r only has ∼ % sequence homology with at r [ ] . although the at r has been described to have two subtypes (at a and at b) in some animal models [ ] , only one gene has been found to codify at r in the human genome [ ] . physiological responses attributed to ang ii-at r binding include stimulation of kidney tubular sodium reabsorption, aldosterone release from the adrenal glomerulosa, smooth muscle cell contraction, and stimulation ang ii binds to at receptors and produces the traditional actions of ras classical axis, including stimulation of sns tone, of vasoconstriction, of inflammation, and of fibrosis and decrease of baroreflex sensitivity, of natriuresis, and of no release. the alternative axis, however, is activated mostly due to ang-( - ) binding to mas receptors, promoting counter-regulatory actions, including decrease of sns tone, of blood pressure, and of cardiac hypertrophy; increase of baroreflex sensitivity, of no release, and of natriuresis; and stimulation of vasodilatation. ang - , angiotensin - ; ang ii, angiotensin ii; masr, mas receptor; at r, at receptor; sns, sympathetic nervous system; no, nitric oxide of hypothalamic thirst sensors [ ] . the at r is expressed in multiple organs involved in the regulation of bp, including the kidney, vasculature, adrenal gland, heart, and both central and peripheral nervous systems. particularly in the kidneys, at r is expressed in vasculature, the juxtaglomerular apparatus (jga), podocytes, mesangial cells, and tubular epithelial cells [ ] . this receptor is part of a gaphaq-linked signaling pathway involving phospholipase c, inositol triphosphate (ip ), and increased intracellular calcium levels [ ] . at r has also been linked to janus kinase, a signal transducer and activator of transcription, and β-arrestin-dependent pathways, linked to extracellular signal-regulated kinases (erk) activation [ ] . moreover, at r can transactivate the epidermal growth factor receptor [ ] . this pathway might contribute to chronic kidney injury and kidney epithelial cell hypertrophy [ ] . once activated, the ace/ang ii/at r axis triggers many systemic and local actions which include ( ) increased aldosterone production, ( ) stimulation of anti-diuretic hormone (adh) production [ ] , ( ) activation of sympathetic nervous system (sns) tone, ( ) elevation of bp ( ) vasoconstriction, ( ) cardiac hypertrophy, ( ) fibrosis, ( ) inflammation, ( ) vascular smooth muscle cell (vsmc) dedifferentiation, ( ) reactive oxygen species (ros) production [ ] , and ( ) tissue injury. on the other hand, activation of the ace/ang ii/ at r axis decreases: ( ) parasympathetic nervous system (psns) tone, ( ) baroreflex sensitivity, ( ) nitric oxide (no) production, and ( ) natriuresis. ang ii-at r binding has opposite effects from at r-mediated activities, including vasodilation and natriuresis. despite being upregulated by sodium depletion and downregulated by ang ii, pdgf, and egf, at r expression is significantly lower during the lifespan, except in fetal and neonatal life [ ] . in the kidney, ace/ang ii/at r axis has synergetic local and systemic actions, both resulting in glomerular hypertension due to preferential vasoconstriction of the efferent arteriole. the systemic actions of ang ii include ( ) stimulation of the sympathetic nervous system, causing renal vasoconstriction and increased renal tubular sodium reabsorption, and ( ) aldosterone secretion, by stimulating adrenal glomerulosa cells in the early and late steps of the steroid biosynthetic cascade. the intrarenal effects of ang ii are ( ) vasoconstriction of efferent arterioles, ( ) sodium reabsorption in the kidney tubules, and ( ) reduction of kidney medullary blood flow [ , ] . the intrarenal stimulation of ang ii synthesis is consistently associated with arterial hypertension and chronic kidney disease (ckd). indeed, studies point to a key role of ang ii in the development of glomerulosclerosis. indeed, the deletion of the at a receptor in animal models resulted in the attenuation of ang ii-induced hypertension [ ] . the expression of at r in leukocytes and lymphocytes may also trigger several pathological events, including enhanced inflammation and kidney fibrosis by activation of epidermal growth factors, increasing tgf-β and nf-Κb levels, which leads to collagen i deposition [ ] . ang ii effects on cardiac structure and function are well established. in the cardiovascular system, ang ii leads to fibrosis, arrhythmias, endothelial dysfunction, and cardiac remodeling. ang ii infusion was associated with concentric left ventricular (lv) hypertrophy due to proliferation of cardiac myocytes. despite this observation being contested in in vivo models, lv hypertrophy may also be the result of arterial hypertension, endothelial dysfunction, and perivascular fibrosis [ ] . recent studies suggest a key role of ang ii in activating the epidermal growth factor receptor (egfr) via a desintegrin and metalloproteinase (adam ). adam expression is enhanced in atherosclerosis, in lv hypertrophy with ang ii infusion, and in vascular neointima after angioplasty [ ] . additionally, deletion of adam in human vsmc culture prevented cardiac and vascular hypertrophy, as well as perivascular fibrosis [ ] . with respect to cardiac remodeling secondary to ang ii-enhanced inflammation, its infusion resulted in elevated tnf-α, il- β, nf-Κb, and inos levels in the heart, which was successfully suppressed by pentoxifylline, a phosphodiesterase inhibitor [ ] . moreover, ang ii infusion in vsmc culture enhanced nox and nox activity and consequently induced hypertrophy and fibrosis [ ] . nox , however, was also linked to enhanced contractile function due to increased ca + uptake [ ] . other possible pathways to ang ii-induced cardiac hypertrophy include β-arrestin, ip , and rhoa [ ] . all cited remodeling mechanisms might exert a major role in atrial fibrillation (af) development, the most common arrhythmia. accordingly, the value and trial studies showed the beneficial effects of at r blocking in preventing af, reinforcing the key role of ace/ang ii/at r axis in cardiac hypertrophy [ ] . the deleterious pulmonary actions of ang ii have become more well-known since the recently described beneficial effects of ace /ang-( - )/masr axis in lung tissue. additionally, this mechanism has recently been associated with the pathophysiology of infection with sars-cov- [ ] (see the discussion below). the role of ang ii in lung inflammation and fibrosis was clarified after the manipulation of the ace gene in transgenic mice. mice with deletion of the ace gene experienced enhanced vascular permeability, increased lung edema, and neutrophil accumulation in sars-cov lung infection [ , ] . accordingly, sodhi et al. [ ] detected increased il- and neutrophil infiltration amplified by star pathway in a mouse model of pseudomonas aeruginosa lung infection. this effect was suppressed by administration of angiotensin receptor blockers (arb) [ ] . ang ii is also associated with excessive deposition of extracellular matrix, resulting in pulmonary fibrosis [ ] . this event is particularly harmful, as it results in thickened alveolar walls, reduced lung compliance, and decreased oxygen diffusion. the activity of ang ii in the central nervous system (cns) has been associated with control of vascular and body fluid homeostasis, stimulation of vasopressin release, thirst, salt intake, and enhancement of the sympathoadrenal system [ ] . the raas in the brain is influenced by both endogenous and peripheral circulation of its molecules, as at r has been found in circumventricular organs, cerebrovascular endothelial cells and neurons [ ] . other recently described functions of ang ii in the cns include the response to stress, stroke susceptibility, and development of certain neuropsychiatric diseases [ ] . the concentration of at receptors in the hypothalamic areas belonging to the hypothalamic-pituitary-adrenal (hpa) axis, including the area responsible for corticotrophin-releasing hormone (crh) synthesis, is associated with ang ii signaling in response to stress [ ] . the at r is also abundant in the pituitary and adrenal glands, corroborating this hypothesis [ ] . indeed, peripheral sympathetic nerve stimulation led to increased renin activity and therefore increased levels of ang ii [ ] . thus, prophylactic at r blockade outside and inside the blood-brain barrier in mice has been shown to reduce activation of the hpa axis and increase catecholamine synthesis [ ] . selective inhibition of at r in spontaneously hypertensive rats with middle brain artery occlusion significantly reduced ischemia and tissue swelling, besides diminishing bp due to ang ii-mediated no release. moreover, the expression of at r in the limbic system also suggests a key role of the classical raas axis as a neuroendocrine modulator in neuropsychiatric disorders. agtdeficient mice presented less depressive-like behavior than wild-type animals [ ] . arb administration has also been reported to exert anti-depressant-like effects in mice subjected to the forced swim test [ ] . the raas also has important influences on metabolism. for example, in vitro studies have demonstrated that ang ii stimulates lipogenesis in human adipocytes [ ] . mice overexpressing agt in adipose tissue exhibited systemic insulin resistance and inflammation [ ] . similarly, agt gene deletion reduced weight gain of animals under a high-fat diet compared with wild-type mice [ ] . ang ii-at r binding negatively regulates insulin-induced phosphorylation of the insulinreceptor (ir) [ ] . in addition, ang ii directly interferes in ampk signaling, causing insulin resistance and inhibiting adiponectin secretion [ ] . in the liver, ang ii is able to enhance hepatic fibrosis due to upregulation of tgf-β and activation of kupffer cells [ ] . accordingly, the arb candesartan and the ace inhibitor (acei) perindopril significantly decreased hepatic fibrosis in animal models [ ] . furthermore, several studies proved the major role of raas axes imbalance in cirrhotic patients and rats with liver fibrosis [ ] [ ] [ ] . in the hepatorenal syndrome, ang ii also seems to promote kidney vasoconstriction secondary to chronic liver disease and advanced hepatic failure [ ] . several of the previously mentioned ace/ang ii/at r physiological effects rely on immune system activation. the pro-inflammatory effects of ang ii-at r binding have specific signaling pathways for each cell line, immune and nonimmune mechanisms. ang ii-mediated macrophage activation, for example, increases tnf, il- beta, il- , and il- , as well as ros production [ ] . lymphocytes, which express raas components such as the at r, are crucial for the ang ii-induced vascular inflammation, ros production, and hypertension [ ] . t lymphocytes activated by ang ii increase cc chemokine receptors (ccr), whose binding increases lymphocyte and leucocyte recruitment and is associated with thrombotic events. neutrophils are also increased by ang ii stimulation and play a key role in ang ii-induced cardiac and pulmonary fibrosis [ , ] . the alternative raas axis, with its main biologically active product ang ( - ) has only been well characterized relatively recently. since the initial description of ang ( - ) binding to the mas receptor, our understanding of the alternative or counterregulatory raas axis has grown [ ] . ang-( - ) can be produced by two different pathways: in the extracellular compartment, as blood and interstitial fluid of several organs, or in the intracellular environment, as inside the mitochondria. the most common pathway is the conversion of ang ii into ang-( - ) by means of ace (fig. ). ang-( - ) may also be synthesized by other endopeptidases, including prolyl-endopeptidase (pep) and neprilysin (nep), forming ang-( - ) directly from ang i [ ] . ace , a monocarboxypeptidase responsible for converting ang ii into ang-( - ), is an ace homologue ( % homology) and formed of amino acids (~ kda). as with ace, the ace protein sequence reveals a hydrophobic site near the c-terminus and a potential signal peptide at the n-terminus, which together classify ace as a type-i integral membrane protein containing the zinc metallopeptidase catalytic site [ ] . thus, when the membrane-bound ectodomain of ace is cleaved by the metalloproteinase adam , ace is released in the circulation [ ] . the specificity of ace is not limited to ang ii or raas peptides, as this enzyme has a multifunctional nature and may also exert effects on many other vasoactive peptides, including apelin- , apelin- , kinin metabolites kallidin, neurotensin, kinetensin, and opioid peptides [ ] . other enzymes, such as nep, pep, and prolyl-carboxy-peptidase (pcp), have been described as biochemically capable of producing ang-( - ) from ang ii and ang i [ ] . nep is a membrane-anchored metalloendopeptidase [ ] . despite its initial description as an opiate pentapeptide encephalin hydrolyzer, nep is capable of forming ( ) ang-( - ) from ang ii, ( ) ang i from ang-( - ), and ( ) ang-( - ) from ang i [ ] . this last conversion is due to the hydrolysis of the pro -phe from ang i, generating ang-( - ). nep is expressed in several peripheral tissues [ ] and its actions on the kidney appear to be of critical importance [ ] . pep, in turn, is a soluble intracellular serine peptidase that cleaves ang i and ang ii to form ang-( - ). nonetheless, this enzyme has already been found in transmembrane and nuclear forms [ ] , although its actions are still unclear. lastly, pcp is a monocarboxypeptidase, with specificity for the c-terminal hydrolysis of the pro-x bond, where x is a hydrophobic residue and is also responsible for converting ang ii into ang-( - ) [ ] . ang-( - ) is a heptapeptide (asp-arg-val-tyr-ile-his-pro) formed from ang i or ang ii, as previously mentioned. the most important difference between ang ii and ang-( - ) is a phenylalanine amino acid on the n-terminal of the first molecule. this phenylalanine is crucial to ang ii binding to at r, explaining the lower affinity of ang-( - ) to at r and higher affinity to the mas receptor [ ] . the signaling mechanism of ang-( - ) is still poorly understood, but it has been postulated to activate the phosphatidylinolsiol- -kinase-akt pathway and/or the mapks (mitogen-activated protein kinase), and/or the c-amp-dependent protein kinase (pka) in different tissues [ ] . pharmacokinetic experiments determined that, in humans, ang-( - ) half-life is~ . h [ ] , normally being hydrolyzed by aminopeptidases, forming ang-( - ) and ang-( - ), or by ace, forming ang-( - ) [ ] . despite being able to bind to the at r, the main effects of ang-( - ) are related to its binding to the mas receptor. the mas receptor was first described in as a protooncogene due to its capability to induce cell transformation via small gtpase p -rac [ ] . afterwards, computational analysis of the amino acid sequence of the mas receptor revealed that it is indeed a g protein-coupled receptor with seven transmembrane domains. the mas receptor gene, mas , is located in close proximity to the imprinted igf r gene in the human genome, being biallelically expressed [ ] . the masr is mainly expressed in the brain, testicles, kidneys, and heart [ ] . its signaling pathways require better understanding. however, the proposed mechanisms include the inhibition of ros production, as well as map kinases, and the activation of phosphatases, phosphorylation of akt/ enos, and counter-regulation of ang ii actions [ ] . the primary effects elicited by the binding of ang-( - ) to mas receptor include ( ) decreased aldosterone production, ( ) stimulation of anti-diuretic hormone release [ ] , ( ) inhibition of sympathetic tone, ( ) reduction of bp, ( ) vasodilation [ ] , ( ) decrease of cardiac hypertrophy, ( ) anti-fibrosis, ( ) anti-inflammatory actions [ ] , ( ) inhibition of vsmc growth, ( ) suppression of ros production, and ( ) promotion of tissue protection [ ] . moreover, ang-( - ) also enhances insulin sensitivity, increases glycogen synthesis, and stimulates lipid metabolism [ ] . experimental studies have pointed to an important nepmediated production of ang-( - ) in the kidney [ ] . however, human production of ang-( - ) is mainly attributed to ace [ ] . the activation of ace /ang-( - )/mas receptor axis exerts protective roles by reducing oxidative stress, inflammation, cell proliferation, and fibrosis [ ] . several studies have shown that the renal actions of ang-( - ) may vary depending on the part of the nephron, type of study, or intervention applied. sometimes ang-( - ) acts as a natriuretic molecule, while, in other studies, it exerts anti-diuretic effects [ ] . moreover, studies support that ang-( - ) has an important physiological role for the regulation of glomerular filtration, water, and sodium handling [ ] . by opposing ang ii effects, ang-( - ) reduces glomerulosclerosis and bp, attenuating plasma elevations of urea and creatinine and preserving cardiac function [ ] . the cardiovascular actions of ang-( - ) are also important. the inhibition of the classical raas axis by therapy with acei and arbs improves the outcomes of patients with hypertension, acute myocardial infarction, and chronic heart failure [ ] . the beneficial effects of the alternative axis in cardiac tissue can be attributed to bradykinin potentiation or counterregulation of ang ii actions, leading to anti-fibrotic, anti-inflammatory, and anti-proliferative responses [ ] . in the heart, ang-( - ) can modulate heart rate, maintain calcium homeostasis, and promote coronary vasodilation, mostly due to prostacyclin and no release [ ] . in blood vessels, ang-( - ) causes vasodilation, increases no and prostaglandin release, reduces thrombus size, and raises stem/progenitor cell proliferation and migration to vascular repair, among other actions [ ] . in the respiratory system, the alternative raas axis has been identified as crucial for the reduction of inflammatory, fibrogenic, and proliferative effects of ace/ang ii/at axis in several pulmonary disorders [ ] . ace administration reduces oxidative stress and inflammatory mediators in lung tissue [ ] . moreover, experimental and clinical evidence indicate the classical raas axis activation and subsequent increase of ang ii take part in the pathophysiology of allergic pulmonary diseases, including asthma [ ] . regarding pulmonary fibrosis, ang-( - ) acts as an anti-fibrotic pulmonary survival factor by many mechanisms: cardiac protection, lung ischemia prevention, inhibition of ros generation, and opposition to ang ii inflammatory and fibrogenic effects [ ] . ang-( - ) has been described as playing an important role in the brain as a neuromodulator, mostly in areas related to tonic and reflex control of arterial pressure. guimarães et al. [ ] demonstrated that ang-( - ) is able to diminish bp in doca-salt hypertensive rats due to enhancement of the baroreflex bradycardia, restoration of baroreflex control of renal sympathetic activity, and regaining the balance of cardiac autonomic tone. these findings are reinforced by the improvement of baroreflex control of heart rate in normotensive and in hypertensive animal models [ ] . other studies suggest that the activation of ace /ang-( - )/mas axis in the brain might play an important protective role against cardiovascular and metabolic disorders [ ] . mecca et al. [ ] have shown the association between central administration of ang-( - ) and reduction in brain damage, as well as improved neurological outcomes in animals with ischemic stroke. these beneficial effects may be due to the stimulation of angiogenesis and the antiinflammatory effects of ang-( - ) [ ] . moreover, it has a l s o b e e n p r o p o s e d t h a t a n g -( - ) p o s s e s s e s neuromodulator actions by influencing the release of certain neurotransmitters (kow and pfaff ), including norepinephrine, gaba, substance p, glutamate, taurine, and dopamine [ ] . recent studies have suggested that the alternative raas axis exerts several metabolic actions, including modulation of hormone secretion in pancreas, adipose tissue, liver, and reproductive organs [ ] . ang-( - ) improves whole-body insulin resistance, while its direct action on the glucagon pathway is still unclear [ ] . in adipose tissue, high levels of ang-( - ) are associated with increased adiponectin and reduced leptin levels. in addition, researchers aim to clarify the actions of raas and complications of metabolic disorders associated with increased body adiposity, like diabetes mellitus (dm) [ ] . in the reproductive system, ace /ang-( - )/ mas axis molecules were found in ovarian follicles and testicle cells [ ] . although the exact effects on those organs are still unclear, evidence suggests the participation of the raas alternative axis in the regulation of gonadotropic hormones [ ] . ang-( - )-masr binding has also been related to restoration of liver function, as shown in experimental studies [ ] . santos et al. [ ] showed the association between masr genetic deletion in fvb/n mice and dyslipidemia, increased abdominal fat, enhanced muscle triglycerides, glucose intolerance, and reduced insulin sensitivity. in addition, studies with arbs, such as captopril and losartan, showed the role of these drugs in preventing tissue fibrosis and restoring liver function both in experimental and clinical studies [ ] . preclinical studies also pointed to ace activity as an important endogenous negative regulator of chronic liver injury [ ] . these results are reinforced by the downregulation of key genes involved in liver fibrosis after ang-( - ) infusion and the aggravation of liver fibrosis after pharmacological blockage of the mas receptor [ , ] . more recently, studies suggest that low levels of ace may be a useful indicator of poor prognosis in patients with cirrhosis [ ] . in terms of immune response, the ace /ang-( - )/masr axis inhibits the biological pro-inflammatory effects of ang ii, causing anti-inflammatory, anti-proliferative, and anti-fibrotic responses [ , , ] . in inflammatory diseases, including arthritis, acute lung injury, asthma, and diabetic nephropathy, several studies have concluded that ( ) ang-( - ) reduced leukocyte recruitment and the production and expression of chemokines, cytokines, and adhesion molecules and ( ) the mas receptor increased neutrophil apoptosis and stimulated the alternative macrophage activation [ ] . in addition, studies using the mas receptor agonist ave demonstrated reduced accumulation of neutrophils and pro-inflammatory cytokines [ ] . table summarizes the main effects of the two raas axes in different organs, systems, and disease conditions, including several not directly discussed here. discovery of several novel molecules and pathways of the raas cascade has led to the present view of the raas as comprising two main axes (fig. ) . indeed, components of the raas, including angiotensin-( - ) (ang-( - )) and alamandine, for instance, show relevant biological effects, but the physiological role of these molecules in the context of the counter-regulation of the classical raas axis remains unclear, especially in humans. it should be mentioned that the endogenous and physiological relevance of a molecule encompasses the existence of an enzyme that synthesizes the molecule and a specific receptor that mediates the biological effects of it. therefore, the activity of an axis depends mostly on ( ) the peptide's concentration, ( ) the enzyme-substrate affinity, ( ) the peptide-receptor affinity, and ( ) the expression of its receptor. despite the existence of angiotensin peptides that have their own receptors such as alamandine and the mas-related g protein-coupled receptor member d (mrgd), for example, the physiological role of this molecule has not been clearly established. alamandine concentration is physiologically low, as reported in several experimental studies [ ] . further investigation is needed for better understanding of the functions and pharmacological potential of these novel raas fragments as an escape route for both classical and alternative arms. alamandine is mainly produced following the hydrolysis of ang-( - ), in which the aspartate radical group in the amino terminal sequence is cleaved by aspartate decarboxylase [ , ] . other possible pathways to form alamandine are from angiotensin a (ang a), following the enzymatic action of ace [ ] . alamandine binds to the mrgd, a receptor previously reported in relation to neuropathic pain and the perception of itching [ ] . alamandine-mrgd interaction has been shown to exert cardiovascular effects, as studies observed vasodilation after the administration of alamandine in spontaneously hypertensive rats [ ] . alamandine also increases enos expression and promotes both central and local no-mediated vasodilation, capable of reversing endothelial dysfunction [ ] . de jesus et al. [ ] also demonstrated the anti-hypertrophic counter-regulatory role of the ace /alamandine/mrgd axis in ang ii hypertrophy rat cardiomyocytes due to activation of the ampk/no pathway. furthermore, in vitro studies have demonstrated the anti-fibrotic effects of alamandine administration in isoproterenol-treated rats [ ] . although patients with ckd seem to have higher plasma levels of alamandine, the pathophysiological implications of this finding require further investigation [ ] . angiotensin iii (ang iii), formed by the action of aminopeptidase a on ang ii, is capable of binding to both at r and at r [ ] [ ] [ ] . some research groups have postulated that ang iii is, in fact, the biological ligand to the at r, after the cleavage of the n-terminal aspartate residue of ang i [ ] . along those lines, padia et al. [ ] observed increased natriuresis after ang iii infusion in rats under arb treatment. although effects on decreasing arterial pressure through natriuresis have been reported, other studies have demonstrated an important role of ang iii in the release of atrial natriuretic peptide and vasopressin [ ] . this finding may be due to the higher expression of at r in comparison to at r in adults, as ang iii possesses greater at r-at r selectivity than ang ii [ ] . indeed, more evidence is needed to define the physiological and pharmacological properties of ang iii in vivo. angiotensin iv (ang iv) is an interesting raas peptide with a variety of described physiological properties in the cns. this fragment is formed following the cleavage of the arginine from the n-terminus of ang iii by aminopeptidases n and b [ ] . ang iv has relevant affinity for both at r and at r, with its affinity to at r being superior to that of ang-( - ) for the same receptor [ ] . in addition, ang iv has its own receptor, the angiotensin iv g protein-coupled receptor (at r) [ ] . lochard et al. [ ] concluded that ang iv is only able to bind to at r in the presence of ang ii. the study was conducted with transgenic mice chronically releasing ang iv in the brain, which experienced an increase in bp due to the mobilization of calcium by ang iv via at r [ ] . this finding supports an allosteric mechanism between at r and at r, since the administration of the at r antagonist candesartan reversed ang iv effect [ ] . ang iv-at r binding was reported to prevent endothelial dysfunction in aortic vessels due to the reduction of no bioavailability in the development of atherosclerotic lesions [ ] . the most promising properties regarding ang iv, however, are related to its binding to at r, whose expression in the cns is increased in the neocortex, hippocampus, and cerebellum [ ] . albiston et al. [ ] concluded in that at r is actually the insulin-regulated aminopeptidase (irap), which substrate in vivo lacks identification. since ang iv has been reported to increase learning and memory recall, one current hypothesis proposes that binding of this peptide to irap increases the endogenous peptide's half-life by inhibiting its degradation [ ] . moreover, knowledge about the association between irap and the glucose transporter type (glut ) suggests that ang iv might play a distinct role in this protein translocation [ ] . angiotensin-( - ) is directly formed by ace activity on ang i and is able to produce ang-( - ) after being hydrolyzed by either ace or nep [ , ] . this peptide was primarily thought to be inactive, exerting counter-regulation to the ace/ang ii/at r axis by diminishing the conversion of ang i into ang ii and consequently producing ang-( - ) by nep activity [ ] . however, new evidence points to physiological implications due to ang-( - ) binding to the at r receptor. ocaranza et al. [ ] were the first to identify its biological effects in the cardiovascular system, leading to anti-hypertrophic effects in neonatal and adult heart. the molecular mechanisms of ang-( - ) were clarified by sotomayor-flores et al. [ ] . considering the main role of mitochondrial dynamics and ca + handling in cardiac hypertrophy, ang-( - ) administration increased mitochondrial fusion and reduced mitochondrial fission and intracellular calcium dysregulation, therefore exerting anti-hypertrophic effects [ ] . previous studies also associated ang-( - ) and modulation of pro-fibrotic pathways, decreasing cardiac fibrosis [ ] . moreover, cha et al. [ ] observed beneficial results after the administration of ang-( - ) in pulmonary hypertensive rat models, by altering the expression of apoptosis-related proteins via at r. although ang-( - ) affinity to the at r receptor is only moderate (~ -fold lower than ang ii), this peptide has also been described to enhance bradykinin action and arachidonic acid release [ ] . angiotensin-( - ) (ang-( - )) plays an intriguing role in understanding the noncanonical pathways of the raas [ ] . ang-( - ) can be cleaved by nep and ace to form ang i, at least in the kidney [ ] . although ang-( - ) can be formed from angiotensin-( - ), ahmad et al. [ ] proposed that ang-( - ) might be produced after cleavage of agt by a member of the kallikrein enzyme system. well-established data have demonstrated renin-independent cardiac ang ii formation and function throughout the years, mainly by the chymase-mediated hydrolysis of ang-( - ) and ang i in rat models and normal human left ventricles. in this matter, the translational research comparing rodents and human cardiac tissue reveals that the chymase rather than renin might be the primary pathway to form ang ii, which may explain the limited results regarding acei and arb treatment for preventing ang ii production in cardiovascular disease [ ] . further investigation on ang-( - ) might unravel a novel perspective on pharmacology related to the raas. lastly, angiotensin a (ang a) was identified by jankowski et al. in [ ] . this octapeptide is very similar to ang ii's amino acid sequence, differing only by an alanine (ala-arg-val-tyr-ile-his-pro-phe) instead of an aspartate [ ] . although some affinity to the at r receptor has been described, this molecule is considered to have intrinsic vasoconstrictive and pro-hypertensive effects via the at r, with similar binding affinity as ang ii [ ] . physiological plasma concentration of ang a is about % of the concentration of ang ii, and its potency of vasoconstriction is one tenth that of ang ii [ ] . however, similarly to alamandine, patients with stage ckd showed higher ang a/ii ratio than healthy controls [ ] . this finding corroborates the supposed escape route of other raas fragments in compromising diseases of the kidney parenchyma. again, the existence of alternative peptides could help understanding the partial results of classical raas blocker therapy in treating cardiovascular and kidney diseases. in this context, an interesting perspective might be the evaluation of the angiotensin a effects via at r after the administration of arbs. the role of the kidney in hypertension is mainly based on sodium and water retention, changes in pressure natriuresis, impaired kidney autoregulation, and systemic and local activation of the classical raas axis [ ] . however, literature regarding htn and the raas are controversial. some research groups have found higher ang-( - ) levels in hypertensive adults, while other studies have reported normal levels [ ] . in the only study to include children, we found that levels of ang ii in patients with renovascular hypertension were higher than levels of ang-( - ) [ ] . in addition, after intervention and correction of renal artery stenosis, levels of raas molecules were restored to normal. in contrast, the study showed that patients with primary htn had significant elevation of circulating levels of ang-( - ), while the levels of ang i and ang ii were within the normal range [ ] . ang-( - ), in this sense, might play a key role as a biological marker of primary htn, as the elevation of ang-( - ) plasma levels may be conceived as a compensatory mechanism to oppose the deleterious effects of ace/ang ii/at r upregulation. the childhood obesity epidemic has highlighted the importance of excess weight as a risk factor for htn development [ ] . a cross-sectional analysis conducted by south et al. [ ] investigated the association between very low birth weight in preterm newborns, plasma and urinary ang ii and ang-( - ), and body weight. results from participants showed that obesity was associated with high bp, increased ang ii, and reduced ang-( - ) both in circulation and in the kidney [ ] . therefore, obesity may increase the risk of htn and cardiovascular diseases in individuals born prematurely by further augmenting the prematurity-associated imbalance of the raas. another intriguing issue related to renal arterial hypertension is the potential protective role of a low-sodium/high-potassium diet [ , ] . high-salt intake is clearly associated with an elevation of bp, a greater risk of albuminuria and of adverse kidney and cardiovascular outcomes compared with low or moderate sodium intake. on the other hand, a potassium-rich diet has been associated with a lower incidence of htn, stroke, nephrolithiasis, and kidney disease [ , ] . several mechanisms may be involved in the responses to lowsodium and high-potassium intake, including decreased sensitivity of vsmcs to ang ii and catecholamines, modulation of baroceptor sensitivity, reduction of sympathetic activity, increased urinary sodium excretion, decreased renal vascular resistance, and increased gfr [ , ] . in a recent study of fetal programming, ace /ang-( - )/ mas receptor axis and cardiovascular events were associated with persistent alterations in ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) in the circulation and kidney during adolescence and abnormal kidney sodium handling, htn, and cardiovascular disease in adulthood [ ] . therefore, both raas axes not only exert a critical physiological role on bp homeostasis but also take part in the pathophysiology of arterial htn in pediatric patients. the raas exerts a central role in the pathophysiology of glomerular disease, especially in the progression to ckd, as previously shown in several studies. therefore, treatment with raas blockers has been shown to reduce proteinuria and exert renoprotective effects in almost all glomerular diseases [ ] . pinheiro et al. [ ] reported significant changes in kidney function and histology secondary to the genetic deletion of mas receptor gene in mice, including ( ) reduced renal blood flow; ( ) glomerular hypertension with consequent increased urinary albumin excretion; ( ) reduction in the diameters of the glomerular tuft and bowman's capsule; ( ) increased deposition of collagen iii, collagen iv, and fibronectin; and ( ) increase of at r and tgf-β rna expression in kidney tissue. these results corroborate previous studies by showing that ang-( - ) may act as a physiological regulator of intraglomerular pressure and may oppose ang ii's proinflammatory and pro-fibrotic effects [ ] . additionally, silveira et al. [ ] demonstrated beneficial effects of mas receptor activation in a murine model of adriamycin-induced nephropathy. treatment with ave , an agonist of the mas receptor, improved kidney function parameters, reduced urinary protein loss, attenuated histological changes on kidney tissue, and decreased urinary levels of fibrogenic cytokines such as tgf-β [ ] . the study also showed that mrna expression of at r and mas receptors were both decreased in adriamycin-induced nephropathy. more importantly, the renoprotective effects of losartan in this experimental model were dependent on mas receptor activation [ ] . a recent study by silva-filha et al. [ ] analyzed the role of both raas axes in pediatric patients with primary nephrotic syndrome (ns). thirty-one patients with primary ns and healthy controls underwent urine collection for measurement of raas molecules [ ] . the analysis showed that primary ns patients had reduced urinary levels of ace , but increased urinary concentrations of ang ii, ang-( - ), and ace [ ] . additionally, reduced ace levels were negatively correlated with proteinuria in patients with primary ns [ ] . previous studies had already indicated that acquired or genetic deficiency of ace increased kidney injury and proteinuria in other kidney diseases [ ] , probably due to potentiation of ang ii effects. furthermore, nadarajah et al. [ ] showed that the overexpression of ace in glomerular podocytes attenuated the development of diabetic nephropathy in mice, apparently as a result of delaying the development of albuminuria. this study also demonstrated that ace overexpression attenuates histological changes, preserves podocyte proteins, avoids podocyte loss, and reduces tgf-β expression [ ] . taken together, these findings support a renoprotective role of ace in glomerular diseases. the apparently counterintuitive finding of high urine concentrations of ang-( - ) in parallel with lower urinary levels of ace in pediatric patients with primary ns, might be explained by the compensatory response to kidney damage triggered by high-level activation of the classical raas axis [ ] . other explanations include the synthesis of ang-( - ) by other enzymes, such as nep and pep, and the diminished masr expression in kidney tissue, as reported by ng et al. [ ] . another important aspect in regard to glomerular diseases and the role of both raas axes are the interactions between components of the raas and inflammatory mediators in the context of glomerular diseases. therefore, current evidence strongly supports the important role of inflammation for kidney injury in glomerular diseases. pediatric patients with primary ns had significantly higher concentrations of mcp- /ccl , a pro-inflammatory chemokine related to macrophage activation than controls without ns [ ] . in addition, urine concentrations of ip- /cxcls- , a cytokine responsible for chemotactic effects, apoptosis, and cell growth, were positively correlated to proteinuria [ ] . moreover, urinary levels of ang-( - ) and ace were negatively correlated with mcp- /ccl and ip- /cscl- , respectively, suggesting that both raas molecules interact with inflammatory pathways in glomerular diseases. well-established data associate low birth weight with systemic diseases in childhood and adult life, including htn, insulin resistance, and coronary heart disease [ ] . previous studies with animal models demonstrated that intrauterine growth restriction (iugr) impairs kidney development and leads to a reduced number of nephrons, with consequent development of glomerular hyperfiltration after birth and ultimately, kidney damage in adulthood [ ] . analysis of serum levels of raas components in a rat model of nephrotic syndrome induced by prenatal ethanol exposure demonstrated ( ) increased serum levels of ang ii, probably due to higher proportion of ace in relation to ace ; ( ) reduced at r expression; and ( ) reduced ace and mas receptor expression. additionally, prenatal ethanol exposure was shown to inhibit normal kidney development, probably due to overexpression of cdh and consequent growth retardation of ureteric bud (ub) branching. finally, intrauterine programming might be impaired by ethanol damage at critical stages of development. in other words, epigenetic modifications might also be responsible for modulating the expression of raas molecules, resulting in, for instance, low expression of renal at r [ ] . the classical raas axis seems to have an important role in cakut. high levels of ang ii lead to reduced renal blood flow, causing ischemia and kidney growth arrest of the impaired kidney. although renal blood flow usually normalizes weeks after the relief of temporary obstruction, kidney growth remains altered, suggesting that other factors are responsible for growth impairment, including the reduction of cell proliferation, increased cell apoptosis, and progression of interstitial fibrosis [ ] . ang ii also increases the expression of tnf-α mrna in obstructed kidneys, enhancing the interest in acei drugs as an effective prevention of kidney fibrosis in cakut [ ] . specifically, in ureteropelvic junction obstruction (upjo), the agtr gene, responsible for at receptor expression, appears to have two main actions in relation to urethral development: ( ) the regulation of the apoptotic process in undifferentiated mesenchymal cells surrounding the developing ureter and ( ) inhibition of ectopic ureteral budding [ ] . additionally, increased urinary levels of ace and of ang-( - ) as well as lower ace urinary levels were detected in fetuses with posterior urethral valves (puv) when compared with healthy neonates [ ] . the explanation for the increased levels of molecules of the alternative raas axis relies on the potential role of ace and ang-( - ) as a compensatory mechanism against the intense inflammatory response process triggered by puv itself [ ] . chronic kidney disease in pediatrics mostly results from cakut and glomerular and cystic diseases and evolves with htn. results from the chronic kidney disease in children study group showed that % of ckd pediatric patients had systolic or diastolic bp measurements above the th percentile for correspondent age and sex and % met the diagnostic criteria for htn [ ] . the loss of glomerular autoregulation in htn leads to development of intraglomerular hypertension, which may contribute to proteinuria and glomerular tissue damage [ ] . in this setting, high bp is the consequence of disease progression and a risk factor for stage ckd. enhanced classical raas axis activation results in increased ang ii production and higher stimulation of at receptors, which may contribute to systemic and glomerular capillary htn, provoking kidney injury and progression of ckd [ ] . ang- ( - ) , on the other hand, induces dilatation of pre-constricted renal afferent arterioles and increases kidney blood flow, neutralizing ang ii effects [ ] . several mechanisms may explain how ang-( - ) counteracts kidney effects of ang ii, including ( ) competition for the binding of ang ii to at receptors; ( ) deregulation of ang ii/at signaling transduction; ( ) interference of at receptor syntheses; and ( ) stimulation of no release, mobilization of anti-apoptotic pathways, and reduction of oxidative stress by the activation of mitochondrial raas alternative axis [ ] . as previously mentioned, we have found high levels of both ang ii and ang-( - ) in children with renovascular htn, whereas patients with primary htn had a selective elevation of plasma ang-( - ) [ ] . these findings suggest upregulation of the classical raas axis in renovascular htn that differ from the response of primary hypertensive pediatric patients [ ] . later studies have demonstrated that plasma levels of ang-( - ) were even more pronounced in pediatric patients with stage ckd, pointing to a deviation of raas metabolism toward ang-( - ) synthesis at more advanced stages of the disease [ ] . in addition, this finding might also be due to an impairment and/or downregulation of mas receptor in patients with stage ckd [ ] . the pro-inflammatory and pro-fibrotic effects of ang ii may also contribute to ckd progression [ ] . ang ii increases the population of inflammatory monocytes by ( ) facilitating differentiation of hematopoietic stem cells; ( ) stimulating adhesion molecules; ( ) enhancing leukocyte rolling flux, adhesion and migration; ( ) upregulating plasma levels of il- , ifn-γ, tnf-α and il- β; and ( ) activating calcium mobilization, among other actions [ ] . moreover, ang ii contributes to fibrogenesis and organ remodeling, while ang-( - ) has anti-fibrogenic and anti-proliferative effects [ , ] . hence, the ang ii pro-inflammatory environment induces tissue damage, which eventually leads to progressive loss of kidney function. ng and colleagues [ ] demonstrated reduction of mas receptor expression in rats with ckd. this group also observed an association between indoxyl sulfate (is) administration and diminished mas receptor kidney expression, as well as increased tgf-β in cultured human proximal tubular cells [ ] . sun et al. [ ] showed the effect of classical raas axis inhibitors like losartan in preventing is-induced fibrosis on targeting organs. these data indicate that the proinflammatory reactions and enhancement of oxidative stress of this uremic toxin may be the result of classical raas axis activation [ ] . moreover, the inhibition of these pathways might slow the development of ckd through indirect raas suppression. consistent with these findings, ma et al. [ ] found that mice with genetic deletion of the at receptor gene have ( ) decreased cortical ace activity, mas expression, and ang-( - ) levels in kidney tissue; ( ) increased production of ace, ang ii, and at receptor in kidney cortex; ( ) high bp, increased indices of kidney injury, and mesangial matrix expansion score; and ( ) microalbuminuria. ali et al. [ ] showed that at and mas receptors have molecular interactions, mainly concerning no release and diuretic responses. the main conclusion is that the downregulation of the alternative raas axis can extend kidney tissue damage. regarding acute kidney injury (aki), increased kidney vascular resistance due to stimulation of sympathetic nervous activity was responsible for triggering renin secretion and consequently elevation of ang ii levels [ ] . the exact role of the classical raas axis in aki, however, is yet to be clarified. barroso et al. [ ] detected renoprotective effects of ave , a mas receptor agonist, in experimental aki by improving kidney function, decreasing tissue damage, preventing leukocyte infiltration, and reducing cytokine release. it should also be mentioned that aki can lead to ckd and the activation of classical raas axis may also play a role in this process. several studies have demonstrated that kidney raas is altered in dm patients and that raas inhibitors can reduce the incidence of vascular complications in these patients, probably by means of the protection of skeletal muscle and pancreatic islets, enhancement of insulin sensitivity, and reduction of adipocyte size [ ] . a recent study by alves et al. [ ] showed that hyperglycemia was responsible for increased urinary levels of cytokines and kidney gene expression of il- , suggesting a dm modulation of kidney inflammation. moreover, previous studies indicated the benefits of acei and arbs as first-line treatments for patients with dm and htn, mostly due to their cardiac and renoprotective effects [ ] . in line with these findings, mori et al. [ ] showed that ang-( - ) ameliorates diabetic nephropathy through decreasing kidney inflammation and fibrosis, attenuating kidney ros production, and reducing kidney lipotoxicity. sickle cell anemia (sca), the most common hemoglobinopathy worldwide, is capable of triggering sickle cell nephropathy (snc) as a result of a poorly understood inflammatory state [ ] . there is a clear association between albuminuria in children with scn and urinary levels of cytokines and chemokines, including il- , tnf, and mcp- /ccl . these molecules might be possible biomarkers for poorer outcomes in scn [ ] . in addition, high hemoglobin f and platelet levels were also associated with increased estimated glomerular filtration rate and albuminuria, respectively [ ] . as the platelets from scd patients can secrete higher amounts of inflammatory molecules, sca progression is closely related to the development of scn. although some studies on acei administration showed significant reduction of albuminuria in patients with sca, none had evaluated raas components in these patients. in this regard, belisário et al. [ ] performed a cross-sectional study with children and showed lower levels of ace and ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) in sca children with persistent albuminuria. these results were the first to provide evidence for a role of raas molecules in human scn, suggesting that sca patients had raas imbalance towards the classical axis [ ] . the raas is also involved in the physiopathology of autoimmune diseases, including rheumatoid arthritis and lupus nephritis (ln). ln is the result of acute kidney inflammation triggered by the deposition of autoantibodies and immune complexes in the kidney [ ] . accordingly, several studies have shown that ang ii has pro-inflammatory effects in autoimmune diseases due to regulation of chemokines, cytokines, and chemotactic agents, including tgf-β , tnf, il- , osteopontin, and nf-kappa b [ ] . similar to glomerular diseases, ln can have a wide spectrum of presentations, from minimal mesangial injury to advanced glomerular sclerosis. likewise, both diseases might be attenuated by the use of acei and arbs, in order to provide renoprotection and reduction of proteinuria [ ] . albuquerque et al. [ ] showed that captopril was able to reduce tgf-β expression in the glomeruli and tgf-β expression in the periglomerular region of mice with lupus. furthermore, it was proposed that captopril changes the cytokine profile of ln patients through inhibition of ang ii effects on lymphoid cells and apoptosis signal complexes [ ] . all of these data support the main role of classical raas axis activation in the pathophysiology of kidney damage triggered by systemic diseases and open the possibility of novel therapeutic approaches by stimulation of the alternative raas axis. the outbreak of sars-cov- virus infection in wuhan, china, in has led to increased scrutiny of the role of the raas in patients with novel coronavirus disease . early in the pandemic, several studies pointed to a pivotal role of transmembrane ace as a viral receptor in nasal and oropharyngeal epithelia [ ] . hence, researchers postulated about the function of the raas in covid- , where a hypothesis of raas imbalance might explain the disease's clinical findings [ ] . the intersection between covid- , raas, and pediatrics is particularly intriguing, due to the epidemiological pattern confirmed in several reports worldwide: lower frequency, severity, and fatality case rates in the pediatric population [ ] . although covid- occurs in all age groups, the number of cases in children is overall low. in china, data from the chinese center for disease control and prevention reported a percentage of . % confirmed cases of covid- among patients aged < years old [ ] . in the usa, a report from the center for disease control (cdc) showed that only % of covid- cases were under years old [ ] . in europe, the incidence is even lower, with . % of italian patients and . % of spanish cases being < years of age [ ] . a comparison with adult patients made by wu et al. [ ] showed a milder clinical presentation of children positive for covid- in the following signs and symptoms: fever ( % for pediatric cases versus % for adult patients), cough ( % vs. %), elevated c reactive protein ( % vs. %), and pneumonia ( % vs. %). this finding was also confirmed by the american cdc regarding fever ( % for pediatric cases versus % for adult patients), cough ( % vs. %), and dyspnea ( % vs. %) [ ] . furthermore, american information on hospitalization status of covid- pediatric cases under the age of presented a percentage of . % of hospitalization and . % of admission to the intensive care unit (icu). in comparison, among the , adult cases, % were hospitalized and . % went to icu [ ] . mortality of children with covid- has been rare, consistent with the seemingly milder course of the infection in those < years old. the significance of these data is counterintuitive in light of the well-established higher susceptibility rates of infants to respiratory infections [ ] . therefore, the theoretical protection of children from sars-cov- infection suggests the reevaluation of covid- as a systemic condition with pulmonary involvement, rather than a pulmonary disease with systemic implications [ ] . the various manifestations of covid- in pediatric patients might be a result of different and multifactorial defense mechanisms involving the raas dynamism throughout life, as well as its implications on disease course, and the naïve immune system, which is not fully developed in these patients. pediatric symptomatology supports this hypothesis, as systemic complications have frequently been reported, including sore throat, vomiting, diarrhea, and abdominal pain [ ] . indeed, the pathophysiology of covid- might rely greatly on imbalance of the raas. binding of the viral spike glycoprotein s to membrane-bound ace leads to the entrance of ace into the cell and as a consequence reduces the amount of active enzyme [ ] . since the raas has two counter-regulatory and dynamic arms, diminished ace results in ( ) higher ang ii levels, as well as classical arm ace/ ang ii/at r upregulation and ( ) ang-( - ) depletion and consequent ace /ang-( - )/masr downregulation. the deleterious effects of ang ii in pulmonary, inflammatory conditions have been previously discussed in this article. hence, the imbalance of both raas axes may be responsible for disease progress in three main phases: hematological, pulmonary, and inflammatory [ ] . the first phase might be the result of sars-cov- invasion of hematopoietic stem/progenitor cells (hspc) expressing ace , leading to the significantly high frequency of lymphopenia and viral induced hypoxia [ ] . secondly, the pulmonary involvement possibly related to ace/ang ii/at r activation, which allegedly induces lung inflammation, endothelial dysfunction, fibrosis, and pulmonary arterial htn, leading to development of the typical acute respiratory distress syndrome (ards) [ ] . moreover, severe hypoxemia might be a result of hypoxic vasoconstriction and reduced ventilation-perfusion ratio [ ] . this explains the widespread need for mechanical ventilation in adults with covid- . the third phase is a hyper-inflammatory state named cytokine storm syndrome which plays a key role in determining disease severity. a range of immune-related cascades might be activated by the exacerbation of ace/ang ii/at r, including the synthesis and release of pro-inflammatory cytokines, including il- , il- , and tnf-α [ ] . this process, added to viral intrinsic activation of both innate and adaptive immune systems, may explain the higher levels of il- , il- r, il- , and tnf-α, as well as lower cd + and cd + levels in covid- patients [ ] . pediatric patients, in particular, presented increased serum creatine kinase mb, procalcitonin and c reactive protein, lymphocytopenia, and leukopenia associated with higher disease severity [ ] . the three presented mechanisms result in alveolar edema and vascular leakage, both responsible for hypoxia and dyspnea [ ] . due to the relevance of the raas imbalance hypothesis in the pathophysiological course of the disease, researchers have pursued the presumable beneficial effects of raas blocker therapy in covid- . the main purpose is to prevent the exacerbation of the ace/ang ii/ at r axis, thought to be responsible for the aggravation of the condition [ ] . in this sense, the national health institute (nih) included several clinical trials using low doses of raas blocker therapy, arbs and acei for instance, to evaluate the effects on preventing disease progression. additionally, studies on ang-( - ) analogues and recombinant human ace also seem promising. the dynamic changes of raas expression throughout life have been considered an important modulator of development of kidney and cardiovascular systems (fig. ) . this physiological modulation process might be divided in three principal stages: fetal, postnatal, and puberty [ ] . in fetal life, the expression of at and at receptors in vasculature and kidneys is greater than at any other period of life in response to renal and cardiovascular demands [ ] . subsequently, postnatal ontogeny implicates a decrease of at r expression due to kidney maturation, activation of which by ang ii plays a protective role in newborns fighting infections [ ] . similarly, higher expression of at r might exert a beneficial effect in covid- due to its anti-inflammatory properties [ ] . research conducted by bunyavanich et al. [ ] showed an age-dependent expression of ace in nasal epithelium, which was relatively low in children younger than years old and progressively increased until adulthood. this may also explain the lower infectivity of covid- in the pediatric group. lastly, during puberty, sexual dimorphism of arterial pressure is closely related to differential modulation of the cardiovascular system by sex hormones, which may impact upon raas expression. testosterone, for instance, has been demonstrated to increase the expression of at receptor and decrease the expression of at in male rodents [ ] . additionally, greater ace and masr expression has been demonstrated in female rodents [ ] and higher plasma ang-( - ) levels have been seen in women than in men [ ] . these findings might explain the male predominance of covid- cases in both children and adults [ ] . on the other hand, specific aspects of the immune system in childhood may also exert an unexpected protection in covid- due to its immaturity, as disease progression and severity are correlated with a hyperimmune response. immunity is constructed and modulated by several genetic and environmental factors from the womb until late childhood [ ] . maternal diet, vaginal health, birth period, breastfeeding, diet, hygiene, and antibiotic medication are some of the defining aspects that determine the child's capacity to fight pathogens through the improvement of the adaptive immune system. despite depending greatly on the innate response due to the poor immunologic memory, studies on respiratory infections showed an inadequate activation or suppression of some innate components in infants-inf-α/β, il- , and tnf-α for instance [ ] . furthermore, the naïve adaptive immune system has been described to present weaker t helper (th ) responses, limited b cell repertoire and inefficient generation of somatic hypermutations [ ] . in common viral infections, these findings might contribute to greater disease severity. in covid- , however, the presumed disadvantage might actually be a protective mechanism, preventing cytokine storm and leading to the large number of asymptomatic and milder cases in children. the emergence of covid- represents a unique challenge for clinicians caring for patients from all age groups. better understanding of the disease is vital so that improved clinical management and therapeutic strategies can be developed. the dynamism of the raas throughout life and the immature immune system in pediatric patients may be responsible for the milder clinical presentation and the epidemiological profile of covid- in the young. further investigation is necessary to completely solve the pathophysiological mechanism of the disease. the raas is crucial for kidney homeostasis. the balance between the classical and alternative raas axes is complex and depends on several cascades of events and effector molecules to exert its effects throughout the body. in this regard, raas inhibitors have grown in clinical relevance due to the multiplicity of beneficial applications. the role of the raas in pediatric kidney diseases has been investigated by several research groups in several different conditions. their findings corroborate the well-known deleterious effect of ace/ang ii/ at r axis upregulation in worsening disease prognosis. in this regard, stimulation of the alternative axis seems promising for reversing the inflammatory and fibrotic processes common in these conditions. meanwhile, several new raas fragments have been discovered and must be closely analyzed to aid further identification of its escape mechanisms and their pharmacological potential, mainly in cardiovascular and kidney diseases. although the pathophysiology of covid- remains obscure and requires further study, the raas hypothesis may help explain the disease's natural history. ace inhibition, ace and angiotensin-( - 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) in adolescents born preterm the association between serum chloride levels and chronic kidney disease progression: a cohort study increasing potassium intake to prevent kidney damage: a new population strategy? fetal programming and the angiotensin-( - ) axis: a review of the experimental and clinical data simões e silva ac ( ) genetic deletion of the angiotensin-( - ) receptor mas leads to glomerular hyperfiltration and microalbuminuria teixeira mm ( ) beneficial effects of the activation of the angiotensin mas receptor in a murine model of adriamycin-induced nephropathy evidence for a role of angiotensin converting enzyme in proteinuria of idiopathic nephrotic syndrome podocyte-specific overexpression of human angiotensinconverting enzyme attenuates diabetic nephropathy in mice indoxyl sulfate downregulates expression of mas receptor via oat /ahr/stat pathway in proximal tubular cells role of the kidney in the prenatal and early postnatal programming of hypertension the expressional disorder of the renal ras mediates nephrotic syndrome of male rat offspring induced by prenatal ethanol exposure interactions between cytokines, congenital anomalies of kidney and urinary tract and chronic kidney disease the protective arm of the renin-angiotensin system may counteract the intense inflammatory process in fetuses with posterior urethral valves blood pressure in children with chronic kidney disease: a report from the chronic kidney disease in children study hypertension in children with chronic kidney disease circulating renin angiotensin system in childhood chronic renal failure: marked increase of angiotensin-( - 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) between men and women publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments the authors thank amanda braga costa for her assistance with the creation of the figures. key: cord- - bc zeww authors: ratajczak, mariusz z.; bujko, kamila; ciechanowicz, andrzej; sielatycka, kasia; cymer, monika; marlicz, wojciech; kucia, magda title: sars-cov- entry receptor ace is expressed on very small cd (−) precursors of hematopoietic and endothelial cells and in response to virus spike protein activates the nlrp inflammasome date: - - journal: stem cell rev rep doi: . /s - - -z sha: doc_id: cord_uid: bc zeww angiotensin-converting enzyme (ace ) plays an important role as a member of the renin–angiotensin–aldosterone system (raas) in regulating the conversion of angiotensin ii (ang ii) into angiotensin ( – ) (ang [ – ]). but at the same time, while expressed on the surface of human cells, ace is the entry receptor for sars-cov- . expression of this receptor has been described in several types of cells, including hematopoietic stem cells (hscs) and endothelial progenitor cells (epcs), which raises a concern that the virus may infect and damage the stem cell compartment. we demonstrate for the first time that ace and the entry-facilitating transmembrane protease tmprss are expressed on very small cd (+)cd (+)lin(−)cd (−) cells in human umbilical cord blood (ucb), which can be specified into functional hscs and epcs. the existence of these cells known as very small embryonic-like stem cells (vsels) has been confirmed by several laboratories, and some of them may correspond to putative postnatal hemangioblasts. moreover, we demonstrate for the first time that, in human vsels and hscs, the interaction of the ace receptor with the sars-cov- spike protein activates the nlrp inflammasome, which if hyperactivated may lead to cell death by pyroptosis. based on this finding, there is a possibility that human vsels residing in adult tissues could be damaged by sars-cov- , with remote effects on tissue/organ regeneration. we also report that ace is expressed on the surface of murine bone marrow-derived vsels and hscs, although it is known that murine cells are not infected by sars-cov- . finally, human and murine vsels express several raas genes, which sheds new light on the role of these genes in the specification of early-development stem cells. [figure: see text] the sars-cov- pandemic, with its high mortality, has become an urgent clinical problem. this infection damages several organs, including lungs, heart, blood vessels, kidneys, and intestines and may lead to a fatal complication known as a "cytokine storm", which results in uncontrolled hyperactivation of the innate immunity-initiated response. sars-cov- may i) directly infect human cells and lead to their lysis or damage or ii) upregulate mediators of the renin-angiotensin-aldosterone system (raas), which may eliminate cells in a nlrp inflammasome hyperactivation-mediated manner by pyroptosis [ ] [ ] [ ] [ ] [ ] . it is well established that sars-cov- enters human cells after binding to the angiotensin-converting enzyme (ace ) receptor and utilizes a spike protein (s) for attachment and entry into the cells [ , ] . the viral s protein must be primed by transmembrane protease (tmprss ) to facilitate interaction with ace and the subsequent fusion of viral and cellular membranes [ ] . the ace receptor has been found on the surface of many cells, and its physiological role is to processes conversion of angiotensin ii (ang ii) to angiotensin ( - ) (ang [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) [ ] [ ] [ ] ] . these two members of the raas family have opposite biological effects on target cells and activate the angiotensin receptor (at r) and masr, respectively [ ] . activation of at r during sars-cov- infection has detrimental effects, inducing fibrosis, an increase in reactive oxygen species (ros) release, vasoconstriction, and gut dysbiosis. by contrast, the effect of masr activation is overall protective, ant-fibrotic, antioxidant, and vasodilatory. it has already been demonstrated that hyperactivation of at r by ang ii may lead to excessive activation of the nlrp inflammasome and cell death by pyroptosis in lung epithelium cells, endothelium, and cardiomyocytes [ ] [ ] [ ] [ ] . by contrast, after binding to masr, ang ( - ) displays the opposite effect and has been demonstrated to stimulate proliferation of skeletal muscle and hematopoietic cells [ , ] . unfortunately, because of ace internalization during sars-cov- infection ang ii is not processed to ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the nlrp inflammasome triggers an inflammatory immune response via intracellular caspase , which leads to i) release of the potent pro-inflammatory cytokines interleukin β (il- β) and interleukin and ii) mediates the release of several biologically active danger-associated molecular pattern molecules (damps) by creating gasdermin d (gsdmd) pore channels in cell membranes [ ] [ ] [ ] . this initiates a sequence of events leading to amplification of the innate immune system response and activation of its major humoral arm, the complement cascade (comc) [ , ] . based on the aforementioned, sars-cov- may enter and damage cells that express ace entry receptor or damage them by hyper-activation of the ang ii-at r axis [ ] , which may lead to excessive nlrp signaling and pyroptosis [ , ] . since many types of cells, including hscs and epcs, express both ace and at r, this mechanism suggests that the stem cell compartment may be a direct target for damage by the virus. this also raises the question of potential long-term deleterious effects of infection on organ and tissue regeneration. to address this important question, we became interested in the potential effect of sars-cov- on the population of cd − cells, known as very small embryonic-like stem cells (vsels), residing in postnatal tissues. these cells, which have been described and confirmed by several independent laboratories [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , may become specified into hscs and epcs and thus may revive the old concept of the presence of postnatal hemangioblasts in bone marrow [ ] [ ] [ ] [ ] [ ] . interestingly, several previous reports have indicated that hemangioblasts may be regulated by raas mediators [ ] . here we demonstrate for the first time that human vsels express ace , the spike protein-priming protease tmprss , and several raas genes. moreover, we show that interaction of the ace receptor with the sars-cov- spike protein activates the nlrp inflammasome in human cells, which, if hyperactivated, may lead to cell death by pyroptosis, as is well known. based on this mechanism, early-development human stem cells residing in adult tissues may be affected by sars-cov- , which could lead to long-term defects in tissue/organ regeneration. we also report that ace is expressed on the surface of murine bone marrow-derived vsels and hscs, although it is known that murine cells are not infected by sars-cov- . finally, human and murine vsels express several raas genes, which sheds new light on their role in the specification of early-development stem cells. pathogen-free, - -week-old female c bl/ j mice were purchased from the jackson laboratory (bar harbor, me; usa) at least weeks before experiments. animal studies were approved by the animal care and use committee of the university of louisville (louisville, ky, usa) and warsaw medical university (warsaw, poland). clinical-grade ucb research units were shipped from the cleveland cord blood center. this study was performed in accordance with the guidelines and approval of the institutional review board at the university of louisville school of medicine (louisville, kentucky). cells were obtained by flushing experimental mouse tibias and femurs. red blood cells (rbcs) were removed by lysis in bd pharm lyse buffer (bd biosciences, san jose, ca, usa), washed, and resuspended in appropriate media. vsels (lin − /cd + /cd − or lin − /cd + /cd − ) and hscs (lin − /cd + /cd + or lin − /cd + /cd + ) were isolated from human umbilical cord blood (hucb) samples from healthy full-term newborns. samples were obtained through a partnership with the cleveland cord blood bank center under irb approval for scientific research purposes. briefly, mononuclear cells (mncs) were isolated from hucb samples by density-gradient centrifugation on a ficoll-paque gradient (ρ = . g/ml; ge healthcare). mncs were then labelled with anti-cd or anti-cd microbeads (miltenyi biotec) and separated on magnetic columns (miltenyi biotec). the cell population of cd + or cd + -enriched cells was stained with fluorescence-labeled antibodies (all becton dickinson) for the hematopoietic lineage marker (lin), cd , and cd or cd . the following murine antihuman antibodies were employed for staining: anti-cd a staining was performed in rpmi- medium supplemented with % fbs on ice for min. the cells was subsequently washed, resuspended in rpmi containing % fbs, and sorted using a moflo xpd cell sorter (beckman coulter) as a highly purified population of lin − /cd + /cd − vsels and lin − /cd + /cd + hsc using the strategy presented in fig. a or lin − /cd + /cd − vsels and lin − /cd + / cd + hscs using the strategy presented in fig. b . lin-/sca- + /cd − cells (vsels) and lin − /sca- + /cd + cells (hscs) were isolated from the bm of adult c bl/ mice (five weeks old; jackson laboratory, usa). bone marrow was flushed from the cavities of tibias and femurs, and the cell suspension was collected and filtered through a -μm strainer (bd bioscience). the population of total nucleated cells (tncs) was obtained after lysis of rbcs using × bd pharm lyse buffer (bd pharmingen, usa), washed with phosphate-buffered saline (dpbs; w/o ca + or mg + ; life technologies), and resuspended in pbs containing % fetal bovine serum (fbs, thermo fisher scientific). staining for vsels and hscs was performed with fluorescence-labeled antibodies for cd , the hematopoietic lineage marker (lin), and sca- . the following anti-mouse antibodies were employed for staining: rat anti-cd (allophycocyanin [apc]-cy ; clone -f ), anti-cd r/b (phycoerythrin [pe]; clone ra - b ), anti-gr- (pe; clone rb - c ), anti-tcrαβ (pe; clone h - ), anti-tcrγδ (pe; clone gl ), anti-cd b (pe; clone m / ), anti-ter (pe; clone ter- ), and anti-ly- a/e (also known as sca- ; biotin; clone e - . , with streptavidin conjugated to pe-cy ). all antibodies were obtained from bd biosciences. staining was performed in pbs with % fbs on ice for min. the cells were subsequently washed, resuspended in dmem plus % fbs, and sorted using a facs melody cell sorter (becton dickinson). the sca- + lin − cd − cells (vsels) and sca- + lin − cd + cells (hscs) were isolated according to the gating and sorting strategy described in fig. . ace expression was detected on human ucb-derived vsels and hscs. briefly, mononuclear cells (mncs) were isolated from hucb samples by density-gradient centrifugation on a ficoll-paque gradient (ρ = . g/ml; ge healthcare). mncs were then labelled with anti-cd or anti-cd microbeads (miltenyi biotec) and separated on magnetic columns (miltenyi biotec). the cell population of cd + or cd + -enriched cells was stained with fluorescence-labeled antibodies for the hematopoietic lineage marker (linlisted in vsels and hscs sorting method), cd (pe-cy ; clone hi , becton dickinson), and cd (apc; clone , beckman dickinson) or cd (apc; clone ac , beckman dickinson). additionally, cells were stained with ace antibody (pe, clone e- , santa cruz). staining was performed in rpmi- medium supplemented with % fbs on ice for min. the cells were subsequently washed, resuspended in rpmi containing % fbs, table list of primer sequences used for real-time qpcr analysis of cells isolated from human umbilical cord blood. and analyzed on bd lsr ii flow cytometer (becton dickinson). vsels, hscs, and mncs facs-sorted from murine bone marrow as well as vsels, hscs, and mncs isolated from ucb were lysed, and then total rna was isolated using the mirneasy mini kit (qiagen, ca). cdna was prepared from rna template using the iscript cdna synthesis kit (bio-rad) according to the manufacturer's recommendations. real-time pcr was performed using a cfx touch™ real-time pcr detection system (bio-rad) with itaq univer sybr green supermix (bio-rad) reagent. all pcrs were performed using the following conditions: predenaturation at °c for min, cycles of denaturation at °c for s, and annealing at °c for s. a melting curve was generated to confirm primer specificity and to avoid the possibility of amplifying dna contamination. the mrna levels of target genes were normalized to the β microglobulin mrna level. the average cycle threshold (ct) value of three technical replicates was used for the comparative ct (ΔΔct) method. the primer sequences used for rt-qpcr are listed in tables and . total rna was isolated using the rneasy mini kit, including treatment with dnase i (both from qiagen inc., germantown, md, usa). the purified mrna ( ng) was afterwards reverse transcribed into cdna using the first strand cdna synthesis kit (thermo scientific, waltham, ma, usa) according to the manufacturer's instructions and using a mixture of oligo(dt) and random hexamers. amplification of the synthesized cdna fragments was carried out using takara experiments ucb-derived hsc were plated in -well plates and stimulated with ncp-cov ( -ncov) spike protein (s + s ecd, with his-tag; sino biological) at a concentration of nm alone or together with angiotensin fragment - (millipore sigma) at a concentration of μg/ml. all results are presented as mean ± sd. statistical analysis of the data was done using student's t test for unpaired samples, with p ≤ . considered significant. like hscs, highly purified human vsels express mrna for ace , tmprss , and raas peptides and receptors as well as components of the nlrp inflammasome complex. we sorted very small cd + lin − cd − cells (vsels) and cd + lin − cd + cells (hscs) from ucb by facs (fig. ) and phenotyped them by real-time pcr for expression of mrnas for the ace entry receptor for sars-cov- , the spike protein-processing enzyme timprss , the receptors for ang ii (at r and at r), and the ang ( - ) receptor (masr, fig. ) . we found that all of these receptors were expressed in human vsels and hscs. in addition, both populations of cells expressed the cma gene, encoding chymase, a chymotryptic serine proteinase, which is also involved in the processing of ang i into ang ii. interestingly, we also detected expression of renin, an enzyme that processes conversion of angiotensinogen into ang i, indicating that raas is involved in regulating the biology of both types of cells. importantly, expression of sars-cov- entry receptor ace was subsequently confirmed at the protein level by facs on both cd + and cd + vsels and hscs ( fig. a and b) . expression of ace was slightly higher on the surface of cd + vsels (~ %) than on cd + vsels (~ %). at the same time cd + hsc~ % expressed ace and cd + hsc~ %. highly purified murine vsels and hscs express mrnas for ace , tmprss , and raas peptides and receptors as well as components of the nlrp inflammasome complex. although murine cells are not infected by sars-cov- , we became interested in whether the murine counterparts of human vsels and murine hscs express the virus-entry receptor and components of the raas. to address this question, we purified murine bone marrow-derived vsels and hscs by facs sorting (fig. ) , and the mrna expression for all of these genes was evaluated by real-time pcr. figure a shows that murine vsels express, at much higher levels than hscs and mononuclear cells (mncs), mrnas for ace , at r, at r, at r, mas, and transmembrane protease (tmprss ), which in human cells primes the sars-cov- spike protein (s) for better interaction with ace . interestingly, fig. b demonstrates that murine vsels and hscs highly express mrnas for angiotensinogen, renin, and the ace receptor, which indicates that these cells may be regulated by intrinsic raas. because they also express at r and masr, they may respond to ang ii and ang ( - ) stimulation. stimulation of human hscs and vsels with sars-cov- spike protein (s) activates the nlrp inflammasome. we have proposed that hyperactivation of the nlrp inflammasome is the culprit in the induction of cytokine storms in sars-cov- infected patients, leading to multi-organ damage. there are different mechanisms that may lead to this problem, involving extensive stimulation of at r by ang ii or enhanced activity of the complement cascade, which recognizes viral proteins by the lectin pathway, immune complexes by the classical pathway, and responds to toll-like receptor activation by activating the alternative pathway. we recently hypothesized that the nlrp inflammasome is activated after stimulation of ace by viral spike protein (s). to test this hypothesis, we exposed human ucb-isolated cd + lin − cd + and cd + lin − cd + hscs to recombinant s protein for h, and activation of the nlrp inflammasome was evaluated for changes in mrna expression of nlrp , il- β, il- , and asc. we also evaluated changes in expression of mrnas for two other inflammasomes, aim and nlrp . we show for the first time that exposure of ucb hscs to spike protein enhances transcription of all mrnas evaluated in our studies. at the same time activation of nlrp inflammasome in response to spike protein was attenuated in presence of ang - (fig. ) . we also detected elevated levels of secreted il- β, as determined by elisa, in the conditioned media from cells stimulated by spike protein, which is an important indicator of nlrp inflammasome activation. importantly, we exposed human ucb-purified vsels to spike protein, and as shown in fig. we found upregulation of nlrp mrna. interestingly, murine vsels also highly express mrnas for elements of the nlrp inflammasome and raas genes (fig. ) . nevertheless, they are not infectable by sars-cov- . however, they may be regulated by raas similarly as have been proposed for putative hemangioblasts [ ] . the most important message of this report is that human earlydevelopment stem cells deposited in adult tissues, known as very small embryonic-like stem cells (vsels), like specified hscs or epcs, express on their surface the ace receptor for entry of sars-cov- as well as the entry-facilitating protease tmprss . in addition, these cells express at r, which indicates thattheycanbechallengedbyangiistimulation.wealsoreportfor the first time that the virus-derived recombinant spike protein (s) activates the nlrp inflammasome in human vsels and hscs. our results are highly relevant to the potential long-term effects of sars-cov- infection, as the virus may damage vsels by i) direct entry and cell lysis or ii) induction of cell death by pyroptosis due to hyperactivation of the nlrp inflammasome in response to s protein or to excessive ang ii stimulation by at r. nlrp inflammasome-induced cell death by pyroptosis is a result of activation of caspase , the release of il- β and il- , the perturbation of mitochondrial function, the creation of pores in the cell membrane by gasdermin d, and the release of several alarmines or dangerassociated molecular pattern molecules (damps) that subsequently amplify an uncontrolled immune response [ , [ ] [ ] [ ] . this response involves secretion from other cells of several pro-inflammatory cytokines such as. il- or tnf-α and mediators as well as activation of the complement and the relative expression of mrnas for ace , raas peptides and receptors, and components of the nlrp inflammasome in highly purified murine bone marrow-derived vsels and hscs. real-time pcr quantitation of facs-sorted murine vsels and hscs in comparison with mononuclear cells; *p < . , **p < . , ***p < . . in order to evaluate relative expression, the comparative Δct method was employed. results are presented as mean ± sem. panel a. differences in the expression of mrnas for angiotensin-converting enzyme (ace ), the type angiotensin ii receptor (at ), the type angiotensin ii receptor (at ), the proto-oncogene mas (mas), the type angiotensin ii receptor (at ), and transmembrane protease (tmprss ). panel b. differences in the expression of mrnas for renin (ren), angiotensinogen (agt), and angiotensin-converting enzyme (ace) coagulation cascades [ ] [ ] [ ] . if uncontrolled, this process may end in a cytokine storm and fatal organ damage. it is well known that the innate immune response and activation of the nlrp inflammasome are important defense mechanisms during the first days of infection, until acquired immunity responds with the production of antibodies. however, on the other hand hyperactivation of this intracellular protein complex may induce a cytokine storm, with detrimental effects, leading to multi-organ failure. we have proposed three scenarios for how this could happen. first, the sars-cov- spike protein (s), after binding to cell surfaceexpressed ace , directly triggers nlrp inflammasome activation. in fact, we show for the first time that s protein binding to ace on human cells contributes to activation of the nlrp inflammasome. importantly, we found that activation of the nlrp inflammasome in human cells after the interaction of ace with the sars-cov- s protein is ameliorated in the presence of ang ( - ) . second, as previously reported, after engaging the at receptor, ang ii activates the nlrp inflammasome in lung, kidney cells, and cardiomyocytes, and excessive activation of the ang ii-at r axis in these cells may lead to pyroptosis [ , , ] . since, as shown in our studies, both vsels and hscs highly express at r, there is i) an increase in ang ii activity in patients infected with sars-cov- and ii) a lack of protective ang ( - )-mas signaling due to blockade and downregulation of ace by viral proteins, which may hyperactivate the ang ii-at r axis. third, recognition and interaction of the complement cascade with sars-cov- releases several potent cleavage fragments, including c a and c a anaphylatoxins as well as the c bc membrane attack complex (mac), which may also directly trigger activation of the nlrp inflammasome in target cells including population of stem cells. therefore, the potential damage of vsels after virus entry or exposure to nlrp inflammasome hyperactivating mediators may have a negative effect on the regenerative potential of sars-cov- -infected individuals and create potential long-term health problems in survivors. this, however, will require further study. in support of this possibility, ace function and ang ( - )-masr signaling, which are perturbed by sars-cov- infection, play a role in the proliferation of several stem cell types, including hscs, epcs, and skeletal muscle cells [ , , ] . moreover, since several complications from endothelium have been already reported, it is important to assess the effects of infection on hematopoiesis and lymphopoiesis and also take into account damage of more primitive precursors of these cells that are vsels. another important observation is that raas mediators may play a role in the development of vsels. as mentioned in the introduction, the role of raas in the development of putative hemangioblasts has been reported in several old publications [ , , , ] . these cells were purified for example from human para-aortic splanchnopleura as cd + , cd − , and cd − real-time pcr quantitation of facs-sorted murine vsels and hscs in comparison with mononuclear cells; *p < . , **p < . , ***p < . , ****p < . . in order to evaluate relative expression, the comparative Δct method was employed. the relative quantity of target gene was normalized to the endogenous β microglobulin gene. results are presented as mean ± sem. graphs represent differences in the expression of mrnas for nacht, lrr, and pyd domain-containing protein (nlrp ); caspase (casp ); interleukin β (il- β); interleukin (il- ); gasdermin (gsdm); and high-mobility group protein b (hmgb ) protein (s + s ecd, his tag) on the expression of inflammasome related genes. real-time pcr quantitation of facs sorted human ucb derived vsels in comparison to mononuclear cells; *p < . , **p < . , ***p < . . in order to evaluate relative expression, comparative Δct method was employed. results are presented as mean ± sem. differences in the expression of mrnas for nlrp after h exposure to sars-cov- spike protein population [ ] . they expressed the ace receptor (cd ), however they were not evaluated for the expression of sars-cov- entry receptor ace . in our studies human vsels, which may correspond to hemangioblasts in in vitro assays, are present among the population of human ace + , very small cd + cd + lin − cd − cells. these cells, which have been isolated from adult bone marrow, mobilized peripheral blood, and umbilical cord blood, according to several independent reports, have been proposed to serve in postnatal tissues as a backup stem cell population involved in tissue or organ rejuvenation [ , [ ] [ ] [ ] ] . importantly in appropriate experimental models they can become specified into functional hscs and epcs. in conclusion, since we still do not have an effective sars-cov- vaccine in hand, the results presented in our current work suggest that inhibition of the nlrp inflammasome by the smallmolecule inhibitor mcc or application of nlrp inflammasome inhibitors, such as ang ( - ) or heme oxygenase activators, could find potential clinical applications to prevent onset of a cytokine storm and cell pyroptosis [ ] . in further support of this possibility, encouraging results have already been obtained in employing antibodies against a nlrp inflammasome-activation product, il- β. finally, based on our results demonstrating ace expression on the surface of vsels, further studies of infection with live virus will be needed to address whether the virus may enter these cells. finally, the expression of several genes involved in raas in vsels raises the possibility that this system is involved in the development and specification of early-development stem cells. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. signaling by the angiotensin-converting enzyme sars-cov- receptor and regulator of the renin-angiotensin system: celebrating the th anniversary of the discovery of ace network modeling reveals steps in angiotensin peptide processing angiotensin-converting enzyme is a functional 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publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -moxhk e authors: novaes rocha, vinicius title: viral replication of sars-cov- could be self-limitative - the role of the renin-angiotensin system on covid- pathophysiology date: - - journal: med hypotheses doi: . /j.mehy. . sha: doc_id: cord_uid: moxhk e currently, the world is suffering with one of the biggest pandemics of recent history. caused by severe acute respiratory syndrome coronavirus (sars-cov- ), the coronavirus disease (covid- ) is provoking devastating consequences on economic and social fields throughout all continents. therefore, pathophysiological knowledge about covid- is imperative for better planning of preventive measures, diagnosis, and therapeutics of the disease. based on previous studies, this work proposes new hypothesis related to the role of the renin-angiotensin system on the pathophysiology of covid- , and its purpose is to enrich the discussion and to offer alternative ways for experimental and clinical studies aiming at the formulation of new diagnosis and / or treatment methods. the renin-angiotensin system (ras) is currently one of the focuses of worldwide research due to coronavirus (covid- ), a pandemic that has destabilized the world and created devastating consequences for economic and social areas [ ] [ ] [ ] . amongst the components of rennin-angiotensin system (ras), the angiotensin-converting enzyme (ace ) has gained great prominence for being directly associated with severe acute respiratory syndrome coronavirus (sars-cov- ) infection, the coronavirus related to covid- [ , ] . thus, the protagonism of ace is being debated amongst researchers with the aim of establishing a role of ras on the pathophysiologic context of covid- [ ] [ ] [ ] . ras was initially described as a regulating component of blood pressure and hydric balance, being called "classic pathway". subsequently, with the discovery of new components and finding of a "local tissue ras", the existence of a more complex system was observed with endocrine, paracrine and intracrine characteristics [ ] [ ] [ ] . ras elucidation continues to expand, new components are being discovered and also new ways of interaction between the local and systemic pathways, as well as interactions with the external environment [ ] . nowadays, ras is known as a complex system, with systemic and local (tissue) activities which communicate between themselves and involve different sign pathways, leading to different results depending on the chosen pathway [ , ] . this system's balance is imperative for homeostasis, in which the angiotensinconverting enzyme (ace) and ace are the main weights in this scale ( figure ). when the scale plate tends to the ace side, the production of angiotensin ii (ang ii) and the activation of its respective receptors (at ) increases. the activation of the detrimental pathway (ace -ang ii -at r) stimulates actions such as cell proliferation, inflammation, fibrosis, and thrombosis. on the other hand, when the scale plate tends to the ace side, the production of angiotensin - (ang - ) increases, as well as the activation of its respective receptors (mas) [ ] . the activation of the protective pathway (ace -ang - -mas) neutralizes the detrimental pathway, stimulating actions such as antiinflammatory, anti-fibrotic, and anti-thrombotic effects [ , ] . coronaviruses are classified into four different genus, in which species (α hcov-nl , sars-cov, and sars-cov- ) have ace as a receptor [ ] . the process of cell invasion occurs similarly amongst the different species. briefly, the virus-cell interaction occurs through the ace receptor coupling, and the internalization of the virus then occurs through fusion or endocytosis, with the participation of other components (clathrin, tmprss , and others) [ ] [ ] [ ] . although they belong to the same subfamily coronavirinae and share the same receptor, these viruses have significant differences, as presented in table . amongst these differences, it is important to highlight in sars-cov- (the virus related to covid- ) the longer incubation period and the faster viral peak, observed a few days after the beginning of symptoms. such characteristics might be related to the fact that sars-cov- presents the most extreme deficiency of cpg amongst the known betacoronavirus genomes. the reduction of the cpg index may have allowed the virus to escape from immune response mediated by human zap (zinc finger antiviral protein), thus becoming a severe human pathogen [ ] . furthermore, sars-cov- has a much higher infection and replication capacity than sars-cov, however without inducing in a significant way the increase in interferons type i, ii or iii, and inflammatory mediators (pro-inflammatory cytokines / chemokines) [ ] . such characteristics of sars-cov- are imperative for the understanding of covid- pathophysiology. the high replication rate associated with low inflammation leads to the viral peak even at the onset of symptoms, which makes early disease identification difficult, and as a consequence, makes treatment with antivirals ineffective because the effectiveness of their action occurs during the rise of the replication curve, the most recommended phase for such medicine [ ] . although sars-cov and sars-cov- use ace as cell coupling receptor, the pathogenic characteristics of sars-cov- provide a great difference on disease progression, as shown in figure . ace is a fundamental piece in the pathophysiology of covid- , since the high replication capacity of sar-cov- is directly related to the coupling to ace and cell infection. studies show that the virus causes a reduction of ace in the infected organs [ ] . therefore, the higher the amount of virus, the higher will be the use of ace by it, creating an inverse correlation between the amount of sars-cov- and ace . at a certain time, the constant and gradual reduction of ace will cause a ras imbalance, increasing the action of ace in detriment of the decrease in the action of ace , thus prevailing the harmful factors ( figure ). the ace level reduction caused by sars-cov- infection may be directly related to the pathogenesis of covid- [ ] . the ang - decrease and the lower activation of mas receptors and / or the ang ii production increase, with subsequent activation of at receptor, is widely known for the triggering of inflammation and fibrosis [ ] . for example, a study with mice has shown that acute lung lesion results in a marked decrease of ace , and the use of recombinant ace has a protective effect against the lung lesion. the same study noted that ace , ang ii, and the ang ii receptor (at ) provoke the disease pathogenesis. thus, the important role of ras on acute lung lesion pathogenesis is demonstrated [ ] . histologically, patients with covid- present a pattern of diffuse alveolar damage and perivascular lymphocyte infiltration, similar to what is observed in influenza cases. a surprising histopathological finding on covid- was the lung angiogenesis, which is . times greater than on the lungs of patients with influenza [ ] . angiogenesis, a complex process by which new blood vessels are formed from existing, is induced by hypoxia. hypoxia is a condition in which tissues are not properly oxygenated, resulting in considerable cell stress and adaptive responses. the transcriptional responses to hypoxia are mostly mediated by the hypoxiainducible factor (hif), a transcription factor that acts as oxygen sensors and are related to the activation of vegf-a expression [ , ] . under hypoxia, hif influences ace and ace in different ways, positively regulating the expression of ace and negatively of ace [ , ] . in covid- , such circumstances may worsen the clinical condition, for stimulating even more the inflammatory, fibrotic, and thrombogenic pathways of ras. although covid- is clinically classified as an acute respiratory syndrome, the increased angiogenesis suggests a chronic cell adaptation towards progressive hypoxia. the chronic adaptation to hypoxia could explain the higher resistance of some patients towards low o saturation, in which such patients do not present an involuntary increase of ventilation, even when great lung damage is present ("ground-glass opacity"). the replicative cycle of sars-cov- causes a local immune response as a result of cell death and tissue damage. in some cases, the immune response occurs in an unregulated manner, triggering a cytokine storm, and consequently, generalized pulmonary inflammation [ ] . as with covid- , other acute respiratory diseases (sars, mers, influenza) can also present the clinical picture of cytokine storm [ ] . high virus titers and dysregulation of the cytokine / chemokine response cause an inflammatory cytokine storm, mainly related to the influx of inflammatory mononuclear macrophages. the activation of these macrophages by interferon-α / β produces more chemotactic factors for monocytes (ccl , ccl and ccl ), resulting in the additional accumulation of mononuclear macrophages, and subsequently, an increase in the levels of pro-inflammatory cytokines (tnf, il- , il -β and inducible nitric oxide synthase), thus increasing the severity of the disease [ ] . in severe cases of covid- , patients demonstrate elevated plasma levels of il- , il- , il- , granulocyte colony stimulating factor (g-csf), ip- , mcp , macrophage inflammatory protein α (mip α) and tumor necrosis factor (tnf) [ ] . viral infection and replication in airway epithelial cells can cause high levels of pyroptosis. pyroptosis is a highly inflammatory form of programmed cell death seen in cytopathic viruses such as sars-cov- . il- β, an important cytokine released during pyroptosis, is elevated in infection by sars-cov- , thus being a possible trigger for the cytokine storm [ ] . the excessive or uncontrolled release of pro-inflammatory cytokines, characteristic of the cytokine storm, is also associated with non-infectious diseases, such as autoimmune diseases [ ] . yiguo qiu et al. analyzed sars in mice with autoimmune uveitis. the study demonstrated that the administration of ang - antagonist reversed the protective effects of ace on inflammatory signals and on the production of inflammatory cytokines, as well as on the regulation of local immune responses. the inhibition of ang - increased the production of the pro-inflammatory cytokines il- , il- β, tnf-α, and mcp- [ ] . this study demonstrates the importance of ang - in mediating the inflammatory process, as its inhibition can cause an increase in the production of pro-inflammatory cytokines, similar to that observed in the cytokine storm in covid- . ace plays a crucial role in ras because it neutralizes ace activity by degrading ang i in ang ( - ), as well as hydrolyzing ang ii, producing ang ( - ) [ ] . thus, ace is essential to stimulate the beneficial effects of the protective axis of ras, ace / ang ( - ) / mas, and to mitigate the deleterious effects of the harmful axis of ras, ace / ang ii / at . maintaining normal levels of ace in the lung is beneficial for combating inflammatory lung disease. the reduction in ace expression may be related to pulmonary inflammation and subsequent cytokine storm seen in patients with severe covid- . ace maintains the proper function of the heart and kidneys, and the negative regulation of ace by sars-cov- can compromise this protective characteristic and contribute to the damage caused by the infection of these organs [ ] . renin-angiotensin system and the self-limitative replication of sars-cov- ras is classified as systemic (classic) and local, and the interaction between them is fundamental to homeostasis [ , , ]. this interaction between local and systemic ras probably occurs through the circulation of soluble ace and ace . although they are transmembrane proteins, ace and ace may undergo a cleavage process by ace secretase and adam , respectively, releasing an active extracellular domain. this soluble component is capable of converting angiotensin i into ang ii (ace) and ang ii into ang - (ace ) [ , ] . the balance between the amount of transmembrane ace and soluble ace is imperative, because the excessive release of the soluble form to the extracellular environment could lead to local tissue injury, since it will cause misbalance on the production of local ang ii and ang - . the local ras misbalance makes the harmful pathway effects prevail (ace -ang ii -at r) on the referred organ or tissue [ ] . in an ace receptor-dependent viral infection (coronavirus nl ), the reduction of ace cellular level occurs without changes in ace levels [ ] . this finding, plus the fact that sars-cov- elicits a low immune and inflammatory response, even when compared to sars-cov [ ] , is more evidence that covid- pathogenesis is more related to ras misbalance than to the viral action itself. mossel although the protease activity of adam over ace is observed, the same is not observed in ace [ ] [ ] [ ] . interestingly, ace can regulate the activity of adam , since it can be triggered from the activation of the at receptor by ang ii [ ] . thus, ace directly regulates the formation of ang ii from ang i, and interferes via the action of adam in ace , in the tissue formation of ang - from ang ii. therefore individuals with greater expression of tissue ace, and consequently, greater production of ang ii, will present a decrease in tissue ace and an increase in soluble ace (systemic), due to greater activation of adam and ace cleavage. this interaction between ace, adam and ace can explain the difference in covid- involvement between men and women. androgens increase renin levels and ace activity, while estrogen decreases renin levels, ace activity, and at receptor density, and is also related to increased levels of ang - [ ] , an important actor in the protective axis of ras. this explains the fact that men have higher circulating (soluble) ace levels than women [ ], due to the greater action of adam via androgens / eca / ang ii / at . thus, due to higher levels of circulating ace , men may present a lower level of tissue ace , becoming more vulnerable to local sars imbalance after sars-cov- infection. this argument gains more strength when we observe that children [ ] and the elderly [ ] do not show a difference in the involvement of covid- between genders, probably due to the lack of hormonal interference before puberty and also due to the loss of protection from estrogen in women after menopause. show similar responses to the disease, presenting a great increase in the production of ang - when compared to the control group [ , ] . in sick adults, the ang - level is also high when compared to healthy subjects [ , ] , but the response intensity is much lower when compared to dogs and covid- brings the world economy to its knees if the world fails to protect the economy, covid- will damage health not just now but also in the future developing a sustainable exit strategy for covid- : health, economic and public policy implications system blockers and the risk of covid- renin-angiotensin-aldosterone system inhibitors in patients with covid- ace : the key molecule for understanding the pathophysiology of severe and critical conditions of covid- : demon or angel? viruses association between reninangiotensin system inhibitors and covid- complications covid- : the renin-angiotensin system imbalance hypothesis renin-angiotensin system revisited a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - aceh/ace is a novel mammalian metallocarboxypeptidase and a homologue of angiotensinconverting enzyme insensitive to ace inhibitors ectodomain shedding of angiotensin converting enzyme in human airway epithelia angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas renin-angiotensin-aldosterone system inhibitors in covid- . reply mechanisms of coronavirus cell entry mediated by the viral spike protein entry of human coronavirus nl into the cell efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss coronavirus membrane fusion mechanism offers a potential target for antiviral development clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study insight into novel coronavirus -an updated interim review and lessons from sars-cov and mers-cov sars-cov- viral load in upper respiratory specimens of infected patients differential downregulation of ace by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl extreme genomic cpg deficiency in sars-cov- and evasion of host antiviral defense comparative replication and immune activation profiles of sars-cov- and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid- the pivotal link between ace deficiency and sars-cov- infection targeting the renin-angiotensinaldosterone system in fibrosis angiotensin-converting enzyme protects from severe acute lung failure pulmonary vascular thrombosis, and angiogenesis in covid- roles of runx in hypoxia-induced responses and angiogenesis hypoxia inducible factor pathway and physiological adaptation: a cell survival pathway? role of hif- alpha in the regulation ace and ace expression in hypoxic human pulmonary artery smooth muscle cells mirna let- b promotes the development of hypoxic pulmonary hypertension by targeting ace the trinity of covid- : immunity, inflammation and intervention the pathogenesis and treatment of the `cytokine storm' in covid- into the eye of the cytokine storm aav -mediated angiotensin-converting enzyme gene delivery prevents experimental autoimmune uveitis by regulating mapk, nf-κb and stat sars-cov- pandemic and research 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and meta-analysis baseline chronic comorbidity and mortality in laboratory-confirmed covid- cases: results from the precovid study in spain polymorphism interacts with dietary n- polyunsaturated fatty acids to modulate obesity risk in the genetics of lipid lowering drugs and diet network study role of adam in kidney disease a score including adam substrates correlates to recurring cardiovascular event in subjects with atherosclerosis the egfr-adam axis in chronic obstructive pulmonary disease and cystic fibrosis lung pathology the shedding protease adam : physiology and pathophysiology tumor necrosis factor-alpha convertase (adam ) mediates regulated ectodomain shedding of the severeacute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensinconverting enzyme- (ace ) the secretases that cleave angiotensin converting enzyme and the amyloid precursor protein are distinct from tumour necrosis factor-alpha convertase receptor signaling pathways in the cardiovascular system gender and the reninangiotensin-aldosterone system circulating angiotensin-converting enzyme activity in patients with chronic kidney disease without previous history of cardiovascular disease epidemiology of covid- among children in china patients with covid- aged years and older in a university hospital in risks to children during the covid- pandemic: some essential epidemiology covid- in children: epidemiology, presentation, diagnosis and management spike protein recognition of mammalian ace predicts the host range and an optimized ace for sars-cov- infection infection of dogs with sars-cov- suessenbach fk, burckhardt bb. levels of angiotensin peptides in healthy and cardiovascular/renal-diseased paediatric population-an investigative review relationship between circulating levels of angiotensin-converting enzyme -angiotensin-( - )-mas axis and coronary heart disease. heart vessels roles of angiotensin peptides and recombinant human ace in heart failure circulating angiotensin converting enzyme activity as a biomarker of silent atherosclerosis in patients with chronic kidney disease plasma and tissue angiotensin-converting enzyme activity and plasma equilibrium concentrations of angiotensin peptides in dogs with heart disease the author has no conflict of interest or commercial sponsor for this work thanks to isabela tanuri bessa for her helpful comments. thanks also to the researchers from different knowledge areas for all their work undertaken as the basis for the construction of this article. the author has no conflict of interest or commercial sponsor for this work. key: cord- -pgr l b authors: sato, teruki; kadowaki, ayumi; suzuki, takashi; ito, hiroshi; watanabe, hiroyuki; imai, yumiko; kuba, keiji title: loss of apelin augments angiotensin ii-induced cardiac dysfunction and pathological remodeling date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: pgr l b apelin is an inotropic and cardioprotective peptide that exhibits beneficial effects through activation of the apj receptor in the pathology of cardiovascular diseases. apelin induces the expression of angiotensin-converting enzyme (ace ) in failing hearts, thereby improving heart function in an angiotensin – -dependent manner. whether apelin antagonizes the over-activation of the renin–angiotensin system in the heart remains elusive. in this study we show that the detrimental effects of angiotensin ii (ang ii) were exacerbated in the hearts of aged apelin-gene-deficient mice. ang ii-mediated cardiac dysfunction and hypertrophy were augmented in apelin knockout mice. the loss of apelin increased the ratio of angiotensin-converting enzyme (ace) to ace expression in the ang ii-stressed hearts, and ang ii-induced cardiac fibrosis was markedly enhanced in apelin knockout mice. mrna expression of pro-fibrotic genes, such as transforming growth-factor beta (tgf-β) signaling, were significantly upregulated in apelin knockout hearts. consistently, treatment with the ace-inhibitor captopril decreased cardiac contractility in apelin knockout mice. in vitro, apelin ameliorated ang ii-induced tgf-β expression in primary cardiomyocytes, accompanied with reduced hypertrophy. these results provide direct evidence that endogenous apelin plays a crucial role in suppressing ang ii-induced cardiac dysfunction and pathological remodeling. apelin is an endogenous peptide ligand for dreceptor (or aplnr) and has potent positive inotropic activity and vasodilatory action [ ] [ ] [ ] . apj is a g protein-coupled receptor that shares significant homology with the angiotensin ii type receptor (at r) [ ] . the apelin-apj axis regulates cardiovascular functions including blood pressure, cardiac contractility, and fluid balance, and thereby exerts beneficial effects on cardiovascular systems [ , ] . treatment with apelin peptide in vivo improves the cardiac contractility and output in myocardial infarcted rats and normal mice, and protects the heart from pressure overload, isoproterenol-induced injury, or ischemia reperfusion injury [ ] [ ] [ ] . in studies of apelin knockout and apj knockout mice, we and others have demonstrated that the endogenous apelin-apj axis regulates the heart contractility associated with aging, exercise, and pressure overload; in the absence of apelin or apj expression, these mutant mice show contractile we first examined whether ang ii levels were affected in apelin ko mice by subjecting the plasma to an analysis of liquid chromatography with tandem mass-spectrometry (lc-ms/ms), also called a ras fingerprint. however, ang ii levels were not significantly changed in aged apelin ko mice ( figure s a,b) , though there was a trend of decreasing ang - /ang ii ratios ( figure s c,d) . we thus set out to directly examine the effects of ang ii signaling in apelin ko mice, and we treated -month-old apelin ko mice and wild type (wt) mice with ang ii or vehicle for weeks using osmotic minipumps. in vehicle-treated apelin ko mice, the blood pressure was slightly but significantly decreased compared with the wt mice ( figure a ). when the mice were continuously injected with ang ii for weeks, both wt and apelin ko mice showed elevated blood pressure in a comparable manner, and there was no difference in hypertension between the mice ( figure a -c). thus, endogenous apelin has little or no effects on ang ii-induced hypertension. figure . blood pressure measurements in ang ii-treated mice. wild type (wt) and apelin knockout (apelin ko) mice at months of age were continuously infused with either vehicle or angiotensin ii (ang ii) for weeks using osmotic minipumps (ang ii, mg/kg/day) and measured for blood pressure using the tail-cuff method. systolic (a), diastolic (b), and mean (c) blood pressures are shown. n = - per group. all values are mean ± sem. † p < . ; * p < . ; ** p < . . ns: not significant. since the apelin ko mice showed reduced ace expression in the hearts of aged mice or mice with pressure overload stress to the left ventricle, we examined the cardiac expressions of ace and ace mrnas. in vehicle-treated mice, ace expression in the heart was downregulated by apelin depletion, whereas ace expression was not altered (figure a ,b), consistent with previous results [ , ] . on the other hand, after ang ii treatment, apelin ko mice showed a trend of decreased expression of ace in the heart, but this did not reach statistical significance ( figure b ). when we calculated the ratio of ace to ace expression, apelin ko mice showed an increased ratio of ace to ace expression compared with wt mice after ang ii infusion as well as after vehicle treatment ( figure c ). thus, the loss of apelin enhances the cardiac effects of exogenous ang ii by shifting the balance of ace and ace toward the ace dominant state. . mrna expression of ace and ace in the heart. the heart tissues harvested from ang iitreated wt and apelin ko mice were subjected to qrt-pcr analysis to measure mrna expression levels. mrna expression of ace (a), ace (b), and the ratio of ace expression to ace expression (ace/ace ) (c) are shown. n = - per group. all values are mean ± sem. * p < . . when cardiac function was assessed with echocardiography, the percent of fractional shortening (%fs) was decreased in aged apelin ko mice treated with vehicle compared to the age-matched wt control mice that received vehicle treatment ( figure a ,b, table ), which was consistent with our previous results [ ] . after weeks of continuous ang ii infusion, wt mice showed non-significant reduction of %fs compared with vehicle-treated wt mice ( figure a ,b, table ). however, ang ii- figure . blood pressure measurements in ang ii-treated mice. wild type (wt) and apelin knockout (apelin ko) mice at months of age were continuously infused with either vehicle or angiotensin ii (ang ii) for weeks using osmotic minipumps (ang ii, mg/kg/day) and measured for blood pressure using the tail-cuff method. systolic (a), diastolic (b), and mean (c) blood pressures are shown. n = - per group. all values are mean ± sem. † p < . ; * p < . ; ** p < . . ns: not significant. since the apelin ko mice showed reduced ace expression in the hearts of aged mice or mice with pressure overload stress to the left ventricle, we examined the cardiac expressions of ace and ace mrnas. in vehicle-treated mice, ace expression in the heart was downregulated by apelin depletion, whereas ace expression was not altered (figure a ,b), consistent with previous results [ , ] . on the other hand, after ang ii treatment, apelin ko mice showed a trend of decreased expression of ace in the heart, but this did not reach statistical significance ( figure b ). when we calculated the ratio of ace to ace expression, apelin ko mice showed an increased ratio of ace to ace expression compared with wt mice after ang ii infusion as well as after vehicle treatment ( figure c ). thus, the loss of apelin enhances the cardiac effects of exogenous ang ii by shifting the balance of ace and ace toward the ace dominant state. wild type (wt) and apelin knockout (apelin ko) mice at months of age were continuously infused with either vehicle or angiotensin ii (ang ii) for weeks using osmotic minipumps (ang ii, mg/kg/day) and measured for blood pressure using the tail-cuff method. systolic (a), diastolic (b), and mean (c) blood pressures are shown. n = - per group. all values are mean ± sem. † p < . ; * p < . ; ** p < . . ns: not significant. since the apelin ko mice showed reduced ace expression in the hearts of aged mice or mice with pressure overload stress to the left ventricle, we examined the cardiac expressions of ace and ace mrnas. in vehicle-treated mice, ace expression in the heart was downregulated by apelin depletion, whereas ace expression was not altered (figure a ,b), consistent with previous results [ , ] . on the other hand, after ang ii treatment, apelin ko mice showed a trend of decreased expression of ace in the heart, but this did not reach statistical significance ( figure b ). when we calculated the ratio of ace to ace expression, apelin ko mice showed an increased ratio of ace to ace expression compared with wt mice after ang ii infusion as well as after vehicle treatment ( figure c ). thus, the loss of apelin enhances the cardiac effects of exogenous ang ii by shifting the balance of ace and ace toward the ace dominant state. when cardiac function was assessed with echocardiography, the percent of fractional shortening (%fs) was decreased in aged apelin ko mice treated with vehicle compared to the age-matched wt control mice that received vehicle treatment ( figure a ,b, table ), which was consistent with our previous results [ ] . after weeks of continuous ang ii infusion, wt mice showed non-significant reduction of %fs compared with vehicle-treated wt mice ( figure a ,b, table ). however, ang ii- figure . mrna expression of ace and ace in the heart. the heart tissues harvested from ang ii-treated wt and apelin ko mice were subjected to qrt-pcr analysis to measure mrna expression levels. mrna expression of ace (a), ace (b), and the ratio of ace expression to ace expression (ace/ace ) (c) are shown. n = - per group. all values are mean ± sem. * p < . . when cardiac function was assessed with echocardiography, the percent of fractional shortening (%fs) was decreased in aged apelin ko mice treated with vehicle compared to the age-matched wt control mice that received vehicle treatment ( figure a ,b, table ), which was consistent with our previous results [ ] . after weeks of continuous ang ii infusion, wt mice showed non-significant table ). however, ang ii-treated apelin ko mice showed a marked reduction of %fs compared with ang ii-treated wt mice ( figure a ,b, table treated apelin ko mice showed a marked reduction of %fs compared with ang ii-treated wt mice ( figure a ,b, table ). in echocardiography, the thickness of the interventricular septum (ivs) was significantly increased in ang ii-infused apelin ko mice ( figure c ), suggesting that ang ii-mediated cardiac hypertrophy was enhanced in apelin ko mice. consistently, apelin ko mice with ang ii infusion showed a significantly increased heart weight to body weight (hw/bw) ratio, compared with ang ii-infused wt mice ( figure d ,e). we next examined cardiac fibrosis, which is the well-known outcome of enhanced ang ii signaling in the heart. mild fibrosis of interstitial and perivascular areas were observed in ang ii-treated wt mouse hearts ( figure a ). in contrast, apelin ko mice with ang ii infusion exhibited massive cardiac fibrosis ( figure a ). quantitative analysis of fibrotic areas in the hearts showed that fibrotic areas in ang ii-treated apelin ko mouse hearts were significantly larger than those in wt hearts with ang ii treatment ( figure b ). consistently, mrna expression of the fibrosis-associated genes collagen a (col a), transforming growth-factor beta (tgfb ), and latent transforming growth-factor beta-binding protein (ltbp ) were significantly increased in ang ii-treated apelin ko mice compared with wt mice ( figure c -e), whereas in vehicle-treated apelin ko mice those gene expressions showed a trend to decrease, but did not reach statistical significance. thus, ang ii augments cardiac dysfunction, hypertrophy, and fibrosis in apelin ko mice. in echocardiography, the thickness of the interventricular septum (ivs) was significantly increased in ang ii-infused apelin ko mice ( figure c ), suggesting that ang ii-mediated cardiac hypertrophy was enhanced in apelin ko mice. consistently, apelin ko mice with ang ii infusion showed a significantly increased heart weight to body weight (hw/bw) ratio, compared with ang ii-infused wt mice ( figure d ,e). we next examined cardiac fibrosis, which is the well-known outcome of enhanced ang ii signaling in the heart. mild fibrosis of interstitial and perivascular areas were observed in ang iitreated wt mouse hearts ( figure a ). in contrast, apelin ko mice with ang ii infusion exhibited massive cardiac fibrosis ( figure a ). quantitative analysis of fibrotic areas in the hearts showed that fibrotic areas in ang ii-treated apelin ko mouse hearts were significantly larger than those in wt hearts with ang ii treatment ( figure b ). consistently, mrna expression of the fibrosis-associated genes collagen a (col a), transforming growth-factor beta (tgfb ), and latent transforming growthfactor beta-binding protein (ltbp ) were significantly increased in ang ii-treated apelin ko mice compared with wt mice ( figure c -e), whereas in vehicle-treated apelin ko mice those gene expressions showed a trend to decrease, but did not reach statistical significance. thus, ang ii augments cardiac dysfunction, hypertrophy, and fibrosis in apelin ko mice. we further examined whether ang ii depletion by ace inhibition affects cardiac dysfunction in aged apelin ko mice. after weeks of treatment with captopril, an ace inhibitor (ace-i), wt and apelin ko mice were subjected to echocardiography. the decreased percentage of fractional shortening (%fs) and an increased end-systolic diameter of the left ventricle (lvesd) in apelin ko mice were significantly improved by ace-i to levels similar to those of the wt mice ( figure a ,b; table ). we further examined whether ang ii depletion by ace inhibition affects cardiac dysfunction in aged apelin ko mice. after weeks of treatment with captopril, an ace inhibitor (ace-i), wt and apelin ko mice were subjected to echocardiography. the decreased percentage of fractional shortening (%fs) and an increased end-systolic diameter of the left ventricle (lvesd) in apelin ko mice were significantly improved by ace-i to levels similar to those of the wt mice ( figure a ,b; table ). to determine whether apelin antagonizes ang ii effects in vitro, we treated the isolated primary cardiomyocytes with ang ii, apelin- , or both peptides. ang ii treatment induced hypertrophic growth of mouse cardiomyocytes, while apelin- did not affect the cell size ( figure a,b) . interestingly, combination treatment of ang ii and apelin- reduced ang ii-mediated hypertrophy in cardiomyocytes ( figure a,b) . consistently, mrna expression of brain natriuretic peptide (bnp), atrial natriuretic factor (anf), and tgfb were upregulated by ang ii treatment, whereas apelin- significantly decreased the elevated expression of bnp, anf, and tgfb in ang ii-treated cells to the to determine whether apelin antagonizes ang ii effects in vitro, we treated the isolated primary cardiomyocytes with ang ii, apelin- , or both peptides. ang ii treatment induced hypertrophic growth of mouse cardiomyocytes, while apelin- did not affect the cell size ( figure a,b) . interestingly, combination treatment of ang ii and apelin- reduced ang ii-mediated hypertrophy in cardiomyocytes ( figure a,b) . consistently, mrna expression of brain natriuretic peptide (bnp), atrial natriuretic factor (anf), and tgfb were upregulated by ang ii treatment, whereas apelin- significantly decreased the elevated expression of bnp, anf, and tgfb in ang ii-treated cells to the levels of control vehicle-treated cardiomyocytes ( figure c-e) . thus, apelin antagonizes ang ii-mediated hypertrophy and gene expression in cardiomyocytes. levels of control vehicle-treated cardiomyocytes ( figure c-e) . thus, apelin antagonizes ang iimediated hypertrophy and gene expression in cardiomyocytes. in this study, we demonstrated that endogenous apelin improves exogenous ang ii-induced cardiac dysfunction and pathological remodeling, as well as antagonizing endogenous ang iimediated impairment of heart contractility in aged mice. we also showed that apelin suppresses cellular hypertrophy and pro-fibrotic gene expression in in vitro cardiomyocytes. these data provide direct evidence that endogenous apelin is crucial to antagonize the ang ii-at r axis in cardiac muscle cells. while endogenous apelin antagonizes ang ii-induced heart pathology and upregulates ace expression, it is interesting to observe that ang ii-stimulated elevation of blood pressure was not further increased in apelin ko mice but comparable to wt mice. this is a sharp contrast to the marked elevation of blood pressure in ace knockout mice when treated with ang ii [ ] . although treatment with the apelin peptide antagonizes ang ii-stimulated blood pressure elevation [ ] , the physiological effects of endogenous apelin on the ras seem to be biased to the heart. consistently, our initial study of apelin ko mice first identified the phenotype of cardiac dysfunction. in addition, ex vivo measurements of cardiac contractility showed that ace-inhibitor treatment partly rescued the impaired contractility of apelin ko mouse hearts (unpublished results), implicating that intrinsic cardiac ras activation is causative of heart dysfunction. despite numerous reports on the variety of functions of apelin, these evidences suggest that the heart is one of the major target organs for endogenous apelin. in this study, we demonstrated that endogenous apelin improves exogenous ang ii-induced cardiac dysfunction and pathological remodeling, as well as antagonizing endogenous ang ii-mediated impairment of heart contractility in aged mice. we also showed that apelin suppresses cellular hypertrophy and pro-fibrotic gene expression in in vitro cardiomyocytes. these data provide direct evidence that endogenous apelin is crucial to antagonize the ang ii-at r axis in cardiac muscle cells. while endogenous apelin antagonizes ang ii-induced heart pathology and upregulates ace expression, it is interesting to observe that ang ii-stimulated elevation of blood pressure was not further increased in apelin ko mice but comparable to wt mice. this is a sharp contrast to the marked elevation of blood pressure in ace knockout mice when treated with ang ii [ ] . although treatment with the apelin peptide antagonizes ang ii-stimulated blood pressure elevation [ ] , the physiological effects of endogenous apelin on the ras seem to be biased to the heart. consistently, our initial study of apelin ko mice first identified the phenotype of cardiac dysfunction. in addition, ex vivo measurements of cardiac contractility showed that ace-inhibitor treatment partly rescued the impaired contractility of apelin ko mouse hearts (unpublished results), implicating that intrinsic cardiac ras activation is causative of heart dysfunction. despite numerous reports on the variety of functions of apelin, these evidences suggest that the heart is one of the major target organs for endogenous apelin. the pathological response during the progression of heart failure involves myocyte hypertrophy and cardiac fibrosis, which is crucially mediated by ang ii signaling. in this study, we showed that apelin inhibits ang ii-induced cellular hypertrophy and pro-fibrotic gene expression in cardiomyocytes in vitro. the results can be explained by two mechanisms: one is apelin-mediated downregulation of ang ii-at r signaling in the levels of receptors or intracellular signaling [ ] , and the other is apelin-mediated downmodulation of the ace/ace ratio and subsequent ang ii downregulation [ ] . while apelin suppresses ang ii-stimulated pro-fibrotic signaling in cardiomyocytes, such as the downregulation of tgf-β expression, it was postulated that apelin directly inhibits fibrogenic responses of fibroblasts. apelin inhibits the tgf-β-induced activation of cardiac fibroblasts and collagen production through a sphingosine kinase [ ] . recently, the tissue-specific deletion of the apj receptor in endothelial cells or myocardial cells was reported [ ] . in tac (transverse aortic constriction) pressure overload models, apj deletion in endothelial cells increased cardiac fibrosis and decreased heart contractility, whereas the cardiomyocyte-specific deletion of apj suppressed cardiac hypertrophy and improved heart function [ ] . therefore, the favorable effects of apelin in the heart are likely mediated through complex cellular interactions and signaling. further analysis of other cell types in the heart, such as fibroblasts, neurons, or atrial cells would be warranted for a precise understanding of apelin signaling in the heart. our current findings establish that endogenous apelin antagonizes the ang ii-at r axis in aged hearts. apelin has been recently proposed to be an anti-aging peptide, which in mice extends the healthy life span and prevents sarcopenia, an aging-associated muscle weakness [ , ] . currently the drugs targeting ras, such as ace inhibitors and angiotensin receptor blockers, are widely used in the clinic, but ras inhibition per se does not have any inotropic affects and thus the efficacies on cardiac dysfunction in aged patients would be relatively limited. the development of therapeutic strategy to stimulate apelin-apj signaling, which exerts positive inotrope and protective effects in the heart, would contribute to making a new class of cardiovascular medicine for aged societies. apelin knockout (apelin-/y) mice were generated as described [ ] and backcrossed to c /bl j mice for more than generations for ang ii treatment, -month-old wt and apelin ko mice were subcutaneously infused with either vehicle or ang ii (sigma-aldrich, st. louis, mi, usa) at mg/kg/day for weeks by osmotic minipumps (alzet model , alzet corp., cupertino, ca, usa). for ace-inhibitor treatment, -months-old wt and apelin ko male mice received either vehicle or captopril ( mg/l) in their drinking water. two weeks after treatment, blood pressure measurement and echocardiography were performed and the mice were sacrificed for analysis. echocardiographic measurements were performed as previously described [ ] . briefly, after mice were anesthetized with isoflurane ( %)/oxygen, echocardiography was performed using vevo (fujifilm, tokyo, japan) equipped with a -mhz linear transducer. %fractional shortening (fs) was calculated as follows: fs = [(lvedd − lvesd)/lvedd] × . m-mode images were obtained for measurement of wall thickness and chamber dimensions with the use of the leading-edge conventions adapted by the american society of echocardiography. blood pressure was measured in conscious mice by a programmable sphygmomanometer (bp- , softron co. ltd., tokyo, japan) using the tail cuff method after days of daily training, as described previously [ ] . blood samples were collected with units of heparin or with an optimized protease inhibitor cocktail. plasma was kept at − • c until further measurement. metabolomic profiling of angiotensin peptides was conducted by attoquant diagnostics gmbh (vienna, austria) using technologies described previously [ ] . to evaluate the activities of all ras-peptide converting enzymes present in the plasma, we equilibrated the heparin plasma ex vivo by incubating it for hour at • c to obtain steady-state angiotensin peptide levels, followed by addition of a protease inhibitor cocktail (ex vivo ras fingerprinting). samples were then subjected to lc-ms/ms analysis using a reversed-phase analytic column (acquity uplc ® c , waters co., milford, ma, usa) operating in line with a xevo tq-s triple quadrupole mass spectrometer (waters) as described [ ] . for each peptide and corresponding internal standards, two different mass transitions were measured [ ] . ten angiotensin peptide metabolites could be simultaneously evaluated by this method: ang i, ang - , ang ii, ang iii, ang iv, ang - , ang - , ang - , ang - , and ang - . heart tissues were fixed with % formalin and embedded in paraffin. five µm-thick sections were prepared and stained with hematoxylin and eosin, or masson's trichrome. rna was extracted using trizol reagent (invitrogen, carlsbad, ca, usa) and cdna synthesized using the primescript rt reagent kit (takara). sequences of the forward and reverse primers of the genes studied are shown in supplementary table s . real-time pcr was run in well plates using a sybr premix extaq ii (takara bio., shiga, japan) according to the instructions of the manufacturer. relative gene expression levels were quantified by using the thermal cycler dice real time system ii software (takara). mouse cardiomyocytes were isolated from prenatal mouse hearts of wild type mice as described previously [ ] . briefly, prenatal mice (e . ) were removed from pregnant mice euthanized by cervical dislocation, and prenatal mouse hearts were harvested and rapidly minced in mss buffer ( mm hepes, mm nacl, mm glucose, mm kcl, mm kh po , ph . ). after digestion with collagenase (wako, osaka, japan) for min at • c, cardiomyocytes were collected, pre-plated to exclude non-cardiomyocytes, and plated on gelatinized culture dishes or plates with dmem/f- (gibco, palo alto, ca, usa) supplemented with % fetal bovine serum (equitech bio, inc. kerrville, tx, usa). data are presented as mean values ± sem. statistical significance between two experimental groups was determined using student's two-tailed t-test. comparisons of parameters among more than groups were analyzed by one-way anova, followed by turkey's post-hoc test. p < . was considered significant. isolation and characterization of a novel endogenous peptide ligand for the human apj receptor characterization of apelin, the ligand for the apj receptor apelin, the novel endogenous ligand of the orphan receptor apj, regulates cardiac contractility a human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome the endogenous peptide apelin potently improves cardiac contractility and reduces cardiac loading in vivo acute cardiovascular effects of apelin in humans: potential role in patients with chronic heart failure apelin protects myocardial injury induced by isoproterenol in rats impaired heart contractility in apelin gene-deficient mice associated with aging and pressure overload apelin- protects the heart against ischemia-reperfusion injury through inhibition of er-dependent apoptotic pathways in a time-dependent fashion endogenous regulation of cardiovascular function by apelin-apj regulatory roles for apj, a seven-transmembrane receptor related to angiotensin-type receptor in blood pressure in vivo apj acts as a dual receptor in cardiac hypertrophy lactation is a risk factor of postpartum heart failure in mice with cardiomyocyte-specific apelin receptor (apj) overexpression gpcr signaling and cardiac function arrestins in the cardiovascular system: an update prognostic implications of echocardiographically determined left ventricular mass in the framingham heart study p -induced inhibition of hif- causes cardiac dysfunction during pressure overload fibrosis and heart failure targeting pathological remodeling: concepts of cardioprotection and reparation impairment of the myocardial ultrastructure and changes of the cytoskeleton in dilated cardiomyopathy a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase angiotensin-converting enzyme is an essential regulator of heart function angiotensin ii-mediated oxidative stress and inflammation mediate the age-dependent cardiomyopathy in ace null mice deletion of angiotensin-converting enzyme accelerates pressure overload-induced cardiac dysfunction by increasing local angiotensin ii loss of angiotensin-converting enzyme- leads to the late development of angiotensin ii-dependent glomerulosclerosis ace deficiency modifies renoprotection afforded by ace inhibition in experimental diabetes angiotensin-converting enzyme protects from severe acute lung failure trilogy of ace : a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury angiotensin-converting enzyme is a functional receptor for the sars coronavirus down-regulation of cardiac apelin system in hypertrophied and failing hearts: possible role of angiotensin ii-angiotensin type receptor system apelin is a positive regulator of ace in failing hearts apelin signaling antagonizes ang ii effects in mouse models of atherosclerosis sustained elabela gene therapy in high salt-induced hypertensive rats altered blood pressure responses and normal cardiac phenotype in ace -null mice apelin prevents cardiac fibroblast activation and collagen production through inhibition of sphingosine kinase elabela, a new endogenous ligand of apj, functions in embryos and adults organisms apelin and apj orchestrate complex tissue-specific control of cardiomyocyte hypertrophy and contractility in the hypertrophy-heart failure transition elabela-apj axis protects from pressure overload heart failure and angiotensin ii-induced cardiac damage elabela deficiency promotes preeclampsia and cardiovascular malformations in mice the ccr -not deadenylase complex controls atg -dependent cell death and heart function murine recombinant angiotensin-converting enzyme : effect on angiotensin ii-dependent hypertension and distinctive angiotensin-converting enzyme inhibitor characteristics on rodent and human angiotensin-converting enzyme we thank all members of our laboratories for technical assistance and helpful discussions. the authors declare no conflict of interest. angiotensin-converting enzyme at rangiotensin type receptor ras renin-angiotensin system tgf-β transforming growth-factor β key: cord- -hgggkr y authors: hisatake, shinji; kiuchi, shunsuke; kabuki, takayuki; oka, takashi; dobashi, shintaro; fujii, takahiro; ikeda, takanori title: the serum angiotensin-converting enzyme and angiotensin-( - ) concentrations after optimal therapy for acute decompensated heart failure with reduced ejection fraction date: - - journal: biosci rep doi: . /bsr sha: doc_id: cord_uid: hgggkr y objective: elucidation of the role of angiotensin-converting enzyme (ace) (ace )/angiotensin (ang)-( - )/mas receptor axis in heart failure is necessary. no previous study has reported serial changes in ace and ang-( - ) concentrations after optimal therapy (ot) in acute heart failure (ahf) patients. we aimed to investigate serial changes in serum ace and ang-( - ) concentrations after ot in ahf patients with reduced ejection fraction (ef). methods: ace and ang-( - ) concentrations were measured in ahf patients with reduced ef immediately after admission and and months after ot. these parameters were compared with the healthy individuals at three time points. results: in the acute phase, ang-( - ) and ace concentrations was statistically significantly lower and higher in ahf patients than the healthy individuals ( . ± . vs. . ± . ng/ml, p< . and . ± . vs. . ± . ng/ml, p< . ), respectively. at month after ot, ang-( - ) concentration remained lower in ahf patients than the healthy individuals ( . ± . vs. . ± . ng/ml, p< . ); however, there was no statistically significant difference in ace concentration between ahf patients and the healthy individuals. at months after ot, there were no statistically significant differences in ang-( - ) and ace concentrations between ahf patients and the healthy individuals. conclusion: ace concentration was equivalent between ahf patients and the healthy individuals at and months after ot, and ang-( - ) concentration was equivalent at months after ot. studies have shown that the renin-angiotensin system (ras) is associated with worsening of hypertension and various types of organopathies via the angiotensin-converting enzyme (ace)/angiotensin (ang) ii (ang ii)/ang ii type receptor axis [ , ] . long-term activation of the neurohormonal response, especially of the sympathetic nervous system and the renin-angiotensin-aldosterone system, is a major molecular hallmark of adverse lv remodeling. in fact, the levels of both plasma catecholamines and aldosterone are important predictors of cardiovascular mortality in post-mi patients [ , ] . recent studies have established the existence of a new cascade in the ras called the ace /ang-( - )/mas receptor axis [ , ] . ang-( - ) is a degradation product of ang i and ang ii associated with ace . studies in mice have reported that ang-( - ) stimulates the mas receptor and induces ace was identified as a major ang-( - )-forming enzyme. ang i serves as a substrate for both ace and ace . ang ii is known to have vasoconstrictor and fibrotic and hypertrophic effects in vivo. both ace and ace are involved in the production of ang-( - ), which binds the mas receptor and induces vasodilation, anti-fibrosis and anti-hypetrophy. abbreviations: ace, angiotensin converting enzyme; at, angiotensin type; nep, neutral endopeptidase. hypotensive effects and organ-protective effects primarily through vasodilation, natriuresis, regulation of myocardial hypertrophy and anti-inflammation [ ] [ ] [ ] [ ] [ ] [ ] . therefore, ang-( - ) has antagonistic influences on the effects induced by the ang ii type receptor (figure ). we previously reported that acute heart failure (ahf) patients requiring hospitalization had higher serum ace and lower ang-( - ) concentrations in the acute phase when compared with the concentrations in healthy volunteers [ ] . furthermore, it is very interesting to evaluate the variability of serum ace and ang-( - ) concentrations in the process of compensating for heart failure. however, no study has reported serial changes in serum ace and ang-( - ) concentrations after optimal therapy (ot) in these patients. because of serum ace and ang-( - ) concentrations may be biomarkers that reflect the pathogenesis of heart failure, significance of measuring ace and ang-( - ) concentrations in patients with compensated heart failure, is very important. the present study aimed to investigate the serial changes in the serum ace and ang-( - ) concentrations after ot in ahf patients requiring inpatient care. also, due to the recent worldwide pandemic of covid- , investigation regarding role of ace has been in the limelight. among patients who were urgently admitted to our hospital because of ahf with reduced left ventricular ejection fraction (lvef) between november and september , we enrolled patients who consented to participate in the present study and were followed for at least months. ahf was diagnosed according to the framingham criteria [ ] . patients received optimal drug therapy according to previous guidelines [ ] , during hospitalization and after discharge as outpatients. the exclusion criteria were as follows: ( ) lvef over %; ( ) age < years; ( ) need for mechanical support; ( ) renal failure (serum creatinine level > . mg/dl); ( ) pregnancy, possible pregnancy, nursing and desire to become pregnant during the study period; ( ) judgement as inappropriate for the present study by responsible doctors. the patients were treated in accordance with the declaration of helsinki after obtaining informed consent. the study protocol was approved by the ethics committee of toho university omori hospital, japan (application number - ). the reference values of serum ace and ang-( - ) concentrations have not been established so far. therefore, the value of the concentrations in healthy volunteers [ ] was defined as the healthy individuals. the primary end points of the present study were the serum ang-( - ) and ace concentrations at and months later after ot in the patients were compared with the healthy individuals. upper limb venous blood samples were percutaneously collected while ahf patients maintained the sitting posture for more than min after they became capable of sitting in the acute phase. similarly, upper limb venous blood samples were percutaneously collected and months after ot for heart failure while the patients maintained the sitting posture for more than min. the serum ace concentration, ang-( - ) concentration plasma aldosterone concentration and plasma brain natriuretic peptide (bnp) concentration were measured. the reagents and detailed method for each test are described in past report [ ] . additionally, serum ace and ang-( - ) concentrations at three time points were compared with the healthy individuals, which have been reported previously [ ] . after collection, all blood samples were immediately centrifuged at rpm for min in our laboratory. dispensed specimens were frozen in dry ice. on the day of blood collection, specimens were brought to lsi medience corporation and were preserved in a deep freezer until measurement. ace was measured without specimen dilution, and ang-( - ) was measured after diluting the specimen five times. with regard to cross-reactivity, we did not perform enzyme treatment, but all blood samples were immediately frozen. serum ace and ang-( - ) concentrations were measured using elisa kits (peninsula laboratories, llc, san carlos, ca), according to the manufacturer's instructions, except for solid phase extraction (spe). we did not extract ang-( - ) from serum samples using spe. after blood separation, all experiments were performed at lsi medience corporation using the following kits: ace transthoracic echocardiography was performed using echocardiograph on admission (in resting conditions). two dimensional imaging was performed in standard apical views. the lvef was measured by simpson method. two experienced cardiologists unaware of the biochemical data performed the echocardiographic measurements. all data are expressed as mean + − standard deviation (sd). serial changes in the plasma aldosterone concentration and bnp concentration were analyzed using one-way analysis of variance (anova). for the serum ace concentration and ang-( - ) concentration, values at baseline, and months later were analyzed using the unpaired student's t test and were compared with the healthy individuals, which were reported previously [ ] . the unpaired student's t test and anova were performed using spss software (ver. . ; ibm corp., armonk, ny). statistical significance was set at a p-value < . . data are given as mean + − sd or n (%). abbreviations: ace-i, angiotensin converting enzyme inhibitor; arb, angiotensin receptor blocker; bun, blood urea nitrogen; cr, creatinine; egfr, estimated glomerular filtration; hba c, hemoglobin a c; hdl-c, high-density lipoprotein cholesterol; ldl-c, low-density lipoprotein cholesterol; ngsp, national glycohemoglobin standardization program; tc, total cholesterol. the clinical characteristics of the ahf patients are presented in table . the contents of optimal medical therapy are described in table . the clinical characteristics of healthy volunteers are shown in table . the plasma aldosterone concentrations at and months after ot were statistically significantly elevated when compared with the concentration at baseline ( + − and + − vs. + − pg/ml, respectively, p< . ). the plasma bnp concentrations at and months after ot were statistically significantly decreased when compared with the concentration at baseline ( + − and + − vs. + − pg/ml, respectively, p< . ; table ). in the acute phase, the serum ang-( - ) concentration was statistically significantly lower and the serum ace concentration was statistically significantly higher in heart failure patients than the healthy individuals ( . + − . data are given as mean + − sd or n (%). * < . (vs. baseline). serial changes in the new york heart association (nyha) classification are shown in figure . in the present study, all cases of decompensated heart failure with reduced ef were treated with ot following the appropriate guidelines [ ] , and they were all compensated along with a statistically significant decrease in the plasma bnp concentration. we previously reported that acute decompensated heart failure patients had a statistically significantly higher serum ace concentration and a statistically significantly lower serum ang-( - ) concentration when compared with the concentrations in healthy volunteers [ ] . at month after ot, there was no significant difference in the serum ace concentration between heart failure patients and the healthy individuals. at months after ot, the statistically significant difference in the serum ang-( - ) concentration between heart failure patients and the healthy individuals disappeared. although the present study did not elucidate the mechanisms of the changes in ace and ang-( - ) concentrations, it showed that the serum ace concentration declined and the ang-( - ) concentration increased by ot in compensated heart failure cases, and as a result, both concentrations were equivalent to the healthy individuals. to our knowledge, this is the first study to report such findings. it is well known that classical cascade in the ras called the ace/ang ii/ang ii type receptor axis is a facilitating factor that promotes hypertension and damage to a variety of organs [ , ] . on the other hand, the existence of ang-( - ) was first reported in [ ] . crackower et al. reported in that ace is involved in the development of heart failure in animal study [ ] . although the ang-( - ) receptor has long been unknown, it was confirmed by santos et al. in that the mas receptor is the ang-( - ) receptor [ ] . based on these researches, it was shown that the ace /ang-( - )/mas receptor system maintains the mutual relationship by the action of ang ii mediated by ang ii type receptor and has an action to antagonize the ace/ang ii/ang ii type receptor system [ , ] . ang-( - ) is produced from ang i and ang ii catalyzed by ace . ang ii exerts actions such as vasoconstriction, inducing oxidative stress, cell hypertrophy, and fibrosis via ang ii type receptor. ang-( - ) is known to exert anti-hypertensive and organ-protective effects via mas receptor through vasodilation, reduction in oxidative stress, suppression of cell hypertrophy, anti-fibrotic action, natriuresis and anti-inflammation, it is known that the relationship between mas receptor and ang ii type receptors shows an antagonistic action like a seesaw mechanism [ ] [ ] [ ] [ ] [ ] [ ] . a previous animal experiment showed that the ace concentration increases if ang ii increases [ ] . ang-( - ) has been shown to have a cardiovascular protective effect. among the few studies that reported on the hypertensive or hypotensive effects induced by a decrease in the ace concentration, yamamoto et al. reported that ace kinetics does not have a direct effect on blood pressure [ ] . the results of this study suggest that ang- ( - ) , which has a cardiovascular protective effect, is insufficient in the decompensated heart failure stage, and its concentration increases with ot. additionally, an increased ace concentration is thought to decline to an optimal level, which is associated with sufficient ang -( - ) . furthermore, a previous study reported that the ang-( - ) concentration increases according to ace activity in human heart failure [ ] ; however, the ang-( - ) concentration increased during the compensation process of decompensated heart failure in our study. the findings indicate that ang-( - ) is relatively insufficient in decompensated heart failure in the acute phase and the concentration increases along with compensation by ot. these findings are very advanced results that favorably add to the further clarification of the mechanisms of the efficacy of the ang ii type receptor blocker/neutral endopeptidase inhibitor combination [ ] , which has recently been approved for heart failure in europe and the u.s.a. in the present study, % of patients were treated with ace-inhibitor (acei) or angiotensin receptor blocker (arb). therefore, it was expected that the plasma aldosterone concentration after discharge was lower than that at baseline. however, contrary to that expectation, plasma aldosterone concentrations increased after discharge, and the values at and months after ot were similar. we observed a gradual increase in the plasma aldosterone concentration along with compensation of heart failure in the present study. a previous study by takeda et al. [ ] reported that the circulating aldosterone concentration was inversely proportional to the myocardial tissue aldosterone concentration in an animal experiment. it can be assumed that a decrease in the myocardial tissue aldosterone concentration caused by compensation of heart failure was indirectly observed. in the present study, % of patients were treated with mineralocorticoid receptor antagonists (mras). it can be explained that mras administration increased plasma aldosterone concentration in these patients. the mechanism of elevated plasma aldosterone levels in patients who did not receive mra is difficult to explain. according to a report by mizuno et al., aldosterone acts as a cardioprotective effect for a short period [ ] . therefore, it is considered that the concentration increases in the early phase of decompensated ahf, and the transition may be different for each patient due to the damage of myocardium when shifting to the chronic phase. however, there have been almost no reports to date that measured plasma aldosterone concentrations in patients with heart failure from the acute phase to the chronic phase. in addition, the reason for the increase in plasma aldosterone concentration after discharge in present study may be the effect of the aldosterone breakthrough phenomenon [ ] [ ] [ ] . specifically, after the ras cascade is inhibited by ace-i or arb, the concentration of aldosterone at the most downstream of ras is temporarily reduced. after that, the compensatory mechanism (details unknown) raises the aldosterone concentration again. it is considered that the aldosterone concentration changes similarly even when mra is administered, and the aldosterone concentration increases because it does not bind to the mineralocorticoid receptor. recently, it has been known that bnp has an action of suppressing the secretion of aldosterone from the adrenal gland [ ] . the mechanism by which the suppression of aldosterone secretion is relieved by the reduction in bnp by compensating heart failure may also influence the increase in aldosterone levels. moreover, there is also a report that administration of ace-i or arb increases the secretion of aldosterone via ang ii type receptor [ ] , and our results may support this report. however, this cannot be affirmed only by the data of present study. further evidence from studies, including those involving animal experiments, is necessary as causal relationships between the kinetics of biomarkers in circulating blood and the concentrations of various biomarkers in tissue are unclear. finally, with recent pandemic of covid- , some concerns about the use of ace-i and arb have emerged in covid- patients. ace is the molecule used by severe acute respiratory syndrome coronavirus (sars-cov- ) responsible for covid- pandemic to enter the cells [ ] [ ] [ ] [ ] [ ] . therefore, optimal medical treatment in heart failure, by reducing ace , will decrease the entrance of sars-cov- in the cells and thus mitigate the severity of the disease [ ] . contrary to initial reports by mass media that ace-i/arb might be deleterious in covid- , use of either may be associated with a lower risk of in-hospital deaths than nonuse. the present study has some limitations. first, the study included limited number of diseased. second, with regard to ace , the evaluation of activity rather than concentration might have allowed better and more direct understanding of the ace effect; however, we could not obtain an ace activity assay kit for the present study. we consider that the ace concentration should reflect ace activity. moreover, although ace activity was not measured, we believe that measurement of the ang-( - ) concentration, which is a product of ace , supports the significance of the present study. third, in the present study, we could not evaluate the effects of the drugs used for hospitalization treatment. therefore, it is impossible to mention the effects on various parameters of drugs. fourth, the patients were not treated with the modern sglt inhibitors that have demonstrated to improve prognosis [ ] . this is important because sglt inhibitors mitigates this neurohormonal activation [ ] . the serum ace concentration was equivalent between ahf patients and the healthy individuals at and months after ot. furthermore, the serum ang-( - ) concentration was equivalent between ahf patients and the healthy individuals at months after ot. in other words, it was observed that the increased serum ace concentration gradually decreased to normal value, and the decreased serum ang-( - ) concentration gradually increased to normal value, in patients with acute decompensated heart failure with reduced lvef, who were compensated after optimal medical therapy based on guidelines. effects of enalapril on mortality in severe congestive heart failure. results of the cooperative north scandinavian enalapril survival study (consensus) effect of enalapril on survival in patients with reduced left ventricular ejection fractions and congestive heart failure prognostic significance of plasma norepinephrine in patients with asymptomatic left ventricular dysfunction. solvd investigators complementary and incremental mortality risk prediction by cortisol and aldosterone in chronic heart failure recent advances in the angiotensin-converting enzyme -angiotensin( - )-mas axis an ace in the hole alternative pathways of the renin angiotensin system and their potential role in cardiac remodeling angiotensin-converting enzyme is an essential regulator of heart function angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas effects of angiotensin-( - ) on forearm circulation in normotensive subjects and patients with essential hypertension therapeutic implications of the vasoprotective axis of the renin-angiotensin system in cardiovascular diseases mas axis and oxidative stress in cardiovascular disease regulation of angiotensin ii receptors beyond the classical pathway serum angiotensin-converting enzyme concentration and angiotensin-( - ) concentration in patients with acute heart failure patients requiring emergency hospitalization the natural history of congestive heart failure: the framingham study guidelines for treatment of acute heart failure release of vasopressin from the rat hypothalamo-neurohypophysial system by angiotensin-( - ) heptapeptide hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase deletion of angiotensin-converting enzyme accelerates pressure overload-induced cardiac dysfunction by increasing local angiotensin ii increased angiotensin-( - )-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme homologue ace baseline characteristics and treatment of patients in prospective comparison of arni with acei to determine impact on global mortality and morbidity in heart failure trial (paradigm-hf) effects of high sodium intake on cardiovascular aldosterone synthesis in stroke-prone spontaneously hypertensive rats aldosterone production is activated in failing ventricle in humans escape" of aldosterone production in patients with left ventricular dysfunction treated with an angiotensin converting enzyme inhibitor: implications for therapy rise in plasma concentration of aldosterone during long-term angiotensin ii suppression sustained reduction of aldosterone in response to the angiotensin receptor blocker valsartan in patients with chronic heart failure: results from the valsartan heart failure trial inhibitory effect of natriuretic peptides on aldosterone synthase gene expression in cultured neonatal rat cardiocytes gene expression and roles of angiotensin ii type and type receptors in human adrenals covid- and the cardiovascular system structural basis for the recognition of sars-cov- by full-length human ace tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis renin-angiotensin-aldosterone system inhibitors in patients with covid- sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor cardiovascular disease, drug therapy, and mortality in covid- do the sglt- inhibitors offer more than hypoglycemic activity? cardiovasc the authors declare that there are no competing interests associated with the manuscript. the authors declare that there are no sources of funding to be acknowledged. abbreviations ace, angiotensin-converting enzyme; ace-i, angiotensin-converting enzyme inhibitor; ahf, acute heart failure; ang, angiotensin; ang-( - ), angiotensin-( - ); ang ii, angiotensin ii; anova, analysis of variance; arb, angiotensin ii receptor blocker; bnp, brain natriuretic peptide; ef, ejection fraction; lvef, left ventricular ejection fraction; mi, myocardial infarction; mra, mineralocorticoid receptor antagonist; ot, optimal therapy; ras, renin-angiotensin system; sars-cov- , severe acute respiratory syndrome coronavirus ; sglt , sodium glucose cotransporter ; spe, solid phase extraction. key: cord- - pnm fn authors: lubel, john s; herath, chandana b; burrell, louise m; angus, peter w title: liver disease and the renin–angiotensin system: recent discoveries and clinical implications date: - - journal: j gastroenterol hepatol doi: . /j. - . . .x sha: doc_id: cord_uid: pnm fn the renin–angiotensin system (ras) is a key regulator of vascular resistance, sodium and water homeostasis and the response to tissue injury. historically, angiotensin ii (ang ii) was thought to be the primary effector peptide of this system. ang ii is produced predominantly by the effect of angiotensin converting enzyme (ace) on angiotensin i (ang i). ang ii acts mainly through the angiotensin ii type‐ receptor (at( )) and, together with ace, these components represent the ‘classical’ axis of the ras. drug therapies targeting the ras by inhibiting ang ii formation (ace inhibitors) or binding to its receptor (angiotensin receptor blockers) are now in widespread clinical use and have been shown to reduce tissue injury and fibrosis in cardiac and renal disease independently of their effects on blood pressure. in , two groups using different methodologies identified a homolog of ace, called ace , which cleaves ang ii to form the biologically active heptapeptide, ang‐( – ). conceptually, ace , ang‐( – ), and its putative receptor, the mas receptor represent an ‘alternative’ axis of the ras capable of opposing the often deleterious actions of ang ii. interestingly, ace inhibitors and angiotensin receptor blockers increase ang‐( – ) production and it has been proposed that some of the beneficial effects of these drugs are mediated through upregulation of ang‐( – ) rather than inhibition of ang ii production or receptor binding. the present review focuses on the novel components and pathways of the ras with particular reference to their potential contribution towards the pathophysiology of liver disease. most of us can recall the schema of the renin-angiotensin system (ras) taught in physiology lectures (fig. ) . the system is often depicted as a simple enzyme cascade starting with the degradation of angiotensinogen (derived from the liver) by circulating renin (secreted from the juxtaglomerular apparatus of the kidney) to form angiotensin i (ang i). subsequent enzymatic action by angiotensin converting enzyme (ace) in the capillaries of the lung yields the predominant effector peptide of the system, angiotensin ii (ang ii). [ ] [ ] [ ] two receptors for ang ii have been cloned and characterized, the angiotensin ii type- receptor (at ) is the abundant receptor in adult life, whereas the angiotensin ii type- receptor (at ) is present in the fetus and persists in the central nervous system of adults. [ ] [ ] [ ] binding of ang ii to the at receptor mediates a number of diverse effects including vasoconstriction and sodium hemostasis. ang ii also participates in inflammation and wound healing through the release of critical cytokines and production of extracellular matrix. the effect of ang ii on vascular tone and systemic blood pressure has been extensively studied and is mediated through direct effects on vascular smooth muscle cells or indirectly by increasing vascular sympathetic tone. sodiumconserving effects occur via reabsorption of sodium by the renal tubules as well as stimulating the adrenal gland to secrete aldosterone. the effect of ang ii to stimulate thirst is mediated through at receptors in the brain. figure illustrates the conventional view of the 'classical' ras. this schema is useful as it clarifies how drugs like ace inhibitors or at receptor blockers (arb) produce their beneficial therapeutic effects in cardiovascular and renal disease. however, there have been a number of major advances in our understanding of the ras which have made it clear that the system is far more complex than this 'classical' view would suggest (fig. ) . one key point of understanding is that ang ii is just one member of a family of angiotensin peptides produced by the ras. ang ii consists of eight amino acids, which, like other peptides, has a free amino group at one end (n-terminus) and a free carboxyl group (c-terminus) at the other. ang ii can also be denoted as ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , where the first amino acid is at the n-terminus and the eighth amino acid is at the c-terminus. therefore, cleavage of amino acids from either end of the ang ii molecule can generate smaller peptide fragments (fig. ) . for example, removal of the n-terminus amino acid results in the generation of a peptide consisting of seven amino acids starting from the second amino acid of ang ii, and is denoted by ang-( - ) (historically also known as ang iii). of the fragments that can be generated from ang ii, only three are known to be physiologically relevant. two are derived from n-terminus cleavage, ang-( - ) (ang iii) and ang-( - ) (ang iv) and one formed by cleavage of a single amino acid from the c-terminus, angiotensin-( - ) (ang-( - )). ang iii is formed following cleavage of the aspartate-arginine bond of ang ii by aminopeptidase a, and ang iv can be formed by further cleavage of ang iii by aminopeptidase b or n. ang iii shares many of the properties of ang ii with % of the pressor activity and % of the aldosterone stimulating activity. ang iv has its own distinct receptor (at ) and has central nervous system effects together with some opposing actions to ang ii. ang-( - ) is generated from cleavage of either ang ii or ang i and has been the focus of much research since the discovery that it has biological functions that oppose those of ang ii. for decades, the 'classical' arm of the ras was recognized as being the only system of biological relevance. however, this interpretation was challenged in the late s with the discovery of ang-( - ) and description of its diverse biological functions. this was followed by studies which clearly demonstrated new components of the ras, such as ace , and the ang-( - ) receptor, mas. these new components, together with the effector molecule ang-( - ), form the axis which we now recognize as the 'alternative' arm of the ras. the discovery of these new ras components provided some missing connections to the hitherto complex biochemical pathways of the ras. the new components of the alternative arm of the ras are reviewed below with a major emphasis on their potential contribution towards the pathophysiology of liver disease. interest in alternative components of the ras was re-ignited in the year when two groups independently discovered an enzyme similar to ace in human tissue. , this homolog of ace was initially called hace but has subsequently been named angiotensin converting enzyme (ace ). although structurally similar to ace, ace has different substrate affinities and resists inhibition by ace inhibitors. ace is a zinc-metalloproteinase and, like ace, is a type- transmembrane protein. it consists of amino acids with a single transmembrane alpha-helical portion, an external n-terminus portion containing the catalytically active enzyme and an internal inactive c-terminus section. a transmembrane proteinase, adam acts as a 'sheddase' releasing the active enzyme into the extracellular environment (fig. ) . the released ace (soluble ace ) is a carboxypeptidase, capable of cleaving a single amino acid from the c-termini of its various substrates, including, ang ii, ang i, des-arg -bradykinin, neurotensin - and kinetensin (see review by burrell and colleagues). importantly, ace can generate ang-( - ) directly from ang ii or indirectly by cleaving ang i into an inactive intermediate fragment, ang-( - ), which is then cleaved by ace to produce ang-( - ) (fig. ). of these two ace pathways, the conversion of ang ii into ang-( - ) is kinetically favoured -fold compared to the conversion of ang i to ang-( - ). , in addition to its role in the 'alternative' ras, the ace transmembrane protein has, interestingly, been identified as a receptor site for spike proteins of the severe acute respiratory syndrome (sars) coronavirus, thereby facilitating infection of target cells. much of the work on ang-( - ) has been carried out in animals and, to date, this peptide has been shown to have antihypertensive, anti-arrhythmic, and cardioprotective properties [ ] [ ] [ ] as well as anti-trophic properties in vascular endothelial cells, smooth muscle cells, cardiac myocytes and cardiac fibroblasts. [ ] [ ] [ ] [ ] in contrast to ang ii, ang-( - ) also has anti-inflammatory, antifibrotic and anti-thrombotic properties. , as a result of these studies, ang-( - ) has been proposed to represent the effector peptide of a counterbalancing arm of the ras, capable of opposing the deleterious actions of ang ii. the putative receptor for ang-( - ) is the g protein-coupled receptor encoded by the mas proto-oncogene, although other receptors may well exist. thus, ace together with ang-( - ) and the mas receptor represent an 'alternative' arm or axis of the ras which may present a counter- figure 'classical' renin-angiotensin system (ras). the ras is depicted here as a linear cascade leading to the generation of angiotensin ii (ang ii) through the enzymatic action of renin on angiotensinogen and angiotensin converting enzyme (ace) on angiotensin i (ang i). there are two known receptors for angiotensin ii, angiotensin ii type- receptor (at ) and angiotensin ii type- receptor (at ). the at receptor is thought to play a more important role than the at receptor in human disease. balancing system to the deleterious ace/ang ii/at axis (fig. ) . clearly, ace holds a central role in the ras influencing both axes, as it is capable of simultaneously degrading ang ii and generating ang-( - ) (fig. ) . angiotensin converting enzyme is known to participate actively in the kallikrein-kinin system by degrading bradykinin (fig. ) . inhibitors of ace can therefore lead to the accumulation of bradykinin, which may contribute to the antihypertensive properties of these drugs, as well as to some of the observed side-effects, such as chronic cough and angioedema. in the liver, bradykinin binds to the b receptor and causes increases in hepatic resistance and elevation of portal pressure. in other vascular beds, bradykinin induces vasodilatation on binding to the b receptor, and ang-( - ) has been shown to induce bradykinin-mediated relaxation in porcine coronary arteries. a possible explanation for this is that ang-( - ) has ace inhibitory properties that prevent acemediated degradation of bradykinin. in recent years, scientists have departed from the traditionally held view of the ras being exclusively a circulating endocrine system and have realized that many organs, such as the heart, kidney, liver and pancreas, constitutionally express all the 'classical' ras components required for a functioning, autonomous intra-organ contemporary renin-angiotensin system (ras). angiotensin converting enzyme (ace) has a central role in the ras influencing both the 'classical' and 'alternative' axes, as it degrades angiotensin ii (ang ii) while simultaneously generating ang-( - ). ace is important in generating ang ii, but is also responsible for the degradation of ang-( - ) into the inactive peptide fragment ang- ( ) ( ) ( ) ( ) ( ) . the ras interacts with the kinin system through ace degradation of bradykinin. the two axes of the ras and the kinin system are shaded grey. enzymes are shown in yellow boxes and peptides in blue boxes. aminopeptidase a (apa) and aminopeptidase n (apn) sequentially cleave ang ii to form angiotensin iii and angiotensin iv, respectively. neprilysin (nep) is involved in both the ras and the kinin system. possible peptide-receptor interactions are shown by dashed lines. peptide structure and fragments of angiotensin i. angiotensin i is a decapeptide (ang-( - )) which can be fragmented by various enzymes into four peptides with biological activity; angiotensin ii (ang-( - )), angiotensin iii (ang-( - )), angiotensin iv (ang-( - )) and angiotensin - (ang-( - )). further enzymatic degradation of ang - yields the inactive fragment angiotensin - (ang-( - )). aminopeptidases are shown in blue and cleave amino acids from the n-terminus, whereas carboxypeptidases are shown in red and cleave amino acids from the c-terminus. amino acids are given numerical values, where , aspartic acid; , arginine; , valine; , tyrosine; , isoleucine; , histidine; , proline; , phenylalanine; , histidine; , leucine. ras. , these locally generated angiotensin peptide fragments have been demonstrated to have a multitude of actions, being implicated in cell growth, cell proliferation, apoptosis, reactive oxygen species generation, inflammation, and fibrogenesis. although conceptually separate, the local intra-organ ras and the systemic ras must interact and the final peptide products will depend on the interplay between the two. despite the discovery of ang-( - ) and the recognition that many of its actions oppose ang ii, the importance of this heptapeptide fragment of ang ii remained elusive until recently. it is now clear that in the diseased liver, not only are the 'classical' ras components such as renin, ace, ang ii and the at receptor overexpressed, but, importantly, components of the 'alternative' ras, such as ace , ang-( - ) and the mas receptor are also upregulated. , the implication from these studies is that the 'classical' components contribute to the fibrotic process whereas the 'alternative' components may be upregulated in an attempt to restore the status quo. in liver disease, architectural changes to the microscopic structure of the liver occur as a result of inflammation and fibrosis. these changes lead to capillarization of the hepatic sinusoids, increased extracellular matrix (ecm) formation and elevated hepatic resistance; the latter impedes liver blood flow and leads to portal hypertension. stretching of the portal vein (as with increased hepatic resistance to blood flow) and oxidative stress together cause release of vasodilators, including nitric oxide, which induce a number of compensatory mechanisms important for restoring the functional blood volume. these mechanisms are effected via sodium and water preservation and stimulation of the sympathetic nervous system, which together contribute to the development of ascites, edema, hepatorenal syndrome, and a hyperdynamic circulation, all of which are typically seen in patients with advanced liver disease. the ras is involved with all these processes. as the result, manipulation of the ras with either antagonists of the 'classical' pathway, or agonists of the 'alternative' pathway could have potential therapeutic benefits. balanced against the possible benefits are the potential side effects of such therapy, as the compensatory mechanisms activated by the systemic ras are necessary to maintain an adequate circulation. counterbalancing effects of the two axes of the renin-angiotensin system (ras). the ras can be thought of as two counterbalancing axes. the angiotensin converting enzyme (ace)/angiotensin ii/at receptor axis causes vasoconstriction, salt retention, inflammation, fibrosis and thrombosis, whereas the ace /angiotensin - /mas receptor axis has opposing effects. hepatic stellate cells (hsc) are thought to play a pivotal role in fibrogenesis within the liver, and there is a large body of evidence to support the hypothesis that ang ii promotes activation, and dedifferentiation of these cells into myofibroblasts. furthermore, ang ii encourages myofibroblast contraction, proliferation and promotes release of inflammatory cytokines as well as the deposition of extracellular matrix (ecm). although both of the ang ii receptors (at and at ) are expressed in the liver, the at receptor is far in abundance and is thought to be responsible for most of the ang ii-mediated effects. studies using gene-deletion mice have demonstrated that at a receptor-deficient mice are protected from hepatic fibrosis whereas at receptor-deficient mice have worse fibrosis. a great deal of evidence supporting the role of the ras in hepatic fibrosis has come from animal studies using ace inhibitors and angiotensin receptor blockers (arb). numerous studies using a variety of animal models have demonstrated antifibrotic effects of these drugs. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] however, there appear to be some conflicting observations reported in the literature. for example, losartan treatment failed to influence either liver injury or progression of fibrosis in an animal model of non-alcoholic steatohepatitis (nash). [editor's note: a detailed review of animal models of nash has been written by larter and yeh for a later article in this basic science miniseries.] in contrast, a study with a similar model of nash but using the arb olmesartan, demonstrated a % reduction in fibrosis in the arb-treated group. the avid interest in ras-blocking drugs is, in part, related to their relative safety in humans and widespread use in cardiovascular and renal medicine. despite the large number of animal studies, there is a relative paucity of human data to support the use of these drugs in human liver disease. in part, this could be due to the need to perform multiple liver biopsies to histologically confirm resolution of fibrosis, which, outside the setting of posttransplantation recurrent hepatitis c, is rarely indicated in . in addition, the slow progression of fibrosis in most diseases such as hepatitis c and non-alcoholic fatty liver disease (nafld) make it difficult to detect possible beneficial effects of antifibrotic therapy, unless studies are conducted over a number of years. a pilot study examining the effects of months of losartan treatment on liver fibrosis in chronic hepatitis c demonstrated a significant decrease in fibrosis stage in the treated group compared to control patients. in support of this, a study using candesartan for weeks in compensated child a and b cirrhotic patients demonstrated a significant reduction of plasma hyaluronic acid levels, a surrogate marker for fibrogenesis. however, in this study, two of three serum markers of fibrosis used showed no improvement, and there were no histological data provided; this makes it difficult to evaluate any effects on architectural changes. a number of other studies have reported possible antifibrotic effects of ras blockers in patients with hepatitis c. in one study, hepatitis c virus (hcv)-infected patients with mild fibrosis were treated with losartan mg/day and ursodeoxycholic acid mg/day whereas controls received ursodeoxycholic acid alone. there were significant reductions in serum markers of hepatic fibrosis such as transforming growth factor b (tgf-b ) and type iv collagen in the losartan and ursodeoxycholic acid group, but no significant changes in fibrosis score between the groups. another report described outcomes in patients with hepatitis c treated with low-dose interferon (ifn alpha ¥ iu times a week for months) in combination with the ace inhibitor, perindopril ( mg/day). treatment was accompanied by significant improvement in serum markers of fibrosis (hyaluronic acid, type iv collagen s and procollagen iii-n-peptide), but histological analysis was not carried out. although this study did not have a perindopril monotherapy group, a subsequent study by the same group demonstrated that perindopril alone decreased serum fibrosis markers in patients with chronic hepatitis c. the addition of interferon significantly augmented the effect of perindopril monotherapy. finally, a retrospective review compared liver histology in liver transplant patients with recurrent hepatitis c who were taking ras-blocking drugs (n = ) with those who were not (n = ). the group taking ras blockers were less likely to develop severe hepatic fibrosis (bridging fibrosis or cirrhosis) at and years after transplantation than were the control group ( % vs % at year [p < . ], and % vs % at years, respectively). only small studies have looked at ras blockers and nash. one such study (n = ) found that giving losartan ( mg/day for weeks) in hypertensive patients with nash reduced serum tgf-b , serum ferritin and aminotransferase levels. five patients showed improvement in the grade of hepatic necroinflammation. the study design could have been improved had the investigators examined pre-and post-treatment histology and biochemical markers in a placebo group. in a subsequent study, the pre-and post-treatment biopsies of seven patients with nash treated with losartan ( mg/day for weeks) were compared with eight patients with nafld who acted as a control group. the treatment group showed a significant improvement in necroinflammatory grade, stage of fibrosis, significantly fewer activated hsc and a mild increase in quiescent hsc at the end of weeks. however, the lack of a proper randomized control group is a particular problem in studies of patients with nash, as the disease can improve in response to changes in lifestyle. fixed changes in hepatic architecture account for approximately % of the total resistance to portal blood flow in the cirrhotic liver. the remaining % results from a reversible or 'dynamic' resistance caused by the contraction of activated myofibroblasts positioned around the sinusoidal endothelial cells within the space of disse. as portal resistance increases, a number of factors, including distension of the portal venous system, endotoxemia and oxidative stress result in the release of mediators, including nitric oxide, which dilate the mesenteric and systemic vasculature. activation of compensatory mechanisms designed to restore functional blood volume results in sodium and water retention, stimulation of the sympathetic nervous system and the development of a hyperdynamic circulation. this cascade of events contributes to many of the key features and complications of advanced liver disease including development of ascites, edema and the hepatorenal syndrome. the ras is involved with all these processes. manipulation of the ras with either antagonists of the 'classical' pathway, or agonists of the 'alternative' pathway therefore has potential for therapeutic benefit. variceal bleeding is one of the most important causes of morbidity and mortality in patients with portal hypertension. a number of pharmacological approaches have been developed for the prevention and treatment of this problem. non-selective ß-adrenergic antagonists (beta-blockers) lower portal pressure by decreasing cardiac output and constricting the mesenteric vascular bed but have no direct effect on intrahepatic resistance to portal flow. these drugs have become the mainstay of treatment for the prevention of variceal bleeding. however, only % of patients achieve the target reduction in portal pressure of %, as measured by hepatic venous pressure gradient (hvpg), and they are poorly tolerated in patients with severe liver disease. as a result, there is a major interest in the development of other pharmacological therapies which can lower portal pressure. interestingly, betablockers interact with the ras by inhibiting renin release, but have not been shown to impact on the development or progression of hepatic fibrosis. in contrast, the use of either ace inhibitors or arb to reduce portal pressure is an attractive proposition, as these drugs have the additional potential benefit of slowing the progression of hepatic fibrosis. ang ii is a potent vasoconstrictor, and myofibroblasts derived from hsc express the at receptor and contract in response to ang ii. , additionally, cirrhotic rat livers are hyperresponsive to ang ii with an increased portal pressure compared to those from healthy rats as a result of increased expression of at receptors. this finding is of interest given that the relative importance of ang ii as a mediator of increased portal resistance has been questioned, based on a study of hepatic hemodynamics in isolated perfused cirrhotic rat livers which suggested that ang ii-mediated vasoconstriction is attenuated in the cirrhotic liver. following some persuasive animal studies, , the effects of at blockade on portal hypertension have been examined in a number of human studies. despite some encouraging initial studies showing a significant reduction of portal pressure by arb, subsequent well-designed studies have failed to confirm these findings. schneider and colleagues reported a dramatic reduction in hvpg with losartan in both moderate and severe portal hypertensive patients, but with only a mmhg drop in mean arterial pressure (map). these findings were markedly different to a subsequent randomized controlled trial comparing the hemodynamic effects of losartan with propanolol given for weeks following an index variceal bleed. losartan failed to reduce hvpg, yet resulted in a significant reduction of map by %. treatment tolerance was equivalent. the hemodynamic effect of losartan was further corroborated by a recent small study of pre-ascitic patients which also found that losartan had no affect on hvpg, but did cause a drop in map of . %. irbesartan, another arb, produced only modest reduction in portal pressures ( % Ϯ . %, p < . ) in a randomized, placebo-controlled, double-blind study. importantly, however, this was associated with significant arterial hypotension and significant renal impairment in % of patients. in this study, plasma renin activity before treatment was a predictor of patients that would not tolerate treatment. the explanation for this adverse effect is that the ras is known to play a central role in the homeostatic response to vasodilatation in patients with portal hypertension. the ras, together with other compensatory systems, the posterior pituitary (through vasopressin secretion) and the sympathetic nervous system, endeavors to restore circulatory volume and organ perfusion by inducing vasoconstriction and sodium and water retention. in patients with advanced cirrhosis, plasma renin, ang ii, ace and aldosterone levels are all increased and, within the kidney, ang ii is critical for maintenance of renal perfusion pressure and an adequate glomerular filtration rate (gfr). as liver disease progresses, the decrease in effective circulatory volume results in vasoconstriction of the glomerular afferent circulation, renal hypoperfusion and a fall in gfr. in response to renal hypoperfusion, ang ii selectively constricts the efferent glomerular arterioles; this restores glomerular perfusion pressure and gfr. the maintenance of adequate renal perfusion is therefore ace dependent. furthermore, ace inhibition results in a rapid fall of gfr. , this adverse effect of ras inhibition on renal function in patients with advanced cirrhosis represents a major disadvantage for the use of this class of drug for the treatment of portal hypertension. a recent study by debernardi-venon and colleagues examined the effects of candesartan treatment for weeks on compensated child a and b cirrhotic patients. treatment was well tolerated, with a mild but significant reduction in hvpg in more than % of those treated. furthermore, % of patients treated achieved a % reduction in their hvpg. interestingly, the changes in hvpg correlated well with those observed for plasma hyaluronic acid. however, the treatment group was preselected in that patients were excluded from analysis if they had large varices, evidence of significant arterial hypotension or renal impairment. angiotensin receptor blockers have also been studied in portal hypertensive gastropathy; at least one study has reported a positive benefit from their use. the effects of ace inhibitors on portal pressure have also been examined in a few small studies, but the results generally have been disappointing, with poor agreement between studies. - a number of explanations have been proposed to explain the lack of uniformity in results from clinical studies investigating the benefits and adverse effects of ras inhibitors. there are known genetic polymorphisms for the at receptor gene and genes responsible for cleaving angiotensin i, including ace; these may confer patient-to-patient variations in response to these drugs. this has led to the suggestion that genetic testing may help determine which patients are likely to have a positive response to therapy. in addition, chronic ace inhibition may not lead to sustained ang ii suppression because of increased renin activity and upregulation of alternative enzymes, such as hepatic chymase, which is capable of generating ang ii from ang i. , furthermore, chronic use of arb also results in hyper-reninemia and elevated ang ii levels; the latter increasingly compete with the at receptor antagonist for binding sites on the at receptor molecule. , finally, it has also been claimed that there is tissuedependent responsiveness to ace inhibitors and arb and, at current therapeutic dosing, both classes of drug may not completely inhibit their respective targets. to date, no studies have examined the effects on portal pressure of combined therapy with an ace inhibitor and arb; theoretically, this may overcome some of the possible issues of ang ii reactivation with use of ace inhibitors alone. in summary, the use of ras inhibitors (other than betablockers) to reduce portal pressure has been disappointing. at the doses used in clinical trials, these drugs appear to have only minor effects on portal pressure but very significant side-effects, includ-ing systemic hypotension and renal impairment. these complications are a useful reminder of the homeostatic role the ras plays in maintaining map and gfr in the vasodilated patient with severe liver disease. [ ] [ ] [ ] based on the current available evidence, the use of either ace inhibitors or arb for reducing portal pressure remains controversial and cannot be recommended outside clinical trials. as outlined above, there is increasing evidence that both the 'classical' and the 'alternative' ras are upregulated in chronic liver disease. , it has recently been suggested that the progression of liver fibrosis may be influenced by a balance between ace and ace activation. in both an animal model of secondary biliary fibrosis and in humans with hepatitis c, ace gene and activity are upregulated. , as fibrosis worsens, the progressive rise in ace and at gene expressions coincide with an increase in ace and mas expression, together with increased plasma levels of both ang-( - ) and ang ii. , cirrhotic livers have a greater capacity than healthy livers to convert ang ii to ang-( - ) because of upregulated ace gene and protein expression (fig. ). in addition, the hepatic production of ang-( - ) from ang ii is augmented by ace inhibition. , this increased ang-( - ) production in the presence of an ace inhibitor can be explained by the fact that ang-( - ) is cleaved by ace to produce the inactive peptide ang-( - ) (fig. ) . inhibition of ace therefore increases ang-( - ) half-life, leading to an increase in net production and accumulation of ang-( - ). [ ] [ ] [ ] evidence for a beneficial role of ang-( - ) in hepatic fibrosis has been provided by a study examining the effects of the mas receptor antagonist [ -d-ala]-ang-( - ) (a ). treatment with a worsened experimental liver injury with increases in tgf-b and hydroxyproline levels; this infers that mas receptor stimulation plays a protective role in liver fibrosis. further compelling evidence for a beneficial role of ang-( - ) has come from a recent rat study presented at aasld by our group. we demonstrated that ang-( - ) infusion in bile duct-ligated rats attenuated fibrosis as quantified using metavir fibrosis score, hydroxyproline content, and type collagen mrna expression. alpha-smooth muscle actin (a-sma) gene and protein expression were also reduced, indicating that hepatic stellate cell activation was inhibited by ang-( - ). interestingly, ang-( - ) infusion also inhibited ace gene and protein expression, and resulted in downregulation of mas receptor gene expression. the ang-( - ) infusion group also showed decreased mrna expression levels for connective tissue growth factor (ctgf, also known as ccn ) and vascular endothelial growth factor (vegf), two critical growth factors implicated in fibrosis and tissue repair. this is the first direct evidence showing that ang-( - ) can ameliorate hepatic fibrosis. evidence from studies in ace deletion mice further supports a central role of ace in regulating fibrosis in liver disease. despite a number of reports that ang-( - ) is a vasodilator, experiments on rat isolated perfused livers have failed to demonstrate any vasodilatory effect in normal or cirrhotic livers. , , likewise, experiments in isolated vessels from normal and cirrhotic rats also failed to show any direct vasodilatory effect of this peptide. conversely, ang-( - ) has been shown to enhance acetylcholine-mediated vasodilatation in aortic rings from cirrhotic rats. the vasodilatory effects of ang-( - ) are thought to be mediated through increased production of nitric oxide (no). , hence, the absence of a vasodilatory effect by ang-( - ) in the cirrhotic rat liver could be explained by the known general impairment of no-dependent vasodilatation in the cirrhotic liver due to endothelial dysfunction. , in summary, there is considerable evidence supporting the concept that opposing axes of the ras are involved in the pathogenesis of chronic liver injury. on one side, the ace/ang ii/at receptor axis promotes liver injury and deposition of extracellular matrix, on the other, ace /ang-( - )/mas receptor promotes collagen degradation and resolution of inflammation. both axes are upregulated in liver disease, but presumably the balance between the two systems is critical in determining the net effect. for many years researchers in the field of ras have concentrated on blocking components of the 'classical' system in an attempt to reduce fibrosis. however, both ace inhibitors and arb have an impact on other components of the 'classical' ras apart from ang ii, as plasma renin activity and ang i levels increase following chronic therapy. this, in part, explains the phenomenon of 'angiotensin ii reactivation' and 'aldosterone escape' whereby chronic administration of an ace inhibitor fails to completely suppress either plasma ang ii or aldosterone production. [ ] [ ] [ ] the actual mechanism underlying this phenomenon remains elusive, figure effects of angiotensin converting enzyme (ace) inhibitors and angiotensin type- receptor (at ) receptor blockers (arb) on the two axes of the renin-angiotensin system (ras). the ras is shown as a balance with 'classical' and 'alternative' axes counterbalancing each other. ace inhibitors (acei) cause an initial reduction in angiotensin ii (ang ii), but after chronic administration increases in plasma renin activity and plasma angiotensin i (ang i) levels occur. both ang ii and aldosterone levels can subsequently rise as a consequence of non-ace-dependent pathways facilitated by enzymes such as chymase. both acei and arb result in elevated levels of ace and ang-( - ) which possibly contribute to the effects of these drugs. manipulations of the ras aimed at tipping the balance in favour of 'alternative' components represents a potential target for antifibrotic therapies. liver and the renin-angiotensin system js lubel et al. although non-ace-dependent pathways involving enzymes like chymase, which is capable of generating ang ii, may play an important part. , interestingly, ace inhibitors and arb have a profound impact on the 'alternative' system by causing significant increases in ang-( - ). , , , it has been postulated that some of the beneficial effects observed with arb and ace inhibitors are mediated through ang-( - ). , , [ ] [ ] [ ] [ ] [ ] in support of this, ace activity and gene expression are both increased in the heart by arb or ace inhibitors. , the elevated ace activity in such tissues would result in both diminished levels of ang ii and simultaneous elevations in tissue ang-( - ), thus tipping the ras balance in favor of the 'alternative' axis. interestingly, our own studies and those of others have shown that ang-( - ) can inhibit ace activity and gene expression; this would further tend to alter the balance of the two axes towards the 'alternative' axis ( fig. ) . , conclusions and future directions our understanding of the ras has considerably expanded since the discovery of ace . emerging evidence supports the hypothesis that the ras consists of two opposing axes. manipulation of the ras, by either blocking the 'classical' ras or by stimulating the 'alternative' ras represents a potential target for antifibrotic and portal hypertension therapy. limitations to treatment may be the side-effects of such drugs, particularly their impact on arterial blood pressure and renal function. current therapies such as ace inhibitors and arb used in cardiovascular and renal fibrosis have been shown to impact on both the 'classical' and 'alternative' pathways. the elevated ang-( - 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) improves the post-ischemic function in isolated perfused rat hearts. braz angiotensin-( - ) inhibits vascular smooth muscle cell growth angiotensin-( - ) inhibits growth of cardiac myocytes through activation of the mas receptor angiotensin-( - ) reduces smooth muscle growth after vascular injury angiotensin-( - ) binds to specific receptors on cardiac fibroblasts to initiate antifibrotic and antitrophic effects chronic angiotensin-( - ) prevents cardiac fibrosis in doca-salt model of hypertension antithrombotic effect of captopril and losartan is mediated by angiotensin-( - ) the antithrombotic effect of angiotensin-( - ) closely resembles that of losartan evidence for a new angiotensin-( - ) receptor subtype in the aorta of sprague-dawley rats bradykinin, angiotensin-( - ), and ace inhibitors: how do they interact? fate of bradykinin on the rat liver when administered by the venous or arterial route angiotensin - induces bradykinin-mediated relaxation in porcine coronary artery angiotensin-( - 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) levels in experimental biliary fibrosis vasoactive agents in intrahepatic portal hypertension and fibrogenesis: implications for therapy differential response of normal and cirrhotic liver to vasoactive agents. a study in the isolated perfused rat liver hemodynamic and antifibrotic effects of losartan in rats with liver fibrosis and/or portal hypertension angiotensin converting enzyme inhibitors and angiotensin ii antagonists as therapy in chronic liver disease effect of losartan, an angiotensin ii receptor antagonist, on portal pressure in cirrhosis randomized comparison of long-term losartan versus propranolol in lowering portal pressure in cirrhosis chronic administration of losartan, an angiotensin ii receptor antagonist, is not effective in reducing portal pressure in patients with preascitic cirrhosis hemodynamic effects of the angiotensin ii receptor antagonist irbesartan in patients with cirrhosis and portal hypertension the levels of renin activity, angiotensin converting enzyme and angiotensin ii in cirrhotic patients with ascites undergoing portacaval shunt effects of captopril on renal function in patients with cirrhosis and ascites acute effects of captopril on systemic and renal hemodynamics and on renal function in cirrhotic patients with ascites clinical usefulness of the angiotensin ii receptor antagonist losartan in patients with portal hypertensive gastropathy effects of captopril on hepatic venous pressure and blood flow in patients with liver cirrhosis captopril reduces portal pressure effectively in portal hypertensive patients with low portal venous velocity haemodynamic effects of enalaprilat on portal hypertension in patients with hbsag-positive cirrhosis effect of enalapril treatment and sclerotherapy of esophageal varices on hepatic hemodynamics in portal hypertension circulating activities of angiotensin-converting enzyme, its homolog, angiotensin-converting enzyme , and neprilysin in a family study a c angiotensin ii type receptor gene polymorphism may predict hemodynamic response to losartan in patients with cirrhosis and portal hypertension significance of chymase-dependent angiotensin ii formation in the progression of human liver fibrosis hepatic chymase level in chronic hepatitis: colocalization of chymase with fibrosis renin-angiotensin system inhibition: how much is too much of a good thing endogenous angiotensin ii levels and the mechanism of action of angiotensin-converting enzyme inhibitors and angiotensin receptor type antagonists aldosterone and renal haemodynamics in cirrhosis with ascites hepatic hemodynamics and the renin-angiotensin-aldosterone system in cirrhosis diagnostic significance of serum angiotensin-converting enzyme activity in biochemical tests with special reference of chronic liver diseases liver fibrosis: a balance of aces? the renin-angiotensin system in a rat model of hepatic fibrosis: evidence for a protective role of angiotensin metabolism of angiotensin-( - ) by angiotensin-converting enzyme converting enzyme determines plasma clearance of angiotensin-( - ) pathways for angiotensin-( - ) metabolism in pulmonary and renal tissues angiotensin - reduces bile duct proliferation and hepatic fibrosis in the bile duct ligated rat angiotensin-converting enzyme is a negative regulator of chronic liver injury advances in biochemical and functional roles of angiotensin-converting enzyme and angiotensin-( - ) in regulation of cardiovascular function upregulation of the ace /ang( - )/mas receptor axis in the bile duct ligation (bdl) model of hepatic fibrosis does not affect hepatic sinusoidal resistance hepatic conversion of angiotensin i and the portal hypertensive response to angiotensin ii in normal and regenerating liver angiotensin-( - )-stimulated nitric oxide and superoxide release from endothelial cells vascular endothelial dysfunction in cirrhosis influence of caveolin on constitutively activated recombinant enos: insights into enos dysfunction in bdl rat liver reactive hyperreninemia is a major determinant of plasma angiotensin ii during ace inhibition gradual reactivation of vascular angiotensin i to angiotensin ii conversion during chronic ace inhibitor therapy in patients with diabetes mellitus determinants of increased angiotensin ii levels in severe chronic heart failure patients despite ace inhibition how often are angiotensin ii and aldosterone concentrations raised during chronic ace inhibitor treatment in cardiac failure? expression of angiotensin ii type receptor in human cirrhotic livers: its relation to fibrosis and portal hypertension effects of renin-angiotensin system blockade on renal angiotensin-( - ) forming enzymes and receptors divergent regulation of circulating and intrarenal renin-angiotensin systems in response to long-term blockade effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme angiotensin-( - ) contributes to the antihypertensive effects of blockade of the renin-angiotensin system the role of ang ( - ) in mediating the chronic hypotensive effects of losartan in normal rats vasodepressor actions of angiotensin-( - ) unmasked during combined treatment with lisinopril and losartan evidence that prostaglandins mediate the antihypertensive actions of angiotensin-( - ) during chronic blockade of the renin-angiotensin system upregulation of angiotensin-converting enzyme after myocardial infarction by blockade of angiotensin ii receptors pharmacological effects of ave , a nonpeptide angiotensin-( - ) receptor agonist angiotensin-converting enzyme and new insights into the renin-angiotensin system dr john lubel is a recipient of an australia national health and medical research council (nhmrc) scholarship, and peter angus and louise burrell hold an nhmrc project grant ( ). key: cord- -hrgtaunt authors: rabelo, luiza a.; nunes-souza, valéria; bader, michael title: animal models with a genetic alteration of the ace /ang-( - )/mas axis date: - - journal: the protective arm of the renin angiotensin system (ras) doi: . /b - - - - . - sha: doc_id: cord_uid: hrgtaunt the aim of this chapter is to describe the animal models generated by transgenic technology for the functional analysis of the protective axis of the renin–angiotensin system, consisting of angiotensin-converting enzyme (ace ), angiotensin (ang)-( - ), and mas. transgenic overexpression of the components of this axis in general led to an ameliorated cardiac and vascular damage in disease states and to an improved metabolic profile. knockout models for ace and mas, however, show aggravated cardiovascular pathologies and a metabolic syndrome-like state. in particular, the local production of ang-( - ) in the vascular wall, in the heart, and in the brain was found to be of high physiological relevance by the use of transgenic animals overexpressing ace or ang-( - ) in these tissues. the study of hormone systems involved in cardiovascular and metabolic diseases, such as the renin-angiotensin system (ras), can only be performed in whole organisms due to the complex interplay of different organs, which determines cardiovascular physiology. in contrast to pharmacological interventions in these systems, which often lack specificity, the targeted genetic alteration of the expression of single-hormone system components is the most straightforward method to analyze their functions in cardiovascular and metabolic homeostasis and disorders. accordingly, the generation of transgenic and knockout (ko) animals was widely used to study the role of ras components in cardiovascular control and in the pathogenesis of diseases. , the aim of this chapter is to describe the animal models generated by transgenic technology for the functional analysis of the protective axis of the ras, consisting of angiotensin-converting enzyme (ace ), ang-( - ), and mas. in biomedical research, the use of rats and mice has become a major tool, considering the easiness of breeding, growth, and maintenance and the similarity with human organisms in most cardiovascular and metabolic systems. currently, the use of transgenic technologies to access animal physiology is routine, but the pioneering work was performed in the s. the first successful genetic modifications of a mouse were achieved by gordon and colleagues. this group demonstrated that foreign genes can be integrated into the mouse genome by transfer of dna constructs into the pronuclei of zygotes. by the use of specific promoters, the investigator can direct the expression of the transgene into specific tissues. about years later, the same technology was also established for the rat, interestingly first targeting the ras component renin. since , the elimination of gene expression is also possible by homologous recombination-mediated targeted gene ko in embryonic stem (es) cells. [ ] [ ] [ ] to this purpose, firstly, a targeting vector has to be designed and constructed that contains parts of dna sequences homologous to the gene of interest and the intended mutation. following the transfection of this vector into es cells, a few cells will incorporate it into the endogenous gene via homologous recombination and can be selected by appropriate methods. these successfully targeted es cells are injected into host blastocysts, and a chimeric animal is obtained. this animal carries the mutation in part of its cells and will eventually pass it on to its offspring, which will be heterozygous or homozygous (ko) for the mutant gene. the ko animals will present the physiological phenotype caused by the absence of the gene product. in general, this method is still the most commonly used for the targeted alteration of the mouse genome. it took more than years for this technology to also became available for the rat by the discovery of a method to establish germ-line-competent es cells from this species in , and a few ko rat models have been developed since using es cells. , however, recently, novel methods for the targeted alteration of genes in the mouse and rat genome have become available, which will probably replace the relatively complicated es cell method in the near future. they are based on nucleases that are targeted to a certain site in the genome by different methods. zinc finger (zfn) and tale nucleases use protein domains that specifically recognize a dna sequence of choice, while the crispr/cas system uses a guide rna that binds dna by specific base pairing. since only one animal model for the ras has been described yet produced by zfn technology, the renin-ko rat, and since the mas-ko rat has obviously been generated using zfns (http://rgd.mcw.edu/rgdweb/report/strain/main.html?id= ; access . . ) but is not yet published, we will not go into more detail on these novel technologies. our group and others have developed several transgenic and ko rat and mouse models with genetic deletion and/or overexpression of components of the ace /ang-( - )/mas axis. some of these models produced pleiotropic phenotypes depending on the genetic background of the strain they were generated in. in the following, we will list these models and summarize the insights into the physiology of the protective ras axis gained by their analysis (see also table ). the ace gene is located on the x chromosome, and thus, heterozygous deletion (ace −/y ) already results in the complete absence of the enzyme in male animals. deletion of ace in mouse leads to several cardiac abnormalities. in an elegant study, crackower and colleagues provided the first in vivo evidence supporting the hypothesis that the loss of ace promotes heart dysfunction. however, later studies on the function of ace in the heart resulted in contradictory observations. two independent groups showed that the baseline cardiac function and morphology appeared normal in their ace -deficient mouse lines. , nevertheless, ace −/y and even heterozygous female ace +/− mice are more susceptible to pressure overload or diabetes-induced cardiac injury. [ ] [ ] [ ] moreover, the lack of ace also exacerbated diabetic and shock-induced kidney injury. [ ] [ ] [ ] there is also still a debate whether the deletion of ace changes basic blood pressure in mice. most likely, this effect depends strongly on the genetic background of the mice analyzed: mice show hardly any effect, while c bl/ or fvb/n ace −/y mice are clearly hypertensive (our unpublished results). nevertheless, c bl/ ace −/y mice displayed high blood pressure during pregnancy and reduced weight gain and gave birth to smaller pups. besides an upregulation of oxidative stress in the brain and consequently of the sympathetic nervous system, the cause for the hypertensive effect of ace deletion may be an endothelial dysfunction as evidenced by an impaired acetylcholine-induced aortic vasodilatation. ace deletion in apolipoprotein e (apoe) ko mice, a classical model for atherosclerosis, worsened plaque formation and vascular inflammation. , in low-density lipoprotein receptor ko mice, fed a high-fat diet, ace deletion also aggravated atherosclerosis. moreover, loss of ace led to increased arterial neointima formation in response to endovascular injury in the femoral artery accompanied by an overexpression of inflammation-related genes. several studies have shown an important role of ace in metabolism. c bl/ ace −/y mice show impaired glucose homeostasis at different ages. , also, ace gene deletion aggravated liver fibrosis in models of chronic hepatic injury. ace −/y mice displayed aggravated pathologies in the acute respiratory distress syndrome and in bleomycin-induced lung injury rendering ace an important target for inflammatory lung diseases. ace −/y animals were also instrumental for the surprising finding that the protein is not only an enzyme but also a trafficking molecule in the gut being responsible for the functional expression of the amino acid transporter slc a . , ace −/y mice, therefore, show reduced levels of large amino acids, such as tryptophan, in the circulation, altered gut microbiota, and intestinal inflammation. besides being an enzyme and trafficking molecule, ace is also the receptor for the human severe acute respiratory syndrome (sars) coronavirus. in order to study this function, transgenic mouse models have been generated by several groups that overexpress human ace using the mouse ace promoter, the cytomegalovirus promoter, , or the cytokeratin promoter specific for the airway and other epithelia. , as expected, human ace expression led to an increased susceptibility of the transgenic mice to sars virus infection. in the kidney, ace overexpression protected the mice from shock-induced injury. the transgenic mouse overexpressing human ace in the brain using the synapsin promoter has confirmed the important actions of central ang ii in the pathogenesis of cardiovascular diseases and the protective role of ang-( - ) . the animals are protected from hypertension induced by low peripheral infusions of ang ii and doca-salt treatment. moreover, they show an amelioration of cardiac hypertrophy induced by angii, of chronic heart failure induced by coronary ligation, and of stroke induced by middle cerebral artery occlusion. , in most cases, an increase in ang-( - ) and a decrease of ang ii in the brain both influencing the levels of local no and the autonomic nervous system were shown to be instrumental. increased blood pressure during pregnancy increased susceptibility to cardiac damage [ ] [ ] [ ] aggravated atherosclerosis , disturbed glucose homeostasis , aggravated kidney [ ] [ ] [ ] and lung injury , amino acid uptake deficiency in the gut transgenic mice overexpressing human ace in the heart surprisingly showed an increase in ventricular tachycardia and sudden death, which was due to a dysregulation of connexins. the underlying mechanism and the involved peptides could not be elucidated. when human ace was overexpressed in podocytes of mice using the nephrin promoter, the nephropathy induced by diabetes was ameliorated. the authors suggest that this is due to reduced renal ang ii levels leading to a reduced expression of tgf-beta, but they could also not exclude a protective effect of ang- ( - ) . ace is highly expressed in the endothelium and smooth muscle cells (smc), and its expression is reduced in the spontaneously hypertensive stroke-prone rat (shrsp). when human ace was overexpressed in vascular smc of transgenic shrsp, endothelial dysfunction and hypertension were ameliorated, which was accompanied by a reduction in oxidative stress linked to a decrease in ang ii and/or an increase in ang-( - ). in , we generated mas-deficient (mas −/− ) mice on the mixed ×c bl/ background and showed that these animals were healthy in appearance and grew normally and exhibited normal ang ii plasma levels. however, male (but not female ) mas −/− mice displayed increased anxiety on the elevated-plus maze. we also showed not only that long-term potentiation was markedly increased in the hippocampal ca region of mas −/− mice but also that object recognition memory is impaired. collectively, these data support a role of mas in behavior. c bl/ mas −/− mice show a marked cardiac dysfunction both in vitro and in vivo accompanied by an increase in extracellular matrix proteins, such as collagen i, collagen iii, and fibronectin. furthermore, mas −/− animals exhibit vascular oxidative stress, endothelial dysfunction, and high blood pressure at least on the fvb/n genetic background. , consistently, endothelial function is also impaired in isolated mas −/− vessels. consequently, mas −/− mice exhibit a pronounced decrease in blood flow and a marked increase in resistance in different vascular beds. these functional changes in both regional and systemic hemodynamics in mas −/− mice suggest that the ang-( - )/mas axis plays an important role in vascular regulation. a dysregulation of the vascular function in the corpus cavernosum is probably also the reason for the erectile dysfunction observed in mas −/− mice. pinheiro et al. showed an imbalance in renal function in mas −/− mice: reduced urine volume and fractional sodium excretion and increased glomerular filtration rate and proteinuria. surprisingly, a proinflammatory role for ang-( - ) and mas was reported in a model of unilateral ureteral obstruction in mice in contrast to the anti-inflammatory effects of ang-( - ) and mas in other models of kidney nephropathy. certainly, additional studies are needed to clarify the role of ang-( - ) and mas in the kidney. the anti-inflammatory actions of mas were also recently confirmed in a lipopolysaccharide (lps)-induced endotoxic shock model. santos and colleagues have revealed the effects of mas deficiency on lipid and glucose metabolism. they used mas −/− mice on the fvb/n background and demonstrated that loss of mas increases the risk of metabolic complications by causing several features of the metabolic syndrome, such as type diabetes mellitus, hypertension, dyslipidemia, and nonalcoholic fatty liver disease. mas deletion decreased the responsiveness of adipocytes to insulin accompanied by a decreased expression of pparγ in adipose tissue. moreover, silva et al. recently showed that mas deletion in apoe −/− mice leads to dyslipidemia and liver steatosis. when rat mas is overexpressed in the retina of transgenic mice using the opsin promoter, degeneration of photoreceptors is the consequence, which is probably induced by proliferative signaling pathways activated in these cells due to the constitutively active mas protein. prolylcarboxypeptidase (prcp, ec . . . ) is another enzyme that can generate ang-( - ) from ang ii, but it is also not specific for angiotensin peptides. prcp-ko mice show elevated levels of α-melanocyte-stimulating hormone (α-msh), an anorexigenic neuropeptide, in the hypothalamus. the phenotype of these animals is characterized by a decrease in body weight and body length under normal diet, accompanied by decreased white adipose tissue. the lean phenotype is also observed after high-fat diet-induced obesity (schadock et al., unpublished results). moreover, prcp-ko mice display hypertension and vascular dysfunction probably due to an increase in reactive oxygen species and uncoupled enos. in addition, prcp also regulates angiogenesis and vascular repair. how much of these cardiovascular actions are due to alterations in ang ii or ang-( - ) levels remains to be elucidated. based on an elegant system to generate ras peptides by release from an artificial protein, which is processed by furin during secretion, , several transgenic animal models with altered ang-( - ) levels were generated. the transgenic rat tgr(a - ) expresses the ang-( - )-producing protein mainly in the testis. in this model, ang-( - ) levels are chronically elevated in plasma and testis ~ . -fold and ~ . -fold, respectively. surprisingly, this chronic increase in ang-( - ) did not alter basal blood pressure levels measured by telemetry. however, tgr(a - ) rats displayed a marked reduction in isoproterenol-induced heart hypertrophy and an improvement of postischemic systolic function. botelho-santos and colleagues showed changes in systemic and regional hemodynamic parameters in these rats, resulting in an increase of vascular conductance in several tissues and a decreased total peripheral resistance. furthermore, the transgenic rats showed a significant increase in stroke volume and cardiac index and a reduction in basal urinary flow, leading to increased urinary osmolality and osmolal clearance. furthermore, chronic elevation of circulating ang-( - ) levels considerably improved the lipid and glycolytic profile and lowered the fat mass accompanied by a decrease in triglycerides and cholesterol in plasma and an improved glucose tolerance and insulin sensitivity and reduced gluconeogenesis in the liver. , transgenic mice and rats overexpressing ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) in the heart transgenic mice carrying the ang-( - )-releasing construct under the control of the alpha-cardiac myosin heavy-chain promoter exhibit about an eightfold increase of the peptide in the heart and show a normal basic cardiac function but are protected from hypertensive cardiac hypertrophy induced by ang ii infusion but not from myocardial infarction. transgenic rats generated with the same construct showed a slightly improved resting cardiac function and were also protected from hypertrophy in this case induced by isoproterenol. transgenic and ko rodent models were pivotal for our understanding of the protective functions of the novel ras axis, ace /ang-( - )/mas. transgenic overexpression of the components of this axis in general led to ameliorated cardiac and vascular damage in disease states and to an improved metabolic profile. ko models for ace and mas, however, show aggravated cardiovascular pathologies and a metabolic-syndrome-like state. in particular, the local production of ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) in the vascular wall, in the heart, and in the brain was found to be of high physiological relevance by the use of transgenic animals overexpressing ace or ang-( - ) in these tissues. inducible and cell-type-specific ko models for mas and ace will be helpful in the future to deepen our understanding of the ace /ang-( - )/mas axis. mouse knockout models of hypertension genetically altered animal models for mas and angiotensin animal models for metabolic, neuromuscular and ophthalmological rare diseases genetic transformation of mouse embryos by microinjection of purified dna fulminant hypertension in transgenic rats harbouring the mouse ren- gene advances in the use of embryonic stem cell technology introduction of homologous dna sequences into mammalian cells induces mutations in the cognate gene targetted correction of a mutant hprt gene in mouse embryonic stem cells capture of authentic embryonic stem cells from rat blastocysts production of p gene knockout rats by homologous recombination in embryonic stem cells efficient gene targeting by homologous recombination in rat embryonic stem cells barbas rd cf. zfn, talen, and crispr/cas-based methods for genome engineering creation and characterization of a renin knockout rat angiotensin-converting enzyme is an essential regulator of heart function angiotensin converting enzyme- confers endothelial protection and attenuates atherosclerosis altered blood pressure responses and normal cardiac phenotype in ace -null mice angiotensin-converting enzyme deficiency is associated with impaired gestational weight gain and fetal growth restriction deletion of angiotensin-converting enzyme accelerates pressure overloadinduced cardiac dysfunction by increasing local angiotensin ii cardioprotective effects mediated by angiotensin ii type receptor blockade and enhancing angiotensin - in experimental heart failure in angiotensin-converting enzyme -null mice heterozygote loss of ace is sufficient to increase the susceptibility to heart disease genetic ace deficiency accentuates vascular inflammation and atherosclerosis in the apoe knockout mouse deletion of angiotensin-converting enzyme promotes the development of atherosclerosis and arterial neointima formation loss of angiotensin-converting enzyme leads to impaired glucose homeostasis in mice loss of ace exaggerates high-calorie diet-induced insulin resistance by reduction of glut in mice loss of angiotensin-converting enzyme- (ace ) accelerates diabetic kidney injury role of angiotensin-converting enzyme (ace and ace ) imbalance on tourniquetinduced remote kidney injury in a mouse hindlimb ischemia-reperfusion model loss of ace accelerates time-dependent glomerular and tubulointerstitial damage in streptozotocin-induced diabetic mice angiotensin-converting enzyme protects from severe acute lung failure angiotensin converting enzyme abrogates bleomycin-induced lung injury defective intestinal amino acid absorption in ace null mice tissue-specific amino acid transporter partners ace and collectrin differentially interact with hartnup mutations ace links amino acid malnutrition to microbial ecology and intestinal inflammation mice transgenic for human angiotensin-converting enzyme provide a model for sars coronavirus infection lethal infection of k -hace mice infected with severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme virus receptor differential virological and immunological outcome of severe acute respiratory syndrome coronavirus infection in susceptible and resistant transgenic mice expressing human angiotensin-converting enzyme heart block, ventricular tachycardia, and sudden death in ace transgenic mice with downregulated connexins podocyte-specific overexpression of human angiotensin-converting enzyme attenuates diabetic nephropathy in mice brain-selective overexpression of human angiotensin-converting enzyme type attenuates neurogenic hypertension ace -mediated reduction of oxidative stress in the central nervous system is associated with improvement of autonomic function angiotensin converting enzyme /ang-( - )/mas axis protects brain from ischemic injury with a tendency of age-dependence transgenic angiotensin-converting enzyme overexpression in vessels of shrsp rats reduces blood pressure and improves endothelial function sustained long term potentiation and anxiety in mice lacking the mas protooncogene sex specific behavioural alterations in mas-deficient mice impairment of in vitro and in vivo heart function in angiotensin-( - ) receptor mas knockout mice endothelial dysfunction and elevated blood pressure in mas gene-deleted mice ablation of angiotensin ( - ) receptor mas in c bl/ mice causes endothelial dysfunction evidence that the vasodilator angiotensin-( - )-mas axis plays an important role in erectile function genetic deletion of the angiotensin-( - ) receptor mas leads to glomerular hyperfiltration and microalbuminuria mas deficiency in fvb/n mice produces marked changes in lipid and glycemic metabolism murine prolylcarboxypeptidase depletion induces vascular dysfunction with hypertension and faster arterial thrombosis prolylcarboxypeptidase regulates food intake by inactivating alpha-msh in rodents expression of an angiotensin-( - )-producing fusion protein produces cardioprotective effects in rats improved lipid and glucose metabolism in transgenic rats with increased circulating angiotensin-( - ) decreased hepatic gluconeogenesis in transgenic rats with increased circulating angiotensin expression of an angiotensin-( - )-producing fusion protein in rats induced marked changes in regional vascular resistance - ) blunts hypertensive cardiac remodeling by a direct effect on the heart attenuation of isoproterenol-induced cardiac fibrosis in transgenic rats harboring an angiotensin-( - )-producing fusion protein in the heart angiotensin ii-mediated oxidative stress and inflammation mediate the agedependent cardiomyopathy in ace null mice angiotensin-converting enzyme gene targeting studies in mice: mixed messages angiotensin-converting enzyme deficiency in whole body or bone marrow-derived cells increases atherosclerosis in low-density lipoprotein receptor−/− mice angiotensin-converting-enzyme inhibits liver fibrosis in mice brain angiotensin-converting enzyme type shedding contributes to the development of neurogenic hypertension angiotensin-converting enzyme over-expression in the central nervous system reduces angiotensin-ii-mediated cardiac hypertrophy brain-selective overexpression of angiotensin-converting enzyme attenuates sympathetic nerve activity and enhances baroreflex function in chronic heart failure neuronal over-expression of ace protects brain from ischemia-induced damage angiotensin-( - )/mas axis integrity is required for the expression of object recognition memory effects of genetic deletion of angiotensin-( - ) receptor mas on cardiac function during ischemia/reperfusion in the isolated perfused mouse heart endothelial dysfunction through genetic deletion or inhibition of the g protein-coupled receptor mas: a new target to improve endothelial function altered regional blood flow distribution in mas-deficient mice angiotensin-( - ) and the g protein-coupled receptor mas are key players in renal inflammation beneficial effects of the activation of the angiotensin-( - ) mas receptor in a murine model of adriamycin-induced nephropathy receptor mas protects mice against hypothermia and mortality induced by endotoxemia angiotensin-( - ) mas-receptor deficiency decreases peroxisome proliferatoractivated receptor gamma expression in adipocytes mas receptor deficiency is associated with worsening of lipid profile and severe hepatic steatosis in apoe-knockout mice degeneration of cone photoreceptors induced by expression of the mas protooncogene prolylcarboxypeptidase promotes angiogenesis and vascular repair tissue targeting of angiotensin peptides renal function in transgenic rats expressing an angiotensin-( - )-producing fusion protein circulating rather than cardiac angiotensin-( - ) stimulates cardioprotection after myocardial infarction key: cord- -rxmpi o authors: guang, cuie; phillips, robert d.; jiang, bo; milani, franco title: three key proteases – angiotensin-i-converting enzyme (ace), ace and renin – within and beyond the renin-angiotensin system date: - - journal: arch cardiovasc dis doi: . /j.acvd. . . sha: doc_id: cord_uid: rxmpi o the discovery of angiotensin-i-converting enzyme (ace ) and a (pro)renin receptor has renewed interest in the physiology of the renin-angiotensin system (ras). through the ace /angiotensin-( – )/mas counter-regulatory axis, ace balances the vasoconstrictive, proliferative, fibrotic and proinflammatory effects of the ace/angiotensin ii/at axis. the (pro)renin receptor system shows an angiotensin-dependent function related to increased generation of angiotensin i, and an angiotensin-independent aspect related to intracellular signalling. activation of ace and inhibition of ace and renin have been at the core of the ras regulation. the aim of this review is to discuss the biochemistry and biological functions of ace, ace and renin within and beyond the ras, and thus provide a perspective for future bioactives from natural plant and/or food resources related to the three proteases. angiotensine ; inhibiteurs de l'enzyme de conversion ; inhibiteur de l'enzyme de conversion ii ; rénine ; récepteur de la prorénine résumé la découverte de l'enzyme de conversion de l'angiotensine (ac ) et un récepteur à la prorénine est une avancée récente dans la compréhension de la physiologie du système rénine-angiotensine. au sein de l'axe inhibiteur de l'enzyme de conversion de l'angiotensine /angiotensine /mas, l'ac contrebalance l'effet vasoconstricteur, prolifératif, fibrosant et pro-inflammatoire de l'axe ace/angiotensine /at . le récepteur à la prorénine a une fonction angiotensine dépendante, liée à l'augmentation de la production d'angiotensine , et un aspect indépendant de l'angiotensine, lié aux signaux intracellulaires. l'activation de l'ac et l'inhibition de l'ace de la rénine ont été considérées comme au centre de la régulation du système rénine-angiotensine. l'objet de cette revue générale est de discuter les fonctions biochimiques et biologiques de l'ace, de l'ac et de la rénine au sein et au-delà du système rénine-angiotensine et ainsi de proposer une perspective de développement d'agents actifs extraits de plantes naturelles ou d'alimentation, produits liés à ces trois protéases. © elsevier masson sas. tous droits réservés. the renin-angiotensin system (ras) is not only an endocrine but also a paracrine and an intracrine system [ ] . in mammals, the intravascular ras plays a key role in maintaining blood pressure homeostasis and fluid and salt balance, and the tissue or local ras is involved in physiological and pathological processes, such as tissue growth and remodelling, development and inflammation [ ] . in a classical ras, the substrate angiotensinogen (agt), which is released into the circulation from the liver, is degraded by the enzyme renin that originates in the kidney, generating the inactive angiotensin i (ang i). when this decapeptide comes into contact with angiotensin-i-converting enzyme (ace) at the endothelial surface of blood vessels, the c-terminal dipeptide is cleaved, giving rise to angiotensin ii (ang ii), the main effector molecule of the ras. through interactions with specific receptors, particularly its type or at receptor, ang ii stimulates a wide variety of signalling pathways in the heart, blood vessels, kidneys, adipose tissue, pancreas and brain, initiating most of the physiological and pathophysiological effects that have been attributed to the ras [ ] . due to the function of directly generating the main effector ang ii, ace -together with the classical axis ace/ang ii/at -has been at the core of ras studies since its discovery. in , a homologue of ace, known as angiotensin-iconverting enzyme (ace ), was cloned by two independent research groups [ , ] . evidence indicates that ace negatively regulates the activated ras by degrading ang ii to the heptapeptide ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) . moreover, through the mas receptor (a g protein-coupled receptor), the resulting ang-( - ) counterbalances the cardiovascular effects of ang ii by opposing many at receptor-mediated actions [ ] . ace and the axis ace /ang-( - )/mas are becoming the focus of intense research regarding the ras [ ] . the discovery of a (pro)renin receptor ([p]rr) and the introduction of renin inhibitors have also brought (pro)renin back into the spotlight [ ] . far from being a straightforward cascade containing one substrate (agt), two proteases (renin and ace), two peptides (ang i and ang ii) and one receptor (at ), the ras currently consists of several axes upstream and downstream of the classical cascade, which include more than two dozen peptidases, nearly a dozen ang fragments and at least six different receptors [ ] . here, we review three critical proteases (ace, ace and renin) within and beyond the ras and thus intend to find new connections between natural plant and/or food resources and the ras. occurrence, gene encoding and structure of ace ace (ec . . . ) is a monomeric glycoprotein that is distributed in many tissues and biological fluids. there are two isoforms of ace in humans: somatic ace (sace) and germinal ace (gace). somatic ace is found in many types of endothelial and epithelial cells [ ] . germinal ace or testicular ace is present exclusively in germinal cells in the male testis. although ace is a type i integral membrane protein, it can also be released as a soluble enzyme into extracellular fluids, such as plasma and seminal and cerebrospinal fluids, following post-translational proteolytic cleavage by a membrane protein sheddase or secretase [ ] [ ] [ ] . somatic ace and gace are encoded by a single gene containing exons. the promoter for sace is situated in the flanking region of the first exon, whereas that for gace is within intron , which results in different lengths for the two isoforms. the longer sace ( - kda) is transcribed from exon to exon , excluding exon , whereas the shorter gace ( - kda) is transcribed from exon to exon . exon encodes a unique sequence for the n-terminus of gace, whereas downstream exons encode a common sequence for both isozymes [ ] . somatic ace and gace both consist of a -residue hydrophilic c-terminal cytoplasmic domain, a -residue hydrophobic transmembrane domain that anchors the protein in the membrane and an n-terminal ectodomain ( fig. ) that is heavily glycosylated with mannose, galactose, fructose, n-acetylneuraminic acid and n-acetylglucosamine [ ] . the ectodomain of sace is further divided into two similar domains (n domain and c domain) encoded by the homologous exons - and - , respectively, and each domain contains an active his-glu-x-x-his (hexxh) sequence [ ] . somatic ace is the only known metallopeptidase with two homologous active sites [ ] , which implies that there has been a gene duplication event during evolution [ ] . except for a unique sequence constituting its n-terminus, gace is identical to the c-terminal half of sace [ ] . due to cleavage of the membrane-bound residues by ace secretase, soluble circulating ace lacks a transmembrane portion and a cytosolic domain [ ] . the three-dimensional x-ray crystallographic structure of a deglycosylated truncated version of gace (c domain of sace), reveals a preponderance of ␣-helices with a zinc ion and two chloride ions incorporated. a deep narrow channel separates the molecule into two subdomains and the active site is located toward the bottom of this channel. an n-terminal 'lid' on the top of molecule appears to allow only small peptide substrates access to the active site cleft. in fact, the structure bears little similarity to that of carboxypeptidase a (m family) on which the initial drug development of ace inhibitors was based. instead, it resembles rat neurolysin (m family) and pyrococcus furiosus carboxypeptidase (m family), despite sharing little sequence similarity with these two proteins [ ] . corradi et al. [ ] reported the crystal structure of the n domain of sace. similarly, it has an ellipsoid shape with a central groove dividing it into two subdomains, one of which contains the n-terminal region that covers the central binding cavity. but the structure reveals differences in the active site and it contains only one chloride ion, equivalent to chloride ii of gace. the three-dimensional structures of c domains (based on gace) and n domains provide an opportunity to design domain-selective ace inhibitors that may exhibit new pharmacological profiles [ , ] . according to the catalytic mechanism and the critical amino acid residue involved, peptidases are classified into four major types: serine, cysteine, aspartic and metallo [ ] . ace is an m family metallopeptidase: ma(e), the gluzincins [ ] . two histidine residues of the functional motif hexxh and a third distant glutamate positioned - residues further towards the c-terminus are the ligands for the zinc cofactor [ ] . an activated water molecule complexed to zn + serves as the nucleophile to attack the carbonyl group of the targeted peptide bond [ ] . the activity of ace is also chloride dependent. chloride primarily activates the active sites of ace and enhances the binding of substrates [ ] . each active domain of ace displays differences in sensitivity to chloride activation [ ] . the activity of the c domain of sace depends highly on chloride ion concentration and is inactive in its absence, whereas the n domain can be completely activated at relatively low concentrations of this anion and is still active in the absence of chloride [ , ] . germinal ace depends on chloride to a lesser extent compared with the c domain of sace [ ] . cushman and cheung [ ] reported an optimal in vitro ace activity of rabbit rung acetone extract in the presence of mm nacl at ph . - . . the two active domains of sace are also subtly different in substrate specificity. they hydrolyze bradykinin almost equally but the c domain active site can hydrolyze ang i, substrate p [ ] and hippuryl-his-leu [ ] more efficiently, while the n domain active site preferentially hydrolyzes ang-( - ) [ ] , luteinizing hormone-releasing hormone (lh-rh) [ ] , the haemoregulatory peptide n-acetyl-ser-asp-lys-pro (acsdkp) [ ] and alzheimer amyloid ␤-peptide (a␤) [ ] . fuchs et al. [ ] proved that the c-terminal catalytic domain was the main site of ang i cleavage in mice. the differentiation of catalytic specificity might be due to very subtle variation in substrate-specific amino acids [ ] and chlorideinduced conformational alteration of active sites [ ] . ace acts as an exopeptidase to cleave dipeptides from the free c-termini of two typical substrates, ang i and bradykinin. for certain substrates such as cholecystokinin [ ] , substrate p [ ] and lh-rh [ ] , which have amidated c-termini, ace not only displays exopeptidase activity but also acts as an endopeptidase [ ] . the most prominent example of endopeptidase activity is ace hydrolyzing the synthetic a␤-( - ) peptide into four fragments: an a␤- peptide and the others corresponding to products of a␤-( - ) hydrolysis [ ] . thus, ace might have a more general impact on the metabolism of biologically active peptides than previously recognized [ ] . the two substrates used most often for measuring ace activity and inhibition in vitro -hippuryl-his-leu and n-[ -( -furyl)acryloyl]-lphenylalanylglycylglycine (fapgg) -only have the n-termini blocked and substrates with two termini blocked have been developed [ ] . ace was originally isolated in as a 'hypertensinconverting enzyme' [ ] . in the ras, ace cleaves the decapeptide ang i-( - ) (asp-arg-val-tyr-ile-his-pro-phe-his-leu) into the octapeptide ang ii-( - ) by removing the c-terminal dipeptide his-leu. when the ras is overactive, ang ii exerts its harmful effects primarily via the at receptor, whereas the at receptor may oppose and counterbalance those effects mediated by at receptor to exert protective actions [ ] . ang ii is a potent vasoconstrictor, stimulates the release of aldosterone and antidiuretic hormone or vasopressin and increases the retention of sodium and water. these effects act directly in concert to raise blood pressure. a nonapeptide derivative of ang i, des-asp -ang i-( - ), prevents infarction-related and non-infarction-related cardiac injuries and disorders. his-leu can be cleaved from the peptide by ace to produce ang iii-( - ) [ ] , which has % of the vasoconstriction activity of ang ii. ang iii exerts its effects, in principle, in a similar manner to ang ii, and may be equally or even more important in mediating the release of vasopressin [ ] . ace also degrades ang-( - ) to ang-( - ) then further degrades this peptide to the inactive ang-( - ) (fig. ). in addition, ace (also termed kininase ii) inactivates the vasodilators bradykinin-( - ) (arg-pro-pro-gly-phe-ser-pro-phe-arg) and kallidin (lys-bradykinin) in the kallikrein-kinin system, by cleaving the c-terminal dipeptide phe-arg. ace eventually cleaves its primary metabolite bradykinin-( - ) into the shorter fragment bradykinin-( - ) [ ] . through ang ii and aldosterone, ace may also be implicated in the impairment of nitric oxide bioavailability and cell oxidative stress, augmenting the generation of reactive oxygen species and peroxynitrite [ , ] . with the ability to hydrolyze neuropeptides such as enkephalin [ , ] , substrate p, neurotensin [ ] and lh-rh, ace may be involved in the functioning of the brain and nervous system. ace may affect the digestive system by hydrolyzing the peptide hormone cholecystokinin and gastrin [ ] . the in vivo experiment conducted by azizi et al. [ ] proved that acute ace inhibition could increase the level of the natural stem cell regulator acsdkp in plasma. acsdkp substantially inhibits cell cycle entry of normal haematopoietic stem cells and protects haemopoiesis against damage caused by cycleactive cytotoxic agents [ ] . acsdkp can also inhibit the proliferation of hepatocytes [ ] and lymphocytes [ ] and stimulate angiogenesis [ ] in vivo. the in vivo antifibrotic effect of ace inhibition is partially mediated by acsdkp [ ] . ace may also affect susceptibility to alzheimer's disease by degrading a␤ and preventing the accumulation of amyloid plaques in vivo [ ] . in the brains of amyloid precursor protein swedish mutation transgenic mice, ace converts a␤ - to a␤ - and degrades a␤, and chronic inhibition of ace with captopril enhances predominant a␤ - deposition [ ] . however, through the inhibition of brain ace activity in the a␤ - -injected mice, perindopril ameliorates cognitive impairment and may therefore have a beneficial effect on alzheimer's disease as well as hypertension [ ] . using the assumed mechanistic analogy to other zinc metallopeptidases, plus the knowledge that several snake-venom peptides potentiate the action of bradykinin by inhibiting ace, efforts were undertaken to develop orally-active peptide analogues for potential use in the treatment of hypertension [ ] . the first such compound, captopril or d- -mercapto- -methylpropanoyl-l-proline, is an analogue of the ala-pro sequence, with sulphydryl as a strong chelating group for the zinc ion. its adverse effects, which were the same as those caused by mercapto-containing penicillamine, prompted the design of non-sulphydryl ace inhibitors [ ] . the results were two active inhibitors: enalaprilat and lisinopril. they are both essentially tripeptide analogues with a zinc-co-ordinating carboxyl group and a phenylalanine that occupies the s groove in the enzyme. lisinopril is a lysine analogue of enalaprilat but it is hydrophilic, with greater affinity than enalaprilat. the later compounds are all variations of the first three inhibitors, with most of the differences residing in the functionalities that bind the active site zinc and the s pocket. in addition to phosphonates, ketones are also useful as chelators [ ] . currently, there are more than ace inhibitors marketed that are widely used as first-line therapy for cardiovascular diseases, including hypertension, heart failure, heart attack and left ventricular dysfunction. according to the functional moiety, they are divided into three types: thiol (captopril), carboxylate (benazepril, enalapril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril) or phosphate (fosinopril). some ace inhibitors are now administrated clinically as ethyl-ester prodrugs, which have good bioavailability but are inactive in their own right. they are then converted to the active diacid molecules in vivo by esterases. as a drug class, ace inhibitors are very effective, have a relatively low incidence of side effects and are well tolerated. a common side effect of ace inhibitors is a dry cough, which appears in - % of patients and may result in the discontinuation of treatment. another serious problem is angioedema, which affects . - . % of patients and can be life-threatening. the two side effects have generally been attributed to altered concentrations of bradykinin [ ] . use of ace inhibitors during the second and third trimesters of pregnancy is contraindicated because of their association with an increased risk of foetopathy, a group of conditions that includes oligohydramnios, intrauterine growth retardation, hypocalvaria, renal dysplasia, anuria, renal failure and death. exposure to ace inhibitors during the first trimester of pregnancy may place the infant at increased risk for major congenital malformations [ ] . the initial drug development of clinical ace inhibitors was based on the assumption of an active site related to that of carboxypeptidase a but organized to remove a dipeptide rather than a single amino acid from the cterminus of its substrate. it is now known that sace has two active sites, neither of which resembles that of carboxypeptidase a, and that these sites are not identical. clinical ace inhibitors, however, show little discrimination between these two active sites [ ] . ang i is hydrolyzed predominantly by the c domain of sace in vivo [ ] but bradykinin is hydrolyzed by both active sites [ ] ; therefore a c domain-selective inhibitor would allow some degradation of bradykinin by the n domain and this degradation could be enough to prevent the accumulation of excess bradykinin that has been observed during attacks of angioedema. that is, the c domain-selective inhibition could possibly result in specialized control of blood pressure with fewer vasodilatorrelated adverse effects [ ] . a structure-activity study has proved that the group substitution involving the phenyl ring and carbon chain at the sulphonyl and propionyl moieties of captopril is essential for better activity towards the c domain of ace [ ] . there is increasing evidence that the n domain of sace is responsible for the in vivo degradation of the natural haemoregulatory hormone acsdkp [ , , ] . so, n domain-selective inhibition might open up novel therapeutic areas. two phosphinic tetrapeptides, rxpa and rxp , have been found to be highly selective inhibitors of the c and n domains of sace, respectively [ ] . the availability of the three-dimensional structures of the c and n domains of sace makes the structure-based design of active site-specific inhibitors possible [ , ] . ace (ec . . .-) is also a type i transmembrane glycoprotein and its expression has now been recognized as being ubiquitous. it appears to be susceptible to cleavage that releases the catalytical active ectodomain. a disintegrin and metalloprotease (adam ; also known as tumour necrosis factor-alpha cleavage enzyme [tace]) is a major protease for soluble ace shedding, while phorbol ester, ionomycin, endotoxin and the proinflammatory cytokines interleukin- ␤ and tumour necrosis factor-alpha can also acutely induce ectodomain release [ ] . calmodulin binding sites have been identified in the cytoplasmic tail of ace and calmodulin inhibitors are proved to increase ace shedding [ ] . the cleavage site resides between amino acids and near the predicted transmembrane domain and residue lys in the ectodomain plays an important role in dictating ace ectodomain shedding [ ] . ace has amino acids encoded from exons and shares about % sequence identity with the n and c domains of sace [ ] . ace also belongs to the m family of metalloproteases but consists of a single active site domain that, by sequence comparison, more closely resembles the n domain than the c domain of sace ( fig. ) [ ] . in addition to the conserved zinc metallopeptidase consensus sequence hexxh (amino acids - ), there is a conserved glutamate residue residues c-terminal to the second histidine of the zinc motif, which serves as the third zinc ligand [ ] . the threedimensional structure of a truncated extracellular region of human ace shows that the active site domain (residues - ) can be further divided into two subdomains i and ii, which form two sides of a long deep cleft and are connected only at the floor of the active site cleft by a prominent ␣helix. the deeply recessed and shielded proteolytic active site of ace is a common structural feature of proteases and can avoid hydrolysis of correctly folded and functional proteins. the zinc is co-ordinated by his , his , glu and one water molecule in the subdomain i near the bottom, whereas a chloride ion is co-ordinated by arg , trp and lys in the subdomain ii [ ] . the ace transmembrane c-terminal domain shares % sequence identity with collectrin (fig. ) , a non-catalytic protein shown to have a critical role in amino acid reabsorption in the kidney [ ] , pancreatic beta cell proliferation and possibly insulin exocytosis [ ] . unlike sace and gace, which are primarily dipeptidylcarboxypeptidases, ace functions predominantly as a monocarboxypeptidase, with a substrate preference for hydrolysis between proline and a hydrophobic or basic cterminal residue [ ] . it is like carboxypeptidase a in its action model but is different in active structure because the latter has an hxxe zinc-binding motif with the third ligand (histidine) positioned - residues further towards the c-terminus [ ] . ace efficiently cleaves a single residue phenylalanine from ang ii to generate ang- ( - ) , with about -fold higher catalytic efficiency than the conversion of ang i to ang-( - ) by removing the c-terminal leucine residue (fig. ). other substrates with high catalytic efficiency are apelin- , dynorphin a-( - ) and des-arg bradykinin [ ] . arg of ace has been identified as a residue critical to substrate selectivity [ ] . ace activity is also regulated by chloride ions; it has been proposed that chloride binding induces subtle changes in the conformation of the active site, which either facilitate or hinder substrate binding [ ] . for the substrate ang i, ace activity increases with increased [cl − ] and a plateau is reached at approximately mm cl − . for the substrate ang ii, an increase in ace activity is observed as [cl − ] increases from - mm but any further increase in [cl − ] decreases ace activity until a plateau is reached at mm cl − ; ace activity at mm cl − is even lower than that in the absence of cl − ( mm). consequently, an increase in [cl − ] above mm, which is the physiological concentration in human plasma, increases ang i and decreases ang ii cleavage by ace . this has an effect on the localized concentration of ang ii in the kidney, where ace has a high level of expression and extracellular chloride ion levels fluctuate. thus, in vivo cl − sensitivity may serve as a homeostatic regulatory mechanism [ ] . ace activity is unaffected by inhibitors of ace (captopril, lisinopril and enalaprilat) or carboxypeptidase a (benzylsuccinate and potato carboxypeptidase inhibitor) [ ] . the major function of ace is to counter-regulate ace activity by reducing ang ii bioavailability and increasing ang-( - ) formation. as a result, ace plays a crucial role in maintaining the balance between the two axes ace /ang-( - )/mas and ace/ang ii/at of the ras; a chronic and sustained imbalance may lead to pathophysiology of the cardiovascular, renal, pulmonary and central nervous systems [ ] . studies have shown that ace overexpression and recombinant ace treatment can attenuate hypertension in animal models [ , ] , while in humans, there is a strong association between ace polymorphisms and hypertension in han chinese [ ] . in addition to the ang ii system, ace may regulate blood pressure through other peptide systems, such as bradykinin and/or apelin [ ] . ace gene delivery [ ] and ace-( - ) infusion [ ] also have beneficial effects on atherosclerosis, whereas ace deficiency accentuates vascular atherosclerosis and inflammation [ ] in animal models. regarding heart function, ace null mice display impaired cardiac contractility [ ] and the loss of ace in wild-type mice accelerates adverse ventricular remodelling by potentiation of ang ii effects by means of the at receptors [ ] . ang-( - ), through interaction with the mas receptor, can improve atrial tachyarrhythmias [ ] , myocardial performance, cardiac modelling and survival [ , ] in rodent heart failure models. in humans, ace gene variants might be involved in modulation of left ventricular mass in men [ ] and soluble ace activity is increased in patients with heart failure and correlates with disease severity to exert cardioprotective actions [ ] . deletion of the ace gene in mice leads to the development of glomerulosclerosis and increased albuminuria [ ] , while treatment with the ace inhibitor mln can worsen renal damage in streptozotocin-induced diabetic mice [ ] . chronic treatment with ang-( - ) improves renal endothelial dysfunction via the mas receptor in apolipoprotein e-deficient mice, by increasing levels of endogenous nitric oxide [ ] , whereas genetic deletion of the mas receptor in mice leads to a reduction in urine volume, sodium retention, microalbuminuria and reduced renal blood flow, which are associated with upregulation of the at receptor and transforming growth factor-beta messenger ribonucleic acid [ ] . this evidence indicates the protective role of ace /ang-( - )/mas in renal function. ace has also been shown to regulate cardiovascular functions in brain regions. overexpression of ace in the rostral ventrolateral medulla reduces high blood pressure and heart rate in spontaneously hypertensive rats (shrs) [ ] . in the nucleus tractus solitarius of shrs, ace gene transfer improves baroreceptor heart rate reflex [ ] . in the mouse subfornical organ, ace overexpression inhibits at receptor expression and prevents ang ii-mediated pressor and drinking responses [ ] . in addition, ace /ang-( - )/mas can exert cerebroprotective functions in endothelin- -induced ischaemic stroke in rodent models [ ] . ace exerts a host of actions on the cardiopulmonary system, which include prevention of endothelial dysfunction, reduction in pulmonary oxidative stress, attenuation of vascular impairment, anti-inflammatory effects and anticardiac remodelling effects. all these properties are responsible for the protective role of ace against pulmonary arterial hypertension [ ] . structure-based drug screening has identified two ace activators: a xanthenone ( -[( -diethylamino)ethyl-amino]- -(hydroxymethyl)- -[( -methylphenyl)sulphonyloxy]- h-xanthene- -one; xnt) and resorcinolnaphthalein. xnt hydrogen bonds with ace residues lys , tyr , gly and his , and resorcinolnaphthalein is involved in three hydrogen bonds with residues gln , gln and gly . xnt and resorcinolnaphthalein modulate ace activity possibly by two mechanisms. logically, the closed conformation of the enzyme cannot allow the substrate into its active site. in the presence of compound, the free enzyme may be shifted to the open form, effectively increasing the activity coefficient of the enzyme. alternatively, product release is a rate-limiting step in ace turnover. ace activity may be enhanced in the presence of compound, as the enzyme-product complex empties more quickly and ace becomes available to start another cycle. xnt and resorcinolnaphthalein enhance in vitro ace activity in a dose-dependent manner and show no significant effects on ace activity. administration of xnt to shrs can result in a decrease in blood pressure, improvements in cardiac function and reversal of myocardial, perivascular and renal fibrosis [ ] . the protective role of xnt against hypertension-induced cardiac fibrosis is associated with activation of ace , increases in ang-( - ) and inhibition of extracellular signal-regulated kinases [ ] . furthermore, ace activation by xnt attenuates thrombus formation and reduces platelet attachment to vessels [ ] . xnt also prevents pulmonary vascular remodelling and right ventricular hypertrophy and fibrosis in a rat model of monocrotaline-induced pulmonary hypertension [ ] . development of xnt is being pursued to translate the vasoprotective concept of ace into an effective cardiovascular therapy [ ] . diminazine aceturate, a small molecule antiprotozoal agent that shares structural similarity with xnt, has recently been demonstrated to be a potent activator of ace and to decrease mean arterial pressure and myocardial fibrosis in shrs [ ] . intracerebroventricular infusion of diminazine aceturate, prior to and following endothelin- -induced middle cerebral artery occlusion, significantly attenuates cerebral infarct size and neurological deficits in a rat model [ ] . these data suggest that diminazine aceturate may serve as a lead compound for the discovery of the next generation of drugs for the treatment of cardiovascular disease and hypertension [ ] . interestingly, peptidase-independent actions of ace have also been elucidated. ace has been identified as an essential receptor for the coronavirus (cov) that causes severe acute respiratory syndrome (sars). the spike protein of sars-cov attaches the virus to its cellular receptor ace with a binding domain on the spike protein mediating the interaction [ ] . this ace -spike interaction leads to endocytosis of virus particles through internalization with ace , induces the fusion of virus with host cells and establishes sars-cov infection with the release of viral rnas into cytoplasm [ ] . the spike protein of sars-cov also induces tace-dependent shedding of the ace ectodomain, with the involvement of the ace cytoplasmic domain. cellular tace and the ace cytoplasmic tail promote viral entry and infection. these observations indicate that ace shedding and its causative cellular signals may be attributable to sars-cov-induced tissue damage [ ] . an ace inhibitor, n-( -aminoethyl)- aziridine-ethanamine, has been identified as being effective in blocking sars-cov spike proteinmediated cell fusion [ ] . the sars-cov receptor function of ace is independent of its catalytic activities for ang ii degradation but ace -mediated ang ii degradation is still important for lung protection from sars pathogenesis [ , ] . so, the consequence of long-term activation of ace must be determined and the effects of ace activators on the immune competence of animals and their vulnerability to sars-cov infection must be tested before these molecules are ready for preclinical trials [ ] . ace is also the receptor for the human coronavirus nl that was discovered in in the netherlands and was shown to be globally distributed [ ] . additionally, due to the high homology with collectrin at its transmembrane, ace binds to b at -family amino acid transporters and contributes to the absorption of neutral amino acids in animal intestines [ , ] . thus, ace may be involved in multiple biological functions. renin (ec . . . ) is a key enzyme of the ras. since its discovery years ago, an impressive quantity of information about renin has been compiled. it is generally accepted that the active renin found in the circulation of mammals almost exclusively originates from the kidney. in addition to systemic renin, there are a number of extrarenal tissues that express renin as part of the local or tissue-specific rass. within the kidney, renin is predominantly produced by the juxtaglomerular cells. (pro)renin gene transcription in these cells is controlled through several mechanisms, among which cyclic adenosine monophosphate (camp)/protein kinase a/camp response element binding protein (stimulatory) and calcium/protein kinase c (inhibitory) cascades are employed by physiological cues, whereas signal transducers, activators of transcription and nuclear factor b transcription factors (inhibitory) and members of the nuclear receptor superfamily probably become relevant in pathological situations. prorenin is stored in vesicles, activated to renin and then released upon demand. the release of renin is under the control of the intracellular camp (stimulatory), ca + (inhibitory) and cyclic guanosine monophosphate signalling pathways. meanwhile, a great number of intrarenally generated or systemically acting factors have been identified that control renin secretion directly at the level of renin-producing cells, by activating the camp or ca + signalling pathways [ ] . renin is an aspartyl protease with a typical structure made of two lobes. the cleft between the lobes contains the active site, characterized by two catalytic aspartic residues. renin is a highly specific enzyme and has only one known substrate (agt) [ ] . prorenin is the 'inactive' precursor of renin. in addition to the main organ, the kidney, other tissues, such as the brain, the adrenal gland, the submandibular gland, the glands of the reproductive system and adipose tissue, are also able to secrete prorenin in the surrounding milieu and in plasma. in contrast to renin, prorenin is released constitutively; renin and prorenin levels are usually well correlated. under some physiopathological circumstances, such as pregnancy and diabetes, prorenin levels exceed by far those of renin [ ] . prorenin has a prosegment of amino acid residues attached to the n-terminus of mature renin; the prosegment folds into an active site cleft of mature renin to prevent catalytic interaction with agt [ ] . prorenin-renin conversion occurs in the kidney before renin is released from the juxtaglomerular cells. several enzymes, including proconvertase and cathepsin b, have been proposed to be responsible for this irreversible cleavage of the prosegment from prorenin to form mature renin. a (reversible) non-proteolytic activation of prorenin may also occur. at acidic ph and/or low temperature, prorenin is capable of undergoing a conformational change, involving the unfolding of the prosegment from the enzymatic cleft. this non-proteolyticallyactivated prorenin is fully enzymatically active [ ] . under physiological conditions, approximately % of plasma prorenin is in the open form and can also display enzymatic activity [ ] . the traditional assumptions of renin being just an enzyme responsible for the cleavage of agt and prorenin being just an 'inactive' proenzyme were challenged by the cloning of a human (p)rr in [ ] . the (p)rr is a single transmembrane-domain protein of amino acids, with a large unglycosylated and highly hydrophobic n-terminal domain responsible for renin and prorenin binding and a short cytoplasmic tail of about amino acids involved in intracellular signalling [ ] . the (p)rr was first identified on cultured human mesangial cells and bound well to renin and prorenin with a k d in the nanomolar range [ ] . binding of prorenin induced a conformational change in the molecule, increasing its enzymatic activity from virtually zero to values similar to those of active renin in solution without proteolytic removal of the prosegment [ , ] . two regions in the human prorenin segment, namely t p fkr p (a gate that is not accessible by its specific antibodies until it is loosened from the active site cleft) and i p flkr p (a handle, a protruding pentameric segment), have been identified as being crucial for the non-proteolytic activation [ ] . renin bound to the (p)rr displays a -to -fold increase in the catalytic efficiency of agt conversion to ang i compared with free renin [ , ] . in addition to the ang-dependent function on the cell surface related to the increased catalytic activity of receptor-bound (pro)renin (renin activity of activated prorenin and increased activity of renin) that leads to the formation of ang i from agt, the (p)rr system also has ang-independent intracellular effects that are not necessarily related to the ras [ ] . pathological conditions like high blood pressure upregulate the receptor, whereas elevated (pro)renin concentrations downregulate the receptor via translocation of the transcription factor promyelotic zinc finger protein to the nucleus [ ] . in the ang-independent signalling pathways, prorenin-induced activation of mitogen-activated protein kinase p and subsequent phosphorylation of heat shock protein result in actin polymerization, while (pro)renin-induced activation of the extracellular signal-regulated kinase / (p /p ) signalling cascade leads to the intracellular expression of profibrotic genes, giving rise to the synthesis of transforming growth factor-␤, plasminogen activator inhibitor- , collagen and fibronectin. these effects potentially increase cardiac and renal hypertrophy and fibrosis [ , [ ] [ ] [ ] . the discovery of the (p)rr has confirmed the hypothesis that renin is also a hormone. it has been suggested that blocking the (p)rr may be a new target for renal and cardiac end-organ protection [ ] . additionally, the mannose- -phosphate/insulin-like growth factor ii receptor can bind both renin and prorenin with high affinity and is believed to serve as a clearance receptor for renin/prorenin. an intracellular renin-binding protein has been also cloned and found to be an inhibitor of renin activity but its deletion affected neither blood pressure nor plasma renin [ ] . after the discovery of the receptor, a (p)rr antagonist was designed, based on the idea that the prosegment contains a handle region that binds to the receptor, allowing prorenin to become catalytically active in a non-proteolytic manner [ ] . the antagonist (also known as hand region peptide, hrp), consisting of amino acids (nh -rillkkmpsv-cooh), resembles the handle region (i p llkk p ) of rat prorenin prosegment and will thus competitively bind to the (p)rr as a decoy peptide and inhibit the receptor-mediated activation of prorenin [ ] . treatment with the hrp in diabetic mice and rats decreased the renal content of ang i and ii and inhibited the development of nephropathy without affecting hyperglycaemia [ , ] . in stroke-prone shrs, continuous subcutaneous administration of the hrp inhibited activation of the tissue ras without affecting the circulating ras or arterial pressure and significantly attenuated the development and progression of proteinuria, glomerulosclerosis and cardiac fibrosis [ , ] . infusion of the hrp in human (p)rr transgenic rats significantly inhibited the development of glomerulosclerosis, proteinuria, mitogen-activated protein kinase activation and transforming growth factor-␤ expression in the kidneys [ ] . future research should be able to determine to what degree the beneficial in vivo effects of hrp are due to prorenin blockade [ ] and are related to interference with renin binding and the ras [ ] . decoy effects of the hrp, however, were not confirmed by other research groups [ ] [ ] [ ] [ ] . ace inhibitors and at receptor blockers (arbs) are proven to be effective therapeutic agents in the treatment of cvd. however, both ace inhibitors and arbs lead to a substantial compensatory rise in circulating active renin and ang peptides that may eventually limit their therapeutic potential [ ] . moreover, the increased ang i seen with ace inhibitors can subsequently be converted to ang ii by non-ace pathways, mediated by chymase and chymotrypsinlike ang-generating enzyme [ ] . in addition to the side effects of ace inhibitors, such as cough and angioedema, a meta-analysis of randomized controlled trials in suggested that arbs are associated with a modestly increased risk of new cancer diagnosis, although conclusions about the exact risk of cancer associated with each particular drug have not been drawn [ ] . based on this study, the united states food and drug administration is conducting a review to assess the possible link between the use of arbs and cancer. therefore, direct renin inhibition may be an alternative pharmacological approach to ras inhibition. renin is the rate-limiting step of the ras and the concept of blocking the ras at its origin by inhibiting renin has existed for at least years [ ] . the first-generation renin inhibitors were peptide analogues of agt and the secondgeneration compounds were peptidomimetic agents that are dipeptide transition-state analogue inhibitors of the active site. but these renin inhibitors had limited clinical use due to poor metabolic stability and oral bioavailability, short duration of action, weak antihypertensive activity and high cost of synthesis [ ] . a combination of molecular modelling and x-ray crystallographic analysis of the active site of renin led to the development of aliskiren, a new non-peptide low-molecular-weight orally-active renin inhibitor. aliskiren has a high binding affinity for renin and appears to bind to both the hydrophobic s -/s -binding pocket and a large, distinct subpocket that extends from the s -binding site toward the hydrophobic core of the enzyme. aliskiren is a potent competitive inhibitor of renin but very poorly inhibits related aspartic peptidases. it shows no effects on cytochrome p isoenzyme activities and is not bound extensively to blood proteins, therefore having a low potential for drug interactions [ ] . compared with the earlier renin inhibitors, aliskiren is more resistant to intestinal degradation and possesses significantly improved oral bioavailability. the terminal half-life ranges from to hours, which makes it suitable for once-daily dosing. aliskiren monotherapy displays an antihypertensive efficacy similar if not superior to that of other first-line antihypertensive drugs, with a safety and tolerability profile similar to that of arbs. combined with various antihypertensive agents, aliskiren exhibits synergistic effects [ ] . from , aliskiren was approved by regulatory bodies in both europe and the united states, for use alone and with other agents in the treatment of hypertension [ ] . recent studies showed that aliskiren treatment also markedly increased the plasma concentration of (pro)renin in patients and failed to inhibit the non-catalytic effects of (pro)renin [ , ] . the pharmaceutical company novartis reported that the addition of aliskiren to standard therapy for patients recovering from a heart attack did not provide the anticipated effect of limiting adverse changes to the heart's left ventricle. in december , novartis announced the early termination of the aliskiren trial in type diabetes using cardio-renal endpoints (altitude) and advised that aliskiren should not be used in combination with ace inhibitors or arbs in patients with diabetes. the study was conducted in type diabetic patients at high risk of fatal and non-fatal cardiovascular and renal events; aliskiren mg was given in addition to an ace inhibitor or an arb. the overseeing data monitoring committee concluded that study patients were unlikely to benefit from aliskiren and that there was a higher incidence of adverse events related to non-fatal stroke, renal complications, hyperkalaemia and hypotension in this highrisk population after - months of combined therapy (www.novartis.com). aliskiren has been shown to be a very safe antihypertensive after more than , patient-years of data. however, as we have seen in ongoing telmisartan alone and in combination with ramipril global endpoint trial (ontarget) that the combination of telmisartan (an arb) and ramipril (an ace inhibitor) is associated with more adverse events without an increase in benefit in patients with vascular disease or high-risk diabetes without heart failure [ ] , the combination of multiple safe drugs in this class may no longer be a promising strategy. over the last decade, a number of food-derived compounds have been shown to have in vitro ace inhibitory activity. some of them display significant antihypertensive activity in rats and humans. among these compounds, foodderived ace inhibitory peptides have been most widely studied [ , ] . a quantitative structure-activity relationship study indicated that a potent ace inhibitory dipeptide should have a large and hydrophobic amino acid at the c-terminus and a non-polar amino acid or possibly a positively charged amino acid at the n-terminus. for tripeptides, the most favourable residues for the c-terminus are aromatic amino acids, while hydrophobic amino acids are preferred for the n-terminus and positively charged amino acids are preferred for the middle position [ ] . the in vitro half-maximal inhibitory concentration (ic ) values of food-derived ace inhibitory peptides are about fold higher than that of synthetic captopril but they have higher in vivo activities than would be expected from their in vitro activities. it has been suggested that food-derived peptides might act via different antihypertensive mechanisms, possess higher affinities for tissues and be more slowly eliminated than synthetic captopril [ ] . until now, only two lactotripeptides (vpp and ipp) have been successfully commercialized. data from van mierlo et al., however, did not support a blood pressure-lowering effect of the two tripeptides in caucasians [ ] . given the discrepancy between in vivo and in vitro results, further investigation into the in vivo and clinical antihypertensive effects and mechanisms of food-derived ace inhibitors is necessary. three dipeptides (ir, kf and ef) in pea protein hydrolate have been identified as inhibiting renin activity in vitro [ ] . oleic acid and linoleic acid isolated from rice and some cereals also have in vitro renin inhibitory activity [ , ] . a series of studies have shown that saponins from different food/plant sources, primarily from soybean, can inhibit in vitro renin activity [ ] [ ] [ ] [ ] ; among them, orally administered soybean saponin can lead to a reduction of blood pressure in shrs but the direct evidence for saponin suppressing plasma renin activity in vivo is still lacking [ ] . experiments are required to further investigate the biochemical and biological properties of these plant/food-derived non-peptides that are related to renin inhibition. significant conceptual progress made in the last few years leads us to conclude that ace could serve as a new direction for improved therapeutics for cardiovascular disease. compared with ace inhibitor therapy, ace is an endogenous regulator of the ras. targeting ace would not only produce the vasoprotective/antiproliferative peptide ang-( - ) but would also influence the vasoconstrictive/proliferative effects of the ace/ang ii/at axis. as a part of the vasoprotective/antiproliferative axis, ace can effectively control fibrosis and structural remodelling and is extremely beneficial for pulmonary hypertension. ace activation may provide improved protection and reversal of ischaemia-induced neural damage. thus, direct activation of ace could result in better outcomes in cardiovascular disease. additionally, ace is a multifunctional enzyme with many biologically active substrates. the effects of ace on substrates other than ang i and ang ii may hold relevance for the treatment of cardiovascular disease [ ] . xnt analogues exist in many natural plant resources and have shown various physiological functions. using these resources to search for in vitro ace activators would be a good starting point for developing plant food-derived ace activating agents. ongoing experiments would characterize these ace activators. compared with ras regulatory drugs, these plant/food-derived bioactives appear more natural and safer to the consumer. as part of a daily diet, bioactives from food sources may result in a much lower healthcare cost. plant/food-derived ras regulators could be applied in the prevention of hypertension and as initial treatment in mildly hypertensive individuals [ ] . the authors declare that they have no conflicts of interest concerning this article. renin-angiotensin system revisited physiology and pharmacology of the (pro)renin receptor an ace in the hole alternative pathways of the renin angiotensin system and their potential role in cardiac remodeling a novel angiotensinconverting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase trilogy of ace : a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters prorenin and (pro)renin receptor: a review of available data 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design major congenital malformations after first-trimester exposure to ace inhibitors roles of the two active sites of somatic angiotensin-converting enzyme in the cleavage of angiotensin i and bradykinin: insights from selective inhibitors d-qsar studies on angiotensinconverting enzyme (ace) inhibitors: a molecular design in hypertensive agents rxp , a selective inhibitor of the n-domain of angiotensin i-converting enzyme, blocks in vivo the degradation of hemoregulatory peptide acetyl-ser-asp-lys-pro with no effect on angiotensin i hydrolysis ectodomain shedding of angiotensin converting enzyme in human airway epithelia tumor necrosis factoralpha convertase (adam ) mediates regulated ectodomain shedding of the severe-acute respiratory syndromecoronavirus (sars-cov) receptor, angiotensin-converting enzyme- (ace ) ace x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis kidney amino acid transport hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase residues affecting the chloride regulation and substrate selectivity of the angiotensin-converting enzymes (ace and ace ) identified by site-directed mutagenesis therapeutic implications of the vasoprotective axis of the reninangiotensin system in cardiovascular diseases transgenic angiotensinconverting enzyme overexpression in vessels of shrsp rats reduces blood pressure and improves endothelial function targeting the degradation of angiotensin ii with recombinant angiotensin-converting enzyme : prevention of angiotensin ii-dependent hypertension correlation of angiotensin-converting enzyme gene polymorphisms with stage hypertension in han chinese angiotensin converting enzyme- confers endothelial protection and attenuates atherosclerosis genetic ace deficiency accentuates vascular inflammation and atherosclerosis in the apoe knockout mouse candesartan ameliorates cardiac dysfunction observed in angiotensin-converting enzyme -deficient mice loss of angiotensin-converting enzyme accelerates maladaptive left ventricular remodeling in response to myocardial infarction the angiotensin-( - )/mas receptor axis is expressed in sinoatrial node cells of rats association of angiotensinconverting enzyme (ace ) gene polymorphisms with parameters of left ventricular hypertrophy in men. results of the monica augsburg echocardiographic substudy detection of soluble angiotensin-converting enzyme in heart failure: insights into the endogenous counter-regulatory pathway of the renin-angiotensin-aldosterone system loss of angiotensinconverting enzyme- leads to the late development of angiotensin ii-dependent glomerulosclerosis ace inhibition worsens glomerular injury in association with increased ace expression in streptozotocin-induced diabetic mice chronic treatment with angiotensin-( - ) improves renal endothelial dysfunction in apolipoproteine-deficient mice genetic deletion of the angiotensin-( - ) receptor mas leads to glomerular hyperfiltration and microalbuminuria overexpression of angiotensin-converting enzyme in the rostral ventrolateral medulla causes long-term decrease in blood pressure in the spontaneously hypertensive rats gene transfer of angiotensin-converting enzyme in the nucleus tractus solitarius improves baroreceptor heart rate reflex in spontaneously hypertensive rats angiotensin-converting enzyme overexpression in the subfornical organ prevents the angiotensin ii-mediated pressor and drinking responses and is associated with angiotensin ii type receptor downregulation cerebroprotection by angiotensin-( - ) in endothelin- -induced ischaemic stroke ace , a promising therapeutic target for pulmonary hypertension structure-based identification of small-molecule angiotensinconverting enzyme activators as novel antihypertensive agents angiotensin-converting enzyme activation protects against hypertension-induced cardiac fibrosis involving extracellular signal-regulated kinases ace activation promotes antithrombotic activity evidence for angiotensin-converting enzyme as a therapeutic target for the prevention of pulmonary hypertension diminazene aceturate is an ace activator and a novel antihypertensive drug structure of sars coronavirus spike receptor-binding domain complexed with receptor modulation of tnf-alpha-converting enzyme by the spike protein of sars-cov and ace induces tnf-alpha production and facilitates viral entry structure-based discovery of a novel angiotensin-converting enzyme inhibitor a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury differential downregulation of ace by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl tissue-specific amino acid transporter partners ace and collectrin differentially interact with hartnup mutations physiology of kidney renin prorenin and the putative (pro)renin receptor inhibition of diabetic nephropathy by a decoy peptide corresponding to the ''handle'' region for nonproteolytic activation of prorenin prorenin and the (pro)renin receptor-an update pivotal role of the renin/prorenin receptor in angiotensin ii production and cellular responses to renin the (pro)renin receptor: a new addition to the renin-angiotensin system? human prorenin has ''gate and handle'' regions for its non-proteolytic activation binding properties of rat prorenin and renin to the recombinant rat renin/prorenin receptor prepared by a baculovirus expression system renin-stimulated tgf-beta expression is regulated by a mitogen-activated protein kinase in mesangial cells renin increases mesangial cell transforming growth factor-beta and matrix proteins through receptor-mediated, angiotensin ii-independent mechanisms prorenin induces intracellular signaling in cardiomyocytes independently of angiotensin ii prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin ii type a receptor-deficient mice regression of nephropathy developed in diabetes by (pro)renin receptor blockade contribution of nonproteolytically activated prorenin in glomeruli to hypertensive renal damage nonproteolytic activation of prorenin contributes to development of cardiac fibrosis in genetic hypertension slowly progressive, angiotensin ii-independent glomerulosclerosis in human (pro)renin receptor-transgenic rats prorenin is the endogenous agonist of the (pro)renin receptor. binding kinetics of renin and prorenin in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor prorenin and renininduced extracellular signal-regulated kinase / activation in monocytes is not blocked by aliskiren or the handle-region peptide the putative (pro)renin receptor blocker hrp fails to prevent (pro)renin signaling pro)renin receptor peptide inhibitor ''handle-region'' peptide does not affect hypertensive nephrosclerosis in goldblatt rats aliskiren: a new renin inhibitor as antihypertensive new class of agents for treatment of hypertension: focus on direct renin inhibition angiotensinreceptor blockade and risk of cancer: meta-analysis of randomised controlled trials prorenin engages the (pro)renin receptor like renin and both ligand activities are unopposed by aliskiren telmisartan, ramipril, or both in patients at high risk for vascular events plant food-derived angiotensin i converting enzyme inhibitory peptides structural requirements of angiotensin i-converting enzyme inhibitory peptides: quantitative structure-activity relationship study of di-and tripeptides lactotripeptides do not lower ambulatory blood pressure in untreated whites: results from controlled multicenter crossover studies identification and inhibitory properties of multifunctional peptides from pea protein hydrolysate the occurrence of renin inhibitor in rice: isolation, identification, and structure-function relationship renin inhibitory activity in rice and cereals reduction of blood pressure by soybean saponins, renin inhibitors from soybean, in spontaneously hypertensive rats inhibition of human renin activity by saponins human renin inhibitory activity in legumes isolation of human renin inhibitor from soybean: soyasaponin i is the novel human renin inhibitor in soybean this work was supported partly by fundamental research funds for the central universities (jusrp a ) and the self-determined research program of jiangnan university (no. & no. ). key: cord- -ei viq authors: sarzani, riccardo; giulietti, federico; di pentima, chiara; filipponi, andrea; spannella, francesco title: antagonizing the renin–angiotensin–aldosterone system in the era of covid- date: - - journal: intern emerg med doi: . /s - - - sha: doc_id: cord_uid: ei viq nan levels [ ] . ace plays a key role in counterbalancing the negative effects of a hyper-activated raas. indeed, ace cleaves angiotensin (ang) i into a nonapeptide (ang - ), which binds ang ii type receptor (at r), and ang ii into ang - , which binds an endogenous orphan receptor (masr). while the activation of ace/ang ii/ang ii-type receptor (at r) pathway induces vascular permeability, inflammation, and lung fibrosis, previous studies found that ace /ang - /masr pathway can protect lungs from the development of acute respiratory distress syndrome (ards) in several animal models, through opposite mechanisms [ ] . moreover, ace interacts with another branch of raas based on ang peptides in which the aminoterminal aspartate is replaced by alanine (alatensins), leading to the production of ala-ang - (alamandine) that has been found to bind mas-related g protein-coupled receptor d (mrgd) and may also protect against lung injury and fibrosis, improving vascular/endothelial dysfunction [ ] . the down-regulation of ace after the binding of the viral surface-spike protein and the consequent raas hyperactivation result in the worsening of acute lung injury. moreover, ace and the raas dysregulation may also play a key role in the myocardial involvement following the sars-cov- infection. in fact, ace is critical for heart function, preventing oxidative stress, inflammation, left ventricular remodeling, and dysfunction [ ] . raas blockers, especially arb, may attenuate these damage mechanisms (see fig. ), through the reduction of ang ii/at r stimulation, increase in ang ii substrate and increase in ace , leading to a larger increase in both ang - and alamandine. ace-i stop the conversion of ang - to ang - and may facilitate stimulation of at r by ang - , but may also reduce the pathway depending on ang - . therefore, most experimental evidences are currently favoring the use of arb in lung protection. very recently, some clinical studies evaluated the effects of ace-i and arb on covid- outcomes in hospitalized patients, although limited to observational data. a retrospective analysis of chinese hypertensive patients showed significant lower risk of all-cause mortality in those treated with ace-i/arb compared to other anti-hypertensive drugs after adjustment for confounders through propensity score-matched analysis [ ] . same favorable results have been found in a small uk cohort study on patients admitted for covid- , in which treatment with ace-i was associated with a reduced risk of rapidly deteriorating severe disease [pre-print] . in another small sample of covid- patients, ace-i and arb therapy affected both il- and peripheral t cell levels and was associated with lower rates of severe disease [ ] . in another study on hospitalized covid- patients, the percentage of hypertensive patients taking ace-i/arb did not differ between those with severe and non-severe infection or between non-survivors and survivors, with a favorable trend for ace-i/arb, although adjusted analysis was not performed [ ] . randomized controlled trials (rcts) with losartan are ongoing to study its possible benefits for covid- patients (nct ; nct ), based on the above-mentioned mechanisms that have been hypothesized. despite these evidences favoring raas blockers, the speculations based on ace induction forced the major cv scientific societies to intervene with position statements to avoid the de-prescribing of these drug classes. further studies are needed to overcome the current lack of evidence either for or against the use of raas inhibitors as part of the treatment of covid- . high-resolution chest computed tomography (hr-ct) well reflects the evolution of pathological lung changes of covid- [ ] . indeed, several subsequent temporal stages have been identified, starting from an early stage characterized by multilobar ground-glass opacities (ggo) with a peripheral or posterior distribution, up to an increase in the number and size of ggo and progressive transformation into multifocal fig. schematic of the protective role of ace and counter-regulatory renin-angiotensin-aldosterone system (raas) in lung injuries potentially leading to ards. arb therapy, in animal models, counterbalances the down-regulation of ace , like the one caused by the sars-cov- infection in lung. arb could lead to an increase in the protective components of the raas (by reduction of ang ii/at r stimulation, increase in ang ii substrate, increase in ang ii and ang a conversion in ang - and alamandine, respectively) with poten-tial prevention and/or attenuation of ards. ace : angiotensinconverting-enzyme ; ang: angiotensin; arb: angiotensin ii type receptor blockers; at r: angiotensin ii type receptor; at r: angiotensin ii type receptor; sars-cov- : severe acute respiratory syndrome coronavirus- ; masr: mas receptor; mrgd: mas-related g protein-coupled receptor d; ad: aspartate decarboxylase; ards: acute respiratory distress syndrome consolidative opacities, septal thickening, and development of a crazy paving pattern, which clinically corresponds to the development of ards [ ] . it might be interesting to evaluate the effect of an arb, such as losartan that has already proven to be protective against acute lung injury in mice [ ] , on the disease progression. being the older patients with cv comorbidities the most severely hit by the disease, it would be worthwhile to study patients aged ≥ years affected by covid- . hypertensive older patients could be randomized to losartan - mg once daily versus an anti-hypertensive drug other than a raas blocker (i.e., a calcium channel blocker at adequate dosage), while normotensive older patients with a history of hypertension and increased levels of circulating natriuretic peptides (i.e., nt-probnp ≥ pg/ml, suggestive for heart failure) could be randomized to losartan . - mg once daily versus placebo. patients would be enrolled and randomized after performing chest hr-ct on admission to identify the early stage of the disease. the primary outcome would be the search for differences in lung disease progression rates, evaluated with chest hr-ct after - days. secondary outcomes would be the search for differences in the incidence of cv complications. these projects could shed light on the putative beneficial effect of arb therapy in humans with acute lung injury caused by sars-cov- infection, confirming the previous findings on animal models, thanks to the rebalancing of the raas. in conclusion, up to now, as promptly stated by both the esc council on hypertension and the hfsa/acc/aha, patients with hypertension and its related cv sequelae, mainly ischemic heart disease and heart failure, should safely continue to take ace-i and arb, also in the context of the pandemic covid- , to not further increase their cv risk, although possible specific benefits of raas inhibitors on covid- are still to be demonstrated. funding none. conflict of interest the authors declare that they have no conflict of interest. this article does not contain any studies with human participants or animals performed by any of the authors. informed consent for this type of study, formal consent is not required. renin-angiotensin system blockers and statins are associated with lower in-hospital mortality in very elderly hypertensives losartan prevents sepsis-induced acute lung injury and decreases activation of nuclear factor kappab and mitogen-activated protein kinases angiotensin-converting enzyme protects from severe acute lung failure the renin-angiotensin system: going beyond the classical paradigms association of inpatient use of angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid- renin-angiotensin system inhibitors improve the clinical outcomes of covid- patients with hypertension association of renin-angiotensin system inhibitors with severity or risk of death in patients with hypertension hospitalized for coronavirus disease (covid- ) infection in wuhan coronavirus disease (covid- ): a systematic review of imaging findings in patients key: cord- -glb y u authors: domingo, pere; mur, isabel; pomar, virginia; corominas, héctor; casademont, jordi; de benito, natividad title: the four horsemen of a viral apocalypse: the pathogenesis of sars-cov- infection (covid- ) date: - - journal: ebiomedicine doi: . /j.ebiom. . sha: doc_id: cord_uid: glb y u the pathogenesis of coronavirus disease (covid- ) may be envisaged as the dynamic interaction between four vicious feedback loops chained or happening at once. these are the viral loop, the hyperinflammatory loop, the non-canonical renin-angiotensin system (ras) axis loop, and the hypercoagulation loop. severe acute respiratory syndrome (sars)-coronavirus (cov)- lights the wick by infecting alveolar epithelial cells (aecs) and downregulating the angiotensin converting enzyme- (ace )/angiotensin (ang- – )/mas r axis. the viral feedback loop includes evading the host's innate response, uncontrolled viral replication, and turning on a hyperactive adaptative immune response. the inflammatory loop is composed of the exuberant inflammatory response feeding back until exploding in an actual cytokine storm. downregulation of the ace /ang-( – )/mas r axis leaves the lung without a critical defense mechanism and turns the scale to the inflammatory side of the ras. the coagulation loop is a hypercoagulable state caused by the interplay between inflammation and coagulation in an endless feedback loop. the result is a hyperinflammatory and hypercoagulable state producing acute immune-mediated lung injury and eventually, adult respiratory distress syndrome. in december , a new epidemic disease appeared in the huanan seafood wholesale market, wuhan, hubei province, china. it was characterized by an upper respiratory tract infection rapidly evolving to bilateral pneumonia and eventually respiratory failure [ ] . the etiologic agent was a new coronavirus which was named sars-cov- , whereas the disease was called covid- [ ] . the disease quickly expanded from its original nucleus in hubei and by march , the who declared it as a pandemic. as of june , , covid- has affected countries around the world, with . . confirmed cases worldwide and a death toll of . [ ] . early in the course of the pandemic, clinicians and researchers realized that full-blown covid- evolved in at least three phases: the first phase with cough, fever, wheezing, fatigue, headache, diarrhea, and dyspnea, reminiscent of an upper tract respiratory infection. the second phase, with the rapid appearance of bilateral pneumonia, infiltrates with variable degrees of hypoxemia, and omit in the third phase in which some patients developed respiratory failure leading to death [ ] . around % of people have sars-cov- infection asymptomatic or with mild to moderate illness, mostly restricted to the upper and conducting airways. the other % will develop symptomatic infection needing hospital admission, and % will require ventilatory support in the intensive care unit (icu) [ ] . the clinical phases of the infection reflect the pathogenic events starting with the virus gaining access to the lungs. the clinical manifestations and pathogenic events of any infectious disease, and covid- in particular, should be viewed in the light of the damageresponse framework in which several factors and forces may tip the scales to the host or pathogen side [ ] . therefore, sometimes the . the first horseman: a sneaky virus sars-cov- is a previously unknown b-coronavirus which shows % identity to the sequences of two bat-derived sars-like coronaviruses, . % identity to sars-cov, and about % identity to middle east respiratory syndrome (mers)-cov [ ] . the genome of sars-cov- is a positive-sense, single-stranded rna with a size of . kb, containing at least ten open reading frames (orfs) [ ] . recently, noncanonical orfs and at least rna modifications with an unknown function, were identified [ ] . the first orfs represent two-thirds of the viral rna. they are translated into two large polyproteins, which are later processed into non-structural proteins (nsp to nsp ) that form the viral complex replicase-transcriptase [ ] . these nsps rearrange endoplasmic reticulum into double-membrane vesicles, where viral replication and transcription take place [ , ] . the other third of the genome encodes four main structural proteins; spike (s), envelope (e), nucleocapsid (n), and membrane (m) proteins, and several accessory proteins whose functions are currently unknown but unrelated to viral replication [ ] . sars-cov- , like sars-cov, requires the ace as a receptor to enter the cells [ , ] . coronavirus s protein is a determinant of virus entry into host cells by binding the envelope spike glycoprotein to its cellular receptor ace [ , ] . although it was initially thought that sars-cov achieved entry by membrane fusion, a critical proteolytic cleavage at sars-cov s protein, mediated by type ii transmembrane serine protease (tmprss ), brings about membrane fusion and viral infectivity [ ] . after the virus entry, the rna genome is released into the cytoplasm and translated into two polyproteins and structural proteins [ ] . the survival of sars-cov in host cells is eased by strategies to evade the immune response. the evolutionarily conserved microbial structures called pathogen-associated molecular patterns (pamps) are recognized by pattern recognition receptors (prrs) such as tolllike receptor (tlrs), retinoic acid-inducible gene-i (rig-i)-like receptors, nucleotide-binding oligomerization domain (nod)-like receptors, and c-type lectin-like receptors [ ] . sars-cov induces the signal transducer and activator of transcription tace tnf-a converting enzyme tbk tank-binding kinase tlr toll-like receptor tmprss type ii transmembrane serine protease tnf-a tumor necrosis alpha traf tnf receptor-associated factor xcr xcl (chemokine [c motif] ligand ) and xcl (chemokine [c motif] ligand ) receptor production of double-membrane vesicles that lack prrs and can then replicate in these vesicles [ ] . furthermore, several structural and nsps encoded by sars-cov and mers-cov antagonize antiviral innate immune response. interferon (ifn) and interferon-stimulated genes (isgs) responses are counteracted by nsp , nsp macrodomain, nsps-deubiquitinase, and orf b, orf , and orf , thus overthrowing antiviral response [ ] [ ] [ ] [ ] [ ] [ ] . nsp inhibits ifn responses by three mechanisms, inactivation of host translational machinery, degradation of host mrnas, and inhibiting signal transducer and activator of transcription (stat ) phosphorylation [ , ] . part of the nsp is a papain-like protease that antagonizes ifn and cytokine production by blocking phosphorylation of ifn regulation factor (irf ) and disrupting nf-kb signaling [ ] . nsp and nsp are also ifn antagonists by an unknown mechanism [ ] . orf b exerts ifn antagonism through inhibition of ifnb induction by transcription factors irf and nf-kb, whereas orf antagonizes ifn by inhibiting signaling through the jak-stat pathway [ ] . m and n proteins flatten ifn signaling by inhibiting tank-binding kinase (tbk )/ikb kinase e (ikke), and the negative regulation of traf / -tbk -irf /nf-kb/ ap signals [ , ] . antagonism of ifn responses further promotes free virus replication resulting in increased viral pamps and damps that additionally dampen ifn signaling and stimulate prrs to induce an aberrant inflammatory response. the replicative capacity of sars-cov- is . folds more than that of sars cov in infected human lung tissue without significantly inducing types i, ii, and iii ifns [ ] . since innate immunity is the frontline defense against sars-cov- , a slow and poorly coordinated response may result in higher viral replication. this sequence of events, namely aecs infection, ifn signaling inhibition, and free viral replication depicts the viral vicious loop (fig. ). . the second horseman: a gathering storm sars-cov- infects primarily airway and alveolar aecs, especially type ii pneumocytes, the cells that produce alveolar surfactant and are predecessors of type i pneumocytes. however, it can infect any cell expressing the receptor ace , such as endothelial cells, pericytes, vascular smooth muscle cells, macrophages, fibroblasts, t-cells, cardiomyocytes, enterocytes, basal cell epidermal cells, and epithelial tubular distal cells [ ] [ ] [ ] . sars-cov- , in the face of unchecked replication because of dampened innate immunity, can replicate in high titres early after the infection [ , , ] . high viral replication in aecs induces cytopathic effects, as shown by the necropsy findings of multinucleated cells (syncytia), cytoplasmic viral inclusions, and apoptosis, an ultimate cellular response to stop virus replication [ , ] . these events are followed by the production of increased levels of proinflammatory cytokines and chemokines by aecs [ , ] . moreover, sars-cov nucleocapsid activates interleukin- (il- ) expression in lung epithelial cells via cellular transportation of nuclear factor kappa b (nf-kb) [ ] . massive infiltration of inflammatory cells into the lungs is, in turn, mounted by these cytokines and chemokines [ ] (fig. ) . although tissue-resident macrophages of the lungs localize to the airspace within alveoli, they do not seem to be the predominant subset in this response [ ] . accumulation of inflammatory monocyte-macrophages and neutrophils in the lungs following sars-cov- infection promotes the additional release of cytokines and chemokines [ ] (fig. ) . besides, the sars-cov spike promotes the upregulation of il- and tumor necrosis alpha (tnf-a) in macrophages [ ] . cytokines spill over from local inflammation to the systemic circulation. covid- patients have high serum levels of inflammatory cytokines, including interleukin (il)- , il- , il- , granulocyte-colony stimulating factor (g-csf), interferon gamma-induced protein (ip)- , monocyte chemoattractant protein (mcp)- , macrophage sars-cov- infects primarily type ii pneumocytes through binding to the ace receptor. the infected and surrounding pneumocytes secret cytokine and chemokines, which attract monocyte-macrophages and neutrophils to the alveolar space, which secrete additional cytokines and chemokines. ultimately the pneumocytes suffer apoptosis/pyroptosis releasing large amounts of proinflammatory factors. endothelial cells are infected, overexpress adhesion molecules, and release chemokines and cytokines. endothelial cells undergo apoptosis, which, together with alveolar cell apoptosis, increases vascular leakage and breaks the alveolar-capillary barrier. the hyperinflammatory milieu and endothelial dysfunction activate coagulation cascades through tissue factor expression, platelet activation, and netosis all of them promoting microcirculatory thrombi formation. the break of endothelial-alveolar barrier further promotes vascular leakage resulting in interstitial and alveolar space flooding. downregulation of the ace /ang-( À )/mas r axis contributes to increasing vasoconstriction, inflammatory signals, endothelial dysfunction, vascular leakage, and prothrombotic state. sars-cov- = severe acute respiratory syndrome coronavirus ; tnf-a = tumor necrosis factor alpha; il- = interleukin ; mcp- = macrophage chemoattractant protein ; mip- a = macrophage inhibitory protein a; il- = interleukin ; il- b = interleukin beta; nf-kb = nuclear factor kappa b. inflammatory protein (mip)- a, and tnf-a. these cytokine/chemokine levels correlate with disease severity [ , ] . covid- patients with severe disease have increased levels of il- more often than those with the mild or moderate disease [ ] . although viremia is not a prominent feature in covid- and is usually short-lived, the degree and duration of sars-cov- viremia relates to the severity of disease and the serum levels of il- [ ] . endothelial cells (ecs) are infected very early in the course of infection. because of speedy viral replication and exuberant proinflammatory cytokine/chemokine response they may suffer apoptosis [ , ] . this apoptotic phenomenon takes place via fas/fasl or trail-dr- -dependent mechanisms [ ] . besides, inflammatory monocyte-macrophages release tnf-a which also promotes apoptosis of both lung ecs and aecs [ ] . ecs and aecs apoptosis compromise lung microvascular bed and alveolar cell-capillary barrier integrity, thereby resulting in vascular leakage and alveolar edema [ ] (fig. ) . pericytes play an essential role in maintaining endothelial cell function in capillary vessels and are among the cells with the highest ace expression. their infection by sars-cov- may add to endothelial cell dysfunction leading to microcirculation disorders [ ] . a striking feature of full-blown covid- is severe lymphopenia. cov-specific t-cells are decisive for viral clearance and limitation of additional damage to host tissues since they can dampen hyperreactive innate immune response [ , ] . however, when exuberant inflammatory response induced by sars-cov- takes place, t-cell response is decreased because of tnf-a-mediated cell apoptosis, thus resulting in uncontrolled inflammatory responses [ ] (fig. ) . besides, normal t-cell activation can be suppressed by il- , further contributing to lymphopenia [ ] . in severe covid- patients with lymphocyte subsets examined, there is intense cd and particularly cd lymphopenia [ ] , both negatively correlating with tnf-a and il- serum levels [ ] . cd cells promote the production of virus-specific antibodies by activating t-dependent b cells, whereas cd cells are cytotoxic and can kill virus-infected cells. since cd cells account for about % of the total inflammatory lung interstitial infiltrate, highly cytotoxic cd lymphocytes can arbitrate immune-mediated tissue damage [ ] . also, in covid- patients, these cells exhibit markers of functionally exhausted t-cells such as programmed cell death protein . there is upregulation of apoptosis, autophagy, and p pathways in pbmc of covid- patients [ ] . mers-cov can induce t-cell apoptosis through activation of the intrinsic and extrinsic apoptosis pathways [ ] , and sars-cov e protein can also promote t-cell apoptosis mediated by cbl-xl binding [ ] . although sars-cov- can non-productively infect t lymphocytes, whether this infection induces t-cell apoptosis is not yet clear [ ] . alternatively, pyroptosis has been suggested as a cause of lymphopenia since covid- patients have increased serum il -b levels, which is the downstream indicator of cell pyroptosis [ ] . in sars-cov infection, viroporin a triggers the activation of nod-like receptor protein (nlrp ) inflammasome and the secretion of il- -b by macrophages [ ] . pyroptosis can release large amounts of proinflammatory factors [ ] . whatever the cause, during the late stages of infection, depletion of t-cells may promote viral survival and, consequently, may prolong the infection. essential to control the persistent phase of sars-cov- infection is the appearance of humoral immunity, in which antiviral neutralizing antibodies play a significant role. however, in animal models, anti-s protein-neutralizing antibodies (anti-s-igg) may cause severe lung injury by altering inflammatory responses [ ] . in sars-cov infection, the development of acute respiratory disease coincides with antiviral igg seroconversion in % of patients [ ] and patients who died developed anti-s-neutralizing antibodies faster [ ] . the presence of anti-s-igg promotes proinflammatory monocyte-macrophage lung accumulation and the production of mcp- and il- . such proinflammatory cytokine release would be mediated through the binding of the virus-anti-s-igg complex to the monocytes-macrophages fcgriia receptor since its blockade reduces the production of ifn-g, tnf-a, il- , and il- [ ] . it would also be possible that such complexes activate the classical pathway of the complement system or induce antibody-dependent cell-mediated cytotoxicity, thus leading to cellular damage. therefore, a possible underlying mechanism would be antibody-dependent enhancement (ade) of viral infection that occurs in some patients with early, sub-optimal antibody activity that cannot completely clear the virus, leading to persistent viral replication and inflammation [ ] . uncontrolled viral replication, because of a delayed innate immunity response, will cause cellular damage leading to an overexuberant and dysregulated immune kickback. this hyper reaction affecting the innate and adaptative immune responses will pave the way for immune-mediated damage of tissues and organs. this sequence of events conforms the inflammatory hurtful feedback loop (fig. ). the ras plays a critical role in the control of cardiovascular and renal functions by maintaining blood pressure homeostasis and hydro-electrolyte balance [ ] . initially, the ras was conceived as a linear hormonal system in which angiotensinogen synthesized in the liver is converted into the active peptide angiotensin i (ang i) through the action of renin [ ] . afterward, ang i is cleaved by the ace generating ang ii [ ] . two g protein-coupled receptors (gpcr) mediate the actions of ang ii, angiotensin ii receptor type (at r), and type (at r) [ ] . the primary role of this canonical or classical ras pathway (ace/ang ii/at r) is to increase the sympathetic nervous system tension, to cause vasoconstriction, increase blood pressure, and promote inflammation, fibrosis and myocardial hypertrophy [ ] . the ras also possesses a non-canonical, counter-regulatory branch composed of ace /ang-( À )/mas r. the activity of the system will depend on the balance between the two branches. ace is the main synthesizer of ang-( À ) by removing a single residue from ang i to generate ang-( À ) and by cleaving a single residue from ang ii to generate ang-( À ) [ , ] . the functional receptor for ang-( À ) is the gpcr mas r [ ] . the conformation of the negative or counter-regulatory axis is relevant not only because it downgrades the vasoconstrictive/ proliferative peptide ang ii to form the vasodilator heptapeptide ang-( À ), but also because it degrades ang i to ang-( À ), thereby limiting the availability of the substrate for ace. ang-( À ) binds to mas r, inducing vasodilation, inhibition of cell growth, anti-thrombotic, and anti-arrhythmogenic effects [ ] . ace activity is controlled by a disintegrin and metalloproteinase domaincontaining protein (adam- , also called tnf-a-converting enzyme, tace). adam- proteolytically cleaves ace causing the shedding of ace into the interstitium, which leads to decreased ace activity in the tissue and elevates circulating ace activity [ ] (fig. ) . since blood and urine measurement of ace levels is feasible, they could potentially be used as a prognostic biomarker in covid- [ ] . ras plays an essential role in the pathogenesis of inflammatory diseases in which most of the proinflammatory actions are caused by ang ii [ ] . ang ii activates several cellular functions and molecular signaling pathways related to tissue injury, inflammation, and fibrosis. they involve calcium mobilization, free radical generation, activation of protein kinases and nuclear transcription factors, recruitment of inflammatory cells, adhesion of monocyte and neutrophils to endothelial and mesangial cells, upregulation of adhesion molecules and stimulation of expression, synthesis, and release of cytokines and chemokines [ ] . at r mediates most of these actions [ ] . the counterregulatory ace /ang-( À )/mas r axis negatively modulates leukocyte migration, cytokine expression and release, and fibrogenesis pathways. hence, ace deficiency increases vascular inflammation by increasing the gene expression of vascular adhesion molecules, cytokines, chemokines, and matrix metalloproteases [ ] . the loss of ace results in higher increases in ang ii-induced expression of inflammatory factors, enhanced vascular permeability, increased lung edema, and neutrophil accumulation [ ] . the ace / ang-( À )/mas r axis also plays an essential role in haemostasis, since it stimulates prostacyclin (pgi ) production and nitric oxide (no) release by ecs and modulates platelet activity which is less adherent having, thus anti-thrombotic activity [ ] (fig. ) . ras exhibits high activity in lung tissue, which is the leading site of ang ii synthesis. ace is a zinc metallopeptidase, type i integral membrane glycoprotein orientated with the n-terminal, and the catalytic site facing the extracellular space [ ] . the union of ace with sars viral spike protein triggers enzyme internalization downregulating activity from the cell surface. once sars-cov binds to its receptor, the abundance on the cell surface, mrna expression, and the enzymatic activity of ace are significantly reduced [ ] . proteolytic shedding of its extracellular domain is a second mechanism for downregulating ace at the cell surface. s protein of sars, once it binds to ace , induces shedding by activating adam (tace) as do bacterial endotoxin and lipopolysaccharide (lps) [ ] (fig. ) . releasing ace from the cell membrane is a critical step in catalyzing substrates and implies that attenuation of ace activity might contribute to disease pathogenesis. the recently described induction of ace expression by type i ifn in human nasal epithelial cells, thus behaving as an isg, highlight an additional mechanism of ace downregulation by sars-cov- [ ] . since ifn-induced isgs are crucial for host antiviral response, the absence of ace induction due to hampered ifn responses will further cause tissue unprotection. therefore, in covid- , ace plays a pivotal role because of its multifaceted task as a facilitator of entry into aecs and its potential role in the pathogenesis of acute lung injury (ali) [ ] . in the mouse model of sars, downregulation of ace protein expression resulted in worse pneumonia, increased ang ii levels, increased vascular permeability, enhanced lung edema, neutrophil infiltration, and further worsened lung function [ , ] . catalytically active ace protein alleviated the symptoms, and active protein improved the outcome of respiratory failure [ ] . in covid- patients, plasma concentrations of ang ii were significantly higher than in healthy individuals and ang ii levels correlated with viral load and lung injury [ ] . owing to the widespread expression of ace , covid- is a disseminated infection. ace is highly expressed in the gut and sars-cov- can productively infect enterocytes [ , ] . despite ace the lung, and other organs, lose the protection of the non-canonical ras system as a result ace downregulation after sars-cov- -induced endocytosis. consequently, the canonical ace/ang ii/at r becomes dominant, levels of ang ii increase with the subsequent promotion of fibrosis, myocardial hypertrophy, increased ros, vasoconstriction, inflammation, and endothelial dysfunction. at r mediates most of these actions. endocytosed sars-cov- spike proteins mediates adam -mediated proteolytic cleavage of ace . adam- activity is enhanced through the activated at r by increased levels of ang ii. tnf-a is the primary substrate of adam . adam cleaves tnf-a releasing soluble tnf-a extracellularly where it has autocrine and paracrine functionalities. activation of tnf-a receptor by tnf-a also enhances adam activity. ace = angiotensin-converting enzyme ; sars-cov- = severe acute respiratory syndrome coronavirus ; ang ii = angiotensin ii; ros = reactive oxygen species; at r = angiotensin receptor; adam = a disintegrin and metalloproteinase domain ; tnf-a = tumor necrosis factor alpha; tmprss = transmembrane protease serine . expression in gut being higher than in the lung, only about À % of patients with covid- experience gastrointestinal symptoms [ ] . the contribution of the digestive system to the pathogenesis of covid- through impairment of mucous membrane barrier and increased inflammatory cytokine production has not been determined yet. similar to mers-cov and owing to ace expression in the brush borders of the proximal tubules and in podocytes, kidney injury in covid- is characterized by diffuse proximal tubule damage with virus-like particles in tubular epithelial cells and podocytes which is indicative of direct sars-cov- infection [ ] . these findings translate clinically into acute kidney injury and proteinuria which affect from . % to % of covid- patients [ ] . the consequence of impaired ace activity in the lung because of sars-cov- infection is a reduction of ang-( À ) production. ang-( À ) binding mas r promotes an array of biological responses to counteract ang ii-mediated processes such as apoptosis, angiogenesis, vasoconstriction, and inflammation in the lung [ , ] . consequently, the attenuation of ace catalytic function perturbs the pulmonary ras balance, resulting in enhanced inflammation and vascular permeability, leaving the lung defenceless in the face of the forthcoming raging cytokine storm. besides, infection of type ii pneumocytes will reduce the production of alveolar surfactant subsequently reducing pulmonary elasticity. moreover, the loss of type ii pneumocytes decreases restoration of type i pneumocytes which ultimately impacts on gas exchange and fibrosis [ ] . the above event sequence depicts the ras vicious feedback loop (fig. ). the association between covid- and coagulation disorders was beheld early during the pandemic when chinese physicians noticed that patients treated mainly with low-molecular-weight heparin had a decreased -day mortality [ ] . this mortality improvement was in patients with a sepsis score higher than four or a markedly elevated d-dimer [ ] . covid- is associated with coagulation disorders that include increases in procoagulant factors such as fibrinogen and d-dimers, both associated with poor prognosis [ , ] . patients admitted to the icu had an increased incidence of venous thromboembolic events ranging from % to % [ ] [ ] [ ] . moreover, standard prophylaxis for venous thromboembolism failed in . % of the patients [ ] . some found that most thrombotic complications were venous and primarily isolated pulmonary embolism, which suggests that it may be primary pulmonary thrombosis instead of embolic phenomena [ , ] . in line with that, microcirculatory thrombosis is a constant finding in lung pathologic studies [ , ] (fig. ) . infection of ecs, together with the derangements caused by cellular infiltration and high exposure to cytokines/chemokines, eventually leads to ecs dysfunction and apoptosis [ ] . all of them contribute to microvascular prothrombotic effects [ ] . there is an intense interplay between haemostasis and innate immunity, called thrombo-inflammation [ ] . both the intrinsic and extrinsic coagulation pathways can activate during inflammation. ecs and macrophages activate the extrinsic pathway through expression of tissue factor [ ] . the intrinsic pathway can be activated by neutrophil extracellular traps (nets) released by polymorphonuclear neutrophils (pmn) in a process called netosis. nets activate ecs, platelets, and the complement system and release proteases that inactivate endogenous anticoagulants [ ] . however, the role of nets in covid- is still a matter of discussion [ ] . platelets play a dual role. first, a proinflammatory role by secreting alpha granules that recruit pmn and macrophages, which are an essential source of il- b [ ] . besides, platelets stimulate pmn to undergo netosis which in turn activates platelets, creating a feedback loop. the second role of platelets is to activate the coagulation pathway by assembling enzyme-cofactor-substrate complexes on their exposed surface [ ] (fig. ) . complement activation, which has been seen in the mouse model of sars, contributes to immune-mediated pathology [ ] . activation of c and c promotes mast cell degranulation and recruitment of pmn and macrophages [ ] . the prothrombotic effects of activated c and c include platelet and ecs activation, together with increasing tissue factor and von willebrand factor expression [ ] . to close the loop, thrombin, and other components of the coagulation cascade can, in turn, activate c and c [ ] . the primary function of thrombin is to promote clot formation by activating platelets and by converting fibrinogen into fibrin [ ] . however, thrombin is a pleiotropic molecule and can increase inflammation via a proteinase-activated receptor (par), principally par- [ ] (fig. ) . the generation of thrombin is controlled by negative feedback loops and physiological anticoagulants such as antithrombin iii, tissue factor pathway inhibitor and the protein c system [ ] . il- b, il- , and tnf-a promote the release of ultra-large von willebrand multimers, and the production of tissue factor and factor vii/activated factor vii, leading to increased thrombin generation while decreasing the levels of endogenous anticoagulants [ ] . the ace /ang-( À )/mas r axis exerts antithrombotic effects through activation of mas r in platelets, which then release no and pgi and by protecting from endothelial dysfunction [ , ] . since this branch of the ras is not working properly in covid- , this protective mechanism is lost ( figs. and ) . in severe covid- , similar to other acute viral infections, a high prevalence od antiphospholipid antibodies was found, although the role of these antibodies in the prothrombotic state of sar-cov- infected patients is still a matter of debate [ ] . the progression of thrombo-inflammation may result in widespread thrombosis, which may be further enhanced by hypoxemia, hyperthermia, and hypovolemia [ ] . hypoxemia triggers increased expression of hypoxia-inducible factors, which may promote additional inflammation and may activate platelets and coagulation factors. they increase tissue factor expression, increase plasminogen activating inhibitor- , and inhibit the endogenous anticoagulant protein s [ ] . in the setting of a hyperinflammatory state and endothelial injury, activation of coagulation occurs whereas the counterregulatory force ace /ang-( À )/mas r axis is inactive, leaving the field to the full expression of a hypercoagulable state. this state may clinically translate into pulmonary thrombosis, venous thromboembolism, or other thrombotic events. if these events affect microvascular lung bed, they may further promote ali and impair gas exchange. whatever the location of the thrombotic event is, it worsens the patient's prognosis. hyperinflammatory state and defective ace /ang-( À )/mas r functioning activate the fourth hurtful feedback loop. hyperinflammation induces hypercoagulation and vice versa, while ace /ang-( À )/mas r axis avoidance maximizes both (fig. ) . the clinical spectrum of covid- is broad. not everyone who acquires sars-cov- becomes sick and the state that emerges after infection can vary among patients or within the same patient over time. consequently, it is envisaged that virus-dependent, hostdependent, and environment-dependent factors may modify the virus-host interaction explaining not only the individual susceptibility to infection but also the broad scale of damage seen in clinical disease. the initial viral titre in the airways could explain the different evolving patterns of covid- , since this will condition the intensity of cytopathic changes, which in turn will shape the strength of immune responses [ ] . sars-cov- replicates in high numbers very early after infection, and in turn, the magnitude of viral replication will impact on the extent of antiviral response [ ] . in humans, there is a strong correlation between sars-cov and mers-cov titres and disease severity [ ] . in animal models, the disease behaves differently if the virus infects airway epithelial cells or both airway and aecs (type i and type ii pneumocytes) predominantly. the viral antigen is mainly located in airway epithelial cells in mouse models permissible to infection, but which do not develop clinical disease. in contrast, in highly susceptible mice, the antigen is detected in both airways and alveolar type i and ii pneumocytes [ ] . consequently, infection of aecs seems critical for both host susceptibility and the development of lung pathology. an aspect that influences sars-cov- infection is the state of differentiation of human airway epithelia, which, in turn, correlates positively with the expression level of ace in these cells [ , ] . it is noteworthy that ace nasal gene expression is lower in children [ ] . this fact is connected to the striking age distribution of covid- in which children are often spared, affecting adults with enhanced severity and mortality as age increases [ , ] . however, the increasingly poor outcome with advancing age is influenced by the presence of common comorbidities, such as hypertension, cardiovascular disease, and diabetes, which bear a poor prognosis by themselves [ ] . besides, these comorbidities relate to a decreased activity of ace in elderly patients, a deficit further exacerbated by sars-cov- infection [ ] . ade is a potentially harmful, pro-inflammatory mechanism which occurs when suboptimal titres of neutralizing antibodies against sars-cov- are present. they are unable to control infection but instead facilitate viral entry into macrophages by a trojan horse mechanism. ade tends to happen when the time interval between coronavirus infections is long enough for antibody fall, which could be a possible mechanism for severe covid- in the aged [ ] . females with covid- usually present with milder disease than males. females exhibit higher ifn and ifn regulator factor and il- production from pbmc, lower production of tnf-a, lower expression of tlr in pmn, lower numbers of nk cells, and lower pmn phagocytic activity than males [ ] . oestrogens downregulate ang ii and upregulate ang-( À ) pathways, which makes apparent gender differences in expression, activity, and tissue responsiveness of ras components [ ] . besides, mas r expression was increased in female rats but not in males after the infusion of ang ii [ ] . in animal models of obesity, females appear to maintain circulating ang-( À ) levels and are protected from hypertension and metabolic complications induced by angiotensinogen, renin, angiotensin ii, and at r activation [ ] . stimulation of counterregulatory at r appears metabolically protective in female rodents, whilst there are inconsistent effects in males [ ] . in the uk, % of covid- patients in the icu were either overweight or obese [ ] , and % of dead north italians had obesity, hypertension, diabetes, heart disease, kidney damage, or cancer [ ] . the frequent occurrence of obesity as a factor of adverse outcome is frequently shadowed by other high prevalent comorbidities in obese people making the identification of the independent role of obesity steep [ ] . the association of covid- with obesity has been attributed to the chronic inflammatory status, the ineffective immune response or to interaction with the ras system. the immune pathways are all susceptible to genetic polymorphisms with functional consequences such as variability in cytokine expression, antigen-binding affinities, receptor ligation strength, and downstream signaling [ , ] . high interconnection is a prominent feature of immune pathways and thus functional resultant polymorphisms may hamper the growth of an optimal immune response to covid- . responses triggered by pamps recognition and its downstream molecules such as myeloid differentiation primary response (mdy ) may be altered by tlr polymorphisms [ , ] . hla genes present extreme allelic polymorphism. since they present viral peptides to host hla molecules to trigger an adaptative immune response, their polymorphisms may cause unevenness in antigen binding and presentation, and consequently in immune response. hla-b* : has been associated to the development and increased severity of sars-cov [ ] , and it has the fewest predicted binding affinity of sars-cov- peptides [ ] . il- plays a central role in the hyperactive immune response in covid- patients. since there are functional polymorphisms in the il- gene that modify its protein level expression, they may affect the severity of the disease [ ] . the role of ras in the pathogenesis of covid- is essential. single nucleotide polymorphisms and haplotypes in ace genes, such as polymorphism a/d in the ace gene, have been associated with circulating and tissue concentrations of ace levels and reduced expression of ace [ , ] . interestingly, the prevalence of covid- in europe correlates inversely with ace d allele frequency [ ] . a genetic variant of the ifn-induced transmembrane protein- gene is associated with covid- severity. ifn-induced transmembrane protein- is an immune effector protein that acts restricting membrane fusion [ ] . recently, a genomewide association study in covid- patients with respiratory failure identified an association signal at locus p . , which includes the genes slc a , lztfl , ccr , fyco , cxcr and xcr , while there was no association signals at the hla complex [ ] . lately, there has been a contention about the beneficial or detrimental role of ace inhibitors (acei) and angiotensin receptor blockers (arb) in the outcome of patients with covid- . currently, there is no evidence to support an advantageous or harmful effect of concomitant therapy with acei or arb in covid- patients [ ] . covid- is a systemic infection since it may impact any tissue or organ expressing ace . however, the most dreadful, often lifethreatening conditions, are ali and ards. therefore, the main challenge is to avoid their development to prevent icu admission and mechanical ventilatory support. we could envisage covid- as a tree in which aecs viral infection and ace downregulation represent the roots. the tree trunk would be the hyperinflammatory and hypercoagulable state. the branches would be an end-organ disease, such as ali, myocarditis, neurological disease, liver injury, gastrointestinal involvement, and skin disease. since the chain of events triggered by sars-cov- infection evolves quickly, any planned intervention must come as early as possible. besides, since the pathogenesis of covid- involves non-viral mechanisms, any intervention planned must also address the correction or modulation of these disbalances. hence, any therapeutic intervention must be early and combine antiviral and adjuvant therapies. however, the moment of diagnosis and eventual hospital admission will mark the timeframe of interventions. to tackle the roots of the disease, potential therapeutic interventions for covid- should first address the viral entry into aecs. the entry of sars-cov- into aecs takes place after binding of the spike to the receptor ace . specifically, the binding takes place in the receptor-binding domain of the s protein. thus, developing neutralizing monoclonal antibodies for this domain is a rational strategy to prevent the viral union and subsequent events [ ] . another possible way of targeting the interaction between ace and s protein may be the use of soluble recombinant ace , which may prevent the binding of the viral particle to the surface-bound, full-length ace [ ] . in the vero-e monkey cell line, a soluble form of ace blocks sars-cov replication and reduced sars-cov- recovery by a factor of À [ , ] . besides, since sars-cov- downregulates the ace /ang-( À )/mas r axis, recombinant human ace (rhace ) could prevent the development of ali in covid- . rhace attenuated arterial hypoxemia in a piglet model of lps-induced ali [ ] . in phase ii, open-label trial in humans with ards rhace was well tolerated, ang ii levels decreased, whereas ang-( À ) and surfactant protein d increased [ ] . however, the study was not powered to detect changes in acute physiological or clinical outcomes [ ] . there is a randomized controlled trial to assess rhace in patients with severe covid- (nct ). apart from ace , sars-cov- entry involves tmprss , whose inhibitor, camostat mesylate, significantly reduced lung cell line infection with sars-cov- [ ] . endocytosis is a crucial step in sars-cov- infection. ap- -associated protein kinase (aak ) regulates this process [ ] . baricitinib, a janus-kinase inhibitor, has been claimed as a candidate drug for covid- since it inhibits aak [ ] . arbidol inhibits viral entry by inhibiting the fusion between viral and cellular membranes [ ] . however, in a small retrospective study, arbidol did not meet noninferiority versus the combination of arbidol and lopinavir/ritonavir (lpv/r) [ ] . chloroquine and its safer derivative hydroxychloroquine are effective against sars-cov- in vitro [ ] . however, recent news from the large recovery trial showed that there is no beneficial effect of hydroxychloroquine in patients hospitalised with covid- ; therefore, that arm of the study was stopped [ ] . other planned large trials, such as solidarity, stopped enrolling patients to the hydroxychloroquine arm, and the national institutes of healthsponsored orchid study was also stopped [ , ] . numerous antivirals agents are being tested in clinical trials. lpv/r could not demonstrate enough efficacy when compared with placebo [ ] . the combination of ifn, lpv/r, and ribavirin showed a shorter time to negativize nasopharyngeal swabs and superiority versus lpv/ r in alleviating symptoms [ ] . in two double-blind, placebo-controlled trials, remdesivir was not associated with statistically significant clinical benefits in one, whereas in the other shortened the time to recovery in hospitalized adults [ , ] . as of now, there is no antiviral drug with proven efficacy for treating patients with covid- . another strategy tries to modulate the exuberant inflammatory response in covid- . the use of corticosteroids is controversial and not supported by previous experience in sars and mers [ ] . however, in the recovery trial, dexamethasone reduced deaths by one third in patients receiving invasive mechanical ventilation and by one fifth in patients receiving oxygen without invasive mechanical ventilation [ ] . tocilizumab, a specific il- receptor antagonist, is promoted to treat the hyperinflammatory state of covid- because of the pathogenic role il- plays. two observational studies have shown a clinical benefit of therapy with tocilizumab in covid- pneumonia with hyperinflammatory syndrome [ , ] . anakinra, a recombinant il- receptor antagonist, has proven useful in a small retrospective study of covid- patients with ards and hyper inflammation [ ] . there are additional trials in progress with tocilizumab, anakinra, and sarilumab. however, when trying to modify the cytokine response by targeting a single molecule or receptor, it should be recalled that the cytokine network is an intricate complex with a high degree of overlap, redundancy, and alternate pathways. this may explain therapy escape and eventually lack of response. therapeutic interventions for the consequences of hyperinflammatory and hypercoagulable states associated with covid- , such as ali, ards or thromboembolic events, are beyond this review's scope. knowledge of pathophysiology is the first step to address the management of a disease appropriately. it is familiar with the mechanism that the virus uses to evade host immune defense mechanisms or those that uses to harm will permit the design of appropriate strategies to neutralize the dysfunctions or disbalances generated either by the virus or by the consequences of the infection. from the knowledge gathered, it seems that most organ damage in severe covid- is done through an immune-mediated mechanism, although sars-cov- is the necessary initiator. the spectrum of disease is comprehensive, and since not all the patients will share the same evolving pattern, the search for predictive factors to promptly identify patients more prone to evolve to life-threatening disease is of the utmost importance. in severe cases, the quick evolving pattern of the disease makes early treatment imperative, at least until reliable predictive factors become widely available. the implication of viral and host-dependent mechanisms in covid- pathogenesis suggests that any therapeutic strategy must combine antiviral drugs and adjuvant therapy to modulate the host's responses. all these goals will be achieved through the broad effort of basic, translational, and clinical scientists and clinicians, and will demand a high degree of commitment from patients and their families, allied professionals, and everyone engaged in the fight against covid- . among them, politicians and health administration officers will play a unique role, since such a gigantic task will need the allocation of a vast amount of resources to overcome a health challenge to mankind like none other in recent times. while engrossed amid the pandemic, there was progress on the physiopathology of covid- . however, gaps regarding viral, environmental, and transmissibility aspects remain-the dynamic interplay between the host and the virus and how to modify it to improve disease prognosis not being the lesser. there is a big difference in transmissibility, which is highest for sars-cov- , among b-coronaviruses despite similar structure and functioning. asymptomatic viral shedding is the main factor. however, the role of newly described orfs and rna modifications and their functional correlations are not evident yet. although tmprss is involved in viral entry into the host cell, the involvement of other host proteins is still under discussion. the role of the different epithelial cells along the bronchial tree and the alveolar space needs to be ascertained. the virus's mechanisms to invade other organs beyond the lung are already poorly known. clinical disease progression is somewhat unpredictable. therefore, the identification of prognostic clinical and biological markers would optimize patients' care and resource consumption, which may be of utmost importance in pandemic times. this effort must include which role comorbidities and gender play. the definition and timing of the optimal therapeutic approach to covid- will represent a colossal effort, which can be accomplished only by randomized clinical trial performance. these should include concerted actions and a combination of diverse disciplines, resources, expertise, and techniques to contribute to advances in prevention, diagnosis, and therapy. this set makes up an almost flawless meaning of translational medicine defined by the european society for translational medicine (eustm) as "an interdisciplinary branch of the biomedical field supported by three main pillars: benchside, bedside, and community." for this review, our search strategy involved the review of original records, either journals or books, mainly from european and american sources, from to . from these sources, we hand searched reference lists of identified additional articles to retrieve additional studies. preference was given to most relevant research, but we were also keen to highlight the breadth of the topic and hence selected some publications that showcase particular areas of interest. we have searched pubmed and google scholar from database inception to may , , for records, journals, and books for the terms "sars-cov- , "covid- , "coronavirus", "raas system", "angiotensin-converting enzyme", "angiotensin-converting enzyme , "cytokine storm", "cytokine", "chemokine", "acute lung injury", "adult respiratory distress syndrome", "interferon", "interleukin ", "middle east respiratory syndrome", and "severe acute respiratory syndrome". references were examined in english. we declare no competing interests. the concept of the manuscript was devised by pd who also performed the overall literature searches. im, vp, hc, and jc designed the search strategy with inputs from pd and ndb. im, vp, hc, and jc carried out the literature searches and screening, and any discrepancies were discussed with pd and ndb. pd wrote the first draft of the review with inputs from all the authors. this work was partially supported by grant cov / . instituto de salud carlos iii, madrid, spain. the funding source was not involved in the design of the study or in writing the report. all authors had access to the data used in the analyses, and the lead author reviewed the full report. the full study data were available to all authors. pd, ndb made the decision to submit the paper for publication. who. novel coronavirus À china genomic characterization and epidemiology of novel coronavirus: implications for virus origins and receptor binding pathogenesis of covid- from a cell biologic perspective characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention the damage-response framework as a tool for the physician-scientist to understand the pathogenesis of infectious diseases the architecture of sars-cov- transcriptome the molecular biology of coronaviruses sars-coronavirus 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class i alleles and haplotypes suggests genetic susceptibility to influenza a (h n ) pandemic: a case-control study pattern recognitions receptors in immunodeficiency disorders the r q polymorphism in toll-like receptor (tlr ) attenuates innate immune responses to mycobacteria and impairs myd adapter recruitment to tlr association of hla class i with severe acute respiratory syndrome coronavirus infection human leukocyte antigen susceptibility map for sars-cov- circulating interleukin- and rheumatoid arthritis: a mendelian randomization meta-analysis relationship between genetic variants of ace gene and circulating levels of ace and its metabolites the host's angiotensin-converting enzyme polymorphism may explain epidemiological findings in covid- infections interferon-induced transmembrane protein- genetic variant rs -c is associated with disease severity in covid- genomewide association study of severe covid- with respiratory failure use of reninÀangiotensinÀaldosterone system inhibitors and risk of covid- requiring admission to hospital: a case-population study novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov- compared to sars-cov soluble angiotensin-converting enzyme : a potential approach for coronavirus infection therapy? inhibition of sars-cov- infections in engineered human tissues using clinical-grade soluble human ace recombinant angiotensin-converting enzyme improves pulmonary blood flow and oxygenation in lipopolysaccharide induced lung injury in piglets a pilot clinical trial of recombinant human angiotensin-converting enzyme in acute respiratory distress syndrome sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor ap- -associated protein kinase and cyclin g-associated kinase regulate hepatitis c virus entry and are potential drug targets baricitinib as potential treatment for -ncov acute respiratory disease arbidol as a broad-spectrum antiviral: an update arbidol combined with lpv/r versus lpv/r alone against corona virus disease : a retrospective cohort study remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro statement from the chief investigators of the randomised evaluation of covid- therapy (recovery) trial on hydroxychloroquine solidarity" clinical trial for covid- treatments study shows treatment does no harm, but provides no benefit a trial of lopinavir-ritonavir in adults hospitalized with severe covid- triple combination of interferon beta- b, lopina-virÀritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial remdesivir for the treatment of covid- -preliminary report clinical evidence does not support corticosteroid treatment for -ncov lung injury low-cost dexamethasone reduces death by up to one third in hospitalised patients with severe respiratory complications of covid- tocilizumab for the treatment of severe covid- pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of patients in pilot prospective open, single-arm multicentre study on off-label use of tocilizumab in patients with severe covid- interlekin- blockade with high-dose anakinra in patients with covid- , acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study we are indebted to jordi mancebo and m ant onia mangues for critical reading of the manuscript, and to richard pike for reviewing its writing. key: cord- -ggx v bz authors: dalan, rinkoo; bornstein, stefan r.; el-armouche, ali; rodionov, roman n; markov, alexander; wielockx, ben; beuschlein, felix; boehm, bernhard o. title: the ace- in covid- : foe or friend? date: - - journal: horm metab res doi: . /a- - sha: doc_id: cord_uid: ggx v bz covid- is a rapidly spreading outbreak globally. emerging evidence demonstrates that older individuals and people with underlying metabolic conditions of diabetes mellitus, hypertension, and hyperlipidemia are at higher risk of morbidity and mortality. the sars-cov- infects humans through the angiotensin converting enzyme (ace- ) receptor. the ace- receptor is a part of the dual system renin-angiotensin-system (ras) consisting of ace-ang-ii-at ( ) r axis and ace- -ang-( – )-mas axis. in metabolic disorders and with increased age, it is known that there is an upregulation of ace-ang-ii-at ( ) r axis with a downregulation of ace- -ang-( – )-mas axis. the activated ace-ang-ii-at r axis leads to pro-inflammatory and pro-fibrotic effects in respiratory system, vascular dysfunction, myocardial fibrosis, nephropathy, and insulin secretory defects with increased insulin resistance. on the other hand, the ace- -ang-( – )-mas axis has anti-inflammatory and antifibrotic effects on the respiratory system and anti-inflammatory, antioxidative stress, and protective effects on vascular function, protects against myocardial fibrosis, nephropathy, pancreatitis, and insulin resistance. in effect, the balance between these two axes may determine the prognosis. the already strained ace- -ang-( – )-mas in metabolic disorders is further stressed due to the use of the ace- by the virus for entry, which affects the prognosis in terms of respiratory compromise. further evidence needs to be gathered on whether modulation of the renin angiotensin system would be advantageous due to upregulation of mas activation or harmful due to the concomitant ace- receptor upregulation in the acute management of covid- . coronaviruses (cov) are a large family of viruses that cause illness ranging from the common cold to more severe diseases such as middle east respiratory syndrome (mers-cov) and severe acute respiratory syndrome (sars)-cov. the sars-cov- , has caused a rapidly spreading outbreak (covid- ) with over infected cases and more than deaths globally [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , (https://coronavirus.jhu.edu/map.html). the sars-cov- , a positive strand rna virus, has been seen to infect humans through the angiotensin converting enzyme - (ace- ) receptor [ ] . in covid- infections, emerging evidence demonstrates that older individuals and people with underlying metabolic conditions of diabetes mellitus, hypertension, and hyperlipidemia are at higher risk of morbidity and mortality [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in individuals with hypertension, diabetes, and other cardiovascular disorders with vascular complications, the renin angiotensin system (ras) is known to be activated with an increase in ace activity and a downregulation of ace- . modulation of this system by ace-inhibitors or at -receptor blockers is now considered as the first-line therapy as well as for prevention and management of vascular complications. in this regard, the questions arise if (i) differences in ace- may explain the exacerbated course of disease in patients with metabolic diseases and (ii) if ace modulation in covid- patients is neutral, beneficial, or harmful. the latter question may have immediate therapeutic consequences for millions of patients. moreover, ace- -based therapy has been proposed as a potential therapeutic approach in covid- pneumonia [ ] . the angiotensin converting enzyme (ace- ), a single pass type membrane monocarboxypeptidase, discovered decades ago [ ] consists of an n-terminal peptidase domain and c-terminal collectrin like domain [ ] . it is the peptidase domain that is responsible for the main functions of the renin angiotensin system (ras) [ ] . the ace- shares % homology with the n-terminal catalytic domain of ace, and a hydrophobic region near the c-terminus likely to serve as a membrane anchor [ , ] . the ace- protein is encoded by the ace- gene located on chromosome xp . these ace- proteins are more abundantly expressed on the apical surface of the well-differentiated and mostly ciliated airway epithelium of the lungs (alveolar type- cells), and enterocytes of the small intestine [ ] . furthermore, ace- protein is expressed in arterial and venous endothelial cells and arterial smooth muscle cells, in the heart, kidneys, adrenal glands, pancreas, skeletal muscle, and adipose tissues [ ] . the coronavirus sars-cov- , a single stranded rna virus, has been seen to infect humans through their envelope spike glycoprotein (s-protein), which is responsible for cov cell entry and hostto-host transmission. during viral infection, this s-protein cleaves into s and s [ ] . the furin cleavage site in the sars-cov- s protein may provide a priming mechanism [ ] . the ectodomain s binds to the peptidase domain of the ace- enzyme, while the s is cleaved further by the host cell serine protease tmprss [ ] resulting in membrane fusion. both these steps are essential for the viral entry into the cells. an in vivo study shows that the infection of human airway epithelia by sars coronavirus correlated with the state of cell differentiation and ace- expression and localization [ ] . the infection tends to occur more readily through well differentiated ciliated cells with higher ace- expression [ ] . it has been observed that ace- membrane expression and plasma levels are reduced after infection with sars coronavirus [ ] . further, sars cov spike protein has been found to reduce ace- expression and to augment pulmonary injury [ ] . however, treatment with blockers of the renin-angiotensin system reduced the pulmonary injury by activating the ace- -ang-( - )-mas axis. in the classical endocrine model of the ras, renin cleaves its substrate, angiotensinogen (agt), to produce the inactive peptide, angiotensin i (ang i), which is then converted to angiotensin ii (ang ii) by endothelial angiotensin-converting enzyme (ace). the catalytic activity of ace to activate ang ii occurs most extensively in the lung. ang ii mediates vasoconstriction as well as aldosterone release from the adrenal gland, resulting in sodium retention and an increase in blood pressure through the angiotensin receptor (at r). however, recent evidence suggests that ras also includes local systems with autocrine (cell-to-same cell) and paracrine (cell-to-different cell) effects in addition to the classical circulating ras with its well-known classical endocrine effects. in particular, ang ii generation at the tissue level by the tissue specific ras appears to have physiologic effects that are as important as circulating/systemic ang ii and, under some circumstances, more important than circulating ang ii. therefore, the ras system is not only involved in controlling blood volume and blood pressure but with the tissue specific local systems it directs tissue remodeling, endothelial dysfunction, and fibrosis [ ] [ ] [ ] [ ] . simplistically, it can be seen as a dual function system, which acts through two apparently opposite arms: the one responsible for the main actions of this system is constituted by the ace ang ii-at receptor axis and the other, a counterregulatory arm, is formed by the ace- -ang-( - )-mas axis. the catalytic activity of ace results in increased ang ii levels and increased catabolism of ang-( - ) whereas the catalytic activity of ace- is predominantly on ang i and ang ii and leads to formation of ang-( - ). ang-( - ) acts on the g-protein coupled receptor mas and is known to have counterregulatory actions on ang ii resulting in vasoconstriction and growth inhibitory effects [ ] [ ] [ ] [ ] (see ▶ fig. ). a common mechanism by which diabetes, hypertension, and other metabolic disorders cause vascular complications is endothelial dysfunction, inflammation, and atherosclerosis. in these disorders, the ace-ang ii-at receptor axis is activated with a downregulation of ace- -ang-( - )-mas axis [ ] [ ] [ ] , which leads to increased local and circulatory ang ii with a decrease in ang-( - ) and subsequent increased oxidative stress, activation of the endothelium, smooth muscle cell migration, growth, proliferation and thrombosis [ ] [ ] [ ] . deficiency in ace- leads to increased formation of atherosclerotic plaques and blocking of ace- results in prevention of atherosclerosis [ ] (▶ fig. ) . ace- is present in multiple organs and has various functions. in the heart, ace- prevents progressive cardiac fibrosis associated with aging and/or cardiac pressure overload and is beneficial in heart failure [ , ] . in most forms of chronic kidney disease including diabetic and hypertensive nephropathy, expression of ace- has been reported to be reduced in kidney tubules with an increased expression in the glomerula [ ] . this imbalance has been postulated as a potential cause of diabetic nephropathy. in the adrenal glands, the local secretory ras stimulates aldosterone production and serves as an amplification system for circulating ang ii. importantly the regulation of the secretory adrenal ras is independent of the circulatory ras and the activity of the adrenal ras correlates with aldosterone production and regulation of potassium serum concentrations [ ] . severe diabetes has been associated with hyporeninaemic hyperactivity of the adrenal gland [ ] . in the pancreas, the ace- -ang-( - )-mas axis has been described to protect the function of insulin-producing beta cells by improving the function of islet microvascular endothelial cells. further, activation of endothelial nitric oxide synthase and no signaling pathways via the ace- -ang-( - )-mas axis may have anti-inflammatory beneficial effects in pancreatitis [ , ] . in the skeletal muscle, the ace- -ang-( - )-mas axis has been described to decrease insulin resistance [ ] . ace- may exert potential anti-obesity effects via stimulating brown adipose tissue formation and induction of browning in white adipose tissue [ ] . a study done in the survivors of the sars infections, the organ involvement correlated with the organ expression of ace- with significantly higher immunostaining in the lung, kidney, heart, and islets of pancreas [ ] . in this series, of the patients had diabetes during hospitalization, which resolved subsequently suggestive of acute islet cell damage and diabetes, probably due to the use of the ace- receptor domain for viral entry [ ] . the respiratory system is a major site of ace-activity and source of systemic ang ii synthesis. locally produced ang ii may trigger increasing vascular permeability facilitating pulmonary edema. the ace- -ang-( - )-mas axis that is highly expressed in the lungs, may potentially induce pulmonary vasoconstriction in response to hypoxia, which is an important process in preventing shunting in patients with pneumonia or lung injury [ ] . in acute respiratory distress syndrome (ards) mouse models, ace- knockout mice displayed more severe ards symptoms compared with wildtype mice, while overexpression of ace- appeared to be protective [ ] . ▶fig. schematic representation to show the renin angiotensin system in diabetes and the interaction of the sars-cov with the ace- . . the sars-cov interacts with the ace- through the spike proteins after priming by tissue serene proteases. it uses the ace- protein to enter the alveolar cells in the lungs. . the renin angiotensin system consists of renin which catalyzes the conversion of angiotensinogen to angiotensin (ang ). the subsequent axis depends on the balance between the angiotensin converting enzyme (ace) and ace- . ace converts ang to ang ii and this acts in the angiotensin receptor (at r), whereas ace- converts it to ang-( - ), which acts on the mas receptor. . in the respiratory system activation of ace leads to a proinflammatory, pro-fibrotic , pro-hyperresponsiveness response in the respiratory system, whereas ace- -ang-( - )-mas induces a protective mechanism of anti-inflammatory, anti-fibrotic and anti-hyperresponsiveness. a lower ace- will put these individuals at higher risk of respiratory distress. . in hypertension, diabetes, and cvd, the ace related pathway is activated with downregulation of the ace- pathway. these results in the multi-organ complications seen in metabolic diseases with endothelial dysfunction promoting atherosclerosis, increased cardiac fibrosis and lv remodeling, diabetic nephropathy, hyperactivity of adrenal gland, and it decreases insulin release and increases insulin resistance. . infection with covid- may exacerbate the ace- deficiency in these patients in all organs and maybe responsible for the multiorgan failure. thus, stimulation of the ace- -ang-( - )-mas axis may have anti-inflammatory and antifibrotic effects in the pulmonary system, and these actions could potentially be favorable in the recovery of patients with pulmonary inflammation. it is possible that an ace/ ace- imbalance is one of the potential mechanisms explaining why patients with cardio-metabolic problems are at higher risk for respiratory failure [ ] . in an animal model study, it has been reported that infusion of sars cov spike protein reduced ace- expression and augmented pulmonary injury. however, treatment with blockers of the renin-angiotensin system reduced the pulmonary injury by activating the ace- -ang-( - )-mas axis [ ] . therefore, it may be assumed that the inherent downregulation of the ace- -ang-( - )-mas axis (as seen in metabolic conditions) is exacerbated in the covid- state because (i) the virus uses the peptidase domain of the enzyme for entry into the cells and (ii) there is a decrease in ace- with an increase in ace [ ] . in covid- infections, chest radiography typically shows patchy or diffuse asymmetric airspace opacities bilaterally more in the peripheral zones and in later stages ground glass opacities. ct scan typically shows bilateral involvement, which is different from sars and mers which starts unilaterally. moreover, it shows bilateral multifocal ground-glass opacities with a peripheral lung involvement in milder cases with more widespread consolidation in patients who go to the intensive care unit. pleural effusion, cavitation, pulmonary nodules, and lymphadenopathy have not been reported in patients with covid- . an important clinical observation of covid- infection in china was that some individuals with early infection exhibited no symptoms. interestingly, they even had negative swab results (due to absence of infection in the upper respiratory tract) and normal chest x-rays, however, showed significant changes in the ct scan of the lungs [ ] . histopathological examination of early covid- infected lungs, which were serendipitously detected in specimens operated for lung cancer, revealed exudative and proliferative phases of acute lung injury, edema and congestion of alveolar septa with inflammatory infiltrates (macrophages), type ii pneumocytes hyperplasia with viral inclusions, organization of inflammatory exudates and interstitial fibrosis [ ] . these pathological findings corroborated with the ct findings of patchy ground glass opacities. intriguingly, these pathological findings of inflammatory and fibrotic changes are known to be exacerbated in animal models of ace- knockout mice [ ] . histopathological examination from autopsies of individuals who have succumbed from covid- infections showed degenerated and necrotized cells in the myocardium and blood vessels with interstitial inflammatory infiltrates and focal necrosis. in the kidneys, proteinaceous exudates in the bowman's capsule surrounding the glomerulus, degeneration and shedding of the renal tubular epithelial cells and hyaline casts, microthrombi and fibrotic foci have been reported [ , ] . some of these changes of myocardial fibrosis and renal changes are already seen in the chronic phase of metabolic disorders due to ace- downregulation and these may get exacerbated [ , ] . the vast majority of covid- patients have mild symptoms. however, a significant percentage is critically ill with admission to an intensive care unit (icu, approx. %) with % case fatality rate globally (john hopkins coronavirus resource center: https://coronavirus.jhu.edu/). mortality is higher in patients with underlying hypertension, type- diabetes mellitus, or cardiovascular disease [ ] [ ] [ ] . to address this further, we performed a cumulative analysis of published data from china and south korea, which showed that a disproportionately higher percentage of patients with a higher morbidity (requiring icu care, ards) had hypertension ( %), type- diabetes ( %), cvd ( %) as an underlying disease. moreover, patients admitted to the icu had a higher systolic bp when compared to asymptomatic cases [ ] [ ] [ ] [ ] and in a recent series reported none of the nonsurvivors had hypotension [ ] (▶table ). a limitation of the data summarized in ▶ table is the posibility that some cases were reported twice in different series. interestingly, however, in a recent case report on a mild case extensively followed in australia, a low level of the monocyte chemoattractant protein- (mcp- ), a key biomarker of type- diabetes related vascular complications, was found to be associated with minor disease pathology [ ] . in a recent preprint study, hypokalemia due to renal potassium wasting has been reported with % of the critically ill patients having hypokalemia independent of gastrointestinal symptoms [ ] . the presence of higher expression of ace- receptors or its upregulation although initially thought to be a risk factor for infection is unlikely the case. in a recent preprint study, wherein they integrated public genomics, epigenomics, and transcriptomics data, they found that the expression of ace- is relatively high in asian females and young people while it is lower in males, and further decreases with age and with the progression of type- diabetes [ ] . this contradicts the hypothesis that higher expression is directly related to increased susceptibility as we know that sars-cov- infections are more common in males and in elderly patients with metabolic disorders. taken together, these observations suggest that patients with underlying type- diabetes, hypertension and/or cardiovascular diseases are at higher risk for requiring critical care and ventilation. there is evidence that although they are critically ill, they tend to demonstrate normotension to hypertension and hypokalemia, which are classical features of an activated ace related ras with a downregulation of the ace- pathway. as per current data, it does not appear that higher ace- expression is associated with an immediate predilection as individuals at risk will tend to have lower ace- expression. since ace- is the entry receptor for cellular infection by sars-cov- , blocking entry using ace- -related therapy could be feasible to prevent the spreading of infection in the lungs and the whole body. convalescent sera containing neutralizing antibodies against sars-s protein offer protection against sars-cov- infection [ ] . therefore, it was suggested to use recombinant human ace- protein to saturate the viral s-protein and thus prevent cellular entry of sars-cov- [ ] . this is especially of interest, since ace- has already been de-veloped as an experimental drug (apn , gsk ) for adult respiratory distress syndrome (ards) and is therefore available for clinical trials. it was shown that ace- could successfully prevent lung injury by the original sars-cov- [ ] . for ards, it had favorable effects in mice and piglets, but the results from a recent study in humans has been disappointing [ ] [ ] [ ] [ ] [ ] . another crucial step in cellular infection involves the serine protease tmprss that cleaves the viral s-protein. the tmprss inhibitor camostat mesylate has been approved in japan for pancre-atic inflammation and has been shown to prevent cellular infection by sars-cov- [ ] . consequently, it could prove being useful in covid- . both ace inhibitors and at receptor blockers have been used in diabetes and cardiovascular disorders to modulate this system in preference for vasodilation and maintenance of endothelial integrity. furthermore, in the diabetic kidney, reduced ace- protein expression could be prevented by ace inhibitor therapy suggestive of an upregulation with ace inhibitor [ ] . it is worthwhile men-▶table mortality (death) and morbidity (icu stay) associated with cardiometabolic disease based on reported cases in the literature. tioning that ace- receptor upregulation due to angiotensin receptor blockers that are commonly used in hypertension could be considered to be risk factor for increased transmission of sars-cov- . however, it has been seen that the at receptors tends to associate with ace- in these cases, which prevent the internalization of the ace- receptor and hence may not be amenable to viral entry [ ] . moreover, as mentioned earlier, higher expression of ace- was not associated with higher susceptibility to infection with sars-cov. according to the scientific data detailed above, it is tempting to speculate that blocking the ace-ang ii-at receptor axis with at blockers commonly used as antihypertensives may tilt the balance in favor of the ace- -ang-( - )-mas axis, which may help to accelerate respiratory recovery in patients suffering from covid- [ ] . however, it is intriguing that the patients most likely to be treated with ace-i or at blockers are the ones with the poorest prognosis in covid- [ ] [ ] [ ] [ ] [ ] [ ] [ ] . moreover, ace- upregulation concomitant with ras inhibitions may ease viral infection. consequently, it is also possible, that ras blockade may have a detrimental effect in covid- . currently, there is no clinical data to support one or the other direction. thus, the heart failure society of america, the american college of cardiology, and the american heart association have all issued statement to suggest that there is no definitive evidence of harm or benefit with the use of ace-inhibitors or at receptor blockers and that patients should continue to take their medications as usual [ ] . it is of the utmost priority to evaluate the growing stock of clinical data from china and worldwide to determine if ras inhibitors are beneficial or deleterious in covid- . only then, we can know how to counsel our patients taking ras inhibitors . in fact some researchers are planning a clinical trial to study losartan in covid- patients [ ]. individuals with underlying hypertension, type diabetes, or cardiovascular disease are at higher risk for respiratory failure and mortality in covid- . one of possible mechanisms for this predilection may be the imbalance in the ace pathways and therapeutics targeting viral entry through the ace- receptor or the ace-ang ii-at receptor axis as well as stimulating the ace- -ang-( - )-mas axis may be beneficial in these individuals. further evidence needs to be gathered on whether modulation of the renin angiotensin system would be advantageous due to upregulation of mas activation or harmful due to the concomitant ace- receptor upregulation in the acute management of covid- . special expert group for control of the epidemic of novel coronavirus pneumonia of the chinese preventive medicine association korean society for antimicrobial therapy, korean society for healthcare-associated infection control and prevention clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study clinical features of patients infected with novel coronavirus in wuhan, china clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china for the china medical treatment expert group for covid- . clinical characteristics of coronavirus disease in china risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease endocrine and metabolic link to coronavirus infection structural basis for the recognition of the sars-cov- by full length human ace- angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis sars-cov- receptor ace and tmprss are predominantly expressed in a transient secretory cell type in subsegmental bronchial branches (preprint); biorxiv sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor ace receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury angiotensin-converting enzyme and new insights into the renin-angiotensin system the renin-angiotensin system and diabetes: an update angiotensin-converting enzyme (ace ) is a key modulator of the renin angiotensin system in health and disease ace , angiotensin-( - ) and mas receptor axis in inflammation and fibrosis upregulation of angiotensin converting enzyme by shear stress reduced inflammation and proliferation in vascular endothelial cells local renin-angiotensin systems in the adrenal gland adrenal renin-angiotensin-aldosterone system in streptozotocin-diabetic rats local renin-angiotensin system regulates the differentiation of mesenchymal stem cells into insulin -producing cells through angiotensin type receptor the role of the renin-angiotensin system in the development of insulin resistance in skeletal muscle ace exerts anti-obesity effect via stimulating brown adipose tissue and induction of browning in white adipose tissue binding of sars coronavirus to its receptor damages islets and causes acute diabetes radiology perspective of coronavirus disease (covid- ): lessons from severe acute respiratory syndrome and middle east respiratory syndrome pulmonary pathology of early-phase novel coronavirus (covid- ) pneumonia in two patients with lung cancer chinese clinical guidance for covid- pneumonia diagnosis and treatment pathological findings of covid- associated with acute respiratory distress syndrome breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid- hypokalemia and clinical implications in patients with coronavirus disease (covid- ) individual variation of the sars-cov receptor ace gene expression and regulation the convalescent sera option for containing covid- ace- protects from severe acute lung failure mechanical stress and the induction of lung fibrosis via the midkine signalling pathway acute respiratory distress syndrome leads to reduced ratio of ace/ace- activities and is prevented by ang-( - ) or an ang ii receptor antagonists a pilot clinical trial of recombinant human angiotensin-converting enzyme in acute respiratory distress syndrome angiotensin ii mediates angiotensin converting enzyme type internalization and degradation through an angiotensin ii receptor type receptor-dependent mechanism hfsa/acc/aha statement addresses concerns re: using raas antagonists in covid- the authors declare that they have no conflict of interest. key: cord- - a tblt authors: chow, jonathan h.; mazzeffi, michael a.; mccurdy, michael t. title: angiotensin ii for the treatment of covid- –related vasodilatory shock date: - - journal: anesth analg doi: . /ane. sha: doc_id: cord_uid: a tblt nan ; ang- = angiotensin i; ang- = angiotensin ii; apache = acute physiology and chronic health evaluation; ards = acute respiratory distress syndrome; at = angiotensin type ; at = angiotensin type ; athos- = angiotensin ii for the treatment of high output shock; ci = confidence interval; covid- = coronavirus disease ; ecmo = extracorporeal membrane oxygenation; edhf = endotheliumderived hyperpolarizing factor; icu = intensive care unit; mas = mitochondrial assembly protein; no = nitric oxide; raas = renin-angiotensin-aldosterone system; rrt = renal replacement therapy; sars = severe acute respiratory syndrome c oronavirus disease (covid- ) first appeared in wuhan, china, in early december . since then, the world health organization has classified it as a pandemic and, as of april , , countries have reported over million confirmed cases and , deaths. in the cohort of patients with severe disease, . % were hospitalized and . % died. in the subgroup of patients admitted to the intensive care unit (icu), required mechanical ventilation, or died from the disease, . % required continuous renal replacement therapy (rrt), . % developed septic shock, and . % developed acute respiratory distress syndrome (ards). given the high morbidity and mortality in this cohort, we must utilize medications that are already available today to alter the pathophysiology and clinical course of this disease. doing so may improve outcomes while awaiting the development of targeted antiviral therapies and vaccines. [ ] [ ] [ ] [ ] [ ] ards increases alveolar-capillary barrier permeability, reduces surfactant production, amplifies cytokine and interleukin production, and increases the risk of septic shock, which all culminate in severe pulmonary endothelial damage. because angiotensin-converting enzyme (ace) is also located on the pulmonary endothelium, these proinflammatory processes severely disrupt ace function. ace is integral to the renin-angiotensin-aldosterone system (raas), which is one of the physiologic pathways that function in concert with the arginine-vasopressin and sympathetic nervous systems to autoregulate hemodynamics in humans. dysfunction in ace (hazard ratio . ; % confidence interval [ci], . - . ; p = . ) and raas (estimated fixed effect of renin . and . , % ci, . - . ; p = . ) has been associated with decreased survival in septic shock. , without functional ace in covid- -associated ards, angiotensin i (ang- ) cannot be hydrolyzed into angiotensin ii (ang- ), which contributes to hypotension via distinct mechanisms. first, inadequate production of ang- directly leads to decreased angiotensin type (at ) receptor agonism (figure ), leading to decreased vascular smooth muscle constriction, decreased free water and sodium reabsorption by the kidney, and decreased aldosterone, cortisol, and vasopressin release by the hypothalamicpituitary-adrenal axis. , second, it leads to excessive accumulation of ang- , which is metabolized into angiotensin-( - ) (ang-( - )) and angiotensin-( - ) (ang-( - )) to agonize the vasodilatory mitochondrial assembly protein (mas) and angiotensin type (at ) receptors ( figure ). third, ang-( - ) directly activates nitric oxide (no) synthase, stimulating production of no, another potent vasodilator. fourth, it impairs ace-dependent hydrolysis of bradykinin into bradykinin-( - ) and bradykinin- ( ) ( ) ( ) ( ) ( ) , which leads to excessive accumulation of bradykinin (figure ) . this vasodilatory substance agonizes b receptors and causes release of prostacyclin, no, and endotheliumderived hyperpolarizing factor (edhf). because of these changes, a strong physiologic rationale exists for utilizing exogenous ang- to treat covid- -associated vasodilatory shock. exogenous ang- targets the raas by replacing depleted endogenous ang- stores and agonizing at receptors to increase vascular tone. furthermore, by increasing renal perfusion and decreasing renin secretion, exogenous ang- decreases ang- production and mitigates secondary mas, at , b , no, and bradykinin-induced vasodilatation. the angiotensin ii for the treatment of high output shock (athos- ) trial found that ang- was effective at increasing mean arterial pressure and decreasing background norepinephrine dose. one study found that patients with vasodilatory shock who rapidly responded to exogenous ang- , defined as the ability to down-titrate to a dose ≤ ng/kg/min within minutes of initiation, had significantly lower levels of baseline endogenous ang- (mean ang- . ± . pg/ml rapid responders versus . ± . pg/ml nonrapid responders; p < . ) and subsequently had decreased -day mortality ( % for rapid responders versus % nonrapid responders; p < . ) than those who did not rapidly respond. in addition, ang- was associated with decreased -day mortality in patients with an acute physiology and chronic health evaluation (apache) ii score > ( . % mortality for ang- versus . % for conventional vasopressors; p = . ) and in patients with acute kidney injury (aki) on rrt ( % mortality for ang- versus % for conventional vasopressors; p = . ). , furthermore, ang- -treated patients experienced an increased rate of liberation from rrt by day ( % for ang- versus % for conventional vasopressors; p = . ) compared to those who only received conventional vasopressors. with up to . % of critically ill covid- patients requiring rrt and with the continued exponential increase in the number of covid- cases worldwide, a large number of patients might benefit from earlier ang- utilization. although the physiologic effects of ang- on the raas are known, many questions remain. current evidence suggests that severe acute respiratory syndrome [sars]-cov- , the virus that causes covid- , binds to the angiotensin-converting enzyme (ace ) receptor with - times the affinity of sars-cov, identified in , and that ace is required for cell entry and viral replication. exogenous ang- has been shown to downregulate ace by internalization and degradation in animal models and in vitro studies of human cells. , it is unknown whether these downregulatory effects on ace and can modulate the rate of covid- cell entry and viral replication. viral load and ace enzyme activity should be measured in patients who receive ang- or other vasopressors to better characterize their effects in covid- -infected patients. the disruption of ace function in ards and sepsis makes early exogenous ang- administration a physiologically rational choice for the treatment of covid- -associated vasodilatory shock. with the anticipated widespread shortage of life-sustaining equipment such as ventilators, continuous rrt machines, and extracorporeal membrane oxygenation (ecmo) circuits, critical care personnel such as rrt-trained nurses, intensivists, and respiratory therapists, and hospital resources such as critical care beds, emergency department beds, and personal protective equipment, every single rrt-free, hypotension-free, ventilator-free, and icu-free day will matter. although there are no current figure . normal function of ace. ace hydrolyzes ang- into ang- , which then acts on at receptors to cause vasoconstriction. ace is also required at points in the hydrolysis of bradykinin into bradykinin-( - ) and bradykinin- ( ) ( ) ( ) ( ) ( ) . ace indicates angiotensin-converting enzyme; ang- , angiotensin i; ang- , angiotensin ii; at , angiotensin type . figure . effect of ace dysfunction on metabolite accumulation. dysfunction in ace as a result of endothelial damage, ards, and septic shock prevents the hydrolysis of ang- to ang- from occurring. ang- accumulates, and the excess is metabolized into ang-( - ) and ang-( - ). ang-( - ) leads to activation of nitric oxide synthase and agonism of at , b , and mas receptors, which all lead to vasodilatation. in addition, ace dysfunction prevents the degradation of bradykinin into bradykinin-( - ) and bradykinin-( - ), which results in an excessive accumulation of bradykinin and potent vasodilatation. the figure was created with motifolio toolkit (motifolio inc, ellicott city, md). ace indicates angiotensinconverting enzyme; ang-( - ), angiotensin-( - ); ang-( - ), angiotensin-( - ); ang- , angiotensin i; ang- , angiotensin ii; ards, acute respiratory distress syndrome; at , angiotensin type ; mas, mitochondrial assembly protein; raas, renin-angiotensin-aldosterone system. clinical characteristics of coronavirus disease in china world health organization. coronavirus disease (covid- ) outbreak angiotensin i and angiotensin ii concentrations and their ratio in catecholamine-resistant vasodilatory shock sensitivity to angiotensin ii dose in patients with vasodilatory shock: a prespecified analysis of the athos- trial effect of disease severity on survival in patients receiving angiotensin ii for vasodilatory shock athos- ) investigators. outcomes in patients with vasodilatory shock and renal replacement therapy treated with intravenous angiotensin ii baseline angiotensin levels and ace effects in patients with vasodilatory shock treated with angiotensin ii the acute respiratory distress syndrome angiotensin converting enzyme defects in shock: implications for future therapy reversal of vasodilatory shock: current perspectives on conventional, rescue, and emerging vasoactive agents for the treatment of shock renin as a marker of tissue-perfusion and prognosis in critically ill patients bradykinin, angiotensin-( - ), and ace inhibitors: how do they interact? role of bradykinin in mediating vascular effects of angiotensin-converting enzyme inhibitors in humans angiotensin ii for the treatment of vasodilatory shock cryo-em structure of the -ncov spike in the prefusion conformation angiotensin ii mediates angiotensin converting enzyme type internalization and degradation through an angiotensin ii type i receptor-dependent mechanism angiotensin ii up-regulates angiotensin i-converting enzyme (ace), but down-regulates ace via the at -erk/p map kinase pathway key: cord- -autprmr authors: burrell, louise m.; johnston, colin i.; tikellis, christos; cooper, mark e. title: ace , a new regulator of the renin–angiotensin system date: - - journal: trends in endocrinology & metabolism doi: . /j.tem. . . sha: doc_id: cord_uid: autprmr abstract angiotensin-converting enzyme (ace) is a zinc metalloproteinase and a key regulator of the renin–angiotensin system (ras). ace is a newly described enzyme identified in rodents and humans with a more restricted distribution than ace, and is found mainly in heart and kidney. ace cleaves a single residue from angiotensin i (ang i) to generate ang – , and degrades ang ii, the main effector of the ras, to the vasodilator ang – . the importance of ace in normal physiology and pathophysiological states is largely unknown. ace might act in a counter-regulatory manner to ace, modulating the balance between vasoconstrictors and vasodilators within the heart and kidney, and playing a significant role in regulating cardiovascular and renal function. angiotensin-converting enzyme (ace) is a zinc metalloproteinase and a key regulator of the renin-angiotensin system (ras). ace is a newly described enzyme identified in rodents and humans with a more restricted distribution than ace, and is found mainly in heart and kidney. ace cleaves a single residue from angiotensin i (ang i) to generate ang - , and degrades ang ii, the main effector of the ras, to the vasodilator ang - . the importance of ace in normal physiology and pathophysiological states is largely unknown. ace might act in a counter-regulatory manner to ace, modulating the balance between vasoconstrictors and vasodilators within the heart and kidney, and playing a significant role in regulating cardiovascular and renal function. angiotensin-converting enzyme (ace), a dipeptidyl carboxypeptidase, is a key enzyme in the renin -angiotensin system (ras); it converts the inactive decapeptide, angiotensin i (ang i; or ang - ), to the active octapeptide and potent vasoconstrictor ang ii (or ang - ) (figure ), and inactivates the vasodilator bradykinin [ ] . ang ii is thought to be responsible for most of the physiological and pathophysiological effects of the ras, and inhibitors of ace that reduce the formation of ang ii have been highly successful in the management of hypertension, are standard therapy following myocardial infarction to delay the development of heart failure, and reduce the rate of progression of renal disease [ , ] . recently, however, the classical view of the ras has been challenged by the discovery of the enzyme ace [ , ] , in addition to the increasing awareness that many angiotensin peptides other than ang ii have biological activity and physiological importance [ ] . the reported vasodilatory actions of ang - [ ] , along with the potential involvement of ace in both ang ii degradation and ang - production, add another level of complexity to the ras [ , ] . it is predicted that cardiovascular disease will be the leading cause of death by , and although current approaches to block the ras have been of major benefit in this area, it is now clear that other pathways and enzymes within the ras can modulate its main effector, ang ii. we must question our understanding of the 'classical' ras, and work towards unravelling the complexity of the 'new' ras ( figure ). this should result in alternative strategies for the treatment of cardiovascular disease in the future. ace structure and function ace is the first known human homologue of ace, and was cloned from a human heart failure cdna library [ ] and a human lymphoma cdna library [ ] . analysis of the genomic sequence of ace has revealed that the gene contains exons and maps to chromosomal location xp [ ] . the full-length, human ace cdna predicts a protein of amino acids that has % homology with the n-terminal catalytic domain of ace, and a hydrophobic region near the c-terminus, which probably serves as a membrane anchor. like ace, ace is predicted to have the topology of a type membrane protein, with the catalytic domain on the extracellular surface. unlike somatic ace, ace has only one active enzymatic site and functions as a carboxypeptidase rather figure . the renin -angiotensin system (ras) pathway. schematic diagram of the classical ras (left), showing the main pathway for angiotensin ii (ang ii) generation from ang i via angiotensin-converting enzyme (ace). in the classical view, ang ii mediates all known effects via the at receptor. on the right is the updated view of the ras, showing the role of ace in degrading ang i to ang - , and ang ii to the vasodilator ang - . in this version of the ras, ang ii also mediates effects via the g-protein-coupled at receptor, whereas ang - acts through the mas receptor. than a dipeptidyl carboxypeptidase. thus, ace removes a single c-terminal leu residue from ang i to generate ang - , a peptide with no known function. although ace was described originally for its ability to generate ang - from ang i [ ] , it also degrades ang ii to the biologically active peptide, ang - [ ] ( figure ). indeed, in vitro studies indicate that the catalytic efficiency of ace for ang ii is -fold greater than for ang i [ ] , indicating that the major role for ace is the conversion of ang ii to ang - . the potential role of ang - as a cardioprotective peptide with vasodilator, anti-growth and anti-proliferative actions has been recognized relatively recently [ , ] . taken together, the data suggest that ace might function to limit the vasoconstrictor action of ang ii through its inactivation, in addition to counteracting the actions of ang ii through the formation of the agonist, ang - . recent studies [ ] have identified the g-protein-coupled receptor encoded by the mas protooncogene, mas, as the receptor for ang - [ ] . genetic deletion of the mas receptor abolished the binding of ang - to whole mouse kidney, whereas the functional significance of ang - and its receptor was demonstrated by studies showing that mas-deficient mice could not respond with antidiuresis to ang - after a water load, and that the aortas of mas-deficient mice were unable to relax in response to ang - [ ] . in addition, an unexpected function of ace has recently been identified and characterized; ace is a functional receptor for coronaviruses, including the coronavirus that causes severe acute respiratory syndrome, and is involved in mediating virus entry and cell fusion [ , ] . although not directly relevant to cardiovascular function, this would indicate that the ras including ace has multiple roles in physiology and various pathophysiological states [ ] . ace distribution ace has a much more restricted distribution compared with ace, and in humans ace transcripts have been identified in the heart, kidney and testis [ , ] . like ace, ace is found in endothelial cells, and is present to a lesser degree in vascular smooth muscle cells. in the kidney, ace is found in the proximal tubular epithelium. recently, ace has been identified in the gastrointestinal tract, brain and lung [ ] , suggesting a more ubiquitous distribution than initially thought. the precise physiological function of ace is currently under intense investigation. there has been increasing interest in peptides that are either cleaved or generated by this enzyme. in addition to effects on the angiotensin peptides, ace cleaves the c-terminal residue of the peptides des-arg -bradykinin, neurotensin - and kinetensin [ ] , and hydrolyses apelin- and dynorphin a - with as high a catalytic efficiency as ang ii [ ] . ace has no effect on bradykinin, in contrast to ace, emphasizing the specificity of ace [ , ] . many of the in vitro substrates of ace have actions that are relevant to cardiovascular regulation; apelin is a potent cardiac inotrope [ ] , dynorphin a is an endogenous opioid neuropeptide and des-arg -bradykinin binds to the bradykinin b receptor, which is activated by inflammation and tissue injury [ ] . it is not yet known whether the in vitro substrates of ace are also physiological in vivo substrates, and further studies are needed that address in vivo changes in the levels of putative substrates or products of ace using ace -knockout mice [ ] , ace transgenic animals [ ] and ace agonists, in addition to potent, selective ace inhibitors [ , ] . interestingly, the in vitro enzymatic activity of ace is unaffected by ace inhibitors [ , ] , but there are no data as to the effect of angiotensin receptor blockers on ace activity. in the future, it will be of great interest to assess the in vivo effects of ace inhibitors, particularly in light of the interesting findings with regard to cardiac function, which have been seen in both the ace knockout and transgenic animals [ , ] . it has been hypothesized that ace might protect against increases in blood pressure and that ace deficiency leads to hypertension. in several rat models of experimental hypertension, the gene for ace maps to a defined quantitative trait locus on the x chromosome previously identified as a quantitative locus for blood pressure [ ] . an association between ace and blood pressure has also been reported; in the spontaneously hypertensive rat (shr) and shr stroke prone rats, renal ace levels are reduced compared with normotensive wistar-kyoto rats (wky) [ ] , and we have confirmed that renal ace mrna is reduced in shr compared with wky [ ] . it is not clear whether ace deficiency plays a pathophysiological role to cause hypertension or is simply a consequence of increased blood pressure, and the precise role of ace in hypertension requires clarification. ace and the heart evidence of the biological role of ace in angiotensin peptide degradation comes from studies in ace -knockout mice, which lack ace protein [ ] . these mice develop abnormal heart function, with severely impaired cardiac contractility, and the decrease in function is both sex and time dependent, with more severe abnormalities in male than in female mice, and a more pronounced phenotype in older animals. the importance of ace in cleaving and/or inactivating ang ii is indicated by the increase in plasma, cardiac and kidney ang ii levels in the ace -knockout animals, and confirmed by studies in which genetic ablation of aceon an ace mutant background completely rescued the cardiac phenotype [ ] . hypoxia markers were also upregulated in the heart of the ace -knockout mice, suggesting that loss of ace from the vascular endothelial cells resulted in coronary vessel constriction and reduced oxygen delivery to the myocytes [ ] . certainly, the localization of ace in the rat and human heart to endothelial cells of intramyocardial blood vessels and smooth muscle cells supports a role for ace in the control of local vasodilation. although it was reported that ace protein synthesis was similar in normal hearts and a failing heart from a single patient with idiopathic cardiomyopathy [ ] , in a study of review subjects with idiopathic cardiomyopathy, there was an increase in functional cardiac ace activity assessed by the ex vivo formation of ang - [ ] . to date, there have been no published studies on cardiac ace in the context of ischaemic heart disease. given that marked stimulation of ace and ang ii occurs after myocardial infarction [ ] , ace probably increases after myocardial infarction; this increase would limit the adverse effects of raised cardiac ang ii by increasing levels of the vasodilator ang - . support for this idea comes from studies in the rat myocardial infarction (mi) model, in which the development of heart failure was associated with increased ang - immunoreactivity [ ] . furthermore, infusion of ang - attenuated the development of heart failure after mi, confirming the functional significance of ang - [ ] . it is possible that the relative balance of vasoconstrictor and vasodilator angiotensin peptides is important in the modulation of both haemodynamic and trophic effects of these peptides in the context of ischaemic heart disease. a word of caution is needed with regard to ace and the heart. although the data presented suggest that ace and ang - might have cardioprotective effects after myocardial injury, in transgenic mice with increased cardiac ace expression [ ] , there was a high incidence of sudden death, which correlated with transgene expression levels. electrophysiology revealed severe, progressive conduction and rhythm disturbances, with sustained ventricular tachycardia, that progressed to fibrillation and death. clearly, further studies are needed to determine the functional relevance of ace in the heart. immunohistochemical studies by our group [ ] and others [ ] have shown that in the kidney, both ace and ace protein are localized predominantly to epithelial cells of the distal tubule. the ras has been implicated in the pathogenesis of diabetic complications, in particular diabetic nephropathy [ ] , and ace inhibition provides significant renoprotection. we have recently characterized ace in the kidney of a rodent model of type diabetes mellitus and compared and contrasted it with ace [ ] . previous studies in the kidney using this model of diabetes demonstrated that ace is downregulated in the renal tubules and upregulated in the glomerulus. in a recent study, we found that both ace and ace mrna levels were decreased in diabetic renal tubules by , %. we also found that ace protein synthesis is reduced in the diabetic kidney, and this reduction is prevented by ace inhibitor therapy, suggesting that ace might have a renoprotective role in diabetes [ ] . although there have been no other studies on the expression of ace after renal tissue injury, the synthesis of collectrin, a protein with significant homology to ace is upregulated in a model of progressive renal injury [ ] . the ras has proved to be an important regulator of cardiovascular and renal structure and function, in addition to salt and water balance. blockade of the ras with ace inhibitors or ang ii type receptor antagonists has clearly established its key role in the pathophysiology of an increasing number of diseases, including hypertension, heart failure, ventricular remodelling, renoprotection and diabetic complications. these beneficial results are thought to result from blocking the vasoconstrictor, hypertrophic and proinflammatory actions of ang ii. the discovery of a new enzyme in the ras pathway, ace , which produces the vasodilatory and antihypertrophic peptide ang - , gives rise to the hypothesis that ace provides a counter-regulatory system to ang ii, which might also contribute to the beneficial effects of ras blockade. the identification of ace in the heart and kidney, its modulation in heart failure and diabetes, and the recent description that this enzyme plays a biological role in the generation and degradation of angiotensin peptides provides a rationale for the further exploration of its role in pathophysiological states, including myocardial ischaemia, renal failure, atherosclerosis, and diabetic complications. many questions remain to be answered about ace (box ), in particular the impact of ace on the generation of bioactive peptides, but clarification of the role of ace in health and disease will be assisted by the development of agents that modulate ace activity. the discovery of ace has opened up an exciting new area of cardiovascular and renal physiology, in addition to providing the possibility of identifying novel therapeutic targets. conversion of angiotensin i to angiotensin ii tissue angiotensin converting enzyme in cardiac and vascular hypertrophy, repair, and remodeling tissue angiotensin and pathobiology of vascular disease -a unifying hypothesis a novel angiotensin-converting enzymerelated carboxypeptidase (ace ) converts angiotensin i to angiotensin - a human homolog of angiotensin-converting enzyme -cloning and functional expression as a captopril-insensitive carboxypeptidase counterregulatory actions of angiotensin exploring the structure and function of zinc metallopeptidases: old enzymes and new discoveries the role of ace in cardiovascular physiology hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase vasopeptidase inhibition and ang-( - ) in the spontaneously hypertensive rat angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas angiotensin-converting enzyme is a functional receptor for the sars coronavirus the secret life of ace as a receptor for the sars virus quantitative mrna expression profiling of ace , a novel homologue of angiotensin converting enzyme novel role for the potent endogenous inotrope apelin in human cardiac dysfunction vasoactive potential of the b- bradykinin receptor in normotension and hypertension angiotensin-converting enzyme is an essential regulator of heart function heart block, ventricular tachycardia, and sudden death in ace transgenic mice with downregulated connexins novel peptide inhibitors of angiotensinconverting enzyme substrate-based design of the first class of angiotensin-converting enzyme-related carboxypeptidase (ace ) inhibitors renal ace- expression in development, hypertension and diabetes increased angiotensin-( - )-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme homologue ace effects of angiotensin-converting enzyme inhibition on angiotensin and bradykinin peptides in rats with myocardial infarction cardiac angiotensin-( - ) in ischemic cardiomyopathy angiotensin-( - ) attenuates the development of heart failure after myocardial infarction in rats characterization of renal angiotensinconverting enzyme in diabetic nephropathy effects of losartan on renal and cardiovascular outcomes in patients with type diabetes and nephropathy commentary on tikellis. et al. there is more to discover about angiotensin-converting enzyme collectrin, a collecting duct-specific transmembrane glycoprotein, is a novel homolog of ace and is developmentally regulated in embryonic kidneys key: cord- -iqepytyd authors: han, su-xia; he, guang-ming; wang, tao; chen, lei; ning, yun-ye; luo, feng; an, jin; yang, ting; dong, jia-jia; liao, zeng-lin; xu, dan; wen, fu-qiang title: losartan attenuates chronic cigarette smoke exposure-induced pulmonary arterial hypertension in rats: possible involvement of angiotensin-converting enzyme- date: - - journal: toxicol appl pharmacol doi: . /j.taap. . . sha: doc_id: cord_uid: iqepytyd chronic cigarette smoking induces pulmonary arterial hypertension (pah) by largely unknown mechanisms. renin–angiotensin system (ras) is known to function in the development of pah. losartan, a specific angiotensin ii receptor antagonist, is a well-known antihypertensive drug with a potential role in regulating angiotensin-converting enzyme- (ace ), a recently found regulator of ras. to determine the effect of losartan on smoke-induced pah and its possible mechanism, rats were daily exposed to cigarette smoke for months in the absence and in the presence of losartan. elevated right ventricular systolic pressure (rvsp), thickened wall of pulmonary arteries with apparent medial hypertrophy along with increased angiotensin ii (ang ii) and decreased ace levels were observed in smoke-exposed-only rats. losartan administration ameliorated pulmonary vascular remodeling, inhibited the smoke-induced rvsp and ang ii elevation and partially reversed the ace decrease in rat lungs. in cultured primary pulmonary artery smooth muscle cells (pasmcs) from - and -month smoke-exposed rats, ace levels were significantly lower than in those from the control rats. moreover, pasmcs from -month exposed rats proliferated more rapidly than those from -month exposed or control rats, and cells grew even more rapidly in the presence of dx , an ace inhibitor. consistent with the in vivo study, in vitro losartan pretreatment also inhibited cigarette smoke extract (cse)-induced cell proliferation and ace reduction in rat pasmcs. the results suggest that losartan may be therapeutically useful in the chronic smoking-induced pulmonary vascular remodeling and pah and ace may be involved as part of its mechanism. our study might provide insight into the development of new therapeutic interventions for pah smokers. pulmonary arterial hypertension (pah) is a group of diseases characterized by a progressive increase of pulmonary vascular resistance that finally cause right ventricular failure and premature death (chaouat et al., ) . it has been generally accepted and acknowledged that alterations in the pulmonary vasculature, commonly marked by vascular proliferation/fibrosis, remodeling, and vessel occlusion, leads to most, if not all, forms of pulmonary arterial hypertension (humbert et al., ) . as a key factor blamed for the alterations in the pulmonary vasculature, cigarette smoking has been reported to result in muscularization of pulmonary vessels in the presence or absence of chronic obstructive pulmonary disease (wright et al., (wright et al., , churg et al., ; santos et al., ) . moreover, increased expression of vasoactive mediators (e.g., endothelin- and vascular endothelial growth factor) have been detected in the guinea pig lungs after chronic smoke exposure and those mediators are reported to be associated with vascular remodeling and elevated pulmonary arterial pressure . however, the precise mechanisms by which chronic cigarette smoke exposure produces pah are still poorly understood. the renin-angiotensin system (ras) has been implicated in the pathogenesis of pulmonary vascular remodeling and pah in a number of studies. for instance, increased expression of angiotensin-converting enzyme (ace) in pulmonary arteries has been reported in pah patients (orte et al., ) . increases in angiotensin ii (ang ii) and ang ii type (at ) receptors were both demonstrated in hypoxic and monocrotaline-treated pulmonary hypertensive rats (chassagne et al., ; ferreira et al., ). in the ras, ace plays a central role in generating ang ii from angiotensin i (ang i) (turner & hooper, ) , and ang ii exerts a prominent role in the development of pulmonary vascular remodeling and pah (jeffery & wanstall, ) , particularly in promoting the growth of pulmonary artery smooth muscle cells (pasmcs) via at receptors (morrell et al., ) . moreover, medication with either ace inhibitor or at receptor antagonist can attenuate pulmonary arterial remodeling and pah in toxicology and applied pharmacology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] chronic hypoxia or monocrotaline-treated rats (nong et al., ; kishi et al., ) . additionally, nicotine, an important component of cigarette smoke, and its metabolites can increase both the activity and the expression of ace in cultured human endothelial cells (saijonmaa et al., ; ljungberg & persson, ) . furthermore, inhibition of ace may improve endothelial function in chronic cigarette smokers (butler et al., ) . based on these previous findings, we hypothesized that activated ras might also contribute to the development of chronic smoking-induced pah. losartan, a specific at receptor blocker, exerts its influence on the ras via inhibiting the stimulation of the at receptor by ang ii. it has been reported that losartan treatment may attenuate pulmonary vascular remodeling and pah in hypoxic rats and in piglets with left-to-right shunts (chassagne et al., ; rondelet et al., ) . however, it has not been determined whether losartan has a therapeutic effect on chronic cigarette smoking-induced pah. in addition, losartan has shown a modulatory effect on the expression of angiotensin-converting enzyme- (ace ) in hypertensive and cardiovascular diseases (ishiyama et al., ; koka et al., ) . ace , with a main role in cleaving ang ii into angiotensin ( - ) [ang ( - )] (ferrario et al., b) , is a recently found regulator of the ras and has been implicated in multiple lung diseases, e.g., severe acute respiratory syndrome-associated coronavirus (sars-cov) infection, acute lung injury, pulmonary fibrosis and monocrotalineinduced pah kuba et al., ; li et al., ; ferreira et al., ). however, it has not been elucidated whether ace has a role in chronic smoking-induced pah. a recent study shows that the antihypertensive action of at antagonists may be partially due to increased ang ii metabolism by ace (ferrario et al., a) . thus, in the present study, we sought to investigate whether losartan is therapeutically useful in smoke-induced pulmonary arterial remodeling and pah and the possible participation of ace in the mechanism. animals. male sprague-dawley (sd) rats weighing to g (supplied by experimental animal center, west china hospital, sichuan university) were used. all experimental protocols were approved by the institutional animal care and use committee of sichuan university (chengdu, china). experimental design. rats were exposed to the whole smoke of commercial, nonfilter cigarettes (five oxen, chengdu cigarette factor, china, per cigarette contains nicotine mg and tar mg) in ventilated whole-body smoking chambers ( cm× cm × cm) for minutes each time, twice per day for up to months as previously described ) with minor modifications. the smoke total particulate matter (tpm) concentration inside the exposure chambers was ± mg/m , determined by gravimetric analysis of filters at the exhaust port for the duration of the exposure. the control groups were exposed to fresh air under similar conditions. all rats were treated once a day, by oral gavage, with either saline or mg/kg losartan or mg/kg losartan . hour before the first smoke exposure each day. hemodynamic analysis. after an observation of months, rats were anaesthetized with pentobarbitone ( mg/kg i.p.) and were placed in a supine position, breathing room air. the right ventricular systolic pressure (rvsp) was measured as previously described (ferreira et al., ) . the cannula, filled with heparin-saline solution ( u/ml), was gently introduced through the right external jugular vein down to the right ventricle. the rvsp was recorded using a miniature liquid pressure transducer (biopac system inc., usa) and a computerized data acquisition system (mp , biopac system inc., usa). data was obtained from steady-state waveform for minutes. histological analysis and tissue preparation. after the rvsp measurement, all rats were killed by exanguination via external iliac artery and the lungs were harvested. the right lung was fixed in % polyformaldehyde (ph . ) overnight for paraffin embedding, sectioning, and histological staining. the left lung was dissected, and snap-frozen in liquid nitrogen, then stored at − °c for biochemical analysis. the paraffin sections ( µm thick) were stained with hematoxylin and eosin (h&e) and van gieson's elastic stain. assessment of vascular morphology was carried out as previously described (kishi et al., ) . briefly, the medial wall thickness (mwt) in fully muscularized arteries with an external diameter of to µm was evaluated by calculating the percentage of medial wall thickness as (medial thickness × / external diameter) × % along the shortest curvature. at least muscular arteries per section were examined using image plus . system (media cybernetics, silver spring, usa) in a blinded fashion by a skilled investigator. for the immunohistochemical detection of ace , the sections were stained with anti-rat ace polyclonal antibody (santa cruz). images were acquired by an optical microscope (h , nikon, tokyo, japan) fitted with a rt spot digital camera (nikon, tokyo, japan). western blotting analysis of ace and ace in lung homogenates. lung homogenates were prepared in lysis buffer, containing mm tris-hcl, mm nacl, % np- , . % sodium deoxycholate, mm naf, mm edta, . % sds, and a protease inhibitor cocktail tablet (roche applied science, indianapolis, usa) as previously described ). the protein concentration in the lung homogenate was determined by the bicinchoninic acid method (pierce, rockford, il, usa). equivalent amounts of protein samples ( µg) were separated on % polyacrylamide gels and then transferred onto . µm polyvinylidene difluoride (pvdf) membranes (millipore, bedford, ma). the membranes were incubated with a : dilution of ace polyclonal antibody and a : dilution of ace polyclonal antibody (santa cruz biotechnologies, santa cruz, ca). western blots were developed by enhanced chemiluminescence (pierce, rockford, il) according to the manufacturer's instructions. measurement of ang ii levels. tissue ang ii levels were measured by iodine- radioimmunoassay (ria) using the ang ii ria kit (beijing north institute of biological technology, beijing, china) according to the manufacturer's instructions as previously described (chen et al., ) . briefly, lung tissue was washed with cold saline, minced and heated in . m hcl at °c for minutes, and then homogenized. after centrifugation at , × g for minutes, the supernatant was lyophilized and redissolved in μl assay buffer, and the radioactivity was measured by a γ counter. measurement of ace levels and cell proliferation in cultured primary pulmonary artery smooth muscle cells from control and smoke-exposed rats. segments of pulmonary arteries were obtained from lungs of sd rats exposed to fresh air or cigarette smoke for months or months. pulmonary artery smooth muscle cells (pasmcs) were isolated and cultured as previously described (ogawa et al., ) with minor modifications. the arteries were excised and isolated, cut into small pieces and immersed in ml of phosphate-buffered saline (pbs) with . mg of collagenase for minutes. the endothelium and adventitia were gently removed. and the remainder of the vascular tissues was placed in culture plates. cells were maintained in dulbecco modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs), u/ml penicillin, and μg/ml streptomycin. and cell immunostaining was performed to confirm that cells were smooth muscle cells with anti-α-smooth muscle actin (α-sma) monoclonal antibody (neomarkers, lab vision, fremont, ca, usa). for the detection of ace in pasmcs, cells were lysed in the lysis buffer as described above in the ace and ace assay in lung homogenates. equivalent amounts of total protein ( μg/sample) were analyzed by western blot with ace antibody. cell proliferation was assessed by [ h]-thymidine incorporation as previously reported (ogawa et al., ) . when pasmcs were cultured to reach to % confluence, cells were detached, seeded and grown for hours at a first density of × cells/ml. subsequently, cells were incubated in low-serum culture media (dmem, . % fbs) to be kept quiescent for hours. then, dx ( . μm), an ace inhibitor purchased from phoenix pharmaceuticals (belmont, ca, usa) was added to the culture media of the cells from cigarette smoke-exposed rats. in another hours of incubation, cells were labeled with [ h]-thymidine at mci/ml for the last hours. then the cells were washed twice with ice-cold pbs, % trichloroacetic acid, and % ethanol, followed by lysis in . m naoh. finally, aliquots of cell lysates were neutralized with hcl, and the radioactivity was measured by a liquid scintillation counter. measurement of cell proliferation and ace levels in cigarette smoke extract-challenged rat pasmcs. an extract of cigarette smoke in dmem was prepared freshly for each experiment. commercial, nonfilter cigarettes (the same cigarettes as used in experimental design) were used. cigarette smoke extract (cse) was prepared as previously described (oltmanns et al., ) with a few modifications. briefly, cigarette smoke derived from one cigarette was drawn slowly into a -ml syringe and bubbled through ml of dmem. and one cigarette yielded draws of ml of the syringe, with each individual draw taking approximately seconds to complete. the resulting solution, which represented " %" strength, was then adjusted to ph . with concentrated naoh and filtered before being diluted in dmem to the required strength for application to pasmcs cultures. cell growth was assessed by cell counting kit- (cck- , dojindo, tokyo, japan). pasmcs from control rats were plated in -well microplates at a first density of × cells/ml per well. before experiment, cells were incubated in low-serum culture media (dmem, . % fbs) to be kept quiescent for hours. then cells were left untreated or pretreated with μm or μm losartan for minutes before exposure to %, . %, %, %, %, or % cse for hours, respectively. after addition of μl of the cck- reagent and hours of incubation, the plates were read at nm in a model microplate reader (bio-rad laboratories, usa). all experiments were repeated at least three times, and each experimental condition was repeated at least in quintuplicate wells in each experiment. ace levels were measured by western blotting analysis as mentioned above. statistical analysis. statistical analysis was carried out using either student's t test (for two-group comparison) or one-way anova (for multiple-group comparison) followed by the least significant difference (lsd) test (spss for windows version . , chicago, usa). a value of p b . was considered statistically significant. values were expressed as means ± sd. to determine the effect of chronic cigarette smoking and losartan treatment on rat pulmonary arteries, we first examined the histopathology and hemodynamics of rat lungs. the percent medial wall thickness (%mwt) of pulmonary arteries, which is a marker of pulmonary arterial remodeling, was calculated. as presented in figs. a and b, thick-walled pulmonary arteries with apparent medial hypertrophy were observed in smoke-exposed rats as compared to the normal pulmonary vascular structure in control rats. in addition, hemodynamic assessment shows a notable rise in rvsp after a month smoke exposure in rats (p b . , fig. ). in contrast, medication with losartan apparently reduced the smoke-induced increase in pulmonary arterial medial thickness and rvsp elevation, suggesting that losartan administration may attenuate pulmonary artery remodeling and pah induced by chronic cigarette smoke exposure in rats. ang ii accumulation induced by cigarette smoke exposure and the effect of losartan treatment activated ras and increased ang ii levels have been detected in hypoxic and monocrotaline-treated pulmonary hypertensive rats (chassagne et al., ; ferreira et al., ) . to investigate whether ang ii accumulation also exists in smoke-induced pah, we measured the ang ii concentration in rat lungs after a -month smoke exposure using ria. as illustrated in fig. , chronic smoke exposure induced a marked increase in ang ii levels in the lung, which was significantly inhibited by losartan treatment (p b . ). to further examine the mechanism for ang ii accumulation in smoke-induced pah in rats, we examined the ace and ace levels in smoke-exposed rats. western blotting analysis for ace and ace protein expression and ace immunohistochemical staining were performed. immunohistochemistry results showed a notable decrease of ace positive staining in lung sections in the smoke-exposed rats as compared to the control rats (fig. a ). densitometric analysis of western blotting bands indicates that smoke exposure induced a significant elevation in ace but a decrease in ace levels in lung tissue (figs. b and c). losartan administration in rats significantly increased the abundance of the band identified by ace antibody, while no significant difference was found in ace expression between the smoke-exposed group and the group treated with smoke plus losartan. the results imply that losartan could partially reverse the smoke-induced ace reduction but had no effect on ace protein expression. to compare the expression levels of ace in rat primary pasmcs from the smoke-exposed group and from the control group, cell lysis samples were used for western blotting analysis. the results showed that ace protein levels were significantly decreased in the pasmcs from the smoke-exposed rats (p b . vs con), especially from the month exposed rats (figs. a and b) , which was consistent with the findings of ace expression in lung homogenates in our in vivo study. enhanced proliferation of vascular smooth muscle cells has been reported in pah patients (yi et al., ) . since pasmc proliferation is a major contributor and an important characteristic of pah, rat primary pasmcs were isolated, and cell proliferation was evaluated by [ h]-thymidine incorporation in vitro. as shown in fig. c , the [ h]-thymidine incorporation rates in pasmcs from the smoke-exposed rats, especially from -month exposed rats, were significantly higher than that from the control group, implying that smoke exposure may simulate pasmc proliferation in rat lungs. furthermore, cells proliferated even more rapidly in the presence of dx , an ace inhibitor, implying a possible involvement of ace in the proliferation of pasmcs. there was no significant difference in [ h]-thymidine incorporation between the pasmcs from -month air-exposed rats and those from -month airexposed rats. to investigate the effect of cse on cell proliferation of pasmcs in vitro, pasmcs from control rats were challenged with %, . %, %, %, %, or % cse and cell proliferation was evaluated. as shown in fig. a , cse at concentrations of . %, %, and % caused a significant increase in cell number compared with the control samples (p b . ), and the peak increase in pasmc viability was observed at a concentration of % cse. in contrast, cse at higher concentrations (n %) appeared to be toxic to pasmc. based on the cell proliferation results, the effects of losartan on cell proliferation and ace expression in % cse-challenged pasmcs were evaluated. as shown in fig. b , μm losartan pretreatment significantly inhibited % cse-induced pasmc proliferation. in addition, % cse also reduced ace protein levels in pasmcs, which could be partially reversed by μm losartan pretreatment (p b . vs. cse; figs. c and d) . these results were consistent with our findings in the in vivo study that losartan treatment may attenuate chronic smoking-induced pulmonary artery remodeling and ace reduction in rat lungs. the present study mainly demonstrated that chronic cigarette smoke exposure significantly increased rat pulmonary arterial wall thickness and rvsp, along with an increase in ang ii and a decrease in ace levels. losartan treatment markedly reduced the pulmonary artery remodeling and rvsp elevation caused by cigarette smoke exposure and partially reversed the smoke-induced ace decrease and ang ii elevation in rat lung. consistent with our in vivo study results, in pasmcs from smoke-exposed rats, ace levels were significantly lower than in those from the control rats in an exposure time-dependent manner. moreover, pasmcs from -month exposed rats proliferated more rapidly than those from -month exposed or control rats, and cells grew even more rapidly by the ace inhibition with dx . in cultured pasmcs from control rats, in vitro cse challenge could also stimulate cell proliferation and reduce ace levels, which could be significantly inhibited by losartan pretreatment. western blotting analysis for ace in pasmcs from control rats (con), -month smoke-exposed rats (sm, month), and -month smoke-exposed rats (sm, month) rats. images are representative of three independent experiments. (b) semiquantification of ace levels by densitometry (ace /β-actin ratio). (c) measurement of cell proliferation rate by [ h]-thymidine incorporation. values are presented as mean counts per minute (cpm) of triplicate samples. dx ( . μm), an ace inhibitor was added to the culture media of the cells from sm rats. open bars: pasmcs from rats exposed to fresh air or cigarette smoke for three months ( month); solid bars: pasmcs from rats exposed to fresh air or cigarette smoke for months ( month). ⁎ p b . , as compared with the con group ( month); # p b . , as compared with the con group ( month); & p b . , as compared with sm group ( month); $ p b . , as compared with the sm group ( month). thus, our results suggest that losartan may have a therapeutic role in the smoking-induced pulmonary artery remodeling and pah, in which ace may serve as part of the mechanism. it has been reported that guinea pigs exposed to cigarette smoke for months produced an increase in pulmonary arterial pressure and enhanced muscularization of small pulmonary arteries (wright et al., . similarly, elevated mean pulmonary arterial pressure and increased muscularized pulmonary vessels have also been detected in rats after -week cigarette smoke exposure (lee et al., ) . consistent with these findings, in the present study, months of smoke exposure induced a marked medial wall thickening in rat pulmonary arteries and significantly increased rvsp in rats. these results suggest that chronic cigarette smoking may directly lead to pulmonary artery remodeling and pah. recently, it has been found that both the expression and activity of ace in human endothelial cells can be increased after the exposure to nicotine, an important component of cigarette smoke (saijonmaa et al., ; ljungberg & persson, ) . since ace plays a key role in converting ang i into ang ii in the ras, we hypothesized that cigarette smoke exposure may increase ang ii production in vivo by activating the ras. in the present study, we found a significant elevation in ace expression and an almost two-fold increase in ang ii levels in the lungs from the pah rats exposed to cigarette smoke for months as compared to those from the control rats. since ang ii is believed to play an important role in pah via binding to at receptors (jeffery & wanstall, ) , our results suggest that activated ras may also be involved in the smoke-induced pulmonary artery remodeling and pah. losartan is a specific ang ii antagonist that directly blocks angii at the at receptor. therapeutic effects of losartan on pah in hypoxia rats and shunted piglets have been reported (rondelet et al., ) . our study showed that losartan treatment effectively suppressed pulmonary artery medial wall thickening and rvsp elevation in smoke-exposed rats as well as the cse-induced cell proliferation in rat pasmcs. the inhibitory effect of losartan may be due to its property of directly blocking the at receptor, which, as we speculate, might result in the accumulation of ang ii via an undetermined mechanism. but interestingly, in the rats treated with smoke exposure plus losartan administration, we observed a significant decrease in ang ii levels in the lung as compared to the rats exposed to smoke alone, implying the existence of additional potential mechanisms for losartan-attenuated pah in the smokeinduced rat model. in other words, losartan can not only inhibit the effect of ang ii but may also decrease ang ii levels in cigarette smoking-induced pah. in the ras, ace cleaves ang i to generate the potent vasoconstrictor ang ii, whereas ace hydrolyses ang ii to inactive ang ( - ) and is a negative regulator of the system. in the present study, we found a significant decrease in ace levels in the cigarette smoke-induced pah rats and in the cultured cse-challenged rat pasmcs, which could be partially restored by losartan treatment. on the contrary, losartan had no effect on the smoke-induced ace elevation. in a previous study, ferrario et al. ( a) also found that losartan treatment ( mg/kg) showed no effect on plasma ace activity in normotensive lewis rats. similarly, in another study in wistar rats with experimental congestive heart failure (chf), ace activity in the myocardial homogenate did not differ between the treatment with and without eprosartan (another at receptor antagonist) administration (karram et al., ) . indeed, several studies have demonstrated that losartan can efficiently increase both the expression and activity of ace in rat heart or human kidney (koka et al., ; xia et al., ) . increased ace may augment the conversion of ang ii into ang ( - ), leading to the decrease in ang ii concentration. thus, these findings suggest that losartan treatment may have more influence on ace than on ace, and the reduction of ang ii in the attenuation of losartan-treated pah is probably due to the losartan-induced elevation in ace levels. as a result, the stimulation of the at receptor in response to ang ii is reduced, which may also contribute to the attenuation of smokeinduced pah by losartan treatment. there is now growing evidence to suggest that ace may play an important part in pah. it has been found that ace plays a protective role but its expression is decreased in human and experimental pulmonary fibrosis (li et al., ) . in a very recent study, chronic treatment with xnt, a synthetic activator of ace , prevented the elevation of rvsp, right ventricular hypertrophy, and thickness of pulmonary vessel wall in monocrotaline-induced pah rat model (ferreira et al., ). in our present study, we found that ace protein expression was significantly decreased in the lung along with apparent pulmonary arterial remodeling and pah in the -month smoke-exposed rats. moreover, in our in vitro study, western blotting results also showed that ace was markedly decreased in the pasmcs from the smoke-exposed rats compared with those from the control rats. in addition, ace inhibition with dx significantly increased pasmc proliferation rate above the level induced by smoke exposure alone, indicating a possible involvement of ace in the smokeinduced growth of pasmcs. there is recent evidence that ang ii downregulates ace via at receptor-mediated erk/p map kinase signaling pathway (koka et al., ) , therefore the decrease in ace protein expression induced by chronic cigarette smoke exposure may be mediated by a mechanism dependent on ang ii and its receptors. the detailed mechanism itself warrants further study. in summary, chronic cigarette smoke exposure significantly induced pulmonary artery wall thickening, rvsp elevation, as well as ang ii accumulation and ace decrease in rat lungs. losartan administration effectively attenuated the smoke-induced pulmonary arterial remodeling and pah, reduced ang ii elevation, and reversed ace reduction. ace inhibition with specific inhibitor in vitro further increased rat pasmc proliferation induced by smoke exposure. similarly, losartan treatment significantly suppressed cse-induced cell proliferation and ace decrease in cultured rat primary pasmcs. these results suggest that losartan may attenuate the chronic smoking-induced pulmonary vascular remodeling and pah in which ace may serve as part of the mechanism. this study might provide insight into the development of new therapeutic interventions for pah smokers. lisinopril improves endothelial function in chronic cigarette smokers clinical classification and epidemiology of pulmonary arterial hypertension modulation of angiotensin ii receptor expression during development and regression of hypoxic pulmonary hypertension transgenic study of the function of chymase in heart remodeling cigarette smoke drives small airway remodeling by induction of growth factors in the airway wall effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme advances in biochemical and functional roles of angiotensin-converting enzyme and 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pulmonary vascular remodeling and of decreased bmpr- expression by losartan therapy in shuntinduced pulmonary hypertension regulation of angiotensin-converting enzyme production by nicotine in human endothelial cells characterization of pulmonary vascular remodelling in smokers and patients with mild copd the angiotensin-converting enzyme gene family: genomics and pharmacology effect of sildenafil on acrolein-induced airway inflammation and mucus production in rats a neutrophil elastase inhibitor reduces cigarette smoke-induced remodelling of lung vessels cigarette smoke induces persisting increases of vasoactive mediators in pulmonary arteries vasoactive mediators and pulmonary hypertension after cigarette smoke exposure in the guinea pig angiotensin ii type receptor-mediated reduction of angiotensin-converting enzyme activity in the brain impairs baroreflex function in hypertensive mice distribution of obstructive intimal lesions and their cellular phenotypes in chronic pulmonary hypertension. a morphometric and immunohistochemical study this study was supported by grant no. from the national natural science foundation of china and - , - from the china medical board of new york and a research fund for the doctoral program of higher education from the ministry of education, pr china, to dr. f.q. wen. key: cord- - quigar authors: nan title: posters date: - - journal: j pept sci doi: . /psc. sha: doc_id: cord_uid: quigar no abstract is available for this article. laboratory of molecular biology and immunology, department of pharmacy, university of patras, patras, greece antimicrobial peptides (amps) are an important component of innate immune system of most living organisms. they have recently gained much attention as new anti-infective drugs with new modes of actions and few or no side effects. their antimicrobial spectrum covers gram-positive and -negative bacteria as well as fungi and certain viruses . fish have proven to be a rich source of antimicrobial peptides. three chrysophsin peptides (chrysophsin- , - , - ) have been identified in the gills of the red sea bream, chrysophrys major, which are all bactericidal to pathogenic bacteria at low concentrations . they are cationic α-helical peptides, rich in histidine residues and all end in an unusual rrrh motif. however, in addition to its high antimicrobial potency, chrysophsins have considerable hemolytic activity. the development of new analogues which would preserve high antimicrobial potency, but would lack the undesired hemolytic activity, could be a useful tool with possible commercial and clinical applications. in the present study, we synthesized a series of analogues of chrysophsin- with different ratios of lys and leu residues, utilizing the fmoc/but solid phase methodology . the synthesized analogues were purified and isolated by rp-hplc. the antimicrobial properties of the above peptide analogues are currently testing in gram positive (s. aureus, s. epidermidis, e. faecium) and gram negative (e. coli, p. aeruginosa) bacteria. the goal is to identify the minimum bacteriostatic and bactericidal concentrations of the analogues, under conditions that simulate the best possible that of the human organism. hemolytic or cytotoxic activity of the peptides will also be determined. the rise of antibiotic resistance demands the development of new antimicrobial agents. these should exhibit a novel mechanism of action so as to overcome the resistance and be invulnerable to 'not yet acquired resistance mechanisms'. such criteria are difficult to meet. however, cationic host defence peptides (hdps) have emerged as promising candidates. hdps target and disrupt bacterial membranes. in order to evade such a threat a bacterium would need to make substantial changes to its membrane composition disfavouring the development of resistance ( ) . however, exact role and mechanism of hpds in the regulation and monitoring of microbial invasions remain to be established. herein we will present new potential mechanisms of antimicrobial regulation by helical hdps using de novo ( ) and native systems ( ) . biophysical and microbiology aspects of the experimental designs will be discussed. the low number of the newly discovered antibiotics, emergence of multiple-drug resistance, and the alarming death rate due to the infection disease led to development the alternative means to combat the infections. the researchers accumulate information about antimicrobial drugs that could be result of the innate immunity mechanisms. armed only with the innate immunity, the insect has developed into the most widespread class in living kingdom. they produce several antimicrobial peptides with complementary and rapid mode of action. so far there are hundreds of antimicrobial peptides isolated from insect and lot of them are waiting to be discovered. the fleshly neobellieria bullata was chosen for isolation of these active compounds. its larvae in the third instar were squeezed to collect the haemolymph, which was gradually centrifuged and precipitated by acidified methanol. supernatant was subsequently separated by chromatographic methods (spe column, rp-hplc) to obtain fractions of short peptides. identification and characterization of these fractions were performed by tricine electrophoresis, mass spectrometry maldi-tof analysis and n-terminal sequencing. several fractions showed antimicrobial activity against institute of chemical kinetics and combustion, novosibirsk, russian federation in this work, we extracted d-structural information on newly synthesized, medium-length, double spin-labeled peptaibiotics using peldor spectroscopy. we investigated the magnetic dipole-dipole interactions between spin labels and the orientation selectivity effects. in particular, the medium-length peptaibiotics tylopeptin b , and heptaibin , double spin-labeled with the nitroxyl probe toac ( -amino- -oxyl- , , , -tetramethylpiperidine- -carboxylic acid), were studied by means of x-band peldor spectroscopy. this study was conducted on tylopeptin labeled at positions and (t ) and heptaibin labeled at positions and (h ) in frozen glassy methanol solutions at Κ. peldor data analysis was carried out using the theory developed for short interspin distances. the distance distribution functions between spin labels for Τ (maximum at . nm, halfwidth of . nm) and Η (maximum at . nm, half-width of . nm) were determined. the intramolecular distances observed between the labels allowed us to assign an essentially α-helical conformation to Τ and a largely prevailing -helical structure to Η under the aforementioned experimental conditions. are amidated at the c-terminus, as a result of a posttranslational enzymatic reaction. temporins are particularly active against gram-positive bacteria and are not toxic to eukaryotic cells. in this study we designed a series of analogues of tb with the aim to improve the peptide antimicrobial activity against both gram negative and gram positive strains and then to structurally elucidate the mechanism of interaction of active peptides with lps. the peptides have been synthesized substituting one or two amino acids with an alanine and lengthening the sequence with positively charged amino acids. among the designed peptides, one of the analogues, tb_kkg a, showed highly increased activity against gram negative bacteria and also a slightly increased activity against gram positive bacteria with a total lack of hemolytic activity. to develop ll- -derived short amps with prokaryotic selectivity and lipolysaccharide (lps)neutralizing activity, a series of amino acid-substituted analogs based on ig- (residues - of ll- ) were synthesized. analog a showed the highest prokaryotic selectivity, but much lower lps-neutralizing activity compared to ll- . the analogs, a , a , a and a with higher hydrophobicity displayed lps-neutralizing activity comparable to that of ll- , but much lesser prokaryotic selectivity. these results indicated that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of lps-neutralizing activity and prokaryotic selectivity. to increase lps-neutralizing activity of the analog a , we synthesized trp-substituted analogs (a -w and a -w ), in which phe or phe of a is replaced by trp. despite their same prokaryotic selectivity, a -w displayed much higher lps-neutralizing activity compared to a -w . this result suggested that the effective site for trp-substitution when designing novel amps with higher lps-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. furthermore, d-enantiomeric peptides (a -w -e and a -w -e) of a -w and a -w possessed not only more improved prokaryotic selectivity and retained lpsneutralizing activity compared to a -w but also protease stability. taken together, a -w -e and a -w -e can serve as promising templates for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection. department of zoology, faculty of science, charles university, prague, czech republic antimicrobial peptides (amps) are among the most promising lead compounds for developing medicines in the fight against resistant pathogenic bacteria. we have already shown that the venom of wild bee is a rich source of pharmacologically interesting antimicrobial peptides [ ] [ ] [ ] [ ] . from the venom of solitary bee macropis fulvipes, we isolated and characterized the novel antimicrobial peptide named macropin (mac- ). by edman degradation and mass spectrometry, its primary sequence was established as gfgmalkllkkvl-nh . mac- possesses potent antimicrobial activity against both gram-positive andnegative bacteria and moderate hemolytic activity against human red blood cells. cd spectra confirmed that mac- can form an amphipathic α-helical secondary structure in the presence of membrane-mimicking substances as sodium dodecyl sulfate or organic solvents like trifluoroethanol. we prepared a series of mac- analogs to study the effect of incorporating d-amino acid residues into the sequence in various positions on antimicrobial and hemolytic activity, α-helicity and serum stability. the substitution of l-amino acid residues at n-terminal part of sequence by d-amino acid residues led to the improving hemolytic activity with maintaining or increasing antimicrobial activity. these modifications increased peptide stability in human serum. effect of the incorporation of d-amino acid residues into the mac- sequence on its α-helical structure will be discussed. the neutralization of endotoxins (lipopolysaccharide, lps) by suitable compounds has been shown to be a key step in the treatment of infectious diseases, in particular in the case of gram-negative bacteria. the active endotoxic center of lps is lipid a, its lipophilic part. an effective antimicrobial peptide against gram-negative bacteria is magainin , which was originally found in the skin of an african frog. here, we studied the interaction of hexa-acyl bisphosphoryl lipid a prepared from erwinia carotovora lps with magainin with some minor substitutions in the amino acid pattern. by using fourier-transform infrared spectroscopy, the gel to liquid crystalline phase transition of the acyl chains of lipid a, the conformation of their phosphate groups due to peptide binding, and the profile of the secondary structure of the peptides was investigated. the zeta potential of lipid a aggregates in the presence of the peptides was determined by measuring the electrophoretic mobility. small-angle x-ray scattering was performed for the elucidation of the aggregate structures in the absence and presence of the peptides, and isothermal titration calorimetry was applied for evaluating the thermodynamics of binding between peptides and lipid a. the data show that asp-or glusubstituted peptides improved the binding activity to lipid a correlated with characteristic changes in the physical parameters, which were stronger expressed for the aspsubstituted peptide. the new hydrogen bond connection between glu and asp by carboxylic acids apparently leads to a more pronounced -structure of the peptide. the conformation change of the peptide enhanced the activity of incorporation into the lipid a aggregates, along with changes in biochemical and biophysical parameters. royal college of surgeons, dublin, ireland cationic antimicrobial peptides (caps) have been reported to exhibit anticancer activity . one such peptide, p , has been shown to inhibit the growth of several cancer cell lines, with inhibiting concentration (ic ) in the range of to μm . however the concentration at which p and other caps act is too high to be clinically relevant. the enhancement of their activity can be achieved through the modification of their amino acid composition or the addition of other molecules. conjugation of naturally produced hydroxylated fatty acids to p showed a -fold improvement in its anticancer activity on a variety of human-derived cell lines. in addition to the enhancement of activity we wished to understand the mechanism of action of the peptide and conjugates. we investigated the uptake of conjugated and unconjugated peptides into hela (cervical) and miapaca (pancreatic) human cancer cells and the localisation of the peptide in the cell once taken up. we investigated the effect of altering the carbon number of the hydroxylated fatty acids ranging from hydroxyhexanoic acid (r ) to hydroxydodecanoic acid (r ) conjugated to p peptide and tested on hela and miapaca cell lines. circular dichroism studies were performed to investigate the effect on α-helical content due to amino acid composition alteration and hydroxyalkanoic acid conjugation. the effect of the position of the hydroxyl moiety on enhancement of activity was also investigated. in the current study p and its derivatives also lacked haemolytic activity with concentrations up to fold higher than ic values needed to observe any haemolysis. when current antibiotics become less efficient, there is a promise that some antibiotics can be replaced by other nature's substances, e.g. peptides. halictines are novel antimicrobial peptides isolated from the venom of the eusocial bee halictus sexcinctus. we obtained four analogues of the native peptide hal from iocb av cr. they already characterized structural properties of these peptides and their antimicrobial activity against selected bacteria . the analogues were prepared by point mutations of native peptide, which could increase antimicrobial activity and decrease undesirable hemolytic activity. our aim was to characterized membrane permeation activity of halictines through the use of a basic model of biological cells -large unilamellar phospholipid vesicles luvs. we prepared two basic types of leakage assays based on luvs with free dyes entrapped inside and one assay with laurdan content. we used classical steady state fluorescence spectroscopy and advanced fluorescence methods for study of dyes escape from luvs and we also used laurdan generalized polarization technique gp for better understanding peptide insertion into membrane. in this way we received complementary information and we can conclude that the most active peptides are the native hal and analogue hal / . however hal / requires presence of negatively charged phospholipids in membrane which may explain its higher selectivity against bacteria. furthermore, fcs results have shown that the leakage happens via pore formation. results from gp revealed that peptide insertion in the membrane do not lead directly to formation of pores. against a wide range of microorganisms, mainly by perturbing the permeability of bacterial membranes through the formation of pores. however, amps effects on membrane properties probably extend beyond poreformation. we performed a systematic spectroscopic analysis of the effects on membrane structure and dynamics of two very different amps: the cationic pmap- , which creates pores according to the "carpet" model , and alamethicin, which forms "barrel-stave" channels . by using fluorescence anisotropy measurements on liposomes comprising probes localized at different depths in the bilayer, we measured peptide effects on membrane fluidity and order. laurdan spectral shifts provided indications about water penetration in the bilayer. in the case of pmap- , it was possible to focus specifically on the lipids surrounding the peptide by following the membrane-probe fluorescence due to fret from the peptide trp residues. finally, peptide-induced perturbation of lateral mobility and domain formation were determined by several methods. all experiments were compared with liposome-leakage measurements: while for pmap- all membrane-perturbing effects are correlated with the vesicle leakage process, alamethicin does not significantly influence membrane dynamics at the concentrations in which it forms pores. surprisingly, in all cases the most significant peptide-induced effect is a reduction in membrane fluidity. we have reinvestigated -residue peptaibols named metanicins from an ascomycetous fungus originally described as metarhizium anisopliae strain cbs . (cbs = centraalbureau voor schimmelcultures, utrecht, the netherlands). however, due to unusually shaped conidia and based on rna-sequencing of its internal transcribed spacer (its) region, the identification of cbs . as metarhizium has been withdrawn and this particular strain is currently under taxonomic reinvestigation . sequencing of four isolated peptides by fab-ms, esi-ms and edman degradation of partial hydrolysates revealed structural relationship to -residue peptaibol antibiotics paracelsins from trichoderma reesei (=hypocrea jecorina). sequences determined are: ac-u-a-u-a-u-a(u)-q-u-v-u-g-l-u-p-v-u-u(j)-q-q-fol (exchange positions in parenthesis; ac, acetyl; u, aib, α-aminoisobutyric acid; j, d-isovaline; fol, l-upmc univ paris laboratoire des biomolécules; cnrs umr ; ens lbm; address: laboratoire des biomolécules, ens dpt de chimie, , rue lhomond f- , paris, france current data suggest that the cellular uptake of cellpenetrating peptides (cpps) occur by two processes: direct translocation across the plasma membrane and endocytosis . the large diversity of cpp sequences described in the literature (derived either from fragments of proteins, structurally constrained synthetic peptides, peptide libraries or dendrimers) has hampered the identification of general rules for their efficacy of internalisation. we have used a reductionist approach, restricting the cpp functional groups (amide and guanidinium) and tailoring cpp amphiphilic properties. two families of cpps have been designed: ) primary amphiphilic cpps corresponding to tetra-arginines functionalised with fatty acid chains of different lengths and ) secondary amphiphilic cpps containing arginine and alanine or tryptophan residues . these cpps were linked by a disulfide bridge to a peptide inhibitor of protein kinase c (pkci). the efficiencies of internalisation of the conjugates were quantified by a method based on maldi-tof mass spectrometry previously developed in our group . the mechanism of internalisation was studied by comparing the amounts of cell-surface bound and internalized pkci cargo on cho-k cells and glycosaminoglycan-deficient cho cells at o c and o c. conjugates were found to enter by both direct translocation and glycosaminoglycandependent endocytosis. in addition, the primary amphipathic cpps were found to be more efficient than the secondary amphipathic ones. furthermore, structural or mechanistic novelty does not guarantee immunity from resistance, with strains resistant to linezolid identified prior to fda approval. therefore, modifying existing antibiotics to overcome resistance mechanisms presents an opportunity to rationally develop effective new drugs more rapidly than screening for new structures. vancomycin is a glycopeptide commonly used as a front line treatment for infections caused by methicillinresistant staphylococcus aureus (mrsa). the emergence of vancomycin-resistant enterococci (vre), vancomycinintermediate s. aureus (visa) and vancomycin-resistant s. aureus (vrsa) has prompted the development of semisynthetic glycopeptides . we have generated a variety of glycopeptide derivatives that show superior antibacterial activity against mrsa and vre compared to vancomycin and second generation lipoglycopeptides. this was undertaken by employing a combination of solid phase and solution phase chemistry to attach a membraneassociative element that selectively binds to bacterial membranes in preference to eukaryotic membranes, thus increasing the local concentration at the lipid ii d-ala-d-ala peptidoglycan cell wall precursor target site. three novel antimicrobial peptides, named panurgines (png), were isolated from the venom of wild bee panurgus calcaratus. one of them is dodecapeptide with sequence lnwgailkhiik-nh (png- ). the next two peptides are almost identical. these are cyclic peptides containing amino acid residues and two intramolecular disulfide bridges ldvkkiicvackixpnpackkicpk-oh (x=k png-k and x=r png-r). all peptides exhibited antimicrobial activity against gram-positive bacteria and gram-negative bacteria, antifungal activity and low haemolytic activity against human erythrocytes. we prepared analogues of α-helical amphipathic png- with the aim to improve its biological properties and a linear analogue of png-r to elucidate the importance of disulfide bridges for its activity. in the second part of the study, we followed the effect of panurgines on the degree of membrane disruption by observing the leakage of fluorescence dye (calcein) entrapped in artificial phospholipids vesicles [ ] . specifically, we investigated membrane interactions of pngs with the vesicles made from negatively charged : dopc/dppg and : dopc/dopg vesicles as a general model of bacteria membrane and : : dopc/dopg/cl as a possible model for a membrane of bacillus subtilis. the membrane interaction of pngs was also investigated on uncharged dopc vesicles as potential model membrane for erythrocytes. pngs exhibited weak dye-leakage activity for neutral vesicles, while they effectively induced dye leakage in the presence of negatively charged vesicles. these results indicate that pngs have stronger potency to disrupt bacteria-mimicking anionic membranes than those which mimic eukaryotic cell membrane. department of biochemistry and toxicology, university "lucian blaga", sibiu, romania a common tool to bias the conformation of linear peptides is the insertion of side-chain modified amino acids or sidechain/main-chain conformationally restricted building blocks. an alternative approach is a simple backbone modification. in this connection, backbone amide replacements with (almost) isosteric surrogates were extensively used. these modifications may impart resistance to enzymatic degradation and better bioavailability to the peptides, but also influence the secondary structure. a thioamide (ψ[cs-nh]) is perhaps the closest structural mimic of an amide. however, it possesses different and attractive features: (i) its nh group forms stronger hydrogen bonds, being more acidic than that of the amide. (ii) its c-n bond undergoes cis/trans isomerization by irradiation at nm (π→π* transition). (iii) it may act as a "minimalist" fluorescence quencher. for all these reasons, we started a programme aimed at exploring how the endothioamide bond affects peptide folding and bioactivity. in this communication, we describe the synthesis and conformational results of the three analogs of the membrane-active peptaibiotic trichogin ga iv listed below: n-octanoyl-aib-gly-ψ[cs-nh]-leu-aib-gly-gly-leu-aib-gly-ile-leu-ome ( / ) n-octanoyl-aib-gly-leu-aib-gly-ψ[cs-nh]-gly-leu-aib-gly-ile-leu-ome ( / ) n-octanoyl-aib-gly-leu-aib-gly-gly-leu-aib-gly-ψ[cs-nh]-ile-leu-ome ( / ) the syntheses of the three peptides were accomplished in solution according to a fragment condensation approach. appropriate thioamide-containing tri-or tetrapeptides were prepared by treating the corresponding all-amide precursors with the lawesson reagent. ft-ir absorption, d-nmr and cd conformational investigations on the three analogs were conducted in comparison with the naturally occurring peptaibiotic. all three analogs maintain the capability to interact with the dope/dopg model phospholipid membranes and exhibit a comparable bioactivity against s. aureus. peptide-peptide interaction of lactococcin g class iib two peptide bacteriocin h. etayash, w.soliman and k. kaur* faculty of pharmacy and pharmaceutical sciences, university of alberta, edmonton, alberta, t g e lactococcin g, a class iib two-peptide bacteriocin, consists of two complementary peptides lcng-α and lcng-β that act as one functional unit with optimal antimicrobial activity achieved by the presence of both peptides in approximately equal amounts. in this study we have investigated the mechanism of pairing of the two complementary peptides as well as explored any specific interaction that could take place between the peptides. molecular dynamics (md) simulation was employed to study the interactions at the atomistic level. four different md simulations with the peptides in a lipid bilayer system were conducted. md results from these simulations confirmed and pointed out that (i) the two putative gxxxg motif, g xxxg in lcng-α and g xxxg in lcng-β, were attracted and came closer to each other, showing the role of these motifs in attracting the two peptides to each other. closer views, however, showed no clear interactions between these two motifs. most likely, nonspecific interactions play a role in bringing the two peptides together; (ii) variations and loss in the secondary structure in both the peptide fragments were confirmed among the four simulations. on the contrary, stability of helical regions was identified between residues w -g and d -q in lcng-α and v -e in lcng-β; and (iii) role of tryptophan at the n-terminal regions in positioning and setting the peptide orientations were confirmed which matched the previous reported results. faculty of pharmaceutical sciences, unesp -univ. estadual paulista, araraquara, sa͂ o paulo, brazil antibiotic resistant bacterial strains represent a global health problem. antimicrobial peptides (amps) are promising novel antibiotics because they have displayed little or no resistance effects. it is well known that the charge, amphipathicity, hydrophobicity and helicity of the peptide are fundamental for the biological activity. in addition, covalent dimerization appears as a new parameter to be studied. in this way, several bioactive sequences were dimerized obtaining pharmacotechnics advantages like enhanced antimicrobial activity, solubility and proteases resistant. however, the effect of this modification is unclear since dimeric versions of some amps are toxic . to evaluate the effects of dimerization on the structure and biological activity of the amp aurein . , the monomeric version (au) and the c-and n-terminal dimers, (au) k and e(au) , respectively, were synthesized. circular dichroism results indicated that dimeric versions showed more defined structures in aqueous solution. e(au) showed "coiled coil" structure while (au) k an αhelix structure. in contrast, au displayed typical spectra for disordered structures. in tfe and lpc, all the peptides acquired a high amount of α-helix structure. the antimicrobial activity against bacteria and yeast decreased with dimerization. however, dimeric peptides promoted the aggregation of c. albicans. hemolytic and vesicle permeabilization assays showed that au has a concentration dependence activity, an effect that can be assigned to a "carpet" like mechanism peptide, whereas this effect was less pronounced for dimeric versions, suggesting that dimerization may change the mechanism of action. in conclusion, our studies showed that the effects of amp dimerization are complex and still unclear. , the first antimicrobial peptide generated in vivo and isolated from the gut contents of the cattle tick boophilus microplus . we have shown that these peptides are equally lethal to candida albicans mdm and practically not active on human erythrocytes . to examine the properties and mode of action of hb - a, we synthesized it and its fluorescently labeled analogue (fam-hb - a) by the solid-phase method at o c, purified them by rp-hplc and characterized their purified forms by lc-esims. at low salt concentration, both peptides were found to inhibit the growth of candida albicans atcc , candida parapsilosis atcc and candida krusei atcc , but hb - a was two-fold more active (mics of . ; . and . μm, respectively). at those concentrations, both peptides also kill the fungi. assays with human erythrocytes showed that, likewise hb - a, fam-hb - a present activity lower than % at μm. apparently, hb - a targets the membrane cell because confocal microscopy analysis revealed that, at the half of mic value, fam-hb - accumulates on the fungal cell membrane. in contrast, fluorescence activated cell sorting (facs) analysis revealed that, at the mic, more than % of the fam-hb - a penetrates into the cell. membrane permeability assay using hb - a, c. albicans atcc and the kit live/dead funga light confirmed progressive membrane damage associated with an increase in peptide concentrations. the use dibac ( ) and facs analysis showed that hb - a alters the plasma membrane potential, leading to cell death. supported by fapesp, cnpq and capes. lasso peptides form a growing class of to residue ribosomally-synthesized and post-translationally modified peptides produced by bacteria. they share a rigid and compact interlocked structure consisting of a macrolactam ring at the n-terminus and a c-terminal tail that is looped back and threaded through the ring, forming a typical [ ] rotaxane , . the macrolactam is formed by condensation of an asp or glu side-chain with the free amino group of a gly or cys . the lasso fold is stabilized either by steric hindrance assumed by bulky amino acid side-chains and/or by disulfide bonds between cysteines from the tail and the ring. given this structure, lasso peptides display a high stability against proteolytic and chemical degradation. they are biologically active on various enzymatic targets, which confer them in some cases an interesting antimicrobial activity. given its characteristics, the lasso scaffold thus represents a promising tool for biotechnological application in the development of bioactive peptides. until now, nine peptides had been structurally characterized as lasso peptides. based on a genomics-based approach, we identified a novel lasso peptide from streptomyces sviceus that we termed sviceucin. it was produced in high yield by heterologous expression in s. coelicolor and submitted to structural analysis by mass spectrometry and nmr. sviceucin is residue long and stabilized by two disulphide bonds. their connectivities were identified mainly from the typical noes between the beta-protons of the cysteines. the lasso structure of sviceucin was obtained by nmr-based molecular modelling. sviceucin was shown to exhibit antibacterial activity directed against gram positive bacteria, while gram-negative bacteria and fungi showed resistant. the penicillium chrysogerum antifungal protein (paf) is a cysteine-rich, cationic protein that inhibits the growth of a variety of filamentous fungi without toxic effect on mammalian cells . although paf is used to be produced in p. chrysogerum or a similar microorganism, preparation of analogues of the protein for structural and functional investigations requires an efficient chemical method. the unsuccessful continuous synthesis of the -mer small protein prompted us to use native chemical ligation . the syntheses of the fragments were performed by solid-phase method applying tboc chemistry. using the acid-labile tboc protecting group, the thioester end of the n-terminal fragment remains intact during the course of the synthesis. the first attempt was the synthesis of peptides with pmethylbenzyl groups on the side chains of all of the six cysteine residues. under no circumstances oxidative folding provided the natural disulphide bridge pattern. the failed attempts led us to orthogonal protection of the sulphydryl groups. different sets of protecting groups were tried and evaluated. our experiments showed that basic treatment triggered rearrangement of the previously formed disulphide pattern. thus, base-labile protecting groups (such as -fluorenylmethyl, fm) have to be avoided in the synthesis of paf. the alarming increase and spread of antibiotic resistance among bacterial pathogens has stimulated the development of new antibacterial agents with innovative mode of action. antimicrobial peptides with broad spectrum activity are widely distributed in nature and play an important role in innate immunity in several species, including humans. tigerinins are a unique family of -to -residue antimicrobial peptides found in skin secretion of the indian frog rana tigerina , . characterized by a disulfide-bridged loop composed of nine amino acids, tigerinins do not show primary structural homology to any known antimicrobial peptides from amphibians. tigerinins could provide novel lead compounds for the design of effective antimicrobial peptides with a new mode of action. the peptide murdp has been identified after the screening of phage display libraries against pseudomonas aeruginosa cell wall biosynthesis murd amide ligase enzyme . murdp is a low micromolar range inhibitor of murd enzyme and showed good antimicrobial activity. composed of nine amino acids, it is also characterized by a nine residues disulfide-bridged loop containing two prolines. this great similarity with tigerinins, led us to investigate if murd enzyme could be a potential target for these peptides. in silico analyses using modelling, molecular dynamics and docking with p. aeruginosa murd showed that murdp and tigerinin- and - make similar interactions in the binding site. these results suggest that murd may be an intracellular target for tigerinin- and tigerinin- . synthesis, murd enzymatic inhibition assay, antibacterial activity evaluation and structure-activity relationships of murdp and tigerinins analogs will be presented. h. etayash, l. norman, t. thundat*, k. kaur* faculty of pharmacy and pharmaceutical sciences, department of chemical and materials engineering, university of alberta, edmonton, alberta t g e , canada listeria monocytogenes is a gram positive bacterium that accounts for about % of the deaths resulting from food borne illnesses in north america. moreover, l. monocytogenes is considered one of the most difficult bacteria to detect in contaminated food products. while standard microbiological and biochemical assays currently used are accurate and sensitive, they are time consuming and often require specialized instruments operated by a trained user making on-site testing difficult. to this end, we propose the development of an antimicrobial peptide (amp) or peptide fragment sensor for the on-site detection of l. monocytogenes. leucocin a, which is a naturally occurring amp consisting of a amino acid sequence, is known to exhibit specific activity against l. monocytogenes at pico to nanomolar concentrations. for this reason, we have synthesized a shorter peptide fragment of leucocin a consisting of amino acids using solid phase peptide synthesis. the peptide was purified by reversed phase hplc and maldi-tof mass spectrometry indicates the desired biological entity was achieved. by including an n-terminal cysteine group, the tailored amp was readily immobilized at a gold interface. the resulting thickness and molecular orientation, determined by ellipsometry and grazing angle infrared spectroscopy, respectively, indicate that the helical peptides were adsorbed on the interface with a preferred orientation parallel to the surface. the bacterial specificity of the anchored leucocin a fragment was tested against three gram positive bacteria and results reveal that the adsorbed amp exhibits a limit of detection of approximately one bacterium/μl which is a clinically useful detection range. faculty of science, university of south bohemia, České budějovice, czech republic during the last few years we have identified three novel defensins from arthropods. two of them, lucifensin and lucifensin ii were purified from various tissues of lucilia sericata and l. cuprina larvae, respectively. larvae of these flies are routinely used in the hospitals around the world for the treatment of non-healing infected wounds in the procedure known as maggot therapy. these amino acid residues and three disulfide bridges peptides differ from each other only in one amino acid residue in position (val-ile). linear precursor of lucifensin was prepared by fmoc-spps chemistry which was then subjected to the oxidative folding yielding a peptide with a pattern of disulfide bridges identical to that of native lucifensin and other insect defensins. this was examined by the identification of the fragments resulting from the thermolysin digestion of lucifensin by means of mass spectrometry. however, this cyclization reaction proceeded via an intermediate having incorrect pairing of disulfide bridges. from the hemolymph of blood sucking tick dermacentor marginatus (d.m.) we purified defensin containing amino acids and three disulfide bridges. its sequence determined by edman degradation and mass spectrometry was identical to that previously determined by molecular biology methods . sequence of d.m.defensin shows no homology to insect defensins. by spps prepared linear precursor of d.m.-defensin was subjected to oxidative folding under the open air. the linear peptide was straightforwardly folded into cyclic one which was identical to the native peptide. in antimicrobial assay using a set of different bacteria all three studied defensins show activity preferentially against gram-positive bacteria including staphylococcus aureus but are inactive against gram-negative ones. the importance of disulfide bridges on tertiary structure of defensins and their antimicrobial activity will be presented. recently, the chemical structure and conformation of pseudodesmin a has been determined through x-ray diffraction and nmr spectroscopic analysis . in this way pseudodesmin a was identified as a new member of the viscosin group of antimicrobial peptides (amps). in addition, it was demonstrated that individual molecules self-assembly in apolar environment into a supramolecular pore-like structure, providing structural support for its biological activity , . to further explore the structure-function relationship, a viable synthesis strategy for pseudodesmin a analogues was developed, based on side-chain attachment of the first amino acid to the solid support, followed by stepwise fmoc solid-phase synthesis of the linear peptide precursor and on-resin head-to-tail cyclization. nmr study confirmed the molecular structure and thus the development of an efficient and successful synthesis of this type of amp's. these results and the synthesis route will be presented. trichogin ga iv, isolated from the fungus trichoderma longibrachiatum , is the prototype of lipopeptaibols, a subclass of short-length peptaibiotics exhibiting membranemodifying properties. its primary structure is as follows: n-oct-aib -gly-leu-aib-gly-gly-leu-aib-gly-ile -lol, where n-oct is n-octanoyl, aib is α-aminoisobutyric acid, and lol is the , -amino alcohol leucinol. this peptaibol is predominantly folded in a mixed -/αhelical conformation with a clear, albeit modest, amphiphilic character . in this work, we synthesized by solution and solid-phase methodologies a set of trichogin ga iv analogs in which the four gly residues, lying on the poorly hydrophilic face of the helical structure, are substituted by one (or more) strongly hydrophilic lys residues. moreover, we synthesized another set of analogs where one (or more) aib residues are replaced by leu. the conformational preferences of these analogs were assessed by x-ray diffraction, cd, and d-nmr techniques . we tested the role played by the substitutions on the peptide bioactivity, e.g. protease resistance, cytotoxicity, and hemolysis. cytotoxicity was tested using three in vitro cell-based assays: (i) human red-blood cells lysis; (ii) cell mortality in total human blood leukocytes and in separate subpopulations; (iii) cell mortality in three tumor-derived stable cell lines (hela, a , and a ). our data show that some of our trichogin analogs are active against tumor cells, leaving the leukocytes unaffected. a convenient post-screening ring opening approach for the decoding of one-bead one-compound cyclic peptide libraries a. girard, e. biron* faculty of pharmacy, université laval and chuq research center, quebec, canada combinatorial chemistry has been widely used as an effective method for the generation and screening of synthetic peptide libraries. amongst the different combinatorial methodologies to discover new bioactive peptide-based compounds, we were particularly interested in the one-bead one-compound (oboc) approach . this powerful approach fully exploit the great molecular diversity accessible with peptides and has been used to identify a great number of ligands and modulators for a wide variety of biological targets. however, its use with cyclic peptides is limited by difficulties in sequencing hit compounds by edman degradation or tandem mass spectroscopy due to the lack of free n-terminal amine and complicated fragmentation patterns, respectively. this problem has been overcome by pei and coworkers by using a bead segregation strategy in which the outer layer exposes the cyclic peptides and the inner layer the linear counterpart for sequencing . more recently, lim et al. reported an elegant method to prepare and sequence oboc cyclic peptoid libraries without encoding by using a ring opening approach with triazine-based cyclic derivatives . unfortunately this method is incompatible with amino acids bearing some functionalized side chains. based on this strategy, we have developed an efficient method to prepare oboc cyclic peptide libraries that does not require encoding by using a simultaneous ring opening/cleavage approach. the procedure is compatible with commonly used amino acids and allows rapid and efficient sequencing of selected hits after on-bead screening. the synthesis of an oboc cyclic peptide library, ring opening methodology and sequencing by mass spectrometry will be presented. cyclotides are a very abundant class of plant peptides that display immense sequence variability around a conserved cystine knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability . their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals . however, laborious isolation and purification prior de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development . here we extend the knowledge about their sequence diversity by analyzing the cyclotide content of a violet species native to western asia and the caucasus region . using an experimental approach, which we named 'sequence fragment assembly' by maldi-tof/tof-based peptidomics, we were able to characterize novel cyclotides from viola ignobilis. amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analyzing mixtures containing multiple peptides. using in-source decay and high energy collision induced dissociation of digested cyclotides allowed to distinguish isobaric residues ile and leu. overall this work underlines the immense structural diversity and plasticity of the unique cyclotide framework. the presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides. glycation is a nonenzymatic reaction occurring between reducing sugars and reactive amino groups of biomolecules. the reaction leads to a formation of a heterogeneous mixture of compounds which are classified as early, intermediate or advanced glycation end products (age). these compounds, especially advanced glycation end products, are involved in many pathological processes, mainly diabetic complications, and could be markers of certain diseases. detection of early products of glycation (amadori products) is a relatively easy task and can be performed by various methods including e.g. ms/ms techniques, isotopic labeling and affinity chromatography on immobilized boronic acid , . however, the diverse structures of ages make detection of these compounds more challenging. the aim of the study was testing a new method of ages identification based on isotopic c labeling. a model protein (hen egg lysozyme) was modified with an equimolar mixture of [ c ]glc and [ c ]glc. then the glycated protein was subjected to reduction of the disulfide bridges followed by enzymatic hydrolysis. the obtained digest was analyzed by lc-ms methods. the glycation products were identified on the basis of characteristic isotopic patterns resulting from the use of isotopically labeled glucose. this method allowed for identification of early maillard reaction products and different structures of the glycation end products. isotopic labeling technique combined with lc-ms is a new and very sensitive method for identification of the advanced glycation end products even if their structures are unknown. this method could be also used as an alternative method of detection of amadori products. in the course of a project aimed to assess the significance of antibiotics for the producing organism(s) in the natural habitat, we screened a specimen of the fungicolous fungus hypocrea phellinicola growing on its natural host phellinus ferruginosus . using a peptaibiomics approach , , we detected -and -residue peptide sequences by (u)hplc/hr-esi-qqtof-ms. structures of peptaibiotics found were independently confirmed by analyzing the peptaibiome of an agar plate culture of h. phellinicola cbs (ex-type) grown under laboratory conditions. notably, h. phellinicola could be identified as a potent producer of -, -, (culture) and -residue (specimen) peptaibiotics of the suzukacillin-type . minor components of the -residue peptaibols, herein named suzukacillins c, are assumed to carry a c-terminal residue tentatively assigned as tyrosinol (tyrol). in addition, the previously isolated suzukacillin b was sequenced and shown to be a microheterogeneous mixture of -residue peptaibols. in order to further investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we screened specimens of the fungicolous fungus hypocrea pulvinata growing on its natural hosts piptoporus betulinus and fomitopsis pinicola . using a peptaibiomics approach , we detected -, -, -(major sequences), and -residue peptide sequences in the five specimens analyzed by (u)hplc/hr-esi-qqtof-ms. structures of peptaibiotics found were independently confirmed by analyzing the peptaibiome of pure agar cultures obtained by single-ascospore isolation from the specimens . major, -residue peptaibols were assigned as deletion sequences of the trichosporins b lacking the ala/aib residue in position . our results corroborate that: i) peptaibiotics are, indeed, biosynthesized in the natural habitat, thus, ii) their membrane-perturbing formation of ion channels may support the parasitic life style of a fungicolous fungus. based on methodology that we have developed in our lab , we identified specific and selective substrates for these serine proteases. we used a , membered pnaencoded peptide library to screen , possible peptide substrates in a single experiment. the library was incubated with the protease of interest and then hybridized on a custom designed dna microarray. microarray scanning and data analysis allowed the measurement of the changes in fam/tamra ratios resulting from the protease activity and the determination of the protease specificity. to verify the predicted activity and specificity, fret peptides were synthesized, incubated with the enzymes and the hydrolysis reaction was followed by monitoring fluorescence emission. specificity constants kcat/km were calculated and the cleavage sites of the peptides were identified. dubs were, moreover, found to be associated with several diseases and as such are emerging as potential therapeutic targets . several directions have been pursued in the search for lead anti-dub compounds. however, none of these strategies have delivered inhibitors reaching advanced clinical stages due to several challenges in the discovery process, such as the absence of a highly sensitive and practically available high-throughput screening assay . in this study, we report on the design and preparation of a fret-based assay for dubs based on the application of our recent chemical method for the synthesis of ub bioconjugates . in the assay, the ubiquitinated peptide was specifically labeled with a pair of fret labels and used to screen a library comprising compounds against uch-l . such analysis identified a novel and potent inhibitor able to inhibit this dub in time-dependent manner with kinact = . μm and ki = . μm. our assay, which was also found suitable for the uch-l enzyme, should assist in the ongoing efforts targeting the various components of the ubiquitin system and studying the role of dubs in health and disease. . more recent work based on rna interference experiments on a mouse model suggested that isoform-specific inhibitors against nmt might be effective anti-cancer agents as a knockdown of nmt inhibits the tumour growth, whereas knockdown of nmt has no effect . if residual nmt activity can compensate for loss of nmt function in healthy cells, potential toxicity may also be minimised. we developed a method to identify peptide or protein substrates of nmt and/or nmt . peptides/ proteins are exposed to nmt and/or nmt and an alkyne-tagged analogue of myristoyl coa. subsequent azide-alkyne "click" cycloaddition allows visualisation of the myristoylated substrates in fluorescence or chemiluminescence, using a fluorescent or a biotin moiety on the capture reagent. this labelling technology was applied to peptide libraries prepared on microarrays to investigate nmt / isozyme substrate specificity using recombinant nmt and nmt . peptides made of the first or amino acids at the nterminus of known myristoylated proteins were functionalised with a biotin moiety at the c-terminus and immobilised on an avidin-functionalised glass plate before being screened for activity. selective peptide substrates will be developed as isozyme-specific inhibitors and applied in cancer cell lines. using chemical proteomics and the labelling technology, a selective nmt or nmt inhibitor could also be used to identify protein substrates of one isozyme. for this purpose computer programs are created which can generate fragments of one compared structure and to reveal homology by their scanning along the amino acid sequence of another. our analysis was performed by comparing the primary structures of all possible protein fragments with the amino acid sequences of all presently known natural regulatory oligopeptides. the oligopeptides were extracted from the erop-moscow database which at the time of analysis contained data on the structures and functions of more than , natural oligopeptide regulators. the structure-function analysis was performed using a specialized software package. the input data were the complete amino acid sequences of the proteins used as a source of fragments with a specified length. then the initial sequence was fragmented in a stepwise manner. for example, in the case of dipeptide fragments, this procedure produced fragments with the following numbers of amino acids from the n-terminus - - , - , and so on until the fragment that started at the second residue from the c-terminus. the cases when the amino acid sequence of a fragment coincided with part of the primary structure of a natural oligopeptide were recorded in the total protein chemical synthesis requires a case by case design and optimization which is governed by factors such as the solubility of the individual peptide segments, their primary sequence and in particular the presence of "difficult" amino acid residues at ligation junctions such as proline or the location of cysteines. usually, a subset of chemical tools are selected among a vast array of methodologies to match the specificities of the target protein. in this context, methods enabling the assembly of three peptide segments in the n-to-c and c-to-n direction play a central role and must be considered as complementary as they can be selected for building subdomains of the target protein. to date, most of the proteins were assembled in the c-to-n direction. only few methods are available for the n-to-c sequential assembly of proteins, whose design is highly challenging. we have recently reported that sea ligation, that is the reaction of a bis( -sulfanylethyl)amido group (called sea) with a cysteinyl peptide, allows the formation of a native peptide bond in water and at neutral ph . in this communication we will show that native chemical ligation and the unique chemical properties of sea group , can be combined in order to design a highly efficient one-pot three segments protein assembly procedure, working in the n-to-c direction amylin is one of the most amyloidogenic peptides, its fibrils are responsible for causing type ii diabetes. amyloid formation mechanism is investigated both to find amyloid inhibitors as potential medical drugs, and to use amyloids as potential self-assembling biomaterials [ ] . amyloid formation of amylin - , its reverse and designed analogue beta-sheets and beta-sheet stacks was studied by molecular dynamics (md), amber . , f force field. md revealed that for amylin - and its reverse analogue both the parallel and antiparallel beta-sheet and beta-sheet stack structures are stable suggesting that this could explain the high tendency of amylin to form amyloid fibrils. parallel amylin - beta-sheet stacks are kept together by two hydrophobic cores, while for the antiparallel system the dominating is the backbone hydrogen bonding between neighbor strands. also the bent form of the amylin - beta-sheet is stable. this is in concordance with transmission electron microscopy (tem) experiments stating that all three peptides, amylin - , its reverse and designed analogues, exhibited significant fibrillar polymorphism [ ] university of gdansk, poland molecular dynamics (md) of two peptides dlsfmkge (mk) and dlsfkkge (kk) not related to any known disease was run to investigate the mechanism of the amyloid formation. the parallel and antiparallel [ ] betasheets of mk and kk peptides were simulated by molecular dynamics (md), amber . , f force field, ntp protocol. it was found that antiparallel beta-sheets both of mk-and kk-peptides show much higher stability than the corresponding parallel beta-sheets. this md result was supported by atr-ftir spectroscopy [ ] . the betasheet stacks built from six ten stranded antiparallel beta-sheets of mk-and kk-peptides: x xmk and x xkk, were subjected to md. it was found that the mk-system, x xmk, is strongly kept together due to hydrophobic core built from two metionines, two phenylalanines and two leucines, but the kk-system, x xkk, which differs only by one mutation m k dissolves already at ns of md run, because the separate beta-sheets don't hold togather in the betasheet stack due to lost hydrophobic core. the hydrophobic core of the mk-system consists of hydrophobic units centered on the two phenylalaninetwo metionine hydrophobic interactions, and two leucines from the both sides stabilize the unit. this mechanism could be used in amyloid based biomaterials. urokinase plasminogen activator (upa) is a serine protease involved in the metastasis of several tumor types. upa is therefore an interesting target in cancer therapy. upain- is a new analogue of a highly specific peptidic inhibitor (upain- ) of upa. the peptide contains twelve amino acids and is cyclized through the cysteines at its termini (s -s cyclo-ac-cswrglenhaac-nh ). upain- inhibits upa with a ki of approximately μm. one method to improve binding affinity is multivalent exposure of the inhibitor, where the local concentration at the binding site is increased. fusion of upain- to the trimeric tetranectin showed improved binding affinity compared to the single peptide. here, we report efforts towards novel chemically linked upain- peptides to allow multivalent display. the ki value of an upain- dimer, linked by a short peg chain through the n-termini, was almost halved compared to that of the single peptide ( μm). this motivated us to explore the role of the site (n-or cterminal) and the size of the linking segment on the binding affinity. additionally, the influence of the number of upain- peptides in the molecule (two vs. four) was investigated by synthesizing a carboprotein that displayed four upain- peptides. we present two novel nmr spectroscopic approaches to study reversible self-assemblies in solution. both methods were applied on the self-assembling pseudodesmin a, a pseudomonas produced cyclic lipodepsipeptide that has the capacity to form pores in cellular membranes. , the first method is based on the dependence of the c α relaxation rate constants on the anisotropy of the assembly. when the monomer conformation is known and the multiple ch bonds in the monomer sufficiently sample all orientations, the rotational diffusion coefficients can be assessed, revealing assembly shape information. in addition, the orientation of the monomer within the assembly is obtained. the second method is based on fitting translational diffusion coefficient data as a function of concentration in a model-free way, i.e. without assuming an oligomer shape beforehand. here, it is assumed that the diffusion coefficient's dependence on the oligomer size behaves as a power law, which dramatically simplifies the expression for the average diffusion coefficient (measured by pfg-nmr) as a function of concentration. the fitted value of the exponent of the power law fully embeds all shape information of the assembly, and may be related to the socalled fractal dimension of the oligomer. moreover, this approach reveals mechanistic information concerning the assembly formation. both methods thus allow structural information of the assembly to be obtained, even when there is little or no prior knowledge available on the mechanism of the selfassembly. nucleotides and α-amino acids are crucial building blocks for living organisms. these chiral molecules are the biosynthetically precursors of two of the most important classes of biopolymers, dna and proteins, respectively. the d-structures of biomolecules are currently studied using a variety of techniques, while helical handedness is routinely detected by means of light pulses of opposite circular polarization. the difference in the uv absorption of these two circularly polarized pulses is called electronic circular dichroism (ecd). in nature, biomolecules explore a wide range of conformations with intrinsically strong ecd signals in the - nm region, but these signals are essentially absent in the visible. nanomaterials such as metallic nanoparticles (depending on their sizes) display absorptions in the visible region but are achiral. as a result, when biomolecules are co-assembled with nanomaterials their chirality is transferred to create a plasmon-induced ecd signal in the visible region. in this work, we present our results which underscore the occurrence of moderately strong ecd bands in the range - nm resulting from a series of appropriately thiolfunctionalized peptide oligomers (based on alternating l-ala and aib residues) covalently anchored to - . nm sized gold nanoparticles. we related the (positive or negative) signs of the ecd plasmonic signal with the oligopeptide length, that in turn is strictly associated with their secondary structure. this latter property was simultaneously monitored via ecd in the - nm range. we believe that in our systems a peptide-tometallic surface chirality transfer would take place. light can be controlled with high temporal and spatial precision. if a specific molecule is made light-sensitive, then a precise spatiotemporal control of some of its properties can be achieved. azobenzene is the most widely used photochromic group due to its propensity to pass reversibly from the cis to the trans state under irradiation with light of the appropriate wavelength. the cisand transazobenzene isomers exhibit different spatial arrangement of the aromatic moieties that give rise to significantly distinct physical and chemical properties. the design of novel azobenzene-based molecules with precisely placed photochromic groups able to induce photomodulation of macroscopic properties is currently attracting much interest. in this work, we explored the behaviour of the conjugate formed by linking each of the four hydroxyl groups of pentaerythritol to the carboxylic function of bis[p-(phenylazo)benzyl]glycine. this c α -tetrasubstituted α-amino acid bears two side-chain azobenzene groups. the resulting system exhibits tetragonal symmetry, with a total of eight azobenzene moieties, and can be viewed as a central core surrounded by a shell of azobenzene groups at the periphery. up to eleven (out of the possible fifteen) discrete states produced by sequential trans-to-cis isomerization of the individual azobenzene units have been observed depending on the time of exposure to uv-light. this process is fully reversible (cis-to-trans) under vis-light irradiation for several cycles. in addition, this compound has been shown to exhibit photomodulated physical properties, such as polarity and hydrodynamic volume. moreover, it shows a high propensity to self-assemble in aqueous solution, giving rise to supramolecular vesicles. light-scattering and electron microscopy experiments confirmed that a conformational reorganization of the vesicles can be triggered under exposure to uv or vis light. the total chemical synthesis of native or modified proteins is gaining increase importance in the study of protein function, but also in the development of protein therapeutics. it is usually achieved by assembling in water unprotected peptide blocks using so-called native peptide ligation methods. recently, our group has developed a novel native peptide ligation method based on a peptide featuring a bis( sulfanylethyl)amido (sea) group on its c-terminus in reaction with a cysteinyl peptide in water at ph . we will discuss in this communication the scope and limitations of sea native peptide ligation. for this, model sea peptides featuring all the possible proteinogenic amino acids were synthesized. their rate of sea native peptide ligation with a model cys peptide were determined in the absence of presence of guanidinium hydrochloride or other additives frequently used in ncl. we will present also experiments intended to clarify the mechanism of sea ligation such as the effect of ph on the rate of ligation, or the ability of the transient thioester sea form produced by in situ n,s-acyl shift to participate in thiol-thioester exchange , . overall, the data show that sea ligation is an interesting method for native peptide ligation at various x-cys junctions, and thus an interesting alternative to ncl. plga copolymers were used as the support for inducing controlled biomarkers releasing system. visualization of the penetration in the hippocampus of mice with confocal microscope was carried out by testing both peptide-free and peptide-bearing nanoparticles, previously labeled with the phthalocyanine fluorescent probe. the encapsulation degree of the larger ( - ) segment was less effective than the others thus stressing the importance of the peptide length to this internalization process. the results showed that all peptide-containing nanoparticles were able to cross the blood-brain-barrier thus indicating improved bioavailability and uptake for peptide delivery into the brain. in regard to the radiolabeling approach, the m tc radioisotope was used to label the peptide sequences at his residues, as previously described . stable metal-peptide complexes were obtained in - - - m peptide concentration range. noteworthy, higher metal labeling yield was achieved with peptide segments bearing his residues at peptide c-terminal position, thus pointing to a positiondependent effect for the m tc coupling reaction. in conclusion, the findings indicate potentials for the proposed encapsulation and radiolabeling strategies applicable for in vitro and in vivo diagnostic assays with these peptides for the study of amyloid plaques. we have used bifunctional short peptides (ac-cg n c-nh , n= , , ) to selectively link gold nanorods in an end-to-end manner. additionally, we have manipulated the gap distance between the rods by changing the length of the peptide linker. the presence of the peptide in the gaps was shown by incorporating a propargylglycine residue in the sequence, which was detected with surface-enhanced raman spectroscopy (sers). the acetylene moiety will allow further chemical modification of the linker in the gaps, opening a wealth of interesting molecular systems to be placed and studies inside self-assembled nanogaps. in this work, the fragmentation pathways of alitame, neotame and andvantame in comparison to those of aspartame and aspartame-d , were studied by negative ion electrospray ionization (esi) high resolution mass spectrometry (thermo orbitrap mass analyzer). accurate mass spectra of the dipeptides allowed proposing specific fragment ions. neotame and advantame, which are the n-( , dimethylbutyl) and n-[ -( -hydroxy- -methoxyphenyl)propyl] derivatives of aspartame, presented similar fragmentation to that of aspartame. for neotame and advantame, the "diketopiperazine'' pathway seemed to be the major one, while a pathway resulting to the formation of a pyrrolidine- , -dione derivative, through the involvement of the side chain carboxyl group of aspartate, was also observed. for alitame, the "pyrrolidine- , -dione" pathway was recorded. similarities in the fragmentation using either orbitrap or triple-quadrupole mass spectrometry have been observed. elucidation of the fragmentation is very useful for the trace-level determination of the artificial dipeptide sweeteners in complex matrices. generation of silver nanoparticles in the presence of oligoproline derivatives p. feinäugle, h. wennemers* eth zürich, switzerland in the last years, the generation of silver nanoparticles (agnps) attracts, due to its unusual physical and chemical properties, more and more attention. agnps offer great opportunities for applications in molecular electronics, catalysis, imaging and for antimicrobial coatings. the characteristics depend on their shape and size. many efforts have been made to optimise the generation process by, for example, varying the reducing agents, which usually are used for the synthesis or using manifold additives which should guide the nucleation and also stabilize the resulting particles. nevertheless, the generation of agnps in defined sizes and shapes still remains a challenge. we address this goal by utilizing functionalized oligoprolines that form a conformationally well-defined and rigid helical secondary structure (ppii) as additives. recently, we showed that by decorating this template with aldehydes which allow for in situ reduction of the silver, they act as scaffolds in the generation process and allow the formation of defined nanoparticles. we will report the results of the generation of agnps with various oligoprolines as additives, which differ in the attached functional groups as well as in the length of the peptides. laser desorption/ionization mass spectrometry (ldi-ms) using specific inert surfaces to promote ion formation has been widely investigated the last decade [ ] . in addition to porous silicon through the original dios technique, different materials were tested as potent ldi-promoting agents. we explored a variety of inert silicon-based uvabsorbing materials that were presenting different physico-chemical properties for the analysis of peptides [ ] [ ] [ ] . both material architecture (amorphous powders, structured particles, structured surfaces) and material hydrophilic/hydrophobic character tuned by specific chemical derivatization (oxidation, silanization) were probed as crucial parameters for achieving efficient and robust detection of an home-made array of model peptides covering a wide structural and mass diversity. through this set of experiments, we were able to compare the performances of all investigated silicon-based supports, especially taking into account peptide detection sensitivity (down to femtomolar concentrations) and reproducibility/repeatability (intra-spot/inter-spot signal variations) as well as the method robustness using conventional maldi-tof/tof instrument. having illustrated the capability to achieve both peptide detection and sequencing on these ionizing surfaces in the same run, high-throughput identification of protein tryptic digests by a rapid ms profiling and subsequent ms/ms analyses was achieved. comparison of the ms and ms/ms data with those obtained with sample conditioned in organic matrix [ , ] showed a great behavior for low mass responses demonstrating the capability of ldi on nanostructured silicon supports to be a complementary method to maldi in proteomic workflow. the dipeptides aspartame, alitame, neotame and advantame are low caloric artificial sweeteners. advantame , which is the n-[ -( -hydroxy- -methoxyphenyl)propyl] derivative of aspartame, is the most recent among them. an application for its approval has been applied in usa, australia and new zealand. such sweeteners are used in food products and beverages and they can help in managing body weight and disorders like obesity and diabetes. in this work, the simultaneous determination of aspartame, alitame, neotame and advantame by negative and positive electrospray ionization (esi), under hydrophilic interaction chromatography (hilic), is presented. advantame, neotame and intermediates were synthesized in our laboratories for the present application. the key-step for the synthesis of advantame and neotame was the reductive amination of h-asp(obu t )-phe-ome with -( -hydroxy- methoxyphenyl)propanal and , -dimethylbutanal, respectively. the chromatographic behavior of the artificial sweetener dipeptides was studied on two hilic columns: kinetex hilic (a fused core silica column) and zic-hilic column (a sulfoalkylbetaine column). the separation of dipeptides was achieved on kinetex hilic using mm ammonium formate buffer ph . / methanol / acetonitrile ( / / ), with a flow rate of μl/min at o c column oven temperature. at this ph, silica is neutral and the dipeptides are in positively charged form. the retention mechanism of all analytes seems to be partition to the water layer as well as hydrogen bonding. département de pharmacologie, université de sherbrooke, sherbrooke, qc, canada plasma and in vivo stability are essential requirements for the successful development of potential drug candidates or diagnostic imaging probes. rapid degradation of compounds in plasma may result in insufficient concentration to produce the desired pharmacological activity or to be used as a diagnostic agent. there are several strategies to improve plasma half life of peptides including pegylation, modification of nand cterminal fragments of peptide, replacement of labile amino acids, and cyclization . we have previously reported on probes which specifically detect matrix metalloproteinase- (mmp- ) activity with magnetic resonance and optical imaging , . mmps are zincdependent endopeptidases degrading the extracellular matrix (ecm) and involved in cancer progression. the main goal of this work was to find more stable probes without sacrificing enzyme specificity. we have selected specific mmp- substrates and their stability was evaluated in three different conditions: in plasma, in plasma with a mmp inhibitor and in a mmp- solution. the samples were analyzed by hplc to detect the degradation pattern of our compounds and by lc-ms to determine the molecular mass of peptide fragments. based on these studies, the most stable peptide was selected and incorporated in a solubility switchable probe with radiolabelled ( )ga-dota. its in vivo stability was estimated up to minutes, making it a suitable candidate for further investigations. cancer of thyroid gland is the most common malignancy of the endocrine system. the treatment improvement could be achieved by early diagnosis. the aim of the study was to identify cancer specific markers using the libraries of artificial receptors immobilized on the cellulose. an array of supramolecular structures formed from n-lipidated peptides attached to cellulose via aminophenylamino- , , -triazine was prone to formation of monolayer of "holes" and "pockets" in dynamic equilibrium. this selforganized structures were found capable of binding small guest molecules very efficiently recognizing the shape, size, and polarity of ligands, thus resembling arti cial receptors . recognition and binding properties of guest molecules by artificial receptors depends mainly on the character of peptidic pockets and structure of the fatty acid. proper construction of the binding pocket allows selective binding components of mixtures of compounds from a living organism . the preliminary data indicates that it is possible to construct an array of artificial receptors with diversified structures of peptidic pockets which are able to distinguish between components of homogenates from tumor and normal tissue. century. most of opioid alkaloids and their derivatives have μ-opioid affinity, while endogenous enkephalins are rather δ-than μ-selective. morphine is still the drug of choice for treating severe pain caused by cancer or surgical operation, but its side effects are the reason for the searching and development of new, selective mor agonists. the aim of our study is to choose within recently published crystallographic structures templates for homology modeling of the human μ-opioid receptor. we generated several models using different templates and all of them were evaluated by docking procedure (gold . ) ligands used in this investigation were synthesized and evaluated for their biological activity in our previous studies. they are enekphalin analogues with substitutions in second position. the best model of the human mu-opioid receptor was chosen according to data obtained from docking and in vitro biological activity of analogues and endogenous enkephalins. acknowledgments: this work was supported by nfsr of bulgaria project dvu / and cost action cm project do - / . . . pneumoniae, h. pylori, proteus sp. are considered as important factor contributory to development of rheumatoid arthritis (ra). the aim of this study was to investigate the level and specificity of antibodies binding to the synthetic peptides corresponding to the bacterial ureases "flap" region sequences in the rheumatoid arthritis patient's sera. for these investigations, peptides with amino acid sequences derived from "flap" regions of different ureases were synthesized using -( , -dimethoxy- , , -triazin- -yl)- -methylmorpholinium tetrafluoroborate (dmt/nmm/bf ) as coupling reagent. peptides were immobilized on a cellulose membrane. the level of antibody binding as well as specificity of them was analyzed by quantitative dot blot method using sera sera from rheumatoid arthritis patients (rap) and sera from volunteer blood donors (vbd). the results of studies suggest that "flap" region may be involved in arising antibodies participating in autoimmunological processes but not to fight infection. this effect indicates that the peptides analyzed by us could be useful for investigation of ra pathogenesis. this suggestion was confirmed by the antibodies absorption experiment which indicates that specificity of antibodies present in rap serum is slightly lower in comparison with vbd serum. it has been found that antibodies present in rap serum recognize not only a specific peptide but also peptides containing fragments with different amino acid sequences. it means that immune system of rap is unstable and may produce a wide spectrum of antibodies recognizing not only a specific epitope but also a set of similar structures. autoantigen-specific t-cells also play a crucial role in the initiation and perpetuation of dsg /dsg -specific t-cell responses. t-cells recognize epitopes from dsg protein and produce different cytokines, e.g. interferon-γ (ifnγ). functional t-cell epitopes of dsg protein have outstanding importance in immunopathological research, development and the design of novel diagnostic tools. our previous studies have shown that certain t-cell epitope peptides are able to stimulate the peripheral blood monomorphonuclear cells (pbmc) of pv patients more effectively than those of healthy donors. our aim was to select a set of t-cell epitope peptides as potential synthetic antigens which are reliably able to distinguish between donors based on the in vitro t-cell stimulating activity. we have prepared synthetic dsg oligopeptides by fmoc/tbu solid phase methodology. after cleaving from the resin with tfa the peptides were purified by rp-hplc, and they were characterized by rp-hplc, mass spectrometry and amino acid analysis. pbmc of pv patients and healthy donors were isolated; and the cultures were stimulated by dsg peptides in a concentration of . mm for hours, and the rate of ifnγ production was determined from the supernatants in sandwich elisa. synthetic dsg oligopeptides induced different in vitro ifnγ production rate on pbmc obtained from pv patients and healthy controls determined by elisa. our approach identified a synthetic antigen set as a promising biomarker for pemphigus vulgaris. [ ] . in particular, cap b (pelyafprvamide) has been shown to elicit antidiuretic activity in the green stink bug acrosternum hilare [ ] , an important pest of cotton and soybean in the southern united states. analogs of cap b containing either an (e)-alkene, cispro or a transpro isosteric component [ ] were synthesized and evaluated in an in vitro stink bug diuretic assay, which involved measurement of fluid secretions of malpighian tubules isolated from a. hilare [ ] . at a concentration of μm, the conformationally constrained transpro analog demonstrated significant antidiuretic activity, whereas the cispro analog failed to elicit any activity. the results provide strong evidence for adoption of a trans orientation for the pro in cap b neuropeptides during interaction with the receptor associated with the antidiuretic process in the stink bug. the work further identifies a scaffold with which to design biostable mimetic cap b analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting cap bregulated diuretic systems. the enkephalins are pentapeptides (tyr-gly-gly-phe-met/leu) with a proven antinociceptive action. it is believed that the interaction between them and the lipids composing the membranes is important for converting the peptides into a "bioactive" conformation , . using langmuir's monolayer technique the interaction of a synthetic methionine-enkephalin (met-enk) and its amidated derivative (met-enk-nh ) with mixed lipid monolayers composed of palmitoleoylphosphatidylcholine (popc), sphingomyelin and cholesterol was studied. the surface pressure-area (π-a) isotherms with regard to πmin, π max and the hysteresis curve shape of the pure lipid monolayers and after the addition of the respective enkephalins were detected. in addition, by using brewster angle microscopy (bam), the surface morphology of the mixed lipids-enkephalins monolayers were determined. our results suggest that there is a strong penetration effect of the enkephalins studied into the mixed monolayers. moreover, our results demonstrate the potential of lipid monolayers formed in langmuir's through in combination with bam to be successfully used as an elegant and simple membrane models to study lipid-peptide interactions at the air/water interface. acknowledgments: this work was supported by bulgarian ministry of education, youth and science, projects n do - / , drg / and my-fs- / . dept pharmacolgy, temple univ, philadelpha, pa , usa bioinformatic algorithms has predicted the existence of several potential hormone-like peptides transcribed from the ecrg gene . previous publications indicated a highly level of gene expression of ecrg products has been found in the pancreas , choroids plexus, epithelial cells, leukocytes, and macrophages . however, the presence in the hypothalamus and the major form of derived peptides in each tissue haves not been clearly identified. knowingledge of the precise peptide generatesd within a given tissue is essential to understanding its functions. we have generated the peptide specific antibodies to against ecrg -derived pprepro-augurin( - ) and developed a specific ria kit for the quantification of the such peptide in question. a method for the purification of endogenous ecrg -derived peptides from bovine hypothalamus also has been established. using ria to monitor the immunoreactive fractions and maldi-tof to identify the endogenous peptides, we foundhave detected the presence of ecrg derived molecular of bovine preproaugurin( - ) from the homogenates of bovine hypothalamus. immunohistochemistrycal staining by antibody aalso confirmed the presence of thee peptide in some of the hypothalamic cells. of hypothalamus. the amount of prepro-augurin( - ) in the hypothalamus although not soas high as pancreas, but is one third of the augurin level of the pituitary. conclusions: the native peptide derived from augurin preproteinecrg has been discovered.identified. we have confirmed the property of purified peptide,s prepro-augurin( - ), along with the synthetic peptide standards. the present study provides the necessary procedures such as the elution from ( ) c column, ( ) p sizing column, and ( ) a further purification conditions for hplc in order to enhance the immunoreactivity from tissue fractions and yield enough amount for identification. this dsip-related peptide (kn-dsip or knd) differs from dsip by only amino acid residues in positions and . we do not consider the homology between dsip and knd as accidental, bearing in mind functional significance of histone demethylases of the jmjc-group. methylation-demethylation of histones is known as an important mechanism of posttranslational modification playing a prominent role in epigenetic regulation of chromatin structure and gene transcription. dsip is also known as an effective "normalizer" and protector from homeostatic disorders induced by stress related disturbances. we suggest that histone demethylase of the jmjc-group containing dsip-related region can be considered as a possible protein precursor of endogenous peptides with dsip-like activity. in order to test our hypothesis we synthesized knd and studied its biological effects. in a preliminary assay cited below [ ] knd showed similar and probably more pronounced effects than dsip as an agent that stimulates endurance and stress-resistance of animals in the forced swimming test. also knd provided a more active detoxifying action after administration of a semi-lethal dose of the cytostatic agent. in the present work we assessed neuroprotective and antioxidative potency of both peptides in vivo and confirmed the higher efficiency of knd. this study is supported by the moscow government. is a tridecapeptide (pglu -leu -tyr -glu -asn -lys -pro -arg -arg -pro -tyr -ile -leu ) highly expressed in the central nervous system. this peptide elicits an analgesic response following peripheral or central administration. importantly, nt exerts a more potent analgesia than morphine at an equimolar dose, without having the associated side effects of opioid drugs. structure-activity studies have identified the c-terminal fragment nt( - ) as the biologically active minimal sequence. however, nor the full or truncated peptides cross the blood-brain barrier (bbb), thus hampering its clinical development. the substitution of pro by an unnatural amino acid silaproline (sip) increased bioavailability and plasma stability. structural properties conferred by the pro were also retained as determined by nmr and ir. aiming at delineating the mode of action of cl, three new cl derivatives bearing suitable labeling moieties, i.e the fluorescent molecule fitc, the streptavidin-counterpart biotinyl-group and the m tc-radiometal chelating unit dimethylgly-ser-cys, were designed, synthesized, purified, and characterized to be applied in in vitro and in vivo evaluation studies. the structure of the cl derivatives in aqueous solutions was studied with nmr, in parallel and in comparison with the parent molecule cl, in order to examine whether the presence of the labeling moieties has induced changes to the structure of the biologically active part of cl. cell survival assays with cl and the cl derivative bearing the fitc moiety were conducted in the pc cell line in order to explore their rescue effect. in parallel, the cl derivative bearing the dimethylgly-ser-cys moiety was successfully radiolabeled with m tc and its stability was assessed over time in its synthesis reaction mixture and in plasma. this m tc-radiolabeled derivative was subsequently administered to swiss albino mice in order to determine the biodistribution of cl in the living organism and its route of excretion, a study that has not been carried out so far for any peptide of the humanin family. furthermore, the potential interaction of cl with β-amyloid peptide, the hallmark of ad pathogenesis, was explored with circular dischroism. the results of this multifaceted approach to the biological action of cl will be presented. institute of biochemistry and biotechnology, martin-luter university, halle-wittenberg, germany kinins, such as the nonapeptide bradykinin, are important mediators of various physiological and pathophysiological responses including inflammatory disease, asthma, rhinitis, cell division, pain, vascular permeability, allergic reactions, pathogenesis of septic and endotoxic shock. there are two types of receptors for kinins, known as b and b . b receptors are constitutively expressed in wide variety of cells and required entire bk sequence for recognition, while b receptors have normally very limited expression and respond to [desarg ]bk. b receptors gene is turned on following either tissue damage or inflammation. accumulated evidence indicates that most of the clinically relevant effects of bk are functions of b receptors this being the reason why research on their antagonists is a topic of great interest. in our previous study we described the synthesis and some pharmacological properties of four new analogues of bradykinin (bk), designed by substitution of position or of the known [d-arg ,hyp ,thi , ,d-phe ]bk antagonist with l-pipecolic acid (l-pip) (both analogues were also prepared in n-acylated form with -adamantaneacetic acid (aaa)). our results showed that presence of l-pip in position slightly increased antagonistic potency in the blood pressure test, but it turned the analogue into an agonist in the rat uterus test. replacement of thi by l-pip in position also enhanced antagonism in the rat pressure test but preserved the antagonism in the rat uterus test. in the present study we continue our previous investigations to find structural requirements which in the case of bk analogues result in high b antagonistic activity. several new bradykinin analogues modified in their cterminus with d-pipecolic acid were synthesized using spps method. the biological properties of the analogues were assessed by their ability to inhibit vasodepressor response of exogenous bk in conscious rats and by their ability to inhibit the contractions of isolated rat uterus evoked by bk. acknowledgements this work was supported by the university of gdansk (ds/ - - - ). peptides with beta-turn structure in peptide/mhc complexes a. stavrakoudis department of economics, university of ioannina, greece major histocombatibility complex (mhc) molecules interact with small peptides and form complexes. in most of the cases, peptide's structure in these complexes is found in extended conformation. however, notable exceptions exist where the peptide forms a beta-turn structure. this happens mainly in the central part of the peptide in class i complexes [ ] , or at the c-terminal of class ii complexes [ ] . several peptide/mhc complexes were, derived with xray studies, were extensively subjected to molecular dynamics simulations [ ] in order to investigate the stability of this turn-like structural feature and to explore the factors that possibly contribute to this stability. it was found that both intra-peptide and peptide/mhc interactions might be responsible for peptide's conformation. the peptides were found to undergo several structural transitions indicating conformational plasticity and not a completely rigid structure inside the mhc groove. the results might be of special importance in designing defective peptide vaccines and beta-turn pharmaceuticals. the heptapeptide met-enkephalin-arg -phe (merf) with the sequence of yggfmrf is a potent endogenous opioid located at the c-terminus of proenkephalin-a (penk), the common polypeptide precursor of met-and leuenkephalin. our systematic bioinformatic survey revealed considerable sequence polymorphism at the heptapeptide region of different penk prepropeptides among vertebrate animals. four orthologous heptapeptides with single or double amino acid replacements were identi ed among animals, such as yggfmgy (zebra sh), yggfmry (newt), yggfmkf (hedgehog tenrek) and yggfmri (mudpuppy). each novel hepta-peptide, together with the mammalian consensus merf and metenkephalin, were chemically synthesized and subjected to functionality studies, using radioligand binding competition and g-protein activation assays in rat brain membranes [ ] . equilibrium binding af nities changed from good to modest as measured by receptor type selective [ h]opioid radioligands. the relative af nities of the heptapeptides reveal slight mu-receptor (mop) preference over the delta-receptors (dop). [ s]gtpγs assay, which measures the agonist-mediated g-protein activation, has demonstrated that all the novel heptapeptides were also potent in stimulating the regulatory g-proteins. all peptides were effective in promoting the agonist induced internalization of the green uorescence protein-tagged human mu-opioid receptor (hmop-egfp) stably expressed in hek cells. thus, the c-terminally processed penk heptapeptide orthologs exhibited satisfactory bioactivities, moreover they represent further members of the so-called "natural combinatorial neuropeptide library" emerged by evolution. corticotropin releasing factor (crf) exerts most of its physiological and pathophysiological actions by interacting with its type receptor (crf ) and activating different intracellular signalling pathways. the crf is a plasmamembrane protein, which belongs to the family b of g-protein coupled receptors (gpcrs) and like the other gpcrs consists of an amino-terminal extracellular region, a carboxyl-terminal intracellular tail and seven, mostly hydrophobic, membrane-spanning segments (tm -tm ), connected by alternating intracellular (il) and extracellular loops (el). binding of crf and its related peptides, such as sauvagine, to the extracellular regions of crf is associated with receptor activation and subsequent activation of different g-proteins and regulation of diverse signalling pathways. using a mutagenesis approach in combination with a radioligand binding study we found that trp and phe in the second extracellular loop of crf interacted with the amino-terminal portion of crf and sauvagine. interestingly only the interaction of sauvagine with trp and phe is important for crf -mediated stimulation of camp accumulation. in marked contrast the interaction between crf and the residues trp and phe was unimportant for the activation of adenylate cyclase. thus it is possible for trp and phe of crf to regulate distinct signalling pathways, or different sets of them, after their interaction with different peptides. we are now performing experiments to fully elucidate the signalling pathways that are regulated by the interaction of crf and sauvagine with trp and phe . these studies will advance the development of crf -selective selective signalling-specific peptides that would be extremely useful for the elucidation of the role of crf in many physiological and pathophysiological situations, and possibly for the treatment of several crf -related diseases. thymus humoral factor gamma- (thf-γ ), an octapeptide, purified from crude thf, retains essentially all the biological properties of thf [ ] [ ] . it regulates clonal expansion, differentiation and maturation of t-cell precursors, stimulates the production of lymphokine, maitains the normalization of impaired ratios between helper(cd +) and suppressor / cytotoxic (cd +) subsets and augments il- production in spleen cells. thf-γ has a calculated molecular weight of and has the following amino acid sequence: leu-glu-asp-gly-pro-lys-phe-leu. its poor stability towards protein enzyme limits its extensive application. with the inte ntion to promote its bioavailability, bioactivity and develop ideal immunoregulatory drug candidat, four series of derivatives of thf were designed and synthesized: . n-and cterminal acylation. .restitution the flexible segment gly-pro by unnatural amino acids -aminohexanoic acid (aca) in order to shorten the synthetic steps and simultaneity improve the bioavailability and biostability of peptide; . reserve protected group of some amino acid residus as spot mutation. . mannich-based cyclization was carried out on resin [ ] , phe was replaced by tyr serving as the active hydrogen component, a proline was introduced at the n terminal as the amine component and formaldehyde was used as the only component in solution. the bioactivity of synthesized products were detected. the leukocytopenia model in mice was induced by cyclophosphamide intraperitioneal injection. white blood cell count, thymus index and spleen index were detected to evaluate the immune function of compounds in mice. the results show that those compounds play a significant role in improving immune function in mice. the activity of compound lhl and lhl are also better than authentic compound tp- and tα . marine organisms have been recognized as a promising source for the development of new pharmaceuticals. in the course of screening for antitumor substances from marine organisms, we found cyclic peptides containing many nonribosomal amino acids such as hydroxyasparagine, hydroxyleucine, or other supporting a hydrophobic side chain that were shown to be a key element for their biological activity. the laxaphycine b, a cyclic lipopeptide isolated from marine cyanobacteria anabaena torulosa harvested in french polynesia constitutes an example of this peptide class. this compound has attracted our attention because of its micromolar cytotoxic activities on different cancer cell lines as well as its antiangiogenic properties which seems to be due to an interaction with the vegf receptor- - . the synthesis of the non-natural amino acids - and of laxaphycine b analogues will be presented along with their preliminary biological activities. immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases including dermatology. cyclolinopeptide a (cla) c(pro -pro -phe -phe -leu -ile -ile -leu -val -), a cyclic, hydrophobic nonapeptide isolated from linseed, possesses strong immunosuppressive and antimalarial activity. it has been suggested that both the pro-pro cis-amide bond and an 'edge-to-face' interaction between the two aromatic rings of adjacent phe residues in tetrapeptide unit are important for biological activity. this edge-to-face interaction can be influenced when phenyl rings are replaced by naphtyl substituent. in this communicate new analogues of cla modified by naphtylalanine ( -nal) in positions or or both and ( - linear analogues, - cyclic analogues) will be presented. the synthetic strategy and biological activity as well as conformational analysis will be evaluated. the onset of type ii diabetes mellitus (t dm) coincides with the deposition of fibrillar material in the islet of langerhans in the pancreas that is a clinical hallmark of more than % of patients suffering this disease. the main component of the pancreatic amyloid deposits is a -residues polypeptide hormone called islet amyloid polypeptide (iapp) or amylin. in this work we have examined, by means of cd spectroscopy and tht-fluorescence, the conformational polymorphism of both full-length - hiapp, and the related fragment hiapp - , and compared the results with the respective rat counterparts. moreover, the cytotoxic activity was determined toward different pancreatic β-cells lines in the attempt to correlate iapp's fibrillogenic properties with the ability to mediate cell death. together the results suggest that β-sheet conformational transition, that generally preludes to fibril formation, is not a prerequisite for eliciting toxicity toward β-cells cultures. interestingly, confocal microscopy indicated that both hiapp - and hiapp - can enter the cell and might exert their toxic action at intracellular level. acknowledgments: this work was supported by miur, firb-merit project rbne hwlz. due to its physiological functions, s proteasome is considered the target molecule in overcoming several diseases [ ] . its core particle s has three types of active sites: chymotrypsin-, trypsin-and caspase-like. many natural and synthetic compounds were tested for their ability to inhibit proteasome. a recent report describing the inhibition of s by the serine proteases inhibitor -bovine pancreatic trypsin inhibitor -was considered by us with great attention [ ] . our scientific interest is focused on peptide inhibitors and their interaction with serine proteases. sunflower trypsin inhibitor (sfti- ) is the smallest and the most potent peptide inhibitor in the bowman-birk family. owing to its size and the rigid structure (disulfide bridge and "head to tail" cyclisation) sfti- is willingly chosen as the lead structure in the search for new inhibitors [ ] . its sequence is shown below (lys the p residue responsible for specificity): & gly-arg-cys(& )-thr-lys -ser-ile -pro -pro-ile-cys(& )-phe-pro-asp& since native sfti- is not able to inhibit s [ ] , we have designed its monocyclic analogues (with disulfide bridge only) with lys or arg in position (p ) and at least one basic amino acid (lys or arg) in positions (p ') and/or (p '). all analogues inhibit chymotrypsin -(ic at the range of ÷ μm) and caspase-like (ic at the range of . ÷ μm) activities in vitro, whereas their activity towards trypsin-like specificity is much weaker. in several rat tissues, our view on ras has changed. metabolism of the ang-( - ) may represent alternative pathway of ang ii formation, importantly, independent on renin and ace activity , . ahmad et al. have described metabolism of ang-( - ) by human atrial tissues and showed that ang ii is formed mainly by chymase. this renin-inependent ang ii production could explain the "resistance" regarding use of ace inhibitors in patients with hypertension or diabetic nephropathy. noteworthy, the role of ang-( - ) in circulation is still unclear and there are no information about possible pharmacological modulation of its metabolism. in our study, we compared the ex vivo metabolism of angiotensinogen (fragment - ) in hypertensive (shr) and normotensive (wky) rats in organ bath of aorta and heart using lc-ms method . surprisingly, we identified ang-( - ) formed via reninindependent pathway to be a main product of angiotensinogen metabolism in rat aortic tissue and heart. in this setting, ang-( - ) appeared to be not only prevalent metabolite of angiotensinogen, but also served as a substrate for generation of ang i and ang ii. as compared to wky rats, formation of ang ii, from ang-( - ), was much higher in shr aortas but not in the heart. the functional consequences of these findings require further investigation. this study was supported by the grant n n polish ministry of science and higher education. the lysosomal cysteine protease cathepsin c (cat c), also known as dipeptidyl peptidase i (dppi), activates a number of granule-associated serine proteases with proinflammatory and immune functions by removal of their inhibitory n-terminal dipeptides. activity of this protease is associated with several pathologies in human body [ ] . in this work the characterization of cat c specificity using combinatorial chemistry methods will be described. the main goal of this work was to determine of substrate specificity of the prime region of this enzyme. the chemical synthesis and deconvolution of two libraries will be described. the hemostatic mechanism has the crucial role to prevent loss of blood from injured blood-vessels. this loss is prevented by the integrity of the vessel walls, by platelets aggregation or by blood coagulation, which in normal conditions is limited onto the local trauma of the vessel wall. in generally, the blood coagulation mechanism is important for maintaining vascular integrity and thus for the precaution of an organism from bleeding, which may also occur by blood coagulation caused by thrombin production. the diversion rate of this production leads to an expansion of thrombin to the general blood circulation. thus, when thrombin generation is not controlled by the mechanisms of inhibition, a widespread undesirable intravascular thrombosis is occurred. the whole process of platelets adhesion requires the presence of clotting factor viii (fviii), a necessary for the blood coagulation cascade glycoprotein, which takes part in the intrinsic pathway and acts as a coenzyme for the activation of factor ix, a serine protease depended on the thrombin production. the target of the present research is the synthesis of biologically active cyclic, head to tail, peptides, analogs of the sequence - of a subunit of fviii, which are potentially capable to block fviiia-fixa complex, reducing the thrombin production and thus the blood coagulation. the synthesized peptides are investigated for their inhibitory activity and tested for clotting deficiency by measuring the chronic delay in the activated partial thromboplastin time (aptt) and the reduction of the % value of the fviiia, which they generate in samples containing recombinant fviiia, in vitro. the blood coagulation is part of an important host defence mechanism, which under pathological conditions results in inappropriate intravascular coagulation when thrombin is produced. clotting sequence is the result of a cascade of two biochemical pathways, intrinsic pathway, so called because all components are present in blood, and extrinsic pathway, in which tissue factor is required in addition to circulating components. the activated form of factor viii (fviiia) is a key component of the fluid phase of the blood coagulation and plays an important role formatting a trimolecular complex with factor ixa, ca + and negatively charged phospholipids of the cells membrane. this complex is called tenase and participates in activation of prothrombin, which acts on fibrinogen to generate fibrin monomer, polymerized rapidly to form fibrin clot. the fviii is comprised of a heavy (a -a -b) and a light (a -c -c ) peptide chain, both cleaved by proteases at three sites, resulting in alteration of its covalent structure and conformation. its deficiency is known as haemophilia a. our research effort is focused on the synthesis, identification and biological evaluation of peptide analogs, expected to inhibit selectively the increasing of thrombin production. their sequence is based on the regions in which the fviii interacts with fix, specifically on the sequence - of the a subunit. the synthesized peptides are examined for their activity and tested for clotting deficiency by measuring the chronic delay in the activated partial thromboplastin time (aptt) and the reduction of the % value of the fviiia, which they generate in samples containing recombinant fviiia, in vitro. inhibitor with the following structure: ac-llllrvkr-nh , which has potent effects on the proliferation of prostate cancer cells. the potency and stability of this compound was subsequently enhanced by substitution of arg residue in position p with its conformationally restricted mimetic - -amidinobenzylamine (amba). nevertheless, the specificity toward pace was significantly reduced by this modification. thus, in order to improve its selectivity without sacrificing inhibitory potency we decided to use positional scanning approach. in this study we present synthesis of two series of peptide libraries, which were designed by substitution of leu in the p , p position of our control peptide (ac-llllrvkr-amba) with each of nineteen amino acid residues in order to verifying its influence on activity and selectivity of the resulting analogues. all peptides were synthesized by a combination of solid phase peptide synthesis and solution synthesis and tested for their inhibitory potency against furin and pace . the p -p fragments were synthesized by fmoc/tbu spps strategy on hydrazinobenzoyl or acid labile chlorotrityl chloride resin. then coupling of the -amidinobenzylamine · hcl was performed. the best modifications were combined to give as several multipoint substituted inhibitors. we believed that our work, will provide new important information about structure-activity relationship of these class of analogs in order to obtain potent and highly specific pace inhibitor. institute for research in biomedicine, parc cientific de barcelona, barcelona -spain a bacterial toxin-antitoxin (ta) system is composed of two genes organized in an operon encoding a toxin and an antitoxin that regulate the growth and death bacterial cell under various stress conditions. the operon parde encode a ta system formed by pare toxin and its antitoxin pard. pare is a kda protein that inhibits dna gyrase activity and thereby blocks dna replication. however the pare-gyrase interactions and the gyrase activity inhibition mechanism have not been explored. as an approach for understanding of this mechanism and to elucidate the pare region responsible for protein-protein interactions we have designed and synthesized a series of linear analogues of pare and investigated the ability of peptides to inhibit dna topoisomerases activity. so, based on structural data inferred from pare three-dimensional model , peptides were synthesized by solid-phase method. four peptides (parelc , parelc , parelc and parelc ), showed complete inhibition of dna gyrase supercoiling activity, by gel electrophoresis assay , with an ic of to μmol.l - . in addition, intrinsic fluorescence and fluorescence anisotropy assays showed that inhibition process must occur by interaction with the gyra subunit. differently of wild type pare, the peptide analogues were able to inhibit the dna relaxation of topoisomerase iv with lower ic values. interesting was that only parelc displayed inhibition of the relaxation activity of human topoisomerase ii. our results suggest a new class of molecules with simultaneous inhibitory activity in dna gyrase and topoisomerase iv. furthermore, we have obtained the first example of a synthetic peptide from a bacterial toxin with inhibitory activity on human topoisomerase ii. institute of experimental endocrinology, slovak academy of sciences, bratislava, slovakia the renin-angiotensin system (ras) has long been recognized as an important regulator of systemic blood pressure and electrolyte homeostasis. our understanding of ras has experienced remarkable change over the past two decades. besides, angiotensin ii, the new biologically active peptides [e.g. ang-( - ), ang-( - ), ang iv, ang-( - )] and pathways [e.g. angiotensin converting enzyme -ace ] have been described ; some of them, like ang-( - ) may oppose many actions of ang ii. importantly, despite all components of classical ras are found in adipose tissue , the data about fat formation of various angiotensins remain scarce. in our study, we compared the ex vivo metabolism of angiotensinogen, ang-( - ) and ang i in hypertensive (shr) and normotensive (wky) rats in organ bath of retroperitoneal and periaortic fat tissue using lc-esi-ms method. additionally, qpcr measurements of mrna expression of main enzymes involved in ang i metabolism were performed. both in the periaortic and epidydymal fat, the formation of ang-( - ) was higher than production of ang ii. fat tissue formation of two main ang i conversion products, ang ii and ang-( - ), differed significantly between shr and wky rats. compared to wky rats, the formation of ang-( - ) in periaortic fat tissue was decreased in shr. in opposite, in epidydymal fat tissue formation of ang-( - ) and ang ii was higher in shr. interestingly, there were no differences in aorta formation of ang ii and ang-( - ) between shr and wky rats. our results suggest that in hypertension visceral fat production of angiotensin peptides is increased, while generation of "beneficial" ang-( - ) in periaortic fat is decreased. however, the functional importance of such finding require further investigation. department of chemistry and biochemistry, university of washington, usa phospholipases a (pla ) are a superfamily of enzymes involved in various inflammatory diseases. in particular, human secreted giia spla is an attractive target for the development of novel medicines. we have shown that oxoamides based on γ-or δ-amino acids are potent inhibitors of cytosolic giva pla . very recently, we have demonstrated that a long chain -oxoamide based on (s)leucine displays inhibition of human and mouse giia spla s (ic nm and nm, respectively). a combined experimental/computational study was undertaken to further understand the role of the α-amino acid of -oxoamides for the inhibitor-enzyme binding. the crystal structure of giia spla s reveals a highly conserved ca + -binding loop and a catalytic dyad consisting of his /asp . -oxoamides based on hydrophobic αamino acids showed better binding score prediction compared to polar α-amino acid derivatives. a number of new -oxoamides based on α-amino acids were synthesised and tested for their inhibitory activity against giia, gv and gx pla . the -oxoamide based on (s)-valine displayed potent inhibition of giia spla (ic nm) in accordance with the predicted docking score. docking results reveal that (s)-valine-based inhibitor forms key interactions with the active site of the enzyme. the carboxylic group participates in a hydrogen bonding with gly and lys , and -carbonyl group with gly . furthermore, both carbonyl groups are in the proximity with ca + . the side chain of (s)-valine adopts a suitable orientation to interact with tyr and lys . the long aliphatic -oxoacyl chain is accommodated in the hydrophobic region of the active site and creates proximal contacts with leu , ile , his and phe . the search for novel classes of pharmaceutical molecules with enhanced therapeutic power has been the subject of numerous research groups all over the world. moreover, systems of immobilization and controlled release which are adapted to these new classes of molecules, has proven to be an area of extreme importance to provide the same therapeutic efficacy. using solid-phase chemistry a series of ccdb toxin analogous peptides were synthesized and were synthesized and tested against the capacity of inhibition of bacterial enzymes dna gyrase and topoisomerase iv (topo iv). subsequently those peptides were detained in drug delivery systems (dds) to be tested against the inhibition of growth of different bacterial species. in this data we could observed that the analogue ccdbsg could inhibit only dna gyrase and not the topoisomerase iv. in the other hand the analogue ccdbsg presents a hard inhibition potential against topo iv specially because of their structural difference. is possible conclude that topoisomerase iv presents the tertiary structure very similar to dna gyrase, but those mechanisms of action must be clearly distinct . in the in vitro studies, as expected, results revealed that the drug delivery systems are the key to the power efficiency of peptide analogues against the bacterial growth inhibition which cannot be observed when the peptides are free in solution. some of the different lipid compositions of the dds are demonstrating to be more efficient in the membrane cell transverse and this data previously assumes that it is possible to apply different types of dds to promote the peptide molecules transport across the cellular membranes according to several specific therapies. with this studies we have obtained more knowledge about the interaction system of enzyme-toxin and hopes which helps in future studies to development a new antimicrobial molecules class. it is urgent to develop less toxic and more efficient treatments for leishmaniases and trypanosomiases. we propose to target an ancestral form of the proteasome, the hslvu protease, which is present in the parasite's single mitochondrion, essential for the growth of these organisms and has no analogue in the human host. originally discovered in eubacteria, this complex is constituted by two central hexameric hslv protease rings sandwiched between two hexameric hslu atp-ase rings. as hslv shares a similar enzymatic mechanism with the host proteasome, we propose to inhibit the assembly of the complex in order to be selective. according to studies on bacterial hslvu, , the c-terminal segment of hslu is essential in hslv activation and in complex assembly, therefore representing a privileged target. we produced recombinant hslv, which is inactive alone, and showed that a synthetic c-terminal hslu peptide was able to induce the digestion by hslv of a fluorogenic substrate that we developed. with this enzymatic test in hands, we started the characterization of the interaction of the c-terminal portion of hslu with hslv. we will present the results obtained with various series of analogues of the original c-terminal hslu peptide, including truncated forms, ala scan, constrained analogues and multivalent constructions. helped by molecular modelling studies, the aim is to establish structural requirements, which could lead to high affinity and stable ligands able to inhibit the interaction between the hslu and hslv rings, obligatory for the degradation of proteins by the hslvu complex. finally, we checked that hslv was inhibited by classical active-site directed proteasome inhibitors like bortezomib. f. babos a,d , e. szarka b , gy. nagy c , z. majer d , g. saŕmay b , a. magyar a , f. hudecz a,d citrullinated filaggrin peptide (ccp) were detected in ra sera and anti-ccp positivity is widely used for diagnostic purposes. identification of new epitopes of filaggrin would be useful in the diagnosis of anti-ccp seronegative patients. in order to achieve optimal immune recognition of biotinylated epitope peptides it is important to analyse the effect of the labelling moiety on antibody binding. for these studies -as well as -mer peptides with nor c-terminal biotin were synthesised manually by spps, using fmoc/ t bu strategy. biotinylation was performed by using biotin, biotinyl- -aminohexanoic acid or , , -trioxa- , -tridecanediamino succinic acid linker modified biotin. labelled peptides were used in an indirect elisa, on neutravidin pre-coated plates and the binding was detected by anti igg-hrp. to examine the role of the presence/position of biotin in the secondary structure of the peptides, electronic circular dichroism (cd) method was used. we found that the ccp + serum samples specifically recognized the c-terminally biotinylated -mer filaggrin peptides, while showed no binding with the n-terminally biotinylated compounds. in case of the -mer epitope peptides there was no difference between the recognition of nand c-terminal biotinylated analogues. data presented suggest that the position of the biotin in case of the short filaggrin epitope peptides markedly influence the serum antibody binding. upon activation process, they are released from the granules and then involved in immunoresponse of the organism. when out of the cell those enzymes remain in free form or become associate with the cell membrane. the physiological role of this proteases is manifestated in several processes such cytokine and chemokine processing, platelet activation, and degradation of extracellular matrix's proteins [ ] . in this work results of the specificity of two members of nsps pr and hne evaluated using the combinatorial chemistry methods will be presented . both enzymes share primary specificity and to obtain the selective substrate that will be recognized only by one enzyme, the prime sites should be investigated. the general formula of the designed library is as follows: where in positions x ', x ' and x ', the set of proteinogenic amino acids (except cys) was introduced. abz is -amino benzoic acid served as donor of fluorescence and -nitro-l-tyrosine as acceptor. eukaryotic proteasome is a highly organized protease complex comprising a catalytic s core particle (cp) and two s regulatory particles (rp), which together form the s structure. the main function of this large intracellular protease is to degraded ubiquitine labeled proteins. the catalytic particle of the proteasome displays three distinct enzymatic activities: trypsin-like, chymotrypsin-like and glutamyl-like. the increase activity of the proteasome is associated with several disease including cancer [ ] . the main aim of this work is to synthesized the cell permeable fret displaying peptides that will selective cleaved by single proteasome activity. additionally each peptide when independently cleaved by the proteasome subunit, should emit the fluorescence energy in a different spectral region. our intention was designing substrates which would allow to monitor simultaneously (in a single experiment) and independently of three proteasome activities in this report, we will describe the chemical synthesis of several peptides modified at on cand n-termini by synthetic fluorescent amino acids the general formula of these peptides is as follows: where x is a non proteinogenic amino acid that serve as a donor of fluorescence, y amino acid that is a acceptor of fluorescence. the obtained fluorescent peptides were examined for their ability to cross the cell membrane. also kinetic parameters (k cat, km, kcat/km) with proteasome will be presented. approximately dubs are encoded in the human genome and are involved in a variety of regulatory processes, such as cell-cycle progression, tissue development, and differentiation. recently, several groups have introduced various methods for linking ubiquitin to different substrates via nonhydrolyzable isopeptide bonds, which resist the action of dubs. using these methods, one could explore the function and the mechanism of dubs and apply them in activity based profiling. here we present a new and convenient strategy for preparing nonhydrolyzable ubiquitinated peptides and proteins by nmethylating the isopeptide bond. using this method we prepared several nonhydrolyzable ubiquitinated peptides with different lengths derived from ubiquitinated h b and examined their affinity to different dubs. f.i. nollmann, c. dauth, d. reimer, h.b. bode* goethe universität, frankfurt, germany bacteria of the genus xenorhabdus and photorhabdus are gram negative gamma proteobacteria that live in symbiosis with nematodes of the genus steinernema. undergoing their partly entomopathogenic life cycle these bacteria not only produce antibiotics , and insecticides but also several different small molecular compounds and peptides. for the most part the biological benefits of these secondary metabolites have not fully been understood yet. with the help of inverse feeding experiments, hr-ms and nmr as well as molecular engineering we were able to characterize and/or isolate some of these peptides. since they are mostly produced in trace amounts, we synthesized them in order to make them accessible to continuative testing. given that not only linear but also highly methylated or cyclic peptides are produced, the synthesis was quite challenging. nevertheless, we were able to establish in our laboratory a general synthesis route for cyclic peptides and depsipeptides , as well as highly methylated hydrophobic linear sequences. testing several of these peptides has revealed activity against insect cells and against the causative organisms of neglected tropical diseases. cyclotides are a large class of plant peptides defined by a head-to-tail cyclized backbone and three conserved disulfide bonds in a knotted arrangement. these unique structural features confer them with remarkable stability and due to a range of bioactivities they are extensively investigated as templates in drug discovery . based on the use of oldenlandia affinis in traditional african medicine for its uterotonic principle we investigated crude plant extracts and semi-pure cyclotide fractions for the ability to induce uterine contractions using a collagen-gel contractility model . pharmacological analysis of the effects led to the identification of the oxytocin receptor, a representative of the g-protein coupled receptor (gpcr) family, as a molecular target for cyclotides. mass spectrometry-based sequence analysis of 'active' fractions revealed cyclotides with high similarity to the human oxytocin (h-ot) peptide that exhibited weak binding to the human oxytocin receptor. we further analyzed synthetic cyclotide-derived small ot-like peptides and grafted the h-ot sequence into the stable cyclotide frame. these peptides showed increased binding and activation as compared to native cyclotides. these findings may open new avenues for the discovery of gpcr ligands from natural peptide sources. gpcrs are promising drug targets and ~ % of currently used drugs act via binding to these receptors. natural combinatorial peptide libraries are likely to play an important role in identifying novel gpcr ligands . particularly plant cyclotides cover a large chemical space based on their high sequence diversity. together with their range of bioactivities and unique stable structure suggests that cyclotides are of current and future interest for drug discovery and development. acknowledgements: this work is funded by the austrian science fund fwf (p ). drosha and dicer are two key endonucleases for biogenesis of micrornas (mirnas) that regulate target mrna. drosha converts pri-mirna to ~ nucleotide (nt) pre-mirna in nucleus and dicer converts pre-mirna to linear ~ nt single-stranded mirnas in cytosol. even though dicer is potentially important to control availability of mature trans-acting rnas in cytosol, the enzyme itself does not seem to be the suitable target controlling mirna processing due to the lack of its substrate specificity. nature, however, might be intelligent enough to differentiate a variety of pre-mirna, so that a certain specific pre-mirna is converted to mature mirna in case it needs. therefore, other component(s) in the enzyme complex could be involved in recognition of auxiliary proteins from out sources to give extra specificity. we have synthesized trp-containing amphiphilic peptides against several pre-mirna. peptide b showed a picomolar binding affinity and a large specificity against pre-let a- . in vitro mirna processing, dicer activity was also selectively enhanced in the presence of this peptide. on treatment with this peptide on hct colon cancer and p ec cell lines, let a- mirna was more processed than reference mirnas. the toxicity of furan is known to rely on its selective oxidation in the liver by cyt p enzymes transforming it into the very reactive butenedial, which quickly reacts with proximate nucleophiles. this principle was used in our laboratory to develop a high yielding dna interstrand crosslinking methodology. in view of the demonstrated site-selectivity, the method further holds promise for sitespecific crosslinking dna to its binding proteins, which is highly relevant in the study of transient protein-dna interactions. furthermore irreversible dna binding can be achieved through such a covalent linkage, which is potentially useful for new generation therapeutics. the reactive furan moiety can in principle be incorporated either in the dna or in the protein. in the former case, a furan modified nucleotide was built into an oligonucleotide positioning the furan moiety at the periphery of the dna, to avoid interstrand crosslinking. for the latter approach, we initially chose to synthetically access a furan modified dna binding protein mimic. next to a previously described non-covalent gcn mimicking dimer, we have also investigated a new type of steroid-based dipodal dna binders. synthesis of the latter constructs has proven challenging in view of the immobilization of two peptide chains with helix forming tendency at close distance on the template. results, showing the power of microwave assistance will be discussed. in an alternative approach, a full length protein was modified with furan by amber suppression based on the structural similarity between a furan modified amino acid and pyrrolysine. pharmaceutical institute, university of bonn, an der immenburg , bonn, germany human matriptase- is a kda protein with trypsin like specificity. this protein exhibits a domain organization similar to family of membrane-bound serine proteinases known as type ii transmembrane serine proteinases. among many ascribed function in human body, this enzyme is a potent negative regulator of hepcidin, the peptide involved in iron homeostasis [ ] . matriptase- has a similar fold as other tmsp members, however their detailed specificity still remain unclear. the aim of this study was to determine the substrate specificity of this physiological important enzyme using combinatorial chemistry approach. in order to characterize the matriptase- specificity, the tetrapeptide library with c-terminal amide of aminocoumarin (acc-nh ) that serve as a fluorophore, was synthesized. its general formula is given below: x -x -x -x -acc-nh , where in position x -x the set of proteinogenic amino acid residues are present, whereas in position x lys or arg was introduced. deconvolution of such library was performed using iterative approach in solution. the results obtained indicate that matriptase- display diverse p -p specificity as compare to matriptase- . the most efficient hydrolyzed amino acid residue in position p appear to be ile, that is followed by arg in p and ser in p . the arg in position p is % faster hydrolyzed then lys. for selected substrates, the kinetic parameters (kcat, k m ) were determined. amyotrophic lateral sclerosis (als) is a chronic progressive disease. it is characterized by degeneration of upper or lower motor neurons, but its pathogenesis is still unknown and no effective treatment currently exists. it is known that antibodies to gangliosides have been found in some als patients, and these antibodies are also well known to be present in the patients affected by a variety of autoimmune diseases including multiple sclerosis. up to now anti-gangliosides antibodies are detected in clinical immunology laboratories using isolated non consistent antigen mixtures. therefore, we are interested in developing reliable and univocally characterized synthetic antigens for efficient antibody detection. csf (glc) is a family of structure-based designed glycopeptides that we previously developed as multiple sclerosis (ms) synthetic probes. these n-glucosylated peptides are able to detect specific autoantibodies in the sera of an antibody-mediated form of ms. autoantibody recognition was favored because of the exposition of the sugar amino acid on the tip of type ' β turn structures. aim of this study is the introduction, in the type ' β turn peptide structure, of the sugar moiety specific for anti-gangliosides antibody recognition by synthesizing specific building blocks. these building blocks are amino acids carrying glycans mimicking the biological activity of complex oligosaccharides. we selected sialic acids (in particular the n-acetylneuraminic acid -neu ac) because they are involved in a significant number of biological events. neuraminic acid and its derivates are widely distributed in animal tissues and in bacteria, especially in glycoproteins and gangliosides. therefore, we synthesized fmoc-l-asn(neu ac)-oh and fmoc-l-ser(neu ac)-oh. these building blocks will be introduced in the type ' β turn structure for the detection of anti-gangliosides antibodies in als. as a distinct pattern of ms could involve an antibodymediated demyelination, identification of autoantibodies as specific biomarkers is a relevant target. even if interesting data focused on the diagnostic and prognostic role of the detection of antibodies to myelin oligodendrocyte glycoprotein (mog) in adults' serum, its value remains dubious due to many other contrasting results. our research group identified csf (glc), an nglucosylated peptide, able to detect disease-specific autoantibodies in the sera of a statistically significant number of ms patients. , since this synthetic antigen may be considered as a mimic of aberrant post-translational modification (i.e. n-glucosylation) of myelin protein(s) triggering autoimmunity in ms, our goal is to obtain the extracellular domain of mog properly glucosylated thanks to a simplified native chemical ligation approach. for this purpose, the n-glucosylation will be introduced in a synthetic peptide fragment following the building-block approach by spps. the other protein fragment bearing an n-terminal cysteine will be expressed in e. coli after introduction of a selective point mutation into mog. finally, our aim is to test the semi-synthetic protein by sp-elisa to study the ability to detect autoantibodies in ms patients' sera and to find a potential cross-reactivity with csf (glc). this peptide is an endogenous ligand of the opioid receptorlike (orl ), previously referred to as "orphan" receptor, structurally and functionally related to the classical opioid receptors. also the hexapeptide ac-ryyrwk-nh is shown to be a selective ligand for the nop receptor with marked analgesic effect. with a view to developing ligands for the nop receptor with more potent analgesic activity, new series of the ac-rfmwmk-nh and ac-ryyrwk-nh , modified at position and respectively with newly synthesized β tryptophan analogues were synthesized . the aim of the present study was to examine the effects of naloxone (nal) and jtc- (nop receptor antagonist) in the analgesic activity of newly synthesized hexapeptide analogues. all peptides ( μg/kg), nal ( mg/kg) and jtc- ( , mg/kg) were injected intraperitoneally (i.p.) in male wistar rats. antinociceptive effects were evaluated by two nociceptive tests -paw-pressure (pp) and hot-plate (hp) and statistically accessed by anova. the results will be discussed compared to the referent compound in both tests used and mechano-and thermo-receptors are involved. [ ] . socs and socs have many similarities as well as some intriguing differences. both can block signalling by direct inhibition of jak enzymatic activity yet apparently require different anchoring points within the receptor complex. while the primary socs interaction is with a critical py residue within the jak catalytic loop [ ] it interacts also with py residues in the ifnαr and ifn r subunits in a jak -independent manner; the socs -sh domain also interact with y in jak , albeit with slightly lower affinity, but subsequent studies demonstrated a high affinity interaction with py residues located within receptor subunits [ ] . mutagenesis studies identified small regions at the n-termini of the socs and socs -sh domains, and at the c-terminus of the socs -sh domain, which were critical for phosphotyrosine binding. in order to gain insights in molecular discriminants for the interaction of both socs and socs toward jak and tyk we designed and synthesized peptides encompassing regions involved in proteins recognition. we set up a spr assay to evaluate the affinities of complexes formation. then through an alascanning approach we have designed new peptide sequences containing un-natural amino acids that are able to better recognize wild sequences and whole proteins. cellular experiments on stat activation signaling suggest their potential application as modulators of disorders involving socss overexpression. targeting proapoptotic death receptors (drs) to trigger apoptosis in cancer cells is a promising anticancer therapeutic approach. trail (tnf-related apoptosis inducing ligand) is a transmembrane homotrimeric protein belonging to the tnf family that triggers selective tumour cell apoptosis upon binding to its cognate receptors dr and dr . several strategies are being developed to exploit the unique cancer selectivity of the trail-dr pathway in therapy, including the use of recombinant trail targeting dr or dr . [ ] recently, a disulfide-bridged macrocyclic -mer peptide (derived from phage display) that binds selectively to dr has been identified. [ ] oligomeric versions of this macrocyclic peptide display increased binding avidity to the receptor and exhibit the capacity to activate the trail apoptotic pathway both in vitro and in vivo. [ ] however, disulfide bonds are susceptible to reduction and scrambling in vivo potentially resulting in the loss of the desired biological activity. among alternative linkages with increased redox stabilities, lanthionine thioethers, in which one of the sulfur atoms of the disulfide bond is removed have previously been introduced into biologically active peptides with some success. [ ] disulfide bridges can undergo a -elimination in alkaline conditions, followed by a michael addition to give a thioether bridge. optimization of this reaction led to the desulfurized analogue of the dr -binding peptide. the native dr -binding peptide and its desulfurized analog have been compared for their structural (nmr conformational analysis) and biological properties (affinity to dr and signaling pathways). the apelin/apj complex has been detected in many tissues and is emerging as a promising target for a number of pathophysiological conditions. in the central nervous system, apelin/apj was detected in brain regions involved in spinal and supraspinal control of pain, such as the amygdala, hypothalamus, dorsal raphe nucleus and spinal cord. we propose the hypothesis that apelinergic agonists represent a potential new approach to pain modulation and that the synthesis of stable analogues would lead to compounds with antinociceptive properties. there is currently little information on the structure/activity relationship (sar) of the apelin hormone. in an effort to better delineate sar, we synthesized analogs of apelin- modified at selected positions with unnatural amino acids, with a particular emphasis on the c-terminal portion. analogs were then tested in binding and functional assays by evaluating gi/o mediated reduction in camp levels and by assessing β-arrestin recruitment to the receptor. the plasma stability of new analogs was also assessed. several were found to possess increased binding and higher stability compared to the parent peptide. there is compelling evidence that the neuropeptide rfa and its cognate receptor gpr , are involved in the control of food intake and bone mineralization. among the gpcrs whose structures have been solved, gpr exhibits the highest sequence homology with the beta adrenergic receptor. the aim of this work was to experimentally characterize predicted ligand-receptor interactions by site-directed mutagenesis of gpr and design of point-substituted rfa analogs. starting from the x-ray structure of the beta -adrenergic receptor, a d molecular model of gpr has been built. the bioactive c-terminal octapeptide rfa( - ), kggfsfrf-nh , was subsequently docked in this gpr model and the ligandreceptor complex was submitted to energy minimization. in the most stable complex, the phe-arg-phe-nh part was oriented inside the receptor cavity whereas the n-terminal lysine remained outside. a strong intermolecular interaction was predicted between the arg residue of rfa and the gln residue located in the third transmembrane helix of gpr . in order to study this interaction, we have investigated the ability of rfa and arg-modified rfa analogs to activate the wild-type (wt) and the q amutant receptors transiently expressed in cho cells. the platelet receptor αiibβ plays a critical role in the process of platelet aggregation and thrombus formation. upon platelet activation its conformation changes leading to an increased affinity for fibrinogen. the αiibβ activation is regulated by "outside-in" and "inside-out" signaling. among the protein-protein interactions, which contribute to «inside-out» signaling, the most important is that of talin with the β cytoplasmic tail. it has been recently suggested that talin-mediated αiibβ activation relies on the cooperative interaction of the membrane proximal (mp) and the membrane distal (md) β regions with talin f domain and that the -n ply -motif of β , which can be phosphorylated at y , plays a critical role in this process. to evaluate the interaction of talin with the β tail of integrin we designed and synthesized three peptides corresponding to the md and mp parts of β in their carboxyfluoresceinlabeled form (md: cf-r akwdtannplyke -nh , cf-n nplykea -nh and mp: cf-k llitihdrke -nh ). emission and anisotropy fluorescence spectroscopy was used to quantitatively assess the affinity of these peptides for talin. furthermore, to challenge the role of the y phosphorylation in talin-α iib β interaction we also studied the binding of talin to the modified analogues of md, cf-r akwdtannpl(ptyr)ke -nh and cf-n npl(ptyr)kea -nh . our experiments revealed that the md and mp parts of β bind tightly to talin and that y phosphorylation has an inhibitory effect on this binding. functionalized oligoprolines as multivalent scaffolds in tumor targeting p. wilhelm, h. wennemers* eth zurich, zurich, switzerland oligoprolines are known to be structurally well-defined molecular scaffolds. in aqueous media, even short chain lengths of six proline residues adopt a polyproline ii helix (ppii). this secondary structure is a highly symmetric helix where every third residue is on top of each other in a distance of about . Å. [ ] the incorporation of azidoproline (azp) allows facile and versatile functionalization either via copper-catalysed azide-alkyne cycloaddition (cuaac) or an acylation that followed a staudinger reduction. [ ] based on the structural integrity of the oligoproline scaffold, targeting vectors can be conjugated via coppercatalysed azide-alkyne cycloaddition in defined distances. recent studies on radiolabeled oligoproline-bombesin conjugates, to target the gastrin-releasing peptide receptor (grp-r), showed in vitro and in vivo superior internalization in prostate cancer cells compared to the established monovalent ligands. [ ] a facile route to synthesize alkynylated ligands has been developed successfully. we are currently expanding this concept to the integrinligand c(rgdyk) as well as to [tyr ]-octreotide, which binds to somatostatin-receptors. the monovalent analogue of the latter, dota-[tyr ]-octreotide (dotatoc), is well established for diagnosis [ ] and therapy [ ] of somatostatinpositive tumors such as neuroendocrine tumors. the cu i -catalyzed azide-alkyne addition (cuaaa), the useful variant of "click chemistry," has emerged as a powerful technique for specific addition. that chemistry is also commonly used for conjugation, and cyclization of peptides. it is known that cyclization can increase the metabolic stability of peptides, as well as enhance potency or selectivity. another useful application of the cuaaa, which we are reporting, is the n-terminal crosslink of two synergic peptides to gain their potency. cuaaa reaction is performed on solid phase (merrifield resin) where one of the peptide components with azido group on the linker ( azido-hexanoic acid) is "clicked" with second peptide component in solution, made by fmoc strategy in partially protected form containing at n-terminal side alkyne group (fmoc-l-propargylglicine). cuaaa coupling is performed in dmf/t-buoh/h o with presence of cui and sodium ascorbate when reacting mixture was degassed. linked peptides are cleaved finally from resin and purified. as an application example we picked two endothelin active peptide analogues: bq derivative (a highly potent and selective eta antagonist) and irl- derivative angiogenesis is a key step in the transition of tumors from a dormant state to a malignant state. the vascular endothelial growth factor (vegf) is a major contributor to tumor angiogenesis. its pro-angiogenic activity is mainly mediated through binding to two tyrosine kinase receptors located predominantly on the surface of endothelial cells: vegfr- and vegfr- . vegf binding to these receptors triggers the activation of different signal transduction pathways responsible for the proliferation, survival and migration of endothelial cells . vegf/vegfr system constitutes a target to stop tumour growth. an attractive approach is the development of peptides, or small-molecules, with a high affinity for the extracellular domain of the receptors to prevent vegf binding. based on the x-ray structure of vegf and the d domain of vegfr- , cyclic peptides had been developed in our group . such peptides, mimicking simultaneously the - loop and helix · of vegf, can bind to d domain of vegfr- and inhibit receptors phosphorylation and thus map kinase pathway . we describe here our strategies to optimize peptidic antagonists of vegfr- . chemical modifications are made in order to better mimic peptide conformations and to increase their receptor binding affinities. we introduce a hydrophobic functional group at the c-terminal of the original cyclic peptide , some of such modified peptides reveal improved vegfr- binding affinity. otherwise, as the helix · presents most of the important residues in vegfr binding according to alanine-scan in the literature , we try to stabilize the helical conformation by insertion of aib residues or by peptide cyclisation. the peptides affinities are evaluated by an elisa test developed previously . institute of chemistry and biochemistry, freie universität berlin, thielallee , d- berlin, germany new polypeptide was isolated from the azemiops feae viper venom by combination of gel filtration and reversephase hplc and called azemiopsin. its amino-acid sequence (dnwwpkpphqgprpprprpkp) was determined by means of edman degradation and mass spectrometry. it consists of residues and does not contain cysteine residues. according to circular dichroism measurements, this peptide adopts a β-structure. peptide synthesis was used to verify the accuracy of the determined sequence and to prepare sufficient peptide amount for biological activity studies. azemiopsin efficiently competed with α-bungarotoxin for binding to torpedo nicotinic acetylcholine receptor (nachr) (ic . ± . m) and with lower efficiency to human α nachr (ic ± μm). ala scanning showed that amino-acid residues at positions - , - and - are essential for binding to torpedo nachr. in biological activity azemiopsin resembles waglerin, a specific blocker of muscle-type nachr from tropidechis wagleri venom. however the sequences of these peptides are markedly different, and azemiopsin is the first natural toxin to block nachrs that does not possess disulfide bridges. laboratory of peptide science, nagahama institute of bio-science and technology, nagahama, shiga - , japan while neutrophils infiltrate into damaged sites immediately after tissue injury, endogenous factors which induce their acute transmigration and activation have not been thoroughly elucidated. for the candidates, we recently identified two novel neutrophil-activating cryptides, mitocryptide- (mct- ) and - (mct- ), which were hidden in mitochondrial cytochrome c oxidase and cytochrome b, respectively [ ] [ ] [ ] . in addition, the presence of many neutrophil-activating peptides other than mct- and - was observed during their purification. these findings suggest that neutrophils are regulated by many unidentified peptides. here, we purified a novel neutrophil-activating octadecapeptide whose primary structure was identical to mitochondrial cytochrome c ( - ) from porcine hearts. we named this functional peptide as mitocryptide-cyc (mct-cyc). the structure-activity relationships of cytochrome c on β-hexosaminidase release from neutrophilic differentiated hl- cells demonstrated that cytochrome c ( - ) was the most potent cryptide among cytochrome c-derived peptides. since cytochrome c is known to be involved in the apoptotic process, our present results suggest that cryptides produced from cytochrome c play an important role in scavenging toxic debris from apoptotic cells by neutrophils. anthracis spores are very resistant and can remain dormant in soil for decades. therefore, an effective detection system for b. anthracis is urgently needed. recently, it was found that one of the components of the b. anthracis exosporuim is a collagen like protein whose carbohydrate portion is composed of the tetrasaccharide with the highly specific monosaccharide upstream terminal, named anthrose. since anthrose was not found on other bacterial spores, including those closely related to b. anthracis, this monosaccharide is an attractive target for the development of new b. anthracis detection and identification methods. peptide cyclization represents particularly interesting approach for the design of artificial receptors for anthrose, because cyclic peptides provide the possibility of having a spherical lipophilic binding site of appropriate size and shape for a particular carbohydrate substrate. the presence of hydrogen donor/acceptor groups within a three-dimensional structure permits carbohydrate substrates to be encapsulated, thereby allowing their binding in water. in order to determine whether the cyclic peptide receptor can selectively detect the anthrose, we have successfully prepared cyclic peptide combinatorial library (total peptides) by the process of divide, couple and recombine ("tea-bag" technology) using standard fmoc solid-phase peptide synthesis. prepared combinatorial library is screened for anthrose binding in fluorescence-based assay, and individual cyclic peptides with enhanced affinity toward anthrose are identified by the positional scanning deconvolution process. cyclization of linear sequences is a well-known approach used to restrict the flexibility of peptides. cyclization often increases selectivity of peptides towards one specific receptor type, increases metabolic stability and generally increases lipophilicity, which often improves the bloodbrain barrier permeability of peptides. in our previous study [ ] we have reported on the synthesis of a cyclic endomorphin- (em- ) analog, tyr-c(d-lys-phe-phe-asp)-nh , which elicited analgesia after peripheral administration. encouraged by the fact that this analog was able to cross the blood-brain barrier we designed and aliskiren is the first orally active, direct renin inhibitor to be approved for the treatment of hypertension. its structure and conformational analysis were explored using molecular dynamics (md) simulations. for the first time, md calculations have also been performed for aliskiren at the receptor site, in order to reveal its molecular basis of action. it is suggested that aliskiren binds in an extended conformation and is involved in several stabilizing hydrogen bonding interactions with binding cavity (asp / , gly ) and other binding-cavity (arg , ser , tyr ) residues. of paramount importance is the finding of a loop consisting of residues around ser that determines the entrapping of aliskiren into the active site of renin. the details of this mechanism will be the subject of a subsequent study. additionally molecular mechanics poisson-boltzmann surface area (mm-pbsa) free energy calculations for the aliskiren-renin complex provided insight into the binding mode of aliskiren by identifying van der waals and nonpolar contribution to solvation as the main components of favorable binding interactions. adamantyltripeptides and phospholipids in liposomal bilayers . now, we were primarily interested to study incorporation profile of mannosylated adamantyltripeptides. we have demonstrated that the adamantyl moiety, due to its liphophilic properties, penetrates into the lipid core of the bilayer while the hydrophilic part with the mannosyl moiety is exposed on the liposome surface. after concanavalin a (con a), a lectin, which specifically binds α-d-mannosyl residues, was added to the liposome preparation, increase in liposome size and appearance of aggregates has been observed. the enlargement of liposomes was ascribed to the specific binding of the con a to the mannose present on the surface of the prepared vesicles. the afm analysis revealed that the adamantyltripeptide molecules grouped into small domains that raise above the bilayer surface. the molecule size and molecular geometry, as well as the hydrophilic and hydrophobic surfaces in the structure of mannosylated adamantyltripeptides, are responsible for arrangement of molecules in the lipid bilayer. this approach might be a useful model for investigation of specific protein interactions with membrane receptors. also, the adamantyl moiety may be considered as a potential membrane anchor for different carbohydrate or other molecules of interest, which could be bound on it and thus exposed on liposome surfaces and as such used in targeted drug delivery. the assay is carried out in a well format p and images are captured throughout the course of the assay, thus we can not only determine a ligand's propensity to induce internalization, but also its efficacy and internalization rate. addition of test compound, followed by the standard agonist at a later interval, enables differentiation between agonist or antagonist activities. in the positional scanning format [ ] , while the possibility of agonists and antagonists working against each other within a mixture exists, the effects are minimized in screening the whole library as there are as many arrangements of the sub-libraries as there are defined positions. therefore while an agonist and antagonist might be present in a particular mixture in one sub-library they will be in different mixtures in all other sub-libraries. we have used this assay format to simultaneously screen for novel agonists and antagonists against the orexin receptor. assay development and library screening will be presented. [ ] . since the pro residue in position of em is very important in the proper conformational alignment of the two aromatic residues tyr and phe in em molecule at the receptor site, it is possible that structural modification around the pro residue yields compounds with unique biological properties and improved metabolic stability. in the present study, we synthesized seven em analogues containing isopro or constrained residues with oxopyrrolidine or oxopiperadine ring, instead of pro residue in position . all peptide analogues were synthesized solid phase method. incorporation of oxopyrrolidine and oxopiperadine rings were carried out on a solid support by the methods of gellerman, et al. [ ] and mohamed, et al. [ ] , respectively. opioid receptor binding activity for μ and δ-receptors using the development of resistance to mainstay cancer therapies has become a major limitation for the treatment of many cancers. there is an urgent need to develop new antineoplasic agents with innovative anticancer approaches. to overcome resistance to cancer therapies, our attention has turned to proteins that regulate multiple signalling pathways essential for tumour survival. among the few known nodal proteins upregulated in cancer cells and involved in many hallmarks of cancer, we are interested in survivin. an essential regulator of cell proliferation and apoptosis, survivin is sharply overexpressed in cancer cells and plays a major role in resistance. being a small protein, its bioactivity is relies mainly on protein-protein interactions (ppi) with different partners. a critical point for its multiple functions in cancer is its association with hsp , which is required for its stability. a nonapeptide from survivin called shepherdin has been shown to modulate the interaction of survivin with hsp by binding to hsp and to induce death of tumour cells. unfortunately, shepherdin is not cell permeable, has low proteolytic stability and shows poor bioavailability, limiting its use as anticancer therapeutic agent. to improve pharmacological properties of shepherdin, cyclic and peptidomimetic analogs of shepherdin have been synthesized followed by structure-activity relationship studies. in hsp binding studies, some cyclic hexa-and heptapeptidic analogs showed increased affinity compared to shepherdin. the synthesis of cyclic and peptidomimetic analogs and the results from the binding assays and the conformational analyses will be presented. the hexapeptides with formula ac-ryyr/kw/ir/k-nh have been identified as shortest peptide sequence with high nop receptor affinity, selectivity and marked analgesic effect. it was found that the following peptides act as partial or full agonists or antagonists of nop receptor in different in vivo and in vitro systems. these hexapeptides were used as chemical templates in sar studies , . the aim of the present study was the synthesis and the biological screening of new analogs of ac-rfmwmk-nh and ac-ryyrwk-nh , modified at position and respectively with newly synthesized β -tryptophan analogues . these non natural amino acids were prepared using reaction of asymmetric friedel-crafts alkylation of various indoles with a chiral nitroacrylate to provide optically active β-tryptophan derivatives. the four newly synthesized ligands for the nociceptin/orphanin fq (n/ofq) receptor (nop) have been prepared by solidphase peptide synthesis-fmoc-strategy. these compounds will be tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of wistar rats. bacterial infections are a common problem associated with dermal wounds. these infections can prolong or impair wound healing. hydrogel materials that display inherent activity against bacteria can be used to directly treat accessible wounds to prevent or kill existing infection. in this work, we describe the design and utilization of injectable gels prepared from self-assembling β-hairpin peptides having a high content of arginine. these gels were found to be extremely effective at killing both gram-positive and gramnegative bacteria, including multi-drug resistant p. aeruginosa. importantly, no added antibacterial agents are necessary since the nanostructure of the gel, itself, is the active agent. using self-assembling peptides for material construction allows facile structure-activity relationships to be determined since changes in peptide sequence at the monomer level are directly transposed to the bulk material's antibacterial properties. structure-activity relationships studies show that arginine content largely influences the hydrogel's antibacterial activity, and influences their bulk rheological properties. these studies culminated in an optimized gel, composed of the peptide pep r. pep r gels prepared at . wt % or higher concentration, demonstrate high potency against bacteria, but are cytocompatible towards mammalian mesenchymal stem cells. the general mechanism by which pep r exerts its action was explored and it is suggested that involves membrane disruption that occurs when cells come in contact with the gel's surface. atomic force microscopy (afm) was used to study the effect of the gel on the cell envelope morphology of e. coli. rheological studies indicate that the gel is moderately stiff and displays shear-thin recovery behavior, allowing its delivery via simple syringe. they are intimately involved in the molecular process leading to the delicate nano-patterned silica shells of diatoms. deciphering the mechanisms of silica-biogenesis in diatoms will inspire the development of novel routes for the biomimetic synthesis of silicon-based materials under mild conditions and expand the scope of biotechnological applications, e.g. for immobilization of enzymes in silica matrices. we synthesized silaffin peptides derived from c. fusiformis that carry posttranslational modifications such as phosphorylation or polyamines linked to lysine side chains. a distinct alteration of silica precipitation activity depending on the particular modifications of the silaffins emerged. these modified silaffin peptides were covalently linked to recombinant proteins by expressed protein ligation leading to stable protein-silaffin conjugates. using egfp as model protein, we could show that egfp-silaffin conjugates can induce biomineralization of silica and ensure an efficient and homogeneous immobilization of egfp into silica particles, superior to simple co-biomineralization approaches. moreover, a significant stabilization of immobilized egfp against denaturing agents was observed. we established a method for controlled immobilization of biomolecules based on covalent attachment of silaffin peptides with well-defined silica precipitation properties. currently this method is applied to the immobilization of biotechnological relevant enzymes in order to test their activity and the stabilization effect. herein, we present the covalent functionalization of multiwalled cnts (mwcnts) with organocatalysts based on proline or proline derivatives carrying either a dipeptide or a sulfonamide moiety. two different approaches were followed, namely, covalent grafting of the organocatalysts either at the tips or at the sidewalls of the cnts. for the former approach, mwcnts were oxidized in order to introduce carboxylic units at their tips and make them easily dispersed in aqueous solutions. then, oxidized mwcnts readily reacted with proline-based derivatives carrying a free amino unit yielding the corresponding hybrid materials. for the latter approach, the functionalization methodology based on in-situ generated aryl diazonium salts was followed. in this context, mwcnts were modified with aryl units carrying free amino terminal groups, which were subsequently conjugated with proline-based derivatives carrying a free carboxylic unit. all newly formed hybrid materials were fully characterized with complementary spectroscopic (atr-ir, raman), thermal (tga) and microscopy (tem) techniques. the catalytic evaluation of the activity of the cnt-based organocatalysts in aldol reactions is in progress. financial support from gsrt/ΕΣΠΑ - ΣΥΝΕΡΓΑΣΙΑ through ΣΥΝ- - -ΝΑΝΟΚΑΤΑΛΥΣΗ project is acknowledged. novel organogels based on self assembly of rationally designed pseudopeptides c. pappas, n. sayyad, a.g. tzakos, i. plakatouras section of organic chemistry and biochemistry, department of chemistry, university of ioannina, ioannina, gr- , greece self-assembly is becoming a rather intriguing way to build an array of nano-and micro-structured materials. low molecular weight organogelators can self-assemble into various architectural types in organic solvents through weak intermolecular interactions. such organogelators have potential applications in the generation of novel materials for nanobiotechnology . herein, we report the synthesis of rationally designed pseudopeptides and the conditions to form organogels. the obtained gels are responsive to temperature, and the sol-gel process is thermoreversible. the architecture of the constructed organogels was characterized via tem and spectroscopic techniques. diffusion ordered nmr spectroscopy (dosy) was further utilized to determine differences in the molecular shape of the different pseudopeptides. applications of the resulted compounds in nanotechnology will be reported. since , organocatalysis has met such a great rate of expansion that is nowadays considered the third major branch of modern asymmetric catalysis along with the transition metal catalysis and biocatalysis. after the seminal work of list, lerner and barbas on the enantioselective aldol reaction between acetone and -nitrobenzaldehyde catalyzed by proline, it became clear that amino acids and peptides could serve as an abundant pool full of potential to develop novel organocatalytic motives. following our recent report that the combination of a prolinamide with a thiourea group having as a spacer a chiral diphenylethylenediamine leads to an efficient organocatalyst for the aldol reaction, we recently considered the possibility to couple the prolinamide unit with an urea moiety. one of our main interests was the substitution of the diphenylethylenediamine spacer by a gem diamine derived from an α-amino acid. the gem diamine is easily synthesized via a curtius rearrengement of the corresponding acyl azide. after synthesis and evaluation of a number of potential catalysts, the prolinamide derivative bearing a gem diamine derived from (s)-phenylalanine and an aryl urea moiety proved to provide the best results in the reaction between cyclic ketones and aldehydes. utilizing mol% of our organocatalyst, the aldol products were obtained in high to quantitative yields (up to %), high to excellent diastereoselectivities(up to > : ) and high to excellent enantioselectivities (up to % ee). peptide self-assembled monolayers are of current interest to study physicochemical properties of modified metal (e.g. au) surfaces. rigid peptide scaffolds could enhance the interaction between gold surfaces and labels by reducing and precisely monitoring the distance between the supported monolayers and gold. the c α -tetrasubstituted αamino acid -amino- , -dithiolane- -carboxylic acid (adt) , which contains a cyclic disulfide system, is interesting in this respect because it may allow the parallel binding of the peptide helical chain to the metal surface. adt occurs in nature and has been utilized in medicinal chemistry and in a model compound of [fefe] hydrogenase. we synthesised a series of constrained helical peptides, based on the ala-ala or the ala-aib sequence, containing one or two adt residues. these peptides were functionalised with spectroscopic or opto-electronic labels. among the large number of reactions involving the formation of carbon-carbon bond, the addition of ketones to nitroolefins is a powerful tool for the synthesis of γ-nitrocarbonyl compounds, useful intermediates for pharmaceutical industry. our recently reported primary amine-thioureas based on tert-butyl esters of natural amino acids exhibit excellent performance for the michael reaction of ketones with nitroolefins providing the products quantitatively and almost stereospecifically (> % ee). , using this methodology, enantiopure baclofen and phenibut (analogs of gaba) have been synthesized. polymersupported organocatalysts constitute a great challenge for the michael reaction. in the current study, we report the immobilization of amine-thiourea catalysts containing ( s, s)or ( r, r)-diphenylethylenediamine and tert-butyl aspartate, on various polymer supports, either directly or through spacer units. the solid-supported catalysts evaluated in the reaction between acetone and βnitrostyrene and highlighted the importance of the choice of the polymer as well as the presence of the spacer or not. the direct attachment of the primary amine-thioureaaspartate to a crosslinked polystyrene-divinyl benzene resin containing a uniform distribution of aminomethyl groups provides a supported catalyst that affords the product of the reaction between acetone and β-nitrostyrene quantitatively and in high enantioselectivity ( % ee a. theodorou, g.n. papadopoulos, c.g. kokotos* laboratory of organic chemistry, department of chemistry, university of athens, athens, greece after the pioneering report that proline can catalyze efficiently the intermolecular aldol reaction between acetone and a variety of aromatic aldehydes, it became evident that amino acids and peptides can afford a plethora of different structural scaffolds for novel catalysts. along the first decade of its life, organocatalysis has grown to such an extend that now it is considered the third major branch of asymmetric catalysis. recently, researchers have paid special attention to other amino acids rather than proline. some primary amino acids have already been applied to a number of transformations with success. usually improved catalytic properties are observed when derivatives of primary amino acids are utilised. we have undertaken a study on the application of simple and cheap primary amino acids and amino acid derivatives, either commercially available or easily obtained, as organocatalysts for the asymmetric α-amination of aldehydes. in the present work, we report that the use of simple derivatives of primary amino acids like phenylalanine and aspartic acid can efficiently catalyze this transformation leading to products in high to quantitative yields and enantioselectivities up to % ee. the majority of the organocatalysts developed up to now for asymmetric organic transformations employ more than one functionalities in the catalytic mechanism that act through either covalent or non-covalent interactions. for example, proline employs the pyrrolidine nitrogen and the carboxylic acid group, while chiral thioureas combine the thiourea functionality with a tertiary or a primary amino group. we have recently shown that an amide of proline with a diamine carrying a thiourea group is a very good catalyst for the enantioselective aldol reaction. trying to improve the activity, we have found that a tripeptide-like thiourea having as building blocks (s)-proline, ( s, s)diphenylethylenediamine and (s)-di-tert-butyl aspartate provides the products of the reaction between ketones and aromatic aldehydes in high to quantitative yields and high stereoselectivities (up to : dr and % ee). a number of structural modifications of the catalyst were undertaken in order to understand the role of the hydrogen bond donors of the catalyst, i.e. the prolinamide hydrogen and the two hydrogen atoms of the thiourea group. we have come to the conclusion that the importance of the hydrogen bond donors of the catalyst follows the order: thiourea hydrogen originated from aspartate › amide hydrogen › thiourea hydrogen originated from diphenylethylenediamine. g eldrug s.a., patras , greece a convenient and facile synthesis and in vitro biological evaluation of n-substituted -butylimidazole derivatives as potent angiotensin ii (ang ii) receptor type (at ) antagonists have been reported in the present study. a series of imidazole based compounds bearing the biphenyl moiety at the n- position, a halogen atom at the c- and polar substituents such as hydroxymethyl at the c- position were synthesized. , these compounds were evaluated for binding to human at receptor and for ang ii antagonism in vitro on isolated rat uterus. in particular, butyl- -[[ ΄-( h-tetrazol- -yl)biphenyl- -yl]methyl]imidazole derivatives complexed with the at receptor and showed high binding affinity. these analogues were also found to be active in the rat uterotonic test. importantly, their binding affinities and potencies were comparable to those of losartan. these results indicate that the hydroxymethyl at the c- position of the imidazole ring is favorable for high affinity binding and antihypertensive activity and in line with the activities of the losartan counterparts. experimental findings are in good agreement with docking studies, which were undertaken in order to investigate ligand/at receptor interactions. z-leu-glu-his-asp-aluc, suc-leu-leu-val-tyr-aluc) are good substrates for bioluminescence assays, for example in the detection of caspase activity during apoptosis . these substrates generally offer significant advantages, such as increased sensitivity, ease of use, and high throughput screening capacity. luciferase-based assays are typically -to -fold more sensitive than the comparable fluorescent assays (rhodamine , -amino- -methylcoumarin (amc) and -amino- trifluoromethylcoumarin (afc)). the synthesis of different type peptide-amino-luciferin conjugates and their precursors have been published and some of them are commercially available. however, because of their high price the in vivo application of these conjugates is limited. to solve this problem we successfully worked out a new, easier and more convenient and economical method for the preparing these derivatives starting from -chloro-benzothiazole. moreover this products have excellent purity (> %) and adequate yield ( - %). major health problems arising from bacterial resistance towards existing antibiotics make discovery of antibacterial drugs with new mechanisms of action pertinent. although proof of concept for a novel antimicrobial approach using peptide nucleic acid (pna) antisense targeting of essential bacterial genes was obtained a decade ago, this technology is still limited by the lack of carriers that facilitate effective bacterial delivery and confer optimal pharmacokinetic properties to the prospective drugs. [ , ] in the past two decades, parallel efforts of exploiting naturally occurring antimicrobial peptides (amps) as drugs have been made. the cationic amp subclass appears to be directly involved in the innate immune response towards microbial infections. [ ] so far only few cell-penetrating peptides, with activity on mammalian cells, and other membrane-active peptides, have been investigated as potential vehicles for bacterial delivery. for instance, cationic amps with an internal target appear not to have been investigated for bacterial delivery of antibiotics. the aim of this project is to develop highly potent genetic antibiotics by exploiting naturally occurring antimicrobial peptides as potential delivery vehicles for antisense peptide nucleic acid oligomers. the amps are chosen from amps reported to act via intracellular targets, and thus must possess an inherent ability to permeate bacterial cell membranes without direct killing of the bacteria. faculty of chemistry university of gdansk, gdansk, poland azt ( '-azido- ' '-dideoksythymidine), a modified nucleoside used in antiretroviral therapy and peptide plant hormone -systemin were used as substrates of , -dipolar cycloaddition (click chemistry). systemin is -aa peptide defense hormone released in response to plant (tomato, tobacco) damage or pathogen attack. we examinated whether systemin's fast movement through plant tissues could be used for cargo (azt) transport. the huisgen cycloaddition also known as , -dipolar cycloaddition is a chemical reaction belonging to the larger class of cycloadditions. reaction between organic azide and alkyne appended substrates allows the synthesis of the desired conjugate in high purity and yields irrespective of the sequences and functional groups on either of the two substrates [ , ] . conjugate of azt-systemin has been synthesized by click chemistry, using systemin modified at n-terminus with propiolic group and azt. the conjugation was catalyzed by cu(i). the reaction was fast, efficient and regioselective. its progress was easily monitored by capillary electrophoresis (ce). ce was also applied for characterization of systemin and azt-systemin stability and movement throughout tomato leaf and stem. despite the fact that systemin moves rapidly through tomato tissues, our calorimetric (itc) studies showed that the peptide does not interact with liposomes-cell membrane model. universitätsklinik für nuklearmedizin, inselspital, bern, switzerland regulatory peptides (e.g., somatostatin, bombesin) have been shown to be suitable vectors for the specific delivery of radioactivity to tumors for diagnostic and therapeutic applications in nuclear oncology. a potential drawback of such vectors is their inherent instability in vivo. thus, new strategies are needed for the stabilisation of radiopeptides in order to improve their bioavailability and, consequently, increase their accumulation in the targeted tissue. it has been suggested that , , -triazoles, readily obtained by cuaac, are suitable amide bond surrogates which are resistant to proteases. in the present study, we report the synthesis and pharmacological evaluation of radiolabelled, triazole-containing analogues of the gastrin releasing peptide receptor (grpr) targeting peptide bombesin (bbn). to study the effect of backbone modifications in the minimal grp-binding sequence, we synthesized a series of analogues of [nle ]bbn ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , in which each amide bond is individually replaced by a , -disubstituted , , triazole. after radiolabelling of the peptidomimetics, their binding affinity and internalization kinetics were determined using pc- cells. metabolic stability was evaluated in blood serum. a number of the novel tumor-targeting peptide analogues presented exhibit both a retained high affinity (nm) towards the grpr and an improved serum stability. first preclinical data on the in vivo evaluation of the most promising candidate will be presented. to the best of our knowledge, this is the first report of the systematic replacement of amide bonds with , , triazoles within the binding sequence of linear, high affinity peptides. the methodology can be applied to a variety of peptide vectors and thus, holds great potential for the development of novel, stabilized peptide-based radiopharmaceuticals. dna is the molecular target for many of the drugs that are used in cancer therapeutics, and is viewed as a nonspecific target of cytotoxic agents. although this is true for chemotherapeutics, other agents that were discovered more recently have shown enhanced specificity. the development of new site-specific dna binders, which are associated with the recognition of the dna major groove, are based on the design of transcription factor mimics that bind the dna as a dimer , and prevent specific genes from being transcribed. these could ultimately result in interesting biomedical applications as designed genome interfering agents or diagnostics. in order to approach this biological constructs, we choose the bzip leucine zipper transcription factor as a model to mimic. as the entire structure cannot be synthesized without expensive, complicated and time-consuming biotechnological methods, the substitution of the dimerization domain by a less complex scaffold is the first step in the design. thus, we consider a steroid based scaffold as a candidate. the specific choice of the steroid scaffold as substituent is inspired by its known ability to enhance proteolytic stability of attached peptides, by its conformational properties ensuring correct positioning of the two appended chains and by its potential to increase bioavailability. this transcription factor binds specific dna sequences by dimerization and inserting short α-helices into the dna major groove. in order to attach the peptides to the scaffold, different strategies were studied. firstly, applying the well-known click chemistry, functionalizing the scaffold with an alkyne moiety, the peptide with an azide and viceversa. secondly, via the unknown resin to resin transfer reaction (rrtr), which has not been applied on peptide chemistry so far. this unprecedent methodology consists on the reaction of a peptide, which is attached on a safety-catch resin, with a second resin bearing a nucleophilic amino terminus resulting in amide bond formation. during the process, the peptide on solid support undergoes cleavage. an hexapeptide was synthesized on a preloaded safetycatch resin. deoxycholic acid derived scaffold with orthogonally protected amines was attached to tentagel resin that acts as acceptor resin. rrtr experiments were performed at both c and c positions of the deoxycholic acid derivative. in addition, this convergent strategy can be applied to other different peptide conjugated systems. we recently described a new kind of cyclized peptide in which the cyclization is performed between the side-chains of two diaminoacyl residues via a diversely substituted guanidine bridge. we showed that the degree of bridge substitution could impact on the orientation of the bridge inside the cycle and therefore the peptide conformation. we prepared two series ( and atoms cycle size) of cyclic enkephalin analogues to assess the potential effect of this kind of bridge on the biological activity. the compounds were synthesized on the solid support via the formation of a thiourea bridge and with the variable substituent being introduced at the last step before cleavage. it is noteworthy that the synthesis afforded at least two stable and separable conformers for each analogue of the shortest cycle series. generally, one major and one minor species were recovered. but in the case of di-substituted compounds with a cyclic moiety (pyrrolidine or piperidine substituents), three significant species were obtained. analogues were submitted to various biological assays (binding to μ and δ opioid receptors and functional assays). we observed a significant variation in affinity and selectivity for the receptors as a function of the degree of bridge substitution. a structural analysis by d nmr has been undertaken and correlated the variation in activity with a variation in conformation. the origin of the multiple conformers observed for the analogue with a pyrrolidine susbtituent was also investigated. this kind of cyclization could represent a useful tool to easily modulate the conformation and biological activity of a unique peptide sequence. the t-cell response is triggered by the formation of the trimolecular complex between the major histocompatibility complex (mhc), the immunodominant myelin protein epitopes and the t cell receptor (tcr). herein, we report the design and synthesis of non-peptide analogues with the ability to mimic the immunodominant epitope - of mbp , . the mimetics were designed to block the formation of the trimolecular complex and therefore the t-cell activation , . more specifically, indole analogues were synthesized with substitution at positions and or . these molecules contain a carboxyl or an ethyl ester group in position and a benzylamino or phenylamino group in position or . the synthesis of the indole ring was achieved by fischer reaction followed by catalytic hydrogenation, reductive amination or arylation and ester hydrolysis. the synthesized molecules were purified using liquid chromatography, and they were identified by mass spectrometry and h-nmr. laboratory of peptide science, nagahama institute of bio-science and technology, nagahama, japan amyloid β peptide (aβ), the main component of senile plaques in the brain of alzheimer's disease (ad) patients, is formed by proteolysis of amyloid precursor protein (app). as β-secretase (bace : β-site app cleaving enzyme ) triggers aβ formation by cleavage at the aβ domain nterminus, it is a molecular target for ad therapeutic intervention. previously, we reported potent pentapeptidic and non-peptidic bace inhibitors containing a substrate transition-state mimic. although these inhibitors exhibited potent inhibitory activities, their molecular-sizes appeared a little too big (mw> ) for developing practical drugs. in this study, we designed a series of small molecular peptides, with bace inhibitory activity, lacking the p -p ' region on the basis of the conformational structure bound in bace . design and synthesis of new '-peptidyl-trna analogues, in particular "hydrolysable" analogues, which represent covalent conjugates of peptide-nucleic acid (pna) with "stop-peptides," were carried out. such compounds are of interest as tools to study the ribosome functioning and as inhibitors of protein biosynthesis. ( aminoethyl)glycine pna models '-end trna sequence cca in designed structures. computer simulations showed the formation of watson-crick pairing of the pna cytosine residues with s rrna nucleotides g and g involved in interactions with peptidyl-trna during its specific binding in p site of the ribosomal peptidyl transferase center (ptc). short "stop-peptides" were planned for conjugation with pna. these peptides form stable complexes with the ribosomal tunnel (rt) that leads to ribosome stalling and translational arrest. structures of "hydrolysable" 'peptidyl-trna analogues that could form peptide bond with amino acid residue of aminoacyl-trna in a-site of ptc included '-deoxyriboadenosine instead of the pna adenine containing residue. such conjugates would permit to identify the chemical nature of specific sites localized in rt and responsible for interactions with amino acid residues of the nascent polypeptide chain. pna and "stop-peptide" as well as pna-"stop-peptide" conjugates were prepared by solid phase synthesis on sasrin polymer using fmoc/bhoc(boc) strategy. synthesis of "hydrolysable" conjugates included modification of the 'hydroxyl of '-protected '-deoxyadenosine by n-blocked "stop-peptide", deprotection of the '-hydroxyl, its conjugation with n-protected pna and removal of protecting groups from the resulted conjugate. the binding of the new '-peptidyl-trna analogues with ribosome will be tested by chemical probing and in the cell free translation system. this study was supported by the russian foundation for basic researches (grant - - -a). a close structural similarity of endomorphin- and another atypical opioid peptide, morphiceptin, which both have a phe residue in the third position, encouraged us to study antinociceptive activity of these two peptides and their analogues. in order to improve the affinity and chemical stability of these opioid peptides, we have designed, synthesized, and analyzed novel analogues. the first modification included endomorphin- and morphiceptin analogues, where halogenated phenylalanines in position or were incorporated as surrogates of the native phenylalanine. another important modifying element is non-protein amino acid canavanine (cav) and its analogue (sarg). it is well documented that cav and sarg exhibit strong analgesic activity. two new morphiceptin analogues were synthesized by introducing cav and sarg in position . we further characterized their antinociceptive activities by the paw pressure (pp) test. the experiments were carried out on male wistar rats ( - g), treated with i.p. doses of mg/kg. e eldrug s.a., patras , greece the renin angiotensin system (ras) has been a prime target for the therapy of cardiovascular diseases. angiotensin ii type (at ) receptor mediates vast majority of biologically detrimental actions. non-peptide at receptor blockers are presently the most specific means to block the ras enzymatic cascade. the dupont group was the first to develop losartan (dup ), an orally effective angiotensin ii receptor blocker, which is metabolized in vivo to the more potent antagonist exp . herein, we report on the preparation of e-urocanic acid based analogs, focusing our attention on the introduction and structural modifications of the substituents on the imidazole ring as well as the modifications on the acrylic side chain. in particular, we have designed and synthesized a series of urocanic acid analogs bearing the biphenylmethyl tetrazole moiety at the n- of the imidazole ring. additionally, the rigid acrylic chain was lengthened by esterification resulting in the ethyl ester and on the other hand the latter was readily converted to the corresponding acrylic alcohol or aldehyde which may proved to be effective structural elements for enhancing biological activity. finally, a lipophilic alkyl chain such as the n-butyl group was introduced at the -position of the ring which may possibly enhance the antihypertensive activity. docking studies and biological evaluation of the synthesized analogs are being undertaken. university of athens, department of chemistry, laboratory of organic chemistry, , panepistimiopolis zografou, athens, greece the backbone modification of bioactive peptides with replacement of a scissile peptide bond in enzymatic hydrolysis is a well-established strategy for developing protease inhibitors. in particular, for zinc metalloproteases, which contain a zinc atom in their active site, several successful modifications have been reported over the past years. phosphinic pseudopeptides are among the best candidates when addressing the challenge to potent and selectively inhibit zinc proteases. a thorough search in the literature revealed the absence of any reference regarding thiophosphinic pseudopeptides. we thought that this class of compounds would add a valuable tool in the field of zincbinding groups. in the present study, we describe in detail the first synthesis of a new class of phosphorous compounds, thiophosphinyl dipeptide isosters (tdis). we prepared several fully protected thiophosphinate pseudodipeptides of the general formula pg-phe-Ψ[p(s)(ox)ch ]-gly-pg' starting from the corresponding phosphinate pseudodipeptide using lawesson's reagent. selective deprotection of these compounds was also studied and the results are disclosed. these compounds can be used as building blocks for the synthesis of longer thiophosphinic pseudopeptides after suitable deprotection and elongation as well as transition transition state-mimicking inhibitors for several zinc metalloproteases. in the last decade, trypsin inhibitor sfti- isolated from sunflower seeds [ ] has become one of the most studied peptidic inhibitors of serine proteases. owing to its small size and a strong trypsin inhibitory activity (ka = . × m - ), sfti- is considered to be a very attractive template for designing proteinase inhibitors with the potential use as pharmacological agents [ ] . it could also serve as an affinity probe for the isolation of trypsin like (sfti- ) or chymotrypsin like ([phe ]sfti- ) proteinases. following this idea, we decided to synthesize a set of cell-permeable monocyclic sfti- analogues with a fluorophore moiety attached at their n-termini. the presence of the fluorophore in the molecule enabled us to show that the analogues can cross the cell membrane. the cell penetration assay was performed using multiple cell lines (hela cells and human fibroblasts cell line ( br. n) was obtained from european collection of cell cultures (ecacc)). for all the obtained peptidomimetics, we determined the association constants with cognate proteinases. selected peptides were also used as a probes for the detection of inhibitor -proteinase complex, which was achieved by the means of gel filtration chromatography equipped with fluorescence detector and acrylamide native gel electrophoresis. the functional reconstruction of folded protein surfaces with peptide-based mimics is an enormous scientific challenge. the majority of proteins show activity through a small area of their folded surface: "the binding site". however, linear peptides are too flexible and seldomly adopt the correct d-structure of the binding site spontaneously. therefore, they show limited or no activity at all . crucial for activity is to control the secondary (αhelix, β-sheet and/or β-turn) and tertiary structure (relative orientation of subdomain structures). we present the development of a new type of watersoluble scaffolds that have the potential to control both secondary and tertiary structure of discontinuous (i.e. double-loop) protein mimics. the new scaffolds contain a first pair of reactive functionalities to constrain the linear peptide conformation via a 'clips' reaction , stabilizing the secondary structure. next to this, a second functionality allows for ligation of two dissimilar constrained peptides to form a discontinuous binding site mimic via oxime-ligation or click-reaction. these ligations offer the ability to position different peptide loops in d, thus mimicking the tertiary structure of the native protein. most unique to our approach is the fact that all chemical conversions are performed in aqueous media, using side-chain unprotected peptides . growth hormone-releasing peptide (ghrp- ) is a synthetic hexapeptide (his-d-trp-ala-trp-d-phe-lys-nh ), which interacts with two kinds of receptors: growth hormone secretagogue receptor a (ghs-r a) and cluster of differentiation (cd ). the latter is a membrane glycoprotein member of the class b scavenger family, and decreases the internalization of oxidized lipids into macrophages, as well as causes inhibitory effects on angiogenesis associated with binding to thrombospondin. to increase activity and selectivity for the cd receptor, different analogues of ghrp- were synthesized. in particular, substitution of trp in ghrp- by aza-amino acids has given selective analogs, due likely to induction of a β-turn secondary structure. for aza-peptide synthesis, a submonomer solid-phase approach has proven effective to introduce side chains onto the semicarbazide residue. studying influences of benzylidene, benzhydrylidene and fluorenylidene residues during the alkylation of the semicarbazide, superior conversion was observed with fluorenone derivative, and mild alkylation conditions employing et noh as base have improved yields and minimized racemisation. our presentation will focus on the improved submonomer synthesis method for optimization of selective and potent cd- ligands with antiatherosclorotic and anti-angiogenic effects. for instance, the integrin αvβ , vitronectin receptor, is expressed in a number of cell types and has been shown to mediate adhesion of osteoclasts to bone matrix, vascular smooth muscle cell migration, and angiogenesis. integrin αvβ also play a significant role in tumor growth, invasion and metastasis, and is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartic (rgd) tripeptide sequence. rgd has been shown to be potent antagonist of the integrin αvβ , and has excellent anti-angiogenic properties including its suppression of tumor growth in animal models. in this context, drug design based on the rgd structure may provide new treatments for diseases such as thrombosis, osteoporosis, and cancer. we designed and synthesized series of short rgdmimetics containing the sequence xaa-gd, where xaa is arg-mimetic. as promising candidates we have chosen canavanine (cav) and canaline (can) instead of the basic residue arg. in order to improve antitumor activity of the parent molecule, c-terminal modifications were also applied. their cellular uptake was determined on human breast (mcf ) cancer cell lines. furthermore, the in vitro cytostatic effect was evaluated by mtt assay on human liver hepatocellular carcinoma (hepg ) and human breast (mcf ) cancer cell lines after , and hours of treatment. in the case with the human tumor cell lines (hepg , mcf ) and c-modified analogues, statistically reliable results were achieved for the most of concentrations used. acknowledgements: this work was supported by bulgarian ministry of education and science, project my-fs- / . microwave assisted solid phase synthesis of urea and urea/amide based foldamers k. pulka, c. douat-casassus, g. guichard* european institute of chemistry and biology, university of bordeaux -cnrs umr , pessac, france foldamers are fully arti cial molecules that structurally and functionally mimic variety of biopolymers. among them, aliphatic n,n'-linked oligoureas with proteinaceous side chains can adopt extremely robust helical folds stabilized by intramolecular three-centred h-bonds. owing to their resistance to enzymatic degradation, diversity of side chains and structural predictability urea-based foldamers represent unique scaffolds to elaborate functional mimetics of α-polypeptides. of note, heterogenous oligo(urea/γamides) backbones obtained by substituting nh groups by ch display very similar folding propensities. in our laboratory we are investigating the solid phase synthesis of urea and urea/γ-amide oligomers. urea bonds are incorporated into the growing chain by reaction of active succinimidyl carbamates. previously we have applied two different strategies involving fmoc-or bocchemistry, but both methodologies suffer some limitations. therefore a new strategy (compatible with the use of tfa sensitive linkers and side chain protecting groups) featuring azide as a masked amine group has been developed. the synthesis of new azido protected succinimidyl carbamate building blocks is reported. they were obtained in steps from α-amino acids ( - % overall yield). the staudinger reduction with pme was successfully applied to restore the amine group after urea formation on solid support. in addition, microwave irradiation has been found to dramatically accelerate the synthesis. overall, this azide-strategy combined with microwave irradiation was found to be very effective for the solid phase synthesis of oligoureas and related hybrids, surpassing previously developed approach utilizing fmoc chemistry. these antibiotics should have a mechanism different from currently used antibiotics to circumvent existing resistance mechanisms . previous results have shown that "genetic" antibiotics operating by gene silencing in bacteria via rna interference may be successful new candidates. efficient silencing requires efficient crossing of cell membrane. this step can be alleviated using cell penetrating peptides (cpp) as carrier of drug candidates, such as peptide nucleic acids (pnas) which inherently have poor internalization properties . the aim of this study is to elucidate mechanisms of uptake in bacteria using pna-cpp conjugates, which previously have shown promising antibacterial effects . the fate of the pna and cpp parts of the conjugates, once inside the cell, is investigated regarding localization and possible degradation within the cell. furthermore, a method for toxicity testing of pna-cpps is being developed using histamine release in rbl- h cells as a quantitative measure of allergenicity of pna-cpps. the prospect of this information is to define boundaries within which cpps can be found, thereby rationally designing novel efficient antibacterial biomolecular drug delivery systems. oxytocin and its fragments have the potential to influence behavioral and cognitive functions, including their disturbances in some brain disorders. therefore, there is an interest to synthesize new peptide-steroids chimeras for potential therapeutic use. oxytocin analogue was synthesized in solution by coupling azido-phenylalanyl residue or p-azidopegylated handle to the n-terminal end of oxytocin molecule. its c-terminal fragment pro-leu-gly-nh (mif- ) was elongated at proline residue by the same type of azido handles as well. both peptides were marked for fluorescent detection of their possible binding on brain slices. peptide chimeras with the suitable steroids were prepared via azide click to the triple bond on the modified steroid counterpart like ( α)- -hydroxypregn- -en- -yn- -one, -norchol- -en- -yn- β-ol. steroidyl-peptides were then used in the trials using rat-brain slices. the sites of the peptide-steroids chimeras bound to the brain tissue were identified with the aid of fluorescent microscopy. the suitable chimeras will be tested for their penetration through blood brain barrier for the pharmacological effects. indicating that the orientation of the n-butyl group is of primary importance. docking studies revealed that the highly active analog affords an additional hydrophobic binding feature compared to losartan which fits to an extra hydrophobic cavity. these results may contribute to the discovery of new biologically active molecules by a convenient and cost effective synthetic strategy. the context of pain research, the co-administration of opioid agonists and nk antagonists previously led to an enhanced antinociceptive potency, and recently largent-milnes and co-workers have shown that a hybrid opioid-nk octapeptide was able to attenuate tolerance development, related to sustained opioid treatment. our group has prepared a compact opioid agonist-nk antagonist peptidomimetic chimera dmt-d-arg-aba-gly-nme- ', '-bn(cf ) that served as a lead structure. we report a solid phase method for the synthesis of the amino- , , , -tetrahydro- -benzazepin- -one (aba) structure, which is used as a central unit in the investigated dual ligands. this method allowed the rapid assembly of new bifunctional ligands containing the aba structure. variations of the d-arg , gly and n-benzyl substituents were made. the introduction of d-cit , a gly → β-ala substitution and the removal of the trifluoromethyl substituents in caused considerable shifts in receptor binding. the obtained structure-activity relationships will be presented. hence, a promising approach for the treatment of dmd is the use of drugs to force ptc readthrough. (+)-negamycin is a dipeptidic antibiotic containing a hydrazide structure. although (+)- was not clinically developed due to some toxicity, it was recently reported that (+)- restore dystrophin expression in the muscles of mdx mice, an animal model of dmd. therefore, (+)- is a promising therapeutic candidate for diseases caused by nonsense mutations. based on our own efficient total synthetic method of (+)- , structure-activity relationship (sar) study was perfromed to discover derivatives with a potent readthrough-promoting activity. we found a derivative, ( r)- -hydroxy- -aminohexanoyl-glycine exhibited not antimicrobial activity but a similar readthrough activity to (+)- , suggesting that the ptc readthrough mechanism can be distinguished from the antimicrobial mechanism. moreover, we synthesized -epi-negamycin and found that this analog exhibited a similar activity to (+)- in in vitro readthrough assay. this result hence prompted us to synthesize a -dehydro-derivative, e.g., -dehydro- -epinegamycin , which is a natural product with little antimicrobial activity. surprisingly, we found that showed a higher in vitro readthrough-promoting activity than (+)- . this result suggests that mother nature independently evolved readthrough-promoting products like suppressor trna, in distinction from aminoglycosides, which show both antimicrobial and readthrough-promoting activities. agricultural university of athens, athens, greece high interest has been paid to synthetic structural motifs that promote specific conformations because of their importance for the development of new therapeutic peptidomimetics. in addition, such motifs may show catalytic activity for asymmetric organic transformations. during the last two decades, various synthetic structural motifs that promote reverse turns have been studied. following our interest on chiral prolinamide-thioureas that present interesting organocatalytic activity, we have undertaken a combined experimental/computational study to understand the structural features that may stabilize a reverse turn in short-length peptidomimetics containing a thiourea functionality. compounds with the sequence r-pro-diphenylethylenediamine-thiourea-asp(obut)-obut (r: boc or fmoc, or boc-ala), were synthesized and studied by nmr spectroscopy (tocsy, h- c hsqc, noesy, roesy spectra) for the sequential assignment and the exploration of the dipolar connectivities. sampling of the conformational space was driven by the noe intensities while molecular dynamics simulations were further applied to the consistent with the experimental data conformers in order to monitor the stability of the formed hydrogen bonding interactions in the course of time. energy refined produced conformers were subsequently modified by applying all combinations of d-and l-amino acids at each site in a stepwise manner. the modelled structures were studied in silico aiming to explore the combinations of heterochiral residues which would promote a folded structure and would favour the potential of β and γ turn motif. the most promising combinations were chosen for synthesis and subsequent nmr characterization. in this research project we will deal with chemical strategies to produce suitable surface modifications in order to induce multidirectional cellular migration along gold surfaces. to achieve this objective we want to use and characterize self-assembled monolayers (sams) of thiolated dna chains (dna-sh) adsorbed on gold surfaces through the hybridization with complementary modified single-stranded pnas. pna is a structural dna mimic obtained by polymerization of n-( aminoethyl) glycine monomers that replace the ribose-phosphate backbone characteristic of natural nucleic acids. it is an achiral, uncharged, and relatively rigid biopolymer of high biological and chemical stability, and it can bind complementary dna strands with higher affinity than the corresponding dna sequences.for all these reasons we have chosen pna as a key molecule to promote and assist the movement of cells. by producing a chemical gradient of dna-sh along a gold surface in the presence of a chemotactic molecule it will be possible to obtain and control a directed cellular migration. the norwegian structural biology centre and the centre for theoretical and computational chemistry, department of chemistry, university of tromso, troms , norway renin is a highly selective aspartic protease which catalyzes the hydrolysis angiotensinogen, a protein secreted from the liver, to the decapeptide angiotensin-i. angiotensin-i is further processed by the relatively nonspecific angiotensin converting enzyme (ace) to give the octapeptide angiotensin-ii, a potent vasoconstrictor and the dominant peptide produced by the reninangiotensin system. renin catalyses the rate determining step in the formation of angiotensin-ii, and has for several decades been an established therapeutic target for drug development in relation to hypertension. in the search for renin inhibitors, substituted piperidine derivatives have been identified as promising, - and piperidines have proven to be efficient scaffolds for the development of novel non-peptide aspartic protease inhibitors, particularly towards renin. [ ] [ ] [ ] we herein describe a series of -triazolyl substituted piperidine derivatives that have been synthesized from n-boc protected trans- -ethynyl- -hydroxy piperidine and tested as novel renin inhibitors. piperidine derivatives containing a -substituted , , -triazol- -yl substituent were found to be most active and molecular docking experiments provides a rank order that is in very good agreement with experimental data. the cxcr /sdf- axis is involved in many biological processes such as hematopoiesis, immune cell migration, as well as in cancer metastasis. cxcr also mediates the infection of t-cells with x -tropic hiv functioning as a coreceptor for the viral envelope protein gp . cxcr , as a pharmaceutical target, is of utmost importance but the lack of synthetic agonists has seriously slowed down drug development. it has been recently described by our research group , that grafting the sdf- n-terminus onto a side-chain of the inverse agonist t . generated high affinity synthetic agonists as well as partial agonists for the chemokine receptor cxcr . to remain stable towards proteases and act as useful pharmaceutical tools, the pk-adme properties need to be improved with a gradual transition to peptidomimetic structures. medicinal chemistry witnessed major advances with the discovery of small synthetic molecules that mimic the natural peptidic substrates. these small molecules do not undergo proteolytic degradation, an advantage they hold over natural counterparts. in order to improve stability against proteases, part of the sdf- chain was replaced with variable lengths of polyethylene glycol and unnatural amino acids at differents positions. here, we have produced a series of compounds, most of which showing nanomolar affinities for cxcr and some are displaying partial agonistic properties. tlrs are the innate immunity receptors that recognize the epitopes found on surfaces of various cells and therefore they initiate and sustain the atherogenic inflammatory response [ , ] . we assume that the use of small stat mrna−binding pna−inhibitors to manipulate the activity and expression of stat could prove an attractive therapeutic strategy in treatment of atherosclerosis. to that end we synthesized a specific stat mrna−binding pna inhibitor as well as a non-specific pna to compare their inhibition of gene expression. in our work we developed effective method of synthesis of pna−peptides conjugates by means of "click chemistry". determination of optimal conditions for conjugation (connection of pna with the peptide) will allow for the design of compounds useful in gene therapy. the specificity of pna hybridization to complementary dna fragment was verified by capillary electrophoresis (ce). as an artificially synthesized somatostatin analogue, tyr octreotate (toca) can specifically bind to somatostatin receptor (sstr), which are usually over-expressed on many tumor cells. carbohydration of n-terminus of toca has resulted in improved pharmacokinetics and tumor targeting ( ) . f is an ideal nuclide for positron emission tomography (pet) imaging; there may be significant uses of f labeled glucitol-toca and its analogues as tumor probes for the diagnosis of sstr-positive tumors. in order to explore a novel pet probe for diagnosis of sstrpositive tumors, we designed a synthetic route to synthesize n-gluc-lys(nota)-toca, which uses , , -triazacyclononane- , , -triacetic acid (nota) as the chelating reagent. n-gluc-lys([al f]nota)-toca is radiosynthesized quickly and efficiently using the chelation reaction of al f complex and n-gluc-lys(nota)-toca. the aim of this study is to develop an efficient method for the synthesis of monomers of triazolic nucleic acid (tna), a new class of artificial nucleic acids. but- -yne- , -diol and nucleobases derivatives will be substrates of the monomers synthesis. tna oligomers could be used as specific inhibitor of tar rna hiv- , the regulatory rna structure crucial for hiv replication. "click chemistry" based on , -dipolar cycloaddition will be used to conjugate an alkyne and azide derivatives of monomers subunits. a ru (ii) complex will be used as a catalyst of internal alkyne (but- -yn-based) cycloaddition. the reaction gives exclusively of , , -trisubstituted derivative of triazole ring . the monomers will be characterized using rp hplc, capillary electrophoresis (ce) and h and c nmr. the resulting monomers containing fmoc-protected amino group and a free carboxyl group will be used for the classical spps method to synthesize tna oligomers. tna sequences will be designed against tar's bulge and an external loop. through the recognition that the repertoire of polypeptide conformations can be greatly expanded by the creation of structures incorporating β-amino acids. moreover, the numerous advantages of hybrid (mixed α-and β-) backbone peptidomimetics with respect to homogeneous ones were quite recently outlined. we describe here various β-amino acid-based β-hphe-β-hphe dipeptide derivatives, also conformationally constrained, and their application to the synthesis and biological evaluation of hybrid analogues of the opioid endogenous peptide endomorphin- (em- ). the opioid system mediates a wide variety of pharmacological and physiological processes, including pain perception and modulation. the amidated tetrapeptide em- has been shown to be μ-opioid receptor (mor) agonist exhibiting a very high μ-receptor affinity and selectivity, and it is an important model in the search towards new analgesics. structural investigation of em- reveals the high conformational freedom of the phe side chains and also the inherent flexibility of the peptide backbone, indicating many probable bioactive conformations, ranging from βturns to extended conformations. with the aim of better clarify the relevant role of the proper spatial orientation of the aromatic rings and in particular of the benzyl side chains at position and , h nmr studies, molecular modelling, and molecular docking to a homology mor model of our hybrid analogues are currently under way. the lantibiotics represent a class of antimicrobial peptides, in which the unusual amino acids dehydroalanine and dehydrobutyrine and the intramolecular thioether bridges (lanthionines) are important structural features for bioactivity.the lipid ii -nisin complex is responsible for pore-formation since the c-terminal part of nisin is inserted into the bacterial cell membrane which ultimately results in cell leakage and collapse of vital ion gradients. in order to increase the metabolic stability of nisin, the oxidationsensitive thioether bridges can be replaced by metabolically stable dicarba moieties, as successfully demonstrated by the synthesis of nisin ab(c) analogs containing alkane/alkene bridges [ ] . to obtain more insight into the importance of the cross-bridged de-ring structure (i→i+ , i+ →i+ connectivity) on nisin's bioactivity, we synthesized a series of all four diastereomers of the crossed alkene-bridged de-ring mimic, using ring-closing metathesis. all four diastereoisomers were obtained by hplc and structurally characterized by nmr spectroscopy. an orthogonal protection scheme was used, to enable the independent n-or c-terminal modification of the bicyclic hexapeptides with azide/alkyne functionalities. via cu(i)-catalyzed cycloaddition chemistries, alkyne-functionalized natural abc-fragments of nisin, which were obtained by tryptic digestion of full length nisin followed by hplc purification, have been conjugated to synthetic de-ring mimics to obtain novel nisin derivatives and their affinity toward lipid ii and pore-forming capacity have been studied. herein, we report on the details of the synthesis and characterization of the geometric isomers of the synthetic de-ring mimics, and their use as synthons in cu(i)-catalyzed click chemistry to obtain newly designed nisin hybrids as potential novel peptide antibiotics. università di ferrara, dipartimento di biochimica e biologia molecolare, ferrara, italy mirnas play an important role in regulation of gene expression, being involved in numerous processes such as cell proliferation, cell differentiation, apoptosis and also in the progress of diseases as cancer and cardiovascular disorders. mirnas associated to diseases recently become targets for the development of new drugs based on antisense oligonucleotides or analogues complementary to the chosen mirna, in order inhibit the binding of the mirna to its mrna target. therapeutic silencing of mirna has been also observed in several animal disease model. in this work we propose a new approach to interfere in the mirna function, based on peptide nucleic acid (pna) oligomers designed to be complementary to selected regions of the mirna precursor (pre-mirna). as the pre-mirna bases belonging to the stem are not perfectly complementary, we hypothesized that the mismatched duplex of the pre-mirna could be opened by pnas inhibiting of its maturation into mirna. two pna sequences, targeting respectively the "sense region" and the " ' end region" of the pre-mir were designed. pnas were conjugated to different carrier peptides, hiv-tat, r , k and two nuclear localization signal (nls and binls), in order to increase their cellular uptake. to verify the ability of the designed pnas to give strand invasion on the pre-mirna, we conjugated also pnas to the thiazole orange, a probe which lights-up upon hybridization the development of privileged molecular scaffolds efficiently mimicking reverse turn motifs has attracted remarkable interest when structural constraints are exploited to increase both binding and selectivity of model peptides. one of the successful approaches to restrict peptide conformation is the disubstitution in the α position of an α-amino acid, leading to a conformational constraint and a stereochemically stable quaternary carbon center. in particular, spirocyclic scaffolds are able to provide, upon the attachment of appropriate functional groups, useful high-affinity ligands, relevant to the field of drug discovery. at present, we are interested to spirocyclic tryptophan (trp) analogues, in order to develop new reverse turn nucleating moieties able to be inserted into pharmacologically relevant peptidomimetic compounds. among peptides sharing a tryptophan-containing β-turn motif of which the trp residue is critical for binding, we looked at the hormone peptide somatostatin, acting in various organ systems as a neuromodulator and a neurotransmitter, as well as a potent inhibitor of various secretory processes and cell proliferation. somatostatin and its analogue octreotide (sandostatin® drug, clinically used for the treatment of endocrine tumors and acromegaly) are thought to interact with the sst - receptors mainly by inserting a β-turn substructure, carrying a lysine (lys) and a trp side chain into a pocket of the g protein-coupled somatostatin receptor. we report here the preparation and structural characterization of a new , , , -tetrahydro-β-carboline (thbc)-based spirocyclic lactam as type-ii β-turn model compound and the application of its core structure to the synthesis of a somatostatin mimetic, whose biological evaluation is under way. the analogues of sfti- modified in the p position by, βand γ-amino acids and n-substituted β-alanines r. lukajtis, a m. filipowicz, a a. legowska, a d. debowski, a a. lesner, a k. rolka a a faculty of chemistry, university of gdansk, - gdansk, poland serine proteinases play very important roles in many physiological processes in humans, such as: food digestion, fertilization of the ovum, blood clotting and dissolution of blood clots, immune response. however, their uncontrolled activity can evoke serious pathological conditions. therefore, serine proteinase inhibitors are considered to be a promising class of therapeutic agents. trypsin inhibitor sfti- , on which we focused our attention in the last decade, is an attractive template for the design of such compounds. its primary structure is shown below: & gly-arg-cys(& )-thr-lys -ser -ile-pro-pro-ile-cys(& )-phe-pro-asp& the inherent feature of natural peptides and proteins is their low stability towards proteases, which seriously reduces their bioavailability. there is a growing need for the development of artificial biopolymers with diverse side chains, capable of mimicing peptide function. β-and γpeptides are an interesting class of peptidomimetics with significant chemical and biological properties. the present communication describes the chemical synthesis and inhibitory activity of a series of trypsin inhibitor sfti- monocyclic analogues (with disulfide bridge only) modified in p position by βand γamino acids and n-substituted β-alanine (β-peptoid units). the following mimetics of proteinogenic lys or phe were used: β hlys, β hphe, γ hhlys, γ hhphe, βhnlys, βhnphe. all compounds were synthesized manually on solid support. β-peptoid monomers were introduced into the peptide structure by two steps method [ ] . newly obtained sfti- analogues modified in p position by β-derivatives of lys and phe were able to inhibit bovine β-trypsin and bovine αchymotrypsin, respectively, whereas the remaining ones (except for [βhnphe ]sfti- ) appeared to be inactive. the notion that early soluble aß intermediates are endowed with cytotoxic effects suggests that a major effort should be directed toward the inhibition of amyloid aggregation at very early stages. inhibiting aß self-oligomerization could, therefore, provide a useful approach to treating and controlling the pathogenic pathways underlying alzheimer's disease (ad). likely, agents that target the basic molecular recognition process preceding the formation of early intermediates are the most valuable candidates. we have conjugated a trehalose moiety to the known ß-sheet breakers pentapeptides lpffd. trehalose has received a special interest because it has been found to be effective in the treatment of neurodegenerative diseases associated with peptide or protein aggregation. the glycosidic moiety was covalently linked to different regions of the peptides' primary sequence, including the n-terminus or c-terminus or the aminoacid side chain. this new class of peptides showed an increased resistance to proteases. in this work, the inherent ability of these peptides to recognize and bind the monomeric form of recently reported a d-amino acid-containing hiv protease inhibitor with a sulfonyl group showed an activity enhancement against drug resistant viruses. x-ray crystallographic study of the derivative revealed existence of four bridging water molecules. we suggest that the additional indirect interactions through water molecules induced the inhibitor's flexibility in binding conformation, keeping the affinity with the mutated proteases. oxalyamide, so-called oxamide, has two carbonyl oxygen atoms as hydrogen bonding acceptor similar to sulfonyl group, which is promising to interact with water molecules. to increase the numbers of bridging water molecules, we built-in two oxamide structures to both terminals of pseudo-symmetric compounds with hydroxymethylcarbonyl-hydrazide isostere. the derivatives were tested for inhibitory activity using wildtype hiv protease and a highly mutated protease with lopinavir resistance. we found that the loss of potency against the mutated protease was relatively small in the oxamide derivatives. the molecular dynamic simulations suggested the ability of bridging water formation of the two oxamide groups. optimization of the pna-synthesis using different bases for fmoc-deprotection s. rawer , k. braun , r. pipkorn life technologies, darmstadt, germany dkfz, heidelberg,germany pna (peptide nucleic acids) are considered as highly sensitive and specific tools for antisense strategies especially conjugated with cell penetrating peptides. individual designed shuttle systems can be applied in cancer diagnostics and possible therapy ( ) . it is, however, undisputed that proper pnas' syntheses prove to be a challenge for coupling and fmoc-deprotection. due to the structure-formation the success of the synthesis depends strongly from parameters, like activator's quality and deproctection kinetics correlating to the length of the pna polymer spps product. using the example of the spps pna synthesis' results of the coding sequence of c-myc human exon ii, different bases, acting as fmoc-deprotection reagents, are compared and analyzed aiming at optimizing the pna synthesis strategy ( ) peptidoglycans are central structural components of the cell wall of bacteria. several plant receptors are known to recognize peptidoglycan fragments. it is believed that these receptors form part of the defense mechanism against bacterial infections in several plant species. peptidoglycans consist of long chains of alternating β( - )linked glcnac and murnac moieties that are crosslinked by short, non-ribosomal peptides. these peptides consist of several d-amino acids and the symmetrical (r,s)diaminopimelic acid (meso-dap). in particular, the latter complicates the synthesis of peptidoglycan fragments due to the requirement for individually addressing the two pairs of functional groups. some chemical syntheses of peptidoglycan fragments have been reported [ ] [ ] [ ] [ ] , hhich involved multi-step formation of an orthogonally protected dap moiety, and elaborate oligosaccharide synthesis. here we present a new and simple approach to peptidoglycan synthesis which is based on the use of commercially available building blocks for the dap and oligosaccharide components. this allows easy access to a range of peptidoglycan fragments for structure-activity studies. the introduction of solid-phase peptide synthesis (spps) and the subsequent refinement of resins, linkers, coupling reagents and amino acid protecting groups allowed access to a wide range of peptides. therapeutic peptides, in particular, have benefitted from the maturation of spps, as complex peptides can be synthesized more efficiently in comparison to conventional solution phase synthesis. however, peptides containing multiple disulfide bonds often still remain difficult to make due to a lack of orthogonal cysteine protecting groups that can be used in routine spps. the cysteine protecting group s-tertbutyl mercapto (s-tbu) is commercial and orthogonal to other cysteine protecting groups. removal of the protecting group is facilitated by reducing agents (e.g. thiols or phosphines) and is stable to tfa and piperidine, hence compatible with fmoc/o-tbu peptide synthesis. however, the protecting group cannot be used in routine spps due to long deprotection times ( - h) . in certain cases it has been shown to be impossible to remove due to proximity of bulky protecting groups and sensitivity to certain sequences. additionally, reports of desulfurization of s-tbu protected cysteine to dehydroalanine, by the use of prolonged exposure to reducing agents, show the limitations of this protecting group. the concept of cysteine protecting groups labile to reducing agents is promising due to orthogonality to other cysteine protecting groups and the limitations of s-tbu initiated an investigation into novel reductive cysteine protecting groups. herein, we introduce s-tmp as a novel cysteine protecting group that is very labile to reducing agents. the increased lability, in comparison to s-tbu, allows utilization of reducing agent labile protecting groups in routine peptide synthesis of disulfide containing peptides. as modern automated spps protocols allow the assembly of larger and increasingly complex peptides, a precise control of the coupling reactions is a crucial prerequisite in peptide synthesis. monitoring the progress of synthesis allows the detection of undesirable products caused by side reactions, incomplete couplings or deprotections. although different methods have been developed for monitoring spps, we observed that the use of colorimetric test or continuous-flow uv absorbance of the reaction column effluent was not informative enough to identify difficult steps in the synthesis. in this study we demonstrate the usefulness of the combination of a mw-assisted mini-cleavage protocol and the uplc-esi-ms analysis for monitoring the quality of the reaction steps. as a proof of concept, based on this strategy, we monitored the synthesis of pthrp( - )nh (synthesised by fmoc/tbu rt-spps, liberty™, cem), characterised by a cluster of arginine residues in the - region. by the use of mw irradiation during the mini-cleavage protocol, we optimized time for mini-cleavages particularly in case of multi-arginine containing peptides, protected by pbf group. the results obtained by uplc-esi-ms showed that the complete removal of the pbf groups from the arginine sidechain residues required h at rt. on the other hand, the mw-assisted mini-cleavage monitoring let us to obtain final results just in min, confirming that the use of microwave irradiation in mini-cleavages is an efficient strategy to monitor also difficult peptide couplings, such as multiarginine peptides. identification of some deletion sequences was helpful to recognise critical couplings in order to adopt more efficient coupling strategies and therefore to optimise the final yield and purity of the crude peptide. development of green sustainable chemistry is currently regarded as a challenge in science and technology to reduce the use of organic solvents and utilize less toxic solvents instead. water and aqueous-based solvent systems represent an increasingly significant choice for replacing traditional solvents in synthetic chemistry. until recently, peptide synthesis in aqueous solution has remained largely unexplored. this is because the most common building blocks are sparingly soluble in water and are considered inappropriate for in-water peptide synthesis. we have developed a method for solid-phase peptide synthesis in water, which utilizes water-insoluble fmoc-amino acids that are converted to water-dispersible nanoparticles. in this way, the solubility problem is overcome. our technology, which uses suspended nanoparticle reactants for the coupling reaction, offers many advantages in terms of reaction efficiency over inwater synthesis using water-soluble or non-disperse reactants. however, there are two main problems with this nanoparticle approach; (i) slow reaction rates compared to general peptide synthesis in ordinary organic solvents (ii) poor yields for the synthesis of long peptides because the protected peptide chains on the resin have a tendency to aggregate in water. mw assisted spps is particularly attractive because of the widespread availability of the new technology, including automated peptide synthesizers. a trial of mw assisted inwater solid-phase synthesis using non-disperse boc-amino acids has been reported by albericio previously. currently, fmoc-amino acids are routinely used as building blocks for solid-phase peptide synthesis. with this in mind, we have developed a mw irradiation procedure aimed at reducing reaction time and increasing reaction yield for in-water solidphase synthesis using water-dispersible fmoc-amino acid nanoparticles. and we demonstrated in-water solid-phase synthesis of difficult sequence peptide with mw irradiation. m. lebl, z. flegelova spyder institute praha, czech republic cotton was shown as a convenient solid phase support earlier - , but did not find wide acceptance by the peptide community. we decided to try its application as (i) a support of choice for the synthesis driven by combination of capillary forces and gravity, (ii) support for synthesis utilizing in situ neutralization boc based protocol, (iii) support for combinatorial synthesis based on easy labeling and physical separability of cotton substrate, and (iv) support for multisupport synthesis. -we have built a simple synthesizer in which the cotton carrier (functionalized thread) is placed inside the capillary tubing and the appropriate reagents are introduced by connecting the inlet with appropriate reagents. the speed of "pumping" the reagents is driven by the difference between the elevation of the inlet and outlet of the capillary tubing. -we have shown that boc solid phase synthesis utilizing in situ neutralization is compatible with cotton substrate and provides high quality products. combining with the fact that cotton by itself acts as the self-association breaking agent, makes cotton a suitable carrier for synthesis of "difficult" sequences. -labeling of individual solid support particles can be easily based on the length of the cotton thread pieces, number and positions of knots, or their attachment to a secondary carrier. in addition, it is possible to synthesize peptides differing by the partial structure (alternative linkers, terminal modifications, etc.) in a mixture of classical resin with labeled cotton carriers, which are easily separable at the end of the synthesis. . use of microwave irradiation provides peptides in a fraction of time compared to conventional methods, and the peptides are also often generated in higher yield and purity. while microwave technology is particularly suited for the synthesis of "difficult" to synthesize peptides, this tool can routinely be used for the synthesis of a wide variety of peptides without the need for extensive method optimization. the focus of this study is to demonstrate how a peptide can be synthesized on a small scale (for example μmol) up through larger scales (> mmol) with ease. as a biologically relevant model peptide the last residues of the human platelet factor protein (hpf - ) was selected due to its significant antimicrobial activity. however, the problem of developing a robust fmoc thioester method is that the deprotection of the fmoc group with base at each cycle is not compatible with an active ester at the c-terminus. many ingenious approaches have been developed to generate the required thioester peptide. , , the most popular has been to use an nacylsulfonamide as a base and acid stable (safety-catch) linker for peptide synthesis. alkylation of the sulfonamide after peptide assembly makes the linker labile to cleavage with nucleophiles. whilst popular, it has been plagued by notoriously low yields which originate from the incomplete acylation of the resin-bound sulfonamide with the c-terminal residue, incomplete alkylation of the sulfonamide and the incomplete thiolysis of the resin-bound protected peptide. in this poster we describe the development of a novel dual linker strategy , involving anchoring of the sulfonamide linker to a standard acid-labile resin. this variation overcomes many of the current limitations of the sulfamylbutyryl linker approach and provides a simpler and scalable method for peptide ligation via fmoc spps. m. ziovas, d. tataraki, p. manousou, n. parveri, f. satoglou, d. gatos and k. barlos department of chemistry, university of patras, patras, greece solid phase peptide synthesis is traditionally performed by the attachment of the c-terminal amino acid through its α-carboxyl function on a suitable solid support and elongating peptide chain towards the amino terminal of the peptide by adding sequentially the amino acid residues in the gradually growing peptide chain. several thousands of publications and patents describe this methodology and its application for the production of peptide pharmaceuticals. in contrary to the attachment of the c-terminal carboxyl function, attachment of amino acids and peptides through an amino acid side-chain on suitable resins and their application in spps is described in a small number of publications and patents. most of these publications describe the attachment of the amino acids through a side-chain carboxyl group of asp and glu. in the present work peptides were synthesized very efficiently in high yield and purity by anchoring of side-chain hydroxyl, amino or thiol groups of amino acids, amino acid amides, amino alcohols or small peptides on resins of the trityl or benzhydryl type. several peptides of pharmaceutical interest, such as exenatide, octreotide, pramlintide, calcitonin, bivalirudin, insulin b-chain and others were produced as examples of this technology, either by the step-by-step procedure or by fragment condensation in solution and on solid phase. step given that some of these diseases are caused by a mutation and/or malfunction of an essential protein, a better understanding of the structure and function of such proteins will allow us to prevent, slow down or even cure these diseases. to increase our knowledge, the synthesis of the target protein, a fragment involved in its activity or interacting peptides that modulate the protein activity is often required. in some cases the preparation of a protein analogue that improves its efficacy is envisage. however, conventional solid-phase peptide synthesis methods have some limitations when attempting to achieve these complex sequences of considerable length. using novel technologies, such as a microwave-assisted solid phase synthesis, commonly found in many peptide synthesis labs, here we performed the step-wise solidphase synthesis of a protein holding more than residues (d-vegf). this synthetic achievement indicates the suitability of this approach for synthesis of long proteins or their analogues. the detailed synthesis of the enatiomeric version of vegf and the selenomethionine substituted analogues of huprp ( - ), proteins involved in angiogenesis and prion protein amyloidoses respectively, are described as study cases, where the use of microwaves allow us to obtain them in a fast and efficient manner. therefore, the development of novel peptide analogues with enhanced in vivo stability could potentially provide therapeutic alternatives. the pharmacological evaluation of a bioactive peptide [des-gly ,tyr (ome),d-leu ,aze-nhet ]gnrh, analogue , is presented herein. in vitro (kidney mouse membranes) and in vivo (clinically relevant pharmacokinetic mouse model) bioassays were coupled to liquid chromatographytandem mass spectrometry. analogue , an agonist of the gnrh receptor with a binding affinity in the nanomolar range, caused testosterone release in mice that was acutely dose-dependent, an effect blocked by cetrorelix. repeated dosing studies in mice demonstrated that analogue was well tolerated and had potency similar to that of leuprolide, based on plasma and testis testosterone reduction and histopathological findings. analogue also shared with leuprolide similar significant antiproliferative activity on androgen-dependent prostate cancer (lncap) cells. on the basis of pharmacokinetic advantages, we expect that analogue or analogues based on this new design will be therapeutically advantageous for the treatment of cancer and endocrine disorders. cortexin is a polypeptide drug isolated from cattle and porcine brain cortex. cortexin is effective in monotherapy and in combination with traditional methods of treatment. cortexin produces tissue-specific, regulatory, and reparative effects on the brain cortex and contains active low-molecular-weight neuropeptides (< kda) penetrating through the blood-brain barrier. cortagen is a tetrapeptide h-ala-glu-asp-pro-oh (geropharm) produces nootropic and neuroprotective effects. oleylcortagen is a lipophylic analog of cortagen c h o-ala-glu-asp-pro-oh was created for increased proteolytic stability and increased penetrating through the blood-brain barrier. the main aim of our investigation is the analysis of psychopharmacological profile of peptide preparations in comparison with piracetam. have been shown oleylcortagen ( mg/kg) and piracetam ( mg/kg) possess activating effect on motor and research components of behavior in «open field» test. two of peptides (oleylcortagen, cortexin) decreased period of immobilisation and demonstrated antidepressant effects on rat behavior in the porsolt's test, on other hand cortagen demonstrated depressant action. therefore, the significant psychoactivating properties are typical for oleyl-cortagen, cortexin. the mechanism of the action of these peptides can be explained from the viewpoint of the regulatory cascade. they produce a direct information impact on cell structures of the brain, and then promote release of the regulatory peptides, which in turn, induce the release of the next group of peptides. neurology, queen square, london wc n bg, uk one of the hypotheses of alzheimer's disease neuropathology involves beta-amyloid (βa) binding with proteins on neuronal cell surface which leads to cell lysis and amyloid plaque formation. according to the latest data α -type of the nicotinic acetylcholine receptor (achr) and the prion protein can be the target for betaamyloid toxicity [ , ] . aggregated βa causes many pathological changes in cultures of mixed neurons and astrocytes such as sporadic cytoplasmic intracellular ca + -signal, activation of reactive oxygen species (ros) production and cell death. in the present work we demonstrated the ability of affinity purified antibodies to synthetic fragment - of α -subunit of the achr (achrabs) or to peptide - of the prion protein (prpabs) to protect cells from βa induced cell death. we also showed that both antibodies did not block βa induced ca + -signal in astrocytes. however, preincubation of cortical co-culture of neurons and astrocytes with achrabs or prpabs significantly reduced the rate of caspase activation and the rate of βainduced ros production via modulating nadph-oxidase. more detailed research of involvement of α -type achr revealed that α-bungarotoxin was also very effective in the inhibition of caspase activation and superoxide production. the observed positive effect of antibodies to α -type achr or to the prion protein gives an additional explanation regarding the involvement of these proteins in ad pathology and provides new approach into an anti-ad vaccine design. capturing and macrophage-aided clearance of amyloid beta by surface modified proteineous particles m. richman, s. rahimipour department of chemistry, bar-ilan university, ramat-gan , israel imbalanced homeostasis and oligomerization of amyloidβ (aβ) peptide in the brain are hallmarks of alzheimer's disease (ad). microglia and macrophages play a critical role in ad progression by clearing aβ from the brain or inducing inflammation. recent evidence suggests that the phagocytic pathway of aβ may be defective in ad microglia/macrophages that contributes to the build-up concentration of aβ in the brain. , therefore, efforts have been directed toward developing treatments that trigger these cells to clear aβ through alternative mechanisms. we have recently demonstrated that protein microspheres modified at their surface with multiple copies of an aβrecognition motif can strongly bind aβ, inhibit its aggregation and directly reduce its toxicity by sequestering it from the medium. here, we describe how the aβ-bound microspheres can stimulate microglial cells and be phagocytosed through a mechanism that is distinct from that of aβ. the phagocytosis was mostly effective with microspheres having diameter size of about . - mm and introduction of polyethylene glycol to the surface of the microspheres changed the kinetics of the phagocytosis. moreover, while aggregated aβ induced a significant inflammatory response that was manifested by the release of tnf-α from the microglial cells, the aβ-bound microspheres dramatically reduced the amount of the released cytokine. our data suggest that surface-modified microspheres could be utilized to detoxify other pathogenic or misfolded proteins that their accumulation may lead to genesis of other diseases. vasoactive intestinal peptide (vip) and its derivatives have been thought to be promising drug candidates for airway inflammatory diseases. however, the therapeutic potential of vips is highly limited because of rapid metabolic degradation and systemic side effects following systemic administration. previously, to overcome these drawbacks, our group developed a novel vip derivative, [arg , , , leu ]-vip-grr (ik ), with improved metabolic stability ( ), and respirable powder (rp) formulation of ik (ik -rp) for pulmonary administration ( ) . these attempts successfully led to enhanced pharmacological effects of ik in the airway system and reduced systemic exposure; however, further chemical modification of ik with a focus on metabolic stability might provide better clinical outcome. the present study aimed to design a pegylated vip derivative with improved metabolic stability and to develop its respirable powder (rp) formulation for inhalation therapy. ik was chemically conjugated with peg ( kda, p k), the physicochemical and biochemical properties of which were characterized by cd spectral analysis, binding assay, and metabolic stability. the rp formulation of pegylated ik (ik /p k) was prepared with a jet mill, and in vitro inhalation performance and in vivo pharmacological effects in antigen-sensitized rats were also evaluated. the cd spectral analysis demonstrated that peg conjugation had no impact on the solution structure of ik . although receptor-binding activity of the ik /p k (ic : nm) was estimated at ca. -fold less than that of ik (ic : . nm), metabolic stability for the ik /p k was highly improved. according to the laser diffraction and cascade impactor analyses, ik /p k-rp had fine in vitro inhalation performance. insufflation of ik /p k-rp ( μg of ik /p k) in antigen-sensitized rats resulted in marked attenuation of inflammatory events, as evidenced by significant decrease of inflammatory biomarkers and granulocyte recruitment in pulmonary tissue at h after antigen challenge. from these findings, pegylation of vip derivative, as well as its strategic application to the rp formulation, might be a viable approach to improve its therapeutic potential for treatment of airway inflammatory diseases. the previous studies have shown that trkb (tropomyocin receptor kinase) acts as an oncogenic agent and its binding to bdnf (brain derived neurotrophic factor) activates signaling angiogenesis of tumor proliferation [ ] . for finding the most stable and potentially effective peptides against the trkb, we applied the following protocol. at the first step of this protocol we designed a peptide library by using sequence tolerance method in rosetta . package, then peptide energy optimization performed by backrub protocol for finding the most stable peptides. the five best peptides in energy optimization selected based on backrup scores by using r package [ ] . d-structure prediction of the selected peptides was performed by using molecular dynamic in hyperchem software. docking of peptides with trkb receptor was carried out in haddock software. we used cyclotraxin, a selective trkb inhibitor as positive control for this protocol. cyclotraxin and the peptides were compared by anova or t-test. the peptides are going to be tested against the trkb in an in vitro model. dirucotide (mbp - ) is a synthetic peptide analog of , that consists of amino acids and tested in a phase trial were failed to reach his tolerance level on previous phase ii in rpms patients. one of the major disadvantages of peptide therapy is the activation of proteolytic enzymes, leading to peptide degradation. to address this problem cyclic peptide analogues have been synthesized. thus we synthesise a linear and cyclic analogues of dirucotide. the two analogues were synthesized by changing the amino acid residue at position from to the synthesis of the linear peptide, as well as of the cyclic one, was carried out by the fmoc/tbu methodology, utilizing the -chlorotrityl chloride resin (cltr-cl). the purification was achieved using semi-preparative rp-hplc and the identification was assessed by analytical rp-hplc and by mass spectrometry (esi-ms). the linear and cyclic peptide analogues will be used in human t-cell cultures to test their immunogenicity in patients versus healthy controls. in the first approach a reporter moiety was introduced to diagnose and treat cxcr related diseases. therefore, an anchor point to attach additional molecules to the ligand was elucidated. using sar studies to optimize the linker from the ligand to the detectable moiety the excellent receptor affinity could be retained. in a final step the reporter moiety was introduced to give a ligand for diagnosis via pet imaging and for possible endoradiotherapeutic applications. originating from the dimeric motif present in many active cxcr ligands several dimers were prepared using a monomeric ligand identified in the prior study. comparison of monomers and dimers yielded a possible subsite binding mode explaining why the dimers exhibit enhanced affinity using a model derived from the origins of the monomer. several peptidomimetic modifications were introduced to the ligand to reduce the peptidic structure. in a conformationally guided approach introduction of a peptoid motif could enforce a single active conformer that was enhanced through subsequent modifications. this yielded a compound times better than the original cxcr antagonist which is the most affine cxcr ligand reported so far. our previous studies have demonstrated that pace represents a potential therapeutic target for the treatment of prostate cancer . moreover, we have developed potent and selective pace inhibitor, ( -fold specificity over furin), known as multi-leu or ml peptide, which has a significant inhibitory effect on the proliferation of prostate cancer cell lines. peptide-based drug candidates can be limited by their poor metabolic stability and low bioavailability. thus, we performed structure-activity relationship studies to improve the pharmacokinetic properties of our ml inhibitor. we have designed and synthesized new ml peptide analogs having various chemical modifications. first, based on our previous results, we combined the most effective modifications of position p (d-amino acids) and p the arginine mimetic amidinobenzylamine (amba) to improve overall properties of our leading compound. second, the n-terminus of the resulting analogs was modified with a fatty acid, in order to enhance their cell permeability properties. third, we modified the inhibitors with a peg moiety to increase their stability and bioavailability. we tested the inhibitory activity, stability in plasma, cellular uptake, and cytotoxicity of each inhibitor. the results of this study demonstrate that the presence of the n-terminal extension (c or peg ) does not affect activity of our inhibitors. on the other hand, we show that introduction of the peg moiety does not increase cytotoxicity of ml analogs. it is interesting to note that the pegylated analog ac-peg -d-leu-lllrvkr-amba has better cell-permeability activity than its counterpart without peg unit. this combination of pharmacological properties makes our new ml analogs promising candidates for the development of potential anti-prostate cancer agents. [ ] peripheral coadministration of rf with opioid analgesics led to confirm the involvement of npff receptors as a part of the antiopioid system. indeed, rf was able to reverse the opioid induced hyperalgesia in rat (randall selitto test). then, a complete structure-activity relationships analysis was performed with rf , assessing the involvement of each moiety for affinity and selectivity towards both npff receptors. a first exploration of the n-terminus part of rf (> synthesized derivatives) led to replace the hydrophobic adamantane moiety by a hindered aromatic group, providing a subnanomolar npff ligand with more than two log-units selectivity against npff . then, the removal of the cterminal amide function led to reduce the dipeptide arg-phe-nh into an arginine derivative. in spite of an initial loss of affinity, optimization of the phenethyl moiety at the cterminus part of arginine led to non-selective nanomolar ligands for both npff & receptors. next, we applied efficient methodologies in order to synthesize non-natural analogs of arginine, leading to various compounds exhibiting selectivity for either npff or npff receptors. in particular, compound rf was identified as a selective npff antagonist (npff , ki = nm; npff : ki > μm). lacking of analgesia properties, oral administration of rf ( mg/kg per os) to rats was able to fully reverse fentanylinduced hyperalgesia. rf is the first orally available npff antagonist capable of reversing opioid induced hyperalgesia at low dose. moreover, this result allows identifying for the first time npff receptor as a key-partner of the anti-opioid system. administration of multiple antiplatelet agents has become the mainstay in the treatment of acute coronary syndromes in everyday clinical practice. we have previously reported significant antiplatelet effects of novel synthetic peptides' single administration on experimental carotid artery thrombosis in rabbits . in the present study we sought to investigate the peptides' effects when administered in marginally effective doses (significantly lower than those utilized in the past), in animals that had previously received low doses of aspirin. the peptides when co-administered with aspirin preserved the carotid artery's blood flow, in contrast to the total artery occlusion observed in animals receiving aspirin and placebo. blood flow at min after electrical stimulation was reduced to . ± . % and . ± . % in the ymesradr and psrcdcr-nh groups respectively (p< . vs aspirin and control). thrombus weight was significantly reduced in animals receiving ymesradr and psrcdcr-nh versus aspirin and control ( . ± . mg and . ± . mg, vs . ± . mg and . ± . mg respectively, p< . ). platelet aggregation was significantly inhibited in the ymesradr and psrcdcr-nh groups by . ± . % and . ± . % for adp (p< . vs aspirin and control), and . ± . % and . ± . % for aa (p< . vs aspirin and control), respectively. blood loss did not significantly differ among the various groups. administration of novel synthetic peptides, even at marginally effective doses, in animals previously treated with low doses of aspirin results in enhanced antiplatelet effects in an experimental model of arterial thrombosis. the study of peptide metabolism is particularly important when examining anticancer peptides; it can provide pivotal clues for the evaluation and improvement of stability of a peptide drug leading to enhanced pharmacokinetics and efficacy. gnrh analogues are used for the treatment of prostate cancer. as with other peptides a drawback is their short half-life due to their metabolic degradation. in order to examine the stability of these analogues we have developed several in vitro peptide stability and metabolism assays using specific tissues, isolated membranes, cancer cells that are analyzed subsequently using lc-ms/ms based approaches. such in vitro studies are followed up with pharmacokinetic studies in mice in order to establish the correlation between in vitro and in vivo approaches. the kidney is the main metabolic organ for peptide metabolism and for that reason we employed a peptide stability and metabolism assay previously described by our group using isolated mouse kidney membranes for the evaluation and comparison of different gnrh analogues. we tested gnrh analogues in other tissues such as mouse plasma, which is the "distributing" tissue for these drugs and mouse brain homogenate, a tissue known for its abundance in peptidases and relevance for centrally mediated effects. stability studies were also performed in cancer cells. in all cases lc-ms/ms based assays were developed for measurement of peptide drug and the resulting metabolites. for triptorelin, and a series of gnrh analogues, degradation and metabolite formation was studied by our mouse kidney membranes assay. studies of coadministering the peptide of interest with inhibitors that are presumed to block the metabolism in mice are ongoing. the vulnerability of gnrh analogues was verified after incubation with plasma and brain homogenate and by metabolite identification we obtained clues about the key cleavage sites. the described in vitro/in vivo protocols provide valuable information that could lead to the synthesis of more stable anticancer peptides with improved anticancer efficacy. in this work, we explored the use of a high-throughput synthesis and screening approach with peptide arrays to identify and structurally optimize shortened hiv- fusion inhibitors. the peptide array technology involves miniaturized synthesis of immobilized peptides, followed by affinity testing with a five-helix bundle, as a mimetic of the fusogenic gp protein. this exercise resulted in the identification of a class of truncated peptides which demonstrates a surprisingly high antiviral potency, despite the absence of the pocket-and the lipid-binding domain. the propensity of these peptides to adopt the bioactive α-helical conformation in solution as determined by circular dichroism, could be the key factor for this unexpected potency. these peptides are promising leads for the treatment and prevention of hiv. the pathological role of platelets in cardiovascular disease (cvd) is well established. platelets secrete adp to recruit additional platelets to a thrombotic site. we have previously identified novel cell-permeable peptide modulators of platelet function by using a bioinformatic screen based on patterns of evolutionary conservation in transmembrane proteins . in this study we further explored peptides derived from cadherin cell adhesion molecules. we explored a range of overlapping peptides derived from different cadherins varying from - amino acids long. peptides are synthesized and analyzed in a high-throughput platelet adp secretion assay. peptides ( . - μm) were assessed in the presence and absence of thrombin receptor activating peptide (trap; μm). we identified cadh- and proteins, but not cadh or in human platelets using western blotting and mass spectrometric analysis. peptides derived from paralogous juxta-membrane, cytoplasmic regions of these cadherins are potent modulators of platelet secretion. by systematically deleting amino acids from c or n-terminus of active peptides, we established that the minimal functional sequence for biological activity was a short six-residue motif, which corresponds to the known catenin-binding region of cadherin tails (kepllp) . peptides alone have no biological activity. however, they potentiate the response induced by the platelet agonist trap at low doses. thus we have identified a cadherin-derived peptide that can modulate platelet secretion events. this highlights a previously unknown role of cadherins in the regulation of platelet function. agents that interfere with cadherin signaling in platelets might have a therapeutic role in cvd. ageing of the brain leads to impairments in cognitive and motor skills, and is the main risk factor for several common neurological disorders such as alzheimer's disease (ad) and parkinson's disease (pd). altered protein handling (proteolysis, repair system, chaperones) forms a basis for a large number of protein conformational disorders. extra-and intracellular, as well as intranuclear accumulation of abnormal proteins, in the form of protein inclusions and aggregates, and dysfunction of the quality control mechanisms are common in all these disorders. alterations in protein homeostasis occur with age, causing molecular changes such as protein misfolding and aggregation. many biologically active proteins lack stable secondary and tertiary structure, these are called intrinsically disordered proteins (idps), some of them (e.g. β-amyloid, α-synuclein) are coupled to neurodegenerative disorders. idps exist as assemblies of rapidly fluctuating structures undergoing coupled folding and aggregation process. protein aggregation is characterized by polymorphism, where a mixture of soluble oligomers, amyloid fibrils and amorphous aggregates is the final product. soluble oligomers are inevitable formed during the self-association process and might initiate the neurodegenerative cascades of ad, pd and similar diseases. the emerging consensus that protein misfolding (leading to idps) is the cause of several neurodegenerative disorders now offers the opportunity to develop a general therapy. soluble oligomers with id regions are potential drug targets. recently short peptide fragments and small peptidomimetic molecules have been found also in our laboratory; these molecules bind to the id regions of β-amyloid and are putative drug candidates. precise control of bleeding is ensured by anticoagulant mechanisms, which under normal conditions prevail over coagulants mechanisms. disrupting the balance between procoagulant and anticoagulant systems due to congenital or acquired defects leading to thrombotic disorders. anticoagulants are substances that inhibit blood clot formation. their action consists in inhibiting the synthesis of prothrombin, the substances forming thrombus as well as some coagulation factors. many peptides and proteins with different molecular weight, such as antistasin (ats), ghilantens, hirudin, etc. showing high anticoagulant activity are isolated from salivary glands of ticks and leeches. they inhibit the action of serine proteinases from blood coagulation cascade. this creates an opportunity for targeted synthesis of low molecular weight analogues of some of these proteins, which can be used in the prevention and treatment of thrombotic disorders. ats is competitive, slow-binding inhibitor of factor xa. it is well known that blood coagulation could be blocked at different stages of the coagulation cascade through inhibition of various enzymes. therefore, it is interesting to determine the place of action of newly synthesized antithrombotic agents in the blood coagulation cascade. this can be done by determining the inhibitory constants of newly synthesized peptides on different enzymes of this cascade. herein we report on our kinetic investigation of newly synthesized peptide amides, analogues of isoform of ats . ki, km, vmax and type of inhibition on the factor xa, thrombin and plasmin were determinate. some interesting differences between type of inhibition of ats, free acids and amide analogues of ats were revealed. to evaluate the relative anti-platelet aggregation activities of each peptide, the lebetins were chemically synthesized and fully characterized. here we described the synthesis, the solution structure of lebetin g -g from the venom of vepera lebetina by h bidimensional nmr and the relation structure-activity. this peptide has been demonstrated to be associated with a potent anti-platelet aggregating activity. the g -g three dimensional structure consists in a compact β-bulged hairpain core from which emerges one loop and the cterminus and the n-terminus. we report on an approach whereby ligands are designed to bind and stabilize the - region of aβ in an α-helical conformation. these ligands reduce aβ toxicity to cells in culture and to hippocampal slice preparations. in addition, when these inhibitors are administered to drosophila melanogaster expressing human aβ ( - ) in the central nervous system, a prolonged lifespan, increased locomotor activity, and reduced neurodegeneration is observed . stabilization of the central aβ α-helix appears to counteract polymerization into toxic assemblies and indicates that this approach holds promise for the development of orally available compounds against alzheimer's disease. encouraged by the above results we are currently developing a second generation of designed ligands. this involves synthesis of different new peptoids and unnatural amino acids. additional support for the concept comes from recent molecular dynamics simulations that also uncover details of the mechanism of unfolding of the aβ central helix as well as retardation of the folding in presence of ligands designed to interact with the native helical conformation . synthesis and methodology for new ligands, which includes synthesis of novel amino acids, will also be presented. triostin a is a well-known natural product with antibiotic, antiviral, and antitumor activities. it inhibits rna synthesis by bifunctional intercalation into dna base pairs through its two quinoxaline units showing cpg selectivity. triostin a must adopt an altered conformation upon dna bisintercalation that is substantially different than its preferred native x-ray or solution conformation. this fact suggests that the destabilizing conformational change in the cyclic octadepsipeptide counteracts much of the gains derived from a second intercalation. nonetheless, the wide range of pharmacological activities exhibited by this compound prompted interests in identifying novel and additionally potent lead compounds with improved pharmacokinetic properties for clinical applications. herein, a library of twelve simplified triostin a analogues has been synthesized by solid-phase peptide synthesis. the introduction of the key quinoxaline units was carried out in solution. the analogues' conformation corresponds to the staple form that bisintercalator cyclic (depsi)peptides adopt when binding to dna and, in addition, some of the synthesized compounds showed improved solubility. our library was evaluated for its antiproliferative activity against four human cancer cell lines and one analogue showed greater cytotoxicity than triostin a and even comparable activity to doxorubicin, a very commonly used drug in cancer chemotherapy nowadays. surprisingly, little is known about its mechanism of degradation in solution or the degradation products. a recent study identified monomeric polysulfides and dimeric degradation products, postulated to derive from β-elimination followed by deamidation and dimerization. we recently reported that degradation of oxytocin and its analogues in aqueous solution at ph . produced monomeric polysulfides with up to sulfur atoms as well as dimeric products. unexpectedly, incubation of ot or of various analogues modified in position resulted in identical dimeric degradants. we concluded that β-elimination via breakage of the c-s bond of cys must be a key step of the process, and that the resulting Δala residue would have to undergo further modifications to yield the same dimeric products independently of the substitution of the n-terminal nitrogen. here we further clarify the degradation mechanism and propose a structure for the dimers. we postulate that hydrolysis of the Δala residue yields an n-pyruvoyl linear peptide, which loses one sulfur atom and subsequently forms dimers, which we found are linked by one disulfide bridge and one non-reducible bond. the putative linear n-pyruvoyl oxytocin intermediate was synthesized and found to degrade to the same dimers as the ones in the incubations of ot. a [u- c]cys ot analogue gave degradation products with c-nmr spectra consistent with a non-stereospecific aldol-type condensation. detailed experimental procedures, structures of the degradants and the postulated mechanism of ot degradation in near neutral solutions will be presented. inserm u paris, france αiibβ is the main platelet integrin and is responsible for platelet aggregation. a lipid-modified peptide corresponding to αiib intracellular sequence - (palmitoyl-k-l eeddeege , pal-k- - ), is platelet permeable and inhibits human platelet aggregation induced by thrombin . ymesradr, a peptide corresponding to the extra-cellular sequence - of αiib, is a platelet activation and aggregation inhibitor . the aim of the present study was to investigate the cooperativeness of the intracellular and extra-cellular peptides on platelet aggregation and their effect on the phosphorylation of fak and erk. pal-k- - together with the extracellular ymesradr peptide, at concentrations lower than their ic values, showed cooperative inhibition of platelet aggregation. the peptide combination inhibited also fibrinogen and pac- binding to activated platelets. fak phosphorylation is a postaggregation event related to outside-in activation of the receptor. the combination of peptides inhibited fak phosphorylation. erk phosphorylation is independent from platelet aggregation, and is enhanced by rgd-peptide inhibitors. the combined peptides inhibited erk phosphorylation. ovarian cancer (oc) is considered a rare disease and represents the fifth most common cause of death from cancer in women. the standard first-line treatment consists of a combination of paclitaxel and carboplatin (ddp) or carboplatin alone. in the case of progressive disease or drug resistance to platinum-based agents, either alone or in combination, investigational compounds should be used ( ) . the mechanisms behind acquired resistance to ddp and its derivatives are not clear yet, although it is evident that the process is multifactorial, including enhanced dna repair processes. some peptides designed from the interface subunit of the human thymidylate synthase (ts) have been identified recently ( ), as effective anticancer agents against sensitive and resistant oc cells. one of them was also able to recover the cellular sensitivity towards cisplatin in resistant oc cells in the μm range. to improve its potency and selectivity structural studies have been performed in combination with cellular assays aimed at understanding the mechanism of action. a label-free quantitative proteomic approach has been undertaken to study the effects of the peptide on the proteins involved in the modulated metabolic pathways, in particular those involved in the folate metabolism. structure-activity relationships (sar) have been performed to improve the lead peptide pharmacodynamics. all the compounds have been assayed and a protein profile set was studied to mark and validate their behavior as inhibitor of oc cell growth. hepatitis c is a liver disease provoked by a virus known as hcv. the disease is insidious. hcv causes anorexia, nausea, vomiting, fever, fatigue and jaundice. in about % of sufferers the disease is short, but others become chronic. in the chronic form in about % of cases the final result is cirrhosis of the liver and in the remaining % it leads to liver cancer. hcv is a very serious problem today. about % of people infected with hcv worldwide, i.e. about million are residents of europe. million people carry the disease as a chronic illness with the potential to develop into cancer in their liver. all these people represent a "reservoir" for storage and distribution of hcv. ribavirin, the nucleoside analog -β-d-ribofuranosyl- , , triazole- -carboxamide, known by the trade name virazole (also known as rebetron in combination with interferon-α), exhibits antiviral activity against a variety of rna viruses (paramyxoviruses, flaviviruses, etc.) as well as some dna viruses. in humans ribavirin is currently used in combination with interferon-α to treat hcv infections. this lack of strict specificity and a broad spectrum of activity are due to its multifunctional mechanism of action against viruses. these characteristics have made ribavirin a drug of substantial research interest. unfortunately, ribavirin shows a significant toxicity, causing bleeding in accumulation [ ] . herein, we report a total synthesis of modified in position ' of ribose residue, ribavirin in order to be further linked to cell penetrating peptides (cpps). in addition the synthesis of some cpps as well as bonding between two parts of final hybrid molecules will be reported. in our case the design of new hybrid molecules is done in order to: (a) vectorize ribavirin into liver cells; (b) transport ribavirin molecule trough cell membrane and (c) decrease toxicity of ribavirin. to obtain oligomeric aβ peptide, our laboratory uses a precursor depsipeptide of aβ. this precursor, termed as "iso-aβ" has an ester bond between the side chain of ser and gly . at physiological ph this ester bond becomes an amide bond via an o→n acyl shift. binding partners by which the oligomeric aβ mediates its toxic effect has not been yet investigated in the proteome or subproteome level. we used protein array technology to study the interaction of oligomeric aβ with recombinant human proteins, immobilized on a protein chip. aβ binding proteins were identified with the aid of a monoclonal aβ antibody. altogether proteins were found to interact with our aβ-oligomers. these proteins were grouped according to their function. one of the major groups contained proteins participating in translation. these proteins were found in ribosomes. to prove our proteomic results ribosomes from rat hippocampus were isolated. elisa experiment revealed that aβ binds to ribosomes in a dose-dependent manner. using the sequence of the aβ-binding proteins a homology search was performed to find oligopeptides, that possibly bind to aβ. based on these sequences a peptide chip containing hexapeptides was prepared. aβ interacting peptides were identified with a monoclonal antibody. several peptides were synthesized and tested on mtt assay. two out of four compounds inhibited the toxicity of aβ on rat hippocampal slices. summarizing our results aβ binding proteins and peptides were identified. knowledge about aβ binding proteins can help to understand the pathogenesis of ad, such us the possible involvement of ribosomes. oligopeptides can be lead compounds of future drug development. huge proteolytic complex named proteasome catalyzes protein degradation in every eukaryotic cell. it consists of subunits forming four stacked rings and one or two regulatory caps. two inner rings of the proteolytic part contain three catalytic β-subunits that possess different substrate specificity. higher vertebrates can express γinterferon-inducible immuno-β-subunits. proteasome plays an essential role in continual turnover of intracellular proteins and in antigen processing. autoimmune diseases such as multiple sclerosis and its murine model eae are believed to rise from breakdown of tolerance of the immune system. it assumed that immunoproteasome could play an important role in autoimmune diseases. several classes of chemicals proved to be inhibitors of proteasome and the most active are boronate peptide derivatives. these inhibitors totally inactivate proteasome and result in full stop of intracellular protein turnover and cell death via apoptosis. another class of inhibitors, epoxy ketones, was shown to be more selective for immunoproteasome and could be used not for full stop of proteasome function, but for fine tuning of altered proteasome functioning. we examined properties of several inhibitors of four different classes, namely peptide boronate bortezomib, peptide aldehyde mg , lactam lactacystin, and peptide epoxyketones epoxomicin, mg ek, uk and pr- . for inhibition experiments we used proteasome isolated from eukaryotic cell lines cho, nso and hek, treated and non-treated with γ-interferon, as a model cells contatinig constitutive and immunoproteasome. the upregulation of proteasome immunosubunits was revealed in cho and nso cells treated with γ-interferon. the ic values for all studied inhibitors were obtained, and ki in some cases were calculated. the epoxyketones were shown to selectively inhibit in submicromolar concentrations the proteasome sample which contain high amount of immunosubunits. in order to find an effective antimalarial, this study refers to some angiotensin ii (aii) analogues which were considered the important physicochemical characteristics described by silva et al. to verify the biological activity against plasmodium gallinaceum and to understand the hydrophobic cluster influence, explained by tzakos et al. these analogues were synthesized and characterized as described by silva , as well as the biological assays and comprises, to verify: the hydrophobic cluster activity -a) drvyhipf; b) drvypr; c) ryhipf and d) fphiyvrd; the importance of these residues in aii molecule -e) rypf; the importance of aromatic residues -f) yhpf and the action of these hydrophobic residues, when interacting with the parasite membrane -g) vipf. it was observed that in a ( % of bioactivity), the phenol group of tyr is close to imidazole group of his that could promote a hydrogen bond formation. besides that, could occur van der waals interactions between ile and phe residues due its proximity and non-polar characteristic. these interactions could not be effective in native aii ( %) , because ile residue promote a steric influence on the organization of his and tyr residues that not exist in b ( %). in c ( %) and e ( %) analogues, the influence of the arg residue could promote a cation-π interaction with tyr residue and the cluster may have suffered slight destabilization and its antiplasmodial activity was compromised subtly. in d ( %), the electrostatic change, obtained with the total inversion can have disordered its interaction with parasite membrane, since it is not related to membrane receptors, because d-aii presented % of biological activity. moreover, hydrophobic and aromatic residues importance was confirmed through the results obtained % and % of activity, with g and f, respectively. we conclude that hydrophobic cluster modifications and interactions of amino acid side-chain influences in the biological activity. closed joint-stock company "vertex", st-petersburg, russia creatine (cr), a small molecule synthesized in the kidney, liver and pancreas plays important role in atp synthesis, replenishing its store even in the absence of oxygen. cr is able to protect brain cells against ischemic damage; however it has poor ability to penetrate the blood-brain barrier without specific carrier protein. thus, synthesis of stable hydrophobic derivatives capable of crossing the bbb by alternative pathway is of great importance for the treatment of different neurological diseases including stroke, traumatic brain injury and hereditary crt deficiency. here we describe the synthesis and biological activity of new hybrid compounds -creatinyl amino acids. originally the title compounds were synthesized by guanidinylation of sarcosyl peptides. however, for large scale synthesis better results can be obtained using direct cr conjugation with amino acid or peptide derivatives by isobutyl chloroformate method. addition of equivalent amount of ptoluenesulfonic acid as lipophilic counterion ensures efficient cr dissolution in dmf along with its simultaneous protection towards intramolecular cyclization. it excludes the application of expensive guanidinylating reagents and permits to simplify the synthetic procedure. purification of final product and its conversion into appropriate salt form can be achieved by iec followed by crystallization from organic solvents. synthesized creatinyl amino acids and peptides exhibited significant biological activity in different assays including platelet aggregation test, ischemic stroke and nano -induced hypoxia model. one of the most effective compounds -creatinyl-glycine ethyl ester increases life span of experimental animals more than two times in hypoxia model and has neuroprotective action in brain stroke model when applied both before and after ischemia. these data evidenced that creatinyl amino acids can represent promising candidates for the development of new drugs useful in stroke treatment. the efficient recognition and destruction of tumor cells via specific cellular markers is a major goal in cancer therapy. various growth factor receptors such as egfr, hgfr, vgfr and their downstream signaling networks have been proven to be effective molecular targets, as they are frequently involved in cancer proliferation and metastasis. downregulation of these receptors and/or blocking their signaling pathways have clear anti-tumoral effects. drugs based on monoclonal antibodies (mab) targeting such cell surface receptors have attracted a lot of attention as a new generation of therapeutics. however, their production is costly and identifying new, variable routes to modified molecules with similar properties is currently a major focus. , here we present an approach to chemically synthesize a molecule that combines the mode of action of antibodies with the advantages of smaller, chemically accessible molecules. these "synthetic antibody" (sab) molecules contain a chemoattractant that activates the innate immune response and resembles the fc domain of a typical antibody. specificity is imparted by two binder peptides that assume the function of the variable antibody domains and bind to a cell surface target. the fc and fab domains of the sab molecules are connected via polyethylene glycol linkers. sab molecules are prepared by solid phase synthesis, a flexible technique that allows fast production, full control of their properties and targeting two different cell surface receptors (bispecific tumor targeting). they are currently tested in vitro and in vivo for their effect on the innate immune system, general toxicity and selective binding to cancer cells. the key enzyme in the processing of polyproteins translated by viral rna genome of sars-cov is a kda protease called c-like protease ( cl protease). sars cl protease is a cysteine protease containing a cys-his catalytic dyad, and cleaves precursor poly proteins at as many as conserved site involved a conserved gln at the p position and a small amino acid (ser, ala, or gly) at the p' position. due to its functional importance in the viral life cycle, sars cl protease is considered to be an attractive target for drug design against sars. recently, we found tetrapeptide aldehyde, ac-thr-val-cha-his-h, showed high inhibitory activity with ic value of nm toward cl-r i mutant protease , . to compare the inhibitory activity of small compounds with those containing active functional groups, we synthesized serine-derivatives within the essential functional groups and evaluated its inhibitory activity. the synthetic scheme was started from fmoc-ser(tbu)-oh, following modification of c-terminal carboxyl group with p , n-terminal amine with p and side chain alcohol with p functionalities. steps overall reaction led to obtain novel serine derivatives for the small molecular inhibitors of sars cl protease. the assay with cl r i mutant protease was examined to evaluate the inhibitory activity of the synthetic serine derivatives. then, molecular docking study of complex of cl protease with the ligand was carried out. docking simulation experiment with r i (pdb id: aw ) and the inhibitor, which has the best activity in the serine derivatives, indicated that p fitting s ' pocket. at the result of assay, p , p and p positions of the inhibitor should be modified by benzoyl group, cyclohexyl group and cinnamoyl group, respectively. their bioactivities are underpinned by their distinctive structure with exceptional stability, thus making cyclotides exciting, not only for agricultural and pharmaceutical purposes, but also as a template in drug design. in all of the reported activities, cell membranes seem to be the primary target for cyclotide activity. to unravel the importance of lipid membranes on the reported activities of cyclotides, a set of cyclotides belonging to möbius and bracelet subfamilies were compared in their mode of action. the lipid selectivity and membrane affinity were compared with their efficiency against different target cells (e.g. red blood cells, bacteria, hiv particles). we have found that the bioactivity of cyclotides is dependent on the lipid composition of the target cell membrane and independent of a protein chiral receptor. in particular, all the native cyclotides tested target the cell membrane through specific binding to phospholipids containing phosphatidylethanolamine (pe)headgroups, but the membrane binding affinity is further modulated thorough non-specific peptide-lipid hydrophobic interactions, which are dependent on the specific cyclotide. in addition, the bioefficiency of cyclotides broadly correlate with their ability to target and disrupt the cell membrane. overall, we have shown that even with a common specificity for membranes containing pe-phospholipids, a fine selection was found across the family. in particular, each cyclotide inserts and disturbs the membrane in a distinct way, which explains the diversity of this family but also their distinct activities. the observation that all the tested cyclotides have a preference for a specific lipid makes this family truly intriguing and brings insights to optimize the use of the cyclotide template in drug design. malaria is a disease that affects around million people causing . - million of deaths annually. based on our previous studies, angiotensin ii (aii) presented antiplasmodial activity against plasmodium gallinaceum, but due to pressure activity, it cannot be used as an antimalarial drug. in an attempt to increase antiplasmodial activity and reduce hypertensive activity, we synthesized by solid phase method, cyclic analogues of aii with i-(i+ ) and i-(i+ ) lactam bridge scaffold using asp and lys residues. the bridge was more effective when inserted next to n-terminal extremity , probably this insertion, on another portion of the peptide, provides a change in the conformation of the molecule and its hydrophobic cluster formed by tyr, ile and his , which may have influence in the peptide-membrane interaction. thus, we have focused in the n-terminal extremity, testing new analogues, using glu/asp/orn/lys residues as bridgeheads components in i-(i+ ) lactam bridge scaffolds, which showed that antiplasmodial activity is increased using glu residue and that larger lactam rings are better to biologically active. therefore, new restrict peptides by i-(i+ ) and i-(i+ ) lactam bridge were designed, using glu residue as bridgehead element, but the same effect was not verified, getting a maximum of % of bioactivity. on the other hand, we promoted an increase in the hydrophobic character of the molecule, replacing the asp residue of aii sequence by fmoc-glu and asp(ofm), in order to improve the interaction of these compounds in the sporozoite membrane. the replacement by fmoc-glu provided a decrease of activity, while that asp(ofm) kept the aii activity, because there are changes of charge in the peptide, which may have modified the conformation in physiological medium. this kind of approach may offer the basis for development of new drugs and chemotherapy against malaria. animal venoms are complex chemical cocktails, comprising a wide range of biologically active reticulated peptides that target with high selectivity and efficacy a variety of membrane receptors such as ion channels or g-protein coupled receptors. venoms can therefore be seen as large natural libraries of biologically active molecules that are continuously selected and highly refined by the evolution process. the vision associated with the venomics project is to investigate in depth the enormous structural and pharmacological diversity of venom peptides through the development, integration and implementation of a novel research paradigm combining cutting-edge "omics" technologies in a high-throughput workflow. this new paradigm enclosed in venomics aims at replicating in vitro the diversity of venoms to generate original peptide banks to be used in drug discovery programs. herein, we show the different strategies we adopted for efficient solid phase synthesis and folding with an easy purification of peptides rich in cysteine and containing posttranslational modifications (ptm). angiogenesis depends on the adhesive interactions of vascular cells. the adhesion receptor integrin av b was identified as a marker of angiogenic vascular tissue. the αν β integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis, mainly by interacting with matrix proteins through recognition of an arg-gly-asp (rgd) motif. inhibition of the αν β integrins with a cyclic rgd peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. the aim of this study was to investigate the effects of replacement of a-amino acids by aza-β -amino acid analogs in cyclic rgd-peptides as αν β -integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. the selectivity profile of these antiadhesive cyclopeptide is rationalized by a special presentation of the pharmacophoric groups. we synthesized cyclic rgd peptidomimetics that include aza-β -amino acid residues. modifications were added to the rgd skeleton in order to optimize the peptide activity. then, we investigated the pharmacokinetics activity of these pseudopeptides in hek (human embryonic kidney ) and endothelails cells huvec (human umbilical vein endothelial cells) cell by analyzing cell viability and protein involved in the angiogneisis processes. since tenascin c is a factor expressed highly in the tumorassociated matrix, targeting it would be a desirable first step for targeting the tumor-specific microenvironment in fact, a high level of tenascin c expression has been reported in most solid tumors, including lung cancer, colon cancer and glioblastoma. therefore, the targeted binding of tenascin c in tumor stroma would inhibit tumor metastasis by modulating cancer cell growth and migration. we isolated a peptide that bound to tenascin c by phage display peptide library selection, and the selected peptide specifically recognized tenascin c protein in xenograft mouse tissue. we also observed exclusive staining of tenascin c by the selected peptide in tumor patient tissues. moreover, the peptide reduced tenascin c-induced cell rounding and migration. we propose that the tenascin c targeting peptide may be useful as a specific anti-cancer diagnostic and therapeutic tool for most human solid tumors. radiolabeled pansomatostatins are expected to enhance hsst - tumor-uptake and to broaden clinical indications as compared to currently established sst -prefering radioligands. previous experience has revealed [ in-dota ,dtrp ]ss- ([ in]at s) as a true pansomatostatin analog, exhibiting however poor in vivo stability. in order to enhance metabolic stability, we introduced a second disulfide bridge to the at s motif by formation of extra / -amino acid (aa) or / -aa ring generating at s and at s, respectively. the orthogonally protected sequences were assembled on the solid support, deprotected and cleaved from the resin with tfa. the first cys -cys (at s) or cys -cys (at s) cyclization was performed in dmso, while the second was completed with iodine oxidation after in situ deprotection of cys (acm) and cys (acm). during hsst - +-autoradiography, at s showed unexpected total loss of sst - affinity, whereas at s showed high affinity (ic in nm) to all hsst - (hsst = . ± . ; hsst = . ± . ; hsst = . ± . ; hsst = . ± . ; and hsst = . ± . ). consistent with this finding, only at s stimulated sst internalization during immunofluorescence-based internalization assays, showing agonistic properties for sst . furthermore, [ in]at s internalized rapidly and specifically in sst + ar - j and hek -hsst +-cells. hplc analysis of min ex-vivo mouse blood samples revealed that > % [ in]at s remained intact. after injection in scid mice bearing ar - j and hek -hsst + tumors [ in]at s specifically localized in the rsst a+ ( . ± . %id/g vs. . ± . %id/g + nmol tate at h postinjection (pi)) and in hsst + implants ( . ± . %id/g vs. . ± . %id/g + nmol ke at h pi). this study has shown that introduction of an extra disulphide bridge in at s confers high metabolic stability. however, in a / member ring combination it leads to total loss of affinity. the reasons for this effect are currently investigated by nmr conformational studies. transporter compounds are useful tools to solubilise and increase the delivery of therapeutic molecules in the human body. one system to improve the cellular uptake of such therapeutic molecules are cell-penetrating peptides (cpps). these short peptide chains are either polycationic (containing several arg and lys) or show a more amphiphatic structure. it is known that the multivalency effect -the presentation of several copies of a cpp motif on a single molecule -can increase the cellular uptake. peptide dendrimers represent a group of tree-like, multivalent macromolecules, which are synthesized for different chemical and biological applications in our group. we now combine linear cpps with peptide dendrimers to get a well defined branched molecule made up of only natural amino acids. in our systematic study of peptide dendrimers decorated with different cpps we found that the potency of the single cpp as a transporter for small molecules can be increased and that these peptides show usually low cytotoxicity. additionally we designed new dendritic cell penetrating peptides with similar activities like linear cpps. all compounds are covalently linked to fluorescein for visualization with flow cytometry and confocal analysis. the results show that the peptides can transport efficiently a hydrophobic cargo into the cells. chemical stability of esters of acyclovir with amino acid and cholic acids k. chuchkov, r. chayov, i. g. stankova* south-west university "neofit rilski", blagoevgrad, bulgaria amino acid esters of antiviral drugs are a very good solution for improving oral bioavailability of the actual medicine. one of the most effective and tolerant prodrugs is valine ester of acyclovir -valaciclovir. taken orally exhibits three to four times higher bioavailability of acyclovir. the chemical stability of amino acids ( -fphenylalanine) (r,s) and bile acids (deoxycholic acid and chenodeoxycholic acid) esters of acyclovir was studied in experimental conditions simulating some relevant biological medias (ph . and . , °c).the chemical stability experiments revealed that the examined amino acid ester of acyclovir were relatively unstable in acidic ph, but bile acid ester is stable in the same ph. the examined amino acid and bile acid esters of acyclovir in neutral ph are relatively stable. in ph , all of tested compounds are more stable than valacyclovir (t / = h) -the first effective prodrug of acyclovir. in acidic ph acyclovirdeoxycholat and acyclovirchenodeoxycholat are more stable than valacyclovir. acyclovirchenodeoxycholat is the most promising anti-ebv prodrug candidate with high activity and satisfying chemical stability. cell-penetrating peptides (cpp) have become efficient tools for the cellular internalization of bioactive molecules due to their ability to cross the plasma membrane of diverse cells and cell lines. [ ] we recently reported that the cpp sc , which consists of the residues - of the c-terminal region of the cationic antimicrobial peptide cathelicidin (cap ), is an effective carrier peptide for small organic molecules like fluorophors and toxic peptide sequences into various cell lines [ ] . however, in general linear peptides are more susceptible to proteolytic degradation than their cyclic analogs [ ] . therefore, we investigated the cyclization of cpp derived from sc by means of cui-mediated azidealkyne cycloaddition (cuaac) [ ] . furthermore, we examined their conformation and proteolytic stability as well as their internalization efficiency and toxicity against various cell lines, in comparison to their linear equivalent and to other cpp. looking for the proper prodrug: a peptidomimetic approach to identify and inactivate bacterial mono-adp-ribosyltransferase toxins m. beich-frandsen, r. jørgensen division of microbiology and diagnostics, statens serum institute, copenhagen, denmark mono-adp-ribosylation is an endogenous posttranslational modification in eukaryotic cells, simultaneously utilized as virulence strategy by deadly secreted bacterial toxins. many bacterial toxins have been found to act as mono-adp-ribosylating enzymes, targeting anything from g-proteins to the actin skeleton. the diphtheria toxin from c. diphtheriae and exotoxin a from p. aeruginosa, both target the diphthamide-group of a unique modified histidine in eef , inhibiting protein synthesis by ribosome mimicry , . we aim to inactivate these nad+-utilizing toxin enzymes by nad-conjugated peptidomimetics, in a target-specific prodrug-approach. the adp-ribosylation reaction follows a random third-order s n mechanism. in the proposed model for the transition state of the reaction, the cleavage of the n c -nn bond of nad + releases strain and generates a oxacarbenium ion intermediate with a positively charged nicotinamide (n)ribose, subject to a nucleophilic attack from the substrate , , . adp-ribosylating toxins are commonly characterized by a artt-motif involved in substrate recognition . studies suggests conformational rearrangement of the residues surrounding the substrate binding site to be required for optimal geometry of the initial glycosidic nc -nn bond cleavage within nad + . subtype specific nad-conjugated peptides, designed based on previous structural analysis of the adpribosylation reaction, act as substrate for the enzymatic adp-ribosyl-transfer, and hereby attach covalently to and inactivate the nad + -utilizing toxin. relying on previous structural studies, and established ligand-binding and kinetic data, an initial peptide library, designed by bioinformatics and evaluated for specificity of common targets in-silico identifies initial leads. lead-scaffolds are implemented in rational peptide-design, based on high-resolution structural-and biophysical studies of multiple peptide-enzyme complexes, to identify possible prodrug-strategies for enzyme inactivation. nanoparticles play a crucial role in medicine for their potential application as in vivo carriers of active principles [ ] . liposome display unique pharmacokinetic properties slowly releasing drugs loaded in the inner aqueous cavity. in the last years we have developed supramolecular aggregates labeled by bioactive peptides able to recognize overexpressed receptors on tumour cells membrane delivering doxorubicin chemiotherapeutic drug [ ] . neurotensin(nt), a amino acid peptide, has dual functions of neurotransmitter or neuromodulator. the cterminus short fragment - preserve the activity but the half life of wild type form in vivo is very short. nt receptor type (nts ) is overexpressed in severe malignancies such as small cell lung cancer and colon, pancreatic, and prostate carcinomas. we have designed new amphiphilic molecules containing in the hydrophobic moiety two aliphatic chains and in the hydrophilic moiety a the bioactive portion able to aggregates with phospholipid molecules achieving liposome. we have synthesized neurotensin wild type sequence, the truncated form and the tetra-branched neurotensin(nt - ) or a truncated form(nt - ) tetrabranched peptides(nt ) adopting an opportune synthetic strategy on solid phase. all liposome were formulated adding the neurotensin amphiphilic monomer in ratio : with dopc in order to evaluate the capability to recognize selectively receptors overexpress on cell membrane surface. the liposomes size was determined by dynamic light scattering measurements, values for the hydrodynamic radius(rh). the selective internalization and cytotoxicity of fully doxorubicin loaded liposomes as compared to pure dopc liposomes, was tested in ht human colon adenocarcinoma and te human rhabdomyosarcoma cells. recently, small interfering rna (sirna), one kind of rna interference (rnai) technology represent the most common and, to date, the most effective method to inhibit target gene expression in human cells. it is also a common recognition that non-toxic delivery of sirna is an urgent problem for the therapeutic application of sirna. for the efficient gene silencing in vivo, prolonged circulation of sirna with take efficient and non-toxic cellular uptake and resistance against enzymatic degradation are indispensably required. ) telomerase activity has been regarded as a critical step in cellular immortalization and carcinogenesis and because of this, regulation of telomerase represents an attractive target for anti-tumor specific therapeutics. in this paper, we present the efficient and non-toxic cellular uptake of sirna using novel amphiphilic peptides and the application to silencing of htert in human cancer cell lines. in the present study, we investigated the intracellular delivery of sirna using some amphiphilic peptides and the silencing effect of sirna targeting htert mrna in human cancer cell lines, jurkat, hela and k . the complex of sirna and a specific amphiphilic peptide or its hybrid with an intracellular transport signal peptide could be effectively taken up into cells. the complex also showed a high silencing effect against htert mrna. moreover, the combination of sirna-nes conjugates and the amphiphilic peptides improved silencing effects up to . %. the amphiphilic peptides and their hybrids showed almost no cyto-toxicity and protected sirna against intracellular nuclease digestion in % fbs (half life time was over h). tumor targeting with the decapeptide gonadotropinreleasing hormone (gnrh) or its analogues is based on the discovery that gnrh receptors are overexpressed in many tumor cells, compared with their expression in normal tissues. using these peptides as carriers/targeting moieties in a conjugate with therapeutic agents can increase the selectivity and the stability of the conjugates, or eliminate the toxic side effects of the drug. gnrh-iii (<ehwshdwkpg-nh ) is especially favoured as a targeting moiety because of its antiproliferative effect and weak endocrine activity in mammals. the aim of our work was to synthesize novel short-chain fatty acid (scfa) acylated daunorubicin-gnrh-iii bioconjugates with enhanced enzymatic stability, cellular uptake, in vitro and in vivo antitumor activity. ser in position of gnrh-iii was replaced by lys and its ε-amino group was acylated with various fatty acids. gnrh-iii( lys(x), in conclusion, glu - /dglu - substitution in gi-derived radiopeptides enhanced stability and reduced renal values by ~ . fold. on the other hand, removal of the (glu) sequence in mg suppressed renal uptake but undermined stability. both modifications, however, compromised tumor targeting, demonstrating that more efficacious structural changes in the gi motif are warranted to improve the in vivo profile of resulting radioligands. synthesis and characterization of a mtc-labeled rhodamine-conjugated angiotensin peptide as a potential cardiac function imaging agent s.m. okarvi king faisal specialist hospital & research centre, cyclotron and radiopharmaceuticals department, riyadh , saudi arabia angiotensin ii (ang ii) is an -amino acid peptide that has been known to play a vital role in cardiovascular system. the actions of ang ii have been implicated in many cardiovascular conditions, such as coronary heart disease and heart failure. in an effort to develop a cardiac imaging agent with enhanced targeting capacity, we attached ang ii to rhodamine (rh), a lipophilic cation that specifically accumulates in the myocardium. the rh conjugated ang ii was evaluated for its potential as a heart imaging agent. rh-lys-gly-cys-asp-arg-val-tyr-ile-his-pro-phe-conh was prepared by solid-phase peptide synthesis according to fmoc/hbtu methodology. nhs-rh was attached to the ang ii peptide via the free amino group of lys residue by manual synthesis. rh-ang ii conjugate was labeled with tc- m by the stannous-tartrate exchange method. in vitro stability was determined in human plasma and in excess of cysteine. in vivo biodistribution and biokinetics were performed in balb/c mice and sprague dawley rats. the rh-ang ii conjugate radiolabeled efficiently with tc- m (> % labeling efficiency) as determined by hplc analysis. tc- m-rh-ang ii exhibited good chemical stability against cysteine transchelation and sufficient metabolic stability in human plasma. in mice, the bioconjugate displayed efficient clearance from the blood and excreted mainly through the renal route with some excretion by the hepatobiliary pathway. the uptake in the heart was . ± . % id/g as early as min post-injection; whereas, the uptake in the lungs, liver, stomach and kidneys varied between - % id/g. in rats, the bioconjugate displayed relatively better pharmacokinetic characteristics, with low uptake in the major organs (< % id/g). the uptake in the heart ( . ± . % id/g) was found to be higher than the uptake in the blood and muscle, resulting in good heart-to-blood and heart-to-muscle uptake ratios. this initial study towards the development of an effective cardiac imaging agent advocates that the use of hybrid conjugates appears to hold a great promise as a new and attractive approach for rapid and efficient imaging of heart. in humans two isoforms of gnrh are exist, gnrh-i (<ehwsyglrpg-nh , where <e is pyroglutamic acid) and gnrh-ii (<ehwshgwypg-nh ). in contrast to gnrh-i, exact function of gnrh-ii is not well identified. however, it was indicated that gnrh-ii derivatives have more potent antiproliferative activity on human endometrial and ovarian cancer cells in vitro than gnrh-i analogs. some kinds of tumor cells (e.g. breast, prostate, colon) produce gnrh-i and gnrh-ii, and express their receptors. however, the presence of functionally active gnrh-ii receptors on cancer cells is still not clear - . on the other hand, gnrh-ii analogs, both agonists and antagonists, induce apoptosis in many different cell types. in contrast to gnrh-i agonists, gnrh-ii agonist [d-lys ]gnrh-ii does not activate nfκb, therefore does not protect the cancer cells from apoptosis [ ] [ ] [ ] . in the regulation of apoptosis, not only the pro-and antiapoptotic proteins play a role , but other molecules also attend (e.g. ck , casein kinase ii enzyme) . our aim was to synthesize new proapoptotic peptide or enzyme inhibitor containing [d-lys ]gnrh-ii based derivatives and investigate their in vitro cytotoxic, cytostatic and apoptotic effects. the sythesis of [d-lys ]gnrh-ii, which is one of the most potent gnrh-ii agonist, was carried out on solid phase using fmoc/tbu strategy. different proapoptotic peptides or enzyme inhibitors were attached directly to the gnrh-ii derivative. in vitro cytostatic and cytotoxic effects of gnrh-ii conjugates were investigated by mtt-assay using different human breast (mcf- and t -d) and colon carcinoma (ht- ) cell cultures. early apoptotic effect of gnrh-ii conjugates was studied by flow cytometry on mcf- , t- d and ht- cell lines. according to our results, these conjugates might be potential candidates for cancer treatment, because the combination of a targeting moiety (gnrh derivative) with proapoptotic peptides or enzyme inhibitors and drug molecules might have synergistic activity resulting in highly potent multifunctional therapeutic agents. department of pharmaceutical and pharmacological sciences, university of padua, padua, italy melanoma is the most deadly skin cancer with an increasing incidence. its therapy continues to be a challenge since, regardless of the treatment used, longterm survival is quite uncommon. melanoma cells are characterized by high levels of glutathione s-transferase p - (gstp - ) isozyme. this enzyme is also highly expressed in solid tumours and drugresistant cells, where a role for this protein in the regulation of cell proliferation has been described. in the past, lyttle et al. synthesised glutathione (gsh)linked anticancer prodrugs activated by physiological concentration of gsts. structure and mechanistic studies showed that in the gst-gsh complex, the gst tyr residue placed near the s atom of the cys in gsh, is in the phenoxide form to facilitate the deprotonation of the sulfhydryl group in gsh, making it able to react with electrophilic species. by taking advantage of this active-site geometry, we synthesised two tyrosinase activated melanoma prodrugs ( -methoxyphenol and n-acetyl- -s-cysteaminylphenol) linked to gsh. in these compounds an ethoxycarbonyl group was placed between the gsh sulphur, oxidate to sulfone, and the tyrosinase-activated prodrugs. in this way the tyr phenoxide (tyr in gstp and tyr in gstm isozyme) would be able to abstract one of the acidic methylene protons linked to the sulfone moiety. such a deprotonation should trigger a beta-elimination, resulting in the release of melanoma prodrugs. the stability of the obtained putative prodrugs has been evaluated in different ph buffers and in rat liver and kidney cytosolic fractions. synthetic peptides represent first choice biomolecules, with respect to proteins and mab, for the monitoring of population variability and receptor functionality associated to a tumor pathology due to their favorable pharmacokinetic profile. it has been demonstrated that the surface molecule integrin alfa-v beta- could be an ideal target for the interaction with melanoma directed radiolabeled peptides. this integrin is indeed overexpressed both in tumor cells and in tumor vessels endothelium, while it is not expressed in the majority of normal tissues. cyclic peptides with the sequence arg-gly-asp (rgd), are known to bind the integrin alpha-v beta- with high affinity and selectivity and could conveniently target a radiotherapeutic to melanoma cells. we previously demonstrated that the cyclic rgdfk peptide interact with high affinity to a human melanoma cells. the aim of this work was to conjugate the mtc to the peptide by using the [ m tc(n)(pnp)] + system (pnp=aminodiphosphines) to obtain a radiolabeled peptide useful in melanoma imaging. , spectrum. a nh(i)→βch(i- ) cross peak more intense than the nh(i)→βch(i) cross peak is observed when the peptide adopts the . -helixl conformation. by contrast, an opposite trend of intensities of the same noe cross peaks indicates the occurrence of a -helical conformation. by using this method, we were also able to determine that polar solvents, such as mecn or meoh, may induce a helical structure in deg homo-peptides which otherwise adopt the . -helix conformation in cdcl . this finding allows us to rapidly switch these peptide series from "short" ( -helix) to "long" ( . -helix) by changing the solvent only, thus making them extremely attractive tools. school of biological sciences, university of auckland, auckland , new zealand naturally occurring anti-freeze proteins (afps) enable organisms like polar fish to survive the freezing temperatures of their natural habitat. as well as being cryoprotective, afps exhibit properties of ice recrystallization inhibition (ri). the ability of afps to influence the size, morphology and aggregation of ice crystals can be used in food technology, where the growth of ice crystals in frozen foods is of primary concern. chemical synthesis of afps enables access to pure material for structural and functional studies. our new research programme in the 'food and health' area, prompted us to develop tailor made anti-freeze peptides for potential applications in the frozen food industry. 'multiple salt-bridge analogues' of the naturally occurring winter flounder afp were synthesized and evaluated for their antifreeze activity in comparison to the natural sequence. details on the molecular modelling, structural characterization and anti-freeze activity analysis on the synthetic afps will be presented. several disorders are now believed to be conformational diseases caused by misfolding in the structure of specific proteins leading to the gradual aggregation and deposition of these abnormal proteins. attempts to develop effective therapies for these proteopathies (such as alzheimer's, parkinson's and huntington's disease) have been directed toward reducing the production of the proteins, inhibiting their aggregation, or promoting their removal. conformation-dependent antibodies that specifically target misfolded proteins are proving to be useful tools to study the pathogenesis and as possible treatments of these diseases. we have previously shown that active immunization with tetrapalmitoylated aβ - peptide embedded in liposomes (aci- ) was capable of eliciting a conformation-specific immune response in mice. the antigenic peptide was shown to mimic the aggregated β-sheet conformation of the pathological a β peptide on the surface of the liposomes by cd, ssnmr and tht assay. we have also reported the modulation of the conformation of aβ - derived sequences by means of different lipidation patterns, lipid chain lengths and liposome surface charge. here we present the characterization of this panel of liposomal peptide constructs by atr-ir and correlate the results with previous studies by cd. in summary, atr-ir secondary structure analysis of a battery of amyloid-derived lipopeptides allowed to study the key parameters responsible for the adopted conformation on the lipid bilayer. the presented strategy to control the conformation of liposomal peptide immunogens could be applied to the rational design of vaccines against a range of protein misfolding diseases, such as alzheimer's disease. cyclic lipodepsipeptides (clps) are secondary metabolites produced by various bacteria, most prominently bacillus and pseudomonas. these often display potential antimicrobial properties, frequently ascribed to the ability to disrupt cellular membranes or form ion-pores. clps are a structurally diverse group of molecules always consisting of an oligopeptide chain cyclized by a lacton bond and a fatty acid moiety. they are divided into several groups based on sequence similarity. one such group is the viscosin group, which contains clps produced by pseudomonas that all feature nine amino acids and a conserved sequence pattern of hydrophobic and hydrophilic residues. recently, we have extensively described the self-assembly properties of one viscosin group member, pseudodesmin a. , we have shown that in non-polar organic solvents this clp forms anisotropic supramolecular structures of unrestricted size, reminiscent of the ability to form ionpores in the non-polar cellular membrane environment. here, we report on how the most prominent structural variations within the viscosin group affect the conformation and self-assembly properties. since the well-defined monomer conformation of pseudodesmin a is key to this self-assembly, the most striking variation within the viscosin group is the variability of the leu stereochemistry. for the first time, a conformational study will be reported for clps featuring an l-leu residue, such as viscosin and viscosinamide. the results of this study contribute to a deeper understanding of the structure-function relationship of clps in general. effective design of peptide mimicking protein helical segments is a great challenge. an intriguing approach to improve the helical content in a peptide sequence is achieved through the "peptide stapling" strategy using a hydrocarbon cross-linker (staple) . in this study we have investigated the effect of the introduction and location of staples on secondary structure of sequences derived from a reported peptide, mimicking cullin binding region to kctd , , to gain an insight into the designing of new molecular entities able to modulate the kctd oncosuppressor activity. indeed, kctd plays a crucial role in the ubiquitination of hdac by acting, in complex with cullin , as an e ubiquitin ligase, thereby affecting hedgehog activity and medulloblastoma growth. in the designed peptides the stapling has been achieved by ring-closing olefin metathesis between two s- -( 'pentenyl) alanine residues incorporated at i and i+ positions and into different sites within the peptide chains. the stapling position has been chosen on the basis of cullin -kctd complex model. circular dichroism studies have highlighted that the stapled peptides exhibit remarkable and different changes in secondary structure contents in comparison with the linear precursors and the unmodified sequence. a nmr conformational analysis will allow the determination of the conformational preferences of the stapled peptides at atomic resolution with also relevant implications for the kctd-cullin recognition. based on our findings from protease-mediated ligation of cleavage-sensitive peptides and proteins using ionic liquids (ils) as additives and from chemical acylation reactions of peptides in ils, we hypothesized direct and unique interactions between biomolecules and ils as the molecular reason for il-mediated structural effects. to verify this, we performed systematic structural investigations of suc-ala-xaa-pro-phe-pna and ala-xaa-pro-phe model peptides dissolved in aqueous imidazolium-based ils using high resolution magic angle spinning (hr-mas) nmr spectroscopy. highly resolved nmr spectra are obtained that allow for complete h resonance assignments of the peptides as solutes and the ils as solvents. the changes in the proton chemical shifts Δδ( Η) of a peptide on dissolution in ils arise from direct interactions between both, including il-induced structural/conformational changes of the peptide molecules. , the strength, position, and type of these interactions are influenced by the nature of the amino acid residues in the peptide sequence as well as the nature and type of the il anions. downfield shifts indicate strong interactions between the ils and the peptide backbone, whereas upfield shifts point to hydrophobic interactions and stacking effects with the il imidazolium ring. the pattern of Δδ( Η) along the peptide chain reveals the sites and the type of interactions. the extent of Δδ( Η) implies a significant effect on the structure of the peptides. different Δδ( Η) were obtained for cis-and trans-configured peptide isomers and point to a conformer-specific il/peptide interaction. calcitonin is a -amino acid polypeptide hormone, which takes part in calcium metabolism in bones. it belongs to a protein family whose members may form amyloid fibrils. these fibrils (or pre-fibrillar aggregates) are related to serious diseases known as amyloidoses, whereas amyloid-like fibrils may have natural functional roles in many organisms. the amyloid form of human calcitonin is related to medullary thyroid carcinoma. using our online consensus prediction tool for 'amyloidogenic propensity' of protein molecules "amylpred", a novel hexapeptide of human calcitonin was predicted as a novel, possible amyloidogenic peptide. we investigated experimentally its ability to form amyloid fibrils, utilizing tem, x-ray fibre diffraction, atr ft-ir spectroscopy and polarized light microscopy. analysis of the results shows that this peptide self-assembles into amyloid-like fibrils and that amyloid fibrillogenesis is mediated via nuclei of liquid crystalline nature, known as spherulites. we thank the university of athens for the financial support. school of pharmacy, university of norwich nr tj ; inserm u , université de rouen, mont saint aignan rfa, a rfamide neuropeptide, is the ligand of gpr and the system rfa/gpr is involved in feeding behaviour [ ] . a structural study by nmr showed that rfa, in a membrane mimetic medium, possesses a helix p -r -linker k -g -turn f -r motif. sar studies on truncated fragments of rfa indicate that, in addition to the turn motif, additional residues are required to maintain a correct biological activity ( rfa ec = , nm; rfa - ec = , nm; rfa - ec = , nm; rfa - ec = nm) [ ] . our aim is to synthesize new gpr ligands using an iterative process combining rational conception, peptide and modified amino acid syntheses, biological and structural analyses. structural and biological results will allow us to find new modifications and conceive more potent analogues. using nmr, we demonstrated that rfa - encompasses a nascent helix and a turn while rfa - possesses only the c-terminal turn structuration. the destabilization of the helical structure on fragments with a shorter n-terminal region could explain the decrease of the biological activity of rfa fragments. we decided to focus on the importance of a stabilized helix on the biological activity. we chose the hydrogen bond surrogate method which stabilizes a helix by replacing the main hydrogen bond with a covalent link without modifying the side-chains [ ] . syntheses, nmr characterization and molecular modelling of analogues will be discussed. acknowledgments: this work is supported by erdf funding through the is:ce-chem project and interreg iv a france-(channel)-programme. firstly, it induces a constraint by restricting the ı o-dihedral angle to values around - °. secondly, the xaa-pro peptide bond is subject to cis-trans isomerization characterized by an increased cis population and an activation energy that is low when compared to the other amino acids. besides, the five-membered ring of the pro residue can adopt two main distinct conformations (up-puckered or downpuckered) that are almost equally abundant in peptides and proteins. a variety of mimics and analogs have been designed in order to control the conformation of the peptide backbone and/or to alter the cis/trans ratios and the rotational barriers for cis-trans isomerization. in this presentation, nmr studies and theoretical calculations have been performed on model peptides ac-ser(Ψpro)-nhme, (s,s)-ac-ser(Ψh, cf pro)-nhme, and (r,s)-ac-ser (Ψ cf ,hpro)-nhme. their thermodynamic and kinetic features have been analyzed in chloroform, dmso, and water, allowing a precise description of their conformational properties. we found that trifluoromethyl c δ -substitutions of oxazolidine-based pseudoprolines can strongly influence the cis-trans rotational barriers with only moderate effects on the cis/trans population ratio. in chcl , the configuration of the cf -c δ entirely controls the ψ-dihedral angle, allowing the stabilization of γ-turn like or ppi/ppii-like backbone conformations. moreover, in water and dmso, this c δ -configuration can be used to efficiently constrain the ring puckering without affecting the cis/trans population ratio. theoretical calculations have ascertained the electronic and geometric properties induced by the trifluoromethyl substituent and provided a rational understanding of the nmr observations. in contrast to formation of the native state, aggregation of misfolded protein molecules into so-called amyloid fibrils is a strongly irreversible, kinetically-controlled process which may lead to structural variants of fibrils composed of chemically-identical protein chains. this poorly understood aspect of polymorphism of amyloid fibrils has important implications in vivo in the course of amyloidassociated neurological disorders -e.g. in the form of prion strains in creutzfeldt-jakob disease. in this paper, we examine cross-seeding behavior of a model amyloidogenic polypeptide -bovine insulin (bov-ins) and recombinant lys b -arg b insulin (kr-ins). fibrils of bov-ins and kr-ins exhibit characteristic "fingerprint" features in the amide i' infrared region. these spectral traits are transferred to daughter generations of fibrils upon cross-seeding of native bov-ins with kr-ins fibrils and vice versa. this study highlights an interesting aspect of a non-anfinsenian behavior of aggregating insulin: the memory effect. our results indicate that fine infrared features in the amide i region of fibrils from closely related proteins may not reflect different amino acid composition but rather the fact that a particular amino acid sequence favors an amyloidogenic pathway that determines a certain and spectrally-distinct stacking mode of β-strands. an encounter with an alien seed on the amyloidgenic pathway would hijack the yetnative monomers and force them to follow the assembly pattern (and resulting ir characteristics) of the preformed fibril. phosphorylation and oglcnacylation are dynamic intracellular protein post-translational modifications that often are alternatively observed on the same protein serine and threonine residues. phosphorylation and oglcnacylation commonly occur on residues in natively disordered regions of proteins, and often have opposing functional effects. in the microtubule-associated protein tau, hyperphosphorylation is associated with protein misfolding and aggregation as the neurofibrillary tangles of alzheimer's disease, whereas oglcnacylation is stabilizing. a series of peptides derived from the prolinerich domain (residues - ) of tau was synthesized, with the free ser/thr hydroxyls, with phosphorylated ser/thr, with oglcnacylated ser/thr, and with the dietylphosphorylated ser/thr. phosphorylation and oglcnacylation were found by cd and nmr to have opposing structural effects, with phosphorylation favoring polyproline helix (ppii) formation, with oglcnacylation opposing ppii, and with the free hydroxyls intermediate. phosphorylation of serine and threonine residues in prolinerich sequences was found to induce ppii in other proline-rich sequences, with significant conformational restriction at phosphoserine and phosphothreonine residues observed by nmr. notably, serine and threonine residues are common in proline-rich domains of proteins, and ppii is a common conformation observed for phosphorylated ligands in protein-protein interactions. the ser/thr diethylphosphate was found to be a practical mimic of oglcnacylation, as an easily synthesized neutral amino acid with similar sterics to oglcnac and strongly opposing ppii. dept. of biology, university of padova, padova , italy we recently established that designed hairpin peptides interfere with the amyloidogenesis of both human pancreatic amylin and α-synuclein ( α-syn), two 'unstructured' polypeptides associated with human diseases. in the case of α-syn, the most potent 'amyloid inhibitors' act by diverting α-syn to non-amyloid aggregates. the binding sites for these agents and nonhairpin peptides of similar sequence have been determined by d-nmr-monitored titration studies using universally n-labeled α-syn. in the case of the hairpins that produce non-amyloid aggregates from α-syn, initial binding occurs in the non-amyloidogenic c-terminal segment of α-syn and the hairpin peptide is incorporated in the resulting aggregates. nmr studies also serve to define binding interactions and conformational changes that may be essential early steps in aggregation and pre-amyloid state processes. mt-ii is a long acting, non-selective super-agonist of melanocortin gpcr receptors, characterized by lactam bridge between residues i and i+ stabilizing a type-ii β turn structure. the recently introduced cui-catalyzed azide-alkyne , -dipolar cycloaddition, presents a promising opportunity to develop a new paradigm for an orthogonal bio-organic and intramolecular side chain-toside chain cyclization. we introduced this [ , , ]triazolyl-mediated cyclization in a pthrp model peptide, and demonstrated that i-to-i+ side chain-to-side chain cyclization offers a powerful approach for generating stable helix mimetic structures. , , in the current study we explored the potential of the i-to-i+ side chain-to-side chain , -disubstituted [ , , ] triazolyl-bridge to stabilize -turn conformations. we designed a series of [ , , ]-triazolyl containing cyclo-heptapeptides, derived from the model peptide lactam mt-ii, differing in the size of the bridge, the location and orientation of the [ , , ]-triazolyl moiety. conformational and biological studies, performed on this series demonstrate that [ , , ] triazolyl-mediated side chain-to-side chain cyclization results in the conformational stabilization of type-i β turn generating heterodetic cyclopeptides with enhanced in vitro potency and improved receptor subtype selectivity. this study confirms the versatile nature of the [ , , ] triazolyl-mediated side chainto-side chain cyclization. moreover the chemical accessibility, functional compatibility, and metabolic stability suggest that the [ , , ] international associated laboratory samuel de champlain, regional platform for cell imaging of haute-normandie (primacen), institute for medical research and innovation (irib), university of rouen, mont-saint-aignan, france scorpion toxins are polypeptidic molecules capable of selectivity binding to ion channels with the highest affinities and represent natural peptide blockers that are good tools to characterize new or particular types of ion channels. astroglial cells appear to express all ion channels species even voltage-gated ion channels previously suspected to be present only in excitable cells; but these channels are poorly studied and their function are not still completely elucidated.thus, the purpose of the present study was to investigate for the first time, the global effect and the potential protective effect of one scorpion toxin acting on kv channels, the pi ,on h o -induced oxidative stress and cell death in rat astrocytes. pi is a -residue toxin crosslinked by disulfide bridges, isolated from the venom of pandinus imperator and is one of the most potent and selective inhibitor of shaker b and kv . channels . in our study, pi was obtained by solid phase peptide chemical synthesis(spps) and named spi . the identity and homogeneity of spi were also verified by hplc, mass spectrometry analysis(maldi-tof) and an enzyme-based cleavage to determine the assignment of spi half-cystine pairings.the synthetic spi was found to be indistinguishable from the natural toxin. incubation of cultured astrocytes with graded concentrations of h o for h provoked a dose-dependent reduction of the number of living cells as evaluated by flow cytometric analysis and lactate dehydrogenase assay. interestingly, pre-treatment of astrocytes with very low concentrations of spi ( - to - m),dose-dependently prevented cell death induced by h o , and the effect of spi was accompanied by a marked attenuation of ros accumulation. also, spi stimulated sod and catalase antioxidant activities in a concentrationdependent manner,and blocked h o -evoked inhibition of sod and catalase activities. in addition, at low concentration ( - to - m) the toxin exhibits no toxic effect on astrocytes.taken together these data indicate that the spi acts as a protective agent on astrocytes from oxidative assault and then might have therapeutic value in the potential treatment of a number of neurodegenerative diseases. [ ] . in order to select small active peptides, the library was kept at the minimum size and was built using only selected d-amino acids to avoid structural redundancies. to further increase the chemical diversity [ ] a set of different carboxylic acids were coupled to the n-terminus. the iterative screening led to the identification of an n-terminally modified tetrapeptide with a very high radical-scavenging activity. by analyzing several peptide variants, we found that antioxidant properties were strongly sequence-and structure dependent, because scrambled or proline-scan variantswhose secondary structures are deeply altered -are much less effective. data on peptide structure-activity relationship will be presented. an investigation of the in vitro effects produced in cellular models of oxidative stress are also in progress. structural analysis of peptide-analogues of zp proteins with amyloidogenic properties: insights into mammalian zona pellucida formation. n.n. louros, v.a. iconomidou, s.j. hamodrakas* department of cell biology and biophysics, faculty of biology, university of athens, athens , greece a filamentous structure surrounding mammalian oocytes, called zona pellucida, is responsible for species-specific sperm binding. this extracellular porous structure is composed of - glycoproteins, termed zp - , which assemble into filamentous polymers that are cross-linked by disulfide bonds. all zp proteins present a unit with high sequence homology at their c-terminal end, designated as the zp domain. this structural module is found in a family of extracellular proteins with various functions and from a wide variety of organisms (zp domain proteins). it is a binary structure of approximately amino acids, divided into two domains, the n-terminal region named zp-n and the c-terminal region known as the zp-c domain. the zp-c block is associated with protein function, whereas the zp-n domain is responsible for protein polymerization. structural analysis revealed a possible polymerization model based on backbone interactions between specific parts of the zp-n units. peptide-analogues of the aforementioned segments were synthesized, since the ''amylpred'' application, an 'amyloidogenic determinant' prediction algorithm developed in our lab, predicted them as being potentially 'aggregation prone'. our experimental data clearly indicate that these peptides do actually fold and self-assemble into amyloid-like fibrils. therefore, all the derived data suggest that mammalian zona pellucida is a novel functional amyloid, similar to its biological equivalent, silkmoth chorion that has been established to be a natural, protective amyloid. we thank the university of athens for the financial support. biostrucutres and bioimmaging institute of national research council, naples, italy the definition of the molecular basis of human diseases is one of the most important goals of structural biology. nucleophosmin (nmp ) is an ubiquitously expressed protein is a nucleolar chaperon and has a key role in several biological processes from ribosome biogenesis, to cell-cycle regulation and is a frequently target of oncogenic mutations in acute myeloid leukaemia (aml) [ ] . these mutations occur predominantly at the c-terminal domain of npm compromising its stability and causing the aberrant translocation of npm to the cytosol. this domain is structurally characterized by the presence of three alpha helices, and the folding of cter-npm proceeds via an extended nucleus and the denatured state retains significant malleable structure at the interface between the second and third helices [ ] . here, we have further investigated the structural determinants in the folding process of the c-term npm domain and on the basis of available structural and composition data, several peptides covering the three helices were designed and synthesized. their conformational properties were investigated by cd and nmr spectroscopy: these studies demonstrated that once removed from the protein architecture helix and helix substantially miss fully ordered conformations while helix retain a high helical content also in mutated variants. our observations may help structure-based drug-design inserm u university of evry-val d'essonne, bâtiment maupertuis, evry, france sl (stop-like protein with mass of kda) is a neuronal protein with a calmodulin-binding and microtubulestabilizing activity. microtubule binding is inhibited by calmodulin , . the lack of the stops gives rise to defects in synaptic plasticity, as observed in schizophrenia . to study the sl protein's structure and its interaction with calmodulin, we have used a residue peptide derived from sl , containing the microtubule binding motif mn. the structure of the peptide has been analyzed using circular dichroism (cd) spectropolarimetry. the cd spectra showed that the peptide's secondary structure consists in a predominantly β-sheets, while the tertiary structure consists in rather folded d-structure. the temperature dependence of cd indicated that the secondary structure of the peptide remained very stable between °c and °c, while the tertiary structure changed at about °c. the interactions between the sl peptide and calmodulin have been studied using the isothermal titration calorimetry (itc). the results showed that calmodulin binds the peptide with the affinity of m- , in a ca + -independent manner, the reaction being driven by entropic contributions. our results might contribute to understanding the microtubule stabilizing activity of the sl protein. peptides , . based on the structure in polar environment a transition from -helical to an α-helical conformation at the water/lipid interface in bilayer membranes is hypothesized the efficient method development for the analysis of proteins, peptides, and synthetic oligonucleotides with a novel hybrid reversed phase column f. becker * , n. shoji * , a. matsui * , c. yokoyama * , t. sato * , t. takai * , and n. kuriyama * * ymc europe gmbh, * ymc co., ltd. the recent development of bio-pharmaceutical industry has been remarkable and an effective analytical method with higher sensitivity, superior selectivity, and increased speed has been required in the characterization of peptides, proteins and oligonucleotides by highperformance liquid chromatography (hplc). the method development of reversed phase hplc requires optimization of several conditions, such as bonded-phase, column efficiency, solvent type, ph and temperature. especially ph and buffer type is a most important parameter to control retention of biomolecules which have multiple ionic functional groups. furthermore, temperature often becomes a key tool to achieve better peak shapes and resolution for larger molecular-weight compounds. although silica based reversed phase columns have been widely used for separation of biomolecules, they have low stability under alkaline conditions and a limited usable ph range. to improve the chemical stability at an expanded ph and temperature range , we have developed a new type of organic/inorganic hybrid reversed phase column, ymc-triart c and ymc-triart c . the novel technologies of manufacturing particles and surface modification provide outstanding chemical stability and excellent peak shape for many types of compounds under a variety of mobile phase conditions. in this poster, we will show characteristics of this new hybrid column, and some example cases of efficient method development in separation of the biomolecules such as peptides, proteins and synthetic oligonucleotides. the fully-extended peptide conformation ( . -helix) is the longest possible for a single α-amino acid as it is characterized by an axial translation per residue of about . Å. therefore, it is extremely attractive for its application as a molecular bridge. however, it is known that the stability of this type of helical structure (relative to the competing, much shorter -helix) appears to be dramatically governed by the choice of the c-terminal functionality (only esters seem to be appropriate). in this work, we examined the series pyrac-(deg)n-o-(pno )bzl [where pyrac is the fluorophore -pyrenylacetyl, deg is cα,α-diethylglycine, n= - , and o-(pno )bzl is para-nitrobenzoxy] with the deg homo-peptides as fully-extended bridges in a static fluorescence study. to perform a very stringent comparison with our ft-ir absorption and nmr results, the solvent used was chcl . the trend observed in the quenching efficiencies for the shortest members of the series, the (deg) - peptides, steadily decreases from . (n= ) to . (n= ), and . (n= ). this finding is compatible with the conclusion that these three peptides are all essentially fully-extended in chcl solution. however, the quenching efficiencies for the longest deg homo-peptides. . for n= and . for n= , are not further reduced. the inevitable conclusion is that the tetra-and pentapeptides populate at least two different conformations (most probably, the . -helix and the helix), in which the probe dyads are located at different distances. notably, these results fit well with those extracted from our ft-ir absorption and nmr studies in the same solvent of low polarity. we conclude that a combination of terminally aromatic groups, as in the deg homo-peptide series investigated in this work, is not eligible for the fabrication of a stable fully-extended conformation. the surfactant protein c (sp-c) is a -residue highly hydrophobic peptide with two covalently linked fatty acyl chains that adopts a mainly helix structure while associated with the membrane. however, it misfolds into a β-rich structure under certain environmental conditions, which suggests that sp-c is in a metastable state . the slow α→β transitions are likely to be caused by the membranedissociation, deacylation and exposure to the neutral ph of the alveolar subphase, leading to the formation of amyloid aggregates associated with pulmonary alveolar proteinosis. we have been studying the ph-dependent conformational behaviour of both the acylated and deacylated isoforms of sp-c in a membrane mimetics chloroform/methanol/water mixture using a constant-ph molecular dynamics method [ ] [ ] [ ] . our simulations identify the increase of ph as a promoter of peptide-peptide interactions, which may contribute to the α→β transitions observed at neutral and alkaline ph conditions in the experimental results . we also observe that the loss of structure is more prone to begin at the c-terminal region and that the β motifs start to form between the n and cterminal disordered regions of the sp-c. in addition, the presence of the acyl groups results in a slightly higher structural stabilization of the sp-c helix and in a decrease of the n-terminal plasticity at acidic and neutral ph. since a comprehensive conformational analysis and the elucidation of the mode of association between sp-c and the membrane are essential to understand the interactions involved in structure stabilization and in some specific functions of the peptide, we are now studying both sp-c isoforms in a membrane environment. these studies will not only be the most relevant for the in vivo situation but will also answer to some questions left open by the previous work. were rather small. vp plays a very important role in controlling resorption of water by the distal tubules of the kidney and in regulating the osmotic pressure of blood in mammals. vt, while exhibiting both oxytocin and vasopressin activities, has not changed over the last million years until appearance of the mammals. currently, it occurs naturally in the non-mammal vertebrates [ ] . here we report on studies of arginine-vasopressin (cyfqncprg-nh , avp) and arginine-vasotocin ([ile ]avp, avt) in a mixed dodecylphosphocholine (dpc) and sodium dodecylsulfate (sds) micelles. dpc micelles are considered good models of eukaryotic cell membrane [ ] , while the sds micelle improves peptide/micelle solubility [ ] . for these studies we use d nuclear magnetic resonance (nmr) supported with molecular modelling methods. conformation-activity considerations relationships and the role of the peptide-micelle interactions on the structure of the analyzed peptides will be described and discussed. vibrational and chiroptical spectroscopic investigation on diamide peptide models k. knapp, a m. hollósi, a e. vass, a zs. majer a a eötvös lor nd university, institute of chemistry, laboratory for chiroptical structure analysis, budapest, hungary understanding of peptide folding has great importance in many fields of chemistry. since amino acid residues are the simplest building blocks of peptides, the investigation of their conformational properties could help us in understanding the structures of peptides or in designing peptides of specific d structure. describing the conformational building blocks of even the simplest peptide models (e.g., amino acid diamides) can serve as a good starting point to decipher higher-level structural properties of their polymers. among the simplest compounds which include the essential structural elements of polypeptide backbone are the n-methylamides of n'acetylamino acids. these compounds have been the subject of extensive spectroscopic analyses (ftir, vcd, ecd, nmr) [ , ] . the conformational landscape of these model compounds were analyzed by solution phase vcd and ecd spectroscopy and density functional theory (dft) computations. this work reports systematic vcd and ecd spectroscopic studies in different solvents on synthesized diamide models (ac-α/βhomo-xxx-nhch , xxx = ala, pro, phe). vcd and ecd data, combined with quantum chemical calculations performed at the b lyp/ - ++g** and - ++g** level of theory was particularly useful for identifying the preferred dipartimento delle scienze biologiche, università di napoli "federico ii", napoli, italy c dipartimento di scienze ambientali, seconda università di napoli, caserta, italy β-hairpin is an important secondary structural element used by many proteins for biomolecular recognition, and thus is an attractive tool for mimetic design . aβ -hairpin motif consists of two antiparallel strands linked by a turn region. in this work we aimed to stabilize a β-hairpin conformation by an interstrand covalent bridge, made through a click chemistry reaction, which yields , -disubstituted , , , triazole, starting from alkyne and azide reactive groups. in particular, we explored the conformational stability of a series of β-hairpin peptides containing a , -disubstituted , , triazole bridge with variable length. we employed the well structurally characterized trpzip peptide as a convenient b-hairpin model system. as alkyne amino acids were introduced propargylglycine (pra), homopropargylglycine (hpg) or bishomopropargylglycine (bpg), while the following azide amino acids were inserted: lβ-azidoalanine (dap (n )), l-γ-azidohomoalanine (dab (n )), and l-δ-azidoornitine (orn (n )). all peptides were synthesized and purified. successively, the linear unprotected peptides were cyclized in solution by the cu(i)-catalyzed azide alkyne cycloaddition (cuaac) reaction and purified prior to the spectroscopic characterization. linear and cyclic peptides were analyzed by a combination of cd and nmr techniques. have been investigated. their behaviour has been compared with that of proline in homochiral and heterochiral dipeptide sequences including l-or dphenylalanine. nmr and ir studies as well as x-ray diffraction analysis provide evidence that the additional β-phenyl substituent does not disrupt the tendency of proline to occupy the i+ position of a β-turn. moreover, the β-turn conformation is stabilised, with reference to l-pro, when l-cis(βph)pro or l-trans(βph)pro are combined with d-phe, that is, in the heterochiral sequences. the pyrrolidine conformation is significantly affected by the presence of the β-phenyl group. the puckering modes that alleviate most the steric hindrance introduced by the bulky phenyl substituent are preferred. the l-cis(βph)pro residue shows a marked propensity for the cγ-endo/cβexo half-chair arrangement whereas the trans derivative exhibits a higher flexibility, with different pyrrolidine shapes being accessible. interactions between the aromatic ring of (βph)pro and that of the contiguous l-or d-phe residue are observed in all the dipeptides crystallised. in solution, they seem to occur only for the heterochiral sequences. this intramolecular aromatic-aromatic interaction may be responsible for the higher β-turn ratio observed for such sequences and also seems to affect the conformational preferences of the pyrrolidine ring. in fmoc chemistry the trt and acm groups are widely accepted as a protecting group for cys. upon incorporation of these cys derivatives onto the growing peptide chain, however, considerable base-catalyzed racemization of cys is known to always occur at the activating and coupling steps with phosphonium or uronium reagents such as pybop or hbtu, respectively. in addition to this, racemization of the c-terminal cys esterified to resins also occurs during the repetitive n α -fmoc deprotection using piperidine. in order to find an s-protected cys derivative applicable for fmoc chemistry that can efficiently suppress racemization of cys both when its incorporation is conducted by phosphonium or uronium reagents and when it is linked to a resin via ester linkage, we introduced the methoxybenzyloxymethyl (mbom) group into the thiol function on cys. the mbom group was found to substantially supress racemization of cys ( . %) during its incorporation mediated by phosphonium or uronium reagents in comparison with the trt and acm groups ( . % and . %, respectively). even after a -h treatment of the peptide resin, in which its c-terminal cys was linked to a trt-type resin, with % piperidine in dmf, the mbom group caused a significant reduction in the level of racemization with the c-terminal cys ( . %) while trt and acm groups led to a considerable extent ( . % and . %, respectively). next, we demonstrated the usefulness of the mbom group on cys by applying it to ncl chemistry involving the coupling of a peptide thioester and a cys-peptide. in the synthesis of the latter peptide, however, a diastereoisomer with racemization of the nterminal cys tends to be difficult to separate from the intact peptide as its chain length increases. thus, this measure using fmoc-cys(mbom) can facilitate the avoidance of ambiguities in the quality of the synthesized peptides. labeled peptides are important tools in chemical biology to obtain information on their biological function in, for example, cellular communication processes as well as to study the molecular basis of diseases. the functionalization of bioactive peptides with biophysical reporter groups like strongly absorbing chromophores, is most often performed via post-synthetic chemoselective biocojugation reactions, involving amino, thiol, or azide specific reactions. an alternative approach for the installation intense chromophoric organic molecules, is the coupling of nonchromophoric precursor molecules to obtain chromophoric properties in the final product. recently, we have shown that the [ + ] cycloaddition-cycloreversion reaction between peptide-functionalized electron-rich alkynes and electrondeficient cyanovinyl derivatives results in the formation of a new class of π-conjugated peptidic donor-acceptor chromophores. , the chromophoric properties of the final product can be tuned by varying the p-electron conjugation pattern. herein, we describe the synthesis of the versatile building blocks, n-a-( -ethynylphenyl)-n-a-(methyl)-glycine and n-a-( -( , , -tricyanovinyl)benzoyl)-glycine, their subsequent application in spps to functionalize peptides with d-p-a chromophore precursor molecules, and their uvvis absorption characteristics. as a typical example, alkyne-functionalized aβ peptides, val-ala-ile and lys-leu-val-phe-phe-ala-glu, have been reacted with a series of cyanoolefins to obtain far-red intense imaging chromophores for amyloid-rich peptide aggregates. a novel method for regioselective disulfide formation in mini-proteins by kinetic oxidation control t. lindner, u. haberkorn, w. mier department of nuclear medicine, university hospital heidelberg, im neuenheimer feld , heidelberg, germany rigid frameworks are essential for the development of peptide-based alternatives to immunoglobulins. [ ] disulfideconstriction covalently stabilizes tertiary structures, which allows further miniaturization of protein-derived drugs by chemical synthesis. while the amino acid assembly steps in solid phase peptide synthesis are highly efficient, regioselective formation of multiple disulfide bonds is still a challenging and time demanding task. even after applying laboriously optimized folding conditions and extensive purification, a successful synthesis of the correctly folded isomer is not guaranteed. [ ] to circumvent these problems, a procedure that takes advantage of the different reactivity of free vs. acmprotected thiols during iodine oxidation was developed. this protocol to sequentially form two disulfide bridges in a one-pot reaction was applied to different peptides, such as (a) min , a mini-protein scaffold for phage-display assisted drug design; [ ] (b) α-conotoxin imi, a neurotoxic peptide isolated from the venom of marine cone snails; [ ] and (c) endothelin- , a peptide involved in blood pressure regulation. [ ] the presented technique tolerates precarious residues within the to-be-folded-peptide, such as fluorescent dyes or metal chelators as used for bioactivityassays. compared to the individually optimized literature procedures, this novel approach offers significant improvements: overall yields > % are obtained in first attempts and stepwise formation of the disulfide bridges is performed within a few hours instead of days. in recent thirty years, c-terminal modified peptides have been proved to have greater potential as apis (active pharmaceutical ingredients) due to their increased chemical and enzymatic stability and improved pharmacodynamic properties - . a prominent example, octreotide - , an octapeptidoalcohol, has witnessed as a potent anti-cancer agent targeted for gastro-entero carcinomas. in view of synthetic methodology, peptidoalcohol can not be directly prepared by standard spps protocol becouse of the c-terminal structure released from resin are not alcohol but always peptidoacid or peptidoamide. to overcome this problem, a novel protocol of shortened n- coupling cycles on merrifield resin and then the ammonolysis of peptedyl resin by an aminoalcohol as the c-terminal residue getting peptido-alcohol as targetting product has been devoloped in our lab. because of the cleavage treatment of peptidyl merrifield resin is not under acidic condition, such as hf or tfmsa, but ammonolysis, some side-chain producting groups(spg) related to boc chemistry like bzl, clz, tos…; must be avoided in sequence assembly. therefore a hybrid orthogonal protection (hop) of boc/fmoc protocol was adopted for the sake of producing naked peptidyl (without any spg) resin before ammonolysis. fifteen peptidoalcohols with different terminal alcohols were conveniently prepared, most of them released form resin with very good yields. due to its cyclic structure, proline is the coded amino acid with a more restricted conformational flexibility. the incorporation of additional groups into the pyrrolidine ring is a useful means to produce new amino acids that combine the conformational properties of proline with sidechain functionality. this is the case of β-phenylproline, (βph)pro, that can be regarded as a proline-phenylalanine hybrid in which the orientation of the aromatic substituent is dictated by the conformation of the five-membered ring and the cis or trans configuration of the phenyl group relative to the carbonyl moiety. accordingly, cis(βph)pro and trans(βph)pro combine the conformational properties of proline with an aromatic side-chain functionality that is rigidly oriented with respect to the peptide backbone, and this may be useful in the design of biologically active peptides and other applications relying on specificallyoriented side-chain moieties. we have developed synthetic procedures for the preparation of the cis(βph)pro and trans(βph)pro stereoisomers in enantiomerically pure form. the methodology is based on the preparation of racemic precursors of each amino acid and their subsequent hplc resolution on chiral columns. multigram quantities of the target amino acids have been isolated in optically pure form and suitably protected for use in peptide synthesis. the importance of peptide cyclization for studying peptide conformation, creating new structures, or for developing peptide therapeutics is well established. in particular, sidechain lactam bridges linking two amino acid residues that are several residues apart in the linear sequence or headto-tail backbone peptide cyclization enable rigidification of the structure and improvement of in vivo stability. native chemical ligation (ncl) is now an established method for producing backbone-cyclized peptides or proteins. the application of ncl to the synthesis of sidechain cyclized peptides is less frequent. head-to-side-chain cyclization by ligating a c-terminal thioester with a cys residue located on a lysine side-chain was used by few authors. the alternative tail-to-side-chain cyclization mode is rare, probably due to the difficulty of installing a thioester group on amino acid side-chains such as aspartic or glutamic acids the reaction of a bis( -sulfanylethyl)amido (sea on ) group with an n-terminal cysteine residue in water and at neutral ph results in the formation of a native peptide bond. [ ] oxidation of sea on results in a cyclic disulfide called sea off having a , , -dithiazepan- -carbonyl structure. [ ] sea off is a self-protected form of sea on . we show here that bis( -sulfanylethyl)amido side-chain acid(dab), ornithine and lysine were selected as building block; a and n,n'-cbz- -amidinopyrazole ( b) were selected as guanidinylating reagents for specific situation. for synthesis of n-terminus local cyclo-guanidine peptide, designated peptides were assembled on acid labile solid support such as rink amide resin by fmoc strategy. then either fmoc-dab(boc)-oh, fmoc-orn(boc)-oh or fmoc-lys(boc)-oh was incorporated respectively at n-terminus. fmoc was removed followed by guanidinylating by b and then peptide was cleaved by acid. by neutralizing with nmm in acetonitrile solution, side chain amino group and a-guanidine would form , or membered local cycloguanidine. the remaining cbz could be removed by hydrogenation. for synthesis of backbone side chain cyclic peptide, bis-fmoc-daa was introduced in the peptide previously on resin followed by removal of fmoc. selective guanidinylate side chain aminogroup by a followed by peptide assembling with an insertion of orthogonal protected daa at - aa apart from first daa. for synthesis of a-nh sidechain cyclic peptide, first daa should be introduced with orthogonal protected form. b was used to guanidinylate a-nh . after cleavage and neutralization of those two kinds of intermediates, guanidine-bridged marco-cyclic peptide was formed. the resin is also a multipurpose tool for the synthesis of carboxylic acids, esters and thioesters. when the synthesis is completed, the fully protected peptide hydrazide resin is oxidized with either n-bromosuccinimide (nbs) or copper(ii) acetate in pyridine. the resulting acyl diazene resin is then cleaved by peptide displacement at the c-terminus with amine. the fully deprotected peptide amide is finally obtained by treatment with trifluoroacetic acid (tfa). in our approach, we used a -fmoc-hydrazinobenzoyl am novagel resin to synthesize a peptide-substituted amide in the c-terminus. first, the oxidative cleavage was carried out with nbs in pyridine and a nucleophile [a protected (aminomethyl) benzimidamide (amba)]. however, the yield of the reaction was very poor. in the next step, we applied copper(ii) acetate in the presence of pyridine and amba. following optimization, the efficiency of the process was significantly improved. herein we discuss the conditions needed to obtain a reasonably high efficiency of the oxidative cleavage in the synthesis of our c-terminal modified peptides using the aryl hydrazine resin linker. blood vessels on tumor tissues, similarly to integrin receptors. this observation suggests cd as a selective target for targeted delivery of drugs and nanoparticles to tumor neovasculature using ngr peptides as homing motif. in our work, new cyclic-ngr peptides containing a thioether linkage were prepared. the influence of their structure on the speed of succinimide ring formation and deamidation was evaluated and compared with the previously published data on cyclic-ngr derivatives containing amide bond or disulfide bridge in the cycle (c[kngre]-nh and c[cngrc]-nh ). to avoid the deamidation under the conditions used for cyclization, the synthetic routes were optimized. the influence of the ph, ionic strength and temperature of the solution on their chemical stability was investigated. the structure of the cyclic peptides was investigated by circular dichroismand nmr-spectroscopy. receptor binding ability and the influence of the cyclic peptides on the cell adhesion and motility were also evaluated. this work was supported by grants from the hungarian national science fund (otka nk and k ) and the national innovation office (bio_surf, om- / ). clickable peptides and their attachment to oligonucleotides m. wenska, m. alvira, r. strömberg department of biosciences and nutrition, karolinska institutet, novum, se- huddinge methodology for the ready conversion of peptides into "clickable" azido-peptides with the possibility of selecting either n-terminus or c-terminus connection is presented. synthesis of peptide-oligonucleotide conjugates (poc's) include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals. a general procedure, based on a new activated alkyne linker, for the preparation of poc's has been developed. with this linker, conjugation is effective at room temperature in mm concentration and submicromolar amounts. this is made possible since the use of a readily attachable activated triple bond linker speeds up the cu(i) catalyzed , -dipolar cycloaddition ("click" reaction). the main scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of i) an h-phosphonate based aminolinker ii) the triple bond donor p-(npropynoylamino)toluic acid (pata) and iii) azido-functionalized peptides. the method gives excellent conversion of oligonucleotide to the poc on solid support, and only involves a single purification step after complete assembly. the procedure which makes use of a low concentration of copper ions leads to a product with very little copper left (similar or less than in drinking water). the synthesis is flexible and can be carried out in non-specialist laboratories without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. comparison of alternative deprotection reagents to piperidine for the synthesis of a poly-alanine peptide on the tribute® peptide synthesizer m.a. onaiyekan,* j.p. cain, c.a. chantell, m. menakuru protein technologies, inc. tucson, az, usa in peptide synthesis, piperidine is a common agent for fmoc removal. however, piperidine is a controlled substance which requires special handling and cannot be used in some countries. therefore, it would be useful to identify alternative deprotection reagents to piperidine for fmoc removal. it is well known that poly-alanine sequences have a high propensity to aggregate after the fifth residue. in this application, (a) k-oh was synthesized using the tribute®'s intellisynth uvmonitoring and feedback system to compare the efficiency of fmoc removal by piperidine vs. three alternative bases (pyrrolidine, cyclohexylamine, and tertbutylamine) in the last cycles of the synthesis. it was found that pyrrolidine produced a higher purity product with fewer deprotection repeats and shorter deprotection times per cycle than piperidine, proving it to be a highly efficient, viable alternative to piperidine for fmoc removal. the endogenous tripeptide gpe also nammed "glypromate" is made up by the three n-terminal residues (glycine-proline-glutamate) of the insulin-like growth factor (igf ). this tripeptide is a partial glutamate antagonist and showed good results in different neuroprotective in vitro and in vivo experiments. , gpe also binds to glial cells regulating neurotransmitter levels in the brain. , however, gpe suffers from poor lipophilicity and a short half-life in vivo. that's why there is a need for more lipophilic and protease resistant analogues of gpe. in this poster we present the synthesis of trifluoromethylated analogues of gpe based on the or -cf -pseudoproline residues. introduction of fluorine atoms on bioactive compounds is known to deeply modify their physico and biochemical properties increasing lipophilicity and resistance to protease. thus, developing a trifluoromethylated analogues, we intend to increase the bioavailability of gpe, keeping the benefit of its neuroprotective properties. our research team is strongly involved in the synthesis of trifluoromethylated alpha-amino acids. recently we published the synthesis of -trifluoromethyl- , oxazolidines derived from fluoral and (l)-serine and we demonstrated that these five membered ring -cf pseudoprolines are hydrolytically stable and can be considered as proline analogues. that's the reasons why we are interested to replace the proline residue of gpe by those trifluoromethylated compounds. the development of original coupling conditions and the detailed synthesis of two pseudoprolines analogues of gpe will be presented in this poster. modifications. in combination with automated spps, unprecedented access to large peptides and small proteins for biological research has been achieved. we demonstrate the application of this methodology to the synthesis of a variety of peptides on the prelude® peptide synthesizer. exploring the space of fluorine-labeled α-amino acids for solid state f-nmr structure analysis of peptides: rational design, synthesis and applications p. solid state f-nmr is a powerful method to study membrane-active peptides, as it can reveal their conformation, orientation and dynamics when embedded in biomembranes. for this purpose the native peptide has to be selectively labeled with a suitable f-containing amino acid at several different positions. the resulting battery of singly f-labeled analogues is then analyzed by solid state f-nmr. the main limitation to this approach currently lies in the poor arsenal of available f-labels. we have therefore rationally designed and synthesized several specific amino acids bearing a cf -reporter group, which fulfil all strict criteria to a "proper" f-label. , to allow a geometry-based structure calculation, the cf group has to be rigidly attached to the peptide backbone. we thus rigidified the side chain using either a [ . . ]bicyclopentane moiety, a cyclobutane ring, or the intrinsic proline framework. this way, suitable cf -labeled analogues were created as substitutes for bulky hydrophobic amino acids (leu/ile/val/met), for aromatic residues (phe), for polar side chains (ser/thr), and for proline (pro). by now we have applied the developed f-labels for a comprehensive structure analysis of more than ten different membrane-active peptides (gramicidin s, pgla, mag , kigaki, sap, temporin a, bp , etc). recently, several new activators have been introduced into the market, and they were evaluated along with some older activators for their ability to synthesize a range of peptides with shorter and longer reaction times on the symphony® peptide synthesizer. it was found that hdmc, pyclock, comu, hctu, and hatu worked well at shorter reaction times ( x min), but pyoxim and tffh only worked well at longer reaction times. the performance of pybop at shorter reaction times was poor only for more difficult sequences. these results are important for selecting an appropriate activator for fast spps applications. the plant cyclotides form the largest family of cyclic peptides . they contain a signature motif referred to as the cyclic cystine knot, which is derived from the cyclic backbone and three inter-knotted disulfide bonds. intriguingly, cyclotides can be boiled, treated with chemicals or enzymes without disrupting their overall fold. thus, they are sometimes labeled as ultra-stable proteins. in addition, cyclotides are tolerant to mutations, and as a scaffold they can successfully accommodate foreign bioactive epitopes of variable sequences . cyclotides share many of these properties with another disulfide containing cyclic plant peptide, the sunflower trypsin inhibitor (sfti- ) . emerging evidence indicates that cyclotides and sfti- are valuable not only as peptide stabilizing scaffolds; in combination with their cell penetrating properties, these disulfide rich cyclic peptides have significance as intracellular drug carriers. although both peptides are genetically encoded, studies to ascertain the exact mechanisms of their biosynthesis are currently on going. thus, the synthesis of cyclotides and sfti- are currently restricted to chemical means. we have recently adapted a fmoc-spps method for cyclic peptide synthesis, via n-acylurea intermediates with the assistance of microwave irradiation. this method is a safe and convenient alternative to boc-spps and has the ability to be automated conveniently. using this method, parent scaffolds as well as several cyclotide and sfti- analogues with potential antimicrobial and matrix metalloprotease activities were synthesized. with the rising interest in the cyclization concept as a tool to impart stability on unstable peptides, the cyclic peptide synthesis method adapted herein is anticipated to have numerous applications. fixed configuration. the nonnatural oligomers have an extended conformational space and are supposed to adopt non-canonical secondary structures . in addition, the backbone modification makes these molecules more stable towards proteolytic degradation. the majority of proteins in nature are post-translationally modified, and the most abundant modification is the protein glycolysation, which introduces wide structural variety to proteins. glycoproteins have an important role in the biological recognition process, such as immunodifferentiation, cell adhesion, cell differenciation and regulation cell growth . new aza-β -amino acids bearing either an azide instead of amine on lys and orn chain or an alkyne group will be described and used in solid phase synthesis to finally performed a click chemistry to cyclize pseudopeptides or to introduced a glycosylated function . true for a series of peptides that display strong corticotropin releasing factor (crf) antagonistic activity. seminal studies by rivier et al. have shown that the incorporation of a lactam bridge in the crf-sequence resulted in an enormous increase in activity and potency, due to stabilization of the bioactive a-helical conformation of the peptide; and the newly designed peptide was called astressin. based on the astressin sequence, we started a truncation and deletion study to arrive at astressin analogs with a reduced size but still remain active as crf antagonists. this study resulted in the smallest active crf antagonist, astressin( - ). this sequence was further optimized by the introduction of novel covalent constraints, other than the well-known lactam bridge. as a first approach, the alkene/alkane bridge, which can be introduced via ring-closing metathesis via alkenesubstituted amino acid side chains, and as a second approach, the triazole bridge ('click' macrocyclization), via either a cu(i)-or a ru(ii)-catalyzed cycloaddition reaction between azide-and alkyne-derivatized amino acid residues were explored. herein, we will present the details of the synthesis of the alkene-, azide-, and alkyne-functionalized amino acids, their use in spps, and the optimized approaches for macrocyclization. furthermore, the peptides have been characterized by hplc, nmr, lcms, and studied by circular dichroism spectroscopy to obtain insight into the helical propensity of the peptides in relation to the cyclic constraint. the synthesis of a nitronyl nitroxide, c α -tetrasubstituted αamino acid (a class of sterically restricted amino acids that promote the formation of peptide β-turns and helical structures) was achieved by derivatisation of racemic amino- -cyano-indan- carboxylic acid [aic(cn)]. racemic boc-aic(nn)-oh was prepared by bis(alkylation) of ethyl isocyanoacetate under phase transfer conditions with , -(bis)bromomethyl benzonitrile as alkylating agent, followed by acidic hydrolysis, n α -boc protection, and saponification of the ester function. resolution was achieved through formation of the diastereomeric amides of (s)-phenylglycinol with chromatographic separation and mild acidic hydrolysis. reduction of the nitrile group to an aldehyde was carried out with raney nickel in the presence of sodium hypophosphite. condensation with , -diamino- , -dimethylbutane gave the corresponding tetramethylimidazolidine, which was oxidised with chloroperbenzoic acid to the desired nitronyl nitroxide. the uv-vis absorption and epr spectra of the amino acid were recorded and its magnetic properties were examined. in order to develop the synthesis of this peptide using the fmoc solid-phase peptide synthetic methodology, orthogonally protected β-hydroxyaspartic acid was needed. more precisely we wish to dispose of ( r, r)-n -fmoc- -tbdm-silyloxy-aspartic acid α -allyl ester instead of the recently reported dmab ester - indeed, in preliminary assay using this protective group we experienced difficulties during the final cyclisation step . the synthesis was developed starting from inexpensive l(+) dimethyltartrate and extended to the others stereoisomers of the β-hydroxyaspartic acid. structure. for that, we chose to replace proline by silaproline to afford polysilaproline. this study shows the comparison of two polyamino acids: polyproline and polysilaproline polymers. homopolypeptides were synthesized by polymerization of corresponding amino acid n-carboxyanhydride . multicomponent reactions (mcrs) represent a chemical process involving at least three reactants for the formation of several covalent bonds in one operation . by definition mcrs are chemo-and regioselective, convergent stepefficient procedures and take place with high atom economy. the copper(i)-catalyzed , -dipolar cycloaddition of organic azides and terminal alkynes (cuaac) reported by meldal and sharpless has been involved in various fields of chemistry and biochemistry research. however only few reports describe the implementation of cuaac and mcrs. , recently our research focused on a novel threecomponent reaction based on a cu(ii)-triggered aminolysis of peptide hydrazide resin and an azide-alkyne cycloaddition sequence. copper(ii)-induced oxidative aminolysis of hydrazides generates cu(i), catalyst of the azide-alkyne cycloaddition. this feature was exploited to design a solid phase detaching three-component reaction. the mcr process requires a peptide hydrazide resin, an amino azide linker and an alkyne, resulting in the formation of peptide modified at the c-terminus through an amino , , -triazole linker. this method can potentially be applied to the synthesis of a large variety of peptide derivatives starting from fmoc-spps assembled peptidyl resins. furthermore, it is not practical to compare hplc spectra from different resin samples (e.g., before and after reaction) directly. a comparison by analyzing the same (mg) amount of resin would involve tedious sample preparation that is extremely error-prone and would be impractical because factors resulting from the increase or decrease of the molecular weight of the resin-bound compounds may have a significant influence on the results. the use of internal reference compounds allows rapid assessment of reactions performed on solid supports. the internal reference compound is bound to the resin together with the substrate and cleaved with the products after completion of the reaction. commercially available compounds can be used for this purpose, or likewise, the reference compound can be generated from the substrate by partial capping of a functionality. the peak integration of the reference compound in the hplc-uv spectra can be correlated directly to those of the rest of the compounds present in the reaction mixture and therefore a quantitative interpretation of the spectra with respect to conversion and yield is possible. here we demonstrate the proof of principle as well as the accuracy of this method. modifier proteins such as ubiquitin are conjugated to protein substrates in cells and thereby mediate various biological processes. of high interest, is the ubiquitin fold modifer (ufm , residues) which has structural similarity to ubiquitin but has no sequence similarity. unlike ubiquitin, ufm has not been extensively studied and little is known about its biological role. to understand ufm 's biological functions, access to pure, homogeneous natural and modified ufm protein is essential. chemoselective ligation techniques are suitable for providing such proteins. recently, a variation of the α-ketoacid-hydroxylamine (kaha) ligation was developed, which utilizes the chemoselective reaction between a c-terminal peptide αketoacid and a n-terminal -oxaproline. this modified form of the kaha ligation furnishes a native peptide bond and a homoserine residue. this ligation is useful for the synthesis of proteins from two unprotected protein segments in aqueous buffers. for the synthesis of larger proteins, a sequential ligation strategy is necessary. using ufm as the model system we have developed a sequential ligation procedure using kaha ligation with -oxaproline. applying the new sequential ligation strategy we have prepared ufm by total chemical synthesis. we have also prepared a cterminal thioester surrogate of ufm protein, which is suitable for conjugation to proteins of interest. the syntheses required the development of a bifunctional peptide segment bearing an α-ketoacid and an orthogonally protected -oxaproline. the preparation of the protein segments, their intermediates, the deprotection, and sequential kaha ligations towards the syntheses of ufm protein and c-terminal modified thioester ufm protein will be discussed. affinity and biological activity, we have designed and synthesized new analogues by multiple n-methylation of hut-ii( - ) backbone amide bonds. all the peptides were performed by a novel synthetic approach, in which the introduction of n-methyl groups occur during regular solidphase peptide synthesis. on these new ligands we evaluated the binding affinity and biological activity at the ut receptor and performed preliminary nmr conformational studies. since that time a number of different machines have been used to automate peptide synthesis. modern machines are following two general setups; the so called "single approach" and the "parallel approach". in the single approach, the machine is developed to synthesize one or few peptides simultaneously. the user is able to optimize the synthesis conditions on each single peptide and each single coupling step. the maximum product quality regarding purity and yield is the major task of this approach. in the parallel approach, the machine is developed to synthesize a huge number of different peptides in the same single setup and time-frame. the user always has to find a synthesis protocol appropriate for the needs of each peptide to reach the maximum quality, knowing that there will be always a number of failed peptides. as a result you will find both types of peptide synthesizers in laboratories all over the world: the single machine, for the complicated peptides, and the parallel machine, allowing generation of multiple peptides with standardized protocols for each. the tetras is the first instrument combining the advantages of both machine types and allows the user to synthesize up to different peptides in parallel. each peptide can have its own individual synthesis protocols, separate of all others. the user can combine different synthesis scales, peptide lengths, and activator reagents in one run. finished peptides can be removed and new peptides can be started while the tetras is still running. the tetras allows the user to establish an uninterrupted production shop using one instrument only. siemion, i. z.; peptide res the peptides: analysis, synthesis and biology monitoring peptide folding in membrane-active peptides: a time-resolved spectroscopic study e. gatto a cordopatis p. st european peptide symposium sar studies of triazolyl-containing cyclopeptides: a defined -turn structure increases potency and selectivity to melanocortin receptor subtypes c. testa, a,b proc. natl. acad. sci proc. natl. acad. sci usa multicomponent reactions microglobulin: a "difficult" protein s. abel, m. beyermann the protein ß -microglobulin constitutes the noncovalently bound light chain of the major histocompatibility complex class i (mhc) and plays an essential role in the dialysisrelated amyloidosis. [ , ] to examine the amyloid fibrils of the ß -microglobulin (ß -m) via infrared spectroscopy we intended to synthesize -c-labeled ß -m. [ ] due to the two cysteine residues in positions and we used the native chemical ligation (ncl) strategy for assembling the -mer protein. this necessitates the synthesis of three segments which was accomplished on solid phase using the fmoc/t-bu chemistry. the preparation of the segments had to be optimized with respect to aggregation, aspartimide and piperidide formation, trifluoracetylation, and s-tert-butylsulfonium formation. additionally, ncl steps had to be optimized, because of "internal" thioester formation, dimerization and the formation of side-products of the activated n-terminal segment peptaderm inc., krakowskie przedmie cie str. , warsaw, poland immunosuppressors, such as cyclosporine a (csa) and tacrolimus®, are routinely used in prevention of graft rejection after organ transplantation and in therapy of some autoimmune diseases, including skin inflammation. a naturally occurring in linseed oil cyclolinopeptide a (cla, c(-pro-pro-phe-phe-leu-ile-ile-leu-val) possesses a strong immuno-suppressive activity, comparable at low doses with that of csa , but is much less toxic. we synthesized new cla analogs, containing instead of one proline residue its six-membered mimics, pipecolic acid (pip): c(-pip-pro-phe-phe-leu-ile-ile-leu-val) ( ) and c(-pro-pip-phe-phe-leu-ile-ile-leu-val) ( ). the incorporation of pipecolic acid residue led to different conformational behavior of the nonapeptide cycle. nmr experiments in cdcl solution showed that cla analogue with the pipecolic acid residue in position was much more flexible than cyclopeptide . the new peptides were devoid of toxicity up to μg/ml with regard to human peripheral blood mononuclear cells (pbmc), did not inhibit tumor necrosis factor alpha production in blood cell culture, but exhibited dosedependent, anti-proliferative actions for phytohemagglutinin a-activated pbmc. since peptide was more potent it was tested for growth inhibition of l- lymphatic leukemia. the peptide was found to strongly inhibit the cell growth even at low concentration ( % inhibition at μg/ml). hiv- has emerged as the largest and the most devastating public pandemic in our days, affecting approximately million people worldwide . development of an effective, safe and preventive hiv vaccine remains an urgently needed priority. epitopes for hiv-specific antibodies in elite controllers, a subgroup of long term non progressors, encompassing segments of mper of gp and for the v loop of gp were identified using the phage display technology . immunization experiments with epitopes conjugated to an artificial sequential oligopeptide carrier (soc ), formed by four repeats of the tripeptide lys-aib-gly in tandem, or to the palmitoyl group are currently in progress. all syntheses were performed on a rink amide resin following the fmoc technology. conjugation of epitopes to the soc carrier was realized via a chemoselective ligation approach, which generates an oxime bond between the h n-o-groups of the modified lysine residues and the aldehyde group of each epitope institute of immunology and experimental therapy, polish academy of sciences, - wrocław, poland peptaderm inc., krakowskie przedmieście , - warszawa, poland nonproteinogenic amino acids have been a tool to modify the structures of natural peptides since a long time . bioactive peptides involved in a physiological and biochemical processes cannot be applied in the therapy because of their instability in physiological conditions. that's why the synthesis of their stable active analogues is a challenge for medicinal chemistry nowadays. -trans-hydroxyproline (hyp) is an important building block of natural collagen. it is responsible for the stabilization of collagen super helix, forcing the trans amide bonds configuration with preceding amino acids . at the same time the impact of trans- -hydroxyproline on the conformation other than the collagen peptide chains of biologically important compounds is little known. it is known that immunosuppressive activity of cla is comparable with cyclosporine a and is associated with the presence of the tetrapeptide fragment pro-pro-phe-phe containing pro-pro cis amide bond. now we present synthesis, conformation and biological activity of new analogues of cyclolinopeptide a (cla), containing -transhydroxyproline instead of proline residues in position or . we expected that the introduction of the hydroxyl group in the pyrrolidine ring might influence the biological activity and conformation of the native peptide due to its hydrophilic character and hydrogen bonding ability. the linear precursors of modified cla analogues were prepared manually by standard solid-phase procedure "step by step" on wang resin using fmoc/tbu strategy and tbtu as coupling reagent. the cyclizations of linear peptides have been made under high dilution conditions by means of edc/hobt coupling reagents. the biological activity of newly synthesized compounds as well as the conformational study will be evaluated. dip. di scienze ambientali, seconda università di napoli, caserta, italy nmr spectroscopy is a powerful method to perform structural studies on peptides. to completely fulfill the potential of nmr, peptides labeled with stable isotopes ( n, c, h) are essential. peptides are easily prepared on solid-phase but chemical synthesis becomes prohibitively expensive when applied to the incorporation of isotopes. an alternative cost-effective strategy is the recombinant expression of peptides in e. coli as fusion constructs with carrier proteins. the main problem of this approach is the need of chemical reagents or proteases to cleave the target peptide from its fusion partner after purification. proteases may determine the heritage of extrasequence amino acids at the peptide n-or c-terminus, while chemical reagents require harsh reaction condition that may modify target peptides. an interesting solution is represented by the use of inteins as fusion partner. inteins are protein elements that can catalyze their self-excision from a flanking sequences in mild conditions, by adding nucleophilic agents such as thiols or simply by shift of ph and temperature, bypassing the use of proteases or chemical reagents. we used the self-cleaving mxegyra mini-intein as fusion partner for the preparation by recombinant means of two isotope labeled peptides, hplw and qk. , the two peptides target vascular endothelial growth factor receptor (vegfr) and have been described to modulate vegf-dependent angiogenesis. our expression and purification scheme allows to obtain homogeneously isotope labeled peptides. the availability of isotope labeled hplw and qk opens the way to nmr studies aimed to characterize the folding dynamics of the two peptides and their structures in complex with vegfr. an nmr method to discriminate between the fullyextended and different helical conformations in a spacer peptide c. peggion*, m. crisma, f. formaggio, c. toniolo icb, padova unit, cnr, department of chemistry, university of padova, padova, italy the ideal fully-extended, α-peptide conformation, also known as . -helix, is characterized by φ = ψ = ω = °t orsion angles. the repeating motif of this foldamer is a pentagonal (pseudo)cyclic structure (called c ), stabilized by an intraresidue h-bond. the n-h and c=o groups in the . -helix are not involved in intermolecular h-bonds. multiple c conformations were observed in homopeptides made up of c α,α -dialkylated glycines with both side chains longer than a methyl. this is the case for c α,αdiethylglycine (deg), the residue studied in this work. it is known that deg homo-peptides can adopt the . -helix or the -helix depending on environmental factors and nand/or c-terminal moieties. , in this communication, we introduce an nmr method to discriminate between the . -helix and the -helix based on the observation of cross-peak intensities in the noesy human serum amyloid a (saa) is a highly conserved apolipoprotein produced by the liver under inflammatory conditions accompanying e.g. atherosclerosis, cancer and amyloidosis [ ] . it is also known that saa α isoform has the amyloidogenic properties [ ] . till now it is little known about structure of human saa, as it hampers structural studies due to its facile aggregation. the analysis of protein sequence and cd data together with theoretical studies revealed a typical globular structure of the protein [ ] . the c-terminal sequence of saa contains three proline residues, which probably are responsible for the unordered structure. recent in vitro studies involving saa and human cystatin c (hcc) revealed direct interactions between the ( - ) fragment of saa and the ( - ) sequence of hcc. the results of elisa test for the ( - ) saa fragment have shown that it binds to hcc very well. the nmr studies for the wild ( - ) sequence found an unordered structure in phosphate buffer. based on these data we decided to check how the point mutations pro→ala in ( - ) saa fragment could influence the peptide's structures. we synthesized four peptides with pro→ala point mutations and we performed cd experiments at different conditions. the results show that two of them contain disordered structure and two α-helical structures. in this project we analyze the solution structures of these peptides at the atomic resolution using d nmr supported with molecular dynamics. design and conformational analysis of stapled peptides mimicking cullin binding region to kctd . i. de paola, a l. pirone, a e. pedone, a s. di gaetano, a l. vitagliano, a r. fattorusso, b g. malgieri, b l. zaccaro*, a acknowledgement: this study was supported by eu within the european regional development fund (poig. . . - - / - ). model of angiotensin ii bound to the at receptor in the lipid bilayer environment m.t. matsoukas, t. tselios* department of chemistry, university of patras, gr- , patras, greece the renin-angiotensin system () plays a major role in blood pressure regulation. a sequence of enzyme reactions leads to the release of angiotensin ii which interacts principally with the type- angiotensin ii receptor (at ), a -residue, which belongs to the g protein-coupled receptor family. in the present study, the human at d model was constructed using modeler for the sequence alignment and loop refinement tools. on this basis, the crystal structure of bovine rhodopsin, (pdb code u ), was used as a d template. the gromacs software and amber sb forcefield were utilized for molecular dynamics calculations [ ] in order to evaluate the binding mode of angiotensin ii. the role of the critical amino acids of the binding site v , n , l , a , k , s , h , n and y is being studied. moreover, newest information on the role of the nd extracellular loop by unal et. al. [ ] have been implemented on the model, therefore we propose the contribution mechanism of the residues f -q for binding of angiotensin ii to the at receptor for activation and signaling. a. stavrakoudis department of economics, university of ioannina, greece one key step in the immune response against infected or tumor cells is the recognition of the t-cell receptor (tcr) by class i major histocompatibility complexes. it has been found [ , ] that such peptide/mhc complexes can interact with antibodies as well. this happens mainly in the central part of the peptide in class i complexes [ ] , or at the cterminal of class ii complexes [ ] . in some cases, the same peptide/mhc complex has been found to interact with both tcr and antibodies [ ] . in these study a series of supermolecular complexes have been studied with stateof-art molecular dynamics simulations [ ] the dipeptide kyotorphin (tyr-arg, kyo) plays a role in pain modulation in the mammalian central nervous system (cns), and is one of the most investigated neuropeptides. the tyr-arg motif exists widely throughout the brain not only as kyotorphin, but also as the n-terminal part of several endogenous analgesic peptides , . also, this peptide is very rapidly degraded by aminopeptidases . one of the successful strategies in the design of neuropeptides with enhanced stability and improved delivery to the cns is that with the use of non-protein amino acids, like canavanive (cav), a structural analogue and antimetabolite of arginine (arg trichogin ga iv (noct-aib-gly-leu-aib-gly-gly-leu-aib-gly-ile-lol, in which noct is n-octanoyl and lol is leucinol) is an antimicrobial lipopeptaibol, a unique group of membraneactive compounds of fungal origin, characterized by a high content of the nonproteinogenic ca,a-disubstituted glycine aib (a-aminoisobutyric acid). owing to the gem-dimethyl substitution on the c a atom, aib exhibits a strong propensity to induce β-turns and /α-helical conformations in peptides. we have previously reported on a fluorescent analog of trichogin ga iv, the primary structure (and acronym) of which are: fmoc-aib-gly-leu-aib-gly-gly-leu-toac-gly-ile-leu-ome (f t ) where fmoc is fluorenyl- -methyloxycarbonyl, toac is , , , -tetramethylpiperidine- -oxyl- -amino- -carboxylic acid, and ome is methoxy. the double substitution of an energy donor (fmoc) at the n-terminus and an acceptor (toac) in the trichogin sequence enabled us to make use of time-resolved optical spectroscopies, spanning from the nanosecond to the microsecond time regime, to investigate the conformational propensity and the dynamical features of f t . experimental and computational results indicated that the d-structural and dynamical properties of f t are characterized by a transition from an elongated helix to a more compact conformation mimicking a helix-turn-helix motif. to further investigate the role of the flexible gly -gly central motif we synthesized a new trichogin analog having the gly residue substituted by aib: fmoc-aib-gly-leu-aib-gly-aib-leu-toac-gly-ile-leu-ome (f a t ) experimental and computational results indicated that also the f a t peptide populate two conformations, the dynamics of which were studied at different temperatures using time-resolved spectroscopic measurements. this replacement was demonstrated to stiffen the peptide backbone by reducing the flexibility around the crucial -gly -gly -dipeptide unit. the antigen α β , a member of the integrin family, is involved in the migration of lymphocytes through endothelium to the site of inflammation. thus, α β antagonists may be useful tools for the treatment of various inflammation disorders such as asthma and inflammatory arthritis. in addition, recent studies indicate that α β integrin promotes angiogenesis by allowing the invasion of myeloid cells into tumors, while α β antagonists prevent monocyte-induced angiogenesis, macrophage colonization of tumors and tumor angiogenesis. aiming to the discovery of novel α β antagonists, a series of new peptide analogues cyclized through cysteine disulphide bonds were synthesized and tested in vivo against angiogenesis in chicken embryo chorioallantoic membrane (cam model) . sar results indicated that: yr-c(cdpc)-conh promoted angiogenesis at the higher studied concentration and showed slight inhibition at the lower one, sal-r-c(cdpc)-oh, sal=salicylic acid, showed important inhibition of angiogenesis at dose-dependent manner, yr-c(cdpc)-oh and sal-yr-c(cdpc)-oh both showed no activity on angiogenesis. nmr spectroscopy was applied for the sequential assignment as well as for the elucidation of specific conformational features. experimental noe data were further imposed as distance constraints to a thorough conformational search by applying molecular dynamics simulations. energy refined produced conformers were used as template for the generation of the pharmacophore model associated with the antagonistic activity. such studies are intended to drive a rationalized design and development of this class of inhibitors. hynes r. o. cell, , , - . scaffold discovery by phylomers: a novel cd l specific scaffold derived from glycyl trna synthetase s.r. stone [ ] , k. hoffmann [ , ] , n. milech [ , ] , p. t. cunningham [ ] , m. kerfoot [ ] , s. winslow [ ] , y-f, tan [ ] , m. anastasas [ ] , c. hall [ ] , m. scobie [ ] , p.watt [ ] , and r. hopkins [ , ] [ ] drug discovery technology unit, telethon institute for child health research, roberts road, subicao, , western australia [ ] phylogica pty ltd, roberts road, subiaco, , western australia biopanning of phylomer phage display libraries against human cd l yielded a cluster of highly specific overlapping peptide fragments, from three bacterial genomes, corresponding to the highly conserved catalytic domain from the tetrameric gα β class of glycyl trna synthetases. structural analysis of the overlapping peptide fragments described a scaffold consisting of a central βsheet, comprising anti-parallel β-strands, flanked by nand c-terminal α-helices. further structural analysis revealed that these key structural features, which also encompass the crucial atp-binding motifs of the catalytic domain, are conformationally conserved across both tetrameric gα β and dimeric gα glycyl trna synthetases, yet importantly, there is only limited sequence conservation across these classes. given the identical function of the described domain and it's structural conservation, we postulated that members of the dimeric gα class would display similar cd l specific binding as the tetrameric gα β class, despite the sequence dissimilarity. to test this hypothesis, structurally equivalent peptide fragments of representative bacterial, archaeal and eukaryotic genomes comprising the dimeric gα class were tested for cd l binding in a process we termed ortholog scanning. the results showed that both archaeal (p. horikoshii) and eukaryotic (h. sapiens) structurally equivalent peptides bound to cd l with reasonable specificity and inhibited the cd :cd l interaction with comparable ic 's to the primary gα β class sequences. similar results were also observed for the representative bacterial gα class peptides. that the sequentially diverse orthologous peptides display cd l specific binding has important implications to the affinity enhancement strategies to develop the scaffold as a therapeutic agent, and in improving its "drug-like" properties. we have initiated an investigation related to the effect of radical species upon structures of some peptide segments. in the proposed experimental protocol, aqueous peptide solution was submitted to gamma ray irradiation in controlled - kgy doses. the generation of peptide analogues, possibly induced by reactive oxygen species were examined by electrospray triplequadrupole tandem mass spectrometry (collision induced dissociation approach) and amino acid analysis of crude and/or purified by-products. noteworthy, the gamma irradiation process induced, regardless of the peptide sequence, a non-linear and progressive degradation of all peptides assayed. furthermore, these peptides could be classified in some different classes according to their halflife dose. for instance, the vasoactives angiotensin ii (aii), ang ( - ), bradykinin (bk) and some related peptides were more stable than the melanocyte-stimulating hormone α-msh, substance p or the bk 's ( - ) b receptor fragment (lvyvivgkrfrkksrevyqai). usually, the most prominent derivatives generated from this experimental protocol revealed that they are likely induced by oxidation process, yielding a variation of + da in their molecular weight. the main source of peptide modifications seems to lie either on the phe (hydroxyl group insertion at o-, m-or p-positions of its aromatic side chain) or met oxydation. in the former case, only phe and not phe is oxidized in the bk structure whereas substance p generates an analogue bearing metsulfoxide without modifying its phe , residues. thus, collectively, these findings clearly stress the complexity of factors involved in peptide structural modifications induced by gamma ray-type strong electromagnetic irradiation experiment. an additional target of this approach lies indeed, in the production of unusual peptides for further structure-function investigations. university of bern, bern, switzerland linear peptides are typically poor drug candidates due to their low bioavailability and rapid proteolysis. these limitations can be overcome by rigidifying their structure through head-to-tail or side chain-involving cyclizations. cyclic constraints may also increase biological activity by stabilizing secondary structures and by reducing the entropic penalty of binding to a protein target. the use of multiple branching amino acids in a peptide sequence, like diamino acids (as used in peptide dendrimers ) or amino diacids, allows to design peptides resembling polycyclic alkanes, a type of topology only rarely found in nature (e.g. amatoxins and lantibiotics). bicyclic homodetic peptides such as "norbornapeptides" (bicyclo[ . . ]heptapeptides) were prepared using an orthogonal protection scheme: the first cyclization is performed on resin after selective deprotection of a glutamic acid residue, whereas the second ring closure is achieved by amide bond formation at high dilutions. these peptides are structurally well-defined and cover an almost pristine area of peptide topological space. their conformational rigidity was investigated by means of d-nmr and x-ray crystallography and may offer a platform to design drugs tackling protein-protein interactions. the interaction of peptide ligands with protein receptors face peculiar challenge in recognizing binding surfaces due to availability of a multitude of conformations. therefore it is essential to constrain the peptide conformations for the recognition of receptors and thus finding the bioactive conformation. the cell surface receptor protein family integrins recognize "rgd" sequence which is present in different proteins. to determine the bioactive conformation required to bind with receptor αiibβ , the peptide sequence "riprgdmp" from kistrin was inserted into cdr loop region of rei protein (rei-rgd ). it helps out in finding the possible bioactive conformation of peptide by restricting the sampling space. the activity of rei-rgd was studied and found that as the temperature increased rei-rgd showed a higher affinity towards the receptor αiibβ . the proposed mechanisms for the increased activity of rei-rgd at higher temperature were justified in either of two ways. the modified complex forces the restricted peptide to adopt a bioactive conformation or it unfolds the peptide in a way that opens its binding surface with high affinity for receptor. in this study we model the conformational preferences of "rgd" sequence in octapeptide "riprgdmp" at two different temperatures ( o c and o c) using multiple md simulations. we found that at higher temperature "rgd" sequence from "riprgdmp" adopt turn conformation, while a bend conformation was observed at low temperature. the analysis of various pharmacophoric parameters hint that the turn conformation of "rgd" sequences adopted at higher temperature could be the potential bio-active conformation, and helps out in designing of antagonists for cell surface receptor αiibβ . the -residue peptaibol antibiotic trichovirin i- a (tv) of the linear, covalent structure ac-aib-asn-leu-aib-pro-ala-val-aib-pro-aib-leu-aib-pro-leuol (ac, acetyl; aib, α-aminoisobutyric acid; leuol, l-leucinol) has been synthesized and very thin (~ μm) hair-like crystals were obtained from a methanolacetonitrile-water mixture. diffraction data were collected at k at the diamond light source england, using the microfocus beamline i and a x-ray beam focused to a size of μm full-width-half-maximum. two independent molecules (a) and (b) were located in the crystal's asymmetric unit . both chains assume complete turns of a curved right-handed helical conformation stabilized by intramolecular hydrogen bonds. up to now tv represents the longest right-handed -helix of a natural peptaibol sequence complementing those of synthetic, protected homooligo-aib- insulin is a protein hormone that plays a key role in regulation of blood glucose levels and, thus, has widespread impact on lipid and protein metabolism.insulin is known to act through binding to the insulin receptor (ir); however, the structure of the insulin-ir complex is not known. the crystal and nmr structures of insulin represent only inactive storage forms. it is widely acknowledged that insulin must undergo structural changes in the c-terminus of the b-chain upon binding to the ir. in addition, the n-terminus of the bchain may adopt two different conformations in hexamer, known as t-and r-states. the r-state of the n-terminus of the b-chain creates a long b -b central a-helix. the t-state of n-terminus is in an extended conformation. however, the biological relevance of the t/r forms remains elusive . in this study, we have focused on the synthesis of new insulin analogues modified at the nterminus of the b-chain and subsequently correlated their biological activities with their d-structures. the invariant residue glyb seems to be critical for the t/r transition. glycine can adopt wide range of dihedral angles (φ/ψ) and it occupies significantly diverse dihedral angles in t-and r-states. a-aminoisobutyric acid (aib) is an amino acid with a high helical propensity, which often folds into right-or left-handed α-helix. we have introduced aib at position b , b and b with the aim to induce the r-state of the hormone. in contrast, as d-pro and nmeala are not able to adopt the φ/ψ angles of the right-handed α-helix we have introduced these amino acids at position b to obtain the t-state of insulin. peptide dendrimers are tree-like molecules formed by alternating functional amino acids with branching diamino acids such as lysine. unfortunately these molecules have not yielded to structural characterization and little is known about their molecular-level structure. computational methods seem to be an adequate tool to address these issues.herein we present a comprehensive structural characterization of peptide dendrimers using molecular simulation methods. multiple long molecular dynamics (md) simulations were used to extensively sample the conformational preferences of several third-generation peptide dendrimers, including some known to bind aquacobalamine. we used several conformational analysis procedures (clustering, energy landscapes and multivariate analysis) to analyze conformational changes that can be correlated with particular structural trends.the results point to a high conformational flexibility of these molecules, with no clear "folded state", although two markedly distinct behaviours were identified. some dendrimers favour mainly loose conformations, while others prefer more compact configurations. through a series of computational mutations we investigated the influence of the presence and placement of charged residues in dendrimer topology, finding that electrostatic interactions among charged residues are a major determinant in structure acquisition by peptide dendrimers. these conclusions bring new insight into the conformational behaviour of these systems and may provide better routes for their functional design. acid-mediated prevention of aspartimide formation in solid phase peptide synthesis t. michels, a r. dölling, b u. haberkorn a , w. mier*, a a department of nuclear medicine, university hospital heidelberg, heidelberg, germany; b biosyntan gmbh, robert-rössle-straße , berlin, germany aspartimide formation is one of the major obstacles that impede the solid phase synthesis of large peptides and proteins. the main reason for aspartimide formation is the piperidine-catalyzed fmoc cleavage of peptides containing aspartic acid. several side chain protecting groups have been developed but the complete prevention of aspartimide formation can only be achieved using n-( hydroxy- -methoxybenzyl) (hmb) as backbone protecting group. however, hmb-protected building blocks are difficult to synthesize and only the dipeptide containing glycine (fmoc-asp(tbu)-(hmb)gly) is commercially available. until now, no cost effective strategy to suppress this side reaction has been developed. formally, aspartimide is the result of an attack of an amidate species at the carbonyl carbon of the otbu protected side chain carboxylate of aspartic acid, which might be prevented by protonation of the amidates with piperidinium ions. in this work the suppression of aspartimide formation by adding small amounts of organic acids to the deprotection agent piperidine was studied. this procedure was shown to efficiently prevent the formation of aspartimide side products in several peptides, i.e. pres - -y, a -mer peptide derived from the hbv surface antigen and a peptide parathyroid hormone (pth) fragment. the testing of a series of different acids covering a broad range of pka values showed that this effect is virtually independent of the acid strength. since aspartic acid is found in most oligopeptides, the authors recommend to generally add % (v/v) formic acid to piperidine based fmoc cleavage mixtures. decomposition of the resin linkers during tfa cleavage of peptides in fmoc-strategy leads to alkylation of sensitive amino acids . this side product formation is a crucial drawback, especially during the synthesis of biologically important cys-containing peptides on wang support. through a battery of approaches ( h-nmr, uv and lc/esi-ms) we detected an unexpected alkylation of the sulfhydryl group of cysteine side-chain residues by the phydroxyl benzyl group from the wang resin linker. herein, we present the feasibility for s-alkylation of cys-containing peptides from wang linker decomposition. this sidereaction occurs during the final tfa cleavage of the peptide from the solid support, while the position of the cysteine residue within the peptide sequence as well as the resin's substitution influence the extent of cys-alkylation. the stephan angeloff institute of microbiology, bulgarian academy of sciences, sofia, bulgaria influenza viruses cause epidemics and pandemics all over the world. therefore, the development of virus resistance to drugs, leads to search for novel derivatives and approaches to chemotherapy for human influenza infection. antioxidant therapy is known to be one potential approach. the application of combination therapy of antioxidants with antiviral drugs could reduce the complications and lethal effects, caused by an influenza virus . in our study, amino group of neuraminidase inhibitor -oseltamivir, which belong to second generation anti-flu drugs, was covalent conjugated with known antioxidantscysteine, histidine. tryptophan and etc. the study of the role of the modified by antioxidants oseltamivir on proliferation of influenza virus is in progress. recently we reported a short synthesis of or -membered cyclic guanidine via intramolecular reaction of alkyl diamine with n,n'-cbz-methylisothiourea( a). here we report a further application of synthesis two types of cyclo-peptide guanidine-bridged cyclopeptides utilizing this mechanism -n-terminus local cyclo-guanidine peptide and backbone guanidine-bridged marco-cyclic peptide. three n,n'protected diaminoacids (daa) including , -diaminobutyric p . antifreeze glycoproteins (afgps) are found in the deep sea teleost fish in arctic and antarctic oceans. these biomolecules are able to inhibit the growth of ice crystals and depress the freezing temperature of the blood serum in fish enough to keep them from freezing in their sub-zero environments while the melting temperature remains unchanged . despite afgps have been consider as a potent cryopreservation, obstacles to develop afgps as medicinal and industrial application are mainly due to the lack of access to pure form from natural sources and the problem of understanding how afgps inhibit ice crystal growth. as a result, a considerable progress toward the design and synthesis of afgp analogues has been made several groups . in the course of the studies on the structure-activity relationships of afgps, we are interested in peptoids as mimics of α-peptides and synthesized monoglycosylated peptoid analogues by substituting the glyco-thr residue as afgps mimics. in this presentation, we will show our studies on how the insertion of peptoid residue into afgp backbone affects the afgp activity by measuring both thermal hysteresis (th) and ice recrystalliztion inhibition (iri). [ ] , both for diagnosis and endoradiotherapy. for this application the peptides can be attached with chelating agents that bind radioactive metals such as ga, ga or in for imaging or therapeutic radiometals such as y and lu. the chelating agent most frequently applied is the macrocyclic ligand , , , -tetraazacyclododecane-n,n',n'',n'''-tetraacetate (dota), it is commonly introduced as the tris(tbu ester). the cleavage of the tbu protecting groups on dota is known to be sluggish [ ] . several attempts have been made to synthesize dota with protecting groups that can be removed under mild conditions. however, these derivatives have not yet found widespread application. our new approach was to prepare a protecting group for dota-based prochelators that is convergently cleaved under the cleavage conditions of the amino acid protecting groups of the peptide. o-phenylisopropyl (opp) esters are more sensitive towards acid than tbu esters. deprotection occurs with % trifluoroacetic acid in dichloromethane [ ] . therefore, a synthesis of the prochelator dota-tris(opp ester) was developed. the copper-catalyzed azide-alkyne cyclization (cuaac), the most commonly recognized variant of "click chemistry," has emerged as a powerful technique for ligation, conjugation, and cyclization reactions of peptides. it is known that cyclization can increase the metabolic stability of peptides, as well as enhancing potency or selectivity by stabilizing an active conformation. one application of the cuaac that has generated interest is the use of this reaction to replace a disulfide bridge with the product triazole, which among other complementary properties may prevent in vivo redox chemistry. in this poster, we synthesize a new analogue of the cyclic cancertargeting peptide cngrc where we replace the disulfide bond with a triazole linkage using click chemistry and a fully automated, on-resin method using the single-shot delivery feature on the prelude® peptide synthesizer. unnatural amino acids including d-amino acids are manufactured mainly by the enzymatic process. however, one enzyme can produce only one amino acid due to its high specificity and it takes a long time and a lot of expenses to develop the appropriate enzyme itself. arca (alanine racemase chiral analogue) is an organic catalyst which can overcome these drawbacks and can produce almost all kinds of amino acids efficiently. the amine functionality of l-threonine is freely reacted with the aldehyde group of arca to form the corresponding imine, which is easily epimerized in the presence of organic base due to the acidity of the alpha proton of imine. the difference in the stability between the imines of the optical epimers rendered them to be shifted to d-allo-threonine derivative dominantly. once the epimerization reaction reached equilibrium, the reaction mixture was hydrolyzed under acidic condition to give d-allo-threonine and arca, which could be recycled repeatedly without significant loss in yield or purity to produce more d-allo-threonine from lthreonine in excellent yields. optimization of the reaction conditions with various bases and solvents is discussed and mass production of optically active d-allo-threonine including optical purification is described. the manufacuring process for the preparation of arca will be shared as well. our group is interested in the development of efficient synthetic routes for the preparation of enantiopure atrifluoromethylated amino acids (a-tfm-aas) starting from chiral cf -oxazolidines or imines and their incorporation into a peptide chain. these non-natural amino acids are very attractive compounds for the design of biologically active molecules, particularly peptides, due to the unique physical, chemical and biological properties impart by the cf group. as conformationally constrained cyclic amino acids have recently gained considerable interest, we are particularly focused on the preparation of pyrrolidine-type a-tfm aas. , incorporation of proline derivatives is known to restrict the amino acyl-proline cis/trans isomerization, to limit the protein folding and consequently to modulate the biological activity of peptides. based on these observations, mutter's group introduced pseudoproline building blocks (ψpro) into a peptide sequence as reversible protecting groups for ser, thr and cys. the ψpro residues proved to be versatile tools for overcoming the aggregation caused by hydrophobic interactions encountered during solid-phase peptide synthesis (spps). they also turned out to be inducers of βturns containing predominantly cis-amide bond and useful tools in peptide cyclization. here, we report the results obtained for the preparation of various hydrolytically stable trifluoromethylated pseudoprolines (cf -ψpro) as well as the methodological studies developed to optimize the synthesis of various c-and n-terminal cf -ψpro containing dipeptides. rennes, france protein strructure and function rely on a still not fully understood interplay of energetic and entropic constraints defined by the permutation of the twenty genetically encoded amino acids. many attempts have been undertaken to design peptide-peptide interaction pairs and synthetic receptors de novo by using special building blocks. a rational approach starting from hydrazine to create new building blocks based on a tailored metalchelating amino acid analogues was envisaged. to create chemical recognition units, which bind oligohistidine tags with high affinity and stability, several supramolecular entities containing one to three nitrilotriacetic acid analogue (ynta) moieties were synthesized. these new building blocks additionally contained an amino group or an acido group, which can be flexibly introduced into peptide in n or c-termini or into the peptidic chain by solid phase chemistry in fmoc/t-bu strategy. these multivalent chelators were characterized and the corresponding metalchelating peptides could act as metal sensors and synthetic receptors for histidine-tagged proteins. the potential of peptides as drug candidates is often limited by their pharmacokinetic properties. structural modification of the peptide backbone via n-methylation is a powerful medicinal chemistry tool that confers oral bioavailability to these molecules. n-methylation exerts a strong effect on the backbone conformation and, as a result, many n-methylated peptides show enhanced biological activity and higher receptor selectivity. another approach to increase the solubility of peptides is by conjugation of peg to a derivatizable functionality. by combining these two approaches we have developed n-oegylation. this novel form of peptide modification consists of the attachment of oligoethylene glycol (oeg) chains to the amide bonds. many bioactive peptides comprise one or more n-me amino acids which are essential for their activity. thus, we consider that replacement of a backbone n-me group by an oeg chain may imply a minimal structural perturbation and may lead to n-oegylated peptides with preserved biological activity. furthermore, our strategy is a promising way to improve the bioavailability of cyclic peptides that do not have any site where a peg could be attached. as a proof of principle, several n-oeg analogs of two bioactive cyclic peptides were synthesized in spps. first, we performed a full n-oeg scan of the sansalvamide a peptide. next, several analogs of cilengitide were prepared by replacing the n-me of valine by oeg chains of different length. depending on its size, the oeg residue was incorporated by using an n-oeg derivative as building block or, alternatively, using an n-substituted amino acid bearing an attachment site where a peg was conjugated post-synthetically.the biological activity of all the n-oegylated peptides was evaluated. some of the sansalvamide a peptide analogs exhibited cytotoxicity within the same range as the original peptide, which suggests that backbone amide groups may be useful oegylation sites in bioactive cyclic peptides. the modification of peptides is an important step in pharmacology to vary the affinity and the stability of peptidic drugs. whereas a wide range of strategies exists for the functionalization of the n-terminus and the side chains, facile variation of the c-terminus remains an important challenge. we consider peptidyl-phosphoranes as a promising platform to enable orthogonal and mild introduction of a great variety of chemical functionalities at the c-terminus. a convenient method for the synthesis of soluble peptidyl-phosphoranes has been presented by our group recently. in this, -bromo-acetyl bromide was coupled to a wang-resin followed by alkylation of triaryl-or trialkyl phosphine moiety. deprotonation to the phosphorus ylide and subsequent acylation with an fmocamino acid created the basis for assembly of the peptide by spps. final acidic cleavage produced a decarboxylated and unprotected, soluble peptidyl-phosphorane. from this point, a variety of orthogonal modification reactions at the peptides c-terminus is possible, e.g. click reaction with azides allow for the incorporation of triazoles as peptide bond mimetics. the wittig reaction opens up another interesting portal for c-terminal modification, as vinyl ketones are formed by reaction with aldehydes. the described chemistry was applied to modify caspase- inhibitors. in order to address the s site of caspase- , the commonly known devd inhibitor was varied at the c-terminus by introduction of different residues. the devd motif was synthesized as peptidyl-phosphorane and modified in wittig reactions. the resulting c-terminal vinyl ketones were obtained by the reaction of aliphatic and aromatic aldehydes. a small library was generated, and novel compounds were tested for their potential to inhibit caspase- . semmelweis university, department of biophysics and radiation biology, budapest, hungary considering the impact of uv irradiation on the structure and function of proteins , it is a matter of utmost importance to resolve the conditions of photolysis more deeply. we think that a protein, as a complex unit, gives multiple responses to all impacts therefore the analysis of these responses is a rather complex problem. the main goal of our research is the deeper understanding the tryptophan-mediated photolysis of disulphide bridges in bio-active proteins upon near-uv irradiation using cyclic peptide models, as small protein units, to define the caused functional damage. cation -pi interaction is increasingly recognised as an important noncovalent binding interaction which plays a role in establishing the final structures of proteins. within a protein, cation -pi interactions can occur between the cationic side-chains of either lys or arg and the aromatic side-chains of phe, tyr or trp . our earlier results with gla indicate that new covalent bonds are also formed between cys and lys during illumination, which is also a reason why the lys residue is planned to be included in the sequence of the models. our aim is to study whether the cation -pi interaction can have an influence on the ss-bridge splitting in small cyclic pentapeptide models. here we report about the conformational analysis, synthesis and spectroscopic investigation of lys-and arg-containing model peptides. the azide functionality is very popular mainly due to azidealkyne click chemistry used in many peptide ligation strategies. azido-peptides are usually prepared by incorporation of azide containing residues or azide functionalization of aldehyde resins affording c-terminal azido-peptides. alternatively, the n-terminus can be converted into an azide via a cu(ii)-catalyzed diazotransfer reaction using triflyl azide. recently, a number of safer, shelf-stable and easily prepared diazotransfer reagents has been developed, of which imidazole- -sulfonyl azide has been used to introduce azide moieties in proteins under copper-free conditions. typically, it has not been reported to be used on the solid phase. we provide a very easy, fast and efficient method for conversion of amines into azides on a solid phase support which in our opinion has major benefits over earlier reported methods making using of less stable reagents that require a metal ion catalyst. we demonstrate how the diazotransfer reaction can be performed on a solid phase support using the imidazole- sulfonyl azide reagent without the need for a metal ion catalyst. using a model peptide we studied the effect of stoichiometry, added base and solvent. in addition we examined the effect of the nature of the n-terminal residue on the efficiency the diazotransfer reaction. finally, we found that the optimal conditions to perform the reaction also depend on the nature of the solid phase support the reaction is performed on. the novo nordisk foundation center for protein research, university of copenhagen, copenhagen, denmark site-selective strategies for post-translational modification of peptides and proteins are essential tools for many areas of research in the life sciences, yet remain a chemical challenge due to the multiplicity of functional groups present. there are powerful chemoselective reactions, however, they aim at introducing only one functionality at each reaction site. here we present a one-pot, threecomponent dual-functionalization of peptides or proteins based on a , -dipolar cycloaddition between a functionalized malemide, an n-hydroxylamine and a peptide or protein with an n-terminal serine residue at the n-terminus, which is selectively oxidized to a -oxoaldehyde. most common moieties for labeling, e.g. fluorophors and peg-chains, are commercially available as maleimides. nitrones were easily obtained by condensation of peptide-aldehydes and primary nhydroxylamines under aqueous conditions. the chemoselective , -dipolar cycloaddition reaction between the peptide-nitrone, and a functionalized maleimide proceeded in aqueous solution at room temperature or with gentle heating, which provided the stable isoxazolidine product. we envision that this 'one site -two functions' method can be used widely to introduce two separate moieties. the method was used to introduce two separate ligands in a range of other peptides. for example, new multimodal molecular imaging techniques depend on facile chemical methods for site-selective dual-functionalization. we used our new methodology to synthesize a cyclic rgd-peptide for combined pet and optical molecular imaging. finally, the small protein ipb was successfully n-terminally modified, including with a peg-chain, using this new, general method. multiple sclerosis (ms) is the most known chronic, inflammatory, demyelinating disease of the central nervous system (cns), characterized by a progressive neurodegeneration, caused by an autoimmune response to self-antigens in genetically susceptible individuals. it is nowadays known that post-translational modifications may affect the immunogenicity of self-protein antigens, triggering an autoimmune response and creating neoantigens; in particular aberrant glycosylations affect various parts of the immune response and have profound effects on immune tolerance. in previous studies we demonstrated the value of the glycopeptide csf (glc) which, by virtue of the particular type i' β-turn structure, optimally exposes the minimal epitope asn(glc) to autoantibody recognizing in elisa in multiple sclerosis patients' sera . elisa assays allowed to conclude that the ability in detecting autoantibodies in multiple sclerosis sera was stricktly linked to saccharidic moieties and to conformation around minimal epitope of the antigenic glycopeptide. herein, taking advantage of such considerations, we focused our attention on the synthesis of a little library of lysine branched multiple antigen peptides (maps), containing the minimal epitope asn(glc), in an attempt to increase the antigenicity of linear peptide sequences . with this aim, we performed the spps of glucosylated maps via the building block approach, studying the role of different long spacers on the dendrimeric core, and the role of different peptide sequences around the sugar moiety, in order to optimize the synthetic process and to evaluate the influence on the affinity and specificity in sp-elisa. environmentally induced co-or post-translational modifications of autoantigens are hypothesized to break immune tolerance leading to self reactivity in pbc. it has been previously reported that the use of synthetic post-translationally modified peptides, introducing fmoc-l-lys(nε-(±)-α-lipoic acid)-oh, as peptidomimetics of natural neoantigens allowed to detect autoantibodies in the sera of patients affected by pbc, and they might be useful diagnostic tools that can be used in earlier stage patients and possibly to monitor disease activity. only the r-(+)-enantiomer of α-lipoic acid exists in nature and is an essential cofactor of four mitochondrial enzyme complexes. but it remains unclear if the tridimensional structure of the lipoic acid is of any importance in the interactions antibody-peptide during the indirect elisa tests. therefore, it is necessary to synthesize each peptide separately with one absolute configuration of the lipoic acid. herein, we describe the synthesis of the two diastereoisomers fmoc-l-lys(nε -(r)-α -lipoic acid)-oh and fmoc-l-lys(nε -(s)-α-lipoic acid)-oh that have to be used in fmoc/tbu spps as building blocks for the synthesis of post-translationally modified peptides. recently it has been reported the introduction of a new generation of cd diagnostics based on a unique antigen approach, consisting on human ttg cross-linked with gliadin peptides coated on the elisa plates . on the basis of experimental data obtained by mass spectrometry and indicating which are the fragments of these two proteins that are supposed to be involved in the antibody recognition, we were able to select the most representative ttg and gliadin fragments , to design and synthesize by fmoc-spps nine cross-linked eoepitopes. aim of our study was the characterization of autoantigenic epitopes by testing, in celiac patients' sera, the reactivity of these nine synthetic peptides. these neoepitopes were tested in elisa to evaluate the iga and igg response against ttg-gliadin adducts in celiac patients' sera in order to develop a new elisa test based on peptides as an even more powerful diagnostic tool in terms of specificity and sensitivity. more than analogs have already been described, wherein the hydroxy acid and the amino acid constituents were replaced by d-amino acids and/or n-methyl amino acids with preserved or altered side chains. for certain types of cancer cells, several of these analogs were found to be more active than the natural product itself. however, it does appear that many of these compounds have limited solubility in water. b here we report the synthesis of novel analogs of the sansalvamide a peptide bearing an n-oligoethyleneglycyl (oeg) chain attached to the different backbone positions. attachment of this chain is aimed to enhance the hydrophilicity of the original peptide. our synthetic strategy to modify the backbone with the n-oeg group relies on the use of n-oeg amino acids, which were synthesized in solution and then used as building blocks in spps. as expected, couplings to the n-oeg residues were found to require special conditions. methods for the coupling to nmethyl amino acids were applied and this enabled to obtain the different linear pentapeptides, which were cyclized in solution. both the synthetic strategies of these demanding peptides as well as the preliminar evaluation of their biological activity will be deeply discussed. glycoconjugates such as glycoproteins and glycolipids have important roles in cell functions, for example, intercellular recognition, cell proliferation control, and information transmission. in order to study the structurefunction relationship, synthesis of these glycoconjugates is essential. glycoproteins and glycopeptides are classified into two categories: n-and o-glycosylated derivatives. the n-acetyl-α-d-galactopyranosylated ser or thr derivatives [ser/thr(α-d-galnac)] are important intermediates for o-glycopeptide synthesis. however, the synthesis of ser/thr(α-d-galnac) derivatives by chemical glycosylation is difficult because of the decreased nucleophilicity of hydroxy function in the glycosyl acceptor due to an unfavorable hydrogen-bonding pattern between the oh and α-nh groups . several approaches to overcome this problem have been reported , . in addition, the o-glycosidic bond is cleaved easily in acidic conditions. in this study, we assumed that the formation of a cyclic structure containing an α-nh group would increase the reactivity of oh function. thus, we focused on the n, n'isopropylidene derivatives of ser/thr containing dipeptides . we found the reaction of mannopyranosyl trichloroacetimidate and the n, n'-isopropylidene dipeptide in the presence of tmsotf in dichloromethane produced the desired glycosylated dipeptide in good yield. however the selective intermolecular disulfide bond formation is a very difficult and complicated synthetic problem. in this work we report on synthetic approaches for the formation of conjugates with intermolecular thioether or disulfide bonds. for the disulfide bond formation, we use two activation approaches: i) activation of the four cys residues of the carrier testing two activating reagents, in both solid and liquid phase respectively and ii) activation of the cys containing bioactive molecule. as bioactive molecule we selected the r ppleed sequence derived from the intracellular part of the αiibplatelet integrin receptor. this region is critically involved in platelet aggregation and is a target of intervention for developing antithrombotic agents . the ac-[lys-aib-cys(ch co-αiib - )] -nh and ac-[lys-aib-cys(cys-αiib - )] -nh conjugates were synthesized and examined for their ability to inhibit platelet aggregation. the biological assays indicated that the synthesized conjugates penetrate the platelet membrane and inhibit human platelet aggregation, in contrast to the corresponding free peptide analogues. the molecules were reported to exhibit broad-spectrum cytotoxicity against the tumor cell lines. although these peptides contained the novel β-methoxytyrosine, lipton et al. reported the synthesis and cytotoxicity of desmethoxycallipeltin b, in which substitution of d-tyrosine for β-methoxytyrosine did not substantially affect the cytotoxicity of callipeltin b , . however, a structure-activity relationship study of the molecules has not been shown to date in detail. in the course of our recent research regarding the synthetic study of cyclic depsipeptides, we conducted studies on the synthesis of callipeltins supposed to be efficient structures for ccr inhibitors as anti-hiv drugs or anti-cancer agents. in the present study, we report the synthesis of cyclic depsipeptides of callipeltin b analogues consisting of l-, d-amino acids and/or n-methyl amino acids, for a structure-activity relationship study of linear-and cyclic depsi-peptides against hela cells . in the assay of synthetic peptides, all of the synthetic callipeltin b analogues exhibited no cytotoxicity. we supposed that dimethylpyroglutamic acid of callipeltin b was essential structure to show the cytotoxicity against hela cells. monash university, melbourne, australia protein-protein interactions represent a significant portion ( - %) of all interactions within the cell; as such these interactions are ideal targets for drug discovery. while difficult to target using small molecules, these interactions can be disrupted using a small section of the protein's binding partner. these short peptides must retain the defined secondary structure associated with the protein binding interface in order to inhibit their protein targets. as the secondary structure adopted by the parent protein is not always exhibited by its derived peptides, constraints are introduced as necessary to help define the structure of the peptide. inducing secondary structure reduces the energy required for organisation, decreasing the energy of binding and has the potential to increase stability with respect to degradation by proteases. solid phase peptide synthesis was used to make several small peptides corresponding to the structured sections of the binding partners for three protein-protein interactions. these peptides were designed to target heart disease, prostate cancer, and liver cancer respectively. secondary structure was introduced using lactam bridge constraints. for the αhelical peptides, a side chain constraint approach was used to nucleate helix formation. as hydrogen bonding between the c=o of the i th residue and the nh of the (i+ ) th residue stabilises native α-helices, constraints were introduced linking the side chains such that the residues were held in close proximity. for β-pleated peptides, an antiparallel β-sheet arrangement was achieved by introducing turn regions into the peptide in such a way that the β-strands were aligned. constraints were again introduced using lactam bridges between lys and arg or glu side chains. these peptides were characterised by nmr and cd spectroscopy to verify the correct secondary structure had been induced. göttingen, germany different properties can be combined in a single molecule by using a scaffold arranging functional groups in a predefined topology. the tasp (template-assembled synthetic proteins) concept describes templates to reinforce and direct the folding of designed molecules into a predetermined topology. [ ] due to their resistance to proteolytic degradation and their rigid basic structure, cyclic β -tripeptides are suitable carrier molecules for bioactive compounds; they are further known to form tubelike structures by stacking of the peptide rings leading to higher organization of functionalized peptides. [ ] with this scaffold different inhibitory systems were synthesized that feature cell penetrating and fluorescent properties. the signal transducer and activator of transcription (stat ) protein, which has been described as an oncogenic protein, was selected as the first target. [ ] a peptide sequence which targets the sh domain of stat was used in two different approaches. it was either directly attached to the cyclic β-tripeptide via a huisgen [ + ]cycloaddition or the peptide was incorporated into the inhibitor loop of the cystine knot microprotein omcoti-ii, which was also attached to the cyclic-β -tripeptide. [ ] further, sodium channels are addressed usingconotoxines. first, an alkyne functionalized conotoxin siiia was synthesized applying different folding methods. the alkyne linker will be used to attach a fluorophore or to functionalize a cyclic-β-tripeptide. using single molecule imaging the spatial distribution, local concentration and organization of the ion channels in neurons will be imaged. further, the cyclic-β -tripeptide templating effect will be used functionalizing with μ-conotoxines. those proteins are folding helper proteins. together with chaperones, they form receptor complexes. they catalyze the isomerization of prolyl bonds in various folding states of target proteins. indeed, their role has been implicated in refolding of denatured proteins, de novo protein synthesis and the biologically active conformation of proteins . among them, the fkbp subclass comprises the small ppi calstabin and . it is of interest to try to understand the way those proteins act, in order to help the overexpression of various types of membrane proteins, aiming at the renaturation, purification and crystallization attempts of receptors. we chose to work on calstabin because this short ( aa) protein has been described as a sub-family comprising isoforms (from ~ to ~ aminoacids), some of them not being fully described to date. the relative shortness of those proteins together with the fact that the two higher molecular weight ones are catalytically active as prolyl isomerases, facilitate the characterization of the synthetic proteins.in order to obtain the full length calstabin , a native chemical ligation (ncl) approach was chosen . an optimized stepwise elongation allowed the obtention of the c-terminal segment up to the cys . moreover, several methods were compared for the synthesis of peptide - opportunely functionalized at its c-terminus for the ncl.the ligation at thr site between peptide - featuring a bis( -sulfanylethyl)amino the chemical diagnostics of paintings is a relevant topic in the field of chemical sciences applied to the conservation and safeguard of cultural heritage. chromatography is a highly sensitive and suitable technique for accurate methods of analysis of the limited amount of sample material typically available from works of art. paint media deriving from proteins traditionally include egg, milk, animal and fish collagen glue. egg yolk (egg tempera), egg albumin (glair) and casein (a blend of related phosphoproteins commonly found in milk) are traditionally used as pigments binders. we propose the uplc-based amino acid analysis as diagnostics technique on non pre-treated or submitted to extraction processes model samples, showing that good results can be achieved with very scarce sample manipulation and great advantage. we applied the amino acids analysis carried out by the accq•tag™ ultra performance liquid chromatography to the standard and model samples. in particular, after protein hydrolysis ( h, °c, m hcl) of the samples, the amino acid derivatization by -aminoquinolyl-n-hydroxysuccinimidyl carbamate allowed a reproducible amino acids analysis characteristic of the protein type. the results obtained confirmed the reliability of the data achieved and demonstrated that the accq•tag™ ultra uplc method could be a powerful technique to be applied to the relevant field of protein binders diagnostics for paintings conservation. moreover a multivariate analysis that offers a wide variety of tools and methods mainly concerned with mathematical models for the representation of multidimensional data has been proposed and the high model efficiency has been established for sample containing mixture of proteins. reactions performed on solid supports, such as resin, are commonly monitored by hplc-uv after cleaving the products from the support. however, uv-absorption coefficients may differ between compounds, and therefore the relation of the area percentage values of the peaks may not directly reflect the molar concentrations of the corresponding compounds. it is for this reason that, for example, in solid-phase peptide synthesis it is difficult to calculate the yield of the coupling of a fmoc-amino acid or the removal of the fmoc-group because of its high absorbance. recently, we reported the identification of minimal phosphopeptides that specifically interact with the pbd of human plk , but not those of the closely related plk and plk . comparative analyses of the crystal structures of the plk pbd in complex with the minimal phosphopeptides revealed that the c-terminal spt dipeptide functions as a high-affinity to the interaction. in an attempt to obtain the adequate cellular permeability and stability in vivo, we have accomplished the peptide-peptoid hybrid or peptomers cyclization using various methods like formation of amide, thioether and triazole and screened the plk inhibition activity on the first cyclic peptomers liibrary using pbd-binding assay. based on our first screening results, we also carried out the detailed investigation to further increase the activity and also to understand the significance of peptoid mimics as plk inhibitors. the mode of interaction between the cyclic peptomers and pbd might provide a template for designing therapeutic agents that target plk . a synthetic amino acid long peptide corresponding to the minimal metacaspase catalytic domain induces cell death in leishmania major c. servis, h. zalila*, i. gonzalez, l. lozano, n. fasel department of biochemistry, university of lausanne, epalinges, switzerland despite a lot of controversy during the last decade, there is increasing experimental evidence that cell death (cd) is genetically programmed in lower eukaryotes.in the cd proteolytic cascade of plants and protozoa, caspases are likely replaced by metacaspases that are cysteine peptidases recognizing arginines or lysines in p position. metacaspases have been found to control cell death in plants. the human protozoan parasite leishmania major expresses a single metacaspase (lmjmca) harboring a central domain with the catalytic dyad histidine and cysteine as found in caspases. metacaspase could therefore be one of the executioners of the death pathway in leishmania.in this work we showed that, in stress conditions, lmjmca precursor forms were extensively processed into soluble forms containing the catalytic domain and this domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion function. we tested different lengths of the lmjmca catalytic domain and found that the overexpression of the polypeptide corresponding to amino acids - was sufficient to sensitize l. major mitochondria to oxidative stress.we synthetized an aa long peptide corresponding to the minimal metacaspase catalytic domain (aa - ) and showed that it has specific metacaspase activity in vitro.we are currently investigating its activity on possible target proteins, which have been identified in a yeast two-hybrid screen. identifying proteins involved in the metacaspase signaling pathway will shed light on the understanding of cd in leishmania and open new perspectives in drug target investigation to fight leishmaniasis and other major infectious diseases. s. alasibi, g. ashkenasy department of chemistry, ben-gurion university of the negev, beer-sheva, israel various factors can affect the conformations and folding states of protein molecules and as a consequence their activity. these factors include amino acid mutations, interactions with other macromolecules, binding to regulatory molecules, and also external changes such as ph jump or shining light. in order to control the folding states and to modulate the functions of peptides and proteins by light, photocleavable groups are usually incorporated into specific residues to mask critical interactions. for example, introducing caging groups into coiled-coil proteins recognition interface affects complex formation and template-assisted ligation reactions, in which the coiled-coils serve as templates to catalyze the condensation reactions between two short peptide fragments . our research group has been studying peptides replication networks, which were made of coiledcoil peptides and analyzed the response of such networks to light as external trigger . it was shown that even replicating networks made up of a small number of molecules can possess complex behavior, considering the wealth of catalytic pathways and transformations. hence, boolean logic operations can provide valuable means to analyze and interpret their behavior . herein, we describe the use of chemical inputs and uv light to manipulate peptides folding and functionality within new synthetic networks. these networks perform complex behavior and, as a result, selective product formation is used to implement boolean operations that have not been achieved before. institute of bioorganic chemistry of ras, moscow, russia earlier, we have shown that n-acylated amino acid nitriles and amides react with ethylene derivatives forming the amino-and -hydroxypyridines and pyrroles [ ] . a possible reaction mechanism is the geterodienic condensation of aminooxazole derivatives to dienophiles. the higher yields were observed when used the dicarboxylic acids as dienophiles and -amino or alkoxyoxazole as geterodienes. while the same reactions with the fullerene derivatives, as dienophiles, gave low yields. the nitrile groups of specified pyridines possess ability to react with amino groups of peptides and proteins even at room temperature. in view of high activity of nitrile groups such pyridines can form tetrapyridotetrazoporphyrins and self-condensation products giving appropriate dendrimers (possible due to mobile hydrogen in the th position). high molecular weight dendrimers were identified by massspectrometry, gel electrophoresis and dynamic light-scattering. catalytic oxidizing properties of tetrazaporphyrin derivatives and phtalocyanin were used in synthesis of cyclic peptides and for the s-s bonds formation. the transformation of peptides into heterocycles via an intramolecular reaction of nitrile groups was used to determine the sequence of some peptides, which favored the resistance of transformed compounds to hydrolysis and to the electron impact at mass spectrometry. diazotization of peptides and their derivatives facilitates identification of amino acid sequence by mass spectrometry due to the peculiarities of their fragmentation. in addition, an amino acid analysis of the diazotized peptide makes it easy to determine the n-terminal amino acid. we present here a multi-disciplinary approach combining x-ray crystallography, computational analyses, and immunological tests to identify epitopes of the oligopeptide-binding protein a (oppa bp) from the gramnegative pathogen burkholderia pseudomallei, the etiological agent of melioidosis. computational analysis on oppa crystal structure was used to design potential consensus epitopes, that once synthesized as free peptides (comp - ) were found to be immunoreactive against sera from melioidosis patients. notably, one of the predicted peptides allowed to distinguish between seropositive, seronegative and recovered groups, underlining its potential for diagnostic purposes. parallel experimental epitope mapping, based on proteolysis and mass spectrometry, allowed us to identify linear peptide epitopes (exp - ) localized in similar protein regions as comp - . moreover, the match between theoretical and experimental mapping of epitopes was improved by expanding our computational approach, i.e. including an energy based decomposition procedure to divide oppa bp into separate fragments. overall, our results illustrate the successful development of a novel integrated structurebased approach for the discovery, design and preparation of epitopes. nonetheless, given antigen crystal structures, our method is expected to be broadly applicable in the design and generation of new epitope candidates, as being confirmed by on going experiments on different antigens. the application of peptide thioacids as reactive intermediates and building blocks has received considerable attention recently. the chemical ligation reaction between thioacids and azides has been reported for the synthesis of small to larger peptides as well as for the modification of proteins. fmoc based methods for the preparation of peptide thioacids have to our knowledge not been extensively researched and a facile approach to their synthesis is desirable.we have recently shown that t-butyl thioesters are robuster than previously reported, and can be used for the fmoc based solid-phase preparation of peptide thioesters being also easily cleavable with thiolates. peptides attached via a -mercapto -methylpentanol (mmp) resin can be cleaved using -mercaptopropionitrile to obtain protected thioacid peptides with a ß-elminable cyanoethyl group.the thioacid peptides could then be obtained in situ after treatment with dbu ( . % in dmf) and further reacted with sulfonyl azides in the presence of , -lutidine in a one pot reaction. by treating the cyanoethyl peptide thioesters with (nh ) s in a sodium phosphate buffer (ph = ), various model penta-peptide thioacids could be obtained cleanly at room temperature in up to % overall yield based on initial coupling. these peptides were then further ligated with electron deficient sulfonyl azide functionalized peptides.larger peptide thioacids could also be obtained using this protocol. a mer derivative of penetratin- , a cell-penetrating peptide from the third helix of the homeodomain of the antennapedia protein, was prepared as a peptide thioacid in a % yield (based on coupling of the first amino acid). in this report, a sensitive, selective and rapid uplc-ms method was developed for the determination of the [lys-gly] -mog - peptide in order to control the conjugation of mannan with the [lys-gly] -mog - peptide. the separation was performed on an acquity uplc system with a beh c column packed with . μm particles. the total run time was min. calibration curve based on peak area ratio was linear at the concentration range of - μg/ml, with a detection limit of μg/ml. the method showed satisfactory reproducibility and confirmed the entire conjugation between oxidized mannan and peptide sequence. the development of simple, low-cost and fast methods for protein purification is of increasing importance both for academic and industrial applications. a very promising approach is inverse transition cycling (itc) that exploits the temperature dependent aggregation properties of elps. elastin-like polypeptides (elp) are artificial polypeptides composed of pentameric repeats (val-pro-gly-xaa-gly) derived from mammalian elastin. elps are characterized by a specific transition temperature (t t ) that depends on the amino acid composition of the pentarepeat; they are water-soluble below and aggregate reversibly above this narrow temperature range (t t ). these properties are transferred to target proteins by n-or cterminal fusion with elps. during itc these fusion proteins precipitate, while other components remain in solution. repeated cycles of heating and cooling allow simple recovery of the target protein.we synthesized various elps consisting of to pentameric repeats and including different guest residues. the transition temperature of all synthetic elps was determined using photometric assays and measuring turbidity. in order to test if elp properties can be efficiently transferred, we fused elp to a small recombinant protein (ras-binding domain, rbd) by expressed protein ligation. this approach will allow the incorporation of elps with unnatural amino acids and other chemical modifications into target proteins. currently we are focusing on biotechnologically relevant enzymes that constitute a major cost factor in industrial processes. the authors thank süd-chemie/clariant for their financial support. department of pharmacy, university of patras, rio, greece peptides penetrating the cell membrane, known as cell penetrating peptides (cpps), as well as their mimics, used as delivery agents to cells have been reported , . cpps can be natural sequences or artificial constructs designed to capture the features of natural formations. cpps are particularly important in the delivery of peptides, proteins, nucleic acids, small molecule drugs or imaging agents. incorporation of a heterocyclic motif into a peptide or peptide-like backbone introduces conformational constraints and/or latent reactivity related to the heterocycle's structural profile. heterocycle-based cpp mimics are, thus, promising candidates for therapeutics protected synthetic non-ionic peptides, which are for example synthetic intermediates for the production of api's, are often very hydrophobic and not soluble in most common solvents. they are thus difficult to purify by preparative rp-hplc, classically used for industrial production. it is then challenging to develop alternative purification chromatographic processes using suitable solvents and providing good yields, high purity and sufficient productivity. the technique of support free liquid-liquid chromatography , including both its hydrostatic (centrifugal partition chromatography or cpc) and its hydrodynamic (counter-current chromatography or ccc) declensions, are mainly involved in phytochemical studies but has also been applied to peptide purification . the previously developed biphasic solvent systems are not adapted to the purification of highly hydrophobic protected peptides. to overcome this problem, two new scales of biphasic solvents systems and a ternary biphasic solvent system were developed to overcome solubility problems often encountered with those peptides. the new systems composed of heptane/thf/ch n/dmso/water, heptane/me-thf/nmp/water, and cmpe/dmf/water were efficiently used for the cpc purification of a mer protected exenatide and a mer protected peptide intermediate of bivalirudin synthesis. the developed scales show a wide range of polarity and should be useful for general use in cpc for the separation of hydrophobic synthetic free or protected peptides. the progressive aggregation of β-amyloid peptide (β-ap) into insoluble amyloid fibrils ultimately leading to formation of toxic amyloid plaques is widely considered to be the central pathogenic cause of alzheimer's disease. in the last decade accumulating evidence suggests that soluble oligomeric non-fibrillar forms of β-ap are neurotoxic as well. consequently, inhibiting the aggregation of β-ap is one of the therapeutic strategies against alzheimer's disease and a number of small molecules have been identified as inhibitors of β-ap aggregation and neurotoxicity. among these, curcumin, the phenolic yellow pigment and active ingredient of the turmeric herb, is receiving special attention because of its rich pharmacology that includes in vitro and in vivo inhibitory action against alzheimer's disease insults. in the current work the interaction of β-ap( - ) with curcumin is investigated with fluorescence, cd, and nmr spectroscopies in water and water-methanol mixtures and at various β-ap( - ):curcumin ratios. in nmr studies in % methanol curcumin behaves like a macromolecular species with a change in the sign of its noe signal providing direct indication of its association with β-ap( - ). in % methanol the presence of β-ap( - ) results in great broadening of the h peaks of curcumin, indicative of a complete change in its solution state. additionally, the fluorescence of curcumin in % methanol shows a blue shift with enhanced intensity, observations consistent with a hydrophobic modification of curcumin environment upon interaction with β-ap. finally, in water the induced circular dichroism spectrum of curcumin in the near uv region provides clear evidence for the loss of symmetry of curcumin molecule due to changes in its microenvironment generated by interaction with β-ap( - ). our experimental findings support the direct interaction of β-ap( - ) with curcumin and establish its importance as a potential aggregation inhibitor of β-ap. [ ] . based on its sequence, we synthesized h-tyr-d-trp-nh- -ada ( -adamantane) (yo- ) and h-tyr-d-trp-nh- -ada ( -adamantane) (yo- ) and reported they had potent antiproliferative activity on cancer cells (a- and sw ), which were comparable to tt- and cycloheximide. a structure-activity relationship analysis revealed that lipophylicity of yo- and - could be responsible for their antiproliferative activity. now, we described the substitution of tyr of yo- and - by tyr(bzl), phe, -nal( -naphthylalanine), -nal ( -naphthyalanine) and the anticancer and dna polymerase inhibitory activities in order to explore the effect of hydrophobic substituent. among the compounds, yo- and - had the highest lipophilicity judging from their retention time and lipophilicity index (yo- : . min, . ; yo- : . min, . ). yo- and - exhibited strong dna polymerase inhibitory activity as well as antifroliferative activity on hct cells at m. these activities were greater than those of yo- and - . antiproliferative activity of the compounds containing -ada such as yo- , - , - , - and - , was comparable to that of the compounds containing -ada such as yo- , - , - , - and - . these findings suggest that the lipophilicity well correlates with dna polymerase inhibitory activity and antiproliferative activity on hct cells. further structureactivity relationship study is progressing in our group. multiple sclerosis (ms) is a chronic autoimmune disease of the central nervous system (cns) , . our aim was to immunologically control the attack of the myelin sheath in ms patients without the total suppression of the immune system. anthraquinones (mitoxantrone, ametantrone) are widely used in cancer therapy as immunosuppressants.mitoxantrone is also used to treat several forms of advancing ms, including secondary progressive ms, progressive relapsing ms, and advanced relapsingremitting ms . more specifically, mitoxantrone is an inhibitor of the type ii topoisomerase, which disrupts dna synthesis and dna repair in both healthy cells and cancer cells. herein, we report the synthesis of an anthraquinone type compound conjugated to the immunodominant - myelin oligodendrocyte glycoprotein (mog - ) for the selective immunosuppression of the encephalitogenic t cells in ms patients. the anthraquinone was synthesized by a friedel-crafts acylation of hydroquinone from phthalic anhydride, followed by reduction of the resulted quinizarine to its leuco form, addition of the appropriate diamine and air oxidation . the synthesized molecules were purified using liquid chromatography, and they were identified by mass spectrometry and h-nmr. the synthesis of the mog - was performed under microwave irradiation and its conjugation with the anthraquinone was performed in solution. the final analogue was purified by rp-hplc and identified by esi-ms. benzopyrans, diketopiperazines and , benzodiazepin- , -diones are well-known and widely investigated scaffolds, e.g. the latter showing anxiolytic and antiarrhythmic effects. now, we propose a new potential "privileged structure" containing a triazole moiety mimicking the cis-amide bond within the , -benzodiazepin- , -dione motif .molecules based on this [ , , ]-triazolo [ , -d] benzo- , diazepin- -one scaffold are synthesized and decorated via a modular approach on wang resin using α-amino acids, -ethynylaniline building blocks and n-alkylating agents resulting in five points of diversity. the methodology involves the attachment of α-amino acids onto a solid support, subsequent removal of the fmoc group followed by an optimized diazotransfer reaction of the resulting amine yielding a resin-bound azide. conversion of the latter into a , -disubstituted , , -triazole moiety is achieved quantitatively by addition of a range of -ethynylaniline building blocks using a ru(ii)-catalyst. the desired scaffold can be obtained in high crude purities (> %) in solution via an acid catalyzed one-step cyclisation-release strategy. solution-phase n-alkylation finally affords the fully diversified scaffold. interestingly, n-alkylation induces atropisomeric effects which can be studied via h nmr spectroscopy.taking into account future screening results of the synthesized libraries, a well-thought decoration of this scaffold leading to discovery of new lead molecules is within reach. peptide symposium in the wonderful small seaside town of porto carras. maurice manning and lajos balaśpiri were nominated as captains of the two teams, the rest of the world and europe. similar matches were organized at subsequent european peptide symposia. now, in greece, the two captains would like to hand over their roles to younger scientists [professors gabor mezö(hungary) and laśzlóÖtvös (usa)] to continue this tradition at the coming european and possibly american peptide symposia. it seems best to play in the free time (in the evenings after the excursions). necessary conditions: good weather; a nice large soccer field; a soccer ball, preferably new; and jerseys and shorts (different colours), organized as always by the organizer commettee. the captain of the winning team will receive a trophy at the end of the nd european peptide symposium. the teams will remember two earlier excellent referees: professor lajos kisfaludy in porto carras [ ] and the soccer professor + ferenc puskaś (hungary) in budapest ( ). in the poster session, the results from the past years will be presented in about - pictures. these pictures may possibly be bought free, % can be saved at the poster session. all participants are welcome at the new party in athens. conclusion will be presented by the players and fans in athens. the human lactoferrin-derived peptide, hlf - , was proven to be highly active against antibiotic-resistant bacteria . however, the clinical use of this antimicrobial peptide (amps) is hampered by the peptide low stability due to fast degradation or to peptide aggregation, as the use of higher peptide concentrations results on higher toxicity levels. amp immobilization onto a biomaterial surface could be the pathway to overcome these difficulties . the aim of this work is the development of an antimicrobial surface by covalent immobilization of hlf - onto the surface of chitosan thin films. chitosan ultrathin films were prepared through the spincoating of a . % chitosan solution in gold substrates. hlf - immobilization was performed through an ss bound between hlf - terminal cysteine and an n-acetyl cysteine previously coupled at chitosan films. surfaces were characterized using ellipsometry (thickness), infrared reflection absorption spectroscopy (irras) and x-ray photoelectron spectroscopy (xps). bacterial adhesion studies were performed using methicillin-resistant s. aureus (atcc ). chitosan films were incubated with this bacterial suspension at ºc for h and h. the viability of the attached bacteria was evaluated using live/dead® bacterial viability kit (baclight tm ) and fluorescence microscopy. hlf - peptide was successfully covalently immobilized onto chitosan thin films. both soluble and attached peptide presented a higher antimicrobial activity than the control chitosan. identified as a potent vasoconstrictor that binds with high affinity to ut receptor. the cysteine-linked cyclic region, hut-ii( - ), is responsible for the biological activity and has been widely used to elucidate the structure-activity relationship of hut-ii. with the aim to investigate the role of hydrogen bond and the effects of a peptide backbone constraint on binding key: cord- -tw cehxv authors: ferrario, carlos m. title: commentary on “angiotensin-converting enzyme inhibitors and angiotensin receptor blockers may be harmful in patients with diabetes during covid- pandemic” date: - - journal: diabetes metab syndr doi: . /j.dsx. . . sha: doc_id: cord_uid: tw cehxv nan dear sir: i wish to clarify the accuracy of certain conclusions advanced in a recently published commentary by cure and cure [ ] . in their interesting article which outlines concerns regarding the potential adverse evolution of sars-cov- infection in diabetic patients medicated with blockers of the renin angiotensin system (ras), it is incorrectly stated that angiotensin converting enzyme (ace ; ec . . . ) hydrolyzes angiotensin-( - ) [ang-( - )] into angiotensin-( - ) [ang-( - )]. despite a % sequence similarity with its homologue angiotensin converting enzyme (ace; ec . . . ), ace functions as a mono-carboxypeptidase cleaving one single amino acid sequentially at the c-terminal amino acid from susceptibles substrates [ ] . as shown in figure , ace cleaves the penultimate amino acid in angiotensin i (ang i) to generate ang-( - ). the nonapeptide is then processed into ang-( - ) by ace. in contrast, ace, functioning as a dipeptidyl carboxypeptidase cleaves the proline (pro )-phenylalanine (phe ) bond of ang ii to liberate ang-( - ). the second point that needs correction is the suggestion that angiotensin receptor blockers (arbs) cause a reduction in ang ii levels (plasma, tissue?) in part by upregulation of ace gene transcription and increased enzymatic activity [ ] . while original studies from my laboratory first documented increased cardiac [ , ] and vascular [ , ] ace mrna and ace activity in response to administration of arbs, the effect was associated with increased, not decreased, plasma ang ii. the increase in ang ii in response to arb administration is the result of blockade of at -r and consequent prevention of the uptake of the peptide into the cell. multiple studies have confirmed ang-( - ) antihypertensive and reno protective actions in adults treated with ras inhibitors [ , ] since we first reported ang-( - ) vasodilator response in the areflexic rat [ ] . lastly, a similar robust literature has documented that ang-( - ) leads to peripheral sympathetic nerve activity tone-down via its actions in the central nervous system [ ] . we appreciate the opportunity to clarify these issues without negating the interesting points raised by the authors of the above referred commentary. angiotensin-converting enzyme inhibitors and angiotensin receptor blockers may be harmful in patients with diabetes during covid- pandemic hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensinconverting enzyme effect of angiotensin ii blockade on a new congenic model of hypertension derived from transgenic ren- rats increased expression of angiotensin converting enzyme in conjunction with reduction of neointima by angiotensin ii type receptor blockade angiotensin ii at receptors regulate ace and angiotensin-( - ) expression in the aorta of spontaneously hypertensive rats role of the vasodilator peptide angiotensin-( - ) in cardiovascular drug therapy. vasc health risk manag characterization of angiotensin-( - ) in the urine of normal and essential hypertensive subjects cardiovascular actions of angiotensin( - ) the ace /angiotensin-( - )/mas axis of the renin-angiotensin system: focus on angiotensin the research studies reported in this letter to the editor from our laboratory have been conducted with grant hl- from the national heart and lung institute (nhlbi) of the national institutes of health (nih). the author declares no conflict of interest. key: cord- -ounktxxv authors: varagic, jasmina; trask, aaron j.; jessup, jewell a.; chappell, mark c.; ferrario, carlos m. title: new angiotensins date: - - journal: j mol med (berl) doi: . /s - - - sha: doc_id: cord_uid: ounktxxv accumulation of a large body of evidence during the past two decades testifies to the complexity of the renin–angiotensin system (ras). the incorporation of novel enzymatic pathways, resulting peptides, and their corresponding receptors into the biochemical cascade of the ras provides a better understanding of its role in the regulation of cardiovascular and renal function. hence, in recent years, it became apparent that the balance between the two opposing effector peptides, angiotensin ii and angiotensin-( - ), may have a pivotal role in determining different cardiovascular pathophysiologies. furthermore, our recent studies provide evidence for the functional relevance of a newly discovered rat peptide, containing two additional amino acid residues compared to angiotensin i, first defined as proangiotensin- [angiotensin-( - )]. this review focuses on angiotensin-( - ) and its important contribution to cardiovascular function and growth, while introducing angiotensin-( - ) as a potential novel angiotensin precursor. it seemed for a long time that all components of the reninangiotensin system (ras) and their physiological roles were well defined. in this traditional view, the ras is viewed as a classical endocrine system with the octapeptide angiotensin ii (ang ii; asp -arg -val -tyr -ile -his -pro -phe ) as an effector hormone expressing its vasoconstrictor, sodium retention, mitogenic, and proliferative effects upon its binding to ang ii type receptors (at ). renin and angiotensin converting enzyme (ace) were thought to be the only enzymes responsible for angiotensin i (ang i; asp -arg -val -tyr -ile -his -pro -phe -his -leu ) and ang ii synthesis, respectively. however, over the last two decades, increasing evidence has accumulated that indicates an exceeding complexity of the system, particularly in tissues such as the heart and kidneys. the evidence for a fully operational ras in local tissues with tissue-specific enzymatic pathways for the processing of ang i and ang ii has been detailed in a number of publications from this laboratory [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, the pleiotropic actions of the resulting fragment of ang i or ang ii, the heptapeptide ang-( - ), have been gradually appreciated over the last decade. in general, ang-( - ) [asp -arg -val -tyr -ile -his -pro ] counterbalances biological actions of ang ii, and in that way, an inadequate balance between these two peptides may determine different cardiovascular pathophysiological states. interestingly, the spectrum of novel peptides within ras continues to expand showing that a peptide containing two amino acids more than ang i, the dodecapeptide angiotensin- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) [ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ; rat sequence: asp -arg -val -tyr -ile -his -pro -phe -his -leu -leu -tyr ], could also be a key player in the regulation of cardiovascular function. this review will therefore focus on ang-( - ), its important contribution to cardiovascular function and growth, while introducing ang-( - ) as a potential novel angiotensin precursor. biochemical pathways for ang-( - ) synthesis and degradation ang-( - ) may be derived from either ang i or ang ii (fig. ) . different tissue-specific endopeptidases, including neprilysin (nep), thimet oligopeptidase (top), and prolyl oligopeptidase (pop) catalyze the hydrolysis of the decapeptide ang-i at the pro -phe bond to release the three terminal amino acids and ang-( - ) [ ] [ ] [ ] . both pop [ ] and top [ ] have been reported by us to mediate ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) formation in cultures of vascular endothelial and smooth muscle cells. neprilysin has been shown to be particularly abundant in the kidney [ ] . importantly, as nep is a membrane-bound enzyme, its localization on the luminal side of the endothelium makes it accountable for most of the ang-( - ) production in the circulation [ ] [ ] [ ] . neprilysin degrades vasodilatory atrial natriuretic peptide as well, but its high substrate specificity for ang-( - ) formation in hypertensive humans may explain, at least in part, the lack of significant beneficial effects of its inhibitors in the treatment of hypertension [ ] . however, neprilysin also degrades ang-( - ) into ang-( - ) [ ] , and further studies are necessary to clarify its role in hypertensive disease. it has been only recently that direct conversion of ang ii into ang-( - ) by a newly discovered homolog of ace, angiotensin converting enzyme (ace ), was demonstrated ( fig. ) [ , ] . as a carboxypeptidase, ace also mediates the conversion of ang i into ang-( - ), which can be further metabolized into ang-( - ) by ace. however, the higher substrate preference of ace towards ang ii than ang i underscores the significance of this enzyme in the regulation of tissue ang ii/ang-( - ) balance [ , ] . consequently, higher cardiac ang ii level was associated with genetic deletion of ace in mice and resulted in the development of severe cardiac dysfunction [ ] . on the other hand, local ace overexpression by systemic lentiviral delivery was followed by an attenuation of cardiac remodeling in hypertensive rats [ ] . furthermore, a recent report from our laboratory showed that the hypertensive heart predominantly depends on ace for the production of ang-( - ) [ ] . together with evidence for increased ace expression in failing human [ ] and rat [ ] hearts, our study suggests a preserved compensatory response of injured hearts to maintain ang-( - ) levels even in the advanced stage of the disease, although it was obviously not sufficient to counteract the deleterious effects of ang ii. besides breaking down bradykinin and acetyl-ser-asp-lys-pro, ace hydrolyzes ang-( - ) as well. it acts upon the ile -his bond to form the inactive metabolite ang-( - ) [ ] [ ] [ ] , and ace inhibitors increase the short half-life of ang-( - ) in the circulation [ ] . on the other hand, neprilysin hydrolysis of the tyr -ile bond of ang-( - ) to form ang-( - ) seems to be the predominant pathway for ang-( - ) metabolism in the kidney [ , [ ] [ ] [ ] . ang-( - ) receptor and signaling mechanisms prior to the identification of a specific ang-( - ) receptor, a modified form of ang-( - ), d-ala -ang-( - ) was designed as a selective antagonist for the putative ang-( - ) receptor. thus, d-ala -ang-( - ) inhibited ang-( - )-induced systemic and renal vasodilation, did not block pressor or contractile response to ang-ii, and did not compete for binding of i-ang ii to rat adrenal at or at receptors [ ] . subsequent studies from our group identified specific non-at /at ang-( - ) binding sites on bovine aortic endothelial cells [ ] and endothelium of coronary artery rings [ ] that were selectively competed by d-ala -ang-( - ). this finding was in agreement with nitric oxide (no) release from bovine aortic endothelial cells stimulated by ang-( - ) that was blocked by d-ala -ang-( - ) [ ] . it was also consistent with previously demonstrated ang-( - )-induced vasodilation of endothelium-intact coronary arteries through release of kinins and no [ , ] . however, it was not before the discovery that endothelium-mediated vasodilation by ang-( - ) was abolished in mas-knockout mice that the "orphan" mas proto-oncogene receptor was linked to the intracellular signaling of ang-( - ) [ ] [ ] [ ] . more recent studies revealed that ang-( - ), acting on this g protein-coupled receptor, activated endothelial nitric oxide synthase and no production via aktdependent pathways [ ] . furthermore, we showed recently [ ] that the presence of an antisense probe directed against mas abolished the ang-( - )-induced inhibition of protein synthesis in cardiomyocytes [ ] . this study also revealed that ang-( - ) decreased serum-stimulated erk /erk mitogen-activated protein kinase activity, a response that was blocked by d-ala -ang-( - ). these findings agree with the observation that genetic deletion of mas elicits cardiac dysfunction [ , ] . thus, it is clear that a reduction in the counterbalancing arm of the reninangiotensin system via the ace /ang-( - )/mas axis may have a major influence in determining cardiac structural and functional development [ , , ] . in addition, a recent report suggests another ang-( - ) receptor subtype sensitive to the ang-( - ) antagonist [d-pro ]-ang-( - ) but not d-ala -ang-( - ) [ ] . this finding, as well as an intriguing interaction between at and mas [ , ] , clearly warrants further investigation. cardiovascular and renal effects of ang-( - ) a series of studies after our initial characterization of ang-( - ) actions in brain [ ] established the basis for exploring the systemic and regional vasodilatory and hypotensive effects of this peptide [ , , ] . in these studies, it was demonstrated that the vasodilator effect of ang-( - ) was mediated through different vasoactive autocoid release [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . moreover, it was also shown that ang-( - ) potentiated bradykinin vasodilatory action [ , ] and that this interaction was exaggerated after ace inhibition. although the precise mechanisms of this potentiation remains controversial [ , ] , data suggest that the release of prostaglandins, no, endothelium-derived hyperpolarizing factor [ ] [ ] [ ] as well as the ability of ang-( - ) to inhibit ace activity [ , , ] may be involved. early studies from our laboratory strongly suggested that ang-( - ) may represent an intrinsic counterbalancing factor to the pressor and trophic actions of ang ii [ ] . this unique concept was confirmed in the experiments in which hyperreninemia was stimulated through induction of renovascular hypertension [ ] or a low-salt diet [ ] . despite increased levels of ang ii during salt depletion, blood pressure remained unchanged, at least in part, due to the opposing actions of ang-( - ). indeed, ang-( - ) blockade by either the selective ang-( - ) receptor antagonist d-ala -ang-( - ) or specific ang-( - ) antibodies caused a dose-dependent increase in arterial pressure in salt-restricted rats [ ] , underscoring the importance of ang-( - ) in counterbalancing the effects of ang ii. the significance of the alternative arm of the ras comprising ace , ang-( - ), and mas in blood pressure regulation was further underscored by the demonstration of a considerable contribution of ang-( - ) to the hypotensive effects of ras blockade [ , , , [ ] [ ] [ ] . importantly, chronic antihypertensive effects of captopril or omapatrilat in hypertensive patients were also associated with increased urinary levels of ang-( - ) [ , ] . the importance of this observation was magnified by the concurrent observation that plasma and urinary excretion levels of ang-( - ) are reduced in untreated essential hypertensive subjects [ ] . more recently, we showed that chronic administration of irbesartan to normotensive subjects was associated with large increases in plasma ang-( - ) [ , ] . these results suggest an important contribution of ang-( - ) in mediating the antihypertensive effects of both ace inhibitors and at receptor antagonists. it was then in our laboratory that the effects of ras blockade on the ang-( - )-forming enzyme, ace , were evaluated for the first time [ ] . from the preceding study, we knew that heart failure due to coronary artery ligation was associated with compensatory increase in cardiac ang-( - ) levels [ ] . it was in this experimental model that we subsequently showed that at receptor antagonism further augmented plasma ang-( - )/ang ii ratio suggesting increased formation of ang-( - ) from ang ii [ ] . indeed, at receptor antagonism attenuated cardiac remodeling and dysfunction, and these changes were associated with a threefold increase in ace mrna expression in the left ventricle. the changes in the cardiac ace gene activity and the profile of plasma angiotensin peptides after ras inhibition were confirmed in following experiments including different strains of normotensive and hypertensive animals [ ] [ ] [ ] . the pathophysiological relevance of ang-( - ) in the heart was further highlighted by studies demonstrating that chronic infusion of either ang-( - ) [ ] or its stable non-peptide analog ave- [ ] was cardioprotective in experimental heart failure. finally, several studies demonstrated that ang-( - ) was protective against cardiac ischemia-induced injury and arrhythmias [ ] [ ] [ ] . the beneficial antiarrhythmic effects of ang ( - ) on the failing heart result from the combined effect of the peptide on the sodium pump, hyperpolarization of cardiac cell membranes, and increased conduction velocity [ ] . however, in isolated hearts, supra-pharmacological concentrations of ang-( - ) enhanced reperfusion arrhythmias [ ] . we also showed that ang-( - ) at higher concentrations ( − m), induces early-after depolarization [ ] ; therefore, an optimal tissue concentration of ang ( - ) must be achieved to permit a protective role of the heptapeptide on cardiac arrhythmias. numerous studies indicate that the ang-( - ) effects on the kidney are opposite to those of ang ii. thus, ang-( - ) infusion induced vasodilation of pre-constricted afferent arterioles [ ] , increased glomerular filtration rate, and induced natriuresis and diuresis [ ] [ ] [ ] by inhibiting the na + -k + -atpase [ ] . these vascular and tubular effects were attenuated by the selective ang-( - ) antagonist d-ala -ang-( - ). interestingly, these counterbalancing effects of ang-( - ) were noticeable under conditions of ras activation, such as during salt depletion or renal hypertension, but not in the salt-replete state [ ] . ang-( - ) functions in the brain ang-( - ) is present in brain tissue, and its distribution throughout the hypothalamus, medulla oblongata, and amygdala underlines its importance in the regulation of blood pressure, fluid balance, and osmoregulation [ ] . although the action of ang-( - ) in the brain sometimes mimics the action of ang ii, such as stimulation of vasopressin release [ ] , their overall effects are in general opposite. the involvement of different receptors, neurotransmitter pathways, and complex integrative regulatory brain mechanisms implicated in the action of the two angiotensins have been already reviewed elsewhere [ ] . in brief, intracerebroventricular administration of an ang-( - ) antibody elevated arterial pressure, while endogenous neutralization of ang ii had an opposite effect [ ] . ang-( - ) at the nucleus of the solitary tract evoked bradycardic and depressor response [ ] , augmented baroreceptor reflex control of heart rate [ ] [ ] [ ] , and these effects were enhanced in hypertensive animals when compared to the controls [ , ] . in the rostral ventrolateral medulla, ang-( - ) elicited pressor responses [ ] ; however, in the caudal ventrolateral medulla, ang-( - ) lowered arterial pressure by inhibiting the pressor action of the rostral ventrolateral medulla [ , ] . more unexpected actions of ang-( - ) include its ability to enhance long-term potentiation, a process thought to be involved in learning and memory [ ] . ang-( - ) relevance in cardiovascular and cancerous growth similar to their actions in the circulation and the control of blood pressure, ang ii and ang-( - ) elicit opposing effects on tissue growth as well. ang-( - ) inhibited proliferation of aortic vascular smooth muscle cells in culture [ ] , and this antiproliferative effect was later confirmed in in vivo studies. indeed, ang-( - ) infusion reduced neointimal proliferation after vascular injury in rat carotid arteries [ ] . recent reports demonstrated that ang-( - ) also inhibited ang ii-induced protein synthesis in neonatal cardiomyocytes by activating mas [ ] . consistently, ang-( - ) infusion reduced myocyte surface area in rats subjected to coronary artery ligation [ ] . these results are in keeping with the beneficial effects of ras blockade on cardiac remodeling and dysfunction after myocardial infarction where activation of ace /ang-( - ) system has been verified [ ] . in addition, ang-( - ) inhibited collagen synthesis in adult rat cardiac fibroblasts acting on receptors that are distinct from the at and at receptors [ ] . subsequent studies confirmed that ang-( - ) prevented an excessive accumulation of cardiac collagen fibrils in different models of experimental hypertension [ , ] . excitingly, the antiproliferative and antiangiogenic ability [ ] of ang- ( - ) found an important application in inhibiting cancerous growth as well. thus, experimental evidence that ang-( - ) inhibited lung [ ] and breast cancer growth in vitro [ ] as well as in vivo [ ] now provides a solid foundation for the initiation of clinical trials in which the chemotherapeutic potential of ang-( - ) is being tested. ang-( - ) in pregnancy all components of the ras are expressed in placenta including ang-( - ) and ace [ ] , and activation of the ras during normal pregnancy has been described in plasma and urine [ , ] . adequate balance between the two opposing arms of ras might be of extreme importance in normal pregnancy, as the predominance or deficit of either one might lead toward adverse outcomes. for example, unopposed antiangiogenic properties of ang-( - ) may have a harmful effect, particularly in early pregnancy during which vascularization of tissue beds is critical. on the other hand, decreased plasma levels of ang-( - ) were associated with preeclamptic pregnancies characterized by elevated arterial pressure and proteinuria [ ] . to further confirm this relationship, the most recent study from our group related an experimental model of preeclampsia with failure to increase ang-( - ) in kidney as well [ ] . further expansion of the complexity of the reninangiotensin system: angiotensin- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in line with expanding data on the newer angiotensin peptide, ang-( - ), nagata and colleagues [ ] recently identified another new angiotensin peptide, the dodecapeptide ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the authors were probing for analogs of ang ii when they discovered an unidentified immunoreactive peak by high-performance liquid chromatography (hplc), which the authors found to be a -amino acid derivative of angiotensinogen, two amino acids larger than the traditional intermediate peptide ang i. the dodecapeptide produced pressor responses both in isolated rat aorta and acutely in intact wistar rats-a finding that was abrogated by coadministration of both an ace inhibitor or an angiotensin receptor blocker (arb). these data suggested that "proangiotensin- ," as the authors named it, was exerting its actions through rapid metabolism into ang ii. recent data from our laboratory provided further evidence for a biological role of ang-( - ) as a new endogenous peptide of the ras. because ang-( - ) was identified endogenously by ria in different organs and tissues [ ] (fig. ) , we first undertook studies that investigated the immunolocalization of the dodecapeptide in the hearts and kidneys of normal wistar-kyoto (wky) and spontaneously hypertensive rats (shr). jessup et al. [ ] found that ang-( - ) was localized by immunohistochemistry predominantly in cardiac myocytes, while staining in the medial and endothelial layers of the coronary arteries appeared more faint (fig. ) and failed to be detected in all vessels examined. the distribution of ang-( - ) within the hearts of shr was more robust than that found in wky. this observation was confirmed by tissue content analysis, which revealed significantly higher levels of cardiac ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in shr compared to wky. renal ang-( - ) was localized to the proximal and distal tubules and the collecting duct, but it was scantily observed in glomeruli or intra-renal vessels. these data, in accordance with those from nagata and colleagues [ ] show that ang-( - ) is indeed localized endogenously within tissues, and the distribution of the new angiotensin peptide may reflect the state of the health of that tissue, as shown by differences in distribution between wky and shr. further enhancements towards the understanding for a biological role for ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) were made by studies from our laboratory which illustrated the metabolic capacity for ang-( - ) to yield known downstream bioactive angiotensin peptides. intriguingly, chappell et al. [ ] found that serum exclusively formed ang ii from ang-( - ) by ace, and renal neprilysin activity converted ang-( - ) to ang-( - ). both of these pathways were independent of renin activity. moreover, we [ ] showed that ang-( - ) could be metabolized into ang i, ang ii, and ang-( - ) in isolated hearts from five different normotensive and hypertensive rat strains. collectively, these data provide strong evidence that ang-( - ) may be an alternate precursor substrate for the formation of bioactive angiotensin peptides in the heart, kidney, and circulation that may depend on the localization of one of its processing enzymes, ace, but not renin. in conclusion, a large body of evidence emerging from experimental and human studies clearly reveals pathophysiological importance of novel peptides and related enzymes incorporated recently into the biochemical ras cascade. the consequences of an altered ace /ang-( - )/mas axis in hypertensive disease and heart failure is now well recognized. in addition to the potential therapeutic application in the treatment of cardiovascular disease, the ace / ang-( - )/mas axis emerges further as a prospective therapeutic target in cancer and preeclamptic patients. thus, there is a great potential for genetic and pharmacological modulation of the ace /ang-( - )/mas axis in the treatment of various diseases that, however, warrants further meticulous investigation. future studies will certainly provide us with better understanding of the relevance ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in the heart of wky and shr rats. note the more robust distribution of ang-( - ) within the hearts of shr than that assessed in wky. this observation was confirmed by tissue content analysis, which revealed significantly higher levels of cardiac ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in shr compared to wky. adapted from jessup et al. [ ] of ace function as a receptor for severe acute respiratory syndrome (sars) virus. they will also expand our comprehension of the signaling mechanisms and a potential angiotensin receptor interaction. building upon the complexity unveiled thus far on the ras as documented by data on the ace /ang-( - )/mas axis, the demonstration of endogenous ang-( - ) is indeed novel and important. although the data are still limited to the rat, forthcoming research may provide insight onto anomalies within ras physiology that we cannot yet explain. conceptually, ang-( - ) may serve as an alternate substrate for the production of bioactive angiotensin peptides as shown in our preliminary studies. moreover, in lieu of the specific sequence requirements for the generation of ang i by renin, ang-( - ) may be formed directly from angiotensinogen in a renin-independent manner. in support of this notion, oparil and colleagues [ ] found that coadministration of aliskiren and the arb valsartan produced additive reduction in blood pressure in patients when compared to each drug administered alonea finding not expected if renin is the limiting step in the formation of angiotensin peptides from angiotensinogen. identification of a biological role for ang-( - ) requires further work; however, expression of the peptide throughout the body argues strongly that this angiotensin intermediate may add further unrecognized complexity to the renin-angiotensin system. counterregulatory actions of angiotensin angiotensin-( - ) and antihypertensive mechanisms angiotensin-( - ): a bioactive fragment of the renin-angiotensin system angiotensin-( - ). its contributions to arterial pressure control mechanisms advances in biochemical and functional roles of angiotensin-converting enzyme and angiotensin-( - ) in regulation of cardiovascular function angiotensin-converting enzyme and angiotensin-( - ): an evolving story in cardiovascular regulation conversion of angiotensin i to angiotensin-( - ) by thimet oligopeptidase (ec . . . ) in vascular smooth muscle cells evidence that prolyl endopeptidase participates in the processing of brain angiotensin a comparison of the properties, and enzymatic activity of three angiotensin processing enzymes: angiotensin converting enzyme, prolyl endopeptidase and neutral endopeptidase . production of angiotensin-( - ) by human vascular endothelium exploring the structure and function of zinc metallopeptidases: old enzymes and new discoveries effects of neutral endopeptidase inhibition and combined angiotensin converting enzyme and neutral endopeptidase inhibition on angiotensin and bradykinin peptides in rats release of angiotensin-( - ) from the rat hindlimb: influence of angiotensin-converting enzyme inhibition angiotensin-( - ) contributes to the antihypertensive effects of blockade of the renin-angiotensin system neutral endopeptidase versus angiotensin converting enzyme inhibition in essential hypertension evaluation of angiotensin-converting enzyme (ace), its homologue ace and neprilysin in angiotensin peptide metabolism hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase angiotensinconverting enzyme is an essential regulator of heart function protection from angiotensin ii-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ace in rats primary role of angiotensin-converting enzyme- in cardiac production of angiotensin-( - ) in transgenic ren- hypertensive rats - )-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme homologue ace augmented ace expression in ischemic cardiomyopathy - ) metabolism in pulmonary and renal tissues metabolism of angiotensin-( - ) by angiotensin converting enzyme an n-domain specific substrate and c-domain specific inhibitor of angiotensin converting enzyme: angiotensin-( - ) and keto-ace converting enzyme determines the plasma clearance of angiotensin-( - ) pathways of angiotensin-( - ) metabolism in the kidney effects of omapatrilat on the renin angiotensin system in salt sensitive hypertension vasopeptidase inhibition and ang-( - ) in the spontaneously hypertensive rat characterization of a new angiotensin antagonist selective for angiotensin-( - ): evidence that the actions of angiotensin -( - ) are mediated by specific angiotensin receptors bovine aortic endothelial cells contain an angiotensin-( - ) receptor angiotensin-( - )-stimulated nitric oxide and superoxide release from endothelial cells angiotensin-( - ) dilates canine coronary arteries through kinins and nitric oxide release of nitric oxide by angiotensin-( - ) from porcine coronary endothelium: implications for a novel angiotensin receptor angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas evidence for a functional interaction of the angiotensin-( - ) receptor mas with at and at receptors in the mouse heart impairment of in vitro and in vivo heart function in angiotensin-( - ) receptor mas knockout mice angiotensin-( - ) through receptor mas mediates endothelial nitric oxide synthase activation via akt-dependent pathways angiotensin-( - ) inhibits growth of cardiac myocytes through activation of the mas receptor effects of genetic deletion of angiotensin-( - ) receptor mas on cardiac function during ischemia/reperfusion in the isolated perfused mouse heart evidence for a new angiotensin-( - ) receptor subtype in the aorta of sprague-dawley rats up-regulation of the angiotensin ii type receptor by the mas proto-oncogene is due to constitutive activation of gq/g by mas g-proteincoupled receptor mas is a physiological antagonist of the angiotensin ii type receptor release of vasopressin from the rat hypothalamo-neurohypophysial system by angiotensin-( - ) heptapeptide vasodilator action of angiotensin-( - ) on isolated rabbit afferent arterioles vasodepressor actions of angiotensin-( - ) unmasked during combined treatment with lisinopril and losartan contribution of angiotensin-( - ) to blood pressure regulation in salt-depleted hypertensive rats evidence that prostaglandins mediate the antihypertensive actions of angiotensin-( - ) during chronic blockade of the renin-angiotensin system angiotensin-( - ) augments bradykinin-induced vasodilation by competing with ace and releasing nitric oxide opposing actions of angiotensin-( - ) and angiotensin ii in the brain of transgenic hypertensive rats angiotensin-( - ) and nitric oxide interaction in renovascular hypertension characterization of angiotensin-( - ) receptor subtype in mesenteric arteries angiotensin-( - ) potentiates the hypotensive effect of bradykinin in conscious rats angiotensin-( - ) potentiates responses to bradykinin but does not change responses to angiotensin i evidence for mas-mediated bradykinin potentiation by the angiotensin-( - ) nonpeptide mimic ave in normotensive rats potentiation of bradykinin by angiotensin-( - ) on arterioles of spontaneously hypertensive rats studied in vivo angiotensin - induces bradykinin-mediated relaxation in porcine coronary artery synergistic effect of angiotensin-( - ) on bradykinin arteriolar dilation in vivo bradykinin potentiation by angiotensin-( - ) and ace inhibitors correlates with ace c-and n-domain blockade effect of angiotensin converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin converting enzyme effects of renin angiotensin system blockade on renal angiotensin-( - ) forming enzymes and receptors effect of angiotensin ii blockade on a new congenic model of hypertension derived from transgenic ren- rats effects of captopril related to increased levels of prostacyclin and angiotensin-( - ) in essential hypertension characterization of angiotensin-( - ) in the urine of normal and essential hypertensive subjects comparison of inhibitory effects of irbesartan and atorvastatin treatment on the renin angiotensin system (ras) in veins: a randomized double-blind crossover trial in healthy subjects role of the vasodilator peptide angiotensin-( - ) in cardiovascular drug therapy upregulation of angiotensin-converting enzyme after myocardial infarction by blockade of angiotensin ii receptors cardiac angiotensin-( - ) in ischemic cardiomyopathy prevention of angiotensin ii-induced cardiac remodeling by angiotensin isoproterenolinduced impairment of heart function and remodeling are attenuated by the nonpeptide angiotensin-( - ) analogue ave angiotensin ( - ) re-establishes impulse conduction in cardiac muscle during ischaemia-reperfusion. the role of the sodium pump angiotensin-( - ): cardioprotective effect in myocardial ischemia/reperfusion angiotensin-( - ) improves the post-ischemic function in isolated perfused rat hearts beneficial versus harmful effects of angiotensin ( - ) on impulse propagation and cardiac arrhythmias in the failing heart effect of angiotensin-( - ) on reperfusion arrhythmias in isolated rat hearts natriuretic action of angiotensin ( - ) effect of intrarenal infusion of angiotensin-( - ) in the dog blocks renal actions of angiotensin - in the anesthetized rat renal actions of angiotensin-( - ) in vivo and in vitro studies effects of angiotensin-( - ) blockade on renal function in rats with enhanced intrarenal ang ii activity identification of angiotensin-( - ) in rat brain: evidence for differential processing of angiotensin peptides cardiovascular effects of angiotensin-( - ) injected into the dorsal medulla of rats pressor and reflex sensitivity is altered in spontaneously hypertensive rats treated with angiotensin differential baroreceptor reflex modulation by centrally infused angiotensin peptides changes in the baroreflex control of heart rate produced by central infusion of selective angiotensin antagonists in hypertensive rats modulation of the baroreflex control of heart rate by angiotensin-( - ) at the nucleus tractus solitarii of normotensive and spontaneously hypertensive rats deficiency of endogenous angiotensin-( - ) in the nucleus tractus solitarii of (mren ) transgenic rats may account for diminished baroreceptor reflex function cardiovascular effects produced by microinjection of angiotensins and angiotensin antagonists into the ventrolateral medulla of freely moving rats the cardiovascular effects of angiotensin-( - ) in the rostral and caudal ventrolateral medulla of the rabbit does angiotensin ii have a significant tonic action on cardiovascular neurons in the rostral and caudal vlm? angiotensin-( - ) enhances ltp in the hippocampus through the g-proteincoupled receptor mas angiotensin-( - ) inhibits vascular smooth muscle cell growth angiotensin-( - ) reduces smooth muscle growth after vascular injury angiotensin-( - ) attenuates the development of heart failure after myocardial infarction in rats angiotensin-( - ) binds to specific receptors on cardiac fibroblasts to initiate antifibrotic and antitrophic effects chronic administration of angiotensin-( - ) attenuates pressure-overload left ventricular hypertrophy and fibrosis in rats opposing actions of angiotensins on angiogenesis inhibition of human lung cancer cell growth by angiotensin inhibition of human breast cancer cell growth by angiotensin-( - ) angiotensin-( - ) inhibits growth of human lung adenocarcinoma xenografts in nude mice through a reduction in cyclooxygenase- distribution of angiotensin-( - ) and ace in human placentas of normal and pathological pregnancies angiotensin-( - ) in normal and preeclamptic pregnancy urinary vasodilator and vasoconstrictor angiotensins during menstrual cycle, pregnancy, and lactation temporal-spatial expression of angiotensin-( - ) and angiotensin converting enzyme in the kidney of normal and hypertensive pregnant rats isolation and identification of proangiotensin- , a possible component of the renin-angiotensin system in the heart and kidney of hypertensive and normotensive rats distinct processing pathways for the novel peptide angiotensin-( - ) in the serum and kidney of the hypertensive mren angiotensin-( - ) is an alternate substrate for angiotensin peptide production in the heart efficacy and safety of combined use of aliskiren and valsartan in patients with hypertension: a randomised, double-blind trial angiotensin-( - ): pharmacology and new perspectives in cardiovascular treatments acknowledgments the studies summarized here were supported by awards from the national institutes of health (hl- to cmf, hl- to mcc), american heart association, mid-atlantic affiliate ( u to jv and u to ajt), and wfusm venture fund (to jv). in addition, the authors gratefully acknowledge grant support in part provided by unifi, inc., greensboro, nc, and farley-hudson foundation, jacksonville, nc. key: cord- -m tn qu authors: lambert, daniel w.; hooper, nigel m.; turner, anthony j. title: angiotensin-converting enzyme and new insights into the renin–angiotensin system date: - - journal: biochem pharmacol doi: . /j.bcp. . . sha: doc_id: cord_uid: m tn qu components of the renin–angiotensin system are well established targets for pharmacological intervention in a variety of disorders. many such therapies abrogate the effects of the hypertensive and mitogenic peptide, angiotensin ii, by antagonising its interaction with its receptor, or by inhibiting its formative enzyme, angiotensin-converting enzyme (ace). at the turn of the millennium, a homologous enzyme, termed ace , was identified which increasingly shares the limelight with its better-known homologue. in common with ace, ace is a type i transmembrane metallopeptidase; however, unlike ace, ace functions as a carboxypeptidase, cleaving a single c-terminal residue from a distinct range of substrates. one such substrate is angiotensin ii, which is hydrolysed by ace to the vasodilatory peptide angiotensin – . in this commentary we discuss the latest developments in the rapidly progressing study of the physiological and patho-physiological roles of ace allied with an overview of the current understanding of its molecular and cell biology. we also discuss parallel developments in the study of collectrin, a catalytically inactive homologue of ace with critical functions in the pancreas and kidney. angiotensin-converting enzyme (ace) is a critical regulator of the renin-angiotensin system and the target of a number of highly effective therapeutic agents used to treat cardiovascular and renal diseases. ace is a metalloproteinase which converts the inactive decapeptide angiotensin i (ang i) into the potent vasoconstrictor and mitogen angiotensin ii (ang ii), while also metabolising the hypotensive peptide bradykinin. although ace inhibitors such as captopril and lisinopril have become mainstays in the treatment of cardiovascular disease, hypertension and congestive heart failure remain leading causes of death in the developed world. due to this continuing morbidity and mortality, significant efforts have been made to identify new drug targets in the renin-angiotensin system (ras). a new member of the ras ace was identified in by two groups simultaneously using distinct methodologies [ , ] . it is an amino acid type i components of the renin-angiotensin system are well established targets for pharmacological intervention in a variety of disorders. many such therapies abrogate the effects of the hypertensive and mitogenic peptide, angiotensin ii, by antagonising its interaction with its receptor, or by inhibiting its formative enzyme, angiotensin-converting enzyme (ace). at the turn of the millennium, a homologous enzyme, termed ace , was identified which increasingly shares the limelight with its better-known homologue. in common with ace, ace is a type i transmembrane metallopeptidase; however, unlike ace, ace functions as a carboxypeptidase, cleaving a single c-terminal residue from a distinct range of substrates. one such substrate is angiotensin ii, which is hydrolysed by ace to the vasodilatory peptide angiotensin - . in this commentary we discuss the latest developments in the rapidly progressing study of the physiological and patho-physiological roles of ace allied with an overview of the current understanding of its molecular and cell biology. we also discuss parallel developments in the study of collectrin, a catalytically inactive homologue of ace with critical functions in the pancreas and kidney. # elsevier inc. all rights reserved. transmembrane glycoprotein with an extracellular catalytic domain. ace displays homology to two quite distinct proteins; its amino-terminal domain shares approximately % sequence identity with ace, whereas its cytoplasmic and transmembrane domains display % homology to collectrin, a non-catalytic protein recently shown to have a critical role in amino acid absorption in the kidney [ , ] , pancreatic beta cell proliferation [ ] and insulin exocytosis [ ] . as predicted from analyses of its peptide sequence, heterologously expressed ace localises predominantly to the plasma membrane [ ] . here, like ace and collectrin [ , ] it is subject to a juxtamembrane cleavage event (shedding) which releases the catalytically active ectodomain [ , ] . this process is stimulated by phorbol ester, an event involving a promiscuous 'sheddase', adam (or tace, tnf-a converting enzyme; fig. ) [ ] . in common with ace, the physiological role of ace ectodomain shedding remains elusive as a role for circulating ace has yet to be identified. indeed, it is possible that shedding is a mechanism to regulate ace activity at the cell surface. when expressed in polarised epithelial cells, ace is predominantly trafficked to the apical surface; this is in contrast to ace, which is evenly distributed between the apical and basolateral membranes [ ] . the mechanisms underlying this difference are as yet unknown, but given the lack of homology between the cytoplasmic domains of the two enzymes, it is likely that this region may contain distinct targeting motifs. in vivo, ace is predominantly expressed in the heart, kidneys and testes, and at a lower level in a wide variety of tissues, particularly the colon and lung [ ] . in the heart, ace is essentially confined to the endothelia, in kidney to the luminal surface of the tubular epithelial cells [ , ] and, in testes, to the adult leydig cells [ ] . substrate specificity of ace unlike ace, ace functions as a carboxypeptidase, cleaving a single carboxy-terminal residue from its peptide substrates [ ] . a wide variety of potential substrates have been identified for ace , both within the ras and elsewhere [ ] . it is able to cleave both ang i and ang ii, to ang ( - ) and ang ( - ), respectively. whilst the affinity for the former is poor in comparison with ace, making it unlikely to be a physiological substrate (except under conditions in which ace activity is inhibited), ace efficiently cleaves ang ii to ang ( - ) [ ] . ang ( - ) is increasingly becoming recognised as a key peptide within the ras [ ] . ang ( - ) is known to potentiate the vasodilatory effects of bradykinin [ ] , stimulate no and prostaglandin release [ ] , and antagonise the actions of ang ii [ ] (fig. ) . hence, the ability of ace to degrade ang ii and simultaneously increase ang ( - ) would effectively oppose the actions of ace, suggesting the balance of the levels of the two enzymes would be critical in pathologies in the aetiologies of which ang ii is implicated ( fig. a ). in addition to its activity on ang ii, ace is also able to hydrolyse other non-ras peptides which have roles in maintaining cardiovascular homeostasis such as [des-arg ] bradykinin (fig. b) , a member of the kininogen-kinin system [ ] . [des-arg ] bradykinin is formed from bradykinin by the action of carboxypeptidases and is an agonist for the b receptor, which is induced upon tissue injury [ ] . bradykinin, a vasodilator which acts through the b receptor, is produced from its precursor kininogen by kallikrein and is degraded by ace [ ] (fig. b ). whilst the degradation of bradykinin by ace is known to be an important aspect of blood pressure regulation, the significance of the degradation of [des-arg ] bradykinin by ace remains to be established. in addition to [des-arg ] bradykinin, ace is also able to degrade apelin- , a peptide proposed to cause vasoconstriction and known to regulate fluid homeostasis, and other non-ras peptides such as kinetensin, dynorphin a and neurotensin [ ] . structural considerations ace and ace belong to the gluzincin clan of metalloproteinases, all of which catalyse reactions by utilising zinc, coordinated by conserved histidines within the active site, to facilitate nucleophilic attack on the carbonyl bond of the substrate by a water molecule, forming a non-covalently bound intermediate. in addition to the two histidines (located within the hexxh motif), a further glutamate residue is involved in coordinating the zinc ion; this is located amino acids c-terminally to the hexxh motif in both ace and ace [ , ] . despite these similarities, however, ace and ace function differently (the former normally releasing a cterminal dipeptide from its substrate; peptidyl dipeptidase action) and the latter releasing a single amino acid (a strict carboxypeptidase). in addition, the activity of ace is unaffected by a range of ace inhibitors, such as captopril and lisinopril [ ] , but is sensitive to inhibition by the dipeptide pro-phe [ ] and by the peptide analogues dx [ ] and mln ( [ ] . mln was the first rationally designed inhibitor of ace , based on the c-terminal dipeptide of ang i (his-leu), and has high potency (k i = . nm) and specificity. molecular modelling studies [ ] and the resolution of the ace crystal structure [ ] revealed that these substrate specificity differences are a result of the smaller binding pocket in ace due to arginine- making a salt-bridge with the c-terminus of the substrate, whereas in ace this residue is substituted by the smaller glutamine. comparison of the structure of ace in the presence and absence of mln revealed a large 'hingebending' motion, in which movement of the catalytic subdomains induced by inhibitor binding repositioned key residues for catalysis [ ] . physiological and patho-physiological roles of ace the high level of expression of ace in the heart together with its ability to hydrolyse angiotensin peptides have suggested a role for ace in maintaining cardiovascular physiology from the outset, a hypothesis subsequently supported by experimental data. crackower et al. [ ] showed that deletion of ace in mice resulted in elevated cardiac and plasma ang ii together with impaired cardiac contractility which increased with age. these changes were associated with an upregulation of hypoxia-induced genes, consistent with a role for ang ii in the ace null phenotype. this hypothesis is given further credence by the observation that the defects in the ace -null mice were reversed by deletion of ace in the same mice [ ] , an intervention which also abrogated the increased ang ii levels. furthermore, a recent study by the same group showed that the cardiomyopathy observed in ace -null mice involves ang ii acting through the at receptor to activate pi kgmediated pathways [ ] . it should be noted, however, that a similar study by gurley et al. [ ] failed to detect any changes in cardiac function in a distinct strain of ace -null mice but instead observed enhanced susceptibility to ang ii-induced hypertension. furthermore, deletion of ace did not cause abnormalities which might be expected as a result of increased ang ii, such as cardiac hypertrophy, fibrosis and altered conductance [ ] , suggesting that ace may not exert its effects in the heart solely via ang ii. interestingly, overexpression of ace in cardiac myocytes did cause changes in cardiac conductivity, resulting in arrest and sudden death [ ] . these contradictory data suggest that the role of ace in the cardiovascular system may be more complex than is immediately apparent. further evidence for a role of ace in maintaining cardiovascular homeostasis via ang ii regulation is provided by studies conducted by zisman et al. [ ] which detected increased ace and ang ( - ) forming activity in failing human hearts. increases in ace expression and activity have been observed in rats following arterial ligation and at receptor blockade [ , ] . in a separate study, arterial ligation of rats resulted in increased ang ii and ang ( - ) levels alongside cardiac hypertrophy, an effect reversed by treatment with at receptor antagonists, which also increased cardiac ace levels [ ] . in this study, however, no change was observed in ace levels following arterial ligation. whilst these studies again highlight some conflicting data, it is likely that ace may play a protective role in the early stages of heart failure by elevating ang - levels. in addition to its postulated roles in the heart, the ace gene which is present on the x chromosome maps to a quantitative trait locus (qtl) in a number of rat models of hypertension. furthermore, saltsensitive hypertensive rats have reduced ace transcript and protein expression [ ] . in humans, single nucleotide polymorphisms associated with increased risk of cardiovascular disease have been identified within the ace gene locus [ ] . a role for the ras in the development of lung disease has been suggested by studies in rodents showing at receptor antagonists protect against experimentally induced pulmonary fibrosis [ ] , and an increased mortality rate in acute respiratory distress syndrome (ards) patients carrying the ace dd polymorphism [ ] . ards can be triggered by a variety of insults including acid aspiration, peritoneal sepsis and acute pancreatitis, and has a high mortality rate due to associated pulmonary oedema, inflammation and hypoxia. a recent study by kuba et al. [ ] identified a protective role for ace in mice in which ards had been experimentally induced by distinct insults. in this study, ace knockout mice developed more severe ards than wild-type littermates, an effect which was reversed in mice in which the ace gene had also been disrupted, or upon treatment with recombinant ace protein. in addition, at r knockout mice also had less severe disease and both ace À/y and wild-type mice responded positively to at receptor antagonists, suggesting the involvement of ang ii in ards progression. it can therefore be postulated that ace exerts its protective role by metabolising ang ii and thereby abrogating its deleterious effects. in , the surprising discovery was made that ace is a functional receptor for the causative agent of severe acute respiratory syndrome (sars), the sars coronavirus [ ] . sars achieved widespread notoriety for its high mortality rate (approaching %) in infected individuals due to an atypical pneumonia resulting in respiratory failure due to ards [ ] . ace was serendipitously identified as a receptor for sars-cov in vitro using co-immunoprecipitation techniques [ ] and has subsequently been shown to be essential for sars infection in vivo [ ] . interaction of sars-cov with ace occurs via trimers of the sars spike protein, a loop of which extends into a hydrophobic pocket of ace ; here a methyl group of threonine of the spike interacts with lysine of ace [ ] . the acquisition of a threonine at position appears to have been critical in the adaptation of the sars virus to humans; in the civet (believed to be the source of the outbreak), this position is occupied by a serine residue which renders it unable to bind human ace [ ] . in the first study to demonstrate that ace functions as the sars receptor in vivo [ ] , infection of mice with sars resulted in downregulation of ace and a concomitant increase in ang ii, and increased severity of lung damage which could be improved by treatment with at antagonists. this downregulation of ace in response to sars infection, leading to increased disease severity resulting from increases in ang ii, may be a significant contributor to the high mortality rates resulting from sars. it has therefore been suggested that administration of recombinant ace may be an effective therapy [ ] . the renal ras plays a vital role in maintaining electrolyte and fluid homeostasis and the kidney is host to high levels of ras components, including renin [ ] , ace [ ] , at receptors [ ] and ace [ ] . alterations of ace levels are associated with differing susceptibility to experimental renal damage and elevated ang ii levels have been detected in diseased kidneys in humans [ ] , suggesting that regulating ang ii expression may be critical for maintaining renal function. ace is expressed on the luminal surface of proximal and distal tubules and at a lower level in glomeruli [ , , ] . in this regard it displays a similar pattern of expression to ace, and may, like ace, regulate the interstitial levels of angiotensin peptides. evidence for this is provided by li et al. [ ] , who showed that isolated proximal tubules, when incubated with ang i, generated ang ( - ), a process which was blocked by addition of the ace inhibitor dx . the results of this study are surprising as they indicate that ace in kidney is generating ang ( - ) indirectly from ang i (ace is able to convert ang i to ang ( - ) which can then be converted to ang ( - ) by ace, but the kinetics for this are not favourable [ ] ) rather than directly from ang ii, but still indicate a role for ace in generating interstitial ang ( - ) . in addition, ace levels are elevated in the kidneys of young diabetic mice in parallel with reduced ace expression [ ] . this is in contrast with a separate study showing markedly reduced levels of ace in a model of diabetic nephropathy [ ] ; the reasons for this difference are not immediately apparent, but may indicate that ace is protective in the early stages of the disease but then a decline in its expression allows elevated ang ii levels to contribute to the development of nephropathy. this is supported in part by the observation that some ace deficient mice develop ang ii dependent glomerulosclerosis with aging [ ] . furthermore, infusion of the specific ace inhibitor mln resulted in marked albuminuria in diabetic mice [ ] . ace levels are also known to rise together with ang ( - ) during gestation in rats [ ] ; this may be a protective mechanism to obviate the effects of hypertension in the kidney during pregnancy. a role for ace has also been proposed in the development of liver fibrosis and subsequent cirrhosis. paizis et al. [ ] identified an increase in ace levels in response to bile-duct ligation in rats, and in human cirrhotic liver. in both cases, ace levels were low in corresponding controls, with expression confined to endothelial cells and perivenular hepatocytes, but were widespread in the parenchymal tissue of diseased livers. intriguingly, ace expression was high in cells exposed to low oxygen levels, and ace could be induced by hypoxia in vitro. this is in keeping with studies showing increased ace levels in myocardial ischaemia, and raises the possibility that ace may exert a protective effect in tissue injury by increasing local oxygenation levels by abrogating ang iimediated vasoconstriction. this hypothesis has been further strengthened by a recent study showing that the increase in ace levels following bile-duct ligation is accompanied by increases in ang - and its receptor, mas [ ] . this study, however, did not detect any direct protective role for ang - , suggesting that ace may exert positive effects in liver injury predominantly by reducing ang ii levels. the ace homologue, collectrin (also known as tmem ), was first identified as a kda kidney-specific developmentally regulated transmembrane protein of unknown function shortly after the discovery of ace [ ] . recently collectrin has been implicated as a critical regulator of amino acid uptake in the proximal tubules of the kidney [ , ] . disruption of collectrin in mice results in downregulation of a variety of renal apical amino acid transporters and associated aminoaciduria. a recent study by zhang et al. [ ] provided evidence of a role for hepatocyte nuclear factor b (hnf- b) in regulating renal collectrin expression, and showed collectrin is essential for the maintenance of primary cilia and polarity of collecting duct cells. mutations in hnf- b are associated with polycystic kidney disease and mody (maturity-onset diabetes mellitus of the young, type ), sufferers of which also develop renal disease. these findings provide a link with studies of the role of collectrin in the pancreas, where it has recently been shown to regulate pancreatic islet cell growth and insulin exocytosis. akpinar et al. [ ] showed that collectrin levels were reduced in mice in which the related hepatocyte nuclear factor a (hnf- a) had been deleted; mutations in this transcription factor are associated with the development of mody. this reduction in expression correlated with decreased islet mass; in keeping with this, overexpression of collectrin resulted in increased islet mass. similar results were obtained by fukui et al. [ ] , however this study also provided evidence of a role for collectrin in regulating insulin secretion by facilitating snare complex formation. additionally, akpinar et al. [ ] showed that collectrin, like ace , is subject to ectodomain shedding. in the case of collectrin, however, this shedding event appeared to occur exclusively in pancreatic b-cells, suggesting the involvement of a sheddase with very restricted distribution and therefore presumably via a mechanism distinct from that of ace . whilst there is much work to be done elucidating the mechanisms underlying the interaction of collectrin with renal amino acid transporters and its actions in the pancreas, given the range of diseases in which collectrin is now implicated, such as hartnup disease and diabetes, it is clear that these discoveries are likely to provide new opportunities for therapeutic intervention. the importance of ace as a regulator of the local ras is becoming increasingly apparent. through its ability to metabolise ang ii to ang ( - ) it is able to regulate local ang ii levels thereby modulating its effects. disturbance of the balance of expression of ace and its homologue ace could alter the levels of ang ii and contribute to the development of a range of pathologies, from myocardial infarction to sars. furthermore, the recent explosion of research into the ace homologue, collectrin, has revealed new possible physiological functions for ace independent of its catalytic activity. while there still exists a myriad of opportunities for further research, recent findings have revealed intriguing possibilities for novel avenues of pharmacological intervention, most probably involving strategies to upregulate ace activity. a novel angiotensin converting enzymerelated carboxypeptidase (ace ) converts angiotensin i to angiotensin - a human homolog of angiotensin converting enzyme: cloning and functional expression as a captoprilinsensitive carboxypeptidase essential role for collectrin in renal amino acid transport aminoaciduria and altered renal expression of luminal amino acid transporters in mice lacking novel gene collectrin tmem : a cleaved and shed plasma membrane protein that stimulates pancreatic beta cell proliferation the hnf- target collectrin controls insulin exocytosis by snare complex formation angiotensin-converting enzyme (ace ), but not ace, is preferentially localized to the apical surface of polarized kidney cells exploring the structure and function of zinc metallopeptidases: old enzymes and new discoveries tumor necrosis factor-alpha convertase (adam ) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme- (ace ) the novel angiotensin-converting enzyme (ace) homolog, ace , is selectively expressed by adult leydig cells of the testis hydrolysis of biological peptides by human angiotensinconverting enzyme-related carboxypeptidase angiotensin-converting enzyme- (ace ): comparative modeling of the active site, specificity requirements, and chloride dependence contribution of angiotensin-( - ) to blood pressure regulation in salt-depleted hypertensive rats angiotensin-( - ) potentiates responses to bradykinin but does not change responses to angiotensin i angiotensin-( - ) enhances anti-aggregatory effects of the nitric oxide donor sodium nitroprusside prevention of angiotensin ii-induced cardiac remodeling by angiotensin-( - ) the angiotensin-converting enzyme gene family: genomics and pharmacology bradykinin b and b receptors both have protective roles in renal ischemia/reperfusion injury identification of critical active-site residues in angiotensinconverting enzyme- (ace ) by site-directed mutagenesis ace x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis evaluation of angiotensin-converting enzyme (ace), its homologue ace and neprilysin in angiotensin peptide metabolism novel peptide inhibitors of angiotensin-converting enzyme substrate-based design of the first class of angiotensin-converting enzyme-related carboxypeptidase (ace ) inhibitors angiotensin-converting enzyme is an essential regulator of heart function loss of angiotensin-converting enzyme- leads to the late development of angiotensin ii-dependent glomerulosclerosis altered blood pressure responses and normal cardiac phenotype in ace -null mice heart block, ventricular tachycardia, and sudden death in ace transgenic mice with downregulated connexins increased angiotensin-( - )-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme homologue ace advances in biochemical and functional roles of angiotensin-converting enzyme and angiotensin-( - ) in regulation of cardiovascular function myocardial infarction increases ace expression in rat and humans upregulation of angiotensin-converting enzyme after myocardial infarction by blockade of angiotensin ii receptors association study of ace (angiotensin i-converting enzyme ) gene polymorphisms with coronary heart disease and myocardial infarction in a chinese han population essential roles for angiotensin receptor at a in bleomycin-induced apoptosis and lung fibrosis in mice angiotensin-converting enzyme insertion/deletion polymorphism is associated with susceptibility and outcome in acute respiratory distress syndrome a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury angiotensin-converting enzyme is a functional receptor for the sars coronavirus the acute respiratory distress syndrome structure of sars coronavirus spike receptor-binding domain complexed with receptor receptor and viral determinants of sars-coronavirus adaptation to human ace lessons from sars: control of acute lung failure by the sars receptor ace the renin content of kidney angiotensin-converting enzyme activity in postmortem human tissues identification of an angiotensin receptor in rabbit renomedullary interstitial cells in tissue culture: correlation with prostaglandin biosynthesis angiotensin ii and renal fibrosis glomerular localization and expression of angiotensinconverting enzyme and angiotensin-converting enzyme: implications for albuminuria in diabetes the role of angiotensin converting enzyme in the generation of angiotensin - by rat proximal tubules ace and ace activity in diabetic mice characterization of renal angiotensinconverting enzyme in diabetic nephropathy ace inhibition worsens glomerular injury associated with increased ace expression in streptozotocin-induced diabetic mice enhanced renal immunocytochemical expression of ang-( - ) and ace during pregnancy chronic liver injury in rats and humans upregulates the novel enzyme angiotensin converting enzyme upregulation of hepatic angiotensin-converting enzyme (ace ) and angiotensin-( - ) levels in experimental biliary fibrosis collectrin, a collecting duct-specific transmembrane glycoprotein, is a novel homolog of ace and is developmentally regulated in embryonic kidneys the role for hnf- beta-targeted collectrin in maintenance of primary cilia and cell polarity in collecting duct cells we would like to acknowledge the financial support provided by the biotechnology and biological sciences research council (uk). r e f e r e n c e s key: cord- - d j jh authors: choi, marcelo; aiello, ernesto alejandro; ennis, irene l.; villa-abrille, maría celeste title: el sraa y el sars-cov- : el acertijo a resolver date: - - journal: hipertens riesgo vasc doi: . /j.hipert. . . sha: doc_id: cord_uid: d j jh resumen el de diciembre de se reportó el primer caso de covid- en wuhan, china, y desde entonces ha habido un interés creciente y sin precedentes por conocer todos los aspectos vinculados con esta nueva enfermedad. uno de los temas que ha generado debate se vincula con la asociación entre la terapia antihipertensiva con inhibidores del sistema renina-angiotensina-aldosterona (sraa) y la infección por el virus sars-cov- . si bien muchas preguntas siguen hoy día sin poder ser respondidas, la intención de este comunicado es informar a los profesionales de la salud acerca del estado actual de conocimiento. dado que este es un tema en constante evolución, se recomienda su actualización a medida que se presenten nuevas evidencias. a continuación, daremos revisión a los estudios preclínicos y clínicos que relacionan el coronavirus con el sraa. abstract the first case of covid- was reported on december in wuhan, china. ever since there has been unprecedented and growing interest in learning about all aspects of this new disease. debate has been generated as to the association between antihypertensive therapy with renin-angiotensin-aldosterone system (raas) inhibitors and sars-cov- infection. while many questions as yet remain unanswered, the aim of this report is to inform health professionals about the current state of knowledge. because this is an ever-evolving topic, the recommendation is that it be updated as new evidence becomes available. below, we provide a review of pre-clinical and clinical studies that link coronavirus to the raas. nivel respiratorio, la principal célula diana para el virus es la célula alveolar tipo ii o neumocito tipo ii, que representa el % de la superficie alveolar. este tipo celular es el responsable de secretar el surfactante y es progenitor de los neumocitos tipo i en caso de lesión tisular ( ) . a diferencia de la clásica eca (enzima convertidora de angiotensina), la eca degrada la angiotensina ii (ang ii) uniéndose a ella con una afinidad veces mayor que a la ang i (ver figura ) ( ) . cabe aclarar que, a pesar de compartir un % de homología estructural con la eca, sus sitios catalíticos son diferentes, por lo que los fármacos inhibidores de la eca (ieca) no inhiben la eca ( ) . la eca es una proteína transmembrana con actividad de monocarboxipeptidasa que regula negativamente el sraa al convertir ang ii en ang - . también degrada ang i para generar ang - (sin actividad biológica conocida) y otros péptidos como desarg-bradiquinina, neurotensina - , kinetensina, apelin- y dinorfina a - ( ) . la conversión de ang ii a ang - por parte de la eca trae como consecuencia la disminución del efecto vasoconstrictor a través de dos acciones principales ( ): -reduciendo los niveles del efector primario del sraa, ang ii y por ende la vasoconstricción. -aumentando la formación de ang - con acciones vasodilatadoras a través de su unión al receptor mas. la eca en su forma completa se encuentra anclada en la membrana plasmática mientras que su forma acortada (o soluble) circula en bajos niveles en la sangre ( ) . existe un gran interés en dilucidar la función de eca en la enfermedad cardiovascular, tanto el de la enzima transmembrana como también el de la forma soluble. para ingresar a las células el coronavirus interactúa, utilizando como receptor, con la eca y serina-proteasas transmembrana de tipo ii (tmprss ) ubicadas en la superficie celular del huésped ( ) . el virus, a través de su proteína s (spike o pico), se une al dominio catalítico de la eca con alta afinidad, incluso a veces más que el sars-cov ( ) . estudios sobre el comportamiento de su congénere sars-cov, permitieron conocer el mecanismo de la infección celular. la unión del virus a la eca desencadena un cambio conformacional en la proteína s del coronavirus permitiendo la degradación proteolítica por las serina-proteasas tmprss , exponiendo una subunidad de la proteína s que permite su fusión a la membrana celular y facilita el ingreso del virus al interior de la célula huésped ( ) . por lo enunciado, las tmprss serían también un potencial objetivo terapéutico para disminuir la infección viral (figura ). existe una vía alternativa de entrada del virus a la célula huésped que no implica la activación de las tmprss (vía independiente de tmprss ). en este caso, luego de la unión del virus a la eca , se forma un endosoma dentro del cual la proteína s es clivada y activada por la cisteína-proteasa catepsina l dependiente de ph ( ) . por otro lado, resulta de especial interés la participación de la proteína transmembrana disintegrina y metaloproteasa (adam ), que es activada por la proteína s viral y cliva a la eca produciendo su eliminación de la superficie celular ( ) . normalmente la eca protege del daño pulmonar ocasionado por la ang ii al degradarla a ang - ( ) . debido a que se ha informado que los niveles de eca durante una infección por sars-cov se encuentran disminuidos, la proteína adam jugaría un rol esencial en el daño pulmonar al promover la eliminación de eca ( ) . la observación de que los pacientes con covid- presentan niveles bajos de potasio en sangre, permite especular que la disminución de la eca llevaría a un incremento de los niveles de ang ii y por ende de aldosterona, con la consecuente pérdida de potasio ( ) . consistentemente, ha sido reportado recientemente en pacientes con covid- que los niveles plasmáticos de ang ii se correlacionan positivamente con la carga viral de sars-cov ( ) . adicionalmente, el receptor at de ang ii regula el alza de la proteína adam , incrementando así los niveles circulantes de la fracción soluble de eca (figura ) ( ). relación sars-cov- e hipertensión arterial: hipótesis postuladas: se han postulado diversas hipótesis para explicar una mayor severidad de la enfermedad del sars-cov- en pacientes hipertensos: ) una de estas hipótesis postula que la mayor expresión de eca en un tejido dado como el pulmón incrementaría la infectividad del virus y empeoraría el curso de la enfermedad. esto fue demostrado en cultivos celulares en donde la mayor expresión de eca se asociaba a una mayor tasa de infección por el virus ( ) . resulta interesante la observación de que las personas mayores y los hombres presentan peor evolución de la enfermedad y mayores tasas de mortalidad por covid- ( ) . esto plantearía el interrogante de cómo varían los niveles de eca en el pulmón con la edad y el género. a nivel experimental, se han demostrado mayores niveles de expresión pulmonar de eca en ratas sprague dawley jóvenes comparadas con ratas envejecidas, contribuyendo a una mayor tasa de infección en las jóvenes ( ) . este último hallazgo nos permitiría sugerir que la mayor infectividad en personas mayores no se relaciona con los niveles de expresión de eca , aunque deberían evaluarse otras variables. por otra parte, las evidencias acerca de diferencias en la expresión de eca plasmática según sexo y edad en seres humanos son controvertidas. un estudio reporta que no existen diferencias en la expresión de eca plasmática entre hombres y mujeres, aunque sí mayores niveles de expresión en mujeres mayores que en jóvenes ( ) . en oposición, en otro trabajo se ha determinado que una mayor expresión de eca plasmática se asocia a hombres de mayor edad ( ) . en conclusión, hasta el momento, ningún estudio clínico ha demostrado fehacientemente la validez de esta hipótesis. ) otra hipótesis relaciona la infección del sars-cov- con ciertos agentes antihipertensivos como los inhibidores del sraa. existen hallazgos controvertidos al respecto. algunos estudios experimentales demuestran que efectivamente los ieca (lisinopril, enalapril) y los antagonistas del receptor de ang ii, ara-ii (losartan, telmisartan) pueden aumentar los niveles de expresión de eca ( ) ( ) ( ) . no obstante, otros trabajos demuestran lo contrario ( , ) o incluso la no asociación. adicionalmente, unos pocos estudios clínicos demostraron que no existía ninguna asociación entre los niveles circulantes de la fracción soluble de eca y el uso de ieca y ara-ii ( , ) . dado que el tratamiento con ieca o ara-ii tiene un impacto diferente en varios componentes del sraa, ya sea directamente o mediante bucles de retroalimentación, se ha postulado la posibilidad de que ambos grupos de inhibidores del sraa tengan un efecto diferencial en pacientes con covid- . sin embargo, faltan estudios que demuestren fehacientemente un efecto de los iecas o ara-ii sobre la expresión o actividad de eca en los pulmones en humanos. los estudios clínicos llevados a cabo hasta el día de hoy no han demostrado que existen diferencias entre ambos tratamientos en términos de aumento del riesgo de infección por sars-cov- o de desarrollo de resultados graves en pacientes con covid- ( ) ( ) ( ) ( ) ( ) . no obstante es de destacar que la mayoría de estos estudios son observacionales y retrospectivos, siendo necesarios datos a partir de estudios controlados y aleatorizados para confirmar estos hallazgos. ) la tercera hipótesis postula que el uso de los ieca y ara-ii conllevaría un beneficio en relación a la infección y gravedad por covid- . se ha demostrado que la eca interactúa con el receptor at formando un complejo que dificultaría la interacción de la eca con la proteína s viral. la ang ii al unirse al receptor at disminuye los niveles de expresión de eca al promover su internalización en lisosomas (figura ) ( ). de esta manera, el uso de ieca o ara-ii evitaría la disociación del complejo eca /at al prevenir la internalización y degradación de eca ( ) . esto trae como consecuencia, por un lado la persistencia del complejo eca -at que dificulta la interacción entre el virus y la eca , y por otra lado, que la eca pueda degradar la ang ii a ang - , disminuyendo así los efectos deletéreos de la ang ii sobre el pulmón. en términos de beneficios en la evolución de la enfermedad por covid- , el estudio de mehra mr y cols. ha mostrado que los ieca, a diferencia de los ara-ii, se asocian con una mayor probabilidad de supervivencia al alta hospitalaria ( ) . ) otra hipótesis posible plantea la importancia que tiene la eca para prevenir el daño pulmonar asociado a la infección viral. altos niveles de ang ii a nivel pulmonar se asocian a mayor daño e injuria tisular por su efecto pro-inflamatorio y pro-oxidante, mientras que la presencia de ang - , a través de la activación del receptor mas, tendría el efecto contrario. se demostró de esta manera que ratones knockout para la eca presentaban mayor severidad y mortalidad por neumonía viral. en este sentido, la presencia de la eca sería crucial para disminuir los niveles de ang ii e incrementar los de ang - ( , ) . ) en base a la evidencia mencionada, sommerstein r y cols. han postulado que las drogas que inhiben el sraa podrían jugar un rol durante el curso severo de la enfermedad covid- . los autores plantean así una especie de arma de doble filo, dependiendo de la fase de la enfermedad en la que nos situemos: el aumento de la expresión basal de eca podría aumentar potencialmente la infectividad por lo que el uso de iecas y/o ara-ii sería un factor de riesgo, mientras que una vez establecida la infección, existe una disminución de la eca por lo que el uso de estos inhibidores del sraa podría resultar beneficioso ( ) . como posibles estrategias a futuro para combatir la enfermedad covid- se encuentran, entre otras: -desarrollo de una vacuna basada en la proteína spike s -producción de anticuerpos neutralizantes contra la proteína s -inhibición de la serina-proteasa transmembrana (tmprss ) (mesilato de camostato) -administración de la forma soluble de la eca -bloqueo de la entrada del sars-cov- (arbidol) -inhibición de la proteinquinasa asociada a ap , proteína reguladora de la endocitosis viral (baricitinib). -bloqueo de la replicación sars-cov- por inhibición de arn polimerasa viral (análogo nucleosídico remdesivir) o de la endonucleasa cap-dependiente (baloxavir marboxilato) o de la proteína básica de polimerasa (favipiravir). -inhibición de proteasas virales necesarias para su replicación (darunavir; lopinavir/ritonavir) -bloqueo de la glicosilación del receptor eca y de la producción de citoquinas inflamatorias (cloroquina e hidroxicloroquina) -bloqueo del receptor de il- (tocilizumab) -inhibición de producción de citoquinas pro-inflamatorias (nitazoxanida) actualmente están en marcha estudios clínicos cuyo objetivo es evaluar las consecuencias y seguridad del bloqueo farmacológico del sraa en la enfermedad covid- : -administración de eca humana recombinante vs placebo: nct ; euctr - - -dk -tratamiento con losartan vs placebo: nct ; nct -tratamiento con telmisartan vs placebo: nct -tratamiento con valsartán vs placebo: euctr - - -nl; nct conclusiones: si bien los datos disponibles de estudios en china y en italia demuestran inequívocamente la asociación entre enfermedad cardiovascular, hipertensión y diabetes con una mayor mortalidad por covid- , ( ) ( ) ( ) ( ) ( ) aún no es clara la relación entre la hipertensión arterial y el bloqueo del sraa en la enfermedad covid- . en humanos, la evidencia actual proviene de estudios no ajustados o ajustados de forma incompleta, lo que implica sesgos de selección, de clasificación y presencia de variables que generan confusión (edad, duración y tipo de medicación antihipertensiva, carga de comorbilidad). si bien existe evidencia in vitro de que el sars-cov- se une a los receptores eca y que éstos se encuentran aumentados en presencia de ieca o ara-ii, no hay evidencia al momento de que la exposición a estos fármacos facilite la entrada del coronavirus ni que produzcan un mayor riesgo de covid- . los beneficios de ieca y ara-ii en la prevención primaria y secundaria de las enfermedades cardiovasculares son indudables y han sido demostrados por numerosos ensayos clínicos controlados. por ello, en base a la evidencia disponible hasta la fecha no hay elementos que sugieran la necesidad del reemplazo ni de la suspensión de ieca o ara-ii en pacientes expuestos o con enfermedad covid- demostrada. estas drogas deben ser iniciadas o mantenidas en pacientes con hipertensión, fallo cardíaco, infarto de miocardio y nefropatía diabética de acuerdo a las guías actuales, independientemente del estado de sar-scov- . la suspensión y/o reemplazo del tratamiento en pacientes que reciban ieca o ara-ii no debe recomendarse como estrategia preventiva ni de tratamiento de la infección por sars-cov- . al día de hoy, las principales sociedades científicas (entre ellas la sociedad europea de cardiología, la sociedad europea de hipertensión, la sociedad internacional de hipertensión, la sociedad española de hipertensión y la asociación americana del corazón, además de la propia sociedad argentina de hipertensión arterial) se han posicionado con indicaciones claras al respecto, recomendando continuar con el tratamiento de ieca o ara-ii dada la falta de evidencia que apoye lo contrario ( - ). sin embargo, esta conclusión debe considerarse en constante revisión. esquema de las posibles vías para la invasión del sars-cov a la célula huésped. adam : proteína transmembrana disintegrina y metaloproteasa ; tmprss : serinaproteasas transmembrana de tipo ii. figura . rol de la proteína transmembrana disintegrina y metaloproteasa , adam , en la formación de la forma soluble de eca . tmprss : serina-proteasas transmembrana de tipo ii; rcat : receptor de ang ii tipo . figura . relación entre el receptor at (rcat ) y la eca : efectos del bloqueo del sraa. panel a: estado basal: la ang ii formada se une al rcat , disminuyendo la interacción de este con la eca , fenómeno que favorece la endocitosis y posterior degradación. disminuyendo el nivel de eca en la membrana. cuando se utilizan inhibidores de la enzima convertidora de angiotensina, ieca, o antagonistas de los rcat , ara-ii, disminuye la cantidad de ang ii formada y aumenta su degradación (por eca ) estabilizándose la interacción del rcat y la eca . por consiguiente, disminuye la endocitosis de eca y; se favorece la conversión de ang ii a ang - (panel b). tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis from gene to proteinexperimental and clinical studies of ace in blood pressure control and arterial hypertension a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - angiotensin receptor blockers as tentative sars-cov- 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systematic review and meta-analysis case-fatality rate and characteristics of patients dying in relation to covid- in italy sitios de acción de la eca y eca en la cascada del sistema renina angiotensina. rcat : receptor de ang ii tipo ; rcat : receptor de ang ii tipo key: cord- - xrehbd authors: wang, hanyin; das, subhraleena; wieruszewski, patrick m.; taji, jamil; bartlett, brian; azad, nabila; chowdhury, arnab; kolar, gururaj; jain, nitesh; subla, mir r.; khan, syed anjum title: unexpected bp sensitivity to angiotensin ii in a patient with coronavirus disease , ards, and septic shock date: - - journal: chest doi: . /j.chest. . . sha: doc_id: cord_uid: xrehbd we report the case of an -year-old man with coronavirus disease (covid- ) who presented with ards and septic shock. the patient had exquisite bp sensitivity to low-dose angiotensin ii (ang- ), allowing for rapid liberation from high-dose vasopressors. we hypothesize that sensitivity to ang- might be related to biological effect of severe acute respiratory syndrome coronavirus infection. the case is suggestive of a potential role for synthetic ang- for patients with covid- and septic shock. further studies are needed to confirm our observed clinical efficacy. of march , , the outbreak of novel coronavirus (sars-cov- ) had , confirmed cases and , deaths globally. an estimated . % of patients with coronavirus disease (covid- ) required icu admission, . % underwent mechanical ventilation, and . % had septic shock. angiotensin ii (ang- ) is a synthetic vasopressor that received us food and drug administration approval in for treatment of refractory vasodilatory shock. we report our experience with ang- for septic shock in a critically ill patient with covid- . an -year-old man with a history of hypertension, coronary artery disease, and type diabetes mellitus presented to clinic with a -day history of cough and shortness of breath. he denied any travel history outside of minnesota. the patient was febrile to . c, tachycardic, and tachypneic with oxygen saturation of % on room air. he was transferred to the ed and received intubation. laboratory tests were notable for lymphopenia, leukocytosis, mildly increased creatinine, and markedly increased c-reactive protein and d-dimer (table ) . ct chest angiogram showed diffuse perihilar ground-glass interstitial opacities with consolidation at lung bases. a nasopharyngeal swab was sent for sars-cov- polymerase chain reaction (mayo medical laboratories, rochester, mn). the patient promptly received ml/ kg of crystalloid resuscitation and guideline-concordant broad-spectrum antibiotics and was admitted to the icu. he became hypotensive and required abbreviations: ace = angiotensin-converting enzyme; ace = angiotensin-converting enzyme ; ang- = angiotensin ii; covid- = coronavirus disease ; sars-cov = severe acute respiratory syndrome-related coronavirus; sars-cov- = novel coronavirus on icu day , the sars-cov- polymerase chain reaction result was positive. the infectious diseases team started hydroxychloroquine and azithromycin. the patient began neuromuscular blocker therapy for ventilator dyssynchrony and was placed in prone position to improve dorsal lung aeration. throughout the day, his vasopressor requirements worsened severely. transthoracic echocardiography showed an ejection fraction of %, normal right ventricular systolic function, and an estimated right ventricular systolic e novel report pressure of mm hg. noninvasive cardiac output monitor (flotrac; edwards lifesciences corp) showed severely reduced vascular resistance and high cardiac output. although corticosteroids are not recommended for covid- , stress dose methylprednisolone was added for refractory septic shock. ang- ( ng/kg/min) was added as the third vasopressor. soon after initiation of ang- therapy, we observed considerable reduction in norepinephrine and vasopressin requirements (fig ) . on icu day , he was weaned of all vasopressors. the patient's respiratory status improved, with a better pao to fio ratio and less peep requirement. he had right upper extremity catheter-associated dvt and received therapeutic heparin. on icu day , neuromuscular blocker and methylprednisolone were discontinued. on icu day , the patient developed acute kidney injury with peak creatinine . mg/dl but did not require dialysis. he remained off vasopressor with stable ventilator requirement. on icu day , family elected to transition to comfort measures and the patient died after compassionate extubation. physiologically, angiotensin-converting enzyme (ace) converts angiotensin i to ang- , which stimulates ang- type receptors in the systemic vasculature and causes potent vasoconstriction. in the phase approval study athos- , synthetic ang- effectively increased bp for patients with vasodilatory shock unresponsive to highdose vasopressor therapy. the patient with covid- was critically ill with ards and septic shock. his acute sensitivity to low-dose ang- treatment allowed for rapid liberation from high-dose vasopressors. although he received hydroxychloroquine and corticosteroids before ang- , we would not expect these medications to have such marked hemodynamic effects. heterogeneous bp sensitivity to ang- has been reported. in athos- , . % of patients ( of ) in the treatment group were able to have ang- downtitration to # ng/kg/min at min. this subgroup of patients had significantly lower endogenous serum ang- levels, but unlike the present patient, they had lower baseline norepinephrine-equivalent requirements. the present patient had ards and was taking an ace inhibitor (lisinopril). severe ards has been shown to disrupt ace function, and an ace inhibitor directly inhibits ace activity. these factors lead to endogenous ang- insufficiency and were suspected to be related to ang- sensitivity in prior studies. , however, in athos- , no difference was observed in ards or exposure to ace inhibitor between the different ang- sensitivity subgroups. treatment with ang- in covid- may have special biological consideration. sars-cov- recognizes angiotensin-converting enzyme (ace ) as a receptor for cell entry. ace converts ang- to heptapeptide angiotensin ( - ), counteracting ace effects. in vitro, ang- has been shown to downregulate ace expression. , a recent paper promoted early use of ang- for covid- -associated vasodilatory shock. chow et al asked whether ang- could cause downregulation of ace in vivo and, subsequently, modulate cell entry and viral replication of sars-cov- . ace is the same receptor used by severe acute respiratory syndrome-related coronavirus (sars-cov). in animal models, sars-cov infection resulted in considerable reduction of ace expression in the lung, with subsequent increase in ang- level that promoted lung injury. we expect sars-cov- infection to cause a similar biological process. however, exquisite sensitivity to ang- in the present case raised the question of possible ang- deficiency during sars-cov- infection. in the patient, we did not observe worsening ards with ang- treatment. our case report has several limitations. first, ang- hypersensitivity has been observed during septic shock without covid- and is thought to be caused by relative ang- insufficiency. although it is possible our observation may not have been directly related to sars-cov- infection, it is suggestive the patient had a dysregulated renin-angiotensin system. second, although it is unlikely that the reduction in vasopressor requirements was secondary to receipt of corticosteroid, this cannot be entirely ruled out. third, the biological effects of ang- in patients with covid- remain unknown, and are beyond the scope of our case report. our report highlights an interesting observation of ang- treatment of covid- -related septic shock. further studies are needed to confirm our observed clinical efficacy. measurement of serum ang- , lung ace activity, and sars-cov- viral load in patients with covid- treated with ang- may provide important insight. in italy, compassionate use of ang- for covid- -associated septic shock was approved. we look forward to hearing about their experience. world health organization clinical characteristics of coronavirus disease in in china an update of the role of renin angiotensin in cardiovascular homeostasis angiotensin ii for the treatment of vasodilatory shock sensitivity to angiotensin ii dose in patients with vasodilatory shock: a prespecified analysis of the athos- trial pulmonary capillary endothelium-bound angiotensin-converting enzyme activity in acute lung injury endogenous angiotensin ii levels and the mechanism of action of angiotensin-converting enzyme inhibitors and angiotensin receptor type antagonists intravenous angiotensin ii for the treatment of high-output shock (athos trial): a pilot study the use of angiotensin ii in distributive shock a pneumonia outbreak associated with a new coronavirus of probable bat origin angiotensin ii up-regulates angiotensin i-converting enzyme (ace), but down-regulates ace via the at -erk/p map kinase pathway angiotensin ii mediates angiotensin converting enzyme type internalization and degradation through an angiotensin ii type i receptor-dependent mechanism angiotensin ii for the treatment of covid- -related vasodilatory shock angiotensin-converting enzyme is a functional receptor for the sars coronavirus a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury la jolla pharmaceutical company to provide giaprezaÔ (angiotensin ii) in italy for compassionate use in patients with septic shock associated with covid- financial/nonfinancial disclosures: q none declared.other contributions: editing, proofreading, and reference verification were provided by scientific publications, mayo clinic. chest worked with the authors to ensure that the journal policies on patient consent to report information were met. key: cord- -pfsztifl authors: clarke, nicola e.; hooper, nigel m.; turner, anthony j. title: chapter angiotensin-converting enzyme- date: - - journal: handbook of proteolytic enzymes doi: . /b - - - - . - sha: doc_id: cord_uid: pfsztifl the third edition of the handbook of proteolytic enzymes aims to be a comprehensive reference work for the enzymes that cleave proteins and peptides, and contains over chapters. each chapter is organized into sections describing the name and history, activity and specificity, structural chemistry, preparation, biological aspects, and distinguishing features for a specific peptidase. the subject of chapter is angiotensin-converting enzyme- . keywords: angiotensin, angiotensin-converting enzyme (ace ), apelin, bradykinin, carboxypeptidase, cardiovascular, collectrin, renin-angiotensin system, sars virus, shedding, transmembrane, vasoactive, zinc-binding motif. angiotensin-converting enzyme- two independent approaches converged in the discovery of ace or angiotensin-converting enzyme homologue (aceh) in . ace was identified from screening a human genome database for novel zinc metallopeptidases, hence recognized as a close homolog of ace (see chapter ), then cloned from a human lymphoma cdna library [ ] . in parallel the same sequence was identified from a human heart failure cdna library [ ] . despite high similarity in sequence to ace, particularly around the active site, ace functions as a carboxypeptidase, rather than a peptidyl dipeptidase, cleaving the c-terminal amino acid from susceptible substrates. the distinct binding specificity of ace was demonstrated by the failure of ace inhibitors to block its activity. given the overall similarity to ace, the first potential substrates to be examined were angiotensin peptides and bradykinin [ ] . the decapeptide angiotensin i (ang i) is converted by ace to ang( À ) but not further metabolized whereas the vasoconstrictor octapeptide, ang ii, is converted to the vasodilatory ang( À ). subsequent detailed kinetic analysis revealed that ang ii was highly preferred as an ace substrate over ang i, being hydrolyzed fold more efficiently: ang ii (k m μm, k cat . s ), ang i (k m . μm, k cat . s ) [ ] . bradykinin is not hydrolyzed by ace but des-arg -bradykinin, the natural ligand for the bradykinin b receptor, was a substrate. when screened against a panel of biologically active peptides, only were hydrolyzed by purified ace , in all cases by single carboxypeptidase action. the three most efficiently hydrolyzed substrates were angiotensin ii, apelin- and dynorphin a-( À ). only for ang ii as a substrate is a physiological role for ace established, its cleavage product being angiotensin-( À ) (ang ( À )), which acts on the mas receptor to oppose the actions of ang ii [ ] . however, there is emerging evidence that ace may further control blood pressure by controlling the degradation of apelin- , another vasoactive peptide [ , ] . comparison of sequences around the cleavage site of susceptible substrates suggested a consensus sequence for carboxypeptidase action of pro-x(( À residues))-pro-hydrophobic/basic [ ] . ace has an optimum ph of . and, as a zinc peptidase, is inhibited by metal chelating agents [ , ] . several selective ace inhibitors have been developed since its discovery, the first such being mln- [ ] , which was designed exploiting the knowledge that ang i was an ace substrate. it is a non-peptide inhibitor with which the inhibitor-bound crystal structure was subsequently solved [ ] . mln- (also known as gl ) was originally, but unsuccessfully, developed as a potential anti-obesity drug and more recently is being examined as an anti-inflammatory agent [ ] . a peptide inhibitor of ace , dx , was identified from phage libraries and displayed a k i of . nm [ ] . more recently a phosphinic-peptide inhibitor has been developed, which mimics the enzyme transition state. this pseudo-peptide inhibitor was designated f and has a k i of . nm [ ] . the ace gene is located on chromosome xp and contains exons, many of which resemble the corresponding exons in the ace gene. the complete cdna sequence of ace encodes a amino acid protein, which resembles a chimera composed of a single acelike catalytic ectodomain fused to the transmembrane protein, collectrin, or tmem [ ] , which plays a role in regulation of renal amino acid transport and has been implicated in insulin exocytosis and β-cell proliferation [ ] . the homology of ace and ace is particularly striking around the hexxh zinc-binding motif which is identical (hemgh) in the two proteins. the crystal structure of ace was solved within years of its discovery [ ] . this established the structural basis for the unique catalytic activity of ace compared to ace, which is the result of subtle differences in their active sites. for example, the presence of an arginine at position in ace compared with glutamine in ace removes the s pocket present in the active site of ace, thereby eliminating the peptidyl dipeptidase activity [ ] . upon substrate or inhibitor binding the two catalytic subdomains of ace undergo a hinge-bending movement of Å towards each other, which closes the active site cleft to initiate substrate hydrolysis [ ] . key active site residues of ace ( figure . ) were identified by site-directed mutagenesis based on the structure of the single domain testicular form of ace [ ] . the fidelity of this model was verified by the inhibitorbound crystal structure. the formation of a salt bridge by arg was found to be critical for substrate recognition since its mutation abolished enzymatic activity. like ace, a histidine in ace (his ) stabilizes the tetrahedral peptide intermediate by acting as the hydrogen bond donor/acceptor. mutation of his to alanine reduced ace activity by -fold and therefore also established his as a critical active site residue. the activities of both ace and ace are chloridedependent, which is due to the presence of cl and cl sites in ace and a comparable cl site in ace . this chloride regulation is, however, substrate-dependent [ , ] . the chloride dependency of the ace cl site is mediated by several critical residues: arg , trp and lys , which correspond to arg , trp and arg in testicular ace. additionally, arg in ace was identified by mutagenesis as a residue important for substrate selectivity. effective inhibition of ace is also dependent on the presence of chloride, the ic for mln- being -fold lower in the presence of mm nacl compared to its absence [ ] . preparation ace protein has been expressed in functional form in mammalian cells (chinese hamster ovary) [ ] and in a baculovirus-infected insect cell (sf ) expression system [ ] . characterization of ace has involved the use of a truncated, secreted form of the protein which lacks the transmembrane and cytosolic domains [ , ] . the baculovirus-expressed protein was purified to homogeneity by sequential chromatography on a qae anion exchanger, hydrophobic interaction chromatography, mono-q ion exchange and gel filtration. this form was subsequently used for the crystallization of the protein [ ] . the main tissue sites of expression of ace were originally identified as testis, heart and kidney [ ] , where it was shown to be localized on the apical membrane of polarized cells [ ] . however, it has now been identified more widely, for example in liver, intestine and lung [ , ] . more recently ace has been localized in the brain [ ] , where it appears to act as a central regulator of cardiovascular function [ À ]. the major identified physiological function of ace is the conversion of the vasoconstrictor ang ii to ang( À ), a vasodilator, antifibrotic and anti-inflammatory peptide [ , ] . deletion of the ace gene in mice was shown to produce a major defect in cardiac contractility and an upregulation of hypoxia-inducible genes in the heart [ ] . deletion of both ace and ace genes led to a rescue of the observed cardiac phenotype [ ] . however, subsequent gene deletion studies of ace have produced mixed results with much less effect on baseline cardiac function [ ] . overall, it appears that the actions of ace may primarily be relevant in pathological states such as hypertension and cardiac hypertrophy. for example, levels of ace have been shown to increase in the failing heart [ ] and post-myocardial infarction (mi) in rat models [ , ] , as well as in failing human heart tissue. viral over-expression of ace can attenuate heart hypertrophy and preserve cardiac function post-mi [ ] . overall, therefore, ace does appear to play a role in cardiac homeostasis and these observations have led to the consensus view that ace plays a cardioprotective role acting as a counterbalance to the actions of ace [ , ] . additionally, a decrease in ace expression is associated with diabetic nephropathy [ ] and ace plays a major anti-fibrotic role in the liver [ ] . unexpected roles for the ace protein independent of its catalytic activity have emerged. firstly, the cellular receptor for the severe acute respiratory syndrome (sars) coronavirus, which causes severe acute lung failure, was identified as ace [ ] . it also serves as a receptor for another coronavirus, nl [ ] . binding of either virus causes downregulation of surface expression of ace . the loss of surface ace on sars infection results in a diminished ability of the lung to respond to inflammatory lung cell damage due to an increased ratio in the levels of ang ii:ang( À ). administration of recombinant human ace in vivo can protect from severe acute lung injury [ ] . ace also functions as a chaperone for the small neutral amino acid transporter (b at ) in the intestine, transporting it from intracellular sites of synthesis to the cell surface. this chaperone role is mediated by the collectrinlike domain of ace , with collectrin serving as the main transporter of b at in the kidney. in hartnup disorder, an inherited disease leading to severe aminoaciduria, a mutation in b at reduces its affinity for ace and collectrin leading to its intracellular sequestration [ ] . like ace, ace is found as a soluble, circulating form in plasma and is also detectable in urine as a result of renal excretion. the soluble form represents the ectodomain, which is shed from the membrane-bound form following proteolytic cleavage by adam [ ] . at present it is unclear if the circulating form is physiologically active since its activity in plasma seems to be masked by an endogenous inhibitor. the retention of ace on the cell membrane is regulated by calmodulin binding and calmodulin inhibitors increase the cellular release of ace [ ] . given the generally protective role of ace in cardiovascular, hepatic and lung pathologies, its upregulation would seem to have therapeutic potential. several strategies have been investigated to date to achieve these ends, including viral delivery and the administration of recombinant soluble ace , in a range of in vivo disease models [ À ]. ace activators have also been developed using a rational drug design approach [ ] . a molecular docking model based on the open (substrate-free) and closed conformations of ace led to the identification of xanthenone as a compound that binds at the hinge site on ace between subdomains and . this compound increased ace activity by approximately -fold in vitro and was shown to reduce the blood pressure of spontaneously hypertensive rats [ ] and attenuate thrombus formation in the same model of hypertension [ ] . ace is a multifaceted enzyme with a broad range of biological functions. as discussed above, ace resembles ace in many structural aspects but differs from it in specificity since it acts as a carboxypeptidase. the cytoplasmic domains of ace and ace show no homology and have distinct functions. ace does not hydrolyze bradykinin or the synthetic ace substrate hip-his-leu. ace is not inhibited by inhibitors of ace or cp-a and specifically designed ace inhibitors such as mln- do not inhibit ace. other metallocarboxypeptidases known to contain an hexxh zinc binding motif are bacterial in origin and include carboxypeptidase taq [ , ] (chapter ) and the cobalt-activated carboxypeptidase from the hyperthermophilic archaeon pyrococcus furiosus [ , ] (chapter ). ace shares some similarities in specificity with these carboxypeptidases but their characterization remains somewhat limited. a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captoprilinsensitive carboxypeptidase a novel angiotensin-converting enzymerelated carboxypeptidase (ace ) converts angiotensin i to angiotensin À evaluation of angiotensin-converting enzyme (ace), its homologue ace and neprilysin in angiotensin peptide metabolism À ) is an endogenous ligand for the g proteincoupled receptor mas emerging roles of apelin in biology and medicine genetic variants in the apelin system and blood pressure responses to dietary sodium interventions: a family-based association study angiotensin-converting enzyme- (ace ): comparative modeling of the active site, specificity requirements., chloride dependence hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase substrate-based design of the first class of angiotensin-converting enzyme-related carboxypeptidase (ace ) inhibitors ace x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis effects of the ace inhibitor gl on acute dextran sodium sulfate-induced colitis in mice development of potent and selective phosphinic peptide inhibitors of angiotensin-converting enzyme collectrin, a collecting duct-specific transmembrane glycoprotein, is a novel homolog of ace and is developmentally regulated in embryonic kidneys identification of critical active-site residues in angiotensinconverting enzyme- (ace ) by site-directed mutagenesis residues affecting the chloride regulation and substrate selectivity of the angiotensinconverting enzymes (ace and ace ) identified by site-directed mutagenesis angiotensin-converting enzyme (ace ), but not ace, is preferentially localized to the apical surface of polarized kidney cells tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis chronic liver injury in rats and humans upregulates the novel enzyme angiotensin converting enzyme differential expression of neuronal ace in transgenic mice with overexpression of the brain renin-angiotensin system ace /ang-( À )/ mas pathway in the brain: the axis of good injections of angiotensin-converting enzyme inhibitor mln into nucleus tractus solitarii reduce baroreceptor reflex sensitivity for heart rate control in rats brain-selective overexpression of human angiotensin-converting enzyme type attenuates neurogenic hypertension angiotensin ii type receptor-mediated reduction of angiotensin-converting enzyme activity in the brain impairs baroreflex function in hypertensive mice anti-inflammatory effects of the activation of the angiotensin-( À ) receptor, mas, in experimental models of arthritis angiotensin( À ) blunts hypertensive cardiac remodeling by a direct effect on the heart angiotensin-converting enzyme is an essential regulator of heart function angiotensin-converting enzyme gene targeting studies in mice: mixed messages increased angiotensin-( À )-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme homologue ace enalapril attenuates downregulation of angiotensin-converting enzyme in the late phase of ventricular dysfunction in myocardial infarcted rat myocardial infarction increases ace expression in rat and humans cardiac overexpression of angiotensin converting enzyme protects the heart from ischemia-induced pathophysiology angiotensin-converting enzyme : cardioprotective player in the renin-angiotensin system? loss of angiotensin-converting enzyme accelerates maladaptive left ventricular remodeling in response to myocardial infarction characterization of renal angiotensin-converting enzyme in diabetic nephropathy angiotensinconverting-enzyme inhibits liver fibrosis in mice angiotensinconverting enzyme is a functional receptor for the sars coronavirus crystal structure of nl respiratory coronavirus receptor-binding domain complexed with its human receptor angiotensin-converting enzyme protects from severe acute lung failure tissue-specific amino acid transporter partners ace and collectrin differentially interact with hartnup mutations tumor necrosis factor-alpha convertase (adam ) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme- (ace ) calmodulin interacts with angiotensin-converting enzyme- (ace ) and inhibits shedding of its ectodomain human recombinant ace reduces the progression of diabetic nephropathy prevention of angiotensin ii-mediated renal oxidative stress, inflammation and fibrosis by angiotensin-converting enzyme targeting the degradation of angiotensin ii with recombinant angiotensinconverting enzyme : prevention of angiotensin ii-dependent hypertension structure-based identification of small-molecule angiotensin-converting enzyme activators as novel antihypertensive agents ace activation promotes antithrombotic activity carboxypeptidase taq, a thermostable zinc enzyme molecular cloning, sequencing and expression of the encoding gene in escherichia coli the active site of carboxypeptidase taq possesses the active-site motif his-glu-x-x-his of zinc-dependent endopeptidases and aminopeptidases crystal structure of a novel carboxypeptidase from the hyperthermophilic archaeon pyrococcus furiosus purification and characterization of a cobalt-activated carboxypeptidase from the hyperthermophilic archaeon pyrococcus furiosus the emerging role of ace in physiology and disease not just angiotensinases: new roles for the angiotensin-converting enzymes trilogy of ace : a peptidase in the renin-angiotensin system, a sars receptor., a partner for amino acid transporters email: a.j.turner@leeds.ac.uk handbook of proteolytic enzymes, rd edn © we thank the british heart foundation, the uk medical research council and the biotechnology and biological sciences research council for support of our work in this area. for reviews, see hamming et al. [ ] , lambert et al. [ ] and kuba et al. [ ] . key: cord- -zg f p authors: chung, mina k.; karnik, sadashiva; saef, joshua; bergmann, cornelia; barnard, john; lederman, michael m.; tilton, john; cheng, feixiong; harding, clifford v.; young, james b.; mehta, neil; cameron, scott j.; mccrae, keith r.; schmaier, alvin h.; smith, jonathan d.; kalra, ankur; gebreselassie, surafel k.; thomas, george; hawkins, edward s.; svensson, lars g. title: sars-cov- and ace : the biology and clinical data settling the arb and acei controversy date: - - journal: ebiomedicine doi: . /j.ebiom. . sha: doc_id: cord_uid: zg f p background: sars-cov- enters cells by binding of its spike protein to angiotensin-converting enzyme (ace ). angiotensin-converting enzyme inhibitors (aceis) or angiotensin ii receptor blockers (arbs) have been reported to increase ace expression in animal models, and worse outcomes are reported in patients with co-morbidities commonly treated with these agents, leading to controversy during the covid- pandemic over whether these drugs might be helpful or harmful. methods: : animal, in vitro and clinical data relevant to the biology of the renin-angiotensin system (ras), its interaction with the kallikrein-kinin system (kks) and sars-cov- , and clinical studies were reviewed. findings and interpretation: sars-cov- hijacks ace to invade and damage cells, downregulating ace , reducing its protective effects and exacerbating injurious ang ii effects. however, retrospective observational studies do not show higher risk of infection with acei or arb use. nevertheless, study of the ras and kks in the setting of coronaviral infection may yield therapeutic targets. in the unprecedented crisis of the covid- pandemic, we must define the epidemiology, predictors of complications and mortality, and potential modifiable risk factors that might prevent or decrease the severity of the disease. recently there has been controversy over whether use of angiotensin-converting enzyme inhibitors (aceis) or angiotensin ii receptor blockers (arbs) might be harmful in severe acute respiratory syndrome coronavirus (sars-cov- ) infection in patients with cardiovascular disease, hypertension, or diabetes mellitus under treatment with these agents. in contrast, it has been suggested that arbs could be protective in the setting of sars-cov- infection. sars-cov- , the coronavirus causing covid- , enters host cells via binding of the virus spike protein to angiotensin-converting enzyme (ace ). aceis or arbs have been reported to increase expression of ace in animal models [ ] . this has led to speculation that use of aceis or arbs might contribute to a higher risk of contracting the infection and worse outcomes of covid- in patients with cardiovascular diseases, hypertension and diabetes [ ] , as these drugs are commonly used in these conditions. moreover, these comorbid conditions are increased with age, which is itself also associated with worse outcomes. as we await evidence from and plan clinical studies, it is essential to understand the biology of the renin-angiotensin system (ras) and its modulation by the sars-cov- virus. . brief primer on ace, ace and the renin-angiotensin system ( fig. ) angiotensin converting enzyme (ace) catalyzes the removal of two residues from the decapeptide angiotensin i (ang i) to form the ace and ace also have intimate roles with the plasma kallikreinkinin system (kks), a hormonal pathway that modulates the intrinsic blood coagulation system, endothelial cell growth and angiogenesis, the complement pathway and ras. the kks consists of plasma and tissue kallikreins, plasma high (hk) and low (lk) molecular weight kininogens, their derivative kinin peptides, including bradykinin (bk) and des-arg -bk, and two g-protein-coupled bradykinin receptors (b r and b r) [ ] . plasma prekallikrein (pk) is activated by blood coagulation factor xii or an endothelial cell serine protease, prolylcarboxypeptidase, to preferentially cleave high molecular hk to liberate bk; the residual cleaved kininogen (chk) is stable in plasma and may be used as a biomarker of kks activation [ ] . bk binds to its receptor b r, which is constitutively expressed in the intravascular compartment, and des-arg -bk binds to the b r, which arises in inflammatory states. separately, tissue kallikreins have preference to cleave lk, releasing lys-bk, which when acted on by several carboxypeptidases, generates des-arg -lys-bk, which also activates b r receptor. the vasodilatory effects of bk are predominantly mediated through b r, which is abundant in vascular endothelium and constitutively expressed in most tissues. b r activation causes a cascade involving nitric oxide synthase (nos), leading to synthesis of nitric oxide (no) and cgmp [ ] . bk and its active metabolite des-arg -bradykinin also agonise b r, which is minimally expressed in healthy tissue, but induced by tissue injury and inflammatory stimuli, playing a role in chronic pain and inflammation [ , ] . activation of both the b and b receptors mediate massive vascular permeability and inflammation, causing marked increases in the levels of inflammatory cytokines, such as il- , il- , il- , il- , and tnf-alpha that have been renin from the kidneys converts angiotensinogen to ang i. ace, and to a lesser extent secreted proteases, such as chymase, catalyzes the conversion of ang i to ang ii. ang ii binds to the angiotensin ii receptor at r, leading to vasoconstriction and cellular injury pathways. aceis inhibit the conversion of ang i to ang ii, reducing ang ii production, while arbs block the ang ii receptor, at r. the ace-ang ii-at r pathway is balanced by ace , which degrades ang ii and ang i to produce ang À and ang i to ang À . ang À and ang À pathways exert protective effects via the receptors mas and at r, respectively. downregulation of ace is associated with an increase in ang ii and activation of the ang ii / at r pathway. upregulation of ace degrades ang i, limiting the substrate for ace, degrades ang ii, limiting its adverse effects, and generates ang À and ang À , leading to protective effects. ace is shed from the cell surface by the action of adam , which is dispensable, but which releases a soluble active form of ace and reduces membrane-bound ace . ang i -angiotensin i. ang ii -angiotensin ii. ang À -angiotensin À . ace -angiotensin converting enzyme. ace -angiotensin converting enzyme . at r -angiotensin receptor. at r -angiotensin receptor. mas r -mitochondrial assembly receptor. acei -angiotensin converting enzyme inhibitor. arb -angiotensin receptor blocker. implicated in the cytokine storm observed with sars-cov- ards [ , ] . the crosstalk between the ras and the kks is profound [ ] . plasma kallikrein converts prorenin to renin. ace is the major intravascular peptidase of bk, producing des-arg -bk and several inactive intermediates, including bk - . ace inactivates des-arg -bk by cleaving its c-terminal residue, but has no effect on bk [ ] . bk receptors are also known to heterodimerise with angiotensin receptors at r, at r, and mas that may augment or diminish their activity [ À ] . benefits of ace inhibition can also be attributed to an intracellular signaling cascade that prevents b r desensitisation. aceis inhibit bk and des-arg -bk degradation, potentiating their effects [ ] . prolylcarboxypeptidase, an enzyme that also produces ang À from ang ii is a pk activator. finally, in relation to influence of thrombosis risk mediated in part through regulation of vessel wall tissue factor, the b r, at r, and mas work in concert to counterbalance the prothrombotic influence of the at r [ , ] . animal models of acute lung injury, sepsis or inflammation (including acid aspiration, lipopolysaccaride challenge, cecal ligation and perforation sepsis) cause increased lung vascular permeability reminiscent of sars-induced acute respiratory distress syndrome (ards). in these models lung ace is decreased and ace-dependent ang ii production is increased, implicating loss of ace protective effects. indeed, chemically induced lung injury leads to severe pathology in ace knockout mice, which is rescued by treatment with the arb losartan [ ] . dual genetic knockdown of ace and ace also attenuates lung injury and is associated with decreased ang ii, suggesting that the beneficial effects of ace in this system are mediated through modulation of ace effects. over-expression of ace or administration of recombinant catalytically active ace in lung injury models has been associated with partial attenuation of injury indices [ , ] . animal ards models also report increased ace, high ang ii, decreased ace levels [ ] and ang ii/at r pathway mediated apoptosis and activation of nf-kb and jak /stat pathways that may be ameliorated by the arb losartan or acei captopril [ ] . in ards models studying the ace /ang À /masr axis, decrease in lung injury with supplemental ang À and rhace have also been reported [ ] . ang À /masr reduces apoptosis and cytokine secretion by inhibiting phosphorylation of jnk-nf-kb. treatment with compound (c ), an at r agonist, also reduced fibrosis, inflammatory cytokines, macrophage infiltration, tnf-alpha and il- in pulmonary hypertension or lung injury models [ ] . these studies suggest a protective effect of ace in the lung, as well as an adverse effect of ang ii. while it appears here that accumulation of excessive ang ii is deleterious, ang ii and at r also have vital and life-preserving roles, for example in maintaining adequate blood pressure and water-electrolyte balance. at r knock-out is lethal, and aceis and arbs are beneficial as they restore more normal ras homeostasis. involvement of the kks, particularly b r activity, in pulmonary injury and ards has been under study for decades. components of the system have been found to be activated irrespective of etiology of lung injury [ , ] . bronchoalveolar lavage (bal) fluid in patients with ards have been found to have increased levels of activated factor xii (fxii), prekallikrein (pk) and high molecular weight kininogen (hk) along with plasminogen and complement proteins [ ] . hk activity but not antigen has been found to be significantly reduced in patients with both sepsis and trauma-induced ards [ ] . in vitro studies indicate that bk stimulates il- , il- , il- and il- production by lung parenchyma [ ] . bk is also known to stimulate type ii pneumocytes to release neutrophil and monocyte chemotactic molecules [ ] . a decrease in ace in lung injury would reduce metabolism of des-arg -bk, potentially increasing its effect via the b r to increase vascular permeability and fluid extravasation. b r antagonism attenuates lipopolysaccharide-induced neutrophil influx in murine models of acute lung injury [ ] . sars-cov was the coronavirus causing the sars outbreak in . the highly glycosylated viral spike proteins form club-shaped projections extending from the surface of the virions, giving the defining appearance of the "corona" around all covs, including sars-cov and sars-cov- , the causative agent of covid- . the spike protein is a key determinant for virus attachment and entry into target cells. animal studies confirm ace as the important receptor for the sars-cov spike protein. in ace knockout mice, only a very small amount of virus could be recovered from lung tissue, supporting the importance of ace as the sars-cov receptor [ ] . infection of wild type mice with sars-cov reduces ace expression [ ] . sars spike protein bound to ace induces shedding of ace with downregulation of ace (fig. ) [ ] . intraperitoneal injection of a sars-cov spike-fc fusion protein into mice with acute acid-induced lung injury worsens acute lung failure that is attenuated by the at receptor blocker losartan [ ] . combining the infection and lung injury studies, the data suggest that both cell surface and released ace catalytic activity producing ang À is protective against lung injury. as sars-cov binding to ace is associated with shedding and downregulation of ace that may worsen injury, loss of ang À protective effects and increased ang ii and des-arg -bk as a result of diminished ace activity may also lead to deleterious effects. injury in these models was attenuated with at receptor blockade. ace expression is lower in rat lung tissues with age [ ] , kidney tissues in type diabetes interactions between the renin-angiotensin system and kallikrein-kinin pathways. factor xii (fxii) autoactivates and then cleaves plasma prekallikrein to activate plasma kallikrein. fxii and plasma kallikrein continue to activate each other, augmenting their activity. plasma kallikrein cleaves high molecular weight (hmw) kininogen to form bradykinin (bk). the renin-angiotensin system angiotensin converting enzyme (ace) or kininase enzymes are involved in kinin metabolism. bk is hydrolyzed by ace into des-arg -bk and inactive products. ace breaks down des-arg -bk into inactive peptides. bk and des-arg -bk interact with two g-protein-coupled bradykinin receptors (b r and b r) at the cellular membrane to cause inflammation with release of il- , il- , il- , and il- cytokines; vasodilation via the nitric oxide synthase (nos), nitric oxide (no) and cyclic gmp pathway; contraction of non-vascular smooth muscle; increased vascular permeability; and fluid extravasation. plasma kallikrein inhibitors and b receptor antagonists (e.g. icatibant) are two categories of medications used to treat hereditary angioedema, a rare genetic condition. with renal disease [ ] and post-mortem brain tissue in alzheimer's disease [ ] . given the novel emergence of sars-cov- , studies on cellular and animal models are just emerging. similar to sars-cov, the receptor for sars cov- is ace . the early availability of sequence information of virus isolates facilitated structural studies confirming the binding of sars cov- spike protein to ace . the sars-cov- spike protein has significant structural homology to the spike protein of sars-cov. both spike proteins bind to ace , but sars-cov- spike protein has been reported to bind with tighter affinity than sars-cov [ ] , so the lessons from sars-cov are expected to apply to sars-cov- , perhaps to an even higher degree. the contribution of the enhanced binding to ace to the infectivity of sars-cov- is not well understood, and binding affinity may reflect genetic variation in ace , but the distribution of ace in lung alveolar cells, mouth, intestines, heart, endothelium, kidneys, testes, and brain may explain effects on lung injury, gastrointestinal symptoms, cardiac damage, acute kidney injury, and reports of late potentially neurally mediated cardiorespiratory depression (fig. ) . like sars-cov, sars-cov- spike protein requires priming by the serine protease tmprss for optimal cell entry [ ] . lung and intestines show ace and tmprss expression and are primary sites of viral entry. the heart shows high levels of ace , but low levels of tmprss expression, which calls into question the mechanism of injury and myocarditis observed in severe cases of covid- . however, a polybasic furin cleavage site has been recently identified in the sars-cov- spike protein [ ] ; furin-like proteases that may contribute to sars-cov- spike protein processing are more ubiquitously expressed and may explain an expanded cell and tissue tropism of sars-cov- compared to sars-cov, which lacks this site [ À ] . we now know that sars-cov- cell entry involves two spike protein subunits, which mediate distinct functions. the s subunit mediates ace attachment through the receptor binding domain, whereas the s subunit, containing the fusion peptide and transmembrane domains, drives fusion of viral and host cell membranes. in addition to attachment, viral entry is determined by spike protein cleavage at two proteolytic cleavage sites, termed s /s and s subunits. unlike sars-cov, the s /s site of the sars-cov- spike protein is processed by the cellular protease furin [ ] . subsequently, processing of the s ' site by the cellular serine protease tmprss (transmembrane protein serine protease ) occurs, and both furin and tmprss are required for entry into human lung cells [ ] . spike protein priming by tmprss was also shown to be essential for spread of sars-cov in infected mouse models [ À ] . although sars-cov- fusion is thought to occur in the endosomes of target cells, the requirement of cathepsins b and l in acidic lysosomes for optimal membrane fusion efficiency in vivo remains unclear (fig. ) [ ] . sars-cov- induced shedding of ace may also reduce its ability to metabolise des-arg-bk, which could contribute to pulmonary inflammation, vascular permeability, and cytokine generation via b r, which is increased in the setting of tissue injury. thrombotic disorders, including mi and stroke, are common features in patients with sars-cov- infection [ ] . sars-cov- virus has been found in endothelium and leads to vessel apoptosis, a risk factor for thrombosis [ ] . the coagulopathy associated with covid- is like disseminated intravascular coagulation (dic) with elevated d-dimer, but high fibrinogens and, in the majority of the patients in the usa, lacking strict criteria for dic. the cross-talk between the kks and coagulation system via the activation of factor xii by kallikrein may contribute to the pro-coagulant state. kallkrein has also been shown to stimulate activation of the complement system through c activation, which likely contributes to the associated coagulopathy [ ] . in experimental models of thrombosis, ace expression was detected in thrombus extract raising the possibility that ace may play a role in the regulation of both thrombotic and hemostatic functions of circulating platelets [ ] . activation of the ace / ang À /mas pathway and/or reduction of ang ii by use of an ace activator (xnt) demonstrated antithrombotic activity in an animal fig. . sars-cov- interaction with ace and tmprss . the spike protein around sars-cov- binds to its receptor, ace , driving fusion of viral and host cell membranes. viral entry is also dependent on spike protein priming at its s /s cleavage site (e.g. by furin) and then at its s site by tmprss , a process inhibited by camostat mesilate and serine protease / furin inhibition. although sars-cov- fusion is thought to occur in the endosomes of target cells, the requirement of cathepsins b and l for optimal membrane fusion efficiency in vivo remains unclear. chloroquine increases the ph of lysosomes and is thought to inhibit the activity of proteases that promote membrane fusion and viral release into the cell. ang i -angiotensin i. ang ii -angiotensin ii. ang À -angiotensin À . ace -angiotensin converting enzyme. ace -angiotensin converting enzyme . at r -angiotensin receptor. mas r -mitochondrial assembly receptor. acei -angiotensin converting enzyme inhibitor. arb -angiotensin receptor blocker. tmprss -transmembrane protein serine protease . model [ ] . sars-cov- replicates in lung tissue, and the lung is a major site for extra-medullary thrombopoiesis [ ] . single-stranded rna (ssrna) viruses, including influenza, were recently demonstrated to augment platelet activation and platelet-to-leukocyte recruitment through the platelet toll-like receptor (tlr ) [ ] . since sars-cov- , like influenza, is also a ssrna virus, the possibility exists that sars-cov- may promote dysregulated platelet activity directly through surface receptor-mediated pathways or indirectly by secreting platelet-derived molecules that regulate the coagulation cascade. lastly, angiotensin receptors are expressed on the surface of platelets, and medications inhibiting the ras attenuate platelet activation [ , ] . therefore, the impact of anti-platelet medications and aceis/arbs on platelet function and thrombotic events in patients with sars-cov- needs further investigation. helpful or harmful? though arbs and aceis may be associated with an increase in ace expression, which theoretically may enhance viral infection, their inhibition of the ras with increase in ace expression, reduction of ang ii or ang ii effects, and increase in ang À and ang À may have protective effects. arbs may increase ang ii by competing with binding to at r, but this may create more available substrate for ace and formation of ang À with its downstream protective effects. binding of substrates to ace may induce conformational changes in ace ; it is unknown whether these interactions would enable or reduce sars-cov- spike protein binding. specific effects of aceis and arbs on this process are not known. genetic factors, including genetic variability in ace polymorphisms, may also determine functional roles of ace for aceis and arbs, as well as in its interaction with the cov- spike protein, and will be important to dissect in the future [ ] . finally, acei could reduce metabolism of bk, leading to b r-and b r-mediated inflammatory, vasodilatory, vascular permeability and fluid extravasation effects. initial clinical data showing conflicting outcomes in covid- associated with arb or acei use were confounded by lack of adjustment for co-morbidities (table) . in a study of covid- patients from wuhan, china, acei or arb use was higher in patients with myocardial injury and elevated troponin t (tnt) levels ( %) compared to patients with normal tnt ( %), p = [ ] . in patients with covid- on antihypertensive therapy, severe disease was observed in % on and % not on an acei or arb, but this was not significant due to the small sample size [ ] . a pre-publication report of covid- + patients with hypertension reported arb use (n = ) was associated with lower occurrence of severe disease (or , % ci À , p = ). in a study of hospitalized covid- patients with hypertension, acei and/or arb use (n = ) was not significantly different between patients with severe vs. non-severe illness or in non-survivors vs survivors [ ] . in a report of acute inpatients from the uk acute hospital trust, died or were transferred to a critical care unit within days of symptom onset. a lower rate of this endpoint occurred in patients on an acei or arb (or , ci À , p < ), adjusting for age, gender, hypertension, diabetes mellitus, chronic kidney disease, ischaemic heart disease and heart failure [ ] . in a preprint from the veterans administration birth cohort, patients tested positive for covid- among tested, and % were on an acei or arb; in adjusted analyses there were no differences in covid- test positivity, hospitalisation, or intensive care in patients receiving or not receiving an acei or arb [ ] . recently studies using adjusted and/or propensity-score adjusted or matched analyses have been published (table) . three tested associations of acei and/or arb use on covid- test positivity and found no significant differences. disease severity and hospital outcomes were assessed in studies. in a study of patients with hypertension, were on an acei or arb; propensity score matched cohorts showed a lower risk of all-cause mortality (hr , % ci À , p = ) and septic shock in patients on an acei or arb. in a study of , patients tested, were positive; acei and/or arb use was associated with a higher rate of hospital admission with acei and acei or arb use was associated with a higher risk of admission to the icu, but with no significant difference in need for mechanical ventilation [ ] . in a study of covid- positive patients, arb or acei use was not associated with degree of disease [ ] . another study of , tested patients, including who tested positive, acei, arb, or either were not associated with test positivity or severe covid- disease [ ] . in a study of hospitalized patients with covid- , had hypertension of which were treated with renin-angiotensin-aldosterone system inhibitors (raasi) and were not; raasi use was not associated with severity of disease or mortality [ ] . lastly, in a study of patients with covid- in danish national administrative registries, prior acei or arb use was not associated with death or severe covid- ; in a nested case-control study of patients with hypertension, acei/ arb use was not significantly associated with covid- diagnosis [ ] . taken together, it is now consistent clinical evidence that acei or arb use does not appear to predispose to sars-cov- infection, which was the main concern raised due to postulated effects of aceis or arbs in raising ace expression. recent studies have shown no association of acei or arb use with sars-cov- test positivity. studies do not indicate harm from acei or arb use in terms of severity of disease but with some conflicting results regarding the benefit vs. risk of aceis. however, the overall the balance appears to be in favor of no significant harm from acei or arb use in covid- , though larger studies are needed to assess the relative effects of aceis versus arbs, whether continuation or withdrawal of these agents impact outcomes, or if acei/arb use may actually be beneficial in alleviating lung or other organ injury in patients with covid- . given the current reassuring data showing no significant association of acei or arb use with test positivity, lack of consistent or convincing evidence as to the risk or benefit of an acei or arb, as well as the potential harm that may occur with withdrawing of aceis or arbs in patients with cardiovascular and other diseases [ ] , findings support the european society of cardiology, american heart association, american college of cardiology, and the heart failure society of america recommendations that patients on these therapies should be continued as clinically indicated. the recent data show consistent lack of an association with sars-cov- positivity. however, there remains a need to further assess impact of aceis and arbs on severity of disease, potentially through larger or randomized studies. the spike protein is a target for drug discovery and vaccine development. blocking the spike protein-ace interaction sites may be targetable with antibodies or small molecules, and use of soluble ace may competitively bind to the spike protein [ ] . strategies to increase ace shedding from cells may be protective against viral infection [ ] . furin inhibitors or other serine protease inhibitors may inhibit sars-cov- replication via the s /s cleavage site [ ] . tmprss is dispensable for homeostatic function and blocked by the serine protease inhibitor camostat mesilate, a drug approved in japan for unrelated conditions [ ] . our recent systems biology study suggested several repurposable drugs for potential treatment of covid- , including melatonin and arbs (i.e., irbesartan) [ ] . melatonin regulates expression of several cellular targets of human cov, including ace , ang ii and at r [ ] . hydroxychloroquine and chloroquine have been commonly tried for treatment of covid- . besides inhibiting viral-endosome fusion and release of viral particles to the cell by reducing endosomal acidification, chloroquine impairs terminal glycosylation of ace , which may have effects on binding affinity between ace and co-v spike protein [ ] . however, efficacy remains to be established, and randomized trials are ongoing. therapeutic application of ang À and ang À is limited because of the short half-life of these peptides and unavailability of fda-approved drugs that can substitute for the potential benefits attributed to these peptides. interventions directed at blocking bk and the pathways leading to its formation may also be of benefit. hereditary angioedema, a rare genetic disorder causing predisposition to attacks of angioedema, is treated with medications to suppress activity in the kks. these medications largely consist of direct kallikrein inhibitors, b r antagonists, and replacement with c inhibitor. bk's role in covid- is under investigation, and use of these suppressive medications is being explored. until such time when there is a highly effective antiviral or a vaccine, these adjunctive approaches need to be developed. in clinical practice the protective effects of arbs and aceis are thought to be associated with an increase in ace expression and their inhibition of the overactive renin-angiotensin system through reduction of ang ii effects. coronavirus infection hijacks ace expression to invade cells and spread infection-associated damage, downregulating ace expression, reducing its protective effects and exacerbating the injurious ang ii effects. retrospective observational studies do not show associations with higher risk of infection for persons receiving aceis or arbs. however, controlled clinical trials would be needed to determine the risks or benefits of these agents in treating covid- . studies in sars-cov- models and clinical retrospective and prospective studies in patients might further clarify these important questions. such studies may also identify plausible therapeutic agents for targets within the ras and kks in the setting of coronaviral infection. important questions remaining for future research include whether drugs targeting components of the ras or the kks might be helpful in the treatment of patients with covid- . prospective controlled clinical trials are needed. basic research on mechanisms to determine if ace expression affects viral infectivity in vitro and expression of components of the ras and kks in infected tissues are needed to help clarify the role of cell-bound or shed ace in covid- pathophysiology. investigations into specific cell types vulnerable to sars-cov- infection may help focus targeting of therapies. search strategy and selection criteria. data for this review were identified by searches of pubmed with search terms including combinations of ace inhibitors, arbs, covid- , sars-cov- , renin-angiotensin system, kallikrein-kinin system. md -writing, revisions, literature search, figures, responsibility for the manuscript sadashiva karnik, phd -ras expertise, critical writing and revisions, figures joshua saef, md -kks expertise, critical writing and revisions, figure cornelia bergmann, phd -coronavirus expertise, critical writing and revisions john barnard phd -cell biology and immunology expertise, critical revisions james b. young, md -acei/arb, heart failure/cardiology expertise, critical comments/revisions neil mehta, md -acei/arb expertise, contributed data/studies on ace/arbs scott j. cameron md, phd -expertise in platelets and thrombosis in covid- , critical writing, revisions keith r. mccrae, md -expertise in thrombosis, critical revisions alvin h. schmaier, md -expertise in thrombosis, kks, critical revisions jonathan d. smith, phd -revisions, insights in ace shedding ankur kalra, md -acei/arb expertise in covid- , review and suggestions for manuscript surafel k. gebreselassie, md -ras revisions/suggestions for manuscript george thomas, md -ras revisions/suggestions for effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme clinical characteristics of coronavirus disease in china role of the ace /angiotensin - axis of the renin-angiotensin system in heart failure significance of angiotensin - coupling with mas receptor and other gpcrs to the renin-angiotensin system: iuphar review trilogy of ace : a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters map kinase/phosphatase pathway mediates the regulation of ace by angiotensin peptides ace : angiotensin ii/angiotensin-( - ) balance in cardiac and renal injury ectodomain shedding of angiotensin converting enzyme in human airway epithelia detection of soluble angiotensin-converting enzyme in heart failure: insights into the endogenous counter-regulatory pathway of the renin-angiotensin-aldosterone system bradykinin receptor ligands: therapeutic perspectives the contact activation and kallikrein/kinin systems: pathophysiologic and physiologic activities counter-regulatory renin-angiotensin system in cardiovascular disease expression cloning of a rat b bradykinin receptor expression cloning of a human b bradykinin receptor a hypothesized role for dysregulated bradykinin signaling in covid- respiratory complications release of cytokines from isolated lung strips by bradykinin the kallikrein-kinin and the renin-angiotensin systems have a multilayered interaction hereditary angioedema angiotensin ii type receptor-bradykinin b receptor functional heterodimerization at -receptor heterodimers show enhanced gprotein activation and altered receptor sequestration beta-arrestin prevents preeclampsia by downregulation of mechanosensitive at -b receptor heteromers serum metabolism of bradykinin and des-arg -bradykinin in patients with angiotensin-converting enzyme inhibitor-associated angioedema angiotensin - and mas decrease thrombosis in bdkrb -/-mice by increasing no and prostacyclin to reduce platelet spreading and glycoprotein vi activation reduced thrombosis in klkb -/-mice is mediated by increased mas receptor, prostacyclin, sirt , and klf and decreased tissue factor angiotensin-converting enzyme protects from severe acute lung failure ace exhibits protective effects against lps-induced acute lung injury in mice by inhibiting the lps-tlr pathway angiotensin-converting enzyme prevents lipopolysaccharide-induced rat acute lung injury via suppressing the erk / and nf-kappab signaling pathways losartan, a selective antagonist of at receptor, attenuates seawater inhalation induced lung injury via modulating jak / stats and apoptosis in rat reninangiotensin-system, a potential pharmacological candidate, in acute respiratory distress syndrome during mechanical ventilation activation of the contact system of plasma proteolysis in the adult respiratory distress syndrome studies on the pathogenesis of the adult respiratory distress syndrome bradykinin stimulates type ii alveolar cells to release neutrophil and monocyte 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genetic variant and influences monocyte transendothelial migration furin, a transcriptional target of nkx - , has an essential role in heart development and function a multibasic cleavage site in the spike protein of sars-cov- is essential for infection of human lung cells tmprss and furin are both essential for proteolytic activation and spread of sars-cov- in human airway epithelial cells and provide promising drug targets tmprss contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection protease inhibitors targeting coronavirus and filovirus entry tmprss and adam cleave ace differentially and only proteolysis by tmprss augments entry driven by the severe acute respiratory syndrome coronavirus spike protein clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study ponatinib treatment promotes arterial thrombosis and hyperactive platelets kallikrein cleaves c and activates complement ace activation promotes antithrombotic activity the lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors the role of platelets in mediating a response to human influenza infection angiotensin ii at receptor antagonists inhibit platelet adhesion and aggregation by nitric oxide release novel angiotensin ii at ( ) receptor antagonist irbesartan prevents thromboxane a ( )-induced vasoconstriction in canine coronary arteries and human platelet aggregation comparative genetic analysis of the novel coronavirus ( -ncov/sars-cov- ) receptor ace in different populations cardiovascular implications of fatal outcomes of patients with coronavirus disease (covid- ) renin-angiotensin system inhibitors improve the clinical outcomes of covid- patients with hypertension association of renin-angiotensin system inhibitors with severity or risk of death in patients with hypertension hospitalized for coronavirus disease (covid- ) infection in wuhan, china angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers are not associated with severe covid- infection in a multi-site uk acute hospital trust covid- testing, hospital admission, and intensive care among , , united states veterans aged association of use of angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers with testing positive for coronavirus disease (covid- ) renin-angiotensin-aldosterone system blockers and the risk of covid- reninangiotensin-aldosterone system inhibitors and risk of covid- association of hypertension and antihypertensive treatment with covid- mortality: a retrospective observational study association of angiotensin-converting enzyme inhibitor or angiotensin receptor blocker use with covid- diagnosis and mortality renin-angiotensin-aldosterone system inhibitors in patients with covid- angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target characterization of the receptor-binding domain (rbd) of novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine network-based drug repurposing for novel coronavirus -ncov/sars-cov- idiopathic pulmonary fibrosis (ipf) signaling pathways and protective roles of melatonin remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro anti-hypertensive angiotensin ii receptor blockers associated to mitigation of disease severity in elderly covid- patients association of inpatient use of angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid- this work was supported by national institutes of health (nih) grants r hl (mkc, jb, js); r ns , r ns (ccb); r ai (jct); u hl (krm); k hl (sjc); r hl and hl (sk); the nih national center for research resources for case western reserve university and cleveland clinic clinical and translational science award ul -rr ; and the cleveland clinic center of excellence for cardiovascular translational functional genomics, funded by the cleveland clinic heart, vascular and thoracic institute and lerner research institute philanthropy funds. the funders had no role in paper design, data collection, data analysis, interpretation, or writing of the paper.the authors gratefully acknowledge the graphics support provided by mary ann citraro and secretarial support provided by anastasia harris. key: cord- -wtxja i authors: cabbab, iris louise n.; manalo, rafael vincent m. title: anti-inflammatory drugs and the renin-angiotensin-aldosterone system: current knowledge and potential effects on early sars-cov- infection date: - - journal: virus res doi: . /j.virusres. . sha: doc_id: cord_uid: wtxja i severe acute respiratory syndrome coronavirus (sars-cov- ) is the etiologic agent of coronavirus disease (covid- ), and is genetically related to the sars and middle east respiratory syndrome (mers-cov) coronaviruses. recent studies have reported that similar to sars-cov, this strain expresses a spike protein (s) with a receptor binding domain (rbd) that binds to angiotensin-converting enzyme (ace ) – an enzyme expressed mostly in the endothelium, kidneys, heart, gastrointestinal tract and lungs – to facilitate viral entry and intracellular replication. incidentally, the renin-angiotensin-aldosterone system (raas) is integral to physiologic control of both ace and ace expression, and is an essential system utilized by sars-cov- , albeit with varying schools of thought on how it can affect viral entry. in this paper, we will review current knowledge on the raas and how it can be affected by non-steroidal anti-inflammatory drugs (nsaids) and corticosteroid use at the organ and cellular levels. we will then discuss the relevance of these interactions on organ-specific ace expression, and provide scientific insights on how this mechanism can potentially affect sars-cov- infection in the early phases of disease. from the standpoint of other known viruses, we will then aim to discuss the potential uses or restrictions of these drugs in viral infection, and provide an update on relevant studies about covid- . coagulation (dic). likewise, it is prudent to assume that cfr is better interpreted after agestandardization is done, or when it is adjusted for the presence of comorbidity, which has been done in some reports including that of the centers for disease control and prevention (cdc) [ ] . a possible contributor to the risk of severity or to variations in the cfr, however, may also come from the patient's medications. there have been studies proposing that covid- morbidity and mortality are a result of excessive inflammation, such as airway inflammation and cytokine storms [ ] , and infected patients in the early phases of disease may experience malaise or fever and may seek symptomatic relief through analgesics like paracetamol and non-steroidal anti-inflammatory drugs (nsaids) such as ibuprofen. similarly, patients with chronic inflammatory diseases may take nsaids or corticosteroids as management for their disease, which may increase their susceptibility to microbial infection. since covid- is a viral illness, there have been concerns that anti-inflammatory drug use can aggravate sars coronavirus infections [ , ] , but evidence on this speculation has not been unified and is even contradicting. interestingly, the membrane receptor of sars-cov- , which is ace , is an essential component of the regulatory mechanism of the renin-angiotensin-aldosterone system (raas), and this hormone pathway may interact with the pathophysiology of covid- . to this end, this paper will review the mechanisms of the raas and its coincidence with the anti-inflammatory pathways of nsaids and corticosteroids both at the cellular and organ levels. we will discuss current evidence on the effects of these anti-inflammatory drugs on sars-cov- infection, as well as the potential interactions of pathways upstream or downstream of ace on the raas, in an effort to bridge these pathways in the setting of covid- . based on the evidence collated, we will then compare the anti-inflammatory drug classes included in this review to determine the potential uses or restrictions of these drugs in early sars-cov- infection. for this review, we did a literature search at ncbi pubmed and medline databases to gather current studies on the covid- -raas-anti-inflammatory drug interactions using the terms 'sars-cov- ' or 'covid- ', matched with 'renin-angiotensin system', 'renin-angiotensin-aldosterone-system', 'raas' or 'ras', which yielded unique results. we further narrowed the search by including only the studies which investigated the effects of anti-inflammatory drugs or corticosteroids on covid- , using additional terms for the string such as 'corticosteroids', 'nsaid', 'steroids', 'anti-inflammatory' or 'inflammation', and by narrowing the publication type to 'journal article', 'clinical trial', 'review', 'systematic review', 'meta-analysis', or 'randomized controlled trial'. we then analyzed each article to include only those that proposed either the mechanisms of action of sars-cov- on the raas or of the drugs that interact with sars-cov- or the raas, to a final count of . from the following studies, all journal articles, clinical trials, systematic reviews and meta-analyses, excluding literature reviews, were included only if they provided experimental or clinical evidence, which narrowed the inclusion to five articles (two retrospective cohort studies, one case series, one in silico analysis, and one in vitro study). a second, less-stringent search was done which included studies on anti-inflammatory or corticosteroid drug use for covid- with or without associations to the raas, using the similar terms as mentioned above. this search returned seven studies which were either systematic reviews or meta-analyses or both, and were included in this review (supplementary file ). for the said searches, we considered articles that are not only in the english language, but also those written in chinese, dutch, french and russian. the two literature searches were accomplished on august . on the other hand, we included studies on the epidemiology of covid- and the molecular biology of sars-cov- based on chronology to reflect the turn of events during the pandemic. for most of the review, basic science researches that explain or complete mechanistic pathways on the physiology and biology of the raas or of the mechanisms of anti-inflammatory drugs and corticosteroids were referenced from pubmed and google scholar at our discretion. in addition, pertinent references j o u r n a l p r e -p r o o f suggested by the reviewers were included as needed, especially those that were not covered at the time of the literature search. it is important to note that since the approach of this paper is to provide current knowledge on the anatomic, physiologic and molecular bases of anti-inflammatory drug and corticosteroid action on the raas, this paper will not demonstrate a systematic review or meta-analysis of current clinical evidence, but will only provide insight on the probable influences of the discussed pathways on early sars-cov- infection. further, since there is a present lack of definitive studies investigating the association between nsaid or corticosteroid drug use and covid- outcome severity, such clinical analyses are only warranted as more evidence is obtained. in late of december , dr. wang and researchers from the institute of pathogen biology in china obtained the complete sequence of sars-cov- from bronchoalveolar lavage specimens of patients in wuhan presenting with acute respiratory distress syndrome (ards) [ ] . a subsequent study then extracted sars-cov- dna from nine patients with covid- , which found a % and % identity with sars-cov and mers-cov, respectively [ ] . similarly, another study reported an % similarity between the sars-cov and sars-cov- genomic sequences, with high degrees of homology to the sars-like coronaviruses isolated from bats [ ] . since sars-cov was known to bind to human angiotensinconverting enzyme (ace ) for viral entry, these data suggested that covid- may also utilize its high affinity binding for human ace to enter host cells, by virtue of its conserved receptor binding domain (rbd), which was later confirmed to be true [ , , ] . in , khan et. al. developed a pilot clinical trial using human recombinant soluble ace , also known as gsk , as a potential treatment for acute lung injury. the two-part clinical trial, although showed a successful interaction of hrsace and its targets by evaluating the modulation of ras, did not elaborate any clinical changes or improvements j o u r n a l p r e -p r o o f upon administration of gsk [ ] . it did, however show an increase in ang( - ) synthesis from ang ii, offering protection against heart failure with reduced and preserved ejection fraction largely due to ace deficiency, as well as preventing ang ii-induced myocardial hypertrophy, diastolic dysfunction, and myocardial fibrosis [ , ] . earlier this year, monteil et. al did a similar study where they engineered human blood vessel organoids and human kidney organoids, both of which expressed a sufficient amount of ace , to emulate sars-cov- infection in human tissues and organs. this idea was built on the knowledge that sars-cov- mainly uses ace as the entry point into cellular invasion. by introducing human recombinant soluble ace (hrsace ), the s-protein region of sars-cov- was competitively bound by the enzyme, which effectively limited the binding and entry of sars-cov- thereby preventing infection in a dose-dependent manner [ ] . several more clinical trials emerged which suggested the importance of hace to sars-cov- infection, and which led hrsace to become one of most promising therapeutics for the early prevention of covid- to date. there were six amino acids identified in the rbd that is crucial for ace binding, and while five of these differed between sars-cov and sars-cov- , the binding affinity of sars-cov- rbd to human ace receptors was found to be times higher than its counterpart spike protein in sars-cov [ , ] ). this ability of sars-cov- to bind the ace enzyme with a stronger affinity than sars-cov and hcov-nl under a non-ideal configuration strongly implies that prior mutations have been occurring as a form of natural selection. that is, sars-cov- may have been circulating the wet animal markets for several years before the outbreak and infecting individuals without triggering any symptoms [ , ] . the downstream effect of the ace ligand-receptor complex is the degradation of angiotensin ii (ang ii) to produce angiotensin ( - ), initiating a negative feedback mechanism that inhibits further production of ang ii. decreased ang ii results in decreased salt and water reabsorption, which lowers blood pressure and vascular tone, and effectively prevents the excessive effects of ang ii [ , ] . when the sars-cov- viral spike s protein binds the human ace enzyme, it fuses with the cell membrane to j o u r n a l p r e -p r o o f initiate viral entry by activating signal transduction pathways, resulting to cell uptake of the virusreceptor complex via a process called clathrin-mediated endocytosis. moreover, the s protein of sars-cov- possesses an s /s site, which is cleaved by furin, to encourage entry of sars-cov- into the cell while evading the host immune response [ , , , ] . in the process, ace is inhibited and internalized, and reduced membrane ace affects the homeostasis of raas due to decreased ang ii breakdown. ang ii is a direct vasoconstrictor known for its predominantly vasoactive properties, and its excessive production is associated with acute hemodynamic changes, such as hypertension and a drop in glomerular filtration rate (gfr). this triggers chronic inflammation if left unaddressed, and eventually leads to end-organ failure due to fibroid formation [ , , ] . ace is an active homologue of ace seen in a diverse group of tissues including the oral and nasal mucosa, the lungs, the gastrointestinal tract, blood forming organs, liver, kidney, brain, and endothelial cells, with the majority seen localizing in the tongue and esophageal epithelial cells, enterocytes, male reproductive cells, type ii lung alveolar epithelial cells, renal tubules, ductal cells, bladder urothelial cells, endothelial cells, and cardiomyocytes [ , , , ] . interestingly, the number of organs that express ace continues to increase as more data is published, which can be monitored using the covid- cell atlas database (https://www.covid cellatlas.org/). the ramifications on the ability of the virion to provoke cellular or tissue damage remain intriguing, but for one, it is dependent not only on the presence or absence of ace but also on the amount of ace on a certain organ or tissue. one of the most commonly studied regions of sars-cov- entry is the respiratory system, for which it is thought to be the main route of infection, transmission, and pathophysiology. the specialized type ii alveolar cells express an abundant amount of ace , and ace inactivity due to sars-cov- -induced j o u r n a l p r e -p r o o f depletion early in the infection may disturb the ras homeostasis, leading to impaired tissue repair mechanisms, increased vascular permeability, fluid accumulation in extra-alveolar spaces, and oxidant/antioxidant imbalance [ ] . in a study by ackerman et. al., which examined postmortem lungs of patients who died from covid- and compared them to lungs obtained from patients who died from ards secondary to ah n and to age-matched healthy lungs, covid- patients were found to suffer more frequently from severe endothelial injury due to cell membrane damage by presence of sars-cov- . widespread thrombosis with microangiopathy was seen, and patients were times more likely to experience alveolar capillary microthrombi, and . times more likely to experience intussusceptive angiogenesis than with flu [ ] . this phenomenon of thrombosis and other vascular events in covid- is likely the result of an abundance of ace in endothelial cells, which permits sars-cov- infection along the endothelium. this will be further discussed in later sections. sars-cov- has also been studied to disseminate into the cns, by initially invading the peripheral nerve terminals via the synapse-connected route, through the mechanoreceptors and chemoreceptors in the lungs, and into the medullary-cardiorespiratory center [ , ] . however, ace is not particularly abundant in neurons, and other pathways of infection are being investigated. the oral and nasal mucosa also holds a significant distribution of ace , especially on the tongue epithelial cells, which may explain its route of infection [ ] . if and how the virus affects the activity of the tongue epithelial cells were investigated by a cross-sectional study in l. sacco hospital in milan, italy, where the prevalence of olfactory and taste disorders in hospitalized covid- patients were obtained. of the hospitalized covid- patients, all patients who were coherent and responsive reported the persistence of olfactory and taste disorders throughout the disease [ ] , indirectly supporting the hypothesis of oral and mucosal routes of invasion to the nervous system. interestingly, ace population is also pervasively seen in pancreatic cells, which in fact shows a higher expression profile than pulmonary cells. this possibly makes the pancreas, by virtue of ace -dependent viral entry, one of the major targets of sars-cov- . complications may include acute pancreatitis resulting from direct injury to pancreatic acinar cells or uncontrollable systemic inflammatory response from cytokine storm syndrome, both of which have been documented in the disease [ ] . acute pancreatitis due to sars-cov- infection could pose an additional threat to people afflicted with diabetes mellitus (dm) types and by promoting metabolic complications as the infection continues to alter pancreatic β-cell function, which may in fact explain the feared risks of severe covid- among patients with dm [ ] . in the kidneys, the ace expression in the proximal tubules remains the highest in concentration, with markedly lower concentrations in the glomeruli [ ] . stz-treated rats in previous experiments were found to have higher ace mrna/ace mrna ratio, a marker of kidney damage that resulted in renal injury similar to human diabetic nephropathy [ , ] . due to a higher ace/ace ratio, there was less ang- ( - ) synthesized in the kidney, which can effectively eliminate its renoprotective effects [ ] . in an experimental study using c bl/l mice models, investigators compared the physiology of normal - week-old c bl/ mice against ace -/y mice which lacked ace expression. the enos expression in both protein and mrna levels and no concentrations in the aortas of ace -/y mice were decreased, along with urine and plasma nitrite concentration. on the other hand, lipid peroxidation was markedly increased, in contrast to a decreased superoxide dismutase level in the aorta homogenates of ace -/y mice subjects -an indication of impaired antioxidant activity leading to the formation of reactive oxygen species (ros) [ ] and observed vascular dysfunction in ace -negative c bl/ mice [ ] . disturbances in the oxidative activity of the vascular system could pose a risk for acquiring inflammation, vascular remodelling and vascular injury, eventually leading to activation of the coagulation cascade that could cause microthrombi formation [ , ] . taken together, these correlate to the negative consequences impacting the vascular homeostasis of sars-cov- -induced ace deficiency, with partial relations to the renin-angiotensin-aldosterone system (raas), which will be discussed. j o u r n a l p r e -p r o o f a correspondence by fang et al published at the lancet this march discussed that hypertensives and diabetics taking ace inhibitor (acei) and angiotensin receptor blocking (arb) drugs may be at an increased risk of infection and severity by sars-cov- and covid- , respectively, citing three studies wherein diabetes and hypertension were major comorbidities of patients with severe covid- and of non-survivors [ ] . several criticisms were received by this article, mainly due to the lack of clinical evidence for many of the author's claims at the time. a descriptive study on the pharmacologic characteristics of covid- patients in the toulouse university hospital intensive care unit (icu) in france showed that many of the patients who were intubated had either taken paracetamol ( . %), arbs ( . ), calcium channel blockers ( . %), corticosteroids ( . %), or metformin ( . %). however, these patients also had pre-existing co-morbid conditions that warranted medication intake, such as obesity, arterial hypertension, and diabetes mellitus, which may regard the evidence as coincidental at best. since the study was also descriptive (case series), the results and their implications can only be suggestive [ ] . however, a drug interaction cannot be ruled out. in terms of the raas, interaction between arb/acei drugs and viral entry is plausible, since these drugs either inhibit ace or the receptor of its enzymatic product angiotensin ii, and a known physiologic response of cells to inhibition or internalization is feedback upregulation [ ] . since the known human receptor of sars-cov- is ace , then it is possible for hypertensives chronically taking aceis to be susceptible to sars-cov- infection, if feedback upregulation of ace expression does occur, or due to the patients' impaired ability to decrease ang ii secondary to renin production, all of which may increase membrane ace levels [ , ] (fig. ) . a similar pathway is expected for arbs, since inhibition of angiotensin receptors can trigger a feedback mechanism to increase ang ii synthesis resulting to an upregulation of ace expression ( fig. ), and this has been shown in previous studies [ , , , ] . several other studies have since followed this hypothesis closely, and have in the process unraveled the role of the renin-angiotensin-j o u r n a l p r e -p r o o f aldosterone system (raas) on the observed clinical outcomes of some covid- patients, albeit with a surprisingly different mechanism. two experimental studies, one in silico and another in vitro, have clarified and suggested the role of the raas, as follows. an in silico approach, which utilized the coxpresdb v , kyoto encyclopedia of genes and genomes (kegg) pathway and the human protein atlas databases to determine the co-expression, molecular interactions and organ-specific expression of ace , neprilysin and carbonic anhydrase (ca), have shown that these three enzymes were highly coexpressed in the local ras, with important overlapping functions on the production of ang-( - ) and ang-( - ) for the regulation of systemic vascular resistance and the conversion of co in times of hypoxia. further, the authors found that these enzymes were most likely co-expressed at organs of interest such as digestive, renal, respiratory, and reproductive systems, with the exception of ca which is ubiquitous [ ] . likewise, an in vitro study showed that lung epithelial cells infected with sars-cov expressed unique ras genes within a -hr period, which correlated well with the time of viral infection. these genes were divided into those that were significantly upregulated during first hrs (ace, ace , insulin-like growth factor receptor (igf r), angiotensinogen (agt), epidermal growth factor receptor (egfr), matrix metalloendopeptidase (mme), alanyl aminopeptidase (anpep)) and those that were significantly upregulated by the th hr (arginyl aminopeptidase (arpep), egfr, anpep, neurolysin, ace , igfr, leucyl and cysteinyl aminopeptidase (lnpep) and cathepsin d), all of which has clearly shown the early response of the raas on sars infection [ ] . studies have also recently reported that cleavage of ace due to the interactions with the viral s protein leads to inhibition of ace function through the ras ace/ang ii/at a arm [ , ] . another study has suggested that excessive activation of raas by sars-cov- plays a role in the onset of acute respiratory distress syndrome (ards), a severe form of acute lung injury [ ] . in the presence of ards, fluid builds up in the alveoli, damaging the surfactant and promoting inflammation, eventually leaving fibrotic scars that prevents complete oxygen uptake. it is important to note that the binding of the sars-cov- spike rbd to ace effectively inhibits j o u r n a l p r e -p r o o f the enzyme receptor, leading to endocytosis and early ace depletion. this mechanism, which can interact with many other pathways, can also lead to the onset of ards, by virtue of feedback activation of the raas or by other mechanisms dependent solely on ace . to date, there seems to be no sufficient clinical evidence that can confirm the biological relevance of these experimental results. in a retrospective multicenter cohort study of covid- -positive geriatric patients in belgian nursing homes, a non-significant association was observed between arb/acei use and severe or poor outcomes (or . ; ci . - . ) [ ] . in another retrospective cohort study of geriatric covid- patients with diabetes mellitus (dm) in daegun, south korea, use of acei/arb drugs did not significantly affect the severity of outcome of covid- , apart from an incidental protective effect against acute cardiac injury (or . ), which was expected from the mechanism of action of the drugs [ ] . another review article discussing the interaction between raas inhibitors and ace in the context of covid- claimed that although angiotensin-receptor blockers and mineralocorticoid blockers increased ace expression in a number of experimental and clinical models, ace inhibitors, while increasing cardiac ace mrna levels, had no effect on ace activity and expression. a direct inhibitor of renin, aliskiren, in contrast was associated with a decrease in ace expression but the mechanism of action remains to be studied further [ ] . in contrast, wysocki and authors investigated the effects of aceis and arbs on membrane and total ace expression in lung and kidney cells and lysates, respectively, and found that use of these drugs do not significantly affect both the membrane and total ace protein levels in mouse lungs. likewise, acei (captopril) and arb (telmisartan) administration did not increase membrane ace levels in mouse idneys, but rather decreased them. although preliminary, this study provided direct evidence supporting continued use of aceis and arbs in covid- , which also implied that feedback upregulation of ace does not occur even if the ras pathway is inhibited, at least for a span of weeks [ ] . it will also be interesting to j o u r n a l p r e -p r o o f determine similar responses to aceis and arbs in mouse models of hypertension, either genetic or nongenetic, to determine the consistency of these results in the setting of compensatory mechanisms. besides these studies, others that directly assessed the clinical effects of acei/arb drugs on covid- outcome severity were not found during the literature search. hence, the direct negative effects of acei/arb drugs on covid- outcome, as earlier hypothesized by clinicians and researchers, are at best experimental and, to the best of our knowledge, not yet supported by clinical evidence. a more in-depth discussion on the interactions between sars-cov- entry and acei/arb drugs can be found elsewhere, and readers are referred to those materials for more information. the renin-angiotensin-aldosterone system (raas), or simply the renin-angiotensin system, is a strong physiologic pathway that controls various processes relating to salt and water homeostasis (fig ) . classically, the chronological order of the raas begins with the cleavage of angiotensinogen into ang i by renin, followed by the conversion of ang i into ang ii by ace which occurs extensively in the lungs. ace catalyzes both ang i and ang ii to produce ang ( - ) and ang ( - ), which provides negative feedback to ang ii thereby modulating its effects. prior to ace interaction, ang ii modulates the raas promotes vasopressin release from the posterior pituitary gland, and directs the adrenal gland to produce aldosterone, which then promotes na + and h o retention in normal physiology. in patients with cardiovascular disease, available ang ii reacts with angiotensin receptor (at r) to stimulate pathologic behavior such as vasoconstriction, oxidative stress and profibrotic events. ang ii is then converted into ang ( - ) by ace , followed by the activation of at r and masr. elevation of ang ii may increase plasma aldosterone levels through the regulatory action of adrenocorticotropic hormone contrast, ang-( - ) increases urinary na + excretion, endothelial oxide activity, insulin sensitivity [ ] . this further shows that the ace /masr/at r axis performs an opposite reaction with the ace/ang ii/at r axis, offering protection against hypertension, fibrosis, and oxidative stress [ ] . as mentioned above, sars-cov- exploits and depletes these ace receptors to facilitate viral host cell entry and increase its pathogenicity. the raas is dysregulated following ace depletion, promoting effects downstream of ang ii and therefore increasing aldosterone secretion. aldosterone acts by inhibiting sodium excretion in the distal convoluted tubule and collecting ducts via the mineralocorticoid receptors. while na + is reabsorbed and h o is retained, potassium is consequently excreted, and an excess can lead to wastage and hypokalemia. this is consistent with the findings of chen et. al. from a cohort study of covid- -positive patients, wherein they classified patients as either being severely hypokalemic ( . %), hypokalemic ( . %), or normokalemic ( . %). they found that patients with higher hypokalemia also had increased creatinine kinase (ck), lactate dehydrogenase (ld) and c-reactive protein (crp) levels, along with a higher body temperature, which were consistent with inflammatory and catabolic pathways [ ] . some suggest that the observed hypokalemia among covid- patients is not isolated but rather is part of a general electrolyte imbalance. on the other hand, aldosterone in the setting of a dysregulated raas may or may not be the cause of hypokalemia in some covid- cases, but rather tubular dysfunction, such as renal tubular acidosis. however, studies on this aspect remain elusive. nonetheless, there is evidence that potassium levels, which are mainly regulated by aldosterone, are commonly deranged in covid- patients, and this could be due to interactions with the raas. in the same correspondence published at the lancet recommending the exercise of caution for covid- patients under acei and arb drugs, ibuprofen and thiazolidinediones were mentioned to potentially increase ace expression [ ] . ibuprofen is under the class known as non-steroidal anti-inflammatory drugs (nsaids), and acts by non-selectively inhibiting cyclooxygenase enzymes (cox) and , which confers its anti-inflammatory and antipyretic effects through the inhibition of prostaglandin synthesis from arachidonic acid [ , ] . of the four prostaglandins synthesized by this pathway, pge and pgi , along with the prostanoid thromboxane txa , are produced specifically in the kidneys, while pgd is produced in the airways, and they act by interacting with rhodopsin-like g-protein coupled receptors or gpcrs [ ] . interestingly, prostaglandins, particularly pge and pgi , have been well-studied and have been shown to affect kidneys by improving blood flow through vasodilation. under conditions of reduced renal flow, this mechanism leads to increased tubular flow, increased renal perfusion, and increased glomerular filtration rate, with a concomitant increase in the secretion of potassium ( fig. and ). this is supported by the fact that pge regulates sodium and water reabsorption, while pgi increases the production of renin --with the latter increasing salt and water retention and hence blood volume, while pge counteracts this process via volume/pressure natriuresis [ ]. since pge is the main prostaglandin in the kidney, cox inhibition leads to an overall increase in sodium and water reabsorption. because ibuprofen and other nsaids decrease renal perfusion by increasing systemic vascular resistance, a vicious cycle can be formed by tricking the body to sense that there is reduced renal perfusion, leading to an inappropriate upregulation of ace to return kidney perfusion back to normal via ang ii (fig. ) . concomitantly, an upregulation of ace is expected by virtue of its role as a physiologic antagonist of ang ii, by catabolizing ang ii to ang ( - ) to reduce vasoconstriction, reduce sodium and water retention, and provide reno-and cardio-protective effects [ , , , ] . hence, the reduction in prostaglandin e and i syntheses is the main mechanism by which nsaids directly contribute to increased ace expression, which can be utilized by sars-cov- (fig. ) . studies have shown that the raas is implicated in acute pulmonary injury, pulmonary hypertension, and pulmonary fibrosis [ ] . likewise, raas is indeed activated in idiopathic pulmonary arterial hypertension [ ] , and the expression of angiotensin receptor , ace, and ace is upregulated in the lungs as a response to lung pathology [ , , ] . hence, ace may also be increased in pulmonary tissues secondary to ace-induced promotion of the raas. further, it has been shown that cox- and pge mediate an antiviral response in lung epithelial cells infected with human coronavirus, and their expression results in decreased coronavirus replication [ ] . similarly, pgd , a prostaglandin known to be expressed in the brain and airways, mediate an anti-inflammatory response that may protect against acute respiratory distress and acute lung injury [ ] . hence, the use of nsaids not only decreases pge and pgi -mediated regulation of kidney function, but also pgd -mediated protection against acute lung injury and pge -mediated antiviral responses, which may lead to an enhanced coronavirus replication in the lung epithelia. a review of clinical evidence has shown that ibuprofen and naproxen improved the symptoms of influenza, and naproxen combined with clarithromycin and oseltamivir reduced both the rate and the duration of hospitalization of patients with influenza pneumonia. however, no evidence was provided for covid- [ ] . the observed benefits in influenza infection may be due to the reduction in airway inflammation which is common in colds, and which is also observed in severe influenza. however, regarding viral entry and replication, this clinical evidence does not directly support nor reject any of the mechanisms discussed, especially with regards to sars-cov- infection. a similar systematic review of various nsaids, corticosteroids, and specific immunosuppressive or immunestimulating drugs have shown no strong evidence supporting the use of nsaids for covid- ; however, indomethacin was noted to have shown direct antiviral activity against sars-cov and canine coronavirus (ccov) in vitro [ ] . the resulting benefit is not expected to contradict the mechanisms discussed; j o u r n a l p r e -p r o o f rather, it must have been a function of the structure of indomethacin, which is reflective of the authors' comment that the results were unexpected since indomethacin is a cox inhibitor [ ] . a retrospective cohort study published in showed that patients in hong kong infected with sars-cov, which were given corticosteroids to manage acute respiratory distress, experienced higher rates of adverse outcomes despite favorable baseline characteristics. further, analysis showed that sars patients treated with corticosteroids (hydrocortisone, methylprednisolone) were at a . -fold higher risk of icu admissions or mortality, independent of age and severity of disease [ ] . this study alone, albeit based on sars-cov, showed that corticosteroid use may in fact increase the risk of unfavorable outcomes for coronavirus-infected patients. however, data supporting the validity of this conclusion on other coronavirus infections are indeed warranted. to note, a systematic review and meta-analysis of studies on the management of coronavirus pneumonia showed that patients with severe coronavirus symptoms were more likely to require corticosteroid therapy (rr . ; ci . - . ), but that they were also twice as likely to contract bacterial infection (rr . ; ci . - . ) and were twice as likely to succumb to the disease (rr . ; ci . - . ) [ ] . however, this systematic review and meta-analysis was not only limited to covid- ( studies), but included other coronavirus infections such as those caused by sars-cov ( studies) and mers-cov ( studies), which may allow for differences in the prognosis and clinical outcomes of covid- patients. another systematic review and meta-analysis of studies on corticosteroid use in coronavirus infections showed a statistically significant delay in virus clearing of . days (ci . - . ), but with no significant changes in hospitalization duration, use of mechanical ventilation, or survival [ ] . in these two studies, the results may have differed mainly because the included studies were mostly retrospective in nature. hence, baseline characteristics and management may not have been the same within a singular study, and this may have affected the recommendations assessment, development, and evaluation) approach, which was mainly due to the nature of the studies included (mostly retrospective cohort studies). in addition, the inclusion of outcomes from non-coronavirus infections may have significantly confounded and affected the analysis of outcomes. on the other hand, a systematic review showed that corticosteroids may help reduce immunopathological damage and may improve outcomes when given high-dose with a quinolone early in the infection, but these results were specific for sars-cov and not for sars-cov- [ ] . to resolve the contradicting reports, randomized controlled trials of corticosteroid use and its benefits or risks to covid- outcomes are needed, especially if more accurate results are to be generated. to date, there seems to be emerging clinical evidence that caution must be exercised in the use of corticosteroids for coronavirus-infected patients. however, due to the varying and conflicting results of their effects on clinical outcome, more specific and better designed studies are required for a better evaluation of current recommendations. notwithstanding, the observed risks are supported experimentally and pharmacologically as will be discussed. first, note that corticosteroids are divided into the mineralocorticoids and the j o u r n a l p r e -p r o o f glucocorticoids. mineralocorticoids comprise a group of proinflammatory hormones that primarily act on the kidney to modulate renal-sodium balance and blood pressure by activating mineralocorticoid receptors (mr) [ ] (fig. ) . it is thought to be proinflammatory, acting through the upregulation of inflammatory cytokines to influence t and b lymphocytes, monocytes, neutrophils, and dendritic cells to mount an immune response. this interaction subsequently promotes inflammation and increased levels of reactive oxygen species (ros) following the activation of hormonal response elements (hres) inside the nucleus (fig ) [ ] . in pathological conditions, the mineralocorticoid receptors (mrs) can remain activated regardless of ang ii levels, interfering with normal salt and water retention and electrolyte regulation. this eventually leads to the exacerbation of hypertension, which if not promptly addressed, can lead to progressive organ injury and finally organ failure. glucocorticoids on the other hand affect insulin metabolism, hepatic synthesis, and muscle protein catabolism by increasing amino acid production, and increase the availability of glycogen stores to buffer blood glucose concentration. insulin resistance triggers an increase in blood glucose, which causes plasma volume depletion secondary to osmotic diuresis (fig. ) . in response to resulting plasma volume reduction, the juxtaglomerular cells of the kidneys will produce renin to act upon liver angiotensinogen to form ang i, followed by an ace-mediated conversion to ang ii in an effort to activate the pathway called the renin-angiotensin system to return plasma volume back to normal. subsequently, ace can increase proportionally with ang ii, acting to produce ang ( - ), which we hypothesize is the mechanism for glucocorticoid-enhanced sars-cov- virulence (fig. ) . on the other hand, glucocorticoids suppress inflammation by binding to glucocorticoid response elements (gres) inside the nucleus to interact with genes and affect gene transcription in three basic ways (transactivation, trans-repression, and cis-repression) to suppress inflammation or to prevent an immune response from occurring altogether [ ] (fig. ) . j o u r n a l p r e -p r o o f recent guidelines released by the uk national health service (nhs) on the intake of ibuprofen for mild covid- symptoms has drawn flak from some health experts as it may pose risks on increased severity [ ] . reduction of prostaglandin levels in the body, which is the main action of nsaids like ibuprofen, effectively reduces the immune response, and previous reports have shown no benefit of taking ibuprofen in symptom control and clinical outcome for respiratory infections, and have been shown to worsen the prognosis of community-acquired pneumonia [ , ] . in a randomized, placebo-controlled study for ibuprofen use in managing respiratory syncytial virus (rsv) infection, ibuprofen was not able to prevent lung consolidation and was shown to increase viral shedding [ ] . from these studies, we can infer the classical mechanism by which anti-inflammatory drugs aggravate infection, which is to reduce the degree of immune response that the body can mount against a virus and to allow uncontrolled progression. when a virus infects its host, it most often remains intracellular as viruses are obligate intracellular organisms, and extracellular viruses are most likely seen only during the lytic phase. when viruses infect a cell, antigen-presenting cells alert nearby immune cells through major histocompatibility complexes (mhcs) of the virus' intracellular presence. concomitantly, toll-like receptors (tlrs) recognize viral nucleic acids intracellularly and in effect promotes the cellular expression of interferons and inflammatory cytokines that act in an autocrine and paracrine fashion to induce an antiviral state in the host and in nearby, uninfected cells via the jak/stat pathway, and to alert the immune cells of an ongoing invasion [ , ] . type i interferons have also been shown to promote prostaglandin synthesis [ ] , the latter acting as a mediator between the thymus and the bone marrow to promote cellular and humoral types of immunity [ ] . a previous study has shown that positive-strand rna viruses, to which the coronaviruses belong, induce the formation of the endoplasmic reticulum double membrane vesicles (er-dmvs) that promote viral replication and protect viruses from immune detection, and j o u r n a l p r e -p r o o f treatment with interferon-beta significantly reduces these viral-induced membrane structures [ ] . hence, suppression of prostaglandin synthesis by the use of nsaids may effectively reduce cellmediated and humoral immunity against sars-cov- , and may modulate immune cells towards the production of il- for t-cell suppression and suppression of phagocytosis, particularly by pge [ , ] . it is however noted that prostaglandins such as pge act differently in different viral infections, and may promote viral replication and shedding in other viruses, such as herpes simplex virus (hsv), enterovirus , and cytomegalovirus, to name a few [ , ] . hence, the effect of nsaids on different viral infections may differ, but for sars-cov- and for other viruses such as hepatitis b and human immunodeficiency virus (hiv), taking ibuprofen and other nsaids of similar action may aggravate viral replication. additional risks by nsaids however lie in its ability to increase ace expression by activating a gene called adam , a metalloprotease that upregulates ace by enabling ace shedding, resulting in increased covid- infection [ ] . from here, severity and susceptibility against covid- is expected to increase as viral load relatively increases. only recently, a prothrombotic state has been associated with covid- infection, and this has led to complicated clinical evaluations as covid- is slowly added to the ever increasing list of differentials for thromboembolism. studies included in a scoping review by al-ani et al showed that venous thromboembolism (vte) occurs in about % of covid- patients, with around . % of those who died from the disease being classified as having disseminated intravascular coagulation (dic), according to one single-center study [ ] . similarly, a systematic review and meta-analysis of of the included studies showed vte as a rather common complication of covid- in % of the total patients from three different studies [ ] . these suggest that covid- , apart from being a mainly respiratory disease, is an ailment of the vasculature frequently complicating into vte, which is expected from its likely tropism for the endothelium. since nsaids, especially the selective cox- inhibitors, likely affect hemostatic balance by inhibiting prostacyclin synthesis without inhibiting cox- -induced thromboxane j o u r n a l p r e -p r o o f production, a prothrombotic state is likely to occur especially among susceptible patients. this is the reason why rofecoxib was withdrawn from the market, after a clinical trial found an increased risk of myocardial infarction and stroke in participants after months of use for colon cancer prevention, which is likely to be a class effect [ ] . this points to a possible risk of vte or other thromboembolic events in covid- patients with multiple co-morbidities taking nsaids, although evidence is not yet clear. to the best of our knowledge, there is no direct evidence to date of nsaid use and induced thromboembolism among covid- patients, but serious caution is advised because associations between thromboembolism and nsaid use have been well established in previous years, albeit in different contexts of disease. the reader is referred to this article for an overview [ ] . similarly, corticosteroids may enhance viral entry when provided for symptomatic treatment of sars infection. as aforementioned, a . -fold increase in the risk for adverse outcomes (icu admission/mortality) was observed among patients in hong kong treated with corticosteroids for sars, which was independent of age and disease severity [ ] . this may suggest the promotion of coronavirus replication secondary to immunosuppression, or of increased ace expression secondary to osmotic diuresis, which we hypothesized earlier above (fig. ) . it is important to note, however, that the proposed effects of corticosteroids and nsaids on sars-cov infection is applicable only for the early stages of the disease. during the late stages, such as in acute respiratory distress syndrome (ards) or acute airway inflammation, treatment with corticosteroids or other more targeted therapies, such as monoclonal antibodies, may be lifesaving. recently, a large randomized clinical trial called the randomised evaluation of covid- therapy (recovery) trial conducted the world's largest clinical trial on covid- treatments. in this study they selected , covid- patients and subjected them to low to moderate dose ( mg/day) dexamethasone treatment, which then compared them to , controls who received the standard care for coronavirus infection. as a result, the investigators discovered a significant decrease in patient deaths. death among severely-ill covid- patients on ventilators were j o u r n a l p r e -p r o o f reduced by %, while death among patients requiring oxygen decreased by % [ ] . in contrast, there was no clinical significance seen in patients with less severe cases, suggesting that the dexamethasone might be more useful in patients experiencing cytokine-mediated inflammation. this shows the potential of corticosteroids to provide benefit in severe covid- ; however, it is noted that great caution must be exercised when using dexamethasone, as it is neither indicated for mild cases nor with methylprednisolone [ ] , the latter being consistent with our previous statement. however, it is not clear in the review if corticosteroids are indeed detrimental for covid- , as the study they included, which showed a higher rate of icu admissions among covid- patients treated with corticosteroids, was only a case series and is at best suggestive [ ] . further, the administration of corticosteroids among the studies varied in terms of chronology, with one study being given before ards and the other during ards. meanwhile, a rapid systematic review of publications on inhaled corticosteroid (ics) use in covid- have found no sufficient evidence of benefit or harm on clinical outcomes [ ] , which reflects the scarcity of covid- clinical studies to date. alternatively, paracetamol may be given as first-line for the management of fever without targeting inflammation, due to its effectiveness and safety profile. unlike the nsaids, paracetamol is a weak prostaglandin inhibitor, and in contrast to cox- /cox- inhibition, it inhibits the cox- isoenzyme, which is a cox- splice variant abundant in cns rather than in the peripheral tissues. this provides paracetamol with analgesic and antipyretic properties, but with little to no peripheral anti-inflammatory properties [ , ] . further, paracetamol employs the serotonergic pathway to provide analgesia. by stimulating the descending serotonergic pathway, mediated by the -ht a/ b receptors in the cns and acting on afferent nerve fibers, paracetamol largely inhibits noxious stimuli [ , , ] . in addition, paracetamol also demonstrates an opioid sparing effect of % that may increase analgesia in patients under opioid analgesics [ ] . hence, by virtue of these pathways, paracetamol may be a safer and more effective alternative for early symptomatic management of headache, fever and pain in covid- . j o u r n a l p r e -p r o o f covid- is a respiratory disease caused by the coronavirus sars-cov- , and has caused a pandemic by infecting more than million people in more than countries to date, while claiming hundreds of thousands of lives as it impacts heavily on both economy and lifestyle. initial clinical reports have shown correlations between adverse outcomes for covid- and hypertension, which they attributed to increased expression of ace . this was followed shortly by anecdotal reports of increased symptom severity among patients taking anti-inflammatory drugs like ibuprofen early in the disease, which was later hypothesized to be associated with ace . in this paper, we provide concrete mechanisms of how nsaids and corticosteroids may aggravate sars-cov- infection in the early stages of disease, through their interaction with prostaglandin synthesis and the raas, apart from the conventional pathway of immunosuppression at a time when the body needs it the most. however, as evidence is often conflicting and scarce, more evidence is needed to gain a definitive understanding of covid- . this research did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors. the authors do not have any conflicts of interest to disclose. we would like to thank the healthcare workers and scientists in this pandemic for continually providing care to covid- patients and for providing reports on the clinical and molecular aspects of this disease. dr. rafael a. manalo and emma manalo are acknowledged for providing great inspiration in this work. revised fig. : (arb-acei mismatch was now addressed) the raas is an important homeostatic mechanism of the body that involves the kidney, liver, lungs and adrenal glands, which responds mainly to electrolyte and volume changes as well as to renal blood flow. in times of reduced plasma volume, reduced perfusion to the kidneys and to a concomitant loss of na + , the juxtaglomerular cells of the kidney produce renin, which catalyzes liver angiotensinogen to produce angiotensin i (ang i). the lungs then upregulate angiotensinconverting enzyme (ace) that converts ang i to ang ii, which influences the adrenal glands to synthesize more aldosterone. in addition, ang ii has a direct vasoconstrictive effect. aldosterone (mimicked by other mineralocorticoids), which is released from the adrenal glands then influences the kidneys to increase na + and h o reabsorption in the distal convoluted tubules and collecting ducts. under normal conditions, prostaglandins e and i are formed which, in the kidneys, generally vasodilate the afferent renal arterioles to increase the glomerular filtration rate (gfr) and renal perfusion. in addition, pge predominantly inhibits na + and h o reabsorption in the thick ascending loop of henle (tal) to induce volume or pressure natriuresis, which harmonizes with the raas to maintain homeostasis. in hypertension and other conditions of reduced renal blood flow, the effects of pgi are marked and there is increased reabsorption of na + and h o. chronic use of ace inhibitors (aceis) and angiotensin receptor type blockers (arbs) can lead to a compensatory upregulation of ace and ace expression, which is the probable mechanism for the aggravation of sars-cov- infection among chronic hypertensives taking these drugs. arachidonic acid is converted to the prostaglandins and thromboxanes by the action of cyclooxygenases cox- and cox- . of the four prostaglandins, pge and pgi promote optimal renal function by dilating afferent renal arterioles and promoting natriuresis in normal conditions, with pgi -mediated na + and h o reabsorption seen more evidently in unhealthy individuals with reduced renal perfusion. when nsaids like ibuprofen or naproxen are taken continuously, the net effects of cox inhibition are ( ) increased na + and h o reabsorption, ( ) reduced renal afferent arteriolar dilatation, ( ) reduced glomerular filtration rate (gfr), and ( ) an increase in systemic vascular resistance. due to reduced gfr and renal perfusion, the body produces greater amounts of angiotensin ii (ang ii) to increase blood flow via increased plasma volume. however, increased ang ii levels leads to inappropriate vasoconstriction and increased systemic vascular resistance, which may ultimately result in a vicious cycle due to feedback compensation from ang ii in susceptible patients. subsequently, an upregulation of ace expression is expected to counteract ang ii providing its catabolic pathway to produce vasodilator and natriuretic angiotensins ( ) ( ) ( ) ( ) ( ) ( ) ( ) . this compensatory increase in ace expression from nsaid intake, which is aggravated in unhealthy individuals such as those with hypertension, is the proposed mechanism by which sars-cov- can more aggressively infect the cell, by binding to greater amounts of ace for viral entry. the concomitant immunosuppression by nsaids also promotes viral replication and shedding of sars-cov , which can complicate the early phases of covid- . corticosteroids can be categorized into the mineralocorticoids and the glucocorticoids. the mineralocorticoids, which mimic the actions of aldosterone, affect kidney function by increasing the na + and h o reabsorption. because this mechanism does not promote ang ii formation, it is not expected to contribute majorly to increased ace expression. on the other hand, corticosteroids affect the liver by increasing its capacity for gluconeogenesis. muscles enhance protein breakdown into amino acids, which are carried to the liver for conversion into glucose. further, glucocorticoids promote the actions of glycogen, and inhibit the actions of insulin. this gluconeogenic action coupled with insulin resistance leads to hyperglycemia in susceptible persons, which result in osmotic diuresis that can substantially reduce plasma volume. the body compensates by upregulating ace to increase ang ii levels, thereby increasing na + and h o reabsorption and vasoconstricting blood vessels. an expected increase in ace to counteract increased ang ii action is proposed to be the mechanism of how corticosteroids, precisely 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thought to cause end-organ damage resulting in hypertension and end-organ failure. meanwhile, glucocorticoids mediate anti-inflammatory pathways that result in the immunosuppressive actions of most corticosteroid drugs in medicine. glucocorticoids bind to their nuclear glucocorticoid receptors (gr), but also to mr, and affect gene transcription via two important mechanisms: ( ) transactivation, which is the direct interaction of the glucocorticoid-gr complex with genetic material, or ( ) trans-repression, in which the complex interacts with other proteins such as creb-binding protein (cbp) and nf-κb. trans-activation leads to an upregulation of anti-inflammatory proteins, while trans-repression leads to decreased expression of inflammatory proteins. together, these pathways converge to result in immunosuppression, which may aggravate covid- by promoting sars-cov- replication and shedding, especially in the early stages of the disease key: cord- -ngwgb y authors: abassi, zaid; higazi, abd al roof; kinaneh, safa; armaly, zaher; skorecki, karl; heyman, samuel n. title: ace , covid- infection, inflammation, and coagulopathy: missing pieces in the puzzle date: - - journal: front physiol doi: . /fphys. . sha: doc_id: cord_uid: ngwgb y engulfed by the grave consequences of the coronavirus disease (covid- ) pandemic, a better understanding of the unique pattern of viral invasion and virulence is of utmost importance. angiotensin (ang)-converting enzyme (ace) is a key component in covid- infection. expressed on cell membranes in target pulmonary and intestinal host cells, ace serves as an anchor for initial viral homing, binding to covid- spike-protein domains to enable viral entry into cells and subsequent replication. viral attachment is facilitated by a multiplicity of membranal and circulating proteases that further uncover attachment loci. inherent or acquired enhancement of membrane ace expression, likely leads to a higher degree of infection and may explain the predisposition to severe disease among males, diabetics, or patients with respiratory or cardiac diseases. additionally, once attached, viral intracellular translocation and replication leads to depletion of membranal ace through degradation and shedding. ace generates ang - , which serves a critical role in counterbalancing the vasoconstrictive, pro-inflammatory, and pro-coagulant effects of ace-induced ang ii. therefore, ang - may decline in tissues infected by covid- , leading to unopposed deleterious outcomes of ang ii. this likely leads to microcirculatory derangement with endothelial damage, profound inflammation, and coagulopathy that characterize the more severe clinical manifestations of covid- infection. our understanding of covid-ace associations is incomplete, and some conceptual formulations are currently speculative, leading to controversies over issues such as the usage of ace inhibitors or ang-receptor blockers (arbs). this highlights the importance of focusing on ace physiology in the evaluation and management of covid- disease. additionally, ace , through the modulation of the renin-ang-aldosterone system (ras), plays an important physiologic role in the homeostasis of tissue microcirculation and inflammation (crackower et al., ; hamming et al., ; santos et al., ; clarke and turner, ; datta et al., ) . this minireview will address the role of ace within the ras, and the inter-association of ace and sars-cov- , with their plausible combined impact on the clinical manifestations of covid- disease. we shall further address knowledge gaps that require elucidation in order to better understand the pathophysiology and clinical features of covid- in order to develop effective means for disease prevention and management. ace : an important component of ras figure a illustrates our current understanding of the complexity of the ras. until recently, most clinicians were familiar with only one axis, namely renin-mediated proteolysis and conversion of angiotensinogen to the -amino-acid peptide ang i, followed by a further cleavage by ace, principally present in the lungs to form the bioactive -amino-acid compound ang ii (crackower et al., ; hamming et al., ; santos et al., ; clarke and turner, ) . the covid- pandemic shifted our attention to another component of ras, namely ace , which plays a role in sars-cov- virulence. ang ii could be further cleaved by ace to form the bioactive -amino-acid peptide ang - . in addition, ace converts ang i into ang ( - ), which can be further converted to ang - by ace. a third pathway of ang - generation involves neprilysin (neural endopeptidase-nep), which converts ang i directly into ang - (tipnis et al., ; crackower et al., ; vickers et al., ; hamming et al., ; santos et al., ; clarke and turner, ). an alternative degradation pathway with conversion of ang i to ang ii takes place by the proteolytic enzyme, chymase, explaining ongoing generation of ang ii in patients on ace inhibitors (miyazaki and takai, ) . importantly, ang derivatives differ by their downstream physiologic properties and are mediated by diverse signal transduction mechanisms ( figure a) . ang ii acts principally as a potent vasoconstrictor, pro-inflammatory, pro-fibrotic, and anti-diuretic agent. these actions are mediated by ang ii binding to ang t receptors (at r) on affected cell membranes. opposing activities may be initiated via attachment of ang ii to ang t receptors (at r; li et al., ) . indeed, ang ii-mediated vasoconstriction or vasodilation at the renal cortex and medulla, respectively, reflects diverse receptor distribution and activity, predominantly at r in the cortex and at r in the medulla (duke et al., ) . as also shown in figure a , unlike ang ii, ang - exerts unequivocal vasodilatory, antiinflammatory, anti-fibrotic, and natriuretic actions by binding to a g-protein-coupled mas receptor (masr; li et al., ; santos et al., ) . thus, a tight physiologic balance exists by the opposing effects of ang derivatives whenever this system undergoes perturbations, with the aim of preventing extreme vasoactive deviations or uncontrolled inflammation and remodeling, with ang - serving to counterbalance the undesired adverse effects of unbridled ang ii action. angiotensin-converting enzyme is expressed on the plasma membranes of various cell types, including alveolar and intestinal epithelia, vascular endothelial cells in the heart, kidney, and testis, and on macrophages, where it catalyzes the production of ang - and its likely paracrine activity (crackower et al., ; hamming et al., ; santos et al., ; clarke and turner, ; abassi et al., c) . unfortunately, cell-membranebound ace also serves as a binding site for the viral spike proteins of sars-cov- and sars-cov- (li et al., ; hamming et al., ; hoffmann et al., ; walls et al., ; wan et al., ; wu et al., ; zou et al., ) . the viral attachment to ace with subsequent internalization is facilitated by additional modifications and cleavage of the s /s spike proteins by convertases, such as transmembrane protease serine (tmprss ) and related proteases (furin and corin; heald-sargent and gallagher, ; coutard et al., ; hoffmann et al., ; shang et al., ; walls et al., ) , and probably by activated factor x (xa), which was shown to cleave recombinant and pseudoviral s protein into s and s subunits (du et al., ) , all exposing the fusion sites in the viral spike protein (figure ) . two principal sites of sars-cov- invasion include the gastrointestinal and respiratory tracts, which express abundant ace . while intestinal homing is clinically more pronounced in children, manifested by gastrointestinal symptoms, the lungs conceivably serve as the principal port of entry, with viral attachment to type ii alveolar cells (at ), and to alveolar macrophages coated by membranal ace (abassi et al., c,d) . interestingly, conditions identified as predisposing to severe covid- disease are characterized by enhanced pulmonary expression of ace . first, chronic airway disease, smoking, and pollution are associated with expansion of the population of alveolar macrophages expressing ace (abassi et al., d) . furthermore, ace expression is increased in males (la vignera et al., ; papadopoulos et al., ) . indeed, bioinformatics analyses revealed higher abundance of ace expressing at cells in men than women (wei et al., ) , potentially enhancing viral susceptibility among men. in this context, testosterone has been described to induce ace expression, the receptor entry of the sars-cov- infection, but also exerts protective effect against lung injury (kuba et al., ) . enhanced ace is also found in diabetes (muniyappa and gubbi, ) and heart failure (zisman et al., ; goulter et al., ; chen et al., ) , and possibly with the administration of ras inhibitors (li et al., ) . diabetes is also associated with increased expression of furin (fernandez et al., ) . thus, while testosterone levels decline with aging among men (harman et al., ; feldman et al., ) , the presence of comorbidities like obesity, diabetes mellitus, and cardiovascular diseases, possibly counterbalance the decline in viral homing capacity related to age-dependent testosterone drop (camacho et al., ; rastrelli et al., ) . in addition, testosterone enhances at r expression in male, whereas estrogen preferentially upregulates at r expression in females (chanana et al., ) . finally, hypogonadal males are characterized by low t cell count which may provide unrestrained environment for severe responses to sars-cov- infection (papadopoulos et al., ) . in sum, it is tempting to assume that enhanced expression of ace in target organs and also of other molecules permissive to viral binding to ace facilitate viral invasion and augment viral load (figure ) , although the details of this formulation require validation in further studies. figure | physiology of coronavirus disease (covid ) homing to target host cells expressing ace : viral spike-domains enable attachment to cellmembrane-bound ace . attachment is further enabled by furin, corin, tmprss , and factor xa. following attachment the virus undergoes internalization and replication in host cells, a process associated with degradation of internalized ace . ang - synthesis consequently declines. unopposed ang ii action triggers inflammation which activates adam , leading to shedding of membranal ace , further depleting cell-bound ace and local ang - synthesis. viral attachment to target host cells may be attenuated by its competitive binding with rising titers of circulating ace . frontiers in physiology | www.frontiersin.org october | volume | article as illustrated in figures , , sars-cov- invasion unbalances the ras. viral cellular internalization is coupled with degradation of membranal ace . furthermore, circulating ang ii, combined with internalized ace activates a sheddase named adam metallppeptidase domain (adam ) also called tumor necrosis factor-α-converting enzyme (tace; lambert et al., ) , which in turn triggers shedding of membranal ace into the circulation with the formation of soluble ace (sace ), further depleting membranal ace along enhanced tnf-α production (figure ) . thus, viral cellular invasion and replication, initially facilitated by ace and in particular under conditions characterized by enhanced ace expression, later lead to diminution of cell membrane-attached ace , and likely increase circulating sace (figures , ) . at the microcirculatory and tissue level, this is expected to result in unbalanced paracrine action of ang compounds, with a local depletion of ang - leaving ang ii activity unopposed ( figure b) . likely, this has a role in microcirculatory dysfunction, intense inflammation, hypercoagulability, tissue damage, and fibrosis (figure ) . lung inflammation in sars cov- disease exemplifies the outcome of ang ii/ang - imbalance: ang ii enhances vascular permeability along infiltration of neutrophils into alveolae and indirectly via induction of interleukin (il- ; diamond, ) . accumulation of neutrophils and their accompanied prooxidative role lead to loss of alveolar epithelial cells and the development of ards. nevertheless, this ang ii-derived lung injury is prevented by ang - as was evident in ace deficient mice (zou et al., ) . additional adverse aspect of unrestricted ang ii action during sars-cov- infection is the increased tendency of thrombosis documented in large number of hospitalized covid- patients (bikdeli et al., ; klok et al., ) . although this phenomenon is multifactorial, as outlined below, at r activation plays an important role where it leads to enhancement of tissue factor (tf) expression on endothelial cells and sequentially initiation of clotting cascade along increased permeability and neutrophils mobilization (dielis et al., ) . many sections in the preceding paragraphs are based on in vitro and animal studies, some with inconsistent and even conflicting interpretations. furthermore, some fundamental concepts are currently being re-evaluated. for instance, previously reported ace expression on vascular endothelial cells (hamming et al., ) has recently been questioned, based on the measurement of single-cell rna (batlle et al., ) . human data based on patients infected by sars-cov- are sparse and are now being intensively studied as we write these lines. it is evident that the foregoing statements should be further examined in the human clinical scenario of covid- disease. second, the role of altered ace physiology detailed above in subsequent clinical features of the disease requires in-depth evaluation (essig et al., ) . there are several hypothetical mechanisms, outlined in figure , that warrant consideration. possibly, unopposed ang ii due to depletion of cell membranebound ace results in altered regional microcirculation and hypoxia, with the generation of reactive oxygen species and endothelial damage, glycocalyx degradation, and disseminated coagulopathy (abassi et al., a) . this may further compromise figure | a summarizing scheme of suggested covid- /ras interactions: see text for details. highlighted are factors enhancing ace expression and viral binding to target host-cells, as are mechanisms leading to declining membranal ace and ang - synthesis. the impact of shedded sace on tissue ang - production and on inhibiting viral homing to target cells expressing ace by means of competition require further elucidation. ras inhibitors potentially can enhance viral invasion by enhancing ace expression, yet they may attenuate the unfavorable outcome of ang - depletion by a parallel inactivation of functionally opposing ang ii activity. potential hazardous feed-forward loops are at r-mediated enhanced ace shedding and intensification of viral attachment via proteases activated by vasoconstriction and ischemia, inflammation, and coagulopathy. frontiers in physiology | www.frontiersin.org october | volume | article the regional microcirculation with a feed forward loop, leading to organ failure including the heart (abassi et al., b) , lungs (abassi et al., d) , and kidneys (batlle et al., ) . furthermore, intense inflammation and coagulopathy may result from unopposed ang ii and by adam -mediated activation of tnf-α/il- / stat- pathways (hirano and murakami, ) as well as uncontrolled heparanase activity (li and vlodavsky, ) together with the induction of defensins (abu-fanne et al., ) . concerning the latter, preliminary findings from our group indicate that alpha-defensin- , released from polymorphonuclear cells as a part of the inflammatory response, plays a pivotal role in the hypercoagulopathy associated with covid- disease, as its rising titers parallel increasing plasma levels of d-dimers (higazi aar, submitted manuscript). regarding the interplay between ang ii and adam /tnf-α/il- /stat- pathways, it was found that ang ii activates nf-κb and release of proinflammatory cytokines (dandona et al., ; benigni et al., ) . specifically, induction of adam by ang ii initiates the conversion of interleukin- (il- rα) to the soluble form (sil- rα) along activation of signal transducer and activator of transcription (stat ) via the sil- rα-il- complex in various nonimmune cells including fibroblasts, endothelial cells, and epithelial cells (hirano and murakami, ) . moreover, stat , essential for the nf-κb pathway, is principally stimulated by il- during inflammation (murakami et al., ) . since il- plays a key role in the recruitment of lymphoid cells and myeloid cells, including activated t cells and macrophages (murakami et al., ) , and likely enhances defensin release (higazi aar, unpublished data), its elevated levels during senescence may contribute to the enhanced covid- mortality in aged people and to coagulopathy. interestingly, at r density is increased, while at r abundance declines under inflammatory conditions (diamond, ) . collectively, these results may explain proinflammatory cytokine release and hypercoagulopathy during sars-cov- infection via the associated ang ii pathway and a possible therapeutic target via the il- -stat axis (diamond, ) . reduced inherent expression of ace in the lungs with aging, as demonstrated in rats (xie et al., ; alghatrif et al., ) may reduce the risk for sars-cov- infection on the one hand, whereas its further suppression to very low levels during viral infection, on the other hand, could amplify ang ii/ang - imbalance, leading to more profound deleterious pulmonary consequences. conversely, younger individuals with higher inherent ace expression may have a higher incidence, yet less severe sars-cov- infection, since ace depletion would not be as severe as in aged patients, with ang - generation sufficient to counteract ang ii (alghatrif et al., ) . deranged vascular reactivity will likely be affected by other mediators, such as inos-activation and intense nitric oxide production (plausibly with abundant formation of the toxic-free radical peroxynitrite), and by altered endothelial production of endothelin and prostaglandins. notably, there are additional plausible inherent feed-forward loops in the scheme of sars-cov- infection and inflammation, including hypoxia-driven perpetuation of endothelial damage and tissue damage. furthermore, as illustrated in figures , , ang ii suppresses ang - generation secondary to downregulation of membranal ace via adam activation. moreover, factor xa, generated during disseminated coagulation, is expected to expose attachment sites on viral spikes and enhance viral attachment to target cells expressing ace (du et al., ) . interestingly, in vitro studies illustrate that heparin interferes with ace binding to the s viral spike protein, reducing viral internalization (mycroft-west et al., ) . thus, enhanced heparanase activity in infected patients might damage endothelial cover by heparin-like proteoglycans and further facilitate viral endothelial invasion. third, discussions regarding the potential impact of medications affecting ras are currently based on inconsistent observations and educated guesses (essig et al., ) . we really do not know for sure if blocking steps in the ras cascade indeed results in enhanced ace expression in humans, and whether this promotes viral attachment and invasion. on the other hand, discontinuation of ras inhibitors might further intensify the uncontrolled action of ang ii, shown in figure b , leaving it unopposed once ang - generation is hampered. those in favor of uninterrupted administration of ras inhibitors would argue that, as illustrated in figure c , depleting ang ii or blocking its action on at r [by ace inhibitors or ang-receptor blockers (arbs), respectively] would balance the exhaustion of ang - caused by viral invasion and might prevent consequent vasoconstriction . furthermore, it is also likely that the profile of ang derivatives may differ in patients treated by arbs, by ace inhibitors or by spironolactone (malha et al., ) . that is why blanket reassurance regarding continuation of ras inhibitors during the current pandemic (vaduganathan et al., ) should be regarded with caution. a cautious approach might consider the avoidance of ace inhibitors or arbs during an active epidemic in non-infected and hemodynamically-stable patients in order to reduce ace expression, permissive to viral attachment, but consideration of ace inhibitors, or arbs at advanced stages of covid- disease to prevent ang ii predominance due to depleted ang - . most of the clinical trials and data analysis are performed on adults, however potential differences between adults and children may exist, thus coronavirus-related research should be undertaken in children as well, including the impact of ace-i and arbs on covid- evolvement among this subpopulation. hopefully, this may provide clues for the question why children are at decreased risk of severe covid- disease . furthermore, we have no idea about the function or malfunction of circulating sace following its shedding from cell membranes. does it exert systemic vasodilation or improve the microcirculation? can it compete with cell-membrane-bound ace (ciaglia et al., ) and reduce viral attachment to target cells as suggested in figure ? nor can we tell if diverse inherent expression and activity of circulating or cell-bound ace or its capacity to attach to viral spike proteins affects infection, infectivity, or susceptibility to severe and complicated disease. we also are not sufficiently knowledgeable of plausible changes in ace transcription in various tissues in response to sars-cov- infection. indeed, rice et al. ( ) reported that up to % of the phenotypic variation in circulating ace could be accounted for by genetic factors. these findings may partially explain the different mortality rate among the various ethnic groups, and strongly support studies of genetic analysis of ace polymorphisms as a reliable approach for precision medicine in the prevention, diagnosis, and therapy of covid- disease. evidence is currently lacking as to whether levels of circulatory sace may have diagnostic and prognostic implication when monitoring patients infected by sars-cov- , as it does in patients with heart failure (epelman et al., ; ortiz-perez et al., ) . with so many pieces of data missing, the need for vigorous clinical studies guided by physiology-based questions and hypotheses are most urgent. such a question includes the continuation or even introduction, rather than cessation of ras inhibitors in patients infected by sars-cov- (kai and kai, ) , or can we inhibit binding of sars spike proteins to ace , for instance by antibodies, without hampering its catalytic capacities to generate ang - ? is there a role for the application of ang - or masr agonists or for the administration of intravenous sace , with an available proof of concept for such postulated approaches (yang et al., ; hemnes et al., ) ? it is likely that many of the above options will be considered and examined in the near future. meanwhile, we are challenged by epidemiologic aspects, by issues of supportive and critical care for very sick individuals, and by minimizing the risk to healthcare providers. the ultimate solution probably will be effective vaccination. yet, until we reach this goal, studying and manipulating ace -viral association is a plausible approach, along with the development of effective anti-viral agents. the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. zab, ah, zar, ks, and sh equally participated in the design, execution of the view of point, drafted the manuscript, participated in critical discussions, and revised the manuscript. sk, sh, and zab prepared the figures. sh supervised the project. all authors contributed to the article and approved the submitted version. zab acknowledges research support from the israel science foundation grant / . glycocalyx degradation in ischemia-reperfusion injury letter to the editor: angiotensin-converting enzyme : an ally or a trojan horse? implications to sars-cov- -related cardiovascular complications the lung macrophage in sars-cov- infection: a friend or a foe? covid- infection and mortality: a physiologist's perspective enlightening clinical features and plausible interventional strategies neutrophil alpha-defensins promote thrombosis in vivo by altering fibrin formation, structure, and stability the dilemma of coronavirus disease , aging, and cardiovascular disease: insights from cardiovascular aging science acute kidney 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)-forming activity in failing human heart ventricles-evidence for upregulation of the angiotensin-onverting enzyme homologue ace single-cell rna-seq data analysis on the receptor ace expression reveals the potential risk of different human organs vulnerable to -ncov infection angiotensinconverting enzyme protects from lethal avian influenza a h n infections the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © abassi, higazi, kinaneh, armaly, skorecki and heyman. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -wj xeqj authors: zhang, chao; chen, shuaiyin; zhou, guangyuan; jin, yuefei; zhang, rongguang; yang, haiyan; xi, yuanlin; ren, jingchao; duan, guangcai title: involvement of the renin-angiotensin system in the progression of severe hand-foot-and-mouth disease date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: wj xeqj background: hand-foot-and-mouth disease (hfmd) is generally considered as a mild exanthematous disease to infants and young children worldwide. hfmd cases are usually mild and self-limiting but for few cases leads to complicated severe clinical outcomes, and even death. previous studies have indicated that serum ang ii levels in patients with h n infection were related to the severity of infection. however, the mechanisms underlying the pathogenesis of severe hfmd remain unclear. this study was undertaken to clarify the role of the renin-angiotensin system (ras) in the progression of severe hfmd. methods: in the present study, children including hfmd patients and healthy controls were recruited. the data was analyzed by time-series fashion. concentrations of angiotensin ii (ang ii) and noradrenaline (na) in serum of patients were measured with elisa. we established a mouse model for enterovirus (ev ) infection and determined concentrations of ang ii, na in tissue lysates at , and days post infection (dpi). results: the concentrations of ang ii and na in serum of the hfmd patients with mild or severe symptoms were significantly higher than that in healthy controls. additionally, the concentrations of ang ii and na in serum of severe cases were significantly higher than those mild cases and the increased concentrations of ang ii and na showed the same time trend during the progression of hfmd in the severe cases. furthermore, the concentrations of ang ii and na in target organs of ev -infected mice including brains, skeletal muscle, and lungs were increased with the progression of ev infection in mice. histopathological alterations were observed in the brains, skeletal muscle and lungs of ev -infected mice. conclusion: our study suggested that activation of the ras is implicated in the pathogenesis of severe hfmd. in the present study, children including hfmd patients and healthy controls were recruited. the data was analyzed by time-series fashion. concentrations of angiotensin ii (ang ii) and noradrenaline (na) in serum of patients were measured with elisa. we established a mouse model for enterovirus (ev ) infection and determined concentrations of ang ii, na in tissue lysates at , and days post infection (dpi). the concentrations of ang ii and na in serum of the hfmd patients with mild or severe symptoms were significantly higher than that in healthy controls. additionally, the concentrations of ang ii and na in serum of severe cases were significantly higher than those mild cases and the increased concentrations of ang ii and na showed the same time trend during the progression of hfmd in the severe cases. furthermore, the concentrations of ang ii and na in target organs of ev -infected mice including brains, skeletal muscle, and lungs were increased with the progression of ev infection in mice. histopathological alterations were observed in the brains, skeletal muscle and lungs of ev -infected mice. plos in recent years, several hand-foot-and-mouth disease (hfmd) outbreaks have occurred in asia-pacific region and europe [ ] [ ] [ ] [ ] . among them, enterovirus (ev ) and coxsackie a (ca ) infection is main cause of hfmd outbreaks, and patients with ev infection are inclined to develop into severe symptoms [ , ] . hfmd is generally considered as a mild exanthematous disease. generally, most of hfmd cases are usually mild and self-limiting but for few cases viral infection leads to complicated clinical outcomes including brainstem encephalitis, aseptic meningitis, encephalitis, and acute flaccid paralysis (afp), and even fatal cardiopulmonary failure [ ] [ ] [ ] .numerous studies have shed light on the etiology and epidemiology of hfmd, and have helped medical professionals and public health officials worldwide understand this condition. previous studies have found that ev enters into the digestive tract first and then enters the lymphatic system, and ultimately into the central nervous system (cns) [ , ] . cns injury can lead to a rapid increase in intracranial pressure, increased sympathetic tone, and excessive activation of local renin-angiotensin system (ras), further results in over-secretion of many cytokines into circulation, leading to peripheral vasoconstriction. peripheral vasoconstriction will cause vascular endothelium injury and increased permeability, further inducing blood accumulation in the lower pulmonary region [ ] [ ] [ ] [ ] . the ras plays a critical action regulating the circulation in the human body in response to low blood pressure or decrease in serum sodium levels [ ] .a component of this system is angiotensinogen (agt) that is synthesized and released by the liver into the general circulation. agt is converted through another reaction into angiotensin i (ang i) by the protein renin from the renal juxtaglomerular apparatus. subsequently, ang i is converted to angiotensin ii (ang ii) by the pulmonary angiotensin-converting enzyme (ace). angii is an active octapeptide that acts primarily on ang ii receptor type (at r) [ ] . angiotensin ii (ang ii) is a key factor to activate at r, further inducing water and sodium storage by promoting the release of aldosterone and excessive activation of other neurohormonal system components by promoting the secretion of endothelin and noradrenaline (na) [ , ] . high concentration of na in circulation is thought to associate with pulmonary edema [ ] , which may involve with fatal pulmonary edema in severe hfmd. the ras can also activate related cells and regulates the expression of many mediators concerning cell growth and inflammatory responses [ , ] . however, the role of ras in the process of hfmd has not been well characterized yet. in the present study, we assumed that ras participated in the progression of hfmd and tried to uncover the ras-related potential mechanism in the progression of severe hfmd. the study was reviewed and approved by the life sciences and ethics committee of zhengzhou university and the ethics committee of the zhengzhou children's hospital. written informed consent was obtained from each case's guardian before enrollment. total subjects ( - months age) including hfmd cases with mild (n = ) or severe symptoms (n = ) and healthy controls (n = ) were recruited from zhengzhou children's hospital during april through june . all hfmd cases were divided into different subsets of , , , , days post infection (dpi) based on the hospitalization and initial onset time. according to the "diagnosis and treatment guideline on hand-foot-and-mouth disease ( )", patients younger than months with severe symptoms including meningitis, pulmonary edema, and mild cases without any nervous system lesions or pulmonary edema were included in this study. the children without any disease were classified as control. the patients with congenital disease, acute or chronic hepatitis, cardiovascular disease, intestinal diseases, and other infectious diseases were excluded from this study. balb/c mice (spf degree) were purchased from the medical animal center in zhengzhou university, henan, china, and raised in individual ventilation cage (ivc) system. as described in our previous study [ , ] , -day-old balb/c mice were i.p. inoculated with ev strain ( × pfu/mouse) and sacrificed with isoflurane anesthesia on , and days post infection (dpi). the -day-old mice injected with the same volume of rd cell culture supernatants were used as controls and sacrificed with isoflurane on , and dpi. the brains, skeletal muscle and lungs of mice were ground into tissue homogenate with cold phosphate-buffered saline (pbs) at ˚c. tissues were quickly removed and stored at - ˚c. tissues were grounded at ˚c and repeated freezing and thawing for three times, followed by centrifugation at , ×g for min at ˚c. mice were sacrificed on , , dpi, and the brains, skeletal muscle and lungs of mice were immediately fixed in % paraformaldehyde at ˚c overnight. after fixation, paraffin-embedded tissues of μm in thickness were stained with h&e. the concentrations of ang ii and na in serum or tissue lysates were measured by enzymelinked immunosorbent assay (elisa) kits (tsz, boston, usa). spss . (ibm, nc, usa) was used for the statistical analysis. one-way anovas or student's t test depending on the validity of the normality assumption and the homogeneity of variance and a pearson correlation analysis were performed. a significance level < . was used for this study. one hundred thirty-two patients with hfmd were identified, including mild hfmd patients, severe hfmd patients, and healthy children were used as controls. there was no significant difference in sex ratio (male ratio, controls: . / ; mild: . / ; severe: . / , p> . ) and age (mouths, controls: . ± . ; mild: . ± . ; severe: . ± . , p> . ) among these three groups. the concentrations of ang ii and na are presented in fig . the concentrations of ang ii and na in serum of the hfmd patients with mild or severe symptoms were significantly higher than that in healthy controls (p< . ). additionally, the concentrations of ang ii and na in serum of severe cases were significantly higher than those mild cases, p< . . pearson correlation analysis (fig ) indicated that the concentration of ang ii was positively correlated to the concentration of na during the progression of hfmd in both mild and severe (r = . , p< . for the mild; r = . , p< . for the severe). together, our results suggested that the concentrations of ang ii and na in serum were increased in hfmd cases. as shown in fig , the concentration of ang ii in serum of the severe cases was significantly higher than that in the mild cases from dpi to dpiduring the progression of hfmd. the concentrations of ang ii among the subgroups of the severe were significantly different and increased from the dpi to dpi (p< . ). the highest level of ang ii in serum of the severe cases occurred at dpi. the concentrations of ang ii among the subgroups in mild cases were not significantly different. the concentration of na in serum of severe cases was significantly higher than those in mild cases during the progression of hfmd from dpi to dpi. the concentrations of na among the subgroups in severe cases were significantly different and increased from dpi to dpi (p< . ). the highest level of na in severe cases occurred as shown in fig , expression of ang ii and na presented similar temporal trends in the corresponding tissues of ev -infected mice. the concentrations of ang ii and na in ev -infected mice were significant increased in brains from dpi to dpi, skeletal muscle at dpi and dpi, lungs at dpi compared to controls. the results demonstrated that ev infection increased ang ii and na levels in target organs. histopathological alterations of mice at , , dpi were examined with haematoxylin and eosin (h&e) staining. as shown in fig , the brain tissues from ev -infected mice exhibited pathological changes including perivascular cuffing compared with controls at , and dpi. mice were sacrificed on , , dpi. the removed organ or tissue were sliced and stained with h&e. brain from ev -infected mice exhibited perivascular cuffing (indicated by red solid arrows). skeletal muscle from ev -infected mice appeared necrotizing myositis with muscle fibers rupture (indicated by orange solid arrows) at dpi and dpi. erythrocyte-filled fluid in the alveolar spaces was found in lungs (indicated by black solid arrows) from ev -infected mice at dpi. bar = μm. https://doi.org/ . /journal.pone. .g skeletal muscle from ev -infected mice demonstrated necrotizing myositis with muscle fibers rupture and inflammatory cells infiltration at dpi and dpi. lung lesions such as swollen alveoli and erythrocyte-filled fluid in the alveolar spaces were detected in lungs of ev infected mice at dpi. the above results indicated that ev infection induced obvious histopathological alterations in target organs. hfmd is a common viral infectious disease mainly caused by enterovirus (ev ) and coxsackievirus (cv) a , which is mostly prevalent in infants and young children [ ] . from to , the mortality of severe hfmd is . %, which is extremely higher than total mortality of hfmd [ , ] . to date, no effective treatment for severe cases has been found. thus, to explore early biomarkers to identify cases with potential to develop into severe symptoms will be beneficial to direct early clinical intervention and reduce mortality of hfmd. in the present study, we found that the concentrations of ang ii and na were increased in serum of hfmd cases with mild or severe symptoms. increasing concentrations of ang ii and na in tissue lysates of mice were also determined during ev infection. the current understanding of ras is far more complex than its classic point, and two significant concepts have been added. first, the system is not only expressed at a systemic level but also works locally in a paracrine function in the vasculature, kidney, heart, lungs and cns et al. [ ] . in our study, we found that serum concentrations of ang ii and na were elevated in hfmd cases, especially in severe cases, suggesting ras activation. the concentration of ang ii was positively related to the concentration of na during the progression of hfmd in both the mild and severe cases. the highest levels of ang ii and na occurred at dpi in hfmd cases. as shown in our previous study, the highest levels of some inflammatory cytokines occurred at dpi in hfmd cases. in terms of above results, ras activation may induce large amounts of pro-inflammatory cytokines [ ] . numerous studies on the pathogenesis of other viral diseases have also shown that pathogens can affect the stability of the ras [ ] [ ] [ ] [ ] . thus evidence support our results, and activation of ras may involve with the development of hfmd. to further confirm the involvement of ras in the pathogenesis of hfmd, we establish an ev -infected mouse model [ , ] . in our study, we found that ev infection induced ang ii and na expression in the brains, skeletal muscle, and lungs of ev -infected mice. we also observed histopathological alterations in these target organs, implying ras activation may participate in the process of ev infection. furthermore, pulmonary edema was also determined in ev -infected mice. previous studies have indicated that serum ang ii levels in patients withh n infection were higher than controls and were related to the severity of infection [ , ] . the s protein of sars-cov virus can down regulate the expression of ace in the lungs and cause an accumulation of ang ii and acute lung injury (ali). after the intervention of the ace protein, symptoms of ali were alleviated [ ] . zou, z et al. found that with h n infection, the ras was involved and final concentration of ang ii were able to be used as prognostic indicators [ ] .these studies suggested that with viral infection, the pathogens could damage the homeostasis of the lung ras, alter the local blood pressure and vascular permeability, and aggravate the imbalance of pro-inflammatory and anti-inflammatory mediators, thus affecting the occurrence and development of lung injury. yumiko i et al. found that an excessive expression of ang ii can enhance the permeability of the lung through at r, resulting in pulmonary edema [ ] . together, above evidence suggest that ev infection-induced pulmonary edema may be an outcome of ras activation. in summary, our study for the first time finds that activation of the ras may involve in the progression of severe hfmd. an epidemic of enterovirus infection in taiwan. taiwan enterovirus epidemic working group. the new england journal of medicine the enterovirus epidemic in -public health implications for hong kong epidemiology and control of hand cyclical patterns of hand, foot and mouth disease caused by enterovirus a in malaysia effects of relative humidity on childhood hand, foot, and mouth disease reinfection in hefei, china. the science of the total environment spatiotemporal cluster patterns of hand, foot, and mouth disease at the county level in mainland china severe hand, foot and mouth disease in shenzhen, south china: what matters most? epidemiology and infection update on hand-foot-and-mouth disease in children; an epidemic associated with coxsakie virus a- foot and mouth disease in singapore: a comparison of fatal and non-fatal cases immunodeficient mouse models with different disease profiles by in vivo infection with the same clinical isolate of enterovirus role of the renin-angiotensin system in ventilator-induced lung injury: an in vivo study in a rat model pathologic characterization of a murine model of human enterovirus encephalomyelitis involvement of inducible nitric oxide synthase and mitochondrial dysfunction in the pathogenesis of enterovirus infection pulmonary edema following central nervous system lesions induced by a non-mouse-adapted ev strain in neonatal balb/c mice renin angiotensin system and its role in biomarkers and treatment in gliomas intensity of hydration changes the role of reninangiotensin-aldosterone system blockers in contrast-induced nephropathy risk after coronary catheterisation in patients with chronic kidney disease changes in angiotensin ii and angiotensin-converting enzyme of different tissues after prolonged hyperoxia exposure. undersea & hyperbaric medicine : journal of the undersea and hyperbaric medical society effect of norepinephrine dosage on mortality in patients with septic shock role of renin-angiotensin aldosterone system on short-term blood pressure variability in hypertensive patients effects of calcium channel blockers comparing to angiotensin-converting enzyme inhibitors and angiotensin receptor blockers in patients with hypertension and chronic kidney disease stage to and dialysis: a systematic review and meta-analysis. plos one hand, foot, and mouth disease in china, - : an epidemiological study. the lancet infectious diseases serum inflammatory cytokine levels correlate with hand-foot-mouth disease severity: a nested serial case-control study angiotensin ii plasma levels are linked to disease severity and predict fatal outcomes in h n -infected patients a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury angiotensin-converting enzyme (ace ) mediates influenza h n virus-induced acute lung injury angiotensin-converting enzyme protects from lethal avian influenza a h n infections angiotensin-converting enzyme protects from severe acute lung failure the authors thank doctor yuanfang shen and doctor yunping qiao, zhengzhou children's hospital, for allowing testing of the patients. key: cord- -glbr kxh authors: naik, george o a title: covid- and the raas date: - - journal: clin infect dis doi: . /cid/ciaa sha: doc_id: cord_uid: glbr kxh nan m a n u s c r i p t dear editor: further to thomas hanff et al [ ] timely call for epidemiological and clinical investigations of covid- infectious disease, measurements of the renin angiotensin aldosterone system (raas) components, as sub-studies would be insightful of this pandemic. angiotensin-converting enzyme (ace ) participates in the coronavirus (sars-cov- ) cell entry. this infection down regulates ace . drugs that block raas also affect ace expression: it is down regulated by renin inhibition (ri) and up regulated by angiotensin-converting enzyme inhibitors (aceis), angiotensin receptor blockers (arbs) ( ) and mineralocorticoid receptor antagonists (mras) [ ] . other likely regulatory factors are age, type ii diabetes and sex difference [ ] . these interactions would directly affect the balance between the beneficial and deleterious angiotensins (angs), such as, ang ( - ) and ang ( - ) versus excess ang ii. such perturbations would also indirectly influence other raas components, and the coordination between circulating and local tissue expressions, as shown in figure ace is distributed throughout the body and is abundantly expressed in the lung, small intestine, and in blood vessels of many organs including the brain, heart, kidney and testis [ ] . these organs and blood vessels are potential sites of infection. the downregulation of ace would reduce the production of ang ( - ) and ang ( - ), and concurrently prevent the reduction of the ang ii, tilting the balance to ang ii accumulation that may lead to toxicity [ ] , figure (b). such dysregulation likely contributed to reported cases of acute respiratory distress syndrome (ards) [ ] , a c c e p t e d m a n u s c r i p t inflammation, myocardial injury [ ] , neurological incidences [ ] and gastrointestinal manifestations [ ] . other components and the crosstalk between the systemic circulation and local tissue renin angiotensin system would also be disrupted. changes in circulating ang ii concentration alter renin secretion through a negative feedback loop, figure (a), as ang ii decreases renin secretion increases and consequently affect renin concentration and plasma renin activity (pra). renin converts angiotensinogen to angiotensin i (ang i) and pra is a measure of this rate. renin catalytic activity is enhanced when bound to its receptor (ppr) [ ] . the inhibition of renin or ace reduces circulating ang ii with an increase in renin concentration. it is conceivable that circulating renin could bind to ppr, where expressed, and activate local tissue renin angiotensin system. ang ii changes would also affect aldosterone stimulation and angiotensin iv (ang iv) production. ang iv through its receptor at has opposite biological effects to ang ii via receptor at . we therefore suggest the measurement [ ] - ) ). ang ii is further transformed to ang iii, ang iv and ang v. a negative feedback loop controls ang ii concentration changes with renin secretion that responds in the opposite direction. ang ii stimulates the release of aldosterone. however, excessive ang ii is deleterious and is associated with hypertension, congestive heart failure and chronic kidney disease. ace transforms ang i and ang ii, that is, ang i to ang ( - ), and ang ii to ang ( - ). ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and ang ( - ) have protective effects balancing the deleterious ang ii, when in excess. a c c e p t e d m a n u s c r i p t figure is there an association between covid- mortality and the renin-angiotensin system-a call for epidemiologic investigations mineralocorticoid receptor blocker increases angiotensin-converting enzyme activity in congestive heart failure patients individual variation of the sars-cov receptor ace gene expression and regulation tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis association of coronavirus disease (covid- ) with myocardial injury and mortality neurologic manifestations of hospitalized patients with coronavirus disease prevalence and clinical characteristics of mild severity patients with digestive symptoms the (pro)renin receptor: an emerging player in hypertension and metabolic syndrome specific and non-specific measurements of tissue angiotensin ii cascade members key: cord- -q e nf authors: gembardt, florian; sterner-kock, anja; imboden, hans; spalteholz, matthias; reibitz, franziska; schultheiss, heinz-peter; siems, wolf-eberhard; walther, thomas title: organ-specific distribution of ace mrna and correlating peptidase activity in rodents date: - - journal: peptides doi: . /j.peptides. . . sha: doc_id: cord_uid: q e nf biochemical analysis revealed that angiotensin-converting enzyme related carboxy-peptidase (ace ) cleaves angiotensin (ang) ii to ang-( – ), a heptapeptide identified as an endogenous ligand for the g protein-coupled receptor mas. no data are currently available that systematically describe ace distribution and activity in rodents. therefore, we analyzed the ace expression in different tissues of mice and rats on mrna (rnase protection assay) and protein levels (immunohistochemistry, ace activity, western blot). although ace mrna in both investigated species showed the highest expression in the ileum, the mouse organ exceeded rat ace , as also demonstrated in the kidney and colon. corresponding to mrna, ace activity was highest in the ileum and mouse kidney but weak in the rat kidney, which was also confirmed by immunohistochemistry. contrary to mrna, we found weak activity in the lung of both species. our data demonstrate a tissue- and species-specific pattern for ace under physiological conditions. in the regulation of heart function and blood pressure, different peptide systems are involved, e.g. the renin-angiotensin system (ras), the kallikrein-kinin system, and the natriuretic peptide system. in these systems, proteases like angiotensin-converting enzyme (ace) or neutral endopeptidase (nep) have the distinction of generating or catabolizing biologically active peptides [ , , ] . the newly discovered angiotensin-converting enzyme-related carboxypeptidase (ace ) has considerable sequence homology to ace ( % identity and % similarity), contains a hexxh zinc-binding domain, and conserves other critical residues typical of the ace family [ , ] . the first step in generating angiotensin peptides is the cleavage of angiotensinogen to angiotensin (ang) i by renin. ang i is hydrolyzed by either ace or chymase to ang ii, which mediates its biological actions via the at and at receptors [ , ] . ang i is also metabolized by nep to ang-( - ) [ ] , which mediates distinct effects through its receptor mas [ ] . importantly, ang-( - ) can also be directly metabolized from ang ii by ace , whereas aminopeptidase a converts ang ii to ang iii [ ] . ace also hydrolyzes ang i to ang-( - ), although there is no hydrolysis of ang-( - ), ang-( - ), and ang- ( ) ( ) ( ) ( ) ( ) . moreover, ace hydrolysis is also specific for des-arg [ ] bradykinin and its shorter fragments, although it cleaves neither bradykinin nor bradykinin-( - ) [ ] . ace mrna is expressed in many tissues but shows a less ubiquitous profile than ace. first studies in mice detected the highest expression in the ileum by quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) [ ] . ace is an important part of the ras, which counteracts the function of ace. it was also shown that ace expression can be upregulated by blockade of at -receptors [ ] . the importance of ace in cardiovascular regulation was confirmed by targeted disruption of ace in mice. the absence of ace in mice leads to a severe cardiac contractility defect, increased ang ii levels, and upregulation of hypoxia-induced genes in the heart [ ] . in addition to its peptidolytic function, recent investigations have discovered that ace is a functional receptor for the coronavirus, which causes the severe acute respiratory syndrome (sars) [ ] . in this investigation, we (i) measured the mrna distribution of ace through different tissues in both species. moreover, we (ii) quantified ace protein by western blot using a commercial polyclonal antibody to ace . we (iii) measured ace activity in different tissues of mice and rats. we (iv) established a monoclonal antibody against ace to complete the investigation of tissue distribution by immunohistochemistry. finally, we compared (v) the distribution of ace in both species on the mrna and protein level. all experiments were done according to the guidelines of the federal law on the use of experimental animals in germany and were approved by the local authorities. for this investigation we used c bl/ mice and sprague-dawley (sd) rats in an age of - months. animals were killed by cervical dislocation. for rnase protection assay (rpa), ace activity assay and western blot, the tissues were snap frozen in liquid nitrogen. the samples were stored at − • c until further processing (all organs in total, heart divided into atria and ventricles). the tissues for immunohistochemistry were put in % formalin. after h they were embedded and processed to paraffin sections. the polymerase chain reaction (pcr) amplified a bp fragment (probe: mmace ) from mouse kidney cdna using the -primer ctc agt gga tgg gat ctt gg (mmace ) and the -primer tgt agc cat ctg ctc cct ct (mmace ), respectively a bp fragment (probe: rnace ) from rat lung cdna using the -primer cgg gga aag atg tca agc tcc tgc (rnace ) and the -primer ctt gtc tgg tga cag cgc (rnace ), which were subcloned in a t-vector (promega gmbh, mannheim, germany). a sp polymerase transcribed a radioactive probe complementary to mmace (resp. rnace ) mrna, and a rna complementary to nucleotides of the rl mrna was used as positive control [ ] . ace -specific mrna for mouse and rat were identified by rnase protection assay (rpa) using the ambion rpa ii kit (ambion (europe) ltd., huntingdon, uk). total rna was isolated from tissues using the trizol reagent (invitrogen gmbh, karlsruhe, germany) with subsequent chloroform-isopropanol extraction according to the manufacturer's instructions. a g total rna fraction of each sample was hybridized with approximately cpm for ace and cpm for rl of the radiolabeled antisense probes in the same assay. equal loading has been insured by mrna measurements and mrna gel electrophoresis using g of each sample (not shown). the hybridized fragments protected from rnase a + t digestion were separated by electrophoresis on a denaturing gel ( %, w/v polyacrylamide, m urea) and analyzed using a fujix bas phospho-imager system (raytest gmbh, straubenhardt, germany) to perform quantitative analysis by measuring the intensity of the ace bands. the blots of each species were calculated to ace mrna expression in kidney, which was present on both blots of each species. the expression level in the lung was set to %. ace activity was measured similar to the method by vickers et al. [ ] . tissue was homogenized in assay buffer ( mm -morpholinoethanesulfonic acid, mm nacl, m zncl , . % brij- , ph . ). protein concentration was determined using roti-quant (carl roth gmbh and co. kg, karlsruhe, germany) by the manufacturers instruction. we used mca-apk(dnp) (biosynthan gmbh, berlin, germany) dissolved in dmso ( m, final concentration) as the ace substrate. the assay was performed in assay buffer and was started by adding l of tissue homogenate. after h incubation at ambient temperature ( • c), the reaction was suppressed by adding m o-phenanthrolin (final concentration). parallel control tests were performed in the presence of m dx (data not shown) [ ] . after centrifugation ( min, × g) the fluorescence was measured at nm (excitation) and nm (emission) with the perkin-elmer fluorescence reader lambda (perkin-elmer las gmbh, rodgau, germany). the molecular standardization was performed with mca-ap (biosynthan gmbh, berlin, germany) and calculated per mg protein. the functionality of the assay was proven by a standardized solution with defined, recombinant ace activity (r&d systems gmbh, wiesbaden, germany). tissue was homogenized in phosphate-buffered solution (pbs) containing protease inhibitor mixture (complete, roche diagnostics gmbh, mannheim, germany). protein concentration was determined with bca protein assay kit (perbio science gmbh, bonn, germany). sample proteins ( g/lane) and a prestained protein-weight marker (amersham biosciences gmbh, freiburg, germany) were size fractionated by sds-polyacrylamide gels ( %) and transferred to pvdf membranes with a pegasus semidry-blotter (phase gmbh, lübeck, germany). equal loading has been insured by staining control gels with simply-blue safe stain (invitrogen gmbh, karlsruhe, germany) using g of each sample (not shown). the membranes were blocked at room temperature in % dry milk powder (blotting grade, non-fat dry milk, bio-rad laboratories gmbh, munich, germany) prepared with tris-buffered saline containing . % tween (ttbs) for h, incubated with goat polyclonal antibody against ace (santa cruz biotechnology inc., heidelberg, germany, : diluted in % dry milk powder ttbs, h), and then washed three times with ttbs ( min each). subsequently, the membranes were incubated with horseradish peroxidase-conjugated antigoat igg (dakocytomation a/s, glostrup, denmark, : , h) and washed three times. specific immunoreactive proteins were detected by enhanced chemiluminescence (amersham biosciences gmbh, freiburg, germany). the bands on the x-ray film were quantified by densitometry scanning and expressed as percentage of the kidney protein signal. monoclonal antibodies against the synthetic peptide avgeimslsaat (aa - of murine ace ) have been raised. for immunization of the mice peptide was cross-linked with glutaraldehyde to albumin fraction v from bovine serum. balb/cj female mice were injected with the conjugate. following four booster injections the spleen lymphocytes were fused with fo myeloma cells by using polyethylene glycol (roche diagnostics gmbh, mannheim, germany) following the manufacturers instructions. the different hybridoma supernatants were screened for specific antibodies by using the synthetic peptide in the nctest [ ] . for production of monoclonal antibodies, positive hybridoma cells were grown in celline incubators (integra biosciences gmbh, fernwald, germany). the mouse monoclonal antibodies were affinity purified on a mabtrap g ii column (amersham-pharmacia gmbh, otelfingen, switzerland) from cell culture supernatants. immunoglobulin class and subclasses were determined with the immuno type kit (sigma-aldrich chemie gmbh, taufkirchen, germany). paraffin sections of mouse tissues were prepared and stained using standard histology procedures. for immunostainings, deparaffinized and rehydrated tissue slides were first treated for min with % h o to block the endogenous peroxidase. after rinsing in ddh o and soaking in pbs for - min, slides were treated with % (w/v) bsa in pbs to eliminate non-specific protein binding sites. the slides were then exposed (overnight, • c) to the monoclonal ace antibodies (clone e , d ) at concentrations of and g/ml, respectively. after removing excess antibody, slides were treated with biotin-labeled anti-mouse (dianova gmbh, hamburg, germany) antibody for min at • c and finally with horse-radish peroxidase (hrp) labeled streptavidine (zymed laboratories inc., san francisco, usa) for min at • c. after washing, slides were incubated in aminoethylcarbazol (sigma-aldrich co., st. louis, usa) for min at room temperature. slides were counterstained with hematoxylin, and cover slipped according to conventional procedures. slides were examined under a conventional microscope after removing the excess substrate in ddh o. negative controls were performed without the primary antibody, just applying dilution buffer of the primary antibody. data were analyzed by t-test using spss software (spss benelux bv, gorinchem, the netherlands). each value was expressed as the mean ± s.e.m., and statistical significance was accepted for p < . . ace mrna could be detected in all investigated organs, but with profound distinction between different organs. in both species, only a low amount was found in ventricle, liver, testis, forebrain, and spleen ( figs. and ) , whereas in the lungs a moderate and comparable expression of ace mrna was found and set to %. the highest levels were found in the ileum of both species (fig. ) . between the species several differences in tissue specific expression of ace mrna were found. the expression in mouse was most pronounced higher than in rat in kidney (∼ . -fold), colon (∼ . -fold), and ileum (∼ . -fold) (fig. ) , whereas in bladder (∼ . -fold) and ventricle (∼ . -fold) ace expression in rat exceeded the mouse. in accordance with the rna expression data, highest activity for ace was found in the ileum of mouse and rat (table ) , whereas the activity in the mouse was . - fold higher. lowest ace activity was found for both species in spleen. low activity was also found for liver of mice and thymus of rats. corresponding to the differences on mrna levels in the kidney the ace activity fig. . quantification of the rpas of mice (white columns) and rats (black columns). the mrna amount of the lungs is set to % (n ≤ ) . the values are shown as mean + s.e.m. . ventricle, . kidney, . lung, . liver, . testis, . bladder, . forebrain, . spleen, . thymus, . stomach, . ileum, . colon, . brainstem, . atrium, . adipose tissue. * p < . , ** p < . , *** p < . compared mouse vs. rat. was much higher in mice than in rats (∼ . -fold). the activity of ace in the lung was different to mrna and . -fold higher in rats than in mice. in contrast to rpa data the activity in colon was comparable between both species. using a commercial polyclonal antibody in western blot for the quantification of protein levels in mouse and rat tissues (fig. ) a pattern completely different from rna expression and ace activity was found. a moderate and comparable expression could be detected in the kidney of both species and was set to %. thus, the highest amount of protein could be detected in atrium of both species (mouse: . %; rat: . %) and ventricle (mouse: . %; rat: . %). for the mouse less ace protein was found in lung ( . %) and testis ( . %), whereas no protein was detectable in these two tissues in rat. in thymus (mouse: . %; rat: . %) and forebrain (mouse: . %; rat: . %) of both species a moderate expression was detectable, whereas no ace protein was found in spleen of mouse and rat. to further clarify the discrepancy between rpa and activity on one side and western blot on the other, immunohistochemistry was performed in lung, kidney (fig. ) , and testis (data not shown) of mice and rat with new monoclonal ace antibodies (clones e and d ), we generated. the antibodies were determined to belong to the igg subclass. in the lungs of both species alveolar macrophages and type cells (fig. , upper row) were stained with both monoclonal ace antibodies (data for clone d not shown). the epithelium of the renal tubuli was strongly stained (fig. , lower row, left) in the kidney of mice. in rats only a weak signal, but the same pattern as in mouse, was detected, what aligned with mrna and ace activity (fig. , lower row, right) . in recent investigations it was shown that peptidases like ace and nep are important regulators of cardiovascular and endothelial function as well as myocardial remodelling [ , , , ] . consequently, after its discovery in , ace became an enzyme of interest for scientific investigation of its impact in cardiovascular physiology and pathophysiology [ , , ] . to elucidate some of its physiological functions we investigated the tissue distribution of mrna and protein in a variety of tissues of c bl/ mice and sprague-dawley rats. while we could see correlating patterns of mrna and ace activity in most of the examined tissues, we also found significant divergences between the investigated species. the huge difference between mrna and protein levels in the lung may be due to shedding as demonstrated for ace [ , , ] . this shedding leads to an increased secretion of ace and lowered its protein content in the lung by even high mrna expression. the significant differences that we found between the species on ace protein and mrna levels in kidney could be explained by the varying interspecies regulation and expression of peptidases, as shown in the literature for nep activity in rat and rabbit kidneys [ ] . comparing our mrna and activity data with the western blot pattern, we have to conclude that the commercial polyclonal antibody is not detecting ace protein in organ homogenates and is not suitable for ace staining. in contrast, using immunohistochemistry our new monoclonal ace antibodies produce staining patterns comparable to our mrna and activity data. we have shown that ace expression in rodents is highest in ileum among the examined organs. it was shown for other peptidases of ras like ace and nep that they are also present at high levels in the intestine [ ] . however, the distinct function of these peptidases in the ileum is not yet known. further investigations have to clarify the physiological and pathophysiological functions of the peptidases in the gastrointestinal tract. beside its physiological function as a peptidase, ace is used by coronavirus as a co-receptor in severe acute respiratory syndrome (sars) [ ] . it was shown that the sars coronavirus only can enter cells which express ace [ ] . ace distribution in the small intestine, lung and vascular endothelium may offer a point of entry for the sars coronavirus, but does not reflect its basic function [ , , ] . interestingly, the distribution patterns we found for mrna and ace activity contradict investigations using a commercial northern blot for detecting mrna [ , ] but have been confirmed by recent papers using rt-pcr [ ] . this discrepancy may be a species-specific alteration of tissue distribution, since they used human tissue for northern blot, or it may be due to technique differences (commercial northern versus rpa and activity assay). the first possibility is at least supported by our finding that significant differences in ace expression patterns exist between the close relatives mouse and rat. recent investigations revealed biological activity for angiotensin peptides other than angii, like ang-( - ) [ , , ] . ace can generate ang-( - ) by cleaving the cterminal amino acid from angii [ ] . ace is also involved in another pathway leading to the generation of ang-( - ). it cleaves angi to ang-( - ) [ ] . ang-( - ) is then hydrolyzed by ace to ang-( - ) . we demonstrated that ang-( - ) is an endogenous ligand for the g protein-coupled receptor (gpcr) mas [ ] . mrna of the gpcr mas was found at high levels in testis and certain brain regions and at fig. . immunohistochemical visualization of ace positive cells. sections of lungs (upper row) and kidneys (lower row) from mouse (left panel) and rat (right panel). in the lungs of both species alveolar macrophages and type cells were stained positive. the tubulus epithelium in mouse kidney was stained positive, whereas in the rat kidney only weak staining was seen. moderate levels in kidney and heart [ , , ] . it was shown that high concentrations of ang-( - ) were present in heart, kidney, and brain [ , , , ] . in recent investigations, it was demonstrated that ace , mas, and its endogenous ligand ang-( - ) are present in the same cells of the kidney [ ] . as we recently postulated, this indicates a relevant impact of the ace /ang-( - )/mas axis on blood pressure regulation and cardioprotection. actual investigations indicate an upregulation of ace in heart failure, pointing to the relevance of ace in cardiac function [ , , ] . however, there was a high incidence of sudden death in animals overexpressing ace . electrophysiology revealed severe, progressive conduction and rhythm disturbances with sustained ventricular tachycardia that progressed to fibrillation and death [ ] . while anti-arrhythmic actions were demonstrated for ang-( - ) in low concentra-tions ( . nm) by stimulating its own receptor, -fold higher concentrations of ang-( - ) lead to arrhythmias by stimulating the at receptor [ , ] . therefore, the overexpression of ace may lead to a high increase in the production of ang-( - ), turning its cardioprotective actions into effects causing arrhythmias by unspecific at stimulation. in future studies, the actions of ang-( - ) and its concentrationdependency on ace expression on heart rhythm have to be proven in in vivo experiments with at -and mas-deficient animals. our data on tissue and species-specific ace expression point to the fact that the ras becomes increasingly complex. since we identified an expression pattern markedly different from ace, we conclude that the expression levels of the involved peptidases like ace, ace , and nep that generate and/or degrade the bioactive peptides of the ras are predic-tive of either the occurrence of vasoconstriction or dilatation or the dominance of pathophysiological stimuli over beneficial conditions. the acute infarction ramipril efficacy (aire) study investigators, effect of ramipril on mortality and morbidity of survivors of acute myocardial infarction with clinical evidence of heart failure imprinting of the murine mas protooncogene is restricted to its antisense rna cell type-specific expression of the mas proto-oncogene in testis a point mutation in the juxtamembrane stalk of human angiotensin i-converting enzyme invokes the action of a distinct secretase cardiac angiotensin-( - ) in ischemic cardiomyopathy immunocytochemical localization of angiotensin-( - ) in the rat forebrain vasopeptidase inhibitors: an emerging class of cardiovascular drugs enhanced renal immunocytochemical expression of ang-( - ) and ace 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up-regulated in the human failing heart insights into angiotensin ii receptor function through at receptor knockout mice tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis quantitative mrna expression profiling of ace , a novel homologue of angiotensin converting enzyme susceptibility to sars coronavirus s protein-driven infection correlates with expression of angiotensin converting enzyme and infection can be blocked by soluble receptor novel peptide inhibitors of angiotensin-converting enzyme immunocytochemistry in brain tissue upregulation of angiotensin-converting enzyme after myocardial infarction by blockade of angiotensin ii receptors angiotensin-( - ) immunoreactivity in the hypothalamus of the (mren- d) transgenic rat burrell lm. differential tissue and enzyme inhibitory effects of the vasopeptidase inhibitor omapatrilat in the rat angiotensin-converting enzyme is a functional receptor for the sars coronavirus expression of the mouse and rat mas proto-oncogene in the brain and peripheral tissues the role of ace in cardiovascular physiology vasodilator action of angiotensin-( - ) on isolated rabbit afferent arterioles angiotensin-( - ): an update angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas remodeling of myocardium and arteries by chronic angiotensin converting enzyme inhibition in hypertensive patients a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase exploring the pathogenesis of severe acute respiratory syndrome (sars): the tissue distribution of the coronavirus (sars-cov) and its putative receptor, angiotensin-converting enzyme (ace ) the angiotensin-converting enzyme gene family: genomics and pharmacology hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase at receptor blockade increases cardiac bradykinin via neutral endopeptidase after induction of myocardial infarction in rats vasopeptidase inhibitors: will they have a role in clinical practice? increased angiotensin-( - )-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme homologue ace florian gembardt is paid by a grant from the "deutsche forschungsgemeinschaft" (german research foundation)[grk ]. this study was also supported by the "stiftung zur förderung der wissenschaftlichen forschung an der universität bern". we thank helmut würdemann and susanne gygax for their technical assistance. key: cord- -npob n authors: gheblawi, mahmoud; wang, kaiming; viveiros, anissa; nguyen, quynh; zhong, jiu-chang; turner, anthony j.; raizada, mohan k.; grant, maria b.; oudit, gavin y. title: angiotensin-converting enzyme : sars-cov- receptor and regulator of the renin-angiotensin system: celebrating the th anniversary of the discovery of ace date: - - journal: circ res doi: . /circresaha. . sha: doc_id: cord_uid: npob n ace (angiotensin-converting enzyme ) has a multiplicity of physiological roles that revolve around its trivalent function: a negative regulator of the renin-angiotensin system, facilitator of amino acid transport, and the severe acute respiratory syndrome-coronavirus (sars-cov) and sars-cov- receptor. ace is widely expressed, including, in the lungs, cardiovascular system, gut, kidneys, central nervous system, and adipose tissue. ace has recently been identified as the sars-cov- receptor, the infective agent responsible for coronavirus disease , providing a critical link between immunity, inflammation, ace , and cardiovascular disease. although sharing a close evolutionary relationship with sars-cov, the receptor-binding domain of sars-cov- differs in several key amino acid residues, allowing for stronger binding affinity with the human ace receptor, which may account for the greater pathogenicity of sars-cov- . the loss of ace function following binding by sars-cov- is driven by endocytosis and activation of proteolytic cleavage and processing. the ace system is a critical protective pathway against heart failure with reduced and preserved ejection fraction including, myocardial infarction and hypertension, and against lung disease and diabetes mellitus. the control of gut dysbiosis and vascular permeability by ace has emerged as an essential mechanism of pulmonary hypertension and diabetic cardiovascular complications. recombinant ace , gene-delivery of ace , ang – analogs, and mas receptor agonists enhance ace action and serve as potential therapies for disease conditions associated with an activated renin-angiotensin system. rhace (recombinant human ace ) has completed clinical trials and efficiently lowered or increased plasma angiotensin ii and angiotensin - levels, respectively. our review summarizes the progress over the past years, highlighting the critical role of ace as the novel sars-cov- receptor and as the negative regulator of the renin-angiotensin system, together with implications for the coronavirus disease pandemic and associated cardiovascular diseases. k nowledge of the underlying biology and physiology of ace (angiotensin-converting enzyme ) has accumulated over the last years since its discovery and has provided a major stimulus to further our understanding of the renin-angiotensin system (ras). [ ] [ ] [ ] [ ] ace has distinct roles ranging from catalytic activities with various substrates, as functional receptors for severe acute respiratory syndrome (sars) coronaviruses (sars-cov), and as an amino acid transporter. [ ] [ ] [ ] [ ] ace functions as a master regulator of the ras mainly by converting ang (angiotensin) i and ang ii into ang - and ang - , respectively. , both loss-of-function and gain-of-function approaches in experimental models of human diseases have defined a critical role for ace in heart failure (hf), systemic and pulmonary hypertension (ph), myocardial infarction (mi), and diabetic cardiovascular complications. gut dysbiosis and altered gut permeability have emerged as an important mechanism of disease controlled by the ace axis in both vascular and lung diseases, , as well as in diabetes mellitus. clinical and experimental studies support a physiological and pathophysiological role for ace in cardiovascular disease (cvd), and increasing/activating ace may elicit protective effects against hypertension and cvd, although this has yet to be proven clinically. , [ ] [ ] [ ] more recently, ace has garnered widespread interest as the cellular receptor of sars-cov- , the causative virus of the coronavirus disease (covid- ) pandemic, which emerged from wuhan, china, in late . , , ace offers protection in acute lung injury, suggesting that, although it facilitates viral entry at the epithelial surface, the ace /ang - axis can be carefully manipulated to mitigate sars-induced tissue injuries, which represents a potential target for therapeutic intervention. , in experimental models of lung disease, catalytically active ace alleviates pulmonary injury and vascular damage , and prevent ph, decreased lung fibrosis, arterial remodeling, and improved right ventricular performance due to a combination of direct action in the lungs and via the ace -dependent gut-lung axis. , in phase ii clinical trials, administration of ace was shown to reduce systemic inflammation and shifted the ras peptide balance away from ang ii toward ang - . , ongoing global efforts are focused on manipulating the ace /ang - axis to curtail sars-cov- infection while affording maximal protective effects against lung and cardiovascular damage in patients with in this review, we summarize the diverse roles of ace , highlighting its role as the sars-cov- receptor and negative regulator of the ras, and the implications for the covid- pandemic. we also provide a framework for developing novel therapeutic strategies exploring the ace pathway as it relates to cvd and covid- . following the initial and seminal discovery of renin in by tigerstedt and bergman, the ras now encompasses a complex network of enzymes, peptides, and receptors ( figure ). , , , , [ ] [ ] [ ] [ ] [ ] while many metallopeptidases cluster in small inter-related gene families (eg, the neprilysin [nep] family), unusually, no human homolog of the vasoactive zinc-peptidase ace (angiotensin converting enzyme) had been identified at the turn of the century. almost simultaneously, in , independent approaches searching for such ace homologs revealed the existence of a close relative of the ace gene designated aceh or ace . aceh was cloned from a human lymphoma cdna library and the identical ace from a human hf ventricular cdna library, the latter emphasizing a potential role for ace in cardiovascular pathologies. expression of the ace gene was initially established in the heart, kidney, and testis, but subsequent studies have shown a much broader distribution, including the upper airways, lungs, gut, and liver ( figure a ). sequence comparison of ace and ace strongly suggested that ace , like ace, was an integral transmembrane protein (and ectoenzyme) with a transmembrane anchor close to the c-terminus (type i membrane protein). a close evolutionary relationship existed between the ace and ace , genes and it was presumed that the proteins would have similar substrate specificities and involvement in the ras. as it turned out, important differences occur, particularly in the active site regions of the enzymes, such that the enzymes counterbalance rather than reinforce each other's actions. many subsequent studies over the next years have revealed their inter-relationship, respective roles in the ras, and multiple physiological and pathological actions from vasoactive peptide metabolism, importantly including not only ang ii but also apelin, to intestinal amino acid transport affecting innate immunity, to lung function and brain amyloid metabolism (converting aβ to aβ , a substrate for ace). , , another unexpected twist in ace biology was its identification in as the cell-surface receptor for the then newly identified sars-cov that led to > cases of sars and almost deaths, and as the receptor for sars-cov- that is currently devastating many countries worldwide. , the ace gene and basic biochemistry unlike the ace gene, which is located on human chromosome , the kb ace gene is located on chromosome xp and contains exons, most of which resemble exons in the ace gene. whereas somatic ace contains active sites, ace possesses only a single catalytic domain. both ace and ace act as zinc metallopeptidases but of differing substrate specificities defining their distinct and counterbalancing roles in the ras. whereas ace cleaves c-terminal dipeptide residues from susceptible substrates (a peptidyl dipeptidase), ace acts as a simple carboxypeptidase able to hydrolyze ang i, forming ang - and ang ii to ang - ( figure b ). ace does not cleave bradykinin, further distinguishing its specificity from that of ace while it is also insensitive to conventional ace inhibitors. , the c-terminal domain of ace , which has no similarity with ace, is a homolog of a renal protein, collectrin, which regulates the trafficking of amino acid transporters to the cell surface, endowing ace with multiple and distinctive physiological functions. it is the multiplicity of physiological roles that ace plays that has allowed it to be hijacked by sars-cov- as a receptor, resulting in the covid- pandemic. , structural studies have revealed the structures of both the sars-cov and much more recently, the sars-cov- in complex with ace ( figure b ). , in the case of sars-cov- , the major spike glycoprotein (s ) binds to the n-terminal region of ace . the knowledge of the biology and physiology of ace accumulated over the last years since its discovery should provide a major stimulus to understanding some of the key steps in sars-cov- infection and its ultimate prevention. on march , , the world health organization declared the outbreak of sars-cov- a global pandemic, reporting community scale transmissions occurring in every continent outside antarctica. since then, the outbreak has escalated to well over one million cases and caused over deaths worldwide by the start of april . however, before the emergence of sars-cov in , coronaviruses were conventionally viewed as inconsequential pathogens circulating in nature throughout various host and intermediate species that occasionally infected humans causing only mild upper respiratory tract infections and symptoms of the common cold. [ ] [ ] [ ] as such, to better understand the renin was the first component of the ras discovered following the finding that extracts from rabbit kidney produced pressor effects (tigerstedt and bergman, ). constriction of the renal artery was then found to lead to hypertension (htn), thus driving the discovery of hypertensin and angiotonin (and later termed angiotensin; goldblatt et al ; page and helmer ). ang (angiotensin) was subsequently purified, and forms were resolved: ang i and ang ii. therefore, the existence of a converting enzyme was predicted (ace) and subsequently isolated and characterized (skeggs et al severity of global health risks posed by sars-cov- and optimize treatment for infected patients, we must recognize the role of ace in sars-cov- pathogenesis. in addition to respiratory involvement, multiorgan dysfunction occurs in response to sars-cov- infections. [ ] [ ] [ ] while respiratory symptoms are predominant, acute cardiac and kidney injuries, arrhythmias, gut, and liver function abnormalities have all been documented in infected patients, suggesting myocardial, renal, enteric and hepatic damage in covid- . similarly, sars-cov also resulted in systemic manifestations with damages to the heart, gastrointestinal, liver, kidney, and other tissues. , ace as the receptor for sars-cov- sars-cov- differs from the original sars-cov by amino acid substitutions, which translates to differences in five of the six vital amino acids in the receptor-binding domain between the viral spike (s) protein with surface expressed human ace . viral s-proteins are well established as a significant determinant of host tropism and represents a key target for therapeutic and vaccine development. additionally, host cell proteases are important for sars-cov- entry and infection of cells as both s-proteins and ace are proteolytically modified during the process. the binding affinity of sars-cov- with ace seems stronger than sars-cov, with alterations in several amino acid residues allowing for enhanced hydrophobic interactions and salt bridge formations, which may explain the considerably larger global influence of covid- than the initial sars. , moreover, sars-cov- has evolved to utilize a wide array of host proteases including cathepsin l, cathepsin b, trypsin, factor x, elastase, furin, and tmprss (transmembrane protease serine ) for s-protein priming and facilitating cell entry following receptor binding. so far, tmprss and cathepsin l/b mediates s-protein priming of sars-cov- , and camostat mesylate, a serine protease inhibitor combined with cathepsin l/b inhibitor, e- d blocked sars-cov- entry. the entry of both sars-cov and sars-cov- into cells is facilitated by the interaction between viral s-protein with extracellular domains of the transmembrane ace proteins, followed by subsequent downregulation of surface ace expression ( figure ). ace -derived peptides, small molecule inhibitors, ace antibody or single chain antibody fragment against ace . in postmortem autopsy heart tissues from patients who succumbed to sars-cov, heart samples had detectable viral sars-cov genome, which was characterized by increased myocardial fibrosis, inflammation, and reduced myocardial ace expression. these patients also had a much more aggressive illness associated with earlier mortality. additionally, bilateral pleural effusions were frequently observed during autopsy of sars-cov patients, further supporting the evidence of cardiac involvement. individuals with preexisting diabetes mellitus, hypertension, and lung disease are at particular risk of covid- infection , and this is likely due to dysregulated ras that occurs in these conditions. , significance of the sars-cov- infection in the cardiovascular system is reflected through incidences of acute myocardial injury (elevated high sensitivity troponin i levels and/or new ecg/echocardiogram abnormalities), arrhythmias, cardiac arrest, sepsis, septic shock, viral myocarditis, and hf (elevated nt-probnp levels, systolic dysfunction on cardiac magnetic resonance imaging). [ ] [ ] [ ] [ ] further abnormalities from laboratory tests, including elevation in d-dimers reflective of increased thrombosis risk, may lead to acute coronary syndrome, and sustained increased inflammatory cytokines levels throughout the clinical course suggest ongoing systemic and tissue inflammation in patients with covid- . , , gut dysbiosis and a possible link to disease progression in covid- patients ubiquitous expression of ace throughout the luminal surface of the gastrointestinal tract, and most prevalently in enterocytes, may serve as a secondary site for enteric sars-cov- infection (figure a ). leaky gastrointestinal ace -mediated cardiovascular protection is lost following endocytosis of the enzyme along with severe acute respiratory syndrome-coronavirus (sars-cov- ) viral particles. ang ii (angiotensin ii) levels elevate with increased activity of angiotensin receptors (at r) at the cost of ace / ang - driven pathways leading to adverse fibrosis, hypertrophy, increased reactive oxygen species (ros), vasoconstriction, and gut dysbiosis. adam (a disintegrin and metalloproteinase )-mediated proteolytic cleavage of ace is upregulated by endocytosed sars-cov- spike proteins. activation of the at r by elevated ang ii levels also further increases adam activity. adam correspondingly also cleaves its primary substrate releasing soluble tnf-α (tumor necrosis factor-α) into the extracellular region where it has auto-and paracrine functionality. tnf-α activation of its tumor necrosis factor receptor (tnfr) represents a third pathway elevating adam activity. tnf-α along with systemic cytokines released due to sars-cov- infection and in conjunction with comorbidities such as diabetes mellitus and hypertension can lead to a cytokine storm. tmprss indicates transmembrane protease serine . conditions in experimental models of human disease can be ameliorated and worsened with either the gain or loss of ace , respectively. , patients with covid- also suffer from gastrointestinal discomfort and diarrhea, which may arise earlier than respiratory conditions concurrent with the detection of viral rna in feces, as seen with previous coronavirus outbreaks. , , [ ] [ ] [ ] [ ] moreover, common comorbidities of cvd, including diabetes mellitus and obesity, are known to affect the integrity of the gastrointestinal-blood barrier and result in gut dysbiosis, bacteremia, and systemic inflammation ( figure ). development of gastrointestinal leakage and gut dysbiosis have correspondingly been linked to the onset of ph through the gut-lung axis and is closely related to hyperactivation of the ace/ang ii/at r (angiotensin ii type receptor) axis from ace loss. , continued viral production by host enterocytes perpetuates this situation and deteriorates conditions in the gut-lung axis. , evidence supports that sars-cov- infection potentially leads to degeneration of the gut-blood barrier leading to systemic spread of bacteria, endotoxins, and microbial metabolites likely affecting the host's response to covid- infection and cumulating in multisystem dysfunction and septic shock. , , enteric involvement and associated worsening in patient outcomes were documented from the initial sars-cov outbreak in the early s. fecal viral rna was detected in up to % of patients with viral shedding from the gastrointestinal tract associated with a more aggressive clinical course. , in a separate study, sars-cov particles were detected within the cytoplasm and surface microvilli of apical enterocytes in the ileum and colon while in patients with covid- , sars-cov- was detected in feces suggesting fecal-oral transmission. as such, the gastrointestinal tract of sars-cov, and possibly sars-cov- patients, acts as a staging ground for sustained viral replication concurrent with disruption of the enteric ace axis and adverse outcomes. , , , , , in addition to the direct impact of the virus on the microbiome, the predisposing disease states such as diabetes mellitus and pulmonary disease have their own adverse effects on the gut microbiome, , which may be worsened by sars-cov- infection. ang iidependent hypertension in animal models and humans is associated with gut dysbiosis, increased gut leakiness, and gut wall pathology. , , , there is broad support for these observations in pulmonary diseases including ph, copd, and asthma , and in type diabetes mellitus where dysbiosis characterized by decreased microbial richness and diversity, altered representation of bacterial metabolic pathways and modifications in the composition of firmicutes (f) and bacteroidetes (b). [ ] [ ] [ ] ace disruption in biomedical models has shown us that gut dysbiosis is quite prevalent and that this change in microbial profiles can alter systemic pathways exacerbating diabetes mellitus and hypertension. we recently showed that ace deficiency magnifies diabetes mellitus-induced dysbiosis characterized by an increase in peptidoglycan-producing bacteria and loss of gut barrier integrity in ace −/y -akita mice. we also identified a new role of bone marrow cells in the gut. in the ace −/y -akita or akita mice, the disrupted gut barrier was associated with reduced levels of circulating angiogenic cells, hematopoietic cells with reparative function. giving exogenous circulating angiogenic cells from wild-type mice corrected gut barrier dysfunction in ace −/y -akita or akita mice. thus, decreased enteric ace expression from sars-cov- infection may similarly reduce circulating angiogenic cells and compromise the integrity of the endothelium and gut epithelium leading to dysbiosis. further examination is required to validate this link and whether it is a direct or indirect effect of viral infection. link between ace , adam , and inflammation proteolytic cleavage of ace by adam- tnf-α (tumor necrosis factor-α) is a cytokine implicated in chronic inflammation, and its extracellular domain shedding and activation is driven by the membrane-bound protease coined tace (tnf-α-converting enzyme), also known as adam- (a disintegrin and metalloproteinase ). , adam- is a type i transmembrane protein belonging to the adamalysin subfamily of zn-dependent metalloproteases. following the discovery that adam- cleaves tnf-α, the substrate specificity of the enzyme has expanded to include various cytokines and receptors, many of which contribute to initiating and exacerbating inflammation. , importantly, adam- was also found to mediate proteolysis and ectodomain shedding of ace . enhanced ace shedding resulting from ras overactivation, and subsequent adam- upregulation drives pathogenesis in hf, atrial fibrillation, coronary artery disease, and thoracic aortic aneurysm. , , , ang ii-mediated activation of at r triggers a signaling cascade, which culminates in the activation of p mapk (mitogen-activated protein kinase) and adam- phosphorylation by napdh oxidase -induced reactive oxygen species formation. , phosphorylation enhances the catalytic activity of adam- , thus increasing ace shedding, resulting in loss of ace at the membrane and impaired conversion of ang ii (into ang - ), leading to ras-mediated detrimental effects in a positive feedback cycle. , importantly, depletion of ace at the cell surface is a critical pathological outcome of sars-cov- infection. sars-cov- is endocytosed by cells in complex with ace ; thus, the initial detrimental effects of viral infection begins with a loss of ace -mediated tissue protection. adam- activity is upregulated upon binding of sars-cov to ace and facilitates viral entry, while knockdown of adam- by sirna severely attenuated sars-cov cellular entry. the molecular mechanisms of sars-cov and another human coronavirus that only causes mild respiratory symptoms, hnl -cov, were compared. interestingly, although hnl -cov also utilizes ace as a receptor for cellular entry, it does not induce adam- activation and ace ectodomain shedding. , therefore, this study elucidates the unique role of adam- mediated shedding of ace in sars-cov infectivity and may inform the disparity in severity between coronavirus subtypes. furthermore, loss of membrane ace promotes ang ii accumulation, which also activates adam- activity, thus perpetuating membrane shedding of ace , ras overactivation, and inflammation. the modulatory effects on the ang ii/at r and ang - /masr axes make ace a plausible target in preventing and treating chronic inflammation and inflammatory diseases, as highlighted by the recent covid- pandemic. patients with covid- develop pneumonia with acceleration of injury in susceptible patients to multiple organ failure , driven in part by an inflammatory cytokine storm and is a notable cause of death in patients who are critically ill. , when the immune system is activated due to factors such as sars-cov- infection, there is an imbalance of th /treg cell function and overactivation of immune cells, which secrete a large number of proinflammatory cytokines. , , imbalance in the ras system and the loss of ace in patients with covid- are further contributing factors to tissue and systemic inflammation. , lipopolysaccharide-induced acute lung injury decreased expression of ace , precipitated inflammatory injury, and upregulated expression of renin, ang ii, ace, and at receptors. after injection of rhace , lung function and pathological injury improved with attenuation of inflammation. in addition, rhace is beneficial and improves acute lung injury caused by sars-cov, acid inhalation, and sepsis. , , ace knockout (ko) mice showed very severe acute respiratory distress syndrome (ards)/acute lung injury pathology, increased vascular permeability, increased pulmonary edema, neutrophil accumulation, and deterioration of lung function compared with normal wt control mice. , ace deficiency partially rescued the severe phenotype of mice with a single mutation of ace in acute lung injury by further deletion of the ace gene, suggesting that the balance of ace /ace levels is the key to lung injury/lung protection during an inflammatory storm. arbs (at r blockers) induce ace , ang - , and mas expression in line with the reduction of proinflammatory cytokines and induction of il- , an anti-inflammatory cytokine. we showed that ace ko hypertensive mice exhibited enhancement of proinflammatory cytokines, il- β, il , tnf-α, and chemokine (c-c motif) ligand while administration of rhace rescued ang ii-induced t-lymphocyte-mediated inflammation. , blockade of mas receptor by d-ala -ang - (a- ) completely inhibited the ang - mediated anti-inflammatory effects while ave , the agonist of ang - receptors, mimicked the actions of ang - . negative regulator of the ras discovery of ace resulted in a paradigm-changing concept in all aspects of the ras. ace is a monocarboxypeptidase that converts ang i into a nonapeptide, ang - , and ang ii into a heptapeptide, ang - (figure a ). this distinct enzymatic pathway for degradation of ang i and ang ii negatively regulates ras activation and mitigates the deleterious actions mediated by ang ii and at r. this is of particular significance in pathological conditions where the ras is overstimulated. ang - is a biologically active peptide whose vast array of effects are opposite to those attributed to ang ii. [ ] [ ] [ ] [ ] [ ] [ ] [ ] furthermore, ace can antagonize ace-independent formation of ang ii, such as from mast cell chymase. , in , an endogenous orphan receptor, mas receptor (masr), was identified as the ang - receptor, and a , a masr antagonist was shown to inhibit the majority of ang - effects. , - ang - has also shown beneficial biological effects via the at r that result in cardioprotection. [ ] [ ] [ ] [ ] thus, the ace /ang - /masr axis has emerged as a physiological antagonist that counter-regulates the activated ras. , [ ] [ ] [ ] [ ] [ ] the cardioprotective effects of ace taken together can be attributed to ( ) degradation of ang i to ang - , whereby limiting action of ace on its substrate, ( ) reducing ang ii detrimental effects through degradation of the peptide, and ( ) formation of ang - which exercises cardioprotective effects. formation of ang - is an important mechanism of ace mediated protection, as antagonism of ang - using a prevented beneficial effects of rhace in murine model of systolic dysfunction. diminished ace activity results in activation of the ang ii/at r axis, contributing to the increased progression of cvd. elevated ace level and activity result in the formation of ang - and ang - , leading to protection against cvd ( figure a ). the apelin family of peptides act through the apelin receptors mediating protection against cvd. , the x-linked apln gene encodes a amino acids prepro-apelin that is subsequently cleaved by endopeptidases to various bioactive peptides from to amino acids in length. cvd, including hf and hypertension, is characterized by an apelin deficient state in both human myocardium and plasma. [ ] [ ] [ ] apelin ko mice exhibit increased infarct size and systolic dysfunction following coronary ligation and reduced myocardial contractility concomitant with increased susceptibility to hf in pressure-overload models. , reduced myocardial ace mrna and ace protein levels in apelin ko mice, which were rescued by infusion of apelin- , suggest a crucial regulatory role of apelin in ace gene expression. apelin signaling through the apelin receptors specifically increased ace promoter activity leading to an increase in ace mrna and protein. [ ] [ ] [ ] these effects are consistent with the ability of the pyr-apelin- peptide to negatively regulate ang ii-mediated superoxide production, myocardial hypertrophy, dysfunction, and fibrosis and analogs of apelin- preventing abdominal aortic rupture in lowdensity lipoprotein receptor ko models induced by ang ii infusion. however, ace , through its monocarboxypeptidase activity, cleaves and inactivates bioactive apelin peptides apelin- and apelin- through a negative feedback mechanism in the heart and vasculature (figure b) . , due to the short half-life of endogenous apelin peptides in the plasma, synthetic apelin peptide analogs resistant to ace degradation and retaining their binding capability to endogenous apelin receptors elicit protection in the cardiovascular system are being explored as potential new therapies. , ace as a chaperone protein for the amino acid transporter, b at (slc a ) b at is highly expressed in the intestines and kidneys with function in the absorption of neutral amino acids. the ace -b( )at complex is assembled as a dimer of heterodimers, with the collectrin-like domain of ace mediating homo-dimerization. ace has a rasindependent function, regulating intestinal amino acid homeostasis, expression of antimicrobial peptides, and the gut microbiome. ace is necessary for the expression of the hartnup transporter in the intestine, and the differential functional association of mutant b( )at transporters with ace in the intestine regulates the phenotypic heterogeneity of human hartnup disorder. cardiovascular disease is the leading cause of death worldwide and a major public health concern. heart disease is characterized by the activation of several signaling pathways associated with pathological hypertrophy and maladaptive ventricular remodeling. in the heart, ace is localized to cardiomyocytes, cardiac fibroblasts, epicardial adipose tissue, and the coronary vascular endothelium , , ; ang - /masr is also present on cardiomyocytes, cardiac fibroblasts, and endothelial and vascular smooth muscle cells. , [ ] [ ] [ ] genetic ace deletion resulted in exacerbation of ang ii-mediated cardiorenal fibrosis and oxidative stress in the heart and kidney of hypertensive mice while administration of rhace (recombinant human ace ) remarkably rescued the ang ii-induced hypertension, pathological hypertrophy, oxidant injury, and cardiac dysfunction. , various ace polymorphisms are linked to cvd. post-mi remodeling and coronary artery disease are common causes of hf. , notably, mi increases ace mrna expression in human, mice, and rat hearts, , whereas genetic ace deletion results in worsening of mi-induced cardiac dysfunction, infarct size, mmp (matrix metalloproteinase) /mmp activation, and extracellular matrix disruption. , loss of ace leads to increased neutrophilic infiltration in the infarct and peri-infarct regions, resulting in upregulation of inflammatory cytokines, interferon-γ, il (interleukin)- , and the chemokine, mcp- (monocyte chemoattractant protein- ), as well as increased phosphorylation of erk / (extracellular signal-regulated kinase / ) and jnk / signaling pathways, changes that were blocked with an figure . ace (angiotensin converting enzyme ) role in the renin-angiotensin system peptide cascade and its interaction with the apelinergic peptide system. a, angiotensinogen is processed by renin into ang i (angiotensin i), which is further cleaved by ace or mast cell chymase into ang ii. ang ii can go on to affect the cardiovascular system predominantly through the angiotensin type receptor (at r) or via the angiotensin type receptor (at r). alternatively, ang ii can be degraded by the carboxypeptidase ace or the pcp (prolyl carboxypeptidase) into ang - (angiotensin - ). ang - mediates protective effects throughout tissues which host the mas receptor (masr). ang - can be further formed through the activity of ace on ang i forming ang - which is then cleaved by either ace or nep (neprilysin). b, stimulation of the apelin receptor by apelin peptides leads to cardiovascular protective effects while disrupting ang ii signaling by sequestration of the at r through receptor heterodimerization. apelin is inactivated by ace cleavage of its c-terminal phenylalanine while stimulation of the apelin receptor promotes ace mrna transcription presenting apelin's role as a positive regulator of ace . ros indicates reactive oxygen species. arb ultimately resulting in improvement in myocardial function. in contrast, overexpression of ace and the action of ang - ameliorates mi-induced cardiac remodeling. , importantly, heterozygote loss of ace , as seen in explanted human hearts from patients with dilated cardiomyopathy, was sufficient to increase susceptibility to heart disease. hf with preserved ejection fraction is a proinflammatory state closely linked to obesity-related cardiac and microvascular dysfunction for which there are no approved therapies. , , epicardial adipose tissue is a primary source of inflammatory cytokines that could have detrimental effects on the heart. loss of ace increases macrophage polarization to proinflammatory m -phenotype (alternatively activated, cd c + ) in epicardial adipose tissue from patients with hf with preserved ejection fraction, with decreases in polarization to anti-inflammatory, m -phenotype macrophages, and worsening of hf with preserved ejection fraction in response to diet-induced obesity. importantly, ang - decreased macrophage polarization in epicardial adipose tissue and preserved the cardiac function of obese ace ko mice. , ang - has potent anti-inflammatory effects in adipose tissue of obese type diabetic mice and protects against diabetic cardiomyopathy and nephropathy. [ ] [ ] [ ] the ace /ang - axis also promotes browning of adipose tissue leading to improved metabolic effects and weight loss, which can confer further benefits to the cardiovascular system. , blockade of the deleterious arm of the ras has been the mainstay of the therapeutic management of hypertensive individuals. an increase in ace and the vasoprotective axis of the ras by ace inhibitors and angiotensin receptor blockers (arbs) clearly reinforces this view (see section pharmacological antagonists of the ras and ace expression below). furthermore, increased ace expression protects against hypertension, while ace deficiency exacerbates hypertension. renal ace expression is inversely related to blood pressure in experimental models of hypertension. in the spontaneously hypertensive rat and stroke-prone spontaneously hypertensive rat, renal ace mrna levels are reduced compared with normotensive wistar-kyoto rats. these studies support the essential role of ace in maintaining healthy blood pressure. lentiviral overexpression of ace results in increased expression of antihypertensive components of the ras and attenuates elevated blood pressure. , pretreatment with rhace prevented hypertension induced by ang ii and decreased plasma ang ii while increasing plasma ang - levels. ace and adam were selectively knocked down from all neurons (ac-n), which revealed a reduction of inhibitory inputs to ac-n presympathetic neurons relevant for blood pressure regulation. mice with ace selectively knocked down from sim neurons in mice exhibited a blunted blood pressure elevation and preserved ace activity during the development of salt-sensitive hypertension. the metalloproteinase adam is responsible for mediating ace shedding from the cell membranebound domain, which can be promoted by ang ii, and release of ace as a soluble form in plasma , , impairing brain ace compensatory activity and thus contributing to the development of neurogenic hypertension. genetic ace deficiency is associated with the upregulation of putative mediators of atherogenesis and enhances responsiveness to proinflammatory stimuli suggestive of a key role of ace in suppressing vascular inflammation and atherosclerotic disease. in addition, ace inhibition blocks neuropeptide catestatin-mediated protective effects in the development of atherosclerosis in apoe −/− mice fed a high-fat diet. the counter-regulatory role of the ace /ang - / masr axis of the ras has been well characterized in the progression of diabetic complications, including cardiovascular and kidney disease. , , support for the importance of ace in diabetes mellitus comes from its impact on diabetic complications wherein diabetes mellitus-induced vascular dysfunction is strongly associated with a shift in the ras axis toward the profibrotic, proinflammatory arm of ras with a reduction in the protective arm ( figure ). loss of the protective effects of the ras is related to the regulation of tissue and circulating levels of ang ii and their sequelae in the context of diabetes mellitus. , alterations within the ras are considered pivotal for the development of both diabetic micro and macrovascular complications. , the blockade of the proinflammatory and profibrotic arms of the ras provides significant renoprotection in both experimental models of diabetes mellitus and in patients. while the loss of ace worsens diabetic kidney injury, rhace is therapeutic in an animal model of diabetic nephropathy and experimental alport syndrome. ace inhibitors in t d and angiotensin receptor blockade with losartan and irbesartan in t d retard the progression of nephropathy. in diabetic renal tubules, ace gene expression is decreased by ≈ %, which would reduce ang - formation and allow ang ii accumulation hence directly increasing the expression of tgf-β and ctgf (connective tissue growth factor), leading to tubulointerstitial fibrosis. ras blockade retards renal damage and ace inhibitor therapy, as mentioned above, resulting in a compensatory increase in ace , leading to renoprotection. therefore, support for the loss of ace contributing to vascular complications in diabetes mellitus comes from strong clinical and experimental evidence. retinopathy, the most common complication of diabetes mellitus and one of the leading causes of blindness in working-age adults is linked to activation of oxidative stress, profibrotic, and proinflammatory arm of the ras, which can be effectively curtailed by the ace /ang - axis in experimental models. , increased secretion of proinflammatory cytokines by bone marrow mesenchymal stem cells skews hematopoiesis toward the generation of an increased number of myelo-monocytic cells. target tissues of diabetic complications secrete ccl in response to high glucose-induced stress facilitating the homing of ccr + cells to these regions and promoting the development of vascular complications. [ ] [ ] [ ] [ ] [ ] [ ] in addition to an increase in myeloidosis, diabetics with complications have reduced bone marrow-derived vascular reparative cells and circulating angiogenic cells (cd + cells). levels of ace mrna were also a significant predictor of the presence of microvascular disease in diabetic patients. diabetic individuals who remained free of retinopathy despite > years of poor glycemic control had higher mrna levels for genes of the vasoprotective axis (ace /mas) compared with age, sex, and glycemia-matched diabetics with retinopathy. in dysfunctional cd + cells from diabetic individuals, activation of the protective arm of ras, by exposing the cells to ang - corrected their dysfunction by restoring bioavailable no and reducing reactive oxygen species. ang - gene modification of cd + cells restored the in vivo vasoreparative function of these cells in a mouse retinal ischemia-reperfusion injury model. moreover, intraocular administration of aav-ace or ang - reduced diabetes mellitus-induced retinal vascular leakage and inflammation, thus preventing retinopathy. patients with diabetes mellitus have a dysregulated ras, which may influence their vulnerability to sars-cov- . guan et al examined individuals with confirmed covid . of these individuals, had severe disease, and of this, . % were diabetics. zhang et al studied patients who were hospitalized due to the severity of their covid- infection, of these individuals, % had diabetes mellitus. it is interesting to speculate why diabetics may be more at risk for sars-cov- infection than the general population, and this may be due to the reduced ace levels that are typically observed in the vasculature of diabetic individuals and diabetic animal models. indeed, loss of ace was associated with marked gut dysbiosis, which was further worsened in a model with type diabetes mellitus. lung epithelial cells express high levels of ace , which positively correlates with airway epithelial differentiation. , , involvement of ace in ards, which is triggered by multiple diseases including sars-cov and sars-cov- , has been established in multiple animal models. , ace ko mice exhibit severe pathology of ards. , additional ace deficiency, or treatment with ar r blockers of ace ko mice rescues them from ards implicating the benefit of ace and the critical balance of the protective versus proinflammatory and fibrotic axes of the ras. these findings are consistent with evidence of a beneficial effect of rhace on pulmonary blood flow and oxygenation in a pig model of lipopolysaccharide-induced ards. age-related loss of ace in the lungs correlates with the increased mortality and worsened phenotype in elderly patients with covid- . ace has been implicated in acute lung injury by inducing an imbalance in the ras. evidence includes that in acute lung injury ( ) a decrease in pulmonary ace and an increase in ang ii levels occurs; ( ) supplementation with ace or inhibition of ang ii improves outcomes; and ( ) a lack or decrease of pulmonary ace aggravates viral-induced acute lung injury. ace is also involved in ph and fibrosis. increasing ace activity using rhace reduced bleomycin-induced inflammation and fibrosis, resulting in improved lung function and exercise capacity, and the ace activator, dize, protects animals from ph and fibrosis. moreover, oral feeding of a bioencapsulated form of ace protects and arrests the progression of ph. validation of this protective effect comes from a small human study that showed that ph is characterized by reduced ace activity and supplementation of these individuals with rhace improved pulmonary hemodynamics and reduced oxidative and inflammatory markers. collectively, these studies unequivocally establish the conceptual framework that ace is a central player in normal pulmonary function, and its imbalance leads to pulmonary diseases. pathological neurohormonal activation of the ras drives the development and progression of cvd. current pharmacotherapies aim to achieve multilevel ras inhibition through distinct modes of action. although ace is not the direct cellular target of these therapies, ace gene transcription, translation, and ultimately catalytic activities are modified due to the intricate nature of the ras. blocking the ace/ang ii/at r axis through limiting the formation and actions of ang ii potentiates the effects of ace as the endogenous ras counter-regulator. the arbs consistently increased ace mrna expression, protein levels, and catalytic activities in the heart, kidneys, and thoracic aorta, but the translation to protein levels and activity differs between experimental models and tissues for ace inhibitors (table) . , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] combination of lisinopril and losartan treatment in normotensive lewis rats abolished the increase in ace mrna levels observed individually but retained losartan induced rise in ace activity in the heart. moreover, lisinopril in normotensive lewis rats increased ace mrna without affecting ace activity in the heart, but the opposite was observed in the kidneys. , these findings could be attributed to tissue-specific regulation of ace , as higher ace protein levels were reported in the heart, but ace activity was higher in the kidneys of sprague-dawley rats, adding to the complexity of the tissue ras. in type diabetic akita angiotensinogen-transgenic mice, dual ras blockade with perindopril and losartan normalized diseasemediated reduction in kidney ace mrna expression and protein levels. these findings suggest that the accumulation of ang ii in pathological conditions contributes to the modulatory effects of ras blockade on ace . ang ii can regulate ace expression through the at r. healthy hearts and kidneys are characterized by high levels of ace mrna and protein expression, with moderate expression of ace. ras overactivation in cvd increases at r stimulation by ang ii, promoting erk / and p mapk signaling pathways to downregulate ace while upregulating ace expression. activation of p mapk upregulates adam activity though posttranslational phosphorylation of the cytoplasmic domain results in shedding of surface ace in a positive feedback loop and could explain the observed effects of arbs in increasing ace protein levels and activity. , , mechanisms behind the augmentation of ace mrna levels by ace inhibitors and arbs require further characterization. moreover, mineralocorticoid receptor antagonists increased ace mrna expression and activity in samples from patients with chronic hf, wild-type mice, and rats to varying degrees among different tissues but not in the heart of a rat hypertensive disease model (table) . [ ] [ ] [ ] spironolactone, a nonselective mineralocorticoid receptor antagonist, prevented the increase in both ace and at r mrna levels, and the associated increase in at r density from aldosterone signaling in cardiomyocytes. , activation of mineralocorticoid receptors also stimulates overlapping downstream signaling pathways with at r, including the erk / and p mapk pathways mentioned before. , blocking these signaling pathways contributes to the observed effect of mineralocorticoid receptor antagonist on ace gene expression, surface protein levels, and activity. promoting the ace /ang - /mas signaling by rhace or the ang - receptors agonist ave can have salutary therapeutic effects in cvd and lung disease from diverse etiologies. the ang - receptors agonist ave has been shown to exert cardiorenal and pulmonary protective effects, and treatment with rhace improved the symptoms of acute lung injury, cvd, and kidney injury in various preclinical models. , , , maintaining ace levels in patients with or predisposed to common cvd states such as diabetes mellitus, hypertension, and obesity wards off the advancement of these comorbidities in instances where the patient contracts sars-cov- by maintaining a level of ace /ang - / masr negative counter-regulation. rhace functionally sequesters circulating viral particles to prevent s-protein interactions with endogenous ace , while simultaneously regulating the systemic ras may provide therapeutic benefits in covid- and is moving into phase ii clinical trials in europe. a potential limitation of rhace is the restricted penetrance and activity against tissue ras owning to its large molecular size. pharmacological ras blockade agents, arbs, in particular, are capable of modulating both systemic and tissue ras, and simultaneously increasing ace expression and activity in experimental models. the direct implications of ras inhibition in patients with covid- with hypertension remain elusive, and clinical evidence is desperately needed to determine the relative benefits and risks associated with usage of these medications. nonetheless, introducing arbs to patients already infected by sars-cov- may be an effective therapeutic option in addressing the viral-mediated ras imbalance and is currently under investigation in several clinical trials (www.clinicaltrial.gov number nct , nct , and nct ). [ ] [ ] [ ] potential for ace as a therapy is also facilitated by using the probiotic species lactobacillus paracasei (lp), which can be engineered to express recombinant ace indicates angiotensin-converting enzyme; and ras, renin-angiotensin system. proteins. mice treated with the recombinant lp expressing the secreted ace in fusion with the nontoxic subunit b of cholera toxin (acts as a carrier to facilitate transmucosal transport) showed increased ace activities in serum and tissues and reduced diabetic retinopathy. these results provide proof of concept for using bioengineered probiotic species as live vectors for delivery of human ace with enhanced tissue bioavailability for treating diabetic complications but could potentially be repurposed for treating cvd and covid- infection. since the discovery of ace in , tremendous progress has been made in elucidating its biochemical actions and fundamental role in cvd and, more recently, as the sars-cov- receptor. ace is a dominant mechanism for negative regulation of the ras by metabolizing ang ii into the beneficial peptide ang - , and this important biochemical and physiological property is being harnessed as a potential therapy in patients with hf. the activation of the ras axis due to binding of sars-cov- to ace , leading to direct loss of ace and indirectly via proteolytic processing and shedding, partly drives the systemic manifestations of covid- . careful targeting of the ras axes is needed in these patients to optimize their clinical outcomes, including the use of at receptor blockers (arb). role of the ace /angiotensin - axis of the renin-angiotensin system in heart failure a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase a novel angiotensinconverting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - angiotensin converting enzyme : a double-edged sword angiotensin-converting enzyme is a functional receptor for the sars coronavirus ace : from vasopeptidase to sars virus receptor angiotensin-converting enzyme : the first decade ace links amino acid malnutrition to microbial ecology and intestinal inflammation gut microbiota: potential for a unifying hypothesis for prevention and treatment of hypertension hypertension-linked pathophysiological alterations in the gut bone marrow-derived cells restore functional integrity of the gut epithelial and vascular barriers in a model of diabetes and ace deficiency oral delivery of angiotensin-converting enzyme and angiotensin-( - ) bioencapsulated in plant cells attenuates pulmonary hypertension roles of angiotensin peptides and recombinant human ace in heart failure ace and adam interaction regulates the activity of presympathetic neurons structure, function, and antigenicity of the sars-cov- spike glycoprotein structural basis for the recognition of sars-cov- by full-length human ace angiotensin-converting enzyme protects from severe acute lung failure a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury angiotensin converting enzyme abrogates bleomycin-induced lung injury altered gut microbiome profile in patients with pulmonary arterial hypertension a potential therapeutic role for angiotensin-converting enzyme in human pulmonary arterial hypertension a pilot clinical trial of recombinant human angiotensin-converting enzyme in acute respiratory distress syndrome six truisms concerning ace and the renin-angiotensin system educed from the genetic analysis of mice angiotonin-activator, renin-and angiotonin-inhibitor, and the mechanism of angiotonin tachyphylaxis in normal, hypertensive, and nephrectomized animals angiotensinconverting enzyme is an essential regulator of heart function the preparation and function of the hypertensin-converting enzyme studies on experimental hypertension: i. the production of persistent elevation of systolic blood pressure by means of renal ischemia evaluation of angiotensin-converting enzyme (ace), its homologue ace and neprilysin in angiotensin peptide metabolism a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study structure of sars coronavirus spike receptor-binding domain complexed with receptor cryo-em structure of the -ncov spike in the prefusion conformation coronaviruses post-sars: update on replication and pathogenesis structural and functional analysis of the surface protein of human coronavirus oc recovery in tracheal organ cultures of novel viruses from patients with respiratory disease epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china clinical features of patients infected with novel coronavirus in wuhan organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways multiple organ infection and the pathogenesis of sars genome composition and divergence of the novel coronavirus ( -ncov) originating in china structural basis of receptor recognition by sars-cov- host cell proteases: critical determinants of coronavirus tropism and pathogenesis blunting of cardioprotective actions of estrogen in female rodent heart linked to altered expression of cardiac tissue chymase and ace clinical and biochemical indexes from -ncov infected patients linked to viral loads and lung injury sars-coronavirus modulation of myocardial ace expression and inflammation in patients with sars china medical treatment expert group for covid- . clinical characteristics of coronavirus disease in china sars-cov : should inhibitors of the renin-angiotensin system be withdrawn in patients with covid- ? cardiac involvement in a patient with coronavirus disease (covid- ) available at jamanetwork association of cardiac injury with mortality in hospitalized patients with covid- in wuhan coronavirus disease (covid- ) and cardiovascular disease available at ahajournals.org cardiovascular implications of fatal outcomes of patients with coronavirus disease (covid- ) a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome enteric involvement of severe acute respiratory syndrome-associated coronavirus infection human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus brain-gut-bone marrow axis: implications for hypertension and related therapeutics prolonged presence of sars-cov- viral rna in faecal samples enteric involvement of coronaviruses: is faecaloral transmission of sars-cov- possible? tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis restructuring of the gut microbiome by intermittent fasting prevents retinopathy and prolongs survival in db/db mice microbiome and diabetes: where are we now? emerging pathogenic links between microbiota and the gut-lung axis chronic control of high blood pressure in the spontaneously hypertensive rat by delivery of angiotensin type receptor antisense intestinal permeability biomarker zonulin is elevated in healthy aging microglial cells impact gut microbiota and gut pathology in angiotensin ii-induced hypertension ace and microbiota: emerging targets for cardiopulmonary disease therapy pulmonary hypertension: pathophysiology beyond the lung churning out safer microbes for drug delivery genetically engineered probiotics lactic acid bacteria for delivery of endogenous or engineered therapeutic molecules a metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha adams: focus on the protease domain adam- : the enzyme that does it all cardiomyocyte a disintegrin and metalloproteinase (adam ) is essential in post-myocardial infarction repair by regulating angiogenesis angiotensin ii induced proteolytic cleavage of myocardial ace is mediated by tace/adam- : a positive feedback mechanism in the ras cell-specific functions of adam regulate the progression of thoracic aortic aneurysm detection of soluble angiotensin-converting enzyme in heart failure: insights into the endogenous counter-regulatory pathway of the renin-angiotensin-aldosterone system reactive oxygen species and p mitogen-activated protein kinase mediate tumor necrosis factor α-converting enzyme (tace/adam- ) activation in primary human monocytes direct activation of tace-mediated ectodomain shedding by p map kinase regulates egf receptor-dependent cell proliferation clinical relevance and role of neuronal at receptors in adam -mediated ace shedding in neurogenic hypertension sars coronavirus entry into host cells through a novel clathrin-and caveolaeindependent endocytic pathway modulation of tnf-alpha-converting enzyme by the spike protein of sars-cov and ace induces tnfalpha production and facilitates viral entry human coronavirus nl employs the severe acute respiratory syndrome coronavirus receptor for cellular entry receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars trilogy of ace : a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters pulmonary angiotensin-converting enzyme (ace ) and inflammatory lung disease ace exhibits protective effects against lps-induced acute lung injury in mice by inhibiting the lps-tlr pathway angiotensin-converting enzyme suppresses pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction prevention of angiotensin iimediated renal oxidative stress, inflammation, and fibrosis by angiotensin-converting enzyme angiotensin( - ) blunts hypertensive cardiac remodeling by a direct effect on the heart recombinant human angiotensin-converting enzyme as a new renin-angiotensin system peptidase for heart failure therapy genetically altered animal models for mas and angiotensin-( - ) and mas: the other side of the coin the renin-angiotensin system: going beyond the classical paradigms emerging evidence for a functional angiotensin-converting enzyme -angiotensin-( - )-mas receptor axis: more than regulation of blood pressure? angiotensin-( - ) contributes to the antihypertensive effects of blockade of the renin-angiotensin system multifunctional role of chymase in acute and chronic tissue injury and remodeling angiotensin-( - ) through receptor mas mediates endothelial nitric oxide synthase activation via akt-dependent pathways angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas angiotensin-( - ) inhibits growth of cardiac myocytes through activation of the mas receptor alteration of cardiac ace /mas expression and cardiac remodelling in rats with aortic constriction adenoviral delivery of angiotensin-( - ) or angiotensin-( - ) inhibits cardiomyocyte hypertrophy via the mas or angiotensin type receptor angiotensin-( - ) attenuates cardiac fibrosis in the stroke-prone spontaneously hypertensive rat via the angiotensin type receptor angiotensin-( - ) regulates cardiac hypertrophy in vivo and in vitro rho kinase inhibition activates the homologous angiotensinconverting enzyme-angiotensin-( - ) axis in experimental hypertension the role of ace in cardiovascular physiology angiotensin-converting enzyme antagonizes angiotensin ii-induced pressor response and nadph oxidase activation in wistar-kyoto rats and spontaneously hypertensive rats angiotensin ii-mediated oxidative stress and inflammation mediate the age-dependent cardiomyopathy in ace null mice cardioprotective effects mediated by angiotensin ii type receptor blockade and enhancing angiotensin - in experimental heart failure in angiotensinconverting enzyme -null mice angiotensin-converting enzyme ii in the heart and the kidney antagonism of angiotensin - prevents the therapeutic effects of recombinant human ace targeting the apelin pathway as a novel therapeutic approach for cardiovascular diseases international union of basic and clinical pharmacology. lxxiv. apelin receptor nomenclature, distribution, pharmacology, and function plasma concentrations of the novel peptide apelin are decreased in patients with chronic heart failure novel role for the potent endogenous inotrope apelin in human cardiac dysfunction reduced circulating apelin in essential hypertension and its association with cardiac dysfunction impaired heart contractility in apelin gene-deficient mice associated with aging and pressure overload loss of apelin exacerbates myocardial infarction adverse remodeling and ischemia-reperfusion injury: therapeutic potential of synthetic apelin analogues apelin is a positive regulator of ace in failing hearts the apelin receptor inhibits the angiotensin ii type receptor via allosteric trans-inhibition apelin is a negative regulator of angiotensin iimediated adverse myocardial remodeling and dysfunction apelin protects against abdominal aortic aneurysm and the therapeutic role of neutral endopeptidase resistant apelin analogs angiotensinconverting enzyme metabolizes and partially inactivates pyr-apelin- and apelin- : physiological effects in the cardiovascular system tissue-specific amino acid transporter partners ace and collectrin differentially interact with hartnup mutations angiotensin-converting enzyme is a critical determinant of angiotensin ii-induced loss of vascular smooth muscle cells and adverse vascular remodeling ace deficiency worsens epicardial adipose tissue inflammation and cardiac dysfunction in response to diet-induced obesity impairment of in vitro and in vivo heart function in angiotensin-( - ) receptor mas knockout mice angiotensin-( - ) counterregulates angiotensin ii signaling in human endothelial cells angiotensin-( - ) binds to specific receptors on cardiac fibroblasts to initiate antifibrotic and antitrophic effects from gene to protein-experimental and clinical studies of ace in blood pressure control and arterial hypertension navigating the crossroads of coronary artery disease and heart failure myocardial infarction increases ace expression in rat and humans loss of angiotensin-converting enzyme accelerates maladaptive left ventricular remodeling in response to myocardial infarction angiotensin-converting enzyme inhibits high-mobility group box and attenuates cardiac dysfunction post-myocardial ischemia circulating rather than cardiac angiotensin-( - ) stimulates cardioprotection after myocardial infarction heterozygote loss of ace is sufficient to increase the susceptibility to heart disease epicardial adipose tissue as a metabolic transducer: role in heart failure and coronary artery disease epicardial adipose tissue may mediate deleterious effects of obesity and inflammation on the myocardium ace /ang - axis: a critical regulator of epicardial adipose tissue inflammation and cardiac dysfunction in obesity angiotensin - ameliorates diabetic cardiomyopathy and diastolic dysfunction in db/db mice by reducing lipotoxicity and inflammation angiotensin - mediates renoprotection against diabetic nephropathy by reducing oxidative stress, inflammation, and lipotoxicity angiotensin - stimulates brown adipose tissue and reduces diet-induced obesity ace exerts anti-obesity effect via stimulating brown adipose tissue and induction of browning in white adipose tissue ace gene transfer attenuates hypertension-linked pathophysiological changes in the shr transgenic angiotensin-converting enzyme overexpression in vessels of shrsp rats reduces blood pressure and improves endothelial function targeting the degradation of angiotensin ii with recombinant angiotensin-converting enzyme : prevention of angiotensin ii-dependent hypertension tumor necrosis factor-alpha convertase (adam ) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme- (ace ) brain angiotensinconverting enzyme type shedding contributes to the development of neurogenic hypertension genetic ace deficiency accentuates vascular inflammation and atherosclerosis in the apoe knockout mouse decreased circulating catestatin levels are associated with coronary artery disease: the emerging anti-inflammatory role human recombinant ace reduces the progression of diabetic nephropathy loss of angiotensin-converting enzyme- exacerbates diabetic cardiovascular complications and leads to systolic and vascular dysfunction: a critical role of the angiotensin ii/at receptor axis interaction of diabetes and ace in the pathogenesis of cardiovascular disease in experimental diabetes role of angiotensin-converting enzyme (ace ) in diabetic cardiovascular complications optimizing treatment of hypertension in patients with diabetes loss of angiotensin-converting enzyme- (ace ) accelerates diabetic kidney injury murine recombinant angiotensin-converting enzyme attenuates kidney injury in experimental alport syndrome effects of losartan on renal and cardiovascular outcomes in patients with type diabetes and nephropathy characterization of renal angiotensin-converting enzyme in diabetic nephropathy ace and ang-( - ) confer protection against development of diabetic retinopathy adeno-associated virus overexpression of angiotensin-converting enzyme- reverses diabetic retinopathy in type diabetes in mice diabetic retinopathy is associated with bone marrow neuropathy and a depressed peripheral clock long-term type diabetes influences haematopoietic stem cells by reducing vascular repair potential and increasing inflammatory monocyte generation in a murine model ccl /mcp- modulation of microglial activation and proliferation ccl induces a pro-inflammatory profile in microglia in vitro role of chemokines in cns health and pathology: a focus on the ccl /ccr and cxcl / cxcr networks critical roles for ccr and mcp- in monocyte mobilization from bone marrow and recruitment to inflammatory sites chronic expression of monocyte chemoattractant protein- in the central nervous system causes delayed encephalopathy and impaired microglial function in mice the role of cc chemokine receptor on microglia activation and blood-borne cell recruitment after transient focal cerebral ischemia in mice activation of the ace / angiotensin-( - )/mas receptor axis enhances the reparative function of dysfunctional diabetic endothelial progenitors decreased glomerular and tubular expression of ace in patients with type diabetes and kidney disease age-and gender-related difference of ace expression in rat lung role of angiotensin-converting enzyme (ace) and ace in a rat model of smoke inhalation induced acute respiratory distress syndrome recombinant angiotensin-converting enzyme improves pulmonary blood flow and oxygenation in lipopolysaccharide-induced lung injury in piglets diminazene aceturate improves autonomic modulation in pulmonary hypertension upregulation of angiotensin-converting enzyme after myocardial infarction by blockade of angiotensin ii receptors effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme effects of renin-angiotensin system blockade on renal angiotensin-( - ) forming enzymes and receptors angiotensin ii at receptors regulate ace and angiotensin-( - ) expression in the aorta of spontaneously hypertensive rats effect of angiotensin ii blockade on a new congenic model of hypertension derived from transgenic ren- rats enalapril attenuates downregulation of angiotensin-converting enzyme in the late phase of ventricular dysfunction in myocardial infarcted rat localization of ace in the renal vasculature: amplification by angiotensin ii type receptor blockade using telmisartan ace deficiency enhances angiotensin ii-mediated aortic profilin- expression, inflammation and peroxynitrite production organ-specific distribution of ace mrna and correlating peptidase activity in rodents dual ras blockade normalizes angiotensin-converting enzyme- expression and prevents hypertension and tubular apoptosis in akita angiotensinogen-transgenic mice mineralocorticoid receptor blocker increases angiotensin-converting enzyme activity in congestive heart failure patients aldosterone, but not angiotensin ii, reduces angiotensin converting enzyme gene expression levels in cultured neonatal rat cardiomyocytes effects of aldosterone and angiotensin ii receptor blockade on cardiac angiotensinogen and angiotensin-converting enzyme expression in dahl saltsensitive hypertensive rats angiotensin ii up-regulates angiotensin i-converting enzyme (ace), but down-regulates ace via the at -erk/p map kinase pathway aldosterone induces angiotensin-converting-enzyme gene expression in cultured neonatal rat cardiocytes angiotensin at receptor subtype as a cardiac target of aldosterone: role in aldosterone-salt-induced fibrosis aldosterone-induced osteopontin expression in vascular smooth muscle cells involves mr, erk, and p mapk aldosterone increases vegf-a production in human neutrophils through pi k, erk / and p pathways angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target renin-angiotensin-aldosterone system inhibitors in patients with covid- losartan for patients with covid- requiring hospitalization. . www.clinicaltrials.gov identifier: nct losartan for patients with covid- not requiring hospitalization. . www ace inhibitors, angiotensin ii type-i receptor blockers and severity of covid- (covid-ace) expression of human ace in lactobacillus and beneficial effects in diabetic retinopathy in mice we acknowledge patients and their families for participating in our studies. none. key: cord- -n zb am authors: postlethwait, john h.; farnsworth, dylan r.; miller, adam c. title: an intestinal cell type in zebrafish is the nexus for the sars-cov- receptor and the renin-angiotensin-aldosterone system that contributes to covid- comorbidities date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: n zb am people with underlying conditions, including hypertension, obesity, and diabetes, are especially susceptible to negative outcomes after infection with the coronavirus sars-cov- . these covid- comorbidities are exacerbated by the renin-angiotensin-aldosterone system (raas), which normally protects from rapidly dropping blood pressure or dehydration via the peptide angiotensin ii (ang ii) produced by the enzyme ace. the ace paralog ace degrades ang ii, thus counteracting its chronic effects. ace is also the sars-cov- receptor. ace, the coronavirus, and covid- comorbidities all regulate ace , but we don’t yet understand how. to exploit zebrafish (danio rerio) as a disease model to understand mechanisms regulating the raas and its relationship to covid- comorbidities, we must first identify zebrafish orthologs and co-orthologs of human raas genes, and second, understand where and when these genes are expressed in specific cells in zebrafish development. to achieve these goals, we conducted genomic analyses and investigated single cell transcriptomes. results showed that most human raas genes have an ortholog in zebrafish and some have two or more co-orthologs. results further identified a specific intestinal cell type in zebrafish larvae as the site of expression for key raas components, including ace, ace , the coronavirus co-receptor slc a , and the angiotensin-related peptide cleaving enzymes anpep and enpep. results also identified specific vascular cell subtypes as expressing ang ii receptors, apelin, and apelin receptor genes. these results identify specific genes and cell types to exploit zebrafish as a disease model for understanding the mechanisms leading to covid- comorbidities. summary statement genomic analyses identify zebrafish orthologs of the renin-angiotensin-aldosterone system that contribute to covid- comorbidities and single-cell transcriptomics show that they act in a specialized intestinal cell type. orthologs and previously unrecognized co-orthologs of important components; and second, that a specific under-characterized cell type expresses many raas components and is hence a focal cell type for the raas that merits further exploration. an apparently similar cell type in humans allows sars-cov- infection, the production of infectious virus, and likely some covid- pathologies (stanifer et al., ) . these studies support zebrafish as a model for investigating the relationship of the raas to covid- pathologies. to identify raas components in the zebrafish genome and to detect potential covid- -related cellular systems, we combined our ongoing efforts in defining the relationships of zebrafish and human genomes (braasch et ang i sequences vary more among ray-finned than lobe-finned vertebrates (fig. ) . because coelacanth and several basally diverging ray-finned vertebrates (sturgeon, elephant fish) have the sequence nrvyvhpfnl, this is likely the ancestral ang i sequence for all bony vertebrates. several ray-finned vertebrates have isoleucine at position , representing independent mutations that happen to match placental mammals. position is highly variable in ray fins as in lobe fins. zebrafish ang i is nrvyvhpfnl, differing from the human form at positions , , and . the angiotensin system was likely already active in stem vertebrates because chondrichthys (cartilaginous fish) and even agnathans (jawless vertebrates) possess angiotensinogen genes (takei et al., ) . at position , some cartilaginous fish have arginine like most ray-finned vertebrates, but others have asparagine or tryptophan, and position is variable. the ancestral ang i sequence in jawed vertebrates was likely the same as in ancestral bony fishes (nrvyvhpfnl). angiotensinogen genes in jawless vertebrates appear to encode an angiotensin that shares the amino- terminal four residues with mammals but varies in the carboxy-terminal six residues (wong and takei, ). at position , lampreys have either aspartic acid or glutamic acid, but the conserved isoleucine or leucine at position is replaced with methionine followed by glutamine replacing the otherwise invariant histidine. lamprey ang ii alters cardiovascular dynamics in live lampreys but teleost ang ii (nrvyvhpf) did not (wong and takei, ) angiotensin ii (ang ii) forms when ace cleaves two c-terminal amino acid residues from ang i (fig. s . ). ang ii contributes to hypertension, an important covid- comorbidity, and likely promotes the inflammation that leads to poor disease outcomes. human and zebrafish ang ii differ only at the first and fifth residues (drvtihpf vs. nrvtvhpf, fig. ) . importantly, the fish and human peptides act about equally in zebrafish has a single ortholog of human agtr with conserved syntenies (fig. g) conserved syntenies suggest a mechanism for this situation. in human, mas lies in the gene sequence: . importantly, recent analyses showed that slc a is at the peak of a genome wide association study for poor outcomes from covid- (ellinghaus et al., ). these findings raise the novel hypothesis that slc a variants contribute to variation in covid- outcomes due to differences in expression or protein function related to interactions with ace , the sars- cov- receptor. adam (fig. s . ) is a metalloendopeptidase that cleaves the membrane isoform of ace to make the soluble protein sace (lambert et al., ) . zebrafish has two copies of adam in double conserved synteny with human adam (fig. a) , showing that they are co-orthologs of human adam from the tgd. the zebrafish atlas showed expression of adam a (ensdarg ) stronger in embryonic intestinal epithelium than in larval ace -expressing c cells (fig. b ). in addition, several cells in the vascular endothelium expressed adam a (fig. c) . the tgd duplicate adam b (ensdarg ) was expressed in the atlas in a few widely dispersed individual cells. because expression of adam paralogs was not detected in c , zebrafish may not have soluble ace ; alternatively, a different enzyme in zebrafish might cleave ace to make the soluble form or expression levels may be too low for detection. analysis of the zebrafish aplnra and aplnrb genes in the same tree (ensgt ) showed that most teleost clades have orthologs of each gene, that the bonytongues, which branch deep in the teleosts, root each clade; furthermore, spotted gar and reedfish serve as pre-tgd outgroups, as expected from historical species relationships (fig. b) . the tree showed that the sister group of the ray-finned aplnra+alpnrb clade is a lobe-finned vertebrate clade rooted on coelacanth and amphibia as expected and includes 'reptiles' and birds (fig. b ). this clade, which we tentatively here call the aplnrl clade, contains only monotremes and marsupials among mammals, indicating that this gene was lost in eutherian mammals (zhang et al., ) . conserved syntenies showed that chicken (gallus callus) chromosome gga , which contains aplnrl (ensgalg ), has orthologs on both dre and dre , the sites of aplnra and aplnrb, respectively, as well as dre (fig. d) . these conserved syntenies independently verify that: ) aplnrl was present in the last common ancestor of human and zebrafish; ) aplnrl was lost from eutherians after they diverged from marsupials; and ) the tgd produced aplnra and aplnrb paraogs. these analyses support the hypothesis that the last common ancestor of zebrafish and human had at least two aplnr-related genes; one became aplnr in human and 'aplnr ' in teleosts and the other was lost in eutherians but retained in other tetrapods, and subsequently duplicated in the tgd, becoming 'aplnra' and 'aplnrb' in zebrafish. renaming aplnr (ensdarg ) to aplnr, aplna (ensdarg ) to aplnrla, and aplnrb (ensdarg ) to aplnrlb would better connect zebrafish to human biology. atlas cells expressing aplnr (ensdarg ) occupied midline fates, including prominently the floorplate (c ), hypochord (c ), and scleroderm (c ), as well as an embryonic intestinal epithelium cell type (c ). expression of alpnr was detected in the spleen and heart of zebrafish adults by qpcr (zhang et conserves genes encoding ang ii receptors agtr and agtr . zebrafish also has orthologs encoding slc a , which binds ace and adam , the enzyme that creates the soluble form of ace . furthermore, zebrafish has an ortholog encoding tmprss , which activates coronavirus spike protein for binding to ace and bringing the virus into human cells. zebrafish also has the ligand and receptors for the apelin signaling system. zebrafish has a single ortholog of most raas genes but has duplicates of some. many zebrafish duplicates of human raas genes derive from the teleost genome duplication event, including ) agtr a and agtr b, ) anpepa and anpepb, ) slc a a and slc a b, and ) aplnrla and aplnrlb. other duplicated raas genes appeared by tandem duplication, including ) anpep and anpepl, ) anpepla. and anpepla. ; and ) slc a a. and slc a a. . the significance of these discoveries is that the actions of both zebrafish co- orthologs must be considered when translating zebrafish science to human biology. the raas/apelin system also provides an example of 'ohnologs gone missing', in which one ohnolog zebrafish express covid- -related raas genes in tissues similar to human. results showed that, like humans and other mammals, zebrafish liver cells express angiotensinogen. in the atlas, dpf zebrafish larvae had three types of hepatocytes, two of which expressed agt, adding cellular precision to expression studies in adult zebrafish (cheng et al., ) . induction of agt expression in mammals relies on cortisol and inflammation (brasier and li, ace, which cleaves ang i to ang ii, was surprisingly shown by our scrna-seq analysis to be co-expressed significantly, slc a is at the peak of the strongest of two genome wide association study loci for undesirable covid- outcomes (ellinghaus et al., ) . we suggest the hypothesis that genetic variants near slc a affect the severity of covid- symptoms due to variations in interactions with ace . angiotensin peptides are related to covid- comorbidities because they bind to agtr and agtr receptors on vascular cells to help regulate vasoconstriction, and on the adrenal to stimulate secretion of aldosterone, leading to salt and water retention related to the comorbidity of obesity-related kidney damage (fyhrquist and saijonmaa, ). as in humans, zebrafish scrna-seq analysis showed that agtr b is expressed in endothelial cells and confirmed that agtr is also expressed in endothelial cells (wong et al., ). the conserved expression of zebrafish raas-related genes supports the contention that raas regulation the zebrafish raas is pharmacologically similar to that of mammals. the ace inhibitor lisinopril blocks the effects of ang i on sodium uptake in zebrafish, as predicted if ace were required to convert ang i to ang ii (kumai et al., ) . zebrafish cultured from hpf to dpf in water containing the ace inhibitor captopril do not differ in survival from controls and neither do fish in water with % of the normal salt concentration (rider (e), anpepa (f), and dpp (g). all were expressed in c , the same intestinal epithelium cell type that expressed ace, except anpeplb, which was expressed in blood vessels at dpf and a few neural crest cells, but not in intestinal epithelium. dipeptidyl peptidase- inhibitors can inhibit angiotensin converting enzyme zebrafish hox clusters and vertebrate genome evolution further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus angiotensin ii stimulation of dpp activity regulates megalin in the proximal tubules mas and its related g protein-coupled receptors presumed asymptomatic carrier transmission of covid- zebrafish models of cardiovascular disease the zebrafish model of tuberculosis -no lungs needed the spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons a new model army: emerging fish models to study the genomics of vertebrate evo-devo mechanisms for inducible control of angiotensinogen gene transcription evolution of hormone-receptor complexity by molecular exploitation analyses of the cyp b gene family in the guinea pig suggest the existence of a primordial cyp b gene with aldosterone synthase activity the renin angiotensin aldosterone system in obesity and hypertension: roles in the cardiorenal metabolic syndrome automated identification of conserved synteny after whole-genome duplication hnf factors form a network to regulate liver-enriched genes in zebrafish kidney disease is associated with in-hospital death of patients with covid- ace orthologues in non-mammalian vertebrates (danio, gallus, fugu, tetraodon and xenopus) dichotomous role of the macrophage in early mycobacterium marinum infection of the zebrafish full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus small molecule screen for compounds that affect vascular development in the zebrafish retina slc neurotransmitter transporters: structure, function, and regulation a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury angiotensin-ii promotes na+ uptake in larval zebrafish, danio rerio, in acidic and ion-poor water in vivo modulation of endothelial polarization by apelin receptor signalling neuroendocrine control of ionic balance in zebrafish developing zebrafish disease models for in vivo small molecule screens the sh -domain-containing inositol -phosphatase (ship) limits the motility of neutrophils and their recruitment to wounds in zebrafish tumor necrosis factor-alpha convertase (adam ) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme- (ace ) possible bat origin of severe acute respiratory syndrome coronavirus notch signaling is required for arterial-venous differentiation during embryonic vascular development genomic characterization and expression analysis of the first nonmammalian renin genes from zebrafish and pufferfish structure and functions of angiotensinogen genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding translational power of the zebrafish: an interspecies analysis of responses to cardiovascular drugs. front pharmacol epigenetic control of intestinal barrier function and inflammation in zebrafish zebrafish angiotensin ii receptor-like a (agtrl a) is expressed in migrating hypoblast, vasculature, and in multiple embryonic epithelia inferring the mammal tree: species-level sets of phylogenies for questions in ecology, evolution, and conservation renin-angiotensin-aldosterone system inhibitors in patients with covid- angiotensin ii stimulation of the basolateral located na+/h+ antiporter in eel (anguilla anguilla) enterocytes human intestine luminal ace and amino acid transporter expression increased by ace-inhibitors antigenicity of the sars-cov- spike glycoprotein does comorbidity increase the risk of patients with covid- : evidence from meta-analysis molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-b and its receptor eph-b immunolocalization of subtype angiotensin ii (at ) receptor protein in rat heart members of the undiagnosed diseases facilitate rare disease diagnosis and therapeutic research the zebrafish book. a guide for the laboratory use of summary of probably sars cases with onset of illness from host gut motility promotes competitive exclusion within a model intestinal microbiota identification of vasculature-specific genes by microarray analysis of etsrp/etv overexpressing zebrafish embryos characterization of a native angiotensin from an anciently diverged serine protease inhibitor in lamprey angiotensin at receptor activates the cyclic-amp signaling pathway in eel high expression of ace receptor of -ncov on the epithelial cells of oral mucosa structural basis for the recognition of sars-cov- by full-length human ace apelin- ( - ) is a biologically active ace metabolite of the endogenous cardiovascular peptide the pathogenesis and treatment of the `cytokine storm' in covid- deletion of the angiotensin type receptor (at r) reduces adipose cell size and protects from diet-induced obesity and insulin resistance apelin and its receptor control heart field formation during zebrafish gastrulation the cytokine release syndrome (crs) of severe covid- and interleukin- receptor (il- r) antagonist tocilizumab may be the key to reduce the mortality characterization of the apelin/elabela receptors (aplnr) in chickens, turtles, and zebrafish: identification of a novel apelin-specific receptor in teleosts. front endocrinol (lausanne) uxt potentiates angiogenesis by attenuating notch signaling an affective disorder in zebrafish with mutation of the glucocorticoid receptor key: cord- -mmpox authors: izumi, yasukatsu; iwao, hiroshi title: angiotensin ii peptides date: - - journal: handbook of biologically active peptides doi: . /b - - - - . -x sha: doc_id: cord_uid: mmpox abstract much evidence supports the notion that angiotensin ii (ang ii), the central product of the renin–angiotensin system (ras), may play a central role not only in the etiology of hypertension but also in the pathophysiology of cardiovascular diseases in humans. ang ii, via the ang ii type receptor, directly causes cellular phenotypic changes and cell growth, regulates the gene expression of various bioactive substances, and activates multiple intracellular signaling cascades in cardiac myocytes and fibroblasts, as well as vascular endothelial and smooth muscle cells. recently, new factors have been discovered, such as angiotensin-converting enzyme , angiotensin-( - ), and its receptor mas. this section summarizes the current knowledge about the broad ras in the pathophysiology of cardiac hypertrophy and remodeling, heart failure, vascular thickening, and atherosclerosis. research on the renin-angiotensin system (ras) began with the discovery of renin from the kidney by tiegerstedt and bergman in . in , a peptide that had vasoconstrictive effects in the ras was discovered, and it was named hypertensin by braun-menendez in argentina, and angionin by page and helmer in the united states. these two terms persisted for about years, until it was agreed to rename the pressor substance angiotensin. in the s, two forms of angiotensin were recognized, the first a decapeptide (angiotensin i: ang i) and the second an octapeptide (angiotensin ii: ang ii). skeggs et al. in the united states reported that ang ii was formed by the enzymatic cleavage of ang i by another enzyme, termed angiotensin-converting enzyme (ace). schwyzer and bumpus succeeded in the synthesis of ang ii in . gross suggested, in , that the ras was involved in the regulation of aldosterone secretion, and then davis, genet, laragh et al. proved his hypothesis. in the early s, polypeptides either inhibiting the formation of ang ii or blocking ang ii receptors were discovered, but these were not orally active. cushman and ondetti succeeded in the development of captopril, the orally active ace inhibitor in . after that, many experimental and clinical studies with ace inhibitors uncovered additional roles for the ras in the pathophysiology of hypertension, heart failure, and vascular diseases. furthermore, losartan (dup ), an orally active, highly selective and potent nonpeptide ang ii receptor blocker (arb), was developed in , and the cloning of ang ii receptors, type (at r) and type (at r) was accomplished in the early s. angiotensin-( - ) [ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ] was discovered in by santos et al., and angiotensin-converting enzyme (ace ) was identified in , which catalyzes the conversion of ang i [ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ] to ang-( - ) by the removal of a single carboxy-terminal amino acid. ace is an essential regulator of heart function and a functional receptor for the sars coronavirus. the ras plays an important role in the regulation of arterial blood pressure. renin is an enzyme that acts on angiotensinogen to catalyze the formation of ang i. ang i is then cleaved by ace to yield ang ii. a representation of the biochemical pathways of ras is shown in fig. . the major element of the rate of ang ii production is the amount of renin released by the kidney. renin is synthesized, stored, and secreted into the renal arterial circulation by the granular juxtaglomerular cells. the secretion of renin is controlled predominantly by three pathways: the first mechanism controlling renin release is the intrarenal macula densa pathway and the second is the intrarenal baroreceptor pathway. the third mechanism is the β-adrenergic receptor pathway, which is mediated by the release of norepinephrine from postganglionic sympathetic nerve terminals. an increase in renin secretion enhances the formation of ang ii, and ang ii stimulates the at r on juxtaglomerular cells to inhibit renin release. the substrate for renin is angiotensinogen, an abundant α -globulin that circulates in the plasma. the primary structure of angiotensinogen has been deduced by molecular cloning. angiotensinogen is synthesized primarily in the liver, although mrna that encodes the protein is abundant in fat, certain regions of the central nervous system, and the kidney. the rate of ang ii synthesis can be influenced by changes in angiotensinogen levels. ace was discovered in plasma as the factor responsible for conversion of ang i to ang ii. the ace gene contains, in intron , an insertion/deletion polymorphism that explains % of the phenotypic variance in serum ace levels. individuals homozygous for the deletion allele have higher levels of serum ace. ang i is rapidly converted to ang ii, when given intravenously. ang iii [ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ] can be formed by the action of aminopeptidase on ang ii. ang iii ang ii cause qualitatively similar effects. ang iii is approximately as potent as ang ii in stimulating the secretion of aldosterone. however, ang iii is only % as potent as ang ii in elevating blood pressure. ang iv [ang- ( ) ( ) ( ) ( ) ( ) ( ) ] is generated by the sequential cleavage of two amino acid residues from the amino terminus of ang ii by aminopeptidases localized to the endothelial surface. there are bypass pathways in the ras to produce ang ii besides the main enzymes such as renin and ace. arakawa et al. showed that trypsin and kallikrein can produce ang ii. one of the most famous enzymes in such bypass pathways is a heart chymase that is secreted by mast cells. circulating renin of renal origin acts on circulating angiotensinogen of hepatic origin to produce ang i in the plasma, and circulating ang i is converted by plasma ace and pulmonary endothelial ace to ang ii. then, ang ii is delivered to its target organs via blood flow. this traditional pathway is called the circulating ras. however, heart, blood vessels, and several other tissues contain and/or synthesize components of the ras, called the tissue (local) ras. many tissues, including heart, blood vessels, brain, kidney, and adrenal gland, express mrnas for renin, angiotensinogen, and/or ace. furthermore, several other ang i cleavage products have been found in the cultured cell types from these tissues, for example, ang-( - ), ang-( - ), ang iii, and ang iv (fig. ) . ang i and ang ii have been localized in atria and ventricles. therefore, it seems that the tissue ras exists independently of the renal/hepatic-based system. several serine proteases, such as tonin and cathepsin g, have been shown to hydrolyze ang ii precursors. an aspartyl protease with cathepsin d-like properties was shown to convert angiotensinogen to ang i. conversion of ang i to ang ii in human and dog cardiac ventricles may occur by heart chymase. unlike ace, human heart chymase shows high specificity for ang i and does not degrade bradykinin or vasoactive intestinal peptide. it is likely that the relative contribution of ace and chymase to cardiac ang ii formation varies with the cardiac chamber in the human heart, because ace levels are the highest in the atria and chymase levels are the highest in ventricles. it is recently believed that chymase plays a crucial role in various tissues because chymase can not only produce ang ii but also convert precursors of transforming growth factor (tgf)-β and matrix metalloproteinase- to their active forms, whose increases damage various organs. the effects of angiotensins are exerted through specific cell surface receptors. there are two subtypes of ang ii receptors, types and (at r and at r). the successful cloning of the at r in and the at r in allowed the development of further research on the structure and function of this receptor. the at r consists of two subtypes, at a r and at b r, which have % homology with regard to amino acid sequence and have similar pharmacological properties and tissue distribution patterns. the at r is a member of the seven-transmembrane-spanning, g protein-coupled receptor (gpcr) family; it binds to heterotrimeric g proteins and lacks intrinsic tyrosine kinase activity. the human at r gene is mapped to chromosome , and the at a r and at b r genes in rats are mapped to chromosomes and , respectively. the at r is ubiquitously and abundantly distributed in adult tissues, including blood vessel, heart, kidney, adrenal gland, liver, brain, and the lung. the at r mediates all the classic well-known effects of ang ii, such as elevation of blood pressure, vasoconstriction, increase in cardiac contractility, aldosterone release from the adrenal gland, facilitation of catecholamine release from nerve endings, and renal sodium and water absorption. therefore, the at r has a role in atherosclerosis, congestive cardiac failure, and several acute and chronic inflammatory diseases, conditions in which inflammation is known to play a significant role (fig. ). numerous selective and potent nonpeptide at r antagonists, such as losartan, candesartan, valsartan, irbesartan, eprosartan, telmisartan, tasosartan, have been developed. in contrast, the at r has high affinity for pd , pd , cgp , l- , , l- , , and cgp a. the cdna and genomic dnas of human, rat, and mouse at rs have been cloned. the at r is mainly expressed in developing fetal tissues, and it is believed to have an essential role in physiological vascular development. the at r expression rapidly decreases after birth, and, in the adult, expression of this receptor is limited mainly to the uterus, ovary, certain brain nuclei, heart, and adrenal medulla. unlike the at r, which has been shown to have subtypes in rats and mice, there is no evidence for subtypes of the at r. although a comparison of amino acid sequences of at a r and at r in rats, deduced from nucleotide sequences, shows a low homology between these receptors ( %), the at r is also a seven-transmembrane domain receptor. in various cell lines, the at r-activated protein tyrosine phosphatase was shown to inhibit cell growth or induce programmed cell death (apoptosis). the at r inhibited at r-mediated cell growth, demonstrating an antagonistic action. it is believed that the expression balance of at and the at is important for cardiovascular diseases. there have also been conflicting findings regarding these receptors. for example, it is controversial whether cardiac hypertrophy is promoted by the at r. however, the at r plays a cardioprotective role on left ventricular function after myocardial infarction. in contrast to extensive data on the molecular and cellular functions and pathophysiological significance of the at r, the role of the at r in cardiovascular diseases remains to be defined. at present, the implications of overstimulation of at r by long-term use of arb are being investigated in clinical trials. it has been suggested that ang-( - ) mediates its effects by interacting with the gpcr mas, a prototypic seventransmembrane domain receptor, which is predominantly expressed not only in the brain and testis but is also found in the kidney, heart, and blood vessels. the ang at receptor was originally defined as the specific, high-affinity binding site for ang iv. therefore, it was suspected to be classically a gpcr. and then, albiston et al. have identified it as the transmembrane enzyme, insulinregulated aminopeptidase (irap), an abundant protein that is found in specialized vesicles containing the insulinsensitive glucose transporter glut . generally, pathological left ventricular hypertrophy is characterized not only by an increase in myocyte size (quantitative change) but also by myocyte gene reprogramming (qualitative change), as shown by enhanced expression of fetal phenotypes of genes such as β-myosin heavy chain (β-mhc), skeletal α-actin, and atrial natriuretic peptide (anp). in the cardiac ventricle of most mammalian species, mhc consists of two isoforms, α-and β-mhcs. in the rat, α-mhc is the predominant isoform in adult hearts, whereas β-mhc is the predominant isoform in fetal hearts. therefore, changes in the ratio of β-mhc to α-mhc in the cardiac ventricle significantly alter the contractile properties of the heart. cardiac sarcomeric actin is also composed of two isoforms: cardiac α-actin and skeletal α-actin. cardiac α-actin is predominantly expressed in adult rat hearts, whereas skeletal α-actin is normally expressed in fetal and neonatal rat hearts. the ratio of skeletal α-actin to cardiac α-actin in the ventricle plays a significant role in cardiac function, because skeletal α-actin has greater contractility than cardiac α-actin. furthermore, in addition to showing enhanced expression of β-mhc and skeletal α-actin, hypertrophic ventricular myocytes are also characterized by significant upregulation of anp, which is scarcely expressed in normal adult ventricular myocytes. another important property of pathological cardiac hypertrophy is increased accumulation of extracellular matrix (ecm) proteins such as collagen (particularly collagen types i and iii) and fibronectin in the interstitium and around blood vessels within the heart. these changes play a central role in ventricular fibrosis or remodeling. , increased interstitial collagen deposition in the heart enhances cardiac stiffness and results in diastolic dysfunction. fibronectin is localized on the surface of cardiac myocytes, connects cardiac myocytes to perimyocytic collagen and is believed to affect cardiac systolic and diastolic functions. thus, increased ecm accumulation, and the abovementioned ventricular myocyte gene reprogramming play critical roles in the impairment of cardiac performance and pathophysiology of cardiac failure. ecm proteins within the heart are predominantly produced by fibroblasts. unlike cardiac myocytes, cardiac fibroblasts proliferate and increase the production of ecm proteins when the heart is exposed to hypertrophic stimuli such as hemodynamic overload. thus, cardiac fibroblasts and cardiac myocytes play key roles in the development of pathological cardiac hypertrophy and dysfunction. accumulating in vitro and in vivo evidence supports the concept that ang ii is involved in all these important processes of pathological cardiac hypertrophy, including myocyte hypertrophy, myocyte gene reprogramming, fibroblast proliferation, and ecm protein accumulation. effect of at r on cardiac remodeling (fig. ) activated at r can activate multiple signaling pathways. the acute vasoconstrictor function of ang ii is primarily mediated through at r by classical g protein-dependent signaling mechanisms. investigations of the effects of ang ii on cardiac intracellular signaling cascades are essential to elucidate the molecular mechanism underlying ang iiinduced pathological cardiac hypertrophy. accumulating in vitro evidence on cultured cardiac myocytes or fibroblasts suggests that mitogen-activated protein (map) kinases, including extracellular signal-regulated kinase (erk), c-jun amino terminal kinase (jnk) and p map kinase (p mapk), may be responsible for myocyte hypertrophy and gene reprogramming or fibroblast proliferation. ang iiinduced cardiac activation of jnk occurs in a more sensitive manner than that of erk. the erk cascade activates platelet-derived growth factor (pdgf), epidermal growth factor receptor (egfr), insulin receptor pathways, and nonreceptor tyrosine kinases belonging to the c-src family, prolinerich tyrosine kinase , focal adhesion kinase, and janus kinases (jaks). besides the g protein-dependent effects of ang ii activated at r, the activated at r can also stimulate g protein-independent signal transduction mechanisms by directly associating with signaling molecules, such as β-arrestins, jak, cdc , and src. jnk activation by ang ii without erk activation is followed by activation of activator protein- (ap- ). importantly, ap- regulates the expression of various genes by binding the ap- consensus sequence present in their promoter regions. interestingly, fetal phenotypes of cardiac genes such as skeletal α-actin and anp, and cardiac fibrosisassociated genes such as tgf-β and collagen type i, have ap- responsive sequences in their promoter regions. indeed, ap- activation has been demonstrated to lead to increased promoter activity of skeletal α-actin and tgf-β . therefore, it is intriguing to postulate that jnk activation, in part through the activation of ap- , may be implicated in ang ii-induced cardiac hypertrophic response in vivo. an in vivo study showed that apoptosis signal-regulating kinase (ask ), one of the map kinase kinase kinases, plays an important role in ang ii-induced cardiac hypertrophy and remodeling, including cardiomyocyte hypertrophy, cardiac hypertrophy-related mrna upregulation, cardiomyocyte apoptosis, interstitial fibrosis, coronary arterial remodeling, and collagen gene upregulation. the ras, tgf-β, and β-adrenergic system network mediates this myocardial remodeling. tgf-β mrna and protein expression is augmented in cardiomyocytes and cardiac fibroblasts, which in these situations transdifferentiate to myofibroblast phenotype, following progressive diastolic dysfunction. ang ii caused a significant increase in tgf-β mrna. chronic administration of ang ii-induced tgf-β protein expression in myocardium. ang ii-induced hypertrophic growth of cardiomyocytes is mediated by tgf-β , which is downstream to ang ii in network. mice expressing constitutively hyperactive tgf-β had a chronic hypertrophy, fibrosis, and cardiac dysfunction that was resistant to the arb telmisartan but blocked by tgf-β antagonist. the ang ii-mediated cardiac remodeling mechanism is thus dependent on tgf-β . from all the studies performed so far, it is clear that ang ii/tgf-β induced autocrine-paracrine cellular responses in cardiac fibroblasts, in the myocardial interstitium, and cardiomyocytes cause cardiac hypertrophy. ang ii-induced tgf-β signaling also induces translocation of smad proteins into the nucleus to drive transcription of fibrotic marker proteins such as collagen, fibronectin, and connective tissue growth factor (ctgf). ctgf is a profibrotic factor that stimulates tgf-β responses mediating fibrosis and apoptosis. the inflammatory cytokines such as interleukin- , interleukin- , vascular cell adhesion molecule- , and monocyte chemoattractant protein- , are believed to have important roles in the coupling acute ischemic episodes and chronic myocardial remodeling with chronic angiotensin receptor stimulation. ang ii facilitates the release of norepinephrine from cardiac sympathetic nerve terminals. in ang ii-infused rats, surgical cardiac sympathectomy or treatment with β -adrenergic receptor blocker significantly prevented cardiac myocyte necrosis, showing that ang ii-induced cardiac damage is, at least in part, mediated by catecholamine release from cardiac sympathetic neurons. thus, the activation of cardiac sympathetic neurons by ang ii also contributes to pathological cardiac hypertrophy. ang ii, via the at r, is also known to be a potent mediator of oxidative stress and reactive oxygen species (ros)mediated signaling. a large body of evidence indicates that ros plays a major part in the initiation and progression of cardiovascular dysfunctions associated with diseases such as hyperlipidemia, diabetes mellitus, hypertension, ischemic heart disease, and chronic heart failure. in addition, ang ii signaling activates membrane nad(p)h oxidases in vsmcs to produce ros, which mediate the pleiotrophic effects of ang ii. ang ii signaling mediated activation of nad(p)h oxidases involves the upstream mediators src/ egfr/pi k/rac- and pld/pkc/p phox phosphorylation. , transcription factors such as nuclear factor-κb, ap- , and nrf that are implicated in the pathogenesis of atherosclerosis are activated by ros. ros may cause vessel inflammation by inducing the release of cytokines and causing an increase in the expression of the leukocyte adhesion molecules in the cell membranes. this may increase recruitment of monocytes to the area of endothelial damage. thus, activated at r-induced ros production can lead to changes in structural and functional properties of the vasculature and is the central aspect of vascular pathobiology in hypertension, diabetes, and cardiovascular diseases. numerous in vivo experiments have shown that ang ii can induce vsmc proliferation in vivo. ang ii infusion increased mesenteric vascular media width, media crosssectional area, and media/lumen ratio. despite detailed investigations into the molecular mechanism of ang ii-mediated vsmc growth in vitro, this process is poorly understood. ang ii infusion, at least in part independent of its blood pressure-elevating effect, increased aortic mrna and protein expression of fibronectin, which is an ecm protein that induces phenotypic change of vsmcs from a contractile to a synthetic phenotype. basic fibroblast growth factor (bfgf) may play a key role in ang ii-mediated vsmc replication in vivo, as shown by the observation that the injection of anti-bfgf antibody significantly inhibited the mitogenic effect of ang ii infusion on rat carotid arteries. ang ii infusion in rats doubled superoxide production in rat aorta by activation of nad(p)h oxidase. on the other hand, norepinephrine infusion did not increase vascular superoxide production, despite a hypertensive effect comparable to that of ang ii, suggesting that vsmc growth due to ang ii may be specifically mediated by increased superoxide generation. ang ii infusion in rats increased heme oxygenase- (ho- ) mrna and protein in the endothelium. because ho- is an oxidant-sensitive gene, it is possible that increased oxidative stress is a trigger for ho- mrna upregulation in the ang ii-infused rat aorta and that ho- may serve to abrogate this increased stress caused by ang ii. ang ii infusion stimulated aortic thrombin receptor mrna expression in rats, which was blocked by either arb or the heparin-binding chimera of human cu/zn superoxide dismutase but not by normalization of blood pressure with hydralazine treatment, suggesting that ang ii increases vascular thrombin receptor by at r-mediated superoxide production and may be implicated in the pathophysiology of atherosclerosis by thrombin cascade activation. the injection of ang ii resulted in increased oxidized low-density lipoprotein uptake by peritoneal macrophages and increased macrophage proteoglycan content, suggesting that ang ii may accelerate atherosclerosis by promoting foam cell formation and cholesterol accumulation in the vascular wall. endothelial dysfunction in the context of hypertension and cardiovascular diseases is partly dependent on the production of ros. at r-induced ros not only burns nitric oxide (no) but also reduces no formation in the endothelium by oxidizing tetrahydrobiopterin, a crucial cofactor for endothelial no synthase (enos). superoxide anion, a highly reactive ros, is known to cause breakdown of endothelial-derived relaxing factor and dysfunction of endothelium. regenerated endothelium has an impaired ability to release endothelial-derived relaxing factors, in particular no. activation of angiotensin receptors triggers an efflux of cytosolic calcium in endothelial cells (ecs). calcium efflux activates pla- to release arachidonic acid, which is metabolized by cyclooxygenase (cox) to generate various eicosanoids including endoperoxides and various prostaglandins that activate thromboxane a /prostanoid receptors located on the vsmcs causing contractions. endothelial cox activity also produces ros. in addition, uric acid, despite its known antioxidant effects, may cause human vascular ec dysfunction by local activation of ras, inducing oxidative stress. ros may, in turn, activate local ras of whole vasculature, including ec, smooth muscle cells, fibroblast-enhancing ang ii production and concomitant stimulation of at r to create a positive feedback loop between local ras and ros. thus, in hypertensive cardiovascular diseases, endothelial dysfunction is characterized by blunted endothelium-dependent vasodilations or enhanced endothelium-dependent contractions. , generally, at r is believed to promote apoptosis and inhibit proliferation and hypertrophy. unlike at r, at r contributes to maintenance of blood pressure by controlling the vascular tone through vasodilatation. available evidence indicates links between at r and bradykinin b receptor (b r) in no production in vasodilation. , at r stimulation by ang ii leads to an increase in cgmp levels through a mechanism involving b r, causing no release. at r activation stimulates bradykinin formation by activating kininogenases. chronic infusion of ang ii into at r overexpressing mice completely abolished the at r-mediated pressor effect, suggesting vasodilatation by at r. this was blocked by one of the inhibitors of b r and nos. in addition, aortic explants from these transgenic mice overexpressing at r showed greatly increased cgmp/no production and diminished at r-induced vascular constriction. removal of endothelium or treatment with the inhibitors of b r and nos abolished these at r-mediated effects, indicating that endothelium is the primary site of effect manifestation by at r. the at r in aortic smcs also stimulates the production of bradykinin, which stimulates the no/cgmp system to promote vasodilation. in the at r-deficient mice, bradykinin-induced dilation is seriously impaired. bradykinininduced dilation is inhibited by at r antagonist but not by losartan, an arb. thus, at r and b r may form functional heterodimers, as recently shown, and this might lead to increased no and cgmp production. the physical association between the dimerized at r and b r initiates changes in intracellular phosphoprotein signaling activities and enhanced production of no and cgmp. , growth and apoptosis are also two opposite components of tissue remodeling. cardiac remodeling happens in certain physiological or pathological circumstances, such as exercise, aging, hypertension, and myocardial infarction. it is known that although at r expression decreases sharply after birth, it can increase again in some pathophysiological conditions. stimulation of at r expression may inhibit neointima formation, cell proliferation, inflammation in vascular injury, myocardial infarction, and ischemic diseases, suggesting its protective role. conversely, in at r-deficient mice, recovery from acute myocardial infarction was reduced compared to the wild-type mice. although neointima formation and vsmc proliferation after vascular injury were exaggerated in at r-deficient mice, they were suppressed in at a r-deficient mice. at r possibly exerts antiproliferative effects and proapoptotic effects in vsmcs by controlling at a r. the number of apoptotic cells in the injured artery was increased significantly in at a r-deficient mice but decreased in at rdeficient mice, indicating that at r mediates apoptosis. selective at r stimulation also inhibited restenosis after balloon angioplasty. thus, experimental studies have shown a beneficial role for the reexpression of the at r after vascular injury leading to inhibition of cell growth and promotion of apoptosis. gene transfer studies in vivo have also supported that at r expression attenuated neointima accumulation in injured carotid arteries by antagonizing growth-promoting effects of at r. vascular injury studies using mice with disrupted at r expression showed enhanced neointimal formation compared with wild-type mice, suggesting a protective role for at r. at r expression is upregulated in several disease conditions. in the left ventricle-specific at r overexpression mice, elevated left ventricular endodiastolic pressure, increased systolic and diastolic dimensions, severely depressed left ventricular fractional shortening, and wall thinning were observed, suggesting that ventricular myocytespecific expression of at r promotes the development of dilated cardiomyopathy and heart failure in vivo. in vivo ectopic at r overexpression in postnatal left ventricular myocardium is thus detrimental, explaining the sharp decline of at r after birth. if prolonged arb treatment turns out to be completely beneficial through clinical trials, then it is either a dosage effect or it depends on the time and site of at r expression. the implications of overstimulation of at r by long-term use of arb are being investigated in clinical trials. ang-( - ) mediates its effects by interacting with the grcr, mas, a prototypic seven-transmembrane domain receptor, which is not only predominantly expressed in the brain and testis but is also found in the kidney, heart, and blood vessels. moreover, several studies have shown that the interaction of ang-( - ) with mas induces the protective cardiovascular actions such as no release, akt phosphorylation and vasodilation. ang-( - ) also functions as an antithrombotic, antiproliferative, and antioxidative agent. although the mechanisms of action in the vascular system has not yet been well established, several studies have focused on control of the no and o − balance, which regulates cardiovascular function. a reduction in superoxide dismutase and catalase activity in mas-deficient mice demonstrates impaired antioxidant properties in these animals. moreover, ros levels are increased in mas-deficient animals. the masdeficient mice exhibit impaired in vivo endothelial function and increased blood pressure. the transgenic rats overexpressing human ace in vsmcs, showed improved endothelial function and decreased vasoconstriction response to ang ii. furthermore, the infusion of ave , a mas agonist, increases the hypotensive effect of bradykinin in rats; one plausible mechanism for this effect involves an increase in no levels. ace protects endothelial cells from ang ii-mediated macrophage infiltration and oxidative stress in an ang-( - )-dependent manner in atherosclerosis. the long-term ang-( - ) treatment induces atheroprotective effects in apoe-deficient mice, an animal model for atherosclerosis. the improvement in endothelial function resulted from an increase in no bioavailability, as the heptapeptide increased nos expression and decreased o − production. the genetic deletion of ace significantly increases plaque formation in apoe-deficient mice, which is decreased by targeted vascular ace overexpression. consequently, these actions of the ace -ang-( - )-mas system may be exploited in the treatment of atherosclerosis and other vascular diseases. thus, the ace -ang-( - )-mas system has opposite functions of the classical ras in tissues responsible for blood pressure regulation and cardiovascular homeostasis. it decreases the production of ros and increases enos, improving the antioxidant capacity of cardiovascular tissues. the ace -ang-( - )-mas system may become an important therapeutic target for cardiovascular protection. angiotensin ii type receptor-bradykinin b receptor functional heterodimerization angiotensin ii type receptor deficiency exacerbates heart failure and reduces survival after acute myocardial infarction in mice evidence that the angiotensin iv (at( )) receptor is the enzyme insulin-regulated aminopeptidase genetically altered animal models for mas and angiotensin flow-dependent dilation mediated by endogenous kinins requires angiotensin at receptors evidence for mas-mediated bradykinin potentiation by the angiotensin-( - ) nonpeptide mimic ave in normotensive rats angiotensin ii receptors and drug discovery in cardiovascular disease international union of pharmacology. xxiii. the angiotensin ii receptors gene expression signals involved in ischemic injury, extracellular matrix composition and fibrosis defined by global mrna profiling of the human left ventricular myocardium pleiotropic at receptor signaling pathways mediating physiological and pathogenic actions of angiotensin ii apoptosis signal-regulating kinase plays a pivotal role in angiotensin ii-induced cardiac hypertrophy and remodeling at( ) receptor-dependent vasodilation is mediated by activation of vascular kinin generation under flow conditions molecular and cellular mechanisms of angiotensin ii-mediated cardiovascular and renal diseases angiotensin ii directly increases transforming growth factor beta and osteopontin and indirectly affects collagen mrna expression in the human heart the angiotensin ii type receptor in cardiovascular disease heme oxygenase attenuates angiotensin ii-mediated increase in cyclooxygenase- activity in human femoral endothelial cells role of transforming growth factor-beta in the progression of heart failure angiotensin ii cell signaling: physiological and pathological effects in the cardiovascular system ligand-independent signals from angiotensin ii type receptor induce apoptosis hyperoxia stimulates an nrf -are transcriptional response via ros-egfr-pi k akt/erk map kinase signaling in pulmonary epithelial cells role of extracellular matrix proteins in heart function ccn proteins: multifunctional signalling regulators ace -angiotensin-( - )-mas axis and oxidative stress in cardiovascular disease tgf-beta and angiotensin networking in cardiac remodeling converting enzyme activity and angiotensin metabolism in the dog brainstem tgf-beta mediates the hypertrophic cardiomyocyte growth induced by angiotensin ii nitric oxide, tetrahydrobiopterin, oxidative stress, and endothelial dysfunction in hypertension angiotensin ii stimulation of nad(p)h oxidase activity: upstream mediators role of angiotensin ii-regulated apoptosis through distinct at and at receptors in neointimal formation new approaches to blockade of the reninangiotensin-aldosterone system: chymase as an important target to prevent organ damage endothelial dysfunction: a strategic target in the treatment of hypertension? reactive oxygen species in the vasculature: molecular and cellular mechanisms p phox associates with the cytoskeleton through cortactin in human vascular smooth muscle cells: role in nad(p)h oxidase regulation by angiotensin ii extracellular matrix remodeling in heart failure: a role for de novo angiotensin ii generation endothelial dysfunction and elevated blood pressure in mas gene-deleted mice key: cord- - ese c e authors: zisman, lawrence s. title: ace and ace : a tale of two enzymes date: - - journal: eur heart j doi: . /eurheartj/ehi sha: doc_id: cord_uid: ese c e nan the cardiac renin-angiotensin-aldosterone system (raas) is an endocrine cascade, which results in the conversion of the inactive pro-hormone angiotensin i (ang i) to the active peptide hormone ang ii, and may also function as an autocrine/paracrine system to modulate cardiac function and growth. renin, the initial enzyme of this cascade, cleaves the amino terminus of the pre-prohormone angiotensinogen, thereby releasing the decapeptide pro-hormone ang i. angiotensin-converting enzyme (ace) removes two additional amino acids to yield the active octapeptide hormone ang ii. ang ii, acting through the at receptor, is a potent vasoconstrictor and stimulates cardiac growth. this cardiac growth may be related to myocyte hypertrophy and/or fibroblast proliferation with a concomitant alteration in the extracellular matrix. ang ii, by interaction with the neuroadrenergic and endothelin systems may cause an increase in local noradrenaline and endothelin levels, which in turn may have direct trophic and toxic effects on the cardiac myocyte. in addition, ang ii stimulates aldosterone secretion from the adrenal glands, which may further stimulate cardiac remodelling. the cellular events triggered by increased ang ii formation, while initially compensatory, may lead to eventual loss of cellular function, contractility, and viability. aceinhibitors have been shown to improve survival and cardiac function in patients with left heart failure. in response to raas activation there is an increase, at the level of both gene and protein expression, of components which decrease the local concentration of ang ii and which generate bioactive compounds that counteract ang ii-mediated effects. ace , the recently described homologue of ace, is a pivotal enzyme in this counter-regulatory response. , ace contains a single hexxh zinc-binding domain which is homologous to one of the active sites of ace and has % overall identity to ace. however, ace is not inhibited by captopril or other 'classical' ace-inhibitors. recombinantly expressed ace was first reported to hydrolyse the his-leu bond in ang i to release ang-( - ). ang-( - ) was a substrate for ace, which hydrolysed it to ang-( - ). of note, ace did not hydrolyse ang-( - ) to form ang ii. subsequent to these initial characterizations it was found that ace had substrate preference for ang ii, which it hydrolysed to ang-( - ) at a high rate. indeed, ace purified from human heart hydrolysed ang ii, but not ang i. thus, ace should function to decrease ang ii concentration where it is present and active. northern blot analysis demonstrated that, in contrast to the near ubiquitous expression of somatic ace, ace gene expression was localized predominantly to the heart, kidney, and testes. subsequent studies using quantitative pcr have shown that ace gene expression also occurs in the gastrointestinal tract and, to a lesser extent, in other organs. ace may be important as a counter-regulatory enzyme not only because it decreases local cardiac ang ii concentrations, but also because its product ang-( - ) mediates specific effects through its recently identified receptor, the mas oncogene product (mas). through this receptor, ang-( - ) may stimulate nitric oxide synthase (nos) and counteract the effects of ang ii. the effects of ang-( - ) may also involve cross-talk with the angiotensin type receptor (at r), and the bradykinin type two receptor (bk r). the importance of ace in regulating cardiac function is highlighted by the phenotype of the ace knockout mouse, which showed left ventricular dilation and impaired contractility. however, this cardiac dysfunction occurred in the absence of ventricular hypertrophy or the myosin heavy chain isoform switch typically found in other animal models of heart failure as well as in human heart failure. the phenotype was rescued by crossing with the ace knockout mouse. this finding suggests that increased local cardiac ang ii was the cause of the cardiac abnormalities in the ace knockout. however, it remains unclear why the model did not show any evidence for cardiac hypertrophy. one possible explanation is that ace is critical in the development of the microvascular capillary bed of the developing heart. indeed, hypoxia-induced genes were highly up-regulated in the hearts of ace knockout mice. burrell et al. quantify ace gene expression in the rat myocardial infarction model. the authors found a marked increase in ace gene expression in the border/infarct zone as well as in viable myocardium in the post-myocardial infarction rat heart. however, ace expression in viable myocardium increased later than in the border/infarct zone. these findings suggest that ace gene expression can be activated by two distinct processes: (i) an acute inflammatory or wound-healing response focused at the border/infarct zone, and (ii) a relatively chronic response induced by such mechanisms as increased wall stress or other factors associated with remodelling. how such processes lead to an increase in ace gene expression remains to be elucidated. the initial description of ace suggested that it was expressed solely in endothelial cells in the human heart. however, in the setting of myocardial infarction, it now appears that ace protein is found in multiple cell types including macrophages, smooth muscle cells, and cardiac myocytes in addition to endothelial cells. furthermore, burrell et al. identified ace protein in cardiac myocytes from failing human hearts. however, it remains unclear whether ace protein is actually produced by translation of mrna in cardiac myocytes or is released from other cell types and diffuses to cardiac myocytes. our laboratory has recently found that ace interacts with integrin beta d, a cardiac specific integrin isoform, a finding which raises this mechanism for localization as a distinct possibility. techniques to sort out cell-type specific gene expression, such as in situ rt-pcr, or laser-capture micro-dissection, will be required to answer this question. although there are many peptidases which can hydrolyse ang ii to ang-( - ), ace appears to be the major pathway for ang-( - ) formation from ang ii in the intact human heart. furthermore, it has been shown that ace gene expression and activity are markedly increased in failing human heart. , if ace is the worst of enzymes, is ace the best of enzymes? cardiac specific over-expression of ace resulted in abnormalities of connexin architecture and conduction. caution must be used in extrapolating the results of this over-expression model to the role of ace in adult heart failure. both the ace transgenic over-expression model, and the ace knockout model may have generated a cardiac phenotype by altering key steps in cardiac morphogenesis; effects that would not occur if ace regulation was modified only after birth. ace may have other important functions aside from its activity as an angiotensinase. a truncated form of ace , found in the kidney, did not have angiotensinase activity, but nevertheless was found to play a role in renal organogenesis. in addition, ace has recently been shown to be a 'receptor' for the sars virus (not a good thing). it is possible that ace may exert non-angiotensinase functions via binding to integrins. further research will be required to understand the importance of this interaction. based on the initial characterization of ace , it was believed that inhibiting the enzyme could be a therapeutic strategy for cardiovascular disease. however, as our knowledge of this enzyme has grown, it appears that increasing its activity, and thereby the local formation of ang-( - ), may actually be beneficial under certain circumstances. burrell et al. have made an important contribution to our understanding of ace and its role in the counter-regulatory response to myocardial infarction. as these authors observe, the next key step is to develop strategies for modulating ace expression and activity in adult animal models of heart failure. a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase hydrolysis of biological peptides by human angiotensinconverting enzyme-related carboxypeptidase interaction of ace and integrin beta in failing human heart quantitative mrna expression profiling of ace , a novel homologue of angiotensin converting enzyme angiotensin-( - ) is an endogenous ligand for the g proteincoupled receptor mas angiotensin-converting enzyme is an essential regulator of heart function myocardial infarction increases ace expression in rat and humans angiotensin-( - ) formation in the intact human heart: in vivo dependence on angiotensin ii as substrate ace gene expression is up-regulated in the human failing heart increased angiotensin-( - ) forming activity in failing human heart ventricles: evidence for upregulation of the angiotensinconverting enzyme homologue heart block, ventricular tachycardia, and sudden death in ace transgenic mice with downregulated connexins collectrin, a collecting ductspecific transmembrane glycoprotein, is a novel homolog of ace and is developmentally regulated in embryonic kidneys angiotensin-converting enzyme is a functional receptor for the sars coronavirus key: cord- - jf fnqe authors: wu, jie; deng, wei; li, shumin; yang, xiuhong title: advances in research on ace as a receptor for -ncov date: - - journal: cell mol life sci doi: . /s - - -x sha: doc_id: cord_uid: jf fnqe currently, a novel coronavirus (sars-cov- , also called -ncov) has triggered pandemic coronavirus disease (covid- ), an acute infectious respiratory disease that first became epidemic in wuhan (china) and is now spreading worldwide. although -ncov and sars-cov are very similar viruses genomically and structurally, the huge number of severe cases and deaths now being caused by -ncov infections has understandably prompted intense research on the receptor used by it to enter human cells. angiotensin converting enzyme (ace ), a functional receptor for sars-cov, now appears likely to mediate -ncov entry into human cells. in this review, we describe the roles performed by ace as an enzymatic catalyst and as a receptor for this novel coronavirus. we also summarize the latest research pertaining to the changes noted in ace expression after viral binding, and the relationships relating to virus transmission and population susceptibility to it. lastly, we speculate on the pathogenesis of covid- and provide a useful reference for drug development against this aggressive virus. the new sars-like acute infectious respiratory disease has now reached pandemic levels. first emerging in wuhan in december in the hubei province of china, it was temporarily called "novel coronavirus pneumonia (ncp)" by china's national health commission. studies have shown that the disease is caused by a novel coronavirus (cov), officially named -ncov, and now referred to as sars-cov- by scientists [ ] . the us food and drug administration named this new epidemic infectious disease coronavirus disease . epidemiological studies have shown that -ncov can spread rapidly through person-to-person transmission routes, which include coughs, sneezes, droplet inhalation, and other contact transmission. the disease, which is now rapidly spreading around the globe, has become an international public health emergency. the clinical symptoms of covid- include fever, dry cough, fatigue, dyspnea, respiratory failure, and even death in severe cases. although preliminary experiments by researchers or research institutions have reported that some agents may be effective against -ncov, there are currently no effective drugs targeting -ncov/sars-cov- [ ] . mammalian angiotensin-converting enzyme (ace ) was first discovered in the human lymphoma cdna library by the tipnis team [ ] and a human heart failure ventricle cdna library by donoghue team [ ] in . during the sars outbreak in , ace was identified by li et al. [ ] as a functional receptor for sars-cov in that it mediated the viral invasion of host cells, a finding later confirmed in animal experiments [ ] . some recent studies have suggested cellular and molecular life sciences * xiuhong yang yangxiuhong@ncst.edu.cn that -ncov may infect host cells through the ace receptor, as has already been established for sars-cov [ ] [ ] [ ] [ ] . accordingly, ace , as a -ncov receptor, may be a potential target for the treatment of covid- . in this review, the latest advances in our understanding of the roles played by ace in enzyme catalysis, cov invasion, cellular expression changes after viral-host cell binding, and its relationships with viral transmission and population susceptibility are described in the context of the pathogenesis of covid- . this review aims to provide a reference for the development of ace -targeted drugs for -ncov. in , a protein highly homologous to mammalian somatic angiotensin-converting enzyme (ace), was isolated and purified from drosophila melanogaster embryos and named ance [ ] . in , researchers identified another drosophila gene whose predicted translation product shared homology with mammalian testicular ace and with ance and called it ace-related (acer), after which it was renamed ace-like protein [ ] . insect ance and ace-like protein then became classified as "ace-like enzymes" [ ] . in , scientists also discovered ace-like enzymes in mammals. tipnis et al. [ ] were the first researchers to clone a human metalloproteinase with high homology to ace, calling it "ace homolog (aceh)" or "ace related carboxypeptidase", latterly known as "angiotensin-converting enzyme (ace )" by donoghue team [ ] . the sequence identity between ace and ace is about %, with a similarity score of % [ ] , while the sequence similarity between mouse and human ace is about % [ ] . the human ace gene is located on the short arm of the x chromosome and contains exons. the complete human ace cdna encodes an amino acid protein with a molecular weight of kd [ ] . membrane-associated ace is a type i transmembrane protein consisting of a signal peptide at the amino terminal, a single metalloproteinase active site containing a zinc ion binding motif (i.e., hemgh), a transmembrane domain, and a small cytoplasmic domain at the carboxyl terminus. a variety of transmembrane proteins can be cleaved by proteolytic enzymes to release the catalytically functional and free-standing extracellular domain, thereby regulating their activities [ , ] . ace , like ace, is a cleavable, extracellular enzyme located on the cell surface membrane [ ] . adam , a metalloproteinase family member, can cleave membrane-bound ace and release its extracellular portion into the circulation as soluble ace (sace ), which lacks the transmembrane and cytoplasmic domains but retains its activity [ ] . it has been shown that the activity of sace is suppressed by the presence of an endogenous inhibitor in the form of a currently unknown positively charged small molecule [ ] . ras, one of the most important hormonal mechanisms for maintaining homeostasis in the human body, regulates blood pressure, fluid volume, and sodium-potassium balance. ras, which is located in the circulation and in local tissues, is linked with many diseases such as cardio and cerebral vascular diseases, and diabetes mellitus, among others [ ] . the classical ras encompasses ace, ang ii, the at receptor, and other molecules, collectively forming the ace-ang ii-at axis. as a dicarboxypeptidase, ace can catalyze the conversion of ang i to ang ii, after which ang ii binds to the at receptor to play roles in promoting vasoconstriction, inflammation and proliferation. the discovery of ace suggests that there is another ace -ang-( - )-mas receptor pathway in the ras, which is contrary to the classical pathway [ ] . ace , a monocarboxyl peptidase, contains an n-terminal peptidase domain (pd) and a c-terminal collectrin-like domain (cld) [ ] . the n-terminal pd directly hydrolyzes ang ii into ang-( - ), or firstly ang i into ang-( - ), which can later be hydrolyzed to ang-( - ) by ace or other enzymatic molecules. ang-( - ) exerts the opposite effect of ang ii (i.e., vasodilation, anti-inflammation and anti-proliferation) by activating mas receptor [ ] . in fact, ras metabolic pathways are not only limited to the two above-mentioned pathways, but some alternative pathways as well, such as ang ii formation via the chymase pathway and ang-( - ) formation directly from ang i by prolyl endopeptidase or neutral endopeptidase [ ] (fig. ). covs are a large group of spherical, - nm in diameter, positive-sense viruses with single-stranded rna genomes. cov genomes, ranging from to kb in size, are the largest among the known rna viruses. the rna genomes of covs can serve as messenger rnas and can be translated directly into proteins in the ribosomes of host cells [ ] . to date, seven cov types are known to infect humans, among which hcov- e, hcov-nl , hcov-oc and hcov-hku only cause common cold symptoms, whereas the other three types, sars-cov, mers-cov and -ncov, are highly transmissible with high fatality rates [ ] . the cov genome comprises a ′-cap structure and ′poly-a tail. the ′-terminal-located two-thirds of cov genes mostly encode the non-structural proteins that form polymers and perform replication and translation functions. one-third of the genome near the ′-terminus encodes at least four main structural proteins; namely, spike (s), envelope (e), membrane (m) and nucleocapsid (n) [ ] . the s protein forms radially protruding trimers on the viral envelope, which mediate virus-receptor binding and membrane fusion. so, the s protein is a key protein for determining the host range and transmission ability of covs [ ] . the s protein is structurally divided into two functionally distinct subunits called the s and s subunits. while the s subunit is responsible for receptor binding and includes the n-terminal domain and c-terminal receptor binding region (rbd), the s subunit facilitates membrane fusion and anchors s into the viral membrane [ ] . annotation of the -ncov genome shows that it possesses open reading frames (orfs) encoding proteins [ ] . although hcov-nl , -ncov and sars-cov all invade host cells via ace receptors, only the latter two viruses share homology and similarity in their genome sequences [ , ] . -ncov and sars-cov are both β covs, sharing the highest nucleotide sequence identity ( . %) across their whole genomes [ ] . the main difference between them lies in orf a and the gene sequence encoding the s protein. homology modeling shows that the receptor domain of -ncov has a similar receptor-binding domain as sars-cov [ , ] . structural analyses have revealed that the e and n proteins in -ncov show evolutionary conservation, with sequence identities of % and . %, respectively, as compared with sars-cov [ ] . in contrast, multiple mutations in -ncov are present in its s and m structural proteins when compared with bat-cov, revealing that different selection pressures have been exerted on -ncov during its evolutionary history [ ] . previous studies have indicated that positions , , , , and are important amino acid residues interacting with ace in the sars-cov s protein and are considered to be crucial for the cross-species and person-to-person transmission of sars-cov [ ] . although the rbd domain of the s protein from -ncov is highly conserved in its sequence, as compared with sars-cov, amino acids at four important positions in five key residues are replacements with only tyr being retained [ ] . the -ncov and sars-cov s proteins, nevertheless, still share an almost identical d structure in the rbd domain. thus, similar van der waals forces and electrostatic properties are still predicted to occur at the interaction interfaces of both viruses, although the binding affinity of -ncov for ace is significantly higher than that of sars-cov [ ] . during the sars outbreak in , li et al. [ ] were the first research group to identify ace as a functional receptor for sars-cov. they first found that t cells transfected with ace formed multinucleated syncytia in cells expressing the s protein. they also found that sars-cov could replicate effectively in non-susceptible cells transfected with ace , while an anti-ace antibody was found to block viral replication in e vero cells. the above in vitro experiments led them to conclude that ace is a functional receptor for sars-cov, and mediates viral entry into host cells. three in vivo experiments using humanized ace transgenic (hace ) mice infected with sars-cov were reported in . under the regulation of cag-complex promoters, including cytomegalovirus immediate-early (cmv-ie) enhancers and chicken β-actin promoters, the hace gene was expressed in the lungs, small intestines, and livers (at the mrna level) of ac transgenic mice. the mice showed clinical manifestations such as weight loss after being attacked by virus, and all died within days [ ] . under the control of the cytokeratin promoter, the hace gene was expressed in alveolar epithelial cells and the liver, kidneys and gastrointestinal epithelial cells of another transgenic mouse, and rapid death also occurred after viral infection [ ] . the transgenic mouse with the endogenous mace promoter was found to have hace expression in its lungs, heart, kidneys and intestines at both the mrna and protein levels. dissimilar to the above two mice showing fig. the metabolic pathway of the rennin-angiotensin system. the ras system mainly encompasses two axes: the classical ras ace-ang ii-at regulatory axis and the ace -ang-( - )-mas counter-regulatory axis. (p) rr, (pro) renin receptor; ang, angiotensin; ace, angiotensin-converting enzyme; ace , angiotensin-converting enzyme ; nep, neutral endopeptidase; pep, prolyl endopeptidase; pcp, prolyl carboxypeptidase lethal sars-cov infections, none of the hace transgenic mice challenged with sars-cov died; however, the viral titers in their lung tissues had significantly increased above normal and serious pathological changes in their lung tissues and multiple organ damage was apparent, thereby establishing a susceptible animal model for sars-cov [ ] . recently, some researchers have shown that ace can be used as a -ncov receptor using molecular models and in vitro experiments. zhou et al. [ ] first performed viral transfection experiments using hela cells expressing or not expressing ace . the results showed that -ncov could only enter hela cells expressing human ace , thus confirming that -ncov is likely the cell receptor for -ncov [ ] . another study demonstrated that expression of human or bat ace in non-susceptible bhk- cells can enable -ncov and sars-cov to enter cells, and that anti-serum raised against human ace can block sars-s and -ncov s-driven cell entry [ ] . a genomic characterization and homology modeling study on the origins of -ncov revealed the existence of structural similarities in the receptor binding domains of sars-cov and -ncov [ ] . in other research on the potential receptor for -ncov, the sequence of -ncov rbd, including its receptor-binding motif that directly contacts ace , was found to be similar to that of sars-cov [ ] . ace -binding affinity has been shown to be one of the most important factors determining the infectivity of sars-cov [ ] . recently, yan et al. [ ] analyzed the interface between ace and -ncov-rbd and sars-rbd complexes to reveal variations that may determine the different affinities of the rbds in the two related viruses. some researchers have used molecular models to show that the -ncov rbd domain penetrates the deep hydrophobic pocket of ace , resulting in a stronger interaction with ace than sars-cov [ ] . wrapp et al. [ ] analyzed the structure of the -ncov s protein trimer by cryoelectron microscopy and found that the binding capacity of the s protein from -ncov to the ace receptor was -to -fold higher than that of sars-cov. interestingly, coutard et al. [ ] identified a peculiar furin-like cleavage site in the s protein from -ncov, possibly explaining why -ncov seems to be readily transmitted between humans. recently, humanized transgenic animal modeling has further demonstrated that ace can mediate host cell invasion by -ncov. in an experiment where hace transgenic mice were infected with -ncov, viral antigens were observed in bronchial epithelial cells, alveolar epithelial cells and macrophages in the transgenic mice, while no viral antigens were found in their infected wild-type counterparts [ ] . therefore, it is speculated that like sars-cov, -ncov infects host cells via the mediating effects of its s protein and ace receptors on the surfaces of human cells. as a point of reference, hcov-nl , which was discovered in the netherlands in , shares no structural homology with sars-cov, but also utilizes the ace receptor when invading host cells. however, the crystal structure has showed that the hcov-nl rbd domain binds to a narrower region of ace , involving fewer amino acids, which would lead to a weaker interaction. however, nl -cov does not have as aggressive transmission as sars-cov or the newer-discovered -ncov, so its pathogenicity is weak and any relevant research on it is scarce [ ] . ace as a receptor may mediate the entry of cov into host cells in two independent ways (fig. ) . the first way involves ace -receptor-mediated clathrindependent endocytosis. the rbd of the virus is recognized by the extracellular pd of ace mainly through polar residues. when cov is connected to ace , the ace extracellular domain controlling the catalytic effect is cleaved off by specific proteases, such as metalloproteinase adam , and the transmembrane domain is internalized. next, with the assistance of clathrin [ ] , viral particles and host cells fuse, and the intracellular structure of ace aids viral transport from the cell membrane to the cytoplasm. in vitro studies have shown that adam inhibitors can attenuate virus replication [ ] . although the physiological function of ace extracellular shedding has not been fully elucidated, it has been shown to be associated with virus invasion and replication [ , ] . sace retains catalytic activity and can partially block sars-cov binding to receptors on target cells, and one in vitro study has shown that sace could block the association between the s domain of sars-cov and e vero cells and reduce viral replication [ ] . cells expressing ace non-catalytic mutants can still be infected by sars-cov, and the enzymatic activity of ace did not contribute to s-protein-mediated infection by this virus [ ] , indicating that the peptidase action of ace is not necessary for viral entry into host cells. elsewhere, structural analysis has shown that the metallopeptidase domain of ace can be divided into two subdomains (i and ii), with the cov s protein contacting the top of subdomain i of the ace catalytic domain, but the s protein does not contact subdomain ii or seal the peptidase activity sites in ace [ ] . the second way involves ace -receptor-mediated transmembrane serine protease (tmprss )-dependent membrane fusion. one study found that ace -mediated viral invasion involves tmprss , which is employed for s protein priming and activation of membrane fusion processes [ ] , and tmprss s priming role has recently been confirmed for -ncov [ ] . when the sars-s protein binds to ace , processing by tmprss is thought to allow fusion at the cell surface or upon uptake into cellular vesicles but before virion transport into cell endosomes. tmprss has also been found to compete with the adam metalloprotease for ace processing, but only cleavage by tmprss was found to enhance sars-s protein-driven entry [ ] . the molecular mechanism underlying proteaseenhanced cell entry and the potential role of ace cleavage upon sars-s protein activation are unclear [ ] . research using the -ncov s protein pseudovirus system and different inhibitors has shown that -ncov enters / hace cells mainly by endocytosis, and that phosphatidylinositol -phosphate -kinase, two pore channel subtype , and cathepsin l are critical for viral entry. another study revealed that -ncov s protein can trigger syncytia in /hace cells independent of an exogenous protease [ ] . recent resolution of the full-length structure of human ace using cryo-electron microscopy by yan et al. [ ] indicated that ace exists as a dimer with both "open" and "closed" conformations. conversion of the two conformations is achieved by rotation of the pd domains in ace , and both conformations contain the mutual recognition interface with covs. further studies have shown that ace dimers can accommodate two s protein trimers through each ace monomer [ ] . ace is also a chaperone protein for membrane transportation of the amino acid transporter b at (also called slc a ) [ ] , which mediates the uptake of neutral amino acid-like substances into intestinal cells in a sodium-dependent manner. these findings suggest that b at may play a regulatory role in intestinal infection with some covs [ ] . further research indicates that the presence of b at may block tmprss s access to the cutting site on ace ; therefore, the role played by the ace /b at dimer structure in conformational binding of the -ncov s protein appears to affect the viral invasion of host cells. whether b at can inhibit -ncov infection by blocking ace cleavage remains an unanswered question [ ] . ace gene expression is regulated at both the transcriptional and post-transcriptional levels by a variety of mechanisms. ang ii/at can upregulate ace mrna and protein expression in human cardiac fibroblasts via the att tgg a promoter sequences in the − to − regions of the gene [ ] . while ang ii/at promotes extracellular ace internalization followed by lysosomal degradation [ ] , adam cleaves membranes bearing ace , thereby releasing sace into the circulation [ ] . furthermore, micror-nas such as mir- can mediate ace gene silencing in cardiac fibroblasts and a variety of human tissue cell lines [ ] . thus, these mechanisms of expression and activity regulation of ace play an important role in cardiovascular and other diseases. interestingly, one study has suggested that ace expression is downregulated after viral infection. in their study, kuba et al. [ ] showed that sars-cov infection and sars-cov s proteins reduce the expression of ace in lungs (but not ace), lead to increased ang ii levels, signaling through the at receptor, increased pulmonary vascular permeability, and acute lung injury induction. notably, ace is not only a receptor for viral invasion of host cells during sars-cov infection, but viral binding to it deregulates its protective effect on the lungs. this may explain why sars-cov is more lethal in humans than other covs [ ] . oudit et al. [ ] demonstrated that sars-cov was also strongly equipped to infect the heart and modulate ace expression in this organ. pulmonary infections with sars-cov in wild-type mice subsequently led to downregulation of myocardial ace mrna and loss of ace protein in an ace -dependent manner. furthermore, the presence of sars-cov in the heart in patients who died from sars was associated with macrophage infiltration and myocardial damage in association with decreased myocardial ace protein expression. collectively, these findings suggest that myocardial ace downregulation may be an underlying pathophysiological mechanism for sars-associated heart disease. recently, some researchers have speculated that -ncov causes ace downregulation by binding to the ace receptor, and they have proposed strategies to treat covid- infections with ace as the target, which involves using the tmprss inhibitor to prevent cov s protein-ace binding [ , ] , blocking the ace receptor using anti-ace antibodies or peptides, or using sace to attenuate viral invasion through competitively binding with -ncov, thereby protecting against lung injury through its unique enzymatic properties [ ] . supporting such strategies is the finding from wang et al. [ ] , who showed that, individually, sars-cov or -ncov s proteins can interact with ace on the surface of host cells and become separately internalized together with ace through endocytosis, resulting in reduced surface ace expression [ ] . this endocytic event also upregulates adam activity, which in turn cleaves ace on cell membrane surfaces, perpetuating the loss of the ace protective effect in tissue ras. loss of ace leads to imbalance of ras homeostasis and increased ang ii levels, causing damage to the organism through at receptors, while also upregulating adam activity in a well characterized positive feedback loop leading to further cleavage of cell surface attached ace [ ] . one study reported that the level of ang ii in the plasma of -ncov-infected patients is significantly higher than in uninfected individuals, a finding linearly correlated with viral load and the degree of lung injury [ ] . adam also mediates the liberation of the membrane bound precursors of tumor necrosis factor α (tnfα), interferon (ifn)-γ, and il- (interleukin- ) proinflammatory cytokines into the circulation [ ] , which also downregulates ace expression on the cell surface and decreases ace mrna levels. soluble recombinant human ace (rhace ) inhibits viral invasion of host cells by competitive binding with -ncov, limits fig. the proposed mechanism underlying -ncov-induced downregulation of cell surface ace expression. cov interacting with ace becomes internalized together with ace through endocytosis, which upregulates adam -mediated proteolytic cleavage of ace . loss of ace leads to accumulation of angiotensin ii, which also increases adam activity through at receptors. rhace inhibits viral invasion of host cells by competitive binding to cov with membrane ace , limits the activities of ang ii, and increases protective ang-( - ) levels the activities of ang ii, and increases protective ang-( - ) levels [ , ] (fig. ) . elsewhere, monteil et al. [ ] found that rhace can prevent -ncov from infecting human blood vessel organoids and human kidney organoids engineered via the induction of pluripotent stem cells and addition of rhace also showed no cellular toxicity. however, because it has only been shown that rhace can block the early stages of -ncov infections, this finding requires confirmatory clinical evidence. however, some researchers have the opposite viewpoint, suggesting that ace expression is upregulated by viral infection. a team at shandong university (china) searched databases for cells and tissues known to be stimulated by viral infections or inflammatory cytokines, and they clearly showed that ace can be significantly upregulated by virus infection and inflammatory cytokine stimulation [ ] . the ace gene is responsive to viral infections, and its expression can be stimulated by a variety of viruses. when upregulated, ace is more receptive to viral invasion and transmission, while its induction by inflammatory cytokines also implies that the "cytokine storm" caused by -ncov not only damage host tissues, but may also accelerate viral spread [ ] . their analysis also showed that there are multiple transcription factor binding sites related to immune and cytokine responses in the promoter region of ace . it is speculated that the activated immune system induces the expression of a variety of cytokines, including ifn, after viral infection, which promotes the transcription and expression of ace by activating downstream signaling pathways such as the jnk (c-jun n-terminal kinase) pathway. ziegler et al. [ ] showed that ifns increase ace in human nasal epithelia and lung tissue, and ace belongs to a large family of interferon-stimulated genes (isgs), and suggested that -ncov may exploit the ace -mediated tissue-protective response to provide further cellular targets for entry. in conclusion, the changes occurring in the expression level of ace and its regulatory mechanism after the s protein of cov binds to the ace receptor remain unclear. if viral invasion downregulates ace via adaptive protective mechanisms in the host this will inevitably cause ras homeostasis impairment, thereby weakening the protective effect of ace and leading to acute lung injury. if, on the other hand, ace is upregulated, that will provide more receptors for viral entry and cause more viruses to invade host cells. therefore, either upregulation or downregulation of ace may have adverse consequences for the body. perhaps during the period when cells are fighting the virus, ace expression may be continuously changing (i.e., either up-or downregulated), and while human immune functioning is particularly important at this time, only those with robust immune functioning can defeat the virus (fig. ) . ace is expressed in many different tissues at different levels, with an organ-specific distribution. it is highly expressed in the heart (coronary vascular endothelial cells, vascular smooth muscle, and cardiomyocytes, among other cells), kidneys (renal vessels and renal tubular epithelial cells) and testes. it is also found in the gastrointestinal tract and lungs [ , ] . ace expression levels and expression patterns in different tissues may be critical to the susceptibility, symptom manifestations, and outcome of infection with covid- . therefore, it is hugely important to explore the expression characteristics of ace in human tissues and its relationship with virus transmission, as well as determining the possible infection pathways for understanding the pathogenesis and treatment options for covid- . the distributional expression of ace in human tissues was reported as early as the sars outbreak in . it has been shown that the epithelial expression of ace in lung and small intestine provided possible routes for sars-cov entry, and ace is also expressed in vascular endothelium [ ] . during their recent analysis of covid- , based on , cells derived from normal lung tissue of eight adult donors by single-cell rna sequencing (rna-seq), the team at tongji university medical college (china) determined that more than % of ace in the lungs was distributed on the surface of type ii alveolar epithelial cells [ ] . other researchers found that ace mrna is mainly distributed in the small intestine, colon, duodenum, kidneys, testes and gallbladder by searching the human protein atlas database, but its expression level was low in the lungs, suggesting that some cells may upregulate ace expression under certain conditions [ ] . that ace is expressed in different tissue types suggests that cov infections may also involve the intestines, liver, kidneys, testes and other organs [ ] . previous studies have found that the proportion of patients infected sars-cov with diarrhea was as high as % [ ] . a recent case report showed the presence of -ncov in the feces from a patient who initially presented with diarrhea symptoms [ ] , and ace has been found to be highly expressed in the upper esophagus, stratified epithelial cells, and absorptive enterocytes from the ileum and colon, findings consistent with the possibility of a fecal-oral transmission route [ , ] . liver dysfunction or injury in patients with ncp may be caused by the inappropriate use of antiviral drugs or systemic inflammatory reactions [ ] . recently, zhao et al. [ ] showed that -ncov could infect cholangiocytes expressing human-specific ace using the human liver ductal organoids model, with resultant barrier and bile acid transport function impairment. thus, liver damage in covid- patients might partly result from direct cholangiocyte injury and consequent bile acid accumulation during -ncov infection. acute kidney injury has also been seen in patients with -ncov infections [ ] . rna-seq has shown that ace is abundantly expressed in various kidney proximal tubule cell subtypes. in addition to renal injury from host immune responses, renal injury may be caused by the virus directly attacking target cells where ace is expressed [ ] . some researchers have indicated that -ncov may enter renal tubular cells by binding to ace , thereby inducing cytotoxicity and renal dysfunction [ ] . one place where ace is highly expressed is the testes, mainly in the spermatogonia, sertoli cells and interstitial cells. although there are no studies to establish whether -ncov infection causes testicular damage, some experts have highlighted the importance of evaluating reproductive function in recovered male patients, especially young ones [ ] . it has been reported that -ncov was present in blood from patients with covid- , suggesting that infection sometimes may be systemic [ ] . guo et al. [ ] analyzed the single cell rna-seq data for normal whole lung tissue samples and fibrotic lung tissue samples and found that ace was mainly expressed in the arterial vascular cells of fibrotic lungs. the first destination of pulmonary circulation outflow is the heart, and the failing human heart has a higher percentage of ace expressing cardiomyocytes. therefore, it is assumed that -ncov may attack the heart through the blood flow, which may explain the high incidence of cardiac injury in critically ill patients [ , ] . the % incidence of thrombotic complications in icu patients with covid- infections is remarkably high [ ] . in short, the flow of virus into the blood stream is a key step for its spread to other organs, body fluids and excreta, and provides the blood transmission pathways for -ncov. in conclusion, in addition to the respiratory, digestive and genitourinary systems, the blood system may also be a potential route of infection for -ncov. more recent researches [ , ] revealed that ace and tmprss were co-expressed in ocular surface cells and nasal epithelium which could therefore serve as a portal of initial infection and transmission. thus, it is important to clarify the mechanism of virus transmission for public health administration departments to formulate the best health policies regarding the prevention of -ncov infections. the world is currently in a severe emergency state regarding -ncov, and the analysis of ace in different species can help with providing a warning system for the cross-species transmission of -ncov. variation in the ace gene sequence in different species is closely linked with viral susceptibility. during the sars outbreak, li et al. [ ] used pseudovirus to infect t cells transfected by human, mouse or rat ace . their results showed that mouse and rat ace -transfected cells were both less efficiently infected by pseudovirus than human ace -transfected cells, and the infection efficacy was significantly enhanced by incubation with human ace -transfected mouse t cells. in human ace , amino acid sites - , - and - were shown to be involved in the interaction with the sars-cov s protein, with residues , , , and being the key positions [ ] . based on the latter residue positions, some researchers explored the effect of ace variation in different species on their susceptibility to the virus. holmes et al. [ ] identified variation at the m n position in rat ace compared with that in the human ortholog, which contains a large n-linked glycan at this position. the same study found that a k h mutation resulted in the lack of a hydrophobic pocket. in mouse ace , there are m s and k h mutations. therefore, ace mutations in rat and mouse ultimately lead them to lack susceptibility to sars-cov. moreover, research on rhesus monkeys experimentally infected with a pathogenic sars-cov strain showed that there are natural non-synonymous changes in the rhesus ace gene, and further mutagenesis analysis showed that y n, a natural mutation, caused ace expression to be dramatically downregulated and reduced the viral entry [ ] . another study found an enhanced interaction between civets and sars-cov caused by an additional region covering residues - in ace [ ] . a recent study on hace transgenic mice and wildtype mice infected with -ncov reported that viral antigens were found in the trachea, alveolar epithelial cells and macrophages of the hace mice, but not in the wild-type mice, suggesting that mice are insusceptible to -ncov [ ] . zhou et al. [ ] used hela cells expressing human, civet, pig, chinese chrysanthemum bat or mouse ace to conduct in vitro viral infection experiments, observing that all ace -expressing cells except those from mouse were infected by the virus. it is worth mentioning here that some researchers have compared the amino acid sequences of ace from different species and found that human and non-human primates share identical sequences in some regional residues. the above critical residues , , , and of companion animals, domestic animals and wildlife animals are relatively conserved among them, while certain ones are variable. changes in amino acid residues may lead to lower affinity viral binding, and certainly the presence of other variable regions cannot be excluded to compensate for such changes [ ] . thus, generic variation in the ace gene can influence the susceptibility of different species to cov, which should alert us to the potential interspecies transmission of -ncov. exploring whether different populations are potentially susceptible to -ncov by analyzing ace expression in different people could provide a valuable reference for effective and timely epidemic prevention. a recent rna-seq analysis of the ace expression patterns in eight normal human lung transplant donors showed no association between the number of cells expressing ace and donor age or smoking status. however, men had a higher proportion of ace -expressing cells than women [ ] , a finding highly consistent with an epidemiological investigation showing that most patients diagnosed with -ncov infection were male [ ] . asian males were also found to have a higher percentage of ace -expressing cells than white and african americans in one study [ ] . but in the current situation, the original epidemic has become a global outbreak, not only in asia, but in europe and america also. one study that analyzed large-scale transcriptomic datasets of normal lung tissue found no significant differences in ace gene expression between race, age, or sex, whereas in smokers, ace gene expression was upregulated [ , ] . elsewhere, ace genes were found to be expressed in specific cell types related to smoking history and location. ace was reported to be actively expressed in the goblet cells of smokers and club cells of non-smokers in the bronchial epithelium [ ] . in the lungs, ace expression is abundant in the remodeled type ii alveolar epithelial cells of smokers. but the reasons for such differences in ace expression related to tobacco smoking are obscure [ ] . some researchers have reported that ace expression decreases with increasing age, and because ace is on the x chromosome its level is higher in women than in men. the ace level is low in men and the elderly, but this pattern does not match the characteristic of severely ill covid- patients being mostly elderly males. therefore, whether or not ace expression is high or low is not a crucial factor affecting the prognosis of patients with covid- [ ] . some researchers have investigated the genetic backgrounds of different populations through coding-region variants in ace in terms of its diversity and variability, which may affect its function. quantitative trait locus variations that may affect ace expression were analyzed using the gtex database (https ://www.gtexp ortal .org/home/datas ets), which enabled the genomic characteristics of ace in different populations to be compared, and the results indicated that ace expression may also show potential differences among different groups and races in asia. however, there is no genomic evidence to support the existence of ace mutants in different populations that can resist its binding to the cov s protein [ ] . recently, othman et al. [ ] found that none of the eight ace mutants they investigated had disrupted interactions with the -ncov rbd; hence, they proposed that the genetic polymorphism they investigated for ace had a marginal effect on the affinity with -ncov rbd. nevertheless, this research area would benefit from further in-depth investigations. recent studies on the epidemiological characteristics of covid- have revealed that people with chronic diseases such as hypertension, diabetes, chronic obstructive lung disease and coronary heart disease are more likely to have serious infections [ , , ] . applying statistical methods to collect clinical cases, we previously found that the serum concentration levels of ace were significantly elevated in patients with essential hypertension [ ] . recently, chen et al. [ ] showed that the level of myocardial ace mrna and protein in patients with heart failure had increased significantly above normal levels by rna-seq. another study analyzed lung transcriptome samples from more than covid- patients with comorbidities, and found that ace was highly expressed in these patients, as compared with the control group, and that ace may be regulated by histone modifying enzymes, including kdm b (lysine-specific demethylase). this finding suggests that patients with such comorbidities may have higher chances than normal of developing severe covid- [ ] . in conclusion, whether ace is up-or downregulated, patients with underlying diseases may have more imbalanced ras homeostasis, lower immunity and greater susceptibility to -ncov. hence, more attention should be paid to this population to reduce their infection risk and disease severity. it has been suggested that -ncov, like sars-cov, invades host cells through ace receptors, but there are also cd l co-receptors for sars-cov [ ] . some scholars noted that ace is widely expressed across a variety of organs and is expressed moderately but not highly in lung, which, however, is the major infected organ [ ] . in addition to the factors that connect the respiratory system with the external environment, is there any receptor other than ace that mediates the invasion of -ncov into the host? one study using molecular model fitting may have an answer to this in showing that ang ii receptor type (at ), a g-protein-coupled receptor, is abundantly expressed in lung tissues and may be another receptor for -ncov [ ] . at plays an antagonistic role with at in the ras system. invasion of host cells by -ncov affects the expression of ace , and if ace is re-incorporated into cells with at as a possible co-receptor, it would accelerate the imblance of the ras system, worsening the situation for patients. it remains unclear whether the at co-receptor leads to a more serious imbalance between the classical ace-angii-at receptor axis and the new ace -ang-( - )-mas homeostasis in the ras system. clinical studies have shown that "cytokine storms", or the massive release of inflammatory factors, occur in critically ill patients, causing bodily tissue damage and multiple organ failure in critical patients [ ] . cov invades host cells through ace , which changes ace expression and leads to increased levels of ang ii, a proinflammatory factor. ang ii contributes to "cytokine storms". recently, studies have shown that -ncov directly affects the human spleen and lymph nodes by infecting tissue-resident cd + macrophages. -ncov also triggers macrophages to produce il- , a proinflammatory cytokine [ ] . -ncov may directly infect lymphocytes, especially t cells, leading to lymphopenia and impaired antiviral responses. but the lymphocytes lack ace expression, and whether alveolar macrophages can phagocytose the viral particles, transfering to lymphocytes remains unknown [ ] . the internal mechanism by which the immune system and ras system interact and regulate each other when -ncov infects host cells is unclear, and this lack of clarity needs to be solved urgently. in effect, ace plays a dual role in covid- infections by first acting as a receptor for -ncov, and then as a protective molecule in the ras system. it is currently unclear whether ace acts as a human friend or foe during infection with -ncov. this review has attempted to summarize the up-to-date research on ace in cov disease, mainly focusing on cov invasion, changes in the expression of ace after viral binding, the effects of -ncov distribution on viral transmission, and genetic susceptibility to -ncov in humans. it is hoped that the information in this review will provide a clearer picture of the pathogenic mechanism of -ncov, and act as a useful reference on ace as a potential therapeutic target for covid- . clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective 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medrxiv: detection of sars-cov- in different types of clinical specimens single-cell rna analysis on ace expression provides insights into sars-cov- potential entry into the bloodstream and heart injury clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan incidence of thrombotic complications in critically ill icu patients with covid- duh ej ( ) ace and tmprss are expressed on the human ocular surface, suggesting susceptibility to sars-cov- infection sars-cov- entry factors are highly expressed in nasal epithelial cells together with innate immune genes efficient replication of severe acute respiratory syndrome coronavirus in mouse cells is limited by murine angiotensin-converting enzyme adaptation of sars coronavirus to humans rhesus angiotensin converting enzyme supports entry of severe acute respiratory syndrome coronavirus in chinese macaques structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections analysis of angiotensin-converting enzyme (ace ) from different species sheds some light on cross-species receptor usage of a novel coronavirus -ncov chinese center for disease control and prevention ( ) the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- ) in china tobacco smoking increases the lung gene expression of ace , the receptor of sars-cov- bulk and single-cell transcriptomics identify tobacco-use disparity in lung gene expression of ace , the receptor of -ncov. medrxiv: organ-protective effect of angiotensin-converting enzyme and its effect on the prognosis of covid- comparative genetic analysis of the novel coronavirus ( -ncov/sars-cov- ) receptor ace in different populations interaction of the spike protein rbd from sars-cov- with ace : similarity with sars-cov, hot-spot analysis and effect of the receptor polymorphism epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of coronavirus disease in china association between circulating angiotensin-converting enzyme and cardiac remodeling in hypertensive patients the ace expression in human heart indicates new potential mechanism of heart injury among patients infected with sars-cov- . cardiovasc res ace expression is increased in the lungs of patients with comorbidities associated with severe covid- cd l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus agtr , one possible novel key gene for the entry of -ncov into human cells. preprints: the novel severe acute respiratory syndrome coronavirus (sars-cov- ) directly decimates human spleens and lymph nodes. medrxiv: sars-cov- and viral sepsis: observations and hypotheses we thank sandra cheesman, phd, from liwen key: cord- -vh dpf e authors: calò, lorenzo a; davis, paul a title: are the clinical presentations (phenotypes) of gitelman’s and bartter’s syndromes gene mutations driven by their effects on intracellular ph, their “ph” enotype? date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: vh dpf e gitelman’s syndrome (gs) and bartter’s syndrome (bs) are rare inherited salt-losing tubulopathies whose variations in genotype do not correlate well with either clinical course or electrolyte requirements. using gs/bs patients as nature’s experiments, we found them to be a human model of endogenous ang ii antagonism with activated renin-angiotensin system (ras), resulting in high ang ii levels with blunted cardiovascular effects. these patients are also characterized by increased and directly correlated levels of both angiotensin converting enzyme (ace ) and ang - . understanding the myriad of distinctive and frequently overlapping clinical presentations of gs/bs arises remains challenging. efforts to find a treatment for covid- has fueled a recent surge in interest in chloroquine/hydroxychloroquine and its effects. of specific interest are chloroquine/hydroxychloroquine’s ability to inhibit sars-cov infection by impairing ace , the sars-cov entry point, through terminal glycosylation via effects on tgn/post-golgi ph homeostasis. several different studies with a gs or a bs phenotype, along with a nonsyndromic form of x-linked intellectual disability linked to a mutated slc a , provide additional evidence that specific gene defects can act via misregulation of tgn/post-golgi ph homeostasis, which leads to a common mechanistic basis resulting in overlapping phenotypes. we suggest that linkage between the specific gene defects identified in gs and bs and the myriad of distinctive and frequently overlapping clinical findings may be the result of aberrant glycosylation of ace driven by altered tgn/endosome system acidification caused by the metabolic alkalosis brought about by these salt-losing tubulopathies in addition to their altered intracellular calcium signaling due to a blunted second messenger induced intracellular calcium release that is, in turn, amplified by the ras system. gitelman's syndrome (gs) is a genetic tubulopathy caused by loss-of-function mutations in the slc a gene, which encodes the na + -cl − cotransporter and is characterized by hypokalemic metabolic alkalosis, hypocalciuria, hypomagnesemia, activated renin-angiotensin system (ras) and high angiotensin ii (ang ii) levels. however, ang ii cardiovascular effects are blunted as gs patients are either normo-or hypotensive and represent a model of endogenous ang ii antagonism [ , ] . patients are either normo-or hypotensive and represent a model of endogenous ang ii antagonism [ , ] . gs resembles the abnormalities induced by thiazide diuretics (inhibitors of nacl cotransporter in the distal convoluted tubule of the nephron), thereby suggesting ncc/slc a gene as a possible candidate. mutations have been found to result in the loss of ncc/slc a function inducing nacl wasting, hypovolemia, and metabolic alkalosis [ , ] (figure ), making it an autosomal recessive disease as well. despite the growing number of causative mutations identified, up to % of patients are still found to carry only one slc a mutant allele; therefore, large genomic rearrangement may account for unidentified mutations [ ] . bartter's syndrome (bs) includes five different types of inherited salt-losing tubulopathies all characterized by hypokalemia, hypochloremic metabolic alkalosis, activated ras, high ang ii levels yet normotension or hypotension, and a blunted cardiovascular effect of ang ii [ ] (figure ). the electrolyte abnormalities of bs are similar to those induced by treatment with furosemide or other drugs that inhibit the na-k- cl cotransporter of the thick ascending limb of henle's loop, which prompted investigations of the gene nkcc /slc a encoding the na-k- cl cotransporter. variants of the coding region were identified in affected patients, which resulted in loss of the cotransporter function, along with na and k wasting in the thick ascending limb of henle's loop and hypovolemia. these patients were thereafter classified as bs type . in patients in whom nkcc mutations were not detected, loss-of-function mutations in the apical atp-sensitive k channel (renal outer medullary potassium channel, romk) were found and classified as bs type . as romk recycles k from the cell back into the lumen, the resultant fall of luminal k shuts down na-k- cl cotransporter activity, resulting in salt wasting and hypovolemia, the same phenotype as bs type . some patients with a bs phenotype show mutations in neither nkcc nor romk genes, but rather in the chloride channel clcnkb. the chloride channel clcnkb mediates cl reabsorption across the basolateral membrane of the renal tubular cells, and the resulting intracellular chloride accumulation inhibits na-k- cl cotransporter activity and manifests in salt-wasting and hypovolemia. these patients are classified as bs type . two additional genetic changes have been identified as conferring a bs phenotype. bs type a results from a mutation of the regulatory protein barttin (bsnd) required for location of the basolateral membrane cl channels, clckb and clcka, and bs type b results from a deletion mutation affecting both clckb and clcka, which are adjacent on chromosome . the final consequence is, as noted above for bartter's syndrome type iii, intracellular chloride accumulation, which inhibits na-k- cl cotransporter activity with ensuing salt wasting and hypovolemia. finally, mutations in maged have been identified in a severe antenatal form of bs (bs type ), which spontaneously resolves during the first week of life. bartter's syndrome (bs) includes five different types of inherited salt-losing tubulopathies all characterized by hypokalemia, hypochloremic metabolic alkalosis, activated ras, high ang ii levels yet normotension or hypotension, and a blunted cardiovascular effect of ang ii [ ] (figure ). the electrolyte abnormalities of bs are similar to those induced by treatment with furosemide or other drugs that inhibit the na-k- cl cotransporter of the thick ascending limb of henle's loop, which prompted investigations of the gene nkcc /slc a encoding the na-k- cl cotransporter. variants of the coding region were identified in affected patients, which resulted in loss of the cotransporter function, along with na and k wasting in the thick ascending limb of henle's loop and hypovolemia. these patients were thereafter classified as bs type . in patients in whom nkcc mutations were not detected, loss-of-function mutations in the apical atp-sensitive k channel (renal outer medullary potassium channel, romk) were found and classified as bs type . as romk recycles k from the cell back into the lumen, the resultant fall of luminal k shuts down na-k- cl cotransporter activity, resulting in salt wasting and hypovolemia, the same phenotype as bs type . some patients with a bs phenotype show mutations in neither nkcc nor romk genes, but rather in the chloride channel clcnkb. the chloride channel clcnkb mediates cl reabsorption across the basolateral membrane of the renal tubular cells, and the resulting intracellular chloride accumulation inhibits na-k- cl cotransporter activity and manifests in salt-wasting and hypovolemia. these patients are classified as bs type . two additional genetic changes have been identified as conferring a bs phenotype. bs type a results from a mutation of the regulatory protein barttin (bsnd) required for location of the basolateral membrane cl channels, clckb and clcka, and bs type b results from a deletion mutation affecting both clckb and clcka, which are adjacent on chromosome . the final consequence is, as noted above for bartter's syndrome type iii, intracellular chloride accumulation, which inhibits na-k- cl cotransporter activity with ensuing salt wasting and hypovolemia. finally, mutations in maged have been identified in a severe antenatal form of bs (bs type ), which spontaneously resolves during the first week of life. confusion remains regarding bs type as there are two different mutations that produce a phenotype designated type . one "type" of bs type , i.e., a severe antenatal form of bs (bs type ), arises as a result of a mutation in maged , which affects the delivery of nkcc to the luminal membrane of loop of henle cells, which spontaneously resolves during the first week of life. the other type of bs type , now more clearly designated as "autosomal dominant hypocalcemia with renal salt wasting" results from a gain-of-function mutation in the casr that blunts romk channel k efflux along with activity of the na-k- cl cotransporter. the simultaneous presence in gs of hypokalemia with hypomagnesemia and hypocalciuria in addition to the occurrence in early adulthood and a generally milder clinical presentation distinguishes gs from bs [ ] . there is significant overlap in clinical manifestations between gs and bs and laboratory findings [ ] . mutations in the clcnkb gene encoding the human voltage-gated chloride clc-kb (hclc-kb) channel cause a classic type bs, which shows phenotypic overlap with gs. whereas mutations in clcnkb are tied to clinical phenotype switching, the mechanism(s) is not well understood, and it is not always possible to correlate a genotype with severity of disease [ , ] . lee et al. recently investigated a group of patients with chronic hypokalemic metabolic alkalosis [ ] . they found wide variability in clinical phenotype, whereas variations in genotype did not correlate with either clinical course or electrolyte requirements. although sex, genotypes, or the number of slc a mutant alleles did not predict severity or response to treatment, hypocalciuria and hypomagnesemia were useful markers to differentiate gs from classical type bs [ ] . cheng et al., using a group of classic type bs patients carrying homozygous missense mutations with well-described functional consequences and clinical presentations, found significant correlations of mutant chloride current activity with the age at diagnosis, plasma chloride concentration and urine calcium excretion rate [ ] . these measurements were possible due to the rescue from accelerated degradation of expressed mutant protein by co-expression of barttin. wojciechowski et al. noted that several cases of dent disease (dd ), another renal salt-wasting tubulopathy arising from mutations in the cl − /h + antiporter clc- , had atypical hypokalemic metabolic alkalosis and hyperaldosteronism, findings usually associated with bs [ ] . this overlap of dd and bs phenotypes suggested that barttin might regulate clc- transport and they found, using barttin cotransfection, that barttin impaired clc- 's complex glycosylation. of note, pathologic barttin mutants are responsible of bs type a and the overlap of dd and bs phenotypes, despite clearly different genetic defects; this suggested that their overlapping phenotypes are a product of a disturbance of pathways common to both, in particular, intracellular glycosylation. de jong et al. investigated the effects of several different mutations found in gs patients to uncover the underlying pathogenic mechanism [ ] . specifically, they examined the effects of ncc mutations of differing types (missense, frameshift, nonsense, and splice-site mutations) at various portions of the entire protein coding sequence on metolazone-sensitive na + uptake, subcellular localization, and glycosylation of human ncc expressed in xenopus laevis oocytes [ ] . the gs mutations examined either had improperly glycosylated nonfunctional proteins that failed to exit the protein secretory pathway or functional mutants that were normally glycosylated but with partly impaired delivery to the plasma membrane. de jong et al.'s results documented that mutations in ncc are associated with changes in ncc processing and that these defects are part of the underlying pathogenic mechanism in gs. however, although providing additional insight into the processing effects of the mutations, their report did not provide an explicit mechanism linking these ncc defects to the myriad of clinical effects that characterize gs. two other recent clinical studies investigating two unrelated ion transport mutations are relevant to our hypothesis, as they have been linked to changes in golgi ph and glycosylation. khayat et al. reported a multigenerational nonsyndromic intellectual disability that is the result of mutation in the alkali cation/proton exchanger gene slc a (also commonly referred to as nhe ) located on human x chromosome [ ] . the gene is widely transcribed in various secretory tissues with prominent enrichment in the trans-golgi network and post-golgi vesicles. mass spectrometry analysis showed an abnormal n-glycosylation profile for transferrin, a clinical diagnostic marker for congenital disorders of glycosylation. this then led khayat et al. to conclude that misregulation of tgn/post-golgi ph homeostasis and glycosylation of exported cargo likely underlay the cellular pathophysiology associated with nonsyndromic form of x-linked intellectual disability linked to a mutated slc a . another study by bastug et al. [ ] was the case of a six-year-old girl who presented with hypokalemic metabolic alkalosis that had prompted an initial diagnosis of bs. however, the patient failed to thrive, and upon being reexamined two years later, slit-lamp cornea examination found punctate needle-shaped crystals and her leukocyte cystine content level was . mg/ml. the cystinosis diagnosis was confirmed by finding a homozygous c. - g>a novel splice mutation in cystinosin. bastug's review of the literature showed that cystinosis with hypochloremic metabolic alkalosis was first reported in a five-year-old boy by berio in [ ] with only further cases with nephropathic cystinosis initially presenting as bs. nephropathic cystinosis is a lysosomal disorder caused by functional defects of ctns, a lysosomal -transmembrane protein h + -driven cystine transporter that mediates cystine efflux into the cytosol. taranta et al. characterized cystinosin protein isoforms resulting in alternative splicing of exon , which directs the protein to other cell compartments, including the plasma membrane, the golgi apparatus, the endoplasmic reticulum (er), and cytosolic vesicles resembling endosomes [ ] . activation of ras and production of ang ii lead to adverse cardiovascular remodeling as well as many other cardiovascular pathologies. ras activation, as shown by the increased ang ii levels, typically accompanies increasing heart failure and systemic and tissue ras dysregulation, which contribute strongly to end-stage heart failure via maladaptive cardiac remodeling, cardiac hypertrophy, apoptosis, fibrosis, and endothelial dysfunction [ ] . ang ii-mediates up-regulation of apelin (apln), which in turn up-regulates ace , leading to conversion of ang ii by ace into the protective ang - peptide [ , ] . the apln pathway has emerged as a major peptide hormone pathway with apln widely expressed in mammals [ ] and ace is an important target of apln action in the vasculature [ ] . the regulatory loop also functions such that apelin is required for ace expression [ ] [ ] [ ] . ace clearly serves as a major negative ras regulator in part by converting ang ii into the vasculoprotective, antiatherosclerotic, antioxidant, and anti-inflammatory peptide, ang - . using gs/bs patients as nature's experiments, we found them to be a human model of endogenous ang ii antagonism with activated ras and high ang ii levels, yet blunted cardiovascular effects [ ] . these patients, despite increased ang ii levels, have normotension or hypotension and lack of cardiovascular remodeling in terms of lack of cardiac left ventricular hypertrophy and carotid intima-media thickness, reduced intracellular calcium signaling, reduced rho kinase activity, and activation of antiatherosclerotic, anti-inflammatory, and antioxidant defenses [ ] . of particular relevance is our demonstration in gs/bs patients of increased levels of both ace and ang - , the levels of which were directly correlated, in contrast to either hypertensive or healthy subjects [ ] . the ace /ang - system was also shown be protective in hyperoxic lung injury [ ] . clearly, ras has at least two counterregulatory axes, ace/ang ii and ace /ang - , and their balance appears fundamental in maintaining cardiovascular homeostasis [ ] . of particular interest are the reports detailing the effects of chloroquine and other members of this class of drugs. chloroquine (cq), n -( -chloro- -quinolinyl)-n ,n -diethyl-pentane- , -diamine) is a weak base that, when unprotonated, can diffuse across membranes and accumulate in acidic cellular compartments. in these compartments, it is protonated and becomes trapped, causing elevated ph and swelling. for example, basque et al. [ ] examined multiple lysosomotropic drugs that alkalinized the trans-golgi network (tgn)/endosome system and their effects on tgfβ production, which requires precursor protein cleavage in the tgn/endosomes for activity. they found that chloroquine, hydroxychloroquine, and azithromycin all suppressed processing of pro-tgfβ, the tgfβ precursor protein and reduced production of mature bioactive tgfβ. chloroquine may also directly affect other cellular trafficking processes as well. kavaliauskiene et al. reported chloroquine interferes with shiga toxin subunit stxa translocation, potentially via effects on the sec channel, as its knockdown also protects cells against shiga toxin [ ] . sec is part of the protein translocon, an ensemble of proteins involved in the er targeting of precursor polypeptides [ ] . mcgill et al. reported that low dose chloroquine in patients with metabolic syndrome resulted in decreased blood pressure [ ] as well as a decrease in jnk activation. li et al. showed that mln an ace inhibitor abolished captopril-induced blockade of jnk phosphorylation along with proinflammatory cytokines secretion and activation of p mapk and erk / [ ] . these findings all suggest that chloroquine's effect may be mediated via effects on ace . endosomes have an acidic interior due to the activity of a proton pump, and endosomal acidification is closely interlinked with endosomal, intracellular messenger (nicotinic acid adenine dinucleotide phosphate, naadp)-mediated calcium release via a specific channel-the two-pore channel tpc [ ] . inhibition of the proton pump prevents calcium release from endosomes and reduction of extracellular calcium blocks endosomal acidification [ ] . these processes have been shown to be critical for sars-cov- entry in the cell, which has been shown to be blocked when the proton pump is inhibited and, at least in part, explained by the reduced/lack of the intracellular messenger's naadp activity on tcp [ ] . kellokumpu suggested that golgi-localized glycosylation is a ph-sensitive process [ ] . for example, axelsson et al. found that treatment with nh cl caused an inhibition of o-glycan synthesis that paralleled the mislocalization of several glycosylation enzymes into endosomes with no effect on golgi morphology [ ] . later, kellokumpu et al. used chloroquine to change intracellular ph and found a . unit increase in ph interfered with both mucin type o-glycosylation and terminal a- , -sialylation of n-linked glycans without changing overall golgi morphology [ ] . vincent et al. reported that in addition to chloroquine inducing elevations of endosomal ph, it interfered with terminal glycosylation of ace , the sars-cov cellular receptor, negatively influencing the virus receptor binding, resulting in the inhibition of infection [ ] . looking at all the above-mentioned reports led us to think about some of the effects noted and we were specifically struck by the blood pressure effects and the noted changes in ace glycosylation. we have sought to understand the basis for the disparate group of clinical effects that characterize gs and bs, as well as the differing levels of clinical severity that both gs and bs can exhibit. we have sought to understand how those clinical effects relate to the mutations in ion transport identified as causal in these syndromes. we would like to suggest that the linkage between the specific gene defects identified in gs and bs and the myriad of alterations noted in clinical effects may be the result of aberrant glycosylation driven by altered tgn/endosome system acidification caused by the chronic metabolic alkalosis induced by their genetic defects. we think that either ion transport issues or chronic state of metabolic alkalosis in gs/bs patients, jointed with the blunted/reduced intracellular calcium signaling due to reduced second messenger induced intracellular calcium release [ ] , drive changes in the ph of the tgn/endosome system and result in altered ace protein glycosylation (figure ). the blood pressure effects, alongside the activation of antiatherosclerotic, anti-inflammatory, and antioxidant defenses found in gs and bs patients [ ] , are mirrored by chloroquine effects. this suggests that altered glycosylation of ace , found upon chloroquine treatment, occurs in gs/bs and drives the multiplicity of gs/bs effects. the central role of ace in the counterregulatory system found in ras then suggests that the effect of altered glycosylation is apt to be amplified by the ras system. similar clinical findings hint at common pathways for chloroquine and gs/bs effects with respect to cardiac electrophysiology. the recent surge of interest in hydroxychloroquine or chloroquine has led to the u.s. fda issuing a safety announcement regarding their use being associated with qt interval changes and ventricular tachycardia [ ] . these types of cardiac effects have also been found associated with gs/bs patients [ , , ] . a mechanism whereby ph-driven altered glycosylation of ace tgn/endosome system, combined with amplification by ras, provides an explanation as to how gs and bs phenotypes can exhibit overlapping clinical findings despite their different mutations. of note, this mechanism also potentially provides an explanation for our findings regarding the absence of covid- in gs/bs patients despite having an increased level of ace [ ] , which has been shown to be the entry point for sars-cov- . increased levels of ace have been linked to increased covid morbidity and mortality [ ] and thus should presumably result in an increased susceptibility to covid- infection in gs/bs patients. however, our cohort of gs and bs patients provided evidence against that hypothesis, as a telephone survey of our over gs and bs patients, all from northern italy (veneto, lombardia, and emilia romagna), the hotspots of the covid- pandemic in italy, found none of them infected with covid- [ , ] . the very low incidence of these rare diseases limited our enrollment and the result of our survey was not significant ( % confidence interval (ci) - %) using the reported covid- prevalence in northern italy ( . %, % ci . - . %). however, given the likely covid- underreporting [ ] , our result becomes significant (pearson's chi-squared test p = . ) compared to the estimated true covid- prevalence in northern italy ( . %, % ci . - . %) [ ] . an altered terminal glycosylation of ace in the tgn/endosome system caused by the gene mutation induced chronic metabolic alkalosis of these patients in addition to the altered intracellular the blood pressure effects, alongside the activation of antiatherosclerotic, anti-inflammatory, and antioxidant defenses found in gs and bs patients [ ] , are mirrored by chloroquine effects. this suggests that altered glycosylation of ace , found upon chloroquine treatment, occurs in gs/bs and drives the multiplicity of gs/bs effects. the central role of ace in the counterregulatory system found in ras then suggests that the effect of altered glycosylation is apt to be amplified by the ras system. similar clinical findings hint at common pathways for chloroquine and gs/bs effects with respect to cardiac electrophysiology. the recent surge of interest in hydroxychloroquine or chloroquine has led to the u.s. fda issuing a safety announcement regarding their use being associated with qt interval changes and ventricular tachycardia [ ] . these types of cardiac effects have also been found associated with gs/bs patients [ , , ] . a mechanism whereby ph-driven altered glycosylation of ace tgn/endosome system, combined with amplification by ras, provides an explanation as to how gs and bs phenotypes can exhibit overlapping clinical findings despite their different mutations. of note, this mechanism also potentially provides an explanation for our findings regarding the absence of covid- in gs/bs patients despite having an increased level of ace [ ] , which has been shown to be the entry point for sars-cov- . increased levels of ace have been linked to increased covid morbidity and mortality [ ] and thus should presumably result in an increased susceptibility to covid- infection in gs/bs patients. however, our cohort of gs and bs patients provided evidence against that hypothesis, as a telephone survey of our over gs and bs patients, all from northern italy (veneto, lombardia, and emilia romagna), the hotspots of the covid- pandemic in italy, found none of them infected with covid- [ , ] . the very low incidence of these rare diseases limited our enrollment and the result of our survey was not significant ( % confidence interval (ci) - %) using the reported covid- prevalence in northern italy ( . %, % ci . - . %). however, given the likely covid- underreporting [ ] , our result becomes significant (pearson's chi-squared test p = . ) compared to the estimated true covid- prevalence in northern italy ( . %, % ci . - . %) [ ] . an altered terminal glycosylation of ace in the tgn/endosome system caused by the gene mutation induced chronic metabolic alkalosis of these patients in addition to the altered intracellular calcium signaling due to a blunted second messenger induced intracellular calcium release, both of which increase endosomal ph [ ] , might reproduce the same ph-dependent effect on ace glycosylation and resulting inhibition of sars-cov- by cq and nh cl [ ] and presumably covid- infection. cheng et al. just published an article that argues that we should focus on increasing ace as a method of treating covid- [ ] . similar studies on the use of human recombinant soluble ace (hrsace ) [ ] support these findings. hrsace has already passed through phase i and ii clinical trials (nct , nct ) for acute respiratory distress syndrome and has received regulatory approval (nct ) for continued study in the fight against covid- [ ] . the proposed mechanism of ace alterations in gs/bs requires much more intensive investigation and a study is ongoing in our laboratory to directly examine the terminal glycosylation of ace in gs/bs patients to further strengthen our hypothesis. the linkage between the specific ion transport gene defects and the noted lack of correlation between genotype and phenotype in these patients might be the result of altered tgn/endosome system acidification causing aberrant glycosylation, which is then amplified by the ras system. in other words, their ph enotype drives their phenotype. funding: this study was supported in part by the grant dor / from university of padova, to l.a.c. the authors declare no conflict of interest. understanding the mechanisms of angiotensin ii signaling involved in hypertension and its long-term sequelae: insights from bartter's and gitelman's syndromes, human models of endogenous angiotensin ii signaling antagonism consensus and guidance from a kidney disease: improving global outcomes (kdigo) controversies conference. kidney int encyclopedia of endocrine diseases a novel mutation 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infection in the general population rho kinase inhibition and the potential role of vitamin d against covid- organ-protective effect of angiotensin-converting enzyme and its effect on the prognosis of covid- inhibition of sars-cov- infections in engineered human tissues using clinical-grade soluble human ace an ace therapy for covid- key: cord- -sma e authors: maldonado, valente; loza-mejía; chávez alderete, jaime title: repositioning of pentoxifylline as an immunomodulator and regulator of the renin-angiotensin system in the treatment of covid- date: - - journal: med hypotheses doi: . /j.mehy. . sha: doc_id: cord_uid: sma e pentoxifylline (ptx) is a phosphodiesterase inhibitor that increases cyclic adenosine monophosphate levels, which in turn activate protein kinase, leading to a reduction in the synthesis of proinflammatory cytokines to ultimately influence the renin-angiotensin system (ras) in vitro by inhibiting angiotensin receptor (at r) expression. the rheological, anti-inflammatory, and renin-angiotensin axis properties of ptx highlight this drug as a therapeutic treatment alternative for patients with covid- by helping reduce the production of the inflammatory cytokines without deleterious effects on the immune system to delay viral clearance. moreover, ptx can restore the balance of the immune response, reduce damage to the endothelium and alveolar epithelial cells, improve circulation, and prevent microvascular thrombosis. there is further evidence that ptx can improve ventilatory parameters. therefore, we propose repositioning ptx in the treatment of covid- . the main advantage of repositioning ptx is that it is an affordable drug that is already available worldwide with an established safety profile, further offering the possibility of immediately analysing the result of its use and associated success rates. another advantage is that ptx selectively reduces the concentration of tnf-α mrna in cells, which, in the case of an acute infectious state such as covid- , would seem to offer a more strategic approach. the immune dysregulation observed in covid- clinically translates into a secondary hemophagocytic syndrome or lymphohistiocytosis, acute respiratory distress syndrome (ards), and multiple organ failure. however, multiple studies have indicated that immunosuppression with corticosteroids impairs the immune response to various respiratory viruses as it affects the induction of antiviral interferon (ifn) type i. therefore, the decision to immunosuppress a hospitalized patient with covid- due to the possible benefits of reducing inflammation must be carefully weighed against the potential deleterious effect of delaying virus clearance or promoting bacterial superinfection. in addition to the adverse effects of immunosuppression therapy that further complicates the management of a hospitalized patient, the use of systemic steroids may worsen lung injury, which is particularly relevant in the case of treating patients with covid- . similar risks of perpetuating viral infection and bacterial or fungal superinfection must be considered when administering a drug that influences ifn-mediated signalling, including jak inhibitors and interleukin (il)- blockers, because the il- r-jak-stat signalling pathway is an essential component of the type i ifn pathway. moreover, il- can play an essential role in initiating the response against virus infections by promoting viral clearance by neutrophils; this was demonstrated in a study where deficiency of il- or il- r in mice favoured the persistence of influenza infection leading to death in the experimental group. therefore, in the face of this global emergency that is witnessing rapid degeneration in patients' clinical conditions toward more serious situations that can be overwhelming to primary physicians (despite the best standards of care), the opportunity to redirect medications offers an ethical and legal solution as medications outside of their usual indications can be prescribed as long as there is sufficient justification to do so. we here propose the repositioning of pentoxifylline (ptx) in the treatment of covid- . ptx is a phosphodiesterase (pde) inhibitor that increases cyclic adenosine monophosphate (camp) levels, which in turn activate protein kinase (pka), leading to a reduction in the synthesis of the proinflammatory cytokines il- , il- , and tumour necrosis factor-alpha (tnf-α). ptx has also been shown to influence the renin-angiotensin system (ras) in vitro by inhibiting angiotensin receptor (at r) expression. the effects of ptx on restoration of glutathione levels, maintenance of mitochondrial viability, inhibition of tnf-α production, and preservation of microvascular blood flow, along with reports of improvement in endothelial function and coagulation have encouraged its use in the treatment of neonatal sepsis, leading to a reduction in hospital stay and mortality. moreover, in the context of covid- , ptx has shown evidence of improvement in experimental ards models. moreover, since it is a short-lived drug, the effect of ptx can be rapidly suppressed if severe adverse reactions arise due to excessive suppression of tnf-. furthermore, the rheological properties of ptx could be useful when faced with the atypical presentation of ards associated with covid- , characterized by marked hypoxemia with preservation of the ventilatory mechanics. this suggests that the loss of regulation of pulmonary perfusion and hypoxic vasoconstriction (vasoplegia) can be associated with microvascular obstructive inflammatory thrombus syndrome of the lungs (i.e., microclots). overall, the rheological, anti-inflammatory, and renin-angiotensin axis properties of ptx highlight this drug as a therapeutic treatment alternative for patients with covid- , which can help reduce the production of the inflammatory cytokines tnf-α, il- , ifn-γ, and il- and increase the anti-inflammatory cytokine il- . ptx also favours restoring the balance of the immune response, reducing damage to the endothelium and alveolar epithelial cells, improving circulation, and avoiding microvascular thrombosis. moreover, ptx decreases the neutrophil/lymphocyte ratio and restores the ratio of treg/th lymphocyte subpopulations. ptx decreases c-reactive protein (crp), ferritin, lactic dehydrogenase, and d-dimer, with consequent improvement in the ventilatory parameters pao /fio and sato . in view of this evidence, it seems reasonable to reposition a drug with a proven safety profile and that is widely available as an alternative treatment of covid- to reduce inflammation without the trade-off of conventional immunosuppression. moreover, such repositioning avoids ethical or legal barriers allowing for immediate application in an acute setting. the sars-cov- spike protein (spike s) binds only to human angiotensin-converting enzyme type (ace ) as a cell entry receptor [ ] . this interaction partially explains why the lung is the organ most vulnerable to infection with sars-cov- . zhao et al. [ ] demonstrated that % of the cells in the healthy lung tissue expressing ace are type ii alveolar epithelial cells (aecii cells), suggesting that these cells can serve as a reservoir for viral infection. moreover, genetic analysis revealed that ace -expressing aecii cells have high levels of regulatory genes related to multiple viral processes, including the viral life cycle, assembly, and genome replication, further supporting that ace expressed in aecii cells facilitates the replication of coronavirus in the lungs [ ] . similarly, other extrapulmonary tissues, including the heart, kidney, endothelium, and intestine, express ace [ ] [ ] [ ] [ ] [ ] . this broad distribution ace tissue expression could partially explain the multiorgan dysfunction seen in patients with severe sars-cov infection [ ] [ ] [ ] . compounding these effects, the coupling of s protein to its cell-binding site of ace leads to the downregulation of ace , resulting in excess production of angiotensin (ang) by angiotensin-converting enzyme (ace). the reduction in ace concentrations in turn decreases the production of the heptapeptide vasodilator or ang - , which contributes to lung injury, since the stimulation of angiotensin type receptors (at rs) by ang ii increases the permeability of the pulmonary vasculature [ , ] . the ras plays an essential role in maintaining blood pressure homeostasis, as well as in fluid and electrolyte balance [ ] [ ] [ ] , whereas ace functions as a negative regulator of the ras [ , , ] . specifically, ace cleaves the ang i decapeptide into the ang ii octapeptide [ , ] , ace cleaves a single ang i residue to generate ang - [ , ] and a single ang ii residue to produce ang - [ ] , thereby negatively regulating ras by inactivating ang ii [ ] . imai et al. [ ] demonstrated that ang ii receptor subtypes are responsible for ace/ace regulated acute lung injury (ali) based on experiments of a mouse model with genetic loss of at a receptor (rat a) expression, which showed markedly improved lung function and reduced oedema formation. conversely, inactivation of the at receptor (rat ) was shown to aggravate ali [ ] . moreover, they attempted to prevent ali in the ace -knockout mice using rat -and rat -specific antagonists. pharmacological inhibition of rat attenuated the severity of acid-induced lung injury, whereas rat inhibition had no apparent effect on the phenotypes of ali in ace knockout mice [ , ] . collectively, these data show that ang ii-rat a interactions play a role in the development and severity of ali [ ] . ace contributes to an increase in vascular permeability and a decrease in the survival of pneumocytes [ ] . at the same time, ang ii is considered to function as a growth factor that regulates cell proliferation/apoptosis and fibrosis, as well as a mediator that attracts inflammatory cells to sites of tissue injury [ ] . all of the evidence accumulated to date has led to the widely accepted belief that ang ii is the primary factor responsible for the exacerbation of lung tissue lesions through the activation of rat [ ] [ ] [ ] . similarly, ang ii exerts its fibrotic effects by inducing the production of transforming growth factor-beta (tgf-β ), thereby triggering fibroblast proliferation and differentiation in collagen-secreting cells [ ] . accordingly, it would be reasonable to consider that dysregulation of the ras could partially explain the observations made by a group of doctors in italy that patients with covid- pneumonia who meet the berlin criteria of ards present an atypical form of ards. the main characteristic of these patients was a dissociation between their relatively wellpreserved lung mechanics and the severity of hypoxemia. these doctors suggested that this disconnect may be explained by the loss of regulation of pulmonary perfusion and hypoxic vasoconstriction (i.e., vasoplegia) [ ] . the obstructive inflammatory thrombus syndrome of the lungs that occurs in patients with covid- could offer another explanation for this atypical presentation of ards involving inflammation of the endothelial vascular lesions, which may involve dysregulation of the ras (i.e., microclots) [ ] . that is, the high concentration of ace in the endothelial cells of the arteries, veins, and arterial smooth muscle cells favour viral replication along with secondary cell damage, consequently releasing alarmins that are generated during cell death, thereby triggering the host's innate immune response through various mechanisms, including activation of complement via lectin and alveolar macrophages through tnf-α and other mediators [ ] . complement activation would not only cause direct endothelial damage but also participates in leukocyte recruitment through c a and c a formation, leading to massive local production of the cytokines il- , il- , il- , and ifn-γ. along with this massive host immune response, lymphocytes, resident macrophages, monocytes, and neutrophils perform their inflammatory functions, causing increased endothelial and alveolar epithelial cell damage and microvascular thrombosis, as reflected in elevations of lactic dehydrogenase and dimer d [ ] . this endothelial damage could be progressive and potentially involve the microvascular beds of the kidney, brain, and other organs [ ] . an attractive alternative to deal with the covid- pandemic, which may even be able to handle the emergence of new strains due to acquired mutations of sars-cov- , is to search for agents that could block the activity of the at rs [ ] while simultaneously reducing the vascular injury mediated by ang ii characterized by inflammation and oxidative stress. since nuclear factor-kappa b (nf-kb) is stimulated after the coupling of rat and ang ii, it would be beneficial to activate the expression of various cytokines such as tnf-α, il- , il- , mcp- , and vcam- by generating oxidative stress, altering endothelial nitric oxide, and inducing the activity of nadph oxidase that increases the production of superoxide and peroxynitrite [ ] [ ] [ ] [ ] . therefore, increasing ace expression through at r blockade [ ] may affect viral replication and the release of useful viral materials. this would confer protection against the ali generated by sars-cov- due to the counterbalance of the excess production of ang ii and its potentially harmful effects [ , ] . however, a limitation in the use of available at r blockers is the risk or potential of deleterious hemodynamic effects of arterial hypotension [ ] . although there is currently a lack of detailed information on hypotension rates among hospitalized patients with sars-cov- during this pandemic [ ] , yu and colleagues [ ] reported that half of the patients with sars-cov infection developed hypotension during their hospitalization. in this regard, ptx, as the drug we are considering repositioning for covid- treatment, has shown atr blocking effects in experimental studies, along with clinical evidence in humans showing no secondary effect on blood pressure [ ] [ ] [ ] . all available reports and anecdotal information indicate that the pathological process contributing to severe cases of covid- is largely related to dysregulation of the immune system owing to a storm of cytokine production, characterized by increased expression levels of inflammatory cytokines and chemokines with a concomitant decrease in naive cd + t cells, cd + t cells, and regulatory t cells (tregs) [ , ] . these findings may be related to previous descriptions of an association of higher levels of serum inflammatory cytokines with extensive lung damage in patients with other coronavirus infections in addition to sars-cov- , including sars-cov and mers-cov [ ] . this is due to induction of the production of large amounts of chemokines and cytokines (il- , il- , il- , il- , tnf-α, and mcp- ) by cov-infected cells, followed by the recruitment of lymphocytes [ ] . coronaviruses also infects macrophages, which present the antigens of the virus to the t cells, thereby generating their activation and differentiation into the different subtypes of t lymphocytes. this results in the massive release of cytokines to amplify the immune response ( ) . the continuous production of these mediators due to viral persistence ultimately harms cd + t cells and natural killer cells [ , ] . the cd + t cells in sars-cov infection play a vital role in the elimination of infected cells and in generating inflammatory immune damage, accounting for % of the total inflammatory cells in the pulmonary interstitium in patients infected with sars-cov [ , ] . in experimental models, t-cell-deficient balb/c mice (transduced with ad -hdp ) were compared with controls and b-cell-deficient mice, demonstrating that t cells could survive in the infected lungs and destroy infected cells [ ] . using these same models, the depletion of cd + t cells did not affect viral replication during sars-cov infection [ , ] , whereas the depletion of cd + t cells was associated with reduced numbers of lung lymphocytes, decreased production of neutralizing antibodies and cytokines, and strong immune-mediated interstitial pneumonitis with delayed clearance of sars-cov from the lungs [ ] . moreover, t-helper cells (th) produce inflammatory cytokines through the nf-b signalling pathway [ ] , and il- cytokines recruit monocytes and neutrophils to the site of infection with inflammation to activate other downstream signalling pathways for cytokines and chemokines, such as il- , il- , il- , il- , tnf-, and mcp- [ , ] . a study of patients with covid- in wuhan, china demonstrated an increase in the neutrophil/lymphocyte ratio, with a high frequency of lymphopenia of t cells and cd + t cells [ ] . this study further showed that patients with severe cases of covid- had higher serum levels of inflammatory cytokines (tnf-α, il- , and il- ) compared to those of individuals of the same age suffering from a milder form of the disease [ ] . host immune responses may play an essential role in the disease severity of covid- since subsets of naive cd + t cells were found to be elevated with a smaller percentage of memory cells and tregs [ , ] . in particular, cd + t cells and cd + t cells play an essential role in attenuating innate immune responses during viral infection [ ] . by contrast, tregs attenuate the activation, proliferation, and effector functions of a wide range of immune cells for the maintenance of auto-tolerance and immune homeostasis [ ] [ ] [ ] . moreover, a histopathological study of biopsy specimens from patients that died of covid- in china revealed that the lungs were full of a gelatinous substance [ ] that was suspected to contain hyaluronic acid (ha) given its reported dysfunction and deregulation in the case of sars-cov infection [ ] . the levels of inflammatory cytokines (e.g., il- and tnf-α) are elevated in patients with covid- , and these cytokines are important inducers of hasynthase (has ) in cd + endothelial cells, epcam+ alveolar epithelial cells, and fibroblasts [ ] . the chinese national health commission reported that patients infected with sars-cov- showed high leukocyte numbers and plasma levels of the inflammatory markers crp, globular sedimentation rate, and d-dimer [ ] . another research group [ ] found a correlation between viral rna in the blood with the incidence of ground-glass opacities and acute cardiac injury in patients with covid- . they further reported high blood levels of in another study [ ] , the viral load of sars-cov- in serum (rnaemia) was positive only in critically ill patients with more severe lymphopenia and a higher number of neutrophils, and the degree of rnaemia was positively correlated with both il- levels and the mortality rate. notably, the authors acknowledged that they did not measure other cytokines due to the lack of available reagents; however, this association of rnaemia with disease severity suggests a correlation with other inflammatory markers [ ] . this possibility is particularly relevant given our suggestion of not limiting immunomodulatory strategies in the treatment of covid- to the current trend of focusing on the il- r jak-stat pathway, whose cost-benefit analysis would also be questionable. xu et al. [ ] documented the characteristics of a patient in china who died of severe sars-cov- infection, indicating substantial reduction in cd + and cd + t-cell counts. they also found that these cells were overactive, showing high proportions of hla−dr (cd - %) and cd (cd . %) double-positive fractions and a high concentration of proinflammatory cd +ccr + th cells [ ] . furthermore, this patient had high concentrations of cytotoxic granules, which is another marker of hyperactive cd + t cells, . % of which were perforin-positive, . % were granulysin-positive, and . % were both perforin-and granulysin-positive. this dysregulation of the immune system clinically manifests as respiratory failure with extensive lung damage, which is histopathologically related to the massive infiltration of neutrophils and macrophages, diffuse alveolar damage with the formation of hyaline membranes, and diffuse thickening of the alveolar wall [ , ] . in addition to the pathological findings of patients who died from sars-cov- infection, splenic atrophy and necrosis of the lymph nodes have been observed, further suggesting the progression to shock and multiple organ failure due to tissue damage to the heart, liver, and kidneys [ , ] . dysregulation of the immune response in covid- involves an immunosuppression stage with a subsequent inflammatory state characterized by a substantial reduction in peripheral lymphocyte counts that is correlated with disease severity [ ] . as cd + and cd + t cells are the most affected subpopulations in this process, the immune response to the virus is consequently compromised, thereby increasing the risk of bacterial superinfection [ , ] . although the detailed mechanism underlying the pathogenesis of lymphopenia is not fully understood, one of the pathways involved in lymphopenia induced by respiratory viral infections is cell death triggered by activation of the fas-fas-ligand interaction, as well as induction of the apoptosis axis from tnf-α ligands [ ] [ ] [ ] . overall, these compounding effects of sars-cov- infection on the immune response and inflammation lead to the conclusion that it is necessary to search for appropriate immunomodulatory strategies to dampen inflammatory responses without simultaneously suppressing the immune response to avoid any deleterious effects. in this regard, in , bermejo et al. [ ] suggested the use of ptx for the treatment of sars-cov infection due to its anti-inflammatory effect by reducing tnf-α and ifn-γ. this proposal was further based on the ability of ptx to regulate activation of the transcription factors nf-b and nfat and to inhibit various viruses such as herpes simplex virus, human immunodeficiency virus, tick- ptx is a drug with multifactorial action, including pde inhibition [ ] and increasing camp levels, which in turn activate pka [ ] , leading to a reduction in the synthesis of the inflammatory cytokines il- , il- , and tnf-α [ ] [ ] [ ] . these inhibitory effects on pdes result in an overall reduction of inflammation [ , ] . similarly, ptx modulates ifn-γ [ ] [ ] [ ] and other molecules such as intracellular adhesion molecule type (icam- ), vascular cellular adhesion molecule type (vcam- ), and crp [ , ] . based on these foundations, several models of kidney disease and clinical studies have shown that ptx is capable of attenuating proteinuria through modulating inflammatory cytokines [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition, clinical trials with diabetic nephropathy patients demonstrated that ptx enhanced the antiproteinuric effect of angiotensin receptor blockade [ , ] . the effect of ptx on the ras appears to be primarily related to its inhibition of at r expression. this speculation was supported by studies using a rat model of heart failure induced by coronary artery ligation, in which ptx infusion to the paraventricular hypothalamic nucleus attenuated the increase in at r expression [ ] and ptx treatment could prevent the overexpression of at r mrna [ ] . in addition, azhar and el-bassossy [ ] showed that ptx significantly decreased at r expression in the aortic tissue in a rat model of metabolic syndrome, and counteracted the induced hypertension by reducing the degree of inflammation, based on significantly lower levels of tnf-α and higher levels of the anti-inflammatory cytokine adiponectin. further, ptx shows promise as a useful therapeutic tool for covid- because the ras is one of the most critical systems activated during oxidative stress. upon binding of ang ii with at rs, the secondary messengers inositol triphosphate and diacylglycerol are produced, resulting in the production of reactive oxygen and vasoconstriction [ ] . a meta-analysis showed that ptx had an anti-inflammatory effect in adults with a variety of disorders, including coronary artery disease, type diabetes mellitus, idiopathic or ischemic cardiomyopathy, and chronic kidney disease. the statistically significant differences were corroborated by a reduction in plasma concentrations of tnf- and crp and no deleterious effects on blood pressure [ ] . from a practical perspective to reposition ptx for covid- , li et al. [ ] proposed using ptx as a treatment strategy for ards in based on its ability to specifically interfere with camp signalling [ ] . considering that an increase in both tregs and th lymphocytes producing interleukin il- (th cells) along with an imbalance of the treg/th ratio are among the main immune alterations observed in ards [ ] [ ] [ ] [ ] [ ] [ ] , targeting camp signalling is a reasonable strategy to induce immune tolerance. in particular, camp signalling plays a critical role give in the proliferation and function of tregs and effector t cells; that is, camp suppresses the adaptive differentiation of tregs mediated by tgf-β to reduce the treg content [ , ] . using a mouse model of cephalic ligation and puncture-induced ards, li et al. [ ] demonstrated that ptx pre-treatment attenuated lung injury and reduced the mortality rate. the authors further observed an increase in the camp levels of the spleens of the ptx-pretreated mice, whereas the number of tregs and th cells decreased. more interestingly, overexpression of stat , which is required for th differentiation, restored the treg/th ratio, accompanied by a decrease in il- , il- , il- , and il- levels, and significant inhibition of the expression of foxp and rorγt [ ] . considering that these transcription factors are essential regulators of the differentiation and function of tregs/th cells [ , ] , li and colleagues concluded that ptx-induced increases in camp might have partially restored the treg/th balance by modulating foxp and rorγt transcription through the stat pathway. therefore, regulating the treg/th balance and the subsequent immune response through camp signalling was proposed as a feasible treatment strategy for ards [ ] . moreover, ptx was shown to inhibit tnf- production by alveolar macrophages [ , ] . because pulmonary sarcoidosis is a chronic inflammatory disease, interactions between an antigen-presenting cell and an unknown antigen are perceived by naive cd + lymphocytes (th cells) and alveolar macrophages, leading to the activation and proliferation of both cell types and the consequent release of il- , tnf-, and ifn-γ [ ] . in support of these effects, adding ptx to a systemic steroid regimen allowed for a steroid dose reduction [ ] . park et al. [ ] confirmed this steroid-sparing effect of ptx in a randomized controlled clinical trial in patients with pulmonary sarcoidosis, in which ptx improved the pulmonary diffusion of carbon monoxide and arterial blood oxygen pressure during exercise, especially in patients who were naive to steroid treatment. in addition to these anti-inflammatory properties, ptx has also been reported to suppress tissue fibrosis by blocking tgf-β and preventing the deposition of type i collagen [ , ] . several in vitro studies have shown that ptx inhibits fibroblast proliferation and extracellular matrix production [ ] [ ] [ ] , and a clinical study demonstrated that administration of ptx to obese patients decreased the plasma levels of plasminogen activator inhibitor- (pai- ) [ ] . these findings motivated lee and colleagues [ ] to test the effect of ptx administration in an experimental model of radiation-induced pulmonary fibrosis in rats, and found a decrease in the expression levels of both fibronectin and pai- . this was considered to be particularly relevant in light of evidence that pai- expression is elevated in fibrotic pathological conditions. indeed, pai- contributes to a reduction in fibrinolysis rates and a subsequent decrease in the degradation of components of the extracellular matrix, including fibronectin, leading to tissue fibrosis [ ] . since there are no clinical guidelines for the management of pulmonary fibrosis, ptx is currently recommended for the prevention and treatment of this condition [ ] . moreover, ptx was shown to prevent the development of pneumonitis in patients with breast and lung cancers [ , ] . taken together, the evidence highlighted herein strongly points to the benefits of redirecting ptx as an ethical and legal attempt in the treatment of covid- , which can help support patients in critical care and overwhelmed hospital resources in the face of this pandemic. the authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. a pneumonia outbreak associated with a new coronavirus of probable bat origin single-cell rna expression profiling of ace , the putative receptor of wuhan covid- angiotensin-converting enzyme is an essential regulator of heart function essential role for collectrin in renal amino acid transport multiple organ infection and the pathogenesis of sars organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways tissue distribution of ace 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pentoxifylline downregulates profibrogenic cytokines and procollagen i expression in rat secondary biliary fibrosis pentoxifylline attenuates tubulointerstitial fibrosis by blocking smad / -activated transcription and profibrogenic effects of connective tissue growth factor pentoxifylline inhibits normal human dermal fibroblast in vitro proliferation, collagen, glycosaminoglycan, and fibronectin production, and increases collagenase activity pentoxifylline, pentifylline, and interferons decrease type i and iii procollagen mrna levels in dermal fibroblasts: evidence for mediation by nuclear factor down-regulation pentoxifylline lowers plasminogen activator inhibitor levels in obese individuals: a pilot study pentoxifylline regulates plasminogen activator inhibitor- expression and protein kinase a phosphorylation in radiation-induced lung fibrosis pai- , fibrosis, and the elusive provisional fibrin matrix radiation-induced lung injury: assessment and management pentoxifylline in prevention of radiationinduced lung toxicity in patients with breast and lung cancer: a double-blind randomized trial randomized, placebo-controlled trial of combined pentoxifylline and tocopherol for regression of superficial radiation-induced fibrosis we would like to thank ms. alejandra maldonado for guidance and support. key: cord- - sh eksm authors: garg, m.; angus, p. w.; burrell, l. m.; herath, c.; gibson, p. r.; lubel, j. s. title: review article: the pathophysiological roles of the renin–angiotensin system in the gastrointestinal tract date: - - journal: aliment pharmacol ther doi: . /j. - . . .x sha: doc_id: cord_uid: sh eksm background: the renin‐angiotensin system (ras) is a homeostatic pathway widely known to regulate cardiovascular and renal physiology; however, little is known about its influence in gastrointestinal tissues. aim: to elicit the anatomical distribution and physiological significance of the components of the ras in the gastrointestinal tract. methods: an extensive online literature review including pubmed and medline. results: there is evidence for ras involvement in gastrointestinal physiology and pathophysiology, with all the components required for autonomous regulation identified throughout the gastrointestinal tract. the ras is implicated in the regulation of glucose, amino acid, fluid and electrolyte absorption and secretion, motility, inflammation, blood flow and possibly malignant disease within the gastrointestinal tract. animal studies investigating the effects of ras blockade in a range of conditions including inflammatory bowel disease, functional gut disorders, gastrointestinal malignancy and even intestinal ischaemia have been encouraging to date. given the ready availability of drugs that modify the ras and their excellent safety profile, an opportunity exists for investigation of their possible therapeutic role in a variety of human gastrointestinal diseases. conclusions: the gastrointestinal renin‐angiotensin system appears to be intricately involved in a number of physiological processes, and provides a possible target for novel investigative and therapeutic approaches. the renin-angiotensin system (ras) is a homeostatic pathway widely known to regulate cardiovascular and renal physiology; however, little is known about its influence in gastrointestinal tissues. to elicit the anatomical distribution and physiological significance of the components of the ras in the gastrointestinal tract. an extensive online literature review including pubmed and medline. there is evidence for ras involvement in gastrointestinal physiology and pathophysiology, with all the components required for autonomous regulation identified throughout the gastrointestinal tract. the ras is implicated in the regulation of glucose, amino acid, fluid and electrolyte absorption and secretion, motility, inflammation, blood flow and possibly malignant disease within the gastrointestinal tract. animal studies investigating the effects of ras blockade in a range of conditions including inflammatory bowel disease, functional gut disorders, gastrointestinal malignancy and even intestinal ischaemia have been encouraging to date. given the ready availability of drugs that modify the ras and their excellent safety profile, an opportunity exists for investigation of their possible therapeutic role in a variety of human gastrointestinal diseases. the gastrointestinal renin-angiotensin system appears to be intricately involved in a number of physiological processes, and provides a possible target for novel investigative and therapeutic approaches. the renin-angiotensin system (ras) plays a central role in regulating cardiovascular and renal physiology. the contemporary view of the ras has evolved from that of a simple linear pathway involving the conversion of angiotensinogen to angiotensin ii (ang ii) via a two-step process facilitated by renin and angiotensin converting enzyme (ace), to a much more complex system involving homologues of ace and multiple angiotensin peptides which play supplementary and counter-regulatory roles ( figure ). the ras was, for many years, thought of as an endocrine system with enzymes and peptides released into the systemic circulation to act on target organs. more recently, it has been recognised that most organs including the brain, kidney, heart, liver, pancreas, reproductive organs, skin and the gastrointestinal tract constitutively express all the components required to allow autonomous function of a local intra-organ ras, where it performs both paracrine and autocrine functions. table summarises the current view of the ras, the key components and their physiological and clinical effects. essentially, the relative activity of two counterbalancing pathways determines the predominant tissue effect. the proinflammatory, profibrotic pathway includes the classical ras components ace and ang ii, and renin, prorenin, chymase and neutral endopeptidase (nep, also known as neprilysin). renin, a glycoprotein derived predominantly from the juxtaglomerular apparatus in the kidney, is an aspartyl protease that cleaves the liver-derived angiotensinogen to angiotensin i. both renin and its proenzyme prorenin, which was previously considered physiologically inactive, have now been demonstrated to have independent pro-inflammatory and pro-fibrotic effects via signalling through the pro(renin) receptor (prr). the classical ras comprising the zinc metalloproteinase ace and ang ii induces vasoconstriction, salt and figure | the contemporary renin-angiotensin system (ras). ace, angiotensin converting enzyme; nep, neutral endopeptidase; am, aminopeptidase; at r, angiotensin type receptor; at r, angiotensin type receptor; at r, angiotensin type receptor; prr, (pro)renin receptor. water retention, thirst response, cardiac hypertrophy, tissue inflammation and fibrosis through the g-protein coupled seven-transmembrane domain receptor angiotensin type i receptor (at r). ang ii also stimulates adrenal gland secretion of aldosterone resulting in renal sodium and water retention. inhibition of this pathway with either ace inhibitors or at r antagonists has beneficial effects in hypertension, cardiac failure, ischaemic heart disease, diabetic nephropathy and renal fibrosis. chymase expressed in the heart and vascular wall and secreted by activated mast cells, acts as an alternative enzyme to ace to generate ang ii from ang i. - nep, a membrane bound zinc metalloproteinase with a structure distinct from ace, was discovered in the s as a key enzyme involved in the cleavage of bradykinin. , in recent years, it has been shown to also have a role in the formation of ang ( - ) from ang i, as an inactivator of atrial natriuretic peptide and in the degradation of amyloid b peptide, a protein involved in the pathogenesis of alzheimer's disease. the net effect of nep inhibition is vasodilatation and natriuresis, a property encompassed by vasopeptidase inhibitors that target both ace and nep and may have additional anti-hypertensive effects to ace inhibitors. in contrast, the alternative ras, comprising ace and ang ( - ), acting via the g-protein coupled seventransmembrane receptor mas, , has vasodilatory, antihypertensive, anti-thrombotic, cardioprotective, antiinflammatory and anti-fibrotic effects in a variety of tissues. - ace is a zinc metalloproteinase and homologue of ace, which cleaves a single amino acid from ang ii to form the heptapeptide ang ( - ). indeed, part of the clinical benefit attributed to ace inhibitors and at r blockers (arbs) may be through the diversion of the classical ras components towards ang ( - ) with subsequent mas receptor activation. [ ] [ ] [ ] the complexity of the ras is further highlighted by recent findings regarding the actions of other angiotensin peptides including angiotensin iii [ang iii, also denoted as ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) ], angiotensin iv [ang iv, also known as ang ( ) ( ) ( ) ( ) ( ) ( ) ] and the at and at receptors. ang iii is formed by cleavage of ang ii by aminopeptidase a, and ang iv results from further conversion by aminopeptidase b or n ( figure ). the at receptor (at r) has affinity for ang ii, ang iii, ang iv and ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) , and is also thought to have effects counteracting the at r and analogous to those of the mas receptor, with vasodilatory, anti-inflammatory and anti-proliferative downstream actions. previously recognised largely for an important role in foetal development, more recently, the at r has been shown to be upregulated in atherosclerotic disease, cutaneous wounds and pancreatic fibrosis, and to stimulate neurite outgrowth, a marker of neuronal regeneration. , ang iii is believed to have actions analogous to ang ii. ang iv appears to have opposing effects to ang ii, and acts predominantly via the at r and the at r, formerly known as insulin regulated aminopeptidase (irap). the greatest role of ang iv is in the central nervous system (cns), where it has a positive effect on neuronal development, learning and memory. , concept of local ras there is considerable evidence that most or all of the components of the ras are present in a variety of organs, supporting the theory that local expression and modulation of the ras play important roles in tissue homeostasis. these roles may be summarised as involving ( ) fluid and electrolyte transport, ( ) regional blood flow regulation and ( ) promoting the wound healing response, including cell proliferation, inflammation and fibrosis. some of the regional effects of the ras are listed below: (i) in the kidney, local angiotensinogen is converted by renin to ang i, which in turn is cleaved by tubular brush border ace to ang ii to facilitate sodium and fluid absorption via luminal at r. this may influence blood pressure independent of systemic ang ii levels and vascular tone. , (ii) the heart expresses renin, prr, ace, chymase, angiotensinogen, at r and at r, and these components modulate myocyte proliferation and cardiac remodelling. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] (iii) the brain has been shown to express renin, angiotensinogen, ang ii, ang iii, ang iv, ang ( - ), at r, at r and at r, with these components regulating blood pressure, fluid and electrolyte balance, thirst, maintenance of the blood-brain barrier and neuronal development including learning and memory processes. [ ] [ ] [ ] [ ] [ ] [ ] [ ] (iv) the liver expresses renin, angiotensinogen, ang ii, ace, at r, ang ( - ), ace and mas receptors, all of which are upregulated in the diseased liver. , furthermore, arbs and ang ( - ) have been demonstrated to reduce liver fibrosis in animal models. [ ] [ ] [ ] (v) in the pancreas, ang ii has been shown to inhibit glucose stimulated insulin secretion, and via at r and at r, regulates exocrine enzyme secretion and the microcirculation. [ ] [ ] [ ] (vi) a local ras has been identified and shown to be involved in tissue homeostasis in the reproductive organs, skin and even adipose tissue. [ ] [ ] [ ] [ ] [ ] [ ] detailed reviews of these and other local ras effects are published elsewhere. , , , , [ ] [ ] [ ] localisation and functionality of the ras in the gastrointestinal tract our understanding of the involvement of the ras in the gastrointestinal tract has gradually evolved over the past five decades since the formulation of the hypothesis that ang ii had a direct effect on intestinal smooth muscle in addition to an indirect effect via myenteric plexus cholinergic neurons. [ ] [ ] [ ] since then, many of the components of the ras have been identified throughout the gastrointestinal tract. an overview of the current state of knowledge is illustrated in figure . the regions of the gut are addressed separately here. most attention has been paid to the small intestine (figure a) , as outlined below: (i) ace, ace and neutral endopeptidase: ace has been shown in humans to be located in abundance on the brush border of epithelial cells and in the mesenteric microvascular endothelium. ace mrna and protein is present in large amounts in small intestinal epithelial brush border, muscularis mucosa and muscularis propria, as well as microvascular endothelium and vascular smooth muscle cells. remarkably, the highest tissue concentrations in the human body of ace and ace mrna are found in the terminal ileum, duodenum and colon. , expression of nep has been demonstrated in the rat intestinal wall, and is suppressible by administration of the combined ace/nep inhibitor omapatrilat. (ii) angiotensin receptors: at r has been localised to the epithelial brush border. the circular and longitudinal muscle layers and the myenteric plexus also strongly express at r, but the at r appears to be largely restricted to the myenteric plexus. , small vessels in the muscularis propria also express at r. , in early studies in the rat intestine, ang ii binding sites were reported to be confined to the muscularis, but subsequent reports have identified expression of at r and a lesser amount of at r in the muscularis mucosa and mucosa, including in epithelial cells. , (iii) renin: mrna for renin has been detected in the human small intestine. (iv) angiotensin peptides: ang ii has been detected in the crypt and crypt-villus junction epithelial cells. to date, the expression of angiotensinogen, ang i or ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) in the human small intestine has not been reported. however, angiotensinogen has been widely localised in the rat brush border, epithelial cells, lamina propria, muscularis mucosa, submucosal blood vessels and muscularis propria. angiotensinogen mrna has been isolated in concentrations of over one-third that of the liver in the rat mesentery, and a high level of proangiotensin- , a precursor of ang i, has been located in the rat intestine. thus, all of the required components for local production and action of ang ii appear to be present in small intestine. there is now evidence of important roles for the ras in a variety of intestinal processes: (i) bicarbonate secretion: this is stimulated by ang ii via at r and at r in the duodenum. (ii) sodium and water absorption: in the jejunum and ileum, this process appears to be modulated by ang ii in conjunction with the enteric sympathetic nervous system. [ ] [ ] [ ] when applied in low dose to rat jejunum, ang ii stimulates sodium and water absorption through at r, but in high dose, it unexpectedly inhibits absorption through at r. both ang ii and ang iii may also increase sodium and water absorption via stimulation of release of noradrenaline from sympathetic neurons, which in turn may act through adrenergic receptors on the basal surface of epithelial cells. , , (iii) glucose absorption: ang ii has also been shown to inhibit rat jejunal sodium-dependent glucose transporter (sglt )-mediated glucose uptake in vitro. (iv) digestion and absorption of peptides: both brush border ace and ace are thought to function as peptidases, allowing for mucosal digestion and absorption of peptides. , ace increases the activity of the neutral amino acid transporter b at , which is mutated in a rare amino acid deficiency disorder, hartnup disorder, clinically manifested by cerebellar ataxia and pellagra-like skin rash. (v) secretion: a role for ace in active secretion has been suggested by the observation that ace is the target for the coronavirus mediating severe acute respiratory syndrome, sars-cov. some patients with this infection suffer from watery diarrhoea, but the exact mechanism remains to be determined. there have been limited studies of ras components in the colonic wall (figure b) . by a combination of rt-pcr and immunohistochemistry, renin was found in the surface epithelium, lamina propria mesenchymal cells, microvascular walls and muscularis mucosa. at r was detected on surface epithelial cells and in crypt bases, lamina propria macrophages, myofibroblasts and mucosal vessel walls, and weak expression of at r has been found on surface epithelium, in crypts and in some mesenchymal cells. ace was also weakly expressed in parts of the surface epithelium, and more prominently in mesenteric microvascular walls, lamina propria and submucosal mesenchymal cells. ace appears to be localised to the mesenteric microvascular endothelium in the colon and is not present in the epithelium. angiotensinogen mrna has also been isolated in homogenised rat colon, but its expression has not been examined in the human colon. a more limited range of functional roles has been attributed to components of the ras in the colon than in small intestine. ang ii has been shown to increase sodium and water reabsorption in rats through nacl coupled transport. the response of circular and longitudinal muscle contraction to ang ii also suggests a role in normal colonic motility. as detailed below, the ras may also be involved in the inflammation associated with ibd, as mucosal levels of ang i and ang ii are higher in patients with active crohn's colitis compared with normal controls and patients with ulcerative colitis. components of the ras are present in the mucosal biopsy specimens of gastric antrum and body from healthy adults (figure c ). renin and angiotensinogen were both seen in lamina propria mesenchymal cells and vascular endothelial cells. at r and at r were both observed in gastric epithelium (mainly in the basal surface), lamina propria mesenchymal cells and vascular endothelium. at r were noted in a subgroup of endocrine cells in the base of antral mucosal glands, and ace and nep in vascular endothelial cells, but not in other parts of the mucosa. other investigators, however, have noted ace in fundic chief cells and mucin secreting cells of the antrum. longitudinal and circular muscle of the stomach has been demonstrated to respond in vitro to ang ii, suggesting the presence of appropriate receptors on gastric myocytes. to date, few functional or pathogenic roles have been attributed to the ras in the stomach. a role of local ras in gastric inflammation has been suggested by higher expression of at r expression in helicobacter pylori positive than h. pylori negative patients and the potentiation of ulceration in animal models by ang ii. , oesophagus immunoreactive ace, at r and at r have been found in the lamina propria microvascular walls, and at r and at r were identified in the superficial stratified epithelium and circular and longitudinal muscle of the oesophagus (figure d ). ang ii caused contraction of isolated oesophageal smooth muscle in vitro, and the at r antagonist candesartan inhibited swallow-induced peristaltic contractions in the distal oesophagus. the expression of other ras components has not been reported. the presence of the various components of the ras in the gastrointestinal tract raise the possibility that modification of this system locally may be a potential therapeutic target in a myriad of gastrointestinal diseases where current strategies are suboptimal. these include inflammatory bowel disease (ibd), gastrointestinal cancer, gut motility disorders and mesenteric ischaemia. although clinical data are sparse, results from animal models and pre-clinical studies provide support for further investigation. information relevant to ibd has arisen from studies in crohn's disease (cd) and ulcerative colitis (uc) and animal models of both ibd and other chronic inflammatory conditions. studies in patients with crohn's disease and ulcerative colitis. two components of the ras have been studied. the first is ace, which was subject to intense interest from the 's in its role in sarcoidosis and other granulomatous conditions. studies of serum ace concentrations yielded largely conflicting findings in ibd, with many studies showing reduced levels [ ] [ ] [ ] [ ] and some finding no difference. [ ] [ ] [ ] many of these studies have been limited by relatively small numbers of patients. serum ace levels are associated with ace gene insertion/deletion (i/d) polymorphisms, with higher levels seen with the dd polymorphism than id or ii. matsuda et al. showed that the ace gene polymorphism variation was similar in patients with cd and patients with uc to controls, but that serum ace levels were lower in patients with ibd after adjusting for polymorphisms. furthermore, they demonstrated that ace levels significantly increased in all of nine patients with active cd when they achieved clinical remission. a larger study involving uc patients and cd patients also found no difference in ace gene polymorphisms when compared with normal controls, but a subgroup analysis revealed a higher proportion of dd genotype in uc patients with extra-intestinal manifestations, with an odds ratio (or) of . . indeed, it is difficult to reconcile these findings into a unifying hypothesis regarding the role of ace in ibd pathogenesis. it is possible that inflammatory cytokines such as tnf-a and il- downregulate systemic endothelial ace production, whereas ang ii produced via local intestinal ace contributes to tissue inflammation. tissue ace levels have not been measured in active ibd. the other major area of investigation has focussed on angiotensin peptide levels in cd. genotype analyses have revealed a significant association of angiotensinogen- aa genotype (or . ) in a cohort of patients with ibd. this genotype results in increased production of angiotensinogen via a substitution in its gene promoter. indeed, mucosal levels of ang i and ang ii are elevated in rectosigmoid biopsies in patients with crohn's colitis compared with patients with uc or normal controls, and a significant correlation was noted between these levels and endoscopic grade of colitis. although these findings demonstrate that components of the local tissue ras probably play a part in inflammation, they do not prove any causal link in the pathogenesis of ibd. nonetheless, they suggest that inhibition of the local ras provides a potential avenue for targeting inflammation and fibrosis. studies in animal models. angiotensin converting enzyme inhibition or angiotensin receptor antagonism have been shown to produce a number of beneficial anti-inflammatory effects in rodent models of intestinal inflammation. ang ii and at a receptor expression are both upregulated in dextran sodium sulphate (dss)induced colitis, a widely studied mouse model of colitis, and inflammation was significantly ameliorated in at a receptor-deficient mice. the ace inhibitor enalaprilat, given parenterally, reduced inflammation and tnf-a, and topical enalaprilat reduced tgf-b expression and fibrosis in mice with dss-colitis. administration of the arb, valsartan, significantly reduced macroscopic inflammation, tnf-a, tgf-b and il- in mice with trinitrobenzene sulphonic acid (tnbs) induced colitis, reduced microscopic inflammation and raised il- (an anti-inflammatory cytokine secreted by regulatory t cells) in dss-colitis. , the ace inhibitor captopril reduced macroscopic and microscopic inflammation, fibrosis and tgfb mrna expression in mice with tnbs-induced colitis. homozygous deficiency of angiotensinogen protects against tnbs-induced colitis, with reduced il -b, ifn-c and greater il- and il- production than wild-type mice. animal studies have not been restricted to the classical ras pathway. nep knockout mice have been shown to have more severe colitis in response to dinitrobenzene sulphonic acid than wild-type mice, which is prevented by the administration of recombinant nep. other evidence to suggest that ras dysfunction may potentiate immune-based diseases such as ibd and provide a target for therapy comes from recent studies involving models for multiple sclerosis (ms). t helper type (th ) and type (th ) cells are strongly implicated in the pathogenesis of ibd, especially cd, and lisinopril and candesartan have been demonstrated to suppress th and th cytokine expression and induce foxp + regulatory t cells in experimental autoimmune encephalitis (eae), a mouse model of ms. other groups have shown a crucial role for ang ii and at r in eae , and murine autoimmune nephritis. the possible involvement of perturbed ras components in solid organ malignancies represents a fascinating expansion of our insight into local tissue ras. early epidemiological studies demonstrated a reduced risk of incident and fatal cancer in patients on ace inhibitors versus those on other anti-hypertensive medication. , in contrast, a recent meta-analysis reported a higher risk of lung cancer in patients taking arbs ; however, two large meta-analyses since then have reported no increased risk. , all these studies have been limited by their retrospective design. increasing in vitro and pre-clinical data suggest a protective effect of at r inhibition against cancer cell proliferation, invasion and metastasis in a variety of solid organs. in gastric cancer, at r and ang ii are expressed to a greater extent than in adjacent normal tissue. the at r and ace gene polymorphism d allele increase risk of nodal metastasis and tumour stage in patients with intestinal-type gastric cancer. in gastric cancer cell cultures, ang ii stimulates map kinase, nfjb and survivin activation, increasing proliferation. the most likely mechanism by which the ras may influence cancer biology is through increasing angiogenesis, , via increased expression of vascular endothelial growth factor (vefg) signalling. [ ] [ ] [ ] in mouse models, ace inhibition and arbs reduced colorectal cancer liver metastases and prolonged survival in peritoneal carcinomatosis. indeed, ace inhibitor use independently protected against distal metastasis in a single centre retrospective review of patients with colorectal carcinoma. also, patients treated with ace inhibitors had a non-significant trend towards reduction in risk of oesophageal adenocarcinoma in a retrospective study of the uk general practice research database. recently, there have been intriguing insights into the involvement of the ras in peutz-jeghers syndrome (pjs). shorning and colleagues have shown that in mice lkb gene deletion, which is associated with pjs, results in marked transcriptional upregulation of the renin gene ren , and also increased ace expression and ang ii production. in human pjs tumour tissue, at r was noted to be increased in stromal tissue, but reduced in the epithelium. gut motility disorders the functional role of the ras in smooth muscle contraction suggests that it might be a target in motility dysfunction. for example, the ability of ang ii blockade to inhibit contraction of oesophageal body and lower oesophageal sphincter (les) smooth muscle, together with the demonstrated reduction in the amplitude of contraction of primary peristaltic oesophageal waves and les on manometry in vivo, suggests a possible role in treatment of hypercontractile oesophageal disorders such as diffuse oesophageal spasm, nutcracker oesophagus and achalasia. furthermore, selective at r-mediated contraction of the les may be an option for treatment of gastro-oesophageal reflux disease, a condition that affects up to - % of the population. the contribution of at r to small and large intestinal muscle contractility also provides an opportunity to intervene in functional intestinal and motility disorders through at r agonism or blockade. furthermore, the role of ace and ace in intestinal fluid and electrolyte absorption suggests a potential mechanism for modulating fluid shifts across the brush border, with subsequent effects on stool consistency and frequency. the ras plays an important role in regulation of the smooth muscle tone of the mesenteric vasculature. in acute hypovolaemia and systemic sepsis, splanchnic vasoconstriction occurs as a homeostatic response to preserve cerebral and renal blood flow, predisposing the gut to ischaemia. this splanchnic response has been shown to correlate with a markedly increased expression of ang ii. furthermore, lower-body negative pressure induction in normal human volunteers has also been shown to raise serum ang ii and reduce intestinal mucosal nitric oxide production. the administration of candesartan maintained jejunal and mucosal perfusion during severe hypovolaemia in pigs and reduced mortality. , a further porcine study reported improved mucosal oxygen delivery, but not an improvement in intestinal mucosal acidosis in pigs administered candesartan during endotoxic shock. these results were replicated by tardos et al. in pigs with burn and endotoxin induced gut ischaemia. in this model, intestinal permeability and bacterial translocation were reduced with the administration of the ang ii inhibitor dup . the obvious limiting factor in applying ang ii blockade in humans at risk of mesenteric ischaemia is the potential for current arbs and ace inhibitors to cause further hypotension and kidney injury, although no adverse renal consequences were noted in one porcine study. manipulation of local gastrointestinal tract ras -potential targets and limitations as a ubiquitous system with a wide array of homeostatic roles, investigation into therapies that manipulate the ras has been extensive. apart from the well-established roles of at receptor blockade and ace inhibition in the treatment of hypertension, cardiovascular and kidney disease, new drugs targeting the mas receptor, at receptor, at r, renin and nep, as well as a recombinant ang ( - ), are under trial for various applications ( table ) . there is limited knowledge of the effect of currently available ace inhibitors and arbs on gastrointestinal function at doses employed for cardiovascular and renal effect at a molecular level. most of these drugs may result in adverse effects including nausea, diarrhoea or constipation in an idiosyncratic manner in between and per cent of patients, often noted in similar numbers of patients in the placebo arm in randomised con-trolled trials. it is likely that gastrointestinal tissue concentrations achieved by these medications at current doses are insufficient to note clinical effect. furthermore, the effect of these drugs in pathological states, like functional gut disorders or ibd, has not been described. for applicability to human gastrointestinal disease, drugs manipulating the ras will need to target the relevant areas of the gut to obtain satisfactory tissue effect without systemic adverse reactions. ideally, this will comprise delivery to the mucosa, absorption and binding of cellular receptors and sufficient first pass hepatic conversion to inactive metabolites to limit systemic effect. there is now a significant body of literature demonstrating the existence and pathophysiological relevance of local tissue renin-angiotensin systems. given the current evidence of involvement of the ras in gastrointestinal fluid and electrolyte homeostasis, smooth muscle control, inflammation and malignancy, it follows that manipulation of this system could be of benefit in a range of gi pathologies. therapies targeting the gastrointestinal ras are attractive, given their excellent safety and tolerability profile and confirmed benefits in other organs and diseases. it is hoped that the recent emergence of further experimental evidence supporting a role for the local ras in intestinal disorders will provide greater impetus for the initiation of well-conducted clinical trials in human disease. renin, (pro)renin and 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therapeutic treatments with at and at receptor antagonists and their effects on changes in the severity of pancreatitis protective effect of candesartan in experimental ischemic stroke in the rat mediated by at and at receptors beneficial effects of angiotensin ( - ) in diabetic rats with cardiomyopathy the angiotensin-( - )/mas receptor axis is expressed in sinoatrial node cells of rats impairment of cardiac function and remodeling induced by myocardial infarction in rats are attenuated by the nonpeptide angiotensin-( - ) analog ave anti-inflammatory effects of the activation of the angiotensin-( - ) receptor, mas, in experimental models of arthritis the nonpeptide ave attenuates myocardial hypertrophy as induced by angiotensin ii through downregulation of transforming growth factor-beta /smad expression ave -angiotensin-( - ) receptor agonist, inhibits atherogenesis in apoeknockout mice angiotensin-converting enzyme suppresses pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction recombinant human angiotensinconverting enzyme as a new reninangiotensin system peptidase for heart failure therapy effects of ace inhibition in the post-myocardial infarction heart comparison of vasopeptidase inhibitor, omapatrilat, and lisinopril on exercise tolerance and morbidity in patients with heart failure: impress randomised trial comparison of omapatrilat and enalapril in patients with chronic heart failure: the omapatrilat versus enalapril randomized trial of utility in reducing events (overture) blood-pressure reduction with lcz , a novel dual-acting inhibitor of the angiotensin ii receptor and neprilysin: a randomised, doubleblind, placebo-controlled, active comparator study inhibition of diabetic nephropathy by a decoy peptide corresponding to the "handle" region for nonproteolytic activation of prorenin involvement of receptor-bound prorenin in development of nephropathy in diabetic db/db mice prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin ii type a receptordeficient mice prorenin receptor blockers: effects on cardiovascular complications of diabetes and hypertension pathologic roles of prorenin and (pro)renin receptor in the eye role of nonproteolytically activated prorenin in pathologic, but not physiologic, retinal neovascularization suppression of ocular inflammation in endotoxin-induced uveitis by inhibiting nonproteolytic activation of prorenin key: cord- -uujny dm authors: lumpuy-castillo, jairo; lorenzo-almorós, ana; pello-lázaro, ana maría; sánchez-ferrer, carlos; egido, jesús; tuñón, josé; peiró, concepción; lorenzo, Óscar title: cardiovascular damage in covid- : therapeutic approaches targeting the renin-angiotensin-aldosterone system date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: uujny dm coronavirus disease (covid- ) is usually more severe and associated with worst outcomes in individuals with pre-existing cardiovascular pathologies, including hypertension or atherothrombosis. severe acute respiratory syndrome coronavirus- (sars-cov- ) can differentially infect multiple tissues (i.e., lung, vessel, heart, liver) in different stages of disease, and in an age- and sex-dependent manner. in particular, cardiovascular (cv) cells (e.g., endothelial cells, cardiomyocytes) could be directly infected and indirectly disturbed by systemic alterations, leading to hyperinflammatory, apoptotic, thrombotic, and vasoconstrictive responses. until now, hundreds of clinical trials are testing antivirals and immunomodulators to decrease sars-cov- infection or related systemic anomalies. however, new therapies targeting the cv system might reduce the severity and lethality of disease. in this line, activation of the non-canonical pathway of the renin-angiotensin-aldosterone system (raas) could improve cv homeostasis under covid- . in particular, treatments with angiotensin-converting enzyme inhibitors (acei) and angiotensin-receptor blockers (arb) may help to reduce hyperinflammation and viral propagation, while infusion of soluble ace may trap plasma viral particles and increase cardioprotective ang-( – ) and ang-( – ) peptides. the association of specific ace polymorphisms with increased susceptibility of infection and related cv pathologies suggests potential genetic therapies. moreover, specific agonists of ang-( – ) receptor could counter-regulate the hypertensive, hyperinflammatory, and hypercoagulable responses. interestingly, sex hormones could also regulate all these raas components. therefore, while waiting for an efficient vaccine, we suggest further investigations on the non-canonical raas pathway to reduce cardiovascular damage and mortality in covid- patients. coronavirus disease (covid- ) is a global pandemic caused by the new severe acute respiratory syndrome coronavirus- (sars-cov- ). compared to the previous sars-cov- and the middle east respiratory syndrome coronavirus (mers) originated in and respectively [ , ] , sars-cov- is less lethal, though it has claimed more lives than both combined due to the higher transmission rate [ ] . however, the epidemiology of sars-cov- remains largely unknown. most publications are small, single-center studies, and focused on hospitalized patients. consequently, the ability to accurately estimate infectivity, symptom burden, and non-fatal and fatal complications is limited, leading to a lack of an effective therapy. also, a substantial undocumented infection ( % of all cases) that facilitates the rapid dissemination of the virus has been documented [ ] . sars-cov- infection initiates in the respiratory system, when the s protein of its external layer binds the angiotensin-converting enzyme- (ace ) at the plasma membrane of host cells [ ] . this interaction also needs the cellular serine protease transmembrane serine protease- (tmprss ) to prime the s protein, allowing the fusion of membranes [ ] . sars-cov- might also bind to other potential receptors such as dipeptidyl peptidase- , sialoglycans, or the extracellular matrix metalloproteinase inducer (emmprin-cd ), enhancing viral infectivity [ , ] . moreover, after cleavage of the short for a disintegrin and metalloproteinase domain (adam- ), ace can be released to the circulation, keeping the ability to interact with viral particles (see below) [ ] . importantly, ace has been mainly localized in lung alveolar epithelium and gastrointestinal tract, but also in bone marrow, kidneys, spleen, myocardium, and the vasculature [ ] . its ubiquitous location explains the multi-organ injury affectation, especially dangerous in the cardiovascular (cv) system [ ] . also, high levels of plasma leukocytes and an enhanced neutrophil/lymphocyte ratio have been described in post-infection stages [ ] . both cellular and humoral immune mechanisms are responsible for this hyperinflammatory response. t-cells initiate a cytokine storm by releasing proinflammatory cytokines (i.e., tnf-α, interleukins il- β, il- , and il- , interferon gamma ifn-γ, and mcp- ), and reducing anti-inflammatory factors (i.e., il- , ifn-γ-induced protein- ; ip- , il receptors; il- rα, il- ra) [ ] . however, sars-cov- can also infect and kill t-cells and peripheral white blood cells, leading to immune deficiency [ ] . in addition, viral rna activates the innate immune system through two main major classes of pattern recognition receptors. the toll-like receptors (tlrs)- and - trigger downstream signaling effectors like nuclear factor-κb (nf-κb) [ ] , which enhances the expression of cytokines, which activate t-cells for secretion of ifn-γ and il- . also, nucleotide-binding and oligomerization domain (nod)-like receptors induce il β upregulation via the nod-, lrr-and pyrin domain-containing protein- (nlrp )-inflammasome [ ] . in turn, this complex cleaves pro-caspase- into caspase- to activate cell death by pyroptosis [ ] . consequently, the uncontrolled release of pro-inflammatory factors and the unbalanced immune response produce an aggressive inflammation and injury in peripheral tissue [ ] . around % of covid- patients exhibit a mild form of the disease, whereas % of them are presenting a severe pathology with respiratory failure, septic shock, and multi-organ dysfunction, with half of these being at extreme risk of death [ ] . the primary causes of morbidity and mortality in covid- are lung injury with acute respiratory stress syndrome (ards), thromboembolism, and heart failure or cardiac dysfunction [ , ] . also, the most prevalent comorbidities are hypertension ( . %), diabetes ( . %), and cv diseases ( . %) [ ] . the heterogeneity of responses among individuals with sars-cov- infection may depend on the development of cv pathologies [ ] . in most cases, cv abnormalities occur around two weeks after infection, suggesting that they may not be caused by a direct viral infection but rather by a hyperinflammatory reaction from hosts cells [ ] . however, due to the systemic nature of the disease and the lack of cv studies, especially histopathological examinations, the diagnosis of cv damage induced by sars-cov- is insufficient. endothelial and perivascular cells, and cardiomyocytes and cardiac macrophages, can be dramatically altered by sars-cov- , leading to irreversible consequences [ ] . both endothelial dysfunction and acute myocardial injury are central events in the cv failure of covid- patients [ ] . endothelial dysfunction is characterized by a change in the physiological actions of the endothelium towards a vasoconstrictor, proinflammatory, and prothrombic state, which frequently precede the development of cv disease [ ] . covid- has been considered as an eminently endothelial disease, though the effects of sars-cov- on vascular homeostasis are still poorly understood ( figure ). in post-mortem samples from covid- patients, a diffuse endothelial inflammation (endothelitis) with accumulation of inflammatory cells has been observed [ ] . after infection, endothelial cells become inflamed and activated by the excessive release of cytokines derived from the exaggerated immune response [ ] . then, endothelial nf-κb is activated and propagates inflammation by upregulation of a wide range of pro-inflammatory cytokines, chemokines, and adhesion molecules, promoting recruitment of leukocytes. persistent inflammation causes endothelial dysfunction and profound vascular disturbances [ ] . moreover, the alteration of cell adherent junctions produces vascular permeability and edema. of interest, some evidence suggests that endothelial cell activation might also be directly induced by sars-cov particles. the endothelium expresses high levels of ace , tmprss , and cd [ , , ] , which provide a dock-way in cells for viral infection [ ] . also, perivascular cells that communicate with endothelial cells to upregulate inflammatory responses, such as pericytes, show high ace levels and can potentially be infected, contributing to increased inflammation [ ] . indeed, post-mortem analysis revealed the presence of viral structures in vascular beds from different organs [ ] . thus, endothelial cells may propagate the inflammatory wave to the surrounding tissues, including lung and heart [ , ] . in addition, post-mortem analysis showed an increase of endothelial cell death [ ] . in covid- , endothelial dysfunction may lead to cellular apoptosis and other types of cell death, such as pyroptosis and necrosis [ , ] . the loss of cells disrupts the endothelial barrier and increases vascular leakage and protein extravasation. it also allows for the exposure of sub-endothelial collagen to circulating platelets, which initiate a pro-coagulant response [ ] . indeed, post-mortem specimens exhibited microvascular platelet-rich thrombotic depositions [ ] . venous thromboembolism in lungs is a common complication in severe covid- [ ] , and arterial thromboembolism with ischemic events in large vessels or limb have been also reported, even in young patients [ , ] . hypercoagulation and thrombosis are devastating consequences of sars-cov infection ( figure ). above % of non-survivor patients had findings compatible with disseminated intravascular coagulation (dic) [ ] . after injury or excessive activation, endothelial cells release pro-thrombotic and pro-coagulant molecules, including von willebrand factor and tissue factor, while reducing anticoagulant agents such as antithrombin-iii, protein c, or plasminogen activator inhibitor type- (pai- ) [ ] . over-expression of platelet adhesion molecules (i.e., pecam- ) and secretion of reactive oxidant species (ros) also contribute to the formation of fibrin clots. in fact, a high proportion of hospitalized patients presented elevated concentrations of d-dimer, a degradation product of cross-linked fibrin that correlates with the severity of the disease and mortality [ ] . altogether, sars-cov- may be able to induce damage of endothelium and thereby initiate platelet aggregation and consecutive vessel occlusion. finally, endothelial dysfunction shifts the vascular tone towards vasoconstriction. pro-inflammatory mediators and vasoconstrictors (e.g., angiotensin-ii (ang-ii)) stimulate intracellular signals that increase ros formation, which quench nitric oxide (no) and coenzyme tetrahydrobiopterin (bh ) as main vasodilator factors. the sustained vasoconstriction may lead to hypoperfusion, particularly relevant for pulmonary and cardiac function [ ] . hyperinflammation, dic, and vasoconstriction contribute to the massive capillary congestion, collapse, and ischemia, as observed in lung, mesentery, and heart from covid- individuals [ , ] . we believe that therapeutic stabilization of the endothelium may be essential during covid- , particularly in those patients with previous endothelial dysfunction due to comorbidities like hypertension, diabetes, and obesity [ , ] . causes endothelial dysfunction and profound vascular disturbances [ ] . moreover, the alteration of cell adherent junctions produces vascular permeability and edema. of interest, some evidence suggests that endothelial cell activation might also be directly induced by sars-cov particles. the endothelium expresses high levels of ace , tmprss , and cd [ , , ] , which provide a dockway in cells for viral infection [ ] . also, perivascular cells that communicate with endothelial cells to upregulate inflammatory responses, such as pericytes, show high ace levels and can potentially be infected, contributing to increased inflammation [ ] . indeed, post-mortem analysis revealed the presence of viral structures in vascular beds from different organs [ ] . thus, endothelial cells may propagate the inflammatory wave to the surrounding tissues, including lung and heart [ , ] . figure . covid- and the cardiovascular (cv) system. the initial step of disease begins with sars-cov- infection in the upper respiratory system, which leads to fever or cough as the main symptomatology. then, in some cases, viral infection leads to lung injury characterized by dyspnea, lung inflammation, and pneumonia, with or without associated hypoxia. later on, if the infection progress, the acute respiratory distress syndrome (ards) and extrapulmonary manifestations can figure . covid- and the cardiovascular (cv) system. the initial step of disease begins with sars-cov- infection in the upper respiratory system, which leads to fever or cough as the main symptomatology. then, in some cases, viral infection leads to lung injury characterized by dyspnea, lung inflammation, and pneumonia, with or without associated hypoxia. later on, if the infection progress, the acute respiratory distress syndrome (ards) and extrapulmonary manifestations can appear. systemic and local hyperinflammation may provoke endothelial dysfunction, vascular permeability, thrombogenesis, and altogether, acute cardiac injury characterized by arrhythmias, acute coronary syndrome, or type- myocardial infarction. also, in covid- subjects, diverse underlaying comorbidities, such as hypertension, coronary artery disease (cad), and obesity, could accelerate these events. in addition, cv cells may be directly infected by viral particles, reinforcing myocarditis and vasculitis processes. ards and cad stand for acute respiratory distress syndrome and coronary artery disease, respectively. acute cardiac injury has been reported to affect between % and % of hospitalized covid- patients, being associated with worse outcomes and mortality, even in the absence of ards [ ] . the diagnosis of cardiac injury was mainly approached by detection of specific plasma biomarkers. as early as days after infection, cardiac troponin-i (ctni) is elevated in most patients, with or without the presence of alterations in electrocardiogram and echocardiography [ , ] . cardiac damage may be secondary to lung or systemic pathologies under an inflammatory milieu [ ] (figure ). vasculitis, endothelial dysfunction, or micro-thrombosis can damage the myocardium. as a reflection of the cytokine storm, patients showed a positive correlation between ctni and inflammatory plasma biomarkers, such as d-dimer, ferritin, il- , and lactate dehydrogenase [ ] . particularly, il- was also associated with the development of cardiac fibrosis and heart failure [ ] . furthermore, these patients displayed higher concentrations of plasma creatine kinase (ck)-mb, myohemoglobin, and n-terminal pro-b-type natriuretic peptide (nt-probnp), in parallel to augmented leukocyte count, c-reactive protein, and procalcitonin [ ] . patients showed an atherosclerosis-like immune response with lower lymphocyte count but hyper-activated t-cells, mainly hla-dr + , cd + cd + /cd + , and ccr + th cd + [ ] . in this sense, though there is no evidence of acute coronary syndrome or epicardial plaque rupture, these pathologies can be developed in covid- subjects with or without previous atherosclerosis [ ] . in fact, in a cohort of hospitalized patients with st-segment elevation on electrocardiography, % of them needed coronary angiography [ ] . in addition, the presence of ace receptors on the myocardium provides a theoretical mechanism for direct sars-cov- infection [ , ] (figure ). in sars-cov- , viral rna and monocyte/lymphocyte infiltration with stromal cardiac edema were detected at the myocardium in parallel to clinical myocarditis [ ] . thus, sars-cov- could also enter into cardiac cells, leading to a hyperinflammatory response within the cardiac tissue, together with myocarditis [ ] . a recruitment of inflammatory cells typical of viral infection (ccr +/th -and cd +t-cells) has been observed in hearts from covid- individuals [ ] . however, more clinical evidence, such as the presence of viral particles inside cells or cardiomyocyte necrosis, has not been reported [ ] . furthermore, sars-cov- could infect resident macrophages that interact with conducting cells of the atrio-ventricular node expressing ace receptors [ ] . this contributes to viral spread and hyperinflammation and may alter cardiac rhythm. in covid- , patients with arrhythmias frequently suffered from myocardial injury and ischemia, hypoxia, widespread systemic inflammation, shock, electrolyte disturbances, and/or receive cardiotoxic drugs (i.e., hydroxychloroquine and antivirals). arrhythmias were developed as a consequence of systemic illness and not only due to direct effects of infection [ ] . nevertheless, the prevalence of arrhythmias varies from different reports. in a study of hospitalized patients, arrhythmias were exhibited in % of cases, whereas this percentage rose up to % when considering only patients admitted to an intensive care unit [ ] . the most common electrocardiographic finding in covid- patients was sinus tachycardia, whereas atrial fibrillation and monomorphic or polymorphic ventricular tachycardia were the most deleterious arrhythmias [ ] . atrial arrhythmias were, however, more frequent among patients requiring mechanical ventilation [ ] . finally, acute cardiac injury may be caused by stress cardiomyopathy as a result of hypoxia, increased metabolic demands, fever, or hypotension in the presence of severe infection and hyperinflammation [ ] . the presence of type- myocardial infarction secondary to a mismatch between oxygen supply and metabolic demands was described in severe infection with sepsis or ards associated with hypoxia [ , ] . altogether, the presence of myocarditis, arrhythmias, stress cardiomyopathy, myocardial infarction, inflammation, or vasculitis and endothelial dysfunction may favor the generation of myocardial dysfunction and, in turn, heart failure [ ] (figure ). remarkably, heart failure has been described in % of survivors and in % of non-survivors in a population of covid- patients [ ] . the raas is a complex system that plays an important role in the control of cv functions. its overstimulation can increase vessel contraction and activate pro-hypertrophic and pro-fibrotic responses of cv cells [ ] . the raas plays a crucial role in immune functions by regulating pro-and anti-inflammatory molecules, participating in cell recruitment. in the canonical pathway, the octapeptide hormone ang-ii is produced by a two-step cleavage process from angiotensinogen through renin and angiotensin convertase enzyme (ace). to a lesser extent, ang-ii can also be converted by aminopeptidase-a to ang-iii, which conserves similar properties to the octapeptide [ ] . ang-ii (and ang-iii) actions are mainly mediated by two distinct g protein-coupled angiotensin receptors, at r and at r. in the cv system, at r overactivity has been associated with the development of several pathological conditions, including hypertension, vascular inflammation, and remodeling, atherosclerosis, and heart failure [ ] . stimulation of at r triggers nf-κb to upregulate proinflammatory and pro-coagulant factors by resident cells and endothelial cells. moreover, this ace/ang-ii/at r axis also induces epithelial to mesenchymal transformation, which enhances pro-inflammatory factors and decreases endothelial permeability [ ] . in the non-canonical axis of the raas, an ace -dependent deletion of ang-ii produces another raas peptide, termed ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) . this heptapeptide can also be synthesized from ang-i by neprilysin, or by degrading ang-i into ang-( - ) and then to ang-( - ) by sequential actions of ace and ace [ ] . importantly, this secondary axis can counter-regulate many actions provoked by the ace/ang-ii/at r. in fact, ang- - bind at r to produce vasodilatory and anti-inflammatory outcomes, and ang-( - ) via mas receptor (masr) (or mas-related receptor, mrg-d) reduces inflammation and blood pressure [ ] . this ace /ang-( - )/masr arm also promotes anti-hypertrophic and anti-fibrotic factors by diminishing mapk-and tgfß-dependent actions, while it stimulates vasoactive prostaglandins and reduces senescence and redox imbalance [ ] . ang-( - ) enhances no production by activation of the pi k/akt/enos pathway and increases ppar and src homology region- domain-containing phosphatase (shp)- [ ] . parallelly, ang-( - ) protects ace-degradation of bradykinins, which possess potent effects on endothelium-dependent vasodilatation, vascular permeability, and blood pressure control. interestingly, stimulation of no by ang-( - ) may involve downstream interactions with the bradykinin b receptor, at r, or at r, suggesting a complex interaction of the ace /ang-( - )/masr pathway with other receptor systems [ ] . in this line, degradation of ang-( - ) is governed predominantly by the amino-terminal catalytic domain of ace, which catalyzes ang-( - ) in ang-( - ) [ ] . altogether, both overstimulation and imbalance of the raas can be associated with cv responses observed in covid- patients, including hypertension, inflammation atherosclerosis, thrombosis, and myocarditis. both the cv and systemic ace/ace ratio could be crucial in the evolution of covid- individuals, favoring either the cardiotoxic ace/ang-ii/at r axis or the cardioprotective ace /ang-( - )/masr pathway [ ] . therefore, an adequate equilibrium of these axes in specific tissues and along the different phases of covid- could ameliorate the severity of disease and mortality in affected subjects. furthermore, the influence of sex hormones on the raas may be of considerable interest for covid- patients. the components of the circulating raas as well as those of tissue raas are markedly affected by sex hormones, mainly androgens (i.e., testosterone), estrogen (i.e., β-estradiol), and progesterone [ ] . in general, testosterone seems to increase renin levels and ace activity, while estrogen elevates angiotensinogen concentrations, decreases renin, ace, at r, and aldosterone, and activates at r, masr, and ang-( - ) [ ] . also, progesterone competes with aldosterone for mineralocorticoid receptor. all these actions explain some of the differences between the cv systems of men and women [ ] . in this context, epidemiological data showed that men are more prone to die ( - % of cases) of sars-cov- infection [ ] . a meta-analysis of studies ( , patients from different countries) showed an association between the male sex and covid- fatality [ ] . in covid- , androgens may increase the levels of ace and tmprss and potentially favor viral uptake into the cells [ ] . in contrast, estrogen could trigger different effects on ace levels according to tissue. estrogen increased ace in cardiac cells [ ] but reduced this expression in airway epithelial cells [ ] . in any case, ace is located on the x-chromosome and thus, in women, more ace could be free to catalyze ang-ii into ang-( - ) after sars-cov- infection [ ] . in addition, androgens generally suppress the inflammatory responses by decreasing the activity of the peripheral blood mononuclear cells, as well as the release of inflammatory factors and cytokines (il- β, tnfα, ifn-γ) [ ] . in contrast, estrogen can robustly stimulate the innate, humoral, and cellular immunity but in a more balanced and adaptive fashion to the cytokine storm [ ] . therefore, the raas can be differentially modulated in males and females affected by covid- , leading to distinctive cv consequences. further research is needed to unveil the potential regulation of the raas components by sex hormones on cv cells during covid- development, in men and women, before and after andropause or menopause, respectively. after sars-cov- binding to ace , the availability of ace to produce ang-( - ) could be decreased and the overactive raas may shift to the deleterious ace/ang-ii/at r arm [ ] . interestingly, ace inhibitors (acei) are widely prescribed as maintenance therapy in several chronic cv diseases, including arterial hypertension, diabetic cardiomyopathy, and heart failure [ ] . although ace shares - % identity and % homology with ace [ ] , acei can specifically inhibit ace and the subsequent ang-ii release, likely leading to ace upregulation and ang-( - ) formation [ ] . similarly, the angiotensin receptor blockers (arb) effectively block at r, antagonizing the main ang-ii actions and exhibiting protective pleiotropic effects against hypertension and cv inflammation, fibrosis, and thrombosis [ ] . like acei, arb could also rise ace levels, thus augmenting ang-( - ) levels. indeed, arb attenuated inflammation, oxidative stress, and blood pressure through stimulation of the ace /ang( - )/masr in experimental myocarditis and human hypertension [ , ] . it was originally suggested that elevation of ace might favor sars-cov- infection and replication in covid- patients with underlying cv disease and acei/arb treatment [ ] . however, the european society of cardiology and the american heart association advise against stopping these maintenance treatments in covid- , especially when hypertension or heart failure were present [ ] . ace internalization is dependent on heterodimerization with at r (under activation with ang-ii), and thus, both acei and arb may prevent ace -mediated viral internalization and consequent propagation [ ] (figure ). in addition, increasing evidence supports advantageous effects of both therapies against cv damage in covid- . after more than twenty clinical trials, the available data indicate that acei and arb decrease the viral load, avoid peripheral t cell depletion, and reduce plasma il- , crp, and procalcitonin levels [ ] [ ] [ ] . acei and arb could diminish the severity of covid- , mainly in the hyperinflammatory phase or in subjects with previous cv failures (i.e., hypertension) ( figure ). further research is required to analyze whether short-and long-term periods with these therapies may trigger different consequences in specific tissues. in fact, a prolonged use of arb increased ace expression in kidney, thought this was not maintained in cv cells [ ] . inhibit ace and the subsequent ang-ii release, likely leading to ace upregulation and ang-( - ) formation [ ] . similarly, the angiotensin receptor blockers (arb) effectively block at r, antagonizing the main ang-ii actions and exhibiting protective pleiotropic effects against hypertension and cv inflammation, fibrosis, and thrombosis [ ] . like acei, arb could also rise ace levels, thus augmenting ang-( - ) levels. indeed, arb attenuated inflammation, oxidative stress, and blood pressure through stimulation of the ace /ang( - )/masr in experimental myocarditis and human hypertension [ , ] . it was originally suggested that elevation of ace might favor sars-cov- infection and replication in covid- patients with underlying cv disease and acei/arb treatment [ ] . however, the european society of cardiology and the american heart association advise against stopping these maintenance treatments in covid- , especially when hypertension or heart failure were present [ ] . ace internalization is dependent on heterodimerization with at r (under activation with ang-ii), and thus, both acei and arb may prevent ace -mediated viral internalization and consequent propagation [ ] (figure ). in addition, increasing evidence supports advantageous effects of both therapies against cv damage in covid- . after more than twenty clinical trials, the available data indicate that acei and arb decrease the viral load, avoid peripheral t cell depletion, and reduce plasma il- , crp, and procalcitonin levels [ ] [ ] [ ] . acei and arb could diminish the severity of covid- , mainly in the hyperinflammatory phase or in subjects with previous cv failures (i.e., hypertension) ( figure ). further research is required to analyze whether short-and long-term periods with these therapies may trigger different consequences in specific tissues. in fact, a prolonged use of arb increased ace expression in kidney, thought this was not maintained in cv cells [ ] . of pro-oxidation, pro-inflammation, pro-thrombosis, pro-fibrosis, and vasoconstriction. however, administration of acei or arb drugs may block ang-ii actions, unbalancing the raas toward the ace /ang-( - )/masr axis, which conserves cardioprotective properties. also, in infected cells, acei and arb could reduce at r heterodimerization with ace , lessening internalization of the ace -sars-cov- complex and viral replication. ang stands for angiotensin. nep and ace mean neprilysin, and angiotensin converting enzyme, respectively. masr and at r stand for mas receptor and angiotensin receptor type i, respectively. acei and arb mean ace inhibitors and angiotensin receptor blockers, respectively. the full-length ace is highly expressed in the cellular plasma membrane of several tissues, while its extracellular domain generated by proteolytic cleavage can be found in urine and rarely in blood [ ] . interestingly, this soluble isoform can also be a receptor for the s-protein of sars-cov- , acting as a neutralizer [ ] . thus, an increase of plasma ace may reduce sars-cov- infectivity and covid- evolution (figure ). in fact, the administration of recombinant human (rh) ace is being tested against ards and acute lung injury [ ] . more data about biologic, physiologic, and clinical actions are expected from a new randomized clinical trial, where rhace is intended to block viral entry and replication [ ] . in the cv system, by using engineered human blood vessel organoids, rhace was able to reduce the early infection of sars-cov- [ ] . moreover, increasing plasma ace may also help to balance the raas system toward the full-size ace isoform. in fact, infusion of rhace rapidly ( h) triggered high levels of plasma ang-( - ) and ang-( - ), while it diminished ang-ii and il- concentrations [ , ] . also, in human explanted hearts from patients with heart failure, administration of rhace normalized ang-ii and augmented ang-( - ) and ang-( - ) levels (see below) [ ] . ang-( - ) hydrolyzes more slowly than ang-( - ) and could also encourage cardioprotective actions via at r [ ] . nevertheless, more studies are required to assess the expression and regulation of ace in other tissues, like liver and intestine, during the different stages of covid- . furthermore, some underlaying comorbidities such as smoking and pulmonary disease can upregulate ace levels in airways, and these levels also increase with advanced age [ ] . besides, type-i and type-iii ifn can upregulate ace in human airway epithelial cells, potentially contributing to an increased sars-cov- binding in upper respiratory tract [ ] . however, ifn also promotes tissue-defending properties against viral invasion, and ace could be protective for the cv system by counterbalancing the ang-ii actions [ ] . whether ifn increases soluble ace in circulation is unknown. nevertheless, beneficial or detrimental effects of ifn may depend on the cell type, stage of infection, presence of co-morbidities, and host sex and age [ ] . more preclinical and clinical data are required to clarify ifn's role in the pathogenesis of covid- and in its potential treatment. in this line, the ace gene locates in chromosome xp and includes exons with a significant number of polymorphisms. remarkably, an association of ace single nucleotide polymorphisms (snps) with higher susceptibility and prognosis to covid- or to its related cv pathologies has been postulated [ , ] . four ace variants termed rs , rs , rs , and rs , changed amino-acids , , , and respectively, and presented reduced interaction with the s-protein of sars-cov- [ ] . similarly, in silico analysis predicted variations in specific amino-acids such as and (n s and d n, respectively) that could also induce lower interactions with s-protein [ , ] . in addition, changes in position and (leu val and pro his, respectively) could diminish the internalization of the s-ace complex into the host cells [ ] . in contrast, alterations of other amino-acids such as and (s p and h r, respectively) may trigger higher binding to s-protein [ , ] . therefore, cellular weakness toward sars-cov- could be modified with genetic edition of ace sequence [ ] . in addition, other snps in ace have been associated with common cv pathologies of covid- . for instance, rs and rs correlated with increased ldl-c, cholesterol, and triglycerides [ ] [ ] [ ] (table ) , and rs and rs variants associated with decreased hdl-c. development of t dm was linked to the presence of rs and rs [ ] , while rs and rs showed higher risk of ventricular hypertrophy in women [ ] . furthermore, the rs variant was associated with atrial fibrillation in men [ ] , and rs and rs correlated with ischemic stroke [ , ] . intriguingly, both rs and rs were also concomitant with a reduction of circulating ang-( - ) [ ] (table ) . again, these snps could be corrected by genetic tools to avoid cv complications in covid- subjects. additionally, they may be useful to detect more vulnerable covid- patients with higher risk of cv damage. table . ace variants and related cv alterations. specific snps of ace can associate with abnormalities in lipid profile (total cholesterol (tc), high-density lipoprotein-cholesterol (hdl-c), low-density lipoprotein-cholesterol (ldl-c), or triglycerides (tg)), in blood pressure (hypertension or elevation of systolic blood pressure (sbp)) and cv injury (ischemic stroke, ventricular hypertrophy, or atrial fibrillation), and/or t dm. other variants have also correlated with decreased levels of plasma ang-( - ) (rs and rs ). all these alterations may worsen covid- evolution. a, g, c, and t stand for adenine, guanine, cytosine, and thymine, respectively. however, ifn also promotes tissue-defending properties against viral invasion, and ace could be protective for the cv system by counterbalancing the ang-ii actions [ ] . whether ifn increases soluble ace in circulation is unknown. nevertheless, beneficial or detrimental effects of ifn may depend on the cell type, stage of infection, presence of co-morbidities, and host sex and age [ ] . more preclinical and clinical data are required to clarify ifn's role in the pathogenesis of covid- and in its potential treatment. - )). on the other hand, some ang-( - ) mimetics (cyclic ang-( - ), hpβcd-ang-( - ), npa , txa ) and masr agonists (ave , cgen- , cgen- ) could also trigger protective actions for the cv system in covid- subjects. ang and ace stand for angiotensin and angiotensin converting enzyme, respectively. sace and nep stand for soluble ace and neprilysin, respectively. masr and at r stand for mas receptor and angiotensin receptor type-ii, respectively. finally, hpβcd-ang-( - ) means hydroxypropyl β-cyclodextrin-ang-( - ). ang-( - ) can be used as a therapeutic agent of the raas that preserves ace expression and activity. ang-( - ) can minimize inflammatory tissue damage and disease by reduction of cytokine release and leukocyte recruitment. thus, we [ ] and others [ , ] have recently suggested that activation of masr can be considered a valid treatment for the deleterious responses prompted by sars-cov- ( figure ). ang-( - )/masr induced cardioprotective actions against hypertension, atherosclerosis, diabetes, heart failure, and stroke by potential downregulation of pi k/akt, p mapk, and nf-κb pathways [ ] . since ang-( - ) possesses a short half-life in plasma, alternative ang-( - ) mimetics (cyclic ang-( - ), hpβcd-ang-( - ), npa , txa ) and masr agonists (ave , cgen- , cgen- ) have been successfully tested in experimental models against oxidation, thrombogenesis, fibrosis, inflammation, endothelial function, and high blood pressure [ , ] (figure ). in humans, hpβcd-ang-( - ) (hydroxypropyl β-cyclodextrin-ang-( - )) also improved the recovery from a supramaximal physical exercise [ ] , and npa (a fusion peptide of bnp (b-type natriuretic peptide) and ang-( - )) reduced blood pressure and cardiac uploading [ ] . in turn, txa has been catalogued as an orphan drug by the food and drug administration (fda) for the treatment of pulmonary arterial hypertension, while is being tested in the txa covid- clinical trial to prevent multi-organ failure in patients with moderate to severe covid- [ ] . another trial is evaluating the potential effect of the ang-( - )-derived plasma on mortality of covid- subjects [ ] , whereas in the angiotensin-( , ) treatment in covid- (atco) trial [ ] , the efficacy, safety, and clinical impact of intravenous ang-( - ) infusion in patients with ards will be analyzed. unfortunately, the cv pathophysiology is not being specifically studied in these trials. we believe that ang-( - ) may confer cardioprotective actions in covid- patients with and without underlying cv failures by attenuating the hyperinflammatory and hypercoagulable state. cv pathologies are the most prevalent comorbidities during covid- and major causes of morbidity and mortality in patients, even in the absence of ards. cv injury may result from lung or systemic pathologies or likely, after direct infection by sars-cov- . current clinical trials based on antivirals or immunomodulators are aimed to reduce infectivity or related-systemic anomalies. although their effects on the cv system are not being evaluated, potential deleterious actions might be found (i.e., by antivirals and hydroxychloroquine). while waiting for effective anti-sars-cov- vaccines, we consider that restoring an adequate balance between the ace/ang-ii/at r and ace /ang-( - )/masr axes of the raas could provide positive therapeutic outcomes. acei and arb can be safe and may help to reduce hyperinflammation and viral propagation. soluble ace might neutralize viral particles in plasma and increase the levels of cardioprotective ang-( - ) and ang-( - ) peptides. moreover, gene edition on specific snps for ace could reduce viral infectivity and/or related cv complications, preserving the biological activity of enzymes. in this context, local and systemic activation of masr by specific stimuli (i.e., txa , ave ) might counterbalance at r-associated actions, such as hypertension, inflammation, and coagulation. also, sex should be taken into account when designing and analyzing clinical trials of covid- , and sex differences may reveal novel therapeutic approaches such as estrogen-related compounds and androgen receptor antagonists. finally, a potential combination of these strategies could also be tested to better combat both the infective and hyperinflammatory phases of the disease. the authors declare no conflict of interest. nod-, lrr-and pyrin domain-containing protein- nod nucleotide-binding and oligomerization domain nt-probnp n-terminal pro-b-type natriuretic peptide pai- plasminogen activator inhibitor type- raas renin-angiotensin-aldosterone system ros reactive oxidant species sace soluble ace sars-cov- severe acute respiratory syndrome coronavirus- sars-cov- severe acute respiratory syndrome coronavirus- snps single nucleotide polymorphisms tc total colesterol tg triglycerides tmprss serine protease 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susceptibility to sars-cov- , hypertension, multi-organ failure, and covid- disease outcome ace gene variants may underlie interindividual variability and susceptibility to covid- in the italian population the expression and polymorphism of entry machinery for covid- in human: juxtaposing population groups, gender, and different tissues molecular simulation of sars-cov- spike protein binding to pangolin ace or human ace natural variants reveals altered susceptibility to infection human ace receptor polymorphisms predict sars-cov- susceptibility. biorxiv a crispr-cas system designed to introduce point mutations into the human ace gene to weaken the interaction of the ace receptor with the sars-cov- s protein association of ace polymorphisms with susceptibility to essential hypertension and dyslipidemia in xinjiang, china correction: association of ace genetic polymorphisms with hypertension-related target organ damages in south xinjiang correlation of angiotensin-converting enzyme gene polymorphisms with stage hypertension in han chinese ace polymorphisms associated with cardiovascular risk in uygurs with type diabetes mellitus hypertension and hypertensive left ventricular hypertrophy are associated with ace genetic polymorphism polymorphisms of angiotensin-converting enzyme gene confer a risk to lone atrial fibrillation in chinese male patients a relationship between genetic variants of ace gene and circulating levels of ace and its metabolites substituting angiotensin-( - ) to prevent lung damage in sars-cov- infection? circular urgent need for evaluating agonists of angiotensin-( - )/mas receptor axis for treating patients with covid- role of the ace /angiotensin - axis of the renin-angiotensin system in heart failure eccentric overload muscle damage is attenuated by a novel angiotensin-( - ) treatment synthesis, and actions of an innovative bispecific designer peptide key: cord- - k eb rk authors: ogunlade, blessing; guidry, jessie j.; mukerjee, snigdha; sriramula, srinivas; lazartigues, eric; filipeanu, catalin m. title: the actin bundling protein fascin- as an ace -accessory protein date: - - journal: cell mol neurobiol doi: . /s - - -x sha: doc_id: cord_uid: k eb rk we have previously shown that angiotensin-converting enzyme (ace ), an enzyme counterbalancing the deleterious effects of angiotensin type receptor activation by production of vasodilatory peptides angiotensin (ang)-( – ) and ang-( – ), is internalized and degraded in lysosomes following chronic ang-ii treatment. however, the molecular mechanisms involved in this effect remain unknown. in an attempt to identify the accessory proteins involved in this effect, we conducted a proteomic analysis in ace -transfected hek t cells. a single protein, fascin- , was found to differentially interact with ace after ang-ii treatment for h. the interactions between fascin- and ace were confirmed by confocal microscopy and co-immunoprecipitation. overexpression of fascin- attenuates the effects of ang-ii on ace activity. in contrast, downregulation of fascin- severely decreased ace enzymatic activity. interestingly, in brain homogenates from hypertensive mice, we observed a significant reduction of fascin- , suggesting that the levels of this protein may change in cardiovascular diseases. in conclusion, we identified fascin- as an ace -accessory protein, interacting with the enzyme in an ang-ii dependent manner and contributing to the regulation of enzyme activity. the renin-angiotensin system (ras) represents one of the most important therapeutic targets for the treatment of cardiovascular diseases. during the last years, angiotensinconverting enzyme (ace ) has been shown to contribute to the maintenance of normal cardiovascular function. its major role includes hydrolysis of angiotensin (ang)-i and ang-ii to vasodilatory peptides ang-( - ) and ang-( - ), respectively (donoghue et al. ; vickers et al. ; xu et al. ) . ang-( - ) and ang-( - ) acting putatively through ang-ii type receptor (at r) and mas receptor (mas r) (filipeanu and lazartigues ; karnik et al. ; mendoza-torres et al. ) attenuate the proliferative and contractile effects of at r activation by ang-ii and ultimately opposing the development of hypertension (xu et al. (xu et al. , . recently, ace was also identified as a "receptor" for the sars-cov- virus responsible for the covid- pandemic. ace contains a single hexxh zinc-binding domain which is homologous to one of the active sites of ace and shares % overall identity to ace but its enzymatic activity is not sensitive to typical ace inhibitors (donoghue et al. ; vickers et al. ) . overexpression of ace has been shown to prevent the development of hypertension (feng et al. (feng et al. , , whereas its downregulation leads to chronic increase in blood pressure levels (deshotels et al. ; xu et al. ) . ace can also be shed and enter the circulation under action of metalloproteases such as adam (de queiroz et al. ; xu et al. ) . however, hydrolysis of ang-ii is mediated in large part by cellular ace (mukerjee et al. ; pedersen et al. ) and requires the carboxypeptidase to be present on the cell surface (deshotels et al. ). our group originally demonstrated that ang-ii induces ace internalization and degradation into lysosomes through an ang-ii type receptor (at r)-dependent mechanism and this process leads to a decrease in the expression levels and activity of the enzyme (deshotels et al. ). however, the molecular mechanisms controlling ace trafficking to lysosomes remain unclear. intracellular protein trafficking is a highly regulated process controlled by interactions with specific accessory proteins (also named molecular chaperones) (ciruela et al. ; filipeanu ; rodemer and haucke ) . indeed, at r-associated protein (atrap) was shown to interact with at r (lopez-ilasaca et al. ) and microtubuleassociated tumor suppressor gene (mtus )/ at r interacting protein (atip) with at r (bozgeyik et al. ) . interestingly, at least one mtus /atip isoform, atip a was reported to be co-localized with microtubules, which form the cell cytoskeleton (bozgeyik et al. ) . no such proteins are yet known for ace and ace , but because both enzymes are internalized from plasma membrane to endosomes (deshotels et al. ; lucero et al. ) , the availability of such accessory proteins can be predicted. identification of these binding partners may offer additional pharmacological and therapeutic tool to target ras activity and sars-cov- infection. therefore, the aim of the present work was to identify and characterize accessory proteins involved in ace internalization. plasmids used in this study were obtained as follows: human gfp-tagged ace was from origene, human fascin- (fusionred-fascin-c- ) was a gift from michael davidson (addgene plasmid # ), human ha-tagged at r was a gift from dr. guangyu wu (medical college of georgia). cell culture medium, antibiotics, lipofectamine , and fetal bovine serum (fbs) were from thermofisher; normal donkey serum was from sigma-aldrich; antibodies against ace (sc- ), gfp (sc- ), β-actin (sc- ) and fascin- (sc- ), as well as lentiviral shrna particles against fascin- (sc- -v) were obtained from santa cruz biotechnology. the α-tubulin antibody (t ) and deoxycorticosterone acetate (doca) were from sigma-aldrich. hek t cells (atcc crl- ) were used in this study between passage number and . hek t were cultured in dulbecco's modified eagle's medium containing % fetal bovine serum, units/ml penicillin, and μg/ml streptomycin. the cells were plated in -well plates and transfected at ~ % confluency with . µg of ace -gfp, . µg at r-ha, and/or . µg fascin- using lipofectamine reagent in dulbecco's modified eagle's medium with no antibiotics and no fetal bovine serum. after h, the cells were cultured in full medium for h, serum starved for another h before performing the experiments. for confocal microscopy, hek t cells were grown on coverslips pre-coated with poly-l-lysine in -well plates and transfected. the cells were fixed with a % paraformaldehyde/ % sucrose mixture in pbs for min and stained with , -diamidino- -phenylindole for min. for plasma membrane staining, cells were incubated with wga (thermofisher) prior to fixation. the cells were permeabilized in pbs and blocked with normal donkey serum. the cells were then incubated with antibodies dilution of : overnight at °c. after washing with pbs ( × min), the cells were incubated with alexa fluor -labeled secondary antibody ( : dilution) for h at room temperature. fluorescent images of fixed samples were captured using a nikon ti-e-pfs inverted spinning-disk confocal microscope equipped with a × . na plan apo lambda objective. transfected cells were collected in loading buffer ( x laemmle buffer) then samples were separated by % sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene fluoride (pvdf) membranes. the membranes were then exposed to specific primary and secondary antibodies then protein levels were visualized using licor digital blot scanner (licor bioscience) and quantified using licor software. for mouse samples, hypothalamic proteins ( - μg) were processed for western blotting as previously described, (xia et al. ) using a mouse anti-fascin monoclonal primary antibody (cell signal technology s, : ) and a goat anti-mouse igg, hrp conjugate (sigma-aldrich a , : ) as secondary antibody. α-tubulin (sigma-aldrich t , : , ) was used as a loading control. the cells were lysed in a co-immunoprecipitation buffer ( mm tris-hcl, ph . , mm nacl, % np- , . % sodium deoxycholate, . % sodium dodecyl sulfate and complete mini protease inhibitor cocktail) two days after transfection. the samples were centrifuged ( , rpm) and the supernatant was collected and subsequently incubated with μl of protein a/g sepharose at °c overnight, followed by incubation with anti-gfp antibodies ( μg/mg protein) at °c for h. the protein complexes were recovered with μl protein a/g sepharose and further processed by western blotting. transfected hek t cells were extracted in ace reaction buffer ( . % triton x- in ace reaction buffer containing m nacl, . mm zncl , mm trishcl and μm mca-yvadapk (dnp, from anaspec inc, freemont, ca) and were sonicated × s and centrifuged at , ×g at °c for min. the supernatant was recovered, and the protein concentration was determined by bradford method. each reaction used μg of cell lysates. fluorescence emission was detected at nm, after excitation at nm in a biotek cytation spectrometer, and the slope of fluorescence development was calculated between and min of incubation. blank values were obtained with extraction buffer only and were subtracted from the hydrolysis rates of the sample extracts. hydrolysis rates were quantified as fluorescence units per minute per amount of protein and are reported as percentage of the values obtained in control (untreated) cells. the control values in ace -transfected cells amounted to , ± arbitrary units (a.u.). the cells were transfected and processed as above, followed by treatment with ang-ii for h. cellular lysates were obtained as in co-immunoprecipitation experiments and subsequently two-dimensional difference gel electrophoresis labeling and analysis was performed as previously described (filipeanu ) . fluorophore-labeled protein gels were scanned using a typhoon variable mode imager (ge healthcare, piscataway, nj) at μm resolution. cydye (ge healthcare) difference gel electrophoresis labeling was detected using the following wavelength settings: cy , excitation nm, emission nm; cy , excitation nm, emission nm; and cy , excitation nm, emission nm. spot detection and quantification were performed using the decyder differential analysis software dia, version . (ge healthcare) and individual gels were imported into decyder biologic variation analysis for t test analysis. spots of interest were determined to have a difference greater than % or a t test p value of less than . . gels were then post stained with sypro ruby (bio-rad), and images were captured again using the typhoon imager (ge healthcare). spots of interest were excised and trypsin (promega, madison, wi) digested using the automated spot handling workstation (ge healthcare). the mass spectrometry (ms) data were acquired using a thermo ltq-xl linear ion trap mass spectrometer (thermo fisher scientific, waltham, ma) coupled to an eksigent nanolc (dublin, ca). peptide samples were loaded onto a dionex c pepmap trap column with dimensions of μm (i.d.) × mm (dionex, sunnyvale, ca) and were separated by a reversed-phase c picofrit emitter with dimensions of μm (i.d.) × cm (bed length) with μm tip size (new objective, woburn, ma). peptides were loaded at nl/min using a mobile phase of % acetonitrile and . % formic acid and then eluted using a gradient of - % acetonitrile and . % formic acid over min, with a ramp to % acetonitrile and . % formic acid for min, and finally a ramp to % acetonitrile and . % formic acid for min. a top five data-dependent scan strategy was used. the ms scan range was between m/z and . the top most abundant peptides in this ms scan were chosen for ms/ms. the ms/ms parameters were the following: isolation window set to da, % relative collision energy, dynamic exclusion enabled with repeat count set to , repeat duration of s, and an exclusion size of with an exclusion duration of s. the ms data were then analyzed by proteome discoverer . (thermo fisher scientific) against the human swissprot database (https :// www.unipr ot.org/prote omes/). c bl /j male mice ( - weeks old, n = /group) were anesthetized with isoflurane ( %) and implanted subcutaneously with a doca ( mg/g body weight) pellet and switched to a % nacl drinking solution, as described previously (sriramula and lazartigues ) . after weeks, mice were euthanized and the hypothalamus was dissected for protein extraction and western blotting analysis, as mentioned above. all procedures conformed to the national institutes of health guide for the care and use of laboratory animals and were approved by the louisiana state university health sciences center (# ) institutional animal care and use committee. each experiment was run in triplicate and repeated at least in three different passages. data are presented as means ± s.e.m. statistical analysis was performed using one-way anova, followed by the tukey post-analysis and p < . was considered as statistically significant. to identify new proteins interacting with ace , we performed a proteomic experiment in ace -transfected hek t cells. from the two-dimensional difference gel electrophoresis, seven spots appeared different between control and ang-ii-treated cells ( nm, h, fig. a ). however, after trypsin digestion and mass spectrometry analysis, we found that all these spots identified a single protein, namely fascin- (fig. b) . the different spots represent electronegative shifts caused by isoelectric focusing point changes due to post-translational modifications and most likely represent phosphorylation differences. known phosphorylation sites for fascin- include serine- , serine- , serine- , serine- , threonine- , and threonie- . to confirm that indeed ace interacts with fascin- , we first performed co-localization experiments by confocal microscopy. in hek t cells co-transfected with gfp-tagged ace , ha-at r, fusionred-fascin- , we found that in control conditions ace and fascin- are colocalized at the plasma membrane level (fig. a, top panel) . however, after treatment with ang-ii ( nm, h), both proteins displayed perinuclear localization, but very little, if any, co-localization. no co-localization between ace and fascin- was observed in absence of at r transfection (data not shown). co-immunoprecipitation experiments demonstrated interactions between ace and fascin- (fig. b) . ang-ii treatment ( nm, h) did not have an effect on ace /fascin- interactions when immunoprecipitation was performed in cells without overexpression of fascin- . however, in fascin- -overexpressing cells, ang-ii treatment appears to decrease ace /fascin- interactions (fig. b , last two lanes). next, we investigated if changes in cellular levels of fascin- are modulating ace activity and expression levels. in untransfected hek t cells, ace activity was below detection limit ( ± a.u., n = ). in hek t cells transfected only with ace plasmid, ace activity in control conditions amounted to , ± a.u., and, in agreement with our previous report (deshotels et al. ), ang-ii treatment ( nm, h) did not have any effect ( , ± a.u., n = ). however, in cells underlined peptides are those that were obtained by trypsinization and subsequent mass spectrometry co-transfected with at r, ang-ii treatment ( nm, h) induced a significant reduction of ace activity ( . ± . % from the values obtained in the untreated cells which was , . ± . a.u. n = , fig. a , p < . ). overexpression of fascin- did not change basal ace activity ( . ± . %, fig. a ), but prevented the effects of ang-ii on this parameter ( . ± . %, fig. a ). when we tested the effects of a longer ang-ii treatment ( nm, h), which we have shown to induce ace degradation in lysosomes (carvalho-galvao et al. ; deshotels et al. ) in control cells, we found the expected decrease in total cellular levels (fig. b) . however, in cells overexpressing fascin- , this effect was abolished (fig. b) . to confirm these results, we generated stable hek t cells with reduced fascin- cellular levels. using puromycin selection after treatment with fascin- shrna lentivirus, we generated a clone with ~ % reduction in total fascin cellular levels (fig. a) . in these cells, we found a major reduction in ace activity in basal conditions (~ % inhibition) and treatment with ang-ii ( nm, h) did not produce a further inhibitory effect (fig. b). a b fig. a co-localization of ace and fascin- at subcellular level. hek t cells were co-transfected with gfp-tagged ace and fusionred-fascin-c- , processed as described in material and methods and examined by confocal microscopy. the far-right panels represent × magnification of the boxes shown the merged column. the pictures shown are representative from three independent transfections, each performed in triplicate. b interactions between ace and fascin- revealed by co-immunoprecipitation. hek t cells were processed as described in material and methods, subject to the indicated treatments, lysed and immuno-precipitated with gfp antibody. gfp immuno-precipitates ( μg/lane) were separated by % sds-page and the ace levels were revealed by western blotting. the band corresponding to the molecular weight of fascin- ( . kda) is indicated by white arrow. the inputs indicate / from the initial cell lysates which were not subject to immunoprecipitation. the experiment shown is representative from three independent transfections fig. a the effects of ang-ii on ace activity in cells transfected with gfp-ace ( . μg) ha-at r ( . μg) and co-transfected with control plasmid (pcdna . ) or fascin- plasmid ( . μg each). ang-ii treatment ( nm) lasted for h and it was performed after starving the cells for h. the data are presented as % from ace activity determined in untreated cell, which was , . ± . a.u. mean ± s.e.m. from five independent transfections, each performed in triplicate. *indicates statistical significant differences with p < . , using a two-tail student's t test. b the effects of ang-ii on total to investigate whether these in vitro results can be related to pathological effects observed in vivo, we tested the levels of fascin- in brain homogenates from hypertensive mice. mice undergoing a standard doca-salt protocol develop chronic hypertension within days which remains stable for several weeks (xia et al. ) . the hypothalamus of these doca-salt-treated hypertensive mice was assessed for fascin- protein expression. as shown in fig. , fascin- levels were significantly reduced in chronically hypertensive mice. ace was discovered in , as a homologue of ace with which it shares about % protein sequence identity in the catalytic domains (donoghue et al. ; vickers et al. ) . however, in contrast to ace, ace hydrolyses ang-i and ang-ii to produce the vasodilator peptides ang-( - ) and ang-( - ) (ocaranza et al. ; xu et al. ) , which counteracts the adverse effects of an overactive ras (feng et al. (feng et al. , . in addition, ace is also the plasma membrane receptor for sars-coronaviruses responsible for severe acute respiratory syndrome and covid- , (da costa et al. ; giannis et al. ; petrosillo et al. ) . while ace shedding has been demonstrated as a mechanism contributing to the development of hypertension (carvalho-galvao et al. ; xia et al. ; xu et al. ) , much less is known about the regulation of de novo ace synthesis and its intracellular trafficking in normal and pathological conditions. intracellular trafficking is a strictly regulated process, involving several steps which include interactions with specific proteins designated accessory proteins or molecular chaperones (ciruela et al. ; filipeanu ; rodemer and haucke ) . ace -interacting proteins have not been investigated thus far and our group previously demonstrated that ang-ii induces ace internalization and degradation into lysosomes through an at r-mediated mechanism (deshotels et al. ) . in the present work, we identify fascin- as the only protein which in hek t cells has differential interactions with ace after h treatment with ang-ii, a time point that coincides with the lysosomal targeting of this enzyme (deshotels et al. ) . fascin belongs to the large family of actin bundling proteins and has three different isoforms, fascin- being mostly expressed in retinal cells (wada et al. ) , fascin- only in testes (adams ; fagerberg et al. ) , whereas fascin- is expressed ubiquitously but with a b fig. a downregulation of fascin- levels by lentiviral shrna. top: fascin- cellular levels in puromycin-selected hek t clones treated with gfp-lentivirus (sc- , left two lanes) and fascin- shrna lentivirus (sc- -v, right two lanes). these clones were selected using puromycin resistance as described in material and methods and subjected to western blotting. bottom: β-actin cellular levels in the same clones. similar results were obtained in three independent experiments. b reduced cellular fascin- levels diminished ace activity. ace activity was determined in stable hek t clones shown in a, obtained after treatment with gfp-or fascin- shrna lentivirus in the absence or in presence of ang-ii ( nm, h). mean ± s.e.m., from two separate experiments, each performed in triplicate. *indicate statistical significant differences with p < . , vs. control cells using a two-tail student's t test: # indicates statistical significant decrease in ace activity in fascin- -downregulated cells using one-way anova a b fig. a the effects of doca-salt-treatment on fascin- cellular levels in vivo. hypothalamus proteins from sham and doca-salt-treated mice were analyzed by western blotting. b quantification of experiments (n = ) shows that hypertension is associated with a reduction in fascin- levels. *indicates statistical significance (p < . ) vs. sham group using a two-tail student's t test. data are expressed as mean ± s.e.m greater extent in mesenchymal and nervous tissues (jayo and parsons ) . fascin- plays important roles in cell adhesion, migration, and neuronal growth cone morphogenesis and reorganization (adams ; gallop ; wei et al. ). phosphorylation of fascin- by protein kinase c has been shown to abolish its actin bundling activity (adams ; jayo and parsons ) , whereas interactions of fascin- with rac-and rab gtp-ases were suggested to play a role in vesicular trafficking (fagerberg et al. ; parsons and adams ; zhang et al. ). fascin- is overexpressed in many human cancers and its expression levels positively correlates with aggressive tumors (jayo and parsons ; kulasingam and diamandis ; tan et al. ) . however, the present study shows that fascin- can also play a role in the regulation of cardiovascular activity, particularly in regulating expression levels and subcellular localization of ace . our results using proteomics, confocal microscopy, co-immunoprecipitation, and enzymatic activity demonstrate that fascin- interacts with ace . importantly, when fascin- intracellular levels are increased, the effects of ang-ii on ace internalization are greatly reduced (fig. ) . in contrast, a decrease in fascin- cellular levels leads to a major decline in ace activity (fig. ) , strongly suggesting that an optimal expression level of fascin- is required for maintaining the compensatory role of this enzyme against an overactive ras. even more interesting, we found that in the hypothalamus of doca-salt-induced hypertensive mice, a region known for harboring pre-sympathetic neurons involved in autonomic regulation (mukerjee et al. ) , the levels of fascin- were reduced. cytoskeletal rearrangements by contractile stimuli in vascular smooth muscle cells have been reported (ishida et al. ) , and a decrease in fascin- cellular levels was observed in rapid ischemic neuronal response as well as in renal podocytes after mechanical stress (kliewe et al. ; meller et al. ). however, the present work is the first observation that fascin- is decreased in chronic neurogenic hypertension. we propose that in normal conditions ace is maintained to the plasma membrane through interactions with fascin- , but in the presence of enhanced circulating ang-ii, these interactions are attenuated and the enzyme is internalized and degraded, a process that is enhanced in cells with reduced fascin- levels (fig. ), in agreement with our previous observations that ace is reduced in this model (xia et al. ) . rearrangement of actin cytoskeleton has been shown to play important roles in intracellular traffic (gallop ) and various proteins have been shown to specifically regulate the transport of receptors like tubulin for at r and α badrenergic receptors (duvernay et al. ; zhang et al. ) , filamin a for somatostatin receptor type (treppiedi et al. ) , and erm proteins for tyrosine kinase receptors and viruses (neisch and fehon ) . our data reveal that beside the previously reported functions of fascin- (adams ; gallop ; jayo and parsons ) , this actin bundling protein also mediates the traffic from plasma membrane to endo-lysosomal system. whether or not this role is limited to ace or fascin- may interfere with the traffic of other proteins remains to be determined. in conclusion, we identified fascin- as an ace -accessory protein, interacting with the enzyme in an ang-iidependent manner. moreover, fascin- expression levels represent an additional mechanism involved in the regulation of ace activity and ultimately in the regulation of autonomic and cardiovascular functions. fig. schematic diagram of the proposed involvement of fascin- in the regulation of ace subcellular localization and expression levels. in normal conditions, fascin- binds to ace and supports the enzyme localization to the plasma membrane (left). increase in extracellular ang-ii concentrations leads to interactions between at r and ace followed by internalization of the complex in endosomes, where it dissociates followed by at r recycling to the plasma membrane and the enzyme transport to lysosomes (deshotels et al. ). in addition, decreases in fascin- cellular levels further contribute to ace degradation (right) roles of fascin in cell adhesion and motility mtus , a gene encoding angiotensin-ii type (at ) receptor-interacting proteins, in health and disease, with special emphasis on its role in carcinogenesis central administration of trv improves baroreflex sensitivity and vascular reactivity in spontaneously hypertensive rats adenosine receptors interacting proteins (arips): behind the biology of adenosine signaling the emergence of sars, mers and novel sars- coronaviruses in the st century lazartigues e ( ) alpha-lipoic acid reduces neurogenic hypertension by blunting oxidative stress-mediated increase in adam angiotensin ii mediates angiotensin converting enzyme type internalization and degradation through an angiotensin ii type i receptor-dependent mechanism a novel angiotensin-converting enzymerelated carboxypeptidase (ace ) converts angiotensin i to angiotensin - alpha b-adrenergic receptor interaction with tubulin controls its transport from the endoplasmic reticulum to the cell surface analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics brain-selective overexpression of human angiotensin-converting enzyme type attenuates neurogenic hypertension angiotensin-converting enzyme overexpression in the subfornical organ prevents the angiotensin ii-mediated pressor and drinking responses and is associated with angiotensin ii type receptor downregulation temperature-sensitive intracellular traffic of alpha c-adrenergic receptor from cell surface to nucleus: mas transportation in hypertension filopodia and their links with membrane traffic and cell adhesion coagulation disorders in coronavirus infected patients: covid- , sars-cov- mers-cov and lessons from the past agoniststimulated cytoskeletal reorganization and signal transduction at focal adhesions in vascular smooth muscle cells require c-src fascin: a key regulator of cytoskeletal dynamics significance of angiotensin - coupling with mas receptor and other gpcrs to the renin-angiotensin system: iuphar review studying the role of fascin- in mechanically stressed podocytes fascin- is a novel biomarker of aggressiveness in some carcinomas the angiotensin ii type i receptor-associated protein, atrap, is a transmembrane protein and a modulator of angiotensin ii signaling cell signaling, internalization, and nuclear localization of the angiotensin converting enzyme in smooth muscle and endothelial cells ubiquitin-proteasome-mediated synaptic reorganization: a novel mechanism underlying rapid ischemic tolerance protection of the myocardium against ischemia/reperfusion injury by angiotensin-( - ) through an at r and akt-dependent mechanism ace and adam interaction regulates the activity of presympathetic ezrin, radixin and moesin: key regulators of membrane-cortex interactions and signaling recent insights and therapeutic perspectives of angiotensin-( - ) in the cardiovascular system rac regulates the interaction of fascin with protein kinase c in cell migration dynamics of adam -mediated shedding of ace applied to pancreatic islets of male db/db covid- , sars and mers: are they closely related? clathrin/ap- -dependent endocytosis: a novel playground for the pharmacological toolbox? kinin b receptor promotes neurogenic hypertension through activation of centrally mediated mechanisms association of fascin- with mortality, disease progression and metastasis in carcinomas: a systematic review and meta-analysis cytoskeleton protein filamin a is required for efficient somatostatin receptor type internalization and recycling through rab and rab sorting endosomes in tumor somatotroph cells hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase mutation of human retinal fascin gene (fscn ) causes autosomal dominant retinitis pigmentosa rufy , a protein specifically expressed in neurons, interacts with actin-bundling protein fascin to control the growth of axons angiotensin ii type receptor-mediated reduction of angiotensin-converting enzyme activity in the brain impairs baroreflex function in hypertensive mice brain angiotensin-converting enzyme type shedding contributes to the development of neurogenic hypertension clinical relevance and role of neuronal at receptors in adam -mediated ace shedding in neurogenic hypertension ace /ang-( - )/mas pathway in the brain: the axis of good rab controls actin bundling by recruiting fascin as an effector protein the angiotensin ii type receptor c-terminal lys residues interact with tubulin and modulate receptor export trafficking acknowledgements this work was supported by nih r hl - key: cord- - eyi authors: wysocki, jan; gonzález-pacheco, francisco r.; batlle, daniel title: angiotensin-converting enzyme : possible role in hypertension and kidney disease date: - - journal: curr hypertens rep doi: . /s - - - sha: doc_id: cord_uid: eyi the discovery of angiotensin-converting enzyme (ace) adds a new level of complexity to the understanding of the renin-angiotensin system. the high catalytic efficiency of ace for the generation of angiotensin (ang)- - from ang ii suggests an important role of ace in preventing ang ii accumulation, while at the same time enhancing ang- - formation. ace and ace may have counterbalancing functions and a regulatory role in fine-tuning the rate at which ang peptides are formed and degraded. by counterregulating the actions of ace on ang ii formation, ace may play a role in maintaining a balanced status of the renninangiotensin system. this review focuses on the function of ace and its possible roles in kidney disease and hypertension. studies using models of ace ablation and the pharmacologic administration of an ace inhibitor suggest that decreased ace activity alone or in combination with increased ace activity may play a role in both diseases. the first human homologue of angiotensin-converting enzyme (ace), termed angiotensin-converting enzyme (ace ) was identified in by two separate groups using genomics-based strategies [ , ] . the gene encoding ace is located on the x chromosome. like ace, ace is a type integral membrane protein; however, ace contains only a single active site domain and consists of amino acids [ , ] . ace acts as a carboxypeptidase, removing single amino acids from the c-terminus of its substrates, whereas ace acts predominantly as a peptidyl dipeptidase removing c-terminal dipeptides. the metalloprotease catalytic domains of ace and ace are % identical, and comparison of the genomic structures indicates that the two genes arose through duplication from a common ancestor [ , ] . the subsequent elucidation of the three-dimensional structure of the extracellular domain of ace [ ] revealed that the catalytic mechanism of ace closely resembles that of ace. however, the substrate-binding pockets differ significantly [ ] , explaining the differences in substrate specificity between the two enzymes and the failure of ace inhibitors to bind to and inhibit ace [ ] . ace is the only known enzymatically active homologue of ace in the human genome. the carboxyl end of ace is homologous to collectrin [ ] . collectrin, unlike ace and ace , lacks carboxypeptidase catalytic properties and was initially localized in the collecting tubule [ ] . more recently, however, it has been localized to the proximal tubule, where it is involved in regulating amino acid uptake [ •, ] . collectrin also has been suggested to have a role in cystogenesis by interacting with ciliaspecific membrane integral proteins [ •] . another gene has been identified in the genomes of several mammalian species that encodes a novel, single-domain ace-like protein that was named ace . in several species ace seems to lack catalytic activity as a zinc metalloprotease [ ] . moreover, in humans, no evidence could be found that the ace gene is expressed, and the presence of deletions and insertions in the sequence suggests that in humans ace is a pseudogene [ ] . ace was initially found in heart, kidney, and testis with lesser amounts in colon, small intestine, and ovary [ ] . ace also has been found in the lungs where it plays an important role in angiotensin (ang) ii metabolism [ , ] . ace protects mice from severe lung injury induced by acid aspiration, sepsis, and severe acute respiratory syndrome (sars) virus infection [ , ] . we first suggested that ace could be renoprotective, particularly in combination with low levels of ace [ ] . recent work by our laboratory is consistent with this hypothesis [ ••, ••, •] . in this review we discuss recent publications dealing with ace as a pathway for ang ii metabolism and its possible role in hypertension and diabetic kidney disease. the discovery of ace adds a new level of complexity to the understanding of the renin-angiotensin system (ras). ace is a carboxypeptidase that facilitates the conversion of ang ii to ang-( - ) and the conversion of ang i to ang-( - ) [ , , ] (fig. ). ang-( - ) has no known effects on blood vessels but can be converted by ace to a shorter peptide, ang-( - ), which is a blood-vessel dilator (fig. ). ang i (with amino acids) is an intermediate peptide without known biologic effects. ang i is converted to ang-( - ) (with nine amino acids) by ace , but is converted to the eight-amino acid ang ii by ace (fig. ) . the actions of ace should act to prevent the accumulation of ang ii, a vasoconstrictor, and lead to formation of ang-( - ), a vasodilator. ace and ace may have counterbalancing functions and a regulatory role in fine-tuning the rate at which ang peptides are formed and degraded. ace has a high catalytic efficiency for the generation of ang-( - ) from ang ii [ ] . this suggests an important role of ace in preventing ang ii accumulation, while at the same time enhancing ang-( - ) formation. recently, it was shown that ang - inhibits ang ii-stimulated mitogen-activated protein kinase (mapk) phosphorylation in proximal tubular cells [ •] . generation of ang-( - ) by proximal tubular ace could therefore also have a role in counteracting the effects of locally generated ang ii [ •] . ace -mediated degradation of ang ii to ang-( - ) has been documented in studies using kidney cortex preparations or isolated proximal tubules [ , •, •]. ace affinity for ang ii conversion has been generally found higher than for ang i to ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) [ , •, •]. in freshly dissected rat proximal tubular segments, however, li et al. [ ••] observed ace -dependent formation of ang-( - ) from ang i, which depended on conversion of ang i to ang-( - ), followed by ace-mediated cleavage to ang-( - ). in another study in cardiac tissue, investigators suggested that ace can act through ang-( - ) instead of ang-( - ), as a counterregulator of ace [ ] . the knowledge of ace biologic peptide substrates is useful in providing an initial understanding of the substrate specificity of the protease and the physiologic role of ace . if ace is a "converting enzyme," as ace is, then its peptidase activity either produces or degrades (or both) a peptide with biologic activity [ ] . therefore, an understanding of the biologic activity of ace substrates and products suggests putative physiologic roles for ace [ ] . biologically active substrate peptides that are cleaved by ace are shown in table . although the biologic peptides ang i and ang ii are the main known ace substrates, ace can hydrolyze several other target peptides. ace does not cleave bradykinin but inactivates both des-arg bradykinin and lys-des-arg -bradykinin [ ] . ace can also remove the c-terminal residue from apelin and other vasoactive peptides such as neurotensin, kinetensin (a neurotensin-related peptide), and des-arg bradykinin [ ] . ace has high catalytic efficiency to hydrolyze apelin- and apelin- peptides [ ] . apelin also induces an increase in myocardial contractility and a reduction of vasomotor tone [ ] . two opioid peptides, dynorphin a and -casamorphin, are also cleaved by ace [ , ] . these two ace substrate peptides stimulate and opioid g-protein-coupled receptors, and may have negative effects on cardiomyocyte contractility [ ] . angiotensin i angiotensin - angiotensin ii angiotensin - a non figure . the vertical arrows show the classic angiotensin (ang) ii-forming pathway driven by angiotensin-converting enzyme (ace) and non-ace mechanisms. horizontal arrows show the actions of the more recently discovered ace . through the action of ace , less ang i substrate is available for ace to generate ang ii because ang i is derived to ang-( - ) (upper horizontal arrow). ang ii is converted to ang-( - ) by ace (lower horizontal arrow). the net effect of ace is less ang ii accumulation and more ang-( - ) formation. [ •, , - ] . in these assays, the substrate peptide contains a fluorescent -methoxycoumarin group (mca), which is quenched by energy transfer to a , -dinitrophenyl moiety (dnp). this reaction is based on the cleavage of an amide bond between the fluorescent and quencher groups, resulting in an increase in fluorescence [ ] . it can be used to measure the activity of ace and of other peptidases [ ] . the fluorescence signal of mca-yvadapk(dnp) is partially quenched by specific inhibitors of both ace and ace , but not by an inhibitor of another metalloprotease, carboxypeptidase a [ •]. we took advantage of this dual action to develop an assay to measure ace and ace activity concurrently [ •] . the combination of specific inhibitors for ace and ace quenched the signal almost completely and to the same degree as ethylenediaminetetraacetic acid (edta), a metal ion chelator, indicating that both metalloenzymes, ace and ace , are involved in the degradation of the substrate. as noted previously, the dual cleavage of this substrate by ace and ace makes it possible to measure activity of these two carboxypeptidases concurrently in tissue samples [ •] . this is particularly important because ace and ace colocalize in numerous tissues where both enzymes have been shown to be enzymatically active [ ] . the assessment of ace activity, the only active homologue of ace, in combination with ace activity is useful in evaluating the ras in the pathophysiology of different disease states [ ••] . ace activity has also been measured using a different fluorogenic substrate, mca-apk(dnp) [ ] [ ] [ ] . this approach detected ace activity in serum samples from mice overexpressing ace (but not in wild-type mice) [ ] , and in rat heart tissue [ ] and rat testes [ ] . using this substrate in mouse tissues, however, we could not quench the fluorescence with mln- . moreover, the mca-apk(dnp) substrate is highly selective for ace [ ] . accordingly, it cannot be used for dual measurements of ace and ace activity. ferrario et al. [ ••] used an hplc-based method to measure ace activity in cardiac membranes; this method was later extended to measurements in other tissues and body fluids [ • ]. the assay is performed in two steps: ) radiolabeled peptide precursors are incubated with ace ; and ) hplc is used to separate the peptide products. the hplc method utilizes the ability of ace to hydrolyze ang ii to ang-( - ) [ ] . the conversion rate of the exogenous radioactively labeled ang ii to ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) in the presence and absence of ace inhibitor is the equivalent of the ace enzymatic activity [ ••] . because this method requires hplc measurements, the process involved is relatively time consuming, but it provides information on ang-( - ) formation directly. another newly developed assay also uses the natural substrate of ace , ang ii; this method utilizes surface-enhanced laser desorption/ionization time-of-flight (seldi-tof) ms with proteinchip array technology (ciphergen biosystems, fremont, ca) [ •] . in this assay, mouse kidney samples are mixed with ang ii and incubated for a specified time. ace converts ang ii to ang-( - ), which both can be quantified by seldi-tof ms. based on the observation of a linear relationship between recombinant ace concentrations and the quotient of the formed ang ( - ) to the hydrolyzed ang ii, the ratio of ang ( - )/ang ii was proposed as a measure of tissue ace activity [ • ]. the levels of ace activity vary widely within tissues. for instance, using our fluorogenic assay the levels of ace activity in mouse kidney cortex are about -to -fold higher than in the heart, whereas ace activity is barely detectable in serum [ •] . studies using other measurement methods found very low or undetectable ace activity in plasma from rodents [ •, ] . similarly, in healthy humans, the levels of circulating ace are undetectable or very low [ ] . rice et al. [ ] found that circulating ace was -fold lower than ace, and ace was detectable in less than % of the study population. low circulating ace levels could result from less shedding of ace as compared with ace from the plasma membrane of endothelial cells [ ] . the zinc metalloprotease adam is responsible for the regulated shedding of ace ; there is little constitutive shedding of ace as compared with ace [ •]. ace activity and protein have been detected in human urine from healthy subjects [ •] , and ace activity has been measured in urine and serum from sheep [ • ]. thus, ace , like ace, appears to be secreted as a soluble protein in vivo. it would be important to determine whether circulating or urinary ace increases in certain disease states, either due to increased expression, increased shedding from the membrane, or decreased clearance from the circulation [ ] . the pattern of ace and ace expression in the glomerulus of db/db mice is just the opposite of what we find in cortical tubules [ ] . consequently, the final urine may not reflect the site of the nephron where the alteration primarily resides. in kidney cortex, a good correlation was found between ace protein abundance and enzymatic activity [ • ]. this indicates that the level of functional activity is highly dependent on the level of protein expression. in contrast, we found a lack of positive correlation between renal ace activity and renal ace mrna levels in the db/db as well as streptozotocin (stz)-induced diabetic mice. these findings illustrate the importance of not relying solely on mrna levels when assessing whether ace is altered. measurements of ace mrna alone may not reflect existing differences occurring at a posttranscriptional level [ •] . investigators must keep this in mind when interpreting data from studies reporting ace gene polymorphisms in diseases such as hypertension or diabetes. this further underscores the importance of having reliable assays to measure ace activity targeting the sites of possible ace alterations in the kidney and other organs. immunohistochemical analysis of ace distribution within the kidney demonstrated that renal tubules clearly show the highest intensity of immunostaining [ , ••, ] . in both kidney sections and cultured polarized renal epithelial cells, ace localizes predominantly to apical surface [ ••, •] where it can undergo regulated proteolytic shedding [ •] . ace is also present in the glomerulus, although the level of expression in mouse kidneys is reduced as compared with renal tubules (see below). li et al. [ ••] have systematically examined the relative distribution of ace in the rat kidney at the mrna level. in microdissected rat nephron segments, semiquantitative reverse transcriptase-polymerase chain reaction (rt-pcr) revealed that ace mrna was widely expressed, with relatively high levels in the proximal straight tubule. the high level of ace expression in the proximal tubule suggests that it may regulate the local levels of ang ii, and generate ang-( - ). in rats, ace has been localized by immunohistochemistry to the proximal tubule [ ] . interestingly, ace colocalizes with ang- ( - ) , and the proximal tubular staining for ace and ang-( - ) increases in normal pregnancy [ ] . this correlated with increased urinary excretion of ang-( - ); however, ace in urine was not measured [ ] . lely et al. [ ] reported that in humans ace is expressed in tubular and glomerular epithelium, as well as in vascular smooth muscle cells and the endothelium of interlobular arteries. in renal biopsies from patients with primary and secondary kidney diseases, these authors found neo-expression of ace in glomerular and peritubular capillary endothelium [ ] . our laboratory recently showed that ace colocalizes with glomerular epithelial cell (podocyte) markers, and its presence in the podocyte/slit diaphragm complex was confirmed by immunogold labeling [ ••] . the presence of ace was further demonstrated in immortalized podocytes grown in culture by western blot and immunofluorescence [ ] . velez et al. [ ] examined the processing of angiotensin substrates by cultured glomerular epithelial cells. they showed that podocytes express a functional intrinsic ras characterized by neprilysin, aminopeptidase a, ace , and renin activities, which predominantly lead to ang-( - ) and ang-( - ) formation and ang ii degradation [ ] . our laboratory has characterized the pattern of glomerular staining for ace and ace in control and diabetic mice [ ••] . in diabetic kidneys, glomerular immunostaining for ace is attenuated in db/db mice of weeks of age. in sharp contrast, ace expression in glomeruli from diabetic db/db mice is increased as compared with glomeruli from their respective age-matched nondiabetic controls [ ••] . tikellis et al. [ ] found the opposite pattern in rats made diabetic by stz. however, glomerular staining for ace in mice is weaker than in rats; thus, there may be a species difference. in humans, as in mice, glomerular staining is weaker than tubular staining [ ] . what may be the significance of reduced ace and increased ace at the glomerular level? we reasoned that in diabetic mice such a combination could foster renal injury by resulting either in ang ii accumulation, decreased ang-( - ) formation, or both [ ••] . to test this hypothesis, we used a specific ace inhibitor, mln- , to reduce ace activity. the administration of mln- for weeks exacerbated albuminuria in the db/db mice [ ••] . this was associated with increased glomerular expression of fibronectin. in a different model of diabetes (stz-treated mice), soler et al. [ ••] found both glomerular mesangial expansion and increased albumin excretion after mln- treatment. we further found that the glomerular expression of ace was increased in stz-treated mice and further increased after mln- administration [ ••] . as outlined in figure , changes in ace and ace can work in concert to regulate the level of angiotensin peptides. the scheme outlined in figure predicts that a pattern of low ace and high ace would lead to less ang ii and more ang-( - ) formation locally. a combination of increased ace and decreased ace in the glomerulus is apt to favor increased glomerular ang ii accumulation and foster an increase in glomerular permeability manifested by albuminuria. this hypothesis does not necessarily conflict with prevailing views of enhanced formation of ang ii in the diabetic kidney. rather, both enhanced ang ii formation and decreased ang ii degradation may occur in glomeruli of diabetic mice, thereby contributing to produce more damage. studies in animals with ace genetic ablation have shown the development of renal lesions, particularly within the glomerulus, although glomerular injury was seen only in older animals. in male mice with genetic ace ablation, early accumulation of fibrillar collagen in glomerular mesangium was followed by the development of glomerulosclerosis by months of age. female ace mutant (ace -/-) mice were relatively protected. urinary albumin excretion was increased as compared with age-matched control mice [ ••] . these structural and functional changes in the glomeruli of male ace mutant mice were prevented by treatment with the ang ii type receptor antagonist irbesartan. the glomerular injury in male mice associated with the deletion of the ace gene were further accentuated by diabetes [ ••] . in this more recent study, ace knockout mice were crossed with akita mice, a model of type diabetes mellitus. diabetic ace knockout mice (ace -/yins wt/c y) exhibited a twofold increase in urinary albumin excretion compared with akita mice not depleted in the ace gene. increased mesangial matrix scores and glomerular basement membrane thicknesses in ace -/yins wt/c y mice were accompanied by increased fibronectin and -smooth muscle actin staining in the glomeruli. there were no differences in blood pressure or heart function to account for the exacerbation of kidney injury. although kidney levels of ang ii were not increased in the diabetic mice, treatment with an ang ii receptor blocker reduced urinary albumin excretion rate in ace -/yins wt/c y mice, suggesting that acceleration of glomerular injury in these mice is ang ii mediated [ ••]. a seminal paper by crackower et al. [ ] , primarily describing an ace knockout and its associated cardiac pathology, also reported that ace was reduced at the gene and protein level in kidneys from three separate rat models of spontaneous and diet-induced hypertension. in various recombinant rat models, several quantitative trace loci (qtl) for hypertension have been identified [ ] . ace maps to the x chromosome in humans and a qtl has been mapped to the x chromosome in several rat models of hypertension [ ] . the finding by crackower et al. [ ] that the ace gene maps to a defined qtl on the x chromosome suggests ace as a candidate gene underlying the loci linked to hypertension. more recently, tikellis et al. [ •] showed that the developmental pattern of ace expression in the spontaneously hypertensive rat (shr) kidney was altered before the onset of hypertension. specifically, the expression and activity of ace were increased in tubules from the shr, before the onset of hypertension. with the increase in blood pressure at weeks of age, the tubular expression of ace is reduced in shr compared to wistar-kyoto rats (wky). over the course of renal development, ace expression does not significantly change in the shr kidney, whereas ace expression increases in the wky kidney [ •] . this finding seems to be consistent with the concept that ace is downregulated in kidneys from hypertensive rats. in humans, few studies have been conducted to examine possible association of ace gene polymorphisms with hypertension. in three studies performed on chinese cohorts, an association was found between single-nucleotide polymorphisms in ace gene and blood pressure in women with the metabolic syndrome [ ] and in women with essential hypertension [ , ] . moreover, one of the ace alleles associated with high blood pressure seemed to confer a risk for reduced antihypertensive response to ace inhibitors as well [ ] . in another study, no link was reported between ace gene polymorphisms and essential hypertension in australian individuals of white anglo-celtic origin [ ] . collectively, the role of the ace gene in conferring the predisposition to hypertension is far from clear; whether the discrepancies in genetic linkage analyses could be related to differences in ethnic origin of the cohorts studied needs further investigation. polymorphisms in the ace gene were also sought in the general population regarding echocardiographically determined parameters of left ventricular mass, structure, or function [ ] . this study provides some evidence that genetic variants in the ace gene may be associated with left ventricular mass and left ventricular hypertrophy in men, but no association with blood pressure was found in either men or women. in a study exploring human renal biopsy specimens, a significant correlation was found between the mrna levels of ace and ace in a variety of renal conditions [ ] . the correlation was highly significant (p < . ) but relatively weak (r = . ). interestingly, the ace to ace ratio was significantly higher in subjects with hypertension than in subjects without hypertension. these findings are in keeping with the idea that ace might play a role in maintaining a balanced status of local renal ras by acting to counterregulate the actions of ace. evidence is also emerging that ace may be altered in pregnancy-related hypertension [ ] . joyner et al. [ ] examined the questions of whether ang-( - ) and ace colocalize and whether they change in parallel during normal and hypertensive pregnancies. the authors found that during normal pregnancies, concurrent changes of ace and ang-( - ) occur, suggesting that ace plays a role in regulating the renal levels of ang-( - ) at mid-to late gestation [ ] . in contrast, in hypertensive pregnant rats, the ace activity in cortex and medulla were unchanged, whereas ang-( - ) levels were reduced. it was therefore concluded that the decrease in renal ang-( - ) content in the absence of a concomitant decrease in ace implicates the participation of other ang-( - ) forming or degrading enzymes during hypertensive pregnancy [ ] . genetic ablation of ace may not result in spontaneous hypertension. two different ace knockouts have been described, and hypertension is not an overt feature of either phenotype [ , ••] . in a study by gurley et al. [ ••] in mice on the c bl/ background, ace deficiency was associated with a modest increase in blood pressure, whereas the absence of ace had no effect on baseline blood pressures in /svev mice. however, this does not mean that ace is not important in blood pressure regulation. after acute ang ii infusion, plasma concentrations of ang ii were almost threefold higher in ace -deficient mice than in controls; moreover, blood pressures were substantially higher in the ace -deficient mice than in wild-type mice [ ••] . severe hypertension in ace -deficient mice was associated with exaggerated accumulation of ang ii in the kidney, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry [ ••] . this study provided strong evidence that complete ace insufficiency can increase blood pressure under conditions of ang ii excess. based on this study, one cannot conclude whether relative ace deficiency or surplus affects blood pressure. to more directly assess the role of ace on blood pressure regulation, we recently used recombinant ace (race ) administered over a period of days via osmotic minipumps, with or without ang ii infusion [ ] . the increase in blood pressure produced by ang ii alone was prevented by the concomitant administration of ace [ ] . moreover, plasma ang ii levels were markedly reduced by race administration, thereby demonstrating the important role of this enzyme in the degradation of ang ii [ ] . this suggests that the administration of race may have a role in the treatment of ang ii-dependent hypertension, and opens the way for the development of therapies based on ace modulators capable of selectively increasing ace activity. ace is the only enzymatically active homologue of ace, and it plays a significant role in maintaining a balanced status of the ras. ace could act by either preventing ang ii accumulation or enhancing ang-( - ) formation, or both. there is also increasing evidence that alterations in ace may be involved in disease states, such as experimental diabetic kidney disease and possibly hypertension. investigating the role of ace in those two prevalent diseases and whether its effects are mediated by ang ii or ang-( - ) and other biologically active peptides, which are also substrates of ace , opens the way for developing new therapeutic targets in hypertension. no potential conflict of interest relevant to this article was reported. papers of particular interest, published recently, have been highlighted as: • of importance •• of major importance a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase a novel angiotensin-coverting enzyme-related carboxypeptidase (ace ) converts antiogensin i to angiotensin - ace x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis angiotensinconverting enzyme- (ace ): comparative modeling of the active site, specificity requirements, and chloride dependence collectrin, a collecting duct-specific transmembrane glycoprotein, is a novel homolog of ace and is developmentally regulated in embryonic kidneys essential role for collectrin in renal amino acid transport revealed a novel function of the ace homologue, collectrin, as an important regulator of renal amino acid uptake and showed that its primary localization is the proximal tubule aminoaciduria and altered renal expression of luminal amino acid transporters in mice lacking novel gene collectrin the role for hnf- beta-targeted collectrin in maintenance of primary cilia and cell polarity in collecting duct cells the ace homologue, collectrin, forms complexes with proteins related to vesicle transport and fusion, and regulates cell polarity. this study provided evidence that collectrin is decreased in polycystic kidneys and suggested its role in primary cilia formation in kidney epithelium identification and characterisation of the angiotensin converting enzyme- (ace ) gene: a novel mammalian homologue of ace angiotensin-converting enzyme ii in the heart and the kidney a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury angiotensin-converting enzyme protects from severe acute lung failure increased ace and decreased ace protein in renal tubules from diabetic mice: a renoprotective combination? hypertension ace inhibition worsens glomerular injury in association with increased ace expression in streptozotocin-induced diabetic mice the first study to show accentuated histologic damage at the glomerular level after pharmacologic ace inhibition in stz model of diabetes. also, administration of ace inhibitor to diabetic mice was associated with increased glomerular ace expression, suggesting that both decreased ace activity and augmented ace expression may be responsible for exaggerated glomerular injury in mice receiving ace inhibitor glomerular localization and expression of angiotensin-converting enzyme and angiotensin-converting enzyme: implications for albuminuria in diabetes determined the localization of ace within mouse glomeruli, and specifically within podocytes. further showed that diabetic db/db mice have decreased glomerular expression of ace . also, pharmacologic ace inhibition was associated with increased albuminuria, suggesting a role of glomerular ace in diabetic kidney injury ace and ace activity in diabetic mice describes the development of a microplate-based fluorometric method for the concurrent determination of ace and ace activity in mouse tissues, and shows that both ace protein and enzymatic activity are increased in two murine models of diabetes hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase angiotensin-( - ) inhibits angiotensin ii-stimulated phosphorylation of map kinases in proximal tubular cells - ), which binds to the mas receptor, suggesting that ace -mediated generation of ang-( - ) may counterbalance the effects of locally formed ang ii. . ferrario cm new mass spectrometric assay for angiotensin-converting enzyme activity describes ms-based assay to measure tissue ace activity angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: evidence for ace -dependent processing of angiotensin ii the role of angiotensin converting enzyme in the generation of angiotensin - by rat proximal tubules this important study provides information on levels of ace expression throughout the rat nephron at the mrna level and demonstrates that ace in proximal straight tubules facilitates conversion of ang i to ang enalapril attenuates downregulation of angiotensin-converting enzyme in the late phase of ventricular dysfunction in myocardial infarcted rat effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme provides evidence that ace inhibition and at blockade result in increased cardiac ace expression. suggests that the antihypertensive action of at antagonists may be due to increased ang ii metabolism by ace myocardial infarction increases ace expression in rat and humans heart block, ventricular tachycardia, and sudden death in ace transgenic mice with downregulated connexins the novel angiotensin-converting enzyme (ace) homolog, ace , is selectively expressed by adult leydig cells of the testis novel peptide inhibitors of angiotensin-converting enzyme angiotensin-converting enzyme (ace ) and ace activities display tissue-specific sensitivity to undernutrition-programmed hypertension in the adult rat circulating activities of angiotensin-converting enzyme, its homolog, angiotensinconverting enzyme , and neprilysin in a family study tumor necrosis factor-alpha convertase (adam ) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensinconverting enzyme- (ace ) this cell culture study provided new insight into the way ace secretion might occur in vivo angiotensin-converting enzyme (ace ), but not ace, is preferentially localized to the apical surface of polarized kidney cells examined cellular localization and cell fate of ace in comparison with ace in nonpolarized chinese hamster ovary (cho) cells and polarized mdckii cells. the results suggest that ace and ace have a distinct secretion pattern from the cell surface of kidney epithelium and distinct cellular localization characterization of renal angiotensin-converting enzyme in diabetic nephropathy enhanced renal immunocytochemical expression of ang-( - ) and ace during pregnancy renal ace expression in human kidney disease? ace is critically important for angiotensin ii metabolism in podocytes characterization of renin-angiotensin system enzyme activities in cultured mouse podocytes the emerging role of ace in physiology and disease loss of angiotensin-converting enzyme- leads to the late development of angiotensin ii-dependent glomerulosclerosis examined effect of ace gene ablation on kidney function, histology, and alteration in signaling pathways in the mouse kidney. this important contribution demonstrated for the first time that lack of ace is associated with sex-dependent glomerular injury in aging animals loss of angiotensinconverting enzyme- (ace ) accelerates diabetic kidney injury the akita model of diabetes was used to determine the possible effect of ace gene ablation on development of diabetic nephropathy in mice. ace knockouts with diabetes had increased urinary albumin excretion and exaggerated glomerular pathology as compared with diabetic mice not depleted in ace gene. these alterations could be reversed by administration of ang ii receptor blockade. this was the first demonstration that genetic ace deficiency in diabetes is associated with ang iix-mediated acceleration of glomerular injury angiotensinconverting enzyme is an essential regulator of heart function developmental expression of ace in the shr kidney: a role in hypertension? described the developmental pattern of ace expression in the kidney of hypertensive shr rats compared with normotensive wky rats. ace expression and activity are initially increased in the shr kidney at birth, but with the onset of hypertension, the tubular expression of ace falls in shr compared with wky association of angiotensinconverting enzyme gene a/g polymorphism and elevated blood pressure in chinese patients with metabolic syndrome association study of angiotensin-converting enzyme gene (ace ) polymorphisms and essential hypertension in northern han chinese polymorphisms of ace gene are associated with essential hypertension and antihypertensive effects of captopril in women no association of angiotensin-converting enzyme gene (ace ) polymorphisms with essential hypertension association of angiotensinconverting enzyme (ace ) gene polymorphisms with parameters of left ventricular hypertrophy in men. results of the monica augsburg echocardiographic substudy synergistic expression of angiotensin-converting enzyme (ace) and ace in human renal tissue and confounding effects of hypertension on the ace to ace ratio temporal-spatial expression of ang-( - ) and angiotensin-converting enzyme in the kidney of normal and hypertensive pregnant rats altered blood pressure responses and normal cardiac phenotype in ace -null mice this outstanding study demonstrated that ace deficiency in mice challenged with exogenous ang ii is associated with impaired blood pressure regulation. it also suggested that impaired blood pressure response could be related to decreased ang ii disposal, as shown by elevated plasma and kidney levels of ang recombinant ace attenuates angiotensin ii induced hypertension key: cord- -o atz c authors: xiao, li; sakagami, hiroshi; miwa, nobuhiko title: ace : the key molecule for understanding the pathophysiology of severe and critical conditions of covid- : demon or angel? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: o atz c recently, the sars-cov- induced disease covid- has spread all over the world. nearly % of the patients have severe or critical conditions. sars-cov- exploits ace for host cell entry. ace plays an essential role in the renin–angiotensin–aldosterone system (raas), which regulates blood pressure and fluid balance. ace also protects organs from inflammatory injuries and regulates intestinal functions. ace can be shed by two proteases, adam and tmprss . tmprss -cleaved ace allows sars-cov- cell entry, whereas adam -cleaved ace offers protection to organs. sars-cov- infection-caused ace dysfunction worsens covid- and could initiate multi-organ failure. here, we will explain the role of ace in the pathogenesis of severe and critical conditions of covid- and discuss auspicious strategies for controlling the disease. since december , the new coronavirus (severe acute respiratory syndrome coronavirus , sars-cov- ) induced disease, covid- , has spread rapidly all over the planet. as shown in covid- dashboard by the center for systems science and engineering (csse) at johns hopkins university (jhu), up to date , , people have been infected and , deaths have been confirmed worldwide [ ] .who officially declared covid- a pandemic on march th, . according to a report based on , cases (test confirmed cases: , ( %) from the chinese center for disease control and prevention, % of covid- patients have cold-like symptoms and mild pneumonia, % have severe respiratory inflammation, and % have critical conditions including respiratory failure, septic shock, and/or multiple organ dysfunction or failure. the mortality is . % ( . % in critical cases) [ ] . since the rates of severe and critical cases are much higher than seasonal influenza, it is essential to understand the pathophysiology and then devise strategies to fight the disease. sars-cov- is a positive-sense single-stranded rna virus with a crown-like appearance of spike proteins (s proteins) that project from their envelope. similar to sars (severe acute respiratory syndrome, [ ] [ ] coronavirus (sars-cov) [ ] , sars-cov- primarily uses the s protein to invade host cells through ace , an enzyme which is known to be important in the renin-angiotensin-aldosterone system (raas) [ , ] . cellular ace , adam , and tmprss are all expressed on the cell membrane. after being shed by adam , soluble ace is released from its full-length form to counteract the effects of ang ii signaling. alternatively, cellular ace is also shed by tmprss , which results in sars-cov- -cell membrane fusion. sars-cov- rna is then released into the cytoplasm and viral replication is efficiently processed. because soluble ace contains the virus binding site, it can also bind to the virus. however, without an intracellular environment, the virus cannot be duplicated. ace is a close homologue of human ace [ , ] . as is well-known, ace cleaves angiotensin i (ang i) to ang ii. ang ii binds to ang ii receptor (at ) and then mediates numerous systemic and local effects (such as promoting vasoconstriction, fibrosis, and salt retention) in the cardiovascular system ( figure ). in the raas, ace exerts the opposite role of ace. ace catalyzes the conversion of ang i to ang-( - ) and ang ii to ang-( - ). the converting efficiency of ace on substrate ang ii is -fold higher than that on ang i [ ] . ang-( - ) binds to the g protein-coupled receptor mas to mediate various effects including vasorelaxation, cardioprotection, anti-oxidative action [ ] , antiinflammation [ ] , and the inhibition of ang ii-induced signaling [ , ] . the ace -ang-( - ) axis is considered an important therapeutic target in cardiovascular disorders [ ] . a large cohort study showed that circulating ace was only detectable in the serum of among test subjects, and its concentration was approximately -fold lower than that of circulating ace [ ] . more evidence showed that circulating ace is increased in patients with type or type diabetes, hypertension, heart failure, and chronic kidney diseases [ ] [ ] [ ] . the reason for high levels of ace in these patients is that increased ace is a defensive response to counteract the adverse effect of ang ii. since ang ii-at receptor signaling also promotes autoimmune response, ace may control immune functions through the ang-( - )-mas axis [ , ] . cellular ace , adam , and tmprss are all expressed on the cell membrane. after being shed by adam , soluble ace is released from its full-length form to counteract the effects of ang ii signaling. alternatively, cellular ace is also shed by tmprss , which results in sars-cov- -cell membrane fusion. sars-cov- rna is then released into the cytoplasm and viral replication is efficiently processed. because soluble ace contains the virus binding site, it can also bind to the virus. however, without an intracellular environment, the virus cannot be duplicated. ace is a close homologue of human ace [ , ] . as is well-known, ace cleaves angiotensin i (ang i) to ang ii. ang ii binds to ang ii receptor (at ) and then mediates numerous systemic and local effects (such as promoting vasoconstriction, fibrosis, and salt retention) in the cardiovascular system ( figure ). in the raas, ace exerts the opposite role of ace. ace catalyzes the conversion of ang i to ang-( - ) and ang ii to ang-( - ). the converting efficiency of ace on substrate ang ii is -fold higher than that on ang i [ ] . ang-( - ) binds to the g protein-coupled receptor mas to mediate various effects including vasorelaxation, cardioprotection, anti-oxidative action [ ] , anti-inflammation [ ] , and the inhibition of ang ii-induced signaling [ , ] . the ace -ang-( - ) axis is considered an important therapeutic target in cardiovascular disorders [ ] . a large cohort study showed that circulating ace was only detectable in the serum of among test subjects, and its concentration was approximately -fold lower than that of circulating ace [ ] . more evidence showed that circulating ace is increased in patients with type or type diabetes, hypertension, heart failure, and chronic kidney diseases [ ] [ ] [ ] . the reason for high levels of ace in these patients is that increased ace is a defensive response to counteract the adverse effect of ang ii. since ang ii-at receptor signaling also promotes autoimmune response, ace may control immune functions through the ang-( - )-mas axis [ , ] . angiotensinogen is produced in the liver and released into the blood stream. the renal juxtaglomerular apparatus-secreted renin cleaves angiotensinogen to ang i. ang i is further converted to ang ii by ace which is mostly produced in the lungs. ang ii binds to both at and at receptors to regulate the blood pressure and inflammation. most actions of ang ii occur via the at receptor. meanwhile, cellular ace is cleaved by adam . after the active form of ace being released into the extracellular environment, ace converts ang i to ang-( - ) and ang ii to ang-( - ). ace also converts ang-( - ) to ang-( - ). ang-( - ) binds to mas receptor to mediate the opposite effects of ang ii. genetic ace deficiency is associated with upregulation of inflammatory mediators, elevated inflammatory responsiveness to proinflammatory stimuli, and enhanced ang ii-induced cardiac and aortic remodeling [ , ] . the anti-inflammatory effects of ace are exerted mostly through the ace -ang-( - ) axis against ang ii-at activities [ ] . ace also has a raas-independent function. in the intestine, cellular ace (not circulating ace ) regulates the absorption of amino acids and the intestinal bacterial balance to reduce intestinal inflammation [ ] . cellular ace is required for expression of the neutral amino acid transporter b at in intestinal epithelial cells [ ] . without ace -b at complex, serum levels of the neutral amino acids valine (val), threonine (thr), and tyrosine (tyr), and the essential amino acid tryptophan (trp) were markedly reduced and resulted in severe intestinal inflammation and microbial imbalance in ace knockout mice. administration with trp or nicotinamide (the metabolic product of trp) could increase the expression of antimicrobial peptide α-defensins and reverse the above-mentioned microbial imbalance and inflammation in the gut. structural modelling suggested that the ace -b at complex could bind to the s-protein of sars-cov- simultaneously [ ] . the intestinal cellular angiotensinogen is produced in the liver and released into the blood stream. the renal juxtaglomerular apparatus-secreted renin cleaves angiotensinogen to ang i. ang i is further converted to ang ii by ace which is mostly produced in the lungs. ang ii binds to both at and at receptors to regulate the blood pressure and inflammation. most actions of ang ii occur via the at receptor. meanwhile, cellular ace is cleaved by adam . after the active form of ace being released into the extracellular environment, ace converts ang i to ang-( - ) and ang ii to ang-( - ). ace also converts ang-( - ) to ang-( - ). ang-( - ) binds to mas receptor to mediate the opposite effects of ang ii. genetic ace deficiency is associated with upregulation of inflammatory mediators, elevated inflammatory responsiveness to proinflammatory stimuli, and enhanced ang ii-induced cardiac and aortic remodeling [ , ] . the anti-inflammatory effects of ace are exerted mostly through the ace -ang-( - ) axis against ang ii-at activities [ ] . ace also has a raas-independent function. in the intestine, cellular ace (not circulating ace ) regulates the absorption of amino acids and the intestinal bacterial balance to reduce intestinal inflammation [ ] . cellular ace is required for expression of the neutral amino acid transporter b at in intestinal epithelial cells [ ] . without ace -b at complex, serum levels of the neutral amino acids valine (val), threonine (thr), and tyrosine (tyr), and the essential amino acid tryptophan (trp) were markedly reduced and resulted in severe intestinal inflammation and microbial imbalance in ace knockout mice. administration with trp or nicotinamide (the metabolic product of trp) could increase the expression of antimicrobial peptide α-defensins and reverse the above-mentioned microbial imbalance and inflammation in the gut. structural modelling suggested that the ace -b at complex could bind to the s-protein of sars-cov- simultaneously [ ] . the intestinal cellular ace might be another viral entry point for sars-cov- as well as tmprss -sheded ace in the lungs. covid- is a lower respiratory tract disease. the first anatomic pathology report of a covid- death indicated that the most seriously injured organs were the lungs. exudative lesions and fibrosis were observed in the lungs. phlegm and exudates filled up the lower respiratory tract and pulmonary alveolus. compared to sars, the exudative lesions were far worse but the fibrosis was much lighter. notably, segmental dilatation and stenosis of the small intestine were observed in the cadaver suggesting that the small intestine was also severely injured by sars-cov- infection. lesions on other organs were not obvious [ ] . the pathological findings of another covid- victim showed that the bilateral diffuse alveolar damage with cellular fibromyxoid exudates, desquamation of pneumocytes, and hyaline membrane formation occurred in the lungs [ ] . this evidence suggests that sars-cov- invades the human body mostly through the respiration system, and possibly also through the intestine and other tissues. in , ding et al. detected organ distribution of sars-cov by immunohistochemistry and in situ hybridization. sars-cov was mainly found in the respiratory system, stomach, small intestine, kidneys, and sweat glands [ ] . the organ distribution of sars-cov- is probably similar to sars-cov. based on the evidence described above, we hypothesize that sars-cov- invades the lungs and intestine through tmprss -cleaved ace . if the immune system is not able to defeat the infection, sars-cov- will be massively replicated, occupy cellular ace , and destroy the host's cells. as a consequence, the ang ii-at signaling cannot be inactive. together, the intestinal function is ruined and the inflammation exacerbated. as a result, a cytokine storm occurs and eventually the respiration system, cardiovascular system and other organs lose function ( figure ) . clinical data showed that among covid- inpatients, about % have underlying diseases. these patients have an increased risk of death. hypertension was the most common, followed by diabetes and coronary heart disease [ , ] . since the ang ii-at axis is hyperactive in these diseases already [ , ] , sars-cov- further decreases the production of functional ace . therefore, in patients with those underlying diseases it is much easier to develop severe and critical conditions. cellular ace can be shed by both adam and tmprss . the adam -cleavage is a normal path which results in the production of circulating ace . circulating ace can prevent severe pathological conditions and protect organs during sars-cov- infection. in contrast, tmprss -shed ace allows the sars-cov- to invade cells in the lungs and intestine. tmprss -cleavage path might inhibit adam -cleavage path. if the immune system is not able to defeat the virus, sars-cov- will be massively reproduced, occupy cellular ace , and destroy the host's cells in the lungs and intestine. as sars-cov- reduces ace expression, there is not enough adam -shed circulating ace against the ang ii signaling-induced inflammatory injuries, and inflammation is accelerated until the immune system is overwhelmed. meanwhile, cellular ace /b at in the intestine is ruined by the virus. essential amino acids cannot be absorbed, the antimicrobial peptides are reduced, and the ecology of the gut microbiome is damaged. these intestinal changes will precipitate inflammation. as a result, a cytokine storm occurs and eventually induces multiple organ dysfunction or failure. cellular ace can be shed by both adam and tmprss . the adam -cleavage is a normal path which results in the production of circulating ace . circulating ace can prevent severe pathological conditions and protect organs during sars-cov- infection. in contrast, tmprss -shed ace allows the sars-cov- to invade cells in the lungs and intestine. tmprss -cleavage path might inhibit adam -cleavage path. if the immune system is not able to defeat the virus, sars-cov- will be massively reproduced, occupy cellular ace , and destroy the host's cells in the lungs and intestine. as sars-cov- reduces ace expression, there is not enough adam -shed circulating ace against the ang ii signaling-induced inflammatory injuries, and inflammation is accelerated until the immune system is overwhelmed. meanwhile, cellular ace /b at in the intestine is ruined by the virus. essential amino acids cannot be absorbed, the antimicrobial peptides are reduced, and the ecology of the gut microbiome is damaged. these intestinal changes will precipitate inflammation. as a result, a cytokine storm occurs and eventually induces multiple organ dysfunction or failure. as addressed above, the strategies for preventing and reversing severe and critical covid- should include: since tmprss plays a very important role in sars-cov- cell entry and ace dysfunction, blocking the activity of tmprss should be the primary strategy for preventing severe and critical conditions of covid- . hoffmann et al. discovered that a serine protease inhibitor, camostat mesylate, partially blocked tmprss -ace -mediated sars-cov- entry [ ] . similarly, nafamostat mesylate inhibited the tmprss -ace -mediated sars-cov- envelope-plasma membrane fusion, and then showed times higher efficiency than camostat mesylate in preventing sars-cov- cell entry [ ] . both camostat mesylate and nagamostat mesylate are clinically approved medicines with verified safety, and thus can be applied for covid- treatment in clinical practices immediately. nafamostat mesylate also has been used as a short-acting anticoagulant due to its ability to prevent the proteolysis of fibrinogen into fibrin. clinical reports of covid- showed that increased d-dimer (a fibrin degradation product) levels in severe and critical patients were often observed [ , , ] . high levels of d-dimer (> µg/ml) are related to higher risk of mortality [ ] . thus, nafamostat mesylate not only can block the viral entry, but also prevent thrombosis and disseminated intravascular coagulation (dic) in covid- patients. a clinical trial of covid- with nafamostat mesylate treatment in japan started in march . because sars-cov- exploits ace for entry, some scientists are concerned that the expression of ace might provide possible advantageous routes for virus entry. they suggest that patients with hypertension, diabetes, and cardiovascular diseases should reduce the use of ace inhibitors and ang ii-at blockers because these medicines can increase the expression of ace [ ] . ibuprofen and thiazolidinediones were also suspected of increasing the risk of covid- for the same reason [ ] . however, there is no evidence that those medicines can facilitate sars-cov or sars-cov- infection. on the contrary, sars-cov infection could reduce ace expression and worsen acute lung failure, which could be attenuated by blocking the raas pathway [ ] . ace expression was also shown to protect against severe acute lung failure in a mouse model [ ] . in addition, cell tropism of sars-cov and sars-cov- did not firmly associate with ace expression. ace is expressed on both type i and type ii pneumocytes, whereas sars-cov and sars-cov- only make use of type ii pneumocytes because the infection requires co-expression of ace and tmprss as we described before [ , , ] . therefore, increasing ace (especially circulating ace ) or ang-( - ) is a potential approach to reduce sars-cov- -induced severe damage and to protect organs. recently, an international research team showed that clinical-grade human soluble ace could combine with sars-cov- and decrease its infection rate to -times in engineered human blood vessel organoids and human kidney organoids [ ] . evidence showed that administration with trp or nicotinamide could reverse severe intestinal inflammation in ace knockout mice [ ] . as a nutrient enhancer, trp and its metabolites play crucial roles in gut microbiota maintenance, microbial metabolism, the host's immune system, the host-microbiome interface, and host immune system-intestinal microbiota interactions. [ ] . thus, supplementation with trp or nicotinamide may regulate the gut microbiome and increase antimicrobial peptides to convert sars-cov- -induced lesions in the intestine and further improve systemic conditions. in summary, ace plays essential roles in sars-cov- cell entry and makes an impact on the progress and prognosis of severe and critical conditions of covid- . regulating ace -related enzymes and amino acid intake would be desirable for disease control. however, more experimental and clinical studies are required. moreover, pathogenesis of severe and critical conditions of covid- is very complicated. other molecules, such as il- , also play important roles in the disease. therefore, therapeutic strategies should be flexibly changed according to the patient's clinical inspection results. funding: this research received no external funding. characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention angiotensin-converting enzyme is a functional receptor for the sars coronavirus sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor enhanced isolation of sars-cov- by tmprss -expressing cells a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase tumor necrosis factor-alpha convertase (adam ) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme- (ace ) receptor and viral determinants of sars-coronavirus adaptation to human ace tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis angiotensin-converting enzyme is an essential regulator of heart function angiotensin-converting enzyme overexpression in the subfornical organ prevents the angiotensin ii-mediated pressor and drinking responses and is associated with angiotensin ii type receptor downregulation tmprss and adam cleave ace differentially and only proteolysis by tmprss augments entry driven by the severe acute respiratory syndrome coronavirus spike protein efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry differential downregulation of ace by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection tmprss contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection cloning and characterization of the cdna and gene for human epitheliasin prostate-localized and androgen-regulated expression of the membrane-bound serine protease tmprss sars-cov- receptor ace is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - aceh/ace is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ace inhibitors. can hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase angiotensin-( - ) prevents activation of nadph oxidase and renal vascular dysfunction in diabetic hypertensive rats angiotensin-( - ) inhibits allergic inflammation, via the mas receptor, through suppression of erk / -and nf-κb-dependent pathways angiotensin-( - ) - ) is an endogenous ligand for the g protein-coupled receptor mas angiotensin-converting enzyme as a therapeutic target for heart failure circulating activities of angiotensin-converting enzyme, its homolog, angiotensin-converting enzyme , and neprilysin in a family study circulating angiotensin-converting enzyme activity in patients with chronic kidney disease without previous history of cardiovascular disease expression of ace and ace in patients with hypertensive nephrosclerosis a review of urinary angiotensin converting enzyme in diabetes and diabetic nephropathy angiotensin ii revisited: new roles in inflammation, immunology and aging identification of splenic reservoir monocytes and their deployment to inflammatory sites genetic ace deficiency accentuates vascular inflammation and atherosclerosis in the apoe knockout mouse enhanced angiotensin ii-induced cardiac and aortic remodeling in ace knockout mice the anti-inflammatory potential of ace /angiotensin-( - 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[crossref] . identification of an existing japanese pancreatitis drug, nafamostat, which is expected to prevent the transmission of new coronavirus infection (covid- ) diagnostic utility of clinical laboratory data determinations for patients with the severe covid- clinical features and treatment of covid- patients in northeast chongqing covid- and the cardiovascular system are patients with hypertension and diabetes mellitus at increased risk for covid- infection? a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury angiotensin-converting enzyme protects from severe acute lung failure sars-cov replicates in primary human alveolar type ii cell cultures but not in type i-like cells histopathologic changes and sars-cov- immunostaining in the lung of a patient with covid- inhibition of sars-cov- infections in engineered human tissues using clinical-grade soluble human ace this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to nathaniel green's proofreading. the authors declare no conflict of interest. key: cord- - nxsrod authors: marquez, alonso; wysocki, jan; pandit, jay; batlle, daniel title: an update on ace amplification and its therapeutic potential date: - - journal: acta physiol (oxf) doi: . /apha. sha: doc_id: cord_uid: nxsrod the renin angiotensin system (ras) plays an important role in the pathogenesis of variety of diseases. targeting the formation and action of angiotensin ii (ang ii), the main ras peptide, has been the key therapeutic target for last three decades. ace‐related carboxypeptidase (ace ), a monocarboxypeptidase that had been discovered years ago, is one of the catalytically most potent enzymes known to degrade ang ii to ang‐( ‐ ), a peptide that is increasingly accepted to have organ ‐protective properties that oppose and counterbalance those of ang ii. in addition to its role as a ras enzyme ace is the main receptor for sars‐cov‐ . in this review, we discuss various strategies that have been used to achieve amplification of ace activity including the potential therapeutic potential of soluble recombinant ace protein and novel shorter ace variants. the renin-angiotensin system (ras) in its traditional view entails an enzymatic cascade of reactions leading to the generation of angiotensin ii (ang ii), the main peptide of the ras, which has a variety of biological effects [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . angiotensin ii is a potent vasoconstrictor and also promotes renal sodium retention, actions that sustain blood pressure and are part of the stress response triggered to maintain circulating volume when survival is threatened by bleeding and other hypovolemic situations [ ] [ ] [ ] . in addition, ang ii has hemodynamic actions that are key to maintain the renal circulation. this peptide, however, exerts a myriad of actions at the tissue level that can be deleterious particularly when sustained chronically [ ] [ ] [ ] . such adverse actions include pro-inflammatory, pro-proliferative and pro-atherosclerotic effects that are independent of its effect on systemic blood pressure and renal hemodynamic actions [ ] [ ] [ ] [ ] [ ] . angiotensin ii also increases the production of reactive oxygen species (ros). this action results from activation of nicotinamide adenine dinucleotide phosphate (nadph). the increase in ros contributes to the unwanted effects of this peptide , . ras blockade based on inhibiting the formation of ang ii with ace inhibitors or blocking the activation of the ang ii type (at ) receptor is a widely used therapy for kidney and cardiovascular disease. pathways that regulate the degradation of ang ii may also be important for determining levels of ang ii, particularly at the tissue level . until recently, however, little attention had been paid to enhancing ang ii degradation as a way to counteract ras over-activity which is usually present in kidneys from experimental models of diabetic kidney disease and likely in patients with many causes of ckd. several enzymes are involved in the degradation of ang ii (figure ) . in this review we will focus on an ace-related carboxypeptidase (ace ), a homolog of ace, described in , . ace shares % homology with the metalloprotease catalytic domains of ace . ace , unlike ace, contains only one active domain , . in its full length form ace has amino acids whereas the soluble form of ace has only amino acids. ace is not inhibited by any of the existing ace inhibitors. ace acts by this article is protected by copyright. all rights reserved removing single amino acids from the c terminus of its peptide substrates , . it is one of the more catalytically potent enzymes known to convert the vasoconstrictor ang ii to ang-( - ) , , ang - is increasingly accepted to have vascular-protective and reno-protective properties that oppose and counterbalance those of ang ii, such as vasodilation and oxidative stress [ ] [ ] [ ] [ ] . within the renin angiotensin system, the other known target peptide for ace cleavage is ang i with the subsequent formation of ang-( - ) [ ] [ ] [ ] . recent interest in ace has increased dramatically as a result of the recognition that it is the main receptor for sars-cov , the coronavirus responsible for the current covid pandemic. studies in experimental models of either genetically or pharmacologically-induced ace ablation have generally reported deleterious effects in various organs. therefore, it is not surprising that over the past several years approaches aimed at augmentation of ace activity had gained a significant interest for their therapeutic potential in a variety of pathological conditions. in this review, we will focus on presenting research done in our laboratory and others using various strategies to achieve amplification of ace activity and discuss its therapeutic implications. two studies in reported the existence of a new enzyme that was termed ace , . this discovery now years ago created an interest in the ace /ang-( - ) axis. in general, ace and ang ( - ) are felt to exert beneficial actions that are organ protective , , . ace and two other peptidases, prolylcarboxipeptidase (prcp) and prolylendopeptidase (pop) are the known enzymes responsible for the formation of ang - from ang ii ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . [ ] [ ] [ ] [ ] these three enzymes cleave the phenylalanine amino acid from the c-terminal end of ang ii-( - ) to form ang - (figure ) . the relative importance varies from tissue to tissue. for instance, ace is very critical for this action in the kidney whereas pop is the dominant enzyme in lungs and systemic circulation. ang i ( - ) can also contribute to the formation of ang ( - ). the conversion to ang - i from ang i is produced by neprilysin. , , ace contributes to the formation of ang ( - ) not only by enhancing the formation ang ( - ) from this article is protected by copyright. all rights reserved ang ii ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) but also by increasing the formation of ang-( - ) from ang i. angiotensin ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) could be then converted to ang - by ace , , neprilysin or cathepsin a . interestingly, ang ( - ) could also contribute to the protective effects since this peptide has been proposed to activate the at r (figure ) . while this review is limited to the formation of ang-( - ) from ang ii by ace it should be noted that ace hydrolyzes several other peptide substrates. those include apelins (i.e. apelin- and apelin- ), the opioid peptide dynorphin a, the kallikrein-kinin-system peptide des- arg bradykinin, and ghrelin, a growth hormone secretagogue . in addition to the catalytic properties of ace this protein may also exert non-enzymatic functions . of great interest , the membrane-bound ace is known to be the receptor for the severe acute respiratory syndrome-associated coronavirus (sars-cov) and recently for sars-cov , the coronavirus responsible for covid- , . (figure ) given the rapid emergence of the cov- pandemic, there is increasing interest in ace and its tissue distribution. this enzyme is only present in small amounts in the circulation but heavily expressed in certain organs such as kidneys and in the intestines . in comparison with these organs the expression of ace in the lungs is low but present in alveolar type ii pneumocytes . unlike the full length ace , which is anchored to the plasma membrane, the soluble form of ace circulates but in very small amounts. this article is protected by copyright. all rights reserved ). most of the work so far has been done in experimental animals in humans healthy volunteers recombinant ace reduced the level of ang ii. even though ang ( - ) infusion failed to demonstrate vascular effects in humans. sasaki et al reported that ang ( - ) infusion increased forearm blood flow in healthy subjects whereas in patients with hypertension there was a minimal effect. in human adipose and atrial micro vessels, ang ( - ) induces vasodilation via nitric oxide (no)-dependent and telomerase-dependent processes through masr, effects that seems to be absent in patients with coronary artery disease. finally, ace /ang - /mas receptor axis undoubtedly seems to have a protective effect on different organ and systems. interestingly different studies, however, reported that ang ( - ) has deleterious effects to increase blood pressure and exacerbate cardiac fibrosis in subtotal nephrectomy rats kidney disease model in association with increased cardiac ace activity , several approaches can be used experimentally to increase ace activity ( viral delivery systems using adenovirus, adeno-associated virus or lentivirus, have been used as proof-of-concept approaches to augment ace expression in vivo at the central nervous system and variety of peripheral tissues. huentelman et al first used lentiviral vector encoding mouse ace (lenti-mace ) to amplify ace activity in the heart. lenti-mace was injected intracardiac in -day-old sprague-dawley rats. angiotensin ii administration for weeks to control rats resulted in the expected increase in systolic blood pressure, increased weight to body weight ratio and increased myocardial fibrosis. transduction with lenti-mace resulted in a decrease in the heart weight to body weight ratio and a reduction in the myocardial fibrosis caused by infusion of angiotensin ii . this improvement in cardiac hypertrophy was associated with increased expression of ace in cardiac tissue. similar to ang ii-induced hypertension, in the spontaneously hypertensive rat (shr) murine ace gene transfer into the heart using lentiviral transduction attenuated hypertension and the associated pathological changes, such as left ventricular wall thickness and of perivascular fibrosis . in addition, an improvement of cardiac function, as evidenced by an increase in left ventricular end diastolic and end systolic diameters in shr rats after ace overexpression was observed . the use of lentivirus and adeno-associated viral systems also allowed to generate ace overexpressing transgenic animals for studies examining effects of cardiac-specific ace amplification in adult animals. these viral systems, similar to conditional transgenic models, have the advantage of long-term in vivo ace over-expression that could be "turned on" in adult life and therefore avoid the interference from the effect of developmental ace overexpression. in this respect it should be noted that initial study in traditional transgenic mice with cardiac overexpression of human full-length ace was not encouraging . even though transgenic mice appeared healthy, they died prematurely. their diminished survival rates correlated with the extent of ace overexpression in two transgenic lines suggesting a transgene dose effect . hearts from both transgenics, however, were essentially normal, this article is protected by copyright. all rights reserved without hypertrophy and similar to wild type. in addition, transgenic and non-transgenic littermates were similar to each other by echocardiography. moreover, by cardiac catheterization ventricular performance, was similar . the mortality of the transgenic mice could ultimately be explained by electrophysiological analyses that revealed conduction disturbances and lethal ventricular arrhythmias in hace transgenic mice. it still remains to be examined whether or not the discrepancy between the cardiac effects by virally-induced ace overexpression in adult animals and those reported in ace transgenic mice are due to developmental issues. yamazato m et al showed the long terms effect of ace on blood pressure. in this study, shr rats had a relative deficiency in ace protein expression within the cardiovascular regulatory neurons of the rostral ventrolateral medulla (rvlm) when compared with normotensive wistar-kyoto rats. attempted correction of this deficiency in the shr rats by ace overexpression by lentivirus injection into rvlm resulted in long-term reduction in blood pressure .therefore central ace overexpression could correct its intrinsic decrease in the rvlm and, similarly to the above discussed cardiac ace overexpression , leading to a substantial blood pressure reduction in the shr rat . in lungs, the involvement of ras in the pathogenesis of certain conditions such as pulmonary hypertension (ph) has been inferred from the high abundance of ace in the pulmonary vasculature , high ace levels likely contribute to excessive generation of ang this article is protected by copyright. all rights reserved ph-induced lung injury suggesting support for ace as a possible target for upregulating strategies for the treatment of this disease it is also important to mention the protective role of ace on the nervous system. feng et al studied the effects of ace using a transgenic mouse model with high expression of hace protein in the brain. in this study they concluded that over-expression of ace would attenuate the development of neurologic hypertension as a consequence of attenuation of parasympathetic tone and spontaneous baroreflex sensitivity. of note, neurological deficits improvement and cerebral infarct size reduction after a neurological ischemic event have been attributed antioxidative and anti-inflammatory effects of ace /ang ( - )/mas axis. therefore, ace could also be a target to prevent and treat ischemic stroke in the future. various studies have shown a role of ace in kidney disease. nadarajah et al. generated a model of glomerular ace overexpression using a podocyte-specific ace transgenic mice and showed partial protection against the early development of albuminuria . preservation of podocyte proteins and podocyte number, were seen in stz-induced diabetes transgenic mice with overexpression of the human ace protein . that kidney over-expressed ace can ameliorate glomerular injury in diabetic animals was also suggested by a study using adenoviral kidney ace (ad-ace ) overexpression in stz rats . compared with control, the ad-ace treated group showed a reduction in systolic blood pressure and improvement in urinary albumin excretion, creatinine clearance and glomeruli sclerosis index. ad-ace also had decreased tgf-β , vascular endothelial growth factor and collagen iv protein expression. no additional benefit of ace inhibition was noticed in the combined use of ad-ace and acei . overall these studies suggest that kidney ace amplification may represent a therapeutic target in the treatment of glomerular injury in diabetic kidney disease. this article is protected by copyright. all rights reserved minicircle dna vectors consist of a circular expression cassette devoid of the bacterial plasmid dna backbone that provides sustained transgene expression in quiescent cells/tissues . we studied the effects of murine recombinant ace in streptozotocininduced diabetes in mice as well as the effect of increasing circulating ace using minicircle dna delivery. this approach resulted in sustained increase in serum ace activity and enhanced ability to degrade infused angiotensin ii ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . . in mice with streptozotocininduced diabetes pretreated with ace , minicircles, plasma ace protein increased as shown by western blot and ace serum activity increased more than -fold. urinary ace activity and kidney ace , however, did not increase despite the profound augmentation of ace in plasma. moreover, the glomerular lesions and hyperfiltration seen in this diabetic model of experimental kidney disease were not affected at all l . from these findings we concluded that, targeting kidney ace , rather than circulating plasma ace , might be necessary to effectively treat early diabetic kidney disease as seen in the stz-model. ace in its full-length form is a - kda-protein comprising amino-acids (aa). it is a type-i transmembrane protein that contains a major extracellular domain (aa - ), and the much smaller: transmembrane region (aa - ), and intracellular tail ( - ) this article is protected by copyright. all rights reserved of acute ang ii dependent hypertension. (figure ). imai et al. used hrace to examine its effect on experimental acute lung injury. using aspiration-induced acute lung injury murine model they injected human recombinant ace (hrace ) into acid-treated ace knockout mice and observed a decrease in the degree of acute lung injury, and pulmonary edema formation. hrace has also been proposed as a potential candidate to treat diastolic and systolic heart failure . zhong et al showed that hrace ameliorated pressure overload induced as well as ang ii-induced myocardial remodeling. hrace has also shown to reduce the level of angiotensin ii in healthy human volunteers. despite the ace blockade the levels of angiotensin ii could remain elevated via chymase system. all of the above suggest that therapies using hrace for heart failure seem promising. human recombinant ace has been evaluated in human subjects in limited clinical studies .the pharmacokinetics, pharmacodynamics, safety, and tolerability of hrace were determined in healthy volunteers . this study was randomized, double-blind, and placebo- this article is protected by copyright. all rights reserved encouraging but still very preliminary findings were drawn from a recent phase iia, openlabel pilot study which suggested a potential therapeutic role for hrace in pulmonary arterial hypertension . this study found a reduced plasma ace activity in subjects with ph. this was inferred, from higher plasma ang ii to ang ( - ) ratio however, the ratio is not specific as it can potentially be affected by changes in other enzymes that affect the conversion of ang ii to ang ( - ) . at baseline in subjects with ph, increased expression in six out of nine measured cytokines as compared with controls (interleukin (il)- , il- β, tumor necrosis factor (tnf)-α, il- , il- and il- ) was found. reduced plasma superoxide dismutase (sod ), which is considered an anti-oxidant enzyme, and increased oxidant stress parameters were also observed . after hrace , cytokines such as il- , il- β, il- and tnf-α were decreased hours after administration. human race administration was reported to also beneficially influence sod levels, and reduce plasma oxidant stress. these findings were based on a limited number of subjects. further assessment of hrace as a potential therapeutic in ph certainly will require larger studies. table provides a summary of studies so far that have used human soluble recombinant ace . while the human race is the ultimate form of the protein that can be used in clinical studies, it has some limitations for preclinical research in rodents. human race can certainly be used in acute studies in rodents. there is limited value, however, for chronic studies due to the development of antibodies that decrease the enzymatic activity of ace this article is protected by copyright. all rights reserved race of - aa might not be effective. consistent with this notion, we have found that in stz-treated mice, a diabetic mouse model with local kidney but not systemic ras overactivity , long-term augmentation of circulating ace activity was not sufficient to beneficially alter albuminuria, gfr or kidney histology. glomerular filtration of administered race is a prerequisite for a subsequent tubular uptake of the protein from the urinary space , . kidney targeting of biologics is complex and can be affected by many factors including their molecular size . we reasoned that in forms of kidney disease with an overactive ras within the kidney , ace amplification would require forms of recombinant ace that are short enough to be able to pass the glomerular filtration barrier and be consequently reabsorbable by the kidney to be able to exert any direct kidneyspecific therapeutic effect. based on these considerations, we have generated ace variants of shorter molecular size that still retain enzymatic activity (see below). we have generated and tested two novel recombinant mouse ace proteins of a molecular size (~ - kd) which are much shorter than the original soluble race of - this article is protected by copyright. all rights reserved longer than that of ace this article is protected by copyright. all rights reserved soluble race . in addition, ex vivo kidney cortex lysates from ace - -injected mice were able to form significantly more ang ( - ) from ang ii than kidney lysates from pbs-or original soluble race -injected mice). in the aggregate these data demonstrate that short ace variants are active, and sufficiently small to be filtered by the kidney and moreover capable to increase kidney ace activity to an extent that ang ( - ) formation from ang ii is increased. the crystal structure of ace was solved by hernández prada et al and putative small molecule binding pockets were identified. these authors carried out in silico screening of a small molecule library followed by in vitro studies and found that out of the sites, two of them were inhibitor sites and the third one was a presumed activator site. the same investigators identified then two small-molecular compounds as ace activators: xnt and resorcinolnaphthalein. xnt was preferred over resorcinolnaphthalein since xnt solubility properties appeared to be more favorable. three years later an agent commonly used in to treat some parasitizes in animals called diminazene (dize), was proposed as an ace activator . both xnt and dize have been used experimentally to serve as potential treatment for various conditions such as certain types of hypertension - , pulmonary hypertension [ ] [ ] [ ] . cardiac and renal fibrosis and glaucoma .the therapeutic benefits of these two components should be attributable to conversion of ang ii to ang ( - ) as a consequence of ace activation , , [ ] [ ] [ ] [ ] . one of the caveats with many of these studies is, however, that the effect on ace activity was usually not reported in vivo , , - , - . in these studies moreover it was not shown that the use of these presumed ace activators had taken place by demonstrating the enhanced conversion of ang ii to ang ( - ). our group reported low levels plasma ace activity in both vehicle and xnt infused mice , . furthermore, after ang ii infusion the plasma levels ang ( - ) , the peptide generated by the cleavage of ang ii and plasma levels ang ii, the substrate of ace , were not affected by the administration of xnt. therefore, it was suggested that the effect of xnt on ang ii- this article is protected by copyright. all rights reserved in a cell free system and also to upregulate ace mrna expression in cultured cells . there have been no follow up studies, to our knowledge, with the irw compound which would independently assess its potency as an ace activator. as of today there is no convincing evidence, in our opinion, that the presumed ace activators studied exert their otherwise undisputable biologic effects by activating ace as their main mechanism of action from the forgoing, it is logical to postulate that ace amplification within the kidney is a very attractive therapeutic approach to promote the metabolism of ang ii and reduce its detrimental actions. some of the potential diseases that could benefit from ace amplification have been mentioned already while discussing the methods up to attempt to increase ace activity. as noted earlier, partial kidney protection against stz-induced diabetic kidney disease could be shown in a transgenic model where ace overexpression was limited to glomerular podocytes . in one study human recombinant ace was shown to improve diabetic kidney disease in akita mice . when these mice were injected with hrace for weeks, hrace normalized blood pressure and reduced albuminuria. unlike other models of early dkd the akita mouse is hypertensive and is very possible that the improvement in albuminuria noted in this study was related to lowering of blood pressure by ace by decreasing ang ii which is elevated in this model. in keeping with previous studies in mice by ye et al. in ace activity as well as an enhanced ability to metabolize acute load of ang ii resulted from ace minicircle delivery however, this augmented ace activity achieved by this technique did not affect urine ace activity. moreover, there were no improvements in albuminuria, glomerular expansion, glomerular cellularity or glomerular size when compared to vehicle -treated diabetic controls. (figure ) therefore, this study emphasized the importance of targeting the kidney rather the circulating ras in order to treat diabetic nephropathy. in other study using col a -/-mouse model of alport's syndrome, murine recombinant (mr) ace also decreased markers of kidney injury. ace plasma activity was increased using mrace mini-pumps leading to an improvement in albuminuria, reduced erk /erk this article is protected by copyright. all rights reserved signaling and amelioration of inflammation, fibrosis and oxidative stress . in this study renal function did not improve as measured by blood urea nitrogen and gfr was not measured. in this model the profound defect in glomerular permeability allows the infused race , despite its large molecular size, to get filtered [ ] [ ] [ ] [ ] [ ] , but also in aki and are associated with adverse outcomes both in experimental and clinical studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we think that aki could be an initial target to explore in humans the potential preventative effect of our short ace variants. finally, this review comes at a time that the world is grappling with the covid pandemic caused by sars-cov virus. from the sars outbreak in we have known that sars-cov spike glycoprotein recognized ace as a receptor on the cell surface for host entry .consequently, there have already been several reports looking at the association of sars-cov and ace , . it is known that sars-cov does bind to ace to gain host cell entry causing its cell internalization and likely reducing membrane ace expression in murine models the dysregulation of ace is associated with cardiac, pulmonary and kidney alterations . there is preliminary data from covid- subjects in whom elevated levels of plasma angiotensin ii correlated with degree of lung injury . further prior preclinical studies in respiratory syncytial virus and avian h n influenza this article is protected by copyright. all rights reserved suggested that restoration of ace by recombinant ace administration appeared to reverse worsening lung injury , , . therefore, there is now significant interest in looking at recombinant ace protein to rebalance the ras network and potentially help mitigate the pulmonary, cardiac and kidney damage done by covid . we have proposed that soluble ace may act as a competitive interceptor of sars-cov and sars-cov by preventing binding of the viral particle to the surface-bound, full-length ace . .in this context, administration of soluble recombinant human ace proteins might be beneficial as a novel biologic therapeutic to limit or even combat the infection progression caused by coronaviruses that utilize ace as a receptor. regulation of blood pressure by the type a angiotensin ii receptor gene renin-angiotensin system and atherothrombotic disease: from genes to treatment role of the renin-angiotensin system in vascular diseases: expanding the field the renin-angiotensin system and the neurodegenerative diseases: a brief review 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the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis accepted article this article is protected by copyright. all rights reserved soluble angiotensin-converting enzyme : a potential approach for coronavirus infection therapy tissue-specific amino acid transporter partners ace and collectrin differentially interact with hartnup mutations angiotensin converting enzyme (ace) and ace bind integrins and ace regulates integrin signalling role of the ace /angiotensin - axis of the renin-angiotensin system in heart failure angiotensin-( - ) does not affect vasodilator or tpa responses to bradykinin in human forearm vascular actions of angiotensin - in the human microcirculation: novel role for telomerase adverse cardiac effects of exogenous angiotensin - in rats with subtotal nephrectomy are prevented by ace inhibition angiotensin-( - ) infusion is associated with increased blood pressure and adverse cardiac remodelling in rats with subtotal nephrectomy protection from angiotensin ii-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ace in rats ace gene transfer attenuates hypertension-linked pathophysiological changes in the shr accepted article this article is protected by copyright. all rights reserved heart block, ventricular tachycardia, and sudden death in ace transgenic mice with downregulated connexins overexpression of angiotensin-converting enzyme in the rostral ventrolateral medulla causes long-term decrease in blood pressure in the spontaneously hypertensive rats role of angiotensin-converting enzyme and angiotensin ii in development of hypoxic pulmonary hypertension expression of pulmonary vascular angiotensin-converting enzyme in primary and secondary plexiform pulmonary hypertension prevention of pulmonary hypertension by angiotensin-converting enzyme gene transfer brain-selective overexpression of human angiotensin-converting enzyme type attenuates neurogenic hypertension ace -ang-( - )-mas 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species-specific activation of the alternative renin-angiotensin-system regulation of urinary ace in diabetic mice angiotensin-converting enzyme -independent action of presumed angiotensin-converting enzyme activators: studies in vivo, ex vivo, and in vitro novel ace -fc chimeric fusion provides long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation novel variants of angiotensin converting enzyme- of shorter molecular size to target the plant tannin immobilized fe o @sio microspheres: a novel and green magnetic bio-sorbent with superior adsorption capacities for gold and palladium urine ras components in mice and people with type diabetes and chronic kidney disease characterization of proteinuria and tubular protein uptake in a new model of oral l-lysine administration in rats urinary renin in patients and mice with diabetic kidney disease accepted article this article is protected by copyright. all rights reserved structure-based identification of smallmolecule angiotensin-converting enzyme activators as novel antihypertensive agents diminazene aceturate (berenil) modulates the host cellular and inflammatory responses to trypanosoma congolense infection prediction of off-target effects on angiotensin-converting enzyme activation of angiotensin-converting enzyme /angiotensin-( - )/mas axis attenuates the cardiac reactivity to acute emotional stress evidence for angiotensin-converting enzyme as a therapeutic target for the prevention of pulmonary hypertension diminazene aceturate improves autonomic modulation in pulmonary hypertension diminazene attenuates pulmonary hypertension and improves angiogenic progenitor cell functions in experimental models antiglaucomatous effects of the activation of intrinsic angiotensin-converting enzyme diminazene aceturate enhances angiotensin-converting enzyme activity and attenuates ischemia-induced cardiac pathophysiology angiotensin-converting enzyme activation protects against hypertension-induced cardiac fibrosis involving extracellular signal-regulated kinases angiotensin-converting enzyme activation improves endothelial function accepted article this article is protected by copyright. all rights reserved ace activation promotes antithrombotic activity. molecular medicine activation of angiotensin-converting enzyme /angiotensin-( - )/mas axis attenuates the cardiac reactivity to acute emotional stress chronic activation of endogenous angiotensin-converting enzyme protects diabetic rats from cardiovascular autonomic dysfunction oral administration of an angiotensin-converting enzyme activator ameliorates diabetes-induced cardiac dysfunction. regulatory peptides antiglaucomatous effects of the activation of intrinsic angiotensin-converting enzyme diminazene aceturate enhances angiotensin-converting enzyme activity and attenuates ischemia-induced cardiac pathophysiology diminazene aceturate prevents nephropathy by increasing glomerular ace and at receptor expression in a rat model of type diabetes egg white-derived tripeptide irw (ile-arg-trp) is an activator of angiotensin converting enzyme human recombinant ace reduces the progression of diabetic nephropathy glomerular localization and expression of angiotensin-converting enzyme and angiotensin-converting enzyme: implications for albuminuria in diabetes accepted article this article is protected by copyright. all rights reserved american journal of kidney diseases : the official journal of the national kidney foundation decreased glomerular and tubular expression of ace in patients with type diabetes and kidney disease murine recombinant angiotensin-converting enzyme attenuates kidney injury in experimental alport syndrome ace as therapy for glomerular disease: the devil is in the detail role of the intrarenal renin-angiotensin-aldosterone system in chronic kidney disease the intrarenal renin-angiotensin system and diabetic nephropathy the renin-angiotensin system and diabetic nephropathy renal renin-angiotensin system in diabetes: functional, immunohistochemical, and molecular biological correlations the role of renin-angiotensin-aldosterone system genes in the progression of chronic kidney disease: findings from the chronic renal insufficiency cohort (cric) study angiotensinogen as a biomarker of acute kidney injury role of at angiotensin ii receptors in renal ischemic injury ace -angiotensin-( - )-mas axis in renal ischaemia/reperfusion injury in rats differential actions of renal ischemic injury on the intrarenal angiotensin system fiend and friend in the renin angiotensin system: an insight on acute kidney injury acute kidney injury intracardiac and intrarenal renin-angiotensin systems: mechanisms of cardiovascular and renal effects intrarenal angiotensin and bradykinin peptide levels in the remnant kidney model of renal insufficiency renin-angiotensin-aldosterone system inhibitors in patients with covid- a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury clinical and biochemical indexes from -ncov infected patients linked to viral loads and lung injury angiotensin-converting enzyme protects from lethal avian influenza a h n infections angiotensin-converting enzyme inhibits lung injury induced by respiratory syncytial virus accepted article key: cord- -htwlfqos authors: liu, qiang; du, jianchao; yu, xuezhong; xu, jun; huang, fengming; li, xiaoyun; zhang, cong; li, xiao; chang, jiahui; shang, daozhen; zhao, yan; tian, mingyao; lu, huijun; xu, jiantao; li, chang; zhu, huadong; jin, ningyi; jiang, chengyu title: mirna- c- p is crucial in acute respiratory distress syndrome date: - - journal: cell discov doi: . /celldisc. . sha: doc_id: cord_uid: htwlfqos influenza infection and pneumonia are known to cause much of their mortality by inducing acute respiratory distress syndrome (ards), which is the most severe form of acute lung injury (ali). angiotensin-converting enzyme (ace ), which is a negative regulator of angiotensin ii in the renin–angiotensin system, has been reported to have a crucial role in ali. downregulation of ace is always associated with the ali or ards induced by avian influenza virus, severe acute respiratory syndrome-coronavirus, respiratory syncytial virus and sepsis. however, the molecular mechanism of the decreased expression of ace in ali is unclear. here we show that avian influenza virus h n induced the upregulation of mir- c- p, which was then demonstrated to target the ′-untranslated region of ace . then, we found that nonstructural protein and viral rna of h n contributed to the induction of mir- c- p during viral infection. additionally, the synthetic analog of viral double-stranded rna (poly (i:c)), bacterial lipopolysaccharide and lipoteichoic acid can all markedly increase the expression of mir- c- p in a nuclear factor-κb-dependent manner. furthermore, markedly elevated plasma levels of mir- c- p were observed in severe pneumonia patients. the inhibition of mir- c- p ameliorated the ali induced by h n virus infection in vivo, indicating a potential therapeutic target. therefore, we identify a shared mechanism of viral and bacterial lung infection-induced ali/ards via nuclear factor-κb-dependent upregulation of mir- c- p to reduce ace levels, which leads increased angiotensin ii levels and subsequently causes lung injury. acute respiratory distress syndrome (ards), the most severe form of acute lung injury (ali), is the main predisposing factor in highly pathogenic avian influenza virus-induced death cases [ , ] . the ali caused by influenza infection can facilitate bacterial superinfection, which is a major factor that promotes mortality and disease severity [ ] . from to , highly pathogenic avian h n influenza virus has caused human infection cases worldwide with a high mortality rate of . % (http:// . . . /www. who.int/entity/influenza/human_animal_interface/ _ _ _tableh n .pdf). currently, china is undergoing its fifth epidemic of human infections of avian h n influenza virus. until january , h n infected human cases with at least deaths have been reported to world health organization (http:// . . . /www.who.int/influenza/human_animal_inter face/influenza_summary_ira_ha_interface_ _ _ _final.pdf?ua = ). with high mutation and reassortment rates, influenza viruses have evolved with a rapid resistance to the current vaccines and anti-viral drugs, making an avian influenza pandemic an urgent unresolved threat to human health [ ] [ ] [ ] . angiotensin-converting enzyme (ace ) was firstly identified from human cardiac left ventricle complementary dna (cdna) library and lymphoma cdna library by two separate groups [ , ] . ace inactivates angiotensin ii (ang ii) by cleaving it to produce ang - [ ] . ang ii binds to the ang ii type and type receptors with strong affinity, mediating regulation of blood pressure, body fluid balance, inflammation, cell proliferation, hypertrophy and fibrosis [ ] [ ] [ ] . reduced expression levels of ace have been reported in hypertension and chronic kidney disease [ , ] . in addition, ace was identified as a key regulator of dietary amino-acid homeostasis and gut microbial ecology [ ] . we previously demonstrated that ace counteracts the development of severe ali or ards induced by avian influenza virus, severe acute respiratory syndrome-coronavirus spikes, sepsis and acid aspiration in mice [ ] [ ] [ ] . administration of recombinant ace or the ang ii receptor blocker losartan can ameliorate h n virus infection-induced ali [ , ] . moreover, ace was also reported to have a protective role in respiratory syncytial virus-induced lung injury [ ] . notably, plasma ang ii levels can indicate disease severity in h n infections [ ] . downregulation of ace appears to be a shared phenomenon in ali or ards induced by viral or bacterial infection [ , [ ] [ ] [ ] [ ] . micrornas (mirnas) are small non-coding rnas that function primarily in regulating gene expression by binding to the ′-untranslated region ( ′-utr) of the targeted mrnas [ ] . widespectrum diseases, such as pulmonary diseases, diabetes and cardiac disorders, can be attributed to mirna dysfunction [ ] . in recent years, multiple mirna-based drugs have been explored and have entered clinical testing [ , ] . mir- family consists of two clusters: the mir- a/b/ cluster containing mir- a- p, mir- a- p, mir- b- p, mir- b- p and mir- on chromosome p . , and the mir- c/ cluster containing mir- c- p, mir- c- p, mir- - p and mir- - p on chromosome p . . studies on this mirna family revealed that it has versatile roles in cancer progression, drug resistance and oxidative stress [ , ] . in our study, we proposed to explore the mechanism behind the downregulation of ace presenting in ali or ards induced by avian influenza virus or other pathogens. we demonstrated that mir- c- p was markedly upregulated by the infection of h n virus and the treatment of synthetic double-stranded rna poly (i:c), bacterial lipopolysaccharide (lps) and lipoteichoic acid (lta) in a nuclear factor-κb (nf-κb)-dependent manner. mir- c- p directly targets the ′-utr of ace and downregulates ace protein expression. moreover, elevated levels of mir- c- p were found in plasma samples of severe pneumonia patients. our in vivo results demonstrated that mir- c- p may be a therapeutic target for ali. to identify mirnas potentially regulating ace protein expression, the mirna expression profile in a cells (human lung adenocarcinoma epithelial cell line) challenged with h n or h n influenza virus was compared with that in a cells mock infected with allantoic fluid (af). with human mirnas at a meaningful level (read count ), we listed the top highly expressed mirnas in h n -infected cells compared with h n -infected cells (figure a ). among them, mir- c- p and mir- - p were predicted to target ace using targetscan (supplementary figure s a) . quantitative real-time pcr (qrt-pcr) analysis confirmed the upregulation of mir- c- p and mir- - p in h n -infected a cells (figure b and c) . moreover, we observed that the expression of mir- , which was reported to target ace [ ] , was not upregulated after h n and h n virus infection (supplementary figure s b) . the pattern of the upregulation of mir- c- p and mir- - p was also validated in hek t cells (human embryonic kidney cells) (supplementary figure s c and d) . additionally, the ace protein expression levels were downregulated in a cells (supplementary figure s e) and hek t cells (supplementary figure s f) after infection with h n influenza virus, which were consistent with our previous observations in the h n -infected mice lung tissues [ ] . interestingly, the h n -induced expression of mir- c- p and mir- - p was highly correlated with both cell viability (figure d and f) and viral replication (figure e and g) . however, the expression of neither mir- c- p nor mir- - p was correlated with cell viability or viral replication (supplementary figure s g and h) . then, we tested the effect of the different multiplicity of infection (moi) on the expression of mir- c- p and mir- - p. a cells were challenged with h n virus at different viral titers (moi = . , . and ), and the expression of mir- c- p and mir- - p was detected. the larger the infective dose administered, the higher the expression of mir- c- p and mir- - p (supplementary figure s i) . successful viral infection was confirmed by detecting the expression of matrix protein (m ) (supplementary figure s j) decreased the abundance of ace protein, whereas mir- c- p inhibitors increased it ( figure a ). however, we did not observe any significant change of ace expression in the group transfected with mir- - p mimics or inhibitors. previously, mir- - p has been reported to regulate tumor growth factor-β by targeting its transcript during h n infection [ ] . to demonstrate the interaction between mir- c- p and ace transcripts, we constructed a reporter vector in which the ′-utr of ace was fused downstream of renilla luciferase coding sequence (supplementary figure s a) . in hek t cells transfected with mir- c- p mimics rather than mir- c- p mimics, renilla luciferase activity significantly decreased (figure b ). successful transfection of mir- c- p mimics and mir- c- p mimics was confirmed by qrt-pcr (supplementary figure s b) . additionally, the activity was inhibited by mir- c- p mimics in a concentration-dependent manner (supplementary figure s c) . when the predicted mir- c- p binding site on ace ′-utr was deleted, the inhibiting effects of mir- c- p mimics on the luciferase activity were diminished ( figure c ). moreover, mutation of the site on mir- c- p binding to ace ′-utr abolished the inhibiting effects of mir- c- p on renilla luciferase activity (supplementary figure s d) . taken together, these results indicate that mir- c- p, rather than mir- c- p and mir- - p, directly targets the ′-utr of ace and downregulates the expression of ace protein. to elucidate how h n influenza virus induces the upregulation of mir- c- p, vectors containing the h n influenza viral protein coding genes, hemagglutinin, neuraminidase (na), nonstructural protein (ns ), nonstructural protein (ns ), polymerase complex pa, polymerase complex pb , polymerase complex pb , m , matrix protein (m ) and nucleocapsid protein (np) were each transfected into hek t cells for h. in hek t cells transfected with ns expressing vector, the upregulation of mir- c- p and mir- - p was observed ( figure d and supplementary figure s a ). in addition, m and np also induced the upregulation of mir- c- p. however, ns induced a much higher level of mir- c- p than m and np. therefore, in the subsequent experiments, we focused on the function of ns . compared with the ns of h n virus, the ns of h n and h n viruses induced much higher expression of mir- c- p and mir- - p ( figure e and supplementary figure s b ). however, ns of neither h n nor h n caused any significant change of the expression of mir- (supplementary figure s c ). transfection of h n -ns and h n -ns expression vectors into hek t cells resulted in the downregulation of ace protein levels ( figure f ). our results suggest that ns of pathogenic avian influenza virus has an important role in the induction of mir- c- p and mir- - p. to examine whether the induction of mir- c- p to downregulate ace is a specific mechanism of pathogenic avian influenza viruses, we challenged a cells with poly (i:c). mir- c- p was upregulated in a concentration-dependent manner in poly (i:c)-transfected a cells (figure a ). in addition, transfection of poly (i:c) also resulted in the downregulation of ace protein expression, which was rescued by the transfection of inhibitors of mir- c- p (figure d ), indicating that downregulating ace protein expression by mir- c- p may be a shared mechanism for rna viruses. induction of interleukin- , interleukin- and interferon-β by poly (i:c) was confirmed by qrt-pcr analysis (supplementary figure s a ). previous studies have reported that lps, which is the major constituent of the outer membrane of gram-negative bacteria, can induce the downregulation of ace and the upregulation of ang ii [ , ] . however, the mechanisms are still unclear. by exposing a and thp cells (human acute monocytic leukemia cell line) to lps, we found that viruses and bacteria are usually the precipitating factors of severe pneumonia, which may induce severe respiratory failure by triggering ards [ , ] , a clinical syndrome characterized by pulmonary edema, hypoxemia and the accumulation of inflammatory cells [ ] . ards has a high mortality rate of - %, thus demanding accurate prediction models [ , ] . because h n virus, poly (i:c), lps and lta can all induce the expression of mir- c- p to downregulate ace protein expression, we speculate that the expression of mir- c- p may be aberrant in severe pneumonia infection. to investigate whether mir- c- p is involved in severe pneumonia infection, we collected plasma from severe pneumonia patients and healthy volunteers and then detected the expression of mir- c- p in the plasma. the characteristics of the study subjects are shown in supplementary table s . of the patients, ( . %) fulfilled ards criteria [ ] . in accordance with our speculation, the plasma levels of mir- c- p were significantly elevated in severe pneumonia patients (figure g ). downregulation of ace may result in the elevated plasma level of ang ii, which is a major substrate of ace . additionally, ang ii is claimed considerably increased in ards patients [ ] . consistent with this report, the plasma levels of ang ii were also markedly upregulated in severe pneumonia patients (figure h ). notably, the plasma level of ang ii changed over mir- c- p in severe pneumonia patient (patient th) whose plasma samples were consecutively collected (figure i ), suggesting that mir- c- p may participate in the upregulation of plasma ang ii and then induces lung injury through ang ii type receptor. consequently, our results raise the possibility that mir- c- p can serve as a biomarker or therapeutic target for severe pneumonia. nf-κb signaling pathway mediates the upregulation of mir- c- p induced by h n , poly (i:c), lps and lta the transcription factor nf-κb heterodimer (p /rela and p ) has an important role in cellular responses to viral and bacterial infections [ , ] . besides, the determinant role of nf-κb in the regulation of mirna expression has been revealed by previous studies [ ] [ ] [ ] . it is well known that the activation of nf-κb signaling is essential in h n infection and propagation [ ] [ ] [ ] . disabling the function of nf-κb heterodimer with small interfering rnas (sirnas) (si-p and si-p ) or its specific inhibitors, caffeic acid phenethyl ester (cape) and jsh- , we found that the induction of mir- c- p by h n infection was significantly repressed (figure a and b and supplementary figure s a ). considering that ns was reported to inhibit nf-κb activation, we proposed that there is an nf-κb-dependent manner for influenza virus to induce the expression of mir- c- p besides ns -mediated induction of mir- c- p [ ] . figure s g) . these data suggest that the induction of mir- c- p by avian influenza a virus h n , double-stranded rna poly (i:c), bacterial lps and lta is nf-κb-dependent. to figure s a) . ards induced by h n virus in mice was used to mimic and investigate the pathogenesis of h n -induced human ards [ ] . h n virus infection results in lung edema, inflammatory cellular infiltration and lung injury in mice [ ] . our results showed that the expression of mir- c- p in the lung tissue of h n infected mice was also markedly upregulated ( figure a) . previously, we reported that the expression of ace protein was downregulated in h n -infected mouse lung tissue [ ] . as inhibition of mir- c- p resulted in recovery of ace protein level in h n -infected a cells, mir- c- p may be a therapeutic target for h n -infected mice. in fact, administration of mir- c- p antagomir ( h before, and h after infection, intraperitoneally mg kg − ) indeed improved wet-to-dry lung tissue weight ratio, which indicates the degree of lung edema (figure b) . in mice administered with mir- c- p antagomir, lung injury scores (including alveolar wall thickening, hyaline membranes formatting and airspaces filling with proteinaceous debris) [ ] were decreased, and inflammatory cell infiltration was alleviated (figure c figure s c) . administration of mir- c- p antagomir also resulted in the recovery of ace protein level in the lung tissue (figure f ). accordingly, treatment with mir- c- p antagomir markedly reduced the plasma level of ang ii of h n -infected mice as well (figure g ). in summary, our data demonstrated that inhibiting the function of mir- c- p improves lung injury and ards induced by h n virus infection, rendering mir- c- p as a potential therapeutic target. mir- c- p in viral and bacterial lung infections although influenza infection and severe pneumonia are known to cause much of their mortality by inducing ards, the mechanisms are still not fully understood, and effective drugs are still lacking [ , , ] . our study indicates that mir- c- p has a crucial role in the regulation of ace in ali or ards induced by h n virus infection and severe pneumonia. our previous studies have demonstrated that ace protects mice against ali induced by severe acute respiratory syndrome-coronavirus spikes, acid aspiration, sepsis and avian influenza virus [ , ] . in this study, we showed that avian influenza virus, poly (i:c), bacterial lps and lta use a similar approach to reduce ace level: the elevated mir- c- p directly downregulates ace expression. moreover, plasma levels of mir- c- p and its downstream effector ang ii were significantly elevated in severe pneumonia patients. while ns was reported to inhibit the activation of nf-κb, it is a matter of common observation that nf-κb signaling pathway is still significantly activated upon h n virus infection and multiple nf-κb-dependent genes are expressed [ ] [ ] [ ] [ ] . interestingly, here we found that pathogenic avian influenza virus can induce the upregulation of mir- c- p through both the expression of ns protein and the accumulation of vrna ( figure ). ns is a multifunctional protein that interacts with more than proteins to regulate innate immune pathways, splicing and mrna export [ , ] . unlike ns of human pandemic h n / , ns of avian highly pathogenic h n effectively inhibits the polyadenylation of cellular pre-mrna in a cells [ ] . batista et al. [ ] have shown that the transcription of mir- c/ can be induced by the bypass of the usual ptpn polyadenylation. ns of pathogenic avian influenza viruses may induce the expression of mir- c- p and mir- - p by bypassing the usual ptpn polyadenylation, which needed to be further investigated. moreover, a study has reported that na protein of influenza a/puerto rico/ / (h n , pr ) can downregulate ace [ ] ; therefore, whether na of h n and h n viruses can downregulate ace requires further investigation. nf-κb is a critical regulator of defensive response to diverse pathogens. viruses, bacteria, parasites and injury initiate distinct signal-transduction pathways to activate nf-κb. influenza virus can induce the activation of nf-κb by the accumulation of viral products, including viral protein and vrna [ , ] . in this study, we found that transfection of rna extracted from h n virus-infected cells elicited the upregulation of mir- c- p, which can be suppressed by the loss of p function ( figure ). however, the detailed molecular mechanism needs to be further investigated. the expression levels of mir- c- p have been found to be markedly upregulated in the livers of hepatocellular carcinoma patients [ ] . among these patients, nine patients were diagnosed with hbv infection and five patients were diagnosed with hcv infection, thus suggesting that hbv and hcv infection may induce the expression of mir- c- p. whether hcv or hbv rna can induce the upregulation of mir- c- p requires further study. as we know, poly (i:c), lps and lta can induce the activation of nf-κb via toll-like receptor , toll-like receptor and toll-like receptor , respectively [ ] [ ] [ ] . in fact, we also noticed that poly (i:c), bacterial lps and lta can induce the upregulation of mir- c- p to downregulate ace protein expression in an nf-κb signaling pathway-dependent manner ( figure ) . previous study has shown that overexpression of ace protects against lps-induced lung injury in rat [ ] . the downregulation of ace might be mediated by mir- c- p and inhibition of mir- c- p might protect against lps-induced lung injury as well. the observations represented in this study may provide an example how pathogenic avian influenza viruses and bacteria use nf-κb activity. compared with healthy controls, the plasma levels of both mir- c- p and its downstream effector ang ii were much higher in severe pneumonia patients. as previous studies have reported that angiotensin- converting enzyme inhibitors, which reduce the production of ang ii, and ang ii receptor blockers, which block the function of ang ii, do have some beneficial effects on pneumonia-related clinical outcomes [ , ] , inhibition of mir- c- p may also produce a positive clinical outcome for pneumonia. in addition, previous study has reported that the mir- c- p level was positively related to the severity of interstitial lung disease [ ] . further studies are needed to explore whether plasma levels of mir- c- p or ang ii can serve as a biomarker or therapeutic target for severe pneumonia. in recent years, mirna-based strategies for exploring novel therapeutic drugs have experienced rapid development. several mirna-targeting drugs have now entered clinical testing or are even close to gaining access to markets [ ] . we found that administration of antagomirs of mir- c- p can ameliorate mir- c- p in viral and bacterial lung infections ali and lung edema in mice induced by h n virus infection, suggesting a potential mirna-targeting drug for avian influenza viruses-mediated ali. one study has demonstrated that administration of anti-mir- c- p oligonucleotide attenuates pulmonary inflammatory responses and lung injury by inhibiting the downregulation of dual-specificity phosphatase in an lps-induced mouse model [ ] . in accordance with our result, ace was not a target of mir- c- p (figure b) . collectively, we found that ns and vrna of avian influenza virus can induce the upregulation of mir- c- p to downregulate ace protein expression. nf-κb-dependent induction of mir- c- p is a shared mechanism of h n -vrna, poly (i:c), lps and lta induced ace reduction, suggesting a molecular mechanism of ali/ards caused by bacterial and viral infections: the elevated mir- c- p downregulated ace expression, increased ang ii levels and caused lung injury through ang ii type receptor. consistently, blocking the function of mir- c- p protects h n -infected mice from ali, indicating mir- c- p as a potential therapeutic target. the influenza viruses a/jilin/ / (h n ) and a/new caledonia/ / (h n ) were used in this study. all live influenza virus experiments were performed in biosafety level facilities according to governmental guidelines. a cells (human lung adenocarcinoma epithelial cells) were purchased from the american type culture collection (atcc, rockville, md, usa) and cultured in ham's f nutrient medium (hyclone, logan, ut, usa). hek t and thp cells were purchased from the peking union medical college cell culture center (beijing, china). hek t cells were propagated in dulbecco's modified eagle's medium (hyclone). thp cells were cultured in rpmi- medium (hyclone). cells were cultured in the appropriate medium supplemented with % (v v − ) fetal bovine serum (gibco, grand island, ny, usa), u ml − penicillin and u ml − streptomycin at °c. infected with h n or h n virus (moi = ) or af control for h, a cells were collected using trizol reagent (invitrogen, carlsbad, ca, usa). total rna of the cells was isolated following the standard protocol. rna libraries were sequenced using an illumina hiseq platform (illumina, san diego, ca, usa) with single-end (se) nt (se ), and each sample yielded an average of m reads. the software fastx_toolkit (version: fastx_toolkit_ _ _ ) was used to preprocess small rna-seq raw data. the software bowtie (version: bowtie - . . ) and rfam database (http://rfam.xfam. org/) were used to filter ribosomal rna or transfer rna. the commands mapper.pl and mirdeep .pl in the software mirdeep (version: mirdeep _ _ _ ) were used for mapping sample reads to the human genome (version hg , http:// hgdownload.cse.ucsc.edu/downloads.html) and detecting conserved mirna. following the standard protocol, total rna was isolated in rnase-free environment using trizol reagent (invitrogen). for gene expression analysis, cdna was synthesized from . μg of total rna with random primers using the high-capacity cdna reverse transcription kit (applied biosystems, foster city, ca, usa). for mirna expression analysis, cdna was synthesized with stem-looped mirnaspecific reverse transcription primers by using the same kit. amplified products were detected using the faststart universal sybr green master mix ii (roche, basel, switzerland) on a lightcycler pcr system (roche). the relative gene expression levels were normalized to the reference gene, human glyceraldehyde- -phosphate dehydrogenase (gapdh). the relative mirna expression levels were normalized to small non-coding rna u . the specific primers used were synthesized by invitrogen and listed in supplementary table s . the a cells were infected with h n or h n influenza virus titrated to an moi of or an equal volume of vehicle. cell viability was determined by an mtt assay (promega, madison, wi, usa) at , , , , , , and h postinfection in the a cell line. a cells were transfected with nm mimics of nc ( ′-uuguacuacacaaaaguacug- ′), mir- c- p ( ′-uaauacugccggguaaugaugga- ′) and mir- - p ( ′-uaacacugucugguaaagaugg- ′), or inhibitors of nc ( ′-caguacuuuuguguaguacaa- ′), mir- c- p ( ′-uccaucauuacccggcaguauua- ′) and mir- - p ( ′-ccaucuuuaccagacaguguua- ′) for h. the mimics and inhibitors were synthesized by genepharma (shanghai, china). then, the cells were collected with ripa lysis buffer. protein was detected by western blotting following the procedures described previously [ ] . anti-ace antibody was purchased from abcam (cambridge, ma, usa; ab ). antibodies against β-actin (clone ac- , a ) and flag tag (f ) were purchased from sigma-aldrich, st louis, mo, usa. the wild-type and mutant-type ′-utrs of the ace transcript were cloned into the psicheck- vector (promega) through the noti and xhoi restriction enzyme cutting sites. mirna mimics were transfected into hek t cells using lipofectamine rnaimax (invitrogen). after h, μg of wild-type vector or mutant vector was transfected into severe pneumonia patients were recruited at the peking union medical college hospital (pumch, beijing, china). healthy volunteers were recruited as controls. all participants had written informed consent. with the approval of the institutional review board of pumch, this study was performed following the ethical guidelines of the declaration of helsinki. the concentration of ang ii in plasma was detected using the elisa kit (rapidbio lab, calabasas, ca, usa). plasma mirna extraction and detection mirnas were extracted from μl plasma using an mirneasy serum/plasma kit (qiagen, hilden, germany). a synthetic spike-in control, caenorhabditis elegans mir- (cel-mir- ), was added to the lysed samples for internal normalization. cdna was synthesized using the taqman microrna assays kit and high-capacity cdna reverse transcription kit (applied biosystems). pcr reactions were performed on a lightcycler pcr system (roche) using the taqman universal master mix ii (applied biosystems). nf-κb p /rela-specific sirna, p -specific sirna and nonspecific control sirna were synthesized by genepharma. the p sirna duplexes (sirna : ′-ggacauaugaga ccuucaa- ′; sirna : ′-cuuccaaguuccuauag aa- ′), the p sirna ( ′-cgccaucuaugacaguaaa uu- ′) or a control sirna were transfected into cells (with a final concentration as μm) with lipofectamine rnaimax (invitrogen) for h, and then subsequent experiments were performed. inhibition of nf-κb transcriptional activity cape (s ) and jsh- (s ) were purchased from selleck chemicals (houston, tx, usa) and resolved in dimethyl sulfoxide. 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protein prevents activation of nf-kappab and induction of alpha/beta interferon double-stranded rna activates binding of nf-kappa b to an inducible element in the human beta-interferon promoter inducibility of kappa immunoglobulin enhancer-binding protein nf-kappa b by a posttranslational mechanism staphylococcus aureus and derived exotoxins induce nuclear factor kappa b-like activity in murine bone marrow macrophages acute respiratory distress syndrome induced by avian influenza a (h n ) virus in mice an official american thoracic society workshop report: features and measurements of experimental acute lung injury in animals clinical characteristics of human cases of highly pathogenic avian influenza a (h n ) virus infection in china avian influenza a (h n ) infection in humans suppression of the antiviral response by an influenza histone mimic influenza virus non-structural protein ns : interferon antagonism and beyond differential effects of ns proteins of human pandemic h n / , avian highly pathogenic h n , and low pathogenic h n influenza a viruses on cellular pre-mrna polyadenylation and mrna translation regulation of mir- c/ expression by intergenic dna-looping and transcriptional readthrough mir- c- p in viral and bacterial lung infections downregulation of angiotensin-converting enzyme by the neuraminidase protein of influenza a (h n ) virus influenza virus-induced nf-kappab-dependent gene expression is mediated by overexpression of viral proteins and involves oxidative radicals and activation of ikappab kinase fat is critical in influenza a virus replication by inhibiting type i ifn microrna profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations recognition of double-stranded rna and activation of nf-kappab by toll-like receptor differential roles of tlr and tlr in recognition of gram-negative and gram-positive bacterial cell wall components nf-kappab regulation in the immune system effects of an angiotensin-converting enzyme inhibitor-based regimen on pneumonia risk populationbased study of statins, angiotensin ii receptor blockers, and angiotensin-converting enzyme inhibitors on pneumonia-related outcomes the correlation between mir- c and the severity of interstitial lung disease associated with different connective tissue diseases mir- regulates alveolar macrophage inflammatory cytokine production and is involved in lps-induced acute lung injury neuraminidase of influenza a virus binds lysosome-associated membrane proteins directly and induces lysosome rupture unpaired t-tests were used for the cell culture and luciferase reporter assay data analysis. pearson's correlation analysis was used to analyze the relationship between mirnas and virus-infected cell viability or viral replication. the mann-whitney u-test was used for the analysis of clinical data. all statistical tests were calculated using graphpad prism . (la jolla, ca, usa). a two-tailed p-value o . was considered statistically significant. the authors declare no conflict of interest. key: cord- - mdh jmo authors: tamanna, sonia; clifton, vicki l.; rae, kym; van helden, dirk f.; lumbers, eugenie r.; pringle, kirsty g. title: angiotensin converting enzyme (ace ) in pregnancy: preeclampsia and small for gestational age date: - - journal: front physiol doi: . /fphys. . sha: doc_id: cord_uid: mdh jmo introduction: an imbalance in angiotensin (ang) peptides could contribute to the pathophysiology of preeclampsia (pe) and poor fetal growth. methods: we measured maternal plasma levels of ang peptides and converting enzymes in non-pregnant women (n = ), in normal pregnant women (n = ), women delivering small for gestational age babies (sga, n = ) across gestation ( – weeks) and in women with pe (n = ) in their third trimester. results: plasma ace, ace , and ang-( - ) levels, and ace activity were significantly higher in normal pregnant women compared with non-pregnant women; neprilysin (nep) levels were not changed. in sga pregnancies, ace and ace levels were higher in early-mid pregnancy compared with normal pregnant women. in women with pe, plasma ace, ace , nep, and ang-( - ) levels and ace activity were lower than levels in normal pregnant women. conclusion: the higher plasma ace levels and activity in pregnancy could be driving the higher ang-( - ) levels. the early gestation increases in ace and ace levels in sga pregnancies highlights the possibility that these enzymes could be used as potential early biomarkers of poor fetal growth. in women with pe, the reduced ace and nep levels at term, could be contributing to the reduction in ang-( - ) levels. these findings suggest that dysfunctional relationships between two key enzymes in the circulating ras are involved in the pathogenesis of pe and sga. since soluble ace can prevent binding of the novel coronavirus, sars-cov- , to membrane bound ace , the interplay between ace and the coronavirus and its impact in pregnancy requires further investigation. during pregnancy, the renin-angiotensin system (ras) plays a significant role in the regulation of blood pressure and salt and water balance. it is activated by estrogen-induced increases in angiotensinogen (agt) and, subsequently, by the reduction in systemic vascular resistance caused by the ovarian hormone, relaxin (lumbers and pringle, ) . it is perhaps not surprising then that alterations in the circulating and intrauterine ras are associated with the development of preeclampsia (pe) and pregnancies in which the infant is born small for gestational age (sga) (irani and xia, ; zhou et al., ; delforce et al., ) . the circulating ras cascade (figure ) begins with the release of active renin from the kidney, which cleaves angiotensin i (ang i) from agt. angiotensin ii (ang ii), the major effector molecule of the ras cascade, is cleaved from ang i by angiotensin converting enzyme (ace). ang ii elicits its effects by binding to the ang ii type receptor (at r). interactions between ang ii and the at r stimulate sodium retention (both directly and via stimulating the release of aldosterone) and raise blood pressure, both by central actions and direct effects on blood vessels. alternatively, ang ii can bind to the at r, which has opposing actions than those of ang ii binding to the at r (escobar et al., ) . the actions of ang ii/at r are counter-balanced by an alternate ras axis (figure ). this axis encompasses ace , which can form ang-( - ) by removing a single amino acid from ang ii. in conjunction with ace, ace can also form ang-( - ) from ang i, as can neprilysin (nep) by acting on ang i. ang-( - ) acting through the mas receptor, opposes the actions of the ang ii/at r pathway, as do interactions between ang ii and the at r (warner et al., ) . ace plays a role in balancing the vasoconstrictor and vasodilator arms of the ras (gallagher et al., ) and exerts a protective role in end organ damage (heart, lung, kidney, etc.) (oudit et al., ; wang et al., ) . ace can undergo ectodomain shedding to secrete a soluble form of ace , sace (lambert et al., ) . ace has an almost -fold higher catalytic efficiency in forming ang-( - ) from ang ii than in converting ang i to ang-( - ) (vickers et al., ) , which is then cleaved by ace. ace is insensitive to ace inhibitors such as captopril, lisinopril, and enalapril (donoghue et al., ) , and there is evidence that ace counteracts the effects of the ras mediated by over activity of ang ii and the at r (chhabra et al., ) . plasma ang-( - ) is increased in pregnancy, whereas ace concentrations are decreased (merrill et al., ) . in women with pe, maternal plasma ang-( - ), ang ii and plasma renin activity are reduced compared with normotensive pregnant women (merrill et al., ; velloso et al., ) . ace concentrations are however, increased in women with pe (merrill et al., ) . women who gave birth to sga babies also had higher concentrations of plasma ace at weeks' gestation compared with women who delivered babies of normal birth weight (zhou et al., ) . to the best of our knowledge, there are no data on changes in maternal ace and nep levels in either normal pregnant women or women suffering from complicated pregnancies. it is possible for ang-( - ) to be formed via the activity of both these pathways (see figure ) . ace is the critical receptor for the highly pathogenic novel sars-cov- virus, which causes covid- . the sars-cov- spike protein binds with human ace with substantially higher affinity than does the spike protein of sars-cov (walls et al., ; wan et al., ; wrapp et al., ) . therefore, our particular interest in measuring sace in pregnant women could be important in understanding the pathogenesis of sars-cov- in pregnancy. in this study, we measured ace, sace , ang-( - ) and nep levels in plasma from women with uncomplicated (normal) pregnancies and compared them to levels found in healthy non-pregnant women. we also measured plasma levels of these components of the ras in women with pregnancies complicated by either pe or sga. data and biobanked plasma samples from a cohort study undertaken at the lyell mcewin hospital, adelaide, australia, that was used to study the effects of asthma during pregnancy on the mother, placenta and baby, were examined (dickinson et al., ) . the study was approved by the queen elizabeth hospital and lyell mcewin hospital human research ethics committee and the university of adelaide human research ethics committee. samples were collected from (n = ) women with uncomplicated pregnancies at , , , and weeks. samples were also collected from women with sga pregnancies ( , , , and weeks) and women who developed pe (range: - weeks). we used stored plasma samples from healthy non-pregnant women (n = ), and women who developed preeclampsia (n = , - weeks of gestation, supplementary table ) collected at the john hunter hospital and tamworth base hospital, nsw, australia. the collection and use of these samples were approved by the university of newcastle research ethics committee and hunter new england health human research ethics committee. the normal pregnancy group included individuals who delivered appropriately grown infants (> th centile for gestational age) at term (> weeks) who did not have hypertension, diabetes, infections, or renal dysfunction. women were classified as having pe based on the somanz definition (lowe et al., ) . however, some samples were collected prospectively, at which time we measured blood pressure. pregnancies complicated by sga had babies whose birth weights were < th centile for gestational age calculated using the grow (gestation related optimal weight) method (https://www.gestation.net/). for all samples, maternal venous blood was collected in lithium/heparin tubes, centrifuged at g for min, aliquoted and stored at − • c until assays were performed. figure | schematic representation of the ras cascade. ace, angiotensin converting enzyme; ace , angiotensin converting enzyme ; nep, neprilysin; at r, angiotensin ii type receptor, at r; angiotensin ii type receptor. maternal plasma ace and nep concentrations were determined using commercially available enzyme-linked immunosorbent assay kits according to the instructions of the manufacturer (duoset, r&d systems, minneapolis, mn, united states). concentrations of maternal plasma ace were measured using commercial enzyme−linked immunosorbent assay (elisa) kits (cloud−clone corp, houston, tx, united states). all samples were assayed in duplicate. the mean intra-and inter-assay coefficients of variations were . % (n = ) and . % ( plates) for ace, . % (n = ) and . % ( plates) for ace , and . % (n = ) and . % for nep ( plates), respectively. the ace activity assay was carried out according to a published protocol with minor modifications (shao et al., ; xiao and burns, ) . briefly, µl of each plasma sample was diluted with an enzyme buffer consisting of m nacl, mm tris-hcl, . mm zncl , ph . with protease inhibitors, which were µm captopril, µm amastatin, and µm bestatin (all from sigma aldrich, st. louis, missouri, united states) and µm z-prolyl-prolinal (enzo life sciences, inc., ny, united states). the ace -specific quenched fluorescent substrate, mca-ala-pro-lys- , dinitrophenyl (anaspec, inc., san diego, ca, united states) diluted in enzyme buffer were added to the samples in a final concentration of µm in µl on a black -well microplate. the plate was covered with aluminum foil to protect from light and incubated at room temperature for h on a plate shaker. we have used recombinant human ace (r&d systems, minneapolis mn) as the standard instead of using a fluorescent product to measure ace activity. ace cleaves the pro-lys bond of the substrate, and the relative fluorescence units (rfu) of the released mca-ala-pro was measured over a period of h. the plate was read on a fluorescence reader (clario star, bmg labtech, gmbh, ortenberg, germany) with an excitation wavelength of nm and emission wavelength of nm. the specificity of the enzyme activity was checked using µm of an ace inhibitor (dx ; anaspec, inc., san diego, ca, united states), which is specific for human ace . we calculated how much ace activity there was in a sample comparing the rfu to the rfu of known concentrations of recombinant ace , that were treated in an identical manner. thus ace activity is reported as the activity/ng/ml of ace . all samples were assayed in duplicate. the mean intra and interassay coefficients of variations were . % (n = ) and . % ( plates), respectively. a direct radioimmunoassay (ria) was used to measure plasma ang-( - ) levels by prosearch pty ltd (malvern, vic, australia) as described previously (sykes et al., b) . data for all ras enzyme levels and activity, as well as ang-( - ) levels, ace /ace ratio, and ace activity/ace ratio, were expressed as median and interquartile ranges. a non-parametric kruskal-wallis test (with dunn's multiple comparison test) was performed to compare ace, ace , nep and ang-( - ) levels and ace activity, ace /ace ratio, and ace activity/ace ratio between non-pregnant women and women with normal pregnancies (at , , , and weeks of gestation). mixed effect analysis (with fisher's lsd multiple comparison test) was used to compare differences between normal pregnancies and pregnancies complicated by sga at each gestational age. a mann-whitney test was used to determine differences between normal pregnancies and women with pe in the third trimester. spearman's correlations were used to determine any association between ace and ang-( - ) levels and ace levels as well as birth weight centile. differences in demographic characteristics of different pregnant groups were identified by the chi-square test. differences were considered significant if p < . . a total of non-pregnant women were recruited in this study. the median age was . years (interquartile range (iqr): - years) and bmi was . kg/m (iqr: . - . ). three of the ten non-pregnant women were taking oral contraceptives, and a further four were using hormonal contraceptive devices (e.g., implanon and mirena). table outlines the baseline characteristics of each pregnancy group. maternal age, bmi and ethnicity were not significantly different between the study groups. the median birth weights and birth weight centiles of sga infants were less (p < . and p < . , respectively), as were the birth weights and birth weight centiles of infants whose mothers had pe (p < . and p = . , respectively) than those from normal pregnancies. women with pe delivered at an earlier gestational age compared with those who had normal pregnancies (p < . ), which could account for their lower birth weights. no significant difference was observed in the gestational age at delivery of sga babies compared with those born from women with normal pregnancies. the number of smokers was significantly higher in women delivering sga babies compared to those with normal pregnancy outcomes (p = . ), whereas there was no significant differences between the prevalence of smokers between the pe and normal pregnancy groups. the number of male babies was higher (p = . ) in the pe group compared to normal pregnant group. two of the women in the pe group were classified as having early-onset pe (developing < weeks gestation) and seven of the women delivered a baby who was sga. there were no significant differences in the systolic or diastolic blood pressures of women with normal pregnancies and those with sga at any gestational age ( , , , or weeks, table ). there were no differences in systolic and diastolic blood pressures in the pe group compared with the normal (normotensive) pregnancy group at the time of sampling in the third trimester (table ) . associations between ace activity and ace levels as well as between ace and ang-( - ) in non-pregnant and pregnant women figure shows the association between plasma ace activity and ace levels as well as the association between plasma ace and ang-( - ) in non-pregnant and pregnant women (normal, pe and sga). ace activity was positively correlated with ace levels (r = . , p = . ). plasma ang-( - ) levels were positively correlated with ace levels and activity (r = . , p = . and r = . , p = . , respectively). ace, ace , nep, and ang-( - ) levels, ace activity and ace /ace, ace activity/ace ratio in normal pregnancies plasma ace levels were significantly higher in women with normal pregnancies compared with non-pregnant women (p < . ) and remained high throughout gestation ( figure a) . women with normal pregnancies also had elevated ace levels (p = . ) and activity (p < . ) compared with non-pregnant women (figures b,c) . however, ace levels and activity did not change across gestation. nep levels in non-pregnant and pregnant women were similar and did not change across gestation (p = . ; figure d ). in women with normal pregnancies, we determined if the known rise in plasma ang ii in pregnancy was counter-balanced by elevated ang-( - ) levels ( figure e ). plasma ang-( - ) levels were significantly increased in normal pregnant women compared with non-pregnant women (p = . , figure e ), this was statistically significant at all gestational ages examined except at weeks of gestation. we also examined the ace /ace ratio ( figure f ) as well as the ratio between ace activity/ace levels ( figure g ) in normal pregnant women. in pregnancy, the ace /ace ratio did not change across gestation (p = . ). however, the ace activity/ace ratio was significantly higher in normal pregnant women (p = . ) and remained elevated throughout gestation ( figure g) . women delivering babies who were born sga had levels of ace and ace that were significantly higher than those in women with normal pregnancies (p = . and p < . ; figures a,b) . when examining the differences at each gestational age, the only statistical differences were at weeks for ace levels and at , , and weeks for ace levels. ace activity however was not significantly - ) were measured by radioimmunoassay in np and pregnant women (e). plasma ace /ace ratio and ace activity/ace ratio in np and pregnant women (f-g). data are expressed as median and interquartile range. n = - samples for the np group (black box), n = - samples/group for , , , and weeks of normal pregnancy (white box). p-values were calculated using a kruskal-wallis test (with dunn's multiple comparison test). *p < . , **p < . , ***p < . versus np. different between normal and sga pregnancies (figure c ). nep and ang-( - ) levels in sga pregnancies were similar to those found in women with normal pregnancies, as was the ace /ace ratio (figures d-f) . however, the ratio between ace activity and ace levels was significantly decreased in women with sga pregnancies compared with normal pregnancies (p = . ; figure g) , this was significant at , , and weeks of gestation but not at weeks. ace levels were significantly decreased in women with preeclampsia compared with levels measured in women with normal pregnancies (p < . ; figure a ). both plasma ace figure | plasma levels and activity of (a) ace, (b,c) ace , and (d) nep as well as levels of (e) ang-( - ), (f) ace /ace ratio, and (g) ace activity/ace ratio in normal and sga pregnancies. data are expressed as median and interquartile range. n = - samples/group for normal pregnancies (gray box) and n = - samples/group for sga pregnancies (white box). p-values were calculated using a two-way anova (mixed effect model with fisher's lsd multiple comparison test). * p < . , * * p < . , * * * p < . versus normal pregnancy group of the same gestational age. levels (p < . ) and activity (p = . ) were significantly lower in women with preeclampsia compared with women with normal pregnancies (figures b,c) . there was no significant difference between the pe alone and pe with sga samples in terms of ace levels (data not shown). plasma nep levels were also decreased in women with pe (p = . ; figure d ). figure e shows the plasma ang-( - ) levels in women with normal pregnancies and women with pe. women with pe had reduced levels of plasma ang-( - ) compared with levels in women with normal pregnancies (p = . ; figure e ). the ace /ace ratio was increased in pe compared with normal pregnancies (p = . ; figure f ). table shows the associations between ace, ace levels, and ace activity and birth weight centile (all pregnant groups). significant negative correlations were found between both ace and ace levels and birth weight centile (ace: r = − . , p = . , r = − . , p = . at weeks and weeks of gestation, ace : r = − . , p = . , r = − . , p < . , r = − . , p = . for , , and weeks of gestation; table ). ace activity was not associated with birth weight centile. in this study, we showed for the first time that ace and ace levels and activity were increased in the maternal circulation during pregnancy and remained high throughout gestation, however nep levels were unchanged. plasma ace levels were approximately % to % higher across gestation in women with normal pregnancies compared with non-pregnant women. we ( - ) , and (f) ace /ace ratio in the third trimester (≥ weeks of gestation) in women with normal pregnancies (gray box) and women with preeclampsia (pe; white box). data are expressed as median and interquartile range. n = - for normal pregnancy group and n = - samples for the pe group. *p < . , **p < . , ***p < . versus normal pregnancy group. also confirmed that ang-( - ) levels were increased in pregnancy compared with non-pregnant women. in addition, we found that ace levels were positively associated with ace activity and that ang-( - ) levels were positively associated with both ace levels and activity. this latter observation suggests that the amount and activity of ace in the plasma is rate-limiting in terms of the production of ang-( - ). whether or not this also applies to membrane bound ace needs to be determined. the increased activity of ace in normal pregnancy suggests that it may play a role, through the production of ang-( - ), in the regulation of maternal blood pressure. previous studies carried out in ace knockout mice show that ace deficiency is associated with an increase in maternal blood pressure during pregnancy (yamaleyeva et al., ) . several studies have also shown that polymorphisms in the ace gene are associated with an increased risk of hypertension (malard et al., ; patnaik et al., ) . in women who gave birth to sga neonates, circulating ace levels were increased at weeks of gestation compared with those found in women with normal pregnancies. elevated levels of maternal plasma ace have also been documented by zhou et al. in women at weeks' gestation who went on to deliver sga neonates (zhou et al., ) . we found increased ace levels in sga pregnancies at , , and weeks of gestation, but not in late pregnancy ( weeks). it is possible, since ace levels were increased at weeks of gestation, that there was an elevation in ace levels in early-mid gestation in order to regulate the activity of the ang ii via the at r pathway. since ace levels were not elevated at term, there was a corresponding reduction in ace levels. ace activity was not significantly different across gestation in women who later delivered sga neonates compared with women with normal pregnancies. the increase in ace levels in the absence of any change in ace activity could be explained by the presence of an endogenous inhibitor, which has been described in human plasma (lew et al., ) . further studies are obviously required to investigate if this inhibitor is elevated in sga pregnancies compared with normal pregnancies. interestingly, a negative correlation was found in early pregnancy between birth weight centiles and ace and ace levels (table ) suggesting that the increased production of angiotensin enzymes in early gestation could influence birth weight. however, in late gestation, any such effect was no longer apparent. we also acknowledge that bmi and parity might influence the levels and activity of ras enzymes as well as ang-( - ). there were however no significant correlations between bmi and ace, ace levels, ace activity, and ang-( - ) (data not shown). this is the first study to measure plasma ace in sga pregnancies. ace mrna is present in almost all human tissues in particular in the kidney, heart, lung, testis and gastrointestinal tissues, where it has critical regulatory functions (harmer et al., ) . indeed, ace metabolizes ang ii and reduces its levels and produces the vasodilator peptide, ang-( - ). therefore, it is critical for regulating the actions of ang ii mediated by its at r. a previous study from our group showed that placental ace mrna expression was lower in pregnancies associated with fetal growth restriction (delforce et al., ) , suggesting that reduced placental ace levels and reduced ang-( - ) production might contribute to the etiology of sga. the elevation in plasma ace levels in sga, taken together with the reduction in placental ace , suggests, however, that placental ace is not contributing to the high levels of sace in the maternal circulation in sga pregnancies. other tissues that could contribute ace to the maternal circulation include the kidney, which has high levels of ace and a % increase in renal blood flow during pregnancy (brosnihan et al., ) . further studies are required to investigate the relationships between placental ace and ace mrna expression and activity and plasma ace and ace levels and activity in matched samples from sga pregnancies to fully elucidate the effects of these two enzymes in the etiology of sga. these studies could also be used to determine if ace and ace are early biomarkers for sga or fetal growth restriction (see zhou et al., ) . samples from women with pe were collected from two different locations in australia (new south wales and south australia). although the methods of sample collection were very similar at the sites we acknowledge that it could have influenced the levels of ace , ace, nep and ang-( - ). however, the clinical characteristics of these pe subgroups were similar (supplementary table ) and there were no significant differences in levels of ras enzymes and ang peptides in samples collected from these two sites (supplementary figure ) . we also showed that there were no significant changes in levels/activity of ras enzymes and ang-( - ) across gestation in women with pe (supplementary figure ) . another limitation of the study is that in some cases samples were collected before the onset of pe and, in other cases, women may have been taking antihypertensive medications to control blood pressure. both of these factors could influence the levels and activity of ras components. unfortunately, we do not have access to bp measurements from women after pe was diagnosed nor information on medications taken by the women. therefore, some degree of caution should be taken in interpreting our findings. women with pe were found to have lower ace and ace (levels and activity) than women with normal pregnancies. this decrease in the angiotensin processing enzymes would be expected to result in altered ang peptide levels in women with pe. we found that ang-( - ) levels were reduced in women with pe compared with women with normal pregnancies, and it is well known that levels of ang ii are suppressed in pe (merrill et al., ; velloso et al., ; brosnihan et al., ) . the decrease in ang ii could be due to the reduction in ace and the increased amount of ace relative to ace (figure f ). in addition, we have shown for the first time that there is decreased levels of nep in women with pe. decreased levels of both ace as well as nep could account for the decreased levels of ang-( - ) in women with pe. importantly, some of the women in the pe group delivered sga babies. in the sga group, ace was elevated in early gestation but in the pe group, maternal circulating ace levels and activity were suppressed at term. this could be due to the timing of sampling (early gestation vs. term), since we have previously shown that ang-( - ) levels are similarly increased at weeks of gestation in women who go on to develop preeclampsia (sykes et al., a) , whereas at term ang-( - ) levels are reduced. ace protects tissues from the pro-inflammatory effects of ang ii by metabolizing it and producing ang-( - ) (jia, ) . as well, ace is the receptor for sars-cov- . ang ii has proinflammatory effects similar to those caused by the sars virus. in acute respiratory distress syndrome recombinant human ace reduces sars-induced lung injury (imai et al., ) and has been shown to prevent cell infection by sars-cov- (monteil et al., ; tai et al., ) . therefore, the high levels of sace seen in normal pregnant women might protect the lungs from sars-cov- induced lung injury and slow down viral entry into cells thus reducing viral spread zhang et al., ) . conversely, the low levels of sace found in pregnant women with pe might increase their susceptibility to covid- . in conclusion, our findings on the relationships between ace and ace in normal pregnancy and pregnancies associated with pe or sga show that dysfunctional relationships between these two key enzymes in the circulating ras could be involved in the pathogenesis of pe and sga. furthermore, increased levels of sace in pregnant women could be significant in understanding the clinical effects of covid- . the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by the queen elizabeth hospital and lyell mcewin hospital human research ethics committee, the university of adelaide human research ethics committee, the university of newcastle research ethics committee, and the 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biological peptides by human angiotensin-converting enzymerelated carboxypeptidase structure, function, and antigenicity of the sars-cov- spike glycoprotein receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus protective role of ace -ang-( - )-mas in myocardial fibrosis by downregulating kca . channel via erk / pathway angiotensin-converting enzyme- : a molecular and cellular perspective cryo-em structure of the -ncov spike in the prefusion conformation measurement of angiotensin converting enzyme activity in biological fluid (ace ) uterine artery dysfunction in pregnant ace knockout mice is associated with placental hypoxia and reduced umbilical blood flow velocity angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target the association of maternal ace a g with small for gestational age babies is modulated by the environment and by fetal sex: a multicentre prospective case-control study st substantially contributed to the design of the study, acquisition of data, analysis and interpretation of data, revision of the draft article for content, and the final approval of the version submitted for publication. vc and kr provided plasma samples and contributed to the revision of the draft article for content and final approval of the version submitted for publication. dh contributed to the interpretation of the data, revision of the drafted article, and the final approval of the version to be published. el substantially contributed to the interpretation of data, article drafting and revision for important intellectual content, and the final approval of the version to be published. kp substantially contributed to the design of the study, analysis and interpretation of data, revision of the draft article for content, and the final approval of the version submitted for publication. all authors contributed to the article and approved the submitted version. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fphys. . /full#supplementary-material key: cord- -yh ndtgm authors: mohammed el tabaa, manar; mohammed el tabaa, maram title: targeting neprilysin (nep) pathways: a potential new hope to defeat covid- ghost date: - - journal: biochem pharmacol doi: . /j.bcp. . sha: doc_id: cord_uid: yh ndtgm covid- is an ongoing viral pandemic disease that is caused by sars-cov , inducing severe pneumonia in humans. however, several classes of repurposed drugs have been recommended, no specific vaccines or effective therapeutic interventions for covid- are developed till now. viral dependence on ace- , as entry receptors, drove the researchers into ras impact on covid- pathogenesis. several evidences have pointed at neprilysin (nep) as one of pulmonary ras components. considering the protective effect of nep against pulmonary inflammatory reactions and fibrosis, it is suggested to direct the future efforts towards its potential role in covid- pathophysiology. thus, the review aimed to shed light on the potential beneficial effects of nep pathways as a novel target for covid- therapy by summarizing its possible molecular mechanisms. additional experimental and clinical studies explaining more the relationships between nep and covid- will greatly benefit in designing the future treatment approaches. coronavirus disease (covid- ) is an infectious viral disease that is caused by a newly discovered coronavirus, namely severe acute respiratory syndrome coronavirus- (sars-cov- ) [ ] . it is a virus belonging to order nidovirales of family coronaviridae, which are singlestranded rna viruses [ ] and broadly distributed in both humans and other mammals [ ] . majority of the people infected with novel coronavirus ( -ncov) may recover without requiring special treatment [ ] , except the elderly people and those with some medical problems such as hypertension, diabetes [ ] and chronic respiratory disease [ ] . such patients are more liable to be infected with covid- and might develop a fatal pneumonia with clinical presentation greatly resembling that caused by the previous beta-coronavirus sars-cov appeared in ; severe acute respiratory syndrome (sars) [ ] . in such cohort, patients firstly complain of fever, dry cough, and at late stage, may suffer from dyspnea that makes them in a desperate need of intensive care unit (icu) admission and oxygen therapy [ ] . however, the time between icu admission and ards development is recorded to be as short as days [ ] , being the reason for the high mortality rate associated with covid- at this stage [ ] . till present, no covid- -specific vaccines or medications are available, although some national medical authorities recommend testing the efficacy of antiviral medication in especially sever clinical trials [ ] . in addition, several medical researchers suggest starting the supportive and symptomatic treatment till the new medications are developed [ , ] . early evidences have shown that the pathogenicity for covid- may be enhanced with increased the body susceptibility to induce a phenomenon called cytokine storm, in which the immune system gears up by overreacting and producing more inflammatory mediators to fight the infection [ ] . as a result, a severe inflammatory reaction is produced accompanied with significant damage, including organ failure [ , ] . receptors [ ] ; disrupting, of course, the normal physiological function of ace- /ang ( - )/masr axis of the renin-angiotensin system (ras) pathway [ ] . therefore, researchers suggested that the use of angiotensin converting enzyme inhibitors (aceis) and/or angiotensin receptor blockers (arbs), may show a positive trend towards the severe inflammatory reactions and endothelial dysfunction caused by stimulating the function of ace/ang ii/at- axis and thereby, towards the bad pulmonary effects associated with the covid- infection [ , ] . however, no clinical trial, till this time, has been proved its efficacy in preventing or even reducing the covid- severity [ , ] . pursuing clinical trials with the absence of whole data describing the structure of covid- virus and its related mechanisms may lead to unsatisfied outcomes. as well, understanding such data will greatly help in determining the most appropriate effective treatment protocols. consequently, the first step to prevent and slow down the disease progression should involve the deduction of proper pathophysiology of that novel viral disease. identifying ace- as a viral entry receptor will emphasize on the important role of classical ras pathway in covid- pathophysiology, however, neither the link between ace- and other ras components nor their exact roles in the covid- pathogenesis have been neatly studied till now. one ras component, namely neprilysin (nep), has been established to potentiate the physiological beneficial role ascribed to the ace- /ang ( - )/masr axis; by an alternative pathway [ ] . nowadays, nep has emerged as a pharmaceutical target for many drugs; especially those used in treating cardiovascular diseases and alzheimer's disease (ad) [ , ] . considering the respiratory system, nep was reported to play a vital role in protecting lungs from inflammation and fibrosis [ ] [ ] [ ] . a long time ago, mentioned that rats infected with common respiratory tract pathogens, such as parainfluenza virus type- , rat coronavirus, and mycoplasma pulmonis showed low nep activity; resulting in an increase in their susceptibility to inflammatory responses [ ] . however, the precise nep's role against life-threatening lung injuries has not been investigated yet. consequently, this current review aims to examine the cellular signaling pathways involving nep in covid- pathogenesis and to summarize the evidences supporting the potential beneficial effects of nep as a novel target for therapy. all previous and recent studies agreed with the fact that the surface of all human pathogenic coronaviruses is covered with crown like projections called spike (s) glycoproteins; giving the viruses their name [ ] . in addition to s protein, another three main structural proteins have been recognized in sars-cov- , namely envelope (e), nucleocapsid (n) and membrane (m) proteins [ ] , figure ( ) . specifically, s proteins are very important for sars-cov- infection to get started [ ] . they possess two functional subunits by which the virus can enter into its target host cells involving; s subunit, which enables it to bind with receptors on the host cell surface and s subunit, that allows the virus to fuse into the cellular membrane [ ] . unlike other coronaviruses, several experimental analyses proved that sars-cov- does not use the common known viral entry receptors, such as aminopeptidase n (apn) [ ] and dipeptidyl peptidase (dpp ) [ ] , but, instead, can utilize ace- receptor [ ] . additionally, it was revealed that transmembrane protease serine (tmprss ) is very critical for the host cell entry of sars-cov- [ ] and its plasma membrane fusion [ ] resembling sars-cov [ ] . firstly, the virus starts its destructive journey with binding of s subunit to ace- enzyme receptor on the cell membrane surface, which in turn activates tmprss . activation of tmprss is followed by cleavage of both virus's s subunits from its s subunits and so of the ace- receptors. secondly, activated tmprss can also act on the s subunit, activating it through prompting an irreversible conformational change that will facilitate the virus-cell fusion [ , ] . within this view, understanding the precise involvement ace- , as a member of pulmonary ras pathway, in covid- pathophysiology may open new therapeutic possibilities for management. for years, ras was depicted as a hormonal circulating system involved in fluid and electrolyte balance, systemic vascular resistance and blood pressure regulation [ , ] . however, a wealth of data showed that lung epithelial cells, fibroblasts and alveolar macrophages could also express the major constituents of the ras [ , ] supporting the existence of a "local" pulmonary ras that can be differentiated from the "systemic" circulating ras [ ] . as regards "local" ras in the lung, it has been reported to involve multiple cellular mechanisms affecting the vascular permeability, fibroblast activity and alveolar epithelial cells [ , ] . by the time, it is well recognized that activating pulmonary ras can influence the pathogenesis of lung injury in different lung diseases such as pulmonary hypertension, acute respiratory distress syndrome (ards), asthma and pulmonary fibrosis [ ] . as a consequence, pulmonary ras has been described to participate in disease process or play a protective role against tissue injury [ ] and thus, modulating ras in the lung can successfully play a good role in the treatment of inflammatory lung disease [ , ] . the functional steps of ras was firstly initiated by hepatic synthesis of a plasma globulin called renin substrate (or angiotensinogen) which could be enzymatically converted through renin secreted by juxtaglomerular cells of the kidney, into the biologically inactive decapeptide, namely angiotensin (ang) i [ ] . subsequently, ras exhibits two main axes based on two distinct enzymes responsible for cleavage of ang i into angiotensin (ang) ii or ang - [ ] , first axis involves generation of ang ii as the main effector via the angiotensin converting enzyme (ace) and named the classical vasopressor axis ace/ ang ii/ ang ii type receptor (at ) [ ] . for the second axis, angiotensin converting enzyme ii (ace- ) was identified as a depressor for ras activation through producing ang - [ ] . henceforth, this axis ace- / ang - / mas- has become an important area of scientific research interest [ ] . the ace/ang ii/at- axis has been well documented to drive a set of deleterious reactions in the lung through generating ang ii, the principal effector of this classical axis, involved in ras mediated vasoconstriction, sodium retention, fluid overload, inflammation and fibrosis [ , ] . in addition, ace has been reported to evoke bradykinin and substance p degradation, two local pro-inflammatory and protussive peptides which can stimulate cough reflex and nitric oxide (no) release [ , ] . specifically, pulmonary vascular inflammation contributes to a phenomenon called ace "shedding," in which endothelial surface-bound ace is released into the interstitium resulting in significant increase in the level of ang ii [ ] , which explains the detection of higher ang ii level in covid- patients than normal [ ] . several studies declared that ang ii could act through two distinct g protein-coupled receptors (gpcr) subtypes, angiotensin ii receptor type (at r) and type (at r), that were found to be expressed in human lung tissue; proving the local generation of ang ii in lung [ , ] . the majority of actions evolved by ang ii could be mediated by at receptor via enhancing many complex intracellular signaling pathways including mapk/erk, plcb/ip /diacylglycerol, tyrosine kinases, and nf-kb [ ] . activating at receptor had been shown to further stimulate monocytes, macrophages and vascular smooth muscle cells to produce the proinflammatory cytokines such as tnf-α and il- [ ] [ ] [ ] . on the contrary, at receptor was documented to have a number of counterregulatory interactions against lung tissue injury via inhibiting inflammation, and fibrosis [ , ] . in addition to the known ang ii effects on promoting vasoconstriction, and pro-inflammatory cytokine release, there is also an increasing evidence that ang ii could induce vascular endothelial dysfunction [ , ] , promoting the activation and aggregation of platelets and thereby, pro-thrombotic milieu [ ] . ang ii could be associated with noticeable rise in the plasma level of endothelin- (et- ) [ ] , which is a peptide secreted mainly from vascular endothelial cells (ecs), and basically works through the endothelin type a receptor (eta) in vascular smooth muscle cells and the endothelin type b receptor (etb) in ecs [ ] . consequently, et- is described as the most potent bronchoconstrictor [ ] and vasoconstrictor in the airways [ ] . practically, et- acts as a key player of angiotensin ii-induced endothelial dysfunction and platelets activation via inducing il- release [ , ] , which was documented to be correlated directly with the extent of endothelial dysfunction [ ] . endothelial dysfunction is generally characterized by an imbalance between endotheliumdependent relaxing and contracting factors, attributed to reduced production/availability of nitric oxide (no); namely endothelium-derived relaxing factor (edrf) [ , ] . no acts as a potent endogenous vasodilator that could prevent platelet aggregation and leukocyte adhesion to ecs [ , ] . since il- would inactivate endothelial nitric oxide synthase (enos), it could disrupt no production [ ] , decreasing its level and inducing a state of oxidative stress that may lead to ang ii-induced impairment in endothelial responses [ ] postulating impaired endothelium functions as a principal factor in the pathogenesis of heart failure, hypertension and diabetes, it will be expected to classify the patients of such diseases as high risk groups for covid- development [ ] [ ] [ ] . furthermore, platelets activation and aggregation may be noticed as a result of inflammatory reactions aggravated by endothelial dysfunction, leading to imbalance between coagulation and fibrinolysis [ ] . it is clearly evident that continuing release of il- can enhance hepatic thrombopoietin (tpo) mrna expression resulting in thrombopoiesis stimulation and increase in circulatory platelets' numbers, known as (inflammatory thrombocytosis). within this context, et- can also mediate the synthesis of platelet activating factor (paf), a potent phospholipid mediator of platelet activation and aggregation, that may activate platelets to stick together and aggregate. as a result, a platelet plug is formed as an initiator for blood clotting and intravascular thrombus formation [ , ] , which is considered as a starting point for developing stroke [ ] . even though the high incidence of inflammatory thrombocytosis in covid- patients, the laboratory results of severe cases showed the opposite; suffering from thrombocytopenia [ ] .the possible explanation is the depletion of both platelets and megakaryocytes as a result of multiple blood clots formed at the injured site, leading to less platelet production with more consumption as the disease severity increases [ ] . interestingly, endothelial dysfunction and platelet activation can successively together worsen the severity of covid- infection. as known, both et- and activated platelets, associated with endothelial dysfunction, [ ] could promote leukocytes rolling, adherence to endothelium, activation and migration into the inflammatory sites, sharing in enhancement of leukocytes recruitment [ , ] . in addition, endothelial dysfunction can drive the fibrotic consequences following sars-cov- infection, developing pulmonary fibrosis as a result of releasing transforming growth factor-β (tgf-β ), the main fibrogenic cytokines implicated in pulmonary fibrosis, which could be induced by et- action [ , ] . taken into consideration the numerous harmful effects possibly induced by ang ii during covid- pathogenesis, we found that most novel studies aim to use the anti-hypertensive drugs which act either by inhibiting the ace activity or by blocking at receptor, suggesting that action may mitigate the disease severity in covid- patients. on the other side, it has been proposed that enhancing the counter regulatory axis composed by ace- /ang ( - )/masr axis may be the most helpful. ace- /ang ( - )/masr axis, the depressor arm of ras, was identified to mitigate the deleterious actions mediated by ace/ ang ii/ at [ ] . within this axis, ace- competes with ace by hydrolyzing ang i into the nonapeptide angiotensin (ang - ) which is further cleaved by the action of ace into heptapeptide angiotensin (ang - ), thus decreasing the amount of ang i available for ang ii generation by ace [ , ] . to the same extent, ace- could also hydrolyze ang ii converting it into ang ( - ) [ , ] . until the year , ace has emerged as the key enzyme in the pulmonary ras, but this was challenged by the discovery of its homologue; ace- [ ] which was found out to be broadly expressed in almost all types of lung cells, within the vascular endothelial and smooth muscle cells, types i and ii alveolar epithelial cells and bronchial epithelial cells [ , ] . ace- could negatively regulate the ras in the lung through reducing ang ii/at receptor signaling and activating the counterregulatory ang ( - )/ mas receptor pathway [ ] . that finding was compatible with the animal studies showed that the use of ras inhibitors could effectively relieve the symptoms of acute severe pneumonia and respiratory failure [ ] . consequently, the increased ace- was addressed as a target for protection from predisposition to inflammatory lung diseases such as, acute respiratory distress syndrome (ards) [ , ] . concerning covid- pathogenesis, binding of sars-cov- to ace- resulted in exhaustion of ace- , breaking the balance of the ras system within the lung and then, exacerbation of pulmonary inflammatory reactions [ , ] . being ace- receptors widely expressed on vascular endothelial cells (ec) within the lungs [ ] , sars-cov- can exploit them to induce and diffuse inflammatory cascades within endothelial cells disrupting their function; evidenced by the existence of viral elements and inflammatory cells within endothelial cells [ ] . thus, distributing ace- , as the main functional receptor for sars-cov- , within human tissues will be the determinator for spreading of viral infection within the lung and other organs [ , ] . both alveolar and bronchial membranes were reported to highly express ace- , which may explain the higher tendency of sars-cov- virus to harmfully affect lower airways than the upper ones [ , ] . however, recent reports indicate that few covid- patients may also develop some signs and symptoms of upper respiratory tract infection (e.g. rhinorrhea, sneezing, or sore throat) [ , , ] . furthermore, it was reported that ace- is also expressed in many epithelial cells of other organs than lung including kidney, blood vessels, intestine [ ] and brain [ ] . the fact which may explain the existence of some extra-pulmonary co-morbidities as myocardial dysfunction [ ] and acute kidney injury (aki) [ ] , gastrointestinal manifestations (diarrhea, vomiting or abdominal pain) [ ] , neurologic manifestations (altered mental status or seizures) [ , ] . therefore, increased ace- may be useless by promoting viral entry into lung cells, potentiating its devastating effect and enhancing mortality [ ] . consistent with these findings, suggesting ace- activators such as, diminazene aceturate (dize) [ ] , -[ -( carbamimidoyl phenyl) imino hydrazinyl] benzene carboximidamide and xanthenone (xnt) [ ] for counteracting covid- pathogenesis will be in vain. as a consequence, in order to diminish sars-cov- entry and its subsequent lung injury, pulmonary ace- activity should be reduced. however, at the same time, reduced ace- activity may contribute to worsening of the lung inflammation, by unopposed angiotensin ii accumulation [ ] and forcing the ras to continuously increase the expression of cytokines [ ] . simultaneously, a previous data indicated that the decrease in the ace- activity in a rat model of ards was paralleled by low amounts of ang-( - ) [ ] , which has been reported to play a beneficial role against pulmonary inflammation and fibrosis [ , ] . in this context, another study stated that treatment with protease-resistant, a cyclic form of ang ( - ) (cang - ), could restore the ace- activity; attenuated the inflammatory response; decreased lung injury and improved lung function [ , ] . that confirms the positive relationship between ang - and preserving the depressor role of ace- /ang ( - )/masr axis in pulmonary ras. hence, these data will attract the attention to the pivotal role of ang ( - ) in covid- pathophysiology and therapy. ang ( - ) is a biologically active metabolite of the ras that had become a peptide of interest in the last decade, because of its effective role in activating a number of crucial events for the homeostasis of normal physiological functions [ ] . it was reported that ang ( - ) could exert various effects, which are greatly in opposition to those of at- receptor activation such as vasodilator, anti-inflammatory, anti-hypertrophy, anti-proliferative, anti-fibrosis and antioxidant effects [ , ] . clinical and epidemiological studies have revealed the existence of a powerful link between the vasodilator effect of ang - and its higher plasma levels in females; making them less liability to hypertension than males [ , ] . several mechanisms have been attributed to ang - in lowering blood pressure which can be explained as follows (i) triggering enos to stimulate the release of no, which could play a critical role in promoting the relaxation of blood vessels and inhibiting the platelet aggregation [ , ] (ii) inducing natriuresis/diuresis [ ] , and (iii) activating peroxisome proliferator activator receptors (ppars), which in turn supports the availability of no [ ] . interestingly, ang - was documented to directly blunt the activation of pro-inflammatory signaling pathways induced by the ang ii-associated phosphorylation of mapks and nf-kb signaling [ , ] , suggesting also the anti-hypertrophic effects of ang - through normalization of mapks activity [ ] . moreover, ang ( - ), by counteracting the ang ii effects, could also preserve the endothelial function through increasing nitric oxide bioavailability and inhibiting oxidative stress [ ] . taken together, ang - could also reduce lung injury by suppressing the expression of fibrogenic molecules such as tgf-β [ ] , which acts as a key mediator involved in pulmonary fibrosis [ ] . moreover, several researches have pointed out the antioxidant role of ang - [ , ] that might be established by; (i) limiting the activation of nadph oxidase, which is a membranebound enzyme complex involved in triggering ros production through generating superoxide radicals [ ] , and/or (ii) normalizing the expression of antioxidant enzymes such as catalase and heme oxygenase- (ho- ) [ ] , as well as the nuclear factor erythroid -related factor (nrf ), that acts as an emerging regulator of cellular resistance to oxidants [ ] . other observation and experimental evidence suggested that endogenous aceis effects of ang ( - ) can be mediated by its unique membrane bound g protein-coupled receptor known as mas (masr) [ ] , which was revealed to be found in the thin areas of the bronchial epithelium and smooth muscle [ ] . masr could direct the ang - biological responses via the masr/camp/protein kinase a (pka) signaling [ ] , which was previously validated by administrating ave (a nonpeptide mimetic of ang - as a specific ligand (agonist) for masr [ ] . subsequently, attention should be drawn to specifically trigger the ang ( - ) as a potential therapeutic agent able to mitigate the lung injury in patients with covid- infection, without increasing ace- activity. there are different formulations of ang - are being developed e.g. ave- [ ] , hpβcd/ang - [ ] , cgen- [ ] , norleu -a [ ] and cyclic ang - [ ] and used to demonstrate its therapeutic potential in numerous animal models of human diseases, including hypertension, heart failure, stroke, diabetes mellitus, atherosclerosis, renal disease and pulmonary arterial hypertension [ , , ] . however, therapeutic attempts and clinical trials are still underway because of ang ( - ) rapid in vivo degradation by ace [ ] . on the other hand, previous studies emphasized that ang ( - ) could induce vasodilatation in rats of mas-deficient vessels [ ] and in rats pretreated with a (masr blocker) [ ] . the former observed data suggested that ang ( - ) might also interact with an additional specific receptor other than masr to elicit vasodilatation [ ] . indeed, ang ( - ) have been shown to stimulate also the bradykinin (bks) pathway via preventing the bk hydrolysis [ ] . bks are one of the formed kinins that can play significant roles in regulating tissue injury, inflammatory responses and vascular permeability [ ] . they have a little direct impact on the activation and recruitment of inflammatory cells, but they could work indirectly through stimulating the airway epithelial cells and lung fibroblasts through producing a wide array of cytokines, including il- , il- , il- , granulocyte colony stimulating factor (g-csf), gm-csf and macrophage chemoattractant protein- (mcp- ) [ ] [ ] [ ] [ ] . bk action was mediated by g-coupled receptors, namely bk receptor (br) [ ] that amazingly found to be interacted with the mas receptors in order to regulate the vasodilator effect of the ang ( - ) [ ] . at the same time, it was found that the reduction in ace- mrna expression within the lungs of stz diabetic rats was linked with an increase in circulating ang ii, but without any significant change in the production of ang ( - ) [ , ] , ensuring that pulmonary ace- was not the only enzyme responsible for ang ( - ) production, but there might be another enzyme contributing to its synthesis. by the time, the classical view of ras has been further expanded and become well established than previously conceived. as a consequence, it was discovered that there was another alternative pathway by which ang ( - ) is produced, instead of that based on ace- . that way was disclosed to degrade ang i directly into ang ( - ) by another ras member, called nep. nep (neutral endopeptidase or neprilysin, previously known as cd ) is a member of transmembrane zink-metalloendopeptidase that particularly highly expressed in both kidney and lung [ ] [ ] [ ] . nep was also found in a number of other tissues, as epithelia of breast, prostate, stomach and in the central nervous system [ ] [ ] [ ] . nep had been also shown to be present in a soluble circulating form (cnep) within several body fluids including urine, cerebrospinal fluid and plasma [ ] . although nep can discriminately hydrolyze a broad spectrum of physiologically relevant substrates, it was found to possess an obvious substrate specificity. classically, nep exhibits a size-related specificity that enables it to hydrolyze only peptides with a small molecular weight generally at or below , dalton [ ] . as well, nep has been also described to show a distinction between substrates being cleaved 'in vitro' and that being cleaved 'in vivo'. initially, nep was reported to specifically cleave more than peptides 'in vivo' including natriuretic peptides (nps) (atrial natriuretic peptide (anp), c-type natriuretic peptide (cnp), and b-type natriuretic peptide (bnp)), bks, neuropeptides (substance p, enkephalins), gastrin, chemotactic peptide formyl-met-leu-phe (fmlp), versus peptides 'in vitro' such as il-lβ, oxytocin, gastrin-releasing peptide (grp), et- , ang i and ii……etc [ ] [ ] [ ] . by time, more substrates had been proposed with varying levels of 'in vitro' and/or 'in vivo' proof of cleavage. among that, grp, et- , ang i which have been proved to be additionally metabolized 'in vivo' by nep [ , , ] , emerging the evidence that nep could play an important role in many physiological and pathological conditions [ ] , table for cvs patients, nps were known to be of therapeutic importance in lowering blood volume by inducing natriuresis, in which excess sodium can be excreted in urine with accompanying water by the renal tubules [ ] . previous clinitcal trials proved that administering synthetic nps might be associated with some limitations especially on the long run [ , ] , that pushed them to depend on using neprilysin inhibitors (nepi) such as (thiorphan or candoxatrilat) to prolong and potentiate the beneficial effects of vasoactive/nps via inhibiting endogenous nps degradation [ ] . nepi have been used for decades to treat acute diarrhea [ ] . however, they are now developed and emerged as a pharmaceutical target for different cvs diseases. nepi have been used mainly in people with congestive heart failure and a reduced left ventricular ejection fraction (lvef) [ ] . however, using nepi (e.g. candoxatril) solely for hypertension treatment was ineffective, because nepi could inhibit ang ii degradation, increasing its associated simultaneous detrimental effects. therefore, combining nepi either with acei (e.g. omapatrilat) or arbs (e.g. lcz ) become a necessity to overcome this limitation [ , ] . another critical aspect for the pharmacological role of nepi was achieved in treating diabetes via inhibiting the breakdown of some substrates known to modulate glucose metabolism, such as incretin glucagon-like peptide- (glp- ), nps and bks [ ] . within the brain, nep had also proved to hold a great beneficial role in the neurological disorders such as alzheimer's disease (ad) on both in vitro and in vivo studies [ ] [ ] [ ] . multiple lines of evidence highlighted the presence of many amyloid plaques in the form of βamyloid (aβ) peptide in the brains of ad patients with dementia [ ] . it was worthy mentioned that nep was one of the major aβ peptide-degrading enzymes in the brain, whose expression was documented to be lower in the brain of ad patients [ ] supporting the role that nep can play in the prevention and treatment of ad. in addition to the above mentioned, nep seems to play a protective role in the pathogenesis of lung injury since significant decrease in the nep enzymatic activity in the lung of mice with ali was associated with inactivation of the tachykinins degradation pathway and consequently, reducing uncontrolled inflammation in ali/ards and in other neurogenic respiratory diseases [ , , ] . of both respiratory bronchioles and alveoli [ , ] , resulting in excessive production of grp [ ] . grp was known to be one of the bombesin-like peptides that can be expressed and released by pnecs into the surrounding airway parenchyma in response to various stimuli like hypoxia or irritation [ , ] to regulate the neutrophil chemotaxis and macrophage infiltration within the lung tissue [ , ] . grp could act via stimulating gastrin-releasing peptide receptor (grpr) at the surface of macrophages, which in turn, would enhance the release of early inflammatory mediators contributing to the recruitment of neutrophils [ , ] . more neutrophil infiltration within the lung is usually associated with high tendency for lung tissue damage [ , ] , since they would be involved in breakdown of basement membrane integrity within the bronchiolar/alveolar architecture and thereby, diminution of pulmonary function. moreover, during the deleterious inflammatory reactions, as in pneumonia, neutrophil lifespan is prolonged to generate more superoxide radicals resulting in damage of the surrounding normal tissue [ ] . interestingly, nep can play a vital role during lung inflammation through its catabolic effect on (grp) [ ] in addition to its presence on the plasma membrane of neutrophils modulating their chemotactic responsiveness via cleaving the chemotactic peptide formyl-met-leu-phe (fmlp) which resembles an effective chemotactic agonist in response to grp [ , ] . thus, breaking both grb and fmlp peptide by nep will cut the way for recruiting more neutrophils into site of injury and consequently, grab the reins. on the other hand, once neutrophils being activated at inflammatory sites, they could secrete high concentration of several serine proteases into the extracellular environment to degrade host pathogens, recruit more cytokines and stimulate further tissue damage [ , ] . cathepsin g was reported to be one of the neutrophil-derived serine proteases that is abundantly found in the azurophil granules and known to degrade both angiotensinogen and ang i into ang ii [ , ] . so far, nep can additionally take a part in decreasing the pro-inflammatory, oxidative and profibrotic effects of ang ii by minimizing the release of cathepsin g and consequently, its action on ang i [ ] [ ] [ ] . as nep was reported to have more catalytic activity than ace- in cleaving ang i into ang ( - ), it could also effectively enhance the protective activities associated with ang ( - ) in the lung [ ] . as well, nep could not affect lung ang ( - ) metabolism because it was involved in the metabolism of ang ( - ) within tissues other than pulmonary tissues, as renal cortex. on the other hand, ace was recorded to be the major enzyme responsible for ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) metabolism in the pulmonary membranes by hydrolyzing it to ang ( - ), and then, into ang ( ) ( ) ( ) by the action of aminopeptidases [ ] . besides, nep shows higher activity than ace towards bks degradation, resulting in inhibition of the bradykinin-induced inflammatory cells influx [ , ] . despite multiple data confirming the expected role of nep in relieving the pulmonary inflammatory response, its effect on reducing exacerbation of acute severe pneumonia in covid patients has not been highlighted yet. hence, the question now, may the reviewed actions of nep pathways be sufficient to impose a new effective strategy for covid- management as compared to the suggested repurposed drugs? guidelines only provide supportive care. however, many drugs repurposed based on host response in order to defeat covid- , table [ ]. recently, there is an empirical use of anti-inflammation therapy in critical patients of covid- presented with severe complications in order to prevent further injury and suppress cytokine storm manifestations as ards and other organs damage till even death. main antiinflammatory medications include, non steroidal anti-inflammatory drugs (nsaids), corticosteroids, chloroquine and statins [ ] . numerous observational data support a strong association between use of nsaids in management of lower respiratory tract infections and higher incidence of complications including fulminating pneumonia, pleural effusions and dissemination of infection [ ] . nsaids may also induce nephrotoxicity among susceptible covid- patient groups and is exacerbated by fever and dehydration [ ] . consequently, some studies recommend avoiding use of nsaids e.g. ibuprofen and diclofenac as the first choice for fever control and pain symptoms in covid- infection and using paracetamol instead [ , ] . previous study demonstrated that corticosteroids-treated asthmatic patients showed enhanced nep expression in their airway epithelium as compared to nonsteroid-treated ones. this fits the hypothesis that the anti-inflammatory effect of corticosteroids within the airways may be partially mediated by upregulating nep [ ] . despite of possible benefits gained from the anti-inflammatory effect of corticosteroids, they will be associated with serious impairment in the immune system of severe covid- cases [ ] . corticosteroids may delay the viral elimination and increase susceptibility for the secondary infection resulting in deterioration of the disease especially with immune system impairment [ ] . other side effects associated with corticosteroid treatment include hyperglycemia, central obesity and hypertension which represent an obstacle against their use in people at higher risk for covid- especially diabetic, cardiac and hypertensive patients [ ] . auyeung et al., reported that there is no survival benefit for treatment of sars patients with corticosteroids [ ] . another study on corticosteroid therapy of mers patients has revealed no mortality difference with delayed clearance of mers-cov from lower respiratory tract [ ] . moreover, there is no evidence from any clinical trials supporting its administration for covid- [ ] . in order to overcome the immune suppression induced by corticosteroids, a variety of other therapies have been developed to act directly as specific anti-cytokines (e.g. anakinra or tocilizumab) [ ] or anti-inflammatory cytokines (e.g. il- or il- ) [ ] without targeting the immune system and have proven to be effective in treating several syndromes that triggered by cytokine storm [ ] . according to previous studies, chloroquine (cq) and its less toxic metabolite, hydroxychloroquine (hcq) possess anti-inflammatory and immunomodulatory benefits by affecting cell signaling in viral infections. cq/hcq also exhibit a wide variety of antiviral reactions against several viruses including members of the flaviviruses, retroviruses, and coronaviruses [ ] . cq and hcq can prevent the attachment of viral particles to their cell surface receptor, modulate ph in order to inhibit ph-dependent steps of viral replication or interfere with posttranslational modifications (ptms) of viral proteins [ , ] . an enough pre-clinical evidence considering cq effectiveness for treatment of covid- showed reduction in the pneumonia exacerbation, improving lung imaging findings and high rate of virus nucleic acid test negativity. accordingly, cq phosphate in guidelines (version ) for treatment of covid- has been recommended with oral administration twice daily at a dose of mg ( mg for chloroquine) for adults and no more than days [ ] . however, there is a narrow margin between cq/hcq therapeutic and toxic dose. its poisoning has been favored and life-threatening in patients with cardiac disorders [ ] . it is also contraindicated for people with retinopathy, elevated liver enzymes, heart rhythm disorders (as qt prolongation) or allergy to cq/hcq [ ] . further, efficiency and safety of cq/hcq for covid- is still unclear and needs a confirmation depending on more preclinical and clinical trials [ ] . statins are the widely used cholesterol lowering drugs, that were also reported to improve endothelial functions via lipid-independent mechanisms; mediated by their anti-inflammatory and anti-oxidant properties as well as their ability to restore vascular no bioavailability [ ] . because of their immunomodulatory effect, they have also proven to be beneficial as adjuvant therapy in patients with different auto-immune inflammatory conditions (e.g. systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis) [ ] . as a consequence, some hospitals included statins in the treatment protocol of covid- . although statin therapy is usually well tolerated, their use in covid- patients may increase the incidence and severity of myopathies and acute kidney injury [ ] . moreover, statin drugs may increase il- levels which can promote severe pneumonia, deteriorating sars-cov- -induced ards and mortality, especially in elderly patients who are more likely to use these drugs [ ] . sex differences in health outcomes following covid- may be attributed to sex-dependent production of steroid hormones. estrogen has been reported to attenuate inflammation which might protect women compared to men [ ] . estrogen signaling are also known to downregulate mcp- expression and promote adaptive t cell response by stimulating neutrophil recruitment [ ] . it has been also reported that serms, like toremifene, exhibits potential effects in blocking various viral infections as sarscov and merscov virus [ ] through interacting with and destabilizing the virus membrane glycoprotein resulting in inhibition of its replication [ ] . wang et al., suggests that treatment with estrogens and estrogen-related compounds as estradiol (e ) could suppress the expression of tmprss in the lung resulting in decreased mortality to sars-cov infection [ ] . an estrogen receptors/ras interaction has been demonstrated in several studies. e was detected to drive ras to increase ang-( - ) production through estrogen receptor (er α) mediated stimulation of ace and ace mrna expression and activity. a sex difference has been revealed in the expression of ras components [ ] . there is a controversy about the role of ras blockers including, ace inhibitors and/or angiotensin ii type receptor blockers (arbs) in treating covid- and their exact roles still remain unclear [ , , ] . their use has been suggested to be of a value through increasing ace- that may have a protective effect against virus-induced lung injury by preserving ace- in competition with sars-cov- entry into the cells [ ] . another mechanism by which ace- can prevent lung damage and attenuate the pulmonary fibrosis, will be via enhancing the ace- /ang-( - )/mas axis to increase the production of ang ( - ), which in turn can counteract the activity of the ace/angii/at r axis [ ] . on the other hand, it was known that ace inhibitors could block the breakdown of bradykinin increasing its level and then, promoting its associated inflammatory reactions resulting in more deterioration in the health state [ ] . additionally, it has been expected that patients receiving arbs may show upregulation in the membrane bound ace- facilitating the coronavirus entry and worsen then its course [ ] . the suggested explanation may be attributed to the increase in angiotensin ii level that probably pushes it to act as an increased substrate loaded on the ace enzyme, resulting in shifting a part of ang ii to be converted by the action of ace into ang ( - ) , that may be associated with ace- upregulation [ ] . other agents acting on the ras, such as beta-blockers and direct renin inhibitors (dri) to lower angi formation and thereby, angii and ang ( - ). however, till now, no one discussed their impact on the severity and prognosis of covid- [ ] . on the contrary, lacking ang ( - ) will be of negative effect on lung health. so, there is an urgent need to suppose a pathway that can ensure the increase of ang ( - ) level without upregulating ace- . that effect may be attained by keeping ace activity to enhance the pulmonary metabolism of ang ( - ) and at the same time, shifting the ras system away from ace/ang ii/at- axis to avoid its associated inflammatory and oxidative activities. we suggest that nep may achieve this complicated equation. based on previous literature that addressed several beneficial and protective effects displayed by nep during lung injury, we postulate that increasing nep activity may mitigate covid- pathogenesis. lung of covid- patients showed pneumocyte hyperplasia with inflammatory cellular infiltration [ ] , confirming the release of excessive grp into the surrounding airway parenchyma as a result of pnecs hyperplasia [ , , , ] . considering the documented links between high grp level and both neutrophil chemotaxis and infiltration as well as reduction in food and water intake [ ] , it is not surprising to detect high neutrophil count [ ] and anorexia in severe covid- patients [ ] . thus, we expect that grp is the first spark in initiating neutrophils recruitment as well as cytokine storm, which are the main pillars in covid- pathophysiology. our suggested hypothesis herein depends on two main aspects, figure ( nep may abrogate grp-induced neutrophil chemotaxis via cleaving grp and degrading fmlp peptide that can modulate the chemotactic responsiveness of neutrophils. on the other aspect, nep may withstand the potent cytokine storm, which was prescribed to be one of causes for lung damage progression and death in covid- patients. we suggest that nep can diminish the release of inflammatory cytokines that may increase sensitivity of target cells for further stimulation by sara-co- virus [ , ] . thus, nep can improve lung histopathology and enhance tissue survival through two mechanisms: firstly, interfering with ang ii formation via preventing the proteolytic cleavage of angiotensinogen and ang i into ang ii by neutrophil-derived cathepsin g [ ] [ ] [ ] that is expected to be released continuously in response to uncontrolled neutrophil activation associated with covid- patients and via regenerating the synthesis of endogenous ang ( - ), expected to be minimized because of ace- exhaustion by sara-co- virus [ ] . ang ( - ) by itself may protect against pulmonary fibrosis through reducing tgf-β expression [ ] . secondly, breaking bks and thereby, inhibiting its role in activation and recruitment of the inflammatory cells. however, recorded findings suggest that lung damage caused by covid- is induced by an alternative mechanism rather than hyperinfammatory injury. it likely seems that endothelial activation and associated pulmonary intravascular coagulopathy are the contributing factors in covid- pathogenesis [ ] . within this context, nep may exert a critical role in suppressing et- that mediates angiotensin ii-induced endothelial and platelets dysfunction. yet, nep may minimize the chance for ang ii formation, which was reported to be a potent stimulator of et- in endothelial cells [ ] . even, nep can additionally degrades et- , preventing its associated inflammatory injury and eventual fibrotic cascade in the lung [ , ] . furthermore, we speculate that nep may be helpful in dealing with individuals at high risk groups for covid- that exhibit many obstacles in their management. nep may regulate blood pressure in cardiovascular and hypertensive patients indirectly via decreasing both blood and tissue levels of ang ii by: (i) increasing ang i substrate availability a result of inhibiting cathepsin g-mediated neutrophil release, and (ii) augmenting the rate of ang i conversion into ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) , that previously reported to exert a stimulatory effect on anp secretion via masr [ ] . for diabetic patients, we demonstrate that nep may regulate pancreatic ras flux to improve glycemic conditions. in response to nep-mediated degradation 'in vitro', nep may evoke the insulin secretory ability of ang ( - ) via hydrolyzing it into the biologically active ang ( ) ( ) dipeptide in pancreatic islets [ , ] . numerous 'in vivo' and 'in vitro' experiments have been made to increase nep expression. one study speculated that dexamethasone could enhance nep expression in airway epithelial cells via promoting its transcription and synthesis [ ] . another prior finding demonstrated that valproic acid could reduce plaque formation and improve learning deficits via up-regulating nep in app/ps transgenic mice [ ] . recently, serotonin and its derivatives were explored to ameliorate symptoms of ad induced in mouse via enhancing nep up-regulation [ ] . several lines of evidence identified that hormones such as androgens [ ] and estrogen [ ] could also positively regulate nep expression, suggesting that decline in levels of androgen or estrogen associated with aging would accompany by decrease in nep synthesis, which may be an important factor for increasing the risk of covid- infection among elderly. on the other hand, several findings investigated the up-regulating effect of some natural products on nep expression such as apigenin, luteolin, and curcumin, (-)-epigallocatechin- -gallate as well as resveratrol [ ] [ ] [ ] . in china, naoerkang (nek), a traditional chinese herbal medicine, improved the ability of learning and memory in rats model of ad by increasing nep expression in their hippocampal tissues [ ] . however, some scientists aimed to produce recombinant nep (r nep) instead, as park et al., who prepared recombinant soluble nep from insect cells to be intracerebrally injected into ad mice [ ] . therefore, we finally suggest that therapeutic strategies aimed to increase nep expression and/or activity may be of great benefit for prevention and treatment of covid- . despite the high widespread of covid- contagion worldwide, there is no specific efficient treatment that has been proved till now. several pharmacological interventions for covid- have been suggested targeting the host's immune response. following sars-cov- exposure, there is an overproduction of grp within lung tissue resulting in increased release of inflammatory cytokines such as il- β, il- , tnf-α, vegf, gm-csf and mcp- . such cytokines are widely known to enhance neutrophil infiltration which, in turn, induce lung inflammation and respiratory distress as reported in covid- infected patients. as well, cytokines release is also presumed to develop sars-cov- -associated pulmonary fibrosis. novel findings define covid- as one of the pulmonary diseases associated with endothelial and platelets dysfunction. now, it is definitely known that viral invasion can be mediated by one of the ras signaling system components, namely ace- . hence, covid- occurrence and progression can be attributed to imbalance in the pulmonary ras signaling resulting from sars-cov- -induced ace- drain. interestingly, the decrease in ace- activity will be accompanied with a decrease in the generation of ang - which was known to be the light side of ras. regarding the data emphasized on the protective role of ang ( - ) in lung injury, it may be recommended as a covid- therapy, but because of its short half-life, ang ( - ) exhibits a limitation for its use. since nep is a more potent alternative way than ace- for producing ang - , it is suggested to assess the possible beneficial role of nep in covidinduced lung injury. few studies have discussed nep-mediated protective pathways in experimental models of lung injury and fibrosis, however its actual role as a lung protective therapy has not been yet recognized. nep has been involved in degradation of many peptides that may be incorporated in covid- pathophysiology. so, we expect that nep can effectively interfere with the chemotactic responsiveness and recruitment of neutrophils by degrading both fmlp peptide and gpr, respectively. furthermore, we suggest that nep can minimize cytokine storm associated with sars-cov- invasion through inhibiting ang ii formation by neutrophil-derived cathepsin g and directing ang i for generating ang ( - ) which can in turn suppress tgf-β expression and its fibrogenic action, protecting against fibrosis. degrading both bks and et- by nep may be associated with low il- levels, which will be beneficial for stabilizing endothelium and restoring its function. in addition to its catabolic properties, we postulate that nep may possess an advantage for covid- high risk patients through modulating blood pressure and glucose homeostasis. practically, numerous invivo and in-vitro experimental manipulations were made to upregulate nep expression either by using drugs (dexamethasone and valproic acid), hormones (androgens and estrogen) or natural substances (apigenin, luteolin, curcumin and (-)-epigallocatechin- -gallate). however, others directed their efforts towards preparing the recombinant nep (r nep). because most pre-clinical and clinical studies within the medical field are interested in studying nep inhibitors, there is a little data concerning use of nep as a therapeutic agent. consequently, its associated adverse effects have not yet been studied well. finally, we hope our hypothesis will be somewhat enough to direct a future work towards the therapeutic role of nep in modulating covid- pandemic and to target the subsequent therapies for enhancing nep activity in covid- patients. the authors declare no conflict of interest. the authors and their institutions are the only responsible for the financial support and the content of this work in the submitted manuscript. all other authors have no conflict of interests to disclose. 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severe acute respiratory syndrome: clinical and laboratory manifestations biologics for targeting inflammatory cytokines, clinical uses, and limitations side effects of anticytokine strategies chloroquine and hydroxychloroquine: current evidence for their effectiveness in treating covid- , cent. evidence-based med new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? the discovery and development of selective estrogen receptor modulators (serms) for clinical practice statpearls [internet aliskiren, the first direct renin inhibitor: assessing a role in pediatric hypertension and kidney diseases angiotensin-converting enzyme /angiotensin-( - )/mas axis protects against lung fibrosis by inhibiting the mapk/nf-kappab pathway efficacy and safety of aliskiren, a direct renin inhibitor, compared with ramipril in asian patients with mild to moderate hypertension interaction between raas inhibitors and ace in the context of covid- a blind, randomized comparison of racecadotril and loperamide for stopping acute diarrhea in adults racecadotril for childhood gastroenteritis: an individual patient data meta-analysis role of neprilysin inhibitors in heart failure lcz therapy reduces ventricular tachyarrhythmia inducibility in a myocardial infarction-induced heart failure rat model angiotensin receptor neprilysin inhibitor attenuates myocardial remodeling and improves infarct perfusion in experimental heart failure antihypertensive and antihypertrophic effects of omapatrilat in shr omapatrilat versus lisinopril effects of omapatrilat on blood pressure and renal injury in l-name and l-name plus doca-treated rats omapatrilat and enalapril in patients with hypertension: the omapatrilat cardiovascular treatment vs enalapril (octave) trial influence of sacubtril/valsartan on glycemic control in patients with type diabetes and heart failure with reduced ejection fraction angiotensin-neprilysin inhibition confers renoprotection in rats with diabetes and hypertension by limiting podocyte injury recombinant soluble neprilysin reduces amyloid-beta accumulation and improves memory impairment in alzheimer's disease mice neutral endopeptidase (ec . . . ) terminates colitis by degrading substance p neutral endopeptidase (ec . . . ) downregulates the onset of intestinal inflammation in the nematode infected mouse effects of recombinant neutral endopeptidase (ec . . . ) on the growth of lung cancer cell lines in vitro and in vivo recombinant human neutral endopeptidase ameliorates pancreatic elastase-induced lung injury recombinant human enkephalinase (neutral endopeptidase) prevents cough induced by tachykinins in awake guinea pigs tachykinin-induced dyspnea in conscious guinea pigs blocking its action on its receptors on mast cell, inhibiting release of inflammatory cells and thereby, fibroblasts activation that may participate in the development of lung fibrosis, (iii) degrading endothelin- and consequently, inhibiting tgf-β release and (iv) stabilizing ang ii-induced endothelial dysfunction as well as suppressing platelet activation and aggregation that initiate blood clot formation ace- = angiotensin-converting enzyme ang ( - ) = angiotensin at- = angiotensin ii type receptor et- = endothelin- ; fmlp = formyl methionyl-leucyl-proline grpr = gastrin-releasing peptide receptor pnecs = pulmonary neuroendocrine cells; sars-cov- = severe acute respiratory syndrome coronavirus-