key: cord-0034413-8vaxw441
authors: Ferrario, Carlos M.; Jessup, Jewell A.; Varagic, Jasmina
title: Newer Insights into the Biochemical Physiology of the Renin–Angiotensin System: Role of Angiotensin-(1-7), Angiotensin Converting Enzyme 2, and Angiotensin-(1-12)
date: 2009-08-21
journal: The Local Cardiac Renin-Angiotensin Aldosterone System
DOI: 10.1007/978-1-4419-0528-4_2
sha: 2139a6fd14cc9256b4507ccdcb2e1bda75d562bc
doc_id: 34413
cord_uid: 8vaxw441

Knowledge of the mechanisms by which the rennin–angiotensin system contributes to cardiovascular pathology continues to advance at a rapid pace as newer methods and therapies uncover the nature of this complex system and its fundamental role in the regulation of blood pressure and tissue function. The characterization of the biochemical pathways and functions mediated by angiotensin-(1-7) [Ang-(1-7)], angiotensin converting enzyme 2 (ACE2), and the mas receptor has revealed a vasodepressor and antiproliferative axis that within the rennin–angiotensin system opposes the biological actions of angiotensin II (Ang II). In addition, new research expands on this knowledge by demonstrating additional mechanisms for the formation of Ang II and Ang-(1-7) through the existence of an alternate form of the angiotensinogen substrate [angiotensin-(1-12)] which generates Ang II and even Ang-(1-7) through a non-renin dependent action. Altogether, this research paves the way for a better understanding of the intracellular mechanisms involved in the synthesis of angiotensin peptides and its consequences in terms of cell function in both physiology and pathology.

Knowledge of the mechanisms contributing to the pathogenesis of cardiovascular disease is today at a crossroad, possibly one of its most important stages, due to rapid advances in genetics, cellular signaling mechanisms, and the addition of new therapies. Concepts, often heavily weighted by a reductionist approach to accepting the multi-faceted nature of the mechanisms contributing to organ changes in the evolution of chronic disease processes, have been confronted by new discoveries that do not match previous tenets 1, 2 . Advances in molecular biology are bringing to the physician new and sometimes bewildering views, which he has to learn and judge in relation to clinical facts and to pressures derived from the new problems generated by advancing technology, earlier diagnosis, and longer survival. The evolution of knowledge on the contribution that the renin-angiotensin system has in the regulation of tissue perfusion in both health and disease is a good example. The past decade brought to the forefront the complexity of the renin-angiotensin system, which is more elaborate than originally accepted. The basic research knowledge of the role of angiotensin II (Ang II) in hypertension, vascular disease, and lipid and carbohydrate metabolism does not necessarily match with the outcomes of clinical trials testing the system's contribution by way of using angiotensin converting enzyme (ACE) inhibitors 1,2 , Ang II receptor blockers (ARBs) 3 , and now the new class of direct renin inhibitors [4] [5] [6] [7] [8] . It will be inappropriate to assume that this relative gap between the lessons that are learnt from testing the effects of these agents in the clinical setting and the information gained from meticulous studies of the renin-angiotensin system in animal models and cell systems suggests that one or the other has gone astray. What we need to remember is that homeostasis, in both health and disease processes, depends on the interplay of multiple regulatory mechanisms, which in the normal state act in coordination while they may become discordant in disease states.

In this chapter, we will address these issues from a viewpoint that for one of us (CMF) originates from perspectives gained from his association with Dr. Irvine H Page and from the research we conducted since the first demonstration of the biological actions of angiotensin-(1-7). Throughout this time, the slow process of unraveling pieces of this puzzle provides a more cogent understanding of the harmonious and dis-harmonious ways by which the renin-angiotensin system works to regulate normal blood pressure and its contribution to the expression of the disease, we call, essential hypertension.

Although a discussion of the biochemical pathways accounting for the formation of angiotensin peptides should begin with a description of the role of renin in the formation of angiotensin I (Ang I), for our objective we will begin with the discovery and analysis of the functions of the heptapeptide angiotensin-(1-7) [Ang-(1-7)], since its characterization became the stepping stone for a new understanding of the renin-angiotensin system. At the time of the first report of a biological effect of Ang-(1-7) 9 , investigators were adamantly focused on finding a receptor for the actions of Ang II. Work on Ang II analogs and Ang II peptide antagonists suggested that the Pro 7 -Phe 8 bond of the Ang II molecule was an essential requisite for binding to the as yet to be identified receptor [10] [11] [12] . Therefore, our first report that Ang-(1-7), having a truncated C-terminus, showed biological activity did not meet with any enthusiasm. Over the ensuing years, and as reviewed elsewhere, our laboratory continued to unravel the participation of Ang-(1-7) in the regulation of blood pressure , characterize the effects of Ang-(1-7) in blocking the proliferative actions of Ang II [34] [35] [36] , and decipher the biochemical pathways of Ang-(1-7) processing and metabolism 18, [37] [38] [39] [40] . Our studies and those of others further demonstrated a role for Ang-(1-7) in mediating a part of the antihypertensive actions of ACE inhibitors and ARBs [21] [22] [23] 31, 41, 42 . As work progressed, the concept advanced that Ang-(1-7) may act in tissues as a paracrine hormone opposing the actions of Ang II 43, 44 . A paracrine role for this component of the renin-angiotensin system explains the relative higher concentrations required for Ang-(1-7) effects, as it is known that at the vicinity of a tissue receptor, the concentration of the ligand may be in the nanomolar range. The characterization of the mas-receptor as the binding site expressing the cellular actions of Ang-(1-7) completed a critical step in defining the role of Ang-(1-7) in cardiovascular homeostasis and opened new avenues for exploring alternate approaches in competing against the pathological actions of Ang II 45, 46 .

A second critical step in gaining acceptance for the functional activity of Ang-(1-7) came about from the identification of angiotensin converting enzyme 2 −HO− −, a form of active oxygen species or free radical; AKT, protein kinase B; MEK 1, dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK); ERK 1 and ERK 2, mitogen-activated protein or extracellular signal-regulated kinase p 44ERK1 and p 42ERK2 (ACE2) by two separate laboratories 47, 48 . These studies led to the demonstration of a high efficiency of ACE2 in converting Ang II into Ang-(1-7) 49-51 , the demonstration that Ang II negatively regulated ACE2 gene expression [52] [53] [54] [55] and that genetic or molecular approaches to suppress ACE2 function were associated with cardiac abnormalities, vascular proliferative responses, and worsening of type 2 diabetes [56] [57] [58] [59] [60] [61] [62] . Several review articles detail the knowledge that was gained from exploring the role of ACE2 in cardiovascular pathology, its interaction with Ang- (1-7) , and its role as the cellular entry point for the severe acute respiratory syndrome (SARS) virus [63] [64] [65] . Figure 2 .1 illustrates the biochemical pathways leading to the formation of the biologically active angiotensin peptides.

Our original view of the RAS as a complex system entailing several levels of regulation and processing expanded with the identification of proangiotensin-12 [angiotensin-(1-12), Ang-(1-12)] as an upstream propeptide to Ang I. Nagata et al. 66 first isolated this novel angiotensinogen-derived peptide from the rat small intestine. Consisting of 12 amino acids, this peptide was termed proangiotensin-12 [Ang-(1-12)], based on its possible role as an Ang II precursor. Ang-(1-12) constricted aortic strips and, when infused intravenously, raised blood pressure in rats 66 . The vasoconstrictor response to Ang-(1-12) was abolished by either captopril or the Ang II type I receptor blocker, CV-11974. Over the years, questions arose regarding the capability of tissues other than the kidneys to synthesize Ang II, in part because gene expression for some of the RAS components occurs at low levels (i.e., renin and Aogen). The heart is a critical example. Although a large body of evidence suggests a participation of local tissue RAS in the regulation of cardiac function and remodeling [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] , most studies showed low levels of gene expression for both cardiac renin and Aogen. Neither the identification of renin in cardiac mast cells 77 nor the finding of renin activation by prorenin binding to the prorenin/renin receptor [78] [79] [80] can be construed as evidence for local production of cellular renin, as an uptake mechanism from the blood compartment cannot be excluded.

Many cell types in myocardial tissue, including cardiomyocytes, contain receptors for Ang II, but as indicated above the activation of these receptors requires angiotensin concentrations in the micromolar range, which do not occur in plasma in vivo. However, angiotensins formed locally in the heart can activate these receptors in a paracrine and autocrine mode 81 . Indeed, recent studies have provided compelling evidence for the existence of an intracellular RAS that functions as an autocrine system 67, 68, [74] [75] [76] 81 . It has been suggested that intracellular generation of Ang II may occur via a non-renin pathway with rerouting of Aogen to other subcellular structures 67, 68, 71, [74] [75] [76] [81] [82] [83] [84] [85] [86] [87] [88] [89] . With this in mind, we began to explore the potential role of Ang- (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in the formation of angiotensin peptides as well as to inquire into the mechanisms that may regulate the processing of Aogen into Ang- (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) . Work in progress documents the expression of Ang- (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in cardiac myocytes of Wistar Kyoto (WKY) rats, increased expression of Ang- (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in the spontaneously hypertensive rat (SHR) 90 , and evidence that Ang-(1-12) is a functional substrate for the formation of Ang I, Ang II, and even Ang-(1-7) in the isolated heart from several rat strains 91 . In rodents, the sequence of Ang- (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) is Asp 1 -Arg 2 -Val 3 -Tyr 4 -Ile 5 -His 6 -Pro 7 -Phe 8 -His 9 -Leu 10 -Leu 11 -Tyr 12 . Since renin specifically cleaves the Leu 10 -Leu 11 bond of rat Aogen to form Ang I, the cleavage between the two aromatic residues Tyr 12 -Tyr 13 for the liberation of Ang-(1-12) may not be accounted for by the action of renin. Assessment of the processing of Ang-(1-12) into angiotensin peptides in the isolated heart confirmed that renin is not involved in processing Ang-(1-12) into Ang I. In addition, we have recently extended these observations in rats in which bilateral nephrectomy was employed as a tool for elimination of renal renin 92 . The anephric state resulted in divergent effects on circulating and cardiac content of Ang-(1-12), Ang I, and Ang II since these peptides fell in the plasma, but increased markedly in the left ventricle of 48 h bilateral nephrectomized WKY rats compared to sham-operated controls. A 34% decrease in plasma Ang-(1-12) levels 48 h post-nephrectomy was associated with a 78 and 66% decrease in plasma Ang I and Ang II, respectively (p < 0.05 versus sham-animals). In contrast, cardiac content of Ang-(1-12) in anephric rats averaged 276 ± 24 fmol/mg compared to 144 ± 20 fmol/mg in sham-operated controls (p < 0.005). A representative example of Ang-(1-12) in cardiac myocytes is illustrated in Fig 2. 2. Further research on Ang-(1-12) may resolve the nature of the precursor protein accounting for the local synthesis of angiotensins (Ang II and Ang-(1-7)) in cardiovascular tissues, particularly the heart. The lower molecular weight Ang-(1-12) peptide (compared to Aogen) may represent a stored form of a precursor to angiotensins formation since its amino acid sequence is composed of 12 rather than the 458 amino acids of the parent Aogen compound. These findings are also germane to the demonstration by others of the existence of an intrinsic mechanism for the intracellular generation of Ang II in cardiac myocytes that is independent from uptake from the circulation or the interstitial environment 68, 82 .

Our past work on Ang-(1-7) and ACE2 has been seminal in bringing about a better understanding of the biochemical and functional role of the renin-angiotensin system in cardiovascular regulation. Furthermore, the discovery of the biological actions of Ang-(1-7) provided the underpinning for the latter characterization of ACE2 and the establishment of the mas-receptor as a component of the system. Altogether, these studies contributed much to the affirmation of a role of this system in tissues and established a newer understanding of how the counter regulatory actions of Ang-(1-7) oppose the effects of Ang II on arterial pressure and cardiovascular remodeling. A new phase in unraveling the biochemical pathways that determine the generation of angiotensin peptides arises from studies now identifying Ang-(1-12) as an alternate Ang I forming substrate for the cellular processing of Ang II and even Ang-(1-7). The discovery of Ang-(1-12) as an endogenous substrate for the formation of angiotensin peptides may be critical to the understanding of intracellular mechanisms associated with the actions of the renin-angiotensin system in health and disease. It will be incorrect to argue that characterization of this potentially alternate substrate may have no major consequences in terms of the functions of the biologically active angiotensins. First, characterization of Ang-(1-12) as a cellular substrate may explain the observations (both clinical and experimental) of incomplete blockade of Ang II formation with the currently approved orally active renin inhibitor [aliskiren (Tekturna R )]. We have reviewed in detail the data 93 that demonstrates that the antihypertensive effects of aliskiren are not greater than those obtained with amlodipine 94 and that the addition of an angiotensin receptor blocker (ARB) markedly potentiates the response to renin inhibition 4, 7 . These data suggest that aliskiren does not eliminate formation of Ang II. Further evidence for an incomplete blockade of Ang II generation was obtained in normal volunteers in whom the reduction of plasma Ang II following administration of Ang II was not complete as plasma Ang II levels returned toward control values within 12 h after oral administration 95 . To date no studies of the fate of Ang II following direct renin inhibition have been performed in essential hypertensive subjects. Thus, our recent studies on Ang-(1-12) have much significance in determining the efficacy of direct renin inhibition as the current data suggest that Ang-(1-12) generation is not dependent on renin.

If we are correct in assuming that the primary factor involved in the expression of hypertension and the remodeling of the heart and blood vessels originates from the rupture of the equilibrium between on one hand the ACE/Ang II/AT 1 receptor axis and on the other the state of the counterbalance actions of ACE2/Ang-(1-7) /mas-receptor axis, a further inquiry into the mechanisms that result in reduced expression of the later regulatory arm of the renin-angiotensin system should lead to new therapies and a better understanding of the cellular processes at which these two systems act to maintain homeostasis.

Lessons from ONTARGET

Telmisartan, ramipril, or both in patients at high risk for vascular events. Results of the ONTARGET trial

VALUE trial: long-term blood pressure trends in 13,449 patients with hypertension and high cardiovascular risk

Long-term safety, tolerability and efficacy of aliskiren in combination with valsartan in patients with hypertension: a 6-month interim analysis

What is the purpose of dual or triple inhibition of the renin-angiotensin-aldosterone system?

Aliskiren combined with losartan: Thor s hammer or Sigurd s sword?

Dual inhibition of the renin system by aliskiren and valsartan

Aliskiren fails to lower blood pressure in patients who have either low PRA levels or whose PRA falls insufficiently or reactively rises

Release of vasopressin from the rat hypothalamo-neurohypophysial system byangiotensin-(1-7) heptapeptide

Synthesis of some analogs of angiotensin II as specific antagonists of the parent hormone

Factors that influence the antagonistic properties of angiotensin II antagonists

Agonist and antagonist relationships in 1-and 8-substituted analogs of angiotensin II

Cardiovascular actions of angiotensin(1-7)

Antihypertensive actions of angiotensin-(1-7) in spontaneously hypertensive rats

Pressor and reflex sensitivity is altered in spontaneously hypertensive rats treated with angiotensin-(1-7)

Angiotensin-(1-7): a novel vasodilator of the coronary circulation

Differential actions of angiotensin-(1-7) in the kidney

Pathways of angiotensin-(1-7) metabolism in the kidney

Angiotensin-(1-7): a new hormone of the angiotensin system

Counterregulatory actions of angiotensin-(1-7)

Characterization of angiotensin-(1-7) in the urine of normal and essential hypertensive subjects

Effects of omapatrilat on the renin-angiotensin system in salt-sensitive hypertension

Vasopeptidase inhibition and Ang-(1-7) in the spontaneously hypertensive rat

Contribution of angiotensin-(1-7) to cardiovascular physiology and pathology

Renal actions of angiotensin-(1-7): in vivo and in vitro studies

Vasodepressor actions of angiotensin-(1-7) unmasked during combined treatment with lisinopril and losartan. Hypertension

Angiotensin-(1-7) contributes to the antihypertensive effects of blockade of the renin-angiotensin system

Evidence that prostaglandins mediate the antihypertensive actions of angiotensin-(1-7) during chronic blockade of the reninangiotensin system

Angiotensin-(1-7). A member of circulating angiotensin peptides

Angiotensin(1-7) in the spontaneously hypertensive rat

Effects of captopril related to increased levels of prostacyclin and angiotensin-(1-7) in essential hypertension

Angiotensin-(1-7) and nitric oxide interaction in renovascular hypertension

Characterization of angiotensin-(1-7) receptor subtype in mesenteric arteries

Angiotensin-(1-7) inhibits vascular smooth muscle cell growth

Alterations in prostaglandin production in spontaneously hypertensive rat smooth muscle cells

Angiotensin-(1-7) reduces smooth muscle growth after vascular injury

Metabolism of angiotensin-(1-7) by angiotensin-converting enzyme

Release of angiotensin-(1-7) from the rat hindlimb: influence of angiotensin-converting enzyme inhibition

Evidence that prolyl endopeptidase participates in the processing of brain angiotensin

A comparison of the properties and enzymatic activities of three angiotensin processing enzymes: angiotensin converting enzyme, prolyl endopeptidase and neutral endopeptidase 24.11

Influence of gender and genetic variability on plasma angiotensin peptides

Role of the vasodilator peptide angiotensin-(1-7) in cardiovascular drug therapy. Vasc Health Risk Manag

Advances in biochemical and functional roles of angiotensin-converting enzyme 2 and angiotensin-(1-7) in regulation of cardiovascular function

Angiotensin-converting enzyme 2 and angiotensin-(1-7): an evolving story in cardiovascular regulation

Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas

Recent advances in the angiotensin-converting enzyme 2-angiotensin(1-7)-Mas axis

A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9

ACEH/ACE2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ACE inhibitors

Evaluation of angiotensin-converting enzyme (ACE), its homologue ACE2 and neprilysin in angiotensin peptide metabolism

Circulating activities of angiotensin-converting enzyme, its homolog, angiotensin-converting enzyme 2, and neprilysin in a family study

Hydrolysis of biological peptides by human angiotensinconverting enzyme-related carboxypeptidase

Distinct roles for ANG II and ANG-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes

Regulation of ACE2 in cardiac myocytes and fibroblasts

MAP kinase/phosphatase pathway mediates the regulation of ACE2 by angiotensin peptides

Upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin II receptors

Angiotensin-converting enzyme 2 is an essential regulator of heart function

ACE2 overexpression inhibits hypoxia-induced collagen production by cardiac fibroblasts

Functional angiotensin-converting enzyme 2 is expressed in human cardiac myofibroblasts

Protection from angiotensin II-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats

Increased expression of angiotensin converting enzyme 2 in conjunction with reduction of neointima by angiotensin II type 1 receptor blockade

ACE2: a new target for prevention of diabetic nephropathy?

Angiotensin converting enzyme-2 confers endothelial protection and attenuates atherosclerosis

The angiotensin-converting enzyme gene family: genomics and pharmacology

ACE2: from vasopeptidase to SARS virus receptor

Hypothesis: ACE2 modulates blood pressure in the mammalian organism

Isolation and identification of proangiotensin-12, a possible component of the renin-angiotensin system

Evidence of a novel intracrine mechanism in angiotensin II-induced cardiac hypertrophy

Intracellular angiotensin II induces cell proliferation independent of AT1 receptor

Tissue renin-angiotensin system: sites of angiotensin formation

Is there an internal cardiac renin-angiotensin system?

Angiotensin II and the heart: on the intracrine renin-angiotensin system

The cardiac renin-angiotensin system: conceptual, or a regulator of cardiac function?

The cardiac renin-angiotensin system: novel signaling mechanisms related to cardiac growth and function

The intracellular renin-angiotensin system: implications in cardiovascular remodeling

Intracellular angiotensin ii production in diabetic rats is correlated with cardiomyocyte apoptosis, oxidative stress, and cardiac fibrosis

Activation of the intracellular renin-angiotensin system in cardiac fibroblasts by high glucose: role in extracellular matrix production

Cardiac mast cell-derived renin promotes local angiotensin formation, norepinephrine release, and arrhythmias in ischemia/reperfusion

Renal renin-angiotensin system

Nonproteolytic activation of prorenin contributes to development of cardiac fibrosis in genetic hypertension

Prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin II type 1a receptor-deficient mice

The cardiac renin-angiotensin system: physiological relevance and pharmacological modulation

In vitro evidence for an intracellular site of angiotensin action

Nuclear accumulation of the AT1 receptor in a rat vascular smooth muscle cell line: effects upon signal transduction and cellular proliferation

Opposite effects of angiotensin II and angiotensin (1-7) on impulse propagation, excitability and cardiac arrhythmias. Is the overexpression of ACE2 arrhythmogenic?

Intracellular signaling pathways of angiotensin II receptor type 1 involved in the development of cardiovascular diseases

Tissue angiotensin II generating system by angiotensin-converting enzyme and chymase

Intracellular renin-angiotensin system: the tip of the intracrine physiology iceberg

Intracellular renin and the nature of intracrine enzymes

Renin excess after renin inhibition: malefactor or innocent bystander?

Localization of the novel angiotensin peptide, angiotensin-(1-12), in heart and kidney of hypertensive and normotensive rats

Angiotensin-(1-12) is an alternate substrate for angiotensin peptide production in the heart

Differential regulation of angiotensin-(1-12) in plasma and cardiac tissue in response to bilateral nephrectomy

Renin inhibitor pharmacotherapy for hypertension

Antihypertensive efficacy of the oral direct renin inhibitor aliskiren as add-on therapy in patients not responding to amlodipine monotherapy

Angiotensin II suppression in humans by the orally active renin inhibitor Aliskiren (SPP100): comparison with enalapril