key: cord-0979013-hrgtaunt
authors: Rabelo, Luiza A.; Nunes-Souza, Valéria; Bader, Michael
title: Animal Models with a Genetic Alteration of the ACE2/Ang-(1-7)/Mas Axis
date: 2015-04-24
journal: The Protective Arm of the Renin Angiotensin System (RAS)
DOI: 10.1016/b978-0-12-801364-9.00022-5
sha: ec1385d8356cf57df6df25a5aa247ebdab148b91
doc_id: 979013
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 2 (ACE2), angiotensin (Ang)-(1-7), 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 ACE2 and Mas, however, show aggravated cardiovascular pathologies and a metabolic syndrome-like state. In particular, the local production of Ang-(1-7) 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 ACE2 or Ang-(1-7) 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. 1, 2 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 2 (ACE2), Ang-(1-7), 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. 3 Currently, the use of transgenic technologies to access animal physiology is routine, but the pioneering work was performed in the 1980s. The first successful genetic modifications of a mouse were achieved by Gordon and colleagues. 4 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 10 years later, the same technology was also established for the rat, interestingly first targeting the RAS component renin. 5 Since 1986, the elimination of gene expression is also possible by homologous recombination-mediated targeted gene KO in embryonic stem (ES) cells. [6] [7] [8] 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. 6 In general, this method is still the most commonly used for the targeted alteration of the mouse genome. It took more than 20 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 2008, 9 and a few KO rat models have been developed since using ES cells. 10, 11 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. 12 Zinc finger (ZFN) and TALE nucleases use protein domains that specifically recognize a DNA sequence of choice, while the CRISPR/Cas9 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, 13 and since the Mas-KO rat has obviously been generated using ZFNs (http://rgd.mcw.edu/rgdweb/report/strain/main.html?id=5131952; access 25.04.2014) 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 ACE2/Ang-(1-7)/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 1 ).

The ACE2 gene is located on the X chromosome, and thus, heterozygous deletion (ACE2 −/y ) already results in the complete absence of the enzyme in male animals. Deletion of ACE2 in mouse leads to several cardiac abnormalities. In an elegant study, Crackower and colleagues provided the first in vivo evidence 14 supporting the hypothesis that the loss of ACE2 promotes heart dysfunction. 60 However, later studies on the function of ACE2 in the heart resulted in contradictory observations. 61 Two independent groups showed that the baseline cardiac function and morphology appeared normal in their ACE2-deficient mouse lines. 16, 18 Nevertheless, ACE2 −/y and even heterozygous female ACE2 +/− mice are more susceptible to pressure overload or diabetes-induced cardiac injury. [18] [19] [20] Moreover, the lack of ACE2 also exacerbated diabetic and shock-induced kidney injury. [25] [26] [27] There is also still a debate whether the deletion of ACE2 changes basic blood pressure in mice. Most likely, this effect depends strongly on the genetic background of the mice analyzed: 129 mice show hardly any effect, while C57BL/6 or FVB/N ACE2 −/y mice are clearly hypertensive 61 (our unpublished results). Nevertheless, C57BL/6 ACE2 −/y mice displayed high blood pressure during pregnancy and reduced weight gain and gave birth to smaller pups. 17 Besides an upregulation of oxidative stress in the brain and consequently of the sympathetic nervous system, 41 the cause for the hypertensive effect of ACE2 deletion may be an endothelial dysfunction as evidenced by an impaired acetylcholine-induced aortic vasodilatation. 15 ACE2 deletion in apolipoprotein E (ApoE) KO mice, a classical model for atherosclerosis, worsened plaque formation and vascular inflammation. 21, 22 In low-density lipoprotein receptor KO mice, fed a high-fat diet, ACE2 deletion also aggravated atherosclerosis. 62 Moreover, loss of ACE2 led to increased arterial neointima formation in response to endovascular injury in the femoral artery accompanied by an overexpression of inflammation-related genes. 22 Several studies have shown an important role of ACE2 in metabolism. C57BL/6 ACE2 −/y mice show impaired glucose homeostasis at different ages. 23, 24 Also, ACE2 gene deletion aggravated liver fibrosis in models of chronic hepatic injury. 63 ACE2 −/y mice displayed aggravated pathologies in the acute respiratory distress syndrome 28 and in bleomycin-induced lung injury 29 rendering ACE2 an important target for inflammatory lung diseases.

ACE2 −/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 SLC6A19. 30, 31 ACE2 −/y mice, therefore, show reduced levels of large amino acids, such as tryptophan, in the circulation, altered gut microbiota, and intestinal inflammation. 32

Besides being an enzyme and trafficking molecule, ACE2 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 ACE2 using the mouse ACE2 promoter, 33 the cytomegalovirus promoter, 36, 37 or the cytokeratin 18 promoter specific for the airway and other epithelia. 34, 35 As expected, human ACE2 expression led to an increased susceptibility of the transgenic mice to SARS virus infection. In the kidney, ACE2 overexpression protected the mice from shock-induced injury. 26 

The transgenic mouse overexpressing human ACE2 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-(1-7) . The animals are protected from hypertension induced by low peripheral infusions of Ang II 40 and DOCA-salt treatment. 64 Moreover, they show an amelioration of cardiac hypertrophy induced by AngII, 65 of chronic heart failure induced by coronary ligation, 66 and of stroke induced by middle cerebral artery occlusion. 42, 67 In most cases, an increase in Ang-(1-7) 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. 16 Increased blood pressure during pregnancy 17 Increased susceptibility to cardiac damage [18] [19] [20] Aggravated atherosclerosis 21, 22 Disturbed glucose homeostasis 23, 24 Aggravated kidney [25] [26] [27] and lung injury 28, 29 Amino acid uptake deficiency in the gut 30 

Transgenic mice overexpressing human ACE2 in the heart surprisingly showed an increase in ventricular tachycardia and sudden death, which was due to a dysregulation of connexins. 38 The underlying mechanism and the involved peptides could not be elucidated.

When human ACE2 was overexpressed in podocytes of mice using the nephrin promoter, the nephropathy induced by diabetes was ameliorated. 39 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- (1-7) .

ACE2 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 ACE2 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-(1-7). 43

In 1998, we generated Mas-deficient (Mas −/− ) mice on the mixed 129×C57BL/6 background and showed that these animals were healthy in appearance and grew normally and exhibited normal Ang II plasma levels. 44 However, male (but not female 45 ) 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 CA1 region of Mas −/− mice 44 but also that object recognition memory is impaired. 68 Collectively, these data support a role of Mas in behavior. C57BL/6 Mas −/− mice show a marked cardiac dysfunction both in vitro 69 and in vivo 46 accompanied by an increase in extracellular matrix proteins, such as collagen I, collagen III, and fibronectin. 46 Furthermore, Mas −/− animals exhibit vascular oxidative stress, endothelial dysfunction, and high blood pressure at least on the FVB/N genetic background. 47, 48 Consistently, endothelial function is also impaired in isolated Mas −/− vessels. 70 Consequently, Mas −/− mice exhibit a pronounced decrease in blood flow and a marked increase in resistance in different vascular beds. 71 These functional changes in both regional and systemic hemodynamics in Mas −/− mice suggest that the Ang-(1-7)/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. 49 Pinheiro et al. 50 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-(1-7) and Mas was reported in a model of unilateral ureteral obstruction in mice 72 in contrast to the anti-inflammatory effects of Ang-(1-7) and Mas in other models of kidney nephropathy. 73 Certainly, additional studies are needed to clarify the role of Ang-(1-7) and Mas in the kidney. The anti-inflammatory actions of Mas were also recently confirmed in a lipopolysaccharide (LPS)-induced endotoxic shock model. 74 Santos and colleagues 51 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 2 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. 75 Moreover, Silva et al. recently showed that Mas deletion in ApoE −/− mice leads to dyslipidemia and liver steatosis. 76 

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. 77 

Prolylcarboxypeptidase (PRCP, EC 3.4.16.2) is another enzyme that can generate Ang-(1-7) 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. 53 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. 52 In addition, PRCP also regulates angiogenesis and vascular repair. 78 How much of these cardiovascular actions are due to alterations in Ang II or Ang-(1-7) 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, 58,79 several transgenic animal models with altered Ang-(1-7) levels were generated.

The transgenic rat TGR(A1-7)3292 expresses the Ang-(1-7)-producing protein mainly in the testis. 80 In this model, Ang-(1-7) levels are chronically elevated in plasma and testis ~2.5-fold and ~4.5-fold, respectively. Surprisingly, this chronic increase in Ang-(1-7) did not alter basal blood pressure levels measured by telemetry. However, TGR(A1-7)3292 rats displayed a marked reduction in isoproterenol-induced heart hypertrophy and an improvement of postischemic systolic function. 54 Botelho-Santos and colleagues 57 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 57 and a reduction in basal urinary flow, leading to increased urinary osmolality and osmolal clearance. 80 Furthermore, chronic elevation of circulating Ang-(1-7) 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. 55, 56 Transgenic Mice and Rats Overexpressing Ang- (1) (2) (3) (4) (5) (6) (7) in the Heart Transgenic mice carrying the Ang-(1-7)-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 58 but not from myocardial infarction. 81 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. 59

Transgenic and KO rodent models were pivotal for our understanding of the protective functions of the novel RAS axis, ACE2/Ang-(1-7)/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 ACE2 and Mas, however, show aggravated cardiovascular pathologies and a metabolic-syndrome-like state. In particular, the local production of Ang- (1) (2) (3) (4) (5) (6) (7) 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 ACE2 or Ang-(1-7) in these tissues. Inducible and cell-type-specific KO models for Mas and ACE2 will be helpful in the future to deepen our understanding of the ACE2/Ang-(1-7)/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-2 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 p53 gene knockout rats by homologous recombination in embryonic stem cells

Efficient gene targeting by homologous recombination in rat embryonic stem cells

Barbas 3rd CF. Zfn, talen, and crispr/cas-based methods for genome engineering

Creation and characterization of a renin knockout rat

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

Angiotensin converting enzyme-2 confers endothelial protection and attenuates atherosclerosis

Altered blood pressure responses and normal cardiac phenotype in ace2-null mice

Angiotensin-converting enzyme 2 deficiency is associated with impaired gestational weight gain and fetal growth restriction

Deletion of angiotensin-converting enzyme 2 accelerates pressure overloadinduced cardiac dysfunction by increasing local angiotensin II

Cardioprotective effects mediated by angiotensin II type 1 receptor blockade and enhancing angiotensin 1-7 in experimental heart failure in angiotensin-converting enzyme 2-null mice

Heterozygote loss of ace2 is sufficient to increase the susceptibility to heart disease

Genetic ace2 deficiency accentuates vascular inflammation and atherosclerosis in the apoe knockout mouse

Deletion of angiotensin-converting enzyme 2 promotes the development of atherosclerosis and arterial neointima formation

Loss of angiotensin-converting enzyme 2 leads to impaired glucose homeostasis in mice

Loss of ace2 exaggerates high-calorie diet-induced insulin resistance by reduction of glut4 in mice

Loss of angiotensin-converting enzyme-2 (ace2) accelerates diabetic kidney injury

Role of angiotensin-converting enzyme (ace and ace2) imbalance on tourniquetinduced remote kidney injury in a mouse hindlimb ischemia-reperfusion model

Loss of ace2 accelerates time-dependent glomerular and tubulointerstitial damage in streptozotocin-induced diabetic mice

Angiotensin-converting enzyme 2 protects from severe acute lung failure

Angiotensin converting enzyme 2 abrogates bleomycin-induced lung injury

Defective intestinal amino acid absorption in ace2 null mice

Tissue-specific amino acid transporter partners ace2 and collectrin differentially interact with hartnup mutations

Ace2 links amino acid malnutrition to microbial ecology and intestinal inflammation

Mice transgenic for human angiotensin-converting enzyme 2 provide a model for sars coronavirus infection

Lethal infection of k18-hace2 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 ace2

Severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 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 2

Heart block, ventricular tachycardia, and sudden death in ace2 transgenic mice with downregulated connexins

Podocyte-specific overexpression of human angiotensin-converting enzyme 2 attenuates diabetic nephropathy in mice

Brain-selective overexpression of human angiotensin-converting enzyme type 2 attenuates neurogenic hypertension

Ace2-mediated reduction of oxidative stress in the central nervous system is associated with improvement of autonomic function

Angiotensin converting enzyme 2/ang-(1-7)/mas axis protects brain from ischemic injury with a tendency of age-dependence

Transgenic angiotensin-converting enzyme 2 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-(1-7) receptor mas knockout mice

Endothelial dysfunction and elevated blood pressure in mas gene-deleted mice

Ablation of angiotensin (1-7) receptor mas in c57bl/6 mice causes endothelial dysfunction

Evidence that the vasodilator angiotensin-(1-7)-mas axis plays an important role in erectile function

Genetic deletion of the angiotensin-(1-7) 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-(1-7)-producing fusion protein produces cardioprotective effects in rats

Improved lipid and glucose metabolism in transgenic rats with increased circulating angiotensin-(1-7)

Decreased hepatic gluconeogenesis in transgenic rats with increased circulating angiotensin

Expression of an angiotensin-(1-7)-producing fusion protein in rats induced marked changes in regional vascular resistance

-7) blunts hypertensive cardiac remodeling by a direct effect on the heart

Attenuation of isoproterenol-induced cardiac fibrosis in transgenic rats harboring an angiotensin-(1-7)-producing fusion protein in the heart

Angiotensin II-mediated oxidative stress and inflammation mediate the agedependent cardiomyopathy in ace2 null mice

Angiotensin-converting enzyme 2 gene targeting studies in mice: mixed messages

Angiotensin-converting enzyme 2 deficiency in whole body or bone marrow-derived cells increases atherosclerosis in low-density lipoprotein receptor−/− mice

Angiotensin-converting-enzyme 2 inhibits liver fibrosis in mice

Brain angiotensin-converting enzyme type 2 shedding contributes to the development of neurogenic hypertension

Angiotensin-converting enzyme 2 over-expression in the central nervous system reduces angiotensin-II-mediated cardiac hypertrophy

Brain-selective overexpression of angiotensin-converting enzyme 2 attenuates sympathetic nerve activity and enhances baroreflex function in chronic heart failure

Neuronal over-expression of ace2 protects brain from ischemia-induced damage

Angiotensin-(1-7)/Mas axis integrity is required for the expression of object recognition memory

Effects of genetic deletion of angiotensin-(1-7) 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-(1-7) and the g protein-coupled receptor Mas are key players in renal inflammation

Beneficial effects of the activation of the angiotensin-(1-7) mas receptor in a murine model of adriamycin-induced nephropathy

Receptor mas protects mice against hypothermia and mortality induced by endotoxemia

Angiotensin-(1-7) 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 mas1 protooncogene

Prolylcarboxypeptidase promotes angiogenesis and vascular repair

Tissue targeting of angiotensin peptides

Renal function in transgenic rats expressing an angiotensin-(1-7)-producing fusion protein

Circulating rather than cardiac angiotensin-(1-7) stimulates cardioprotection after myocardial infarction