Keywords

Introduction

Transgenic and knockout animal models are the most effective tools to study cardiovascular hormone systems, since they reveal effects of changes in single components of these systems on the whole physiology. In particular, studies on the renin-angiotensin systems (RAS) have profited from this technology in recent decades [3, 5, 58]. Therefore, it was warranted to establish such models also for the novel RAS consisting of ACE2, Ang-(1-7), and Mas (Table 1). Despite that these three components comprise a common axis, distinct phenotypes of models with one of the components altered are expected since each of the three components has distinct additional functions independent from the two other molecules. ACE2, in particular, is a protein with several functions, a carboxypeptidase metabolizing a multitude of peptides, such as AngII and apelins, thereby either activating or inactivating them [104], a protein with a collectrin domain, which is involved in amino acid uptake in the gut [34, 95], and the receptor for the severe acute respiratory syndrome (SARS) coronavirus [45]. Moreover, also Ang-(1-7) may interact with other receptors than Mas and Mas may have other ligands or exert ligand-independent effects [4, 86].

Table 1 Independently generated transgenic and knockout mouse and rat models for the ACE2/Ang-(1-7)/Mas axis of the RAS

ACE2 Models

ACE2 Knockout Mice

Since the ACE2 gene is localized on the X-chromosome, male mice with ACE2 gene deletion (ACE2−/y) are already deficient in the enzyme in the hemizygous state. Based on the pleiotropic actions of this protein, mice lacking ACE2 are expected to exhibit increased levels of AngII, decreased levels of Ang-(1-7) and tryptophan, as well as alterations in other peptide levels, which all may contribute to observed phenotypes. ACE2−/y mice and also heterozygous female ACE2+/− mice were more susceptible to cardiac injury induced by pressure overload, AngII infusion, or diabetes [69, 109, 115, 125] and ACE2−/y mice developed cardiac abnormalities at older age [17] probably due to an increased level of Ang II [68]. However, the spontaneous appearance of cardiac alterations could not be confirmed by another group and therefore remains controversial [31, 32, 125]. However, obesity-induced epicardial inflammation was worsened and caused cardiac dysfunction in ACE2−/y mice [70]. Furthermore, in heart and skeletal muscle, ACE2 was involved in training-induced physiological hypertrophy [59].

There were also inconsistencies in the reports about hypertension in ACE2−/y mice, but it is now accepted that this phenotype is depending on the strain of mice appearing in C57BL/6 and FVB/N but not in 129 mice [32, 38, 77, 101]. ACE2-deficient mice on C57BL/6 background even developed a pre-ecclampsia-like syndrome when pregnant [8] and placental hypoxia and uterine artery dysfuncion caused fetal growth restriction in these animals [124]. In ACE2−/− female mice, estrogen cannot inhibit obesity-induced hypertension in contrast to wild-type controls [114]. We and others have described AngII-dependent endothelial dysfunction in ACE2-deficient mice [49, 77], which probably mediated the prohypertensive phenotype. However, an increased sympathetic outflow may have also contributed [119]. On the other hand, ACE2 also degrades the vasodilator apelin peptides which consequently accumulate in ACE2−/y mice and counteract the effects on the RAS [110]. Nevertheless, there were several other vascular effects of genetic ACE2-deletion such as a worsening of atherosclerosis and aortic aneurysm in apolipoprotein E (ApoE)- and low-density lipoprotein receptor-deficient mice [63, 81, 99, 100] and an increased neointima formation after vascular injury [81], to which the endothelial dysfunction was a major contributor.

In double knockout mice for ACE2 and ApoE, also the renal injury induced by atherosclerosis was aggravated [38]. Moreover, ACE2−/y mice spontaneously developed glomerulosclerosis in older age [67] and were more susceptible to renal ischemia/reperfusion injury due to increased cytokine expression, inflammation, and oxidative stress [23]. Accordingly, genetic ACE2 deficiency led to accelerated nephropathy in streptozotocin (STZ)-induced and Akita diabetic mice [91, 115]. Furthermore, knockout mice for ACE2 infused with AngII showed enhanced collagen I deposition in renal glomeruli and expression of genes related to fibrosis, such as smooth muscle actin, transforming growth factor β (TGF-β), and procollagen I, probably through activation of ERK1/2 and enhancement of protein kinase C levels [133]. ACE2-deficient mice also showed a worse outcome in shock-induced kidney injury [127], chronic hepatic injury [66], liver steatosis [12, 64], and cerulein-induced pancreatitis [48].

The lung is a major site of ACE2 expression. Accordingly, ACE2−/y mice exhibited an aggravated pathogenesis of lung injury induced by cigarette smoke, air pollution, bleomycin, influenza virus or respiratory syncytial virus [30, 36, 46, 80, 134], of pulmonary hypertension [129], and of acute respiratory distress syndrome [37]. In most of these injury models, the increased oxidative stress observed in kidneys [116], livers [12, 64], and vessels [71] of ACE2−/y mice contributed to the exacerbation.

ACE2 in the gut with its collectrin domain is part of the amino acid uptake system and, therefore, mice lacking this protein showed reduced tryptophan in the blood, an altered gut microflora, and intestinal inflammation [34, 95]. These results were recently confirmed in a novel ACE2-deficient mouse model on an outbred genetic background generated by transcription-activator-like effector nucleases (TALEN) [47]. Whether the collectrin-domain-dependent effects contributed to the metabolic alterations shown in ACE2-deficient mice, such as insulin resistance and impaired glucose homeostasis [12, 63] in particular under a high-fat diet [15, 50, 90, 92, 123] needs still to elucidated [7]. However, in the liver, the carboxypeptidase function of ACE2 was more relevant for these metabolic effects since they could be ameliorated by Ang-(1-7) infusion [12].

ACE2 in the brain also influences behavior since ACE2-deficient mice showed impaired performance in cognition and memory tests [111].

ACE2 S680D Knockin Mouse

Recently, it was discovered that serine 680 of mouse ACE2 is phosphorylated by AMP kinase, leading to increased stability of the protein. When this phosphorylation was mimicked (S680D) in knockin mice by CRISPR/Cas9 technology, the resulting animals were partially resistant to a pulmonary hypertension model [129].

ACE2 Knockout Rats

ACE2 knockout rats have recently been established using TALEN technology [130]. These animals exhibited cardiac hypertrophy and impaired heart function; however, their blood pressure was not reported. Therefore, it remains unclear whether the cardiac effects are direct or caused by hypertension.

Inducible Mouse ACE2 Overexpression in Mouse

In order to allow tissue-specific activation of ACE2 expression, the mouse ACE2 coding region was knocked into the Rosa26 locus of mice with a Stop-lox cassette in front of it, which inhibits transcription. This cassette can be removed by Cre-recombinase expression and then ACE2 gets highly expressed in the cells expressing Cre-recombinase. When Cre-recombinase was expressed in the germline, ubiquitously ACE2 overexpressing mice resulted, which were protected from post-infarction cardiac dysfunction [75] and exhibited less anxiety-related behavior [107]. The same behavioral effects were also observed when the gene was only activated in CRH (corticotropin-releasing hormone) expressing cells using the corresponding Cre-recombinase-expressing mouse for breeding with the ACE2/Rosa26 animals [108].

Human ACE2 Overexpression in Mouse

Human ACE2 is hijacked by the SARS virus as a receptor to enter cells. In order to create a model for this disease, mice were “humanized” by several groups by inserting human ACE2 transgenes in their genome either using the ACE2 promoter itself [126], the ubiquitously active cytomegalovirus (CMV) promoter [102, 128], or the airway-specific cytokeratin 18 promoter [53, 62]. These animals were also suitable for studies on the role of ACE2 in other diseases and therefore the first model was tested in a kidney injury model and showed a protected phenotype [127]. Moreover, it was shown to be protected from AngII-induced hypertension and myocardial fibrosis [109].

Human ACE2 Overexpression in Mouse Heart

When human ACE2 was overexpressed in hearts of transgenic mice, surprisingly ventricular tachycardia and sudden death was observed accompanied by a dysregulation of connexin expression [21]. Apelin, which is also a substrate for ACE2 [104], may in this case be lacking and this deficiency may have caused the cardiac dysfunction [41].

Human ACE2 Overexpression in Mouse Podocytes

When human ACE2 was overexpressed in kidneys of transgenic mice, particularly in podocytes using the nephrin promoter, the animals became protected from diabetes-induced renal injury [61]. The authors provided evidence that the relative amounts of AngII and Ang-(1-7) are critical for the phenotype by increased AngII upregulating TGF-β.

Human ACE2 Overexpression in Mouse Brain

When human ACE2 was overexpressed in brains of transgenic mice using the synapsin promoter, a protective phenotype is observed for several cardiovascular diseases. This included hypertension induced by peripheral infusions of AngII [26] and by desoxycorticosterone acetate (DOCA)/salt treatment [118], cardiac hypertrophy elicited by AngII [25], coronary ligation-induced chronic heart failure [120], and stroke triggered by middle cerebral artery occlusion [14, 132]. In another model, the ACE2 transgene was flanked by loxP sites and it could therefore be specifically deleted in distinct brain regions by the local injection of Cre-recombinase-expressing adeno-associated viruses to assess the relevance of these areas for the blood pressure increase after DOCA/salt treatment. Such experiments revealed the paraventricular nucleus of the hypothalamus and the subfornical organ as important but not exclusive contributors to hypertension development [117]. The shift in the balance between Ang-(1-7) and AngII in brain regions important for cardiovascular control modulated local NO and ROS production as well as cyclooxygenase-mediated neuroinflammation [97] and likely caused the beneficial effects of ACE2 in the brain. Accordingly, the AngII-dependent deleterious effects on brain tissues observed in double transgenic mice expressing human angiotensinogen and human renin were mitigated in triple transgenic animals additionally expressing human ACE2 [14, 131].

Human ACE2 Overexpression in Rat Vascular Smooth Muscle

When we overexpressed human ACE2 in vascular smooth muscle of transgenic rats of the spontaneously hypertensive stroke-prone (SHRSP) strain using the smooth muscle myosin heavy chain promoter, blood pressure was significantly reduced [79]. This confirmed a study postulating that reduced ACE2 is an important genetic determinant for hypertension in this strain [17]. Reduced blood pressure was accompanied by decreased oxidative stress and improved endothelial function [79].

Mas Models

Mas Knockout Mice

When we generated Mas-deficient (Mas−/−) mice, it was not yet known that it is the receptor for Ang-(1-7) [105]. Therefore, phenotyping concentrated on the brain as major Mas-expressing organ. Male (but not female [106]) Mas-deficient mice showed increased anxiety-like behavior and long-term potentiation (LTP) in the hippocampus [105]. Surprisingly, despite the improved LTP, object recognition memory was impaired [43]. However, Mas−/− mice showed delayed extinction of fear memory [42] and were protected from cognitive impairments induced by ischemia but only in the presence of the AngII AT2 receptor [35] supporting a role of the dimerization of both receptors in brain function [44].

After our discovery that Mas is the receptor for Ang-(1-7) [85], we performed comprehensive cardiovascular phenotyping. Mas-deficient mice on the C57BL/6 background exhibited spontaneous cardiac fibrosis and dysfunction [13, 72, 83, 113]. Increased oxidative stress and endothelial dysfunction were observed on all genetic backgrounds studied (C57BL/6 and FVB/N) [33, 78, 121], but only resulted in hypertension in FVB/N mice. Possibly, an autonomic dysbalance in Mas−/− mice also contributed to the increased blood pressure [76]. Moreover, regional blood flow and local vascular resistance were differentially altered in different tissues of Mas−/− mice [10], which may also be the cause for the increased vascular resistance in the corpus cavernosum and the resulting erectile dysfunction observed in these mice [29].

Mas−/− mice showed an impaired renal function with increased urinary volume and proteinuria [74]. However, Esteban and coworkers found that Mas knockout mice presented an attenuation of renal damage in the unilateral ureteral obstruction and in the renal ischemia/reperfusion model [22]. The authors reported that Ang-(1-7) infusion led to NF-κB activation and inflammation via Mas. In contrast, Kim et al. showed protective effects of Ang-(1-7) infusion in the same model [40] and no aggravation of renal injury produced by kidney ischemia/reperfusion was observed in Mas−/− mice [6]. Moreover, Mas−/− mice were protected from adriamycin-induced renal injury, again confirming the protective actions of the ACE2/Ang-(1-7)/Mas axis of the RAS in the kidney [94]. The discrepancy between the studies remained unresolved, but anti-inflammatory and protective actions of Mas have repeatedly been described also in other organs: Ang-(1-7) protected from intracranial aneurysm only in wild-type but not in Mas−/− mice [73]. Mas deficiency promoted atherosclerosis and autoimmune encephalitis by affecting macrophage polarization and migration [33] and by increasing vascular intima proliferation [2]. The effects on macrophages and other leukocytes were probably also the reason for the higher susceptibility of Mas−/− mice in an endotoxic shock model [65, 96]. Moreover, Mas−/− mice presented aggravated inflammatory pain [16] and allergic pulmonary inflammation [51].

Mas−/− mice are also a model for metabolic syndrome since they developed metabolic abnormalities, such as type 2 diabetes mellitus and dyslipidemia [88], besides their hypertensive phenotype. On the mechanistic level, this was accompanied by decreased PPARγ expression in fat tissue [52] and a change in the relative amounts of α and β cells in pancreatic islets [24]. Ang-(1-7), mainly via Mas, stimulated insulin secretion from β cells [82]. Furthermore, Mas−/− mice developed liver steatosis when bred with ApoE-deficient mice [93] and Mas−/− female mice were more susceptible to obesity-induced hypertension [113]. Ang-(1-7) and Mas were involved in vascular repair, which is deficient in diabetes, and hindlimb ischemia-induced progenitor cell mobilization was absent in Mas−/− mice [103].

In skeletal muscle, Ang-(1-7) and Mas protected from atrophy since Mas−/− mice were more susceptible to a Duchenne muscular dystrophy model (mdx) [1] and to immobilization-induced atrophy [56].

Mas Knockout Rats

Mas knockout rats have been established using Zinc-finger nuclease technology but their phenotype is only partially reported on the Rat Genome Database website (https://rgd.mcw.edu/rgdweb/report/gene/main.html?id=3049).

Mas Overexpression in Retina

Transgenic mice overexpressing Mas in the retina under the control of the opsin promoter developed degeneration of photoreceptors [122]. This surprising phenotype may have been caused by the ligand-independent constitutive activity of Mas [4] causing proliferative effects in cells when the gene is overexpressed.

Ang-(1-7) Models

Transgenic Rats Overexpressing Ang-(1-7)

The group of Timothy Reudelhuber invented a method to express and secrete peptides from an artificial protein without the need of specific proteases in transgenic animals [54, 55]. Using this method, Ang-(1-7) was overexpressed in transgenic rats (TGR(A1-7)3292) using the CMV promoter [28]. These animals mainly expressed the peptide in the testis, which nevertheless significantly increased plasma levels of Ang-(1-7). As a consequence, total peripheral resistance was decreased together with increases in the blood flow to several organs. Nonetheless, the animals remained normotensive, probably since they exhibited an improved pumping function of the heart [11]. These cardiac effects also protected the heart from pressure and ischemia-induced damage [84] as well as from DOCA-induced diastolic dysfunction [19]. A part of these effects may be due to alterations in autonomic regulation observed in these rats [18]. The increased levels of plasma Ang-(1-7) exerted antinatriuretic actions in the kidney resulting in reduced urinary flow and increased urinary osmolality [28]. Furthermore, TGR(A1-7)3292 rats exhibited metabolic improvements such as decreased plasma lipid levels, improved glucose tolerance, less fat tissue, decreased lipogenesis, and less cafeteria-diet-induced obesity [9, 57, 87, 89]. Moreover, these rats presented a reduction in anxiety-like behavior [39] and in the response to stress [60].

Transgenic Mice and Rats Overexpressing Ang-(1-7) in the Heart

We also generated transgenic mice and rats expressing the Ang-(1-7) release protein specifically in the heart using the α cardiac myosin heavy chain promoter. Both lines showed a slightly improved heart function at baseline and were protected from cardiac hypertrophy [27, 54], but, interestingly, not from myocardial infarction [112].

Conclusions

As summarized in this chapter, several genetically altered rat and mouse models have been generated changing the expression of components of the ACE2/Ang-(1-7)/Mas axis of the RAS (Table 1). With the help of these models, physiological and pathophysiological functions of this axis have been elucidated. Nevertheless, novel models are warranted with cell-type-specific deficiency of ACE2 or Mas to further delineate their tissue-specific effects. The already collected findings are the basis for the development of novel therapeutic strategies for cardiovascular and metabolic diseases by targeting ACE2 or Mas [86, 98].