Angiotensin-converting Enzyme 2 Knockout Mice Research Articles

High-salt diet increases glomerular ACE/ACE2 ratio leading to oxidative stress and kidney damage

Angiotensin II (AngII) contributes to salt-driven kidney damage. In this study, we aimed at investigating whether and how the renal damage associated with a high-salt diet could result from changes in the ratio between angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2). Forty-eight rats randomly allocated to three different dietary contents of salt were studied for 4 weeks after undergoing a left uninephrectomy. We focussed on kidney functional, structural and molecular changes. At the same time, we studied kidney molecular changes in 20 weeks old Ace2-knockout mice (Ace2KO), with and without ACE inhibition. A high salt content diet significantly increased the glomerular ACE/ACE2 ratio. This was associated with increased oxidative stress. To assess whether these events were related, we measured renal oxidative stress in Ace2KO, and found that the absence of ACE2 promoted oxidative stress, which could be prevented by ACE inhibition. One of the mechanisms by which a high-salt diet leads to renal damage seems to be the modulation of the ACE/ACE2 ratio which in turn is critical for the cause of oxidative stress, through AngII.

545 Cardioprotective effects mediated by AT1 receptor blockade and enhancing ang 1-7 in experimental heart failure in ACE2-null mice

Loss of ACE2, and inability to metabolize Ang II to Ang 1-7, perpetuates the actions of Ang II after biomechanical stress and is associated with early adverse myocardial remodeling and enhanced fibrosis. Angiotensin receptor blockers (ARBs) are known to antagonize the effect of Ang II by blocking AT1 receptor (AT1R) and also by upregulating the ACE2 expression. We directly compare the benefits of AT1R blockade versus enhancing Ang 1-7 action in pressure-overload induced heart failure in ACE2 knockout mice. AT1R blockade and Ang 1-7 both resulted in marked recovery of systolic dysfunction in pressure-overloaded ACE2-null mice. ARB and Ang 1-7 supplementation increased expression of the Ang 1-7 Mas receptor. Similarly, both therapies attenuated the increase in NADPH oxidase activation, by downregulating the expression of Nox2 and p47phox subunits and also by limiting the p47phox phosphorylation. Biomechanical stress-induced increase in PKC-a expression and phosphorylation of ERK1/2, STAT-3, Akt and GSK-3b was normalized by irbesartan and Ang 1-7. ARB and Ang 1-7 effectively reduced MMP2 activation and MMP9 levels. Thus, treatment with AT1R blocker, irbesartan, and Ang 1-7 prevented the cardiac hypertrophy and improved cardiac remodeling in pressure-overloaded ACE2-null mice in part by upregulating the Mas receptor, suppressing NADPH oxidase and normalizing pathological signaling pathways.

Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays

Angiotensin-converting enzyme 2 (ACE2) is a component of the renin-angiotensin system, and its expression and activity have been shown to be reduced in cardiovascular diseases. Enzymatic activity of ACE2 is commonly measured by hydrolysis of quenched fluorescent substrates in the absence or presence of an ACE2-specific inhibitor, such as the commercially available inhibitor DX600. Whereas recombinant human ACE2 is readily detected in mouse tissues using 1 μM DX600 at pH 7.5, the endogenous ACE2 activity in mouse tissues is barely detectable. We compared human, mouse, and rat ACE2 overexpressed in cell lines for their sensitivity to inhibition by DX600. ACE2 from all three species could be inhibited by DX600, but the half maximal inhibitory concentration (IC(50)) for human ACE2 was much lower (78-fold) than for rodent ACE2. Following optimization of pH, substrate concentration, and antagonist concentration, rat and mouse ACE2 expressed in a cell line could be accurately quantified with 10 μM DX600 (>95% inhibition) but not with 1 μM DX600 (<75% inhibition). Validation that the optimized method robustly quantifies ACE2 in mouse tissues (kidney, brain, heart, and plasma) was performed using wild-type and ACE2 knockout mice. This study provides a reliable method for measuring human, as well as endogenous ACE2 activity in rodents. Our data underscore the importance of validating the effect of DX600 on ACE2 from each particular species at the experimental conditions employed.

Prevention of Angiotensin II–Mediated Renal Oxidative Stress, Inflammation, and Fibrosis by Angiotensin-Converting Enzyme 2

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase capable of metabolizing angiotensin (Ang) II into Ang 1 to 7. We hypothesized that ACE2 is a negative regulator of Ang II signaling and its adverse effects on the kidneys. Ang II infusion (1.5 mg/kg⁻¹/d⁻¹) for 4 days resulted in higher renal Ang II levels and increased nicotinamide adenine dinucleotide phosphate oxidase activity in ACE2 knockout (Ace2(-/y)) mice compared to wild-type mice. Expression of proinflammatory cytokines, interleukin-1β and chemokine (C-C motif) ligand 5, were increased in association with greater activation of extracellular-regulated kinase 1/2 and increase of protein kinase C-α levels. These changes were associated with increased expression of fibrosis-associated genes (α-smooth muscle actin, transforming growth factor-β, procollagen type Iα1) and increased protein levels of collagen I with histological evidence of increased tubulointerstitial fibrosis. Ang II-infused wild-type mice were then treated with recombinant human ACE2 (2 mg/kg⁻¹/d⁻¹, intraperitoneal). Daily treatment with recombinant human ACE2 reduced Ang II-induced pressor response and normalized renal Ang II levels and oxidative stress. These changes were associated with a suppression of Ang II-mediated activation of extracellular-regulated kinase 1/2 and protein kinase C pathway and Ang II-mediated renal fibrosis and T-lymphocyte-mediated inflammation. We conclude that loss of ACE2 enhances renal Ang II levels and Ang II-induced renal oxidative stress, resulting in greater renal injury, whereas recombinant human ACE2 prevents Ang II-induced hypertension, renal oxidative stress, and tubulointerstitial fibrosis. ACE2 is an important negative regulator of Ang II-induced renal disease and enhancing ACE2 action may have therapeutic potential for patients with kidney disease.

Vascular Angiotensin-Converting Enzyme 2

See related article, pages 888–897 Through the actions of its main biological peptide, angiotensin (Ang) II, the renin–angiotensin system (RAS) has been implicated in atherosclerosis.1,2 The proatherogenic effect of Ang II may involve a variety of proinflammatory mechanisms that are largely independent from its effect on blood pressure.1 Ang II promotes monocyte and endothelial cell activation, as well as leukocyte recruitment and adhesion to the atherosclerotic plaque.3 This peptide also increases vascular oxidative stress, an effect that may also contribute to atherogenesis.4 The formation of Ang II by angiotensin-converting enzyme (ACE)-dependent and ACE-independent pathways has been viewed as the main determinant of systemic and local Ang II levels. With the discovery of ACE2, an enzyme that degrades Ang II, there has been increased interest in the regulation of Ang II levels through the degradation pathway.5,6 ACE2 is a homolog of ACE that cleaves the octapeptide Ang II into Ang-(1-7) by removing a single amino acid phenylalanine from its C-terminal end.7,8 Because of its ability to degrade Ang II, ACE2 is viewed as a counter-regulator of Ang II overactivity.8,9 This property could have therapeutic implications. We suggested that ACE2 could be renoprotective, especially when associated with low levels of ACE.10 Increasing evidence suggests that ACE2 plays a protective role in cardiovascular disease and other pathologies.5,11 In this issue of Circulation Research , Thomas et al12 report their findings of a cross between atherosclerosis-prone apolipoprotein (Apo)E knockout (KO) and ACE2 KO mice to study the effects of ACE2 deficiency on the development of atherosclerosis. Genetic ACE 2 ablation led to enhanced vascular inflammation and plaque formation in ApoE KO mice. In ApoE-replete mice, ACE2 deficiency was also associated with increased expression of …

Angiotensin-Converting Enzyme 2 Suppresses Pathological Hypertrophy, Myocardial Fibrosis, and Cardiac Dysfunction

Angiotensin-converting enzyme 2 (ACE2) is a pleiotropic monocarboxypeptidase capable of metabolizing several peptide substrates. We hypothesized that ACE2 is a negative regulator of angiotensin II (Ang II)-mediated signaling and its adverse effects on the cardiovascular system. Ang II infusion (1.5 mg x kg(-1) x d(-1)) for 14 days resulted in worsening cardiac fibrosis and pathological hypertrophy in ACE2 knockout (Ace2(-/y)) mice compared with wild-type (WT) mice. Daily treatment of Ang II-infused wild-type mice with recombinant human ACE2 (rhACE2; 2 mg x kg(-1) x d(-1) IP) blunted the hypertrophic response and expression of hypertrophy markers and reduced Ang II-induced superoxide production. Ang II-mediated myocardial fibrosis and expression of procollagen type I alpha 1, procollagen type III alpha 1, transforming growth factor-beta1, and fibronectin were also suppressed by rhACE2. Ang II-induced diastolic dysfunction was inhibited by rhACE2 in association with reduced plasma and myocardial Ang II and increased plasma Ang 1-7 levels. rhACE2 treatment inhibited Ang II-mediated activation of protein kinase C-alpha and protein kinase C-beta1 protein levels and phosphorylation of the extracellular signal-regulated 1/2, Janus kinase 2, and signal transducer and activator of transcription 3 signaling pathways in wild-type mice. A subpressor dose of Ang II (0.15 mg . kg(-1) . d(-1)) resulted in a milder phenotype that was strikingly attenuated by rhACE2 (2 mg x kg(-1) x d(-1) IP). In adult ventricular cardiomyocytes and cardiofibroblasts, Ang II-mediated superoxide generation, collagen production, and extracellular signal-regulated 1/2 signaling were inhibited by rhACE2 in an Ang 1-7-dependent manner. Importantly, rhACE2 partially prevented the development of dilated cardiomyopathy in pressure-overloaded wild-type mice. Elevated Ang II induced hypertension, myocardial hypertrophy, fibrosis, and diastolic dysfunction, which were exacerbated by ACE2 deficiency, whereas rhACE2 attenuated Ang II- and pressure-overload-induced adverse myocardial remodeling. Hence, ACE2 is an important negative regulator of Ang II-induced heart disease and suppresses adverse myocardial remodeling.

Loss of ACE2 Accelerates Pressure Overload-Induced Myocardial Oxidative Stress and Ventricular Dysfunction

Introduction: Pressure overload activates the cardiac renin-angiotensin system (RAS) in association with myocardial oxidative stress and left ventricular dysfunction. The key peptidase action of angiotensin converting enzyme 2 (ACE2) is degradation of angiotensin (Ang) II to Ang (1–7), functioning effectively as a negative regulator of the RAS. Hypothesis: We hypothesized that ACE2 exerts its protective roles against myocardial oxidative stress and ventricular dysfunction by modulation of NADPH oxidase activity. Methods: Male wildtype (WT) mice, ACE2 knockout (KO) and ACE2/p47phox double KO mice of 10 weeks of age underwent aortic banding model of pressure-overload for 3 weeks. We characterized the functional, structural and molecular changes in the heart in response to pressure overload by echocardiography, TaqMan real-time PCR, Western blot analysis and assessed NADPH oxidase activity. Results: Compared with WT mice, loss of ACE2 resulted in a marked increase in pressure overload-induced myocardial oxidative stress in banded ACE2 KO mice, associated with decreased cardiac function, increased expression of hypertrophy markers (ANF and BNP) and elevated NADPH oxidase activities including p47phox, and Nox2 and Nox4 mRNA expression. These changes were linked with activation of PKCα protein and phosphorylation of the extracellular signal-regulated protein kinase (ERK1/2), Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling in ACE2 KO mice. Loss of p47 subunit prevented the increase in NADPH oxidase activity and attenuated the adverse remodeling in the ACE2 KO mice. Conclusions: Loss of ACE2 accelerates pressure-overload induced myocardial oxidative stress, ventricular dysfunction by activation of the NADPH oxidase subunits with the p47 subunit playing a key role. ACE2 suppresses the myocardial NADPH oxidase system possible by reducing Ang II levels. Introduction: Pressure overload activates the cardiac renin-angiotensin system (RAS) in association with myocardial oxidative stress and left ventricular dysfunction. The key peptidase action of angiotensin converting enzyme 2 (ACE2) is degradation of angiotensin (Ang) II to Ang (1–7), functioning effectively as a negative regulator of the RAS. Hypothesis: We hypothesized that ACE2 exerts its protective roles against myocardial oxidative stress and ventricular dysfunction by modulation of NADPH oxidase activity. Methods: Male wildtype (WT) mice, ACE2 knockout (KO) and ACE2/p47phox double KO mice of 10 weeks of age underwent aortic banding model of pressure-overload for 3 weeks. We characterized the functional, structural and molecular changes in the heart in response to pressure overload by echocardiography, TaqMan real-time PCR, Western blot analysis and assessed NADPH oxidase activity. Results: Compared with WT mice, loss of ACE2 resulted in a marked increase in pressure overload-induced myocardial oxidative stress in banded ACE2 KO mice, associated with decreased cardiac function, increased expression of hypertrophy markers (ANF and BNP) and elevated NADPH oxidase activities including p47phox, and Nox2 and Nox4 mRNA expression. These changes were linked with activation of PKCα protein and phosphorylation of the extracellular signal-regulated protein kinase (ERK1/2), Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling in ACE2 KO mice. Loss of p47 subunit prevented the increase in NADPH oxidase activity and attenuated the adverse remodeling in the ACE2 KO mice. Conclusions: Loss of ACE2 accelerates pressure-overload induced myocardial oxidative stress, ventricular dysfunction by activation of the NADPH oxidase subunits with the p47 subunit playing a key role. ACE2 suppresses the myocardial NADPH oxidase system possible by reducing Ang II levels.

Angiotensin Converting Enzyme 2 Suppresses Pathological Hypertrophy, Myocardial Fibrosis and Diastolic Dysfunction

Background: Angiotensin-converting enzyme 2 (ACE2) is a pleiotropic monocarboxypeptidase capable of metabolizing several peptide substrates. We hypothesized that ACE2 is a negative regulator of angiotensin II (Ang II)-mediated signaling and its adverse effects on the cardiovascular system. Methods and Results: Ang II infusion (1.5 mg.kg−1.d−1) for 14 days resulted in worsening cardiac fibrosis and pathological hypertrophy in ACE2 knockout (Ace2-/y) mice compared to wildtype (WT) mice. Daily treatment of Ang II-infused WT mice with recombinant human ACE2 (rhACE2; 2 mg.kg−1.d−1; i.p.) blunted the hypertrophic response, expressions of hypertrophy markers and reduced the Ang II-induced superoxide production. Ang II-mediated myocardial fibrosis and expression of pro-collagen type Ia1, pro-collagen type IIIa1, TGFb1 and fibronectin were also suppressed by rhACE2. Ang II-induced diastolic dysfunction was normalized by rhACE2 in association with reduced plasma and myocardial Ang II and increased plasma Ang 1–7 levels. rhACE2 treatment reversed Ang II-mediated activation of protein kinase C (PKC)α and PKCβ1 protein levels and phosphorylation of the ERK1/2, JAK2 and STAT3 signaling pathways in WT mice. A subpressor dose of Ang II (0.15 mg.kg−1.d−1) resulted in a milder phenotype which was also completed suppressed by rhACE2 (2 mg.kg−1.d−1; i.p). In cultured adult murine ventricular cardiomyocytes and cardiofibroblasts, Ang II-mediated superoxide generation, collagen production and ERK1/2 signaling were suppressed by rhACE2 in an Ang 1–7 dependent manner. Importantly, rhACE2 partially prevented the development of dilated cardiomyopathy in pressure-overloaded wildtype mice. Conclusions: Elevated Ang II induces hypertension, myocardial hypertrophy, fibrosis and diastolic dysfunction which were exacerbated by ACE2 deficiency while rhACE2 prevents this Ang II-induced and pressure-overload induced myocardial adverse remodeling. Hence, ACE2 as an important negative regulator of Ang II-induced heart disease can have therapeutic potentials for patients with heart disease. Background: Angiotensin-converting enzyme 2 (ACE2) is a pleiotropic monocarboxypeptidase capable of metabolizing several peptide substrates. We hypothesized that ACE2 is a negative regulator of angiotensin II (Ang II)-mediated signaling and its adverse effects on the cardiovascular system. Methods and Results: Ang II infusion (1.5 mg.kg−1.d−1) for 14 days resulted in worsening cardiac fibrosis and pathological hypertrophy in ACE2 knockout (Ace2-/y) mice compared to wildtype (WT) mice. Daily treatment of Ang II-infused WT mice with recombinant human ACE2 (rhACE2; 2 mg.kg−1.d−1; i.p.) blunted the hypertrophic response, expressions of hypertrophy markers and reduced the Ang II-induced superoxide production. Ang II-mediated myocardial fibrosis and expression of pro-collagen type Ia1, pro-collagen type IIIa1, TGFb1 and fibronectin were also suppressed by rhACE2. Ang II-induced diastolic dysfunction was normalized by rhACE2 in association with reduced plasma and myocardial Ang II and increased plasma Ang 1–7 levels. rhACE2 treatment reversed Ang II-mediated activation of protein kinase C (PKC)α and PKCβ1 protein levels and phosphorylation of the ERK1/2, JAK2 and STAT3 signaling pathways in WT mice. A subpressor dose of Ang II (0.15 mg.kg−1.d−1) resulted in a milder phenotype which was also completed suppressed by rhACE2 (2 mg.kg−1.d−1; i.p). In cultured adult murine ventricular cardiomyocytes and cardiofibroblasts, Ang II-mediated superoxide generation, collagen production and ERK1/2 signaling were suppressed by rhACE2 in an Ang 1–7 dependent manner. Importantly, rhACE2 partially prevented the development of dilated cardiomyopathy in pressure-overloaded wildtype mice. Conclusions: Elevated Ang II induces hypertension, myocardial hypertrophy, fibrosis and diastolic dysfunction which were exacerbated by ACE2 deficiency while rhACE2 prevents this Ang II-induced and pressure-overload induced myocardial adverse remodeling. Hence, ACE2 as an important negative regulator of Ang II-induced heart disease can have therapeutic potentials for patients with heart disease.

Loss of ACE2 accelerates time-dependent glomerular and tubulointerstitial damage in streptozotocin-induced diabetic mice

As angiotensin-converting enzyme-2 (ACE2) was identified as a negative regulator of the renin-angiotensin system, there have been many reports concerning its role in several tissues, including the kidney. However, the role of ACE2 during the development of diabetic nephropathy remains undetermined, as previous reports did not necessarily support a protective role against renal injury. Thus, we performed detailed observations of kidneys in ACE2-knockout (ACE2-KO) mice at early (4 weeks) and advanced (18 weeks) stages of diabetes. ACE2-KO and wild-type C57BL/6 mice were rendered diabetic by intraperitoneal injection of streptozotocin. Diabetic ACE2-KO mice showed earlier onset and more severe progression of albuminuria than those did wild-type mice. The elevation of serum creatinine and urea nitrogen levels at 18 weeks of diabetes was more prominent in ACE2-KO mice. Periodic acid-Schiff-stained cross-section of diabetic ACE2-KO mice showed a more severe time-dependent increase in glomerular/tubulointerstitial damage than did that of wild-type mice, confirmed by the immunostaining of alpha-smooth muscle actin, collagen IV and F4-80 antigen. Glomeruli of diabetic ACE2-KO mice showed earlier and more severe decrease in the expression of nephrin, whose degradation is involved in the onset of albuminuria, and more potent increase of vascular endothelial growth factor expression. In addition, treatment with AT1 receptor blocker olmesartan significantly, but not totally, ameliorated the functional and morphological deterioration of diabetic nephropathy in ACE2-KO mice. These results suggest that ACE2 might continuously protect from both glomerular and tubulointerstitial injury during the development of diabetic nephropathy. The renal-protective effect of ACE2 might involve more than just suppressing angiotensin II-mediated AT1 receptor signaling.

Angiotensin-converting-enzyme 2 inhibits liver fibrosis in mice #

The renin-angiotensin system (RAS) plays a major role in liver fibrosis. Recently, a homolog of angiotensin-converting-enzyme 1 (ACE1), termed ACE2, has been identified that appears to be a negative regulator of the RAS by degrading Ang II to Ang(1-7). The aim of this study was to characterize the long-term effects of gene deletion of ACE2 in the liver, to define the role of ACE2 in acute and chronic liver disease, and to characterize the role of Ang(1-7) in hepatic stellate cell (HSC) activation. Ace2 knockout (KO) mice and wild-type (wt) littermates underwent different models of acute and chronic liver injury. Liver pathology was analyzed by histology, immunohistochemistry, alpha smooth muscle actin (alpha-SMA) immunoblotting, and quantitative polymerase chain reaction (qPCR). Murine HSCs were isolated by collagenase-pronase-perfusion, and density gradient centrifugation. One-year-old ace2 KO mice spontaneously developed an inflammatory cell infiltration and mild hepatic fibrosis that was prevented by treatment with irbesartan. Ace2 KO mice showed increased liver fibrosis following bile duct ligation for 21 days or chronic carbon tetrachloride (CCl(4)) treatment. In contrast, ace2 KO mice subjected to acute liver injury models did not differ from wt littermates. Treatment with recombinant ACE2 attenuated experimental fibrosis in the course of cholestatic and toxic liver injury. HSCs express the Ang(1-7) receptor Mas and Ang(1-7) inhibited Ang II-induced phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in cultured HSCs. ACE2 is a key negative regulator of the RAS and functions to limit fibrosis through the degradation of Ang II and the formation of Ang(1-7). Whereas loss of ACE2 activity worsens liver fibrosis in chronic liver injury models, administration of recombinant ACE2 shows therapeutic potential.

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

This study aimed to investigate the role of angiotensin-converting enzyme 2 (ACE2) in regulating glucose homeostasis. Immunohistochemistry was used to investigate ACE2 expression in the pancreas. Glucose tolerance test, insulin secretion test, and insulin tolerance test were performed in age-matched male ACE2 knockout (KO) and wild-type (WT) mice. We found that ACE2 was positively expressed in the pancreas. Male ACE2 KO mice displayed a selective decrease in first-phase insulin secretion in response to glucose and a progressive impairment of glucose tolerance compared with age- and sex-matched WT mice. On the other hand, insulin sensitivity of the peripheral tissue in age-matched ACE2 KO and WT mice showed no difference. These findings suggest that ACE2 might play an important role in glucose homeostasis as well as type 2 diabetes.

The sweeter side of ACE2: physiological evidence for a role in diabetes.

Diabetes mellitus is a growing problem in all parts of the world. Both clinical trials and animal models of type I and type II diabetes have shown that hyperactivity of angiotensin-II (Ang-II) signaling pathways contribute to the development of diabetes and diabetic complications. Of clinical relevance, blockade of the renin–angiotensin system prevents new-onset diabetes and reduces the risk of diabetic complications. Angiotensin-converting enzyme (ACE) 2 is a recently discovered mono-carboxypeptidase and the first homolog of ACE. It is thought to inhibit Ang-II signaling cascades mostly by cleaving Ang-II to generate Ang-(1–7), which effects oppose Ang-II and are mediated by the Mas receptor. The enzyme is present in the kidney, liver, adipose tissue and pancreas. Its expression is elevated in the endocrine pancreas in diabetes and in the early phase during diabetic nephropathy. ACE2 is hypothesized to act in a compensatory manner in both diabetes and diabetic nephropathy. Recently, we have shown the presence of the Mas receptor in the mouse pancreas and observed a reduction in Mas receptor immuno-reactivity as well as higher fasting blood glucose levels in ACE2 knockout mice, indicating that these mice may be a new model to study the role of ACE2 in diabetes. In this review we will examine the role of the renin–angiotensin system in the physiopathology and treatment of diabetes and highlight the potential benefits of the ACE2/Ang-(1–7)/Mas receptor axis, focusing on recent data about ACE2.

Modulation of mouse intestinal epithelial cell turnover in the absence of angiotensin converting enzyme

Angiotensin converting enzyme (ACE) has been shown to be involved in regulation of apoptosis in nonintestinal tissues. This study examined the role of ACE in the modulation of intestinal adaptation utilizing ACE knockout mice (ACE-/-). A 60% small bowel resection (SBR) was used, since this model results in a significant increase in intestinal epithelial cell (EC) apoptosis as well as proliferation. Baseline villus height, crypt depth, and intestinal EC proliferation were higher, and EC apoptosis rates were lower in ACE-/- compared with ACE+/+ mice. After SBR, EC apoptosis rates remained significantly lower in ACE-/- compared with ACE+/+ mice. Furthermore, villus height and crypt depth after SBR continued to be higher in ACE-/- mice. The finding of a lower bax-to-bcl-2 protein ratio in ACE-/- mice may account for reduced EC apoptotic rates after SBR in ACE-/- compared with ACE+/+ mice. The baseline higher rate of EC proliferation in ACE-/- compared with ACE+/+ mice may be due to an increase in the expression of several EC growth factor receptors. In conclusion, ACE appears to have an important role in the modulation of intestinal EC apoptosis and proliferation and suggests that the presence of ACE in the intestinal epithelium has a critical role in guiding epithelial cell adaptive response.

New insights into the role of angiotensin-converting enzyme obtained from the analysis of genetically modified mice

Angiotensin-converting enzyme (ACE) has been well-recognized for its role in blood pressure regulation. ACE is made by many tissues, though it is most abundantly expressed on the luminal surface of vascular endothelium. ACE knockout mice show a profound phenotype with low blood pressure, but also with hemopoietic and developmental defects, which complicates understanding the biological functions of ACE in individual tissue types. Using a promoter-swapping strategy, several mouse lines with unique ACE tissue expression patterns were studied. These include mice with ACE expression in the liver (ACE 3/3), the heart (ACE 8/8), and macrophages (ACE 10/10). We also investigated mice with a selective inactivation of either the N- or C-terminal ACE catalytic domain. Our studies indicate that ACE plays a role in many other physiologic processes beyond simple blood pressure control.

The discovery of angiotensin‐converting enzyme 2 and its role in acute lung injury in mice

During several months of 2002, severe acute respiratory syndrome (SARS) caused by SARS‐coronavirus (SARS‐CoV) spread rapidly from China throughout the world, causing more than 800 deaths due to the development of acute respiratory distress syndrome (ARDS), which is the severe form of acute lung injury (ALI). Interestingly, a novel homologue of angiotensin‐converting enzyme, termed angiotensin‐converting enzyme 2 (ACE2), has been identified as a receptor for SARS‐CoV. Angiotensin‐converting enzyme and ACE2 share homology in their catalytic domain and provide different key functions in the renin–angiotensin system (RAS). Angiotensin‐converting enzyme cleaves angiotensin I to generate angiotensin II, which is a key effector peptide of the system and exerts multiple biological functions, whereas ACE2 reduces angiotensin II levels. Importantly, our recent studies using ACE2 knockout mice have demonstrated that ACE2 protects murine lungs from ARDS. Furthermore, SARS‐CoV infections and the Spike protein of the SARS‐CoV reduce ACE2 expression. Notably, injection of SARS‐CoV Spike into mice worsens acute lung failure in vivo, which can be attenuated by blocking the renin–angiotensin pathway, suggesting that the activation of the pulmonary RAS influences the pathogenesis of ALI/ARDS and SARS.

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

As a major regulator of blood pressure homeostasis, the renin-angiotensin system (RAS) has been the subject of extensive scientific investigation. While the RAS was first discovered more than 100 years ago, several novel components of the system have been identified only in the last decade. One of these newer members of the RAS family is angiotensin-converting enzyme 2 (ACE2). Among the approaches used to establish a physiological role for ACE2 has been the generation of ACE2-null mouse lines using homologous recombination in embryonic stem cells. In the literature, there have been at least three lines of ACE2 knockout mice generated by gene targeting by different investigative groups. Interestingly, there are significant differences in some of the reported phenotypes of these distinct lines, especially with regard to their cardiovascular physiology. In this paper, we will review the results of published experiments using these ACE2-null mouse lines, highlighting similarities and differences in these studies and summarizing their contributions to our understanding of the physiological functions of this novel member of the RAS.

ACE2 Deficiency Modifies Renoprotection Afforded by ACE Inhibition in Experimental Diabetes

The degradation of angiotensin (Ang) II by ACE2, leading to the formation of Ang 1-7, is an important step in the renin-angiotensin system (RAS) and one that is significantly altered in the diabetic kidney. This study examines the role of ACE2 in early renal changes associated with diabetes and the influence of ACE2 deficiency on ACE inhibitor-mediated renoprotection. Diabetes was induced by streptozotocin in male c57bl6 mice and ACE2 knockout (KO) mice. After 5 weeks of study, animals were randomized to receive the ACE inhibitor perindopril (2 mg x kg(-1) x day(-1)). Wild-type mice were further randomized to receive the selective ACE2 inhibitor MLN-4760 (10 mg x kg(-1) x day(-1)) and followed for an additional 5 weeks. Markers of renal function and injury were then assessed. Induction of diabetes in wild-type mice was associated with a reduction in renal ACE2 expression and decreased Ang 1-7. In diabetic mice receiving MLN-4760 and in ACE2 KO mice, diabetes-associated albuminuria was enhanced, associated with an increase in blood pressure. However, renal hypertrophy and fibrogenesis were reduced in diabetic mice with ACE2 deficiency, and hyperfiltration was attenuated. Diabetic wild-type mice treated with an ACE inhibitor experienced a reduction in albuminuria and blood pressure. These responses were attenuated in both diabetic ACE2 KO mice and diabetic mice receiving MLN-4760. However, other renoprotective and antifibrotic actions of ACE inhibition in diabetes were preserved in ACE2-deficient mice. The expression of ACE2 is significantly modified by diabetes, which impacts both pathogenesis of kidney disease and responsiveness to RAS blockade. These data indicate that ACE2 is a complex and site-specific modulator of diabetic kidney disease.

Tissue specific expression of angiotensin converting enzyme: A new way to study an old friend

Angiotensin-converting enzyme (ACE) plays a central role in blood pressure regulation by producing the vasoconstrictor angiotensin II. When ACE knockout mice were studied, they presented with a complicated phenotype, including cardiovascular, reproductive, hematologic and developmental defects. The complexity of an ACE knockout mouse emphasizes the advantages and disadvantages of the classic knockout strategy. An animal lacking all ACE is very different from a wild type animal, and can be modeled as representing an extreme phenotype. To understand the role of ACE in a tissue and organ specific fashion, our group used targeted homologous recombination to create mouse models in which a promoter swapping strategy results in very restricted tissue patterns of ACE expression. Mice with ACE expression only in the heart, termed ACE 8/8 mice, present with atria enlargement and electrical conduction defects, but normal ventricular function. Mice with ACE expression only in monocytes and macrophages, termed ACE 10/10 mice, have a marked resistance to the growth of melanoma due to an enhanced immune response characterized by increased tumor specific CD8+ T cells and increased proinflammatory cytokines. These mice may define a new means of augmenting the immune response, potentially useful in human clinical situations. The promoter swapping strategy permits scientific investigation of questions unapproachable by other experimental approaches.

Does angiotensin interact with dopaminergic mechanisms in the brain to modulate prepulse inhibition in mice?

Changes in the levels of angiotensin-converting enzyme (ACE) have been found in brains of schizophrenia patients, suggesting a possible involvement of angiotensin in the illness. Prepulse inhibition (PPI) is a measure of sensorimotor gating and is disrupted in patients with schizophrenia. In a previous study, a reduction of ACE activity, either in ACE knockout mice or after ACE inhibitor treatment, markedly inhibited the disruption of PPI caused by the dopamine receptor agonist, apomorphine. ACE is not specific for the angiotensin system and, therefore, in the present study we assessed pharmacological regulation of PPI in two other, more specific genetic mouse models of altered angiotensin activity. We used renin-enhancer knockout (REKO) mice, which display reduced renin activity, and neuron-specific enolase (NSE)-AT 1A mice, which selectively over-express angiotensin AT 1A receptors in the brain. Treatment of these mice with apomorphine, the dopamine releaser, amphetamine or the NMDA receptor antagonist, MK-801, significantly disrupted PPI. There was, however, no difference in these effects between the genotypes. These data suggest that genetically induced changes in the activity of the angiotensin system do not alter regulation of PPI in mice. Our previous results on the effects of reduced ACE activity could be explained by mechanisms other than angiotensin, such as effects on enkephalin or bradykinin metabolism.

Angiotensin converting enzyme 2 activity is not detectable in mouse plasma

Angiotensin converting enzymes (ACE & ACE2) are critical in metabolism of Ang peptides. There is evidence for ACE and ACE2 activity in heart, kidney and brain. In addition, there is abundant ACE activity in plasma which is activated in diabetic mice and humans. Data is limited regarding the presence and activity of circulating plasma ACE2 in normal and in disease states. Further, there is a need for a reliable and specific ACE2 assay. The aim of this study was to develop, screen and quantify ACE2 activity in plasma using a new mass spectrometric (MS) method. Plasma from normal C57BL/6, streptozotocin (STZ) and db/db diabetic mice and kidney from wildtype and ACE2 knockout mice were tested. Plasma (1 μl) or tissue extracts (1–2 μg protein) were incubated with Ang II (0.1–10 μM) in the presence of protease inhibitors. Substrates and generated peptides were analyzed in the Ciphergen ProteinChips® Reader using selective enhanced lazer desorption time of flight MS (SELDI-TOF-MS). ACE2 activity was expressed as the ratio of the generated Ang (1–7) (899, m/z) to Ang II (1045, m/z). ACE2 activity was not detected in plasma obtained from normal or diabetic mice. It was present in kidney from control wild type mice, but was absent in tissue from ACE2 knockout mice. Unlike ACE, tissue ACE2 activity was not inhibited by captopril (10 μM), but was blocked with selective antagonists MLN 4760 (100 μM) and DX 600 (10 μM). We demonstrate the development of a novel assay for the measurement of ACE2 activity using endogenous peptide substrates and MS technology. Results show 1) specificity of the method since ACE2 was absent in tissues from gene deletion mice and could be blocked with an ACE 2 inhibitor and 2) absence of detectable plasma ACE2 activity in normal, STZ or genetically diabetic mice.