Angiotensin-converting Enzyme 2 Knockout Mice Research Articles

Gonadectomy prevents the increase in blood pressure and glomerular injury in angiotensin-converting enzyme 2 knockout diabetic male mice. Effects on renin-angiotensin system.

Angiotensin-converting enzyme 2 (ACE2) deletion worsens kidney injury, and its amplification ameliorates diabetic nephropathy. Male sex increases the incidence, prevalence, and progression of chronic kidney disease in our environment. Here, we studied the effect of ACE2 deficiency and gonadectomy (GDX) on diabetic nephropathy and its relationship with fibrosis, protein kinase B (Akt) activation, and the expression of several components of the renin-angiotensin system (RAS).Mice were injected with streptozotocin to induce diabetes and followed for 19 weeks. Physiological and renal parameters were studied in wild-type and ACE2 knockout (ACE2KO) male mice with and without GDX. Diabetic ACE2KO showed increased blood pressure (BP), glomerular injury, and renal fibrosis compared with diabetic wild-type. Gonadectomized diabetic ACE2KO presented a decrease in BP. In the absence of ACE2, GDX attenuated albuminuria and renal lesions, such as mesangial matrix expansion and podocyte loss. Both, α-smooth muscle actin accumulation and collagen deposition were significantly decreased in renal cortex of gonadectomized diabetic ACE2KO but not diabetic wild-type mice. GDX also reduced circulating ACE activity in ACE2KO mice. Loss of ACE2 modified the effect of GDX on cortical gene expression of RAS in diabetic mice. Akt phosphorylation in renal cortex was increased by diabetes and loss of ACE2 and decreased by GDX in control and diabetic ACE2KO but not in wild-type mice. Our results suggest that GDX may exert a protective effect within the kidney under pathological conditions of diabetes and ACE2 deficiency. This renoprotection may be ascribed to different mechanisms such as decrease in BP, modulation of RAS, and downregulation of Akt-related pathways.

OS 15-09 DOES ACE2-ANGIOTENSIN-(1–7) AXIS PROTECT FROM AGING-ASSOCIATED LOSS OF SKELETAL MUSCLE FUNCTION?

Objective: Angiotensin converting enzyme2 (ACE2), an enzyme that produces the Angiotensin 1–7(A1–7) from Angiotensin II, is considered to suppress organ damage by inhibition of the activation of renin-angiotensin system. We recently found that ACE2 deficiency in mice ameliorated insulin sensitivity of skeletal muscle with reduced expression of glucose transporter 4 and myocyte enhancer factor 2, an important transcription factor to maintain homeostasis of skeletal muscle. In this study, we investigated whether ACE2-A1–7 axis plays a protective role in aging-associated loss of skeletal muscle function in mice. Design and Method: To assess serial change in motor function, we performed grip strength test in 3, 6, 12, 18, and 24 month old wildtype and ACE2 knockout (KO) mice. Grip strength at each period was calculated by average of 12 times measurements (6 times for 2days). At 24 month old, saline or A1–7 (100ng / kg / min) was administered for 4 weeks by osmotic mini pumps and grip strength was compared before and after administration. Results: At 3 months, there was no significant difference in grip strength between ACE2KO and wildtype mice. While grip strength of wildtype mice remained unchanged until 18 month old, ACE2KO mice reduced their grip strength after 6 month old. Wildtype mice reduced grip strength at 24 month, but the difference in grip strength between wildtype and ACE2KO mice was still observed. Administration of A1–7 at 24 month old mice significantly improved loss of grip strength in wildtype and ACE2KO mice. There was no difference in grip strength between A1–7-administrated wildtype and ACE2KO mice. There was no difference in weight and size of skeletal muscle of lower limbs between wildtype and ACE2KO mice at 24 months. Conclusions: ACE2-Angiotensin(1–7) axis may protect from age-associated loss of skeletal muscle function without no obvious alternation in muscle mass.

Angiotensin-Converting Enzyme 2 Metabolizes and Partially Inactivates Pyr-Apelin-13 and Apelin-17: Physiological Effects in the Cardiovascular System.

Apelin peptides mediate beneficial effects on the cardiovascular system and are being targeted as potential new drugs. However, apelin peptides have extremely short biological half-lives, and improved understanding of apelin peptide metabolism may lead to the discovery of biologically stable analogues with therapeutic potential. We examined the ability of angiotensin-converting enzyme 2 (ACE2) to cleave and inactivate pyr-apelin 13 and apelin 17, the dominant apelin peptides. Computer-assisted modeling shows a conserved binding of pyr-apelin 13 and apelin 17 to the ACE2 catalytic site. In ACE2 knockout mice, hypotensive action of pyr-apelin 13 and apelin 17 was potentiated, with a corresponding greater elevation in plasma apelin levels. Similarly, pharmacological inhibition of ACE2 potentiated the vasodepressor action of apelin peptides. Biochemical analysis confirmed that recombinant human ACE2 can cleave pyr-apelin 13 and apelin 17 efficiently, and apelin peptides are degraded slower in ACE2-deficient plasma. The biological relevance of ACE2-mediated proteolytic processing of apelin peptides was further supported by the reduced potency of pyr-apelin 12 and apelin 16 on the activation of signaling pathways and nitric oxide production from endothelial cells. Importantly, although pyr-apelin 13 and apelin 17 rescued contractile function in a myocardial ischemia-reperfusion model, ACE2 cleavage products, pyr-apelin 12 and 16, were devoid of these cardioprotective effects. We designed and synthesized active apelin analogues that were resistant to ACE2-mediated degradation, thereby confirming that stable apelin analogues can be designed as potential drugs. We conclude that ACE2 represents a major negative regulator of apelin action in the vasculature and heart.

Angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis activates Akt signaling to ameliorate hepatic steatosis.

The classical axis of renin-angiotensin system (RAS), angiotensin (Ang)-converting enzyme (ACE)/Ang II/AT1, contributes to the development of non-alcoholic fatty liver disease (NAFLD). However, the role of bypass axis of RAS (Angiotensin-converting enzyme 2 (ACE2)/Ang-(1–7)/Mas) in hepatic steatosis is still unclear. Here we showed that deletion of ACE2 aggravates liver steatosis, which is correlated with the increased expression of hepatic lipogenic genes and the decreased expression of fatty acid oxidation-related genes in the liver of ACE2 knockout (ACE2−/y) mice. Meanwhile, oxidative stress and inflammation were also aggravated in ACE2−/y mice. On the contrary, overexpression of ACE2 improved fatty liver in db/db mice, and the mRNA levels of fatty acid oxidation-related genes were up-regulated. In vitro, Ang-(1–7)/ACE2 ameliorated hepatic steatosis, oxidative stress and inflammation in free fatty acid (FFA)-induced HepG2 cells, and what’s more, Akt inhibitors reduced ACE2-mediated lipid metabolism. Furthermore, ACE2-mediated Akt activation could be attenuated by blockade of ATP/P2 receptor/Calmodulin (CaM) pathway. These results indicated that Ang-(1–7)/ACE2/Mas axis may reduce liver lipid accumulation partly by regulating lipid-metabolizing genes through ATP/P2 receptor/CaM signaling pathway. Our findings support the potential role of ACE2/Ang-(1–7)/Mas axis in prevention and treatment of hepatic lipid metabolism.

Abstract WP266: Deficiency of Angiotensin Converting Enzyme 2 Causes Deterioration of Cognitive Function

Introduction: The classical renin-angiotensin system (RAS), known as the angiotensin (Ang) converting enzyme (ACE)/Ang II/Ang II type 1 (AT1) receptor axis, induces various organ damage including cognitive decline. On the other hand, the ACE2/Ang-(1-7)/Mas receptor axis has been highlighted as exerting antagonistic actions against the classical RAS axis in the cardiovascular system. However, the roles of the ACE2/Ang-(1-7)/Mas axis in cognitive function largely remain to be elucidated, and we therefore examined possible roles of ACE2 in cognitive function. Methods: Male 10-week-old C57BL6 (wild-type: WT) mice and ACE2 knockout (KO) mice were subjected to the Morris water maze task to evaluate spatial cognitive function. Vascular dementia model were induced by bilateral common carotid artery stenosis (BCAS). The Morris water maze task was performed 6 weeks after BCAS operation. Results: ACE2KO mice exhibited significant impairment of spatial cognitive function, compared with that in WT mice, without significant difference in cerebral blood flow determined by laser speckle flowmetry and morphological changes in the hippocampus between both strains. Superoxide anion production in the hippocampus tended to be increased in ACE2KO mice, with increased mRNA levels of NADPH oxidase subunit in the hippocampus of ACE2KO compared with WT mice. Protein level of superoxide dismutase (SOD) 3 tended to be decreased in ACE2KO mice compared with WT mice. AT1 receptor mRNA level in the hippocampus was higher in ACE2KO mice compared with WT mice. In contrast, AT2 receptor mRNA level in the hippocampus did not differ between the two strains. Mas receptor mRNA was highly expressed in the hippocampus compared with the cortex, with no significant difference between ACE2KO and WT mice. Brain-derived neurotrophic factor (BDNF) mRNA was lower in the hippocampus in ACE2KO mice compared with WT mice. Escape latency after BCAS was prolonged in WT mice compared with sham operated mice, whereas impaired cognitive function in ACE2 KO mice was not further exaggerated after BCAS. Conclusion: Taken together ACE2 deficiency resulted in impaired cognitive function probably due to enhanced oxidative stress and a decrease in BDNF.

Alternative Roles of STAT3 and MAPK Signaling Pathways in the MMPs Activation and Progression of Lung Injury Induced by Cigarette Smoke Exposure in ACE2 Knockout Mice.

Inflammation-mediated abnormalities in the renin-angiotensin system (RAS) and expression of matrix metalloproteinases (MMPs) are implicated in the pathogenesis of lung injury. Angiotensin converting enzyme II (ACE2), an angiotensin converting enzyme (ACE) homologue that displays antagonist effects on ACE/angiotensin II (Ang II) axis, could also play a protective role against lung diseases. However, the relationship between ACE2 and MMPs activation in lung injury is still largely unclear. The purpose of this study is to investigate whether MMPs activity could be affected by ACE2 and which ACE2 derived signaling pathways could be also involved via using a mouse model with lung injury induced by cigarette smoke (CS) exposure for 1 to 3 weeks. Wild-type (WT; C57BL/6) and ACE2 KO mice (ACE2-/-) were utilized to study CS-induced lung injury. Increases in the resting respiratory rate (RRR), pulmonary immunokines, leukocyte infiltration and bronchial hyperplasia were observed in the CS-exposed mice. Compared to WT mice, more serious physiopathological changes were found in ACE2-/- mice in the first week of CS exposure. CS exposure increased pulmonary ACE and ACE2 activities in WT mice, and significantly increased ACE in ACE2-/- mice. Furthermore, the activity of pulmonary MMPs was decreased in CS-exposed WT mice, whereas this activity was increased in ACE2-/- mice. CS exposure increased the pulmonary p-p38, p-JNK and p-ERK1/2 level in all mice. In ACE2-/- mice, a significant increase p-STAT3 signaling was detected; however, no effect was observed on the p-STAT3 level in WT mice. Our results support the hypothesis that ACE2 deficiency influences MMPs activation and STAT3 phosphorylation signaling to promote more pulmonary inflammation in the development of lung injury.

High-mobility group box 1 protein, angiotensins, ACE2, and target organ damage.

High-mobility group box 1 (HMGB1) protein is an important chromatin protein. In the nucleus, HMGB1 interacts with nucleosomes, transcription factors, and histones. The nuclear protein organizes the DNA and regulates transcription [1]. After binding to DNA, HMGB1 bends DNA, thereby facilitating the binding of other proteins. HMGB1 supports transcription of many genes in interactions with numerous transcription factors. HMGB1 also interacts with nucleosomes to loosen packed DNA and remodel the chromatin. Contact with core histones changes the structure of nucleosomes. The presence of HMGB1 in the nucleus depends on posttranslational modifications. When the protein is not acetylated, it remains within the nucleus; however, hyper acetylation on lysine residues causes HMGB1 to translocate into the cytosol [2]. Here, HMGB1 assumes quite different roles. Immune cells can secrete the protein. For instance, dendritic cells, macrophages, and monocytes secrete HMGB1 in response to cytokines to mediate inflammation. The mechanism of inflammation and damage is HMGB1 binding to receptor for advanced glycation end products (RAGE), Toll-like receptor 2 (TLR2), and TLR4 to mediate nuclear factor kappaB (NF-κB) activation [3]. This state-of-affairs positions HMGB1 at the intersection of sterile and infectious inflammatory responses [4, 5]. The multifaceted activities of HMGB1 (Fig. 1) have been reviewed (5). Evidence has been presented that angiotensinconverting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARB) can reduce secretion of HMGB1 [6, 7]. This observation would be consistent with possible interactions between HMGB1 and the renin-angiotensin system. The angiotensin-converting enzyme (ACE) is a matrix metalloproteinase cleaving decapeptide angiotensin (Ang) I to the octapeptide Ang II. ACE, and its orthologs possess 2 peptidase M2 domains. ACE2, discovered more recently but probably a teleological older enzyme, and its orthologs possess a single peptidase M2 domain. Ang I and II can be cleaved by ACE2 into nonapeptides (Ang1–9) and septapeptides (Ang1–7), respectively. The ACE2 protein also serves as a receptor for corona viruses. The ACE metalloproteinases have been around on the planet for a long time. Similar sequences are found in flies and even in bacteria, while renin first enters the scene at the bony-fish level [8]. The generation of Ang1–7 by ACE2 is said to have a vascular protective function. Nevertheless, ACE2 targeting in mice resulted in mixed messages. There have been at least three lines of ACE2 knockout mice reported by various investigators [9]. Interestingly, there are significant differences in the phenotypes of these distinct lines, especially with regard to their cardiovascular physiology. ACE2 appears to have only modest effects on baseline cardiovascular functions and blood pressure control. These effects can be substantially modulated by genetic and, perhaps through environmental factors. In contrast, the activity of ACE2 may have more profound effects on susceptibility to pathological states, such as hypertension and cardiac hypertrophy. In this issue of J Mol Med, Qi et al. [10] studied mice harboring a FLAG-tagged mouse ACE2 gene * Friedrich C. Luft luft@charite.de

Abstract P113: Angiotensin Converting Enzyme 2 Modulates Bradykinin B1 Receptor Function During DOCA-Salt Hypertension

DOCA-salt hypertension is associated with reduced angiotensin converting enzyme 2 (ACE2) and increased bradykinin B1 receptor (B1R) expression in the brain. ACE2 hydrolyzes des-Arg(9)-bradykinin, the endogenous B1R agonist, into inactive metabolites. Therefore, we hypothesized that ACE2 overexpression or deletion modulates B1R function in the brain during neurogenic hypertension. To test this hypothesis, we used mice overexpressing ACE2 in neurons (SA), ACE2 knockout and B1R knockout mice. Blood pressure (BP) was monitored using telemetry probes in conscious animals. While baseline BP was not different between strains, DOCA-salt treatment (1 mg/g body weight DOCA, 1% NaCl for 3 weeks) resulted in a significantly lower BP in B1R knockout mice (121 ±2 mmHg, n=5) compared to wildtype (WT) mice (138 ±3 mmHg, n=8). DOCA-salt hypertension resulted in 27% decrease (74 ±6 vs. 54 ±2 Fluorescence Units (FU)/min/μg of protein, p<0.05) in ACE2 activity in the hypothalamus, but not in B1R knockout mice with DOCA (69 ±6 vs. 65 ±3 FU/min/μg of protein). In DOCA-treated WT mice, B1R mRNA (Real time PCR) and protein expression (Western blot) in the hypothalamus were increased by 3 and 2 fold (n=6, p<0.01), respectively. This increased B1R expression was blunted in SA mice with DOCA (3.2 ±0.4 vs. 0.9 ±0.1 fold, p<0.001) but not in ACE2 knockout mice with DOCA (3.2 ±0.4 vs. 3.1 ±0.9 fold). Moreover, DOCA-salt treatment resulted in an increased expression of pro-inflammatory ADAM17 in the brain by 2.2 fold (n=6, p<0.01 vs. WT). ACE2 overexpression or B1R knockdown blunted this ADAM17 expression (1.4 ±0.1 and 1.1 ±0.6 fold, respectively, n=3, p<0.01 vs. WT+DOCA), while it was remain increased in ACE2 knockout mice (2.6 ±0.7 fold, n=3, p<0.01) compared to control mice. Together, our data provide novel evidence to support a role for ACE2 in the modulation of central B1R function in the development of DOCA-salt hypertension.

Mesenchymal Stem Cells Overexpressing Angiotensin-Converting Enzyme 2 Rescue Lipopolysaccharide-Induced Lung Injury

Bone marrow-derived mesenchymal stem cells (MSCs), which have beneficial effects in acute lung injury (ALI), can serve as a vehicle for gene therapy. Angiotensin-converting enzyme 2 (ACE2), a counterregulatory enzyme of ACE that degrades angiotensin (Ang) II into Ang 1-7, has a protective role against ALI. Because ACE2 expression is severely reduced in the injured lung, a therapy targeted to improve ACE2 expression in lung might attenuate ALI. We hypothesized that MSCs overexpressing ACE2 would have further benefits in lipopolysaccharide (LPS)-induced ALI mice, when compared with MSCs alone. MSCs were transduced with ACE2 gene (MSC-ACE2) by a lentiviral vector and then infused into wild-type (WT) and ACE2 knockout (ACE2(-/y)) mice following an LPS-induced intratracheal lung injury. The results demonstrated that the lung injury of ALI mice was alleviated at 24 and 72 h after MSC-ACE2 transplantation. MSC-ACE2 improved the lung histopathology and had additional anti-inflammatory effects when compared with MSCs alone in both WT and ACE2(-/y) ALI mice. MSC-ACE2 administration also reduced pulmonary vascular permeability, improved endothelial barrier integrity, and normalized lung eNOS expression relative to the MSC group. The beneficial effects of MSC-ACE2 could be attributed to its recruitment into the injured lung and enhanced local expression of ACE2 protein without changing the serum ACE2 levels after MSC-ACE2 transplantation. The biological activity of the increased ACE2 protein decreased the Ang II amount and increased the Ang 1-7 level in the lung when compared with the ALI and MSC-only groups, thereby inhibiting the detrimental effects of accumulating Ang II. Therefore, compared to MSCs alone, the administration of MSCs overexpressing ACE2 resulted in a further improvement in the inflammatory response and pulmonary endothelial function of LPS-induced ALI mice. These additional benefits could be due to the degradation of Ang II that accompanies the targeted overexpression of ACE2 in the lung.

Uterine artery dysfunction in pregnant ACE2 knockout mice is associated with placental hypoxia and reduced umbilical blood flow velocity.

Angiotensin-converting enzyme 2 (ACE2) knockout is associated with reduced fetal weight at late gestation; however, whether uteroplacental vascular and/or hemodynamic disturbances underlie this growth-restricted phenotype is unknown. Uterine artery reactivity and flow velocities, umbilical flow velocities, trophoblast invasion, and placental hypoxia were determined in ACE2 knockout (KO) and C57Bl/6 wild-type (WT) mice at day 14 of gestation. Although systolic blood pressure was higher in pregnant ACE2 KO vs. WT mice (102.3 ± 5.1 vs. 85.1 ± 1.9 mmHg, n = 5-6), the magnitude of difference was similar to that observed in nonpregnant ACE2 KO vs. WT mice. Maternal urinary protein excretion, serum creatinine, and kidney or heart weights were not different in ACE2 KO vs. WT. Fetal weight and pup-to-placental weight ratio were lower in ACE2 KO vs. WT mice. A higher sensitivity to Ang II [pD2 8.64 ± 0.04 vs. 8.5 ± 0.03 (-log EC50)] and greater maximal contraction to phenylephrine (169.0 ± 9.0 vs. 139.0 ± 7.0% KMAX), were associated with lower immunostaining for Ang II receptor 2 and fibrinoid content of the uterine artery in ACE2 KO mice. Uterine artery flow velocities and trophoblast invasion were similar between study groups. In contrast, umbilical artery peak systolic velocities (60.2 ± 4.5 vs. 75.1 ± 4.5 mm/s) and the resistance index measured using VEVO 2100 ultrasound were lower in the ACE2 KO vs. WT mice. Immunostaining for pimonidazole, a marker of hypoxia, and hypoxia-inducible factor-2α were higher in the trophospongium and placental labyrinth of the ACE2 KO vs. WT. In summary, placental hypoxia and uterine artery dysfunction develop before major growth of the fetus occurs and may explain the fetal growth restricted phenotype.

Plasma and Kidney Angiotensin Peptides: Importance of the Aminopeptidase A/Angiotensin III Axis

The renin-angiotensin system is a complex regulatory hormonal network with a main biological peptide and therapeutic target, angiotensin (Ang) II (1-8). There are other potentially important Ang peptides that have not been well evaluated. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for concurrent evaluation of multiple Angs downstream of Ang I (1-10) and Ang II (1-8) in kidney and plasma from wild-type (WT) mice. Angiotensin converting enzyme 2 knockout (ACE2KO) was also used as a way to examine the Angs profile in the absence of ACE2, an enzyme that cleaves both Ang I (1-10) and Ang II (1-8). In plasma from both WT and ACE2KO, levels of Ang I (1-10), Ang III (2-8), and Ang (2-10) were the highest of all the renin-angiotensin system (RAS) peptides. The latter two peptides are products of aminopeptidase A cleavage of Ang II (1-8) and Ang I (1-10), respectively. In contrast, plasma levels of Ang II (1-8), and Ang (1-7), the product of Ang II (1-8) cleavage by ACE2, were low. In kidney from both WT and ACE2KO, Ang II (1-8) levels were high as compared to plasma levels. In the ACE2KO mice, a significant increase in either Ang II (1-8) or a decrease in Ang (1-7) was not observed in plasma or in the kidney. RAS-focused peptidomic approach revealed major differences in Ang peptides between mouse plasma and kidney. These Ang peptide profiles show the dominance of the aminopeptidase A/Ang (2-10) and aminopeptidase A/Ang III (2-8) pathways in the metabolism of Ang I (1-10) and Ang II (1-8) over the ACE2/Ang (1-7) axis. Ang III (2-8) and other peptides formed from aminopeptidase A cleavage may be important therapeutic RAS targets.

Upregulation of Angiotensin (1-7)-Mediated Signaling Preserves Endothelial Function Through Reducing Oxidative Stress in Diabetes.

Angiotensin-converting enzyme 2 (ACE2)-angiotensin (1-7) [Ang (1-7)]-Mas constitutes the vasoprotective axis and is demonstrated to antagonize the vascular pathophysiological effects of the classical renin-angiotensin system. We sought to study the hypothesis that upregulation of ACE2-Ang (1-7) signaling protects endothelial function through reducing oxidative stress that would result in beneficial outcome in diabetes. Ex vivo treatment with Ang (1-7) enhanced endothelium-dependent relaxation (EDR) in renal arteries from diabetic patients. Both Ang (1-7) infusion via osmotic pump (500 ng/kg/min) for 2 weeks and exogenous ACE2 overexpression mediated by adenoviral ACE2 via tail vein injection (10(9) pfu/mouse) rescued the impaired EDR and flow-mediated dilatation (FMD) in db/db mice. Diminazene aceturate treatment (15 mg/kg/day) activated ACE2, increased the circulating Ang (1-7) level, and augmented EDR and FMD in db/db mouse arteries. In addition, activation of the ACE2-Ang (1-7) axis reduced reactive oxygen species (ROS) overproduction determined by dihydroethidium staining, CM-H2DCFDA fluorescence imaging, and chemiluminescence assay in db/db mouse aortas and also in high-glucose-treated endothelial cells. Pharmacological benefits of ACE2-Ang (1-7) upregulation on endothelial function were confirmed in ACE2 knockout (ACE2 KO) mice both ex vivo and in vitro. We elucidate that the ACE2-Ang (1-7)-Mas axis serves as an important signal pathway in endothelial cell protection in diabetic mice, especially in diabetic human arteries. Endogenous ACE2-Ang (1-7) activation or ACE2 overexpression preserves endothelial function in diabetic mice through increasing nitric oxide bioavailability and inhibiting oxidative stress, suggesting the therapeutic potential of ACE2-Ang(1-7) axis activation against diabetic vasculopathy. Antioxid.

Increased ADAM17 Expression in ACE2 Knockout Mice is Associated with Increased Excitability of Paraventricular Nucleus Pre‐sympathetic Neurons

We previously reported that ACE2 knockout (KO) mice, like humans, develop age‐dependent hypertension. In addition, a disintegrin and metalloprotease 17 (ADAM17) was recently shown to contribute to neurogenic hypertension. Accordingly, we hypothesized that ADAM17 in the paraventricular nucleus (PVN) of hypothalamus contributes to enhanced sympathetic tone in mice lacking ACE2. Adult 10‐12 week‐old wildtype (WT) and ACE2 KO male mice (n=2‐4/group) were injected in the kidney with a pseudorabies virus encoding a green fluorescent protein, for retrograde labeling of sympathetic neurons. After 4 days, whole‐cell patch‐clamp recordings were conducted from labeled pre‐sympathetic PVN neurons. The resting membrane potential of these PVN neurons in WT mice was ‐59.7 ±1.06 mV, while ‐52.7 ±1.6 mV in ACE2 KO mice, which may suggest an increase threshold for excitability in pre‐sympathetic PVN neurons. In another set of mice, the hypothalamus was dissected from WT and KO animals (n=4/group) for western blot analysis. ADAM17 expression showed a 2‐fold increase in KO compared to WT (P<0.05). Finally, using immunohistochemistry, ADAM17 expression was confirmed in parvocellular neurons. Taken together, our data suggest that enhanced expression of ADAM17 in the parvocellular PVN could be associated with increased excitability of pre‐sympathetic neurons in ACE2 KO mice. This dysregulation in neuronal activity might alter the intricate pressor homeostasis leading to increased blood pressure. These results suggest that ADAM17 could be a potential new target for the reduction of enhanced sympathetic activity in neurogenic hypertension.Support: NIH HL093178

Generation of outbred Ace2 knockout mice by RNA transfection of TALENs displaying colitis reminiscent pathophysiology and inflammation.

The angiotensin I converting enzyme 2 (ACE2) is a key factor in the maintenance of intestinal homeostasis. Dysregulation of homeostasis can lead to inflammation of the colon (colitis), which can cause life-threatening enfeeblement or even cancer. Animal models are valuable surrogates in deciphering the pathology behind such human conditions and for screening of putative therapeutic targets or treatment paradigms. However, development of disease models can be time-consuming and technical demanding, which might hamper their application-value. In this study, we genetically disrupted the mouse Ace2 gene by direct injection of in vitro transcribed mRNA coding for transcription activator-like effector nucleases (TALENs) into the cytoplasm of outbred Kunming mouse zygotes. Consequently, somatic mutations were induced with an efficiency of 57 %, of which 39 % were frameshift mutations. Moreover, all modifications were stably transferred during germline transmission. In Ace2-knockout male mice (Ace2 −/y), we observed severe chemical induced colitis, characterized by considerable weight loss, diarrhea and a shortened colon length. Histologically, Ace2 mutations resulted in the infiltration of leukocytes and the overt damage of the intestinal mucosal barrier. In addition, we detected an increased expression of inflammatory cytokines in the colon tissue of Ace2 −/y mice. Collectively, the data indicate that high targeting efficiency and heritability can be achieved in an outbred mouse model by zygote injection of TALEN mRNA. Furthermore, the generated Ace2 −/y mice display phenotypic traits reminiscent of colitis and we anticipate that such mice can be of value in studies of the intestinal microbiome or fecal transplantation.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-014-9855-3) contains supplementary material, which is available to authorized users.

ACE2 deficiency shifts energy metabolism towards glucose utilization

BackgroundThis study aimed at investigating the effects of genetic angiotensin-converting enzyme (ACE) 2 deficiency on glucose homeostasis in the pancreas and skeletal muscle and their reversibility following ACE inhibition. ProceduresACE2-knockout and C57bl6J mice were placed on a standard diet (SD) or a high-fat diet (HFD) for 12weeks. An additional group of ACE2-knockout mice was fed a SD and treated with the ACE inhibitor, perindopril (2mgkg−1day−1). Glucose and insulin tolerance tests, indirect calorimetry measurements and EchoMRI were performed. Non-esterfied ‘free’ fatty acid oxidation rate in skeletal muscle was calculated by measuring the palmitate oxidation rate. β-cell mass was determined by immunostaining. Insulin, collectrin, glucose transporter protein, and peroxisome proliferator-activated receptor-γ expression were analysed by RT-PCR. Markers of mithocondrial biogenesis/content were also evaluated. Main FindingsACE2-knockout mice showed a β-cell defect associated with low insulin and collectrin levels and reduced compensatory hypertrophy in response to a HFD, which were not reversed by perindopril. On the other hand, ACE2 deficiency shifted energy metabolism towards glucose utilization, as it increased the respiratory exchange ratio, reduced palmitate oxidation and PCG-1α expression in the skeletal muscle, where it up-regulated glucose transport proteins. Treatment of ACE2-knockout mice with perindopril reversed the skeletal muscle changes, suggesting that these were dependent on Angiotensin II (Ang II). Principal ConclusionsACE2-knockout mice display a β-cell defect, which does not seem to be dependent on Ang II but may reflect the collectrin-like action of ACE2. This defect seemed to be compensated by the fact that ACE2-knockout mice shifted their energy consumption towards glucose utilisation via Ang II.

Abstract 480: ACE2 Independent Pathways of Angiotensin 1-7 Formation in the Murine Kidney

Fluid balance, salt retention and tissue remodeling is hormonally regulated by the Renin-Angiotensin-System. The sophisticated enzyme network serially cleaves peptide hormones generating various effector hormones including the vasoconstrictor Angiotensin 1-8 and its counterbalancing downstream metabolite Angiotensin 1-7. LC-MS/MS based analysis of kidney and plasma of ACE2 knockout mice revealed unchanged Angiotensin 1-7 concentrations compared to wild type animals pointing to alternative pathways of Angiotensin 1-7 formation and suggesting a minor role for ACE2 in the formation of Angiotensin 1-7 in vivo. Moreover, we observed a decrease in the Angiotensin 1-8/Angiotensin 1-10 ratio in ACE2KO-mice indicating a changed conversion rate from Angiotensin 1-10 to Angiotensin 1-8. We analyzed Angiotensin 1-7 metabolism in kidney homogenates by spiking natural substrates in the absence and presence of specific enzyme inhibitors. The metabolism of ex vivo spiked Angiotensins in kidney homogenates in presence and absence of the Prolyl-Carboxypepdidase (PCP) and Prolyl-Endopeptidase (PEP) Inhibitor ZPP and the ACE2-Inhibitor MLN-4760 revealed stronger contribution of PCP and PEP to Angiotensin 1-7 formation than ACE2. Analyzing the formation of Angiotensin 1-8 from Angiotensin 1-10 in renal cortex and medulla homogenates of WT and ACE2KO mice in the absence and presence of an ACE-Inhibitor confirmed our observations on endogenous peptide levels and the ratio between Angiotensin 1-8 and Angiotensin 1-10 in plasma, revealing a reduced ACE activity in the renal cortex of ACE2KO. Our study raises questions concerning the relevance of renal ACE2 for the local formation of Angiotensin 1-7. Moreover, the reduced ACE expression and activity in renal cortex might be a physiologically and therefore phenotype relevant consequence of the knockdown of ACE2 in these mice. The possible crosstalk between ACE and ACE2 expression and activity might give rise to further studies addressing the underlying mechanisms and might be important for our understanding of RAS dynamics in regard to therapeutic interventions.

The angiotensin-converting enzyme 2/angiotensin (1-7)/Mas axis protects the function of pancreatic β cells by improving the function of islet microvascular endothelial cells.

In the diabetic state, the local rennin-angiotensin system (RAS) is activated in the pancreas, and is strongly associated with islet dysfunction. The angiotensin-converting enzyme2 (ACE2)/angiotensin(1-7) [Ang(1-7)]/Mas axis is a protective, negative regulator of the classical renin-angiotensin system. In this study, we assessed the role of the ACE2/Ang(1‑7)/Mas axis in pancreatic βcell survival and function. ACE2 knockout and wild-type mice were fed a high-fat diet for 16weeks. We then performed terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays, and determined the expression levels of interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) in the pancreatic islets. The effects of Ang(1-7) or Mas receptor silencing on endothelial function were assessed in MS-1 cells. MIN6 cells were then co-cultured with the MS-1 cells to evaluate the effects of ACE2 on insulin secretion. The ACE2 knockout mice were more susceptible than the wild-type mice to high-fat diet-induced βcell dysfunction. The TUNEL-positive area of the pancreatic islets and the expression levels of IL-1β and iNOS were markedly increased in the ACE2 knockout mice compared with their wild-type littermates. The Mas-silenced MS-1 cells were more sensitive to palmitate-induced dysfunction and apoptosis invitro. Ang(1-7) increased the activity of the Akt/endothelial NOS/nitric oxide (NO) pathway in the MS-1 cells, protected MIN6 cells against palmitate-induced apoptosis, and improved MIN6 insulin secretory function in the co-culture system. In conclusion, this study demonstrates that the ACE2/Ang(1-7)/Mas axis is a potential target for protecting the funcion of βcells by improving the function of islet microvascular endothelial cells.

Angiotensin-Converting Enzyme 2–Independent Action of Presumed Angiotensin-Converting Enzyme 2 Activators

Angiotensin (Ang)-converting enzyme 2 (ACE2) is a key enzyme in the metabolism of Ang II. XNT (1-[(2-dimethylamino)ethylamino]-4-(hydroxymethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one) and diminazene have been reported to exert various organ-protective effects, which are attributed to the activation of ACE2. To test the effect of these compounds, we studied Ang II degradation in vivo and in vitro as well as their effect on ACE2 activity in vivo and in vitro. In a model of Ang II–induced acute hypertension, blood pressure (BP) recovery was markedly enhanced by XNT (slope with XNT, −3.26±0.2 versus −1.6±0.2 mm Hg/min without XNT; P <0.01). After Ang II infusion, neither plasma nor kidney ACE2 activity was affected by XNT. Plasma Ang II and Ang (1–7) levels also were not significantly affected by XNT. The BP-lowering effect of XNT seen in wild-type animals was also observed in ACE2 knockout mice (slope with XNT, −3.09±0.30 versus −1.28±0.22 mm Hg/min without XNT; P <0.001). These findings show that the BP-lowering effect of XNT in Ang II–induced hypertension cannot be because of the activation of ACE2. In vitro and ex vivo experiments in both mice and rat kidney confirmed a lack of enhancement of ACE2 enzymatic activity by XNT and diminazene. Moreover, Ang II degradation in vitro and ex vivo was unaffected by XNT and diminazene. We conclude that the biological effects of these compounds are ACE2-independent and should not be attributed to the activation of this enzyme.

Angiotensin‐II mediates ACE2 internalization and degradation through an angiotensin‐II type I receptor‐dependent mechanism (1066.9)

ACE2 mediates the conversion of ang‐II to ang‐(1‐7) and by this mechanism prevents the pathological effects related to the activation of rennin‐angiotensin system (RAS). We previously reported that decreased ACE2 expression and activity leads to the development of Ang‐II‐mediated hypertension in mice. The aim of the present study was to characterize the mechanisms involved in ACE2 down‐regulation during neurogenic hypertension. In ACE2‐transfected Neuro‐2A cells, Ang‐II treatment resulted in a significant attenuation of the enzyme activity. Confocal microscopy experiments demonstrated that in presence of ang‐II, ACE2 is internalized and co‐localizes with Rab7, a lysosomal marker. These effects were prevented by the AT1R blocker losartan and the lysosomal inhibitor leupeptin. In contrast, in HEK293T cells, which lack endogenous AT1R, Ang‐II failed to promote ACE2 internalization. This effect was restored after AT1R transfection. Further, ang‐II enhanced ACE2 ubiquitination and decreased the interactions between AT1R and ACE2. In contrast, ACE2 activity was not changed by transfection of AT2R or Mas1R. In vivo, Ang‐II‐mediated hypertension was blunted by chronic infusion of leupeptin in C57Bl/6, but not in ACE2 knockout mice. This is the first demonstration that elevated Ang‐II levels reduce ACE2 expression and activity by stimulation of lysosomal degradation through an AT1R‐dependent mechanism.Grant Funding Source: P20‐RR‐018766, RO1‐HL093178, Howard University

ACE2 deficiency increases NADPH-mediated oxidative stress in the kidney.

Angiotensin‐converting enzyme 2 (ACE2) is highly expressed in the kidney and hydrolyzes angiotensin II (Ang II) to Ang(1–7). Since Ang II is a strong activator of oxidative stress, we reasoned that ACE2 could be involved in the regulation of renal oxidative stress by governing the levels of Ang II. We, therefore, assessed levels of oxidative stress in kidney cortex of ACE2 knockout and wild‐type littermate mice under baseline conditions. We found multiple markers of increased oxidative stress in ACE2KO mice. NADPH oxidase activity was increased in kidney cortex from ACE2KO mice as compared to WT (227 ± 24% vs.100 ± 19%, P < 0.001). However, kidney catalase and superoxide dismutase activities were not different between groups. Exogenous Ang II was degraded less efficiently by kidneys from ACE2KO mice than WT mice, and administration of an AT1R blocker (losartan 30 mg/kg/day) resulted in normalization of NADPH oxidase activity in the ACE2KO. These findings suggest that an AT1R‐dependent mechanism contributes to increased ROS observed in the ACE2KO. This study demonstrates that genetic deficiency of ACE2 activity in mice fosters oxidative stress in the kidney in the absence of overt hypertension and is associated with reduced kidney capacity to hydrolyze Ang II. ACE2KO mice serve as a novel in vivo model to examine the role of overactivity of NADPH oxidase in kidney function.