Angiotensin-converting Enzyme 2 Knockout Research Articles

Male bias in ACE2 basic science research: missed opportunity for discovery in the time of COVID-19.

Throughout the world, including the United States, men have worse outcomes from COVID-19 than women. SARS-CoV-2, the causative virus of the COVID-19 pandemic, uses angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. ACE2 is a member of the renin-angiotensin system (RAS) and plays an important role in counteracting the harmful effects mediated by the angiotensin type 1 receptor. Therefore, we conducted Ovid MEDLINE and Embase database searches of basic science studies investigating the impact of the biological variable of sex on ACE2 expression and regulation from 2000, the year ACE2 was discovered, through December 31, 2020. Out of 2,131 publications, we identified 853 original research articles on ACE2 conducted in primary cells, tissues, and/or whole mammals excluding humans. The majority (68.7%) of these studies that cited the sex of the animal were conducted in males, while 11.2% were conducted solely in females; 9.26% compared ACE2 between the sexes, while 10.8% did not report the sex of the animals used. General findings are that sex differences are tissue-specific and when present, are dependent upon gonadal state. Renal, cardiac, and adipose ACE2 is increased in both sexes under experimental conditions that model co-morbidities associated with worse COVID-19 outcomes including hypertension, obesity, and renal and cardiovascular diseases; however, ACE2 protein was generally higher in the males. Studies in Ace2 knockout mice indicate ACE2 plays a greater role in protecting the female from developing hypertension than the male. Studying the biological variable of sex in ACE2 research provides an opportunity for discovery in conditions involving RAS dysfunction and will shed light on sex differences in COVID-19 severity.

ACE2 CONTRIBUTES TO THE NORMAL REGULATION OF ARTERIAL PRESSURE AND IMMUNITY IN FEMALES OF REPRODUCTIVE AGE

Objective: Hypertension and cardiovascular disease are age and sex dependent. These differences may, in part, be mediated by the depressor/pressor balance of the renin angiotensin system. Here we determined the role of angiotensin converting enzyme 2 (ACE2) in the regulation of arterial pressure in females of reproductive age and investigated whether targeting deficits in ACE2-generated angiotensin (Ang)-(1–7) restores the normal regulation of arterial pressure during pregnancy. Design and method: Mean arterial pressure (MAP) was measured via telemetry in 14 week old wild-type (WT) and ACE2 knockout (ACE2-KO) male mice and WT and ACE2-KO female mice receiving vehicle or the MasR agonist, AVE-0991 (24 ug/kg/min s.c) prior to and during pregnancy. FACS analysis was used to determine circulating immune cell activation and infiltration into kidneys (baseline and Gd18) and placentae (Gd18). Results: Basal MAP was lower in WT females than ACE2-KO females, WT males and ACE2-KO males (91 ± 2 vs 100 ± 1, 98 ± 1 and 102 ± 2 mmHg, respectively; all P < 0.05 vs WT female). In ACE2-KO females, AVE-0991 lowered basal MAP by 5 ± 1 mmHg (P = 0.03). In WT females, MAP decreased during pregnancy reaching a nadir at Gd9 before returning to pre-conception levels during late gestation. In contrast, in ACE2-KO mice, MAP increased significantly during late gestation (P < 0.0001 vs WT) and this effect was prevented by AVE-0991 (P < 0.05 vs vehicle). This effect of AVE-0991 on MAP was due to changes in diastolic rather than systolic arterial pressure. ACE2-KO mice had smaller litters but greater birth/pup weight than WT mice. AVE-0991 normalised litter size and birth/pup weight in ACE2-KO mice to that observed in WT mice. Circulating and renal T-regulatory cells were lower in non-pregnant and pregnant female and male ACE2-KO mice than their WT counterparts. Treatment with AVE-0991 did not alter the proportion of T-regulatory cells. Conclusions: These data indicate that ACE2 plays an important role in the regulation of arterial pressure and immunity in females of reproductive age. A corollary of this is that deficits in ACE2-generated Ang-(1–7) may contribute to an increased risk of hypertension in non-pregnant and pregnant premenopausal females and therefore may be a novel therapeutic target.

A pressor dose of angiotensin II has no influence on the angiotensin-converting enzyme 2 and other molecules associated with SARS-CoV-2 infection in mice.

In the early phase of the Coronavirus disease 2019 (COVID‐19) pandemic, it was postulated that the renin‐angiotensin‐system inhibitors (RASi) increase the infection risk. This was primarily based on numerous reports, which stated that the RASi could increase the organ Angiotensin‐converting enzyme 2 (ACE2), the receptor of Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), in rodents. RASi can theoretically antagonize the potential influence of angiotensin II (Ang II) on ACE2. However, while Ang II decreases the ACE2 levels in cultured cells, there is little evidence that supports this phenomenon in living animals. In this study, we tested whether Ang II or Ang II combined with its antagonist would alter the ACE2 and other molecules associated with the infection of SARS‐CoV‐2. Male C57BL6/J mice were administered vehicle, Ang II (400 ng/kg/min), or Ang II with losartan (10 mg/kg/min) for 2 weeks. ACE2 knockout mice were used as a negative control for the ACE2 assay. We found that both Ang II, which elevated blood pressure by 30 mm Hg, and Ang II with losartan, had no effect on the expression or protein activity of ACE2 in the lung, left ventricle, kidney, and ileum. Likewise, these interventions had no effect on the expression of Transmembrane Protease Serine 2 (TMPRSS2) and Furin, proteases that facilitate the virus‐cell fusion, and the expression or activity of Tumor Necrosis Factor α‐Convertase (TACE) that cleaves cell‐surface ACE2. Collectively, physiological concentrations of Ang II do not modulate the molecules associated with SARS‐CoV‐2 infection. These results support the recent observational studies suggesting that the use of RASi is not a risk factor for COVID‐19.

Berberine Reshapes the Balance of the Local Renin-Angiotensin System by Modulating Autophagy under Metabolic Stress in Pancreatic Islets.

Results Prolonged exposure to palmitate increased the expression of ACE and AngII type 1 receptor (ATR1) and decreased the ACE2 expression, which was partly offset by berberine. In ob/ob mice, berberine increased in tolerance to glucose, improved abnormal β-cell and α-cell distributions, upregulated ACE2 expression, and decreased autophagosomes and the expression of LC3 and SQSTM1/p62. Autophagosomes and expression of LC3 and SQSTM1/p62 were increased in ACE2KO mice. Conclusions We demonstrated that berberine may improve the pancreatic islet function by regulating local RAS-mediated autophagy under metabolic stress.

ACE2 contributes to the maintenance of mouse epithelial barrier function

BackgroundThe whole world was hit hard by the coronavirus disease-19 (COVID-19). Given that angiotensin I converting enzyme 2 (ACE2) is the viral entry molecule, understanding ACE2 has become a major focus of current COVID-19 research. ACE2 is highly expressed in the gut, but its role has not been fully understood and thus COVID-19 treatments intending to downregulate ACE2 level may cause untoward side effects. Gaining insight into the functions of ACE2 in gut homeostasis therefore merits closer examination, and is beneficial to find potential therapeutic alternatives for COVID-19. Methods: We took advantage of Ace2 knockout out mice and isolated intestinal organoids to examine the role of ACE2 in intestinal stemness. Inflammatory bowel disease (IBD) mouse model was established by 4% dextran sodium sulfate. LGR5 and KI67 levels were quantitated to reflect the virtue of intestinal stem cells (ISCs). FITC-dextran 4 (FD-4) assay was used to assess intestinal barrier function. Results: Western blotting identified the expression of ACE2 in colon, which was consistent with the results of immunofluorescence and RT-PCR. Moreover, Ace2−/− organoids showed decreased LRG5 and KI67 levels, and elevated calcium concentration. Furthermore, the permeability of ace2−/− organoids was markedly increased compared with ace2+/+ organoids. Collectively, ace2−/− mice were more susceptible than ace2+/+ mice to IBD, including earlier bloody stool, undermined intestinal architecture and more pronounced weight loss. Conclusions: Our data reveal that ACE2 contributes to the proliferation of intestinal stem cells and hence orchestrates the mucosal homeostasis.

Dual deficiency of angiotensin-converting enzyme-2 and Mas receptor enhances angiotensin II-induced hypertension and hypertensive nephropathy.

Angiotensin‐converting enzyme‐2 (ACE2) and Mas receptor are the major components of the ACE2/Ang 1‐7/Mas axis and have been shown to play a protective role in hypertension and hypertensive nephropathy individually. However, the effects of dual deficiency of ACE2 and Mas (ACE2/Mas) on Ang II‐induced hypertensive nephropathy remain unexplored, which was investigated in this study in a mouse model of hypertension induced in either ACE2 knockout (KO) or Mas KO mice and in double ACE2/Mas KO mice by subcutaneously chronic infusion of Ang II. Compared with wild‐type (WT) animals, mice lacking either ACE2 or Mas significantly increased blood pressure over 7‐28 days following a chronic Ang II infusion (P < .001), which was further exacerbated in double ACE2/Mas KO mice (P < .001). Furthermore, compared to a single ACE2 or Mas KO mice, mice lacking ACE2/Mas developed more severe renal injury including higher levels of serum creatinine and a further reduction in creatinine clearance, and progressive renal inflammation and fibrosis. Mechanistically, worsen hypertensive nephropathy in double ACE2/Mas KO mice was associated with markedly enhanced AT1‐ERK1/2‐Smad3 and NF‐κB signalling, thereby promoting renal fibrosis and renal inflammation in the hypertensive kidney. In conclusion, ACE2 and Mas play an additive protective role in Ang II‐induced hypertension and hypertensive nephropathy. Thus, restoring the ACE2/Ang1‐7/Mas axis may represent a novel therapy for hypertension and hypertensive nephropathy.

Sex-Specific Modulation of Blood Pressure and the Renin-Angiotensin System by ACE (Angiotensin-Converting Enzyme) 2.

We showed ACE (angiotensin-converting enzyme) 2 is higher in the kidney of male compared with female mice. To further investigate this sex difference, we examined the role of ACE2 in Ang-[1-8] (angiotensin [1-8])-induced hypertension and regulation of the renin-angiotensin system in the kidney of WT (wild type) and Ace2 KO (knockout) mice. Mean arterial pressure rose faster in WT male than WT female mice after Ang-[1-8] infusion. This sex difference was attenuated in ACE2 KO mice. Ang-[1-8] infusion reduced glomerular AT1R (angiotensin type 1 receptor) binding in WT female mice by 30%, and deletion of Ace2 abolished this effect. In contrast, Ang-[1-8] infusion increased glomerular AT1R binding in WT male mice by 1.2-fold, and this effect of Ang-[1-8] persisted in Ace2 KO male mice (1.3-fold). ACE2 also had an effect on renal protein expression of the neutral endopeptidase NEP (neprilysin), the enzyme that catabolizes Ang-[1-10] (angiotensin [1-10]), the precursor of Ang-[1-8]. Ang-[1-8] infusion downregulated NEP protein expression by 20% in WT male, whereas there was a slight increase in NEP expression in WT female mice. Deletion of Ace2 resulted in lowered NEP expression after Ang-[1-8] infusion in both sexes. These findings suggest sex-specific ACE2 regulation of the renin-angiotensin system contributes to female protection from Ang-[1-8]-induced hypertension. These findings have ramifications for the current coronavirus disease 2019 (COVID-19) pandemic, especially in hypertension since ACE2 is the SARS-CoV-2 receptor and hypertension is a major risk factor for poor outcomes.

Impaired Breakdown of Bradykinin and Its Metabolites as a Possible Cause for Pulmonary Edema in COVID-19 Infection.

A growing body of clinical evidence shows that vascular leakage leads to pulmonary edema in coronavirus disease 2019 (COVID-19) patients. Coronaviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) use membrane ectopeptidases to invade cells, most notably dipeptidyl peptidase 4 (DPP4; https://www.uniprot.org/uniprot/P27487), aminopeptidase N (APN; https://www.uniprot.org/uniprot/P15144), and angiotensin-converting enzyme 2 (ACE2; https://www.uniprot.org/uniprot/Q9BYF1).[1] It is important to distinguish this latter protein from ACE, which is the main target for conventional blood pressure lowering drugs: ACE inhibitors do not inhibit ACE2.[2] As a result of virus entry, these membrane ectopeptidases are internalized and their activity becomes downregulated. The common denominator between these membrane ectopeptidases is their enzyme specificity: they degrade peptide hormones, for instance, angiotensin II. For SARS-CoV-2 (as well as SARS-CoV), ACE2 is the predominant target for cellular uptake in vivo.[3] It is present on lung alveolar epithelial cells, enterocytes of the small intestine, endothelial cells, and arterial smooth muscle cells.[4] In an acid aspiration lung injury mouse model, ACE2 knockout (KO) worsened the resulting edema.[5] This was accompanied by increased angiotensin II levels[6] and was prevented by angiotensin II receptor KO, leading to the conclusion that the lack of ACE2-mediated angiotensin II degradation was responsible for the observed increase pulmonary vascular permeability. The same observations were made when downregulating ACE2 with the spike protein of the SARS CoV prior to acid aspiration.[6] Yet, the authors did not measure renin in these studies. This is crucial since normally angiotensin II rises result in rapid renin suppression,[7] thereby normalizing its levels. Moreover, ACE2 is just one of many angiotensin-degrading enzymes (angiotensinases). In full accordance with this concept, Gurley et al did not find increased angiotensin II levels after ACE2 KO.[8] Hence, whether the ACE2 KO dependent permeability changes are entirely due to ACE2-mediated angiotensin II degradation remains uncertain. ACE2 has multiple other substrates. Among these are the kinins, which are also degraded by DPP4.[9] We and others believe that this deserves attention, as it offers an alternative view, and forms a basis on which novel therapeutic opportunities might be proposed.[10]

Liraglutide Attenuates Non-Alcoholic Fatty Liver Disease in Mice by Regulating the Local Renin-Angiotensin System.

The renin-angiotensin system (RAS) is involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and represents a potential therapeutic target for NAFLD. Glucagon-like peptide-1 (GLP-1) signaling has been shown to regulate the RAS within various local tissues. In this study, we aimed to investigate the functional relationship between GLP-1 and the local RAS in the liver during NAFLD. Wild-type and ACE2 knockout mice were used to establish a high-fat-induced NAFLD model. After the mice were treated with liraglutide (a GLP-1 analogue) for 4 weeks, the key RAS component genes were up-regulated in the liver of NAFLD mice. Liraglutide treatment regulated the RAS balance, preventing a reduction in fatty acid oxidation gene expression and increasing gluconeogenesis and the expression of inflammation-related genes caused by NAFLD, which were impaired in ACE2 knockout mice. Liraglutide-treated HepG2 cells exhibited activation of the ACE2/Ang1-7/Mas axis, increased fatty acid oxidation gene expression, and decreased inflammation, which could be reversed by A779 and AngII. These results indicate that the local RAS in the liver becomes overactivated in response to NAFLD. Moreover, ACE2 knockout increases the severity of liver steatosis. Liraglutide has a negative and antagonistic effect on the ACE/AngII/AT1R axis, a positive impact on the ACE2/Ang1-7/Mas axis, and is mediated through the PI3K/AKT pathway. This may represent a potential new mechanism by which liraglutide improves NAFLD.

ACE overexpression in myeloid cells increases oxidative metabolism and cellular ATP

ACE overexpression in myeloid cells increases oxidative metabolism and cellular ATP

ACE overexpression in myeloid cells increases oxidative metabolism and cellular ATP.

Angiotensin-converting enzyme (ACE) affects blood pressure. In addition, ACE overexpression in myeloid cells increases their immune function. Using MS and chemical analysis, we identified marked changes of intermediate metabolites in ACE-overexpressing macrophages and neutrophils, with increased cellular ATP (1.7-3.0-fold) and Krebs cycle intermediates, including citrate, isocitrate, succinate, and malate (1.4-3.9-fold). Increased ATP is due to ACE C-domain catalytic activity; it is reversed by an ACE inhibitor but not by an angiotensin II AT1 receptor antagonist. In contrast, macrophages from ACE knockout (null) mice averaged only 28% of the ATP levels found in WT mice. ACE overexpression does not change cell or mitochondrial size or number. However, expression levels of the electron transport chain proteins NDUFB8 (complex I), ATP5A, and ATP5β (complex V) are significantly increased in macrophages and neutrophils, and COX1 and COX2 (complex IV) are increased in macrophages overexpressing ACE. Macrophages overexpressing ACE have increased mitochondrial membrane potential (24% higher), ATP production rates (29% higher), and maximal respiratory rates (37% higher) compared with WT cells. Increased cellular ATP underpins increased myeloid cell superoxide production and phagocytosis associated with increased ACE expression. Myeloid cells overexpressing ACE indicate the existence of a novel pathway in which myeloid cell function can be enhanced, with a key feature being increased cellular ATP.

Different effects of the deletion of angiotensin converting enzyme 2 and chronic activation of the renin-angiotensin system on muscle weakness in middle-aged mice.

Inhibition of the renin-angiotensin system (RAS) has been shown to alleviate muscle atrophy both under pathological conditions and during physiological aging. We recently reported that the deletion of angiotensin converting enzyme 2 (ACE2), which converts Angiotensin II to Angiotensin-(1-7) in mice, leads to the early manifestation of aging-associated muscle weakness along with the increased expression of p16INK4a, a senescence-associated gene, and increased central nuclei in the tibialis anterior (TA) muscle in middle age. As ACE2 is multifunctional and functions beyond its role in the RAS, we investigated whether activation of the RAS primarily contributes to muscle weakness in ACE2 knockout (KO) mice by comparing these mice to Tsukuba hypertensive (TH) mice that overproduce human angiotensin II. The grip strength of young (6 months) and middle-aged (15 months) TH mice was consistently lower than that of wild-type mice at the same ages. Middle-aged TH mice were continuously lean with extremely reduced adiposity. Central nuclei in the gastrocnemius (GM) muscle were increased in ACE2KO mice, while no apparent morphological change was observed in the GM muscles of TH mice. Increased expression of p16INK4a along with alterations in the expression of several sarcopenia-associated genes were observed in the GM muscles of ACE2KO mice but not TH mice. These findings suggest that chronic overactivation of the RAS does not primarily contribute to the early aging phenotypes of skeletal muscle in ACE2KO mice.

Angiotensin-converting enzyme 2 regulates endoplasmic reticulum stress and mitochondrial function to preserve skeletal muscle lipid metabolism

ObjectiveEndoplasmic reticulum (ER) stress and mitochondrial function affected intramuscular fat accumulation. However, there is no clear evident on the effect of the regulation of ER stress and mitochondrial function by Angiotensin-converting enzyme 2 (ACE2) on the prevention of intramuscular fat metabolism. We investigated the effects of ACE2 on ER stress and mitochondrial function in skeletal muscle lipid metabolism.MethodsThe triglyceride (TG) content in skeletal muscle of ACE2 knockout mice and Ad-ACE2-treated db/db mice were detected by assay kits. Meanwhile, the expression of lipogenic genes (ACCα, SREBP-1c, LXRα, CPT-1α, PGC-1α and PPARα), ER stress and mitochondrial function related genes (GRP78, eIF2α, ATF4, BCL-2, and SDH6) were analyzed by RT-PCR. Lipid metabolism, ER stress and mitochondrial function related genes were analyzed by RT-PCR in ACE2-overexpression C2C12 cell. Moreover, the IKKβ/NFκB/IRS-1 pathway was determined using lysate sample from skeletal muscle of ACE2 knockout mice.ResultsACE2 deficiency in vivo is associated with increased lipid accumulation in skeletal muscle. The ACE2 knockout mice displayed an elevated level of ER stress and mitochondrial dysfunctions in skeletal muscle. In contrast, activation of ACE2 can ameliorate ER stress and mitochondrial function, which slightly accompanied by reduced TG content and down-regulated the expression of skeletal muscle lipogenic proteins in the db/db mice. Additionally, ACE2 improved skeletal muscle lipid metabolism and ER stress genes in the C2C12 cells. Mechanistically, endogenous ACE2 improved lipid metabolism through the IKKβ/NFκB/IRS-1 pathway in skeletal muscle.ConclusionsACE2 was first reported to play a notable role on intramuscular fat regulation by improving endoplasmic reticulum and mitochondrial function. This study may provide a strategy for treating insulin resistance in skeletal muscle.

Angiotensin 1-7 alleviates aging-associated muscle weakness and bone loss, but is not associated with accelerated aging in ACE2-knockout mice.

The angiotensin-converting enzyme 2 (ACE2)-angiotensin 1-7 (A1-7)-A1-7 receptor (Mas) axis plays a protective role in the renin-angiotensin system (RAS). We recently found that ACE2 knockout (ACE2KO) mice exhibit earlier aging-associated muscle weakness, and that A1-7 alleviates muscle weakness in aging mice. In the present study, we investigated the role of the A1-7-Mas pathway in the effect of ACE2 on physiological aging. Male wild-type, ACE2KO, and Mas knockout (MasKO) mice were subjected to periodical grip strength measurement, followed by administration of A1-7 or vehicle for 4 weeks at 24 months of age. ACE2KO mice exhibited decreased grip strength after 6 months of age, while grip strength of MasKO mice was similar to that of wild-type mice. A1-7 improved grip strength in ACE2KO and wild-type mice, but not in MasKO mice. Muscle fibre size was smaller in ACE2KO mice than that in wild-type and MasKO mice, and increased with A1-7 in ACE2KO and WT mice, but not in MasKO mice. Centrally nucleated fibres (CNFs) and expression of the senescence-associated gene p16INK4a in skeletal muscles were enhanced only in ACE2KO mice and were not altered by A1-7. ACE2KO mice, but not MasKO mice, exhibited thinning of peripheral fat along with increased adipose expression of p16INK4a A1-7 significantly increased bone volume in wild-type and ACE2KO mice, but not in MasKO mice. Our findings suggest that the impact of ACE2 on physiological aging does not depend on the endogenous production of A1-7 by ACE2, while overactivation of the A1-7-Mas pathway could alleviate sarcopenia and osteoporosis in aged mice.

Angiotensin-converting enzyme 2 inhibits endoplasmic reticulum stress-associated pathway to preserve nonalcoholic fatty liver disease.

Previous works indicated that the stress on the endoplasmic reticulum (ER) affected nonalcoholic fatty liver disease (NAFLD). However, there is no clear evident on the effect of the regulation of ER stress by angiotensin-converting enzyme 2 (ACE2) on the prevention of NAFLD. HepG2 cells were treated with thapsigargin (Tg) or palmitic acid (PA). We analysed ACE2 expression using Western-blotting analyses. ER stress-related proteins were detected in ACE2 knockout mice and Ad-ACE2-treated db/db mice by immunofluorescence or Western-blotting analyses. In ACE2-overexpression HepG2 cells, the triglyceride (TG), total cholesterol (TC), and glycogen content were detected by assay kits. Meanwhile, the expression of hepatic lipogenic proteins (ACCα, SREBP-1c, FAS, and LXRα), enzymes for gluconeogenesis (PEPCK, G6Pase, and IRS2), and IKKβ/NFκB/IRS1/Akt pathway were analysed by Western-blotting analyses. ACE2 was significantly increased in Tg/PA-induced cultured hepatocytes. Additionally, ACE2 knockout mice displayed elevated levels of ER stress, while Ad-ACE2-treated db/db mice showed reduced ER stress in liver. Furthermore, activation of ACE2 can ameliorate ER stress, accompanied by decreased TG content, increased intracellular glycogen, and downregulated expression of hepatic lipogenic proteins and enzymes for gluconeogenesis in Tg/PA-induced hepatocytes. As a consequence of anti-ER stress, the activation of ACE2 led to improved glucose and lipid metabolism through the IKKβ/NFκB/IRS1/Akt pathway. This is the first time documented that ACE2 had a notable alleviating role in ER stress-induced hepatic steatosis and glucose metabolism via the IKKβ/NFκB/IRS1/Akt-mediated pathway. This study may further provide insight into a novel underlying mechanism and a strategy for treating NAFLD.

Angiotensin-converting enzyme inhibitor works as a scar formation inhibitor by down-regulating Smad and TGF-β-activated kinase 1 (TAK1) pathways in mice.

Angiotensin-converting enzyme (ACE), an important part of the renin-angiotensin system, is implicated in stimulating the fibrotic processes in the heart, lung, liver and kidney, while an ACE inhibitor (ACEI) promotes physiological tissue repair in these organs. The mechanism is closely related to TGF-β1 pathways. However, the reported effects of applying ACEIs during scar formation are unclear. Hence, we explored the anti-fibrotic effects of an ACEI and the molecular mechanisms involved in a mouse scar model. After a full-thickness skin wound operation, ACE wild-type mice were randomly assigned to receive either ramipril, losartan or hydralazine p.o. ACE knockout (KO) mice and negative control mice only received vehicle (water). Wound/scar widths during wound healing and histological examinations were recorded at the final day. The ability of ACEI to reduce fibrosis via TGF-β1 signalling was evaluated in vitro and in vivo. ACE KO mice and mice that received ramipril showed narrower wound/scar width, reduced fibroblast proliferation, decreased collagen and TGF-β1 expression. ACEI attenuated the phosphorylation of small mothers against decapentaplegic (Smad2/3) and TGF-β-activated kinase 1 (TAK1) both in vitro and in vivo. The expression of ACE-related peptides varied in murine models with different drug treatments. ACEI showed anti-fibrotic properties in scar formation by mediating downstream peptides to suppress TGF-β1/Smad and TGF-β1/TAK1 pathways. These findings suggest that dual inhibition of Smad and TAK1 signalling by ACEI is a useful strategy for the development of new anti-fibrotic agents.

A12839 Angiotensin 1-7 functionally recovers aging-induced locomotive dysfunction, but is not associated with accelerated aging phenotypes in angiotensin-converting-enzyme 2 knockout mice

Objectives: The angiotensin-converting enzyme 2 (ACE2)-angitotensin 1-7 (A1-7)-A1-7 receptor (Mas) axis has been shown to have a protective role in the renin-angiotensin system (RAS). We recently found that ACE2 knockout (KO) mice exhibit earlier aging-associated muscle weakness, and A1-7 restores muscle weakness in aging mice. As ACE2 has a multifunctional role outside the RAS, we investigated the dependence on the A1-7-Mas pathway in the effect of ACE2 on physiological aging including locomotive dysfunction with muscle weakness and osteoporosis in mice. Methods: Wildtype (WT), ACE2KO, and MasKO mice were subjected to periodical grip strength measurement, followed by administration of A1–7 (100 ng/kg/min) or saline for 4 weeks with an osmotic pump in 24-month-old mice. Results: ACE2KO mice exhibited decreased grip strength compared with WT mice after 6 months of age, while MasKO mice had grip strength similar to WT mice during aging. A1-7 infusion at 24 months old improved grip strength with increased muscle size in WT and ACE2KO mice but not in MasKO mice. A1–7 significantly increased bone volume in WT and ACE2KO mice, but not in MasKO mice. Histological and gene expression analysis revealed that tissue aging including skin and adipose tissue was accelerated in aging ACE2KO mice, but not in MASKO mice, compared to WT mice. Conclusion: A1-7 improves muscle weakness and osteoporosis in aged mice via a Mas-dependent pathway. In contrast, the different aging phenotypes between ACEKO and MasKO mice suggest the A1-7-independent effect of ACE2 on senescence.

A12686 The sirtuin 6 attenuates myocardial injury and dysfunction by regulating ADAM17/ACE2 pathway in hypertensive rats

Objectives: The sirtuin 6 (SIRT6) is an important modulator of cardiovascular functions in health and diseases. However, the exact role of SIRT6 in hypertension is poorly defined. Methods: The male Sprague-Dawley rats were used to the normal control or randomized to preparation of hypertensive rats by Ang II (200 ng/kg/min) infusion with treatment of recombinant pAAV-SIRT6 or pAAV-GFP for 4 weeks. Results: There were reduced expression of SIRT6 and ACE2, and augmented levels of TGF-β, osteopontin (OPN), MMP-2 and MMP-9 in hypertensive rats, contributing to elevated cardiac fibrosis and exacerbated ultrastructural injury. Enhanced phosphorylation of ADAM-17 and impaired heart function were observed in both hypertensive rats and ACE2 knockout rats, characterized by increased LVPWT. Treatment of pAAV-SIRT6 significantly prevented Ang II-mediated pressor response and attenuated myocardial fibrosis and ultrastructural injury in hypertensive rats. These changes were associated with marked increases in LVEF and LVFS as well as downregulated levels of OPN, MMP-2 and MMP-9 in hypertensive rats. In cultured neonatal rat cardiac fibroblasts, exposure of Ang II resulted in reduction in ACE2 expression and phosphorylation of p-ADAM-17 as well as increases in cellular proliferation and migration, which were rescued by treatment of pAAV-SIRT6. Conclusion: The SIRT6 prevents hypertension-mediated pathological hypertrophy, impaired heart function and ultrastructural injury in hypertensive rats. These results indicate cardiac protective roles of SIRT6 in the prevention of cardiac dysfunction and injury via activation of ADAM-17-ACE2 pathway and suppression of the TGF-β-OPN signaling. Targeting SIRT6 and ACE2 has potential therapeutic importance for controlling myocardial injury and dysfunction in hypertension.

AMP-activated Protein Kinase Phosphorylation of Angiotensin-Converting Enzyme 2 in Endothelium Mitigates Pulmonary Hypertension.

Endothelial dysfunction plays an integral role in pulmonary hypertension (PH). AMPK (AMP-activated protein kinase) and ACE2 (angiotensin-converting enzyme 2) are crucial in endothelial homeostasis. The mechanism by which AMPK regulates ACE2 in the pulmonary endothelium and its protective role in PH remain elusive. We investigated the role of AMPK phosphorylation of ACE2 Ser680 in ACE2 stability and deciphered the functional consequences of this post-translational modification of ACE2 in endothelial homeostasis and PH. Bioinformatics prediction, kinase assay, and antibody against phospho-ACE2 Ser680 (p-ACE2 S680) were used to investigate AMPK phosphorylation of ACE2 Ser680 in endothelial cells. Using CRISPR-Cas9 genomic editing, we created gain-of-function ACE2 S680D knock-in and loss-of-function ACE2 knockout (ACE2-/-) mouse lines to address the involvement of p-ACE2 S680 and ACE2 in PH. The AMPK-p-ACE2 S680 axis was also validated in lung tissue from humans with idiopathic pulmonary arterial hypertension. Phosphorylation of ACE2 by AMPK enhanced the stability of ACE2, which increased Ang (angiotensin) 1-7 and endothelial nitric oxide synthase-derived NO bioavailability. ACE2 S680D knock-in mice were resistant to PH as compared with wild-type littermates. In contrast, ACE2-knockout mice exacerbated PH, a similar phenotype found in mice with endothelial cell-specific deletion of AMPKα2. Consistently, the concentrations of phosphorylated AMPK, p-ACE2 S680, and ACE2 were decreased in human lungs with idiopathic pulmonary arterial hypertension. Impaired phosphorylation of ACE2 Ser680 by AMPK in pulmonary endothelium leads to a labile ACE2 and hence is associated with the pathogenesis of PH. Thus, AMPK regulation of the vasoprotective ACE2 is a potential target for PH treatment.

Angiotensin-Converting Enzyme 2 Deficiency Increases Translocation of Gut Bacteria by Depletion of Bone Marrow-Derived Circulating Angiogenic Cells

A healthy microbiome is central to good nutrition and efficient metabolism both of which can be altered in diabetes. Disruption of gut-vascular integrity may lead to translocation of microbial antigens and bacteria into the circulation. Bone marrow-derived circulating angiogenic cells (CACs) are essential to maintain vascular integrity. We tested the hypothesis that angiotensin-converting enzyme 2 (ACE2), the key enzyme in the vasoprotective axis of renin-angiotensin system (RAS), acts to regulate the infiltration of bone marrow-derived CACs in the gut and to maintain gut-vascular integrity in diabetes and could influence the systemic translocation of pathogenic antigens and bacteria from the gut. Diabetic ACE2 knockout (ACE2-/y)/C57BL/6-Ins2WT/C96Y (Akita) mice were examined at 9 months after the onset of diabetes and compared to age-matched controls. Beta diversity analysis of the gut microbiome revealed a distinct bacterial profile among the different genotypes (n=9-17 per group, p=0.002). The fecal samples from ACE2-/y-Akita group compared to Akita group were enriched with a great diversity of bacterial taxa that have been reported to contribute to the pathogenesis of diabetes and showed activation of peptidoglycan biosynthesis pathways by metatranscriptome analysis (n=4 per group, p=0.005). Flow cytometry analysis showed that loss of ACE2 led to reduced infiltration of bone marrow-derived CACs (CD45+Flk-1+CD31+) into the gut (n=8-14 per group, p&amp;lt;0.0001), without modifying levels of proinflammatory or immune cell phenotypes. The CAC depletion mediated by ACE2 deficiency was associated with increased levels of gut bacteria in the blood as assessed by SourceTracker analysis (n=9 per group). These data suggested a loss of the protective arm of RAS contributes to the depletion of CACs and disruption of gut-vascular barrier that may promote bacterial translocation in diabetes. Disclosure Y. Duan: None. D. Feng: None. E. Beli: None. S. Li Calzi: None. A.F. Longhini: None. R. Lamendella: None. J.R. Wright: None. T.A. Markel: None. A.G. Obukhov: None. M.B. Grant: None.