Angiotensin-converting Enzyme 2 Knockout Mice Research Articles

ACE2 Rescues Sepsis-Associated Encephalopathy by Reducing Inflammation, Oxidative Stress, and Neuronal Apoptosis via the Nrf2/Sestrin2 Signaling Pathway.

Neuroinflammation and oxidative stress contribute to the progression of sepsis-associated encephalopathy (SAE). Angiotensin-converting enzyme 2 (ACE2) is considered to be a neuroprotective factor due to its anti-inflammatory and antioxidant properties. However, the role of ACE2 on myeloid cells in regulating SAE and the underlying mechanism warrants further exploration. SAE was induced in ACE2 transgenic (TG), knockout (KO), and bone marrow (BM) chimeric mice by cecal ligation and puncture (CLP). The expression levels of apoptosis-, oxidation- and neuroinflammation-associated mediators and morphological changes were monitored by quantitative real-time PCR analyses and histological examinations in the cortex of septic mice. The contents of angiotensin (Ang) II and Ang-(1-7) along with the activity of ACE2 were examined with commercial kits. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and Sestrin2 was detected by immunoblotting analysis. Our results indicated that the expression of cortical ACE2 was significantly reduced in the early phase of CLP-induced sepsis. Moreover, ACE2 overexpression in TG mice conferred neuroprotection against sepsis, as evidenced by alleviated neuronal apoptosis, oxidative stress, and proinflammatory M1-like microglial polarization, accompanied by upregulation of the Ang-(1-7), Nrf2, and Sestrin2 protein levels. Conversely, ACE2 deficiency in KO mice exacerbated SAE. The neuroprotective effects of ACE2 were further confirmed in wild-type mice transplanted with ACE2-TG and KO BM cells. Therefore, our data suggest that myeloid ACE2 exerts a protective role in the pathogenesis of SAE, potentially by activating Ang-(1-7)-Nrf2/sestrin2 signaling pathway, and highlight that upregulating ACE2 expression and activity may represent a promising approach for the treatment of SAE in patients with sepsis.

Untargeted metabolomics uncovers metabolic dysregulation and tissue sensitivity in ACE2 knockout mice

Angiotensin-converting enzyme 2 (ACE2) polymorphisms are associated with increased risk of type 2 diabetes mellitus (T2DM), obesity and dyslipidemia, which have been determined in various populations. Consistently, ACE2 knockout (ACE2 KO) mice display damaged energy metabolism in multiple tissues, especially the key metabolic tissues such as liver, skeletal muscle and epididymal white adipose tissue (eWAT) and show even more severe phenotype under high-fat diet (HFD) induced metabolic stress. However, the effects of ACE2 on global metabolomics profiling and the tissue sensitivity remain unclear. To understand how tissues independently and collectively respond to ACE2, we performed untargeted metabolomics in serum in ACE2 KO and control wild type (WT) mice both on normal diet (ND) and HFD, and in three key metabolic tissues (liver, skeletal muscle and eWAT) after HFD treatment. The results showed significant alterations in metabolic profiling in ACE2 KO mice. We identified 275 and 168 serum metabolites differing significantly between WT and ACE2 KO mice fed on ND and HFD, respectively. And the altered metabolites in the ACE2 KO group varied from 90 to 196 in liver, muscle and eWAT. The alterations in ND and HFD serum were most similar. Compared with WT mice, ACE2 KO mice showed an increase in N-phenylacetylglutamine (PAGln), methyl indole-3-acetate, 5-hydroxytryptophol, cholic acid, deoxycholic acid and 12(S)-HETE, while LPC (19:0) and LPE (16:1) decreased. Moreover, LPC (20:0), LPC (20:1) and PC (14:0e/6:0) were reduced in both ND and HFD serum, paralleling the decreases identified in HFD skeletal muscle. Interestingly, DL-tryptophan, indole and Gly-Phe decreased in both ND and HFD serum but were elevated in HFD liver of ACE2 KO mice. A low level of l-ergothioneine was observed among liver, muscle, and epididymal fat tissue of ACE2 KO mice. Pathway analysis demonstrated that different tissues exhibited different dysregulated metabolic pathways. In conclusion, these results revealed that ACE2 deficiency leads to an overall state of metabolic distress, which may provide a new insight into the underlying pathogenesis in metabolic disorders in both ACE2 KO mice and in patients with certain genetic variant of ACE2 gene.

ACE2 Expressed on Myeloid Cells Alleviates Sepsis-Induced Acute Liver Injury via the Ang-(1-7)-Mas Receptor Axis.

Sepsis-induced acute liver injury (ALI) is common in intensive care units. Angiotensin-converting enzyme 2 (ACE2) plays a vital role in hepatic fibrosis and steatosis; however, its role in sepsis-induced ALI remains unclear. This study found that hepatic ACE2 expression in cecal ligation and puncture (CLP)-treated mice significantly decreased 24h after CLP. ACE2-transgenic (TG) mice exhibited a significant improvement in CLP-induced ALI, accompanied by the inhibition of hepatocyte apoptosis, oxidative stress, and inflammation, while ACE2-knockout mice demonstrated an opposite trend. During sepsis-induced ALI, ACE2-TG could also elevate the Ang-(1-7) and Mas receptor (MasR) levels in liver tissues. Interestingly, the MasR inhibitor A779 abrogated the favorable effects of ACE2 on CLP-induced ALI. In a bone marrow transplantation experiment, the ACE2-TG transplantation group showed significantly improved inflammation and liver dysfunction, less hepatocyte apoptosis, and reduced oxidative stress after CLP compared with the wild-type transplantation group. In contrast, the ACE2-knockout group showed poor inflammatory response and liver dysfunction, significantly more hepatocyte apoptosis, and elevated oxidative stress than the wild-type transplantation group after CLP. ACE2 protects against sepsis-induced ALI by inhibiting hepatocyte apoptosis, oxidative stress, and inflammation via the Ang-(1-7)-Mas receptor axis. Thus, targeting ACE2 may be a promising novel strategy for preventing and treating sepsis-induced ALI.

Myeloid ACE2 protects against septic hypotension and vascular dysfunction through Ang-(1–7)-Mas-mediated macrophage polarization

Angiotensin converting enzyme 2 (ACE2) is a new identified member of the renin-angiotensin-aldosterone system (RAAS) that cleaves angiotensin II (Ang II) to Ang (1–7), which exerts anti-inflammatory and antioxidative activities via binding with Mas receptor (MasR). However, the functional role of ACE2 in sepsis-related hypotension remains unknown. Our results indicated that sepsis significantly reduced blood pressure and led to disruption between ACE-Ang II and ACE2-Ang (1–7) balance. ACE2 knock-in mice exhibited improved sepsis-induced mortality, hypotension and vascular dysfunction, while ACE2 knockout mice exhibited the opposite effects. Bone marrow transplantation and in vitro experiments confirmed that myeloid ACE2 exerted a protective role by suppressing oxidative stress, NO production and macrophage polarization via the Ang (1–7)-MasR–NF–κB and STAT1 pathways. Thus, ACE2 on myeloid cells could protect against sepsis-mediated hypotension and vascular dysfunction, and upregulating ACE2 may represent a promising therapeutic option for septic patients with hypotension.

Involvement of ACE2 in the intestinal transport of amino acids: Possible health and nutritional consequences in altered expression

Background:Absorption of neutral amino acids across the intestinal cells is mediated by the broad neutral amino acid transporter B0AT1. Intestinal expression of B0AT1 depends on the coexistence with the membrane-attached angiotensin-converting enzyme 2 (ACE2) which binds to the B0AT1 or the sodium-dependent imino-acid transporter 1 (SIT1) in the intestinal epithelial cells. Objectives:The objective of this article is to provide an overview of the involvement of ACE2 in the transport of amino acids from the intestinal lumen and possible harmful effects on health and nutrition due to altered ACE2 expression. Methods: PubMed, Scopus, and Google Scholar were searched using the keywords ACE2 paired with intestinal and gut B0AT1, SIT1, amino acid transport or absorption, health, and nutrition. Expression of B0AT1 in the intestine was completely absent in ACE2 knockout mice. Lack of ACE2 and B0AT1 expression lead to low plasma and muscle tryptophan, niacin deficiency, decreased intestinal antimicrobial peptide, and increased susceptibility to inflammatory bowel disease in animal models. Results: Expression of ACE2 in the small intestine and its association with amino acid transporters play a crucial role in the absorption of amino acids, maintenance of structural and functional gut integrity and overall health and nutrition status of an individual. Bangladesh Journal of Medical Science Vol. 22 No. 04 October’23 Page : 729-733

420 A novel mouse model of COVID-19

OBJECTIVES/GOALS: Rodents are the most widely used experimental animals to study disease mechanisms due to their availability and cost-effectiveness. An international drive to investigate the pathophysiology of COVID-19 is inhibited by the resistance of rats and mice to SARS-CoV-2 infection. Our goal was to establish an appropriate small animal model. METHODS/STUDY POPULATION: To recreate the cytokine storm that is associated with COVID-19, we injected angiotensin converting enzyme 2 knockout (ACE2KO) mice (C57BI/6 strain) with lipopolysaccharide (LPS) intraperitoneally and measured the expression of multiple cytokines as a function of time and LPS dose. We then chose a minimum dose (500ug/kg) and time (3h) when multiple cytokines were elevated to measure lung injury scores using a point-counting technique on tissue sections stained with hematoxylin and eosin. The data are expressed as mean percentage of grid points lying within the peribronchial and superficial area in up to 20 fields. Percentage of peribronchial and superficial intrapulmonary hemorrhage, congestion, neutrophil infiltration and area of alveolar space were all assessed. RESULTS/ANTICIPATED RESULTS: Compared to the wildtype group (WT-G), the LPS-injected ACE2KO mice (LPS-G) exhibited a higher percentage of peribronchial intrapulmonary hemorrhage [(%): LPS-G, 10.56 ± 2.06 vs. WT-G, 5.59 ± 0.53; p DISCUSSION/SIGNIFICANCE: Establishing this novel mouse model of COVID-19 will facilitate studies investigating tissue-specific mechanisms of pathogenesis in this disease. This model can also be used to discover novel therapeutic targets and the design of clinical trials focusing on diagnostics, treatments and outcomes in COVID-19.

Intestinal ACE2 regulates glucose metabolism in diet-induced obese mice through a novel gut-islet axis mediated by tryptophan.

Angiotensin-converting enzyme 2 (ACE2) plays a vital role in regulating intestinal tryptophan (Trp) transport and maintaining Trp homeostasis. This study aimed to investigate the functional relationship between intestinal ACE2 and Trp in the regulation of glucose metabolism under metabolic stress. ACE2-knockout mice and mice with adeno-associatedvirus-mediated overexpression of ACE2 were fed with a high-fat diet for 12 weeks to establish a high-fat-induced metabolic stress model. They were subjected to a Trp gavage intervention for another 4 weeks. Here, it is reported that ACE2 regulates intestinal Trp absorption by stabilizing neutral amino acid transporter B0 AT1. Notably, in ACE2-knockout mice, it was found that B0 AT1 and serum Trp levels were significantly reduced, which was not reversed by Trp supplementation. However, mice receiving adeno-associatedvirus-ACE2 did the opposite and showed significantly improved glycolipid metabolism. It was then confirmed that Trp potentiated glucagon-like peptide 1 production from intestinal and islet α-cells. Meanwhile, Trp-treated MIN6 cells ameliorated mitochondrial function and safely guarded MIN6 cells against reactive oxygen species exposure. This study highlights an essential role of ACE2 in the maintenance of systemic metabolism to optimize the function of the islets through a novel gut-islet axis mediated by Trp. These results provide proof-of-concept evidence for treating obesity and diabetes.

Molecular imaging on ACE2-dependent transocular infection of coronavirus.

A transocular infection has been proved as one of the main approaches that severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) invades the body, and angiotensin‐converting enzyme 2 (ACE2) plays a key role in this procedure. Dynamic and quantitative details on virus distribution are lacking for virus prevention and drug design. In this study, a radiotraceable pseudovirus packed with an enhanced green fluorescent protein (EGFP) gene, 125I‐CoV, was prepared and inoculated in the unilateral eye of humanized ACE2 (hACE2) mice or ACE2‐knockout (ACE2‐KO) mice. Single‐photon emission computed tomography/computed tomography images were acquired at multiple time points to exhibit ACE2‐dependent procedures from invasion to clearance. Positron emission tomography (PET) and western blot were performed to quantify ACE2 expression and verify the factors affecting transocular infection. For the transocular infection of coronavirus (CoV), the renin–angiotensin–aldosterone system (RAAS), lungs, intestines, and genital glands were the main targeted organs. Due to the specific anchor to ACE2‐expressed host cells, virus concentrations in genital glands, liver, and lungs ranked the top three most and stabilized at 3.75 ± 0.55, 3.30 ± 0.25, and 2.10 ± 0.55% inoculated dose (ID)/mL at 48 h post treatment. Meanwhile, ACE2‐KO mice had already completed the in vivo clearance. In consideration of organ volumes, lungs (14.50 ± 3.75%ID) and liver (10.94 ± 0.71%ID) were the main in‐store reservoirs of CoV. However, the inoculated eye (5.52 ± 1.85%ID for hACE2, 5.24 ± 1.45%ID for ACE2‐KO, p > 0.05) and the adjacent brain exhibited ACE2‐independent virus infection at the end of 72 h observation, and absolute amount of virus played a key role in host cell infection. These observations on CoV infection were further manifested by infection‐driven intracellular EGFP expression. ACE2 PET revealed an infection‐related systematic upregulation of ACE2 expression in the organs involved in RAAS (e.g., brain, lung, heart, liver, and kidney) and the organ that was of own local renin–angiotensin system (e.g., eye). Transocular infection of CoV is ACE2‐dependent and constitutes the cause of disturbed ACE2 expression in the host. The brain, genital glands, and intestines were of the highest unit uptake, potentially accounting for the sequelae. Lungs and liver were of the highest absolute amount, closely related to the respiratory diffusion and in vivo duplication. ACE2 expression was upregulated in the short term after infection with CoV. These visual and quantitative results are helpful to fully understanding the transocular path of SARS‐CoV‐2 and other CoVs.

Angiotensin-converting enzyme 2 particapates in ozone-induced lung inflammation and airway remodeling in mice

To investigate the roles of angiotensin-converting enzyme 2 (ACE2) in ozone-induced pulmonary inflammation and airway remodeling in mice. Sixteen wild-type (WT) C57BL/6J mice and 16 ACE2 knock-out (KO) mice were exposed to either filtered air or ozone (0.8 ppm) for 3 h per day for 5 consecutive days. Masson's staining and HE staining were used to observe lung pathologies. Bronchoalveolar lavage fluid (BALF) was collected and the total cell count was determined. The total proteins and cytokines in BALF were determined by BCA and ELISA method. The transcription levels of airway remodeling-related indicators in the lung tissues were detected using real-time quantitative PCR. The airway resistance of the mice was measured using a small animal ventilator with methacholine stimulation. Following ozoneexposure ACE2 KO mice had significantly higher lung pathological scores than WT mice (P < 0.05). Masson staining results showed that compared with ozone-exposed WT mice, ozone-exposed ACE2 KO mice presented with significantly larger area of collagen deposition in the bronchi [(19.62±3.16)% vs (6.49±1.34)%, P < 0.05] and alveoli [(21.63±3.78)% vs (4.44±0.99)%, P < 0.05]. The total cell count and total protein contents in the BALF were both higher in ozone-exposed ACE2 KO mice than in WT mice, but these differences were not statistically significant (P > 0.05). The concentrations of IL-6, IL-1β, TNF-α, CXCL1/KC and MCP-1 in the BALF were all higher in ozone-exposed ACE2 KO mice than in ozone-exposed WT mice, but only the difference in IL-1β was statistically significant (P < 0.05). The transcription levels of MMP-9, MMP-13, TIMP 4, COL1A1, and TGF-β in the lung tissues were all significantly higher in ozone-exposed ACE2 KO mice (P < 0.01). No significant difference was found in airway resistance between ozone-exposed ACE KO mice and WT mice after challenge with 0, 10, 25, or 100 mg/mL of methacholine. ACE2 participates in ozone-induced lung inflammation and airway remodeling in mice.

Angiotensin-Converting Enzyme 2 Activation Mitigates Behavioral Deficits and Neuroinflammatory Burden in 6-OHDA Induced Experimental Models of Parkinson's Disease.

Hypertension is reported to cause major brain disorders including Parkinson's disease (PD), apart from cardiovascular and chronic kidney disorders. Considering this, for the first time, we explored the effect of modulation of the ACE2/Ang (1-7)/MasR axis using diminazene aceturate (DIZE), an ACE2 activator, in 6-hydroxydopamine (6-OHDA) induced PD model. We found that DIZE treatment improved neuromuscular coordination and locomotor deficits in the 6-OHDA induced PD rat model. Further, the DIZE-mediated activation of ACE2 led to increased tyrosine hydroxylase (TH) and dopamine transporters (DAT) expression in the rat brain, indicating the protection of dopaminergic (DAergic) neurons from 6-OHDA induced neurotoxicity. Moreover, 6-OHDA induced activation of glial cells (astrocytes and microglia) and release of neuroinflammatory mediators were attenuated by DIZE treatment in both in vitro as well as in vivo models of PD. DIZE exerted its effect by activating ACE2 that produced Ang (1-7), a neuroprotective peptide. Ang (1-7) conferred its neuroprotective effect upon binding with the G-protein-coupled MAS receptor that led to the upregulation of cell survival proteins while downregulating apoptotic proteins. Importantly, these findings were further validated by using A-779, a MasR antagonist. The result showed that treatment with A-779 reversed the antioxidative and anti-inflammatory effects of DIZE by decreasing glial activation and neuroinflammatory markers. Although the role of ACE2 in PD pathology needs to be additionally confirmed using transgenic models in either ACE2 overexpressing or knockout mice, still, our study demonstrates that enhancing ACE2 activity could be a novel approach for ameliorating PD pathology.

Role of angiotensin-converting enzyme 2 in fine particulate matter-induced acute lung injury

Fine particulate matter (PM2.5) pollution poses significant health concerns worldwide and can cause respiratory diseases. However, how it causes health problems is still poorly understood. Angiotensin-converting enzyme (ACE)2 is a terminal carboxypeptidase implicated in the functions of renin-angiotensin system (RAS) and plays a crucial role in the control of lung inflammation. To investigate whether ACE2 functions in PM2.5-induced lung inflammation, wild-type (WT) C57BL/6J mice and ACE2 knock-out (KO) mice were intratracheally instilled with PBS or PM2.5 suspension for 3 consecutive days, respectively. The concentrations of cytokines in bronchoalveolar lavage fluid (BALF) were determined by ELISA. The expression of ACE2 and ACE and activation of inflammatory signaling pathways in lung tissues were evaluated by immunofluorescence staining and Western blotting. We found that PM2.5 exposure increased ACE2 expression. Loss of ACE2 significantly elevated the levels of total proteins, total cells, and the concentrations of MCP-1, IL-1β in BALF after PM2.5 challenge. Additionally, loss of ACE2 enhanced lung pathologies, airway resistance, and inflammatory signaling activation. Collectively, loss of ACE2 exacerbates PM2.5-induced acute lung injury in mice.

ACE2 pathway regulates thermogenesis and energy metabolism.

Identification of key regulators of energy homeostasis holds important therapeutic promise for metabolic disorders, such as obesity and diabetes. ACE2 cleaves angiotensin II (Ang II) to generate Ang-(1-7) which acts mainly through the Mas1 receptor. Here, we identify ACE2 pathway as a critical regulator in the maintenance of thermogenesis and energy expenditure. We found that ACE2 is highly expressed in brown adipose tissue (BAT) and that cold stimulation increases ACE2 and Ang-(1-7) levels in BAT and serum. Ace2 knockout mice (Ace2-/y) and Mas1 knockout mice (Mas1-/-) displayed impaired thermogenesis. Mice transplanted with brown adipose tissue from Mas1-/- display metabolic abnormalities consistent with those seen in the Ace2 and Mas1 knockout mice. In contrast, impaired thermogenesis of Leprdb/db obese diabetic mice and high-fat diet-induced obese mice were ameliorated by overexpression of Ace2 or continuous infusion of Ang-(1-7). Activation of ACE2 pathway was associated with improvement of metabolic parameters, including blood glucose, lipids, and energy expenditure in multiple animal models. Consistently, ACE2 pathway remarkably enhanced the browning of white adipose tissue. Mechanistically, we showed that ACE2 pathway activated Akt/FoxO1 and PKA pathway, leading to induction of UCP1 and activation of mitochondrial function. Our data propose that adaptive thermogenesis requires regulation of ACE2 pathway and highlight novel potential therapeutic targets for the treatment of metabolic disorders.

Ang-(1\u20137) protects skeletal muscle function in aged mice

BackgroundThe angiotensin-converting enzyme 2 (ACE2)/angiotensin 1–7 (Ang-(1–7)) axis has been shown to protect against the age-associated decline in skeletal muscle function. Here, we investigated the protective effects of ACE2 in mitigating the age-associated decline of skeletal muscle function and to identify the potential underlying molecular mechanisms.MethodsWe measured the expression levels of Ang-(1–7) in C57BL/6J mice of different ages and correlated these levels with measures of skeletal muscle function. We also investigated the expression of myocyte enhancer factor 2 A (MEF2A) in ACE2 knockout (ACE2KO) mice and its relationship with muscle function. We then treated aged ACE2KO mice for four weeks with Ang-(1–7) and characterized the levels of MEF2A and skeletal muscle function before and after treatment. We assessed the impact of Ang-(1–7) on the growth and differentiation of C2C12 cells in vitro and assessed changes in expression of the glucose transporter type 4 (Glut4).ResultsAged mice showed reduced skeletal muscle function and levels of Ang-(1–7) expression in comparison to young and middle-aged mice. In ACE2KO mice, skeletal muscle function and MEF2A protein expression were significantly lower than in age-matched wild-type (WT) mice. After one month of Ang-(1–7) treatment, skeletal muscle function in the aged ACE2KO mice improved, while MEF2A protein expression was similar to that in the untreated group. In C2C12 cells, Ang-(1–7) was shown to promote along with the upregulated expression of Glut4.ConclusionsThe ACE2/ Ang-(1–7) axis has a protective function in skeletal muscle and administration of exogenous Ang-(1–7) can delay the age-related decline in the function of skeletal muscle.

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.

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.

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.

ACE2 modulates glucose homeostasis through GABA signaling during metabolic stress.

The angiotensin-converting enzyme 2 (ACE2)/angiotensin 1-7 (A1-7)/MAS axis and glutamate decarboxylase 67 (GAD67)/gamma-aminobutyric acid (GABA) signal both exist in the islet and play important roles in regulating blood glucose metabolism. It has been reported that the activation of ACE2 in the brain increases GABA expression to improve biological effects; however, it is unclear whether there is functional correlation between the ACE2/A1-7/MAS axis and GAD67/GABA signal in the islet. In this study, we showed that the ACE2/A1-7/MAS and GABA signaling systems decreased in the islet of different metabolic stress models. In ACE2-knockout mice, we found that GAD67 and GABA expression decreased significantly, which was reversed by exogenous administration of A1-7. Furthermore, A1-7 mediated PDX1 and AKT activation was inhibited by allylglycine (a specific GAD67 inhibitor) in MIN6 cells. Moreover, giving A1-7 and GABA could significantly reduce beta-cell dedifferentiation and improved glucose metabolism during metabolic stress in vivo and in vitro. In conclusion, our study reveals that the ACE2/A1-7/MAS axis improves beta-cell function through regulating GAD67/GABA signal in beta cells and that up-regulating the ACE2/A1-7/MAS axis and GABA signals delays the development of obesity-induced diabetes.

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.

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.