SARS-CoV-2 Spike Protein Regulation of Angiotensin Converting Enzyme 2 and Tissue Renin-Angiotensin Systems: Influence of Biologic Sex.

Angiotensin converting enzyme 2 (ACE2) is an enzyme that limits activity of the renin-angiotensin system (RAS) and also serves as a receptor for the SARS-CoV-2 Spike (S) protein. Binding of S protein to ACE2 causes internalization which activates local RAS. ACE2 is on the X chromosome and its expression is regulated by sex hormones. In this study, we defined ACE2 mRNA abundance and examined effects of S protein on ACE2 activity and/or angiotensin II (AngII) levels in pivotal tissues (lung, adipose) from male and female mice. In lung, ACE2 mRNA abundance was reduced following gonadectomy (GDX) of male and female mice and was higher in XX than XY mice of the Four Core Genotypes (FCG). Reductions in lung ACE2 mRNA abundance by GDX occurred in XX, but not XY FCG female mice. Lung mRNA abundance of ADAM17 and TMPRSS2, enzymes that shed cell surface ACE2 and facilitate viral cell entry, was reduced by GDX in male but not female mice. For comparison, adipose ACE2 mRNA abundance was higher in female than male mice and higher in XX than XY FCG mice. Adipose ADAM17 mRNA abundance was increased by GDX of male and female mice. S protein reduced ACE2 activity in alveolar type II epithelial cells and 3T3-L1 adipocytes. Administration of S protein to male and female mice increased lung AngII levels and decreased adipose ACE2 activity in male but not female mice. These results demonstrate that sex differences in ACE2 expression levels may impact local RAS following S protein exposures.

Therapeutic Assessment of Combination Therapy with a Neprilysin Inhibitor and Angiotensin Type 1 Receptor Antagonist on Angiotensin II-Induced Atherosclerosis, Abdominal Aortic Aneurysms, and Hypertension.

Combined neprilysin (NEP) inhibition (sacubitril) and angiotensin type 1 receptor (AT1R) antagonism (valsartan) is used in the treatment of congestive heart failure and is gaining interest for other angiotensin II (AngII)-related cardiovascular diseases. In addition to heart failure, AngII promotes hypertension, atherosclerosis, and abdominal aortic aneurysms (AAAs). Similarly, NEP substrates or products have broad effects on the cardiovascular system. In this study, we examined NEP inhibition (with sacubitril) and AT1R antagonism (with valsartan) alone or in combination on AngII-induced hypertension, atherosclerosis, or AAAs in male low-density lipoprotein receptor-deficient mice. Preliminary studies assessed drug delivery via osmotic minipumps for simultaneous release of sacubitril and/or valsartan with AngII over 28 days. Mice were infused with AngII (1000 ng/kg per minute) in the absence (vehicle) or presence of sacubitril (1, 6, or 9 mg/kg per day), valsartan (0.3, 0.5, 1, 6, or 20 mg/kg per day), or the combination thereof (1 and 0.3, or 9 or 0.5 mg/kg per day of sacubitril and valsartan, respectively). Plasma AngII and renin concentrations increased 4-fold at higher valsartan doses, indicative of removal of AngII negative feedback on renin. Sacubitril doubled plasma AngII concentrations at lower doses (1 mg/kg per day). Valsartan dose-dependently decreased systolic blood pressure, aortic atherosclerosis, and AAAs of AngII-infused mice, whereas sacubitril had no effect on atherosclerosis or AAAs but reduced blood pressure of AngII-infused mice. Combination therapy with sacubitril and valsartan did not provide additive benefits. These results suggest limited effects of combination therapy with NEP inhibition and AT1R antagonism against AngII-induced hypertension, atherosclerosis, or AAAs. SIGNIFICANCE STATEMENT: The combination of valsartan (angiotensin type 1 receptor antagonist) and sacubitril (neprilysin inhibitor) did not provide benefit above valsartan alone on AngII-induced hypertension, atherosclerosis, or abdominal aortic aneurysms in low-density lipoprotein receptor-deficient male mice. These results do not support this drug combination in therapy of these AngII-induced cardiovascular diseases.

Monosomy X in Female Mice Influences the Regional Formation and Augments the Severity of Angiotensin II-Induced Aortopathies.

OBJECTIVE: Turner syndrome women (monosomy X) have high risk of aortopathies consistent with a role for sex chromosomes in disease development. We demonstrated that sex chromosomes influence regional development of Ang II (angiotensin II)-induced aortopathies in mice. In this study, we determined if the number of X chromosomes regulates regional development of Ang II-induced aortopathies. Approach and Results: We used females with varying numbers of X chromosomes (XX female mice [XXF] or XO female mice [XOF]) on an C57BL/6J (ascending aortopathies) or low-density lipoprotein receptor deficient (Ldlr-/-) background (descending and abdominal aortopathies) compared with XY males (XYM). To induce aortopathies, mice were infused with Ang II. XOF (C57BL/6J) exhibited larger percent increases in ascending aortic lumen diameters than Ang II-infused XXF or XYM. Ang II-infused XOF (Ldlr-/-) exhibited similar incidences of thoracic (XOF, 50%; XYM, 71%) and abdominal aortopathies (XOF, 83%; XYM, 71%) as XYM, which were greater than XXF (XXF, 0%). Abdominal aortic lumen diameters and maximal external diameters were similar between XOF and XYM but greater than XXF, and these effects persisted with extended Ang II infusions. Larger aortic lumen diameters, abdominal aortopathy incidence (XXF, 20%; XOF, 75%), and maximal aneurysm diameters (XXF, 1.02±0.17; XOF, 1.96±0.32 mm; P=0.027) persisted in ovariectomized Ang II-infused XOF mice. Data from RNA-seq demonstrated that X chromosome genes that escape X-inactivation (histone lysine demethylases Kdm5c and Kdm6a) exhibited lower mRNA abundance in aortas of XOF than XXF (P=0.033 and 0.024, respectively). Conversely, DNA methylation was higher in aortas of XOF than XXF (P=0.038). CONCLUSIONS: The absence of a second X chromosome promotes diffuse Ang II-induced aortopathies in females.

XX sex chromosome complement promotes atherosclerosis in mice.

Men and women differ in circulating lipids and coronary artery disease (CAD). While sex hormones such as estrogens decrease CAD risk, hormone replacement therapy increases risk. Biological sex is determined by sex hormones and chromosomes, but effects of sex chromosomes on circulating lipids and atherosclerosis are unknown. Here, we use mouse models to separate effects of sex chromosomes and hormones on atherosclerosis, circulating lipids and intestinal fat metabolism. We assess atherosclerosis in multiple models and experimental paradigms that distinguish effects of sex chromosomes, and male or female gonads. Pro-atherogenic lipids and atherosclerosis are greater in XX than XY mice, indicating a primary effect of sex chromosomes. Small intestine expression of enzymes involved in lipid absorption and chylomicron assembly are greater in XX male and female mice with higher intestinal lipids. Together, our results show that an XX sex chromosome complement promotes the bioavailability of dietary fat to accelerate atherosclerosis.

Pancreatic AT1aR Deficiency Decreases Insulin Secretion in Obese C57BL/6 Mice.

BACKGROUND: Previously, we demonstrated that obese mice have marked elevations in systemic concentrations of angiotensin II (AngII). Drugs that inhibit the renin-angiotensin system (RAS), including angiotensin type 1 receptor (AT1R) antagonists, have been reported to delay the onset of type 2 diabetes (T2D), suggesting improvements in insulin sensitivity or regulation of pancreatic insulin secretion. Pancreatic islets possess components of the RAS, including AT1R, but it is unclear if AngII acts at islets to regulate insulin secretion during the development of T2D. METHODS: We deleted AT1aR from pancreatic islets and examined effects on insulin secretion in mice fed a low-fat (LF) or high-fat (HF) diet. In separate studies, to exacerbate the system, we infused HF-fed mice of each genotype with AngII. RESULTS: Pancreatic AT1aR deficiency impaired glucose tolerance and elevated plasma glucose concentrations in HF, but not LF-fed mice. In HF-fed mice, high glucose increased insulin secretion from islets of AT1aRfl/fl, but not AT1aRpdx mice. In AngII-infused mice, following glucose challenge, plasma glucose or insulin concentrations were not significantly different between genotypes. Moreover, high glucose stimulated insulin secretion from islets of AT1aRfl/fl and AT1aRpdx mice, presumably related to weight loss, and improved insulin sensitivity in both groups of AngII-infused HF-fed mice. CONCLUSIONS: Our results suggest that during the adaptive response to insulin resistance from HF feeding, AngII promotes insulin secretion from islets through an AT1aR mechanism. These results suggest the timing of initiation of AT1R blockade may be important in the progression from prediabetes to T2D with ß-cell failure.

Sex Chromosome Complement Defines Diffuse Versus Focal Angiotensin II-Induced Aortic Pathology.

OBJECTIVE: Aortic pathologies exhibit sexual dimorphism, with aneurysms in both the thoracic and abdominal aorta (ie, abdominal aortic aneurysm [AAA]) exhibiting higher male prevalence. Women have lower prevalence of aneurysms, but when they occur, aneurysms progress rapidly. To define mechanisms for these sex differences, we determined the role of sex chromosome complement and testosterone on the location and progression of angiotensin II (AngII)-induced aortic pathologies. APPROACH AND RESULTS: We used transgenic male mice expressing Sry (sex-determining region Y) on an autosome to create Ldlr (low-density lipoprotein receptor)-deficient male mice with an XY or XX sex chromosome complement. Transcriptional profiling was performed on abdominal aortas from XY or XX males, demonstrating 1746 genes influenced by sex chromosomes or sex hormones. Males (XY or XX) were either sham-operated or orchiectomized before AngII infusions. Diffuse aortic aneurysm pathology developed in XY AngII-infused males, whereas XX males developed focal AAAs. Castration reduced all AngII-induced aortic pathologies in XY and XX males. Thoracic aortas from AngII-infused XY males exhibited adventitial thickening that was not present in XX males. We infused male XY and XX mice with either saline or AngII and quantified mRNA abundance of key genes in both thoracic and abdominal aortas. Regional differences in mRNA abundance existed before AngII infusions, which were differentially influenced by AngII between genotypes. Prolonged AngII infusions resulted in aortic wall thickening of AAAs from XY males, whereas XX males had dilated focal AAAs. CONCLUSIONS: An XY sex chromosome complement mediates diffuse aortic pathology, whereas an XX sex chromosome complement contributes to focal AngII-induced AAAs.

Female Mice With an XY Sex Chromosome Complement Develop Severe Angiotensin II-Induced Abdominal Aortic Aneurysms.

BACKGROUND: Abdominal aortic aneurysms (AAAs) are a deadly pathology with strong sexual dimorphism. Similar to humans, female mice exhibit far lower incidences of angiotensin II-induced AAAs than males. In addition to sex hormones, the X and Y sex chromosomes, and their unique complements of genes, may contribute to sexually dimorphic AAA pathology. Here, we defined the effect of female (XX) versus male (XY) sex chromosome complement on angiotensin II-induced AAA formation and rupture in phenotypically female mice. METHODS: Female low-density lipoprotein receptor (Ldlr) deficient mice with an XX or XY sex chromosome complement were infused with angiotensin II for 28 days to induce AAAs. Abdominal aortic lumen diameters were quantified by ultrasound, whereas AAA diameters were quantified at study end point. DNA microarrays were performed on abdominal aortas. To mimic males, female mice were administered a single dose of testosterone as neonates or as adults before angiotensin II infusions. RESULTS: Female Ldlr-/- deficient mice with an XX and XY sex chromosome complement had similar sex organ weights and low serum testosterone concentrations. Abdominal aortas from female XY mice selectively expressed Y chromosome genes, whereas genes known to escape X inactivation were higher in XX females. The majority of aortic gene differences in XY versus XX females fell within inflammatory pathways. AAA incidences doubled and aneurysms ruptured in XY females. AAAs from XY females exhibited inflammation, and plasma interleukin-1ß concentrations were increased in XY females. Moreover, aortas from XY females had augmented matrix metalloproteinase activity and increased oxidative stress. Last, testosterone exposure applied chronically, or as a single bolus at postnatal day 1, markedly worsened AAA outcomes in XY in comparison with XX adult females. CONCLUSIONS: An XY sex chromosome complement in phenotypic females profoundly influenced aortic gene expression profiles and promoted AAA severity. When XY females were exposed to testosterone, aneurysm rupture rates were striking. Mechanisms for augmented AAA severity in XY females include increased inflammation, augmented matrix metalloproteineases, and oxidative stress. Our results demonstrate that genes on the sex chromosomes regulate aortic vascular biology and contribute to sexual dimorphism of AAAs. Sex chromosome genes may serve as novel targets for sex-specific AAA therapeutics.

Exogenous 17-ß estradiol administration blunts progression of established angiotensin II-induced abdominal aortic aneurysms in female ovariectomized mice.

BACKGROUND: Abdominal aortic aneurysms (AAAs) occur predominately in males. However, AAAs in females have rapid growth rates and rupture at smaller sizes. Mechanisms contributing to AAA progression in females are undefined. We defined effects of ovariectomy, with and without 17-ß estradiol (E2), on progression of established angiotensin II (AngII)-induced AAAs in female mice. METHODS: We used neonatal testosterone exposures at 1 day of age to promote susceptibility to AngII-induced AAAs in adult female Ldlr (-/-) mice. Females were infused with AngII for 28 days to induce AAAs, and then stratified into groups that were sham, ovariectomized (Ovx, vehicle), or Ovx with E2 administration for 2 months of continued AngII infusions. Aortic lumen diameters were quantified by ultrasound and analyzed by linear mixed model, and maximal AAA diameters were analyzed by one-way ANOVA. Atherosclerosis was quantified en face in the aortic arch. AAA tissue sections were analyzed for cellular composition. We quantified effects of E2 on abdominal aortic smooth muscle cell (SMC) growth, α-actin and transforming growth factor-beta (TGF-ß) production, and wound healing. RESULTS: Serum E2 concentrations were increased significantly by E2. Aortic lumen diameters increased over time in sham-operated and Ovx (vehicle) females, but not in Ovx females administered E2. At day 70, E2 administration decreased significantly aortic lumen diameters compared to Ovx vehicle and sham-operated females. Compared to Ovx females (vehicle), maximal AAA diameters were reduced significantly by E2. AAA tissue sections from Ovx females administered E2 exhibited significant increases in α-actin and decreases in neutrophils compared to Ovx females administered vehicle. In abdominal aortic SMCs, E2 resulted in a concentration-dependent increase in α-actin, elevated TGF-ß, and more rapid wound healing. E2 administration to Ovx females also significantly reduced atherosclerotic lesions compared to sham-operated females. This effect was accompanied by significant reductions in serum cholesterol concentrations. CONCLUSIONS: E2 administration to Ovx females abolished progressive growth and decreased severity of AngII-induced AAAs. These effects were accompanied by increased SMC α-actin, elevated TGF-ß, and reduced neutrophils. Similarly, E2 administration reduced AngII-induced atherosclerosis. These results suggest that loss of E2 in post-menopausal females may contribute to progressive growth of AAAs.