The two axes of the renin–angiotensin system include the classical ACE/Ang II/AT1 axis and the counter-regulatory ACE2/Ang-(1-7)/Mas1 axis. ACE2 is a multifunctional monocarboxypeptidase responsible for generating Ang-(1-7) from Ang II. ACE2 is important in the vascular system where it is found in arterial and venous endothelial cells and arterial smooth muscle cells in many vascular beds. Among the best characterized functions of ACE2 is its role in regulating vascular tone. ACE2 through its effector peptide Ang-(1-7) and receptor Mas1 induces vasodilation and attenuates Ang II-induced vasoconstriction. In endothelial cells activation of the ACE2/Ang-(1-7)/Mas1 axis increases production of the vasodilator’s nitric oxide and prostacyclin’s and in vascular smooth muscle cells it inhibits pro-contractile and pro-inflammatory signaling. Endothelial ACE2 is cleaved by proteases, shed into the circulation and measured as soluble ACE2. Plasma ACE2 activity is increased in cardiovascular disease and may have prognostic significance in disease severity. In addition to its enzymatic function, ACE2 is the receptor for severe acute respiratory syndrome (SARS)-coronavirus (CoV) and SARS-Cov-2, which cause SARS and coronavirus disease-19 (COVID-19) respectively. ACE-2 is thus a double-edged sword: it promotes cardiovascular health while also facilitating the devastations caused by coronaviruses. COVID-19 is associated with cardiovascular disease as a risk factor and as a complication. Mechanisms linking COVID-19 and cardiovascular disease are unclear, but vascular ACE2 may be important. This review focuses on the vascular biology and (patho)physiology of ACE2 in cardiovascular health and disease and briefly discusses the role of vascular ACE2 as a potential mediator of vascular injury in COVID-19.

Rationale: Even in antiretroviral therapy-treated patients, HIV continues to play a pathogenic role in cardiovascular diseases. A possible cofactor may be persistence of the early HIV response gene Nef, which we have demonstrated recently to persist in the lungs of HIV+ patients on antiretroviral therapy. Previously, we have reported that HIV strains with Nef, but not Nef-deleted HIV strains, cause endothelial proinflammatory activation and apoptosis. Objective: To characterize mechanisms through which HIV-Nef leads to the development of cardiovascular diseases using ex vivo tissue culture approaches as well as interventional experiments in transgenic murine models. Methods and Results: Extracellular vesicles derived from both peripheral blood mononuclear cells and plasma from HIV+ patient blood samples induced human coronary artery endothelial cells dysfunction. Plasma-derived extracellular vesicles from antiretroviral therapy+ patients who were HIV-Nef+ induced significantly greater endothelial apoptosis compared with HIV-Nef-plasma extracellular vesicles. Both HIV-Nef expressing T cells and HIV-Nef-induced extracellular vesicles increased transfer of cytosol and Nef protein to endothelial monolayers in a Rac1-dependent manner, consequently leading to endothelial adhesion protein upregulation and apoptosis. HIV-Nef induced Rac1 activation also led to dsDNA breaks in endothelial colony forming cells, thereby resulting in endothelial colony forming cell premature senescence and endothelial nitric oxide synthase downregulation. These Rac1-dependent activities were characterized by NOX2-mediated reactive oxygen species production. Statin treatment equally inhibited Rac1 inhibition in preventing or reversing all HIV-Nef-induction abnormalities assessed. This was likely because of the ability of statins to block Rac1 prenylation as geranylgeranyl transferase inhibitors were effective in inhibiting HIV-Nef-induced reactive oxygen species formation. Finally, transgenic expression of HIV-Nef in endothelial cells in a murine model impaired endothelium-mediated aortic ring dilation, which was then reversed by 3-week treatment with 5 mg/kg atorvastatin. Conclusions: These studies establish a mechanism by which HIV-Nef persistence despite antiretroviral therapy could contribute to ongoing HIV-related vascular dysfunction, which may then be ameliorated by statin treatment.

Mas and ET B receptors physically interact in endothelial cells (ECs) and are involved in the protective actions of angiotensin 1-7 (Ang (1-7)). We assessed whether the MAS/ET B R interaction plays a role in EC signalling and whether strategies to enhance MAS/ET B R association influence vascular responses. Human ECs were stimulated with Ang (1-7) (10 -7 M) in the presence/absence of A779 (Mas receptor antagonist, 10 -5 M) and BQ788 (ET B R antagonist, 10 -5 M). Protein expression and signalling activation were assessed by immunoblotting. NO production was evaluated by DAF-FM fluorescence and ROS production by chemiluminescence (superoxide anion) or amplex red (hydrogen peroxide (H 2 O 2 )). mRNA expression was assessed by qPCR. Endothelial function was assessed in mouse intact arteries by myography. Ang (1-7), through Mas and ET B R induced phosphorylation of eNOS (35%); followed by an increase in NO production (2.0 fold) (p<0.05 vs ctl). High throughput screening of protein:protein interaction compounds in an in-house library identified 23 potential enhancers of the MAS/ET B R interaction. Fluorescence polarization assays were used to further select the most potent enhancers and define their working concentration for testing in ECs (Enh1-4: 10 -5 M). Enh4 increased superoxide anion (55.6±26.3% vs ctl, p<0.05) and H 2 O 2 production (54.7±11.1% vs ctl, p<0.05), while Enh3 increased H 2 O 2 generation (48.1±15.4% vs ctl, p<0.05) in ECs. Moreover, Enh4 increased Nrf2 (3.0 fold), Sod1 (2.0 fold) and Nqo1 (3.1 fold) mRNA expression (p<0.05 vs ctl). Enh3 and Enh4 increased NO production (Enh3: 21.2±7.4%; Enh4: 23.6±8.2% vs veh, p<0.05) in ECs. Acetylcholine (Ach) curves were performed to assess endothelium-dependent relaxation in the absence and presence of enhancers. Enh4 increased ACh-induced relaxation (Emax%: 96.7±4.6 vs ctl: 70.4±3.3, p<0.05), while other enhancers did not improve endothelial function. Taken together, increasing MAS/ET B R interaction with specific enhancers augments protective signalling in ECs and promotes endothelial-dependent vasorelaxation, particularly with Enh4. In conclusion, enhancing interactions between MasR and ET B R may be a new vasoprotective strategy to improve vascular function in cardiovascular disease.

We previously reported that Nox5 regulates contraction through mechanisms involving ROS and Ca 2+ channels in the endoplasmic reticulum (ER). In young mice expressing human Nox5 in VSMCs (Nox5+SM22+), we observed hypercontractility, without changes in blood pressure. Here we tested the hypothesis that Nox5 influences ER Ca 2+ homeostasis and vascular function and that Nox5 amplifies aging-associated vascular dysfunction through processes involving ER stress. Female WT and Nox5+SM22+ mice aged 20 and 35weeks were studied. Blood pressure (BP) was assessed by tail-cuff and vascular function/structure by myography. BP was similar in all groups. Vascular contraction to U46619, a TXA2 analogue, was increased in aged Nox5+SM22+ (EMax - %KCl: 114±2.8 vs WT 95±2.4, p<0.05). Hypercontractility was reversed by NAC (antioxidant - 0.01 mM, EMax: 92±5%), melittin (Nox5 inhibitor - 0.1 μM, EMax 92±3.2%) and dantrolene (RyR Ca 2+ channel blocker - 0.01 mM, EMax: 67±4.2%) (p<0.05). VSMCs isolated from 20 and 35 wk WT and Nox5+SM22+ mice were used to study molecular mechanisms whereby Nox5 influences the contractile machinery, focusing on the ER. Expression of BIP, a marker of ER stress, was increased only in VSMCs from aged Nox5+SM22+ mice (AU: 0.13±0.01 vs WT 0.05±0.002, p<0.05). 4-PBA, an inhibitor of ER stress (1 mM), reversed the hypercontractile responses in 35 wk Nox5+SM22+ mice (EMax: 87±3%, p<0.05). We identified calreticulin, important in ER Ca 2+ homeostasis and channel function, as a molecular target of Nox5. As Nox regulates signalling by oxidation, we assessed calreticulin oxidation by pulldown using dimedone based probe (DCP-Bio). Calreticulin oxidation was increased in mesenteric arteries, aorta and VSMCs from 35 week Nox5+SM22+. Moreover, expression of calreticulin (23.5±2%) and BIP (27.6±9%) was increased by U46619 in VSMCs from Nox5+SM22+ (p<0.05); an effect inhibited by melittin and 4-PBA. Our study highlights molecular mechanisms whereby Nox5 regulates contraction, through oxidation of calcium regulatory proteins, such as calreticulin, and ER stress in aged Nox5 mice. These age related changes may predispose Nox5 mice to cardiovascular damage when challenged with factors associated with hypertension.