Membrane-bound Angiotensin-converting Enzyme 2 Research Articles

Angiotensin-Converting Enzyme 2 Expression and Severity of SARS-CoV-2 Infection.

Angiotensin-converting enzyme 2 (ACE2), first discovered in 2000, serves as an important counterregulatory enzyme to the angiotensin II-mediated vasoconstrictive, pro-inflammatory, and pro-fibrotic actions of the renin-angiotensin system (RAS). Conversion of angiotensin II to the peptide angiotensin 1-7 (ANG 1-7) exerts protective vasodilatory, anti-inflammatory, and anti-fibrotic actions through interaction with the MasR receptor. There are many important considerations when noting the role of ACE2 in the pathogenesis and sequelae of COVID-19 infection. ACE2, in the role of COVID-19 infection, was recognized early in 2020 at the beginning of the pandemic as a cell membrane-bound and soluble binding site for the viral spike protein facilitating entering into tissue cells expressing ACE2, such as the lungs, heart, gut, and kidneys. Mechanisms exist that alter the magnitude of circulating and membrane-bound ACE2 (e.g., SARS-CoV-2 infection, viral variants, patient characteristics, chronic disease states, and the degree of cell surface expression of ACE2) and the influence these mechanisms have on the severity of disease and associated complications (e.g., respiratory failure, systemic inflammatory response syndrome, acute myocarditis, acute kidney injury). Several medications alter the ACE2 receptor expression, but whether these medications can influence the course of the disease and improve outcomes is unclear. In this review, we will discuss what is known about the interrelation of SARS-CoV-2, ACE2 and the factors that may contribute to the variability of its expression and potential contributors to the severity of COVID-19 infection.

Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection.

Angiotensin-converting enzyme 2 (ACE2) is the cell-surface receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). While its central role in Coronavirus Disease 2019 (COVID-19) pathogenesis is indisputable, there remains significant debate regarding the role of this transmembrane carboxypeptidase in the disease course. These include the role of soluble versus membrane-bound ACE2, as well as ACE2-independent mechanisms that may contribute to viral spread. Testing these roles requires in vivo models. Here, we report humanized ACE2-floxed mice in which hACE2 is expressed from the mouse Ace2 locus in a manner that confers lethal disease and permits cell-specific, Cre-mediated loss of function, and LSL-hACE2 mice in which hACE2 is expressed from the Rosa26 locus enabling cell-specific, Cre-mediated gain of function. Following exposure to SARS-CoV-2, hACE2-floxed mice experienced lethal cachexia, pulmonary infiltrates, intravascular thrombosis and hypoxemia-hallmarks of severe COVID-19. Cre-mediated loss and gain of hACE2 demonstrate that neuronal infection confers lethal cachexia, hypoxemia, and respiratory failure in the absence of lung epithelial infection. In this series of genetic experiments, we demonstrate that ACE2 is absolutely and cell-autonomously required for SARS-CoV-2 infection in the olfactory epithelium, brain, and lung across diverse cell types. Therapies inhibiting or blocking ACE2 at these different sites are likely to be an effective strategy towards preventing severe COVID-19.

Inhibiting the Deubiquitinase UCHL1 Reduces SARS-CoV-2 Viral Uptake by ACE2.

Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a significant public health burden with limited treatment options. Many β-coronaviruses, including SARS-CoV-2, gain entry to host cells through the interaction of SARS-CoV-2 spike protein with membrane-bound ACE2 (angiotensin-converting enzyme 2). Given its necessity for SARS-CoV-2 infection, ACE2 represents a potential therapeutic target in COVID-19. However, early attempts focusing on ACE2 in COVID-19 have not validated it as a druggable target nor identified other ACE2-related novel proteins for therapeutic intervention. Here, we identify a mechanism for ACE2 protein modulation by the deubiquitinase (DUB) enzyme UCHL1 (ubiquitin carboxyl-terminal hydrolase isozyme L1). ACE2 is constitutively ubiquitinated and degraded by the proteasome in lung epithelia. SARS-CoV-2 spike protein cellular internalization increased ACE2 protein abundance by decreasing its degradation. Using an siRNA library targeting 96 human DUBs, we identified UCHL1 as a putative regulator of ACE2 function as a viral receptor. Overexpressed UCHL1 preserved ACE2 protein abundance, whereas silencing of the DUB in cells destabilized ACE2 through increased polyubiquitination. A commercially available small molecule inhibitor of UCHL1 DUB activity decreased ACE2 protein concentrations coupled with inhibition of SARS-CoV-2 infection in epithelial cells. These findings describe a unique pathway of ACE2 regulation uncovering UCHL1 as a potential therapeutic target to modulate COVID-19 viral entry as a platform for future small molecule design and testing.

Angiotensin converting enzyme 2 level and its significance in COVID-19 and other diseases patients.

Angiotensin-converting enzyme 2 (ACE2) expressions and its modulation are of great interest as being a key receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) and the protective arm of the rennin-angiotensin axis, maintaining cardiovascular homeostasis. However, ACE2 expressions and their modulation in the healthy and disease background are yet to be explored. We performed a meta-analysis, extracting the data for ACE2 expression in human subjects with various diseases, including SARS-CoV2 infection without or with co-morbidity. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed. Out of 203 studies, 39 met the inclusion criteria with SARS-CoV2 patients without co-morbidity, SARS-CoV2 patients with co-morbidity, cardiovascular (CVD) patients, diabetes patients, kidney disorders patients, pulmonary disease patients, and other viral infections patients. Angiotensin-converting enzyme 2 expression was significantly increased in all diseases. There was an elevated level of ACE2, especially membrane-bound ACE2, in COVID-19 patients compared to healthy controls. A statistically significant increase in ACE2 expression was observed in CVD patients and patients with other viral diseases compared to healthy subjects. Moreover, subgroup analysis of ACE2 expression as soluble and membrane-bound ACE2 revealed a remarkable increase in membrane-bound ACE2 in CVD patients, patients with viral infection compared to soluble ACE2 and pooled standard mean difference (SMD) with the random-effects model was 0.37 and 2.23 respectively. It was observed that utilizing the ACE2 by SARS-CoV2 for its entry and its consequence leads to several complications. So there is a need to investigate the underlying mechanism along with novel therapeutic strategies.

Why Angiotensin II is a Poor Choice for Circulatory Support of Ventilated COVID-19 Patients Compared to Vasopressin

Early in the COVID-19 pandemic when it was first reported that SARS-CoV-2 used membrane-bound angiotensin-converting enzyme-2 (ACE2) as its receptor for entry into cells, warnings were raised against the use of angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) because of their potential to increase ACE2 expression. These reports ignored the adverse effects that the renin-angiotensin system (RAS) exerts on the cardiovascular system and kidneys via its primary hormone angiotensin (Ang) II acting upon AT1 receptors that could exacerbate the cytokine storm induced by SARS-CoV-2 1. At one point it was even recommended that COVID-19 patients suffering from cardiovascular collapse be administered Ang II to restore blood pressure rather than norepinephrine or vasopressin 2. An alternative strategy for treating COVID-19 was the administration of soluble ACE2 (sACE2) to act as a decoy receptor for the virus, misdirecting it away from vulnerable cells expressing membrane bound ACE2 3-5. However, a paper published in early 2021 6 described a scenario in which sACE2 and vasopressin played essential roles in SARS-CoV-2 infection of cells vulnerable to the virus. This commentary challenges both the 2 and 6 reports based upon their misconceptions and technical errors that pose a threat to the administration of life-saving therapies for severely affected COVID-19 patients.

The role of angiotensin-converting enzyme 2 (ACE2) in SARS-CoV-2 (severe acute respiratory syndrome Coronavirus-2) infection process

SARS-CoV-2 can cause severe acute respiratory illness and multi-organ injury. Angiotensin-converting enzyme 2 (ACE2) is an important enzyme of the renin-angiotensin system (RAS), which is the main receptor for entry of SARS-CoV-2 into host cells. Both cell surface ACE2 and soluble ACE2 (sACE2) can interact with the receptor binding domain (RBD) in SARS-CoV-2 S protein. After binding to cell surface ACE2, SARS-CoV-2 enters host cells by direct fusion of the viral with host cell membranes with the support of host’s TMPRSS2 (transmembrane serine protease 2) and furin. In contrast, sACE2-bound SARS-CoV-2 enters host cells through AT1 (Angiotensin type 1 receptor) and AVPR1B (arginine vasopressin receptor 1B). The entery process strongly down regulates membrane-bound ACE2, promoting an activation of the canonical pathway of RAS (ACE-Ang II-AT1 receptor axis). Consequently, this promotes an intense activity of NF-kB (nuclear factor kappa B), which in turn increases the transcription of IL-6, and triggers the cytokine storm. Moreover, the decreasing of ACE2 function in lung leed to the activation of DABK/BKB1R axis signaling, contributing to the pathogenesis of acute lung inflammation. This review summarizes information about the role of ACE2 in SARS-CoV-2 infection and Covid-19, also the research directions for prevention and treatment of Covid-19 which based on ACE2.

Celecoxib, Glipizide, Lapatinib, and Sitagliptin as potential suspects of aggravating SARS-CoV-2 (COVID-19) infection: a computational approach

COVID-19 caused by SARS-CoV-2 has emerged as a potential threat to human life, especially to people suffering from chronic diseases. In this study, we investigated the ability of selected FDA-approved drugs to inhibit TACE (tumor necrosis factor α converting enzyme), which is responsible for the shedding of membrane-bound ACE2 (angiotensin-converting enzyme2) receptors into soluble ACE2. The inhibition of TACE would lead to an increased population of membrane-bound ACE2, which would facilitate ACE2-Spike protein interaction and viral entry. A total of 50 drugs prescribed in treating various chronic diseases in Saudi Arabia were screened by performing molecular docking using AutoDock4.2. Based on docking energy (≤ −9.00 kcal mol−1), four drugs (Celecoxib, Glipizide, Lapatinib, and Sitagliptin) were identified as potential inhibitors of TACE, with binding affinities up to 106–107 M −1. Analysis of the molecular docking suggests that these drugs were bound to TACE's catalytic domain and interact with the key residues such as His405, Glu406, and His415, which are involved in active site Zn2+ ion chelation. Molecular dynamics simulation was performed to confirm the stability of TACE-drugs complexes. RMSD (root mean square deviation), RMSF (root mean square fluctuation), Rg (radius of gyration), and SASA (solvent accessible surface area) were within the acceptable limits. Free energy calculations using Prime-MM/GBSA suggest that Celecoxib formed the most stable complex with TACE, followed by Glipizide, Sitagliptin, and Lapatinib. The finding of this study suggests a mechanism for drugs to aggravate SARS-CoV-2 infection and hence high mortality in patients suffering from chronic diseases. Communicated by Ramaswamy H. Sarma

Protocol for the Controlled evaLuation of Angiotensin Receptor blockers for COVID-19 respIraTorY disease (CLARITY): a randomised controlled trial

BackgroundSARS-CoV-2 binds to membrane-bound angiotensin-converting enzyme 2 (ACE2) which may result in downregulation of membrane-bound ACE2. ACE2 is a key regulator of the renin-angiotensin system (RAS) and is responsible for degrading angiotensin II and thereby counteracting its pro-inflammatory, pro-fibrotic effects mediated through the angiotensin II type 1 receptor (AT1R). As AT1R is directly blocked by angiotensin receptor blockers (ARBs), these agents may offer a safe, low-cost solution for reducing COVID-19 respiratory outcomes.Methods and discussionCLARITY is a pragmatic, adaptive, two-arm, multi-centre, comparative effectiveness phase III randomised controlled trial that examines whether ARBs reduce COVID-19 severity among high-risk patients. Recruiting in India and Australia, the trial will compare treatment with a maximum tolerated daily dose of an ARB to standard of care. Treatment allocation is blinded in India but open-label in Australia due to interruptions to placebo supply in the latter. The primary endpoint is a 7-point ordinal scale of clinical states, ranging from no limitation of activities (category 1) to death (category 7), assessed on day 14. Secondary outcomes include the 7-point scale assessed at day 28 and 28- and 90-day mortality. The design adapts the sample size based on accumulating data via frequent interim analyses and the use of predictive probability to determine whether the current sample size is sufficient or continuing accrual would be futile. The trial commenced recruitment on 18 August 2020.Trial registrationClinicalTrials.gov, NCT04394117. Registered on 19 May 2020. Clinical Trial Registry of India: CTRI/2020/07/026831)

Role of ACE 2 and Vitamin D: The Two Players in Global Fight against COVID-19 Pandemic

AbstractThe global pandemic of coronavirus disease 2019 (COVID-19) has spread across the borders, gaining attention from both health care professional and researchers to understand the mode of entry and actions induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), its causative agent in the human body. The role of angiotensin-converting enzyme–2 (ACE2) in facilitating the entry of the virus in the host cell by binding to it is similar to SARS-CoV-1, the causative agent for severe acute respiratory syndrome (SARS) which emerged in 2003. Besides the role of ACE2 as a molecular target for the virus, the review displays the potential benefits of ACE2 enzyme and various agents that modify its activity in curbing the effects of the deadly virus, thus unfolding a dual character of ACE2 in the current pandemic. As evident by the differences in the susceptibility toward viral infection in children and geriatric population, it must be noted that the older population has limited ACE2 levels and greater infection risk, whereas the situation is reversed in the case of the pediatric population, demonstrating the defensive character of ACE2 in the latter, despite acting as receptor target for SARS-CoV-2. Also, the upregulation of ACE2 levels by estrogen has indicated greater resistance to infection in females than in the male human population. ACE2 is a carboxypeptidase, which degrades angiotensin II and counteracts its actions to protect against cardiovascular risks associated with the virus. Another contribution of this enzyme is supported by the role of circulating soluble ACE2, which acts as a receptor to bind the virus but does not mediate its actions, therefore blocking its interaction to membrane-bound ACE2 receptors. The review also shares the enhanced risks of developing COVID-19 infection by using ACE inhibitors and ARBs. However, both these agents have been reported to upregulate ACE2 levels; yet, adequate evidence regarding their role is quite inconsistent in human studies. Furthermore, the role of vitamin D has been highlighted in regulating the immune system of the body through renin-angiotensin-aldosterone system (RAAS) inhibition, by downregulating host cell receptor expression to prevent virus attachment. Besides, vitamin D also acts through several other mechanisms like upregulating antimicrobial peptides, fighting against the proinflammatory milieu created by the invading virus, and interfering with the viral replication cycle as well as calcitriol-mediated blockage of CREB protein. Hypovitaminosis D is attributed to elevated risks of acute respiratory distress syndrome (ARDS), lung damage, and cardiovascular disorders, further increasing the severity of COVID-19 infection.

The Duplicitous Nature of ACE2 in COVID-19 Disease.

Our understanding of the renin-angiotensin-system (RAS) has been revolutionized over the last 20 years with the discovery of angiotensin converting enzyme (ACE)2 and its principal product – angiotensin (Ang)-(1–7) [[1]Santos R.A.S. Oudit G.Y. Verano-Braga T. Canta G. Steckelings U.M. Bader M The renin-angiotensin system: going beyond the classical paradigms. American journal of physiology.Heart Circul Physiol. 2019; 316: H958-H970Crossref PubMed Scopus (146) Google Scholar]. ACE2 is a cell-membrane-bound protease, also appearing as a soluble form in the circulation, which converts Ang II to Ang-(1-7) [[2]Sampaio W.O. dos Santos R.A.S. Faria-Silva R. Machado L.T.D. Schiffrin E.L. Touyz R.M. Angiotensin-(1-7) through receptor Mas mediates endothelial nitric oxide synthase activation via Akt-dependent pathways.Hypertension. 2007; 49: 185-192Crossref PubMed Scopus (409) Google Scholar,[3]Gheblawi M. Wang K. Viveiros A. Nguyen Q. Zhong J.C. Turner A.J. Raizada M.K. Grant M.B. Oudit G.Y. Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin system: celebrating the 20th anniversary of the discovery of ACE2.Circ Res. 2020; 126: 1456-1474Crossref PubMed Scopus (1095) Google Scholar]. Ang-(1–7) binds to Mas receptor (MasR) and angiotensin AT2-receptors (AT2R) and induces vasorelaxation through nitric oxide [[1]Santos R.A.S. Oudit G.Y. Verano-Braga T. Canta G. Steckelings U.M. Bader M The renin-angiotensin system: going beyond the classical paradigms. American journal of physiology.Heart Circul Physiol. 2019; 316: H958-H970Crossref PubMed Scopus (146) Google Scholar,[2]Sampaio W.O. dos Santos R.A.S. Faria-Silva R. Machado L.T.D. Schiffrin E.L. Touyz R.M. Angiotensin-(1-7) through receptor Mas mediates endothelial nitric oxide synthase activation via Akt-dependent pathways.Hypertension. 2007; 49: 185-192Crossref PubMed Scopus (409) Google Scholar]. It also suppresses inflammation, oxidative stress, apoptosis, fibrosis and coagulation [[1]Santos R.A.S. Oudit G.Y. Verano-Braga T. Canta G. Steckelings U.M. Bader M The renin-angiotensin system: going beyond the classical paradigms. American journal of physiology.Heart Circul Physiol. 2019; 316: H958-H970Crossref PubMed Scopus (146) Google Scholar,[2]Sampaio W.O. dos Santos R.A.S. Faria-Silva R. Machado L.T.D. Schiffrin E.L. Touyz R.M. Angiotensin-(1-7) through receptor Mas mediates endothelial nitric oxide synthase activation via Akt-dependent pathways.Hypertension. 2007; 49: 185-192Crossref PubMed Scopus (409) Google Scholar]. This tissue-protective arm of RAS is essential in counterbalancing the devastating effects of the RAS pressor axis, mediated by Ang II and AT1 receptor (AT1R), that exerts vasoconstriction and induces inflammation, apoptosis, fibrosis, coagulation and renal salt and fluid retention [[1]Santos R.A.S. Oudit G.Y. Verano-Braga T. Canta G. Steckelings U.M. Bader M The renin-angiotensin system: going beyond the classical paradigms. American journal of physiology.Heart Circul Physiol. 2019; 316: H958-H970Crossref PubMed Scopus (146) Google Scholar,3Gheblawi M. Wang K. Viveiros A. Nguyen Q. Zhong J.C. Turner A.J. Raizada M.K. Grant M.B. Oudit G.Y. Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin system: celebrating the 20th anniversary of the discovery of ACE2.Circ Res. 2020; 126: 1456-1474Crossref PubMed Scopus (1095) Google Scholar, 4Abassi Z. Higazi A.A.R. Kinaneh S. Armaly Z. Skorecki K. Heyman S.N.ACE2 COVID-19 infection, inflammation, and coagulopathy: missing pieces in the puzzle.Front Physiol. 2020; 11574753Crossref PubMed Scopus (30) Google Scholar, 5Heyman S.N. Walther T. Abassi Z. Angiotensin-(1-7)—A Potential Remedy for AKI: insights derived from the COVID-19 pandemic.J Clin Med. 2021; 10: 1200Crossref PubMed Scopus (10) Google Scholar]. Thus, homeostasis requires balanced opposing actions of the pressor and depressor-protective arms of RAS. Indeed, altered generation of Ang-(1–7) has been associated with hypertension and tissue damage, leading in particular to renal and cardiac fibrosis and dysfunction [[1]Santos R.A.S. Oudit G.Y. Verano-Braga T. Canta G. Steckelings U.M. Bader M The renin-angiotensin system: going beyond the classical paradigms. American journal of physiology.Heart Circul Physiol. 2019; 316: H958-H970Crossref PubMed Scopus (146) Google Scholar,[3]Gheblawi M. Wang K. Viveiros A. Nguyen Q. Zhong J.C. Turner A.J. Raizada M.K. Grant M.B. Oudit G.Y. Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin system: celebrating the 20th anniversary of the discovery of ACE2.Circ Res. 2020; 126: 1456-1474Crossref PubMed Scopus (1095) Google Scholar,[5]Heyman S.N. Walther T. Abassi Z. Angiotensin-(1-7)—A Potential Remedy for AKI: insights derived from the COVID-19 pandemic.J Clin Med. 2021; 10: 1200Crossref PubMed Scopus (10) Google Scholar]. Moreover, the induction of ACE2 or the administration of Ang-(1–7)- mimetics that activate MasR have recently been proposed in the management of various systemic and organ disorders [[5]Heyman S.N. Walther T. Abassi Z. Angiotensin-(1-7)—A Potential Remedy for AKI: insights derived from the COVID-19 pandemic.J Clin Med. 2021; 10: 1200Crossref PubMed Scopus (10) Google Scholar]. COVID-19 disease stirred the interest in the ACE2/Ang-(1-7)/MasR axis, since cell-membrane-bound ACE2 is essential for viral invasion, as it binds to Sars-CoV-2 spike proteins, permeating its attachment to target host [[6]Yan R. Zhang Y. Li Y. Xia L. Guo Y. Zhou Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2.Science. 2020; 367: 1444-1448Crossref PubMed Scopus (2911) Google Scholar]. Additional convertases such as circulating furin and transmembrane protease serine (TMPRSS2) strengthen viral attachment to membranal ACE2 by modification and cleavage of the S1/S2 spike proteins [[6]Millet J.K. Whittaker G.R. Host cell proteases: critical determinants of coronavirus tropism and pathogenesis.Virus Res. 2015; 202: 120-134Crossref PubMed Scopus (564) Google Scholar]. Thus, primary viral invasion likely occurs in epithelial cells expressing ACE2, such as the nasal mucosa, alveolar epithelial cells, pulmonary macrophages and the gut mucosa. Viral dissemination following replication in target homing tissues conceivably also requires ACE2 expression in the vasculature and various tissues such as the myocardium and renal tubular cells [[1]Santos R.A.S. Oudit G.Y. Verano-Braga T. Canta G. Steckelings U.M. Bader M The renin-angiotensin system: going beyond the classical paradigms. American journal of physiology.Heart Circul Physiol. 2019; 316: H958-H970Crossref PubMed Scopus (146) Google Scholar,[3]Gheblawi M. Wang K. Viveiros A. Nguyen Q. Zhong J.C. Turner A.J. Raizada M.K. Grant M.B. Oudit G.Y. Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin system: celebrating the 20th anniversary of the discovery of ACE2.Circ Res. 2020; 126: 1456-1474Crossref PubMed Scopus (1095) Google Scholar]. It has been suggested that enhanced expression of ACE2 increases susceptibility to SARS-CoV-2 infection [[8]Hofmann H. Geier M. Marzi A. Krumbiegel M. Peipp M. Fey G.H. Gramberg T. Pohlmann S. Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor.Biochem Biophys Res Commun. 2004; 319: 1216-1221Crossref PubMed Scopus (199) Google Scholar]. Indeed, Genetic factors and male gender, as well as acquired conditions, such as air pollution and chronic lung disease, diabetes, heart failure, and the use of ACE inhibitors, all leading to enhanced expression of ACE2 in target organs, are likely associated with predisposition to SARS-CoV-2 infection [[3]Gheblawi M. Wang K. Viveiros A. Nguyen Q. Zhong J.C. Turner A.J. Raizada M.K. Grant M.B. Oudit G.Y. Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin system: celebrating the 20th anniversary of the discovery of ACE2.Circ Res. 2020; 126: 1456-1474Crossref PubMed Scopus (1095) Google Scholar]. Viral attachment to membranal ACE2 is followed by viral internalization together with ACE2 and ACE2 degradation, leading to depletion of membranal ACE2 [[5]Heyman S.N. Walther T. Abassi Z. Angiotensin-(1-7)—A Potential Remedy for AKI: insights derived from the COVID-19 pandemic.J Clin Med. 2021; 10: 1200Crossref PubMed Scopus (10) Google Scholar]. Additionally, activation of ADAM-17 (shedase), triggered by Ang II, further reduces cell-bound ACE2 [[9]Patel V.B. Clarke N. Wang Z. Fan D. Parajuli N. Basu R. Putko B. Kassiri Z. Turner A.J. Oudit G.Y. Angiotensin II induced proteolytic cleavage of myocardial ACE2 is mediated by TACE/ADAM-17: a positive feedback mechanism in the RAS.J Mol Cell Cardiol. 2014; 66: 167-176Summary Full Text Full Text PDF PubMed Scopus (224) Google Scholar]. This leads to depletion of ACE2 and Ang-(1–7) at the tissue level, altering the balance between the pressor and depressor axes of RAS, in favour of the ACE/Ang II/AT1R pathway [[5]Heyman S.N. Walther T. Abassi Z. Angiotensin-(1-7)—A Potential Remedy for AKI: insights derived from the COVID-19 pandemic.J Clin Med. 2021; 10: 1200Crossref PubMed Scopus (10) Google Scholar]. This is likely manifested by the clinical characteristics of COVID-19 disease, namely profound inflammation, oxidative stress, tissue injury, multi-organ dysfunction and coagulopathy [[3]Gheblawi M. Wang K. Viveiros A. Nguyen Q. Zhong J.C. Turner A.J. Raizada M.K. Grant M.B. Oudit G.Y. Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin system: celebrating the 20th anniversary of the discovery of ACE2.Circ Res. 2020; 126: 1456-1474Crossref PubMed Scopus (1095) Google Scholar,[5]Heyman S.N. Walther T. Abassi Z. Angiotensin-(1-7)—A Potential Remedy for AKI: insights derived from the COVID-19 pandemic.J Clin Med. 2021; 10: 1200Crossref PubMed Scopus (10) Google Scholar]. A logical interventional approach might therefore be the restoration of balanced RAS by the stimulation of the ACE-2/Ang-(1–7)/MasR axis, in parallel with the inhibition of AT1R activation [[5]Heyman S.N. Walther T. Abassi Z. Angiotensin-(1-7)—A Potential Remedy for AKI: insights derived from the COVID-19 pandemic.J Clin Med. 2021; 10: 1200Crossref PubMed Scopus (10) Google Scholar]. Taken together, high expression of membranal ACE2 facilitates viral homing, but its loss by degradation and shedding leads to Ang-(1–7) depletion, promoting the devastating clinical manifestations of COVID-19 disease [[5]Heyman S.N. Walther T. Abassi Z. Angiotensin-(1-7)—A Potential Remedy for AKI: insights derived from the COVID-19 pandemic.J Clin Med. 2021; 10: 1200Crossref PubMed Scopus (10) Google Scholar]. Yet, many parts are still missing in the puzzle of SARS-CoV-2 and ACE2/Ang-(1–7) interactions. For that reason, the study of Nikiforuk et al., published in EBioMedicine [[10]Nikiforuk A.M. Kuchinski K.S. Twa D.D.W. Lukac C.D. Sbihi H. Basham C.A. Steidl C. Prystajecky N.A. Jassem A.N. Krajden M. Patrick D.M. Sekirov I. The contrasting role of Nasopharyngeal Angiotensin Converting Enzyme 2 (ACE2) expression in SARS-CoV-2 infection: a cross-sectional study of people tested for COVID-19 in British Columbia.E Bio Med. 2021; 66103316Scopus (12) Google Scholar] is important, providing new perspectives, by looking at the association of SARS-CoV-2 positivity and the transcription of ACE2 and TMPRSS2 in nasopharyngeal samples, comparing 212 SARS-CoV-2–positive patients and 212 SARS-CoV-2-negative matched controls. The major findings reported are that among SARS-CoV-2 carriers, the transcription of membrane-bound ACE2 was proportional to viral replication, as opposed to an inverse association of viral load and the transcription of the ACE2 isoform lacking the trans-membrane domain. These observational findings are interesting, but cautious interpretation is needed due to present methodological limitations. Firstly, gene transcription in samples obtained by swabs or washout may not reflect true tissue expression, as it is affected by the composition of nasopharyngeal excretion and by tissue damage and turnover. Furthermore, it is believed that circulating ACE2 (lacking the transmembrane domain) is generated from cell-membrane expressed ACE2, shed by ADAM-17 and other proteases [[9]Patel V.B. Clarke N. Wang Z. Fan D. Parajuli N. Basu R. Putko B. Kassiri Z. Turner A.J. Oudit G.Y. Angiotensin II induced proteolytic cleavage of myocardial ACE2 is mediated by TACE/ADAM-17: a positive feedback mechanism in the RAS.J Mol Cell Cardiol. 2014; 66: 167-176Summary Full Text Full Text PDF PubMed Scopus (224) Google Scholar], a conversion that is accelerated during SARS-CoV-2 infection. Nikiforuk et al. checked de novo synthesis of mRNA putatively encoding truncated ACE2, using two distinct primers that span different sites along the ACE2 transcript. It is important to consider the existence of alternatively spliced isoforms (Fig. 1) and to note that the detection of the two primer products does not necessarily indicate the existence of an independent formation of soluble ACE2 at the protein level. Thus, we believe the findings reported by Nikiforuk et al. and their conclusions will require further confirmation, looking at ACE2 protein abundance and not just transcription. This would revolutionize current concepts and may suggest an active mechanism of preventing viral invasion by using a synthesized form of circulating ACE2 to compete with cell-bound isoforms. Meanwhile, a more solid finding provided by Nikiforuk et al. is the positive association between viral load and ACE2 transcription in SARS-CoV-2 positive carriers, that underscores the importance of ACE2 expression in SARS-CoV-2 homing and infectivity. All authors contributed to conceptualization, writing, reviewing, editing and have read and agreed to the published version of the manuscript. The authors declare no conflict of interest. The contrasting role of nasopharyngeal angiotensin converting enzyme 2 (ACE2) transcription in SARS-CoV-2 infection: A cross-sectional study of people tested for COVID-19 in British Columbia, CanadaNasopharyngeal ACE2 transcription plays a dual, contrasting role in SARS-CoV-2 infection of the upper respiratory tract. Transcription of the transmembrane ACE2 isoform positively correlates, while transcription of the soluble isoform negatively correlates with viral RNA load after adjusting for age, biological sex, and transcription of TMPRSS2. Full-Text PDF Open Access

This Month's Highlights.

### Inhibiting SARS-CoV-2 with a Soluble ACE2 Variant Use of a soluble angiotensin-converting enzyme 2 (ACE2) protein has been proposed as a way to inhibit binding of the S spike of SARS-CoV-2 to the full-length membrane-bound ACE2 receptor. Wysocki et al. developed a newly bioengineered soluble

Potential Detrimental Role of Soluble ACE2 in Severe COVID-19 Comorbid Patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) takes its entry to host cell via angiotensin converting enzyme 2 (ACE2) receptor. Biochemical and structural analysis indicate the roles of other important proteins and/or enzymes involved in this process including disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) also known as tumor necrosis factor-α-converting enzyme (TACE) and trans membrane serine protease 2 (TMPRSS2). As a biological protective mechanism ACE2 converts angiotensin II (Ang II) to angiotensin (1-7) and helps to balance renin angiotensin system (RAS). Being the key receptor for virus spike binding and subsequent role in viral entry, the detrimental effect of ACE2 is also of great concern. Membrane-bound ACE2 ectodomain is shedded by ADAM17 upon viral spike binding, Ang II overproduction and in some disease condition. The shedded soluble ACE2 (sACE2) retains its catalytic activity, but its exact role in viral entry and pathogenesis is still unclear. It has been claimed that therapeutic sACE2 which mimic the ectodomain can exert dual effects; reduction of excess Ang II and blocking the viral entry by masking spike protein. But the important concern is why SARS-CoV-2 comorbid patients having higher amount of sACE2 cannot attain such benefit in viral infection. At this context, in this perspective we partially argue about the protective role of sACE2 and hypothesize that sACE2 can convert Ang II but we additionally speculate a series of events where protease primed or non-primed virus-sACE2 complex can possibly take entry inside the host cell. As floating virus can bind a good number of sACE2, upon endocytosis by host cell, extracellular sACE2 level could be reduced drastically. As a consequence, rapid rise in Ang II level can potentially aggravate disease severity through Ang II-angiotensin receptor 1 (AT1R) axis in comorbid patients. Thus, therapeutic recombinant sACE2 should be designed accordingly to achieve maximum outcome in treating COVID-19.

Potential detrimental role of soluble ACE2 in severe COVID-19 comorbid patients.

SummarySevere acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) enters the host cell by binding to angiotensin‐converting enzyme 2 (ACE2) receptor. Other important proteins involved in this process include disintegrin and metalloproteinase domain‐containing protein 17 (ADAM17) also known as tumour necrosis factor‐α‐converting enzyme and transmembrane serine protease 2. ACE2 converts angiotensin II (Ang II) to angiotensin (1–7), to balance the renin angiotensin system. Membrane‐bound ACE2 ectodomain shedding is mediated by ADAM17 upon viral spike binding, Ang II overproduction and in several diseases. The shed soluble ACE2 (sACE2) retains its catalytic activity, but its precise role in viral entry is still unclear. Therapeutic sACE2 is claimed to exert dual effects; reduction of excess Ang II and blocking viral entry by masking the spike protein. Nevertheless, the paradox is why SARS‐CoV‐2 comorbid patients struggle to attain such benefit in viral infection despite having a high amount of sACE2. In this review, we discuss the possible detrimental role of sACE2 and speculate on a series of events where protease primed or non‐primed virus–sACE2 complex might enter the host cell. As extracellular virus can bind many sACE2 molecules, sACE2 level could be reduced drastically upon endocytosis by the host cell. A consequential rapid rise in Ang II level could potentially aggravate disease severity through Ang II‐angiotensin II receptor type 1 (AT1R) axis in comorbid patients. Hence, monitoring sACE2 and Ang II level in coronavirus disease 2019 comorbid patients are crucial to ensure safe and efficient intervention using therapeutic sACE2 and vaccines.

Correcting the imbalanced protective RAS in COVID-19 with angiotensin AT2-receptor agonists.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is responsible for the global corona virus disease 2019 (COVID-19) pandemic enters host cells via a mechanism that includes binding to angiotensin converting enzyme (ACE) 2 (ACE2). Membrane-bound ACE2 is depleted as a result of this entry mechanism. The consequence is that the protective renin-angiotensin system (RAS), of which ACE2 is an essential component, is compromised through lack of production of the protective peptides angiotensin-(1-7) and angiotensin-(1-9), and therefore decreased stimulation of Mas (receptor Mas) and angiotensin AT2-receptors (AT2Rs), while angiotensin AT1-receptors (AT1Rs) are overstimulated due to less degradation of angiotensin II (Ang II) by ACE2. The protective RAS has numerous beneficial actions, including anti-inflammatory, anti-coagulative, anti-fibrotic effects along with endothelial and neural protection; opposite to the deleterious effects caused by heightened stimulation of angiotensin AT1R. Given that patients with severe COVID-19 exhibit an excessive immune response, endothelial dysfunction, increased clotting, thromboses and stroke, enhancing the activity of the protective RAS is likely beneficial. In this article, we discuss the evidence for a dysfunctional protective RAS in COVID and develop a rationale that the protective RAS imbalance in COVID-19 may be corrected by using AT2R agonists. We further review preclinical studies with AT2R agonists which suggest that AT2R stimulation may be therapeutically effective to treat COVID-19-induced disorders of various organ systems such as lung, vasculature, or the brain. Finally, we provide information on the design of a clinical trial in which patients with COVID-19 were treated with the AT2R agonist Compound 21 (C21). This trial has been completed, but results have not yet been reported.

The flexibility of ACE2 in the context of SARS-CoV-2 infection

The coronavirus disease 2019 (COVID-19) pandemic has swept over the world in the past months, causing significant loss of life and consequences to human health. Although numerous drug and vaccine development efforts are underway, there are many outstanding questions on the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral association to angiotensin-converting enzyme 2 (ACE2), its main host receptor, and host cell entry. Structural and biophysical studies indicate some degree of flexibility in the viral extracellular spike glycoprotein and at the receptor-binding domain (RBD)-receptor interface, suggesting a role in infection. Here, we perform explicitly solvated, all-atom, molecular dynamics simulations of the glycosylated, full-length, membrane-bound ACE2 receptor in both an apo and spike RBD-bound state to probe the intrinsic dynamics of the ACE2 receptor in the context of the cell surface. A large degree of fluctuation in the full-length structure is observed, indicating hinge bending motions at the linker region connecting the head to the transmembrane helix while still not disrupting the ACE2 homodimer or ACE2-RBD interfaces. This flexibility translates into an ensemble of ACE2 homodimer conformations that could sterically accommodate binding of the spike trimer to more than one ACE2 homodimer and suggests a mechanical contribution of the host receptor toward the large spike conformational changes required for cell fusion. This work presents further structural and functional insights into the role of ACE2 in viral infection that can potentially be exploited for the rational design of effective SARS-CoV-2 therapeutics.

Downregulation of Membrane-bound Angiotensin Converting Enzyme 2 (ACE2) Receptor has a Pivotal Role in COVID-19 Immunopathology.

The Coronavirus Disease 2019 (COVID-19) is becoming the major health issue in recent human history with thousands of deaths and millions of cases worldwide. Newer research and old experience with other coronaviruses highlighted a probable underlying mechanism of disturbance of the renin-angiotensin system (RAS) that is associated with the intrinsic effects of SARS-CoV-2 infection. In this review, we aimed to describe the intimate connections between the RAS components, the immune system and COVID-19 pathophysiology. This non-systematic review article summarizes recent evidence on the relationship between COVID-19 and the RAS. Several studies have indicated that the downregulation of membrane-bound ACE2 may exert a key role for the impairment of immune functions and for COVID-19 patients' outcomes. The downregulation may occur by distinct mechanisms, particularly: (1) the shedding process induced by the SARS-CoV-2 fusion pathway, which reduces the amount of membrane-bound ACE2, stimulating more shedding by the high levels of Angiotensin II; (2) the endocytosis of ACE2 receptor with the virus itself and (3) by the interferon inhibition caused by SARS-CoV-2 effects on the immune system, which leads to a reduction of ACE2 receptor expression. Recent research provides evidence of a reduction of the components of the alternative RAS axis, including ACE2 and Angiotensin-(1-7). In contrast, increased levels of Angiotensin II can activate the AT1 receptor in several organs. Consequently, increased inflammation, thrombosis and angiogenesis occur in patients infected with SARS-COV-2. Attention should be paid to the interactions of the RAS and COVID-19, mainly in the context of novel vaccines and proposed medications.

Angiotensin-Converting Enzyme Inhibitors Versus Angiotensin II Receptor Blockers

HomeCirculation: Cardiovascular Quality and OutcomesVol. 13, No. 10Angiotensin-Converting Enzyme Inhibitors Versus Angiotensin II Receptor Blockers Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBAngiotensin-Converting Enzyme Inhibitors Versus Angiotensin II Receptor BlockersA Comparison of Outcomes in Patients With COVID-19 Ankur Kalra, Edward S. Hawkins, Amy S. Nowacki, Vardhmaan Jain, Alex Milinovich, Joshua Saef, George Thomas, Surafel K. Gebreselassie, Sadashiva S. Karnik, Lara Jehi, James B. Young, Lars G. Svensson, Mina K. Chung and Neil Mehta Ankur KalraAnkur Kalra Ankur Kalra, MD, Department of Cardiovascular Medicine, Heart, Vascular and Thoracic Institute, Cleveland Clinic, 225 W Exchange St, Suite 225, Akron, OH 44302. Email E-mail Address: [email protected] https://orcid.org/0000-0003-0080-1660 Department of Cardiovascular Medicine, Heart, Vascular and Thoracic Institute (A.K., J.S., M.K.C.), Cleveland Clinic, OH. Section of Cardiovascular Research, Heart, Vascular and Thoracic Department, Cleveland Clinic Akron General, OH (A.K.). Search for more papers by this author , Edward S. HawkinsEdward S. Hawkins https://orcid.org/0000-0003-3169-1513 Center for Emergency Medicine-West, Cleveland Clinic Medina Hospital, Medina, OH (E.S.H.). Search for more papers by this author , Amy S. NowackiAmy S. Nowacki Department of Quantitative Health Sciences, Lerner Research Institute (A.S.N., A.M.), Cleveland Clinic, OH. Search for more papers by this author , Vardhmaan JainVardhmaan Jain https://orcid.org/0000-0001-7265-380X Department of Internal Medicine, Cleveland Clinic Foundation, OH (V.J.). Search for more papers by this author , Alex MilinovichAlex Milinovich https://orcid.org/0000-0003-0585-1480 Department of Quantitative Health Sciences, Lerner Research Institute (A.S.N., A.M.), Cleveland Clinic, OH. Search for more papers by this author , Joshua SaefJoshua Saef Department of Cardiovascular Medicine, Heart, Vascular and Thoracic Institute (A.K., J.S., M.K.C.), Cleveland Clinic, OH. Search for more papers by this author , George ThomasGeorge Thomas Department of Nephrology and Hypertension (G.T.), Cleveland Clinic, OH. Search for more papers by this author , Surafel K. GebreselassieSurafel K. Gebreselassie Department of Nephrology and Hypertension, Cleveland Clinic Florida, Weston, FL (S.K.G.). Search for more papers by this author , Sadashiva S. KarnikSadashiva S. Karnik https://orcid.org/0000-0003-0746-2753 Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute (S.S.K., M.K.C.), Cleveland Clinic, OH. Search for more papers by this author , Lara JehiLara Jehi Cleveland Clinic Foundation, OH (L.J.). Search for more papers by this author , James B. YoungJames B. Young Department of Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University (J.B.Y., N.M.), Cleveland Clinic, OH. Search for more papers by this author , Lars G. SvenssonLars G. Svensson https://orcid.org/0000-0002-5749-4588 Department of Thoracic and Cardiovascular Surgery, Heart, Vascular and Thoracic Institute (L.G.S.), Cleveland Clinic, OH. Search for more papers by this author , Mina K. ChungMina K. Chung https://orcid.org/0000-0002-7835-6045 Department of Cardiovascular Medicine, Heart, Vascular and Thoracic Institute (A.K., J.S., M.K.C.), Cleveland Clinic, OH. Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute (S.S.K., M.K.C.), Cleveland Clinic, OH. Search for more papers by this author and Neil MehtaNeil Mehta Department of Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University (J.B.Y., N.M.), Cleveland Clinic, OH. Search for more papers by this author Originally published28 Aug 2020https://doi.org/10.1161/CIRCOUTCOMES.120.007115Circulation: Cardiovascular Quality and Outcomes. 2020;13Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: September 28, 2020: Ahead of Print August 28, 2020: Ahead of Print Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes membrane-bound ACE2 (angiotensin-converting enzyme 2) as a functional receptor to gain entry into host cells. An early finding of the coronavirus disease 2019 (COVID-19) pandemic was the high mortality observed in patients with hypertension, diabetes mellitus, and coronary heart disease,1 raising concerns with regard to the potential for increased risk of SARS-CoV-2 infection and adverse outcomes in patients taking either ACE inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs). These concerns largely stemmed from the possible upregulation of ACE2 expression by these medications, although literature exists to both support and refute this notion.2,3 Observational studies in patients with COVID-19 have since shown no association between the use of either ACEIs or ARBs and testing positive for SARS-CoV-2.4,5 Whether a differential impact of ACEIs compared with ARBs on outcomes in COVID-19 exists remains uncertain. We sought to report on the association of ACEIs compared with ARBs on the risk of hospital admission, intensive care unit (ICU) admission, and need for mechanical ventilation in patients diagnosed with COVID-19.The data that support the findings of this study are available from the corresponding author upon request. A retrospective cohort analysis of an observational, institutional review board-approved registry of all patients who were tested for COVID-19 in the Cleveland Clinic Health System between March 8, 2020 and May 8, 2020 was conducted. Data regarding baseline characteristics, medications, test results, use of ACEI or ARB, and outcomes were extracted from EPIC electronic medical record system. Subjects recorded as being on both an ACEI and an ARB were excluded from the analysis. To account for the heterogeneity in the medical comorbidities among patients taking ACEI and ARB, an overlap propensity-score weighting was performed, as previously published.4 The primary outcome of interest was a head-to-head comparison of incidence of a severe disease process as assessed by hospital admission, ICU admission, and requirement for mechanical ventilation in patients with COVID-19 who were on ACEI versus ARB. All statistical analyses were performed using SAS version 9.4 (SAS Institute). P values were 2-sided, with a significance threshold of 0.05.The data set consisted of 39 042 patients tested for SARS-CoV-2. A total of 117 patients were tested multiple times for COVID-19. Data regarding only the index test was used for analysis in such cases to avoid duplication. The mean (SD) age was 65 (14) years, 2758 (51%) were male, and 3867 (72%) were White. Among all tested patients, 3094 (58%) were taking ACEIs and 2277 (42%) were taking ARBs. Among these 5371 tested patients, 381 (7.1%) tested positive (219 on ACEIs and 162 on ARBs). After overlap propensity-score weighting, the test positivity rate was 7.0% in ACEIs compared with 7.4% in ARBs group (overlap propensity score–weighted odds ratio, 0.95 [95% CI, 0.77–1.17]; Figure). Among patients with positive test results, overlap propensity-score weighting demonstrated the following: 58% taking ACEIs (versus 59% taking ARBs) were admitted to the hospital (odds ratio, 0.95 [95% CI, 0.62–1.45]); 22% taking ACEIs (versus 19% taking ARBs) were admitted to an ICU (odds ratio, 1.15 [95% CI, 0.69–1.92]); and 12% taking ACEIs (versus 11% taking ARBs) required mechanical ventilation (odds ratio, 1.12 [95% CI, 0.59–2.15]; Figure). A total of 21 deaths (5.6%) occurred. Fifteen of the 219 patients (6.9%) were taking an ACEI and 6 of 162 (3.7%) were taking an ARB.Download figureDownload PowerPointFigure. Comparison of ACEIs (angiotensin-converting enzyme inhibitors) vs angiotensin II receptor blockers (ARBs) for incidence of hospital admission, intensive care unit admission, and requirement for mechanical ventilation in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; primary outcome) and testing positive for SARS-CoV-2 (secondary outcome): overlap propensity-score weighted analysis odds ratios (ORs) with 95% CIs.This study did not show a difference in the rates of hospitalization, ICU admission, and need for mechanical ventilation between patients taking ACEIs versus ARBs. Although both classes of medications act on the renin-angiotensin-aldosterone system, their mechanisms of action differ. Both of these medications, in theory, could enhance viral entry into the respiratory epithelial cell. Interestingly, angiotensin II has previously been studied for its prothrombotic and proinflammatory role in patients with hypertension.6 It is also increasingly becoming clear that patients with severe COVID-19 experience coagulopathy with increased thromboembolic events. This raises a question regarding the possible differential effects of the 2 agents in patients with severe COVID-19. The differential effects of ACEIs versus ARBs on bradykinin levels via their interaction with the prekallikrein/kallikrein system may be another driver of their differential outcomes in patients with COVID-19. Higher bradykinin levels lead to increased nitric oxide and prostaglandin levels, inhibiting platelet activation and release of von Willebrand factor, which may play a role in COVID-19 coagulopathy.7 At the same time, prior studies have shown a possible vasoprotective role of bradykinin in mediating endothelium-mediated vasodilation,8 making such estimates conjectural. Although our study did not show any difference in the odds of testing positive for COVID-19, hospital and ICU admission, and subsequent need for mechanical ventilation, it underscores the need for a much-needed randomized controlled trial to address any differential effects on clinical outcomes in COVID-19 between the 2 drug classes.Our study has several limitations, including the potential for bias given the observational nature of the analysis. The majority of our cohort was White, which limits the external validity of our findings, especially given the differential expression of ACE2 receptor among different ethnicities.Sources of FundingThis study was supported by the Cleveland Clinic Lerner Research Institute (operations account) and National Institutes of Health/National Center for Advancing Translational Sciences (grant UL1TR002548). Role of the Funder/Sponsor: the funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.DisclosuresNone.FootnotesAnkur Kalra, MD, Department of Cardiovascular Medicine, Heart, Vascular and Thoracic Institute, Cleveland Clinic, 225 W Exchange St, Suite 225, Akron, OH 44302. Email [email protected]org

Insights on SARS-CoV-2 Molecular Interactions With the Renin-Angiotensin System.

The emergence of SARS-CoV-2/human/Wuhan/X1/2019, a virus belonging to the species Severe acute respiratory syndrome-related coronavirus, and the recognition of Coronavirus Disease 2019 (COVID-19) as a pandemic have highly increased the scientific research regarding the pathogenesis of COVID-19. The Renin Angiotensin System (RAS) seems to be involved in COVID-19 natural course, since studies suggest the membrane-bound Angiotensin-converting enzyme 2 (ACE2) works as SARS-CoV-2 cellular receptor. Besides the efforts of the scientific community to understand the virus’ molecular interactions with human cells, few studies summarize what has been so far discovered about SARS-CoV-2 signaling mechanisms and its interactions with RAS molecules. This review aims to discuss possible SARS-CoV-2 intracellular signaling pathways, cell entry mechanism and the possible consequences of the interaction with RAS components, including Angiotensin II (Ang II), Angiotensin-(1-7) [Ang-(1-7)], Angiotensin-converting enzyme (ACE), ACE2, Angiotensin II receptor type-1 (AT1), and Mas Receptor. We also discuss ongoing clinical trials and treatment based on RAS cascade intervention. Data were obtained independently by the two authors who carried out a search in the PubMed, Embase, LILACS, Cochrane, Scopus, SciELO and the National Institute of Health databases using Medical Subject Heading terms as “SARS-CoV-2,” “COVID-19,” “Renin Angiotensin System,” “ACE2,” “Angiotensin II,” “Angiotensin-(1-7),” and “AT1 receptor.” Similarly to other members of Coronaviridae family, the molecular interactions between the pathogen and the membrane-bound ACE2 are based on the cleavage of the spike glycoprotein (S) in two subunits. Following the binding of the S1 receptor-binding domain (RBD) to ACE2, transmembrane protease/serine subfamily 2 (TMPRSS2) cleaves the S2 domain to facilitate membrane fusion. It is very likely that SARS-CoV-2 cell entry results in downregulation of membrane-bound ACE2, an enzyme that converts Ang II into Ang-(1-7). This mechanism can result in lung injury and vasoconstriction. In addition, Ang II activates pro-inflammatory cascades when binding to the AT1 Receptor. On the other hand, Ang-(1-7) promotes anti-inflammatory effects through its interactions with the Mas Receptor. These molecules might be possible therapeutic targets for treating COVID-19. Thus, the understanding of SARS-CoV-2 intracellular pathways and interactions with the RAS may clarify COVID-19 physiopathology and open perspectives for new treatments and strategies.

Pathogenesis and preventive approach of hypercoagulopathy and microvascular thrombosis induced mortality from COVID-19

Background: COVID-19 is the current pandemic infection caused by severe acute respiratory syndrome coronavirus-2 (SARS Cov-2). Pulmonary collapse in severe critical COVID-19 patients may be due to development of multiple micro thrombi within pulmonary vasculature. As COVID-19 pandemic is accelerating, it is important to understand the molecular mechanism through which SARSCov2 induces hypercoagulopathy and intravascular thrombosis to be able to design more appropriate therapy. Focus of this review is to identify mechanisms through re-analysis of publicly available data by which SARS-Cov2 infection induce mortality by augmenting intravascular thrombosis and attempt to understand therapeutic approach to it. Findings: SARS Cov-2 accesses host cells via membrane bound angiotensin converting enzyme-2 (ACE2). This leads to imbalance of renin angiotensin system (RAS) increase ratio of Ag-II: Ag-1-7. Ag-II stimulates release of IP-10 from endothelium which upregulates local renin angiotensin system in endothelial cell by positive feedback process. Therefore it is suggested that angiotensin-II of renin angiotensin systemr in endothelial cells sustained proinflammatory signal and developed microvascular thrombosis. During inflammation both extrinsic and intrinsic coagulation pathways are activated. Fibrinolysis is suppressed by imbalance activity of two system: Ang-II-PAI-1 (plasminigen activator inhibitor-1) and Bradykinin-tPA (tissue plasminogen activator) system. Therapy with low molecular weight heparin (LMWH), which have anticoagulant and anti-inflammatory property, is associated with better prognostic in patients with severe COVID-19. ACE inhibitors decrease production of Ag-II and increases availability of bradykinin and consequence reduces coagulopathy Conclusion: Thus it is concluded that SARS-Cov2 infection induces microvascular thrombosis from hyperinflammation, misbalance between Ag-II and Ag-1-7 and imbalance activity of two system: Ang-II-PAI-1 and bradykinin-tPA system. ACE inhibitor and anticoagulant mainly LMWH and UFH may serve potential role in COVID-19 therapy particularly in patients with hypercoagulopathy and microvascular thrombosis. Keywords: COVID19; angiotensin converting enzyme; renin angiotensin system; fibrinolysis; microvascular thrombosis; ACE inhibitor; heparin

Protein Kinase C-δ Mediates Shedding of Angiotensin-Converting Enzyme 2 from Proximal Tubular Cells.

Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin (Ang) II to Ang-(1–7), and protects against diabetic renal injury. Soluble ACE2 fragments are shed from the proximal tubule, and appear at high levels in the urine with diabetes. High glucose-induced shedding of ACE2 from proximal tubular cells is mediated by the enzyme “a disintegrin and metalloproteinase-17″ (ADAM17). Here, we investigated the mechanism for constitutive shedding of ACE2. Mouse proximal tubular cells were cultured and ACE2 shedding into the media was assessed by enzyme activity assay and immunoblot analysis. Cells were incubated with pharmacologic inhibitors, or transfected with silencing (si) RNA. Incubation of proximal tubular cells with increasing concentrations of D-glucose stimulated ACE2 shedding, which peaked at 16 mM, while L-glucose (osmotic control) had no effect on shedding. In cells maintained in 7.8 mM D-glucose, ACE2 shedding was significantly inhibited by the pan-protein kinase C (PKC) competitive inhibitor sotrastaurin, but not by an inhibitor of ADAM17. Incubation of cells with the PKC-α and -β1-specific inhibitor Go6976, the PKC β1 and β2-specific inhibitor ruboxistaurin, inhibitors of matrix metalloproteinases-2,-8, and -9, or an inhibitor of ADAM10 (GI250423X) had no effect on basal ACE2 shedding. By contrast, the PKC-δ inhibitor rottlerin significantly inhibited both constitutive and high glucose-induced ACE2 shedding. Transfection of cells with siRNA directed against PKC-δ reduced ACE2 shedding by 20%, while knockdown of PKC-ε was without effect. These results indicate that constitutive shedding of ACE2 from proximal tubular cells is mediated by PKC-δ, which is also linked to high glucose-induced shedding. Targeting PKC-δ may preserve membrane-bound ACE2 in proximal tubule in disease states and diminish Ang II-stimulated adverse signaling.