Effect of Angiotensin-Converting-Enzyme Inhibitor and Angiotensin II Receptor Antagonist Treatment on ACE2 Expression and SARS-CoV-2 Replication in Primary Airway Epithelial Cells

Abstract

Rationale: SARS-CoV-2 gains entrance to airway epithelial cells (AECs) through binding of the viral spike protein to the angiotensin-converting enzyme 2 (ACE2) on the cell surface. However, ACE2 also converts angiotensin II into angiotensin-(1-7) and counterbalances the renin-angiotensin-aldosterone system, with resultant protective effects in the cardiovascular system. Some data suggest that two common antihypertension medications (angiotensin II receptor antagonists, ARBs; and angiotensin-converting-enzyme inhibitors, ACEIs) may increase ACE2 expression in heart and kidney cells, fueling debate about how these widely used medications may modulate SARS-CoV-2 infectivity and risk of COVID-19.Aim: Determine whether exposure of bronchial AECs to the ARB losartan or the ACEI captopril modulate expression of ACE2 by AECs, SARS CoV2 replication, or expression of proinflammatory cytokines and type I and III interferon (IFN) responses.Methods: Primary bronchial AECs from children and adults (n = 19; Ages 8–75 yrs) were differentiated ex vivo at an air-liquid interface to generate organotypic cultures. Cultures were treated with captopril (1 μM) or losartan (2 μM) with culture media changes starting 72 h before infection with SARS-CoV-2. In a biosafety level 3 (BSL-3) facility, cultures were infected with SARS-CoV-2 isolate USA-WA1/2020 at a multiplicity of infection (MOI) of 0.5. At 96 h following infection, RNA and protein were isolated. SARS-CoV-2 replication in cultures was assessed with quantitative PCR (qPCR). ACE2, IL-6, IL-1B, IFNB1, and IFNL2 expression were assessed by qPCR.Results: Neither captopril nor losartan treatment significantly changed ACE2, IL-6, IL-1B, IFNB1, or IFNL2 expression by AECs as compared to SARS-CoV-2 infected AEC cultures without captopril or losartan treatment. At 96 h following infection, SARS-CoV-2 copy number/ng RNA was not significantly different between untreated AEC cultures, cultures treated with captopril, or cultures treated with losartan.Conclusion: These findings suggest that at the level of the airway epithelium neither the ACEI captopril or ARB losartan significantly modify expression of the SARS-CoV-2 entry factor ACE2, nor does either medication increase replication SARS-CoV-2 replication. This ex vivo data is reassuring and is consistent with evolving clinical data suggesting ACEIs and ARBs do not increase the risk for poor prognosis with COVID-19 and may actually reduce the risk of COVID-19 disease.

Commentary: Effect of Angiotensin-Converting-Enzyme Inhibitor and Angiotensin II Receptor Antagonist Treatment on ACE2 Expression and SARS-CoV-2 Replication in Primary Airway Epithelial Cells

“…Nevertheless, the principal message of both papers is similar, suggesting the apparent clinical safety of ACEI/ARB in COVID-19 patients (Okoloko et al, 2021;Pedrosa et al, 2021).…”

“…The experiments showed that neither captopril nor losartan modified membrane-bound ACE2 (mACE2) expression in respiratory epithelial cell cultures, nor did these RAS inhibitors increase SARS-CoV-2 replication and the level of inflammatory cytokines or interferon type I/III in epithelial cell cultures exposed to SARS-CoV-2 compared to a SARS-CoV-2-treated cell line without RAS-inhibition. However, treatment with captopril/losartan, significantly increased mACE2 expression when compared to untreated control epithelial cell cultures (without SARS-CoV-2 or RAS inhibitors), a fact that remained unexplained by the authors (Okoloko et al, 2021).…”

“…Okoloko et al, in a paper recently published in Frontiers in Pharmacology (Okoloko et al, 2021), investigated whether captopril or losartan were able to modulate the expression of ACE2 in airway epithelial cell cultures from children or adults, as well as SARS-CoV-2 replication, or the expression of inflammatory mediators. The experiments showed that neither captopril nor losartan modified membrane-bound ACE2 (mACE2) expression in respiratory epithelial cell cultures, nor did these RAS inhibitors increase SARS-CoV-2 replication and the level of inflammatory cytokines or interferon type I/III in epithelial cell cultures exposed to SARS-CoV-2 compared to a SARS-CoV-2-treated cell line without RAS-inhibition.…”

SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1

“…AngII causes vasoconstriction initiated by interaction with the AT1 receptor, and decline in opposing vasodilation, due to reduced production of Ang1-7 when ACE2 is bound by the SARS-CoV-2 spike protein, may predispose to unopposed signaling events and significant pathophysiology [24][25][26][27]. The impact of inhibition of the renal angiotensin system (RAS) by angiotensin-converting enzyme (ACE) inhibitors or by angiotensin receptor blockers (ARBs) has been considered in relation to treatment and outcome of COVID-19 infection, and studies are ongoing [28][29][30].…”

Relation Between Renin–Angiotensin–Aldosterone System Inhibitors and COVID-19 Severity

“…Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) may increase the expression of ACE2, resulting in concerns that patients with COVID-19 who are receiving these agents may be at increased risk of severe disease [5][6][7]. However, some recent studies concluded that ACEIs and ARBs do not modify the expression ACE2 and that they do not increase the replication of SARS-CoV-2 [8,9]. Moreover, contrary to the hypnotized negative effects of ACEIs and ARBs on COVID-19 patients, there are conflicting data that they might be beneficial.…”