Angiotensin II Receptor Blockers (ARBs Antihypertensive Agents) Increase Replication of SARS-CoV-2 in Vero E6 Cells

Abstract

Several comorbidities, including hypertension, have been associated with an increased risk of developing severe disease during SARS-CoV-2 infection. Angiotensin II receptor blockers (ARBs) are currently some of the most widely-used drugs to control blood pressure by acting on the angiotensin II type 1 receptor (AT1R). ARBs have been reported to trigger the modulation of the angiotensin I converting enzyme 2 (ACE2), the receptor used by the virus to penetrate susceptible cells, raising concern that such treatments may promote virus capture and increase their viral load in patients receiving ARBs therapy. In this in vitro study, we reviewed the effect of ARBs on ACE2 and AT1R expression and investigated whether treatment of permissive ACE2+/AT1R+ Vero E6 cells with ARBs alters SARS-CoV-2 replication in vitro in an angiotensin II-free system. After treating the cells with the ARBs, we observed an approximate 50% relative increase in SARS-CoV-2 production in infected Vero E6 cells that correlates with the ARBs-induced up-regulation of ACE2 expression. From this data, we believe that the use of ARBs in hypertensive patients infected by SARS-CoV-2 should be carefully evaluated.

Keywords: COVID-19; SARS-CoV-2; Vero E6 cells; angiotensin I converting enzyme 2; angiotensin II receptor blockers; antihypertensive.

Figure 1

Expression of Angiotensin II receptor type 1 (AT1R) and Angiotensin-converting enzyme 2 (ACE2) mRNAs and proteins by VERO E6 cells following 72 hours incubation with different Angiotensin II Receptor Blockers (ARBs). Non-cytotoxic concentrations of ARBs (Azil, Azilsartan 15µM; Epr, Eprosartan 30µM; Irb, Irbesartan 60µM; Los, losartan 7µM; Olm, Olmesartan 15µM; Tel, Telmisartan 7µM; Val, Valsartan 7µM) were previously defined by the MTT assay and cells were treated with the various ARBs. (A) Relative expression of the ACE2/ACEH gene in untreated (Unt) and ARB-treated Vero E6 cells. (B) Relative expression of the ATR1/AGTR1 gene in untreated versus ARB-treated Vero E6 cells. (C) Expression of ACE2 protein in untreated versus ARB-treated Vero E6 cells (the membrane-anchored ACE2 glycosylated protein that acts as receptor for SARS-CoV-2 is the 110kDa form of the molecule).

Figure 2

Modulation of cell surface-expressed ATR1 and ACE2 molecules in Vero E6 cells non-infected and treated with Azilsartan (15 µM) for 72 hours by immunofluorescence microscopy. (A) The panel presents non-infected cells after incubation with Azilsartan (15 µM) and evaluating the fluorescence corresponding to the ATR1, ACE2, Actin, and the nucleus of the cells. The merge of the images is displayed at the right of the panel. Images were acquired using a confocal microscope (Zeiss LSM 800) with a 63X/1.4 oil objective. (B) Quantitative representation of Mean Fluorescence corresponding to ATR1 and ACE2 molecule expression on VERO E6 cells treated or not treated with Azilsartan. **P < 0.01.

Figure 3

Effects of pre-treating Vero E6 cells in cells with different ARBs on the SARS-CoV-2 production. (A) Schematic flow of the analysis: Vero E6 cells were treated with different non-cytotoxic concentrations of ARBs (Azilsartan 15µM; Eprosartan 30µM; Irbesartan 60µM; Losartan 7µM; Olmesartan 15µM; Telmisartan 7µM; Valsartan 7µM) were previously defined by the MTT assay and cells were treated with the different ARBs.) for 72 hours and were subsequently infected and produced virus evaluated by RT-qPCR and Western Blotting 24 hours post-infection (h.p.i.). (B) Relative SARS-CoV-2 genome quantification in supernatant of treated and infected Vero E6 cells by RT-qPCR: Relative viral quantification was performed compared to the untreated control (viruses without drugs) using the 2^(–ΔCT) method, where ΔCt = {[(Ct 48 h.p.i) treated well] – [mean (Ct 0 h.p.i)]}; (C) Viral protein detection in treated and infected Vero E6 cells by Western Blotting performed in JessTM Simple Western system (automated Western immunoblotting), in which every column represents individual runs (D) TCID50 (six days post-infection) from the supernatant of the treated with different ARBs Vero E6 cells recovered 24 h.p.i. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Modulation of cell surface-expressed ATR1 and ACE2 molecules in Vero E6 cells infected with SARS-CoV-2 (24 h.p.i) and treated with Azilsartan (15 µM) for 72 hours by immunofluorescence microscopy. (A) Schematic flow of the analysis: Vero E6 cells were treated with various ARBs (the MTT assay previously defined non-cytotoxic concentrations: Azilosartan 15µM; Eprosartan 30µM; Irbesartan 60µM; Losartan 7µM; Olmesartan 15µM; Telmisartan 7µM; Valsartan 7µM) for 72 hours and were subsequently infected for analysis of ACE2 and ATR1 on treated and infected cells, 24 hours post-infection (h.p.i.). (B) The panel presents SARS-CoV-2 infected cells after incubation with Azilsartan (15 µM) and evaluation of fluorescence corresponding to the ATR1, ACE2, viral spike protein, and the nucleus of the cells. The merge of the images is displayed at the right of the panel. Images were acquired using a confocal microscope (Zeiss LSM 800) with a 63X/1.4 oil objective. (C) Quantitative representation of Mean Fluorescence corresponding to ATR1 and ACE2 molecules expression on VERO E6 cells treated or not treated with Azilsartan and SARS-CoV-2 in the cells. ***P < 0.001; ****P < 0.0001.