key: cord-0742578-ropkw784 authors: de Ligt, Marlies; Hesselink, Matthijs K.C.; Jorgensen, Johanna; Jocken, Johan W.E.; Blaak, Ellen E.; Goossens, Gijs H. title: The angiotensin II type 1 receptor blocker valsartan in the battle against COVID‐19 date: 2021-05-05 journal: Obesity (Silver Spring) DOI: 10.1002/oby.23221 sha: 336826e5aca869e99c6e8bca81497f866d238bf8 doc_id: 742578 cord_uid: ropkw784 OBJECTIVE: SARS‐CoV‐2 uses the hosts angiotensin‐converting enzyme 2 (ACE2) as cellular entry point. Hence, modulating ACE2 might impact SARS‐CoV‐2 viral replication, shedding and COVID‐19 severity. Here, we investigated if the angiotensin II type 1 receptor (AT1R) blocker valsartan alters the expression of renin‐angiotensin system (RAS) components, including ACE2, in human adipose tissue (AT) and skeletal muscle. METHODS: We performed a randomized, double‐blind, placebo‐controlled clinical trial, in which 36 participants (BMI 31.0±0.8 kg/m(2)) with impaired glucose metabolism received either valsartan or placebo for 26 weeks. Before and after 26 weeks treatment, abdominal subcutaneous AT and skeletal muscle biopsies were obtained, and gene expression of RAS components was measured by qRT‐PCR. RESULTS: Valsartan treatment did not significantly impact the expression of RAS components, including ACE2, in AT and skeletal muscle. CONCLUSIONS: Given the pivotal role of ACE2 in SARS‐CoV‐2 spread and the clinical outcomes in COVID‐19 patients, our data suggest that the putative beneficial effects of ARBs on the clinical outcomes in patients with COVID‐19 may not be mediated through altered ACE2 expression in abdominal subcutaneous AT. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the hosts angiotensinconverting enzyme 2 (ACE2) as cellular entry point. Hence, ACE2 is pivotal in SARS-CoV-2 replication and viral shedding. ACE2 is part of the renin-angiotensin system (RAS), which appears a central player in the clinical outcomes of patients with coronavirus disease 19 (COVID-19) (1, 2) . Angiotensin (Ang)II is the main effector peptide of RAS, which is converted by cleavage of AngI into AngII by the angiotensinogen-converting enzyme (ACE) (3) . AngII has a high binding affinity for the AngII type 1 receptor (AT1R), thus increasing blood pressure, inflammation, fibrosis and oxidative stress. ACE2 is the rate-limiting enzyme in the degradation of AngII, counteracting deleterious effects of AngII by converting AngII into Ang(1-7). SARS-CoV-2 reduces ACE2 activity, resulting in elevated (tissue) AngII concentrations, which appears to be associated with poor clinical outcomes. Hence, modulating ACE2 expression/activity might impact SARS-CoV-2 viral replication, shedding and the severity of complications in COVID-19 patients (2) . At the beginning of the SARS-CoV-2 pandemic it was suggested that pharmacological blockade of RAS, by commonly used ACE inhibitors or AT1R blockers (ARBs), might increase the risk of SARS-CoV-2 infection, since ACE inhibition and AT1R blockade might increase ACE2 expression/activity (1, 2) . However, results from large observational and experimental studies suggest that ACE inhibitors or ARBs are not associated with the risk of SARS-CoV-2 infection (2). Rather, RASblockade might prove advantageous in COVID-19, since RAS-blockade decreases detrimental effects of the ACE-AngII-AT1-axis and may be beneficial through the ACE2-Ang(1-7)-Mas receptor-axis. ARB treatment might be a promising strategy to also prevent SARS-CoV-2 infection, since ACE2 can only enter cells by AT1R-mediated endocytosis (2, 4) . Currently, RASmodulating drugs are being investigated as therapeutic strategy in several clinical trials to prevent SARS-CoV-2 infection and to alleviate disease severity (5) . Notably, excess adipose tissue (AT) in people with obesity might serve as a reservoir for SARS-CoV-2 spread, thus providing a link between obesity, the susceptibility to and severity of COVID-19 (2, 6). Hence, the effects of RAS modulators on AT are worthwhile exploring, since RAS components are highly expressed in human AT. Since it is unknown whether RAS blockade alters tissue-specific expression of RAS components in humans, we here investigated if treatment with the ARB valsartan for 26 weeks alters the expression of RAS components, including ACE2, in human AT and skeletal muscle. We previously performed a randomized, double-blind, placebo-controlled clinical trial, in which participants with impaired glucose metabolism received either the ARB valsartan (160mg for 2wks and 320mg for subsequent 24wks) or placebo for 26wks (ISRCTN Registry: ISRCTN42786336) (7). Before and after 26wks treatment, abdominal subcutaneous AT and skeletal muscle (m. vastus lateralis) biopsies were collected after an overnight fast (n=36; BMI 31.0±0.8 kg/m 2 ; SBP 128±2 mmHg; DBP 80±1 mmHg). Total RNA was extracted and stored at -80C. Here, we performed qRT-PCR analyses to determine the effects of ARB treatment on gene expression of RAS components in abdominal subcutaneous AT and skeletal muscle. Total RNA was extracted from the biopsies and stored at -80 degrees Celsius until analyses. Next, total RNA was reverse transcribed using the High-Capacity-RNA-to-cDNA-kit (Applied Biosystems) and qRT-PCR was performed in the CFX384 Real-Time System (Biorad). For both tissues the geometric mean of ribosomal protein, large, P0 (RPLP0), ribosomal protein L26 (RPL26) and 18S RNA was used as internal control. Both SensiMix SYBR Hi-ROX mastermix (Bioline) and HOTFIREPol Probe qPCR Mix Plus (ROX) (Solis Biodyne) were used for quantification of mRNA expression levels. Taqman primers ACE (Hs001741179_m1), ACE2 (Hs01085333_m1) and AT1R (Hs00258938_m1) (Applied Biosystems) were used with HOTFIREPol Probe qPCR Mix Plus (ROX). Angiotensinogen (AGT), 18S, RPLPO and RPL26 were amplified using SensiMix SYBR Hi-ROX mastermix and genespecific forward and reverse primers. Gene expression was defined using a derivative of the ΔΔCt method. The expression of the housekeeping genes was presented as 2−ΔCt. This article is protected by copyright. All rights reserved The effects of valsartan treatment were tested using repeated-measures ANOVA with time as within-subject factor and treatment as between-subject factor. Statistical analyses were performed using IBM SPSS Statistics for Mac, Version 24.0 (Armonk, New York, IBM Corp.), and p<0.05 was considered statistically significant. Values are presented as mean ± SEM. We found no significant differences in the expression of AGT, ACE, ACE2 and AT1R in AT after 26 wks of valsartan treatment compared to placebo (Figure 1A) . In skeletal muscle also no differences were found AGT and ACE expression ( Figure 1B) . Unfortunately, expression of ACE2 and AT1R could not be quantified reliably in muscle. So far, data on plasma ACE2 activity and Ang-(1-7) levels in patients without COVID-19 who have been treated with ACE inhibitors or ARBs are inconsistent; some studies show increased circulating ACE2 activity while others do not (1) . Our finding that ARB treatment did not alter AT ACE2 expression is in agreement with a recent report showing that ARB treatment has no significant effect on systemic ACE2 levels, neither in COVID-19 patients nor in controls (8). A limitation of the present study is the lack of data on systemic RAS components due to unavailability of plasma samples. Notably, we cannot exclude that valsartan treatment alters RAS components, including ACE2, in people with (morbid) obesity, who usually present higher RAS activity in AT (2) . Likewise, it would be interesting to assess the effects of RAS blockade on AT expression of RAS components in patients with COVID- In conclusion, we provide the first evidence that the ARB valsartan does not affect the expression of RAS genes in AT and skeletal muscle in overweight humans. Our findings may help to better understand the results of ongoing clinical trials investigating the effects of RAS blockade on clinical outcomes in patients with confirmed COVID-19. Given the increased risk of worse clinical outcomes after SARS-CoV-2 infection in people with obesity, further studies are warranted to investigate the effects of RAS blockade on ACE2 in AT in people with obesity and COVID-19. Renin-Angiotensin-Aldosterone System Inhibitors in Patients with Covid-19 Obesity and COVID-19: A Perspective from the European Association for the Study of Obesity on Immunological Perturbations, Therapeutic Challenges, and Opportunities in Obesity Possible involvement of the adipose tissue reninangiotensin system in the pathophysiology of obesity and obesity-related disorders The mechanistic overview of SARS-CoV-2 using angiotensin-converting enzyme 2 to enter the cell for replication: possible treatment options related to the renin-angiotensin system Ongoing Clinical Trials for the Management of the COVID-19 COVID 19 and the Patient with Obesity -The Editors Speak Out This article is protected by copyright. All rights reservedWe would like to acknowledge the contribution of Prof. Michaela Diamant † , Dr. Chantalle Moors and Dr. Nynke van der Zijl to the execution of the original study. This article is protected by copyright. All rights reserved