key: cord-0771960-uppmn9hy
authors: Wang, Yu; Takeshita, Hikari; Yamamoto, Koichi; Huang, Yibin; Wang, Cheng; Nakajima, Tsuneo; Nozato, Yoichi; Fujimoto, Taku; Yokoyama, Serina; Hongyo, Kazuhiro; Nakagami, Futoshi; Akasaka, Hiroshi; Takami, Yoichi; Takeya, Yasushi; Sugimoto, Ken; Rakugi, Hiromi
title: A pressor dose of angiotensin II has no influence on the angiotensin‐converting enzyme 2 and other molecules associated with SARS‐CoV‐2 infection in mice
date: 2021-02-10
journal: FASEB J
DOI: 10.1096/fj.202100016r
sha: 2e66a0dd3665f1f559551372e6695100c58b268a
doc_id: 771960
cord_uid: uppmn9hy

In the early phase of the Coronavirus disease 2019 (COVID‐19) pandemic, it was postulated that the renin‐angiotensin‐system inhibitors (RASi) increase the infection risk. This was primarily based on numerous reports, which stated that the RASi could increase the organ Angiotensin‐converting enzyme 2 (ACE2), the receptor of Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), in rodents. RASi can theoretically antagonize the potential influence of angiotensin II (Ang II) on ACE2. However, while Ang II decreases the ACE2 levels in cultured cells, there is little evidence that supports this phenomenon in living animals. In this study, we tested whether Ang II or Ang II combined with its antagonist would alter the ACE2 and other molecules associated with the infection of SARS‐CoV‐2. Male C57BL6/J mice were administered vehicle, Ang II (400 ng/kg/min), or Ang II with losartan (10 mg/kg/min) for 2 weeks. ACE2 knockout mice were used as a negative control for the ACE2 assay. We found that both Ang II, which elevated blood pressure by 30 mm Hg, and Ang II with losartan, had no effect on the expression or protein activity of ACE2 in the lung, left ventricle, kidney, and ileum. Likewise, these interventions had no effect on the expression of Transmembrane Protease Serine 2 (TMPRSS2) and Furin, proteases that facilitate the virus‐cell fusion, and the expression or activity of Tumor Necrosis Factor α‐Convertase (TACE) that cleaves cell‐surface ACE2. Collectively, physiological concentrations of Ang II do not modulate the molecules associated with SARS‐CoV‐2 infection. These results support the recent observational studies suggesting that the use of RASi is not a risk factor for COVID‐19.

Angiotensin-converting enzyme (ACE) 2, named for its counter-regulatory role in the renin-angiotensin system (RAS), has been the target of diverse studies outside of the RAS owing to its multifunctional roles in many pathological and physiological conditions. 1 ACE2 is a zinc metalloprotease first reported in 2000 which shares homology with angiotensin-converting enzyme (ACE) in its catalytic domain and provides different key functions in the renin-angiotensin system (RAS). 2, 3 Recently, the importance of ACE2 has gained new interest, as it is the functional receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the new respiratory disease, Coronavirus disease 2019 (COVID- 19) , the pandemic that is currently ravaging through the world. 4 In the early phase of the COVID-19 pandemic, an argument was put forth to understand if antihypertensive treatment using renin-angiotensin-system inhibitors (RASi), including ACE inhibitors and Angiotensin receptor blockades (ARBs), is associated with increased susceptibility or severity of COVID-19 in hypertensive patients. [5] [6] [7] This hypothesis is primarily based on the notion that RASi could increase the expression or activity of ACE2. Previous studies indicate that RASi increase ACE2 expression or activity in multiple organs, including the heart, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] kidney, 8, 12, 13, 18, 19, 20 aorta, [21] [22] [23] [24] and lung, [25] [26] [27] in rodents. Theoretically, increased tissue ACE2 could increase the chances of virus invasion, resulting in the initiation of infection or local inflammation of each organ. However, recent observational studies indicate that the use of RASi is not associated with an increased risk of COVID-19 in patients with hypertension. [28] [29] [30] [31] Therefore, it is desirable to find a theoretical basis to explain why the use of RASi might not increase the risk of COVID-19 despite its potential influence on ACE2. In our review, we raised several issues that need to be addressed in order to understand the actual impact of RASi on ACE2 in relation to SARS-CoV-2 infection. First, it is unclear how RASi increase tissue ACE2 in living animals. A simple interpretation of the in vivo phenomenon is conceivable that RASi antagonize the effects of angiotensin II (Ang II) on the transcriptional regulation of ACE2. It is reported that Ang II decreased the mRNA and protein levels of ACE2 in cultured rat astrocytes 32, 33 and human kidney epithelial cells. 34 However, the concentration of Ang II used in these studies (10 −7 -10 −6 M) was far higher than the physiological levels in the plasma of mice (<1 × 10 −11 M), 35 rats (<1 × 10 −10 M), 36 and humans (<1-2 × 10 −11 M). 37 Interestingly, Petel et al reported that a 2-week infusion of Ang II in mice increased the mRNA expression of ACE2 in the heart, but paradoxically decreased the cardiac protein levels of ACE2. This was accompanied by the increased activity of the tumor necrosis factor-α conversion (TACE) that decreases the levels of cell surface full-length ACE2 by ectodomain shedding. 38 However, it should be noted that the concentration of Ang II (1.5 mg/kg/day) used in the study was high enough to increase BP by approximately 1.5-fold, and plasma Ang II concentration around 15-fold. 38 In contrast to the positive influence of Ang II on the mRNA expression of ACE2 reported by Petel et al. 38 Sriramula et al reported that Ang II infusion at 200 ng/kg/min for 2 weeks reduced mRNA and protein expression of ACE2 in the periventricular nucleus accompanied by 1.6-fold increase of BP in male Sprague-Dawley (SD) rats. 39 Gonzalez et al also reported that Ang II infusion at 400 ng/kg/min for 2 weeks reduced protein expression of ACE2 in the renal cortex and medulla accompanied by an increase of systolic BP up to over 200 mm Hg in male SD rats. 40 Collectively, Ang II at a level that could induce a vigorous increase in BP appears to decrease protein expression or activity of tissue ACE2 with the inconsistent transcriptional effect on this gene in rodents. Nevertheless, it remains to be understood whether the influence of Ang II on ACE2 is biologically relevant in animals at concentrations that could mimic physiological conditions. Second, the cellular invasion of SARS-CoV-2 depends not only on ACE2 but also on proteases, including Transmembrane Protease Serine 2 (TMPRSS2) and Furin. Spike proteins of SARS-CoV-2 bind to the cell surface ACE2, followed by proteolytic cleavage by these proteases, which results in the endocytosis of the virus. 41 It was recently reported that an ARB, losartan, or an ACE inhibitor, enalapril did not affect the organ expression of ACE2 and TMPRSS2 in male C57BL/6J mice. 42 However, it remains unknown whether Ang II or Ang II in combination with RASi would affect the expression of these proteases. Third, the effect of Ang II or RASi on the expression or activity of TACE, which is also a determinant of the localization of ACE2 on the cell surface, needs to be examined to understand the net influence of RASi on SARS-CoV-2 infection. Finally, as most studies analyzed the effect of RASi in each organ individually, there is little knowledge about the relative effects of RASi on ACE2 in different organs.

To address these issues, we conducted a cross-organ analysis of the lung, small intestine, heart, and kidney in mice infused with a lower dose of Ang II than previously reported, 38 to understand the effect of Ang II or ARB on ACE2 and the associated molecules involved in the tissue invasion of SARS-CoV-2. Samples from ACE2 knockout mice were used as negative controls to confirm the assay specificity of ACE2 in the present study.

angiotensin II, angiotensin-converting enzyme 2, ARB, COVID-19, SARS-CoV-2 | 3 of 9 WANG et Al.

Wild-type C57BL/6 mice were purchased from CLEA Japan. ACE2 knockout (ACE2KO) mice with a C57/BL6 background were generated as previously described. 43 Mice were fed standard chow and water ad libitum. A schematic of the study protocol is shown in Figure 1A . At 14 to 15 week of age, male wild-type mice were randomly assigned to three groups, and they received either Ang II (400 ng/kg/min) (n = 9) or Ang II with losartan (10 mg/kg/min) (n = 8) or saline (n = 8) for 2 weeks via subcutaneous implantation of osmotic minipumps (model 1004, ALZET Corp, Palo Alto, CA). The concentration of losartan was determined based on previous papers. [44] [45] [46] After 2 weeks of infusion, tissues were excised, weighed, and subjected to further analysis. Blood pressure (BP) was measured before and after the administration of reagents using the tail-cuff method (Softron Co, Tokyo, Japan). Three 19-week-old and one 27-week-old ACE2KO mice were used as a negative control for the evaluation of ACE2 gene expression and activity, and tissues were collected in the same manner as that for wild-type mice. During the procedure of the implantation of osmotic mini-pumps and tissue collection, mice were anesthetized with intraperitoneal injection of a mixture of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg) following the guideline of Osaka University Institutional Animal Care and committee.

The study protocol was approved by the Osaka University Institutional Animal Care and Use Committee and was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

The left ventricle, lung, kidney, and ileum were collected, weighed, and divided into three parts for RNA extraction, and the evaluation of ACE2 and TACE protein activity. For RNA extraction, tissues were stored in RNAlater according to the manufacturer's instructions (Thermo Fisher Scientific, USA). For the activity evaluation, tissues were immediately frozen in liquid nitrogen and stored at −80°C until further use.

ACE2 activity was measured using an MCA-based peptide substrate in the ACE2 assay kit (ab273297, Abcam, UK) according to the manufacturer's instructions with the modification based on previous reports. 38, 47 Briefly, tissues were homogenized in the assay buffer containing a protease inhibitor cocktail (78415, Thermo Fisher Scientific, USA), using TissueLyser LT (Qiagen, Germany). Thereafter, tissue homogenates were incubated with protease inhibitors, including 10 µmol/L of captopril (07134-21, Nacalai Tesque, Japan), 5 µmol/L of amastatin (A1276, Sigma-Aldrich, USA), 10 µmol/L of bestatin (B8385, Sigma-Aldrich, USA), and 10 µmol/L of Z-Pro-prolinal (BML-PI112-0005, Enzo Life Sciences, USA) in the absence or presence of 10µmol/l of MLN-4760 (CS-0015513, Chemscene, USA), an ACE2 inhibitor at 37°C for 30min. Fluorescence intensity was monitored every 5 min for 30 min and measured using a Tecan SPARK multimode microplate reader with appropriate filters (Ex/Em = 320/420 nm) (Tecan Trading AG, Switzerland). The protein concentration of the homogenate was F I G U R E 1 Schematic overview and the influence of Ang II infusion on blood pressure (BP). A, Schematic overview. B, BP before and after the infusion of Ang II (400 ng/kg/min), Ang II with losartan (10 mg/kg/min) or saline. Difference between BP before and after the infusion in each group was analyzed with paired t test. Difference in the before-after change in BP among the groups was analyzed by repeated measures ANOVA with the Tukey post hoc test. Ang II, angiotensin II; Los, losartan determined with the Bradford protein assay (Bio-Rad Protein Assay Reagent, Bio-Rad Laboratories). Tissue ACE2 activity was defined as the amount of enzyme that catalyzes the release of 1 pmol of MCA per min per mg protein from the substrate under the assay conditions at 25°C (pmol/min/mg protein). The final ACE2 activity was determined by subtracting the measurement value in the presence of MLN-4760 from that in the absence of MLN-4760.

TACE activity was assessed using the SensoLyte 520 TACE activity assay (AnaSpec, San Jose, CA) kit according to the manufacturer's instructions. Tissues were homogenized in the assay buffer using TissueLyser LT (Qiagen, Germany). Fluorescence intensity was monitored every 5 min for 15 min and measured using a Tecan SPARK multimode microplate reader with filters to detect the fluorescence (Ex/ Em = 490/520 nm). (Tecan Trading AG, Switzerland). The protein concentration of the homogenate was determined using the Bradford protein assay (Bio-Rad Protein Assay Reagent, Bio-Rad Laboratories). Tissue TACE activity was calculated to represent the amount of enzyme that catalyzes the release of 1 μmol of 5-FAM per min per mg protein from the substrate under the assay conditions at 25°C (μmol/min/mg protein).

Total RNA from the lung, ileum, and kidney was isolated using Sepasol (R)-RNA Super G (09379-55; Nacalai Tesque, Japan) after treatment with DNase (Code No. 2270A, Takara Bio Company, Japan). Total RNA of the left ventricle was isolated using the ReliaPrepTM RNA Tissue Miniprep System (Z6110, Promega, USA). cDNA synthesis was performed with a ReverTra Ace qPCR RT kit (FSQ-101, Toyobo, Japan) according to the manufacturer's instructions. All genes were evaluated using the SYBR green qPCR system (Applied Biosystems 7900HT Fast real-time PCR system, Thermo Fisher Scientific, USA) with the primers listed in Table S1 . Relative expression was calculated using the ΔΔCt method with normalization to the 18S gene.

The minimal sample size in each group was calculated as 7 by Power and Sample Size Calculation 3.1 to detect a 50% difference in the real-time PCR analysis, with a standard deviation of 0.3 when the expression level of vehicle treatment was determined as 1 (α error = 0.05, β error = 0.2). All data are presented as mean ± SEM. Difference in BP before and after the administration of Ang II, Ang II with losartan, or saline was analyzed by paired t test, and difference in the beforeafter change in BP among the groups was analyzed by repeated measures ANOVA. Differences among multiple groups were analyzed by one-way ANOVA with Bonferroni testing. An outlier in each group in each analysis was excluded when determining significance by Grubbs' test with α error < 0.01.

Two-week continuous infusion of Ang II at 400 ng/kg/min increased the systolic BP by approximately 30 mm Hg (before to after the infusion, 111 ± 4 mm Hg to 139 ± 5 mm Hg, respectively, P < .01) ( Figure 1B ). Co-administration of losartan blunted the pressor effect of Ang II (109 ± 2 mm Hg to 112 ± 2 mm Hg, P = .15) ( Figure 1B ). There were no differences in the body weight and organ weight of the lung, ileum, kidney, and left ventricle among the groups (Table S2 ).

The expression of ACE2 and the associated genes involved in the tissue invasion of SARS-CoV-2 was analyzed using real-time PCR in the lung, ileum, kidney, and left ventricle. We confirmed that the expression of ACE2 was the highest in the ileum and the lowest in the left ventricle ( Figure 2A) . In all the organs tested, neither Ang II nor Ang II with losartan altered the expression of ACE2, which was absent in ACE2 knockout mice ( Figure 2B ). Consistent with previous reports, the expression of TMPRSS2 was very low in the left ventricle ( Figure 2A ). 48 We did not find any difference in the expression of TMPRSS2, Furin, and TACE in response to Ang II or Ang II with losartan in the tested organs ( Figure 2B ).

We measured tissue ACE2 activity using the conventional MCA-based peptide substrate under the treatment with several protease inhibitors including an ACE inhibitor, captopril. 38, 47, 49 In all the organs, neither Ang II nor Ang II with losartan altered the ACE2 activity in wild-type mice ( Figure 3A ). In addition, the TACE activity was also not altered by Ang II or Ang II with losartan in the lung, ileum, kidney, and left ventricle ( Figure 3B ). F I G U R E 2 RT-PCR analysis of tissue expression of ACE2, TMPRSS2, Furin, and TACE in mice. A, Comparison of the expression of the corresponding genes across the organs in vehicle-treated wild-type mice. Y-axis represents log-transformed ΔΔCt of each gene normalized to 18S. B, The expression of the corresponding genes among wild-type mice with Ang II (400 ng/kg/min), Ang II with losartan (10 mg/kg/min) or saline, and ACE2KO mice. Differences were analyzed with a one-way ANOVA using the Bonferroni post hoc test. *P < 0.05 vs. wild-type mice with saline. † P < 0.05 vs. wild-type mice with Ang II. ‡ P < 0.05 vs. wild-type mice with Ang II and losartan

In the current study, we found that a 2-week administration of Ang II increased the systolic BP by approximately 30 mm Hg. However, this did not alter the mRNA abundance of ACE2 in multiple organs, including the lung and ileum, which are the vulnerable sites for the initial invasion of SARS-CoV-2. The same was the case for the left ventricle and kidney, where local inflammation by direct viral invasion is associated with the severity of COVID-19. [50] [51] [52] [53] [54] Ang II also did not alter the expression of TMPRSS2 and Furin, proteases that facilitate viral invasion after binding to ACE2, and TACE, which regulates the surface expression of ACE2 by proteolytic cleavage. Losartan, an ARB that antagonized the pressor effect of Ang II, did not affect the tissue expression of ACE2 and the associated genes in all the tissues tested. In accordance with the transcript analysis, we found that neither Ang II nor Ang II with losartan had an effect on the tissue activity of ACE2 and TACE. Collectively, the current findings clearly excluded the possibility that a pressor dose of Ang II is a potent regulator of ACE2 and the genes associated with the SARS-CoV-2 infection in rodents ( Figure 4) . Accordingly, ARBs did not appear to alter these molecules by antagonizing the effect of Ang II. Considering the numerous reports on the effects of RASi on the tissue levels of ACE2, it is safe to assume that factors other than the increase in Ang II are involved in the effect of these drugs on ACE2 expression.

High doses of Ang II downregulate ACE2 in cultured cells. [32] [33] [34] Koka et al reported that Ang II at 0.5 μM, but not 0.25 μM, decreased the expression of ACE2 in cultured human kidney tubular cells. 34 This suggests that Ang II downregulates ACE2 not simply by provoking specific downstream cellular signaling but by boosting vigorous cellular inflammation. Conversely, it is assumed that RASi could normalize the expression of ACE2, which is reduced because of cellular inflammation. This hypothesis is in line with previous reports where ACE2 was reduced by experimental intervention to provoke tissue injury in rodents, including experimental autoimmune myocarditis, 9 experimental CHF induced by an aortocaval fistula, 14 subtotal nephrectomy, 20 myocardial infarction by ligation of the left coronary artery, 11 and pulmonary injury induced by lipopolysaccharide 26, 27 or cigarettes. 25 RASi restored the expression or activity of ACE2 in these experimental models, accompanied by the alleviation of the pathological condition. 9, 11, 14, 20, [25] [26] [27] In addition, the hypothesis that the effect of RASi on ACE2 depends on their ability to attenuate tissue inflammation is also consistent with a previous report that high concentrations of Ang II decreased cardiac ACE2 protein levels despite the paradoxical increase in transcript levels as a result of increased TACE activity, which is commonly activated by inflammatory stimuli. 38, 55 Therefore, the potential effect of RASi on tissue ACE2 can be attributed to their ability to alleviate organ inflammation F I G U R E 3 Tissue activity of ACE2 and TACE in mice. The activity of (A) ACE2 and (B) TACE among wild-type mice with Ang II (400 ng/ kg/min), Ang II with losartan (10 mg/kg/min) or saline, and ACE2KO mice. Differences were analyzed with a one-way ANOVA using the Bonferroni post hoc test. *P < .05 vs. wild-type mice with saline. † P < 0.05 vs. wild-type mice with Ang II. ‡ P < .05 vs. wild-type mice with Ang II and losartan and damage (graphical abstract in Figure 4 ). This is consistent with recent observational studies suggesting that the use of RASi is not a risk in the treatment of COVID-19. [28] [29] [30] As we used 15-weeks-old young male mice in the study, our findings should be interpreted cautiously when considering the effect of angiotensin II or RASi on female or aged animals. Particularly, aging is considered a great risk for COVID-19 and the effect of angiotensin II on ACE2 and associated molecules might be different depending on the age of animals.

In summary, we found that the 2-week infusion of Ang II in normal mice increased the BP but did not alter the expression or activity of ACE2 and other molecules associated with SARS-CoV-2 infection. Accordingly, the inhibition of Ang II by ARB had no influence on the expression or activity of these molecules. This study clearly indicated that Ang II is not a direct transcriptional regulator of ACE2 in vivo, and the effect of RASi on ACE2 might depend on their ability to repair organ injury. Therefore, this study could provide a theoretical basis for the fact that the use of RASi is not a risk in hypertensive patients in this COVID-19 era.

We are grateful to Yoshinori Koishi and Giovanni Bravin for their excellent technical assistance.

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The authors have no financial conflicts of interest.