Angiotensin Type 1 Receptor-Dependent Internalization of SARS-CoV-2 by Angiotensin-Converting Enzyme 2
2021; Lippincott Williams & Wilkins; Volume: 77; Issue: 4 Linguagem: Inglês
10.1161/hypertensionaha.120.16795
ISSN1524-4563
AutoresBlessing Ogunlade, Eric Lazartigues, Catalin M. Filipeanu,
Tópico(s)Computational Drug Discovery Methods
ResumoHomeHypertensionVol. 77, No. 4Angiotensin Type 1 Receptor-Dependent Internalization of SARS-CoV-2 by Angiotensin-Converting Enzyme 2 Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBAngiotensin Type 1 Receptor-Dependent Internalization of SARS-CoV-2 by Angiotensin-Converting Enzyme 2 Blessing O. Ogunlade, Eric Lazartigues and Catalin M. Filipeanu Blessing O. OgunladeBlessing O. Ogunlade https://orcid.org/0000-0002-4256-4547 From the Department of Pharmacology, College of Medicine, Howard University, Washington, DC (B.O.O., C.M.F.) , Eric LazartiguesEric Lazartigues Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans (E.L.) Southeast Louisiana Veterans Health Care Systems, New Orleans (E.L.) Cardiovascular Center of Excellence, Louisiana State University Health Sciences Center, New Orleans (E.L.). and Catalin M. FilipeanuCatalin M. Filipeanu Correspondence to: Catalin M. Filipeanu, Department of Pharmacology, College of Medicine, Howard University, Washington, DC 20059. Email E-mail Address: [email protected] https://orcid.org/0000-0002-7298-9327 From the Department of Pharmacology, College of Medicine, Howard University, Washington, DC (B.O.O., C.M.F.) Originally published20 Jan 2021https://doi.org/10.1161/HYPERTENSIONAHA.120.16795Hypertension. 2021;77:e42–e43Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 20, 2021: Ahead of Print The current coronavirus disease 2019 (COVID-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus, which infects the cells by interaction of its envelope S1 spike protein (S1) with ACE2 (angiotensin-converting enzyme 2).1 ACE2 is a carboxypeptidase and a negative regulator of the renin-angiotensin system, reducing the levels of Ang II (angiotensin II) and its pathological actions in cardiovascular diseases. Early on, conflicting views were expressed regarding the use of AT1R (angiotensin type 1 receptor) blockers in patients with COVID-19,2 and incertitude remains regarding the role of the renin-angiotensin system in SARS-CoV-2 infection.We previously reported that cellular ACE2 activity is strictly dependent on its plasma membrane localization. The enzyme is internalized by Ang II, and this effect depends on AT1R expression.3 In HEK293T cells, which do not endogenously express AT1R, treatment with S1 (10–300 ng/mL) failed to induce ACE2 internalization (Figure [A], top) and consequently, we detected no decrease in enzymatic activity (Figure [B], left). Upon AT1R transfection ACE2 internalization was observed following Ang II and S1 exposure (Figure [A], bottom). The effects of Ang II and S1 were not additive, similar reduction in ACE2 activity being observed after simultaneous addition of both treatments (29.6±2.5%). Like for Ang II, S1-driven internalization depended on ACE2/AT1R ratio (Figure [B]). Pretreatment with the typical AT1R blocker losartan (1 μmol/L) or the β-arrestin biased AT1R ligand TRV0274 (1 μmol/L) fully prevented the effects of Ang II or S1. These drugs have no effect on ACE2 activity in the absence of AT1R (data not shown).Download figureDownload PowerPointFigure. The effects of Ang II (angiotensin II) and S1 spike protein (S1) on ACE2 (angiotensin-converting enzyme 2) in HEK293T cells. A, Subcellular localization of ACE2 (green) in HEK293T cells cotransfected with 0.5 μg GFP (green fluorescent protein)-tagged ACE2 and 1 μg pcDNA3.1 (control vector, top) or HA-tagged AT1R (angiotensin type 1 receptor; bottom) after vehicle (left), Ang II (1 μmol/L, middle) or S1 (30 ng/mL, right) treatment for 4 h. B, ACE2 activity in HEK293T cells cotransfected with GFP-tagged ACE2 (0.5 μg) and HA-tagged AT1R (0, 0.5, and 1 μg). The total transfected cDNA was kept at 1.5 μg by addition of necessary amounts of pcDNA3.1. ACE2 activity was determined in vehicle-treated cells or after 4 h (corresponding to maximal effect on enzyme activity) treatment with Ang II (1 μmol/L) or S1 (30 ng/mL). C, Pretreatment (30 min) with the AT1R antagonist losartan (1 μmol/L,) or β-arrestin biased agonist TRV027 (100 nmol/L) blocked Ang II– or S1-mediated reduction of ACE2 activity in HEK293T cells transfected with 0.5 μg GFP-ACE2 and 1 μg AT1R. D, Effects of treatment for 18 h (corresponding to maximal effect on protein levels) with Ang II (1 μmol/L) or S1 (30 ng/mL) on total ACE2 cellular levels determined by Western blotting. E, Pretreatment (30 min each) with losartan (1 μmol/L), HCQ (hydroxychloroquine; 100 μmol/L) or sucrose (100 mmol/L) blocks the decrease of ACE2 activity induced by Ang II (1 μmol/L) or S1 (30 ng/mL). The data are presented as mean ±SEM with n=3–4 from at least 3 independent transfections. The experiments were performed as described previously5 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456754/).As observed for Ang II, S1-induced ACE2 internalization resulted in enzyme degradation (Figure [D]), an effect also prevented by losartan. Accordingly, the lysosomal inhibitor leupeptin prevented the loss of ACE2 activity (Figure [E]) and its degradation (data not shown). Hydroxychloroquine (100 μmol/L), which inhibits lysosomal fusion to endosomes and the activity of lysosomal enzymes, reversed the effects of Ang II and S1 on ACE2 activity (Figure [E]). The use of sucrose as a hyperosmolar agent prevented the loss of ACE2 activity (Figure [E]), thus confirming the importance of clathrin-coated pits in ACE2 internalization.In summary, this is the first demonstration that S1 binding to ACE2 induces enzyme internalization through clathrin-coated pits, followed by lysosomal degradation, as previously reported by our group for Ang II.3 This process requires AT1R expression and higher AT1R levels potentiate the internalization of S1, component of SARS-CoV-2 envelope. One can envision that S1 stimulates the formation of AT1R/ACE2 complexes which are required for enzyme internalization, but further experiments are required to validate this hypothesis.Blockade of lysosomal function by leupeptin or hydroxychloroquine fully prevented the effects of S1 on ACE2 internalization and degradation. Importantly from a clinical point of view, our cellular experiments strongly indicate that losartan, an extensively used antihypertensive drug, blocked ACE2 internalization induced by S1. Therefore, unlike initial concerns,2 the use of AT1R blockers in COVID-19 may produce unexpected benefits. However, clinical studies are necessary to confirm these in vitro results.Nonstandard Abbreviations and AcronymsACE2angiotensin-converting enzyme 2Ang IIangiotensin IIAT1Rangiotensin type 1 receptorCOVID-19coronavirus disease 2019AcknowledgmentsThe use of RCMI core facilities at Howard University is gratefully acknowledged.Sources of FundingThis work was supported by the National Institutes of Health (NIH)/National Heart, Lung, and Blood Institute (NHLBI) HL150592 Grant (to C.M. Filipeanu and E. Lazartigues), Merit Award (BX004294) from the US Department of Veterans Affairs (to E. Lazartigues), and bridge funds from Howard University (to C.M. Filipeanu).Disclosures None.FootnotesFor Sources of Funding and Disclosures, see page e43.Correspondence to: Catalin M. Filipeanu, Department of Pharmacology, College of Medicine, Howard University, Washington, DC 20059. Email catalin.filipeanu@howard.eduReferences1. Lan J, Ge J, Yu J, Shan S, Zhou H, Fan S, Zhang Q, Shi X, Wang Q, Zhang L, et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor.Nature. 2020; 581:215–220. doi: 10.1038/s41586-020-2180-5CrossrefMedlineGoogle Scholar2. Danser AHJ, Epstein M, Batlle D. Renin-angiotensin system blockers and the COVID-19 pandemic: at present there is no evidence to abandon renin-angiotensin system blockers.Hypertension. 2020; 75:1382–1385. doi: 10.1161/HYPERTENSIONAHA.120.15082LinkGoogle Scholar3. Deshotels MR, Xia H, Sriramula S, Lazartigues E, Filipeanu CM. Angiotensin II mediates angiotensin converting enzyme type 2 internalization and degradation through an angiotensin II type I receptor-dependent mechanism.Hypertension. 2014; 64:1368–1375. doi: 10.1161/HYPERTENSIONAHA.114.03743LinkGoogle Scholar4. Carvalho-Galvão A, Ogunlade B, Xu J, Silva-Alves CRA, Mendes-Júnior LG, Guimarães DD, Cruz JC, Queiroz TM, Balarini CM, Braga VA, et al. Central administration of TRV027 improves baroreflex sensitivity and vascular reactivity in spontaneously hypertensive rats.Clin Sci (Lond). 2018; 132:1513–1527. doi: 10.1042/CS20180222CrossrefMedlineGoogle Scholar5. Ogunlade B, Guidry JJ, Mukerjee S, Sriramula S, Lazartigues E, Filipeanu CM. The actin bundling protein fascin-1 as an ACE2-accessory protein [published online August 31, 2020].Cell Mol Neurobiol. doi: 10.1007/s10571-020-00951-x. https://link.springer.com/article/10.1007/s10571-020-00951-xCrossrefGoogle Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.Sign In to Submit a Response to This Article Previous Back to top Next FiguresReferencesRelatedDetailsCited By Zorad S, Skrabanova M, Zilkova M, Cente M, Turic Csokova N, Kovacech B, Cizkova D and Filipcik P (2024) Angiotensin I and II Stimulate Cell Invasion of SARS-CoV-2: Potential Mechanism via Inhibition of ACE2 Arm of RAS, Physiological Research, 10.33549/physiolres.935198:1/2024, (27-35) Kang S, Kim Y and Cho H (2024) COVID-19 Outcome and Tobacco Product Use: Case-Control and Retrospective Cohort Studies Using Nationwide Samples, Journal of Korean Medical Science, 10.3346/jkms.2024.39.e103, 39:11 Papadopoulos K, Papadopoulou A and Aw T (2023) Live to die another day: novel insights may explain the pathophysiology behind smoker's paradox in SARS-CoV-2 infection, Molecular and Cellular Biochemistry, 10.1007/s11010-023-04681-8, 478:11, (2517-2526), Online publication date: 1-Nov-2023. 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Altman N, Berning A, Saxon C, Adamek K, Wagner J, Slavov D, Quaife R, Gill E, Minobe W, Jonas E, Carroll I, Huebler S, Raines J, Messenger J, Ambardekar A, Mestroni L, Rosenberg R, Rove J, Campbell T and Bristow M Myocardial Injury and Altered Gene Expression Associated with SARS-CoV-2 Infection or mRNA Vaccination, SSRN Electronic Journal, 10.2139/ssrn.4137991 April 2021Vol 77, Issue 4 Advertisement Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/HYPERTENSIONAHA.120.16795PMID: 33470144 Originally publishedJanuary 20, 2021 Keywordsangiotensin IIangiotensin-converting enzyme 2cardiovascular diseasescoronavirusreceptor, angiotensin, type 1PDF download Advertisement SubjectsACE/Angiotensin Receptors/Renin Angiotensin SystemBasic Science ResearchMechanisms
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