{"id":3143,"date":"2021-11-29T17:32:37","date_gmt":"2021-11-29T17:32:37","guid":{"rendered":"http:\/\/wp.cov19longhaulfoundation.org\/?p=3143"},"modified":"2021-11-29T17:32:37","modified_gmt":"2021-11-29T17:32:37","slug":"sars-cov-2-spike-protein-impairs-endothelial-function-via-downregulation-of-ace-2-2","status":"publish","type":"post","link":"https:\/\/cov19longhaulfoundation.org\/?p=3143","title":{"rendered":"SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE 2"},"content":{"rendered":"\n

Authors: Yuyang Lei<\/a>,1,2,*<\/sup>Jiao Zhang<\/a>,1,2,5,*<\/sup>Cara R. Schiavon<\/a>,8,9<\/sup>Ming He<\/a>,5<\/sup>Lili Chen<\/a>,2<\/sup>Hui Shen<\/a>,5,10<\/sup>Yichi Zhang<\/a>,5<\/sup>Qian Yin<\/a>,2<\/sup>Yoshitake Cho<\/a>,5<\/sup>Leonardo Andrade<\/a>,8<\/sup>Gerald S. Shadel<\/a>,9<\/sup>Mark Hepokoski<\/a>,6<\/sup>Ting Lei<\/a>,3<\/sup>Hongliang Wang<\/a>,4<\/sup>Jin Zhang<\/a>,7<\/sup>Jason X.-J. Yuan<\/a>,6<\/sup>Atul Malhotra<\/a>,6<\/sup>Uri Manor<\/a>,8,\u2020<\/sup>Shengpeng Wang<\/a>,2,\u2020<\/sup>Zu-Yi Yuan<\/a>,1,\u2020<\/sup> and John Y-J. Shyy<\/a>5,\u2020<\/sup><\/p>\n\n\n\n

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection relies on the binding of S protein (Spike glycoprotein) to ACE (angiotensin-converting enzyme) 2 in the host cells. Vascular endothelium can be infected by SARS-CoV-2,1<\/a><\/sup> which triggers mitochondrial reactive oxygen species production and glycolytic shift.2<\/a><\/sup> Paradoxically, ACE2 is protective in the cardiovascular system, and SARS-CoV-1 S protein promotes lung injury by decreasing the level of ACE2 in the infected lungs.3<\/a><\/sup> In the current study, we show that S protein alone can damage vascular endothelial cells (ECs) by downregulating ACE2 and consequently inhibiting mitochondrial function.<\/p>\n\n\n\n

We administered a pseudovirus expressing S protein (Pseu-Spike) to Syrian hamsters intratracheally. Lung damage was apparent in animals receiving Pseu-Spike, revealed by thickening of the alveolar septa and increased infiltration of mononuclear cells (Figure [A]). AMPK (AMP-activated protein kinase) phosphorylates ACE2 Ser-680, MDM2 (murine double minute 2) ubiquitinates ACE2 Lys-788, and crosstalk between AMPK and MDM2 determines the ACE2 level.4<\/a><\/sup> In the damaged lungs, levels of pAMPK (phospho-AMPK), pACE2 (phospho-ACE2), and ACE2 decreased but those of MDM2 increased (Figure [B], i). Furthermore, complementary increased and decreased phosphorylation of eNOS (endothelial NO synthase) Thr-494 and Ser-1176 indicated impaired eNOS activity. These changes of pACE2, ACE2, MDM2 expression, and AMPK activity in endothelium were recapitulated by in vitro experiments using pulmonary arterial ECs infected with Pseu-Spike which was rescued by treatment with N-acetyl-L-cysteine, a reactive oxygen species inhibitor (Figure [B], ii).<\/a><\/a>Open in a separate window<\/a>Figure.<\/a><\/p>\n\n\n\n

SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) Spike protein exacerbates endothelial cell (EC) function via ACE (angiotensin-converting enzyme) 2 downregulation and mitochondrial impairment.<\/strong> A<\/strong>, Representative H&E histopathology of lung specimens from 8- to 12 wk-old male Syrian hamsters 5-day post administration of pseudovirus overexpressing Spike protein (Pseu-Spike) or mock virus in control group (n=3 mice per group, 1\u00d7108<\/sup> PFU). Thickened alveolar septa (red arrowhead) and mononuclear cell (red arrow). Scale bar=20 \u03bcm. B<\/strong>, Pseu-Spike (n=4) or mock virus (n=4)\u2013infected hamster lungs were subjected to Western blot analysis for pAMPK (phospho-AMPK) T172, AMPK, pACE2 (phospho angiotensin-converting enzyme) S680, ACE 2, MDM2, peNOS S1176, peNOS T494, eNOS (endothelial NO synthase), and \u03b2-actin (B<\/strong>, i). Human pulmonary arterial EC (PAECs) were infected with Pseu-Spike or mock virus for 24 h with or without N-acetyl-L-cysteine (NAC; 5 mmol\/L) pretreatment for 2 h. The protein extracts were analyzed by Western blot using antibodies against proteins as indicated (n=4; B<\/strong>, ii). C<\/strong>, Representative confocal images of mitochondrial morphology of ECs treated with human recombinant S1 protein or IgG (4 \u03bcg\/mL) for 24 h (C<\/strong>, i) or infected with human adenovirus ACE2 S680D (ACE2-D) or ACE2 S680L (ACE2-L; 10 MOI) for 48 h (C<\/strong>, ii). Mitochondria were visualized using TOM20 antibody (n=4, 50 cells counted for each replicate). Scale bar=2.5 \u03bcm. Tubular: the majority of mitochondria in ECs was >10 \u03bcm in length; Intermediate: the mitochondria were <\u224810 \u03bcm; Fragment: the majority of mitochondria were spherical (no clear length or width). D<\/strong>, Measurement of oxygen consumption rate (OCR, D<\/strong>, i and iii) and extracellular acidification rate (ECAR, D<\/strong>, ii and iv) in ECs infected with ACE2-D vs ACE2-L (10 MOI) for 48 h (n=3) or treated with IgG vs S1 protein (4 \u03bcg\/mL) for 24 h (n=3). E<\/strong>, Real-time quantitative polymerase chain reaction analysis of the indicated mRNA levels in lung ECs from ACE2-D (n=4) and ACE2-L (n=4) knock-in mice. Eight-week-old ACE2-D and ACE2-L male mice with C57BL\/6 background were used. F<\/strong>, Dose-response curves of acetylcholine (ACh, left<\/strong>)- and sodium nitroprusside (SNP, right<\/strong>)\u2013mediated relaxation on the tension of phenylephrine (1 \u03bcmol\/L) precontracted intrapulmonary artery stripes from Pseu-Spike-(ACh n=8, SNP n=5) or mock (ACh n=6, SNP n=5) virus\u2013infected Syrian hamsters (1\u00d7108<\/sup> PFU; F<\/strong>, i) and ACE2-D (n=6) or ACE2-L (n=5) mice (F<\/strong>, ii). The animal experiments were approved by the ethical committee of Xi\u2019an Jiaotong University. 2-DG indicates 2-Deoxy-D-glucose; ACE2-D, a phospho-mimetic ACE2 with increased stability; ACE2-L, a dephospho-mimetic ACE2 with decreased stability; AMPK, AMP-activated protein kinase; AA\/R, antimycin A&Rotenone; ENO2, enolase 2; FCCP, carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone; H&E, Hematoxylin and Eosin; HK2, hexokinase 2; HO1, heme oxygenase-1; MDM2, murine double minute 2; MOI, multiplicity of infection; NRF1, nuclear respiratory factor 1; peNOS, phospho-eNOS; PFKFB3, 6-phosphofructo-2-kinase\/fructose-2,6-biphosphatase 3; Resp, respiration; and TFAM, transcription factor A, mitochondrial.<\/p>\n\n\n\n

We next studied the impact of S protein on mitochondrial function. Confocal images of ECs treated with S1 protein revealed increased mitochondrial fragmentation, indicating altered mitochondrial dynamics (Figure [C], i). To examine whether these mitochondrial changes were due, in part, to the decreased amount of ACE2, we overexpressed ACE2 S680D (ACE2-D, a phospho-mimetic ACE2 with increased stability) or S680L (ACE2-L, a dephospho-mimetic with decreased stability)4<\/a><\/sup> in ECs. As shown in Figure [C], ii, ECs with ACE2-L had a higher number of fragmented mitochondria when compared to those with ACE2-D. Performing oxygen consumption rate and extracellular acidification rate assays, we found that ECs overexpressing ACE2-L had reduced basal mitochondrial respiration, ATP production, and maximal respiration compared to ECs overexpressing ACE2-D (Figure [D], i). Moreover, ACE2-L overexpression caused increased basal acidification rate, glucose-induced glycolysis, maximal glycolytic capacity, and glycolytic reserve (Figure [D], ii). Also, ECs incubated with S1 protein had attenuated mitochondrial function but increased glycolysis, when compared with control cells treated with IgG (Figure [D], iii and iv). We also compared the expressions of mitochondria- and glycolysis-related genes in lung ECs isolated from ACE2-D or ACE2-L knock-in mice.4<\/a><\/sup> Shown in Figure [E], the mRNA levels of NRF1<\/em>, HO1<\/em>, and TFAM<\/em> (mitochondria biogenesis-related genes) were increased, whereas those of HK2<\/em>, PFKFB3<\/em>, and ENO2<\/em> (glycolysis-related genes) were decreased in lung ECs in ACE2-D mice, as compared to those in ACE2-L mice.<\/p>\n\n\n\n

SARS-CoV-2 infection induces EC inflammation, leading to endotheliitis.1<\/a>,5<\/a><\/sup> Because S protein decreased ACE2 level and impaired NO bioavailability, we examined whether S protein entry is indispensable for dysfunctional endothelium. As shown in Figure [F], i, the endothelium-dependent vasodilation induced by acetylcholine was impaired in pulmonary arteries isolated from Pseu-Spike-administered hamsters, whereas the endothelium-independent vasodilation induced by sodium nitroprusside was not affected. We also compared the acetylcholine- and sodium nitroprusside\u2013induced vasodilation of pulmonary vessels from ACE2-D or ACE2-L mice. As anticipated, acetylcholine-induced vasodilation was hindered in pulmonary arteries isolated from ACE2-L mice in comparison to ACE2-D mice (Figure [F], ii). There was, however, little difference in sodium nitroprusside\u2013induced vasodilation between ACE2-D and ACE-L animals.<\/p>\n\n\n\n

Although the use of a noninfectious pseudovirus is a limitation to this study, our data reveals that S protein alone can damage endothelium, manifested by impaired mitochondrial function and eNOS activity but increased glycolysis. It appears that S protein in ECs increases redox stress which may lead to AMPK deactivation, MDM2 upregulation, and ultimately ACE2 destabilization.4<\/a><\/sup> Although these findings need to be confirmed with the SARS-CoV-2 virus in the future study, it seems paradoxical that ACE2 reduction by S protein would decrease the virus infectivity, thereby protecting endothelium. However, a dysregulated renin-angiotensin system due to ACE2 reduction may exacerbate endothelial dysfunction, leading to endotheliitis. Collectively, our results suggest that the S protein-exerted EC damage overrides the decreased virus infectivity. This conclusion suggests that vaccination-generated antibody and\/or exogenous antibody against S protein not only protects the host from SARS-CoV-2 infectivity but also inhibits S protein-imposed endothelial injury.<\/p>\n\n\n\n

For More Information: https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8091897\/<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"

Authors: Yuyang Lei,1,2,*Jiao Zhang,1,2,5,*Cara R. Schiavon,8,9Ming He,5Lili Chen,2Hui Shen,5,10Yichi Zhang,5Qian Yin,2Yoshitake Cho,5Leonardo Andrade,8Gerald S. Shadel,9Mark Hepokoski,6Ting Lei,3Hongliang Wang,4Jin Zhang,7Jason X.-J. Yuan,6Atul Malhotra,6Uri Manor,8,\u2020Shengpeng Wang,2,\u2020Zu-Yi Yuan,1,\u2020 and John Y-J. Shyy5,\u2020 SARS-CoV-2 (severe acute […]<\/p>\n","protected":false},"author":2,"featured_media":3144,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[144,289,290,337,541,607,608],"tags":[],"class_list":["post-3143","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-endothelium","category-long-haul-disease","category-long-term-effects","category-mrna","category-spike-protein","category-vaccine-news","category-vaccine-safety"],"_links":{"self":[{"href":"https:\/\/cov19longhaulfoundation.org\/index.php?rest_route=\/wp\/v2\/posts\/3143","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/cov19longhaulfoundation.org\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/cov19longhaulfoundation.org\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/cov19longhaulfoundation.org\/index.php?rest_route=\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/cov19longhaulfoundation.org\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=3143"}],"version-history":[{"count":0,"href":"https:\/\/cov19longhaulfoundation.org\/index.php?rest_route=\/wp\/v2\/posts\/3143\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/cov19longhaulfoundation.org\/index.php?rest_route=\/wp\/v2\/media\/3144"}],"wp:attachment":[{"href":"https:\/\/cov19longhaulfoundation.org\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=3143"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/cov19longhaulfoundation.org\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=3143"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/cov19longhaulfoundation.org\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=3143"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}