[Norm’s note: This study ends with the conclusion 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.” That is in a narrow sense true. However, the generation of antibodies is a process and not something that is instantaneous. There is a period of time during which the S protein (spike protein) will be uninhibited by any antibody, namely, in the time it takes the immune system to get up to speed on antibody generation. In that period of time, as Doctors for Covid Ethics contend and as the results of this study actually if somewhat ironically corroborate, any S protein produced intravascularly as a result of vaccination will have a window of opportunity to potentially produce serious coagulopathies.]
Source: Circulation Research
SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE 2
Yuyang Lei, Jiao Zhang, Cara R. Schiavon, Ming He, Lili Chen, Hui Shen, Yichi Zhang, Qian Yin, Yoshitake Cho, Leonardo Andrade, Gerald S. Shadel, Mark Hepokoski, Ting Lei, Hongliang Wang, Jin Zhang, Jason X.-J. Yuan, Atul Malhotra, Uri Manor, Shengpeng Wang, Zu-Yi Yuan, John Y-J. ShyySee fewer authors
Originally published 31 Mar 2021
Circulation Research. 2021;128:1323–1326
Meet the First Author, see p 1239
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 which triggers mitochondrial reactive oxygen species production and glycolytic shift.2 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 In the current study, we show that S protein alone can damage vascular endothelial cells (ECs) by downregulating ACE2 and consequently inhibiting mitochondrial function.
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 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).
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 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 Shown in Figure [E], the mRNA levels of NRF1, HO1, and TFAM (mitochondria biogenesis-related genes) were increased, whereas those of HK2, PFKFB3, and ENO2 (glycolysis-related genes) were decreased in lung ECs in ACE2-D mice, as compared to those in ACE2-L mice.
SARS-CoV-2 infection induces EC inflammation, leading to endotheliitis.1,5 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–induced 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–induced vasodilation between ACE2-D and ACE-L animals.
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 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.
Nonstandard Abbreviation and Acronyms
|eNOS||endothelial NO synthase|
|S protein||Spike glycoprotein|
The data that support the findings of this study, including statistical analyses and reagents used, are available from the corresponding author upon request.
Sources of Funding
This work was supported in part by grants from the National Natural Science Foundation of China (NSFC) grants 81870220 (S. Wang), 81800328 (J.Z.), 81941005 (Z.-Y. Yuan); Shaanxi Natural Science Fund S2020-JC-JQ-0239 (S. Wang); The National Key Research and Development Program (Grant No. 2018YFC1311500; Z.-Y. Yuan); the Clinical Research Award of the First Affiliated Hospital of Xi’an Jiaotong University (Grant No. XJTU1AF-CRF-2016-004; Z.-Y. Yuan); Xi’an Jiaotong University Financial support.
*Y. Lei and J. Zhang contributed equally.
†U. Manor, S. Wang, Z.-Y. Yuan, and J.Y.-J. Shyy contributed equally as senior authors.
For Sources of Funding and Disclosures, see page 1324.Correspondence to: John Y-J. Shyy, PhD, Division of Cardiology, Department of Medicine, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093, Email email@example.comZu-Yi Yuan, MD, PhD, Department of Cardiology, First Affiliated Hospital of Xi’an Jiaotong University, 277 Yanta W Rd, Xi’an 710061, China, Email firstname.lastname@example.org
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