The paper, published on April 30, 2021, in Circulation Research, also shows conclusively that COVID-19 is a vascular disease, demonstrating exactly how the SARS-CoV-2 virus damages and attacks the vascular system on a cellular level. The findings help explain COVID-19’s wide variety of seemingly unconnected complications, and could open the door for new research into more effective therapies.
A lot of people think of it as a respiratory disease, but it’s really a vascular disease,” says Assistant Research Professor Uri Manor, who is co-senior author of the study. “That could explain why some people have strokes, and why some people have issues in other parts of the body. The commonality between them is that they all have vascular underpinnings.Salk researchers collaborated with scientists at the University of California San Diego on the paper, including co-first author Jiao Zhang and co-senior author John Shyy, among others.While the findings themselves aren’t entirely a surprise, the paper provides clear confirmation and a detailed explanation of the mechanism through which the protein damages vascular cells for the first time. There’s been a growing consensus that SARS-CoV-2 affects the vascular system, but exactly how it did so was not understood. Similarly, scientists studying other coronaviruses have long suspected that the spike protein contributed to damaging vascular endothelial cells, but this is the first time the process has been documented.In the new study, the researchers created a “pseudovirus” that was surrounded by SARS-CoV-2 classic crown of spike proteins, but did not contain any actual virus. Exposure to this pseudovirus resulted in damage to the lungs and arteries of an animal model—proving that the spike protein alone was enough to cause disease. Tissue samples showed inflammation in endothelial cells lining the pulmonary artery walls.The team then replicated this process in the lab, exposing healthy endothelial cells (which line arteries) to the spike protein. They showed that the spike protein damaged the cells by binding ACE2. This binding disrupted ACE2’s molecular signaling to mitochondria (organelles that generate energy for cells), causing the mitochondria to become damaged and fragmented.Previous studies have shown a similar effect when cells were exposed to the SARS-CoV-2 virus, but this is the first study to show that the damage occurs when cells are exposed to the spike protein on its own.“If you remove the replicating capabilities of the virus, it still has a major damaging effect on the vascular cells, simply by virtue of its ability to bind to this ACE2 receptor, the S protein receptor, now famous thanks to COVID,” Manor explains. “Further studies with mutant spike proteins will also provide new insight towards the infectivity and severity of mutant SARS CoV-2 viruses.”The researchers next hope to take a closer look at the mechanism by which the disrupted ACE2 protein damages mitochondria and causes them to change shape.Other authors on the study are Yuyang Lei and Zu-Yi Yuan of Jiaotong University in Xi’an, China; Cara R. Schiavon, Leonardo Andrade, and Gerald S. Shadel of Salk; Ming He, Hui Shen, Yichi Zhang, Yoshitake Cho, Mark Hepokoski, Jason X.-J. Yuan, Atul Malhotra, Jin Zhang of the University of California San Diego; Lili Chen, Qian Yin, Ting Lei, Hongliang Wang and Shengpeng Wang of Xi’an Jiatong University Health Science Center in Xi’an, China.The research was supported by the National Institutes of Health, the National Natural Science Foundation of China, the Shaanxi Natural Science Fund, the National Key Research and Development Program, the First Affiliated Hospital of Xi’an Jiaotong University; and Xi’an Jiaotong University.DOI: 10.1161/CIRCRESAHA.121.318902
SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE 2
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).
Figure. 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.A, 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, 1×108 PFU). Thickened alveolar septa (red arrowhead) and mononuclear cell (red arrow). Scale bar=20 μm. B, Pseu-Spike (n=4) or mock virus (n=4)–infected 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 β-actin (B, 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, ii). C, Representative confocal images of mitochondrial morphology of ECs treated with human recombinant S1 protein or IgG (4 μg/mL) for 24 h (C, i) or infected with human adenovirus ACE2 S680D (ACE2-D) or ACE2 S680L (ACE2-L; 10 MOI) for 48 h (C, ii). Mitochondria were visualized using TOM20 antibody (n=4, 50 cells counted for each replicate). Scale bar=2.5 μm. Tubular: the majority of mitochondria in ECs was >10 μm in length; Intermediate: the mitochondria were <≈10 μm; Fragment: the majority of mitochondria were spherical (no clear length or width). D, Measurement of oxygen consumption rate (OCR, D, i and iii) and extracellular acidification rate (ECAR, D, 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 μg/mL) for 24 h (n=3). E, 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, Dose-response curves of acetylcholine (ACh, left)- and sodium nitroprusside (SNP, right)–mediated relaxation on the tension of phenylephrine (1 μmol/L) precontracted intrapulmonary artery stripes from Pseu-Spike-(ACh n=8, SNP n=5) or mock (ACh n=6, SNP n=5) virus–infected Syrian hamsters (1×108 PFU; F, i) and ACE2-D (n=6) or ACE2-L (n=5) mice (F, ii). The animal experiments were approved by the ethical committee of Xi’an 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.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
| ACE | angiotensin-converting enzyme |
| ECs | endothelial cells |
| eNOS | endothelial NO synthase |
| pACE2 | phospho-ACE2 |
| pAMPK | phospho-AMPK |
| S protein | Spike glycoprotein |
Data Availability
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 suppor