SARS‐CoV‐2 spike protein induces degradation of junctional proteins that maintain endothelial barrier integrity

Abstract

Background SARS‐CoV‐2 enters cells through the Angiotensin‐converting enzyme 2 (ACE‐2) receptor present on the cell surface. ACE‐2 is present in many cell types including endothelial cells, where it functions to protect against oxidative damage. Reports suggest that COVID‐19 patients also showed symptoms related to endothelial damage. We hypothesized that these vascular symptoms might be associated with disrupted endothelial barrier integrity. We investigated this in vitro using endothelial cell culture and recombinant SARS‐CoV‐2 spike protein S1 Receptor‐Binding Domain (S1RBD). Materials and Methods Mouse brain microvascular endothelial cells from normal (C57BL/6 mice) and diabetic (db/db) mice were used. The expression of VE‐Cadherin, PECAM‐1, JAM‐A and Connexin 43, was probed by Western blot in normal and diabetic cells, before and after treatment with S1RBD. Results The expression of VE‐Cadherin, an endothelial adherens junction protein, was not affected by diabetes or by S1RBD treatment. PECAM‐1 expression showed a small but significant (~15%) decrease after S1RD treatment in control cells. Junctional Adhesion Molecule (JAM‐A), a tight junction protein, and the gap junction protein, Connexin‐43, decreased by ~50 and 80% respectively in the presence of S1RBD when compared to untreated controls. Expression of PECAM‐1, JAM‐A and connexin‐43 were significantly lower in untreated diabetic endothelial cells, compared to untreated control cells. This expression level further decreased significantly in the presence of S1RBD, with maximal effects observed with JAM‐A and Connexin‐43 expression (~80 and 90% decrease, respectively). These results indicate that healthy and diabetic endothelial cells respond differently when challenged with SARS‐CoV‐2 S1RBD. Conclusion S1RBD‐induces degradation of endothelial junctional proteins that likely affects endothelial barrier function and causes vascular damage.

SARS‐CoV‐2 spike spurs intestinal inflammation via VEGF production in enterocytes

“…In order to validate whether SARS‐CoV‐2 was responsible for the VEGF production in intestinal tissue, we generated an animal model to specifically mimic the intestinal inflammation by intraperitoneally injecting the recombinant spike‐Fc containing the receptor binding domain (RBD) to C57BL/6J mice (Fig EV2A). Co‐localization of murine ACE2 with spike was confirmed by immunofluorescence staining (Fig EV2B–D), which was consistent with previous results (Kuba et al , 2005; Raghavan et al , 2021; Shin et al , 2021). Since intestinal mucosal barrier renders strong protection from the infectious agents (Sharma & Riva, 2020), treating mice with spike RBD alone for a short time only gave rise to mild inflammation (Fig EV2E).…”

“…Here, we provide an alternative route by showing that the ERK/VEGF axis in enterocytes plays a pivotal role in SARS‐CoV‐2‐induced intestinal inflammation. However, we have no adequate evidence to support the mediating role of ACE2 in the SARS‐CoV‐2 spike‐induced effects since the affinity of murine ACE2 and SARS‐CoV‐2 spike is still debatable (Li et al , 2004; Kuba et al , 2005; Zhao et al , 2020; Gu et al , 2021; Nuovo et al , 2021; Raghavan et al , 2021; Shin et al , 2021; Wang et al , 2021). We consider that whether the effect of spike RBD is through or fully through ACE2 is beyond the scope of this work.…”

“…Increased inflammatory cytokines from intestine could overflow to bloodstream and cause lung inflammation (Mjo ¨sberg & Rao, 2018). Current studies proved that SARS-CoV-2 infection could induce vascular barrier dysfunction in vivo and in vitro (Colunga Biancatelli et al, 2021;Raghavan et al, 2021). However, as a critical component of GI barrier (Bouziat & Jabri, 2015), the role and the mechanism of intestinal vascular barrier in GI inflammation and disease progression are still unclear.…”

Adverse effects of COVID-19 mRNA vaccines: the spike hypothesis

“…injection of the S1 subunit in mice results in its localization in endothelia of mice brain microvessels showing colocalization with ACE2, caspase-3, IL-6, tumor necrosis factor α (TNF-α), and C5b-9; it was thus suggested that endothelial damage is a central part of SARS-CoV-2 pathology which may be induced by the S protein alone [75]. Also, the S1 subunit (or recombinant S1 RBD) impaired endothelial function via downregulation of ACE2 [76] and induced degradation of junctional proteins that maintain endothelial barrier integrity in a mouse model of brain microvascular endothelial cells or cerebral arteries; this latter effect was more enhanced in endothelial cells from diabetic versus normal mice [77]. Similarly, the S1 subunit decreased microvascular transendothelial resistance and barrier function in cultured human pulmonary cells [78].…”

Low-Dose Interferon I and Iii-Based Nasal Sprays: A Good-Looking Covid-19 Vaccine Candidate?

“…the venae cavae and the aorta) will be affected in people with more severe forms of COVID-19, as the number of tissues infected by the virus is generally proportional with the intensity of the symptoms. It is important to note that the spike protein is often capable of damaging the endothelial cell barrier and entering the bloodstream (Raghavan et al, 2021), and that the way the novel SARS-CoV-2 vaccines have been designed can often allow the pathogenic protein to reach the endothelial cells and then important blood vessels.…”