Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the Angiotensin converting enzyme 2 (ACE2) receptor present on the cell surface to enter cells. Angiotensin converting enzyme 2 is present in many cell types including endothelial cells, where it functions to protect against oxidative damage. There is growing evidence to suggest that coronavirus disease (COVID-19) patients exhibit a wide range of post-recovery symptoms and shows signs related to cardiovascular and specifically, endothelial damage. We hypothesized that these vascular symptoms might be associated with disrupted endothelial barrier integrity. This was investigated in vitro using endothelial cell culture and recombinant SARS-CoV-2 spike protein S1 Receptor-Binding Domain (Spike). Mouse brain microvascular endothelial cells from normal (C57BL/6 mice) and diabetic (db/db) mice were used. An endothelial transwell permeability assay revealed increased permeability in diabetic cells as well as after Spike treatment. The expression of VE-Cadherin, an endothelial adherens junction protein, JAM-A, a tight junctional protein, Connexin-43, a gap junctional protein, and PECAM-1, were all decreased significantly after Spike treatment in control and to a greater extent, in diabetic cells. In control cells, Spike treatment increased association of endothelial junctional proteins with Rab5a, a mediator of the endocytic trafficking compartment. In cerebral arteries isolated from control and diabetic animals, Spike protein had a greater effect in downregulating expression of endothelial junctional proteins in arteries from diabetic animals than from control animals. In conclusion, these experiments reveal that Spike-induced degradation of endothelial junctional proteins affects endothelial barrier function and is the likely cause of vascular damage observed in COVID-19 affected individuals.

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.