COVID-19 IgG antibodies drive endothelial cell activation and dysfunction Free

The effect of COV-19 infection or post-acute sequelae of SARS-CoV-2 (PASC) increases risk of thrombotic events. The mechanism underlying cardiovascular complications post-COV-19 infections remains incompletely understood. During severe COV-19 infection, viral antigens trigger , initiating and amplifying the immune response. This cytokine storm serves to eliminate the virus but inflicts significant collateral damage on the surrounding cellular environment, especially the vascular endothelium. In this study, we hypothesized that the antibody-mediated response to COV-19 infection activates the vascular endothelium and alters endothelial barrier integrity. We also investigated the mechanism driving endothelial activation and dysfunction which fosters a procoagulant state during COV-19 infection and PASC.

To assess endothelial dysfunction and creation of a procoagulant state post-severe COV-19 infection, primary human umbilical vein endothelial cells (HUVEC) were isolated and incubated with 100 µg/mL IgG for 12 h. We compared the effects of IgG from 56 COV-19 patients with IgG from 12 healthy donors (HD IgG), as controls. Relative to HD-IgG, COV-19 IgG increased surface expression of vascular cell adhesion molecule-1, VCAM-1 (A_450nm = 0.491±0.09 in case of COV-19 IgG compared to A_450nm= 0.15±0.02 in HD IgG; P<0.05) used as a marker of endothelial cell activation. Using an assay of plasma clot formation in which HUVEC treated with HD or COV-19 IgG was exposed to normal human plasma and clot formation measured by turbidity (A_405nm), we observed that 11 COV-19 IgG that induced increased VCAM-1 expression caused enhanced clot formation on HUVEC with maximal V_max of 55±18 and decreased lag time of 18±5 min compared to HD-IgG (V_max=35±5; lag time= 43±7 min). Increased clot formation was associated with increased expression of tissue factor mRNA (fold change increase = 4), plasminogen activator inhibitor type 1, PAI-1 (fold change increase =30), and TMEM-16F mRNA (fold change increase=2.5), though the relative contribution of each of these mediators to the prothrombotic phenotype is under investigation. We also observed delayed clot lysis after initial clot formation on HUVEC, possibly reflecting the effects of PAI-1, which was the highest upregulated HUVEC gene after exposure to COV-19 IgG.

COV-19 infection is also associated with increased vascular permeability that contributed substantially to the clinical manifestations of the disease. To investigate the potential effects of COV-19 on endothelial cell barrier function we stained COV-19 treated HUVEC for VE-cadherin, an important adhesion molecule in endothelial cells. We observed that COV-19 IgG induced internalization and nuclear localization of VE-cadherin, in association with the formation of extensive contractile stress fibers leading to destabilization of cell-cell contacts.

In preliminary analyses we observed that the correlation (r²) between levels of CL IgG and LBPA IgG in CoV -19 IgG fraction was 0.6545, whereas that between the EC activation markers, VCAM-1 or PAI-1 and CL IgG was 0.5479 and 0.6066 respectively. The correlation between VCAM-1 or PAI-1 and LBPA IgG were 0.2256 and 0.3791 respectively.

The nature of the endothelial cell reactive antibodies contained in COV-19 IgG is uncertain at this point, but the effects observed occurred in the absence of added β2GPI. Analysis of proteins immunoprecipitated by COV-19 IgG is currently being undertaken.

Previous studies have shown that IgG fractions from COV-19 patients are prothrombotic, but the mechanisms of these antibodies and the cellular antigens they recognize are uncertain. Our studies begin to address these concerns on a molecular mechanistic level and may lead to better understanding of the endotheliopathy that may underlie several viral syndromes.