RESUMO
Severe coagulopathy has been observed at the level of the microcirculation in several organs including lungs, heart and kidneys in patients with COVID-19, and in a minority of subjects receiving the SARS-CoV-2 vaccine. Various mechanisms have been implicated in these effects, including increases in circulating neutrophil extracellular traps, excessive inflammation, and endothelial dysfunction. Even if a correlation between infection by SARS-CoV-2 and upregulation of coagulation cascade components has been established in the lung, no direct proofs have been yet provided about the transcriptional machinery controlling the expression of these factors. Recent results obtained by us reported a novel transcriptional function of the SARS-CoV-2 Spike (S) viral protein involving a direct protein-protein interaction with the human Estrogen Receptor- (ER). Given the implications of ER in the control of key effectors in the coagulation cascade, we hypothesized that S-protein might increase the pro-coagulation activity of endothelial cells via the transcriptional activity of the ER, thus justifying the enhanced risk of thrombosis. To assess this, we tested the effects of S-protein on the expression of Tissue Factor (TF) and the overall procoagulation activity in a human endothelial cell line and confirmed this finding by overexpressing S-protein by gene transfer in mice. We then designed and tested two-point mutations in the S2 S-protein sequence that abolished the pro-coagulation function of S-protein in vitro and in vivo, without compromising its immunogenicity. In addition to reveal a new potential transcriptional function of S-protein, these results inspire the design of new vaccines with lower risk of thrombogenesis. Indeed, while the benefit/risk ratio remains overwhelming in favor of COVID-19 vaccination, our results shed light on the causal mechanisms of some rare anti-SARS-CoV-2 vaccine adverse events, and are thus essential for current and future vaccination and booster campaigns.
RESUMO
Patients with severe respiratory syndrome caused by SARS-CoV-2 undergo cardiac complications due to hyper-inflammatory conditions. Although the presence of the virus has been detected in the myocardium of infected patients, and infection of cardiac cells may involve ACE2 receptor, the underlying molecular/cellular mechanisms are still uncharacterized. We analyzed expression of ACE2 receptor in primary human cardiac stromal cells using proteomic and transcriptomic methods before exposing them to SARS-CoV-2 in vitro. Using conventional and high sensitivity PCR methods, we measured virus production in the cellular supernatants and monitored the intracellular viral bioprocessing. We performed high-resolution imaging to show the sites of intracellular viral production. We finally used Q-RT-PCR assays to detect genes linked to innate immunity and fibrotic pathways coherently regulated in cells exposed to virus. Our findings indicate that human cardiac stromal cells have a susceptibility to SARS-CoV-2 infection and produce variable viral yields depending on the extent of cellular ACE2 receptor expression. Interestingly, these cells also evolved toward hyper-inflammatory/pro-fibrotic phenotypes independently of ACE2 levels, suggesting a dual cardiac damage mechanism that could account for the elevated numbers of cardiac complications in severe COVID-19 cases.
