O63 SARS-CoV-2 S PROTEIN REDUCES CYTOPROTECTIVE DEFENSES AND PROMOTES HUMAN ENDOTHELIAL CELL DYSFUNCTION AND SENESCENCE: PROTECTION BY KLOTHO AND ANGIOTENSIN-(1-7)

Background and objective: Premature vascular aging and endothelial cell senescence are major risk factors for cardiovascular diseases, including hypertension and atherothrombotic disturbances, which are main complications of both acute and long COVID-19. SARS-CoV-2 S protein, which forms the crown of the coronavirus, can induce endothelial cells inflammation and has been found as an isolated element in both the circulation and human tissues reservoirs months after infection. The aim of this study was to investigate whether S protein is able to directly induce endothelial cell senescence and endothelial dysfunction and to decipher some of the mechanisms involved in such deleterious effect. Methods: Using primary cultures of human umbilical vein endothelial cells (HUVEC), cellular senescence was assessed by senescence-associated-β galactosidase (SA-β-gal) staining and the quantification of DNA damage response markers (γH2AX) and growth-arrest effectors (p53, p21, p16) by Western blot. Cytoprotective proteins were equally determined by Western blot. In isolated murine mesenteric microvessel, myography was used to assess defective vascular relaxation, as a key marker of endothelial dysfunction. Results: In primary cultures of human umbilical vein endothelial cells (HUVEC), SARS-CoV-2 S protein enhanced in a concentration-dependent manner from a threshold of 35 nmol/L the cellular content of senescence and DNA damage response markers, as well as growth-arrest effectors. In parallel, the S protein reduced the availability of cytoprotective proteins, such as the anti-aging protein klotho, Nrf2 or heme oxygenase-1, and caused functional harm by impairing ex vivo endothelial-dependent vasorelaxation in murine microvessels. These effects were prevented by the pharmacological inhibition of the NLRP3 inflammasome with MCC950. Furthermore, the supplementation with either recombinant klotho or angiotensin-(1-7), equally protected against the pro-senescence, pro-inflammatory and pro-oxidant action of the S protein. Conclusions: Globally, this study proposes novel mechanisms of disease in the context of COVID-19 and its vascular sequelae and provides pharmacological clues in order to prevent such complications.