ENDOTHELIAL CELLS AND BLOOD VESSELS – CRITICAL TARGETS FOR COVID-19-INDUCED TISSUE INJURY AND SPREAD TO DISTANT ORGANS. UNDERLYING MECHANISMS INCLUDE ENDOTHELIAL DYSFUNCTION AND INJURY

ABSTRACT

Endothelial cells (ECs) lining the blood vessels of all organs express the SARS-CoV2 receptor. In the absence of preexisting tissue damage, the virus would need to pass through the ECs to blood vessels to infect other tissues. Thus, EC are a target for SARS-CoV-2 infection and a conduit for viral dissemination to distant organs. We hypothesized that ECs infection and/ or injury are the mechanisms of COVID-19 pathology and multi-organ dissemination and injury. Methods: Human studies We used lung, heart, kidney, and small bowel specimens obtained during autopsies (n=5) from COVID-19 patients and uninfected subjects. Studies 1) histologic evaluation of endothelial damage and endotheliitis, 2) immunohistochemistry for vWF, PAI-1, VCAM-1, & ICAM-1. Studies in cultured human microvascular ECs (HMVECs) We cultured lung and cardiac HMVECs in the presence or absence of SARSCoV- 2 S1 and/or S2 protein (10 ng/ml) for 0 - 24 hr. Studies1) cell viability and proliferation;2) angiogenesis on Matrigel and cell migration;3) mitochondrial membrane potential (MMP);4) RNA seq analysis;5) Western blotting for vWF, PAI-1, VCAM-1, and ICAM-1. We examined the protective effect of melatonin, Coenzyme Q10 and nerve growth factor on S1/S2 protein induced HMVEC cell damage. Results: Histopathologic examination revealed presence of endothelial abnormalities and endotheliitis with marked presence of inflammatory cells in vessel wall & lumen, and fibrinous microthrombi) in lung, heart & kidney in autopsy specimens of COVID-19 patients. Immunostaining visualized increased vWF, PAI-1, VCAM- 1, & ICAM-1 in COVID-19. In in vitro study, S1 and S2 proteins induced endothelial injury, reduced angiogenesis and phosphorylated/activated Erk and Akt proteins in cultured HMVECs. Treatment of HMVECs for 1 & 4 hours with S2 but not S1 protein increased ICAM-1 levels by 1.4- to 1.8-fold (P < 0.001). RNA Seq analysis showed that treatment of HMVECs with S1 and S2 proteins upregulated VCAM-1, ICAM-1 and E-selectin mRNA in cultured HMVECs. Melatonin, Coenzyme Q10 and NGF stimulated angiogenesis in HMVECs by 2.4-, 1.3-&1.4-fold (all P < 0.001). Conclusions: 1) Significant endothelial abnormalities, blood vessel damage and endotheliitis are present in lung, heart and kidney autopsy specimens of COVID-19 patients, 2) There is increased expression of vWF, PAI-1, VCAM-1, and ICAM- 1 in lung, heart, and kidney specimens of COVID-19 patients, 3) Treatment of cultured HMVECs with SARS-CoV-2 S1 and S2 proteins upregulates VCAM-1, ICAM-1 and Eselectin expression, 4) SARS-CoV-2 S1 and S2 proteins induce endothelial injury in cultured HMVECs, and 5) melatonin, Coenzyme Q10 and NGF stimulated EC function. These studies uncovered novel mechanism – endothelial dysfunction underlying SARS-CoV-2 and identified melatonin, Coenzyme Q10 and NGF as potential drugs for treatment of COVID- 19-induced EC injury