Abstract 17314: Flow-Mediated Susceptibility of Cerebral Endothelia to Sars-CoV-2 Infection Using Endothelialized 3D Human Vascular Models

Abstract

Introduction: The clinical syndrome associated with Sars-CoV-2 infection is associated with an increased rate of cerebrovascular events, predominantly ischemic stroke. Direct viral invasion of the cerebral endothelium could be a significant driver of Sars-CoV-2-mediated stroke through expression of ACE2 and viral entry co-factors including TMPRSS2. Prior work indicates that ACE2 mRNA and protein are expressed in the endothelial cells of the brain, yet the effect of flow-mediated phenomena on ACE2 expression and the susceptibility of human cerebral endothelia to Sars-CoV-2 infection is unknown. Methods: 3D printed models of the human middle cerebral artery were endothelialized with either human umbilical vein endothelial cells (HUVECs) or human brain microvascular endothelial cells (HBMECs). Endothelialized models were subjected to 3D rotational perfusional culture at variable pulsatile flow rates (0-22.5 dynes/cm 2 ). ACE2 and TMPRSS2 expression were measured by Taqman qPCR. Biotinylated recombinant Sars-CoV-2 S protein was used to judge regional vessel binding of virus using confocal microscopy. Results: In 3D models endothelialized with either HUVECs or HBMECs, ACE2 mRNA expression is significantly modulated by vessel diameter and the presence of of flow (15 dynes/cm 2 ) (p<0.0001 by two-way ANOVA). Pulsatile flow drives ACE2 expression more robustly in HUVECs than in HBMECs (11.5-fold vs. 4.9-fold; p<0.001). Higher flow rates (22.5 dynes/cm 2 ) produced further increases in ACE2 expression in both cell types. Gene expression analysis demonstrated no detection of the Sars-CoV-2 viral entry co-factor TMPRSS2 in HBMECs. Flow-mediated ACE2 expression was associated with binding of recombinant Sars-CoV-2 S protein to the vessel wall suggesting direct cerebrovascular susceptibility to Sars-CoV-2. Conclusion: The Sars-CoV-2 cellular receptor ACE2 is expressed by human cerebral endothelia in a flow-dependent manner. Cerebral vessels are susceptible to Sars-CoV-2 infection and bind viral S protein under flow conditions but likely requires unique co-factors for viral entry. Identification of the molecular mechanisms triggered by Sars-CoV-2 infection in brain vasculature can alleviate the burden of stroke associated with COVID-19.