Abstract P431: SARS-CoV-2-induced Inflammation In Human Endothelial Cells Involves ACE2

COVID-19 is associated with hypertension, where endotheliitis and endothelial dysfunction are risk factors. Considering the relationship between the viral spike protein and ACE2, it is unclear whether this interaction is merely a mechanism of infection or whether contributes to vascular injury induced by SARS-CoV-2. Here we tested whether ACE2 acts as a receptor for SARS-CoV-2 pro-inflammatory responses, independently of ACE2 enzymatic function, in human endothelial cells (ECs). ECs and ACE2-expressing HEK cells were exposed to recombinant SARS-CoV-2 spike protein [(rS1p) 0.66 μg/mL] for 24h. Gene expression was assessed by RT-PCR and cytokine release was assessed by ELISA. Protein expression was assessed by immunoblotting. ACE2 was blocked by MLN4760 (ACE2 inhibitor) and siRNA. Label-free quantitation of full proteome of ECs after rS1p exposure or ACE2 co-immunoprecipitation was performed. Co-localization studies were performed by confocal microscopy. Proteomics identified 1150 proteins associated with NFκB signalling and inflammation. 21 proteins were found to be differentially expressed vs vehicle-treated cells (fold change >2, p<0.05), including upregulation of ICAM-1. rS1p increased levels of IL6 (1221.2±18.3 vs. C:22.77±3.2 pg/mL); ICAM1 (17.7±3.1 vs. C:3.9±0.4 AU); PAI1 (5.6±0.7 vs. C: 2.9±0.2) and NFκB activation (0.13±0.004 vs. C:0.06±0.01 AU); p<0.05. MLN4760 and ACE2 siRNA blocked rS1P effects. rS1p did not alter ACE2 enzymatic activity. HEK-ACE2 cells had increased IL-8 (78.7±8 vs. C:56.2±4 pg/mL), while rS1p induced IL-8 production only in HEK expressing ACE2 (ACE2:112.8±5.3 vs. C:60.3±3.3 pg/mL); p<0.05. ACE2 CO-IP proteomics identified 216 proteins (filtered with ≥1 unique peptide, 1% FDR) along with ACE-2 (11 peptides). Potential candidate interacting proteins include caveolin 1 (Cav 1). Co-localization studies confirmed interactions between ACE2/Cav-1 in ECs, which was lost after rS1p stimulation. SARS-CoV2 spike protein induces pro-inflammatory responses in ECs via receptor-like function of ACE2. These molecular processes may contribute to vascular damage after COVID-19.