(CCSP126) SARS-COV-2 SPIKE PROTEIN INDUCES ENDOTHELIAL CELL INFLAMMATION VIA ACE2 INDEPENDENTLY OF ENZYMATIC FUNCTION

Background: COVID-19 is primarily a respiratory disease associated with cardiovascular damaging effects, where it is predicted that patients may develop cardiovascular disease (CVD), including hypertension, where endotheliitis is a risk factors for these adverse outcomes. Considering the relationship between the viral spike protein and the host’s ACE2, it is unclear whether this interaction is merely a mechanism of infection or whether it also contributes to vascular injury induced by SARS-CoV-2.

METHODS AND RESULTS: Human endothelial cells (EC), previously shown to be resistant to SARS-CoV-2 infection, were exposed to recombinant S1p [(rS1p) 0.66 μg/mL] for 24h. Label-free quantitation of full proteome of ECs with/without rS1p stimulation were performed. Gene expression was assessed by RT-PCR and cytokine release was assessed by ELISA. Protein expression was assessed by immunoblotting and microparticle formation was quantified by Nanosight technology. ACE2 was blocked by MLN4760 (ACE2 inhibitor) and siRNA. Proteome of ECs after exposure to rS1p identified 1150 proteins from pathways associated with NFκB signaling and inflammation, including upregulation of ICAM-1; one of the 21 proteins found to be differentially expressed vs vehicle-treated cells (fold change >2, p< 0.05). SARS-CoV-2 spike protein 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) and PAI1 (5.6±0.7 vs. C: 2.9±0.2) in an ACE2 dependent manner as MLN4760 and ACE2 siRNA blocked rS1P effects, p< 0.05. These effects were also independent of ACE2 enzymatic activity as treatment with diminazine aceturate (DIZE; an ACE2 activator) nor antagonists of Ang II or Ang(1-7) receptors (AT1R, AT2R and MasR) did not influence rS1p-induced inflammatory responses in hECs. Microparticles release, a marker of EC injury, was increased by rS1p. ACE2 and flotilin-2, were present in rS1p-derived microparticles, suggesting that ACE2 may be part of cholesterol-rich domains, an important hub of cell signaling control.

Conclusion: ACE2 regulates SARS-CoV2 spike protein inflammatory signaling in human endothelial cells, independently of virus infection. These molecular processes may contribute to vascular damage in COVID-19.