ACE2 and Proinflammatory Signaling by S1 Protein of SARS‐Cov‐2 in Human Endothelial Cells

Abstract

IntroductionCOVID‐19 is primarily a respiratory disease associated with cardiovascular risk. SARS‐CoV‐2, the virus causing COVID‐19, uses ACE2, an important enzyme in the cardiovascular system that regulates the conversion of Ang II (deleterious/pro‐hypertensive) to Ang 1‐7 (protective/anti‐hypertensive), as a receptor for host cell entry and infection. Considering the relationship between the viral S1‐protein and the host's ACE2, it is unclear whether this interaction is merely a mechanism of infection or whether it also contributes to cardiovascular damage associated with COVID‐19. We hypothesisedthat SARS‐Cov‐2‐ACE2 interaction induces activation of vascular cell inflammatory responses that are influenced by ACE2 dependent and/or independent enzymatic Ang‐(1‐7) production.MethodsHuman microvascular endothelial cells (MEC) were used and stimulated with SARS‐CoV‐2 recombinant S1 protein (rS1p) (0.66 μg/mL) at 10/30 min (acute) and 5/24h (chronic). Activation of pro‐inflammatory signaling pathways (immunoblotting, real‐time PCR), microparticle (MP) generation (NanoSight), and cytokine production (ELISA) were assessed. In some experiments, cells were pre‐incubated with an ACE2 activator (DIZE – 190 nM) and inhibitor (MLN‐4760 – 440 pM).ResultsrS1P increased NFκB activation (Control ©=0.99±0.06 vs. 1.38±0.19 AU; p<0.05) and MP formation (C=1.01±0.17 vs. 2.06±0.21, x109/mL; p<0.05), a marker of endothelial cell damage. mRA expression of IL‐1β (C=1.07±0.13 vs. 50.04±4.63 2^‐ddCT), IL6 (C=1.15±0.0.19 vs. 13.52±2 2^‐ddCT), TNFα (C=1.12±0.16 vs. 20.29±2.15 2^‐ddCT), VCAM‐1 (C=1.21±0.24 vs. 13.39±1.57 2^‐ddCT) and MCP‐1 (C=1.02±0.06 vs. 7.01±2.16 2^‐ddCT) was increased by rS1p (p<0.05). This was associated with an increased production of IL6 (C=22.77±3.27 vs. 1221±18.33 pg/mL) and MCP‐1 (C=876.9±33.47 vs. 1110±13.33 pg/mL, (p<0.05). rS1p did not change gene levels of TGFβ, galectin‐3 or ACE2. While DIZE did not influence rS1p pro‐inflammatory markers; MLN‐4760 potentiated rS1p‐induced increase in IL‐1β gene levels (rS1p=27.59±5.05 vs. MLN=62.11±11.17 2^‐ddCT) and blocked rS1p effects on MCP‐1 (rS1p=5.08±0.47 vs. MLN=2.43±0.6 2^‐ddCT) and VCAM‐1 (rS1p=72.82±13.76 vs. MLN=20.74±4.53 2^‐ddCT). ACE2 inhibition did not interfere with rS1p‐induced increase in IL6 or TNFα mRNA expression. Analysis of MP content revealed the expression of ACE2 short form (60 kDa) and flotillin‐1, suggesting that ACE2 localizes in membrane microdomains.ConclusionsOur data demonstrate that in human endotheial cells rS1P induces a pro‐inflammatory response and stimulates generation of ACE2‐containing microparticles. These findings suggest that the spike protein 1 of SARS‐CoV‐2 has a direct injurious effect on the endothelium that may contribute to endotheliitis in COVID‐19. Whether these processes are associated with viral infection await clarification.