Abstract 11093: Spike Proteins Increase Endothelial Calcium via TRPV4

Abstract

Introduction: Endothelial dysfunction plays a central role in the pathogenesis of acute respiratory distress syndrome (ARDS) with COVID-19. Transient receptor potential vanilloid 4 (TRPV4), a cation channel ubiquitously expressed, can regulate inflammatory cytokines that play key roles in in acute lung injury/ARDS. However, it is unknown whether spike proteins can affect TRPV4 activity and related Ca 2+ signaling in pulmonary microvascular endothelial cells. Hypothesis: We hypothesized that spike protein causes activation of TRPV4 channels, resulting in increases in intracellular Ca 2+ , may lead to pulmonary endothelial dysfunction. Methods: Intracellular Ca 2+ concentrations in human lung microvascular endothelial cells (HLMECs) were measured by calcium imaging in the presence of SARS CoV-2 Spike protein S1, receptor-binding domain (RBD) of S1, or protein S2 with or without co-incubation of the selective TRPV4 antagonist (HC-067047). Results: The intracellular Ca 2+ concentration of HLMECs was significantly increased when incubated with S1 (1, 10nM) or S1 RBD (1, 10nM) for 12, 24, 48 hours, relative to control or S2 (p<0.05, respectively, Fig. A, B). Co-incubation of HC-067047 (500nM) significantly attenuated Ca 2+ intracellular influx upon treatment with S1 (10nM, 24 hours, p<0.05) or S1 RBD (10nM, 24 hours, p<0.05) (Fig. C). TRPV4 sensitive current density was significantly increased when incubated with S1 (10nM) or S1 RBD (10nM) for 24 hours (p<0.05 vs. control, respectively, Fig. D-G), whereas co-incubated with HC-067047 (500nM) significantly reversed the S1 (10nM, 24 hours, p<0.05) or S1 RBD (10nM, 24 hours, p<0.05) induced increases of TRPV4 sensitive current density (Fig. D-G). Conclusions: The of SARS CoV-2 Spike protein S1 and S1 RBD caused the activation of TRPV4 channels, resulting in increased intracellular Ca 2+ , may lead to pulmonary endothelial dysfunction.