D28-22 Ca2+ Influx Through Mechanosensitive Cation Channel Piezo1 Induces the Activation of Noncanonical Inflammasomes in Lung Vascular Endothelial Cells

Abstract

Vascular and perivascular inflammation is implicated in the development and progression of pulmonary vascular remodeling and vascular wall stiffening in patients with pulmonary arterial hypertension and animals with experimental pulmonary hypertension. Inflammasomes are cytosolic multiprotein complexes assembled in response to infection or cellular stress to induce the release of proinflammatory cytokines (IL-1β/IL-18) and to initiate pyroptosis, an inflammatory form of cell death. Noncanonical inflammasome, activated by intracellular lipopolysaccharide (LPS), causes the oligomerization and activation of caspase-4 (in humans) and caspase-11 (in mice) that induce pyroptosis through cleavage of gasdermin D (GSDMD), formation of N-GSDMD channels/pores, and enhancement of K+ efflux. Noncanonical inflammasomes and caspases-4/5 or 11 also trigger a secondary activation of the canonical NLRP3 inflammasome for releasing IL-1β and IL-18. Ca2+ influx and K+ efflux are implicated in the activation of the canonical NLRP3 inflammasomes. However, it remains unknown whether Ca2+ influx through mechanosensitive cation channels is involved in activation of the noncanonical inflammasome. Intracellular LPS increased non-selective cation currents in human lung vascular endothelial cells (LVEC), which is associated with increased GSDMD processing and caspase-4 activation. Inhibition of caspase-4 with a pan-caspase inhibitor had no effect on the LPS-mediated increase in non-selective cation currents, suggesting that Ca2+ influx occurs prior to caspase-4 activation during the process of non-canonical inflammasome activation. Removal of extracellular free Ca2+ with EGTA inhibited intracellular LPS-mediated caspase-4 activation, while activation of mechanosensitive cation channel Piezo1 by Yoda-1 enhanced the GSDMD processing and caspase-4 activation, and induced noncanonical inflammasome activation. Downregulation of Piezo1 with siRNA was sufficient to inhibit Yoda-1-mediated noncanonical inflammasome activation. We, and other investigators, also showed that oligomerized TRAF6 (TNF-associated factor 6) interacts with TRAF6-interacting proteins and caspase-4, causing caspase-4 oligomerization and activation. The oligomerization of TRAF6, monitored by blue native gel electrophoresis, was enhanced by increasing [Ca2+]. These results indicate that Ca2+ influx through mechanosensitive cation channels (e.g., Piezo1) is involved in the early process (prior to caspase-4 oligomerization and activation) that activates noncanonical inflammasomes in human LVEC. The Ca2+- or Ca2+ influx-associated noncanonical inflammasome activation (via Ca2+-TRAF6 interaction to mediate caspase-4 activation) is potentially an important mechanism involved in LVEC pyroptosis and (peri)vascular inflammation in pulmonary arterial hypertension.

This abstract is funded by: NIH (HL171538) and DOD (W81XWH-21/PRMRP/TTDA)