Angiotensin II Triggers NLRP3 Inflammasome Activation by a Ca2+ Signaling-Dependent Pathway in Rat Cardiac Fibroblast Ang-II by a Ca2+-Dependent Mechanism Triggers NLRP3 Inflammasome in CF.

ABSTRACT

Angiotensin II (Ang-II) is a widely studied hypertensive, profibrotic, and pro-inflammatory peptide. In the heart, cardiac fibroblasts (CF) express type 1 angiotensin II receptors (AT1R), Toll-like receptor-4 (TLR4), and the NLRP3 inflammasome complex, which play important roles in pro-inflammatory processes. When activated, the NLRP3 inflammasome triggers proteolytic cleavage of pro-IL-1, resulting in its activation. However, in CF the mechanism by which Ang-II assembles and activates the NLRP3 inflammasome remains not fully known. To elucidate this important point, we stimulated TLR4 receptors in CF and evaluated the signaling pathways by which Ang-II triggers the assembly and activity. In cultured rat CF, pro-IL-1ß levels, NLRP3, ASC, and caspase-1 expression levels were determined by Western blot. NLRP3 inflammasome complex assembly was analyzed by immunocytochemistry, whereas by ELISA, we analyzed NLRP3 inflammasome activity and [Formula see text] release. In CF, Ang-II triggered NLRP3 inflammasome assembly and caspase-1 activity; and in LPS-pretreated CF, Ang-II also triggered [Formula see text] secretion. These effects were blocked by losartan (AT1R antagonist), U73221 (PLC inhibitor), 2-APB (IP3R antagonist), and BAPTA-AM (Ca2+ chelator) indicating that the AT1R/PLC/IP3R/Ca2+ pathway is involved. Finally, bafilomycin A1 prevented Ang-II-induced [Formula see text] secretion, indicating that a non-classical protein secretion mechanism is involved. These findings suggest that in CF, Ang-II by a Ca2+-dependent mechanism triggers NLRP3 inflammasome assembly and activation leading to [Formula see text] secretion through a non-conventional protein secretion mechanism.