Activation of Endothelial NLRP3 Inflammasomes associated with Acid Sphingomyelinase‐dependent Formation of Membrane Raft Redox Signaling Platforms

Abstract

There is evidence that endothelial NLRP3 inflammasomes are activated by atherogenic stimuli such as cholesterol crystals (CHC), 7-ketocholesterol (7- Keto) and visfatin. However, the mechanism by which endothelial NLRP3 inflammasomes remains poorly understood. The present study hypothesized that acid sphingomyelinase (ASM)-mediated ceramide and associated membrane redox signaling platforms may be one of the triggering mechanisms of NLRP3 inflammasomes. We first demonstrated that 7-Keto or CHC markedly increased the formation and activation of NLRP3 inflammasomes in mouse carotid arterial endothelial cells (CAECs), as shown by increased colocalization of NLRP3 with ASC or caspase-1, caspase-1 activity and IL-1β levels. This NLRP3 inflammasome formation and activation were markedly attenuated by caspase-1 inhibitor YVAD, ASC siRNA, ASM inhibitor, amitriptyline or its siRNA and CAECs from ASM-/-. Furthermore, in CAECs with NLRP3 inflammasome formation, we found that membrane raft (MR) clustering with NADPH oxidase subunits was remarkably increased as shown by more than 10 folds enhancement of CTXB and gp91phox or p47phox, indicating the formation of MR redox signaling platforms, which was further confirmed by inhibitory effects of lipid raft disruptor (MCD), ROS scavenger (TEMPOL+catalase) and TXNIP inhibitor (verapamil) on 7-Keto or CHC-induced increase in caspase-1 activity and IL-1β production. These results provide the first direct evidence showing that the activation of endothelial NLRP3 inflammasomes by 7-Keto or CHC may be triggered by ROS derived from MR redox signaling platforms.