Endothelial NLRP3 inflammasome activation and arterial neointima formation associated with acid sphingomyelinase during hypercholesterolemia

Abstract

The NLRP3 inflammasome has been reported to be activated by atherogenic factors, whereby endothelial injury and consequent atherosclerotic lesions are triggered in the arterial wall. However, the mechanisms activating and regulating NLRP3 inflammasomes remain poorly understood. The present study tested whether acid sphingomyelinase (ASM) and ceramide associated membrane raft (MR) signaling platforms contribute to the activation of NLRP3 inflammasomes and atherosclerotic lesions during hypercholesterolemia. We found that 7-ketocholesterol (7-Keto) or cholesterol crystal (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, enhanced caspase-1 activity and elevated IL-1β levels, which were markedly attenuated by mouse Asm siRNA, ASM inhibitor- amitriptyline, and deletion of mouse Asm gene. In CAECs with NLRP3 inflammasome formation, membrane raft (MR) clustering with NADPH oxidase subunits was found remarkably increased as shown by CTXB (MR marker) and gp91phox aggregation indicating the formation of MR redox signaling platforms. This MR clustering was blocked by MR disruptor (MCD), ROS scavenger (Tempol) and TXNIP inhibitor (verapamil), accompanied by attenuation of 7-Keto or ChC-induced increase in caspase-1 activity. In animal experiments, Western diet fed mice with partially ligated left carotid artery (PLCA) were found to have significantly increased neointimal formation, which was associated with increased NLRP3 inflammasome formation and IL-1β production in the intima of Asm+/+ mice but not in Asm-/- mice. These results suggest that Asm gene and ceramide associated MR clustering are essential to endothelial inflammasome activation and dysfunction in the carotid arteries, ultimately determining the extent of atherosclerotic lesions.

Trimethylamine-N-Oxide Instigates NLRP3 Inflammasome Activation and Endothelial Dysfunction

“…The mouse carotid arterial endothelial cells were isolated and characterized as described earlier [20, 21]. For the TMAO stimulation, cells were treated with TMAO (30 μm) and then incubated for overnight.…”

Crosstalk Between Acid Sphingomyelinase and Inflammasome Signaling and Their Emerging Roles in Tissue Injury and Fibrosis

“…Recent study demonstrated that ASM and ceramide contribute to NLRP3 inflammasome formation and activation in hypercholesterolemia mice (Koka et al, 2017). 7-ketocholesterol or cholesterol crystals dramatically induced the formation and activation of NLRP3 inflammasomes, clustering of NLRP3-ASCcaspase-1 complex, activation of caspase-1, and production of IL-1β, these events were dramatically suppressed by gene silence of ASM, pharmacological inhibition, or gene deficiency in mice carotid arterial endothelial cells (CAECs).…”

“…Emerging studies have revealed that acid sphingomyelinase (ASM) plays crucial role in the activation of inflammasomes (Grassmé et al, 2014;Boini et al, 2016;Koka et al, 2017). ASM is an enzyme that functions to catalyze sphingomyelin into ceramide (Gatt, 1963).…”

Medial calcification in the arterial wall of smooth muscle cell‐specific Smpd1 transgenic mice: A ceramide‐mediated vasculopathy

“…Notably, several SL metabolites have been associated with the development of several pathologies, including diabetes, cancer, microbial infections, neurological syndromes and cardiovascular disease . Our laboratory previously reported that acid sphingomyelinase (ASM) plays an important role in glomerular injury and inflammasome activation …”

“…[14][15][16] Our laboratory previously reported that acid sphingomyelinase (ASM) plays an important role in glomerular injury 17,18 and inflammasome activation. 19,20 Acid sphingomyelinase (ASM), a lysosomal enzyme, is present in lysosomes and secretory lysosomes, and its fusion with plasma membrane results in the release of ceramide on the outer leaflet of the plasma membrane that serves to re-organize and cluster receptors and signalling molecules. 21,22 This re-organization of receptors and associated signalling molecules mediates various effects of ceramides at the cellular level.…”