Nucleotide oligomerization domain (NOD)-like receptor protein with pyrin domain containing 3 (Nlrp3) inflammasome has been reported to be activated by atherogenic factors, thereby triggering endothelial injury and consequent atherosclerotic lesions in the arterial wall. However, the mechanism activating and regulating Nlrp3 inflammasomes remains poorly understood. The present study tested whether membrane raft (MR) signaling platforms associated with acid sphingomyelinase (ASM) and its product ceramide (Ce) importantly contribute to the activation of Nlrp3 inflammasomes and atherosclerotic lesions during hypercholesterolemia (HC). By confocal microscopy and biochemical analyses, we demonstrated the formation and activation of Nlrp3 inflammasomes in the intima of the carotid arteries of Asm
+/+
mice with HC (as shown by a 2-fold increase in caspase-1 activity and a 6-fold enhancement of IL-1β positive stain areas), but not in Asm
-/-
mice. In endothelium-specific ASM transgenic mice (EC-Asm
trg
), this inflammasome formation and activation were enhanced. Correspondingly, HC-induced increases in IL-1β production, ASM expression, Ce level and MR-gp91
phox
clustering in the carotid intima were abolished in Asm
-/-
mice, but enhanced in EC-Asm
trg
mice. Functionally, endothelium-dependent vasodilation (EDVD) in carotid arteries
in vivo
(by ultrasound flowmetry) and
in vitro
(in perfused artery) was impaired by HC in Asm
+/+
mice by 33% and 54%, respectively. This endothelial dysfunction was not observed in Asm
-/-
mice. The endothelial tight junction protein, ZO-1 was reduced by HC in both Asm
+/+
and EC-Asm
trg
mice, but not in Asm
-/-
mice. It was also found that HC-increased neointimal formation, T-cell infiltration, and fibrosis in 2-week partially ligated carotid arteries (PLCA) occurred in Asm
+/+
mice, but not in Asm
-/-
mice with HC. EC-Asm
trg
mice even exhibited more severe inflammatory and atherosclerotic lesions. All these results suggest that Asm gene and related MR clustering are essential to endothelial inflammasome activation and dysfunction in carotid arteries, ultimately determining the extent of atherosclerotic lesions.
The Cyclopentenone Prostaglandin 15d-PGJ2 Inhibits the NLRP1 and NLRP3 Inflammasomes