Sensing ceramides by CYSLTR2 and P2RY6 to aggravate atherosclerosis.

Recent evidence shows elevated circulating long-chain ceramide levels predict atherosclerotic cardiovascular disease (ASCVD) independently of cholesterol. Although targeting ceramide signaling may provide therapeutic benefits beyond the treatment of hypercholesterolemia, the underlying mechanism by which circulating ceramides aggravate ASCVD remains elusive. We examined whether circulating long-chain ceramides activate membrane G protein-coupled receptors (GPCRs) to exacerbate atherosclerosis. We performed a systematic screen combining G protein signaling quantification, bioinformatic analysis of GPCRs expression, and functional examination of NLRP3 inflammasome activation, and the results suggested CYSLTR2 and P2RY6 are potential endogenous receptors of C16:0 ceramide-evoked inflammasome activation in both endothelial cells and macrophages. We found that inhibiting CYSLTR2/P2RY6 genetically or pharmacologically alleviated ceramide-induced atherosclerosis aggravation. Additionally, increased ceramide levels correlated with the severity of coronary artery disease in patients with varying degrees of renal impairment. Notably, CYSLTR2/P2RY6 deficiency mitigated chronic kidney disease (CKD)-aggravated atherosclerosis in mice, without affecting cholesterol or ceramide levels. Structural analysis of the ceramide-CYSLTR2-Gq complexes revealed that both C16:0 and C20:0 ceramides bind within an inclined channel-like ligand binding pocket on CYSLTR2. We further revealed an unconventional mechanism underlying ceramides-induced CYSLTR2 activation and CYSLTR2-Gq interface. Overall, our study provided structural and molecular mechanisms that long-chain ceramides initiate transmembrane Gq and inflammasome signaling through directly binding to CYSLTR2 and P2RY6 receptors, and blocking these signaling may provide new therapeutic potential to treat atherosclerosis-related diseases.