The P2RY12 receptor promotes VSMC-derived foam cell formation by inhibiting autophagy in advanced atherosclerosis.

ABSTRACT

Vascular smooth muscle cells (VSMCs) are an important source of foam cells in atherosclerosis. The mechanism for VSMC-derived foam cell formation is, however, poorly understood. Here, we demonstrate that the P2RY12/P2Y12 receptor is important in regulating macroautophagy/autophagy and VSMC-derived foam cell formation in advanced atherosclerosis. Inhibition of the P2RY12 receptor ameliorated lipid accumulation and VSMC-derived foam cell formation in high-fat diet-fed apoe-/- mice (atherosclerosis model) independent of LDL-c levels. Activation of the P2RY12 receptor blocked cholesterol efflux via PI3K-AKT, while genetic knockdown or pharmacological inhibition of the P2RY12 receptor inhibited this effect in VSMCs. Phosphoproteomic analysis showed that the P2RY12 receptor regulated the autophagy pathway in VSMCs. Additionally, activation of the P2RY12 receptor inhibited MAP1LC3/LC3 maturation, SQSTM1 degradation, and autophagosome formation in VSMCs. Genetic knockdown of the essential autophagy gene Atg5 significantly attenuated P2RY12 receptor inhibitor-induced cholesterol efflux in VSMCs. Furthermore, activation of the P2RY12 receptor led to the activation of MTOR through PI3K-AKT in VSMCs, whereas blocking MTOR activity (rapamycin) or reducing MTOR expression reversed the inhibition of cholesterol efflux mediated by the P2RY12 receptor in VSMCs. In vivo, inhibition of the P2RY12 receptor promoted autophagy of VSMCs through PI3K-AKT-MTOR in advanced atherosclerosis in apoe-/- mice, which could be impeded by an autophagy inhibitor (chloroquine). Therefore, we conclude that activation of the P2RY12 receptor decreases cholesterol efflux and promotes VSMC-derived foam cell formation by blocking autophagy in advanced atherosclerosis. Our study thus suggests that the P2RY12 receptor is a therapeutic target for treating atherosclerosis.Abbreviations 2-MeSAMP 2-methylthioadenosine 5'-monophosphate; 8-CPT-cAMP 8-(4-chlorophenylthio)-adenosine-3',5'-cyclic-monophosphate; ABCA1 ATP binding cassette subfamily A member 1; ABCG1 ATP binding cassette subfamily G member 1; ACTB actin beta; ADPßs adenosine 5'-(alpha, beta-methylene) diphosphate; ALs autolysosomes; AMPK AMP-activated protein kinase; APOA1 apolipoprotein A1; APs autophagosomes; ATG5 autophagy related 5; ATV atorvastatin; AVs autophagic vacuoles; CD chow diet; CDL clopidogrel; CQ chloroquine; DAPI 4',6-diamidino-2-phenylindole; dbcAMP dibutyryl-cAMP; DIL-oxLDL dioctadecyl-3,3,3,3-tetramethylin docarbocyanine-oxLDL; EIF4EBP1/4E-BP1 eukaryotic translation initiation factor 4E binding protein 1; EVG elastic van gieson; HE hematoxylin-eosin; HDL high-density lipoprotein; HFD high-fat diet; KEGG Kyoto Encyclopedia of Genes and Genomes; LDL-c low-density lipoprotein cholesterol; LDs lipid droplets; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; Masson masson trichrome; MCPT maximal carotid plaque thickness; MK2206 MK-2206 2HCL; NBD-cholesterol 22-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino)-23,24-bisnor-5-cholen-3ß-ol; OLR1/LOX-1 oxidized low density lipoprotein receptor 1; ORO oil Red O; ox-LDL oxidized low-density lipoprotein; SQSTM1/p62 sequestosome 1; TEM transmission electron microscopy; TIC ticagrelor; ULK1 unc-51 like autophagy activating kinase 1; VSMCs vascular smooth muscle cells.