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

Autophagy. 2021 Apr;17(4):980-1000. doi: 10.1080/15548627.2020.1741202. Epub 2020 Mar 19.

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; ADPβs: 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-3β-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.

Keywords: Atherosclerosis; P2RY12 receptor; VSMC; autophagy; foam cell.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Atherosclerosis / pathology*
  • Atorvastatin / pharmacology
  • Autophagy* / drug effects
  • Cholesterol / metabolism
  • Clopidogrel / pharmacology
  • Drug Synergism
  • Female
  • Foam Cells / drug effects
  • Foam Cells / metabolism
  • Foam Cells / pathology*
  • Foam Cells / ultrastructure
  • Humans
  • Lipolysis / drug effects
  • Lysosomes / drug effects
  • Lysosomes / metabolism
  • Male
  • Mice
  • Middle Aged
  • Muscle, Smooth, Vascular / pathology*
  • Myocytes, Smooth Muscle / drug effects
  • Myocytes, Smooth Muscle / metabolism
  • Myocytes, Smooth Muscle / pathology*
  • Myocytes, Smooth Muscle / ultrastructure
  • Phosphatidylinositol 3-Kinases / metabolism
  • Proto-Oncogene Proteins c-akt / metabolism
  • Receptors, Purinergic P2Y12 / metabolism*
  • Signal Transduction
  • TOR Serine-Threonine Kinases / metabolism

Substances

  • P2RY12 protein, human
  • P2ry12 protein, mouse
  • Receptors, Purinergic P2Y12
  • Cholesterol
  • Atorvastatin
  • Clopidogrel
  • Proto-Oncogene Proteins c-akt
  • TOR Serine-Threonine Kinases

Grants and funding

This work was supported by grants from the National Natural Science Foundation of China (No. 81820108010 to B.H., No. 81571139 and 81974182 to L.M.), the National Key Research and Development Program of China (no. 2018YFC1312200 to B.H.), the National Natural Science Foundation of China (No. 81771249 to Y.P.X. and No. 81671147 to H.J.J.), and the Natural Science Foundation of Hubei Province of China (No. 2016CFB518 to B.H.).