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-3ʹ,5ʹ-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: 4ʹ,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

血管平滑肌细胞 (VSMC) 是动脉粥样硬化中泡沫细胞的重要来源。然而，对 VSMC 衍生的泡沫细胞形成的机制知之甚少。在这里，我们证明 P2RY12/P2Y12 受体在调节晚期动脉粥样硬化中的巨自噬/自噬和 VSMC 衍生的泡沫细胞形成方面很重要。抑制 P2RY12 受体可改善高脂饮食喂养的apoe 中脂质积累和 VSMC 衍生的泡沫细胞形成^-/-小鼠（动脉粥样硬化模型）与 LDL-c 水平无关。P2RY12 受体的激活通过 PI3K-AKT 阻止胆固醇流出，而 P2RY12 受体的基因敲低或药理学抑制抑制了 VSMC 中的这种作用。磷酸化蛋白质组学分析表明 P2RY12 受体调节 VSMC 中的自噬途径。此外，P2RY12 受体的激活抑制了 VSMC 中的 MAP1LC3/LC3 成熟、SQSTM1 降解和自噬体形成。必需自噬基因Atg5 的遗传敲低显着减弱 VSMC 中 P2RY12 受体抑制剂诱导的胆固醇流出。此外，P2RY12 受体的激活导致 VSMC 中通过 PI3K-AKT 激活 MTOR，而阻断 MTOR 活性（雷帕霉素）或降低 MTOR 表达可逆转 VSMC 中 P2RY12 受体介导的胆固醇流出抑制。在体内，该P2RY12受体的抑制促进通过PI3K-AKT-mTOR信号在动脉粥样硬化先进在血管平滑肌细胞的自体吞噬的ApoE ^{- / -}小鼠，这可能会受到自噬抑制剂（氯喹）的阻碍。因此，我们得出结论，P2RY12 受体的激活通过阻断晚期动脉粥样硬化的自噬来减少胆固醇外流并促进 VSMC 衍生的泡沫细胞形成。因此，我们的研究表明 P2RY12 受体是治疗动脉粥样硬化的治疗靶点。

缩写：2-MeSAMP：2-甲基硫腺苷 5'-单磷酸；8-CPT-cAMP：8-(4-氯苯硫基)-腺苷-3ʹ,5ʹ-环单磷酸；ABCA1：ATP结合盒亚家族A成员1；ABCG1：ATP结合盒亚家族G成员1；ACTB：肌动蛋白β；ADPβs：腺苷 5'-（α，β-亚甲基）二磷酸；ALs：自溶酶体；AMPK：AMP 活化蛋白激酶；APOA1：载脂蛋白A1；APs：自噬体；ATG5：自噬相关5；ATV：阿托伐他汀；AVs：自噬泡；CD：食物；CDL：氯吡格雷；CQ：氯喹；DAPI：4ʹ,6-二脒基-2-苯基吲哚；dbcAMP：二丁酰-cAMP；DIL-oxLDL：双十八烷基-3,3,3,3-四甲基多碳花青-oxLDL；EIF4EBP1/4E-BP1：真核翻译起始因子4E结合蛋白1；EVG：弹性范吉森；HE：苏木精-伊红；HDL：高密度脂蛋白；HFD：高脂肪饮食；凯格：京都基因与基因组百科全书；LDL-c：低密度脂蛋白胆固醇；LDs：脂滴；MAP1LC3/LC3：微管相关蛋白1轻链3；马森：马森三色；MCPT：最大颈动脉斑块厚度；MK2206：MK-2206 2HCL；NBD-胆固醇：22-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-23,24-bisnor-5-cholen-3β-ol; OLR1/LOX-1：氧化低密度脂蛋白受体1；ORO：油红O；ox-LDL：氧化低密度脂蛋白；SQSTM1/p62：sequestosome 1；TEM：透射电子显微镜；TIC：替格瑞洛；ULK1：unc-51 类似自噬激活激酶 1；VSMCs：血管平滑肌细胞 MK-2206 2HCL；NBD-胆固醇：22-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-23,24-bisnor-5-cholen-3β-ol; OLR1/LOX-1：氧化低密度脂蛋白受体1；ORO：油红O；ox-LDL：氧化低密度脂蛋白；SQSTM1/p62：sequestosome 1；TEM：透射电子显微镜；TIC：替格瑞洛；ULK1：unc-51 类似自噬激活激酶 1；VSMCs：血管平滑肌细胞 MK-2206 2HCL；NBD-胆固醇：22-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-23,24-bisnor-5-cholen-3β-ol; OLR1/LOX-1：氧化低密度脂蛋白受体1；ORO：油红O；ox-LDL：氧化低密度脂蛋白；SQSTM1/p62：sequestosome 1；TEM：透射电子显微镜；TIC：替格瑞洛；ULK1：unc-51 类似自噬激活激酶 1；VSMCs：血管平滑肌细胞