Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Department of Ultrasound, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Autophagy. 2021 Apr;17(4):980-1000. doi: 10.1080/15548627.2020.1741202. Epub 2020 Mar 19.
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 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 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. , inhibition of the P2RY12 receptor promoted autophagy of VSMCs through PI3K-AKT-MTOR in advanced atherosclerosis in 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. 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.
血管平滑肌细胞（VSMCs）是动脉粥样硬化中泡沫细胞的重要来源。然而，VSMC 衍生的泡沫细胞形成的机制尚不清楚。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中很重要。在高脂饮食喂养的小鼠（动脉粥样硬化模型）中，抑制 P2RY12 受体可改善脂质积累和 VSMC 衍生的泡沫细胞形成，而与 LDL-c 水平无关。P2RY12 受体的激活通过 PI3K-AKT 阻断胆固醇流出，而 VSMCs 中 P2RY12 受体的遗传敲低或药理学抑制则抑制了这种作用。磷酸化蛋白质组学分析表明，P2RY12 受体调节 VSMCs 中的自噬途径。此外，P2RY12 受体的激活抑制了 VSMCs 中 MAP1LC3/LC3 的成熟、SQSTM1 的降解和自噬体的形成。必需自噬基因的遗传敲低显著减弱了 P2RY12 受体抑制剂在 VSMCs 中诱导的胆固醇流出。此外，P2RY12 受体的激活通过 PI3K-AKT 导致 MTOR 在 VSMCs 中的激活，而阻断 MTOR 活性（雷帕霉素）或降低 MTOR 表达则逆转了 P2RY12 受体在 VSMCs 中介导的胆固醇流出的抑制作用。综上所述，在小鼠的晚期动脉粥样硬化中，抑制 P2RY12 受体通过 PI3K-AKT-MTOR 促进 VSMCs 的自噬，而自噬抑制剂（氯喹）可以阻止这种作用。因此，我们得出结论，P2RY12 受体的激活通过阻断自噬来减少胆固醇流出并促进晚期动脉粥样硬化中的 VSMC 衍生的泡沫细胞形成。我们的研究表明，P2RY12 受体是治疗动脉粥样硬化的治疗靶点。2-MeSAMP：2-甲硫腺苷 5'-单磷酸；8-CPT-cAMP：8-（4-氯苯硫基）-腺苷-3'，5'-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：自溶体；AMPK：AMP 激活的蛋白激酶；APOA1：载脂蛋白 A1；APs：自噬体；ATG5：自噬相关 5；ATV：自噬空泡；AVs：自噬溶酶体；CD：标准饮食；CDL：氯吡格雷；CQ：氯喹；DAPI：4'，6-二脒基-2-苯基吲哚；dbcAMP：二丁酰环磷酸腺苷；DIL-oxLDL：二辛基-3,3,3,3-四甲基吲哚羰花青 oxLDL；EIF4EBP1/4E-BP1：真核翻译起始因子 4E 结合蛋白 1；EVG：弹性 van Gieson；HE：苏木精-伊红；HDL：高密度脂蛋白；HFD：高脂饮食；KEGG：京都基因与基因组百科全书；LDL-c：低密度脂蛋白胆固醇；LDs：脂质滴；MAP1LC3/LC3：微管相关蛋白 1 轻链 3；Masson：马松三色染色；MCPT：最大颈动脉斑块厚度；MK2206：MK-2206 2HCL；NBD-胆固醇：22-（N-[7-硝基苯-2-氧代-1,3-二唑-4-基]氨基）-23,24-双降-5-胆甾醇-3β-醇；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：自噬体相关蛋白 1；TEM：透射电子显微镜；TIC：替格瑞洛；ULK1：UNC-51 样自噬激活激酶 1；VSMCs：血管平滑肌细胞。
