The P2RY12 Receptor In VSMCs Promotes Foam Cell Formation And Atherosclerosis By Inhibiting Autophagy

Background and purpose:Atherosclerosis is a progressive disease of large arteries and a leading cause of stroke and cardiovascular diseases.Studies have reported that The P2RY12 receptor mitigates atherosclerosis and a large number of P2RY12-positive VSMCs are found around the necrotic core.Vascular smooth muscle cells(VSMCs)are an important source of foam cells in atherosclerosis.This study aims to investigate whether the P2RY12 receptor in mitigating atherosclerosis and to further clarify the potential mechanism.Methods:We conducted a single-center,small-scale,double-blind,randomized controlled clinical trial.Patients with carotid plaque were randomly assigned to 2groups,receiving 20 mg atorvastatin(ATV)or 75 mg CDL+20 mg ATV daily.Ultrasound was performed before and 6 months after treatment and the changes in the maximal carotid plaque thickness(MCPT)after 6 months of treatment were detected.apoe^-/-mice were fed a high-fat diet(HFD)for 8 weeks to form atherosclerosis.Mice were then randomly divided into indicated groups.The isolated aortic arches of mice were stained with oil red O(ORO),and the proportion of ORO positive area was used to assess the atherosclerostic burden.The cross-sections of aortic root in mice were stained by Histochemical and immunofluorescence to assess the plaque composition.ORO staining,BODIPY staining,and a cholesterol detection kit were used to assess intracellular lipid deposition.DIL-ox LDL and NBD-cholesterol were used to measure lipid uptake and cholesterol efflux by VSMCs,respectively.The expression level of molecules were detected using immunoblotting or real-time PCR.Fluorescence were used to label lysosomal,lipid drops and autophagy-related structure for analyzing the lipid metabolism.Phosphorylation proteomics was used to detect and analysis the phosphorylation of autophagy-related signaling pathways in VSMCs with or without ADP?s(the P2RY12 receptor agonist).Autophagy flow was detected using the autophagy flux reporter adenovirus.Electron microscopy was used to detect LDs and autophagic structures.Results:The decreases in MCPT were more apparent in the CDL+ATV group than in the ATV group.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 P2RY12receptor 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).Conclusion:We conclude that activation of the P2RY12 receptor decreases cholesterol efflux and promotes VSMC-derived foam cell formation by blocking autophagy in advanced atherosclerosis.This study revealed that the P2RY12 receptor is involved in regulating autophagy and lipid metabolism of VSMCs during the process of atherosclerosis,which may provide a new therapeutic strategy for clinical treatment of atherosclerosis-related diseases.