Endothelial G Protein-coupled Receptor 124 Participate In Pathological Process Of Atherosclerosis

Objective:Atherosclerosis(AS)is a chronic inflammatory response and has become the principal pathological process of cardiovascular diseases.The lesions of atherosclerosis are usually found in large and medium-sized arteries.The accumulated evidence have revealed that numerous risk factors involved in the pathological process of atherosclerosis,among which the high serum cholesterol level,particularly the low-density lipoprotein cholesterol(LDL-c)plays the primary role.G protein-coupled receptor 124(GPR124),an orphan receptor of adhesion GPCR subfamily,has been revealed to promote angiogenesis in the brain.In this study,we explored the role of endothelial GPR124 in the development and progress of atherosclerosis.Methods:1)Construction of endothelial-specific GPR124 overexpression mice for the atherosclerotic model.Based on the tetracycline-regulate system,we generated the mice that GPR124 expressed specifically under the control of the Tie-2 promoter.Administration of doxycycline(Dox)sucrose solution(2 mg/mL)to turn on GPR124 expression.The atherosclerotic animal model was constructed by intravenous injection of AAV-PCSK9DY to tetracycline-regulate mice,it can drive PCSK9DY gene expressing in hepatocytes under liver-specific promoter,combined with a high-fat diet fed to the animal for consecutive 16 weeks.2)For the detection of atherosclerotic pathological related indicators serum total cholesterol(TC)and low-density lipoprotein cholesterol(LDL-c)were tested and sections of aortic sinus stained with Oil Red O to detect lipid deposition in the arterial wall.Alpha-SMA were also detected by immunofluorescence to assessed the proliferation in smooth muscle cell.3)To better understand the role and mechanism of GPR124 in pathological process of atherosclerosis,we observe the inflammatory injury in aortic sinus.Firstly,we detected the level of peroxynitrite(ONOO-)and Nitrotyrosine(NT)by using specific probe NP3 and Nitrotyrosine antibody to sections of aorta sinus using immunofluorescence staining.We also assessed the level of macrophages by its marker CD68,Then we checked the expression level of NLRP3 and caspases-1 in aortic sinus.4)Evaluation of cognitive function in GPR124 overexpressed atherosclerotic mice.The behavioral test of these mice was examined using open field test,Y-maze test and novel object recognized test.We also detected the expressions of phospho-CaMKII(Thr286)and GFAP by immunofluorescence analysis in the hippocampus.Results:1)Firstly,DNA extracted from the tail of tetracycline-regulate mice were sent for further genotyping identification.The results confirmed overexpression of GPR124 especially in the endothelium of this transgenic line.The high mRNA levesl of human PCSK9DY demonstrated that the AAV construct drives a stable overexpression of PCSK9DY in the liver.2)A higher TC and LDL-c level in serum and lipid deposition in aortic sinus were found in atherosclerotic model with GPR124 overexpression mice.Abundant smooth muscle cells are found in aortic sinus when GPR124 overexpressed in endothelium using immunofluorescence analysis.3)We observed an elevation level of ONOO-and Nitrotyrosine were found in this model by using immunofluorescence,and the experiments showed that overexpression of GPR124 especially in the endothelium can induce up-regulation of expression of CD68,NLRP3 and caspases-1.4)The behavioral performance test showed no significant difference between atherosclerotic model and endothelial GPR124 overexpressing mice.Furthermore,the expressions level of phospho-CaMKII(Thr286)and GFAP in the hippocampus,appeared with significant difference when compared with atherosclerotic mice.Conclusion:In this study,we constructed a atherosclerotic animal model in tetracycline-regulate mice in which GPR124 was overexpressed in the endothelium.GPR124 not only increase serum cholesterol and lipid deposition but also aggravates smoth muscle cell proliferation.We also found that overexpression of GPR124 especially in the endothelium can induce endothelial dysfunction in aortic sinus.F urthermore,GPR124 overexpression in endothelium have no effects on the cognitive function at least in 16 weeks atherosclerotic model mice.The above results indicate that manipulating the level of GPR124 in endothelium may contribute to delay the pathological process in atherosclerosis.