Study On The Role And Mechanism Of Lypla1 In Atherosclerosis

Background:In recent years,the incidence of acute coronary syndrome has risen year by year.It is a leading cause of death in the world.The formation of coronary atherosclerotic plaques,followed by rupture is an important factor in causing acute cardiovascular events.Therefore,understanding its pathological mechanisms,and developing effective detection and treatment tools to reduce plaque formation or increase the stability of plaque is significance for therapeutic of atherosclerosis.Objective:Palmitoylation is the only reversible lipid-modified post-translational modification that increases the affinity of soluble proteins to membranes and plays an important role in protein transport,localization and function.Acyl protein thioesterase 1（APT1,Lypla1）functions as a homodimer,exhibiting depalmitoylating activity,and involved in the palmitoyl modification of many proteins.Inhibitors of Lypla1 can affect H-Ras localization via depalmitoylation of H-Ras and reverse Ras-induced tumor phenotypechanges.However,studies on the impact of Lypla1 on Ras function have rarely been reported in the cardiovascular system.In this study,atherosclerosis model were established by ox-LDL stimulation of human umbilical vein endothelialcells（HUVECs）and apoE^-/-mice fed with high fat diet.In our study,on the one hand,we identified the upstream regulatory of Lypla1;on the other hand,we studied the expression level and localization of H-Ras and MAPK signaling pathway as the downstream of Lypla1,to try to elucidate the mechanism of action of the Lypla1 in the development of atherosclerosis.Methods:The candidate upstream regulatorys of Lypla1 and miR-138 were screened and verified by bioinformatics,double fluorescence report assay,Realti-me PCR and Western blot assay.Based on the model of atherosclerosis induced by ox-LDL in HUVECs in vitro,the expression level of H19,miR-138,Lypla1,DHHC9 and the level of palmitoylation of H-Ras were detected at different time points by Realtime PCR,Western blot assay and Acyl-Biotin Exchange assay（ABE）.Then up or down regulated the expression level of Lypla1 to measure the expression level and location of H-Ras.After that,the adhesion ability of monocyte,lipid deposition of HUVEC and the expression level of inflammatory factors were detected by confocus,flow cytometry and Realtime PCR to evaluate the influence of Lypla1 on the AS.Finally,the apoE^-/-mice were fed with high fat diet to established the animal model of atherosclerosis and verify the changes of the expression level of H19,miR-138 and Lypla1 by Realtime PCR and Western blot assay in vivo.Results:Expression level of Lypla1 in HUVECs induced by ox-LDL was up-regulated,while the miR-138 was down-regulated（P<0.05）,and the location of Lypla1 and H-Ras was translocated from the cytoplasm to the plasma membrane.Based on bioinformatics analysis,double fluorescence report,realtime PCR and Western blot assay,miR-138 was identified as the upstream regulatory of Lypla1,could negatively regulate Lypla1.The similar results also observed in H19 and miR-138.In addition,knockdown the expression level of Lypla1 by siRNA,we found the downregulated Lypla1 could inhibit the expression level of ICAM-1,VCAM-1and MMP-9（P<0.05）,and decreased adhesion ability of monocyte（P<0.05）,while the dil-ox-LDL aggregation ability increased（P<0.05）.Furthermore,we used LPC to stimulated HUVECs and collected the microparticles（MPs）by ultracentrifugation to detect the Lypla1 mRNA and protein levels by Realtime PCR and Western blot assay.We found Lypla1 expression level was significantly increased after stimulated for 24h,and used the endothelial microparticles（EMP）to stimulated THP-1,the expression level of MMP-9 were increased（P<0.05）too.Finally,In vivo,compare with the normal group,the expression level of H19 and Lypla1 in high fat diet group were significantly increased（P<0.05）,while the expression level of miR-138 was decreased.Conclusion:In atherosclerosis,Lypla1 not only regulate palmitoylation modification and the downstream pathway of H-Ras,but also could leave the endothelial cells through MP and influence the MMP-9 expression level of macrophage,ultimately affect the formation and development of atherosclerosis.