LPI-GPR55 Induces Endothelial Cell Activation And Promotes Atherosclerosis

Section?General IntroductionCardiovascular diseases?CVDs?are a class of diseases that involve the heart or blood vessels.The underlying pathological mechanism of CVDs is atherosclerosis which is a general term describing the pathogenic process of the thickening?Athero-?and hardening?-sclerosis?of arteries.Increasing evidences indicate that atherosclerosis is initiated by activation of endothelial cells?ECs?at the sites of atherosclerotic lesion formation with increased expression of adhesion molecules on the cell surface and secretion of pro-inflammatory chemokines and cytokines.Previous reports showed that when hyperlipidemia occurs,the peroxidation of the phospholipid component of ox-LDL generates bioactive phospholipids that can activate endothelial cells.Therfore,it is essential to reveal the molecular mechanisms involved in endothelial cell activation during the early stages of atherosclerosis,especially the mechanisms mediated by bioactive lipids,which will provide novel insight that may potential identification of novel therapeutic targets to treat atherosclerosis.Section?LPIs Are Induced in the Aorta During Early Atherosclerosis in MiceObjective:To determine the aortic lipidomics level during early atherosclerosisMethods:We fed the WT mice and apolipoprotein E knockout(ApoE^-/-)mice?the best-known atherosclerotic mouse model?with high fat diet?HF?for 3 weeks.Then we performed non-targeted metabolomics analysis on the aortas,hearts,livers and plasmas extracted from ApoE^-/-mice and WT control mice.We probed for different LPI species?cholesterol and triglyceride in WT and ApoE^-/-mice fed with high fat diet for different duration?3 weeks,6 weeks,and 12 weeks?and did correlation analysis between LPIs?lysophosphatidylinositol,LPI?and hyperlipidemia.Moreover,we tested the expression level of synthesis phospholipases PLA and degrading phospholipases PLC.Results:Our lipidomics profile data showed the presence of 5 different LPI species in the aortas of WT and ApoE^-/-mice.Expect 2-arachidonoyl-LPI,which could not be detected in aorta and 1-arachidonoyl-LPI which increased but no significant,other 3 LPI species include palmitoyl-LPI,stearoyl-LPI and oleoylgl-LPI were significantly induced in the aortas of ApoE^-/-compared with WT mice.In addition,the level of palmitoyl-LPI,stearoyl-LPI and oleoylgl-LPI were increased with the duration of high fat diet feeding in both WT and ApoE^-/-mice plasma.And Our results demonstrated that palmitoyl-LPI?16:0?was positively associated with hypercholesterolmia and hypertriglyceridemia.The expression level of PLA was significantly increased,while PLC was significantly decreased in plasma of ApoE^-/-mice fed high fat diet for 3 weeks and 12 weeks in comparison to that of WT mice.Conclusions:LPI was significantly increased during the early stage of atherosclerosis due to hyperlipidemia mediated induction of synthesis phospholipases and suppression degrading phospholipases.In addition,LPI was positively associated with hyperlipidemia,which means LPI play an essential role in promoting atherosclerosis development.Section ? LPI Induces Endothelial Cell Activation and Promotes Atherosclerosis Via combining GPR55Objective: To determine whether LPI mediated endothelial cell activation and atherosclerosis depends on GPR55Methods: To examine our hypothesis,we detected the expression level of adhesion molecules including ICAM-1,VCAM-1 and E-selectin and pro-inflammatory and chemoattractant chemokines by using Western Blot and Chemokine antibody array analysis in LPI treated HAECs.In addition,we detected increased monocyte adhesion to HAECs treated with LPI by conducting monocyte adhension assay.Morever,we generated GPR55-si RNA?small interference ribonucleic acid,si RNA?to knockdown GPR55 expression level determined its effects on ICAM-1 expression level in HAECs treated with LPI.Furthermore,to verify that GPR55 is needed for LPI function,we generated GPR55 deficient?Apo E-/-/GPR55-/-?in Apo E-/-background.The number of rolling leukocyte and adhesion leukocyte the endothelium of cremaster venules were detected by using intravital microscope analysis in Apo E-/-and Apo E-/-/GPR55-/-mice fed with high fat diet for 3 weeks.Atherosclerotic lesion formation in the aorta was assessed by En face analysis with Sudan ? staining in Apo E-/-and Apo E-/-/GPR55-/-mice.Results: Our results demonstrated that LPI monocyte adhesion was significantly increased with LPI treatment in a dose dependent manner,up-regulated adhesion molecules?ICAM-1,VCAM-1 and E-Selectin?expression levels of HAECs on time-depend and promoted chemoattractant production in HAECs.Next,we sought to determine whether LPI-GPR55 axis contributes to HAECs activation in vitro and atherosclerosis development in vivo.Knockdown of GPR55 by si RNA minigated LPI-mediated increase in ICAM-1 expression.In addition to the in vitro data,we generated Apo E-/-/GPR55-/-and Apo E-/-mice and fed with HF for 3 weeks to visualize and quantify in vivo leukocyte rolling and adhesion to endothelium by using intravital microscope.There were no differences in the venule diameters of Apo E-/-/GPR55-/-and Apo E-/-mice.However,we did find that there was a significant decrease in the number of leukocyte rolling and adhered to endothelium of the Apo E-/-/GPR55-/-mice compared to Apo E-/-mice.In addition,we quantified en face atherosclerotic plaque formation in whole aortas of Apo E-/-/GPR55-/-and Apo E-/-mice.We found that GPR55 gene deficiency did significantly reduce lesion area of whole aorta.Conclusions: Endothelial cell activation is the initiation step for atherosclerosis.Mechanistically,during inflammation,locally released pro-inflammatory cytokines and chemokines activate endothelial cell to up-regulate the expression of soluble adhesion molecules,which then guide the adhesion and migration of circulating leukocytes and monocytes across the endothelial cell lining of blood vessels to access the site of injury.Knockdown of GPR55 by small interference ribonucleic acid?RNA?can rescue LPI-mediated EC activation;Knockout of GPR55 can rescue LPI-mediated atherosclerosis,which suggest that LPI may play an important role in the endothelial cell activation and atherosclerosis via GPR55 receptor.Our novel results have provided an insight into the roles of LPI-GPR55 in promoting early of atherogenesis,starting from endothelial cell activation,monocyte-endothelial cell interaction,inflammatory leukocytes rolling and adhesion to vessel inner membrane,to development of atherosclerosis.Section ? LPI-GPR55 mediates ADMA productionObjective: To determine the downstream signaling targets of LPI-GPR55Methods: Conduct correlation between GPR55 and different metabolites using micro-Array database and metabolic database of 60 cell lines.Expression level of GPR55 and ADMA of Apo E-/-and WT mice aorta were detected by micro-Array and chromatography electrospray tandem mass spectrometry method.The m RNA and protein level of ADMA synthesis enzyme,PRMT-1 and degradation enzyme DDAH-2 in HAECs and aorta of Apo E-/-/GPR55-/-and Apo E-/-mice were detected by RT-PCR and Western Blot respectively.Results: Our results showed that GPR55 was positively associated with ADMA in 60 cell lines.In addition,GPR55 and ADMA expression level were increased in aorta of Apo E-/-mice compared with WT mice.LPI treatment up-regulated m RNA and protein expression of PRMT-1 in a dose dependent manner in HAECs,while DDAH-2 m RNA and protein levels were down-regulated.Similarly,LPI induced ADMA production in a dose dependent manner in HAECs.Furthermore,we quantified PRMT-1 and DDAH-2 expression level in aortas of Apo E-/-/GPR55-/-and Apo E-/-mice.We found that GPR55 gene deficiency did significantly reduced PRMT-1 m RNA and protein expression and increased DDAH-2 m RNA and protein expression.ADMA level in aorta extract from Apo E-/-/GPR55-/-mice fed with high fat diet for 8 weeks was significantly decreased in comparison to that of Apo E-/-mice.Conclusions: Our data showed that GPR55 function not only as a G protein receptor,but also as a metabolic regulator.Our results indicated that GPR55 was positively associated with ADMA?Asymmetric dimethylarginine,ADMA?level in both in 60 cell lines and in Apo E-/-mice,which is an independent risk factor for cardiovascular disease.LPI-GPR55 can induced ADMA production in HAECs and mouse aorta due to PRMT-1/ADMA/DDAH-2 pathway.Section ? LPI-GPR55 mediates ADMA-induced mt ROS productionObjective: To determine whether LPI-GPR55 mediated ADMA-induced mt ROS productionMethods: mt ROS level was detected by fowcytometry?electron spin resonance and confocal microscope.Binding of activator protein-1?AP-1?on the promoter of intercellular adhesion molecule-1?ICAM-1?was detected by electrophoretic mobility shift assay?EMSA?.Results: We frst compared the effects of LPI-GPR55 on the production of mt ROS in HAECs simultaneously by using electron spin resonance method.We found that LPI induced mt ROS production in a dose dependent manner.To confirm the results from electron spin resonance,we used another specifc mt ROS fluorescence probe,Mito SOX,combined with the use of flow cytometry and found that LPI dose-dependently increased mt ROS production in HAECs.Finally,we used a third method,confocal microscopy,and confrmed that LPI induced mt ROS production on dose-depend in HAECs.In addition,mito-TEMPO,a special inhibitor of mt ROS,can decrease LPI-GPR55 mediated mt ROS level.Conclusions: Our results suggested that LPI,upregulated in early atherosclerotic aortas,could induce mt ROS in HAECs.Also,mt ROS induced endothelial activation by upregulating nuclear binding of AP-1 on the promoter of ICAM-1.Section ? SummaryTo summarize,our study demonstrated that LPI is induced in the aortas of mice during early atherogenesis.In addition,LPI induced endothelial cells activation to secrete pro-inflammatory chemoattractants and adhesion molecules and enhanced monocyte adhesion to endothelial cells depend on GPR55.Moreover,LPI-GPR55 induced leukocytes to roll and adhere to vascular inner wall and promoted lesion development finally.Mechanically,LPI-GPR55 can induced ADMA production by upregulating PRMT-1 expression,a key synthesis enzyme of ADMA.Furthermore,LPI-GPR55 induced mt ROS which is mediated by ADMA.mt ROS further led to ICAM-1 upregulation by facilitating the nuclear translocation of AP-1 to increase ICAM-1 transcription.In brief,LPI-GPR55 induced endothelial cells activation and promoted AS via inducing mt ROS production meditated by ADMA.Our study will provide new insights for prevention and treatment of atherosclerosis disease.