Effect Of TMEM98 On The Function And Mechanism Of Vascular Endothelial Cells And Smooth Muscle Cells

Background and Objective Atherosclerosis(AS)is a common clinical disease,which is the main pathological basis of cardiovascular and cerebrovascular diseases.In the global high incidence and mortality of many diseases,cardiovascular disease at the top of the list.Whereas AS is the primary cause leading to cardiovascular disease,it is a major chronic inflammatory disease in the large and medium-sized arterial,and the plasma lipoprotein levels are elevate due to abnormal lipid metabolism.In a number of physical and chemical factors and a variety of inflammatory cytokines,endothelial damage will be the first to occur,leading to plasma lipids,inflammatory factors and other ingredients into the intima and deposition in the subintimal space.At this point,due to the damage of the endothelium,monocytes in the blood flow tend to adhere to the damaged endothelial cells(ECs)and enter the endothelium through the damaged endothelium,Its function has also changed,allowing it to take up the lipid deposited on the endothelium and turn it into macrophages,which in turn can take up a large amount of lipids through the scavenger receptor on their cell membranes foam cells form.This is in contrast to the mechanism by which monocytes migrate into the endothelium through the endothelial cell gap under normal physiological conditions.This migration of monocyte due to endothelial cell damage is pathological.In the development of atherosclerosis,endothelial dysfunction is the first step in the development of AS.A further development is that vascular smooth muscle cells(VSMCs)begin to proliferate and migrate toward the intima,under the influence of locally produced growth factors,cytokines,and depositional lipids,and gradually accumulate in the subintimal space,to form the early plaque,while VSMC cells can also take up lipid-forming foam cells.These foam cells,proliferating smooth muscle cells,deposited lipids,etc.together constitute the necrotic center in the plaque,resulting in the formation of atherosclerosis.Platelet derived growth factor(PDGF)is one of the most abundant cytokines produced by plaque.It is a strong mitogenic agent and chemoattractant,target cells are mainly vascular smooth muscle cells.It is not expressed in normal arteries,when the vessel is damaged to medial,it can excrete in large quantities and release into the blood,which binds to its receptor to form dimers,thereby activating downstream PI3K/AKT and MAPK signaling pathways to promote smooth muscle cell proliferation and migration from the middle to subintima.PDGF has five subtypes of PDGF-AA,PDGFAB,PDGF-BB,PDGF-CC and PDGF-DD.PDGF-BB belongs to the strongest chemotactic agent in vitro,therefore,we used PDGF-BB as in vitro induction of this experiment.With the completion of Human Genome Project in 2006,it gives birth to a large number of new genes.In 2008,the National Human Genome Northern Research Center conducted bioinformatic analysis of the human genome database to screen for potential new cytokine-encoding genes and perform functional screening and systematic research to obtain a number of candidate genes to validate a variety of proteins,including human transmembrane protein 98(TMEM98).As a new gene,TMEM98 has no functional articles reported in cardiovascular field.In recent years,it has been found that TMEM98 is closely related to the occurrence,development of cancer,immunity and inflammation.In this study,we aims to explore the role of TMEM98 under the PDGF induced in AS and relevant molecular mechanisms,parsing TMEM98 new gene functions,which provides new thinking for further research AS pathogenesis.Method(1)ELISA and Enzyme linked immunosorbent assay(ELISA)and immunofluorescence were performed to detect,under the stimulation of PDGF-BB,the content of TMEM98 in the supernatant and the expression of TMEM98 in ECs and VSMCs closely related to atherosclerosis.(2)The TMEM98 plasmid and small interfering RNA(siTMEM98)were designed and synthesized,and TMEM98 was upregulated and down-regulated in VSMC and ECs by transient transfection.(3)The effect of siTMEM98 on monocyte-endothelial cell adhesion was detected by cell adhesion assay.(4)The effect of TMEM98 on the migration of VSMCs was detected by Boyden Chamber assay.(5)The effect of TMEM98 on VSMCs proliferation was detected by MTT and CCK-8 assay.(6)The effect of secretory TMEM98 on the formation of pseudopods in VSMCs was analyzed by immunofluorescence.(7)To detect the effect of siTMEM98 on the expression of ICAM-1 and VCAM-1 by real-time quantitative PCR.(8)Western Blot was used to detect the effect of TMEM98 on the AKT/GSK-3β/Cyclin D1 signaling pathway and the β-catenin.Results1.PDGF mediates TMEM98 secretion and expressionPDGF-BB was used to induce rat aortic smooth muscle cells(A7r5),human aortic smooth muscle cells(AOSMC)and human umbilical vein endothelial cells(HUVECs).The cell culture medium was detected by ELISA,and the content of TMEM98 was significantly increased and with time-dependent.The expression of TMEM98 in the three cell lines was significantly increased by using qPCR method.After PDGF-BB stimulated cells 12 h,the expression of TMEM98 in AOSMC and HUVEC was significantly higher than the control group in immunofluorescence anylysis.2.TMEM98 promotes endothelial cell adhesion and molecular mechanismsTransient transfection of siTMEM98 to HUVEC which induced by PDGF-BB could inhibit monocyte-endothelial cell adhesion,and over-expression of TMEM98 could promote monocyte-endothelial cell adhesion.The result showed that the expression of adhesion molecules ICAM-1 and VCAM-1 were significantly inhibited in transient transfection of siTMEM98 into HUVEC by qPCR.3.TMEM98 promotes smooth muscle cell proliferation and migrationThe proliferation of AOSMC and A7r5 mediated by PDGF-BB was detected by MTT and CCK-8.It was found that siTMEM98 could significantly inhibit the proliferation of cells,whereas TMEM98 overexpression could significantly promote the proliferation.Similarly,siTMEM98 was found to inhibit the migration of A7r5 and AOSMC using the Boyden Chamber assay for migration of A7r5 and AOSMC.When added exogenous TMEM98 to cell culture medium,we found that TMEM98 can also induce AOSMC proliferation and migration with time and concentration-dependent.The exogenous TMEM98 can also promote the formation of cell pseudopodia by immunofluorescence.4.The molecular mechanism of TMEM98 promotes smooth muscle cell proliferation and migrationThe mechanism of PDGF-mediated TMEM98 on cell proliferation and migration was explored using rat primary vascular smooth muscle cells,AOSMC and A7r5,respectively.The results showed that siTMEM98 can inhibit the expression of p-AKT(T308),p-AKT(S473)and p-GSK-3β in A7r5 cells.Similarly,siTMEM98 inhibits PDGF-BB-induced cyclin D1 and β-catenin expression in A7r5 cells.AKT agonist(SC79)partially restored siTMEM98 downregulation of primary rat vascular smooth muscle cells and AOSMC proliferation and AOSMC migration.SC79 can increase the phosphorylation of GSK-3β and Cyclin D1 expression.AKT pathway inhibitor(BEZ235)can inhibit the proliferation and migration of AOSMC,but also inhibit the phosphorylation of GSK-3β and the expression of Cyclin D1.Conclusion 1.PDGF-BB mediates the expression and secretion of TMEM98 in ECs and VSMCs,which indicates that TMEM98 is a secreted protein.2.siTMEM98 inhibits monocyte-endothelial cell adhesion by down-regulating the expression of ICAM-1 and VCAM-1.3.siTMEM98 inhibits the proliferation and migration of smooth muscle cells by down-regulating Cyclin D1 and β-catenin.4.TMEM98 promotes the proliferation and migration of smooth muscle cells by activating AKT/GSK-3β/Cyclin D1 signaling pathway.