| BackgroundCardiovascular and cerebrovascular diseases are common and frequentlyoccurring diseases that threaten people’s life and health.In recent years,with the aging trend of China’s population and the change of diet and lifestyle,the incidence of coronary heart disease,myocardial infarction and other diseases is increasing.At present,percutaneous coronary stent implantation is the main clinical treatment for coronary heart disease,however,postoperative restenosis（RS）caused by endothelial injury caused by stent implantation and proliferation and migration of vascular smooth muscle cells（VSMCs）has always been a problem for doctors and patients.With the continuous development of clinical diagnosis and treatment technology,stent implantation has experienced the replacement from bare metal stent（BMS）to drugeluting stent（DES）,but the pathological mechanism of vascular restenosis is still not fully revealed,and the study of its pathogenesis and treatment is of great significance to clinical diagnosis and treatment as well as people’s health.Sema4d is a type I transmembrane protein,which is widely distributed in various tissues and organs such as nerve,skeletal muscle,endothelium and tumor,and is involved in a variety of physiological and pathological processes such as cell proliferation,migration,adhesion,immune response and cardiovascular development.Studies have shown that in heart failure,coronary heart disease and other diseases,its serum expression level is significantly increased,and its receptor,plexin B1,can activate the cascade signal to activate the proliferation-related MAPK signaling pathway.Since the proliferation,migration and phenotypic transformation of vascular smooth muscle are important pathological mechanisms of vascular restenosis,we speculated that Sema4d might be involved in the occurrence of vascular restenosis,and explored the role of Sema4d in this process through this study.ObjectiveTo explore the role of Sema4d in pathological intimal hyperplasia and related signaling pathways.MethodPart one: Cell experiment,the primary rat VSMCs were cultured by tissue block adhesion method in vitro,and the cells were identified by immunofluorescence assayα-SMA,and the concentration of platelet-derived growth factor BB（PDGF-BB）.The proliferation and migration of Sema4d were induced,and the expression of Sema4d before and after induction was observed by immunofluorescence and western blot.In addition,Sema4d expression was reduced by adenovirus（AD-sh Sema4d）,and cells were divided into control group,PDGF-BB stimulation group,PDGF-BB+ AD-GFP group,and PDGF-BB+ AD-sh Sema4d group.Cell proliferation was detected by CCK-8 and PCNA levels.Cell migration was detected by Transwell,wound healing assay and MMP-9 level.Phenotypic transformation was detected by expression levels of α-SMA and OPN.Western blot was used to detect ERK1/2 and P-ERK1/2 levels to explore the effect of Sema4d on MAPK signaling pathway.Part two: Animal experiment,the rat animal model of common carotid artery injury was established by balloon catheter,and the intervention was carried out by local incubation of AD-sh Sema4d adenovirus in the vascular injury area.The animals were divided into sham group,model group,model + AD-GFP group and model +AD-sh Sema4d group.The rats were sacrificed 14 days after the operation.Samples were taken from the injured blood vessels and paraffin-embedded sections or histopathological proteins were extracted.The effect of Sema4d on intimal hyperplasia of rat common carotid artery was investigated by HE staining,and the expression levels of Sema4d,KI67,MMP-9,SM-22α and OPN were investigated by tissue immunofluorescence or immunohistochemistry.ResultPart one:In cell experiment,immunofluorescence cell identification showed that the positive rate of immunofluorescence α-SMA staining of the common carotid artery VSMCs extracted by tissue block adhesion method was more than 95%.Immunofluorescence and Western blot showed that sema4 D expression level was significantly increased after PDGF-BB（ng/ m L）induction（P < 0.05）.CCK-8 cell proliferation assay showed that the proliferation activity of PDGF-BB stimulation group（group B）and PDGF-BB+ AD-GFP group（group C）was significantly higher than that of control group（group A）（P < 0.05）.The proliferation activity of PDGFBB+AD-sh Sema4d group（group D）was lower than that of PDGF-BB stimulation group（group B）and PDGF-BB+ AD-GFP group（group C）（P < 0.05）,and there was no significant difference in proliferation level between group B and C（P > 0.05）.Western blot showed that the expression level of PCNA in group B and C was higher than that in group A（P < 0.05）,and the expression level of PCNA in group D was significantly lower than that in group B and C（P < 0.05）,but there was no significant difference between groups B and C.Transwell assay results showed that the number of cells coming from the chamber in group B and C was significantly higher than that in group A.Compared with groups B and C,the number of cells in group D was significantly lower（P < 0.05）.The results of wound healing experiment showed that compared with group A,cell mobility of group B and C was significantly increased（multiple?）.The cell migration rate of group D was significantly lower than that of group B and group C（P < 0.05）.Western blot showed that the expression of MMP-9in group B and C was significantly higher than that in group A（P < 0.05）,and the expression of MMP-9 in group D was significantly lower than that in group B and C（P < 0.05）.Western blot analysis of α-SMA showed that the expression of α-SMA in group B and C was significantly lower than that in group A（P < 0.05）,and the expression of α-SMA in group D was significantly higher than that in group B and C（P < 0.05）.Western blot analysis of OPN showed that the expression of OPN in group B and C was significantly higher than that in group A,and that in group D was significantly lower than that in group B and C.Western blot analysis of ERK1/2 and P-ERK1/2 showed that there was no significant difference in the total ERK1/2 level of the four groups,while the p-ERK1/2 level of group B and C was significantly higher than that of group A（P < 0.05）,and group D was significantly lower than that of group B and C（P < 0.05）.Part two : Animal experiments: HE staining of vascular sections showed that compared with sham group（group A）,the intima hyperplasia area and intima/media ratio of model group（group B）and model + AD-GFP group（group C）were significantly increased（P < 0.05）.The intima hyperplasia area and intima/media ratio of model + AD-Shsema4 D group（GROUP D）were significantly lower than those of group B and group C（P < 0.05）.Immunohistochemistry of vascular sections showed that the expression of Sema4d in model group was significantly higher than that in Sham group,and the expression of Sema4d was concentrated in intima hyperplasia region.Immunofluorescence analysis of KI67,MMP-9 and OPN expression showed that the fluorescence intensity of KI67,MMP-9 and OPN in group B and group C was higher than that in group A,and the fluorescence intensity of KI67,MMP-9 and OPN in group D was lower than that in group B and group C（P < 0.05）.Immunofluorescence assay showed that the expression of SM-22α in group B and C was significantly lower than that in group A（P < 0.05）,and the expression of SM-22αin group D was significantly higher than that in group B and C（P < 0.05）.Western blot analysis of Sema4d,ERK1/2 and P-ERK1/2 expressions of vascular tissue proteins showed that the Sema4d expression of group B and C was significantly higher than that of group A（P < 0.05）,and the Sema4d expression of group D was significantly lower than that of group B and C（P < 0.05）.There was no significant difference between group B and group C.ConclusionSema4d expression was increased in PDGF-BB induced rat carotid vascular smooth muscle cell model and Intimal hyperplastic vessels.Down-regulated Sema4d expression can inhibit the proliferation,migration and phenotypic transformation of PDGF-BB-induced vascular smooth muscle cells,and significantly inhibit the pathological hyperplasia of intima after vascular injury,the mechanism may be related to the promotion of MAPK pathway activation by Sema4d. |
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