Effect Of TFPIct32 On Proliferation And Migration Of Vascular Smooth Muscle Cells And Its Mechanism

Background:Atherosclerosis cardio-cerebrovascular diseas is a frequently occurring disease that seriously threatens human health.Atherosclerosis（As）is a complex chronic inflammatory disease involving multiple factors.Among them,vascular smooth muscle cells,macrophages and endothelial cells are closely related to the formation of As plaques.The main methods to treat arterial stenosis caused by As include balloon dilation,stent formation and bypass surgery.However,vascular wall damage,abnormal proliferation and migration of smooth muscle cells,and endothelial dysfunction caused by stent implantation are important factors that promote As plaque formation and vascular restenosis,involving the participation of a variety of intracellular signal transduction molecules.Among them,the PI3K/AKT signaling pathway is closely related to As plaque formation and vascular restenosis.Abnormal activity can not only lead to abnormal proliferation and migration of vascular smooth muscle cells,but also be related to the degradation of extracellular matrix（ECM）,but the exact mechanism remains to be elucidated.Tissue factor pathway inhibitor（TFPI）is a physiological inhibitor of the exogenous blood coagulation pathway mediated by tissue factor（TF）,which has anti-thrombosis and anti-As effects.The inhibitory effect of TFPI on plaque development is related to its inhibition of vascular endothelial cell activation,vascular smooth muscle cell proliferation and migration,and the expression of inflammatory factors.At the carboxyl end of human TFPI,there is a small fragment（TFPIct32）composed of 32 amino acid sequences that has a TFPI-like effect in reducing As and can reduce bleeding tendency,but it inhibits vascular endothelial activation,vascular smooth muscle cell proliferation and migration.The mechanism of action is unclear.The previous research of our group showed that after treatment with TFPIct32,the plaque area of the aortic sinus of Apo E^-/-mice was reduced.In vitro experiments showed that its mechanism of action was related to the promotion of the migration of macrophages from the plaque.However,this mechanism is not yet sufficient to explain the anti-As effect of TFPIct32,indicating that there are other mechanisms in the effect of TFPIct32 antagonizing the formation and development of As.This study aims to further clarify the new mechanism of anti-As by observing the effect of TFPIct32 on PDGF-BB-induced proliferation and migration of vascular smooth muscle cells and its preliminary exploration of the mechanism.Objective:Observe the effect of TFPIct32 on proliferation and migration of vascular smooth muscle cells and its mechanism.Methods:The experimental groups were as follows:control group（PDGF-BB）,TFPI negative control peptide group（PDGF-BB+TFPIs）and experimental group（PDGF-BB+TFPIct32）.In order to clarify that TFPIct32 regulated PDGF-BB-induced proliferation and migration of vascular smooth muscle cells and was related to AKT activity,the AKT agonist SC79 was used to treat the cells.The experimental groups were as follows:control group（PDGF-BB）,AKT agonist group（PDGF-BB+SC79）,TFPIct32 group（PDGF-BB+TFPIct32）and AKT agonist+TFPIct32 group（PDGF-BB+SC79+TFPIct32）.CCK-8 method and PCNA protein expression was detected by Western blot to determine the proliferation ability of vascular smooth muscle cells.Scratch test and Transwell method were used to determine the migration ability of vascular smooth muscle cells;Western blot was used to detect the expression levels of PI3K and p-AKT/AKT.Results:1.CCK-8 method was used to detect the proliferation ability of PDGF-BB-induced vascular smooth muscle cells.The results showed that TFPIct32 alone had no effect on the proliferation of vascular smooth muscle cells.After PDGF-BB treatment,the proliferation ability of PDGF-BB treatment group is significantly increased compared with the control group（P<0.05）.After treatment with TFPIct32,the proliferation of PDGF-BB-stimulated vascular smooth muscle cells was inhibited in a concentration-dependent manner,and the proliferation of 50μg/m L TFPIct32 group was decreased by 16.34%（P<0.05）,and the inhibition effect was the most obvious.Meanwhile,the expression of proliferation-related protein PCNA in TFPIct32 group decreased by25.48%compared with the control group（P<0.05）,which was consistent with the results of CCK-8.2.The scratch test was used to detect the migration ability of PDGF-BB-induced vascular smooth muscle cells.The results showed that the cell migration rate of TFPIct32 group was decreased by 62.96%compared with the control group（P<0.05）.The migration ability of vascular smooth muscle cells was again measured with the Transwell assay,and the results showed that the number of cells crossing the compartment was reduced by 68.99%in the TFPIct32 group compared to the control group（P<0.05）.3.PDGF-BB,TFPIs and TFPIct32 cells were grouped according to the experimental design,and the expression levels of PI3K and p-AKT/AKT in vascular smooth muscle cells were detected by Western blot.The results showed that compared with the control group,the expression of PI3K in TFPIct32 group was decreased by 18.71%（P<0.05）,and the expression of p-AKT/AKT was decreased by 32.15%（P<0.05）.4.After the cells were treated with AKT agonist SC79,the proliferation ability of vascular smooth muscle cells induced by PDGF-BB was detected by CCK-8 assay.The results showed that the cell proliferation ability of PDGF-BB+SC79 group was increased by 13.24%compared with PDGF-BB group（P<0.05）.The proliferation ability of vascular smooth muscle cells in PDGF-BB+SC79+TFPIct32 group was decreased by 7.94%compared with PDGF-BB+SC79 group（P<0.05）.5.After cells were treated with AKT agonist SC79,PDGF-BB induced vascular smooth muscle cell migration ability was detected by scratch test.The results showed that the cell migration ability of PDGF-BB+SC79 group was increased by 50.06%compared with PDGF-BB group（P<0.05）.The migration ability of vascular smooth muscle cells in PDGF-BB+SC79+TFPIct32 group was decreased by56.67%compared with PDGF-BB+SC79 group（P<0.05）.Transwell assay was used to detect the migration ability of vascular smooth muscle cells,and the results showed that the migration ability of PDGF-BB+SC79 group was increased by 25.28%compared with PDGF-BB group（P<0.05）.The migration ability of vascular smooth muscle cells in PDGF-BB+SC79+TFPIct32 group was decreased by21.24%compared with PDGF-BB+SC79 group（P<0.05）.Conclusion:TFPIct32 can significantly inhibit the proliferation and migration of vascular smooth muscle cells induced by PDGF-BB,and this effect is related to the inhibition of AKT activity.