The Effects And Mechanisms Of Fluvastain To TF Expression And RhoA/Rho Kinase Pathway

Background Tissue factor(TF)was closely related to the atherothrombosis.Statins were proved to have multiple effects of anti-inflammation,anti-oxidation,and anti-thrombosis etc,which were beyond reducing lipids level.Mevalonic acid(MVA)was the intermediate metabolic product of cholesterol.In the meanwhile of competitively inhibiting the limit-control enzyme of cholesterol formation,statins indirectly inhibited the formation of intermediate products as isoprenoid in the MVA pathway.While these intermediate products were both important media in cell signal transduction and complicated intracellular correlation,and important lipo-adhesive molecules in the post-transcription of small G proteins such as Rho.How did statins influence the TF expression? Whether TF expression was enhanced by the activation of RhoA signal transduct pathway? Whether the effects of statins to TF was regulated by RhoA signal transduct pathway? All of these questions remained not clear.Our research firstly proved that serum TF level was very important to prognostic judgement for acute myocardial infarction(AMI)patients,and then we observed that the inhibitive effect of various concentrations of fluvastatin(Flu)to the tumor necrosis factor-a(TNF-a)induced expression of human umbilical venous endothelium cells(HUVECs)and RhoA activation at TF activity, gene and protein level.In addition,its possible mechanisms of RhoA/Rho kinase pathway were investigated at protein level.Objectives1.To study the relations among serum TF level,C-reaction protein (CRP),CK-MB,LVEF and related clinical events in AMI patients,so as to explore the clinical significance of serum TF level in predicting the AMI prognosis.2.To investigate the effect of various concentrations of fluvastatin to the TNF-a induced HUVECs TF content,activity,to the expression of TFmRNA,and to the TF protein level,so as to explore the effect of statins in anti-thrombosis formation.3.To observe the regulatory effect of RhoA/Rho kinase in TF protein expression influenced by fluvastatin,so as to explore the noval target point of fluvastatin in anti-artherosclerosis and anti-thrombosis.Methods1.ELISA method was used to examine the TF levels of AMI patients, stable angina pectoris(SAP),and normal controls.AMI patients were divided into high TF group and low TF group according to serum TF level,and CRP,CK-MB,LVEF,etc,were examined meanwhile.All patients were followed up for 30 days,mainly for recording the incidence of cardiovascular events such as cardiac sudden death,congenital heart disease,non-fatal cardiac re-infartion,etc.Statistical correlation analysis was done between TF and CRP,CK-MB,LVEF and related clinical events.2.The same human umbilical vein endothelial cells(HUVECs)was cultured and passage cultured,and was then divided into control group, TNF-a group,Flu group,and TNF-a+Flu group.In TNF-a+FIu group, different concentrations(0.01,0.1,1,10,100μmol/L)of fluvastatin was added.ELISA method was used to examine the TF levels, chromatogenous substrate assay was used to examine TF activity. RT-PCR method was used to examine the expression of TFmRNA. Western-Blot method was used to examine the TF protein expression and RhoA activity.TF protein expression of HUVECs in control group, TNF-a+AngⅡgroup,AngⅡgroup group,TNF-a group and TNF-a+Y27632 group was examined using Western-Blot method.3.Statistical analysis was done using SPSS13.0 software. Quantitative data was expressed by X±S.Normal distribution analysis was done for the main parameters,t-test was done between 2 groups. P＜0.05 was considered to be statistically important.Results1.TF level in SAP and AMI group was significantly increased compared with normal health control[(28.34±17.58)pg/ml vs(16.73±8.28)pg/ml;(42.76±25.14)pg/ml vs(16.73±8.28)pg/ml,P＜0.05]. Compared to SAP,TF level and activity of AMI patients was significantly increased[(42.76±25.14)pg/ml vs(28.34±17.58)pg/ml,P＜0.05].In AMI patients,main cardiac event of high TF group was significantly more than that of low TF group(48.4%vs 19.4%,P＜0.05).Logistic regression analysis showed that serum TF level was positively related to cardiac events in AMI patients(OR为1.45,P=0.026).Compared with low TF group,CRP[(12.7±5.9)mg/l vs(9.3±4.9)mg/l,P＜0.05]and CK-MB [(84.4±30.2)U/L vs(65.9±29.7)U/L,P＜0.05]of high TF group was significantly increased,and LVEF[(34.9±11.4)%vs(51.9±8.9)%,(P＜0.05]was significantly decreased.Multiple regression analysis showed that TF was positively related to CRP(r=0.702,P＜0.05)and CK-MB(r=0.519,P＜0.05),and was negatively related to LVEF(r=-0.312, P＜0.05).2.The TF level[(9.64±0.58)pg/ml vs(2.30±0.20)pg/ml,P＜0.05] and activity[(32.56±3.12)pM vs(11.36±0.67)pM,P＜0.05],TF expression[(0.88±0.26)vs(0.02±0.01),P＜0.05]and its protein level [(0.94±0.32)vs(0.00±0.02),P＜0.05]in TNF-a group was significantly higher than that of control group(P＜0.05).Compared to TNF-a group, the TF content,activity,TFmRNA expression,and TF protein level of different concentration of fluvastatin intervention group was significantly decreased.There was significant difference(Fig2-2,P＜0.05).The inhibitive effect of Flu was concentration dependent.At concentration of 1/μmol/L,Flu significantly inhibited the TNF-a induced TF expression and activity[(4.99±0.24)pg/ml vs(9.64±0.58)pg/ml,(20.27±1.26)pg/ml vs(32.56±3.12)pM,P＜0.05].Further increase of the Flu concentration made no difference in the above effect(P＞0.05).3.In the meanwhile of inducing the HUVECs TF expression, TNF-a also activated RhoA.Fluvastatin inhibited the activation of RhoA. AngⅡ(RhoA kinase activator)could enhanc the the TF expression in HUVECs which induced by TNF-a,while Y27632(RhoA kinase inhibitor)could inhibit the TF expression in HUVECs which induced by TNF-a.Conclusions1.TF was the key factor in the formation of thrombosis in artherosclerosis plaque rupture.Serum TF level was positively correlated to inflammation,extent of disease and severity in AMI patients.Early examination of serum TF level may considered to be a practical predicting factor in the assessment of future malprognosis such as cardiac events in AMI patients.2.Fluvastatin effectively inhibited the TF content,activity,gene and its protein expression level in HUVECs which induced by TNF-a,thus indicating a possible external anti-thrombosis effect of Fluvastatin besides its anti-cholesterol effects.3.The inhibitive effect of fluvastatin to the TF expression might be achieved through the RhoA/Rho signal regulatory pathway at small G protein level.