The Research Of Endothelial KLF15 Regulate TM Active Protein C Anticoagulation Mechanism And MCP-1 Induce Inflammatory Reaction With Deep Venous Thrombosis

Our research group has establish rat and rabbit DVT model, also use gene chip analysis of thrombosis/non-thrombosis relative genes. We found that venous endothelial cell activation, coagulation/anticoagulation system imbalance and inflammatory reaction had close relationship with DVT. In this research we study the relationship and regulatory relationship between KLLF15,inflammatory gene MCP-1, anticoagulatory gene TM and APC with DVT in animal and cell model. In animal model research we discuss the expression changes and functions of KLF15, MCP-1, TM genes in inferior vena cava stenosis DVT C57 mice venous wall and APC protein expression in mice venous wall and plasma; in thrombin induced HUVECs cell model we used siRNA technique inhibited KLF15 expression then study the regulatory relationship with down stream TM gene; over-expression/interference MCP-1 gene of HUVECs to investigate the association between the down stream genes and signaling pathways exchange with DVT.Objective:1. Established inferior vena cava stenosis DVT C57 mice model, harvested inferior vena cava venous tissue and plasma separeted by blood 24 hours after stenosis, observed the DVT formation by HE dyeing, detected KLF15,MCP-1.TM,APC expression of venous tissue and also detected APC expression of plasma before and after the formation of DVT then analysis the relationship between these genes with DVT.2. To investigate the effect of thrombin on KLFs and down stream inflammatory, coagulation/anticoagulation and adhension genes expression of HUVECs and then to decide the optimal action time course and concentration of thrombin induced KLF15 expression; in thrombin induced HUVECs cell model after siRNA technique inhibited KLF15 expression to study the regulatory relationship with down stream TM gene.3. To constructed MCP-1 over-expression/interference vector then transfected HUVECs, we investigate the association between the down stream genes and signaling pathways exchange with DVT.Method:1.120 C57 mice were randomly divided into normal control group(n=40)、sham operation group(n=40)、DVT group(n=40), DVT group was built by stenosis inferior vena cava harvested inferior vena cava venous tissue and blood separation of the plasma 24 hours after stenosis, observed the DVT formation by HE dyeing, we detected KLF15、MCP-1、TM expression of venous tissue by real-time PCR test then we detected APC expression of venous tissue and plasma by ELISA test.2. We measured the expression of KLF2、KLF4、KLF5、KLF6、KLF10、KLF11、 KLF15 induced by thrombin at 0、2、4u/ml concentrations 8 hours in HUVECs cells. The optimal action time course and concentration of thrombin induced KLF15 expression was determined by different concentration (0,2u/ml,4u/ml, 8u/ml) pretreated HUVECs at 8 hours also used 4u/ml thrombin at different action time(0,4h,8h,12h) pretreated HUVECs. We measured the expression of anti-coagulation gene TM, anti-fibrinolytic factor (PAI-1), adhesion gene (VCAM) induced by thrombin at 4u/ml concentrations at 8 hours in HUVECs. We used siRNA technique inhibited KLF15 expression then study the regulatory relationship with down stream TM, VCAM, PAI-1 genes.3. The cultured HUVECs were tested by immunofluorescence and co-immunoprecipitation then constructed MCP-1 over-expression/interference MCP-1-pCDH-GFP/MCP-l-LMP shRNAmirlvector transfected virus then infected HUVECs, detected efficiency of transfection, and the change of transcription profile were detected by microarray assay and biological information technology analysis.Result:1. DVT group thrombosis rate 85.3%, motality rate 15%, stenosis rate 90.8%±0.8%, residual lumen cross-sectional area percentage 9.2%±0.8%, thrombus length 5.85±0.25mm, thrombus wet weight 0.27±0.02g, IVC diameter 0.99±0.04mm; real-time PCR detected mice venous tissue KLF15、MCP-1、TM mRNA expression in three groups. KLF15、MCP-1 expression were higher in DVT group compare with normal control and sham operation group(P<0.05).TM expression were higher in DVT group compare with normal control group(P<0.05).3 genes expression were no differences between normal control and sham operation group(P>0.05).ELISA detected mice plasma APC protein expression in three groups. APC expression were higher in DVT group compare with normal control and sham operation group(P<0.05) but no differences between normal control and sham operation group(P>0.05). ELISA detected mice venous tissue APC protein expression in three groups. APC expression were lower in DVT group compare with normal control and sham operation group(P< 0.05) but no differences between normal control and sham operation group(P>0.05).2. Thrombin pretreated HUVECs at 0、2、4u/ml concentrations 8 hours, KLF2、 KLF4、KLF5、KLF6、KLF10 expression significant down-regulate and KLF11、 KLF15 expression up-regulate compare with control gourp(P<0.05). Different concentration(0,2u/ml,4u/ml,8u/ml) pretreated HUVECs at 8 hours and used 4u/ml thrombin at different action time(0,4h,8h,12h) pretreated HUVECs, the optimal action time course and concentration of thrombin induced KLF15 expression was 4 u/ml and 8hours(P<0.05).Thrombin pretreated HUVECs at 4u/ml concentrations 8 hours, TM、PAI-1 expression significant up-regulate(P< 0.05) and VCAM has no difference compare with control gourp(P> 0.05).KLF15、TM expression in Thrombin+KLF15 siRNA group lower than Thrombin group(P<0.05), PAI-1 expression in Thrombin+KLF15 siRNA group had no statistic difference compare with Thrombin group (P>0.05), VCAM down-expressed in all three group, KLF15 had not regulated PAI-1 and VCAM expression.3. After MCP-1 over-expression/interference in HUVECs. According to the microarray analysis we found that MCP-1 over-expression group with normal cell have 158 genes down-expression 18 signaling pathways down-regulated and 71 genes up-expression 7 signaling pathways up-regulated; Compared with normal group cells, in MCP-1 shNRAmirl group has 1390 genes down-expressed 60 signaling pathways down-regulated and 250 genes showed up-expressed 15 signaling pathways up-regulated. IL-6、MMP-3、MMP-19、VEGF in MCP-1 over-expression group up-expressed and in MCP-1 shNRAmirl group down-expressed compare with normal cell. Compared between MCP-1 over-expression group, MCP-1 shNRAmirl group and normal cell we found MAKP and PI3K-AKT signaling pathway has significant difference change.Conclusion:1. Use 30G needle to establish inferior vena cava stenosis DVT C57 mice model has higher thrombosis rate and lower mortality rate, at 24h there were complete thrombus formation in veins. This animal DVT model is stable and reliable IVC stenosis DVT C57 mice model.2. In initial stage of DVT, coagulation/anticoagulation system out of balance, protein C anticoagulation system has been activated. MCP-1、TM may play an important raole in thrombus formation.3. In thrombin induced HUVECs model KLF15 could regulated down stream TM gene expression.4. In DVT inflammatory reaction MCP-1 may coordinate or regulate effect with IL-6, in DVT resolution MCP-1 may coordinate or regulate effect with MMP-3,MMP-19 and in venous remodeling MCP-1 may coordinate or regulate effect with VEGF.MCP-1 may regulated MAKP and PI3K-AKT signaling pathways in DVT endothelial cell inflammation, venous remodeling and thrombus resolution.