Effect Of Fibrinogen, Fibrin, Fibrinogen Degradation Products On The Migration And Adhesion Of Human Umbilical Vein Endothelial Cells

Objective:To investigate the role of fibrinogen(Fg),fibrin(Fb),fibrinogen degradation products(FDPs)on the migration of human umbilical vein endothelial cells(HUVECs)and contribution to express of ICAM-1 and the molecular mechanism of their action.Methods:Part 1:HUVECs in primary culture were grown on the side of Transwell membrane,and smooth muscle cells(SMCs)of rabbit were grown on the bottle of culture, so that two kinds of coculture system were established,and detail observation on the coculture systems was carried out by fluorescence inverted microscope and Immunocytochemical stain.Part 2:The coculture system of HUVECs and SMCs were treated with different concentrations(0mg/ml,0.5 mg/ml,1.5 mg/ml,3.0 mg/ml,4.5 mg/ml,6.0 mg/ml)Fg,Fb,FDPs,the migration of HUVECs were performed using the Sarkar's technique and the Transwell cell culture apparatus.Part 3:The coculture system of HUVECs and SMCs were treated with different concentrations(0mg/ml,0.5 mg/ml,3.0 mg/ml,6.0 mg/ml)Fg,Fb,FDPs,among these models the inhibitor of I-κB(BAY11-7082) and PKC(Staurosporine)were used,which concentrations were 20μM and 50nM.And then total cellular RNA was prepared by the Trizol extraction from confluent cells.ICAM-1 mRNA levels expressed by HUVECs was determined by Reverse transcription polymerase chain reaction(RT-PCR)method and the antigen level of ICAM-1 was detected by Enzyme linked immunosorbent assay(ELISA)method.Result:Part 1:HUVECs in primary culture were positive ofⅧfactor by histochemistry staining.HUVECs and SMCs were grown well on both sides of Transwell membrane, relative to HUVECs monolayer of "cobblestone appearance",SMCswere multilayer of "hills and valleys appearance".HUVECs and SMCs on both sides of Transwell membrane can form the gap junctions by micropores.Part 2:Fg Group:The higher concentration of Fg(≥3.0mg/ml)can stimulated HUVECs migration in a dose-dependent manner.In the subunits of 3.0mg/ml and 6.0 mg/ml Fg group enhanced significantly the expression of ICAM- 1 mRNA and protein.Both the antagonist inhibit the expression of ICAM-1;when BAY11-7082 were added in 3.0mg/ml and 6.0mg/ml Fg group,it has a sepecial effect of inhibition.However,only Staurosporine dropped in 3.0 mg/ml Fg group down-regulated the expression of ICAM-1.Fb Group:The concentration of Fb which is higher than 1.5mg/ml can stimulated HUVECs migration in a dose-dependent manner.Only in 6.0mg/ml group,the expression of ICAM-1 mRNA and protein were obviously up-regulate, as the drops after BAY11-7082 and Staurosporine were respectively added in hyper-concentration(6.0mg/ml),the expression of ICAM-1 mRNA and protein were obviously down-regulated FDPs Group:The concentration of FDPs which is higher than 0.5mg/ml can stimulated HUVECs migration in a dose-dependent manner.Only in 6.0mg/ml group,the expression of ICAM-1 mRNA and protein were obviously up-regulate, as the drops after BAY11-7082 and Staurosporine were respectively added in hyper-concentration(6.0mg/ml),the expression of ICAM-1 mRNA and protein were obviously down-regulated.Conclusion:PartⅠ:The coculture systems of HUVECs and SMCs were established successefully by modeling the structural relationship of vascular wall.The coculture systems have a good biological compatibility which was no effect to normal activity and growth of both cells.PartⅡ:Fg,Fb and FDPs of certain concentration can promote the migration of HUVECs,and play an important role in the development of atherosclerosis. PartⅢ:Fg,Fb and FDPs may play important roles in the inflammation and adhesion process of atherosclerosis via influencing the expression of ICAM-1 in HUVECs,in which the effect of Fg,Fb and FDPs may be mediated by PKC and NF-κB pathway.