Molecular Mechanism Of Fibrinogen, Fibrin And Fibrin(ogen) Degradation Products Promoting Atherosclerosis

Objective:To investigate the role of different concentrations of fibrinogen(Fg),fibrin(Fb) and fibrin(ogen) degradation products(FDPs) in the pathogenesis of atherosclerosis and the molecular mechanism of their action.Methods:Part I:Endothelial cells(ECs) of rabbit aorta in primary culture were grown on the side of Transwell membrane,and smooth muscle cells(SMCs) were grown on the other side or the botter of culture well,so that two kinds of coculture systems were established,and the growth characteristic of both cells and the normal phenotypic transition of SMCs were observed.Part II:The coculture systems of ECs and SMCs were treated with different concentrations(0mg/ml,0.5mg/ml,1.5mg/ml,3.0mg/ml,4.5mg/ml and 6.0mg/ml) of Fg,Fb and FDPs,the protein concentrations,α-SM-actin mRNA,proliferating cell nuclear antigen(PCNA)protein expression in the coculture systems were detected by BCA method,reverse transcription polymerase chain reaction(RT-PCR)and western blot method,and the migration rate of SMCs in the coculture systems was observed by a wounding model.PartⅢ-Ⅳ:The coculture systems of ECs and SMCs were treated with different concentrations of Fg,Fb and FDPs,in this coculture systems,tissue plasminogen activator(tPA)mRNA,plasminogen activator inhibitor-1(PAI-1)mRNA,matrix metalloproteinase-2(MMP-2)mRNA and vascular endothelial growth factor(VEGF)mRNA were detected by RT-PCR method,the antigen level of tPA,PAI-1,MMP-2,MMP-8 and VEGF in cell culture medium were assayed with enzyme linked immunosorbent assay kit(ELISA),and the activity of tPA,PAI-1,MMP-2 and VEGF in cell culture medium were assayed by substrate chromogenic assays(tPA and PAI-1),gelatin zymography(MMP-2)and MTT assay(VEGF),while nuclear factor-κB pathway activation of ECs and SMCs in part groups was detected by nuclear translocation assay kit.PartⅤ:The histopathologic changes of human atherosclerotic unstable plaques were observed by hematoxylin and eosin(HE)stainning,and the coexpression in situ of MMP-2,MMP-8 and VEGF in human atherosclerotic unstable plaque were observed by double fluoresent immunochemistry technology and confocal microscopy.Results:Part I:ECs of rabbit aorta in primary culture were positive ofⅧfactor by histochemistry staining.ECs and SMCs were grown well on both sides of Transwell membrane,relative to ECs monolayer of"cobblestone appearance",SMCs were multilayer of"hills and valleys appearance".ECs and SMCs on both sides of Transwell membrane can form the gap junctions by micropores.Proliferation phenotype of SMCs appeared after three days of growth cycle,while six days later SMCs phenotype switched to contraction type.Part II:The higher concentration of Fg(≥3.0mg/ml)can facilitate greatly the protein synthesis,PCNA expression,chemotaxis and migration of SMCs,while inhibitα-SM-actin transcription of SMCs;From 0.5mg/ml concentration,Fb can promote significantly the protein synthesis of SMCs。1.5-4.5mg/ml concentration of Fb enhanced significantly PCNA protein expression,on the contrary,hyper-concentration(6.0mg/ml)of Fb could inhibit PCNA expression of SMCs.Fb of 3.0-6.0mg/ml can promote greatly the chemotaxis,and down-regulateα-SM-actin mRNA of SMCs,but accelerate significantly migration of SMCs from 1.5mg/ml concentration;At the range of 0.5-6.0mg/ml,FDPs can enhance the protein synthesis,PCNA expression,chemotaxis and migration of SMCs in a concentration-dependent manner,meanwhile the higher concentration of FDPs(3.0-6.0mg/ml)could inhibit SMCs greatly switching to contraction type.PartⅢ:There was no significant effect of Fg on tPA expression.The higher concentration of Fg(3.0-4.5mg/ml)enhanced significantly the expression of PAI-1 mRNA,protein and activity,while the highest concentration of Fg(6.0mg/ml)could inhibit the expression of PAI-1;Fb of 3.0-4.5mg/ml can up-regulate tPA mRNA and antigenic content and down-regulate the activity;The higher concentration of Fb(1.5-4.5mg/ml)can up-regulate the expression of PAI-1 at the level of mRNA,protein and activity;FDPs of 3.0-6.0mg/ml down-regulated the expression of tPA,while FDPs of 1.5-6.0mg/ml enhanced the expression of PAI-1 greatly.PartⅣ:4.5-6.0mg/ml of Fg can down -regulate significantly the transcription,protein expression and activity of MMP-2;3.0-6.0mg/ml concentration of Fb enhanced significantly protein expression and activity of MMP-2;At the range of 0.5-6.0mg/ml,MMP-2 mRNA was up-regulated in a concentration-dependent manner;In the subgroups of FDPs of 1.5-6.0mg/ml,the antigen level and activity of MMP-2 was obviously rised.1.5-6.0mg/ml of Fg,Fb and FDPs can enhance over-expression of VEGF at the level of mRNA,protein and activity.3.0-6.0mg/ml of Fg,1.5-6.0mg/ml of Fb and FDPs up-regulated significantly he antigen level of MMP-8 in cellculture medium,while dreased lightly in all hyper-concentration(6.0mg/ml)subgroups.Under the treatment of Fb and FDPs of 3.0-6.0mg/ml,NF-κB pathway in ECs and SMCs can be activated,SN50(the special antagonist of NF-κB pathway)can inhibit the expression of MMP-2 and VEGF,while no effect for expression of MMP-8.PartⅤ:The human atherosclerotic plaques in 6 cases have typical histopathologic unstability,which is classified as super-Ⅳtype unstable plaques.The MMP-2 coexpression was most obvious in the smooth muscle cells of fibrous cap infiltrated by monocyts,and in the monocytes of shoulder of plaques,and more expression of MMP-2 in the microvascular endothelial cells at the edge of shoulder and lipid necrosis;MMP-8 coexpress obviously with the monocytes in the fibrous cap and lipid cores of plaques,and next to coexpressing in the smooth muscle cells of fibrous cap,while coexpression in endothelial cellvs was very little;VEGF coexpression was significant in the proliferative microvascular endothelial cells of plaques;The fibrous cap,which consist of the smooth muscle cells mainly,and the edge of lipid necrosis infiltrated by more monocyts,were over-expression areas of VEGF.Conclusion:Part I:The coculture systems of ECs 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.Part II-Ⅲ:Fg,Fb and FDPs of certain concentration can promote the proliferation,abnormal phenotype transition,chemotaxis and migration of SMCs,and drease the fibrinolysis activity by regulating the expression of tPA and PAI-1 in vascular ECs.As a result,development of atherosclerosis was accelerated.PartⅣ:Fg,Fb and FDPs may play important roles in the unstable process of atherosclerosis via influencing the expression of MMP-2,-8 and VEGF in ECs and SMCs,in which the effect of Fb and FDPs may be mediated by NF-кB pathway.PartⅤ:MMP-2,-8 and VEGF can coexpress with monocytes,SMCs and ECs in unstable plaques,and the major expression areas were in the fibrous cap,shoulder and micro-vessel at the edge of lipid necrosis,which were infiltrated by monocytes.Moreover,the outer membrane of vessel was involved in the pathogenesis of atherosclerosis.As a result,it was confirmed that Fg,Fb and FDPs could promote the unstable process of atherosclerosis in vivo by regulating the expression of MMP-2,-8 and VEGF.