The Effect Of Tissue Factor Pathway Inhibitor On Apoptosis Of Cultured Rat Mesangial Cells And Its Relative Mechanisms

IntroductionGlomerulosclerosis is a final common outcome of glomerular injury in various types of human glomerular diseases,which is characterized with the proliferation of glomerular mesangial cell(MsC) and abnormal accumulation of extracellular matrix(ECM) in glomeruli.There is increasing evidence that the imbalance of ECM synthesis and degradation may be an important mechanism of glomerulosclerosis.Glomerular MsC plays an important role in the regulation of ECM metabolism.Recent research points are focused on the regulation of MsC apoptosis and its correlation with the development of glomerulosclerosis.As resident cells in glomerulus,mesangial cells play an important role not only in maintaining the normal structure and functions of glomerulus,but also in acute glomerulonephritis and progressive glomerulosclerosis.In many glomerular sclerosing diseases,mesangial cell proliferation is a major important pathological feature in progressive glomerular injuries.Apoptosis is the major cell clearance mechanism for removal of large numbers of excess mesangial cells in experimental mesangial proliferation. Recent researches have demonstrated that apoptosis may be limited increases in MsC number,to mediate removal of surplus MsCs and to resume the structure and function of glomerular.Tissue factor pathway inhibitor(TFPI) is a Kunitz-type serine protease inhibitor which exhibits a strong and specific inhibitory activity against the tissue factor(TF)-mediated initiation of the blood coagulation cascade.The function of TFPI as the physiologic inhibitor of TF-initiated coagulation has promoted interests in applying recombinant TFPI (rTFPI) as a substitution therapy in some inflammations and cancers.Growing evidence indicates that TFPI has antiangiogenic and antitumor activities and may play a role in cell anti-proliferation and apoptosis.Some studies show that human recombinant TFPI(hrTFPI) prevented the proliferation of cultured human neonatal aortic smooth muscle cells and induced apoptosis in cultured human endothelial cells.In addition to endothelial cells, human MsCs also have the ability to produce TFPI which inhibits fibrin formation.Some animal tests and clinical trials also show that TFPI is strongly expressed in crescentic glomerulonephritis and has the potential to be of therapeutic benefit in the management of fibrin dependent human glomerulonephritis.This suggested us TFPI may has the function in the therapeusis of glomerulonephritis.The aim of the present study is to observe the expression of TFPI in the progression of mesangial proliferative glomerulonephritis;to investigate the effect of rTFPI on the apoptosis of rat cultured MsCs and find the functional domain of rTFPI which leads to apoptosis of MsC and to analyze the possible signaling pathways involved in rTFPI-induced apoptosis in MsC.The purpose of our research was to elucidate the function and significance of rTFPI in mesangial proliferative glomerulonephritis and provide new sight and experiments data for the therapeusis of these glomerulonephritis.PartⅠExpression and purification of recombinant tissue factor pathway inhibitorObjective To obtain the purified recombinant TFPI.Methods pET-28a(+) and TFPI gene was treated with the restriction endonucleases of NcoI and NotI.The recombinant plasmid of pET-28a(+)-TFPI was transformed to E.coli. rTFPI was purified by ion exchange chromatography and characterized by Western blot. Dilute-prothrombin time(dPT) was used to examined the anti-coagulation activity of rTFPI.Results Recombinant TFPI(rTFPI) was expressed successfully in E.coli as inclusion bodies.Compared with control,rTFPI prolonged prothrombin time for 16.2s.Conclusions After refolding and purification,recombinant TFPI was successfully obtained with purity of more than 95%with highly anti-coagulation activity.PartⅡExpression of TFPI in rat anti-Thy-1 nephritis model and human glomerulonephritis and its effect on MsC apoptosisObjective To explore the expression of TFPI in glomerulus in rat anti-Thy-1 glomerulonephritis model and human glomerulonephritis,determinate the effect of rTFPI on apoptosis in cultured rat MsC and find the functional domain of rTFPI which leads to MsC apoptosis.Methods Rabbit anti-rat Thy-1 serum was used to construct rat anti-Thy-1 glomerulonephritis model.Immunohistochemistry and image analysis software were used to analyze the expression of TFPI in glomerulus in rat anti Thy-1 glomerulonephritis model and human glomerulonephritis with different pathologic types.Hoechst 33258,flow cytometry,DNA fragmentation and Western blot were used to analyze apoptosis of MsC which stimulated by rTFPI,rTFPI_1-161.MBP-TFPI_162-276,MBP-TFPI KD3 and MBP-TFPI C terminal,respectively.Results In rat anti-Thy-1 glomerulonephritis,TFPI was begun to express obviously in the glomeruli at day 14,markedly increased at day 21 and peaked at day 28.Compared with glomerular minor lesion,TFPI in glomerulus was highly expressed in human mesangial proliferative glomerulonephritis.Apoptotic MsCs were increased after stimulated by rTFPI in a dose-and time-dependent manner.The characteristic pattern of fragmentation of DNA in multiple of 180-200bp was found after MsCs were treated with rTFPI,rTFPI has an effect on MsC apoptosis when MsC is proliferating induced by PDGF.The expression of active Caspase-3 was also increased in a dose-dependent manner after added rTFPI.There was no apoptotic MsCs after treated with rTFPI_1-161 and MBP-TFPI KD3,whereas typical images of apoptotic MsCs were found in which stimulated by rTFPI,MBP-TFPI_162-276 and MBP-TFPI C terminal.Conclusions In rat anti-Thy-1 glomerulonephritis,TFPI in glomerulus was expressed in the late period and increased with the progress of nephritis.TFPI was expressed higher in glomerulus in human mesangial proliferative glomerulonephritis than that in glomerular minor lesion,rTFPI induced apoptosis in cultured rat MsC in a dose-and time-dependent manner by its C terminal even though MsC was proliferating.PartⅢStudy of the related signaling pathway on rTFPI-induced apoptosis in cultured rat MsCObjective To explore whether rTFPI induced MsC apoptosis via binding to TF and the possible signaling pathway involved in rTFPI-induced MsC apoptosis.Methods Immunofluorescence was used to determine whether rTFPI induced MsC apoptosis via binding to TF.Western blot was used to analyze the expressions of Bcl-2,Fas, phospho-Akt,total Akt,phospho-IκB.Hoechst 33258 was used to determine apoptosis after blocked the Fas pathway by its neutralizing antibody.RT-PCR,restriction endonucleases digestion and ligation were used to reconstructing eukaryotic expression plasmid pEGFPc1-Akt1.Lipofectamine^TM 2000 was used to stably transfect Akt1 vectors into MsC. Western blot analysis were performed to verify the transfection.Hoechst 33258 and flow cytometry were used to analyze apoptosis in normal MsC,pEGFPcl-MsC and pEGFPc1-Akt1-MsC treated by rTFPI.Results The red fluorescence which represented rTFPI didn't merge in the green fluorescence represented TF in MsC cytomembrane.Treated with rTFPI in MsC,there were no changes in the Bcl-2 protein expression,but the expression of Fas was increased in a dose-dependent manner.After blocked Fas signalling pathway by its neutralizing antibody, apoptotic MsCs induced by rTFPI were decreased obviously.By contraries,phospho-Akt and phospho-IκB,which represented the activation of PI3K/Akt pathway,were decreased in a dose-dependent manner after treated with rTFPI.After over-expressed Akt1 gene, apoptosis was prevented in MsC which were transfected with Akt1 although treatment with rTFPI.However,the normal MsCs and MsCs which were transfected pEGFPc1 were undergone apoptosis after rTFPI treatment.Conclusions rTFPI-induced apoptosis of MsCs is not via binding to TF.Bcl-2 did not involve in the signaling pathway which induces apoptosis in MsCs by rTFPI stimulation. Death receptor Fas/FasL signalling pathway involved in the progress of apoptosis in MsC induced by rTFPI.Blocked this pathway,apoptotic MsCs were decreased obviously. PI3K/Akt signalling pathway was one of the significant pathways which mediated rTFPI-induced MsC apoptosis.Up-regulated Akt was able to prevent the apoptosis progress.Conclusions1.MsC is one kind of cells which expresses and secretes TFPI.In rat anti-Thy-1 glomerulonephritis,TFPI in glomerulus was expressed in the late period and increased with the progress of nephritis.TFPI was expressed higher in glomerulus in human mesangial proliferative glomerulonephritis than that in glomerular minor lesion.This suggests that TFPI may involve in the pathological progress of mesangial proliferative glomerulonephritis.2.rTFPI induced apoptosis in cultured rat MsC in a dose- and time-dependent manner by its C terminal. 3.rTFPI-induced apoptosis of MsCs is not via binding to TF.Bcl-2 did not involve in the signalling pathway which induces apoptosis in MsCs by rTFPI stimulation.Death receptor Fas/FasL signalling pathway involved in the progress of apoptosis in MsC triggered by rTFPI.PI3K/Akt signalling pathway was one of the significant pathways which mediated rTFPI-induced MsC apoptosis.