Functions And Mechanisms Of Tissue Factor Pathway Inhibitor 2 In Atrial Fibrosis

Part ?:Preliminary study on phenotypes and mechanisms of TFPI-2 in primary atrial cardiomyocytes and fibroblastsObjective:To explore phenotypes and mechanisms of TFPI-2 in primary atrial cardiomyocytes and atrial fibroblasts initially.Methods:The atrial tissues of SD neonatal rats were separated,cultured and identified to obtain primary atrial cardiomyocytes and atrial fibroblasts by enzymatic hydrolysis and immunofluorescence assay;CCK-8 assay was used to detect the effect of TFPI-2 on the viability of both cells;flow cytometry was used to detect the effects of TFPI-2 on apoptosis in two kinds of cells;transwell test was used to detect the effects of TFPI-2 on the atrial fibroblasts migration;western blot was used to detect the levels of cell viability and migration-related protein p-Erk1/2,and cell apoptosis-related protein PARP as well as profibrotic proteins(Col-?,Col-?,MMP-9 and MMP-2).Results:SD neonatal rat atrial cardiomyocytes and atrial fibroblasts was isolated,cultured,and identified successfully with higher purity;after TFPI-2 was administrated,the cells viability of both cells were reduced in a concentration gradient-dependent manner;atrial fibroblasts migration were inhibited and its apoptosis were induced;the expression of pro-fibrotic proteins(Col-?,Col-?,MMP-9 and MMP-2)were down-regulated in both cells;the expression of apoptosis protein PARP was up-regulated.Conclusion:TFPI-2 exerted anti-fibrotic effects,and its functions could be achieved by reducing the phosphorylation level of p-Erk1/2 to reduce the viability and migration of atrial fibroblasts,up-regulating the expression of PARP to induce apoptosis of atrial fibroblasts,down-regulating the expression of cells promoting fibrosis proteins(Col-I,Col-III,MMP-9 and MMP-2).Part II:Effects of TFPI-2 on fibrosis-related phenotypes in murine HL-1 atrial cardiomyocytes and murine NIH/3T3 fibroblastsObjective:To study the effects of TFPI-2 on fibrosis-related phenotypes in HL-1 cells and NIH/3T3 cells.Methods:We constructed a TFPI-2 up-regulated stable transfection strain in HL-1 atrial myocytes by lentivirus-mediated method;the proliferation functions of TFPI-2 and TGF-?1 on HL-1 cells and NIH/3T3 cells were determined via MTT assay;the effects of TFPI-2 on apoptosis of HL-1 cells and NIH/3T3 fibroblasts were determine via flow cytometry;HL-1-TFPI-2 cells and NIH/3T3 cells were co-cultured to measure the effect of HL-1-TFPI-2 supernatants on migration of NIH/3T3 fibroblasts;TFPI-2 was administrated to NIH/3T3 fibroblasts for its effects on the migration of NIH/3T3 fibroblasts via scratch assay.Results:Green fluorescent protein GFP expression was observed by fluorescence microscopy and q-RT-PCR assay was used for TFPI-2 mRNA to confirme the construction of HL-1-TFPI-2 stable cell line;the proliferations of HL-1 atrial myocytes and NIH/3T3 fibroblasts were inhibited by TFPI-2 in MTT assay;the migration of NIH/3T3 fibroblasts was inhibited by TFPI-2 in transwell co-culture and scratch assay;flow cytometry double staining showed that TFPI-2 induced NIH/3T3 fibroblasts apoptosis.Conclusion:The HL-1-TFPI-2 cells were successfully constructed;the proliferations of HL-1 cells and NIH/3T3 cells were promoted by TGF-?1,while the proliferations of both cells were inhibited by TFPI-2;TFPI-2 inhibited the migration and induced apoptosis in NIH/3T3 cells.Part ?:The anti-atrial fibrosis mechanisms of TFPI-2Objective:To study the anti-fibrosis mechanism of TFPI-2 in HL-1 atrial cardiomyocytes and NIH/3T3 fibroblasts.Methods:HL-1-TFPI-2 cells culture medium supernatant was collected,and the effect of TFPI-2 on MMP-9 and MMP-2 activity was detected by gelatin zymography.The effects of TFPI-2 on the transcription levels of pro-fibrotic molecules(Col-?,Col-?,MMP-9,MMP-2,TGF-?1)were determined by quantitative real-time PCR in HL-1 atrial cardiomyocytes and NIH/3T3 fibroblasts.The effect of TFPI-2 on the transcription activity of MMP-2 and MMP-9 gene promoters were obtained via dual-luciferase reporter gene experiment in HL-1 cells and NIH/3T3 cells.RNA interference technology was used for knocking down TFPI-2 in both cells to detect the expressions of pro-fibrotic protein(Col-?,Col-?,MMP-9,MMP-2).Western blot experiments to detect:the effect of TFPI-2 on fibrogenic proteins(Col-?,Col-?,MMP-9 and MMP-2)expression in HL-1-TFPI-2 cells;the regulation of TFPI-2 on MAPK signaling pathway,NF-?B/MMP signaling pathway,TGF-?1/Smad/Collagen signaling pathway,Bcl-2/Bax in NIH/3T3 fibroblasts;the effects of TGF-?1 on the expression of pro-fibrotic proteins(Col-I,Col-III,MMP-9 and MMP-2)in HL-1 atrial cardiomyocytes and NIH/3T3 fibroblasts.Results:The activities of MMP-9 and MMP-2 were directedly inhibited by TFPI-2;the pro-fibrosis molecules(Col-?,Col-?,MMP-9,MMP-2,TGF-?1)mRNA transcription level were suppressed by TFPI-2 in HL-1 cells and NIH/3T3 cells;the transcription activity of MMP-2 and MMP-9 gene promoters were inhibited by TFPI-2 in HL-1 atrial cardiomyocytes and NIH/3T3 fibroblasts;western blot experiments showed that:the expressions of fibrosis-promoting proteins(Col-?,Col-?,MMP-2,MMP-9)were down-regulated in HL-1-TFPI-2 cells;knocking down TFPI-2 promoted the expression of fibrosis-promoting proteins(Col-?,Col-?,MMP-2,MMP-9)in in HL-1 cells and NIH/3T3 cells;TFPI-2 inhibited p-Erk1/2 level and promoted p-JNK and p-P38 levels by regulating MAPK signaling pathway,TFPI-2 down-regulated the levels of NF-?B,p-NF-?B,MMP-2 and MMP-9 by regulating NF-?B/MMPs signaling pathway,TFPI-2 declined the levels of TGF-?1,Col-I,Col-III,p-Smad2,p-Smad3 by regulating TGF-?1/Smad/Collagen signaling pathway,TFPI-2 down-regulated Bcl-2 expression and up-regulated Bax protein expression;TGF-?1 promoted pro-fibrosis protein(Col-?,Col-?,MMP-9)expression in HL-1 cells,TGF-?1 promoted NIH/3T3 fibroblasts pro-fibrosis proteins(Col-?,Col-?,MMP-9,MMP-2,?-SMA)expression.Conclusion:TGF-?1 stimulated the expression of fibrosis-promoting proteins(Col-?,Col-?,MMP-2,MMP-9)in HL-1 atrial cardiomyocytes and NIH/3T3 fibroblasts;in HL-1 atrial cardiomyocytes,TFPI-2 exerted its anti-fibrosis function by inhibiting fibrosis-promoting molecules(Col-?,Col-?,MMP-2,MMP-9,TGF-?1)transcription and expression levels;in NIH/3T3 fibroblasts,the anti-fibrosis effects of TFPI-2 were achieved by the following ways:in the MAPK signaling pathway,TFPI-2 inhibits fibroblast proliferation and migration by down-regulating p-Erk1/2 level and induces fibroblast apoptosis by up-regulating p-JNK and p-P38 levels;in the NF-?B/MMPs signaling pathway,TFPI-2 not only inhibited fibrosis by down-regulating the levels of NF-?B,p-NF-?B,MMP-2 and MMP-9,but also directly inhibited the activities of MMP-2 and MMP-9;in the TGF-?1/Smad/Collagen signaling pathway,TFPI-2 down-regulated the levels of TGF-?1,Col-?,Col-?,p-Smad2,p-Smad3 to play an anti-fibrotic role;TFPI-2 induced NIH/3T3 fibroblasts apoptosis through up-regulating Bax protein and down-regulating Bcl-2 expression.