TNAP Promotes Cardiac Fibrosis After Myocardial Infarction By Mediating TGF-?1/Smads And ERK1/2 Signal Pathway

PART ONE RELATIONSHIP BETWEEN TNAP AND MORTALITY IN PATIENTS WITH ISCHEMIC HEART DISEASE AND THE EXPRESSION FEATURES OF TNAP IN HEART TISSUEAims: The purpose of this part was to evaluate the effect of serum tissue non-specific alkaline phosphatase(TNAP)on the mortality in patients with ischemic heart disease(IHD)by a meta-analysis.Then,the expression features of TNAP in heart tissue with myocardial infarction(MI)were examined.Methods: We conducted a comprehensive online search of published literature using Medline,Cochrane and EMBASE databases(to December31,2020)to identify all publications and to assess the effect of serum TNAP on mortality in patients with IHD.The primary endpoints were all-cause mortality and cardiovascular mortality.Dichotomous data were analyzed using relative risk(RR)with 95% confidence interval(CI).Pooled analyses were calculated using random-effect models.Human heart biopsies with MI and the mouse of MI model(the ligation of left anterior descending coronary artery,LAD)were used to detect the expression and distribution of TNAP by Masson staining,immunohistochemistry(IHC)and Western Blot.Results: After searching the main databases and detailed full-text articles assessed,eight studies with 14,816 individuals were included in the quantitative analysis.Meta-analysis showed that the high level of serum TNAP was associated with increased risk of all-cause mortality(RR: 1.89;95% CI,1.48 to 2.42;P<0.001)and cardiovascular mortality(RR: 1.85;95% CI,1.33 to 2.59;P<0.001)in patients with IHD.TNAP was also associated with increased risk of all-cause mortality in patients with MI(RR: 1.71;95% CI,1.36 to 2.16;P<0.001).In different subgroups,high serum TNAP was still associated with increased risk of all-cause mortality.Both in humans and animals,heart tissues with MI exhibited a significantly increased expression of TNAP,and the distribution of TNAP was consistent with the distribution of ?-SMA and Collagen ?.TNAP expression reached the peak within 14 days after MI surgery,then maintained parallel levels with myofibroblasts marker ?-SMA activation and Collagen ? deposition.Conclusions: High serum TNAP level was associated with an increased mortality risk in patients with IHD and MI.The expression of TNAP was upregulated in heart tissues with MI,and the distribution of TNAP was consistent with the distribution of ?-SMA and Collagen ?.PART TWO TNAP REGULATS CARDIAC FUNCTION AND CARDIAC FIBROSIS AFTER MYOCARDIAL INFARCTIONAims: To explore the role of TNAP in cardiac function and cardiac fibrosis after MI.Methods: The adenovirus-mediated TNAP gene knockdown and TNAP gene overexpression plasmids were constructed.In vivo studies,mouse model of MI was established by the ligation LAD coronary artery.Enzyme digestion was used to isolate cardiac fibroblasts(CFs)from C57BL/6J(1-3days)for in vitro studies,and then stimulated by hypoxia.The parameters of cardiac function were evaluated by cardiac ultrasound.Masson staining was used to examine the deposition of collagen.The IHC and Western Blot were used to detect the expression protein of fibrosis.Wound healing assay was used to examine the ability of migration.The CCK-8 assay was performed to test the ability of proliferation.Results:(2)In vivo studies(1)TNAP knockdown exhibited a significant improvement in left ventricular ejection fraction(LVEF),fractional shortening(FS),left ventricular internal diameter at diastole(LVIDd),left ventricular internal diameter at systole(LVIDs)in MI mice.Contrary to the findings of TNAP knockdown,TNAP overexpression exhibited a dramatic reduction in LVEF and FS,enlarged LVIDd and LVIDs.(2)TNAP knockdown reduced the infarct area of ventricular,overall collagen deposition and the expression of ?-SMA and Collagen ? compared with sham group.But TNAP overexpression aggravated the infarct area of ventricular,overall collagen deposition and the expression of?-SMA and Collagen ?.In vitro studies(1)the expression of TNAP increased with the prolongation of hypoxia time in CFs,and the difference was significant at 24 hours,which was similar to the expression of transforming growth factor-?1(TGF-?1)and Collagen ?.(2)TNAP knockdown markedly inhibited the expression of ?-SMA and Collagen ? induced by hypoxia.However,TNAP overexpression significantly promoted the expression of ?-SMA and Collagen ?.(3)Wound healing assay found that TNAP knockdown significantly inhibited migration of CFs induced by hypoxia.In contrast,TNAP overexpression significantly enhanced the migration of CFs.(4)CCK-8 assay showed that TNAP knockdown significantly inhibited proliferation of CFs induced by TGF-?1.In contrast,the overexpression of TNAP significantly accelerated the growth rate of CFs.Conclusions: In vivo studies,the knockdown of TNAP improved cardiac function and ameliorated cardiac fibrosis after MI.In contrast,the overexpression of TNAP aggravated cardiac fibrosis and worsened cardiac function in vivo after MI.In vitro studies,TNAP knockdown inhibited the differentiation,migration and proliferation of CFs.TNAP overexpression promoted the differentiation,migration and proliferation of CFs.PART THREE TNAP PROMOTES CARDIAC FIBROSIS BY MEDIATING TGF-?1/SMADS AND ERK1/2 SIGNALAims: To explore the potential mechanisms of TNAP in cardiac fibrosis after MI.Methods: In vivo studies,mouse model of MI was established by the ligation of LAD coronary artery.Enzyme digestion was used to isolate CFs from C57BL/6J(1-3days)for in vitro studies.The expression of ?-SMA,Collagen ?,TGF-?1,Smad2,p-Smad2,Smad3,p-Smad3,ERK1/2,p-ERK1/2,Cyclin D1,Cyclin E1 and CDK2 were quantified by Western Blot.The proliferation of CFs was evaluated by CCK-8 assay.The cell cycle distribution of CFs was detected by flow cytometry(FCM).The relative expression of ?-SMA and vimentin was examined by immunofluorescence.Results:(1)In vivo studies(1)Mice heart with MI exhibited a greater of TGF-?1 expression and the phosphorylation of Smad2 and Smad3.TNAP knockdown suppressed these increase,while TNAP overexpression enhanced them.(2)Mice heart with MI exhibited increased phosphorylation level of ERK1/2.TNAP knockdown suppressed the phosphorylation level of ERK1/2.In contrast,TNAP overexpression enhanced the phosphorylation level of ERK1/2.(2)In vitro studies(1)TNAP knockdown dramatic limited the conversion of hypoxia-induced CFs into myofibroblasts,accompanied by suppressing TGF-?1 and the phosphorylation of Smad2 and Smad3.In contrast,TNAP overexpression enhanced the expression of TGF-?1 and the phosphorylation of Smad2 and Smad3 induced by hypoxia.(2)TNAP knockdown marked attenuated the phosphorylation level of ERK1/2 induced by TGF-?1 in CFs.In contrast,TNAP overexpression enhanced the phosphorylation level of ERK1/2.(3)TNAP knockdown increased the number of cells in G0/G1 phase and reduced in S phase,suppressing the expression of Cyclin E1,Cyclin D1 and CDK2.In contrast,TNAP overexpression reduced the number of cells in G0/G1 phase and increased in S phase,promoting the expression of Cyclin E1,Cyclin D1 and CDK2.(4)CCK-8 assay showed that U0126(10?mo L/L,a specific inhibitor of ERK1/2)inhibited the proliferation of CFs induced by TNAP overexpression.(5)Western Blot and immunofluorescence exhibited that U0126 inhibited the expression of?-SMA,Collagen ? and vimentin induced by TNAP overexpression.Conclusions: Combined with the vivo and vitro experiments,we found that TNAP promoted the cardiac fibrosis,CFs migration,differentiation and proliferation at least in part by activating TGF-?1/Smads and ERK1/2 signal pathways.Additionally,TNAP promoted the conversion of G1 phase to S phase by enhancing the expression of Cyclins.