Study Of The Signaling Pathways Of Crosstalk Between Ang Ⅱand TGF-β1 Mediated In The Myocardium Remodeling

PartⅠThe dynamic observation of the expression of TNF-αand TGF-β1 in the ventricular remodeling of rat after myocardial infarctionObjective To clarify the relation of inflammation mediator such as cytokine and ventricular remodeling after myocardial infarction and elucidate the pathophysiology of left ventricular remodeling.Method One hundred and twenty male Sprague Dawley rats were randomized to MI groups(75) and sham-operated(45) groups. MI model was achieved by ligating coronary artery. The sham groups were performed the similary methods with the exception that the coronary ligature was not tied.hemodynamic measurement and histomorphology such as LVmass (ratio of left ventricular weight to body weight ) and size of the MI were determined before the rat were decapitated. Myocardial remodeling models of myocardial infarction in SD rats were sacrificed to detected the gene expression of marker of ventricular remodeling by RT-PCR. We investigate the mRNA levels of embryonic gene(beta-MHC), alpha-MHC ,collagen typeⅠ,Ⅲand several cytokines such as tumor necrosis factor alpha(TNF-α)and transforming growth beta1(TGFβ1 )at different times( 3d, 1, 4, 24weeks). The immunolocations of the collagen typeⅠ,Ⅲwere analyzed by immunohistochemistry in the rat heart at the same time.Results In the non-infarcted myocardial aera, the ration of beta/alpha MHC and collagen typeⅠ/Ⅲw ere increased at 3 days, the increase was sustained for 4 weeks compared with those in sham group( P<0.01), followed by a decrease to sham control level.While thelevel of collagen typeⅠa nd collagen typeⅢis predominant in the infracted zone than that of in the non-infarcted zone at acute phase(4 weeks) , even if at phase(24 weeks) the level of collagen was still higher than that of sham operated. Compared with the sham operated groups, the levels of TGFβ1 and TNF-αwere higher and reached their peak , and decreased gradually. The cytokine levels in the 24w were significantly higher than the sham operated in the non infracted zone(P <0.01).The dynamic of the ratio of Collagen type I and Collagen typeⅢin the rat heart can reflect the remodeling of myocardial infarction. Transforming growth factor-beta1 and tumor necrosis alpha could perform their important role by regulating the gene expression of contractile protein isoforms, fetal gene and collagen. Correlation analysis of the mRNA expression indicated that fetal gene beta/alpha MHC, cardiac collagen subtypeⅠ,Ⅲlevels were increased correlated positively to pro- inflammatory factor(TGFβ1) as well as pro-fibrosis(TNF-α) cytokine levels significantly wherever in infracted and non infracted zone.(P<0.01).Conclusion Cytokines participated in the myocardial remodeling of MI. Interfering with expression of cytokines maybe the potentially preventative method in the myocardial remodeling.PartⅡThe study of myocardium remodeling mediated by cross talk between AngⅡand TGF-β1 via STAT and Smads signaling pathwaysObjective To study the signal transduction crosstalk between the cardiomyocyte and fibroblast in neonatal rat in vitro, and investigate the mechanism of ventricular remodeling.Method Cultured neonatal rat cardiomyocytes and cardiac fibroblasts were used to evaluate the effects of AngⅡa nd TGF-β1 on ventricular remodeling with or without their inhibitor valsartan and staurosporine, respectively. Protein content, beat frequency per minute, cell surface area detection were used to evaluate the cardiomyocyte hypertrophy. Cardiomyoctye apoptosis were measured by flow cytometry analysis. The proliferation of cardiomyocyte and fibroblast were measured by MTT assay. The mRNA of ANF,β-MHC, Bax, Collagen typeⅠ,Ⅲ, MMP2, Smad3 were measured by RT-PCR. Ratios of phospho-Stat1/total Stat1, phospho-Stat3/total Stat3 and Smad2/3 were detected by Western blotting. MMPs activity in the supernatant of cultured cardiac fibroblast were measured by Gelatine Zymography. AngⅡconcentration in the supernatant of cardiac fibroblast was analyzed by radioimmunoassay. TGFβ1 released from the cultured cardiomyocyte and cardiac fibroblast culture media was performing by ELISA.Results Protein content, beat frequency per minute, cell surface area were elevated in cardiomyocytes treated with AngⅡ(10-7 mol/L) or TGF-β1(3ng/ml) in a time-dependent manner compared with those control groups. MTT and flow cytometric method revealed that the pro-apoptotic or the pro-survival induced by AngⅡa ndTGF-β1 in a concentration- and time-dependent manner. The hyperplasia effects of AngⅡor TGF-β1 on the cardiac fibroblasts showed a concentration- and time-dependent manner was analyzed by MTT method. The hallmark of ventricular remodeling such as ANF,β-MHC, Bax, Collagen typeⅠ,Ⅲ, MMP2, and Smad3 mRNA levels were elevated in the AngⅡor TGF-β1 treated with groups compared to those without drug interfered with groups. The augmentation of ratios of phospho-Stat1/total Stat1, phospho-Stat3/total Stat3 and Smad2/3 in the two specific cells stimulated with AngⅡor TGF-β1 were reversed by the selective AT1 receptor antagonist, Valsartan, and Staurosporine, a PKC inhibitor in a time-dependent manner. MMPs activity in the supernatant of cultured cardiac fibroblast was enhanced by treated with AngⅡo r TGF-β1 in a time-dependent manner. Elevated TGF-β1 released from the cultured cardiomyocyte and cardiac fibroblast supernatant stimulated with AngⅡwas abolished by the pretreatment with the AT1 recepor blocker, i.e. Valsartan. In addition, TGF-β1 promoted the cardiomyocyte and cardiac fibroblast secreting AngⅡvia the PKC pathway, all of which were significantly inhibited by Staurosporine treatment.Conclusion These findings indicate the AngⅡand TGF-β1 mediated the hypertrophy, apoptosis of cultured neonatal rat cardiomyocyte and hyperplasia of cardiac fibroblast via the common Stat1, Stat3, and Smad2/3 signal pathway. The cross talk between the cardiomyocyte and cardiac fibroblast may provide an effective mean of ameliorating ventricular remodeling.PartⅢObservation of the effect of administration of valsartan on cytokine and AngⅡin patients with acute myocardial infarctionObjective To study the effects of valsartan on the cytokine, RAS, and cardiac function in patients with AMI, and therefore, provide new theory for the immunoregulation mechanism of valsartan in the AMI.Method We prospectively studied 79 patients underwent acute myocardial infarction, who were random designated to valsartan treated group and control without valsartan treated group. Proinflammatory cytokine such as TNFα, Profrobtic cytokine such as TGF-β1, anti-inflammatory cytokine such as IL-10,hsCRP(high-sensitivity C-reactive protein), RAS and blood lipids were measured on admission, and 1 weeks after AMI. Meanwhile the cardiac function was measured through echocardiography.Results The administration of 160 mg/day of valsartan from the time of diagnosis of AMI is associated with lower LVEDd and cytokine plasma concentrations after 7 days of treatment compared to admission and the control group. The amelioration of LVEF and up-regulated ratio of IL-10/TNF-αand AngⅡin the valsartan treated group showed significant statistical difference compared to the control group at day 7, which verified the immunoregulation role of valsartan in the AMI.Conclusion Regulation of cytokine and RAS and cardiac function by valsartan, suggesting a possible role of valsartan in the prevention of ventricular remodeling after AMI. Our results demonstrate better clinical evolution in the treated patients, although this should be corroborated by new and larger patient population studies.