| Myocardial fibrosis is the main pathological changes after myocardial infarction (MI ), which involving hypertrophy of myocardium, proliferation of fibroblasts, excessive deposition of collagen. These pathological changes lead to stiffness of myocardium, impairment of ventricle diastolic function, disorder of myocardium electrophysiology, arrhythmia, decreasing coronary flow reserve and even sudden death. The machanism of myocardial fibrosis is very complicated and a variety of cytokines and growth factors are involved in it. TGF-β1 is considered the most important and powerful cytokine relating to myocardial fibrosis, which expression is increased in myocardial fibrosis. BMP-7 is anther number of TGF-β1 superfamily which is studied mostly in bone defect, renal fibrosis, eye fibrosis, liver fibrosis, etc. BMP-7 and TGF-β1 regulate the development and progression of fibrosis together as they share the similar signal transduction mechanism. There are no reports about the effect and mechanism of BMP-7 on myocardial fibrosis after MI. Our study designed to (1) investigate the effects and mechanisms of rhBMP-7 on the TGF-β1 induced CFs proliferation and collagen production; (2) compare the effects of rhBMP-7 with benazepril on myocardial fibrosis and left ventricle remodeling after MI.Part I The cultivation and identification of cardiac fibroblasts of neonatal SD ratsObjective:More than three quarters of cells of normal heart are non-cadiocyte, 90% of which are fibroblasts. CFs are the cells not only generating collagen but also effecting myocardial fibrosis. CFs had important effect on the formation of extracellular matrix (ECM) and myocardial fibrosis. TGF-β1 is the common pathway of various factors leading to myocardial fibrosis, and it had important effects on the proliferation, differentiation and immigration of cadiocyte, as well as deposition of ECM. In this study, we established the in vitro model for myocardial fibrosis, observed the effect of TGF-β1 on the proliferation of CFs.Methods:CFs were isolated with trypsin digestion method from neonatal SD rats,2nd and third passages of the cells were used in experiments. The cells were identified under inverted microscope and by immunocytochemistry stain. The livability of the cultured cells were estimated by trypanblue. MTT method was used to obeserve the proliferation of CFs.Results: Under microscrope, the primary cardiac fibroblasts isolated appeared irregular triangular in morphology, the cytoplasm was transparent and thin, with 2-3 nucleus which were bigger than normal. No spontaneous beat could be seen. Trypanblue staining showed that viable cells were>97%. When stained by immunohistochemistry, the cells showed positive for vimentin, and negative for alpha smooth muscle actin (α-SMA). That suggested the cells isolated were cardiac fibroblasts and purity of the cells was about 98%. The effect of TGF-β1 on the proliferation of CFs was performed by MTT method, and the result showed that TGF-β1 at the concentrations of 0.1ng/ml, 1ng/ml, 10ng/ml and 100ng/ml promoted the proliferation of CFs, and this effect was dosage-dependent, with most significant effect at concentration of 10ng/ml. The effect of TGF-β1 at the concentrations of 10ng/ml on the proliferation of CFs was performed for 24h, 48 h, 72h, and the result showed the effect was time-dependant.Conclusion: The results above suggested that trypsin digestion combined with differential attachment method could be used successfully to culture neonatal rats cardiac fibroblasts; TGF-β1 at the concentration of 10ng/ml for 48h could enhance the proliferation of CFs and this system could be used as a in vitro model for the further investigation of drugs on the myocardial fibrosis. Part II Effects and molecular mechanisms of rhBMP-7 on the proliferation and collagen production of CFs in TGF-β1 induced neonatal rat cardiac fibroblastsObjective: In this part of study, we observed the effects of rhBMP-7 on the proliferation and collagen production in TGF-β1 induced cardiac fibroblast with various concentration, in order to exploit whether rhBMP-7 could prevent or regress myocardial fibrosis in cellular level. We also add Jun activation domain-binding protein 1(the inhibiter of Smad5,which is the downstream signal protein of BMP-7)to observe the change of cells proliferation and callagen production and explore the possible mechaniam of rhBMP-7.Methods: CFs were were isolated with trypsin digestion method from neonatal SD rats. The 2nd and third passage of CFs were used in experiment. When the cells growth to 80% confluent, the medium was discarded and the cells were washed with PBS for two times and then the different stimulate factors were added according to the differently treated groups: (1) control group: CFs were cultured with free serum medium (FSM) + TGF-β1(10ng/ml); (2) TGF-β1+rhBMP-7 group: TGF-β1(10ng/ml)+rhBMP-7(1,10,100ng/ml)+FSM; (3) rhBMP-7+Jab-1 group: TGF-β1(10ng/ml)+rhBMP-7(1,10,100ng/ml)+Jab-1 (20ng/ml)+FSM. The OD were measured using MTT method to investigate the proliferation of CFs in different groups. ELISA was used to detect the expression of PICP,PIIINP. The expression of Smad2, Smad3 and CTGF mRNA and protein were detected by Real Time-PCR and Western-Blot.Results: (1) The results derived from cell counting showed that rhBMP-7 could dose-dependently inhibit the proliferation of CFs induced by TGF-β1, and the same results could be obtained by MTT method. rhBMP-7 could supressed the proliferation of CFs at the concentration between 1 and 100ng/ml in the concentration dependent manner. Compared with TGF-β1, rhBMP-7 at the concentration of 100ng/ml could significantly supress the proliferation of CFs. (2)Compared with TGF-β1 group, rhBMP-7 at different concentrations inhibited the collagen production of the cells stimulated by TGF-β1 and the effect was concentration-dependent. The results of Real Time-PCR and Western-Blot showed that rhBMP-7 could supress the expression of Smad2, Smad3, CTGF, and the effects was concentration-dependent. (3) Compared with the group of TGF-β1, there was no significantly difference between the group of rhBMP-7 and Jab-1 on the MTT results, the production of PICP, PIIINP, the mRNA and protein expression of Smad2, 3, CTGF.Conclusion : (1) The results suggested that rhBMP-7 could inhibit the proliferation of CFs and collagen production induced by TGF-β1; rhBMP-7 could decreased the expression of mRNA and protein of Smad2, Smad3, CTGF, which might be the mechanism for rhBMP-7 to suppress the proliferation, collagen sythesis of CFs. (2) rhBMP-7 could suppress the proliferation of CFs through the Smad5.Part III Effects of rhBM-7 on the myocardial fibrosis and ventricular remodeling after myocardial infarctionObjectives: To observed the effect of rhBMP-7 on myocardial fibrosis and left ventricle remodeling after myocardial infarction in ratsMethods: 60 adults SD rats that survived ligatation of the left coronary artery were randomized to following group: Sham group (Sham-operated animals were treated similarly except that the suture around the coronary artery was not tied), MI group and treated group (treated group were divided into BMPA/BMPB/Ben subgroup). We administered saline 1ml/qod, rhBMP-7 80ug/kg/qod, rhBMP-7 320ug/kg/qod, Benazepril 10mg/Kg/qd respectively starting at 1d after surgery and lasting until the end of study (28d). Six rats of each group were sacrificed at 14 and 28 days post-MI. We assessed the myocardial tissue injury by HE staining; observed the change of collagen by Masson staining and quantitative evaluated the deposition of collagen by calculating the CVF and PVCA; detected the level of Hyp, PICP and PIIINP by ELISA; we also observed the expression of MMP-9 and TIMP-2 by Immunohistochemistry; the mRNA and protein expression of Smad2 Smad3 and CTGF by Real Time-PCR and Western-Blot; the left ventricular remodeling and cardiac function by echocardiography.Results: (1) rhBMP-7 could decrease the level of CVF and PVCA after MI , there was no difference between BMPA and Ben group, there was significant difference between BMPB,BMPA,Ben group; (2) rhBMP-7 could attenuated the level of Hyp, PICP, PIIINP of myocardial in a dose-dependent manner. There was no difference between BMPA and Ben group, significant difference between BMPB and Ben group (P<0.05); (3) rhBMP-7 could significantly suppressed the expression of MMP-9, decrease the MMP-9/TIMP-2 ratio. There was no significant difference between BMPA and Ben group, significant difference between BMPB and Ben group. (4) rhBMP-7 therapy could significantly improved cardiac systolic and diastolic function, there was no difference between BMPA and Ben group, significant difference between BMPB and BMPA Ben group.Conclusion:(1) rhBMP-7 could reduce the degree of myocardial fibrosis and reverse the left ventricular remodeling; (2) The anti-fibrosis effect of rhBMP-7 was dosage-dependent. (3) The therapeutic effect of 320ug/Kg rhBMP-7 preceded that of benazepril.
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