Bone Marrow Mesenchymal Stem Cell Transplantiation Combined With Perindopril Treatment Attenuates Infarction Remodelling In A Rat Model Of Acute Myocardial Infarction

Despite enormous progress in the medical technology in the past decade, acute myocardial infarction(AMI) remains the major cardiovascular disease which affect public health.To reduce the mortality rate of AMI,people make afforts to study, with the establishment of CCU.the technique for placing pulmonary artery catheter to hemodynamic monitoring .thrombolytic therapy,percutaneous transluminal coronary angioplasty ,multidrug therapy,but there are still approximately one thirds patients died from AMI.Althrough those therapy might save the myocardical cell, but in vain for myocardical cell died.When the myocardical infarction occured ,the heart adapts through structural changes ,called remodeling ,which include ventricular dilation ,compensatory opposite ventricular wall hypertrophies,and myocardium would continue to deteriorate, and affect the prognosis .The Survival and Ventricular Enlargement trial(SAVE) show,angiotensin-converting enzyme(ACEI) could improve survival.But fundamental study display that ACEI only partial improve ventricular remodeling.Stem cell is a kind of cell that has self-renewal and/or differentiation properties.and become importance in regenerative medicine.Cardiiomyocyte injury is irreversible after infarction,does it possible to repalace myocardial scar with functional tissue?In vitro studies in animals show myoblasts merge into myotubes in the scar. Marrow stromal cells(MSCs) augment collateral and anti-apotosis through release of several cytokine.On the basis of the property of the stem cell,We study the affection of bone marrow mesenchymal stem cell transplantiation on infarction remodeling, especially for matrix remolding ,include collagen and matrix metalloproteinase,we also explore the expression of generelate to arteriogensis and hypertrophy in myocardial infarction.In this study,we compare the effection on attenuate infarction remodeling between MSC and ACEI.Part one Bone marrow mesenchymal stem cell transplantiation combined with perindopril treatment attenuates infarction matrix remodelling in a rat model of acute myocardial infarctionVentricular remodeling after acute myocardial infarction include the topography change ,for example, ventricular enlargement and lengthening of the noncontractile region; in the view of microstructure , include myocardial apoptosis , myocardial hypertrophy .increasing content of collagen and expression of MMP . This remodelling can lead to decline of ventricular function and advesely affect the prognosis for survival. Thus ,reversing the procedure of ventricular remodelling would be desirable for the treatment of AMI.Activation of the circulating and tissure rennin-angiotensin-aldosterone system(RAAS) could lead to ventricular remodelling.The therapeutic use of ACEI is beneficial for reducing ventricular remodeling. Optimization studies demonstrated that angiotensin-converting enzyme inhibitors attenuate ventricular remodelling after acute myocardial infarction.MSCs are pluripotent stem cells within the marrow microenvironment. Clinical studies showed that MSC significantly improved left ventricular function after intracoronary injection of patient with AMI.However,little information is available about the infarction remodelling after bone marrow mesenchymal stem cell transplantiation .The purpose of this study was to investigate: (1) whether transplantation of MSC could reduce the fibrillar collagen content the expression of MMP as compared to treatment of ACEI; (2)whether transplantation of MSC could enhance the beneficial effect of ACEI in reducing ventricular remodelling.MATERIALS AND METHODS 1 Preparation of MSCThe experimental rats were anesthetized with intraperitonneal injection of 4% chloral hydrate solution (1 ml/100g) .Tibia and thigh bone were collected and pulverized,then transferred to a sterile tube and mixed with 5 ml phosphate-buffered saline (PBS) solution containing 7500 U heparin ,then added into Dulbcco's Modifed Eagle Medium(DMEM). The tube was centrifuged at 900g for 20 min .The cell suspension was loaded on Percoll solution. The cells were centrifuged at 900g for 25 min. The middle one third of total volume were transferred into a tube containing DMEM, and centrifuged at 900g for 25 min.The cell pellet was then resuspended in culture medium and seeded in culture flasks,and maintained at 37℃ in 5% CO₂ humidified air..After washing off the unattached cells during the period of 48 hours ,the adherent cells were cultured in DMEM containing 20% fetal bovine serum and antibiotics .The medium was changed once every 5 d.2 DAPI labellingWhen MSCs completely occupied the flask bottom ,they were incubated in medium containing DAPI (4'6-Diamidino-2-Phenylindole, 50 μg/ml, Sigma, USA) for 24 h .3 AMI model were made by ligation of the left anterior descending coronary artery. Forty rats were divided into four groups: control group, MSC group, ACEI group, MSC+ACEIgroup.. PBS was injected into control group and ACEI group. Perindopril was administered( p.o. 2mg/kg body weight) to ACEI group ,and MSC+ACEI group.PBS medium containing MSC (total 10⁷ cells, 3 sites×0.05 ml per site ) or medium alone was injected into the left anterior descending coronary artery risk area (ischemic border) with a 24-gauge needle. Six weeks after MSC implantation,the rats were killed and tissure from ischemic area were collected.4 HistologyTissure samples from the infraction area were collected at 6 weeks after transplantation and fixed in 4% PBS formaldehyde for histological study. The samples were embedded and cut to yield 10-μm thick sections and stained with meliorative Masson's trichome stain .Interstitial fibrosis was stained blue. Some samples were frozened and cut into 4-μm thick sections.then observed under the fluorescence microscopy.5 Western Blottingprimary antibodies diluted in TBST (1:200 for MMP2,MMP9; 1:1000 for β-actin).6 RT-PCRDetection expression of MMP2,MMP9 and TIMP1 mRNA7 Statistical AnalysisNumerical data presented as mean ± SD.Comparisons among groups were performed with one-way ANOVA and LSD comparison. Statistical significance was accepted while the null hypothesis was rejected at P value<0.05.RESULTSMasson's trichrome staining revealed that collagen(blue) existed in the ischemic area. The area of collagen was decreased in MSC+ACEI group ACEI group MSC group compared with PBS group.MMP2, MMP9 were highly expressed in the infraction area in the PBS group. The Western blot for MMP2, MMP9 showed the band of 72 kDa. and 92 kDa.The band of 72kDa was barely seen in the MSC +ACEI group.MMP2 Expression was also reduced by ACEI. MSC, ACEI and MSC +ACEI lowered MMP9 expression.The PCR products of MMP2.MMP9 and TIMP1 were observed at the expected locations on agarose gels. Statistical analysis indicated that the expression of MMP2,MMP9mRNA in 3 treatment groups was significant different from that in the control group.In the MSC+ACEI,ACEI,MSC and PBS groups,the densitometry ratio of MMP2/β-actin was 0.43 ± 0.20, 0.45 ±0.19,0.46 ±0.14 and 0.78 ± 0.22,respectively,P<0.05), the densitometry ratio of MMP9/β-actin was 0.40 ± 0.13,0.51 ± 0.10, 0.48 ± 0.09,and 0.70 ± 0.20 .respectively, P<0.05).However, no significant difference was detected in the expression of TIMP1mRNA among the 4 groups.ConclusionBoth ACEI and MSC could attenuates infarction matrix remodling in a rat model of AMI by reducing the expression of mRNA of MMP2 MMP9,ACEI could enhance the effect of MSC .Part TwoThe Effection of Bone Marrow Mesenchymal Stem Cell Transplantiation Combine Perindopril Treatment on Gene Expression Associate With Remodeling in a Rat Model of Acute Myocardial InfarctionThis study was performed to evalute the effection of bone marrow mesenchymal stem cell and ACEI on the expression of oncogenne in noninfaction tissure,arteriogenic cytokine genes in infaction tissure and TGFβ-smad pathway n infaction tissure during acute myocardial infarction.MethodsAs described at the first part of the paper, 40 rats were divided into four groups:group control,group MSC,group ACEI,group MSC+ACEI.Bone marrow stem cell derived rat were injected immediately into a zone made ischemic by coronary artery ligation in group MSC and group MSC+ACEI . Phosphate-buffered saline (PBS) were injected in Group control. Perindopril was administered p.o.in group ACEI and group MSC+ACEI. Six weeks after implantation,rat were killed and sample of heart were collected, and echo were performed to evaluate the left ventricular geometry before killed the rats.Western blot was employed to evaluated the protein expression of VEGF and TGFβ, the transcriptional level of c-myc and smad2 was detected by reverse transcriptase PCR(RT-PCR) analysis.ResultThe transcriptional level of c-myc mRNA in noninfarction zone increased in PBS group,but seems to no significance. The expression of TGFβ also increased in PBS group.While smad2 seems to no change.There were no significant difference of left ventricular geometry by echo examination and the expression of VEGF among 4 groups by western blot six months after acute myocardial infarction.ConclusionBoth MSC and ACEI could reduce the expression of TGFβ,therefore,reduce the fibrosis in infarction area.