BMMNCs Implantation Attenuates Deleterious Ventricular Remodeling And Results In The Related Gene Changes After Myocardial Infarction In Dogs

Objectives:1. To explore a method for the isolation, labeling, and identification of bone marrow mononuclear cells (BMMNCs) in vitro and prepare the cell material for implantation.2. To establish acute myocardial infarction (AMI) model in dogs and observe the changes of post-infarction ventricular remodeling.3. To observe the changes in morphology, structure, and ventricular function of infarct heart after BMMNCs implantation.4. To evaluate the effect of BMMNCs implantation on myocardial collagen and the related gene such as matrix metalloproteinase (MMP), tissue inhibitor of MMP(TIMP) and transforming growth factor beta 1(TGFβ1), and investigate the molecular mechanism of inhibition effect on post-infarction ventricular remodeling by transplanting BMMNCs.Methods:1. BMMNCs were isolated by ficoll gradient, the cell viability was assesed by trypan blue exclusion test, the cells were observed in morphology by Wright's Staining, and were labeled with 4, 6 diamidino-2-phenylindole (DAPI) to be traced with fluorescence. Flow cytometry was applied to calculate the amounts of CD34 positive cell in BMMNCs, cells cultured and passaged in vitro were observed to identify mesenchymal stem cells.2. AMI in dog was induced by modified ligation of left anterior descending coronary artery via thoracotomy. After myocardial infarction (MI), serial ultrasoundcardiograms (UCG) were performed to observe the change of heart morphology and function, and myocardium radioisotope scanning was usedto examine the myocardial perfusion. After six weeks of myocardial infarction, the canine model was sacrificed and the heart was harvested for morphological and histological study.3. 24 dogs were randomized into four groups: AMI-CONTROL group and AMI-BMMNC group (transplantation at 1 hour after MI), OMI-CONTROL and OMI-BMMNC group (transplantation at 4 weeks after MI). Autologous BMMNCs were injected into infarct and peri-infarct myocardium for transplantation. UCG were performed to observe the change of heart morphology and function after cell transplantation. At six weeks of cell transplantation, the canine heart was harvested for morphological and histological study.4. At six weeks of BMMNCs transplantation, the myocardial speciemens were harvested in infarct, peri-infarct, and non-infarct myocardium. The amount of myocardial collagen was assesed by the method of chloramines T. The mRNA expression of type I collagen, type III collagen, MMP-2, MMP-9, TIMP-1, TIMP-2, TGF-β, and VEGF was detected by RT-PCR. Nuclear protein was extracted from myocardium for western blot to examine NF-κB.Results:1. The number of 5 to 12× 10⁷ BMMNCs was obtained from 10ml bone marrow by ficoll gradient isolation in each dog. The viability, assessed by trypan blue exclusive test, was about 95%. The cells showed mononuclear cell and a mild difference in size. The efficiency of labeling by DAPI was above 95%. The mounts of CD34 positive cell in BMMNCs was 1.8±0.28% by flow cytometry. 1 week after cell culture, cloned cell colony was observed.2. The appearance of myocardial ischemic was observed in the early phase after MI in each canine model. The infarct region was found by UCG, myocardium radioisotope scanning, morphological and histological examination in the late phase, the incidence rate of ventricular aneurysm was 69.2%, the achievement ratio of modeling was 92.8% in all canine models.Serial UCGs also showed the dimension and volume of left ventricle were enlarged progressively, while the interventricular septum (IVS) and left ventricle post wall (LVPW) were thickening after modeling in all canine MI models.3. After BMMNCs transplantation into myocardium, UCG showed that left ventricular end diastolic dimension (LVDD), left ventricular end diastolic volume (LVDV), the thickness of IVS and LVPW in AMI-BMMNC group and OMI-BMMNC group were significantly less than in AMI-CONTROL group and OMI-CONTROL group; while the value of EF and FS in OMI-BMMNC group were significantly higher than in OMI-CONTROL group. With morphological measurement, the increase of infarct region thickness and the reduction of infarct region length were observed after transplantation, and no ventricular aneurysm was found in AMI-BMMNC group, and the ratio between long axis and minor axis circumference of left ventricle increased in OMI-BMMNC group. Fluorescence expressed in transplantation region was observed, the morphology of most nuclei with fluorescence was irregular, and the differentiated cardiocyte with fluorescence was not found in myocardium after transplantation. The histological examination showed more neovascularization after transplantation both in AMI and in OMI, and significant lymphocyte infiltration in OMI-BMMNC group.4. The amount of collagen in myocardium was decreased after BMMNCs transplantation. The mRNA of type I collagen, MMP-2, MMP-9 and TGF-β₁, in infarct and peri-infarc region after cell transplantation were lower than in control group, while the mRNA of TIMP-1 and TIMP-2 in infarct and peri-infarc region after cell transplantation were higher than in control group. The VEGF mRNA level after transplantation was increased in all myocardium in AMI-BMMNC group, only in peri-infarct region in OMI-BMMNC group. Nuclear protein was extracted from myocardium forwestern blot, which showed the decrease of NF-κB in infarct and peri-infarc region after cell transplantation. Conclusion:1. The amount of BMMNCs by ficoll gradient isolation is enough; the labeling efficiency by DAPI is stable. By identification with flow cytometry and cell culture, BMMNCs include main stem cell type such as HSCs and MSCs, and may be used for cell transplantation.2. By modifying the method of coronary ligation, canine MI model have appearance of transmural MI and ventricular aneurysm, and experience a typical pathological change of post-infarction ventricular remodeling. The model is stable and reliable, may be used to study the pathology of post-infarction ventricular remodeling.3. BMMNCs implantation into infarct myocardium both in AMI and OMI have a beneficial effect, which can attenuate deleterious ventricular remodeling in morphology and structure, and improve neovascularization in histology, and improve the heart function. The differentiated cardiocyte from transplantational cell was not found.4. BMMNCs implantation can inhibits the synthesis of collagen in infarct myocardium. The changes of related gene, such as the down-regulation of MMP-2, MMP-9 and TGF-β₁, the up-regulation of TIMP-1, TIMP-2 and VEGF, and the decrease of nuclear protein NF-κB may take part in the process of cell transplantation alleviating ventricular remodeling. The inhibition to post-infarction ventricular remodeling was mainly on matrix remodeling and vessel remodeling rather than myocyte remodeling.