Myocardium-targeted Transplantation Of Bone Marrow-derived Endothelial Progenitor Cells In Infarcted Myocardium Of Rat Mediated By Diagnostic Ultrasound And Microbubbles

Backgrounds and objectives:Ischemic heart disease (IHD) is a kind of myocardial injury caused by the imbalance between coronary perfusion and requirement of myocardium due to the change of coronary circulation, about 90% of which consists of coronary artery disease. The conventional therapy is to reconstruct the blood supply, but it can not repair the injuried and necrotic myocardium. The stem cells transplantation might regenerate the vessel and myocardium, which lights the hope of treating IHD completely in the future.Recent studies showed that endothelial progenitor cells (EPCs) contributed to angiogenesis involved in the repair of ischemic tissues after birth and had potential to be a kind of therapeutic method for myocardial ischemia (MI). A number of experimental and clinical trials have proved that transplantation of EPCs in ischemic heart disease made an effect. But there were some problems yet limiting the application of EPCs in IHC, such as the invasive operation of implantation, the low efficiency of cell therapy and the targeted homing ability of stem cells needing to improve.The possible mechanisms of the transplantation of EPCs by the ultrasound-mediated microbubble destruction in prompting the homing and the treatment of EPCs in ischemic heart are as following. Firstly, the sonoporation induced by ultrasound-mediated microbubbles destruction leads to the increased permeability of blood vessels in myocardium, which is helpful to the homing of EPCs into ischemic heart, and the shock wave and microstream produced during bubbles'blowing up prompts the EPCs to come into ischemic heart and helps the neovascularization and cell proliferation. Secondly, the ultrasound probe was positioned on the chest, so the regional part of ishemic anterior wall was improved in permeability and the intravenously injected EPCs were easy to home to ischemic heart. What's more, studies showed that cavitation induced by ultrasound and microbubbles might stimulate vessel formation.This research tried to explore a non-invasive and targeted cell transplantation method by applying ultrasound-mediated microbubbles destruction system, find out whether the targeted homing of EPCs would be improved, and evaluate the feasibility and the efficiency of EPCs in the treatment of myocardial infarction.Methods:1. Isolation, culture and identification of bone marrow-derived EPCs:EPCs derived from bone marrow mononuclear cells of rats were isolated by Ficoll gradient centrifugation. Cells were planted on culture dishes coated with human fibronectin and maintained in M199 supplemented with VEGF(50ng/mL),bFGF(5ng/mL) and EGF(10ng/mL) . Cell growth and morphology were observed. The cell specific surface mark CD34 and VEGFR-2 were assessed by fluorescence activated cell sorter ( FACS) analysis.The cell phaenotype vWF, the function of EPCs taking in ac-LDL and binding UEA-I were detected by immunofluorescence.2. Myocardial infarction model building and assessment:Model of myocardial infarction of 40 adult SD rats builded by ligating the left anterior descending coronary artery. Before building and one week after modeling, two-dimensional ultrasound was performed to observe the thickness and motion of infarcted wall; left ventricular function was analyzed with M-mode echocardiography; Tei index was also detected to evaluated the left heart function. HE and Masson's dye were applied to prove the successful building of MI models in pathology.3. Efficacy of intravenous injection of EPCs mediated by the diagnostic ultrasound combined with microbubbles in MI rats:Fourty two MI rats were randomly divided into three groups: EPCs infusion group (n=15), ultrasound (US) + microbubbles (MB) + EPCs group ( n =15) and the control group (NS only) (n=12),. The ultrasound parameters were set as following: VIVID 7 ultrasound system, 3S probe with frequency 5 MHz, Mechanical Index (MI) set as 1.3 for eight-minute irradiation with a time trigger at 1 second. After the model had been builded for 1 week, 1 ml saline was injected, 1×106 cells were infused within 1 ml saline and injected through tail vena caudalis slowly in EPCs infusion group, and lipid microbubbles of 1ml/kg were injected, followed by intravenous injection of EPCs combined with the diagnostic ultrasound irradiation for 8 minutes. Four weeks after cell therapy, capillary density (CD) was counted with HE dye slices in each group, and infarct size was determined;CD34 expression in ischemic myocardium was assessed with immunohistochemistry, and left cardiac function were detected by M-mode and Tei index and compared in three groups.All the data were presented as Mean±SD ( x±s) and one-way ANOVA and paired t test were applied in the statistic analysis, which was accomplished by SPSS 13.0 software in computer, P<0.05 was regarded as having statistical significance.Results:1. Isolation,culture and identification of bone marrow-derived EPCs:Three days later, cells number increased and cell body stretched. Fusiform cells appeared accompanied with some cell processes. Five days later, the number of fusiform cells augmented obviously and cell cluster appeared. Seven days later, cells connected with each other and led to chord, reticulate, or blood capillary-shaped structure. With the culture time lasting, cells mixed together, resulting in the cobble-stone morphology. CD34 and VEGFR-2 and positive cells were 86.02% and 81.37% respectively, assessed by fluorescence activated cell sorter ( FACS) analysis. Cells cultivating expressed vWF phaenotype, capable of intaking acLDL and binding UEA-I.2. Myocardial infarction model building and assessment:MI model was builded successfully in 29 rats and the other 11 rats died of anesthetic accident, respiratory failure, cardia failure and etc. The anterior wall of left ventricle thinningzed and asynersised, observed by 2D ultrasound. Left ventricular function(LVF) detected with ultrasound before and after model establishment showed that there was distinguished statistical significance (P<0.01). HE dye showed that there was a great quantity of cardiac muscle cellular necrosis. Masson's dye showed that the MI area of left ventricle'anterior wall was dyed into blue. 3. Efficacy of intravenous injection of EPCs mediated by the diagnostic ultrasound combined with microbubbles in MI rats:There was significant differences of CD between US+MB+EPCs group(25.3±12.2)and control group(12.6±4.5)and EPCs infusion group (19.2±6.2) respectively, both P<0.01. There were erythrocytes among the myocardial fibers in US+MB+EPCs group, which leaked out of the vessels, while there was nearly none in the other two groups. The result of the infarct size showed that US+MB+EPCs group(25.3±4.5)% was obviously lower than control group (41.9±4.3)% and EPCs infusion group(36.7±3.8)%( P<0.01). CD34 was expressed most in US+MB+EPCs group, with the brown positive blood vessels distributing intensively, while it was less in EPCS and the lest in control. There were significant differences in FS, EF and Tei index between US+MB+EPCs group and the other two groups. FS in US+MB+EPCs group was much higher than those in EPCs infusion group and control group (P<0.05). So were the EF and Tei index (P<0.05, P<0.01).All the data were presented as Mean±SD ( x±s) and one-way ANOVA and paired t test applied in the statistic analysis, which was accomplished by SPSS 13.0 software in computer, P<0.05 was regarded as having statistical significance.Conclusions:1. The mononuclear cells from the bone marrow of rats could be induced into endothelial progenitor cells. The VEGF, bFGF and the EGF can make up the optimal induction condition.2. MI model of rat built with LAD ligation is successful and stable. Tei index is a good parameter to appraise cardiac function after myocardial infarction.3. Intravenous implantation of EPCs mediated by diagnostic ultrasound and microbubbles can improve the heart function of MI rat. It relates to the the effect of cavitation and mechanical effect that ultrasound combined with microbubbles can increase the permeability of the ischemic heart, which facilitates the homing and gathering of EPCs and results in the enhanced neovascularization.