Transplantation Of Bm-mscs After Microenvironment Changes Induced By Ultrasound Targeted Microbubble Destruction Promote Angiogenesis In A Myocardial Infarction Canine Model

Transplantation of stem cell is a promising method for the treatment of myocardial infarction. Mesenchymal stem cells(MSCs) can secrete vascular growth factors,enhance angiogenesis,improve heart function and differentiate into endothelial cells or cardiomyocytes in a specific environment. The myocardial microenvironment plays a decisive role in the survival,migration,and differentiation of stem cells,which may determine the biological effect induced by stem cells. Most of the engrafted stem cells will be killed in the infarcted zone because of the harsh environment such as ischemia/reperfusion injury and inflammatory factors, which may decrease the efficacy of stem cells transplantation. So providing a suitable local environment to allow stem cells to survival and differentiation is therefore the focus of many studies.Ultrasound has specific bio-effects on biological tissues and cells, the effects will be greatly enhanced by ultrasound microbubbles. Ultrasound target microbubble destruction(UTMD)has been found to produce microvascular permeabilization,microvascular ruptures and endothelium interspace widen,it also can stimulate the secretion of endogenous vascular growth factors and promote angiogenesis. The microenvironment will be changed and inflammatory response will be induced after UTMD. Mild inflammatory response may be helpful for the mobilization and homing of stem cells. However, excessive inflammatory cell infiltration may result in apoptosis and necrosis, which is a disadvantage for the cellular therapy. Different ultrasound irradiation parameters produce different biological effects, in a certain condition, UTMD even lead to cardiac hemorrhage and cadiomyocytes necrosis. Therefore, the parameters of ultrasound during UTMD need to be optimized, which may promote the expression of vascular growth factor and chemokines, and not result in excessive inflammatory response and cadiomyocytes necrosis.Based on the above mentioned theoretical basis and problems, the expression of cytokine and adhesion molecule in the infarcted myocardium induced by various intensities of pulse ultrasound target microbubble destruction will be investigated, and the proper ultrasound intensity which is suitable for MSCs transplantation will be determined in this study. MSCs transplantation will be performed after myocardial environment changes induced by UTMD. Myocardial perfusion,angiogenesis,coronary collateral circulation,and heart function will be measured by coronary angiography,radionuclide myocardial perfusion imaging and echocardiography. The microvessle density in the margin of the infarcted myocardium will be observed by CD34 immunohistochemistry. Overall,The study was composed of the following two parts.PART I: EFFECTS ON MYOCARDIAL MICROENVIROMENT BY ULTRASOUND-TARGETED MICROBUBBLES DESTRUCTION IN A CANINE MYCARDIAL INFARTION MODELObjective: To explore the effects on myocardial microenviroment induced by ultrasound-targeted microbubbles destruction (UTMD) with different ultrasound intensities and to determine the proper ultrasound intensity which is suitable for stem cells transplantation. Methods: Twenty mongrel dogs were randomly divided into four groups after the establishment of animal models of myocardial infarction. One week after infarction, dogs in group 1,2 and group3 were received pulse ultrasound (with frequency of 1MHz) targeted microbubble destruction for ten minutes, focused on the anterior left-ventricular wall with intensity of 0.5W/cm2(0.5W/cm2 US+MB),1.0W/cm2(1.0W/cm2 US+MB),1.5W/cm2 (1.5W/cm2 US+MB) respectively , no UTMD in group 4(control group).UTMD was performed every 2 or 3 days, total three times in one week. All dogs were sacrificed and the heart were harvested after the last UTMD in experimental group,The expression of IL-1β,VCAM-1,SDF1,VEGF mRNA and protein were detected by real time PCR,Western Blot and immunohistochemical examination. Results: The results from real time PCR revealed that 1.5W/cm2 US+MB and 1.0W/cm2 US+MB treatment markedly increased the expression of VEGF,VCAM-1,SDF-1 (P<0.01, 5fold and 3.8~4.7fold, respectively) compared with the non-treated control, the expression of IL-1βin 1.5W/cm2 US+MB and 1.0W/cm2 US+MB group were increased 9 and 5 fold over the control group respectively(P<0.01). While 0.5W/cm2 US+MB-treated group showed no significant changes. Western blot analysis and immunohistochemical examination showed that the expression of IL-1β,VCAM-1,SDF1,VEGF in 1.5W/cm2 US+MB and 1.0W/cm2 US+MB group were marked increased than 0.5W/cm2 US+MB and the control group. The expression of IL-1βin1.5W/cm2 US+MB group was obviously higher than those with 1.0W/cm2 US+MB group. Conclusion: Ultrasound-targeted microbubbles destruction with frequency of 1MHz and intensity of 1.0W/ cm2 can increase the expression of vascular growth factor and chemotactic factor resulting in beneficial of the homing and differentiation of stem cells without inducing severe inflammatory reaction. PART II: TRANSPLANTATION OF MSCS AFTER MICROENVIRONMENT CHANGES INDUCED BY ULTRASOUND TARGETED MICROBUBBLE DESTRUCTION PROMOTE ANGIOGENESIS IN A MYOCARDIAL INFARCTION CANINE MODELSECTIONⅠISOLATION, CULTIVATION AND IDENTIFICATION OF CANINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS IN VITROObjective: To investigate a stable method for the isolation, purification, cultivation and identification of bone marrow-derived mesenchymal stem cells in vitro. Methods Bone marrow mononuclearcells were extracted from canine bone marrow and purified via a density gradientcentrifugation on Percoll (density 1.073g/ml), then proliferated in vitro. The expression of CD29,CD31,CD34,CD44,CD45 and vWF were measured by immunohistochemistry. Flow cytometry was used to determine the cycle of BMMSCs by surface maker protein.Results: Immunohistochemistry detection showed the expression of CD29 and CD44 was positive, and the expression of CD31, CD34, CD45 and vWF was negative. 88.88% of the isolated cells stayed in G0/G1 period, which demonstrate that the separated cells were BMMSCs. The cultured stem cells can be expanded to 107 orders of magnitude in 2 weeks, and the purity of more than 99% can be achieved. This may provide sufficient sources of cells for stem cell transplantation in the treatment of ischemic heart disease. Conclusion: High purity BMMSCs can be obtained by isolating the canine bone marrow aspirates on Percoll (1.073 g/ml) and purifying with adherent culture, it can be expanded to 107 orders of magnitude in 2 weeks, which may provide sufficient cell sources for canine cadiomyocytes replacement therapySECTIONⅡ: ULTRASOUND TARGETED MICROBUBBLES DESTRUCTION COMBINE WITH MESENCHYMAL STEM CELLS TRANSPLANTATION PROMOTE ANGIOGENESIS IN A CANINE MODEL OF MYOCARDIAL INFARCTIONObjective: To explore the therapeutic effects of MSCs transplantation to infarcted myocardium after myocardial microenvironment changes induced by ultrasound-targeted microbubbles destruction. Methods: Fifteen mongrel dogs were randomized into 3 groups (n= 5 per group) after the establishment of myocardial infarction model by left anterior descending coronary artery ligation. One week after myocardial infarction, group 1 (UTMD+MSCs)was treated by ultrasound (with frequency of 1MHz and intensity of 1.0W/cm2) targeted microbubble destruction, focused on the anterior left-ventricular wall, UTMD was performed every 2 or 3 days, total three times in one week. No UTMD in group 2 and group 3. MSCs transplantation was performed though coronary infusion 10 minutes after the third time of UTMD. Dogs in group 2(MSCs) and group 3 (Control) were received the same dose of MSCs or the same volume of cell culture medium infusion through coronary infusion 14 d after MI. Cardiac function and myocardial perfusion were evaluated by echocardiography, ECT and coronary angiography before and one month after MSCs transplantation in all groups. The microvessel density was observed by CD34 immunohistochemistry. Results: Myocardial perfusion , coronary collateral circulation and angiogenesis were markedly improved by UTMD compared with MSCs alone and control. At echocardiographic analysis, Left ventricular ejection fraction was significantly improved by UTMD+MSCs treatment (69.8±4.62% vs 60.7±9.18% vs 52.5±3.73%,P<0.05). Conclusion: In a canine model of myocardial infarction, therapeutic effects will be markedly enhanced by MSCs transplantation after the myocardial microenvironment changes induced by ltrasound targeted microbubble destruction, which provide a novel and efficient approach for cellular therapy.