| BackgroundsMyocardial ischemia associated with coronary artery disease is a leading cause of morbidity and mortality in clinical practice. Transplantation of mesenchymal cells has been clinically applied for the patients with ischemic heart diseases via medicines,Percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass graft surgery (CABG) procedures which are effective for revascularization and might improve the myocardial perfusion, but are hard to regenerate the myocytes. Lately, stem cell transplantation is applied in the treatment of coronary artery disease (CHD) and developed rapidly,which can not only generate new vessels,but also regenerate necrotic myocardium.Currently one of the most popular cell types used by cardiomyoplasty investigators is bone marrow derived mesenchymal stem cells(BMSCs). BMSCs transplantation has shown promise for cardiac regeneration not only in animal models of myocardial infarction, but also in patients with CHD. Several studies have indicated that BMSCs offer a novel therapeutic option in the treatment of heart diseases. BMSCs transplantation leads to a significant improvement in the myocardial perfusion and post-infarction left ventricular function through angiogenesis. MSCs are pluripotent cells capable of high proliferation and differentiation into multiple cell types. What has made MSCs particularly appealing to investigators in cellular cardiomyoplasty is that they are easily obtained from bone marrow,can be expanded in culture, have good gene stability and histocompatibility,and are not involved in ethic problems. MSCs have high proliferative and selfrenewal capability. They exhibit multilineage differentiation capacity being capable to give rise to diverse cells like osteoblasts,chondrocytes,adipocytes,myocytes,tenocytes and possibly neural cells.Although the implantation of MSCs showed some favorable effects on cardiac remodeling and function,the efficiency of cell delivery was still limited by some factors,such as that the cell therapies were invasive and the targeted homing ability of implanted cells need to be improved. How to solve these existing problems is critial and pressing in the cell therapy in coronary artery diseases. Zen et al reported that therapeutic ultrasound-mediated albumin microbubbles destruction combined with the intravenous transfusion of BM-MNCs could greatly enhance the neovascularization by an increase in the endothelial attachment of BM-MNCs, leading to an improvement in blood perfusion and cardiac function of BIOTO2 cardiomyopathy, which resulted in an improvement of cardiac function via the inhibition of myocyte apoptosis and interstitial fibrosis. Their results suggest that US + Microbubble + MNC treatment maybe a feasible, efficient and non-invasive system for targeted cell delivery to the myocardium.Objectives1. To study the feasibility of the improvement of targeted homing of bone marrow derived mesenchymal stem cells by diagnostic ultrasound-mediated microbubbles destruction to the ischemic heart and its mechanism.2. To study the efficacy of intravenous MSCs implantation by diagnostic ultrasound-mediated mirobubbles destruction on the improvement of myocardial perfusion and cardiac function after rabbit myocardial infarction.3. To study the preliminary mechanisms of the improvement of the cardiac function by MSCs transplantation by diagnostic ultrasound-mediated microbubbles destruction after myocardial infarction.MethodsDensity gradient centrifugation and adherent culture method were used in the isolation and cultivation of MSCs. The morphological and other characterics were detected. Flow cytometry (FCM) and immune cells chemistry were applied to detect the expression of CD44 and CD45 on the stem cells. Osteogenic and adipogenesis induction of MSCs were performed.Myocardial infarction (MI) model was established by ligation of the left anterior descending coronary artery (LAD). The successful building of MI was assessed with serology, ultasonography and pathology.Sixteen models were equally devided into DAPI-labeled MSCs infusion group and DAPI MSCs + diagnostic US + microbubbles group. After 48 hours of cell transplantation,the survival of implanted cells was identified by the number of DAPI-positive cells in frozen sections (8μm in thickness) made from hearts of MI and lung under fluorescent microscope. Permeability of microvessel in MI area was assessed in sections of the anterior wall (right under the irradiation of diagnostic ultrasound) under Transmission electronic microscope. Expressions of VCAM-1(Vascular cell adhesion molecule 1) and SDF-1(Stromal cell-derived factor-1) in MI area were assessed with IHC after cell therapy.After 4 weeks of treatment, capillary density was assessed with HE staining, CD34 expression with IHC, VEGF level with western blot and myocardial perfusion with myocardial contrast echocardiography (MCE), which would prove the mechanism of angiogenesis. Myocardial collagen fiber area was analyzed with Q-Win image software with Masson staining to give evidence of inhibition in fibrosis by this implantation method. Protein Bax was detected with IHC in 4 groups. Echocardiography was performed blindly to assess the cardiac function. Fractional shortening (FS) and ejection fraction (EF) were detected with M mode echocardiography. Apical two and four chamber views were obtained and EF with biplane Simpson's method was examined, too.ResultsMost of the cultured MSCs were spindle shaped and became more uniform after several passages. These expanded MSCs were uniformly positive for CD44 (96.02%) and negative for CD45 (2.47%). The cultured cells were uniformly negative for CD34 with immunocytochemistry. Adipogenic and osteocyte differentiation was successfully induced in MSCs.MI models were builded by ligation of LAD. Perfusion defect was observed in the anterior and anteroseptal wall of LV with MCE,a blue dye was detected in the MI area with Masson staining,and LV systolic function was attenuated greatly after MI.The DAPI positive cells were located in infarcted and border area,while there was nearly none in the normal myocardium. The number of DAPI positive cells in MI area of US+Buble+MSCs group (214±27) was much more than that of MSCs infusion group(147±19) (P < 0.01). There were some endothelial cells in part of vessel wall impaired in ischemic myocardium under ultrasonic irradiation, the intercellular spaces increased in UMC(US+Microbubble+MSCs) group and UM (US+Bubble)group. The number of VCAM-1 positive cells of group UMC (77±44) was markedly increased compared with MSCs infusion (MSC) group(34±18), UM group (12±3) and PBS group (8±5), all the P<0.01. The scale of SDF-1 analyzed with Q-Win was increased in both MSCs treated groups, especially in UMC group.The number of capillaries stained by HE in UMC group (47±23) was much greater than that of the MSC group (26±7), the UM group (22±5) and the PBS group (19±10) under light microscope. Western blotting results showed that the level of VEGF in infarcted zone was higher in the UMC group (236.59±47.13) than that of the MSC group (151.48±25.07), the UM group (133.47±20.16) and the PBS group (89.43±20.43), with all P < 0.01. CD34 assessed with IHC reached the highest in the UMC group, followed by the MSCs group, the UM group and the PBS group. The collagen percentage area(%) of UMC group (9±4) was decreased by 25.6% compared with MSC group (13±5), decreased by 40.1% with UM group (15±3) and 46.8% with PBS group(17±6). There were significant differences of gray scale (analyzed with histogram of Photoshop) in anterior wall between the PBS group (52±12) and UM group (59±10), MSC group(64±16) and UMC group(71±13), with P = 0.091, 0.032 and 0.000,respectively. There was significant difference of FS between the UMC group (31±4)% and the MSC group [ (27±3)%, P<0.05], the UM group [ (22±3)%,P<0.01] and the PBS group [(20±3)%, P<0.01], respectively. So was the difference of EF between the UMC group [(62±6)%] and the MSC group [(54±5)%, P<0.05], the UM group [(47±4)%, P<0.01] and the PBS group [(43±5.0)%, P<0.01], respectively. And so was the EF(%) of biplane Simpson rule between the UMC group [(49±4)%] and the PBS group [(35±5)%, P<0.01], the UM group [(35±6)%, P<0.01] and the MSC group [(42±5)%, P<0.05], respectively. Conclusions1. Purified MSCs can be obtained via the density gradient centrifugation and adhere culture methods using morphological observation, surface marker identification and inducing differentiation. It is a simple available method for the isolation and culture of MSCs.2. DAPI fluorescent agent is a good stem cell label molecule, which could efficiently track the distribution of DAPI labeled MSCs in heart, lung and other organs.3. The intercellular space of vessel wall was increased with diagnostic ultrasound-mediated microbubble destruction, which facilitated the homing and gathering of MSCs to the MI area. Higher expression of VCAM-1 in the US + Microbubble +MSCs group may be induced by both the US + Bubble-mediated response and the paracrine effect of the implanted MSCs, which caused the enhanced attachment of transfused BM-MSCs onto the targeted endothelial layer.4. Intravenous infusion of rabbits BM-MSCs combined with US+Bubbles can stimulate neovessel formation by supplying angiogenic factors (VEGF) and inducing angiogenesis in ischemic heart and enhance the cardiac perfusion. The upregulation of VEGF, SDF-1, VCAM-1 and inhibition of Bax protein were induced by Paracrine effect of the implanted MSCs, which augmented the adhension, homging and gathering ability to the infarcted myocardium, and resulted in the improvement of cardiac function through the neogenesis and the inhibition of apoptosis.5. Intravenous infusion of BM-MSCs combined with diagnostic US + Bubble may assist in the inhibition of fibrosis and LV remodeling, which causes the improvement in cardiac function.6. Intravenous implantation of BM-MSCs combined with diagnostic US + Bubble can enable targeted delivery of BM-MSCs to the infarcted myocardium and induce the regional angiogenic response, resulting in an improvement of LV perfusion and cardiac function of ischemic heart associated with the inhibition of cardiac fibrosis and remodeling.7. Myocardium-targeted homing of intravenously implantated mesenchymal stem cells by diagnostic ultrasound-mediated microbubble destruction may be feasible as a non-invasive and efficient angiogenic cell therapy to the ischemic heart disease. |
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