| BackgroundsThe incidence and mortality of ischemic heart diseases and their complications remain at a high level in the world. Impaired cardiac function after acute myocardial infarction (AMI) is at least in part ascribed to loss of myocytes and development of fibrosis leading to unfavorable left ventricular remodeling. Interventional strategies for myocardial repair (drug treatment, percutaneous coronary intervention, coronary artery bypass grafting) have failed to ameliorate these pathophysiological complications. Cellular cardiomyoplasty is a newly developed cell-based therapy, whereby stem cells are transplanted into dysfunctional areas of the heart to replace the damaged myocardium. Especially, owning to the ease of preparation, immunologic privilege, and no ethics problem, bone-marrow-derived mesenchymal stem cells (MSCs) are emerging as an extremely promising therapeutic agent for cardiac repair.MSCs of engraftment differentiate into cardiomyocytes. At same time, MSCs are known to secrete soluble paracrine factors that have been demonstrated to contribute to endogenous cardiomyogenesis and angiogenesis. However, many animal experiments and clinical studies involving MSCs therapy demonstrated improvement left ventricular (LV) function only partly. One of reasons may be that the level of growth factors concentration in target tissues such as VEGF, TGF did not reach sufficient levels and/or did not persist long enough to trigger relevant vascular growth.Previous studies have demonstrated arterial blood vessels are specifically aligned with peripheral sensory nerves, and the effect of the nerve promoting arteriogenesis may be mediated by local Schwann cells (SC) secretion of VEGF. In vitro studies show high VEGF concentration led to the expression of arterial markers, and low VEGF concentration contributed to venous differentiation. SC can secret a diversity of cell molecules, extracellular matrix molecules, and neurotrophic factors. We speculate that there must be some other growth factors involved in the process of repair. So we hypothesized that transplanting MSC and SC combined might produce a synergistic effect for treatment of MI.However, SC xenotransplantation approaches may be affected by the rejection of the host. It poses a significant obstacle to the transplantation of xenogeneic cells. To overcome this problem, we introduce the microencapsulation technique to shelter the implanted SC from the recipient's immune system. Cell microencapsulation is a promising tool to overcome these drawbacks of cell transplantation. It consists of surrounding cells with a semi-permeable polymeric membrane. The latter permits the entry of nutrients and the exit of therapeutic protein products, for sustained delivery of the desired molecule.Research ObjectivesWe aimed to investigate whether transplantation of microencapsulated SC and MSCs combined in the ischemic myocardium could augment angiogenesis and improve heart function.Materials and Methods1. Isolation and culture of MSCsBone-marrow cells were obtained by flushing the tibiae and femurs with low-glucose DMEM supplemented with10%fetal bovine serum (FBS) and1×penicillin/streptamycin. Then cells were collected and incubated in an incubator with5%CO2at37℃. The culture medium was replaced with fresh medium every48-72h. After12-14days, MSCs at the primary passage were detached with0.25%EDTA-trypsin and passaged. Three passage were used for transplantation and cells were labeled with sterile BrdU.2. Isolation, culture and identification of SCCultures of pure SC were obtained from newborn1-3day old SD rats. Their sciatic nerves were harvested with the rats under anesthesia and pealed from the epineurium under a surgical microscope. The nerves were cut into0.5-1mm pieces and incubated in0.25%trypsin and0.125%collagenase at37℃for15-20min. Then cells were collected and incubated in an incubator with5%CO2at37℃for24h. The following day, the cells were treated with cytosine arabinoside (5μg/ml) for3days to eliminate proliferating fibroblasts. The culture medium was replaced with fresh medium supplemented with basic fibroblast growth factor (bFGF;20ng/ml) and forskolin (4μM) to allow the cells to expand. After7-8days, the primary passage of SC was detached with0.25%EDTA-trypsin and passaged. Differentiated MSCs cultured on chamber slides were fixed in4%paraformaldehyde at4℃for20min. Immunostaining was performed with antibodies to S100.3. Preparation of microencapsulated SCExponentially growing SC were harvested and re-suspended in a1.5%filter-sterilized sodium alginate solution (Sigma-Aldrich) at4×106ml-1cells. Then the cell suspension was extruded through a0.4-mm needle into a100-mM CaCl2solution by use of an electrostatic droplet generator to form calcium alginate gel beads. The needle tip was2cm above the CaCl2solution, and flow-rate of the extruding cell suspension was set at10ml h-1. After being gelled for20min, the micro-beads were incubated with0.05%poly-L-lysine (Sigma-Aldrich) to form an alginate-poly-L-lysine membrane around the surface. An amount of0.15%alginate solution was added to counteract excess charges on membranes for5min. The membrane-enclosed gel beads were further suspended in55mM sodium citrate to liquify the alginate gel core. Blank microcapsules were prepared by substituting the cell suspension with1.5%sodium alginate solution. The microcapsules containing SC were cultured as usual. The supernatant of the discharged culture medium was collected and analyzed for VEGF by use of an ELISA kit.4. AMI model and transplantation of MC-SC and MSCAMI was surgically induced by ligation of the left anterior descending artery. At30min after AMI induction, rats were randomly divided into4groups for injection of cells:MC-SC+MSC (n=10), MC+MSC (n=9), MSC (n=8), MC-SC (n=9) and controls (n=10). Cells were injected intra-myocardially at4sites in and around the infarcted region with a29-gauge insulin syringe. 5. Transthoracic echocardiography and MI size assessmentAt3days and2and4weeks after transplantation, LV dimension and function were assessed by2-D transthoracic echocardiography on isoflurane-anesthetized animals. LV end-systolic dimension (ESD) and end-diastolic dimension (EDD) were measured from the short-axis view of the LV at the papillary muscle level. Fractional shortening (FS) as a sensitive marker of systolic function was calculated as FS (%)=((EDD-ESD)/EDD)×100. All measurements were averaged on3consecutive cardiac cycles and analyzed by2independent observers. After the right ventricle was removed the left ventricle was cooled for30minutes and then cut into four1mm thick slices along its short axis. All pieces were kept in TTC at37℃for30min. TTC sections were photographed, digitized, and analyzed6. Immunocytochemistry and immunofluorescenceAt4weeks after transplantation, rats were killed and hearts were harvested and fixed in4%paraformaldehyde, embedded in paraffin and sectioned into5-μm sections. The antibody used was rabbit polyclonal antibody against rat von Willebrand factor to detect vascular endothelial cells. The capillary density was determined as the mean number of vWF-positive small vessels. Mouse monoclonal antibody against rat cardiac troponin I was used for labeling myocardia and rabbit monoclonal antibody against BrdU was used to detect transplanted BrdU-labeled MSC. Nuclei were stained with DAPI. BrdU-positive cells were counted in randomized fields of each specimen, with data expressed as mean±SD for capillary density and BrdU-positive cells.7. Expression of vWF and VEGF in tissueTo detect expression of vWF and VEGF in tissue, Western blot analysis was performed.Results1.SC and MC-SC AssessmentMore than95%of cells were positive for S-100during the entire culture process.SC survived and proliferated normally in microcapsules in vitro. VEGF was increasingly secreted into the supernatant of the culture medium with SC proliferation. At day8, the concentration of VEGF was elevated to813.43±86.50and821.65±83.30 pg.ml-1/48h for MC-SC and SC, respectively (P>0.05).2. Heart Function Assessment and MI sizeEchocardiography revealed the FS value decreased gradually, with an increase in EDD and ESD after LAD ligation and transplantation in all groups. At4weeks after transplantation, the decrease in FS and LV enlargement was significantly less for the MC-SC+MSC group than the other groups (P<0.01) and was less for MC+MSC and MSC groups than the control group (P<0.05). Intramyocardial injection of MSC or MC+MSC was associated with23%and24%decrease in infarct size compared with the control group (38.2±3.8%,37.7±3.5%vs.49.6±4.6%, respectively). However, injection of MC-SC+MSC was associated with the smallest infarct size (26.8±2.3%) among all groups (P<0.05).3. MSCs SurvivalBrdU-labeled MSCs were found in all hearts that received MSCs transplantation. Compared with the MSC and MC+MSC groups, the MC-SC+MSC group showed more BrdU-positive cells (13.7±2.14,12.95±1.92vs.20.79±2.89/HPF, P<0.01). Thus, transplantation with the combination of MC-SC and MSC enhanced the survival of transplanted MSC.4. Vessel DensityImmunohistochemical staining for vWF revealed that the number of capillaries in the infarcted areas was higher in the treatment groups than the control group at4weeks after transplantation (P<0.01). Furthermore, the MC-SC+MSC group had greater vessel density than the other transplant groups (P<0.01). As well, the MC-SC group had greater number of new vessels than the MC-MSC and MSC groups (P<0.05).5. vWF and VEGF protein levelsAs for vessel density, western blot analysis revealed increased protein levels of vWF and VEGF for the MC-SC+MSC group than the other transplantation groups (P<0.01). Thus, VEGF may be associated with angiogenesis in AMI.Conclusions and significanceOur data showed that transplantation of MC-SC and MSC combined could augment angiogenesis in the ischemic myocardium and improve heart function in rat. The combination could produce a synergistic effect in improving heart function in AMI. Our data supports for a possible use of MC-SC in cardiac therapeutic angiogenesis. More mechanism studies are needed for this technique. |
PDF Full Text Request