Therapeutic Effects Of Bone Marrow Mesenchymal Stem Cells Transfected Ex Vivo By Sphingosine Kinase 1 (SPK1) Gene In A Rat Acute Myocardial Infarction Model

Background and ObjectivesMesenchymal stem cells (MSC) have been increasingly tested experimentally and clinically for cardiac repair, and cumulative data have proven that more than 90% of grafted cells would die in the ischemic heart as the result of death cues including hypoxia, inflammatory cytokines, and proapoptotic factors, which are known as the limiting factors that restrict their clinical utilization. Though inasmuch, MSC remain attractive as a cellular vehicle for gene delivery, providing an alterative and applicable path by modification with well-designed exogenous gene(s). This strategy is to improve the survival of transplanted cells and thus, therapeutic efficiency might be appropriately exhibited.Human sphingosine kinase 1 (SPK1) is a key enzyme in modulating sphingolipids balance, which is important in the regulation of cell growth, differentiation and programmed cell death. Sphingosine-1 -phosphate, as one of its key product, mediates proliferative and angiogenic responses. In our previous study, we have proved that adenovirus-mediated SPK1 gene transfer could efficiently prevent Ischemia/ Reperfusion-induced myocardial injury and attenuate postischemic heart failure. This led us to hypothesize that SPK-1 gene transduction could offer cellular protection to MSC and thereby potentially improve therapeutic efficiency for ischemic heart disease. The main objects of this study are to identify whether adenovirus-mediated SPK1 gene transfer can enhance the survival of grafted MSC and better preserve ischemic heart function.Methods1. MSC were isolated from the femoral and tibial bones of 2-week-old Wistar rats (about 15-20g) by density gradient centrifugation and purified on the basis of their ability to adhere to plastic. The morphology of MSC was observed with microscope constantly and their osteogenic and adipogenic differentiation potentials were identified by histological and molecular methods. MSC were infected by an adenoviral vector carrying green fluorescent protein gene (Ad-GFP) and the infection efficiency was assessed with flow cytometry.2. The recombinant adenoviruses expressing SPK1 were generated by in vivo homologous recombination in 293 cells and purified by cesium chloride density gradient centrifugation. The titer of Ad-SPK was measured by TCID50 assay. MSC were exposed to adenoviral vectors at MOI of 50, 150 or 300 pfu per cell for 48h, then the expression of SPK1 in the transfected cells was detected by Western Blot Analysis, cellular SPK1 activity was measured by [gamma-(32)P] adenosine 5'-triphosphate incorporation. The optimal MOI was chosen for both highest SPK1 protein expression and enzyme activity of MSC transfected with Ad-SPK. Thereafter, the lasting time of SPK1 expression was tested by measuring protein and enzyme activity at 0, 2, 3, 5, 7 and 10 days after transfection.3. To observe if SPK1 gene transfer affect the properties of MSC, including their proliferation status, osteogenic and adipogenic differentiation potentials and their resistance to serum deprivation, MTT assay, cell cycle analysis with flow cytometric techniques, histological staining of inductive agents treated cells, and apoptosis indicated by annexin V and propidium iodide staining were performed.4. Recombinant retroviral vector carrying firefly luciferase reporter gene (rv-fluc) was constructed by subcloning, then it was transfected into GP+E86 and PA317 incasing cells by ping-pong infection method and selected with G418. The stable expression of the fluc gene in positive clones was identified by detecting luciferase enzyme activity.5. Murine bone marrow MSC were transfected with rv-fluc and those with stable expression of fluc were selected with G418. Then, mu-MSC/fluc were transplanted into murine skeletal muscle, and the in situ expression was noninvasively tracked by in vivo bioluminescence imaging.6. Retrovirally transduced rat MSC carrying fluc were infected with Ad-SPK or Ad-GFP, and 48 hours later injected into ischemic zone of myocardial infarction rats. The survival rate of graft cells was estimated by detecting luciferase enzyme activity of myocardium.7. Acute myocardial infarction model was developed in 30 8-week-old male inbred Wistar rats (about 250g) by permanent ligation of the left anterior descending coronary artery. Then 1×10~6 SPK1 modified MSC (MSC-SPK) or green fluorescent protein transfected MSC (MSC-GFP) or PBS only (AMI control) were injected intramyocardially in the free wall of left ventricle at multiple sites 30 minutes after myocardial infarction (n =10/group). And other 5 non-ligated rats (SHAM group) were used as the normal control. Echocardiographic studies of LV function, plasma atrial natriuretic peptide (ANP) level were measured at pre-infarction, 14 and 28 days after transplantation. Finally, all the rats were performed invasive left ventricular (LV) hemodynamic measurement in closed chest preparation with multichannel physiologic recorder four weeks after cell transplantation. Middle transverse section was stained with Masson's trichrome to measure the infarct size and collagen volume density fraction (CVF) with the use of image analysis software. Factor V! was identified by immunohistochemical staining to evaluate the angiogenesis in the injured heart area.Results1. MSC were successfully isolated and cultured by density gradient centrifugation followed by adherence-separation. The cultured rat MSC were fibroblastic in morphology and attached to the culture dish tightly. After 7 to 10 days of primary cultivation, rat MSC were nearly 80% in confluence. MSC proliferated readily in the culture medium, typically 3 days for one passage. They could be induced to differentiate into osteoblasts and adipocytes under appropriate conditions. Ad-GFP infected MSC with high efficiency of 97.82% around.2. Ad-SPK was successfully generated and purified with a titer of 6×10(10) TCID50/ml. The optimal MOI chosen by balancing transfection efficiency and cell viability was 150pfu/cell. Transduction efficiencies and the expression levels of SPK1 gene in MSC were found to be higher with the adenovirus vector and able to be maintained in culture for at least 10 days.3. In vitro, SPK1 Overexpression did not affected MSC in cell proliferation, cell cycle and the osteogenic or adipogenic differentiation potential. Interestingly, SPK1 gene modification reduced MSC apoptosis by 30.71% under serum starvation conditions.4. Recombinant retroviral vector carrying firefly luciferase reporter gene (pL (fluc) SN) was successfully constructed. The fluc gene was integrated into the PA317 and GP+E86 genome and expressed stably in the host cells.5. No more than 10 percent of flue-labeled murine bone marrow MSC survived 3 days after transplantation into normal skeletal muscle, as shown by the in vivo bioluminescence imaging assay.6. When compared with GFP transfected MSC/fluc, SPK1 modified MSC/fluc survived in greater numbers in the ischemic region of the myocardium at the early stage after transplantation. The survived number of MSC/fluc-SPK was 1.83-fold and 1.69-fold higher than those of GFP-MSC at 3 days and 5days respectively.7. Four weeks after acute myocardial infarction, the rats' hemodynamics deteriorated, left ventricular dialated and sphericity change exhibited, collagen deposited in non-infarcted area, plasma ANP level increased and left ventricular function greatly damaged. Both GFP and SPK1 transfected MSC transplantation can effectively improve hemodynamics, decrease LVEDP, reduce collagen deposition in non-infarted area of LV, enhance angiogenesis, lower ANP level and improve LV function. However, in contrast to the MSC-GFP group, the MSC-SPK group showed enhanced angiogenesis (by 32%), reduced infarction area (by 6%) , lower LVEDP (by 10%) and relative normal LV appearance.Conclusions1. Density gradient centrifugation and adherent separation provide a simple and efficient way to obtain highly purified Wistar rat's MSC from bone marrow. These cells can be easily proliferated, exhibit differentiation potentials along osteogenic and adipogenic pathways, and can be efficiently transferred by adenovirus, endowing them as ideal vectors for tissue and gene engineering.2. Adenovirus -mediated gene transfer strategies provide an efficient tool for ex vivo modification of MSC. And the gene modification does not interfere with cell cycle, proliferation and differentiation.3. Grafted bone marrow MSC remain high death rate whether they are transplanted into normal or ischemic tissue, more than 90% died in the early stage of transplantation. Although SPK1 modification strategy provide MSC enhanced tolerance to serum deprived injury in vitro, their survival rate in ischemic myocardium showed no difference with MSC-GFP 5 days later after transplantation.4. Both GFP and SPK1-modified MSC transplantation improved heart function, inhibited left ventricular remodeling, enhanced angiogenesis 4 weeks after AMI. SPK1 modified MSC decreased LVEDP and infarct area, enhanced angiogenesis significantly, when compared with MSC-GFP only.