Anoxic Preconditioned Bone Marrow Mesenchymal Stem Cells Transplantation Reduced Cardiaomyocyte Apoptosis And Improved Heart Function After Myocardial Infarct

Myocardial infarction(MI) is the irreversible necrosis of heart muscle secondary to prolonged ischemia.This usually results from an imbalance of oxygen supply and demand,leading to irreversible loss of cardiomyocytes and scar formation,is the leading cause of congestive heart failure.Initial therapy for acute myocardial infarction is directed toward restoration of perfusion as soon as possible to salvage as much of the jeopardized myocardium as possible.This may be accomplished through medical or mechanical means,such as percutaneous coronary intervention or coronary artery bypass grafting.Further treatment is based on(1) restoration of the balance between the oxygen supply and demand to prevent further ischemia,(2) pain relief,and(3) prevention and treatment of any complications that may arise.The identification of stem cells capable of contributing to tissue regeneration has raised the possibility that cell therapy could be employed for repair of damaged myocardium,and mesenchymal stem cells(MSCs) are pluripotent,which have the potential for differentiating into cardiomyocytes and vascular endothelial cells within the microenvironment of the heart.They can release angiogenic,antiapoptotic,and mitogenic factors such as vascular endothelial growth factor(VEGF),hepatic growth factor(HGF) and insulin-like growth factor(IGF),which stimulate the endogenous repairing mechanisms.MSCs are considered to be an effective therapeutic approach for MI both in basic researches and clinical trials,but the effect is not so distinct partly because of the low grafted cell survival rate.Some researches using gene-modified MSCs to increase the cardio-protective effect,however,there is much debate over the safety and efficiency of this method.Recently,it is reported that the cardio-protective effect of MSCs is still related to paracrine effect which could be enhanced by hypoxia.Bone marrow is also a hypoxic place of residence of MSCs;we hypothesized treating MSCs with anoxia preconditioning(AP) before transplantation may increase the cardioprotective effect of MSCs.We,therefore,hypothesized that MSCs could protect diabetic myocardium and AP might enhance this protective effect.MethodsIn vivo:Donor rats were 8 weeks old male Sprague-Dawley rats.We harvested bone marrow by crushing and flushing their femurae and tibiae with phosphate-buffered saline(PBS).A small percentage of MSCs isolated from the density interface of 1.077 g/ml were cultured.The medium was replaced every 4 days and nonadherent hematopoietic cells were removed.The adherent,spindle-shaped MSCs were expanded and purified with 3 to 5 passages after initial plating.MSCs were tested through fluorescence-activating cell sorting before transplantation,by using directly conjugated antibodies against anti-rat CD44,anti-CD45 and anti-CD90.Before transplantation, MSCs were incubated in nuclear dye DAPI at a concentration of 50μg/ml at 37℃for 30min and then washed three times with PBS.AP-MSCs were exposed to 3 hours of anoxia.The male SD rats(250g) were enrolled in the studies and anesthetized with 4% chloral hydrate(4 mg/kg,administered intraperitoneally),then mechanically ventilated by using a small animal ventilator.MI was set up by permanent ligation of left anterior descending coronary artery.Successful performance of coronary occlusion was verified by blanching of the myocardium distal to the coronary ligation.The sham-operated group received the same procedure of thoracotomy without coronary ligation.The chest was closed,and rats were weaned from the ventilator and allowed to recover for a week. Then the MI rats were randomly divided into 3 groups(16 rats per group):control animals that received DMEM injection,and cell-treated animals that received either MSCs(5×10⁶) or AP-MSCs(5×106).The cells suspended in 150ul DMEM or an equivalent volume of DMEM were prepared individually and injected into periinfarcted regions of left anterior ventricles at 3-5 sites.Sham group were received the same volume of DMEM injection as transplant subjects.The rats were then bred for another 1 or 4 weeks.Apoptotic rate of cardiomyocytes in periinfracted area was assessed by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling(TUNEL) assay 1 week after transplantation.Cardiac function was assessed by echocardiography 4 weeks after transplantation.Infarct size was measured by hematoxylin and eosin staining 1 and 4 weeks after transplantation.The expression of bcl-2,bax and cleaved-caspase-3 was analyzed by western-blot.In vitro:Primary culture of cardiomyocytes was prepared by multi-digestion method.After incubation of dispersed cells on 25 cm2 flask for 60 min in normoxic incubator(95%air/ 5%CO2),unattached viable cells were collected and seeded into 25 cm2 flask or tanswell lower plates and incubated.The cells were then incubated with DMEM supplemented with 20%fetal calf serum plus 0.1 mmol/L 5-bromo-2-deoxyuridine(Sigma,USA) for 72 h to prevent low-level nonmyocardial cell proliferation,and then replaced with DMEM plus 20%calf serum.Cardiomyocytes in the lower plates of transwells were randomly divided into 3 groups:DMEM group, MSCs group and AP-MSCs group.For co-culture experiments,MSCs(or AP-MSCs) and cardiomyocytes were cultured in 2 individual chambers separated by a semipermeable membrane(3um hole).This system allowed sharing the culture medium in 2 chambers but prevented cells contacts.Each was treated with H/R:To mimic hypoxia,the cardiomyocytes were incubated at 37℃in the anoxic chamber for 24 h, where normal air was replaced by 95%N2/5%CO2.Then cardiomyocytes were moved into normoxic incubator(95%air/ 5%CO₂) for 3 h to mimic reoxygenation process. Control group was incubated in DMEM supplemented with 20%fetal calf serum under standard cell culture conditions(95%air/5%CO2).Apoptosis of cardiomyocytes was determined by Annexin V-FITC Apoptosis Detection Kit.Cells were visualized directly on glass slides under a fluorescence microscopy and analyzed by flow cytometry. Results1,Morphology of cardiomyocytes,MSCs,AP-MSCsCardiomyocytes were attached to culture dishes and displayed a polygon or spindle-like shape,with the capability to beat.MSCs were attached to culture dishes and the majority displayed a spindle-like shape.AP-MSCs changed little in morphology and CD markers,hypoxia for up to 6h didn't cause irreversible damage.2.MSCs,especially AP-MSCs prevented cardiomyocytes apoptosis induced by H/RNormal cultured cardiomyocytes showed a low apoptotic rate(2.16±0.78%),which increased after H/R(21.5±2.71%,vs Nonnal,P<0.05).The rate reduced while co-cultured with MSCs(17.47±1.62%,vs Normal and DMEM,P<0.05),and more obviously while co-cultured with AP-MSCs(11.01±2.10%,vs normal,DMEM and MSCs,P<0.05).3.The affection of MSCs and AP-MSCs on apoptotic related proteins expression in cardiomyocytesCo-culture with MSCs or AP-MSCs increased the bcl-2/bax ratio and prevented the activation of caspase-3(vs DMEM,P<0.05),and that was more obvious in AP-MSCs group(vs MSCs,P<0.05).4.Identification of engrafted MSCsThe cells before transplantation were labeled(≈100%) with DAPI and the implanted cells do survive in the peri-infarct region for at least 4 weeks after transplantation.5.MSCs,especially AP-MSCs prevented cardiomyocytes apoptosis after MI TUNEL labeling was performed 1 week after transplantation and the results showed that the number of TUNEL-positive cells was significantly reduced by treating with MSCs or AP-MSCs(MSCs group:11.18±0.88%;AP-MSCs group:9.03±0.42%; vs.DMEM group:14.54±0.60%,P<0.05),and AP-MSCs had the predominant effect(vs MSCs,P<0.05).We further examined the expression of cleaved-caspase-3,bcl-2 and bax in infarcted heart,and found MSCs and AP-MSCs increased the bcl-2/bax ratio(vs. DMEM,P<0.05),prevented the activation of caspase-3(vs.DMEM,P<0.05), AP-MSCs were predominant(vs.MSCs,P<0.05).6.MSCs,especially AP-MSCs improved the cardiac function post MIThe results of the cardiac function measurements were as follows:(1) Fractional shortening and ejection fraction significantly increased;left ventricular diastolic dimension and systolic dimension significantly decreased 4 weeks after cell transplantation(vs DMEM,P<0.05).AP-MSCs were predominant(vs MSCs,P<0.05).7.MSCs,especially AP-MSCs transplantation reduced infarct sizeInfarct size was measured 4 weeks post transplantation.The results were as follows: Infarct size significantly decreased in cell translation group(MSCs group:38.76±1.42%,AP-MSCs group:32.8±1.67%;vs DMEM group:47.30±2.63%,P<0.05) 4 weeks after transplantation,and there was trend of smaller infarct size in AP- MSCs group as compared with MSCs group(P<0.05).8.AP-MSCs transplantation increased the arteriole density and the expression of VEGFArteriole density was significantly greater in AP-MSCs group than in other groups (AP-MSCs:6.10±0.23,vs DMEM:4.88±0.37;MSCs:4.78±0.27,P<0.05).The expression of VEGF was the most intense in AP-MSCs group.ConclusionAP-MSC is antiapoptotic in our rat model of MI,possibly mediated through upregulating Bcl-2/Bax ratio and inhibition of activation of caspase-3,thus reduce the apoptosis of cardiomycyts.AP may be an effective and easy way to enhance the cardioprotection of MSCs.