Improved Anti-apoptotic And Anti-remodeling Potency Of Bone Marrow Mesenchymal Stem Cells By Anoxic Preconditioning In Diabetic Cardiomyopathy

Diabetic cardiomyopathy(DCM)is a clinical condition,diagnosed when ventricular dysfunction develops in patients with diabetes mellitus(DM)in the absence of coronary artery disease,valvular heart disease or hypertension,which is characterized by early diastolic dysfunction and late systolic heart failure.Pathologically,DCM is characterized by microcirculatory defects,apoptosis and necrosis of cardiomyocytes and progressive interstitial fibrosis,which lead to poor myocardial perfusion and myocardium weakness.Thus,restoration of the microvascular homeostasis and impaired myocardium would be desirable.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 also inhibit myocardial fibrosis in rat models of ischemic and dilated cardiomyopathy and attenuate the increased expression of matrix metalloproteinase(MMP)-2 and MMP-9.Anoxic preconditioning(AP)of MSCs increased their effectiveness against ischemic injury by reducing myocardilal apoptosis and remodeling.We,therefore,hypothesized that MSCs could protect diabetic myocardium and AP of MSCs might enhance this protective effect.Methods7-8 weeks old male Sprague-Dawley rats with body weight between 160g and 180g were given a single intraperitoneal injection of streptozotocin(STZ)(60mg/kg)to induce DM.Tail blood glucose was measured every week after injection and all animals with blood glucose level less than 300mg/dl were excluded from the DM group.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. Immunofluorescence staining was performed to detect the VEGF expression of MSCs. Before transplantation,MSCs were incubated in membrane dye CM-DiI at a concentration of 2μg/ml at 37℃for 20min and then washed three times with PBS. AP-MSCs were exposed to 3 hours of anoxia.4 months after STZ injection,diabetic rats were randomly given an intramyocardial injection of one of the followings: 150μL of DMEM(n=10),5×10~6 MSCs/150μL(n=10),or 5×10~6 AP-MSCs/150μL (n=10).Injections were performed at 5 sites:right ventricular free wall,basal and midanterior wall,lateral wall,and posterior wall.2 months,3 months after STZ injection and 2 weeks after transplantation,we evaluated the cardiac function by echocardiography.We also evaluated the cardiac conditions of DMEM,MSC and AP-MSC group by histopathologic analysis including capillary density and and fibrosis area,western blot analysis for apoptosis related proteins,growth factors and signal pathways,and zymography analysis for MMP-2 activity 2 weeks after transplantation.Results1,Blood Glucose Increased and Cardiac Function Decreased in DM Rats 2 months after STZ injection,DM rats had obvious higher blood glucose(520±12 vs 113±5mg/dl,p＜0.01)and lower body weight(274±9 vs 347±11g,p＜0.01)than control group.Left ventricular posterior wall thickness(LVPW)and fractional shortening (FS)were lower in DM rats(LVPW:1.6±0.1 vs 1.8±0.1mm,p＜0.05;FS:38.7±1.2 vs 46.2±1.2%,p＜0.01).Left atrial dimension(LAD)was a little higher in DM rats,while the difference didn't reach statistical significance.3 months after STZ injection with DM progression,LVPW and FS were further decreased in DM rats (LVPW:1.1±0.1 vs 1.85±0.1mm,p＜0.01;FS:32.9±2.3 vs 44.5±2.0%, p＜0.01).It's indicated that cardiac function has already decreased in rats with DM for 2 months and further deterioration of heart function was seen with DM progression.2,Characterization of Cultured MSCsMost cultured MSCs expressed CD44 and CD90,whereas they were negative for CD45.MSCs express VEGF.Red fluorescence-labeled MSCs were found in the diabetic myocardium 3 days after transplantation.3,Improved cardiac function of DCM by MSCs2 weeks after transplantation,the levels of blood glucose were still high in 3 groups (ns).Heart weight normalized for body weight was lower in AP-MSC group(p＜0.01). Interventricular septal diastolic thickness(IVSd),LVDd and LVPW were similar in 3 groups,while left ventricular systolic dimension was lower in AP-MSC group (p＜0.01 vs DMEM).MSCs transplantation,especially AP-MSCs increased FS of diabetic heart(p＜0.01 vs DMEM respectively).4,Increasesd Capillary Density and Decreased Myocardial Fibrosis by AP-MSCs As shown in alkaline phosphatase staining,AP-MSCs greatly increased the capillary density of diabetic myocardium(2904±149 capillaries/mm~2 vs 2210±86 capillaries/mm~2 of DMEM group and 2402±89 capillaries/mm~2 of MSC group, p＜0.01 respectively).Masson's trichrome staining demonstrated much more severe fibrosis in the myocardium of DMEM group compared with MSC and AP-MSC group.Quantification of fibrosis area showed significant decrease of fibrosis in MSC (7.4±0.2%vs 8.2±0.2%,p＜0.05)and AP-MSC group(6.7±0.2%vs 8.2±0.2%, p＜0.01)compared with DMEM group.The activity of MMP-2 were both increased in MSC and AP-MSC group compared with DMEM group(-fold increase versus control;MSC,1.31±0.07,not significant;AP-MSC,1.94±0.17,p＜0.01).5,AP-MSCs Increased Bcl-2/Bax Ratio and Decreased Caspase3 Activation 2 weeks after transplantation,western blot showed similar expression of Bcl-2 in 3 groups and lower expression of Bax in AP-MSC group compared with DMEM and MSC group,although the change did not reach the statistical significance.However, the Bcl-2/Bax ratio was higher in AP-MSC group(p＜0.05 vs DMEM).The expression of caspase-3 was similar in 3 groups,while cleaved caspase-3 was lower in AP-MSC group(p＜0.01 vs DMEM and MSC group respectively).6,AP-MSCs Decreased the Expression of Transforming Growth Factorβ-1 No obvious difference was seen in the cardiac expression of VEGF in 3 groups 2 weeks after transplantation,while cardiac expression of TGF(transforming growth factor)β-1 was lower in AP-MSC group(p＜0.01 versus DMEM and MSC group respectively).7,AP-MSCs induced the phosphorylation of Akt and ERKWestern blot analysis of the molecular pathways involved in apoptosis revealed increased activity of Akt and ERK in AP-MSC group.Increased activity of ERK was also seen in MSC group. ConclusionAP-MSC is antiapoptotic in our rat model of DCM,possibly mediated through upregulating Bcl-2/Bax ratio and inhibition of expression and activation of caspase-3, thus reduce the apoptosis of endothelial cells and improve myocardial microcirculation.AP-MSCs transplantation increased MMP-2 activity and inhibited the expression of TGFβ-1 in diabetic myocardium,leading to the decreased fibrosis area in diabetic myocardium.Intramyocardial transplantation of MSCs has a protective effect on diabetic myocardium and AP can enhance this protective effect.AP-MSCs transplantation improved cardiac function in the rat model of DCM,possibly through an antiapoptotic effect on diabetic myocardium and attenuation of cardiac remodeling.