DMOG Enhances Mesenchymal Stem Cells Survival And Its Mechanisms

Background:Despite great strides in medical strategies aimed at post-infarct remodeling,the development of aggressive reperfusion strategies such as interventional diagnosis and therapy,as well as common drug therapy,heart failure persists as an emerging public health concern in the developing world and remains the leading cause of death and hospitalization in industrialized countries.In recent years,cell-based therapy has emerged as a promising therapeutic approach for restoration of heart function after myocardial infarction.Circumventing ethical and immune rejection issues,autologous mesenchymal stem cells(MSCs) transplantation is an ideal option for clinical cell therapy use.To date,it has been demonstrated that MSCs are capable of ameliorating the cardiac function after myocardial infarction.However,the majority of transplanted cells usually die a few days after transplantation into the harsh host environment.It was reported that over 90%of MSCs died in the first 24 h and only 0.44%survived 4 d after transplantation to ischemic heart.Poor cell viability after transplantation into infarcted myocardium obviously restricts the efficacy of this attractive therapy.Therefore, strategies to enhance MSCs survival are important and urgently needed.Under hypoxic conditions,prolyl hydroxylase activity decreases because they require oxygen as a co-factor and the HIF-1αprotein is stabilized.HIF-1αstabilization is followed by activation of the transcription of numerous target genes such as vascular endothelial growth factor(VEGF),erythropoietin(EPO),and glucose transporter 1 (Glut-1) that are involved in angiogenesis,erythropoiesis,energy metabolism and cell survival.It has been demonstrated that hydroxylases play an important role in heart diseases.Inhibition of prolyl hydroxylase activity or expression can be effected using competitive inhibitors such as dimethyloxalylglycine(DMOG) or siRNA leading to the stabilization of HIF and other beneficial effects.In ischemia-reperfusion(I/R) injury and acute myocardial infarction animal models,inhibition of prolyl hydroxylases has been observed to prevent deleterious remodeling and to attenuate the acute inflammatory response.Additionally,hydroxylase inhibition followed by HIF-1αup-regulation participates in the protection of cell survival and regulation of apoptosis which have been shown in cancer cells and neurons under hypoxic condition,serum deprivation or tropic factor deprivation.The precise role of HIF-1αin regulation cell death and apoptosis remains controversial because of the concern that HIF-1αmay promote cell death.The role of prolyl hydroxylase inhibition by DMOG in regulation of cell survival has not been investigated in MSCs.The purpose of this study is to evaluate the effect of DMOG on the apoptosis and cell death induced by serum deprivation in MSCs and to delineate the underlying signaling mechanisms.Objectives:The objective of this study is to evaluate the effect DMOG on MSCs survival against serum deprivation and to explore the possible mechanisms.Methods:The typical apoptotic model of serum deprivation was used in this study to mimic the microenvironment of ischemic heart diseases in vivo.Three groups,Control group,serum deprivation group(SD) and serum deprivation with DMOG group (SD+DMOG),were divided.According to the concentration of DMOG application, SD+DMOG group was sub-divided into 100μM,500μM and 1000μM groups. Western-blot was applied to detect HIF-1αand its downstream Glut-1 expression of MSCs.Trypan blue was used to detect the cell toxicity in the normal culture condition and MSCs death under serum deprivation.Apoptosis of MSCs in different groups was observed by morphological changes,caspase-3 activation.Morphological anlysis was detected by phase-contrast microscope and Hoechst staining which was checked under fluorescent microscope to detect the nuclear condensation and fragmentation.Apoptotic rate was calculated by cleaved caspase-3 staining and western-blot of cleaved caspase-3 was used to confirm staining results.To evaluate the mechanisms beside HIF-1αpathway,mitochondrial and survival pathways were detected.To study involvement of the mitochondrial pathway in the mechanism,western-blot was used to detect cytochrome C release and apoptosis inducing factor(AIF) translocation to evaluate caspase dependent or caspase independent pathway,respectively.Akt and ERK phosophorylation level were detected by western-blot to evaluate the possible effect of PI3K/Akt and MEK/ERK pathway.Wortmannin,a specific inhibitor of PI3K,was used to block PI3K/Akt pathway and then Akt activation,cleaved caspase-3,and trypan-blue staining were re-evaluated to confirm the involvement of PI3K/Akt pathway in the protective effect of DMOG on MSCs.Results:DMOG remarkably increased MSCs HIF-1αstablizaion and Glut-1 expression without obvious toxicity.DMOG ameliorated the morphological changes by serum deprivation and decreased nuclear condensation detected by Hoechst staining.DMOG significantly reduced the apoptotic rate calculated by cleaved caspase-3 staining in a dose dependent manner which was confirmed by western-blot analysis of cleaved caspase-3 activation.What's more,DMOG prevented caspase-3 activation in a time course from 6h to 24h.Similarly,DMOG decreased MSCs death in a dose-dependent manner and in a time course from 12h to 72h.Serum deprivation induced cytochrome C release from mitochondria into cytosol and AIF translocation from cytosol into nucleus. DMOG decreased both of of them indicating both caspase dependent and independent apoptotic pathway were possibly involved in the protective effect.Furthermore,serum deprivation significantly decreased Akt and ERK activation compared with control group,while DMOG reversed Akt but not ERK phosphorylation,indicating PI3K/Akt but not MEK/ERK pathway maybe involved in the mechanism.To confirm this, wortmannin,a specific PI3K inhibitor,was used to block PI3K/Akt pathway and Akt activation was inhibited.Meanwhile,wortmannin abrogated the beneficial effect of DMOG on MSCs apoptosis and cell death re-evaluated by cleaved caspase-3 and trypan-blue staining,respectively.Conclusions:The prolyl hydroxylase inhibitor DMOG inhibited apoptosis and cell death induced by serum deprivation in MSCs concurrent with HIF-1αstabilization, mitochondrial protection,and activation of the PI3K/Akt pathway.