Hypoxic Preconditioning And Cyclosporine A Attenuates Hypoxia/reoxygenation Induced Apoptosis In Mesenchymal Stem Cells

Although there are many therapeutic advances in myocardial infarction,the irreversible loss of cardiomyocytes remains a key problem to resolve,and this forms the cellular basis of cardiac dysfunction.Mesenchymal stem cell(MSC)transplantation is a promising strategy.MSC can differentiate into vascular endothelial cells and cardiomyocytes as well as improve heart function.However,this cell replacement therapy is limited by their poor viability after transplantation.Approximately 99%of transplanted MSC are readily lost during 4 days after transplantation.Apoptosis is thought to be a major factor in their demise.Therefore,protecting MSC against apoptosis in a pro-apoptotic microenvironment of the infarcted heart is critical for improving the efficiency of cell therapy.Hypoxia preconditioning(HPC)increases cellular resistance against subsequent lethal hypoxia injury.However,the effect of HPC on the apoptosis in MSC is still unclear.Thus,we aimed to study the protective effect of HPC on MSC against hypoxia/reoxygenation(H/R)-induced injuries.TUNEL and MTT were examined to determine whether HPC could protect MSCs from H/R injury.Mitochondrial membrance potential,bcl-2,ERK,AKT,HIF-1a and VEGF were examined to reveal the mechanism of HPC.Several experimental and clinical studies suggest that cyclosporin A (CsA)treatment reduces apoptosis in human endothelial cells and neurocytes.So,the effects on MSCs of CsA were examined.TUNELand MTT were examined to determine whether CsA could protect MSCs from H/R injury.Mitochondrial membrance potential,Bcl-2,Bax,ERK and Bad were examined to reveal the mechanism of CsA too.PartⅠHypoxic preconditioning attenuates hypoxia/reoxygenation-induced apoptosis in mesenchymal stem cellsWe humanely killed male Sprague-Dawley rats(80 g)and harvested the bone marrow by flushing their femoral and tibial cavities with phosphate-buffered saline(PBS). Bone marrow cells were prepared by gradient centrifugation at 900×g for 30 min on a at a density of 1.073 g/mL and cultured in Dulbecco's modified Eagle's medium supplemented with 10%fetal bovine serum.Cells were determined by fluorescence-activating cell sorting analysis before this experiment using directly conjugated antibodies against anti-rat CD44,anti-CD45,and anti-CD90.Cultured MSC of passage 3 were divided into six groups:(ⅰ)group 1:normal MSC.(ⅱ)group 2:H/R. (ⅲ)group 3:cyclosporine A(CsA)+H/R.(ⅳ)group 4:HPC 10 min+H/R.(ⅴ)group 5: HPC 20 min+H/R,and(ⅵ)group 6:HPC 30 min+H/R.The apoptosis of MSCs were assessed by Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling(TUNEL)assay.Cell viability in each group was assessed by MTT assay.For the detection of the transduction signal of HPC antiapoptosis,we detected the protein expression using antibodies as follows:antibodies specific to Bcl-2,Akt,phospho-Akt, HIF-1a,VEGF,β-actin,ERK,and phosphor-ERK.Results1.Surface analysis of MSC line by flow cytometryThe surface markers for MSC were identified.The putative MSC used in the experiments expressed CD44 and CD90 at moderate to high levels(86.5%and 99.4%, respectively),but CD45 was at a low level(4.47%).2.HPC prevented MSC from H/R-induced apoptosis and increased cell viabilityApproximately 3%of normal MSC of the 3 generations were TUNEL-positive. H/R markedly increased TUNEL-positive MSC compared with normal cells (48.2%±3.1%,P＜0.05 vs normal cells).However,when the MSC were pretreated by HPC for 10,20,and 30 min before H/R,the number of TUNEL-positive MSC significantly decreased in a time-dependent manner(HPC 10 min 20.4%±1.3%,HPC 20 min 16.8%±1.5%,and HPC 30 min 14.2%±0.5%,P＜0.05 vs H/R).HPC also increased the viability of the MSC in a time-dependent manner in response to H/R(P＜0.05 vs H/R).To further investigate whether HPC has long-term beneficial effects on MSC,we tested the apoptosis induced by 24 h hypoxia and 24 h reoxygenation on the MSC.HPC 10 min and 30 min reduced the apoptotic index from 83%±5.6%to 56%±3.5%,and 42%±4.1% (P＜0.05 vs H/R).3.Influence of HPC on H/R-inducedΔΨmNormal MSC exhibited punctate red staining indicative of coupled mitochondria with a normalΔΨm.The MSC in the H/R group developed a diffuse green staining pattern,representative of reducedΔΨm.4.Effect of HPC on the translocation of Bcl-2 and bax in mitochondriaThe results showed that Bcl-2 levels were upregulated after H/R treatment by 2.3±0.18-fold versus normal cells(P＜0.05),but HPC significantly increased the expression of the protein Bcl-2 compared with the H/R group(HPC 10 min 3.3±0.11, HPC 20 min 3.4±0.23,and HPC 30 min 3.8±0.11-fold vs normal cells;P＜0.05,HPC vs H/R).Pre-incubation with 0.5μmol/L CsA upregulated the Bcl-2 expression by 2.68±0.18-fold versus normal cells(P＜0.05,CsA vs H/R).This suggested that the Bcl-2 upregulation induced by H/R could be enhanced by HPC treatment.5.Effects of HPC on the activation of ERK-1/2 induced by H/R in MSCsThe results showed that the phospho-ERK content decreased in the MSC maintained after H/R by 0.44±0.17-fold versus normal cells(P＜0.05,H/R vs normal cells). However,these effects induced by H/R were reversed by HPC,and HPC could time-dependently upregulate phospho-ERK(HPC 10 min 0.80±0.16,HPC20 min 0.91±0.03,and HPC 30 min 1.04±0.04-fold vs normal cells;P＜0.05,HPC vs H/R). However,phospho-ERK in the 0.5uM CsA group was not higher than that of the H/R group(P＞0.05).HPC or CsA did not alter the ERK1/2 expression.6.Effects of HPC on the activation of Akt induced by H/R in MSCs The result showed that the phospho-Akt content decreased in the MSCs after H/R treatment by 0.39±0.14-fold versus normal cells(P＜0.05,H/R vs normal cells). However,these effects induced by H/R were reversed by HPC.HPC time-dependently upregulated the phospho-Akt level(HPC 10 min 0.43±0.13,HPC 20 min 0.47±0.12, and HPC 30 min 0.73±0.014-fold vs normal cells;P＜0.05,HPC vs H/R).The level of phospho-Akt in the CsA group decreased(P＜0.05,CsA vs H/R).HPC or CsA did not alter the Akt expression.7.Effects of HPC on HIF-1a and VEGF expressions induced by H/R in MSCAfter 6 h hypoxia and 12 h reoxygenation,the expression of HIF-1a did not significantly change in the MSCs subjected to H/R or HPC.The VEGF content was increased in the MSCs after H/R by 1.89±0.15-fold versus normal cells(P＜0.05,HR vs normal cells).Meanwhile,these effects induced by H/R were enhanced by HPC.HPC time-dependently upregulated VEGF(HPC 10 min 2.37±0.12,HPC 20 min 2.40±0.10, and HPC 30 min 2.44±0.11-fold vs normal cells;P＜0.05,HPC vs H/R).The VEGF expression in the CsA group had no difference compared with the H/R group(P＞0.05, CsA vs H/R).PartⅡCyclosporine A preineubation attenuates hypoxia/reoxygenation-indueed apoptosis in mesenchymai stem cellsWe humanely killed male Sprague-Dawley rats(80 g)and harvested the bone marrow by flushing their femoral and tibial cavities with phosphate-buffered saline(PBS). Bone marrow cells were prepared by gradient centrifugation at 900×g for 30 min on a at a density of 1.073 g/mL and cultured in Dulbecco's modified Eagle's medium supplemented with 10%fetal bovine serum.Cells were determined by fluorescence-activating cell sorting analysis before this experiment using directly conjugated antibodies against anti-rat CD44,anti-CD45,and anti-CD90.Cultured MSC of passage 3 were divided into 4 groups:(ⅰ)group 1:normal MSCs.(ⅱ)group 2:H/R.(ⅲ) group 3:0.5 uM cyclosporine A(CsA)+H/R.(ⅳ)group 4:5 uM cyclosporine A +H/R. The apoptosis of MSCs were assessed by Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUYP nick-end labeling(TUNEL)assay.Cell viability in each group was assessed by MTT assay.For the detection of the transduction signal of CsA antiapoptosis,we detected the protein expression using antibodies as follows: antibodies specific to Bcl-2,Bax,Bad,phospho-Bad,cytochrome C,ERK,and phosphor-ERK.Results1.Surface analysis of mesenchymal stem cell line by flow cytometry analysesSurface markers for MSCs were identified.The putative MSCs used in the experiments expressed CD44 and CD90 at moderate to high levels(86.5%and 99.4%,respectively),but CD45 was at low level(4.47%).2.CsA Prevents MSCs from H/R-induced apoptosis and increases cell viability About 3%of normal MSCs were positive.When MSCs cultured in serum-free media and insulted hypoxia for 6 hours and reoxygenation for 12 hours,the number of TUNEL-positive MSCs was significantly increased(48.2%,p＜0.05 vs normal cell).When MSCs were preincubated with CsA for 30 minutes before treatment by H/R,the number of TUNEL-positive MSCs was significantly decreased in a dose-dependent manner(0.5 uM CsA 22±1.4%,1 uM CsA 18±1.5%,2.5 uM CsA 17±1.6%,5uM 15±1.4%,p＜0.05 vs H/R).0.5-5uM CsA also protected the loss of MSCs viability in a dose-dependent manner in response to H/R(p＜0.05 vs H/R).3.Influence of CsA on H/R-induced mitochondrial membrane potential(ΔΨm).Normal MSCs exhibited punctate red staining indicative of coupled mitochondria with a normalΔΨm.After insulted by H/R,MSCs developed a diffuse green staining pattern,representative of reducedΔΨm.CsA had a marked effect on JC-1 staining.4.Effect of CsA on translocation of cytochrome cCytochrome c translocation from mitochondria to cytosol was increased when MSCs were subjected to H/R(1.4-fold vs normal cells,P＜0.05 vs normal cell).CsA blocked the H/R-induced increase of cytochrome c expression in the cytosol fraction.At the same time,total expression of cytochrome c was unaffected after H/R or CsA preincubation (p＞0.05).5.Effect of CsA on expression of Bcl-2 and bax.Total Bcl-2 expression were upregulated at H/R by 4.5-fold vs normal cells(P＜0.05 vs normal cell),but 0.5 and 5 uM CsA preincubation showed a higer expression of the protein Bcl-2 than in H/R group(0.5 uM CsA 6.6,5uM 5.3-fold vs normal cells,P＜0.05 vs H/R group).Total bax levels were upregulated at H/R by 1.9-fold vs normal cells(P＜0.05 vs normal cell),but 0.5 and 5 uM CsA preincubation showed no significantly changed expression of the protein bax(0.5 uM CsA 2.02,5uM 1.9-fold vs normal cells,P＞0.05). 6.Effects of CsA on activation of ERK-1/2 induced by H/R in MSCsERK phosphorylation decreased in MSCs maintained after H/R by 0.8 fold compared with normal cells(P＜0.05 vs normal cell).0.5 uM CsA did not affect ERK phosphorylation(p＞0.05).However,ERK phosphorylation in 5 uM CsA group decreased (P＜0.05 vs H/R group).7.Effects of CsA on BAD induced by H/R in MSCsWhen MSCs insulted by H/R,Bad phosphorylation decreased.(p＜0.05 vs normal MSCs cells).CsA prevented the H/R-induced decreased in Bad phosphorylation(0.5 uM CsA 0.85,5uM 0.88-fold vs normal cells,p＜0.05 vs H/R group).ConclusionsThe results of the present study demonstratethat HPC inhibits the H/R-induced apoptosis of MSC in a time-dependent manner by TUNEL assay.The protective effect of HPC was also confirmed by MTT,which was in agreement with the results of the TUNEL analysis.The mechanism underlying the protective effect of HPC appeared to involve the stabilization of mitochondria,therefore reducingΔΨm loss.HPC also increased anti-apoptotic protein Bcl-2 expression.At the same time,this study demonstrated that the phosphorylation of Akt and ERK1/2 markedly decreased in the H/R group.The downregulating effect of H/R on the levels of phospho-ERK1/2 and phospho-Akt was fully reversed by HPC.In our study,HPC also stimulated the phosphorylation of Akt to reduce the apoptosis of MSCs.VEGF was dramatically increased by H/R.The VEGF expression in the HPC groups was greater than the H/R group,which revealed that VEGF was involved in anti-apoptosis effect of HPC on MSC.In contrast,we found that CsA had no effect on VEGF expression.By contrast, although CsA could reduce MSC apoptosis,stabilize mitochondrial potential,and increase Bcl-2 expression in mitochondria,0.5 mol/L CsA did not promote ERK and Akt phosphorylation.This may be the difference in the protective effects between HPC and CsA.In summary,the present investigation demonstrates that apoptosis induced by H/R insult was partially attenuated by HPC.Apoptosis suppression by HPC correlated with the prevention of mitochondrial dysfunction and promotion of ERK and Akt phosphorylation and upregulation of Bcl-2 and VEGF expression in H/R-induced apoptosis signaling.This evidence is the first using HPC for antiapoptosis of MSCs to promote cell transplantation efficiency.The results of our study demonstrate CsA inhibits H/R-induced apoptosis in MSCs in a dose-dependent manner by TUNEL assay.The protective effect of CsA was also confirmed by MTT.MTT data were in accordance with the TUNEL analysis,showing that mitochondrial viability was significantly decreased when the MSCs were insulted by H/R and CsA could preserve it.The mechanism underlying the protective effect of CsA appeared to involve stabilization of mitochondria,therefore reducedΔΨm loss and mitochondrial cytochrome C release.CsA also increased anti-apoptotic protein Bcl-2.As H/R also induced Bcl-2 expression,increased Bcl-2 by CsA does not seem to be the main mechanism by which CsA prevented apoptosis.CsA prevented the H/R-induced decreased in Bad phosphorylation.This suggested that BAD dephosphorylation might be involved in anti-apoptosis of CsA in H/R- induced apoptosis of MSCs.In summary,the present investigation demonstrates that apoptosis induced by H/R was partially attenuated by CsA.Apoptosis suppression by CsA correlated with the prevention of mitochondrial dysfunction and translocation of cytochrome c.Meanwhile, CsA could promote bcl-2 and p-BAD expression in H/R-induced apoptosis signaling. CSA preincubation could be an effective MSC death-prevention strategy.