Myocardial Ischemia/Reperfusion Induced Cardiac Stem Cell Homing

PartⅠIschemia/Reperfusion induced cardiac stem cell homing to theinjured myocardium by stimulating stem cell factor expressionBackgroundIschemia/reperfusion (I/R) is a major cause of heart failure. Recently cardiac stem cells(CSCs) were proposed as the most appropriate cell type for heart disease therapy. However,it is still unclear whether I/R can stimulate the CSCs homing to the injured myocardium.The activation and migration of stem cells, as we know, were usually regulated by somechemoattractant factors, such as stem cell factor (SCF), the ligand of the c-kit receptor. Inthis study, we speculated that SCF/c-kit signaling pathway may be involved in themigration of CSCs, subsequently repairing the injured myocardium and contributing to theimprovement of cardiac function.ObjectiveIn this study, we utilized the I/R model to investigate the expression of SCF and thehoming of CSCs during I/R.MethodsMale Sprague-Dawley rats were subjected to a 30-min ischemia followed byreperfusion of different intervals. RT-PCR, western blotting and immunohistochemistrywere performed to detect SCF expression at mRNA and protein levels, respectively. Toassess the homing of CSCs in vivo, BrdU-labeled CSCs were injected into AV-groovebefore induction of ischemia and examined by immunofluorenscent staining in the injured myocardium after I/R. In simulated I/R (SI/R) in vitro, RT-PCR and ELISA was used todetect the expression of SCF in the cultured neonatal rat cardiomyocytes.ResultsSignificant up-regulation of SCF was occurred in the I/R group after 3 days ofreperfusion examined by RT-PCR, western blotting and immunohistochemistry respectively.From day 3 to day 6 after reperfusion, the accumulation of CSCs was significantly elevatedin the injured area, which was matched with the increased SCF expression during I/R. Theresults of cardiac function showed injection of CSCs into the AV-groove resulted in therecovery of cardiac function after I/R. Furthermore, the study in vitro revealed that SI/Rstimulated the SCF expression of cardiomyocytes.ConclusionOur results demonstrated that I/R induced CSCs homing to the injured myocardium bystimulating myocardial SCF expression and improved the cardiac function. PartⅡIschemia/Reperfusion induced stem cell factor expression viaactivation of NF-κB pathwayBackgroundNowadays, it remains entirely unknown about the signal transduction pathwaysresponsible for regulating the SCF expression during I/R. Da Silva's study firstly identifieda NF-κB responsive element located in the first intron of the SCF gene, which provided thepossibility of NF-κB involved in the regulation of SCF transcription. NF-κB is composed ofP65 and P50. Under normal conditions, NF-κB in the cytoplasm is maintained in aninactive form because of binding to the inhibitor proteins known as IκBα, which retainNF-κB dimers in the cytoplasm. Upon stimulation, IκBαis rapidly phosphorylated,leading to the ubiquitination and subsequent degradation of IκBα. The liberated NF-κBdimers then translocate to the nucleus, where they regulate the transcription oftarget genes. Accordingly, in this study, we wondered whether NF-κB activation can reallyplay a role in regulating SCF expression during I/R.ObjectiveIn this study, we utilized the I/R model to investigate the activation of NF-κB and theexpression of IκBαduring I/R, examining its role in SCF expression as well as CSCshoming.MethodsActivation of Nuclear factor-κB (NF-κB) was determined by electrophoretic mobilityshift assay (EMSA). Western blot was performed to detect the expression of IκBαandphosphor-IκBα. 30 min prior to the induction of ischemia and at the beginning ofreperfusion, rats were twice treated with the inhibitor of NF-κB, NAC (200 mg/kg) to detect its role in NF-κB activation, SCF expression and CSCs homing. In order to confirmthe causative role of NF-κB activation in the myocardial SCF expression induced by SI/R,the cardiomyocytes were transiently transfected with the NF-κB decoyoligodeoxynucleotide (ODN,1mM) or treated with NAC (10mM) before SI/R, and thenNF-κB activity as well as SCF expression were examined in the cells.ResultsThe results of EMSA showed NF-κB activation was evident at 30 min of reperfusionand reached its maximum at 2 h of reperfusion. The level of phospho-IκBαwassignificantly elevated in the injured myocardium after 15 min of reperfusion and the totalprotein levels of IκBαwere decreased examined by western blot. Pretreatment of rats withNAC not only suppressed NF-κB activation induced by I/R but also attenuated SCFexpression and CSCs homing. Furthermore, pretreatment with NAC led to the decreasedcardiac function compared with non-NAC treatment. The study in vitro revealed treatmentwith NF-κB decoy ODN or NAC markedly contributed to the blockage of the NF-κBactivation, and subsequently attenuated the SI/R-induced myocardial SCF expression.ConclusionOur data documented that I/R markedly induced the expression of SCF, in whichNF-κB activation was greatly involved.Combination the results of partⅠand partⅡ, in this study we reported that CSCs can berecruited to repair the injured myocardium by the increased endogenous myocardial SCFvia NF-κB activation during I/R.