The Study On The Anti-apoptotic Effect Of Hypoxic Preconditioning In Cardiac Progenitor Cells And DNMT1-related Mechanisms

Part oneIsolation, culture and purification of CPCs from adult mouse heart tissue in vitro Objective The aim of this study was to explore the effective methods for isolation,culture and purification of c-kit(+) cardiac progenitor cells (CPCs) from the adult mouse heart tissue.Methods The hearts were obtained from two months old adult C57BL/6 mice, minced and digested thrice with 0.25% trypsin and 0.1% collagenase ?. After digestion, the remained heart tissue fragments (named cardiac explants) were cultured in complete explants medium (CEM). After one to two weeks of growth,a layer of fibroblast-like cells was generated from the adherent cardiac explants,and over which small,round,phase-bright cells emerged. These cells were collected using mild enzymatic digestion.c-kit(+) were purified using magnetic-activated cell sorting (MACS). Then the cells were seeded on poly-D-lysine coated flasks in cardiosphere growing medium (CGM)?Cells were characterized using phase contrast microscopy evaluating morphology, and flow cytometry (FCM) to examine the expression of stem cell surface markers.Results After about two weeks in culture, a layer of fibroblast-like cells emerged from adherent plated adult cardiac explants and some small,round and phase-bright cells migrated. Inverted phase contrast microscope examinations showed that CPCs presented clone-like proliferation. The purity of sorted c-kit(+) CPCs were characterized by flow cytometric analysis. After MACS,80.17% ±5.32% were positive of c-kit(+) CPCs for c-kit expression,40.37% ± 4.61% were positive for Sca-1 expression,1.77% ± 0.08% were positive for CD34 expression,and 1.22% ± 0.21%were positive for CD45 expression.Conclusions An adequate amount of c-kit(+) CPCs could be obtained from adult mouse heart tissue using enzymatic digestion and purified with magnetic-activated cell sorting.Part twoAnti-apoptotic effects of hypoxia preconditioning on CPCsObjective The objective of the study was to investigate whether hypoxia preconditioning (HP) could inhibit apoptosis and improve survival of c-kit(+) CPCs,and the underlying mechanism.Methods c-kit(+) CPCs were cultured under normoxic (HO) or hypoxic (0.1 %O25%CO2 94.9%N2) conditions for 6 hours (H6) prior to oxygen-serum deprivation for 24 hours (24h). We measured the viability of c-kit(+) CPCs by flow cytometry and MTT assay. Myocardial infarction model mice received an intramyocardial injection of c-Kit(+) CPCs. Cardiac function was measured seven days post cell transplantation by transthoracic echocardiography. Hearts were used for Masson staining analysis 4 weeks after cell transplantation to observe infarct size. WB (Western blot) were performed to revealed protein levels of p-Akt.Results The apoptosis rate of CPCs in the H6+24h group (15.05±1.54%) was obviously reduced compared with the H0+24h group (28.8211.43%) (P<0.05).LY294002 (a specificPI3k inhibitor) almost completely inhibited the pro-survival effect of HP on CPCs in the H6+24h group (31.07±1.44%),whereas insulin-like growth factor-1 (IGF-1, a PI3K-specific agonist) decreased the CPC apoptosis rate in the H0+24h group (17.26±2.13%). The MTT assay indicated that an analogous trend was evident among the six groups. Echocardiographic results showed that the LVEF and LVFS were both significantly greater in hearts administrated with c-kit(+) CPCs than in the HO group and in the PBS group (P<0.05) . And the LVEDD and the LVESD were both less in the H6 group than in the HO group and in the PBS group(P<0.05 ) . Mice transplanted with hypoxia preconditioned c-kit(+) CPCs demonstrated greater cardiac function improvement than those treated with normoxic cultured CPCs. Similarly,infarct size was smaller in hearts from mice administered with hypoxia preconditioned c-kit(+) CPCs than the HO group (29.4%±2.13% VS 40.3%±4.28%, P<0.05). Infarct size in hearts from the H6 group and the HO group were both smaller than the PBS group ((49.2%±5.64%) (P<0.05). It was found that phosphorylated Akt (p-Akt) expression was significantly up-regulated in CPCs that underwent HP for 6 hours (H6) compared with CPCs cultured under normoxic conditions (HO) (P<0.01).Conclusions HP could inhibit apoptosis and improve CPCs survival, and the PI3K/Akt pathway may play a pivotal role in the cytoprotective effects of HP.Part threeHP of CPCs modulates the expression of DNMTs and p53 and the underlying mechanismsObjective The aim of the study is to determine how HP of CPCs influences the expression of DNMTs and p53, and further to investigate the underlying mechanism.Methods CPCs were cultured under normoxic or hypoxic (0.1 %O2 5%CO2 94.9%N2)conditions prior to oxygen-serum deprivation for 24 hours. WB and quantitative real-time PCR (qPCR) were performed to revealed protein and mRNA expression levels of DNMT1, DNMT3? and p53. After DNMT1 was knocked down via transfection with siRNA-DNMT1, and the catalytic activity of DNMTs was blocked by 5-aza-2'-deoxycytidine (5-azadc),p53 protein level was measured by WB. We investigated the p53 promoter region encompassing the 2000 bp upstream of the p53 transcription start site. Bisulphite sequencing analysis was performed to quantify the methylation status of the CpG islands. A reporter gene assay was performed using the pGL4.17-basic vector containing the p53 promoter from +157 to -2150 bp. The putative DNMT1 binding site, approximately 2k bp upstream of the p53 transcription start site, was mutated to construct a mutated promoter. Chromatin immunoprecipitation (ChIP) analysis was performed to further investigate whether DNMT1 could bind to the promoter region of p53 in hypoxia-preconditioned CPCs.Results WB and qPCR, respectively, revealed that DNMT1 and DNMT3P protein and mRNA expression levels were both significantly up-regulated in the H6+24h group compared with the H0+24h group (P<0.01). However, DNMT1 and DNMT3? protein and mRNA expression levels decreased sharply when LY294002 was added to the medium of the H6+24h group. DNMT3a protein expression was unaltered in all 6 groups (P>0.05).WB and qPCR confirmed that p53 mRNA and protein were highly expressed in the H0+24h group. However, p53 mRNA and protein expression was obviously reduced in the H6+24h group compared with the H0+24h group (P<0.01).siRNA-DNMT1-transfected CPCs in the H6+24h group exhibited increased p53 protein and mRNA expression levels compared with cells in the H6+24h group without transfection (P<0.05). When CPCs in the H6+24h group were incubated with 5-azadc, DNMT1 expression was slightly reduced without obvious significance compared with the H6+24h group (P>0.05), whereas p53 expression was significantly elevated (P<0.05). When CPCs in the H6+24h group were treated with both 5-azadc and siRNA-DNMTl, p53 protein and mRNA expression levels were completely restored and exhibited no significant difference compared with the H6+siDNMTl+24h group or the H6+5-azadc +24h group (P>0.05). We found the p53 promoter region encompassing the 2000 bp upstream of the p5 3 transcription start site and identified 3 candidate CpG islands in this region. Quantitative analysis revealed that the methylation levels in all 3 promoter regions were minimally altered in the H6+24h group compared with the H0+24h group (P>0.05). Dual-luciferase assay results showed that HP-treated samples in the p53 promoter group showed lower promoter activity than untreated samples (P<0.01) and that the degree of HP time dependency of promoter activity was consistent with the observed DNMT1 protein expression ratios. We also observed a significant change in promoter activities in the DNMT1-binding site mutant group compared with the p53 promoter group (P<0.01).ChIP analysis showed that DNA sequence fragments from the p53 promoter onto which DNMT1 was recruited in all groups were amplified by PCR using specific primers.Conclusions Hypoxic preconditioning represses p53 and up-regulates DNA methyltransferases via the PI3K/Akt pathway, and p53 could be modulated by DNMT1. DNMTI does not hypermethylate the p53 promoter in hypoxia-preconditioned CPCs. DNMT1 might directly repress the transcription of p53 by binding to its promoter locus as a transcription factor.