Dai Ying, Xu Meifeng, Wang Yigang, Pasha Zeeshan, Li Tingyu, Ashraf Muhammad
Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, OH 45267, USA.
J Mol Cell Cardiol. 2007 Jun;42(6):1036-44. doi: 10.1016/j.yjmcc.2007.04.001. Epub 2007 Apr 6.
Hypoxia inducible factor-1alpha (HIF-1alpha) is a proangiogenic transcription factor stabilized and activated under hypoxia. It regulates the expression of numerous target genes, including vascular endothelial growth factor (VEGF) and other cytoprotective proteins. In this study, we hypothesized that bone marrow stem cells (BMSCs) secrete growth factors which protect cardiomyocytes via HIF-1alpha pathway. BMSCs were obtained from transgenic mice overexpressing green fluorescent protein (GFP). The study was carried out in vitro using co-culture of BMSCs with cardiomyocytes. LDH release, MTT uptake, DNA fragmentation and annexin-V positive cells were used as cell injury markers. The level of HIF-1alpha protein as well as its activated form and VEGF were measured by ELISA. The expression of HIF-1alpha and VEGF in BMSCs was analyzed by quantitative PCR and cellular localization was determined by immunohistochemistry. LDH release was increased and MTT uptake was decreased after exposure of cardiomyocytes to hypoxia for 30 h, which were prevented by co-culturing cardiomyocytes with BMSCs. Cardiomyocyte apoptosis induced by hypoxia and H(2)O(2) was also reduced by co-culture with BMSCs. VEGF release from BMSCs was significantly increased in parallel with high level of HIF-1alpha in BMSCs following anoxia or hypoxia in a time-dependent manner. Although no significant up-regulation could be seen in HIF-1alpha mRNA, HIF-1alpha protein and its activated form were markedly increased and translocated to the nucleus or peri-nuclear area. The increase and translocation of HIF-1alpha in BMSCs were completely blocked by 2-methoxyestradiol (2-ME2; 5 mumol), a HIF-1alpha inhibitor. Moreover, the protection of cardiomyocytes by BMSC and VEGF secretion was abolished by neutralizing HIF-1alpha antibody in a concentration dependent manner (200-3200 ng/ml). Bone marrow stem cells protect cardiomyocytes by up-regulation of VEGF via activating HIF-1alpha.
缺氧诱导因子-1α（HIF-1α）是一种在缺氧条件下稳定并激活的促血管生成转录因子。它调节众多靶基因的表达，包括血管内皮生长因子（VEGF）和其他细胞保护蛋白。在本研究中，我们假设骨髓干细胞（BMSC）分泌生长因子，通过HIF-1α途径保护心肌细胞。BMSC取自过表达绿色荧光蛋白（GFP）的转基因小鼠。该研究在体外通过BMSC与心肌细胞共培养进行。乳酸脱氢酶（LDH）释放、MTT摄取、DNA片段化和膜联蛋白-V阳性细胞用作细胞损伤标志物。通过酶联免疫吸附测定（ELISA）测量HIF-1α蛋白及其激活形式以及VEGF的水平。通过定量聚合酶链反应（PCR）分析BMSC中HIF-1α和VEGF的表达，并通过免疫组织化学确定细胞定位。心肌细胞在缺氧30小时后，LDH释放增加，MTT摄取减少，而与BMSC共培养可防止这种情况发生。与BMSC共培养也可减少缺氧和过氧化氢（H₂O₂）诱导的心肌细胞凋亡。缺氧或缺氧后，BMSC中VEGF的释放与BMSC中高水平的HIF-1α平行显著增加，且呈时间依赖性。虽然在HIF-1α信使核糖核酸（mRNA）中未见明显上调，但HIF-1α蛋白及其激活形式显著增加并转移至细胞核或核周区域。BMSC中HIF-1α的增加和转移被HIF-1α抑制剂2-甲氧基雌二醇（2-ME2；5微摩尔）完全阻断。此外，中和HIF-1α抗体以浓度依赖性方式（200 - 3200纳克/毫升）消除了BMSC对心肌细胞的保护作用以及VEGF的分泌。骨髓干细胞通过激活HIF-1α上调VEGF来保护心肌细胞。
