Singla Dinender K, McDonald Debbie E
Department of Medicine, Division of Cardiology, Cardiovascular Research Institute, University of Vermont, College of Medicine, Colchester, Vermont, USA.
Am J Physiol Heart Circ Physiol. 2007 Sep;293(3):H1590-5. doi: 10.1152/ajpheart.00431.2007. Epub 2007 Jun 1.
Our recent study (Singla DK, Hacker TA, Ma L, Douglas PS, Sullivan R, Lyons GE, Kamp TJ, J Mol Cell Cardiol 40: 195-200, 2006) suggests that transplanted embryonic stem (ES) cells subsequent to myocardial infarction differentiate into the major cell types in the heart and improve cardiac function. However, the extent of regeneration is relatively meager compared with the observed functional improvement. The mechanisms underlying their improved function are completely unknown. In this report, we provide evidence using a cell culture model system for novel mechanisms that involve the release of cytoprotective, anti-apoptotic factor(s) from ES cells and inhibit H(2)O(2)-induced apoptosis in the rat cardiomyocyte-derived cell line H9c2. Conditioned medium (CM) from growing mouse ES cells treated with and without H(2)O(2) was generated. Apoptosis was induced after exposure to H(2)O(2) in H9c2 cells for 2 h followed by replacement with fresh cell culture or ES cell-CM. After 24 h, H9c2 cells treated with both ES cell-CMs demonstrated significantly decreased apoptosis, as determined by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining, apoptotic ELISA, caspase-3 activity, and DNA ladder. Next, using Luminex technology, we examined the presence of antiapoptotic proteins cystatin c, osteopontin, and clusterin and anti-fibrotic, tissue inhibitor of metalloproteinase-1 (TIMP-1) in both ES cell-CMs. The levels of released factors were 2- to 170-fold higher than those in H9c2 cell-CM. Antiapoptotic effects of ES cell-CM were significantly inhibited with TIMP-1 antibody, suggesting that TIMP-1 is an important factor to inhibit apoptosis. Furthermore, we used CM from an TIMP-1-overexpressing cell line and demonstrated that H(2)O(2)-induced apoptosis in the H9c2 cells was significantly inhibited. These observations demonstrate that factors released from ES cells contain antiapoptotic factors and that the effects are mediated by TIMP-1. Moreover, these findings suggest that released factors might be useful for therapeutic applications in ischemic heart disease as well as for many other diseases.
我们最近的研究（Singla DK, Hacker TA, Ma L, Douglas PS, Sullivan R, Lyons GE, Kamp TJ, 《分子与细胞心脏病学杂志》40: 195 - 200, 2006）表明，心肌梗死后移植的胚胎干细胞（ES细胞）可分化为心脏中的主要细胞类型并改善心脏功能。然而，与观察到的功能改善相比，再生程度相对较低。其功能改善的潜在机制完全未知。在本报告中，我们使用细胞培养模型系统提供证据，证明了涉及ES细胞释放细胞保护、抗凋亡因子并抑制大鼠心肌细胞衍生细胞系H9c2中H₂O₂诱导的细胞凋亡的新机制。制备了来自经H₂O₂处理和未经处理的生长中的小鼠ES细胞的条件培养基（CM）。在H9c2细胞中用H₂O₂处理2小时后诱导细胞凋亡，然后用新鲜细胞培养基或ES细胞 - CM替换。24小时后，通过末端脱氧核苷酸转移酶dUTP介导的缺口末端标记染色、凋亡ELISA、半胱天冬酶 - 3活性和DNA梯带分析确定，用两种ES细胞 - CM处理的H9c2细胞显示凋亡显著减少。接下来，使用Luminex技术，我们检测了两种ES细胞 - CM中抗凋亡蛋白胱抑素c、骨桥蛋白和簇集蛋白以及抗纤维化的金属蛋白酶组织抑制剂 - 1（TIMP - 1）的存在情况。释放因子的水平比H9c2细胞 - CM中的高2至170倍。ES细胞 - CM的抗凋亡作用被TIMP - 1抗体显著抑制，表明TIMP - 1是抑制细胞凋亡的重要因子。此外，我们使用来自过表达TIMP - 1细胞系的CM，并证明H9c2细胞中H₂O₂诱导的细胞凋亡被显著抑制。这些观察结果表明，ES细胞释放的因子含有抗凋亡因子，且其作用由TIMP - 1介导。此外，这些发现表明，释放的因子可能在缺血性心脏病以及许多其他疾病的治疗应用中有用。
