Peng Chengfei, Pei Haifeng, Wei Feipeng, Tian Xiaoxiang, Deng Jie, Yan Chenghui, Li Yang, Sun Mingyu, Zhang Jian, Liu Dan, Rong Jingjing, Wang Jie, Gao Erhe, Li Shaohua, Han Yaling
Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China; Cardiovascular Research Institute, Department of Cardiology, General Hospital of Shenyang Military Region, Shenyang 110016, China.
Department of Cardiology, Chengdu Military General Hospital, Chengdu 610083, China.
Int J Cardiol. 2015 Mar 15;183:232-41. doi: 10.1016/j.ijcard.2015.01.059. Epub 2015 Jan 27.
Bone mesenchymal stem cell (BMSC) therapy has modest success in ischemic heart disease but has been limited by poor survival in diseased microenvironments. Cellular repressor of E1A-stimulated genes (CREG) can prevent BMSCs from apoptosis in vitro; however, the effects of CREG-modified BMSCs on ischemic heart disease and the related mechanism remain undefined. Therefore, we designed to study the cardioprotective effects of CREG overexpression in BMSCs ((CREG)BMSCs) after transplantation into infarcted heart of rats.
In vivo studies, 50 μl PBS or 1.5×10(6)(Norm)BMSCs, (GFP)BMSCs or (CREG)BMSCs were implanted intramyocardially in myocardial infarction rat models. 3 or 14 days later, cardiac function, fibrosis, apoptosis and angiogenesis were analyzed by echocardiography, masson, western blot and immunofluorescence staining, respectively. ELISA, western blot and matrigel assay were used in vitro to detect vascular endothelial growth factor (VEGF) secretion, signaling molecule expression, and angiogenic tube formation.
In vivo, prolonged cardiac function (14d LVEF: 50.87 ± 0.94%; LVFS: 23.41 ± 1.12%), decreased fibrosis (14d Fibrotic area: 27.37 ± 1.03%) and apoptosis and increased angiogenesis were observed in (CREG)BMSCs, compared with other groups. In vivo and in vitro, VEGF secretion from (CREG)BMSCs was markedly enhanced. In vitro, angiogenic tube formation in (CREG)BMSC supernatants significantly increased. Moreover, CREG activated hypoxia-inducible factor-1α (HIF-1α), but not HIF-1β. Knockdown of HIF-1α with siRNA decreased VEGF secretion and angiogenic tube formation. Notably, CREG did not influence HIF-1α mRNA synthesis but inhibited the expression of Von Hippel-Lindau (VHL), a key protein that regulates HIF-1α degradation.
The (CREG)BMSC transplantation, directly or indirectly, may promote VEGF's anti-apoptosis and angiogenesis via the inhibition of VHL-mediated HIF-1α degradation, consequently protecting against myocardial infarction.
骨间充质干细胞（BMSC）治疗在缺血性心脏病中取得了一定成效，但在病变微环境中的低存活率限制了其应用。E1A刺激基因的细胞抑制因子（CREG）可在体外防止BMSC凋亡；然而，CREG修饰的BMSC对缺血性心脏病的影响及其相关机制仍不明确。因此，我们旨在研究将过表达CREG的BMSC（(CREG)BMSC）移植到大鼠梗死心脏后对心脏的保护作用。
在体内研究中，将50 μl PBS或1.5×10⁶个正常BMSC、绿色荧光蛋白标记的BMSC（(GFP)BMSC）或过表达CREG的BMSC（(CREG)BMSC）心肌内注射到心肌梗死大鼠模型中。3天或14天后，分别通过超声心动图、Masson染色、蛋白质免疫印迹和免疫荧光染色分析心脏功能、纤维化、细胞凋亡和血管生成情况。在体外，采用酶联免疫吸附测定（ELISA）、蛋白质免疫印迹和基质胶实验检测血管内皮生长因子（VEGF）分泌、信号分子表达及血管生成管形成情况。
在体内，与其他组相比，(CREG)BMSC组心脏功能得到改善（14天左室射血分数：50.87±0.94%；左室短轴缩短率：23.41±1.12%），纤维化程度降低（14天纤维化面积：27.37±1.03%），细胞凋亡减少，血管生成增加。在体内和体外，(CREG)BMSC分泌的VEGF均显著增加。在体外，(CREG)BMSC培养上清液中的血管生成管形成明显增加。此外，CREG激活缺氧诱导因子-1α（HIF-1α），但不激活HIF-1β。用小干扰RNA（siRNA）敲低HIF-1α可降低VEGF分泌和血管生成管形成。值得注意的是，CREG不影响HIF-1α mRNA合成，但抑制了调节HIF-1α降解的关键蛋白冯·希佩尔-林道蛋白（VHL）的表达。
(CREG)BMSC移植可能直接或间接通过抑制VHL介导的HIF-1α降解来促进VEGF的抗凋亡和血管生成作用，从而对心肌梗死起到保护作用。
