Suicide Gene Reveals the Myocardial Neovascularization Role of Mesenchymal Stem Cells Overexpressing CXCR4 (MSCCXCR4)

BackgroundOur previous studies indicated that MSCCXCR4 improved cardiac function after myocardial infarction (MI). This study was aimed to investigate the specific role of MSCCXCR4 in neovascularization of infarcted myocardium using a suicide gene approach.MethodsMSCs were transduced with either lentivirus-null vector/GFP (MSCNull as control) or vector encoding for overexpressing CXCR4/GFP. The MSC derived-endothelial cell (EC) differentiation was assessed by a tube formation assay, Dil-ac-LDL uptake, EC marker expression, and VE-cadherin promoter activity assay. Gene expression was analyzed by quantitative RT-PCR or Western blot. The suicide gene approach was under the control of VE-cadherin promoter. In vivo studies: Cell patches containing MSCNull or MSCCXCR4 were transduced with suicide gene and implanted into the myocardium of MI rat. Rats received either ganciclovir (GCV) or vehicle after cell implantation. After one month, the cardiac functional changes and neovascularization were assessed by echocardiography, histological analysis, and micro-CT imaging.ResultsThe expression of VEGF-A and HIF-1α was significantly higher in MSCCXCR4 as compared to MSCNull under hypoxia. Additionally, MSCCXCR4 enhanced new vessel formation and EC differentiation, as well as STAT3 phosphorylation under hypoxia. STAT3 participated in the transcription of VE-cadherin in MSCCXCR4 under hypoxia, which was inhibited by WP1066 (a STAT3 inhibitor). In addition, GCV specifically induced death of ECs with suicide gene activation. In vivo studies: MSCCXCR4 implantation promoted cardiac functional restoration, reduced infarct size, improved cardiac remodeling, and enhanced neovascularization in ischemic heart tissue. New vessels derived from MSCCXCR4 were observed at the injured heart margins and communicated with native coronary arteries. However, the derived vessel networks were reduced by GCV, reversing improvement of cardiac function.ConclusionThe transplanted MSCCXCR4 enhanced neovascularization after MI by boosting release of angiogenic factors and increasing the potential of endothelial differentiation.

背景 我们既往的研究表明，MSCCXCR4可改善心肌梗死（myocardial infarction, MI）后的心脏功能。本研究旨在利用自杀基因策略，探究MSCCXCR4在梗死心肌血管新生中的具体作用。
方法 将间充质干细胞（mesenchymal stem cells, MSCs）分别转导至慢病毒空载体/绿色荧光蛋白（green fluorescent protein, GFP，作为对照，记为MSCNull），或转导至过表达CXCR4/GFP的载体。通过管形成实验、Dil标记的乙酰化低密度脂蛋白（Dil-ac-LDL）摄取实验、内皮细胞（endothelial cell, EC）标志物表达检测以及血管内皮钙粘蛋白（VE-cadherin）启动子活性实验，评估间充质干细胞来源的内皮细胞分化情况。采用定量实时聚合酶链反应（quantitative RT-PCR）与蛋白质印迹（Western blot）分析基因表达水平。本研究中的自杀基因策略受VE-cadherin启动子调控。体内实验：将转导了自杀基因的MSCNull或MSCCXCR4细胞补丁植入心肌梗死大鼠的心肌组织中，大鼠于细胞植入后分别给予更昔洛韦（ganciclovir, GCV）或赋形剂处理。于术后1个月，通过超声心动图、组织学分析以及微型计算机断层扫描（micro-CT）成像评估心脏功能变化与血管新生情况。
结果 缺氧条件下，MSCCXCR4组的血管内皮生长因子A（vascular endothelial growth factor A, VEGF-A）与缺氧诱导因子1α（hypoxia-inducible factor 1α, HIF-1α）表达水平显著高于MSCNull组。此外，缺氧条件下MSCCXCR4可促进新生血管形成与内皮细胞分化，并增强信号转导与转录激活因子3（signal transducer and activator of transcription 3, STAT3）的磷酸化水平。缺氧条件下，STAT3参与调控MSCCXCR4中VE-cadherin的转录，而该过程可被STAT3抑制剂WP1066抑制。此外，GCV可特异性诱导携带激活自杀基因的内皮细胞死亡。体内实验结果显示：MSCCXCR4移植可促进心脏功能恢复、缩小梗死面积、改善心脏重构，并增强缺血心肌组织的血管新生。在受损心脏边缘可观察到源自MSCCXCR4的新生血管，且这些血管可与本土冠状动脉形成连通。然而，GCV处理可减少此类衍生血管网络，逆转心脏功能的改善效果。
结论 移植的MSCCXCR4可通过促进血管生成因子的释放以及增强内皮分化潜能，改善心肌梗死后的血管新生。