Genetically Altered Mesenchymal Stem Cell; Paracrine Effect on Neovascularization

描述(申请人提供)：我们先前已经证明，当心肌梗死(MI)后将间充质干细胞(MSC)注射到啮齿动物心脏中时，可以增强心肌修复和恢复心功能。此外，通过转导带有细胞保护基因Akt的MSC(Akt-MSC)，这种保护作用得到了增强。此外，我们的数据显示，这些细胞表达旁分泌介质，从而减少心肌损伤。在这一应用中，我们假设Akt-MSC增强心肌修复，部分是通过其对血管生成的旁分泌作用。事实上，我们的实验室已经证明，Akt-MSCs表达多种血管生成细胞因子，如血管内皮生长因子和成纤维细胞生长因子。此外，从这些细胞收集的培养液在体外诱导内皮细胞迁移和管状形成。重要的是，我们最近证明了Akt-MSC可以在梗塞的心脏中诱导新生血管。由于已经证明血管生成信号动员了定位于缺血心肌的骨髓来源的内皮祖细胞(EPC)，我们假设(I)Akt-MSC通过旁分泌机制增加梗死后心脏的血管生成，并有助于改善心肌修复和功能(Ii)Akt-MSC部分通过募集骨髓来源的EPC增加血管生成，(Iii)Akt-MSC介导的EPC募集受HIF 11控制。为了验证这些假设，我们将研究从这些细胞收集的Akt-MSCs或从这些细胞收集的培养液在体外刺激EPC迁移和管状形成的能力，以及在体内促进梗死后心脏新生血管的能力。我们还将研究潜在的Akt-MSC衍生因子对这些过程的贡献。接下来，我们将使用基因标记的供体细胞在辐射/骨髓移植模型中研究骨髓前体细胞在Akt-MSC诱导的缺血心肌新生血管中的作用。此外，我们将使用“自杀”基因传递方法选择性地清除可能在Akt-MSC介导的新生血管中发挥作用的骨髓前体细胞。最后，我们将探讨缺氧和Akt调节的转录因子HIF11在调节Akt-MSC诱导的骨髓细胞向缺血心肌募集的分子开关中的作用。这些研究应该有助于阐明Akt-MSCs对梗塞心肌提供如此戏剧性和长期保护的机制，并可能确定潜在的治疗方法。公共卫生相关性成人骨髓干细胞对受损心脏的保护作用已被证明，然而，确切的机制尚不清楚。在这项研究中，我们将研究这些骨髓细胞在梗死心脏新血管发育中的作用。心脏新血管的增强将极大地帮助治疗冠状动脉疾病和心力衰竭。

DESCRIPTION (provided by applicant): We have previously demonstrated that mesenchymal stem cells (MSC), when injected into rodent hearts following myocardial infarction (MI), enhance myocardial repair and restore cardiac function. Moreover, this protective effect is enhanced by transduction of the MSC with the cytoprotective gene Akt (Akt-MSC). Furthermore, our data showed that these cells express paracrine mediators that reduce myocardial injury. In this application we hypothesize that Akt-MSC enhance myocardial repair, in part, through its paracrine effects on angiogenesis. Indeed we have shown in our laboratory that Akt-MSCs express multiple angiogenic cytokines such as VEGF and FGF. Moreover, media collected from these cells induce endothelial cell migration and tube formation in vitro. Importantly, we have recently demonstrated that Akt-MSC induces neovascularization in the infarcted heart. Since it has been shown that angiogenic signals mobilize bone marrow derived endothelial progenitor cells (EPC) that home to the ischemic myocardium, we hypothesize that (i) Akt-MSC increase angiogenesis in the post infarct heart through paracrine mechanisms and contribute to improved myocardial repair and function (ii) Akt-MSC increase angiogenesis in part through the recruitment of bone marrow derived EPCs and (iii) Akt-MSC mediated EPC recruitment is controlled by HIF 11. To test these hypotheses, we will investigate the capability of Akt-MSCs or media collected from these cells to stimulate EPC migration and tube formation in vitro and the ability to enhance neovascularization in the post infarcted heart in vivo. We will also examine the contribution of the potential Akt-MSC derived factors towards these processes. Next we will study the role of bone marrow progenitor cells in Akt-MSC induced neovascularization in ischemic myocardium in irradiated/bone marrow transplantation models using genetically marked donor cells. Moreover we will use a "suicide" gene delivery approache to selectively eliminate the bone marrow progenitor cells which may play a role in Akt-MSC mediated neovascularization. Finally, we will investigate the role of hypoxia and Akt regulated transcription factor HIF11 as a molecular switch which may regulate Akt-MSC induced bone marrow cell recruitment to the ischemic myocardium. These studies should help elucidate the mechanism by which Akt-MSCs provide such dramatic and long term protection of the infarcted myocardium and may identify potential therapeutic approaches. PUBLIC HEALTH RELEVANCE The protective effects of adult bone marrow stem cells in the injured heart have been demonstrated, however, the exact mechanism is unknown. In this study, we will examine the effects of these bone marrow cells in the development of new blood vessels in the infarcted heart. Enhancement of new vessels in the heart will greatly aid in the treatment of coronary vessel disease and heart failure.