Smarter exosomes derived from engineered MSCs promote neo-vascularization

项目总结/摘要 间充质干细胞（MSC）治疗已经显示出巨大的前景，使心脏组织修复后， 缺血性损伤干细胞的治疗作用是由旁分泌因子介导的。一些研究表明， 来源于干细胞的外泌体（exosomes，EXO）在干细胞介导的缺血性脑血管病治疗中起着关键作用， 心肌通过增加血管生成。从翻译的角度来看，EXO有更大的好处 治疗效果比全细胞：EXO具有更少的潜在不良反应，更少的免疫排斥反应， EXO货物可以通过预处理或遗传操作进行修饰。加塔- 4，心脏转录因子，促进心脏形态发生，延长心肌细胞（CM）存活， 通过调节各种生物活性分子和激活心脏保护性miRs来保护心脏功能。 我们发表的数据表明，MSC过表达加塔-4（MSC加塔-4）增加MSC存活，保护天然细胞， CM和促进血管生成，相比，载体转染MSC（MSCnull）。EXO派生EXO派生 来自过表达加塔-4的加塔-4（Exo加塔-4）比来自载体转染的MSC的EXO更有效 （Exonull）在保护CM免受缺血性损伤中的作用。在我们的初步研究中，ExoGATA-4富含促血管生成蛋白， miR，其调节血管形成。ExoGATA-4下调血小板反应蛋白1的表达，这是一个众所周知的 新生血管形成的内源性抑制剂。该项目的总体目标是利用 与普通EXO相比，加塔-4使EXO具有Exo加塔-4的可修复效果。我们的核心假设是 ExoGATA-4富含转移到受体细胞中的促血管生成miR和蛋白质， 激活多种信号通路，导致血管生成和心脏修复。三个具体目标是 目的1，确定EXO携带的miR在应答加塔-4转染中的作用。目标2， 证明转移的生物活性分子是否在ExoGATA-4介导的血管生成中起关键作用。目的 3、为了验证来源于过表达加塔-4的MSC的EXO比来源于过表达GATA-4的MSC的EXO更有效的假设， 载体转染的MSCs促进血管生成和心肌再生。拟议的研究是 创新，因为以前没有研究系统地检查了ExoGATA-4的有效性， 特别关注EXO介导的血管生成和缺血性心脏修复的功效， 局静脉内注射提供了能够重复治疗而没有压力的优点。 反复心肌内注射。这项拟议的研究非常重要，因为它将评估 细胞治疗的有效性可以通过无细胞EXO重现的概念，并将探索这种新的 通过静脉内给药的未来临床应用模式。这个项目将是第一个系统的努力， 目的是开发一种全新的策略，使用EXO给药代替细胞， 彻底改变细胞疗法其结果将提供一个变革性的验证，并指导翻译研究， 基于来自基因组编辑的MSC的EXO的增强的血管生成特性的推定疗法。 1

Project Summary/Abstract Mesenchymal stem cell (MSC) therapy has shown tremendous promise for enabling heart tissue repair after ischemic injury. The therapeutic effects of stem cells are mediated by paracrine factors. Several studies illustrate that exosomes (EXO) derived from stem cells play a critical role in stem cell mediated therapy of ischemic myocardium via increasing angiogenesis. From the translational perspective, EXO have greater salutary therapeutic effects than whole cells: EXO have fewer potential adverse effects, less immune rejection, are more amenable to manipulation, and EXO cargo can be modified by preconditioning or genetic manipulation. GATA- 4, a cardiac transcription factor, promotes cardiac morphogenesis, extends cardiomyocyte (CM) survival and preserves cardiac function via regulating various bioactive molecules and activating cardiac protective miRs. Our published data indicate that MSC overexpressing GATA-4 (MSCGATA-4) increase MSC survival, protect native CM and promote angiogenesis, compared to vector-transfect MSC (MSCnull). EXO derived from EXO derived from GATA-4 overexpressing GATA-4 (ExoGATA-4) are more efficient than EXO from vector-transfected MSC (Exonull) in protecting CM from ischemic injury. In our pilot study, ExoGATA-4 are enriched with pro-angiogenic miRs, which regulate vascularization. ExoGATA-4 downregulate the expression of thrombospondin 1, a well-known endogenous inhibitor of neovascularization. The overarching goal of this project is to exploit effectiveness of GATA-4 conferred EXO than the ordinary EXO in repairable effects of ExoGATA-4. Our central hypothesis is that the ExoGATA-4 are enriched with pro-angiogenic miRs and proteins that are transferred into recipient cells and active multiple signaling pathways, leading to angiogenesis and cardiac repair. Three Specific Aims are proposed: Aim 1, to determine the role of miRs carried by EXO in response to GATA-4 transfection. Aim 2, to demonstrate if the transferred bioactive molecules play a critical role in ExoGATA-4 mediated angiogenesis. Aim 3, to test the hypothesis that EXO derived from GATA-4 overexpressing MSCs are more effective than that from vector-transfected MSCs in promoting angiogenesis and myocardial regeneration. The proposed studies are innovative because no previous study has systematically examined the effectiveness of ExoGATA-4, with particular focus on the efficacy of EXO mediated angiogenesis and ischemic heart repair following systemic administration. Intravenous injection offers the advantage of enabling repeated treatments without the stress of repeated intramyocardial injections. The proposed study is highly significant because it will evaluate the concept that the effectiveness of cellular therapy can be reproduced by cell-free EXO and will explore this new modality for future clinical application by intravenous administration. This project will be the first systematic effort aimed at developing a completely new strategy in use of EXO administration in lieu of cells, which would revolutionize cell therapy. The outcome will offer a transformative validation and guide translational research and putative therapies based on enhanced angiogenic properties of EXO from genome-edited MSCs. 1