Exosome-mediated cardioprotection and regeneration

描述（由申请人提供）：心球源性细胞（CDCs）在心肌梗死（MI）后减少疤痕大小和再生存活心肌方面有效，并在心肌梗死动物模型中发挥急性心脏保护作用。CDCs的主要益处似乎是通过间接机制介导的。在这里，我们测试了cdc的间接影响是由外泌体介导的这一总体假设。外泌体由多种细胞类型分泌，是30-100 nm的脂质双分子层囊泡，富含rna，包括microRNAs （miRs）。我们的初步数据表明，在小鼠急性心肌梗死模型中，人类cdc分泌的外泌体复制了cdc的治疗益处，并且对于cdc的治疗益处是不可或缺的。将CDC外泌体注射到损伤心脏中，可以模拟CDC移植在结构和功能上的益处；相反，cdc对外泌体分泌的抑制抵消了移植cdc的治疗益处。并不是所有的外泌体都是有益的：注射来自真皮成纤维细胞的外泌体，对照细胞不能改善心肌梗死后的预后，没有治疗效果。cdc -外泌体降低心肌细胞急性死亡和炎症细胞因子释放，同时减轻心肌梗死后左室重构和纤维化。来自microRNA阵列的初步数据揭示了在CDC-外泌体中高度上调的几种“签名miRs”。相比之下，质谱分析表明，cdc -外泌体的蛋白质组成是常规的，与成纤维细胞外泌体相当。因此，我们假设：i) cdc -外泌体含有一种独特的miRs补体，它们共同介导许多或全部cdc的治疗效果；ii) cdc -外泌体及其组成的mir有利于调节心肌梗死后心脏的细胞凋亡、炎症和纤维化；iii) cdc -外泌体改善心肌梗死后的功能恢复并增加组织活力；iv) cdc -外泌体代表了一种可行的“无细胞”心脏修复治疗候选体。在这里，我们建议对cdc -外泌体的全部RNA含量进行测序，以确定cdc -外泌体治疗益处的分子基础，并优化体内递送策略。我们的最终目标是开发cdc外泌体作为治疗心肌梗死和心衰的生物制品。由于外泌体与活细胞不同，活细胞可以主动向靶细胞迁移，因此我们也寻求测试新的递送方法，以选择性地将cdc -外泌体靶向损伤的肌细胞。该提案既是假设驱动的，也是以产品为导向的。我们寻求详细回答机制和翻译问题，依靠最先进的科学方法和完善的临床前疾病模型。cdc（自体和异体）已经在人体试验中，但细胞作为治疗药物有其局限性。因此，我们的建议，专注于cdc外泌体（作为cdc的无细胞衍生物），具有潜在的重要翻译价值。该结果也将为急性心肌梗死后细胞介导的心脏保护和心脏再生的基本机制开辟新的见解。

DESCRIPTION (provided by applicant): Cardiosphere-derived cells (CDCs) are effective in reducing scar size and regenerating viable myocardium in patients after myocardial infarction (MI), and exert acute cardioprotective effects in animal models of MI. The major benefits of CDCs appear to be mediated by indirect mechanisms. Here we test the overarching hypothesis that the indirect effects of CDCs are mediated by exosomes. Secreted by a wide range of cell types, exosomes are 30-100 nm lipid bilayer vesicles that are enriched in RNAs including microRNAs (miRs). Our preliminary data indicate that exosomes secreted by human CDCs reproduce the therapeutic benefits of CDCs, and are indispensable for the therapeutic benefits of CDCs, in a mouse acute MI model. Injection of CDC-exosomes into the injured heart mimics the structural and functional benefits of CDC transplantation; conversely, inhibition of exosome secretion by CDCs abrogates the therapeutic benefits of transplanted CDCs. Not all exosomes are salutary: Injection of exosomes from dermal fibroblasts, control cells which do not improve post-MI outcomes, had no therapeutic benefit. CDC-exosomes decreased acute cardiomyocyte death and inflammatory cytokine release, while attenuating LV remodeling and fibrosis in the post-MI heart. Preliminary data from microRNA arrays reveal several "signature miRs" that are highly up-regulated in CDC- exosomes. In contrast, mass spectrometry indicates that the protein composition of CDC-exosomes is conventional and comparable to that of fibroblast-exosomes. Thus, we hypothesize that: i) CDC-exosomes contain a unique complement of miRs that, collectively, mediate many, or all, of the therapeutic effects of CDCs; ii) CDC-exosomes and their constituent miRs favorably modulate apoptosis, inflammation and fibrosis in the post-MI heart; iii) CDC-exosomes improve functional recovery and increase tissue viability post-MI; iv) CDC-exosomes represent a viable "cell free" therapeutic candidate for cardiac repair. Here we propose to sequence the full RNA content of CDC-exosomes, to determine the molecular basis underlying the therapeutic benefit of CDC-exosomes, and to optimize delivery strategies in vivo. Our ultimate goal is to develop CDC-exosomes as a biologic product to treat MI and HF. Because exosomes are unlike live cells that can migrate actively towards a target, we also seek to test novel delivery methods to target CDC-exosomes selectively to injured myocytes. The proposal is both hypothesis-driven and product-oriented. We seek to answer both mechanistic and translational questions in detail, relying upon state-of-the-art scientific methods and well-established preclinical disease models. CDCs (both autologous and allogeneic) are already in human trials, but cells have their limitations as therapeutic agents. Thus, our proposal, focusing on CDC-exosomes (as cell-free derivatives of CDCs), is of potentially significant translational value. The results will also open up novel insights into the fundamental mechanisms of cell-mediated cardioprotection and cardiac regeneration after acute MI.