Enhancing endothelial cell engraftment via transplantation of exogenous mitochondria

通过外源线粒体移植增强内皮细胞植入

• 批准号: 10320796
• 负责人: Juan M Melero-Martin
• 金额: $ 53.34万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-20 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10320796
• 关键词: Ablation; Affect; Aftercare; Apoptosis; Autophagosome; BCL2/Adenovirus E1B 19kd Interacting Protein 3-Like; Biogenesis; Blood Vessels; Blood flow; Cell Line; Cell Therapy; Cells; Centrifugation; Clinical Trials; Complement; Cytolysis; Cytoprotection; Data; Disease; Echocardiography; Endocytosis; Endothelial Cells; Endothelium; Engraftment; Evaluation; Fibrosis; Genetic; Goals; Human; Immunodeficient Mouse; Impairment; Infarction; Lasers; Measures; Mediating; Mediator of activation protein; Medicine; Methods; Microcirculatory Bed; Mitochondria; Mitochondrial DNA; Modeling; Mus; Myocardial Infarction; Myocardial Ischemia; Nanotubes; Natural regeneration; Nude Rats; Operative Surgical Procedures; Organ; PINK1 gene; Parkin; Pathway interactions; Persons; Pharmaceutical Preparations; Process; Property; Publishing; Rattus; Recovery; Reperfusion Injury; Research; Role; Supporting Cell; TBK1 gene; Technology; Testing; Tissues; Transplantation; United States; base; cell type; critical limb Ischemia; effective therapy; experience; heteroplasmy; imager; implantation; improved; in vivo; insight; limb ischemia; mesenchymal stromal cell; novel strategies; pressure; therapy development

PROJECT SUMMARY/ABSTRACT Ischemic diseases, including critical limb ischemia and myocardial infarction, afflict millions of people in the United States. Currently, these diseases are predominately treated by surgical interventions. However, the inability to regenerate microvascular beds in ischemic tissues remains a challenge. Alternatively, the development of therapies based on transplanting endothelial cells (ECs) continues to be a priority in vascular medicine. Unfortunately, engrafting ECs is not trivial. Studies have repeatedly shown that in order to achieve significant engraftment resulting in functional new blood vessels, ECs require co-transplantation with supporting cells such as mesenchymal stromal cells (MSCs). However, this paradigm is problematic because it increases the complexity of clinical trials exponentially. Previously, we have published extensively on all aspects of human EC+MSC engraftment. However, the underlying mechanisms by which MSCs facilitate EC engraftment remain incompletely understood. Recently, we found that upon implantation, MSCs transfer mitochondria to ECs via tunneling nanotubes and that when this transfer was genetically abrogated, EC engraftment was drastically impaired. Based on this insight, we propose a new concept: artificially transplanting mitochondria into human ECs as a means to preemptively enhance their ability to engraft without a secondary cell type. Indeed, our preliminary data show that transplanting exogenous mitochondria into ECs renders the cells (termed mitoT-ECs) capable of forming functional vessels in vivo in ischemic tissues, without the support of MSCs. We also found that transplanted mitochondria co-localized with LC3B-marked autophagosomes and that genetic ablation of PINK1 and Parkin (both central players in mitophagy) eliminated the enhanced engraftment ability of mitoT-ECs. Together, our overarching hypothesis is that transplanting exogenous mitochondria into ECs renders transient cytoprotection via mitophagy; this, in turn, enhances the engraftment ability of the cells. To test this hypothesis and to determine the efficacy of mitoT-ECs to treat ischemic diseases, we propose two specific aims. In Aim-1, we will determine conditions (e.g., concentration and timing) for optimal EC engraftment in immunodeficient mice. We will dissect the role of mitophagy and will examine the fate and persistence of the transplanted mitochondria. We will also determine if selective drugs with mitophagy-enhancing properties could also enhance EC engraftment. In Aim-2, we will determine the efficacy of mitochondrial transplantation-enabled EC therapy in two well-established models of ischemic diseases: critical hind limb ischemia (in mice) and myocardial ischemia/reperfusion injury (in rats). In summary, we propose studies to develop a novel approach to engraft ECs more successfully. We envision this research could become the basis for a new strategy in vascular cell therapies.

项目总结/摘要 缺血性疾病，包括严重肢体缺血和心肌梗死，困扰着数百万人 美国的目前，这些疾病主要通过外科手术治疗。但 不能再生缺血组织中的微血管床仍然是一个挑战。或者， 基于移植内皮细胞（EC）的疗法的开发仍然是血管内皮细胞移植中的优先事项。 药不幸的是，移植EC并不简单。研究一再表明，为了实现 显著的植入导致功能性新血管，EC需要与支持物共移植 细胞如间充质基质细胞（MSC）。然而，这种模式是有问题的，因为它增加了 临床试验的复杂性呈指数级增长。在此之前，我们已经广泛地发表了关于人类各个方面的文章， EC+MSC植入。然而，MSC促进EC植入的潜在机制仍然存在， 不完全理解。最近，我们发现，在植入后，MSC通过线粒体转移到EC， 隧道纳米管，当这种转移被遗传废除，EC植入急剧 受损基于这一认识，我们提出了一个新的概念：将线粒体人工移植到人体内 内皮细胞作为一种手段，先发制人地提高他们的能力，植入没有第二细胞类型。的确，我们的 初步数据显示，将外源性线粒体移植到EC中使细胞（称为mitoT-EC） 能够在缺血组织中体内形成功能性血管，而无需MSC的支持。我们还发现 移植的线粒体与LC 3B标记的自噬体共定位， PINK 1和Parkin（mitophagy的核心参与者）消除了mitoT-EC增强的植入能力。 总之，我们的总体假设是，将外源性线粒体移植到EC中， 通过线粒体自噬的细胞保护作用;这反过来又增强了细胞的植入能力。为了验证这一假设 为了确定mitoT-EC治疗缺血性疾病的功效，我们提出了两个具体目标。在Aim-1中， 我们将确定条件（例如，浓度和时间）以在免疫缺陷小鼠中实现最佳EC植入。 我们将剖析线粒体自噬的作用，并将检查移植的线粒体的命运和持久性。 我们还将确定是否选择性药物与线粒体自噬增强性能也可以提高EC 移植。在Aim-2中，我们将确定线粒体移植支持的EC治疗在两个患者中的疗效。 缺血性疾病的完善模型：严重后肢缺血（小鼠）和心肌缺血（小鼠） 缺血/再灌注损伤（大鼠）。总之，我们提出了研究开发一种新的方法， EC更成功。我们设想这项研究可以成为血管细胞新策略的基础。 治疗