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: 10520043
• 负责人: Juan M Melero-Martin
• 金额: $ 57.55万
• 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/10520043
• 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; Ischemia; Lasers; Measures; Mediating; Mediator; Medicine; Methods; Microcirculatory Bed; Mitochondria; Mitochondrial DNA; Modeling; Mus; Myocardial Infarction; Myocardial Reperfusion Injury; Nanotubes; Natural regeneration; Nude Rats; Operative Surgical Procedures; Organ; PINK1 gene; Parkin; Pathway interactions; Perfusion; Persons; Pharmaceutical Preparations; Pinocytosis; Process; Property; Publishing; Rattus; Recovery; Research; Role; Supporting Cell; TBK1 gene; Technology; Testing; Tissues; Transplantation; United States; Vascularization; cell type; critical limb Ischemia; effective therapy; efficacy evaluation; experience; heteroplasmy; imager; implantation; improved; in vivo; insight; limb ischemia; mesenchymal stromal cell; novel strategies; oxidation; 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.

项目摘要/摘要 缺血性疾病，包括严重的肢体缺血和心肌梗死，困扰着世界上数百万人 美国。目前，这些疾病的治疗主要是通过手术干预。然而， 在缺血组织中再生微血管床的能力仍然是一个挑战。或者，也可以使用 基于血管内皮细胞移植的治疗方法的开发仍然是血管领域的优先事项 医药。不幸的是，移植ECs并非易事。研究一再表明，为了实现 大量植入导致功能正常的新血管，内皮细胞需要在支持下共同移植 间充质基质细胞(MSCs)等细胞。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。此前，我们已经发表了大量关于人类各个方面的文章 EC+MSC移植。然而，MSCs促进EC植入的潜在机制仍然存在 不完全理解。最近，我们发现骨髓间充质干细胞在植入后，通过 当这种转移在基因上被废除时，EC的植入就大大减少了 受伤了。基于这一认识，我们提出了一个新的概念：人工将线粒体移植到人类体内 ECS作为一种先发制人的手段，增强了他们在没有次级细胞类型的情况下移植的能力。的确，我们的 初步数据显示，将外源性线粒体移植到内皮细胞内可产生细胞(称为mitoT-ECs)。 能够在体内缺血组织中形成功能性血管，而不需要MSCs的支持。我们还发现 移植的线粒体与LC3B标记的自噬小体共定位，以及基因消融 PINK1和Parkin(都是丝裂原吞噬作用的中心角色)消除了MITOT-ECs增强的植入能力。 总而言之，我们的主要假设是，将外源性线粒体移植到内皮细胞中会产生短暂的 通过有丝分裂来保护细胞；这反过来又增强了细胞的植入能力。为了检验这一假说 为了确定有丝分裂T-ECs治疗缺血性疾病的疗效，我们提出了两个具体的目标。在AIM-1中， 我们将确定在免疫缺陷小鼠体内最佳EC植入的条件(例如，浓度和时间)。 我们将剖析有丝分裂的作用，并将检查移植的线粒体的命运和持久性。 我们还将确定具有促进丝裂原吞噬作用的选择性药物是否也可以增强EC 嫁接。在AIM-2中，我们将确定线粒体移植使能EC治疗的疗效在两个 已建立的缺血性疾病模型：严重的后肢缺血(小鼠)和心肌 缺血/再灌注损伤(大鼠)。综上所述，我们建议开展研究以开发一种新的嫁接方法 ECS更成功。我们预见这项研究将成为血管细胞新策略的基础。 治疗。