Mesenchymal stem cell extracellular vesicles for ischemic retinal damage

视网膜中央动脉阻塞(CRAO)是一种眼科急症，很少得到证实。 治疗。基于干细胞的视网膜细胞移植是一种非常鼓舞人心的实现方法 视网膜神经保护和挽救视网膜疾病的视力。但是，它的限制包括 整合的细胞很少，免疫反应不良，生长异常，是一种替代无细胞的细胞 方法是必需的。EVS是一种纳米泡状体，当被靶细胞内吞时， 触发特定的响应。在这里，电动汽车的microRNA(MiRNA)货物起着关键作用。这 提案旨在使用具有特定功能的工程化MSC-EVS恢复视网膜功能 MiRNA。我们的研究表明，EVS可以挽救受到严重损害的视网膜细胞 缺氧或缺血是导致CRAO细胞死亡的关键机制。我们还发现， 骨髓间充质干细胞低氧预适应诱导细胞保护性增强的血管内皮细胞 包括抗细胞凋亡和抗炎作用。一些miRNAs在H-EV中过度表达 在视网膜细胞中具有细胞保护特性。 我们的中心假设是在MSC-EVS中针对EV特异性的关键miRNAs的表达 将使H-EVS的抗细胞凋亡和抗炎作用重新降温。我们指定这样的人 电动汽车作为功能工程电动汽车(收费)。为了促进MSC-EV疗法的临床翻译， 我们确定了关键的知识缺口：(1)肠道病毒miRNA与其抗细胞凋亡的关系 性质；(2)EV miRNA及其在视网膜MSC-EVS抗炎作用中的作用；以及(3) 能否通过改造MSC-EVS的miRNA货物来增强其靶向功能？目标1 将产生过度表达miR-424(费用-424)和146b(费用-146b)的费用。我们将评估 它们的作用机制，以及它们在视网膜神经节细胞产生费用的候选资格， 小胶质细胞、穆勒细胞和视网膜血管内皮细胞的功能丧失和功能获得研究 在体外模拟缺血。这些结果将作为开发的原则证明 用于改善视网膜细胞损伤的费用。在目标2中，含有miR-424和-146b的费用 将在一种啮齿动物模型中测试抗凋亡和炎症机制的特异性靶向 CRAO。拟议的研究有望提供变革性的结果，从而使MSC-EVS 用于治疗CRAO的缺血事件后用于视网膜保护作用的改良和交付。 《多样性补编》将培训研究生进行数据收集和分析， 批判性地回顾文献，设计实验，提交和撰写研究报告。 我们的目标是提高学生在执行翻译基本技能方面的能力 科学研究，并确保有更多的机会使科学工作者多样化。

Central retinal artery occlusion (CRAO) is an ophthalmological emergency with few proven therapies. Stem cell-based retinal cell replacement is a highly encouraging approach to achieve retinal neuroprotection and to save vision in retinal diseases. However, with limitations including few cells integrated, adverse immune responses, and aberrant growth, an alternative cell-free approach is required. EVs are nano-vesicular bodies that, when endocytosed by target cells, trigger specific responses. Here, the microRNA (miRNA) cargo of the EVs plays a key role. This proposal targets restoration of retinal function using engineered MSC-EVs with function-specific miRNA. Our studies indicate that EVs can rescue retinal cells that have been acutely subjected to hypoxia or ischemia, the key mechanism that starts cells dying in CRAO. We also found that hypoxic preconditioning of MSCs resulted in EVs (H-EVs) with enhanced cytoprotective properties including anti-apoptosis and anti-inflammation. A number of miRNAs overexpressed in the H-EVs have cytoprotective properties in retinal cells. Our central hypothesis is that targeted EV-specific expression of key miRNAs in MSC-EVs will re-capitulate the anti-apoptosis and anti-inflammatory actions of H-EVs. We designate such EVs as Functionally Engineered EVs (FEEs). To facilitate clinical translation of MSC-EV therapy, we identified key knowledge gaps: (1) The relationship between EV miRNA and its anti-apoptotic properties; (2) EV miRNA and its role in anti-inflammatory actions of MSC-EVs in retina; and (3) Can MSC-EVs be enhanced for targeted functionality by engineering their miRNA cargo? Aim 1 will produce FEEs overexpressing miR-424 (FEE-424) and 146b (FEE-146b). We will evaluate their mechanisms of action, and their candidacy for generation of FEEs in retinal ganglion cells, microglia, Muller cells, and retinal vascular endothelial cells using loss and gain of function studies in simulated ischemia in vitro. These results will serve as proof-of-principle for development of FEEs for amelioration of cell damage in the retina. In Aim 2, FEEs containing miR-424 and -146b will test specific targeting of anti-apoptotic and inflammatory mechanisms in a rodent model of CRAO. Proposed studies are expected to provide transformative results whereby MSC-EVs are modified and delivered for retinal protective action after the ischemic event to treat CRAO. The Supplement for Diversity will train a graduate student in data collection and analysis, reviewing literature critically, designing experiments, and presenting and writing research reports. The goal is for this experience to increase the student’s skills in performing translational basic science research and to ensure more opportunities for diversification of the scientific workforce.