Exosome treatment-induced mechanisms in chronic wound beds - Supplement

项目摘要 慢性伤口带来了昂贵的社会和医疗困境，特别是在II型糖尿病患者中， 没有有效的治疗方法。这些伤口的特点是慢性炎症，严重 微血管并发症，因此缺乏必要的肉芽组织扩张和增殖 来治愈伤口。最近，外泌体给药已成为促进创伤的有效疗法 但治疗效果的机制大多是未知的。外泌体是分泌的 可以从多能基质细胞的细胞培养物中分离的膜纳米囊泡。在临床前类型中 在II型糖尿病模型中，外泌体的单次局部施用是非常有效的，并且减少了闭合时间， 接近野生型小鼠。我们发现在外泌体处理的肉芽组织中广泛的新生血管形成 伤口和存在大量巨噬细胞对β-淀粉酶1呈免疫反应性，通常表明 促愈合表型。来自多能基质细胞的外泌体携带纤溶酶原激活物抑制剂-1（派-1） 以及其他血管生成相关蛋白。当我们同时应用派-1抑制剂和 当外来体处理后，外来体处理的有益效果被部分减轻。外来体可以具有多个 信号通路的靶点，派-1是主要成分。我们的长期目标是了解 我们需要解决的分子机制来改变慢性伤口状态， 结束像这样的方法对于理解外泌体治疗的原理和安全性是必要的。 在这项研究中，我们正在研究骨髓MPSC外泌体中的派-1介导 对慢性伤口床中的巨噬细胞和EC的适应性作用，从而促进肉芽扩张 有效改变伤口愈合轨迹的组织。在目标1中，我们将确定是否过表达或 外来体中派-1的损失影响典型的延迟的糖尿病伤口愈合。我们将评估时空 巨噬细胞和内皮细胞的分布。在目标2中，我们将分析伤口床的变化 巨噬细胞和内皮细胞在单细胞分辨率，使用整合表位和转录组学 approach.此外，在炎症条件下的体外培养中，我们将确定细胞中的分子变化。 巨噬细胞和微血管内皮细胞，外泌体摄取的下游。结果将证明 协调外泌体治疗在使炎症正常化和促进炎症反应中的功效的机制， 糖尿病伤口缝合我们将在布鲁斯·克朗斯坦博士、安·玛丽博士的指导下进行研究 施密特和Thorsten Kirsch博士，并获得在临床前研究设计中建模慢性疾病的专业知识， 高度炎症性疾病状态、巨噬细胞测定和外泌体介导的机制以及细胞免疫学 慢性皮肤伤口床中的通信。纽约大学的协作和科学学习环境 将提供最佳设置，以获得基于证据的转化研究的合理设计的专业知识。这 培训期间将提供科学和职业发展的标尺，为一个独立的职业生涯的研究。

PROJECT SUMMARY Chronic wounds present a costly social and medical dilemma, particularly in patients with type II diabetes, but there are no effective treatments. These wounds are characterized by chronic inflammation, severe microvascular complications, and therefore lack expansion of the granulation tissue and proliferation necessary to heal the wound. Recently, exosome administration has emerged as a potent therapy for promoting wound healing but the mechanisms underlying the therapeutic effect are mostly unknown. Exosomes are secreted membranous nanovesicles that can be isolated from cell culture of multipotent stromal cells. In a preclinical type II diabetic model, a single local administration of exosomes was very effective and reduced time to closure to nearly that of wild type mice. We found extensive neovascularization in granulation tissue of exosome-treated wounds and presence of large numbers of macrophages immunoreactive for arginase1, typically indicative of a pro-healing phenotype. Exosomes from multipotent stromal cells carry plasminogen activator inhibitor-1 (PAI-1) among other angiogenesis-associated proteins. When we applied a PAI-1 inhibitor simultaneously with exosomes, the beneficial effect of exosome treatment was partially mitigated. Exosomes can have multiple signaling pathway targets, and PAI-1 is a major component. Our long term objective is to understand the molecular mechanisms we need to address to alter the chronic wound state, and actually promote wound closure. An approach like this is necessary to understand the rationale and safety of exosome treatment. In this study, we are investigating the hypothesis that PAI-1 in bone marrow MPSC exosomes mediates adaptive effects on macrophages and ECs in chronic wound beds, thereby promoting expansion of granulation tissue with effective changes in wound healing trajectory. In Aim 1, we will determine whether overexpression or loss of PAI-1 in exosomes affects the typically delayed diabetic wound healing. We will assess spatiotemporal distribution of macrophages and endothelial cells. In Aim 2, we will analyze the changes in wound bed macrophages and endothelial cells at a single cell resolution, using an integrated epitope and transcriptomics approach. Additionally, in in vitro culture with inflammatory conditions, we will determine molecular changes in macrophages and microvascular endothelial cells, downstream of exosome uptake. The results will demonstrate the mechanisms orchestrating the efficacy of exosome treatment in normalizing inflammation and promoting diabetic wound closure. We will perform the study under the mentorship of Dr. Bruce Cronstein, Dr. Ann Marie Schmidt, and Dr. Thorsten Kirsch, and gain expertise in modeling chronic diseases in preclinical study designs, hyper-inflammatory disease states, macrophage assays, and exosome-mediated mechanisms and cellular communication in a chronic cutaneous wound bed. The collaborative and scientific learning environment at NYU will provide the optimal setting to gain expertise in rational design of evidence-based translational research. This training period will provide the scientific and career development rubric for an independent career in research.