Exosome treatment-induced mechanisms in chronic wound beds - Resubmission - 1

项目总结 慢性伤口是一个代价高昂的社会和医疗困境，特别是在II型糖尿病患者中，但 目前还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，因此缺乏肉芽组织的扩张和必要的增殖 来治愈伤口。最近，外周给药已成为促进伤口的一种有效疗法。 治愈，但其治疗效果的机制大多尚不清楚。外切体是分泌的 可从多能基质细胞培养中分离出来的膜性纳米囊泡。在临床前类型 II型糖尿病模型，局部单次给药外切体非常有效，缩短了闭合时间 几乎和野生型小鼠一样。我们发现外切体处理的肉芽组织中有广泛的新生血管。 创伤和大量精氨酸酶1免疫反应的巨噬细胞的出现，通常表明 促进愈合的表型。多能基质细胞外切体携带纤溶酶原激活物抑制物-1 在其他与血管生成相关的蛋白中。当我们同时应用PAI-1抑制剂和 外切体，外切体处理的有益效果被部分缓解。Exosome可以有多个 信号通路为靶点，而PAI-1是其主要成分。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，并实际上促进伤口 结案了。像这样的方法对于理解外渗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外体中PAI-1介导的假说。 对慢性创面床巨噬细胞和内皮细胞的适应性作用，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，我们将确定过度表达或 PAI-1在外切体中的丢失影响典型的糖尿病伤口愈合延迟。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 单细胞分辨率的巨噬细胞和内皮细胞，使用整合的表位和转录组学 接近。此外，在炎性条件下的体外培养中，我们将确定 巨噬细胞和微血管内皮细胞，位于外切体摄取的下游。结果将证明 外切治疗消炎促炎的作用机制 糖尿病伤口闭合。我们将在布鲁斯·克朗斯坦博士、安·玛丽博士的指导下进行这项研究 Schmidt和Thorsten Kirsch博士，并在临床前研究设计中建立慢性疾病模型方面获得了专业知识， 高炎症性疾病状态、巨噬细胞分析、外切体介导的机制和细胞 慢性皮肤伤口床上的交流。纽约大学的协作和科学学习环境 将提供最佳环境，以便在循证翻译研究的合理设计方面获得专业知识。这 培训期间将为独立从事研究工作提供科学和职业发展准则。

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.