Understanding the Mechanisms of Fibrosis in Muscular Dystrophies

项目摘要 肌营养不良症（MD）是一种遗传性神经肌肉疾病，导致进行性虚弱和损失 肌肉的再生过程不能充分应对进行性肌肉损伤。这最终 导致功能性肌肉组织被病理性细胞外基质（ECM）替代， 纤维化和脂肪，称为纤维基质，这是最常见的MD的突出特征， MD（DMD）。有效的治疗，以打击纤维基质的发展和促进肌肉再生是一个 MD患者的主要未满足的临床需求，然而，负责这些问题的机制尚不清楚 明白该项目研究导致再生失败和纤维化的细胞机制 在营养不良的肌肉中发育。具体地，NAD（P）H氧化酶4（NOX 4）已被鉴定为抗- 肌肉中纤维化和促再生靶点。肌肉纤维化的显著减少证明了这一点 和有益的肌肉重塑的严重营养不良的肌肉，模型DMD，其中NOX 4的目标 使用遗传学和药理学方法。据推测，肌成纤维细胞、细胞中的NOX 4表达 在组织损伤后产生ECM，通过阻止肌成纤维细胞清除而导致肌肉纤维化 肌肉再生后。这种现象被称为肌成纤维细胞持久性。的实验 该项目将严格调查导致和病理后果的机制 从肌成纤维细胞持久性的发展，使用创新的遗传模型，体外试验， 转录组学分析。目的1将研究肌纤维母细胞动力学在营养不良的骨骼肌， 肌成纤维细胞命运定位和N0X4的条件性消融。肌成纤维细胞持久性将在 将采用一种新开发的使用从营养不良肌肉中分离的细胞的测定法和几种体外测定法 探讨肌成纤维细胞在分化和发育过程中的行为。目标2将评估 肌成纤维细胞对成肌细胞、成纤维细胞和成纤维细胞的基因表达和细胞行为的影响 营养不良和再生肌肉中的免疫细胞。体外模型将用于区分物理与 这些肌纤维母细胞驱动的表型的扩散线索负责。本项目的最终目标是 定义慢性肌肉疾病中肌成纤维细胞的病理后果， 机制的洞察力，负责有效的影响，NOX4靶向作为一个有益的重塑 用于治疗MD和潜在的其他形式的肌肉病理学的治疗策略。

PROJECT SUMMARY Muscular dystrophies (MDs) are heritable neuromuscular diseases that cause progressive weakness and loss of muscle as regenerative processes fail to adequately respond to progressive muscle damage. This ultimately results in the replacement of functional musculature with a pathological extracellular matrix (ECM) composed of fibrosis and fat, known as a fibro-matrix, which is a prominent feature of the most common of the MDs, Duchenne MD (DMD). Effective therapeutics to combat fibro-matrix development and facilitate muscle regeneration are a major unmet clinical need for MD patients, however, the mechanisms responsible for these issues are not well understood. This project investigates cellular mechanisms contributing to the failed regeneration and fibrosis development in dystrophic muscle. Specifically, NAD(P)H oxidase 4 (NOX4) has been identified as an anti- fibrotic and pro-regenerative target in muscle. This was demonstrated by marked reductions in muscle fibrosis and beneficial muscle remodeling of severely dystrophic muscle that models DMD, where NOX4 was targeted using genetic and pharmacological approaches. It is hypothesized that NOX4 expression in myofibroblasts, cells that produce ECM following tissue injury, contributes to muscle fibrosis by preventing myofibroblast clearance following muscle regeneration. This phenomenon is known as myofibroblast persistence. The experiments of this project will rigorously investigate the mechanisms leading to and the pathological consequences resulting from the development of myofibroblast persistence using innovative genetic models, in vitro assays, and transcriptomic analyses. Aim 1 will investigate myofibroblast dynamics in dystrophic skeletal muscle using myofibroblast fate-mapping and conditional ablation of NOX4. Myofibroblast persistence will be assessed in a newly-developed assay using cells isolated from dystrophic muscle, and several in vitro assays will be employed to investigate myofibroblast behavior during differentiation and development of persistence. Aim 2 will evaluate the influence that myofibroblasts exert on gene expression and cellular behavior of myogenic, fibroblastic, and immune cells in dystrophic and regenerating muscle. In vitro models will be used to discern physical versus diffusible cues responsible for these myofibroblast-driven phenotypes. The ultimate goal of the current project is to define the pathological consequences of myofibroblasts in chronic muscle disease and provide solid mechanistic insight responsible for the efficacious impact of NOX4-targeting as a beneficial remodeling therapeutic strategy for the treatment of MDs and, potentially, other forms of muscle pathology.