Function of Fibro-Adipogenic Progenitors in Heterotopic Ossification of Skeletal Muscle

项目摘要 异位骨化(HO)，即骨骼肌和相关软组织中的骨形成，可以 由创伤或疾病造成的。HO最极端的形式表现在罕见的常染色体- 显性遗传性疾病，进行性骨化纤维发育不良(FOP)，在FOP中，HO继续进行性 在整个生命中，对健康和预期寿命造成毁灭性的影响。我们开发了一种新的遗传模型 基于致病BMP受体ACVR1(R206H)的条件表达。使用这个模型， 我们鉴定了肌肉组织中的纤维成脂前体细胞(FAPs)，它是一种多潜能的间充质祖细胞， 致病细胞群。值得注意的是，我们已经证明了将BMP2肌肉注射到野生型 小鼠也导致FAP衍生的HO，提示FOP患者和FAP患者的HO发病机制相似 普通民众。对HO的研究几乎完全集中在骨生长的机制上，但也很严重 肌肉损失可能是导致患者发病率的一个重要因素。这个项目的总体目标是 研究是为了1)了解FAP编程如何被颠覆以形成病理性骨形成；2) 了解对HO敏感的骨骼肌再生是如何被抑制的；3)确定HO是否 骨骼肌完全依赖于FAP。通过使用pdgrfαCreer将Acvr1R206H表达靶向FAP 和Tie2-Cre驱动因素，Aim 1将量化再生损伤，并将确定肌肉干细胞 (卫星细胞；SC)功能障碍是再生缺陷的原因。这些研究将量化SC的增殖 和肌肉损伤后的细胞凋亡，并将定义SC mRNA转录组。目标1还将决定是否 ACVR1(R206H)在FAP中的表达破坏了FAP-SC的相互作用。AIM 2将使用RNA-Seq来定义FAP 转录组在损伤后的早期、关键时刻识别ACVR1(R206H)信号的基因靶点。Gro-Seq (全基因组连续)分析将量化活跃基因转录的变化，将确定候选基因 由启动子-近端聚合酶暂停调节，并将识别潜在的增强子靶点 ACVR1(R206H)信令。血统追踪将确定异常的肌肉环境是否存在差异 影响正常和突变FAP的命运。在目标3中，DTA消融方法将决定FAP 是肌肉中成骨细胞的唯一来源，这是评估可能的疗效所必需的信息 细胞特异性疗法。激活素抑制最近已成为治疗FOP的一种有效的潜在疗法。目标4 将使用血统追踪来解决激活素阻滞剂如何影响增殖、生存和发育能力 FAP和SC。RNA-Seq将定义激活素抑制使FAP和SC“正常化”的程度 抄本。这项拟议的研究将大大有助于理解细胞和细胞 相互作用导致HO和相关的肌肉再生抑制，并可能导致 发展以细胞为基础的治疗策略。

Project Summary Heterotopic ossification (HO), the formation of bone in skeletal muscle and associated soft tissues, can result from traumatic injury or disease. The most extreme form of HO is manifested in the rare, autosomal- dominant genetic disorder, Fibrodysplasia ossificans progressiva (FOP), in which HO continues progressively throughout life, resulting in devastating effects on health and life expectancy. We developed a new genetic model of FOP based on conditional expression of the disease-causing BMP receptor, Acvr1(R206H). Using this model, we identified fibro-adipogenic progenitors (FAPs), a multipotent mesenchymal progenitor in muscle tissue, as the disease-causing cell population. Notably, we have shown that intramuscular injection of BMP2 into wild type mice also leads to FAP-derived HO, suggesting mechanistic similarities of HO pathogenesis in FOP patients and the general population. Studies of HO have focused almost entirely on mechanisms of bone growth, yet severe muscle loss can be a significant contributing factor to patient morbidity. The overarching objectives of this research are to 1) understand how FAP programming becomes subverted for pathological bone formation; 2) understand how regeneration is inhibited in skeletal muscle susceptible to HO; 3) determine whether HO of skeletal muscle is entirely dependent on FAPs. By targeting Acvr1R206H expression to FAPs using PdgrfαCreER and Tie2-Cre drivers, Aim 1 will quantify regeneration impairment, and will determine whether muscle stem cell (satellite cell; SC) dysfunction is responsible for regeneration deficits. These studies will quantify SC proliferation and apoptosis after muscle injury and will define the SC mRNA transcriptome. Aim 1 will also determine whether Acvr1(R206H) expression in FAPs disrupts FAP-SC interactions. Aim 2 will use RNA-Seq to define the FAP transcriptome at early, critical, times after injury to identify gene targets of Acvr1(R206H) signaling. Gro-Seq (Genome-wide Run-On) analyses will quantify changes in active gene transcription, will identify candidate genes regulated by promoter-proximal polymerase pausing, and will identify potential enhancer targets of Acvr1(R206H) signaling. Lineage tracing will determine whether the abnormal muscle environment differentially affects the fate of normal and mutant FAPs. In Aim 3, DTA ablation approaches will determine whether FAPs are the sole source of osteogenic cells in muscle, information that is essential for evaluating the possible efficacy of cell-specific therapies. Activin inhibition has recently emerged as a powerful potential therapy for FOP. Aim 4 will use lineage tracing to address how Activin blockade affects proliferation, survival and developmental capacity of FAPs and SCs. RNA-Seq will define the extent to which Activin inhibition “normalizes” FAP and SC transcriptomes. The proposed research will contribute significantly to an understanding of the cells and cellular interactions responsible for HO and associated inhibition of muscle regeneration, and may lead to the development of strategies for cell-based therapies.