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

纤维脂肪祖细胞在骨骼肌异位骨化中的功能

• 批准号: 9366782
• 负责人: DAVID J GOLDHAMER
• 金额: $ 34.4万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9366782
• 关键词: ACVR1 gene; Ablation; Activins; Address; Affect; Alleles; Amino Acids; Apoptosis; Area; BMP2 gene; Blocking Antibodies; Bone Formation Inhibition; Bone Growth; Candidate Disease Gene; Cartilage; Cell Communication; Cell Proliferation; Cell Therapy; Cells; Coculture Techniques; Development; Diphtheria Toxin; Disease; Dominant Genetic Conditions; Enhancers; Environment; Exhibits; Fiber; Fibrosis; Functional disorder; Gene Expression; Gene Targeting; General Population; Genetic Models; Genetic Transcription; Glycine; Goals; Health; Hereditary Disease; Heterotopic Ossification; Hypersensitivity; Immunofluorescence Immunologic; Impairment; Individual; Infiltration; Injection of therapeutic agent; Injury; Intramuscular Injections; Kinetics; Lead; Life; Life Expectancy; Mesenchymal; Messenger RNA; Modeling; Molecular; Morbidity - disease rate; Mus; Muscle; Muscle satellite cell; Muscular Atrophy; Natural regeneration; Osteogenesis; Pathogenesis; Pathologic; Pathway interactions; Patients; Physiologic Ossification; Physiological; Polymerase; Population; Research; Running; Serine; Signal Transduction; Skeletal Muscle; Source; Specificity; Therapeutic; Time; Traumatic injury; Wild Type Mouse; activin A; alpha Toxin; base; bone; bone morphogenetic protein receptors; experimental study; genome-wide; global run on sequencing; muscle regeneration; mutant; osteogenic; prevent; progenitor; progressive myositis ossificans; promoter; regenerative; response; satellite cell; soft tissue; tibialis anterior muscle; transcriptome; transcriptome sequencing

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 PdgrfaCreER 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.

项目概要 异位骨化（HO），即骨骼肌和相关软组织中骨的形成，可以 由外伤或疾病引起。 HO 最极端的形式表现为罕见的常染色体- 显性遗传性疾病，进行性骨化性纤维发育不良 (FOP)，其中 HO 持续进行性发展 终生，对健康和预期寿命造成毁灭性影响。我们开发了一种新的遗传模型 FOP 基于致病 BMP 受体 Acvr1(R206H) 的条件表达。使用这个模型， 我们鉴定了纤维脂肪生成祖细胞（FAP），这是肌肉组织中的多能间充质祖细胞， 致病细胞群。值得注意的是，我们已经证明，将 BMP2 肌肉注射到野生型体内 小鼠也导致 FAP 衍生的 HO，这表明 FOP 患者和 FOP 患者的 HO 发病机制具有相似性。 普通民众。 HO 的研究几乎完全集中在骨生长机制上，但严重 肌肉损失可能是导致患者发病的一个重要因素。本次活动的总体目标 研究目的是 1) 了解 FAP 编程如何因病理性骨形成而被破坏； 2） 了解对 H2O2 敏感的骨骼肌再生是如何受到抑制的； 3) 判断HO是否为 骨骼肌完全依赖于 FAP。使用 PdgrfaCreER 将 Acvr1R206H 表达靶向 FAP 和 Tie2-Cre 驱动程序，目标 1 将量化再生损伤，并确定肌肉干细胞是否 （卫星细胞；SC）功能障碍是导致再生缺陷的原因。这些研究将量化 SC 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 还将确定是否 FAP 中的 Acvr1(R206H) 表达会破坏 FAP-SC 相互作用。目标 2 将使用 RNA-Seq 来定义 FAP 在损伤后的早期、关键时刻进行转录组鉴定 Acvr1(R206H) 信号传导的基因靶标。格罗测序 （全基因组运行）分析将量化活性基因转录的变化，识别候选基因 受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) 信号传导。谱系追踪将确定异常的肌肉环境是否有差异 影响正常和突变 FAP 的命运。在目标 3 中，DTA 消融方法将确定 FAP 是否 是肌肉中成骨细胞的唯一来源，这些信息对于评估可能的功效至关重要 细胞特异性疗法。激活素抑制最近已成为 FOP 的一种强大的潜在疗法。目标 4 将使用谱系追踪来解决激活素阻断如何影响增殖、生存和发育能力 FAP 和 SC。 RNA-Seq 将定义激活素抑制“正常化”FAP 和 SC 的程度 转录组。拟议的研究将极大地有助于了解细胞和细胞 相互作用负责 HO 和相关的肌肉再生抑制，并可能导致 开发基于细胞的治疗策略。