Developing New Diagnostic and Timed, TAK1 Specific Treatment Strategies for Trauma Induced Heterotopic Ossification

项目总结 病理起源于过度的异位骨形成，或创伤诱导的异位骨化(HO)， 20%的髋关节置换、肌肉骨骼创伤、 脊髓损伤、截肢和烧伤。HO患者经历慢性疼痛，关节受限 功能和开放性伤口；他们经常接受外科手术切除病变的骨骼，但这些 手术不能逆转关节痉挛和活动范围受限，并可能导致复发。 由于我们对HO的理解存在这些局限性，而且我们无法阻止它的发展，我们着手 阐明负责的细胞(间充质细胞和巨噬细胞)和涉及的主要信号通路。 整个转录组测序和早期HO位点的免疫荧光成像显示 间充质中转化生长因子-β激活蛋白1(TAK1)信号的显著增加 细胞。然而，间充质细胞分化的阶段，其中TAK1是必需的，以及什么细胞 刺激间充质细胞TAK1的作用尚不清楚。初步数据表明，基因和治疗 TAK1基因敲除减轻了导致HO的早期间充质凝聚和分化。 重要的是，我们还证明了巨噬细胞的中心作用及其激活的TAK1在 间充质细胞。我们计划验证一种新的细胞特异性药物传递系统，以阻断a的分泌 负责HO的初级细胞(巨噬细胞)的初级配体(转化生长因子1β)。 以下目标旨在测试我们的假设，即HO的总体治疗范例将是 通过使用高频频谱超声和定时、特定途径和特定细胞进行早期检测而得到改进 抑制TAK1信号转导。 目的1：明确创伤后间充质细胞中TAK1信号转导途径并验证 用于预防异位骨化的新型TAK1途径抑制剂。这一目标将证明 TAK1信号在间充质凝聚和基因敲除过程中上调 特别是在间充质细胞或早期TAK1药物抑制小分子NG-25将 通过Arkadia/Smad7介导的过程减轻HO。这个目标将利用新的成像技术来实现 检测早期HO前的变化，并验证预防HO的早期定时治疗策略。 ·目的2：确定巨噬细胞特异性转化生长因子β1在HO中的作用，并验证新的 使巨噬细胞中的Tgfb1沉默的微粒。这一目标将证明受伤 巨噬细胞及其产生的转化生长因子β1是异位间充质细胞TAK1信号转导的关键。 软骨生成与HO。这一目标还将优化微粒的巨噬细胞特异性摄取和 药物传递，以管理Tgfb1 siRNA和防止HO。

PROJECT SUMMARY Pathology stemming from excess ectopic bone formation, or trauma-induced heterotopic ossification (HO), presents a substantial barrier to recovery in 20% of patients with hip replacements, musculoskeletal trauma, spinal cord injury, amputations and burn injuries. Patients with HO experience chronic pain, restricted joint function, and open wounds; they often undergo surgical procedures to excise the offending bone, but these procedures fail to reverse the joint contractures and restricted range of motion and can lead to recurrence. With these limitations in our understanding of HO and our inability to prevent its development, we set out to clarify the cells responsible (mesenchymal cells and macrophages) and primary signaling pathway involved. Whole transcriptome sequencing and immunofluorescent imaging of the early HO site have demonstrated a marked increase in Transforming Growth Factor-beta activating kinase 1 (TAK1) signaling in the mesenchymal cells. However, the stage of mesenchymal cell differentiation in which TAK1 is necessary and what cells stimulate mesenchymal cell TAK1 remains unknown. Preliminary data demonstrate that gene and therapeutic knockdown of TAK1 mitigates early mesenchymal condensation and differentiation responsible for HO. Importantly, we have also demonstrated the central role of macrophages and their activation of TAK1 in mesenchymal cells. We plan to validate a novel cell specific drug delivery system to block secretion of a primary ligand (Transforming Growth Factor 1β) from a primary cell (macrophage) responsible for HO. The following aims are designed to test our hypothesis that the overall treatment paradigm of HO will be improved with early detection using high frequency spectral ultrasound and timed, pathway and cell specific inhibition of TAK1 signaling. · Aim 1: To define the pathway of TAK1 signaling in mesenchymal cells after trauma and to validate novel TAK1 pathway inhibitors to prevent heterotopic ossification. This aim will demonstrate that TAK1 signaling is upregulated during mesenchymal condensation and that genetic knockout of TAK1 specifically in mesenchymal cells or early TAK1 pharmacologic inhibition with small molecule NG-25 will mitigate HO through an Arkadia/SMAD7 mediated process. This aim will utilize novel imaging to allow detection of early pre-HO changes and validate an early timed treatment strategy to prevent HO. · Aim 2: To define the role of macrophage-specific TGFβ1 production on HO and to validate novel microparticles that silence Tgfb1 specifically in macrophages. This aim will demonstrate that injury site macrophages and their production of TGFβ1 is critical for ectopic mesenchymal cell TAK1 signaling, chondrogenesis and HO. This aim will also optimize microparticles for macrophage-specific uptake and drug delivery to administer Tgfb1 siRNA and prevent HO.