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

项目摘要 病理学源于异位骨形成过多，或创伤诱导的异位骨化（HO）， 在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冒犯的骨头，但这些 手术不能逆转关节挛缩和活动范围受限，并可能导致复发。 由于我们对HO的理解存在这些局限性，而且我们无法阻止其发展，因此我们着手 阐明相关细胞（间充质细胞和巨噬细胞）和主要信号通路。 早期HO位点的全转录组测序和免疫荧光成像已经证明了 间充质细胞中转化生长因子-β激活激酶1（TAK 1）信号传导显著增加 细胞然而，需要TAK 1的间充质细胞分化阶段和需要哪些细胞 刺激间充质细胞TAK 1仍然未知。初步数据表明，基因和治疗 TAK 1的敲低减轻了负责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及其TAK 1活化在肿瘤细胞中的中心作用。 间充质细胞我们计划验证一种新的细胞特异性药物递送系统，以阻断 来自负责HO的原代细胞（巨噬细胞）的原代配体（转化生长因子1β）。 以下目的旨在检验我们的假设，即HO的总体治疗模式将是 通过使用高频光谱超声和定时、途径和细胞特异性的早期检测来改善 抑制TAK 1信号传导。 ·目的1：确定创伤后间充质细胞中TAK 1信号传导的途径，并验证 预防异位骨化的新型TAK 1通路抑制剂。这一目标将表明， TAK 1信号在间充质凝聚过程中上调，TAK 1基因敲除 特别是在间充质细胞中，或者用小分子NG-25的早期TAK 1药理学抑制将 通过Arkadia/SMAD 7介导的过程减轻HO。这一目标将利用新的成像技术， 检测早期HO前变化，并验证早期治疗策略以预防HO。 ·目的2：确定巨噬细胞特异性TGFβ1产生在HO中的作用，并验证新的TGFβ1表达。 在巨噬细胞中特异性沉默Tgfb 1的微粒。这一目标将表明， 位点巨噬细胞及其产生TGFβ1对于异位间充质细胞TAK 1信号传导至关重要， 软骨形成和HO。该目的还将优化用于巨噬细胞特异性摄取的微粒， 药物递送以施用Tgfb 1 siRNA并预防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.