CGRP-CLR mediated regulation of bone healing

骨膜是骨折愈合反应的主要部位，其高度血管化且密度高 神经支配对骨折的研究主要集中在生长因子和血管化的作用上 在愈合过程中，但我们假设感觉神经信号是调节的关键部分， 启动骨折骨痂形成所需的干/祖细胞反应的机制。一些 有证据表明，感觉神经支配，或与感觉神经相关的信号， 骨积累和愈合。例如，化学感觉去神经后骨折愈合受损， 但感觉神经衍生的信号，促进愈合尚未确定。我们假设 感觉神经通过降钙素基因相关的 肽（CGRP）-降钙素样受体（CGRP）信号传导。这一点很重要，因为多种CGRP FDA最近批准了用于预防和治疗偏头痛的抑制剂。 在目标1中，我们将评估CGRP抑制剂对骨愈合的影响。考虑到CGRP在 由于CGRP在骨转换和潜在愈合中的作用，重要的是要了解CGRP抑制对 骨折愈合在目标2中，我们将确定哪个细胞谱系或哪些谱系对CGRP信号有反应 在愈合过程中使用靶向删除的E2受体。在骨折愈合早期， 使用诱导型Cre靶向以下谱系：MPC（αSMA-CreER），软骨细胞（Acan）-CreER， 在骨折愈合过程中，成骨细胞使用Col2.3CreER和内皮细胞（Cdh 5-CreER）。的效果 将测定愈伤组织的形成和强度以及愈伤组织的分化。我们将研究 利用体内方法研究MPC的扩增、分化、 和血管化。我们建议通过区分配体的作用来剖析信号传导。主要 配体是CGRP和肾上腺髓质素，它们通过CGRP和RAMP起作用，其中CLR-RAMP 1是主要的复合物 而CLR-RAMP 2和RAMP 3负责ADM信号激活。在目标3中， 使用ADMf 1/f1小鼠评估ADM缺失在间充质群体中的作用。我们还将定义 使用10 x基因组学在MSC和内皮细胞中缺失的下游信号传导机制。 我们的研究还将提供新批准的CGRP抑制剂的作用的关键信息， 信号传导在骨愈合中的表现以及通过β受体影响愈合的细胞机制最后， 调节CGRP/CGRP感觉单一性的测试方法可能会导致治疗策略， 骨愈合

Periosteum, a primary site of the fracture healing response, is highly vascularized and densely innervated. Studies on bone fracture have primarily focused on the role of growth factors and vascularization in the healing process, but we postulate that sensory nerve signals are a critical part of the regulatory mechanism that initiate the stem/progenitor cell responses required for fracture callus formation. A number of lines of evidence point to sensory innervation, or signals associated with sensory nerves as promoters of bone accrual and healing. For example, fracture healing is impaired following chemical sensory denervation, but the sensory nerve-derived signals that promote healing as not yet defined. We hypothesize that damage to sensory nerves in the periosteum orchestrates the bone-healing cascade through calcitonin gene-related peptide (CGRP) – calcitonin like receptor (CLR) signaling. This is important given that multiple CGRP inhibitors were recently approved by the FDA for prevention and treatment of migraines. In Aim 1 we will evaluate the effects of CGRP inhibitors on bone healing. Given that CGRP plays a role in bone turnover and potentially healing, it is important to understand the impact of CGRP inhibition on fracture healing. In the Aim 2, we will determine which cell lineage or lineages responsive to CGRP signals during healing using targeted deletion of the CLR receptor. CLR deletion in early fracture healing will be targeted to the following lineages using inducible Cre’s: MPCs (αSMA-CreER), chondrocyte (Acan)-CreER, osteoblasts using Col2.3CreER and in endothelial (Cdh5-CreER) during fracture healing. The effects on callus formation and strength, as well as differentiation in the callus will be determined. We will examine cellular mechanisms of CGRP/CLR action utilizing in vivo approaches to study MPC expansion, differentiation and vascularization. We propose to dissect CLR signaling by distinguishing effects of the ligands. The main ligands are CGRP and Adrenomedullin that act via CLR and RAMP, of which CLR-RAMP1 is main complex for CGRP signaling while CLR-RAMP2 and RAMP3 is responsible for ADM signal activation. In Aim 3 we will evaluate effects of ADM deletion in mesenchymal population using ADMfl/fl mice. We will also define downstream signaling mechanism of CLR deletion in MSCs and endothelial cells using 10x genomics. Our study will also provide critical information on the effects that newly approved inhibitors of CGRP signaling exhibit on bone healing and what cellular mechanisms affect healing via CLR receptor Finally, testing approaches to modulate CGRP/CLR sensory singling may lead to a therapeutic strategy to enhance bone healing.