CGRP-CLR mediated regulation of bone healing

骨膜是骨折愈合反应的主要部位，血管化程度高，密度大。 神经紧张。关于骨折的研究主要集中在生长因子和血管生成的作用上。 但我们推测感觉神经信号是调节 启动骨折骨痂形成所需的干/祖细胞反应的机制。一批 一系列证据表明，感觉神经或与感觉神经相关的信号是 骨量增加和愈合。例如，化学感觉神经丧失后，骨折愈合受到损害， 但促进愈合的感觉神经衍生信号尚未定义。我们假设这种损害 骨膜中的感觉神经通过降钙素基因相关来协调骨愈合级联反应 肽(CGRP)-降钙素样受体(CLR)信号传导。这一点很重要，因为许多CGRP 抑制剂最近被FDA批准用于预防和治疗偏头痛。 在目标1中，我们将评估降钙素基因相关肽抑制剂对骨愈合的影响。鉴于CGRP扮演着一个 在骨转换和潜在愈合中的作用，了解CGRP抑制对骨转换和潜在愈合的影响非常重要 骨折愈合。在目标2中，我们将确定哪个或哪些细胞系对CGRP信号有反应 在愈合过程中使用靶向删除CLR受体。CLR缺失在骨折早期愈合中的作用 使用可诱导的Cre靶向下列谱系：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(CDH5-Creer)。对以下方面的影响 将确定愈伤组织的形成和强度，以及在愈伤组织中的分化。我们将研究 用体内方法研究CGRP/CLR作用于MPC扩增、分化的细胞机制 和血管重建化。我们建议通过区分配体的作用来剖析CLR信号。主 通过CLR和RAMP发挥作用的配体是CGRP和肾上腺髓质素，其中CLR-RAMP1是主要的络合物 而CLR-RAMP2和RAMP3负责ADM信号的激活。在《目标3》中，我们将 利用ADMfl/fl小鼠评估间充质细胞中ADM缺失的影响。我们还将定义 用10x基因组学研究间充质干细胞和内皮细胞CLR缺失的下游信号机制。 我们的研究还将提供关于新批准的降钙素基因相关肽抑制剂的影响的关键信息 信号在骨愈合中的作用以及最终通过CLR受体影响愈合的细胞机制。 调节CGRP/CLR感觉信号的测试方法可能导致一种治疗策略，以增强 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.