Notch signaling and Bone Fracture Healing

临床上迫切需要开发新的治疗方法来促进骨的愈合。虽然大多数人 骨损伤可以愈合，很多不能，尤其是大的缺损处。了解细胞信号机制 调节正常的愈合，可以引导我们找到新的治疗靶点。Notch信号调节细胞的扩张和 间充质祖细胞(MPC)的分化和调节许多组织的血管形成， 包括骨头。我们的研究以及其他研究人员发表的研究表明，Notch信号是一种 骨愈合过程中的关键调控途径。事实上，我们的初步和公布的结果表明， 增加MPC中的Notch信号可以促进骨再生，并且使用Notch的全局抑制 各种各样的模型，有害地影响了治疗。充分提高我们对Notch信号的理解 在骨愈合方面，并转化这些机械观察，将需要强有力的实验，包括 相关损伤模型的临床前研究。我们的长期目标是开发一种与临床相关的方法 增加Notch信号，促进骨骼愈合。 我们假设Notch信号促进MPC的扩张和骨痂血管化，导致 促进骨形成。我们将询问在骨愈合过程中的Notch信号通路，以揭示 更深入地了解在愈合过程中发挥作用的配体和受体，以及细胞类型特异性 这些信号成分的表达。这项工作将在两个具体目标下完成，使用状态 艺术老鼠模型。在第一个目标中，我们将研究Notch配体的作用。我们的工作以前 证实Jagged1是MPC和成骨软骨细胞表达的主要Notch配体 血统。我们将在骨痂中的MPC、软骨细胞、成骨细胞和骨细胞中特异性地干扰Jag1 在骨折愈合过程中。此外，由于内皮细胞产生的Jag1和DLL4调节血管形成， 我们将通过有条件地删除两者来确定哪一种是调节血管形成的主要配体 使用CDH5-Creer从血管内皮细胞中提取配体。一系列骨折愈合结果，包括 血管形成以及对内皮细胞、MPC增殖和MPC分化的影响 在活体内测定。我们将利用我们使用可诱导的Cre小鼠的丰富经验来确保正常 发育，从而绕过了配体破坏的发育效应。这些研究将是 补充了一项翻译研究，在该研究中，Jag1蛋白单独或与现有的 治疗，骨形态发生蛋白2，将提供在愈合的关键大小的股骨缺损。在第二个目标中，我们将 使用Notch1或Notch2系小鼠检测Notch受体在MPC和内皮细胞上的作用。我们 将决定这些受体是否对BMP2或Jag1驱动的缺陷修复至关重要。这项研究将 通过阐明配体和受体在骨愈合过程中的细胞特异性作用，极大地推动了该领域的发展， 为局部激活Notch信号通路促进骨缺损愈合提供了临床前指导意义。

There is an urgent clinical need to develop new therapeutics to promote healing of bone. While most bone injuries heal, many do not, particularly large defects. Understanding cellular signaling mechanisms that regulate normal healing, can lead us to new therapeutic targets. Notch signaling regulates the expansion and differentiation of mesenchymal progenitor cells (MPC) and regulates vascularization of many tissues, including bone. Our studies, and published studies from other investigators, show that Notch signaling is a key regulatory pathway during bone healing. Indeed, our preliminary and published results show that increasing Notch signaling in MPCs improves bone regeneration, and that global inhibition of Notch using various models, deleteriously impacts healing. To sufficiently advance our understanding of Notch signaling in bone healing, and translate these mechanistic observations, will require robust experimentation, including preclinical studies in relevant injury models. Our long-term goal is to develop a clinically relevant approach to increase Notch signaling that enhances bone healing. We hypothesize that Notch signaling promotes expansion of MPCs and callus vascularization, leading to enhanced bone formation. We will interrogate the Notch signaling pathway during bone healing to reveal a deeper understanding of ligands and receptors that are at play during healing, and the cell-type specific expression of these signaling components. This work will be completed in two specific Aims, using state of the art mouse models. In the first Aim, we will study the role of Notch ligands. Our work has previously demonstrated that Jagged1 is the dominant Notch ligand expressed in MPCs and the osteochondrogenic lineage. We will disrupt Jag1 specifically in MPCs, chondrocytes, osteoblasts and osteocytes in the callus during fracture healing. Additionally, as Jag1 and Dll4 produced by endothelial cells regulate vascularization, we will determine which is the dominant ligand regulating vascularization using conditional deletion of both ligands from endothelial cells using Cdh5-CreER. A spectrum of fracture healing outcomes, including vascularization, as well as effects on endothelial cell and MPC proliferation and MPC differentiation will be determined in vivo. We will capitalize on our extensive experience using inducible Cre mice to ensure normal development thereby by-passing developmental effects of ligand disruption. These studies will be complemented with a translational study in which Jag1 protein, alone or in combination with an existing therapy, BMP2, will be delivered during healing of critical sized femoral defects. In the second Aim, we will examine the role of Notch receptors on MPC and endothelial cells using Notch1 or Notch2 floxed mice. We will determine whether these receptors are critical for defect healing driven by BMP2 or Jag1. This study will significantly advance the field by clarifying the cell-specific role of ligand and receptor during bone healing, and provide the preclinical relevance for local activation Notch signaling to increase bone defect healing.