Regulation of Fracture Healing by Macrophage-Derived Wnt Ligands

巨噬细胞衍生的 Wnt 配体对骨折愈合的调节

• 批准号: 10079397
• 负责人: Jefferson Overlin Abaricia
• 金额: $ 4.65万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-25 至 2023-12-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10079397
• 关键词: Ablation; Animals; Anti-Inflammatory Agents; Architecture; Automobile Driving; Biological; Bone Injury; Bone callus; Cell Differentiation process; Cells; Coculture Techniques; Complex; Data; Development; Effectiveness; Enterobacteria phage P1 Cre recombinase; Exhibits; Exposure to; Flow Cytometry; Fracture; Fracture Healing; Gene Expression; Genes; Heart; Histology; Homeostasis; Immune; Immune system; Implant; In Vitro; Individual; Inflammation; Inflammatory; Injury; Intestines; Kidney; Ligands; Literature; Liver; Lymphangiogenesis; Measures; Mechanics; Mediating; Mediator of activation protein; Mesenchymal Differentiation; Mesenchymal Stem Cells; Modeling; Mus; Myeloid Cells; Names; Natural regeneration; Neoplasms; Osseointegration; Osteoblasts; Pathway interactions; Pharmacology; Phase; Phenotype; Play; Population; Process; Production; Regulation; Reporter; Role; Signal Transduction; Signaling Molecule; Source; Surface; Tamoxifen; Testing; Time; Tissues; Titanium; WNT Signaling Pathway; Wild Type Mouse; Work; angiogenesis; autocrine; bone; bone healing; cell behavior; cytokine; experimental study; extracellular vesicles; healing; hydrophilicity; in vivo; macrophage; macrophage product; microCT; monocyte; novel; osteogenic; paracrine; prevent; progenitor; protein transport; recruit; stem; stem cell differentiation; stem cell proliferation; stem cells; success; tissue regeneration; trafficking

PROJECT SUMMARY Fracture healing is a complex bone healing process regulated by various classes of cells and signaling mechanisms. Of these, Wnt signaling has long been known to be critically important in directing the differentiation of mesenchymal stem cells (MSCs) into osteoblasts during bone healing. However, the source of these Wnt ligands is not known. Recently, a body of literature has arisen finding both beneficial and detrimental effects of macrophage-derived Wnt ligands on the regeneration of other tissues (liver, kidney, heart, and intestine), often by their modulation of progenitor cell behavior. As macrophages have been well-characterized as orchestrators of healing, their use of Wnt signaling to regulate tissue regeneration is unsurprising. However, no studies have yet identified the contribution of macrophages to Wnt signaling during fracture healing. Interestingly, recent unpublished data suggests that macrophages represent a major source of Wnt ligands during bone healing and underscore the importance of macrophages in driving this process. In the work proposed here, the expression of Wnt ligands by macrophages will first be measured, as well as the cells targeted by Wnt ligands, during fracture healing. Then, Wls, the gene encoding a necessary Wnt ligand trafficking protein, will be deleted in Csf1r- expressing cells (monocytes and macrophages), inhibiting the secretion of all Wnt ligands by these cells after induction of Cre recombinase with tamoxifen. Following deletion of macrophage Wls, the consequences of loss of macrophage-derived Wnt ligands will be characterized during the inflammatory, soft callus, and hard callus phases of healing. Appropriate transition through these phases, and ultimately successful healing, will be evaluated using qPCR, flow cytometry, micro-computed tomography, and histology. Finally, the mechanism of Wnt trafficking to MSCs, and the consequences of their effects on MSC behavior, will be evaluated through in vitro co-culture experiments. The studies defined in this proposal will elucidate the importance of macrophage Wnt ligands on bone healing and potentially identify Wnt signaling as a targetable pathway to enhance successful osseous healing.

项目总结 骨折愈合是一个受多种细胞和信号调控的复杂的骨愈合过程。 机制。在这些信号中，Wnt信号早已被认为是指导分化的关键 在骨愈合过程中，骨髓间充质干细胞(MSCs)转化为成骨细胞。然而，这些WNT的来源 配体尚不清楚。最近，出现了一批文献，它们既有好处，也有坏处。 巨噬细胞来源的Wnt配体对其他组织(肝、肾、心脏和肠道)的再生，通常 通过它们对祖细胞行为的调节。因为巨噬细胞一直被认为是交响乐团 在愈合方面，他们使用Wnt信号来调节组织再生并不令人惊讶。然而，没有研究表明 尚未发现巨噬细胞在骨折愈合过程中对Wnt信号的作用。有趣的是，最近 未发表的数据表明，巨噬细胞是骨愈合和骨修复过程中Wnt配体的主要来源 强调巨噬细胞在推动这一过程中的重要性。在这里提出的工作中，表达 首先将测量巨噬细胞的Wnt配体的数量，以及在骨折过程中Wnt配体靶向的细胞 治愈。然后，编码必要的Wnt配体运输蛋白的基因Wls将在CSF1R- 表达细胞(单核细胞和巨噬细胞)，抑制这些细胞分泌所有Wnt配体 三苯氧胺诱导Cre重组酶。巨噬细胞WLS缺失后，丢失的后果 巨噬细胞来源的Wnt配体在炎症、软性骨痂和硬性骨痂中的特征 治疗的各个阶段。通过这些阶段的适当过渡，并最终成功治愈，将是 采用定量聚合酶链式反应、流式细胞仪、微型计算机断层扫描和组织学方法进行评估。最后，提出了一种新的运行机制 向MSCS贩运WNT及其对MSC行为的影响的后果将通过 体外共培养实验。本提案中定义的研究将阐明巨噬细胞的重要性。 WNT配体在骨愈合中的作用，并潜在地将WNT信号识别为促进成功的靶向途径 骨性愈合。