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: 10330448
• 负责人: Jefferson Overlin Abaricia
• 金额: $ 4.7万
• 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/10330448
• 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; 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 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 fracture repair; bone healing; cell behavior; cytokine; directed differentiation; 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.

项目概要 骨折愈合是一个复杂的骨愈合过程，受各类细胞和信号传导的调节 机制。其中，Wnt 信号传导长期以来一直被认为在指导分化方面至关重要 骨愈合过程中间充质干细胞（MSC）转化为成骨细胞。然而，这些Wnt的来源 配体未知。最近，大量文献的出现发现了有益和有害的影响 巨噬细胞衍生的 Wnt 配体对其他组织（肝、肾、心脏和肠）再生的影响，通常 通过它们对祖细胞行为的调节。由于巨噬细胞已被充分描述为协调者 在愈合过程中，他们利用 Wnt 信号传导来调节组织再生也就不足为奇了。然而，没有研究表明 尚未确定巨噬细胞在骨折愈合过程中对 Wnt 信号传导的贡献。有趣的是，最近 未发表的数据表明巨噬细胞是骨愈合过程中 Wnt 配体的主要来源 强调巨噬细胞在驱动这一过程中的重要性。在这里提出的工作中，表达 在骨折过程中，首先测量巨噬细胞的 Wnt 配体以及 Wnt 配体靶向的细胞 康复。然后，编码必要的Wnt配体运输蛋白的基因Wls将在Csf1r中被删除- 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 用他莫昔芬诱导 Cre 重组酶。删除巨噬细胞 Wls 后，损失的后果 巨噬细胞衍生的 Wnt 配体将在炎症、软愈伤组织和硬愈伤组织过程中进行表征 愈合的阶段。通过这些阶段的适当过渡以及最终成功的治愈将是 使用 qPCR、流式细胞术、微型计算机断层扫描和组织学进行评估。最后，机制 Wnt 向 MSC 的运输及其对 MSC 行为的影响的后果将通过以下方式进行评估： 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 配体对骨愈合的影响，并可能将 Wnt 信号传导确定为增强成功的靶向途径 骨质愈合。