Diabetes, Macrophages and Bone Regeneration

糖尿病、巨噬细胞和骨再生

• 批准号: 10371527
• 负责人: MIYA KANG
• 金额: $ 15.93万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10371527
• 关键词: Affect; American; Applications Grants; Attention; Bioinformatics; Biological Assay; Bone Regeneration; CRISPR/Cas technology; Calvaria; Cells; Chronic; Complex; Conditioned Culture Media; Defect; Dentistry; Diabetes Mellitus; Diabetic mouse; Disease; Engineering; Epidemic; Evaluation; Gene Expression Regulation; Genes; Immune response; Immune system; Immunologics; Impaired wound healing; Impairment; Implant; In Vitro; Individual; Inflammation; Knock-out; Knowledge; Link; Measures; Mediating; Mesenchymal Differentiation; Mesenchymal Stem Cells; Messenger RNA; MicroRNAs; Modeling; Natural regeneration; Nature; Non-Insulin-Dependent Diabetes Mellitus; Oral; Osseointegration; Osteogenesis; Outcome; Paracrine Communication; Pathology; Pathway interactions; Phenotype; Physiological; Physiology; Play; Population; Procedures; Process; Publishing; Reporter Genes; Role; Scientist; Selection Criteria; Shapes; Signal Pathway; Signal Transduction; Standardization; Systemic disease; Testing; Therapeutic Intervention; Training; Undifferentiated; Wild Type Mouse; Work; base; bone; bone fracture repair; bone healing; bone repair; craniofacial bone; cytokine; diabetic; experimental study; extracellular vesicles; immunoregulation; in vitro Assay; indexing; innovation; knock-down; macrophage; morphometry; novel; osteogenic; paracrine; targeted treatment; therapeutic target; wound; wound healing

Project Summary/Abstract Both soft and hard tissue wound healing are impaired in type 2 diabetes (T2DM). Diabetes negatively impacts fracture healing, bone regeneration and osseointegration of endosseous implants. The complex physiological changes associated with diabetes are often manifest in immunological responses to wounding and repair where macrophages play a prominent role in determining outcomes. Recent discoveries have demonstrated that the immune system is tightly linked to bone physiology and immunomodulation of bone repair is affected by key interaction involving macrophages and mesenchymal stem cells (MSCs). Yet, a fundamental knowledge gap exists with respect to the nature of and the mechanisms that govern this interaction in the presence of T2DM. My recent published study has revealed that the conditioned medium from diabetic mouse macrophages impairs osteogenic differentiation of MSCs. My studies also show that macrophages secrete phenotype-dependent extracellular vesicles (EVs) that affect the level of bone repair. Here, I hypothesize that diabetic macrophage EVs mediate specific paracrine control of osteogenesis. To test this hypothesis, I propose two independent but thematically related aims. In Aim 1, I will characterize and compare wild type mouse macrophage EVs (wtEVs) and diabetic mouse macrophage EVs (dbEVs) at the structural and functional level and define the effects of these EVs on MSC osteogenic differentiation quantitatively. I will demonstrate the role of macrophage miRNAs cargo on osteoinduction by interfering miRNA function in Argonaute 2 (involved in RISC complex formation and miRNA function) knockdown MSCs. In addition, I will validate that dbEVs contain miRNAs that negatively influence the process of osteogenesis by generating EVs from DICER (required for mature miRNA formation) knockout macrophages. In Aim 2, I will identify significantly distinct miRNAs in dbEVs, their target genes and signaling pathways involved in osteogenesis by bioinformatic approach. I will evaluate the functionality of these miRNAs on osteoinduction by utilizing mimics/antagomirs that increase or eliminate the effects of identified miRNAs. I will study selected target genes at the level of miRNA interaction to affirm the direct effects on gene regulation and downstream effects of these key miRNAs on osteoinductive pathways. Translationally, I will generate functionally engineered EVs (FEEs) by engineer the candidate miRNAs (promote osteogenesis) that rescue dbEV effects into MSC EVs. The FEEs will be characterized structurally and functionally in vitro. Further I will utilize calvarial bone defect model to evaluate the function of selected miRNAs within FEEs on bone healing in diabetic mice. Overall, these mechanistic studies will explore the significance of the macrophage EV-mediated immunomodulation that occurs between macrophages and MSCs in the context of bone healing in the presence of T2DM. These studies will refine knowledge of diabetic pathology and provide potential targets for therapeutic intervention.

项目概要/摘要 2 型糖尿病 (T2DM) 的软组织和硬组织伤口愈合均受到损害。糖尿病带来负面影响 骨折愈合、骨再生和骨内植入物的骨整合。复杂的生理 与糖尿病相关的变化通常表现在对损伤和修复的免疫反应中，其中 巨噬细胞在决定结果方面发挥着重要作用。最近的发现表明， 免疫系统与骨生理学密切相关，骨修复的免疫调节受到关键因素的影响 涉及巨噬细胞和间充质干细胞（MSC）的相互作用。然而，基本知识差距 存在于 T2DM 存在时控制这种相互作用的性质和机制。 我最近发表的研究表明，来自糖尿病小鼠巨噬细胞的条件培养基会损害 MSC 的成骨分化。我的研究还表明巨噬细胞分泌表型依赖的 影响骨修复水平的细胞外囊泡（EV）。在这里，我假设糖尿病巨噬细胞 EVs 介导成骨的特异性旁分泌控制。为了检验这个假设，我提出了两个独立但 主题相关的目标。在目标 1 中，我将表征并比较野生型小鼠巨噬细胞 EV (wtEVs) 和糖尿病小鼠巨噬细胞 EVs (dbEVs) 在结构和功能水平上并定义 这些 EV 对 MSC 成骨分化的定量影响。我来演示一下巨噬细胞的作用 通过干扰 Argonaute 2 中的 miRNA 功能（参与 RISC 复合物），miRNA 负载在骨诱导上 形成和 miRNA 功能）敲低 MSC。此外，我将验证 dbEV 包含的 miRNA 通过从 DICER（成熟 miRNA 所需）生成 EV 对成骨过程产生负面影响 形成）敲除巨噬细胞。在目标 2 中，我将识别 dbEV 中显着不同的 miRNA，它们的目标 通过生物信息学方法研究参与成骨的基因和信号通路。我将评估 通过利用模拟物/拮抗物来增加或消除这些 miRNA 对骨诱导的功能 已识别的 miRNA 的影响。我将在 miRNA 相互作用水平上研究选定的靶基因，以确认 对基因调控的直接影响以及这些关键 miRNA 对骨诱导途径的下游影响。 从翻译上讲，我将通过设计候选 miRNA 来生成功能工程 EV（FEE）（促进 成骨）将 dbEV 效应拯救到 MSC EV 中。 FEE 的特点是结构和 在体外发挥功能。此外，我将利用颅骨骨缺损模型来评估所选 miRNA 的功能 在糖尿病小鼠骨愈合的费用范围内。总的来说，这些机制研究将探讨 巨噬细胞 EV 介导的免疫调节发生在巨噬细胞和 MSC 之间 T2DM 存在时骨愈合的影响。这些研究将完善糖尿病病理学知识并提供 治疗干预的潜在目标。