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.

项目摘要/摘要 在2型糖尿病（T2DM）中，软组织伤口愈合均受损。糖尿病对糖尿病产生负面影响 骨折愈合，骨再生和腹腔植入物的骨整合。复杂的生理 与糖尿病相关的变化通常表现在对伤害和修复的免疫反应中 巨噬细胞在确定结果中起着重要的作用。最近的发现表明 免疫系统与骨生理密切相关，骨修复的免疫调节受钥匙的影响 涉及巨噬细胞和间充质干细胞（MSC）的相互作用。但是，基本知识差距 存在于T2DM存在下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性 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 EV介导特异性旁分泌对成骨的控制。为了检验这一假设，我提出了两个独立但 主题相关的目标。在AIM 1中，我将表征和比较野生型小鼠巨噬细胞EV （WTEVS）和糖尿病小鼠巨噬细胞EV（DBEVS）在结构和功能水平上，并定义 这些电动汽车对MSC成骨分化的影响定量分化。我将展示巨噬细胞的作用 miRNA货物通过干扰miRNA功能在Argonaute 2中（参与RISC COMFFELK中的miRNA功能） 形成和miRNA功能）敲低MSC。此外，我将验证dbevs包含miRNA 通过产生迪切尔（成熟miRNA所需的EV）来负面影响成骨的过程 形成）敲除巨噬细胞。在AIM 2中，我将在DBEV中确定明显不同的miRNA 通过生物信息学方法参与成骨的基因和信号通路。我将评估 这些miRNA在骨诱导的功能通过使用模仿/antagomirs增加或消除 鉴定的miRNA的影响。我将在miRNA相互作用水平上研究选定的靶基因，以肯定 对这些关键miRNA对骨诱导途径的基因调节和下游影响的直接影响。 翻译上，我将通过工程师候选人miRNA生成功能设计的电动汽车（费用）（促进 成骨）将DBEV效应源成MSC EV。费用将在结构上进行特征，并 在功能上体外。此外，我将利用颅骨缺陷模型来评估选定的miRNA的功能 在糖尿病小鼠的骨骼愈合费用之内。总体而言，这些机械研究将探讨 巨噬细胞EV介导的免疫调节在巨噬细胞和MSC之间发生 在T2DM存在下的骨骼愈合。这些研究将完善有关糖尿病病理学的知识，并提供 治疗干预的潜在目标。