Macrophages, Cell-Cell Communication, Ischemic Injury in Diabetes and the RAGE/DIAPH1 Signaling Axis

项目总结：总体 缺血是糖尿病心血管(CVD)和外周动脉病变(PAD)的并发症 通过单核/巨噬细胞的募集、渗透和激活(MΦS)，进入受影响的组织。在……里面 糖尿病，MΦ的特性受到干扰，修复显著减轻，导致器官衰竭。这个 心脏和骨骼肌中的微环境对缺血表现出独特的反应，但 机制还没有完全弄清楚。晚期糖基化终产物受体(RAGE)的配体， 例如非酶糖化和氧化晚期糖基化终末产物；S100/钙颗粒蛋白和更高 迁移率组框1，它在非糖尿病和糖尿病的CVD和PAD组织中蓄积，并本身RAGE， 有助于MΦ和特定的生态位对缺血的反应。在全球范围内缺乏Ager(编码基因)的小鼠 AGE)或无髓系老化(致命性照射/骨髓移植)可免受不利影响 结扎冠状动脉左前降支和股动脉对心肌梗死模型的影响 脑梗塞(MI)和后肢缺血(HLI)。在MI和HLI模型中，伴随Ager缺失 组织MΦ含量明显减少，炎性介质表达减少。令人惊讶的是，在 HLI，AGER缺失伴随M-Φ含量和炎性介质表达增加 骨骼肌。然而，在这两种情况下，Ager缺失预示着修复。我们的发现是胞质结构域 RAGE与福尔明DIAPH1相互作用，介导信号转导，氧化应激的产生和 线粒体功能障碍(由于我们新发现DIAPH1与Mfn2结合在一起 缺血组织MΦS，心肌细胞和内皮细胞)，可能是这些RAGE依赖的关键 调查结果。该计划的三个项目将使用新的小鼠模型、最先进的分子生物学 技术、新型RAGE-DIAPH1相互作用小分子拮抗剂、核磁共振波谱和细胞内 荧光分析验证RAGE/DIAPH1参与M-Φ细胞固有和M-Φ-1的假设 心肌梗死中的心肌细胞串扰和HLI中的TO和MΦ-内皮细胞串扰，从而放大组织 损坏。我们假设RAGE-DIAPH1和DIAPH1-Mfn2相互作用控制M-Φ炎症，并且 药理阻断RAGE-DIAPH1-Mfn2相互作用和/或单核细胞给药/MΦS缺失 AGER或DIAPH1将促进从促损伤向适应性MΦ炎症的转变，从而加速 糖尿病心脏和外周动脉系统的组织修复。体内生物学的精心整合 而结构生物学的分子机制研究(项目1和2)(项目3)确保了创新， 这项工作对开发新的糖尿病治疗策略的意义和最终相关性 和脑缺血。

Project Summary: OVERALL Ischemia, a complication of diabetic cardiovascular (CVD) and peripheral arterial disease (PAD), is accompanied by the recruitment, infiltration and activation of monocytes/macrophages (MΦs), into affected tissues. In diabetes, MΦ properties are perturbed and repair is significantly mitigated, leading to organ failure. The microenvironments in the heart vs. the skeletal muscle display unique responses to ischemia, but the mechanisms are not fully understood. The ligands of the receptor for advanced glycation endproducts (RAGE), such as nonenzymatically glycated and oxidized advanced glycation endproducts; S100/calgranulins and high mobility group box 1, which accumulate in non-diabetic and diabetic CVD and PAD tissue, and RAGE itself, contribute to MΦ and niche-specific responses to ischemia. Mice globally devoid of Ager (the gene encoding RAGE) or devoid of myeloid Ager (lethal irradiation/bone marrow transplantation) are protected from the adverse effects of ligation of the left anterior descending coronary artery and the femoral artery, models for myocardial infarction (MI) and hind limb ischemia (HLI), respectively. In MI and HLI models, Ager deletion is accompanied by a marked reduction in tissue MΦ content and reduced expression of inflammatory mediators. Surprisingly, in HLI, deletion of Ager is accompanied by increased MΦ content and expression of inflammatory mediators in the skeletal muscle. Yet, in both cases, Ager deletion augured repair. Our discovery that the cytoplasmic domain of RAGE interaction with the formin, DIAPH1, mediates signal transduction, generation of oxidative stress and mitochondrial dysfunction (on account of our novel discovery that DIAPH1 binds to Mitofusin2 (MFN2) in ischemic tissue MΦs, cardiomyocytes and endothelial cells), may hold the key to these RAGE-dependent findings. The three Projects of this Program will use novel mouse models, state-of-the-art molecular biology techniques, novel small molecule antagonists of RAGE-DIAPH1 interaction, NMR spectroscopy and in-cell fluorescence assays to test the hypothesis that RAGE/DIAPH1 contributes to MΦ cell-intrinsic and MΦ- cardiomyocyte cross talk in MI and to and MΦ-endothelial cell cross talk in HLI, thereby amplifying tissue damage. We posit that RAGE-DIAPH1 and DIAPH1-MFN2 interactions control MΦ inflammation and that pharmacological blockade of RAGE-DIAPH1-MFN2 interaction and/or administration of monocytes/MΦs devoid of Ager or Diaph1 will facilitate the transition from pro-injury to adaptive MΦ inflammation and, thereby, hasten tissue repair in the diabetic heart and peripheral arterial systems. The meticulous integration of in vivo biology and molecular mechanisms studies (Projects 1 and 2) with structural biology (Project 3) assures the innovation, significance and ultimate relevance of this work for the development of novel therapeutic strategies for diabetes and ischemia.