Project 2: Diabetes, RAGE/DIAPH1 and Hind Limb Ischemia

项目摘要：项目2 1型和2型糖尿病的发病率正在上升，这将导致大血管和微血管的增加 并发症。糖尿病是外周动脉疾病(PAD)的主要原因，也是 截肢指或四肢的截肢到目前为止，还没有有效的治疗方法。先进细胞因子受体的配体 糖基化终产物(RAGE)，如非酶糖化蛋白(AGEs)、S100/钙颗粒蛋白和HIGH 迁移组盒1(HMGB1)，在非糖尿病患者体内蓄积，但在糖尿病患者的PAD组织中尤为明显。在人类中 受试者，RAGE及其配体在心血管疾病(CVD)和PAD组织中上调，在 细胞类型，但尤其是单核/巨噬细胞(MΦS)和内皮细胞。小鼠后肢缺血模型 (HLI)是一种单侧结扎和切除周围血管系统对周围血管系统造成缺血性损伤的模型。 股动脉(FAL)，全球缺失Ager(编码RAGE的基因)的小鼠显示出显著的增加 糖尿病和非糖尿病与野生型(WT)骨骼肌病变的血流和血管生成 老鼠。同时，令人惊讶的是，AGER缺失增加了炎性单核细胞亚群，巨噬细胞(MΦ) 受影响的骨骼肌中的含量和炎症。相反，在动脉粥样硬化的小鼠和心肌梗死中 组织(项目1)，显著减少MΦ组织含量和伴随组织修复的炎症，从而 揭示了调节愤怒依赖的炎症反应的新的利基特定力量。细胞质 RAGE的结构域与转导RAGE配体刺激的信号转导的Forin DIAPH1结合； 初步数据显示，全球缺乏DIAPH1的小鼠在HLI后表现出显著的血流量增加 与WT小鼠比较。此外，我们新发现的DIAPH1与Mitofusin2(Mfn2)结合将RAGE/DIAPH1与 线粒体特性和缺血后组织动态平衡的各种后果。本计划 项目首次显示RAGE、DIAPH1和MΦS在人类冠状动脉粥样硬化中共定位 动脉。我们假设RAGE/DIAPH1/Mfn2特异性信号来自渗透的免疫细胞和/或细胞 微环境介导M-ΦS和组织内皮细胞固有和/或细胞-细胞串扰机制 HLI/FAL中的细胞(ECs)，加重组织损伤，抑制修复。我们将采用新颖的AGER和DIAPH1 小鼠，RAGE-DIAPH1相互作用的小分子拮抗剂和最新的分子技术 揭示糖尿病PAD的发病机制，寻找新的治疗靶点和策略。项目2将 与项目1和3以及两个核心密切合作，以实现这些目标。

Project Summary: Project 2 The incidence of types 1 and 2 diabetes is on the rise, which will lead to increased macro- and microvascular complications. Diabetes is a leading cause of peripheral arterial disease (PAD), a significant risk factor for amputations of digits or limbs. To date, there are no effective therapies. The ligands of the receptor for advanced glycation endproducts (RAGE), such as nonenzymatically glycated proteins (AGEs), S100/calgranulins and high mobility group box 1 (HMGB1), accumulate in non-diabetic, but especially in diabetic PAD tissues. In human subjects, RAGE and its ligands are upregulated in cardiovascular disease (CVD) and PAD tissues, in multiple cell types, but especially in monocytes/macrophages (MΦs) and endothelial cells. In murine hind limb ischemia (HLI), a model of ischemic injury to the peripheral vascular system by unilateral ligation and excision of the femoral artery (FAL), mice globally devoid of Ager (the gene encoding RAGE) display significant increases in blood flow and angiogenesis in the affected skeletal muscle in diabetes and non-diabetes vs. wild type (WT) mice. In parallel, and surprisingly, Ager deletion increased inflammatory monocyte subsets, macrophage (MΦ) content and inflammation in affected skeletal muscle. In contrast, in atherosclerotic mice and in myocardial infarct tissue (Project 1), significantly reduced MΦ tissue content and inflammation accompanied tissue repair, thereby unveiling novel niche-specific forces that regulate RAGE-dependent inflammatory responses. The cytoplasmic domain of RAGE binds to the formin, DIAPH1, which transduces RAGE ligand-stimulated signal transduction; preliminary data show that mice globally devoid of Diaph1 display significant increases in blood flow after HLI vs. WT mice. Further, our novel observation that DIAPH1 binds to Mitofusin2 (MFN2) links RAGE/DIAPH1 to mitochondrial properties and the myriad consequences for tissue homeostasis after ischemia. This Program Project shows for the first time that RAGE, DIAPH1 and MΦs co-localize in human atherosclerosis in the coronary artery. We hypothesize that RAGE/DIAPH1/MFN2-specific cues from infiltrating immune cells and/or the cellular microenvironment mediate cell-intrinsic and/or cell-cell cross-talk mechanisms in MΦs and in tissue endothelial cells (ECs) in HLI/FAL, which aggravate tissue damage and quell repair. We will employ novel Ager and Diaph1 floxed mice, small molecule antagonists of RAGE-DIAPH1 interaction and state-of-the-art molecular techniques to uncover mechanisms of diabetic PAD and to identify novel therapeutic targets and strategies. Project 2 will work closely with Projects 1 and 3 and the two Cores to achieve these goals.