Mechanisms regulating VEGF receptors in diabetic angiogenesis

项目摘要/摘要 在美国，糖尿病影响着3000多万人，每年因糖尿病造成的直接损失高达3270亿美元 医疗成本和生产力损失。糖尿病对血管有不良影响，表现为高血糖和胰岛素 抵抗力是动脉粥样硬化、周围神经病变、视网膜病变和 外周动脉疾病。外周动脉疾病是一种慢性疾病，脂肪沉积称为斑块。 在通往腿部的动脉中积聚，导致溃疡和感染，这是85%糖尿病的先兆- 相关截肢。我们的目标是了解慢性高血糖如何损害血管内皮细胞 识别分子靶点的功能，这些靶点将形成治疗溃疡的新治疗方法的基础 以及糖尿病的其他血管并发症。血管内皮生长因子受体2和3 血管内皮生长因子受体(VEGFR2/3)是血管生长或血管生成的重要调节因子。这些受体显著地 糖尿病患者血管内皮细胞减少，导致血管生成不足。在我们的 在之前资助的申请中，我们表明糖尿病条件诱导自噬小体的表达 蛋白和促进VEGFR2/3的降解。特别是，我们鉴定了UNC-51蛋白样的自噬 激活激酶1(Ulk1)作为重要的血管生成抑制因子刺激自噬小体形成 导致VEGFR2/3的选择性降解。内皮细胞Ulk1的丧失提高了VEGFR2/3的水平 增强血管生成反应，如内皮细胞增殖、迁移和管状形成。在这 在竞争续订申请时，我们提供了令人信服的初步数据，展示了叉头箱O1 转录因子FoxO1在体内和体外糖尿病模型中调控内皮细胞Ulk1的表达 内皮型FoxO1的缺失抑制了自噬小体的形成。我们还向您展示了 非编码RNA miR183-3p抑制内皮细胞FoxO1表达及内皮蛋白1和2接头缺失 蛋白质促进FoxO1泛素化和糖尿病的降解。这些发现有力地表明 靶向FoxO1保护VEGFR2/3不被降解可能是一种新的治疗策略 糖尿病溃疡的血管生成不足。有鉴于此，我们将调查以下具体目标 利用独特的突变小鼠和体外糖尿病模型：1)确定分子机制 FoxO1介导的糖尿病新生血管抑制的基础，2)确定分子 调节糖尿病内皮细胞FoxO1活性的机制，以及3)决定治疗潜力 通过基因缺失或miR183-3p介导的抑制糖尿病血管生成来靶向FoxO1。我们的发现 将加强对VEGFR2/3缺失和FoxO1激活背后的细胞机制的理解 调节糖尿病的血管损伤。我们预计针对FoxO1的疗法可能对 恢复外周血管生成，以改善糖尿病相关的血管并发症。

PROJECT SUMMARY/ABSTRACT Diabetes affects over 30 million people in the United States and costs a staggering $327 billion a year in direct medical costs and lost productivity. Diabetes adversely affects blood vessels as hyperglycemia and insulin resistance are key players in the development of atherosclerosis, peripheral neuropathy, retinopathy, and peripheral artery disease. Peripheral artery disease is a chronic condition where fatty deposits called plaques build up in the arteries to the legs, resulting in ulcerations and infections, which precedes 85% of diabetes- related amputations. Our goal is to understand how chronic hyperglycemia impairs vascular endothelial cell function to identify molecular targets that will form the basis for new therapeutic approaches to treat ulcerations and the other vascular complications of diabetes. Vascular endothelial growth factor receptors 2 and 3 (VEGFR2/3) are critical regulators of blood vessel growth or angiogenesis. These receptors are significantly reduced in the vascular endothelium of diabetic patients, resulting in inadequate angiogenesis. In our previously funded application, we showed that diabetic conditions induced expression of autophagosome proteins and promoted degradation of VEGFR2/3. In particular, we identified the protein Unc-51-like autophagy activating kinase 1 (Ulk1) as an important inhibitor of angiogenesis by stimulating autophagosome formation causing selective degradation of VEGFR2/3. Loss of endothelial Ulk1 elevated VEGFR2/3 levels and enhanced angiogenic responses such as endothelial cell proliferation, migration, and tube formation. In this competing renewal application, we present compelling preliminary data demonstrating the Forkhead box O1 transcription factor (FoxO1) controls expression of endothelial Ulk1 in both in vivo and in vitro diabetic model systems and that deficiency of endothelial FoxO1 inhibits autophagosome formation. We also show the noncoding RNA miR183-3p inhibits endothelial FoxO1 expression and the deficiency of epsin 1 and 2 adaptor proteins promotes FoxO1 ubiquitination and degradation in diabetes. These findings strongly suggest that targeting FoxO1 to protect VEGFR2/3 from degradation may represent a novel therapeutic strategy to prevent inadequate vascularization in diabetic ulcers. In view of that, we will investigate the following Specific Aims using unique mutant mice as well as in vitro models of diabetes: 1) determine the molecular mechanisms underlying FoxO1-mediated inhibition of neovascularization in diabetes, 2) determine the molecular mechanisms regulating FoxO1 activity in the diabetic endothelium, and 3) determine the therapeutic potential of targeting FoxO1 by genetic deletion or miR183-3p-mediated inhibition in diabetic angiogenesis. Our findings will enhance understanding of the cellular mechanisms behind VEGFR2/3 loss and activation of FoxO1 in regulating blood vessel damage in diabetes. We anticipate that therapies targeting FoxO1 may be useful for restoring peripheral angiogenesis to ameliorate the vascular complications associated with diabetes.