Mechanisms regulating VEGF receptors in diabetic angiogenesis

 描述（由申请人提供）：糖尿病是一种慢性和复杂的代谢性疾病。慢性高葡萄糖暴露在伤口愈合期间产生受损的血管生成/淋巴管生成，导致下肢皮肤溃疡（非创伤性下肢截肢的主要原因），并使糖尿病成为发病率和死亡率的主要原因。血管内皮生长因子受体（VEGFR 2/3）是血管生成和淋巴管生成的关键调节因子。重要的是，糖尿病患者血管内皮中的VEGFR 2/3显著减少。然而，负责VEGFR 2/3损失的机制仍然知之甚少。据报道，长时间的高葡萄糖暴露诱导高尔基体定位的VEGFR 2的配体非依赖性降解;然而，降解途径尚未确定。有趣的是，我们揭示了糖尿病状况会提高自噬体组分Ulk 1和LC 3B，并诱导VEGFR 2/3靶向自噬体。鉴于VEGFR 2/3耗竭导致糖尿病伤口愈合受损，自噬体是否介导高尔基体定位的VEGFR 2的降解是一个非常重要和开放的问题。我们的最新数据显示，在生理条件下，对于配体诱导的VEGFR 2/3内化和降解至关重要的内吞衔接蛋白epsins被上调，并与糖尿病自噬体中的Ulk 1/LC 3B和VEGFR 2/3相互作用。Epsin缺乏抑制糖尿病诱导的细胞表面VEGFR 2/3的损失，而不依赖于VEGF。因此，我们假设，胰蛋白酶通过将VEGFR 2/3靶向自噬体来促进细胞表面VEGFR 2/3的降解，并且自噬降解下调新合成的高尔基体定位的和细胞表面VEGFR 2/3以有效地降低糖尿病内皮中的VEGFR 2/3水平。最后，我们假设破坏epsins和Ulk 1以保护VEGFR 2/3免受糖尿病诱导的自噬降解可能提供一种新的治疗策略来对抗延迟的糖尿病伤口愈合。在目标1中，我们将确定糖尿病诱导的细胞内VEGFR 2/3降解的机制。拟议的研究将确定LC 3B和Ulk 1介导糖尿病内皮细胞中高尔基体定位的VEGFR 2/3降解的新机制。在目标2中，我们将确定糖尿病诱导的细胞表面VEGFR 2/3自噬降解的机制。这些研究将提供关于epsins和Ulk 1/LC 3B如何协同调节配体非依赖性细胞表面VEGFR 2/3降解的新信息。在目标3中，我们将在我们的新型db/db和秋田糖尿病小鼠模型中使用体外血管生成测定和体内糖尿病伤口愈合和基质胶塞测定来检查内皮细胞epsin和Ulk 1缺失动物在糖尿病血管生成中的治疗潜力，所述糖尿病小鼠模型具有内皮细胞epsin和Ulk 1的组合条件性耗竭。这些研究将揭示epsin和Ulk 1丢失在糖尿病中的有益作用，并测试epsin和Ulk 1抑制在治疗缺陷性糖尿病外周血管生成中的组合治疗潜力。

 DESCRIPTION (provided by applicant): Diabetes is a chronic and complex metabolic disease. Chronic high glucose exposure produces impaired angiogenesis/lymphangiogenesis during wound healing resulting in skin ulcerations of the lower extremities (leading cause of non-traumatic lower limb amputation) and making diabetes a leading cause of morbidity and mortality. Vascular endothelial growth factor receptors (VEGFR2/3) are critical regulators of angiogenesis and lymphangiogenesis. Importantly, VEGFR2/3 is significantly reduced in the vascular endothelium of diabetic patients. However, the mechanisms responsible for the VEGFR2/3 loss remain poorly understood. Prolonged high glucose exposure reportedly induces ligand-independent degradation of Golgi-localized VEGFR2; however the degradative pathway has not been defined. Intriguingly, we unveil that diabetic conditions elevate autophagosome components, Ulk1 and LC3B and induce VEGFR2/3 targeting to autophagosomes. Given that VEGFR2/3 depletion drives impaired diabetic wound healing, whether autophagosomes mediate degradation of Golgi-localized VEGFR2 is a highly significant and open question. Our latest data show that epsins, endocytic adaptor proteins critical for ligand-induced VEGFR2/3 internalization and degradation in physiologic conditions, are upregulated and interact with Ulk1/LC3B and VEGFR2/3 in autophagosomes in diabetes. Epsin deficiency inhibits diabetes-induced loss of cell surface VEGFR2/3 independent of VEGF. Thus, we hypothesize that epsins promote the degradation of cell surface VEGFR2/3 by targeting VEGFR2/3 to autophagosomes, and autophagic degradation downregulates both new synthesized Golgi-localized and cell surface VEGFR2/3 to effectively decrease VEGFR2/3 levels in diabetic endothelium. Lastly, we hypothesize that disrupting epsins and Ulk1 to protect VEGFR2/3 from diabetic-induced autophagic degradation may offer a new therapeutic strategy to combat retarded diabetic wound healing. In Aim 1, we will determine mechanisms underlying diabetes-induced degradation of intracellular VEGFR2/3. The proposed studies will identify novel mechanisms by which LC3B and Ulk1 mediate degradation of Golgi-localized VEGFR2/3 in the diabetic endothelium. In Aim 2, we will determine mechanisms of diabetes-induced cell surface VEGFR2/3 autophagic degradation. These studies will provide novel information on how epsins and Ulk1/LC3B cooperatively regulate ligand-independent cell surface VEGFR2/3 degradation. In Aim 3, we will examine the therapeutic potential of epsins and Ulk1 null animals in diabetic angiogenesis using in vitro angiogenesis assays and in vivo diabetic wound healing and Matrigel plug assays in our novel db/db and Akita diabetic mouse models with combinatorial conditional depletion of endothelial epsins and Ulk1. These studies will shed light upon the beneficial effects of epsins and Ulk1 loss in diabetes, and test the combinatory therapeutic potential of epsin and Ulk1 inhibition in the treatment of defective diabetic peripheral angiogenesis.