Role of VEGFR2 trafficking in pathological angiogenesis in age related macular degeneration

摘要 湿性老年性黄斑变性(AMD)是导致老年患者视力丧失的主要原因。在湿性AMD中， 血管内皮生长因子(VEGF)过多会导致异常血管生成和血管渗漏， 这反过来又会损害视网膜。这项提议的总体目标是确定机制的过度程度 血管内皮生长因子通过以下途径诱导湿性AMD从静止血管向病理性渗漏血管生成转变 研究VEGFR2贩运的基本作用。静止期健康人的血管生成受到抑制 血管内皮细胞(ECs)，其中VEGFR2的运输受到与黏附蛋白VE-cadherin相互作用的限制 连接点(AJ)。相反，在血管生成的内皮细胞中，明显的VEGFR2转运是明显的，其中VEGFR2 移位到丝状足尖，向血管内皮生长因子配体延伸。我们提供了第一个证据表明VEGFR2 是由Kinesin-3家族蛋白KIF13B(一种微管+端马达)直接运输到丝状足部的 萌芽中的ECs。基于我们的发现，我们将检验我们的中心假设，即KIF13B介导VEGFR2 从AJ向外贩运以导致AJ解体和血管渗漏，以及定向贩运 VEGFR2丝状伪足诱导湿性AMD病理性血管生成我们的 特定的目标将检验以下假设：1)KIF13B介导的来自AJ的VEGFR2贩运打破了 VEGFR2和VE-cadherin之间的关键相互作用参与稳定AJ，从而诱导AJ解体 和血管渗漏。2)血管内皮细胞生长因子信号转导KIF13B介导的VEGFR2向丝足的定向转运 延伸到血管内皮生长因子，而运输是萌芽血管生成所必需的。3)KIF13B介导的VEGFR2 贩运是湿性AMD的发病机制，因此抑制贩运是一种很有前途的治疗策略。 湿性AMD。为了严格测试这些假说，我们的实验室已经开发出了强大的工具(遗传老鼠模型 和多肽抑制剂)，这导致了概念上的进步。EC特异性KIF13B基因敲除小鼠显示 病理背景下的选择性血管生成缺陷。一种干扰KIF13B/VEGFR2的小肽抑制剂 相互作用，称为KAI，抑制了湿性AMD模型小鼠的脉络膜新生血管(CNV)。使用这些 强大的工具，我们将研究KIF13B在血管内皮生长因子诱导的眼血管通透性中的作用 血管内皮生长因子受体2在体外脉络膜萌发中定向转运的成像和湿性AMD的病理， 以血管生成异常、血管渗漏、炎症为特征，采用激光诱导的CNV模型。 如果成功，我们提出的研究提供了通过靶向VEGFR2来调节血管生成的新概念 贩卖人口。

ABSTRACT Wet age-related macular degeneration (AMD) is a leading cause of vision loss in old patients. In wet AMD, excessive vascular endothelial growth factor (VEGF) causes abnormal angiogenesis and vascular leakage, which in turn damages the retina. The overall goal of this proposal is to determine the mechanism how excessive VEGF induces transition from quiescent blood vessels to pathological leaky angiogenesis in wet AMD by studying the fundamental roles of VEGFR2 trafficking. Angiogenesis is restrained in quiescent healthy endothelial cells (ECs), where VEGFR2 trafficking is limited by the interaction with VE-cadherin at adherens junctions (AJs). In contrast, marked VEGFR2 trafficking is evident in angiogenic ECs, where VEGFR2 translocates to filopodia tips that extend towards the VEGF ligand. We provided the first evidence that VEGFR2 is directly transported by the kinesin-3 family protein, KIF13B, a microtubules plus-end motor, to filopodia of sprouting ECs. Based on our finding, we will test our central hypothesis that KIF13B mediates VEGFR2 trafficking away from AJs to induce AJ disassembly and vascular leakage, and the directional trafficking of VEGFR2 to filopodia induces pathological angiogenesis in response to excessive VEGF in wet AMD. Our Specific Aims will test the following hypotheses; 1) KIF13B-mediated VEGFR2 trafficking from AJs breaks the critical interaction between VEGFR2 and VE-cadherin involved in stabilizing AJs, thus induces AJ disassembly and vascular leakage. 2) VEGF signaling induces KIF13B-mediated directional trafficking of VEGFR2 to filopodia extending toward VEGF, and the trafficking is required for sprouting angiogenesis. 3) KIF13B-mediated VEGFR2 trafficking is pathogenesis in wet AMD, thus the inhibition of the trafficking is a promising strategy for the therapy of wet AMD. To rigorously test these hypotheses, our lab has generated powerful tools (genetic mouse models and peptide inhibitors) that have led to conceptual advances. EC specific KIF13B knockout mice display a selective angiogenic defect in the pathological setting. A small peptide inhibitor disrupting the KIF13B/VEGFR2 interaction, termed KAI, inhibited choroidal neovascularization (CNV) in wet AMD model in mice. Using these powerful tools, we will examine the roles of KIF13B in VEGF-induced permeability of ocular blood vessels, live imaging of directional VEGFR2 trafficking in choroidal sprouting ex vivo, and pathology of wet AMD, characterized by abnormal angiogenesis, vascular leakage, and inflammation, using laser-induced CNV model. If successful, our proposed studies provide the novel concept of angiogenesis regulation by targeting VEGFR2 trafficking.