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

摘要 湿性年龄相关性黄斑变性（AMD）是老年患者视力丧失的主要原因。在湿性AMD中， 过量的血管内皮生长因子（VEGF）引起异常的血管生成和血管渗漏， 这反过来又会损害视网膜。这一建议的总体目标是确定该机制如何过度 VEGF诱导湿性AMD中从静止血管到病理性渗漏血管生成的转变 研究VEGFR 2贩运的基本作用。血管生成在静止的健康状态下受到抑制 内皮细胞（EC），其中VEGFR 2的运输受到粘附细胞上与VE-钙粘蛋白相互作用的限制。 连接（AJs）。相比之下，在血管生成EC中明显的VEGFR 2运输，其中VEGFR 2 易位到向VEGF配体延伸的丝状伪足尖端。我们提供了第一个证据， 由驱动蛋白-3家族蛋白KIF 13 B（一种微管加端马达）直接转运至 新生内皮细胞基于我们的发现，我们将检验我们的中心假设，即KIF 13 B介导VEGFR 2 从AJ运输出来，诱导AJ分解和血管渗漏， 丝状伪足的VEGFR 2诱导湿性AMD中响应于过量VEGF的病理性血管生成。我们 具体目的将检验以下假设：1）来自AJs的KIF 13 B介导的VEGFR 2运输打破了细胞的增殖能力。 VEGFR 2和VE-钙粘蛋白之间的关键相互作用参与了AJ的稳定，从而诱导AJ解体 和血管渗漏2)VEGF信号转导诱导KIF 13 B介导的VEGFR 2向丝状伪足的定向运输 向VEGF延伸，并且运输是萌芽血管生成所需的。3)KIF 13 B介导的VEGFR 2 在湿性AMD的发病机制中，运输是一个有前途的治疗策略，因此抑制运输是一个有前途的策略 湿性AMD为了严格验证这些假设，我们的实验室已经开发出了强大的工具（遗传小鼠模型 和肽抑制剂），这导致了概念上的进步。EC特异性KIF 13 B敲除小鼠显示出 病理环境中的选择性血管生成缺陷。破坏KIF 13 B/VEGFR 2的小肽抑制剂 相互作用，称为KAI，抑制小鼠湿性AMD模型中的脉络膜新生血管形成（CNV）。使用这些 作为一种强有力的工具，我们将研究KIF 13 B在VEGF诱导的眼血管通透性、活细胞通透性和血管通透性中的作用。 离体脉络膜发芽中定向VEGFR 2运输的成像，以及湿性AMD的病理学， 其特征在于异常血管生成、血管渗漏和炎症。 如果成功的话，我们提出的研究提供了通过靶向VEGFR 2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.