A critical role for intracellular VEGF receptor translocation in ocular angiogenesis

集体证据表明，血管内皮生长因子(VEGF)家族是 在糖尿病视网膜病变和AMD等条件下，对眼血管生成至关重要。 对血管内皮生长因子作用的研究主要集中在受体结合事件和激活 经典的下行信号转导级联。我们和其他人的研究表明，血管内皮生长因子 受体(VEGFR)信号要复杂得多，另一种途径包括 血管内皮生长因子受体的细胞内运输(图1)。我们有初步证据表明a)内体 分选、神经粘连蛋白、分泌酶和总甲基化似乎是VEGFR的关键调节因子 贩运，b)细胞核内的血管内皮生长因子受体起源于细胞内池，c)移位 血管内皮生长因子受体和亚细胞内血管生长因子受体的相对水平决定了血管生成 结果和d)粘附性连接(AJ)和紧密连接的VEGFR1/VEGFR2比率 (TJS)对促血管生成因子和抗血管生成因子的反应变化。基于这些观察，我们 提出以下范式转换假说：血管内皮生长因子驱动的血管通透性和 新生血管高度依赖于靶向亚细胞转位 血管内皮细胞中特异的VEGFRs以及血管内皮细胞中VEGFRs的相对水平 亚细胞室决定血管的通透性和血管生成的结果。我们 进一步假设血管内皮细胞通过血管内皮细胞生长因子受体1：2的比例 基因治疗将降低血管通透性并抑制视网膜和脉络膜的异常 新生血管。我们将通过以下目标来检验这一假设。在目标1中，我们 将a)确定VEGFR贩运的来源和路线，b)确定核 VEGFR1和VEGFR2的靶点，并评估这些靶点如何促进血管生成，c) 评估VEGFRs的核水平如何决定血管生成结果和d)确定 分泌酶和/或相思作用调节血管内皮生长因子受体运输的机制。在目标2中， 我们将a)确定VEGFRs是通过膜扩散还是通过膜扩散转移到AJ和TJ 以及b)评估在AJ和TJ的VEGFR1和VEGFR2的比率 对促血管生成因子和抗血管生成因子的反应变化，所涉及的连接结合伙伴， 以及这是如何影响渗透率的。在目标3中，我们将评估内皮细胞特异性的结果 细胞内VEGFR水平对血管通透性和视网膜及 小鼠脉络膜血管生成。拟议的研究将极大地推动我们的机械化 了解异常眼血管生成并将发现新的潜在治疗靶点 用于抑制病理性眼血管生成。

The collective evidence dictates that the vascular endothelial growth factor (VEGF) family is critical for ocular angiogenesis in conditions such as diabetic retinopathy and AMD. Investigation of VEGF action has largely focused on receptor binding events and activation of classical downstream signal transduction cascades. Work from us and others show that VEGF receptor (VEGFR) signaling is much more complex and an alternative pathway involves intracellular trafficking of VEGFRs (Fig 1). We have preliminary evidence that a) endosomal sorting, neuropilin, secretases and sumoylation appear to be key regulators of VEGFR trafficking, b) VEGFRs in the nucleus originate from an intracellular pool, c) translocation of VEGFRs and the relative levels of VEGFRs within subcellular compartments dictates angiogenic outcome and d) the VEGFR1:VEGFR2 ratio at adherens junctions (AJs) and tight junctions (TJs) changes in response to pro- and antiangiogenic factors. Based on these observations we propose the following paradigm shifting hypothesis: VEGF-driven vascular permeability and neovascularization are highly dependent on the targeted subcellular translocation of specific VEGFRs in endothelial cells and that the relative levels of VEGFRs within subcellular compartments dictates vascular permeability and angiogenic outcome. We further postulate that endothelial-specific manipulation of the VEGFR1:2 ratio through gene therapy will reduce vascular permeability and inhibit aberrant retinal and choroidal neovascularization. We will test this hypothesis through the following aims. In Aim 1, we will a) To characterizing the origin and routes of VEGFR trafficking, b) identify the nuclear targets of VEGFR1 and VEGFR2 and assess how these targets contribute to angiogenesis, c) assess how the nuclear level of VEGFRs dictates the angiogenic outcome and d) identify the mechanism by which secretases, and/or sumoylation regulate trafficking of VEGFRs. In Aim 2, we will a) determine if translocation of VEGFRs to AJs and TJs is via membrane diffusion or endosomal trafficking and b) assess how the ratio of VEGFR1 and VEGFR2 at AJs and TJs changes in response to pro- and antiangiogenic factors, the junctional binding partners involved, and how this affects permeability. In Aim 3, we will assess the outcome of endothelial-specific genetic manipulation of intracellular VEGFR levels on vascular permeability and retinal and choroidal angiogenesis in mice. The proposed studies will significantly advance our mechanistic understanding of aberrant ocular angiogenesis and will identify new potential therapeutic targets for the inhibition of pathological ocular angiogenesis.