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 在糖尿病性视网膜病变和AMD等疾病中对眼部血管生成至关重要。 对VEGF作用的研究主要集中在受体结合事件和VEGF的激活。 经典的下游信号转导级联。我们和其他人的工作表明，VEGF 受体（VEGFR）信号传导要复杂得多，并且替代途径涉及 VEGF的细胞内运输（图1）。我们有初步证据表明a）内体 分选、神经纤毛蛋白、分泌酶和类小泛素化似乎是VEGFR的关键调节因子 运输，B）细胞核中的VEGF来源于细胞内库，c）VEGF的易位， 亚细胞区室中的VEGF和VEGF的相对水平决定了血管生成 结果和d）粘附连接（AJs）和紧密连接处的VEGFR 1：VEGFR 2比率 (TJs)对促血管生成因子和抗血管生成因子反应的变化。根据这些观察，我们 提出了以下范式转换假说：VEGF驱动的血管通透性， 新血管形成高度依赖于靶向的亚细胞易位， 血管内皮细胞中特异性VEGF的表达以及血管内皮细胞中VEGF的相对水平 亚细胞区室决定血管渗透性和血管生成结果。我们 进一步假设，内皮特异性操纵VEGFR 1：2比率， 基因治疗将降低血管通透性，抑制异常的视网膜和脉络膜 新生血管形成我们将通过以下目标来检验这一假设。目标1： a）为了表征VEGFR贩运的来源和途径，B）鉴定核 VEGFR 1和VEGFR 2的靶点，并评估这些靶点如何促进血管生成，c） 评估VEGF的核水平如何决定血管生成结果，以及d）鉴定VEGF的核水平， 分泌酶和/或类小泛素化调节VEGF运输的机制。在目标2中， 我们将a）确定VEGF向AJs和TJ的易位是否是通过膜扩散，或 内体运输和B）评估AJs和TJ处的VEGFR 1和VEGFR 2的比率如何 对促血管生成因子和抗血管生成因子反应的变化，涉及的连接结合配偶体， 以及这对渗透性的影响。在目标3中，我们将评估内皮特异性 细胞内VEGFR水平对血管通透性和视网膜和 小鼠脉络膜血管生成。这些研究将大大推进我们的机制。 了解异常的眼部血管生成，并将确定新的潜在治疗靶点 用于抑制病理性眼部血管生成。

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.