Non-canonical VEGF receptor signaling regulates retinal neovascularization

描述(申请人提供)：眼部新生血管疾病包括早产儿视网膜病变、增殖性糖尿病视网膜病变和渗出性或“湿性”形式的老年性黄斑变性(AMD)。这些疾病加在一起影响所有年龄段，是发达国家视力障碍的主要原因。大量证据表明，血管内皮生长因子(VEGF)家族在眼血管生成中起关键作用，已成为治疗干预的主要靶点。对血管内皮生长因子作用的研究主要集中在质膜上或质膜附近的受体结合事件以及随后经典的信号转导级联反应的激活。然而，从我们和其他人的工作中可以明显看出，通过与VEGFR结合的配体(VEGFR1和VEGFR2)介导的信号传递要复杂得多，涉及VEGFR的细胞内运输。我们的数据显示，VEGFRs的靶向性亚细胞转位到黏附/紧密连接(AJs/TJs)导致VEGFRs调节血管通透性，或者，如果转位到细胞核，VEGFRs可以调节促血管生成调节因子和抗血管生成调节因子的转录。初步数据还表明，特定细胞部位(如AJs/TJs和细胞核)的VEGFR1/VEGFR2的特定比率在决定血管通透性和血管生成方面至关重要。此外，内体分选、分泌酶和SUMO化似乎是VEGFR运输的关键调节因素。基于这些观察，我们提出以下假设：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特定血管内皮生长因子受体的靶向性亚细胞转位。药物或基因操作内体转运途径的组成部分、分泌酶复合体和/或SUMO化将降低血管通透性，并抑制异常的视网膜和脉络膜新生血管。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)通过表征VEGFR内化和贩运的途径来确定核VEGFRs的来源，b)确定核目标 这些研究包括：(A)检测VEGFR1和VEGFR2的表达，并评估这些靶点如何促进血管生成；c)评估核VEGFR1/VEGFR2的比例如何决定血管生成的结果；以及d)确定β-分泌酶、早老素和/或总糖基化调节VEGFR运输的机制。在目标2中，我们将a)确定VEGFRs向AJ和TJ的转位是通过膜扩散还是内体转运，以及b)评估AJ和TJ的VEGFR1和VEGFR2的比例如何随促血管生成因子和抗血管生成因子的反应而变化，所涉及的连接结合伙伴(如VE-钙粘蛋白、β-连环蛋白、claudin-5)以及这如何影响通透性。在目标3中，我们将a)评估药物或遗传调节跨膜蛋白水解酶和/或SUMO化的小鼠VEGFR亚细胞定位和VEGFR1/VEGFR2比例的变化，并确定这是否可以防止VEGF诱导的小鼠视网膜血管通透性和/或视网膜或脉络膜血管生成，以及b)在亚目标1B中发现的新的核信号通路在小鼠血管生成模型中的调制。了解这一新的非典型血管内皮生长因子信号通路(S)将提供关于血管生成的新信息，并有助于开发可持续治疗AMD和糖尿病视网膜病变的策略。 公共卫生相关性：眼部新生血管疾病包括早产儿视网膜病变、增殖性糖尿病视网膜病变和渗出性或“湿性”形式的老年性黄斑变性(AMD)。这些疾病加在一起影响所有年龄段，是发达国家视力障碍的主要原因。大量证据表明，血管内皮生长因子(VEGF)家族在眼血管生成中起关键作用，已成为治疗干预的主要靶点。我们发现了一条新的非规范的血管内皮细胞生长因子信号通路，它需要血管内皮生长因子受体1和2的亚细胞靶向转位。我们认为，亚细胞内VEGFR1/VEGFR2的比例决定了血管生成的结果，这依赖于内体的分选，并被β-分泌酶和总甲基化“微调”。药物或基因操作将降低血管通透性，抑制视网膜和脉络膜新生血管的形成。了解血管内皮生长因子信号通路将为AMD和糖尿病视网膜病变的发病机制提供新的信息，并可能有助于为这些衰弱的疾病制定可持续的治疗策略，这是NEI的优先领域。

DESCRIPTION (provided by applicant): Neovascular diseases of the eye include retinopathy of prematurity, proliferative diabetic retinopathy, and the exudative or "wet" form of age-related macular degeneration (AMD). Together these diseases affect all age groups and are the leading causes of vision impairment in developed nations. The collective evidence suggests that the vascular endothelial growth factor (VEGF) family is critical for ocular angiogensis and this has become a major target for therapeutic intervention. Investigation of VEGF action has largely focused on receptor binding events at or near the plasma membrane and subsequent activation of classical signal transduction cascades. However, it is now apparent from our work and others that the signaling mediated by ligands binding to the VEGFRs (VEGFR1 and VEGFR2) is much more complex and involves intracellular trafficking of VEGFRs. Our data shows that targeted subcellular translocation of VEGFRs to adherens/tight junctions (AJs/TJs) results in the VEGFRs regulating vascular permeability or, if translocated to the nucleus VEGFRs can regulate transcription of pro- and anti-angiogenic regulators. Preliminary data also indicate that the specific ratio of VEGFR1:VEGFR2 at specific cell sites (such as AJs/TJs and the nucleus) is critical in determining vascular permeability and angiogenesis. Furthermore, endosomal sorting, ¿-secretase and SUMOylation appear to be key regulators of VEGFR trafficking. Based on these observations we propose the following hypothesis: VEGF-driven vascular permeability and neovascularization are highly dependent on the targeted subcellular translocation of specific VEGFRs. Pharmacological or genetic manipulation of components of the endosomal trafficking pathway, ?-secretase complex and/or SUMOylation 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) determine the origin of nuclear VEGFRs by characterizing the routes of VEGFR internalization and trafficking, b) identify the nuclear targets of VEGFR1 and VEGFR2 and assess how these targets contribute to angiogenesis, c) assess how the ratio of nuclear VEGFR1:VEGFR2 dictates the angiogenic outcome and d) identify the mechanism by which ?- secretase, presenilin 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 (e.g. VE-cadherin, ?- catenin, claudin-5) involved and how this affects permeability. In Aim 3, we will a) assess the changes in VEGFR subcellular localization and VEGFR1:VEGFR2 ratio in mice which have undergone pharmacological or genetic modulation of transmembrane proteases and/or SUMOylation and determine if this can prevent VEGF-induced retinal vascular permeability and/or retinal or choroidal angiogenesis in mice and b) evaluate the modulation of novel nuclear signaling pathways identified in sub Aim 1B in mouse models of angiogenesis. Understanding this novel non-canonical VEGF signalling pathway(s) will provide new information on angiogenesis and allow development of a sustainable treatment strategy for AMD and diabetic retinopathy. PUBLIC HEALTH RELEVANCE: Neovascular diseases of the eye include retinopathy of prematurity, proliferative diabetic retinopathy, and the exudative or "wet" form of age-related macular degeneration (AMD). Together these diseases affect all age groups and are the leading causes of vision impairment in developed nations. The collective evidence suggests that the vascular endothelial growth factor (VEGF) family is critical for ocular angiogensis and this has become a major target for therapeutic intervention. We have identified a novel non-canonical VEGF signalling pathway that requires targeted subcellular translocation of VEGF receptors 1 and 2. We believe that the ratio of VEGFR1:VEGFR2 within subcellular compartments dictates angiogenic outcome and that this is dependent on endosomal sorting and is "fine tuned" by ?-secretase and sumoylation. Pharmacological or genetic manipulation at different points along this non-canonical pathway will reduce vascular permeability and inhibit aberrant retinal and choroidal neovascularization. Understanding the VEGF signalling pathways will provide new information on the pathogenesis of AMD and diabetic retinopathy and may help develop a sustainable treatment strategy for these debilitating conditions which is a priority area for the NEI.