Investigating the role of active Rap1a binding IQGAP1 in choroidal neovascularization

项目摘要 新生血管性年龄相关性黄斑变性（nvAMD）是个体中心视力丧失的主要原因 超过50岁的人当脉络膜内皮细胞（CEC）穿过视网膜时，发生视力丧失。 色素上皮（RPE）进入神经感觉视网膜并形成脉络膜新生血管（CNV）。电流 标准治疗包括抑制血管内皮生长因子（VEGF）生物活性的药物。 虽然抗VEGF药物可以减少CNV并改善患者的视力，但仍存在一些问题。 关于长期VEGF抑制对视网膜神经元和神经胶质健康可能产生的不良影响。因此，在本发明中， 更好地理解CEC中的下游VEGF信号传导是必要的， 治疗方法。下游效应子之一是Rac 1，它是一种Rho GTP酶，在GTP结合状态下 CEC调节迁移。VEGF介导的Rac 1GTP通过结合含有GTP的IQ基序来维持 激活蛋白1（IQGAP 1），一种多结构域GTP酶结合蛋白，在特定结构域。因此，我们认为， 干扰Rac 1GTP/IQGAP 1结合可能是抑制CEC迁移和阻止视力的一种方法。 威胁CNV培养的CEC表达组成型活性Rap 1a，一种也结合 IQGAP 1降低了VEGF介导的Rac 1GTP和Rac 1GTP/IQGAP 1相互作用。这些结果一起 提示CEC中Rap 1aGTP/IQGAP 1结合增加减少VEGF诱导的Rac 1GTP，CEC 通过干扰Rac 1GTP/IQGAP 1结合来抑制细胞迁移和CNV。该提案将确定机械 Rap 1GTP结合IQGAP 1的IQ结构域对Rac 1GTP/IQGAP 1相互作用在特异性目的1和 特异性靶向2中的体外血管生成。在具体目标3中，这一建议将确定以下机制的作用： Rap 1aGTP/IQGAP 1在CNV发展过程中的相互作用方法包括：分离 来自成人供体眼的CEC;用突变IQGAP 1构建体转染CEC;药理学 Rac 1和Rap 1激活;免疫共沉淀;蛋白质印迹;基质胶迁移试验;管形成 试验;增殖试验; Rap 1b缺失小鼠和他莫昔芬诱导的内皮-IQGAP 1敲除小鼠; 小鼠激光诱导的CNV模型;玻璃体内注射激活Rap 1的试剂;流式细胞术; RPE/脉络膜平片和冷冻切片免疫组织化学; Micron IV成像;和光谱域 光学相干断层扫描这些研究将阐明一种潜在的调节机制， Rac 1GTP/IQGAP 1相互作用，在VEGF诱导的CEC迁移和CNV中起重要作用 发病机制，并可能确定有针对性的治疗方法，可以降低风险，从目前的 VEGF抑制剂的标准治疗。此外，该奖学金申请概述了一项详细的研究， 临床培训计划，以帮助候选人成为一个成功的眼科医生，科学家。

PROJECT SUMMARY Neovascular age-related macular degeneration (nvAMD) is a leading cause of central vision loss in individuals over the age of 50 years. Vision loss occurs when choroidal endothelial cells (CECs) transmigrate the retinal pigment epithelium (RPE) into the neurosensory retina and form choroidal neovascularization (CNV). Current standard of care includes agents that inhibit the bioactivity of vascular endothelial growth factor (VEGF). Although anti-VEGF agents have reduced CNV and improved visual acuity in patients, there are concerns about possible adverse effects on neuronal and glial health in the retina from long-term VEGF inhibition. Thus, better understanding of downstream VEGF signaling in CECs is necessary to identify more targeted therapeutic approaches. One of the downstream effectors is Rac1, a Rho GTPase that in the GTP-bound state regulates CEC transmigration. VEGF-mediated Rac1GTP is sustained by binding IQ motif containing GTPase activating protein 1 (IQGAP1), a multidomain GTPase-binding protein, at a specific domain. Therefore, interfering with Rac1GTP/IQGAP1 binding may be an approach to inhibit CEC migration and prevent vision- threatening CNV. Cultured CECs that express constitutively active Rap1a, a Ras GTPase that also binds to IQGAP1, had reduced VEGF-mediated Rac1GTP and Rac1GTP/IQGAP1 interactions. Together, these results suggest that increased Rap1aGTP/IQGAP1 binding in CECs reduces VEGF-induced Rac1GTP, CEC migration and CNV by interfering with Rac1GTP/IQGAP1 binding. This proposal will determine the mechanistic role of Rap1GTP binding the IQ domain of IQGAP1 on Rac1GTP/IQGAP1 interactions in Specific Aim 1 and in vitro angiogenesis in Specific Aim 2. In Specific Aim 3, this proposal will determine the mechanistic role of Rap1aGTP/IQGAP1 interactions in endothelial cells during CNV development. Methods include: isolation of CECs from adult human donor eyes; transfection of CECs with mutant IQGAP1 constructs; pharmacologic activation of Rac1 and Rap1; co-immunoprecipitation; western blot; Matrigel migration assay; tube formation assay; proliferation assay; Rap1b null mice and tamoxifen-inducible endothelial-IQGAP1 knockout mice; murine laser-induced CNV model; intravitreal injections of agents that activate Rap1; flow cytometry; immunohistochemistry of RPE/choroid flat mounts and cryo-sections; Micron IV imaging; and spectral domain optical coherence tomography. These studies will elucidate a potential mechanism of regulating Rac1GTP/IQGAP1 interactions, which has an important role in VEGF-induced CEC migration and CNV pathogenesis, and potentially identify targeted therapeutic approaches that can reduce risks from current standard of care VEGF inhibitors. Furthermore, this fellowship application outlines a detailed research and clinical training plan to help the candidate become a successful physician-scientist in ophthalmology.