Investigating the role of active Rap1a binding IQGAP1 in choroidal neovascularization

项目总结 新生血管性老年性黄斑变性(NvAMD)是导致个人中心视力丧失的主要原因 年龄在50岁以上。当脉络膜内皮细胞(CECs)移行视网膜时会发生视力丧失 色素上皮(RPE)进入神经感觉视网膜，形成脉络膜新生血管(CNV)。当前 标准护理包括抑制血管内皮生长因子(VEGF)生物活性的药物。 尽管抗血管内皮生长因子药物减少了患者的CNV，提高了患者的视力，但仍存在一些问题 关于长期抑制血管内皮生长因子对视网膜神经元和神经胶质健康可能产生的不利影响。因此， 有必要更好地了解CEC中的下游血管内皮生长因子信号，以确定更有针对性的 治疗方法。下游效应器之一是rac1，它是一种Rho GTP酶，处于GTP结合状态 调节CEC的迁移。通过结合含有GTP酶的IQ基序维持血管内皮生长因子介导的rac1GTP 激活蛋白1(IQGAP1)，一个多结构域的GTP酶结合蛋白，位于特定的结构域。因此， 干扰rac1GTP/IQGAP1结合可能是抑制CEC迁移和防止视力下降的一种途径。 威胁到CNV。培养的CEC表达组成活性的Rap1a，Ras GTP酶也结合到 IQGAP1抑制了血管内皮生长因子介导的rac1GTP和rac1GTP/IQGAP1的相互作用。总而言之，这些结果 提示CEC中Rap1aGTP/IQGAP1结合增加可降低血管内皮生长因子诱导的rac1GTP、CEC 通过干扰rac1GTP/IQGAP1结合，迁移和CNV。这项提议将决定机械的 Rap1GTP结合IQGAP1的IQ结构域在特定目的1和IQGAP1中对rac1GTP/IQGAP1相互作用的影响 体外血管生成在特定的目标2。在特定的目标3，这一建议将确定的机制作用 新生血管发育过程中内皮细胞中Rap1aGTP/IQGAP1的相互作用方法包括：分离 成人供眼CECs；突变IQGAP1结构的CECs的转染；药理学 Rac1和Rap1的激活；免疫共沉淀；蛋白质印迹；Matrigel迁移试验；管形成 实验；增殖实验；Rap1b基因缺失小鼠和他莫昔芬诱导的内皮-IQGAP1基因敲除小鼠； 激光诱导的小鼠CNV模型；玻璃体内注射激活RAP1的药物；流式细胞术； RPE/脉络膜扁平铺片和冰冻切片的免疫组织化学；Micron IV成像；和光谱域 光学相干层析成像。这些研究将阐明一种潜在的调控机制 Rac1GTP/IQGAP1相互作用在血管内皮生长因子诱导的CEC迁移和CNV中的重要作用 发病机制，并有可能确定可以降低当前风险的靶向治疗方法 标准护理血管内皮生长因子抑制剂。此外，这份奖学金申请概述了详细的研究和 临床培训计划，以帮助应聘者成为一名成功的眼科内科科学家。

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.