Investigating the role of active Rap1a binding IQGAP1 in choroidal neovascularization

研究活性 Rap1a 结合 IQGAP1 在脉络膜新生血管形成中的作用

• 批准号: 10513814
• 负责人: Aniket Ramshekar
• 金额: $ 3.65万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2025-09-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10513814
• 关键词: Adult; Adverse effects; Age; Binding; Binding Proteins; Biological Assay; Blindness; Blood Vessels; Choroid; Choroidal Neovascularization; Clinical; Co-Immunoprecipitations; Cyclic AMP; Development; Endothelial Cells; Endothelium; Event; Exudative age-related macular degeneration; Eye; Fellowship; Flow Cytometry; GTP Binding; Generations; Goals; Growth; Growth Factor Inhibition; Guanosine Triphosphate Phosphohydrolases; Health; Homeostasis; Human; IQ motif containing GTPase activating protein 1; Image; Immunohistochemistry; In Vitro; Individual; Knockout Mice; Laser injury; Lasers; Lead; Lectin; Lesion; Mediating; Methods; Migration Assay; Modeling; Mus; Neural Retina; Neurons; Ophthalmology; Optical Coherence Tomography; Pathogenesis; Pathologic; Patients; Pharmacology; Physicians; Physiological; Regulation; Research; Retina; Risk; Role; Scientist; Signal Transduction; Stains; Structure of retinal pigment epithelium; Tamoxifen; Testing; Therapeutic; Training; Transfection; Tube; Vascular Endothelial Growth Factors; Vision; Visual Acuity; Western Blotting; angiogenesis; cell motility; improved; inhibitor; intravitreal injection; knock-down; matrigel; migration; mouse model; mutant; neurosensory; novel; prevent; relating to nervous system; rho GTP-Binding Proteins; standard care; standard of care; targeted treatment

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.

项目概要 新生血管性年龄相关性黄斑变性（nvAMD）是导致个体中央视力丧失的主要原因 50岁以上。当脉络膜内皮细胞 (CEC) 迁移到视网膜时，就会发生视力丧失 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）。当前的 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 VEGF 药物可以减少 CNV 并提高患者的视力，但仍存在一些担忧 长期抑制 VEGF 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， 更好地了解 CEC 中的下游 VEGF 信号传导对于识别更有针对性的 治疗方法。下游效应器之一是 Rac1，一种处于 GTP 结合状态的 Rho GTPase 调节 CEC 的迁移。 VEGF 介导的 Rac1GTP 通过结合含有 GTPase 的 IQ 基序来维持 激活蛋白 1 (IQGAP1)，一种多结构域 GTP 酶结合蛋白，位于特定结构域。所以， 干扰 Rac1GTP/IQGAP1 结合可能是抑制 CEC 迁移和预防视力的一种方法 威胁CNV。培养的 CEC 表达组成型活性 Rap1a，一种 Ras GTP 酶，也结合 IQGAP1 减少了 VEGF 介导的 Rac1GTP 和 Rac1GTP/IQGAP1 相互作用。综合起来，这些结果 表明 CEC 中 Rap1aGTP/IQGAP1 结合的增加可减少 VEGF 诱导的 Rac1GTP、CEC 通过干扰 Rac1GTP/IQGAP1 结合来抑制迁移和 CNV。该提案将确定机制 Rap1GTP 结合 IQGAP1 的 IQ 域对特定目标 1 和中 Rac1GTP/IQGAP1 相互作用的作用 具体目标 2 中的体外血管生成。在具体目标 3 中，该提案将确定 CNV 发育过程中内皮细胞中 Rap1aGTP/IQGAP1 的相互作用。方法包括： 隔离 来自成人捐献者眼睛的 CEC；用突变型 IQGAP1 构建体转染 CEC；药理学 Rac1 和 Rap1 的激活；免疫共沉淀；蛋白质印迹；基质胶迁移测定；管形成 化验；增殖测定； Rap1b 缺失小鼠和他莫昔芬诱导内皮-IQGAP1 敲除小鼠； 小鼠激光诱导CNV模型；玻璃体内注射激活 Rap1 的药物；流式细胞术； RPE/脉络膜平片和冷冻切片的免疫组织化学；微米 IV 成像；和谱域 光学相干断层扫描。这些研究将阐明调节的潜在机制 Rac1GTP/IQGAP1 相互作用，在 VEGF 诱导的 CEC 迁移和 CNV 中发挥重要作用 发病机制，并可能确定可以降低当前风险的靶向治疗方法 VEGF 抑制剂的治疗标准。此外，该奖学金申请概述了详细的研究和 临床培训计划帮助候选人成为一名成功的眼科医师科学家。