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.

项目摘要 新生血管相关的黄斑变性（NVAMD）是个体中央视力丧失的主要原因 超过50岁。当脉络膜内皮细胞（CEC）转移视网膜时，就会发生视力丧失 颜料上皮（RPE）进入神经感觉视网膜并形成脉络膜新生血管形成（CNV）。当前的 护理标准包括抑制血管内皮生长因子（VEGF）生物活性的药物。 尽管抗VEGF药物的CNV降低并提高了患者的视力，但仍有担忧 关于长期VEGF抑制作用对视网膜中神经元和神经胶质健康的不良影响。因此， 必须更好地了解CEC中下游VEGF信号传导，以识别更多针对性的 治疗方法。下游效应器之一是Rac1，一种在GTP结合状态下的Rho GTPase 调节CEC转移。 VEGF介导的RAC1GTP通过包含含有GTPase的iQ基序来维持 在特定结构域中激活蛋白1（IQGAP1），一种多域GTPase结合蛋白。所以， 干扰Rac1GTP/IQGAP1结合可能是抑制CEC迁移并防止视力的一种方法 - 威胁CNV。培养的CECs表达组成性活性RAP1A，这是一种RAS GTPase，也与 IQGAP1减少了VEGF介导的Rac1GTP和Rac1GTP/IQGAP1相互作用。在一起，这些结果 建议在CEC中增加RAP1AGTP/IQGAP1结合可以减少VEGF诱导的Rac1GTP，CEC 通过干扰Rac1GTP/IQGAP1结合来迁移和CNV。该建议将决定机械 RAP1GTP在Rac1GTP/IQGAP1相互作用中的RAP1GTP绑定IQGAP1的IQ域在特定AIM 1和IN中的作用 特定目的2中的体外血管生成。在特定的目标3中，该建议将确定 CNV开发过程中内皮细胞中的RAP1AGTP/IQGAP1相互作用。方法包括：隔离 成人人类捐助者眼睛的CEC；用突变体IQGAP1构建体转染CEC；药理学 Rac1和Rap1的激活；共免疫沉淀；蛋白质印迹；矩阵迁移测定；管形成 测定；扩散测定； RAP1B无效的小鼠和他莫昔芬诱导的内皮内皮iqGAP1敲除小鼠； 鼠激光诱导的CNV模型；玻璃体内注射激活Rap1的药物；流式细胞仪； RPE/脉络膜平坦坐骑和冷冻部门的免疫组织化学；微米IV成像；和光谱域 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中具有重要作用 发病机理，并有可能识别有针对性的治疗方法，可以降低当前的风险 护理标准VEGF抑制剂。此外，此研究金应用程序概述了一项详细的研究和 临床培训计划，以帮助候选人成为眼科的成功医师科学家。