Endothelial Transmigration in Neovascular Age-related Macular Degeneration

新生血管性年龄相关性黄斑变性中的内皮细胞迁移

基本信息

批准号：
9037013
负责人：
Mary Elizabeth Ruth Hartnett
金额：
$ 37.25万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-04-01 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9037013
关键词：
Adherens Junction Age related macular degeneration Blindness Blood Vessels Blood-Retinal Barrier CCR3 Signaling Pathway Cadherins Cell Communication Cell Survival Cells Chemicals Choroid Choroidal Neovascularization Coculture Techniques Development E-Cadherin Endothelial Cells Engineering Etiology Event Exudative age-related macular degeneration Eye Family Fluorescein Functional disorder Funding GTP Binding Gene Transfer Generations Goals Guanosine Triphosphate Phosphohydrolases Health Homeostasis Human Hypoxia In Vitro Injection of therapeutic agent KDR gene Knockout Mice Knowledge Laser injury Lasers Lead Mediating Methods Modeling Molecular Monomeric GTP-Binding Proteins Mus NADPH Oxidase Neurons Pathway interactions Phosphorylation Proliferating Protein Isoforms Protein Tyrosine Phosphatase Proteins Reactive Oxygen Species Retina Retinal Retinal Ganglion Cells Risk Role Sensory Signal Pathway Signal Transduction Stress Stress Fibers Structure of retinal pigment epithelium Techniques Testing Time Toxic effect Transgenic Animals Transgenic Model Transgenic Organisms Vascular Endothelial Growth Factors Vision Visual Acuity Wild Type Mouse angiogenesis base bevacizumab cell motility cell type gene therapy improved in vivo knock-down member migration mouse model novel novel strategies nutrient deprivation prevent promoter rho standard care

项目摘要

Interactions between the retinal pigment epithelium (RPE) and choroidal endothelial cells (CECs) are important in neovascular age-related macular degeneration (AMD). Events that occur prior to the development of choroidal neovascularization (CNV) in AMD include the activation of CECs to migrate toward a chemotactic gradient in the sensory retina and the loss of integrity of the blood retinal barrier created by RPE. Two complementary sets of findings during the previous funding period - (a) that the active GTP-bound form of Rap1, a GTPase of the Ras superfamily, is important in RPE barrier integrity, whereas knockdown of Rap1 isoforms lead to larger CNV in laser-induced models; and (b) that in CECs, active Rac1, a member of the small GTPases of the Rho family, is important in CEC motility and migration and is a common downstream effector of two signaling pathways in human AMD, VEGF/VEGFR2 and CCl11/CCR3 - provide the bases for the following hypothetical framework that will be tested in the next funding period: In health, RPE junctions disassemble and reassemble as part of homeostasis and the RPE cells release VEGF basally. In early AMD, stresses cause RPE to produce more VEGF. This VEGF activates Rac1 in CECs causing the CECs to migrate and contact the RPE. As a result of CEC-RPE contact, Rap1a has reduced associations with cadherin in adherens junctions, with p22phox, and with protein tyrosine phosphatases, and all these contribute to reduce RPE barrier integrity. CECs then migrate into the sensory retina along a VEGF gradient and proliferate into CNV. Specific Aim 1 is to test how Rap1a associates with junctional proteins to increase RPE barrier integrity, reduce CEC motility and stress fiber formation, and reduce CNV. Specific Aim 2 is to test how active Rap1a regulates endogenous generation of reactive oxygen species and junctional protein phosphorylation in RPE to increase RPE barrier integrity. Specific Aim 3 is to determine mechanisms of crosstalk between CCR3 and VEGF in Rac1-mediated signaling and CEC migration, and the effect of CCR3 inhibition on retinal ganglion and neural cell survival. Methods include: physiologically relevant human RPE-CEC coculture and transmigration models to determine signaling pathways that cause CEC transmigration; engineered adenoviral constructs to test mechanisms in vitro; transgenic Rap1 isoform knockout mice; gene therapy techniques using scAAV and promoters specific to RPE; laser-induced models of CNV; and Micron III fluorescein Interactions between the retinal pigment epithelium (RPE) and choroidal endothelial cells (CECs) are important in neovascular age-related macular degeneration (AMD).

视网膜色素上皮 (RPE) 和脉络膜内皮细胞 (CEC) 之间的相互作用很重要新生血管性年龄相关性黄斑变性（AMD）。开发之前发生的事件 AMD 中的脉络膜新生血管 (CNV) 包括激活 CEC 向趋化性迁移感觉视网膜梯度和 RPE 产生的血视网膜屏障完整性丧失。二上一个资助期间的补充研究结果 - (a) 活跃的 GTP 结合形式 Rap1 是 Ras 超家族的一种 GTP 酶，对于 RPE 屏障完整性很重要，而 Rap1 的敲低同工型导致激光诱导模型中的 CNV 更大； (b) 在 CEC 中，活跃的 Rac1，小细胞的成员 Rho 家族的 GTP 酶在 CEC 运动和迁移中非常重要，并且是常见的下游效应子人类 AMD 中的两条信号通路，VEGF/VEGFR2 和 CCl11/CCR3 - 为以下假设框架将在下一个资助期间进行测试：在健康方面，RPE 连接作为体内平衡的一部分，RPE 细胞会分解和重新组装，并且基础上释放 VEGF。在AMD早期，压力导致 RPE 产生更多 VEGF。该 VEGF 激活 CEC 中的 Rac1，导致 CEC 迁移并联系 RPE。由于 CEC-RPE 接触，Rap1a 减少了与钙粘蛋白的关联粘附连接，与 p22phox 和蛋白酪氨酸磷酸酶，所有这些都有助于减少 RPE 屏障完整性。然后 CEC 沿着 VEGF 梯度迁移到感觉视网膜并增殖到 CNV。具体目标 1 是测试 Rap1a 如何与连接蛋白结合以提高 RPE 屏障完整性，减少 CEC 运动和应力纤维形成，并减少 CNV。具体目标2是测试Rap1a的活跃程度调节 RPE 中活性氧的内源性生成和连接蛋白磷酸化，提高 RPE 屏障完整性。具体目标 3 是确定 CCR3 和 VEGF 在 Rac1 介导的信号传导和 CEC 迁移中的作用，以及 CCR3 抑制对视网膜神经节的影响和神经细胞的存活。方法包括：生理相关的人 RPE-CEC 共培养和迁移模型以确定导致 CEC 迁移的信号通路；工程腺病毒构建体外测试机制；转基因 Rap1 亚型敲除小鼠；基因治疗技术使用 scAAV 和 RPE 特异性启动子；激光诱导的 CNV 模型；和 Micron III 荧光素相互作用视网膜色素上皮细胞 (RPE) 和脉络膜内皮细胞 (CEC) 之间的相互作用对于新生血管性年龄相关性黄斑变性（AMD）。