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：
8451297
负责人：
Mary Elizabeth Ruth Hartnett
金额：
$ 35.42万
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/8451297
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 Knockout Mice Knowledge Laser injury Lasers Lead Mediating Methods Modeling Molecular Monomeric GTP-Binding Proteins Mus NADPH Oxidase Neurons Nutritional 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 Factor Receptor-2 Vascular Endothelial Growth Factors Vision Visual Acuity Wild Type Mouse angiogenesis base bevacizumab cell motility cell type deprivation gene therapy human CYBA protein improved in vivo member migration mouse model novel novel strategies 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在新血管相关的黄斑变性（AMD）中。开发之前发生的事件 AMD中的脉络膜新血管形成（CNV）包括CEC的激活以迁移到趋化性 RPE产生的感官视网膜中的梯度和血液视网膜屏障的完整性丧失。二在上一个资金期间的互补发现集 - （a）主动GTP结合形式的 RAP1是RAS超家族的GTPase，在RPE屏障完整性中很重要，而RAP1的敲低同工型在激光诱导的模型中导致更大的CNV；（b）在CEC中，Active Rac1，小型成员 Rho家族的GTPases在CEC运动和迁移中很重要，并且是常见的下游效应器人类AMD，VEGF/VEGFR2和CCL11/CCR3中的两个信号通路的基础遵循将在下一个资金期间进行测试的假设框架：在健康方面，RPE交界处作为体内平衡的一部分，拆卸并重新组装，RPE细胞基本上释放了VEGF。在AMD早期应力导致RPE产生更多的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与钙粘蛋白的关联减少了具有P22Phox和蛋白酪氨酸磷酸酶的粘附连接，所有这些都有助于减少 RPE屏障完整性。 CEC然后沿VEGF梯度迁移到感觉视网膜中，并扩散到 CNV。具体目标1是测试RAP1A如何与连接蛋白相关以增加RPE屏障完整性，减少CEC运动和应力纤维形成，并减少CNV。特定目标2是测试活性RAP1A 调节RPE中的活性氧和连接蛋白磷酸化的内源性产生增加RPE屏障完整性。具体目的3是确定CCR3和CCR3之间的串扰机制 Rac1介导的信号传导和CEC迁移中的VEGF，以及CCR3抑制对视网膜神经节的影响和神经细胞存活。方法包括：生理相关的人类RPE-CEC共培养和转移模型，以确定导致CEC转移的信号通路；工程腺病毒体外测试机制的构造；转基因RAP1同工敲除小鼠；使用基因治疗技术 SCAAV和针对RPE的启动子；激光诱导的CNV模型；和微米III荧光素相互作用视网膜色素上皮（RPE）和脉络膜内皮细胞（CEC）之间很重要新生血管相关的黄斑变性（AMD）。