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

• 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
• 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

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）之间的相互作用是重要的 新生血管性年龄相关性黄斑变性（AMD）。发生在发展之前的事件 AMD中的脉络膜新生血管形成（CNV）包括CEC的活化以向趋化因子迁移。 视网膜色素上皮细胞（RPE）是视网膜感觉层的一个重要组成部分，它可以降低视网膜感觉层的梯度，降低视网膜血屏障的完整性。两 （a）积极的全球贸易点约束形式， Rap 1是Ras超家族的一个GTP酶，在RPE屏障完整性中很重要，而Rap 1的敲低 同种型在激光诱导的模型中导致较大的CNV;和（B）在CEC中，活性Rac 1，小分子的成员， Rho家族的GTP酶在CEC运动和迁移中很重要，并且是常见的下游效应物 VEGF/VEGFR 2和CCl 11/CCR 3是AMD的两条信号通路之一，它们为AMD的治疗提供了基础。 以下假设框架将在下一个资助期内进行测试： 作为体内平衡的一部分，RPE细胞分解和重新组装，并且RPE细胞基本上释放VEGF。在早期的AMD中， 压力导致RPE产生更多的VEGF。这种VEGF激活CEC中的Rac 1，导致CEC迁移 联系皇家警察由于CEC-RPE的接触，Rap 1a与钙粘蛋白的相关性降低， 粘附连接，与p22 phox，和蛋白酪氨酸磷酸酶，所有这些有助于减少 RPE屏障完整性。CEC然后沿着VEGF梯度沿着迁移到感觉视网膜中，并增殖成血管内皮细胞。 CNV.具体目标1是测试Rap 1a如何与连接蛋白结合以增加RPE屏障完整性， 减少CEC运动性和应力纤维形成，并减少CNV。具体目标2是测试活性Rap 1a 调节RPE中活性氧的内源性产生和连接蛋白磷酸化， 增加RPE屏障完整性。具体目标3是确定CCR 3和CCR 3之间的串扰机制。 VEGF在Rac 1介导的信号传导和CEC迁移中的作用以及抑制CCR 3对视网膜神经节的影响 和神经细胞的存活方法包括：生理学相关的人RPE-CEC共培养和 确定导致CEC迁移的信号通路的迁移模型;工程化腺病毒 体外机制测试构建体;转基因Rap 1同种型敲除小鼠;使用基因治疗技术 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）之间的相互作用在 新生血管性年龄相关性黄斑变性（AMD）。