Endothelial Transmigration in Neovascular Age-related Macular Degeneration

描述（由申请人提供）：视网膜色素上皮（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）中很重要。在AMD中脉络膜新生血管形成（CNV）发展之前发生的事件包括CEC的活化以向感觉视网膜中的趋化梯度迁移以及由RPE产生的血视网膜屏障的完整性的丧失。在上一个资助期内有两组互补的发现-（a）Rap 1的活性GTP结合形式，Ras超家族的一种GTP酶，在RPE屏障完整性中很重要，而Rap 1亚型的敲低导致激光诱导模型中更大的CNV;和（B）在CEC中，活性Rac 1，Rho家族的小GTP酶的成员，在CEC运动和迁移中是重要的，并且是人AMD中两种信号传导途径的常见下游效应物，VEGF/VEGFR 2和CCl 11/CCR 3-为以下假设框架提供了基础，这些框架将在下一个资助期进行测试：在健康的情况下，RPE连接作为稳态的一部分分解和重新组装，并且RPE细胞基本释放VEGF。在早期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迁移并接触RPE。由于CEC-RPE接触，Rap 1a与粘附连接中的钙粘蛋白、p22 phox和蛋白酪氨酸磷酸酶的相关性降低，所有这些都有助于降低RPE屏障的完整性。CEC然后沿沿着VEGF梯度迁移到感觉视网膜中并增殖成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如何调节RPE中活性氧和连接蛋白磷酸化的内源性产生以增加RPE屏障完整性。具体目标3是确定CCR 3和VEGF在Rac 1介导的信号传导和CEC迁移中的串扰机制，以及CCR 3抑制对视网膜神经节和神经细胞存活的影响。方法包括：生理学相关的人RPE-CEC共培养和迁移模型，以确定引起CEC迁移的信号传导途径;工程化的腺病毒构建体，以测试体外机制;转基因Rap 1同种型敲除小鼠;使用scAAV和RPE特异性启动子的基因治疗技术;激光诱导的CNV模型;视网膜色素上皮（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）中是重要的。 公共卫生相关性：新生血管性年龄相关性黄斑变性是世界范围内致盲的主要原因。目前的治疗涉及抑制血管生成的方法，这可能导致营养缺乏和外部视网膜缺氧，并且随着时间的推移效果降低。我们使用新的方法来理解为什么血管在新生血管性AMD的感觉视网膜内生长，然后测试将血管维持在眼睛适当区域内的策略。通过这些方法，我们努力寻找安全、有效的方法来代替或补充目前的策略，以减少新生血管性AMD的视力丧失。

DESCRIPTION (provided by applicant): 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 co culture 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). PUBLIC HEALTH RELEVANCE: Neovascular age-related macular degeneration is a leading cause of blindness worldwide. Current treatments involve methods to inhibit angiogenesis, which can lead to nutritional deprivation and hypoxia to the outer retina and over time have reduced effect. We use novel approaches to understand why blood vessels grow within the sensory retina in neovascular AMD and then test strategies to maintain vessels within appropriate compartments of the eye. With these approaches we strive to find safe, effective methods that may be used instead of or in addition to current strategies to reduce visual acuity loss in neovascular AMD.