Endothelial Transmigration in Neovascular Age-related Macular Degeneration

视网膜色素上皮(RPE)和脉络膜内皮细胞(CECs)之间的相互作用是重要的 新生血管性老年性黄斑变性(AMD)。在开发之前发生的事件 AMD的脉络膜新生血管(CNV)包括CEC的激活以向趋化方向迁移 视网膜感觉的倾斜和RPE造成的血-视网膜屏障完整性的丧失。二 上一次供资期间的补充调查结果--(A)有效的GTP约束性形式 RAP1是RAS超家族的一个GTP酶，在RPE屏障的完整性中起重要作用，而敲除RAP1 在激光诱导的模型中，异构体导致更大的CNV；以及(B)在CEC中，活性的rac1，小分子 Rho家族的GTP酶在CEC的运动和迁移中起重要作用，是常见的下游效应因子 血管内皮生长因子/血管内皮生长因子2和CCL11/CCR3这两条信号通路在人AMD中的表达 以下假设框架将在下一个资助期进行测试：在卫生方面，RPE交界处 作为动态平衡的一部分，RPE细胞拆解和重组，基本释放出血管内皮生长因子。在AMD早期， 压力会导致RPE产生更多的血管内皮生长因子。这种血管内皮生长因子激活CECs中的rac1，导致CECs迁移 并联系RPE。由于CEC-RPE接触，Rap1a减少了与钙粘附素的相关性 与p22Phox和蛋白酪氨酸磷酸酶结合，所有这些都有助于减少 RPE屏障完整性。然后CEC沿着血管内皮生长因子梯度迁移到感觉性视网膜，并增殖到 CNV.具体目标1是测试Rap1a如何与连接蛋白结合以增加RPE屏障的完整性， 减少CEC运动和应力性纤维形成，减少CNV。具体目标2是测试Rap1a的活性 调节RPE内源性产生的活性氧和结合蛋白的磷酸化以 提高RPE屏障的完整性。具体目标3是确定CCR3和CCR3之间的串扰机制 血管内皮生长因子在rac1介导的信号转导和CEC迁移中的作用及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 确定引起CEC迁移的信号通路的移行模型；工程化腺病毒 体外测试机制的构建；转基因Rap1亚型基因敲除小鼠；使用 RPE特异性的单链AAV和启动子；激光诱导的CNV模型；以及Micron III荧光素相互作用 视网膜色素上皮(RPE)和脉络膜内皮细胞(CECs)之间的关系在 新生血管老年性黄斑变性(AMD)。

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).