Endothelial Transmigration in Neovascular Age-related Macular Degeneration

描述（由申请人提供）：我们对年龄相关性黄斑变性（AMD）中脉络膜新生血管（CNV）的机制感兴趣，AMD是美国老年人失明的主要原因。我们关注脉络膜内皮细胞（CEC）在迁移到神经感觉视网膜之前与视网膜色素上皮（RPE）接触的步骤，因为该步骤占与CNV相关的视力下降的>50%。我们假设CEC被刺激向RPE及其细胞外基质（ECM）迁移并进行“接触”，这对RPE和CEC中的后续信号传导事件至关重要，所述后续信号传导事件使得CEC能够穿过RPE迁移并形成感觉神经CNV。我们发现，RPE和CEC之间的“接触”释放生长因子，包括VEGF，其损害RPE屏障并促进CEC迁移;增加RPE表达的细胞相关VEGF 189;并引起CEC迁移的PI 3-激酶依赖性激活和CEC中的Rac 1激活。我们有数据支持Rap 1作为一种可能的介质，以加强细胞的屏障特性，在RPE抑制CEC的迁移。我们将使用原代人RPE和CEC的共培养模型，该模型概括了“接触”和迁移以及适当的体内血管生成测定，以研究三个目的：1）RPE和CEC接触对RPE细胞相关VEGF 189表达和CEC跨RPE迁移的影响;（2）CEC与RPE接触对CEC信号通路PI 3-kinase/Akt 1和Rac 1的作用，以及对CEC迁移和CNV形成的影响; Rap 1亚型对RPE屏障特性、CEC迁移和CNV持续性的作用。方法将包括真实的时间PCR; VEGF同种型的蛋白质印迹; GTP酶Rac 1和Rap 1的活性测定;短发夹RNA的RNAi转染和原代人CEC和人RPE的病毒感染;共培养和移行测定;以及血管生成（角膜！微袋、激光诱导的CNV模型和基质胶塞测定）。我们还将使用从仅表达VEGF 188同种型的小鼠中分离的RPE。AMD中CNV的机制复杂，涉及多个通路。有效的管理将需要针对疾病发病机制中不同步骤的治疗，包括预防。我们的目标是确定预防感觉神经CNV的机制。

DESCRIPTION (provided by applicant): We are interested in the mechanisms of choroidal neovascularization (CNV) in age-related macular degeneration (AMD), the leading cause of blindness in elderly Americans. We focus on the step when choroidal endothelial cells (CECs) make contact with the retinal pigment epithelium (RPE) prior to their transmigration into the neurosensory retina, because this step accounts for >50% of vision decline associated with CNV. We hypothesize that CECs are stimulated to migrate toward the RPE and its extracellular matrix (ECM) and to make "contact", which is crucial to subsequent signaling events in RPE and CECs that enable CEC transmigration across the RPE and the formation of neurosensory CNV. We found that "contact" between RPE and CECs released growth factors, including VEGF that compromised the RPE barrier and facilitated CEC transmigration; increased cell-associated VEGF189 expressed by RPE; and caused PI 3-kinase dependent activation of CEC transmigration and Rac1 activation in CECs. We have data that support Rap1 as a possible mediator to strengthen cellular barrier properties in RPE to inhibit CEC transmigration. We will use a coculture model of primary human RPE and CECs that recapitulates "contact" and transmigration and appropriate in vivo angiogenesis assays to study three aims: 1) the effect of RPE and CEC contact on the expression of RPE cell-associated VEGF189 and on CEC transmigration across the RPE; 2) the role of CEC and RPE contact on CEC signaling through PI 3-kinase/Akt1 and Rac1 pathways, and the effect on CEC transmigration and CNV formation; and 3) the role of Rap1 isoforms on RPE barrier properties, CEC transmigration, and CNV persistence. Methods will include real time PCR; Western blot of VEGF isoforms; activity assays for GTPases Rac1 and Rap1; RNAi transfection of short hairpin RNAs and viral infection of primary human CECs and human RPE; coculture and transmigration assays; and in vivo models of angiogenesis (cornea! micropocket, laser-induced CNV model, and matrigel plug assay) in wild type and knockout mice (Akt1-/-, Rapla-/-, Raplb-/-). We will also use RPE isolated from mice that only express the VEGF188 isoform. The mechanisms of CNV in AMD are complex and involve multiple pathways. Effective management will require treatments targeting different steps in the disease pathogenesis, including prevention. Our goal is to determine mechanisms to prevent neurosensory CNV.