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：
7389477
负责人：
Mary Elizabeth Ruth Hartnett
金额：
$ 28.48万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
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 2012-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7389477
关键词：
1-Phosphatidylinositol 3-Kinase Accounting Age related macular degeneration Agonist American Basement membrane Binding Biological Assay Blindness Bruch&apos s basal membrane structure Cell Proliferation Cells Cellular Structures Characteristics Choroidal Neovascularization Coculture Techniques Complex Cornea Data Development Disease Elderly Endothelial Cells Event Extracellular Matrix Exudative age-related macular degeneration Funding Goals Growth Factor Guanosine Triphosphate Phosphohydrolases Human In Vitro Knockout Mice Laboratories Laser injury Lasers Learning Measures Mediator of activation protein Methods Modeling Mus Other Finding Oxygen measurement, partial pressure, arterial Pathogenesis Pathway interactions Polymerase Chain Reaction Prevention Property Protein Isoforms Proteins RNA Interference Retina Retinal Role Signal Pathway Signal Transduction Source Stimulus Structure of retinal pigment epithelium Testing Time Transfection VEGF189 Vascular Endothelial Growth Factor Receptor-2 Vascular Endothelial Growth Factors Virus Diseases Vision Western Blotting angiogenesis cell motility cell type in vitro Model in vivo in vivo Model interest matrigel migration prevent release factor small hairpin RNA viral RNA

项目摘要

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.

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。我们重点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细胞 (RPE) 接触的步骤，因为该步骤占与 CNV 相关的视力下降的 50% 以上。我们假设 CEC 受到刺激向 RPE 及其细胞外基质 (ECM) 迁移并进行“接触”，这对于 RPE 和 CEC 中随后的信号事件至关重要，这些信号事件使 CEC 能够跨 RPE 迁移并形成神经感觉 CNV。我们发现RPE和CEC之间的“接触”释放了生长因子，包括破坏RPE屏障并促进CEC迁移的VEGF； RPE 表达的细胞相关 VEGF189 增加；并导致 CEC 迁移的 PI 3 激酶依赖性激活和 CEC 中的 Rac1 激活。我们有数据支持 Rap1 作为一种可能的介质，可以增强 RPE 中的细胞屏障特性，从而抑制 CEC 迁移。我们将使用原代人 RPE 和 CEC 的共培养模型来概括“接触”和迁移以及适当的体内血管生成测定，以研究三个目标：1）RPE 和 CEC 接触对 RPE 细胞相关 VEGF189 表达的影响以及对 RPE 细胞相关 VEGF189 表达的影响。 CEC 穿越 RPE 的迁移； 2) CEC和RPE接触通过PI 3-激酶/Akt1和Rac1途径对CEC信号传导的作用，以及对CEC迁移和CNV形成的影响； 3) Rap1 同工型对 RPE 屏障特性、CEC 迁移和 CNV 持久性的作用。方法包括实时 PCR； VEGF 同种型的蛋白质印迹； GTPases Rac1 和 Rap1 的活性测定；短发夹RNA的RNAi转染以及原代人CEC和人RPE的病毒感染；共培养和迁移分析；以及野生型和基因敲除小鼠（Akt1-/-、Rapla-/-、Raplb-/-）中血管生成的体内模型（角膜微囊、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 VEGF188 同工型的小鼠中分离出的 RPE。 AMD中CNV的机制很复杂，涉及多种途径。有效的管理需要针对疾病发病机制的不同步骤进行治疗，包括预防。我们的目标是确定预防神经感觉 CNV 的机制。