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：
8035291
负责人：
Mary Elizabeth Ruth Hartnett
金额：
$ 28.47万
依托单位：
UNIVERSITY OF UTAH
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 2012-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8035291
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 Oxygen measurement, partial pressure, arterial Pathogenesis Pathway interactions 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 effective therapy in vitro Model in vivo in vivo Model interest matrigel migration prevent release factor small hairpin 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）：我们对与年龄相关的黄斑变性（AMD）的脉络膜新生血管形成（CNV）的机制感兴趣，这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）在将视网膜上皮（RPE）接触到神经感觉视网膜之前与视网膜色素上皮（RPE）接触的步骤，因为此步骤占与CNV相关的视力下降的50％。我们假设CEC被刺激为朝向RPE及其细胞外基质（ECM）迁移，并进行“接触”，这对于RPE和CEC中的后续信号事件至关重要，这些信号事件和CEC在RPE中促进CEC跨RPE及其神经感知CNV的形成至关重要。我们发现，RPE和CEC之间的“接触”释放了生长因子，包括损害RPE屏障并促进CEC转移的VEGF； RPE表达的与细胞相关的VEGF189增加；并导致CEC转移和RAC1激活CEC的PI 3-激酶依赖性激活。我们有支持RAP1作为增强RPE中细胞屏障特性以抑制CEC迁移的可能介体的数据。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”和移民，并适当的体内血管生成测定法研究了三个目的：1）RPE和CEC接触对RPE细胞相关的VEGGF189和CEC对CEC跨RPE跨RPE的影响的影响； 2）CEC和RPE接触通过PI 3-激酶/AKT1和RAC1途径在CEC信号传导上的作用，以及对CEC跨移民和CNV形成的影响； 3）RAP1同工型在RPE屏障性质，CEC转移和CNV持久性中的作用。方法将包括实时PCR； VEGF同工型的蛋白质印迹； GTPases Rac1和Rap1的活动测定； RNAi对原发性人CEC和人RPE的短发夹RNA和病毒感染的RNAi转染；共培养和移民测定；野生型和敲除小鼠（Akt1 - / - ，Rapla-/ - ，Raplb - / - ）中的血管生成（角膜！我们还将使用仅表达VEGF188同工型的小鼠分离的RPE。 AMD中CNV的机制很复杂，涉及多个途径。有效的管理将需要针对疾病发病机理（包括预防）的不同步骤的治疗。我们的目标是确定防止神经感觉CNV的机制。