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. ? ? ?
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