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L.J. Peterson, P. Geisen, J.R. McColm, E. Wittchen, K. Burridge, M.E. Hartnett; The Role of RPE Matrix and RPE Contact in Choroidal Endothelial Cell Migration . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5533.
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Vision loss in neovascular AMD is due in part to transmigration of choroidal endothelial cells (CECs) across Bruch’s membrane and the retinal pigment epithelium (RPE) into the neurosensory retina. We have shown that coculture of RPE and CECs increased PI 3–kinase (PI3K) and activated Rac1 and that these were important for CEC transmigration. We now investigate whether contact between CECs and RPE increases PI3K, activates Rac1 and increases CEC transmigration across RPE. We also investigated the role of RPE generated ECM on CEC spreading and cytoskeleton.
CECs isolated from human donor eyes (up to passage 3) and ARPE–19 (RPE, ATCC) were grown in solo or coculture assays using Transwells. For coculture assays, RPE (1.8x105 cells/cm2) were plated on the underside of a Transwell with 1 µm pores, the insert inverted, and RPE grown until a monolayer with ZO–1 localization occurred. Human CECs (1x105 cells/cm2) were plated into the inserts. Cells were separated and analyzed for phosphorylated Akt1 by Western blot at different time points. Controls were cocultures of CECs and RPE grown without contact. In transmigration assays, RPE stained red and CECs green were cocultured but on Transwell inserts with 5–8 µm pores. RPE plated in the well provided a chemotactic gradient. Green CECs on the underside of the insert and in the well were counted. CECs were pulsed for 30 min with 50µM of the PI3K inhibitor, LY294002. In coculture assays, CECs were removed and assayed for phosphorylated Akt1; in transmigration assays, transmigrated CECs were counted in the well. CECs were grown on extracellular matrix (ECM) generated by RPE or on coverslips blocked with 2% heat–inactivated bovine serum albumin (BSA) and stained for actin and paxillin.
In coculture assays, contact between RPE and CECs resulted in significantly more phosphorylated Akt1 and increased Rac1 activity in CECs at 20 minutes compared to CECs in coculture with RPE without contact (p=0.001 and p=0.022 respectively) . CEC transmigration was inhibited by the PI3K inhibitor LY294002 (p<0.009). CECs grown on ECM generated by RPE adhered to and spread more readily than CECs plated onto coverslips.
Contact between CECs and RPE activates Rac1 in CECs through a PI3K dependent pathway and increased CEC transmigration, a step necessary in the development of neurosensory retinal CNV. Changes in CEC cytoskeletal structure when CECs are plated onto RPE–generated ECM indicates that some activation of signaling pathways in CECs start with CEC contacting the RPE–ECM.
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