Purchase this article with an account.
M.E. Hartnett, P. Geisen, J.R. McColm, E.S. Wittchen, K. Burridge; PI 3–kinase Mediates Choroidal Endothelial Cell Transmigration of the Retinal Pigment Epithelium and Involves Active Rac . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3023.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the role of phosphatidylinositol 3 kinase (PI3–kinase) in human choroidal endothelial cell (CEC) transmigration of a monolayer of human retinal pigment epithelial cells (ARPE–19). Methods: To provide a chemotactic gradient in transmigration (TM) assays, ARPE–19 were plated in the wells of Transwells with 8 micron pore inserts that permit CEC transmigration. For coculture (CC) assays, Transwells with 0.4 micron pore inserts were used to permit heterotypic cell contact without cell migration. In both TM and CC assays, ARPE–19 were plated on the underside of the Transwell inserts and permitted to attach for 4 hours. The inserts were then placed upright. After 72 hours, primary human CECs stained with CellTracker Green (TM assays) or unstained (CC assays) were plated into the inserts. After 72 hours, CEC and ARPE–19 were collected from the underside of the insert and either 1) measured for Rac GTPase activity by Western blot analysis (CC assay) or 2) counted (TM assay). Migrated CECs were counted as green cells from the insert and transmigrated CECs were green cells within the well media counted prior to trypsinization of the Transwell inserts. Solo CEC migration assays with CEC plated in the insert were also performed. CECs in TM or CC assays were exposed to 30 minutes of either the PI3 kinase inhibitor LY294002 (50µM) or vehicle (DMSO) and then washed with media. The proliferation of CECs exposed to different concentrations of LY294002 or to vehicle (DMSO) was also tested. Results: In the TM assay, exposure of CECs to LY294002 resulted in significantly fewer migrated CECs (4737 ± 284 vs 176 ± 104, p<0.001, students T–Test). LY294002 significantly reduced CEC migration in the solo migration assays (15,593 ± 3052 control vs 3200 ± 363 LY294002, p<0.018, student’s T–test). In the CC assay, CECs exposed to LY294002 had decreased Rac activity compared to those exposed to vehicle. Varying concentrations of LY294002 did not alter CEC count significantly compared to vehicle control. Conclusions: PI3 kinase plays a role in CEC transmigration to a monolayer of ARPE–19, which is in part mediated by downstream activation of Rac.
This PDF is available to Subscribers Only