Figure 6.

RPE-derived VEGF₁₈₉ stimulates CEC transmigration mediated by Rac1 activation. Activities of Rac1 (A) and PI-3K (B) were measured in CECs grown in contact with ARPE-19 cells with reduced VEGF₁₈₉. Twenty-four hours after contact, CECs were collected for the detection of active Rac1 and phospho-Akt by GST pull-down and immunoprecipitation. Whole cell lysates were used to detect total Rac1 and Akt by Western blot analysis. Knockdown of VEGF₁₈₉ decreased Rac1 activity compared with control siRNA, whereas p-Akt remained unchanged. The bar graph on the right shows active-Rac1 or phospho-Akt band density normalized to total Rac1 or total Akt1. *P < 0.05 vs. control siRNA (n = 3). (C) CEC transmigration assay was performed using hfRPE and CECs in which Rac1 activity had been inhibited by transfection with GFP-POSH-RBD. Twenty-four hours after transfection, CECs were added, and transmigration was measured after 48 hours. During the last 16 hours of contacting coculture, hfRPE was incubated with 600 μM H₂O₂. CECs in which Rac1 activity was inhibited by expressing GFP-POSH did not exhibit increased transmigration when cocultured with H₂O₂-treated hfRPE compared with cells expressing GFP alone. ***P < 0.0001 vs. GFP alone (n = 6).