The Role of RPE Cell-Associated VEGF₁₈₉in Choroidal Endothelial Cell Transmigration across the RPE

To determine the role of vascular endothelial growth factor 189 (VEGF 189 ) in choroidal endothelial cell (CEC) migration across the retinal pigment epithelium (RPE) and to explore the molecular mechanisms involved. METHODS. Using real-time PCR, the expression of VEGF splice variants VEGF 121, VEGF 165, and VEGF 189 was determined in human RPE from donor eyes, cultured human RPE in contact with CECs exposed to hydrogen peroxide (H 2 O 2 ) or hypoxia, and RPE/choroid specimens from mice treated with laser to induce choroidal neovascularization (CNV). Activation of VEGF receptors (VEGFRs), phosphoinositol 3-kinase (PI-3K) or Rac1 was measured in CECs cocultured in contact with RPE exposed to peroxide or silenced for VEGF 189 expression. Migration of CECs across the RPE was determined using fluorescence microscopy. RESULTS. VEGF 189 expression was increased in human RPE from aged compared with young donor eyes and from mouse RPE/ choroids after laser to induce CNV. VEGF 189 was also upregulated in human RPE challenged with peroxide, hypoxia, or cultured in contact with CECs. CEC migration across RPE was greater after RPE exposure to peroxide to induce VEGF 189 ; VEGFR2 and Rac1 activities were also increased in these CECs. When CECs were cocultured with RPE silenced for VEGF 189 , VEGFR2 and Rac1 activities in CECs were significantly reduced, as was CEC migration across the RPE. Inhibition of Rac1 activity significantly inhibited CEC transmigration without affecting PI-3K activity. CONCLUSIONS. RPE-derived cell-associated VEGF 189 facilitates CEC transmigration by Rac1 activation independently of PI-3K signaling and may have importance in the development of neovascular AMD.