The role of RPE cell-associated VEGF₁₈₉ in choroidal endothelial cell transmigration across the RPE

RPE-derived cell-associated VEGF&sub1;₈₉ facilitates CEC transmigration by Rac1 activation independently of PI-3K signaling and may have importance in the development of neovascular AMD.

Using real-time PCR, the expression of VEGF splice variants VEGF&sub1;&sub2;&sub1;, VEGF&sub1;₆₅, and VEGF&sub1;₈₉ was determined in human RPE from donor eyes, cultured human RPE in contact with CECs exposed to hydrogen peroxide (H&sub2;O&sub2;) 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&sub1;₈₉ expression. Migration of CECs across the RPE was determined using fluorescence microscopy.

To determine the role of vascular endothelial growth factor 189 (VEGF&sub1;₈₉) in choroidal endothelial cell (CEC) migration across the retinal pigment epithelium (RPE) and to explore the molecular mechanisms involved.

VEGF&sub1;₈₉ expression was increased in human RPE from aged compared with young donor eyes and from mouse RPE/choroids after laser to induce CNV. VEGF&sub1;₈₉ 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&sub1;₈₉; VEGFR2 and Rac1 activities were also increased in these CECs. When CECs were cocultured with RPE silenced for VEGF&sub1;₈₉, 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&sub1;₈₉ facilitates CEC transmigration by Rac1 activation independently of PI-3K signaling and may have importance in the development of neovascular AMD.