The Role of RPE Cell-Associated VEGF189 in Choroidal EC Transmigration in Neovascular Age-Related Macular Degeneration

Abstract

Purpose: : To determine the molecular mechanisms involved in RPE-derived vascular endothelial growth factor 189 (VEGF₁₈₉) mediation of choroidal endothelial cell (CEC) transmigration across the retinal pigment epithelium (RPE), an important step in the development of neovascular age-related macular degeneration (AMD).

Methods: : Using real time PCR, the expression of VEGF splice variants, VEGF_121, VEGF_165, and VEGF_189, was determined in human fetal RPE (hfRPE) exposed to hydrogen oxide (H₂O₂), hypoxia, or growth in contact with human CECs. mRNA level of analogous VEGF splice variants was determined in elderly and young human eyes and in mice induced to develop choroidal neovascularization (CNV) by laser treatment. Knockdown of VEGF₁₈₉ was performed by transfection of small interference RNA (siRNAs) into ARPE-19 cells. Activation of PI-3 kinase or Rac1 was measured in CECs grown in contacting coculture with hfRPE or ARPE with transfection of siRNA. Transmigration of CECs across the RPE was determined using fluorescence microscopy.

Results: : VEGF₁₈₉ was upregulated in hfRPE challenged with H₂O_2, or when grown in contact with CECs and, in both conditions, a greater number of transmigrated CECs were found in transmigration assays compared to respective controls (p<0.05). Compared with control siRNA, ARPE transfected with siRNA to VEGF₁₈₉ had >70% knockdown of endogenous VEGF₁₈₉ and reduced CEC transmigration to 40% of control. VEGF_188/189 was upregulated in elderly human RPE compared to young RPE and murine RPE-choroids treated with laser to induce CNV compared to control. Rac1 was activated when CECs were grown in contact with hfRPE exposured to H₂O₂, in which VEGF₁₈₉ was upregulated (P<0.05). When CECs were cocultured with ARPE-19 transfected with siRNA to VEGF₁₈₉, Rac1 activity in CECs was significantly reduced (P<0.05). However, phosphorylated Akt was unaffected.

Conclusions: : Cell associated VEGF₁₈₉ significantly increased CEC transmigration across the RPE by activating Rac1 in CECs, but did not appear to trigger downstream signaling of PI-3 kinase.

Keywords: age-related macular degeneration • choroid: neovascularization • vascular endothelial growth factor