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H. Wang, P. Geisen, E. Wittchen, K. Burridge, M. Hartnett; The Role of RPE Cell-Associated VEGF189 in Choroidal EC Transmigration in Neovascular Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6189.
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© ARVO (1962-2015); The Authors (2016-present)
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).
Using real time PCR, the expression of VEGF splice variants, VEGF121, VEGF165, and VEGF189, 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.
VEGF189 was upregulated in hfRPE challenged with H2O2, 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. VEGF188/189 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.
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.
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