Gene profiling of human VEGF signaling pathways in human endothelial and retinal pigment epithelial cells after anti VEGF treatment

Abstract

Background: Ranibizumab (Lucentis®) is a Fab-antibody fragment developed from Bevacizumab, a full-length anti-VEGF antibody. Both compounds are used for treating age-related macular degeneration (AMD). The influence of bevacizumab and ranibizumab on genes involved in signal transduction and cell signaling downstream of VEGF were compared in order to detect possible differences in their mode of action, which are not related to their Fab-antibody fragments.

Methods: Human umbilical vein cell lines (EA.hy926) and retinal pigment epithelial cells (ARP-19) were exposed to oxidative stress. The cells were treated with therapeutic concentrations of bevacizumab (0.25 mg/mL) and ranibizumab (125 mg/mL) for 24 hours prior to all experiments, and their effects on gene expressions were determined by RT- PCR.

Results: After exposure to bevacizumab, more genes in the endothelial cells were up-regulated (KDR, NFATc2) and down-regulated (Pla2g12a, Rac2, HgdC, PRKCG) compared to non-treated controls. After exposure to ranibizumab, fewer genes were up-regulated (PTGS2) and down-regulated (NOS3) compared to controls. In comparison between drugs, more genes were up-regulated (NFATc2 and KDR) and more were down-regulated (Pla2g12a, Pla2g1b, Ppp3r2, Rac2) by bevacizumab than by ranibizumab. In RPE cells, NOS3 and PGF were up-regulated and Pla2g12b was down-regulated after exposure to ranibizumab, while PIK3CG was up-regulated and FIGF was down-regulated after exposure to bevacizumab, but the differences in gene expression were minor between drugs (PIK3CGand PGF were down-regulated more by ranibizumab than by bevacizumab).

Conclusions: The different gene expressions after exposure to ranibizumab and bevacizumab in endothelial and RPE cells may indicate a somewhat different biological activity of the two compounds.

Figure 1

Heatmap of the altered expression of gens in the different groups compared. A. Heatmap of the results showing genes with significantly altered transcription levels as a function of exposure to bevacizumab versus non-treated control in RPE cells. B. Heatmap of the results showing ranibizumab versus human VEGF treated control in retinal pigment epithelial cells. C. Heatmap of the results showing bevacizumab versus ranibizumab in RPE cells. D. Heatmap of the results showing bevacizumab versus human VEGF-treated control in endothelial cells. E. Heatmap of the results showing ranibizumab versus human VEGFreated control in endothelial cells. F. Heatmap of the results showing bevacizumab versus ranibizumab in endothelial cells.