Figure 2. Retinal CFH expression is regulated by VEGF.

RPE cells showed a dose-dependent increase in CFH protein (red, DAPI blue) after VEGF treatment, but this effect was inhibited by VEGF antagonism using bevacizumab or the fab fragment ranibizumab (A). Quantification of the IF images is shown in the graph. Results were validated using Western blotting of cell lysates (B, n = 4) and qPCR (C, n = 4). VEGF pretreatment reduced C3d deposits (green, DAPI blue) on RPE cells after complement activation, while VEGF antagonism by bevacizumab or ranibizumab caused increased complement deposits (D). Three days after RPE-induced deletion of Vegfa in adult mice, reduced CFH RNA (E, CFH red, DAPI blue, n = 8) was detected by in situ hybridization and qPCR (F, n = 3). Dissection of the choroid/RPE from mutant mice showed a significant reduction in VEGF (G, n = 4), but significantly more C5b-9, indicating complement activation (H, n = 4). (A and D) Representative images from 4 independent experiments. Ten images obtained for each condition. MFI was measured and corrected for cell number. (A) Two-way ANOVA. (D) One-way ANOVA with Bonferroni’s post hoc analysis. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 100 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ^##P < 0.01; ^###P < 0.001. Statistics comparing media alone with VEGF treatments are shown by asterisks, while statistics showing the effect of adding the anti-VEGF agent are shown by hatch marks