The matricellular protein CCN5 prevents anti-VEGF drug-induced epithelial-mesenchymal transition of retinal pigment epithelium

Abstract

Age-related macular degeneration (AMD) is one of the major causes of blindness in the elderly worldwide. Anti-vascular endothelial growth factor (VEGF) drugs have been widely used to treat the neovascular type of AMD (nAMD). However, VEGF acts not only as a pro-angiogenic factor but also as an anti-apoptotic factor in the eyes. In this study, we found that anti-VEGF drugs, including bevacizumab (Bev), ranibizumab (Ran), and aflibercept (Afl), induced epithelial-mesenchymal transition (EMT) in ARPE-19 cells in vitro, accompanied by the induction of CCN2, a potent pro-fibrotic factor. Similarly, intravitreal injection of Afl into mouse eyes resulted in EMT in the retinal pigmented epithelium (RPE). Co-treatment with CCN5, an anti-fibrotic factor that down-regulates CCN2 expression, significantly attenuated the adverse effects of the anti-VEGF drugs both in vitro and in vivo. Inhibition of the VEGF signaling pathway with antagonists of VEGF receptors, SU5416 and ZM323881, induced EMT and up-regulated CCN2 in ARPE-19 cells. Additionally, knock-down of CCN2 with siRNA abolished the adverse effects of the anti-VEGF drugs in ARPE-19 cells. Collectively, these results suggest that anti-VEGF drugs induce EMT in RPE through the induction of CCN2 and that co-treatment with CCN5 attenuates the adverse effects of anti-VEGF drugs in mouse eyes.

Figure 1

Anti- VEGF drugs induce EMT in ARPE-19 cells. (A) An experimental scheme. The ARPE-19 cells were treated with anti-VEGF drugs, bevacizumab (Bev), ranibizumab (Ran), and aflibercept (Afl), at 2× clinical doses. (B) Cell lysates from ARPE-19 cells were immunoblotted with antibodies against CCN2, α-smooth muscle actin (α-SMA), fibronectin, and GAPDH. Data are representatives of more than three independent experiments. (C) Protein levels were quantified using Image-J software and plotted. Bars show the mean ± SD. (n = 4–6 per group; one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001) (D) Immunofluorescence images of ARPE-19 cells stained with anti-α-SMA and anti-zonula occludens-1 (ZO-1) antibodies, and Hoechst 33342. Representative images are shown. Scale bar, 50 μm. (E) The intensity of α-SMA per cell was quantified and plotted using Image-J software. Bars show the mean ± SD. (n = 3 per group; one-way ANOVA; ***p < 0.001).

Figure 2

Recombinant CCN5 protein inhibits EMT induced by anti-VEGF drugs in ARPE-19 cells. (A) An experimental scheme. The ARPE-19 cells treated with anti-VEGF drugs, Bev, Ran, and Afl, at 2× clinical doses in the absence or presence of recombinant CCN5 protein (500 ng/mL). (B) Cell lysates from ARPE-19 cells were immunoblotted with antibodies against CCN2, α-SMA, fibronectin, and GAPDH. Data are representatives of more than three independent experiments. (C) Protein levels were quantified using Image-J software and plotted. Bars show the mean ± SD. (n = 4 per group; one-way ANOVA; *p < 0.05, ***p < 0.001) (D) Immunofluorescence images of ARPE-19 cells stained with anti-α-SMA and anti-ZO-1 antibodies, and Hoechst 33342. Representative images are shown. Scale bar, 50 μm. (E) The intensity of α-SMA per cell was quantified and plotted using Image-J software. Bar show the mean ± SD. (n = 3–4 per group; one-way ANOVA; ***p < 0.001).

Figure 3

Recombinant CCN5 protein inhibits EMT induced by aflibercept (Afl) in mouse eyes. (A) An experimental scheme. Normal human IgG (40 mg/eye), Afl (40 μg/eye), and Afl and CCN5 (40 ng/eye) were intravitreally injected. Mice were sacrificed, and eyes were enucleated at 14 days post-injection. (B) Flat mounts of RPE/choroid complex were stained with anti-α-SMA antibody. Representative images are shown. Scale bar, 1000, 500, and 250 μm. (C) The intensity of α-SMA positive areas was quantified by Image-J software and plotted. (D) Flat mounts of RPE/choroid complex were stained with anti-α-SMA and anti-ZO-1 antibodies, and Hoechst 33342. Representative images are shown. Scale bar, 50 μm. (E) The mean number of RPE cells in 100 × 100 μm area was manually counted and plotted. (F) The mean size of RPE cells in 100 × 100 μm area was manually calculated and plotted. Error bars = SEM. (n = 8, Cont; n = 8, IgG; n = 10, Afl; n = 13, Afl+CCN5; one-way ANOVA; ***p < 0.001).

Figure 4

Antagonists of VEGF receptors induce EMT in ARPE-19 cells. (A) An experimental scheme. The ARPE-19 cells were treated with 5 μM of SU5416, an antagonist of VEGFR-1/2, or 10 nM of ZM323881, an antagonist of VEGFR-2. (B) Cell lysates from ARPE-19 cells were immunoblotted with antibodies against CCN2, α-SMA, fibronectin, and GAPDH. Data are representatives of more than three independent experiments. (C) Protein levels were quantified using Image-J software and plotted. Bars show the mean ± SD. (n = 4 per group; one-way ANOVA; **p < 0.01, ***p < 0.001) (D) Immunofluorescence images of ARPE-19 cells stained with anti-α-SMA and anti-CCN2 antibodies, and Hoechst 33342. Representative images are shown. Scale bar, 50 μm. (E) The intensity of CCN2 or α-SMA per cell was quantified and plotted using Image-J software. Bars show the mean ± SD. (n = 4 per group; one-way ANOVA; ***p < 0.001).

Figure 5

Afl-induced EMT is ameliorated by CCN2 siRNA in ARPE-19 cells. (A) An experimental scheme. The ARPE-19 cells were treated with CCN2 siRNA (1 μM) or CCN5 (500 ng/mL) in the presence of 2× Afl for 5 days. (B) Cell lysates from ARPE-19 cells were immunoblotted with antibodies against CCN2, α-SMA, fibronectin, and GAPDH. Data are representatives of more than three independent experiments. (C) Protein levels were quantified using Image-J software and plotted. Bars show the mean ± SD. (n = 5 per group; one-way ANOVA; ***p < 0.001) (D) Immunofluorescence images of ARPE-19 cells were stained with anti- α-SMA and anti-CCN2 antibodies, and Hoechst 33342. Representative images are shown. Scale bar, 50 μm. (E) The intensity of CCN2 or α-SMA per cell was quantified and plotted using Image-J software. Bars show the mean ± SD. (n = 4 per group; one-way ANOVA; ***p < 0.001).