Different effects of anti-VEGF drugs (Ranibizumab, Aflibercept, Conbercept) on autophagy and its effect on neovascularization in RF/6A cells.
Choroidal neovascularization (CNV) is the main pathological change of wet age-related macular degeneration. Anti-VEGF drugs are the most commonly used treatment for CNV. The biggest drawback of anti-VEGF drugs is the recurrence of CNV, which requires repeated therapy several times. Autophagy activation may be involved in reducing the therapeutic effect of anti-VEGF drugs. So, this study aims to elucidate the effect and mechanism of anti-VEGF drugs on endothelial autophagy and neovascularization in vitro.
RF/6A cells were randomly divided into five groups: The control group, hypoxia group (1% O2, 5% CO2, 94% N2), anti-VEGF group (group1: Ranibizumab 100 μg/ml; group2: Aflibercept, 400 μg/ml; group3: Conbercept, 100 μg/ml). Autophagy-related proteins were examined by Western blot. RFP-GFP-LC3 was used to detect autophagy and autophagic flow. Subsequently, we used autophagy inhibitors (3-MA or CQ) to inhibit Conbercept induced autophagy and to observe its effect on angiogenesis in vitro. Proliferation, migration, and tube formation of endothelial cells can be used to study neovascularization in vitro. In this research, the CCK-8 assay was used to detect cell proliferation. Cell migration and tube formation were assessed by wound assay and matrix method, respectively. Flow cytometry and Tunel were used to detect cell apoptosis. Finally, the mechanism of Conbercept activated autophagy was studied. Western blot was used to detect the expression of p53 and DRAM (damage-regulated autophagy modulator), upstream activators of autophagy.
The protein levels of Beclin-1 and LC3-2/1 in Ranibizumab and Conbercept groups were significantly higher than in the hypoxia group(P < 0.05). While the expression of P62 was decreased (P < 0.05). The autophagic flux was showed the same results. However, Aflibercept showed the opposite effect on autophagy. Compared with the Conbercept group, autophagy inhibitor 3-MA or CQ can further inhibit cell proliferation and promotes cell apoptosis (P < 0.05). Conbercept significantly inhibited cell migration compared with the hypoxia group (633.08 ± 72.52 vs. 546.33 ± 24.61), while the autophagy inhibitor group (3-MA or CQ) had a more obvious inhibition effect (309.75 ± 86.36 and 263.33 ± 68.67) (P < 0.05). For tube formation, the number of tube formation was decreased significantly in the Conbercept group (32.00 ± 2.00) compared to the hypoxia group (39.00 ± 1.53) and even further reduced in 3-MA or CQ group (24.00 ± 3.61, 20.00 ± 2.65). The length of master segments in the hypoxia group was 15,668.00 ± 894.11. It was decreased in Conbercept (13,885.34 ± 730.03). In 3-MA or CQ group, the length of master segments dropped further (11,997.00 ± 433.66, 10,617.67 ± 543.21). Compare with the hypoxia group, the expression P53 and DRAM were increased in the Conbercept group (P < 0.05). Autophagy-related proteins LC-3, Beclin-1, and DRAM were inhibited by P53 inhibitor Pifithrin-α (PFTα) (P < 0.05).
Ranibizumab and Conbercept can trigger the autophagy of vascular endothelial cells while Aflibercept can inhibit it. The combination of Conbercept and autophagy inhibitor can significantly inhibit the formation of angiogenesis in vitro. The mechanism of autophagy activation is related to the activation of the p53/DRAM pathway.
Wang Y
，Yao Y
，Li R
，Wu B
，Lu H
，Cheng J
，Liu Z
，Du J
... -
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Decorin inhibits angiogenic potential of choroid-retinal endothelial cells by downregulating hypoxia-induced Met, Rac1, HIF-1α and VEGF expression in cocultured retinal pigment epithelial cells.
Choroidal neovascularization (CNV) is one of the most common causes of severe vision loss. Decorin, a multiple receptor tyrosine kinase inhibitor, has been recently shown to play an important regulatory role in angiogenic response. This study aims to investigate whether the overexpression of decorin in retinal pigment epithelial (RPE) cells under hypoxia alters the in vitro angiogenic ability of cocultured choroid-retinal endothelial cells and to explore the possible mechanisms involved. Human RPE cells (ARPE-19) were subjected to hypoxia with or without decorin pretreatment, and RNA interference technique was used to knock down the Met gene in ARPE-19 cells. Cell viability was determined using the Cell Counting Kit-8 assay. Expression of Met, Rac1 and hypoxia-inducible factor-1 alpha (HIF-1α) was evaluated by western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Vascular endothelial growth factor (VEGF) expression was evaluated by enzyme-linked immunosorbent assay (ELISA) and qRT-PCR. We then constructed a recombinant lentiviral vector carrying the decorin gene to transduce ARPE-19 cells. The overexpression of decorin in transduced RPE cells was confirmed by qRT-PCR and western blot. The transduced RPE cells were then cocultured with rhesus macaque choroid-retinal endothelial cells (RF/6A) in a transwell coculture system to observe the effects of decorin overexpression in ARPE-19 cells on the proliferation, migration and tube formation of RF/6A cells. In response to hypoxia, the VEGF concentrations in the culture supernatants increased greatly at 24 and 48 h, and this effect was inhibited significantly and nearly equally in the presence of 50-200 nM decorin. Decorin pretreatment before hypoxia exposure effectively reduced the hypoxia-induced expression of Met, Rac1, HIF-1α and VEGF in ARPE-19 cells. Transfection of small interfering RNA against Met to ARPE-19 cells also resulted in significant downregulation of Rac1, HIF-1α and VEGF under hypoxia, and this effect was similar to that noted with decorin pretreatment alone or with their combination. Results from the coculture system showed that the overexpression of decorin in ARPE-19 cells significantly inhibited the proliferation, migration and tube formation of RF/6A cells. These results indicate that Met pathway activation plays an important role in the upregulation of VEGF in RPE cells under hypoxia. Decorin may interfere with angiogenesis by downregulating hypoxia-induced Met, Rac1, HIF-1α and VEGF expression in RPE cells, which suggests a potential strategy for the inhibition of CNV.
Du S
，Wang S
，Wu Q
，Hu J
，Li T
... -
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