P53/DRAM/ autophagy - a new target for improving the therapeutic effect of anti-VEGF on intraocular neovascularization
Research Square (Research Square)(2020)
Abstract
Backgroud Recurrence of intraocular neovascularization is a major clinical problem. Anti-VEGF drugs are the main treatment for intraocular neovascularization currently. However, anti-VEGF drugs can activate endothelial autophagy and weaken the therapeutic effect. This study aims to elucidate the effect and mechanism of anti-VEGF drugs on autophagy of vascular endothelial cells. Methods 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 100ug/ml; group2: Aflibercept, 400ug/ml; group3: Conbercept, 100ug/ml) and autophagy inhibition group(3-MA or CQ) which was corresponding to anti-VEGF group. Autophagy-related proteins were examined by Western blot. RFP-GFP-LC3 was used to detect autophagy and autophagic flow. CCK-8 assay was used to detect cell proliferation. Flow cytometry and Tunel was used to detect cell apoptosis. Cell migration and tube formation were assessed by wound assay and matrix method, respectively. Results Ranibizumab and Conbercept can triger autophagy in hypoxia condition in RF/6A cells, while Aflibercept can inhibit autophagy. Conbercept combined with autophagy inhibitor (3-MA or CQ) could inhibit cell migration and tube formation of RF/6A cells more effectively in hypoxia condition. For mechanism, p53 and DRAM proteins paly an important role in Conbercept induced autophagy. Inhibition of P53 can suppressed the autophagy induced by Conbercept. Conclusion Ranibizumab and Conbercept can triger the autophagy of vascular endothelial cells while Aflibercept can inhibit it. The combination of ranibizumab/ Concept and autophagy inhibitor can significantly inhibit the formation of angiogenesis in vitro. The mechanism of autophagy activation is related to the activation of p53 / DRAM pathway.
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Key words
intraocular neovascularization,p53/dram/,anti-vegf
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