Different effects of anti-VEGF drugs (Ranibizumab, Aflibercept, Conbercept) on autophagy and its effect on neovascularization in RF/6A cells.

Abstract

INTRODUCTION:

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.

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 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.

RESULTS:

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).

CONCLUSION:

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.

• Authors: Yi Wang, Yang Yao, Rong Li, Binghui Wu, Huiqin Lu, Jing Cheng, Zhe Liu, Junhui Du
• Journal: Microvascular research (Microvasc Res) Vol. 138 Pg. 104207 (11 2021) ISSN: 1095-9319 [Electronic] United States
• PMID: 34119535 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2021. Published by Elsevier Inc.

Chemical References

• Angiogenesis Inhibitors
• Recombinant Fusion Proteins
• Tumor Suppressor Protein p53
• Vascular Endothelial Growth Factors
• aflibercept
• KH902 fusion protein
• Receptors, Vascular Endothelial Growth Factor
• Ranibizumab

Topics

• Angiogenesis Inhibitors (pharmacology)
• Animals
• Apoptosis (drug effects)
• Autophagy (drug effects)
• Cell Line
• Cell Movement (drug effects)
• Cell Proliferation (drug effects)
• Choroidal Neovascularization (drug therapy, metabolism, pathology)
• Endothelial Cells (drug effects, metabolism, pathology)
• Macaca mulatta
• Neovascularization, Physiologic (drug effects)
• Ranibizumab (pharmacology)
• Receptors, Vascular Endothelial Growth Factor
• Recombinant Fusion Proteins (pharmacology)
• Signal Transduction
• Tumor Suppressor Protein p53 (metabolism)
• Vascular Endothelial Growth Factors (antagonists & inhibitors, metabolism)