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AIM: To investigate the effect of anti-vascular epithelial growth factor (VEGF) agents on the expression of fibrosis-related inflammatory mediators under normoxic and hypoxic conditions, and to further clarify the mechanism underlying fibrosis after anti-VEGF therapy. METHODS: Human retinal pigment epithelial (RPE) cells were incubated under normoxic and hypoxic conditions. For hypoxia treatment, CoCl2 at 200 µmol/L was added to the media. ARPE-19 cells were treated as following: 1) control group: no treatment; 2) bevacizumab group: bevacizumab at 0.25 mg/mL was added to the media; 3) hypoxia group: CoCl2 at 200 µmol/L was added to the media; 4) hypoxia+bevacizumab group: CoCl2 at 200 µmol/L and bevacizumab at 0.25 mg/mL were added to the media. The expression of interleukin (IL)-1ß, IL-6, IL-8 and tumor necrosis factor (TNF)-α were evaluated using real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) at 6, 12, 24 and 48h. RESULTS: Both mRNA and protein levels of IL-1ß, IL-6 and IL-8 were statistically significantly higher in the bevacizumab group than in the control group at each time point, and TNF-α gene and protein expression was only significantly higher only at 24 and 48h (P<0.05). Under hypoxic conditions, bevacizumab significantly increased the expression of IL-1ß, IL-6, IL-8 and TNF-α at 6, 12, 24 and 48h (P<0.05). IL-1ß, IL-8 and TNF-α peaked at 24h and IL-6 peaked at 12h after the bevacizumab treatment under both normoxic and hypoxic conditions. CONCLUSION: Treatment of ARPE-19 cells with bevacizumab can significantly increase the expression of fibrosis-related inflammatory mediators under both normoxic and hypoxic conditions. Inflammatory factors might be involved in the process of fibrosis after anti-VEGF therapy, and the up-regulation of inflammatory factors induced by anti-VEGF drugs might promote the fibrosis process.
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AIM: To investigate the effect of bevacizumab treatment on Notch signaling and the induction of epithelial-of-mesenchymal transition (EMT) in human retinal pigment epithelial cells (ARPE-19) in vitro. METHODS: In vitro cultivated ARPE-19 cells were treated with 0.25 mg/mL bevacizumab for 12, 24, and 48h. Cell morphology changes were observed under an inverted microscope. The expression of zonula occludens-1 (ZO-1), vimentin and Notch-1 intracellular domain (NICD) was examined by immunofluorescence. The mRNA levels of ZO-1, α-SMA, Notch-1, Notch-2, Notch-4, Dll4, Jagged-1, RBP-Jk and Hes-1 expression were evaluated with quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of α-SMA, NICD, Hes-1 and Dll-4 expression were examined with Western blot. RESULTS: Bevacizumab stimulation increased the expression of α-SMA and vimentin in ARPE-19 cells which changed into spindle-shaped fibroblast-like cells. Meanwhile, the mRNA expression of Hes-1 increased and the protein expression of Hes-1 and NICD also increased, which Notch signaling was activated. The mRNA expression of Notch-1, Jagged-1 and RBP-Jk increased at 48h, and while Dll4 mRNA and protein expression did not change after bevacizumab treatment. CONCLUSION: Jagged-1/Notch-1 signaling may play a critical role in bevacizumab-induced EMT in ARPE-19 cells, which provides a novel insight into the pathogenesis of intravitreal bevacizumab-associated complication.
RESUMO
BACKGROUND: Fibrosis is the most common side effect after anti-vascular epithelial growth factor (VEGF) therapy (intravitreal bevacizumab) for retinal or choroidal neovascularization. This study was to investigate the efficacy of bevacizumab on the expressions of fibrosis-related cytokines in human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: Cultured HUVECs were divided into groups of controls (group 1), hypoxia (group 2) and hypoxia combined with bevacizumab (group 3). No treatment was given in group 1. In group 2, cobalt(II) chloride (CoCl2) (200 µm) was added to the medium. In group 3, in addition to CoCl2, bevacizumab was mixed in the medium, with a final concentration of 0.25 mg/mL, roughly equal to the concentration used clinically. The expressions of connective tissue growth factor (CTGF), transforming growth factor-ß2 (TGF-ß2) and basic fibroblast growth factor-2 (bFGF-2) were evaluated by SYBR green real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay at 6 h, 12 h, 24 h and 48 h. Matrix metalloproteinases (MMP)-2 was detected by SYBR green real-time PCR and Western blotting at each time point. RESULTS: Both messenger RNA and protein levels of CTGF, bFGF, TGF-ß2 and MMP-2 in group 2 were higher than group 1 (P < 0.05). In group 3, the expressions of CTGF, bFGF, TGF-ß2 and MMP-2 were upregulated compared with group 2 (P < 0.05). CONCLUSIONS: Bevacizumab at clinical doses can exert pro-fibrotic effects on HUVECs by upregulating the expressions of CTGF, bFGF, TGF-ß2 and MMP-2. This may be involved in fibrosis after anti-VEGF therapy.