Inhibitory effects of NADPH oxidase 4 inhibitor on VEGF expression in hypoxia-induced human RPE cells

Objective

To observe the inhibitory effects of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) on vascular endothelial growth factor (VEGF) expression in hypoxia-induced human retinal pigment epithelial cells.

Methods

The cultured APRE-19 cells were divided into Avastin group and VAS2870 group, and then the Avastin group was subdivided into the normoxic control group, hypoxia control group, 0.25 mg/ml Avastin intervention group, 0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group, and the VAS2870 group was subdivided into 1 μmol/ml VAS2870 intervention group, 3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group.CoCl₂ of final concentration of 300 mol/L was added to the medium to establish the cytochemical hypoxia model.The expressions of NOX4 and VEGF in human retinal pigment epithelial cells were located and evaluated by immunofluorescence staining, and relative expressing levels of NOX4 and VEGF proteins were compared by Western blot assay.

Results

The relative expression of NOX4 was 0.657±0.153, 1.000±0.200, 1.206±0.300, 1.260±0.200 and 1.413±0.273, and the relative expression of VEGF-A was 0.821±0.110, 1.210±0.100, 0.672±0.100, 0.340±0.120 and 0.300±0.130 in the normoxic control group, hypoxia control group, 0.25 mg/ml Avastin intervention group, 0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group, respectively, with statistically significant differences among the groups (F=17.631, P<0.001; F=4.777, P<0.05). The relative expression of NOX4 protein in 0.75 mg/ml Avastin intervention group was significantly lower than that in normoxia control group (P<0.001). The relative expression of VEGF-A protein in the cells of the 0.25, 0.50 and 0.75 mg/ml Avastin intervention group was significantly lower than that in hypoxia control group (P<0.05). The expression of NOX4 protein in the cells was 0.970±0.120, 1.060±0.130, 0.880±0.130, 0.567±0.135 and 0.450±0.120, and the relative expression of VEGF-A protein was 0.387±0.135, 0.627±0.125, 0.370±0.140, 0.363±0.140 and 0.160±0.100 in the normoxia control group, hypoxia control group, 1 μmol/ml VAS2870 intervention group, 3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group, respectively, with statistically significant differences among them (F=12.933, P<0.001; F=4.948, P<0.05). The relative expression of VEGF-A protein in the 1, 3 and 5 μmol/ml VAS2870 intervention group was significantly lower than that in hypoxia control group (P<0.05).

Conclusions

NOX4 inhibitor can inhibit the expression of VEGF-A protein in hypoxia-induced human RPE cells by down-regulating the NOX4 level.

Hypoxia; Retinal pigment epithelium, human; Nicotinamide adenine dinucleotide phosphate oxidase 4; Vascular endothelial growth factor; Inhibitor