Rice Bran and Vitamin B6 Suppress Pathological Neovascularization in a Murine Model of Age-Related Macular Degeneration as Novel HIF Inhibitors

Abstract

Pathological neovascularization in the eye is a leading cause of blindness in all age groups from retinopathy of prematurity (ROP) in children to age-related macular degeneration (AMD) in the elderly. Inhibiting neovascularization via antivascular endothelial growth factor (VEGF) drugs has been used for the effective treatment. However, anti-VEGF therapies may cause development of chorioretinal atrophy as they affect a physiological amount of VEGF essential for retinal homeostasis. Furthermore, anti-VEGF therapies are still ineffective in some cases, especially in patients with AMD. Hypoxia-inducible factor (HIF) is a strong regulator of VEGF induction under hypoxic and other stress conditions. Our previous reports have indicated that HIF is associated with pathological retinal neovascularization in murine models of ROP and AMD, and HIF inhibition suppresses neovascularization by reducing an abnormal increase in VEGF expression. Along with this, we attempted to find novel effective HIF inhibitors from natural foods of our daily lives. Food ingredients were screened for prospective HIF inhibitors in ocular cell lines of 661W and ARPE-19, and a murine AMD model was utilized for examining suppressive effects of the ingredients on retinal neovascularization. As a result, rice bran and its component, vitamin B6 showed inhibitory effects on HIF activation and suppressed VEGF mRNA induction under a CoCl₂-induced pseudo-hypoxic condition. Dietary supplement of these significantly suppressed retinal neovascularization in the AMD model. These data suggest that rice bran could have promising therapeutic values in the management of pathological ocular neovascularization.

Keywords: age-related macular degeneration; food ingredients; hypoxia-inducible factor; retinal pigment epithelium; rice bran; vascular endothelial growth factor; vitamin B6.

Figure A1

An inhibitory effect of panax ginseng on HIF activity. Quantitative analysis of HIF-reporter luciferase assay using ARPE-19 cells (n = 3 per group) showed that ginseng inhibited CoCl₂-induced HIF activity. *** p < 0.001, ### p < 0.001, compared with no treatment and 200 µM of CoCl₂ treatment, respectively. A bar graph was presented as mean ± standard deviation. The data were analyzed using one-way ANOVA followed by a Bonferroni post hoc test. Solvent, ginseng: water.

Figure A2

Inhibitory effects of components of vitamin B in rice bran on HIF activity. Quantitative analysis of HIF-reporter luciferase assay using ARPE-19 cells (n = 3 per group) showed that vitamin B6 dramatically inhibited CoCl₂-induced HIF activity more than any other components in rice bran. *** p < 0.001, ### p < 0.001, compared with no treatment and 200 µM of CoCl₂ treatment, respectively. A bar graph was presented as mean ± standard deviation. The data were analyzed using one-way ANOVA followed by a Bonferroni post hoc test. Solvents, rice bran: DMSO; vitamin B: water.

Figure A3

No cytotoxicity of rice bran and vitamin B6. Quantitative analyses (n = 4 per group) showed that a significant change in mitochondrial activity was not seen in ARPE-19 cells 12 h after rice bran or vitamin B6 treatment under a CoCl₂-induced pseudo-hypoxic condition. However, high-dose vitamin B6 (1 mg/mL) tended to damage mitochondrial activity. ** p < 0.01, *** p < 0.001, compared with no treatment. Bar graphs were presented as mean ± standard deviation. The data were analyzed using one-way ANOVA followed by a Bonferroni post hoc test. Solvents, rice bran: DMSO; vitamin B6: water.

Figure A4

No effect of rice bran and vitamin B6 on HIF-1α stabilization. Representative immunoblot images and quantitative analyses (n = 4 per group) for HIF-1α and β-Actin showed that HIF-1α was stabilized in 661W cells under a CoCl₂-induced pseudo-hypoxic condition. 1 mg/mL of rice bran and vitamin B6 did not significantly decrease stabilized HIF-1α expression. * p < 0.05, compared with no treatment. Bar graphs were presented as mean ± standard deviation. The data were analyzed using one-way ANOVA followed by a Bonferroni post hoc test. Solvents, rice bran: DMSO; vitamin B6: water.

Figure A5

No effect of rice bran and vitamin B6 on HIF-2α stabilization. Representative immunoblot images and quantitative analyses (n = 4 per group) for HIF-2α and β-Actin showed that HIF-2α was not significantly stabilized in ARPE-19 cells under a CoCl₂-induced pseudo-hypoxic condition. 1 mg/mL of rice bran and vitamin B6 did not significantly change its expression. Bar graphs were presented as mean ± standard deviation. The data were analyzed using one-way ANOVA followed by a Bonferroni post hoc test. Solvents, rice bran: DMSO; vitamin B6: water.

Figure A6

Evaluation of a mouse model of LIR. Representative waveforms of a- and b-waves (2 cd.s/m²) and quantitative analyses showed that the light exposure (3000 lux) significantly decreased the amplitudes of a-wave and b-wave in the retina (n = 6 per group, 12 eyeballs per group). ** p < 0.01, *** p < 0.001. Bar graphs were presented as mean ± standard deviation. The data were analyzed using Student’s t-test.

Figure 1

Inhibitory effects of rice bran and vitamin B6 on hypoxia-inducible factor (HIF) activity. (A) A process of drug screenings for HIF inhibitors. After the first screening, 10 samples were shown to be positive. After the second screening, four food ingredients (lactoferrin, rice bran, panax ginseng and Garcinia cambogia) with their expected 2 component compounds (hydroxycitric acid and vitamin B6) were selected as HIF inhibitors. (B) Quantitative analyses of HIF-reporter luciferase assay using ARPE-19 cells (n = 3 per group) showed that rice bran (1 mg/mL) and vitamin B6 (1 mg/mL) inhibited HIF activity induced by 200 µM CoCl₂. *** p < 0.001, ### p < 0.001, compared with no treatment and 200 µM of CoCl₂ treatment, respectively. Bar graphs were presented as mean with the ± standard deviation. The data were analyzed using one-way ANOVA followed by a Bonferroni post hoc test. Solvents, rice bran: DMSO; vitamin B6: water.

Figure 2

Suppressive effects of rice bran and vitamin B6 on HIF-1α stabilization. Representative immunoblot images and quantitative analyses (n = 4 per group) for HIF-1α and β-Actin showed that HIF-1α was stabilized in ARPE-19 cells under a CoCl₂-induced pseudo-hypoxic condition. Rice bran (1 mg/mL) and vitamin B6 (1 mg/mL) significantly decreased stabilized HIF-1α expression. *** p < 0.001, compared with no treatment, ## p < 0.01, ### p < 0.001, compared with CoCl₂ treatment. Bar graphs were presented as mean ± standard deviation. The data were analyzed using one-way ANOVA followed by a Bonferroni post hoc test. Solvents, rice bran: DMSO; vitamin B6: water.

Figure 3

Suppression of hypoxia-responsive gene expressions by rice bran and vitamin B6. Quantitative analyses (n = 4–6 per group) showed significant changes in HIF-1α, VEGF, BNIP3 and PDK1 mRNA expressions 8 h after incubation of CoCl₂ in ARPE-19 cells. Upregulated VEGF mRNA expression was significantly reduced by rice bran treatment. There was a decreasing tendency of upregulated VEGF mRNA expression by vitamin B6 treatment. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with no treatment, # p < 0.05, ## p < 0.01, ### p < 0.001, compared with CoCl₂ treatment. Bar graphs were presented as mean with ± standard deviation. The data were analyzed using one-way ANOVA followed by a Bonferroni post hoc test. Solvents, rice bran: DMSO; vitamin B6: water.

Figure 4

Suppression of neovascularization by rice bran and vitamin B6. (A) A schematic illustration demonstrates the murine choroidal neovascularization (CNV) model procedure and administration of rice bran or vitamin B6 to mice. (B) An immunoblot image for HIF-1α and β-Actin in the retina with or without the supplement of rice bran or vitamin B6, 3 days after the laser irradiation. (C) Representative images of CNV in the whole mount staining with isolectin B4 and quantitative analyses (n = 5–6 per group, n = 42–49 laser spots in the eyes per group) showed that the volume of CNV was significantly reduced by administration of rice bran (587.5 mg/kg diet weight) and vitamin B6 (9 or 35 mg/kg diet weight), respectively. Scale bars, 100 μm. * p < 0.05, ** p < 0.01. Dot plot graphs were presented as mean. The data were analyzed using Student’s t-test.

Figure 5

Direct retinal protection by rice bran and vitamin B6. (A) A schematic illustration demonstrates the murine light-induced retinopathy (LIR) model procedure and administration of rice bran or vitamin B6 to mice. (B,C) Representative waveforms of a- and b-waves (2 cd.s/m²) and quantitative analyses showed that rice bran (587.5 mg/kg diet weight) or vitamin B6 (9 mg/kg diet weight) did not change the amplitudes of a-wave and b-wave in LIR mice (n = 9–10 per group, 18–20 eyeballs per group). There was a significant decrease in the amplitudes of a-wave and b-wave in high-dose vitamin B6 (35 mg/kg diet weight)-administered LIR mice. * p < 0.05, ** p < 0.01, compared with control. The data were analyzed using Student’s t-test.