Modulation of Hypoxia-Inducible Factors and Vascular Endothelial Growth Factor Expressions by Superfood Camu-Camu (Myrciaria dubia) Treatment in ARPE-19 and Fetal Human RPE Cells

Abstract

Background: Anti-vascular endothelial growth factor (anti-VEGF) therapy via intravitreal injection is an effective treatment for patients with abnormal ocular neovascularization, such as age-related macular degeneration (AMD) and diabetic macular edema (DME). However, prolonged and frequent anti-VEGF treatment is associated with a risk of local and systemic adverse events, including geographic atrophy, cerebrovascular disease, and death. Furthermore, some patients do not adequately respond to anti-VEGF therapy. Hypoxia-inducible factor (HIF) is a transcription factor that controls the expression of hypoxia-responsive genes involved in angiogenesis, inflammation, and metabolism. The HIF/VEGF pathway plays an important role in neovascularization, and the inhibition of HIF activation could be an effective biomolecular target for neovascular diseases. The demand for disease prevention or treatment using functional foods such as superfoods has increased in recent years. Few reports to date have focused on the antineovascular effects of superfoods in the retinal pigment epithelium (RPE). In light of the growing demand for functional foods, we aimed to find novel HIF inhibitors from superfoods worked in RPE cells, which could be an adjuvant for anti-VEGF therapy.

Methods: Seven superfoods were examined to identify novel HIF inhibitor candidates using luciferase assay screening. We used the human RPE cell line ARPE-19 and fetal human RPE (fhRPE) to investigate the biomolecular actions of novel HIF inhibitors using quantitative PCR and western blotting.

Results: Under CoCl₂-induced pseudohypoxic condition and 1% oxygen hypoxic incubation, camu-camu (Myrciaria dubia) showed HIF inhibitory effects determined by luciferase assays. Camu-camu downregulated HIF-1α and VEGFA mRNA expressions in a concentration-dependent manner. Camu-camu also inhibited HIF-1α protein expressions, and its inhibitory effect was greater than that of vitamin C, which is present at high levels in camu-camu.

Conclusion: The camu-camu extract suppressed the activation of HIF and VEGF in RPE cells. This could assist anti-VEGF therapy in patients with abnormal ocular neovascularization.

Figure 1

Inhibitory hypoxia-inducible factor (HIF) activity effects of superfoods. Of seven superfoods (camu-camu, coconut, broccoli sprout, chia seed, hemp, maca, and cacao nib) after the first screening, four samples (camu-camu, chia seed, maca, and cacao nib) were shown to be positive (a). After the second screening, camu-camu was identified as a new HIF inhibitor candidate (b). Quantitative analyses of the HIF-reporter luciferase assay using ARPE-19 cells (n = 3 per group, biological). The superfoods were added at 1 mg/mL each. (a) HIF activity induced by 200 μM CoCl₂ or (b) 1% O₂ hypoxic incubation. The control was no induction of HIF activity. ^###p < 0.001 compared with no treatment. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 compared with MQ by (a) 200 μM of CoCl₂ treatment or (b) 1% O₂ hypoxic incubation, respectively. The bar graphs present means with the ± standard deviation. The data were analyzed using two-tailed Student's t-test for comparison. MQ: Milli-Q® pure water; Topo: topotecan.

Figure 2

Inhibitory hypoxia-inducible factor (HIF) activity effects of camu-camu. Camu-camu was evaluated at various concentrations. (a, b) Quantitative analyses of the HIF-reporter luciferase assay using RH-ARPE19 cells (n = 3 per group, biological). Camu-camu was added from 1 to 1000 μg/mL each. HIF activity induced by 200 μM CoCl₂. HIF activity was not induced in the control group. ^###p < 0.001 compared with no treatment. ^∗p < 0.05 and ^∗∗∗p < 0.001 compared with MQ by 200 μM of CoCl₂ treatment. (a) Relative luciferase activity reflecting HIF-1α activity. (b) Relative Renilla activity reflecting cell viability. The bar graphs present means with  ± standard deviation. The data were analyzed using two-tailed Student's t-test for comparison. MQ: Milli-Q® water; Topo: topotecan; DXR: doxorubicin.

Figure 3

Suppression of hypoxia-responsive gene expressions by camu-camu. (a–f) Quantitative analyses (n = 3–5 per group, biological) show significant changes in HIF-1α, GLUT1, BNIP3, PDK1, and VEGFA mRNA expression after 7 hours under camu-camu treatment from 0.1 μg/mL to 10 μg/mL each in ARPE-19 cells. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 compared with no treatment. The bar graphs present means with ± standard deviation. The data were analyzed using two-tailed Student's t-test for comparison.

Figure 4

Comparison of hypoxia-responsive gene expression by camu-camu and vitamin C. Quantitative analyses (n = 3–5 per group, biological) using ARPE-19 cells reveal significant changes in HIF-1α, HIF-2α, and BNIP3 mRNA expression after 7 hours of 10 μg/mL and 30 μg/mL camu-camu treatment. Vitamin C was added at 1.755 μg/mL. Low-dose camu-camu treatment comprised 10 μg/mL of camu-camu, and high-dose camu-camu treatment (30 μg/mL) contained 1.755 μg/mL of vitamin C. HIF-1α, HIF-2α, and BNIP3 mRNA-suppressive effects of camu-camu treatment were generally greater than those of vitamin C treatment. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared with no treatment. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 compared with camu-camu treatments and vitamin C treatment. NS: no significant. The bar graphs present means with ± standard deviation. The data were analyzed using two-tailed Student's t-test for comparison.

Figure 5

Comparison of camu-camu and vitamin C effects on suppression of HIF-1α protein expression. HIF-1α activity induced by 200 μM CoCl₂ and suppression by doxorubicin. Treatment with 1 μg/mL camu-camu comprises 5.85 ×10⁻² μg/mL of vitamin C. (a) Camu-camu suppresses HIF-1α protein expression in ARPE-19 cells. (b) Quantification of the blots shows that the administration of camu-camu suppressed increased HIF-1α protein expression under CoCl₂ in ARPE-19 cells (n = 3, biological and technical). (c) HIF-1α expression under CoCl₂ in fhRPE cells. (d) Quantification of the blots in fhRPE (n = 3, biological and technical). ^#p < 0.05 compared with CoCl₂ + MQ. ^∗p < 0.05 and ^∗∗p < 0.01 compared with no CoCl₂ treatment. The bar graphs present means with ± standard deviation. The data were analyzed using two-tailed Student's t-test for comparison. MQ: Milli-Q® pure water; Topo: topotecan; DXR: doxorubicin; Vit C: vitamin C.