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

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.

Humans consume a variety of nutrients in a daily life.Interests in the relationship between food and health and the prevention of diseases through food in recent years have increased as healthcare costs, life expectancy, and health concerns have been issued among people in developed countries [23,24].In general, superfoods are ingredients consumed for health promotion purposes for a long time, with excellent nutritional balance or exceptionally high amounts of certain nutrients [24,25].Medical uses of superfoods, such as the prevention of metabolic syndrome through the consumption of superfoods, have also been reported [26][27][28].Previously, ten clinical trials, which investigated broccoli sprout supplementation and cardiometabolic health, showed that the dietary intake of broccoli sprouts signifcantly reduced systolic and diastolic blood pressures [29].Te health efect of camu-camu is still in controversial status.However, daily 70 mL of 100% camucamu juice taken for 7 days could reduce oxidative stress markers, such as levels of urinary 8-hydroxydeoxyguanosine and infammatory markers, including serum levels of high-sensitivity C-reactive protein, interleukin (IL)-6, and IL-8 in male with smoking [14].
In recent fundamental studies using superfoods, the polyphenol velutin of acai fruit has been shown to downregulate HIF-1α expression in RAW 264.7 mouse monocyte macrophage cells [30], and wolfberry polysaccharides could inhibit HIF-1α expression in the mouse retina [31].Few studies to date have focused on the antineovascular efects of superfoods on RPE cells.In this study, we screened seven superfoods (camu-camu, coconut, broccoli sprout, chia seed, hemp, maca, and cacao) that have been suggested to have efcacies in human health in interventional clinical trials [28,29,[32][33][34] for new inhibitors for the HIF/VEGF pathway in RPE cells.
Cacao nibs were homogenized with zirconia balls (As One, Osaka, Japan) at 6,000 rpm for 20 s three times in MQ.All samples were adjusted immediately before use.Camu-camu powder used in this study was made from camu-camu pulps and contains 5,850 mg of vitamin C per 100 g.

Statistical Analysis.
Statistical signifcance was calculated using two-tailed Student's t-test for comparison of two groups.p values of less than 0.05 were considered statistically signifcant.

Results
RH-ARPE19 cells were treated with seven superfood samples to test for the HIF inhibitory activity.For the frst luciferase assay screening, 200 μM CoCl 2 was used to induce the HIF activity, and 1 mM of topotecan was used as the positive control for HIF inhibition [37,38] (Figure 1(a)).Compared to the control containing only pure water, the relative luciferase activity was increased in the MQ group loaded with CoCl 2 -induced pseudohypoxic conditions (Figure 1(a)).Te four superfood samples camu-camu, chia seeds, maca, and cacao nibs exhibited HIF-suppressive effects compared to MQ (Figure 1(a)) under CoCl 2 -induced pseudohypoxic conditions.In the second luciferase screening, to better mimic hypoxic conditions, we used 1% O 2 hypoxic incubation to stabilize HIF expression (Figure 1(b)).Te four superfood samples selected by the frst trial of the luciferase assay screening were used in the second luciferase screening (Figure 1(b)).Camu-camu had a statistically signifcant inhibitory efect on HIF activation.Camu-camu treatment inhibited the HIF activity under CoCl 2 -induced pseudohypoxic conditions in a dosedependent manner (from 1 to 1,000 μg/mL) (Figure 2(a)).Cell toxicity of camu-camu treatment was increased between 300 and 1000 μg/ml regarding internal control Renilla expression (Figure 2(b)).From the series of screening tests, we found that camu-camu may have the potential to destabilize HIF expression in human RPE cells.

Discussion
In this study, we found that camu-camu treatment could inhibit the stabilization of nonphysiologic HIF-1α proteins in ARPE-19 cells.Among the seven superfood candidates, camu-camu was found to be a novel HIF inhibitor based on luciferase assay screening under pseudohypoxic conditions using CoCl 2 and 1% O 2 (Figure 1).Camu-camu is a native Amazonian bush-bearing, round, redberry-like fruit.Camucamu pulps are used not only in the Amazon region but also in Japan and Europe as juice, sherbet, and extracts [41].Camu-camu contains natural antioxidants, such as vitamin C, carotene, phenolic compounds, favonols, anthocyanins, ellagic acid conjugates, ellagitannins, gallic acid derivatives, and proanthocyanidins [41,42,46].Tese bioactive substances have been reported to possess antioxidant and free radical scavenging abilities [47][48][49].Camu-camu has been reported to contain approximately 1,882-2,061 mg of vitamin C in 100 g of fresh mature fruit [46,50].Te camu-camu powder used in this study contained 5,850 mg of vitamin C per 100 g, indicating that 5.85% of the powder contained vitamin C by weight.
Vitamin C is a strong antioxidant [43] that inhibits HIF expression in cancer [51] and lens epithelial cells [52].Furthermore, the mode of action of vitamin C on HIF-1α suppression has been suggested to involve in prolyl hydroxylation [45].Te camu-camu extract suppressed HIF-1α and HIF-2α expressions to a greater extent than vitamin C in ARPE-19 cells (Figures 4 and 5).Tese results suggest that components other than vitamin C in camu-camu may synergize with the inhibitory efects of HIF.In our current study, water-soluble substances in camu-camu were only Te ellagic acid has been reported to have HIF-1α suppressive efects on the human urinary bladder carcinoma cell line (ECV304) [53].Aqueous HIF-inhibitory substances can reach the choroidal blood vessels that nourish RPE cells, as abundant blood fows into the choroid blood vessels from the short posterior ciliary artery, the long posterior ciliary artery, and the anterior ciliary artery which are from the internal carotid artery.Taken together, the investigation on substances in camu-camu that may have additional HIFinhibitory efects will be further studied.Intravitreal anti-VEGF therapy is an important treatment option for patients with vision loss due to abnormal neovascularization, including AMD, macular edema secondary to retinal vein occlusion (RVO), diabetic macular edema (DME), myopic choroidal neovascularization (mCNV), and retinopathy of prematurity (ROP).More than 2.5 million intravitreal injections are used annually in the United States [54].Anti-VEGF therapy appears to be an efective treatment for the retina; however, prolonged and/or frequent treatments may be associated with an increased risk of ocular local and/or systemic adverse events, including geographic atrophy [55], cerebrovascular disease, and death [56].Occasionally, patients do not respond adequately to anti-VEGF therapy [57,58].Terefore, it is necessary to explore treatment options other than anti-VEGF therapy for these diseases.In this regard, our camu-camu extract might be helpful.
Increased HIF-1α expression in RPE cells promotes the production of VEGF, and increased VEGF expression promotes the development of abnormal neovascularization [59][60][61].Muller cells have possibilities to play an important role in the production of VEGF and HIF-1α, which are associated with infammation of the inner retinal layers, such as in diabetic retinopathy [62].Although we focused on HIF and VEGF expressions in RPE cells in our current study, it may be necessary to consider Muller cells for the further work.Based on the role of HIF-1α in angiogenesis, HIF-1α may represent a molecular therapeutic target for ocular neovascularization diseases in addition to VEGF, as noted in previous reports [63][64][65].We showed that the camu-camu extract inhibited HIF-1α and HIF-2α expressions in ARPE-19 cells.Te HIF-2α mRNA level was decreased by the 10 μg/ml and 30 μg/ml camu-camu treatments (Figure 4).Te 10 μg/ml and 30 μg/ml camu-camu treatments inhibited the transcription of HIF-2a, but the translation is unknown from this experiment because we were unable to detect translation changes in HIF-2α protein.Regarding HIF-1α, the camu-camu treatments inhibit transcription and translation (Figures 3-5).VEGF and its receptors VEGF receptor-1 and VEGF receptor-2 are directly induced by HIF-2α under hypoxic conditions through their identifed HRE [66,67].Inhibition of excessive
HIF-2α is expressed in the subretina in patients.Deferoxamine (DFO), an iron chelator, causes RPE atrophy as adverse efects.Clinically inhibiting upregulation of HIF-2α by α-ketoglutarate relieved DFO-related RPE atrophy [69].Camu-camu, which suppresses both HIF-1α and HIF-2α expressions in RPE cells, could become an adjuvant therapy to assist current treatments for patients with abnormal neovascularization and subsequent RPE atrophy.Because camucamu can readily be consumed in the form of juice or food, it is considered acceptable to patients mentally and economically.However, the dosage and administration need to undergo further study.

Conclusions
Although the in vivo efects need to be further investigated, we found that among the seven superfood candidates, camucamu treatment inhibited upregulation of HIF/VEGF expressions in ARPE-19 cells.Camu-camu could become an adjuvant therapy to assist anti-VEGF therapy in patients with abnormal neovascularization and subsequent RPE atrophy in an era of rising expectations regarding functional foods and superfoods.