7,8-Dihydroxyavone Inhibits VEGF-Induced in Vitro Angiogenesis of RF/6A Cells via the Blocking of VEGFR2 Signaling Pathway

Background: This study aimed to investigate the anti-angiogenesis effect of 7,8-dihydroxyavone (7,8-DHF) and its potential molecular mechanism. Methods: The rhesus macaque choroid-retinal endothelial (RF/6A) cells were treated with different concentrations (from 0 to 100 μM) of 7,8-DHF and/or 40 ng/ml VEGF. The morphology, proliferation, migration, capillary-like tube formation (cid:0) and apoptosis of RF/6A cells were evaluated by Giemsa staining, CCK-8 assay, transwell migration assay, matrigel tube formation assay, and ow cytometry/hoechst33342 staining, respectively. The protein content of VEFGR2 and p-VEGFR2 was assessed by western blotting. Results: 7,8-DHF signicantly inhibited the proliferation, migration, and tube formation of RF/6A cells and promoted their apoptosis in vitro. The expression of VEGFR2 in RF/6A cells was constant whether or not to administer 7,8-DHF. However, the phosphorylation of VEGFR2 signicantly decreased after the administration of 7,8-DHF. Conclusions: 7,8-DHF could inhibit RF/6A angiogenesis in vitro. The inhibitory mechanism of 7,8-DHF in angiogenesis was attributed to the suppression of VEGFR2 phosphorylation and thus blocking of VEGF/VEGFR2 signal pathway.

repressive effect of 7, 8-DHF on pathologic neovascularization in vitro and explored its possible mechanisms, intending to identify a novel compound that would simultaneously inhibit angiogenesis and protect neurons.

Media and Reagents
Recombinant human VEGF165 was purchased from Novoprotein (Shanghai, China). Rabbit anti-human phosphor-VEGF receptor 2 and rabbit anti-human VEGF receptor 2 were obtained from Sigma-Aldrich (MO, USA). TRIZOL reagent was purchased from Invitrogen Life Technologies (Maryland, USA). Growth factorreduced Matrigel™ Matrix was purchased from BD Biosciences Labware (Massachusetts, USA). All other reagents and solvents were obtained from Beyotime (Shanghai, China).
Cell viability assay RF/6A cells in 100 µl of medium were seeded with a density of 2000 per well in a 96-well plate and allowed to attach for 24 hours before the addition of drugs. Immediately after that, cells in a 96-well plate were pretreated with different concentrations (from 0 to 100 µM) of 7,8-DHF for 1 hour, then together with VEGF (40 ng/ml) or not for 24 hours. After that, the assay medium was aspirated from the well without disturbing the formazan crystals. The cell proliferation was detected with CCK-8 kit according to the manufacturer's instructions. The optical density was measured with a microplate reader at 450 nm.
Giemsa Staining RF/6A cells were seeded in 24-well plates at a density of 5 × 10 5  Cell migration assay Cell migration assay was performed in a 24-well plate with 8.0-µm pore-size transwell inserts (Corning, MA, USA). Cells were also divided into 4 groups: the control group, 7,8-DHF group, VEGF group, and 7,8-DHF plus VEGF treated groups. Firstly, the RF/6A cells were placed in the upper chamber (5 × 10 4 /chamber) in a nal volume of 200 µl of serum-free medium containing or not 25 µmol/L 7,8-DHF. Then the bottom chamber was added with a nal volume of 600 µl of serum-free medium containing or not 40 ng/ml VEGF. After incubation at 37 °C for 24 h, the inserts were xed with 4% paraformaldehyde, and the cells were stained with Giemsa solution. Cell numbers from 5 random visual elds were counted under a phase-contrast microscope (Olympus BX40, Olympus) at 100 × magni cation.
Tube formation assay RF/6A cells in this assay were divided into 4 groups as previously described. Aliquots (250 µl) of Matrigel (Bedford, Massachusetts, USA) were added into a 24-well plate and incubated at 37 °C for 60 min. Flow Cytometry RF/6A cells in this assay were also divided into 4 groups. These cells were treated according to their respective grouping for another 24 h after being starved in the serum-free medium for 24 h. Trypsinized and oating cells were collected and washed in PBS. Then these cells were treated according to the manufacturer 's instructions of Apoptosis Detection Kit (Abcam plc., Cambridge, MA). Finally, the treated suspensions of RF/6A cells were analyzed by ow cytometry. Cells stained with both PI and FITC were considered late apoptotic while singly stained with FITC were early apoptotic, and these two parts were counted as apoptotic cells.
Hoechst33342 staining RF/6A cells were seeded in 24-well plates at a density of 5 × 10 5 cells/well and stabilized for 24 h. Then cells in this assay were divided into 4 groups as previously described. After incubation at 37 °C for 24 h, cells were incubated with 10 mol/L of Hoechst33342 for 10 min at room temperature and then washed with the medium for 3 times. Finally, cells were observed under a uorescence microscope. The apoptotic cell rate was calculated by the following formula: apoptotic nucleus rate = (apoptotic nucleus/ total nucleus) × 100%. Western blots were carried out according to the standard protocol and bands were analyzed by ImageJ software (NIH, Bethesda, MD, USA). The band intensities of p-VEGFR2 and VEGFR2 were quanti ed and normalized with VEGFR2 and GAPDH respectively.

Statistics
Statistical analysis was conducted with SPSS 19.0 software (IBM, Chicago, IL, USA). Data were expressed as the mean ± SD. The signi cance of the differences between the groups was determined using the independent-samples t-test or one-way ANOVA analysis. A p value < 0.05 was considered statistically signi cant. RF/6A cells were displayed a cobblestone-like appearance and structural integrated (Fig. 2a). VEGF (40 ng/ml) could increase RF/6A cell density and reduce extracellular space (Fig. 2b). After administration of 7,8-DHF, whether or not in the presence of VEGF, RF/6A cell density decreased with a reduction in cytoplasm density, and extracellular space became increased. Furthermore, the appearance of the RF/6A cells became irregular with the naked nucleus, apoptosis body, and cell atrophy and debris ( Fig. 2c-d).

Effects of 7,8-DHF on RF/6A migration
The migration study was assessed by the Transwell assay. As shown in Fig. 3

Discussion
Choroidal and retinal angiogenesis are the primary causes of vision loss in patients with neovascular AMD, proliferative DR, retinopathy of prematurity, and other diseases. It should be noted that these retinal vascular disorders are always accompanied by neuronal damage [11]. Although anti-VEGF treatments have been extensively administered for neovascular eye diseases [12], they are likely to aggravate this neuronal injury and thus deteriorate visual function [13]. To solve this dilemma, a treatment that can protect neurons and simultaneously inhibit angiogenesis is urgently needed. 7,8-DHF, a naturallyoccurring avone, has been reported to possess multiple bene cial effects, such as neuroprotective effect, anti-in ammatory, and vasorelaxing [14]. Recently, growing evidence suggests that 7, 8-DHF may also inhibit pathological neovascularization [7], although this has not yet been con rmed. In the present study, we observed that 25 µM 7,8-DHF signi cantly prevented the proliferation, migration, and tube formation of RF/6A cells and promoted their apoptosis in vitro regardless of in the presence of VEGF, indicating that 7,8-DHF has inhibitory effects on angiogenesis in vitro. This result, together with the previous ndings that low concentration (500 nM) of 7,8-DHF provides neuroprotection via the activation of TrkB receptor while high concentration (25 µM) of that via its potent antioxidant activity [15][16][17], make 7,8-DHF a potential candidate for the treatment of retinal vascular disease co-existing with neuropathy.
Many studies have revealed that 7,8-DHF at the concentration of 25 µM is nontoxic and even protective to several types of cells in vitro [16][17][18], whereas in the present experiment we discovered that 25 µM 7,8-DHF signi cantly inhibits the viability of RF/6A cells. The varying effects of 7, 8-DHF in these cell types may be attributed to its different pharmacological actions. VEGF is a potent proangiogenic factor that stimulates the proliferation, migration, and tube formation of endothelial cells and inhibits their apoptosis [19]. These biological VEGF signaling pathways are mainly mediated by VEGFR-2 receptor, which is abundant on the surface of vascular endothelial cells [20]. Phosphorylation of VEGFR-2 induces multiple downstream signals that subsequently promote angiogenesis in endothelial cells [21]. Thus, VEGF/VEGFR2 signal pathway is an important target for studying the angiogenesis inhibiting effects of 7, 8-DHF. In the present study, we demonstrated that although VEGFR2 expression was relatively constant, phosphorylated VEGFR2 levels were signi cantly decreased in RF/6A cells pretreated with 7, 8-DHF for 2 hours, in both the presence and absence of VEGF, indicating that 7, 8-DHF modulates the VEGFR2 signaling pathway by deactivating VEGFR2 rather than down-regulating its expression. There are two possible explanations for the observed 7, 8-DHF-induced signal deactivation. One is that 7, 8-DHF may down-regulate the expression of VEGF-A in RF/6A cells and thus inhibit signal transmission. According to this hypothesis, the extent of angiogenesis and level of phosphorylated VEGFR2 protein should be signi cantly different between the 7, 8-DHF-treated and 7, 8-DHF plus VEGF-treated groups, which contradicts our ndings in this study. The other explanation is that 7, 8-DHF prevents VEGFR2 activation by interfering with the binding of VEGF to VEGFR2. This well explains why even if VEGF levels are elevated in RF/6A cells, the levels of angiogenesis and phosphorylated VEGFR2 protein remain unchanged in the presence of 7,8-DHF (25 µM). This assumption is also supported by a previous study using a combination of molecular docking, computational mapping tools, and molecular dynamics, which showed that 7, 8-DHF may exert its effects on VEGFR2 via the intracellular kinase domain [7]. Collectively, the above evidence suggests that 7, 8-DHF inhibits angiogenesis in RF/6A cells in vitro by suppressing the phosphorylation of VEGFR2, although the exact mechanism of VEGFR2 dephosphorylation induced by 7, 8-DHF remains to be elucidated.
In this study, we comprehensively investigated the antiangiogenic effects of 7, 8-DHF on RF/6A cells, which were mainly used in the study of chorioretinal neovascular diseases. However, whether this medicine has similar inhibitory effects on other types of vascular endothelial cells, such as human umbilical vein endothelial cells (HUVEC), remains to be studied. Furthermore, in vivo studies are needed to obtain a comprehensive understanding of the observed effect and the mechanism underlying the effects of this compound on choroidal and retinal angiogenesis.

Conclusions
In summary, the present study revealed the inhibitory effect of 7, 8-DHF on angiogenic properties in vitro. This nding, combining with the previous discoveries that low concentration (500 nM) of 7,8-DHF provides neuroprotection via the activation of TrkB receptor while high concentration (25 µM) of that via its potent antioxidant activity [15][16][17], suggests the great potential of 7,8-DHF for the treatment of ocular diseases with coexisting angiogenesis and neuropathy, such DR, AMD, neovascular glaucoma, etc. Since there are numerous patients with these diseases, medicine with the above pharmacological activities could provide great bene ts. Availability of data and materials The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors do not have any con ict of interest to report and have no proprietary interest in any of the materials mentioned in this article