Ampelopsin was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mM as a stock solution. Endothelial growth medium-2 (EGM-2) was obtained from Lonza (Walkersville, MD, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Grand Island, NY, USA). Recombinant human vascular endothelial growth factor (VEGF), Matrigel and Transwell chamber systems were obtained from Koma Biotech (Seoul, Korea), BD Biosciences (San Jose, CA, USA) and Corning Costar (Acton, MA, USA), respectively. Anti-hypoxia inducible factor-1α (HIF-1α) antibody was purchased from BD Biosciences. Anti-phospho-VEGFR2, anti-VEGFR2, anti-phospho-STAT3, anti-STAT3, anti-phospho-AKT, anti-AKT, anti-phospho-ERK1/2, anti-ERK1/2, cyclin D1, MMP-2, MMP-9 and anti-β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

The fresh branches of Hovenia dulcis Thunb. (Jangheung, Korea) were fragmented and stored in a closed container. Ethanol extract was obtained by dissolving 150 g of raw material in 1 liter of 100% (vol/vol) ethanol and shaken for 24 h at room temperature. The mixture was filtered and the clear filtrate was concentrated, vacuum dried and kept at −20°C.

Human umbilical vein endothelial cells (HUVECs) and HepG2 (human hepatocarcinoma) cells were obtained from the Korean Cell Line Bank (KCLB) and grown in EGM-2 and DMEM supplemented with 10% FBS, respectively. All cells were maintained at 37°C in a humidified 5% CO₂ incubator. For hypoxic condition, cells were incubated in a hypoxic chamber (Forma Scientific, Marietta, OH, USA) under 5% CO₂ and 1% O₂ balanced with N₂.

Serum-starved HUVECs (3×10³ cells/well) seeded in a 96-well culture plate were stimulated by VEGF (30 ng/ml) with or without various concentrations of Hovenia dulcis Thunb. extract and ampelopsin for 72 h. Cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Briefly, 50 µl of MTT solution (2 mg/ml; Sigma-Aldrich) was added to each well followed by incubation for 3 h at 37°C. To dissolve formazan crystals, the culture medium was removed and an equal volume of DMSO was added to each well. The absorbance of each well was determined at a wavelength of 540 nm using a microplate reader (Thermo Scientific, Vantaa, Finland).

The invasiveness of HUVECs was investigated using a Transwell chamber system with polycarbonate filter inserts with a pore size of 8.0 µm. Briefly, the lower side of the filter was coated with 10 µl gelatin (1 mg/ml) and the upper side was coated with 10 µl Matrigel (3 mg/ml). Serum-starved HUVECs (8×10⁴ cells) were placed in the upper chamber of the filter and Hovenia dulcis Thunb. extract and ampelopsin were added to the lower chamber in the presence of VEGF (30 ng/ml). The chamber was incubated at 37°C for 18 h, and then the cells were fixed with methanol and then stained with hematoxylin and eosin (H&E). The total number of cells that invaded the lower chamber of the filter was counted using an optical microscope (Olympus, Center Valley, PA, USA) at a magnification of ×100.

Serum-starved HUVECs (4×10⁴ cells) were inoculated on a surface containing Matrigel (10 mg/ml) and were incubated with Hovenia dulcis Thunb. extract and ampelopsin for 6 h in the presence of VEGF (30 ng/ml). Morphological changes in the cells and tube formation were visualized under a microscope and photographed at a magnification of ×100 (Olympus). Tube formation was quantified by counting the total number of branched tubes in randomly selected fields at a magnification of ×100.

The confluent monolayer HUVECs were scratched using a tip and each well was washed with PBS to remove non-adherent cells. The cells were treated with Hovenia dulcis Thunb. extract and ampelopsin in the presence of VEGF (30 ng/ml) and then incubated for up to 48 h. The perimeter of the area with a central cell-free gap was confirmed at the time intervals 0, 24, and 48 h under an optical microscope (Olympus).

Fertilized chick eggs were maintained in a humidified incubator at 37°C for 3 days. Approximately 5–6 ml egg albumin was removed with a hypodermic needle, allowing the CAM and yolk sac to drop away from the shell membrane. After 2 days, the shell was punched out and peeled away. Thermanox coverslips (Nalge Nunc International, Rochester, NY, USA) with or without Hovenia dulcis Thunb extract and ampelopsin were air-dried and applied to the CAM surface, respectively. Two days later, 1 ml of 10% intralipid (Sigma-Aldrich) was injected into the chorioallantois and the CAM was observed under a microscope.

Cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) using standard electroblotting procedures. The blots were blocked and immunolabeled with primary antibodies against phospho-VEGFR2, VEGFR2, phospho-STAT3, STAT3, phospho-AKT, AKT, phospho-ERK1/2, ERK1/2, cyclin D1, MMP-2, MMP-9, HIF-1α and β-actin overnight at 4°C. Immunolabeling was detected with an enhanced chemiluminescence (ECL) kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions.

ROS levels were detected with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Eugene, OR, USA). For the assay, serum-starved HUVECs seeded at a density of 1×10⁵ cells/well in 96-black well culture plates were pretreated with Hovenia dulcis Thunb. extract and ampelopsin for 3 h. After incubation with H2DCFDA (10 µM) for 5 min, the cells were stimulated with VEGF (30 ng/ml) for 5 min. The fluorescence intensity of DCF was detected using an Optinity KI-2000F fluorescence microscope (Korea Lab Tech) or a multimode microplate reader (Thermo Scientific, Vantaa, Finland) at the excitation and emission wavelengths of 495 and 529 nm, respectively.

VEGF concentration in the media from Hovenia dulcis Thunb. extract- or ampelopsin-treated cells was determined using a VEGF immunoassay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The results were expressed as the concentration of VEGF relative to the total amount of protein from each well.

The results are expressed as the mean ± standard error (SE). Student's t-test was used to determine statistical significance between the control and the test groups. A P-value of <0.05 was considered to indicate a statistically significant difference.

First, to determine the optimum dose of 100% ethanol extract of Hovenia dulcis Thunb. (HDT) with no cytotoxicity for angiogenesis assays, various concentrations of HDT were applied to human umbilical vein endothelial cells (HUVECs) for 72 h. The viability of the HUVECs was not influenced up to 100 µg/ml of HDT treatment (Fig. 1A). Based on this result, in vitro angiogenesis assays were performed in a concentration range (0–100 µg/ml) of HDT at which no cytotoxicity was observed. To assess the antiangiogenic activity of HDT, we investigated the effect of HDT on the proliferation of HUVECs induced by VEGF. Serum-starved HUVECs were stimulated by VEGF with or without HDT and then cell growth was measured using the MTT colorimetric assay. As shown in Fig. 1B, HDT inhibited the VEGF-induced proliferation of HUVECs. We next examined the effect of HDT on key angiogenic phenotypes of endothelial cells such as cell invasion, tube formation and migration. HDT dose-dependently decreased the VEGF-induced invasiveness, tube forming ability and migration of HUVECs at subtoxic doses (Fig. 1C-E). These results suggest that Hovenia dulcis Thunb. extract significantly inhibits VEGF-induced angiogenesis of HUVECs in vitro.

The antiangiogenic activity of HDT was also validated in vivo using a chorioallantoic membrane (CAM) assay. Coverslips containing HDT were placed on the CAM surface, and angiogenesis zones were observed under a microscope. As shown in Fig. 1F, the inhibition of neovascularization on control coverslips was 16% (n=18), whereas HDT much more potently inhibited the angiogenesis of the CAM (68% at 2 µg/egg, n=16) without toxicity against pre-existing vessels. Taken together, these results demonstrated that Hovenia dulcis Thunb. extract efficiently inhibits angiogenesis without affecting endothelial cell viability both in vitro and in vivo.

VEGFR2 is considered the major mediator of VEGF-induced downstream signaling in endothelial cells, including survival, mitogenesis and especially angiogenesis (27–29). To examine the underlying molecular mechanism of HDT on angiogenesis, we evaluated the effect of HDT on VEGF-mediated VEGFR2 signaling pathways in HUVECs. As shown in Fig. 2A, HDT decreased VEGF-induced VEGFR2, STAT3, AKT and ERK1/2 phosphorylation in a dose-dependent manner. In addition, HDT reduced the total protein levels of VEGFR2, indicating that the inhibitory effect on VEGFR2 expression affected VEGF-induced VEGFR2 phosphorylation. VEGFR2 signaling induces the expression of endothelial cell-derived matrix metalloproteases (MMPs), including MMP-2 and MMP-9, which degrade the matrix to allow for endothelial sprouting. The cell cycle regulatory protein, cyclin D1, also plays a key role in endothelial cell proliferation during angiogenic process (6,7). As shown in Fig. 2B, HDT suppressed the protein expression of MMP-2, MMP-9 and cyclin D1 in a dose-dependent manner. Furthermore, HDT remarkably reduced the VEGF-stimulated ROS generation in HUVECs (Fig. 2C). Thus, these data suggest that HDT exhibits the antiangiogenic activity by inhibiting VEGFR2-mediated downstream signaling cascades in HUVECs.

HIF-1α is a crucial transcription factor of the angiogenesis in tumor cells, which controls transcription of hypoxia-regulated genes such as VEGF (10,11). Thus, we evaluated the HIF-1α inhibitory activity of HDT in the human hepatoma cell line HepG2, a hypervascularized tumor. HDT-treated HepG2 cells attentively reduced the hypoxia-induced accumulation of HIF-1α protein at 100 µg/ml (Fig. 3A). We further assessed the effect of HDT on the expression of VEGF during hypoxia. As shown in Fig. 3B, treatment with HDT under hypoxia decreased the VEGF production in HepG2 cells, indicating that HDT inhibits tumor angiogenesis by downregulating HIF-1α and VEGF expression.

Ampelopsin is a major active ingredient of Hovenia dulcis Thunb. extract (23) (Fig. 4A). The high-performance liquid chromatography (HPLC) analysis also revealed that the ethanol extract of Hovenia dulcis Thunb. branches used in the present study contained ampelopsin (Fig. 4B). Thus, we assessed whether ampelopsin is one of the possible pharmaceutically active compounds contributing to the antiangiogenic activity of Hovenia dulcis Thunb. extract. Unlike HDT, ampelopsin did not affect the VEGF-induced proliferation of HUVECs (Fig. 4C). However, ampelopsin significantly inhibited the VEGF-induced invasiveness, tube forming ability and migration of HUVECs (Fig. 4D-F). Furthermore, ampelopsin effectively inhibited the angiogenesis of the CAM (72% at 10 µg/egg, n=18) without showing any rupture of or toxicity against pre-existing vessels, when compared to the control (15%, n=20) (Fig. 4G). These results demonstrated that ampelopsin suppresses both in vitro and in vivo angiogenesis without affecting endothelial cell proliferation.

To elucidate whether ampelopsin affects VEGFR2 signal transduction, we investigated the effect of ampelopsin on VEGF-induced VEGFR2 signaling pathways in HUVECs. As shown in Fig. 5A, ampelopsin effectively reduced the phosphorylation of VEGFR2, AKT and ERK1/2 induced by VEGF without affecting the total protein levels. In addition, we found that ampelopsin dose-dependently reduced the generation of ROS induced by VEGF in HUVECs, indicating that the blockade of VEGFR2 signaling by ampelopsin may be associated with the downregulation of ROS generation (Fig. 5B). We next evaluated the HIF-1α inhibitory activity of ampelopsin in HepG2 cells. Ampelopsin prominently inhibited the expression of HIF-1α and its transcriptional target, VEGF, during hypoxia (Fig. 5C and D). Taken together, these results suggest that ampelopsin shows antiangiogenic activity through the blockade of both VEGFR2 signaling and HIF-1α expression and, therefore, the antiangiogenic effect of Hovenia dulcis Thunb. extract is at least in part due to ampelopsin (Fig. 6).