Primary cultures of human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Walkersville, MD, USA) and used between passages 4 and 6 for all the experiments. The cells were cultured in EGM-2^® BulletKit media, according to the manufacturer’s instructions (Lonza).

The following pharmacological agents and antibodies were purchased from commercial sources: vascular endothelial growth factor-A (VEGF-A) and LY294002 (Merck Millipore, Billerica, MA, USA); rapamycin (Sigma-Aldrich, St. Louis, MO, USA); PD98059, anti-phospho-ERK (T202/Y204), anti-phospho-Akt (S473), anti-phospho-p70^S6K (T421/S424), anti-phospho-p38^MAPK (T180/Y182), anti-phospho-pRb (S780) and anti-MMP-2 (Cell Signaling Technology, Beverly, MA, USA); anti-VEGFR-2, anti-ERK, anti-Akt, anti-p70^S6K, anti-p38^MAPK, anti-TIMP-2, anti-Cdk4, anti-Cdk2, anti-cyclin D, anti-cyclin E, anti-actin antibodies and mouse and rabbit IgG-horseradish peroxidase conjugates (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

The ethanol extract was prepared by mixing 100 g of twigs of BK with 1 liter of 100% ethanol and stirring for 72 h. The extract was then filtered through a filter paper (Advantec No. 1; Toyo Roshi Kaisha, Ltd., Tokyo, Japan), and the filtrate was concentrated using a rotary evaporator (Tokyo Rikakikai Co., Ltd., Tokyo, Japan) at 40°C under vacuum. The yield of the dried extract was ~1.37%.

HUVECs, plated on 6-well plates (1×10⁵ cells/well; BD Biosciences, Bedford, MA, USA), were serum-starved for 14 h to synchronize cells in the G₁/G₀ phase of the cell cycle, and pretreated with BK (0.1–10 μg/ml) for 30 min in the presence or absence of PD98059 (25 μM), LY294002 (10 μM) or rapamycin (50 nM) as indicated, and then incubated with VEGF-A (10 ng/ml) for 24 h. Following culture for 24 h, cell viability was determined by a Muse™ cell analyzer using a cell count and viability assay kit (Merck Millipore), and the cell proliferation was quantified as previously described (20–22). The results from triplicate determinations (mean ± standard deviation) are presented as the number of cells per culture.

HUVECs in 100-mm dishes (1×10⁶ cells/dish; BD Biosciences) were serum-starved for 14 h in endothelial cell basal medium (EBM-2; Lonza) and replaced with fresh media, followed by treatments for different time-points, as indicated in the figure legends, at 37°C. The cells were rinsed twice with ice-cold phosphate-buffered saline (PBS) and lysed by incubation in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 100 μg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml pepstatin A, 0.5 μg/ml leupeptin, 80 mM β-glycerophosphate, 25 mM sodium fluoride and 1 mM sodium orthovanadate for 30 min at 4°C. Cell lysates were clarified at 12,500 × g for 20 min at 4°C, and the supernatants were subjected to western blot analysis as previously described (23,24). All the western blots were performed in triplicate and representative gels were shown.

Cell migration was quantified in the in vitro wound-healing assay as previously described (25,26). Cells were plated on 48-well plates (4×10⁴ cells/well), grown to confluence, and a single wound was created in the center of the cell monolayer by the gentle removal of the attached cells with a sterile plastic pipette tip. Following serum starvation with EBM-2 for 2 h, the cells were pretreated with BK (0.1–10 μg/ml) for 30 min, followed by VEGF-A (10 ng/ml) stimulation for 16 h. The cells were fixed with methanol, and then stained with 0.04% Giemsa solution (Sigma-Aldrich). The migration of the cells into the wound was observed using images captured at the indicated time-points.

Matrigel^® basement membrane matrix (10.4 mg/ml; BD Biosciences) was thawed overnight at 4°C, and each well of pre-chilled 24-well plates was coated with 200 μl Matrigel^® and then incubated at 37°C for 30 min. Following serum starvation with EBM-2 for 2 h, the cells (4×10⁴ cells/ml) were added to Matrigel^®-coated plates and pretreated with BK (0.1–10 μg/ml) for 30 min, followed by VEGF-A (10 ng/ml) for 6 h. Tube formation was observed with an Olympus CKX41 inverted microscope (CAchN 10/0.25php objective) and ToupTek Toupview software (version x86, 3.5.563; Hangzhou ToupTek Photonics Co., Ltd., Zhejiang, China).

Activities of MMPs were measured by zymography (27,28). Aliquots of conditioned medium collected from HUVECs treated with BK (10 μg/ml) and VEGF-A (10 ng/ml) for 16 h were diluted in sample buffer, and applied to 8% polyacrylamide gels containing 1 mg/ml gelatin (Sigma-Aldrich) as a substrate. After electrophoresis, the gels were incubated in 2.5% Triton X-100 for 1 h to remove SDS and allow the re-naturalization of MMPs, and then incubated in developing buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM CaCl₂, and 150 mM NaCl for 16 h at 37°C. The gels were stained with 0.5% Coomassie brilliant blue R-250 in 30% methanol-10% acetic acid for 3 h and followed by destaining with 30% methanol-10% acetic acid. Gelatinolytic activities were detected as unstained bands against the background of the Coomassie blue-stained gelatin.

Statistical analysis was performed using the Student’s t-test, and was based on at least three different experiments. The results were considered to be statistically significant when P<0.05.

We first examined the ability of BK to regulate proliferation in HUVECs. BK treatment suppressed VEGF-A-stimulated cell proliferation in a dose-dependent manner (Fig. 1A) and did not alter cell viability (Fig. 1B), suggesting that BK inhibition of cell proliferation is not mediated by the induction of apoptosis or cytotoxicity. Based on these findings, we examined the regulatory effect of BK on cell cycle progression by analyzing the changes of cyclin-dependent kinases (Cdks) and cyclins. Cell cycle progression requires activation of Cdks through formation with cyclins and subsequent phosphorylation of retinoblastoma protein (pRb) (29). As shown in Fig. 1C, BK treatment markedly suppressed the expression of Cdks and cyclin D, resulting in pRb hypophosphorylation in VEGF-A-treated HUVECs. These findings indicate that BK downregulates the expression of cell cycle-related proteins, resulting in inhibition of cell cycle progression and proliferation.

Endothelial cell migration and tubular formation are coordinately controlled by the interactions from ECM molecules to the intracellular and intercellular components of cells, and play important roles in angiogenic responses associated with cancer growth and progression (2). BK treatment dose-dependently inhibited VEGF-A-stimulated cell migration and tubular formation in HUVECs (Figs. 2 and 3). Collectively, these findings suggested that the bioactive components from the ethanolic extracts of BK act on multiple targets and mechanisms involved in cell proliferation, migration and tubular formation, resulting in the regulation of angiogenic responses in vitro.

The expression and activation of VEGFR-2 and MMP-2 have been reported to promote angiogenic responses including endothelial cell proliferation, migration, invasion and tubular formation (1,2,30). To understand the molecular mechanism underlying the regulatory effects of BK on endothelial cell proliferation, migration and tubular formation, we analyzed the changes in the expression of VEGFR-2, MMP-2 and TIMP-2, an endogenous inhibitor of MMPs, in VEGF-A-treated HUVECs. BK treatment markedly suppressed the VEGF-A-induced expression of VEGFR-2 and MMP-2 (Fig. 4A). As shown in Fig. 4B, BK treatment also inhibited VEGF-A-induced activation of MMP-2 in conditioned medium of HUVECs. By contrast, the levels of TIMP-2 in HUVECs were not altered by VEGF-A or BK treatment (Fig. 4A). Taken together, these findings indicates that the inhibitory effects of BK on endothelial cell proliferation, migration and tubular formation may be mediated at least in part through the suppression of VEGFR-2 and MMP-2 expression.

To investigate the molecular mechanisms by which BK regulates endothelial cell responses, we examined the changes in the activation of signaling pathways including extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38^MAPK), phosphatidylinositol 3-kinase (PI3-K)/Akt and mammalian target of rapamycin (mTOR)/p70^S6K, which play pivotal roles in cell fate (31). As shown in Fig. 5A, VEGF-A stimulation markedly increased the phosphorylation/activation of ERK, Akt and p70^S6K, but not that of p38^MAPK, when compared with unstimulated controls. By contrast, BK treatment significantly inhibited the VEGF-A-stimulated phosphorylation of ERK, Akt, and p70^S6K in HUVECs. Pretreatment of cells with PD98059 (an inhibitor of the ERK pathway), LY294002 (an inhibitor of the PI3-K/Akt pathway) or rapamycin (an inhibitor of the mTOR/p70^S6K pathway) suppressed the expression of VEGFR-2 and MMP-2 in response to VEGF-A stimulation, suggesting that BK may contain pharmacologically effective components similar to these inhibitors, and share the roles and mechanisms of action in regulating endothelial cell proliferation, migration and tubular formation (Fig. 5B). Taken together, these findings demonstrated that the suppression of endothelial cell proliferation, migration and tubular formation by BK ethanol extracts might be mediated through the inactivation of VEGFR-2 downstream signaling pathways and subsequent downregulation of VEGFR-2 and MMP-2.