Vaccarin was purchased from Shanghai Shifeng Technology Co., Ltd. (Shanghai, China). The HMEC-1 cell line was obtained from the French Institute of Health and Medical Research (Paris, France). Matrigel™ was purchased from Becton-Dickinson (Oxford, UK). MCDB-131, rhodamine B, epidermal growth factor (EGF), fetal bovine serum (FBS) and trypsin were purchased from Sigma-Aldrich (St. Louis, MO, USA). CD31 rabbit polyclonal antibody was purchased from Sangon Biotech (Shanghai) Co., Ltd. (cat. no. AB60701a; Shanghai, China). P-Erk (ab76165) and p-Akt (ab66138) rabbit polyclonal antibodies were purchased from Abcam (Hong Kong, China). Goat anti-rabbit secondary immunoglobulin (Ig)G antibody was purchased from Sangon Biotech (Shanghai) Co., Ltd. The SABC kit and Masson dye were obtained from Nanjing Jiancheng Biological Technology, Inc. (Nanjing, China).

HMEC-1 cells were cultured in MCDB-131 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin at 37°C in 5% CO₂. After the treatments were administered, the cells were harvested for further analysis.

The effects of vaccarin on HMEC-1 growth promotion were measured using a sulforhodamine B (SRB) assay. Briefly, adherent cells were seeded into each well of 96-well plates and cultured at 37°C in a 5% CO₂ saturated humidity condition for 24 h, then incubated with different concentrations of vaccarin for 48 h. Following removal of the culture medium (10% FBS), 200 μl trichloroacetic acid solution was added into each well at 4°C for 40 min. Subsequently, the mixture was cleaned and dried at 37°C, then 100 μl SRB was added into each well for 20 min. Following washing, 150 μl Tris solution was added into each well. The mixture was agitated and measured at 540 nm using a Multiskan MK3 microplate reader (Thermo LabSystems, Inc., Milford, MA, USA) and the cell viability was expressed as an optical density. The control was defined as 100%. The average level was calculated using the proliferation rate formula: Percent proliferation = Test well signal / Control group signal × 100%.

Briefly, HMEC-1 cells (8×10³ cells/well) were seeded onto each well and cultured at 37°C in a saturated humidity containing 5% CO₂ for 24 h. Once the cells had completely attached, the middle of the cell plate was scraped with a line ~1 mm wide and then changed to MCDB medium containing 10% FBS and two different concentrations of vaccarin (1.08 and 2.15 μM). The cells were incubated and images were captured of randomly selected fields (magnification, ×100) under a microscope video system (Olympus IX70; Olympus, Tokyo, Japan). The width of each gap in the three different sections was measured and averaged.

The capillary tube formation assay was performed as described previously (19). Briefly, the 96 well plate was coated with 0.1 ml Matrigel and the material was allowed to solidify and polymerize at 37°C for 30 min. Each well was seeded with 4×10⁵ HMEC-1 cells and incubated in MCDB medium containing 10% FBS and different concentrations of vaccarin (1.08 and 2.15 μM) for 6, 12, 24 and 48 h. Subsequently, images were captured of the tubular structures from five randomly selected fields using an inverted fluorescence microscope (Olympus IX70; Olympus) at different time points.

The present study was approved by the ethics committee of Jiangnan University (JN no. 20130327-0702; Jiangsu, China) and was conducted in accordance with the guidelines issued by the State Food and Drug Administration (Beijing, China). The Matrigel plug assay in imprinting control region (ICR) mice was performed as described previously with specific modifications (20). Female ICR mice, aged 4–5 weeks old and weighing 17–20 g, were subcutaneously injected with 500 μl of Matrigel containing heparin (113 units) and mouse EGF (120 ng) into the left hypogastric region. Matrigel mixed with heparin alone was included as a blank control. The next day following implantation of Matrigel, mice were orally administered a single dose of saline. In addition, mice were divided into four groups (six mice per group), two groups were treated with oral vaccarin at doses of 5 and 10 mg/kg/day, respectively. The EGF control and blank control groups were treated with saline. After two weeks, the Matrigel plugs were carefully removed and images were captured. They were then formalin-fixed and paraffin-embedded. To determine the number of endothelial cells, hematoxylin and eosin (H&E) and Masson’s Trichrome (MT) staining were performed to visualize endothelial infiltration. Briefly, 5 μm sections were stained with H&E and MT solution. The number of blood vessels in the high power field was quantified by counting vessel infiltration in the plugs.

Matrigel tissue sections of the same volume were placed onto treated slides. According to sequences, sections were fixed, deparaffinized and rehydrated in distilled water. Tissues were boiled in 2% citrate buffer at 95°C for 20 min, then treated with 3% hydrogen peroxide in order to block the activity of endogenous peroxidase. The slides were incubated with protein-blocking agent, then treated with the primary antibodies, including CD31 (1:25), p-Erk (1:40) and p-Akt (1:40) at 4°C overnight. The tissues were then incubated with the secondary biotinylated IgG antibodies (1:2,000) and finally stained with 3,3′-diaminobenzidine according to the manufacturer’s instructions (EnoGene Biotech Co., Ltd., New York, NY, USA). Counterstaining was performed with hematoxylin. Microvascular density (MVD) was determined, as specified by Weidner (21). p-Erk and p-Akt expression was determined using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).

The results are presented as the mean ± standard deviation. Statistical analysis was performed using analysis of variance/Dunnett’s t-test for multiple group comparisons and Student’s t-test for two group comparisons using SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The effects of vaccarin on HMEC-1 cells were evaluated. The results revealed that vaccarin increased the proliferative activity of HMEC-1 cells. The optimum concentration of vaccarin was 2.15 μmol/l⁻¹ (proliferation ratio / 125.62±1.14% of control; P<0.01). The proliferation ratio of vaccarin at 0.54, 1.08, 4.30, 8.60, 17.20 and 34.40 μM was 103.15±0.95, 109.71±0.85, 117.54±1.65, 114.54±1.85 and 110.21±1.27% of the control, respectively (P<0.05; Fig. 2A).

As shown in Fig. 2B and C, the effects of vaccarin on HMEC-1 migration and tube formation in vitro were analyzed. It was demonstrated that vaccarin significantly promoted migration, when treated with concentrations of 1.08 and 2.15 μM, compared with the control group, for 24 and 48 h, respectively (Fig. 2D). As shown in Fig. 2C and Db, the control group did not form a complete, enclosed and robust network of tubes at 6 h, however, vaccarin stimulated the angiogenic differentiation of HMEC-1 cells (tube formation) on Matrigel at the same time at doses of 2.15 μM (P<0.01). Furthermore, after 24 and 48 h, the tube formation numbers of the vaccarin groups significantly increased compared with those of the control groups (P<0.05) (Fig. 2Db); however, the group treated with 2.15 μM vaccarin maintained a complete, enclosed and strong network of tubes as shown in Fig. 2C.

To examine whether the angiogenic function of vaccarin exhibits a response to neovascularization activity in vivo, Matrigel allografts were implanted subcutaneously into ICR mice to establish an angiogenesis assay in vivo (22). As shown in Fig. 3A, Matrigel plugs containing EGF alone were light red, however, the EGF with oral treatment of vaccarin (5 mg and 10 mg/kg/day) were dark red, particularly the higher dose. This suggested that functional vasculature had formed in the EGF group and a greater density of blood vessels were present in the Matrigel plugs of mice subjected to oral vaccarin treatment. To confirm this conclusion, the number of endothelial cells in the Matrigel plugs were compared using MT and H&E staining. MT stained the Matrigel blue and the endothelial cells/vessels red and H&E stained cells purple and the Matrigel pink (Fig. 3A). More vessels and endothelial cells were observed in the Matrigel plugs treated with EGF and vaccarin than in those plugs treated with EGF alone (Fig. 3A, 3Ca and b). The results demonstrated that vaccarin at concentrations of 5 and 10 mg/kg significantly enhanced EGF-induced angiogenesis after 14 days of Matrigel implantation and this was consistent with the results of the tube formation assay.

To further examine whether vaccarin may promote angiogenesis, Matrigel tissues were stained with antibodies against CD31, p-Erk and p-Akt (Fig. 3B). An increased number of vessels and endothelial cells were observed in the Matrigel plugs treated with both EGF and vaccarin compared with those plugs treated with EGF alone (Fig. 3A, 3Ca and 3Cb). These results suggested that vaccarin at the concentrations of 5 and 10 mg/kg remarkably enhanced the EGF-induced angiogenesis following 14 days of matrigel implantation; in addition, these results are consistent with the results of tube formation assay.

The neovascularization activity was measured based upon the MVD via detecting expression of CD31. Compared with the controls, the MVD of vaccarin-treated groups at concentrations of 5 and 10 mg/kg exhibited a significant increase (Fig. 3Cb). In addition, activation of p-Erk and p-Akt has been verified to correlate with angiogenesis (23). It was found that vaccarin treatment markedly upregulated the expression of p-Erk and p-Akt in blood vessels (Fig. 3Cc), further indicating that vaccarin was able to promote angiogenesis, possibly through the MAPK/Erk and PI3K/Akt signaling pathways.