HUVECs, endothelial cell basal medium (HuMedia-EB2), human epidermal growth factor (hEGF), human fibroblast growth factor B (hFGF B), hydrocortisone, heparin, VEGF, amphotericin B, and gentamicin were purchased from Kurabo (Osaka, Japan). Fetal bovine serum (FBS) was purchased from HyClone Laboratories (South Logan, UT). Collagen type I (Cellmatrix type I-C) was purchased from Nitta Gelatin Inc. (Osaka, Japan). GM6001 was purchased from SIGMA-Aldrich (St. Louis, MO, USA). RJ, 10 HDA, bee pollen, Chinese red propolis, and CAPE were gifted by Api Co. Ltd. (Gifu, Japan). Ruboxistaurin was gifted from Sanwa Kagaku Kenkyusho Co., Ltd.
The RJ, produced by Apis mellifera, of Chinese origin, was a freeze-dried product. The bee pollen used in the present study originated from Jara pringosa (Cistus ladanifer L.) and Jara blanca (Cistus albidus L.) shrubs in Spain, and was extracted with 95% ethanol at room temperature for 24 h, and then filtrated to obtain the ethanol extract. The propolis used in this study was Chinese red propolis from Shandong, China, and was also extracted with 95% ethanol at room temperature for 24 h, and then filtered to obtain its ethanolic extract. RJ, bee pollen, Chinese red propolis, and CAPE were dissolved in dimethylsulfoxide (DMSO). DMSO, at the final concentration reached in each examination (0.1%), was added to each bee product, the non-drug control, and VEGF alone.
The main constituents in ethanol extracts of Chinese red propolis were analyzed by high performance liquid chromatography (HPLC), the samples being injected into an HPLC system (Waters, Washington, NJ, USA) fitted with a Shim-pack CLC-ODS (Shimazu, Kyoto, Japan) C18 column (φ 6.0 ×150 mm). The mobile phase consisted of 1% acetic acid in 55% methanol. All constituents of Chinese red propolis were measured at a wavelength of 290 nm. Inject samples into HPLC system fitted with an Inertsil ODS-3 (φ 4.0 ×150 mm). The mobile phase consisted of 10 mM PBS in methanol. The constituent was measured at a wavelength of 210 nm.
HUVECs were cultured in growth medium (HuMedia-EG2) at 37°C in a humidified atmosphere of 5% CO2 in air. The HuMedia-EG2 consists of basal medium (HuMedia-EB2) supplemented with 2% FBS, 10 ng/ml hEGF, 5 ng/ml hFGF B, 1 μg/ml hydrocortisone, 10 μg/ml heparin, 50 ng/ml amphotericin B, and 50 μg/ml gentamicin. Subconfluent monolayers of HUVECs, from passages 3 to 8, were used in the experiments.
In vitro tube formation assay
An angiogenesis assay kit (Kurabo) was used according to the manufacturer's instructions. This kit consists of a 24-well cluster dish in which HUVECs and fibroblasts have been seeded in the optimal condition for capillary tube formation. The optimized angiogenesis medium in each well was changed on days 1, 4, 7, and 9 with fresh medium containing VEGF (10 ng/ml) plus various concentrations of RJ (30 to 300 μg/ml), GM6001 (10 μM), bee pollen (30 to 300 μg/ml), Chinese red propolis (0.3 to 3 μg/ml), or CAPE (1 to 10 μM). Bee products and CAPE were dissolved in DMSO and diluted with culture medium. DMSO, at the final concentration reached in each examination (0.1%), was added to the non-drug control, and samples containing VEGF alone.
On day 11, cells were fixed in 70% ethanol and stained with anti-CD31 antibody. For the evaluation of capillary tube formation (the stained tube-like structures), 100 mm2 areas of each well were photographed using a CCD camera (HS all-in-one fluorescence microscope; Keyence, Osaka, Japan). These photographs were then used for measurement of the tube area (the total area of the tubes), tube length (the total length of the tubes), joints (the number of capillary connections), and paths (the number of tubes branching from the capillary-like network) of the stained tube-like structures, using angiogenesis image analyzer version 2 (Kurabo).
In vitro cell proliferation assay
Subconfluent (~80%) HUVECs were trypsinized, seeded into a 96-well plate at 2000 cells/well, and incubated in HuMedia-EG2 for 24 h. The culture medium was then changed to HuMedia-EB2 with 2% FBS, and incubation allowed to proceed for 24 h. Fresh medium containing VEGF (10 ng/ml) was then added with or without various concentrations of RJ (100 to 300 μg/ml), bee pollen (30 to 300 μg/ml), Chinese red propolis (0.3 to 3 μg/ml), CAPE (1 to 10 μM), or ruboxistaurin (1 μM) and the incubation was continued for a further 72 h. Cell proliferation was estimated by measuring cell metabolic activity using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions. The viable cell numbers were measured using a water-soluble tetrazolium salt, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) and 1-methoxy-phenazine methosulfate. At the end of the drug treatments, the medium was replaced, then 10 μl of WST-8 assay solution was added to each well, and incubation allowed to proceed for 3 h at 37°C. Finally, the absorbance of the culture medium at 450 nm was measured using a microplate reader (Varioskan Flash, Thermo Electron Corporation, Vantaa, Finland).
In vitro wounding-healing assay
An in vitro wound-healing assay was performed to measure unidirectional migration by HUVECs. For this, we partially modified the procedure described by Izuta et al. (2007) . Briefly, HUVECs (4 × 104/well) were seeded in 12-well plates coated with collagen type I, and incubated at 37°C until they attached. The HUVECs were then washed twice with PBS and incubated in Humedia-EB2 with 1% FBS for 24 h at 37°C. The monolayers of HUVECs were scratch-wounded to a 1 mm depth in a straight line using a 10-200 μl pipette-tip, and incubated with VEGF (10 ng/ml), with or without various concentrations of RJ (100 to 300 μg/ml), GM6001 (10 μM), bee pollen (100 to 300 μg/ml), Chinese red propolis (1 to 3 μg/ml), or CAPE (3 to 10 μM) for 24 h. To measure the number of endothelial cells that had migrated from the edge of the injured monolayer, images were photographed both immediately after wounding and after 24 h incubation, using a phase-contrast microscope (OLYMPUS, Tokyo, Japan). At least four points in each of three fields were examined at random for two independent wounds.
Data are given as mean ± SEM. Statistical analysis was performed using a Dunnett's multiple-comparison test, with P < 0.05 being considered to indicate statistical significance.