Antiangiogenic Effect of Ethanol Extract of Vigna angularis via Inhibition of Phosphorylation of VEGFR2, Erk, and Akt

Though dietary azuki bean (Vigna angularis) seed containing antioxidant proanthocyanidins was known to have multibiological activities including antioxidant, hypotensive, anti-inflammatory, and immunomodulatory activities, the antiangiogenic activity of ethanol extract of Vigna angularis (EVA) was never reported so far. In the present study, the antiangiogenic mechanism of EVA was examined in human umbilical vein endothelial cells (HUVECs). EVA showed weak cytotoxicity in HUVECs, while it significantly suppressed the VEGF induced proliferation of HUVECs. Consistently, wound healing assay revealed that EVA inhibited the VEGF induced migration of HUVECs. Also, EVA abrogated the VEGF induced tube formation of HUVECs in a concentration dependent fashion. Furthermore, Matrigel plug assay showed that EVA significantly reduced the hemoglobin level of Matrigel plug in mice compared to untreated control. Of note, EVA effectively attenuated the phosphorylation of VEGFR2, Erk, and Akt in VEGF-treated HUVECs. Overall, our findings suggest that EVA inhibits angiogenesis in VEGF-treated HUVECs via inhibition of phosphorylation of VEGFR2, ERK, and Akt.


Cell
Culture. Human umbilical vein endothelial cells (HUVECs, passages between 4 and 6) were obtained from fresh human umbilical cord veins after collagenase treatment as described previously [22,40]. The cells were maintained in M199 (Invitrogen, Carlsbad, CA) with 20% fetal bovine serum (FBS), 5 U/mL heparin and 3 ng/mL basic fibroblast growth factor (R&D Systems, Minneapolis, MN), and 100 U/mL of antibiotic-antimycotic in 0.1% gelatin coated flasks. The cells were cultured at 37 ∘ C in a humidified atmosphere containing 5% CO 2 .

Proliferation Assay.
Cell proliferation was determined using a 5-bromo-2 -deoxyuridine (BrdU) colorimetric assay kit (Roche, Mannheim, Germany). Briefly, HUVECs (5 × 10 3 cells/well) were seeded into 0.1% gelatin coated microplates and incubated in a humidified atmosphere containing 5% CO 2 at 37 ∘ C. After starvation for 6 h in M199 containing 5% heat-inactivated FBS, the cells were treated with various concentrations of EVA (10, 20, and 30 g/mL) in the presence or absence of VEGF (50 ng/mL) and incubated at 37 ∘ C for 48 h. 10 L of BrdU (100 g/mL) was added; the cells were incubated at 37 ∘ C for 6 h. The cells were fixed with anti-BrdU and measured by the substrate reaction. 25 L of 1 M H 2 SO 4 was added to stop the reaction and the absorbance was calculated by using an ELISA reader (Molecular Devices Co., USA) (450 nm with 690 nm correction).

Wound Healing Migration
Assay. The migratory activity of HUVECs was tested by a wound healing assay. The cells (4 × 10 5 cells/2 mL) were exposed to various concentrations of EVA (10 and 20 g/mL) in a 6-well plate in the presence or absence of VEGF (50 ng/mL) and incubated in a humidified atmosphere containing 5% CO 2 at 37 ∘ C. The plates were scratched with a 200 L pipet tip and washed with PBS for 3 times. Then, cells were treated with EVA extracts (0, 10, and 20 g/mL) in media. The cells were fixed and stained with Diff-Quick after 24 h, and randomly chosen fields were photographed with a fluorescence microscope (AXIO observer A1, Zeiss, Germany). The migrated cells were calculated.

Matrigel Plug Assay.
Six-week-old C57BL/6 mice (Chungang animal expt. Co., Seoul, Republic of Korea) were given subcutaneous injection of 0.5 mL of growth factor reduced Matrigel containing VEGF (50 ng/mL) and heparin (10 U/mL). EVA extracts (40 g/mL in PBS) were orally administrated (300 g/kg) after 7 days of Matrigel injection (three mice per group). Ten days later, the mice were sacrificed, and the matrigel plugs were detached. To indirectly quantify the formation of new blood vessel, the amount of hemoglobin (Hb) was calculated using the hemoglobin assay kit (YD Diagnostics, Republic of Korea). The concentration of Hb was measured from standard curve made from the known amount of Hb provided according to the manufacturer's protocol.

Statistical Analyses.
All data were expressed as mean ± SD (standard deviation). The statistically significant differences between untreated control and EVA-treated cells were calculated by ANOVA test followed by a post hoc analysis (Tukey -or Dunnettes multiple comparison test) using Prism software 5 (GraphPad Software, Inc., San Diego, CA, USA).

EVA Shows Weak Cytotoxicity in HUVECs and Suppressed
VEGF Induced Proliferation of HUVECs. Cytotoxicity of EVA was evaluated in HUVECs using MTT assay. As shown in Figure 1, EVA decreased the viability of HUVECs to ∼60% at the concentration of 50 M for 48 h. EVA treatment showed cytotoxicity in VEGF-treated HUVECs in a concentration dependent manner (Figure 2).

EVA Significantly Inhibits the VEGF Induced Migration and Tube Formation in HUVECs.
It was well known that endothelial cells migrate in tandem using adhesion molecules such as integrins and form loops to become a full-fledged vessel lumen like tube formation [42]. To check the physiological angiogenic formation, wound healing assay and tube formation assay were carried out in HUVECs. As shown in Figures 3(a) and 3(b), EVA significantly reduced the number of tube forming endothelial cells, whereas tube-like formation by endothelial cells was enhanced by VEGF in HUVECs. Similarly, EVA significantly suppressed the VEGF induced migratory activity of HUVECs, while the gap physically made by tip was narrowed via repair through migratory activity of VEGF-treated HUVECs (Figures 4(a) and 4(b)). Cell proliferation was determined using a BrdU colorimetric assay kit. Cells (5 × 10 3 cells/well) were seeded onto 96-well plates and incubated for 24 h. After 6 h starvation, the cells were exposed to various concentrations of EVA (10, 20, and 30 g/mL) in the presence or absence of VEGF (50 ng/mL) and incubated for 48 h at 37 ∘ C. Data are presented as means ± SD. * ,# < 0.05 versus VEGF only treated group.

EVA Significantly Reduces Hemoglobin Content in VEGF Induced Angiogenesis of Matrigel Plugs of C57BL/6 Mice.
To confirm the antiangiogenic activity of EVA, Matrigel plug assay was carried out. EVA (300 g/kg) extract was administered orally to C57BL/6 mice for 7 days before growth factor reduced Matrigel was subcutaneously injected into right flank of mice. As shown in Figures 5(a) and 5(b), EVA significantly reduced hemoglobin content in VEGF induced angiogenesis of Matrigel plugs of C57BL/6 mice, while dark red color was shown in Matrigel plugs of untreated control group.

EVA Effectively Attenuates the Phosphorylation of VEGFR2, Erk, and Akt in VEGF-Treated
HUVECs. Since Dr. Judah Folkman suggested the importance of tumor angiogenesis, a variety of angiogenesis related factors such as VEGF, bFGF, PDGF, angiopoietin, and transforming growth factor-(TGF-) were studied for years [1,43]. To elucidate antiangiogenic molecular mechanism of EVA, Western blot analysis was performed in VEGF-treated HUVECs. As shown in Figure 6, EVA effectively attenuated the phosphorylation of VEGFR2 and Erk and Akt in VEGF-treated HUVECs.

Akt, Erk Inhibitors, and EVA Attenuate Tube Formation Enhanced by VEGF in HUVECs.
To investigate the roles of Erk and Akt in angiogenesis, tube formation assay was conducted with HUVECs in the presence of LY294002 (Akt inhibitor) or PD98059 (Erk inhibitor). As shown in Figures  7(a) and 7(b), Akt, Erk inhibitors, and EVA treatments inhibited tube formation enhanced by VEGF. Particularly, LY294002 and EVA significantly attenuated the tube formation.

Discussion
Azuki bean (Vigna angularis) has been used in east Asian food, and its coat of seed was known to contain watersoluble anthocyanins, catechins, and flavonols [44]. Despite multibiological activities of azuki bean, its antiangiogenic activity was never reported until now. So, this study elucidates the antiangiogenic mechanism of azuki bean in VEGFtreated HUVECs.
Though EVA exerted weak cytotoxicity in HUVECs, EVA significantly suppressed the VEGF induced proliferation of HUVECs at nontoxic concentrations, implying that EVA has antiangiogenic potential by blocking pathological angiogenic process via VEGF induced proliferation of endothelial cells, since these endothelial cells become motile and invasive and protrude filopodia, which drives new vessel formation [14,25,42].
Also, EVA inhibited VEGF induced migration of HUVECs in wound healing assay and suppressed VEGF induced tube formation of HUVECs in a concentration dependent manner in tube formation assay, strongly indicating that EVA could inhibit the motility and tube formation of proliferative endothelial cells.
To confirm in vivo antiangiogenic activity of EVA, Matrigel plug assay was performed in C57BL6 mice orally administered by EVA extracts for 1 week earlier. Here, we observed less red color in Matrigel plugs isolated from mice treated by EVA, while black red color was shown in those of untreated control group. The treatment of EVA significantly reduced the hemoglobin level of Matrigel plug in mice compared to untreated control, demonstrating antiangiogenic activity of EVA. However, it requires further in vivo angiogenesis studies using Matrigel plug assay via direct EVA treatment in the increased number of mice or CAM assay in the near future.
It was well documented that several molecules were involved in angiogenesis process. Angiogenesis is processed via the interaction of the VEGF family of proangiogenic cytokines and their receptors. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, and placental growth factor (PGF) with three receptors including VEGFR1 (Fmslike tyrosine kinase 1/Flt-1), VEGFR2 (Flk-1/KDR), and VEGFR3 (Flt-4) [20,45,46]. Also, VEGFR2 expression is typically limited to vessel endothelial cells, whereas VEGFR1 can be usually found in bone-marrow derived progenitors [47]. Furthermore, there was evidence that Akt and Erk mediate angiogenesis in endothelial cells [48][49][50][51][52][53]. sc-221226, an  Akt inhibitor, attenuated sphingosine-1-phosphate induced angiogenesis in endothelial progenitor cells [54]. Also, low molecular weight fucoidan inhibited angiogenesis in vitro and in vivo evidenced by attenuation of tube formation of hypoxic HUVECs and suppressing Akt activity reduced hypoxia-induced VEGF secretion in T24 cells, supporting the involvement of Akt in the induction of HIF-1 and VEGF and angiogenesis [55]. Epidermal growth factor-like domain 7 promotes angiogenesis via ERK signaling pathway in endothelial cells [56]. And fractalkine, a large cytokine protein of 373 amino acids, stimulates angiogenesis by activation of ERK and Akt [57]. These results support our conclusions.
In summary, EVA significantly suppressed the VEGF induced proliferation, migration, tube formation, and angiogenesis in HUVECs, and the molecular mechanism is the phosphorylation inhibition of VEGFR2 and Erk and Akt in VEGF-treated HUVECs. Overall, our findings suggest that EVA inhibits angiogenesis in VEGF-treated HUVECs via phosphorylation inhibition of VEGFR2 and Erk and Akt. Our study suggests that EVA is a promising natural agent which has antiangiogenic activity in HUVECs. However, further study is required to find out a leading antiangiogenic constituent from EVA and its potency for clinical application as a functional food.