HUVECs were purchased from Innopharma Screen (Chungnam, Korea). Matrigel was from Collaborative Biomedical Products (Bedford, MA, USA) for the mouse Matrigel plug assay. Basic fibroblast growth factor (bFGF) and heparin were from Peprotech (Gaithersburg, MD, USA). FBS, penicillin and streptomycin were purchased from JBI (Daegu, Korea). Drabkin reagent kit 525 was purchased from Sigma (St. Louis, MO, USA). Trans-well filter chambers (8-μm pores) were purchased from Corning-Costar (Cambridge, MA, USA). Antibodies for NF-κB, phospho-NF-κB, Akt and phospho-Akt were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Betaine and gelatin were purchased from Sigma.

HUVECs were grown in M199, supplemented with heat-inactivated 20% fetal bovine serum (JBI), 20 ng/Ml of bFGF, 100 U/Ml of penicillin and 100 μg/Ml of streptomycin in a 37°C incubator with a humidified atmosphere containing 5% CO₂.

Seven-week-old, specific pathogen-free (SPF) male C57BL/6 mice were supplied from Hyochang Science and Samtako (Daegu, Korea). They were provided with autoclaved tap water and lab chow ad libitum and were housed at 23±0.5°C, 10% humidity under a 12 h light-dark cycle. The animal protocol used in this study was reviewed on the ethical procedures and scientific care, and approved by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC).

HUVECs (2x10⁴ cells) were seeded on a layer of previously polymerized matrigel and treated with or without betaine. Matrigel culture was incubated at 37°C. After 18 h, changes in cell morphology were captured through a phase contrast microscope and photographed at x40 magnification. Each sample was assayed in duplicate, and independent experiments were repeated three times.

HUVECs were seeded onto 24-well culture plates until confluence and left overnight. Media were aspirated the next day, and cells were scratched with a 200 μl pipette tip along the diameter of the well. Cells were washed twice with PBS and incubated at 37°C and 5% CO₂. After wounding, the cultures were washed with serum-free medium and further incubated in M199 with 1% serum, 1 mM thymidine, and/or betiane. These culture conditions minimized proliferation of HUVECs. Wound diameters were photographed at 18 to 24 h. Wound closure was determined by measurement with optical microscopy at x40 magnification. Migration was quantitated by counting the number of cells that moved beyond the reference line. Each sample was assayed in duplicate, and independent experiments were repeated three times.

Invasiveness of HUVECs was performed in vitro using a trans-well chambers system (Corning-Costar) with 8.0-μm pore polycarbonate filter inserts. The upper side was coated with 10 μl of matrigel (0.5 mg/Ml) at room temperature for 1 h. Cells (2x10⁴ cells) and betaine in serum-free media were placed in the upper part of the filters, and full media was treated in the lower parts. Cells were incubated at 37°C for 24 h, fixed with methanol, and then stained with H&E. Cells on the upper surface of the membrane were removed by wiping with a cotton swab. Cell invasion was determined by counting whole cell numbers in a single filter by optical microscopy at x40 magnification. Each sample was assayed in duplicate, and independent experiments were repeated three times.

C57BL/6 mice (7 weeks of age) were injected subcutaneously into 500 μl of matrigel (Collaborative Biomedical Products) containing bFGF (100 ng/Ml) and heparin (50 U/Ml) without or with betaine. After injection, the matrigel rapidly formed a plug. After 7 days, skin of the mouse was pulled back to expose the matrigel plug, which remained intact. After quantitative differences were noted and photographed, hemoglobin content was measured using the Drabkin reagent kit 525 (Sigma) for quantification of blood vessel formation. The amount of hemoglobin was calculated from a known amount of hemoglobin assayed in parallel. Independent experiments were repeated twice and at least five mice in each experiment were used.

Total-RNA from HUVECs was isolated using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer’s instructions. First-stranded cDNA was synthesized by M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA) with 2 μg each of DNA-free total-RNA sample and oligo (dT)15 (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions. Equal amounts of cDNA were subsequently amplified by PCR in a 20 μl reaction volume containing 1X PCR buffer, dNTP mixture, 10 μM of each specific primer and i-Taq™ DNA polymerase (iNtRON Biotechnology, Korea). Amplification products were electrophoresed on 2% agarose gels and visualized by GelRed (Biotium Inc, Hayward, CA, USA) staining under ultraviolet trans-illumination.

HUVECs were treated with or without betaine for 24 h in medium. Total cell lysates were prepared by addition of PRO-PREP Protein Extraction Solution (iNtRON Biotechnology), including 1 mM sodium orthovanadate. Equal amounts (30 μg) of samples were resolved by electrophoresis on a 10% SDS-polyacrylamide gel, transferred to a membrane, and sequentially probed with antibody. The following primary antibodies were used at the indicated dilutions: total NF-κB and anti-phospho-NF-κB, total Akt and anti-phospho-Akt, 1:1,000 in 5% BSA in TBS-T.

Data are presented as means ± SD or as percentage of control. Statistical comparisons between groups were performed using Student’s t-test. ^*p<0.05 was considered statistically significant.

We carried out tube formation assay to examine the effects of betaine on the morphological differentiation of endothelial cells into a capillary-like structure, an important step in the process of angiogenesis. HUVECs were placed on a matrigel-coated plate and incubated. Endothelial cells formed hollow capillaries on matrigel beds, and these tubes became stronger and more robust with elongated networks between 6–24 h. As shown in Fig. 2, HUVECs on matrigel formed a typical blood vessel network in the absence of betaine. However, treatment with betaine for 18 h resulted in broken, shortened and much narrower tube networks. This finding demonstrates that betaine inhibits tube formation of HUVECs.

The migration and invasion of endothelial cells are critical features in the formation of new blood vessels and in the repair of injured vessels. Therefore, we investigated the effects of betaine on the movement of HUVECs from a wounded edge to an open area using wounding migration assay (Fig. 3). Treatment with betaine for 20 h profoundly decreased the migration of HUVECs compared with that of control in a dose-dependent manner (Fig. 3A). Migration of endothelial cells was decreased by about 70% by betaine treatment compared with that of control.

To examine the effects of betaine on the invasiveness of HUVECs, we performed invasion assay with a trans-well system. Trans-wells were prepared such that the upper sides of the filter were coated with matrigel and used for invasion assay. Betaine suppressed the invasiveness of HUVECs compared with that of control in a dose-dependent manner after 24 h of incubation (Fig. 3B). The invasiveness of endothelial cells was inhibited by about 60% by betaine treatment compared with that of control. Taken together, betaine strongly suppressed migration and invasion of HUVECs, which are crucial steps of angiogenesis.

We demonstrated the in vitro anti-angiogenic activities of betaine as above. We then performed the mouse Matrigel plug assay to examine the effects of betaine on angiogenesis in vivo. As shown in Fig. 4A, matrigel plugs containing bFGF were abundantly filled with intact red blood cells, which indicates the formation of a functional vasculature inside the matrigel, whereas vessels were not observed in matrigel alone (blank). Matrigel plug containing betaine produced fewer vessels compared with plug containing bFGF, indicating that betiane inhibited the formation of bFGF-induced neomicrovessels. In addition, we measured the hemoglobin contents inside the matrigel plugs in order to quantify the anti-angiogenic effects of betaine. The hemoglobin content of bFGF-treated plug was 32 g/dl, whereas that of 1 mM betaine-treated plugs was profoundly diminished to about 2 g/dl (Fig. 4B). These results indicate that betaine has strong anti-angiogenic activity in vivo.

To determine which factors are involved in the anti-angiogenic activity of betaine, we examined the mRNA expression levels of angiogenic factors (VEGF and bFGF) in HUVECs treated with betaine using RT-PCR. As shown in Fig. 5, treatment with betaine decreased the mRNA expression of bFGF in a dose-dependent manner. However, treatment with betaine did not produce any significant change in mRNA levels of VEGF (date not shown).

Regulation of extracellular proteolytic activity is important in the process of endothelial cell migration and invasion through the basement membrane and capillary morphogenesis. Thus, the mRNA expression levels of both MMP-2 and MMP-9, which are major functional molecules in the degradation of the extracellular membrane, were examined. The mRNA expression levels of both MMP-2 and MMP-9 were markedly downregulated by treatment with betaine in a dose-dependent manner (Fig. 5). These data suggest that the downregulated expression of bFGF, MMP-2 and MMP-9 mRNA may be responsible for the anti-angiogenic activity observed in HUVECs treated with betaine.

Since NF-κB and Akt activation are two of the main signaling pathways for tumor progression and angiogenesis, we examined the effects of betaine on NF-κB and Akt activation in HUVECs. In this experiment, total proteins were prepared and analyzed using western blotting for detection of NF-κB and Akt phosphorylation. As shown in Fig. 6A, NF-κB phosphorylation was suppressed by betaine in a dose-dependent manner. As NF-κB activation is mediated by the Akt signaling pathway, we next examined Akt activation. Phosphorylation of Akt was inhibited by the presence of betaine in a dose-dependent manner (Fig. 6B). These results indicate that betaine interferes with NF-κB phosphorylation and Akt phosphorylation in HUVECs.