HUVECs were purchased from InnoPharmaScreen (Chungnam, Korea). Matrigel was obtained from Collaborative Biomedical Products (Bedford, MA, USA) for the mouse Matrigel plug assay. Basic fibroblast growth factor (bFGF) and heparin were obtained from PeproTech (Gaithersburg, MD, USA). Fetal bovine serum (FBS), penicillin and streptomycin were purchased from JBI (Daegu, Korea). Drabkin reagent kit 525 was purchased from Sigma (St. Louis, MO, USA). The 8-μm pore Transwell filter chambers were purchased from Corning-Costar (Corning, NY, USA). Antibodies for NF-κB, phosphor-NF-κB, Akt, and phosphor-Akt were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Xylitol 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, 10 U/ml of heparin, 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 by Hyochang Science and Samtako (Daegu and Kyung-gi, 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 (2×10⁴ cells) were seeded on a layer of previously polymerized Matrigel and treated with or without xylitol. Matrigel culture was incubated at 37°C. After 6 h, changes in cell morphology were captured through a phase contrast microscope and photographed at ×40 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 was 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 xylitol. 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 ×40 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 Transwell 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 (2×10⁴ cells) and xylitol in serum-free medium was placed in the upper part of the filters, and full medium was treated in the lower parts. Cells were incubated at 37°C for 24 h, fixed with methanol, and then stained with hematoxylin and eosin. 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 ×40 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, Bedford, MA, USA) containing bFGF (100 ng/ml) and heparin (50 U/ml) without or with xylitol. 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, Sungnam, Korea). Amplification products were electrophoresed on 1% agarose gels and visualized by GelRed (Biotium Inc., Hayward, CA, USA) staining under ultraviolet trans-illumination.

HUVECs were treated with or without xylitol 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.

Because differentiation of endothelial cells into a capillary-like network is important for the process of angiogenesis, we tested the effect of xylitol on the morphological differentiation of endothelial cells in vitro. HUVECs were placed on a Matrigel-coated plate and incubated. Endothelial cells formed weak capillaries on Matrigel beds, and these tubes became stronger and more robust with elongated networks over 6–24 h. HUVECs on Matrigel formed a blood vessel-like network in the absence of xylitol (Fig. 2), whereas treatment with xylitol for 18 h resulted in broken, shortened and narrow tube networks. This result shows that xylitol inhibits tube formation of HUVECs.

Migration and invasion in endothelial cells is a critical feature in forming new blood vessels and repairing injured vessels (19,20). Thus, we examined the effect of xylitol on the movement of HUVECs from a wounded edge to the open area by using a wound migration assay. Exposure to xylitol for 20 h significantly decreased HUVEC migration compared with that of control cells in a dose-dependent manner (Fig. 3A). To examine the effect of xylitol on HUVEC invasiveness, we performed an invasion assay by using a Transwell system. As shown in Fig. 3B, xylitol suppressed HUVEC invasiveness compared with that of control cells in a dose-dependent manner after 24 h of incubation. These inhibitory activities of xylitol on the migration and invasion of HUVECs indicate that xylitol suppresses in vitro angiogenesis.

To examine the effect of xylitol on in vivo angiogenesis, we performed a mouse Matrigel plug assay, an established in vivo angiogenesis model. As shown in Fig. 4A, Matrigel plugs containing bFGF were abundantly filled with intact red blood cells, indicating formation of a functional vasculature inside the Matrigel, whereas vessels were not observed in the Matrigel alone (blank). Matrigel plugs containing xylitol produced fewer vessels compared with plugs containing bFGF, indicating that xylitol inhibits formation of bFGF-induced neo-microvessels. We then measured the hemoglobin content inside the Matrigel plugs to quantify the anti-angiogenic effect of xylitol. The amount of hemoglobin indicates the degree of formation of a functional vasculature inside the Matrigel. The hemoglobin content of bFGF-treated plugs was 31.8 g/dl, whereas that of 1 mM xylitol-treated plugs was profoundly lowered to approximately 1.4 g/dl (Fig. 4B). These results indicate that xylitol has strong anti-angiogenic activity in vivo.

To determine which molecules are involved in the anti-angiogenic activity of xylitol, we examined mRNA expression of angiogenic factors and their receptors in HUVECs following xylitol treatment by using RT-PCR. As shown in Fig. 5A, VEGF mRNA expression was significantly reduced in the presence of xylitol. VEGFR-II (KDR) mRNA expression was also downregulated following treatment with xylitol in a dose-dependent manner. mRNA expression of other angiogenic molecules, including bFGF and bFGR-II, was remarkably reduced by xylitol (Fig. 5B).

Regulation of extracellular proteolytic activity is important for endothelial cell migration and invasion, and MMP-2 and MMP-9 are major functional molecules involved in the degradation of the extracellular membrane (21). Thus, we examined the effect of xylitol on the mRNA expression of MMP-2 and MMP-9. Expression of both molecules was markedly downregulated following treatment with xylitol in a dose-dependent manner (Fig. 5C). These data suggest that downregulation of VEGF, KDR, bFGF, bFGFR-II, MMP-2 and MMP-9 mRNA expression may be responsible for the anti-angiogenic action observed in HUVECs treated with xylitol.

NF-κB and Akt are the main signaling pathways involved in tumor progression and angiogenesis (22,23). Therefore, we examined the effect of xylitol on NF-κB and Akt activation in HUVECs. In this experiment, total proteins were prepared to detect NF-κB and Akt phosphorylation and then analyzed by western blot analysis. As shown in Fig. 6A, NF-κB phosphorylation was suppressed by xylitol in a dose-dependent manner. Because NF-κB activation is known to be mediated by the Akt signal pathway, we then examined Akt activation (17,24). Akt phosphorylation was inhibited in the presence of xylitol in a dose-dependent manner (Fig. 6B). These results indicate that xylitol interferes with NF-κB and Akt phosphorylation in HUVECs.