Figure 4. Oligosaccharides inhibit growth factor-induced endothelial tube formation.

(A) HUVEC coated beads were embedded in fibrin gels and treated with either FGF2 or VEGF₁₆₅ alone or with FGF2 in the presence of PD173074 (0.5 µM) and VEGF₁₆₅ in the presence of SU4312 (1 µM) for 6 days. Tubes were visualised by staining with Calcein-AM. Scale bar, 100 µm. (B) HUVEC coated beads were treated with FGF2 or VEGF₁₆₅ mixed with oligosaccharides at 50 µg/ml concentration (equivalent molar concentrations are shown in Table 1). PD173074 and SU4312 were used at 0.5 µM and 1 µM concentration, respectively. The average number of tubes per bead (20–50 beads were evaluated) was quantified. FGF2 or VEGF₁₆₅ alone treatment represents a control (100%). Percentage in number of tubes per bead in each treatment when compared to the control is shown as mean ± SD (n = 2–4). *, P<0.035; †, P<0.02. (C) Oligosaccharide dose-dependent inhibition of FGF2- or VEGF₁₆₅-induced endothelial tube formation. Response to 1, 10 and 50 µg/ml concentration (Table 1) of oligosaccharides was evaluated as in B. The mean ± SD (n = 2) is shown. *, P<0.035; †, P<0.02. (D) Mouse aortic rings were embedded in fibrin gels supplemented with the medium with no growth factors (0.1% FBS) or with FGF2, FGF2 plus 12-mer 2SNS (50 µg/ml; 14.7 µM), VEGF₁₆₅ or VEGF₁₆₅ plus 12-mer 2SNS (50 µg/ml; 14.7 µM). Tube growth was evaluated after 6 days. Aortas were stained with Calcein-AM. Scale bars, 300 µm. (E) Quantification of total aortic tube length was performed using MetaMorph software and expressed as a percentage of a total tube length induced by the growth factor alone. 12-mer 2SNS was used at 50 µg/ml (14.7 µM), PD173074 at 0.5 µM and SU4312 at 1 µM concentration. RTKi – receptor tyrosine kinase inhibitors. The mean ±SD (n = 3–5) is shown. *, P = 0.01; †, P≤0.003; ‡, P<0.0001. (F) Tubular structures derived from aortas show endothelial cell-specific staining with FITC-conjugated isolectin B4. Scale bar, 300 µm.