Synthetic oligosaccharide stimulates and stabilizes angiogenesis: structure-function relationships and potential mechanisms

Abstract

To determine the proangiogenesis effect of series of saccharides and a synthetic oligosaccharide and potential mechanisms, an in vitro 3-dimensional endothelial cell sprouting (3D-ECS) assay and the chick chorioallantoic membrane (CAM) model were used. We demonstrated that a sulfated oligosaccharide significantly promotes the endothelial capillary network initiated by vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF). Furthermore, although the capillary network initiated by VEGF and b-FGF lasts no more than 7 days, addition of a sulfated oligosaccharide significantly amplifies angiogenesis and stabilizes the capillary network of new blood vessels. In the CAM model, sulfated oligosaccharide also stimulated angiogenesis. In both the CAM and the 3D-ECS assay, structure-function studies reveal that increased saccharide chain length up to the hexa- to decasaccharide show optimal proangiogenesis efficacy. In addition, the sulfation and molecular shape (branched vs linear) of oligosaccharide are important for sustained proangiogenesis efficacy. Data indicate that chemically defined synthetic oligosaccharides can play an important role in regulation of capillary structure and stability, which may contribute to future advances in therapeutic angiogenesis. The proangiogenesis efficacy of an oligosaccharide is mediated via integrin alphavbeta3 and involves mitogen-activated protein kinase signaling mechanisms.

FIGURE 1

In vitro angiogenesis assay of structurally defined, homogenous heparin-derived oligosaccharides in the presence of VEGF and b-FGF. A, Oligosaccharide structure. B, Beads with disaccharide (dp 2). C, Beads with tetrasaccharide (dp 4). D, Beads with hexasaccharide (dp 6). E, Beads with octasaccharide (dp 8). F, Beads with decasaccharide (dp 10). CAM data for the same oligosaccharide afforded 115 ± 6, 135 ± 9, 160 ± 11, 161 ± 8, 165 ± 12 (new branch points ± SEM) for oligosaccharides dp 2, dp 4, dp 6, and dp 8, respectively, as shown in Fig. 6A.

FIGURE 2

Evaluation of linear synthetic oligosaccharides using 3D on ovo angiogenesis assay in the presence of subthresh-old concentration of VEGF (1.25 ng/mL) and b-FGF (2.5 ng/mL). A, Structure of Ch 1, Ch 4, Ch 5, Ch 6, and Ch 7. B, Proangiogenesis activity of Ch 1, Ch 4, Ch 5, Ch 6, and Ch 7 in the 3D HMDEC sprouting assay. Data represent mean ± SD, n = 3.

FIGURE 3

Comparison of linear and branched sulfated synthetic oligosaccharides in 3D on ovo angiogenesis assay in the presence of VEGF and b-FGF. A, Structures of Ch 2 and 3. B, Control with VEGF and b-FGF but no added oligosaccharide, Ch 2, or Ch 3 with subthreshold b-FGF (2.5 ng/mL) and VEGF (1.25 ng/mL). Data showed significant increase in tube length (P < 0.01) with the sulfated hexasaccharides Ch 2 but not Ch 3 as compared to control.

FIGURE 4

Concentration response of the proangiogenic activity of Ch 2 measured using the 3D in vitro assay in the presence of VEGF and b-FGF at day 7. A, Activity at 0 μg/mL. B, Activity at 1 μg/mL. C, Activity at 10 μg/mL. D, Activity at 100 μg/mL. E, Table demonstrating the concentration-dependent effect on the proangiogenesis efficacy of the saccharides vs subthreshold VEGF (1.25 ng/mL) plus b-FGF (2.5 ng/mL; control) in the 3D angiogenesis sprouting assay.

FIGURE 5

The average tube length measured using the 3D in vitro assay as a function of time. VEGF (50 ng/mL) and b-FGF (25 ng/mL) control (■) and VEGF (1.25 ng/mL), b-FGF (2.5 ng/mL) plus Ch 2 (◆). b = b-FFG; V = VEGF.

FIGURE 6

A, Effect of heparin oligosaccharide chain length on angiogenesis (mean branch points) as compared to b-FGF (1 μg/mL) in the on ovo CAM model. Data represent mean ± SD, n = 8/group. B, Representative illustration for the proangiogenesis effect of sulfated saccharide (Ch 2) vs b-FGF or VEGF in the CAM model. CAMs (n = 8/group/experiment) were exposed to sulfated Ch 2 analogs at 1 or 10 μg vs 1.0 μg b-FGF or 20 μg VEGF for 3 d. Sulfated saccharide Ch 2, VEGF, or b-FGF resulted in maximal increase in blood vessel branching, as shown in these representative images.