Preparation and characterization of hyaluronan oligosaccharides for angiogenesis study.

Low molecular weight hyaluronan (HA) is proved to be active in stimulating endothelial cell (EC) proliferation. The study is to obtain biological active HA fragments of defined length from high molecular weight HA for angiogenesis research. A novel method, by a high-voltage polyacrylamide-gel electrophoresis (PAGE) that uses specific fluorophore labeling, was developed to identify the degraded products digested from native high molecular weight HA. The polymer HA was digested by hyaluornidase partially, and the enzymatic fragments of HA or HA oligosaccharides (o-HAs) were analyzed for molecular sizes by a combination of PAGE, high-performance liquid chromatography (HPLC), and mass spectrometry (MS). The purified HA oligomers were pooled and the biological effects of the mixtures on EC proliferation were studied, by separating the digests of HA fragments by size exclusion chromatography on a Bio-Gel P-10 column. The prepared o-HAs were collected and analyzed by a series of methods, PAGE, HPLC, and MS, which indicated a mixture of oligosaccharides covering a range of different sizes from 4 to 20-mer. The minimal molecular size determined by the special PAGE was 2 disaccharide, and the method can determine o-HA mass as high as 10 disaccharide (seeing the least clear band as much as possible on gel). Fractions containing HA fragments with sizes from 4 to 20-mer were pooled by distinct oligosaccharide species and added to the culture of pig endothelial cell (PIEC) for biological activities study. A significant increase in EC proliferation was found in comparison with nondigested high molecular weight HA. Our results suggested that o-HA can be obtained by a method of fluorophore-labeled olyacrylamide gel electrophoresis for identifying as small as 4-mer oligomer HA from high molecular weight HA-digested products. This method was proved to be sensitive, simple, and rapid in characterizing saccharides, which may be used by replacing the complicated ways of HPLC and MS. The prepared o-HA was found to be active in stimulating EC proliferation.