title: Identification of core active disaccharides in heparin for HGF-inducing activity.
abstract: OBJECTIVE
  To ascertain the positions of sulfated groups for HGF-inducing activity using differently sulfated heparin disaccharides and to investigate whether the heparin disaccharide elevates HGF levels in plasma in vivo and exerts protective effects on acute liver injury.
  
  
  METHODS
  The heparin disaccharides ΔUA-GlcNS, ΔUA (2S)-GlcN, ΔUA-GlcNAc (6S), ΔUA-GlcNS (6S), ΔUA (2S)-GlcNS, ΔUA (2S)-GlcNAc (6S), ΔUA-GlcNAc and ΔUA (2S)-GlcNS (6S) were added to MRC-9 fibroblasts and HGF concentrations in culture media were determined by enzyme-linked immunosorbent assay. Furthermore, ΔUA-GlcNS (100 μg/head) was injected into C57BL/6 mice and plasma levels of HGF measured at 12 h. After acute hepatitis was induced by CCl4 (15 mg/kg) in mice, liver specimens were stained with hematoxylin and eosin (H and E). Levels of aspartate aminotransferase and alanine aminotransferase were measured at 24 h.
  
  
  RESULTS
  Among the disaccharides investigated, ΔUA-GlcNS, ΔUA-GlcNAc (6S) and ΔUA-GlcNS (6S) stimulated HGF production in MRC-9 fibroblasts. However, none of the 2-O-sulfated disaccharides [ΔUA (2S)-GlcNS, ΔUA (2S)-GlcNAc (6S) and ΔUA (2S)-GlcNS (6S)] showed any activity despite the presence of N-sulfated and/or 6-O-sulfated disaccharides. Thus, 2-O-sulfation of hexuronic acid has an inhibitory effect. Moreover, ΔUA-GlcNS administration increased plasma levels of HGF in normal mice and prevented CCl4-induced liver injury in mice.
  
  
  CONCLUSIONS
  N-sulfation and/or 6-O-sulfation of glucosamine with nonsulfated hexuronic acid provides a structural basis for the HGF-inducing activity of disaccharides. ΔUA-GlcNS increases plasma levels of HGF and protects against CCl4-induced acute liver injury.
authors.affiliation: Department of Pathology, Faculty of Medicine, Kinki University, 377-2 Ohno-Higashi, Osaka Sayama, Osaka, 565-0871, Japan ; Department of Clinical Engineering, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kato
authors.affiliation: Research and Development, Neurogen Inc., 7-7-20 Saito-asagi, Ibaraki, Osaka 567-0085, Japan.
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Sakiyama
authors.affiliation: Division for Regenerative Drug Discovery, Center for Advanced Science and Innovation, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Kiyomasa
authors.initials: K
authors.lastname: Oka
authors.affiliation: Division for Regenerative Drug Discovery, Center for Advanced Science and Innovation, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Nakamura
keywords: Disaccharide; HGF; heparin; sulfation