title: Synthetic heparan sulfate oligosaccharides inhibit endothelial cell functions essential for angiogenesis.
abstract: BACKGROUND
  Heparan sulfate (HS) is an important regulator of the assembly and activity of various angiogenic signalling complexes. However, the significance of precisely defined HS structures in regulating cytokine-dependent angiogenic cellular functions and signalling through receptors regulating angiogenic responses remains unclear. Understanding such structure-activity relationships is important for the rational design of HS fragments that inhibit HS-dependent angiogenic signalling complexes.
  
  
  RESULTS
  We synthesized a series of HS oligosaccharides ranging from 7 to 12 saccharide residues that contained a repeating disaccharide unit consisting of iduronate 2-O-sulfate linked to glucosamine with or without N-sulfate. The ability of oligosaccharides to compete with HS for FGF2 and VEGF165 binding significantly increased with oligosaccharide length and sulfation. Correspondingly, the inhibitory potential of oligosaccharides against FGF2- and VEGF165-induced endothelial cell responses was greater in longer oligosaccharide species that were comprised of disaccharides bearing both 2-O- and N-sulfation (2SNS). FGF2- and VEGF165-induced endothelial cell migration were inhibited by longer 2SNS oligosaccharide species with 2SNS dodecasaccharide activity being comparable to that of receptor tyrosine kinase inhibitors targeting FGFR or VEGFR-2. Moreover, the 2SNS dodecasaccharide ablated FGF2- or VEGF165-induced phosphorylation of FAK and assembly of F-actin in peripheral lamellipodia-like structures. In contrast, FGF2-induced endothelial cell proliferation was only moderately inhibited by longer 2SNS oligosaccharides. Inhibition of FGF2- and VEGF165-dependent endothelial tube formation strongly correlated with oligosaccharide length and sulfation with 10-mer and 12-mer 2SNS oligosaccharides being the most potent species. FGF2- and VEGF165-induced activation of MAPK pathway was inhibited by biologically active oligosaccharides correlating with the specific phosphorylation events in FRS2 and VEGFR-2, respectively.
  
  
  CONCLUSIONS
  These results demonstrate structure-function relationships for synthetic HS saccharides that suppress endothelial cell migration, tube formation and signalling induced by key angiogenic cytokines.
authors.affiliation: School of Cancer and Enabling Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Claire L
authors.initials: CL
authors.lastname: Cole
authors.forename: Steen U
authors.initials: SU
authors.lastname: Hansen
authors.forename: Marek
authors.initials: M
authors.lastname: Baráth
authors.forename: Graham
authors.initials: G
authors.lastname: Rushton
authors.forename: John M
authors.initials: JM
authors.lastname: Gardiner
authors.forename: Egle
authors.initials: E
authors.lastname: Avizienyte
authors.forename: Gordon C
authors.initials: GC
authors.lastname: Jayson
mesh_terms: D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects*; D006497:Heparan Sulfate / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D009844:Oligosaccharides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D012235:Rhodamines / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014945:Wound Healing / Q000187:drug effects
chemical_list: D016207:Cytokines; D006497:Heparan Sulfate; D009844:Oligosaccharides; D012235:Rhodamines; C022027:lissamine rhodamine B