Synthetic heparan sulfate oligosaccharides inhibit endothelial cell functions essential for angiogenesis.
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.
Cole
Claire L
CL
School of Cancer and Enabling Sciences, The University of Manchester, Manchester, United Kingdom.
Hansen
Steen U
SU
Baráth
Marek
M
Rushton
Graham
G
Gardiner
John M
JM
Avizienyte
Egle
E
Jayson
Gordon C
GC
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
D016207:Cytokines; D006497:Heparan Sulfate; D009844:Oligosaccharides; D012235:Rhodamines; C022027:lissamine rhodamine B