Background: Heparan sulfate (HS) is an essential regulator of multiple angiogenic growth factors. Results: Down-regulation of 6-O-sulfation in endothelial cell HS affects FGF2-and VEGF-mediated endothelial cell functions. Conclusion:The level of 6-O-sulfation in specific HS domains regulates endothelial cell responses to angiogenic growth factors. Significance: The relationships between 6-O-sulfation and endothelial phenotypes could help to design HS sequences inhibiting angiogenic growth factors.

No abstract

BackgroundHeparan 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.Methodology/Principal FindingsWe 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.Conclusion/SignificanceThese results demonstrate structure-function relationships for synthetic HS saccharides that suppress endothelial cell migration, tube formation and signalling induced by key angiogenic cytokines.

Multigram-scale synthesis of a sulfation-site programmed dodecasaccharide is described. CXCL8- and CXCL12-mediated in vitro and in vivo biology is shown to be regulated by a single sulfation site change.

A concise chemical synthesis of a series of structurallydefined heparin-like oligosaccharides is described. This work provides an efficient entry to octa-, deca-, and 10 dodecasaccharides, including the first synthesis of (GlcNS6S-IdoA2S) 5 and (GlcNS6S-IdoA2S) 6 . Evaluation of the in vitro activity of these species against FGF2-and VEGF 165 -dependent endothelial cell proliferation and migration establishes that octa-and decasaccharides are more potent in 15 targeting FGF2-induced effects, where cell migration is affected more significantly than proliferation. These structure-activity relationships exemplify the significance of 6-O-sulfation in regulating the activity of angiogenic growth factors. 20Heparin and heparan sulphate (H/HS) are highly-charged, ubiquitous, naturally-occurring glycosaminoglycans (GAGs) which are involved in regulating a wide range of biologically important cellular signalling events that control a variety of 25 biological functions, including angiogenesis.1 Amongst these, angiogenic signalling pathways that control angiogenesis are regulated by pro-angiogenic and anti-angiogenic cytokines, many of which depend on H/HS for their biological activity.Fibroblast Growth Factor 2 (FGF2) and Vascular Endothelial demonstrating the potency of octa-and deca-saccharides. There is considerable interest in developing synthetic, structurally-defined H/HS sequences as tools to further probe these angiogenic signalling pathways and for other structural 40 interaction studies. Efficient synthetic routes, as well as access to a diversity of functionality, are essential to provide such agents to interrogate a range of biological targets and also with relation to potentially developing new anti-angiogenic therapies. 3A number of reports concerning the construction of various 45 H/HS architectures are known and address variation of sequence length and sulfation pattern. 4 The majority of these target the (IS) n repeating sequence, 5 with disaccharide-based strategies typically introducing the S-I anomeric linkage or employing iditol-based rather than iduronate donors. Noteworthy also are 50 recent approaches utilising chemoenzymatic methodologies 6 and efforts towards sequences containing mixed (GS/IS) oligomers. 60The synthesis of these novel deca-and dodecassaccharides compliments the synthesis of the alternative dodecasaccharide sequence (IS) 6 reported by the Bonnaffé group 1d and an (SI) 4 octasaccharide, similar to 26, reported by Martin-Lomas' group. 4d Furthermore, our optimized approach provides rapid iterative 65 access to multi-hundred mg quantities of octasaccharide 15, scalability which is pivotal to further elongations up to and including novel dodecasaccharide 19. The work was underpinned by developing a reliable 2+(2) n disaccharide iteration strategy for oligosaccharide chain elongation using stable thioglycoside

Background: Numerous angiogenic growth factors depend on heparan sulfate for their activity. Results: Heparan sulfate 6-O-sulfotransferases induce angiogenesis through HB-EGF/EGFR signaling and angiogenic cytokine expression in ovarian cancer cells. Conclusion: Ovarian cancer cell 6-O-sulfation levels influence angiogenic responses. Significance: HS6ST inhibitors and HS mimetics should be explored in the development of new anti-angiogenic agents.

Heparin-like oligosaccharides mediate numerous important biological interactions, of which many are implicated in various diseases. Synthetic improvements are central to the development of such oligosaccharides as therapeutics and, in addition, there are no methods to elucidate the pharmacokinetics of structurally defined heparin-like oligosaccharides. Here we report an efficient two-cycle [4+4+4] tetrasaccharide-iteration-based approach for rapid chemical synthesis of a structurally defined heparin-related dodecasaccharide, combined with the incorporation of a latent aldehyde tag, unmasked in the final step of chemical synthesis, providing a generic end group for labelling/conjugation. We exploit this latent aldehyde tag for 3H radiolabelling to provide the first example of this kind of agent for monitoring in vivo tissue distribution and in vivo stability of a biologically active, structurally defined heparin related dodecasaccharide. Such studies are critical for the development of related saccharide therapeutics, and the data here establish that a biologically active, synthetic, heparin-like dodecasaccharide provides good organ distribution, and serum lifetimes relevant to developing future oligosaccharide therapeutics.

Several angiogenic growth factors including fibroblast growth factors 1 and 2 (FGF1 and FGF2) depend on heparan sulphate (HS) for biological activity. We previously showed that all cellular elements in ovarian tumour tissue synthesised HS but biologically active HS (i.e. HS capable of binding FGF2 and its receptor) was confined to ovarian tumour endothelium. In this study, we have sought to explain this observation. Heparan sulphate sulphotransferases 1 and 2 (HS6ST1 and HS6ST2) attach sulphate groups to C-6 of glucosamine residues in HS that are critical for FGF2 activation. These enzymes were strongly expressed by tumour cells, but only HS6ST1 was found in endothelial cells. Immunostaining with the 3G10 antibody of tissue sections pretreated with heparinases indicated that HS proteoglycans were produced by tumour and endothelial cells. These results indicated that, in contrast to the endothelium, HS produced by tumour cells may be modified by cell-surface heparanase (HPA1) or endosulphatase (SULF). Protein and RNA analysis revealed that HPA1 was strongly expressed by ovarian tumour cells in eight of ten specimens examined. HSULF-1, which removes specific 6-O-sulphate groups from HS, was abundant in tumour cells but weakly expressed in the endothelium. If this enzyme was responsible for the lack of biologically active HS on the tumour cell surface, we would expect exogenous FGF2 binding to be preserved; we showed previously that this was indeed the case although FGF2 binding was reduced compared to the endothelium and stroma. Thus, the combined effects of heparanase and HSULF could account for the lack of biologically active HS in tumour cells rather than deficiencies in the biosynthetic enzymes.