Synthetic Heparan Sulfate Oligosaccharides Inhibit Endothelial Cell Functions Essential for Angiogenesis

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.

Ovarian Cancer Cell Heparan Sulfate 6-O-Sulfotransferases Regulate an Angiogenic Program Induced by Heparin-binding Epidermal Growth Factor (EGF)-like Growth Factor/EGF Receptor Signaling

“…The ability of HS to regulate the expression and function of HB-EGF, FGF2, IL-8, and IL-6 presents an opportunity to target these cytokines with HS mimetics. We have developed an efficient chemical synthesis that generates synthetic oligosaccharides with specific sulfation patterns (39 -41) and characterized structure-inhibition relationships for FGF2 and VEGF 165 (18). Our work has demonstrated a feasibility of designing structurally distinct synthetic HS mimetics that target multiple angiogenic cytokines.…”

“…In Vitro Endothelial Cell Assays-CM from ovarian cancer cells were used in HUVEC migration and three-dimensional tube formation assays as described before (18,26). The aortic ring assay was performed as previously described (18 4 cells/well) were plated on confluent monolayers of NHDF in RPMI media containing 10% FBS in a 96-well plate in a final volume of 50 l. Two hundred microliters of CM were added to each well.…”

“…The aortic ring assay was performed as previously described (18 4 cells/well) were plated on confluent monolayers of NHDF in RPMI media containing 10% FBS in a 96-well plate in a final volume of 50 l. Two hundred microliters of CM were added to each well. When required, neutralizing antibodies against FGF2 (5 g/ml; mouse monoclonal IgG1; Millipore), IL-6, and IL-8 (both at 0.5 g/ml; polyclonal goat IgG; R&D) were added to the media.…”

“…Some growth factors, for example VEGF 165 and IL-8, require longer HS sequences organized into domains for their activity (8,16), whereas others, such as FGF2, require shorter activating sequences with critical 6-O-sulfation moieties along the HS chain (5-7). Conversely, sulfation at 2-O-and N-positions are essential for effective inhibition of FGF2 signaling by HS oligosaccharides that compete with cellular HS for binding to FGF2 (17,18).…”

Endothelial Heparan Sulfate 6-O-Sulfation Levels Regulate Angiogenic Responses of Endothelial Cells to Fibroblast Growth Factor 2 and Vascular Endothelial Growth Factor

“…For example, FGF2 binds preferentially to HS that contains iduronate-2-O-sulfation and glucosamine-N-sulfate, whereas 6-O-sulfation is essential for the formation of the FGF2/FGFR/HS signaling complex and its mitogenic activity (17)(18)(19). Moreover, we previously showed that a synthetic structurally-defined 2-O-and N-sulfated dodecasaccharide (12-mer 2SNS) inhibited FGF2-dependent endothelial cell functions and signaling (20). In contrast, VEGF dimer formation requires highly sulfated HS domains, where sulfates at 2-O-, 6-O-, and N-positions significantly contribute to VEGF binding (21).…”