A new co-operative effect of low molecular weight polysaccharides and cell surface heparin sulfate in enhancing bFGF-induced endothelial cell differentiation

Abstract number: OC302

Chabut^* D., Fischer^* A. M., Colliec-Jouault† S., Laurendeau‡ I., Matou† S., Bros^* A., Helley^* D.

†IFREMER, France; ^*INSERM U428, France; ‡UPRES EA 3618, France

Basic fibroblast growth factor (bFGF) exerts its biological activities through both high and low affinity receptors, FGFRs and heparin sulfate (HS) proteoglycans, respectively. Exogenous and extra-cellular glycosaminoglycans are able to modulate the angiogenic activity of bFGF in vitro and in vivo. Thus, we investigated the effect and mechanism of action on in vitro angiogenesis of a low molecular weight fucoidan (LMWF), a new anti-thrombotic polysaccharide from marine origin, as compared to a low molecular weight heparin (LMWH). Human umbilical vein endothelial cells (HUVEC) were treated with bFGF and LMWF or LMWH. By addition of LMWF or LMWH to bFGF, we observed: (i) a twofold increase of bFGF-induced a 6 overexpression measured by flow cytometry (P < 0.001 and P < 0.05, respectively); (ii) an increase of a 6 mRNA, measured by a real time quantitative reverse transcriptase PCR assay, higher for LMWF than for LMWH: 421% and 206% of bFGF control value (P < 0.001 and P < 0.05, respectively); (iii) an increase of bFGF-induced vascular tube formation observed 18 h later on Matrigel, with a higher amount of capillary-like structures with LMWF. While the a 6 overexpression is identical for both polysaccharides, the more intense vascular tube formation observed with LMWF could be explained by the higher level of a 6 mRNA observed 18 h before. The correlation between a 6 overexpression and vascular tube formation was confirmed by the suppression of the capillary network on Matrigel by an anti-a6 antibody. To understand the mechanism involved in the enhancing effect of LMWF and LMWH on bFGF-induced a6 overexpression, we evaluated the relative contribution of FGFRs and HS. Anti-FGFRs blocking antibody had no effect on a6 overexpression, whereas heparitinases (which deplete HS from endothelial cell surface), induced a complete inhibition of a6 overexpression. These data suggest a not yet described co-operative effect of LMW polysaccharides and HS in modulating bFGF signaling; it differs greatly from that described for unfractionated heparin and other heparin-like molecules which compete with HS for bFGF binding. Several studies have reported an increase in HS expression after an ischemic injury. As a result of its HS-dependent proangiogenic effect, associated with its anti-thrombotic properties in animals, LMWF is interesting as a potential drug for revascularization of ischemic areas.