Inhibition of bFGF activity by size-defined oligosaccharide derivatives of low molecular weight heparin in the sponge angiogenesis assay

4101 bFGF is a potent stimulator of tumour angiogenesis and is dependent on its interaction with heparan sulfates for its biological effect. Previous studies have shown that oligosaccharide derivatives from bovine heparin can inhibit the biological activity of bFGF in vitro, an effect that is determined by the length of the oligosaccharide chain (Jayson et al, BJC 1997). In this study we attempted to establish the potential anti-bFGF activity of size-defined oligosaccharides derived from LMWH by inhibiting bFGF induced neoangiogenesis in the sponge angiogenesis assay. Size-defined heparin oligosaccharides were obtained from Tinzaparin sodium (Innohep) by size-separation gel chromatography. Oligosaccharides ranging from tetrasaccharides to tetradecasaccharides (dp 4- dp14) were used for the study. Two experiments were performed to enable the quantification of angiogenesis at two separate time points (one week and two weeks). Cohorts of 5 mice bearing subcutaneous sponge implants (8mm x 4mm, type 611-9) were given intra-sponge injections of the following for 7 days and 14 days respectively. (i) Saline only (ii) bFGF (100ng/day) (iii) dp4 (20mg/kg/day) + bFGF (iv) dp6 (20mg/kg/day) + bFGF (v) dp8 (20mg/kg/day) + bFGF (vi) dp10 (20mg/kg/day) + bFGF (vii) dp12 (20mg/kg/day) + bFGF (viii) dp 14 (20mg/kg/day( + bFGF The animals were sacrificed and sponges harvested on the day following the last injection. Paraffin-embedded sections were stained with a pan-endothelial marker (von Willebrand factor) and neoangiogenesis quantified by light microscopy (x400 magnification). Microvessels were counted in the new granulation tissue that envelopes and invades the sponge. Microvessel counts were performed independently by two observers in a double-blinded fashion to minimise bias. The results were analysed using a linear mixed effects model. At one week no systematic differences in microvessel counts were noted between the different treatment groups (p=0.45). At the two week end point, the tetrasacccharides, octasaccharides and decasaccharides significantly inhibited neoangiogenesis compared to the bFGF control (p values: 0.01, 0.01 and 0.001 respectively). The hexasaccharide and tetradecasaccharide derivatives also demonstrated a trend towards inhibition of bFGF induced neoangiogenesis whilst the dodecasaccharides potentiated the angiogenic response at a level that was not statistically significant. Conclusion: The shorter length oligosaccharide derivatives of Tinzaparin inhibit the angiogenic effects of bFGF in an invivo angiogenesis assay and could be potentially developed as anti-FGF agents.