Synergistic effect of hyaluronan oligosaccharides and vascular endothelial growth factor on angiogenesis in vitro

The aim of the present study was to determine whether hyaluronan (HA) degradation products, which have been shown to be angiogenic in vivo, influence endothelial cell invasion of a 3-dimensional matrix, an essential component of the neovascularization process. Using a previously described in vitro assay, we demonstrate that like the angiogenic cytokines basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), HA oligosaccharides (OHA) induce bovine microvascular endothelial cells to invade a 3-dimensional collagen gel within which they form capillary-like tubes, with an optimal effect at approximately 0.5 to 2 micrograms/ml. Strikingly, co-addition of OHA (0.5 - 2 micrograms/ml) and VEGF (30 ng/ml), but not co-addition of OHA and bFGF (10 ng/ml), induced an in vitro angiogenic response that was greater than the sum of the effects elicited by either agent separately. In contrast to OHA, native high molecular weight HA was consistently inactive, whether added alone or in combination with VEGF or bFGF. Because endothelial cell invasion is believed to require extracellular proteolytic activity, we also investigated the effect of OHA on the plasminogen activator (PA)-plasmin system. OHA (0.01 to 1 microgram/ml) but not native high molecular weight HA induced a dose-dependent increase in mRNA levels of urokinase type PA (uPA), urokinase type PA receptor and PA inhibitor type 1, and a parallel increase in the functional activity of urokinase type PA and PA inhibitor type 1, as determined by zymography and reverse zymography, respectively. The effects of OHA on proteolytic activity were additive with those of VEGF, but not with those of bFGF. Taken together, these results demonstrate that OHA modulate the invasive and proteolytic properties of bovine microvascular endothelial cells and synergize specifically with VEGF in the induction of angiogenesis in vitro. We suggest that the synergism between OHA and VEGF plays a role in the regulation of angiogenesis and that it may be exploited therapeutically in situations that would benefit from stimulation of new blood vessel growth.