Angiogenic effect of fibroblast growth factor-1 and vascular endothelial growth factor and their synergism in a novel in vitro quantitative fibrin-based 3-dimensional angiogenesis system

Background. We have developed an in vitro 3-dimensional angiogenesis system in which the length, distribution, and ultrastructure of induced capillary sprouts were analyzed in response to concentration ranges of fibroblast growth factor (TGF)-1 and vascular endothelial growth factor (VEGF) and synergistic activity quantitated. Methods. Bovine aorta endothelial cell aggregates were embedded in fibrin gel (FG) supported by a nylon mesh ring. The formed disks were cultured in 24-well plates in assay media. The test growth factors FGF-1, VEGF, or both (0 to 100 ng/mL) with 100 KIU/mL aprotinin were added to the media. The disks (n = 8/group) were digitally photographed and capillary sprouts quantitated. Assay disks were then fixed and sectioned for morphology. Results. In aprotinin-stabilized FG, aggregated ECs invaded FG radially, forming sprouts and capillary networks. Neovessel lumens surrounded by ECs were confirmed on hematoxylin and eosin and transmission electron microscopy and by formation of cell junctions by transmission electron microscopy. The angiogenic effects of FGF-1 and VEGF were dose-dependent in the range from I to 100 ng/mL. Significant activity of FGF-1 started at 1 ng/ml, and of VEGF at 2 ng/mL. The greatest effect was at the highest concentration (100 ng/mL) for both cytokines. The combination of 10 ng/mL of each FGF-1 and VEGF induced a significantly greater effect than the additive effects of FGF-1 (10 ng/mL) or VEGF (10 ng/mL) alone when analyzed with SAS system for mixed model (P < .0001), and that also exceeded the effects by 20 ng/mL of either FGF-1 or VEGF Conclusions. A 3-dimensional capillary network can be induced in aprotinin-stabilized FG using FGF-1 or VEGF with synergism between the 2 angiogens.