Fig. 2. Angiogenic factors and tumour-derived factors increased HUVEC tubulogenesis within fibrin gels. (A) HUVECs were grown within fibrin gels in standard serum-containing medium and stimulated with the angiogenic factors as indicated: VEGF (25 ng/ml), FGF-2 (10 ng/ml), TNF- (10 ng/ml), TGF-ß1 (1 ng/ml), EGF (50 ng/ml), TGF- (10 ng/ml), HGF/SF (300 U/ml), IL1 (10 ng/ml), and angiogenin (100 ng/ml). The factors present in the angiogenic cocktail are a combination of all the above listed angiogenic factors. The cells were incubated at 37°C and 5% CO₂ (v/v) for 3 days, after which time the cells were photographed and the tubules quantified with the LUCIA G/Comet software. (B) HUVECs were cultured within a fibrin gel as a drop culture in the bottom of a well. Where indicated, equal amounts of U87 glioma cells or MDCK cells, or twice the amount of U87 glioma cells, were also incubated within a separate fibrin gel as a drop culture within the same well as the HUVECs. Standard serum-containing EC medium (with no exogenous angiogenic factors) was added to each well with both gels covered with the same medium, allowing diffusible molecules produced by the cells from one gel to reach the cells in the other gel. Aprotinin (100 µg/ml) was also added to each well in order to prevent gel degradation by soluble serine protease. The cells were incubated at 37°C and 5% CO₂ (v/v) for 3 days, after which time the cells were photographed and the tubules quantified with the LUCIA G/Comet software. Quantification of tubular structures was performed by measuring the total length of structures per field. Five fields were quantified per gel and the average and s.d. of each result are shown. ^* indicates statistically significant differences (P<0.05) compared with the buffer control (^** P<0.0001) using the student's t-test assuming equal variance, two tail.