Fig. 5. Modulation of 3D sprouting angiogenesis (A) and endothelial alignment on Matrigel (B) by EphB4-Fc and ephrinB2-Fc. (A) Quantitative analysis of the cumulative sprout length (CSL, quantitated after 48 hours) originating from 10 collagen gel embedded HUVEC spheroids (one out of three independent experiments with similar results). Representatives of each experimental group are shown below the bar graph (bar, 100 µm). EphB4-Fc (1 µg/ml) as well as VEGF (50 ng/ml) induce capillary-like sprouting. By contrast, ephrinB2-Fc (1 µg/ml) inhibits baseline sprouting as well as VEGF-induced sprouting angiogenesis. (B) Quantitative cellular alignment analysis of ECs grown on Matrigel. HUVECs were grown on Matrigel for 24 hours after which the circumferential length of tube-like structures was quantitated by automated image analysis. EphrinB2-Fc significantly inhibits alignment of HUVECs. **P<0.01; ***P<0.01 (compared with baseline control); P<0.01 (compared with VEGF induction).