Characterization of antiangiogenic capability of ephrinB2-specific scFvs. (A) HUVEC migration (Transwell) assays in the absence or presence of B11 or 2B1 and/or clustered EphB4-Fc are shown. FBS in the bottom chamber was used as migration stimuli. Cells crossing the Transwell membrane are in green, whereas the blue signal indicates cells that remain at the upper compartment. Results are expressed as the percentage ± SD of migrated cells relative to total cells. *P < .001 and **P < .01 as determined by an unpaired Student t test. (B) Analysis of lateral migration of HUVECs by wound healing assays in vitro. HUVECs were grown to confluence when scratches were done. Cells were incubated with starving media in the absence (Control) or presence of 100 ng/mL VEGF as migrating stimulus. Cell migration was monitored over 24 hours with 100 μg/mL ephrinB2-specific scFvs (B11 or 2B1) or an irrelevant scFv as indicated. Representative still photographs (4× magnification) taken 24 hours after scratching are shown (original wound areas at time 0 are indicated by dotted lines). Quantification of lateral migration after 24 hours is shown in the bottom right panel. Each scFv was assayed at least 3 times and the corresponding values (means ± SD) were represented as the percentage of migrated area from time 0. *P < .01 versus control. (C) Tubular formation assay. HUVECs were cultured on standard Matrigel in VEGF-stimulated conditions in the absence (control) or presence of anti-ephrinB2 (B11 or 2B1) scFvs or an irrelevant scFv. Representative microphotographs of tube formation after 6 hours of culture (4× magnification) are shown. Graph shows quantitative measure of tube formation. Each treatment was assayed at least 3 times, and the corresponding values (means ± SE) were plotted as percentages of tube formation in the respective condition. *P < .001 versus control.