Fig. 3. EphrinB2-Fc-induced detachment of ECs from 3D co-culture spheroids of HUVECs and HUASMCs (EC/SMC; left) and from explanted fragments of umbilical vein (right). (A,C,E) EC/SMC co-culture spheroids were treated for 24 hours with ephrinB2-Fc (2 µg/ml) or EphB4-Fc (2 µg/ml), fixed and whole mount stained for the EC marker CD31. Control spheroids (A) and EphB4-Fc-treated spheroids (E) have an intact surface monolayer of CD31⁺ ECs. EphrinB2-Fc (C) disintegrates the surface endothelial monolayer and induces EC detachment (arrows). (B,D,F) Explanted fragments of freshly isolated human umbilical cords were cultured for 24 hours in the presence of ephrinB2-Fc (2 µg/ml) or EphB4-Fc (2 µg/ml) after which they were fixed, paraffin embedded and stained for the EC marker CD34. Control (B) and EphB4-Fc-treated (F) umbilical veins have an intact monolayer of CD34⁺ ECs. By contrast, ephrinB2-Fc (D) induces detachment and denudation of umbilical veins. (G) Quantitation of umbilical vein denudation induced by ephrinB2-Fc. Umbilical vein integrity was assessed by automated image analysis quantitating the relative SMC surface area that is covered by CD34⁺ ECs. *P<0.05 compared with control.