Contact-dependent interactions between endothelial cells (ECs), as well as between ECs and mural cells, play a key role in the formation of a regular vascular system and the assembly of the vessel wall. Recent studies have identified ephrinB2 and EphB4 as markers and makers of arteriovenous differentiation during vascular development. On the basis of these findings,we hypothesized that Ephephrin interactions in the vascular system mediate distinct propulsive and repulsive effector functions that provide guidance signals for the proper spatial organization of the developing vasculature. Utilizing a set of specialized endothelial differentiation and angiogenesis assays, the present study was aimed at studying vascular morphogenic functions of endothelial EphB4 and ephrinB2 activation. EphrinB2-Fc acts anti-adhesively and induces detachment of ECs, as demonstrated by (1) inhibition of adhesion to ephrinB2-Fc-coated culture dishes, (2) detachment of ECs grown as differentiated 3D spheroids, and (3) endothelial denudation of explanted fragments of umbilical vein. Conversely, soluble ephrinB2-Fc inhibits lateral cell migration, vascular endothelial growth factor (VEGF) gradient-driven chemotaxis, capillary-like network formation and sprouting angiogenesis. In turn, soluble EphB4-Fc is pro-adhesive and stimulates EC migration and sprouting angiogenesis. EphrinB2-mediated repulsive signals are transduced by EphB4, as demonstrated by EphrinB2-Fc inhibition of sprouting angiogenesis of constitutively EphB4-overexpressing ECs. Confrontation experiments of EphB4-overexpressing ECs with ECs overexpressing full-length or truncated ephrinB2 that lacks the cytoplasmic catalytic domain demonstrated that forward EphB4 signaling with EphB4 tyrosine phosphorylation restricts intermingling of cells and supports cellular segregation. Taken together, these data identify distinct propulsive and repulsive effector functions of endothelial ephrinB2 and EphB4 that mediate spatial positional signals during angiogenesis and vessel assembly.

Objective-The EphB ligand ephrinB2 has been identified as a critical determinant of arterial endothelial differentiation and as a positive regulator of invading endothelial cells during angiogenesis. This study was aimed at identifying determinants of endothelial cell ephrinB2 expression. Methods and Results-Arteriovenous asymmetrical endothelial cell ephrinB2 expression in vivo is lost on transfer into culture with aortic endothelial cells becoming partially ephrinB2-negative and saphenous vein endothelial cells becoming partially ephrinB2-positive. Contact with smooth muscle cells and angiogenic stimulation by vascular endothelial growth factor lead to an increased endothelial cell ephrinB2 expression. Quiescent, smooth musclecontacting endothelial cells express ephrinB2 uniformly on their luminal surface. In contrast, monolayer endothelial cells translocate ephrinB2 to interendothelial cell junctions, which is strongly enhanced by EphB4-Fc-mediated receptor body activation. Junctional ephrinB2 colocalizes and coimmunoprecipitates with CD31. Conclusions-This study identifies distinct regulatory mechanisms of endothelial ephrinB2 expression and cellular distribution in quiescent and activated endothelial cells. The data demonstrate that endothelial cell ephrinB2 expression is controlled by microenvironmental determinants rather than being an intrinsic endothelial cell differentiation marker.