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 muscle-contacting 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.