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Eph-receptors represent the largest family of receptor tyrosine kinases. Eph-receptors and their cognate ephrin-ligands are cell-surface proteins, which are able to generate bidirectional signaling. Eph/ephrin interactions are essential in a variety of processes like tumor biology and inflammation. However, the impact of Eph/ephrin-interactions in the pathophysiology of atherosclerosis is still not well understood. The aim of the present study was to investigate the involvement of the Eph/ephrin-system in the TNF-α mediated monocyte adhesion.Human umbilical vein endothelial cells (HUVEC) were treated with TNF-α and the expression of different ephrin-ligands and Eph-receptors was analyzed on mRNA and protein level. EphrinA1 was found to be highly induced by TNF-α stimulation. This induction is mediated by NFkB, as overexpression of a constitutive active IkB mutant completely abolished the ephrinA1 induction. Previous results of our group showed an involvement of ephrinA1 in the process of monocyte adhesion to endothelial cells. Therefore, the impact of TNF-α mediated ephrinA1 induction in monocyte adhesion was studied. The siRNA-mediated silencing of ephrinA1 in endothelial cells, leads to a reduction of monocyte adhesion to TNF-α stimulated endothelial cells. Using a Single-Cell-Force-Spectroscopy approach we could confirm these results. The detachment forces of monocytes from endothelial cells increase after TNF-α stimulation and more importantly were decreased in ephrinA1-silenced endothelial cells. The decrease in monocyte adhesion was accompanied by reduced cell-surface expression of VCAM-1 and ICAM-1 in TNF-α-stimulated and ephrinA1-silenced cells compared to control-transfected cells. Interestingly, the overall expression of VCAM-1 and ICAM-1 on mRNA and protein level was not influenced by ephrinA1 silencing. In contrast, the overexpression of ephrinA1 in endothelial cells shows contrary effects. Ephrin-A1 overexpression enhances the TNF-α mediated monocyte adhesion to endothelial cells as well as the detachment forces.In conclusion, these data demonstrate that endothelial ephrinA1 is induced by TNF-α in a NFkB dependent manner. This induction of ephrinA1 by TNF-α in endothelial cells represents a crucial part of the proadhesive effect of TNF-α on monocytes. Mechanistically it can be shown, that ephrinA1 regulates the trafficking of adhesion molecules and therefore the presentation on the cell surface of endothelial cells. These results might open perspectives by defining a new role of ephrinA1 in TNF-α induced inflammatory processes like monocyte adhesion in atherosclerotic plaques. [ABSTRACT FROM PUBLISHER]

The Eph-family, consisting of Eph-receptors and ephrin-ligands represents the largest class of receptor tyrosine-kinases. The role of Eph/ephrins in elementary physiological processes as re-endothelialisation is still not well understood. The aim of the present study was to investigate the regulation of the ligand ephrin-A1 and its potential impact on proliferation and migration of human umbilical venous (HUVEC) and arterial endothelial cells (HUAEC).Initially, it could be shown, that ephrin-A1 expression was positively correlated with the density of the endothelial cells. Thus, a significant induction of ephrin-A1 in endothelial cells was observed after contact inhibition. The impact of ephrin-A1 on endothelial proliferation and migration was studied using siRNA and adenoviral overexpression. The siRNA-mediated silencing of ephrin-A1 in HUVEC increased the proliferation. In contrast, adenoviral overexpression of ephrin-A1 decreased the proliferation, suggesting an involvement of ephrin-A1 in endothelial proliferation. To study the role of ephrin-A1 in processes associated with an endothelial defect, a wound healing assay was performed. Ephrin-A1-silenced HUVEC showed a faster gap area closure in comparison to cells transfected with a scrambled control siRNA. Interestingly, ephrin-A1-overexpressing endothelial cells showed a faster gap area closure compared to lacZ-control as well. Using live cell imaging it could be visualized that the silencing of ephrin-A1 influences the direction of the migration of HUVEC resulting in disorientation and a missing polarization of the cells. Overexpression of ephrin-A1 leads to a straight forward and faster migration compared to the controls. By using baculoviral expression of actin-GFP and talin-RFP accordingly both the silencing and the overexpression of ephrin-A1 resulted in an increased number of endothelial focal adhesions which also influenced the actin-cytoskeleton in endothelial cells. A migration assay was established to investigate endothelial response to contact with an ephrin-A1-coated surface. A temporary stop of migration was observed after surface coating with ephrin-A1-Fc. Taken together, these results show that ephrin-A1-expression depends on cell-density and is a critical determinant of endothelial proliferation. Furthermore, ephrinA1 is involved in the regulation of migration of cells, by modulating the speed of migration and more importantly the direction of migration. These results show, that ephrinA1 is highly involved in the process of wound healing which is amongst others of great importance for re-endothelialisation. [ABSTRACT FROM AUTHOR]