Authors Ende, G. ; Poitz, D. ; Augstein, A. ; Wiedemann, E. ; Friedrichs, J. ; Werner, C. ; Strasser, R. H. ; Jellinghaus, S.
Title Involvement of ephrinA1 in TNF-alpha-mediated monocyte adhesion
Date 31.12.2013
Number 41742
Abstract The Eph-receptors represent the largest family of receptor tyrosine kinases. Both, Eph-receptors and their ephrin-ligands are cell-surface proteins, which are able to generate bidirectional signaling. Eph/ephrin mediated cell-to-cell communications are essential in a variety of processes like tumor biology and inflammation. However, the impact of Eph-receptors and ephrin-ligands in the pathophysiology of atherosclerosis, as a vascular proliferative and inflammatory disease, is currently not well understood. The aim of the present study was to investigate the involvement of the Eph/ephrin-system in the TNF-a mediated monocyte adhesion. <br /><br />Results: Human umbilical vein endothelial cells (HUVEC) were treated with TNF-a. Analysis of mRNA and protein expression of different ephrin-ligands (ephrinA1, -A4, -B1, -B2) and Eph-receptors (EphA2, -A4, -B1, -B4) revealed striking changes in the response to TNF-a. The most pronounced effect was observed in the case of ephrinA1, which was induced 4-fold after 24 h of TNF-a stimulation. The impact of NFkB in TNF-a mediated ephrinA1 induction was shown by overexpression of a constitutive active IkB mutant. This inhibitor of NFkB was able to completely block the ephrinA1 induction. For monocyte adhesion a siRNA-based approach was tested. The siRNA-mediated silencing of ephrinA1 in endothelial cells, leads to a reduction of monocyte adhesion to TNF-a stimulated endothelial cells. To verify this result in-vitro a Single-Cell-Force-Spectroscopy approach was applied. This method demonstrated that the detachment forces of monocytes from endothelial cells increase after TNF-a stimulation and, more importantly, that the detachment forces in ephrinA1-silenced endothelial cells is decreased after TNF-a stimulation, confirming the results of the adhesion assay. The decrease in monocyte adhesion was accompanied by reduced expression of VCAM-1 in TNF-a-stimulated and ephrinA1-silenced cells compared to control-transfected cells. <br /><br />Conclusion: In conclusion, these data demonstrate that TNF-a induces the expression of ephrinA1 (and other Eph-receptors and ephrin-ligands) in endothelial cells in a NFkB dependent manner. This induction of ephrinA1 by TNF-a represents a crucial part of the TNF-a mediated influence on monocyte adhesion. These results might open perspectives by defining a new role of ephrinA1 in TNF-a induced inflammatory processes like monocyte adhesion in atherosclerotic plaques.
Publisher European Heart Journal
Citation European Heart Journal 34 (2013) P596, 114

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