Abstract 1734: Ephrin-B1 and Its Cognate Receptor EphB2 Are Expressed on Normal Adult Arterial Endothelium and Both Inhibit Monocyte Migration: New Molecular Insight into Atherogenesis
Background: Transmigration of monocytes through endothelium is the initial step of their transformation to macrophages, key players in atherogenesis. We have previously found that macrophages within human atherosclerotic plaque express ephrin-B1 and EphB2, both of which are transmembrane proteins regulating cell migration in embryogenesis. In this study, we aimed to elucidate the functional roles of ephrin-B1 and EphB2 in adult monocytes, especially in terms of monocyte-to-endothelium interaction.
Methods: We enriched human peripheral blood monocytes from healthy adult volunteers by Lymphoprep reagent followed by counterflow centrifugal elutriation system. By RT-PCR and immunofluorescence study, we examined the expression of ephrin-B1 and EphB2 in the monocytes, monocytic cell line of THP-1, and primary culture of coronary arterial endothelial cells from normal adult humans. Monocyte-to-endothelium interaction was mimicked by Boyden’s chamber assay. Coulter counter was used to count the number of THP-1 cells migrating through the chamber membranes, which were coated with the ephrin-B1 or EphB2 extracellular-domain fusion proteins or IgG-Fc as negative control.
Results: In all of these monocytes, THP-1 cells and endothelial cells, RT-PCR and immunofluorescence study detected mRNA and cell surface protein, respectively, for both ephrin-B1 and EphB2. In Boyden’s chamber assay, ephrin-B1 inhibited spontaneous and SDF-1 (stromal-derived factor-1)-induced migration of THP-1 cells by 83.1 % (P < 0.0001, n =6) and 72.6% (P <0.0001, n =6), respectively. EphB2 also inhibited spontaneous and SDF-1-attracted migration of THP-1 cells by 75.2 % (P =0.0001, n =6) and 45.2 % (P =0.0028, n =6), respectively.
Conclusions: In contrast to diffusible factors like chemokines, ephrin-B1 and EphB2, anchored to cell surface, are considered to work locally through cell-to-cell interaction. As far as we know, ephrin-B1 and EphB2 with so-called reverse signaling are the first molecules to inhibit monocyte migration. We suggest that, in atherogenesis, detachment or dysfunction of endothelium might decrease its ephrin-B1 and/or EphB2 as barriers against monocytes, further promoting monocyte infiltration into atherosclerotic lesions.