2008 Annual Meeting Abstracts
Eph-B4 Stimulates Endothelial Cell Migration by an Akt1-Dependent Mechanism
Tiffany T. Fancher, MD1, Akihito Muto, MD, PhD2, Tamara N. Fitzgerald, MD, PhD2, Dania Magri, BS2, Stanley J. Dudrick, MD, FACS1, Alan A. Dardik, MD, PhD3.
1St. Mary's Hospital, Waterbury, CT, USA, 2Yale University, New Haven, CT, USA, 3Yale University, New Haven, CT, USA.
Objective: Venous endothelial cell migration is thought to be critical for successful vein graft adaptation. Eph-B4 is an endothelial cell surface receptor tyrosine kinase and determinant of venous identity during development. We recently described persistence of Eph-B4 on adult venous endothelium. Therefore, we examined whether Eph-B4 mediates venous endothelial cell migration.
Design: Mouse Lung Endothelial Cells derived from adult wildtype (EC) or Akt1-knockout (EC-Akt-KO) mice were stimulated with the ligand Ephrin-B2/Fc, and cell migration was counted with a Boyden chamber. Akt phosphorylation was determined with Western blotting. In some experiments EC were preincubated with wortmannin (10 nM) for 30 min. ANOVA was used for statistical analysis.
Setting: Academic medical center
Main Outcome Measures: Quantify migration of mouse lung endothelial cells to Eph-B4.
Results: Stimulation of Eph-B4 with its ligand Ephrin-B2/Fc resulted in dose-dependent EC migration (maximum 10 mcg/ml; n=4; p<.0001). Stimulation of EC Eph-B4 resulted in time-dependent Akt phosphorylation on both Ser-473 and Thr-308 (maximum 30 min; n=5). Eph-B4 mediated EC migration was inhibited by 95% in EC-Akt-KO, but was completely restored in EC-Akt-KO reconstituted with a retrovirus encoding Akt1 (n=4; p<.0001). Inhibition of PI3-kinase, the upstream activator of Akt1, with wortmannin inhibited Eph-B4 mediated EC migration by 85% (n=3; p<.0001).
Conclusion: Activation of the venous determinant Eph-B4 with its ligand stimulates adult venous endothelial cell migration via the Akt1 pathway. These results suggest that strategies to optimize vein graft adaptation by altering Eph-B4 signaling may also stimulate signaling pathways downstream to induce endothelial cell migration.