Ephrin type-B receptor 4 activation reduces neointimal hyperplasia in human saphenous vein in vitro

BackgroundVein bypass is an essential therapy for patients with advanced peripheral and coronary artery disease despite development of neointimal hyperplasia. We have shown that stimulation of the receptor tyrosine kinase ephrin type-B receptor 4 (Eph-B4) with its ligand ephrin-B2 prevents neointimal hyperplasia in murine vein grafts. This study determines whether Eph-B4 in adult human veins is capable of phosphorylation and activation of downstream signaling pathways, as well as functional to release nitric oxide (NO) and prevent neointimal hyperplasia in vitro.MethodsDiscarded human saphenous veins were taken from the operating room and placed in organ culture without or with ephrin-B2/Fc (2 μg/mL) for 14 days, and the neointima/media ratio was measured in matched veins. Primary human umbilical vein endothelial cells were treated with ephrin-B2/Fc (2 μg/mL) and examined with quantitative polymerase chain reaction, Western blot, immunoassays, and for release of NO. Ephrin-B2/Fc (2 μg/mL) was placed on the adventitia of saphenous veins treated with arterial shear stress for 24 hours in a bioreactor and activated Eph-B4 examined with immunofluorescence.ResultsThe baseline intima/media ratio in saphenous vein rings was 0.456 ± 0.097, which increased to 0.726 ± 0.142 in untreated veins after 14 days in organ culture but only to 0.630 ± 0.132 in veins treated with ephrin-B2/Fc (n = 19, P = .017). Ephrin-B2/Fc stimulated Akt, endothelial NO synthase and caveolin-1 phosphorylation, and NO release (P = .007) from human umbilical vein endothelial cells (n = 6). Ephrin-B2/Fc delivered to the adventitia stimulated endothelial Eph-B4 phosphorylation after 24 hours of arterial stress in a bioreactor (n = 3).ConclusionsEph-B4 is present and functional in adult human saphenous veins, with intact downstream signaling pathways capable of NO release and prevention of neointimal hyperplasia in vitro. Adventitial delivery of ephrin-B2/Fc activates endothelial Eph-B4 in saphenous veins treated with arterial shear stress in vitro. These results suggest that stimulation of Eph-B4 function may be a candidate strategy for translation to human clinical trials designed to inhibit venous neointimal hyperplasia.

Clinical RelevanceNeointimal hyperplasia remains the Achilles' heel of vein grafts, with the disappointing failure of the Project of Ex-vivo Vein Graft Engineering via Transfection (PREVENT) trials showing our incomplete knowledge of endothelial cell biology. Promising results in mice suggest that stimulation of ephrin type-B receptor 4 (Eph-B4), the embryonic determinant of veins in undifferentiated cells, may prevent neointimal hyperplasia in adult vein grafts. This study shows that adult human veins also have functional Eph-B4 receptors and that stimulation of these Eph-B4 receptors maintains activation even under arterial flow conditions. These results suggest that stimulation of Eph-B4 function may be a worthwhile treatment to test in human vein grafts.