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

Abstract

Background Vein 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 Eph-B4 with its ligand Ephrin-B2 prevents neointimal hyperplasia in murine vein grafts. This study determines whether Eph-B4 in human veins is capable of phosphorylation, activation of downstream signaling pathways, and functional to release nitric oxide and prevent neointimal hyperplasia in vitro. Methods Discarded human saphenous veins were taken from the operating room and placed in organ culture without or with Ephrin-B2/Fc (2 μg/ml; 14 days) and the neointima:media ratio was measured in matched veins. Primary human umbilical vein endothelial cells (HUVEC) were treated with Ephrin-B2/Fc (2 μg/ml) and examined with qPCR, Western blot, immunoassays and for release of nitric oxide. Ephrin-B2/Fc (2 μg/ml) was placed in pluronic gel on the adventitia of saphenous veins treated with arterial shear stress for 24 hours in a bioreactor and activated Eph-B4 examined with immunofluorescence. Results The 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 (p=.017; n=19). Ephrin-B2/Fc stimulated Akt, eNOS and caveolin-1 phosphorylation and NO release (p=0.007) from HUVEC (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). Discussion Eph-B4 is present and functional in adult human saphenous veins, with intact downstream signaling pathways capable of nitric oxide 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.

Molecular identity of arteries, veins, and lymphatics

“…5 Throughout adult life, ephrin-B2 and Eph-B4 remain segregated to arterial or venous endothelium, respectively. 11…”

Improving the Outcome of Vein Grafts: Should Vascular Surgeons Turn Veins into Arteries?

“…To determine whether Eph-B4 is functional in human veins, as it is in adult murine veins, we stimulated discarded human saphenous veins with ephrin-B2/Fc. 62 , 76 ) Eph-B4 activation was associated with reduced neointimal thickening in a human saphenous vein ring assay. Stimulation of Eph-B4 in human endothelial cells induced phosphorylation of Eph-B4 and Cav-1, and release of NO.…”

“… 73 ) Impairment of eNOS-derived NO accelerates smooth muscle cell proliferation and migration that result in NIH. 74 , 75 ) Eph-B4 regulates NO release in endothelial cells; after ephrin-B2/Fc stimulation, eNOS phosphorylation and NO production are increased in human endothelial cells, 76 ) while mouse endothelial cells derived from heterozygous Eph-B4 mice have less NO release compared with WT EC. 70 )…”

Membrane‐mediated regulation of vascular identity

“…Many studies have shown that EphB4 regulates NO release in endothelial cells. With ephrinB2/Fc stimulation, eNOS phosphorylation and NO production are increased in human endothelial cells (Wong et al, 2014), while mouse endothelial cells derived from heterozygous EphB4 mice have less NO release compared with wild type (EphB4+/+) endothelial cells (Jadlowiec et al, 2013). …”