Reduced adult endothelial cell EphB4 function promotes venous remodeling

Abstract

Reduced EphB4 expression is observed during vein graft adaptation and is associated with increased venous wall thickening. These findings suggest that EphB4 may mediate normal adult venous endothelial cell (EC) function and vein graft adaptation. We therefore tested the functional significance of EphB4 using EC with genetically reduced EphB4 signaling. EC were isolated from EphB4(+/+) and EphB4(+/-) mice. In vitro function was assessed through EC proliferation, migration, nitric oxide (NO) synthesis, and chemokine production. A mouse vein graft model was used to correlate in vitro findings with in vivo vein grafts. Smooth muscle cells (SMC) were subjected to proliferation and migration assays using EphB4(+/+) and EphB4(+/-) EC-conditioned medium. EphB4(+/-) EC exhibited diminished proliferation (P < 0.0001, n = 6), migration (P < 0.0001, n = 3), and NO production (P = 0.0012, n = 3). EphB4(+/-) EC had increased VEGF-A mRNA (P = 0.0006, n = 6) and protein (P = 0.0106, n = 3) as well as increased secretion of VEGF-A (P = 0.0010, n = 5), PDGF-BB (P < 0.0001, n = 6), and TGF-β1 (P < 0.0001, n = 6). EphB4(+/-)-conditioned medium promoted SMC proliferation (P < 0.0001, n = 7) and migration (P = 0.0358, n = 3). Vein grafts and EphB4(+/-) EC showed similarity with regard to VEGF-A and eNOS mRNA and protein expression. In conclusion, reduced venous EC EphB4 function is associated with a proangiogenic and mitogenic phenotype. EphB4(+/-) EC have increased secretion of SMC mitogens and reduced NO production that correlate with the thickened neointima formed during vein graft adaptation. These findings suggest that EphB4 remains active in adult venous EC and that loss of EphB4 plays a role in vein graft adaptation.

Eph-B4 mediates vein graft adaptation by regulation of endothelial nitric oxide synthase

“…8 Our finding that eNOS is phosphorylated with stimulation of Eph-B4 in adult venous endothelial cells is consistent with previous reports that have shown that activation of Eph-B4 enhances NO production in endothelial cells in vitro . 8, 25 We previously showed that reduced endothelial Eph-B4 signaling in endothelial cells derived from heterozygous Eph-B4 mice 27 was associated with diminished basal levels of eNOS protein expression and NO production but increased tube formation, 15 consistent with our data in vitro (Figure 3), but emphasizing the differences between endothelial cell function in vitro and in 3-dimensions, especially during vein graft adaptation in vivo .…”

“…19–22 We have previously shown that Eph-B4 expression is diminished during vein graft adaptation in adults, suggesting that successful vein graft adaptation, characterized by increased venous wall thickness and adaptive outward remodeling, is associated with loss of venous identity. 7, 8 Our data also suggests that Eph-B4 is a negative regulator of eNOS during vein graft adaptation in vivo , with reduced Eph-B4 expression during vein graft adaptation associated with enhanced eNOS phosphorylation after 3 weeks (Figure 4), although not at 1 week postoperatively, 15 consistent with successful vein graft adaptation at 3 weeks in this model. Improving vein graft adaptation with increased NO production has led to the use of eNOS as a potential therapeutic agent.…”

“…14 We have previously shown that reduced Eph-B4 function is associated with increased basal Akt phosphorylation in endothelial cells but not in whole vein grafts, 15 suggesting a link from Eph-B4 to eNOS via the Akt pathway, at least in endothelial cells. In addition, caveolin-1 interacts with eNOS, and negatively regulates eNOS phosphorylation, 30, 31 and we have previously shown that caveolin-1 is a mediator of vein graft adaptation, 8 eg the Eph-B4-eNOS pathway is quite complex, 10 and the activity of this pathway during vein graft adaptation is likely to be have additional layers of regulation in vivo .…”

“…15 Briefly, WT and eNOS KO cells were starved for 15 hours and placed in a 0.1% Gelatin (Sigma-Aldrich) coated transwell upper chamber. Cells were pretreated with L-NAME (1 mM, 30 mins) as indicated.…”

Eph-B4 Mediates Vein Graft Adaptation By Regulation of eNOS

“…[19][20][21][22] We have previously shown that Eph-B4 expression is diminished during vein graft adaptation in adults, suggesting that successful vein graft adaptation, characterized by increased venous wall thickness and adaptive outward remodeling, is associated with loss of venous identity. 7,8 Our data also suggests that Eph-B4 is a negative regulator of eNOS during vein graft adaptation in vivo, with reduced Eph-B4 expression during vein graft adaptation associated with enhanced eNOS phosphorylation after 3 weeks (Figure 4), although not at 1 week postoperatively, 15 consistent with successful vein graft adaptation at 3 weeks in this model. Improving vein graft adaptation with increased NO production has led to the use of eNOS as a potential therapeutic agent.…”