br Introduction Vein graft implantation into the
Introduction Vein graft implantation into the arterial environment for surgical bypass is the gold standard to treat severe cardiovascular occlusive disease. After placement of a vein into the higher pressure, flow, and oxygen tension of the arterial circulation, the vein adapts to the arterial environment , . Vein graft LKB1 is characterized by wall thickening with the deposition of smooth muscle cells and extracellular matrix; this thickening occurs in all layers of the vein graft and especially in the intima . We have previously shown that vein graft adaptation is also characterized by the loss of venous identity without gain of arterial identity, that is, diminished EphB4 expression without induction of EphrinB2 expression , . EphB4, a member of the transmembrane receptor tyrosine kinase family, is a determinant of venous fate during embryonic development, whereas its ligand EphrinB2 is a determinant of arterial fate; interestingly, both EphB4 and EphrinB2 persist on adult veins and arteries, respectively , . Although the functions of EphB4 and EphrinB2 in adult cells are unknown, we have previously shown that EphB4 inhibits neointimal thickening of vein grafts, suggesting an active role for EphB4 in the limitation of venous wall thickness in adult veins . Vascular endothelial growth factor (VEGF) is a family of indispensable signal proteins particularly prominent in all aspects of vascular development, with their normal function to stimulate both angiogenesis and arteriogenesis , . VEGF-A is the earliest discovered member of the VEGF family and plays critical functions in both vasculogenesis and angiogenesis. VEGF-A is an upstream stimulus of EphrinB2–EphB4 signaling , ,  and is a critical determinant of arterial endothelial specification during embryogenesis  and arteriogenesis in adult organisms . However, the role of VEGF-A and its potential ability to regulate EphrinB2 and EphB4 during vein graft adaptation to the arterial environment is not well understood. We have previously shown that vein graft adaptation is characterized by both sustained downregulation of EphB4 expression and by transient upregulation of VEGF-A expression (24–72 h) before subsequent downregulation . Furthermore, Luo et al. have shown that VEGF-A reduces vein graft intimal hyperplasia in a rabbit model. Based on this data, we hypothesized that VEGF-A is an upstream inhibitor of EphB4 expression and venous identity during vein graft adaptation. Therefore, we examined the response of adult endothelial cells (EC) to VEGF-A treatment in vitro.
Materials and methods
Discussion We show that VEGF-A decreases expression of EphB4 in adult venous EC, consistent with diminished venous identity, without concomitant induction of EphrinB2 expression (Fig. 2); these changes recapitulate those changes during vein graft adaptation in human, rat, and mouse vein grafts , . Interestingly, osteopontin expression is induced during vein graft adaptation , , and osteopontin expression is stimulated by VEGF-A in EC (Fig. 3A). We also show that VEGF-A stimulates dll4 (Fig. 2) but nothing downstream of it (Fig. 3), consistent with a block in the delta–notch pathway in adult EC. In addition, we show that VEGF-A downregulation of EphB4 is VEGFR2- and ERK1/2 dependent but is Akt independent (Fig. 4). Finally, we show that EphB4 negatively regulates this pathway (Fig. 5), although the exact point of regulation is not clear (Fig. 6). These results confirm the importance of VEGF-A not just in promotion of arterial identity but in repression of venous identity. VEGF-A downregulation of EphB4 has been previously reported in embryonic stem cells, human umbilical vein endothelial cells, and transformed EC , ; however, we show that VEGF-A can also downregulate EphB4 in adult EC. Interestingly, we also show that VEGF-A stimulates dll4 expression, but neither notch nor EphrinB2 expression, in adult EC. These results are consistent with some plasticity of adult venous EC, with ability to lose venous identity but inability to gain arterial identity. Although VEGF-A stimulates arterial differentiation and prevents venous fate in embryogenesis , , , , VEGF-A fails to stimulate arterial identity in adult EC. The VEGF-A–dll4–notch–EphrinB2 pathway appears to be blocked at the dll4–notch step. These results show that, at least in adult EC, loss of venous identity does not automatically confer an arterial identity. Our study is limited in that we only tested a single isoform of VEGF-A; it is possible that other VEGF family members may participate in Eph regulation or may stimulate notch and EphrinB2 expression. However, VEGF-A is the major isoform implicated in arterial differentiation , and a delta–notch block has been previously reported in adult cells . Because both dll4 and notch-4 quiescence have been reported during vein graft adaptation in aged vein grafts , we believe that the differences between these findings may reflect differences between the in vitro and in vivo models; nevertheless, both models suggest that delta–notch signaling may be active during vein graft adaptation.