pDuring pregnancy, VEGF (vascular endothelial growth factor) regulates in part endothelial angiogenesis and vasodilation. In the present study we examine the relative roles of VEGFRs (VEGF receptors) and associated signalling pathways mediating the effects of VEGFsub165/sub on eNOS (endothelial nitric oxide synthase) activation. Despite equal expression levels of VEGFR-1 and VEGFR-2 in UAECs (uterine artery endothelial cells) from NP (non-pregnant) and P (pregnant) sheep, VEGFsub165/sub activates eNOS at a greater level in P- compared with NP-UAEC, independently of Akt activation. The selective VEGFR-1 agonist PlGF (placental growth factor)-1 elicits only a modest activation of eNOS in P-UAECs compared with VEGFsub165/sub, whereas the VEGFR-2 kinase inhibitor blocks VEGFsub165/sub-stimulated eNOS activation, suggesting VEGFsub165/sub predominantly activates eNOS via VEGFR-2. Although VEGFsub165/sub also activates ERK (extracellular-signal-regulated kinase)-1/2, this is not necessary for eNOS activation since U0126 blocks ERK-1/2 phosphorylation, but not eNOS activation, and the VEGFR-2 kinase inhibitor inhibits eNOS activation, but not ERK-1/2 phosphorylation. Furthermore, the inability of PlGF to activate ERK-1/2 and the ability of the VEGFR-2 selective agonist VEGF-E to activate ERK-1/2 and eNOS suggests again that both eNOS and ERK-1/2 activation occur predominately via VEGFR-2. The lack of VEGFsub165/sub-stimulated Akt phosphorylation is consistent with a lack of robust phosphorylation of Sersup1179/sup-eNOS. Although VEGFsub165/sub-stimulated eNOS phosphorylation is observed at Sersup617/sup and Sersup635/sup, pregnancy does not significantly alter this response. Our finding that VEGFsub165/sub activation of eNOS is completely inhibited by wortmannin but not LY294002 implies a downstream kinase, possibly a wortmannin-selective PI3K (phosphoinositide 3-kinase), is acting between the VEGFR-2 and eNOS independently of Akt./p
展开▼
