Flk-1/KDR mediates ethanol-stimulated endothelial cell notch signaling and angiogenic activity

Abstract

We previously reported that ethanol (EtOH) stimulates endothelial angiogenic activity mediated via a notch- and angiopoietin- 1 (Ang-1) pathway. As crosstalk exists between notch and vascular endothelial growth factor (VEGF) signaling, we examined whether the VEGF receptor (VEGFR) Flk-1 (fetal liver kinase 1) mediates EtOH-stimulated notch signaling and angiogenic activity. Methods and Results: Treatment of human coronary artery endothelial cells (HCAECs) with EtOH (1-50 m M , 24 h) dose-dependently increased Flk- 1 expression with a maximum increase observed at 25 m M EtOH. Ethanol treatment activated both Flk-1 and Flt-1 (FMSlike tyrosine kinase 1) as indicated by their phosphorylation, and subsequent stimulation of Akt. EtOH activation of Flk-1 was inhibited by the VEGFR inhibitor SU5416. Gene silencing of Flk-1 using small interfering RNA inhibited the EtOH-induced increase in notch receptors 1 and 4 and notch target gene (hairy enhancer of split-related transcription factor 1) mRNA. Knockdown of Flk-1 inhibited EtOH-induced Ang-1/ Tie-2 mRNA expression and blocked EtOH-induced HCAEC network formation on Matrigel, a response that was restored by notch ligand, notch ligand delta-like ligand 4, treatment. In vivo, moderate alcohol feeding increased vascular remodeling in mouse ischemic hindlimbs. Conclusions: These data demonstrate that EtOH activates Flk-1 and Flt-1 receptors in HCAECs and promotes angiogenic activity via an Flk-1/notch pathway. These effects of EtOH may be relevant to the influence of moderate alcohol consumption on cardiovascular health.