Hydrogen peroxide induces S1P1 receptors and sensitizes vascular endothelial cells to sphingosine 1-phosphate, a platelet-derived lipid mediator

Sphingosine 1-phosphate (S1P) is a platelet-derived angiogenic lipid growth factor, modulating G-protein-coupled S1P₁ receptors (S1P₁-R) to activate endothelial nitric oxide synthase (eNOS), as well as MAPK pathways in endothelial cells. We explored whether and how hydrogen peroxide (H₂O₂), a representative reactive oxygen species, alters S1P₁-R expression and influences S1P signaling in cultured bovine aortic endothelial cells (BAECs). When BAECs are treated with pathophysiologically relevant concentrations of H₂O₂ (150 µM for 30 min), S1P₁-R protein expression levels are acutely augmented by 30-fold in a dose-dependent fashion. When BAECs have been pretreated with H₂O₂, subsequent S1P stimulation (100 nM) leads to a higher degree of eNOS enzyme activation (assessed as intracellular cGMP content, 1.7 ± 0.2-fold vs. no H₂O₂ pretreatment groups, P < 0.05), associated with a higher magnitude of phosphorylation responses of eNOS and MAPK ERK1/2. PP2, an inhibitor of Src-family tyrosine kinase, abolished the effects of H₂O₂ on both S1P₁-R protein upregulation and enhanced BAEC responses to S1P. H₂O₂ does not augment S1P₁ mRNA expression, whereas VEGF under identical cultures leads to increases in S1P₁ mRNA signals. Whereas H₂O₂ attenuates proliferation of BAECs, addition of S1P restores growth responses of these cells. These results demonstrate that extracellularly administered H₂O₂ increases S1P₁-R expression and promotes endothelial responses for subsequent S1P treatment. These results may identify potentially important points of cross-talk between reactive oxygen species and sphingolipid pathways in vascular responses. sphingolipids; G protein-coupled receptors; reactive oxygen species; signal transduction