Abstract 854: 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 NO synthase (eNOS) as well as MAP kinase pathways in vascular endothelial cells. We explored whether and how hydrogen peroxide (H2O2), a representative reactive oxygen species (ROS), alters S1P1-R expression and influences S1P signaling in cultured bovine aortic endothelial cells (BAEC). When BAEC are treated with pathophysiologically relevant concentration of H2O2 (150 μM for 30 min), S1P1-R protein expression levels are acutely augmented by ~30 folds in a dose-dependent fashion (western blot analyses, P<0.01). When BAEC have been pre-treated with H2O2, subsequent S1P stimulaion (100 nM for 5 min) leads to higher degree of eNOS enzyme activation (assessed as i[cGMP], 1.7 ± 0.2 folds vs. no H2O2 pretreatment groups, p<0.05), associated with higher magnitudes of phosphorylation responses of eNOSSer1179 as well as those of MAP kinases ERK1/2Thr183/Tyr185 (phopho-western assays). PP2, an inhibitor of Src-family tyrosine kinase pathways, abolished the effects of H2O2 on both S1P1-R protein up-regulation and enhanced eNOS/ERK1/2 responses to S1P. H2O2 does not augment S1P1 mRNA expression, while vascular endothelial growth factor under identical cultures leads to increases in S1P1 mRNA signals (Semi-quantitative RT-PCR assays). While prolonged incubation for 2 days with H2O2 (150 μM) attenuates proliferation of BAEC by 39 % (cell counting assay, p<0.05), stimulation with S1P (100 nM) 30 min after H2O2 addition markedly restores growth responses of these cells. These results demonstrate that extracellularly administered H2O2 increases S1P1-R protein expression in a manner dependent on Src-family tyrosine kinase pathways, without appearing to increase S1P1-R transcripts. H2O2 also promotes endothelial responses for subsequent S1P treatment at both levels of acute activation of effector molecules (eNOS/ERK1/2) and of delayed proliferation responses. These results may identify potentially important point of cross-talk between ROS and sphingolipid pathways in vascular responses, where S1P may counteract deleterious effects of H2O2 via induction and activation of S1P1-R.