2002;108:98C106. siRNA) and Gq-independent (not blocked by glycoprotein [GP] 2A, a Gq inhibitor, or Gq siRNA). PLC- activation and cell migration was blocked by siRNA to G12/13. Oxygen free radical generation induced by S-1-P, as measured by dihydroethidium staining, was significantly inhibited by U7 but not by InactiveU7. Inhibition of oxygen free radicals with the inhibitor diphenyleneiodonium resulted in decreased cell migration Cytochalasin H to S-1-P. VSMC mitogen-activated protein kinase activation and VSMC migration in response to S-1-P was inhibited by PLC- inhibition. Conclusion S-1-P induces oxygen free radical generation through a G12/13, PLC–mediated mechanism that facilitates VSMC migration. To our knowledge, this is the first description of PLC-mediated oxygen free radical generation as a mediator of S-1-P VSMC migration and illustrates the need for the definition of cell signaling to allow targeted strategies in molecular therapeutics for restenosis. Clinical Relevance Activation of vascular easy muscle cells by growth factors leads to cell proliferation and migration, which are integral features of the healing response in a vessel that leads to the development of intimal hyperplasia after bypass grafting, angioplasty, and stenting. Sphingosine-1-phosphate (S-1-P) is usually a common phospholipid, released from activated platelets at sites of vessel injury. It is a G-proteinCcoupled receptor agonist that induces easy muscle cell migration, a key event in the development of intimal hyperplasia. Mechanisms of cell migration are not well defined, and understanding the mechanisms of signal transduction is important in defining potential targets for therapeutic intervention. The present study shows that S-1-P induces oxygen free radical generation through a G12/13, PLC-Cmediated mechanism that facilitates smooth muscle cell migration. Targeting choke points in cell signaling, such as membrane G-proteins, is an attractive molecular target in developing therapeutic strategies to moderate restenosis. Activation of vascular smooth muscle cells (VSMC) by growth factors leads to cell proliferation and migration, which are integral features of the healing response in a vessel.1 Targeting key molecules at the level of the membrane, within the cell and within the nucleus, appears to be a novel way to achieve local control of the activated SMCs. Multiple pathways, including mammalian target of rapamycin, mitogen-activated protein kinase (MAPK), and cell-cycle proteins, have been successfully targeted by therapeutic agents. In human restenotic lesions, migration rather than proliferation may be a primary biologic event leading to intimal expansion, and the signaling leading to these events is not fully defined.1 Sphingosine-1-phosphate (S-1-P) MTC1 is a bioactive sphin-golipid released in large amounts from activated platelets that has been shown to have numerous biologic effects in a large number of vascular cell types. It can act as a secondary mediator for cell responses mediated by platelet-derived growth factor (PDGF) and tissue plasminogen activator (tPA).2,3 S-1-P has been identified as a chemoattractant for VSMCs expressing G-proteinCcoupled receptors S-1-P1, S-1-P2 and S-1-P3 receptors.2,4C7 S-1-P1 couples exclusively to G-proteins of the Gi type, while S-1-P2 and S-1-P3 couple to G-proteins of Gi, Gq, and G12/13 types.8,9 We have previously demonstrated that migration stimulated by S-1-P in VSMCs involves PI3-kinase, extracellular signal-regulated kinases 1/2 (ERK1/2), and p38MAPK.4C6 Others have shown that exogenous S-1-P will stimulate oxygen free radical generation in association with an increase in intracellular Ca2+ concentration and an increase in inositol phosphate production, reflecting activation of phospholipase C (PLC). Very limited data are available on the role of PLC- in arterial Cytochalasin H injury. One report has shown that PLC- Cytochalasin H activities are not significantly elevated 24 hours of balloon injury in the rat aorta, but PLC- activity has increased twofold by 14 days.10 Thus, it appears that PLC- activity is not temporally associated with VSMC apoptosis.