Sphingosine kinase-1 mediates TNF-alpha-induced MCP-1 gene expression in endothelial cells: upregulation by oscillatory flow.

Atherosclerosis is a focal inflammatory disease and preferentially occurs in areas of low fluid shear stress and oscillatory flow, whereas the risk of atherosclerosis is decreased in regions of high fluid shear stress and steady laminar flow. Sphingosine kinase-1 (SphK1) catalyzes the conversion of sphingosine to sphingosine-1 phosphate (S1P), a sphingolipid metabolite that plays important roles in angiogenesis, inflammation, and cell growth. In the present study, we demonstrated that exposure of human aortic endothelial cells to oscillatory flow (shear stress, +/-5 dyn/cm(2) for 48 h) resulted in a marked increase in SphK1 mRNA levels compared with endothelial cells kept in static culture. In contrast, laminar flow (shear stress, 20 dyn/cm(2) for 48 h) decreased SphK1 mRNA levels. We further investigated the role of SphK1 in TNF-alpha-induced expression of inflammatory genes, such as monocyte chemoattractant protein-1 (MCP-1) and VCAM-1 by using small interfering RNA (siRNA) specifically for SphK1. Treatment of endothelial cells with SphK1 siRNA suppressed TNF-alpha-induced increase in MCP-1 mRNA levels, MCP-1 protein secretion, and activation of p38 MAPK. SphK1 siRNA also inhibited TNF-alpha-induced cell surface expression of VCAM-1, but not ICAM-1, protein. Exposure of endothelial cells to S1P led to an increase in MCP-1 protein secretion and MCP-1 mRNA levels and activation of NF-kappaB-mediated transcriptional activity. Treatment of endothelial cells with the p38 MAPK inhibitor SB-203580 suppressed S1P-induced MCP-1 protein secretion. These data suggest that SphK1 mediates TNF-alpha-induced MCP-1 gene expression through a p38 MAPK-dependent pathway and may participate in oscillatory flow-mediated proinflammatory signaling pathway in the vasculature.

Selective inhibition of endothelial and monocyte redox-sensitive genes by AGI-1067: a novel antioxidant and anti-inflammatory agent.

Atherosclerosis is a disease of oxidative stress and inflammation. AGI-1067 [butanedioic acid, mono[4-[[1-[[3,5-bis(1,1-dimethylethyl)-4-,hydroxyphenyl]thio]-1-methylethyl]thio]-2,6-bis (1,1-dimethylethyl)phenyl] ester] is a metabolically stable derivative of, yet pharmacologically distinct from, the antioxidant drug probucol. It is a member of a novel class of orally active, antioxidant, anti-inflammatory compounds termed vascular protectants and exhibits antiatherosclerotic properties in multiple animal models and in humans. To elucidate its antiatherosclerotic mechanisms, we have evaluated several cellular and molecular properties of AGI-1067 in vitro. AGI-1067 exhibited potent lipid peroxide antioxidant activity comparable with probucol yet demonstrated significantly enhanced cellular uptake over that observed with probucol. AGI-1067, but not probucol, inhibited basal levels of reactive oxygen species (ROS) in cultured primary human endothelial cells and both basal and hydrogen peroxide-induced levels of ROS in the promonocytic cell line, U937. Furthermore, AGI-1067 inhibited the inducible expression of the redox-sensitive genes, vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1, in endothelial cells as well as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 production in peripheral blood mononuclear cells, whereas probucol had no effect. cDNA array hybridization experiments demonstrated that AGI-1067 selectively inhibited the expression of only a subset of TNF-alpha-responsive and nuclear factor-kappaB (NF-kappaB)-inducible genes in endothelial cells. The inhibitory effect of AGI-1067 on inducible VCAM-1 gene expression occurred at the transcriptional level, yet AGI-1067 had no effect on the activation of the redox-sensitive transcription factor NF-kappaB. These studies suggest that the anti-inflammatory and antiatherosclerotic properties of AGI-1067 may be due to selective inhibition of redox-sensitive endothelial and monocyte inflammatory gene expression. These studies provide a molecular basis for understanding the mechanism of action of this new class of therapeutic antiatherosclerotic compounds.

Laminar flow induction of antioxidant response element-mediated genes in endothelial cells. A novel anti-inflammatory mechanism.

Atherosclerotic lesions preferentially develop in areas of the vasculature exposed to nonlaminar blood flow and low fluid shear stress, whereas laminar flow and high fluid shear stress are athero-protective. We have identified a set of genes including NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), ferritin (heavy and light chains), microsomal epoxide hydrolase, glutathione S-transferase, and gamma-glutamylcysteine synthase, whose expression is induced by exposure to prolonged physiological levels of steady laminar flow (shear stress = 20 dyn/cm(2)) in endothelial cells (EC). These genes contain an antioxidant response element (ARE) or ARE-like transcriptional regulatory sequence in their promoters and generally function to protect cells against oxidant stress. We demonstrate that exposure of EC to laminar flow activates ARE-mediated transcriptional activity. Mutation of the ARE from either the NQO1 or HO-1 promoter abolished laminar flow-induced NQO1 and HO-1 transcriptional activation. Expression of antisense Nrf2 (a transcriptional factor for ARE), a dominant negative Nrf2, or the cytoplasmic inhibitor of Nrf2 (Keap1/INrf2) inhibited laminar flow-induced NQO1 promoter activation in EC. In addition, expression of NQO1 or Nrf2 inhibited tumor necrosis factor-alpha-induced activation of VCAM-1 (vascular cell adhesion molecule-1) gene expression in EC. These data define the ARE as a novel endothelial shear stress response element. Furthermore, laminar flow activation of antioxidant genes via an ARE-dependent transcriptional mechanism may represent a novel athero-protective and anti-inflammatory mechanism in the vasculature.