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 ␥-glutamylcysteine Vascular endothelial cells are exposed to a tangential shearing force resulting from the flow of blood over the lumenal surface of the vessel wall (1). The nature and magnitude of this fluid shear stress play a key role in the maintenance of vascular integrity and in the development of vascular diseases. For example, the nonrandom distribution of atherosclerotic lesions is due at least in part to local alterations in hemodynamic forces impinging on the vasculature (2-4). At sites vulnerable to lesion formation such as branch points, bifurcations, and curvatures, unidirectional laminar flow is disturbed, with areas characterized by complex flow patterns such as nonlaminar flow and flow reversal. In contrast, lesion-protected areas of the vasculature are characterized by more uniform laminar flow patterns with relatively high levels of fluid shear stress (2-4).

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, Ϯ5 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-␣-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-␣-induced increase in MCP-1 mRNA levels, MCP-1 protein secretion, and activation of p38 MAPK. SphK1 siRNA also inhibited TNF-␣-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-B-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-␣-induced MCP-1 gene expression through a p38 MAPK-dependent pathway and may participate in oscillatory flow-mediated proinflammatory signaling pathway in the vasculature. p38 MAPK ATHEROSCLEROSIS IS A FOCAL inflammatory disease and preferentially occurs in areas of low fluid shear stress and nonlaminar flow within the vasculature. Conversely, the risk of atherosclerosis is decreased in regions of high fluid shear stress and steady laminar flow (3,8,22). Recent studies show that hemodynamic forces play a significant role in determining the functional phenotype of the vascular endothelium. For example, extended exposure of endothelial cells to laminar flow activates expression of antioxidant and cytoprotective genes including heme oxygenase-1, ferritin, manganese, copper-zinc superoxide dismutase, and endothelial nitric oxide synthase (7,9,32). In contrast, extended exposure of endothelial cells to oscillatory flow leads to upregulation of VCAM-1, ICAM-1, and E-selectin gene expression (6), suggesting that oscillatory flow may contribute to the pathogenesis of atherosclerosis by stimulating the expression of proinflammatory genes. Overall, results from these studies suggest that hemodynamic stress modulates the expression of genes that regulate vascular inflammation.Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological processes (25, 31). Recently, it was reported (35) that sphingosine kinase (SphK), the enz...

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-␣ (TNF-␣), interleukin (IL)-1␤, 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-␣-responsive and nuclear factor-B (NF-B)-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-B. These studies suggest that the anti-inflammatory and antiatherosclerotic properties of AGI-1067 may be due to selective inhibition of redoxsensitive 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.

Mitochondrial biogenesis is accompanied by an increased expression of components of the protein import machinery, as well as increased import of proteins destined for the matrix. We evaluated the role of the outer membrane receptor Tom20 by varying its expression and measuring changes in the import of malate dehydrogenase (MDH) in differentiating C2C12 muscle cells. Cells transfected with Tom20 had levels that were twofold higher than in control cells. Labeling of cells followed by immunoprecipitation of MDH revealed equivalent increases in MDH import. This parallelism between import rate and Tom20 levels was also evident as a result of thyroid hormone treatment. Using antisense oligodeoxynucleotides, we inhibited Tom20 expression by 40%, resulting in 40-60% reductions in MDH import. In vitro assays also revealed that import into the matrix was more sensitive to Tom20 inhibition than import into the outer membrane. These data indicate a close relationship between induced changes in Tom20 and the import of a matrix protein, suggesting that Tom20 is involved in determining the kinetics of import. However, this relationship was dissociated during normal differentiation, since the expression of Tom20 remained relatively constant, whereas imported MDH increased 12-fold. Thus Tom20 is important in determining import during organelle biogenesis, but other mechanisms (e.g., intramitochondrial protein degradation or nuclear transcription) likely also play a role in establishing the final mitochondrial phenotype during normal muscle differentiation.

Zidovudine (AZT) and didanosine (ddI), two drugs used in the treatment of AIDS, are also known to cause mitochondrial abnormalities. We investigated the physiological relevance of the mitochondrial defects by measuring in situ skeletal muscle performance and cytochrome c oxidase (CYTOX) enzyme activity in heart muscle, red highoxidative (RG) and white low-oxidative (WG) portions of the gastrocnemius muscle of control (n = 17), AZT-(n = 14), or ddI-treated (n = 11) rats for 28 days. We also evaluated the hypothesis that AZT treatment could alter the expression of the mitochondrial transcription factor A (mtTFA), a key molecule involved in mitochondrial DNA (mtDNA) replication and transcription. AZT had a pronounced effect on blood pressure and skeletal muscle performance, which were significantly decreased during contractile activity at 2 and 5 Hz, compared with control. A significant decrease in CYTOX activity in heart and RG, but not WG muscles, was also evident. In the heart, this was accompanied by an apparent compensatory increase in mtTFA mRNA level that could not be attributed to enhanced transcriptional activation mediated by nuclear respiratory factor 1 (NRF-1). In contrast with AZT, no effect of ddI was found on the extent of fatigue or muscle enzyme activity. These results indicate that AZT induces mitochondrial defects primarily in muscles with the highest oxidative capacities (heart and RG). The long-term effects of AZT on mitochondrial biogenesis have the potential to reduce muscle performance, but the effects on performance in this short-term study were likely due to an inability of the AZT-treated animals to maintain blood pressure during contractile activity.