Atherosclerosis is an inflammatory disorder involving complex interactions between vascular wall cells and invading inflammatory cells (14). Genetically modified mouse models of atherosclerosis, based on two pivotal genes of lipid metabolism, i.e., apolipoprotein E (ApoE) and the low-density lipoprotein receptor (LDLR), have been crossed with mice deficient in a specific gene of the LT cascade, e.g., 5-LO and BLT 1 , to investigate the impact of the gene product on the atherosclerotic process (9,(15)(16)(17).In the present study we examined the expression of 5-LO, FLAP, LTA4H, LTC4S, and the receptors BLT 1 , BLT 2 , CysLT 1 , and CysLT 2 in human carotid plaques and aortic lesions from both ApoE (Ϫ͞Ϫ) and compound ApoE (Ϫ͞Ϫ) ϫ LDLR (Ϫ͞Ϫ) mice. In contrast to the mouse models, we find that both 5-LO and LTA4H are expressed at increased levels and colocalize within human atherosclerotic lesions, with expression levels correlating with recent or ongoing symptoms of plaque instability. We also report that a selective inhibitor of LTA4H can block LTB 4 biosynthesis in plaque tissue, thus identifying LTA4H as a potential target for development of drugs for prevention and treatment of atherosclerosis and acute atherothrombotic events. Results Expression of 5LO, FLAP, LTA4H, and LTC4S mRNA in Human CarotidPlaque and Mouse Atherosclerotic Aorta. In the human carotid atherosclerotic lesions, the transcript levels of 5-LO, FLAP, and LTA4H were high, corresponding to a 7.5-fold (7.5 Ϯ 4.1, P Ͻ 0.001), 2.7-fold (2.7 Ϯ 1.3, P ϭ 0.003), and nearly 2-fold (1.9 Ϯ 1.0, P ϭ 0.03) increase relative to normal iliac arteries, respectively (Fig. 1A). In contrast, the levels of LTC4S mRNA were not significantly different from controls.In the ApoE (Ϫ͞Ϫ) mouse aorta, 5-LO and LTA4H mRNA levels were essentially unaltered, whereas FLAP and LTC4S mRNA showed a tendency to increase relative tissue from wild-type C57BL͞6J mice (Fig. 1B). In ApoE (Ϫ͞Ϫ) ϫ LDLR (Ϫ͞Ϫ) mice, the expression pattern of the enzymes was essentially the same as in ApoE (Ϫ͞Ϫ) mice, except that LTA4H mRNA levels were significantly increased (1.7 Ϯ 0.3, P ϭ 0.01) (Fig. 1C). Expression of 5-LO, FLAP, and LTA4H Protein in Human and MouseAtherosclerotic Lesions. Staining of human carotid plaque demonstrated prominent expression of 5-LO, FLAP, and LTA4H in intimal lesion areas that also stained positively for human macrophage marker CD163 (Fig. 2). The distribution of 5-LO,

Cysteinyl leukotrienes are key mediators in inflammation and have an important role in acute and chronic inflammatory diseases of the cardiovascular and respiratory systems, in particular bronchial asthma. In the biosynthesis of cysteinyl leukotrienes, conversion of arachidonic acid forms the unstable epoxide leukotriene A4 (LTA4). This intermediate is conjugated with glutathione (GSH) to produce leukotriene C4 (LTC4) in a reaction catalysed by LTC4 synthase: this reaction is the key step in cysteinyl leukotriene formation. Here we present the crystal structure of the human LTC4 synthase in its apo and GSH-complexed forms to 2.00 and 2.15 A resolution, respectively. The structure reveals a homotrimer, where each monomer is composed of four transmembrane segments. The structure of the enzyme in complex with substrate reveals that the active site enforces a horseshoe-shaped conformation on GSH, and effectively positions the thiol group for activation by a nearby arginine at the membrane-enzyme interface. In addition, the structure provides a model for how the omega-end of the lipophilic co-substrate is pinned at one end of a hydrophobic cleft, providing a molecular 'ruler' to align the reactive epoxide at the thiol of glutathione. This provides new structural insights into the mechanism of LTC4 formation, and also suggests that the observed binding and activation of GSH might be common for a family of homologous proteins important for inflammatory and detoxification responses.

or Glu-318 were replaced by codons encoding tyrosine, tyrosine, and glutamine, respectively. The mutated cDNAs were expressed inEscherichia coil, and the three mutated proteins were purified to apparent homogeneity. None of these mutants contained sigcant amounts of zinc, as determined by atomic absorption spectrometry, and all of them were practically devoid of both LTA4 hydrolase and peptidase enzyme activities. Nevertheless, the mutated proteins could be positively identified by their immunoreactivities with an antiserum for human LTA4 hydrolase in immunoblot analysis. Site-directed

No abstract

In humans, the antimicrobial peptide LL-37 and the potent chemotactic lipid leukotriene B4 (LTB4) are important mediators of innate immunity and host defense. Here we show that LTB4, at very low (1 nM) concentrations, strongly promotes release of LL-37 peptides from human neutrophils (PMNs) in a time- and dose-dependent manner, as determined by Western blot, enzyme-linked immunoassay (ELISA), and antibacterial activity. The LTB4-induced LL-37 release is mediated by the BLT1 receptor, and protein phosphatase-1 (PP-1) inhibits the release by suppressing the BLT1-mediated exocytosis of PMN granules. Conversely, LL-37 elicits translocation of 5-lipoxygenase (5-LO) from the cytosol to the perinuclear membrane in PMNs and promotes the synthesis and release of LTB4, particularly from cells primed with LPS or GM-CSF. Furthermore, LL-37 stimulates PMN phagocytosis of Escherichia coli particles, a functional response that is enhanced by LTB4, especially in GM-CSF pretreated cells. In these cells, LL-37 also enhances LTB4-induced phagocytosis. Hence, in human PMNs, positive feedback circuits exist between LL-37 and LTB4 that reciprocally stimulate the release of these mediators with the potential for synergistic bioactions and enhanced immune responses. Moreover, these novel lipid-peptide signaling pathways may offer new opportunities for pharmacological intervention and treatment of chronic inflammatory diseases.