As of 2004, >73 million people were prescribed antiinflammatory medication. Despite the extensive number of current products, many people still suffer from their diseases or the pharmacological properties (side effects) of the medications. Therefore, developing therapeutic strategies to treat inflammation remains an important endeavor. Here, we demonstrate that the soluble epoxide hydrolase (sEH) is a key pharmacologic target for treating acute systemic inflammation. Lipopolysaccharide-induced mortality, systemic hypotension, and histologically evaluated tissue injury were substantially diminished by administration of urea-based, small-molecule inhibitors of sEH to C57BL͞6 mice. Moreover, sEH inhibitors decreased plasma levels of proinflammatory cytokines and nitric oxide metabolites while promoting the formation of lipoxins, thus supporting inflammatory resolution. These data suggest that sEH inhibitors have therapeutic efficacy in the treatment and management of acute inflammatory diseases.cyclooxygenase ͉ lipoxin A4 ͉ lipoxygenase ͉ proinflammatory mediators ͉ epoxygenase T he oxidative metabolism of polyunsaturated fatty acids produces potent inflammatory mediators (1). The bulk of research has focused on the arachidonic acid derivatives processed by cyclooxygenase (COX) (prostaglandins) and lipoxygenases (LOX) (leukotrienes), as well as cytokines and oxygen͞ nitrogen radicals. To this end, many pharmaceuticals have been produced to alleviate inflammatory conditions in rheumatoid arthritis, psoriasis, osteoarthritis, and asthma. These drugs include nonsteroidal antiinflammatory drugs (acetylsalicylic acid), specific COX-2 inhibitors (Rofecoxib), and 5-LOX inhibitors (Zileuton).One critical pathway, still relatively unexplored, is mediated by cytochrome P450 enzymes, transforming arachidonic and linoleic acids to various biologically active compounds, including epoxyeicosatrienoic acids (EETs) or hydroxyeicosatrienoic acids (HETEs) (2, 3) and epoxyoctadecenoic acids (EpOMEs), respectively. EETs are endothelium-derived hyperpolarizing factor candidates that mediate vascular relaxation responses (4) and possess antiinflammatory properties (5-8). EETs and EpOMEs are further metabolized by soluble epoxide hydrolase (sEH) to their corresponding diols, dihydroxyeicosatrienoic acids (DHETs; also known as DiHETs) and dihydroxyoctadecenoic acids (DiHOMEs) (Fig. 5, which is published as supporting information on the PNAS web site) (9).Based on the antiinflammatory action of EETs, we surmised that increasing cellular EETs by inhibition of sEH would decrease the inflammatory effects of acute endotoxin [i.e., lipopolysaccharide (LPS)] exposure. Endotoxin exposure is a common model of septicemia, a disease with mortality rates of 40-70% (1). LPS is the primary Gram-negative bacteria surface antigen responsible for eliciting immunologic responses. These responses include leukocyte activation, cytokine production, enhanced proinflammatory gene expression, increased reactive oxygen͞nitrogen species production, and enhanced bi...

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Cyclooxygenase, lipoxygenase and epoxygenase derived oxylipins, especially eicosanoids play important roles in many physiological processes. Assessment of oxidized fatty acid levels is important for understanding their homeostatic and pathophysiological roles. Most reported methods examine these pathways in isolation. The work described here employed a SPE-LC-ESI MS/MS method to monitor these metabolites. In 21 minutes, 39 oxylipins were quantified along with eight corresponding internal standards. The limits of quantification were between 0.07-32 pg (20 pM-10 nM). Finally, the validated method was used to evaluate oxylipin profiles in lipopolysaccharide-exposed mice, an established septic inflammatory model. The method described here offers a useful tool for the evaluation of complex regulatory oxylipin responses in in vitro or in vivo studies.

We previously reported that laminar flow activates peroxisome proliferator-activated receptor ␥ (PPAR␥) in vascular endothelial cells in a ligand-dependent manner that involves phospholipase A2 and cytochrome P450 epoxygenases. In this study, we investigated whether epoxyeicosatrienoic acids (EETs), the catalytic products of cytochrome P450 epoxygenases, are PPAR␥ ligands. Competition and direct binding assays revealed that EETs bind to the ligandbinding domain of PPAR␥ with Kd in the M range. In the presence of adamantyl-ureido-dodecanoic acid (AUDA), a soluble epoxide hydrolase (sEH)-specific inhibitor, EETs increased PPAR␥ transcription activity in endothelial cells and 3T3-L1 preadipocytes. Inclusion of AUDA in the perfusing media enhanced, but overexpression of sEH reduced, the laminar flow-induced PPAR␥ activity. Furthermore, laminar flow augmented cellular levels of EETs but decreased sEH at the levels of mRNA, protein, and activity. Blocking PPAR␥ by GW9662 abolished the EET͞AUDA-mediated antiinflammatory effect, which indicates that PPAR␥ is an effector of EETs. endothelial cells ͉ shear stressA therosclerosis preferentially localizes in branches and curved regions of the arterial tree, where the blood flow is disturbed. In contrast, the straight parts of vessels exposed to nondisturbed laminar flow have few lesions (1). The focal distribution of atherosclerotic lesions has been proposed to be related to the proinflammatory effect of disturbed flow imposed on the endothelium vs. the antiinflammatory effect of laminar flow. In vitro studies using flow channels with cultured endothelial cells (ECs) revealed that disturbed flow induces a number of molecules involved in inflammation, including chemoattractants, adhesion molecules, and cytokines (2, 3). However, prolonged exposure to laminar flow suppresses the cytokine-stimulated or oxidized low-density lipoprotein (LDL)-stimulated inflammatory response in ECs (4).Recent studies showed that the nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR␥) is involved in antiinflammatory effects in the artery wall (5, 6). The activation of PPAR␥ in cultured ECs suppresses the NF-B-mediated expression of molecules such as vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and endothelin 1 that are involved in the inflammatory response (7,8). Troglitazone, a synthetic PPAR␥ ligand, attenuates the formation of lesions in both apolipoprotein E-and low-density lipoprotein receptordeficient mice (7, 9), due in part to the reduction of monocytes͞ macrophages homing to the plaques.We previously demonstrated that laminar flow activates PPAR␥ in a ligand-dependent manner, which exerts an antiinflammatory effect in ECs. Furthermore, we showed that such induction of PPAR␥ ligands involves phospholipase A2 and cytochrome P450 epoxygenases (CYPs) (10). Epoxyeicosatrienoic acids (EETs), the main products of arachidonic acid catalyzed by CYPs, have been reported to dilate coronary arteries by hyperpolarizing vascular smooth muscles (11) and to exe...