Epoxyeicosatrienoic acids (EETs), lipid mediators synthesized from arachidonic acid by cytochrome P-450 epoxygenases, are converted by soluble epoxide hydrolase (SEH) to the corresponding dihydroxyeicosatrienoic acids (DHETs). Originally considered as inactive degradation products of EETs, DHETs have biological activity in some systems. Here we examined the capacity of EETs and DHETs to activate peroxisome proliferator-activated receptor-α (PPARα). We find that among the EET and DHET regioisomers, 14,15-DHET is the most potent PPARα activator in a COS-7 cell expression system. Incubation with 10 μM 14,15-DHET produced a 12-fold increase in PPARα-mediated luciferase activity, an increase similar to that produced by the PPARα agonist Wy-14643 (20 μM). Although 10 μM 14,15-EET produced a threefold increase in luciferase activity, this was abrogated by the SEH inhibitor dicyclohexylurea. 14-Hexyloxytetradec-5( Z)-enoic acid, a 14,15-EET analog that cannot be converted to a DHET, did not activate PPARα. However, PPARα was activated by 2-(14,15-epoxyeicosatrienoyl)glycerol, which was hydrolyzed and the released 14,15-EET converted to 14,15-DHET. COS-7 cells incorporated 14,15-[3H]DHET from the medium, and the cells also retained a small amount of the DHET formed during incubation with 14,15-[3H]EET. Binding studies indicated that 14,15-[3H]DHET binds to the ligand binding domain of PPARα with a Kd of 1.4 μM. Furthermore, 14,15-DHET increased the expression of carnitine palmitoyltransferase 1A, a PPARα-responsive gene, in transfected HepG2 cells. These findings suggest that 14,15-DHET, produced from 14,15-EET by the action of SEH, may function as an endogenous activator of PPARα.

Kip1 (27) may mediate these cellular processes.EETs are rapidly taken up by vascular cells and converted either to the corresponding dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH) or to chainshortened fatty acid epoxides by ␤-oxidation (7,8,10,13,33). These metabolic processes likely play an important role in modulating the bioactivity of EETs. Increased excretion of 11,12-DHET and 14,15-DHET has been observed in patients with pregnancy-induced hypertension (3), suggesting a role for epoxide hydrolase in the regulation of blood pressure. Selective inhibition of sEH by N,NЈ-dicyclohexyl urea (DCU) or N-cyclohexyl-NЈ-dodecyl urea (CUDA) decreased blood pressure in rodent models of hypertension (18, 39), and sEH gene knockout reduced blood pressure in male mice (31). These observations suggest that sEH inhibition could represent a novel approach for the treatment of hypertension.Although sEH has been detected in various human cells and tissues (33), recent studies indicate that ␤-oxidation, rather than sEH, is the primary pathway for EET metabolism in cultured human vascular cells and skin fibroblasts (7, 13). Thus it is necessary to delineate the pathways of EET metabolism in intact human blood vessels to evaluate the potential utility of sEH inhibition in human vascular tissues. In the present study, we examined the metabolism of EETs and tested the effects of a novel sEH inhibitor with enhanced potency and solubility in intact human blood vessels (21,22). Our findings demonstrate that the main EET metabolic pathway in human blood vessels is conversion to DHET, that selective sEH inhibitors are effective in inhibiting this process, and that EET conversion to ␤-oxidation products only occurs in intact human vascular tissue when sEH is inhibited. METHODS Human Intact Vessel Collection and Cell CultureUnused human saphenous veins (HSVs) harvested for coronary artery bypass surgery and human aortas (HAs) and coronary arteries (HCAs) removed at the time of heart transplantation surgery were obtained from the operating room at the University of Iowa Hospitals and Clinics according to a protocol approved by the University of Iowa Human Subjects Office (28). Tissues were maintained overnight in medium 199 (M199) supplemented with 10% FBS, MEM nonessential amino acids, MEM vitamin solution, 2 mmol/l L-glutamate, 50 mol/l gentamicin, and 15 mmol/l HEPES in a humidified atmosphere containing 5% CO 2 at 37°C. HSV endothelial cells (HSVECs) and smooth muscle cells (HSVSMCs) were isolated from HSVs using the Address for reprint requests and other correspondence: X. Fang,

. Human coronary endothelial cells convert 14,15-EET to a biologically active chain-shortened epoxide. Am J Physiol Heart Circ Physiol 283: H2306-H2314, 2002. First published August 22, 2002 10.1152 10. /ajpheart.00448.2002 epoxygenase-derived epoxyeicosatrienoic acids (EETs) play an important role in the regulation of vascular reactivity and function. Conversion to the corresponding dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolases is thought to be the major pathway of EET metabolism in mammalian vascular cells. However, when human coronary artery endothelial cells (HCEC) were incubated with 3 H-labeled 14,15-EET, chain-shortened epoxy fatty acids, rather than DHET, were the most abundant metabolites. After 4 h of incubation, 23% of the total radioactivity remaining in the medium was converted to 10,11-epoxy-hexadecadienoic acid (16:2), a product formed from 14,15-EET by two cycles of ␤-oxidation, whereas only 15% was present as 14,15-DHET. Although abundantly present in the medium, 10,11-epoxy-16:2 was not detected in the cell lipids. Exogenously applied 3 H-labeled 10,11-epoxy-16:2 was neither metabolized nor retained in the cells, suggesting that 10,11-epoxy-16:2 is a major product of 14,15-EET metabolism in HCEC. 10,11-Epoxy-16:2 produced potent dilation in coronary microvessels. 10,11-Epoxy-16:2 also potently inhibited tumor necrosis factor-␣-induced production of IL-8, a proinflammatory cytokine, by HCEC. These findings implicate ␤-oxidation as a major pathway of 14,15-EET metabolism in HCEC and provide the first evidence that EET-derived chain-shortened epoxy fatty acids are biologically active.beta-oxidation; vasorelaxation; inflammation; epoxyeicosatrienoic acid ENDOTHELIUM-DERIVED epoxyeicosatrienoic acids (EETs) produced by arachidonic acid (AA) cytochrome P-450 epoxygenases are thought to play an important role in vascular biology. The four EET regioisomers, 5,6-, 8,9-, 11,12-, and 14,15-EET, potently dilate coronary arteries and other blood vessels through activation of maxiCa 2ϩ -activated K ϩ channels (5, 15, 39). Therefore, EETs may function as endothelium-derived hyperpolarizing factors in some vascular beds (4, 13). In addition, EETs modulate a variety of cellular functions and signaling pathways, including protein kinase C (28), Ca 2ϩ mobilization (12, 23), tyrosine kinases, mitogenactivated protein kinases, extracellular signal-regulated kinases 1 and 2 (6, 16, 20), cyclooxygenase (11, 14), mono-ADP-ribosylation (22), G s ␣ protein (26), and expression of adhesion molecules (25). Thus EETs are involved in regulation of vascular function, smooth muscle cell proliferation, and vascular inflammation (5, 15, 39).The major metabolic fate of EETs in the vasculature is thought to be conversion to the corresponding dihydroxyeicosatrienoic acids (DHETs) catalyzed by epoxide hydrolases, particularly the soluble form of epoxide hydrolase (sEH) (9). Inhibition of sEH by N,NЈ-dicyclohexyl urea (DCU) in rats (37) and sEH knockout in male mice (31) reduced blood pressure. These observa...

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