Adipose tissue is an important endocrine and secretory organ. There is growing evidence that adipocytes from different fat depots have distinct functional properties. Epicardial adipose tissue has been shown to produce inflammatory mediators, but the functional properties of isolated epicardial adipocytes (EA) have not been studied. We characterized human EA isolated from freshly harvested hearts, along with subcutaneous and perirenal adipocytes from the same donors. Differentiation from preadipocytes was associated with greatly increased adipokine and proinflammatory cytokine expression; of note, the proinflammatory mediator CD14 was expressed at 50-fold higher levels by EA compared to preadipocytes. EA have substantial cyclooxygenase and epoxide hydrolase activity. The major COX metabolite is PGE2, which plays an important role in angiogenesis and inflammation. LPS caused increased expression of adiponectin, VEGF, and CD14 in both epicardial adipose tissue explants and cultured EA. Conditioned medium from EA (but not control medium or medium from epicardial preadipocytes or subcutaneous adipocytes) strongly promoted growth and tubule formation by human coronary artery endothelial cells. These findings suggest that EA may play an important role in promoting coronary artery inflammation and growth of vasa vasorum and collateral coronary vessels in ischemia. (Supported by NIH HL70860 [NLW])

Soluble epoxide hydrolase (sEH) plays a major role in regulating vascular epoxyeicosatrienoic acid metabolism and function, and substituted urea derivatives that inhibit sEH activity reduce blood pressure in hypertensive rats. We found that substituted urea derivatives containing a dodecanoic acid group, besides effectively inhibiting sEH, increased peroxisome proliferator-activated receptor (PPAR) alpha activity. In PPARalpha transfected COS-7 cells, treatment with 10 microM N-cyclohexyl-N'-dodecanoic acid urea (CUDA) or N-adamantanyl-N'-dodecanoic acid urea (AUDA) produced 6- and 3-fold increases, respectively, in PPARalpha activation. Neither CUDA nor AUDA activated PPARdelta or PPARgamma directly, indicating selectivity for PPARalpha. CUDA did not alter PPARalpha protein expression, and it competitively inhibited the binding of Wy-14643 (pirinixic acid) to the ligand binding domain of PPARalpha, suggesting that it functions as a PPARalpha ligand. CUDA and AUDA were metabolized to chain-shortened beta-oxidation products, a process that reduced their potency as sEH inhibitors and their ability to bind and activate PPARalpha. N,N'-Dicylclohexylurea and N-cyclohexyl-N'-dodecylurea, sEH inhibitors that do not contain a carboxylic acid group, did not activate PPARalpha. In HepG2 cells, CUDA increased the expression of the PPARalpha-responsive gene carnitine palmitoyltransferase 1A. We conclude that CUDA and AUDA, by virtue of their carboxylic acid substitution, activate PPARalpha in addition to potently inhibiting sEH. Further development of these compounds could lead to a class of agents with hypotensive and lipid-lowering properties that may be valuable for the prevention and treatment of cardiovascular disease.

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-alpha (PPARalpha). We find that among the EET and DHET regioisomers, 14,15-DHET is the most potent PPARalpha activator in a COS-7 cell expression system. Incubation with 10 microM 14,15-DHET produced a 12-fold increase in PPARalpha-mediated luciferase activity, an increase similar to that produced by the PPARalpha agonist Wy-14643 (20 microM). Although 10 microM 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 PPARalpha. However, PPARalpha 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 PPARalpha with a Kd of 1.4 microM. Furthermore, 14,15-DHET increased the expression of carnitine palmitoyltransferase 1A, a PPARalpha-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 PPARalpha.

Epoxyeicosatrienoic acids (EETs) are synthesized in the endothelial cells of vascular tissues. They are released from the endothelial cells and produce relaxation of the smooth muscle cells by hyperpolarization. The present findings demonstrate that EETs also regulate aromatase activity in vascular smooth muscle cells. Exposure of cultured rat aortic smooth muscle cells to either 1 microM 14,15-EET or 1 microM 11,12-EET inhibits dibutyryl cAMP-induced aromatase activity by 80-100%. 11,12-Dihydroxyeicosatrienoic acid, the hydration product of 11,12-EET, has no effect on dibutyryl cAMP-induced vascular smooth muscle aromatase activity. In contrast to 14,15-EET, the N-methylsulfanilamide derivative of 14,15-EET (14,15-EET-SA) was neither metabolized nor incorporated into cell lipids, but it retained the ability to inhibit cAMP-induced aromatase activity. Furthermore, the 14,15-EET-SA inhibition of cAMP-induced aromatase activity persisted when the sulfanilamide derivative of 14,15-EET was covalently tethered to silica beads (average diameter, 0.5 microm), which restricted 14,15-EET-SA from entering the cell. These data are consistent with the presence of a receptor for EETs in the plasma membrane and support the hypothesis that the inhibition of aromatase by EETs is initiated by the interaction of EET with the putative plasma membrane receptor.

Prostaglandin (PG) formation by the inducible (type 2) cyclooxygenase (COX-2) and reactive oxygen species (ROS) have been proposed to play important roles in cerebrovascular pathological processes. To explore the relationship between ROS and COX-2 expression, adenovirus (Ad) vectors containing cDNA for human antioxidant enzymes including catalase (AdCAT:), copper/zinc superoxide dismutase (AdCu/ZnSOD), and manganese superoxide dismutase (AdMnSOD) were transferred into murine cerebral microvascular endothelial cells. AdCAT: (100 multiplicity of infection) infection increased the content and enzymatic activity of cellular Cat threefold and decreased the intracellular peroxide level. The expression of COX-2 mRNA and protein in cell lysates was up-regulated, and the amount of PGE(2) formed from exogenous arachidonic acid increased following AdCAT: infection in a dose-dependent manner, paralleling the expression of COX-2 protein. The AdCAT:-induced increase in PGE(2) formation was inhibited by NS-398, a selective inhibitor of COX-2 enzymatic activity. AdCAT: infection did not change the expression of the constitutive (type 1) COX protein. Although AdCu/ZnSOD and AdMnSOD infection increased the expression of superoxide dismutase proteins, COX-2 expression was not induced. An in vitro nuclear transcription assay indicated that overexpression of the Cat gene increases the transcription of the COX-2 gene. Furthermore, the stability of COX-2 mRNA induced by lipopolysaccharide was increased after AdCAT: gene transfer. These results indicate that AdCAT: gene transfer induces the transcriptional activation of the COX-2 gene and increases COX-2 mRNA stability. Therefore, peroxide may have regulatory effect on COX-2 function in the cerebral microcirculation.

We investigated the presence of nitric oxide (NO) synthase in ovarian follicular cells obtained from women undergoing in vitro fertilization procedures. Endothelial NO synthase messenger RNA was demonstrated by polymerase chain reaction amplification of reverse transcribed RNA. NO synthase was localized to granulosa-luteal cells by immunocytochemistry, using a monoclonal antibody. Ovarian follicular cell NO synthase enzyme activity was confirmed by measuring the conversion of L-arginine to citrulline. To investigate the effect of NO on granulosa-luteal cell steroidogenesis, NO synthase inhibitors and NO donors were added to cell cultures. NG-Monomethyl-L-arginine and N-nitro-arginase methyl ester, selective inhibitors of NO synthase, significantly increased estradiol secretion by granulosa-luteal cells. S-Nitroso-L-acetyl penicillamine (S-NAP) and S-nitroso glutathione, NO donors, caused a dose-dependent decrease in both estradiol and progesterone secretion. The decrease by S-NAP was reversed by hemoglobin, which binds free NO. Although S-NAP increased the concentration of cGMP in granulosa-luteal cells, cGMP analogs had no effect on steroidogenesis in cell cultures. S-NAP and native NO in solution decreased cellular and microsomal aromatase activities. We conclude that NO synthase is present in human granulosa-luteal cells and that NO inhibits estradiol secretion independent of cGMP by directly inhibiting aromatase.

Epoxyeicosatrienoic acids (EETs), cytochrome P-450 metabolites of arachidonic acid, have attracted attention because of their effects on stimulus-response coupling in endocrine, renal, and vascular cells. To investigate a possible role for EETs in ovarian physiology, we conducted a series of experiments using human luteinized granulosa cells. Granulosa cell microsomes produce EETs, which are identified by their comigration with known standards using reverse phase high pressure liquid chromatography. EET synthesis by granulosa cells is NADPH dependent and inhibited by ketoconazole, suggesting an enzymatic mechanism of production. Intact granulosa cells synthesize EETs from exogenous arachidonic acid, and EET production is increased by hCG stimulation of the cells. To investigate whether EETs have a role in ovarian steroidogenesis, they were added to cultures of granulosa cells. Varying concentrations of 14,15-EET differentially affected estradiol secretion; 0.001-0.05 microM stimulated estradiol production, whereas 14,15-EET concentrations of 10-50 microM inhibited estradiol production. hCG-stimulated estradiol secretion was also inhibited by 10-50 microM 14,15-EET. In contrast, progesterone secretion was not affected by any concentration of 14,15-EET tested. The cellular concentration of cAMP was not affected by the addition of EETs. These findings suggest that hCG stimulates granulosa cell production of EETs via an NADPH-supported, cytochrome P-450-dependent enzymatic mechanism. EETs may have an important autocrine or paracrine role in regulating ovarian granulosa cell estrogen synthesis.

Objective: To determine the effects of smoking on ovulation induction for assisted reproductive techniques.Design: Matched, retrospective, cohort study.Setting: Outpatient University endocrine/infertility program.Patients: Eighteen smokers and 36 nonsmokers: 2 nonsmokers matched to each smoker for age, weight, and history of ovarian surgery.Main Outcome Measures: During a stimulation cycle, the serum estradiol (E2) level, number of follicles, number of oocytes, number of embryos, and ampules of gonadotropins used were compared in the smoking versus the nonsmoking groups by Wilcoxon's signed rank test for paired data. Follicular fluid (FF), testosterone (T), androstenedione (A), E2, A:E2 ratios, and T:E2 ratios were measured and compared between groups by Mann-Whitney U-tests.Results: Smokers had significantly lower serum E2 levels, fewer follicles, fewer oocytes retrieved, and fewer embryos per cycle than nonsmokers, despite equal amounts of gonadotropin administration. Follicular fluid obtained from mature follicles had a higher A:E2 ratio and a higher T:E2 ratio in smokers compared with nonsmokers.Conclusions: Smoking adversely affects ovulation induction parameters and alters the FF hormonal milieu.