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Department of Entomology (J.W.N., J.E.S., Q.H., B.D.H.), University of California-Davis Cancer Research Center (J.W.N., J.E.S., Q.H., B.D.H.), and Department of Population Health and Reproduction (J.D.V., C.J.C., A.J.C.), University of California, Davis, California 95616
Address all correspondence and requests for reprints to: Dr. Alan Conley, Veterinary Medicine-Population Health & Reproduction, School of Veterinary Medicine, 1131 Tupper Hall, University of California, Davis California 95616. E-mail: ajconley{at}ucdavis.edu.
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Introduction |
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B-mediated signaling (8). Changes in theca interna blood flow (12, 13), COX expression (14), estradiol biosynthesis (15), and plasminogen activator expression (16) are all reported ovulatory events. Therefore, one could anticipate dynamic changes in EET production in the hours leading up to ovulation.
Little is known about EET production and degradation in the ovary during follicular maturation. Cytosolic concentrations of EETs are regulated by their rates of synthesis and degradation (17) along with sequestration and release from phospholipids (18). The synthesis of EETs is primarily catalyzed by enzymes of the CYP2 family, being dominated by 2Cs and 2Js (8). However, the relative abundance of the four regioisomeric EETs produced by each epoxygenase varies considerably (19, 20). In most tissues, the soluble epoxide hydrolase (sEH) dominates epoxy lipid degradation, mediating the formation of the corresponding vicinal- or 1,2-diols; however, metabolism by ß-oxidation and 
-hydroxylation has also been reported in mammals (8). The microsomal epoxide hydrolase has also been reported to hydrolyze epoxy fatty acids, albeit at a greatly reduced rate (21). sEH is primarily localized in the cytosolic compartment (22) and is present in the ovary (10, 23); however, ovarian distribution of this enzyme has not been reported to our knowledge.
To date, in vivo evidence linking EETs with steroidogenesis, ovarian function, or ovulation is scant. The present experiments were designed to document CYP-dependent lipid metabolism in maturing ovarian follicles while testing the hypothesis that CYP-derived lipids are elevated in the preovulatory follicle. These results are related to follicular maturation through correlative analyses with steroid hormone levels. To complement these data, thecal and granulosa tissues were examined for the expression of sEH using polyclonal antibodies directed against recombinant porcine ovarian sEH along with available antibodies for CYP2Cs and CYP2Js. To our knowledge, this is the first study to document the levels and changes in CYP-derived oxylipins in mammalian ovarian follicular fluid and to report the tissue distribution of sEH in the ovarian follicle.
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Materials and Methods |
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Microsome and cytosol preparation
The largest follicles on both ovaries (10–12/animal) were pooled within each animal and frozen immediately on dry ice. Cells and tissues from each pool were homogenized on ice in buffer [0.1 M potassium phosphate (pH 7.4), 20% glycerol, 5 mM ß-mercaptoethanol, and 0.5 mM phenylmethylsulfonylfluoride] at a ratio of approximately 1 ml buffer/0.1 g tissue. Microsomes were enriched by subcellular fractionation as previously described (27, 28). After a brief sonication, cellular debris and mitochondria were removed by centrifugation at 15,000 x g for 10 min. The supernatant was centrifuged at 100,000 x g for 60 min, the supernatant (soluble fraction containing the cytosol) was recovered, the pellet (microsomal fraction) was resuspended in homogenization buffer containing 1 mM (3-[3-cholamidopropyl-dimethylammonio]-1-propanesulfonate), and both were stored frozen. Protein concentrations of microsomal and cytosolic fractions were determined using the bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, IL). The purity of microsomal fractions has been demonstrated previously by immunoblot analysis for microsomal and mitochondrial proteins (28).
RNA isolation, cloning, and expression
Porcine ovarian mRNA was isolated from preovulatory tissues and reverse transcribed into cDNA using SuperScript reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA). Degenerate primers, designed to conserved regions of human, mouse, and rat sEH C-terminal catalytic sites, (5'-GTNTTYATNGGNCAYGAYTGG-3' and 5'-CNACNCCNGGYTCYTGRAA-3') were used to amplify a fragment of porcine sEH employing the following conditions: 35 cycles of 94 C for 45 sec, 55 C for 1 min, and 72 C for 1 min. The pig sEH fragment generated (190 bp) was cloned into pBluescript and sequenced (DNA Sequencing Facility, University of California-Davis). New primers were designed for rapid amplification of cDNA 5'-ends (Invitrogen Life Technologies) and rapid amplification of cDNA 3'-ends (Takara, Madison, WI) to determine the sequence of the entire porcine sEH cDNA. Based on this sequence, PCR primers were then designed to the N and C termini of the porcine sEH to isolate the full-length cDNA (GenBank accession no. AY566232), which was ligated into pCR2.1. The resulting full-length porcine sEH cDNA clone was excised from the pCR2.1 using EcoRI and ligated into pACUW21 baculovirus expression vector (pACUW21-psEH). The recombinant baculovirus was then generated, and porcine sEH protein was overexpressed as previously described (29). Porcine sEH was purified from the cell lysate by affinity chromatography (30). Protein concentration was quantified using the Pierce bicinchoninic acid assay (Pierce Chemical Co.), using fraction V BSA as the calibration standard. Epoxide hydrolase activity was measured using racemic [3H]trans-1,3-diphenylpropene oxide ([3H]tDPPO) (31). The preparation was judged to be pure by SDS-PAGE stained with Coomassie Brilliant Blue. Polyclonal antibodies were raised against recombinant porcine sEH in male New Zealand White rabbits (Animal Resources Service, University of California-Davis). Four weeks after the initial injection (100 µg porcine sEH), the animals were boosted every 2 wk until no additional increase in antibody titer was observed. Titer was analyzed by ELISA and Western immunoblot using the recombinant protein.
Western blot analysis
Microsomal (10 µg) and cytosolic (20 µg) proteins were separated by electrophoresis on 8% SDS-PAGE in electrode buffer (50 mM Tris, 383 mM glycine, 0.1% sodium dodecyl sulfate, and 0.4 mM EDTA). Separated proteins were transferred onto polyvinylidene difluoride membranes (Immobilon P, Millipore Corp., Bedford, MA) and detected with polyclonal antibody raised against recombinant proteins. Antibodies were obtained from various sources: CYP2J courtesy of Dr. Darryl Zeldin (NIEHS, Research Triangle Park, NC), CYP2C8 and CYP2C9 from GenTest (Woburn, MA), and sEH produced as described. Immunoblotting procedures were carried out at room temperature in PBS with 0.1% Tween 20 (Amresco, Solon, OH). After blocking in 10% dried milk, the proteins were visualized by incubating the membranes with donkey antirabbit horseradish peroxidase-linked IgG whole antibody (Amersham Biosciences, Arlington Heights, IL) at a 1:10,000 dilution, washing, then generating a chemiluminescent signal using Luminol reagent (NEN Life Science Products, Boston, MA) detected by autoradiography.
Follicular fluid lipid extraction
Follicular fluid aliquots (50–100 µl) were thawed, spiked with analytical surrogates (
1 pmol/sample), and extracted using modifications of published procedures (32). Briefly, samples were extracted three times with 100 µl ethyl acetate, the organic phases were combined, and solvent was removed under dry nitrogen. The resulting residue was spiked with internal standards, redissolved in 50 µl methanol, and stored at –20 C until analysis.
Tissue oxylipin metabolic capacity
The biochemical activities of a series of polyunsaturated fatty acid oxidases and epoxide hydrolases were determined in microsomal and cytosolic (soluble) fractions of both granulosa and thecal cells. Fatty acid epoxygenase, -hydroxylase, midchain hydroxylase, and prostaglandin synthase activities were obtained simultaneously. Briefly, duplicate aliquots (50 µg) were suspended in 100 µl 0.1 M sodium phosphate buffer (pH 7.4) and were incubated (60 min, 30 C) with arachidonic acid (100 µM) in the presence of an NADPH-regenerating system. Reactions were halted with the addition of 100 µl methanol and centrifuged to remove the protein precipitate. Incubations were then spiked with analytical surrogates, and arachidonate oxidation products were quantified in the isolated supernatant using the lipid chromatography/mass spectrometry/mass spectrometry method described below. sEH activity was determined using tDPPO as the substrate (31). Briefly, 1 µl tDPPO in dimethyl formamide was added to 100 µl tissue homogenate in sodium phosphate buffer (0.1 M; pH 7.4) containing 0.1 mg/ml BSA (final tDPPO concentration, 50 µM) and incubated for 30 min at 30 C. The reactions were quenched by the addition of 60 µl methanol and 200 µl isooctane, followed by vortexing. The diol metabolites were quantified by liquid scintillation counting of the aqueous phase. Assays were performed in triplicate. Controls for background glutathione transferase activity were performed (33) and were subtracted when necessary. Counts per minute were converted to picomoles of product and averaged for triplicate determinations. Specific activities were expressed as picomoles per minute per milligram of protein.
Oxylipins analysis
Oxidized lipids were measured in tissue and assay extracts using negative mode electrospray ionization with a tandem quadrupole mass spectral detector (Quattro Ultima, Micromass, Manchester, UK) operated in multireaction monitoring mode. Follicular fluids were assayed using slight modifications of previously described methods for CYP-dependent metabolite quantification (32, 34). Modifications of HPLC (Table 1
) and mass spectral acquisition parameters (Table 2) allowed detection and quantification of the reported epoxygenase-, lipoxygenase- (i.e. midchain hydroxylation), and cyclooxygenase-dependent metabolites.
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as 6-keto-PGF1.
Statistical analysis
Differences among tissues isolated from follicles at different stages of development were evaluated by one-way ANOVA. A paired t test was used to evaluate differences between thecal and granulosa sEH activity. A one-tailed t test, assuming unequal variance, was used to evaluate differences in oxylipin concentrations and compositions. The potential for type I errors resulting from the multiple ANOVAs performed was controlled using a sequentially rejective method for multiple comparison adjustment. This approach compares calculated, ranked P values with modified levels of significance (* = /n, where n = the number of statistical comparisons and = 0.05); significance was determined when P 
*. Associations between oxylipin concentrations or ratios and follicular fluid estradiol concentrations were calculated after log transformation of the steroid values.
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Follicular fluid oxylipins
The cytochrome P450-derived polyunsaturated fatty acids were readily detected in the follicular fluid samples described above at concentrations up to 150 nM (Fig. 1). Both arachidonate and linoleate metabolites were identified, and the sum of all arachidonate products was within 1 order of magnitude of the total of linoleate products measured. Epoxides, diols, and 20-HETE were detected in each sample (Figs. 1 and 2), consistent with the expression of epoxygenase, epoxide hydrolase, and -hydroxylase enzymes in follicular tissues. Three of the four EET regio-isomers were detected in six of 18 samples. 8(9)-EET was not observed in all groups, and it must be noted that 5(6)-EET recoveries with the applied method are approximately 25% (32). We have since confirmed our earlier supposition that losses are due to 5(6)-
lactone formation, and not 5,6-DHET formation (unpublished observations).
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), the detected 20-HETE was quite stable (20 ± 6%) across all follicle classes. The relative abundance of epoxide regio-isomers were 14(15)>11(12)>5 (6)-[42 (± 21):24 (± 26):3 (± 2)] for the EETs and 12(13)>9 (10)-[67 (± 6):33 (± 6)] for the EpOMEs. The high variability of the EET isomeric ratios was probably due to the low magnitude (<5 nM) of these residues. The relative abundance of the diol regio-isomers generally tracked those observed for epoxides with 14,15>11,12->> 8,9-5,6-[58 (± 7):34 (± 6):5 (± 2):3 (± 3)] for the DHETs, and 12(13)->9(10)-[57 (± 7):43 (± 7)] for the dihydroxy octadeceneoic acids.
In follicular fluid, the concentrations of epoxides (Fig. 1A) were routinely less than those of diols (Fig. 1B) in all groups. When the sum of all epoxides and diols was evaluated, the epoxide:diol ratio was higher (P < 0.05) in samples from the preestrous group (mean, 0.45) compared with all other stages (mean, 0.06–0.07). At estrus, the concentrations of 20-HETE and 14,15-, 11,12-, and 5,6-DHET increased approximately 5-fold, whereas epoxide levels remained constant. 11,12-DHET continued to increase into the post-LH period. The relative abundance of the diols and 20-HETE within each group was then calculated (Fig. 2). These data treatment greatly reduced the variability and allowed visualization of clear trends. At estrus, 14,15-DHET increased from 30% to 50% of the total measured CYP metabolites; this held through the post-LH period and declined at ovulation to a level indistinguishable from the preestrous value. In contrast, the relative abundance of 11,12-DHET was unchanged between preestrus and estrus, but was elevated between estrus and post-LH, and it was significantly different from the preestrous ratios at ovulation. When expressed as a ratio, 14,15-DHET:11,12-DHET was highest in the estrous follicular fluid samples, whereas no change was seen with linoleate-derived diols (Fig. 3A). In addition, although the magnitude was low (1–5 nM), concentrations of 5,6-DHET also showed a phased distribution, with peak concentrations at estrus (Fig. 3B). The relationship between steroid and oxylipin concentrations was complex, and direct correlations were not observed between steroids and oxylipin concentrations. This was in part because steroid levels varied markedly, more than 2 orders of magnitude in the case of estradiol. However, after natural log transformation of the raw estradiol concentrations (Ln E2), correlations were evident between this and certain lipids (Fig. 3C). Specifically, Ln E2 was correlated with the ratio of 14,15-DHET:11,12-DHET (y = 0.24x + 1.16; r2 = 0.80; n = 18; P < 0.01) with all individuals included. In addition, Ln E2 correlated with 5,6-DHET concentrations among samples of the post-LH group, including those from the two ovulating animals (y = 1.02x + 0.64; r2 = 0.86; n = 11; P < 0.01). After eliminating two outlying 5,6-DHET levels, the pre-LH 5,6-DHET concentrations also showed a strong correlation with Ln E2 (y = 0.46x – 0.07; r2 = 0.89; n = 6; P < 0.05), which was distinct from the post-LH regression. No correlations with progesterone were observed.
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) was indicated in both compartments of the follicle by 14,15- and 11,12-DHET synthesis, at similar levels in each and without marked differences between the pre-LH and post-LH specimens. The appearance of DHETs in these incubations is consistent with the presence of sEH in these microsomal preparations as previously reported (37, 38, 39). Synthesis of 20-HETE was not observed, but 15-, 12-, 11-, and 5-HETE were formed in both thecal and granulosa tissues. Activities ranged from 0.10–5.6 pmol/min·mg microsomal protein of the individual samples, with 15-HETE and 5-HETE being the most abundant isolated metabolites. The production of 6-keto-PGF1 was exclusively by the theca and increased 6-fold during the post-LH period (P < 0.01). In contrast, PGE2 synthesis was readily detectable in both thecal and granulosa microsomal incubations. Although granulosa synthesis of PGE2 appeared to increase during the post-LH period, it did not reach statistical significance with the number of samples analyzed (P > 0.05).
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). Although three definitive bands were evident in immunoblots for CYP2J in liver microsomes, only one band was seen in thecal and granulosa microsomes, corresponding in size to the middle band of the hepatic triplet. CYP2J expression was higher in theca than in granulosa and was lost as follicles matured, appearing to decrease in both compartments.
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Using the porcine sEH antibodies for immunoblot analysis indicated that the sEH protein level was lower in tissues from follicles harvested in the post-LH period (Fig. 4). Although sEH activity appeared to decrease in both theca and granulosa in the post-LH period, too few samples were available for a valid statistical analysis by group. However, the sEH activity was consistently higher in the granulosa than in the theca in pairwise comparisons within individual pigs (mean difference, 0.49 ± 0.14 nmol/min·mg; P < 0.01; n = 10) and was correlated positively between the two follicular tissue compartments (r2 = 0.65; P < 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production) was restricted to the theca and increased in the post-LH period, findings consistent with previous reports (44, 45, 46). Therefore, these findings with COX metabolism indicate that thecal and granulosa tissue isolates were pure, and the applied biochemical and analytical evaluations appear valid.
Consistent with previous studies (2, 3, 4, 6), we confirmed the ability of follicular tissue to synthesize epoxy and dihydroxy fatty acids, but also report the presence of -hydroxylation products in follicular fluid. Dynamic changes in follicular fluid CYP-dependent fatty acid metabolites were observed during the preovulatory period examined. Although the concentration of epoxides in follicular fluid was constant, in the low nanomolar concentration range, linoleate and arachidonate diols as well as 20-HETE levels generally increased at estrus and remained elevated through ovulation. Therefore, it would appear that the flux of fatty acids through CYP-dependent metabolism increases approximately 5-fold at estrus. The increase in diol formation was coincident with elevated sEH levels, which also appeared positively correlated with follicular fluid estradiol concentrations. In addition, the expression of sEH in both thecal and granulosa cells along with its post-LH decline are reminiscent of the tissue-specific and temporal regulation of porcine gonadal P450 aromatase (15), the key enzyme metabolizing androgens to estrogens. It is interesting to note that both sEH (47) and the dominant CYP -hydroxylases (48) are induced by androgens, and the declining androgen production in the follicle may be functionally related to the decrease in sEH expression. The failure of sEH-dependent metabolites to decline in follicular fluid along with the drop in sEH expression suggest that these compounds may be selectively retained in this compartment, unlike the steroid hormones.
The functional consequences of the altered oxylipin concentrations are unclear at present; however, the close association with estrus argues for an influence on estradiol synthesis. In fact, in human luteinized granulosa cell cultures, 14(15)-EET was shown to increase estradiol production at 1–5 nM while inhibiting it at 10–50 µM (3). In the present study, concentrations of the sum of 14(15)-EET and DHET in follicular fluid were in the low nanomolar range, suggesting the potential for a stimulatory effect on estradiol synthesis in vivo. A direct stimulatory effect of this or other epoxide isomers on estradiol biosynthesis would predict a positive correlation between the concentration of the causal factor and the responsive agent. In fact, no such relationship was found between any of the observed follicular fluid oxylipins and estradiol. However, the relative abundance of 14,15-DHET and 11,12-DHET showed a strong positive correlation with follicular estradiol concentrations, reaching a maximum of 14,15-DHET over 11,12-DHET when estradiol concentrations peaked and collapsing to a 1:1 ratio at ovulation. These data suggest that the levels of specific epoxygenases may have changed differentially, and that follicular estradiol biosynthesis and arachidonate epoxidation may be mechanistically linked, as previously proposed (3). Although the influence of 11,12-DHET on estradiol synthesis has not been reported, these findings suggest that future studies be conducted to determine whether 11(12)-EET or 11,12-DHET alters estradiol synthesis, as reported for 14(15)-EET.
Other roles for CYP-generated metabolites in the maturing ovary, besides the potential modulation of steroidogenesis, are also likely. The recent finding that EETs, particularly 11(12)-EET and 11,12-DHET, enhance tissue plasminogen activator (tPA) gene expression is particularly relevant to follicular maturation (49). tPA has been implicated in the process of ovarian follicle rupture (16) and may be a critical factor in oocyte maturation (50). In fact, the abundance of tPA mRNA and protein was low in both granulosa and thecal/interstitial cells during early follicular development, but increased thereafter, reaching the highest levels in the preovulatory period (51). These reports parallel our current finding of increasing levels of 11,12-DHET in the follicular fluid of the maturing ovary and implicate 11(12)-EET as a potential regulator of the follicular wall remodeling that occurs before ovulation (51). In addition, the EETs are endothelial-derived hyperpolarizing factors (8) that may act in concert with prostacyclin to regulate thecal blood flow.
As alluded to above, one means of regulating the isomeric ratios of oxylipins would be to alter the relative abundance or activity of different epoxygenases with unique isomeric product ratios. For instance, CYP2C8 activity is consistent with the tissue concentrations and chirality of EETs in human liver, where values of 14(15)-EET exceed those of 11(12)- and 8(9)-EETs (52). In contrast, CYP2C3 in rat kidney cortex may be responsible for the elevation of 11(12)-EET over both 8(9)- and 14(15)-EETs (53). Renal microsomes from spontaneously hypertensive rats also exhibit increased 14(15)- and 11(12)-EET synthesis over normotensive wild-type rats in association with the increase in expression of a CYP2J isoform (54), which, as a family, favor 14,15-epoxidation (55). A similar profile seems to exist in the porcine ovarian follicle.
To test the hypothesis that differences in CYP-dependent oxylipin profiles reflected tissue-specific expression of CYPs with differing regio- and stereo-selectivity for substrate oxidation, microsomal fractions were incubated with AA and an NADPH-regenerating system. Although thecal preparations produced more 14,15- products than 11,12- products in both pre- and post-LH groups, the post-LH, but not pre-LH, granulosa cell preparations showed a 14,15-:11,12-DHET product ratio of 1:1, suggesting that granulosa CYPs may be responsible for the observed changes in follicular fluid arachidonate products.
The failure to find differences in AA metabolite production between pre- and post-LH thecal and granulosa microsomes was not consistent with the changing metabolite profiles in follicular fluid. However, follicular fluid steroid concentrations differed between animals in the preestrous and estrous groups, which were combined for logistic reasons in these analyses. Given the relationship between estradiol concentration and follicular fluid diols, these data can only be interpreted as evidence of the presence of enzyme activity, not the absence of changes with follicle maturation in either tissue compartment, which require more extensive study. Western blots detected a single CYP2J isoform in the maturing ovary, which comigrated with a CYP2J isoform from human granulosa cells (data not shown), and one of three isoforms was expressed in pig liver. Human liver and heart also express three CYP2Js (56). However, rather than peaking at estrus, the ovarian CYP2J protein declined progressively, with the lowest levels observed in the post-LH period. In addition, no change in total epoxygenase activity could be detected in these tissues, suggesting that the observed CYP2J may not be a critical isoform responsible for EET biosynthesis in the maturing ovarian follicle. Attempts to identify other epoxygenases by Western blot (i.e. CYP2C8 and 2C9) were successful in the liver, but not in the ovary, of these pigs. In addition, although 20-HETE was routinely detected in follicular fluids, 20-HETE synthesis was not detected in vitro. Western blots for CYP4A, the dominant AA hydroxylase enzymes, revealed detectable protein in the liver, but not ovary (data not shown).
The failure to detect 20-HETE in microsomal incubations may have been due to a lack of sensitivity for this analyte using current methods (32), where limited tissue mass was available for incubations. It is also possible that the single buffer system that was used for all microsomal P450 activities may not be optimal for some (57). The expression of any of these enzymes may have been below the limits of detection in follicular tissues, but still sufficient to contribute meaningfully to AA metabolism. Therefore, despite shifts in ratios of eicosanoid regio-isomers suggesting changes in the relative levels of specific epoxygenases in the final stages of follicle maturation, the identity of putative CYPs remains unknown. Additional studies are required to detect and define the epoxygenases most relevant to eicosanoid synthesis in follicular tissues. Whatever their identities, however, the enzymes expressed in the preovulatory follicle appear to exhibit a preference for epoxidation at double bonds distal to the carboxyl terminal of both arachidonate and linoleate.
In summary, the role of COX- and LOX-derived eicosanoids in follicular maturation and ovulation is well recognized, but little is known about the role of the epoxygenase arm of the AA cascade in these events. The present study used isolated theca and granulosa from porcine follicles to investigate epoxy eicosanoid synthesis near the time of ovulation. Oxylipin profiling of follicular fluids together with activity and immunoblot analyses identified epoxygenase synthesis and metabolic pathways in preovulatory theca and granulosa. Ratios of regio-isomers changed in concert with follicular fluid estradiol concentrations, suggesting differential regulation of specific epoxygenases during follicular maturation. At the concentrations observed, these agents may play important roles in follicular maturation, including the regulation of estradiol biosynthesis and matrix degradation necessary for the remodeling of the follicular wall before ovulation. These events and their interactions warrant investigation in future research.
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Acknowledgments |
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Footnotes |
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Abbreviations: AA, Arachidonic acid; COX, cyclooxygenase; CYP, cytochrome P450; DHET, dihydroxyeicosatrienoic acid; 14,15-DiHETrE, 14,15-dihydroxyeicostri-(5Z,8Z,11Z)-enoic acid; DHOME, dihydroxy octadeceneoic acid; EET, epoxyeicosatrienoic acid; 14(15)-EpETrE, 14(15)-epoxyeicostri-(5Z,8Z,11Z)-enoic acid; 9(10)-EpOME, 9(10)-epoxyoctadec-(12Z)-enoic acid; 20-HETE, 20-hydroxyeicosatetra-(5Z,8Z,11Z,14Z)-enoic acid; LOX, lipoxygenase; PGE2, prostaglandin E2; sEH, soluble epoxide hydrolase; tDPPO, trans-1,3-diphenylpropene oxide; tPA, tissue plasminogen activator.
Received June 3, 2004.
Accepted for publication August 4, 2004.
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s mediates induction of tissue-type plasminogen activator gene transcription by epoxyeicosatrienoic acids. J Biol Chem 276:15983–15989s mediates induction of tissue-type plasminogen activator gene transcription by epoxyeicosatrienoic acids. J Biol Chem 276:15983–15989
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