©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-1 Enhances the Ability of Cultured Human Umbilical Vein Endothelial Cells to Oxidize Linoleic Acid (*)

(Received for publication, December 20, 1994; and in revised form, May 10, 1995)

Mercedes Camacho , Nuria Godessart , Rosa Antón , Montserrat Garca , Lus Vila (§)

From the Inflammation Mediator Laboratory, Institute of Research of Santa Creu i Sant Pau Hospital, 08025 Barcelona, Spain

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human umbilical vein endothelial cells (HUVEC) were treated with recombinant interleukin (IL)-1, and the metabolism of exogenous linoleic acid was studied. High performance liquid chromatography, gas chromatography-mass spectrometry, and chiral analysis revealed that HUVEC enzymatically convert linoleic acid mainly into 13-(S)hydroxy-9(Z),11(E)-octadecadienoic (13-HODE) and 9-(R)hydroxy-10(E),12(Z)-octadecadienoic acids, which may isomerize toward all-trans compounds. IL-1 increased the formation of all octadecanoids in a time- and dose-dependent manner with similar EC (approximately 1 unit/ml). The apparent K values of linoleic acid were 15.59 ± 8.39 and 152.9 ± 84 µM (p < 0.05) in IL-1-treated cells and controls, respectively, indicating a higher substrate affinity in cells stimulated with IL-1. Ratios of S/R enantiomers for the hydroxyoctadecanoids produced by untreated and IL-1-treated cells were similar to those from isolated cyclooxygenases (COXs), whereas isolated 15-lipoxygenase yielded 13-HODE with a strict S configuration. The formation of octadecanoids was inhibited in a dose-dependent manner by several COX inhibitors in both controls and IL-1-treated cells, COX2 selective inhibitors being more effective on IL-1-treated cells than on controls. COX1 and COX2 protein levels increased less than 2-fold and 8-fold, respectively, after IL-1 treatment. The specificity of COX inhibitors was proven since they did not inhibit 13-HODE formation by human polymorphonuclear leukocytes. Overall, these results indicate that COXs are responsible for the oxidative metabolism of linoleic acid in HUVEC, and IL-1 increases it by inducing the expression of new enzyme, mainly COX2.


INTRODUCTION

Linoleic acid is an essential fatty acid with a cis,cis-pentadiene structure in the molecule which easily reacts with oxygen to yield biologically active compounds. Great amounts of esterified and free hydroperoxyoctadecadienoic acid (HPODE)()and hydroxyoctadecadienoic acid (HODE) have been detected in psoriatic and atherosclerotic lesions(1, 2) , and they are major components of oxidized lipoproteins(3) . Endothelial cells(4, 5, 6) , epidermal cells(7) , platelets(8) , and polymorphonuclear cells (9, 10) convert linoleic acid into 13-HODE and 9-HODE. Aorta(11, 12) , porcine neutrophils(10) , and human and rat epidermis (7) may also form additional metabolites of linoleic acid such as epoxy, trihydroxy, and epoxihydroxy derivatives.

Reports concerning the biological activities of HODEs show that they are active mediators in hemostasis, inflammation, and cancer invasion. Both 9-HODE and 13-HODE induce interleukin (IL)-1 release in macrophages, the latter being less active(13) . 13-HODE modulates the mitogenic response to epidermal growth factor (14, 15) and plays a regulatory role by modulating the activity of several enzymes of the arachidonic acid cascade(16, 17, 18) . One of the most interesting activities reported for 13-HODE is that it modulates the adhesive properties of endothelium by inhibiting the expression of adhesion molecules on the cell surface in basal(4, 5, 19, 20, 21, 22) or stimulated conditions(23, 24) .

A characteristic feature in the immune response is the cooperation between cytokines and lipid mediators in regulating the interaction between immunocompetent cells, tumor cells, and vascular endothelium. IL-1 is a pleiotropic cytokine that plays a major role in the inflammatory response. It orients endothelial cell function in a proinflammatory and prothrombotic sense (25, 26, 27, 28, 29) and induces the expression of adhesion molecules and secondary cytokines that, in turn, induce acute and chronic inflammatory changes(26, 30, 31) . IL-1 also stimulates the release of prostaglandins and promotes the expression of cyclooxygenase (COX)(32, 33, 34, 35) . Two COX isoenzymes COX1 and COX2 encoded by different genes have been characterized. COX1 is expressed in a constitutive manner, and COX2 is the inducible isoenzyme by mitogens, which is overexpressed in many inflammatory processes(36, 37) . In general, enzymatic oxidation of linoleic acid may involve both COX and 15-lipoxygenase activities(9, 38) . In fact, there are conflicting reports concerning the COX and/or 15-lipoxygenase origin of 13-HODE from linoleic acid, and 15-HETE from arachidonic acid, in endothelial cells(4, 5, 6, 21, 22, 39, 40, 41, 42, 43) .

The fact that IL-1 is a crucial mediator of the interactions among endothelial cells, immunocompetent cells, and tumor cells and that part of these interactions may be mediated by octadecanoids prompted us to investigate the effect of this cytokine on the metabolism of linoleic acid in endothelial cells.


EXPERIMENTAL PROCEDURES

Materials

Culture plates of six 35-mm wells were purchased from Costar Europe, Badhoevedorp, NL. Medium 199, fetal bovine serum, glutamine, pyruvate, and penicillin/streptomycin solutions were purchased from Bio-Whittaker, Walkersville, MD. Heparin, soybean lipoxygenase, indomethacin, and nordihydroguaiaretic acid (NDGA) were provided by Sigma, St. Louis, MO. Anti-human von Willebrand factor antibodies were from Dakopatts, Copenhagen, Denmark. Endothelial cell growth supplement, collagenase, human recombinant IL-1 (50,000 units/µg, purity >98%), and BM Chemiluminescence Western blotting kit were obtained from Boehringer Mannheim S.A. Barcelona, Spain. [C]Linoleic acid (50-53 mCi/mmol) was obtained from DuPont NEN. COX1 isolated from ram seminal vesicles, COX2 purified from sheep placenta, COX2 polyclonal antibody, ibuprofen, and authentic standards of unlabeled 9-HODE and 13-HODE were obtained from Cayman Chemical Co, Ann Arbor, MI. COX1 polyclonal antibody and rabbit reticulocyte 15-lipoxygenase were obtained from Oxford Biomedical Research, Oxford, MI. 1-Methyl-3-nitro-1-nitrosoguanidine was purchased from Fluka Chemie, Buchs, Switzerland. BSTFA was purchased from Merck, Darmstadt, Germany. Hydrated platinum(IV) oxide was obtained from ICN Biochemicals, Costa Mesa, CA. Hydrogen gas was obtained from Abelló Oxgeno-Linde S.A., Barcelona. The scintillation mixture was Ready flow III, Beckman, San Ramón, CA. All HPLC solvents were supplied by Scharlau S.A., Barcelona. Polyvinylidene difluoride transference membrane Immobilon-P was supplied by Millipore Ibérica, Barcelona. 6-MNA and NS-398 were synthesized by Laboratorios Almirall S.A., Barcelona. Zileuton was kindly supplied by Laboratorios Esteve S.A., Barcelona.

Endothelial Cell Cultures

Endothelial cells were isolated from human umbilical veins by collagenase digestion as originally described by Jaffe et al.(44) . HUVEC were cultured in plastic tissue culture flasks coated with gelatin and grown to confluence in medium 199 containing 20% fetal bovine serum supplemented with 2 mML-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin, 100 IU/ml streptomycin, 10 USP/ml heparin, and 30 µg/ml endothelial cell growth factor. Cells in confluent state were seeded into six-well plates and maintained without heparin and endothelial cell growth factor for 72 h prior to incubations with linoleic acid. Cells were used at the first passage, and they were routinely characterized by indirect immunofluorescence with rabbit anti-human von Willebrand factor antibodies and by morphological and biochemical criteria as described(44, 45) .

Preparation of Polymorphonuclear Leukocyte (PMN) Suspensions

PMN suspensions were obtained as described before(46) . Peripheral venous blood was obtained from healthy donors without medication for 10 days before extraction. Blood was incubated with 200 µM aspirin for 15 min before the washing procedure was carried out to minimize the contribution of COX from platelets present in the preparation on HODES formation.

Incubation of HUVEC with [C]Linoleic Acid

Semiconfluent cells in six-well plates were incubated at 37 °C in the presence of 0.5 ml of medium 199 containing 10 mM HEPES, and the desired concentration of [C]linoleic acid was added in 5 µl of ethanol. At the indicated periods of time the reactions were stopped by adding 1 N HCl to yield pH 3 followed by a volume of cold methanol. Samples were kept at -80 °C until analysis. The nonenzymatic formation of HODEs was estimated by incubating [C]linoleic acid with boiled cells.

Straight Phase (SP)-HPLC Analysis

Octadecanoids from samples were extracted three times with 1 ml of diethyl ether:n-hexane (1:1) containing 50 µM butylated hydroxytoluene. Extracts were evaporated under a N stream until dryness. Residues were then redissolved in 250 µl of eluent and injected into a normal phase column (Ultrasphere-Si 4 250 mm, 5 µm, Beckman). Recoveries were 88.5 ± 2.5 and 84.62 ± 7.47% for 13-HODE and 9-HODE, respectively. Isocratic elution was performed with diethyl ether:n-hexane:acetic acid (30:70:0.1) at a flow rate of 1 ml/min.

The column was coupled on line with a radioactivity detector (Beckman 171) endowed with a liquid scintillation cell. Eluents were mixed with scintillation mixture pumped at 3 ml/min. UV absorbance was monitored by means of a diode array detector (Beckman 168). Data were processed with System Gold software (Beckman) in a PC coupled with the detectors.

Chiral Analysis

HODEs from samples of HUVEC, isolated COXs, or isolated 15-lipoxygenase were collected after SP-HPLC and dried under a N stream. Residues were redissolved, and methyl esters were obtained as described later. Methyl esters were purified by SP-HPLC using a mixture of diethyl ether:n-hexane:acetic acid (15:85:0.1) as eluent and dried under a N stream. Residues were redissolved in the chromatography eluent (hexane:isopropyl alcohol (100:0.75)), and 20 µl was then injected into an N-3,5-dinitrobenzoyl-(R)-phenyl-glycine chiral column (Sumichiral OA-2000, 4 250 mm, 5 µm, Sumika Chemical Analysis Service, Osaka, Japan). Elution was performed at a flow rate of 1 ml/min, and both 234 nm absorbance and radioactive counts were simultaneously recorded.

Gas Chromatography-Mass Spectrometry (GC-MS)

All mass spectra were recorded on an Incos XL mass spectrometer (Finnigan MAT, San José, CA) coupled directly to a Varian 3400 gas chromatograph. The electron impact (EI) mass spectra were obtained using the standard EI box at 180 °C and an electron energy of 70 eV. GC was performed on a DB-5 fused silica capillary column (30 m length, 0.25 mm inner diameter, 0.25 µm film thickness, J& Scientific, Folsom, CA) with helium as the carrier gas. The GC temperature was programmed from 80 to 200 °C at a rate of 20 °C/min followed by a further increase from 200 to 275 °C at a rate of 5 °C/min. After each run the column was cleaned by leaving the column at 275 °C for 5 min. Samples were injected in the splitless mode.

Derivatization

Peaks collected from SP-HPLC were evaporated under a N stream. Methyl esters were obtained by adding 20 µl of methanol and 100 µl of a freshly prepared solution of diazomethane in diethyl ether and allowed to react for 5 min at room temperature in a N atmosphere and darkness. The reagents were removed under a gentle N stream. Trimethylsilyl ethers were then obtained by adding 25 µl of pyridine plus 25 µl of BSTFA. Afterwards, the samples were incubated at 80 °C for 45 min. After cooling at room temperature the reagents were removed under a N stream, and the samples were redissolved in acetone to inject in the gas chromatograph.

When required, hydrogenation of methyl esters was performed before trimethylsilyl ether derivatization. Dry residue from methyl ester derivatization was redissolved in 500 µl of methanol. Then 1-3 mg of platinum oxide was added, and H was bubbled through the sample solutions for 20 min. Platinum oxide was removed by centrifugation at 15,000 g for 5 min, and supernatants were recovered. Solvent was removed under a N stream, and trimethylsilyl derivatives were obtained as described above.

Incubations with IL-1

HUVEC were incubated for the indicated period of time with 2 ml of medium 199 containing 4% v/v fetal bovine serum and 0-20 units/ml human recombinant IL-1. The medium was removed, and the cells were washed with 2 ml of phosphate-buffered saline. Afterwards the cells were incubated with [C]linoleic acid for 10 min as described above.

Effect of Lipoxygenase and COX Inhibitors

HUVEC were preincubated in the presence or in the absence of 10 units/ml IL-1 for 24 h. Medium was removed, and cells were then washed and treated with 0.5 ml of medium 199 containing the several concentrations of drugs dissolved in ethanol (final ethanol concentration 0.1% v/v) for 5 min. Afterwards cells were incubated with 25 µM [C]linoleic acid for 10 min as described above. The effect of the drugs on HODE formation by PMN was studied by incubating 10 PMN in 0.5 ml of Hanks' buffer containing 2 mM CaCl and 1.5 mM MgCl with the indicated concentrations of drugs for 5 min, after which 5 µM A23187 plus 25 µM [C]linoleic acid were added and incubated for a further 5 min. The reactions were stopped and products analyzed as described for HUVEC incubations.

Effect of the Inhibition of Protein Synthesis on the Action of IL-1 on Linoleic Acid Metabolism

The cells were incubated for 8 h with 10 units/ml IL-1 in the presence or in the absence of 0.75 µg/ml cycloheximide or 0.25 µM actinomycin D. HUVEC were then incubated with 25 µM [C]linoleic acid for 10 min as described above.

Incubation of HUVEC with Labeled HPODEs and HODEs

13-[C]HPODE was obtained by incubating [C]linoleic acid with soybean lipoxygenase. 13-[C]HODE was purified after reduction of 13-[C]HPODE with NaBH by collecting the corresponding SP-HPLC peak. 9-[C]HPODE was obtained by incubating [C]linoleic acid with tomato fruit homogenate according to the method described by Matthew et al.(47) . 9-[C]HPODE was then reduced with NaBH, and 9-[C]HODE was purified by SP-HPLC.

Some plaques of cells untreated and treated with IL-1 for 24 h were heated at 100 °C for 10 min. Intact and boiled cells were incubated for 15 min at 37 °C in 0.5 ml of medium 199 containing 2.5 µMC-labeled 13-HPODE, 9-HPODE, 13-HODE, or 9-HODE. Afterwards, 1 volume of cold methanol was added, and the medium was recovered for H(P)ODEs transformation analysis as described above.

Incubations with isolated COX1, COX2, and 15-Lipoxygenase

For the incubation with isolated COX the reaction mixture was composed of 50 units of COX1 or COX2, 2 mM phenol, 2 mM CaCl, 10 mM HEPES, 25 µMC-linoleic acid, in 1 ml of medium 199. For the incubations with isolated 15-lipoxygenase the reaction mixture was composed of 50 units of rabbit reticulocyte 15-lipoxygenase and 25 µMC-linoleic acid in 1 ml of 0.2 mM borate buffer, pH 9.2. Both COX and 15-lipoxygenase reaction mixtures were incubated at 37 °C for 15 min, and the reactions were stopped by adding 1N HCl to yield pH 3 followed by 1 ml of cold methanol. The products were extracted as indicated above and then subjected to chiral analysis. Products from the incubations with isolated 15-lipoxygenase were extracted, HPODEs were reduced with NaBH, and then HODEs were subjected to chiral analysis.

Western and Dot Blot Analysis

Lysates of control and HUVEC treated with IL-1 for 24 h were prepared by treating washed cells with lysis buffer consisting of 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 mM benzamidine, and 0.1% Triton X-100. Lysates were stirred vigorously and centrifuged at 12,000 g for 10 min to sediment particulate material. Protein concentrations of the supernatants were determined using the method of Bradford(67) . For Western blotting total protein equivalents of each sample were submitted to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (minigels, Miniprotean, Bio-Rad) using the Laemmli buffer system (68) and transferred to polyvinylidene difluoride membranes. After blocking nonspecific binding of antibody, membranes were incubated with anti-COX1 (dilution 1:100) or anti-COX2 (dilution 1:1,000). Immunoreaction and detection were performed by using BM Chemiluminescence Western blotting Kit (rabbit) following the manufacturer's instructions. Dot blotting was performed by placing 2-fold serial dilutions of equivalent amounts of protein from cell lysates and COX1 and COX2 standards directly into the transfer membranes. Spots were developed as described for Western blotting.


RESULTS

Characterization of Linoleic Acid Metabolites

SP-HPLC analysis of the samples from incubations of HUVEC with [C]linoleic acid revealed the presence of four peaks S-I, S-II, S-III, and S-IV as shown in Fig. 1. Peaks S-I and S-III coeluted with the authentic standards of 13-HODE and 9-HODE, respectively. The presence of the two minor peaks (S-II and S-IV) varied, and occasionally they were undetectable in samples from control cells. The UV spectra of the peaks (Fig. 1, inset) shows a = 234-235 for S-I and S-III, which reveals the presence of a conjugated cis,trans-hydroxydiene system in these peaks (48) . The = 231-232, indicates the presence of a conjugated trans,trans-hydroxydiene system in peaks S-II and S-IV(48) .


Figure 1: Representative SP-HPLC from a sample of HUVEC incubated with 25 µM [C]linoleic acid for 10 min. S-I and S-III had the same retention time that authentic standards of 13-HODE (Z,E) and 9-HODE (E,Z), respectively. Inset, UV spectra of peaks S-I, S-II, S-III, and S-IV. S-I and S-III had a = 234-235, which reveals the presence of a conjugated cis,trans-hydroxydiene system in these peaks(48) . Peaks S-II and S-IV had a = 231-232, which indicates the presence of a conjugated trans,trans-hydroxydiene system.



To characterize further the linoleic acid-derived compounds, the material eluting as S-I, S-II, S-III, and S-IV was collected separately from SP-HPLC and derivatized. Each purified compound was derivatized as the trimethylsilyl ether methyl ester and subsequently analyzed by GC-MS. The EI mass spectra of all peaks are shown on the left side of Fig. 2. Spectra of S-I and S-II peaks were identical to the spectrum of the authentic 13-HODE. A molecular ion was detected at m/z 382 (M). Additional informative ions were present at m/z 311 (M-71; loss of (CH)CH) and 225 (M-157; loss of (CH)COOCH). Similar ions occurred in the S-III and S-IV spectra. Although the relative intensities were different, the spectra of these compounds were identical to the spectrum of the authentic 9-HODE. To determine the location of the hydroxyl group, methyl esters were catalytically hydrogenated before trimethylsilyl ether derivatization. The EI mass spectra of the saturated peaks are shown on the right side of Fig. 2. The EI mass spectra of saturated S-I and S-II contained major fragment ions at m/z 315 (M-71; loss of (CH)CH) and 173 (M-213; loss of (CH)COOCH). The fragment ion at m/z 315 suggests the location of the hydroxyl group at C-13 and cleavage of the bonds between C-13 and C-14. The ion at m/z 173 also indicates the presence of a hydroxyl group at C-13 and represents the breaking of the carbon bond between C-12 and C-13. The EI mass spectra of saturated S-III and S-IV contain major fragment ions at m/z 259 (M-127; loss of (CH)CH) and 229 (M-157; loss of (CH)COOCH). The major fragment ion at m/z 259 is consistent with the location of the hydroxyl group at C-9 and cleavage of the bond between C-9 and C-10. The ion at m/z 229 also indicates that the location of the hydroxyl group is C-9 and that fragmentation occurred between C-8 and C-9.


Figure 2: EI mass spectra from GC-MS analysis of peaks of Fig. 1. Each purified compound was derivatized as the trimethylsilyl ether methyl ester and subsequently analyzed by GC-MS (left panels). EI mass spectra from GC-MS analysis of saturated compounds are shown in the panels on the right. Each purified compound was derivatized as methyl ester and subsequently catalytically hydrogenated before trimethylsilyl derivatization. The schemes of fragmentation show the origin of the most characteristic fragments.



The GC-MS analysis together with the UV spectra indicate that S-I corresponds to 13-hydroxy-9(Z),11(E)-octadecadienoic acid (13-HODE(Z,E)), S-II corresponds to 13-hydroxy-9(E),11(E)-octadecadienoic acid (13-HODE(E,E)), S-III corresponds to 9-hydroxy-10(E),12(Z)-octadecadienoic acid (9-HODE(E,Z)), and peak IV corresponds to 9-hydroxy-10(E),12(E)-octadecadienoic acid (9-HODE(E,E)).

To elucidate the origin of all-trans-isomers, experiments were performed incubating intact and boiled cells with 2.5 µM 13-[C]HPODE(Z,E), 9-[C]HPODE(E,Z), 13-[C]HODE(Z,E), or 9-[C]HODE(E,Z) for 15 min, and then the samples were extracted and analyzed as described under ``Experimental Procedures.'' After 15 min of incubation, hydroperoxides were found almost totally transformed, mainly into the corresponding hydroxide, and small amounts of the all-trans-hydroxy isomers were found, even with boiled cells. The chromatograms obtained from incubations of cells with hydroperoxides were essentially identical to those obtained from incubations with the hydroxides. Fig. 3shows an example of chromatograms obtained from incubations of labeled HODEs with boiled cells. Small amounts of 13-[C]HODE(E,E) and 9-[C]HODE(E,E) were observed when cells (boiled or not) were incubated with 13-[C]HODE(Z,E) or 9-[C]HODE(E,Z). Very small amounts of 9-[C]HODE(E,Z) from 13-[C]HODE(Z,E) and 13-[C] HODE(Z,E) from 9-[C]HODE(E,Z) were also observed; however, these transformations represent less than 1% of the total amount of radioactivity added to the cells. None of these results was attributable to the impurities of the initial compounds since their purity was analyzed prior to the incubations. No significant differences were observed between untreated and IL-1-treated cells as regards HODEs isomerization. These results indicate that these isomerizations were nonenzymatic.


Figure 3: Chromatograms showing the all-trans-isomer formation after incubation of boiled cells with: A, 13-[C]HODE(Z,E); B, 9-[C]HODE(E,Z) for 15 min and later extracted and analyzed as described under ``Experimental Procedures.'' The chromatograms obtained incubating 13-[C]HPODE(Z,E) and 9-[C]HPODE(E,Z) in the same conditions were essentially identical.



Effect of IL-1 on Linoleic Acid Metabolism

The increase in the production of HODEs was dependent on concentration and the time of exposure to IL-1 (Fig. 4). The maximum metabolic activity was detected between 6 and 24 h of exposure to the cytokine. The synthesis of HODEs reached a plateau at concentrations between 5 and 20 units/ml IL-1. The EC values of IL-1 for all HODEs were quite similar (between 0.87 and 1.1 units/ml).


Figure 4: A, production of HODEs by HUVEC as a function of time of treatment with IL-1. Cells were treated with IL-1 for the indicated periods of time. Then the cells were washed and incubated with 25 µM [C]linoleic acid for 10 min as described under ``Experimental Procedures.'' B, effect of the concentration of IL-1 on HODE production by HUVEC. Cells were treated with the indicated concentrations of IL-1 for 24 h. Then the cells were washed and incubated with 25 µM [C]linoleic acid for 10 min, and the products were analyzed. Points are the mean ± S.D., n = 3.



The apparent kinetic constants of [C]linoleic acid transformed by HUVEC were determined by incubating cells with increasing concentrations of [C]linoleic acid for 10 min. The kinetic constants of cells treated with IL-1 were statistically different from those of controls (p < 0.05): V, 3,228 ± 1,178 and 2,152 ± 867 pmol/10cell/10 min; and K, 15.59 ± 8.39 and 152.9 ± 84 µM for IL-1-treated and untreated cells, respectively (Fig. 5).


Figure 5: Kinetics of [C]linoleic acid transformation by HUVEC as a function of substrate concentration in untreated and IL-1-treated cells. Inset, Lineweaver-Burk plot. IL-1 treatment was performed by incubating cells with 10 units/ml IL-1 for 24 h before the addition of the indicated concentrations of [C]linoleic acid. Cells were further incubated for 10 min and the products analyzed. Points are the mean ± S.D. of the sum of all HODEs formed, n = 4. V: 3,228 ± 1,178 and 2,152 ± 867 pmol/10cell/10 min. K: 15.59 ± 8.39 and 152.9 ± 84 µM for IL-1-treated and untreated cells, respectively. Significant differences were observed between controls and IL-1-treated cells in terms of apparent V and K (p < 0.05). Statistical significance was evaluated by a paired t test.



Fig. 6shows the effect of different inhibitors on the enzymatic formation of 13-HODE(Z,E) and 9-HODE(E,Z) in controls and IL-1-treated cells. Indomethacin, ibuprofen, NS-398, 6-MNA, and NDGA caused a dose-dependent inhibition of HODE synthesis. All inhibitors were more potent in inhibiting 9-HODE(E,Z) than 13-HODE(Z,E) in terms of IC, particularly in control cells (see Table 1). Only indomethacin caused total inhibition of HODE formation at the concentrations tested. Data in Table 1show that ibuprofen, NDGA, NS-398, and 6-MNA inhibited the transformation of linoleic acid more efficiently in IL-1-treated cells than in controls. This fact was more notable for the formation of 13-HODE(Z,E) than for 9-HODE(E,Z), being particularly dramatic for NS-398 and 6-MNA (IC values were 2 orders of magnitude lower in IL-1-treated cells than in controls). For 9-HODE(E,Z) formation, this selectivity for IL-1-treated cells was only significant in the case of NS-398 but to a lesser extent than for 13-HODE(Z,E) (IC values were 1 order of magnitude lower in IL-1-treated cells than in controls). When 100 µM NS-398 or 100 µM 6-MNA was incubated together with 2 µM indomethacin, formation of both 13-HODE(Z,E) and 9-HODE(E,Z) was inhibited completely (>99.7%) both in controls and IL-1-treated cells.


Figure 6: Effect of the concentration of several inhibitors on the production of HODEs by IL-1-treated and untreated HUVEC. Cells were preincubated at 37 °C with the inhibitor for 5 min before the addition of 25 µM [C]linoleic acid. Afterwards, cells were incubated for another 10 min. Points are the mean of three (indomethacin) or two (other inhibitors) separate experiments. , indomethacin; , NDGA; , ibuprofen; ▾, NS-398; and , 6-MNA. IC values are in Table 1.





To exclude any possible effect of COX inhibitors on 15-lipoxygenase, suspensions of human PMN were incubated with the maximum concentration of COX inhibitors tested on HUVEC. PMN yielded almost exclusively 13-HODE(Z,E), and 9-HODE(E,Z) was not detected. 50 µM indomethacin, 500 µM ibuprofen, 100 µM NS-398, and 1,000 µM 6-MNA did not inhibit 13-HODE(Z,E) formation by PMN at all. To exclude any contribution of 5-lipoxygenase to 13-HODE(Z,E) formation, 20 µM zileuton was also tested on PMN; it suppressed leukotriene generation totally (data not shown) but without effect on 13-HODE(Z,E) production. The same concentrations of NDGA tested on HUVEC were also tested on PMN. NDGA showed a significantly higher efficiency in inhibiting 13-HODE(Z,E) formation by PMN that by HUVEC in terms of IC (Table 1). Furthermore, 25 µM NDGA suppressed 13-HODE(Z,E) formation by PMN totally, whereas 50 µM NDGA produced only partial inhibition of 13-HODE(Z,E) formation by HUVEC.

Fig. 7shows representative chromatograms from samples analyzed by chiral phase HPLC. The analysis of the chirality of the HODEs showed that 9(R)-HODE(E,Z) and 13(S)-HODE(Z,E) were the main isomers formed by HUVEC. To characterize better the enantiomer composition of HODEs formed by HUVEC they were compared with those formed by isolated COX1, COX2, and 15-lipoxygenase (Table 2). The ratios between the enantiomers formed by the cells and by isolated COX1 or COX2 were similar. As expected, all-trans-HODEs were racemic mixtures. 15-Lipoxygenase yielded, after reduction of HPODEs, almost exclusively 13(S)-HODE(Z,E). 13(R)HODE(Z,E) was not detected, and small amounts of racemic 9-HODE(E,Z) were also found (<1% of the amount of 13-HODE(Z,E)), indicating its nonenzymatic origin.


Figure 7: Representative chiral phase chromatograms of the methyl esters of standards and samples from untreated and IL-1-treated cells (10 units/ml for 24 h) incubated with 25 µM [C]linoleic acid for 10 min. Ratios of enantiomers are shown in Table 2.





When HUVEC were incubated with 0.25 µM actinomycin D or 0.75 µg/ml cycloheximide in addition to IL-1, the protein synthesis inhibitors completely blocked the effect of IL-1 on [C]linoleic acid metabolism. No effect of actinomycin D or cycloheximide on the production of HODEs by untreated cells was observed. At the concentrations used, these inhibitors had no effect on cell viability as determined by trypan blue dye exclusion.

To correlate the increase in the ability of HUVEC to form HODEs with the induction of COX expression, three separate experiments of Western and dot blot analysis of COX1 and COX2 were performed. No cross-reactivity was observed between COX1 antiserum and COX2 protein at the concentrations used in this assay, nor did COX2 antiserum recognize COX1 protein (Fig. 8). Results showed that in our experimental conditions both COX1 and COX2 were present in control cells in approximately equivalent amounts.


Figure 8: Representative Western and dot blotting analysis of COX1 and COX2 in controls and cells treated overnight with 10 units/ml IL-1. A, Western blot of COX1. COX1 antiserum did not cross-react with COX2 at the concentrations of protein used in this assay. B, Western blot of COX2. COX2 antiserum did not recognize COX1. C, dot blot of COX1 from 2-fold serial dilutions of 73.5 ng of isolated COX1 and 490 ng of protein from untreated(-) and IL-1-treated (+) cells. D, dot blot of COX2 from 2-fold serial dilutions of 665 ng of isolated COX2 and 1,960 ng of protein from untreated(-) or IL-1-treated (+) cells. In three separate experiments, in IL-1-treated cells the amount of COX1 protein was less than 2-fold higher than in controls, whereas the amount of COX2 protein was approximately 8-fold higher.




DISCUSSION

HUVEC formed 13(S)-HODE(Z,E) and 9(R)-HODE(E,Z) as the mayor enzymatic products from linoleic acid. Minor nonenzymatic isomerizations toward 13- and 9-hydroxy all-trans-isomers from both 13-HPODE(Z,E) and 9-HPODE(E,Z) and/or the corresponding hydroxides may also occur. Isomerization of very small amounts of 9-HODE(E,Z) toward 13-HODE(Z,E) and 13-HODE(Z,E) toward 9-HODE(E,Z) were also observed. These nonenzymatic isomerizations (mainly all-trans, which are thermodynamically favorable) probably occurred due to rearrangements of radical species formed during the incubation and/or further manipulation.

Exposure of HUVEC to IL-1 increases their ability to transform linoleic acid into HODEs enzymatically in a time- and dose-dependent fashion. Blocking the stimulating action of IL-1 on the biosynthesis of HODEs by cycloheximide and actinomycin D indicates that de novo synthesis of protein is required for this effect to occur. Biosynthesis of 13-HODE may be catalyzed by both COX and 15-lipoxygenase(9, 38, 49) , whereas significant amounts of 9-HODE are produced by COX(38, 49) . The absence of significant differences in the stimulating effect of IL-1 in the production of 13- and 9-HODEs in terms of EC suggests the involvement of a common enzymatic pathway in the effect of IL-1 on the production of the two position isomers.

The exact enzymatic pathway for the biosynthesis of HODEs in HUVEC, specially 13-HODE, remains controversial. The location of the hydroxyl group in the molecule of 15-HETE or 13-HODE led other authors to infer the presence of 15-lipoxygenase in HUVEC, but as mentioned before 15-HETE and 13-HODE can also be formed by COX(38, 49, 50) . The 15-lipoxygenase origin of 13-HODE and 15-HETE in nonstimulated HUVEC has been claimed by several authors, although no direct evidence of this has been reported(4, 5, 21, 22, 39, 41, 43) . Buchanan et al.(5) first reported the production by HUVEC of a lipoxygenase-derived product, lipoxygenase X, which inhibited adhesion of platelets to endothelial surface; lipoxygenase X was later identified as 13-HODE(4) . These authors studied the content of free 13-HODE in HUVEC, showing that it is dependent on the turnover of linoleic acid in the triacylglycerol pool(21) . The amount of free 13-HODE present in cells after stimulation with thrombin or calcium ionophore was lower than in controls(4) . Thrombin and calcium ionophore cause the release of free arachidonic acid; interestingly, it was found that when HUVEC were incubated with 200 µM labeled arachidonic acid lipoxygenase X (13-HODE) was formed(5) . The fact that the cytosolic fraction was able to produce 13-HODE, together with the inhibition by ETYA, was supporting evidence for the conclusion that 15-lipoxygenase is the origin of 13-HODE in HUVEC(4, 5) . COX2 protein and COX activity have also been observed in the cytosol(51, 52) .

The fact that lipoxygenase inhibitors such as ETYA and/or NDGA inhibit the production of 15-HETE and 13-HODE(4, 5, 39, 41, 42, 43) and the oxidative action of HUVEC on lipoproteins (53) has been used as the supporting evidence for the 15-lipoxygenase involvement in such events in HUVEC. ETYA actually is a competitive arachidonic acid analogue that inhibits many dioxygenases including COXs(54, 55) . In the present work, we found that 50 µM ETYA completely inhibited HODEs formation by HUVEC (not shown). Furthermore, we found that NDGA inhibited 13-HODE(Z,E) formation by HUVEC in a concentration-dependent manner. However, NDGA showed higher efficiency in inhibiting 9-HODE(E,Z) than 13-HODE(Z,E), especially in resting cells. NDGA is a potent inhibitor of lipoxygenases but also inhibits COX (40) and cytochrome P450(56) . Since among PMN at least eosinophils express 15-lipoxygenase(57, 58, 59) , we used PMN suspensions to examine the specificity of the inhibitors. NDGA was 40-fold and 7-fold less potent, in terms of IC, in inhibiting 13-HODE formation by untreated and IL-1-treated HUVEC, respectively, when compared with its effect on PMN. Based on these results we think that caution should be exercised in defining the role of 15-lipoxygenase in the metabolic pathways based only on inhibitions by NDGA or ETYA.

The limited effect of aspirin in the production of 15-HETE has also been considered as supportive evidence of the 15-lipoxygenase origin of this eicosanoid in endothelial cells(41) . We reported that aspirin, even at 1 mM concentration, was unable to suppress totally 15-HETE formation in HUVEC and human dermal fibroblasts treated with IL-1(40, 52) . This phenomenon can be explained not only by the 15-lipoxygenase origin of 15-HETE but also by the COX2 origin, since 15-HETE is the main eicosanoid produced by COX2 treated with aspirin(60) . However, 100 µM aspirin totally inhibited all octadecanoids, even 13-HODE(Z,E), in both controls and IL-1-treated cells (not shown).

All COX inhibitors tested in the present study inhibited the synthesis of both products 13-HODE(Z,E) and 9-HODE(E,Z) in untreated as well as in IL-1-treated cells in a dose-dependent fashion. The greater ability to inhibit 9-HODE(E,Z) than 13-HODE(Z,E) formation, especially in untreated cells, was a common feature of all inhibitors tested (Table 1). This is consistent with the lower inhibitory strength of indomethacin on 15-HETE than 11-HETE or prostaglandin formation from arachidonic acid by HUVEC and dermal fibroblasts(40, 52) . Nevertheless, only indomethacin was able to suppress HODEs formation totally at the concentrations used. Consistent with reports that 100 µM indomethacin did not inhibit 15-lipoxygenase activity in cells transfected with reticulocyte 15-lipoxygenase(61) , we found that 50 µM indomethacin did not inhibit 13-HODE(Z,E) formation by PMN.

Furthermore, lipoxygenases render compounds with a strict S stereospecificity(62) , whereas COX forms R- and S-isomer mixtures(49) . Results from chiral analysis indicate that the main products formed by HUVEC, irrespective of their treatment or not with IL-1, were 13(S)-HODE and 9(R)-HODE. The ratio of enantiomers in controls or IL-1-treated HUVEC is quite similar to that obtained when HODEs were synthesized by isolated COX1 or COX2 independently and is different from that obtained in the incubations of linoleic acid with reticulocyte 15-lipoxygenase, which yielded only 13(S)-HODE(Z,E). These results agree with those obtained by Hamberg and Samuelsson(49) , Baer et al.(63) , and Reinaud et al.(9) with pure COX, bovine aortic endothelial cells, and human leukocytes, respectively.

Recent studies have shown that 15-lipoxygenase expressed in macrophages is regulated by inflammatory cytokines, and only IL-4 induces specifically 15-lipoxygenase mRNA and enzyme activity in cultured human monocytes(64) . We were unable to find 15-lipoxygenase mRNA in untreated and in IL-4- or IL-1-treated HUVEC, whereas IL-4, but not IL-1, effectively induced 15-lipoxygenase mRNA on cultured monocytes (40) . Overall, these results indicate that formation of both 13-HODE(Z,E) and 9-HODE(E,Z) was mediated by COX rather than by 15-lipoxygenase in resting and IL-1-treated HUVEC.

According to data reported by Jones et al.(35) , both COX1 and COX2 were detected in nonstimulated HUVEC. COX1 was induced slightly by IL-1 (less than 2-fold), whereas COX2 increased 8-fold as a result of IL-1 exposure. The ratio of HODE biosynthetic activity between IL-1-treated cells and controls at substrate concentrations 50 µM (from data in Fig. 5: IL-1/controls 5-8) is consistent, but not absolutely coincident, with the increment of COX proteins. Kinetics of linoleic acid concentration show a 1.5-fold increase of apparent V and a 9.8-fold decrease in the apparent K in IL-1-treated cells with respect to controls. That the increased amount of COX2 caused by IL-1 was observed mainly as a reduction of the apparent Kvalue may be due to a combination of two factors: (i) the higher affinity of COX2 than COX1 for linoleic acid, and (ii) a substrate inhibition at high substrate concentrations which led to an undervalued V, even though the experimental values fit the Michaelis-Menten equation.

The higher strength of COX2-specific inhibitors NS-398 and 6-MNA (60, 65) for inhibiting HODEs in IL-1-treated cells is consistent with the fact that IL-1 induced mainly the expression of new COX2. The strength of COX2-selective inhibitors NS-398 and 6-MNA on HODE formation was similar to those reported with arachidonic acid as substrate(60, 65, 66) . As mentioned before, except indomethacin none of the COX inhibitors totally suppressed formation of HODEs, but when 100 µM COX2-selective inhibitors were used together with 2 µM indomethacin, HODEs were suppressed completely. To exclude any possible effect of NS-398, 6-MNA, or ibuprofen on 15-lipoxygenase in HUVEC, the maximum concentrations of the drugs tested on HUVEC were also tested on PMN suspensions, and no inhibitory effect was observed. An interesting finding is that the selectivity of NS-398 and 6-MNA for COX2 was much more evident in the formation of 13-HODE(Z,E) than in 9-HODE(E,Z). Present results indicate that the selectivity of indomethacin toward COX1 is less accentuated using linoleic acid as substrate than using arachidonic acid, whereas ibuprofen and 6-MNA yield quite similar results using either linoleic acid or arachidonic acid(60) .

Buchanan and co-workers (21, 22) found that IL-1 decreases the basal levels of free 13-HODE in HUVEC, which is in apparent contradiction to our results. This fact could be explained by an increased esterification of 13-HODE in the cellular lipids as a consequence of the exposure of cells to the IL-1.()From our point of view, results on the basal production of 13-HODE should be considered carefully if esterified HODEs are not evaluated(4, 5, 19, 21) . In addition, Buchanan et al.(4) did not mention any data regarding 9-HODE levels, even in samples from incubations with exogenous linoleic acid. Actually all HODEs found in the present work have been detected in high amounts free and esterified in inflammatory lesions such as atherosclerotic and psoriatic plaques(1, 2) . The effect of IL-1 on increasing the ability of endothelial cells to metabolize linoleic acid could contribute to these high levels and suggests that all factors that increase the expression of COX2 may act on HUVEC by increasing their ability to form HODEs. Presumably, the amount of free linoleic acid is the limiting step in the formation of HODEs in in vivo situations. Nevertheless, the fact that high levels of H(P)ODEs are found in some pathophysiological situations probably means that free linoleic acid is available by oxidizing enzymes.

We can conclude from our results that COXs rather than 15-lipoxygenase are responsible for HODE formation, including 13-HODE in HUVEC. The present results show that the increase in the formation of linoleic acid metabolites by IL-1 in HUVEC is a consequence of the effect of the cytokine on the expression of new COXs, mainly COX2. Taking into account that COX2 oxidizes linoleic acid more efficiently than COX1, our results also support the hypothesis that the induction of COX2 by IL-1 may contribute to the indirect generation of minimal oxidized lipoproteins via the transfer of HODE-containing phospholipids and cholesterol esters of HODEs from the endothelium to lipoproteins. Since octadecanoids by themselves and/or octadecanoid-containing molecules can exert important biological activities involved in the inflammatory response and tumor invasion(13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) , they could mediate the effect of IL-1 and other COX2 inducers in such processes.


FOOTNOTES

*
This work was supported by Institut de Recerca of the Santa Creu i Sant Pau Hospital and Grants DGICYT PM92-0183 and FIS 94/1559. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: H. S. Creu i S. Pau (Casa de Convalecencia), S. Antonio M Claret 167, 08025 Barcelona, Spain. Tel.: 34-3-291-9105; Fax: 34-3-455-2331.

The abbreviations used are: HPODE, hydroperoxyoctadecadienoic acid; HODE, hydroxyoctadecadienoic acid; IL, interleukin; COX, cyclooxygenase; 15-HETE, 15-hydroxyeicosatetraenoic acid; NDGA, nordihydroguaiaretic acid; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; HPLC, high performance liquid chromatography; 6-MNA, 6-methoxy-2-naphthylacetic acid; NS-398, N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide; HUVEC, human umbilical vein endothelial cells; PMN, polymorphonuclear leukocyte; SP-HPLC, straight phase HPLC; GC-MS, gas chromatography-mass spectrometry; EI, electron impact; ETYA, eicosatetraynoic acid.

These data were presented as a poster in the 9th International Conference on Prostaglandins and Related Compounds in Florence June 6-10, 1994, M. Camacho and L. Vild, Abstract Book, p. 39.


ACKNOWLEDGEMENTS

We thank the staff of Casa de la Maternidad, Barcelona, for the contribution of umbilical cordons and Cristina Gerbolés, Esther Gerbolés, and Rosa Gaya for technical assistance.


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