(Received for publication, December 20, 1994; and in revised form, May 10, 1995)
From the
Human umbilical vein endothelial cells (HUVEC) were treated with
recombinant interleukin (IL)-1
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)
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.
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.
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
Some plaques of cells untreated and treated
with IL-1
Figure 1:
Representative SP-HPLC from a sample of
HUVEC incubated with 25 µM [
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.
To elucidate the origin of
all-trans-isomers, experiments were performed incubating
intact and boiled cells with 2.5 µM
13-[
Figure 3:
Chromatograms showing the
all-trans-isomer formation after incubation of boiled cells
with: A,
13-[
Figure 4:
A, production of HODEs by HUVEC as a
function of time of treatment with IL-1
Figure 5:
Kinetics of
[
Figure 6:
Effect of the concentration of several
inhibitors on the production of HODEs by IL-1
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
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
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
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
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
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
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
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
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
The higher strength of COX2-specific inhibitors NS-398 and 6-MNA (60, 65) for inhibiting HODEs in IL-1
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
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
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, 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.
(
)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.
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
[
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
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.
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.
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
The cells
were incubated for 8 h with 10 units/ml IL-1 on Linoleic Acid Metabolism
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.
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 µM
C-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
µM
C-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
µM
C-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.
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) .
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.
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)).
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.
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
The increase in the production of HODEs was
dependent on concentration and the time of exposure to IL-1 on Linoleic Acid
Metabolism
(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).
. 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/10
cell/10 min; and K
,
15.59 ± 8.39 and 152.9 ± 84 µM for
IL-1
-treated and untreated cells, respectively (Fig. 5).
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/10
cell/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.
-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.
-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.
. 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.
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.
, 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.
(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).
-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.
, 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.
-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.
(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 K
value 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.
-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) .
.
(
)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.
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.
Claret 167, 08025 Barcelona, Spain. Tel.:
34-3-291-9105; Fax: 34-3-455-2331.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.