© 1997 American Heart Association, Inc.
Articles |
HDL-Induced Prostacyclin Release in Smooth Muscle Cells Is Dependent on Cyclooxygenase-2 (Cox-2)
From the Cardiovascular Research Centre, CSIC-H. Sant Pau-UAB, Barcelona, Spain, and the Cardiovascular Institute, Mount Sinai Medical Center, New York, USA (J.J.B.).
Correspondence to Lina Badimon, Laboratori d'Investigació-Cardiovascular, Hospital de la Santa Creu i Sant Pau, Avda. San Antoni Maria Claret No. 167, 08025 Barcelona, Spain. E-mail lbmucv{at}cid.csic.es
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Abstract |
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Abstract Cyclooxygenase-1 (Cox-1) and Cox-2 are key enzymes in the conversion of arachidonic acid to prostaglandins and other eicosanoids. We studied the effects of plasma HDL and LDL on the synthesis of prostacyclin, Cox-1/Cox-2 mRNA, and protein expression by rabbit aortic smooth muscle cells. Prostacyclin synthesis was measured by enzyme immunoassay (EIA) of the stable metabolite of prostacyclin (PGI2), 6-keto-prostaglandin F1
. HDL (150 µg/mL)
induced release of PGI2 to values 3.46±0.3-fold above
control. Incubations with LDL did not induce release of
PGI2.
N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide
(NS-398), a selective irreversible Cox-2 inhibitor, blocked
the HDL-induced PGI2 synthesis. Cycloheximide, actinomycin
D, and dexamethasone downregulated HDL-induced
PGI2 synthesis; therefore, HDL induced de novo synthesis of
protein and Cox-2 mRNA. In addition, Northern blot analyses did
not reveal differences in Cox-1 mRNA levels between control and
HDL-treated cells, whereas Cox-2 mRNA levels were significantly
increased in treated cells. Western blot analysis also showed
an increase in the levels of Cox-2 protein. Therefore, the effects of
HDL on PGI2 synthesis are mediated via upregulation of
Cox-2 expression.
Key Words: HDL • LDL • cyclooxygenase-1 • cyclooxygenase-2 • NS-398
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Several epidemiologic studies have shown that high-density lipoproteins (HDL) have a protective effect against coronary artery disease (CAD).1 2 3 The mechanism for the beneficial effect of HDL on the prevention of atherosclerosis has not been fully elucidated. The most widely accepted mechanism for this protective effect is through enhanced reverse cholesterol transport.4 5 However, other mechanisms such as induction of prostacyclin (PGI2) synthesis in cultured aortic endothelial and smooth muscle cells have also been postulated.6 7 PGI2 is a vasodilator prostaglandin that is synthesized by blood vessels. The balance between platelet thromboxane A2 production and endothelial cell PGI2 synthesis seems important in the maintenance of vascular tone.8 9 Therefore, the mechanism by which HDL exerts its protective effect against CAD could be related to prostacyclin release. Furthermore, eicosanoids are lipid mediators for intercellular signaling and exert diverse cellular actions, including regulation of platelet and neutrophil function, fibrinolysis, immune response and others.10
Cyclooxygenase (Cox; prostaglandin G/H synthase, E.C. 1.14.99.1) is the rate-limiting enzyme in the conversion of arachidonic acid to prostaglandins and other eicosanoids. Cyclooxygenase-1 (Cox-1) is present in several cells and tissues in relatively stable levels,11 12 although small increases in enzyme content can occur after stimulation with hormones or growth factors.13 14 Cyclooxygenase-2 (Cox-2) is usually absent in resting cells, but its expression is greatly increased by serum, cytokines, mitogens, and conditions that stimulate cell proliferation.11 12 13 Previous reports suggested that distinct pools of arachidonic acid are available to Cox enzymes: a pool of arachidonate utilized for physiological functions by the constitutive Cox-1 and an arachidonate pool released by activation and used as substrate by Cox-2.15 In two in vivo experimental studies, we previously reported that HDL inhibits the development and progression of atherosclerosis in the aortas of cholesterol-fed rabbits.16 17 In this report, we studied the mechanism by which HDL induces eicosanoid production in rabbit smooth muscle cells (rSMC) in culture. Our study shows that HDL-induced PGI2 release is dependent on Cox-2 and that its synthesis is regulated by both transcriptional and translational processes.
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Methods |
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Cell culture medium and reagents were purchased from Gibco Laboratories. [6-3H]thymidine (27 Ci/mmol), [
-32P]dCTP (3000 Ci/mmol),
nylon membrane (Hybond-N), and the RIA
6-keto-PGF1 assay system were purchased from
Amersham. NS-398 and 6-keto-PGF1
enzyme-immunoassay (EIA) kit were obtained from Cayman Chemical Co.
Actinomycin D, cycloheximide, dexamethasone, aspirin,
calphostin C, and lactate-dehydrogenase kit (LDH-L 10) were obtained
from Sigma Chemical Co. Cholesterol kit was obtained from
Medical Analysis Systems, Inc. Phospholipids and
triglyceride kits were from Wako Chemicals. Monoclonal
antibodies anti--smooth muscle actin (clone 1A4) and anti-human von
Willebrand factor (clone F8/86) were from Dako. Ultraspec was purchased
from Biotecx Laboratories, Inc. Oligonucleotides were
obtained from Genosys Biotechnologies. Thermus aquaticus
(Taq) DNA polymerase was from Perkin Elmer. Geneclean-II kit was
from Bio 101. pBluescript SK+ vector and Escherichia coli
XLI-Blue cells were from Stratagene. Other materials and chemicals were
obtained from commercial sources.
Lipoprotein Isolation
Lipoproteins were obtained by sequential
ultracentrifugation of normolipemic rabbit plasma in a
Beckman 50.2 Ti rotor at densities between 1.019 and 1.063 g/mL (LDL)
and 1.063 and 1.255 g/mL to include the high-density and very
high-density lipoprotein fractions (HDL). Lipoproteins were
recentrifuged once at the upper density limit and dialyzed
against three changes of 200 volumes of 1 mmol/L EDTA/0.9% NaCl
pH 7.4 overnight and once against 200 volumes of 0.9% NaCl for at
least 2 hours.16 Lipoproteins were assayed for
protein by the method of Lowry et al18 and for
lipids by colorimetric assay kits. The purity of the
fraction was assessed by SDS-polyacrylamide and agarose gel
electrophoresis. All lipoproteins used in the experiments were less
than 2 weeks old, and they did not contain detectable TBARS.
Apolipoprotein A-I content was established by densitometry of Coomassie Blue–stained bands from SDS-polyacrylamide gel. HDL contained 89% apolipoprotein A-I. Values of cholesterol, triglycerides, and phospholipids expressed as milligrams of lipid per milligram of protein were 0.178±0.012, 0.434±0.048, and 0.136±0.021, respectively, for HDL and 1.183±0.012, 1.115±0.159, and 1.245±0.141, respectively, for LDL.
Rabbit Apo A-I was isolated from HDL (density: 1.063 to 1.21 g/mL). HDL was delipidated by using diethyl-ether:ethanol (3:1) extraction at 4°C. The apolipoproteins were dissolved in 5 mol/L guanidine-HCl, 0.1 mol/L Tris-HCl, and 1 mmol/L EDTA at pH 8 (G-Tris-EDTA). The soluble fraction was then fractionated by gel filtration on Sephadex G-150 column (2.6 x 200 cm) equilibrated with the G-Tris-EDTA buffer. The purity of the apolipoprotein A-I was determined by SDS-polyacrylamide electrophoresis.16
Isolation of Rabbit Aortic Smooth Muscle Cells
rSMC were obtained by gentle scraping of the medial layer of
male New Zealand rabbit aortas after endothelial layer
removal. Cells were incubated at 37°C in a humidified atmosphere of
5% CO2 in Ham's F12-DMEM (8:2) supplemented
with 20% fetal calf serum (FCS). Antibiotics (100 U/mL penicillin and
0.1 mg/mL streptomycin) were added to the culture media. To maintain
exponential growth, cells were subcultured by trypsinization and seeded
at a density 10 000 cells/cm2. rSMC were
identified by their growth behavior, morphology, and
immunofluorescence. Immunocytochemical
identification of cells was performed by specific monoclonal antibodies
for -smooth muscle cell actin and von Willebrand factor.
Cells were fixed with ice-cold methanol at 20°C for 5 minutes. A
solution of BSA at 1% in PBS was used as blocking agent. Monoclonal
antibodies were used diluted in PBS/1% BSA/0.01% Triton X-100.
Finally, a fluorescein-conjugated goat anti-mouse IgG was
used as secondary antibody.19
Cell Culture and Supernatant Determinations
Cells between the second and seventh passages were grown as
described above. At subconfluency, fresh medium was added to the wells,
and 24 hours later, cells were washed three times with FCS-free medium
and incubated in the same medium with lipoproteins at concentrations of
86, 150, 300, or 600 µg of cholesterol/mL, saline
(serum-free control), or 20% FCS (serum control). NS-398
(N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide) (0.5
to 5 µmol/L), a Cox-2 selective
inhibitor,20 21 22 23 24 25 was added either
simultaneously or 2 hours before the addition of
lipoproteins.
To abolish baseline Cox activity, cells were pretreated with 300 µmol/L of aspirin for 30 minutes and then, before HDL treatment, preincubated with dexamethasone (2 µmol/L), NS-398 (5 µmol/L), or aspirin (300 µmol/L) for 2 hours or actinomycin D (1 µg/mL) and cycloheximide (2 µg/mL) for 45 minutes. After 8 or 24 hours of lipoprotein incubation, the culture media was kept at -80°C.
In selected experiments, calphostin C (0.2 µmol/L and 1 µmol/L) was added 45 minutes before incubations with lipoproteins. After 24 hours of incubations, the culture media was kept at -80°C.
PGI2 in the supernatants was measured as
6-keto-PGF 1, its stable hydrolysis
product, by an EIA or RIA kit. Lactate dehydrogenase activity in
media was measured by a Sigma kit. The Bradford
colorimetric assay26 and the
bicinchoninic acid method, when detergent was present in the
samples,27 were used to determine cell protein.
Measurements were performed according to the manufacturer's
instructions.
DNA Synthesis
[6-3H]Thymidine (0.5 µCi/mL) was
simultaneously added with lipoproteins to the cells. After
incubation, plates were placed on ice, the media was removed, and the
cell monolayers were washed once with PBS. One milliliter of ice-cold
95% methanol was added for 5 minutes. It was then removed, and 1 mL of
10% TCA was added for 10 minutes. Cell monolayers were dissolved in 1
mL of 0.3 mol/L NaOH. Aliquots were taken to measure
[3H]thymidine uptake and protein determination
using the Bradford colorimetric
assay.26 The radioactivity incorporated into DNA
was measured as dpm by scintillation spectrophotometry (model 1217
RackBeta, LKB, Wallac, Pharmacia). The results were expressed as dpm in
treated cells normalized by dpm in unstimulated cells.
Western Blot Analysis
rSMC were cultured as described before. Cells were stimulated
with lipoproteins (150, 300 µg/mL) for 24 hours. Cell monolayers were
washed with PBS and lysed with 50 mmol/L Tris, 1mmol/L EDTA, 0.1%
Triton. Twenty micrograms of total protein was separated on 4% to 15%
gradient polyacrylamide gels using mini-PROTEAN II Dual Slab Cell (Bio
Rad). Proteins from polyacrilamide gels were blotted onto
nitrocellulose membranes (Bio Rad) at 40 mA for 1 hour at 4°C. The
residual binding capacity of the membranes was blocked with 5% nonfat
milk in 10 mmol/L Tris pH 7.5, 100 mmol/L NaCl, 0.1% Tween
20. Blots were incubated with monoclonal antibodies against human Cox-2
(C22420, Transduction Laboratories) or Cox-1 (PG22, Oxford Biomedical).
Bound antibody was detected by using the appropriate horseradish
peroxidase–conjugated antibody. Signals were detected with the ECL
chemiluminescent detection system (Amersham) on a standard X-ray
system.
Preparation of cDNA Probes
A specific rabbit Cox-1 cDNA probe was obtained by reverse
transcriptase-polymerase chain reaction (RT-PCR). Briefly,
single-stranded cDNA was synthesized by reverse transcription of rabbit
lung RNA with Moloney leukemia virus reverse transcriptase.
Amplification of Cox-1 cDNA was performed by using the following paired
primers: sense [GATCCATGTT(Y)GC(N)TT(Y)TT(Y)GC(N)CA] and antisense
[CGGATCCAT(N)AC(D)AT(Y)TT(D)AT(N)GT(Y)TC], which correspond to
human Cox-1 amino acid residues 196 to 202 and 338 to 344,
respectively. (Degenerate positions are indicated in parentheses in the
following code: Y is either C or T; D is A, G, or T; and N is A, C, G,
or T). The annealing/elongating/denaturing conditions for the PCR
reaction was 49°C/72°C/95°C.
The PCR-amplified cDNA (pRCox-1) was subcloned into pBluescript SK+ vector and sequenced by the dideoxy chain termination method.28 pRCox-1 cDNA, Cox-2 cDNA, which was kindly provided by Dr Hla,11 and a ribosomal cDNA29 were used as probes in Northern blot experiments. The labeling reaction was performed by the random priming method.30
Southern Blot Analysis
Genomic DNA was obtained from rabbit
lung,31 and Southern blot analysis was
carried out.32 Ten micrograms of DNA were
digested with BamHI, EcoRI, and
HindIII restriction enzymes. DNA fragments were
electrophoretically fractionated on 1% agarose gels in
Tris/Phosphate/EDTA buffer. DNA was transferred by capillarity to a
Hybond C-Extra membrane (Amersham) in 20 x SSC (1 x SSC is
0.15 mol/L NaCl/0.015 mol/L sodium citrate). Filters were prehybridized
and hybridized at 65°C in a solution containing 0.7 mol/L NaCl,
40 mmol/L NaH2PO4 pH
7.6, 4 mol/L EDTA, 2 mg/mL polyvinylpyrrolidone, 2 mg/mL Ficoll 400,
0.1% SDS, 10% dextran sulfate, and heat-denatured salmon sperm DNA
(200 µg/mL). Washes and film expositions were performed as described
below.
RNA Blot Analysis
rSMC were cultured as previously described. After 24 hours of
lipoprotein incubations, stimulations were halted by the addition of
ice-cold isolation reagent Ultraspec RNA (1 mL/21
cm2 dish). RNA samples were fractionated in 1.1%
agarose gels containing formaldehyde. RNA was transferred by
capillarity to Hybond membranes and UV-cross-linked. Nylon filters were
prehybridized for 3 hours at 42°C in 50% formamide, 1 mol/L NaCl,
50 mmol/L NaH2PO4 pH
6.3, Denhardt's solution 7.5x, 1% SDS, 10% dextran sulfate, and 200
µg/mL denatured salmon sperm DNA.32
32 P-labeled probes were added to the
prehybridization mixture. Hybridization was carried out overnight with
the same solution. pRCox-1 and human Cox-2 cDNAs were used as probes.
Washes were carried out under moderate stringency conditions. Filters
were dried and exposed to Agfa Curix RP2 x-ray film at -70°C with
Agfa Curix Blue C2 intensifying screens. To assess the amount of RNA in
the nylon filters, filters were rehybridized with a ribosomal cDNA
probe. The levels of mRNA were measured by densitometric scanning of
the autoradiograms with a Molecular Dynamics computing
densitometer.
Statistical Analysis
Data are presented as the mean±SEM. Means were compared
by using ANOVA. Differences were considered significant at
P<.05.
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Results |
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Effect of Lipoproteins on Prostacyclin Release
The effect of lipoproteins on prostacyclin release was assessed in rSMC exposed to different concentrations of HDL or LDL cholesterol. HDL induced a dose-dependent increase in PGI2 release. At no concentration did LDL-treated cells show differences with respect to control cells (Fig 1
). The subsequent studies were carried
out at a physiological rabbit HDL
cholesterol concentration (150 µg/mL). After 24 hours of
incubation, HDL induced 6-keto-PGF1 levels to
11.003±1.313 ng/mL (3.46±0.3 times control).
6-Keto-PGF1 production was 3.134±0.48
ng/mL for control cells and 4.028±0.7 ng/mL for LDL-treated cells. The
normalization of these results by the protein content of the cell
monolayer did not alter the significance of the HDL-induced response.
Lipoprotein treatment did not change LDH activity, a result indicating
that the different behavior of LDL or HDL in the release of
prostacyclin was not due to a citotoxic effect (Table 1).
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) or HDL (
), and
6-keto-PGF1
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Cells were incubated with HDL or its major apoprotein, Apo A-I, and
6-keto-PGF1 was measured by RIA. Apo A-I
induced a significant release of 6-keto-PGF1
(205% of control), but the degree of stimulation was lower than that
caused by equivalent quantities of Apo A-I in HDL (420% of
control).
FCS, which was used as a comparative agonist, highly increased PGI2 production (137.82±9.04 ng/mL; 37.77±4.3 times control).
[3H]Thymidine incorporation into newly
synthesized DNA was measured as an index of cell proliferation. A time
course study of thymidine incorporation was performed at 2, 4, 10, 24,
and 48 hours. Incorporation at 2, 4, and 10 hours was independent of
the treatment. Cells stopped incorporating
[3H]thymidine after 24 hours of incubation in
serum-free medium, even in the presence of lipoproteins. Only
FCS-treated cells proliferated between 24 and 48 hours. At 24 hours of
incubation, LDL and HDL increased DNA duplication with respect to
control cells, but the effect was shown to be independent of the
lipoprotein type (Table 1
).
Inhibitory Effect of NS-398 on Prostacyclin
HDL-Induced Release
rSMC were incubated with HDL or LDL, kept in serum-free media, or
incubated with 20% serum media and simultaneously treated
with NS-398 (0.5 and 5 µmol/L). After 24 hours,
6-keto-PGF1 levels were measured in the
culture media. NS-398, a Cox-2-selective inhibitor,
significantly reduced PGI2 synthesis in
serum-free control and in lipoprotein-treated cells.
In media containing 20% serum, which in rat aortic SMC has been
reported to increase the steady-state level of Cox-2 mRNA without
affecting that of Cox-1,33 34 35 NS-398 also
inhibited PGI2 release (Table 2).
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Since NS-398 is a specific inhibitor of Cox-2, our results indicate that the effect of HDL on PGI2 synthesis in rSMC is largely mediated by Cox-2.
Effect of Transcription and Translation and Protein Kinase C
Inhibitors on Prostacyclin Release Induced by HDL
rSMC were treated with aspirin (300 µmol/L) for 30 minutes,
extensively washed, and preincubated with cycloheximide (2 µg/mL) or
actinomycin D (1 µg/mL). HDL was then added. In serum-free medium,
aspirin pretreatment abolishes the residual activities of both
isoforms. However, HDL promoted a significant prostacyclin release even
in aspirin-treated rSMC. The aspirin-resistant prostacylin
production promoted by HDL was significantly reduced by
cycloheximide and actinomycin D (Fig 2).
These findings indicate that the effect(s) of HDL on prostacyclin
release require(s) new protein and mRNA synthesis.
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Significantly different from actinomycin D
and cycloheximide treatments (P<.05); 
Significantly
different from cycloheximide (P<.05); §Significantly
different from control (P<.005).PGI2 release induced by HDL was unaffected by calphostin C (0.2 and 1 µmol/L), a protein kinase C (PKC) inhibitor, a result indicating that activation of PKC by HDL is not directly related to the ability of HDL to stimulate prostacyclin release.
Effect of Lipoproteins on Cox-2 Protein Levels
After 8 hours of incubations, protein from rSMC incubated with
lipoproteins (150 or 300 µg cholesterol/mL) or FCS (20%)
was isolated, and Western blot analysis was performed to
evidence whether Cox-2 protein was induced by the treatment. Results
showed that Cox-2 protein was present both in controls and in
treated cells and increased after HDL and FCS incubations. LDL did not
modify Cox-2 protein baseline levels (Fig 3). No bands corresponding to
cyclooxygenase-1 protein were observed in SMC
lysates even with overexposure of the films.
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Effect of Dexamethasone on Prostacyclin Release Induced
by HDL
After aspirin pretreatment, rSMC were extensively washed and
treated with HDL in the presence or absence of aspirin, NS-398, or
dexamethasone. Aspirin, NS-398, and
dexamethasone significantly and similarly inhibited the
release of prostacyclin induced by HDL (Table 3). These results suggest that HDL
regulates PGI2 release by increasing Cox-2
transcription rate.
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Effect of HDL on Cox-1 and Cox-2 mRNA Levels
To obtain a specific rabbit Cox-1 probe, amplification of a cDNA
encoding for rabbit Cox-1 was performed. Comparison of the primary
structure of cyclooxygenases previously
reported12 35 36 37 38 39 40 revealed several highly
conserved regions. A consensus sequence was compiled from this
analysis. Two of the most conserved amino acid motifs were
selected for the synthesis of oligonucleotides to be
used as primers in PCR experiments. After PCR amplification, a cDNA of
the expected size (462 bp) was obtained (Fig 4C). This cDNA was used as a probe in
Southern blot and Northern analysis (Figs 4A and 4B). A genomic
Southern analysis yielded a hybridization pattern showing that
this putative Cox-1 gene is present as a single copy in the rabbit
genome (Fig 4A). Northern blot analysis showed that the size of
the specific transcript corresponding to this gene is 2.8 kb (Fig 4B).
These experiments confirm that the transcript corresponding to this
protein exists and rule out the possibility that the PCR products
were derived from pseudogenes by amplification of contamination
chromosomal DNA. Sequence analysis of this cDNA showed an open
reading frame coding for 148 amino acid residues exhibiting high
identity to other cyclooxygenase sequences (Fig 5). The overall identity between the
cyclooxygenase sequences is 65%, and 87% when
only Cox-1 sequences are considered. Both the size of the transcript
and the higher identity shared with Cox-1 sequences indicated that the
PCR product correspond to a rabbit Cox-1 probe.
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To analyze whether the effect of HDL on prostacyclin release
was mediated by an increased expression of Cox-1 or Cox-2 mRNA,
Northern blot analyses were performed. Total RNA was isolated
from 24-hour-treated rSMC. Results show that in HDL incubated cells,
Cox-2 mRNA levels (transcript size 4.2 kb) were upregulated (Fig 6). In contrast, Cox-1 mRNA levels
(transcript size 2.8 kb) were not modified (Fig 6). Regulation of
PGI2 synthesis by HDL therefore seems to be
mediated by an increase in Cox-2 mRNA levels.
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Discussion |
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Several studies have reported increases in PGI2 generation by HDL in cell culture.43 44 45 46 However, the mechanism of this increase remains unknown. In this study, we observed an effect of HDL on PGI2 production that correlated with a increase in Cox-2 mRNA expression. Our results revealed that at similar cholesterol concentrations, HDL, but not LDL, stimulated prostacyclin synthesis. This effect was not due either to an increase in cell proliferation (thymidine incorporation) or to a citotoxic effect (Table 1
).
On the basis of plasma cholesterol content,
LDL-cholesterol was added in a concentration twofold to
threefold higher than that of HDL; however, HDL were more effective
than LDL in eliciting PGI2 formation, a finding
suggesting that the process is specific for a single class of
lipoprotein and that the process may occur under
physiological conditions (Fig 1). Additionally, the
effect is concentration dependent. The specificity in
PGI2 synthesis induced by HDL suggested that the
effect might reside in the apoprotein moiety. However, in concordance
with other authors,44 45 we found that the major
apoprotein of HDL (apoA-I) caused a weaker stimulation of
PGI2 release than the entire lipoprotein
fraction, implying that the intact micelle might be required to achieve
the full effect.
Previous studies of the effects of LDL on the biosynthesis of
prostanoids have yielded controversial results. LDL either stimulated,
inhibited, or had no effect on prostanoid
synthesis.43 44 45 47 48 49 This variable effect
of LDL on PGI2 synthesis seems to be related to
its degree of oxidation. Unoxidized LDL did not affect prostanoid
synthesis in SMC cultures, whereas mildly oxidized LDL stimulated and
highly oxidized LDL inhibited prostanoid
synthesis.50 Our results, in agreement with the
unoxidized LDL results, showed no effect on PGI2
release caused by native LDL (Table 1).
Previous reports suggested that an arachidonic pool
released after activation is selectively used as a substrate by
Cox-2.15 On the other hand, direct evidence
supporting incorporation of arachidonate from HDL as
substrate for cell prostaglandin synthesis was obtained in
SMC. Experiments with cells preincubated with recombinant cholesteryl
[1-14 C]arachidonate HDL exposed to
A-23187 (a calcium ionophore that induces Cox-2 mRNA
expression51 ) showed that
[1-14C]arachidonate was used for
prostaglandin synthesis.43 46 In
addition, in SMC-derived foam cells, where the induction of Cox-2 mRNA
steady-state levels following exposure to stimulus is
impaired,52 the HDL-induced
PGI2 production is essentially
abrogated.46 NS-398, a new selective irreversible
Cox-2 inhibitor, significantly inhibited HDL induced
PGI2 generation (Table 2). Because NS-398 (0.5
and 5 µmol/L) did not affect the Cox-1
isoenzyme,20 21 22 23 24 25 the effect of HDL seems to be
mediated by means of Cox-2 activity.
NS-398 also inhibited the PGI2 production produced in the presence of saline and LDL. Since the cells were not rendered quiescent before exposure of lipoproteins, Cox-2 protein was present not only in treated but also in control cells, as demonstrated by Western analysis, and therefore was inhibited by the Cox-2 inhibitor.
Since HDL stimulates PKC in a variety of cell types53 54 and Cox-2 expression is potently enhanced by PKC activators,55 we tested the ability of calphostin C to inhibit PGI2 induction by HDL. Calphostin was unable to reduce PGI2 release; therefore, PKC does not seem to be involved in the mechanism of HDL induction. However, we cannot rule out a possible mechanism linked to phosphorylation. It has been reported that HDL provokes a 20-kDa protein-phosphorylation that is not modulated by phorbol ester but seems to be mediated by a calmodulin kinase.56
Actinomycin D and cycloheximide reduced PGI2
levels induced by HDL (Fig 2). The inhibition produced by both
compounds demonstrated that HDL induces new protein and mRNA synthesis.
Furthermore, dexamethasone has been reported to selectively
inhibit new transcription of Cox-2 mRNA without affecting Cox-1
mRNA,57 58 59 and aspirin has been revealed as a
nonselective inhibitor of the two
isoforms.60 Interestingly, the values of
inhibition for dexamethasone, aspirin and also NS-398 were
fairly comparable (Table 3), a result indicating that Cox-2 activity
was increased by inducing transcription of Cox-2 mRNA. In addition,
Northern blot experiments showed that Cox-2 mRNA levels were
upregulated while Cox-1 mRNA levels remain unchanged.
Lack of Cox-2 activity seems to be important in the formation of foam cells.52 Particularly, PGI2 was reported to decrease intracellular levels of cholesterol.61 Other authors as well as ourselves44 45 have observed that the HDL fraction exerts its effect through both apoprotein and lipid content, probably by providing substrate to cells.43 At the same time, we have demonstrated for the first time that the HDL fraction induces Cox-2 mRNA levels. It is thus possible that the HDL fraction plays a protective role in atherogenesis by acting as an intracellular cholesterol-trafficking mediator both by increasing PGI2 levels45 52 through Cox-2 and also by working as a cholesterol acceptor in the reverse cholesterol transport process. Further studies focused on the regulation of protein expression and posttranscriptional regulation will provide a new light on the mechanism by which HDL increases PGI2 production and reduces foam cell formation.
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Acknowledgments |
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This work has been funded in part by grants from PNS SAF 712/94, Fundación Española del Corazón, and Fundación de Investigación Cardiovascular (FIC)-Catalana Occidente.
Received December 20, 1996; accepted July 22, 1997.
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