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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3481-3488

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3481-3488.)
© 1997 American Heart Association, Inc.

Articles

HDL-Induced Prostacyclin Release in Smooth Muscle Cells Is Dependent on Cyclooxygenase-2 (Cox-2)

M. Viñals; J. Martínez-González; J. J. Badimon; ; L. Badimon

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


*    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{alpha}. 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


*    Introduction
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 *Introduction
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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.


*    Methods
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Cell culture medium and reagents were purchased from Gibco Laboratories. [6-3H]thymidine (27 Ci/mmol), [{alpha}-32P]dCTP (3000 Ci/mmol), nylon membrane (Hybond-N), and the RIA 6-keto-PGF1{alpha} assay system were purchased from Amersham. NS-398 and 6-keto-PGF1{alpha} 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-{alpha}-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 {alpha}-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{alpha}, 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.


*    Results
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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 1Down). 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{alpha} levels to 11.003±1.313 ng/mL (3.46±0.3 times control). 6-Keto-PGF1{alpha} 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 1Down).



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  		Figure 1. Dose dependence of lipoproteins on 6-keto-PGF1{alpha} release by smooth muscle cells. rSMC were cultured for 24 hours in the presence of different concentrations of cholesterol LDL ({circ}) or HDL ({bigtriangleup}), and 6-keto-PGF1{alpha} levels in the media were measured by EIA. The results represent the means±SEM of three independent experiments run in triplicate. *Significantly different from control and LDL treatment (P<.05).


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  		Table 1. Effect of Lipoprotein (150 µg/mL Cholesterol) on Prostacyclin Release, LDH Activity, and Smooth Muscle Cell Growth

Cells were incubated with HDL or its major apoprotein, Apo A-I, and 6-keto-PGF1{alpha} was measured by RIA. Apo A-I induced a significant release of 6-keto-PGF1{alpha} (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 1Up).

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{alpha} 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 2Down).


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  		Table 2. Effect of NS-398 on Prostacyclin Release

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 2Down). These findings indicate that the effect(s) of HDL on prostacyclin release require(s) new protein and mRNA synthesis.



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  		Figure 2. Effect of cycloheximide (CHX) and actinomycin D (ACT) on 6-keto-PGF1{alpha} synthesis in response to HDL stimulation. rSMC were treated for 30 minutes with or without 300 µmol/L aspirin (ASP-30 minutes) and then preincubated with actinomycin D (1 µg/mL) or cycloheximide (2 µg/mL) for 45 minutes before addition of medium alone or with HDL (150 µg cholesterol/mL). Cells were harvested after 8 hours of stimulation. Results are expressed as 6-keto-PGF1{alpha} levels referred to control. Results are the mean±SEM of three experiments. *Significantly different from the same treatment but aspirin treated (P<.05); {dagger}Significantly different from actinomycin D and cycloheximide treatments (P<.05); {ddagger}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 3Down). No bands corresponding to cyclooxygenase-1 protein were observed in SMC lysates even with overexposure of the films.



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  		Figure 3. Effect of lipoproteins on Cox-2 protein levels. rSMC were cultured in the presence or absence of lipoproteins (HDL, LDL, 150 or 300 µg cholesterol/mL) or 20% FCS for 8 hours. Protein was isolated, and equal amounts of cell protein (20 µg) were subjected to Western blot analyses using cyclooxygenase-2 specific antibodies. Protein bands were visualized by using horseradish peroxidase–linked secondary antibodies and enhanced chemiluminescence.

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 3Down). These results suggest that HDL regulates PGI2 release by increasing Cox-2 transcription rate.


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  		Table 3. Effect of Various Inhibitors on Eicosanoid Synthesis in Response to HDL Stimulation

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 4CDown). This cDNA was used as a probe in Southern blot and Northern analysis (Figs 4ADown and 4BDown). 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 4ADown). Northern blot analysis showed that the size of the specific transcript corresponding to this gene is 2.8 kb (Fig 4BDown). 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 5Down). 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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  		Figure 4. Validation of the rabbit Cox-1 cDNA obtained by PCR. (A) Southern blot analysis. Rabbit DNA (10 µg/lane) was digested with BamHI (lane 1), EcoRI (lane 2), and HindIII (lane 3) restriction enzyme. Fragments were electrophoretically fractionated, bound to Hybond C-Extra filter, and hybridized to the radiolabeled pRCox-1 insert. DNA fragment sizes are indicated in kilobase pairs on the right. (B) Northern blot analysis. RNA from rabbit lung was run in an agarose/formaldehyde gel (10 and 5 µg, lanes 1 and 2, respectively) transferred to a Hybond membrane, and hybridized to the radiolabeled pRCox-1 insert. RNA markers (Promega) were used to estimate size. Transcripts sizes are indicated in kilobases on the left. (C) Sequence of nucleotides and deduced amino acid sequence from rabbit Cox-1 cDNA. The deduced amino acid sequence is shown in the single-letter code. Underlined are the sequence BamH1.



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  		Figure 5. Alignment of cyclooxygenase sequences. The deduced amino acid sequence from rabbit Cox-1 cDNA obtained by PCR (Rb-C1) was aligned with the corresponding cyclooxygenase-1 sequences from Homo sapiens (Hu-C1),36 Mus musculus (Mo-C1),38 and Ovis aries (Sh-C1)37 and cyclooxygenase-2 sequences from Homo sapiens (Hu-C2)39 Mus musculus, (Mo-C2)35 Rattus norvegicus (Ra-C2),40 and Gallus gallus (Ch-C2)41 by the CLUSTAL V program.42 Amino acid residues are shown in the single-letter code. Stippled boxes indicate residues identical to those of the rabbit enzyme in both Cox-1 and Cox-2. Open boxes indicate residues identical only between rabbit cyclooxygenase and Cox-1 proteins. The arrowheads above primers used in PCR experiments (LB1 and LB2) indicate the direction of polymerization. Presumed axial and distal heme-binding sites as suggested by DeWitt et al38 for ovine cyclooxygenase are underlined with a dotted line.

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 6Down). In contrast, Cox-1 mRNA levels (transcript size 2.8 kb) were not modified (Fig 6Down). Regulation of PGI2 synthesis by HDL therefore seems to be mediated by an increase in Cox-2 mRNA levels.



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  		Figure 6. Effect of HDL on Cox-2 and Cox-1 mRNA levels. rSMC were cultured in the presence or absence of HDL (150 µg/mL of cholesterol). (A) After 24 hours of incubation, RNAs were isolated, and Northern blot analyses for Cox-1, Cox-2, and ribosomal RNA were performed as described in the "Methods" section. (B) Cox-1 and Cox-2 bands were quantified by densitometry and values expressed as a ratio of 28S ribosomal RNA (rRNA). Saline and HDL treatments are indicated by open and shaded bars, respectively. Representative from three experiments.


*    Discussion
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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 1Up).

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 1Up). 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 1Up).

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 2Up). 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 2Up). 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 3Up), 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.


*    Acknowledgments
 
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.


*    References
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 *References
 

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