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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:910-917.)
© 1999 American Heart Association, Inc.

Original Contributions

High-Density Lipoproteins Differentially Modulate Cytokine-Induced Expression of E-Selectin and Cyclooxygenase-2

Gillian W. Cockerill; Jeremy Saklatvala; Simon H. Ridley; Helen Yarwood; Norman E. Miller; Barbaros Oral; Saro Nithyanathan; Graham Taylor; Dorian O. Haskard

From the BHF Cardiovascular Medicine Unit (G.W.C., H.Y., B.O., D.O.H.), National Heart and Lung Institute, the Section on Clinical Pharmacology (G.T.), Imperial College School of Medicine, the MRC Lipid Team (S.N.), Hammersmith Hospital, the Kennedy Institute of Rheumatology (J.S., S.H.R.), and the Department of Cardiovascular Biochemistry (N.E.M.), St Bartholomews and the Royal School of Medicine and Dentistry, London, UK.

Correspondence to G.W. Cockerill, BHF Cardiovascular Medicine, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK. E-mail gcockeri{at}rpms.ac.uk

	Abstract

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Abstract—Atherogenesis is a multifactorial chronic inflammatory disease in which low plasma levels of HDLs are a strong predictor of the condition. Although the mechanism of protection by HDLs is not precisely known, HDLs have been shown to influence many of the events involved in the development of atherosclerosis. Previously we have shown that HDLs inhibited the cytokine-induced expression of adhesion molecules (E-selectin, VCAM-1, and ICAM-1) by endothelial cells (ECs). As the complete transcriptional regulation of all 3 genes requires the NF-B family of transcription factors, we examined the effect of HDLs on activation of NF-B. We also investigated the effect of HDLs on 2 other cytokine-induced genes, granulocyte-macrophage colony-stimulating factor (GM-CSF) and cyclooxygenase (Cox-2; prostaglandin H₂ synthase, EC 0.1.14.99.1). E-selectin expression in response to tumor necrosis factor- (TNF) was, as expected, inhibited in ECs that had been preincubated with HDLs. However, the level of secretion of GM-CSF in the same cultures was no different from control. In a similar manner, although HDLs had no effect on steady-state mRNA levels of GM-CSF, the levels of E-selectin were significantly inhibited by HDLs. In transient cotransfection experiments we found that HDLs inhibited the cytokine-induced expression of a reporter gene driven by the E-selectin proximal promoter (-383 to 80) but had no effect on the expression of a reporter gene driven under the control of the proximal promoter of GM-CSF (-627 to 28). As would be predicted from this differential response, HDLs did not influence the nuclear translocation or DNA binding of NF-B, or alter the kinetics of degradation and resynthesis of the inhibitory protein IB. We found that HDLs synergized with cytokine to enhance the expression of Cox-2 and induce the synthesis of its main EC product, prostacyclin (PGI₂), a potent inhibitor of platelet and leukocyte functions. In conclusion, HDL induces an antiinflammatory phenotype in cytokine-induced ECs, synergizing with cytokine to induce elevation of Cox-2 in addition to inhibiting adhesion molecule expression. Our studies show that these differential effects are mediated in a manner that is likely to be independent of NF-B per se.

Key Words: inflammation • coronary artery disease • prostacyclin • granulocyte-macrophage colony-stimulating factor

	Introduction

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Coronary artery disease remains one of the major causes of death in the Western world today.¹ A great deal of epidemiological research shows a strong inverse correlation between the concentration of plasma HDL and the frequency of coronary artery disease.^{2 3 4} More direct evidence of the protective effects of HDL comes from studies in which elevation of HDL in animal models has led to a dramatic protective effect against early atherosclerotic fatty streak formation.^{5 6 7 8} It is not yet known whether the protective effects of HDL relate to their role in reverse cholesterol transport or to mechanisms unrelated to their lipid transport function.^{9 10 11 12 13 14 15 16 17 18 19}

Normal endothelium is one that maintains a nonadhesive, nonthrombogenic, and nonproliferative interface of cells that separate the blood from the underlying tissues. The phenotype of the endothelium changes in response to certain inflammatory mediators, such as tumor necrosis factor- (TNF) and interleukin-1ß (IL-1ß), to one that expresses and secretes several adhesion molecules and chemoattractants capable of promoting the recruitment of leukocytes across the endothelial barrier.^{20 21} Many cytokine-stimulated genes are regulated at the transcriptional level by transcription factors of the NF-B family.²² Normally, NF-B members are confined to the cytoplasm through association with inhibitory proteins of the IB family.²³ IL-1ß or TNF leads to the phosphorylation and degradation of the IB proteins, resulting in the release, nuclear translocation, and DNA binding of NF-B, with subsequent transcriptional activation of responsive genes. In many instances this process is transitory, as NF-B stimulates the transcription and resynthesis of IB.^{23 24 25}

There is considerable evidence for the importance of leukocyte recruitment into the arterial intima, both for the development of atherosclerosis and for the maintenance of the mature plaque. The earliest observable cellular event in the genesis or development of atheroma is the binding of leukocytes to the endothelium.^{26 27} Furthermore, there are now several studies demonstrating the presence on endothelium of IL-1 or TNF-inducible adhesion molecules, such as P-selectin, E-selectin, VCAM-1, and ICAM-1, both in animal models of atherosclerosis and in human atherosclerotic tissue.^{28 29 30 31 32} In a previous study the possibility was explored that one of the actions of HDLs, contributing to protection from coronary artery disease, might be through inhibition of expression of cytokine-inducible endothelial cell (EC) adhesion molecules.³³ HDLs were found to significantly inhibit the ability of TNF or IL-1ß to stimulate expression of E-selectin, VCAM-1, and ICAM-1 in human umbilical vein ECs (HUVECs), both at the level of cell surface expression and steady-state mRNA. As these adhesion molecules have all been demonstrated to require activation of the NF-B family of transcription factors for complete expression, these experiments were initially designed to explore the possibility that the antiinflammatory effects of HDLs were mediated by inhibiting the activation of the NF-B family of transcription factors. Our results show that inhibition of NF-B is unlikely to be the central mechanism by which HDL inhibits adhesion molecule expression and suggests that HDLs have a more complex effect on cytokine-induced gene expression than was previously appreciated.

	Methods

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Cell Culture
HUVECs were isolated as described previously.³³ All cells were plated onto 1% gelatin-coated culture plastic in medium 199 with Earle's salts (ICN Biomedicals Inc) supplemented with 20% FCS (GIBCO), 2 mmol/L glutamine, penicillin and streptomycin, 50 µg/mL EC growth supplement (Sigma), and 50 mg/mL heparin (Sigma). In typical experiments, confluent HUVECs were incubated for 16 hours with native or reconstituted HDL (1 mg/mL apoAI), after which TNF or IL-1 (1 ng/mL) was added to the culture medium for a further period before ending the experiment. From using this concentration of cytokines, no loss of cell viability was observed.

Isolation of HDL
Blood from normal healthy volunteers, <40 years of age, was collected in Vacutainers containing K-EDTA as the anticoagulant (Beckton Dickinson). Plasma was separated by spinning at 2000 rpm for 20 minutes at 4°C. HDLs were then purified by sequential ultracentrifugation in the 1.07 to 1.21 g/mL density range, according to the previously described method.³⁴ Resulting HDLs were then dialysed against 4 changes of PBS and filter-sterilized by using a 0.2-nm Minisart microfilter (Sartorius). HDL isolated in the presence of EDTA loses paraoxonase,³⁵ hence effects observed in these studies are independent of this enzyme.

Analysis of HDL
Concentrations of apoAI and apoB were determined by immunoturbidimetric methods, using a Cobas-Fara centrifugal analyzer (Roche Diagnostic) with commercially available antibodies (Boehringer Mannheim). HDL particle size was evaluated by electrophoresis through 3% to 30% nondenaturing gradient gels (Pharmacia). All preparations of HDL contained 2 main populations: 1 with a Stokes diameter of 10.4 nm (HDL 2b) and 1 with a Stokes diameter of 8.6 nm (HDL 3a). HDL preparations contained no contaminating particles of the size range of LDL and there was no apoB detectable by immunoturbidimetric analysis. All preparations used were endotoxin free, as determined by the limulus lysate microassay,³⁶ and the inability of native HDL to elevate basal adhesion molecule expression on HUVECs (Figure 1).

View larger version (15K): [in this window] [in a new window]   Figure 1. HDLs inhibit TNF-induced E-selectin expression but have no effect on GM-CSF synthesis. a, After a 16-hour preincubation with HDL (1 mg/mL apoAI), TNF (1 ng/mL) was added to confluent ECs for a further 9 hours in the continuous presence of HDL. Cell surface E-selectin expression was then measured by flow cytometry using monoclonal antibody 1.2B6. b, Supernatants were harvested at the end of the same experiment and levels of GM-CSF (pg/mL) measured by using an ELISA kit (R & D Systems). Data in each panel are representative of 3 experiments showing means and SD, using HDL from 3 separate donors. Differences between means were evaluated by using unpaired t tests. *P, not significant; **P<0.005.

Reconstituted HDL
The reconstituted HDLs used in this study were kindly provided by the Swiss Red Cross, Blood Transfusion Service, Bern, Switzerland. The particles, containing apoAI as the sole protein, and soybean phosphatidylcholine as the sole phospholipid, were prepared by using cholate dialysis according to the reported method.³⁷ Reconstituted discoidal HDL particles were used in parallel experiments to those using native HDL.

Flow Cytometry
HUVECs were plated at confluence in 24-well plates (2x10⁵ cells/400 µL per well), and incubated with HDL and TNF (as above). Cells were then harvested with trypsin and washed in ice-cold PBS. Surface expression of E-selectin was then determined by resuspending the cells in 50 µL of anti-E-selectin monoclonal antibody³⁸ for 45 minutes at 4°C. Cells were then washed in PBS containing 0.02% sodium azide and 5% newborn calf serum (Gibco) at 4°C and incubated for 45 minutes at 4°C in 50 µL of wash buffer containing FITC-labeled rabbit anti-mouse Ig (DAKO). After further washing, the cells were fixed with 2.5% formaldehyde. Expression of E-selectin was measured by using a Coulter Epics Profile II flow cytometer, counting 10⁴ cells per sample. Controls included an isotype-matched irrelevant antibody and no primary antibody.

GM-CSF Enzyme-Linked Immunosorbent Assay
GM-CSF protein concentrations in EC culture supernatants were determined by using a human GM-CSF ELISA kit (R & D Systems Ltd) according to the manufacturer's recommendations.

Northern Analysis
Total RNA was prepared as described by Chomczynski and Sacchi.³⁹ Equal aliquots of total RNA (10 µg/lane) were size-fractionated in a 1% formaldehyde gel and transferred to nylon membrane (Hybond N, Amersham). Blots were hybridized according to Church and Gilbert,⁴⁰ using 10 ng/mL -³²P–labeled cDNA probes. After washing, the blots were exposed on Kodak BIOMAX film (Eastman Kodak). Relative amounts of RNA per lane were normalized to the ethidium bromide–stained ribosomal RNAs.

Western Blotting
After appropriate preincubation with HDL and TNF, ECs were washed twice with ice-cold PBS and then harvested by scraping. ECs were then pelleted and lysed in 50 µL of lysis buffer (50 mmol/L Tris-Cl, 1% NP-40, 0.25% deoxycholate, 5 mmol/L EDTA, 1 mmol/L Na₃VO₄, 10 mmol/L NaF, 5 mmol/L leupeptin, 100 U/mL aprotinin, and 1 mmol/L PMSF) (Sigma) on ice for 15 minutes. Debris was then removed by centrifuging the lysate for 5 minutes at 4°C and the supernatants stored at -70°C until use. Protein concentration was estimated by using the Bio-Rad BCA kit (Bio-Rad). Protein was separated by running 10 µg of protein lysate in a 15% SDS-PAGE, using a Proteanô minigel apparatus. Proteins were then transferred to Immobilon (Millipore) membrane, according to the manufacturer's recommendations, using the Protean minigel wet transfer apparatus. Filters were then blocked overnight in 5% milk solid in PBS and washed once for 15 minutes in PBS with 0.05% Tween (Sigma). After a further 15 minutes wash with PBS alone, the filters were incubated at room temperature in the primary antibody (MAD3-anti-IB; rabbit anti-human COX-2, Oxford Biomedical) at 1:5000 in hybridizing buffer (PBS containing 0.5% milk solid) for 1 hour. After extensive washing (5x15 minutes in PBS containing 0.05% Tween), the protein–antibody complex was identified by incubating the filter for 1 hour in a 1:10 000 dilution of an HRP-conjugated goat anti-rabbit immunoglobulin (Sigma) in hybridizing buffer. After further washes (5x15 minutes with PBS containing 0.05% Tween, and 2x15 minutes with PBS only), the filter was treated with chemiluminescence reagent according to the manufacturer's recommendations (ECL) and exposed to Kodak BIOMAX film. Equivalent loading was monitored by protein staining the filter using Ponceau S.

Prostacyclin Synthesis Assay
The stable hydrolysis product of prostacyclin, 6-oxo-prostaglandin F₁ (6-oxo-PGF₁), was measured by using an immunoabsorbance assay (Cayman Island Chemicals), according to the manufacturer's recommendation. The identity of the antigen quantified by this assay was confirmed by immunoabsorbance analysis of fractions of supernatant separated by HPLC (Waters St Quentin en Yvelines) Model 510 with an Altex ultosphere column (Beckman). The mobile phase was composed of 10% acetonitrile (HPLC Grade, Merck), 5 mmol/L phosphoric acid (solvent 1) and 50% acetonitrile, and 5 mmol/L phosphoric acid (solvent 2). The wavelength detection monitored was 204 nm.

Electrophoretic Mobility Shift Assay (EMSA)
EMSAs were performed as previously described.⁴¹ In brief, 5 µg of nuclear extract with 0.2 ng of ³²P-labeled probe, 2 µg of poly(dI–dC) (Promega) in a binding buffer (30 mmol/L HEPES, pH 7.9, 16 mmol/L KCl, 0.5 mmol/L DTT, 13% glycerol, 8 µg/mL leupeptin, and 8 µg/mL aprotinin) in a final volume of 18 µL for 30 minutes at room temperature. Samples were fractionated by electrophoresis through a 4% low-salt (0.5x 89 mmol/L Tris, 89 mmol/L borate, and 2 mmol/L EDTA, pH 8.0) PAGE and visualized by autoradiography using Kodak BIOMAX.

Nuclear Extract Preparation
Nuclear extracts were made according to the method of Dignam et al,⁴² with minor modification. Confluent HUVEC monolayers were treated as indicated in the text, washed twice with ice-cold PBS, and then harvested by scraping. Cells were then pelleted at 1000 rpm for 3 minutes in a Beckman CS-6R benchtop centrifuge. Supernatants were discarded and the pellet resuspended by the rapid addition of 5 packed cell volumes of hypotonic buffer (10 mmol/L HEPES, pH 7.9, 1.5 mmol/L MgCl₂, 10 mmol/L PMSF, and 0.5 mmol/L DTT). After immediately pelleting the cells at 1000 rpm for 5 minutes at 4°C, the cells were resuspended in 3 packed cell volumes of hypotonic buffer. Cells were then incubated for 15 minutes on ice, during which cell lysis was monitored by viewing aliquots of the reaction with an inverted microscope with Hoffman optics. Nuclei were then pelleted at 4000g, and the supernatant containing cytoplasmic factors carefully removed. The nuclei pellet was then resuspended in half a packed nuclei volume of low-salt buffer (20 mmol/L HEPES, pH 7.9, 40 mmol/L NaCl, 25% glycerol, 1.5 mmol/L EDTA, 0.2 mmol/L PMSF, 0.5 mmol/L DTT, 8 µg/mL leupeptin, and 8 µg/mL aprotinin) followed by 0.5 packed nuclei volume of high-salt buffer (as for low salt, with 1.2 mmol/L KCl), added slowly while stirring the nuclei with a pipette tip. The extraction mixture was then agitated on a shaking platform for 30 minutes at 4°C and nuclear extract recovered by spinning down the nuclei at 13 000 rpm in an Eppendorf microfuge at 4°C. Supernatants containing the nuclear extracts were then recovered and stored at -70°C before use. Protein concentration of the nuclear extracts was measured by using the Bio-Rad BCA kit (Bio-Rad).

Purification and Labeling of the Oligonucleotide Probe
NF-B–specific oligonucleotide duplexes had the following sequence with complimentary single-stranded regions at each end shown in lower case for the upper strand only: E-selectin NF-B, aattCGTTTTTGGATGCCATTGGGATTTCCTCTTTACTGGATGTG; and Ig NF-B, AACAGAGGGGACTTTCCGAGGCCATCT.

The oligonucleotides were labeled by end-filling, using DNA polymerase 1 (Klenow) fragment (Promega) (E-selectin NF-B)⁴³ or kinase end-labeling (Ig NF-B)⁴⁴ and gel-purified on a low-salt 7.5% nondenaturing PAGE.

Plasmids
The luciferase reporter gene plasmid pGM-Luc⁴⁵ includes a 655-bp fragment of the human GM-CSF promoter (-627 to 28) upstream of the luciferase gene (Promega). The chloramphenicol acetyl transferase (CAT) reporter gene plasmid pCAT-Esel contains a 463-bp fragment of the human E-selectin promoter (-383 to 80) upstream from the CAT gene (pCAT-Basic, Promega). The ß-galactosidase vector pCMVß (Clontech) containing the constitutively active cytomegalovirus (CMV) promoter was used as a cotransfectant to correct for variations in transfection efficiency. All of the expression plasmids were purified by CsCl equilibrium centrifugation.⁴⁶ The luciferase reporter gene plasmid pNF-B3 includes 3 concatomerized sequences of the E-selectin NF-B concensus sequence.

Endothelial Cell Transfection
HUVECs were cotransfected with reporter genes using electroporation, according to a method previously described.⁴⁷ In brief, 10⁷ confluent HUVECs were trypsinized into suspension and washed twice in HEPES-buffered saline. Cells were finally resuspended in 300 mL of HEPES-buffered saline containing 50 µg of pGM-Luc, 50 µg of pCAT-Esel, and 5 µg of pCMVß plasmids, then shocked in a 4-mm electroporation cuvette (Bio-Rad) at 250 V, 960 mF, giving a time constant of 20 msec. Cells were then immediately resuspended in normal growth medium and plated into 6x60-mm culture dishes precoated with 1% gelatin (Sigma). Growth medium was changed completely after an overnight incubation, removing dead cells after electroporation (50%). HDL were added to the cultures 32 hours after plating. Reporter gene activity was measured after exposure to HDL and stimulation with TNF, and levels of luciferase and CAT were normalized to ß-galactosidase activity.

Luciferase, CAT, and ß-Galactosidase Assays
Transfected cells were harvested and lysed in reporter lysis buffer (Promega), according to manufacturer's recommendations, which allow extracts to be analyzed for luciferase, ß-galactosidase, and CAT. Luciferase activity was assayed by using the Promega kit according to the manufacturer's specifications, measuring photons using a Turner Design Model 20/20 luminometer. CAT and ß-galactosidase activity was measured using kits, according to the manufacturer's recommendations (Boehringer Mannheim and Promega).

	Results

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The data shown are representative experiments where the HDL preparations used are native heterogeneous samples isolated from different donors as described in Methods. In parallel experiments, the use of reconstituted discoidal HDL particles, in which apoAI is the sole apolipoprotein and phosphatidylcholine is the sole phospholipid, gave similar results (data not shown).

If HDLs were able to inhibit cytokine-induced adhesion molecule expression by inhibiting some element of NF-B activation, then it is likely that most genes known to require the NF-B family of transcription factors should be inhibited by treatment with HDLs. We therefore examined the effect of HDL treatment on a range of cytokine-inducible genes. Preincubation of HUVECs with HDLs resulted in an inhibition of cytokine-induced adhesion molecule expression, as has been shown previously,^{34 48} although clearly having no effect on GM-CSF secretion (Figure 1) or steady-state mRNA level (Figure 2).

View larger version (71K): [in this window] [in a new window]   Figure 2. Northern blot analysis showing effect of HDLs on steady-state mRNA levels of E-selectin and GM-CSF. Confluent ECs were incubated for 20 hours without addition (NIL), or for 16 hours with no addition followed by 4 hours in the presence of TNF (1 ng/mL) (TNF), or for 16 hours with HDL (1 mg/mL apoAI) followed by 4 hours with TNF (1 ng/mL) and HDL (TNF,HDL), or for 20 hours in the presence of HDL (1 mg/mL apoAI) (HDL). In each case, 10 µg of total RNA was extracted and analyzed by northern analysis by using cDNA probes to E-selectin, GM-CSF, and GAPDH. Data are representative of 3 experiments, using HDL from 3 separate donors.

Although the proximal promoters of both E-selectin and GM-CSF have a major requirement for the NF-B family of transcription factors, the GM-CSF gene is also regulated by a distal enhancer region, located at 716 to 625 bp upstream from the transcriptional start site of GM-CSF, controlled via a cyclosporin-sensitive nuclear factor of activated T cells binding site.⁴⁹ Activation of nuclear factor of activated T cells requires both the elevation of Ca²⁺ and mobilization of protein kinase C.⁵⁰ As HDLs have been shown to be capable of inducing both these responses,^{51 52} it was possible that the lack of effect on GM-CSF synthesis is by induction of the calcium-sensitive distal enhancer region, which could mask any inhibition of the proximal promoter region. To overcome this consideration, we compared the effect of HDLs on the TNF-inducible expression of reporter genes controlled by the E-selectin and GM-CSF proximal promoters in transient transfection assays in HUVECs. Whereas HDLs had no effect on the stimulation of luciferase activity under the control of the GM-CSF promoter (-627 to 28), they suppressed, in the same cells, the ability of TNF to stimulate CAT activity under the control of the E-selectin promoter (-383 to 89) (Figure 3). As both these complex promoters have been shown to require NF-B binding for regulation, these findings suggest that the differential effect afforded by HDL treatment is likely to be independent of NF-B per se. However, as both these proximal promoter fragments contain a wide array of other transactivating factor binding sites, we examined the effect of HDL treatment on the ability to influence the cytokine induction of a reporter construct containing a series of 3 consensus E-selectin NF-B binding domains. As shown in Figure 4, HDL was unable to inhibit the cytokine-induced expression of this construct when transiently transfected into ECs.

View larger version (15K): [in this window] [in a new window]   Figure 3. HDLs inhibit the TNF-induced expression of the CAT reporter driven by the E-selectin promoter but have no effect on the expression of a luciferase reporter driven by the proximal promoter of GM-CSF. Forty-eight hours after cotransfection of confluent ECs with the E-selectin promoter (-627 to 28 bp) in a CAT reporter plasmid, the GM-CSF promoter (-388 to 80 bp) in a luciferase reporter plasmid and the ß-galactosidase reporter gene, cells were incubated either with or without HDL (1 mg/mL apoAI) for 16 hours followed by a 9-hour incubation with TNF (10 ng/mL) in the presence (HDL,TNF) or the absence (TNF) of HDL (1 mg/mL apoAI). Levels of gene activation were measured and compared with untreated cells (NIL). Levels of CAT (a) and luciferase activity (b) are expressed as relative values normalized to levels of ß-galactosidase. Values represent the mean and SD and are representative of 6 similar experiments, using HDL from 6 separate donors. Differences between means were evaluated by using Student's unpaired t test. *P, not significant; **P<0.005.

View larger version (13K): [in this window] [in a new window]   Figure 4. HDLs do not alter TNF-induced expression of a luciferase reporter construct driven by a concatomer of 3 E-selectin NF-B domains. Forty-eight hours after cotransfection of ECs with the luciferase plasmid containing 3 E-selectin NF-kB domains and the ß-galactosidase reporter gene, cells were incubated either with or without HDL (1 mg/mL apoAI) for 16 hours followed by 9 hours of incubation with TNF (1 ng/mL) in the presence (TNF,HDL) or the absence (TNF) of HDL. Levels of gene activation were measured and compared with levels of ß-galactosidase. Values represent the means and SD and are representative of 3 similar experiments, using HDL from 3 separate donors. Differences between means were evaluated by using Student's unpaired t test. *P, not significant.

To explore the effect of HDL on NF-B, we examined the effect of HDL on NF-B nuclear translocation, using EMSAs. These showed that preincubation with HDL had no effect on the binding of nuclear extracts from TNF-stimulated ECs either to the E-selectin NF-B consensus probe (Figure 5) or to the Ig NF-B consensus probe (data not shown).

View larger version (42K): [in this window] [in a new window]   Figure 5. HDLs do not alter the ability of NF-B to translocate to the nucleus and bind specific DNA sites. Confluent ECs were either untreated or incubated in the presence or absence of HDL (1 mg/mL apoAI) for 16 hours followed by a 1-hour incubation with TNF (1 ng/mL). Five micrograms of nuclear extract from each group was incubated with a double-stranded 32P-labeled E-selectin/Ig NF-B probe. Autoradiographs of the size-fractionated complexes show an absence in untreated cells (unstimulated) of NF-B complex, which is present in cytokine-stimulated cells (TNF) and not altered by preincubation of the cells with HDL (TNF,HDL). The specific NF-B complex could be competed out in the presence of excess unlabeled E-selectin NF-B (TNF,100x cold probe). The band migrating below the NF-B complex appears to represent a nonspecific constitutive factor. Data representative of 6 experiments, using HDL from 6 separate donors.

Because activation of NF-B is also controlled at the level of modulation of inhibitory binding proteins of the IB family, we examined the effects of HDL on the kinetics of IB degradation and resynthesis in HUVECs after cytokine stimulation. As shown by western analysis (Figure 6), IB was similarly reduced at 15 minutes after TNF activation, whether or not the cells had been preincubated with HDL. In a similar manner, HDL had no detectable effect on the levels of IB that had been resynthesized by 3 hours after cytokine stimulation. Once again, these data support the conclusion that differential gene regulation by HDL is independent of NF-B.

View larger version (39K): [in this window] [in a new window]   Figure 6. HDL do not affect the kinetics of degradation and resynthesis of the inhibitory protein IB. After a 16-hour incubation of confluent ECs in the presence or absence of HDL (1 mg/mL apoAI), cells were stimulated with TNF (1 ng/mL) and cell lysates prepared at times indicated. Cell extracts (10 µg/lane) were western blotted and probed with the MAD-3 anti-IB antibody. Data from each panel are representative of 3 such experiments, using HDL from 3 separate donors.

Because HDL had previously been shown to elevate prostacyclin synthesis in vascular cells,^{53 54 55} we reasoned that HDL might also be able to elevate the levels of the enzymes involved in the synthesis of this eicosonoid. Cyclooxygenase (Cox; prostaglandin G/H synthase, EC 0.1.14.99.1) may be considered the rate-limiting enzyme in the conversion of arachidonic acid to the prostacyclins. Currently, there are 2 isoforms of this enzyme, Cox-1, which is present in several cell types and is constitutively expressed and relatively stable,^{56 57} and Cox-2, which is the inducible form.^{56 57 58} As shown in Figure 7, not only could HDL alone stimulate Cox-2, but HDL synergized with TNF and IL-1ß to augment this increase. Consistent with the findings of others (Dr J. Mitchell, personal communication, 1998), IL-1 was a far more potent activator of the enzyme than TNF. We confirmed that this elevation in the level of Cox-2 was concomitant with an increase in the accumulation of prostacyclin in the culture medium, by measuring the accumulation of the stable breakdown product of PGI₂, 6-oxo-PGF₁ (Figure 8).

View larger version (40K): [in this window] [in a new window]   Figure 7. HDLs synergize with the cytokines TNF and IL-1ß to elevate the level of Cox-2 expression in ECs. Western blot analysis of whole cell lysates (10 µg/lane) prepared from confluent ECs preincubated with HDL (1 mg/mL apoAI) alone for 8 hours or preincubated with HDL for 4 hours and then treated with either TNF (1 ng/mL) or IL-1ß (1 ng/mL) and HDL for a further 4 hours. The lanes have been probed with antibody against Cox-2 (Biogenics Ltd). Results are representative of 3 experiments, using HDL from 3 separate donors.

View larger version (15K): [in this window] [in a new window]   Figure 8. HDL stimulates the accumulation of 6-oxo-PGF1. After preincubation of confluent ECs with HDL (1 mg/mL apoAI) for 16 hours, IL-1ß (1 ng/mL) or TNF (1 ng/mL) was added for a period of 16 hours after which the supernatants were assayed for levels of 6-oxo-PGF1. Levels in supernatants from cells treated with cytokine in the presence of HDL (HDL,IL-1; or HDL,TNF) and absence of HDL (IL-1 or TNF) were compared with cells incubated with HDL alone (HDL) and cells left untreated (NIL). The data are representative of 3 separate experiments, using HDL from 3 separate donors, and show mean and SD values. Differences between means were evaluated by using Student's unpaired t test. *P, not significant; **P<0.001.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study, we have shown that in addition to inhibiting the expression of adhesion molecules for leukocytes, HDL can synergize with TNF and IL-1ß to induce levels of Cox-2 and the secretion of prostacyclin. The in vitro EC phenotype induced by HDL is therefore one of reduced surface adhesiveness for leukocytes, together with increased secretion of a factor that can inhibit platelet and leukocyte activation and induce vasodilation. This phenotype might be expected to contribute to the in vivo mechanisms by which this multipotent lipoprotein reduces the risk of atherosclerosis.

Our findings that HDL have differential effects on expression of E-selectin, GM-CSF, and Cox-2 in response to IL-1ß and TNF stimulation challenge that HDL has a general inhibitory effect on the NF-B pathway. All 3 genes have been shown to have a requirement for the NF-B family of transcription factors for full cytokine-mediated expression.^{59 60 61} The mechanism by which HDLs modulate cytokine-mediated E-selectin expression is at least mediated at the transcriptional level, as we also observed inhibition of steady-state mRNA level and repression of a reporter gene driven by a full E-selectin proximal promoter. Furthermore, by using a reporter construct containing 3 tandem E-selectin NF-B binding domains, we were unable to show inhibition of expression by HDL treatment. Consistent with this, we were unable to detect any inhibition by HDL of TNF-mediated NF-B nuclear translocation or DNA binding, or a difference in the kinetics of degradation and resynthesis of IB, a process known to be NF-B dependent.^{23 24 25} These data therefore suggest that HDL primarily influences transactivating factors other than those of the NF-B pathway itself. Similar conclusions may be drawn from the recent work of Wolle et al,⁶² who have shown that flavone (PD 09806) inhibits VCAM-1 expression by human aortic ECs but does not affect the binding or translocation of NF-B or inhibit the transactivation of a reporter construct containing 2 tandem VCAM-1 NF-B sites. Further deletion analysis of the E-selectin promoter is now required to establish the specific domains involved in HDL-mediated inhibition.

The original observation that HDL could induce EC synthesis of prostacyclin was attributed to the ability of the lipoprotein to provide the substrate, arachidonic acid.^{53 54 55 62 63} Our findings that HDL synergizes with IL-1ß and, to a lesser extent TNF, to increase Cox-2 protein levels and prostacyclin production is novel, and pertinent to atherogenesis, in which both cytokines are likely to play a role.⁶⁴ Our studies offer an additional mechanism whereby prostacyclin is elevated by HDL, but do not preclude the possibility that HDL may also supply exogenous substrate for the synthesis of the eicosonoid. However, as reconstituted discoidal HDL particles, containing only apoAI as the sole protein and phosphatidylcholine as the sole phospholipid, had effects on 6-oxo-PGF₁ accumulation similar to native HDL, it is likely that HDL-induced Cox-2 elevation in confluent ECs is sufficient to stimulate the eicosonoid in the absence of exogenous arachidonic acid. Furthermore, although studies by Stuhlmeier et al⁶⁵ have shown that arachidonic acid can inhibit the TNF-induced expression of E-selectin by bovine aortic ECs, provision of this component cannot explain the broad spectrum of antiinflammatory effects of HDL, where both VCAM-1 and ICAM-1 are also inhibited.^{34 48} As recent work by Vinals et al⁶⁶ has shown that HDL also induce Cox-2 in vascular smooth muscle cells, it will be interesting to see whether the effect is also enhanced by IL-1ß and TNF.

The mechanism whereby HDL synergizes with cytokines to induce Cox-2 remains to be clarified. It has been shown that EC incubation with HDL can result in the production of lysophosphatidylcholine (LPC), and as exogenous addition of LPC to EC has been demonstrated to elevate not only Cox-2, but also ET-1, another vasoactive agent known to be elevated by HDL,⁶⁷ it is tempting to consider that the synergy we have seen is secondary to the elevation in LPC. However, elevation of LPC cannot explain the full actions of HDL, as LPC has been shown to elevate, rather than inhibit, the expression of adhesion molecules that we and others have shown to be inhibited by HDL.^{33 48} In addition, it is possible that HDL may be able to prolong messenger RNA stability of Cox-2, as has been suggested for IL-1 induction of the enzyme.⁶⁸ It is also possible that HDL may mediate its effects transcriptionally. Analyses of the transcriptional requirements of the Cox-2 promoter have been conducted in bovine aortic EC with lipopolysaccharide or 12-O-tetradecanoylphorbol 13-acetate (TPA),⁶⁹ in murine osteoblasts with TNF,⁷⁰ in rheumatoid synovial fibroblasts by using IL1ß,⁷¹ and in A549 by using IL-1ß,⁷² and all provide evidence that NF-B is required for induced expression of the enzyme. Although our data suggest that HDL does not affect NF-B translocation or transactivation in ECs, mutational analysis of the effect of HDL on cytokine-induced activation of the Cox-2 promoter may provide important clues as to the mechanism of the synergy between HDL and cytokine.

Although at face value it might seem paradoxical that HDLs inhibit expression of 1 cytokine-induced response while synergistically increasing another, it may be that the differential effects are functionally complementary. Thus, prostacyclin (PGI₂) has been shown to be a potent inhibitor of leukocyte activation and adhesion, platelet aggregation, and vascular smooth muscle cell contraction, migration, and growth, and it is also shown to inhibit cholesterol ester accumulation in vascular cells.^{73 74} The ability of HDL to enhance TNF- or IL-1ß–mediated prostacyclin production would therefore be expected to act in conjunction with the inhibition of E-selectin, VCAM-1, and ICAM-1 expression to suppress leukocyte–EC interactions and thereby to protect against the progression of atherosclerosis and its thrombotic complications.

	Acknowledgments

 
This work was supported through funding from the British Heart Foundation. G.W.C. is supported by an Intermediate Research Fellowship (FS95/059). D.O.H. is supported by a Professorial Award. The authors wish to thank Dr P. Lerch, Swiss Red Cross, for providing reconstituted discoidal HDL.

Received February 9, 1998; accepted May 11, 1998.

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				S. J. Stanford, J. R. Pepper, and J. A. Mitchell Cyclooxygenase-2 Regulates Granulocyte-Macrophage Colony-Stimulating Factor, but Not Interleukin-8, Production by Human Vascular Cells : Role of cAMP Arterioscler Thromb Vasc Biol, March 1, 2000; 20(3): 677 - 682. [Abstract] [Full Text] [PDF]

				J.-R. Nofer, B. Levkau, I. Wolinska, R. Junker, M. Fobker, A. von Eckardstein, U. Seedorf, and G. Assmann Suppression of Endothelial Cell Apoptosis by High Density Lipoproteins (HDL) and HDL-associated Lysosphingolipids J. Biol. Chem., September 7, 2001; 276(37): 34480 - 34485. [Abstract] [Full Text] [PDF]

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