This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Marx, N. Articles by Plutzky, J. Search for Related Content PubMed PubMed Citation Articles by Marx, N. Articles by Plutzky, J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Related Collections Coagulation Lipid and lipoprotein metabolism Mechanism of atherosclerosis/growth factors Gene expression Gene regulation

(Circulation. 2001;103:213.)
© 2001 American Heart Association, Inc.

Clinical Investigation and Reports

PPAR Activators Inhibit Tissue Factor Expression and Activity in Human Monocytes

Nikolaus Marx, MD; Nigel Mackman, PhD; Uwe Schönbeck, PhD; Nurcan Yilmaz, MS; Vinzenz Hombach, MD; Peter Libby, MD; Jorge Plutzky, MD

From the Department of Internal Medicine II–Cardiology, University of Ulm (N. Marx, N.Y., V.H.), Ulm, Germany; the Departments of Immunology and Vascular Biology, The Scripps Research Institute (N. Mackman), La Jolla, Calif; and the Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School (U.S., P.L., J.P.), Boston, Mass.

Correspondence to Nikolaus Marx, MD, Department of Internal Medicine II, Cardiology, University of Ulm, Robert-Koch-Straße 8, D-89081 Ulm, Germany. E-mail nikolaus.marx{at}medizin.uni-ulm.de

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Tissue factor (TF), expressed on the surface of monocytes and macrophages in human atherosclerotic lesions, acts as the major procoagulant initiating thrombus formation in acute coronary syndromes. Peroxisome proliferator–activated receptor- (PPAR), a nuclear receptor family member, regulates gene expression in response to certain fatty acids and fibric acid derivatives. Given that some of these substances reduce TF activity in patients, we tested whether PPAR activators limit TF responses in human monocytic cells.

Methods and Results—Pretreatment of freshly isolated human monocytes or monocyte-derived macrophages with PPAR activators WY14643 and eicosatetraynoic acid (ETYA) led to reduced lipopolysaccharide (LPS)-induced TF activity in a concentration-dependent manner (maximal reduction to 43±8% with 250 µmol/L WY14643 [P<0.05, n=5] and to 42±12% with 30 µmol/L ETYA [P>0.05, n=3]). Two different PPAR activators (15-deoxy^_12,14-prostaglandin J₂ and BRL49653) lacked similar effects. WY14643 also decreased tumor necrosis factor- protein expression in supernatants of LPS-stimulated human monocytes. Pretreatment of monocytes with WY14643 inhibited LPS-induced TF protein and mRNA expression without altering mRNA half-life. Transient transfection assays of a human TF promoter construct in THP-1 cells revealed WY14643 inhibition of LPS-induced promoter activity, which appeared to be mediated through the inhibition of nuclear factor-B but not to be due to reduced nuclear factor-B binding.

Conclusions—PPAR activators can reduce TF expression and activity in human monocytes/macrophages and thus potentially reduce the thrombogenicity of atherosclerotic lesions. These data provide new insight into how PPAR-activating fibric acid derivatives and certain fatty acids might influence atherothrombosis in patients with vascular disease.

Key Words: leukocytes • thrombosis • genes • coagulation

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Tissue factor (TF) is an integral membrane protein, which binds to factor VII/VIIa and initiates the coagulation cascade.^{1 2} Expressed on the surface of lipid-laden monocytes, macrophages, and foam cells in human atherosclerotic lesions, TF acts as the major procoagulant for thrombus formation in acute coronary syndromes.^{3 4} The expression of TF in monocytes can be rapidly induced by lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate as well as by an inflammatory mediator found in atherosclerotic lesions, CD40L.^{5 6} TF activity of circulating monocytes appears controlled locally by its intrinsic inhibitor, TF pathway inhibitor (TFPI).⁷ LPS-induced monocyte TF expression is regulated through a 56-bp promoter region (-227 through -172 bp) containing 2 activator protein-1 (AP-1) binding sites and a nuclear factor (NF)-B binding site, elements that bind c-fos/c-jun and c-Rel/p65 heterodimers, respectively.^{8 9} LPS-induced TF expression in cells of the monocyte lineage requires functional interaction between these transcription factors.¹⁰

Although the clinical course of acute coronary events may be influenced by reduced monocyte/macrophage TF expression,¹¹ the mechanisms inhibiting TF expression in these cells remain largely unexplored. Animal studies suggest that a diet rich in polyunsaturated fatty acids (PUFAs) reduces TF expression in mononuclear cells,¹² whereas dietary intake of PUFAs in humans reduces TF activity in unstimulated and LPS-stimulated monocytes.¹³ Thus, PUFAs seem to inhibit monocyte TF expression in vivo, although through unknown mechanisms. Interestingly, some of the PUFAs used in those studies (docosahexaenoic acid [DHA] and eicosatetraynoic acid [ETYA]) are ligands for the peroxisome proliferator–activated receptor- (PPAR).¹⁴ PPAR, as well as the other members of the PPAR family, PPAR and PPAR, are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily.¹⁵ PPAR can be activated by PUFAs as well as lipid-lowering fibric acid derivatives, such as fenofibrate or WY14643.¹⁶ Although prior work focused on PPAR as a regulator of genes involved in lipid metabolism and fatty acid oxidation,¹⁴ recent studies have highlighted its anti-inflammatory role in vascular cells.^{17 18} The effect of PPAR activation on monocyte TF expression is unknown.

Given the clinical evidence that fibric acid drugs and PUFA-rich diets may reduce thrombotic complications of atherosclerosis,¹² the present study tested whether PPAR activation limits inducible TF activity and expression in human cells of the monocytic lineage.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cell Culture
Human monocytes isolated from freshly drawn blood of healthy volunteers by sequential Ficoll-Hypaque (Sigma) and Percoll (Sigma) gradient centrifugation were >85% pure. Monocyte-derived macrophages were obtained by culturing isolated human monocytes for 6 days (RPMI media, GIBCO; 5% human serum, Sigma; and 1% penicillin-streptomycin, GIBCO). The monocyte-like cell line THP-1, obtained from the American Tissue Culture Collection, was cultured as described before.¹⁹

Reverse Transcriptase–Polymerase Chain Reaction
Total RNA from freshly isolated monocytes, monocyte-derived macrophages, or THP-1 cells was isolated by the guanidinium thiocyanate–phenol–chloroform method (RNAzol, Tel-Test). Reverse transcriptase (RT)–polymerase chain reaction (PCR) for PPAR and GAPDH was performed as described before.²⁰

TF Activity Assay
For TF activity assays, monocytes, monocyte-derived macrophages, or THP-1 cells were pretreated with PPAR activators (PPAR activators: ETYA, Sigma, and WY14643, Biomol; PPAR activators: 15-deoxy^_12,14-prostaglandin J₂ [15d-PGJ₂], Calbiochem, and BRL49653, SmithKline Beecham) for 30 minutes at the concentrations indicated and then LPS-stimulated (100 µg/L for monocytes, 10 mg/L for THP-1 cells) for 5 hours. TF activity was determined by using a standard chromogenic assay (American Diagnostica).⁶

TNF- ELISA
Monocytes or THP-1 cells (1x10⁶ cells/mL) were pretreated with the PPAR activator WY14643 for 30 minutes before LPS stimulation at the concentrations indicated. Cells were cultured for 24 hours, and a tumor necrosis factor (TNF)- ELISA (R&D Systems) was performed on cell-free supernatants by using a recombinant TNF- standard curve.

Western Blot Analysis
For Western blot analysis of TF and TFPI expression, human monocytes or monocyte-derived macrophages were pretreated with WY14643 (30 minutes) at the concentrations indicated and stimulated with LPS (5 hours). Standard Western blot analysis on total cell lysates was performed by using mouse anti-human TF and mouse anti-human TFPI antibodies (monoclonal antibodies, American Diagnostica). Restaining with -tubulin antibodies ensured equal loading of intact protein.

RNA Extraction and Northern Blot Analysis
For Northern blot experiments, cells were pretreated with the PPAR or PPAR activators for 30 minutes and then LPS-stimulated (100 µg/L, 2 hours). Five micrograms of total RNA was used in standard Northern blot analysis with the use of TF, TNF-, or GAPDH cDNA probes.

TF mRNA half-life was determined by stimulating monocytes with LPS for 2 hours before blocking transcription with actinomycin D (5 µg/L). Cells then received WY14643 for the times indicated, and mRNA levels were compared with untreated cells.

Transient Transfection Assays
To investigate the effect of PPAR activators on TF or TNF- promoter activity, we transiently transfected THP-1 cells with TF promoter constructs containing the luciferase reporter [pTF(-2106)-LUC and pTNF--LUC] as previously described.⁹ To determine the effect of PPAR on NF-B activation, p(B)₄LUC, a luciferase reporter construct containing 4 copies of the TF-B site cloned upstream from the minimal simian virus 40 promoter⁹ was transfected into THP-1 cells. Transfected cells were cultured for 46 hours before stimulation with LPS (10 mg/L) for 5 hours. Cells were harvested, and luciferase activity was measured as described before.¹⁹

Electrophoretic Mobility Shift Assay
Nuclear extracts of THP-1 cells were prepared by using standard procedures.¹⁹ THP-1 cells were stimulated for 2 hours with LPS (10 µg/mL) with or without WY14643 (10 or 100 µmol/L) pretreatment before nuclear extract preparation. Oligonucleotides for the TF-specific NF-B site (5'-GTCCCGGAGTTTCCTACC-3') or the prototypic site from the murine Ig gene were annealed with a complementary primer and radiolabeled with the use of [-³²P]dCTP (ICN) as described.¹⁹ Protein-DNA complexes were separated from free DNA probe by electrophoresis (6% nondenaturing acrylamide gels/0.5x Tris-borate-EDTA), and autoradiography was performed.

Assessment of Total Protein Synthesis
To determine the effects of WY14643 on total protein synthesis in human monocytes, cells were treated with LPS in the absence or presence of WY14643 in media containing [³⁵S]methionine (0.2 Ci/mL) (Table). After 5 hours, cells were harvested, and total protein synthesis in both lysates and supernatants was measured by counting radioactivity after cold trichloroacetic acid precipitation.¹⁸

View this table: [in this window] [in a new window]   Table 1. Effects of WY14643 on Human Monocytes

Statistical Analysis
Results of the experimental studies are reported as mean±SEM or mean±SD. Differences were analyzed by 1-way ANOVA followed by the Fisher least significant difference test. A value of P<0.05 was regarded as significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Cells of Monocyte Lineage Express PPAR
To establish the expression of PPAR in cells of the monocyte lineage used in our experiments, RT-PCR was performed. Freshly isolated human monocytes, monocyte-derived macrophages, and monocyte-like THP-1 cells all contained PPAR mRNA as detected by a 276-bp RT-PCR product (Figure 1).

View larger version (57K): [in this window] [in a new window]   Figure 1. Cells of monocyte lineage express PPAR. RT-PCR reaction of PPAR and GAPDH RNA in freshly isolated human monocytes (Mo), monocyte-derived macrophages (M), and monocytic THP-1 cells (THP) reveals appropriately sized cDNA fragment. Also shown are DNA ladder (MW) and negative control consisting of RT-PCR reactions lacking reverse transcriptase (Co).

PPAR Activators Inhibit LPS-Induced TF Activity and TNF- Expression in Human Monocytes
To determine the effect of PPAR activation on LPS-induced TF activity in freshly isolated human monocytes, purified cells were pretreated for 30 minutes with the PPAR activators WY14643 or ETYA at the concentrations indicated and then stimulated for 5 hours with LPS (100 µg/L) before TF activity assays were performed. As expected, monocyte TF activity markedly increased in response to LPS stimulation. Pretreatment of cells with the PPAR activators WY14643 (Figure 2A) and ETYA (Figure 2B) inhibited LPS-induced TF activity in a concentration-dependent manner, with a maximal reduction at 250 µmol/L WY14643 or 30 µmol/L ETYA to 43±8% (P<0.05, n=5) or 42±12% (P<0.05, n=3), respectively. Two different PPAR activators, the naturally occurring ligand 15d-PGJ₂ and the antidiabetic agent BRL49653 (rosiglitazone), had no significant effect (P=NS, n=3; Figure 2B). PPAR activators alone also had no effect on TF activity (data not shown). Cell viability in all conditions was >95%, as determined by trypan blue exclusion, suggesting that enhanced cell death is not responsible for these observations. In addition, WY14643 did not affect total protein synthesis in LPS-treated human monocytes, thus making toxic effects of PPAR activators an unlikely explanation for our findings (Table).

View larger version (20K): [in this window] [in a new window]   Figure 2. PPAR but not PPAR activators inhibit LPS-induced TF activity and TNF- protein expression in human monocytes. A and B, Freshly isolated human monocytes were pretreated with PPAR (WY14643 [WY] and ETYA) or PPAR (15d-PGJ2 [PGJ2] and BRL49653 [BRL]) activators for 30 minutes at concentrations indicated and then stimulated with LPS (100 µg/L) for 5 hours before TF activity assays were performed. Results are expressed as percentage of LPS-stimulated cells (% control). Bars represent mean±SEM (WY, n=5; ETYA, 15d-PGJ2, and BRL, n=3). *P<0.05 compared with control. C, For analysis of TNF- expression in cell-free supernatants of isolated monocytes, cells were stimulated as described above, and TNF- ELISA was performed. Bars represent mean±SEM (n=3). *P<0.05 compared with LPS-treated cells.

To investigate whether PPAR alters other LPS-induced genes in human monocytes, we measured the effect of WY14643 on monocyte TNF- protein secretion after LPS stimulation. Pretreatment of monocytes for 30 minutes with the PPAR activator WY14643 significantly reduced LPS-induced TNF- protein content in the supernatant (Figure 2C), with a maximal inhibition at 250 µmol/L WY14643 (7696±449 pg/mL in LPS-treated cells versus 4490±197 pg/mL in LPS- and WY14643-treated cells, P<0.05; n=3). As previously reported,²¹ 2 PPAR activators had no effect on LPS-induced TNF- secretion.

PPAR Activation Inhibits TF Protein Expression in Human Monocytes
To test whether the effect of PPAR on TF activity corresponded to changes in TF protein levels, human monocytes were pretreated and stimulated as indicated, and Western Blot analysis was performed on total cell lysates. Stimulation of monocytes with LPS markedly increased TF protein expression, whereas 30-minute pretreatment with the PPAR activator WY14643 reduced cellular TF protein content in a concentration-dependent manner (Figure 3, top). In contrast, WY14643 did not reduce the expression of TFPI, the intrinsic inhibitor of TF activity (Figure 3, middle).

View larger version (54K): [in this window] [in a new window]   Figure 3. PPAR activators inhibit TF protein expression in human monocytes. Western blot analysis of TF and TFPI expression in freshly isolated human monocytes stimulated with LPS (100 µg/L) for 5 hours with or without WY14643 pretreatment (30 minutes). Equal loading of intact protein was confirmed by restaining with -tubulin antibodies. Similar results were obtained in 3 independent experiments.

PPAR, but Not PPAR, Activators Reduce LPS-Induced TF mRNA Levels in Human Monocytes
Northern blot analysis was used for the evaluation of a potential effect of PPAR ligands at the mRNA level. Increased TF mRNA levels after a 2-hour stimulation of human monocytes with LPS (100 µg/L) could be inhibited in a concentration-dependent manner by 30-minute pretreatment with the PPAR activator WY14643 (Figure 4A). In contrast, pretreatment of monocytes with 2 different PPAR activators, 15d-PGJ₂ (10 µmol/L) and BRL49653 (10 µmol/L), had no such effect (Figure 4B). In addition, WY14643 also decreased LPS-induced TNF- mRNA expression (Figure 4C). Stimulation experiments in the presence of actinomycin D revealed that WY14643 did not significantly reduce TF mRNA half-life compared with the control condition (Figure 4D), indicating that PPAR activators affect TF transcription but not mRNA stability.

View larger version (42K): [in this window] [in a new window]   Figure 4. PPAR activator WY14643 inhibits TF and TNF- mRNA expression. A, Northern blot analysis for TF in human monocytes pretreated for 30 minutes with WY at concentrations shown before stimulation with LPS for 2 hours (100 µg/L) (top). B, Northern blot analysis for TF of human monocytes pretreated with PPAR (250 µmol/L WY) or PPAR activators (10 µmol/L PGJ2 and 10 µmol/L BRL) for 30 minutes and then stimulated with LPS for 2 hours. C, TNF- mRNA expression in human monocytes stimulated as indicated above. GAPDH mRNA expression confirmed equal loading of intact RNA in all experiments (A, B, and C, bottom). Three independent experiments yielded similar results. D, Graph showing that PPAR activation does not affect TF mRNA half-life. Actinomycin D and WY were added to human monocytes 2 hours after LPS stimulation (0 hours), and cells were harvested at times indicated. Amount of mRNA at each time point was compared with mRNA levels after 2 hours of LPS stimulation at time 0 (ordinate labeled as relative mRNA level). Co indicates control. Results are shown as mean±SEM of 3 independent experiments.

WY14643 Reduces LPS-Induced TF Activity as Well as TF Protein and mRNA Expression in Monocyte-Derived Macrophages
To determine whether PPAR activators had similar effects on TF activity in a more macrophage-like cell type, human monocyte–derived macrophages were used for stimulation experiments. As expected, these cells had higher baseline TF activity than did monocytes. Pretreatment of monocyte-derived macrophages with WY14643 reduced LPS-induced TF activity in a concentration-dependent manner with a maximal reduction at 100 µmol/L to 40.3±7.6% (P<0.05 compared with LPS-stimulated cells, Figure 5A). In addition, WY14643 also decreased LPS-induced TF protein (Figure 5B) and mRNA expression (Figure 5C) in these cells.

View larger version (26K): [in this window] [in a new window]   Figure 5. PPAR activator WY14643 reduces TF activity and expression in human monocyte–derived macrophages. A, TF activity assays for monocyte-derived macrophages were performed after 30-minute pretreatment with PPAR activator WY14643, followed by 5-hour stimulation with LPS (100 µg/L). Results are expressed as percentage of LPS-stimulated cells (% control). Bars represent mean±SEM (n=3). *P<0.05 compared with control. B, Western blot analysis for TF protein expression in monocyte-derived macrophages (top). Equal loading of intact protein was confirmed by restaining membrane with -tubulin antibodies (bottom). C, Northern blot analysis for TF in human monocyte–derived macrophages pretreated for 30 minutes with WY14643 at concentrations shown before stimulation with LPS (100 µg/L) for 2 hours (top). GAPDH mRNA expression confirmed equal loading of intact RNA (bottom). Three independent experiments yielded similar results.

WY14643 Inhibits LPS-Induced TF Activity and TNF- Protein Expression in Monocyte-Like THP-1 Cells
Because prior studies have successfully used monocytic THP-1 cells for investigating the regulation of TF expression,^{9 19} we used this cell line to study responsiveness to PPAR agonism in anticipation of promoter analysis. Treatment of THP-1 cells with the PPAR activator WY14643 decreased LPS-induced (10 mg/L) TF activity as well as TNF- protein expression in a concentration-dependent fashion (Figure 6). Maximal reduction of TF activity to 9.3±0.4% was achieved in cells treated with 100 µmol/L WY14643 compared with LPS-stimulated cells. WY14643 decreased TNF- protein expression from 1396±58 pg/mL in LPS-treated cells to 144±8 pg/mL. These data indicate that the more readily transfectable monocyte-like THP-1 cells can be used to examine the effects of PPAR activators on TF and TNF-.

View larger version (11K): [in this window] [in a new window]   Figure 6. PPAR activator WY14643 reduces TF activity and TNF- protein expression in THP-1 monocytoid cells. THP-1 cells were pretreated with WY14643 for 30 minutes at concentrations indicated before stimulation with LPS at 10 mg/L. After 5 hours, cellular TF activity and TNF- protein content in cell-free supernatants were measured. Results are expressed in comparison with LPS-treated cells (% control) for TF activity. Bars represent mean±SEM (n=2).

PPAR Activation Inhibits LPS-Induced TF and TNF- Promoter Activity
THP-1 cells were transiently transfected with TF (pTF-LUC) or TNF- (pTNF-LUC) promoter-reporter constructs to determine the effects of PPAR activation on LPS-induced promoter activity. Stimulation of transfected cells with LPS (10 mg/L) increased TF promoter activity 7.8±1.8-fold, whereas 30-minute pretreatment with WY14643 reduced this increase to 3.3±1.1-fold (Figure 7A). Similar results were obtained when cells were transfected with the TNF- promoter construct (Figure 7B). WY14643 alone had no effect on TF or TNF- promoter activity.

View larger version (8K): [in this window] [in a new window]   Figure 7. PPAR activation inhibits TF and TNF- promoter activity. THP-1 cells were transiently transfected with promoter reporter constructs pTF-LUC (A) and pTNF-LUC (B) and stimulated with LPS (10 mg/L) for 5 hours with or without PPAR activator WY (100 µmol/L) pretreatment. Results of luciferase activity are expressed as fold induction compared with unstimulated cells. Bars represent mean±SD (n=3).

PPAR Activator WY14643 Reduces NF-B Activation but Does Not Inhibit DNA Binding of NF-B Transcription Factors p65/c-Rel
To assess the effect of PPAR activation on LPS-induced NF-B activation, transient transfection experiments of reporter construct p(B)₄LUC were performed. Stimulation of transfected cells with LPS (10 µg/mL) induced a 50±5-fold increase in luciferase activity, which was reduced by 30% to 35±7-fold by WY14643 (Figure 8A). To investigate whether PPAR activation inhibits binding of NF-B transcription factors to the TF promoter, we performed gel-shift analysis (electrophoretic mobility shift assay) with the use of oligonucleotides corresponding to the TF NF-B site. PPAR activation did not inhibit LPS-induced nuclear translocation and DNA binding of the NF-B proteins to the TF promoter (Figure 8B, left) nor did PPAR activation alter binding of the NF-B proteins p50/p65, known to regulate monocyte TNF- gene expression (Figure 8B, right).

View larger version (15K): [in this window] [in a new window]   Figure 8. PPAR activator WY14643 (WY) reduces nuclear NF-B activity but does not inhibit DNA binding of NF-B transcription factors. A, THP-1 cells transiently transfected with reporter construct p(B)4LUC containing 4 copies of TF NF-B site. Transfected cells were stimulated as described above, and luciferase activity was determined. Results are expressed as fold induction compared with unstimulated cells. Bars represent mean±SD (n=3). B, Gel-shift analysis of THP-1 cells pretreated with WY14643 (Wy, 100 µmol/L) and then stimulated with LPS (10 mg/L) for 2 hours. Electrophoretic mobility shift assays were performed by using NF-B site of TF promoter (left) and prototypic NF-B site of the murine Ig gene (right).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrates inhibition of LPS-induced TF and TNF- expression by PPAR activators in human monocytes and monocyte-derived macrophages. This effect is mediated through an inhibition of gene transcription, likely through changes in NF-B activation.

Human monocytes and macrophages express PPAR as well as PPAR,^{21 22 23 24} although the amount of PPAR compared with PPAR may be lower in these cells.²⁴ In monocytes, PPAR increases during differentiation, and its stimulation can inhibit the expression of genes implicated in atherosclerosis.^{21 22 23 24} However, we did not observe any effects of PPAR activators on LPS-induced TF or TNF- expression. These findings concur with previous reports showing that PPAR activators inhibit phorbol 12-myristate 13-acetate but not LPS-induced TNF- expression.²¹ The lack of an effect by PPAR activators on TF and TNF- expression might result from lower levels of PPAR in these cells or may indicate different PPAR subtype specificity. The PPAR activators used in the present study, the fibric acid derivative WY14643 and the PUFA ETYA, likely inhibit TF expression via effects on PPAR. Both agents are known to be selective PPAR activators.¹⁶ Our experiments also revealed that lower concentrations of WY14643 yielded similar effects on TF activity and expression in monocyte-derived macrophages (60% reduction at 100 µmol/L WY14643) compared with freshly isolated monocytes (57% reduction at 250 µmol/L WY14643). This finding might reflect higher PPAR expression levels in macrophages as previously described by Chinetti et al.²⁴

The reduction of TF activity, protein, and mRNA expression by PPAR activators likely results from inhibition of gene transcription given its inhibition of LPS-induced TF promoter activity as well as the lack of an effect of WY14643 on TF mRNA half-life. The reduction of NF-B activation by PPAR parallels our observation that PPAR activators also decrease LPS-induced expression and promoter activity of TNF-, another NF-B–regulated gene. Gel-shift experiments suggest that PPAR does not inhibit LPS-induced nuclear translocation or DNA binding of NF-B transcription factors to the TF promoter. Several models might account for the effect of PPAR on functional NF-B activity independent of DNA-protein complex assembly. The NF-B proteins c-Rel/p65 and PPAR have been shown to interact with several transcriptional coactivators, such as cAMP response element–binding protein or p300^{25 26} ; competition for such coactivators might inhibit NF-B signaling through a "sequestering" effect. Alternatively, a direct protein-protein interaction between PPAR and c-Rel/p65 or between PPAR and another coactivator might allow binding of NF-B proteins to the promoter but prevent functional activation of NF-B, as suggested previously.²⁷

Other mechanisms in addition to the 30% inhibition of NF-B activation suggested by the p(B)₄LUC construct experiments might account for the effects of PPAR on TF and TNF-. Because AP-1 is implicated in TF expression, its inhibition by PPAR could also be involved.²⁷ Alternatively, the effects of PPAR might engage a transcriptional repressor complex with corepressors such as N-CoR, as previously suggested in the context of the effects of retinoic acid receptor on TF expression.²⁸

Our findings that PPAR activators reduce monocyte TF expression but not the expression of the intrinsic inhibitor of TF activity, TFPI, suggest a PPAR-mediated shift toward limiting a critical procoagulant in atherosclerosis, an effect with possible important in vivo relevance. Diets enriched in PPAR-activating PUFAs can limit the occurrence and course of acute coronary events,²⁹ and clinical studies with fibric acid derivatives demonstrate reduced cardiovascular events in patients with coronary heart disease.³⁰ The regulation by PPAR of monocyte and macrophage TF expression may contribute to these reported benefits, a particular intriguing possibility given recent data from the Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial (VA-HIT), which demonstrated that fibric acid derivatives decrease cardiac events in coronary heart disease patients with low LDL levels despite only a modest HDL increase.³¹ The observations reported in the present study may offer one molecular mechanism contributing to how PUFA-rich diets and fibrates might reduce acute coronary events.

	Acknowledgments

 
This work was supported by grants from the Deutsche Forschungsgemeinschaft to Dr Marx (MA 2047/2-1), from the National Institutes of Health (NIH) (HL-48872) to Dr Mackman, from an American Heart Association, Massachusetts Affiliate, Paul Dudley White fellowship to Dr Schönbeck, from NIH (MERIT Award, HL-34636) to Dr Libby, and from the American Diabetes Association Research Award and the Partners/Nesson Research Award to Dr Plutzky. We thank Miriam Grüb, Bettina Kehrle (University of Ulm), and Angela Hollis (Scripps Research Institute) for their assistance.

Received August 10, 2000; revision received August 22, 2000; accepted August 23, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Nemerson Y. Tissue factor and hemostasis. Blood. 1988;71:1–8.[Free Full Text]
   
2. Rapaport SI, Rao LV. Initiation and regulation of tissue factor–dependent blood coagulation. Arterioscler Thromb. 1992;12:1111–1121.[Medline] [Order article via Infotrieve]
   
3. Wilcox JN, Smith KM, Schwartz SM, et al. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci U S A. 1989;86:2839–2843.[Abstract/Free Full Text]
   
4. Ardissino D, Merlini PA, Ariens R, et al. Tissue-factor antigen and activity in human coronary atherosclerotic plaques. Lancet. 1997;349:769–771.[Medline] [Order article via Infotrieve]
   
5. Rivers RP, Hathaway WE, Weston WL. The endotoxin-induced coagulant activity of human monocytes. Br J Haematol. 1975;30:311–316.[Medline] [Order article via Infotrieve]
   
6. Mach F, Schonbeck U, Bonnefoy JY, et al. Activation of monocyte/macrophage functions related to acute atheroma complication by ligation of CD40: induction of collagenase, stromelysin, and tissue factor. Circulation. 1997;96:396–399.[Abstract/Free Full Text]
   
7. McGee MP, Foster S, Wang X. Simultaneous expression of tissue factor and tissue factor pathway inhibitor by human monocytes: a potential mechanism for localized control of blood coagulation. J Exp Med. 1994;179:1847–1854.[Abstract/Free Full Text]
   
8. Mackman N, Brand K, Edgington TS. Lipopolysaccharide-mediated transcriptional activation of the human tissue factor gene in THP-1 monocytic cells requires both activator protein 1 and nuclear factor kappa B binding sites. J Exp Med. 1991;174:1517–1526.[Abstract/Free Full Text]
   
9. Oeth PA, Parry GC, Kunsch C, et al. Lipopolysaccharide induction of tissue factor gene expression in monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site. Mol Cell Biol. 1994;14:3772–3781.[Abstract/Free Full Text]
   
10. Mackman N. Regulation of the tissue factor gene. Thromb Haemost. 1997;78:747–754.[Medline] [Order article via Infotrieve]
    
11. Fuster V, Badimon J, Chesebro JH, et al. Plaque rupture, thrombosis, and therapeutic implications. Haemostasis. 1996;26(suppl 4:269–284.
    
12. Harker LA, Kelly AB, Hanson SR, et al. Interruption of vascular thrombus formation and vascular lesion formation by dietary n-3 fatty acids in fish oil in nonhuman primates. Circulation. 1993;87:1017–1029.[Abstract/Free Full Text]
    
13. Tremoli E, Eligini S, Colli S, et al. n-3 fatty acid ethyl ester administration to healthy subjects and to hypertriglyceridemic patients reduces tissue factor activity in adherent monocytes. Arterioscler Thromb. 1994;14:1600–1608.[Abstract/Free Full Text]
    
14. Keller H, Dreyer C, Medin J, et al. Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers. Proc Natl Acad Sci U S A. 1993;90:2160–2164.[Abstract/Free Full Text]
    
15. Schoonjans K, Martin G, Staels B, et al. Peroxisome proliferator-activated receptors, orphans with ligands and functions. Curr Opin Lipidol. 1997;8:159–166.[Medline] [Order article via Infotrieve]
    
16. Forman BM, Chen J, Evans RM. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta. Proc Natl Acad Sci U S A. 1997;94:4312–4317.[Abstract/Free Full Text]
    
17. Staels B, Koenig W, Habib A, et al. Activation of human aortic smooth-muscle cells is inhibited by PPARalpha but not by PPARgamma activators. Nature. 1998;393:790–793.[Medline] [Order article via Infotrieve]
    
18. Marx N, Sukhova G, Collins T, et al. PPAR activators inhibit cytokine-induced vascular cell adhesion molecule 1 expression in human endothelial cells. Circulation. 1999;99:3125–3131.[Abstract/Free Full Text]
    
19. Oeth P, Yao J, Fan ST, et al. Retinoic acid selectively inhibits lipopolysaccharide induction of tissue factor gene expression in human monocytes. Blood. 1998;91:2857–2865.[Abstract/Free Full Text]
    
20. Marx N, Schönbeck U, Lazar MA, et al. Peroxisome proliferator activated receptor gamma activators inhibit gene expression and migration in human vascular smooth muscle cells. Circ Res. 1998;83:1097–1103.[Abstract/Free Full Text]
    
21. Jiang C, Ting AT, Seed B. PPARgamma agonists inhibit production of monocyte inflammatory cytokines. Nature. 1998;391:82–86.[Medline] [Order article via Infotrieve]
    
22. Ricote M, Li AC, Willson TM, et al. The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature. 1998;391:79–82.[Medline] [Order article via Infotrieve]
    
23. Marx N, Sukhova G, Murphy C, et al. Macrophages in human atheroma contain PPARgamma: differentiation-dependent peroxisome proliferator-activated receptor gamma expression and reduction of MMP-9 activity through PPARgamma activation in mononuclear phagocytes in vitro. Am J Pathol. 1998;153:17–23.[Abstract/Free Full Text]
    
24. Chinetti G, Griglio S, Antonucci M, et al. Activation of proliferator-activated receptors alpha and gamma induces apoptosis of human monocyte-derived macrophages. J Biol Chem. 1998;273:25573–25580.[Abstract/Free Full Text]
    
25. Parry GC, Mackman N. Role of cyclic AMP response element-binding protein in cyclic AMP inhibition of NF-kappaB-mediated transcription. J Immunol. 1997;159:5450–5456.[Abstract]
    
26. Dowell P, Ishmael JE, Avram D, et al. p300 functions as a coactivator for the peroxisome proliferator-activated receptor alpha. J Biol Chem. 1997;272:33435–33443.[Abstract/Free Full Text]
    
27. Delerive P, De Bosscher K, Besnard S, et al. Peroxisome proliferator-activated receptor alpha negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappaB and AP-1. J Biol Chem. 1999;274:32048–32054.[Abstract/Free Full Text]
    
28. Chen JD, Evans RM. A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature. 1995;377:454–457.[Medline] [Order article via Infotrieve]
    
29. De Lorgeril M, Salen P, Martin JL, et al. Effect of a Mediterranean type of diet on the rate of cardiovascular complications in patients with coronary artery disease: insights into the cardioprotective effect of certain nutriments. J Am Coll Cardiol. 1996;28:1103–1108.[Abstract]
    
30. Frick MH, Elo O, Haapa K, et al. Helsinki Heart Study. primary-prevention trial with gemfibrozil in middle-aged men with dyslipidemia: safety of treatment, changes in risk factors, and incidence of coronary heart disease. N Engl J Med. 1987;317:1237–1245.[Abstract]
    
31. Rubins HB, Robins SJ, Collins D, et al. Gemfibrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol: Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study Group. N Engl J Med. 1999;341:410–418.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				E. Rigamonti, G. Chinetti-Gbaguidi, and B. Staels Regulation of Macrophage Functions by PPAR-{alpha}, PPAR-{gamma}, and LXRs in Mice and Men Arterioscler. Thromb. Vasc. Biol., June 1, 2008; 28(6): 1050 - 1059. [Abstract] [Full Text] [PDF]

				K. Wang and Y.-J. Y. Wan Nuclear Receptors and Inflammatory Diseases Experimental Biology and Medicine, May 1, 2008; 233(5): 496 - 506. [Abstract] [Full Text] [PDF]

				M. Bouwens, L. A Afman, and M. Muller Fasting induces changes in peripheral blood mononuclear cell gene expression profiles related to increases in fatty acid {beta}-oxidation: functional role of peroxisome proliferator activated receptor {alpha} in human peripheral blood mononuclear cells Am. J. Clinical Nutrition, November 1, 2007; 86(5): 1515 - 1523. [Abstract] [Full Text] [PDF]

				J. D. Brown and J. Plutzky Peroxisome Proliferator Activated Receptors as Transcriptional Nodal Points and Therapeutic Targets Circulation, January 30, 2007; 115(4): 518 - 533. [Abstract] [Full Text] [PDF]

				A. Zambon, P. Gervois, P. Pauletto, J.-C. Fruchart, and B. Staels Modulation of Hepatic Inflammatory Risk Markers of Cardiovascular Diseases by PPAR-{alpha} Activators: Clinical and Experimental Evidence Arterioscler. Thromb. Vasc. Biol., May 1, 2006; 26(5): 977 - 986. [Abstract] [Full Text] [PDF]

				D. Walcher, A. Kummel, B. Kehrle, H. Bach, M. Grub, R. Durst, V. Hombach, and N. Marx LXR Activation Reduces Proinflammatory Cytokine Expression in Human CD4-Positive Lymphocytes Arterioscler. Thromb. Vasc. Biol., May 1, 2006; 26(5): 1022 - 1028. [Abstract] [Full Text] [PDF]

				J. Steffel, T. F. Luscher, and F. C. Tanner Tissue Factor in Cardiovascular Diseases: Molecular Mechanisms and Clinical Implications Circulation, February 7, 2006; 113(5): 722 - 731. [Abstract] [Full Text] [PDF]

				B. Staels and J.-C. Fruchart Therapeutic Roles of Peroxisome Proliferator-Activated Receptor Agonists Diabetes, August 1, 2005; 54(8): 2460 - 2470. [Abstract] [Full Text] [PDF]

				A. C. Li and C. K. Glass PPAR- and LXR-dependent pathways controlling lipid metabolism and the development of atherosclerosis J. Lipid Res., December 1, 2004; 45(12): 2161 - 2173. [Abstract] [Full Text] [PDF]

				N. Marx, H. Duez, J.-C. Fruchart, and B. Staels Peroxisome Proliferator-Activated Receptors and Atherogenesis: Regulators of Gene Expression in Vascular Cells Circ. Res., May 14, 2004; 94(9): 1168 - 1178. [Abstract] [Full Text] [PDF]

				N. Marx, D. Walcher, C. Raichle, M. Aleksic, H. Bach, M. Grub, V. Hombach, P. Libby, A. Zieske, S. Homma, et al. C-Peptide Colocalizes with Macrophages in Early Arteriosclerotic Lesions of Diabetic Subjects and Induces Monocyte Chemotaxis In Vitro Arterioscler. Thromb. Vasc. Biol., March 1, 2004; 24(3): 540 - 545. [Abstract] [Full Text]

				T. F. Luscher, M. A. Creager, J. A. Beckman, and F. Cosentino Diabetes and Vascular Disease: Pathophysiology, Clinical Consequences, and Medical Therapy: Part II Circulation, September 30, 2003; 108(13): 1655 - 1661. [Full Text] [PDF]

				O. Ziouzenkova, S. Perrey, L. Asatryan, J. Hwang, K. L. MacNaul, D. E. Moller, D. J. Rader, A. Sevanian, R. Zechner, G. Hoefler, et al. Lipolysis of triglyceride-rich lipoproteins generates PPAR ligands: Evidence for an antiinflammatory role for lipoprotein lipase PNAS, March 4, 2003; 100(5): 2730 - 2735. [Abstract] [Full Text] [PDF]

				U. Kintscher, C. Lyon, S. Wakino, D. Bruemmer, X. Feng, S. Goetze, K. Graf, A. Moustakas, B. Staels, E. Fleck, et al. PPAR{alpha} Inhibits TGF-{beta}-Induced {beta}5 Integrin Transcription in Vascular Smooth Muscle Cells by Interacting With Smad4 Circ. Res., November 29, 2002; 91 (11): e35 - e44. [Abstract] [Full Text] [PDF]

				K. Takayama, G. Garcia-Cardena, G. K. Sukhova, J. Comander, M. A. Gimbrone Jr., and P. Libby Prostaglandin E2 Suppresses Chemokine Production in Human Macrophages through the EP4 Receptor J. Biol. Chem., November 8, 2002; 277(46): 44147 - 44154. [Abstract] [Full Text] [PDF]