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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:265-272

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:265-272.)
© 1997 American Heart Association, Inc.

Articles

Vastatins Inhibit Tissue Factor in Cultured Human Macrophages

A Novel Mechanism of Protection Against Atherothrombosis

Susanna Colli; Sonia Eligini; Mariagrazia Lalli; Marina Camera; Rodolfo Paoletti; Elena Tremoli

the E. Grossi Paoletti Center, Institute of Pharmacological Sciences, University of Milan, Milan, Italy

Correspondence to Prof Elena Tremoli, Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. E-mail tremoli@isfunix.farma.unimi.it.


*    Abstract
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 *Abstract
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 down arrowMethods
 down arrowResults
 down arrowDiscussion
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We examined the effect of fluvastatin, the first entirely synthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor that is structurally different from other vastatins, on tissue factor (TF) expression in human macrophages spontaneously differentiated in culture from blood monocytes. Fluvastatin decreased TF activity in a dose-dependent manner (1 to 5 µmol/L) in both unstimulated and lipopolysaccharide-stimulated macrophages, and this reduction paralleled the decrease in immunologically recognized TF protein. The same results were obtained with another lipophilic vastatin, simvastatin, but not with hydrophilic pravastatin. The reduction in TF expression was also observed in macrophages enriched in cholesterol after exposure to 50 µg/mL acetylated low density lipoprotein. The inhibitory effect of fluvastatin on TF activity and antigen was fully reversible by coincubation with 100 µmol/L mevalonate or 10 µmol/L all-trans-geranylgeraniol but not with dolichol, farnesol, or geraniol. Suppression of TF antigen and activity was accompanied by a diminution in TF mRNA levels, which was completely prevented by mevalonate. Furthermore, fluvastatin impaired bacterial lipopolysaccharide–induced binding of c-Rel/p65 heterodimers to a {kappa}B site in the TF promoter, indicating that this drug influences induction of the TF gene. We conclude that lipophilic vastatins inhibit TF expression in macrophages, and because this effect is prevented by mevalonate and geranylgeraniol, a geranylgeranylated protein plays a crucial role in the regulation of TF biosynthesis. The suppression of TF in macrophages by vastatins indicates a potential mechanism by which these drugs interfere with the formation and progression of atherosclerotic plaque as well as thrombotic events in hyperlipidemic patients.


Key Words: 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors • procoagulant activity • isoprenoids • atherosclerosis • thrombosis


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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HMG-CoA reductase inhibitors (vastatins) are widely used to suppress plasma LDL cholesterol levels in patients with primary hypercholesterolemia.1 2 3 These drugs appear to reduce plaque progression in coronary and carotid arteries4 5 and prevent restenosis in experimental animals6 7 and humans,8 perhaps by influencing the behavior of smooth muscle or endothelial cells9 10 11 12 and in particular by blocking the biosynthesis of mevalonate.

The synthesis of mevalonate by HMG-CoA reductase is a key event in the biosynthesis of isoprenoids, which are essential for normal cellular proliferation and activity.13 14 Isoprenoids are the common precursors of sterols (eg, cholesterol, ubiquinone, dolichols) and the intermediates FPP and GGPP, which are substrates for posttranslational modifications of prenylated proteins. FPP is the direct precursor for farnesylation of proteins such as lamin B15 and ras16 ; however, after FPP has been elongated to all-trans-GGPP, it becomes a substrate for the geranylgeranylation of proteins, including many low-molecular-weight GTP-binding proteins.17 Although the function of these prenyl modifications is not clearly understood, many prenylated proteins play important roles in the regulation of cell growth, cell secretion, and signal transduction.18 19

Inhibitors of HMG-CoA reductase have been used to establish the functional involvement of isoprenoids in the regulation of cell growth20 21 and proliferation.11 12 22 In addition, these drugs have been shown to reduce both the secretory and the morphological responses of rat basophil leukemia cells23 and to interfere with the regulation of natural killer cell toxicity.24 Recently inhibitors of HMG-CoA reductase have been shown to also inhibit monocyte chemotaxis25 and the release of interleukin-8 by monocytic cells, an effect that is completely prevented by supplementation of the cells with mevalonate.26

Among the products elaborated by monocytes and macrophages, TF, a membrane-bound glycoprotein, plays a prominent role in the extrinsic pathway of blood coagulation and fibrin deposition. TF is considered to be the primary cellular initiator of the coagulation protease cascade, in that it serves as a cellular receptor and cofactor for plasma factor VIIa.27 Although normally absent from all intravascular cells, TF can be induced to appear on circulating monocytes by specific inflammatory mediators.28 In particular, bacterial LPS mediates transcriptional activation of the human TF gene in monocytes by inducing the nuclear translocation of c-Rel/p65 heterodimers and their subsequent binding to a {kappa}B site in the TF promoter.29 30 Monocyte TF activity has been shown to be higher in vivo in various animal models after endotoxin shock.31 32 Furthermore, TF antigen and the corresponding mRNA have been localized in foam cell macrophages of human atherosclerotic plaque.33 Macrophages isolated from human carotid plaques express elevated surface procoagulant activity with the functional characteristics of TF,34 which suggests a role for this protein in atherogenesis.

In this study we demonstrate that fluvastatin, the first entirely synthetic HMG-CoA reductase inhibitor structurally distinct from other HMG-CoA reductase inhibitors,35 inhibits TF expression in human macrophages that have spontaneously differentiated in culture from blood monocytes. Thus, involvement of isoprenoid biosynthesis through the mevalonate pathway has been explored.


*    Methods
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 *Methods
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Reagents
LPS (Escherichia coli 0114:B4) was obtained from Difco Labs; all-E-geranylgeraniol (all-trans-geranylgeraniol) was purchased from American Radiolabeled Chemicals Inc; 1-O-octyl-ß-D-glycopyranoside, PMSF, benzamidine, aprotinin, bovine albumin (fatty acid free and low endotoxin), dolichol, geraniol, and farnesol were from Sigma Chemical Co; mevalonate (as mevalonolactone) was from Aldrich Chemical Co; the interleukin-1ß (human) ELISA system was from Amersham International; pravastatin from Bristol-Meyers-Squibb Pharmaceutical Institute and simvastatin from Merck, Sharp & Dohme Research Laboratories were kindly provided by Dr A. Corsini, Institute of Pharmacological Sciences, University of Milan, Milan, Italy. Pravastatin was dissolved in saline. Simvastatin in lactone form was dissolved in 0.1 mol/L NaOH to generate the active form and the pH adjusted to 7.4 by adding 0.1 mol/L HCl. Fluvastatin as sodium salt (Sandoz Prodotti Farmaceutici SpA) was dissolved in ethanol. A dolichol dispersion was prepared by sonication with equimolar concentrations of phosphatidylcholine (Sigma) into 0.15 mol/L KCl.24

Monocyte Isolation and Macrophage Culture
Venous blood obtained from healthy donors was anticoagulated with 3.8% sodium citrate, and mononuclear cells were separated with Ficoll-Paque solution (Pharmacia Fine Chemicals) at 450g for 20 minutes. To minimize platelet contamination, the cells were washed with PBS (ICN Biomedicals Inc) containing EDTA (5 mmol/L) and suspended (2 to 3x106/mL) in RPMI-1640 (ICN Biomedicals) supplemented with 2 mmol/L glutamine (Bio-Whittaker), 0.5% penicillin-streptomycin-Fungizone (Bio-Whittaker), and 10% autologous serum. Monocytes were isolated from lymphocytes by adherence (2 hours at 37°C, 5% CO2, humid atmosphere) to 35-mm plastic dishes. The cell preparations were >90% monocytes, as determined by nonspecific esterase staining. To obtain differentiated macrophages, monocytes were cultured for 7 days at 37°C in a 5% CO2 humid atmosphere in medium 199 (ICN Biomedicals) supplemented with 2 mmol/L glutamine, 0.5% antibiotics, and 10% autologous serum (filtered through a 0.22-µm filter; Millipore Corp). The average protein content of different macrophage preparations ranged from 50 to 180 µg per well. Macrophages were identified by the presence of CD68 antigen, as detected by a specific monoclonal mouse anti-human macrophage antibody (Dakopatts). Cell viability was >95% as determined by trypan blue exclusion. After culture the medium was removed and replaced with medium 199 from which serum had been omitted. The endotoxin content of all culture materials and reagents was measured with the Limulus amebocyte lysate assay (Bio-Whittaker), and only those free of endotoxin or containing <3 pg/mL were used. This level was shown to be lower than that required for activation of macrophages.

Acetyl-LDL Preparation
LDLs (d=1.024 to 1.050 g/mL) were isolated by sequential preparative ultracentrifugation of plasma obtained from normolipidemic donors after an overnight fast.36 The density of plasma containing aprotinin (100 U/mL) and PMSF (10 mmol/L) was adjusted to 1.020 g/mL with a KBr solution, and samples were ultracentrifuged at 40 000 rpm for 20 hours at 4°C in a Beckman SW50Ti rotor. LDLs were extensively dialyzed at 4°C against phosphate buffer (1.5 mmol/L NaH2PO4 and 3.5 mmol/L Na2HPO4, pH 7.5). After dialysis in 0.15 mol/L NaCl, LDLs were diluted with an equal volume of saturated sodium acetate and treated with acetic anhydride.37 Acetyl-LDLs were concentrated with Centricon filters (CF25, Amicon). The extent of LDL acetylation was determined by agarose gel electrophoresis, and the protein content of acetyl-LDL was determined by the method of Lowry et al38 with BSA as the standard. Acetyl-LDLs were sterilized by passage through 0.22-µm filters, stored in sterile tubes at 4°C, and used within 1 month. Acetyl-LDLs prepared as described were shown to bind and be internalized by macrophages (data not shown). The presence of LPS in acetyl-LDL was tested by the Limulus assay. Only those preparations with LPS levels <0.1 ng/mg protein were used for the study.

Addition of Reagents
Cells (macrophages or adherent monocytes, where specified) were incubated in serum-free medium containing 0.2% fatty acid–free BSA for 20 to 24 hours with different vastatins. The cells were then exposed to fresh medium containing vastatins and LPS (10 µg/mL) for 8 hours or acetyl-LDL (50 µg protein per milliliter) for 24 hours. These incubation times were selected after preliminary experiments of 4 to 24 hours. Mevalonate or isoprenoids were present throughout the incubations where indicated. The medium was removed, and plates were washed with cold PBS and frozen at -80°C until assayed for TF activity.

Evaluation of TF Activity
Immediately before assay, cells were lysed with 15 mmol/L 1-O-octyl-ß-D-glycopyranoside at 37°C for 10 minutes and diluted with 25 mmol/L HEPES-saline. TF activity was determined as procoagulant activity by a one-step clotting assay.39 Clotting times were quantified by comparison with a standard curve based on serial dilutions of a standard human thromboplastin preparation (Thromborel, Behring). The lack of TF activity in cells incubated with 10 µg/mL of a monoclonal antibody against TF (American Diagnostica Inc), which blocked activity by >90%, demonstrated the specificity of the assay. Assays performed with plasma from donors congenitally deficient in factor VII consistently demonstrated no TF activity. Data are expressed as units of TF activity per microgram of protein determined by the Bradford method.40

TF Antigen Determination
TF antigen was determined by ELISA with the Imubind Tissue Factor kit (American Diagnostica Inc). For the ELISA, cells were solubilized with PBS containing 1% Triton X-100, 1 mmol/L PMSF, 100 U/mL aprotinin, and 5 mmol/L benzamidine. Values are expressed as nanograms of TF antigen per microgram of protein.

Northern Blot Analysis
Total cellular RNA was extracted from macrophages with the guanidinium thiocyanate/phenol/chloroform method,41 and TF mRNA levels were estimated by Northern hybridization analysis.42 RNA concentration was determined spectrophotometrically. RNA samples (10 µg) were denatured at 65°C for 10 minutes in 50% formamide, 16% formaldehyde, and 1x formaldehyde gel running buffer; chilled on ice for 5 minutes; and fractionated on denaturing gel (1% agarose and 2.2 mol/L formaldehyde). Samples were then transferred to nylon membranes by capillary blotting. The human TF cDNA probe was a 1200-bp Sal I fragment cloned into pUC 18 obtained from the American Type Culture Collection. The human GAPDH probe was a 1469-bp BamHI fragment cloned into pBR322, kindly provided by Dr P. Castelli (Consorzio Mario Negri Sud, Italy). cDNA probes were labeled with [{alpha}32P]dCTP by the random-primer technique to a specific activity of 5x108 to 1x109 counts per minute per microgram of DNA. Filters were prehybridized for at least 4 hours and hybridized overnight at 42°C in 50% formamide, 5x SSPE (0.75 mol/L NaCl and 0.05 mol/L NaH2PO4·H2O), 5x Denhardt's solution (0.1% Ficoll, 0.1 polyvinylpyrrolidone, and 0.1% BSA), 100 mg/mL dextran sulfate, 100 µg/mL salmon sperm DNA, and 200 µg/mL bakers' yeast tRNA. Blots were then washed at 42°C (two washes with 5x SSPE, one with 1x SSPE and 0.1% SDS, and one with 0.1x SSPE and 0.1% SDS). Membranes were air dried and exposed to autoradiography film (Kodak XAR) with intensifying screens at -80°C.

The density of autoradiographic signals was quantified by densitometry with a scanner equipped with an integrator. GAPDH levels were used to normalize densitometric values.

EMSA
For EMSAs, macrophages were preincubated for 16 to 24 hours with fluvastatin (5 µmol/L) and exposed to 10 µg/mL LPS for 45 minutes.29 Nuclear proteins were prepared from extracts of cells obtained from three to five dishes. The following procedures were performed at 4°C. Macrophages were harvested in cold Tris-buffered saline (pH 7.4) and pelletted at 4000 rpm for 2 minutes. The pellet was resuspended in 100 µL buffer A (10 mmol/L HEPES, pH 7.9; 1.5 mmol/L MgCl2; 10 mmol/L KCl; and 0.5 mmol/L) and incubated on ice for 15 minutes. After addition of 4 µL 10% Triton X-100, samples were vortexed for 10 seconds and centrifuged at 13 000 for 2 minutes. Pellets were resuspended in 50 µL of cold buffer B (20 mmol/L HEPES, pH 7.9; 1.5 mmol/L MgCl2; 0.4 mol/L NaCl; 0.2 mmol/L EDTA; 25% glycerol; 0.5 mmol/L DTT; and 0.5 mmol/L PMSF) and incubated on ice for 30 minutes. After centrifugation (13 000 rpm for 10 minutes), the supernatants were frozen (-80°C). Protein concentrations in nuclear extracts were 0.5 to 1 mg/mL as determined by the Bradford method.

The oligonucleotide containing the TF {kappa}B site (underlined), 5'-GTCCCGGAGTTTCCTACCGGG-3 (Oligo Etc Inc), was annealed with a complementary primer and radiolabeled with [32P]dCTP (Amersham). EMSAs were performed as follows. Nuclear extracts (2 to 5 µg) were incubated with radiolabeled DNA probes ({approx}1x105 cpm) for 20 minutes at room temperature in 18 µL binding buffer containing 60 mmol/L HEPES, 180 mmol/L KCl, 3 mmol/L EDTA, 36% glycerol, 15 mmol/L DTT, and 4 µg poly(dI-dC). Protein-DNA complexes were separated from free DNA probe by electrophoresis through 5% nondenaturing acrylamide gels in 1x Tris-glycine buffer. Gels were dried under vacuum on filter paper and exposed for 16 to 40 hours to Kodak XAR film at -80°C. Specificity of binding was ascertained by competition with excess cold consensus oligonucleotides.

Determination of Intracellular Cholesterol
To determine macrophage cholesterol content, cells were washed with PBS and lipids were extracted by hexane/isopropanol (3:2, vol/vol). Total cholesterol levels were determined in aliquots of the total lipid extract by enzymatic methods (Boehringer Mannheim, GmbH Diagnostica) and expressed as micrograms per milligram of cell protein.

Statistical Analysis
Results are expressed as mean±SEM or as mean percentages of control±SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's test.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Adherent monocytes cultured for 7 days with 10% autologous serum were spontaneously transformed into macrophages and characterized by the presence of CD68 antigen, as detected by a specific monoclonal mouse anti-human macrophage antibody. These cells expressed basal amounts of TF activity (6.8±1.3 units/µg cell protein, n=15), a feature typical of mature macrophages.43 Although they constitutively expressed TF, macrophages retained their capacity to form substantial amounts of TF in response to various agonists. LPS (10 µg/mL) doubled TF activity (to 12.4±2.1 units/µg cell protein, P<.001 versus unstimulated cells, n=15), and the extent of this increase remained almost constant between 4 and 16 hours.

Basal and LPS-induced TF activity levels of macrophages were characterized by clotting and immunologic techniques. TF activity was completely abrogated in the absence of factor VII, and an inhibitory anti-TF monoclonal antibody completely suppressed it, which further confirmed that the measured activity was due to expression of TF by the cells.

Effect of Vastatins on TF Expression in Macrophages
Macrophages were incubated for 20 to 24 hours with vastatins and then cultured for 8 to 24 hours with freshly prepared medium containing either vastatins alone or vastatins plus the stimulus. Under both unstimulated and LPS-stimulated conditions, TF activity of cells treated with 2.5 µmol/L fluvastatin was significantly lower (-45% and -40%, respectively) than that of cells incubated with medium alone (Fig 1Down). A similar degree of inhibition was observed when macrophages preincubated with fluvastatin were exposed to LPS in the presence of serum. Under this experimental condition, the increase in TF activity was higher (133±22%, n=9) than that obtained in serum-starved cells. Experiments performed by adding fluvastatin to lysates of untreated cells ruled out interference of the drug with the clotting assay.



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  		Figure 1. Effect of fluvastatin on TF activity in unstimulated and LPS-stimulated human macrophages. Cells were incubated with fluvastatin (2.5 µmol/L) for 24 hours and then with freshly prepared medium containing fluvastatin and LPS (10 µg/mL) for 8 hours. TF activity was determined as procoagulant activity as described in "Methods." Bars are mean±SEM of triplicate determinations from six experiments. *P<.05 compared with control cells; {ddagger}P<.05 compared with LPS-treated cells (one-way ANOVA followed by Tukey's test).

The inhibitory effect of fluvastatin was concentration dependent in the range 1 to 5 µmol/L (Fig 2Down) and paralleled reductions of immunologically detectable TF antigen (0.36±0.044 in control cells versus 0.14±0.016 ng/µg protein in cells treated with 2.5 µmol/L fluvastatin; n=3, P<.05). Similar results were obtained in LPS-stimulated cells (0.58±0.049 versus 0.44±0.027).



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  		Figure 2. Concentration-dependent effect of fluvastatin on TF activity in unstimulated ({square}) and in LPS-stimulated () macrophages. Incubations were performed as in the legend to Fig 1Up with the indicated concentrations of fluvastatin. Bars represent percent of control and are mean±SEM of triplicate determinations from six experiments. *{ddagger}P<.05, §P<.01 compared with control and LPS-treated cells.

Simvastatin (2.5 µmol/L), a semisynthetic lipophilic vastatin, showed a similar activity profile by reducing both TF antigen and activity in unstimulated and LPS-stimulated macrophages, whereas pravastatin had no such effect (Fig 3Down). Concentrations of pravastatin 100 times greater (250 µmol/L) than those found effective for fluvastatin or simvastatin slightly affected TF activity (-30% and -15% for unstimulated and LPS-stimulated macrophages, respectively; data not shown).



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  		Figure 3. Effects of simvastatin (S) and pravastatin (P), 2.5 µmol/L, on TF activity in unstimulated ({square}) and LPS-stimulated () macrophages. Incubations were performed as in the legend to Fig 1Up. Bars represent percent of control and are mean±SEM of triplicate determinations from four experiments. *P<.05 compared with preceding bar.

The lack of morphological change or cell viability as assessed by trypan blue exclusion, together with unaltered levels of lactate dehydrogenase in cell supernatants, ruled out the possibility that the observed effects were due to cell death. Another macrophage function, the release of interleukin-1ß, was not suppressed but was increased by fluvastatin treatment, confirming the data of Terkeltaub et al26 in THP-1 cells. Fluvastatin also inhibited TF activity in adherent monocytes exposed to LPS (10 µg/mL) for 6 hours but at slightly higher concentrations than those needed for macrophages (-29±10% and -54±6% at 2.5 and 5 µmol/L fluvastatin concentration; n=5).

Effect of Vastatins on TF Expression in Cholesterol-Loaded Macrophages
Acetyl-LDLs bind to and are taken up by the scavenger receptor of monocytes/macrophages,44 with subsequent processing of the lipoprotein that leads to intracellular accumulation of cholesterol, mostly in the form of cholesteryl esters. Incubation of acetyl-LDL (50 µg/mL) for 24 hours with macrophages raised intracellular total cholesterol levels from 27.7±0.8 to 36.5±2.1 µg/mg protein (n=6, P<.02). Concomitantly, TF activity was doubled, in agreement with previous data.43 45

Fluvastatin or simvastatin (2.5 µmol/L) but not pravastatin inhibited this enhancement (Fig 4Down), which was accompanied by reductions in TF antigen (data not shown) that did not affect the increase in intracellular cholesterol levels induced by acetyl-LDL.



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  		Figure 4. Effects of fluvastatin (F), simvastatin (S), and pravastatin (P) on TF activity in macrophages stimulated with acetyl-LDL. Cells were incubated with vastatins (2.5 µmol/L) for 24 hours and then with fresh medium containing vastatins with () or without ({square}) 50 µg/mL acetyl-LDL for 24 hours. Bars represent percent of control and are mean±SEM of triplicate determinations from five experiments. *P<.05 compared with unstimulated cells; {ddagger}P<.05.compared with acetyl-LDL–stimulated and pravastatin-treated cells.

Involvement of the Mevalonate Pathway in TF Expression by Macrophages
Simultaneous addition of mevalonate and fluvastatin to the culture medium completely prevented the inhibitory effect on TF antigen and activity under all experimental conditions (ie, unstimulated, LPS-stimulated, and acetyl-LDL–stimulated cells; Fig 5Down), which indicates that products of the mevalonate pathway are involved in TF biosynthesis. The inhibitory effect of vastatins was not prevented by the addition of cholesterol to cells, as performed by supplementation of the culture medium with autologous serum or acetyl-LDL; this ruled out the possibility that the effect of vastatins on TF was due to intracellular cholesterol depletion. Furthermore, dolichol (100 µmol/L), another end product of the mevalonate pathway involved in glycoprotein assembly, did not restore TF activity in fluvastatin-treated macrophages (data not shown).



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  		Figure 5. Mevalonate (M) prevents the inhibitory effect of fluvastatin (F) on TF activity in unstimulated ({square}), LPS-stimulated (), and acetyl-LDL–stimulated () macrophages. Cells were incubated for 24 hours with 100 µmol/L mevalonate alone or 2.5 µmol/L fluvastatin with or without mevalonate for an additional 8-24 hours with fresh medium containing fluvastatin, LPS, or acetyl-LDL and mevalonate, where indicated. Bars represent percent of control and are mean±SEM of triplicate determinations from six experiments. *P<.05 compared with bar above; {ddagger}P<.05 compared with control.

We then evaluated the involvement of isoprenoid intermediates in the inhibition of TF by vastatins. Farnesol, geraniol, or all-trans-geranylgeraniol (10 µmol/L) was added to macrophages together with vastatins. Among these, only all-trans-geranylgeraniol prevented the effect of fluvastatin on TF activity (Fig 6Down) and antigen, thus mimicking the effect observed with mevalonate. Similar results were obtained with simvastatin (data not shown).



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  		Figure 6. Geranylgeraniol prevents the inhibitory effect of fluvastatin (F) on TF activity in unstimulated ({square}) and in LPS-stimulated () macrophages. Cells were incubated for 24 hours with 2.5 µmol/L fluvastatin and 10 µmol/L isoprenoids and for an additional 8 hours with fresh medium containing fluvastatin, isoprenoids, and LPS, where indicated. Bars represent percent of control and are mean±SEM of triplicate determinations from five experiments. *P<.05 compared with corresponding control; {ddagger}P<.05 compared with unstimulated cells.

Effect of Fluvastatin on TF mRNA Levels
TF mRNA levels in control cells and in cells exposed to fluvastatin were assessed by Northern blot analysis. Exposure of macrophages to 10 µg/mL LPS was associated with TF mRNA accumulation, peaking between 1 and 2 hours. Fluvastatin (10 µmol/L) diminished TF mRNA levels (-70%) in LPS-stimulated cells (Fig 7Down). Mevalonate (100 µmol/L) prevented the reduction of TF mRNA exerted by fluvastatin (Fig 7Down).



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  		Figure 7. Fluvastatin inhibits macrophage TF mRNA levels and this effect is prevented by mevalonate. Cells were treated with 10 µmol/L fluvastatin and 100 µmol/L mevalonate for 24 hours and incubated with fluvastatin, mevalonate, and LPS for an additional 2 hours. Total RNA was extracted and analyzed by Northern blotting with a radiolabeled human TF cDNA probe. The blot was reprobed to determine GAPDH mRNA levels as a measure of RNA loading. Positions of TF and GAPDH mRNAs are indicated. A representative autoradiogram is shown. Lane 1, medium alone; lane 2, LPS; lane 3, LPS+fluvastatin; and lane 4, LPS+fluvastatin and mevalonate. Similar results were observed in three independent experiments.

Effect of Fluvastatin on LPS-Induced Activation of c-Rel/p65 Heterodimers
It has been shown that LPS induction of human TF gene expression in monocytic cells is specifically mediated by binding of c-Rel/p65 heterodimers to a {kappa}B-like site in the TF promoter.30 To determine whether fluvastatin prevented activation of c-Rel/p65 heterodimers, nuclear extracts from macrophages were analyzed by EMSA and a radiolabeled oligonucleotide containing the TF {kappa}B site.30 c-Rel/p65 binding activity to DNA was low in nuclei of unstimulated cells but was present at elevated levels in cells treated with LPS (Fig 8Down). The LPS-induced increase was largely but not completely abrogated by fluvastatin (Fig 8Down). Competition of DNA binding by a 50-fold excess of unlabeled TF {kappa}B oligonucleotide in nuclear extracts of macrophages exposed to LPS verified the specificity of binding to DNA (Fig 8Down, lane 4).



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  		Figure 8. Fluvastatin attenuates LPS activation of c-Rel/p65 heterodimer binding to DNA in human macrophages. Cells were pretreated with 5 µmol/L fluvastatin for 24 hours before the addition of 10 µg/mL LPS for 45 minutes. EMSA was performed on nuclear extracts with the oligonucleotide containing the TF {kappa}B site. Position of c-Rel/p65-DNA complex is indicated. An autoradiogram, representative of three, is shown. Lane 1, medium alone; lane 2, LPS; lane 3, LPS+fluvastatin; lane 4, LPS and a 50-fold excess of unlabeled TF {kappa}B oligonucleotide. fp indicates free probe.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The results show that fluvastatin and simvastatin suppress TF expression in human macrophages, whether unstimulated or exposed to inflammatory stimuli or atherogenic lipoproteins, and that the effect is prevented by mevalonate and by all-trans-geranylgeraniol. The effect of lipophilic vastatins on TF activity coincides with a decrease in immunologically recognized protein in lysed cells, which rules out the possibility that the drugs act by interfering with TF activity. Fluvastatin attenuated not only TF antigen and activity but also TF mRNA levels, indicating that the drug affects the biosynthesis of the protein. Inhibition of TF activity was also observed in adherent monocytes, and this suggests that the effect of fluvastatin is not confined to differentiated cells.

Interestingly, the inhibitory effect of vastatins on TF tightly parallels their ability to inhibit HMG-CoA reductase, the effect of fluvastatin being similar to that of simvastatin and far greater than that of the hydrophilic drug pravastatin, as has also been shown in murine macrophages.46 Thus, as expected, the lipophilic properties of the compounds govern their effects at the cellular level. Inhibitors of HMG-CoA reductase have been reported to affect macrophage sterol biosynthesis in vitro.47 Under our experimental conditions, HMG-CoA reductase inhibitors are as effective in LPS-stimulated and cholesterol-loaded cells, indicating that TF inhibition by vastatins is not mediated by a reduction in sterol synthesis.

Mevalonate prevents the effect of fluvastatin on TF biosynthesis. Mevalonate, the primary product of HMG-CoA reductase, is the precursor of dolichols and two isoprenoid intermediates, FPP and GGPP. The failure of dolichol to prevent TF inhibition by fluvastatin rules out the hypothesis that this end product of the mevalonate pathway is responsible for the effect. FPP and GGPP are substrates for the posttranslational transfer of a polyisoprene chain to a diverse group of cellular proteins, whose C-terminal amino acid determines the type of isoprenylation (attachment of either farnesol or geranylgeraniol to a cysteine residue via a thioether bond). Because the effect of vastatins on TF expression is prevented by geranylgeraniol but not by farnesol, it is likely that a geranylgeranylated protein plays a crucial role in the events preceding TF gene transcription.

The TF gene is positively regulated by the LPS-inducible transcription factor c-Rel/p65 in monocytic cells,29 30 and inhibition of the nuclear translocation of this factor could prevent induction of the TF gene.48 Interestingly mevinolin, the first described HMG-CoA reductase inhibitor originally isolated from cultures of Aspergillus and Monascus,49 has been shown to block nuclear factor {kappa}B and {kappa} gene activation in an LPS-treated murine pre-B cell line.50 Our data demonstrate that fluvastatin impairs activation of c-Rel/p65 induced by LPS in human macrophages. Thus, on the basis of these preliminary studies, it seems likely that vastatins influence TF biosynthesis by interfering with this pathway. Because TF gene expression is regulated by a number of transcription factors that bind to distinct regions of the TF promoter,48 additional studies are needed to elucidate the mechanism through which fluvastatin affects the overall scenario of TF gene transcription.

The finding that vastatins effectively inhibit TF upregulation induced by acetyl-LDL deserves particular attention. Interaction of modified LDL with macrophages induces intracellular cholesterol accumulation, with subsequent formation of lipid-laden foam cells, a characteristic feature of the atherosclerotic lesion.51 Lesnik et al43 showed that TF activity is upregulated in lipid-enriched macrophages, which suggests that the transformation of macrophages into foam cells in the arterial wall might favor thrombin generation and local fibrin deposition, as occurs in atherogenesis and its thrombotic sequelae. The interference of vastatins with the biosynthesis of TF in cholesterol-enriched macrophages might therefore reduce the atherogenic potential of this cell within the vessel wall.

We conclude that the lipophilic vastatins interfere with TF biosynthesis by human macrophages through inhibition of the synthesis of the isoprenoid geranylgeraniol, which is also involved in the posttranslational modification of various other proteins. These results may have important clinical implications. Administration of the HMG-CoA reductase inhibitor simvastatin to patients at high risk of coronary death and nonfatal myocardial infarction reduces the frequency of major coronary events.52 Moreover, vastatins attenuate coronary and carotid artery lesion progression.53 54 TF has been found in the matrix of necrotic cores of atherosclerotic plaques, mostly associated with macrophages.33 Thus, in conclusion, this newly described effect of vastatins, which occurs at concentrations that are reached after their administration in conventional doses (20 to 40 mg/d PO)55 may represent an important mechanism by which these drugs provide protection against atherothrombosis. Animal models for evaluating TF activity in atheromatous plaques will define the in vivo relevance of this newly described effect of vastatins.


*    Selected Abbreviations and Acronyms
 
acetyl-LDL = acetylated LDL
ELISA = enzyme-linked immunosorbent assay
EMSA = electrophoretic mobility shift assay
FPP = farnesyl pyrophosphate
GAPDH = glyceraldehyde-3-phosphate dehydrogenase
GGPP = geranylgeranyl pyrophosphate
HMG-CoA = 3-hydroxy-3-methylglutaryl coenzyme A
LPS = lipopolysaccharide
TF = tissue factor

Received December 28, 1995; revision received November 21, 1996;
*    References
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