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(Circulation. 1995;92:238-243.)
© 1995 American Heart Association, Inc.

Articles

Copper-Induced Tissue Factor Expression in Human Monocytic THP-1 Cells and Its Inhibition by Antioxidants

David J. Crutchley, PhD; Benito G. Que, PhD

From the Miami Heart Research Institute, Miami Beach, Fla.

Correspondence to David J. Crutchley, PhD, Miami Heart Research Institute, 4701 Meridian Ave, Miami Beach, FL 33140.

	Abstract

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Background Transition metals such as copper are known to initiate free radical formation and lipid peroxidation. Recent reports suggest that intracellular reactive oxygen intermediates can induce the transcription of a number of important genes. The present study examines the effects of copper and iron on the ability of monocytic cells to synthesize and express tissue factor, the potent procoagulant factor.

Methods and Results Exposure of human monocytic THP-1 cells to 5 to 10 µmol/L Cu²⁺ led to cell damage and the expression of tissue factor activity to levels up to 70 times higher than control, as measured by a single-stage plasma coagulation assay. These effects were seen only in the presence of a lipophilic chelating agent, 8-hydroxyquinoline, suggesting that intracellular transport of Cu²⁺ was required. The effects of Cu²⁺ were mimicked by ceruloplasmin but not by Fe³⁺ or hemin. The induction of tissue factor activity by Cu²⁺ was slow in onset (6 hours) but sustained (24 hours) and was accompanied by increased tissue factor mRNA levels, measured by reverse transcription/polymerase chain reaction after annealing with oligomer primers. Increases in tissue factor protein, measured by a specific immunoassay, also occurred but were smaller than those in activity. Cu²⁺, therefore, appears to act at both the transcriptional and posttranslational levels. The effects of Cu²⁺ were inhibited by a number of lipophilic antioxidants, including probucol, vitamin E, butylated hydroxytoluene, and a 21-aminosteroid, U74389G.

Conclusions Exposure of monocytes to oxidizing conditions may lead to the expression of high levels of tissue factor activity, with accompanying risk for disseminated intravascular coagulation, and this may be inhibited by lipophilic antioxidants.

Key Words: endothelium • antioxidants • lipids

	Introduction

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It is becoming increasingly apparent that oxidation reactions are important to the development of atherosclerosis. Exposure of LDL to cells of the artery wall, including macrophages, smooth muscle cells, and endothelial cells, can lead to oxidative changes that include degradation of apolipoprotein B, oxidation of polyunsaturated fatty acids, and generation of lysophospholipids. Similar changes occur when LDL is exposed to micromolar concentrations of Cu²⁺. The resulting "oxidized LDL" possesses pronounced atherogenic properties in vitro, including avid uptake by macrophages, chemotaxis of smooth muscle cells and leukocytes, and stimulation of the expression of a number of adhesion molecules and cytokines by endothelial cells (for review, see References 1 and 2). The potential importance of oxidized LDL to atherosclerosis is further emphasized by the detection of oxidized LDL in human blood and atherosclerotic lesions,^{3 4 5} suggesting that oxidation is ongoing in vivo. Furthermore, recent studies have shown that the titer of autoantibodies to oxidized LDL appears to correlate with the progression of carotid atherosclerosis.⁶

Intracellular generation of reactive oxygen species also may play a role in atherosclerosis. Oxygen radicals have been proposed as signaling molecules that facilitate the transcription of several genes important to the atherosclerotic process. The underlying mechanism appears to involve the NF-B and AP-1 nuclear transcription factors, both of which are activated by changes in cellular redox potential.^{7 8 9} For example, expression of the vascular cell adhesion molecule VCAM-1 by endothelial cells and production of the cytokines, tumor necrosis factor-, interleukin-1ß, and interleukin-6 by human monocytes are all thought to proceed via oxidant activation of NF-B.^{10 11 12}

We now report that exposure of monocytic cells to Cu²⁺ induces expression of tissue factor, a membrane-bound glycoprotein that binds coagulation factor VII and thereby initiates blood coagulation.¹³ Induction was observed only in the presence of a lipophilic chelator to facilitate intracellular uptake of Cu²⁺ and was inhibited by lipophilic antioxidants, suggesting that reactive oxygen intermediates were involved. These results therefore suggest a link between intracellular oxidation reactions and blood coagulation and reinforce the concept that antioxidants may play important roles in protection against arteriosclerosis and thrombosis.

	Methods

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Materials
Monocytic tumor THP-1 cells¹⁴ were purchased from the American Type Culture Collection. Antibiotics and materials for the preparation of cell culture media were obtained from GIBCO. Fetal bovine serum was obtained from Hyclone. Cupric acetate, ferric ammonium citrate, ceruloplasmin, 8-hydroxyquinoline, probucol, vitamin E (-tocopherol acetate), butylated hydroxytoluene (BHT), and hemin were obtained from Sigma Chemical Co. Bacterial endotoxin (lipopolysaccharide B; Escherichia coli 0111:B4) was obtained from Difco. Rabbit brain thromboplastin standard was obtained from Ortho. Human plasma deficient in factor VII and tissue factor immunoassay kits were obtained from American Diagnostica.

Cell Treatment
THP-1 cells were grown in RPMI-1640 medium containing 100 µg/mL streptomycin, 100 U/mL penicillin, 10% fetal bovine serum, and 10 mmol/L N-2-hydroxyethylpiperazine-N2'-ethanesulfonic acid (HEPES), pH 7.4. Serum was heated at 56°C for 30 minutes to inactivate complement. Cells were seeded into 24-well dishes at a density of 10⁶ cells/mL. Stock solutions of antioxidants and 8-hydroxyquinoline were prepared in dimethylsulfoxide and ethanol, respectively, and added to growth medium such that the concentration of each vehicle did not exceed 0.1% wt/vol. Control cultures received vehicle alone. After incubation, the cells were collected by centrifugation, washed with Puck's saline A solution, and resuspended in Tris-saline buffer (50 mmol/L tris[hydroxymethyl]aminomethane [Tris] and 110 mmol/L NaCl, pH 7.4). Cells were counted by standard hemocytometry techniques, and cell viability was assessed by exclusion of the vital dye trypan blue.

Tissue Factor Activity
Procoagulant activity on the surface of THP-1 cells was determined by a single-stage clotting assay. Briefly, 50 µL of cell suspension was mixed with 50 µL each of 25 mmol/L CaCl₂ and normal human plasma, and the time for clotting to occur at 37°C was recorded by using a fibrometer. Procoagulant activity was calculated by reference to a rabbit brain thromboplastin standard; a standard curve was constructed by plotting log (units) versus log (clotting time) values. For functional characterization of cellular procoagulant activity, cell suspensions were tested for their ability to shorten the clotting times of recalcified plasma deficient in factor VII. For immunological characterization, cell suspensions were incubated for 1 hour at 37°C with nonimmune mouse IgG or HTF1-7B8, a mouse monoclonal antibody directed against human tissue factor,¹⁵ before testing for procoagulant activity.

Tissue Factor Antigen
THP-1 cells were disrupted by brief sonication on ice and extracted with buffer (0.1 mol/L NaCl, 50 mmol/L Tris, pH 7.4) containing 0.1% Triton X-100 for 18 hours at 4°C. Cell debris was pelleted by centrifugation in a microfuge, and tissue factor in the extracts was measured by using a kit (Imubind, American Diagnostica), according to the manufacturer's instructions. The kit uses a murine monoclonal antibody for antigen capture and a biotinylated rabbit polyclonal antibody for detection.

Tissue Factor mRNA
Total RNA was prepared from THP-1 cells by the method of Chomczynski and Sacchi.¹⁶ One to ten micrograms of RNA was reverse-transcribed with recombinant Moloney murine leukemia virus reverse transcriptase in a reaction mixture containing 20 mmol/L Tris, pH 8.3, 2.5 mmol/L MgCl₂, 50 mmol/L KCl, 100 µg/mL bovine serum albumin, 0.5 mmol/L deoxyribonucleoside triphosphates, and 10 U of placental RNase inhibitor. Reaction mixtures were incubated for 90 minutes at 42°C, heated to 95°C for 5 minutes, and then quickly chilled on ice. One tenth of the reaction mixture was used for polymerase chain reaction amplification,^{17 18} using the following primers specific for tissue factor: 5'-end primer: 5'-CTCGGACAGCCAACAATTCAGAGT-3'; 3'-end primer: 5'-TGTTCGGGAGGGAATCACTGCTTGAACACT-3'. For control purposes, primers for the "housekeeping gene" glyceraldehyde-3-phosphate dehydrogenase (5'-end: 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3'; 3'-end: 5'-CATGTGGGCCATGAGGTCCACCAC-3') were used in a side-by-side amplication with the target gene for tissue factor. Polymerase chain reaction was performed at a final concentration of 10 mmol/L Tris, pH 8.3, 50 mmol/L KCl, 50 µmol/L deoxyribonucleoside triphosphates, 0.1 µmol/L each of 5' primer and 3' primer, and 1 unit of Taq polymerase in a total volume of 50 µL. The mixture was overlayered with mineral oil and then amplified with the Perkin-Elmer Cetus thermal cycler. The amplification profile consisted of denaturation at 95°C for 1 minute, primer annealing at 58°C for 1 minute, and primer extension at 72°C for 2 minutes in a 20- to 50-cycle reaction. Five to ten microliters of each reaction mixture was electrophoresed in 1.5% agarose gels or 8% polyacrylamide gels in Tris/borate/EDTA buffer. Gels were stained with 0.5 µg/mL ethidium bromide and photographed. Film negatives were scanned with a laser densitometer (Ultroscan XL equipped with GelScan software; Pharmacia).

Statistical Analysis
Data were analyzed by the Student's t test. Differences were considered significant at P<.05.

	Results

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Incubation of THP-1 monocytic cells for 18 hours with Cu²⁺, in concentrations up to 20 µmol/L, had no effect on procoagulant activity. However, in the presence of 1 µmol/L 8-hydroxyquinoline, Cu²⁺ produced a dose-dependent expression of procoagulant activity (Table 1). This was accompanied by a decline in cell viability; for example, approximately 80% of cells treated with 5 µmol/L Cu²⁺ excluded trypan blue, as opposed to more than 90% of untreated cells. The effects of Cu²⁺ could be mimicked by the copper transporting protein ceruloplasmin. In contrast, incubation of cells for 18 hours with up to 20 µmol/L Fe³⁺ or hemin had no effect on cell viability or procoagulant activity, even in the presence of 8-hydroxyquinoline.

View this table: [in this window] [in a new window]   Table 1. Induction of Tissue Factor Procoagulant Activity in THP-1 Cells by Cu2+

The Cu²⁺-induced procoagulant activity was due to tissue factor, since cells treated with Cu²⁺ plus 8-hydroxyquinoline failed to shorten the clotting time of plasma deficient in factor VII. In addition, the activity was specifically inhibited by a blocking monoclonal antibody to human tissue factor (HTF1-7B8, generously provided by Dr Steven Carson of the University of Nebraska Medical Center) (Table 2).

View this table: [in this window] [in a new window]   Table 2. Characterization of Procoagulant Activity in THP-1 Cells Treated With Cu2+ and 8-Hydroxyquinoline

Tissue factor activity reached extremely high levels in response to Cu²⁺. Thus, activity was 70 times higher than in untreated cells after exposure to 10 µmol/L Cu²⁺ for 18 hours; this contrasts with the threefold to fivefold increase obtained in response to the well-characterized stimulus, bacterial lipopolysaccharide (LPS). The kinetics of the induction by the two agents were also dissimilar. Thus, increased tissue factor activity in response to LPS was readily observed at 3 hours, peaked at 6 hours, and declined at 24 hours. In contrast, Cu²⁺-induced procoagulant activity was slow in onset, being observed only after 6 hours, and sustained, being readily observable at 24 hours (Fig 1).

View larger version (23K): [in this window] [in a new window]   Figure 1. Bar graph shows time course of induction of tissue factor activity in THP-1 cells. Cells were incubated for 3, 6, or 24 hours with growth medium (control), medium containing 1 µg/mL bacterial lipopolysaccharide (LPS), or medium containing 5 µmol/L Cu2+ in the presence of 1 µmol/L 8-hydroxyquinoline (Cu2+/HQ). Cellular procoagulant activity was then determined. Values are mean±SEM of triplicate determinations. Tissue factor activity in LPS-treated cells was significantly greater than control at 3 hours (P<.005), 6 hours (P<.005), and 24 hours (P<.01), whereas activity in Cu2+/HQ-treated cells was significantly greater than control only at 6 hours and 24 hours (P<.005).

Monocyte tissue factor expression in response to LPS proceeds via increased gene transcription.¹⁹ The effects of Cu²⁺ on steady-state levels of tissue factor mRNA were therefore investigated and compared with those of LPS. Primers were selected according to nucleotides 8496 (exon 4) through 9372 (exon 5) of the tissue factor gene sequenced by Mackman et al²⁰ in order to exclude contamination with genomic DNA. Reverse transcription/polymerase chain reaction of THP-1 cell mRNA with these primers resulted in a 270-bp size fragment, corresponding to amino acids 140 through 229 in the mature tissue factor protein. As shown in Fig 2, exposure of THP-1 cells to LPS or Cu²⁺ led to increased tissue factor mRNA levels. Furthermore, the kinetics of the increases paralleled those in procoagulant activity; LPS effects were readily detected after 2 hours of exposure and declined to basal levels by 6 hours, whereas Cu²⁺-induced increases were clearly detected only after 6 hours of exposure. Neither LPS nor Cu²⁺ affected mRNA levels of the "housekeeping gene" G3PDH.

View larger version (19K): [in this window] [in a new window]   Figure 2. Effects of bacterial lipopolysaccharide (LPS) and Cu2+ on steady-state levels of tissue factor (TF) mRNA in THP-1 cells. Cells were incubated for the times shown with 1 µg/mL LPS or 5 µmol/L Cu2+ in the presence of 1 µmol/L 8-hydroxyquinoline (Cu2+/HQ). RNA was extracted and subjected to reverse transcription/polymerase chain reaction, using primers for TF or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Left, Ethidium bromide staining of 1.5% agarose gel: A, Control; B, LPS; and C, Cu2+/HQ. Right, Line plot: Areas corresponding to TF mRNA were quantitated by densitometry, normalized with respect to G3PDH mRNA, and expressed in relative units, with control cells being assigned a value of 1.0. (), LPS-treated cells; () Cu2+/HQ-treated cells. Results shown are from a representative experiment performed in triplicate.

We next explored the effects of Cu²⁺ on tissue factor protein levels in THP-1 cells. Exposure for 18 hours to 5 µmol/L Cu²⁺ in the presence of 1 µmol/L 8-hydroxyquinoline led to a threefold increase in cellular tissue factor antigen (Fig 3). However, tissue factor activity increased by more than sevenfold in the same cells. Thus, increased tissue factor protein production did not account for all of the increased procoagulant activity after Cu²⁺ treatment.

View larger version (27K): [in this window] [in a new window]   Figure 3. Bar graph: Comparison between tissue factor activity and antigen in Cu2+-treated THP-1 cells. Cells were incubated for 18 hours with growth medium (control) or medium containing 5 µmol/L Cu2+ in the presence of 1 µmol/L 8-hydroxyquinoline (Cu2+/HQ). Tissue factor activity was determined by a single-stage clotting assay, and antigen was determined by ELISA. Values are mean±SEM of triplicate determinations. Activity and antigen levels in Cu2+-treated cells were both significantly higher than control (P<.005 and P<.01, respectively).

Since Cu²⁺ is well known to support lipid peroxidation and free radical generation, we explored the possibility that tissue factor induction was an oxidation-dependent process. Accordingly, the ability of several structurally diverse antioxidants were studied for their ability to inhibit Cu²⁺-induced tissue factor induction. As shown by Fig 4, a number of these lipophilic antioxidants significantly blunted the effect of Cu²⁺, including probucol (20 µmol/L), vitamin E (50 µmol/L), BHT (50 µmol/L), and a 21-aminosteroid ("lazaroid") antioxidant, U74389G (20 µmol/L). These agents were more effective if preincubated with cells for 1 hour before exposure to Cu²⁺; little inhibition was seen if Cu²⁺ and antioxidants were added simultaneously. For comparison, the ability of these antioxidants to inhibit tissue factor expression in response to LPS was also studied. Only vitamin E significantly affected LPS-induced tissue factor expression, producing approximately 50% inhibition at 50 µmol/L with a 1-hour preincubation (data not shown).

View larger version (51K): [in this window] [in a new window]   Figure 4. Bar graph: Suppression of Cu2+-induced tissue factor expression by antioxidants. Cells were incubated for 1 hour with U74389G (20 µmol/L), probucol (20 µmol/L), vitamin E (50 µmol/L), or butylated hydroxytoluene (BHT) (50 µmol/L). 8-Hydroxyquinoline (1 µmol/L) and Cu2+ (5 µmol/L) were then added, and cells were incubated further for 18 hours. Tissue factor activity was then determined by a single-stage clotting assay and compared with that of untreated cells (control). Values are mean±SEM of triplicate determinations. Activity of cells treated with Cu2+ plus antioxidants was significantly lower than that of cells treated with Cu2+ alone (P<.05, U74389G and probucol; P<.005, vitamin E and BHT).

	Discussion

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The present studies show that exposure of human monocytic THP-1 cells to low micromolar concentrations of Cu²⁺ but not Fe³⁺ led to a slowly developing expression of high levels of tissue factor procoagulant activity. Substitution of Cu²⁺ by equivalent concentrations of ceruloplasmin, the major copper-transporting protein in blood, also led to increased tissue factor expression. The effects of ceruloplasmin were less pronounced, however, presumably reflecting the lower availability of Cu²⁺ when bound to ceruloplasmin.

The effects of Cu²⁺ required the presence of 8-hydroxyquinoline, a lipophilic chelating agent that has been used to efficiently transport Fe³⁺ across the plasma membrane of endothelial cells.²¹ We conclude from this that Cu²⁺ exerted its effects intracellularly. The effects were also strongly inhibited by a number of structurally diverse, lipophilic antioxidants, including probucol, vitamin E, BHT, and the 21-aminosteroid "lazaroid" compound U74389G.²² We conclude from this that Cu²⁺ exerted its effects at least in part via an oxidation mechanism. The well-established ability of Cu²⁺ to generate toxic free radicals upon reaction with thiols and to support lipid peroxidation would be consistent with such a mechanism. In previous studies, we have reported that monocyte tissue factor is induced by homocysteine and Cu²⁺, a combination that is known to also generate oxidizing species.²³ However, the effects were extremely variable, suggesting that oxidation is less effective when it occurs extracellularly.

Recent evidence suggests that reactive oxygen intermediates function as important and widespread signaling molecules, facilitating gene expression by activation of the ubiquitous nuclear transcription factor NF-B. Thus, cellular oxidative conditions appear to dissociate NF-B from its cytosolic inhibitor, permitting translocation to the nucleus and binding to the appropriate sites on DNA.^{7 8 9} Activation of NF-B by reactive oxygen intermediates is blocked by antioxidants, resulting in inhibition of gene transcription and hence protein synthesis. For example, cytokine-induced expression of the vascular cell adhesion molecule VCAM-1 by human endothelial cells and LPS-induced cytokine production by human monocytes are inhibited by antioxidants acting at the level of NF-B.^{10 11 12} Endothelial expression of E-selectin is similarly inhibited by antioxidants, although the role of NF-B in this process is less clear.²⁴

Recent studies have shown that the promoter region of the tissue factor gene contains two AP-1 binding sites and one NF-B binding site, the latter binding proteins of the c-Rel/p65 type. Together, they constitute the LPS-responsive element (LRE) and as such are essential for LPS-induced tissue factor gene transcription in human peripheral blood monocytes and THP-1 cells.^{25 26} Activation of NF-B by intracellular reactive oxygen intermediates might therefore be expected to enhance tissue factor gene transcription. The increased tissue factor mRNA levels seen in Cu²⁺-treated cells would be consistent with such a scheme.

The apparent discrepancy between tissue factor antigen and activity in Cu²⁺-treated cells deserves comment. In previous studies on THP-1 cells, we have observed a close correlation between these parameters, both under conditions of upregulation by LPS and downregulation by prostacyclin analogues or K⁺-channel antagonists.^{27 28} However, in the present study, marked increases in activity were associated with substantially lower increases in antigen after exposure to Cu²⁺. In addition, LPS induced at most a fivefold increase in tissue factor activity, whereas Cu²⁺ induced activity to levels 70 times those seen in untreated cells. The polymerase chain reaction method we used is semiquantitative, so that strict correlation of mRNA levels with the other parameters is not justified. Nevertheless, these observations raise the possibility that Cu²⁺ may also have exerted posttranslational effects, perhaps by oxidative modification of tissue factor protein.

Caution must clearly be exercised when attempting to relate acute in vitro studies to the situation pertaining in vivo, particularly in reference to atherosclerosis, a disease that takes many years to develop. Furthermore, although serum copper concentrations are in the 15- to 20-µmol/L range,²⁹ almost all of the copper is bound to ceruloplasmin and albumin, and estimations of free Cu²⁺ indicate that it is unlikely to exceed picomolar concentrations.³⁰ Despite this caveat, the possible relevance of our findings to atherosclerosis and thrombosis deserves comment. Several recent epidemiological studies have suggested that high serum concentrations of iron and especially copper are associated with an increased risk for myocardial infarction.^{29 31 32} A similar relation has been reported for ceruloplasmin.³³ Other epidemiological studies have suggested that antioxidant vitamins may afford protection against coronary heart disease. Thus, plasma concentrations of vitamins A, C, E, and ß-carotene have been inversely related to coronary heart disease mortality and risk for angina,^{34 35 36} while prospective studies suggest that vitamin E consumption may decrease risk for cardiovascular disease.^{37 38} Finally, a number of studies have provided direct experimental evidence that antioxidants, including probucol,³⁹ vitamin E,⁴⁰ and BHT,⁴¹ can protect against the development of atherosclerosis in animal models. Oxidation of LDL has generally been invoked as an explanation for these findings. However, it is possible that intracellular oxidation independent of LDL also may be involved. The finding that Cu²⁺ may lead to an increased thrombotic potential via monocyte tissue factor expression may help in further understanding the role of transition metals and oxidation reactions in cardiovascular disease. Certainly, our results would appear to reiterate the importance of antioxidant defenses such as vitamin E. They also may help explain early observations that treatment with vitamin E leads to significant improvement in occlusive vascular disease.^{42 43 44}

	Acknowledgments

 
This work was supported in part by a grant from the Walter Ross Foundation. We wish to thank Franco M. Arias, Viviana Barria, and Andy W. Toledo for their excellent technical assistance. We also thank Dr Steven D. Carson of the University of Nebraska Medical Center, Omaha, for his generous gift of anti–tissue factor monoclonal antibody. Finally, we wish to thank Garnett Huguley, Medical Services Liaison of the Upjohn Co, for his help in obtaining a sample of U74839G and funding for this project.

Received November 2, 1994; revision received December 27, 1994; accepted January 9, 1995.

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