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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:384-392

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:384-392.)
© 1999 American Heart Association, Inc.

Original Contributions

Cholesterol or Triglyceride Loading of Human Monocyte-Derived Macrophages by Incubation With Modified Lipoproteins Does Not Induce Tissue Factor Expression

Mark M. E. D. van den Eijnden; Jacqueline T. van Noort; Leny Hollaar; Arnoud van der Laarse; Rogier M. Bertina

From the Hemostasis and Thrombosis Research Center, Departments of Hematology (M.M.E.D.v.d.E., J.T.v.N., R.M.B.) and Cardiology (L.H., A.v.d.L.), Leiden University Medical Center (University Hospital), Leiden, the Netherlands.

Correspondence to M.M.E.D. van den Eijnden, Hemostasis and Thrombosis Research Center, Department of Hematology, Leiden University Medical Center (University Hospital), Bldg 1, C2-R, PO Box 9600, 2300 RC Leiden, the Netherlands. E-mail rbertina{at}hematology.azl.nl


*    Abstract
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Abstract—Macrophages/foam cells localized in cholesterol- and triglyceride-rich regions of atherosclerotic plaques express high levels of tissue factor (TF), the essential cofactor and receptor of factor VIIa. It is not clear whether modified lipoproteins, for which several agonistic effects on macrophages have been described, are independent stimuli of TF expression in these cells. Therefore, we studied the effect of short-term (1 day) and long-term (4 to 7 days) incubation of human monocyte-derived macrophages cultured in suspension with modified and native LDLs or VLDLs on the expression of TF mRNA, antigen, and activity. We used native LDL or VLDL, moderately oxidized LDL or VLDL, severely oxidized LDL or VLDL, acetylated LDL, and ß-VLDL at a protein concentration of 100 µg/mL. Cholesterol loading occurred within 9 hours after the addition of acetylated LDL and continued during long-term incubation. Incubation of severely oxidized LDL for 7 days resulted in a slight increase in cholesterol content. Triglyceride loading was observed during short-term and long-term incubation with native and modified VLDLs. Neither cholesterol nor triglyceride loading resulted in expression of TF. Bacterial LPS still could induce TF expression in lipid-laden macrophages. Our results show that incubation with modified lipoproteins or lipid loading does not lead to TF expression in monocyte-derived macrophages cultured in suspension. This suggests that induction of TF expression in foam cells in the atherosclerotic lesion is triggered by additional or other components.


Key Words: monocyte-derived macrophages • lipoproteins • lipopolysaccharide • tissue factor • atherosclerosis


*    Introduction
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The oxidation of lipoproteins that have been transported into the artery wall and trapped in the extracellular matrix of the subendothelial space is considered as one of the crucial steps in the onset of atherogenesis (for recent reviews see References 1 and 21 2 ). Incubation of LDL with endothelial cells, monocytes/macrophages, or smooth muscle cells leads to formation of modified forms of LDL that can be internalized by macrophages (for reviews References 3 and 43 4 ). Also, oxidized LDL has been detected in atherosclerotic lesions using immunohistochemical and other immunological techniques.5 6 7 Therefore, there is strong evidence that oxidation of LDL may occur in vivo. VLDL and VLDL remnants (ß-VLDL) have been found in atherosclerotic plaques as well.8 Oxidized LDL has been shown to possess strong agonistic, modulating, and growth-promoting effects on cells in vitro.9 10 11 12 13 14 15 16 17 18 19 20 On the other hand, the presence of specific receptors for oxidized LDL and apoE-enriched or lipoprotein lipase-enriched VLDL or ß-VLDL on macrophages enable these cells to accumulate cholesterol and triglycerides, leading to their conversion into foam cells.21 22 23 This event is one of the first steps in the formation of a fatty streak that may progress to an atherosclerotic lesion.24 Such a lesion is enriched in macrophages/foam cells and smooth muscle cells that have been found to express high levels of tissue factor (TF) mRNA, antigen, and activity.25 26 27 28 29 30 In addition, these cells would be the source of extracellular TF, which is especially found in the lipid-rich core of the atherosclerotic plaque.25 26 27 31

TF is a membrane-bound glycoprotein of 45 to 50 kDa that is considered to be the major cellular initiator of blood coagulation. It serves as the essential cofactor and receptor of coagulation factor VII/VIIa. The TF-VIIa complex activates coagulation factors IX and X by limited proteolysis, which ultimately leads to thrombin formation and the conversion of soluble fibrinogen into insoluble fibrin32 33 34 . Thus, TF is considered to be the primary initiator of thrombin generation. Therefore, it has been proposed that TF contributes to the high thrombogenicity of the atheromatous core.35 36

Interestingly, monocytes/macrophages, smooth muscle cells, and endothelial cells do not express TF in the quiescent state, but can be stimulated to do so by a variety of agonists, such as endotoxin, cytokines, and growth factors37 (see also Reference 3838 for a recent review). Therefore, the question is which stimuli are responsible for the induction of TF expression in smooth muscle cells and macrophages in the atherosclerotic lesion. A likely candidate is oxidized lipoprotein.39 40 41 42 Oxidized LDL is a potent inducer of inflammatory molecules.43 44 45 It may activate nuclear factor-{kappa}B (NF{kappa}B)–like transcription factors and therefore stimulates transcription of genes containing NF{kappa}B-like binding sites in their promoter.46 47 48 Such a binding site is also present in the promoter of the TF gene and has been demonstrated to be involved in LPS-induced TF expression in monocytic cells.49 On the other hand, it also might be that the accumulation of lipid components by macrophages during their maturation to foam cells will result in a change in the repertoire of expressed genes.

A number of studies have reported on the effects of native LDL and VLDL or LDL modified by acetic anhydride, malondialdehyde, or Cu2+/Fe2+-oxidation on TF expression in adherent monocytes/macrophages, monocytic THP-1 cells, and endothelial cells.20 39 40 41 42 50 51 52 Most of these studies showed an increase in TF activity when cells were incubated with modified lipoproteins.39 40 41 42 50 51 On the other hand, Brand and coworkers20 found no effects at all on TF expression. The reasons for this apparent discrepancy might be related to differences in the lipoproteins used (contamination with endotoxin, degree, and mode of modification), the type of cells that were used, and their actual state of differentiation or activation.

In the present study we have addressed the question of whether modified lipoproteins (LDL and VLDL) themselves directly (agonist action) or indirectly (by lipid accumulation) can induce TF expression. In earlier studies, similar experiments have been performed with monocytes that were isolated by adherence and cultured under adherent conditions.39 40 50 51 Because it is very likely that adherence itself already activates the monocytes/macrophages, we have chosen to use monocyte-derived macrophages, cultured in suspension on Teflon membranes (s-MDM), to examine the effect of native and modified lipoproteins (LDL, moderately oxidized LDL [Ox-LDL], severely oxidized LDL [Ox-LDL+], acetylated LDL [Ac-LDL], VLDL, moderately oxidized VLDL [Ox-VLDL], severely oxidized VLDL [Ox-VLDL+], and ß-VLDL) on TF expression (mRNA, antigen, and activity) and lipid accumulation. Previously, we demonstrated that these cells, which show stable expression of macrophage-specific markers (CD71, the mannose receptor, the scavenger receptors types I and II), do not express significant amounts of TF.53 Although lipid loading in s-MDM incubated with Ac-LDL, Ox-LDL+, or modified VLDLs did occur, no evidence could be obtained for induced or constitutive expression of TF mRNA, antigen, or activity during incubation of the cells with these lipoproteins.


*    Methods
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Isolation and Modification of Lipoproteins
EDTA was added in a final concentration of 0.25 mmol/L to citrate-buffered plasma from healthy donors. VLDL and LDL were isolated according to the method of Havel et al54 using a 2-step density ultracentrifugation. VLDL was obtained by centrifugation of plasma (d=1.017 g/mL) in a fixed-angle rotor (TFT 70.38, Kontron AG) at 117 000g for 22 hours at 4°C using the Centrikon T-2070 ultracentrifuge (Kontron AG). LDL was prepared after separation of VLDL from the plasma. After adjustment of the density to d=1.063 g/mL with solid KBr, the plasma was centrifuged in the same way as described above and the LDL fraction was collected. VLDL and LDL were extensively dialyzed (4 changes) against 80 volumes of PBS (0.9% NaCl, 1.4 mmol/L PO43-, pH 7.5) containing 0.25 mmol/L EDTA and 50 µg/mL gentamycin for 70 hours at 4°C.

LDL was acetylated as described by Basu et al.55 Fifteen milliliters of LDL (3.25 mg protein/mL) was mixed with 15 mL of a saturated sodium acetate solution (10 mol/L) in an ice-water bath. Subsequently, 73 µL acetic anhydride was added in aliquots of 2 µL at 2-minute intervals. Ac-LDL was extensively dialyzed (4 changes) against 100 volumes of PBS, 0.25 mmol/L EDTA, and 50 µg/mL gentamycin for 20 hours at 4°C.

LDL was oxidized as described by Steinbrecher.56 LDL was dialyzed overnight against 300 volumes of PBS supplemented with 50 µg/mL gentamycin at 4°C, diluted with PBS to 0.5 mg protein/mL, and incubated with 5 µmol/L CuCl2 at 37°C for varying intervals before EDTA (final concentration 0.25 mmol/L) was added. Ox-LDL was obtained by oxidation of LDL for 5 hours with 5 µmol/L CuCl2. Ox-LDL+ was obtained by oxidation of LDL for 24 hours with 5 µmol/L CuCl2. Both Ox-LDL and Ox-LDL+ were extensively dialyzed (4 changes) against 100 volumes of PBS, 0.25 mmol/L EDTA, and 50 µg/mL gentamycin for 20 hours at 4°C. A similar procedure was followed for the oxidation of VLDL. Ox-VLDL was obtained by oxidation of VLDL for 7 hours with 50 µmol/L CuCl2. Ox-VLDL+ was obtained by oxidation of VLDL for 24 hours with 50 µmol/L CuCl2. Additionally, ß-VLDL (VLDL/VLDL remnants + ß-VLDL) was isolated from plasma of patients with type III hyperlipoproteinemia (familial dysbetalipoproteinemia) by centrifugation at 256 000g at 15°C for 10 hours in a fixed-angle rotor (TLA 100.3, Beckman) in a tabletop ultracentrifuge (TL-100, Beckman).57

Modified and native LDLs and VLDLs were sterilized by filtration through a 0.2- or a 0.45-µm sterile filter (Schleicher & Schuell GmbH), respectively, and stored at 4°C. The protein concentration was determined by the bicinchoninic acid method (Pierce) using bovine serum albumin as a standard. Total cholesterol (free cholesterol and cholesterol esters) and triglycerides in native and modified lipoproteins were measured enzymatically using commercial kits (Boehringer Mannheim). The CHOD-PAP kit was used for total cholesterol measurements. The GPO-PAP kit was used for triglyceride measurements.

The lipoprotein preparations were tested for endotoxin contamination using a chromogenic assay (Limulus amebocyte lysate test).58 The level of endotoxin in these preparations was between 0.2 and 0.8 pg/µg protein.

Determination of LDL and VLDL Oxidation
The extent of LDL and VLDL oxidation was determined by using the thiobarbituric acid reactive substance (TBARS) assay, which is based on the reaction of malondialdehyde (MDA) with thiobarbituric acid.59 From the LDL or VLDL incubation mixture (0.5 mg protein/mL), 0.1 mL was taken and added to 1 mL TCA-TBA-HCl reagent (15% [wt/vol] trichloroacetic acid, 0.375% [wt/vol] thiobarbituric acid, 0.25 mol/L HCl). This mixture was heated for 15 minutes in a boiling water bath. After cooling, the precipitate was removed by centrifugation at 3000g for 10 minutes. The absorbance of MDA-TBA product was determined at 535 nm. The amount of TBARS was calculated using an extinction coefficient of 1.56x105 L/mol and expressed as nanomoles MDA equivalent per milligram protein. With this method a TBARS concentration of 25 to 30 nmol MDA equivalent/mg protein was determined for Ox-LDL, 35 to 40 nmol MDA/mg protein for Ox-LDL+, 10 to 15 nmol MDA equivalent/mg protein for Ox-VLDL, and 30 to 35 nmol MDA equivalent/mg protein for Ox-VLDL+.

Electrophoresis of Native and Modified Lipoproteins
Native and modified lipoproteins were applied onto agarose plates (Ciba Corning Diagnostics Corp) and electrophoresed for 35 minutes at 90 V. Subsequently, the plates were dried, stained for 10 minutes with Fast Red B, rinsed, and dried again. Native and modified lipoproteins were detectable as a single band. Electrophoresis of the ß-VLDL fraction showed several lipoprotein bands, indicating that these fractions contain both VLDL and VLDL remnants. The modified lipoproteins showed a higher electrophoretic mobility than the native lipoproteins.

Isolation of Monocytes and Preparation of s-MDM
Monocytes were isolated as described previously.53 Peripheral blood mononuclear cells were obtained from pooled citrate-buffered buffy coats from 5 healthy donors by centrifugation on a Ficoll-Amidotrizoate or Ficoll-Paque Plus density gradient (d=1.077 g/mL, 20 minutes at 1000g) following the procedure of Bøyum.60 Monocytes were further purified by countercurrent centrifugal elutriation as described by Plas et al.61 Analysis for CD14 (anti-Leu-M3, Becton Dickinson) expression on a FACScan flow cytometer (Becton Dickinson) showed that the monocytes were 85% to 90% pure.

s-MDM were prepared by culturing monocytes for 1 week on hydrophobic Teflon-FEP film, gauge 25 µm (3P), at 37°C in a humid 5% CO2 atmosphere at a cell concentration of 1.5x106/mL in RPMI 1640 medium supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), 2 mmol/L L-glutamine (Gibco BRL Life Technologies Ltd), and 4% heat-inactivated human AB serum (Sigma Chemical Co). The level of endotoxin in this medium was under the detection limit (<10 pg/mL) of the chromogenic assay (Limulus amebocyte lysate test).58

Previously, we have shown that after 1 week of culture the monocytes have differentiated to macrophages (expression of CD71, the mannose receptor, and the scavenger receptors types I and II), which do not express significant amounts of TF.53 Therefore, on day 7, PBS or the various LDL or VLDL preparations (all at 100 µg protein/mL) were added to the s-MDM. In some experiments, s-MDM were incubated with Ox-LDL or Ac-LDL in the presence of 5 µg/mL bovine lipoprotein lipase (LPL [kindly provided by Dr. L.M. Havekes]).62 After 1, 4, or 7 days of incubation with PBS or modified or native lipoproteins, s-MDM were analyzed for the uptake of lipoproteins (cholesterol or triglyceride loading and lipid staining) and for the presence of TF activity, antigen, and mRNA. In some experiments, cells were incubated for 0.5 to 30 hours with 100 ng/mL Salmonella typhimurium-derived LPS (Sigma). Cell viability, using the trypan blue exclusion assay, varied between 80% and 95%.

Cholesterol, Triglyceride, and Protein Measurements
At the end of the incubation, s-MDM were washed twice with PBS and then resuspended in 1 mL PBS (5 to 10x106 cells/mL). The cells from 0.3 mL cell suspension (1.5 to 3x106 cells) were sonicated (2x 10 seconds), and 0.1 mL cell extract was then used for cholesterol or triglyceride measurements. The amount of total cholesterol (free cholesterol and cholesterol esters) in s-MDM incubated with PBS or modified or native LDLs was measured enzymatically using the CHOD-PAP commercial kit. The amount of triglycerides in s-MDM incubated with PBS or modified or native VLDLs was measured enzymatically using the GPO-PAP commercial kit. The protein content was determined in 10 µL cell extract using the bicinchoninic acid method. The remainder of the cell suspension (0.7 mL) was used for TF antigen and activity measurements.

Statistics
Differences in cholesterol content between 2 different cell preparations for statistical significance was determined by using the 2-tailed Student's unpaired t test. A P value of <0.05 was considered a significant difference.

Lipid Staining
Cytospins (2.5 to 5x104 cells) were fixed for 10 minutes in a container saturated with 40% formaldehyde and rinsed for 5 seconds with 60% isopropanol. Lipids were stained by incubating the cytospins for 10 minutes in an oil red O solution (0.3 g oil red O in 100 mL 60% isopropanol). After rinsing for 5 seconds with 60% isopropanol and washing with water, the cytospins were incubated for 3 to 5 minutes in a Mayer's acid hemalum solution (Merck) and washed for 10 to 20 minutes with water.

TF Antigen and Activity
The cells from 0.7 mL cell suspension (3.5 to 7x106 cells) were used for the analysis of TF antigen and activity. For TF antigen, cells were centrifuged and resuspended in extraction buffer (50 mmol/L TEA, 100 mmol/L NaCl, and 1% Triton X-100, pH 7.5) to a final concentration of 15 to 20x106 cells/mL. TF antigen in the cell extracts was determined by ELISA as described previously.53 63 For the analysis of TF activity, cells were resuspended in a buffer containing 10 mmol/L HEPES, 137 mmol/L NaCl, 4 mmol/L KCl, 2.5 mmol/L CaCl2, 11 mmol/L {alpha}-D-glucose, 5 mg/mL ovalbumin (pH 7.45) to a final concentration of 33x106 cells/mL. From this suspension several dilutions were prepared (final concentrations ranging from 8.5 to 25x106 cells/mL). From the various concentrations of cell suspensions 30 µL was used for the assay. TF activity was measured as factor VIIa-dependent factor X activation following the procedure of Consonni and Bertina.63 Active site-titrated factor Xa (0 to 15 nmol/L) was used for calibration of the factor Xa assay.

TF activity was calculated from the initial rate of factor VIIa-dependent factor Xa formation (duplicate experiments) and expressed as picomoles of factor Xa per minute per 106 cells.

RNA Isolation and Northern Blot Analysis
After incubation with LPS, PBS, LDL, or Ac-LDL, total RNA was isolated from the cells using the TRIzol reagent (GIBCO BRL Life Technologies Ltd), which is based on the guanidinium isothiocyanate method.64 RNA (15 µg per lane) was separated by electrophoresis, transferred to Hybond-N filters, and immobilized by UV irradiation as described previously.53 The filters were hybridized under standard conditions53 with [{alpha}-32P]dCTP (Amersham International)-labeled TF cDNA and GAPDH cDNA probe.65 66 Filters were washed for 10 to 15 minutes in 2x saline–sodium citrate (SSC)/0.1% SDS and/or 1x SSC/0.1% SDS at room temperature or, when necessary, at 42°C. Fuji x-ray films (Fuji Photo Film Co) were exposed to the filters at -80°C.


*    Results
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 *Results
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Characteristics of Modified Lipoproteins
Oxidation of LDL and VLDL leads to the production of aldehydes such as MDA, which can be detected by incubation with TBA.59 To be informed about the extent of lipoprotein oxidation, samples were drawn from the LDL-CuCl2 or VLDL-CuCl2 mixture at different intervals and analyzed for their MDA content. In Figure 1Down the result of an LDL and VLDL oxidation experiment is shown. For LDL a strong increase in MDA levels is observed within 5 hours of oxidation. Enhanced levels of MDA are also found after 7 hours' oxidation of VLDL and is further increased during oxidation for longer times.



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  		Figure 1. Copper-induced oxidation of VLDL ({triangleup}) and LDL ({square}). VLDL and LDL (0.5 mg protein/mL) were incubated at 37°C in the presence of 50 µmol/L (VLDL) or 5 µmol/L (LDL) CuCl2 in PBS, pH 7.5. The extent of oxidation was determined by measuring the MDA levels using the TBARS assay. The level of MDA of the lipoproteins used in the experiments are shown by arrows defined by a small letter between brackets: (a) MDA level of VLDL, (b) MDA level of Ox-VLDL, (c) MDA level of Ox-VLDL+, (d) MDA level of LDL, (e) MDA level of Ox-LDL, and (f) MDA level of Ox-LDL+.

Modification of lipoproteins by oxidation (LDL, VLDL) and acetylation (LDL) results in a net increase of negative surface charges.67 The difference in charge between the native and the modified lipoproteins was monitored by agarose gel electrophoresis. Oxidized lipoproteins (Ox-LDL, Ox-VLDL, Ox-LDL+, Ox-VLDL+) and Ac-LDL migrate faster (ie, are more electronegative) through the agarose gel than LDL and VLDL, as shown in Figure 2Down for LDLs.



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  		Figure 2. Electrophoresis pattern of plasma, native, and modified lipoproteins. From all preparations 1 to 2 µL was applied onto an agarose plate and electrophoresed for 35 minutes at 90 V. After drying, the plate was stained for 10 minutes with Fast Red B, rinsed, and dried again.

Lipid Loading of s-MDM
Monocytes were cultured on Teflon membranes (suspension culture) in RPMI-1640/4% human AB serum until day 7. During this period the monocytes differentiate to mature macrophages (s-MDM), which do not express significant amounts of TF.53 On day 7, PBS or lipoproteins were added to the s-MDM.

Uptake of lipids by s-MDM during incubation with LDL or modified LDLs was determined quantitatively by measuring the total cholesterol accumulation and qualitatively by lipid staining with oil red O. Lipid loading in s-MDM exposed to VLDL or modified VLDLs was determined quantitatively by measuring the accumulation of triglycerides. In s-MDM incubated with Ox-LDL, LDL, or PBS for 1, 4, or 7 days, the cholesterol content was hardly changed. Cellular cholesterol content was slightly increased during incubation with Ox-LDL+ after 4 and 7 days (Table 1Down). When s-MDM were incubated with Ac-LDL, accumulation of cholesterol was already observed after 1 day of incubation. The s-MDM that were cultured for 4 to 7 days in the presence of Ac-LDL showed significant cholesterol accumulation compared with s-MDM incubated with LDL or PBS (Table 1Down). The content of triglycerides in s-MDM incubated with the VLDL preparations for 1, 4, or 7 days was significantly higher than that in the PBS-incubated s-MDM (Table 2Down). The amounts of triglycerides decreased when s-MDM were incubated with VLDL or modified VLDLs for 4 to 7 days, probably as a result of expenditure of triglycerides. Measurements of lactate dehydrogenase in conditioned media and cell extracts indicated that under all conditions more than 90% of the cells remained intact.


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  		Table 1. Analysis of Cholesterol Content


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  		Table 2. Analysis of Triglyceride Content

Recently, it has been reported that the murine macrophage-like J774 cells minimally internalize Ox-LDL.62 However, incubation of Ox-LDL with LPL resulted in LPL-mediated binding and uptake by J774 cells of this modified lipoprotein, thereby leading to stimulation of cholesterol ester accumulation in these cells.62 We examined whether incubation of Ox-LDL (100 µg/mL) with exogenous LPL (5 µg/mL) stimulates the accumulation of cholesterol in human s-MDM. However, no significant differences in lipid loading were observed after 1, 4, or 7 days of incubation of s-MDM with Ox-LDL (132±28 versus 118±13 nmol cholesterol/mg cell protein in control cells after 7 days) or Ox-LDL/LPL (140±26 versus 118±13 nmol cholesterol/mg protein in control cells after 7 days). This suggests that LPL does not stimulate the binding and uptake of Ox-LDL by human s-MDM.

Lipid loading analyzed by oil red O staining showed that intracellular accumulation of lipids did not occur in s-MDM incubated with LDL (Figure 3ADown). A similar result was found for Ox-LDL–incubated s-MDM (Figure 3BDown). Some intracellular lipid accumulation was observed in s-MDM cultured in the presence of OxLDL+ (Figure 3CDown). However, when s-MDM were incubated with Ac-LDL, many cells were stained strongly with oil red O (Figure 3DDown).



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  		Figure 3. Lipid staining by oil red O of s-MDM incubated with 100 µg protein/mL LDL (A), Ox-LDL (B), Ox-LDL+ (C), or Ac-LDL (D) for 7 days.

During monocyte-to-macrophage differentiation, expression of the scavenger receptor types I and II is stably upregulated.53 68 69 70 These receptors are used by macrophages for the internalization of modified lipoproteins.21 The observation that after 7 days of incubation the uptake of Ac-LDL by s-MDM was inhibited 95% by 100 µg/mL polyinosinic acid, an effective inhibitor of the scavenger receptors,21 confirms the involvement of the scavenger receptors in the internalization of modified lipoproteins by these cells.

TF Expression in s-MDM During Incubation With Native or Modified Lipoproteins
The effect of native and modified lipoproteins on the expression of TF in s-MDM was examined at the level of activity (intact cells), antigen (cell extracts), and mRNA. As can be seen in Figure 4Down and in Tables 3Down and 4Down, induction of TF activity and antigen did not occur when s-MDM were exposed to the various LDL and VLDL preparations for 1, 4, or 7 days. The amounts of TF activity and antigen found in these cells were close to the detection limits of the assays. Expression of TF was also not detected at the level of mRNA in s-MDM incubated with PBS, LDL, or Ac-LDL for 1, 4, or 7 days (Figure 5Down). No detectable amounts (<150 pg/mL) could be detected in any of the conditioned media using a very sensitive commercial ELISA for measurement of TF antigen (America Diagnostica).



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  		Figure 4. Effect of PBS, LDL, Ox-LDL, Ox-LDL+, and Ac-LDL on the expression of TF activity (intact cells, A) and antigen (cell extracts, B) in s-MDM. The cells were incubated with PBS (treatment A) or with 100 µg protein/mL native or modified LDLs (treatments B–E) for 1, 4 or, 7 days. Control TF activity and antigen were determined in s-MDM exposed to 100 ng/mL LPS for 4 hours. Results are given as the mean±SD for 4 independent experiments.


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  		Table 3. Effect of PBS, VLDL, Ox-VLDL, Ox-VLDL+, and ß-VLDL on the Expression of TF Activity (Intact Cells)


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  		Table 4. Effect of PBS, VLDL, Ox-VLDL, Ox-VLDL+, and ß-VLDL on the Expression of TF Antigen (Cell Extracts)



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  		Figure 5. Northern blot analysis of TF mRNA in s-MDM incubated with PBS or 100 µg protein/mL LDL or Ac-LDL for 1, 4, or 7 days (d.1, d.4, d.7, respectively) or incubated with 100 ng/mL LPS for 30 minutes and 3 hours (30', 3h, respectively). RNA (15 µg per lane) was separated by electrophoresis and transferred to a Hybond-N filter. TF mRNA was detected by hybridization with a partial TF cDNA probe. The presence of equivalent amounts of RNA in each lane was checked by rehybridization of the blot with a GAPDH cDNA probe.

Although the cells did not show induction of TF expression after incubation with native and modified lipoproteins, exposure of s-MDM to LPS resulted in the induction of TF activity and antigen (Figure 4Up) and TF mRNA (Figure 5Up). Lipid-laden macrophages (s-MDM incubated with Ac-LDL for 7 days) were also responsive to LPS in their expression of TF. TF antigen increased from 0.22±0.07 ng TF/106 cells to 2.76±0.87 ng TF/106 cells after 5 hours' incubation with LPS. Thus, these cells have not lost their ability to express TF in response to external stimuli. Because monocytes/macrophages show a transient TF response to LPS,53 71 72 we wondered whether the initial phase of lipid loading could also induce a transient TF response in s-MDM. Because of the clear accumulation of cholesterol in s-MDM exposed to Ac-LDL for 1 day, we focused on the eventual expression of TF during a 30-hour incubation with Ac-LDL. During this incubation period, lipid loading (accumulation of cholesterol) was observed within 9 hours of incubation. Incubation of s-MDM with PBS, LDL, or LPS for 3 to 30 hours did not result in lipid loading. The s-MDM incubated with PBS, LDL, or Ac-LDL did not show induction of TF activity and antigen, whereas incubation of s-MDM with LPS clearly resulted in a transient TF expression (Figure 6Down).



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  		Figure 6. Analysis of TF activity (intact cells, A) and antigen (cell extracts, B) at different intervals during 30 hours of incubation of s-MDM with PBS (x), 100 µg protein/mL LDL ({triangledown}), 100 µg protein/mL Ac-LDL ({square}), or 100 ng/mL LPS ({triangleup}).

Lesnik et al51 have reported that TF procoagulant activity was about 2 times higher in adherent macrophages exposed to Ac-LDL for 48 hours than in the control cells that already express significant amounts of TF. Monocyte-to-macrophage differentiation is accompanied by a transient burst of spontaneous TF expression.53 We used suspension monocytes of day 4 (Mo-S 4), which spontaneously express TF,53 to examine the effect of modified lipoproteins on TF expression in these cells. Mo-S4 (1.5 to 2 ng TF/106 cells) were incubated with Ac-LDL, LDL, PBS, or LPS for 3 to 30 hours. No significant increase in TF antigen was observed in Mo-S4 incubated with Ac-LDL, LDL, or PBS, whereas incubation of Mo-S4 with LPS clearly resulted in the production of additional TF antigen (data not shown).


*    Discussion
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 up arrowMethods
 up arrowResults
 *Discussion
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The microenvironment of macrophages/foam cells in the atherosclerotic lesion is complex and includes the simultaneous presence of proliferating smooth muscle cells, T lymphocytes, fibers of collagen and fibrinogen, cellular debris, lipids, modified lipoproteins, and a multitude of inflammatory cytokines, chemotactic factors, and growth factors (for reviews, see References 24, 73, and 7424 73 74 ). Many of these have been shown to be able to induce TF expression in monocytes or monocytic cells in culture either directly (agonists) or indirectly (cell–cell contact).34 38 In some but not all studies, it has been reported that modified lipoproteins also stimulate or induce TF expression in monocytes/macrophages,39 40 50 51 although the presence of contaminating endotoxin levels has not been excluded in all studies.40 Most of these studies have examined the effect of modified lipoproteins on TF expression in monocytes/macrophages by using monocytes isolated by adherence to plastic and cultured while adherent.39 40 50 51 Because adherence itself can be considered as an activated state of the monocyte/macrophage75 76 that possibly contributes to its responsiveness to modified lipoproteins, the aforementioned studies give no information on whether or not modified lipoproteins independently can induce TF expression (directly as agonist or indirectly by lipid accumulation).

Recently, we have reported that monocytes cultured for 1 week on Teflon membranes (suspension culture) have differentiated to macrophages that stably express macrophage-specific markers (CD71, the mannose receptor, and the scavenger receptor types I and II) and do not express significant amounts of TF.53 In the present study we have used this well-defined model system to examine the role of modified and native lipoproteins on the expression of TF (mRNA, antigen, and activity) in s-MDM. Our data indicate that under the conditions (short- and long-term incubations) used, TF expression was not induced in s-MDM during incubation with modified lipoproteins, even though Ac-LDL and the modified VLDLs, and to a smaller extent also Ox-LDL+, are accumulated by these cells (Tables 1Up and 2Up). Incubation with native LDL or VLDL also did not result in induction of TF expression in s-MDM. Also, native and modified lipoproteins do not stimulate or modulate the spontaneous TF expression that occurs during the monocyte-to-macrophage transition (data not shown). From these results we conclude that modified lipoproteins themselves do not induce TF expression in s-MDM, neither directly (through activation of signal transduction by agonist action) nor indirectly (through lipid accumulation). This suggests that in the atherosclerotic lesion additional or other combinations of stimuli are needed to explain the high levels of TF expression observed in the macrophages/foam cells of such a lesion. That such cells are still sensitive to such stimuli is illustrated by our observation that lipid-laden macrophages (s-MDM incubated with Ac-LDL for 7 days) still showed LPS-induced TF expression. Also, Lesnik et al51 have found that adherent cholesterol-laden macrophages exposed to LPS express significantly higher levels of TF procoagulant activity than the control cholesterol-loaded cells.

The observation that exposure of s-MDM to modified LDLs for 3 to 30 hours or for several days does not result in induction of TF expression (Figures 4Up, 5Up, and 6Up) is in contrast with some previous reports in which it was shown that Ac-LDL or oxidized LDL can stimulate TF procoagulant activity in adherent macrophages after 6, 18, or 48 hours of incubation.39 40 51 However, the fact that we did not obtain any evidence for effects of modified LDL on TF expression both in cells that already express significant amounts of TF and in s-MDM appears to confirm the results of other studies, which show that modified lipoproteins do not induce TF expression in adherent monocytes.20 77

Endotoxin contamination of the lipoprotein preparations may account for some of these conflicting results. Therefore, the stimulating effect of lipoproteins on TF expression in endotoxin-sensitive cells must be interpreted very carefully. Recently, Brand et al20 have reported that modified lipoproteins that are contaminated with endotoxin will stimulate the expression of LPS-sensitive genes such as the TF gene in monocytes. Although these investigators could not demonstrate an effect of endotoxin-free modified LDL on TF expression, they did show a synergistic interaction between oxidized LDL and LPS for the expression of TF in monocytes.20

The discrepancy between our findings and those of other groups39 40 51 may also be caused by differences in the procedures for isolation of monocytes, conditions of cell culture, TF regulation in adherent monocytes/macrophages versus monocytes/macrophages cultured in suspension, or response to stimuli between human monocytes/macrophages and monocytes/macrophages of other species. We have chosen to culture the cells in suspension to avoid adhesion-related activation of the monocytes/macrophages. In all other studies dealing with the effects of modified lipoproteins on TF expression, adherent cells have been used.39 40 50 51 Therefore, it cannot be excluded that adhesion-related responses play a role in the induction and stimulation of TF expression by lipoproteins. When monocytes or monocytic THP-1 cells were adhered to fibrin or fibronectin, some induction of TF was observed,78 79 suggesting that under certain conditions adhesion itself is already able to induce TF expression to some extent. Also the engagement of the adhesion molecule VLA-4 may lead to the synthesis of TF protein in monocytic cells.79 80 It has been suggested that tyrosine phosphorylation and activation of the mitogen-activated protein (MAP) kinase pathway is involved in this process.79 Interestingly, oxidized LDL also activates MAP kinase in adherent human macrophages.81 Whether the MAP kinase pathway is also activated by modified lipoproteins in s-MDM is not known.

In summary, under our experimental conditions, modified lipoproteins have no effect on TF expression in s-MDM, whereas lipid loading (accumulation of cholesterol and triglycerides) in the cells did occur during incubation with Ac-LDL or modified VLDLs and to some extent during incubation with Ox-LDL+. This indicates that lipid loading per se is not sufficient to induce TF synthesis and activity and suggests that additional or other components play a role in the induction of TF expression in macrophages/foam cells of an atherosclerotic lesion.


*    Acknowledgments
 
We thank Dr H.L. Vos for critical reading of the manuscript, Richard Dirven for the purification of factor X and for the preparation of active site-titrated factor Xa, and Ton Vroom for his technical assistance with the electrophoresis of the lipoproteins. This work was supported by grant 91.066 from The Netherlands Heart Foundation.

Received November 5, 1997; accepted August 19, 1998.


*    References
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