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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departments of Cardiology, Cell Biology, and
Immunology, Lerner Research Institute, Cleveland Clinic Foundation,
Cleveland, OH.
Tissue factor, which is expressed in vascular lesions, increases
thrombin production, blood coagulation, and smooth muscle cell
proliferation. We demonstrate that oxidized low-density lipoprotein (LDL) induces surface tissue factor pathway activity (ie, activity of
the tissue factor:factor VIIa complex) on human and rat smooth muscle
cells. Tissue factor messenger RNA (mRNA) was induced by oxidized LDL
or native LDL; however, native LDL did not markedly increase tissue
factor activity. We hypothesized that oxidized LDL mediated the
activation of the tissue factor pathway via an oxidant-dependent
mechanism, because antioxidants blocked the enhanced tissue factor
pathway activity by oxidized LDL, but not the increased mRNA or protein
induction. We separated total lipid extracts of oxidized LDL using
high-performance liquid chromatography (HPLC). This yielded 2 major
peaks that induced tissue factor activity. Of the known oxysterols
contained in the first peak, 7 Tissue factor is a 47-kd transmembrane cell
surface protein that forms a complex with factor VII, initiating blood
coagulation and leading to the local production of thrombin via the
successive activation of factor IX or factor X and
prothrombin.1-4 Studies using in situ hybridization and
immunohistochemistry reveal significant tissue factor expression in
cells of mesenchymal lineage within atherosclerotic
lesions5 and in vascular smooth muscle cells after
mechanical injury.6 The presence of tissue factor in coronary atherectomy specimens has been correlated with unstable angina,7 as has increased tissue factor expression
in circulating blood monocytes in patients with unstable angina
and acute myocardial infarction.8-10 These data indirectly
support the hypothesis that the tissue factor is responsible for
thrombosis in acute coronary syndrome.
We have recently demonstrated in vitro that native low-density
lipoprotein (LDL) induces tissue factor messenger RNA (mRNA) and cell
surface protein in smooth muscle cells without an increase in cell
surface tissue factor pathway activity,11 defined in our
assay system as the enzymatic activity of the tissue factor:factor VIIa complex of the blood coagulation cascade. We have also
recently identified elements of the tissue factor promoter
required for tissue factor gene induction by native and oxidized
LDL.12 Interestingly, surface tissue factor protein made
in response to LDL led to increased tissue factor pathway activity only
after activation of the pathway by hydrogen peroxide, demonstrating a
novel oxidative mechanism for regulating smooth muscle cells surface
tissue factor pathway activity.11 Our observations on the
induction of tissue factor by LDL in vitro may be related to the recent
finding that in the atheroma of cholesterol-fed rabbits, where
lipoproteins and oxidant-producing phagocytes are known to accumulate,
tissue factor protein expression correlated with plasma cholesterol
levels.13
We hypothesized that oxidized LDL alters the thrombogenicity of an
atherosclerotic lesion via a tissue factor-dependent, oxidant-mediated mechanism. In this study, we evaluated in vitro the effects of oxidized
LDL, a known component of atherosclerotic lesions,14 on
smooth muscle cell surface tissue factor pathway activity and determined whether these effects were altered by antioxidants. From our
results, we propose that oxidized LDL may alter multiple thrombosis-related events by 2 distinct and novel actions. Like native LDL, oxidized LDL induces tissue factor gene expression, synthesis, and presence on smooth muscle cell surfaces,12
but unlike native LDL, oxidized LDL also can activate the tissue factor pathway on the smooth muscle cell surface. Our results further demonstrate that antioxidants blunt the second of these steps, the
increased tissue factor pathway activity, but not the induction of gene
expression. The activation of the cell surface tissue factor pathway by
oxidized LDL can be linked to specific classes of oxidized lipids.
Tissue culture
In those experiments involving antioxidant pretreatment, antioxidants
(N, N'-diphenyl-1,4-phenylene-diamine, DPPD from Aldrich Chemical Co,
Milwaukee, WI; Tiron, Lipoprotein isolation and oxidation
LDL (4 to 8 mg cholesterol/ml) was oxidized during dialysis at 4°C or 22°C against isotonic saline without EDTA, pH 7.4, containing 3 µmol/L CuS04 or 6 µmol/L FeSO4 for up to 2 days, as previously described.18,21 No difference was noted in the tissue factor response of aortic smooth muscle cells to LDL oxidized using either CuS04 or FeSO4 (data not shown). During oxidation LDL underwent a characteristic color change from golden to yellow to translucent as previously reported.22 Numerous oxidized LDL preparations were used in these experiments. The ranges of typical oxidation levels for LDL using these oxidation protocols have been previously reported:18,21,23 Thiobarbituric acid reactivities are 5 to 10 nmol of malondialdehyde (as standard) per milligram LDL cholesterol and total lipid peroxide contents, 190 to 1200 nmol/mg LDL cholesterol. Preparations used in this study were within these ranges. After modification, oxidized LDL samples were assayed for cholesterol and/or protein, dialyzed against 0.5 mmol/L EDTA in isotonic saline, pH 8 to 9, and stored at 4°C until use. A less oxidized preparation of oxidized LDL was made by exposing LDL to smooth muscle cells in culture medium that contained higher amounts of metal ions than DMEM, ie, a mixture of DMEM and Ham F12 (DME/F12: Fe(NO3)3-50 µg/L, FeSO4-417 µg/L, CuSO4-0.13 µg/L vs DMEM: Fe(NO3)3-100 µg/L). We have previously characterized this preparation of oxidized LDL.24 Total lipid extracts were obtained and separated using high-performance
liquid chromatography (HPLC), as described previously.25 Briefly, 5 mg aliquots of either native or oxidized LDL were
lyophilized overnight and extracted in acetone. The total lipid
extracts were dried under nitrogen and redissolved in
isopropanol/acetonitrile, 1:1 (vol/vol). The extracts were separated
using reverse phase HPLC (Waters µBondapak C18 preparative column
[Milford, MA]) over 1 hour using a 5 mL/min flow rate. The initial
solvent elution gradient was water/acetonitrile, 1:1 (vol/vol), which
was increased over 5 minutes to acetonitrile (100%), and then changed
over 45 minutes to isopropanol (100%). Isopropanol (100%) was
continued for the final 10 minutes. Fractions were collected every 3 minutes. Each fraction was then dried under nitrogen and stored at
The 7 SnCl2 reduction of total lipid extracts Lipid hydroperoxides contained in total lipid extracts of lipoprotein preparations were reduced using SnCl2.27 Approximately 5 mg (protein) native or oxidized LDL were treated with SnCl2 (10 mmol/L final concentration) for 2 minutes at room temperature. Lipoproteins were treated with ethanol to serve as a control. The lipoproteins were then lyophilized, extracted in chloroform:methanol, aliquoted, and dried under nitrogen and then resuspended in ethanol:acetone (1:1) before use.Tissue factor assay Cell surface tissue factor pathway activity was assessed using a 2-step amidolytic assay previously described.28 After each well was washed twice with PBS, a reaction mixture containing 0.5 mL of phenol red-free M199, 50 µL of 2 mg/mL S-2222 (Pharmacia-ATPAR, Piscataway, NJ), and 20 µL of Proplex T (containing 1 unit of factor VII, plus factor X; Baxter Biotech, Glendale, CA) was added to each well. Standards containing the same reaction mixture with varying amounts of rabbit brain thromboplastin (Sigma) were also prepared. One milliunit (mU) of tissue factor activity was defined as the amount of activity contained in 1 µL of resuspended rabbit brain thromboplastin after a 1:10 dilution. The reaction mixture remained on cell layers for approximately 20 minutes. Aliquots of the media were pipetted into 96-well plates and read along with the standards on a spectrophotometer at 405 nm. The tissue factor activity in each well was then calculated using the standard curve.Northern hybridization Total cellular RNA was extracted by the guanidine isothiocyanate-cesium chloride method from rat arterial smooth muscles cells grown in 100-mm dishes.29 Samples of total RNA (10 µg) were separated on a 1% agarose/2.2 mol/L formaldehyde gel and subsequently blotted onto Magna nylon membranes with 20 × SSC by capillary transfer, according to previously published methods.30 The RNA was cross-linked to the membrane with an ultraviolet cross-linker (Stratagene, La Jolla, Ca). The blots were prehybridized for 2 to 6 hours at 42°C in 50% formamide, 1% SDS, 5 × SSC, 1 × Denhardt solution (0.02% Ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 0.25 mg/mL denatured salmon sperm, and 50 µmol/L sodium phosphate (pH 6.5) and then hybridized with 2 × 106 cpm/mL of ( -32P) dCTP
radiolabeled complementary DNA (cDNA) plasmid probe at 42°C for 16 to
24 hours. After hybridization, blots were washed with 0.1% SDS, 2 ×
SSC for 30 minutes at 65°C, followed by 2 washes with 0.1% SDS,
0.1 × SSC for 30 minutes at 65°C. The blots were then exposed to
XAR-5 x-ray film with intensifying screens at  70°C. Expression of
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as an
internal control for the quantity of total RNA on each lane of the gel
and this control was applied in all experiments.
Western blot analysis Total cellular protein was obtained from human smooth muscle cells grown in 100-mm dishes. At the end of each incubation cell layers were washed twice with PBS and protein was extracted in ice-cold RIPA buffer, containing protease inhibitors (leupeptin, PMSF, pepstatin; Sigma). Cellular DNA was removed by collecting the supernatant after centrifugation at 10 000 rpm in a microcentrifuge for 10 minutes. The total protein concentration in each sample was determined using bovine serum albumin as a standard.31 SDS-polyacrylamide gel electrophoresis using 10% acrylamide gels was performed with each lane loaded with 50 µg protein per sample under denaturing conditions. Broad range and low range molecular weight standards (Sigma) were run in parallel. Proteins were transferred to membrane paper using 2 mA/cm2 of current. Membranes were blocked with 5% milk powder for 30 minutes, followed by 45 minutes of primary antibody (0.5 µg/mL rabbit antihuman tissue factor, American Diagnostics, Greenwich, CT) in PBS, containing 5% milk powder and 0.1% Tween, then washed 3 times in PBS and 0.1% Tween for 10 minutes. A 1:5000 dilution of a peroxidase-labeled secondary antibody was then applied (Goat antirabbit IgG; Boehringer Mannheim) in 5% milk powder and 0.1% Tween for 30 minutes. The blots were then washed an additional 3 times. The signal was developed with exposure to film for 1 to 3 minutes using ECL (Amersham, Buckinghamshire, England).Quantification of surface tissue factor protein levels was also performed using Western blot analysis, as previously described.11 After the 4-hour treatment with a defined agonist, cell layers were incubated with rabbit-antihuman tissue IgG antibody at 4°C for 60 minutes. Cell layers were harvested, as described previously. Cell lysate (50 µg protein per treatment) was incubated with Protein-A sepharose beads to immunoprecipitate IgG bound tissue factor.11 Immunoprecitates were then separated on 10% SDS-PAGE gels, as described previously.
Effect of oxidized low-density lipoprotein on tissue factor messenger RNA and surface tissue factor pathway activity Figure 1A shows that the rat aortic smooth muscle cells treated with oxidized LDL, but not native LDL exhibited a transient increase in the expression of cell surface tissue factor pathway activity, with peak activity occurring at approximately 4 hours. The transience of the response to oxidized LDL, and the time to reach peak tissue factor pathway activity, are similar to those observed for smooth muscle cells exposed to other agonists.16 The level of cell surface tissue factor activity induced by oxidized LDL was comparable to that induced by 2 agonists previously shown to induce tissue factor gene expression and activity in smooth muscle cells (Figure 1B), FBS and-thrombin,16 at concentrations of the agonists that we
determined to be optimal (data not shown).
The induction of tissue factor mRNA in response to oxidized LDL was
concentration dependent (Figure 2A). Peak
tissue factor mRNA induction occurred at approximately 90 minutes after
exposure to either native or oxidized LDL, as previously
reported12 (data not shown), which is also similar to the
time course of tissue factor mRNA increases in smooth muscle cells
exposed to other agonists, including FBS.16 Interestingly,
although the peak levels of tissue factor pathway activity induced by
serum were comparable to those induced by oxidized LDL (Figure 1B),
mRNA levels achieved after serum treatment were significantly higher than those after oxidized LDL treatment (Figure 2A).
Immunoprecipitation of tissue factor protein on the cell surface
analyzed by Western blot analysis revealed that the surface tissue
factor protein was elevated above control levels after 4 hours of FBS
or oxidized LDL stimulation (Figure 2B).
The protocol that we adapted for assessing agonist induction of tissue factor pathway activity included a 48-hour serum-free pretreatment before the addition of agonist.16 To evaluate the effects of this serum-free period on our results, we compared cells pretreated in serum-free DMEM with those in serum (2% FBS) for the 48 hours before the addition of lipoprotein. We observed similar levels of surface tissue factor mRNA induction at 90 minutes and surface tissue factor pathway activity at 4 hours in response to oxidized LDL in serum-free and 2% FBS conditions (data not shown). Furthermore, native LDL-induced tissue factor mRNA levels at 90 minutes were similar with either serum-free DMEM or FBS (2%) pretreatments (data not shown), and native LDL did not significantly induce tissue factor pathway activity whether cells were maintained in 2% FBS or serum-free DMEM. Role of lipid peroxidation in oxidized low-density lipoprotein activation of cell surface tissue factor pathway Because we have previously shown that oxidized LDL could induce cellular lipid peroxidation,22,26 and that oxidant stress in the form of exogenous H2O2 can enhance latent cell surface tissue factor pathway activity,11 we asked whether cellular lipid peroxidation induced by oxidized LDL is responsible for the activation of the tissue factor pathway involving latent cell surface tissue factor protein. Cells were pretreated overnight and during a 4-hour oxidized LDL treatment with multiple antioxidants (Figure 3) with different inhibitory actions, including N, N'-diphenyl-1,4-phenylene-diamine (DPPD) (1 µmol/L) or-tocopherol (25 µmol/L), peroxyl radical scavengers;32 ebselen (10 µmol/L), an agent that reduces
complex lipid hydroperoxides;33 or Tiron (10 mmol/L) or
desferoximine (3 mmol/L), iron chelators that are active in cells or at
cell membranes.34,35 Cells treated with these antioxidants
were inhibited from the increased surface tissue factor pathway
activity elicited by oxidized LDL (Figure 3A,B). Pretreatment of the
cells with -tocopherol or DPPD was required to achieve inhibition, because the presence of equivalent concentrations solely during the 4 hours of oxidized LDL exposure did not inhibit the activation of the
tissue factor pathway (data not shown). Supporting the hypothesis that
lipoproteins affect tissue factor activity by a novel pathway that is
distinct from that of other known agonists, -tocopherol had no
effect on the induction of surface tissue factor pathway activity by
-thrombin or FBS (Figure 3B). Although antioxidants blocked the
induction of surface tissue factor pathway activity, they did not
inhibit the increases in tissue factor mRNA (Figure
4A) or whole cell tissue factor protein
expression (Figure 4B) induced in response to oxidized LDL.
Multiple lipids in oxidized low-density lipoprotein induce surface tissue factor pathway activity Total lipid extracts of oxidized LDL were separated by HPLC on a C-18 column and collected in 22 fractions. The data in Figure 5 depict a typical tissue factor activity profile obtained by placing each of the 22 fractions on smooth muscle cells and measuring activity 4 hours later. Fractions 7 and 11-12 reproducibly yielded distinct peaks of tissue factor activity in the profiles from the lipid fractions of isolates from multiple preparations of oxidized LDL. Native LDL lipid fractions separated on HPLC failed to enhance tissue factor pathway activity (data not shown).
We focused attention on fraction 7, because we had previously
identified several oxysterols that elute in it.26 The data in Figure 6 demonstrate that DPPD, tiron,
ebselen, and desferoximine all inhibited the activation of the surface
tissue factor pathway by the lipids contained in fraction 7 as they had
for oxidized LDL. We know from prior studies in our laboratory that
fraction 7 from this HPLC protocol contains, among other lipids,
7
We directly added pure preparations of these oxysterols to smooth
muscle cells in an attempt to determine whether they activated the
tissue factor pathway on the smooth muscle cell surface. Figure 7A shows that commercial preparations of
7
To verify the importance of the contribution of lipid hydroperoxides on the activation of the surface tissue factor pathway by oxidized LDL, we exposed rat aortic smooth muscle cells to SnCl2-treated total lipid extracts of native and oxidized LDL. SnCl2 treatment results in the reduction of lipid hydroperoxides to their hydroxy-equivalents.27 Furthermore, SnCl2 partitions to the aqueous phase during lipid extraction, thus it is not present when the lipid extract is added to the cells. SnCl2-treated oxidized LDL resulted in approximately 65% less tissue factor pathway activation than untreated oxidized LDL (data not shown). In an attempt to assess the extent of LDL oxidation that is required
for induction of increased tissue factor pathway activity, we oxidized
LDL relatively mildly by incubating it with smooth muscle cells for 6 hours in a culture medium containing DME/F12 (described in "Materials
and methods"). In the other experiments described in this study, we
used DME medium, devoid of copper and with minimal iron, to minimize
spurious oxidation of LDL in experiments in which we wished to examine
the effects of native LDL on smooth muscle cells. However, previously
we showed that exposure of LDL to smooth muscle cells in DME/F12
facilitated smooth muscle cell oxidation of LDL.24 At
relatively short times of exposure (eg, 6 hours), the oxidation was
mild (minimal changes in thiobarbituric acid reactivity or
electrophorectic mobility).24 The data in Figure
8 show that cells exposed to LDL for 6 hours in DME/F12 exhibited significantly elevated levels of surface tissue factor pathway activity compared with those exposed to LDL in
DME. Our data suggest that the increased activity in the cells exposed
to DME/F12 was due to lipid hydroperoxides, because ebselen treatment
significantly decreased tissue factor activity in the cells. Confirming
that only mild oxidation of LDL occurred at 6 hours (as predicted by
previous results),24 LDL recovered from these wells had
the same electrophoretic mobility as LDL not exposed to cells (data not
shown).
Because native and oxidized LDL both increased tissue factor
mRNA, but 7
In this study, we have demonstrated that, like native LDL,
oxidized LDL increases tissue factor protein expression on the surface
of smooth muscle cells (Figure 2B); however, in contrast to native LDL,
oxidized LDL also significantly increases tissue factor pathway
activity on the cell surfaces (Figure 1A). Furthermore, we have
demonstrated that the cellular pathway by which oxidized LDL increases
tissue factor pathway activity on the cell surface is distinct from
that reported for other agonists (Figure 3B), in that oxidative stress
appears to be a required step in a mechanism in which the cell surface
tissue factor complex exhibits increased activity. Finally, our data
show that there are multiple lipids borne by oxidized LDL that enhance
tissue factor pathway activity in smooth muscle cells. One of the
lipids formed during LDL oxidation, which could be responsible for part
of the activation of the tissue factor pathway by oxidized LDL, is
7 Our data further demonstrate that there exists a second peak of less polar lipids in oxidized LDL (fractions 11-12, Figure 5) that can also induce surface tissue factor pathway activity. Reduction of the total lipid extract of oxidized LDL by SnCl2 did not completely block the induction of surface tissue factor activity, which suggests there may be nonhydroperoxide lipids in oxidized LDL that are capable of inducing surface tissue factor pathway activity. However, because the increased cell surface tissue factor pathway activity induced by oxidized LDL was inhibitable by multiple antioxidants, it is likely that these other nonhydroperoxide lipids also activate the tissue factor pathway via inducing lipid peroxidation. Experiments are underway to identify more of the multiple lipids of oxidized LDL that activate tissue factor and to evaluate their relative potencies and mechanisms of action. Our findings could have important pathophysiologic implications,
because tissue factor is expressed in mesenchymal cells of vascular
lesions5,7 and in smooth muscle cells after vascular injury,6 and because oxidized lipoproteins36
and their constituents, including
7 The pathway of tissue factor induction by lipoproteins in smooth muscle cells is at least in part distinct from that observed in endothelial cells and macrophages.38-43 Drake et al40 reported an increase in tissue factor activity and mRNA accumulation in response to oxidized, but not native LDL in human umbilical vein endothelial cells. Pigeon macrophages in culture have been shown to have enhanced expression of tissue factor in response to oxidized LDL.41 Petit et al42 showed a significant increase in cell surface tissue factor activity in human monocyte-derived macrophages in response to cholesterol and oxidized LDL without an increase in cell surface-associated tissue factor protein. Brand et al,43 using human adherent monocyte cultures, found that neither native nor oxidized LDL altered tissue factor protein expression or activity; however, they found that oxidized LDL potentiated tissue factor expression induced by LPS. Despite the differing mechanisms by which oxidized LDL influences tissue factor expression in these various cell types, it is clear from these studies and ours that oxidized LDL can increase surface tissue factor pathway activity in endothelial cells, monocyte-derived macrophages, and smooth muscle cells, all of which exist in atherosclerotic lesions in close proximity to oxidized lipids. That oxidized LDL, but not native LDL exposure, resulted in increased surface tissue factor pathway activity and antioxidants inhibited it (Figure 3A,B), suggests that oxidative stress causes activation of the tissue factor pathway, involving preexisting tissue factor protein on the cell surface that, before oxidant stress, could not participate in active complex formation. It has been proposed previously that tissue factor may exist in such a latent form on cell surfaces, and that can be induced to initiate tissue factor pathway activity.4 Possible mechanisms proposed for tissue factor pathway latency on the cell surface, which could be potentially reversed by oxidized lipids include (1) sequestration of tissue factor in membrane crypts or caveolae;44,45 (2) surface tissue factor interaction with tissue factor pathway inhibitor;45-47 (3) changes in the quaternary structure of tissue factor on the cell surface; or (4) changes in cell membrane outer leaflet (eg, increased phosphatidylserine content) that enhances tissue factor:factor VIIa activity by, for example, increasing factor X associating with the membrane.48 The exact mechanism of surface tissue factor pathway activation by oxidant stress is unclear from these experiments; however, a general scheme of the propagation of lipid peroxidation could include (1) an initial interaction of cells with lipoprotein-borne lipid hydroperoxides, (2) reduction of lipid hydroperoxides by cellular iron to form alkoxyl radicals, and (3) formation of lipid and peroxyl radical intermediates leading to propagation of peroxidation of cellular lipids22,49 and tissue factor pathway activation. This interpretation is compatible with a report that copper transported via the carrier 8-hydroxyquinoline into human THP-1 monocytes caused cellular lipid peroxidation and significantly increased tissue factor pathway activity.50 Furthermore, Brisseau et al51 demonstrated that LPS-induced tissue factor pathway activity in macrophages and monocytes was inhibited by the antioxidants, N-acetyl-cysteine and pyrrolidine dithiocarbamate, with no decrease in LPS-induced tissue factor mRNA. It is likely that the mechanism of oxidant-induced activation of the tissue factor pathway involves cell surface or extracellular events because we have previously shown that H2O2-induced activation of tissue factor did not require the cytoplasmic tail of tissue factor.11 Our results suggest that oxidized lipoproteins, known to reside in
vascular lesions,36 could be stimulants of tissue factor pathway activity in vivo. One of the oxidized LDL constituents, 7
We thank Dr Robert D. Rosenberg, Massachusetts Institute of
Technology, for encouragement and helpful discussions and Dr Scott Colles and Charles Kaul for the preparation of
7
Submitted February 8, 2000; accepted June 23, 2000.
Supported in part by National Institutes of Health grant HL 29582. M.S.P. is a recipient of a NRSA from the National Institutes of Health (HL 09911). M.-Z.C. is a recipient of a Scientist Development Grant from the American Heart Association (Dallas) (9730039N).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Guy M. Chisolm, Lerner Research Institute, NC10, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195; e-mail:chisolg{at}ccf.org.
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Top
Abstract
Introduction
-hydroxy or 7-ketocholesterol
had no effect on tissue factor pathway activity; however,
7
) or native LDL (
), 150 µg cholesterol/mL,
was added to rat aortic smooth muscle cells at various times before
measuring tissue factor pathway activity. (B) Serum and 
) or vitamin E (25 µmol/L, 
). All antioxidant
treatments were overnight before and during the addition of the
agonists. NT indicates no treatment (solvent control). All cells
received 0.5% ethanol at the time of treatment with antioxidants to
control for those antioxidants that required ethanol as a carrier. Data
represent means ± SD of 4 wells per treatment.
) overnight before and during the addition of LDL. Data represent
fold induction over DMEM, lipoprotein-free control. DMEM and DME/F12
lipoprotein-free controls yielded identical tissue factor pathway
activity. Data represent means ± SD of 4 wells per
treatment.
