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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Smooth muscle cell surface tissue factor pathway activation by oxidized
low-density lipoprotein requires cellular lipid peroxidation
Marc S. Penn, Mei-Zhen Cui, Allison L. Winokur, John Bethea, Thomas A. Hamilton, Paul E. DiCorleto, and Guy M. Chisolm
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␣- or 7␤-hydroxy
or 7-ketocholesterol had no effect on tissue factor pathway activity; however, 7␤hydroperoxycholesterol increased tissue
factor pathway activity without induction
of tissue factor mRNA. Tertiary butyl hy-
droperoxide also increased tissue factor
pathway activity, suggesting that lipid hydroperoxides, some of which exist in atherosclerotic lesions, activate the tissue factor
pathway. We speculate that thrombin production could be elevated via a mechanism
involving peroxidation of cellular lipids, contributing to arterial thrombosis after plaque
rupture. Our data suggest a mechanism by
which antioxidants may offer a clinical benefit in acute coronary syndrome and
restenosis. (Blood. 2000;96:3056-3063)
© 2000 by The American Society of Hematology
Introduction
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 oxidantproducing 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, oxidantmediated 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.
From the Departments of Cardiology, Cell Biology, and Immunology, Lerner
Research Institute, Cleveland Clinic Foundation, Cleveland, OH.
Reprints: Guy M. Chisolm, Lerner Research Institute, NC10, Cleveland Clinic
Foundation, 9500 Euclid Ave, Cleveland, OH 44195; e-mail:[email protected].
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).
3056
Materials and methods
Tissue culture
Smooth muscle cells were prepared from explants of excised aortas of
Sprague Dawley rats or samples of human aorta obtained from transplant
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.
© 2000 by The American Society of Hematology
BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
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BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
donors using procedures analogous to those previously described.15 Cultures from passages 4 to 10 were used in these studies. Twenty-four–well
plates were seeded with approximately 25 000 cells per well in DMEM
containing 10% fetal bovine serum (FBS). One day after seeding, the media
was changed to serum-free DMEM after being washed twice with
phosphate-buffered saline (PBS). All cells were in serum-free DMEM for
48 hours before the addition of the agonist of interest as previously
described.11,16 In select experiments, cells were placed in DMEM containing 2% FBS, instead of serum-free DMEM, for the 48 hours before the
addition of the agonist. Also in select experiments, (described in lipoprotein
isolation and oxidation, below) cells were placed in DME/F12 on the day
of plating, and then for the 48 hours before and after the addition of
lipoproteins.
In those experiments involving antioxidant pretreatment, antioxidants
(N, N⬘-diphenyl-1,4-phenylene-diamine, DPPD from Aldrich Chemical Co,
Milwaukee, WI; Tiron, ␣-tocopherol, and deferoxamine from Sigma
Chemical, St Louis, MO; ebselen from Cayman Chemical Co, Ann Arbor,
MI) were added overnight immediately before the end of the 48-hour
serum-free pretreatment. Antioxidants were also present during the exposure to oxidized LDL, native LDL, or control solvent. The ␣-tocopherol and
ebselen were added in ethanol to a final ethanol concentration of 0.5%.
Solvent concentration was matched in selected controls. In experiments
using cycloheximide to block protein synthesis, cycloheximide (20 ␮mol/L)
was added 30 minutes before and during the addition of lipoprotein or
lipoprotein extract.
Lipoprotein isolation and oxidation
Human LDL was isolated from citrated plasma by differential ultracentrifugation between solvent density limits of 1.019 to 1.063 as previously
described.17 EDTA was present (0.5 mmol/L) throughout the isolation
procedure. Quality of all preparations was checked by assaying endotoxin
level (less than 0.15 endotoxin units/mL) (Whittaker Bioproducts kit No.
QCL-1000), electrophoretic mobility (Corning, Corning, NY) and thiobarbituric acid reactivity.18,19 Preparations were indexed by total cholesterol
(Boehringer Mannheim Diagnostics kit No. 236691; [Indianapolis, IN]) and
total protein20 measurements. Native LDL preparations were stored in 0.5
mM EDTA at 4°C until use.
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 ␮mol/L
CuS04 or 6 ␮mol/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 ␮g/L, FeSO4-417 ␮g/L, CuSO4-0.13
␮g/L vs DMEM: Fe(NO3)3-100 ␮g/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 ␮Bondapak 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
EFFECTS OF OXIDIZED LDL ON TISSUE FACTOR EXPRESSION
3057
final 10 minutes. Fractions were collected every 3 minutes. Each fraction
was then dried under nitrogen and stored at ⫺70°C. Lipids in each fraction
were then dissolved in acetone:ethanol, 1:1 (vol/vol) just before adding the
lipids to the cell layers.
The 7␤-hydroperoxycholesterol was synthesized by incubating cholesterol with soybean lipoxidase and ethyl lineoleate, as previously prescribed.26 Isolation, purification, and quantification was achieved by
sequential HPLC steps, as reported previously.25,26 Other oxysterols were
obtained commercially (Steraloids Inc, Wilton, NH).
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 ␮L of 2 mg/mL S-2222 (Pharmacia-ATPAR, Piscataway, NJ), and 20 ␮L
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 ␮L 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 ␮g) 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
␮mol/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 ␮g protein per
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3058
PENN et al
Figure 1. Oxidized LDL-induced tissue factor pathway activity as a function of
time and compared with other known agonists at 4 hours in smooth muscle
cells. (A) Oxidized LDL (F) or native LDL (E), 150 ␮g cholesterol/mL, was added to
rat aortic smooth muscle cells at various times before measuring tissue factor
pathway activity. (B) Serum and ␣-thrombin or lipoprotein was added to wells of rat
aortic smooth muscle cells at the specified concentrations 4 hours before tissue
factor assay. NT indicates no treatment (control). All cells had been in serum-free
DMEM for 48 hours at the time of assay. Data represent means ⫾ SD of 4 wells per
treatment. Data are from an experiment representative of multiple experiments.
BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
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
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 ␮g/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 rabbitantihuman tissue IgG antibody at 4°C for 60 minutes. Cell layers were
harvested, as described previously. Cell lysate (50 ␮g 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.
Results
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
Figure 2. Effect of oxidized LDL on tissue factor mRNA and protein. (A) Tissue
factor (TF) mRNA from rat aortic smooth muscle cells was assessed by Northern blot
analysis 90 minutes after the addition of fetal bovine serum (FBS, 10%), or oxidized
LDL at 0, 50, 100, and 200 ␮g cholesterol/mL. (B) Tissue factor surface protein
immunoprecipitated from 50 ␮g of cell lysate of human aortic smooth muscle cells
was assessed by Western blot analysis 4 hours after “no treatment” (NT; control),
FBS (10%), or oxidized LDL (100 or 200 ␮g cholesterol/mL). Glyceraldehye
3-phosphate dehydrogenase (GAPDH) was assessed as an internal control to
confirm equal loading in each lane for Northern blot analysis.
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BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
EFFECTS OF OXIDIZED LDL ON TISSUE FACTOR EXPRESSION
3059
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).
Figure 3. Effect of antioxidant treatments on oxidized LDL induction of surface
tissue factor pathway activity. Oxidized LDL or native LDL (200 ␮g protein/mL),
␣-thrombin (50 nmol/L), or FBS (10%) was added to rat aortic smooth muscle cells for
4 hours before tissue factor assessment. (A) Cells were pretreated with DPPD (1
␮mol/L), ebselen (Ebs, 10 ␮mol/L), Tiron (10 mmol/L), or desferoximine (Des, 3
mmol/L). (B) Cells were pretreated with ethanol alone (0.5%, 䡺) or vitamin E (25
␮mol/L, p). 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.
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 ␮mol/L) or ␣-tocopherol (25 ␮mol/L),
peroxyl radical scavengers;32 ebselen (10 ␮mol/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,
Figure 4. Effect of ␣-tocopherol and DPPD pretreatment on oxidized LDL
induction of tissue factor mRNA and protein. Tissue factor (A) mRNA by Northern
blot at 90 minutes and (B) total cellular protein by Western blot at 4 hours was
assessed after the addition of oxidized LDL at 100 ␮g protein/mL to plates of (A) rat or
(B) human aortic smooth muscle cells. Cells were pretreated with either ␣-tocopherol
(25 ␮mol/L) or DPPD (1 ␮mol/L) for 24 hours before the addition of oxidized LDL.
GAPDH was assessed as an internal control to confirm equal loading in each lane for
Northern blot analysis.
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3060
PENN et al
BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
Figure 5. Tissue factor pathway activity at 4 hours induced by serial fractions of
oxidized LDL lipids separated by reverse phase HPLC. Fractions obtained from
HPLC separation of the total lipid extract from oxidized LDL were added to rat aortic
smooth muscle cells at 400 ␮g protein equivalents/mL 4 hours before tissue factor
pathway assay. Data represent means ⫾ SD of 4 wells per treatment. Data are from
an experiment representative of multiple experiments.
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␤-hydroperoxycholesterol,25,26 7␤- and 7␣-hydroxycholesterol, and
7-ketocholesterol.26
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␣- and
7␤-hydroxycholesterol, and 7-ketocholesterol, at concentrations
approximating those in active levels of oxidized LDL,26 failed to
induce surface tissue factor pathway activity in smooth muscle
cells. In contrast, 7␤-hydroperoxycholesterol did induce surface
tissue factor protein expression at 2 hours (Figure 7B) and 4 hours
(data not shown). Because only the hydroperoxide was active, we
tested the effects of another hydroperoxide, t-butyl hydroperoxide
(not found on oxidized LDL) at various times. The t-butyl
hydroperoxide induced surface tissue factor pathway activity at 2
hours (Figure 7C) and 4 hours, with a peak at 2 hours (data not
shown), similar to that which we observed previously for hydrogen
Figure 6. Effect of antioxidant treatments on fraction 7 induction of surface
tissue factor pathway activity. Fraction 7 (see Figure 5) at a level equivalent to 400
␮g protein of intact oxidized LDL per milliliter was added to rat aortic smooth muscle
cells for 4 hours before tissue factor assessment. Cells were pretreated with either
DPPD (1 ␮mol/L), ebselen (10 ␮mol/L), Tiron (10 mmol/L), or desferoximine (3
mmol/L) overnight before and during the addition of fraction 7. NT indicates no
treatment (solvent) control. Data represent means ⫾ SD of 4 wells per treatment.
Figure 7. Cell surface tissue factor pathway activity in response to 7␣- or
7␤-hydroxycholesterol, 7-ketocholesterol, 7␤-hydroperoxycholesterol, or tbutyl hydroperoxide. (A) Oxidized LDL, native LDL (200 ␮g cholesterol/mL), 7␣- or
7␤-hydroxycholesterol (10 ␮mol/L) or 7-ketocholesterol (10 ␮mol/L) was added to rat
aortic smooth muscle cells, and surface tissue factor pathway activity was measured
4 hours later. 7␤-Hydroperoxycholesterol (10 ␮mol/L, B) or t-butyl hydroperoxide (25
␮g/mL, C) was added to rat aortic smooth muscle cells, and surface tissue factor
pathway activity was measured 2 hours later. Data represent means ⫾ SD of 4 wells
per treatment. Data are from experiments representative of multiple experiments at 2
and 4 hours.
peroxide.11 As we have previously demonstrated for hydrogen
peroxide, neither t-butyl hydroperoxide nor 7␤-hydroperoxycholesterol induced tissue factor mRNA expression (data not shown).
However, similar to oxidized LDL and fraction 7, the induction of
smooth muscle cell surface tissue factor pathway activity by t-butyl
hydroperoxide and 7␤-hydroperoxycholesterol was inhibited by
treatment with multiple antioxidants (data not shown).
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
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BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
EFFECTS OF OXIDIZED LDL ON TISSUE FACTOR EXPRESSION
3061
Discussion
Figure 8. Mildly oxidized LDL can induce surface tissue factor pathway activity.
Cells in DMEM or DME/F12 were treated with LDL (200 ␮g protein/mL) for 6 hours
before assessment of tissue factor pathway activity. Cells were pretreated with
solvent (control, 䡺) or ebselen (10 ␮mol/L, f) 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.
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␤-hydroperoxycholesterol (up to 20 ␮mol/L)
did not, it appeared that an unknown lipid (or lipids) in both
native LDL and oxidized LDL, or perhaps a lipid common to
both, induced the gene and that this action is distinct from lipid
hydroperoxide activation. The concept of separate lipids inducing the tissue factor gene and activating its pathway was tested
further in 2 ways. (1) We treated cells with native LDL to induce
new tissue factor protein production11 before the addition of a
lipid hydroperoxide “activator” of tissue factor pathway. The
data in Figure 9A reveal that more tissue factor pathway activity
is observed in cells treated with native LDL before the addition
of t-butyl hydroperoxide than in cells treated with t-butyl
hydroperoxide alone. This shows that a lipid hydroperoxide is
capable of “activating” the latent surface tissue factor pathway,
including that involving tissue factor synthesized in response to
native LDL. (2) The data in Figure 9B demonstrate that oxidized
LDL induces surface tissue factor pathway activity even when
new protein synthesis is decreased by the addition of cycloheximide. These data together are consistent with the concept that
oxidized LDL increases surface tissue factor activity in 2
distinct steps: (1) increasing surface tissue factor protein and (2)
activating the cell surface tissue factor pathway involving
preexisting latent tissue factor protein. The lipoprotein component responsible for (1) is unknown; 7␤-OOH cholesterol is one
of the oxidized LDL components capable of (2).
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␤-hydroperoxycholesterol. That this may be a more general property of hydroperoxides is indirectly suggested by our findings that nonhydroperoxide
oxysterols were ineffective in enhancing tissue factor pathway
activity (Figure 7A), t-butyl hydroperoxide also activated the tissue
factor pathway (Figure 7C), reduction of oxidized LDL lipids by
SnCl2 significantly decreased the oxidized LDL tissue factor
pathway activating capacity, and, as shown previously, hydrogen
peroxide activated the tissue factor pathway.11
Our data further demonstrate that there exists a second peak of
Figure 9. t-Butyl hydroperoxide can activate the tissue factor pathway involving new synthesized protein induced by native LDL; oxidized LDL lipid extracts
can activate surface tissue factor without new protein synthesis. (A) Rat aortic
smooth muscle cells were treated without or with LDL (200 ␮g protein/mL). Two hours
later t-butyl hydroperoxide (25 ␮g/mL) was added to the wells of the groups indicated.
After an additional 2 hours, surface tissue factor activity was assayed in all wells. (B)
Total lipid extracts of oxidized LDL, at a concentration equivalent to 200 ␮g
protein/mL of intact lipoprotein were added to rat aortic smooth muscle cell layers.
Some were treated with cycloheximide (20 ␮mol/L; filled bars) 30 minutes before, and
concurrent with lipid treatment. Tissue factor pathway activity was measured after 2
hours of lipid treatment. Data represent means ⫾ SD of 4 wells per treatment.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
3062
BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
PENN et al
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␤-hydroperoxycholesterol,25,37 have been demonstrated in
atherosclerotic lesions. If oxidized LDL in vivo induces tissue
factor pathway activity, it could result in thrombosis as well as
smooth muscle cell proliferation and thus contribute to atherosclerotic lesion growth, plaque rupture, or intimal smooth muscle
cell proliferation observed in some instances of restenosis after
angioplasty.
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-acetylcysteine 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␤hydroperoxycholesterol, that can enhance tissue factor pathway
activity, is also present in atherosclerotic tissue.25,37 The lipid
peroxidation responsible for tissue factor pathway activation is
antioxidant inhibitable (Figures 3 and 5). Thus, tissue factor
pathway activation by lipid hydroperoxides could explain ways in
which antioxidants may inhibit atherosclerosis, acute coronary
syndrome, and restenosis after angioplasty.
Acknowledgments
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␤hydroperoxycholesterol.
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2000 96: 3056-3063
Smooth muscle cell surface tissue factor pathway activation by oxidized
low-density lipoprotein requires cellular lipid peroxidation
Marc S. Penn, Mei-Zhen Cui, Allison L. Winokur, John Bethea, Thomas A. Hamilton, Paul E.
DiCorleto and Guy M. Chisolm
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