Regulation of Tissue Factor Expression in Human Microvascular Endothelial Cells by
Nitric Oxide
Yinke Yang and Joseph Loscalzo
Circulation. 2000;101:2144-2148
doi: 10.1161/01.CIR.101.18.2144
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Regulation of Tissue Factor Expression in Human
Microvascular Endothelial Cells by Nitric Oxide
Yinke Yang, MD; Joseph Loscalzo, MD, PhD
Background—Tissue factor (TF) is a critical determinant of thrombin generation in normal hemostasis and in
atherothrombotic disease. Nitric oxide has both antithrombotic and antiatherosclerotic actions in the vasculature, yet its
role in the regulation of TF expression has not been examined.
Methods and Results—To study the effect of endogenous endothelium-derived nitric oxide on TF expression and activity,
we induced TF in human microvascular endothelial cells with lipopolysaccharide or interleukin-1␤ and observed a doseand time-dependent increase in TF activity and expression by Northern and Western blotting. L-Arginine, the principal
substrate for nitric oxide synthases, added to the media suppressed the induction of TF activity significantly (by 66%
for lipopolysaccharide induction and by 59% for interleukin-1␤ induction) at 24 hours. These changes in activity were
accompanied by correlative changes in TF protein and steady-state mRNA. D-Arginine had no effect, and inhibition of
endogenous nitric oxide production failed to increase TF expression.
Conclusions—These data suggest that enhanced production of endothelium-derived nitric oxide reduces endotoxin- and
cytokine-induced expression of TF and, thereby, the prothrombotic phenotype of the endothelial cell. (Circulation.
2000;101:2144-2148.)
Key Words: nitric oxide synthase 䡲 proteins 䡲 lipids
T
issue factor (TF) is a recognized critical determinant of
thrombotic responses in vivo. It is a transmembrane
glycoprotein that initiates blood coagulation by binding factor
VII with high affinity, thereby promoting factor X activation,
thrombin generation, and fibrin formation.1–3 Under normal
circumstances, endothelial cells express no or minimal
amounts of TF; however, with endothelial cell activation or
frank dysfunction, TF expression is significantly enhanced.4 – 6 Recent studies suggest that the accumulation of
TF in atherosclerotic plaque plays a major role in determining
plaque thrombogenicity.7–9
Nitric oxide (NO) is produced by normally functioning
endothelial cells as a result of the action of the endothelial
isoform of NO synthase (eNOS), which oxidizes the amino
acid L-arginine to citrulline and NO. Endothelial cells can
also be induced to express the inducible isoform of NO
synthase (iNOS),10 –12 which is a much more catalytically
efficient enzyme that is regulated principally at the level of
transcription. Regardless of its enzymatic source, endothelial
NO has been shown to influence a variety of endothelial
responses that are important in normal and pathophysiological vascular function, including the suppression of inflammatory responses, leukocyte adhesion, and platelet-dependent
thrombosis.13–15
Both TF and NO synthase expression are upregulated when
endothelial cells are activated. Recently, an association be-
tween the coagulation and endothelial NO pathways has been
reported by Papapetropoulos et al,16 who showed that factor
Xa-induced NO release modulates endothelial cell– dependent
vasorelaxation and cytokine gene expression. However, the
relation between the TF pathway and NO synthase systems in
endothelial cells is, as yet, unknown. Because of the antithrombotic effects of NO, the present study was designed to
explore the possible inhibitory role of endothelial NO on
endothelial TF expression in response to endotoxin or cytokine exposure.
Methods
Chemicals
Thromboplastin was purchased from Sigma Diagnostics. Interleukin
1␤ (IL-1␤) was purchased from Gibco, Inc. Protein G Plus-Agarose
was obtained from Calbiochem. L-Arginine, D-arginine, lipopolysaccharide (LPS), N␻-nitroarginine-L-monoethyl ester (L-NAME), and
S2222 were all purchased from Sigma Chemical Co.
Cell Culture
Human microvascular endothelial cells (HMVECs) were obtained
from Cell Systems Corp and cultured in CS-C medium supplemented
with growth factor and attachment factor (Cell Systems Corp) and
with 20% FBS (Gibco). Before experiments, cells were grown to
confluence in 24-well microplates or 100-mm tissue culture dishes.
All experiments were performed on the second to eighth passage of
cells.
Received September 29, 1999; revision received November 22, 1999; accepted December 10, 1999.
From the Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, Mass.
Correspondence to Joseph Loscalzo, MD, PhD, Boston University School of Medicine, Whitaker Cardiovascular Institute, 700 Albany St, W507,
Boston, MA 02118. E-mail [email protected]
© 2000 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
2144
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Yang and Loscalzo
Nitric Oxide and Tissue Factor
2145
Preparation of Barium Citrate Eluate (Containing
Factors VII and X)
Factors VII and X were partially prepared from bovine serum as
described by Pitlick and Nemerson.17 Briefly, 7.6% sodium citrate
and 15% barium chloride were added to the serum. After 20 minutes
of stirring, the slurry was centrifuged at 23 000 g for 20 minutes. The
precipitate was washed twice with 15% barium chloride and once
with water. The precipitate was then resuspended in 75.5 mL water/L
serum. Solid ammonium sulfate (20 g/L serum) was added to the
extract. After centrifugation, the same amount of ammonium sulfate
was added to the supernatant. The precipitate was collected by
centrifugation, resuspended in water (12 mL water/L serum), and
dialyzed overnight against 50 mmol/L imidazole HCl, 100 mmol/L
sodium chloride, pH 7.0. Aliquots were stored at ⫺80°C. Before use,
an aliquot of eluate was dialyzed overnight against the same
dialysate as described above.
Figure 1. TF activity on surface of HMVECs after LPS or IL-1␤
stimulation. HMVECs were grown to confluence in 24-well
microplates. Varying concentrations of LPS (0.01, 0.1, 1.0, and
10.0 ␮g/mL; left panel) or IL-1␤ (0.1, 1.0, 10.0, and 100 ng/mL;
right panel) were added to the medium, and cells were incubated after 24 hours. TF activity was measured on cell surface
by 2-stage amidolytic assay as described in Methods. Data are
mean⫾SEM of 6 experiments, each performed in duplicate.
TF Activity Assay
The surface expression of TF on HMVECs was measured with a
2-stage amidolytic assay as previously described18 with slight
modifications. A 24-well microplate containing confluent HMVECs
was washed four times with CS-C medium. CS-C medium (0.20 mL,
serum free) was added to the test wells. In the first stage of the assay,
10 ␮L barium citrate eluate was added to each well and incubated for
10 minutes on a rotating platform (120 cycles/min). In the second
stage, 0.1 mL of conditioned medium was combined with 0.05 mL
1 mmol/L S2222 in a 96-well microplate. The reaction was carried out
at 37°C for 30 minutes, after which the OD405 was measured with a
Thermomax Microplate Reader (Molecular Devices). TF activity
was obtained from a standard curve derived from serial dilutions of
rabbit brain thromboplastin assayed in medium. After the assay, the
cells were removed from each well with 0.05 trypsin/EDTA and
counted with a hemocytometer.
Western Analysis
Monolayers of HMVECs were lysed with immunoprecipitation
buffer (1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris, pH
7.4, 1 mmol/L EDTA, 1 mmol/L EGTA, 0.2 mmol/L sodium
orthovanadate, 0.2 mmol/L PMSF, and 0.5% NP-40) and sonicated
on ice to disperse any large aggregates. Specific antibodies (mouse
anti-human TF antibody, Calbiochem, and anti-iNOS and anti-eNOS
antibodies, Transduction Laboratories) were added to total lysate.
After 1 hour of incubation at 4°C, Protein G Plus-Agarose was added
to the lysate to collect the antigen-antibody complexes. Agarose
beads were washed twice with immunoprecipitation buffer, resuspended in electrophoresis sample buffer (250 mmol/L Tris, pH 6.8,
4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2%
␤-mercaptoethanol), and then boiled for 5 minutes. The beads were
pelleted by centrifugation at 16 000g at 4°C for 3 minutes. The
supernatant was loaded onto an SDS-PAGE gel to separate the
proteins. Proteins were transferred to nitrocellulose filters and then
immunoblotted with specific antibodies. An anti-mouse horseradish
peroxidase– conjugated antibody was used as a secondary antibody.
The blots were detected with the enhanced chemiluminescence
(ECL) system (Amersham Life Sciences, Inc).
Northern Analysis
Total RNA was isolated from HMVECs with an RNAgents Total
RNA Isolation System (Promega). RNA was probed with a human
TF cDNA kindly provided by Dr Mark Taubman (Mt Sinai School
of Medicine, New York, NY); a human eNOS cDNA (Alexis
Biochem); or an iNOS cDNA (Alexis Biochem). cDNA probes were
[32P]dCTP-labeled to 1⫻106 cpm/␮g of cDNA (Random Primer
Labeling Kit, Stratagene). The blot was prehybridized in a solution
containing 50% formamide, 5⫻ Denhardt’s solution, 5⫻ SSPE,
0.1% SDS, and 100 ␮g/mL salmon sperm DNA at 42°C for 2 hours
before addition of the probe; hybridization was performed at 42°C
overnight. Human ␤-actin cDNA-labeled with [32P] was used as a
control.
Measurement of Total Nitrite and Nitrate
Total nitrite and nitrate were measured in conditioned medium by the
method of Saville with a nitrate reductase step.19
Statistical Analysis
Treatment and time course responses were performed by ANOVA
techniques. Multiple comparisons were made with either Dunnett’s
or Newman-Keuls post hoc tests, where appropriate. Multiple
time-course treatment comparisons were performed by 2-way
ANOVA. Values given represent mean⫾SEM).
Results
TF Activity on HMVEC Surface
Confluent HMVECs were treated with LPS (0 to 10 ␮g/mL)
or IL-1␤ (0 to 100 ng/mL) for 24 hours, after which
cell-surface TF activity was measured. As shown in Figure 1,
LPS and IL-1␤ both induced TF activity. The effects of these
agents were concentration dependent and occurred maximally
after 24 hours of induction. Activity increased ⬇3-fold at the
highest concentration of LPS (10 ␮g/mL), from 34.9⫾4.2 to
93.4⫾5.3 mU/106 cells, or IL-1␤ (100 ng/mL), from
33.9⫾4.3 to 96.2⫾4.1 mU/106 cells.
TF activity increased in a time-dependent fashion, as
shown in Figure 2. L-Arginine suppressed these effects by
66% for LPS (from 70.7⫾2.1 to 50.9⫾2.4 mU/106 cells) and
by 59% for IL-1␤ (from 70.8⫾1.9 to 53.0⫾3.6 mU/106 cells)
and did so maximally by 24 hours of incubation. Neither
␻
D-arginine (data not shown) nor N -nitro-L-arginine methyl
ester (L-NAME) had any significant effect on TF activity
under these conditions; however, L-NAME coincubated with
L-arginine prevented the decrease in TF activity produced by
L-arginine (data not shown). L-Arginine supplementation
increased NO production by HMVECs (measured as nitrite/
nitrate in the conditioned medium) 7.2-fold, from 6.2 ␮mol/L
in control conditions (ie, without L-arginine supplementation)
to 44.2 ␮mol/L (with 1 mmol/L L-arginine supplementation)
after 24 hours. Preliminary data also suggest that incubation
with an NO donor, 10 ␮mol/L S-nitrosoglutathione, also
suppressed TF activity by 60%.
TF Protein Expression
HMVECs were exposed to various concentrations of LPS (0
to 10 ␮g/mL) or IL-1␤ (0 to 100 ng) for 24 hours. Protein
extracts from HMVECs were immunoprecipitated with an
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May 9, 2000
Figure 2. Effects of L-arginine (L-Arg) and L-NAME on LPSinduced and IL-1␤–induced TF activity on surface of HMVECs.
Confluent HMVECs were pretreated with L-Arg (1 mmol/L) or
L-NAME (300 ␮mol/L) for 45 minutes and then exposed to 1
␮g/mL LPS (A) or 1 ng/mL IL-1␤ (B) for 24 hours. Cell-surface
TF activity was measured by 2-stage amidolytic assay as
described in Methods. Data are mean⫾SEM of 6 experiments,
each performed in duplicate. E indicates control; , LPS or
IL-1␤; F, LPS or IL-1␤ plus L-Arg; and f, LPS or Il-1␤ plus
L-NAME. *P⬍0.01 vs LPS or IL-1␤.
Figure 4. Effect of L-Arg on LPS-induced or IL-1␤–induced TF
protein expression in HMVECs. Confluent HMVECs were pretreated with L-Arg (1 mmol/L), D-arginine (D-Arg, 1 mmol/L), or
L-NAME (300 ␮mol/L) for 45 minutes and then exposed to 1
ng/mL IL-1␤ (A) or 1 ␮g/mL LPS (B) or 1 ␮g/mL LPS and 1
ng/mL IL-1␤ (B) for 24 hours. Ctl indicates control. Western
blotting was performed with anti-human TF antibody as
described in Methods and Results.
anti-human TF antibody to quantify TF protein. Both IL-1␤
and LPS increased TF protein expression in a concentrationdependent manner, as shown in Figure 3, and did so by 6-fold
and 5-fold, respectively, at the maximal concentration of the
inducing agent used.
After treatment with L-arginine (1 mmol/L), D-arginine
(1 mmol/L), or L-NAME (300 ␮mol/L) for 45 minutes,
HMVECs were exposed to LPS (1 ␮g/mL) or IL-1␤ (1
ng/mL), and immunoprecipitation and immunoblotting were
repeated. As shown in Figure 4, L-arginine suppressed the
expression of TF protein that resulted from activation with
LPS or IL-1␤, whereas neither D-arginine nor L-NAME had
any effect. In addition, the combination of LPS and IL-1␤
(Figure 4B) further enhanced TF expression, and this enhanced expression was also suppressed by L-arginine.
L-arginine, but not D-arginine. L-NAME had no enhancing effect on
increases in mRNA were suppressed by coincubation with
TF mRNA Expression
Using a human TF cDNA probe, we determined the effect of
LPS and IL-1␤ on TF mRNA expression in HMVECs.
Steady-state TF mRNA levels increased in response to LPS
and IL-1␤ and did so in a dose-dependent manner. Steadystate TF mRNA levels increased by up to 2-fold, relative to
␤-actin mRNA, at the highest concentration of the inducing
agent (10 ␮g/mL LPS or 100 ng/mL IL-1␤) (Figure 5). These
Figure 3. LPS-induced and IL-1␤–induced TF protein expression in HMVECs. Confluent HMVECs were incubated with various concentrations of LPS (0.10, 0.1, 1.0, and 10.0 ␮g/mL) or
IL-1␤ (0.1, 1.0, 10.0, and 100 ng/mL) for 24 hours. Western blotting was performed with anti-human TF antibody as described
in Methods and Results.
TF mRNA expression (Figure 6). Importantly, neither L-arginine,
nor L-NAME had any effect on TF expression in cells
not induced with LPS or IL-1␤ (Figure 7).
D-arginine,
eNOS/iNOS Protein
An anti-eNOS antibody and an anti-iNOS antibody were used
in immunoblots to measure changes in eNOS and iNOS
proteins, respectively, induced by LPS or IL-1␤. There was
no change in eNOS protein expression after incubation of
HMVECs with IL-1␤ or LPS under these experimental
conditions (Figure 8). Similarly, iNOS protein was not
induced by LPS or IL-1␤ used alone under these experimental conditions (data not shown).
eNOS/iNOS Northern Analysis
We used a human eNOS cDNA probe and a mouse iNOS
cDNA probe to blot RNA of HMVECs after coincubation
with LPS and IL-1␤. Similar to the immunoblotting experiments, there were no differences in the steady-state levels of
eNOS mRNA after LPS or IL-1␤ treatment compared with
Figure 5. LPS-induced and IL-1␤–induced TF mRNA expression in HMVECs. HMVECs were grown to confluence and then
exposed to LPS (0.01, 0.1, 1.0, and 10.0 ␮g/mL) or IL-1␤ (0.1,
1.0, 10.0, and 100 ng/mL) for 24 hours. Northern analysis was
performed with human TF cDNA probe as described in Methods
and Results.
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Yang and Loscalzo
Figure 6. Effect of L-Arg on TF mRNA expression in HMVECs.
Confluent HMVECs were pretreated with L-Arg (1 mmol/L),
D-Arg (1 mmol/L), or L-NAME (300 ␮mol/L) and then exposed to
LPS (1 ␮g/mL) or IL-1␤ (1 ng/mL) for 24 hours. TF/␤-actin values are shown as arbitrary units (arb.units). Total RNA was isolated from cells for Northern analysis as described in Methods
and Results.
control untreated cells; iNOS mRNA was not induced by
pretreatment with either of these agents (data not shown).
Discussion
Endothelial cells modulate the balance between thrombosis and
hemostasis.20 This balance, however, can be perturbed by the
synthesis and expression of TF. TF expression is associated with
life-threatening thrombosis in a variety of diseases, including
sepsis, cancer, and atherosclerosis.21 Although quiescent endothelial cells express no or very little TF, TF expression can be
significantly induced on the surface of endothelial cells by
inflammatory stimuli, such as LPS,18 IL-1␤,22 and tumor necrosis factor-␣.23 In the present study, we demonstrate that LPS and
IL-1␤ can induce TF activity on the surface of HMVECs. The
increase in TF activity in response to these agents was concentration dependent. Moreover, Western and Northern analyses
Nitric Oxide and Tissue Factor
2147
Figure 8. Effects of LPS and IL-1␤ on eNOS protein expression
in HMVECs. Confluent HMVECs were treated with LPS (1
␮g/mL) or IL-1␤ (1 ng/mL) for 24 hours. Western blotting was
performed with anti-human eNOS antibody as described in
Methods and Results.
showed that TF protein and mRNA expression, respectively,
were increased in HMVECs in a correlative manner.
NO is a cellular signaling molecule that plays a major role
in a variety of important biological processes, including the
prevention of vascular thrombosis,24 suppression of inflammatory cell-mediated injury,25 and regulation of endothelial
integrity and cell proliferation.26,27 NO is synthesized from
L-arginine by NO synthase, and cellular NO production is
absolutely dependent on the availability of L-arginine.28 The
present study shows for the first time that L-arginine, as a
substrate for NO synthase(s), suppresses endotoxin- and
cytokine-induced expression of TF in HMVECs.
Endothelial cells provide the vascular system with a
nonthrombogenic surface.29 Activation of endothelial cells
enhances NO production. NO synthesized by endothelial cells
not only evokes vasorelaxation but also plays a pivotal role in
preventing thrombosis. The antithrombotic actions of NO
have, to date, largely focused on its antiplatelet effects,30 –33
and deficiencies of endothelium- or platelet-derived NO can
lead to a thrombotic diathesis.34,35 Vascular injury also
induces the expression of TF at local sites, where it becomes
available for plasma factor VII/VIIa binding.36
Dysregulation of NO production had been implicated in the
pathogenesis of a number of vascular diseases, including
essential hypertension and atherothrombosis. The data in the
present study indicate that NO may also reduce thrombotic
propensity by interfering with TF expression and, thereby,
coagulation. Thus, NO may prevent thrombosis not only by
inhibiting platelet function but also by suppressing TFdependent coagulation.31 Interestingly, the lack of effect of
L-NAME in these experiments suggests that basal levels of
NO have little effect on the induction of TF by LPS or IL-1␤.
We conclude that endothelial NO regulates the expression of
TF in endothelial cells. These data suggest that endothelial NO
modulates the prothrombotic phenotype of endothelial cells.
Acknowledgments
This study was supported in part by National Institutes of Health grants
HL-48743, HL-48976, HL-53919, and HL-55993 and by a Merit
Review Award from the US Veterans Administration. We would also
like to thank Stephanie Tribuna for expert technical assistance.
Figure 7. Effects of L-Arg, D-Arg, or L-NAME on TF protein and
mRNA expression in HMVECs. Confluent HMVECs were incubated with L-Arg (1 mmol/L), D-Arg (1 mmol/L), or L-NAME
(300 ␮mol/L). Western (A) and Northern (B) analyses were performed as described in Methods and Results.
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