Original Article
Acta Cardiol Sin 2008;24:151-6
Vascular Medicine
Thrombin Induces the Expression of Tissue
Factor in Human Aortic Endothelial Cells
Huang-Joe Wang,1,2 Huey-Chun Huang,3 Yi-Ching Chuang3 and Haimei Huang2
Background: Atherosclerotic plaque disruption and subsequent thrombosis formation is responsible for many
thrombotic complications in cardiovascular disease. Tissue factor (TF) exposure upon plaque rupture can trigger
thrombin formation. Thrombin, on the other hand, can induce tissue factor expression in human umbilical vein
endothelial cells or saphenous vein endothelial cells. The impact of thrombin in the endothelial cells of arterial side
was less known.
Methods: Human aortic endothelial cells (HAEC) were cultivated and methoxyphenyl tetrazolium inner salt assays
were used to determine the non-toxicity of thrombin to endothelial cells. Real-time PCR was used to determine the
relative TF mRNA quantity. Western blot was used to determine the relative protein level. TF functional activity was
determined by a chromogenic assay.
Results: Thrombin was non-toxic to the HAEC. TF mRNA relative quantity was enhanced 390 ± 114% by thrombin
(P = 0.034). TF protein relative quantity was enhanced 332 ± 39% by thrombin (P = 0.01). TF activity was enhanced
547 ± 54% by thrombin (P < 0.001).
Conclusion: Thrombin is a sufficient stress to induce TF expression in HAEC. The transcriptional control by
thrombin causes an increase in TF mRNA. This increase in mRNA is modestly paralleled by an increase in protein
level and functional activity.
Key Words:
Human aortic endothelial cell · Thrombin · Tissue factor
INTRODUCTION
trinsic coagulation system. In blood vessels, TF is detectable in adventitial fibroblasts under normal circumstance. Atherosclerotic plaque, however, contains significant TF mRNA and antigen, which is associated with
monocytes and smooth muscle cells within the plaque.2
TF in plaque can bind to factor VIIa and is functional.3
Once formed in situations of plaque rupture, TF:VIIa
can activate factor IX or factor X. The Xa can assemble
with factor Va on phospholipid surface to catalyze the
formation of thrombin from prothrombin. Thrombin then
initiates clot formation by converting fibrinogen to fibrin. These chain reactions of coagulation cascades will
result in thrombus formation at the ruptured plaque site.
Previous reports have documented that thrombin
could elicit tissue factor production in endothelial cells
coming from human umbilical cord vein and human
saphenous vein.4-6
The endothelium is a special organ and performs a
Atherosclerotic plaque disruption and subsequent
thrombosis formation is responsible for the onset of
most cardiovascular events. The magnitude of the thrombotic processes triggered upon plaque disruptions is dependent upon tissue factor (TF).1 TF is the principal initiator and propagator of thrombus formation in the ex-
Received: May 20, 2008
Accepted: July 29, 2008
1
Division of Cardiology, Department of Medicine, China Medical
University Hospital and University; 2Institute of Biotechnology, National Tsing Hua University; 3Department of Medical Laboratory Science and Biotechnology, China Medical University.
Address correspondence and reprint requests to: Dr. Haimei Huang,
Institute of Biotechnology, National Tsing Hua University, No.
101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan, R.O.C.
Tel: 886-3-5742479; Fax: 886-3-5742479; E-mail: hmhuang@
life.nthu.edu.tw
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Huang-Joe Wang et al.
template in a 25-ml reaction mixture containing a specific
primer pair of each cDNA. The total cDNA pool obtained
served as template for subsequent PCR amplification
using the SYBR PCR kit (Applied Biosystems) according to the manufacturer’s protocols for the ABI PRISM
7900 HT sequence detection system. The primers were
designed using Primer Express 2.0 (Applied Biosystems)
and are synthesized by Blossom Biotechnologies Inc.
(Taiwan). The primer sequences were as follows:
complex function. A unique characteristic of endothelial
cell is their remarkable heterogeneity in different organs.7 Because of such heterogeneity, we are interested
in determining whether endothelial cells from an arterial
side (i.e human aortic endothelial cells, HAEC) still preserve the same response to human thrombin regarding to
its ability to induce tissue factor expression. The purpose
of the current study was to examine whether exposure of
HAEC to human thrombin could cause sufficient perturbation to alter the expression of tissue factor.
Tissue factor
forward primer:
5¢-TCCCGAACAGTTAACCGGAA-3¢
reverse primer:
5¢-GACCACAAATACCACAGCTCCA-3¢
MATERIALS AND METHODS
Cell culture
HAEC were purchased from Cell Applications, Inc.
(San Diego, CA). The HAEC were cultured in endothelial cell growth medium (Cell Applications, Inc.) according to the supplier’s recommendation. Cells were grown
to near confluence in 10-cm culture dishes before experiment. Before thrombin stimulant was added, the HAEC
were rendered quiescent in Medium-199 supplement
with 1% fetal bovine serum (HyClone) for 24 hours. Human thrombin (Sigma-Aldrich) stimulation (1 unit/ml)
up to 3 hours (for TF mRNA expression) and 5 hours
(for TF protein and activity expression) was done.
GAPDH
forward primer:
5¢-ATCCCTCCAAAATCAAGTGGG-3¢
reverse primer:
5¢-TGAAGACGCCAGTGGACTCC-3¢
The real-time PCR was performed using the following cycling parameters: 95° for 10 minutes for 1 cycle;
95° for 30 seconds, 60° for 1 minute, and 72° for 1 minute, for a total of 40 cycles. Expression of GAPDH
mRNA served as loading control, and a melting curve
analysis was performed after amplification to verify the
accuracy of the amplicon.
Methoxyphenyl tetrazolium inner salt (MTS)
cell viability assay
MTS assays were performed to monitor conversion
of methoxyphenyl tetrazolium inner salt to formazan by
mitochondrial dehydrogenase in viable cells. Briefly,
HAEC were seeded in a 96-well plate with final cell
concentration 1 ´ 104/well. After serum starvation for 24
hours, the HAEC were treated according to the protocol.
Twenty ml MTS solution (0.5 mg/ml, Promega) was then
added to each well and the cells were cultivated at 37 °C
for two hours. The absorbance was then read at 490 nm.
Tissue factor activity assay
TF activity was analyzed in HAEC by a colorimetric
assay (Actichrome TF, American Diagnostica) according
to the supplier’s recommendations. Briefly, HAEC were
grown, serum-starved, and stimulated in 6-well plates
according to the treatment protocols. After stimulation,
cells were washed twice with phosphate-buffered saline
(PBS) and lysed by sonications in a buffer of 50 mM
Tris-HCl, 100 mM NaCl, 0.1% Triton X-100, pH 7.4 for
30 minutes at 37 °C. The extracted tissue factors were
placed in a 96-well microplate and incubated with human Factor VIIa, and human Factor X at 37 °C for 15
minutes. The formed FVIIa/FX complex could cleave a
chromogenic substrate Spectrozyme FXa, which was
added to each well and incubated at 37 °C for 20
minutes. This chromogenic reaction was stopped after
Real-time polymerase chain reaction analysis
Endothelial cells were harvested by trypsinization
and pelleted. Total RNA was extracted from the pellet
using Micro-to-Midi total RNA purification system
(Invitrogen). cDNA was synthesized from total RNA using M-MLV reverse transcriptase (Superscript II, Invitrogen), and PCR was performed using cDNA as the
Acta Cardiol Sin 2008;24:151-6
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Thrombin Induces Tissue Factor Expression
20 minutes by adding glacial acetic acid. The data were
read by the absorbance of the reaction solutions at a 405
nm wavelength. A standard curve with different concentrations of lipidated human TF was constructed for interpolating the TF concentrations of the test samples.
Western blotting for tissue factor
Protein was quantified with BCAÔ protein assay kit
(PIERCE), and 35 mg protein were then loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. Protein was transferred onto PVDF
membrane by semi-dry transfer at 5 volts for 100 minutes. Antibodies against tissue factor (American Diagnostica), GAPDH (Santa Cruz Biotechnology) were used at
1:1500 and 1:3000, respectively. Results were quantified
by densitometry analysis of the bands and then normalization to GAPDH.
Statistical analysis
All data were presented as mean ± SEM. The significance was determined by unpaired t-test. A P < 0.05
was considered statistically significant.
RESULTS
Lack of effects of thrombin stimulation on the
morphology and cell viability of HAEC
HAEC were cultivated in endotheial cell growth medium until the experiment was done. The HAEC were
serum-starved in M-199 supplement with 1% fetal bovine serum for 24 hours and stimulated with human
thrombin (1 unit/ml) for 5 hours. The morphology was
not changed upon thrombin treatment (Figures 1A, 1B).
Cell viability was also not changed by thrombin, as measured using MTS assay (P=NS). This result revealed that
thrombin did not cause damage to HAEC during the experiments (Figure 1C).
Figure 1. Lack of short-term toxicity of thrombin. The morphology of
HAEC after 5 hours of thrombin stimulation was similar without (1A) or
with (1B) thrombin. 1C. MTS cell viability assay revealed no cytotoxic
effect of thrombin on HAEC at 1 unit/ml concentration used (P = NS).
The data is shown as mean ± SEM from three independent experiments
with triplication in each experiment.
Thrombin enhanced TF mRNA expression in
HAEC
To determine whether human thrombin could affect TF mRNA expression, we treated the HAEC with
thrombin (1 unit/ml) for 3 hours. We determined the TF/
GAPDH mRNA relative quantity compared to control.
The thrombin stimulation induced the relative mRNA
quantity to increase 390 ± 114% (n = 5, P = 0.034) (Figure 2).
Thrombin enhanced TF protein production in
HAEC
As expected from the TF mRNA up-regulation by
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Acta Cardiol Sin 2008;24:151-6
Huang-Joe Wang et al.
enhancement of TF activity by 547 ± 54% (n = 5, P <
0.001) (Figure 4).
DISCUSSION
The central player of thrombotic complications in
cardiovascular disease is the generation of thrombin
through the tissue factor pathway.8 Ruptured plaque with
exposure of underlining tissue factor is responsible for
many atherothrombosis events, culminating in ischemic
stroke, myocardial infarction, peripheral arterial occlusive diseases, and death.9 Thrombin, on the other hand,
can exert a positive feedback to augment the tissue factor production. This vicious cycle was shown by our data,
with thrombin proved to be a stress sufficient to trigger
TF activity in endothelial cell origin from arterial side.
Previous study utilizing human saphenous vein endothelial cells (HSVEC) showed that thrombin-induced
TF mRNA expression was time-dependent, with significant mRNA expression from 2 to 4 hours after thrombin
stimulation and rapid decrease to baseline levels after 6
hours.5 The optimal tissue factor activity and protein expression in human umbilical vein cells (HUVEC), however, was 4-6 hours after thrombin stimulation. 4,6,10
Based on the above time-course studies, we selected the
3-hour time point for TF mRNA study and the 5-hour
time point for TF activity and protein expression study.
A previous HSVEC study showed that thrombin treatment for 4 hours could increased TF mRNA to 8 times
Figure 2. TF/GAPDH mRNA relative expression level upon thrombin
stimulation. Real-time PCR demonstrated that relative quantities of TF
mRNA level was enhanced 390 ± 114% (*P = 0.034 vs control). Data
are shown as mean ± SEM from five independent experiments.
the thrombin, TF protein expression could be induced
under thrombin treatment for 5 hours. The relative TF
expression normalized to GAPDH increased 332 ± 39%
(n = 4, P = 0.01) (Figure 3)
Thrombin enhanced functional TF activity
To determine whether thrombin could affect TF activity, we treated HAEC with thrombin for 5 hours to determine if their extracted proteins could trigger TF activity. As anticipated, the TF chromogenic assay showed
Figure 3. Western blot of TF protein expression upon thrombin
stimulation. The results were quantified with densitometry analysis of
the bands and then normalization to GAPDH. The TF protein was
enhanced 332 ± 39% in TF protein expression (*P = 0.01 vs control).
Data are shown as mean ± SEM from four independent experiments.
Bolt was representative of four different experiments.
Acta Cardiol Sin 2008;24:151-6
Figure 4. Functional TF activity upon thrombin stimulation. Human
thrombin significantly increased the relative TF activity to 547 ± 54%
(*P < 0.001 vs control). Data are shown as mean ± SEM from five
independent experiments.
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Thrombin Induces Tissue Factor Expression
the control level, assessing by RT-PCR.5 Our HAEC data
also showed a 3.9-fold significant increase in mRNA
level upon thrombin stimulation. This difference in the
fold changes was likely due to different cell sources, detection methods, and time course. Furthermore, cultured
HUVEC could respond to different thrombin concentrations (10-2 - 10 NIH u/ml) with a 2- to 5-fold increase in
TF activity.4 This result was consistent with our HAEC
result of 5.47-fold increase in TF activity upon 1 unit/ml
thrombin stimulation.
Expression of tissue factor in the endothelium is
very rare in vivo, found, for example, in the splenic
microvasculature in a baboon septic shock model or vascular endothelial cells in patients with invasive breast
cancer.11,12 In-vitro studies, however, occasionally noted
variable TF mRNA and activity expressions, even in
non-perturbed conditions. 10,13 Those stresses incurred
during cell cultures were rendered minimally by 1% fetal
bovine serum starvation to decrease the interference of
endothelial cell culture medium and preserve the viability of endothelial cells in our study design. Therefore,
the tissue factor activity was not totally absent in our
control group, even after 24 hours’ starvation. However,
thrombin stimulation could significant up-regulate the
TF induction in HAEC. This increase in TF mRNA, protein, and functional activity supported that thrombin can
up-regulate the TF gene expression via a transcriptional
control.
Cultured endothelial cells can be induced to express
tissue factor by a variety of agents, including tumor necrosis factor-a, interleukin-1b, CD40 ligand, serotonin,
histamine, oxidized LDL, vascular endothelial growth
factor, and thrombin.1 Thrombin can induce activation of
c-Jun-N-terminal kinase and NF-kB activation in endothelial cells.14,15 Both JNK and NF-kB pathways are important transcription pathways for tissue factor expression in endothelial cells.16 The reported transcription factors involved in the tissue factor expression of HUVEC
are c-Fos/c-Jun and c-Rel/P65.17 Since thrombin also induced TF mRNA expression in thrombin-stimulated
HAEC, both c-Fos/c-Jun and c-Rel/P65 NF-kB transcription factors were considered to be involved.
We conclude that thrombin is a sufficient stress to
induce TF expression in HAEC. Transcriptional control
by thrombin causes an increase in TF mRNA. This increase in mRNA is modestly paralleled by an increase in
protein level and functional activity.
ACKNOWLEDGEMENTS
This study was supported in part by grants from the
National Science Council (NSC 96-2314-B-039-024);
Department of Medical Research, China Medical University Hospital (DMR-96-012, DMR-97-011); and
China Medical University (CMU-96-184).
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