A Novel Class of Antioxidants Inhibit LPS Induction of
Tissue Factor by Selective Inhibition of the Activation of
ASK1 and MAP Kinases
James P. Luyendyk, J. Daniel Piper, Michael Tencati, K. Veera Reddy, Todd Holscher, Rong Zhang,
Jayraz Luchoomun, Xilin Chen, Wang Min, Charles Kunsch, Nigel Mackman
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Objective—Oxidative stress contributes to the pathogenesis of many diseases, including atherosclerosis and sepsis. We
have previously described a novel class of therapeutic compounds with antioxidant and antiinflammatory properties.
However, at present, the intracellular targets of these compounds have not been identified. The purpose of this study was
to elucidate the mechanism by which 2 structurally-related antioxidants (AGI-1067 and AGI-1095) inhibit LPS
induction of tissue factor (TF) expression in human monocytic cells and endothelial cells.
Methods and Results—We found that succinobucol (AGI-1067) and AGI-1095 inhibited LPS induction of TF expression
in both monocytic cells and endothelial cells. These compounds also reduced LPS induction of nuclear AP-1 and
expression of Egr-1 without affecting nuclear translocation of NF-␬B. Importantly, these antioxidants inhibited LPS
activation of the redox-sensitive kinase, apoptosis signal-regulating kinase-1 (ASK1) and the mitogen-activated protein
kinases (MAPKs) p38, ERK1/2, and JNK1/2.
Conclusions—AGI-1067 and AGI-1095 inhibit TF gene expression in both monocytic cells and endothelial cells through
a mechanism that involves the inhibition of the redox-sensitive MAP3K, ASK1. These compounds selectively reduce
the activation/induction of MAPK, AP-1, and Egr-1 without affecting NF-␬B nuclear translocation. (Arterioscler
Thromb Vasc Biol. 2007;27:1857-1863.)
Key Words: tissue factor 䡲 LPS 䡲 oxidative stress 䡲 ASK1 䡲 AGI-1067
O
xidative stress contributes to the pathogenesis of many
inflammatory diseases, including atherosclerosis and
sepsis.1,2 Reactive oxygen species (ROS) are critical regulators of cellular homeostasis. However, disruption of the
intracellular prooxidant/antioxidant balance causes excessive
ROS generation, modulation of intracellular signaling, and
oxidative stress.3 The potential benefit of antioxidant vitamins such as vitamins C and E as therapeutic agents for
cardiovascular disease has been aggressively debated. Although several studies have indicated benefits of antioxidant
therapy in the treatment of various cardiovascular diseases, it
has not been effective in all cases.4
Probucol is an antioxidant drug that exhibits antiatherosclerotic and antirestenotic activity in patients with cardiovascular disease.5 However, treatment with probucol was
associated with several side effects, including prolongation of
the cardiac QT interval and decreased circulating HDL
levels.5 Recently, a new class of chemically and metabolically-stable probucol derivatives has been synthesized that
retain antioxidant activity and display enhanced cellular
antioxidant and antiinflammatory activity compared with
probucol, which may be attributable to enhanced cellular
uptake.6,7 One of these compounds, succinobucol (AGI1067), was found to reduce atherosclerosis in several animal
models including hypercholesterolemic primates.8 In addition, clinical studies with AGI-1067 have demonstrated its
ability to decrease the rate of restenosis and regress coronary
atherosclerosis.9 –11 AGI-1067 is currently being evaluated in
a large phase III clinical trial called The Aggressive Reduction of Inflammation Stops Events (ARISE) trial that will
determine the effect of AGI-1067 on major cardiovascular
events, including stroke, myocardial infarction, and death.
AGI-1067 and related compounds also possess antiinflammatory activity.6,7,12 We have shown that both AGI-1067 and
a related compound, AGIX-4207, inhibited tumor necrosis
factor (TNF)-␣ induction of monocyte chemoattractant
protein-1 (MCP-1) and vascular cell adhesion molecule
(VCAM)-1 expression in endothelial cells and LPS induction
of TNF-␣ and interleukin (IL)-6 expression in peripheral
blood mononuclear cells (PBMCs).6,7 Importantly, nuclear
Original received September 27, 2006; final version accepted May 21, 2007.
From the Department of Immunology (J.P.L., M.T., K.V.R., T.H., N.M.), The Scripps Research Institute, La Jolla, Calif; AtheroGenics Inc (J.D.P., J.L.,
X.C., C.K.), Atlanta, Ga; and the Interdepartmental Program in Vascular Biology and Transplantation, Department of Pathology (R.Z., W.M.), Yale
University School of Medicine, New Haven, Conn.
J.P.L., J.D.P., and M.T. contributed equally to this study.
Correspondence to Nigel Mackman, PhD, The Scripps Research Institute, 10550 North Torrey Pines Road, SP30-3040, La Jolla, CA 92037. E-mail
nmackman@scripps.edu
© 2007 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
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DOI: 10.1161/ATVBAHA.107.143552
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translocation of NF-␬B was not affected by these compounds,
suggesting that the antiinflammatory effects of these compounds are mediated by inhibition of intracellular pathways
other than the NF-␬B pathway.6,7 At present, the precise
mechanism by which these compounds reduce inflammation
has not been elucidated.
Bacterial LPS, inflammatory cytokines, and oxidized lipids
all induce oxidative stress in cells and increase ROS.3 This
leads to the activation of many intracellular signaling pathways, such as the mitogen activated protein kinase (MAPK)
and I␬B kinase pathways, and ultimately activation of transcription factors such as AP-1, Egr-1, and NF-␬B.3 The
MAPK pathways include ERK1/2, p38, and JNK1/2. A key
redox-regulated kinase that controls the activation of MAPK
pathways is ASK1.13 The inactive form of ASK1 is bound to
the reduced form of thioredoxin and to 14-3-3 proteins.
Oxidation of thioredoxin and the release of 14-3-3 results in
the activation of ASK1 and the subsequent activation of
p38.14 Recently, it was shown that LPS-mediated ROS
production leads to the activation of ASK1.15 Moreover, the
ROS-dependent TRAF6-ASK1-p38 axis plays a crucial role
in TLR4-mediated mammalian innate immunity.16
LPS stimulation of monocytes and endothelial cells induces the expression of the procoagulant protein tissue factor
(TF).17 Oxidized low-density lipoproteins (ox-LDL) also
induce TF expression in endothelial cells and pathologic
expression of TF within the vasculature leads to disseminated
intravascular coagulation.18,19 In addition, high levels of TF
are present in atherosclerotic plaques and likely contribute to
thrombosis after plaque rupture.20 Importantly, several studies have shown that antioxidants inhibit LPS induction of TF
expression in monocytes, macrophages, and endothelial
cells.21,22 These results suggest that inhibition of ROSdependent intracellular signaling may be an effective strategy
for reducing TF expression and thrombotic complications
associated with inflammatory diseases, such as atherosclerosis and sepsis.
We and others have characterized the intracellular signaling pathways and transcription factors that mediate LPS
induction of TF gene expression in monocytic cells and
endothelial cells. Activation/induction of the transcription
factors AP-1, NF-␬B, and Egr-1 was required for maximal
induction of TF expression.23–26 In addition, inhibition of the
MAPK pathways, ERK1/2 and p38, reduced LPS-induced TF
expression in monocytic cells and endothelial cells.23,27
In this study, we demonstrate that 2 novel antioxidant
compounds, AGI-1067 and AGI-1095, inhibit LPS induction
of TF expression in human monocytic and endothelial cells.
Importantly, these compounds inhibited LPS activation of the
redox-sensitive kinase ASK1, as well as the downstream
MAPK pathways ERK1/2, JNK1/2, and p38, and the transcription factors AP-1 and Egr-1 without affecting the nuclear
translocation of NF-␬B.
Methods
Materials
LPS (E coli serotype 0111:B4 or 026:B6), dimethylsulfoxide
(DMSO), and pyrrolidine dithiocarbamate (PDTC) were purchased
from Sigma-Aldrich. SP600125 were purchased from EMD Bio-
sciences Inc. The antioxidant compounds AGI-1067 and AGI-1095
were synthesized by AtheroGenics Inc, and their chemical structures
have been described previously.12,28 Compounds were dissolved
in DMSO.
Cell Culture
The human monocytic THP-1 cells were obtained from American
Type Culture Collection (Manassas, Va), and human aortic endothelial cells (HAECs) were obtained from Cambrex (Walkersville, Md).
PBMCs were isolated from citrated blood from healthy volunteers by
buoyant density gradient centrifugation on low endotoxin FicollPaque Plus (GE Healthcare). Cells were pretreated with compounds
for either 30 or 60 minutes before the addition of LPS.
Tissue Factor Activity
TF activity in cell lysates was measured using a 1-stage clotting
assay.29
Western Blotting
Levels of I␬B␣ and Egr-1 were determined using antibodies from
Santa Cruz Biotechnology. Activation of ERK1/2, p38, and JNK1/2
in THP-1 cells was assessed using antiphosphospecific antibodies
(New England Biolabs). Activation of p38 and JNK1/2 in HAECs
was evaluated with an antiphosphospecific p38 antibody (Cell
Signaling Technology) and an antiphosphospecific JNK1/2 antibody
(Biosource International), respectively. Activation of ASK1 was
assessed by measuring phosphorylation of Thr845 using a rabbit
polyclonal anti-ASK1 Thr845 antibody (Cell Signaling Technology).
Nonphosphospecific forms of each protein were used to monitor
loading.
Northern Blotting
The level of TF mRNA was determined by Northern blotting. Blots
were rehybridized with the housekeeping gene glyceraldehyde
3-phosphate dehydrogenase (GAPDH) to monitor loading. Bands
were visualized by autoradiography.
Electrophoretic Mobility Shift Assay
Nuclear extracts were incubated with a radiolabeled double-stranded
oligonucleotide probe (Operon Technologies) containing a prototypic AP-1 site, a ␬B site from the murine Igê gene, or the human
TF ␬B site.24 Protein-DNA complexes were separated from free
probe by electrophoresis through 6% nondenaturing acrylamide
gels (Invitrogen) using 0.5X Tris borate EDTA (TBE) buffer and
visualized by autoradiography.
Evaluation of Cytotoxicity
Cell viability was evaluated by Hoescht staining and by trypan blue
exclusion.
Statistics
All experiments were performed at least 3 independent times.
Statistical analyses were performed using SigmaStat version 3.1
(SPSS Inc). Student t test was used when only 2 groups were
compared. For comparisons of more than 2 groups, data were
analyzed by ANOVA with Tukey post-hoc test. The criterion for
significance for all studies was P⬍0.05.
Results
AGI-1067 and AGI-1095 Inhibit LPS-Induced TF
Activity in Monocytic Cells and Endothelial Cells
In this study, we investigated the effect of AGI-1067 and
AGI-1095 on LPS induction of TF expression in human
monocytic cells and endothelial cells. Both AGI-1067 and
AGI-1095 reduced LPS induction of TF activity in THP-1
monocytic cells and PBMCs in a concentration-dependent
manner (Figure 1). In addition, both compounds inhibited
Luyendyk et al
Antioxidant Inhibition of TF Expression
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Figure 1. AGI-1067 and AGI-1095 inhibit LPS induction of TF activity in THP-1 cells PBMCs and HAECs. TF activity was measured 5
hours after LPS stimulation of THP-1 cells, HAECs, or PBMCs. Cells were pretreated with various concentrations of AGI-1067 or AGI1095 (0 to 10 ␮mol/L) before LPS stimulation. THP-1 cells were treated with 10 ␮g/mL of LPS whereas PBMCs and HAECs were
treated with 1 and 2 ␮g/mL of LPS, respectively. n⫽3 to 5 independent experiments. *P⬍0.05. Data are shown as mean⫾SEM.
LPS-induced TF activity in HAECs (Figure 1). These compounds did not cause cytotoxicity (data not shown). These
results indicate that AGI-1067 and AGI-1095 inhibit LPS
induction of TF activity in monocytic cells and endothelial
cells.
1095 in monocytic cells reduced LPS-induced TF mRNA
expression by 59⫾7% and 68⫾18% (mean⫾SD, n⫽3),
respectively. These results suggest that these antioxidant
compounds inhibit TF expression at the level of gene transcription in monocytic cells.
Inhibition of LPS-Induced TF mRNA Expression
in THP-1 Monocytic Cells by AGI-1067
and AGI-1095
Effect of AGI-1067 and AGI-1095 on the LPSInduced Increase in Nuclear AP-1, Induction of
Egr-1 Expression, and Nuclear Translocation
of NF-␬B
To determine whether AGI-1067 and AGI-1095 inhibited
LPS induction of TF mRNA expression in THP-1 monocytic
cells, we measured levels of TF mRNA. LPS induced TF
mRNA expression in THP-1 cells (Figure 2A). The larger
band is an alternatively spliced transcript that contains the
majority of intron 1.30 We found that both compounds
inhibited the increase in TF mRNA expression in LPSstimulated THP-1 cells (Figure 2A). AGI-1067 and AGI-
The transcription factors AP-1, NF-␬B, and Egr-1 are required for the induction of TF gene expression in monocytic
cells.6,23,26 Therefore, we analyzed the effect of AGI-1067
and AGI-1095 on LPS induction of Egr-1 expression, nuclear
AP-1, and nuclear translocation of NF-␬B. LPS stimulation
induced a time-dependent increase in Egr-1 protein expression, which was inhibited by pretreatment of the cells with
Figure 2. AGI-1067 and AGI-1095 inhibit
LPS induction of TF mRNA expression
and Egr-1 protein expression in THP-1
cells. THP-1 cells were pretreated with
AGI-1067 (5 ␮mol/L), AGI-1095 (5 ␮mol/L),
or vehicle for 30 minutes before stimulation with LPS (10 ␮g/mL). A, TF mRNA
expression was measured by Northern blotting. GAPDH was used as a loading control.
Results from a representative experiment of
3 independent experiments are shown. Normalized levels of TF mRNA are shown
below the blot. B, Egr-1 expression was
measured by Western blotting. Results from
a representative experiment of 3 independent experiments are shown. Actin was
used as a loading control. Levels of Egr-1
are shown below the blot.
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Figure 3. Effect of the antioxidant compounds on LPS induction of I␬B␣ degradation and nuclear levels of AP-1 and NF-␬B
in THP-1 cells. THP-1 cells were pretreated
with AGI-1067 (5 ␮mol/L), AGI-1095
(5 ␮mol/L), or vehicle for 30 minutes before
stimulation with LPS (10 ␮g/mL). A, Nuclear
AP-1 levels were determined by EMSA. B,
I␬B␣ levels were determined by Western
blotting. C, Nuclear NF-␬B levels were
determined by EMSA 1 hour after LPS
stimulation of THP-1 cells. Results from a
representative experiment of 3 independent
experiments are shown.
either of the antioxidant compounds (Figure 2B). AGI-1067
and AGI-1095 reduced LPS-induced Egr-1 expression by
83⫾8% and 74⫾9% (mean⫾SD, n⫽3), respectively. AGI1067 and AGI-1095 also significantly reduced LPS induction
of nuclear AP-1 (Figure 3A). LPS-induced nuclear translocation of NF-␬B requires degradation of the cytoplasmic
inhibitor I␬B␣. Therefore, we first analyzed the effect of
AGI-1067 and AGI-1095 on LPS-induced degradation of
I␬B␣. The antioxidants did not affect degradation of I␬B␣ in
THP-1 cells (Figure 3B). Similar results were observed using
HAECs (data not shown). Next, we analyzed nuclear translocation of NF-␬B by EMSA. AGI-1067 and AGI-1095 did
not affect LPS-induced nuclear translocation of NF-␬B(p50/
p65) or c-Rel/p65 in THP-1 cells (Figure 3C). The c-Rel/p65
heterodimer binds to the TF ␬B site.24 In contrast, and
consistent with a previous study,31 the antioxidant PDTC
(100 ␮mol/L) significantly reduced LPS-induced nuclear
translocation of NF-␬B (data not shown). Taken together,
these data indicate that AGI-1067 and AGI-1095 inhibit LPS
induction of Egr-1 expression and nuclear levels of AP-1
without affecting the nuclear translocation of NF-␬B.
Inhibition of LPS Activation of MAPKs in
Monocytic Cells and Endothelial Cells by
AGI-1067 and AGI-1095
The MAPKs ERK1/2 and p38 regulate LPS induction of TF
gene expression in monocytic and endothelial cells by activating various transcription factors. We found that inhibition
of JNK1/2 with the inhibitor SP600125 (45 ␮mol/L) significantly reduced LPS-induced TF expression in THP-1 cells
(data not shown), indicating that activation of JNK1/2 is
required for LPS induction of TF expression. JNK1/2 and p38
regulate the activation and expression of AP-1, whereas
ERK1/2 regulates Egr-1 expression.32 Therefore, we determined the effect of AGI-1067 and AGI-1095 on LPS activation of various MAPK pathways in monocytic cells and
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Figure 4. AGI-1067 and AGI-1095 inhibit activation of MAPKs in LPS-stimulated THP-1 cells. THP-1 cells were pretreated with AGI1067 (5 ␮mol/L), AGI-1095 (5 ␮mol/L), or vehicle for 30 minutes before stimulation with LPS (10 ␮g/mL). At various times after LPS
treatment (0 to 60 minutes), whole cell lysates were prepared, and phospho-ERK1/2, phospho-p38, and phospho-JNK1/2 levels were
analyzed by Western blotting. Results from a representative experiment of 3 independent experiments are shown. Normalized levels of
the different phosphorylated proteins are shown below the blots.
endothelial cells. The activation of all 3 MAPK pathways was
strongly inhibited by AGI-1067 and AGI-1095 (Figure 4).
Similarly, AGI-1067 and AGI-1095 reduced LPS activation
of both p38 and JNK1/2 in HAECs (supplemental Figure I,
available online at http://atvb.ahajournals.org). LPS activation of ERK1/2 could not be evaluated in LPS-treated HAECs
because of a high basal ERK1/2 phosphorylation in these
cells (data not shown). The levels of inhibition of the different
MAPK pathways by AGI-1067 and AGI-1095 are shown in
supplemental Table I. AGI-1067 (5 ␮mol/L) and AGI-1095
(5 ␮mol/L) also inhibited LPS activation of p38 in PBMCs
(data not shown). These results demonstrate that these antioxidants inhibit LPS activation of MAPK pathways in both
monocytic cells and endothelial cells.
Discussion
In this study, we determined a mechanism by which a new
class of antioxidant compounds derived from probucol inhibits LPS induction of TF expression in human monocytic cells
and endothelial cells. LPS stimulation leads to the activation
LPS Activation of ASK1 Is Inhibited by AGI-1067
and AGI-1095 in Monocytic Cells and
Endothelial Cells
Because ASK1 is a redox-regulated MAP3K that is activated
in cells exposed to LPS and regulates various MAPK pathways, we determined whether LPS activation of ASK1 was
inhibited by AGI-1067 and AGI-1095. LPS treatment increased the phosphorylation of ASK1 in THP-1 cells within
15 minutes, and this activation was inhibited by pretreatment
with either AGI-1067 or AGI-1095 (Figure 5A). Similarly,
LPS activated ASK1 in HAECs, and this activation was
inhibited by treatment with AGI-1067 (Figure 5B). These
data indicate that these antioxidant compounds inhibit LPS
activation of ASK1 in monocytic cells and endothelial cells.
Figure 5. AGI-1067 and AGI-1095 inhibit LPS activation of
ASK1 in monocytic and endothelial cells. A, THP-1 cells were
pretreated with AGI-1067 (5 ␮mol/L), AGI-1095 (5 ␮mol/L), or
vehicle for 30 minutes before stimulation with LPS (10 ␮g/mL).
B, HAECs were pretreated with AGI-1067 (10 ␮mol/L), or vehicle
for 60 minutes before stimulation with LPS (2 ␮g/mL). At various
times after LPS treatment (0 to 60 minutes), whole cell lysates
were prepared and phospho-ASK1 (T845) levels were analyzed
by Western blotting. Each blot was stripped and reprobed for
the nonphosphorylated form of ASK1. Results from a representative experiment of 3 independent experiments are shown.
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Figure 6. Schematic representation of selective inhibition of LPS
activation of the MAPK pathway by AGI-1067 and AGI-1095. The
antioxidants AGI-1067 and AGI-1095 inhibit LPS activation of the
MAPK pathways without affecting IêB␣ degradation or nuclear
translocation of NF-êB. In contrast, antioxidants such as PDTC
inhibit NF-êB. Both types of antioxidants inhibit LPS induction of
TF and cytokine expression in monocytic and endothelial cells.
of various ROS-sensitive intracellular signaling pathways
(eg, MAPKs) and transcription factors (NF-␬B, AP-1, and
Egr-1) that mediate the induction of TF expression.3,23,26,32–34
We found that the antioxidants inhibited LPS induction of
Egr-1, AP-1, and the activation of MAPK pathways. However, these compounds did not reduce the nuclear translocation of NF-␬B. In contrast, the antioxidants PDTC and
N-acetyl cysteine (NAC) inhibit LPS induction of inflammatory genes and TF in monocytes, macrophages, and endothelial cells by reducing NF-␬B activation.6,21,22,31,35,36 These
results indicate that AGI-1067 and AGI-1095 inhibit LPS
induction of gene expression via a mechanism that is distinct
from other antioxidant compounds, such as PDTC and NAC
(Figure 6). Other antioxidant compounds (ie, flavanoids) also
do not inhibit inducible NF-␬B activation37,38 despite their
ability to inhibit inflammatory gene expression in endothelial
cells. Currently, it is not clear what governs the ability of
some antioxidants, but not others, to inhibit inducible NF-␬B
activation.
How do AGI-1067 and AGI-1095 inhibit LPS activation of
the MAPK signaling pathways? Interestingly, a recent article
showed that activation of the redox-sensitive MAP3K ASK1
is critical for LPS induction of inflammatory cytokines, but
not for the activation of the NF-␬B pathway.16 We found that
LPS activated ASK1 in monocytic and endothelial cells, and
that ASK1 activation was inhibited by both AGI-1067 and
AGI-1095. Thus, the inhibition of ASK1 by these compounds
may account for the selective inhibition of the MAPK
pathways without affecting the NF-␬B signaling pathway
(Figure 6). Importantly, we showed that these antioxidants
inhibited LPS activation of the 3 major MAPK pathways,
ERK1/2, JNK1/2, and p38, which control the activation of
AP-1 and Egr-1. A recent study found that LPS activation of
ASK1 was required for p38, but not JNK1/2 signaling.16
However, other studies have found that ASK1 was required
for the activation of JNK1/2 signaling in cells stimulated by
TNF␣.39 Thus, consistent with the inhibition of ASK1 by
AGI-1067 and AGI-1095, both compounds inhibited activa-
tion of JNK1/2 and p38. Although a role for ASK1 in the LPS
activation of ERK1/2 has not yet been established, our data
suggest that activation of ASK1 may be required for the
activation of ERK1/2. Alternatively, these compounds may
inhibit ERK1/2 by affecting another upstream activator of
this pathway. Taken together, our results suggest that unlike
other antioxidants, such as PDTC and NAC, AGI-1067 and
AGI-1095 selectively inhibit LPS activation of ASK1 and
MAPK signaling pathways without affecting the nuclear
translocation of NF-␬B (Figure 6).
Oxidative stress and inflammation contribute to the initiation and progression of atherosclerosis. One mechanism by
which the generation of ROS, such as superoxide, can
contribute to the development of atherosclerotic lesions is
through the formation of oxidized proteins and lipoproteins
such as LDL.40,41 In addition, intracellular ROS have been
shown to modulate intracellular signaling pathways and
inflammatory gene expression in the vasculature.42 We have
previously found that this class of antioxidants inhibited
expression of inflammatory cytokines and adhesion molecules, such as VCAM-1 and MCP-1, in monocytes and
endothelial cells both in vitro and in vivo.6,8 Inhibition of the
expression of these inflammatory mediators may reduce the
accumulation of inflammatory cells, such as macrophages,
into the atherosclerotic lesion. Indeed, AGI-1067 reduced the
size of atherosclerotic lesions in hypercholesterolemic rabbits, LDLR⫺/⫺ mice, ApoE⫺/⫺ mice, hypercholesterolemic
primates, and coronary atherosclerosis in humans.6,8,9 Here,
we show that both AGI-1067 and AGI-1095 reduced MAPK
activity and TF expression in monocytic cells and endothelial
cells. Importantly, the thrombogenicity of atherosclerotic
plaques is associated with an increase in TF expression.20
Plaque rupture results in exposure of TF to circulating
coagulation factors, which can lead to myocardial infarction
and stroke.20 Thus, a reduction in TF expression is one
potential mechanism by which these antioxidants may reduce
the risk of myocardial infarction.
Taken together, these findings indicate that this novel class
of antioxidants may inhibit the development of atherosclerotic lesions, in part, by reducing ROS and inhibiting the
activity of the redox-sensitive kinase ASK1 in both monocytes and endothelial cells, resulting in the inhibition of
inflammatory mediators and TF expression. This study therefore provides a molecular mechanism for how this class of
antioxidants may target redox-sensitive signaling pathways
that modulate inflammatory and prothrombotic processes.
Sources of Funding
These studies were funded, in part, by National Institutes of Health
grant HL048872 (to N.M.), National Institutes of Health NRSA
Fellowship HL085983 (to J.P.L.), and by a Specific Funding
Proposal from AtheroGenics Inc.
Disclosures
None.
References
1. Victor VM, Rocha M, De la FM. Immune cells: free radicals and antioxidants in sepsis. Int Immunopharmacol. 2004;4:327–347.
Luyendyk et al
Downloaded from http://atvb.ahajournals.org/ by guest on October 2, 2016
2. Madamanchi NR, Hakim ZS, Runge MS. Oxidative stress in atherogenesis and arterial thrombosis: the disconnect between cellular studies
and clinical outcomes. J Thromb Haemost. 2005;3:254 –267.
3. Griendling KK, Sorescu D, Lassegue B, Ushio-Fukai M. Modulation of
protein kinase activity and gene expression by reactive oxygen species
and their role in vascular physiology and pathophysiology. Arterioscler
Thromb Vasc Biol. 2000;20:2175–2183.
4. Kris-Etherton PM, Lichtenstein AH, Howard BV, Steinberg D, Witztum JL.
Antioxidant vitamin supplements and cardiovascular disease. Circulation.
2004;110:637–641.
5. Pfuetze KD, Dujovne CA. Probucol Curr Atheroscler Rep. 2000;2:
47–57.
6. Kunsch C, Luchoomun J, Grey JY, Olliff LK, Saint LB, Arrendale RF,
Wasserman MA, Saxena U, Medford RM. Selective inhibition of endothelial and monocyte redox-sensitive genes by AGI-1067: a novel antioxidant and anti-inflammatory agent. J Pharmacol Exp Ther. 2004;308:
820 – 829.
7. Kunsch C, Luchoomun J, Chen XL, Dodd GL, Karu KS, Meng CQ,
Marino EM, Olliff LK, Piper JD, Qiu FH, Sikorski JA, Somers PK, Suen
KL, Thomas S, Whalen AM, Wasserman MA, Sundell CL. AGIX-4207
[2-[4-[[1-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio]-1methylethyl]thio]-2,6-bis(1,1-dimethylethyl)phenoxy]acetic acid], a
novel antioxidant and anti-inflammatory compound: cellular and biochemical characterization of antioxidant activity and inhibition of redoxsensitive inflammatory gene expression. J Pharmacol Exp Ther.
2005;313:492–501.
8. Sundell CL, Somers PK, Meng CQ, Hoong LK, Suen KL, Hill RR,
Landers LK, Chapman A, Butteiger D, Jones M, Edwards D, Daugherty
A, Wasserman MA, Alexander RW, Medford RM, Saxena U. AGI-1067:
a multifunctional phenolic antioxidant, lipid modulator, antiinflammatory and antiatherosclerotic agent. J Pharmacol Exp Ther. 2003;
305:1116 –1123.
9. Tardif JC, Gregoire J, L’Allier PL, Ibrahim R, Anderson TJ, Reeves F,
Title LM, Schampaert E, Lemay M, Lesperance J, Scott R, Guertin MC,
Brennan ML, Hazen SL, Bertrand OF, for the CART-2 Investigators.
Effects of the antioxidant succinobucol (AGI-1067) on human atherosclerosis in a randomized clinical trial. Atherosclerosis. In press.
10. Tardif JC, Gregoire J, Schwartz L, Title L, Laramee L, Reeves F,
Lesperance J, Bourassa MG, L’Allier PL, Glass M, Lambert J, Guertin
MC. Effects of AGI-1067 and probucol after percutaneous coronary
interventions. Circulation. 2003;107:552–558.
11. Tardif JC, Gregoire J, Lavoie MA, L’Allier PL. Vascular protectants for
the treatment of atherosclerosis. Expert Rev Cardiovasc Ther. 2003;1:
385–392.
12. Meng CQ, Somers PK, Hoong LK, Zheng XS, Ye Z, Worsencroft KJ,
Simpson JE, Hotema MR, Weingarten MD, MacDOnald ML, Hill RR,
Marino EM, Suen KL, Luchoomun J, Kunsch C, Landers LK, Stefanopoulos D, Howard RB, Sundell CL, Saxena U, Wasserman MA, Sikorski
JA. Discovery of novel phenolic antioxidants as inhibitors of vascular cell
adhesion molecule-1 expression for use in chronic inflammatory diseases.
J Med Chem. 2004;47:6420 – 6432.
13. Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi T, Takagi
M, Matsumoto K, Miyazono K, Gotoh Y. Induction of apoptosis by
ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science. 1997;275:90 –94.
14. Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y,
Kawabata M, Miyazono K, Ichijo H. Mammalian thioredoxin is a direct
inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J. 1998;
17:2596 –2606.
15. Chiang E, Dang O, Anderson K, Matsuzawa A, Ichijo H, David M.
Cutting edge: apoptosis-regulating signal kinase 1 is required for reactive
oxygen species-mediated activation of IFN regulatory factor 3 by lipopolysaccharide. J Immunol. 2006;176:5720 –5724.
16. Matsuzawa A, Saegusa K, Noguchi T, Sadamitsu C, Nishitoh H, Nagai S,
Koyasu S, Matsumoto K, Takeda K, Ichijo H. ROS-dependent activation
of the TRAF6-ASK1–p38 pathway is selectively required for TLR4mediated innate immunity. Nat Immunol. 2005;6:587–592.
17. Mackman N. Regulation of the tissue factor gene. Thromb Haemost.
1997;78:747–754.
18. Drake TA, Hannani K, Fei H, Lavi S, Berliner JA. Minimally oxidized
low-density lipoprotein induces tissue factor expression in cultured
human endothelial cells. Am J Pathol. 1991;138:601– 607.
19. Drake TA, Cheng J, Chang A, Taylor FB Jr. Expression of tissue factor,
thrombomodulin, and E-selectin in baboons with lethal Escherichia coli
sepsis. Am J Pathol. 1993;142:1–13.
Antioxidant Inhibition of TF Expression
1863
20. Tremoli E, Camera M, Toschi V, Colli S. Tissue factor in atherosclerosis.
Atherosclerosis. 1999;144:273–283.
21. Brisseau GF, Dackiw APB, Cheung PYC, Christie CN, Rotstein OD.
Posttranscriptional regulation of macrophage tissue factor expression by
antioxidants. Blood. 1995;85:1025–1035.
22. Orthner CL, Rodgers GM, Fitzgerald LA. Pyrrolidine dithiocarbamate
abrogates tissue factor (TF) expression by endothelial cells: Evidence
implicating nuclear factor- kB in TF induction by diverse agonists. Blood.
1995;86:436 – 443.
23. Guha M, O’Connell MA, Pawlinski R, Yan SF, Stern D, Mackman N.
Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human
monocytic cells mediates tissue factor and tumor necrosis factor ␣
expression by inducing Elk-1 phosphorylation and Egr-1 expression.
Blood. 2001;98:1429 –1439.
24. Oeth PA, Parry GCN, Kunsch C, Nantermet P, Rosen CA, Mackman N.
Lipopolysaccharide induction of tissue factor gene expression in
monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a
␬B-like site. Mol Cell Biol. 1994;14:3772–3781.
25. Mackman N. Regulation of tissue factor gene expression in human
monocytic and endothelial cells. Haemostasis. 1996;26:17–20.
26. Mackman N, Brand K, Edgington TS. Lipopolysaccharide-mediated transcriptional activation of the human tissue factor gene in THP-1 monocytic
cells requires both activator protein 1 and nuclear factor ␬B binding sites.
J Exp Med. 1991;174:1517–1526.
27. Chu AJ, Wang ZG, Walton MA, Seto A. Involvement of MAPK activation in bacterial endotoxin-inducible tissue factor upregulation in
human monocytic THP-1 cells. J Surg Res. 2001;101:85–90.
28. Meng CQ, Somers PK, Rachita CL, Holt LA, Hoong LK, Zheng XS,
Simpson JE, Hill RR, Olliff LK, Kunsch C, Sundell CL, Parthasarathy S,
Saxena U, Sikorski JA, Wasserman MA. Novel phenolic antioxidants as
multifunctional inhibitors of inducible VCAM-1 expression for use in
atherosclerosis. Bioorg Med Chem Lett. 2002;12:2545–2548.
29. Morrissey JH, Fair DS, Edgington TS. Monoclonal antibody analysis of
purified and cell-associated tissue factor. Thromb Res. 1988;52:247–261.
30. van der Logt CPE, Reitsma PH, Bertina RM. Alternative splicing is
responsible for the presence of two tissue factor mRNA species in LPS
stimulated human monocytes. Thromb Haemost. 1992;67(2):272–276.
31. Ziegler-Heitbrock HWL, Sternsdorf T, Liese J, Belohradsky B, Weber C,
Wedel A, Schreck R, Bäuerle P, Ströbel M. Pyrrolidine-dithiocarbamate
inhibits NF-␬B mobilization and TNF production in human monocytes.
J Immunol. 1993;151:6986 – 6993.
32. Guha M, Mackman N. LPS induction of gene expression in human
monocytes. Cell Signal. 2001;13:85–94.
33. Oeth P, Parry GCN, Mackman N. Regulation of the tissue factor gene in
human monocytic cells. Role of AP-1, NF-␬B/Rel and Sp1 proteins in
uninduced and lipopolysaccharide-induced expression. Arterioscler
Thromb Vasc Biol. 1997;17:365–374.
34. Parry GC, Mackman N. Transcriptional regulation of tissue factor
expression in human endothelial cells. Arterioscler Thromb. 1995;15:
612– 621.
35. Schreck R, Meier B, Männel DN, Dröge W, Baeuerle PA. Dithiocarbamates as potent inhibitors of nuclear factor ␬B activation in intact cells.
J Exp Med. 1992;175:1181–1194.
36. Polack B, Pernod G, Barro C, Doussiere J. Role of oxygen radicals in
tissue factor induction by endotoxin in blood monocytes. Haemostasis.
1997;27:193–200.
37. Gerritsen ME, Carley WW, Ranges GE, Shen CP, Phan SA, Ligon GF,
Perry CA. Flavonoids inhibit cytokine-induced endothelial cell adhesion
protein gene expression. Am J Pathol. 1995;147:278 –292.
38. Wolle J, Hill RR, Ferguson E, Devall LJ, Trivedi BK, Newton RS,
Saxena U. Selective inhibition of tumor necrosis factor-induced vascular
cell adhesion molecule-1 gene expression by a novel flavonoid. Lack of
effect on transcription factor NF-kappa B. Arterioscler Thromb Vasc Biol.
1996;16:1501–1508.
39. Nishitoh H, Saitoh M, Mochida Y, Takeda K, Nakano H, Rothe M,
Miyazono K, Ichijo H. ASK1 is essential for JNK/SAPK activation by
TRAF2. Mol Cell. 1998;2:389 –395.
40. Cathcart MK. Regulation of superoxide anion production by NADPH
oxidase in monocytes/macrophages: contributions to atherosclerosis.
Arterioscler Thromb Vasc Biol. 2004;24:23–28.
41. Patel RP, Moellering D, Murphy-Ullrich J, Jo H, Beckman JS,
Darley-Usmar VM. Cell signaling by reactive nitrogen and oxygen
species in atherosclerosis. Free Radic Biol Med. 2000;28:1780 –1794.
42. Kunsch C, Medford RM. Oxidative stress as a regulator of gene
expression in the vasculature. Circ Res. 1999;85:753–766.
Downloaded from http://atvb.ahajournals.org/ by guest on October 2, 2016
A Novel Class of Antioxidants Inhibit LPS Induction of Tissue Factor by Selective
Inhibition of the Activation of ASK1 and MAP Kinases
James P. Luyendyk, J. Daniel Piper, Michael Tencati, K. Veera Reddy, Todd Holscher, Rong
Zhang, Jayraz Luchoomun, Xilin Chen, Wang Min, Charles Kunsch and Nigel Mackman
Arterioscler Thromb Vasc Biol. 2007;27:1857-1863; originally published online June 7, 2007;
doi: 10.1161/ATVBAHA.107.143552
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Figure I. AGI-1067 and AGI-1095 inhibit LPS induction of TF protein expression in
HAECs. HAECs were pretreated with AGI-1067 (10 µM), AGI-1095 (5 µM) or vehicle for 60
minutes prior to prior to stimulation with LPS (2 µg/ml). At various times after LPS treatment
(0-60 minutes), whole cell lysates were prepared and phospho-p38 and phospho-JNK1/2 levels
were analyzed by western blotting. Each blot was stripped and reprobed for the nonphosphorylated form of each MAPK. For B, western blots were developed using Licor Odyssey
technology.
Table I: Inhibition of LPS activation of MAPK pathways by AGI-1067 and AGI-1095
ERK1/2
1067
THP-1 HAEC
81 ± 3*
—
1095
THP-1
HAEC
39 ± 26*
—
p38
1067
THP-1 HAEC
47 ± 34 70 ± 10*
1095
THP-1 HAEC
45 ± 17 31 ± 10*
JNK1/2
1067
THP-1
HAEC
70 ± 12* 51 ± 11*
1095
THP-1 HAEC
49 ± 39 42 ± 20*
Numbers show percentage inhibition of LPS activation of MAPK pathways using either AGI1067 or AGI-1095 (mean ± SD) at 60 minutes for 3 independent experiments. For THP-1 cells
5µM of each compound was used, whereas for HAEC we used 10µM of AGI-1067 and 5µM of
AGI-1095. *p<0.05
Supplement