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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Vascular Biology and Thrombosis
Research, University of Vienna, Austria, and Department of Medical
Biochemistry and Medical Molecular Biology, University of Graz,
Austria.
Activation of endothelial cells by lipid oxidation products is a
key event in the initiation and progression of the atherosclerotic lesion. Minimally modified low-density lipoprotein (MM-LDL) induces the
expression of certain inflammatory molecules such as tissue factor (TF)
in endothelial cells. This study examined intracellular signaling
pathways leading to TF up-regulation by oxidized
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a biologically active component of MM-LDL. OxPAPC induced TF
activity and protein expression in human umbilical vein endothelial cells (HUVECs). However, OxPAPC neither induced phosphorylation or
degradation of I Tissue factor (TF) is a cell surface
receptor initiating blood coagulation,1 thereby promoting
thrombotic events in atherosclerosis, sepsis, and
cancer.2,3 Enhanced endothelial TF expression has been
demonstrated in atherosclerotic plaques,4,5 a process that
may account for thrombotic events associated with early and advanced
atherosclerosis. TF expression in endothelial cells (ECs) can be
induced by a variety of agonists, including inflammatory cytokines,
angiogenic growth factors, infectious agents, and minimally modified
low-density lipoprotein (MM-LDL).1,6 MM-LDL regulates TF
expression at the level of transcription5; however, the signaling pathways and transcription factors involved in this process
are not known. Some of the effects of MM-LDL can be mimicked by
oxidation of
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC).7 Three biologically active components of oxidized
PAPC (OxPAPC) have been structurally identified as
1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC),
1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC),8 and
1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC).9 Which of these components of MM-LDL is
responsible for induction of TF is not known.
In contrast to interleukin-1 (IL-1) or tumor necrosis factor Apart from NF- In the present study, we investigated signaling pathways and
transcription factors mediating induction of TF expression in human ECs
by biologically active oxidized phospholipids. We show that expression
of TF is elevated by OxPAPC, and that this induction was mainly
mediated by EGR-1- and NFAT-dependent transcription, but was
independent of NF- Materials
Cell culture
Lipid oxidation, preparation, and analysis of POVPC and PGPC PAPC was oxidized by exposure of dry lipid to air for 72 hours. The extent of oxidation was monitored by positive ion electrospray mass spectrometry (ESI-MS) as described previously.8 POVPC and PGPC were prepared by ozonolysis of PAPC essentially as described.12 Purification was performed on a normal phase column (Hypersil, 3 µM, 4 × 150 mm) with UV detection at 270 nm. Fractions were collected and analyzed by ESI-MS. Lipids were stored at 70°C in chloroform and used not longer than 1 week
after testing for purity. OxPAPC, POVPC, and PGPC preparations were
shown negative for endotoxin by the Limulus amebocyte assay
(Biowhittaker, Walkersville, MD).
Clotting assay Confluent HUVECs grown in 6-well plates were incubated for 4 hours in medium 199/1% SCS before the addition of agonists. After 6 hours of stimulation with OxPAPC, cells were washed and scraped into 1 mL clotting buffer (10 mM sodium acetate, 7 mM sodium barbiturate, 130 mM NaCl, pH 7.4). The cells were pelleted in a microcentrifuge and resuspended in 200 µL clotting buffer. Clotting time was measured at 37°C after mixing equal volumes of sample, pig plasma, and 20 mM CaCl2. TF activity equivalents were determined from a standard curve obtained using rabbit brain thromboplastin.Western blotting After stimulation, HUVECs were lysed in Laemmli buffer and proteins separated by electrophoresis on 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels. Proteins were blotted onto polyvinylidene difluoride (PVDF) membrane and, after blocking with 3% dry milk/0.1% Tween-20, incubated with primary antibodies in the same solution. Bound antibodies were detected by anti-IgG conjugated with peroxidase and subsequent chemiluminescent detection.Measurements of intracellular free Ca++ concentration Free intracellular Ca++ concentration was examined in HUVECs grown on coverslips as described previously.18 Single-cell measurements were performed using a deconvolution microscope as described.19 Due to the overall errors in calibration, intracellular Ca++ was expressed as F/F0.20Analysis of NF-  B p65 kit (Active Motif, Rixensart, Belgium)
according to the manufacturer's instructions. Specificity of p65
binding was confirmed by incubation of cell lysates with the
immobilized NF-B consensus probe in the presence of excess wild-type
or mutated oligonucleotide.
Quantitation of NF- B-Luc (Stratagene, Amsterdam, The Netherlands, 0.5 µg/well) and pCMV- (Clontech, Heidelberg, Germany, 0.3 µg/well) using Lipofectamine Plus reagent (Life Technologies,
Lofer, Australia) according to the manufacturer's
instructions. After 48 hours, the medium was changed to medium 199/10%
SCS and cells stimulated with agonists. After 6 hours of incubation,
the cells were washed with PBS, scraped into 0.1 M sodium phosphate, pH
7.8, and lysed by 3 freeze-thaw cycles. Luciferase activity was
determined using adenosine triphosphate (ATP) and
D-luciferin as substrates. Luminescence was
measured in a Wallac 1420 Multilabel Counter (Wallac, Turku, Finland). -Galactosidase activity was determined by
spectrophotometric assay using chlorophenol red
-D-galactopyranoside as substrate. Optical density was
read at 570 nm in a Wallac 1420 Multilabel Counter.
Transfections and fluorescence microscopy The HUVECs were transiently transfected with a green fluorescence protein (GFP)-NFAT construct21 (kindly provided by L. Gerace, The Scripps Research Institute), using the Lipofectamine reagent (Life Technologies) according to the protocol published previously.14 One day after transfection cells were analyzed by fluorescence microscopy on a Nikon Diaphot TMD microscope equipped with a cooled charge-coupled device camera (Kappa, Gleichen, Germany). To induce nuclear import of GFP-NFAT, Ca++ ionophore A23187 (4 µM) or OxPAPC (125 µg/mL) was added to cells at 37°C for 30 minutes.Nuclear extraction and electrophoretic mobility shift assays Preparation of nuclear extracts and electrophoretic mobility shift assays (EMSAs) were performed as described previously,15 except that spermine/spermidine and polyvinylethanol were omitted from the extraction and binding buffers, respectively. Synthetic double-stranded nucleotides were labeled by filling in the overhangs with Klenow enzyme in the presence of [ -32P]dATP. Labeled probes were purified by
polyacrylamide gel electrophoresis. The following oligonucleotides were
used: 5'-aattATAAAATTTTCCAATGTAAAC-3' (NFATp sequence 60 to 80 of
the murine IL-4 promoter), 5'-aattCCGGAGTTTCCTACC-3' (nucleotides 183
to 197 of the human TF promoter) and
5'-aattGGAGGCGGGGCAGGGGTGTGGAACTCG-3' (Sp1 site from the human TF
promoter). Each reaction contained 3 µg nuclear protein. To control
for specificity of binding, 50-fold excess of unlabeled specific or
unrelated (Sp1) oligonucleotide was added. For supershift, 4 µg
antibody was added 15 minutes before addition of labeled
oligonucleotide. Separation of DNA-protein complexes was performed in
5% nondenaturing polyacrylamide gels in 0.5 × Tris borate EDTA buffer (TBE).
Recombinant adenoviral constructs and infection The generation of recombinant adenoviruses expressing NAB2 will be described elsewhere (M.L. and D.M., manuscript submitted). The IB-encoding adenovirus22 was from Rainer de
Martin (Dept of Vascular Biology and Thrombosis Research, University of
Vienna). HUVECs grown in 6-well plates were washed twice with
phosphate-buffered saline (PBS) and incubated at a multiplicity of
infection of 100 with recombinant adenovirus or control adenovirus in
PBS. After 30 minutes, cells were washed with PBS and then cultured in
normal medium. Twenty-four hours after infection, cells were incubated in 1% SCS medium for 4 hours followed by stimulation with OxPAPC for
6 hours.
Phospholipase A2 treatment and thin-layer chromatography of OxPAPC OxPAPC has been treated with phospholipase A2 (PLA2) as described previously9 with minor modifications. Briefly, 5 mg OxPAPC was dried, resuspended by vortexing in 1 mL PBS containing 5 mM CaCl2 and split into 2 portions. Then, 0.1 mL PBS/Ca++ containing 300 U Naja mossambica PLA2 was added to one tube, and the same amount of PBS/Ca++ to the other tube (mock-treated OxPAPC). After incubation for 1 hour at 36°C with periodic vortexing, lipids were extracted with 7 volumes of CHCl3/methanol (2:1) containing 0.01% butylated hydroxytoluene. After vortexing and centrifugation, lower phases were collected and 5 volumes of CHCl3 were added to the residual aqueous phases. After the next cycle of vortexing and centrifugation, lower organic phases were pooled, dried, redissolved in 250 µL CHCl3 and stored at70°C until analysis of activity or separation by thin-layer
chromatography (TLC).
Lipids were separated by TLC into phospholipids, neutral lipids, and
fatty acids on Whatman 60K silica plates developed with hexane/ethyl
ether/acetic acid (70:30:1). Standards were stained by iodine vapors,
and plate areas corresponding to phospholipids and fatty acids were
scraped off and extracted with
CHCl3/methanol/H2O (25:50:20) for 40 minutes at
room temperature with shaking. Supernatants obtained after
centrifugation of silica gel suspension were evaporated, dissolved in
CHCl3, and stored at
OxPAPC induces TF expression in HUVECs Previously, MM-LDL was shown to induce TF expression in ECs.6 It is known that some biologic effects of MM-LDL can be mimicked by OxPAPC, a component of MM-LDL.7 In this work, we tested whether OxPAPC was capable of inducing expression of TF in HUVECs. We found that OxPAPC elevated TF activity in a time-dependent manner (Figure 1A), whereas native phospholipid was inactive (not shown). The increase in activity paralleled increasing levels of TF protein, as demonstrated by Western blotting (Figure 1A, insert). The effects of OxPAPC were concentration dependent and reached apparent saturation at about 100 µg/mL (Figure 1B).
OxPAPC does not activate the NF- or IL-1 elevate TF in ECs
through activation of NF-B-mediated transcription.23
Important steps in this process are phosphorylation and degradation of
the inhibitory IB subunit allowing translocation of active
NF-B to the nucleus. To address a possible involvement of the
NF-B pathway in OxPAPC-induced TF expression, we used 4 different
approaches. First, we demonstrated by Western blotting that OxPAPC, in
contrast to TNF-, did not induce phosphorylation of IB (Figure
2A). Furthermore, OxPAPC did not activate
IB degradation, whereas TNF- induced a rapid decrease in the
levels of IB (Figure 2B). Second, OxPAPC did not induce binding
of p65 to its consensus DNA (Figure 2C). Third, we have found that in
contrast to lipopolysaccharide (LPS), OxPAPC did not stimulate
a luciferase reporter construct bearing 5× NF-B consensus
site (Figure 2D). Fourth, we used an IB-expressing adenovirus
(AdIB) that has been described to block NF-B
activation.22 Overexpression of IB had no influence on the induction of TF by OxPAPC, but significantly inhibited the
effect of TNF- (Figure 2E). Together, these data strongly suggest
that induction of TF expression by OxPAPC is independent of the
classical NF-B pathway in HUVECs.
OxPAPC activates ERK1/2 MAP kinases Another mechanism of induction of gene transcription used by inflammatory cytokines is signal transduction through the MAP kinase cascades.24 Therefore, we examined whether OxPAPC could activate ERK1/2 MAP kinases. Western blotting with antibodies recognizing the active phosphorylated forms of ERK1/2 demonstrated rapid and sustained activation of these kinases after addition of OxPAPC or TNF- (Figure 3). We also
detected a slight activation of p38 MAP kinase; however, we could not
detect activation of JNK by OxPAPC (data not shown).
OxPAPC induces expression of the transcription factor EGR-1 in a MEK/ERK-dependent manner The TF promoter contains binding sites for the EGR-1 transcription factor.25 EGR-1 is required for induction of TF expression by several cytokines and growth factors including TNF- and
VEGF.16,26 It is known that ERK1/2 stimulates expression
of EGR-1.27 Because we demonstrated activation of ERK1/2
by OxPAPC, we tested whether OxPAPC would also induce EGR-1. The data
presented in Figure 4A demonstrate that
OxPAPC induced rapid and transient elevation of EGR-1 protein. The
maximum induction was reached after 60 minutes following treatment with
OxPAPC and dropped to baseline levels within 5 hours. The time course
(Figure 4A) indicated that elevation of EGR-1 preceded the rise of TF
protein and activity levels (Figure 1A). The level of EGR-1 decreased
after 2 hours of stimulation, whereas phosphorylation of ERK1/2
remained apparently constant for up to 8 hours after addition of OxPAPC
(Figure 4A).
Furthermore, inhibition of MEK, which is upstream of ERK1/2, using a specific inhibitor (PD98059), significantly suppressed OxPAPC-induced phosphorylation of ERK1/2 and elevation of EGR-1 (Figure 4B). These results suggest that activation of ERK1/2 is necessary for induction of EGR-1 expression by OxPAPC. Activation of ERK1/2 and elevation of EGR-1 are functionally important for induction of TF expression by OxPAPC To elucidate the functional role of MAP kinases in induction of TF by OxPAPC, we pretreated cells with a MEK inhibitor (PD98059), which suppressed induction of TF expression by OxPAPC in a dose-dependent manner (Figure 5A). To address the role of EGR-1 in induction of TF by OxPAPC, HUVECs were infected with recombinant adenovirus to overexpress NAB2 (AdNAB2), a natural corepressor of EGR-1 capable of inhibiting its transcriptional activity.28 We found that overexpression of NAB2 in HUVECs significantly suppressed induction of TF by OxPAPC (Figure 5B). These results demonstrate that the MEK/ERK/EGR-1 pathway is critically involved in OxPAPC-induced expression of TF in HUVECs.
PKC is necessary for induction of ERK1/2, EGR-1, and TF by OxPAPC The above data show that OxPAPC stimulates TF expression in HUVECs at least in part via activation of the ERK/EGR-1 pathway, which, in turn, may be stimulated by various upstream kinases, such as PKC.29 Treatment of ECs with the specific PKC inhibitor Bis I in a concentration-dependent manner inhibited TF activity induced by OxPAPC (Figure 6A). Furthermore, ERK1/2 and EGR-1 activation were also inhibited by Bis I (Figure 6B). These data indicate that PKC plays an important role in activation of ERK/EGR-1 signaling pathway and up-regulation of TF by OxPAPC.
OxPAPC elevates cytosolic Ca++ levels and induces nuclear translocation of NFAT Phorbol esters, Ca++ ionophore, and VEGF are known to stimulate TF gene expression also through activation of the NFAT transcription factor.15,30 Because elevation of intracellular Ca++ levels is a prerequisite for stimulation of NFAT, we tested whether OxPAPC would induce a Ca++ response in HUVECs. Using the fluorescent Ca++-sensitive probe fura-2, we demonstrate that OxPAPC, but not its nonoxidized precursor PAPC, induces rapid and transient elevation of Ca++ in the cytosol of HUVECs (Figure 7A). To investigate whether OxPAPC is capable of inducing nuclear translocation of NFAT, we studied the intracellular distribution of NFAT tagged with GFP (NFAT-GFP) before and after OxPAPC-treatment. The NFAT-GFP in the transfected cells exhibits the same localization and nuclear translocation as described for endogenous NFAT.31 In unstimulated cells (control), NFAT-GFP is predominantly localized in the cytoplasm (Figure 7B). Addition of OxPAPC induced NFAT-GFP accumulation in cell nuclei within 30 minutes. A similar effect was exerted by calcium ionophore (A23187). Translocation of NFAT-GFP induced by both agonists was inhibited by cyclosporin A, known to suppress activation of NFAT by blocking its upstream phosphatase calcineurin30 (Figure 7B).
OxPAPC induces NFAT/DNA binding activity We further analyzed the ability of OxPAPC to activate NFAT by EMSA using an NFAT site of the murine IL-4 promoter that has been demonstrated to bind NFAT independently of AP-1.32 OxPAPC induced NFAT/DNA-binding activity in HUVECs (Figure 8A). Similar DNA-protein complexes were formed after treatment of HUVECs with a combination of Ca++ ionophore and phorbol ester (Figure 8B). In both cases, the bands were competed with excess of unlabeled specific oligonucleotide, but not by excess of an unlabeled probe for a Sp1 binding site. Moreover, cells pretreated with cyclosporin A failed to form indicated DNA-protein complexes independently whether OxPAPC or Ca++ ionophore/phorbol myristate acetate (PMA) was used for stimulation (Figure 8A,B). Supershift experiments demonstrated the presence of NFATp as well as NFATc in the DNA-protein complexes induced by OxPAPC (Figure 8A). In contrast, incubation with an antibody against the NF-B subunit p50 had no effect. These data
correlate with previous studies showing the presence of NFATc (NFATc1)
and NFATp (NFATc2) in HUVECs15,33 Performing EMSA using
labeled oligonucleotide corresponding to the NFAT-binding site of human TF promoter produced results similar to those obtained with the NFAT-binding oligonucleotide from the IL-4 promoter (Figure
8C).
Cyclosporin A inhibits induction of TF by OxPAPC Because cyclosporin A inhibited activation of NFAT, we tested whether it would also block expression of TF induced by OxPAPC. Cyclosporin A decreased induction of TF activity as well as protein in OxPAPC-treated cells by 50% to 60% (Figure 9). In addition, cyclosporin A blocked induction of TF by Ca++ ionophore; for both OxPAPC and Ca++ ionophore, maximal inhibition was observed at 100 ng/mL cyclosporin A (not shown). These data suggest that, in addition to EGR-1, activation of NFAT plays an important role in OxPAPC-induced TF expression in HUVECs.
PGPC is one biologically active component of OxPAPC responsible for TF up-regulation Oxidation of OxPAPC is known to generate a variety of products, such as oxidized phospholipids, lysophospholipids, and fatty acids.8 To determine which of these substances mediate induction of TF, we separated OxPAPC by TLC into fatty acid and phospholipid fractions and found that the activity was predominantly localized in the phospholipid fraction (Figure 10A). Treatment of OxPAPC with PLA2 significantly reduced biologic activity (Figure 10A), strongly indicating that the fatty acid residue at the sn-2 position of oxidized phospholipids was important for the ability to induce TF expression. Furthermore, we tested the effect of 2 previously identified PAPC oxidation products, namely, POVPC and PGPC,8 on TF expression in HUVECs. We found that PGPC, but not POVPC, induced expression of TF in a concentration-dependent manner (Figure 10B).
The inflammatory effects of MM-LDL can mainly be attributed to its oxidized phospholipid components.7 We used OxPAPC as a surrogate for MM-LDL to study signaling pathways and transcription factors mediating induction of TF expression in HUVECs. The induction of TF activity by OxPAPC paralleled with the increase in TF protein. These data are in good correlation with previous findings demonstrating transcriptional regulation of TF by MM-LDL in ECs5 and suggest that changes in protein expression, rather than in receptor activity, are responsible for the observed effects. In addition, we demonstrate that transcriptional mechanisms mediating
elevation of TF in OxPAPC-treated HUVECs differ from those activated by
classical inflammatory mediators. The TF gene promoter
contains consensus sites for Sp1,26 thought to be
important for basal transcription, and for NF- Another transcription factor capable of binding to the TF promoter is
EGR-1, which is critically involved in TF gene regulation by
inflammatory cytokines, serum, VEGF, and phorbol
esters.14,26 We have found that OxPAPC induced a rapid
increase in EGR-1 levels in HUVECs. We also demonstrate a link between
EGR-1 and TF expression, because overexpression of the EGR-1
corepressor NAB2 inhibited OxPAPC-induced TF expression. Recently, it
was shown that EGR-1 and EGR-1-responsive genes are up-regulated in
atherosclerotic lesions.39 However, the factors inducing
EGR-1 expression in the vascular wall are not known. OxPAPC, which is
present in atherosclerotic lesions,8 could very well be
responsible for increased EGR-1 levels. EGR-1 is known to mediate
induction of several growth factors and their receptors, including
platelet-derived growth factor (PDGF)-A, PDGF-B, fibroblast growth
factor 2, transforming growth factor Analyzing signaling mechanisms induced by OxPAPC, we found that OxPAPC
induced phosphorylation of ERK1/2. These data are consistent with
previous findings of MAP kinase activation by oxidized LDL in other
cell types.41,42 It is known that EGR-1 expression can be
induced by ERK1/2 kinases, which stimulate a ternary complex factor,
leading to induction of EGR-1
transcription.27,43 We demonstrate here that the MEK/ERK
signaling cascade is required for EGR-1 induction by OxPAPC. This
mechanism is different from stress-induced EGR-1
transcription, which was shown to be mediated by p38 MAP kinase and
JNK, but shows striking similarities to TNF- Phosphorylation of ERK1/2 as well as elevation of EGR-1 and TF in OxPAPC-treated cells was suppressed by PD98059, known to block the upstream kinase MEK. These data indicate that activation of the MEK/ERK cascade is necessary for full induction of TF by OxPAPC. Various agonists can stimulate MEK/ERK kinases either through PKC-dependent or PKC-independent mechanisms.29 Taken together, our data show that PKC-MEK-ERK1/2-EGR-1 represents one pathway leading to induction of TF expression by OxPAPC. In addition, the present study provides evidence for the first time that oxidized phospholipids are capable of inducing NFAT-mediated activation of ECs. NFAT was shown to participate in the regulation of TF expression by Ca++ ionophore, phorbol ester, and VEGF.15,30 Here we demonstrate that OxPAPC induces the typical mechanisms leading to NFAT activation, starting from elevation of cytosolic Ca++, next activation of calcineurin (as demonstrated by the inhibitory effect of cyclosporin A), and finally leading to nuclear translocation and DNA binding of NFAT. Furthermore, these data indicate that activation of NFAT by OxPAPC is functionally important for induction of TF, because both NFAT translocation and TF elevation were partially inhibited by cyclosporin A. We also addressed the question with respect to the biologically active components in OxPAPC, which may be responsible for stimulation of TF expression. We found that the TF-inducing activity was associated mainly with the phospholipid fraction. The phospholipid nature of the major active principle was further confirmed by the loss of biologic activity after treatment of OxPAPC with PLA2. This result also indicates that the TF-inducing phospholipids were different from lysophosphatidylcholine. This conclusion is consistent with data demonstrating inhibition, rather than up-regulation, of TF in human monocytes by lysophosphatidylcholine.44 Two identified phospholipids, POVPC and PGPC, are present in OxPAPC and
are capable of activating ECs.8 Both substances are
generated by fragmentation of arachidonic acid in PAPC. POVPC contains
a 5-carbon residue with an Altogether, the data presented in this report are consistent with the following mechanism: OxPAPC activation of HUVECs elevates cytosolic Ca++ levels and activates PKC. Further steps include calcineurin-dependent activation and nuclear translocation of NFAT as well as parallel activation of ERK1/2, resulting in up-regulation of EGR-1. NFAT and EGR-1 bind to their respective sites in the TF promoter and stimulate transcription. Increased TF expression may promote local fibrin formation in the tissue in the course of the atherosclerotic process. In addition, NFAT and EGR-1 are known to regulate expression of other inflammatory genes. OxPAPC may thereby contribute to chronic inflammatory stimulation and thus to the progression of atherosclerosis by activation of signaling pathways in part distinct from the signals triggered by inflammatory cytokines. Our results contribute to the understanding of mechanisms of endothelial cell activation during chronic inflammatory processes, and thus may lead to novel strategies for therapeutic intervention.
We thank Dr Z. Zhegu and R. Ecker for preparation of HUVEC, G. Mitulovic for help with MS analysis of phospholipids, and Dr R. de
Martin for providing the I
Submitted April 24, 2001; accepted September 7, 2001.
Supported by the Austrian Science Foundation, project numbers P13954-MED (N.L.) and SFB-714, P-14586-PHA (W.F.G.), and by the ICP Program of the Austrian Federal Ministry for Education, Science and Culture. V.N.B. and D.M. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Norbert Leitinger, Department of Vascular Biology and Thrombosis Research, University of Vienna, Schwarzspanierstrasse 17, A-1090 Vienna, Austria; e-mail: norbert.leitinger{at}univie.ac.at.
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1997;272:13597-13607 |
Top
Abstract
Introduction
7 M, respectively)
were incubated with 32P-labeled NFAT probe of the murine
IL-4 (A,B) or human TF (C) promoters and then separated by gel
electrophoresis. Where indicated, cells were preincubated for 30 minutes with 500 ng/mL cyclosporin A before addition of OxPAPC or
A23187/PMA. The mobility of the specific OxPAPC-induced/cyclosporin
A-sensitive complex is indicated by an arrow. Supershifted band is
labeled with an asterisk. The results are representative of 4 independent experiments.
-aldehyde group at the sn-2 position, in contrast to the 
a major role for phospholipid oxidation products. In:
Keaney J, ed.
Oxidative Stress and Vascular Disease. Boston, MA: Kluwer Academic Publishers; 2000:119-134.