(Circulation. 1997;96:2262-2271.)
© 1997 American Heart Association, Inc.
Articles |
Advanced Glycation End Product (AGE)–Mediated Induction of Tissue Factor in Cultured Endothelial Cells Is Dependent on RAGE
From the Department of Internal Medicine I, University of Heidelberg (A.B., P.W., R.Z., P.P.N.), Germany; the Institute of Pathology (T.I., T.L., M.M.) and the Institute of Anatomy, Technical University Dresden (M.K.), Germany; the Allgemeines Krankenhaus Wien (P.Q.), Vienna, Austria; and Asta Medica (H.T.), Frankfurt, Germany.
Correspondence to P.P. Nawroth, MD, Medizinische Klinik I, Bergheimer Str 58, 69115 Heidelberg, Germany.
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Abstract |
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Background Binding of advanced glycation end products (AGEs) to the cellular surface receptor (RAGE) induces translocation of the transcription factor NF-
B into the nucleus and
NF-B–mediated gene expression. This study examines the role of RAGE
in the AGE albumin–mediated induction of
endothelial tissue factor, known to be partly
controlled by NF-B.
Methods and Results Endothelial cells (ECs)
were incubated in the presence of an 18-mer phosphorothioate
oligodeoxynucleotide antisense to the 5'-coding sequence of
the RAGE gene (antisense RAGE; 0.1 µmol/L). Sense
oligonucleotides (sense RAGE, 0.1 µmol/L) of the
same region served as control. The cellular uptake of
oligonucleotides was controlled by
immunofluorescence microscopy. RAGE transcription
was suppressed by antisense RAGE, as demonstrated by RT-PCR reactions.
AGE albumin–mediated activation of cultured ECs was studied
after 48 hours of preincubation of ECs with antisense or sense RAGE.
Electrophoretic mobility shift assays and Western blot analysis
demonstrated that the AGE albumin–induced translocation of
NF-B from the cytoplasm into the nucleus was suppressed in the
presence of antisense RAGE but not by sense RAGE. In parallel, AGE
albumin–mediated tissue factor transcription, activity, and
antigen were significantly reduced in ECs exposed to antisense RAGE,
whereas sense RAGE (and nonspecific oligonucleotides)
did not influence tissue factor expression.
Conclusions Activation of ECs and induction of tissue factor by AGE albumin in ECs is dependent on RAGE.
Key Words: arteriosclerosis • atherosclerosis • coagulation • diabetes mellitus • endothelium
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Nonenzymatic glycation of proteins, leading to the formation of irreversible AGEs, has been found to occur during aging and at an accelerated rate in diabetes.1 2 AGEs are believed to play a pivotal role in atherosclerosis,3 4 5 because they are found in the atherosclerotic plaque even in young, nondiabetic animals.4 AGEs modulate EC functions by inducing cytokines,6 growth factors,7 adhesion molecules,8 9 and procoagulant activity.10 These reactions are thought to contribute to the development of diabetic complications, including atherosclerosis and microvascular disease.2 One mechanism through which AGEs exert their cellular effects is by specific interactions with cell surface–associated AGE-binding proteins. To date, AGE-binding proteins designated p60,11 p90,11 RAGE,12 13 lactoferrin,12 14 15 and galectin-316 have been described, one of which, RAGE, has been identified and isolated on ECs.12 13 RAGE has also been found on monocytes/macrophages17 and smooth muscle cells.18 Furthermore, RAGE expression has been localized to areas of atherosclerosis in patients with diabetes mellitus19 and with uremia.20 Recent studies demonstrated that both antibodies directed against RAGE and soluble RAGE, a truncated form of the receptor, inhibited AGE albumin–induced VCAM-1 expression,14 which is regarded as a hallmark of atherogenesis.21 In addition, in vitro and in vivo studies revealed that suppression of RAGE reversed the AGE albumin–induced impairment of endothelial barrier function22 as well as the diabetes-mediated hyperpermeability in diabetic rats.23
TF is a membrane-bound glycoprotein that functions as the
primary cellular initiator of coagulation by its ability to bind factor
VII/VIIa.24 25 ECs,26 27 28 29 30
monocytes/macrophages,27 and smooth muscle
cells,31 all of which participate in the development of
atherosclerotic lesions, have been demonstrated to synthesize TF in
vitro and in vivo. TF expression has consistently been detected
in the atherosclerotic plaque32 and might be associated
with intravascular thrombotic complications.33 34 35 In
addition to its central role in coagulation, high-level TF expression
promotes metastasis33 and tumor
angiogenesis.28 34 In contrast to extravascular cells,
vascular cells do not express TF under
physiological conditions.24 25 26 27 Under
pathological conditions, however, ECs can be induced to synthesize
TF.27 29 30 Thus, inducible endothelial TF
expression is central in promoting thrombogenicity associated with
dysfunctional endothelium,36 as confirmed
by in vivo studies demonstrating endothelial TF
expression in the spleenic microvasculature in lethal Escherichia
coli septicemia,35 in the tumor vasculature of breast
carcinoma,34 in tumor necrosis factor-
–treated Meth-A
sarcoma,29 30 in Kaposi's sarcoma,36 and in
the renal microvasculature in hydronephrosis.37
Expression of endothelial and monocytic TF in vitro is
controlled by the transcription factors AP-1 and
NF-B.29 38 39 40 Somatic gene transfer experiments in a
mouse tumor model confirmed that AP-1 and NF-B also mediate the
induction of TF expression in vivo.29 Because (1) AGEs
bound to RAGE induce cellular oxidant stress and thereby
activate NF-B,41 (2) AGEs increase TF
expression,15 and (3) elevated TF levels have been
reported in patients with diabetes,42 TF seems to be a
likely candidate to be induced by an AGE-activated
RAGE-mediated NF-B–dependent mechanism. In the present study,
we used ODNs antisense to RAGE (antisense RAGE
[PS]ODNs)43 to show that inhibition of RAGE
transcription in part prevents AGE albumin–mediated NF-B
activation and TF induction.
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Methods |
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Reagents
Reagents were obtained as follows: DMEM (4500 mg/L glucose), RPMI 1640, HEPES buffer solution, L-glutamine, penicillin/streptomycin mixture, and PBS (pH 7.4) were from Biowhittaker. FCS, Dotap, DNAse I (RNAse free), proteinase K, dNTPs, and rNTPs were from Boehringer Mannheim. Barbital buffer was obtained from Behring. [
-32P]dCTP (3000 Ci/mmol at 10
Ci/mL), [
-32P]ATP (3000 Ci/mmol at 10 Ci/mL),
[-32P]UTP (3000 Ci/mmol at 10 Ci/mL), Hybond N nylon
filter, ECL nitrocellulose membranes, ECL detection reagents, and
Hyperfilm x-ray films were obtained from Amersham. The EZ-rTth-RT-PCR
kit and Taq polymerase were from Perkin-Elmer Inc. Poly
dI/dC and the cDNA synthesis kit were purchased from Pharmacia.
GAPDH-specific primers were obtained from Clontech. Anti–p65-(sc-109X)
and anti– c-Rel-(sc-70X) polyclonal antibodies were obtained from
Santa Cruz Inc. The antiserum for p50/p105 was a generous gift from Dr
Nancy Rice, Frederick Cancer Research and Development Center,
Frederick, Md. AGE bovine albumin (3.14 mg/mL) was prepared by preincubation of BSA with 200 mmol/L glucose-6-phosphate at 37°C for 4 to 8 weeks in 100 mmol/L phosphate (pH 7.4)/0.5 mmol/L sodium azide. Alternatively, glycated bovine albumin was purchased from Sigma. Nonglycated bovine albumin and heat-inactivated AGE bovine albumin or bovine albumin incubated with the synthetic substrate sorbite served as negative controls.
Antisense [PS]ODNs
Antisense [PS]ODNs (5'-GACCACTGCCCCTGCTGC-3' [bovine];
5'-AACTGCTGTTCCGGCTGC-3' [human]) and sense [PS]ODNs
(5'-GCAGCAGGGGCAGTGGTC-3' [bovine]; 5'-GCAGCCGGAACAGCAGTT-3'
[human]) composing the region bp +13 to bp +30 (bovine) or bp +4 to
bp +21 (human) (Fig 1
) derived from the
published DNA sequences for RAGE13 were synthesized on a
Gene Assembler Plus (Pharmacia) and purified on histidine gels.
FAM-(5'-carboxyfluorescein) phosphoroamitide–labeled
oligonucleotides were purchased from Biometra.
[PS]ODNs were chemically modified at the 5'-boundaries by
introduction of phosphorothioate linkages, in which a nonbridging
phosphate oxygen atom was substituted with a sulfur atom to protect
oligonucleotides from serum- and nuclease-mediated
degradation.44 FCS used throughout the experiments was
routinely heat-inactivated for 30 minutes at 65°C to
minimize nuclease activity.44 45 Uptake of ODNs by the
cells was in a passive manner without any further pretreatment of the
cells.44
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Fluorescence Microscopy
BAECs were cultivated on chamber slides in the presence of
0.1 µmol/L antisense RAGE PS[ODN]s (Fig 2a) or 0.1 µmol/L sense
RAGE PS[ODN]s (data not shown) for 48 hours. The cells were washed
three times with PBS, overlaid with PBS-glycerol (1:9) containing 2.5%
DABCO (Janssen) and a coverslip, and analyzed by
fluorescence microscopy with an Olympus BH2 microscope equipped
with a fluorescence device (Olympus) in the green (FITC)
channel.
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Determination of Oligonucleotide Stability
[PS]ODNs were radiolabeled with T4–polynucleotide
kinase, added to FCS-containing medium in a final concentration of 1
nmol/L, and incubated at 37°C for various times before
aliquots were taken and subjected to gel
electrophoresis.46
Plasmids
The plasmids pGL2 control, pGL2 basic, pSV-ß-Gal, and pCAT
control were obtained from Promega. pSPT18 was purchased from
Boehringer Mannheim. The TF promoter mutants pHTF(-278)Luc
(A1-A2-N), pHTF-N, and pHTF(-111)Luc have been
described.29 47 The human TF cDNA probe 
-HTF8 was
generously provided by Dr E. Sadler (Washington University, St Louis,
Mo).48 The plasmid pGEM-TFay49 was a gift
from Dr R.M.W. de Waal (Institute of Pathology, Nijmegen, Netherlands);
other plasmids mentioned were obtained from ATCC.
Tissue Culture
Tissue culture of BAECs and HUVECs was performed as previously
described in detail.26 50 51 FCS used throughout the
studies was routinely heat-inactivated for 30 minutes at
65°C to minimize interference of nuclease activity with
oligonucleotide application.45
Determination of TF Activity by One-Stage Clotting Assays
One-stage clotting assays were performed as previously
described.50
Nuclear Run-on Transcription Assay
Nuclear run-on transcription assays were performed essentially
according to the procedure of Greenberg and Ziff52 and
have been previously described in detail.29
Reverse Transcription–Polymerase Chain Reaction
RT and PCR for human and bovine RAGE, TF, and GAPDH were
performed basically as described by Pötgens et al49
under the following conditions: RAGE RT: 60 minutes, 60°;
amplification: 1x [94°C, 360 seconds; 80°C, 120
seconds; 70°C, 35 seconds; 70°C, 40 seconds]; 30x [94°C, 60
seconds; 70°C, 35 seconds; 70°C, 40 seconds]; 1x [95°C, 60
seconds; 70°C, 35 seconds; 70°C, 600 seconds]; TF RT: 60
minutes, 60°; amplification: 1x [95°C, 300 seconds; 62°C, 90
seconds; 72°C, 240 seconds]; 33x [95°C, 60
seconds; 62°C, 90 seconds; 72°C, 240 seconds]; 1x
[95°C, 60 seconds; 62°C, 90 seconds; 72°C, 420 seconds]. The
RAGE-specific primers are listed in Fig 1
; the TF-specific primers have
been described.49 For quantitative RT-PCR, the amount of
TF mRNA was determined by competition with different amounts of the TF
control plasmid pGEM-TFay as described in detail by Pötgens et
al.49
Electrophoretic Mobility Shift Assay
For EMSAs, nuclear proteins were harvested as described
elsewhere29 53 and assayed for transcription factor
binding activity by use of oligonucleotides for the
TF-derived NF-B site (5'-AGGGTCCCGGAGTAGTTTCCTACCGGGA-3'), the
NF-B consensus sequence (5'-AGTTGAGGGGACTT TCCCAGGC-3'), and the
SP-1 consensus sequence (5'-ATTC GATCGGGGCGGGGCGAGC-3'). Specificity
of binding was ascertained by competition with a 160-fold molar excess
of unlabeled consensus oligonucleotides.
Western Blot Analysis
Cytoplasmic and nuclear fractions were prepared and probed with
the respective antisera for anti-p65 (1:500), anti c-Rel (1:500), and
anti-p50/p105 (1:2000) as previously described in
detail.53
Transient Transfection of ECs
Logarithmically growing ECs were transfected by the calcium
phosphate method as described previously in detail.29 When
indicated, oligonucleotides (0.1 µmol/L)
were included in the DNA preparation and in the culture medium, and/or
500 nmol/L control albumin, 500 nmol/L AGE
albumin, or 1 µg/µL LPS was added. Data were
analyzed with the aid of Sigma Plot software (Jandel
Scientific). Levels of significance were determined by Student's
t test. Any value of P
.05 was considered to be
significant.
Densitometric Quantification
Densitometry was performed with a Scan-Pack Personal
Densitometer scan (Pharmacia). Determination of the signal area to be
measured and quantitative evaluation were performed independently by
two different investigators (A.B. and T.I.), and the mean of both
measurements was taken for the statistical analysis
provided.
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Results |
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Antisense RAGE Oligonucleotides Downregulate RAGE Expression
We examined whether one of the known receptors for AGE, RAGE,54 mediates AGE albumin–induced endothelial TF expression by an NF-
B–dependent
mechanism. Cultured ECs were incubated in the presence of antisense
[PS]ODNs complementary to the 5'-region of the published sequences
for bovine and human RAGE.18 Sense [PS]ODNs of the same
region and several unrelated [PS]ODNs with similar base-pair
compositions served as controls. The sequences selected for the
antisense [PS]ODNs used (Fig 1) did not contain four contiguous
guanosines, because it has been reported that hybridization-unrelated
effects are mediated by a stretch of guanosines.55
[PS]ODNs were added to the medium at a concentration of 0.1
µmol/L, which is below the concentration recently described to
mediate unspecific effects of [PS]ODNs in ECs.56 Uptake
of [PS]ODNs by ECs was in a passive manner without any further
pretreatment.44 Successful internalization was monitored
by immunofluorescence detection of FAM-labeled
[PS]ODNs and demonstrated a maximum between 12 and 24 hours (Fig 2a).
After 48 hours, lysosomal localization became evident, indicating that
degradation had already started (Fig 2a). Ninety-six hours after
incubation, [PS]ODNs were no longer detectable (Fig 2a). Gel
electrophoresis demonstrated stability of [PS]ODNs in the culture
medium for at least 36 hours (Fig 2b). The doublet band, already
present at time 0, was probably due to secondary structures of the
oligonucleotide,57 because it was not
detected if the oligonucleotide was run in the absence
of serum (Fig 2c). In the following experiments, cells were
preincubated in the presence of 0.1 µmol/L [PS]ODNs for
48 hours, in the course of which [PS]ODNs were renewed after 24
hours, before AGE albumin induction was performed. After AGE
albumin (500 nmol/L) stimulation of HUVECs for 90
minutes, RAGE transcription was monitored in RT-PCR reactions (Fig 2d).
No RAGE transcripts were detected in unstimulated human ECs (Fig 2d).
In contrast, strong induction of RAGE mRNA synthesis was observed in
AGE albumin–induced cells (Fig 2d). Sense [PS]ODNs did not
alter AGE albumin–induced RAGE transcription (Fig 2d), whereas
a successful downregulation of AGE albumin–dependent RAGE mRNA
synthesis could be demonstrated after 48 hours of preincubation with
antisense [PS]ODNs (Fig 2d). Thus, AGE albumin induces
endothelial RAGE in a RAGE-dependent fashion. It is
noteworthy that under the most stringent conditions, RT-PCR detected
not only the expected 480-bp RAGE fragment but also two smaller mRNA
transcripts (Fig 2d). This is in striking contrast to the single 480-bp
banding observed in other human cells, eg, human leukemia cell lines
(data not shown), under the same RT-PCR conditions.
Antisense RAGE Oligonucleotides Inhibit AGE
Albumin–Induced NF-B Activation
Recently it has been demonstrated that inhibition of RAGE by
RAGE-specific antibodies inhibits AGE albumin–induced NF-B
activation.9 41 EMSAs evidenced that AGE albumin
induced binding of NF-B to its consensus motif (Fig 3a, lane 1 versus 2) and to the
TF-derived NF-B–like site (Fig 3b, lane 1 versus 2), previously
characterized as NF-B(p65/c-Rel).29 38 39 40 NF-B
activation was reduced in nuclear extracts from ECs that had been
pretreated with antisense RAGE [PS]ODNs before incubation with AGE
albumin (500 nmol/L) (Fig 3a and 3b, lane 4).
Preincubation with sense RAGE [PS]ODNs did not significantly affect
NF-B–binding activity (Fig 3a and 3b, lane 3). When 0.1
µmol/L antisense RAGE [PS]ODNs was added directly to the
binding reaction, no inhibition of NF-B binding to its respective
binding site was observed (Fig 3a, lane 5). Thus, direct physical
interactions of the antisense [PS]ODN with NF-B or its DNA-binding
subunit could be excluded. Because it has been reported that ODNs,
independent of their base-pair composition, can mediate induction of
the transcription factor SP-1 in diverse cell types,58 the
above nuclear extracts were tested for their SP-1–binding capacity.
However, no significant SP-1 induction by sense RAGE or antisense RAGE
[PS]ODNs was detected (Fig 3c).
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Western blot analysis showed that reduced binding activity of
NF-B was due to suppressed translocation of NF-B proteins from
the cytoplasm (Fig 4, lanes 1 through 4)
into the nucleus (Fig 4, lanes 5 through 8). Although only a little
NF-B(p65) was detected in the nucleus of control ECs (Fig 4a, lane
5), incubation with AGE albumin (30 minutes, 500 nmol/L)
resulted in increased translocation of NF-B(p65) (Fig 4a, lane 6).
This was also evident in ECs pretreated with sense RAGE [PS]ODNs (Fig 4a, lane 7). Only a little NF-B(p65) was detected in ECs that had
been incubated in the presence of antisense RAGE [PS]ODNs (Fig 4a, lane 8). The effect for NF-B(p50) was less pronounced because,
consistent with previous studies,59 considerable
amounts of NF-B(p50) were already present in uninduced nuclear
extracts (Fig 4b, lanes 5 through 8), whereas the presence of its
precursor p105 was restricted to the cytoplasm (Fig 4b, lanes 1 through
4). However, the slight induction observed after AGE albumin
induction in the presence or absence of sense RAGE [PS]ODNs was
abolished in the presence of antisense RAGE [PS]ODNs (Fig 4b, lanes 6
and 7). As reported previously,59 the expected 75-kD
protein NF-B c-Rel was detected mainly in the cytoplasm of ECs,
whereas almost no 75-kD signal was found in the nucleus (Fig 4c, lanes
5 through 8). However, an additional 62-kD protein was selectively
detected by anti–c-Rel antibodies in cytoplasmic and nuclear extracts
(Fig 4c, lanes 1 through 8). Translocation of this protein was
inducible by AGE albumin in untreated or sense RAGE
[PS]ODN–treated ECs (Fig 4c, lanes 6 and 7), whereas antisense RAGE
[PS]ODNS reduced the observed translocation (Fig 4c, lane 8).
Together with the EMSA data demonstrating AGE albumin–induced
binding activity of NF-B(p65/ c-Rel) to the TF-derived
NF-B–like site, these data imply that the 62-kD band might
represent a c-Rel isoform or a c-Rel/p65–containing
protein complex. However, additional studies are necessary to further
characterize the nature of this c-Rel–related protein or protein
complex.
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Antisense RAGE Oligonucleotides Suppress TF
Activity
Next, we asked whether reduction of RAGE expression by antisense
RAGE [PS]ODNs directly reduced TF activity. AGE albumin (500
nmol/L)–induced TF activity, determined in one-stage clotting
assays (Fig 5a), was completely prevented
when ECs were preincubated with 0.1 µmol/L antisense RAGE
[PS]ODNs 48 hours before stimulation (Fig 5b). However, when 0.1
µmol/L antisense RAGE [PS]ODNs was added at the same time as
AGE albumin (500 nmol/L), a moderate TF activation was
observed after 3 hours of AGE albumin stimulation (Fig 5c) but
was downregulated thereafter (Fig 5c). Heat-inactivated AGE
albumin that served as control did not induce TF (Fig 5d). To
demonstrate specificity, several other [PS]ODNs were applied to ECs
at the same concentration (0.1 µmol/L) as antisense RAGE
[PS]ODNs (Fig 5e). Sense RAGE [PS]ODNs, sense TF [PS]ODNs, or
antisense HMWK [PS]ODNs had no effect on AGE albumin–induced
TF activity (Fig 5e).
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Antisense RAGE Oligonucleotides Suppress TF
Transcription
To test whether the inhibition seen at the TF antigen and activity
level was also observed on the transcriptional level, nuclear run-on
experiments (Fig 6) were performed.
Compared with unstimulated cells (Fig 6, top, lane 1), AGE
albumin induced new synthesis of TF mRNA (Fig 6, top, lane 2).
Preincubation with 0.1 µmol/L sense RAGE [PS]ODNs (Fig 6, top, lane 3) had no effect, whereas 0.1 µmol/L
antisense RAGE [PS]ODNs directly reduced AGE
albumin–dependent TF transcription (Fig 6, top, lane 4). The
household gene GAPDH (Fig 6, middle, lanes 1 through 4) served as
control. Signals obtained for TF and GAPDH were evaluated by laser
densitometry and are given below each autoradiograph. Method-dependent
variations in the hybridization signal were normalized by calculating
the ratio of TF and GAPDH signal intensity serving as a measure for the
increase in TF transcription (Fig 6, bottom). RT-PCR with TF-specific
primers (performed with the same RNA template as used for the
RAGE-specific RT-PCR shown in Fig 2c) confirmed that inhibition of RAGE
by 0.1 µmol/L antisense RAGE [PS]ODNs resulted in a
decrease of AGE albumin–induced TF mRNA (Fig 7a). To evaluate the RT-PCR results,
competitive PCR was performed in the presence of decreasing amounts of
a TF-specific competitor plasmid (Fig 7b) consisting of a nonmammalian
sequence surrounded by TF-specific sequences matching the PCR primers
used.49 Different cDNA preparations were adjusted for the
same input of GAPDH cDNA before the 0.3-kb TF-specific signal and the
competing 0.7-kb control fragment of pGEM-TFay49 (data not
shown) were amplified. Greater availability of TF yielded more 0.3-kb
PCR product relative to the 0.7-kb control product. Signals
were quantified by laser densitometry, and the output ratio of the
intensity of the TF-specific signal to the intensity of the
competitor-derived signal was plotted against the input of competitor
DNA49 (Fig 7b). The data confirmed that the AGE
albumin–induced increase in TF mRNA was significantly reduced
after preincubation with 0.1 µmol/L antisense RAGE
[PS]ODNs, whereas sense RAGE [PS]ODNs had no effect (Fig 7b). These
results evidenced that AGE albumin exerts its effect on TF
expression at least in part via a RAGE-dependent upregulation of TF
transcription.
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Antisense RAGE Oligonucleotides Inhibit the AGE
Albumin–Mediated NF-B–Dependent TF Expression
To confirm the significance of these data in functional assays,
ECs were transiently transfected with TF promoter plasmids, as
previously described in detail.29 47 AGE
albumin–induced expression of TF promoter constructs was
observed as long as the TF-derived NF-B–like site was present
(Fig 8a). Loss of the NF-B site
resulted in the loss of inducibility (Fig 8a). When 0.1
µmol/L antisense RAGE [PS]ODNs was included in the
transfection reaction, AGE albumin–mediated TF induction was
significantly decreased (Fig 8a). Application of sense RAGE [PS]ODN
did not reduce AGE albumin induction but resulted in a slight,
statistically insignificant increase in AGE albumin–mediated
TF expression (Fig 8a). The observed effect was specific, because
antisense RAGE [PS]ODNs only blocked AGE albumin–induced TF
expression (Fig 8a) but did not reduce LPS-mediated TF induction (Fig 8b). Thus, AGE albumin–induced TF expression is at least in
part dependent on a RAGE-mediated activation of the transcription
factor NF-B.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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AGEs exert their pleiotropic effects by binding to a heterogeneous class of cell surface binding proteins. These include five proteins called p60,11 p90,11 galectin-3,16 lactoferrin,12 and RAGE.12 13 The latter has been localized on ECs,18 monocytes/macrophages,17 and smooth muscle cells,18 all of which contribute to atherosclerotic plaque formation. In vitro9 and in vivo23 experiments linked AGE/RAGE interactions and endothelial gene expression relevant in atherogenesis by demonstrating that antibodies directed against RAGE inhibited the expression of VCAM-1.21 With respect to the diversity of receptors described, these experiments do not completely exclude the possibility that receptors others than RAGE might also be involved in the AGE-dependent activation of ECs, because an excess of antibodies specific for RAGE might at least partly mask other AGE-binding proteins with related structures.
We used antisense RAGE [PS]ODNs to inhibit RAGE transcription and to
study whether a RAGE-dependent mechanism underlies the AGE
albumin–mediated induction of TF,10 the major
cellular initiator of coagulation and an important trigger of
endothelial dysfunction.24 25 Because the
biological effects of [PS]ODNs could result from both
sequence-specific and hybridization-independent
mechanisms,43 60 the antisense RAGE [PS]ODNs used in
this study were constructed and controlled according to recently
published guidelines for the generation of reliable antisense
ODNs.43 Minimizing the number of phosphorothioate
modifications avoided unspecific inhibition of DNA polymerases and
RNAse H.61 Four guanosines, mediating
hybridization-unrelated effects,55 were evaded. Low
[PS]ODN concentrations ensured that the overall DNA synthesis was not
reduced in a nonspecific manner.56 62 Fluorescence
microscopy demonstrated the cytoplasmic location of [PS]ODNs (Fig 2a)
and principally excluded hybridization to nuclear DNA. But even these
precautions can not unequivocally ensure that antisense is the sole
effect observed. However, because similar effects were demonstrated in
human and bovine ECs with species-specific antisense RAGE [PS]ODNS
that exhibit significant sequence differences13 (see Fig 1), it seems unlikely that the experimental results were influenced by
the choice of the target sequences. Specificity was further underlined
by the findings that (1) antisense RAGE [PS]ODNs inhibited AGE
albumin–induced TF expression but not LPS-dependent
upregulation (Fig 8), (2) antisense RAGE [PS]ODNs suppressed
NF-B– but not SP-1–binding activity (Fig 3), and (3)
RAGE-unrelated [PS]ODNs did not alter AGE albumin–mediated
TF induction (Fig 5). Finally, the data presented here are
confirmed by the experiments of Schmidt et al9 and Wautier
et al,23 because the reduction of AGE
albumin–mediated endothelial TF expression by
antisense RAGE [PS]ODNs is comparable to the effects observed for
VCAM-1 expression in the presence of RAGE-blocking antibodies or a
RAGE-specific competitor called soluble RAGE.
Downregulation of AGE albumin–induced TF was observed after
preincubation with antisense RAGE [PS]ODNS on the transcriptional
level (Figs 6 and 7) and on the activity level (Fig 5). This implies
that RAGE is the most important AGE-binding protein that translocates
signals after AGE albumin stimulation of ECs. When antisense
RAGE [PS]ODNs and AGE albumin were applied
simultaneously, a moderate TF induction was evident after 3
hours but was later downregulated to below basal levels (Fig 5c). The
maintenance of a rapid TF induction might be explained by the
presence of preexisting RAGE on the surface of cultured ECs, because
cultured ECs are moderately activated by the isolation
procedure, culture conditions (including a high glucose content in the
medium), and passaging. This hypothesis is supported by the observation
that a weak TF basal expression, detected in unstimulated ECs cultured
under high-glucose conditions (4500 mg/L), is further
downregulated in the presence of antisense RAGE [PS]ODNs (data not
shown).
Under physiological conditions, moderate RAGE
expression has been demonstrated only in the pulmonary
arterial vessels,18 the aorta, and adrenal
capillary ECs, whereas most of the arteries, arterioles, and
endothelium from vasa vasorum of apparently normal
human vessels exhibited no or only very weak RAGE
expression.18 19 63 AGE albumin strongly induced
RAGE mRNA in vitro (Fig 2c). Although further studies will be needed to
investigate the effects of AGE albumin on RAGE transcription
and expression, these data imply the existence of a circulus vitiosis
in which the ligand induces its receptor and thereby potentiates the
availability of its own binding sites. Because it has recently been
reported that lipid peroxidation enhances AGE formation even under
euglycemic conditions,4 AGE-induced RAGE
expression followed by RAGE-mediated gene activation might be a crucial
step in atherogenesis. In addition, it has been speculated that RAGE
can be induced by such cytokines as tumor necrosis
factor-.19 This might explain the increased expression
of RAGE not only in diabetes mellitus19 and
uremia20 but also in pathological situations in which the
existence of blood-borne AGEs is unlikely, such as inflammatory and
immunological kidney disease63 and vascular occlusive
disease in the absence of diabetes.19
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (DFG) (Dr Nawroth), the state of Baden-Württemberg (Dr Nawroth), and the State of Saxony (Drs Bierhaus and Luther). Dr Nawroth performed this work during his time as a Heisenberg scholar (DFG) and his tenure as a Schilling professor. The authors wish to thank Dr Nigel Mackman (La Jolla, Calif) for the tissue factor promoter constructs pHTF(-278)Luc, pHTFM4(-278)Luc, and pHTF(-111)Luc; Dr J. Evan Sadler (St Louis, Mo) for the human tissue factor cDNA probe; Dr Robert M.W. de Waal (Nijmegen, Netherlands) for the control plasmid pGEM-TFay; Dr Nancy Rice (Frederick, Md) for the anti-p50 antibodies; and Dr Helmut Kühne (Dresden, Germany) for the human umbilical vein endothelial cells. The expert technical assistance of Silke Langer (Dresden) is gratefully acknowledged.
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Footnotes |
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Presented in part at the 39th meeting of the German Society for Thrombosis and Hemostasis, February 15-18, 1995, Berlin, Germany; at the 30th meeting of the German Diabetes Society, May 25-27, 1995, Nürnberg, Germany; and at the 15th Congress of the ISTH, June 11-16, 1995, Jerusalem, Israel.
Received December 6, 1996; revision received April 15, 1997; accepted April 26, 1997.
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 K Thiele, A Bierhaus, F Autschbach, M Hofmann, W Stremmel, H Thiele, R Ziegler, and P P Nawroth Cell specific effects of glucocorticoid treatment on the NF-kappa Bp65/Ikappa Balpha system in patients with Crohn's disease Gut, November 1, 1999; 45(5): 693 - 704. [Abstract] [Full Text] [PDF]
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T. Kislinger, C. Fu, B. Huber, W. Qu, A. Taguchi, S. Du Yan, M. Hofmann, S. F. Yan, M. Pischetsrieder, D. Stern, et al. Nepsilon -(Carboxymethyl)Lysine Adducts of Proteins Are Ligands for Receptor for Advanced Glycation End Products That Activate Cell Signaling Pathways and Modulate Gene Expression J. Biol. Chem., October 29, 1999; 274(44): 31740 - 31749. [Abstract] [Full Text] [PDF]
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C. Renard, O. Chappey, M. P. Wautier, M. Nagashima, J. Morser, J. M. Scherrmann, and J. L. Wautier The Human and Rat Recombinant Receptors for Advanced Glycation End Products Have a High Degree of Homology but Different Pharmacokinetic Properties in Rats J. Pharmacol. Exp. Ther., September 1, 1999; 290(3): 1458 - 1466. [Abstract] [Full Text]
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P. Quehenberger, M. Exner, R. Sunder-Plassmann, K. Ruzicka, C. Bieglmayer, G. Endler, C. Muellner, W. Speiser, and O. Wagner Leptin Induces Endothelin-1 in Endothelial Cells In Vitro Circ. Res., April 5, 2002; 90(6): 711 - 718. [Abstract] [Full Text] [PDF]
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