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Circulation. 1997;96:2262-2271

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(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

Angelika Bierhaus, PhD; Thomas Illmer, MD; Michael Kasper, PhD; Thomas Luther, MD; Peter Quehenberger, MD; Hans Tritschler, PhD; Peter Wahl, MD; Reinhard Ziegler, MD; Martin Müller, MD; ; Peter P. Nawroth, MD

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.


*    Abstract
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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-{kappa}B into the nucleus and NF-{kappa}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-{kappa}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-{kappa}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


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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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-{alpha}–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-{kappa}B.29 38 39 40 Somatic gene transfer experiments in a mouse tumor model confirmed that AP-1 and NF-{kappa}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-{kappa}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-{kappa}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-{kappa}B activation and TF induction.


*    Methods
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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. [{alpha}-32P]dCTP (3000 Ci/mmol at 10 Ci/mL), [{gamma}-32P]ATP (3000 Ci/mmol at 10 Ci/mL), [{alpha}-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 1Down) 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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  		Figure 1. RAGE-specific oligonucleotides. Localization and sequences of various RAGE-specific ODNs used for antisense inhibition, sense control, RT-PCR, and verification of specific RAGE PCR products as well as of two unrelated control oligonucleotides. Position of the phosphorothioate linkages in sense and antisense [PS]ODNs are indicated by -[PS]-.

Fluorescence Microscopy
BAECs were cultivated on chamber slides in the presence of 0.1 µmol/L antisense RAGE PS[ODN]s (Fig 2aDown) 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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  		Figure 2. a, Uptake of antisense RAGE [PS]ODNs by BAECs. BAECs were left untreated (0 h) or incubated with fluorescent-labeled FAM antisense RAGE [PS]ODNs (0.1 µmol/L) added to medium for 12 to 96 hours before cell-associated fluorescence was monitored by fluorescence microscopy in the green (FITC) channel. Control BAECs exhibited weak autofluorescence (top left); BAECs incubated in presence of FAM antisense RAGE [PS]ODNs for 12 to 48 hours demonstrated strong fluorescence signals located in cytoplasm and perinuclear areas but not in nuclei. Lysosomal localization became evident 48 hours after application of [PS]ODNs and indicated beginning degradation that increased after 72 hours. At 96 hours after incubation, [PS]ODNs were no longer detectable by fluorescence microscopy. b, Stability of antisense RAGE [PS]ODNs in culture medium: 32P-end-labeled antisense RAGE [PS]ODNs (1 nmol/L) were added to 10% FCS-containing medium in final concentration of 1 nmol/L46 and incubated at 37°C. At 0, 6, 12, 24, 36, 48, 60, and 72 hours after incubation, 20-µL aliquots of [PS]ODN–containing medium were taken and subjected to gel electrophoresis. Oligonucleotide size markers covering range from 32 to 8 bp in 2-bp intervals demonstrated that observed pattern corresponded to 18-bp antisense RAGE [PS]ODN. c, Doublet band of antisense RAGE [PS]ODNs was not observed in absence of serum (-) but was detected when oligonucleotides were run in presence of 10% FCS (+). Thus, it is probably due to secondary structures formed in presence of serum. d, Antisense RAGE [PS]ODNs downregulate AGE albumin–induced RAGE transcription: HUVECs were left untreated (lanes 1 and 2) or preincubated in presence of 0.1 µmol/L sense (S; lane 3) or 0.1 µmol/L antisense (AS; lane 4) RAGE [PS]ODNs before AGE albumin stimulation (500 nmol/L) was performed for 90 minutes. RAGE-specific RT-PCR reactions were performed independently by two investigators (A.B., T.I.) with identical results and monitored by gel electrophoresis. One representative experiment is shown (top). RT-PCR reactions of household gene GAPDH served as control (bottom). Lane 1, 100-bp DNA ladder; lane 2, unstimulated; lane 3, AGE albumin (500 nmol/L, 90 minutes); lane 4, AGE albumin (500 nmol/L, 90 minutes)+0.1 µmol/L sense RAGE [PS]ODNs; lane 5, AGE albumin (500 nmol/L, 90 minutes)+0.1 µmol/L antisense RAGE [PS]ODNs.

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 {lambda}-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 1Up; 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-{kappa}B site (5'-AGGGTCCCGGAGTAGTTTCCTACCGGGA-3'), the NF-{kappa}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.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
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-{kappa}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 1Up) 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 2aUp). After 48 hours, lysosomal localization became evident, indicating that degradation had already started (Fig 2aUp). Ninety-six hours after incubation, [PS]ODNs were no longer detectable (Fig 2aUp). Gel electrophoresis demonstrated stability of [PS]ODNs in the culture medium for at least 36 hours (Fig 2bUp). 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 2cUp). 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 2dUp). No RAGE transcripts were detected in unstimulated human ECs (Fig 2dUp). In contrast, strong induction of RAGE mRNA synthesis was observed in AGE albumin–induced cells (Fig 2dUp). Sense [PS]ODNs did not alter AGE albumin–induced RAGE transcription (Fig 2dUp), whereas a successful downregulation of AGE albumin–dependent RAGE mRNA synthesis could be demonstrated after 48 hours of preincubation with antisense [PS]ODNs (Fig 2dUp). 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 2dUp). 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-{kappa}B Activation
Recently it has been demonstrated that inhibition of RAGE by RAGE-specific antibodies inhibits AGE albumin–induced NF-{kappa}B activation.9 41 EMSAs evidenced that AGE albumin induced binding of NF-{kappa}B to its consensus motif (Fig 3aDown, lane 1 versus 2) and to the TF-derived NF-{kappa}B–like site (Fig 3bDown, lane 1 versus 2), previously characterized as NF-{kappa}B(p65/c-Rel).29 38 39 40 NF-{kappa}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 3aDown and 3bDown, lane 4). Preincubation with sense RAGE [PS]ODNs did not significantly affect NF-{kappa}B–binding activity (Fig 3aDown and 3bDown, lane 3). When 0.1 µmol/L antisense RAGE [PS]ODNs was added directly to the binding reaction, no inhibition of NF-{kappa}B binding to its respective binding site was observed (Fig 3aDown, lane 5). Thus, direct physical interactions of the antisense [PS]ODN with NF-{kappa}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 3cDown).



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  		Figure 3. Antisense RAGE [PS]ODNs inhibit AGE albumin–induced binding activity of transcription factor NF-{kappa}B. BAECs were left untreated (lane 1), stimulated with AGE albumin (500 nmol/L) for 30 minutes (lane 2), or preincubated for 48 hours with 0.1 µmol/L sense (AGE+S; lane 3) or 0.1 µmol/L antisense (AGE+AS; lane 4) RAGE [PS]ODNs before AGE albumin was added for 30 minutes. Nuclear extracts were prepared and assayed for NF-{kappa}B– or SP-1–binding activity, monitored by EMSA. Radioactive-labeled oligonucleotides spanning (a) consensus NF-{kappa}B recognition motif, (b) TF-specific NF-{kappa}B(p65/c-Rel) site, or (c) SP-1 consensus site were incubated with equal amounts of nuclear extracts, and complexes formed were separated onto nondenaturing 5% polyacrylamide gels. In separate binding reaction, nuclear extract from AGE albumin–induced BAECs was incubated simultaneously with 0.1 µmol/L unlabeled antisense RAGE [PS]ODN and radiolabeled consensus NF-{kappa}B–oligonucleotide (a, lane 5). To confirm NF-{kappa}B binding, observed shifts were competed with 160-fold molar excess of cold consensus NF-{kappa}B oligonucleotides (a, lane 6; b, lane 5). Arrows show position of NF-{kappa}B complexes (a, b), SP-1 complex (c), and nonspecific formed complexes (n.s.). To determine extent of inhibition, signals were evaluated by laser densitometry; signal intensity for each complex formed is given below autoradiograph.

Western blot analysis showed that reduced binding activity of NF-{kappa}B was due to suppressed translocation of NF-{kappa}B proteins from the cytoplasm (Fig 4Down, lanes 1 through 4) into the nucleus (Fig 4Down, lanes 5 through 8). Although only a little NF-{kappa}B(p65) was detected in the nucleus of control ECs (Fig 4aDown, lane 5), incubation with AGE albumin (30 minutes, 500 nmol/L) resulted in increased translocation of NF-{kappa}B(p65) (Fig 4aDown, lane 6). This was also evident in ECs pretreated with sense RAGE [PS]ODNs (Fig 4aDown, lane 7). Only a little NF-{kappa}B(p65) was detected in ECs that had been incubated in the presence of antisense RAGE [PS]ODNs (Fig 4aDown, lane 8). The effect for NF-{kappa}B(p50) was less pronounced because, consistent with previous studies,59 considerable amounts of NF-{kappa}B(p50) were already present in uninduced nuclear extracts (Fig 4bDown, lanes 5 through 8), whereas the presence of its precursor p105 was restricted to the cytoplasm (Fig 4bDown, 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 4bDown, lanes 6 and 7). As reported previously,59 the expected 75-kD protein NF-{kappa}B c-Rel was detected mainly in the cytoplasm of ECs, whereas almost no 75-kD signal was found in the nucleus (Fig 4cDown, lanes 5 through 8). However, an additional 62-kD protein was selectively detected by anti–c-Rel antibodies in cytoplasmic and nuclear extracts (Fig 4cDown, lanes 1 through 8). Translocation of this protein was inducible by AGE albumin in untreated or sense RAGE [PS]ODN–treated ECs (Fig 4cDown, lanes 6 and 7), whereas antisense RAGE [PS]ODNS reduced the observed translocation (Fig 4cDown, lane 8). Together with the EMSA data demonstrating AGE albumin–induced binding activity of NF-{kappa}B(p65/ c-Rel) to the TF-derived NF-{kappa}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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  		Figure 4. Antisense RAGE [PS]ODNs inhibit nuclear import of NF-{kappa}B. BAECs were either left untreated (lanes 1 and 5), stimulated with AGE albumin (500 nmol/L) for 30 minutes (lanes 2 and 6), or preincubated for 48 hours with 0.1 µmol/L sense (AGE+S; lanes 3 and 7) or 0.1 µmol/L antisense (AGE+AS; lanes 4 and 8) RAGE [PS]ODNs before AGE albumin induction was performed. Cytoplasmic (left) and nuclear (right) extracts were prepared and assayed in Western blot analysis for presence of NF-{kappa}Bp65 (a), NF-{kappa}Bp50 (b), or NF-{kappa}B–c-Rel and c-Rel–related protein complexes (c). Arrows show NF-{kappa}B–specific complexes.

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 5aDown), was completely prevented when ECs were preincubated with 0.1 µmol/L antisense RAGE [PS]ODNs 48 hours before stimulation (Fig 5bDown). 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 5cDown) but was downregulated thereafter (Fig 5cDown). Heat-inactivated AGE albumin that served as control did not induce TF (Fig 5dDown). 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 5eDown). Sense RAGE [PS]ODNs, sense TF [PS]ODNs, or antisense HMWK [PS]ODNs had no effect on AGE albumin–induced TF activity (Fig 5eDown).



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  		Figure 5. Antisense RAGE [PS]ODNs inhibit AGE albumin–induced TF activity. a, BAECs were induced with AGE albumin (500 nmol/L) for times indicated or b, preincubated for 48 hours with 0.1 µmol/L antisense RAGE [PS]ODNs before induction with AGE albumin (500 nmol/L). c, AGE albumin (500 nmol/L) and 0.1 µmol/L antisense RAGE [PS]ODNs were added simultaneously; d, BAECs were incubated with heat-inactivated AGE albumin (500 nmol/L); or e, BAECs were left untreated or preincubated with 0.1 µmol/L sense RAGE [PS]ODNs (sense RAGE), 0.1 µmol/L sense TF [PS]ODNs (sense TF), or 0.1 µmol/L antisense HMWK [PS]ODNs (antisense HMWK) before being induced with AGE albumin (500 nmol/L). TF procoagulant activity was determined with a one-stage clotting assay and is expressed as pg TF/106 cells±SD. Each result shown represents mean of three independent experiments performed in triplicate.

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 6Down) were performed. Compared with unstimulated cells (Fig 6Down, top, lane 1), AGE albumin induced new synthesis of TF mRNA (Fig 6Down, top, lane 2). Preincubation with 0.1 µmol/L sense RAGE [PS]ODNs (Fig 6Down, top, lane 3) had no effect, whereas 0.1 µmol/L antisense RAGE [PS]ODNs directly reduced AGE albumin–dependent TF transcription (Fig 6Down, top, lane 4). The household gene GAPDH (Fig 6Down, 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 6Down, bottom). RT-PCR with TF-specific primers (performed with the same RNA template as used for the RAGE-specific RT-PCR shown in Fig 2cUp) 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 7aDown). To evaluate the RT-PCR results, competitive PCR was performed in the presence of decreasing amounts of a TF-specific competitor plasmid (Fig 7bDown) 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 7bDown). 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 7bDown). 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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  		Figure 6. Antisense RAGE [PS]ODNs reduce AGE albumin newly transcribed TF mRNA. BAECs were left untreated (control, lane 1), induced with AGE albumin for 90 minutes (lane 2), or preincubated for 48 hours with 0.1 µmol/L sense (AGE+S, lane 3) or 0.1 µmol/L antisense (AGE+AS, lane 4) RAGE [PS]ODNs before AGE albumin induction. Nuclei were prepared and nuclear run-on experiments were performed to allow in vitro synthesis of [{alpha}-32P]UTP–labeled mRNA. RNA was hybridized to Hybond N nylon filters onto which cDNAs for TF and household gene GAPDH had been fixed. Signals obtained for TF and GAPDH were evaluated by laser densitometry and are given below each autoradiograph. Method-dependent variations were normalized by dividing TF signal by signal obtained for GAPDH; ratio is given at bottom of figure.



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  		Figure 7. Antisense RAGE [PS]ODNs downregulate AGE albumin–induced TF transcription. a, HUVECs were left untreated (lanes 1 and 2) or preincubated in presence of 0.1 µmol/L sense (S; lane 3) or 0.1 µmol/L antisense (AS; lane 4) RAGE [PS]ODNs before AGE albumin stimulation (500 nmol/L) was performed for 90 minutes. TF mRNA transcription was monitored by RT-PCR in same RNA preparations in which RAGE transcription was determined earlier (Fig 2cUp). RT-PCR reactions were performed independently by two investigators (A.B., T.I.) with identical results; one representative experiment is shown (top). RT-PCR reactions of household gene GAPDH served as control (bottom). Lane 1, 100-bp DNA ladder; lane 2, unstimulated; lane 3, AGE albumin (500 nmol/L, 90 minutes); lane 4, AGE albumin (500 nmol/L, 90 minutes)+0.1 µmol/L sense RAGE [PS]ODNs; lane 5, AGE albumin (500 nmol/L, 90 minutes)+0.1 µmol/L antisense RAGE [PS]ODNs. b, To evaluate PCR results, quantitative RT-PCR was performed.49 After RT, cDNA probes were subjected to GAPDH-specific PCR and thereafter adjusted to same amount of GAPDH-cDNA (data not shown). For each preparation, TF-specific PCR was then performed in presence of 2 ng, 2 pg, and 500 fg of the TF control plasmid pGEM-TFay49 containing a nonmammalian DNA sequence flanked by TF sequences that match TF-specific PCR primers.49 Amplification detected the 0.3-kb TF-specific signal and competing 0.7-kb control fragment of pGEM-TFay (data not shown). Signals were evaluated by laser densitometry, and ratio of intensity of TF-specific signal to intensity of competitor-derived signal was plotted against input of competitor DNA.49

Antisense RAGE Oligonucleotides Inhibit the AGE Albumin–Mediated NF-{kappa}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-{kappa}B–like site was present (Fig 8aDown). Loss of the NF-{kappa}B site resulted in the loss of inducibility (Fig 8aDown). When 0.1 µmol/L antisense RAGE [PS]ODNs was included in the transfection reaction, AGE albumin–mediated TF induction was significantly decreased (Fig 8aDown). 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 8aDown). The observed effect was specific, because antisense RAGE [PS]ODNs only blocked AGE albumin–induced TF expression (Fig 8aDown) but did not reduce LPS-mediated TF induction (Fig 8bDown). Thus, AGE albumin–induced TF expression is at least in part dependent on a RAGE-mediated activation of the transcription factor NF-{kappa}B.



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  		Figure 8. Antisense RAGE [PS]ODNs inhibit TF promoter activity. BAECs were transiently transfected with TF promoter plasmids pHTF(-278)Luc, which spans both AP-1 regions and NF-{kappa}B site (A1-A2-N); plasmid pHTFM4(-278)Luc, which contains only functional TF-derived NF-{kappa}B site (N); or pHTF(-111)Luc, in which AP-1 sites and NF-{kappa}B site were deleted (---). Where indicated, oligonucleotides (0.1 µmol/L) were included in DNA preparation and also added to culture medium after transfection. At 36 hours after transfection, part of cells was either left untreated or induced with 500 nmol/L AGE albumin (a) or 1 µg/µL LPS (b). Luciferase activity was determined in cell lysates and normalized for transfection efficiency to amount of ß-galactosidase activity expressed by cotransfected control plasmid pSV-ß-Gal. Results for AGE albumin induction represent mean of three independent experiments±SD that were performed in triplicate. Results for LPS activation are mean of one representative experiment performed in triplicate. All results are given as relative Luc units expressed as % of pHTF(-278)Luc basal activity. Statistical evaluation of data is given beside graph.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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 2aUp) 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 1Up), 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 8Up), (2) antisense RAGE [PS]ODNs suppressed NF-{kappa}B– but not SP-1–binding activity (Fig 3Up), and (3) RAGE-unrelated [PS]ODNs did not alter AGE albumin–mediated TF induction (Fig 5Up). 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 6Up and 7Up) and on the activity level (Fig 5Up). 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 5cUp). 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 2cUp). 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-{alpha}.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


*    Selected Abbreviations and Acronyms
 
AGE = advanced glycation end product
AP-1 = activator protein-1
BAEC = bovine aortic endothelial cell
EC = endothelial cell
EMSA = electrophoretic mobility shift assay
ß-Gal = ß-galactosidase
HMWK = high-molecular-weight kininogen
HUVEC = human umbilical vein endothelial cell
LPS = lipopolysaccharide
NF-{kappa}B = nuclear factor-{kappa}B
ODN = oligodeoxynucleotide
PCR = polymerase chain reaction
[PS]ODN = phosphorothioate oligodeoxynucleotide
RAGE = receptor for AGE
RT = reverse transcription
TBE = Tris-borate-EDTA
TBS = Tris-buffered saline
TF = tissue factor
VCAM-1 = vascular adhesion molecule-1


*    Acknowledgments
 
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.


*    Footnotes
 
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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