Effect of Advanced Glycation End Product–Modified Albumin on Tissue Factor Expression by Monocytes
Role of Oxidant Stress and Protein Tyrosine Kinase Activation
Abstract Diabetes is associated with a hypercoagulable state that contributes to macrovascular complications, including cardiovascular events. The glycation reaction, a consequence of chronic hyperglycemia, has also been implicated in the pathogenesis of diabetic complications. Glycated proteins have receptors on monocytes and generate reactive oxygen species that can regulate the expression of a number of genes. As abnormal monocyte expression of tissue factor (TF), the main initiator of the coagulation cascade, is responsible for thrombosis in a number of clinical settings, we studied the effect of glycated albumin on monocyte TF expression. Mononuclear cells were incubated with glycated albumin for 24 hours, and monocyte TF activity was measured with a plasma recalcification time assay; TF antigen was measured by ELISA and TF mRNA by RT-PCR. Glycated albumin induced blood monocyte expression of the procoagulant protein TF at the mRNA level. Oxidative stress appeared to be involved in this effect, as the antioxidant N-acetylcysteine diminished TF mRNA accumulation in stimulated monocytes. Hydroxyl radicals, which may be generated inside cells from H2O2 via the Fenton reaction, also appeared to be involved in this effect, as hydroxyl radical scavengers downregulated TF activity and antigen levels (but not TF mRNA). Finally, the involvement of activated protein tyrosine kinase in the transmission of the signal from the membrane to the nucleus was suggested by the inhibitory effect of herbimycin A. These results point to a new mechanism for the hypercoagulability often described in diabetic patients and suggest that antioxidants or protein tyrosine kinase inhibitors might be of therapeutic value in this setting.
- Received December 31, 1996.
- Accepted April 15, 1997.
Advanced glycation end products are nonenzymatically glycated adducts that form on proteins with long half-lives and accumulate at an accelerated rate in diabetes. A receptor system for AGEs has been described on monocyte/macrophages; it includes a 60-kD and a 90-kD protein identified in rat liver homogenates,1 the macrophage scavenger receptor,2 and a 35-kD protein named RAGE, which belongs to the immunoglobulin superfamily. RAGE is also expressed by endothelial cells and smooth muscle cells, as well as by certain neurons and mesangial cells.3
In monocytes, the interaction of AGE-modified proteins with AGE receptors triggers the synthesis and release of cytokines, particularly interleukin-1β and TNF-α.4 AGEs also induce human monocytes to produce insulin-like growth factor and platelet-derived growth factor.5 6 The interaction of AGEs with RAGE has also been shown to result in oxidant stress, based on the generation of thiobarbituric acid-reactive substances, induction of heme oxygenase type I and activation of NF-κB.7 The signal pathways responsible for gene expression are not completely understood. AGEs themselves have been shown to generate reactive oxygen intermediates (mainly superoxide anion and hydrogen peroxide [H2O2]) in vitro and in vivo.8 Once generated in proximity to the cell membrane, H2O2 can penetrate rapidly within the cell, whereas other activated oxygen species cannot. Inside the cell, H2O2 can react with iron or copper in the Fenton reaction; the resulting hydroxyl radical may be an important mediator of the H2O2 action on cells.
TF, a transmembrane glycoprotein, is the receptor for plasma factors VII and VIIa. The TF-VIIa complex is now considered as the main initiator of the coagulation cascade. TF is not normally expressed by circulating monocytes, but it can be synthesized in response to a number of inflammatory molecules. Abnormal TF expression by monocytes is responsible for the thrombosis occurring in sepsis, inflammatory diseases, and acute coronary syndromes.9 Excessive activation of coagulation has often been observed in diabetic patients10 and may play a role in the onset of vascular complications.
The first aim of this study was to assess whether AGEs induce monocytes to produce and express TF. The second aim was to determine whether reactive oxygen intermediates, especially hydroxyl radicals, are involved in this effect. The role of PTK activation was also evaluated by using herbimycin A, an inhibitor of these enzymes.
Preparation of AGE-Alb
AGE-Alb was prepared by incubating human serum albumin (Biotransfusion) in PBS (150 mmol/L, pH 7.4) with 250 mmol/L d-glucose at 37°C for 6 weeks in the presence of 1.5 mmol/L PMSF, 0.5 mol/L EDTA, 2 μg/mL aprotinin (Trasylol, Bayer), 0.5 μg/mL leupeptin, and antibiotics (100 μg/mL penicillin and 143 μg/mL streptomycin). Control albumin was incubated in the same conditions without glucose. At the end of the incubation period, both solutions were extensively dialyzed against PBS. The endotoxin content was measured in a chromogenic assay (Coatest Endotoxin, Chromogenix) and was less than 0.05 ng/mL. Glucose content was less than 3.5 mmol/L. AGE-specific fluorescence was determined at 460 nm after excitation at 390 nm by using an AMINCO SPF 500 ratio spectrofluorometer as described by Kirstein et al.5 AGE-Alb contained 25 AGE units per 10 mg of protein, while native albumin contained 0.6 AGE units per 10 mg of protein.
Peripheral blood cells were obtained from healthy donors by venipuncture, using EDTA as anticoagulant. Mononuclear cells isolated by the Ficoll-Hypaque gradient technique contained 20% to 30% monocytes, 60% to 70% lymphocytes, and fewer than 10 platelets per monocyte.11 Mononuclear cell suspensions (1×106 monocytes per milliliter) were incubated with a fivefold dilution of AGE-Alb or control albumin, for 24 hours in RPMI-1640 supplemented with penicillin, streptomycin, pyruvate, l-glutamine, and nonessential amino acids (BioWhittaker). The polypropylene tubes (Polylabo) used for incubating the cells did not allow monocytes to adhere. As monocytes are extremely sensitive to traces of contaminating endotoxin, polymyxin B (2 μg/mL) was added to the culture medium in all experiments. It was verified that this concentration of polymyxin B did not alter basal TF activity. The stimulating effect of phorbol myristate acetate was not modified either. At the end of the incubation, the medium was discarded and cells were washed and stored for less than 1 week at −80°C until TF assay or RNA extraction. Cell viability was respected in all the experimental conditions, as measured by lactate dehydrogenase release or trypan blue exclusion.
Cells were lysed and TF activity was quantified in a one-step plasma recalcification time assay.12 To confirm that procoagulant activity was TF, residual procoagulant activity was measured in some experiments, after incubating the cell lysate for 10 minutes with a pool of neutralizing anti-TF monoclonal antibodies (TF8-5G9, TF8-6B4, and TF9-9C3) generously provided by T.S. Edgington (the Scripps Research Institute, La Jolla, Calif). Neutralizing antibodies were used at a concentration of 10 μg/mL. TF antigen was determined by an ELISA method (Imubind TF kit, American Diagnostica Inc) according to manufacturer’s instructions, with minor modifications.11
RNA was extracted from mononuclear cells with the RNA PLUS reagent (Bioprobe Systems). Five micrograms of total RNA was incubated for 5 minutes at 65°C with 3 μmol/L of random hexamers (Perkin-Elmer). After 10 minutes at room temperature, reverse transcription was carried out for 1 hour at 37°C in a 50-μL reaction volume containing 50 U of murine leukemia virus reverse transcriptase (Perkin-Elmer), 40 U of RNAse inhibitor (Perkin-Elmer), and all four deoxyribonucleoside triphosphates (dNTPs, 1 mmol/L each) in reaction buffer (50 mmol/L KCl, 10 mmol/L Tris HCl, pH 8.3, 5 mmol/L MgCl2). PCR reactions were run in a Trio-Thermoblock apparatus (Biometra) with 2 μL of the retrotranscription reaction product in a total volume of 100 μL with 0.2 mmol/L each dNTP, 0.4 μmol/L each primer, and 1 U of Taq DNA polymerase (ATGC). The reaction cycles were 94°C for 1.5 minutes, 62°C for 3 minutes, and 72°C for 3 minutes (30 cycles). Sense and antisense primers for TF were those described by Bartha et al.13 Semiquantitative competitive RT-PCR was used to correct for variations in amplification efficiency in each reaction and to calculate relative changes in mRNA levels. This was done using the PCR MIMIC construction kit (Clontech Laboratories), which enables a PCR mimic to be generated and used as a competitive internal standard in PCR amplification. The PCR mimic consists of a nonhomologous DNA fragment that bears the recognition sequences for TF primers at each extremity. The PCR mimic and target template thus compete for the same primers in the same reaction. Equal amounts of the mimic were included in each PCR reaction. Amplification products were subjected to electrophoresis in 2% agarose gels containing ethidium bromide and were photographed with Polaroid 665 negative film. RNA was quantitated by scanning the bands with a Macintosh One-Scanner densitometer; the density of each band was normalized to the density of the mimic band and plotted in arbitrary units.
Results, expressed as mean±SEM, were compared using nonparametric tests: the Kruskal-Wallis test was used for analysis of variance, while the Mann-Whitney U test and the Wilcoxon test were used for unpaired and paired data, respectively.
Effect of AGE-Alb on TF Expression by Mononuclear Cells
To study the kinetics of the effect of AGE-Alb on monocyte TF production, the cells were incubated with AGE-Alb for 4, 12, 24, and 48 hours. As shown in Fig 1⇓, inset, TF activity peaked at 24 hours. Subsequent experiments were thus performed at 24 hours. Incubation of mononuclear cell suspensions for 24 hours with AGE-Alb (1 vol in 4 vol of culture medium) was associated with a 5.6-fold increase in procoagulant activity expressed by viable cells (from 30 with control albumin to 170 mU/106 monocytes, n=2); in lysed cells, AGE-Alb induced a significant (P<.05) 5.2-fold increase in procoagulant activity (from 395±90 with control albumin to 2076±336 mU/106 monocytes; Fig 1A⇓). Procoagulant activity was identified as TF, as more than 99% was abolished by a cocktail of neutralizing monoclonal antibodies directed against TF (data not shown). Moreover, when cells were treated with AGE-Alb, TF antigen levels increased from 147±70 pg/106 monocytes (control albumin) to 896±198 pg/106 monocytes (n=6, P<.05; Fig 1A⇓). A good correlation was found between TF activity and TF antigen levels (r=.6, P<.05, y=0.4 x+113.8). The addition of increasing concentrations of AGE-Alb (1:10 and 1:5) to the incubation medium led to a concentration-dependent increase in TF activity (590 and 2000 mU/106 monocytes, respectively, mean of two experiments, versus 85 and 396 with control albumin at the same concentrations). The effect of costimulating the cells with 200 U/mL recombinant TNF-α (Genzyme) was then evaluated. The cells were incubated for 20 hours with AGE-Alb or control albumin, followed by a 4-hour incubation with or without TNF. The addition of TNF had a direct effect on the induction of TF activity in control cells (which increased from 200 to 950 mU/106 monocytes); when TNF was added to AGE-Alb–treated cells the TF activity level increased from 1050 to 2000 mU/106 monocytes, an increase that corresponds to the sum of the activity induced by AGE-Alb and TNF separately (n=2).
To assess the level of action of AGE-Alb, TF mRNA was studied after reverse transcription and amplification by PCR. After treatment with AGE-Alb for 24 hours, TF transcripts were readily detected as the expected band of 235 bp (1.1 arbitrary units), which remained faint (0.2 arbitrary units) in cells incubated with unmodified albumin (Fig 1B⇑). As the primers for the TF gene were separated by one intron (intron 5), the observed band could only have originated from spliced mRNA (not from genomic DNA). To determine whether AGE-Alb induction of TF mRNA required de novo protein synthesis, monocytes were stimulated in the presence of the translational inhibitor cycloheximide. Cycloheximide (10 μg/mL) completely suppressed AGE-Alb–induced TF mRNA accumulation, showing that the induction mechanisms elicited by AGE-Alb required de novo protein synthesis.
Effect of the Antioxidant N-Acetylcysteine on TF Expression
As the interaction of AGEs with RAGE has several effects in monocytes through the enhancement of cellular oxidant stress, the role of reactive oxygen species in AGE-induced TF production was assessed using the antioxidant N-acetylcysteine. N-Acetylcysteine acts mainly by increasing levels of the endogenous antioxidant reduced glutathione, and consequently increases H2O2 metabolism by the glutathione peroxidase system. Incubation in the presence of N-acetylcysteine resulted in concentration-dependent inhibition of TF activity (Fig 2A⇓) and TF antigen expression (Fig 2B⇓); (>90% suppression at 30 mmol/L). Moreover, TF mRNA was almost completely suppressed at this concentration (Fig 2C⇓).
Effect of Hydroxyl Radical Scavengers
The role of hydroxyl radicals, which can be generated intracellularly via the interaction of H2O2 with iron or copper, was evaluated by using two hydroxyl radical scavengers: TU and DMTU. Mononuclear cells were incubated with increasing concentrations (0.5 to 25 mmol/L) of TU or DMTU. These agents caused a concentration-dependent reduction in TF procoagulant activity (Fig 3A⇓ and 3B⇓). At 25 mmol/L, residual TF activity was 49±8% with TU, 16±3% with DMTU, and 114±12% with 25 mmol/L urea (used as a molarity control). No direct effect of these compounds on the TF assay occurred, as their addition to the culture medium at 24 hours did not alter the TF activity expressed by the cells. The effect of these inhibitors on TF protein expression was analyzed by means of ELISA. The residual induction of TF antigen by AGE-Alb was 41±15% and 15±5.9%, at 25 mmol/L TU (n=5) or DMTU (n=6), respectively, while residual TF activity was 55±9.9% and 20±8%, respectively, in paired experiments. We concluded that TF antigen expression fell in parallel with TF activity. By contrast (Fig 3C⇓), TF mRNA expression induced by AGE-Alb was not inhibited when cells were simultaneously treated with TU or DMTU.
Effect of Herbimycin A, an Inhibitor of PTK
As H2O2 activates PTK in B cells, we tested the effect of herbimycin A on AGE-Alb–induced TF expression. As shown in Fig 4A⇓, herbimycin inhibited AGE-Alb TF activity in a concentration-dependent manner; moreover, 1 μmol/L herbimycin A completely suppressed AGE-Alb–induced accumulation of TF antigen and TF mRNA (Fig 4B⇓).
Diabetes is associated with a hypercoagulable state,10 14 which contributes to macrovascular complications, including cardiovascular events. The glycation reaction, a consequence of chronic hyperglycemia, has been implicated in the pathogenesis of diabetic complications. Schiff-base adducts and Amadori products produced through the glycation reaction generate reactive oxygen species (including superoxide anion and H2O28 ) that may regulate the expression of a number of genes.15 16 The results of this study show that glycated albumin induces blood monocyte expression of the procoagulant protein TF at the mRNA level. Oxidative stress may have a role in this effect, as the antioxidant N-acetylcysteine attenuated the accumulation of TF mRNA in AGE-Alb–stimulated monocytes. Hydroxyl radicals, which may be generated inside the cell from H2O2 via the Fenton reaction, also appear to be involved in this effect, as hydroxyl radical scavengers downregulated the expression of TF activity and antigen, but not that of TF mRNA. Finally, the involvement of activated PTK in the transmission of the signal from membrane to nucleus was suggested by the inhibitory effect of herbimycin A.
Incubation of the cells with AGE-Alb led to a time- and concentration-dependent increase in TF activity, which was maximal at 24 hours and decreased thereafter. The TF expression of intact cells was about 10% that of lysed cells. This distribution, which is similar to that obtained with other well-studied agonists such as endotoxin, is considered to reflect the potentiation of TF activity by lysis-induced exposure of anionic phospholipids.17 This 5.2-fold increase in TF activity was accompanied by a 6-fold increase in TF antigen and a similar increase in TF mRNA. Endotoxin contamination had no role in this effect, as all albumin preparations, reagents, and media were discarded if more than 0.05 ng/mL was detected; moreover, polymyxin B was added to all the culture media. Costimulation of the cells with TNF-α and AGE-Alb was associated with a response corresponding to the sum of the activity induced separately by the two agents. Esposito et al18 have reported a similar effect of glycated albumin on binding to endothelial cells; in these cells, TF activity was maximal after 60 hours of incubation with glycated albumin. In these cells, however, TNF and AGE-Alb had a synergistic effect. Interaction of AGEs with mononuclear phagocytes induces an activated phenotype manifested by induction of mRNA corresponding to several inflammatory cytokines and growth factors, including interleukin 1β, TNF,4 19 and insulin-like growth factor.5 TF mRNA accumulation induced by AGE-Alb was completely suppressed by cycloheximide pretreatment of the cells, suggesting the involvement of newly synthesized proteins in this effect. Interleukin 1β and TNF are known TF inducers and might at least partly mediate TF production.20
The antioxidant N-acetylcysteine completely suppressed the induction of TF activity, TF antigen, and TF mRNA expression after 24 hours of monocyte incubation with glycated albumin. This observation strongly suggested that oxidant stress was responsible for TF gene expression. N-Acetylcysteine, a sulfhydryl-group donor, is easily transported into the cell, where it is deacylated and increases the thiol pool (primarily reduced glutathione). Glutathione peroxidase is the key peroxide detoxification system, and the availability of reduced glutathione is essential for this role.21 Previous studies have shown that glycated proteins can generate reactive oxygen intermediates7 8 such as superoxide anion, which is further reduced to H2O2. Monocyte interactions with AGEs appear to be mediated at least in part by RAGE and a lactoferrin-like polypeptide. RAGE is an integral membrane protein that belongs to the immunoglobulin superfamily.3 22 Lactoferrin-like polypeptide binds noncovalently and with high affinity to the extracellular domain of RAGE, and the resulting complex also binds AGEs. Yan and coworkers7 showed that, on binding to RAGE, AGE-Alb induces oxidant stress characterized by generation of thiobarbituric acid reactive substances, activation of NF-κB, and induction of heme oxygenase mRNA. This effect was inhibited by superoxide dismutase and catalase, strongly suggesting a central role of H2O2 via NF-κB activation. In keeping with this sequence of events, a nonconsensus NF-κB sequence is present in the LPS response element of the TF promoter and is involved in transcriptional activation of the gene in response to LPS and cytokines.23
Since Schreck and Bauerle24 first postulated that certain oxygen free radicals may play the role of intracellular messengers, the regulation of the TF gene by oxidant stress has been reported in several studies. In cultured endothelial cells, oxygen free radicals induce TF messenger RNA transcription25 and expression of TF procoagulant activity. Furthermore, isolated rabbit hearts exposed to oxygen free radicals and hearts subjected in vivo to ischemia and reperfusion, showed a marked increase in TF activity that was abolished by oxygen radical scavengers such as superoxide dismutase. The involvement of oxygen free radicals in LPS- and cytokine-induced TF expression by endothelial cells has also been suggested, as PDTC, a specific inhibitor of the NF-κB pathway, abrogated this effect at the mRNA level.26 PDTC scavenges free radicals and chelates transition metal ions. PDTC and N-acetylcysteine inhibited LPS-induced TF activity in monocytes/macrophages, but neither of these agents reduced LPS-stimulated TF mRNA accumulation, thereby suggesting a posttranscriptional mechanism (impaired translation, or degradation of newly translated protein).27 These results also suggest that different regulatory mechanisms might be at work in monocytes and endothelial cells. Our results are in keeping with a report that TNF production by mononuclear phagocytes in response to glycated albumin is inhibited by N-acetylcysteine.19 This latter study also clearly showed that TNF production was dependent on AGE binding to RAGE.
However, it is not clear which form of activated oxygen species underlies the effect of AGEs on the signaling pathway that leads to activation of NF-κB and gene expression. The reactive oxygen intermediates generated outside the cell, in close contact with RAGE-bound AGEs, lead to the formation of H2O2, which, unlike other activated oxygen species, readily crosses cell membranes. Once inside the cell, H2O2 can react with iron or copper in the Fenton reaction to form the hydroxyl radical, which may be an important mediator of H2O2 action. Our results with two hydroxyl radical scavengers, TU and DMTU, suggest that induction of TF activity and antigen expression by AGEs is dependent on hydroxyl radical formation. At 25 mmol/L TU or DMTU, the residual TF activity was 49% and 16%, respectively, relative to values obtained without the inhibitors. TF activity and antigen levels fell in parallel, suggesting that the observed effect was secondary to decreased production of the protein. TU and DMTU did not, however, alter TF mRNA expression, pointing to a posttranscriptional mechanism. TU and DMTU are low-molecular-weight, membrane-permeable hydroxyl scavengers. A suppressive effect of hydroxyl radical scavengers has been reported28 on the expression of interleukin-8 in response to LPS, PHA, and immune complexes; in this case, however, mRNA levels fell in parallel to those of the released cytokine.
Given the growing body of evidence that reactive oxygen species modulate PTK activities,29 we studied the effect of herbimycin A, an inhibitor of PTK, on AGE-Alb–induced TF expression. Herbimycin A clearly reduced both TF activity and TF mRNA accumulation in response to AGE-Alb. This is consistent with the fact that H2O2 induces tyrosine phosphorylation and inhibits protein tyrosine phosphatase activities in several cell types.29 All protein tyrosine phosphatases have a conserved cysteine residue in their catalytic domain, which must be in reduced form for full activity; these enzymes are thus strongly dependent on the intracellular redox state, which is controlled by glutathione. Taken together, our results suggest that the effect of glycated albumin on TF expression by monocytes might be the result of two associated mechanisms. H2O2 (or a metabolite) might act at the mRNA level (increasing mRNA transcription and/or stability) via the activation of PTK, as indirectly suggested by the effect of N-acetylcysteine and herbimycin A; hydroxyl radicals, by contrast, would act through a posttranscriptional mechanism, as their scavenging does not alter TF mRNA expression.
These results also point to a new mechanism for the hypercoagulability often described in patients with diabetes. The induction of TF expression on circulating blood monocytes by glycated proteins confers a procoagulant phenotype that might partly be responsible for the thrombotic complications in these patients. Indeed, recent observations strongly suggest that thrombosis is initiated via the TF pathway in vivo. TF antigen and active TF have been identified in human coronary atheroma30 and were shown to be associated with plaque thrombi, which play an important role in the pathogenesis of acute coronary syndromes. TF-producing monocytes and macrophages of the atherosclerotic plaque31 may represent therapeutic targets for the prevention of thrombotic complications. Furthermore, antioxidants or PTK inhibitors32 might be of therapeutic value in these patients.
Selected Abbreviations and Acronyms
|AGE||=||advanced glycation end product|
|ELISA||=||enzyme-linked immunosorbent assay|
|PTK||=||protein tyrosine kinase|
|RAGE||=||receptor for AGE|
|RT-PCR||=||reverse-transcribed polymerase chain reaction|
|TNF||=||tumor necrosis factor|
These studies were supported by the Fondation de France and by Hoechst Laboratories. Anti-TF monoclonal antibodies TF8-5G9, TF8-6B4, and TF9-9C3 were generously provided by T.S. Edgington of the Scripps Research Institute, La Jolla, Calif.
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