Superoxide Dismutase Inhibits the Expression of Vascular Cell Adhesion Molecule-1 and Intracellular Cell Adhesion Molecule-1 Induced by Tumo
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《动脉硬化血栓血管生物学》
From the Institute of Clinical Medicine (S.-J.L., Y.-H.C., J.-W.C.), Cardiovascular Research Center (S.-J.L.), and the Institute of Anatomy and Cell Biology (Y.-Y.H., H.-H.K., Y.-L.C.), National Yang-Ming University; the Institute of Biomedical Science (S.-K.S., K.-B.T.), Academia Sinica; and the Division of Cardiology (S.-J.L., J.-W.C.), Taipei Veterans General Hospital, Taipei, Taiwan, Republic of China.
Correspondence to Dr Yuh-Lien Chen, Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, No. 1, Section 1, Ren-Ai Rd, Taipei, 100, Taiwan. E-mail ylchen@ha.mc.ntu.edu.tw
Abstract
Objective— Expression of adhesion molecules on endothelial cells and subsequent leukocyte recruitment are critical early events in the development of atherosclerosis. We tried to study possible effects of Cu/Zn superoxide dismutase (SOD) on adhesion molecule expression and its underlying mechanism in the prevention and treatment of cardiovascular disorders.
Methods and Results— Human aortic endothelial cells (HAECs) were transfected with adenovirus carrying the human SOD gene (AdSOD) to investigate whether SOD expression in HAECs attenuated tumor necrosis factor (TNF)-–induced reactive oxygen species production and adhesion molecule expression and to define the mechanisms involved. SOD expression significantly suppressed TNF-–induced expression of vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1 and reduced the binding of the human neutrophils to TNF-–stimulated HAECs. SOD expression suppressed c-JUN N-terminal kinase and p38 phosphorylation. It also attenuated intracellular superoxide anion production and NADPH oxidase activity in TNF-–treated HAECs.
Conclusions— These results provide evidence that SOD expression in endothelial cells attenuates TNF-–induced superoxide anion production and adhesion molecule expression, and that this protective effect is mediated by decreased JNK and p38 phosphorylation and activator protein-1 and nuclear factor B inactivation. These results suggest that SOD has antiinflammatory properties and may play important roles in the prevention of atherosclerosis and inflammatory response.
Superoxide dismutase overexpression in endothelial cells attenuates tumor necrosis factor-–induced superoxide anion production and adhesion molecule expression, and this effect is mediated by decreased JNK and p38 phosphorylation and AP-1 and nuclear factor B inactivation. These results suggest that superoxide dismutase may play an important role in the prevention of atherosclerosis and inflammatory response.
Key Words: superoxide dismutase ? atherosclerosis ? endothelial cell ? adhesion molecule ? MAPKs
Introduction
Vascular endothelial cells (ECs) are very sensitive to oxidative damage mediated by superoxide anion () released from the endothelium itself and from inflammatory cells, and reactive oxygen species, such as , are implicated in the pathogenesis of cardiovascular disorders.1 Superoxide dismutase (SOD), an antioxidant enzyme, may be useful in the augmentation of antioxidant defenses in the endothelium.2 However, the role of SOD in the protection of cells against oxidative injury is controversial. A protective role for SOD was proposed by some investigators, because addition of SOD to cultures has been reported to prevent EC-mediated low-density lipoprotein (LDL) oxidation,3 decrease reoxygenation-induced cellular injury and intercellular cell adhesion molecule (ICAM)-1 expression in ECs,4 and prevent apoptosis of ECs in the presence of free radicals.5 In contrast, others have reported that addition of SOD does not inhibit EC-induced LDL oxidation6 or hyperoxia-induced ICAM-1 expression in ECs.7 The effects produced by addition of exogenous SOD do not firmly establish the role of SOD in ECs, because the added protein may contain impurities; in addition, both SOD and the EC cell surface are negatively charged, which may impede the entry of SOD into the cell. To overcome these problems, several attempts have been made to overexpress SOD enzyme in various cell types and examine its effect in preventing oxidation-related damage.8–10 Transient overexpression of Cu/Zn SOD was highly effective in reducing EC-induced LDL oxidation.8 However, the effect of SOD expression on human ECs exposed to inflammatory cytokines still remains unclear.
Expression of cell adhesion molecules, such as vascular cell adhesion molecule (VCAM)-1 and ICAM-1, on ECs represents one of the earliest pathological changes in immune and inflammatory diseases, such as atherosclerosis.11 Increased expression of adhesion molecules by ECs in human atherosclerotic lesions may lead to further recruitment of leukocytes to atherosclerotic sites. Possible effects of SOD on adhesion molecule expression could play a key role in the prevention and treatment of cardiovascular disorders. We were therefore interested in understanding the underlying mechanism of action of SOD on ECs and determining whether it stimulates or blocks the expression of adhesion molecules. Induction of adhesion molecule expression by tumor necrosis factor (TNF)- and other inflammatory cytokines involves a wide spectrum of host-responsive systems.12 This requires the activation of multiple signaling molecules in transduction pathways (eg, protein tyrosine kinase, TNF- receptor–associated serine/threonine kinase, Ras, Raf-1, IB kinase, mitogen-activated extracellular-regulated kinase, and mitogen-activated protein kinases (MAPKs).13,14 The pathways involving these molecules may converge or diverge and often have "cross-talk" properties, resulting in a complicated signaling network. Subsequently, the signals are transduced to downstream molecules and activate numerous transcriptional factors, including activator protein-1 (AP-1), nuclear factor B (NF-B), and ATF-2,15 which trigger the expression of a large number of genes encoding inflammatory mediators and adhesion molecules. The effects of TNF- and SOD overexpression on the signaling pathways and the events involved in the expression of adhesion molecules on TNF-–treated human arterial ECs (HAECs) are poorly understood. In this study, adenovirus-mediated gene transfer was used to test the effect of SOD overexpression on the expression of adhesion molecules and MAPKs in HAECs. The results showed that SOD overexpression attenuated the TNF-–induced expression of VCAM-1 and ICAM-1, and that this effect was mediated by blockage of the JNK/p38 MAPK pathway as well as NF-B and AP-1 expression. SOD also significantly inhibited the adhesion of the human neutrophils to TNF-–treated HAECs.
Materials and Methods
Culture of HAECs
HAECs were obtained as cryopreserved tertiary cultures from Cascade Biologics (Portland, Ore) and were grown in EC growth medium.16 The detailed preparation of HAECs is described in the expanded Methods section, available online at http://atvb.ahajournals.org.
Preparation of Recombinant Adenoviruses
cDNA containing the entire human SOD coding sequence was subcloned into the adenovirus shuttle plasmid vector pAd-PGK, which contains the human phosphoglycerate kinase gene promoter and the bovine growth hormone gene polyadenylation signal. The detailed preparation of recombinant adenoviruses is described in the online Methods.
Cell Lysate Preparation and Western Blot Analysis
Western blot analyses were performed as described in the online Methods.
Immunocytochemical Localization of SOD
Please see online Methods for the description of immunocytochemical staining of SOD.
SOD Activity Assay
SOD activity was measured spectrophotometrically by monitoring inhibition of the autooxidation of pyrogallol. The details are described in the online Methods.
Detection of Production
The effect of SOD overexpression on production in HAECs was determined by a fluorometric assay using dihydroethidium (Sigma) as probe. Details are described in the online Methods.
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide Assay
Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Details are described in the online Methods.
NADPH Oxidase Assay
Plasma membrane fractions of these cells were prepared and NADPH oxidase activities were measured by lucigenin chemiluminescence assay (5 μmol/L lucigenin, Sigma) in the presence of its substrate NADPH (100 μmol/L, Sigma) according to the previous study.17 Details are described in the online Methods.
Cell ELISA
The cell-surface expression of adhesion molecules was examined using the cell ELISA assay as described in the online Methods.
EC–Leukocyte Adhesion Assay
Human neutrophils were isolated from whole blood of healthy donors using standard dextran sedimentation and centrifugation over Ficoll-Hypaque (Amersham Pharmacia Biotech). The detailed EC–leukocyte adhesion assay is described in the online Methods.
Electrophoretic Mobility-Shift Assay
The preparation of nuclear extracts and the conditions for electrophoretic mobility-shift assay (EMSA) reactions have been described previously.18 The 22-mer synthetic double-stranded oligonucleotides used as the NF-B and AP-1 probes in the gel shift assay were, respectively, 5'-AGTTGAGGGGACTTTCCCAGGC-3', 3'-TCAACTCCCCTGAAAGGGTCCG-5' and 5'-ATTCGATCGGGG-CGGGGCGAGC-3', 3'-TAAGCTAGCCC CGCCCCGCTCG-5'.
Statistical Analysis
Values are expressed as the mean±SEM. Statistical evaluation was performed using 1-way ANOVA followed by the Dunnett test, with P<0.05 considered significant.
Results
Overexpression of SOD Reduces Production in Control and TNF-–Treated HAECs
Transfection with human SOD gene (AdSOD) resulted in a great increase in the amount of functional SOD in HAECs detected by Western blot, enzyme activity assay, and immunofluorescent staining (Figure I, available online at http://atvb.ahajournals.org). When HAECs were transfected for 48 hours with various multiplicities of infection (MOIs) of AdSOD, Western blot analysis showed that SOD was expressed in a dose-dependent manner in AdSOD-infected cells but not in cells infected with the control virus (AdPGK) or in control nontransfected cells. Cellular SOD activity was increased 12-fold in AdSOD-infected cells compared with AdPGK-infected and nontransfected cells. In SOD-transfected cells, strong SOD expression was seen throughout the cytoplasm by immunofluorescent staining, whereas in nontransfected cells immunofluorescence was weak.
To determine whether SOD expression affected production in HAECs, cells were transfected for 48 hours with 25 MOI of AdSOD or AdPGK, then production was measured; a portion of the cells was then incubated with 10 ng/mL of TNF- for 5 to 30 minutes, and production was again measured. The results showed that AdSOD-transfected cells produced significantly less than control or AdPGK-transfected cells (Figure II, available online at http://atvb.ahajournals.org). When the same control or transfected cells were subsequently treated with 10 ng/mL of TNF-, levels increased in a time-dependent manner in both control and transfected cells, but the increase was significantly lower in AdSOD-transfected cells (Figure 1). To examine whether SOD expression affected NADPH oxidase activity under TNF- stimulation, HAECs were treated with TNF- for up to 30 minutes, and membrane fractions were prepared to measure NADPH oxidase activity. TNF- addition resulted in a significant increase in enzymatic activity within 5 minutes then declined to the lower levels by 15 minutes. All of the TNF-–induced increase in the oxidase activity was blocked by SOD overexpression (Figure III, available online at http://atvb.ahajournals.org). Transfection or TNF treatment of HAECs did not result in cell death, as demonstrated by the MTT assay (data not shown).
Figure 1. Effect of AdSOD transfection on production by HAECs. A, Control cells or cells transfected for 48 hours with 25 MOI of AdPGK or AdSOD and subsequently treated with 10 ng/mL of TNF- were tested for production by incubation for 45 minutes with dihydroethidium. The fluorescence readings were taken after 5, 10, and 30 minutes. The data are the mean±SEM of 3 independent experiments. *P<0.05 compared with TNF-–treated non–AdSOD-transfected cells.
SOD Overexpression Decreases TNF-–Induced Expression of VCAM-1 and ICAM-1 in HAECs
The effects of SOD overexpression on TNF-–induced VCAM-1 and ICAM-1 expression by HAECs were studied by incubating control and AdSOD-transfected HAECs with different concentrations of TNF- and measuring cell surface expression by cell ELISA and total expression using Western blots of cell lysates.
Cell surface expression of VCAM-1 and ICAM-1 in control, AdPGK-, and AdSOD-transfected cells was very low, but addition of TNF- resulted in a marked dose-dependent increase in control cells but not in AdSOD-transfected cells (Figure IV, available online at http://atvb.ahajournals.org). SOD overexpression almost completely blocked the effect of TNF- on adhesion molecule expression at all TNF- concentrations tested.
Western blot analysis of cell lysates showed that levels of VCAM-1 and ICAM-1 were very low in control untreated HAECs but were increased, respectively, 9.4-fold or 11.3-fold by TNF- treatment, and pretransfection of the HAECs with AdSOD significantly inhibited the TNF-–mediated induction of VCAM-1 and ICAM-1 expression (Figure 2A and 2B).
Figure 2. SOD overexpression reduces TNF-–induced VCAM-1 and ICAM-1 expression in HAECs. Control cells or cells transfected for 48 hours with 25 MOI of AdSOD were treated for 6 hours with TNF- (10 ng/mL), then the expression of VCAM-1 (A) and ICAM-1 (B) was measured by Western blots. All histograms show the mean±SEM for 3 separate experiments. *P<0.05 compared with control cells; P<0.05, with TNF-–treated nontransfected cells.
Neutrophil Adhesion to TNF-–Stimulated HAECs Is Inhibited by SOD Overexpression
To explore the effects of SOD overexpression on EC–leukocyte interactions, we examined the adhesion of neutrophils to TNF-–treated HAECs. As shown in Figure 3A, control-confluent HAECs showed minimal binding of neutrophils, but adhesion was substantially increased when the HAECs were treated with TNF-. Native SOD (nSOD) had no effect on the binding of neutrophils to TNF-–treated HAECs. In contrast, polyethylene glycol conjugation to SOD (PEG-SOD) and AdSOD-pretransfected HAECs significantly reduced the binding of the neutrophils to TNF-–treated HAECs (78±4% and 32±2%, respectively). In addition, the adhesion of neutrophils to TNF-–treated nontransfected or AdSOD-transfected HAECs was examined by pretreatment of the HAECs with antibodies against VCAM-1 or ICAM-1. As shown in Figure 3B, after pretreatment of HAECs with 5, 20, 40, or 80 μg/mL VCAM-1 or ICAM-1 antibodies, as compared with non–antibody-incubated controls, the binding percentage of the neutrophils to TNF-–treated HAECs was 107±6, 93±8, 86±7, 44±5 and 109±8, 44±3, 24±3, 22±3, respectively. ICAM-1 antibody shows significant effects even under very low dosage (ie, 20 μg/mL). VCAM-1, however, starts to show some effects only at much higher concentration (ie, 80 μg/mL). The result showed that at the same concentration, anti–ICAM-1 antibodies had a greater inhibitory effect on the adhesion of neutrophils to TNF-–treated HAECs than anti–VCAM-1 antibodies.
Figure 3. Effect of SOD overexpression on the adhesion of neutrophils to TNF-–stimulated HAECs. A, HAECs treated with native SOD (nSOD), or with polyethylene glycol–SOD (PEG-SOD), or with 25 MOI of AdSOD for 48 hours and for 6 hours with TNF- (10 ng/mL), then incubated with fluorescein-labeled neutrophils. B, Control HAECs or cells transfected for 48 hours with 25 MOI of AdSOD were treated sequentially for 1 hour with antibodies against VCAM-1 (5, 20, 40, and 80 μg/mL) or ICAM-1 (5, 20, 40, and 80 μg/mL) and for 6 hours with TNF- (10 ng/mL), then incubated with fluorescein-labeled neutrophils. After 30 minutes at 37°C, the wells were washed and adhesion quantified in a fluorescence analyzer. The values are reported as the fluorescent intensity of the bound neutrophils expressed as a percentage of that seen with non–TNF-–treated nontransfected cells. The data are the mean±SEM for 3 separate experiments. *P<0.05 compared with control cells or AdPGK-transfected cells; P<0.05, with TNF-–treated nontransfected cells in the absence of antibody; P<0.05, with TNF-–treated PEG-SOD–treated cells.
Involvement of JNK and p38 in SOD-Transfected TNF-–Stimulated HAECs
TNF- can activate MAPKs in the signaling pathway leading to cytokine expression.19 To examine whether MAPKs were regulated through phosphorylation during the TNF-–mediated processes described above, cell lysates were analyzed by Western blotting. As shown in Figure 4, phosphorylation of extracellular signal regulated kinase (ERK; Figure 4A), JNK1/2 (Figure 4B), and p38 (Figure 4C) in nontransfected HAECs was significantly increased after 20 minutes treatment with TNF-. SOD transfection abolished the JNK and p38 phosphorylation induced by TNF- but had little effect on ERK phosphorylation. In addition, a JNK inhibitor (SP600125) and a p38 inhibitor (SB203580) significantly reduced TNF-–induced VCAM-1 and ICAM-1 expression in nontransfected cells, with SP600125 having a much greater effect than SB203580; in contrast, the ERK inhibitor PD98059 had no effect (Figure 5A and 5B).
Figure 4. Western blot analysis of the effect of SOD overexpression on the phosphorylation of ERK1/2 (A), JNK (B), or p38 MAPKs (C) in TNF-–stimulated HAECs. Control cells or cells transfected for 48 hours with 25 MOI of AdPGK or AdSOD were incubated with or without 10 ng/mL of TNF- for 20 minutes at 37°C, then aliquots of cell lysate containing equal amounts of protein were subjected to immunoblotting with specific antibodies. *P<0.05 compared with control cells; P<0.05, with TNF-–treated cells.
Figure 5. Effects of inhibitors of MAPKs phosphorylation on adhesion molecule expression in TNF-–stimulated HAECs analyzed by cell-ELISA. HAECs were incubated for 1 hour with medium or 30 μmol/L of PD98059 (an ERK inhibitor), SB203580 (a p38 inhibitor), or SP600125 (a JNK inhibitor), then with or without 10 ng/mL of TNF- for 6 hours at 37°C the expression of VCAM-1 (A) and ICAM-1 (B) was measured. *P<0.05 compared with control cells; P<0.05, with TNF-–treated cells in the absence of inhibitor; P<0.05, with TNF-+PD98059-treated cells; P<0.05, with TNF-+SB203580-treated cells.
Effects of SOD Overexpression on AP-1 and NF-B Activity in TNF-–Treated HAECs
Transcriptional regulation involving AP-1 and NF-B activation has been implicated in the TNF-–induced expression of adhesion molecules.20 Gel-shift assays using digoxigenin-labeled AP-1 and NF-B consensus sequences as probes were performed to determine the effect of SOD overexpression on AP-1 and NF-B activation in TNF-–treated HAECs. As shown in Figure 6, low basal levels of AP-1 and NF-B binding activity were detected in nontransfected control cells, and binding was significantly increased by TNF- treatment (10 ng/mL for 30 minutes). The binding activity was blocked by a 100-fold excess of the unlabeled AP-1 and NF-B probes (data not shown). In AdSOD-pretransfected HAECs, the TNF-–induced increase in AP-1 and NF-B binding was reduced by 62% and 40%, respectively.
Figure 6. Effect of SOD overexpression on AP-1 and NF-B activation in TNF-–stimulated HAECs measured by EMSA. Nuclear extracts were prepared from untreated control cells or control cells or AdSOD-transfected cells (25 MOI for 48 hours), incubated with 10 ng/mL of TNF- for 30 minutes at 37°C, and tested for DNA binding activity of AP-1 (A) and NF-B (B). A representative result from 3 separate experiments is shown, and the summarized data for the 3 experiments are shown in the bar chart. *P<0.05 compared with the untreated control cells; P<0.05, with TNF-–treated cells.
Discussion
In this study, we found that SOD overexpression effectively blocked VCAM-1 and ICAM-1 expression in TNF-–stimulated HAECs. In addition, it inhibited the binding of the human neutrophils to TNF-–stimulated HAECs and decreased levels in both control and TNF-–treated HAECs. Our data also demonstrated that the effects of SOD overexpression were mediated through regulation of JNK and p38 phosphorylation and AP-1 and NF-B activity but did not involve ERK.
An elevated oxidative stress level is an important risk factor for atherosclerosis and cardiovascular morbidity.21 Oxidative stress initiates the processes involved in atherogenesis by the continuous interaction between reactive oxygen species and the arterial endothelium. Overexpression of Cu/Zn SOD or catalase in transgenic mice protects LDL against vascular cell–mediated oxidation and reduces oxidized (ox)LDL–induced apoptosis in smooth muscle cells.22 Oxidative stress in humans with coronary artery disease is also exacerbated by a reduction in vascular extracellular SOD, normally an important protective enzyme against .21 These data suggest that alteration of the antioxidant status in the arterial wall may change the pathogenesis of atherosclerosis and that an increase in the activity of antioxidant enzymes may reduce the development of atherosclerosis. The present study demonstrated that transfection of HAECs with AdSOD significantly reduced TNF-–induced production. Furthermore, a role for superoxide anions in mediating TNF-–induced leukocyte–EC adhesion was supported by our observation that SOD expression in HAECs resulted in less adhesion of neutrophils. Our results are consistent with those of previous studies showing that superoxide is involved in leukocyte adhesion to ischemia-reperfusion–treated,23 oxLDL-treated,24 or oxidized chylomicron–treated25 ECs of arterioles and postcapillary venules. SOD inhibited TNF-–induced VCAM-1 and ICAM-1 expression in HAECs in this study by Western blot and cell-ELISA as well as the study of Chen et al26 by Northern analysis. These data suggested that is involved in the redox-sensitive regulation of adhesion molecule expression and agreed with the reports of other investigators showing that SOD can protect ECs against oxidative injury.8,22–24,26
The SOD-induced decrease in neutrophil-EC adhesion has important implications in terms of atherogenic mechanisms, as well as in the treatment of atherosclerosis. In our previous study,27 antioxidant treatment was shown to significantly reduce areas of atheroma, monocyte chemoattractant protein-1 expression, and the area occupied by macrophages and smooth muscle cells in the aorta of cholesterol-fed endothelium-denuded rabbits. Atherosclerotic lesions result from the accumulation of foam cells of monocyte/macrophage origin within the arterial intima.28 Given the probable involvement of VCAM-1 and ICAM-1 in leukocyte recruitment to early atherosclerotic lesions, our findings suggest an additional mechanism by which SOD expression may be involved in preventing the progress of atherosclerosis. In this study, we showed that an increase in endogenous SOD levels in HAECs reduced the binding of neutrophils to TNF-–stimulated HAECs. This observation is consistent with the view that the adherence of leukocytes to oxLDL or oxidized chylomicron–treated ECs can be prevented by prior administration of either SOD or a monoclonal antibody directed against ICAM-1.24,25 Our study also showed that anti–ICAM-1 antibodies had a greater inhibitory effect on the adhesion of neutrophils to TNF-–treated HAECs than anti–VCAM-1 antibodies, suggesting that ICAM-1 plays the major role in neutrophil adherence to ECs.
MAPKs are highly conserved serine/threonine kinases that are activated in response to a wide variety of stimuli, including growth factors, G protein–coupled receptors, and environmental stresses. Our study showed that TNF- caused strong activation of MAPK 3 subtypes in HAECs, as reported in previous studies.14,29 However, the relationship between their activation and the protection effect of SOD remains unclear. ERK1/2 is primarily involved in control of growth and differentiation, whereas JNK and p38 MAPKs are inflammatory and stress-sensitive kinases. Because they have contradictory roles in terms of cell growth/death, the relative activation of these proteins is important for the inflammatory status of the cell. Additionally, there is cross-talk between MAPK cascades, whereby the activity of one MAPK can be influenced by another. Thus, these proteins collectively integrate the pro- and antiinflammatory stimuli acting on the cell to produce appropriate downstream effects.30 To our knowledge, our study is the first to identify specific TNF-–stimulated signal transduction pathways (JNK and p38) which are inhibited by SOD overexpression in HAECs. A JNK inhibitor (SP600125) and a p38 inhibitor (SB203580) markedly inhibited the expression of VCAM-1 and ICAM-1 in TNF-–treated HAECs, whereas an ERK inhibitor (PD98059) had no significant effect. Although SOD inhibited the phosphorylation of both JNK and p38 MAPK in TNF-–treated HAECs, the JNK inhibitor had a greater effect than the p38 inhibitor on TNF-–induced VCAM-1 and ICAM-1, suggesting that this effect was mediated to a greater degree by the JNK phosphorylation pathway. In addition, SB203580 inhibited VCAM-1 expression more noticeably than ICAM-1 expression in this study. This indicates that p38 plays a much more important role in VCAM-1 expression. The result is similar to that of a previous study showing that TNF-–induced expression of VCAM-1 but not ICAM-1 is inhibited by the p38 MAPK inhibitor.31
The binding of TNF- to its receptors causes activation of 2 major transcription factors, AP-1 and NF-B, which in turn induce the expression of genes involved in chronic and acute inflammatory responses.19 NF-B transcriptional activity can be modulated through phosphorylation by MAPKs. AP-1 is a heterogeneous collection of dimeric transcription factors made up of Jun, Fos, and ATF subunits, the activity of which is regulated by multiple mechanisms, including phosphorylation by various MAPKs. JNK and p38 are involved in the TNF-–mediated induction of AP-1 activity.32,33 JNK phosphorylates members of the AP-1 transcription factors (c-Jun, JunB, and JunD) and NF-B. It has been established that JNK regulates AP-1 transcription activity in vivo, and it is likely that increased AP-1 activity mediates, in part, the effects of the JNK signaling pathway.34 It is thought that certain nuclear oncoproteins, such as c-Fos and c-Jun (components of AP-1) as well as NF-B, which all have transcriptional regulatory activity, play important roles in adhesion molecule expression.35 In this study, SOD overexpression suppressed the increase in AP-1 and NF-B binding activity induced by TNF-. These findings raise the possibility that SOD overexpression may reduce adhesion molecule expression through the reduction in AP-1 and NF-B activity.
In summary, this study provides the evidence that SOD overexpression reduces the TNF-–induced expression of VCAM-1 and ICAM-1 by HAECs and TNF-–induced leukocyte adhesion to HAECs. It also shows that these effects of SOD are, in part, because of its inhibitory action on TNF-–induced activation of JNK/p38, NF-B, and AP-1. Because leukocyte recruitment into the vascular wall after their adhesion to ECs is a crucial step in the pathogenesis of atherosclerosis, our study implies that antioxidants may have an as yet unexplored therapeutic potential in the prevention of atherosclerosis. Thus, SOD may have an additional beneficial effect in multiple pathological events involving leukocyte adhesion, including inflammation and atherosclerosis.
Acknowledgments
This work was supported in part by grants from the National Science Council (NSC 92-2320-B010-031 and NSC 92-2314-B010-030), the Academia Sinica (GPCP91-26-6), the Yen Tjing-Ling Medical Foundation (CI-92-7-1), and the Program for Promoting University Academic Excellence (91-B-FA09-2-4), Taiwan.
Received February 5, 2004; accepted November 3, 2004.
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Correspondence to Dr Yuh-Lien Chen, Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, No. 1, Section 1, Ren-Ai Rd, Taipei, 100, Taiwan. E-mail ylchen@ha.mc.ntu.edu.tw
Abstract
Objective— Expression of adhesion molecules on endothelial cells and subsequent leukocyte recruitment are critical early events in the development of atherosclerosis. We tried to study possible effects of Cu/Zn superoxide dismutase (SOD) on adhesion molecule expression and its underlying mechanism in the prevention and treatment of cardiovascular disorders.
Methods and Results— Human aortic endothelial cells (HAECs) were transfected with adenovirus carrying the human SOD gene (AdSOD) to investigate whether SOD expression in HAECs attenuated tumor necrosis factor (TNF)-–induced reactive oxygen species production and adhesion molecule expression and to define the mechanisms involved. SOD expression significantly suppressed TNF-–induced expression of vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1 and reduced the binding of the human neutrophils to TNF-–stimulated HAECs. SOD expression suppressed c-JUN N-terminal kinase and p38 phosphorylation. It also attenuated intracellular superoxide anion production and NADPH oxidase activity in TNF-–treated HAECs.
Conclusions— These results provide evidence that SOD expression in endothelial cells attenuates TNF-–induced superoxide anion production and adhesion molecule expression, and that this protective effect is mediated by decreased JNK and p38 phosphorylation and activator protein-1 and nuclear factor B inactivation. These results suggest that SOD has antiinflammatory properties and may play important roles in the prevention of atherosclerosis and inflammatory response.
Superoxide dismutase overexpression in endothelial cells attenuates tumor necrosis factor-–induced superoxide anion production and adhesion molecule expression, and this effect is mediated by decreased JNK and p38 phosphorylation and AP-1 and nuclear factor B inactivation. These results suggest that superoxide dismutase may play an important role in the prevention of atherosclerosis and inflammatory response.
Key Words: superoxide dismutase ? atherosclerosis ? endothelial cell ? adhesion molecule ? MAPKs
Introduction
Vascular endothelial cells (ECs) are very sensitive to oxidative damage mediated by superoxide anion () released from the endothelium itself and from inflammatory cells, and reactive oxygen species, such as , are implicated in the pathogenesis of cardiovascular disorders.1 Superoxide dismutase (SOD), an antioxidant enzyme, may be useful in the augmentation of antioxidant defenses in the endothelium.2 However, the role of SOD in the protection of cells against oxidative injury is controversial. A protective role for SOD was proposed by some investigators, because addition of SOD to cultures has been reported to prevent EC-mediated low-density lipoprotein (LDL) oxidation,3 decrease reoxygenation-induced cellular injury and intercellular cell adhesion molecule (ICAM)-1 expression in ECs,4 and prevent apoptosis of ECs in the presence of free radicals.5 In contrast, others have reported that addition of SOD does not inhibit EC-induced LDL oxidation6 or hyperoxia-induced ICAM-1 expression in ECs.7 The effects produced by addition of exogenous SOD do not firmly establish the role of SOD in ECs, because the added protein may contain impurities; in addition, both SOD and the EC cell surface are negatively charged, which may impede the entry of SOD into the cell. To overcome these problems, several attempts have been made to overexpress SOD enzyme in various cell types and examine its effect in preventing oxidation-related damage.8–10 Transient overexpression of Cu/Zn SOD was highly effective in reducing EC-induced LDL oxidation.8 However, the effect of SOD expression on human ECs exposed to inflammatory cytokines still remains unclear.
Expression of cell adhesion molecules, such as vascular cell adhesion molecule (VCAM)-1 and ICAM-1, on ECs represents one of the earliest pathological changes in immune and inflammatory diseases, such as atherosclerosis.11 Increased expression of adhesion molecules by ECs in human atherosclerotic lesions may lead to further recruitment of leukocytes to atherosclerotic sites. Possible effects of SOD on adhesion molecule expression could play a key role in the prevention and treatment of cardiovascular disorders. We were therefore interested in understanding the underlying mechanism of action of SOD on ECs and determining whether it stimulates or blocks the expression of adhesion molecules. Induction of adhesion molecule expression by tumor necrosis factor (TNF)- and other inflammatory cytokines involves a wide spectrum of host-responsive systems.12 This requires the activation of multiple signaling molecules in transduction pathways (eg, protein tyrosine kinase, TNF- receptor–associated serine/threonine kinase, Ras, Raf-1, IB kinase, mitogen-activated extracellular-regulated kinase, and mitogen-activated protein kinases (MAPKs).13,14 The pathways involving these molecules may converge or diverge and often have "cross-talk" properties, resulting in a complicated signaling network. Subsequently, the signals are transduced to downstream molecules and activate numerous transcriptional factors, including activator protein-1 (AP-1), nuclear factor B (NF-B), and ATF-2,15 which trigger the expression of a large number of genes encoding inflammatory mediators and adhesion molecules. The effects of TNF- and SOD overexpression on the signaling pathways and the events involved in the expression of adhesion molecules on TNF-–treated human arterial ECs (HAECs) are poorly understood. In this study, adenovirus-mediated gene transfer was used to test the effect of SOD overexpression on the expression of adhesion molecules and MAPKs in HAECs. The results showed that SOD overexpression attenuated the TNF-–induced expression of VCAM-1 and ICAM-1, and that this effect was mediated by blockage of the JNK/p38 MAPK pathway as well as NF-B and AP-1 expression. SOD also significantly inhibited the adhesion of the human neutrophils to TNF-–treated HAECs.
Materials and Methods
Culture of HAECs
HAECs were obtained as cryopreserved tertiary cultures from Cascade Biologics (Portland, Ore) and were grown in EC growth medium.16 The detailed preparation of HAECs is described in the expanded Methods section, available online at http://atvb.ahajournals.org.
Preparation of Recombinant Adenoviruses
cDNA containing the entire human SOD coding sequence was subcloned into the adenovirus shuttle plasmid vector pAd-PGK, which contains the human phosphoglycerate kinase gene promoter and the bovine growth hormone gene polyadenylation signal. The detailed preparation of recombinant adenoviruses is described in the online Methods.
Cell Lysate Preparation and Western Blot Analysis
Western blot analyses were performed as described in the online Methods.
Immunocytochemical Localization of SOD
Please see online Methods for the description of immunocytochemical staining of SOD.
SOD Activity Assay
SOD activity was measured spectrophotometrically by monitoring inhibition of the autooxidation of pyrogallol. The details are described in the online Methods.
Detection of Production
The effect of SOD overexpression on production in HAECs was determined by a fluorometric assay using dihydroethidium (Sigma) as probe. Details are described in the online Methods.
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide Assay
Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Details are described in the online Methods.
NADPH Oxidase Assay
Plasma membrane fractions of these cells were prepared and NADPH oxidase activities were measured by lucigenin chemiluminescence assay (5 μmol/L lucigenin, Sigma) in the presence of its substrate NADPH (100 μmol/L, Sigma) according to the previous study.17 Details are described in the online Methods.
Cell ELISA
The cell-surface expression of adhesion molecules was examined using the cell ELISA assay as described in the online Methods.
EC–Leukocyte Adhesion Assay
Human neutrophils were isolated from whole blood of healthy donors using standard dextran sedimentation and centrifugation over Ficoll-Hypaque (Amersham Pharmacia Biotech). The detailed EC–leukocyte adhesion assay is described in the online Methods.
Electrophoretic Mobility-Shift Assay
The preparation of nuclear extracts and the conditions for electrophoretic mobility-shift assay (EMSA) reactions have been described previously.18 The 22-mer synthetic double-stranded oligonucleotides used as the NF-B and AP-1 probes in the gel shift assay were, respectively, 5'-AGTTGAGGGGACTTTCCCAGGC-3', 3'-TCAACTCCCCTGAAAGGGTCCG-5' and 5'-ATTCGATCGGGG-CGGGGCGAGC-3', 3'-TAAGCTAGCCC CGCCCCGCTCG-5'.
Statistical Analysis
Values are expressed as the mean±SEM. Statistical evaluation was performed using 1-way ANOVA followed by the Dunnett test, with P<0.05 considered significant.
Results
Overexpression of SOD Reduces Production in Control and TNF-–Treated HAECs
Transfection with human SOD gene (AdSOD) resulted in a great increase in the amount of functional SOD in HAECs detected by Western blot, enzyme activity assay, and immunofluorescent staining (Figure I, available online at http://atvb.ahajournals.org). When HAECs were transfected for 48 hours with various multiplicities of infection (MOIs) of AdSOD, Western blot analysis showed that SOD was expressed in a dose-dependent manner in AdSOD-infected cells but not in cells infected with the control virus (AdPGK) or in control nontransfected cells. Cellular SOD activity was increased 12-fold in AdSOD-infected cells compared with AdPGK-infected and nontransfected cells. In SOD-transfected cells, strong SOD expression was seen throughout the cytoplasm by immunofluorescent staining, whereas in nontransfected cells immunofluorescence was weak.
To determine whether SOD expression affected production in HAECs, cells were transfected for 48 hours with 25 MOI of AdSOD or AdPGK, then production was measured; a portion of the cells was then incubated with 10 ng/mL of TNF- for 5 to 30 minutes, and production was again measured. The results showed that AdSOD-transfected cells produced significantly less than control or AdPGK-transfected cells (Figure II, available online at http://atvb.ahajournals.org). When the same control or transfected cells were subsequently treated with 10 ng/mL of TNF-, levels increased in a time-dependent manner in both control and transfected cells, but the increase was significantly lower in AdSOD-transfected cells (Figure 1). To examine whether SOD expression affected NADPH oxidase activity under TNF- stimulation, HAECs were treated with TNF- for up to 30 minutes, and membrane fractions were prepared to measure NADPH oxidase activity. TNF- addition resulted in a significant increase in enzymatic activity within 5 minutes then declined to the lower levels by 15 minutes. All of the TNF-–induced increase in the oxidase activity was blocked by SOD overexpression (Figure III, available online at http://atvb.ahajournals.org). Transfection or TNF treatment of HAECs did not result in cell death, as demonstrated by the MTT assay (data not shown).
Figure 1. Effect of AdSOD transfection on production by HAECs. A, Control cells or cells transfected for 48 hours with 25 MOI of AdPGK or AdSOD and subsequently treated with 10 ng/mL of TNF- were tested for production by incubation for 45 minutes with dihydroethidium. The fluorescence readings were taken after 5, 10, and 30 minutes. The data are the mean±SEM of 3 independent experiments. *P<0.05 compared with TNF-–treated non–AdSOD-transfected cells.
SOD Overexpression Decreases TNF-–Induced Expression of VCAM-1 and ICAM-1 in HAECs
The effects of SOD overexpression on TNF-–induced VCAM-1 and ICAM-1 expression by HAECs were studied by incubating control and AdSOD-transfected HAECs with different concentrations of TNF- and measuring cell surface expression by cell ELISA and total expression using Western blots of cell lysates.
Cell surface expression of VCAM-1 and ICAM-1 in control, AdPGK-, and AdSOD-transfected cells was very low, but addition of TNF- resulted in a marked dose-dependent increase in control cells but not in AdSOD-transfected cells (Figure IV, available online at http://atvb.ahajournals.org). SOD overexpression almost completely blocked the effect of TNF- on adhesion molecule expression at all TNF- concentrations tested.
Western blot analysis of cell lysates showed that levels of VCAM-1 and ICAM-1 were very low in control untreated HAECs but were increased, respectively, 9.4-fold or 11.3-fold by TNF- treatment, and pretransfection of the HAECs with AdSOD significantly inhibited the TNF-–mediated induction of VCAM-1 and ICAM-1 expression (Figure 2A and 2B).
Figure 2. SOD overexpression reduces TNF-–induced VCAM-1 and ICAM-1 expression in HAECs. Control cells or cells transfected for 48 hours with 25 MOI of AdSOD were treated for 6 hours with TNF- (10 ng/mL), then the expression of VCAM-1 (A) and ICAM-1 (B) was measured by Western blots. All histograms show the mean±SEM for 3 separate experiments. *P<0.05 compared with control cells; P<0.05, with TNF-–treated nontransfected cells.
Neutrophil Adhesion to TNF-–Stimulated HAECs Is Inhibited by SOD Overexpression
To explore the effects of SOD overexpression on EC–leukocyte interactions, we examined the adhesion of neutrophils to TNF-–treated HAECs. As shown in Figure 3A, control-confluent HAECs showed minimal binding of neutrophils, but adhesion was substantially increased when the HAECs were treated with TNF-. Native SOD (nSOD) had no effect on the binding of neutrophils to TNF-–treated HAECs. In contrast, polyethylene glycol conjugation to SOD (PEG-SOD) and AdSOD-pretransfected HAECs significantly reduced the binding of the neutrophils to TNF-–treated HAECs (78±4% and 32±2%, respectively). In addition, the adhesion of neutrophils to TNF-–treated nontransfected or AdSOD-transfected HAECs was examined by pretreatment of the HAECs with antibodies against VCAM-1 or ICAM-1. As shown in Figure 3B, after pretreatment of HAECs with 5, 20, 40, or 80 μg/mL VCAM-1 or ICAM-1 antibodies, as compared with non–antibody-incubated controls, the binding percentage of the neutrophils to TNF-–treated HAECs was 107±6, 93±8, 86±7, 44±5 and 109±8, 44±3, 24±3, 22±3, respectively. ICAM-1 antibody shows significant effects even under very low dosage (ie, 20 μg/mL). VCAM-1, however, starts to show some effects only at much higher concentration (ie, 80 μg/mL). The result showed that at the same concentration, anti–ICAM-1 antibodies had a greater inhibitory effect on the adhesion of neutrophils to TNF-–treated HAECs than anti–VCAM-1 antibodies.
Figure 3. Effect of SOD overexpression on the adhesion of neutrophils to TNF-–stimulated HAECs. A, HAECs treated with native SOD (nSOD), or with polyethylene glycol–SOD (PEG-SOD), or with 25 MOI of AdSOD for 48 hours and for 6 hours with TNF- (10 ng/mL), then incubated with fluorescein-labeled neutrophils. B, Control HAECs or cells transfected for 48 hours with 25 MOI of AdSOD were treated sequentially for 1 hour with antibodies against VCAM-1 (5, 20, 40, and 80 μg/mL) or ICAM-1 (5, 20, 40, and 80 μg/mL) and for 6 hours with TNF- (10 ng/mL), then incubated with fluorescein-labeled neutrophils. After 30 minutes at 37°C, the wells were washed and adhesion quantified in a fluorescence analyzer. The values are reported as the fluorescent intensity of the bound neutrophils expressed as a percentage of that seen with non–TNF-–treated nontransfected cells. The data are the mean±SEM for 3 separate experiments. *P<0.05 compared with control cells or AdPGK-transfected cells; P<0.05, with TNF-–treated nontransfected cells in the absence of antibody; P<0.05, with TNF-–treated PEG-SOD–treated cells.
Involvement of JNK and p38 in SOD-Transfected TNF-–Stimulated HAECs
TNF- can activate MAPKs in the signaling pathway leading to cytokine expression.19 To examine whether MAPKs were regulated through phosphorylation during the TNF-–mediated processes described above, cell lysates were analyzed by Western blotting. As shown in Figure 4, phosphorylation of extracellular signal regulated kinase (ERK; Figure 4A), JNK1/2 (Figure 4B), and p38 (Figure 4C) in nontransfected HAECs was significantly increased after 20 minutes treatment with TNF-. SOD transfection abolished the JNK and p38 phosphorylation induced by TNF- but had little effect on ERK phosphorylation. In addition, a JNK inhibitor (SP600125) and a p38 inhibitor (SB203580) significantly reduced TNF-–induced VCAM-1 and ICAM-1 expression in nontransfected cells, with SP600125 having a much greater effect than SB203580; in contrast, the ERK inhibitor PD98059 had no effect (Figure 5A and 5B).
Figure 4. Western blot analysis of the effect of SOD overexpression on the phosphorylation of ERK1/2 (A), JNK (B), or p38 MAPKs (C) in TNF-–stimulated HAECs. Control cells or cells transfected for 48 hours with 25 MOI of AdPGK or AdSOD were incubated with or without 10 ng/mL of TNF- for 20 minutes at 37°C, then aliquots of cell lysate containing equal amounts of protein were subjected to immunoblotting with specific antibodies. *P<0.05 compared with control cells; P<0.05, with TNF-–treated cells.
Figure 5. Effects of inhibitors of MAPKs phosphorylation on adhesion molecule expression in TNF-–stimulated HAECs analyzed by cell-ELISA. HAECs were incubated for 1 hour with medium or 30 μmol/L of PD98059 (an ERK inhibitor), SB203580 (a p38 inhibitor), or SP600125 (a JNK inhibitor), then with or without 10 ng/mL of TNF- for 6 hours at 37°C the expression of VCAM-1 (A) and ICAM-1 (B) was measured. *P<0.05 compared with control cells; P<0.05, with TNF-–treated cells in the absence of inhibitor; P<0.05, with TNF-+PD98059-treated cells; P<0.05, with TNF-+SB203580-treated cells.
Effects of SOD Overexpression on AP-1 and NF-B Activity in TNF-–Treated HAECs
Transcriptional regulation involving AP-1 and NF-B activation has been implicated in the TNF-–induced expression of adhesion molecules.20 Gel-shift assays using digoxigenin-labeled AP-1 and NF-B consensus sequences as probes were performed to determine the effect of SOD overexpression on AP-1 and NF-B activation in TNF-–treated HAECs. As shown in Figure 6, low basal levels of AP-1 and NF-B binding activity were detected in nontransfected control cells, and binding was significantly increased by TNF- treatment (10 ng/mL for 30 minutes). The binding activity was blocked by a 100-fold excess of the unlabeled AP-1 and NF-B probes (data not shown). In AdSOD-pretransfected HAECs, the TNF-–induced increase in AP-1 and NF-B binding was reduced by 62% and 40%, respectively.
Figure 6. Effect of SOD overexpression on AP-1 and NF-B activation in TNF-–stimulated HAECs measured by EMSA. Nuclear extracts were prepared from untreated control cells or control cells or AdSOD-transfected cells (25 MOI for 48 hours), incubated with 10 ng/mL of TNF- for 30 minutes at 37°C, and tested for DNA binding activity of AP-1 (A) and NF-B (B). A representative result from 3 separate experiments is shown, and the summarized data for the 3 experiments are shown in the bar chart. *P<0.05 compared with the untreated control cells; P<0.05, with TNF-–treated cells.
Discussion
In this study, we found that SOD overexpression effectively blocked VCAM-1 and ICAM-1 expression in TNF-–stimulated HAECs. In addition, it inhibited the binding of the human neutrophils to TNF-–stimulated HAECs and decreased levels in both control and TNF-–treated HAECs. Our data also demonstrated that the effects of SOD overexpression were mediated through regulation of JNK and p38 phosphorylation and AP-1 and NF-B activity but did not involve ERK.
An elevated oxidative stress level is an important risk factor for atherosclerosis and cardiovascular morbidity.21 Oxidative stress initiates the processes involved in atherogenesis by the continuous interaction between reactive oxygen species and the arterial endothelium. Overexpression of Cu/Zn SOD or catalase in transgenic mice protects LDL against vascular cell–mediated oxidation and reduces oxidized (ox)LDL–induced apoptosis in smooth muscle cells.22 Oxidative stress in humans with coronary artery disease is also exacerbated by a reduction in vascular extracellular SOD, normally an important protective enzyme against .21 These data suggest that alteration of the antioxidant status in the arterial wall may change the pathogenesis of atherosclerosis and that an increase in the activity of antioxidant enzymes may reduce the development of atherosclerosis. The present study demonstrated that transfection of HAECs with AdSOD significantly reduced TNF-–induced production. Furthermore, a role for superoxide anions in mediating TNF-–induced leukocyte–EC adhesion was supported by our observation that SOD expression in HAECs resulted in less adhesion of neutrophils. Our results are consistent with those of previous studies showing that superoxide is involved in leukocyte adhesion to ischemia-reperfusion–treated,23 oxLDL-treated,24 or oxidized chylomicron–treated25 ECs of arterioles and postcapillary venules. SOD inhibited TNF-–induced VCAM-1 and ICAM-1 expression in HAECs in this study by Western blot and cell-ELISA as well as the study of Chen et al26 by Northern analysis. These data suggested that is involved in the redox-sensitive regulation of adhesion molecule expression and agreed with the reports of other investigators showing that SOD can protect ECs against oxidative injury.8,22–24,26
The SOD-induced decrease in neutrophil-EC adhesion has important implications in terms of atherogenic mechanisms, as well as in the treatment of atherosclerosis. In our previous study,27 antioxidant treatment was shown to significantly reduce areas of atheroma, monocyte chemoattractant protein-1 expression, and the area occupied by macrophages and smooth muscle cells in the aorta of cholesterol-fed endothelium-denuded rabbits. Atherosclerotic lesions result from the accumulation of foam cells of monocyte/macrophage origin within the arterial intima.28 Given the probable involvement of VCAM-1 and ICAM-1 in leukocyte recruitment to early atherosclerotic lesions, our findings suggest an additional mechanism by which SOD expression may be involved in preventing the progress of atherosclerosis. In this study, we showed that an increase in endogenous SOD levels in HAECs reduced the binding of neutrophils to TNF-–stimulated HAECs. This observation is consistent with the view that the adherence of leukocytes to oxLDL or oxidized chylomicron–treated ECs can be prevented by prior administration of either SOD or a monoclonal antibody directed against ICAM-1.24,25 Our study also showed that anti–ICAM-1 antibodies had a greater inhibitory effect on the adhesion of neutrophils to TNF-–treated HAECs than anti–VCAM-1 antibodies, suggesting that ICAM-1 plays the major role in neutrophil adherence to ECs.
MAPKs are highly conserved serine/threonine kinases that are activated in response to a wide variety of stimuli, including growth factors, G protein–coupled receptors, and environmental stresses. Our study showed that TNF- caused strong activation of MAPK 3 subtypes in HAECs, as reported in previous studies.14,29 However, the relationship between their activation and the protection effect of SOD remains unclear. ERK1/2 is primarily involved in control of growth and differentiation, whereas JNK and p38 MAPKs are inflammatory and stress-sensitive kinases. Because they have contradictory roles in terms of cell growth/death, the relative activation of these proteins is important for the inflammatory status of the cell. Additionally, there is cross-talk between MAPK cascades, whereby the activity of one MAPK can be influenced by another. Thus, these proteins collectively integrate the pro- and antiinflammatory stimuli acting on the cell to produce appropriate downstream effects.30 To our knowledge, our study is the first to identify specific TNF-–stimulated signal transduction pathways (JNK and p38) which are inhibited by SOD overexpression in HAECs. A JNK inhibitor (SP600125) and a p38 inhibitor (SB203580) markedly inhibited the expression of VCAM-1 and ICAM-1 in TNF-–treated HAECs, whereas an ERK inhibitor (PD98059) had no significant effect. Although SOD inhibited the phosphorylation of both JNK and p38 MAPK in TNF-–treated HAECs, the JNK inhibitor had a greater effect than the p38 inhibitor on TNF-–induced VCAM-1 and ICAM-1, suggesting that this effect was mediated to a greater degree by the JNK phosphorylation pathway. In addition, SB203580 inhibited VCAM-1 expression more noticeably than ICAM-1 expression in this study. This indicates that p38 plays a much more important role in VCAM-1 expression. The result is similar to that of a previous study showing that TNF-–induced expression of VCAM-1 but not ICAM-1 is inhibited by the p38 MAPK inhibitor.31
The binding of TNF- to its receptors causes activation of 2 major transcription factors, AP-1 and NF-B, which in turn induce the expression of genes involved in chronic and acute inflammatory responses.19 NF-B transcriptional activity can be modulated through phosphorylation by MAPKs. AP-1 is a heterogeneous collection of dimeric transcription factors made up of Jun, Fos, and ATF subunits, the activity of which is regulated by multiple mechanisms, including phosphorylation by various MAPKs. JNK and p38 are involved in the TNF-–mediated induction of AP-1 activity.32,33 JNK phosphorylates members of the AP-1 transcription factors (c-Jun, JunB, and JunD) and NF-B. It has been established that JNK regulates AP-1 transcription activity in vivo, and it is likely that increased AP-1 activity mediates, in part, the effects of the JNK signaling pathway.34 It is thought that certain nuclear oncoproteins, such as c-Fos and c-Jun (components of AP-1) as well as NF-B, which all have transcriptional regulatory activity, play important roles in adhesion molecule expression.35 In this study, SOD overexpression suppressed the increase in AP-1 and NF-B binding activity induced by TNF-. These findings raise the possibility that SOD overexpression may reduce adhesion molecule expression through the reduction in AP-1 and NF-B activity.
In summary, this study provides the evidence that SOD overexpression reduces the TNF-–induced expression of VCAM-1 and ICAM-1 by HAECs and TNF-–induced leukocyte adhesion to HAECs. It also shows that these effects of SOD are, in part, because of its inhibitory action on TNF-–induced activation of JNK/p38, NF-B, and AP-1. Because leukocyte recruitment into the vascular wall after their adhesion to ECs is a crucial step in the pathogenesis of atherosclerosis, our study implies that antioxidants may have an as yet unexplored therapeutic potential in the prevention of atherosclerosis. Thus, SOD may have an additional beneficial effect in multiple pathological events involving leukocyte adhesion, including inflammation and atherosclerosis.
Acknowledgments
This work was supported in part by grants from the National Science Council (NSC 92-2320-B010-031 and NSC 92-2314-B010-030), the Academia Sinica (GPCP91-26-6), the Yen Tjing-Ling Medical Foundation (CI-92-7-1), and the Program for Promoting University Academic Excellence (91-B-FA09-2-4), Taiwan.
Received February 5, 2004; accepted November 3, 2004.
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