Nitric Oxide Regulates Monocyte Chemotactic Protein-1
Background Monocyte chemotactic protein-1 (MCP-1) is a 76-amino-acid chemokine thought to be the major chemotactic factor for monocytes. We and others have demonstrated that NO inhibits monocyte–endothelial cell interactions and atherogenesis. We hypothesize that the antiatherogenic effect of NO may be due in part to its inhibition of MCP-1 expression.
Methods and Results Smooth muscle cells (SMCs) were isolated from normal rabbit aortas by the explant method. Cells were then exposed to LPS (10 μg/mL), native LDL, or oxidized LDL (30 μg/mL) for 6 hours. The expression of MCP-1 in SMCs and chemotactic activity in the conditioned medium were induced by lipopolysaccharide (LPS) or by oxidized LDL but not native LDL. The induction of MCP-1 by cytokines or oxidized lipoproteins was associated with an increased generation of superoxide anion by the SMCs and increased activity of the transcriptional protein nuclear factor-κB (NFκB). The induced expression of MCP-1 and activation of NFκB were reduced by previous exposure of the SMCs to the NO donor DETA-NONOate (100 μmol/L) (P<.05). To determine whether NO exerted its effect at a transcriptional level, SMCs and COS cells were transfected with a 400-bp fragment of the MCP-1 promoter. Promoter activity was enhanced by oxidized LDL, and LPS was inhibited by DETA-NO. Nuclear run-on assays confirmed that the effect of NO occurred at a transcriptional level. To investigate the role of endogenous NO in the regulation of MCP-1 in vivo, New Zealand White rabbits were fed normal chow, normal chow plus nitro-l-arginine (LNA), high-cholesterol diet (Chol), or high-cholesterol diet supplemented with l-arginine (Arg). After 2 weeks, thoracic aortas were harvested and total RNA was isolated. Northern analysis using full-length MCP-1 cDNA demonstrated increased expression in Chol and LNA aortas; this expression was decreased in aortas from Arg animals.
Conclusions These studies indicate that the antiatherogenic effect of NO may be mediated in part by its inhibition of MCP-1 expression.
Within days of exposure to a high-cholesterol diet, monocytes begin to adhere to the vascular endothelium.1 This phenomenon is thought to be mediated by alterations in the adhesiveness of the endothelium due to the induced expression of adhesion molecules and chemotactic proteins, such as MCP-1.2 3 This 76-amino-acid chemokine has been implicated as a major culprit in the enticement of monocytes and T lymphocytes into the vessel wall.4 5 The expression of MCP-1 is induced by LPS, cytokines (tumor necrosis factor-α, interleukins 1 and 4), or minimally modified LDL.6 Human aortic ECs and SMCs exposed to minimally modified LDL express MCP-1; this protein accounts for virtually all of the chemotactic activity produced under these conditions.4 MCP-1 may also activate or increase the expression of adhesion molecules to facilitate monocyte adhesion.7 8 9 The role of this chemokine in human disease has been implicated by immunohistochemical studies of atherosclerotic plaques.10 The weight of the available evidence indicates that MCP-1 is one of the key factors initiating the inflammatory process of atherogenesis.
There are countervailing forces in the vessel wall that oppose atherogenesis. One of these is endothelium-derived NO. In addition to being a potent vasodilator, NO inhibits key events that promote atherogenesis, including alterations in endothelial redox state and monocyte adherence to the vessel wall.11 12 13 However, the salutary influence of this factor wanes in hypercholesterolemia,14 15 and this endothelial dysfunction may promote atherogenesis. Indeed, chronic oral administration of an NOS antagonist reduces vascular NO activity, increases monocyte-EC interaction, and promotes development of lesions in hypercholesterolemic animals.16 17 By contrast, the enhancement of vascular NO activity in hypercholesterolemic rabbits (by chronic administration of the NO precursor l-arginine) inhibits monocyte adherence and accumulation in the vessel wall.18 19 20
We speculate that endogenous NO inhibits atherogenesis by modulating the expression of adhesion molecules and chemokines mediating EC-monocyte interaction.
As a test of this paradigm, we designed this study to determine whether NO suppresses the expression of MCP-1. In addition, we tested the hypothesis that NO exerts its effect by suppressing redox-sensitive transcriptional pathways regulating MCP-1 expression. Finally, we extended our in vitro observations regarding the interaction of NO and MCP-1 into an animal model of atherogenesis.
COS-7 cells were grown in six-well polystyrene culture wells using DMEM+10% FCS. Vascular SMC cultures were prepared from aortas of normal New Zealand White rabbits by the explant method as previously described.21 Briefly, isolated thoracic aortas were cut into 4-mm squares and placed on culture dishes, and DMEM/F12 with 20% FCS, penicillin (100 IU/mL), and streptomycin (100 μg/mL) were added. Cells from passages 3 through 6 were grown to confluence in DMEM/F12 with 10% FCS in a 5% CO2 atmosphere at 37°C. These cells were confirmed to be of vascular smooth muscle origin by immunohistochemistry with a monoclonal antibody directed against α-actin (Sigma Chemical Co).
When SMCs reached confluence, growth medium was changed to HBSS (Irvine Scientific) for 4 hours. Cells were then exposed to oxLDL (30 μg/mL) or LPS (10 μg/mL). In some experiments, the NO donor DETA-NO (100 μmol/L) was added 2 hours before and throughout the time of stimulation. Cells were washed with HBSS and lysed in acid guanidinium isothiocyanate solution, and total RNA was isolated by phenol extraction by the method of Chomczynski and Sacchi.22 Total RNA was denatured by heating, size fractionated on a 1.5% agarose/2.2 mol/L formaldehyde gel, and transferred to a nylon membrane. Hybridizations were performed overnight at 42°C with a 32P-labeled, random-primed full-length rabbit MCP-1 cDNA (kindly provided by Tezio Yoshimura) or human GAPDH (ATCC) in 50% formamide, 10 μg/mL sheared salmon sperm DNA in 6× SSC/5× Denhardt’s solution/0.5% SDS. Blots were then washed twice in 2× SSC/0.1% SDS for 15 minutes at room temperature and then twice in 0.4× SSC/0.1% SDS for 15 minutes at 65°C.
Nuclear run-on assay was performed as previously described. Confluent rabbit SMCs were exposed to the conditions outlined above. Cells were washed twice with HBSS and removed from flasks by scraping with a rubber policeman. Cells were then centrifuged at 300g at 4°C, and the pellet was resuspended in lysis buffer containing 10 mmol/L Tris-HCl (pH 7.4), 10 mmol/L NaCl, 3 mmol/L MgCl2, and 0.5% NP-40; vortexed; and allowed to incubate on ice for 5 minutes. The lysate was recentrifuged at 500g for 5 minutes at 4°C, and the nuclear pellet was resuspended in NP-40 lysis buffer. Nuclear lysates were then centrifuged at 500g for 5 minutes at 4°C, and the nuclear protein pellet was resuspended in 200 μL glycerol storage buffer containing 50 mmol/L Tris-HCl (pH 8.3), 40% glycerol, 5 mmol/L MgCl2, and 0.1 mmol/L EDTA.
Nuclear run-on using the nuclear pellets (100 μL) was carried out at 37°C for 30 minutes in buffer containing 10 mmol/L Tris-HCl (pH 8.0), 5 mmol/L MgCl2, 300 mmol/L KCl, 50 μmol/L EDTA, 1 mmol/L dithiothreitol, 0.5 U RNase inhibitor, 0.5 mmol/L CTP, ATP, and GTP, and 250 μCi [32P]UTP. The reaction was terminated by incubating the reaction mixture with 40 U DNase I for 10 minutes at 30°C.
Equal amounts (1 μg) of purified, denatured full-length MCP-1 or GAPDH cDNA were vacuum-transferred onto nylon membranes with a slot-blot apparatus. Radiolabeled transcripts from nuclear run-on reactions were then resuspended in 4 mL of hybridization buffer containing 50% formamide, 5× SSC, 2.5× Denhardt’s solution, 25 mmol/L sodium phosphate buffer (pH 6.5), 0.1% SDS, and 250 μg/mL salmon sperm DNA. Hybridization of radiolabeled transcripts to the nylon membranes was carried out at 42°C for 48 hours. The membranes were then washed twice with 1× SSC with 0.1% SDS for 30 minutes at 65°C before autoradiography for 72 hours at −80°C.
LDL was isolated by density-gradient ultracentrifugation of normal human plasma collected in EDTA (1 mg/mL). The protein fraction was quantified by Lowry assay with BSA as standard. oxLDL was prepared by incubation of LDL (100 μg/mL) in 2 mL F-10 medium containing CuSO4 (10 μmol/L) in a 37°C incubator for 24 hours. BHT was then added to halt the oxidation process. The extent of oxidation was monitored by measurement of thiobarbituric acid–reactive substances at 550 Å, as previously described.23 Copper oxidation of LDL routinely produced 40 to 60 nmol thiobarbituric acid–reactive substances per milligram LDL.
To test the chemotactic activity of rabbit SMCs, a coculture system was applied using transformed HUVECs grown on fibronectin-coated polytetrafluoroethylene inserts with 3.0-μm pores above rabbit SMCs grown to confluence in the bottom chamber at 37°C. SMCs were stimulated with LPS in the presence or absence of NO donor as indicated above for 6 hours. THP-1 monocytoid cells (3×106 cells/mL) were then added to the top chamber for an additional 4 hours. Nonmigrating leukocytes were subsequently enumerated from the upper chamber by microscopy. Transmigrating monocytes were expressed as a percentage of the total number initially seeded.
To determine whether NO regulated endothelial redox state by reducing superoxide anion production, the following studies were performed. Superoxide anion production by SMCs was monitored by lucigenin chemiluminescence using a modification of the method previously reported.24 After undergoing the described protocols, SMCs were detached from culture dishes with EDTA, washed with PBS, and resuspended in HBSS containing bis-N-methyl-acridinium nitrate (lucigenin) (250 μmol/L). Superoxide was monitored in a Turner Designs luminometer for 1 minute with a 30-second delay. The relative specificity of lucigenin-induced chemiluminescence by superoxide anion is demonstrated by the lack of effect of scavengers of hydrogen peroxide and by the potent effect of Tiron, an intracellular scavenger of superoxide, to block the signal.25
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared as described by Dignam et al.26 Cells from the appropriate conditions were harvested and washed in ice-cold PBS. The remaining steps were performed on ice or at 4°C. Cells were resuspended in Buffer A (in mmol/L: PMSF 0.5, HEPES 10 [pH 7.8], MgCl2 1.5, KCl 10, and DTT 0.5) containing 0.1% Noninet P-40 and disrupted with a tight-fitting Dounce homogenizer. Nuclei were then pelleted by centrifugation (25 000g, 20 minutes, 4°C). Crude nuclei were resuspended in Buffer C (in mmol/L: HEPES 20 [pH 7.8], NaCl2 0.42, MgCl2 1.5, EDTA 0.2, DTT 0.5, and PMSF 0.5, and 25% vol/vol glycerol) and incubated on ice for 30 minutes. The mixture was then spun at 25 000g for 20 minutes at 4°C, the supernatant was collected, and protein was quantified. Nuclear proteins were stored at −85°C until gel shift assay. Binding reactions were carried out by mixing nuclear proteins with a double-stranded oligonucleotide corresponding to the published NFκB binding domain (cs 5′-AGT TGA GGG GAC TTT CCC AGG C). Reactions were performed with 32P-labeled DNA oligonucleotide in the presence of 1 mmol/L MgCl2, 0.5 mmol/L EDTA, 0.5 mmol/L DTT, 50 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 7.5), and 0.05 mg/mL poly-dIdC in 20% vol/vol glycerol. Samples were separated on a 4% nondenaturing polyacrylamide gel and exposed to x-ray film overnight.
To confirm the transcriptional regulation by NO of MCP-1 expression, we used a promoter construct of the MCP-1 gene. Chimeras containing a DNA fragment of 400 bp, which corresponds to the 5′ flanking region of the 540-bp fragment of the MCP-1 gene, were made with a promoterless luciferase reporter gene by ligating the MCP-1 promoter fragment into the Kpn I and Xho I sites of plasmid pGL2-Basic (Promega).27 Two putative consensus TRE sequences and one NFκB binding motif are found in the 5′ flanking region of the MCP-1 gene.
Purified DNA plasmids were transfected into SMCs or COS cells (at 60% confluence) by conventional cationic liposome transfection methods (Lipofectamine, Life Technologies). The pSV-β-galactosidase plasmid, which contains a β-galactosidase gene driven by SV40 promoter and enhancer, was cotransfected to monitor transfection efficiency. After incubation in 5% CO2/95% air at 37°C for 6 hours, cells were washed with PBS and incubated with fresh DMEM for another 48 hours before harvest.
To release the reporter luciferase and β-galactosidase, cells were lysed with Triton X-100. Luciferase activity was measured by incubating cell extract with luciferase assay reagent (Promega) containing coenzyme A (270 μmol/L), luciferin (470 μmol/L), and ATP (530 μmol/L) and measuring the activity by luminescence. Separately, the level of β-galactosidase was assayed by adding the substrate o-nitrophenyl-β-d-galactopyranoside (1.33 mg/mL) to the cell lysate and incubating at 37°C for 1 hour. The reaction was quenched by addition of Na2CO3 (1 mol/L), and the absorbance at 420 nm was recorded. The expression of luciferase is normalized to that of β-galactosidase.
To extend our observation regarding NO regulation of MCP-1 expression to an in vivo model, the following studies were performed. Male New Zealand White rabbits were pair-fed, receiving one of the following dietary interventions for 2 weeks: normal rabbit chow (control group, n=3); rabbit chow enriched with 1% cholesterol (n=3) (ICN Biomedical); or 1% cholesterol chow supplemented with 2.25% l-arginine HCl (Sigma Chemical Co) in the drinking water (n=3) ad libitum throughout the course of the study. We have previously shown that this regimen of l-arginine doubles plasma arginine and enhances vascular NO activity, as demonstrated by bioassay and chemiluminescence. Some animals received a normal chow diet supplemented with the NOS antagonist LNA (10 mg/100 mL; n=5) administered in the drinking water ad libitum throughout the course of the study (for an average daily dose of 13.5 mg · kg−1 · d−1). This dose of LNA has been shown to significantly reduce vascular NO activity.18 Total plasma cholesterol levels and HDL cholesterol were enzymatically measured with a spectrophotometric assay (Sigma). Aliquots of plasma were deproteinized with 2% sulfosalicylic acid and analyzed for free arginine with an automated amino acid analyzer (Beckman 6300). These protocols were approved by the Administrative Panel on Laboratory Animal Care of Stanford University and were performed in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care.
To perform quantitative analysis of whole-vessel mRNA, the following procedure was used. Thoracic aortas were harvested and snap-frozen in liquid nitrogen. Total RNA was isolated by acid guanidinium thiocyanate lysis. RNA (20 μg) from three rabbits in each group was pooled, and 60 μg total RNA was loaded and separated by 1% formaldehyde gel electrophoresis. MCP-1 mRNA expression was determined by Northern analysis with a full-length cDNA probe for rabbit MCP-1. For positive control, isolated aorta from normal rabbits was exposed ex vivo to LPS/ConA for 4 hours.
Band intensities from the Northern and nuclear run-on assay blots were analyzed densitometrically by Image Analyst software (Automatix). Data are expressed as mean±SEM. Comparisons of multiple means were made by ANOVA followed by a Fisher’s exact test. A value of P<.05 was accepted as statistically significant.
NO Donor Decreases Expression and Activity of MCP-1 in SMCs
The expression of MCP-1 was increased in rabbit aortic SMCs incubated with oxLDL (30 μg/mL) or with LPS (10 μg/mL) for 6 hours (Fig 1⇓, top). By contrast, when the cells were previously exposed to the NO donor DETA-NO for 2 hours and then exposed to LPS or oxLDL in the presence of DETA-NO, MCP-1 mRNA levels were markedly reduced. Densitometric analysis of autoradiographic bands showed a 50% to 75% reduction in MCP-1 expression in DETA-NO–treated cells.
To determine whether the effect of NO was at the level of transcriptional regulation, nuclear run-on studies were performed with LPS stimulation. In vitro transcription studies indicated that there is minimal transcriptional activity of the MCP-1 gene in rabbit SMCs grown in standard culture conditions. Treatment with LPS (10 μg/mL) significantly increased MCP-1 mRNA transcription after 6 hours relative to GAPDH gene transcription. This effect was reduced 10-fold by concomitant administration of NO donor (Fig 1⇑, bottom). To further investigate transcriptional regulation of MCP-1, rabbit aortic SMCs and COS cells were transfected with an MCP-1 promoter construct containing the luciferase reporter gene. Cells were stimulated with LPS (10 μg/mL) for 6 hours (Fig 2⇓). Chimeras containing segments of the promoter region responded to stimulation, as reflected by increased luciferase activity (COS cells, 4930±7610 and SMCs, 3510±381 AUC). Coincubation with DETA-NO reduced luciferase activity in cells transfected with these promoter fragments (COS cells, 1276±258 and SMCs, 1802±305 AUC; P<.05 versus cells stimulated by LPS in the absence of DETA-NO).
Increased transcriptional activity of rabbit SMC MCP-1 gene activity was associated with increased chemotactic activity. Fig 3⇓ illustrates that the monocyte chemotactic activity of SMCs stimulated with LPS was significantly increased over baseline conditions. The addition of the NO-donor DETA-NONOate reduced this chemoattraction to near-baseline conditions.
Superoxide Generation and Gel Shift Analysis
The transcriptional pathway mediated by NFκB appears to be activated by cytokine-induced oxidative stress in other cell systems. Molecular cloning of the MCP-1 promoter has provided evidence for an NFκB binding domain at position −148 in the 5′ flanking region. To determine whether a similar mechanism mediated MCP-1 induction by cytokines or oxidized lipoproteins, nuclear extracts from SMCs were isolated and electrophoretic mobility shift analysis was performed with an oligonucleotide containing the putative NFκB binding site. As shown in Fig 4⇓, prior exposure of the SMCs to LPS (10 μg/mL, lane 2) for 6 hours induced activation of NFκB, which is consistent with the hypothesis that an NFκB binding site was required for MCP-1 regulation by oxidized lipid or cytokines. To determine whether NO suppressed MCP-1 induction by regulating NFκB activity, stimulated SMCs were incubated with the NO donor DETA-NO. Stimulation of NFκB activity induced by LPS was greatly reduced in cells that were also exposed to the NO donor (lane 4). The intensity of the bands representing the shifted complexes are reduced by the addition of excess unlabeled oligonucleotide (lanes 3 and 5).
To determine whether NO was exerting its effect on NFκB by reducing oxidative stress, we examined the effect of NO on superoxide production using lucigenin chemiluminescence. oxLDL or LPS stimulation for 6 hours significantly increased the generation of superoxide anion (control, 127±11; oxLDL, 200±12; and LPS, 240±36 AUC/mg protein; P<.05 from control). Coincubation with the NO donor reduced superoxide generation induced by oxidized lipid or cytokines (P<.05) (Fig 5⇓).
Endogenous NO Decreases MCP-1 Expression In Vivo
To determine whether vascular NO regulates MCP-1 expression in vivo, the following study was performed. New Zealand White rabbits received the following dietary interventions: normal chow; 0.5% cholesterol chow; 0.5% cholesterol chow and 2.25% l-arginine in the drinking water; or normal chow and LNA (≈13.5 mg · kg−1 · d−1) in the drinking water. We have previously demonstrated by chemiluminescence and bioassay that this dose of l-arginine has no effect on the lipid profile, doubles the plasma l-arginine concentration, and enhances endothelium-derived NO production.18 The dose of LNA (an NOS antagonist) used in the present study reduces vascular NO production by 50% in the rabbit thoracic aorta.18 After 2 weeks of these interventions, the rabbit thoracic aortas were harvested and total RNA was isolated by guanidinium thiocyanate lysis. MCP-1 mRNA expression was determined by Northern analysis. MCP-1 was undetectable in total RNA derived from the thoracic aortas of normocholesterolemic animals (n=3) (Fig 6⇓). Ex vivo exposure of these aortas to LPS/ConA for 4 hours induced MCP-1 expression. MCP-1 expression was observed in the thoracic aortas of hypercholesterolemic rabbits (n=3). In pair-fed hypercholesterolemic animals receiving dietary arginine supplementation to increase endogenous NO production, the expression of MCP-1 was attenuated. Most remarkably, normocholesterolemic animals exposed to the NOS antagonist LNA expressed MCP-1 at levels that exceeded those of hypercholesterolemic animals.
The salient findings of this study are as follows.
Exogenous or endogenous NO suppresses the induced expression and activity of MCP-1 in rabbit vascular cells. This effect of NO is exerted at the level of transcriptional regulation.
The administration of an NO donor is associated with inhibition of superoxide anion elaboration and a reduction in NFκB activity in vascular SMCs.
This study provides insight into the mechanism by which endogenous NO may act to inhibit atherogenesis. Previous observations by our group and others have revealed that modulation of vascular NO activity can significantly influence the course of lesion formation in experimental hypercholesterolemia.16 17 18 19 Hypercholesterolemia reduces the influence of endothelium-derived NO by its degradation and/or reduced production.14 15 28 NO activity can be restored by administration of the NO precursor l-arginine in hypercholesterolemic animals and humans.29 30 31 Chronic administration of the NO precursor inhibits endothelial adhesiveness for monocytes and reduces lesion formation in the thoracic aortas and coronary arteries of hypercholesterolemic rabbits.18 19 20 By contrast, chronic administration of NOS antagonists reduces the production of endothelium-derived NO, enhances endothelial adhesiveness, and promotes monocyte accumulation in the vessel wall.16 17
Accumulating evidence supports the hypothesis that NO exerts its effect on monocyte adherence and accumulation in part by modulating the activity of redox-responsive transcriptional pathways.11 32 33 In our study, stimulation of SMCs increased superoxide production, NFκB activity, activity of a transfected MCP-1 promoter–luciferase chimera, transcription of MCP-1 mRNA, and chemotactic activity for monocytoid cells. These effects were all suppressed by the NO donor DETA-NO. These observations indicate that NO reduces transcription of MCP-1; it is also possible that NO may reduce the half-life of MCP-1 mRNA, although this was not directly examined in this study.
NO may act by reducing intracellular oxidative stress. In the present study, oxLDL and LPS induced the generation of superoxide anion by SMCs in culture (the condition of serum starvation used in these experiments may have contributed to the increased oxidative stress,34 which raises the question whether NO would play a similar role in a more physiological environment). NO can scavenge superoxide anion, although the product of this reaction, peroxynitrate anion, is itself a highly reactive free radical.35 However, it is possible that peroxynitrate anion could subsequently nitrosate sulfhydryl groups to form S-nitrosothiols. This class of molecules is known to induce vasodilation, inhibit platelet aggregation, and interfere with leukocyte adherence to the vessel wall.36 37
Another mechanism by which NO may ameliorate oxidative stress is by terminating the autocatalytic chain of lipid peroxidation that is initiated by oxLDL or intracellular generation of oxygen-derived free radicals. Indeed, exogenous NO inhibits copper-induced oxidation of LDL cholesterol, causing a lag in the formation of conjugated dienes.38 Finally, NO may directly suppress the generation of oxygen-derived free radicals by nitrosylating, and thereby inactivating, oxidative enzymes. This hypothesis is supported by the observation that the generation of superoxide anion by stimulated neutrophils is reduced by their exposure to exogenous NO.39 This is due to the inactivation of NADPH oxygenase, a multimeric enzyme with cytosolic and particulate components. The particulate component is vulnerable to nitrosylation by NO (at either its heme moiety or sulfhydryl group), which prevents its association with the cytosolic component and reconstitution of the active enzyme. A similar phenomenon may occur in ECs. This would explain the observation of Niu and colleagues,40 who reported that antagonism of endogenous NO production increases oxidative stress in HUVECs, as demonstrated with redox-sensitive fluorophores. Furthermore, Pagano and colleagues25 showed that exogenous NO donors inhibit the generation of superoxide anion by the endothelium of rabbit thoracic aortas treated ex vivo with antagonists of superoxide dismutase.
It is well established that hypercholesterolemia reduces the activity of endothelium-derived NO.14 15 In parallel, the endothelium begins to generate superoxide anion.28 This alteration in endothelial redox state triggers a transcriptional cascade that results in the activation of genes encoding molecules that regulate endothelial adhesiveness.32 41 Cytokines and lysophosphatidylcholine induce the expression by HUVECs of vascular cell adhesion molecule-1, an endothelial immunoglobulin implicated in monocyte adhesion and atherogenesis.33 42 This expression is regulated by an NFκB-mediated transcriptional pathway that is blocked by exposure of the cells to the antioxidant pyridoldiothiocarbamate32 or aspirin.41
The cytokine-induced activation of vascular cell adhesion molecule-1 and macrophage colony–stimulating factor in human saphenous vein ECs is suppressed by NO donors.11 33 43 This effect of NO appears to be due in part to stabilization and/or increased expression of IκBa, which complexes with NFκB to inhibit its transcriptional activity.33
Our data are consistent with this model of an oxidant-responsive NFκB-mediated pathway that is modulated by NO. Recently, Zeiher and colleagues44 have also provided evidence that NO inhibits MCP-1 expression in cytokine-stimulated HUVECs in a cGMP-independent fashion.
To summarize, we provide in vitro and in vivo evidence that NO suppresses the expression of MCP-1, a major chemokine for monocytes. NO appears to exert its effects by reducing intracellular oxidative stress, thereby defusing oxidant-triggered transcription. This study has elucidated one mechanism by which NO can serve as an endogenous antiatherogenic molecule. Therapeutic strategies to enhance vascular NO activity may augment vasodilation and inhibit the progression of atherosclerosis.
Selected Abbreviations and Acronyms
|AUC||=||arbitrary units of chemiluminescence|
|COS||=||SV40-transformed monkey fibroblasts|
|DETA-NO||=||donor ethanamine 2,2′-(hydroxynitrosohydrazonon)bis|
|HUVEC||=||human umbilical vein endothelial cell|
|LPS/ConA||=||lipopolysaccharide, 10 μg/mL–concanavalin A, 5 μg/mL|
|MCP-1||=||monocyte chemotactic protein-1|
|NOS||=||nitric oxide synthase|
|SMC||=||smooth muscle cell|
- Received August 16, 1996.
- Revision received January 31, 1997.
- Accepted February 7, 1997.
- Copyright © 1997 by American Heart Association
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