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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:490-497.)
© 1997 American Heart Association, Inc.

Articles

Tumor Necrosis Factor- Activates Smooth Muscle Cell Migration in Culture and Is Expressed in the Balloon-Injured Rat Aorta

Stefan Jovinge; Anna Hultgårdh-Nilsson; Jan Regnström; ; Jan Nilsson

From the King Gustaf V Research Institute, Karolinska Hospital (S.J., J.R., J.N.), and the Department of Cell and Molecular Biology, Division of Cell Biology, Karolinska Institute (A.H.N.), Stockholm, Sweden.

Correspondence to Dr Stefan Jovinge, King Gustaf V Research Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail jovinge{at}instmed.ks.se.

	Abstract

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Abstract In experimental models of atherosclerosis, activation of smooth muscle cell (SMC) migration from the media to the intima is preceded by intimal accumulation of inflammatory cells, suggesting that cytokines may be involved in this process. The present study demonstrates that tumor necrosis factor- (TNF-) regulates cytoskeletal organization of SMCs by inducing depolymerization of actin stress fibers and dispersion of vinculin from sites of focal adhesion and stimulates the migration of cultured human SMCs in a dose-dependent manner. Moreover, TNF- induces rapid activation of the c-ets-1 gene, which codes a transcription factor known to regulate enzymes important for matrix degradation during cell migration. Balloon catheter injury of the rat femoral artery resulted in medial expression of TNF- within 6 hours. This expression appeared to be localized to SMCs and remained elevated until SMCs began to migrate into the intima 7 days after injury. These findings demonstrate that TNF- has a stimulatory effect on SMC migration and suggest that TNF- may be involved in the intimal recruitment of SMCs during plaque formation.

Key Words: tumor necrosis factor- • smooth muscle cells • migration • ets-1

	Introduction

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SMCs and the ECM produced by them constitute a major part of atherosclerotic lesions.^{1 2} Animal studies of lipid-induced atherosclerosis have shown that most intimal SMCs are recruited from the underlying media and that inflammatory cells play a critical role in this process. Most important are the T cells and monocytes/macrophages, which release potent cytokines and other growth factors that affect the function of surrounding cells.^{3 4} Several of these factors are known to act as mitogens for SMCs and some, such as PDGF-BB and IL-1, have also been shown to affect SMC migration.⁵ Activation of SMC migration is also an important event in neointima formation after mechanical injury of arteries. Studies demonstrating that antibodies against PDGF-BB block some of the balloon injury–induced migration in rat carotid arteries suggest that the PDGF released from platelets may have an important role in this process.⁶

The role of TNF- in atherosclerosis has lately been receiving much attention. TNF- is present in atherosclerotic plaques but not in normal vessels^{7 8} and has important effects on endothelial function, such as regulation of adhesion molecule expression^{9 10 11} and anticoagulant and fibrinolytic capacity.^{12 13} The effects of TNF- on SMCs include stimulation of DNA synthesis¹⁴ and ICAM-1 expression.¹⁵ TNF- was originally regarded as an exclusive product of activated macrophages, but more recently SMCs have been identified as an alternate source of this cytokine.^{7 16} Moreover, in transplantation atherosclerosis, an association between TNF- expression and induction of DNA synthesis in medial SMCs has been described.¹⁷

TNF- has also been found to regulate the migration of other cell types, such as fibroblasts and monocytes.^{18 19} This effect is associated with alterations in the cytoskeletal organization, including the transient disappearance of F-actin stress fibers and loss of vinculin from focal adhesion sites.^{20 21 22} Another important factor in the activation of SMC migration is degradation of the surrounding ECM. This is achieved by release of matrix metalloproteinases, such as interstitial collagenase, gelatinase, and stromelysin.²³ Transcription factor ets-1, which is encoded by proto-oncogene c-ets-1, increases transcription of the stromelysin gene.²⁴ Moreover, transcription of the urokinase plasminogen activator gene is also increased by members of the ets family,²⁵ and by urokinase plasminogen activator's activation of plasminogen, they increase collagenase, gelatinase, and stromelysin activity.²⁶

In this study we examined the role of TNF- in the regulation of SMC migration by analyzing its effect on cytoskeletal organization, expression of the c-ets-1 gene, and cell migration into an artificial wound in culture. We also analyzed the expression of TNF- by SMCs during migration from the media to the intima after balloon injury of the rat femoral artery.

	Methods

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Isolation and Culture of SMCs
SMCs were isolated from the media of a normal saphenous vein obtained from a patient undergoing coronary artery bypass surgery. The specimens were carefully dissected, cut into small pieces, and allowed to become attached to the surface of six-well multiplates by drying for 15 minutes. The explants were then cultivated in F-12 medium containing 10% newborn calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in an atmosphere of 5% CO₂ in air. Cells began to migrate from the explants within 1 to 2 weeks and reached confluence within another 2 weeks. Secondary cultures were established by trypsinization and seeding of the cells in 75-cm² culture flasks. The purity of the SMC populations was determined by the presence of muscle-specific -actin immunoreactivity with the HHF 35 antibody.²⁷ Cultures were growth arrested by incubation in F-12 medium supplemented with antibiotics and 0.1% low-endotoxin BSA (Sigma Chemical Co) for 48 hours. Experiments were performed in F-12 medium containing antibiotics and 0.1% low-endotoxin BSA. All tissue-culture reagents except BSA were obtained from GIBCO BRL. Experiments were performed on cells cultured for up to 10 passages. Endotoxin levels in the experimental media were determined by the Limulus assay (Sigma) and were always <0.5 ng/mL.

Analysis of SMC Migration
Cells were seeded onto glass coverslips and grown to confluence in F-12 medium containing antibiotics and 10% newborn calf serum. After 48 hours of serum starvation, an artificial wound was created in the cultures by gentle pressure for 10 seconds with a 5-mm-wide soft plastic tube. The cultures were then washed in F-12 medium and incubated with the respective experimental medium. Recombinant human TNF- and anti-human TNF- monoclonal neutralizing antibody were obtained from R&D Systems and an isotypic control (mouse IgG2b) was obtained from Immunotech. After 24 hours the cells were fixed in 3% buffered glutaraldehyde for 1 hour at 4°C, dehydrated in ethanol, and stained with methylene blue. The distance between the margin of injury and the point on the migrating cells most distant from that margin was measured with an Optilab 2.03 image analysis program (Graftek) on a Macintosh IIci computer. Only those cells that had migrated from a distinct and continuous border were included in the analysis. All cells (50) that fulfilled this criterion were analyzed in three independent samples.

Analysis of Stress Fiber and Vinculin Distribution in Cultured Human SMCs
Human SMCs were grown on glass coverslips and exposed to experimental medium as described for the migration assay. Staining of F-actin stress fibers was performed with FITC-phalloidin (Sigma) as described by Wulf et al,²⁸ and the coverslips were then mounted onto glass slides with 0.1% p-phenylendiamine/PBS/glycerol.

For vinculin staining, cells were rinsed in PBS and then fixed and permeabilized in 95% ethanol for 5 minutes at 20°C. They were subsequently rinsed in PBS, incubated with 10% normal pig serum for 20 minutes, and incubated with a mouse anti-human vinculin antibody (dilution, 1:400) for 16 hours at 4°C. After they were washed four times in PBS, the sections were incubated with fluorescein-labeled pig anti-mouse IgG (Dako; dilution, 1:30) for 60 minutes. Finally, the samples were washed four times in PBS and mounted in 0.1% p-phenylendiamine PBS/glycerol.

A semiquantitative analysis to determine the fraction of cells with frequent and distinct stress fibers versus those with few, less condensed, or no stress fibers was performed by a blinded investigator. At least 100 cells were counted in each culture.

Analysis of c-ets-1 Expression
RNA was extracted essentially according to the method of Chirgwin et al.²⁹ In brief, the cells were washed twice with PBS at 4°C; lysed with 4 mol/L guanidine isothiocyanate, 0.03 mol/L sodium acetate, and 1.0% ß-mercaptoethanol, and scraped from the dishes with a rubber policeman. The homogenized suspensions were carefully loaded onto 4 mL of 5.7 mol/L CsCl and centrifuged for 20 hours at 33 000 rpm at 20°C in an SW 40 Ti rotor. The supernatant was carefully removed and the RNA pellet resuspended in buffer. The solubilized RNA was precipitated overnight in 0.3 mol/L sodium acetate and 2.5 volumes of ethanol at -70°C. The isolated RNA was pelleted by centrifugation at 13 000 rpm for 30 minutes at 4°C, washed with 70% ethanol, dried, and resuspended in sterile water. Quantity and purity of the RNA preparation was determined spectrophotometrically at 260 and 280 nm. Gel electrophoresis of 20 µg total RNA was performed on 1.4% agarose gels containing 2.2 mol/L formaldehyde according to Lehrach et al.³⁰ Hybridization was performed in 50% formamide, 5x SSC (43.8 g/L NaCl and 22 g/L sodium citrate), 5x Denhardt's solution (1 g/L polyvinylpyrrolidone, 1 g/L BSA, and 1 g/L Ficoll 400), 0.1% SDS, 100 µg/mL salmon sperm DNA, and 10% dextran sulfate for 20 hours at 42°C with ³²P-labeled DNA probes. Probes were labeled with [-³²P]dCTP by the random-primer technique according to the manufacturer's protocol (Stratagene). The probes used were ets-1³¹ and ribosomal RNA.³²

Animals, Surgical Procedures, and Tissue Handling
Adult male Sprague-Dawley rats weighing approximately 400 g were anesthetized with sodium pentobarbital (30 mg/kg SC). The left carotid artery was exposed and a Fogarty arterial embolectomi catheter 2F (Baxter) advanced to the distal part of the femoral artery. The balloon was inflated with 0.7 mL saline for 1 minute and then withdrawn to the aortic bifurcation. This balloon injury was repeated twice. The carotid artery was subsequently ligated. The animals were euthanized at different times after injury, heparinized (2000 U/kg), perfused for 5 minutes with 0.9% NaCl, and perfusion fixed in 4% p-formaldehyde for 10 minutes. The tissues were immersed in 4% p-formaldehyde at 4°C for 6 hours and in 15% sucrose at 4°C overnight. After the samples were embedded in OCT compound, they were frozen at -70°C. Three sections from two animals were analyzed for each time point.

Immunohistochemical Analysis of TNF- Expression in Injured Arteries
Cryostat sections (6 µm) were dried under vacuum for 5 minutes, washed in PBS, and incubated with 0.3% H₂O₂ for 30 minutes followed by a 60-minute incubation in 10% goat serum at room temperature. The sections were then incubated with a mouse monoclonal antibody against rat monocytes/macrophages (ED-1, Serotec) and a polyclonal rabbit anti-mouse TNF- antibody (Genzyme),³³ diluted 1:50 in PBS with 10% goat serum overnight at 4°C. The latter antibody was raised against purified recombinant mouse TNF- and does not cross-react with human TNF-. After three washes in PBS, biotinylated goat anti-rabbit and horse anti-mouse antibodies (Vector) were applied for 30 minutes at room temperature. The sections were washed three times in PBS followed by an avidin-biotin horseradish peroxidase complex for 30 minutes. Antibody binding was visualized with 3,3'-DAB. Sections were counterstained with hematoxylin. Omission of the primary antibody served as a negative control.

Statistical Methods
Values are expressed as mean±SEM. Differences between groups were analyzed by Student t test. A value of P <.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
SMC Migration in Response to TNF-
Microscopic analysis of the wound area 5 minutes after injury demonstrated a distinct demarcation between the cell-free wound area and the remaining intact cell layer. No cells were seen protruding into the wound. At 24 hours after injury, cells had migrated from the confluent layer into the injury zone (Fig 1). In cultures grown in serum-free medium without TNF-, the mean migratory distance was 127±5 µm. PDGF-BB, a potent activator of SMC migration, was used as positive control in the assay. Addition of 10 ng/mL PDGF-BB increased the mean migratory distance at 24 hours after injury to 233±9 µm (P<.001 compared with control cultures). Addition of TNF- increased SMC migration into the injury zone in a dose-dependent manner, and at a concentration of 4 ng/mL, TNF- was as effective as 10 ng/mL of PDGF in activating SMC migration (236±12 µm, P<.001 versus control cells; Fig 2). There was no significant difference in the number of cells migrating into the injury zone between serum-free control cultures and cultures incubated with TNF- (data not shown). Addition of a neutralizing TNF- antibody reduced the migration induced by exogenous TNF- by 87% (P<.0001), whereas basal postinjury migration was reduced by only 7% (NS, Fig 3).

View larger version (89K): [in this window] [in a new window]   Figure 1. Migration of SMCs into an artificial cell-culture wound. Micrographs show the wound area 24 hours after injury in cultures grown in (A) serum-free medium and (B) medium containing 4 ng/mL TNF-. Bar = 150 µm.

View larger version (10K): [in this window] [in a new window]   Figure 2. Migration of SMCs in response to TNF-. Serum-starved, confluent cultures of SMCs were injured by gentle pressure with a rubber policeman. The mean migration distance (MMD) of 50 cells in each culture was determined by image analysis. Values represent the mean and SEM.

View larger version (15K): [in this window] [in a new window]   Figure 3. Effect of TNF-–neutralizing antibody on basal and TNF-–induced migration after 24 hours of treatment. A mouse monoclonal anti-human TNF-–neutralizing antibody (0.05 µg/mL) was preincubated with medium containing 4 ng/mL recombinant human TNF- or control medium. The mean migration distance (MMD) of 50 cells was determined by image analysis as described in "Methods." Values represent the mean and SEM.

Alterations in Cytoskeletal Organization in Response to TNF-
To study the mechanisms whereby TNF- influences SMC migration, we used FITC-labeled phalloidin to analyze F-actin organization in stress fibers. Because of the difficulty in visualizing individual stress fibers in dense cultures, these studies were performed in subconfluent cultures of SMCs. Untreated control cells contained numerous stress fibers that usually extended the entire length of the cells. Exposure of the cells to TNF- resulted in marked reduction of stress fiber expression (Fig 4), similar to that observed in cells incubated with 10 ng/mL PDGF-BB. Quantification of the F-actin response to TNF- confirmed that the effect was both time and concentration dependent (Fig 5). After 24 hours of exposure to TNF-, the normal stress fiber configuration returned, demonstrating that the effect was transient.

View larger version (98K): [in this window] [in a new window]   Figure 4. FITC-phalloidin staining of stress fibers in SMCs. A, Control cells incubated in serum-free medium after 4 hours; B, cells incubated with 10.0 ng/mL PDGF-BB for 1/2 hour; C, cells incubated with 1.0 ng/mL TNF-, and D, cells incubated with 4.0 ng/mL TNF- for 4 hours. Bar=10 µm.

View larger version (18K): [in this window] [in a new window]   Figure 5. Quantitative analysis of the fraction of cells with stress fiber depolymerization. Serum-starved subconfluent cultures of SMCs were incubated with increasing concentrations of TNF- for 30 minutes (open circles) or 4 hours (closed circles). Stress fibers were stained with FITC-phalloidin. At least 100 cells were counted in each culture. Each value represents the mean and SEM of four independent samples. ***=P<.001.

F-actin stress fibers are associated with the plasma membrane at focal contacts through vinculin, paxillin, and tallin, which bind to different integrins.³⁴ To further evaluate the changes in cytoskeletal organization, SMCs were stained with an antibody against vinculin. Control cells contained scattered streaks of vinculin immunoreactivity that are characteristic of vinculin accumulation at focal adhesion sites. Incubation of the cells with TNF- resulted in a reduction of vinculin streaks and the formation of a diffuse, faint cytoplasmic stain (Fig 6), further supporting the notion that TNF- has direct effects on the organization of the cytoskeleton in SMCs. A similar effect was seen in response to 10 ng/mL PDGF-BB. In accordance with the previous observation of F-actin distribution, a normal vinculin stain was seen 24 hours after the addition of TNF-.

View larger version (98K): [in this window] [in a new window]   Figure 6. Vinculin immunostaining in SMCs. Vinculin staining of stress fibers in SMC. A, Control cells incubated in serum-free medium after 4 hours; B, cells incubated with 10.0 ng/mL PDGF-BB for 1/2 hour; C, cells incubated with 1.0 ng/mL TNF- for 4 hours; and D, cells incubated with 4.0 ng/mL TNF- for 4 hours. Bar=10 µm.

ets-1 Transcription
In control cells there were no detectable levels of ets-1 mRNA. Exposure of the cells to 4 ng/mL TNF- resulted in an accumulation of ets-1 mRNA within 1 hour. A peak level of ets-1 mRNA was observed after 2 hours (2.4-fold increase as determined by densitometric scanning and normalization for 28S ribosomal RNA levels) with a subsequent decline at 6 hours (Fig 7).

View larger version (65K): [in this window] [in a new window]   Figure 7. Northern blot analysis of c-ets-1 expression of serum-starved confluent SMC cultures after treatment with 4 ng/mL TNF-. Ribosomal RNA (rRNA) was used as the loading control.

Expression of TNF- in Rat Femoral Arteries After Balloon Injury
Uninjured arteries did not contain detectable TNF- immunoreactivity. At 2 hours after balloon injury, TNF- immunoreactivity was still not detected in the artery wall, whereas 6 hours after injury there was widespread TNF- staining in the media. Most of this staining was found close to the lumen, but areas with an accumulation of TNF- immunoreactivity were also detected close to the external elastic lamina. There was no ED-1 immunoreactivity present in the media, suggesting that macrophages were not involved in the generation of TNF- immunoreactivity. At 1 and 3 days after injury, TNF- immunoreactivity was predominantly expressed by cells underlying the internal elastic lamina. One week after injury, a neointima than was several cell layers thick had developed and contained a few TNF-–positive cells. At 2 weeks after injury, the neointima was several layers thick, with some TNF-–positive cells being present toward the lumen (Fig 8). Overall, TNF- staining was stronger in the media during the first 3 days after injury than in the neointima that subsequently developed.

View larger version (129K): [in this window] [in a new window]   Figure 8. Immunohistochemical analysis of TNF- expression after balloon injury in the rat femoral artery. Micrographs show sections of (A) uninjured artery, (B) TNF- staining 2 hours after injury, (C) control without primary antibody 6 hours after injury, (D) TNF- staining 6 hours after injury, (E) TNF- staining 24 hours after injury, (F) TNF- staining 3 days after injury, (G) TNF- staining 7 days after injury, and (H) TNF- staining 14 days after injury. Arrows indicate internal elastic lamina. Bar=30 µm.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrates that TNF- induces rapid depolymerization of F-actin stress fibers and the disappearance of vinculin from focal adhesion in vascular SMCs. A similar rearrangement of the cytoskeleton has been observed in response to PDGF-BB,³⁵ a factor that regulates the migratory activity of cells and is believed to represent an important early event in the initiation of cell movement. By using a cell migration assay based on the movement of SMCs into an artificial cell culture wound, we confirmed that TNF- enhances SMC migration and that this effect is similar in magnitude to that observed by incubation with 10 ng/mL PDGF-BB. Immunohistochemical analysis of balloon-injured rat femoral arteries showed that injury results in the development of SMC-associated immunoreactivity within 6 hours. The TNF- immunoreactivity remained in the medial SMCs until they began migrating into the intima and was also present in the fraction of intimal cells during growth of the neointima, suggesting that TNF- may also be involved in regulating SMC migration in vivo. However, it should be noted that addition of TNF-–neutralizing antibodies did not decrease basal migration in response to mechanical injury in vitro, suggesting that TNF- does not have a major part in this process. The observation that TNF- activates expression of the protooncogene c-ets-1 suggests another mechanism by which TNF- may participate in the regulation of SMC migration. The 5.3- and 2.3-kb c-ets-1 mRNA bands observed in this study probably correspond to transcripts arising through alternative splicing or differential polyadenylation, as has been shown to occur within the c-ets-1 locus in T cells of different species.^{31 36} Earlier studies also demonstrated the presence of two ets-1 proteins in mesodermal cells.^{37 38} Our findings demonstrate a marked increase in the expression of the 5.3- and 2.3-kb bands, with a peak of both bands at 2 hours. The fact that the 2.3 kb-band is not visible in the pretreatment lane may reflect the fact that the Northern blotting technique is not sensitive enough to detect transcripts at this low level.

The ets family of transcription factors is known to regulate genes involved in matrix degradation, such as the matrix metalloproteinases and their activators.^{24 25} Degradation of the immediately surrounding ECM is believed to be a prerequisite for migration of cells in tissues and is likely to be of key importance for the migration of SMCs from the media to the intima.²³ This notion is further supported by the recent finding of increased expression of c-ets mRNA and protein in balloon-injured rat arteries.³⁷ Moreover, preliminary studies in our laboratory have demonstrated increased expression of c-ets mRNA and ets-1 immunoreactivity after mechanical injury of cultured SMCs (A.H-N. et al, unpublished data).

The mechanisms by which changes in the cytoskeletal network are coordinated to produce cell locomotion are incompletely understood. In fibroblasts, actin filaments are polymerized into stress fibers, which are attached to actin-binding proteins like -actinin, vinculin, paxillin, and talin at focal contacts in the plasma membrane. These actin-binding proteins adhere to the cytoplasmic part of integrin receptors, such as the fibronectin receptor, that anchors the cell to the ECM.³⁴ Diminished attachment of the cell to the surrounding matrix appears to be a critical early step in the activation of cell migration, and several motility-promoting factors have been shown to promote focal adhesion disassembly.³⁴ Our findings suggests that TNF- may affect the SMC migration by similar mechanisms. Accordingly, TNF- has also been shown to induce depolymerization of stress fibers and the disappearance of vinculin from focal contacts in endothelial and mesangial cells.²² In endothelial cells this effect is associated with an increase in the soluble monomeric G-actin pool, and de novo actin synthesis and has been linked to barrier dysfunction rather than activation of migration.^{20 39}

The idea that TNF- may influence the migration of connective tissue cells is not new. TNF- has previously been shown to act as chemoattractant for human fibroblasts, suggesting that TNF- released from macrophages is involved in the recruitment of fibroblasts during wound healing. This concept is also supported by the finding that blocking antibodies against TNF- almost completely inhibit the fibroblast chemotactic activity released from endotoxin-treated monocytes.¹⁸ Similar mechanisms may also be involved in the intimal recruitment of SMCs in atherosclerosis. In the initial stage of the atherosclerotic process, inflammatory cells such as monocytes and T cells accumulate in the arterial intima.^{3 40} Data obtained from experimental animal models of atherosclerosis suggest that intimal inflammation stimulates medial SMCs to modulate from a contractile to a synthetic phenotype and to migrate into the intima. The present findings, taken together with the observation that TNF- antibodies inhibit the chemotactic activity released from activated monocytes, suggest that TNF- may play an important role in this process.

In mechanically injured arteries, TNF- was expressed by medial SMCs until they began migrating into the intima. This observation is compatible with the idea that TNF- is involved in inducing the structural changes of the cell and its surrounding matrix that are required for activation of migration. The ability of TNF- to increase c-ets-1 expression may be of particular importance in this context. The ets-1 transcription factor regulates expression of stromelysin, a proteolytic enzyme responsible for degradation of the basal lamina surrounding medial SMC and for the activation of other matrix-degrading enzymes.²⁶ As discussed above, our findings suggest that SMCs in injured rat arteries produce TNF-. However, it should be noted that we did not use in situ hybridization or Northern blotting analysis to confirm that TNF- was produced by the medial SMCs, and we cannot completely exclude the possibility that this immunoreactivity represents TNF- taken up from the circulation by increased cellular expression of TNF- receptors. However, studies by Tanaka et al¹⁷ on rabbit cardiac allografts during acute rejection have shown that development of medial SMC immunoreactivity to TNF- is associated with the expression of TNF- mRNA, as assessed by in situ hybridization, demonstrating that medial SMCs indeed have the capacity to produce TNF-. Moreover, the same authors recently demonstrated that balloon injury of the rabbit aorta lead to increased SMC expression of both TNF- mRNA and protein levels.⁴¹ Using a transplant model of atherosclerosis, Clausell and coworkers⁴² found that in vivo blockade of TNF- with TNF-–soluble receptors inhibits neointima formation in the arteries of the transplanted heart. Since TNF- is also a potent mitogen for SMCs, this effect may be due to reduced proliferative activity of the cells rather than inhibition of migration of medial cells into the intima. However, it is likely that the mitogenic and migration-promoting effects of TNF- are mediated through the same receptor and at least in part by the same intracellular signal pathways, suggesting that the two processes cannot be separated.

In conclusion, the present investigation shows that TNF- may affect the migratory activity of SMCs, adding further support to the notion that this cytokine plays an important role in atherosclerosis.

	Selected Abbreviations and Acronyms

 

ECM = extracellular matrix ICAM = intercellular adhesion molecule IL = interleukin PDGF = platelet-derived growth factor SMC(s) = smooth muscle cell(s) TNF = tumor necrosis factor

	Acknowledgments

 
This study was supported by grants from the Swedish Medical Research Council, the Swedish Heart and Lung Foundation, the Nanna Svartz' Fund, the Tore Nilssons Fund, the Swedish Society of Medicine, King Gustaf V's 80th Birthday Fund, the Sigurd and Elsa Goljes Foundation, and Förenade Liv Mutual Group Life Insurance Company of Stockholm. PDGF-BB was generously provided by Dr Carl-Henrik Heldin, Uppsala, Sweden.

Received January 4, 1996; accepted June 18, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Faggiotto A, Ross R. Studies of hypercholesterolemia in the nonhuman primate, II: fatty streak conversion of fibrous plaque. Arteriosclerosis.. 1984;4:341-356. [Abstract/Free Full Text]

2. Masuda J, Ross R. Atherogenesis during low level hypercholesterolemia in the non-human primate, II: fatty streak conversion to fibrous plaque. Arteriosclerosis.. 1990;10:178-187. [Abstract/Free Full Text]

3. Hansson GK. Immune and inflammatory mechanisms in the development of atherosclerosis. Br Heart J. 1993;69(suppl):S38-S41.

4. Libby P, Clinton SK. The role of macrophages in atherogenesis. Curr Opin Lipidol.. 1993;4:355-363.

5. Nomoto A, Mutoh S, Hagihara H, Yamaguchi I. Smooth muscle cell migration induced by inflammatory cell products and its inhibition by a potent calcium antagonist, nilvadipine. Atherosclerosis.. 1988;72:213-219. [Medline] [Order article via Infotrieve]

6. Ferns GAA, Raines EW, Sprugel KA, Motani AS, Reidy MA, Ross R. Inhibition of neointimal smooth muscle cell accumulation after angioplasty by an antibody to PDGF. Science.. 1991;253:1129-1132. [Abstract/Free Full Text]

7. Barath P, Fishbein MC, Cao J, Berenson J, Helfant RH, Forrester JS. Tumor necrosis factor gene expression in human vascular intimal smooth muscle cells detected by in situ hybridization. Am J Pathol.. 1990;137:503-509. [Abstract]

8. Tipping PG, Hancock WW. Production of tumor necrosis factor and interleukin-1 by macrophages from human atheromatous plaques. Am J Pathol.. 1993;142:1721-1728. [Abstract]

9. Pohlman TH, Stanness KA, Beatty PG, Ochs HD, Harlan JM. An endothelial cell surface factor(s) induced in vitro by lipopolysaccharide, interleukin 1, and tumor necrosis factor- increases neutrophil adherence by a CDw 18-dependent mechanism. J Immunol.. 1986;136:4548-4553. [Abstract]

10. Wertheimer SJ, Myers CL, Wallace RW, Parks TP. Intercellular adhesion molecule-1 gene expression in human endothelial cells: differential regulation by TNF- and phorbol myristate acetate. J Biol Chem.. 1992;267:12030-12035. [Abstract/Free Full Text]

11. Roebuck KA, Rahman A, Lakshminarayanan V, Janakidevi K, Malik AB. H₂O₂ and tumor necrosis factor- activate intercellular adhesion molecule 1 (ICAM-1) gene transcription through distinct cis-regulatory elements within the ICAM-1 promoter. J Biol Chem.. 1995;270:18966-18974. [Abstract/Free Full Text]

12. Sawdey M, Podor TJ, Loskutoff DJ. Regulation of type 1 plasminogen activator inhibitor gene expression in cultured bovine aortic endothelial cells. J Biol Chem.. 1989;264:10396-10401. [Abstract/Free Full Text]

13. Nawroth PP, Stern DM. Tumor necrosis factor/cachectin-induced modulation of endothelial cell hemostatic properties. Onkologie.. 1987;10:254-258. [Medline] [Order article via Infotrieve]

14. Sawada H, Kan M, McKeehan WL. Opposite effects of monokines (interleukin-1 and tumor necrosis factor) on proliferation and heparin-binding (fibroblast) growth factor binding to human aortic endothelial and smooth muscle cells. In Vitro Cell Dev Biol.. 1990;26:213-216. [Medline] [Order article via Infotrieve]

15. Couffinhal T, Duplàa C, Labat L, Lamaziere J-MD, Moreau C, Printseva O, Bonnet J. Tumor necrosis factor- stimulates ICAM-1 expression in human vascular smooth muscle cells. Arterioscler Thromb.. 1993;13:407-414. [Abstract/Free Full Text]

16. Warner SJC, Libby P. Human vascular smooth muscle cells: target for and source of tumor necrosis factor. J Immunol.. 1989;142:100-109. [Abstract]

17. Tanaka H, Swanson SJ, Sukhova G, Schoen FJ, Libby P. Smooth muscle cells of the coronary arterial tunica media express tumor necrosis factor- and proliferate during acute rejection of rabbit cardiac allografts. Am J Pathol.. 1995;147:617-626. [Abstract]

18. Postlethwaite AE, Seyer JM. Stimulation of fibroblast chemotaxis by human recombinant tumor necrosis factor (TNF-) and a synthetic TNF- 31-68 peptide. J Exp Med.. 1990;172:1749-1756. [Abstract/Free Full Text]

19. Wang JM, Walter S, Mantovani A. Re-evaluation of the chemotactic activity of tumor necrosis factor for monocytes. Immunology.. 1990;71:364-367. [Medline] [Order article via Infotrieve]

20. Camussi G, Turello E, Bussolini F, Baglioni C. Tumor necrosis factor alters cytoskeleton organization and barrier function of endothelial cells. Int Arch Allergy Appl Immunol.. 1991;96:84-91. [Medline] [Order article via Infotrieve]

21. Gronowicz G, Hadjimichael J, Richards D, Cerami A, Rossomando EF. Correlation between tumor necrosis factor- (TNF-)-induced cytoskeletal changes and human collagenase gene induction. J Periodont Res.. 1992;27:562-568. [Medline] [Order article via Infotrieve]

22. Camussi G, Turello E, Tetta C, Bussolino F, Baglioni C. Tumor necrosis factor induces contraction of mesangial cells and alters their cytoskeletons. Kidney Int.. 1990;38:795-802. [Medline] [Order article via Infotrieve]

23. Newby AC, Southgate KM, Davies M. Extracellular matrix degrading metalloproteinases in the pathogenesis of arteriosclerosis. Basic Res Cardiol. 1994;89(suppl 1):59-70.

24. Wasylyk C, Gutman A, Nicholson R, Wasylyk B. The c-Ets oncoprotein activates the stromelysin promoter through the same elements as several non-nuclear oncoproteins. EMBO J.. 1991;10:1127-1134. [Medline] [Order article via Infotrieve]

25. Nerlov C, Rørth P, Blasi F, Johnsen M. Essential AP-1 and PEA3 binding elements in the human urokinase enhancer display cell type-specific activity. Oncogene.. 1991;6:1583-1592. [Medline] [Order article via Infotrieve]

26. Murphy G, Atkinson S, Ward R, Gavrilovic J, Reynolds JJ. The role of plasminogen activators in the regulation of connective tissue metalloproteinases. Ann N Y Acad Sci.. 1992;667:1-12.

27. Gown AM, Vogel AM, Gordon D, Lu PL. A smooth muscle-specific monoclonal antibody recognizes smooth muscle actin isozymes. J Cell Biol.. 1985;100:807-813. [Abstract/Free Full Text]

28. Wulf E, Deboben A, Bautz FA, Faulstich H, Wieland T. Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc Natl Acad Sci U S A.. 1979;76:4498-4502. [Abstract/Free Full Text]

29. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochem J.. 1979;18:5294-5299. [Medline] [Order article via Infotrieve]

30. Lehrach H, Diamond D, Wozney JM, Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry.. 1977;16:4743-4751. [Medline] [Order article via Infotrieve]

31. Belacosa A, Datta K, Bear SE, Patriots C, Lazo PA, Copland NG, Jenkins NA, Tsichlis PN. Effects of provirus integration in the Tp1-1/Ets-1 locus in moloney murine leukemia virus-induced rat T-cell lymphomas: levels of expression, polyadenylation, transcriptional initiation and differential splicing of the Ets-1 mRNA. J Virol.. 1994;68:2320-2330. [Abstract/Free Full Text]

32. Torczynski R, Bollon AP, Fuke M. The complete nucleotide sequence of the rat 18S ribosomal RNA gene and comparison with the respective yeast and frog genes. Nucleic Acids Res.. 1983;11:4879-4890. [Abstract/Free Full Text]

33. Diamond JR, Pesek I. Glomerular tumor necrosis factor and interleukin 1 during acute aminonucleoside nephrosis: an immunohistochemical study. Lab Invest.. 1991;64:21-28. [Medline] [Order article via Infotrieve]

34. Burridge K, Fath K, Kelly T, Nuckolls G, Turner C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol.. 1988;4:487-525.

35. Herman B, Pledger WJ. Platelet-derived growth factor-induced alterations in vinculin and actin distribution in BALB/c-3T3 cells. J Cell Biol.. 1985;100:1031-1040. [Abstract/Free Full Text]

36. Leprince D, Duterque-Coquillaud M, Li RP, Henry C, Flourens A, Debuire B, Stehelin D. Alternative splicing within the chicken c-ets-1 locus: implications for transduction within the E26 retrovirus of the c-ets-1 proto-oncogene. J Virol.. 1988;62:3233-3241. [Abstract/Free Full Text]

37. Hultgårdh-Nilsson A, Cercek B, Wang J-W, Naito S, Lövdahl C, Sharifi B, Forrester JS, Fagin JA. Regulated expression of the Ets-1 transcription factor in vascular smooth muscle cells in vivo and in vitro. Circ Res.. 1996;78:589-595. [Abstract/Free Full Text]

38. Quéva C, Leprince D, Stehelin D, Vandenbunder B. p54 c-ets-1 and p68 c-ets-1, the two transcription factors encoded by the c-ets-1 locus, are differently expressed during development of the chick embryo. Oncogene.. 1993;8:2511-2520. [Medline] [Order article via Infotrieve]

39. Goldblum SE, Ding X, Cambell-Washington J. TNF- induces endothelial cell F-actin depolymerization, new actin synthesis, and barrier dysfunction. Am J Physiol.. 1993;264:C894-C905. [Abstract/Free Full Text]

40. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature.. 1993;362:801-809.[Medline] [Order article via Infotrieve]

41. Tanaka H, Sukhova G, Schwartz D, Libby P. Proliferating arterial smooth muscle cells after balloon injury express TNF- but not interleukin-1 or basic fibroblast growth factor. Arterioscler Thromb Vasc Biol.. 1996;16:12-18. [Abstract/Free Full Text]

42. Clausell N, Molossi S, Sett S, Rabinovitch M. In vivo blockade of tumor necrosis factor- in cholesterol-fed rabbits after cardiac transplant inhibits acute coronary artery neointimal formation. Circulation.. 1994;89:2768-2779.[Abstract/Free Full Text]

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