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Am J Physiol Heart Circ Physiol 293: H1962-H1968, 2007. First published June 29, 2007; doi:10.1152/ajpheart.01366.2006
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The inflammatory cytokine oncostatin M induces PAI-1 in human vascular smooth muscle cells in vitro via PI 3-kinase and ERK1/2-dependent pathways

Svitlana Demyanets,1 Christoph Kaun,1 Kathrin Rychli,1 Gersina Rega,1 Stefan Pfaffenberger,1 Taras Afonyushkin,2 Valery N. Bochkov,2 Gerald Maurer,1 Kurt Huber,3 and Johann Wojta1,4

Departments of 1Internal Medicine II and of 2Vascular Biology and Thrombosis Research, Medical University of Vienna, Vienna; 3Third Medical Department for Cardiology and Emergency Medicine, Wilhelminenhospital, Vienna; and 4Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna, Austria

Submitted 14 December 2006 ; accepted in final form 22 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Plasminogen activator inhibitor-1 (PAI-1) plays a pivotal role in the regulation of the fibrinolytic system and in the modulation of extracellular proteolysis. Increased PAI-1 was found in atherosclerotic lesions, and high PAI-1 plasma levels were associated with coronary heart disease. Smooth muscle cells (SMC) are a major source of PAI-1 within the vascular wall, and PAI-1 was implicated in SMC migration, proliferation, and apoptosis. We treated human coronary artery SMC (HCASMC) and human aortic SMC (HASMC) with the glycoprotein 130 (gp130) ligands cardiotrophin-1, interleukin-6 (IL-6), leukemia inhibitory factor (LIF), or oncostatin M (OSM). Only OSM increased PAI-1 antigen and activity production significantly in these cells up to 20-fold. OSM upregulated mRNA specific for PAI-1 up to 4.5-fold in these cells. HCASMC and HASMC express gp130, OSM receptor, IL-6 receptor, and LIF receptor. OSM induced extracellular signal-regulated kinase (ERK) 1/2 and Akt phosphorylations in HASMC. A phosphatidylinositol 3-kinase inhibitor and a mitogen-activated protein/extracellular signal-regulated kinase inhibitor reduced Akt and ERK1/2 phosphorylation, respectively, and abolished OSM-induced PAI-1 upregulation. A janus kinase/signal transducer and activator of transcription inhibitor, a p38 mitogen-activated protein kinase inhibitor, or c-Jun NH2-terminal kinase inhibitor I did not inhibit the OSM-dependent PAI-1 induction. OSM enhanced proliferation of both HCASMC and HASMC by 77 and 90%, respectively. We hypothesize that, if the effect of OSM on PAI-1 expression in smooth muscle cells is operative in vivo, it could, via modulation of fibrinolysis and extracellular proteolysis, be involved in the development of vascular pathologies such as plaque progression, destabilization and subsequent thrombus formation, and restenosis and neointima formation.

smooth muscle cells; oncostatin M; glycoprotein 130; plasminogen; plasminogen activator inhibitor

BY INHIBITING THE ACTIVATION of tissue-type plasminogen activator (t-PA) and urokinase-type PA (u-PA), the serine protease inhibitor PA inhibitor-1 (PAI-1) regulates fibrinolysis and proteolysis (20). As evidenced by numerous clinical and experimental studies, these two processes are key features in the development and progression of various cardiovascular disease states (27). Thus it is not surprising that epidemiological studies have indicated that high plasma levels of PAI-1 constitute a risk factor for ischemic heart disease that is the result of impaired thrombolysis after plaque rupture (10). Such elevated PAI-1 plasma levels have also been associated with the progression of atherosclerosis, supposedly by affecting matrix remodeling and fibrin clearance (36). In support of these findings, increased expression of PAI-1 was shown in human atherosclerotic lesions, and it was concluded that altered expression of PAI-1 in vessel walls might directly affect the development and progression of such lesions (for a review, see Ref. 41). Smooth muscle cells seem to be a major source of PAI-1 within the vascular wall, and PAI-1 expression in smooth muscle cells has been shown to be upregulated among others by inflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor-{alpha}, or transforming growth factor-beta (14, 18, 37, 46). Furthermore, PAI-1 has been implicated recently in smooth muscle cell migration, proliferation, and apoptosis, which in turn are processes thought to be critically involved in vascular pathologies such as plaque development and destabilization and restenosis and neointima formation (6, 7, 23, 38).

Here we investigate whether a proinflammatory cytokine produced by activated T lymphocytes, monocytes, and macrophages, namely the glycoprotein 130 (gp130) ligand oncostatin M (OSM), which is involved in a variety of physiological and pathophysiological events including, besides inflammation, tissue remodeling and cell growth and which has been shown by us and others to affect the expression of components of the fibrinolytic system in various cell types, regulates the expression of PAI-1 in human vascular smooth muscle cells (9, 13, 21, 24, 33, 44, 45).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
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Cell culture. Human coronary artery smooth muscle cells (HCASMC) and human aortic SMC (HASMC) were isolated from pieces of coronary arteries and aortas obtained from patients undergoing heart transplantation. Such smooth muscle cells were cultured and characterized as described (47).

Treatment of cells with gp130 ligands. SMC were incubated in minimum essential medium (M199; Sigma, St. Louis, MO) containing 0.1% BSA (Sigma) for 24 h before treatment with the respective cytokine. Thereafter, the medium was replaced with fresh M199 containing 0.1% BSA, and recombinant human (rh) cardiotrophin-1 (CT-1), obtained from Calbiochem (La Jolla, CA), rhIL-6, rh leukemia inhibitory factor (LIF), or rhOSM, all obtained from R&D Systems (Minneapolis, MN), were added at the concentrations indicated for time periods between 24 and 72 h. In additional experiments, the cells were preincubated for 2 h with the janus kinase/signal transducer and activator of transcription (JAK/STAT) inhibitor AG-490, with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY-294002, with the mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD-98059, with the c-Jun NH2-terminal kinase (JNK) inhibitor I (all at 30 µmol/l), or the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB-202190 at 5 µmol/l (all from Calbiochem/Merck, Darmstadt, Germany). Similar concentrations of these inhibitors were also used by others in various in vitro studies (4, 16). Thereafter, the cells were treated with OSM at a concentration of 100 ng/ml for 24 h. The culture supernatants were then collected followed by removal of cell debris by centrifugation and stored at –80°C until used. The total cell number of the respective cultures after trypsinization was counted with a hemocytometer.

Antigen and activity assays. PAI-1, t-PA, and u-PA antigen in culture supernatants were measured by specific ELISAs using monoclonal antibodies [PAI-1, u-PA (Technoclone, Vienna, Austria), and t-PA (Bender MedSystems, Vienna, Austria), respectively]. The ELISA for PAI-1 antigen measures free, complexed, and latent PAI-1. The t-PA ELISA detects free and complexed t-PA antigen. The u-PA ELISA measures u-PA in its single- and two-chain form and the latter in its free and complexed form. PAI-1 activity was measured with a PAI-1 Actibind ELISA (Technoclone). t-PA activity was determined by a t-PA Actibind ELISA (Technoclone). All measurements were performed in triplicates.

Total RNA purification. Cells were treated as described, supernatants were removed, and total RNA were isolated using a Gentra PURESCRIPT RNA Purification Kit (Minneapolis, MN) according to the manufacturer's instructions.

Real-time PCR. Specific mRNA levels for PAI-1, u-PA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression were determined by real-time PCR (RealTime-PCR) using LightCycler-RNA Master SYBR Green I (Roche, Basel, Switzerland) according to the manufacturer's instructions. Primers were designed using the LightCycler Probe Design Software version 1.0 (Roche) and the Primer3 Software (http://frodo.wi.mit.edu/), respectively. The amplification conditions consisted of an initial incubation at 61°C for 20 min, followed by incubation at 95°C for 30 s, 50 cycles of 95°C for 1 s, the respective annealing temperature [65°C for GAPDH, PAI-1, and u-PA, 62°C for gp130, OSM receptor (OSMR), and IL-6 receptor (IL-6R), and 52°C for LIF receptor (LIFR)] for 10 s and 72°C for 10 s, a melting step from 45 to 95°C increasing 0.1°C/s, and a final cooling to 40°C. Data were analyzed using LightCycler Software version 3.5 (Roche). mRNA specific for gp130, OSMR, IL-6R, and LIFR were detected by RT-PCR using Titan one step RT-PCR (Roche) according to the manufacturer's instructions. PCR products for gp130, OSMR, IL-6R, and LIFR were analyzed by gel-electrophoresis (3% agarose-gel, stained with ethidium bromide).

Western blotting. HASMC were incubated in M199 containing 0.1% BSA for 24 h before treatment with OSM. Thereafter, the medium was replaced with fresh M199 containing 0.1% BSA, and rhOSM was added at the concentration of 100 ng/ml for 5, 15, 30, or 60 min. In additional experiments, the cells were preincubated for 30 min with the PI 3-kinase inhibitor LY-294002 or with the MEK inhibitor PD-98059 at a concentration of 30 µmol/l. Thereafter, the cells were treated with OSM at a concentration of 100 ng/ml for 20 min. Samples containing equal amounts of protein were separated on SDS-polyacrylamide gels, and subsequently proteins were transferred by electroblotting to Immobilon-P (Millipore, Billerica, MA) membranes. Western blotting was performed as described previously (3). p44/42 MAPK antibody, phospho-p44/42 MAPK (Thr202/Tyr204) antibody, Akt (pan) (11E7) monoclonal antibody, and phospho-Akt (Thr308) antibody (all rabbit) were from Cell Signaling Technology (Danver, MA), and immunodetection was performed according to vendor protocol. The membranes were blocked in 10% dried milk suspension and probed with polyclonal rabbit antibodies followed by development using the LumiGLO Chemiluminescent Substrate (Upstate, Lake Placid, NY).

Proliferation assay. The effect of OSM on proliferation of HCASMC and HASMC was determined using the colorimetric EZ4U cell proliferation assay, which is based on the conversion of tetrazolium salts into formazan derivates by living cells, according to the manufacturer's instructions (Biomedica, Vienna, Austria). Briefly, cells were seeded in 96-well plates and incubated in M199 containing 0.1% BSA for 24 h before treatment with the respective cytokine. Thereafter, the cells were treated with fresh 0.1% BSA M199 alone or with OSM at 100 ng/ml or platelet-derived growth factor-AB (PDGF-AB) at 100 ng/ml for 72 h. Substrate was dissolved in 2.5 ml activator according to the manufacturer's instruction and prewarmed to 37°C. Thereafter, 22.5 ml M199 containing 5% FCS was added. The medium was replaced with 200 µl of this solution per well, and the cells were incubated at 37°C for 3 h. Absorbance was read at 600 nm. Cell numbers were calculated by using a standard curve constructed with known cell numbers.

Determination of cell viability. To determine possible cytotoxic effects of gp130 ligands or transcription pathways inhibitors, lactate dehydrogenase (LDH) leakage was measured in cultures treated with CT-1, IL-6, LIF, OSM, AG-490, LY-294002, PD-98059, SB-202190, and JNK inhibitor I as described above using a commercially available assay for photometric determination of LDH activity (Sigma).

Statistical analysis. Values are expressed as means ± SD. Data were compared by ANOVA. Values of P < 0.05 were considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Human smooth muscle cells express receptors for gp130 ligands. Human smooth muscle cells derived from both coronary arteries and aortas express mRNA specific for gp130, OSMR, IL-6R, and LIFR as demonstrated by RT-PCR (Fig. 1).


Figure 1
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  		Fig. 1. Human aortic and coronary artery smooth muscle cells express receptors for glycoprotein 130 (gp130) ligands. RNA was prepared from confluent monolayers of untreated human aortic smooth muscle cells (HASMC; lanes 1, 3, 5, and 7) and human coronary artery smooth muscle cells (HCASMC; lanes 2, 4, 6, and 8), and RT-PCR using specific primers for gp130 (lanes 1 and 2), oncostatin M (OSM) receptor (lanes 3 and 4), interleukin-6 (IL-6) receptor (lanes 5 and 6), and leukemia inhibitory factor (LIF) receptor (lanes 7 and 8) was performed as described in MATERIALS AND METHODS. Experiments were performed two times. A representative experiment is shown.

 
Effects of gp130 ligands on PAI-1 production by human coronary artery and aortic smooth muscle cells. OSM increased PAI-1 protein production significantly in both human coronary artery and aortic smooth muscle cells. If the cells were incubated with OSM at a concentration of 100 ng/ml for 48 h, PAI-1 production was increased up to 20-fold in HCASMC and up to 7-fold in HASMC. CT-1 (100 ng/ml), IL-6 (100 ng/ml), or LIF (104 U/ml) had no effect on PAI-1 production in human SMC (Fig. 2, A and B). The effect of OSM on PAI-1 production was reproducible in each HCASMC and HASMC derived from three different donors (Table 1). OSM also stimulated PAI-1 activity after 24 h in HCASMC and HASMC derived from three different donors (Table 2). None of the gp130 ligands affected cell viability at concentrations used as determined by LDH leakage (data not shown).


Figure 2
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  		Fig. 2. Effects of gp130 ligands on plasminogen activator inhibitor-1 (PAI-1) production by human coronary artery (A) and aortic smooth muscle cells (B). Confluent monolayers of HCASMC and HASMC were incubated for 48 h in the absence or presence of cardiotrophin-1 (CT-1, 100 ng/ml), IL-6 (100 ng/ml), LIF (104 U/ml), or OSM (100 ng/ml). Conditioned media of such treated cells were collected, and PAI-1 antigen was determined as described in MATERIALS AND METHODS. Values are given in ng·10–4 cells·48 h–1 and represent mean values ± SD of three independent determinations. *P < 0.0001 compared with control. Experiments were performed three times. A representative experiment is shown.

 

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  		Table 1. Effect of OSM on PAI-1 production in human coronary artery and aortic smooth muscle cells isolated from different donors

 

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  		Table 2. Effect of OSM on PAI-1 activity in human coronary artery and aortic smooth muscle cells isolated from different donors

 
When the cells were treated with OSM at the concentrations of 0.01, 0.1, 1, 10, or 100 ng/ml for time periods of 24, 48, and 72 h, a dose- and time-dependent effect on PAI-1 production was observed in both types of smooth muscle cells (Fig. 3, A and B).


Figure 3
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  		Fig. 3. Effect of OSM on PAI-1 production by human coronary artery (A) and aortic smooth muscle cells (B) is dose- and time-dependent. Confluent monolayers of HCASMC and HASMC were incubated for 24, 48, or 72 h in the absence (bullet) or presence of OSM at a concentration 0.01 ({circ}), 0.1 ({triangleup}), 1 ({square}), 10 ({triangledown}), or 100 ({lozenge}) ng/ml. Conditioned media of such treated cells were collected, and PAI-1 antigen was determined as described in MATERIALS AND METHODS. Values are given in ng/104 cells and represent mean values ± SD of three independent determinations. Experiments were performed three times. A representative experiment is shown.

 
OSM at a concentration of 100 ng/ml after incubation time of 24 h upregulated mRNA specific for PAI-1 4.5-fold in HCASMC and 2.4-fold in HASMC as demonstrated by RealTime-PCR. CT-1 (100 ng/ml), IL-6 (100 ng/ml), or LIF (104 U/ml) had no significant effect on mRNA specific for PAI-1 in smooth muscle cells (HCASMC: CT-1 1.4-fold, IL-6 1.4-fold, and LIF 1.1-fold compared with untreated control; HASMC: CT-1 1.4-fold, IL-6 1.1-fold, LIF 1.2-fold compared with untreated control).

Effects of gp130 ligands on t-PA and u-PA production by human coronary artery and aortic smooth muscle cells. CT-1 (100 ng/ml), IL-6 (100 ng/ml), LIF (104 U/ml), or OSM (100 ng/ml) had no significant effect on t-PA protein production in human smooth muscle cells (in ng·10–4 cells·48 h–1: HCASMC: control 9.1 ± 0.3, CT-1 7.9 ± 1.1, IL-6 8.0 ± 0.2, LIF 7.2 ± 0.4, OSM 9.0 ± 0.6; HASMC: control 6.2 ± 0.2, CT-1 7.4 ± 0.7, IL-6 5.8 ± 0.4, LIF 7.4 ± 0.8, OSM 6.8 ± 0.3). t-PA activity and u-PA protein were undetectable under the culture conditions tested. mRNA specific for u-PA was not affected by any of the gp130 ligands tested (data not shown).

OSM induced extracellular signal-regulated kinase 1/2 and Akt phosphorylation. In HASMC, OSM induced extracellular signal-regulated kinase (ERK) 1/2 and Akt phosphorylation with a peak between 5 and 30 min, as shown by Western blot (Fig. 4A).


Figure 4
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  		Fig. 4. OSM induces phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt (A), mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD-98059 and phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY-294002 counteract ERK1/2 and Akt phosphorylation (B), and OSM induced PAI-1 upregulation (C). A: confluent monolayers of HASMC were incubated for 5, 15, 30, or 60 min in the absence or presence of 100 ng/ml OSM. The cells were lysed, and Western blot was performed for phosphorylated (pAkt), total Akt, phosphorylated (pERK1/2) and total ERK1/2, as described in MATERIALS AND METHODS. B: in the additional experiment, the cells were preincubated for 30 min with the PI 3-kinase inhibitor LY-294002 or the MEK inhibitor PD-98059, each 30 µmol/l. Thereafter, the cells were treated with 100 ng/ml OSM for 20 min and lysed, and Western blot for pAkt, total Akt, pERK1/2, and total ERK was performed as described in MATERIALS AND METHODS. C: confluent monolayers of HCASMC were preincubated for 2 h with the janus kinase/signal transducer and activator of transcription (JAK/STAT) inhibitor AG-490, the MEK inhibitor PD-98059, or the PI 3-kinase inhibitor LY-294002 (all at 30 µmol/l). Thereafter, the cells were treated with OSM at a concentration of 100 ng/ml for 24 h. Conditioned media of such treated cells were collected, and PAI-1 antigen was determined as described in MATERIALS AND METHODS. Values are given in ng·10–4 cells·24 h–1 and represent mean values ± SD of three independent determinations. *P < 0.01 compared with control. §P < 0.01 compared with OSM-treated cells without inhibitors. Experiments were performed three times. A representative experiment is shown.

 
PI 3-kinase inhibitor LY-294002 and MEK inhibitor PD-98059 interfere with OSM-induced Akt and ERK1/2 phosphorylation and PAI-1 expression in smooth muscle cells. It is known that gp130-gp130 ligand interaction activates JAK/STAT, MEK, and the PI 3-kinase-Akt pathways (12, 29). To investigate which pathways are involved in the OSM-induced PAI-1 upregulation in human smooth muscle cells, we treated these cells with inhibitors of these pathways before the addition of OSM. Western blot analysis revealed that the PI 3-kinase inhibitor LY-294002 robustly counteracted OSM-induced Akt phosphorylation, whereas the MEK inhibitor PD-98059 showed a slight inhibitory effect on ERK1/2 phosphorylation (Fig. 4B). As can be seen from Fig. 4C, LY-294002 and PD-98059 abolished the OSM-dependent PAI-1 induction almost completely in HCASMC, whereas the JAK/STAT inhibitor AG-490 had no effect. Furthermore, neither the p38 MAPK inhibitor SB-202190 nor JNK inhibitor I had an effect on OSM-induced PAI-1 upregulation (in ng·10–4 cells·24 h–1: control 66.8 ± 6.6, SB-202190 60.6 ± 4.4, JNK inhibitor I 66.3 ± 0.9, OSM 119.4 ± 11.7, SB-202190 + OSM 103.9 ± 10.1, JNK inhibitor I + OSM 119.3 ± 2.9). Similarly, also in HASMC, LY-294002 and PD-98059 reversed the OSM-induced PAI-1 upregulation, whereas AG-490, SB-202190 or JNK inhibitor I had no effect (data not shown). Neither of the blockers affected cell viability at concentrations used, as determined by LDH leakage (data not shown).

Effect of OSM on proliferation of human vascular smooth muscle cells. After 3 days, OSM at the concentration of 100 ng/ml enhanced proliferation of both HCASMC and HASMC by 77 and 90%, respectively. The effect of OSM was comparable to the proliferative action of PDGF-AB, a known mitogen for SMC (cell count mean ± SD: HCASMC control 1,845 ± 250, OSM 3,175 ± 335, PDGF-AB 2,805 ± 355 cells/well, HASMC control 1,280 ± 205, OSM 2,335 ± 325, PDGF-AB 1,925 ± 275 cells/well; see Ref. 32).


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The gp130 ligand OSM, which is mainly produced by activated T lymphocytes, monocytes, and macrophages, is a pleiotropic cytokine that plays a role in a variety of pathophysiological processes such as inflammation, tissue remodeling, and cell growth (21, 45). We and others could show that OSM modulates the expression of various components of the fibrinolytic system. It upregulates PAI-1 expression in human adipocytes, cardiac myocytes, and astrocytes; u-PA expression in human endothelial cells, synovial fibroblasts, and in the hepatoma cell line HepG2; and t-PA expression in lung carcinoma cells (9, 13, 24, 30, 33, 42, 44).

Here we show for the first time that OSM robustly upregulates the production of PAI-1 in human vascular smooth muscle cells isolated from coronary artery and aorta. The effect was reproducible in cell preparations isolated from different donors. The upregulation was dose- and time-dependent, with a significant increase seen at concentrations of >1 ng/ml of OSM. PAI-1 activity was also increased in OSM-stimulated cells. The increase in PAI-1 protein production induced by OSM in human vascular smooth muscle cells was reflected at the level of specific mRNA expression in these cells. OSM caused a significant upregulation of PAI-1-specific mRNA in human coronary artery and aortic smooth muscle cells, as determined by RealTime-PCR. The effect seemed to be specific for OSM, since other gp130 ligands such as IL-6, LIF, or CT-1 did not affect PAI-1 production in human vascular smooth muscle cells. It should be noted that this lack of response could not be attributed to the absence of specific receptors for the respective ligands on smooth muscle cells. In agreement with earlier reports, we could show that both types of smooth muscle cells used in this study expressed detectable amounts of mRNA specific for the receptors gp130, IL-6R, LIFR, and OSMR (15, 17, 43).

It is known that gp130-gp130 ligand interaction activates JAK/STAT, MEK, and the PI 3-kinase-Akt pathways (12, 29). In smooth muscle cells, OSM caused phosphorylation of JAK1 and activation of STAT1 and induced tissue factor and matrix metalloproteinase-9 (MMP-9) expression via activation of ERK1/2 (2, 26, 28). We show here that OSM induced phosphorylation of ERK1/2 and Akt in HASMC. PD-98059, an MEK inhibitor, and LY-294002, a PI 3-kinase inhibitor, not only reduced ERK1/2 and Akt phosphorylation, respectively, but also abolished the OSM-induced PAI-1 upregulation. It should be noted, however, that PD-98059 only slightly reduced OSM-induced phosphorylation of ERK1/2, whereas the same inhibitor robustly counteracted OSM-induced PAI-1 production. This discrepancy might be caused by different treatment times of the cells with the inhibitor. In the phosphorylation assay, the cells were only pretreated with PD-98059 for 30 min, whereas, for determination of PAI-1 antigen, the treatment period was 2 h. However, it could not be conclusively concluded from these data that the minor reduction in phosphorylated ERK1/2 was fully responsible for the large reduction in PAI-1. AG-490, a JAK/STAT inhibitor, SB-202190, a p38 MAPK inhibitor, or JNK inhibitor I did not inhibit the OSM-dependent PAI-1 induction. In that respect, it should be emphasized that PI 3-kinase and ERK1/2 activation have been shown to be essential for upregulation of PAI-1 expression by insulin (1).

Here we provide evidence that OSM increases the proliferation of human smooth muscle cells originating both from coronary artery and aorta. This is in agreement with a report by Grove et al. (8) who showed that OSM induced proliferation of rabbit vascular smooth muscle cells. It should be noted, however, that a slight antiproliferative effect of OSM was reported for HASMC. Different culture conditions could be the reason for this discrepancy, e.g., in that paper proliferation was measured in serum containing cultures of smooth muscle cells, whereas we used serum-free conditions (2).

Recent evidence suggests that PAI-1 present in the vessel wall through its antifibrinolytic and antiproteolytic properties is modulating key events in the pathophysiology of cardiovascular diseases. Increased amounts of PAI-1 have been detected in atherosclerotic lesions (22, 31, 39). Such increased levels of PAI-1 in the tissue have been considered to be protective, since they inhibit the activation of MMPs through u-PA and plasmin, thereby preventing plaque destabilization (40). However, according to a more recent notion, high PAI-1 levels in the vessel wall are thought to be detrimental, since PAI-1 has been shown to inhibit smooth muscle cell migration, thus leading to the development of smooth muscle cell-poor plaques prone to rupture (38). Furthermore, such high PAI-1 levels would create a prothrombotic environment by inhibiting plasminogen activation and subsequently clot resolution (40). Recently, PAI-1 has been also shown to increase smooth muscle cell proliferation and to decrease smooth muscle apoptosis (6, 35). The authors of one of these studies conclude that, by inducing smooth muscle cell proliferation, high PAI-1 levels would favor restenosis (6). Here we show that an inflammatory cytokine, namely OSM, robustly upregulates the expression of PAI-1 in human vascular smooth muscle cells. OSM also enhanced proliferation of these cells in our study. Thus, although not fully and conclusively supported by our data presented here, one could speculate that, via induction of PAI-1, OSM is also involved in the modulation of smooth muscle cell proliferation. It should be emphasized that the major producers of OSM, namely activated T lymphocytes, monocytes and macrophages, are abundantly present in atherosclerotic lesions and that the latter cell type has been shown to express OSM in human aortic aneurysms (5, 11, 19, 25, 34). We speculate that in such lesions OSM could, through the induction of PAI-1 in smooth muscle cells, contribute to the development and progression of vascular pathologies described above.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The work was supported by the Association for the Promotion of Research in Arteriosclerosis, Thrombosis and Vascular Biology and by the Fund for the Promotion of Scientific Research (Grant Number S9409-B11). S. Demyanets was a recipient of a scholarship from the Austrian Cardiologic Society.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Wojta, Dept. of Internal Medicine II, Medical Univ. of Vienna, A-1090 Vienna, Waehringer Guertel 18-20, Austria (e-mail: johann.wojta{at}meduniwien.ac.at)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 DISCUSSION
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