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Circulation. 1997;96:2361-2367

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(Circulation. 1997;96:2361-2367.)
© 1997 American Heart Association, Inc.

Articles

Platelet-Derived Growth Factor–Stimulated Superoxide Anion Production Modulates Activation of Transcription Factor NF-{kappa}B and Expression of Monocyte Chemoattractant Protein 1 in Human Aortic Smooth Muscle Cells

Takeshi Marumo, MD, PhD; Valérie B. Schini-Kerth, PhD; Beate Fisslthaler, PhD; ; Rudi Busse, MD, PhD

From the Zentrum der Physiologie, Klinikum der Johann Wolfgang Goethe Universität, Frankfurt am Main, Germany.

Correspondence to Takeshi Marumo, MD, PhD, Zentrum der Physiologie, Klinikum der Johann Wolfgang Goethe Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. E-mail busse{at}merlin.add.uni-frankfurt.de


*    Abstract
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 *Abstract
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Background Platelet-derived growth factor (PDGF) and superoxide anion (O2·-) have been implicated in vascular diseases. We investigated whether PDGF stimulates the production of O2·- in human aortic smooth muscle cells (HSMCs) and whether O2·- leads in this way to the activation of nuclear factor–{kappa}B (NF-{kappa}B) and induction of monocyte chemoattractant protein 1 (MCP-1) in PDGF-stimulated HSMCs.

Methods and Results PDGF-AB concentration- and time-dependently stimulated O2·- generation from HSMCs. The stimulatory effect of PDGF-AB was mimicked by PDGF-BB but not by PDGF-AA. The generation of O2·- by PDGF-AB was attenuated by the NAD(P)H oxidase inhibitor iodonium diphenyl, the specific protein kinase C (PKC) inhibitor Ro 31-8220, and the phosphatidylinositol 3-kinase inhibitor wortmannin. Allopurinol and nifedipine had no effect on PDGF-AB–induced O2·- release, whereas indomethacin potentiated this response. Gel mobility shift assay revealed that PDGF-AB increased the binding activity of NF-{kappa}B, which contained predominantly the p50/p65 heterodimer in nuclear extracts from HSMCs. Superoxide dismutase as well as iodonium diphenyl, Ro 31-8220, and wortmannin attenuated PDGF-AB–induced activation of NF-{kappa}B and expression of MCP-1 mRNA. In contrast, superoxide dismutase did not inhibit the interleukin-1ß–induced NF-{kappa}B activation.

Conclusions The results demonstrate that PDGF stimulates O2·- generation in HSMCs via PKC-dependent and wortmannin-sensitive pathways involving flavoenzyme(s). This PDGF-induced O2·- production may be involved in vascular lesion formation by mediating, at least in part, NF-{kappa}B activation and MCP-1 induction.


Key Words: growth substances • atherosclerosis • lesion


*    Introduction
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 *Introduction
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 down arrowDiscussion
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Platelet-derived growth factor plays an important role in intimal thickening after experimental angioplasty,1 2 and increased levels of PDGF have been shown in human atherosclerotic lesions,1 although its role in atherogenesis has not been clarified. PDGF activates the expression of a number of genes, including those encoding transcription factors, chemokines, and cytokines that participate in vascular lesion formation.3 Signal transduction pathways stimulated by PDGF include activation of phospholipase C{gamma}, PKC, PI3-kinase, mitogen-activated protein kinase, and signal transducer and activator of transcription proteins.4

Emerging evidence has pointed to the involvement of O2·- in the regulation of vascular functions. O2·- inactivates nitric oxide to yield peroxynitrite,5 6 stimulates the proliferation of VSMCs,7 and increases vascular tone.8 Studies using experimental animals have suggested that increased vascular O2·- production is associated with risk factors of atherosclerosis such as hypercholesterolemia,9 genetic hypertension,10 and diabetes.11 In hypertensive rats after chronic infusion of angiotensin II, the predominant source of increased O2·- generation is the vascular smooth muscle layer.12 Enzymatic sources involved in vascular O2·- production include xanthine oxidase, NAD(P)H oxidase, arachidonic acid–metabolizing enzymes, and NOS.13 14 15 16

Although both PDGF and O2·- have been implicated in vascular diseases, it has not yet been demonstrated whether PDGF can stimulate O2·- production in VSMCs. In the present study, we examined the effect of PDGF on O2·- production in HSMCs. In addition, we investigated the potential role of O2·- in the control of activation of NF-{kappa}B and induction of MCP-1 mRNA in PDGF-stimulated HSMCs.


*    Methods
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 *Methods
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Smooth Muscle Cell Culture
VSMCs isolated from the thoracic aorta of a young, healthy donor for cardiac transplantation by enzymatic disaggregation as described previously17 were kindly provided by Dr T. Scott-Burden (Texas Heart Institute, Houston). Cells were maintained in MEMTH, which consisted of MEM containing Earle's salts, 2 mmol/L glutamine, 5 mmol/L TES-NaOH, 5 mmol/L HEPES-NaOH (both pH 7.3), nonessential amino acids, 50 U/mL penicillin, and 50 µg/mL streptomycin, supplemented with 20% FCS. For experiments, cells at passages 16 through 20 were plated onto 12-well culture plates for measurements of O2·- release, 6-cm dishes for gel mobility shift assay, or 10-cm dishes for Northern blot analysis. After reaching confluence, cells were serum-deprived by 24-hour incubation with MEMTH supplemented with 0.1% BSA before all experiments.

O2·- Measurement
The release of O2·- was determined by measuring SOD-inhibitable reduction of ferricytochrome c. Cells were washed twice with HEPES-modified Tyrode's solution (pH 7.4) and incubated in the same buffer with and without 500 U/mL SOD for 10 minutes at 37°C in humidified air. The composition of HEPES-modified Tyrode's solution was as follows (in mmol/L): CaCl2 1.8, KCl 2.6, MgCl2 0.49, NaCl 137, NaH2PO4 0.36, glucose 5.6, and HEPES 10. In some experiments, HSMCs were pretreated with reagents for 15 minutes. Ferricytochrome c was added to the reaction buffer solution to a final concentration of 81 µmol/L. HSMCs were then stimulated with PDGF with or without reagents as indicated. At the times indicated, the medium was removed, and the absorbance at 550 nm was measured immediately. O2·--specific reduction of ferricytochrome c was calculated from the difference in absorbance between cells incubated with or without SOD by use of an extinction coefficient of 2110 mm-1 · mol/L-1.18 After the exposure of HSMCs to reagents that inhibited O2·- release, >95% of cells were negative with trypan blue.

Gel Mobility Shift Assay
HSMCs were washed twice with HEPES-modified Tyrode's solution and exposed to agents as indicated. In some experiments, HSMCs were pretreated with reagents for 15 minutes. Nuclear proteins were extracted from HSMCs by the method of Schreiber et al19 as described previously.20 Double-stranded oligonucleotides containing the sequence of the binding site for NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG C-3', Promega Corp) were radiolabeled with 30 µCi [{gamma}-32P]ATP with a 5' end-labeling kit (Pharmacia Biotech GmbH). Nuclear proteins (6 µg) were incubated with 3000 counts of labeled oligonucleotide in (mmol/L) HEPES 10 (pH 7.5), sodium chloride 100, EDTA 1, and dithiothreitol 1.5, with 5% (vol/vol) glycerol and 2 µg poly(dI/dC) (Pharmacia Biotech GmbH) for 30 minutes at room temperature. The reaction mixture was loaded onto a native 6% polyacrylamide gel buffered with (mmol/L) Tris 89, boric acid 89, and EDTA 2 and electrophoresed. After vacuum drying, the gel was exposed to x-ray film at -70°C. Densitometric analysis of the autoradiographic results was performed after nonsaturating exposures. Values obtained were normalized and expressed as percentages of control. In some experiments, a supershift analysis was performed by preincubation of nuclear extracts with 1 µg of a specific polyclonal anti-p65 antibody or anti-p50 antibody for 12 hours at 4°C.

Northern Blot Analysis
HSMCs were incubated in MEMTH with 0.1% BSA with or without agents as indicated for 4 hours. In some experiments, HSMCs were pretreated with reagents for 15 minutes. Total RNA was extracted, size-fractionated, and transferred to nylon membranes (Hybond, Amersham-Buchler) as described previously.20 A 740-bp Kpn I restriction fragment from the clone pXM-hJE34 (kindly provided by Dr B.J. Rollins) containing the coding region for MCP-1/JE and the cDNA probe specific for mouse 18S rRNA were labeled by the random priming method with [{alpha}-32P]dCTP as previously described.20 After hybridization with the probe and washing, the blots were exposed to x-ray film at -70°C as described previously.21

Reagents
Recombinant human PDGF-AB was purchased from R&D Systems Inc. Recombinant human PDGF-AA and -BB were from Pepro Tech Inc. Recombinant human IL-1ß was from CIBA-Geigy Co. Ro 31-8220 was from Roche Products Ltd. Ferricytochrome c, wortmannin, allopurinol, indomethacin, and 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron) were from Sigma Chemical Co. Iodonium diphenyl was from Fluka Chemie. SOD was from Boehringer Mannheim GmbH. Polyclonal antibodies to p65 and p50 were from Santa Cruz Biotechnology Inc. FCS was from Biochrom KG. All other chemicals and reagents were obtained from commercial sources and were of reagent or molecular biology grade.

Stock solutions of PDGF were made in MEMTH with 0.1% BSA to a concentration of 10 µg/mL and stored at -20°C.

Statistics
Multiple comparisons were evaluated with ANOVA, followed by Fisher's protected least significant difference method. Data are presented as mean±SEM, and values of P<.05 were considered statistically significant.


*    Results
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 up arrowMethods
 *Results
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Effect of PDGF on O2·- Production
PDGF-AB concentration-dependently stimulated O2·- production in HSMCs as measured after a 1-hour incubation period (Fig 1ADown). The generation of O2·- by HSMCs increased within 20 minutes of treatment and continued to increase for at least the next 40 minutes (Fig 1BDown).



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  		Figure 1. PDGF-AB and PDGF-BB but not PDGF-AA increase O2·- production from HSMCs. HSMCs were treated with various concentrations of PDGF-AB for 1 hour (A) and with 30 ng/mL PDGF-AB ({bullet}) for increasing time intervals (B). Superoxide production with vehicle at 1 hour ({circ}) is also shown in B. C, HSMCs were treated with vehicle, 100 ng/mL PDGF-AB (AB 100), 30 and 100 ng/mL PDGF-AA (AA 30 and AA 100), or 30 and 100 ng/mL PDGF-BB (BB 30 and BB 100) for 1 hour. The values (mean±SEM) in A and B are obtained from two separate experiments, and those in C are representative of data obtained in one of two qualitatively identical experiments with determinations performed in triplicate. Symbol without bars indicates SEM within symbol. *P<.05 vs control values. +P<.05 vs values at 20 minutes.

Significant release of O2·- was also observed with PDGF-BB (30 and 100 ng/mL), whereas PDGF-AA (up to 100 ng/mL) failed to increase O2·- production (Fig 1CUp).

To elucidate the enzymatic source responsible for the PDGF-AB–induced generation of O2·-, the effects of various inhibitors were examined. Iodonium diphenyl (10 µmol/L), a potent inhibitor of flavoenzymes, including NADPH oxidase,22 attenuated PDGF-AB–induced O2·- production (Fig 2Down). In contrast, allopurinol (100 µmol/L), an inhibitor of xanthine oxidase, had no effect (Fig 2Down). Surprisingly, indomethacin (10 µmol/L) significantly enhanced PDGF-stimulated O2·- release, whereas the drug was ineffective when applied alone (Fig 2Down).



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  		Figure 2. Effects of inhibitors of O2·--generating enzymes on PDGF-stimulated O2·- production. HSMCs were incubated with vehicle or 30 ng/mL PDGF-AB with or without 10 µmol/L iodonium diphenyl (ID), 100 µmol/L allopurinol, or 10 µmol/L indomethacin for 1 hour. Inhibitors were dissolved in 0.1% (vol/vol) DMSO. This concentration of DMSO was added to all culture media. Values (mean±SEM) are obtained from two separate experiments, each performed in triplicate. *P<.05 vs control values. +P<.05 vs values with PDGF-AB.

Next, the effects of various inhibitors of the signal transduction pathway activated by PDGF in vascular cells on the PDGF-stimulated O2·- release were examined. Ro 31-8220 (1 µmol/L), a selective PKC inhibitor,23 significantly inhibited PDGF-AB–induced O2·- production (Fig 3ADown). Wortmannin, a PI3-kinase inhibitor that inhibits O2·- release from neutrophils,24 significantly inhibited the PDGF-AB–induced generation of O2·- at a concentration of 0.1 µmol/L and completely abolished the increase at 1 µmol/L (Fig 3BDown). Nifedipine (0.1 and 1 µmol/L), a Ca2+ channel blocker that has been shown to inhibit the PDGF-induced intracellular Ca2+ elevation in VSMCs,25 did not significantly modify the PDGF-AB–induced O2·- generation (Fig 3CDown).



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  		Figure 3. Effects of inhibitors of signal transduction pathways on PDGF-stimulated O2·- production. HSMCs were incubated with vehicle or 30 ng/mL PDGF-AB with or without Ro 31-8220 (A), wortmannin (B), or nifedipine (C) under conditions shown for 1 hour. Concentrations were Ro 31-8220, 0.3 and 1 µmol/L; wortmannin, 0.1 and 1 µmol/L; and nifedipine, 0.1 and 1 µmol/L. Inhibitors were dissolved in 0.1% (vol/vol) DMSO. This concentration of DMSO was added to all culture media. Values (mean±SEM) are obtained from four (A), three (B), and two (C) separate experiments, each performed in triplicate. *P<.05 vs control values. +P<.05 vs values with PDGF-AB.

Effect of PDGF-Stimulated O2·- Formation on NF-{kappa}B Activation
Because reactive oxygen intermediates have been shown to activate NF-{kappa}B,26 it was examined whether PDGF activates NF-{kappa}B in HSMCs via an increase in O2·- generation. With the gel mobility shift assay, at least two bands with specific NF-{kappa}B binding activity were detected in nuclear extracts from unstimulated HSMCs (Fig 4ADown). PDGF-AB significantly increased NF-{kappa}B binding activity at 30 and 60 minutes. The stimulatory effect reached a peak within 30 minutes, then gradually decreased and returned to the basal level at 120 minutes (Fig 4ADown). The induction of NF-{kappa}B binding activity by PDGF-AB was concentration dependent (Fig 4BDown). Treatment of nuclear extracts with a specific anti-p65 antibody resulted in a supershift of the slower but not the faster band in both untreated and PDGF-treated cells, demonstrating the presence of p65 in the slower but not the faster band (Fig 5Down). Addition of a specific anti-p50 antibody to nuclear extracts resulted in a supershift of both the slower and the faster bands. These findings indicate that the slower-migrating band represents the p50/p65 heterodimer and the faster-migrating band contains the p50 subunit of NF-{kappa}B.



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  		Figure 4. Gel mobility shift assay demonstrating activation of NF-{kappa}B binding by PDGF-AB in HSMCs. A, Time-dependent increase of NF-{kappa}B binding activity in nuclear extracts by PDGF-AB. HSMCs were stimulated with 30 ng/mL PDGF-AB for indicated periods, and gel shift assay was performed as described in "Methods." Brackets and arrows indicate positions of specific and nonspecific NF-{kappa}B binding complexes, which were and were not inhibited with excess amounts of unlabeled NF-{kappa}B binding DNA consensus sequence, respectively (data not shown). B, Concentration-dependent activation of NF-{kappa}B binding by PDGF-AB. HSMCs were stimulated with concentrations of PDGF-AB indicated for 30 minutes, and gel shift assay was performed. Representative autoradiogram of three (A) and two (B) different experiments is shown in top panels. Results of densitometric analysis for specific NF-{kappa}B binding activity are shown in bottom panels (mean±SEM in A and mean in B). *P<.05 vs control values.



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  		Figure 5. Supershift of NF-{kappa}B binding complex by anti-p65 and anti-p50 antibodies (Ab). Nuclear extracts obtained from cells stimulated with or without 30 ng/mL PDGF-AB for 30 minutes were treated with anti-p65 or anti-p50 antibody and analyzed for NF-{kappa}B binding activity. Brackets and arrows indicate positions of specific and nonspecific NF-{kappa}B binding complexes. Arrowheads indicate positions of supershifted complexes. A representative autoradiogram from two different experiments with similar results is shown.

The role of O2·- generation in the PDGF-AB–stimulated activation of NF-{kappa}B in HSMCs was investigated next. Incubation of HSMCs with SOD (500 U/mL) attenuated the stimulatory effect of PDGF-AB (Fig 6ADown). Tiron (10 mmol/L), another scavenger of O2·-,15 also attenuated the effect of PDGF-AB (Fig 6ADown). In addition, NF-{kappa}B activation in response to PDGF-AB was inhibited by iodonium diphenyl (50 µmol/L), Ro 31-8220 (1 µmol/L), and wortmannin (1 µmol/L) (Fig 6BDown).



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  		Figure 6. Inhibitory effects of SOD, Tiron (A), and reagents that inhibit O2·- production (B) on PDGF-AB–stimulated NF-{kappa}B binding activity in HSMCs. HSMCs were treated with vehicle (lane 1) or 30 ng/mL PDGF-AB for 30 minutes in the absence (lane 2) or presence (lane 3) of 500 U/mL SOD or 10 mmol/L Tiron (lane 4) in A and 50 µmol/L iodonium diphenyl (ID, lane 3), 1 µmol/L Ro 31-8220 (lane 4), or 1 µmol/L wortmannin (lane 5) in B. Reagents in B were dissolved in 0.1% (vol/vol) DMSO. This concentration of DMSO was added to all culture media in experiments shown in B. Brackets indicate positions of specific NF-{kappa}B binding complexes. A representative autoradiogram of four different experiments is shown in top panels. Results of densitometric analysis for specific NF-{kappa}B binding activity are shown in bottom panels (mean±SEM). *P<.05 vs control values. +P<.05 vs values with PDGF-AB.

We next investigated the effect of PDGF-AB on NF-{kappa}B activation in combination with IL-1ß, a potent activator of NF-{kappa}B. NF-{kappa}B activation stimulated by 300 U/mL IL-1ß, a concentration that elicited maximum NF-{kappa}B binding activity in HSMCs, was significantly augmented by PDGF-AB but was not affected by SOD (Fig 7Down).



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  		Figure 7. Enhanced stimulation of NF-{kappa}B binding activity by PDGF-AB in IL-1ß–treated HSMCs. HSMCs were treated with vehicle (lane 1) or 300 U/mL IL-1ß in the absence (lane 2) or presence (lane 3) of 30 ng/mL PDGF-AB or 500 U/mL SOD (lane 4) for 30 minutes, and gel shift assay was performed. Bracket indicates position of specific NF-{kappa}B binding complexes. Representative autoradiogram of three different experiments is shown in top panel. Results of densitometric analysis for specific NF-{kappa}B binding activity are shown in bottom panel (mean±SEM). *P<.05 vs control values. +P<.05 vs values with IL-1ß.

Effect of PDGF-Stimulated O2·- Formation on MCP-1 mRNA Induction
Because the promoter of the human MCP-1 gene contains functionally active NF-{kappa}B binding sites,27 we tested whether PDGF-induced expression of MCP-1 mRNA is decreased by inhibition of O2·- production in HSMCs. PDGF-AB induced a pronounced expression of MCP-1 mRNA within 4 hours, although the basal level was barely detectable (Fig 8Down). This PDGF-induced MCP-1 mRNA expression was inhibited by SOD, iodonium diphenyl, Ro 31-8220, and wortmannin (Fig 8Down).



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  		Figure 8. Northern blot analysis showing inhibitory effects of SOD and reagents that inhibit O2·- production on induction of MCP-1 mRNA by PDGF-AB in HSMCs. HSMCs were treated with vehicle or 30 ng/mL PDGF-AB for 4 hours in the presence and absence of 500 U/mL SOD, 10 and 50 µmol/L iodonium diphenyl (ID), 1 µmol/L Ro 31-8220, or 1 µmol/L wortmannin. ID, Ro 31-8220, and wortmannin were dissolved in 0.1% (vol/vol) DMSO. This concentration of DMSO was included in lanes 4 through 8. DMSO alone had no effect on MCP-1 mRNA expression (data not shown). 18S rRNA levels are shown in bottom panel. Representative autoradiogram from three different experiments with identical results is shown.


*    Discussion
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 up arrowMethods
 up arrowResults
 *Discussion
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The present study shows that PDGF is a potent stimulant of O2·- generation in HSMCs. The finding that iodonium diphenyl inhibited this O2·- production implicates the involvement of flavoenzyme(s). Because NADH and NADPH oxidases have been shown to generate O2·- in VSMCs,14 15 it is conceivable that PDGF may activate these enzymes. An angiotensin II–induced increase in the activity of these oxidases in VSMCs has recently been shown.15 In this report, the increase in oxidase activity was observed only several hours after stimulation, suggesting the requirement of some induction steps for the activation of this enzyme system. In our study, however, a consistent and substantial production of O2·- was already evident within 20 minutes. Considering the time course required for the generation of O2·-, the pathways involved in oxidase activation in the present study may be different from those observed after stimulation with angiotensin II.

Earlier studies using nonrecombinant PDGF have shown that PDGF stimulates O2·- release from neutrophils and monocytes.28 29 However, a recent study has indicated that neutrophils do not respond to recombinant PDGF, suggesting that the production of O2·- observed in these previous studies might have been caused by contaminants copurified with PDGF.30 Thus, the stimulatory effect of PDGF on O2·- production in VSMCs seems to be a cell-type–specific phenomenon.

PDGF is a dimer of two polypeptide chains, A and B, forming three isoforms, called PDGF-AA, PDGF-AB, and PDGF-BB.4 PDGF isoforms bind and dimerize two PDGF receptors, {alpha} and ß, with different affinities. PDGF-A chain binds only the PDGF {alpha}-receptor, whereas the PDGF-B chain can bind either receptor. In the present study, PDGF-AB and -BB but not -AA increased O2·- production, suggesting that PDGF exerts its effect via the ß-receptor.

The lack of inhibition by indomethacin of O2·- production suggests that arachidonic acid metabolism through cyclooxygenase does not seem to be a major source of O2·- in PDGF-stimulated HSMCs. Interestingly, indomethacin did further augment PDGF-stimulated O2·- production. Indomethacin has been shown to prevent cAMP elevation evoked by PDGF in VSMCs.31 Because cAMP elevation inhibited O2·- production in other cell types,13 it might be possible that the augmented release of O2·- induced by indomethacin is due to the inhibition of cAMP elevation.

PDGF stimulates phospholipase C{gamma}, leading to the activation of PKC,4 which in turn seems to be involved in O2·- generation and the subsequent NF-{kappa}B activation and MCP-1 mRNA induction, because Ro 31-8220 inhibited these responses. The increase in intracellular Ca2+ concentration, however, does not seem to play a major role in O2·- generation in PDGF-stimulated HSMCs, because nifedipine, which has been shown to inhibit PDGF-stimulated elevation of intracellular Ca2+ concentration in VSMCs,25 had no effect on the O2·- generation by PDGF.

Wortmannin inhibited O2·- production, NF-{kappa}B activation, and MCP-1 mRNA induction in PDGF-stimulated HSMCs. However, the concentrations required (0.1 to 1 µmol/L) were higher than those reported to inhibit PI3 to kinase32 33 (IC50 values, 3 to 50 nmol/L) and O2·- release from neutrophils24 (IC50 value, 7.3 nmol/L). Wortmannin has also been reported to inhibit myosin light chain kinase, a novel phosphatidylinositol 4–kinase and DNA-dependent protein kinase at concentrations greater than those required to inhibit PI3-kinase.32 33 Therefore, it is possible that wortmannin exerted its inhibitory effect via a pathway other than the inhibition of PI3-kinase in HSMCs.

The finding that PDGF activates NF-{kappa}B in HSMCs is consistent with recent data obtained in rat aortic smooth muscle cells34 and murine NIH/3T3 cells.35 Our observations that all compounds that inhibited PDGF- induced O2·- generation in HSMCs also attenuated the activation of NF-{kappa}B suggest a close link between O2·- production and NF-{kappa}B activation. Moreover, the inhibitory effect of SOD and Tiron on PDGF-induced NF-{kappa}B activation further supports the notion that O2·- generated in HSMCs acts as a mediator for the activation of NF-{kappa}B. This conclusion is consistent with recent data showing that generation of O2·- activates NF-{kappa}B in mouse mesangial cells.36 However, our finding that SOD and Tiron did not completely inhibit the activation of NF-{kappa}B in response to PDGF suggests that other signal transduction pathways might also be involved.

The IL-1ß–induced NF-{kappa}B activation was augmented by PDGF in HSMCs. Furthermore, activation of NF-{kappa}B by IL-1ß was not inhibited by SOD, and IL-1ß failed to increase O2·- release from HSMCs (vehicle, 0.17±0.10 nmol · million cells-1 · h-1 versus IL-1ß, 0.18±0.07 nmol · million cells-1 · h-1, n=6). These findings underline that PDGF activates NF-{kappa}B through a signaling pathway different from that activated by IL-1ß. NF-{kappa}B activation has been shown to be functionally important in the induction of NOS type II.37 An inhibitor of NF-{kappa}B, pyrrolidine dithiocarbamate, inhibited induction of NOS type II activity in vascular smooth muscle layers.38 Despite its stimulatory effect on IL-1ß–induced NF-{kappa}B activation observed in the present study, PDGF has been shown to inhibit the induction of NOS type II in response to IL-1ß in VSMCs.17 Recently, transforming growth factor-ß1 has been shown to inhibit NOS type II induction in VSMCs by mechanisms distinct from inhibition of NF-{kappa}B activation.39 In conjunction with these findings, it is conceivable that an as yet unknown inhibitory pathway on induction of NOS type II might be switched on by PDGF that can overcome the stimulatory drive of NF-{kappa}B activation.

Treatments that inhibited PDGF-induced O2·- production, as well as incubation with SOD, significantly attenuated MCP-1 mRNA induction in response to PDGF. These changes were associated with parallel changes in PDGF-induced NF-{kappa}B activation, supporting the hypothesis that PDGF-stimulated O2·- production may be involved in the activation of NF-{kappa}B and subsequent induction of MCP-1 mRNA in HSMCs. Recruitment of inflammatory cells to the subendothelial space is one of the early events in atherogenesis. MCP-1, which is secreted by macrophage-derived foam cells and VSMCs in atherosclerotic plaques,40 acts as a potent chemoattractant for monocytes41 and T lymphocytes.42 Furthermore, MCP-1 accounts for virtually all of the monocyte chemotactic activity secreted by VSMCs exposed to minimally modified LDL.43 Increased MCP-1 mRNA is also observed in aorta after balloon injury.44 Recent reports suggest the potential roles of O2·- and NF-{kappa}B in the development of atherosclerosis and the arterial response to injury. Increased vascular O2·- production has been demonstrated in atherosclerotic animal models,9 10 11 and coronary vasospasm after experimental angioplasty is inhibited by SOD.45 Activated NF-{kappa}B is present in human atherosclerotic lesions,46 and administration of antisense oligonucleotides to the p65 of NF-{kappa}B inhibits neointimal formation in balloon angioplasty–treated rat carotid arteries.47 In conjunction with these reports, the present study supports the idea that O2·- generation and subsequent NF-{kappa}B activation in response to PDGF might contribute to vascular lesion formation by stimulating the migration of vascular cells via induction of MCP-1. It is tempting to explore the possibility that PDGF-induced O2·- production might modulate the expression of other NF-{kappa}B–regulated molecules, such as cytokines, adhesion molecules, colony-stimulating factors, and tissue factor, which are implicated in atherogenesis and restenosis after balloon angioplasty.46

Reduction of ferricytochrome c detects extracellular O2·- levels in the present study. It has been shown that O2·- generated intracellularly is capable of crossing the plasma membrane through anion channels48 and reacting in the extracellular space.49 Therefore, it is conceivable that O2·-, which we have measured, also reflects changes in intracellular O2·- levels. Although we cannot exclude the possibility that PDGF-stimulated HSMCs produce O2·- extracellularly like leukocytes,13 a partial equilibrium between extracellular and intracellular O2·- levels might exist through anion channels.

Considering that SOD cannot easily enter the cells because of its high molecular weight, the findings that exogenous SOD blunted the activation of NF-{kappa}B and MCP-1 induction suggest that the concentration of extracellular O2·- modulates those intracellular responses in PDGF-treated HSMCs. In agreement with our findings, extracellular generation of O2·- has been shown to activate NF-{kappa}B36 and increase MCP-1 mRNA50 in mouse mesangial cells. Extracellular O2·- has been suggested to be at least partly involved in other intracellular responses, including intracellular alkalinization by phorbol ester in U937 cells,51 enhanced bradykinin-stimulated intracellular Ca2+ elevation in vascular endothelial cells by high glucose concentration,52 and inhibition of cardiac muscle oxygen consumption by pyrogallol.53 Together with these reports, our findings support the concept that alteration in extracellular O2·- concentration could influence intracellular signaling pathways. Because cytosolic oxidizing conditions appear to be important regulators of NF-{kappa}B activation,54 it is possible that the intracellular redox state might change in parallel with extracellular O2·- levels in PDGF-stimulated HSMCs. However, the molecular target of O2·- and the subsequent signaling pathways leading to NF-{kappa}B activation remain to be elucidated.

Hydrogen peroxide has been shown to be generated and to mediate signal transduction pathways in PDGF-stimulated VSMCs.55 Because hydrogen peroxide serves as a mediator of NF-{kappa}B activation in some cell types,54 it is possible that increased O2·- might lead to NF-{kappa}B activation, in part via generation of intracellular hydrogen peroxide in PDGF-treated HSMCs.

In conclusion, PDGF stimulates O2·- generation in HSMCs via PKC-dependent and wortmannin-sensitive pathways involving activation of flavoenzyme(s). This O2·- generation, which represents a novel biological activity of PDGF, is most likely to be involved in NF-{kappa}B activation and MCP-1 induction, which are important events in the progression of atherosclerosis and restenosis after balloon angioplasty.


*    Selected Abbreviations and Acronyms
 
HSMC = human aortic smooth muscle cell
MCP-1 = monocyte chemoattractant protein-1
NF-{kappa}B = nuclear factor–{kappa}B
NOS = nitric oxide synthase
O2·- = superoxide anion
PDGF = platelet-derived growth factor
PI3 = phosphatidylinositol 3
PKC = protein kinase C
SOD = superoxide dismutase
VSMC = vascular smooth muscle cell


*    Acknowledgments
 
This work was supported by the Deutsche Forschungsgemeinschaft (Bu436/6-1). Dr Marumo is supported by the Uehara Memorial Foundation and Alexander von Humboldt-Stiftung. The authors are indebted to Dr Alexander Mülsch for many helpful discussions.

Received April 2, 1997; revision received May 8, 1997; accepted May 28, 1997.


*    References
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