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(Circulation Research. 1997;81:797-803.)
© 1997 American Heart Association, Inc.

Articles

Pulsatile Stretch Stimulates Superoxide Production and Activates Nuclear Factor-B in Human Coronary Smooth Muscle

Keiichi Hishikawa, Barry S. Oemar, Zhihong Yang, , Thomas F. Lüscher

From Cardiology, Cardiovascular Research, University Hospitals, Bern and Zürich, Switzerland, and the Institute of Physiology, University Zürich.

Correspondence to Thomas F. Lüscher, MD, FACC, FESC, Professor and Head of Cardiology, University Hospital, CH-8091 Zürich, Switzerland.

	Abstract

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Abstract There is increasing evidence that oxidative stress is of pathophysiological importance in cardiovascular disease. Mechanical forces such as pulsatility may also contribute. Using human coronary artery smooth muscle cells (HCAS), we tested the hypothesis that stretch-induced cell proliferation is associated with oxidative stress. Stretch induced DNA synthesis in HCAS, and this was prevented by the antioxidants N-acetylcysteine and pyrrolidinedithiocarbamate (PDTC). Pulsatile stretch also increased superoxide production from HCAS in a time- and stretch-dependent manner. Stretch-induced superoxide production was inhibited by diphenyleneiodoniumchloride, an NADPH oxidase inhibitor, and p-chloromercuriphenylsulfonic acid, an NADH oxidase inhibitor, but not by the xanthine oxidase inhibitor oxypurinol or the cyclooxygenase inhibitor indomethacin. In electrophoretic mobility shift assays, tumor necrosis factor- activated nuclear factor-B (NF-B) with a peak at 3 hours, whereas pulsatile stretch showed sustained activation during stimulation for up to 24 hours. The sustained activation of NF-B was abolished by cotreatment with N-acetylcysteine or PDTC. Furthermore, treatment of HCAS with antisense p65 and p50 oligodeoxynucleotides of NF-B inhibited stretch-induced DNA synthesis. We propose that pulsatile stretch increases oxidative stress and, in turn, promotes DNA synthesis via NF-B in cultured human coronary artery smooth muscle cells.

Key Words: superoxide • stretch • nuclear factor-B • antisense • NADPH oxidase

	Introduction

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Oxygen-derived free radicals have been implicated in the pathogenesis of atherosclerosis and restenosis.^{1 2} Hypercholesterolemia,³ diabetes,⁴ and ischemia followed by reperfusion⁵ are also associated with increased vascular superoxide (O₂^-) production. Recently, native LDL⁶ and angiotensin II⁷ have been reported to stimulate O₂^- production from endothelial and vascular smooth muscle cells. Moreover, the proliferative response of smooth muscle cells to PDGF is mediated by hydrogen peroxide (H₂O₂).⁸

Mechanical forces such as shear stress,^{9 10} hydrostatic pressure,^{11 12 13} and stretch^{14 15} alter function and structure of the blood vessel wall at the cellular and molecular level.¹⁶ In particular, mechanical forces regulate the release of prostacyclin,^{17 18} NO,^{19 20 21 22 23 24} endothelin,^{25 26 27 28} PDGF,^{29 30 31} and fibroblast growth factor³² from the blood vessel wall. Pulsatile stretch stimulates proliferation of vascular smooth muscle cells,^{14 15} but little is known about the cellular mechanism and the role of oxygen-derived radicals.

To test the hypothesis that oxidative stress is involved in stretch-induced DNA synthesis of HCAS, we examined the effects of antioxidants on stretch-induced DNA synthesis and measured O₂^- production by stretch with or without inhibitors of enzymatic pathways for O₂^- generation. To clarify the molecular mechanism of stretch-induced DNA synthesis, we performed an electrophoretic mobility shift assay evaluating the activation of NF-B. Finally, to characterize the contribution of the NF-B subunit in stretch-induced DNA synthesis, we studied the effects of antisense oligodeoxynucleotides for the human NF-B p65 and p50 subunits on stretch-induced DNA synthesis.

	Materials and Methods

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Materials
Cytochrome c, SOD, PDTC, NAC, DPI, oxypurinol, indomethacin, PCMPS, and NCDC were purchased from Sigma Chemical Co. Chelerythrine chloride was purchased from Calbiochem-Nova Biochem Corp. FBS and tissue culture materials were from GIBCO.

Cell Isolation and Culture
HCAS were obtained from organ donors by a modified explant method, as recently reported.¹⁵ In brief, the media of left coronary arteries was isolated under a microscope, cut into 1-mm² pieces, and placed onto plastic culture plates in DMEM supplemented with 20 mmol/L L-glutamine, 10 mmol/L HEPES, 100 U/mL penicillin, 100 µg/mL streptomycin, and 20% FBS. The medium was replaced every 3 days. Experiments were performed between passages 3 and 10. HCAS were characterized by an indirect immunofluorescence immunological staining using specific anti–smooth muscle -actin antibodies.¹⁵ Cell number was determined by Coulter counter (Coulter Electronics).

Application of Pulsatile Stretch on Cultured Cells
HCAS were seeded onto type I collagen-coated Flex I (Flexcell International Corp) culture plates at an initial density of 10⁵ cells/mL. After 24 hours of incubation to allow for cell attachment, culture medium was replaced with serum-free medium (DMEM) containing 0.2% BSA for 48 hours to obtain quiescent nondividing cells. After 48 hours of serum starvation, Flex I culture plates were placed on a computerized Flexcell Strain Unit (Flexcell International Corp). The membranes were subjected to deformation with -5 and -20 kPa of vacuum (maximal and average strain, 12.5%, 6.0%, 25%, and 10% elongation, respectively) at a frequency of 60 cycles/min for up to 24 hours.^{14 15 33 34} The cell viability with and without stretch, as assessed with trypan blue exclusion test, was >90% throughout the experiments.

DNA Synthesis
DNA synthesis was evaluated by [³H]thymidine incorporation. After 24 hours of pulsatile stretch, HCAS were pulsed with [³H]thymidine (1 µCi/mL, 70 to 85 Ci/mol, Amersham) for 4 hours and then washed with PBS and ice-cold 10% TCA at 4°C for 30 minutes. The rubber bottoms of the Flex plates containing the TCA-precipitated material were washed with PBS, removed from the plates, and placed directly into a scintillation vial for counting with a beta counter (LKB Wallac, MBV AG).

Measurement of Superoxide (O₂^-) Production
Superoxide production was measured as the SOD-inhibitable reduction of cytochrome c.^{35 36 37} Briefly, HCAS were preincubated in DMEM without phenol red for 30 minutes at 37°C, and then cytochrome c (final concentration, 1 mg/mL) with or without SOD (final concentration, 500 U/mL) was added and kept in a CO₂ incubator on the Flex Unit. At the indicated time points, the medium was removed from the cells, and the absorbance was read at 550 nm against a distilled water blank. Reduction of cytochrome c in the presence of SOD was subtracted from the values without SOD. The portion of superoxide-specific reduction of cytochrome c was between 30% and 35%. The optical density difference between comparable wells with or without SOD was converted to equivalent O₂^- production by using the molar extinction coefficient for cytochrome c [21.0x10³ (mol/L)^-1 · cm^-1].³⁵

Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared as described.³⁸ The NF-B double-stranded oligodeoxynucleotides corresponding to the palindromic NF-B consensus sequence (AGTTGAGGG GACTTTCCCAGG) were end-labeled with [-³³P]ATP and T4 polynucleotide kinase and purified by the QIA quick nucleotide removal kit (QIAGEN). Nuclear extracts (10 µg) were added to ³³P-labeled NF-B oligonucleotides (20 000 to 50 000 cpm, 0.2 ng) in a buffer containing 1 mmol/L MgCl₂, 0.5 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 50 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 7.5), and 0.05 µg/µL poly dIdC in 20% glycerol. Mixtures were incubated for 30 minutes at room temperature. Then samples were analyzed on native 4% nondenaturing acrylamide gel at 120 V for 3 hours in a low ionic strength buffer (0.5x TBE). The gels were dried and subjected to autoradiography for 24 hours. For supershift assay, specific antibodies to p65 and p50 (Santa Cruz Biotechnology) were supplemented 30 minutes before electrophoresis.

Antisense Oligonucleotides
Phosphorothio analogue oligodeoxynucleotides for the human NF-B p65 and p50 subunits were synthesized by MWG (Biotech). The sequence of oligodeoxynucleotides used in the present study were as follows: antisense p65, 5'-GGGGAA CAGTTCGTCCATGGC-3'³⁹ ; sense p65, 5'-GCCATGGACG AACTGTTCCCC-3'³⁹ ; scramble p65, 5'-CTAGCTCCCGAG GGTGGAGTA-3'; antisense p50, 5'-TGGATCATCTTCTG CATTCT-3'⁴⁰ ; sense p50, AGAATGGCAGAAGATGATC CA-3'⁴⁰ ; and scramble p50, 5'-CAGACAGATGTACAAGT GAGA-3'. These oligodeoxynucleotides were added in a serum-free medium 24 hours before the start of pulsatile stretch. Compared with transfection using cationic compound or the calcium phosphate method, this simple procedure could avoid nonspecific effect, and oligodeoxynucleotides were uptaken effectively by HCAS.

RT-PCR Analysis
Total RNA was extracted using Trizol (GIBCO) and was reverse-transcribed using random hexamers by superscript (II) GIBCO. The p65 NF-B primers (1) 5'-GCGGCCAAGCT TAAGATCTGCCGAGTAAAC-3' and (2) 5'-CGCTGCT CTAGAGAACACAATGGCCACTTGCCG-3' define an amplicon of 150 bp.⁴¹ The p50 NF-B primers (1) 5'-AAAGGT TATCGTTCAGTT-3' and (2) 5'-TTGTAGATAGGCAAG GTC-3' define an amplicon of 250 bp. The human GAPDH primers (1) 5'-CAGGAATTCGGTGAAGGTCGGAGTCA ACGG-3' and (2) 5'-AGTGGATCCGGTCATGAGTCCTT CCACGAT-3' define an amplicon of 540 bp.⁴² PCR reactions were performed in a Biometra Trioblock thermocycler for 40 cycles at an annealing temperature of 60°C for 30 seconds, denaturation was at 94°C for 30 seconds, and primer extension was at 72°C for 1 minute. PCR products were then separated by 1% agarose gel electrophoresis and visualized and photographed using the visionary gel documentation system (Fotodyne).

Statistical Analysis
Data are presented as mean±SEM. Multiple comparisons were evaluated by ANOVA, followed by Fisher's protected least-significant-difference test. Student's paired or unpaired t tests were used for comparisons between two experiments. A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of NAC and PDTC on Stretch-Induced Cell Proliferation and DNA Synthesis
To clarify the role of oxidative stress in stretch-induced cell proliferation and DNA synthesis, we used two different antioxidants, NAC⁴³ and PDTC,⁴⁴ with several pilot experiments to determine the appropriate concentration and incubation time. Although neither NAC (10 mmol/L) nor PDTC (50 µmol/L) showed a significant effect on static cells (data not shown), these compounds significantly inhibited stretch-induced cell proliferation and DNA synthesis (Fig 1).

View larger version (19K): [in this window] [in a new window]   Figure 1. Effect of PDTC and NAC on stretch-induced cell proliferation. HCAS that were growth-arrested by 48-hour serum starvation were stimulated by 20% FBS or pulsatile stretch (ST; 10% elongation, 60 cycles/min). PDTC (50 µmol/L) was added immediately before the exposure to ST. NAC (10 mmol/L) was added 12 hours before ST was initiated. A, Cell number was determined at the indicated time points. indicates control (no ST); , 20% FBS; , ST; , ST+PDTC; and , ST+NAC. B, DNA synthesis was measured after 24 hours of stimulation. Data are mean±SEM (n=6). *P<.05 vs control (no ST). +P<.05 vs ST alone.

Pulsatile Stretch Stimulates O₂^- Production From HCAS
We next examined whether pulsatile stretch stimulates O₂^- production from HCAS. Pulsatile stretch leading to a 10% average elongation for up to 60 minutes enhanced O₂^- production in a time-dependent manner (Fig 2, P<.05). After 60 minutes, O₂^- production reached plateau, and 10% stretch showed continuous enhancement of O₂^- production during stimulation for up to 24 hours. Lower pulsatile stretch (6% average elongation) showed a tendency to enhance O₂^- production, but this did not reach significance.

View larger version (22K): [in this window] [in a new window]   Figure 2. Pulsatile stretch stimulates O2- production from HCAS. A, O2- production was measured in the absence () and presence () of pulsatile stretch (10% average, 60 cycles/min) up to 60 minutes. Data are mean±SEM (n=6). *P<.05 vs absence of stretch. B, Dose-response relation of stretch-induced O2- production from HCAS. HCAS were stimulated by pulsatile stretch (6% and 10% average, 60 cycles/min) for 60 minutes. Data are mean±SEM (n=6). *P<.05 vs control (no stretch).

Possible Enzymatic Pathway of O₂^- Production by Stretch
To clarify the enzymatic pathway by which pulsatile stretch stimulates O₂^- production, HCAS were pretreated with DPI,⁴⁵ PCMPS,⁴⁶ indomethacin,⁴⁷ and oxypurinol.⁴⁸ The effects of these inhibitors were examined between 0.1 and 100 µmol/L, and maximal effects were statistically examined. Stretch-induced O₂^- production was significantly inhibited by DPI and PCMPS (P<.05) but not by indomethacin and oxypurinol (Fig 3A). We further examined the effect of NCDC and chelerythrine chloride. Both of them significantly inhibited stretch-induced O₂^- production (Fig 3B, P<.05).

View larger version (36K): [in this window] [in a new window]   Figure 3. Possible enzymatic pathways of O2- production by pulsatile stretch. A, HCAS were stimulated by pulsatile stretch (ST; 10% average, 60 cycles/min) for 60 minutes in the absence or presence of DPI (10 µmol/L), PCMPS (10 µmol/L), indomethacin (INDO, 1 µmol/L), or oxypurinol (OXY, 1 µmol/L). None of the inhibitors showed a significant effect on O2- production in a static condition. Data are mean±SEM (n=6). *P<.05 vs control (no ST). +P<.05 vs ST alone. B, HCAS were stimulated by ST (10% average, 60 cycles/min) for 60 minutes in the absence or presence of NCDC (100 µmol/L) and chelerythrine chloride (CHEL, 1 µmol/L). None of the inhibitor showed a significant effect on O2- production under static conditions. Data are mean±SEM (n=6). *P<.05 vs control (no ST). +P<.05 vs ST alone.

Activation of NF-B by TNF- and Stretch
To clarify the effects of pulsatile stretch on the activity of NF-B binding protein directly, electrophoretic mobility shift assays were performed. Incubation of ³³P-labeled double-stranded 21-bp oligodeoxynucleotides containing NF-B binding sites with nuclear proteins from HCAS treated with TNF- (10 ng/mL) resulted in retardation of bands, with a peak at 3 hours (Fig 4, left). Although 6% stretch showed no shift (data not shown), 10% stretch showed sustained NF-B activation during stimulation for up to 24 hours. This sustained NF-B activation induced by 24-hour stretch was abolished by both NAC and PDTC (Fig 4, right).

View larger version (90K): [in this window] [in a new window]   Figure 4. Activation of NF-B by TNF- and mechanical stretch. HCAS were starved with 0.2% BSA for 48 hours. HCAS were then treated with TNF- (10 ng/mL) or stretch (10% elongation, 60 cycles/min). Nuclear protein extracts were obtained at varying times, and an electromobility shift assay was performed. Control (cont) refers to no stretch. PDTC (50 µmol/L) was added immediately before the exposure to stretch. NAC (10 mmol/L) was added 12 hours before stretch was initiated. Assays are representative of three independent experiments.

Properties of NF-B Binding Proteins Induced by Stretch
Nuclear proteins from HCAS stretched by 10% for 24 hours were preincubated with a 20- to 100-fold molar excess of unlabeled probe before exposure to the ³³P-labeled oligodeoxynucleotides. Addition of unlabeled probe abolished the bands (Fig 5, left). These results show that stretch-induced nuclear proteins bound specifically to B sequence. Furthermore, supershift assay using antibodies to NF-B subunits (p50 and p65) caused further gel retardation (Fig 5, right).

View larger version (113K): [in this window] [in a new window]   Figure 5. Properties of stretch-induced NF-B binding complex in HCAS. Nuclear proteins from HCAS (lane 1 and 4) stimulated by pulsatile stretch for 24 hours were pretreated with 20-fold (lane 2) and 100-fold (lane 3) excess of unlabeled probe or antibodies to human p50 (lane 5) or p65 (lane 6). Assays are representative of three independent experiments.

Inhibition of Stretch-Induced DNA Synthesis by Antisense to NF-B Subunits
To determine whether the activation of NF-B by stretch is directly associated with stretch-induced DNA synthesis, the effect of antisense oligodeoxynucleotides to NF-B subunits p65 and p50 was examined. Both antisense oligodeoxynucleotides to p65 and p50 concentration-dependently inhibited stretch-induced DNA synthesis, but neither sense nor scramble oligodeoxynucleotides of p65 and p50 showed significant effects up to a concentration of 20 µg/mL (Fig 6). Moreover, these antisense oligodeoxynucleotides to p65 and p50 showed no effect on DNA synthesis in static cells (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 6. Inhibition of stretch-induced DNA synthesis by antisense oligodeoxynucleotides to p65 and p50. HCAS growth-arrested by 48-hour serum starvation were stimulated by pulsatile stretch (10% elongation, 60 cycles) for 24 hours in the presence of various concentrations of antisense oligodeoxynucleotides. indicates sense oligodeoxynucleotides; , antisense oligodeoxynucleotides; and , scramble oligodeoxynucleotides. Data are mean±SEM (n=6). *P<.05 vs no antisense treatment.

Inhibition of p65 and p50 mRNA by Antisense
To ensure that antisense oligodeoxynucleotides of p65 and p50 specifically inhibited the expression of mRNA of p65 and p50, the direct effects of these antisense oligodeoxynucleotides were evaluated by RT-PCR. The p65 antisense oligodeoxynucleotides abolished p65 mRNA expression by stretch, whereas mRNA expression of the housekeeping gene GAPDH remained (Fig 7A). Similarly, the p50 antisense oligodeoxynucleotides abolished p50 mRNA expression, whereas mRNA of GAPDH was unaffected (Fig 7B).

View larger version (53K): [in this window] [in a new window]   Figure 7. Inhibition of p65 and p50 mRNA expression by antisense oligodeoxynucleotide treatment. HCAS were stretched for 24 hours in the presence of p65 antisense oligodeoxynucleotides (A) or p50 antisense oligodeoxynucleotides (B). Total RNA was isolated and analyzed by RT-PCR for p65, p50, and GAPDH expression. Lane 1 indicates no stretch; lane 2, 24-hour stretch; lane 3, 24-hour stretch+antisense oligodeoxynucleotides; lane 4, 24-hour stretch+sense oligodeoxynucleotides; lane 5, 24-hour stretch+scramble oligodeoxynucleotides; lane 6, no reverse transcription; and lane 7, no template. Assays are representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study for the first time demonstrates that pulsatile stretch stimulates O₂^- production in HCAS and activates the nuclear transcription factor NF-B. Our results may in part support the usefulness of NAC and PDTC in clinical medicine and that of p65 antisense oligodeoxynucleotides to inhibit neointimal formation after experimental balloon injury in rat carotid arteries.⁴⁹

Recently, superoxide has been reported to be involved in angiotensin II–induced hypertension⁵⁰ in the rat, platelet activation,⁵¹ and Ras-induced cell cycle progression,⁵² but the mechanisms of O₂^- generation in HCAS are poorly understood. In phagocytic cells, oxidative burst requires the assembly of the flavoprotein containing the NADPH-oxidase complex. In the rabbit aorta, NADPH oxidase is the major source of O₂^- production.⁴⁵ In calf pulmonary artery smooth muscle cells,^{46 47} NADH oxidase appears to be the most important source of O₂^-. In platelets, on the other hand, NADPH oxidase and activation of phospholipase C and cyclooxgenase are involved to produce O₂^-.⁵³ To determine whether functionally similar enzymes were involved in the O₂^- production by HCAS, we treated HCAS with various enzyme inhibitors. DPI inhibits NADPH oxidase but also other flavoprotein-dependent enzymes. In HCAS, DPI almost completely inhibited stretch-induced O₂^- production. This therefore strongly suggests that NADPH oxidase is the enzyme responsible for O₂^- generation in HCAS, but the unspecificity of DPI limits this interpretation.

Protein kinase C is activated by diacylglycerol, which is released by hydrolysis of phosphatidylinositol 4,5-diphosphate by phospholipase C, and inositol 1,4,5-trisphosphate as a coproduct. Interestingly, protein kinase C also activates NADPH oxidase.⁵⁴ Because phospholipase C is located in the cell membrane and can be easily affected by mechanical stretch, we hypothesized that the phospholipase C–protein kinase C system may be involved in stretch-induced O₂^- production. Both NCDC, a phospholipase C inhibitor,⁵⁵ and chelerythrine chloride, a selective protein kinase C inhibitor,⁵⁶ significantly inhibited stretch-induced O₂^- production. These results suggest that phospholipase C and protein kinase C are involved in the activation of NADPH oxidase.

Although NF-B has been extensively investigated in hematopoietic cells and is well known to be activated by oxidative stress,^{57 58} its activity and function in vascular smooth muscle cells has not been elucidated. In bovine smooth muscle cells, the immediate-early gene of cytomegalovirus is activated by NF-B–like activity.⁴³ Recently, mitogenic growth factors (FBS, PDGF-BB, bFGF, EGF, and IGF-1) but not growth inhibitors (TGF-ß and IFN-) have been reported to induce NF-B in rat aortic smooth muscle cells.⁵⁹ However, nothing is known about the activation of NF-B by mechanical forces in vascular smooth muscle cells. This is the first report that demonstrates activation of NF-B by mechanical forces, such as stretch, in vascular smooth muscle cells. Interestingly, NF-B activation by TNF- was transient, as reported for mitogens (FBS, PDGF-BB, bFGF, EGF, and IGF-1),⁵⁹ whereas the activation by stretch was sustained as long as stretch continued for up to 24 hours.

NF-B consists of two major distinct polypeptides of 50 and 65 kD, termed p50 and p65. In mammals, the most widely distributed B binding factor is a heterodimer consisting of p50 and p65. Because a different contribution of p50 and p65 has been reported,^{41 49} we tried to characterize the contribution of p50 and p65 to stretch-induced DNA synthesis. Supershift assays showed a specific contribution of both p50 and p65 in the NF-B activation by stretch but did not clarify the difference between the two. Therefore, we constructed antisense oligodeoxynucleotides to p50 and p65 and tried to characterize their contribution in stretch-induced DNA synthesis. To avoid the nonspecific effect of phosphorothioate oligodeoxynucleotides,⁶⁰ we limited the concentration to 20 µg/mL. Under these conditions, both antisense oligodeoxynucleotides to p50 and p65, but not the sense or scramble oligodeoxynucleotides, showed significant inhibitory effects. However, no clear difference between the two antisense oligodeoxynucleotides was found. To clarify the specificity of antisense oligodeoxynucleotides, we performed RT-PCR. Although Northern blot analysis has a higher selectivity to target than does RT-PCR, we choose the latter method because of its higher sensitivity and because our aim was to document inhibition of mRNA synthesis by the antisense oligodeoxynucleotides. Each antisense oligodeoxynucleotide specifically inhibited its mRNA and had no effect on mRNA of the housekeeping gene GAPDH. These results therefore suggest that the heterodimer of p50 and p65 plays a major role in the activation of NF-B by stretch but that the exact intracellular mechanism of stretch-induced superoxide production and NF-kB activation is still unclear.

Finally, to clarify a possible role of prestored growth factors such as bFGF, which might be released from the cells during stretch, we collected conditioned medium after 24 hours of stretch and added the fluid to another well containing quiescent HCAS. Although we counted cell number up to 4 days after addition of the medium, we were unable to find any proliferative effect (data not shown), suggesting that the contribution of a paracrine release of growth factors is minimal.

In summary, we demonstrated that pulsatile stretch stimulates O₂^- production and activates NF-B in HCAS. Chemical antioxidants and antisense oligodeoxynucleotides to NF-B significantly inhibited stretch-induced DNA synthesis. Taken together, our data provide novel insight into the interaction of mechanical stimulation and oxidative stress and demonstrate the usefulness of antioxidants and antisense oligodeoxynucleotides to prevent proliferation of HCAS by mechanical stimuli.

	Selected Abbreviations and Acronyms

 

bFGF = basic fibroblast growth factor DPI = diphenyleneiodoniumchloride EGF = epidermal growth factor HCAS = human coronary artery smooth muscle cell(s) IFN- = interferon gamma IGF-1 = insulin-like growth factor NAC = N-acetylcysteine NCDC = 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate NF-B = nuclear factor-B PCMPS = p-chloromercuriphenylsulfonic acid PCR = polymerase chain reaction PDGF = platelet-derived growth factor PDTC = pyrrolidinedithiocarbamate RT = reverse transcription SOD = superoxide dismutase TCA = trichloroacetic acid TNF- = tumor necrosis factor-

	Acknowledgments

 
This study was supported by the Swiss National Research Foundation (grant 32-32541.91/2), a grant-in aid from Mobiliar Insurance, Bern, Switzerland, and the Karl Mayer Foundation, Liechtenstein.

Received July 18, 1997; accepted July 31, 1997.

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