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(Circulation. 1997;96:3602-3609.)
© 1997 American Heart Association, Inc.

Articles

Differential Effect of Hydrogen Peroxide and Superoxide Anion on Apoptosis and Proliferation of Vascular Smooth Muscle Cells

Pei-Feng Li, PhD; Rainer Dietz, MD; ; Rüdiger von Harsdorf, MD

From the Max-Delbrück-Center for Molecular Medicine, Berlin, and the Department of Cardiology, Franz-Volhard-Clinic, University Hospitals Rudolf-Virchow, Humboldt-University Berlin (R.D., R. von H.), Germany.

Correspondence to Rüdiger von Harsdorf, MD, Franz-Volhard-Klinik, Universitätsklinikum Rudolf-Virchow, Humboldt-Universität zu Berlin, Wiltbergstr 50, 13125 Berlin, FRG. E-mail rharsdo{at}mdc-berlin.de

	Abstract

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Background Proliferation and apoptosis of vascular smooth muscle cells (VSMCs) are two important components of atherosclerosis, restenosis, and hypertension. Although reactive oxygen species have been demonstrated to participate in the pathogenesis of these diseases, their precise involvement has not been fully understood. We hypothesized that different reactive oxygen species exert distinct effects on proliferation and apoptosis of VSMCs.

Methods and Results Cultured rat VSMCs were exposed to xanthine oxidase/xanthine (XO/X) or H₂O₂-Fe(II). A single exposure to XO/X predominantly resulted in cell proliferation, whereas frequent exposures to high levels of XO/X predominantly resulted in cell death. Administration of superoxide dismutase and catalase revealed that O₂^- but not H₂O₂ was mitogenic to VSMCs, whereas H₂O₂ was responsible for VSMC death. Treatment with H₂O₂-Fe(II) alone or in the presence of different hydroxyl radical scavengers showed that VSMC death occurred in a dose-dependent manner and was mediated by the formation of hydroxyl radicals. Cell death caused by XO/X or H₂O₂-Fe(II) occurred by apoptosis as revealed by condensation of nuclei, appearance of a "DNA ladder," increases in DNA fragmentation, and positive in situ nick-end labeling. Northern blot analysis indicated that bcl-2 and c-fos but not p53 and c-myc may participate in mediating H₂O₂-Fe(II)–induced VSMC apoptosis.

Conclusions Different reactive oxygen species exert distinct effects on VSMCs, with O₂^- inducing proliferation and H₂O₂ causing apoptosis. Thus, reactive oxygen species might participate in atherosclerosis, restenosis, and hypertension in a dual manner by stimulating proliferation and triggering apoptosis of VSMCs.

Key Words: arteriosclerosis • free radicals • muscle, smooth • hypertension

	Introduction

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Vascular remodeling represents the pathophysiological basis of many diseases, including atherosclerosis, hypertension, and restenosis. It is referring to the modulation of the phenotype of VSMCs, which is characterized not only by cell migration and synthesis of extracellular matrix but also by such contrasting phenomena as cell proliferation and cell death.

Abnormal VSMC proliferation has been shown to lead to functional and anatomic alterations of the vessel.^{1 2} Physiologically, VSMCs are in a low proliferating state, but in pathological processes, they can be stimulated to proliferate by a number of factors, including growth factors, cytokines, and angiotensin II.^{3 4} Recently, ROS have been found to be related to VSMC proliferation. In vivo studies show that balloon-injured arteries produce increased amounts of ROS.⁵ Vitamin E, an antioxidant, can attenuate intimal response to balloon injury.⁶ In vitro studies also demonstrate that ROS can stimulate DNA synthesis in VSMCs.^{7 8} Because ROS comprise a group of different molecules, including H₂O₂, O₂^-, and ·OH, it would be important to understand the specific role of each of these species for VSMC proliferation.

There is an increasing body of evidence showing that apoptosis of VSMCs participates in the pathogenesis of atherosclerosis, restenosis, and hypertension^{9 10 11 12} and plays a role in intimal thickening induced by endothelial denudation.¹³ Furthermore, inflammatory components are important for the induction of apoptosis as indicated by the observation that simultaneous treatment with interferon- and tumor necrosis factor- and/or interleukin-1-ß can trigger apoptosis in cultured human and rat VSMCs.¹⁴ Whereas cultured human VSMCs derived from normal vessels undergo apoptosis only on serum withdrawal, VSMCs from coronary atherosclerotic plaques are much more susceptible to apoptotic stimuli, resulting in a significantly elevated rate of apoptosis after serum deprivation.¹⁵ Eukaryotic cells continuously produce ROS in physiological levels. The imbalance between their generation and decomposition has been implicated in many kinds of clinical disorders.^{16 17} In the vascular system, for example, certain circumstances such as ischemia, reperfusion, inflammation, thrombosis, and angioplasty are accompanied by excessive productions of ROS.^{18 19} It is therefore conceivable that ROS might participate in inducing apoptosis of VSMCs. However, whether ROS can trigger VSMC apoptosis remains unknown.

The present study was designed to investigate the specific effect of H₂O₂, O₂^-, and ·OH on proliferation and apoptosis of VSMCs and to determine the effect of H₂O₂ on expression of apoptosis-related genes in VSMCs. Our results provide for the first time evidence showing that different ROS exert distinct effects on VSMCs. Furthermore, H₂O₂ is an important factor for the induction of apoptosis in VSMCs. Finally, bcl-2 and c-fos but not c-myc and p53 may participate in mediating H₂O₂-induced VSMC apoptosis.

	Methods

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Materials
H₂O₂, XO, X, ferrous sulfate, SOD, CAT, DF, DMSO, propidium iodide, and monoclonal anti–-smooth muscle actin antibody were purchased from Sigma Chemical Co. MTT kit, cell death detection ELISA kit, blocking reagent, and anti-digoxigenin antibody were purchased from Boehringer Mannheim. [³H]-thymidine was from Du Pont NEN. In situ apoptosis detection kit was purchased from Oncor. The human c-myc cDNA probe and the mouse p53 cDNA probe were purchased from Calbiochem. The human bcl-2 cDNA was a generous gift from Timothy E. Allsopp,²⁰ and the human c-fos cDNA was kindly provided by Michael Greenberg.

Cell Culture
VSMCs were obtained from the thoracic aortas of 200- to 250-g male Wistar rats by use of the collagenase and elastase digestion method.²¹ Cells were seeded in medium 199 supplemented with 10% heat-inactivated FCS, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified 5% CO₂ atmosphere at 37°C. Cells at passages 8 through 13 were used for experiments. For the experiments with growth-arrested VSMCs, cells were made quiescent by incubating in the above culture medium containing 0.2% FCS for 48 hours before use. VSMCs cultured in 10% FCS are hereafter referred to as growing VSMCs.

Exposure of Cells to ROS Generating Systems
The cultured cells were incubated at 37°C for 1 hour in HBSS containing the indicated concentration of XO/Xor H₂O₂-Fe(II). To remove ammonium sulfate that is present in commercially available preparations of XO, XO was centrifuged for 1 minute at 13 000 rpm at 4°C and washed twice with HBSS before use. Ferrous sulfate was dissolved in double-distilled water, gently stirred, and immediately prepared before use to prevent autoxidation. The reaction was stopped by removing the HBSS containing the ROS generating systems. The cells were further cultured for the indicated time in freshly prepared culture medium as before treatment. The second and the third exposures of VSMCs to XO/X were performed 24 and 48 hours after the first treatment, respectively. For the administration of antioxidants, SOD, CAT, DMSO, ethanol, or mannitol was added simultaneously with XO/X or H₂O₂-Fe(II), whereas DF was preincubated with cells for 2 hours before treatment.

Cell Viability Assay
H₂O₂-Fe(II)–treated cell viability was assessed by MTT test with MTT kits. The assay procedures were followed according to kit instructions. Briefly, the cells were plated in 96-well plates in a final volume of 100 µL of culture medium. After treatment with ROS, 10 µL of the MTT labeling reagent was added to each well and incubated for 4 hours at 37°C. Then 100 µL of the solubilization solution was added to each well and incubated overnight at 37°C. Optical density was determined at 570 nm. XO/X-treated cell viability was determined by trypan blue exclusion.

Measurements of DNA Synthesis and Cell Number
Cells were grown in 12-well plates with a seeding density at 3000 per 1 cm². When cells reached subconfluence, they were made quiescent for 48 hours in low serum conditions. After treatment, cells were further cultured for 24 hours and pulse labeled with [³H]-thymidine (1 µCi/mL) for 2 hours before the completion of the 24-hour incubation period. [³H]-thymidine incorporation into DNA was measured as TCA-insoluble radioactivity as described previously with slight modifications.²² Briefly, cells were washed three times with PBS and then incubated with 15% TCA at 4°C for 30 minutes. After aspiration of TCA, cells were washed twice with distilled water. Then 1 mol/L NaOH was added for 20 minutes and neutralized with 1 mol/L HCl. The contents of the wells were placed in scintillation vials for counting. For the determination of cell numbers, cells were cultured for 6 days after treatment. The media were changed every 48 hours. Cells were suspended with trypsin/EDTA (0.05%/0.5 mmol/L) and counted with a hemocytometer.

Analysis of DNA Fragmentation
Cells were harvested, centrifuged at 1200 rpm for 6 minutes, and then digested with lysing buffer (10 mmol/L Tris-HCl, pH 8.0, 25 mmol/L EDTA, 0.5% SDS (wt/vol), 50 µg/mL proteinase K, 20 µg/mL RNase) at 37°C for 20 hours. The DNA was extracted with equal volumes of phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated at -20°C overnight by adding 0.1 volume of 3 mol/L sodium acetate, pH 5.5, and 2 volumes of ethanol (100%). DNA was subjected to electrophoresis on a 1.8% agarose gel, stained with ethidium bromide, and visualized under UV light.

In Situ Nick-End Labeling and Propidium Iodide Staining
The terminal deoxyribonucleotidyl transferase–mediated TUNEL assay was used to detect DNA fragmentation in situ. The detection procedures were in accordance with the kit instructions. Briefly, the samples were preincubated with equilibration buffer for 5 minutes and subsequently incubated with deoxyribonucleotidyl transferase in the presence of digoxigenin-conjugated dUTP for 1 hour at 37°C. The reaction was terminated by incubating the samples in stopping buffer for 30 minutes. After three rinses with PBS, the fluorescein-labeled anti-digoxigenin antibody was incubated with samples for 30 minutes, and three rinses with PBS were repeated. Finally, the samples were stained for 8 minutes with propidium iodide at 5 µg/mL in PBS, pH 7.4, containing 50 µg/mL RNase A (without DNase) and then washed with PBS and mounted.

Cell Death Detection ELISA
Cell death detection ELISA was performed according to the manufacturer's instruction. Briefly, the anti-histone monoclonal antibody was added to the 96-well ELISA plates and incubated overnight at 4°C. After recoating and three rinses, the cytoplasmic fractions were added and incubated for 90 minutes at room temperature. After washing, bound nucleosomes were detected by the addition of anti–DNA-peroxidase monoclonal antibody and reacted for 90 minutes at room temperature. After the addition of substrate, optical density was read with an ELISA reader at 405 nm.

RNA Isolation and Northern Blot Analysis
Total RNA was isolated from cultured cells with the guanidinium isothiocyanate extraction method.²³ Prehybridization was conducted at 42°C for 4 hours in prehybridization buffer: 50% formamide, 5x SSC, 2% blocking reagent, 50 mmol/L sodium phosphate, pH 7.4, 7% SDS (wt/vol), and 0.1% N-laurylsarkosine (wt/vol). Hybridization was performed in the same buffer and temperature for 30 hours with digoxigenin-labeled specific probes. For chemiluminescent detection, the membrane was blocked for 30 minutes in 2.5% blocking reagent. The membrane was then incubated for 30 minutes with anti-digoxigenin antibody conjugated with alkaline phosphatase. After two washes with 100 mmol/L maleic acid buffer containing 0.3% Tween-20, CSPD substrate solution was added to the membrane and incubated for 10 minutes. The membrane was wrapped in plastic and exposed to film.

Statistical Analysis
The results are expressed as mean±SEM of at least three independent experiments unless stated otherwise. Paired data were evaluated by Student's t test. A one-way ANOVA was used for multiple comparisons. A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Distinct Effects of O₂^- and H₂O₂ on VSMCs With O₂^- Inducing Proliferation and H₂O₂ Causing Cell Death
As Fig 1 shows, a single 1-hour exposure of growth-arrested VSMCs to XO/X resulted in increases of [³H]-thymidine incorporation (Fig 1A) and cell number (Fig 1B). Because XO/X yields both O₂^- and H₂O₂, the effect of XO/X in the presence of SOD or CAT was tested. Neither SOD nor CAT alone had any influence on VSMC DNA synthesis or cell number (data not shown). The effect of XO/X was not altered in the presence of CAT. In contrast, the administration of SOD abolished XO/X-induced increases in DNA synthesis and cell number, resulting in values even below those obtained from unstimulated control cells. It is quite unlikely that the observed effect is due to proteases contaminating preparations of XO, because the stimulation of VSMCs with XO in the absence of xanthine had no effect on VSMCs (see Fig 1) and XO/X-induced cell proliferation could be inhibited by the XO inhibitor allopurinol in a dose-dependent fashion (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 1. Effect of XO/X on proliferation of growth-arrested VSMCs analyzed by [3H]-thymidine incorporation and cell counting. VSMCs were exposed to XO in the presence of 0.1 mmol/L xanthine. XO was 0.01 U/mL when SOD and CAT were administrated. VSMCs in 10% FCS served as positive control. A, [3H]-thymidine incorporation. Each bar represents the mean±SEM of three separate experiments in quadruplicate. *P<.05, **P<.001 vs control. B, Cell counting. Each bar represents the mean±SEM of five to seven separate experiments in duplicate. *P<.05, **P<.02 vs control.

Three consecutive exposures of growing VSMCs to XO/X conducted every 24 hours led to a gradual and dose-dependent decline of VSMC viability (Fig 2A). Interestingly, in growth-arrested VSMCs, XO at 0.0025 or 0.005 U/mL continued to induce proliferation after repeated exposures. None of these effects could be observed when VSMCs were stimulated by XO/X in the presence of allopurinol (data not shown). We conclude that XO/X elicits distinct reactions in VSMCs, depending on the dose and the frequency of exposure resulting in either proliferation or cell death.

View larger version (40K): [in this window] [in a new window]   Figure 2. Effect of three exposures to XO/X on VSMC death assessed by trypan blue exclusion. Cell viability was determined 24 hours after the third exposure to XO/X. Each bar represents the mean±SEM of four separate experiments in duplicate. A, Cell viability of growing and growth-arrested VSMCs exposed to increasing concentrations of XO in the presence of 0.1 mmol/L xanthine. *P<.05 vs unstimulated control cells. B, Effect of antioxidants on viability of growing VSMCs exposed to 0.08 U XO/mL plus 0.1 mmol/L X, SOD, and CAT. *P<.05 vs XO/X alone.

To examine which species of reactive oxygen is responsible for cell death induced by XO/X, SOD and CAT were used in the treatment of growing VSMCs that were exposed to 0.08 U XO/mL three times (Fig 2B). The data show that the administration of SOD at 200 and 1000 U/mL augmented cell death after the third exposure to XO/X. This effect of SOD is most likely related to two effects exerted by SOD: the scavenging of O₂^-, which facilitates proliferation rather than death of VSMCs (see Fig 1), and an increase in H₂O₂ via SOD-catalyzed dismutation of O₂^-. In contrast, CAT at 200 U/mL or 500 U/mL prevented death of VSMCs exposed repeatedly to XO/X. Taken together, these data suggest that O₂^- and H₂O₂ exert distinct effects on VSMCs, with O₂^- inducing proliferation and H₂O₂ triggering cell death.

H₂O₂ Induces VSMC Death in a Dose-Dependent Manner and Via the Formation of ·OH
We tested whether H₂O₂ directly causes death of VSMCs. As can be seen from Fig 3A, a single exposure to H₂O₂ in the presence of 0.1 mmol/L ferrous sulfate resulted in VSMC death in a dose-dependent manner in both growing and growth-arrested VSMCs. To examine whether H₂O₂ exerts its effect directly or through the formation of ·OH formed by the Fenton reaction,²⁴ we used various ROS scavengers (Fig 3B). The results show that CAT and DF attenuated H₂O₂-Fe(II)–induced cell death (Fig 3B) as did DMSO (10 to 20 mmol/L) or ethanol (10 to 20 mmol/L) (data not shown). Administration of SOD failed to inhibit H₂O₂-Fe(II)–induced cell death (Fig 3B). None of these ROS scavengers had any significant effect on cell viability within the concentrations used in the present study. These data indicate that H₂O₂ induces VSMC death in a dose-dependent manner and exerts its effect predominantly through the formation of ·OH. Moreover, growth-arrested VSMCs are more susceptible to H₂O₂ treatment than growing VSMCs.

View larger version (30K): [in this window] [in a new window]   Figure 3. Effect of H2O2-Fe(II) on VSMC death analyzed by MTT test. A, Growing (solid bars) or growth-arrested (hatched bars) VSMCs were exposed to increasing concentrations of H2O2 in the presence of 0.1 mmol/L FeSO4. *P<.05, **P<.02, ***P<.01 vs unstimulated control cells. B, Growing VSMCs were exposed to 0.1 mmol/L H2O2 plus 0.1 mmol/L FeSO4 in the presence of SOD, CAT, or DF, respectively. *P<.05, **P<.02 vs H2O2-Fe(II) alone. Each bar represents the mean±SEM of three separate experiments (n=16 wells in each individual experiment).

VSMC Death Caused by XO/X and H₂O₂ Occurs by Apoptosis
To characterize the nature of VSMC death induced by XO/X and H₂O₂-Fe(II), we used agarose gel electrophoresis of DNA, in situ nick-end labeling (TUNEL method), propidium iodide staining of nuclei, and cell death ELISA. As Fig 4A shows, the DNA pattern of growing VSMCs revealed no difference between untreated VSMCs (lane 1) and VSMCs treated once with 0.08 U XO/mL (lane 2). However, a typical "DNA ladder" became evident after the third exposure to 0.08 U XO/mL (lane 3). Additionally, a gradual increase in the formation of the DNA ladder could be obtained by increasing doses of H₂O₂ (Fig 4B). Identical results were obtained with growth-arrested VSMCs (data not shown).

View larger version (71K): [in this window] [in a new window]   Figure 4. Agarose gel electrophoresis demonstrating DNA fragmentation of growing VSMCs caused by XO/X or H2O2-Fe(II). Left, Lane M, 100-bp DNA marker. Lane 1, untreated control VSMCs. Growing VSMCs were treated with 0.08 U XO/mL in the presence of 0.1 mmol/L X once (lane 2) or three times (lane 3). DNA was extracted 24 hours after the first or after the third treatment. Right, Lane M, 100-bp DNA marker. Lane 1, untreated control VSMCs. Growing VSMCs were treated with H2O2 at 0.01 mmol/L (lane 2), 0.05 mmol/L (lane 3), or 0.1 mmol/L (lane 4) in the presence of 0.1 mmol/L FeSO4. DNA was extracted 24 hours after treatment.

Propidium iodide DNA staining was used together with in situ nick-end labeling (TUNEL method) to characterize apoptosis. As Fig 5A shows, untreated growing VSMCs appear with relatively large and regularly shaped nuclei stained by propidium iodide (red) without being positively labeled by TUNEL. However, condensed and TUNEL-positive nuclei (green or yellow) were observed in growing VSMCs 8 hours after the third exposure to 0.08 U XO/mL plus 0.1 mmol/L xanthine (Fig 5B) and after exposure to 0.1 mmol/L H₂O₂ plus 0.1 mmol/L FeSO₄ (Fig 5C).

View larger version (85K): [in this window] [in a new window]   Figure 5. Propidium iodide staining and in situ nick-end labeling of growing VSMCs exposed to XO/X or H2O2-Fe(II). A, Control culture of untreated VSMCs. B, Cells treated three times with 0.08 U XO/mL in the presence of 0.1 mmol/L X. C, Cells treated with 0.1 mmol/L H2O2 in the presence of 0.1 mmol/L FeSO4. Propidium iodide staining and in situ nick-end labeling were processed 8 hours after treatment with H2O2-Fe(II) or after the third exposure to XO/X. Magnificationx400; arrows indicate apoptotic cells.

The data of the cell death ELISA show that exposure to either XO/X (Fig 6A) or H₂O₂/ferrous sulfate (Fig 6B) led to increases in histone-associated DNA fragments within the cytoplasmic fraction of growing VSMCs as well as growth-arrested VSMCs. The levels of histone-associated DNA fragments were noticeably higher in growth-arrested VSMCs than in growing VSMCs after exposure to H₂O₂-Fe(II) or XO/X. This indicates that growth-arrested VSMCs are more susceptible to ROS-induced apoptosis than growing VSMCs. In summary, all these criteria characterizing apoptosis suggest that cell death caused by H₂O₂-Fe(II) or XO/X occurs by apoptosis.

View larger version (28K): [in this window] [in a new window]   Figure 6. Cell death ELISA determination of the histone-associated DNA fragmentation in VSMCs exposed to XO/X or H2O2-Fe(II). Growing (solid bars) or growth-arrested (hatched bars) VSMCs were exposed to increasing concentrations of XO plus 0.1 mmol/L X (A) or to H2O2 plus 0.1 mmol/L FeSO4 (B). The ELISA detections were processed 8 hours after the third exposure to XO/X or after treatment with H2O2-Fe(II). The histone-associated DNA fragments are presented as the optical density at 405 nm. Each bar represents the mean±SEM of three separate experiments in triplicate. *P<.01, **P<.001 vs unstimulated control cells.

·OH but Not O₂^- Participates in the Induction of VSMC Apoptosis
The cell death ELISA was used to assess the precise role of H₂O₂, O₂^-, and ·OH for the induction of apoptosis in VSMCs. As Fig 7A shows, after the third exposure to 0.08 U XO/mL, histone-associated DNA fragments increased significantly in VSMCs. XO/X still led to DNA fragmentation in the presence of SOD (1000 U/mL). In contrast, CAT (500 U/mL) prevented XO/X-induced fragmentation of DNA. When VSMCs were exposed to 0.1 mmol/L H₂O₂ plus 0.1 mmol/L Fe(II), CAT and DF inhibited DNA fragmentation (Fig 7B), as did DMSO (10 to 20 mmol/L) or ethanol (10 to 20 mmol/L) (data not shown). However, SOD had no protective effects (Fig 7B). None of these ROS scavengers alone had any significant effect on DNA fragmentation within the concentrations used in the present study (data not shown). These data indicate that H₂O_2, but not O₂^- is able to induce apoptosis of VSMCs and H₂O₂ exerts its effect via formation of ·OH.

View larger version (32K): [in this window] [in a new window]   Figure 7. Cell death ELISA determination of the effect of antioxidants on the formation of the histone-associated DNA fragmentation in VSMCs. SOD was 1000 U/mL, CAT was 500 U/mL, and DF was 0.5 mmol/L. ELISA detection was processed 8 hours after the third exposure to XO/X or after treatment with H2O2-Fe(II). The histone-associated DNA fragments were presented as the optical density at 405 nm. Growing (solid bars) and growth-arrested (hatched bars) VSMCs were exposed to (A) 0.08 U XO/mL in the presence of 0.1 mmol/L X (*P<.008, **P<.003, ***P<.002 vs XO/X alone) or to (B) 0.1 mmol/L H2O2 in the presence of 0.1 mmol/L FeSO4 [*P<.002, **P<.001 vs H2O2-Fe(II) alone]. Each bar represents the mean±SEM of three separate experiments in triplicate.

Bcl-2 Downregulation and c-fos Induction Are Part of H₂O₂-Fe(II)–Induced VSMC Apoptosis
To identify genes involved in mediating the H₂O₂-Fe(II)–induced VSMC apoptosis, we determined the expression of bcl-2, p53, c-myc, and c-fos. Northern blot analysis was used to evaluate their expressions after treatment with 0.1 mmol/L H₂O₂ plus 0.1 mmol/L FeSO₄. Densitometric scan analysis showed that exposure to H₂O₂-Fe(II) led to a persistent decrease of bcl-2 mRNA expression after treatment (data not shown; Fig 8A is a representative blot). Surprisingly, even after a prolonged time interval, there was no detectable change regarding the expression of c-myc or p53 (data not shown). However, H₂O₂-Fe(II) induced a transient expression of c-fos detectable as early as 1 hour and peaking 2 hours after exposure (Fig 8B). We conclude that bcl-2 downregulation and c-fos induction may be part of H₂O₂-Fe(II)–induced apoptosis of VSMCs. However, neither p53 nor c-myc seems to be crucial for H₂O₂-Fe(II)–induced apoptosis in VSMCs.

View larger version (52K): [in this window] [in a new window]   Figure 8. Northern blot analysis showing the effect of H2O2-Fe(II) on the expression of bcl-2 (A) and c-fos (B). Growing VSMCs were treated with 0.1 mmol/L H2O2 in the presence of 0.1 mmol/L FeSO4. Total RNA was isolated from cells at the indicated time. Control RNA was isolated at 1 or 2 hours, respectively.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study was designed to test whether H₂O₂ and O₂^- exert distinct effects on the viability of VSMCs. Our data not only provide evidence supporting this hypothesis but for the first time indicate an important role of H₂O₂ for the induction of VSMC apoptosis as it occurs in many vascular diseases.

XO/X was used in our study not only because it gives rise to two distinct reactive oxygen metabolites (H₂O₂ and O₂^-) but also because it plays an important role for vascular dysfunction in vivo.²⁵ The data of our study suggest an important role of O₂^- for the induction of VSMC proliferation. This is consistent with the previous observation that O₂^- generated by naphthoquinedione LY 83583 stimulated DNA synthesis in VSMCs.⁸ Previous experiments have demonstrated that H₂O₂ is effective in stimulating the in vitro growth of several cell types such as rodent fibroblasts^{26 27} and murine osteoblastic cells.²⁸ The contradictory effect of H₂O₂ on VSMCs observed in our study may reflect cell-specific differences caused by oxidative stress. However, human and rat VSMCs have been reported to undergo growth detected by [³H]-thymidine incorporation in response to H₂O₂ stimulation.^{7 29} The inconsistency with our results is most likely due to the different methods used in evaluating cell proliferation. Recently, it has been reported that DNA synthesis in cells exposed to ROS cannot be safely taken as an index for cell proliferation.³⁰ The reason is that H₂O₂-stimulated DNA synthesis in VSMCs is not followed by cell proliferation but rather by cell death.³⁰ Therefore, extracellular H₂O₂ alone does not likely function as a mitogen to VSMCs, at least in vitro.

The observation that XO/X can elicit opposite reactions in VSMCs leading to either proliferation or cell death, depending on the dose and the frequency of exposure, is an intriguing finding. XO/X leads to the production of both H₂O₂ and O₂^-, and thus the effect of XO/X may be related to the interaction of these two compounds with one another. Previous studies revealed that exogenously added O₂^- could cause an increase in intracellular pH within 10 seconds.³¹ Cytoplasmic alkalinization has been considered an important early signal for the initiation of DNA synthesis.³² In contrast, a great body of evidence indicates that intracellular acidification is required for apoptosis.^{33 34} The activity of endogenous endonucleases responsible for internucleosomal DNA degradation is pH dependent, with its optimum pH below 7.0.^{35 36} Also, acidification can be responsible for activating other proteins such as proteases, which are associated with apoptosis.³⁴ On the other hand, O₂^- has recently been found to act as a natural inhibitor of Fas-mediated programmed cell death.³⁷ Therefore, when O₂^- and H₂O₂ coexist, the effect of O₂^- may predominate. However, this situation might be reversed by frequent exposures to high levels of H₂O₂ and O₂^-. First, O₂^- has been shown to travel through anion channels into membranes, whereas H₂O₂ can freely permeate cell membranes.^{31 38} A single dose of H₂O₂ and O₂^- produced by XO/X might not damage the ion channels, but frequent exposures to high levels of H₂O₂ or O₂^- could directly or indirectly influence ion channels.³⁹ Second, lipid peroxidation is the oxidation of polyunsaturated fatty acids of membrane lipids initiated by ROS. It can be triggered not only by H₂O₂ but also by high concentrations of O₂^-.⁴⁰ Particularly, lipid peroxidation could be initiated when both H₂O₂ and O₂^- were produced by XO and X.⁴¹ The progressive accumulation of lipid peroxides, especially its breakdown products, are capable of reducing cell proliferation.⁴² Additionally, lipid peroxides are capable of inducing apoptosis.⁴³ Third, there is evidence that cell proliferation and cell death are regulated by the cellular pro-oxidant state. The intracellular level of reduced glutathione is a good example of this. Growth factors, including O₂^-, that stimulate cell proliferation are accompanied by a progressive decline in cellular glutathione levels. Such effects were observed in many cell types.⁴² In VSMCs, a relationship also exists between glutathione levels and cell proliferation.⁴⁴ Overall, frequent exposures to H₂O₂ and O₂^- could change the cellular redox state, which directs the cell to undergo proliferation or death.

Recently, several studies indicate that p53, c-myc, and bcl-2 are involved in regulating VSMC apoptosis. For example, deregulated c-myc expression in VSMCs is associated with an increased incidence of apoptosis.⁴⁵ On the other hand, elevation of c-myc expression can also induce apoptosis of VSMCs.⁴⁶ Overexpression of p53 results in VSMC apoptosis,⁴⁷ whereas bcl-2 overexpression can inhibit apoptosis of VSMCs.¹⁵ c-myc has been shown to act upstream of p53 to convey cell death signals.⁴⁸ Our data indicate that H₂O₂-Fe(II) does not stimulate the expression of p53 in VSMC apoptosis directly or indirectly via the activation of c-myc. This is supported by recent observations describing that ROS are downstream mediators of p53-dependent apoptosis⁴⁷ and by the existence of p53-dependent or p53-independent apoptosis in VSMCs.⁴⁶ More likely, H₂O₂-Fe(II) appears to induce VSMC apoptosis by directly downregulating bcl-2 expression. In view of the previous finding that bcl-2 prevents apoptosis by decreasing generation of ROS,⁴⁹ our data support an interaction between ROS and bcl-2 in regulating apoptosis. Provoked c-fos expression in H₂O₂-Fe(II)–induced VSMC apoptosis is in agreement with a recent report indicating that induction of c-fos is a harbinger of programmed cell death.⁵⁰ Taken together, bcl-2 downregulation and c-fos induction may be part of H₂O₂-Fe(II)–induced VSMC apoptosis, whereas c-myc and p53 do not appear to participate in this process.

Our findings may have important clinical implications. VSMC proliferation and apoptosis are two important components of atherosclerosis, coronary restenosis after balloon angioplasty, and the vascular remodeling occurring in arterial hypertension.^{1 2 9 10 11 12 13} The data provided by our study not only indicate an important role of O₂^- for VSMC proliferation but also reveal that apoptosis of VSMCs may be triggered by H₂O₂. Moreover, a specific equilibrium between O₂^- and H₂O₂ may decide whether VSMCs undergo proliferation or apoptosis.

In summary, our present study demonstrates that O₂^- and H₂O₂ exert differential effects on VSMCs. Furthermore, the superiority of one compound over the other is variable and depends on the dose and frequency of exposure, resulting in either proliferation or death of VSMCs. Finally, H₂O₂ appears to be a potent inducer of VSMC apoptosis. Our data imply that O₂^- and H₂O₂ may participate in a variety of vascular diseases in a dual manner by stimulating both proliferation and apoptosis of VSMCs. Future studies are needed to unravel the mechanism by which H₂O₂ and O₂^- interact with one another.

	Selected Abbreviations and Acronyms

 

CAT = bovine liver catalase DF = deferoxamine mesylate DMSO = dimethyl sulfoxide FCS = fetal calf serum H2O2 = hydrogen peroxide H2O2-Fe(II) = H2O2/ferrous sulfate HBSS = Hanks' balanced salt solution MTT = 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide O2- = superoxide anion ·OH = hydroxyl radical ROS = reactive oxygen species SOD = bovine erythrocyte superoxide dismutase TCA = trichloroacetic acid TUNEL = dUTP-digoxigenin nick-end labeling VSMC = vascular smooth muscle cell XO/X = xanthine oxidase/xanthine

Received May 14, 1997; revision received June 23, 1997; accepted June 26, 1997.

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