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(Circulation Research. 2000;86:347.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Transcriptional and Posttranscriptional Regulation of Endothelial Nitric Oxide Synthase Expression by Hydrogen Peroxide

Grant R. Drummond, Hua Cai, Michael E. Davis, Santhini Ramasamy, David G. Harrison

From the Division of Cardiology, Emory University, Atlanta, Ga.

Correspondence to David G. Harrison, Division of Cardiology, Emory University, 1639 Pierce Dr, Atlanta, GA 30322. E-mail dharr02{at}emory.edu

	Abstract

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Abstract—Diverse stimuli, including shear stress, cyclic strain, oxidized LDL, hyperglycemia, and cell growth, modulate endothelial nitric oxide synthase (eNOS) expression. Although seemingly unrelated, these may all alter cellular redox state, suggesting that reactive oxygen intermediates might modulate eNOS expression. The present study was designed to test this hypothesis. Exposure of bovine aortic endothelial cells for 24 hours to paraquat, a superoxide (O₂^-·)–generating compound, did not affect eNOS mRNA levels. However, cotreatment with paraquat and either Cu²⁺/Zn²⁺ superoxide dismutase or the superoxide dismutase mimetic tetrakis(4-benzoic acid)porphyrin chloride increased eNOS mRNA by 2.3- and 2.2-fold, respectively, implicating a role for H₂O₂. Direct addition of 100 and 150 µmol/L H₂O₂ caused increases in bovine aortic endothelial cell eNOS mRNA that were dependent on concentration (ie, 3.1- and 5.2-fold increases) and time, and elevated eNOS protein expression and enzyme activity, accordingly. Nuclear run-on and 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole–chase studies showed that H₂O₂ caused a 3.0-fold increase in eNOS gene transcription and a 2.8-fold increase in eNOS mRNA half-life. Induction of eNOS by H₂O₂ was not affected by the hydroxyl radical scavenger DMSO, mannitol, or N-tert-butyl--phenylnitrone, but it was inhibited by the antioxidants N-acetylcysteine, ebselen, and exogenously added catalase. Unlike H₂O₂, the 4.0-fold induction of eNOS by shear stress (15 dyne/cm² for 6 hours) was not inhibited by N-acetylcysteine or exogenous catalase. In conclusion, H₂O₂ increases eNOS expression through transcriptional and post-transcriptional mechanisms. Although H₂O₂ does not mediate shear-dependent eNOS regulation, it is likely to be involved in regulation of eNOS expression in response to other physiological and/or pathophysiological stimuli.

Key Words: paraquat • superoxide dismutase • eNOS mRNA stability • cultured endothelial cells

	Introduction

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Although endothelial nitric oxide synthase (eNOS) was originally thought to be a constitutively expressed enzyme, it has become clear in recent years that its expression can be modulated by a variety of chemical, physical, and developmental stimuli.¹ Regulation of eNOS expression occurs at both the transcriptional and post-transcriptional levels. For example, in cultured human and bovine endothelial cells, cyclic strain,² nordihydroguaiaretic acid (NDGA),³ estrogens,⁴ and the oxidized LDL (oxLDL) components 13-hydroperoxyoctadecadienoate⁵ and lysophosphatidylcholine⁶ have been shown to increase eNOS gene transcription. Likewise, transforming growth factor-ß (TGF-ß)⁷ and laminar shear stress⁸ increased the transcriptional activity of the 5'-promoter region of the eNOS gene as assessed by transfection of bovine aortic endothelial cells (BAECs) with chimeric eNOS promoter/luciferase constructs. In contrast, stimuli such as cell growth⁹ and the 3-hydroxy-3-methylglutaryl–CoA reductase inhibitor, simvastatin¹⁰ increase eNOS expression by prolonging the half-life of the eNOS mRNA.

A common property of many stimuli that increase eNOS expression is the ability to increase the production of reactive oxygen intermediates (ROIs) in endothelial cells. Thus, laminar shear stress,¹¹ cyclic strain,¹² oxLDL,¹³ high glucose,¹⁴ TGF-ß¹⁵ and proliferation¹⁶ have all been associated with an elevated production of ROIs in endothelial cells. Furthermore, for many of these stimuli, this increase in production of ROIs was shown to be important for regulating expression of other genes. For example, induction of heme-oxygenase-1,¹⁷ c-fos,¹⁸ monocyte chemotactic protein-1,¹⁹ and intercellular adhesion molecule-1 (ICAM-1)^{20 21} in human umbilical vein endothelial cells by shear stress and cyclic strain was inhibited by the antioxidants N-acetylcysteine (NAC) and catalase. Similarly, the phenolic antioxidants probucol and vitamin E have been shown to inhibit oxLDL-mediated induction of ICAM-1 and vascular cell adhesion molecule-1 expression.²² Finally, transcriptional induction of macrophage-colony stimulating factor by TGF-ß₁ was inhibited by catalase but not by superoxide dismutase (SOD), suggesting that H₂O₂, rather than superoxide anions (O₂^-·), mediates this effect.²³

These observations raise the possibility that ROIs represent common signaling molecules for modulating eNOS expression. Indeed, the human, bovine, and murine eNOS promoter regions contain putative binding sites for redox-sensitive transcription factors, including activator protein-1 (AP-1), Sp1, and antioxidant-responsive elements.^{24 25 26} Thus, the present study was performed to examine the effects of ROIs on eNOS expression in both BAECs and human aortic endothelial cells (HAECs). Our results indicate that H₂O₂, but not O₂^-· or hydroxyl radicals (OH^·), increases eNOS expression by increasing both the rate of transcription of the eNOS gene and the stability of the eNOS message once it is formed. Finally, because shear stress has been shown to increase both eNOS expression and endothelial cell ROI production, additional studies were performed to determine whether ROIs might mediate increased eNOS expression in response to shear stress.

	Materials and Methods

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Cell Culture
BAECs (Cell Systems) were cultured in medium 199 (GIBCO Laboratories) containing 10% FCS (Hyclone Laboratories) as described earlier.⁵ Postconfluent BAECs between passages 4 and 10 were used for experiments. HAECs (Clonetics) were cultured in endothelial cell growth medium (Clonetics) containing 2% FCS. Postconfluent cells (passages 6 to 10) were used for experiments. In some experiments, endothelial cells were exposed to laminar shear stress (15 dyne/cm²) using a cone-plate viscometer (cone angle 1°, rotational velocity 4 per second).²⁷

Assessment of eNOS Expression
Northern analyses, Western blots, nuclear run-ons, and eNOS enzyme activity assays were performed as previously described.⁵

Drugs and Their Suppliers
Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), N-tert-butyl--phenylnitrone (BPN), 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB), and ebselen were obtained from Calbiochem, and bovine liver catalase was from Boehringer Mannheim. All other chemicals were from Sigma. DMSO was used to dissolve BPN (1 mol/L) and DRB (25 mmol/L). MnTBAP (100 mmol/L) was dissolved in NaHCO₃ (1 mol/L). All other drugs were dissolved in dH₂O.

	Results

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Effect of Paraquat on eNOS mRNA Expression
To increase intracellular ROIs, endothelial cells were treated with paraquat. This xenobiotic redox cycles with intracellular flavins to produce O₂^-·. In BAECs, paraquat (1 mmol/L) treatment for 24 hours had no effect on eNOS mRNA levels (Figure 1). In contrast, cotreatment with paraquat and either Cu²⁺/Zn²⁺ SOD (100 U/mL) or MnTBAP (100 µmol/L) resulted in 2.3- and 2.2-fold increases in eNOS mRNA, respectively (Figure 1). In some experiments, treatment with Cu²⁺/Zn²⁺ SOD alone appeared to cause a small increase in eNOS mRNA expression, although this failed to reach significance (P=0.27; Figure 1). MnTBAP alone had no effect (Figure 1).

View larger version (66K): [in this window] [in a new window]   Figure 1. Northern analysis showing effects of paraquat, Cu2+/Zn2+ SOD, and MnTBAP on eNOS mRNA expression in BAECs. Cells were treated with control medium or medium containing Cu2+/Zn2+ SOD (100 U/mL) or MnTBAP (100 µmol/L) for 1 hour before incubation with paraquat (1 mmol/L) for 24 hours. Top panel is a representative blot, middle panel shows the corresponding 28S bands, and bottom panel contains the grouped densitometric data (mean±SEM) from 5 experiments. Asterisks represent eNOS mRNA levels significantly different from that in control cells (**P<0.01, Tukey-Kramer test after 1-way ANOVA).

H₂O₂ Increases eNOS Expression and Activity
The above results demonstrate that although eNOS expression is not affected by O₂^-·, dismutation of O₂^-· potently stimulates eNOS expression. We therefore examined the effect of directly applied H₂O₂. Treatment of BAECs with 100 and 150 µmol/L H₂O₂ for 24 hours resulted in 3.1- and 5.2-fold increases in eNOS mRNA expression, respectively (Figure 2). The eNOS-inducing effect of H₂O₂ (100 µmol/L) was also time-dependent. After 6 hours of exposure to H₂O₂, eNOS mRNA expression was 1.6 times that in the time-matched controls. eNOS expression continued to increase with time, reaching levels that were 3.1 and 3.6 times those in the time-matched 12- and 24-hour control groups, respectively (Figure 2).

View larger version (28K): [in this window] [in a new window]   Figure 2. Northern analyses showing concentration-dependent (A) and time-dependent (B) effects of H2O2 on eNOS mRNA expression in BAECs. A, Cells were treated with control or H2O2 (50 to 150 µmol/L)–containing medium for 24 hours. B, Cells were exposed to control or H2O2 (100 µmol/L)–containing medium for 1, 3, 6, 12, and 24 hours. In panel A, top panel is a representative blot, middle panel shows the corresponding 28S bands, and bottom panel contains the grouped densitometric data (mean±SEM) from 8 experiments. Panel B shows the grouped densitometric data (mean±SEM) from 3 experiments. Asterisks represent eNOS mRNA levels significantly different from that in control cells (***P<0.001, Tukey-Kramer test after repeated-measures ANOVA).

H₂O₂ also caused a concentration-dependent increase in eNOS protein levels in BAECs as measured by Western analyses. eNOS protein expression in BAECs treated with 100 and 150 µmol/L H₂O₂ was 2.1 and 2.8 times that in control cells, respectively (Figure 3). Likewise, eNOS enzyme activity, as measured by conversion of [¹⁴C]L-arginine to [¹⁴C]L-citrulline, was increased by H₂O₂ (Figure 3).

View larger version (25K): [in this window] [in a new window]   Figure 3. Western analysis (A) and eNOS activity assay (B) showing concentration-dependent increase in expression of functional eNOS protein in BAECs in response to H2O2. Cells were exposed to control or H2O2 (50 to 100 µmol/L)–containing medium for 24 hours before homogenization. In panel A, each lane was loaded with 15 µg of protein. A monoclonal antibody against human eNOS was used to detect bands. A representative blot is illustrated in the upper panel, whereas lower panel represents grouped densitometric data (mean±SEM) from 5 experiments. In panel B, 250 µg protein was used for the assay. Results (mean±SEM) represent grouped data from 5 experiments. Asterisks represent significantly different levels of eNOS protein expression or activity from that in control cells (*P<0.05, ***P<0.001, Tukey-Kramer test after 1-way ANOVA).

Similar to its effects on BAECs, H₂O₂ also increased eNOS protein expression in HAECs. In these cells, treatment with 100 and 200 µmol/L H₂O₂ caused 1.6- and 1.7-fold increases in eNOS protein expression, respectively (Figure 4).

View larger version (41K): [in this window] [in a new window]   Figure 4. Western analysis showing concentration-dependent increase in eNOS expression in HAECs in response to H2O2. Cells were exposed to control or H2O2 (50 to 200 µmol/L)–containing medium and were harvested after 48 hours. Each lane was loaded with 15 µg protein. A monoclonal antibody against human eNOS was used to detect bands. Upper panel is a representative blot, whereas lower panel depicts grouped densitometric data (mean±SEM) from 3 experiments. Asterisks represent significantly different levels of eNOS protein expression from that in control cells (*P<0.05, **P<0.01, Tukey Kramer test after 1-way ANOVA).

Effect of Short-Term Exposure to H₂O₂ on eNOS Expression
Experiments were performed to determine the length of exposure needed for H₂O₂ to stimulate eNOS expression. BAECs were treated with H₂O₂ (100 µmol/L) for 0.25 to 12 hours. In all cases, cells were washed and exposed to control medium and harvested at 12 hours after the beginning of the H₂O₂ exposure. H₂O₂ exposure as short as 0.25 hours caused a 4.4-fold increase in eNOS mRNA expression (Figure 5). Maximal effects (ie, 8.3- and 7.8-fold increases compared with time-matched controls) occurred after 1- and 3-hour exposures (Figure 5).

View larger version (29K): [in this window] [in a new window]   Figure 5. Northern analyses showing effects of short-term exposure to H2O2 on eNOS mRNA expression in BAECs. In panel A, cells were exposed to control (open bars) or H2O2 (100 µmol/L)–containing medium (filled bars) for 0.25, 0.5, 1, 3, or 6 hours. Cells were subsequently washed with control medium and further incubated until 12 hours after the initial exposure to H2O2, at which time they were harvested. Top panel is a representative blot, middle panel shows the corresponding 28S bands, and bottom panel depicts the grouped densitometric data (mean±SEM) from 3 experiments. In panel B, BAECs were exposed to control () or H2O2 (100 µmol/L)–containing medium () for 1 hour, before being washed with control medium, and harvested after 6, 12, 24, 48, and 72 hours. Values represent grouped densitometric data (mean±SEM) from 3 experiments. Asterisks represent significantly different levels of eNOS mRNA expression from those in time-matched controls (*P<0.05, **P<0.01, and ***P<0.001, Student unpaired t test).

Additional experiments were performed to determine how long eNOS expression remained elevated after short-term H₂O₂ exposure. Cells were harvested 6, 12, 24, 48, and 72 hours after a 1-hour-pulse exposure to H₂O₂ (100 µmol/L). At all of the time points tested, eNOS mRNA expression was significantly higher in H₂O₂-treated cells than in time-matched controls (Figure 5). Maximum effects of H₂O₂-pulse treatment occurred after 12 and 24 hours (7.6 and 8.4 times control levels, respectively; Figure 5). However, even at 72 hours after the 1-hour H₂O₂ pulse, eNOS mRNA expression remained elevated at 3.9 times that of control levels (Figure 5).

Effect of Scavengers on H₂O₂ Induction of eNOS
Because the effects of H₂O₂ could be mediated either directly or indirectly by OH^·, produced from H₂O₂ via Fenton chemistry, the effects of scavengers of H₂O₂ and OH^· were examined. Induction of eNOS mRNA expression by H₂O₂ was abolished by catalase (1000 U/mL) and was significantly inhibited by 50% by the glutathione peroxidase mimetic ebselen (40 µmol/L) (Figure 6). NAC (5 mmol/L), whether retained in the medium throughout the H₂O₂ incubation or washed out just before the addition of H₂O₂, also abolished the increase in eNOS mRNA expression caused by H₂O₂ (Figure 6). None of these interventions affected basal eNOS mRNA expression (data not shown). In contrast, the OH^· scavengers DMSO (0.3%), mannitol (20 mmol/L), and BPN (1 mmol/L) had no effect on H₂O₂-induced eNOS mRNA expression (Figure 6). Moreover, cotreatment with H₂O₂ (100 µmol/L) and either of the Fenton chemistry–enhancing agents, EDTA (100 µmol/L) or FeSO₄ (100 µmol/L), was not more effective at inducing eNOS mRNA expression than was H₂O₂ alone (Figure 6). Thus, these data indicate that the effect of H₂O₂ on eNOS expression is unlikely to be mediated by OH^·.

View larger version (31K): [in this window] [in a new window]   Figure 6. Northern analyses showing effects of H2O2 scavengers (A) and OH· scavengers/generators (B) on eNOS mRNA expression in BAECs. Cells were exposed to control medium or medium containing catalase (1000 U/mL), ebselen (40 µmol/L), NAC (5 mmol/L), DMSO (0.3%), mannitol (20 mmol/L), BPN (1 mmol/L), EDTA (100 µmol/L), or FeSO4 (100 µmol/L) for 1 hour before exposure to H2O2 (100 µmol/L) for 24 hours. In all groups except the studies in which NAC was washed out before H2O2 was added (NAC w.o.), the antioxidant/OH· generator was retained in the medium throughout the H2O2 incubation period. In panels A and B, top panels are representative blots, middle panels show the corresponding 28S bands, and bottom panels depict grouped densitometric data (mean±SEM) from 3 experiments. Asterisks indicate significantly different levels of eNOS mRNA expression from those in groups treated with H2O2 (100 µmol/L) alone (*P<0.05, **P<0.01, and ***P<0.001, Dunnett test after 1-way ANOVA).

Effect of H₂O₂ on eNOS Gene Transcription and eNOS mRNA Stability
To examine the effect of H₂O₂ on eNOS mRNA transcription rate, nuclear run-on analyses were performed. BAECs were treated with 100 µmol/L H₂O₂ for 6 hours and nuclei harvested. H₂O₂-treated BAECs produced 3.0 times the amount of radiolabeled eNOS mRNA transcripts of those made by control cells (Figure 7). In contrast, H₂O₂ had no effect on transcription rate of the housekeeping gene, ß-actin (Figure 7).

View larger version (21K): [in this window] [in a new window]   Figure 7. Nuclear run-on analysis and DRB-chase experiments demonstrating increase in both rate of eNOS gene transcription and eNOS mRNA stability after treatment with H2O2 (100 µmol/L). In panels A and B, nuclei, harvested from BAECs after 6-hour exposure to control or H2O2-containing medium, were incubated with [32P]UTP and unlabeled ATP, CTP, and GTP, in reaction buffer for 30 minutes at 30°C. The resulting radiolabeled transcripts from each group were added in equal counts to membranes previously slot-blotted with eNOS, ß-actin, and vector DNA. Hybridization was allowed to proceed for 72 hours at 68°C. Panel A is a representative blot, whereas panel B shows the grouped densitometric data (mean±SEM) from 3 experiments. In panels C and D, BAECs were treated with control or H2O2-containing medium for 6 hours before the addition of DRB (60 µmol/L). Cells were then harvested at the time points indicated and Northern analyses performed to determine the rate of eNOS mRNA decay in each group. In panel C, top panel is a representative blot, whereas bottom panel shows the corresponding 18S bands. Note: Double the amount of RNA was loaded for the control groups to enhance visualization of eNOS bands at the later time points. Panel D shows the grouped densitometric data (mean±SEM) from 4 experiments. Asterisks indicate significant difference from control (***P<0.001; Student unpaired t test).

To determine the effects of H₂O₂ on eNOS mRNA stability, BAECs were incubated with control or H₂O₂ (100 µmol/L)–containing medium for 6 hours. Next, cells from each group were treated with the RNA polymerase II inhibitor DRB (60 µmol/L). The rate of eNOS mRNA decay was significantly slower in H₂O₂-treated cells than it was in control cells (Figure 7). In H₂O₂-treated cells, the half-life of the eNOS message was 20 hours compared with 7 hours in control cells. Thus, H₂O₂ increases eNOS expression both by increasing the rate of transcription and by elevating the half-life of eNOS mRNA.

Effect of H₂O₂ Scavengers on Shear-Induced eNOS Expression
Shear stress has been shown to increase both eNOS expression and production of ROIs by the endothelium. To determine whether H₂O₂ produced in response to shear stress might mediate the increase in eNOS expression, cells were exposed to a laminar shear of 15 dyne/cm² for 6 hours. In the absence of other interventions, shear caused a 4.0-fold increase in eNOS mRNA expression. Addition of exogenous catalase or NAC had no effect on induction of eNOS by shear (Figure 8). Thus, the increase in eNOS expression produced by shear stress is unlikely to be caused by endogenously produced H₂O₂.

View larger version (50K): [in this window] [in a new window]   Figure 8. Northern analyses showing effects of H2O2 scavengers on eNOS induction by shear stress. BAECs were treated for 1 hour with control or with medium containing catalase (1000 U/mL) or NAC (5 mmol/L), before exposure to laminar shear stress (15 dyne/cm2), for 6 hours. Top panel is a representative blot, middle panel shows the corresponding 28S bands, and bottom panel depicts the grouped densitometric data (mean±SEM) from 3 experiments.

	Discussion

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The important finding from this study is that expression of eNOS in endothelial cells is upregulated by H₂O₂. We have shown that short-term exposure of BAECs to H₂O₂, but not to O₂^-· or OH^·, causes a sustained increase in eNOS mRNA and functional protein expression. Furthermore, this effect was mediated both by an increase in transcription rate of the eNOS gene and by an increase in stability of the eNOS message after its formation.

In recent years, it has become apparent that ROIs play an important role in gene regulation in response to several stimuli.²⁸ These effects likely result from stimulation of intracellular phosphorylation cascades, leading to activation of transcription factors. In the present study, we demonstrated a 3-fold increase in transcription rate of the eNOS gene in response to H₂O₂. The 5'-promoters of the human, bovine, and murine eNOS genes contain putative binding sites for a number of redox-regulated transcription factors, including AP-1, Sp1, and antioxidant-responsive elements.^{24 25 26} In the present study, we did not examine the role of the 5'-promoter in the transcriptional induction of eNOS by H₂O₂. However, a previous study showed through gel-shift assays that the H₂O₂ generating system, glucose oxidase, increased binding of BAEC nuclear proteins to a radiolabeled probe containing an eNOS promoter AP-1 binding site.²⁹ It is thus tempting to speculate that AP-1 and perhaps other redox-sensitive cis-acting elements are involved in the response to H₂O₂.

In addition to increasing eNOS gene transcription, H₂O₂ also enhanced the stability of the eNOS message. Modulation of eNOS mRNA stability is now known to represent an important mechanism for regulation of expression of the enzyme by a number of stimuli. For example, tumor necrosis factor- decreases eNOS expression by reducing the stability of the eNOS message.³⁰ In addition, stabilization of the eNOS message augments eNOS expression in response to cell growth⁹ and 3-hydroxy-3-methylglutaryl–CoA reductase inhibitors.¹⁰ This effect of cell growth on eNOS mRNA stability has been found to be due to decreased binding of a 51-kDa mRNA-destabilizing protein to a G+C-rich region near the start of the 3'-untranslated region.⁹ Interestingly, preliminary UV cross-linking studies in our laboratory showed that H₂O₂ similarly decreased binding of a 51-kDa protein to the eNOS mRNA 3'-untranslated region.

Of note, in preliminary studies, we found that the duration of exposure to H₂O₂ required to elicit an increase in eNOS expression was substantially longer in HAECs than in BAECs. Although the explanation for this remains unclear, it may relate to the longer eNOS mRNA half-life in human³⁰ as compared with bovine endothelial cells (ie, >24 hours versus 7 to 8 hours). Therefore, although a 24-hour exposure to H₂O₂ in BAECs is equivalent to 3 eNOS mRNA half-lives, it is only equal to 1 half-life in HAECs. Because at least half of the effects of H₂O₂ on eNOS are mediated by enhancing mRNA stability, longer exposures to H₂O₂ may be necessary to impact on steady-state eNOS mRNA levels in human cells.

Recently, we demonstrated that phenolic antioxidants, including NDGA and probucol, increased eNOS mRNA and protein expression.³ Although these findings may seem to be at odds with those of the present study, it is known that phenolic antioxidants can exert both antioxidant and pro-oxidant effects, depending on the prevailing redox environment. Abstraction of the hydrogen atom from the phenolic group of such antioxidants by O₂^-· results in formation of a phenoxyl radical (itself a pro-oxidant species) along with H₂O₂, which could in turn induce eNOS expression. Augmentation of the effects of NDGA by the reducing compounds NAC and ascorbic acid, observed in our previous study,³ could be explained if these compounds were acting to promote reduction of the phenoxyl radical back to the parent compound,³¹ thus allowing further participation in redox cycling.

Diverse stimuli, including cyclic strain, shear stress, and TGF-ß₁, have been shown to induce endothelial cell expression of different genes in a manner that is abolished by NAC and catalase,^{17 18 19 20 21 23} implying that the upregulation is dependent on generation of endogenous H₂O₂. In these same studies, exogenous H₂O₂ in concentrations of 100 to 150 µmol/L induced a similar magnitude of expression of these genes. Thus, although the concentration of H₂O₂ used in this study and previous studies may seem high, these concentrations likely have physiological and pathophysiological significance. Because of intracellular catalase, glutathione peroxidase, and other antioxidants, the ultimate intracellular concentration of H₂O₂ is likely to be substantially lower than that added exogenously. Importantly, these concentrations of H₂O₂ exerted little or no microscopically visible toxic effects on endothelial cells.

To examine the physiological role of H₂O₂ in regulation of eNOS expression, we studied the effects of antioxidants on eNOS induction by shear stress. Several studies have shown that shear stress increases ROIs within the endothelium,^{8 11 18 20} probably via activation of an NADH-dependent oxidase.¹⁷ Furthermore, activation of heme-oxygenase-1,¹⁷ c-fos,¹⁸ and ICAM-1²⁰ by shear stress was inhibited by catalase and NAC, indicating that ROIs are likely to be important in induction of these genes. Additionally, we have shown that laminar shear stress induces Cu²⁺/Zn²⁺ SOD expression in BAECs,³² which should shift the balance of ROIs from O₂^-· to H₂O₂. We therefore hypothesized that eNOS induction by shear stress may be due to H₂O₂ production. This hypothesis was proven incorrect, however, as shear stress induction of eNOS was not inhibited by NAC or catalase, both of which abolished eNOS upregulation by H₂O₂. Moreover, preliminary studies in our laboratory demonstrate that the intracellular signaling pathways mediating the effects of H₂O₂ and shear stress on eNOS expression are different. For example, eNOS induction by shear stress seems to be inhibited by the c-Src inhibitor PP1. In contrast, H₂O₂-dependent eNOS induction is not blocked by this compound but can be inhibited by tyrphostin A25 and herbimycin A, implicating a role for a non–Src-related tyrosine kinase in the transduction pathway.

Although H₂O₂ does not mediate the effect of shear stress on eNOS expression, it may be important in modulating eNOS expression in other settings. The production of O₂^-· by vascular cells is increased in several pathophysiological conditions, including hypertension, hypercholesterolemia, and diabetes. This increase in O₂^-· production is responsible for decreasing the bioactivity and thus beneficial actions of NO^· in these disease states. Because O₂^-· is a precursor to other ROIs, including H₂O₂, it is likely that this response of increased eNOS expression represents an important compensatory mechanism. Indeed, in several pathological conditions, including early hypercholesterolemia,³³ hypertension,³⁴ and diabetes (T. Münzel, personal communication), eNOS expression is increased. It is therefore interesting to speculate that the increase in ROIs (especially H₂O₂) in these diseases underlies the increase in eNOS expression.

	Acknowledgments

 
This work was supported by an American Heart Association Southeast Affiliate Postdoctoral Fellowship 9840026SE (to G.R.D), NIH Grants HL390006 and HL59248, NIH Program Project Grant HL58000, and a Department of Veterans Affairs merit grant.

Received July 8, 1999; accepted November 22, 1999.

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