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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:1845.)
© 2002 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Dose-Dependent Regulation of NAD(P)H Oxidase Expression by Angiotensin II in Human Endothelial Cells

Protective Effect of Angiotensin II Type 1 Receptor Blockade in Patients With Coronary Artery Disease

Uwe Rueckschloss; Mark T. Quinn; Juergen Holtz; Henning Morawietz

From the Institute of Pathophysiology (U.R., J.H., H.M.) and the Julius-Bernstein-Institute of Physiology (U.R.), Martin Luther University Halle-Wittenberg, Halle, Germany, and the Department of Veterinary Molecular Biology (M.T.Q.), Montana State University, Bozeman.

Correspondence to Henning Morawietz, PhD, Institute of Pathophysiology, Faculty of Medicine, Martin Luther University Halle-Wittenberg, Magdeburger Str. 18, D-06097 Halle, Germany. E-mail henning.morawietz{at}medizin.uni-halle.de

	Abstract

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Objective— Angiotensin II (Ang II)–mediated induction of vascular superoxide anion formation could contribute to the development of endothelial dysfunction, hypertension, and atherosclerosis. An NAD(P)H oxidase has been identified as a major endothelial source of superoxide anions. However, the molecular mechanism underlying the regulation of NAD(P)H oxidase activity in response to Ang II is not well understood.

Methods and Results— We investigated the dose-dependent regulation of superoxide anion formation and of NAD(P)H oxidase subunit expression in response to Ang II in human endothelial cells. Ang II regulates superoxide anion formation and the limiting subunit of endothelial NAD(P)H oxidase, gp91-phox, in a dose-dependent manner via Ang II type 1 (AT₁) receptor–mediated induction and Ang II type 2 receptor–mediated partial inhibition at higher Ang II concentrations. Furthermore, AT₁ receptor blocker therapy before coronary bypass surgery downregulates the gp91-phox expression in internal mammary artery biopsies of patients with coronary artery disease.

Conclusions— Our data support a dose-dependent induction of proatherosclerotic oxidative stress in human endothelial cells in response to Ang II. The expression of NAD(P)H oxidase subunit gp91-phox is critical for endothelial superoxide anion formation. AT₁ receptor blockade has an antiatherosclerotic and antioxidative potential by the reduction of oxidative stress in the vessel wall.

Key Words: angiotensin • atherosclerosis • cardiopulmonary bypass • endothelium • free radicals

	Introduction

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Angiotensin II (Ang II) has been suggested to be involved in the development and progression of atherosclerosis.^1,2 This hypothesis is supported by recent studies with Ang II receptor type 1 (AT₁) inhibitors, which show antiatherosclerotic effects in experimental studies³ and reversal of endothelial dysfunction in patients with coronary artery disease.⁴ Furthermore, ACE inhibitor therapy has a positive effect on the prognosis of patients with coronary artery disease.⁵

A putative risk factor involved in the proatherosclerotic effects of Ang II is increased oxidative stress by an elevated formation of reactive oxygen species, including superoxide anion (O₂^-). Animals made hypertensive by chronic Ang II infusion show augmented O₂^- formation and endothelial dysfunction.^6–8 O₂^- rapidly reacts with NO and reduces the bioavailability of this vasoprotective mediator of endothelium-dependent relaxation. Therefore, Ang II–stimulated increase in vascular O₂^- formation might contribute to the development of endothelial dysfunction and atherosclerosis.⁹

Several studies have been conducted to elucidate the molecular basis of vascular O₂^- formation. In every cell type of the vessel wall, an NAD(P)H oxidase similar to the phagocytic enzyme complex has been identified as a major source of O₂^- formation.^10–13 The NADPH oxidase complex in neutrophils and, most probably, endothelial cells (ECs) involves 4 essential subunits. The subunits gp91-phox and p22-phox reside in the plasma membrane.^14,15 These subunits bind the components of the electron transport chain heme and FAD, forming cytochrome b₅₅₈. The cytosolic NADPH oxidase subunits p47-phox and p67-phox are involved in activation of the enzyme complex. After stimulation, p47-phox is phosphorylated by protein kinase C, forming a complex with p67-phox. Subsequent translocation of this complex to the cytochrome b₅₅₈ induces O₂^- formation.¹⁶

In each vascular cell type, Ang II treatment causes upregulation of O₂^- formation.^12,17–19 The molecular basis for this upregulation of enzyme activity by Ang II is not well understood. In adventitial fibroblasts, induction of p67-phox expression by Ang II was suggested to be responsible for the induced NAD(P)H oxidase-dependent O₂^- formation.¹⁷ The NAD(P)H oxidase subunit p22-phox has been shown to be a critical component in the Ang II–induced hypertrophy in vascular smooth muscle cells (VSMCs).²⁰ Data on the regulation of NADPH oxidase subunit expression by Ang II are lacking for ECs. Furthermore, a dose-dependent bimodal regulation of NAD(P)H oxidase activity has been observed in fibroblasts and ECs.^7,18,19 The molecular mechanism for this bimodal regulation of NAD(P)H oxidase activity is unknown. In addition, the influence of ACE inhibitor or AT₁ receptor blocker therapy on vascular NAD(P)H oxidase expression in patients with coronary artery disease has not been studied to date.

In the present study, we show a dose-dependent bimodal regulation of expression of the limiting NAD(P)H oxidase subunit (gp91-phox) and of corresponding O₂^- formation by Ang II in human ECs. In addition, gp91-phox expression was reduced in internal mammary arteries of patients with coronary artery disease by AT₁ receptor blocker therapy. These data suggest an antioxidative and antiatherosclerotic potential of AT₁ receptor blockade by reduction of endothelial NAD(P)H oxidase-dependent O₂^- formation.

	Methods

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Cell Culture
Primary cultures of human umbilical vein ECs (HUVECs) were isolated by using collagenase IV and grown in medium M199 (Life Technologies) supplemented with 20% calf serum, as described previously.²¹ Confluent cell cultures were incubated with medium containing 0.5% calf serum for 24 hours and subsequently treated with Ang II (1 nmol/L to 1 µmol/L) or with Ang II and the AT₁ receptor antagonist candesartan (1 µmol/L, AstraZeneca) or the Ang II type 2 (AT₂) receptor antagonist PD123319 (1 µmol/L, Parke-Davis).

Patients
Distal remnant specimens of left internal mammary artery (arteria thoracica interna) were obtained after informed consent from 23 patients undergoing elective coronary artery bypass grafting (CABG) surgery. The use of human tissue was approved by the local ethics committee. Long-term ACE inhibitor or AT₁ receptor blocker therapy before surgery was evaluated in a retrospective manner. The following substances were prescribed: ACE inhibitors, including captopril, lisinopril, quinapril, and ramipril (38±10% of target dose in recent heart failure and endothelial function megatrials),^22,23 and AT₁ receptor antagonists, including losartan and valsartan (100% of target dose in recent heart failure megatrials).^24,25 Nine consecutive patients without pharmacological intervention in the renin-angiotensin system were matched with patients receiving preoperative ACE inhibitor (n=9) or AT₁ receptor blocker (n=5) therapy, according to New York Heart Association functional classification. These groups of patients showed no significant differences in systolic or diastolic blood pressure. In addition, no differences in central venous pressure, heart rate, left ventricular ejection fraction, sex, age, weight, or concomitant therapy with calcium antagonists, ß-blockers, diuretics, NO donors, antidiabetics, or lipid-lowering drugs were found (please see online Table 1, available at http://atvb.ahajournals.org).

Cytochrome c Reduction Assay
Cells were preincubated for 6 hours with or without Ang II and specific Ang II receptor antagonists in medium containing 0.5% calf serum. Cells were subsequently incubated for 1 to 4 hours at 37°C in an assay buffer consisting of medium M199 without phenol red supplemented with 40 µmol/L cytochrome c and 500 µmol/L N^G-nitro-L-arginine methyl ester to exclude EC NO synthase as a possible source of detected O₂^- generation.²⁶ To evaluate NAD(P)H oxidase–derived O₂^- generation, the flavin-containing enzyme inhibitor diphenylene iodonium (DPI, 100 µmol/L) was included in some experiments. At indicated time points, aliquots of the supernatant were taken, absorption at 550 nm was determined, and blank was subtracted. The amount of O₂^- generated was estimated by the use of the millimolar extinction coefficient for reduced cytochrome c (29.5 L · mmol^-1 · cm^-1). The O₂^- generation was normalized versus protein concentration of samples determined with the BCA Protein Assay Reagent (Pierce). DPI-inhibited O₂^- generation was estimated as the difference between samples with or without DPI in each group.

RNA Isolation
Total RNA from primary cultures of HUVECs and from biopsies of internal mammary arteries was isolated by guanidinium thiocyanate/cesium chloride centrifugation as previously described.²⁷

Quantification of NAD(P)H Oxidase Subunit mRNA Expression by Multistandard-Assisted RT-PCR
For quantification of mRNA expression of NAD(P)H oxidase subunits gp91-phox, p22-phox, p47-phox, and p67-phox in competitive reverse transcription (RT)–polymerase chain reaction (PCR), a common linker primer, PCR-generated, internal-deleted, and in vitro–transcribed multistandard cRNA was generated. The identity of the amplified RT-PCR fragments was confirmed by DNA sequencing. Competitive RT-PCR was performed by using the NAD(P)H oxidase multistandard as previously described.²⁸ DNA sequences of specific primers and PCR characteristics (PCR protocol: 30 seconds at 95°C, 30 seconds at primer-specific annealing temperature, and 30 seconds at 72°C) are summarized in online Table 2, available at http://atvb.ahajournals.org.

Protein Isolation and Western Blot Analysis
Protein isolation and Western blot analysis was performed as described²¹ with the use of monoclonal gp91-phox–specific antibodies.

Statistical Analysis
Data are given as mean±SEM. An ANOVA followed by the Bonferroni method (multiple comparison) or a Student t test was used for statistical comparison, as appropriate, and correlation was tested by using SigmaStat software (Jandel Corp). A value of P<0.05 was considered statistically significant.

An extended Methods section is available online at http://atvb.ahajournals.org.

	Results

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Dose-Dependent Regulation of O₂^- Formation and NAD(P)H Oxidase Subunit Expression by Ang II in HUVECs
Incubation of primary cultures of HUVECs with 100 nmol/L Ang II for >7 hours resulted in 2-fold induction of endothelial O₂^- formation (Figure 1A). In contrast, HUVECs treated with 1 µmol/L Ang II compared with control cells showed no significant difference in O₂^- formation.

View larger version (18K): [in this window] [in a new window]   Figure 1. Induction of O2- formation and NAD(P)H oxidase subunit gp91-phox expression in human ECs by Ang II. A, DPI-inhibited O2- formation of primary cultures of HUVECs was determined by cytochrome c reduction assay in control cells (•) and cells stimulated with 100 nmol/L ( or 1 µmol/L Ang II (). Incubation with 100 nmol/L Ang II for >7 hours caused an induction of endothelial O2- formation. A higher dose of Ang II (1 µmol/L) had no significant effect on O2- formation. B, Time-dependent regulation of gp91-phox mRNA expression by Ang II was determined by competitive RT-PCR from primary cultures of HUVECs. Cells were incubated with 100 nmol/L Ang II (solid bars) for 1, 3, 7, or 24 hours (control without Ang II, open bars). Maximal induction of gp91-phox expression by Ang II (2.6-fold) was found after 7 hours. *P<0.05 and **P<0.01 vs control.

Because an NAD(P)H oxidase has been previously shown to be a major source of endothelial O₂^- formation, mRNA expression of the 4 essential NAD(P)H oxidase subunits (gp91-phox, p22-phox, p47-phox, and p67-phox) was determined by multistandard-assisted competitive RT-PCR. First, expression of the limiting subunit of endothelial NAD(P)H oxidase gp91-phox was analyzed. Stimulation of HUVECs with 100 nmol/L Ang II caused a time-dependent induction of gp91-phox mRNA expression, reaching its maximum after 7 hours (Figure 1B). Further increase of Ang II concentration up to 1 µmol/L resulted in a partial inhibition of augmented gp91-phox expression (Figure 2A). Expression of NAD(P)H oxidase subunits p22-phox, p47-phox, and p67-phox was 2- to 3-fold induced by 100 nmol/L Ang II as well (maximum at 7 hours; please see online Figure IA, available at http://atvb.ahajournals.org). In contrast to gp91-phox, no decrease in the expression of these subunits could be found after incubation with 1 µmol/L Ang II.

View larger version (16K): [in this window] [in a new window]   Figure 2. Dose-dependent regulation of NAD(P)H oxidase subunit gp91-phox expression by Ang II is correlated with O2- formation in HUVECs. A, mRNA expression of NAD(P)H oxidase subunit gp91-phox was quantified by competitive RT-PCR from HUVECs after incubation without (control, open bar) or with Ang II (7 hours at 10 nmol/L, 100 nmol/L, or 1 µmol/L; solid bars). Expression of gp91-phox is induced by 100 nmol/L Ang II. Further increase of Ang II concentration to 1 µmol/L mediates a partial inhibition of augmented gp91-phox expression. B, NAD(P)H subunit gp91-phox mRNA expression in Ang II–stimulated HUVECs (10 nmol/L, 100 nmol/L, and 1 µmol/L Ang II) after 7 hours was plotted graphically vs DPI-inhibited O2- formation determined by cytochrome c reduction assay in control and stimulated HUVECs at corresponding Ang II concentrations after 8 hours. CIs are shown by dotted lines. *P<0.05 vs control.

The expression of NAD(P)H oxidase subunits in control and Ang II–stimulated HUVECs (10 nmol/L to 1 µmol/L Ang II) after 7 hours was graphically plotted versus DPI-inhibited O₂^- formation at the corresponding Ang II concentrations after 8 hours. The dose-dependent regulation of endothelial gp91-phox mRNA expression and O₂^- formation by Ang II showed a significant correlation (r²=0.976, Figure 2B). In contrast, dose-dependent regulation of the expression of NAD(P)H oxidase subunits p22-phox, p47-phox, and p67-phox in response to Ang II showed no significant correlation to dose-dependent endothelial O₂^- formation (p22-phox, r²=0.636; p47-phox, r²=0.626; and p67-phox, r²=0.351; see online Figure IB).

The endothelial gp91-phox protein expression was induced by 100 nmol/L Ang II (8 hours) as well, whereas no induction could be found with the use of 1 µmol/L Ang II (Figure 3).

View larger version (38K): [in this window] [in a new window]   Figure 3. Dose-dependent regulation of gp91-phox protein expression by Ang II in HUVECs. HUVECs were incubated with Ang II (100 nmol/L and 1 µmol/L) for 8 hours, and gp91-phox protein expression was quantified by Western blot analysis. Incubation of HUVECs with 100 nmol/L Ang II induced gp91-phox protein expression. In contrast, 1 µmol/L Ang II had no effect on gp91-phox. *P<0.05 vs control.

Role of Specific Ang II Receptors in Dose-Dependent Regulation of NAD(P)H Oxidase Expression and Activity
Induction of endothelial gp91-phox mRNA expression by treatment with 100 nmol/L Ang II was inhibited by a specific AT₁ receptor antagonist (1 µmol/L candesartan, Figure 4A). A similar effect of AT₁ receptor antagonism could be demonstrated on the functional level for Ang II–induced endothelial O₂^- formation (Figure 4B).

View larger version (17K): [in this window] [in a new window]   Figure 4. Induction of endothelial gp91-phox expression and O2- formation by Ang II is mediated by AT1 receptors. A, Induction of gp91-phox mRNA expression by 100 nmol/L Ang II (7 hours) is inhibited by treatment with specific AT1 receptor antagonist candesartan (1 µmol/L) in HUVECs (con indicates control without Ang II). B, DPI-inhibited O2- formation in control HUVECs (•) and in HUVECs after stimulation with 100 nmol/L Ang II ( is shown. The induction of endothelial O2- formation after 7 and 8 hours by 100 nmol/L Ang II was inhibited by 1 µmol/L candesartan (). *P<0.05 vs control.

The impact of specific AT₂ receptor antagonism on reduced gp91-phox expression by higher Ang II dose (1 µmol/L) was evaluated. As shown in Figure 5, concomitant treatment of HUVECs with 1 µmol/L Ang II and 1 µmol/L PD123319, a specific AT₂ receptor antagonist, resulted in significant induction of gp91-phox mRNA expression (n=5, panel A) and O₂^- formation (panel B). These data suggest an AT₂ receptor–mediated downregulation of gp91-phox expression and O₂^- formation in response to higher doses of Ang II in HUVECs.

View larger version (19K): [in this window] [in a new window]   Figure 5. Inhibition of augmented gp91-phox expression and O2- formation at higher Ang II concentrations in human endothelial cells is AT2 receptor–mediated. A, Expression of gp91-phox is shown in response to stimulation with Ang II (7 hours) in HUVECs. Application of 1 µmol/L Ang II and specific AT2 receptor antagonist PD123319 (1 µmol/L) resulted in a significant induction of gp91-phox expression compared with control or 1 µmol/L Ang II. *P<0.05 vs control. B, DPI-inhibited O2- formation is shown in control HUVECs (•) and in HUVECs after stimulation with 1 µmol/L Ang II (. PD123319 increased O2- generation after stimulation with 1 µmol/L Ang II in HUVECs after 7 and 8 hours (). *P<0.05 vs control and vs 1 µmol/L Ang II.

Regulation of Vascular gp91-Phox Expression by Therapeutic Intervention in the Renin- Angiotensin System
The impact of preoperative ACE inhibitor or AT₁ receptor blocker therapy before CABG surgery on vascular gp91-phox expression was determined in internal mammary arteries of patients with coronary artery disease (Figure 6). Long-term treatment with ACE inhibitors had, at the prescribed dosages, no effect on vascular gp91-phox expression. In contrast, pharmacological treatment with AT₁ receptor antagonists resulted in significant reduction of gp91-phox expression. These data suggest an antioxidative and vasoprotective potential of AT₁ receptor blocker therapy by downregulation of endothelial NAD(P)H oxidase expression.

View larger version (9K): [in this window] [in a new window]   Figure 6. Expression of gp91-phox mRNA in biopsies of internal mammary arteries of patients undergoing elective CABG surgery. Groups were matched for New York Heart Association classification, hemodynamics, sex, age, weight, and concomitant therapy. Preoperative long-term ACE inhibitor or AT1 receptor blocker therapy was determined in a retrospective manner. In contrast to ACE inhibitor therapy in the prescribed dosages, AT1 receptor blockers reduced vascular gp91-phox expression. *P<0.05 vs control without ACE inhibitor or AT1 receptor blocker.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, regulation of O₂^- formation and NAD(P)H oxidase subunit expression in response to Ang II was investigated in HUVECs. Incubation of HUVECs with Ang II resulted in a time- and dose-dependent induction of O₂^- formation. At higher Ang II concentrations, no significant increase in O₂^- formation could be found. This bimodal regulation of O₂^- formation in HUVECs is in agreement with previous studies in ECs from human umbilical arteries¹⁸ and the guinea pig microcirculation¹⁹ and in adventitial rabbit fibroblasts.⁷

To find a molecular source for this dose-dependent induction of endothelial O₂^- formation, the expression of NAD(P)H oxidase subunits was studied in response to Ang II in HUVECs. A similar dose-dependent bimodal regulation of NAD(P)H oxidase subunit gp91-phox expression in Ang II–treated HUVECs showing a significant correlation with DPI-inhibited O₂^- formation was found. In contrast to gp91-phox, endothelial NAD(P)H oxidase–dependent O₂^- formation and expression of the 3 additional subunits showed no significant correlation. These data provide evidence that the bimodal regulation of endothelial O₂^- formation by Ang II might be the consequence of Ang II–dependent regulation of gp91-phox expression. This is in agreement with recent data from our laboratory showing that gp91-phox is the limiting subunit of endothelial NAD(P)H oxidase.²⁸

In further studies, we sought to determine which Ang II receptor mediates the dose-dependent induction or repression of gp91-phox expression by the use of specific Ang II receptor antagonists. An AT₁ receptor antagonist attenuated the induction of gp91-phox expression and endothelial O₂^- formation by 100 nmol/L Ang II. These data provide evidence that induction of gp91-phox expression and O₂^- formation by Ang II is mediated via AT₁ receptor stimulation. These data are in agreement with previous reports showing that AT₁ receptor antagonists are capable of blocking Ang II–mediated induction of O₂^- formation in ECs,^18,19 VSMCs, ¹² and intact aortic segments.^29,30

Ang II treatment might cause downregulation of AT₁ receptors.³¹ Therefore, a possible explanation for the reduced gp91-phox expression and O₂^- formation in response to high Ang II concentration might be the consequence of a negative-feedback regulation of AT₁ receptor expression. However, O₂^- formation is further induced via AT₁ receptor stimulation with 1 µmol/L Ang II in other cell types.¹² Furthermore, no downregulation of Ang II–induced expression could be found at higher Ang II concentrations in respect to other NAD(P)H oxidase subunits.

On the other hand, in fibroblasts and ECs, concomitant application of the selective AT₂ receptor antagonist PD123319 further augmented Ang II–induced NAD(P)H oxidase activity.^7,32 A similar increase of O₂^- formation after application of 1 µmol/L Ang II in the presence of PD123319 has been found in the present study. The described significant induction of gp91-phox expression at higher Ang II concentrations in the presence of a specific AT₂ receptor antagonist supports the view that repression of gp91-phox expression and O₂^- formation at high Ang II concentration is at least partially mediated by AT₂ receptor stimulation. Because a similar affinity to Ang II was shown for AT₁ and AT₂ receptors,³³ the different threshold levels of AT₁ receptor–mediated induction and AT₂ receptor–mediated repression of gp91-phox expression might result from differences in receptor density. Because cultured ECs express AT₁ receptors in excess compared with AT₂ receptors,³⁴ higher Ang II concentrations might be necessary for AT₂ receptor stimulation. Thus, the cell- and vessel-specific ratio of AT₁ and AT₂ receptor expression might determine an induction or repression of gp91-phox expression at a given Ang II concentration.

The role of the AT₂ receptor in the regulation of vascular tone has been analyzed in several studies. Increased blood pressure after chronic infusion of Ang II in rats was augmented by concomitant application of PD123319.³⁵ Furthermore, an elevated blood pressure even under basal conditions was reported for AT₂ receptor–knockout mice.³⁶ Finally, mice overexpressing the AT₂ receptors showed a blunted pressure response to chronic Ang II infusion.³⁷ Because it is known that elevated vascular O₂^- formation reduces the bioavailability of NO, the bimodal regulation of endothelial gp91-phox expression and corresponding O₂^- formation via AT₁ and AT₂ receptors shown in the present study could contribute to the described changes in blood pressure.

To gain further insight into the regulation of NAD(P)H oxidase expression in vivo, we quantified the expression of gp91-phox in internal mammary arteries of patients undergoing elective CABG surgery. Long-term treatment with ACE inhibitors had no effect on vascular gp91-phox expression. In contrast, long-term therapy with AT₁ receptor antagonists reduced the expression of gp91-phox. These data could be the consequence of the bimodal dose-dependent regulation of gp91-phox by Ang II described in the present study in vitro. In our retrospective study, ACE inhibitor dosages prescribed by the referring physicians were only 38% of the respective target dosage in recent clinical megatrials.^22–25 As a consequence, local Ang II concentration might be decreased below the threshold of AT₂ receptor–mediated repression, but it remains above the threshold level of AT₁ receptor–mediated induction of gp91-phox expression. Therefore, prescribed ACE inhibitor dosages might be critical regarding gp91-phox expression and corresponding O₂^- formation. This is in agreement with clinical studies suggesting beneficial effects of higher doses of ACE inhibitors in patients with heart failure.³⁸ In contrast, AT₁ receptor blocker therapy is effective in reducing gp91-phox expression. This is in agreement with our in vitro data showing an AT₁ receptor–mediated induction of endothelial gp91-phox expression in response to Ang II. Because of the small sample size of internal mammary artery biopsies, we were not able to perform additional protein expression or O₂^- measurements in these tissues. However, our data in HUVECs in the present study support a correlation of gp91-phox mRNA expression and O₂^- formation. Therefore, the decreased gp91-phox expression by AT₁ receptor blocker therapy suggests a reduced O₂^- formation in internal mammary arteries. A critical role of gp91-phox in vivo is further supported by the 2-fold increased O₂^- formation by Ang II infusion in the aortas of wild-type animals but not in gp91-phox–knockout mice.³⁹

In summary, we show that the dose-dependent bimodal regulation of endothelial NAD(P)H oxidase by Ang II is exclusively related to the regulation of gp91-phox expression. Induction of gp91-phox is mediated by the AT₁ receptor, whereas a partial inhibition involves AT₂ receptor stimulation. Furthermore, vascular gp91-phox expression in internal mammary arteries of patients with coronary artery disease was reduced in response to AT₁ receptor blocker therapy. These data suggest an antioxidative and vasoprotective potential of AT₁ receptor blocker therapy by downregulation of vascular NAD(P)H oxidase expression.

	Acknowledgments

 
This study was supported by the Rudolf Thauer Award of the German Cardiac Society, grants from Deutsche Forschungsgemeinschaft (SFB/TR2 C1), the Wilhelm Roux program, the Novartis Foundation for therapeutic research, Bristol-Myers Squibb, AstraZeneca, and a grant from the National Institutes of Health (RO1 HL-66575). We are grateful to R. Gall for excellent technical assistance. We deeply express our appreciation for the cooperation of patients and staff of the Clinic for Cardiothoracic Surgery, Martin Luther University Halle-Wittenberg, Halle, Germany.

Received June 28, 2002; accepted July 14, 2002.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Holtz J, Goetz RM. Vascular renin-angiotensin-system, endothelial function and atherosclerosis? Basic Res Cardiol. 1994; 89: 71–86.[Medline] [Order article via Infotrieve]
2. Kim S, Iwao H. Molecular and cellular mechanisms of angiotensin II-mediated cardiovascular and renal diseases. Pharmacol Rev. 2000; 52: 11–34.[Abstract/Free Full Text]
3. Strawn WB, Chappell MC, Dean RH, Kivlighn S, Ferrario CM. Inhibition of early atherogenesis by losartan in monkeys with diet- induced hypercholesterolemia. Circulation. 2000; 101: 1586–1593.[Abstract/Free Full Text]
4. Prasad A, Tupas-Habib T, Schenke WH, Mincemoyer R, Panza JA, Waclawin MA, Ellahham S, Quyyumi AA. Acute and chronic angiotensin-1 receptor antagonism reverses endothelial dysfunction in atherosclerosis. Circulation. 2000; 101: 2349–2354.[Abstract/Free Full Text]
5. Yusuf S, Sleight P, Pogue J, Bosch J, Davies R, Dagenais G. Effects of an angiotensin-converting-enzyme inhibitor, ramipril, on cardiovascular events in high-risk patients: the Heart Outcomes Prevention Evaluation Study Investigators. N Engl J Med. 2000; 342: 145–153.[Abstract/Free Full Text]
6. Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK, Harrison DG. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation: contribution to alterations of vasomotor tone. J Clin Invest. 1996; 97: 1916–1923.[Medline] [Order article via Infotrieve]
7. Pagano PJ, Clark JK, Cifuentes-Pagano ME, Clark SM, Callis GM, Quinn MT. Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: enhancement by angiotensin II. Proc Natl Acad Sci U S A. 1997; 94: 14483–14488.[Abstract/Free Full Text]
8. Laursen JB, Rajagopalan S, Galis Z, Tarpey M, Freeman BA, Harrison DG. Role of superoxide in angiotensin II–induced but not catecholamine-induced hypertension. Circulation. 1997; 95: 588–593.[Abstract/Free Full Text]
9. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000; 87: 840–844.[Abstract/Free Full Text]
10. Mohazzab KM, Kaminski PM, Wolin MS. NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium. Am J Physiol. 1994; 266: H2568–H2572.[Medline] [Order article via Infotrieve]
11. Gorlach A, Brandes RP, Nguyen K, Amidi M, Dehghani F, Busse R. A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall. Circ Res. 2000; 87: 26–32.[Abstract/Free Full Text]
12. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994; 74: 1141–1148.[Abstract/Free Full Text]
13. Pagano PJ, Ito Y, Tornheim K, Gallop PM, Tauber AI, Cohen RA. An NADPH oxidase superoxide-generating system in the rabbit aorta. Am J Physiol. 1995; 268: H2274–H2280.[Medline] [Order article via Infotrieve]
14. Royer-Pokora B, Kunkel LM, Monaco AP, Goff SC, Newburger PE, Baehner RL, Cole FS, Curnutte JT, Orkin SH. Cloning the gene for an inherited human disorder-chronic granulomatous disease-on the basis of its chromosomal location. Nature. 1986; 322: 32–38.[CrossRef][Medline] [Order article via Infotrieve]
15. Dinauer MC, Orkin SH, Brown R, Jesaitis AJ, Parkos CA. The glycoprotein encoded by the X-linked chronic granulomatous disease locus is a component of the neutrophil cytochrome b complex. Nature. 1987; 327: 717–720.[CrossRef][Medline] [Order article via Infotrieve]
16. Nunoi H, Rotrosen D, Gallin JI, Malech HL. Two forms of autosomal chronic granulomatous disease lack distinct neutrophil cytosol factors. Science. 1988; 242: 1298–1301.[Abstract/Free Full Text]
17. Pagano PJ, Chanock SJ, Siwik DA, Colucci WS, Clark JK. Angiotensin II induces p67phox mRNA expression and NADPH oxidase superoxide generation in rabbit aortic adventitial fibroblasts. Hypertension. 1998; 32: 331–337.[Abstract/Free Full Text]
18. Zhang H, Schmeisser A, Garlichs CD, Plotze K, Damme U, Mugge A, Daniel WG. Angiotensin II-induced superoxide anion generation in human vascular endothelial cells: role of membrane-bound NADH-/NADPH-oxidases. Cardiovasc Res. 1999; 44: 215–222.[Abstract/Free Full Text]
19. Lang D, Mosfer SI, Shakesby A, Donaldson F, Lewis MJ. Coronary microvascular endothelial cell redox state in left ventricular hypertrophy: the role of angiotensin II. Circ Res. 2000; 86: 463–469.[Abstract/Free Full Text]
20. Ushio-Fukai M, Zafari AM, Fukui T, Ishizaka N, Griendling KK. p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells. J Biol Chem. 1996; 271: 23317–23321.[Abstract/Free Full Text]
21. Schubert A, Cattaruzza M, Hecker M, Darmer D, Holtz J, Morawietz H. Shear stress-dependent regulation of the human ß-tubulin folding cofactor D gene. Circ Res. 2000; 87: 1188–1194.[Abstract/Free Full Text]
22. Flather MD, Yusuf S, Kober L, Pfeffer M, Hall A, Murray G, Torp-Pedersen C, Ball S, Pogue J, Moye L, Braunwald E. Long-term ACE-inhibitor therapy in patients with heart failure or left-ventricular dysfunction: a systematic overview of data from individual patients: ACE-Inhibitor Myocardial Infarction Collaborative Group. Lancet. 2000; 355: 1575–1581.[CrossRef][Medline] [Order article via Infotrieve]
23. Kloner RA, Birnbaum Y. Cardiovascular Trials Review. Greenwich, Conn: Le Jacq Communications, Inc; 2000.
24. Pitt B, Segal R, Martinez FA, Meurers G, Cowley AJ, Thomas I, Deedwania PC, Ney DE, Snavely DB, Chang PI. Randomised trial of losartan versus captopril in patients over 65 with heart failure (Evaluation of Losartan in the Elderly Study, ELITE). Lancet. 1997; 349: 747–752.[CrossRef][Medline] [Order article via Infotrieve]
25. Dickstein K. ELITE II and Val-HeFT are different trials: together what do they tell us? Curr Control Trials Cardiovasc Med. 2001; 2: 240–243.[Medline] [Order article via Infotrieve]
26. Xia Y, Tsai AL, Berka V, Zweier JL. Superoxide generation from endothelial nitric-oxide synthase: a Ca²⁺/calmodulin-dependent and tetrahydrobiopterin regulatory process. J Biol Chem. 1998; 273: 25804–25808.[Abstract/Free Full Text]
27. Morawietz H, Rueckschloss U, Niemann B, Duerrschmidt N, Galle J, Hakim K, Zerkowski HR, Sawamura T, Holtz J. Angiotensin II induces LOX-1, the human endothelial receptor for oxidized low-density lipoprotein. Circulation. 1999; 100: 899–902.[Abstract/Free Full Text]
28. Rueckschloss U, Galle J, Holtz J, Zerkowski HR, Morawietz H. Induction of NAD(P)H oxidase by oxidized low-density lipoprotein in human endothelial cells: antioxidative potential of hydroxymethylglutaryl coenzyme A reductase inhibitor therapy. Circulation. 2001; 104: 1767–1772.[Abstract/Free Full Text]
29. Warnholtz A, Nickenig G, Schulz E, Macharzina R, Brasen JH, Skatchkov M, Heitzer T, Stasch JP, Griendling KK, Harrison DG, Bohm M, Meinertz T, Munzel T. Increased NADH-oxidase-mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the renin- angiotensin system. Circulation. 1999; 99: 2027–2033.[Abstract/Free Full Text]
30. Berry C, Hamilton CA, Brosnan MJ, Magill FG, Berg GA, McMurray JJ, Dominiczak AF. Investigation into the sources of superoxide in human blood vessels: angiotensin II increases superoxide production in human internal mammary arteries. Circulation. 2000; 101: 2206–2212.[Abstract/Free Full Text]
31. Hein L, Meinel L, Pratt RE, Dzau VJ, Kobilka BK. Intracellular trafficking of angiotensin II and its AT₁ and AT₂ receptors: evidence for selective sorting of receptor and ligand. Mol Endocrinol. 1997; 11: 1266–1277.[Abstract/Free Full Text]
32. Sohn HY, Raff U, Hoffmann A, Gloe T, Heermeier K, Galle J, Pohl U. Differential role of angiotensin II receptor subtypes on endothelial superoxide formation. Br J Pharmacol. 2000; 131: 667–672.[CrossRef][Medline] [Order article via Infotrieve]
33. Timmermans PB, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JA, Smith RD. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev. 1993; 45: 205–251.[Medline] [Order article via Infotrieve]
34. Li DY, Zhang YC, Philips MI, Sawamura T, Mehta JL. Upregulation of endothelial receptor for oxidized low-density lipoprotein (LOX-1) in cultured human coronary artery endothelial cells by angiotensin II type 1 receptor activation. Circ Res. 1999; 84: 1043–1049.[Abstract/Free Full Text]
35. Munzenmaier DH, Greene AS. Opposing actions of angiotensin II on microvascular growth and arterial blood pressure. Hypertension. 1996; 27: 760–765.[Abstract/Free Full Text]
36. Ichiki T, Labosky PA, Shiota C, Okuyama S, Imagawa Y, Fogo A, Niimura F, Ichikawa I, Hogan BL, Inagami T. Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor. Nature. 1995; 377: 748–750.[CrossRef][Medline] [Order article via Infotrieve]
37. Tsutsumi Y, Matsubara H, Masaki H, Kurihara H, Murasawa S, Takai S, Miyazaki M, Nozawa Y, Ozono R, Nakagawa K, Miwa T, Kawada N, Mori Y, Shibasaki Y, Tanaka Y, Fujiyama S, Koyama Y, Fujiyama A, Takahashi H, Iwasaka T. Angiotensin II type 2 receptor overexpression activates the vascular kinin system and causes vasodilation. J Clin Invest. 1999; 104: 925–935.[Medline] [Order article via Infotrieve]
38. Packer M, Poole-Wilson PA, Armstrong PW, Cleland JG, Horowitz JD, Massie BM, Ryden L, Thygesen K, Uretsky BF. Comparative effects of low and high doses of the angiotensin-converting enzyme inhibitor, lisinopril, on morbidity and mortality in chronic heart failure: ATLAS Study Group. Circulation. 1999; 100: 2312–2318.[Abstract/Free Full Text]
39. Wang HD, Xu S, Johns DG, Du Y, Quinn MT, Cayatte AJ, Cohen RA. Role of NADPH oxidase in the vascular hypertrophic and oxidative stress response to angiotensin II in mice. Circ Res. 2001; 88: 947–953.[Abstract/Free Full Text]

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