This Article Abstract Full Text (PDF) All Versions of this Article: 110/7/856    most recent 01.CIR.0000138743.09012.93v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Haendeler, J. Articles by Dimmeler, S. Search for Related Content PubMed PubMed Citation Articles by Haendeler, J. Articles by Dimmeler, S. Related Collections Cell signalling/signal transduction Oxidant stress Endothelium/vascular type/nitric oxide

(Circulation. 2004;110:856-861.)
© 2004 American Heart Association, Inc.

Original Articles

Antioxidant Effects of Statins via S-Nitrosylation and Activation of Thioredoxin in Endothelial Cells

A Novel Vasculoprotective Function of Statins

Judith Haendeler, PhD; Jörg Hoffmann, PhD; Andreas M. Zeiher, MD; Stefanie Dimmeler, PhD

From Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Frankfurt, Germany.

Correspondence to Stefanie Dimmeler, PhD, Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Theodor Stern-Kai 7, 60590 Frankfurt, Germany. E-mail Dimmeler{at}em.uni-frankfurt.de

Received October 6, 2003; de novo received December 19, 2003; revision received April 8, 2004; accepted April 12, 2004.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— HMG-CoA reductase inhibitors (statins) are lipid-lowering drugs that also exert pleiotropic vasculoprotective effects via activation of the endothelial NO synthesis. NO induces S-nitrosylation of target proteins. S-Nitrosylation of the antioxidant enzyme thioredoxin was recently shown to enhance its activity, thereby reducing intracellular reactive oxygen species. Therefore, we investigated whether statins may exert an antioxidant activity in endothelial cells via S-nitrosylation of thioredoxin.

Methods and Results— Statins dose- and time-dependently increased the overall level of S-nitrosylated proteins in endothelial cells (atorvastatin 0.1 µmol/L, 206±30% increase; simvastatin 1 µmol/L, 214±19% increase; mevastatin 1 µmol/L, 191±10% increase). The increased S-nitrosylation was blocked by an NO-synthase inhibitor and mevalonate. Moreover, S-nitrosylation of thioredoxin was also significantly augmented after atorvastatin treatment. The atorvastatin-mediated increase in S-nitrosylation was associated with an enhanced enzymatic activity of thioredoxin (atorvastatin, 157±9% increase). This resulted in a significant reduction of intracellular reactive oxygen species within the endothelial cells. In contrast, in endothelial cells overexpressing a thioredoxin construct in which the S-nitrosylation acceptor amino acid cysteine 69 was replaced by serine [TRX(C69S)], atorvastatin did not activate the redox-regulatory activity of thioredoxin. Moreover, overexpression of the non-nitrosylatable thioredoxin TRX(C69S) abolished atorvastatin-mediated reduction of reactive oxygen species.

Conclusions— Here, we demonstrate a novel antioxidant mechanism by which statins reduce reactive oxygen species in endothelial cells. Statin-mediated S-nitrosylation of thioredoxin enhanced the enzymatic activity of thioredoxin, resulting in a significant reduction in intracellular reactive oxygen species.

Key Words: antioxidants • nitric oxide • endothelium

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
HMG-CoA reductase inhibitors (statins) are effective lipid-lowering agents. Recent experimental studies suggested that the beneficial effects of statins in patients at risk for cardiovascular disease are not only a result of an improved lipid profile but also mediated by direct vasculoprotective actions.¹ Nitric oxide (NO) produced within the endothelium is a pivotal mediator of vasculoprotective functions.² Importantly, genetic ablation of the endothelial NO synthase (eNOS) abrogates the protective effects of statins on vascular function and ischemic tissue injury, suggesting an important role for eNOS to mediate the beneficial effects of statins.^3–5 Indeed, statins were shown to upregulate eNOS posttranscriptionally in a Rho-GTPase–dependent manner.⁶ Moreover, statins activate the protein kinase Akt,⁷ which in turn phosphorylates and activates the eNOS.^8,9 In addition to its well-established signaling cascade via the cGMP pathway, more recently S-nitrosylation was identified as an important cGMP-independent mechanism of NO. S-Nitrosylation is the reversible covalent binding of NO to an SH-group of a reactive cysteine and contributes to the redox-based signaling mechanisms (for review, see Stamler et al).¹⁰

Oxidant stress of the vascular wall is a hallmark of atherosclerosis (for review, see Harrison et al).¹¹ To defend themselves from oxidative damage, cells possess antioxidative enzymes, including superoxide dismutase, catalase, and the thioredoxin system, to maintain intracellular levels of reactive oxygen species (ROS).^12,13 The thioredoxin system contains 2 oxidoreductases, thioredoxin reductase and thioredoxin-1 (TRX). TRX is expressed ubiquitously in mammalian cells, and TRX deficiency results in a lethal phenotype,^14,15 underscoring the fundamental importance of the enzyme. TRX exerts its enzymatic activity as an oxidoreductase via the cysteines 32 and 35. TRX can directly bind and metabolize ROS. Moreover, TRX interacts directly with other proteins, such as ribonucleotide reductase, protein disulfide isomerase, and several transcription factors (including p53, nuclear factor-B, and activating protein-1 via Ref-1) by forming disulfide bridges.^16–18 In addition to the 2 cysteines at position 32 and 35 in the redox-regulatory domain, TRX contains 3 cysteines at position 62, 69, and 73, which are believed to be structurally important.¹⁹ NO induces the S-nitrosylation of TRX at cysteine 69 in endothelial cells, and this S-nitrosylation is required for the redox-regulatory activity of TRX and its ability to reduce intracellular ROS in endothelial cells.²⁰ Therefore, we investigated the effects of statins on the content of S-nitrosylated molecules in endothelial cells and in particular on the S-nitrosylation of TRX. The results of the present study demonstrate that statins increase S-nitrosylation of TRX at cysteine 69. The increase in S-nitrosylation of TRX is accompanied by an increase in the redox-regulatory activity of TRX and by a decrease in intracellular ROS. Therefore, these data provide evidence for a direct antioxidative effect of statins via S-nitrosylation of TRX in endothelial cells.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cell Culture
Human umbilical vein endothelial cells (endothelial cells) were cultured in endothelial basal medium supplemented with hydrocortisone (1 µg/mL), bovine brain extract (12 µg/mL), gentamicin (50 µg/mL), amphotericin B (50 ng/mL), epidermal growth factor (10 ng/mL), and 10% FCS from passages 2 to 4. After detachment with trypsin, cells were grown for at least 18 hours.^21,22 Atorvastatin (AT) was kindly donated by Pfizer. Mevastatin was activated as described previously.⁶ Simvastatin was obtained from Merck Sharp and Dohme. H₂O₂ (200 µmol/L) was incubated for 6 hours in all the experiments.

Transfection
TRX wild-type (TRXwt) and TRX(C69S) were cloned²⁰ and transfected into endothelial cells as described previously with a transfection efficiency of 40%.²¹

S-NO Content
S-NO content was measured by use of the Saville-Griess assay as described.^20,23 In brief, endothelial cells were lysed in Griess lysis buffer (50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 5 mmol/L KCl, 1% Nonidet-P40, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L bathocuproinedisulfonic acid, 1 mmol/L diethylenetriaminepenta-acetic acid, 10 mmol/L N-ethylmaleimide), and 80 µg of cell lysate was incubated with 1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine in the presence or absence of 3.75 mmol/L p-chloromercuribenzosulfonic acid or 10 mmol/L CuCl₂ for 20 minutes, and S-NO content was measured photometrically at 540 nm. The amount was calculated using defined GSNO concentrations as a standard.

TRX Activity
Endogenous TRX activity was measured as previously described by use of the insulin assay.^24,25 After transfection, cells were lysed in Griess lysis buffer as described above. For TRX activity, 70 µL cell lysates was incubated with 140 mU of thioredoxin reductase, 1 mmol/L DTT, 1 mmol/L EDTA, and 450 nmol/L NADPH. NADPH consumption was measured by use of a fluorescence photometer at 340 nm over a period of 2 minutes with monitoring every 30 seconds. TRX activity was calculated as (OD2–OD1)/(T2–T1)/mg protein.

Detection of S-Nitrosylated TRXwt or TRX Mutants
Detection of S-nitrosylated proteins was performed as described.^20,26 In brief, cells were transfected with vector, TRXwt, TRX(C69S), or TRX(C32/35S) and lysed in 50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 1% Nonidet P 40, 0.5% deoxycholic acid, and 0.1% SDS and subjected to immunoprecipitation by use of an anti-Xpress antibody. Immunoprecipitates were washed twice with lysis buffer and twice with PBS. The pellet was resuspended in 500 µL PBS. After addition of 100 µmol/L HgCl₂ and 100 µmol/L 2,3-diaminonaphthalene, samples were incubated in the dark at room temperature for 30 minutes, and 1 mol/L NaOH was added. The generated fluorescent triazole from the reaction of 2,3-diaminonaphthalene with the NO released from TRXwt or TRX mutants was measured by use of an excitation wavelength of 375 nm and an emission wavelength of 450 nm. As negative control, the Xpress antibody alone in lysis buffer was immunoprecipitated. The resulting background fluorescence intensity was subtracted from each experiment, and the equal expression of TRXwt and TRX mutants was confirmed by immunoblot using an anti-Xpress antibody (1:2000, Invitrogen).

Detection of ROS Formation
For measurement of the intracellular H₂O₂ and O₂^– levels, the adherent cells were loaded with 2',7'-dichlorodihydrofluorescein diacetate (H₂DCF-DA, 20 µmol/L, Molecular Probes) or dihydroethidine (DHE, 5 µmol/L, Molecular Probes) for 30 minutes at 37°C in the dark. Cells were trypsinized for 2 minutes, and the reaction was stopped with PBS/10% FCS. Cells were pelleted by centrifugation and washed twice with PBS. Formation of ROS was detected by the signal obtained from the fluorescent reaction products DCF and ethidine by use of flow cytometry (FACSCalibur; Perkin Elmer; fluorescence 1 [DCF], 530/30 nm; fluorescence 2 [ethidine], 585/42 nm; CellQuest software). All experiments were performed with standardized instrumental settings (BD Biosciences).

Immunoblot
After stimulation with 0.1 µmol/L AT for 24 hours, endothelial cells were scraped off the plates and lysed in RIPA buffer (50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 1% Nonidet-P40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate). For detection of protein expression, membranes were incubated with antibodies against TRX (1:500, overnight, 4°C, Pharmingen) or tubulin (1:1000, 2 hours, room temperature, Neomarkers) as described previously.²⁰

Statistics
Statistical analysis was performed with Student’s t test or ANOVA followed by modified least significant difference (Bonferroni) test (SPSS Software).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of Statins on Intracellular S-NO Content in Endothelial Cells
To address the question of whether statins increase the intracellular content of S-nitrosylated molecules (S-NO content), endothelial cells were incubated with different statins in a concentration-dependent manner for 24 hours. All 3 statins tested significantly increased the S-NO content in a dose-dependent manner (Figure 1A). Next, we determined the time course of increased S-NO content. Incubation with 0.1 µmol/L AT enhanced the S-NO content in a time-dependent manner, with maximum effect observed after 48 hours (Figure 1B). The AT-induced increase in S-NO content was completely inhibited by coincubation with mevalonate (Figure 1C). Likewise, inhibition of the NO synthase with N^G-monomethyl-L-arginine abolished the AT-induced increase in S-NO content (Figure 1D), demonstrating that NOS activation is required for the increase in S-NO content by AT.

View larger version (22K): [in this window] [in a new window]   Figure 1. A, AT, simvastatin, and mevastatin increased S-NO content in endothelial cells. Endothelial cells were incubated with AT, simvastatin, and mevastatin at concentrations indicated for 24 hours. Intracellular S-NO content was measured as described in Methods (data are mean±SEM, n=6, *P<0.01 vs control). B, Endothelial cells were incubated with 0.1 µmol/L AT for indicated times, and S-NO content was measured as described in Methods (data are mean±SEM, n=3, *P<0.01 vs control). C, Endothelial cells were preincubated with 40 µmol/L mevalonate followed by 0.1 µmol/L AT for 24 hours. S-NO content was measured as described in Methods (data are mean±SEM, n=4, *P<0.01 vs AT). D, AT-induced increase in S-NO content is dependent on NOS activation. Endothelial cells were incubated with L-NMMA for 24 hours before treatment with 0.1 µmol/L AT for 24 hours. S-NO content was measured as described in Methods (data are mean±SEM, n=4, *P<0.01 vs AT).

AT Increases S-Nitrosylation of TRX and Thereby TRX Enzyme Activity
TRX was shown to be S-nitrosylated at cysteine 69 in endothelial cells,²⁰ which enhances the redox-regulatory and antiapoptotic functions of TRX. Therefore, we determined whether AT might influence S-nitrosylation of TRX. Indeed, S-nitrosylation of TRX was significantly increased by incubation with AT compared with control cells (Figure 2A). An effect of AT on TRX protein levels was excluded by Western blot analysis (Figure 2B). Taking into account that S-nitrosylation of TRX increased TRX enzyme activity in endothelial cells,²⁰ we next examined the effect of AT on TRX enzyme activity. AT significantly increased TRX activity under basal conditions in endothelial cells, whereas a specific Rho kinase inhibitor, Y 27632, had no effect (Figure 3A). Moreover, AT also inhibited H₂O₂-induced reduction in TRX activity (Figure 3A). Similar results were found in cells overexpressing TRXwt (Figure 3B). In contrast, AT had no effect in endothelial cells overexpressing the non–S-nitrosylatable TRX mutant (TRXC69S) (Figure 3B), indicating that AT-induced S-nitrosylation of TRX contributes importantly to AT-increased TRX enzyme activity.

View larger version (21K): [in this window] [in a new window]   Figure 2. A, AT induced S-nitrosylation of endogenous TRX. After immunoprecipitation with an antibody against thioredoxin, S-nitrosylation of endogenous thioredoxin was detected by use of diaminonaphthalene-assay (data are mean±SEM, n=6, *P<0.05 vs control). B, AT has no effect on TRX protein expression. Endothelial cells were incubated with 0.1 µmol/L AT for 24 hours. Immunoblotting with an anti-TRX antibody was performed. Membranes were reprobed with anti-tubulin antibody. A representative immunoblot is shown (top). TRX/tubulin ratio was quantified by scanning densitometry using Scion Image program (bottom; n=4).

View larger version (18K): [in this window] [in a new window]   Figure 3. A, AT increased endogenous TRX activity. Endothelial cells were incubated with 0.1 µmol/L AT or Rho kinase inhibitor 1 µmol/L Y27632 for 24 hours. TRX activity was measured as described in Methods (data are mean±SEM, n=3, *P<0.05 vs control, **P<0.05 vs H2O2). B, AT-increased TRX activity is dependent on cysteine 69. Endothelial cells were transfected with TRXwt or non–S-nitrosylatable mutant TRX(C69S) and incubated with 0.1 µmol/L AT for 24 hours. Reductase activity of thioredoxin was monitored by a NADPH oxidation assay (data are mean±SEM, n=3 to 5, *P<0.05 vs TRXwt, **P<0.05 vs TRXwt+AT).

AT Reduces Intracellular ROS by Activatory S-Nitrosylation of TRX Under Basal Conditions
We demonstrated previously that S-nitrosylation of TRX enhances its antioxidant capacity, which leads to reduced intracellular ROS.²⁰ Indeed, incubation of endothelial cells with AT reduced intracellular ROS (Figure 4, A and B). To distinguish between superoxide anions and H₂O₂, we used 2 fluorescent dyes, H₂DCF-DA and DHE. H₂DCF-DA is described to detect primarily, but not exclusively, H₂O₂.²⁷ As demonstrated in Figure 4, A and B, AT reduced intracellular ROS to a similar extent, suggesting that O₂^– and H₂O₂ are produced to a similar extent. Moreover, preincubation with AT also reduced H₂O₂-induced ROS levels below basal levels (Figure 4C). To document that AT-induced S-nitrosylation of TRX was required for the ability of AT to decrease intracellular ROS, we transfected endothelial cells with empty vector, TRXwt, or TRX(C69S). As shown in Figure 4B, overexpression of TRXwt significantly reduced intracellular ROS levels, whereas overexpression of TRX(C69S) showed ROS levels comparable to those in vector-transfected cells. Incubation with AT significantly reduced ROS in vector-transfected and in TRXwt-transfected cells (Figure 4D). However, AT did not influence intracellular ROS levels in endothelial cells overexpressing TRX(C69S) (Figure 4D), demonstrating that AT-induced reduction of intracellular ROS is dependent on S-nitrosylation of TRX at cysteine 69 in endothelial cells.

View larger version (23K): [in this window] [in a new window]   Figure 4. A, AT reduced intracellular ROS formation in endothelial cells. ROS were measured with DCF using FACS analysis as described in Methods (data are mean±SEM, n=3, *P<0.05 vs control of original data). B, AT reduced intracellular ROS formation in endothelial cells. ROS were measured with ethidine using FACS analysis as described in Methods (data are mean±SEM, n=3, *P<0.05 vs control of original data). C, AT reduced H2O2-induced intracellular ROS levels. Endothelial cells were incubated with 200 µmol/L H2O2 for 6 hours. ROS were measured with DCF using FACS analysis as described in Methods (data are mean±SEM, n=3, *P<0.05 vs H2O2). D, In endothelial cells overexpressing TRX(C69S), AT did not inhibit intracellular ROS formation. Production of endogenous ROS was measured with DCF using FACS analysis as described in Methods (data are mean±SEM, n=3, *P<0.05 vs TRXwt+AT, **P<0.05 vs TRXwt).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
It is generally accepted that statins exert beneficial effects not only by improving the lipid profile but also because of pleiotropic effects.¹ Recent studies demonstrated that statins have anti-inflammatory and antioxidant effects.^28–30 Because increased ROS contribute to vascular inflammation and may injure endothelial cells, we investigated the effect of statins on endothelial ROS levels under basal conditions. The present study demonstrates that statins increase the overall content of S-nitrosylated proteins and the S-nitrosylation of the redox regulator TRX, thereby reducing the intracellular ROS levels within endothelial cells.

Recent studies demonstrated that statins reduce the generation of intracellular ROS in smooth muscle cells and in cardiac myocytes after stimulation of the NADPH oxidase with growth factors or angiotensin II.^29–31 Moreover, cerivastatin was shown to reduce basal NADPH oxidase activity in human umbilical vein endothelial cells.³² Mechanistically, the reduction of ROS generation by statins was attributed to an inhibition of the rac1 GTPase activation and translocation from the cytosolic compartment to the cell membrane, which is a prerequisite of NADPH oxidase activation.^29,30,33 Associated with the Rac-GTPase inhibition is a reduction of the expression of NADPH oxidase isoforms by statins.^29,30,33 All of these studies focused on induced ROS formation by different stimuli and thereby investigated the effect of statins on ROS-generating enzymes. The present data investigating intracellular O₂^– and H₂O₂ levels under basal conditions in endothelial cells provide novel insights into the regulation of the antioxidant defense by statins. In contrast to catalase, which was shown to be transcriptionally increased after statin treatment in some but not all of the studies,^30,32 TRX was not regulated on a transcriptional level. Instead, statins activated TRX via posttranscriptional activation of the enzymatic activity, which resulted in reduction of intracellular ROS levels. The reduction of intracellular ROS by AT was abolished in endothelial cells overexpressing a thioredoxin construct, in which cysteine 69 was mutated to non–S-nitrosylatable serine. These results demonstrate that AT-mediated reduction of ROS depends on S-nitrosylation of thioredoxin. Thus, S-nitrosylation of TRX may provide an another mechanism in addition to the inhibition of Rac1 GTPase^29,30,33 underlying the antioxidant effects of statins. Interestingly, all effects of statins on the redox system, which include the inactivation of the NADPH oxidase and the activation of thioredoxin, are reversed by mevalonate. Therefore, all these antioxidant actions of statins are dependent on HMG-CoA reductase inhibition.

The role of eNOS in cholesterol-independent vascular and ischemic tissue protection by statins was delineated previously in in vivo models using genetic or pharmacological approaches. Not only did pretreatment with statins increase eNOS activity, but protection by statins was also completely abolished in eNOS-knockout mice or after inhibition of eNOS with N^G-nitro-L-arginine-methyl ester.^3,5 Of note, genetic ablation of eNOS results in a reduction of S-nitrosylated molecules.²⁰ Inhibition of the NO release thus may result in a reduced antioxidant defense capacity. Therefore, NO not only interacts directly with ROS to reduce intracellular ROS via the formation of peroxynitrite but also indirectly targets intracellular ROS by activation of ROS-metabolizing enzymes, such as thioredoxin. Given that ROS are intimately involved in activation of the prototypic inflammatory transcription factor nuclear factor-B,³⁴ it is reasonable to postulate that statin-mediated S-nitrosylation of TRX also contributes to the well-established anti-inflammatory effects of statins by maintaining the redox balance within endothelial cells.

Increased oxidative stress leads to severe cell damage and induction of apoptosis. Several in vitro and in vivo studies demonstrated that TRX can potently reduce oxidative stress-induced cellular damage and apoptosis.^20,35,36 TRX not only inhibits reperfusion-induced arrhythmias in a rat heart model^37,38 but is also protective against adriamycin-induced cardiotoxicity.³⁹ Thus, activation of TRX by NO-dependent S-nitrosylation may provide an important protective mechanism, which may not be restricted to endothelial cells. One may speculate that statin-mediated activation of TRX may also contribute to the direct cardioprotective effects of statins in cardiac myocytes.²⁹

Taken together, the data of the present study demonstrate that statins S-nitrosylate TRX via activation of NO synthesis, resulting in an enhanced redox-regulatory activity of TRX and decreased intracellular ROS levels. Moreover, AT-induced S-nitrosylation of TRX is a novel mechanism by which statins interfere with the antioxidant potential of endothelial cells.

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (SFB 553, C2). Jörg Hoffmann was supported by a fellowship of the Boehringer Ingelheim Fonds. We thank Christine Goy for excellent technical help.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Takemoto M, Liao JK. Pleiotropic effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Arterioscler Thromb Vasc Biol. 2001; 21: 1712–1719.[Abstract/Free Full Text]

2. Berk BC, Abe JI, Min W, Surapisitchat J, Yan C. Endothelial atheroprotective and anti-inflammatory mechanisms. Ann N Y Acad Sci. 2001; 947: 93–109; discussion 109–111.

3. Endres M, Laufs U, Huang Z, Nakamura T, Huang P, Moskowitz MA, Liao JK. Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 1998; 95: 8880–8885.[Abstract/Free Full Text]

4. Lefer DJ, Scalia R, Jones SP, Sharp BR, Hoffmeyer MR, Farvid AR, Gibson MF, Lefer AM. HMG-CoA reductase inhibition protects the diabetic myocardium from ischemia-reperfusion injury. FASEB J. 2001; 15: 1454–1456.[Free Full Text]

5. Wolfrum S, Grimm M, Heidbreder M, Dendorfer A, Katus HA, Liao JK, Richardt G. Acute reduction of myocardial infarct size by a hydroxymethyl glutaryl coenzyme A reductase inhibitor is mediated by endothelial nitric oxide synthase. J Cardiovasc Pharmacol. 2003; 41: 474–480.[CrossRef][Medline] [Order article via Infotrieve]

6. Laufs U, Liao JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem. 1998; 273: 24266–24271.[Abstract/Free Full Text]

7. Kureishi Y, Luo Z, Shiojima I, Bialik A, Fulton D, Lefer DJ, Sessa WC, Walsh K. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med. 2000; 6: 1004–1010.[CrossRef][Medline] [Order article via Infotrieve]

8. Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999; 399: 597–601.[CrossRef][Medline] [Order article via Infotrieve]

9. Dimmeler S, Fisslthaler B, Fleming I, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells via Akt-dependent phosphorylation. Nature. 1999; 399: 601–605.[CrossRef][Medline] [Order article via Infotrieve]

10. Stamler JS, Lamas S, Fang FC. Nitrosylation: the prototypic redox-based signaling mechanism. Cell. 2001; 106: 675–683.[CrossRef][Medline] [Order article via Infotrieve]

11. Harrison D, Griendling KK, Landmesser U, Hornig B, Drexler H. Role of oxidative stress in atherosclerosis. Am J Cardiol. 2003; 91: 7A–11A.[CrossRef][Medline] [Order article via Infotrieve]

12. Orr WC, Sohal RS. Extension of life-span by overexpression of superoxide dismutase and catalase in Drosophila melanogaster. Science. 1994; 263: 1128–1130.[Abstract/Free Full Text]

13. Nordberg J, Arner ES. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic Biol Med. 2001; 31: 1287–1312.[CrossRef][Medline] [Order article via Infotrieve]

14. Holmgren A. Thioredoxin and glutaredoxin systems. J Biol Chem. 1989; 264: 13963–13966.[Free Full Text]

15. Matsui M, Oshima M, Oshima H, Takaku K, Maruyama T, Yodoi J, Taketo MM. Early embryonic lethality caused by targeted disruption of the mouse thioredoxin gene. Dev Biol. 1996; 178: 179–185.[CrossRef][Medline] [Order article via Infotrieve]

16. Tanaka T, Nakamura H, Nishiyama A, Hosoi F, Masutani H, Wada H, Yodoi J. Redox regulation by thioredoxin superfamily: protection against oxidative stress and aging. Free Radic Res. 2001; 33: 851–855.[CrossRef]

17. Hirota K, Matsui M, Iwata S, Nishiyama A, Mori K, Yodoi J. AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. Proc Natl Acad Sci U S A. 1997; 94: 3633–3638.[Abstract/Free Full Text]

18. Schenk H, Klein M, Erdbrugger W, Droge W, Schulze-Osthoff K. Distinct effects of thioredoxin and antioxidants on the activation of transcription factors NF-kappa B and AP-1. Proc Natl Acad Sci U S A. 1994; 91: 1672–1676.[Abstract/Free Full Text]

19. Akamatsu Y, Ohno T, Hirota K, Kagoshima H, Yodoi J, Shigesada K. Redox regulation of the DNA binding activity in transcription factor PEBP2: the roles of two conserved cysteine residues. J Biol Chem. 1997; 272: 14497–14500.[Abstract/Free Full Text]

20. Haendeler J, Hoffmann J, Tischler V, Berk BC, Zeiher AM, Dimmeler S. Redox regulatory and anti-apoptotic functions of thioredoxin depend on S-nitrosylation at cysteine 69. Nat Cell Biol. 2002; 4: 743–749.[CrossRef][Medline] [Order article via Infotrieve]

21. Dimmeler S, Haendeler J, Nehls M, Zeiher AM. Suppression of apoptosis by nitric oxide via inhibition of ICE-like and CPP32-like proteases. J Exp Med. 1997; 185: 601–608.[Abstract/Free Full Text]

22. Dimmeler S, Haendeler J, Galle J, Zeiher AM. Oxidized low-density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-like proteases: a mechanistic clue to the response to injury hypothesis. Circulation. 1997; 95: 1760–1763.[Abstract/Free Full Text]

23. Marzinzig M, Nussler AK, Stadler J, Marzinzig E, Barthlen W, Nussler NC, Beger HG, Morris SM Jr, Bruckner UB. Improved methods to measure end products of nitric oxide in biological fluids: nitrite, nitrate, and S-nitrosothiols. Nitric Oxide. 1997; 1: 177–189.[CrossRef][Medline] [Order article via Infotrieve]

24. Hojo Y, Saito Y, Tanimoto T, Hoefen RJ, Baines CP, Yamamoto K, Haendeler J, Asmis R, Berk BC. Fluid shear stress attenuates hydrogen peroxide–induced c-Jun NH₂-terminal kinase activation via a glutathione reductase–mediated mechanism. Circ Res. 2002; 91: 712–718.[Abstract/Free Full Text]

25. Schulze PC, De Keulenaer GW, Yoshioka J, Kassik KA, Lee RT. Vitamin D₃-upregulated protein-1 (VDUP-1) regulates redox-dependent vascular smooth muscle cell proliferation through interaction with thioredoxin. Circ Res. 2002; 91: 689–695.[Abstract/Free Full Text]

26. Hoffmann J, Haendeler J, Zeiher AM, Dimmeler S. TNFalpha and oxLDL induce a denitrosylation of proteins in endothelial cells. J Biol Chem. 2001; 276: 41383–41387.[Abstract/Free Full Text]

27. Haugland RP. Handbook of Fluorescent Probes and Research Products. 9th ed. Eugene, Ore: Molecular Probes; 2002.

28. Kwak B, Mulhaupt F, Myit S, Mach F. Statins as a newly recognized type of immunomodulator. Nat Med. 2000; 6: 1399–1402.[CrossRef][Medline] [Order article via Infotrieve]

29. Takemoto M, Node K, Nakagami H, Liao Y, Grimm M, Takemoto Y, Kitakaze M, Liao JK. Statins as antioxidant therapy for preventing cardiac myocyte hypertrophy. J Clin Invest. 2001; 108: 1429–1437.[CrossRef][Medline] [Order article via Infotrieve]

30. Wassmann S, Laufs U, Muller K, Konkol C, Ahlbory K, Baumer AT, Linz W, Bohm M, Nickenig G. Cellular antioxidant effects of atorvastatin in vitro and in vivo. Arterioscler Thromb Vasc Biol. 2002; 22: 300–305.[Abstract/Free Full Text]

31. Delbosc S, Cristol JP, Descomps B, Mimran A, Jover B. Simvastatin prevents angiotensin II–induced cardiac alteration and oxidative stress. Hypertension. 2002; 40: 142–147.[Abstract/Free Full Text]

32. Vecchione C, Brandes RP. Withdrawal of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors elicits oxidative stress and induces endothelial dysfunction in mice. Circ Res. 2002; 91: 173–179.[Abstract/Free Full Text]

33. Maack C, Kartes T, Kilter H, Schäfers H-J, Nickenig G, Bohm M, Laufs U. Oxygen free radical release in human failing myocardium is associated with increased activity of Rac1-GTPase and represents a target for statin treatment. Circulation. 2003; 108: 1567–1574.[Abstract/Free Full Text]

34. Gimbrone MA Jr, Topper JN, Nagel T, Anderson KR, Garcia-Cardena G. Endothelial dysfunction, hemodynamic forces, and atherogenesis. Ann N Y Acad Sci. 2000; 902: 230–239.[Medline] [Order article via Infotrieve]

35. Liu Y, Min W. Thioredoxin promotes ASK1 ubiquitination and degradation to inhibit ASK1-mediated apoptosis in a redox activity–independent manner. Circ Res. 2002; 90: 1259–1266.[Abstract/Free Full Text]

36. Didier C, Kerblat I, Drouet C, Favier A, Beani JC, Richard MJ. Induction of thioredoxin by ultraviolet-A radiation prevents oxidative-mediated cell death in human skin fibroblasts. Free Radic Biol Med. 2001; 31: 585–598.[CrossRef][Medline] [Order article via Infotrieve]

37. Reddy PG, Bhuyan DK, Bhuyan KC. Lens-specific regulation of the thioredoxin-1 gene, but not thioredoxin-2, upon in vivo photochemical oxidative stress in the Emory mouse. Biochem Biophys Res Commun. 1999; 265: 345–349.[CrossRef][Medline] [Order article via Infotrieve]

38. Aota M, Matsuda K, Isowa N, Wada H, Yodoi J, Ban T. Protection against reperfusion-induced arrhythmias by human thioredoxin. J Cardiovasc Pharmacol. 1996; 27: 727–732.[CrossRef][Medline] [Order article via Infotrieve]

39. Shioji K, Kishimoto C, Nakamura H, Masutani H, Yuan Z, Oka S, Yodoi J. Overexpression of thioredoxin-1 in transgenic mice attenuates adriamycin-induced cardiotoxicity. Circulation. 2002; 106: 1403–1409.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. Chakrabarti, O. Lekontseva, A. Peters, and S. T. Davidge 17-{beta}-Estradiol induces protein S-nitrosylation in the endothelium Cardiovasc Res, December 14, 2009; (2009) cvp368v2. [Abstract] [Full Text] [PDF]

				C.-M. Lee, J. Pohl, and E. T. Morgan Dual Mechanisms of CYP3A Protein Regulation by Proinflammatory Cytokine Stimulation in Primary Hepatocyte Cultures Drug Metab. Dispos., April 1, 2009; 37(4): 865 - 872. [Abstract] [Full Text] [PDF]

				C.-Y. Wang, M.-S. Wen, H.-W. Wang, I-C. Hsieh, Y. Li, P.-Y. Liu, F.-C. Lin, and J. K. Liao Increased Vascular Senescence and Impaired Endothelial Progenitor Cell Function Mediated by Mutation of Circadian Gene Per2 Circulation, November 18, 2008; 118(21): 2166 - 2173. [Abstract] [Full Text] [PDF]

				E. Ritz and C. Wanner Statin Use Prolongs Patient Survival after Renal Transplantation J. Am. Soc. Nephrol., November 1, 2008; 19(11): 2037 - 2040. [Full Text] [PDF]

				E. M. Hylek, L. Frison, L. E. Henault, and A. Cupples Disparate Stroke Rates on Warfarin Among Contemporaneous Cohorts With Atrial Fibrillation: Potential Insights Into Risk From a Comparative Analysis of SPORTIF III Versus SPORTIF V Stroke, November 1, 2008; 39(11): 3009 - 3014. [Abstract] [Full Text] [PDF]

				L. Guasti, F. Marino, M. Cosentino, R. C. Maio, E. Rasini, M. Ferrari, L. Castiglioni, C. Klersy, G. Gaudio, A. M. Grandi, et al. Prolonged statin-associated reduction in neutrophil reactive oxygen species and angiotensin II type 1 receptor expression: 1-year follow-up Eur. Heart J., May 1, 2008; 29(9): 1118 - 1126. [Abstract] [Full Text] [PDF]

				P. Ferdinandy, R. Schulz, and G. F. Baxter Interaction of Cardiovascular Risk Factors with Myocardial Ischemia/Reperfusion Injury, Preconditioning, and Postconditioning Pharmacol. Rev., December 1, 2007; 59(4): 418 - 458. [Abstract] [Full Text] [PDF]

				Y.-M. Go, P. J. Halvey, J. M. Hansen, M. Reed, J. Pohl, and D. P. Jones Reactive Aldehyde Modification of Thioredoxin-1 Activates Early Steps of Inflammation and Cell Adhesion Am. J. Pathol., November 1, 2007; 171(5): 1670 - 1681. [Abstract] [Full Text] [PDF]

				Y. Hoshino, T. Nakamura, A. Sato, M. Mishima, J. Yodoi, and H. Nakamura Neurotropin Demonstrates Cytoprotective Effects in Lung Cells through the Induction of Thioredoxin-1 Am. J. Respir. Cell Mol. Biol., October 1, 2007; 37(4): 438 - 446. [Abstract] [Full Text] [PDF]

				T.-a. Okabe, K. Shimada, M. Hattori, T. Murayama, M. Yokode, T. Kita, and C. Kishimoto Swimming reduces the severity of atherosclerosis in apolipoprotein E deficient mice by antioxidant effects Cardiovasc Res, June 1, 2007; 74(3): 537 - 545. [Abstract] [Full Text] [PDF]

				A. J Bank, A. S Kelly, D. R Kaiser, W. W Crawford, B. Waxman, D. A Schow, and K. L Billups The effects of quinapril and atorvastatin on the responsiveness to sildenafil in men with erectile dysfunction Vascular Medicine, November 1, 2006; 11(4): 251 - 257. [Abstract] [PDF]

				J. Chen and J. L. Mehta Angiotensin II-mediated oxidative stress and procollagen-1 expression in cardiac fibroblasts: blockade by pravastatin and pioglitazone Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1738 - H1745. [Abstract] [Full Text] [PDF]

				L. Tao, X. Jiao, E. Gao, W. B. Lau, Y. Yuan, B. Lopez, T. Christopher, S. P. RamachandraRao, W. Williams, G. Southan, et al. Nitrative Inactivation of Thioredoxin-1 and Its Role in Postischemic Myocardial Apoptosis Circulation, September 26, 2006; 114(13): 1395 - 1402. [Abstract] [Full Text] [PDF]

				D. E. Handy and J. Loscalzo Nitric Oxide and Posttranslational Modification of the Vascular Proteome: S-Nitrosation of Reactive Thiols Arterioscler Thromb Vasc Biol, June 1, 2006; 26(6): 1207 - 1214. [Abstract] [Full Text] [PDF]

				S. Fichtlscherer, C. Schmidt-Lucke, S. Bojunga, L. Rossig, C. Heeschen, S. Dimmeler, and A. M. Zeiher Differential effects of short-term lipid lowering with ezetimibe and statins on endothelial function in patients with CAD: clinical evidence for 'pleiotropic' functions of statin therapy Eur. Heart J., May 2, 2006; 27(10): 1182 - 1190. [Abstract] [Full Text] [PDF]

				J. Schwartz, S. K. Park, M. S. O'Neill, P. S. Vokonas, D. Sparrow, S. Weiss, and K. Kelsey Glutathione-S-Transferase M1, Obesity, Statins, and Autonomic Effects of Particles: Gene-by-Drug-by-Environment Interaction Am. J. Respir. Crit. Care Med., December 15, 2005; 172(12): 1529 - 1533. [Abstract] [Full Text] [PDF]

				U. Landmesser, F. Bahlmann, M. Mueller, S. Spiekermann, N. Kirchhoff, S. Schulz, C. Manes, D. Fischer, K. de Groot, D. Fliser, et al. Simvastatin Versus Ezetimibe: Pleiotropic and Lipid-Lowering Effects on Endothelial Function in Humans Circulation, May 10, 2005; 111(18): 2356 - 2363. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 110/7/856    most recent 01.CIR.0000138743.09012.93v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Haendeler, J. Articles by Dimmeler, S. Search for Related Content PubMed PubMed Citation Articles by Haendeler, J. Articles by Dimmeler, S. Related Collections Cell signalling/signal transduction Oxidant stress Endothelium/vascular type/nitric oxide