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Endothelin stimulates vascular hydroxyl radical formation:Effect of obesity

Mundy, A L; Haas, E; Bhattacharya, I; Widmer, C C; Kretz, M; Baumann, K; Barton,M

Mundy, A L; Haas, E; Bhattacharya, I; Widmer, C C; Kretz, M; Baumann, K; Barton, M (2007). Endothelinstimulates vascular hydroxyl radical formation: Effect of obesity. American Journal of Physiology - Regulatory,Integrative and Comparative Physiology, 293(6):R2218-R2224.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 2007, 293(6):R2218-R2224.

Mundy, A L; Haas, E; Bhattacharya, I; Widmer, C C; Kretz, M; Baumann, K; Barton, M (2007). Endothelinstimulates vascular hydroxyl radical formation: Effect of obesity. American Journal of Physiology - Regulatory,Integrative and Comparative Physiology, 293(6):R2218-R2224.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 2007, 293(6):R2218-R2224.

Endothelin stimulates vascular hydroxyl radical formation:Effect of obesity

Abstract

Reactive oxygen species (ROS) and endothelin-1 (ET-1) contribute to vascular pathophysiology inobesity. In this context, whether ET-1 modulates hydroxyl radical (*OH) formation and the function ofROS/*OH in obesity is not known. In the present study, formation and function of ROS, including *OH,were investigated in the aorta of lean and leptin-deficient obese ob/ob mice. Hydroxyl radical formationwas detected ex vivo using terephthalic acid in intact aortic rings and the involvement of ROS inET-1-mediated vasoreactivity was analyzed using the antioxidant EPC-K1, a combination ofalpha-tocopherol and ascorbic acid. Generation of either *OH, *O(2)(-), and H(2)O(2) was stronglyinhibited by EPC-K1 (all P < 0.05). In obese mice, basal vascular *OH formation and ROS activity werereduced by 3-fold and 5-fold, respectively (P < 0.05 vs. lean). ET-1 markedly enhanced *OH formationin lean (6-fold, P < 0.05 vs. untreated) but not in obese mice. Obesity increased ET-1-inducedcontractions (P < 0.05 vs. lean), and ROS scavenging further enhanced the response (P < 0.05 vs.untreated). Exogenous ROS, including *OH caused stronger vasodilation in obese animals (P < 0.05 vs.lean), whereas endothelium-dependent relaxation was similar between lean and obese animals. Inconclusion, we present a sensitive method allowing ex vivo measurement of vascular *OH generationand provide evidence that ET-1 regulates vascular *OH formation. The data indicate that in obesity,vascular formation of ROS, including *OH is lower, whereas the sensitivity to ROS is increased,suggesting a novel and important role of ROS, including *OH in the regulation of vascular tone indisease status associated with increased body weight.

Endothelin stimulates vascular hydroxyl radical formation: effect of obesity

Alexa L. Mundy,* Elvira Haas,* Indranil Bhattacharya,* Corinne C. Widmer, Martin Kretz,Karin Baumann, and Matthias BartonMolecular Internal Medicine, Medical Policlinic, Department of Internal Medicine, University Hospital Zurich,Zurich, Switzerland

Submitted 27 April 2007; accepted in final form 19 September 2007

Mundy AL, Haas E, Bhattacharya I, Widmer CC, Kretz M,Baumann K, Barton M. Endothelin stimulates vascular hydroxylradical formation: effect of obesity. Am J Physiol Regul Integr CompPhysiol 293: R2218–R2224, 2007. First published September 26,2007; doi:10.1152/ajpregu.00295.2007.—Reactive oxygen species(ROS) and endothelin-1 (ET-1) contribute to vascular pathophysiol-ogy in obesity. In this context, whether ET-1 modulates hydroxylradical (•OH) formation and the function of ROS/•OH in obesity is notknown. In the present study, formation and function of ROS, includ-ing •OH, were investigated in the aorta of lean and leptin-deficientobese ob/ob mice. Hydroxyl radical formation was detected ex vivousing terephthalic acid in intact aortic rings and the involvement ofROS in ET-1-mediated vasoreactivity was analyzed using the antiox-idant EPC-K1, a combination of �-tocopherol and ascorbic acid.Generation of either •OH, •O2

�, and H2O2 was strongly inhibited byEPC-K1 (all P � 0.05). In obese mice, basal vascular •OH formationand ROS activity were reduced by 3-fold and 5-fold, respectively(P � 0.05 vs. lean). ET-1 markedly enhanced •OH formation in lean(6-fold, P � 0.05 vs. untreated) but not in obese mice. Obesityincreased ET-1-induced contractions (P � 0.05 vs. lean), and ROSscavenging further enhanced the response (P � 0.05 vs. untreated).Exogenous ROS, including •OH caused stronger vasodilation in obeseanimals (P � 0.05 vs. lean), whereas endothelium-dependent relax-ation was similar between lean and obese animals. In conclusion, wepresent a sensitive method allowing ex vivo measurement of vascular•OH generation and provide evidence that ET-1 regulates vascular•OH formation. The data indicate that in obesity, vascular formationof ROS, including •OH is lower, whereas the sensitivity to ROS isincreased, suggesting a novel and important role of ROS, including•OH in the regulation of vascular tone in disease status associated withincreased body weight.

oxidative stress; acetylcholine; vasoconstriction; murine; ob/ob;terephthalic acid; vitamin E; vitamin C

ENDOTHELIN-1 ( ET-1) IS A POTENT vasoconstrictor (57) and mito-gen (30) that has also been implicated in the pathogenesis ofmetabolic diseases, including obesity and diabetes (4, 29, 31,43). Endogenous ET-1 contributes to increased basal vasculartone in human obesity (8, 31), and we and others have shownthat vascular responsiveness to ET-1 is enhanced in obesity (8,50). Recent evidence also suggests that ET-1 stimulates thegeneration of certain reactive oxygen species (ROS) (26, 52).

ROS are short-lived, oxygen intermediates that participate inactivation of intracellular signaling pathways (48) and regulatecell function (14), and they are known to contribute to abnor-mal vascular reactivity in obesity-related diseases such asdiabetes, atherosclerosis, and hypertension (16, 41, 44, 46, 48,55). Although vascular effects of ROS have been often re-

garded as deleterious, resulting in impaired vasodilation and/orenhanced vasoconstriction (45), ROS also have beneficial ef-fects; indeed, superoxide (•O2

�), hydrogen peroxide (H2O2),and the hydroxyl radical (•OH) may also act as vasodilatorsunder certain conditions (40, 53).

Previous studies have investigated vascular interactionsbetween ET-1 and •O2

� or H2O2 (26, 52); however, little isknown about whether ET-1 also interacts with •OH, a highlyreactive ROS (33). Hydroxyl radical is mainly formed byH2O2 via either the Fenton (13) or the Haber-Weiss reaction(23):

Fe2� � H2O2 3 Fe3� � •OH � OH� (Fenton Reaction)

H2O2 � •O2� 3Cu2��

•OH � OH� � O2 (Haber-Weiss Reaction)

At high concentrations, hydroxyl radicals can cause mem-brane damage, DNA and RNA fragmentation, and lipid per-oxidation (9, 49). In the vasculature, •OH regulates vasocon-striction (49) and vasodilation (40) under normal and patho-logical conditions (39, 40).

The aim of the present study was to characterize the inter-actions between ET-1 and ROS/•OH in the vasculature ofnormal C57BL6/J laboratory mice and to determine the possi-ble modulatory effects of obesity on ROS-mediated vasculartone and •OH production using a model of leptin-deficientobesity.

METHODS

Animals

Healthy 40-wk-old male C57BL/6 mice (controls) and severelyobese leptin-deficient mice (B6.V-Lepob/J; Charles River, SulzfeldGermany; obese) were used. Body weight of obese mice was 58 � 4 gcompared with 29 � 1 g for control mice. Animals were held at theinstitutional animal facilities and received standard rodent chow andtap water ad libitum. Housing facilities and experimental protocolswere approved by the local authorities for animal research (Kommis-sion fur Tierversuche des Kantons Zurich, Switzerland) and conformto the Guide for the Care and Use of Laboratory Animals publishedby the U.S. National Institutes of Health (NIH Publication No. 85-23,revised 1996). Mice were anesthetized (xylazine: 100 mg/kg body wt;ketamine: 23 mg/kg body wt; and acepromazine: 3.0 mg/kg body wtip), and exsanguinated via cardiac puncture.

Measurement of Vascular Hydroxyl Radical Formation

To quantify •OH generation in very small specimens of livingvascular tissue ex vivo, we established a method using terephthalic

* These authors contributed equally to this article.Address for reprint requests and other correspondence: M. Barton, Medical

Policlinic, Dept. of Internal Medicine, Univ. Hospital Zurich, Raemistrasse100, 8091 Zurich, Switzerland (e-mail: barton@usz.ch).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Regul Integr Comp Physiol 293: R2218–R2224, 2007.First published September 26, 2007; doi:10.1152/ajpregu.00295.2007.

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acid (TPA), a specific probe for •OH detection, slightly modified frompublished in vitro protocols (3, 42). Hydroxylation of TPA yields astable and highly specific isomer (2-hydroxyl terephthalic acid, TPA-OH) (42) that is not formed by reaction with either superoxide orhydroperoxides (3). The sensitivity and specificity of TPA to detect•OH are comparable to that of the spin trap 5,5 dimethyl-1-pyrrolineN-oxide (27), as detected by electron spin resonance technique (seealso supplemental information, which may be found online at theAmerican Journal of Physiology—Regulatory, Integrative and Com-parative Physiology website). Vascular •OH generation was measuredin aortic rings, which were left unconstricted and placed in gassedKrebs solution (95% O2, 5% CO2, 37°C, pH 7.4) for 4 h containing2.5 mM TPA in the absence (basal •OH formation) or in the presenceof 10�6 mol/l ET-1 (ET-1-stimulated •OH formation). TPA-OH wasquantified fluorometrically (excitation: 326 nm, emission: 432 nm)using a microplate reader (SpectraMax M2) (42). Hydroxyl radicalformation was calculated as relative light units per milligram ofprotein after subtraction of background fluorescence. Protein wasextracted from vascular tissue in a RIPA buffer containing orthovana-date, PMSF, and complete mini protease inhibitor (Roche, Rotkreuz,Switzerland) using a cell shredder (Qiagen, Hombrechtikon, Switzer-land) and incubated for 30 min at 4°C. Insoluble material wasseparated by centrifugation and protein content of the soluble fractionwas quantified using Bio-Rad Protein Assay solution (Bio-Rad, Re-inach, Switzerland), according to the manufacturer’s instructions.

Characterization of the Antioxidant Activities of EPC-K1

The effects of EPC-K1 on •O2� and •OH formation and on the

stability of H2O2 were determined using specific assays (12). Theradical scavenger EPC-K1, a water-soluble compound, is a combina-tion of two known antioxidants, vitamin E (alpha-tocopherol) andvitamin C (ascorbic acid) (54) linked by a phosphate diester (See alsosupplemental information that is available online at the AmericanJournal Physiology—Regulatory, Integrative and Comparative Phys-iology website.). Effects of EPC-K1 (10�1 mg/ml) on xanthine/xanthine oxidase-mediated •O2

� generation were determined byL-012-dependent chemiluminescence, as described (10). The overallmeasuring time was 6 min, and a total of 24 measurements were takenduring this period. Each measurement was for 10 s, and the intervalbetween each reading was 5 s (Lumat LB 9507, Berthold Technolo-gies, Regensdorf, Switzerland). Total superoxide anion formation wasdetermined by cumulative measurements of chemiluminescence over6 min and calculated after subtracting the background. Hydroxylradical was generated from the reaction of Fe2� and ascorbic acid (51)(both at 100 �mol/l) in warm, gassed Krebs solution (37°C, 95% O2,5% CO2), in the presence or absence of EPC-K1 (10�1 mg/ml) (35),and quantified using TPA (2.5 mM) hydroxylation (42). The effect ofEPC-K1 (10�1 mg/ml) on hydrogen peroxide (0.3 �mol/l H2O2) wasassessed using the Amplex Red assay (Molecular Probes, Eugene,OR), as previously described (18). The reaction product resorufin (18)was detected fluorometrically (excitation and emission wavelengths:571 nm and 585 nm, respectively) using a SpectraMax M2 (see alsosupplemental information online at the American Journal of Physiol-ogy—Regulatory, Integrative and Comparative Physiology website).

Vascular Function Studies

The aorta was excised and placed in cold (4°C) Krebs-Ringerbicarbonate solution [in mmol/l: NaCl 118.6; KCl 4.7; CaCl2 2.5;MgSO4 1.2; KH2PO4 1.2; NaHCO3 25.1; ethylenediaminetetraaceticacid calcium disodium salt (EDTANa2Ca) 0.026; glucose 10.1], dis-sected free of connective tissue under a microscope (Olympus SZX9,Volketswil, Switzerland), and cut into rings 3 mm in length. Specialcare was taken not to damage the endothelium during this procedure.Segments of thoracic aorta were mounted onto tungsten wires (100�m in diameter) and transferred to water-jacketed organ baths con-taining Krebs solution (95% O2,-5% CO2 at 37°C, pH 7.4) and

connected to force transducers (Hugo Sachs Elektronik, March-Hug-stetten, Germany) (50). Resting tension was gradually increased to theoptimal level as previously described (50); vessels were repeatedlyexposed to 100 mM KCl until a stable contraction response wasachieved.

Endothelium-dependent and -independent vascular function. Endo-thelium-dependent relaxation to ACh (0.1–10 �mol/l) was determinedin the presence of indomethacin (10 �mol/l, a nonselective cycloox-ygenase inhibitor, 30 min preincubation) in rings preconstricted withphenylephrine to 80% of KCl-induced contraction, as previouslydescribed (5). Endothelium-independent vasodilation was investi-gated using the nitric oxide (NO) donor sodium nitroprusside (10�mol/l).

Reactive oxygen species and basal vascular tone. Rings werepretreated with L-nitro-arginine methyl ester (L-NAME, 300 �mol/l)for 15 min to block effects of endogenous NO (24). To determinewhether endogenous ROS formation contributes to basal vasculartone, the ROS scavenger EPC-K1 (10�1 mg/ml equal to 141.46 �M)(35) was applied to quiescent vascular rings and the resultant con-traction, reflecting basal vascular ROS generation (6, 20) was mea-sured. The contractile responses of quiescent vessels to scavengers orenzyme inhibitors allow us to indirectly quantify basal generation ofmolecules such as NO or •O2

� in a particular vascular bed (6, 20).Vascular responses to ET-1. Vascular rings were exposed to ET-1

(0.01–300 nmol/l) in the presence of L-NAME (300 �mol/l) for 30min, as described (56). To investigate the role of ROS for ET-1-induced contraction, selected rings were also preincubated with theROS scavenger EPC-K1 (10�1 mg/ml) for 15 min.

Effects of ROS on vascular tone. Vascular responses to exog-enously generated ROS/•OH generated by the reaction between Fe2�

and ascorbic acid (51) were investigated in aortic rings preconstrictedwith phenylephrine, as previously described (36).

Gene Expression Measurements

Steady-state mRNA expression levels of vascular nitric oxidesynthase (NOS) isoforms 2 and 3 were determined by real-timequantitative reverse transcriptase-PCR. Aortic total RNA was ex-tracted using the RNeasy micro kit for RNA isolation from fibroustissues (Qiagen, Hilden, Germany). Purity of RNA was controlled byRT(�) reactions (PCR with nontranscribed RNA). RNA was reverse-transcribed with the Omniscript RT kit (Qiagen, Hilden, Germany).Real-time quantitative PCR was used to determine relative expres-sion levels of murine genes encoding for NOS2 (Nos2, locus:NM_010927), NOS3 (Nos3, locus: NM_008713) and the housekeep-ing gene tyrosine-3-monooxygenase (locus: NM_011740). For ampli-fication of a tyrosine-3-monooxygenase-specific cDNA fragment 5�-CGA GCA GGC AGA GCG ATA TG-3� was used as the forward and5�-AGA CGA CCC TCC ACG ATG AC-3� as the reverse primer, fora Nos2-specific fragment 5�-GCA CCG AGA TTG GAG TTC-3� asthe forward and 5�-AGC ACA GCC ACA TTG ATC-3� as the reverseprimer. 5�-CCT AGT CCT CGC CTC CTTC-3� was used as theforward primer for a Nos3-specific fragment and 5�-ACC ACT TCCATT CTT CGT AGC-3� as the reverse primer. Two-step PCR wasperformed with iQ SYBR Supermix PCR kit (Bio-Rad) as follows:activation of the hot start Taq polymerase for 3 min (95°C), followedby 40 cycles of denaturation at 95°C for 15 s (step 1), and annealingand extension at 60°C for 1 min (step 2). Fluorescence was detectedat the end of each extension step. Identity and specificity of ampliconswere confirmed by agarose gel electrophoresis, melting curve analy-sis, and sequencing (Microsynth, Balgach, Switzerland). Steady-statemRNA expression levels were calculated by normalizing the relativeamount of each mRNA to the housekeeping gene tyrosine-3-mono-oxygenase (28) and expressed as arbitrary units (AU).

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Materials

ET-1 and L-NAME were from Alexis Corp (Lausanne, Switzer-land). EPC-K1 was from Senju Pharmaceuticals (Osaka, Japan).L-012 was from Wako Chemicals (Neuss, Germany), and AmplexRed was from Molecular Probes (Eugene, OR). All other chemicalswere from Sigma Chemicals (Buchs, Switzerland).

Statistical Analyses

Data are given as means � SE, and n denotes the number ofanimals used per experiment. Contraction is expressed as a percentageof 100 mM KCl-induced contraction, and dilations are given as apercentage of the maximal contraction. EC50 values (as negativelogarithm pD2) were calculated by nonlinear regression analysis, andarea under the curve was calculated for each individual curve usingSigmaPlot (SPSS Chicago, IL). Data were analyzed using ANOVAfor repeated measurements with Bonferroni correction, the Mann-Whitney U-test, or unpaired Student’s t-test when appropriate. A Pvalue �0.05 was considered significant.

RESULTS

Efficacy of •OH-Generating Systems in Krebs Solution

Hydroxyl radical generation was determined in vitro byTPA, a specific probe for •OH (27). The efficacy of •OHgenerated by ascorbic acid and Fe2� was compared withanother •OH-generating system consisting of H2O2 and Fe2�.The amount of •OH generated by vitamin C and Fe2� was2.6-fold higher compared with H2O2 and Fe2� (P � 0.05 vs.H2O2 and Fe2�, Table 1). DMSO, a known •OH scavenger (1),blocked in either system •OH formation by �80%, as shown in

Table 1 (See also supplemental data for this article, which isavailable online at the American Journal of Physiology—Regulatory, Integrative and Comparative Physiology web-site.).

Vascular Hydroxyl Radical Formation and Acute Effectsof ET-1

TPA was used to quantify vascular •OH formation ex vivo.Formation of •OH was detected under basal conditions inuncontracted aortic rings from control mice [2.5 � 0.1 relativefluorescence units (RFU)/mg protein, Fig. 1A] and markedlyincreased by endothelin-1 (15.0 � 1.0 RFU/mg protein, six-fold, P � 0.0001, vs. untreated, Fig. 1B). Basal •OH formationwas reduced approximately threefold in obese animals (P �0.0001 vs. lean, Fig. 1, A and B) and ET-1 slightly increasedthis attenuated •OH formation (0.6 � 0.1 to 2.3 � 0.3 RFU/mgprotein, P � 0.0001 vs. untreated, Fig. 1B). Interestingly,ET-1-stimulated •OH formation in obese mice was still belowbasal levels of •OH detected in lean mice (2.3 � 0.3 vs. 2.5 �0.1 RFU/mg protein, respectively).

Effects of EPC-K1 on ROS Formation and Vascular Tone

The effects of EPC-K1 on in vitro generation of •O2�, •OH,

and H2O2 were determined. EPC-K1 inhibited xanthine-oxi-dase-mediated •O2

� generation by 74 � 2% (P � 0.0001 vs.control), that of ascorbic acid and Fe2�-mediated •OH forma-tion by 84 � 1% (P � 0.0001 vs. control), and that of H2O2 by61 � 2% (P � 0.0001 vs. control). We next investigated basalROS bioactivity in quiescent aortic rings by measuring changesin vascular tone after applying EPC-K1, which caused contrac-tion in all rings investigated (P � 0.05, vs. untreated, Fig. 2).EPC-K1-induced contractions were markedly reduced in obesemice (5-fold, P � 0.05, vs. control).

Effects of Exogenous ROS on Vascular Tone

The addition of exogenous ROS/•OH to precontracted vas-cular rings caused a vasodilator response (P � 0.0001, vs.untreated, Fig. 3, ROS/•OH in Fig. 3A), that was enhancedthreefold by obesity (P � 0.05 vs. control).

Function and Expression of Vascular NO Synthases

NO-mediated endothelium-dependent relaxation to AChwas similar in lean and obese mice [�94 � 1.6 vs. �94 �

Table 1. Efficacy of in vitro •OH-generating systems

•OH-Generating System DMSO TPA-OH, relative fluorescence units

Control � 0Control � 0H2O2 � Fe2� � 691�39*‡H2O2 � Fe2� � 102�15*Vitamin C � Fe2� � 1804�197*†‡Vitamin C � Fe2� � 388�13*

The amount of •OH-generated by vitamin C and Fe2� (100 �M each) andthat by H2O2 and Fe2� was measured by the hydroxylation of terephthalic acid(TPA-OH). The •OH-generating systems were investigated in Krebs solutionin the presence or absence of the •OH radical scavenger DMSO (3 mM). *P �0.05 vs. control (Krebs buffer). †P � 0.05 vs. H2O2 and Fe2�. ‡P � 0.05 vs.•OH-generating systems in the presence of DMSO.

Fig. 1. Vascular •OH formation in lean control(open bars) and obese ob/ob mice (solid bars), asmeasured by TPA hydroxylation. Under unstimu-lated conditions basal formation of •OH was de-tected. Obesity reduced basal •OH formation bythree-fold (P � 0.001 vs. lean). Exposure to en-dothelin-1 (ET-1) (10�6 mol/l) increased •OHformation in lean control mice but had only asmall effect in obese mice. Values are expressedas fluorescence units per milligram protein; n 6/group *P � 0.001 vs. basal; †P � 0.001 vs.lean.

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3.3%, not significant (ns), Fig. 3B], as was relaxation to sodiumnitroprusside (SNP) (�109 � 1.3 vs. �107 � 1.5%). Simi-larly, vascular expression of NOS3 mRNA was similar be-tween control (504 � 53 AU) and obese mice (471 � 74 AU,ns), as were NOS2 mRNA levels (control, 1.2 � 1 AU, n 4;obese, 0.95 � 0.35 AU, n 7, ns).

Effect of Endogenous ROS on ET-1-Induced Contractility

Contractions to ET-1 were enhanced by obesity (20.4 � 4.9%vs. 10.6 � 1.5%, P � 0.05, Fig. 4, A and B and Table 2). In obesemice, scavenging of ROS by EPC-K1 further enhanced ET-1-induced vasoconstriction (45 � 9%, P � 0.05 vs. untreated);this effect was absent in lean controls (Fig. 4).

DISCUSSION

This study demonstrates interactions between the vasocon-strictor ET-1 and ROS/•OH under normal conditions and inobesity. We present a sensitive method to quantify hydroxylradical formation in small vascular specimens ex vivo, usingTPA hydroxylation, and show that ET-1 acutely stimulatesvascular •OH formation in the aorta of normal laboratory mice.The results provide evidence that basal ROS activity, as well asET-1-stimulated •OH formation were markedly reduced inobesity. Scavenging of endogenous ROS with the nonselectiveradical scavenger EPC-K1 increased vasoconstrictor responsesto ET-1 in aortic rings of obese animals, but not in controls,indicating that ROS antagonize ET-1-mediated vasoconstrictorresponses in obesity.

•OH is a highly reactive ROS, and its role in pathophysiol-ogy has only been clarified in part (17, 25, 48). Investigationson the role of •OH in the pathophysiology of cardiovasculardisorders have, to date, been hindered by the lack of sensitiveand specific methods. Previously, salicylic acid has been usedto detect •OH formation (11). However, because salicylic acidalso detects peroxynitrite (19) and inhibits enzymes involved inROS production (7, 21), salicylic acid cannot be considered aspecific probe for •OH. Therefore, one of the goals of the

present study was to establish a specific and sensitive methodthat would allow us to measure •OH generation in smallvascular specimens ex vivo. We used TPA, which is a highlyspecific and sensitive probe for •OH (3), as shown by electronspin resonance experiments (27) (see also supplemental infor-mation online at the American Journal of Physiology—Regu-latory, Integrative and Comparative Physiology website), andtherefore, no other probes for •OH were used in our experi-ments. Using the TPA-based •OH detection method, thepresent study shows that basal •OH formation can indeed be

Fig. 2. Basal reactive oxygen species (ROS) formation in quiescent aorticrings expressed as a change in basal vascular tone in response to the ROSscavenger EPC-K1. Inhibition of basal ROS resulted in contraction in allvessels studied. In obesity, basal ROS formation was markedly reduced(fivefold, P � 0.05). Contractions to EPC-K1 are expressed as a percentage ofKCl-induced contractions. n 5–14/group. *P � 0.05 vs. lean.

Fig. 3. Dilator responses to exogenous ROS/•OH (A) and endothelium-depen-dent relaxation to ACh (B) in aortic rings of lean and obese animals precon-tracted with phenylephrine. Dilations to ROS/•OH are expressed as a percent-age of precontraction. n 5–14/group. *P � 0.05 vs. lean. Relaxation to AChis expressed as percent of precontraction. n 7–9/group.

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detected in very small specimens of arterial tissue in both leanand obese animals.

Endothelin contributes to the pathogenesis of atherosclero-sis, hypertension, and diabetes (30, 43) and has been previ-ously shown to stimulate the generation of ROS such as •O2

�

and H2O2 (26, 52). Presently, there is no information onwhether ET-1 modulates vascular formation of •OH. UsingTPA as a probe for •OH, we found that ET-1 is a potentstimulus of vascular •OH formation in animals with normalbody weight. An unexpected finding of the present study wasthat this stimulating effect of ET-1 was more or less abrogatedin obese mice. Because of the limitations in tissue and animals,only a single concentration of •OH was used. To our knowl-edge, this is not only the first demonstration that ET-1 modu-lates vascular •OH formation, but also that both basal andstimulated •OH formation are markedly reduced in obesity. Inline with these results, basal vascular activity of ROS, asmeasured indirectly by EPC-K1-induced contractions, was alsoreduced in obese animals. Our study did not identify the natureof ROS mediating the observed effects in response to EPC-K1.One of the potential candidates of ROS could be •O2

� as in amost recent study using young obese db/db mice, whichexpress a mutated leptin receptor and unlike the ob/ob animalsused in our study show high circulating leptin levels, Grien-dling and colleagues (41) demonstrated that aortic membranesuperoxide generation was enhanced compared with lean con-trols.

EPC-K1 has been previously proposed to be a scavenger thatis selective for •OH (35). On the basis of its chemical structurethat combines ascorbic acid and �-tocopherol linked by aphosphodiester, it is very unlikely to be specific for •OHscavenging (54). Indeed, our results now show that EPC-K1not only scavenges •OH but also that it potently inactivates•O2

� and H2O2. Therefore, in the experiments involving EPC-K1, scavenging of any of the ROS mentioned may contributeto the observed changes in vasoreactivity. According to aprevious report, EPC-K1 as a ROS scavenger induces NO-independent relaxation in precontracted canine coronary arter-ies (47). In the present study, however, we demonstrate thatEPC-K1 also causes contraction of quiescent murine aorticrings, reflecting basal ROS vasodilator bioactivity. These dif-ferences in the effects of EPC-K1 are likely to be explained by1) the state of contraction of the vascular rings, 2) by the typeof artery studied and/or 3) by interspecies differences.

To investigate the effects of exogenous ROS/•OH on vas-cular tone, we used ascorbic acid and iron (45, 49) as aROS/•OH generating system. In vitro experiments confirmedthat this system is indeed a strong inductor of •OH formationand that concentration-dependent relaxation can be achieved(see supplemental information for details). The results of thepresent study demonstrate that exogenous ROS/•OH causevasodilation, an effect that is markedly enhanced by obesity.This suggests that obesity modulates the sensitivity of thevasculature to vasodilator activities of ROS. Also, it is likelythat obesity per se and not leptin deficiency enhances theresponsiveness to ROS/•OH, since enhanced vascular relax-ation to ROS/•OH was also observed in a model of diet-induced obesity (36). An unexpected finding was that nodifferences in the NO-mediated endothelium-dependent relax-ation in the presence of cyclooxygenase inhibitors were seenbetween obese and lean animals. The steady-state mRNAexpression levels of endothelial nitric oxide synthase (eNOS/NOS3) was also similar between the groups. Of note, in theseexperiments, a cyclooxygenase inhibitor was used since obe-sity enhances cyclooxygenase-mediated production of vaso-constrictor prostanoids (36, 50), which attenuate endothelium-dependent vascular relaxation in response to ACh in obesity(50). In a recent study, endothelium-dependent relaxation was

Fig. 4. Contractions to ET-1 (0.01–300 nmol/l) in thepresence of L-NAME in aorta of lean (open symbols)and obese mice (solid symbols) in untreated rings (left),and after preincubation with EPC-K1 (right). Obesitywas associated with increased contractions to ET-1, andROS scavenging increased contractions to ET-1 inobese animals only. Data are expressed as a percentageof contraction to 100 mM KCl; n 8–19 /group. *P �0.05 vs. lean. †P � 0.05 vs. untreated.

Table 2. pD2 values and values of area under the ET-1concentration-response curve (AUC)

Lean Obese

Untreated EPC-K1 Untreated EPC-K1

pD2 8.5�0.1 9.2�0.3† 8.5�0.1 9.2�0.3†AUC 21�3 14�2 42�10* 137�39†‡

pD2 values and values of area under the endothelin-1 (ET-1) concentration-response curve (area under the curve, AUC) were calculated for each concen-tation-response curve, and data were averaged. In untreated rings of controland obese mice, pD2 values were similar. EPC-K1 increased AUC threefoldonly in the aorta of obese animals. *P � 0.05, lean vs. obese. †P � 0.05 vs.untreated. ‡P � 0.05 vs. lean, EPC-K1.

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found to be impaired in the aorta of obese db/db mice (41),which is likely to be explained by the lack of a cyclooxygenaseinhibitor in the organ chamber experiments (50). The results ofthe present study are in line with previous studies showing thatin the presence of cyclooxygenase inhibition, NO-dependentrelaxation actually remains unaffected in diet-induced, as wellas in monogenetic forms of obesity in rodents (2, 22, 36).

Similar to our recent study using a model of diet-inducedobesity (50), we found that contractions to ET-1 are alsoincreased in obesity due to leptin deficiency. The role of ROSfor ET-1-induced contractions was investigated using the ROSscavenger EPC-K1. While in lean animals, EPC-K1 had noeffect on contractions to ET-1, the antioxidant markedly en-hanced contractions in obese animals, suggesting that endog-enous ROS antagonize ET-1-mediated vasoconstriction in obe-sity, but not under normal conditions. The present data alsosuggest that this indirect vasodilator activity of ROS in obesityis independent of NO, since all experiments were performed inthe presence of a NO synthase inhibitor. Indeed, reactiveoxygen species have been shown to block vasoconstriction andmediate vasorelaxant effects in the vasculature by 1) activationof endothelium-dependent hyperpolarizing factor (32, 37), 2)by activating cGMP pathway (15, 34), and/or 3) by inhibitionof phosphorylation of myosin light chain protein (38). In thepresent study, because NOS was blocked, cyclic GMP isunlikely to play a role in the ROS-mediated responses ob-served. We would like to again emphasize that the findings ofET-1-induced •OH generation and the observed changes invasoreactivity are two independent observations (tissue speci-mens vs. arterial rings on passive tension), which were madeunder different conditions. Therefore, ET-1-derived •OH gen-eration is unlikely to be the sole mechanistic link for theobserved changes in vasoreactivity.

Perspective

The present study demonstrates novel vascular effects andinteractions between ET-1 and ROS/•OH under normal condi-tions and in states of obesity, in particular. Furthermore, thedata presented here suggest possible “protective” vasodilatoractivities of ROS/•OH, which are altered in obesity. Thepossible pathophysiologicial significance of these observationsin human obesity and/or insulin resistance remains yet to beshown. However, the activation of direct and indirect ROS-mediated vasodilator mechanisms may explain the clinicalobservation that only some, but not all, obese patients develophypertension.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the expert technical assistance ofEmerita Ammann and thank Senju, Osaka, Japan for providing EPC-K1.

GRANTS

This work was supported by the Swiss National Science Foundation(SCORE 3200-058426.99, 3232-058421.99, and 3200-108258/1), and theHanne Liebermann Stiftung, Zurich.

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