pentaerythritol tetranitrate improves angiotensin ii ... · tetranitrate. improvement of vascular...

DaiberFalk Mäthner, Aruni Bhatnagar, Ulrich Förstermann, Huige Li, Thomas Münzel and AndreasKamuf, Tommaso Gori, Thomas Jansen, Maike Knorr, Susanne Karbach, Marcus Hortmann, Swenja Schuhmacher, Philip Wenzel, Eberhard Schulz, Matthias Oelze, Christian Mang, Jens

Induction of Heme Oxygenase-1Induced Vascular Dysfunction via−Pentaerythritol Tetranitrate Improves Angiotensin II

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 2010 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension doi: 10.1161/HYPERTENSIONAHA.109.1495422010;55:897-904; originally published online February 15, 2010;Hypertension.

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Pentaerythritol Tetranitrate Improves AngiotensinII–Induced Vascular Dysfunction via Induction of Heme

Oxygenase-1Swenja Schuhmacher, Philip Wenzel, Eberhard Schulz, Matthias Oelze, Christian Mang, Jens Kamuf,Tommaso Gori, Thomas Jansen, Maike Knorr, Susanne Karbach, Marcus Hortmann, Falk Mathner,

Aruni Bhatnagar, Ulrich Forstermann, Huige Li, Thomas Munzel, Andreas Daiber

Abstract—The organic nitrate pentaerythritol tetranitrate is devoid of nitrate tolerance, which has been attributed to theinduction of the antioxidant enzyme heme oxygenase (HO)-1. With the present study, we tested whether chronictreatment with pentaerythritol tetranitrate can improve angiotensin II–induced vascular oxidative stress and dysfunction.In contrast to isosorbide-5 mononitrate (75 mg/kg per day for 7 days), treatment with pentaerythritol tetranitrate (15mg/kg per day for 7 days) improved the impaired endothelial and smooth muscle function and normalized vascular andcardiac reactive oxygen species production (mitochondria, NADPH oxidase activity, and uncoupled endothelial NOsynthase), as assessed by dihydroethidine staining, lucigenin-enhanced chemiluminescence, and quantification ofdihydroethidine oxidation products in angiotensin II (1 mg/kg per day for 7 days)–treated rats. The antioxidant featuresof pentaerythritol tetranitrate were recapitulated in spontaneously hypertensive rats. In addition to an increase in HO-1protein expression, pentaerythritol tetranitrate but not isosorbide-5 mononitrate normalized vascular reactive oxygenspecies formation and augmented aortic protein levels of the tetrahydrobiopterin-synthesizing enzymes GTP-cyclohydrolase I and dihydrofolate reductase in angiotensin II–treated rats, thereby preventing endothelial NO synthaseuncoupling. Haploinsufficiency of HO-1 completely abolished the beneficial effects of pentaerythritol tetranitrate inangiotensin II–treated mice, whereas HO-1 induction by hemin (25 mg/kg) mimicked the effect of pentaerythritoltetranitrate. Improvement of vascular function in this particular model of arterial hypertension by pentaerythritoltetranitrate largely depends on the induction of the antioxidant enzyme HO-1 and identifies pentaerythritol tetranitrate,in contrast to isosorbide-5 mononitrate, as an organic nitrate able to improve rather than to worsen endothe-lial function. (Hypertension. 2010;55:897-904.)

Key Words: pentaerythritol tetranitrate � isosorbide-5-mononitrate � angiotensin-II � SHR� endothelial dysfunction � vascular oxidative stress

Both arterial hypertension and coronary artery disease areassociated with an activation of the circulating and local

renin-angiotensin system and increased oxidative stresswithin the vascular wall.1,2 Angiotensin-II (AT-II) treatmenthas been shown to cause endothelial dysfunction, which is atleast in part mediated by increased vascular reactive oxygenspecies (ROS) levels.3,4 ROS sources involved may includethe NADPH oxidases,3 an uncoupled endothelial NOsynthase (NOS; eNOS),4 and mitochondrial superoxidesources.5 The crucial role of the NADPH oxidase as animportant superoxide source was further substantiated by thedemonstration that NADPH oxidase 1 overexpression intransgenic mice potentiates AT-II–induced hypertension,6

whereas blood pressure responses to AT-II were reduced in

NADPH oxidase 1–deficient mice.7 Increased vascular ROSproduction and endothelial dysfunction are also accompaniedby increased eNOS expression but decreased vascular NOproduction.8 Recently, we were able to demonstrate thatpharmacological intervention with a statin or an AT-II type 1receptor blocker improved vascular dysfunction and reducedoxidative stress in an experimental model of diabetes melli-tus9,10 and identified the downregulation of tetrahydrobiop-terin (BH4) synthesizing enzymes GTP-cyclohydrolase-I(GCH-I) and dihydrofolate reductase (DHFR) as key eventsfor the development of endothelial dysfunction.8

Organic nitrates act as endothelium-independent vasodila-tors of coronary arteries, venous capacity vessels, and collat-erals. Nitroglycerin (GTN) is one of the most widely used

Received December 24, 2009; first decision December 31, 2009; revision accepted January 19, 2010.From the Johannes Gutenberg University Hospital (S.S., P.W., E.S., M.O., J.K., T.G., T.J., M.K., S.K., F.M., T.M., A.D.), 2nd Medical Clinic,

Molecular Cardiology, Mainz, Germany; Institut fur Pharmakologie (C.M., M.H., U.F., H.L.), Johannes Gutenberg-Universitat Mainz, Mainz, Germany;Experimental Research Laboratory (A.B.), Division of Cardiology, University of Louisville and Jewish Hospital Heart and Lung Institute, Louisville, Ky.

S.S. and P.W. and T.M. and A.D. contributed equally to this study and should therefore be considered as joint first and senior authors.Correspondence to Andreas Daiber, Universitatsmedizin der Johannes Gutenberg Universitat, II. Med Klinik u Poliklinik-Labor fur Molekulare

Kardiologie, Obere Zahlbacher Str. 63, 55101 Mainz, Germany. E-mail [email protected]© 2010 American Heart Association, Inc.

Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.109.149542

897 by guest on August 10, 2013http://hyper.ahajournals.org/Downloaded from


anti-ischemic drugs for more than a century. The chronicefficacy of nitrates, however, is blunted because of adverseeffects, such as the development of nitrate tolerance andendothelial dysfunction. Recent data indicate that GTN-induced ROS formation accounts for both phenomena, asROS formed in response to GTN therapy because bothphenomena can be corrected by treatment with antioxi-dants.11,12 Treatment with mononitrates and dinitrates alsocauses nitrate tolerance and endothelial dysfunction,13 al-though both compounds are clearly not bioactivated by themitochondrial aldehyde dehydrogenase 2.14 These findingsmay explain results from a retrospective analysis indicatingincreased mortality in response to treatment of patients withmyocardial infarction with mononitrates and dinitrates.15

Among all of the organic nitrates, the most frequently usedcompounds in the treatment of coronary artery disease arecomposed of GTN, pentaerythritol tetranitrate (PETN; inGermany), isosorbide dinitrate, and isosorbide-5 mononitrate(ISMN; United States).

More recently, we and others were able to demonstrate thatdifferent organic nitrates have distinct pharmacological ef-fects with respect to their bioactivating process and theircapacity to stimulate vascular superoxide production. Forinstance, we demonstrated that the nitrate PETN, while stillundergoing mitochondrial activation by the aldehyde dehy-drogenase 2,12 does not induce tolerance or stimulate vascularsuperoxide production.12 Although GTN caused a strong oxida-tive stress–mediated inhibition of the mitochondrial aldehydedehydrogenase 2, PETN induced an upregulation of the anti-oxidant enzyme heme oxygenase (HO)-1, which preservedaldehyde dehydrogenase 2 activity. These observations fromexperimental animal studies were substantiated by clinical dataobtained in humans indicating that, in contrast to GTN, PETNdoes not induce tolerance16 or endothelial dysfunction.17

On the basis of these considerations, we investigated in theAT-II infusion model and in spontaneously hypertensive rats(SHR) to what extent different organic nitrates, such as PETNand ISMN, are able to modulate oxidative stress, endothelialdysfunction, and tolerance development.

Methods and MaterialsMaterialsPETN was obtained from Actavis. GTN was used from a Nitrolin-gual infusion solution (Pohl-Boskamp). All of the other chemicalswere purchased from Sigma-Aldrich, Merck, or Fluka.

Animal Models, In Vivo Infusion of AT-II, andSpontaneously Hypertensive RatsAll of the animals were treated in accordance with the Guide for theCare and Use of Laboratory Animals as adopted by the NationalInstitutes of Health and were granted by the University HospitalMainz Ethics Committee. Male Wistar rats (250 g) were treated witheither AT-II (1.0 mg/kg per day) or solvent (0.9% NaCl) for 7 days,as described previously.18 Male spontaneously hypertensive rats(SHRs) and Wistar-Kyoto control rats were obtained from CharlesRiver Laboratories (Sulzfeld, Germany). Animals from both groupswere treated with either PETN (15 mg/kg per day) or ISMN (75mg/kg per day) versus vehicle (dimethyl sulfoxide [DMSO]). MaleHO-1�/� or HO-1�/� mice on a 129sv�BALB/c background weretreated with PETN (75 mg/kg per day; DMSO, 0.5 �L/h) for 7days,19 high-dose AT-II (1.0 mg/kg per day) versus solvent (0.9%NaCl) for 7 days with and without the HO-1 inducer hemin (25mg/kg single IP injection, 12 hours before being euthanized), orlow-dose AT-II (0.1 mg/kg per day) versus solvent (0.9% NaCl) withor without PETN (75 mg/kg per day) for 7 days. For details see theExtended Methods section in the online Data Supplement, availableat http://hyper.ahajournals.org.

Vascular Reactivity StudiesVasodilator responses to the endothelium-dependent vasodilatoracetylcholine (ACh) and the endothelium-independent vasodilatorGTN were determined in organ chambers by isometric tensionstudies using phenylephrine-preconstricted aortic ring segments, asdescribed previously.18 Murine aorta were preconstricted by prosta-glandin F2�, as published previously.19,20

Assessment of Vascular and CardiacOxidative StressVascular and cardiac oxidative stress were assessed by L-012 (aluminol derivative) or lucigenin-enhanced chemiluminescence(ECL) and dihydroethidine (DHE)-dependent fluorescence, as de-scribed elsewhere.10,14,18,21 For details see the Extended Methodssection in the online Data Supplement. For determination of cardiacROS in mice, 2 to 3 hearts were pooled and mitochondria andmembrane fractions were isolated as published recently.11,19

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Figure 1. Effects of in vivo PETN (15 mg/kg per day) and ISMN (75 mg/kg per day) treatment on the concentration response relation-ship to ACh (A) and GTN (B) in aortic rings from AT-II (1 mg/kg per day for 7 days) rats. C, The effects of sepiapterin (100 �mol/L), aBH4 precursor, and polyethylene glycolated-superoxide dismutase (100 U/mL) pretreatment of aortic rings from AT-II–infused rats for 1hour were determined in separate experiments. Data are the mean�SEM of n�36 to 57 aorta from 10 to 15 rats per group (A and B)and n�6 to 8 from 3 rats per group (C). P�0.05 vs *control/DMSO; vs #AT-II�PETN; vs $AT-II/BH4. The statistics were on the basis of1-way ANOVA comparison of pD2 values and efficacies (see Table S1) but also on comparisons of all concentrations in all groups by2-way ANOVA (for sake of clarity, significance is not shown for all of the data points).

898 Hypertension April 2010



Western Blot Analysis and RT-PCRWestern blotting against eNOS, GCH-1, DHFR, and HO-1 wasperformed as described previously.10,21 mRNA expression of HO-1and ferritin (heavy chain) was analyzed with quantitative real-timeRT-PCR using an iCycler iQ system (Bio-Rad Laboratories). Taq-Man Gene Expression assays (Applied Biosystems) for HO-1 andGAPDH were purchased as probe and primer sets, and geneexpression was normalized to the endogenous control GAPDHmRNA as described.21 For details see the Extended Methods sectionin the online Data Supplement.

Statistical AnalysisResults are expressed as mean�SEM. pD2 values (potencies) wereobtained by logit transformation. One-way ANOVA (with Bonfer-roni or Dunn correction for comparison of multiple means) was usedfor comparisons of vasodilator potency and efficacy and vascular andcardiac ROS formation and aortic protein and mRNA expression.P values �0.05 were considered significant.

ResultsEffects of PETN and ISMN Cotherapy onAT-II–Induced Vascular DysfunctionWeight gain in solvent-treated control animals was 50�6 g in1 week, whereas AT-II treatment caused weight loss of61�16 g. Weight loss in AT-II–treated animals was signifi-cantly improved in hypertensive rats by PETN cotherapy(3�17 g), whereas ISMN treatment had no significant effect(�13�18 g). In vivo treatment with PETN rather improvedendothelial dysfunction in AT-II–treated animals (Figure 1Aand Table S1 in the online Data Supplement), whereas ISMNfurther impaired AT-II–induced endothelial dysfunction.AT-II infusion–induced cross-tolerance to GTN was cor-rected by PETN cotreatment but not by ISMN (Figure 1B andTable S1). Treatment of vessels from AT-II–infused rats withthe BH4 precursor sepiapterin normalized endothelial func-tion (Figure 1C). Sensitivity of aorta to vasoconstriction byKCl and phenylephrine was not changed (Table S2). Bloodpressure data are presented in Figure S1 and show thatAT-II–infused rats and SHRs are valid models of experimen-tal hypertension. The beneficial effects of PETN and sepiap-terin on vascular function were almost absent in SHRs, butISMN further impaired this parameter (Figure S2).

Effects of PETN and ISMN Cotreatment onVascular and Cardiac ROS Production, as Well aseNOS Uncoupling in AT-II–Induced HypertensionDHE staining (fluorescent microtopography) demonstratedincreased vascular superoxide throughout the vessel wall andthe endothelium in vessel cryosections from AT-II–treatedanimals compared with controls (Figure 2A and 2B). Al-though vascular superoxide in hypertensive rats was notmodified by ISMN treatment, a marked reduction was ob-served under PETN therapy (Figure 2A and 2B). LucigeninECL in intact aortic ring segments yielded qualitativelysimilar results (Figure 2B). AT-II–stimulated mitochondrialROS formation and NADPH oxidase activity in tissue fromthe heart were normalized by PETN but not by ISMNtreatment (Figure 3A through 3C).

To assess the contribution of an uncoupled eNOS tosuperoxide formation because of eNOS uncoupling, rat aortictissue was incubated with an NOS inhibitor, NG-nitro-L-

arginine methylester (L-NAME). L-NAME increased DHE-derived fluorescence within the endothelial monolayer fromcontrol rats (marked with “E” in Figure 3D), whereas thesignal in the media was not changed. Inhibition of NOS incontrol aorta eliminates basal NO production, leading tohigher superoxide steady-state levels (which is otherwisescavenged by NO). In contrast, NOS inhibition in vesselsfrom AT-II–treated rats with L-NAME decreased DHE-derived fluorescence exclusively within the endothelium,thereby identifying eNOS as a significant source of superox-ide. L-NAME increased the endothelial signal in vesselsections from AT-II/PETN-treated rats (like in the controlgroup), indicating that PETN treatment was able to preventeNOS uncoupling (Figure 3D and 3E). The AT-II/ISMNgroup displayed no difference compared with the AT-IIgroup, indicating that eNOS uncoupling was not prevented byISMN cotherapy. Similar beneficial effects of PETN but notISMN were observed in SHRs (Figure S3).

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Figure 2. Effects of in vivo PETN and ISMN treatment on vascu-lar superoxide levels (A and B) in AT-II rats. A, Transverse aorticcryosections were labeled with DHE (1 �mol/L), which producesred fluorescence when oxidized by ROS. Lamina autofluores-cence is green. Pictures shown are representative for �6 ani-mals per group. E indicates endothelium. B, Densitometricquantification of the DHE-derived ROS signal throughout thevessel wall (left) and lucigenin (5 �mol/L)-ECL in intact aorticring segments (right). The data are mean�SEM of n�18 to 19experiments with tissue from �10 animals per group. P�0.05:vs *control/DMSO; vs #AT-II�PETN. C indicates control; A, AT-IItreated; P, AT-II and PETN treated; I, AT-II and ISMN treated.

Schuhmacher et al PETN Improves Experimental Hypertension 899



Effects of PETN and ISMN Cotreatment onVascular Expression of eNOS, BH4 SynthesizingEnzymes, and the Antioxidative Principle HO-1AT-II treatment has been shown to increase aortic HO-1 geneexpression at the mRNA and protein levels,22 which wasfurther increased by in vivo PETN treatment but decreased byISMN cotherapy (Figure 4A and 4B). As before, we found asignificant increase in the expression of eNOS in AT-II-treated rats, which was normalized by neither PETN norISMN (Figure 4C). It is important to note, however, that, inresponse to PETN treatment, eNOS was recoupled by PETN,whereas, in response to ISMN, the expression of an uncou-pled eNOS was increased. AT-II infusion tended to decreasethe expression of GCH-I and significantly decreased thelevels of DHFR, another important enzyme for synthesis ofBH4 (the so-called rescue pathway for BH2 (dihydrobiopterin)recycling; Figure 4D and 4E). PETN cotreatment significantly

increased expression of both BH4 synthesizing enzymes even tohigher levels as compared with the control identifying anotherimportant property of PETN, that is, how eNOS recoupling wasachieved. The effects on BH4 synthase and BH2 (dihydrobiop-terin) reductase were not shared by ISMN.

The role of HO-1 as the antioxidative principle of PETNwas elucidated by 3 key experiments aiming to prove thishypothesis. The first experimental setup consisted of thetreatment of control (HO-1�/�) and partially deficient (HO-1�/�) mice with PETN. In HO-1�/� but not HO-1�/� mice,PETN infusion induced tolerance against itself, envisaged byimpaired vasodilator potency of the drug and increasedmitochondrial ROS formation (Figure 5A and 5B). Thesecond approach was on the basis of HO-1 induction by theknown inducer of this enzyme, hemin, which improvedAT-II–dependent endothelial dysfunction and prevented ac-tivation of NADPH oxidase (Figure 5C and 5D). The third

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Figure 3. Effects of in vivo PETN and ISMN treatment on mitochondrial ROS formation (A and B), NADPH oxidase activity (A and C),and eNOS-dependent ROS formation (uncoupling; D and E) in AT-II rats. A, ROS formation in isolated cardiac mitochondria was mea-sured by L-012 (100 �mol/L) ECL (left), and ROS production (NADPH oxidase activity) in membrane fractions from hearts was deter-mined by lucigenin (5 �mol/L)-ECL (right). B and C, ROS formation in isolated cardiac mitochondria and NADPH oxidase activity inmembranous fractions were also quantified by 2-hydroxyethidium levels. The inserts show representative high-performance liquid chro-matography chromatograms. E� indicates ethidium; 2-HE, 2-hydroxyethidium. The data are mean�SEM of n�31 to 43 (mitochondriaand NADPH oxidase activity) experiments with tissue from �10 animals per group. The high-performance liquid chromatography dataare mean�SEM of n�9 (mitochondria) and n�15 (NADPH oxidase activity) experiments with tissue from 3 to 5 animals per group. Dand E, Fluorescence microscopy revealed ROS formation by red staining (top column). To determine eNOS-dependent ROS formation,vessels were preincubated with the NOS inhibitor L-NAME (bottom column). Densitometric data are presented by bar graphs (solid,without L-NAME and open, with L-NAME). Pictures and data shown are representative for �4 animals per group. For methodologicaldetails see Figure S4 in the online Data Supplement. P�0.05: vs *control/DMSO; vs #AT-II�PETN. C indicates control; A, AT-II treated;P, AT-II and PETN treated; I, AT-II and ISMN treated.




experiment demonstrated that PETN did not improveendothelial dysfunction and cardiac oxidative stress inAT-II–treated HO-1�/� mice but further impaired vascularfunction and increased ROS formation in this setting(Figure 5E and 5F).

DiscussionThe present studies demonstrate that the organic nitratePETN but not ISMN improves vascular function and reducesoxidative stress via inhibition of vascular superoxide produc-tion in mitochondria and by inhibition of the vascularNADPH oxidase in an experimental model of AT-II–inducedhypertension. In almost all of the animal models whereendothelial dysfunction is encountered, such as atherosclero-sis,23 chronic congestive heart failure,24 AT-II–induced hy-pertension,4 and diabetes mellitus,25 we established thatincreased production of ROS via activation of the vascularNADPH oxidase and xanthine oxidase contributed consider-ably to this phenomenon. Interestingly, in all of the models,eNOS expression was upregulated rather than downregulated,suggesting that eNOS might be dysfunctional, as uncoupledunder these circumstances.4,23–25

Previously, it was conceptualized that treatment with anexogenous source of NO (eg, GTN) could compensate for thediminished endothelial NO availability in atherosclerosis,thereby preventing the consequences of endothelial dysfunc-tion, such as enhanced constriction and increased plateletaggregation. Theoretically, however, NO rapidly reacts withsuperoxide to produce the highly reactive intermediate, per-

oxynitrite, a potent oxidant that has been demonstrated tocause vascular (endothelial) dysfunction by inhibiting pros-tacyclin activity26 and by causing eNOS uncoupling viaoxidation of the important eNOS cofactor BH4.27 Indeed,treatment of atherosclerotic animals with GTN worsenedrather than improved endothelial function, caused consump-tion of plasma antioxidants such as �- and �-carotene,decreased extracellular superoxide dismutase activity, and ledto a dramatic increase in vascular protein tyrosine nitration asa footprint of peroxynitrite formation under GTN therapy.28

In contrast to GTN, PETN is capable of upregulating theimportant antioxidant enzyme HO-I, which has been shownto play a key role for the prevention of nitrate tolerance andendothelial dysfunction under chronic GTN therapy.21

It remained to be established, however, whether PETN, anitrate with antioxidant properties, is able to improve endo-thelial dysfunction in an animal model of endothelial dys-function and oxidative stress. To address this issue, we usedthe model of AT-II infusion, in which the superoxide-producing enzymes are well characterized. For comparison,AT-II–treated animals were treated with the mononitrateISMN. As before, infusion of AT-II led to a marked degree ofendothelial dysfunction, as well as an attenuation of theendothelium-independent nitrovasodilator GTN associatedwith increased oxidative stress in vascular tissue. As super-oxide sources, the NADPH oxidase, the mitochondria, and anuncoupled NOS were identified.

Experiments with isolated mitochondria and membranefractions from the heart revealed that PETN and not ISMN

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Figure 4. Effects of in vivo PETN and ISMN treatment on vascular HO-1 (mRNA and protein), eNOS, GCH-I, and DHFR expression inaortic tissue from hypertensive rats. Expressions of HO-1 mRNA (A) and HO-1 (B), eNOS (C), GCH-I (D), and DHFR (E) protein wereassessed by RT-PCR and Western blot analysis, respectively. Representative blots are shown at the bottom of the densitometric bargraphs. The data are mean�SEM of aortic rings from 6 to 8 animals per group. P�0.05: vs *control/DMSO; vs #AT-II�PETN. C indi-cates control; A, AT-II treated; P, AT-II and PETN treated; I, AT-II and ISMN treated.




treatment significantly reduced mitochondrial ROS produc-tion and to inhibit NADPH oxidase activity. Previously wehave proposed that superoxide production by the vascularNADPH oxidase and mitochondria might represent so-called“kindling radicals,” which may react with NO to formperoxynitrite.25 This intermediate in turn oxidizes BH4 to theBH3

� radical, thereby causing superoxide production byeNOS, the so called “bonfire” radical.27 Thus, all of themeasures that successfully reduce vascular superoxide pro-duction via inhibition of the NADPH oxidase should lead toa prevention of eNOS uncoupling. Indeed, as indicated by theDHE experiments with L-NAME, PETN but not ISMNprevented eNOS uncoupling in this model of oxidative stress.The observed reduction of mitochondrial ROS formationcould be a direct consequence of decreased NADPH oxidaseactivity, because it was demonstrated recently that GTN-induced increases in NADPH oxidase activity can triggermitochondrial ROS formation via KATP (ATP-sensitive po-tassium) channels.11

Downregulation of the BH4 synthesizing enzyme DHFRhas been proposed to contribute substantially to endothelialdysfunction and eNOS uncoupling in the AT-II infusionmodel.29 In 2 recent studies with diabetic rats, we were able

to demonstrate that the prevention of eNOS uncoupling inresponse to atorvastatin or the Ang II type 1 receptor blockertelmisartan was at least in part secondary to an upregulationof BH4 synthesizing enzyme, such as the GCH-I or theDHFR.9,10 With the present studies, we established that PETNand not ISMN was able to substantially upregulate not onlyDHFR but also GCH-I, which may also contribute consider-ably to the recoupling of the enzyme.

As mentioned before, eNOS protein was upregulated ratherthan downregulated in this animal model of hypertension. It isimportant to note that this is likely attributed to increasedH2O2 production, which has been shown previously toincrease eNOS expression at the transcriptional and transla-tional levels.30 Thus, a reduction in vascular oxidative stressshould always result in a normalization of eNOS under thesecircumstances. With the present studies we were able to showthat AT-II–upregulated eNOS was not modified by PETN orISMN treatment. The fact, however, that, in response toPETN but not ISMN treatment, eNOS was recoupled explainswhy PETN treatment and not ISMN treatment improvedendothelial dysfunction.

In one of our recent articles we identified the antioxidantenzyme HO-1 as the key player determining whether an

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Figure 5. Effects of HO-1 deficiency vs HO-1 induction on vascular improvement by PETN. A and B, PETN treatment (75 mg/kg perday for 4 days) had no effect on PETN potency (PETN-induced relaxation) in aorta from control mice (HO-1�/�) but caused nitrate toler-ance in aorta from mice with partial HO-1 deficiency (HO-1�/�). In accordance, cardiac mitochondrial ROS formation (L-012 ECL) wasincreased in PETN-treated HO-1�/� mice. P�0.05: vs *HO-1�/�/DMSO; vs #HO-1�/�/DMSO. S indicates solvent; P, PETN treated. Cand D, Hemin (25 mg/kg IP)-triggered HO-1 induction improved high-dose AT-II (1 mg/kg per day for 7 days)-induced endothelial dys-function (ACh response) in aorta and NADPH oxidase activity in heart (lucigenin-ECL) from control mice (HO-1�/�). P�0.05: vs *AT-II-treated HO-1�/�/DMSO. E and F, PETN (75 mg/kg per day for 7 days) failed to prevent endothelial dysfunction (ACh response) inducedby low-dose AT-II (0.1 mg/kg per day for 7 days) in aorta from HO-1�/� mice. In accordance, PETN did not improve NADPH oxidaseactivity (2-HE formation by high-performance liquid chromatography analysis) in cardiac samples from AT-II–treated HO-1�/� mice.P�0.05: vs *AT-II–treated HO-1�/�/DMSO. All of the data are mean�SEM of aortic rings and hearts from 4 to 5 animals per group.




organic nitrate causes endothelial dysfunction and nitratetolerance.21 HO-1 exerts its beneficial effects on vascularfunction via formation of the sGC stimulator carbon monox-ide, the antioxidant bilirubin, and the chelator protein of freeiron, ferritin.31 Likewise, with the present studies we canshow that AT-II upregulated HO-1 expression at the mRNAand protein levels, which was further stimulated by PETN butnot by ISMN treatment. The results by using HO-1-deficientmice (HO-1�/�) clearly demonstrate that HO-1 largely con-tributes to the pleiotropic protective properties of PETN,because the beneficial effects of PETN on endothelial dys-function in the AT-II hypertension model were almost com-pletely abolished by heterozygous HO-1 deficiency. Other-wise, induction of HO-1 by hemin was able to preventvascular dysfunction by high-dose AT-II treatment of controlmice (HO-1�/�).

To address whether similar effects can be observed in agenetically determined model of hypertension, SHRs werestudied. The results confirm those obtained from the AT-IIinfusion model. PETN but not ISMN treatment markedlyreduced vascular superoxide production, as quantified byDHE staining and by lucigenin-ECL. The effects of PETN onvascular function in SHRs were significantly different fromthose of ISMN although less pronounced as compared withthe effects in AT-II–triggered hypertension. For detailedconsideration of these differences see the Extended Resultssection in the online Data Supplement. Therefore, the obser-vations on the beneficial effects of PETN in both animalmodels of arterial hypertension are in accordance with pre-vious studies, indicating that PETN improves experimentalatherosclerosis in rabbits.32,33 It should be noted that theseauthors have also reported on antiatherosclerotic effects ofISMN and improvement of endothelial dysfunction by thisdrug,34,35 which, however, is at variance with our presentobservations and a human study indicating detrimental effectsof ISMN on endothelial function in healthy volunteers.13

PerspectivesThe results of the present studies show for the first time that,in an animal model of oxidative stress, the organic nitratePETN but not the mononitrate ISMN is able to improveendothelial dysfunction. PETN substantially inhibitedNADPH oxidase activity, inhibited mitochondrial superoxideproduction, and prevented eNOS uncoupling. The recouplingof eNOS may be a consequence of a reduction of oxidativestress secondary to upregulated HO-1 protein but may also beattributed to PETN-mediated upregulation of the key en-zymes for BH4 synthesis, such as the GCH-I and the DHFR.These preclinical studies contribute to the understanding ofwhy PETN treatment does not cause tolerance or endothelialdysfunction. The spectrum of antioxidant features of thiscompound indicates that PETN may not be used only for thetreatment of symptomatic coronary artery disease but also tobeneficially influence the progression of the atheroscleroticprocess.

AcknowledgmentsWe appreciate the expert technical support by Jorg Schreiner andNicole Schramm. This study contains parts of the thesis work of JensKamuf.

Sources of FundingThis study was supported by a vascular research grant fromActavis Deutschland GmbH (to T.M. and A.D.) and the GermanHeart Foundation/German Foundation of Heart Research (F/39/08; to S.S. and P.W.).

DisclosuresA.D. and T.M. received a vascular research grant and honoraria fromActavis Deutschland GmbH (Langenfeld, Germany). T.G. and T.M.received honoraria from Actavis Deutschland GmbH (Langenfeld,Germany).

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11. Wenzel P, Mollnau H, Oelze M, Schulz E, Wickramanayake JM, MullerJ, Schuhmacher S, Hortmann M, Baldus S, Gori T, Brandes RP, MunzelT, Daiber A. First evidence for a crosstalk between mitochondrial andNADPH oxidase-derived reactive oxygen species in nitroglycerin-triggered vascular dysfunction. Antioxid Redox Signal. 2008;10:1435–1447.

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Schumacher et al. – PETN improves experimental hypertension – online supplement 1

Online Data Supplement

Pentaerithrityl tetranitrate improves angiotensin II induced vascular dysfunction via induction of heme oxygenase-1

Swenja Schuhmacher*1, Philip Wenzel*1, Eberhard Schulz1, Matthias Oelze1, Christian

Mang2, Jens Kamuf1, Tommaso Gori1, Thomas Jansen1, Maike Knorr1, Susanne Karbach1, Marcus Hortmann2, Falk Mäthner1, Aruni Bhatnagar3, Ulrich Förstermann2, Huige Li2,

Thomas Münzel1, and Andreas Daiber†1




Extended Methods

Animal models, in vivo infusion of angiotensin-II and SHR All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the U.S. National Institutes of Health and was granted by the Ethics Committee of the University Hospital Mainz. Male Wistar rats (60 in total; weight 250g; Charles River, Sulzfeld, Germany) were anesthetized by isoflurane inhalation and treated with a subcutaneous osmotic minipump (Durect Corp., Cupertino, CA 95014) containing either AT-II (1.0mg/kg/d) or solvent (0.9 % NaCl) for 7d, as described previously 1. Male SHR (20 in total; 6 months of age) and Wistar-Kyoto control rats were obtained from Charles River Laboratories (Sulzfeld, Germany). Animals from both groups were randomized to receive either PETN (15mg/kg/d), ISMN (75mg/kg/d) or vehicle (DMSO) via an additional subcutaneous osmotic minipump. After 7d the rats were sacrificed under isoflurane anesthesia. Male HO-1+/+ or HO-1+/- mice (3-4 months old) on a 129sv x BALB/c mixed genetic background as described 2, were treated with a subcutaneous osmotic minipump containing either PETN (75mg/kg/d) or solvent (DMSO, 0.5µl/h) for 7d 3. The mice were generated as previously reported. Male HO-1+/+ were also treated with high dose AT-II (1.0mg/kg/d) or solvent (0.9 % NaCl) for 7d and the HO-1 inducer hemin (25mg/kg single i.p. injection, 12h prior to sacrifice). Male HO-1+/- were also treated with low dose AT-II (0.1mg/kg/d) or solvent (0.9 % NaCl) and co-treated with either PETN (75mg/kg/d) or solvent (DMSO, 0.5µl/h) for 7d. Determination of blood pressure Systolic blood pressure was obtained on a weekly basis in isoflurane anesthetized rats using a tail cuff non-invasive blood pressure system coupled to a PowerLab system (ML125 NIBP, ADInstruments, Colorado Springs, CO). A minimum of three measurements were obtained from each rat. We used a protocol that was previously published 4. Western Blot analysis and RT-PCR Isolated aortic tissue was frozen and homogenized in liquid nitrogen. Proteins were separated by SDS-Page and blotted onto nitrocellulose membranes. After blocking, immunoblotting was performed with antibodies against α-actinin (100kDa) or actin (42kDa) (1:2500, Sigma-Aldrich) as controls for loading and transfer, eNOS (1:1000, BD Biosciences, USA), GTP-cyclohydrolase-1 (GCH-1: 1µg/ml, Abnova Corp., Germany), dihydrofolate reductase (DHFR: 1µg/ml, RDI Div. of Fitzgerald Ind., USA) 5 and HO-1 (1:5000, monoclonal, Stressgen, San Diego, CA) 6. Detection was performed by ECL with peroxidase conjugated anti–rabbit/mouse (1:10000, Vector Lab., Burlingame, CA) and anti-goat (1:5000, Santa Cruz Biotechnologies, USA) secondary antibodies. The antibody-specific bands were quantified by densitometry as described. mRNA expression of HO-1 and ferritin (heavy-chain) was analyzed with quantitative real-time RT-PCR using an iCyclerTM iQ system (Bio-Rad Laboratories, Munich, Germany). TaqMan® Gene Expression assays (Applied Biosystems, Foster City, CA) for HO-1 and GAPDH were purchased as probe and primer sets and gene expression was normalized to the endogenous control, GAPDH mRNA as described6. Assessment of vascular and cardiac oxidative stress Mitochondria were isolated from heart and mitochondrial ROS formation was detected by L-012 (100µM)-enhanced chemiluminescence (ECL) in the presence of succinate (5mM) as previously described 7. Membrane fractions were isolated from heart and NADPH oxidase activity was determined by lucigenin (5µM) ECL in the presence of NADPH (200µM) according to a previous protocol. Vascular ROS formation was detected in intact aortic ring




segments (0.5cm length) by lucigenin (5µM) ECL and dihydroethidine (1µM)-dependent fluorescence in aortic cryo-sections (fluorescent microtopography) as reported elsewhere8. The functional state of eNOS (coupled or uncoupled) was estimated from dihydroethidine-treated aortic cryo-sections in the presence and absence of the NOS inhibitor L-NAME (0.5mM) by fluorescence microscopy as described 8. Mitochondrial ROS production and NADPH oxidase activity were determined by HPLC-based quantification of the conversion of dihydroethidine to 2-hydroxyethidium as described 9.

Extended Results

Effects of PETN and ISMN co-treatment on vascular function and ROS production in SHR rats Spontaneously hypertensive rats (SHR) had significantly higher levels of aortic ROS formation as compared to corresponding controls (Wistar-Kyoto rats), which was demonstrated by dihydroethidine-dependent fluorescent microtopography in aortic cryo-sections as well as lucigenin ECL in intact aortic ring segments (Figure S5 A-C). Both methods revealed a significant improvement of vascular ROS formation in SHR by PETN but not ISMN therapy. SHR rats had significantly impaired endothelial and smooth muscle function as compared to WKY controls (Figure S2). The effects of PETN on endothelium-dependent (ACh) and –independent (GTN) relaxation in SHR were not as pronounced as those on vascular oxidative stress but still reflected the tendency observed in the ROS determination assays (compare Figure S2 versus S3). Although the improvement of endothelial and smooth muscle function was not significant under PETN therapy, the impairment under ISMN was significant as compared to the SHR+PETN group (Figure S2). These rather small effects of PETN therapy on vascular function may be attributed to the experimental setup. We started the organic nitrate therapy in 6 months old SHR rats. These animals already developed severe hypertension (Figure S1) and vascular remodeling may interfere with pronounced effects of cardiovascular therapeutics as previously observed for statin treatment of SHR 10. Most studies on antihypertensive therapy in SHR rats start in young animals, in the prehypertensive state to prevent vascular remodeling and development of hypertension and its adverse effects 11, 12. Therefore, SHR may not represent the best experimental model of hypertension to study improvement of vascular dysfunction by cardiovascular therapeutics. This consideration is further supported by the weak effect of BH4/SOD pretreatment on endothelial dysfunction in aorta from SHR as compared to the significant beneficial effect of the eNOS cofactor on endothelial dysfunction in aorta from AT-II-infused rats (compare Figure S2 versus Figure 1 in the main manuscript). Obviously, endothelial dysfunction in SHR is rather not dependent on eNOS dysfunction but on structural changes in the vascular wall. However, in a recent study Dovinova et al. have demonstrated that endothelial function of aorta from SHR was improved by treatment with PETN for 6 weeks 13. It should be noted that these authors used a 6.5-fold higher dose of PETN (100 mg/kg/d) than used in the present study and treatment was maintained for 6 weeks instead of 1 week. Effects of PETN and ISMN co-treatment on renal salt handling in SHR and AT-II infused rats As known from Dahl salt-sensitive rats, renal salt handling may largely affect blood pressure. Also in SHR, effects of high salt diet on blood pressure have been observed 14. Since there is




no literature available on the effects of organic nitrates on renal salt handling, we cannot exclude that changes in renal salt handling may contribute to the beneficial effects observed for PETN and likely to the adverse effects of ISMN. This assumption is supported by modulation of renal salt handling by nitric oxide 15.

Extended References

1. Oelze M, Daiber A, Brandes RP, Hortmann M, Wenzel P, Hink U, Schulz E, Mollnau H, von Sandersleben A, Kleschyov AL, Mulsch A, Li H, Forstermann U, Munzel T. Nebivolol inhibits superoxide formation by nadph oxidase and endothelial dysfunction in angiotensin ii-treated rats. Hypertension. 2006;48:677-684

2. Yet SF, Perrella MA, Layne MD, Hsieh CM, Maemura K, Kobzik L, Wiesel P, Christou H, Kourembanas S, Lee ME. Hypoxia induces severe right ventricular dilatation and infarction in heme oxygenase-1 null mice. J Clin Invest. 1999;103:R23-29

3. Mollnau H, Wenzel P, Oelze M, Treiber N, Pautz A, Schulz E, Schuhmacher S, Reifenberg K, Stalleicken D, Scharffetter-Kochanek K, Kleinert H, Munzel T, Daiber A. Mitochondrial oxidative stress and nitrate tolerance--comparison of nitroglycerin and pentaerithrityl tetranitrate in mn-sod+/- mice. BMC Cardiovasc Disord. 2006;6:44

4. Daugherty A, Manning MW, Cassis LA. Angiotensin ii promotes atherosclerotic lesions and aneurysms in apolipoprotein e-deficient mice. J Clin Invest. 2000;105:1605-1612

5. Wenzel P, Schulz E, Oelze M, Muller J, Schuhmacher S, Alhamdani MS, Debrezion J, Hortmann M, Reifenberg K, Fleming I, Munzel T, Daiber A. At1-receptor blockade by telmisartan upregulates gtp-cyclohydrolase i and protects enos in diabetic rats. Free Radic Biol Med. 2008;45:619-626

6. Wenzel P, Oelze M, Coldewey M, Hortmann M, Seeling A, Hink U, Mollnau H, Stalleicken D, Weiner H, Lehmann J, Li H, Forstermann U, Munzel T, Daiber A. Heme oxygenase-1: A novel key player in the development of tolerance in response to organic nitrates. Arterioscler Thromb Vasc Biol. 2007;27:1729-1735

7. Daiber A, August M, Baldus S, Wendt M, Oelze M, Sydow K, Kleschyov AL, Munzel T. Measurement of nad(p)h oxidase-derived superoxide with the luminol analogue l-012. Free Radic Biol Med. 2004;36:101-111

8. Wenzel P, Daiber A, Oelze M, Brandt M, Closs E, Xu J, Thum T, Bauersachs J, Ertl G, Zou MH, Forstermann U, Munzel T. Mechanisms underlying recoupling of enos by hmg-coa reductase inhibition in a rat model of streptozotocin-induced diabetes mellitus. Atherosclerosis. 2008;198:65-76

9. Wenzel P, Mollnau H, Oelze M, Schulz E, Wickramanayake JM, Muller J, Schuhmacher S, Hortmann M, Baldus S, Gori T, Brandes RP, Munzel T, Daiber A. First evidence for a crosstalk between mitochondrial and nadph oxidase-derived reactive oxygen species in nitroglycerin-triggered vascular dysfunction. Antioxid Redox Signal. 2008;10:1435-1447

10. Alvarez de Sotomayor M, Perez-Guerrero C, Herrera MD, Marhuenda E. Effects of chronic treatment with simvastatin on endothelial dysfunction in spontaneously hypertensive rats. J Hypertens. 1999;17:769-776




11. Baumann M, Hermans JJ, Janssen BJ, Peutz-Kootstra C, Witzke O, Heemann U, Smits JF, Boudier HA. Transient prehypertensive treatment in spontaneously hypertensive rats: A comparison of spironolactone and losartan regarding long-term blood pressure and target organ damage. J Hypertens. 2007;25:2504-2511

12. Baumann M, Megens R, Bartholome R, Dolff S, van Zandvoort MA, Smits JF, Struijker-Boudier HA, De Mey JG. Prehypertensive renin-angiotensin-aldosterone system blockade in spontaneously hypertensive rats ameliorates the loss of long-term vascular function. Hypertens Res. 2007;30:853-861

13. Dovinova I, Cacanyiova S, Faberova V, Kristek F. The effect of an no donor, pentaerythrityl tetranitrate, on biochemical, functional, and morphological attributes of cardiovascular system of spontaneously hypertensive rats. Gen Physiol Biophys. 2009;28:86-93

14. Wang C, Chao C, Chen LM, Chao L, Chao J. High-salt diet upregulates kininogen and downregulates tissue kallikrein expression in dahl-ss and shr rats. Am J Physiol. 1996;271:F824-830

15. Ikari A, Kano T, Suketa Y. Magnesium influx enhanced by nitric oxide in hypertensive rat proximal tubule cells. Biochem Biophys Res Commun. 2002;294:710-713

16. Alp NJ, Mussa S, Khoo J, Cai S, Guzik T, Jefferson A, Goh N, Rockett KA, Channon KM. Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic gtp-cyclohydrolase i overexpression. J Clin Invest. 2003;112:725-735




Table S1. Effects of treatment with PETN or ISMN on the potency and efficacy of endothelium-dependent (ACh) and -independent (GTN) vasodilators in isolated aortic segments from hypertensive rats.

Potency (pD2)§ Efficacy (%)

In vivo Treatment ACh GTN ACh GTN

Control + DMSO 7.30±0.05 (n=41) 7.66±0.04 (n=43) 90±1 99±0

AT-II + DMSO 6.79±0.07* (n=57) 7.24±0.06* (n=53) 58±3* 98±1

AT-II + PETN 6.93±0.06* (n=46) 7.51±0.06*† (n=50) 70±2† 99±0

AT-II + ISMN 6.54±0.07*†‡ (n=36) 7.16±0.06*‡ (n=34) 61±3*‡ 97±1*‡

* P<0.05 vs. control; † P<0.05 vs. AT-II; ‡ P<0.05 vs. AT-II + PETN. § Potency is –log EC50 and efficacy is defined as maximal relaxation obtained with the highest employed concentration of the vasodilator. n indicates the number of aortic rings per group.




Table S2. Effects of treatment with PETN or ISMN on sensitivity to vasoconstrictors in isolated aortic segments from hypertensive rats.

Induced Vasoconstriction [g]* In vivo Treatment KCl Phenylephrine

Control + DMSO 3.54±0.16 (n=30) 2.48±0.26 (n=29)

AT-II + DMSO 3.60±0.24 (n=28) 2.91±0.35 (n=30)

AT-II + PETN 3.41±0.24 (n=30) 2.78±0.27 (n=28)

AT-II + ISMN 3.25±0.24 (n=28) 2.77±0.23 (n=30) * 80 mM KCl and 300 nM phenylephrine. n indicates the number of aortic rings per group.




Figure S1. Left panel: Systolic blood pressure in control, AT-II-treated rats or SHR was measured by the tail cuff method. Data shown are the mean±SEM of at least 4 measurements. P < 0.05: * vs. Ctr. Right panel: Weight gain of rats in the different treatment groups (initial weight was 250g. Data shown are the mean±SEM of at least 8 measurements. P < 0.05: * vs. Ctr; + vs. AT-II.

Figure S2. Effects of in vivo pentaerithrityl tetranitrate (PETN) and isosorbide-5-mononitrate (ISMN) treatment on the vasoreactivity of aorta from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) controls. Endothelium-dependent (ACh, A) or –independent (GTN, B) vasodilation was assessed by isometric tension recording. (C) The effect of sepiapterin (100 µM), a BH4 precursor, and PEG-SOD (100 U/ml) preincubation of aortic rings from SHR for 1 h was determined in separate experiments. Data shown are representative for at least 4 animals/group. P < 0.05: * vs. WKY+DMSO; # vs. SHR+PETN. The statistics were based on 1-way-ANOVA comparison of pD2-values and efficacies but also on comparisons of all concentrations in all groups by 2-way-ANOVA analysis (for sake of clarity significance is not shown for all data points).




Figure S3. Effects of in vivo pentaerithrityl tetranitrate (PETN) and isosorbide-5-mononitrate (ISMN) treatment on the reactive oxygen species formation in aorta from spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) controls. Dihydroethidine (DHE, 1µM) staining of aortic cryo-sections (A), densitometric quantification of the DHE-derived reactive oxygen species signal throughout the vessel wall (B) and lucigenin (5µM) enhanced chemiluminescence (ECL) in intact aortic ring segments (C). Pictures and data shown are representative for 3-4 animals/group. P < 0.05: * vs. Ctr/DMSO; # vs. AT-II+PETN. W, Wistar-Kyoto controls; S, spontaneously hypertensive rats; P, PETN-treated SHRs; I, ISMN-treated SHRs.




Figure S4. Effects of PETN tratment on eNOS uncoupling. Effects of in vivo pentaerithrityl tetranitrate (PETN) and isosorbide-5-mononitrate (ISMN) treatment on eNOS-dependent reactive oxygen species formation (uncoupling) in aorta from hypertensive rats. Dihydroethidine (DHE, 1µM)-fluorescent microtopography was used to assess vascular reactive oxygen species formation in aortic cryo-sections which were incubated with dihydroethidine. (A) eNOS uncoupling was assessed by densitometric quantification of DHE staining in the endothelial cell layer which was extracted from the whole microscope image. (B) A fixed area was used for densitometric quantification and the procedure is shown for endothelial cell layer of AT-II (#2) image. eNOS uncoupling was previously assessed by the effects of L-NAME on DHE staining 1, 5. The method of densitometric quantification of endothelial DHE staining was adopted from the protocol of Alp et al. 16.

A

B



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