European Heart Journal Advance Access published May 3, 2012

European Heart Journal BASIC SCIENCE doi:10.1093/eurheartj/ehs100

Chronic therapy with isosorbide-5-mononitrate causes , , and a marked increase in vascular endothelin-1 expression

Matthias Oelze1†, Maike Knorr1,2†, Swenja Kro¨ ller-Scho¨ n1,2, Sabine Kossmann1, Downloaded from Anna Gottschlich1, Robert Ru¨mmler1,AlexandraSchuff1, Steffen Daub1, Christopher Doppler1, Hartmut Kleinert3, Tommaso Gori1, Andreas Daiber1†, and Thomas Mu¨nzel1*† http://eurheartj.oxfordjournals.org/

12nd Medical Clinic, Department of Cardiology, Medical Center of the Johannes Gutenberg University, 55131 Mainz, Germany; 2Center of Thrombosis and Hemostasis (CTH), Medical Center of the Johannes Gutenberg University, Mainz, Germany; and 3Department of Pharmacology, Medical Center of the Johannes Gutenberg University, Mainz, Germany

Received 10 December 2011; revised 8 March 2012; accepted 28 March 2012

Aims Isosorbide-5-mononitrate (ISMN) is one of the most frequently used compounds in the treatment of coronary artery disease predominantly in the USA. However, ISMN was reported to induce endothelial dysfunction, which was cor- rected by C pointing to a crucial role of reactive oxygen species (ROS) in causing this phenomenon. We

sought to elucidate the mechanism how ISMN causes endothelial dysfunction and oxidative stress in vascular tissue. at Universitaetsbibliothek Mainz on July 19, 2012 ...... Methods Male Wistar rats (n 69 in total) were treated with ISMN (75 mg/kg/day) or placebo for 7 days. Endothelin (ET) ¼ and results expression was determined by immunohistochemistry in aortic sections. Isosorbide-5-mononitrate infusion caused significant endothelial dysfunction but no tolerance to ISMN itself, whereas ROS formation and nicotinamide adenine dinucleotidephosphate (NADPH) oxidase activity in the aorta, heart, and whole blood were increased. Iso- sorbide-5-mononitrate up-regulated the expression of NADPH subunits and caused uncoupling of the endothelial synthase (eNOS) likely due to a down-regulation of the -synthesizing GTP-cyclohydrolase-1 and to S-glutathionylation of eNOS. The adverse effects of ISMN were improved in gp91phox knockout mice and normalized by in vivo/ex vivo treatment and suppressed by apocynin. In add- ition, a strong increase in the expression of ET within the endothelial cell layer and the adventitia was observed...... Conclusion Chronic treatment with ISMN causes endothelial dysfunction and oxidative stress, predominantly by an ET-depend- ent activation of the vascular and phagocytic NADPH oxidase activity and NOS uncoupling. These findings may explain at least in part results from a retrospective analysis indicating increased mortality in post-infarct patients in response to long-term treatment with mononitrates. ------Keywords Organic therapy Nitrate tolerance Endothelial dysfunction Reactive oxygen and nitrogen species † † † † NADPH oxidase S-glutathionylation of endothelial Endothelin-1 Bosentan therapy † † †

† M.O. and M.K., and A.D. and T.M. contributed equally and should be considered as first and senior authors, respectively. * Corresponding author: University Medical Center Mainz, II. Medizinische Klinik Langenbeckstrasse 1, 55131, Mainz, Germany. Tel: 49 6131 17 7250, Fax: 49 6131 17 6615, + + Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2012. For permissions please email: [email protected] Page 2 of 11 M. Oelze et al.

Introduction Methods Despite their potent anti-ischaemic effects when given acutely, the A more detailed description of the Methods section is provided in the efficacy of organic is rapidly lost upon chronic administra- Supplementary material online. tion due to the development of tolerance, i.e. the loss of haemo- dynamic effects that invariably occurs when nitrate therapy is Materials protracted for longer as 12–14 h (for review, see Munzel For isometric tension studies, GTN was used from a Nitrolingual infu- et al.1,2). While organic nitrates have long been considered to sion solution (1 mg/mL) from G.Pohl-Boskamp (Hohenlockstedt, Germany). All other chemicals including 1,1-diphenyl-2-picrylhydrazyl have neutral implications beyond their haemodynamic effects, free radical (DPPH) and ISMN were of analytical grade and were recent research points out that these drugs induce oxidative obtained from Sigma-Aldrich, Fluka or Merck. stress and endothelial dysfunction,3 which has been described and well characterized in response to treatment with Animals and in vivo treatment 1,2 (GTN). These phenomena have also been attributed an import- All animals were treated in accordance with the Guide for the Care ant role in the development of nitrate tolerance. Beyond the acti- and Use of Laboratory Animals as adopted by the US National Insti- vation of counterregulatory mechanisms such as neurohormonal tutes of Health and approval was granted by the Ethics Committee Downloaded from activation, an increase in vasopressin levels, and signs for intravas- of the University Hospital Mainz. Wistar rats or mice were anaesthe- cular volume expansion,1,2 a number of specific abnormalities have tized by isoflurane, and a subcutaneous osmotic minipump (model been shown to occur in the setting of prolonged nitrate therapy. 2001 for rat and model 1007D for mice, ALZET, Cupertino, USA) con- These abnormalities include the desensitization of the taining ISMN (75 mg/kg/day), bosentan (80 mg/kg/day), or the vehicle alone (dimethyl sulfoxide) was implanted as described recently.16 GTN-bioactivating enzyme ALDH-2, the desensitization of the http://eurheartj.oxfordjournals.org/ After 7 days of treatment, rats were killed by exsanguination in isoflur- soluble guanlylyl cyclase,4 an increase in phosphodiesterase 1A1 ane anaesthesia, and the blood, aorta, and heart were collected. activity,5 an increase in endothelin (ET) expression,6 and a stimula- 7 tion of vascular production of reactive oxygen species (ROS) Isometric tension studies along with an inhibition of the mitochondrial ALDH-2.8,9 These Concentration–relaxation curves and concentration–constriction phenomena contribute substantially to tolerance but also to endo- curves in response to increasing concentrations of ISMN, acetylcholine thelial dysfunction as observed in response to chronic treatment (ACh), and GTN or KCl, phenylephrine (Phe), and angiotensin-II 6,7 10 with GTN in experimental animals and in humans. In 2010, (AT-II) were performed as described.6,17 Zweier and colleagues11 have proposed a new mechanism of

endothelial nitric oxide synthase (eNOS) regulation that is based Reactive oxygen species formation at Universitaetsbibliothek Mainz on July 19, 2012 on S-glutathionylation of a cysteine in the reductase domain. Re- Reactive oxygen species formation was measured by oxidative burst of cently, we were able to detect S-glutathionylated eNOS in vascular leucocytes in whole blood or NADPH oxidase activity in the heart and 18– 20 cells/tissue from GTN-treated rats and endothelial cells.12 aorta by ECL. For fluorescence [dihydroethidine (DHE), 1 mM] Notably, while GTN and other nitrates such as isosorbide-5- oxidative microtopography, isolated aortic rings were OCT-embedded mononitrate (ISMN) and pentaerythrityl tetranitrate have trad- (Tissue Tek, USA) and stained with DHE (1 mM) with or without L-NG- methyl ester (L-NAME) or apocynin as itionally considered to compose a homogeneous class, several im- reported.16,19 – 22 capacity in serum was measured using portant differences have been demonstrated across these different a DPPH assay.21 compounds. For instance, pentaerythrityl tetranitrate has not been associated with either tolerance or endothelial dysfunction in both Western blot analysis 13 animal and human studies. In the USA, the most popular nitrate Western blot analysis was performed as described previously.16,19 – 21 in addition to GTN is the ISMN. Animal experiments revealed that eccentric treatment with ISMN does not cause endothelial dys- Endothelial nitric oxide synthase function and does not stimulate vascular production.14 immunoprecipitation and S-gluathionylation These animal data contrast however with a recent human report M-280 Sheep anti-Rabbit IgG-coated beads from Invitrogen (Darm- demonstrating that ISMN treatment for 5 days causes endothelial stadt, Germany) were used along with a monoclonal mouse eNOS 15 dysfunction, an effect that was likely due to increased bioavailabil- (Biosciences, USA) antibody as described.12 The beads were loaded ity of ROS, as it was reversed by the intra-arterial application of with the eNOS antibody and cross-linked according to the manufac- .15 Based on this background, we investigated in a well- turer’s instructions. Next, the aortic homogenates were incubated characterized animal model of nitrate tolerance whether chronic with the eNOS antibody beads, precipitated with a magnet, washed ISMN therapy causes endothelial dysfunction, stimulates vascular and transferred to gel, and subjected to SDS–PAGE followed by a ROS production, and uncouples eNOS by tetrahydrobiopterin standard western blot procedure using a monoclonal mouse antibody against glutathionylated proteins from Virogen (Watertown, MA, USA) (BH4)- and/or S-glutathionylation-dependent mechanisms. In this at a dilution of 1:1000 under non-reducing conditions. context, we used nicotinamide adenine dinucleotidephosphate (NADPH) oxidase (Nox2)-deficient mice to define its role in Immunohistochemistry ISMN-mediated endothelial dysfunction. In addition, since GTN The immunohistochemistry protocol was published previously.23 therapy has been demonstrated to cause autocrine stimulation of Aortic segments were OCT-embedded (Tissue Tek, USA) and 6 vascular ET production, we studied whether changes in ET bio- frozen in liquid nitrogen [for anti-ET-1, 1:500, Meridian Life Science, availability also occur in response to chronic ISMN treatment. USA] or fixed in paraformaldehyde (4%), paraffin-embedded [for Mononitrate and endothelial function Page 3 of 11 anti-big ET-1 (big ET-1), 1:10, IBL Co., Japan], and stained with primary Effects of isosorbide-5-mononitrate antibodies; following the species of primary antibodies, appropriate treatment on myocardial and vascular biotinylated secondary antibodies were used after dilution following the manufacturer’s instructions. For immunochemical detection, ABC nicotinamide adenine reagent (Vector) and then DAB (peroxidase substrate Kit, Vector) dinucleotidephosphate oxidase activity reagent as substrates were used. and expression Isosorbide-5-mononitrate treatment substantially increased Statistical analysis NADPH oxidase-driven superoxide production in membrane pre- Results are expressed as mean + SEM. One-way ANOVA (with Bon- parations from the heart (Figure 3A) and the aorta (Figure 3B). Like- ferroni’s or Dunn’s correction for comparison of multiple means) wise, the increased DHE staining throughout the vasculature was was used for comparisons of vasodilator potency and efficacy (see substantially reduced by the inhibitor of the NADPH oxidase apoc- Supplementary material online, Tables S1 and S2), ROS detection by ynin (Figure 3C and D), compatible with an activation of the chemiluminescence or fluorescence as well as protein expression NADPH oxidase in response to ISMN treatment. Accordingly, and antioxidant capacity based on the SigmaStat 3.5 Software. The we found an increase in the expression of Nox1, Nox2, and EC50 value for each experiment was obtained by log-transformation. Downloaded from The isometric tension data were also analysed using two-way Nox4 (see Supplementary material online, Figure S2A–C). The ANOVA with Bonferroni’s post hoc test using GraphPad Prism 5 Soft- observed increase, however, was rather moderate. The increase ware. Haem -1 promoter activity data were analysed using in Nox4 expression was also observed in cultured endothelial GraphPad Prism 5 Software and represent means + SEM. Statistical cells [human umbilical vein endothelial cells (HUVECs)] in re- differences were determined either by t-tests or by factorial analysis sponse to ISMN (see Supplementary material online, Figure S3). of variance (ANOVA) followed by Tukey’s multiple comparison test As a proof of concept for the role of NADPH oxidase in http://eurheartj.oxfordjournals.org/ for comparison of multiple means. P-values ,0.05 were considered causing endothelial dysfunction, we treated gp91phox (the phago- significant. All used tests were two-sided. cytic isoform of NADPH oxidase, also termed Nox2)-deficient mice with ISMN, observed accordingly a normalization of the acti- vation of leucocytes (oxidative burst in whole blood), and Results decreased serum antioxidant capacity, endothelial dysfunction (ACh-elicited relaxation), and membranous NADPH oxidase activ- Effects of isosorbide-5-mononitrate on ity (Figure 4A–E). endothelial function

Treatment with ISMN for 7 days did not modify the responsiveness Effects of isosorbide-5-mononitrate at Universitaetsbibliothek Mainz on July 19, 2012 to ISMN, demonstrating an absence of tolerance to this organic treatment on the nitric oxide/cyclic nitrate (Figure 1A for rat; see Supplementary material online, guanosine monophosphate signalling Figure S1, for mouse). In contrast, a marked shift of the dose– pathway response relationship to the endothelium-dependent vasodilator Isosorbide-5-mononitrate treatment significantly increased super- ACh was observed, compatible with endothelial dysfunction oxide production by the endothelium. This increase was substan- (Figure 1B). Likewise, a small but significant degree of GTN toler- tially decreased by using the eNOS inhibitor L-NAME compatible ance was established, indicated by the significant decrease in with eNOS uncoupling in vessels from ISMN-treated animals ED50 and maximal relaxation to GTN (Figure 1C; see Supplemen- (Figure 5A and B). Isosorbide-5-mononitrate treatment significantly tary material online, Table S1). Constriction in response to KCl, reduced the expression of the BH -synthesizing enzyme GTP- Phe, and AT-II was increased significantly in the ISMN group 4 cyclohydrolase-1 (GCH-1; Figure 5C). Endothelial nitric oxide syn- (Figure 1D–F; see Supplementary material online, Table S2). thase S-glutathionylation, which contributes to eNOS dysfunction/ uncoupling, was significantly increased by 43% in ISMN-treated Effects of isosorbide-5-mononitrate tissue (Figure 5D). Isosorbide-5-mononitrate treatment decreased treatment on total serum antioxidant the activity of the cGK-I [assessed as phosphorylation of the capacity and superoxide production vasodilator-stimulated phosphoprotein (VASP) at serine 239] in inflammatory cells (Figure 5E). The incubation of vessels from ISMN-treated animals with a polyethylene glycolated Cu,Zn superoxide dismutase Isosorbide-5-mononitrate treatment significantly decreased serum (PEG-SOD) normalized the ACh–dose relationship (Figure 5F). antioxidant capacity by 50% (P , 0.05) and markedly stimulated ! Isosorbide-5-mononitrate treatment did neither modify the ex- the superoxide signal of whole blood in the presence of NADPH pression of eNOS, , nor of the sGC subu- (200 mM). This assay mainly reflects superoxide production in nits (see Supplementary material online, Figure S4). inflammatory cells such as neutrophils and monocytes and involves the enzymatic source phagocytic NADPH oxidase (Figure 2A and B) Role for endothelin in since the signal is almost absent in whole blood from gp91phox knockout mice.24 Isosorbide-5-mononitrate and ET-1-stimulated isosorbide-5-mononitrate-induced ROS formation in whole blood and isolated neutrophils, which endothelial dysfunction was suppressed by co-incubation with the ET receptor antagonist Isosorbide-5-mononitrate treatment substantially increased the ex- bosentan (Figure 2C and D). pression of big ET-1 (Figure 6A) and ET-1 (Figure 6B)inthe Page 4 of 11 M. Oelze et al. Downloaded from http://eurheartj.oxfordjournals.org/ at Universitaetsbibliothek Mainz on July 19, 2012

Figure 1 Effects of in vivo isosorbide-5-mononitrate (ISMN, 75 mg/kg/day for 7 days) therapy on vascular reactivity. Vascular sensitivity to isosorbide-5-mononitrate (A), the endothelium-dependent vasodilator acetylcholine (ACh, B), and nitroglycerin (GTN, C) as well as to the con- strictors KCl (D), phenylephrine (Phe, E), and AT-II (F) were tested. Isosorbide-5-mononitrate treatment caused a substantial shift of the dose– response curve to acetylcholine to the right compatible with endothelial dysfunction. The sensitivity to vasoconstrictors was increased in the isosorbide-5-mononitrate group. Data are mean + SEM from at least 6–12 independent experiments. The statistics were on the basis of one-way ANOVA comparison of EC50 values (half maximal relaxation/constriction) and efficacies (maximal relaxation/constriction) (see also # Supplementary material online, Tables S1 and S2); *EC50: P , 0.05 vs. Ctr; % Max.Rel. or Max.Con.: P , 0.05 vs. Ctr.

endothelial and adventitial cell layer. The increase in prepro-ET ex- the suppression of ET-1-triggered ROS formation by bosentan in pression was also observed in cultured endothelial cells (HUVECs) whole blood (Figure 2C). ET-1 and ISMN in vitro incubations also acti- in response to ISMN (see Supplementary material online, Figure S3). vated ROS formation in isolated neutrophils (Figure 2D). Bosentan Bosentan, the non-selective ET receptor blocker treatment normal- in vivo therapy completely abolished endothelial dysfunction in the ized endothelial dysfunction in isolated aortic ring segments from ISMN treatment group and improved ACh-dependent ISMN in vivo treated rats ex vivo (Figure 7A), which went along with in the control group (Figure 7B). As expected, ET-1-dependent Mononitrate and endothelial function Page 5 of 11 Downloaded from http://eurheartj.oxfordjournals.org/ at Universitaetsbibliothek Mainz on July 19, 2012

Figure 2 Effects of in vivo isosorbide-5-mononitrate treatment on serum antioxidant capacity and oxidative burst in whole blood. (A) Isosorbide-5-mononitrate treatment significantly decreased serum antioxidant capacity and (B) stimulated oxidative burst in whole blood compatible with an activation of superoxide-producing such as the nicotinamide adenine dinucleotidephosphate oxidase in neu- trophils and monocytes. Effects of endothelin-1 and isosorbide-5-mononitrate in vitro challenges on leucocyte-derived reactive oxygen species in whole blood (C) and isolated neutrophils (D). Data are mean + SEM from six (A and B) and eight (C) independent experiments. *P , 0.05 vs. Ctr; #P , 0.05 vs. endothelin-1 or isosorbide-5-mononitrate treatment groups.

was significantly shifted at least to three-fold higher source of this free radical, organic nitrates might represent a concentrations by bosentan in vivo therapy (Figure 7C). Isosorbide- therapy for endothelial dysfunction. Several flaws have been iden- 5-mononitrate treatment increased leucocyte-dependent oxidative tified in this hypothesis. First of all, the discovery that endothelial burst in whole blood in response to the phorbol ester PDBu or dysfunction is caused by increased production of ROS such as the fungal endotoxin zymosan A in the ISMN treatment group, all superoxide25 implies that by adding NO, the formation of the of which was normalized by in vivo therapy with bosentan NO/superoxide reaction product is favoured,26 (Figure 7D and E). resulting in the increased of a highly reactive inter- mediate with negative effects on eNOS,27 prostacyclin synthase function28 and NO signalling.29 It is also likely that the induction Discussion of ROS production within tolerant tissue stimulates the expression of one of the most potent vasoconstrictors known, i.e. ET-1,30,31 a Differences across different organic phenomenon which has been observed in response to chronic nitrates GTN treatment.6 The finding of the importance of NO as a mediator of endothelial Other more recent findings by our group and others revealed function leads to the speculation that by providing an exogenous that rather than being ‘simple’ NO donors, organic nitrates are a Page 6 of 11 M. Oelze et al. Downloaded from http://eurheartj.oxfordjournals.org/

Figure 3 Effects of in vivo treatment with isosorbide-5-mononitrate on nicotinamide adenine dinucleotidephosphate oxidase activity. Isosorbide-5-mononitrate increased the activity in the heart (A) and the aorta (B). Dihydroethidine staining revealed that the increases in super- oxide were largely blocked by the nicotinamide adenine dinucleotidephosphate oxidase inhibitor apocynin (Apo) (C and D). Date are mean from four to eight independent experiments; *P , 0.05 vs. Ctr; #P , 0.05 vs. w/o apocynin. at Universitaetsbibliothek Mainz on July 19, 2012

quite heterogeneous group of compounds.32 For instance, GTN as that ISMN treatment for 7 days caused oxidative stress-induced well as pentaerythrithyl tetranitrate (PETN) undergo mitochon- endothelial dysfunction.15 Finally, while GTN has been consistently drial bioactivation by the ALDH-2, a redox-sensitive enzyme.8,33 associated with endothelial dysfunction, superoxide bioavailability, Chronic treatment with GTN has been shown to stimulate super- ET-1 expression, and NADPH oxidase activation, PETN does not oxide production within mitochondria and therefore causes an appear to have these deleterious effects.2,16,36 oxidative stress-mediated inhibition and down-regulation of the enzyme, thereby concurring to tolerance.2,9 A cross-talk mechan- Summary of the present findings ism between mitochondria and the vascular NADPH oxidase has Isosorbide-5-mononitrate treatment for 7 days in rats caused, as been shown to be responsible for the development of endothelial observed in humans, a substantial degree of endothelial dysfunc- dysfunction in response to GTN treatment.34 tion as evidenced by the strong shift to the right of the dose–re- Although less is known regarding the bioactivation of other sponse relationship of the endothelium-dependent vasodilator organic nitrates such as ISMN and its dinitrate ISDN, the cyto- acetylcholine. Interestingly, the ISMN sensitivity of the vasculature chrome P450-dependent mechanisms within the endoplasmatic was not altered by the ISMN pre-treatment, while a small degree reticulum has been proposed as a possible enzyme.1,2 It also of cross-tolerance to GTN was observed. This clearly emphasizes seems conceivable that vascular relaxations in response to ISDN that endothelial dysfunction, rather than tolerance development, and ISMN are mediated by the radical NO, while this mediator represents the major side effect of chronic therapy with the does not appear to be involved in the relaxation in response to mononitrate. GTN.35 Previously, we have demonstrated that endothelial dysfunction While there are numerous manuscripts addressing the effects of to GTN is likely due to a cross-talk between mitochondria and chronic GTN therapy on vascular function in preclinical and clinical the vascular NADPH oxidase since no endothelial dysfunction studies, information on the effects of ISMN on vascular function is developed in response to treatment with this organic nitrate in rather scarce.2,36 Muller et al.14 described that eccentric dosing of an NADPH oxidase knockout animal.34 The present studies dem- high concentrations of ISMN prevent the development of endothe- onstrate that this superoxide source is activated also during ISMN lial dysfunction and the stimulation of ROS production in the rabbit treatment and that increased ROS production in mitochondria is aorta. In contrast, as reported above, human data clearly showed not a prerequisite. Increased NADPH oxidase activity was Mononitrate and endothelial function Page 7 of 11 Downloaded from http://eurheartj.oxfordjournals.org/

Figure 4 Effects of in vivo treatment with isosorbide-5-mononitrate on whole blood and serum oxidative stress and vascular function in wild- type (B6-WT) vs. gp91phox knockout (gp912/2) mice. Isosorbide-5-mononitrate increased whole blood reactive oxygen species formation under basal (A) and phorbol ester (PDBu, B) stimulated conditions in wild-type mice, whereas no increase was observed in gp912/2 mice. Isosorbide-5-mononitrate decreased serum antioxidant activity (C) in B6-WT but not in gp912/2 mice. The isosorbide-5-mononitrate-induced at Universitaetsbibliothek Mainz on July 19, 2012 impairment of acetylcholine (ACh)-dependent relaxation was more pronounced in B6-WT than in gp912/2 mice (D). Isosorbide-5-mononitrate increased nicotinamide adenine dinucleotidephosphate oxidase activity in B6-WT more than in gp912/2 mice (E). Date are mean from four to six independent experiments; *P , 0.05 vs. B6-WT; #P , 0.05 vs. B6-WT isosorbide-5-mononitrate. +

observed in membrane preparations from the heart and from the ISMN-dependent increase in haem oxygenase-1 promoter activity aorta along with an increase in the expression of the NADPH (see Supplementary material online, Figure S5). oxidase subunits Nox1, Nox2, and Nox4. The increase in super- Increased superoxide production, in particular in the endothelial oxide was observed throughout the vessel wall, also including cell layer, leads to increased formation of peroxynitrite, which in the media, all of which was largely blocked by the NADPH turn may cause eNOS uncoupling37 via oxidation of the important 27 oxidase inhibitor apocynin, an observation which points to the vas- eNOS BH4 to the so-called BH3 radical. Intracelluar cular NADPH oxidase as the leading superoxide source. Addition- BH4 depletion may be also a consequence of a decreased expres- al support comes from key experiments in gp91phox (Nox2) sion of the BH4-synthesizing enzyme, the GCH-1. Recently, we knockout mice clearly indicating that the phagocytic (but also vas- were able to demonstrate a down-regulation and an uncoupling cular) Nox2 is a major source of ROS and its deletion prevents of the eNOS in the setting of mellitus,22 in AT-II hyperten- ISMN-dependent adverse effects and significantly improves sion16 as well as in GTN-induced nitrate tolerance.12 Interestingly, ISMN-induced endothelial dysfunction. Interestingly, superoxide treatment with ISMN caused a similar down-regulation of GCH-1 production of whole blood (reflecting NADPH oxidase activity in the present studies. Isosorbide-5-mononitrate therapy also of inflammatory cells such as neutrophils and monocytes18) was shares another eNOS impairing/uncoupling effect with GTN clearly enhanced in response to ISMN treatment, indicating that therapy that is based on S-glutathionylation of a critical cysteine ISMN is able to activate the enzyme in inflammatory cells and residue in the reductase domain as reported by Zweier and collea- this effect was absent when gp91phox knockout mice were gues11 and just recently confirmed for GTN-induced tolerance and treated with ISMN. Moreover, ET-1 and ISMN in vitro challenges type 1 diabetes.12,38 Accordingly, we observed an uncoupling of stimulated the oxidative burst in whole blood leucocytes, which eNOS as demonstrated by the reduction in the endothelial super- was suppressed by bosentan. Finally, in contrast to the tetranitrate oxide signal in response to the eNOS inhibitor L-NAME. Likewise, PETN, ISMN does not confer the induction of the antioxidant eNOS dysfunction was accompanied by a decrease in the activity enzyme system haem oxygenase-1 as shown by the lack of of the cGK-1 as indicated by the decreased phosphorylation of Page 8 of 11 M. Oelze et al. Downloaded from http://eurheartj.oxfordjournals.org/

Figure 5 Effects of in vivo isosorbide-5-mononitrate treatment on endothelial superoxide production with and without incubation with the endothelial nitric oxide synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME) and on the expression and modification of enzymes involved in nitric oxide formation and nitric oxide signalling. Incubation of control vessels increased DHE staining (A, original staining of trans- versal cryosections and B, densitometric quantification) in the endothelial cell layer due to the prevention of superoxide scavenging by nitric oxide. In contrast, the incubation of vessels from animals treated with isosorbide-5-mononitrate decreased endothelial superoxide levels iden- tifying endothelial nitric oxide synthase as a significant superoxide source. Data are presented for the expression of the tetrahydrobiopterin- synthesizing enzyme GTP-cyclohydrolase-1 (GCH-1; C), the level of S-glutathionylated (dysfunctional) endothelial nitric oxide synthase

(eNOS-GSH; D), and the activity of the cyclic guanosine monophosphate-dependent kinase as assessed by the phosphorylation of vasodila- at Universitaetsbibliothek Mainz on July 19, 2012 tor-stimulated phosphoprotein (VASP) at serine 239 (P-VASP, E). (F) Improvement of endothelial function (ACh-elicited relaxation) in the aorta from isosorbide-5-mononitrate-treated rats by polyethylene glycolated Cu,Zn superoxide dismutase (PEG-SOD) (100 U/mL). Date # $ are mean + SEM from 6 to 8 (A–E) and 4 to 14 (F) independent experiments; P , 0.05 vs. w/o L-NAME; *P , 0.05 vs. Ctr; P , 0.05 vs. isosorbide-5-mononitrate.

the VASP at serine 239. The importance of the oxidative stress was further substantiated on a more functional basis by a complete concept was further demonstrated by the improvement of normalization of endothelial dysfunction and whole blood oxidative ISMN-induced endothelial dysfunction by PEG-SOD. The present burst induced by bosentan in vivo therapy. data fit well with recent studies where Sekiya et al.39 demonstrated that ISDN, of which ISMN is a metabolite, causes endothelial dys- function as demonstrated by a decrease in flow-mediated dilation Conclusions and clinical of the brachial artery and also a worsening of the intima–media implications thickness, an observation suggestive of a proatherogenic action Taken together, the present studies indicate that the organic of ISDN.39 nitrate ISMN causes endothelial dysfunction, activates the vascular Finally, oxidative stress in endothelial cells has been demonstrated and phagocytic NADPH oxidase, and stimulates vascular ET-1 pro- to cause a marked stimulation of the expression of ET-1 in cultured duction. Although these findings look strikingly similar to previous endothelial and cells.30,31 Since ISMN treatment observations in response to GTN,6,7 there are several fundamental increased ROS production in the present animal model throughout differences: the vessel wall, we tested whether we can observe an increase in the expression of ET-1 and big ET-1 in ISMN-treated animals as (i) In contrast to GTN, ISMN is not bioactivated by mitochon- described previously by in vitro experiments with cultured endothe- drial ALDH-2, a process, which leads to a marked increase lial and smooth muscle cells.30,31 Indeed, using immunohistochemis- in mitochondrial ROS production.8 try, we clearly found an increase in the expression of ET-1 mainly in (ii) Thus, the NADPH oxidase activation in response to ISMN is the endothelial cell layer and also in the adventitia. The important not dependent at all on the cross-talk between ROS- role of ET-1 in causing vascular dysfunction in response to ISMN producing mitochondria and the enzyme.34 Mononitrate and endothelial function Page 9 of 11 Downloaded from http://eurheartj.oxfordjournals.org/

Figure 6 Effects of in vivo isosorbide-5-mononitrate treatment of vascular endothelin-1 and big-endothelin-1 expression. Immunohistochem- istry revealed that big endothelin-1 (A) and endothelin-1 (B) staining substantially increased in the endothelial cell layer and to a lesser extent in the adventitia. Images are representative for n 4 independent experiments. ¼ at Universitaetsbibliothek Mainz on July 19, 2012

Figure 7 Effects of ex vivo and in vivo bosentan treatment on isosorbide-5-mononitrate-induced vascular dysfunction and whole blood oxi- dative stress. Bosentan (10 mM for 1 h) ex vivo incubation improved isosorbide-5-mononitrate-induced endothelial dysfunction (ACh, A). Bosen- tan (80 mg/kg/day for 7 days) in vivo therapy improved isosorbide-5-mononitrate-induced endothelial dysfunction (ACh, B) and vasoconstrictor sensitivity to endothelin-1 (ET-1, C) as well as the leucocyte-dependent oxidative burst in whole blood in response to a phorbol ester (PDBu, D) or the endotoxin zymosan A (E). Data are mean + SEM from at least 8 to 12 (A), 4 to 14 (B–C) or 6 to 24 (D–E) independent experiments. *P , 0.05 vs. Ctr; #P , 0.05 vs. isosorbide-5-mononitrate. Page 10 of 11 M. Oelze et al.

(iii) This is the first demonstration that ISMN stimulates the 5. Kim D, Rybalkin SD, Pi X, Wang Y, Zhang C, Munzel T, Beavo JA, Berk BC, Yan C. phagocytic NADPH oxidase, a phenomenon, which is com- Upregulation of phosphodiesterase 1A1 expression is associated with the devel- opment of nitrate tolerance. Circulation 2001;104:2338–2343. pletely blocked by the ET receptor blocker bosentan. 6. Munzel T, Giaid A, Kurz S, Stewart DJ, Harrison DG. Evidence for a role of (iv) It is indeed the first demonstration that the non-selective ET endothelin 1 and protein kinase C in nitroglycerin tolerance. Proc Natl Acad Sci receptor blocker bosentan improves endothelial dysfunction USA 1995;92:5244–5248. 7. Munzel T, Sayegh H, Freeman BA, Tarpey MM, Harrison DG. Evidence for induced by an organic nitrate. enhanced vascular superoxide anion production in nitrate tolerance. A novel mechanism underlying tolerance and cross-tolerance. J Clin Invest 1995;95: The results of the present studies have important clinical implica- 187–194. tions. Isosorbide-5-mononitrate is still the most commonly used 8. Daiber A, Oelze M, Coldewey M, Bachschmid M, Wenzel P, Sydow K, Wendt M, oral nitrate worldwide. The demonstration of a marked degree Kleschyov AL, Stalleicken D, Ullrich V, Mulsch A, Munzel T. Oxidative stress and mitochondrial aldehyde dehydrogenase activity: a comparison of pentaerythritol of endothelial dysfunction due to activation of superoxide- tetranitrate with other organic nitrates. Mol Pharmacol 2004;66:1372–1382. producing enzymes such as the NADPH oxidase or an uncoupled 9. Sydow K, Daiber A, Oelze M, Chen Z, August M, Wendt M, Ullrich V, Mulsch A, NO synthase in response to ISMN treatment provides the reason Schulz E, Keaney JF Jr, Stamler JS, Munzel T. Central role of mitochondrial alde- hyde dehydrogenase and reactive oxygen species in nitroglycerin tolerance and for concerns since endothelial dysfunction, as well as oxidative cross-tolerance. J Clin Invest 2004;113:482–489. stress, in patients with coronary artery disease has been demon-

10. Gori T, Mak SS, Kelly S, Parker JD. Evidence supporting abnormalities in nitric Downloaded from strated to have adverse prognostic implications.40 oxide synthase function induced by nitroglycerin in humans. J Am Coll Cardiol Our findings may provide a mechanistic background for the 2001;38:1096–1101. 11. Chen CA, Wang TY, Varadharaj S, Reyes LA, Hemann C, Talukder MA, Chen YR, observation of an increased incidence of a coronary events in Druhan LJ, Zweier JL. S-glutathionylation uncouples eNOS and regulates its cel- patients treated with ISMN or ISDN after a .41 lular and vascular function. Nature 2010;468:1115–1118. As well, the demonstration of endothelial dysfunction and oxida- 12. Knorr M, Hausding M, Kro¨ller-Schuhmacher S, Steven S, Oelze M, Heeren T, Scholz A, Gori T, Wenzel P, Schulz E, Daiber A, Mu¨nzel T. Nitroglycerin-induced http://eurheartj.oxfordjournals.org/ tive stress in response to ISMN treatment and its reversal in endothelial dysfunction and tolerance involves adverse phosphorylation and response to concomitant therapy with bosentan suggests that S-glutathionylation of endothelial nitric oxide synthase—beneficial effects of the combination with and/or ET receptor blocker therapy with the AT1-receptor blocker telmisartan. Arterioscler Thromb Vasc Biol 2011;31:2223–2231. could be helpful to avoid the particular ISMN side effect of endo- 13. Schnorbus B, Schiewe R, Ostad MA, Medler C, Wachtlin D, Wenzel P, Daiber A, thelial dysfunction. More recently, the combination therapy of Munzel T, Warnholtz A. Effects of pentaerythritol tetranitrate on endothelial ISDN and hydralazine has been demonstrated to improve substan- function in coronary artery disease: results of the PENTA study. Clin Res Cardiol 2010;99:115–124. tially the prognosis of African Americans and severe congestive 14. Muller S, Konig I, Meyer W, Kojda G. Inhibition of vascular oxidative stress in 42 (A-HeFT), an observation that may be explained hypercholesterolemia by eccentric . J Am Coll Cardiol by the powerful antioxidant effects of hydralazine.43 2004;44:624–631.

15. Thomas GR, DiFabio JM, Gori T, Parker JD. Once daily therapy with isosorbide-5- at Universitaetsbibliothek Mainz on July 19, 2012 mononitrate causes endothelial dysfunction in humans: evidence of a free-radical-mediated mechanism. J Am Coll Cardiol 2007;49:1289–1295. Supplementary material 16. Schuhmacher S, Wenzel P, Schulz E, Oelze M, Mang C, Kamuf J, Gori T, Jansen T, Knorr M, Karbach S, Hortmann M, Mathner F, Bhatnagar A, Forstermann U, Li H, Supplementary material is available at European Heart Journal Munzel T, Daiber A. Pentaerythritol tetranitrate improves angiotensin II-induced online. vascular dysfunction via induction of oxygenase-1. 2010; 55:897–904. 17. Munzel T, Li H, Mollnau H, Hink U, Matheis E, Hartmann M, Oelze M, Acknowledgements Skatchkov M, Warnholtz A, Duncker L, Meinertz T, Forstermann U. Effects of long-term nitroglycerin treatment on endothelial nitric oxide synthase (NOS The authors would like to thank Margot Neuser, Jo¨rg Schreiner, III) gene expression, NOS III-mediated superoxide production, and vascular Angelica Karpi, and Anja Conrad for technical assistance. This NO bioavailability. Circ Res 2000;86:E7–E12. work contains parts of the doctoral thesis of A.G. 18. 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. 19. Oelze M, Daiber A, Brandes RP, Hortmann M, Wenzel P, Hink U, Schulz E, Funding Mollnau H, von Sandersleben A, Kleschyov AL, Mulsch A, Li H, Forstermann U, The studies were supported in part by a grant by the Federal Ministry Munzel T. inhibits superoxide formation by NADPH oxidase and endo- of Education and Research (BMBF 01EO1003) to T.M. thelial dysfunction in angiotensin II-treated rats. Hypertension 2006;48:677–684. 20. Oelze M, Warnholtz A, Faulhaber J, Wenzel P, Kleschyov AL, Coldewey M, Conflict of interest: Previous work on organic nitrates was sup- Hink U, Pongs O, Fleming I, Wassmann S, Meinertz T, Ehmke H, Daiber A, Munzel T. NADPH oxidase accounts for enhanced superoxide production and ported by vascular research grants from Actavis Deutschland GmbH, impaired endothelium-dependent smooth muscle relaxation in BKbeta12/2 Germany. A.D. and T.M. received honoraries from Actavis Deutsch- mice. Arterioscler Thromb Vasc Biol 2006;26:1753–1759. land GmbH [distributor of PETN (Pentalong)], Germany. 21. Oelze M, Knorr M, Schuhmacher S, Heeren T, Otto C, Schulz E, Reifenberg K, Wenzel P, Munzel T, Daiber A. Vascular dysfunction in streptozotocin-induced experimental diabetes strictly depends on insulin deficiency. J Vasc Res 2011; References 48:275–284. 1. Munzel T, Daiber A, Mulsch A. Explaining the phenomenon of nitrate tolerance. 22. Wenzel P, Schulz E, Oelze M, Muller J, Schuhmacher S, Alhamdani MS, Circ Res 2005;97:618–628. Debrezion J, Hortmann M, Reifenberg K, Fleming I, Munzel T, Daiber A. 2. Munzel T, Daiber A, Gori T. Nitrate therapy: new aspects concerning molecular AT1-receptor blockade by telmisartan upregulates GTP-cyclohydrolase I and action and tolerance. Circulation 2011;123:2132–2144. protects eNOS in diabetic rats. Free Radic Biol Med 2008;45:619–626. 3. Munzel T. Does nitroglycerin therapy hit the endothelium? J Am Coll Cardiol 2001; 23. Schuhmacher S, Foretz M, Knorr M, Jansen T, Hortmann M, Wenzel P, Oelze M, 38:1102–1105. Kleschyov AL, Daiber A, Keaney JF Jr, Wegener G, Lackner K, Munzel T, Viollet B, 4. Schroder H, Leitman DC, Bennett BM, Waldman SA, Murad F. Glyceryl Schulz E. alpha 1AMP-activated protein kinase preserves endothelial function { } trinitrate-induced desensitization of in cultured rat lung fibro- during chronic angiotensin II treatment by limiting Nox2 upregulation. Arterioscler blasts. J Pharmacol Exp Ther 1988;245:413–418. Thromb Vasc Biol 2011;31:560–566. Mononitrate and endothelial function Page 11 of 11

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