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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 endothelial dysfunction, oxidative stress, 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 vitamin 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 nitric oxide synthase (eNOS) likely due to a down-regulation of the tetrahydrobiopterin-synthesizing enzyme GTP-cyclohydrolase-1 and to S-glutathionylation of eNOS. The adverse effects of ISMN were improved in gp91phox knockout mice and normalized by bosentan 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 nitrate therapy Nitrate tolerance Endothelial dysfunction Reactive oxygen and nitrogen species † † † † NADPH oxidase S-glutathionylation of endothelial nitric oxide synthase 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 nitrates 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 nitroglycerin 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-nitroarginine methyl ester (L-NAME) or apocynin as itionally considered to compose a homogeneous class, several im- reported.16,19 – 22 Antioxidant 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 superoxide 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- vitamin C.15 Based on this background, we investigated in a well- turer’s instructions. Next, the aortic homogenates were incubated characterized animal model

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