Vascular Pharmacology 63 (2014) 105–113

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Review

Organic nitrates: Update on mechanisms underlying vasodilation, tolerance and endothelial dysfunction Thomas Münzel ⁎, Sebastian Steven, Andreas Daiber Department of Cardiology and Angiology, University Medical Center, Mainz, Germany

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Article history: Received 8 August 2014 Received in revised form 15 September 2014 Accepted 20 September 2014 Available online 14 October 2014 Chemical compounds studied in this article: Nitroglycerin: Pubchem CID 4510 ISDN: Pubchem CID 6883 ISMN: Pubchem CID 27661 PETN: Pubchem CID 6518 PETrin: Pubchem CID 15353 Lipoic acid: Pubchem CID 864 Folic Acid: Pubchem CID 6037 Captopril: Pubchem CID 44093 Macitentan: Pubchem CID 16004692 Hydralazine: Pubchem CID 3637

a b s t r a c t Given acutely, organic nitrates, such as nitroglycerin (GTN), isosorbide mono- and dinitrates (ISMN, ISDN), and pentaerythrityl tetranitrate (PETN), have potent vasodilator and anti-ischemic effects in patients with acute coronary syndromes, acute and chronic congestive heart failure and arterial hypertension. During long-term treatment, however, side effects such as nitrate tolerance and endothelial dysfunction occur, and therapeutic efficacy of these drugs rapidly vanishes. Recent experimental and clinical studies have revealed that organic nitrates per se are not just nitric oxide (NO) donors, but rather a quite heterogeneous group of drugs considerably differing for mechanisms underlying vasodilation and the development of endothelial dysfunction and tolerance. Based on this, we propose that the term nitrate tolerance should be avoided and more specifically the terms of GTN, ISMN and ISDN tolerance should be used. The present review summarizes preclinical and clinical data concerning organic nitrates. Here we also emphasize the consequences of chronic nitrate therapy on the supersensitivity of the vasculature to vasoconstriction and on the increased autocrine expression of endothelin. We believe that these so far rather neglected and underestimated side effects of chronic therapy with at least GTN and ISMN are clinically important. © 2014 Elsevier Inc. All rights reserved.

Keywords: Nitroglycerin Isosorbide mono- and dinitrate Pentaerythrityl tetranitrate Tolerance Endothelin

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular mechanisms underlying nitrate-induced vasodilation and nitrate-induced supersensitivity to vasoconstrictors . . . . . . . . . . Mechanisms of bioactivation of organic nitrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms underlying tolerance and endothelial dysfunction induced by various organic nitrates . . . . . . . . . . . . . . . . . . . 4.1. GTN causes vascular tolerance and endothelial dysfunction and stimulates vascular superoxide and endothelin production . . . . GTN therapy and neurohormonal activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GTN and desensitization of the soluble guanylyl cyclase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GTN stimulates vascular production of reactive oxygen species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GTN also stimulates ROS production in mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative stress is responsible for increased autocrine endothelin expression and supersensitivity to vasoconstrictors . . . . . . . . . . . 9.1. ISMN causes no vascular tolerance but endothelial dysfunction, increased sensitivity to vasoconstrictors and increased vascular endothelin production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. PETN causes no tolerance, no endothelial dysfunction and upregulates antioxidant enzymes . . . . . . . . . . . . . . . . . . 9.3. ISDN causes …? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author at: II. Medizinische Klinik und Poliklinik, Kardiologie, Johannes Gutenberg Universität, Langenbeckstr. 1, 55131 Mainz, Germany. Tel.: +49 6131 17 7250; fax: +49 6131 17 6615. E-mail address: [email protected] (T. Münzel).

http://dx.doi.org/10.1016/j.vph.2014.09.002 1537-1891/© 2014 Elsevier Inc. All rights reserved.

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10. Conclusions and clinical implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Organic nitrates are present in the market as both short- or long acting formulations, and can be administered as sublingual tablets, capsules, sprays, patches or ointments. Despite their potent vasodilator actions when given acutely, their effects are however less evident when given chronically, and current guidelines recommend nitrate therapy for chronic stable angina only as a third-line approach in patients whose symptoms persist despite therapy with beta-blockers and/or calcium antagonists (and/or coronary percutaneous coronary intervention) [1]. It is important to note that organic nitrates were introduced in the market at a time when no double-blind, randomized long-term studies were required. Thus, the level of evidence for the use of nitrates is 1C, i.e., “expert opinion”, in the absence of studies investigating their impact on clinical endpoints. Nitrate tolerance is defined as the loss of effects, or the need to increase dosages to maintain the effects, of organic nitrates, limiting their efficacy as chronic therapy. While this has long been felt as a limitation to the benefit of nitrates, the concept that nitrate tolerance might reflect the onset of potentially dangerous vascular abnormalities is much more recent. A number of phenomena have been described among these abnormalities. These include systemic changes, such as neurohormonal activation and intravascular volume expansion [2] (called pseudotolerance, because it occurs in response to every vasodilator therapy), as well as more specific vascular disturbances (so called true vascular tolerance), such as a desensitization of the soluble guanylyl cyclase [3] or an increase in phosphodiesterase (PDE) activity [4] leading to cross tolerance to other nitric oxide (NO)-donating substances, increased vascular superoxide (O.− 2 ) production [5] and supersensitivity to vasoconstrictors due to the autocrine expression of endothelin within the vasculature [6,7] (Table 1). Importantly, an increased bioavailability of O.− 2 and its reaction with vascular (or nitrate-derived) NO to form peroxynitrite (ONOO−) might represent the initial trigger of all such changes [8]. Beyond their pharmacological interest at the molecular level, these nitrate-induced changes might also have important implications in patients with coronary artery disease, hypertension and heart failure, in whom oxidative stress has been shown to have negative prognostic implications (for a review see [9]). 2. Cellular mechanisms underlying nitrate-induced vasodilation and nitrate-induced supersensitivity to vasoconstrictors The activation of the enzyme soluble guanylyl cyclase by nitratederived nitric oxide (NO), leading to increased bioavailability of cyclic Table 1 Definitions of terms related to nitrate tolerance. Pseudotolerance Activation of the renin, angiotensin, aldosterone system Increase in circulating catecholamine levels and catecholamine release rates Increase in vasopressin levels Volume expansion True vascular tolerance Impaired biotransformation Increased vascular superoxide production Desensitization of the soluble guanylyl cyclase Increase in phosphodiesterase activity Increased vascular endothelin expression Supersensitivity to vasoconstrictors

110 112 112

guanosine-3′,-5′-monophosphate (cGMP) and activation of cGMPdependent protein kinases, such as the cGMP-dependent protein kinase I (cGK-I), was identified as the principal mechanism of action of these drugs [10]. The relaxation downstream to these processes requires Ca2+-dependent and/or -independent mechanisms. cGK-I inhibits the inositol-1,4,5-trisphosphate [IP3]-dependent calcium release mediated by phosphorylation of the IP3 receptor-associated cGMP kinase substrate (IRAG) and activates the big calcium-activated potassium channel (BKCa) through phosphorylation, leading to hyperpolarization and reduced calcium influx. Further, cGK-I activates the Ca2+-ATPase-pump and thereby the efflux of calcium to the extracellular space. Ca2+-independent relaxation by cGK-I involves phosphorylation of the myosin-binding subunit [e.g. myosin phosphatase targeting subunit 1 (MYPT1)] (Fig. 1). Furthermore, cGK-I might phosphorylate, and thereby inhibit, the small GTPbinding protein RhoA, leading to decreased Rho-kinase (ROK) activity and conserved activity of myosin light chain phosphatase (MLCP), all of which is vasodilatory. ROK can also directly phosphorylate and increase the contractility of myosin light chain (MLC). cGK-I also induces a feedback mechanism (which lowers the intracellular cGMP concentration) by the phosphorylation and activation of phosphodiesterases. More recent studies also put emphasis on epigenetic regulation of nitrate-induced smooth muscle relaxation: GTN increases histone acetylase activity, and Nε-lysine acetylation of contractile proteins influences GTN-dependent vascular responses. Interestingly, tolerance was reversed by proacetylation drugs [11], an effect that might be mediated by changes in the activity of aldehyde dehydrogenase 2 (ALDH-2). In contrast with these effects, when administered chronically, organic nitrates may also trigger supersensitivity to vasoconstrictors, a phenomenon attenuating the vasodilator effects of nitrates and mediated by an increased autocrine levels of endothelin within the vasculature, with the subsequent activation of phospholipase C (PLC) and protein kinase C (PKC) [6,7]. These pathways depend on increased intracellular calcium levels, activate the myosin light chain kinase (MLCK) leading to increased contractility of the myosin-actin-filaments, but also provide the link to cytosolic oxidative stress. Similarly, agonist-driven calciumindependent activation of the RhoA/ROK pathway contributes to vasoconstriction via inhibition of myosin light chain phosphatase (MLCP). 3. Mechanisms of bioactivation of organic nitrates After the discovery of endogenous (endothelium-derived) NO and the demonstration of its outstanding importance in controlling vascular hemostasis, the concept that nitrates may act as NO donor led to the concept that these drugs might be able to compensate for the compromised endothelial function, a characteristic of patients with coronary artery disease, hypertension and heart failure [9]. Evidence now tells us that the story is more complicated. Using electron magnetic resonance spin trapping, we found a striking dissociation between the vascular activity and NO donor characteristics for GTN compared with ISDN and ISMN, suggesting that NO per se can not account for vasodilation induced by GTN [12]. These findings were confirmed by Nunez et al., who demonstrated that the vasodilatory action of GTN is not related to its bioconversion to NO [13]. Additional investigations further indicated that the vasoactive molecule released, e.g., by GTN or PETN must be different. Treatment of experimental animals with these called “NO donors” caused substantially different effects on myocardial gene expression [14]. Treatment with GTN resulted in a substantially larger expression of cardiotoxic genes and inhibition of cardioprotective genes, while PETN enhanced the expression of more beneficial cardiac genes,

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Fig. 1. Mechanisms of NO/nitrate-induced vasodilation of vascular smooth muscle. Upon nitrate bioactivation, NO stimulates the soluble guanylyl cylase, increases cyclic 3′,5′-guanosine monophosphate (cGMP) that activates the cGMP-dependent protein kinase I (cGK-I), and subsequently decreases intracellular calcium levels (via inhibition of IP3-regulated Ca channel, activation of potassium channels with subsequent inhibition of calcium channels, and activation of the calcium pump into the sarcoplasmic reticulum). Decreased calcium levels reduce the contractility of myosin/actin filaments via inhibition of the myosin light chain phosphorylation and therefore cause vasorelaxation (modified from [75]).

therefore with effects in the opposite direction. This clearly points to the fact that both compounds release different vasoactive molecules upon bioactivation, or that large part of GTN-derived NO is directly converted to another species by undesired superoxide formation, most likely peroxynitrite. With respect to GTN bioactivation, this is accomplished by at least 2 different pathways. In high doses a low-affinity pathway accounts for it, and several enzymes might be involved, such as the xanthine oxidoreductase, glutathione-S transferases and the cytochrome P450 enzymes. A high-affinity pathway, presumably the one with clinical relevance, was discovered in 2002 by the group of Stamler [15]. These authors suggested that the mitochondrial ALDH-2, an enzyme, which is also responsible for the catabolism of alcohol, may also have a role in GTN bioactivation. Further studies confirmed this concept by demonstrating a marked attenuation of GTN-induced activation of the cGMPdependent cascade and vasodilatory potency after incubation with ALDH-2 inhibitors [16,17] and extended these findings to PETN. In contrast, ISMN and ISDN were unaffected by ALDH-2 inhibitors (or genetic ALDH-2 deletion) [18,19] (Fig. 2). More recently, it was postulated that the subcellular localization of ALDH-2 might play an important role in explaining GTN potency, with cytosolic ALDH-2 being the preferred pathway of GTN bioactivation [20]. In addition, ex vivo data suggest that cytosolic ALDH isoforms 1A1 [21] and 3A1 [22] may contribute to GTN and ISDN bioactivation at supraphysiological concentrations.

4. Mechanisms underlying tolerance and endothelial dysfunction induced by various organic nitrates 4.1. GTN causes vascular tolerance and endothelial dysfunction and stimulates vascular superoxide and endothelin production Most research focused so far on mechanisms underlying GTN tolerance, which explains why investigations concerning the effects of GTN

on the neurohormonal axis, the desensitization of the target enzyme sGC and the effects on the activity of the cGMP-dependent kinase I (cGK-I) are rather complete, in contrast to what known for the other organic nitrates. 5. GTN therapy and neurohormonal activation In patients with coronary artery disease, nitrate tolerance has been known, since the late XIX century, as the loss of effects on treadmill walking time and time of onset of angina. A similar loss of hemodynamic effects has also been seen in congestive heart failure and hypertension, as a progressive return of blood pressure values to pre-treatment ones despite continuation of nitrate therapy [23]. After initiation of GTN therapy neurohormal adjustments occur, including increases in intravascular volume, increased catecholamine release rates and increased levels of catecholamine [2], plasma vasopressin [2,24], aldosterone [2,24], increased plasma renin activity [2,24], and a paradoxical worsening of anginal symptoms caused by the withdrawal of nitrate therapy in the early morning hours, called “rebound agina” [25]. The activation of the sympathetic nervous system further leads to impaired baroreflex and to the withdrawal of the parasympathetic system [26]. Importantly, these neurhormonal changes are not specific for nitrate therapy, and are also encountered in response to therapy with other vasodilators, simply reflecting counter-regulatory mechanisms in response to the initial hypotensive effects of these compounds. 6. GTN and desensitization of the soluble guanylyl cyclase In the late 80s, the desensitization of the sGC was suggested as a mechanism of tolerance [27,28]. This desensitization is compatible with the evidence that patients treated with one nitrate also show reduced sensitivity to other NO-dependent vasodilators (so called cross tolerance).

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Fig. 2. The organic nitrates isosorbide-5-mononitrate (ISMN) and isosorbide dinitrate (ISDN) are bioactivated by P450 enzymes, xanthine oxidoreductase (XO), glutathione-S-transferase (GST) or cytosolic aldehyde dehydrogenase isoforms (cytALDH = 1A1, 3A1 or cytosolic ALDH-2). ISMN only induces reactive oxygen and nitrogen species formation (cytROS/RNS) in the cytosol. ISDN effects on oxidative stress are largely unknown. Nitroglycerin (GTN), pentaerythrityl tetranitrate (PETN) and its trinitrate metabolite (PETriN) are bioactivated by mitochondrial aldehyde dehydrogenase (ALDH-2) and at higher concentrations also by the above mentioned enzymatic systems (P450, XO, GST, cytALDH). During the bioactivation process, critical cysteine groups in the active center of ALDH-2 are oxidized, and need reactivation by reduced dithiols, such as dihydrolipoic acid (or thioredoxins [Trx]) and their reductase systems (lipoamide reductase [LAR], Trx reductase [TrxR] or glutathione reductase [GR]. GTN induces mitochondrial and cytosolic ROS/RNS formation (mainly superoxide and peroxynitrite) that contributes to the inactivation of the ALDH-2-dependent bioactivation pathway. In contrast, PETN induces antioxidant systems such as heme oxygenase-1 (HO-1), with subsequent expression of ferritin (which decreases free iron levels), bilirubin (a potent antioxidant) and carbon monoxide (CO, a vasodilator and anti-atherosclerotic molecule). Downstream to this, PETN improves endothelial NO synthase function by up-regulation of GTP-cyclohydrolase-1 and extracellular superoxide dismutase, all of which is highly protective and prevents nitrate tolerance and endothelial dysfunction. Similarly, co-therapy with hydralazine improves the efficacy of GTN and ISDN therapy through potent antioxidant properties, inhibiting mitochondrial ROS formation and prostacyclin synthase (PGIS) nitration.

Importantly, it has been shown that S-nitrosylation of sGC results in decreased responsiveness to NO, characterized by loss of NO-stimulated sGC and cGK-I activities [29]. Desensitization of sGC was concentrationand time-dependent on exposure to S-nitrosocysteine, and it was proposed that S-nitrosylation of sGC is a means by which memory of NO exposure is kept in smooth muscle cells, possibly leading to NO tolerance. The authors extended this in vitro evidence to in vivo observations by demonstrating that development of nitrate tolerance and cross tolerance by 3-day chronic GTN treatment correlates with S-nitrosylation and desensitization of sGC in tolerant tissues, and that tolerance was reversed by concomitant treatment with the sulfhydryl donor N-acetylcysteine [30]. In line with this, our group (unpublished observation) just established that GTN-induced tolerance is partially prevented in rats by therapy with a sGC activator.

7. GTN stimulates vascular production of reactive oxygen species In both the resistance and conduit vessels of the forearm, the administration of GTN [31,32] causes endothelial dysfunction. Interestingly, this phenomenon was not directly associated with the development of tolerance, as demonstrated by the persistence of a vasodilatory effect at the level of the brachial and the coronary arteries despite the presence of endothelial dysfunction [32,33]. In large coronary arteries, continuous treatment (5 days) with transdermal GTN leads to enhanced acetylcholine-induced paradoxical constriction instead of endothelium-dependent vasodilation [33,34]. Similarly, evidence of impaired responses to acetylcholine was found in arteries removed from patients undergoing GTN therapy at the time of bypass surgery [35]. Evidence that chronic treatment with GTN stimulates vascular superoxide production leading to increasing bioavailability of this

metabolite and potentially to tolerance and cross-tolerance was first proposed in 1995 [5]. Subsequently, we demonstrated that GTN treatment stimulates the vascular (and particularly endothelial) production of peroxynitrite, a highly reactive intermediate generated from the rapid, diffusion-limited reaction of NO with O.− 2 [8,36]. Evidence of GTN-induced increased ROS production in humans was then obtained ex vivo in arterial segments taken from patients rendered tolerant to GTN and in plasma [35,37–39]. It is now understood that the oxidation of thiol groups in the active site of ALDH-2 observed during chronic GTN therapy may cause inhibition of this enzyme [18,40], and therefore lead to both reduced GTN biotransformation and effectiveness [16]; and that similar changes might explain the desensitization of the sGC. In line with this, treatment of tolerant animals with mitochondria-targeted antioxidants completely prevented or reversed nitrate tolerance [41], whereas genetic deficiency in manganese superoxide dismutase (the mitochondrial isoform) increased the susceptibility to nitroglycerin-induced nitrate tolerance [42].

8. GTN also stimulates ROS production in mitochondria As previously described, these data provide a unifying mechanism for several different pathways proposed to explain the pathophysiology of nitrate tolerance, and are compatible with the original observations published by Needleman and Hunter that incubation of isolated heart mitochondria with high concentrations of nitrates uncouples mitochondrial respiration [43]. The subsequent observation that GTN stimulates primarily mitochondrial ROS production is stimulating, and the co-localization of ROS production and of the GTN bioactivating enzyme provides an interesting evolution in the hypotheses on nitrate

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tolerance, based on either the oxidative inhibition of ALDH-2 by electrophiles or the depletion of essential repair co-factors such as lipoic acid [40,44]. Recent data obtained with purified ALDH-2 also provide evidence that ALDH-2 could be a source of GTN-triggered ROS formation [44]. Importantly, the implications of mitochondrial ROS formation are not confined to the mitochondrial matrix, as ROS leaking into the cytoplasm activate a cross-talk with the vascular NADPH oxidase [45], resulting in further ROS production from membrane oxidases. The consequences of these processes, which also involve the formation of the highly reactive peroxynitrite (ONOO-), include—a mong others—the oxidization of the eNOS cofactor BH4, causing uncoupling of the enzyme, a mechanism that clinically translates into evidence of endothelial dysfunction [32,46]. Further, the tyrosine nitration by peroxynitrite of prostacyclin synthase reduces endothelial PGI2 formation and directly inhibits the activity of the soluble guanylyl cyclase [8]. In line with this, we recently demonstrated increased expression and uncoupling of the eNOS in an animal model of GTN tolerance [47,48]. Interestingly, supplementation of GTN-treated rats with BH4 or of GTN-treated healthy volunteers with folic acid reverted this abnormality [49].

being mediated by increased autocrine levels of endothelin within the vasculature, with subsequent activation of phospholipase C (PLC) and protein kinase C (PKC) [6]. Importantly, this phenomenon is likely due to increased oxidative stress within endothelial and smooth muscle cells, since reactive oxygen species have been shown to increase the expression of endothelin within endothelial and smooth muscle cells [50, 51]. The increased sensitivity to vasoconstriction was shown for norepinephrine, KCL, serotonin, angiotensin II and PKC-activators, and was normalized following inhibition of protein kinase C [6] (Fig. 3). Studies in patients with coronary artery disease demonstrated that long-term infusion for 48 h of GTN causes supersensitivity of forearm arterioles to vasoconstrictors such as angiotensin II and noradrenaline, all of which was corrected by concomitant treatment with the ACEinhibitor captopril [52]. Thus it is tempting to speculate that enhanced vasoconstriction in GTN tolerant patients may quite substantially contribute to the attenuation of the GTN vasodilatory effects (Fig. 3).

9. Oxidative stress is responsible for increased autocrine endothelin expression and supersensitivity to vasoconstrictors

ISMN is currently the most commonly used nitrate worldwide. In patients with chronic congestive heart failure, acute coronary syndromes and stable coronary artery disease chronic ISMN therapy leads to the rapid development of the tolerance phenomenon. The main question to be answered was whether chronic treatment with ISMN causes

GTN has also been shown to trigger supersensitivity to vasoconstrictors, a phenomenon attenuating the vasodilatory effects of nitrates and

9.1. ISMN causes no vascular tolerance but endothelial dysfunction, increased sensitivity to vasoconstrictors and increased vascular endothelin production

Fig. 3. A and B: GTN treatment for 3 days causes nitrate tolerance and a strong increase on sensitivity of aortic tissue of experimental animals to vasoconstrictors such as phenylephrine (PE) and angiotensin II (Ang II) [6]. C and D: Continuous treatment with GTN for 48 h markedly increases the sensitivity of forearm arterioles to phenylephrine (assessed by forearm plethysmography, reduction in forearm blood flow), a phenomenon that is completely prevented by concomitant treatment with captopril [52]. NTG means nitroglycerin.

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true vascular tolerance to the organic nitrates, endothelial dysfunction or supersensitivity to vasoconstrictors, or all of these together. In 2007 the group from Parker reported a strong degree of endothelial dysfunction in subjects treated with ISMN [53]. Interestingly endothelial dysfunction was corrected by the administration of vitamin C, pointing to an involvement of ROS in causing this phenomenon. We recently found that ISMN, interestingly, does not cause “true” tolerance, while inducing a marked degree of endothelial dysfunction and increasing vascular superoxide production, predominantly driven by an activation of the NADPH oxidase and the uncoupling of eNOS [7] . Even more importantly, ISMN treatment was associated with a strong increase in the expression of endothelin-1, mainly within the endothelial cell layer and the adventitia, and by increased sensitivity of the vasculature to vasoconstricting agents such as phenylephrine and angiotensin II, as demonstrated before in GTN tolerant vessels. Incubation of inflammatory cells with ISMN activated the phagocytic NADPH oxidase and caused an oxidative burst, all of which was blocked in vitro by the endothelin receptor blocker bosentan and was normalized in vivo by gp91phox deficiency [7]. Although these adverse effects of ISMN look similar to the previous observations in response to GTN treatment, there are several fundamental differences. First, in contrast to GTN, ISMN is not bioactivated by mitochondrial ALDH-2, a process that leads to a marked increase in mitochondrial ROS production; second, NADPH oxidase activation in response to ISMN is not dependent on the cross-talk between ROS-producing mitochondria and NADPH oxidase, and is an independent phenomenon triggered by unclear mechanisms. Third, ISMN-triggered endothelial dysfunction is mediated by enhanced endothelin production, which was blocked in vivo by endothelin-receptor blockade. These findings are compatible with the evidence that therapy of post-infarct patients with ISMN leads to an increased rate of coronary events [54]. The effects of bosentan on ISMN-induced endothelial dysfunction also suggest that concomitant therapy with an ET-receptor blocker, e.g. the new non-selective endothelin receptor blocker macitentan or with other modern drugs with ancillary antioxidant effects, might modify nitrate pharmacology and potentially prevent the side effects of ISMN treatment. 9.2. PETN causes no tolerance, no endothelial dysfunction and upregulates antioxidant enzymes There is now evidence that tolerance might not be a class effect, and that the nitrate PETN might be a remarkable exception in this regard. In contrast with other long-acting nitrates, studies with PETN in healthy volunteers showed preserved vasodilatory potency, as well as absence of oxidative stress and endothelial dysfunction [38,55] during continuous treatment with this nitrate. In animals, PETN was reported to prevent endothelial dysfunction as well as atherogenesis [56], which might be mediated by its capacity to induce the antioxidant defense protein heme oxygenase-1 (HO-1). This would in turn increase the gene expression and protein levels of ferritin (which binds iron, and therefore prevents hydroxyl radical formation), of the antioxidant molecule bilirubin, and of the vasodilator carbon monoxide [57–59]. A placebo-controlled trial, the PENTA Trial, addressed the effects of chronic PETN therapy on endothelial function in patients with coronary artery disease. With these studies we could demonstrate that PETN 80 mg given twice a day compared with placebo did not attenuate endothelial function. In addition, relative changes in mean flow volume and mean flow velocity significantly increased in the PETN group vs the placebo group. Thus, PETN remains at present the only organic nitrate not causing endothelial dysfunction likely because of an up-regulation of HO-1 [58], of extracellular superoxide dismutase [60], as well as of protective microRNAs (Thomas Thum, personal communication, unpublished) (Fig. 4). On this basis, we recently performed a large randomized, doubleblind, placebo-controlled, multicentre study (the ‘CLEOPATRA’ study)

to investigate the anti-ischemic efficacy of PETN 80 mg b.i.d. (given in the morning and at mid-day) over placebo in patients with chronic stable angina pectoris [61]. A total of 655 patients were evaluated in the intention-to-treat population, were randomized to PETN (80 mg b.i.d.) or placebo, and completed the study. Patients underwent treadmill exercise tests at randomization, and after 6 and 12 weeks of treatment. Treatment with PETN over 12 weeks did not modify the primary endpoint of total exercise duration (TED). In a pre-specified sub-analysis of patients with reduced exercise capacity, however, PETN appeared more effective than placebo treatment. Superiority of PETN over placebo was evident in patients who were symptomatic at low exercise levels. PETN 80 mg b.i.d. was well tolerated, and the overall safety profile was comparable with placebo [61]. Thus, although providing no additional benefit in unselected patients with known coronary artery disease, PETN therapy, administered in addition to modern anti-ischemic therapy, could increase exercise tolerance in symptomatic patients with reduced exercise capacity. 9.3. ISDN causes …? As indicated by the heading of this paragraph, the evidence concerning vascular tolerance, endothelial dysfunction and enhanced vasoconstriction with ISDN is less clear as compared to PETN, ISMN and GTN (see also Table 2). It is accepted that, in patients with CAD, therapy with ISDN causes rapid hemodynamic tolerance and also tolerance to its antianginal effects [62–65]. No animal experiments are available to illustrate whether vascular tolerance, increased oxidative stress and endothelial dysfunction occur in response to chronic ISDN treatment. In patients with stable coronary artery disease, Sekiya et al. demonstrated a marked degree of endothelial dysfunction, as assessed by FMD of the brachial artery, in response to 3 months of treatment with ISDN, but not with the NO donor nicorandil [66]. An interesting aspect is the interaction between ISDN and the arteriolar dilator hydralazine in patients with severe heart failure. Several clinical studies have clearly established that the combination of ISDN with hydralazine improves prognosis [67] compared to prazosin in the V-Heft-I trial, and improves exercise capacity compared to enalapril in the V-HeFT II trial [68]. In addition, the double-blind, randomized African–American Heart Failure Trial (A-HeFT) demonstrated that the combination therapy of ISDN and hydralazine was markedly effective in improving the composite endpoint of the trial, which included death from any cause, a first hospitalization for heart failure and quality of life measures. The substantial reduction in mortality even leads to an early termination of the trial [69] (Fig. 5). Thus, it appears that this particular combination of ISDN and hydralazine is possibly devoid of the development of tolerance and of endothelial dysfunction. Indeed, more recent experimental studies have demonstrated that hydralazine is a powerful inhibitor of nitrate-induced formation of reactive oxygen species, such as superoxide or the NO/superoxide reaction product peroxynitrite (ONOO−) in vitro [70] and in vivo [71] (Fig. 5), thus correcting the disrupted nitroso-redox balance between superoxide and nitric oxide in the cardiovascular system of patients with chronic congestive heart failure [72]. Experimental data on ISDN are scarce. One experimental study demonstrated increased oxidative stress and eNOS uncoupling in ISDNtreated endothelial progenitor cells [73], whereas a clinical study revealed no increased oxidative stress under chronic ISDN therapy [74]. Nevertheless, many questions are still open regarding ISDN, and there is a great need for additional preclinical studies in order to improve our understanding of vascular consequences to long-term treatment with this drug. 10. Conclusions and clinical implications The major progress with respect to organic nitrate research within the last decade were the discovery of the GTN bio-activating enzyme

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Fig. 4. A: In contrast to GTN, PETN strongly up-regulates hemeoxygenase I (HO-I) in vascular tissue [58] and catalyzes the conversion of heme into bilirubin, carbon monoxide (CO) and iron. Bilirubin is one of the strongest antioxidants in the body; ferritin chelates iron and therefore effectively suppresses hydroxyl radical formation; and carbon monoxide is a stimulator of soluble guanylyl cyclase, all of which may be responsible for the lack of development of tolerance to PETN. B: Although the primary endpoint in the Cleopatra trial was missed, a subgroup analysis of a pre-specified subgroup, patients with refractory angina pectoris (defined as at least two episodes of angina per week, self administration of at least two doses of sublingual nitrates per week, and total exercise duration ≤9 min), the change in total exercise duration at 6 and 12 weeks was markedly larger in the PETN group than in the control group [61]. TED means total exercise duration.

Table 2 Mechanisms underlying tolerance and endothelial dysfunction in response to treatment with various nitrates. Drug

GTN

ISMN

PETN

ISDN

Bioactivation

ALDH-2 [16]

Cytochrome P 450 [76]

ALDH-2 [18]

Endothelial Dysfunction Vascular tolerance

Coronary arteries [33] Forearm arterioles [31,32]Aorta [5] Coronary arteries [79]Aorta [5] Arterioles Yes [5] Yes [6] Yes [6]

Forearm arterioles [53] Aorta [7] No

No [78] No [58]

Cytochrome P450 [77] ALDH3A1 [22] Forearm conductance arteries [66] ?

Yes [7,53] Yes [7] Yes [7]

No [58] No (unpublished observation) No

? ? ?

Increase in oxidative stress Increase in autocrine endothelin expression Supersensitivity to vasoconstrictors

ALDH-2 and the knowledge that organic nitrates are not just NO donors, but rather are a heterogeneous group of vasodilators. Since the mechanisms of tolerance vary considerably between the various organic nitrates, it seems wise not to use the term “nitrate tolerance” anymore,

but rather refer to GTN, ISDN and ISMN tolerance. With respect to PETN, this NO donor is unique because it up-regulates the powerful antioxidant enzyme HO-1, which may explain why this compound is devoid of tolerance development and without negative effects on

Fig. 5. The combination therapy of hydralazine and ISDN is successfully used in the treatment of chronic congestive heart failure. The left hand side panel illustrates the powerful inhibitory effect of hydralazine on protein tyrosine nitration in rat smooth muscle cells by in situ generated peroxynitrite (from SIN-1) [70]. The right hand panel illustrates the marked effect of ISDN/ hydralazine on survival, as occurred in the A-HeFT Trial. In addition to the effects on pre- and afterload of this combination therapy, hydralazine, as an effective peroxynitrite inhibitor, may prevent the development of tolerance by correcting the disrupted nitroso-redox balance in the setting of chronic congestive heart failure [69]. SIN-1 means 3-morpholino sydnonimine.

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endothelial function. In the clinical setting the Cleopatra trial indicates that patients who are symptomatic with refractory chest pain may benefit from this organic nitrate. The data presented also indicate that ISMN can cause, both in experimental models and in humans, a severe degree of endothelial dysfunction, which may explain at least in part the higher incidence of acute coronary syndromes in response to prolonged treatment with this nitrate.

Acknowledgments These studies were supported by the Stiftung Mainzer Herz, the Robert Müller Stiftung, and the Center for Translational Vascular Biology (CTVB). T.M. is PI of the German Center for Cardiovascular Research (DZHK).

References [1] Task Force Members, Montalescot G, Sechtem U, Achenbach S, Andreotti F, Arden C, et al. ESC guidelines on the management of stable coronary artery disease: the Task Force on the management of stable coronary artery disease of the European Society of Cardiology. Eur Heart J 2013;34:2949–3003. [2] Parker JD, Farrell B, Fenton T, Cohanim M, Parker JO. Counter-regulatory responses to continuous and intermittent therapy with nitroglycerin. Circulation 1991;84: 2336–45. [3] Rapoport RM, Waldman SA, Ginsburg R, Molina CR, Murad F. Effects of glyceryl trinitrate on endothelium-dependent and -independent relaxation and cyclic GMP levels in rat aorta and human coronary artery. J Cardiovasc Pharmacol 1987;10: 82–9. [4] Kim D, Rybalkin SD, Pi X, Wang Y, Zhang C, Munzel T, et al. Upregulation of phosphodiesterase 1A1 expression is associated with the development of nitrate tolerance. Circulation 2001;104:2338–43. [5] Munzel T, Sayegh H, Freeman BA, Tarpey MM, Harrison DG. Evidence for enhanced vascular superoxide anion production in nitrate tolerance. A novel mechanism underlying tolerance and cross-tolerance. J Clin Invest 1995;95:187–94. [6] Munzel T, Giaid A, Kurz S, Stewart DJ, Harrison DG. Evidence for a role of endothelin 1 and protein kinase C in nitroglycerin tolerance. Proc Natl Acad Sci 1995;92: 5244–8. [7] Oelze M, Knorr M, Kroller-Schon S, Kossmann S, Gottschlich A, Rummler R. Chronic therapy with isosorbide-5-mononitrate causes endothelial dysfunction, oxidative stress, and a marked increase in vascular endothelin-1 expression. Eur Heart J 2013;34:3206–16. [8] Hink U, Oelze M, Kolb P, Bachschmid M, Zou MH, Daiber A, et al. Role for peroxynitrite in the inhibition of prostacyclin synthase in nitrate tolerance. J Am Coll Cardiol 2003;42:1826–34. [9] Munzel T, Sinning C, Post F, Warnholtz A, Schulz E. Pathophysiology, diagnosis and prognostic implications of endothelial dysfunction. Ann Med 2008;40:180–96. [10] Schlossmann J, Feil R, Hofmann F. Insights into cGMP signalling derived from cGMP kinase knockout mice. Front Biosci 2005;10:1279–89. [11] Colussi C, Scopece A, Vitale S, Spallotta F, Mattiussi S, Rosati J, et al. P300/CBP associated factor regulates nitroglycerin-dependent arterial relaxation by N(epsilon)lysine acetylation of contractile proteins. Arterioscler Thromb Vasc Biol 2012;32: 2435–43. [12] Kleschyov AL, Oelze M, Daiber A, Huang Y, Mollnau H, Schulz E, et al. Does nitric oxide mediate the vasodilator activity of nitroglycerin? Circ Res 2003;93:e104–12. [13] Nunez C, Victor VM, Tur R, Alvarez-Barrientos A, Moncada S, Esplugues JV, et al. Discrepancies between nitroglycerin and NO-releasing drugs on mitochondrial oxygen consumption, vasoactivity, and the release of NO. Circ Res 2005;97:1063–9. [14] Pautz A, Rauschkolb P, Schmidt N, Art J, Oelze M, Wenzel P, et al. Effects of nitroglycerin or pentaerithrityl tetranitrate treatment on the gene expression in rat hearts: evidence for cardiotoxic and cardioprotective effects. Physiol Genomics 2009;38: 176–85. [15] Chen Z, Zhang J, Stamler JS. Identification of the enzymatic mechanism of nitroglycerin bioactivation. Proc Natl Acad Sci U S A 2002;99:8306–11. [16] Sydow K, Daiber A, Oelze M, Chen Z, August M, Wendt M, et al. Central role of mitochondrial aldehyde dehydrogenase and reactive oxygen species in nitroglycerin tolerance and cross-tolerance. J Clin Invest 2004;113:482–9. [17] Zhang J, Chen Z, Cobb FR, Stamler JS. Role of mitochondrial aldehyde dehydrogenase in nitroglycerin-induced vasodilation of coronary and systemic vessels: an intact canine model. Circulation 2004;110:750–5. [18] Daiber A, Oelze M, Coldewey M, Bachschmid M, Wenzel P, Sydow K, et al. Oxidative stress and mitochondrial aldehyde dehydrogenase activity: a comparison of pentaerythritol tetranitrate with other organic nitrates. Mol Pharmacol 2004;66: 1372–82. [19] Chen Z, Foster MW, Zhang J, Mao L, Rockman HA, Kawamoto T, et al. An essential role for mitochondrial aldehyde dehydrogenase in nitroglycerin bioactivation. Proc Natl Acad Sci U S A 2005;102:12159–64. [20] Beretta M, Gruber K, Kollau A, Russwurm M, Koesling D, Goessler W, et al. Bioactivation of nitroglycerin by purified mitochondrial and cytosolic aldehyde dehydrogenases. J Biol Chem 2008;283:17873–80.

[21] Tsou PS, Page NA, Lee SG, Fung SM, Keung WM, Fung HL. Differential metabolism of organic nitrates by aldehyde dehydrogenase 1a1 and 2: substrate selectivity, enzyme inactivation, and active cysteine sites. AAPS J 2011;13:548–55. [22] Lin S, Page NA, Fung SM, Fung HL. In vitro organic nitrate bioactivation to nitric oxide by recombinant aldehyde dehydrogenase 3A1. Nitric Oxide 2013;35:137–43. [23] Elkayam U, Kulick D, McIntosh N, Roth A, Hsueh W, Rahimtoola SH. Incidence of early tolerance to hemodynamic effects of continuous infusion of nitroglycerin in patients with coronary artery disease and heart failure. Circulation 1987;76:577–84. [24] Munzel T, Heitzer T, Kurz S, Harrison DG, Luhman C, Pape L, et al. Dissociation of coronary vascular tolerance and neurohormonal adjustments during long-term nitroglycerin therapy in patients with stable coronary artery disease. J Am Coll Cardiol 1996;27:297–303. [25] Parker JD, Parker AB, Farrell B, Parker JO. Intermittent transdermal nitroglycerin therapy. Decreased anginal threshold during the nitrate-free interval. Circulation 1995;91:973–8. [26] Gori T, Floras JS, Parker JD. Effects of nitroglycerin treatment on baroreflex sensitivity and short-term heart rate variability in humans. J Am Coll Cardiol 2002;40:2000–5. [27] Mulsch A, Busse R, Bassenge E. Desensitization of guanylate cyclase in nitrate tolerance does not impair endothelium-dependent responses. Eur J Pharmacol 1988;158: 191–8. [28] Molina CR, Andresen JW, Rapoport RM, Waldman S, Murad F. Effect of in vivo nitroglycerin therapy on endothelium-dependent and independent vascular relaxation and cyclic GMP accumulation in rat aorta. J Cardiovasc Pharmacol 1987;10:371–8. [29] Sayed N, Baskaran P, Ma X, van den Akker F, Beuve A. Desensitization of soluble guanylyl cyclase, the NO receptor, by S-nitrosylation. Proc Natl Acad Sci U S A 2007;104:12312–7. [30] Zhou Z, Sayed N, Pyriochou A, Roussos C, Fulton D, Beuve A, et al. Protein kinase G phosphorylates soluble guanylyl cyclase on serine 64 and inhibits its activity. Arterioscler Thromb Vasc Biol 2008;28:1803–10. [31] Gori T, Harvey P, Floras JS, Parker JD. Continuous therapy with nitroglycerin impairs endothelium-dependent vasodilation but does not cause tolerance in conductance arteries: a human in vivo study. J Cardiovasc Pharmacol 2004;44:601–6. [32] Gori T, Mak SS, Kelly S, Parker JD. Evidence supporting abnormalities in nitric oxide synthase function induced by nitroglycerin in humans. J Am Coll Cardiol 2001;38: 1096–101. [33] Caramori PR, Adelman AG, Azevedo ER, Newton GE, Parker AB, Parker JD. Therapy with nitroglycerin increases coronary vasoconstriction in response to acetylcholine. J Am Coll Cardiol 1998;32:1969–74. [34] Azevedo ER, Schofield AM, Kelly S, Parker JD. Nitroglycerin withdrawal increases endothelium-dependent vasomotor response to acetylcholine. J Am Coll Cardiol 2001;37:505–9. [35] Schulz E, Tsilimingas N, Rinze R, Reiter B, Wendt M, Oelze M, et al. Functional and biochemical analysis of endothelial (dys)function and NO/cGMP signaling in human blood vessels with and without nitroglycerin pretreatment. Circulation 2002;105:1170–5. [36] Warnholtz A, Mollnau H, Heitzer T, Kontush A, Moller-Bertram T, Lavall D, et al. Adverse effects of nitroglycerin treatment on endothelial function, vascular nitrotyrosine levels and cGMP-dependent protein kinase activity in hyperlipidemic Watanabe rabbits. J Am Coll Cardiol 2002;40:1356–63. [37] Sage PR, de la Lande IS, Stafford I, Bennett CL, Phillipov G, Stubberfield J, et al. Nitroglycerin tolerance in human vessels: evidence for impaired nitroglycerin bioconversion. Circulation 2000;102:2810–5. [38] Jurt U, Gori T, Ravandi A, Babaei S, Zeman P, Parker JD. Differential effects of pentaerythritol tetranitrate and nitroglycerin on the development of tolerance and evidence of lipid peroxidation: a human in vivo study. J Am Coll Cardiol 2001;38: 854–9. [39] McVeigh GE, Hamilton P, Wilson M, Hanratty CG, Leahey WJ, Devine AB, et al. Platelet nitric oxide and superoxide release during the development of nitrate tolerance: effect of supplemental ascorbate. Circulation 2002;106:208–13. [40] Wenzel P, Hink U, Oelze M, Schuppan S, Schaeuble K, Schildknecht S, et al. Role of reduced lipoic acid in the redox regulation of mitochondrial aldehyde dehydrogenase (ALDH-2) activity. Implications for mitochondrial oxidative stress and nitrate tolerance. J Biol Chem 2007;282:792–9. [41] Esplugues JV, Rocha M, Nunez C, Bosca I, Ibiza S, Herance JR, et al. Complex I dysfunction and tolerance to nitroglycerin: an approach based on mitochondrial-targeted antioxidants. Circ Res 2006;99:1067–75. [42] Daiber A, Oelze M, Sulyok S, Coldewey M, Schulz E, Treiber N, et al. Heterozygous deficiency of manganese superoxide dismutase in mice (Mn-SOD+/−): a novel approach to assess the role of oxidative stress for the development of nitrate tolerance. Mol Pharmacol 2005;68:579–88. [43] Needleman P, Hunter Jr FE. Effects of organic nitrates on mitochondrial respiration and swelling: possible correlations with the mechanism of pharmacologic action. Mol Pharmacol 1966;2:134–43. [44] Oelze M, Knorr M, Schell R, Kamuf J, Pautz A, Art J, et al. Regulation of human mitochondrial aldehyde dehydrogenase (ALDH-2) activity by electrophiles in vitro. J Biol Chem 2011;286:8893–900. [45] Wenzel P, Mollnau H, Oelze M, Schulz E, Wickramanayake JM, Muller J, et al. First evidence for a crosstalk between mitochondrial and NADPH oxidase-derived reactive oxygen species in nitroglycerin-triggered vascular dysfunction. Antioxid Redox Signal 2008;10:1435–47. [46] Gori T, Burstein JM, Ahmed S, Miner SE, Al-Hesayen A, Kelly S, et al. Folic acid prevents nitroglycerin-induced nitric oxide synthase dysfunction and nitrate tolerance: a human in vivo study. Circulation 2001;104:1119–23. [47] Munzel T, Li H, Mollnau H, Hink U, Matheis E, Hartmann M, et al. Effects of long-term nitroglycerin treatment on endothelial nitric oxide synthase (NOS III) gene

T. Münzel et al. / Vascular Pharmacology 63 (2014) 105–113

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

expression, NOS III-mediated superoxide production, and vascular NO bioavailability. Circ Res 2000;86:E7-12. Knorr M, Hausding M, Kroller-Schuhmacher S, Steven S, Oelze M, Heeren T, Scholz A, Gori T, Wenzel P, Schulz E, Daiber A and Munzel T. Nitroglycerin-induced endothelial dysfunction and tolerance involve adverse phosphorylation and S-Glutathionylation of endothelial nitric oxide synthase: beneficial effects of therapy with the AT1 receptor blocker telmisartan. Arterioscler Thromb Vasc Biol. 31:2223–31. Gruhn N, Aldershvile J, Boesgaard S. Tetrahydrobiopterin improves endotheliumdependent vasodilation in nitroglycerin-tolerant rats. Eur J Pharmacol 2001;416: 245–9. Kahler J, Ewert A, Weckmuller J, Stobbe S, Mittmann C, Koster R, et al. Oxidative stress increases endothelin-1 synthesis in human coronary artery smooth muscle cells. J Cardiovasc Pharmacol 2001;38:49–57. Kahler J, Mendel S, Weckmuller J, Orzechowski HD, Mittmann C, Koster R, et al. Oxidative stress increases synthesis of Big endothelin-1 by activation of the endothelin-1 promoter. J Mol Cell Cardiol 2000;32:1429–37. Heitzer T, Just H, Brockhoff C, Meinertz T, Olschewski M, Munzel T. Long-term nitroglycerin treatment is associated with supersensitivity to vasoconstrictors in men with stable coronary artery disease: prevention by concomitant treatment with captopril. J Am Coll Cardiol 1998;31:83–8. Thomas GR, DiFabio JM, Gori T, Parker JD. Once daily therapy with isosorbide-5mononitrate causes endothelial dysfunction in humans: evidence of a free-radicalmediated mechanism. J Am Coll Cardiol 2007;49:1289–95. Nakamura Y, Moss AJ, Brown MW, Kinoshita M, Kawai C. Long-term nitrate use may be deleterious in ischemic heart disease: a study using the databases from two largescale postinfarction studies. Multicenter Myocardial Ischemia Research Group. Am Heart J 1999;138:577–85. Gori T, Al-Hesayen A, Jolliffe C, Parker JD. Comparison of the effects of pentaerythritol tetranitrate and nitroglycerin on endothelium-dependent vasorelaxation in male volunteers. Am J Cardiol 2003;91:1392–4. Hacker A, Muller S, Meyer W, Kojda G. The nitric oxide donor pentaerythritol tetranitrate can preserve endothelial function in established atherosclerosis. Br J Pharmacol 2001;132:1707–14. Oberle S, Abate A, Grosser N, Hemmerle A, Vreman HJ, Dennery PA, et al. Endothelial protection by pentaerithrityl trinitrate: bilirubin and carbon monoxide as possible mediators. Exp Biol Med (Maywood) 2003;228:529–34. Wenzel P, Oelze M, Coldewey M, Hortmann M, Seeling A, Hink U, et al. Heme oxygenase-1: a novel key player in the development of tolerance in response to organic nitrates. Arterioscler Thromb Vasc Biol 2007;27:1729–35. Schuhmacher S, Wenzel P, Schulz E, Oelze M, Mang C, Kamuf J, et al. Pentaerythritol tetranitrate improves angiotensin II-induced vascular dysfunction via induction of heme oxygenase-1. Hypertension 2010;55:897–904. Oppermann M, Balz V, Adams V, Dao VT, Bas M, Suvorava T, et al. Pharmacological induction of vascular extracellular superoxide dismutase expression in vivo. J Cell Mol Med 2009;13:1271–8. Munzel T, Meinertz T, Tebbe U, Schneider HT, Stalleicken D, Wargenau M, et al. Efficacy of the long-acting nitro vasodilator pentaerithrityl tetranitrate in patients with chronic stable angina pectoris receiving anti-anginal background therapy with betablockers: a 12-week, randomized, double-blind, placebo-controlled trial. Eur Heart J 2014;35:895–903. Parker JO, Fung HL, Ruggirello D, Stone JA. Tolerance to isosorbide dinitrate: rate of development and reversal. Circulation 1983;68:1074–80.

113

[63] Thadani U, Fung HL, Darke AC, Parker JO. Oral isosorbide dinitrate in the treatment of angina pectoris. Dose–response relationship and duration of action during acute therapy. Circulation 1980;62:491–502. [64] Thadani U, Manyari D, Parker JO, Fung HL. Tolerance to the circulatory effects of oral isosorbide dinitrate. Rate of development and cross-tolerance to glyceryl trinitrate. Circulation 1980;61:526–35. [65] Thadani U, Fung HL, Darke AC, Parker JO. Oral isosorbide dinitrate in angina pectoris: comparison of duration of action an dose–response relation during acute and sustained therapy. Am J Cardiol 1982;49:411–9. [66] Sekiya M, Sato M, Funada J, Ohtani T, Akutsu H, Watanabe K. Effects of the long-term administration of nicorandil on vascular endothelial function and the progression of arteriosclerosis. J Cardiovasc Pharmacol 2005;46:63–7. [67] Cohn JN, Archibald DG, Ziesche S, Franciosa JA, Harston WE, Tristani FE, et al. Effect of vasodilator therapy on mortality in chronic congestive heart failure. Results of a Veterans Administration Cooperative Study. N Engl J Med 1986;314:1547–52. [68] Cohn JN, Johnson G, Ziesche S, Cobb F, Francis G, Tristani F, et al. A comparison of enalapril with hydralazine-isosorbide dinitrate in the treatment of chronic congestive heart failure. N Engl J Med 1991;325:303–10. [69] Taylor AL, Ziesche S, Yancy C, Carson P, D'Agostino Jr R, Ferdinand K, et al. Combination of isosorbide dinitrate and hydralazine in blacks with heart failure. N Engl J Med 2004; 351:2049–57. [70] Daiber A, Oelze M, Coldewey M, Kaiser K, Huth C, Schildknecht S, et al. Hydralazine is a powerful inhibitor of peroxynitrite formation as a possible explanation for its beneficial effects on prognosis in patients with congestive heart failure. Biochem Biophys Res Commun 2005;338:1865–74. [71] Munzel T, Kurz S, Rajagopalan S, Thoenes M, Berrington WR, Thompson JA, et al. Hydralazine prevents nitroglycerin tolerance by inhibiting activation of a membrane-bound NADH oxidase. A new action for an old drug. J Clin Invest 1996; 98:1465–70. [72] Hare JM. Nitroso-redox balance in the cardiovascular system. N Engl J Med 2004; 351:2112–4. [73] Thum T, Fraccarollo D, Thum S, Schultheiss M, Daiber A, Wenzel P, et al. Differential effects of organic nitrates on endothelial progenitor cells are determined by oxidative stress. Arterioscler Thromb Vasc Biol 2007;27:748–54. [74] Keimer R, Stutzer FK, Tsikas D, Troost R, Gutzki FM, Frolich JC. Lack of oxidative stress during sustained therapy with isosorbide dinitrate and pentaerythrityl tetranitrate in healthy humans: a randomized, double-blind crossover study. J Cardiovasc Pharmacol 2003;41:284–92. [75] Munzel T, Daiber A, Gori T. More answers to the still unresolved question of nitrate tolerance. Eur Heart J 2013;34:2666–73. [76] Martens D, Kojda G. Impaired vasodilator response to organic nitrates in isolated basilar arteries. Br J Pharmacol 2001;132:30–6. [77] Minamiyama Y, Takemura S, Imaoka S, Funae Y, Okada S. Cytochrome P450 is responsible for nitric oxide generation from NO-aspirin and other organic nitrates. Drug Metab Pharmacokinet 2007;22:15–9. [78] Schnorbus B, Schiewe R, Ostad MA, Medler C, Wachtlin D, Wenzel P, et al. Effects of pentaerythritol tetranitrate on endothelial function in coronary artery disease: results of the PENTA study. Clin Res Cardiol 2011;99:115–24. [79] Munzel T, Holtz J, Mulsch A, Stewart DJ, Bassenge E. Nitrate tolerance in epicardial arteries or in the venous system is not reversed by N-acetylcysteine in vivo, but tolerance-independent interactions exist. Circulation 1989;79:188–97.