Nitrate Therapy

HomeCirculationVol. 123, No. 19Nitrate Therapy Free AccessReview ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessReview ArticlePDF/EPUBNitrate TherapyNew Aspects Concerning Molecular Action and Tolerance Thomas Münzel, MD, Andreas Daiber, PhD and Tommaso Gori, MD, PhD Thomas MünzelThomas Münzel From II. Medizinische Klinik und Poliklinik, Kardiologie, Johannes Gutenberg Universität, Mainz, Germany. , Andreas DaiberAndreas Daiber From II. Medizinische Klinik und Poliklinik, Kardiologie, Johannes Gutenberg Universität, Mainz, Germany. and Tommaso GoriTommaso Gori From II. Medizinische Klinik und Poliklinik, Kardiologie, Johannes Gutenberg Universität, Mainz, Germany. Originally published17 May 2011https://doi.org/10.1161/CIRCULATIONAHA.110.981407Circulation. 2011;123:2132–2144Although the short-term vasodilatory properties of organic nitrates are potent and well known, a number of vascular and extravascular changes have been shown to compromise their hemodynamic effects on long-term administration. Among these changes, systemic phenomena such as neurohormonal activation and intravascular volume expansion1 as well as specific vascular changes such as increased vascular superoxide (O2·−) production,2 increased sensitivity to vasoconstrictors,3 and decreased responsiveness to nitric oxide (NO) donors4,5 have long been identified as playing a role. Several hypotheses have been proposed to explain these abnormalities, and over the last 15 years, our groups have focused on the concept that an inappropriate production of reactive oxygen species (ROS), an impairment in the scavenging of these mediators (Figure 1), or both might have a crucial mechanistic importance in all these modifications.6,7 Independently of the role of ROS in tolerance, the possibility that nitrate-induced oxidative stress might affect patients' prognosis has important implications, and the observation that long-term therapy with most of the drugs in this class causes endothelial dysfunction, the prognostic significance of which is well accepted in patients with coronary artery disease, hypertension, and heart failure,8 should not be taken as an academic curiosity.Download figureDownload PowerPointFigure 1. Free radical biochemistry. The radical nitric oxide reacts with superoxide to form the highly reactive intermediate peroxynitrite. Superoxide is dismutated by superoxide dismutase (SOD), leading to the formation of hydrogen peroxide (H2O2) and molecular oxygen (O2).Beyond these as-yet insufficiently investigated prognostic implications, recognition of the role of ROS suggests that a number of interventions thought to interfere with the vascular redox balance might also retard or prevent the development of nitrate tolerance and nitrate-induced side effects. Evidence that therapy with angiotensin-converting enzyme (ACE) inhibitors, angiotensin-1 receptor blockers, certain β-blockers, statins, and vitamins such as folic acid or vitamin C beneficially influence nitrate tolerance and glyceryl trinitrate (GTN)–induced endothelial dysfunction suggests that nitrate therapy might have profoundly different implications since these therapies have become diffusely available. In addition, the recognition of important differences in the mechanisms triggered by different nitrates should also be attributed clinical relevance, and evidence suggests that, rather than the generic nitrate tolerance, more specific expressions (GTN tolerance, isosorbide mononitrate [ISMN] tolerance, etc) should be used.9 This review summarizes the current concepts underlying tolerance and endothelial dysfunction in response to long-term therapy with different nitrates and addresses the question of whether the use of these drugs remains indicated despite these side effects.The Hemodynamic Effects of NitratesVenous capacitance vessels, large and medium-sized coronary arteries, and collateral vessels are most sensitive to GTN, whereas coronary and peripheral arterioles with a diameter <100 μm are relatively more resistant. In the setting of stable angina, the preferential venodilatation induced by antiischemic doses of GTN results in venous pooling and preload reduction, yielding reduced left ventricular filling pressure and wall tension. As a result of these changes, myocardial workload and oxygen demand decrease (Figure 2). Beyond this effect, the clinical benefit of organic nitrates in the setting of acute coronary syndromes is thought to depend primarily on the dilation of epicardial coronaries and coronary collaterals, improving perfusion and oxygen delivery to subendocardial regions by increasing total coronary conductance. Furthermore, because the tone and caliber of myocardial resistance vessels are almost unaffected, coronary steal phenomena and reflex tachycardia are in general avoided. In summary, the mismatch between oxygen demand and supply in ischemic regions is rapidly relieved on short-term administration of organic nitrates.Download figureDownload PowerPointFigure 2. Antianginal effects of acutely administered glyceryl trinitrate (GTN).Through the same hemodynamic effects, organic nitrates also markedly improve left ventricular function in patients with congestive heart failure, decreasing right atrial pressure with a redistribution of blood from the central circulation into larger capacitance veins, a reduction in impedance to the left ventricle ejection owing to an increase in compliance of the arterial vasculature, and a reduction in the magnitude, frequency, and velocity of reflected waves in the arterial circulation. The resulting increase in cardiac output reduces left ventricular end-diastolic pressure and wall tension, thereby shifting the stroke-volume/left ventricular end-diastolic pressure relationship from a negative to a positive slope.Antiaggregant Effects of Organic NitratesThe effect of nitrates on platelet aggregation and fibrinolysis might also have clinical importance particularly in the setting of acute coronary syndromes in which vascular thrombosis determines low-flow ischemia. Glyceryl trinitrate reduces platelet aggregability ex vivo in healthy volunteers and to a lesser extent10–14 (a situation that parallels the reduced vasodilator potency of GTN observed in these clinical conditions15) in patients with insulin resistance, congestive heart failure, or coronary artery disease. Whether the antiaggregant effects of nitrates have an additional clinical impact on the therapy of coronary artery disease, particularly in light of the introduction and systematic use of targeted antiplatelet agents such as aspirin, thienopyridines, or glycoprotein IIb IIIa inhibitors, is unclear.Mechanistic InsightThe principal mechanism underlying the hemodynamic effects of nitrates is believed to be the activation of the intracellular NO receptor enzyme soluble guanylyl cyclase, leading to increased bioavailability of cGMP and activation of cGMP-dependent protein kinases and/or cyclic nucleotide-gated ion channels (reviewed elsewhere16). The mechanism of GTN-induced inhibition of platelet aggregation is probably more complex in that it might involve both cGMP-dependent and -independent cGMP pathways.17–20 Whatever the mechanism, it appears to be (like the hemodynamic effects [see below]) redox dependent because antioxidants restored GTN responsiveness in heart failure and coronary artery disease patients.21 After the discovery of endogenous (endothelium-derived) NO and the recognition of its importance in controlling vascular homeostasis,22 the concept that nitrates may act as NO donors led to the speculation that these drugs might also be able to compensate for the compromised endothelial function typical of patients with cardiovascular disease. Evidence presented below suggests that this is not the case.No Nitric Oxide From Glyceryl Trinitrate?Evidence demonstrates that NO per se is not the mediator of the hemodynamic effects of GTN. Using spin trapping techniques, we found a striking dissociation between the vascular activity and NO donor properties23 for GTN compared with ISMN and isosorbide dinitrate (ISDN). Likewise, Nunez et al24 reported a similar discrepancy between the hemodynamic effects of GTN administration and NO release, suggesting that the action of GTN is unrelated to its bioconversion to NO. In another study, only suprapharmacological doses of short-term oral GTN evoked a measurable NO release.25 At the time of these observations, however, the mechanism of GTN biotransformation and its relationship with tolerance remained obscure. Evidence consonant with the hypothesis that the mediator is different among different nitrate molecules in this class was also provided by a recent study demonstrating that so-called NO donors such as GTN and pentaerythrityl tetranitrate (PETN) have different effects on myocardial gene expression.26 Treatment with GTN resulted in a larger expression of cardiotoxic genes and inhibition of the expression of cardioprotective proteins, whereas PETN treatment enhanced the expression of genes in the opposite direction. A possible explanation for this differential gene expression profiles in response to both compounds may be the release of different vasoactive molecules on bioactivation26 (Figure 3).Download figureDownload PowerPointFigure 3. Effects of organic nitrates such as glyceryl trinitrate (GTN) and pentaerythrityl tetranitrate (PETN) on myocardial gene expression. Treatment with GTN significantly changed the expression of 532 genes; PETN treatment changed the expression of 1215 genes. Interestingly, only 68 genes were significantly changed by both compounds, raising doubt that a common vasoactive molecule, such as nitric oxide is released from both drugs. Adapted from Pautz et al.26Aldehyde Dehydrogenase, the Enzyme That Metabolizes Alcohol, Also Bioactivates Glyceryl TrinitrateGlyceryl trinitrate is metabolized by at least 2 different pathways (Figure 4). When administered in high doses (low-affinity pathway), GTN is metabolized by several enzymes, including glutathione-S-transferases, xanthine oxidoreductase, and the cytochrome P450. Beyond this low-affinity pathway, the relevance of which for clinically used dosages remains unclear, a high-affinity pathway was identified in 2002 by Chen et al,27 who suggested that the mitochondrial aldehyde dehydrogenase (ALDH-2), the enzyme responsible for the catabolism of alcohol, could also have a role in the bioactivation of GTN. Subsequent studies confirmed a marked attenuation of GTN-induced activation of the cGMP-dependent cascade and vasodilator potency after incubation with ALDH-2 inhibitors28,29 and extended these results to PETN; in contrast, ISMN and ISDN were unaffected by ALDH-2 inhibition (or genetic deletion).30,31Download figureDownload PowerPointFigure 4. Proposed mechanisms underlying bioactivation of organic nitrates. Left, Characterization of the bioactivation of high-potency nitrates such as nitroglycerin (glyceryl trinitrate [GTN]), pentaerythrityl tetranitrate (PETN), and pentaerythrityl trinitrate (PETriN) by mitochondrial aldehyde dehydrogenase (ALDH-2) when used at low, clinically relevant concentrations (<1 μmol/L). The reductase activity converts the organic nitrates to nitrite and the denitrated metabolite (1,2-glyceryl dinitrate, PETriN, or its dinitrate, PEDN). In general, there are 3 proposed mechanisms including nitrogen oxide formed via a reduction of nitrite (NO2), nitric oxide, formed directly in response to interaction with the ALDH-2, and NO2, released from the mitochondria, may be reduced by the xanthine oxidase in the cytoplasm to form NO. Right, The bioactivation of low-potency nitrates such as isosorbide dinitrate (ISDN) and isosorbide-5-mononitrate (ISMN) but also GDN, PEDN, and their respective mononitrates GMN and PEMN by P450 enzyme(s) in the endoplasmic reticulum (ER), directly yielding nitric oxide. The latter mechanism also accounts for the high-potency nitrates when used at high concentrations (>1 μmol/L). Cyt Ox indicates cytochrome c oxidase.The precise active metabolite formed during this process remains obscure; several hypotheses have been proposed. Whatever the nature of this chemical species, these findings found rapid human translation. For instance, incubation of human vessels with an ALDH-2 inhibitor recapitulated the abnormalities associated with the development of tolerance,32 (Figure 5) and a loss-of-function mutation of ALDH-2 particularly frequent in Eastern Asia and Eastern Europe was found to be associated with impaired GTN metabolism and reduced responsiveness to GTN.33Download figureDownload PowerPointFigure 5. Effects of in vivo glyceryl trinitrate (GTN) treatment in patients undergoing bypass surgery on tolerance of mammary artery and vena saphena magna. A and B, in vivo treatment will lead to a marked degree of tolerance in the mammary artery and in vena saphena magna veins (blue lines). The shift to the right was comparable when the mammary artery and the vena saphena were treated in vitro with the aldehyde dehydrogenase (ALDH-2) inhibitor benomyl (green line). C, In vivo and in vitro treatment with GTN and benomyl resulted in comparable inhibition of the activity of ALDH-2 in arteries and veins. D, Long-term treatment with GTN leads to a downregulation of the GTN-bioactivating enzyme ALDH-2. Adapted from Hink et al,32 with permission of the publisher. Copyright © 2007, American College of Cardiology Foundation.Nitrate ToleranceThe clinical introduction of organic nitrates at the end of the 19th century was soon followed by the observation that the hemodynamic and clinical effects of GTN, ISMN, and ISDN invariably wane upon continuous therapy. In the setting of coronary artery disease, nitrate tolerance has been demonstrated as the loss of effects on treadmill walking time and time of onset of angina. In congestive heart failure, it has been described as the loss of hemodynamic effect of the administered nitrate,34 and in hypertension, it is evident as the rapid loss of the hypotensive effects of these drugs. Rather controversial data have been reported for the antiplatelet effects of GTN. A study in dogs showed that tolerance is associated with a paradoxical activation of platelets,35 and another report showed that prior exposure to GTN, even in very low doses, induces tolerance to the antiaggregatory effects of the drug.36 In contrast, other studies in both rats and humans have shown that platelet responsiveness is preserved despite hemodynamic tolerance.37,38 Another issue is the so-called nitrate resistance, ie, the reduced effectiveness of organic nitrates in the setting of cardiovascular disease, which limits nitrate effectiveness independently of prior nitrate use. For instance, McVeigh et al39 reported reduced hemodynamic effects in diabetic patients and (as mentioned above) that GTN-induced inhibition of platelet aggregability is blunted in patients with coronary artery disease or diabetes. To date, it remains unclear whether these different forms of reduced responsiveness to nitrates share common mechanisms (eg, dysfunction of downstream NO signaling pathways) or should rather be considered 2 distinct entities. If the understanding of the mechanism of nitrate bioactivation has proved to be more complex than initially thought, the mechanism of the development of nitrate tolerance is likely even more complex in that it involves neurohormonal counterregulation, expansion of plasma volume (collectively classified as pseudotolerance), and intrinsic vascular processes, defined as true tolerance (the Table). Beyond the loss of the vasodilatory action of nitrates, a typical phenomenon associated with these changes is the worsening of anginal symptoms caused by the withdrawal of nitrate therapy, the so-called rebound effect.40Table. Hypotheses Proposed to Explain the Development of Nitrate TolerancePseudotolerance Activation of the renin-angiotensin-aldosterone system Increase in circulating catecholamine levels and catecholamine release rates Increase in vasopressin levels Volume expansionVascular tolerance Impaired GTN biotransformation Increased vascular superoxide production Desensitization of the soluble guanylate cyclase Increase in phosphodiesterase activity Increased sensitivity to vasoconstrictors Increased endothelin expressionGTN indicates glyceryl trinitrate.Nitrate PseudotoleranceThe vasodilation evoked by intravenous, oral, and transdermal nitrate therapy causes the release of catecholamines41 and plasma vasopressin41,42 and increases plasma renin activity41,42 and aldosterone levels.41,42 Such activation of neurohormonal vasoconstrictor forces has been demonstrated in patients with coronary artery disease, patients with heart failure,43 and healthy subjects.41 In line with these data, long-term continuous transdermal GTN therapy has been associated with altered autonomic neural function, including impaired baroreflex activity and prevalence of sympathetic to parasympathetic tone in the regulation of heart rate.44 In addition, in both animal and human studies, long-term therapy with organic nitrates was associated with increased sensitivity to receptor-dependent vasoconstrictors such as serotonin, phenylepherine, angiotensin II, and thromboxane.3,45A marked increase in intravascular volume, secondary to the transvascular shift of fluid and/or to aldosterone-mediated salt and water retention, has also been observed in patients treated with GTN.41,42 Although these changes could attenuate the preload effect of GTN, evidence suggests that these mechanisms are not sufficient to fully explain the loss of nitrate effectiveness. For instance, there is a difference in the time frame of neurohormonal activation, plasma expansion, and development of tolerance42; furthermore, studies testing the effects of diuretics, β-blockers, or ACE inhibitors did not invariably reverse or prevent tolerance. Thus, although the possible prognostic implications of these changes need to be acknowledged, other mechanisms of tolerance and a hypothesis that explained all these changes had to be sought.Does Oxidative Stress Account for Nitrate Tolerance and Cross-Tolerance?In 1995, we proposed a new molecular mechanism for GTN tolerance and cross-tolerance. Critical to this concept was the evidence that the bioavailability of ROS in tolerant vessels amounted to about twice that in controls, and that this abnormality was corrected by the addition of liposomal superoxide dismutase, which dismutes O2·− to H2O2 and oxygen2 (Figure 1). 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 O2·−.32,46Evidence of GTN-induced increased ROS production in humans was then obtained ex vivo in arterial segments and in blood or platelets taken from patients rendered tolerant to GTN.19,39,48,49 GTN tolerance was also associated with increased markers of free radical–induced lipid peroxidation such as cytotoxic aldehydes and isoprostanes50 and esterified 8-epi-PGF2α51 and with a mild reduction in the responsiveness to the NO donor sodium nitroprusside in healthy volunteers,4 which might also be compatible with ROS-mediated interference with NO signaling. From these findings, we proposed the existence of a unifying hypothesis that, founded on the concept of GTN-induced increased oxidative stress, could be compatible with the multiple different observations associated with long-term nitrate therapy.6,7,52Mechanisms Underlying Tolerance: Impaired Biotransformation Versus Oxidative Stress ConceptSeveral concepts concerning the implications of the oxidative stress hypothesis of nitrate tolerance have been discussed intensively within the last decades. Importantly, the recognition of the role of a mitochondrial enzyme in the biotransformation of organic nitrates and of a role of mitochondrial oxidative stress in the development of tolerance provided a link between 2 only apparently separate hypotheses (reduced bioactivation versus ROS-mediated NO scavenging or ROS-mediated inactivation of NO signaling). This hypothesis is essentially based on the concept that the oxidation of thiol groups may cause inhibition of several and guanylyl and both reduced GTN biotransformation and NO In line with treatment of tolerant with antioxidants or GTN and of markedly tolerance development in response to data the bioactivation and oxidative stress hypotheses and an clinical of the observations by and that incubation with high concentrations of nitrates induced of cardiac mitochondria, oxygen and oxidative all data that are with a mitochondrial of Importantly, however, these to GTN tolerance, but most likely not to ISMN or ISDN tolerance, because these drugs not mitochondrial of the mechanism by which GTN stimulates mitochondrial ROS production (eg, release of reduced oxygen from mitochondrial complex or of lipid of mitochondrial mitochondrial these observations the that oxidative stress may directly GTN by oxidative inhibition of ALDH-2 or by of such as data obtained with ALDH-2 evidence that ALDH-2 could be a of ROS The pathways leading to GTN tolerance in this hypothesis are in figureDownload PowerPointFigure Molecular mechanisms of nitrate tolerance. of continuous glyceryl trinitrate (GTN) therapy, neurohormonal of increased catecholamine and vasopressin plasma increased intravascular volume, and activation of the renin-angiotensin-aldosterone system reduces After 3 endothelial and dysfunction tolerance and by different increased endothelial and superoxide formation from oxidase activation by protein C and from the inhibition of nitric oxide activation by of endothelial caused by resulting from oxidation of and reduced expression of vasoconstrictor caused by increased impaired bioactivation of GTN caused by inhibition of aldehyde dehydrogenase inhibition of soluble guanylate cyclase by superoxide and increased inactivation of cGMP by and inhibition of by peroxynitrite, leading to reduced For of radical in endothelial was from the implications of ROS however, are not to the mitochondrial because ROS into the cytoplasm have been demonstrated to a with the vascular resulting in ROS production and in the formation of the highly reactive peroxynitrite. Although the role of specific free radical is unclear, ROS and/or reactive nitrogen species such as in may the endothelial NO cause of the endothelial and directly the activity of the soluble guanylyl cyclase and/or other in NO impact of ROS on is particularly GTN may cause a phenomenon this rather than which may increase oxidative stress in vascular in a positive Although the existence of negative to be we demonstrated increased expression of an in an animal of nitrate an abnormality that was by of is also important to that changes in ROS production such as an increase in oxidase activity and evidence for an all of which could be corrected by administration of vitamin C, have been observed not only in vascular but also in platelets from observations are with the hypothesis that nitrate-induced oxidative stress, with impairment of endogenous NO might endothelial dysfunction (Figure figureDownload PowerPointFigure Organic nitrates cause endothelial Evidence for the development of endothelial dysfunction in response to nitroglycerin (glyceryl trinitrate in peripheral arterioles and coronary arteries and to isosorbide mononitrate (ISMN) peripheral arteries was tetranitrate caused endothelial dysfunction in the whereas isosorbide dinitrate (ISDN) treatment did Importantly, such as folic which have been shown to cause of an nitric oxide (NO) and the vitamin C were able to improve endothelial dysfunction in patients treated with ISMN and GTN. The mechanisms underlying endothelial dysfunction in response to long-term ISDN therapy have not been indicates blood C, left of to and Adapted from Gori et et Thomas et et and et with permission of the publisher. Copyright © 2007, American College of Cardiology the have provided a new of the of nitrate tolerance leading to a unifying hypothesis of the abnormalities observed in this it to be that nitrate tolerance remains a complex phenomenon by several of which In addition, a number of controversial Although a interference with NO signaling pathways is compatible with to and -independent such as ISMN and other hypotheses based on mechanisms that are of the oxidative stress concept as of the soluble guanylate cyclase via have also been inactivation of ALDH-2 not explain tolerance to ISMN and ISDN or the existence of a certain degree of between GTN and these drugs (the metabolism of which is ALDH-2 In addition, of cGMP via is also a possible explanation for GTN tolerance and the role of ALDH-2 inactivation in GTN tolerance was in an animal study that showed that treatment with inhibitors of ALDH-2 or causes a similar decrease in GTN-induced in both tolerant and In addition, despite a markedly reduced GTN-induced vasodilation in of ALDH-2 and after ALDH-2 human studies suggested that this enzyme accounts for only a of the total bioactivation of the role of abnormalities, et reported an impaired biotransformation of GTN in venous from patients treated with GTN but to other NO donors and to a in this study, although the bioavailability of superoxide was increased in arterial segments from the same short-term exposure to oxidative stress did not GTN the of the mechanisms underlying GTN tolerance, in another study, folic acid preserved but not responsiveness to suggesting the existence of different mechanisms different vascular the importance of the different free radical species peroxynitrite, etc) remains Organic number of of evidence that therapy with most organic nitrates in clinically used doses responsiveness to for the release of NO (Figure This also as endothelial dysfunction, has been observed in animal studies and in humans during GTN, ISMN, and ISDN therapy. In large coronary arteries, continuous treatment with transdermal GTN leads to enhanced paradoxical of Evidence of impaired to was found in arteries from patients undergoing nitrate therapy at the time of bypass and continuous