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Gen. Physiol. Biophys. (2004), 23, 265—295 Endothelial Dysfunction and Reactive Oxygen SpeciesProduction in Ischemia/Reperfusion and Nitrate Tolerance Institute of Experimental Pharmacology, Slovak Academy of Sciences,Bratislava, Slovakia Abstract. Reactive oxygen species (ROS), as superoxide and its metabolites, haveimportant roles in vascular homeostasis as they are involved in various signalingprocesses. In many cardiovascular disease states, however, the release of ROS is in-creased. Uncontrolled ROS production leads to impaired endothelial function andconsequently to vascular dysfunction. This review focuses on two clinical condi-tions associated with elevated ROS levels: ischemia/reperfusion and nitrate toler-ance. Injury caused by ischemia/reperfusion is an important limitation of trans-plantations, and complicates the management of stroke and myocardial infarction.
Nitrates, which are used to treat transient myocardial ischemia (angina pectoris),decrease in efficacy in long-term continuous administration. There are several en-zyme systems, such as xanthine oxidase, cyclooxygenase, uncoupled endothelialnitric oxide synthase, NAD(P)H oxidase, cytochrome P450 and the mitochondrialelectron transport chain, which are responsible for the increased vascular produc-tion of superoxide. The contribution of particular ROS producing enzymes and theeffect of antioxidant treatment are discussed in both pathological conditions.
Key words: Reactive oxygen species — Ischemia/reperfusion — Nitrate tolerance— NADPH oxidases — Nitric oxide synthase Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266Endothelial dysfunction as an indicator of vascular pathology . . . . . . . . 267 Ischemia/reperfusion injuryNitrate tolerance Xanthine oxidaseMitochondrial oxidase Correspondence to: Katalin Sz˝ ocs, Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratislava 4, SlovakiaE-mail: [email protected] Nitric oxide synthaseNAD(P)H oxidase Nitric oxide synthaseNAD(P)H oxidase Reactive oxygen species (ROS) are recognized as important signaling molecules inthe cardiovascular system. By oxidizing cysteine residues ROS alter the activity oftarget proteins (e.g. transcription factors, protein phosphatases) and induce redox-sensitive gene transcription, smooth muscle cell growth and motility (Chiarugi andCirri 2003). Elevated ROS, however, inhibit cell growth and in even higher concen-trations induce apoptosis (Bladier et al. 1997). Excess ROS production may injurevascular tissue by multiple mechanisms. For example, ROS can react with vasoac-tive substances and impair responses to vasodilators. Long-term exposure to ROSoxidizes proteins and lipids and damages DNA. These harmful actions togetherresult in vascular injury (reviewed in Bauer and Bauer 1999). Since the endothe-lium is the most vulnerable component of the vessel wall, it is not surprising thatthe first functional abnormality common to many vascular diseases is endothelialdysfunction.
Enhanced production of ROS, in particular superoxide (O − 2 ), has been impli- cated in the pathogenesis of a number of cardiovascular diseases includingatherosclerosis, coronary artery disease, hypertension and diabetes mellitus (Caiand Harrison 2000). In addition, ROS are frequently elevated in patients with oneor more cardiovascular risk factors, for example in smokers or in patients withhypercholesterolemia. Moreover, increased ROS production is associated with clin-ical conditions like ischemia/reperfusion (I/R). Tissue injury caused by I/R is afrequent complication of surgical procedures, transplantation, stroke, circulatoryshock, coronary artery disease, etc. (Li and Jackson 2002; Carlucci et al. 2002).
Myocardial ischemia (i.e. lack of oxygen due to inadequate perfusion) manifests asangina pectoris, the clinical sign of ischemic heart disease. Although nitrates arevaluable drugs in treatment of recurrent ischemic heart attacks, their continuoususe is complicated by the rapidly developing tolerance. Since elevated ROS con-centrations are also present in nitrate tolerance, they could also play a causal rolein this condition.
Involvement of ROS in the development of endothelial dysfunction is described in the first part of the review. Two pathological states in which increased ROScontribute to impaired vascular relaxation, i.e. injury caused by I/R and nitratetolerance, are then discussed in detail. Contribution of specific ROS producingenzyme systems in these pathophysiological states will be reviewed last.
ROS in Ischemia/Reperfusion and Nitrate Tolerance Endothelial dysfunction as an indicator of vascular pathology The inner layer of blood vessels, the vascular endothelium, is an active organwith paracrine, endocrine, and autocrine activities and it is crucial for the reg-ulation of vascular tone and maintenance of vascular homeostasis. It produces the endothelium-derived relaxing factor nitric oxide ( NO) and several other vasodi-lating agents, including prostacyclin and the endothelium-dependent hyperpolar-izing factor (EDHF). Endothelial cells also produce contracting factors, such as endothelin-1 (ET-1) and thromboxane (Vanhoutte 2000). Recently, O − been classified as a vasoconstrictor agent derived from endothelial cells (Vanhoutte2000; Rey et al. 2001).
NO can interact whith O − 2 , forming peroxynitrite (ONOO−) (Fig. 1) and also by other ROS and lipid radicals. Under physiological conditions, ROS production islow and the endogenous antioxidant systems are sufficient to maintain the balance between O − production and elimination, thus preventing the breakdown of NO.
In disease states in which the production of ROS is increased or the antioxidant capacity of the vessel is decreased, NO is transformed to ONOO−, resulting ininhibition of endothelial-dependent relaxation (O'Donnell et al. 1997) (Fig. 1). Inaddition, reduced expression or activity of endothelial nitric oxide synthase (eNOS) can also decrease NO bioavailability.
Increased production of ROS is thought to be one of the key events in the pathogenesis of endothelial dysfunction. In 1986, before NO was identified asthe endothelium-derived relaxing factor, Rubanyi and Vanhoutte (1986) alreadyshowed that superoxide dismutase (SOD) augmented endothelium-dependent re-laxation. Their observation was confirmed by several later studies demonstratingimproved vascular responses to vasodilators after SOD administration (d'Uscio etal. 2001; Steinhorn et al. 2001; Jung et al. 2003).
Impaired endothelium-dependent vasodilation is often the first sign of adverse cardiovascular events and can predict their long-term outcome (Schachinger et al.
2000; Perticone et al. 2001). Clinical evaluation of endothelial function is thusan important diagnostic tool. This is frequently done by analysis of acetylcholine(ACh)-induced relaxation (Erbs et al. 2003) or flow-dependent dilatation of theradial artery using ultrasound probes (Hambrecht et al. 2000). In endothelial dys-function, the sensitivity and maximal relaxation in response to vasodilators whichact via the endothelium is decreased, while vascular responses to sodium nitroprus-side, which acts directly on vascular smooth muscle cells (VSMC), are unchanged.
Inhibitors of nitric oxide synthase (NOS) or generators of ROS, for example pyro-gallol (Haj-Yehia et al. 1999), decrease endothelium-dependent vasorelaxation to ACh. Thus, both decreased NO and increased ROS levels contribute to endothelialdysfunction.
Altered function of the vascular endothelium is associated with arterial hyper- tension (Taddei and Salvetti 2002), preeclampsia (Page 2002), diabetes (Makimat-tila and Yki-Jarvinen 2002), atherosclerosis (Anderson 2003), heart failure (Linke etal. 2003), coronary artery disease (Schachinger et al. 2000), nitrate tolerance (Mun- Figure 1. ROS production and its consequences during I/R and nitrate- tolerance in vessel wall. Under physiological conditions, eNOS forms NO and citrulline from L-arginine. NO, directly or by inhibiting ET-1 release,promotes vasorelaxation. In I/R and nitrate-tolerance there is an imbalance between NO and O − formation. Enzymes contributing to increased O2 production include the mitochondrial electron transport chain, NAD(P)H oxidase, xanthine oxidase (XO), eNOS. Excess of O − results in NO in- activation through peroxinitrite (ONOO−) formation and consequently todecreased endothelium-dependent vasorelaxation, and eventually to vaso-contriction. ONOO− is also a potent oxidant, it mediates lipid peroxidation and eNOS uncoupling. O − can be dismutated to H2O2 (spontaneously or by CuZnSOD). In the presence of metal ions, H2O2 can be further metab-olized to hydroxyl radical. L-Arg, L-arginine; H2B, dihydrobiopterin; H4B,tetrahydrobiopterin; XD, xanthine dehydrogenase; SOD, superoxide dismu-tase.
ROS in Ischemia/Reperfusion and Nitrate Tolerance zel et al. 1995) and clinical conditions linked to ischemia followed by reperfusion(Sotnikova et al. 1998; Pagliaro et al. 2003). In these disease states, the productionof ROS is increased and the beneficial effect of antioxidants suggests that ROScontribute to the development of endothelial dysfunction (Cai and Harrison 2000).
Administration of probucol to cholesterol fed rabbits, for example, decreased vas- cular O − production and improved endothelium-dependent relaxations (Inoue et al. 1998). The membrane-permeable polyethylene glycol SOD (Mügge et al. 1991),glutathione (GSH) (Prasad et al. 1999), vitamin C (Ting et al. 1995; Gokce et al.
1999) and vitamin E (Heitzer et al. 1999) increase NO bioavailability. Estradiol,which also has indirect antioxidant properties, was found to increase coronary bloodflow in postmenopausal women with coronary artery disease (Blumel et al. 2003).
Antioxidants reverse also endothelial vasomotor dysfunction in patients with riskfactors for coronary artery disease (smokers, hypercholesterolemic patients) andimprove vasodilation of atherosclerotic vessels. Recent clinical data showed thatacute administration of vitamin C to patients with coronary artery disease re-stored peripheral endothelial function by reducing elevated levels of ROS (Erbs etal. 2003).
In summary, endothelial dysfunction is present in many vascular diseases and pathological conditions. The protective role of antioxidants indicates that increased ROS production, and consequently low NO bioavailability, are responsible for theimpaired vasorelaxation. Vascular injury, as caused by I/R, and tolerance to ni-trates, drugs used in the treatment of myocardial ischemia and its prevention, areboth associated with elevated ROS levels. The following sections are focusing onthe involvement of ROS in the pathogenesis of these two conditions.
One of the earliest events following I/R injury is vascular dysfunction linked toaltered function of endothelial cells. Both ischemia (hypoxia) and reperfusion (re-oxygenation) are important in human pathophysiology as they occur in variousclinical conditions, for example during circulatory shock, stroke, organ transplan-tations (Li and Jackson 2002) or coronary artery bypass surgery (Carlucci et al.
2002). These events are associated with increased oxidative stress and endothelialdamage.
Increased ROS levels have been classically attributed to exposure of ischemic tissues to molecular oxygen during reperfusion (reoxygenation) (Zweier et al. 1994;Kim et al. 1998, Bauer et al. 2002). Indeed, the largest increase in ROS production isusually observed within the first 15 min of reperfusion (Kim et al. 1998; Buttemeyeret al. 2002; Bertuglia and Giusti 2003; Bertuglia et al. 2004). Furthermore, increasedROS production was detected by luminol or lucigenin in rat superior mesentericarteries one hour after reperfusion following a 30-min ischemia (Haklar et al. 1998).
This rapid burst of oxygen-derived free radicals during reperfusion coincides withthe time course of endothelial dysfunction. While 90 or 120 min of ischemia alonedid not alter vascular responses, the reactivity of the endothelium to ACh wasattenuated as soon as 2.5 min following initiation of reperfusion (Tsao et al. 1990; Hayward and Lefer 1998). Neither did partial occlusion of the abdominal aorta ofrats for 18 h change vascular responses significantly. However, if prolonged ischemiawas followed by a 30-min reperfusion, endothelium-dependent vasorelaxation wasimpaired (Sotníková et al. 1998). Reperfusion lasting up to 120 min further reducedvascular responses to endothelium-dependent vasodilators, which remained alteredeven more than 4 h after blood flow renewal (Tsao et al. 1990; Hayward and Lefer1998). Similarly, in superior mesenteric arteries of mice and rats, I/R for either45/45 min or 30/60 min decreased vasodilatory responses to ACh by approximately40 %, compared to sham-operated animals (Banda et al. 1997; Chen et al. 2000).
Although initial studies suggested that ROS were predominantly produced during reperfusion, recent discoveries showed that hypoxia itself could also increaseROS production in various tissues (Vanden Hoek et al. 1997; Li and Jackson 2002;Pearlstein et al. 2002). For example, increased superoxide production was detectedin vivo within 10 min of femoral artery occlusion (Buttemeyer et al. 2002). Inarteries injured by ischemia, ROS production returned to basal levels when oxy-gen tension recovered completely, which may take 30 min after clamp removal(Bertuglia and Giusti 2003).
Endothelial dysfunction induced by I/R is manifested with a delay of a few minutes following increased vascular ROS production and is maintained for a longerperiod (hours) than elevated ROS levels. For example, in hamster cheek pouch arte-rioles, while elevated ROS returned to normal as soon as 30 min after ischemia, thearteriolar diameter kept decreasing (Bertuglia and Giusti 2003). Coronary bloodflow was also found significantly decreased after 30 min of reperfusion (Pernow andWang 1999). Similarly, impaired vasodilatory responses to ACh were observed insuperior mesenteric arteries 120 min after reperfusion (Hayward and Lefer 1998).
However, 60/120 min I/R did not affect sensitivity to ACh in rabbit major conduitarteries (Koksoy et al. 2000). This may be due to the higher resistance of some largerather than small arteries to I/R. Thus the small vessels seem to be more sensitiveto hypoxia than the large ones. In arterioles and capillaries, hypoxic conditions aremaintained even after restoration of blood flow in adjacent arteries. Hypoxic injury,manifested by endothelial cell swelling, leads to the "no reflow" phenomenon withinthe first minutes of reperfusion (Menger et al. 1992). If the ischemic period lastsfor several hours, hypoxia-induced continuous ROS release impairs the endothelialbarrier, increases vascular permeability and thus damages nearby tissues (Plateelet al. 1995). Electronmicroscopical examination of 18 h/30 min I/R revealed struc-tural changes, such as edematous mitochondria in both endothelial and vascularsmooth muscle cells, microvilli formation on the surface of endothelial cells, in-creased pinocytic activity and disturbed tight junctions between endothelial cells(Sotníková et al. 1998). 20 min after reperfusion, polymorphonuclear cells adheredto the endothelium (Hayward and Lefer 1998). Adhesion of polymorphonuclearcells to the vessel wall further impairs vascular function. Recent data indicate thatleukocyte adhesion is due to endothelial dysfunction, as a consequence of oxida- tive stress. Both decomposition of NO during reperfusion and inhibition of NOsynthesis increased leukocyte adherence to the venular wall (Bertuglia and Giusti ROS in Ischemia/Reperfusion and Nitrate Tolerance 2003), suggesting a protective role of NO in maintaining a non-adherent endothe-lial surface. These phagocytic cells continue to produce ROS and injure endothelialcells, which contributes to later loss of vasomotor tone control. This could explainthe prolonged endothelial dysfunction and the protective action of SOD long af-ter ROS production in the vessel wall (within endothelial cells and VSMC) hasreturned to normal.
Decreased NO availability, at least in part, has been attributed to the activ- ity of O − formed during reperfusion in endothelial cells (Zweier et al. 1994) and neutrophils (Fabian and Kent 1999). Studies performed in vivo suggest the involve-ment of at least two ROS in I/R injury. As demonstrated with dihydroethidium (DHE) and dichlorofluorescein (DCF) fluorescence, both O − 2 and hydrogen perox- ide (H2O2) were elevated in endothelial cells during hypoxia (Kim et al. 1998; Liand Jackson 2002; Pearlstein et al. 2002). Another mechanism by which ROS injure cells and impair endothelial function involves generation of hydroxyl radical ( OH)by surface-associated iron, which reacts with H2O2. This was demonstrated by the protective effect of iron chelators and OH scavengers on reoxygenated endothe-lial cell permeability (Terada 1996). Pretreatment with stobadine, a pyridoindole derivative, which is known to scavenge OH, exerted protective effect on I/R in-duced injury (Sotníková et al. 1998).
MnSOD overexpression or exogenous SOD administration protects against I/R injury (SOD mimetics were reviewed in Salvemini and Cuzzocrea (2003). For exam-ple, pretreatment with SOD before reperfusion maintained ROS at normal levels,prevented lipid peroxidation, increased arteriolar diameter and decreased leuko-cyte adhesion in hamster microcirculation (Bertuglia and Giusti 2003; Nakae etal. 2003). Similarly catalase, if delivered directly to the endothelium using specificantibodies, protected against oxidative stress during lung transplantation in therat. This antioxidant enzyme restores the barrier function of the endothelium, asshown by decreased lung graft edema and reduced number of intracapillary, inter-stitial and intraalveolar neutrophils (Christofidou-Solomidou et al. 2003; Kozoweret al. 2003).
Upregulation of endogenous antioxidant enzymes is likely to contribute to the beneficial effect of ischemic or hyperthermic preconditioning (Hoshida et al. 2002;Pagliaro et al. 2003). These techniques, involving application of a short ischemia orhyperthermia for several minutes to hours before I/R, improved the ability of thetissue to withstand subsequent ischemia. For example, a brief ischemia applied be-fore prolonged occlusion of the left anterior descending coronary artery attenuatedreperfusion injury in dogs. In this experiment both pre- and postconditioning (ashort reperfusion period during ischemia) decreased oxidative stress and increasedmaximal vasodilatory response to ACh (Zhao et al. 2003). Similarly, a short-termbody temperature elevation prior to I/R preserved endothelium dependent vasodi-lation (Chen et al. 2000). Exercise is another physical stress that induces protec-tion against ischemia. In patients with coronary artery disease, long-term aerobicexercise improves endothelium-dependent vasodilation (Hambrecht et al. 2000).
Following exercise, vascular CuZnSOD activity is induced and NAD(P)H-oxidase expression is decreased. These changes are expected to decrease oxidative stressand thereby to contribute to improved endothelial function (Rush et al. 2003).
.NO, besides acting directly as a vasodilator, inhibited the release of ET-1 (Vanhoutte 2000). This potent vasoconstrictor is produced in arteries by endothelialand vascular smooth muscle cells. Similarly, production of ET-1 was enhanced byI/R injury in the myocardium (Gourine et al. 2001). ET-1 levels were increasedalso in blood samples collected from the coronary sinus 30 min after reperfusion,and they most likely contribute to the vasoconstrictive response (Carlucci et al.
2002).
Thus, I/R is linked to increased ROS production both during the ischemic and early reperfusion period, leading to decreased NO availability, vasoconstriction andleukocyte adhesion. Upregulation of antioxidant enzymes by preconditioning tech-niques or physical exercise improves vascular responses to vasodilators. In additionto non-pharmacological management of coronary artery disease, organic nitratesare generally used to treat transient ischemic attacks. This medication, however, isnot straightforward, since it is accompanied by undesirable vascular reaction, i.e.
nitrate tolerance, as seen in the next section.
Nitrate tolerance Nitrates are frequently used in the management of coronary artery disease. They cause vasorelaxation by releasing NO from the parent molecule in vascular smoothmuscle (Wong and Fukuto 1999). Due to general vasodilation, ventricular volumeand arterial pressure are lowered, thus myocardial oxygen requirement decreases,which manifests as relief from angina. Although acute application of nitrates isa highly efficient vasodilator and anti-ischemic therapeutic, prolonged continuousadministration for more than 12 to 24 h leads to desensitization, an effect referred toas nitrate tolerance (Munzel et al. 1995). In healthy volunteers, a continuous 6-daytreatment with nitroglycerine patches led to decreases in ACh-induced vasodilationand consequently to a decrease in forearm blood flow (Gori et al. 2001). In orderto minimize the tolerance and restore useful responses to the drug, patients areadvised to keep nitrate-free periods daily, at least for 8 h. This approach, however,has two drawbacks. The first is the lack of tissue protection during the nitrate-free period, and the second is rebound ischemia upon nitrate withdrawal (Thadani1997).
Tolerance has most frequently been reported in the case of nitroglycerine (NTG), historically the first therapeutic organic nitrate (Munzel et al. 1995; Leopoldand Loscalzo 2003). However, all other members of this pharmacological class, suchas amyl nitrite and isosorbide dinitrate, induce tolerance and cross-tolerance (Mun-zel 2001). This means that the vascular response is decreased not only to NTG butalso to other endothelium-dependent and -independent nitrovasodilators.
Several mechanisms may account for the phenomenon of nitrate tolerance, or tachyphylaxis. The first one is oxidative changes following nitrate administration.
Intracellular thiols, such as GSH and cysteine, are involved in the conversion of nitrates to their bioactive metabolite ( NO). For example, in rat aortic cytosol, ROS in Ischemia/Reperfusion and Nitrate Tolerance glutathione S-transferases, which catalyze the conversion of NTG, were shown torequire glutathione as a cosubstrate (Nigam et al. 1996). The activity of the mito-chondrial aldehyde reductase, recently identified as another site of NTG bioactiva-tion, seems to depend also on sulfhydryl groups (Chen et al. 2002).
The second mechanism explaining the beneficial effects of thiol donors and other antioxidants on vascular relaxation is based on their O − erty. Thus, exogenous administration of the thiol compound N-acetylcysteine aug-mented the hypotensive effect of NTG even if intracellular arterial and venousthiol levels were similar in nitrate-tolerant and control animals (Boesgaard 1995).
Other antioxidants improving vascular responses to NTG (assessed by forearmplethysmography) in long-term nitrate-treated patients include vitamins C and E(Watanabe et al. 1997; Watanabe et al. 1998). Consistent with this effect of antiox- idants, increased O − generation was observed in nitrate-tolerant rabbit arteries (Munzel et al. 1995). In this model, the main site of its production appears tobe the vascular endothelium, since endothelial denudation decreased ROS levels.
Platelets of healthy volunteers also enhanced O − generation upon 3-day expo- sure to NTG (McVeigh et al. 2002). Both tolerance to nitrates and cross-toleranceto other vasodilators, ACh for example, are specific to long-term treatment, andcan be reversed by SOD administration. Treatment with liposomal SOD, which canact intracellularly, significantly enhanced relaxations to NTG in NTG-tolerant rab-bit aortic segments with endothelium (Munzel et al. 1995). However, as expected,administration of conventional SOD at lower concentration did not significantlyimprove vasodilatory responses of aortic strips in the same model (Nakae et al.
2003).
A third possible mechanism of nitrate tolerance involves downstream targets of .NO (Fig. 2) since impaired endothelium-dependent vasodilation to ACh was also observed following overexpression of eNOS in mice leading to elevated basal NOand cyclic guanosyl monophosphate (cGMP) (Ohashi et al. 1998; Yamashita et al.
2000). The expression of two downstream NO targets, soluble guanylyl cyclase(GC) and the cGMP-dependent protein kinase (cGK), remained unaltered uponlong-term nitrate intake (Schulz et al. 2002). GC activity was found increased ifNTG was administered in combination with cysteine (Artz et al. 2002). Thereforein the presence of thiol groups nitrate tolerance may result from impaired cGK ac-tivity. This enzyme catalyzes vasodilator-stimulated phosphoprotein (VASP) phos-phorylation and mediates vasorelaxation (Lincoln et al. 2001) (Fig. 2). A recentstudy showed decreased phospho-VASP (P-VASP) staining, a marker of cGK activ-ity, in arteries of patients previously treated with nitroglycerine. Their arteries alsoexhibited increased superoxide production (Schulz et al. 2002). Treatment withthe antioxidant vitamin C partially reversed tolerance, correlating with reduced vascular O − and elevated P-VASP levels (Mulsch et al. 2001). The peroxynitrite scavenger, ebselen, showed similar effects. In nitrate-tolerant aortic rings ebselen en-hanced P-VASP formation, decreased ROS levels and improved vascular responsesto NTG (Hink et al. 2003).
Other factors contributing to nitrate tolerance include neurohormonal counter- Figure 2. Downstream targets of NO. NO, produced by NOS, activates the targetenzyme – soluble guanylyl cyclase – and increases tissue levels of cGMP. cGMP activates acGMP-dependent protein kinase that mediates vasorelaxation. NOS, nitric oxide synthase;GC, soluble guanylyl cyclase; cGK, cGMP-dependent protein kinase; VASP, vasodilator-stimulated phosphoproteinf.
regulatory mechanisms activated by the fall in blood pressure upon NO intake.
One of these is the renin-angiotensin system (RAS). Its inhibition by angiotensinconverting enzyme inhibitors or AT1 receptor blockers ameliorated nitrate tolerance(Berkenboom et al. 1999; Kurz et al. 1999).
Thus, several mechanisms may contribute to nitrate tolerance (reviewed in de- tail by Leopold and Loscalzo 2003). These mechanisms are not mutually exclusive,while increased ROS production is considered the main factor. Neurohormonal ac-tivation leads to stimulation of the renin angiotensin system, thus increasing ROSformation, which in turn deplete intracellular sulfhydryl groups. Increased oxida-tive stress is associated with decreased cGK activity, which manifests as endothelialdysfunction.
Sources of ROS in blood vessels Multiple ROS are produced in the vascular wall, such as O − (Fig. 1). These molecules, if present in physiological concentrations, have importantfunctions in homeostasis. The primary oxygen-derived free radical produced in the vasculature appears to be O − 2 , which is formed by univalent reduction of molecular oxygen (3O2). This radical is involved in signal transduction pathways leading forexample to vascular smooth muscle proliferation, migration and hypertrophy (Cai ROS in Ischemia/Reperfusion and Nitrate Tolerance and Harrison 2000). These effects are mediated either directly by O − Dismutation of O − 2 by SOD or nonenzymatic univalent reduction of this rad- ical generates H2O2, which acts as a second messenger (Griendling et al. 2000a)and also regulates vascular tone. Depending on the concentration and the type ofartery, H2O2 can induce either contraction (Sotnikova 1998; Nowicki et al. 2001) orrelaxation (Matoba et al. 2000). H2O2 may be inactivated by conversion to waterand molecular oxygen in a reaction catalyzed by glutathione peroxidase or catalase.
In the presence of metal ions, H2O2 can give rise to OH (Fenton reaction). This very reactive radical participates in lipid hydroperoxidation and leads to injury ofendothelial cells, for example upon reperfusion (Kontos 2001). On the other hand, .OH may also induce relaxation of uninjured, norepinephrine-precontracted aortic strips (Prasad and Bharadwaj 1996). Since this effect of the OH generating systemis concentration-dependent, it is possible that certain physiological amounts of thisradical do not damage the endothelium.
There are many potential sources of cellular O − 2 , including membrane and mitochondrial NAD(P)H oxidases, cyclooxygenase, cytochrome P450, xanthine oxi-dase (XO) and NOS (Fig. 1). In unstimulated systems, non-mitochondrial NAD(P)H oxidases are a major source of O − in the vessel wall (Griendling et al. 2000b). In contrast to the neutrophil enzyme, vascular NAD(P)H oxidases areactivated more slowly and produce superoxide at a lower rate. However, both theneutrophil and vascular oxidases are flavoproteins and most likely have similarsubunit composition (Griendling et al. 2000b; Lassegue and Clempus 2003). Thephagocytic oxidase consists of 5 major subunits: gp91phox (which is also referredto as nox2) and p22phox are the membrane subunits; p47phox, p67phox and rac1are located in the cytoplasm (Babior et al. 2002) (Fig. 3). Most of these subunitsand some of their homologues were recently detected in vascular tissue. Homo-logues of the catalytic subunit gp91phox which are expressed in the vasculatureinclude nox1, nox4, nox5 and duox1 (Lassegue et al. 2001; Lassegue and Clempus2003). Homologues of the cytosolic subunits were also recently described (Banfi etal. 2003; Geiszt et al. 2003) but their expression in the vascular wall has not beenreported.
eNOS is another important source of O − in the vessel wall. Besides being the source of NO, this enzyme can produce O − and H2O2 in the absence of ei- ther L-arginine (L-Arg) or tetrahydrobiopterin (H4B) (Parker et al. 2002). In thisdysfunctional state, known as NOS uncoupling, reductive activation of molecu- lar oxygen to form O − is not followed by oxidation of L-Arg and NO synthesis (Landmesser and Harrison 2001).
Arachidonic acid metabolism is another potential source of ROS. Cyclooxy- genase, which converts arachidonic acid into prostaglandin, as well as lipoxyge-nase, responsible for leukotriene synthesis, can both contribute to ROS production(Kukreja et al. 1986). However, in the vessel wall these enzymes usually seem tobe insignificant sources of ROS, since their inhibitors, indomethacin and eicosate-traenoic acid, do not decrease superoxide production (Mohazzab and Wolin 1994).
Figure 3. Structure of the NAD(P)H oxidases. The phagocytic NAD(P)H oxidase iscomposed of 2 membrane subunits, nox2 (a. k. a. gp91phox, the catalytic component) andp22phox, and of 3 cytosolic subunits, p47phox, p67phox, p40phox, and of a small molecularweight G protein, rac. These components are also expressed in vascular cells, but theinteraction between them remains to be determined. In addition, novel members of the noxfamily of proteins, such as nox1, nox4 and nox5, seem to serve as catalytic components ofthe vascular oxidase. The catalytic component contains flavine adenine dinucleotide (FAD)and heme (H) groups, which participate in electron transfer. P, phosphate group; GTP,guanosyl triphosphate; NADPH, nicotinamide adenine dinucleotide phosphate. (Schemeadapted from Lassegue et al. 2001).
Microsomal cytochrome P450 enzymes (CYP), which convert arachidonic acid toepoxyeicosatrienoic acids (EET), can also generate varying amounts of oxygen-derived free radicals (Puntarulo and Cederbaum 1998). One of the CYP isoforms,CYP 2C9, identified as an EDHF synthase, is another source of ROS in the vas-culature. Expression of this enzyme was detected in porcine coronary endothelialcells, where it most likely contributes to basal ROS production (Fleming et al.
2001).
The xanthine oxidoreductase enzyme, which is involved in purine metabolism, is present in vascular tissue in two forms: xanthine dehydrogenase (XD) and xan-thine oxidase (XO) (Fig. 4). The constitutively expressed XD can be convertedin certain conditions to XO (reviewed in Meneshian and Bulkley 2002), which is capable of generating O − and H2O2 by reducing molecular oxygen.
Finally, the mitochondrial electron transport chain is another possible source of vascular ROS (Fig. 5). During oxidative phosphorylation, electrons are trans-ferred through complexes I–III to cytochrome oxidase (complex IV), which in nor-moxia reduces molecular oxygen to water. Under physiological conditions only asmall fraction of the oxygen consumed by mitochondria is converted to superoxide, ROS in Ischemia/Reperfusion and Nitrate Tolerance Figure 4. ROS formation by xanthine oxidase.
During purine metabolism, ATP is degraded toADP, AMP and adenosine, which is further me-tabolized to inosine and hypoxanthine. XO cat-alyzes the final step of this metabolic pathway, i.e.
the conversion of hypoxanthine to xanthine andxanthine to uric acid. In these reactions O2 servesas an electron acceptor. By its respective univalent or divalent reduction, O − and H2O2 are formed.
(Scheme adapted from Bauer et al. 2002). XD,xanthine dehydrogenase; XO, xanthine oxidase.
which is rapidly dismutated by MnSOD (Raha et al. 2000). In contrast in hypoxia,mitochondria seem to be an important source of ROS in endothelial cells (Pearl-stein et al. 2002). There are multiple possible sites for ROS generation within themitochondrial respiratory chain. For example, complexes I and III were identifiedas main sites of H2O2 generation (Kwong and Sohal 1998).
The enzymes just mentioned have been associated with vascular O − production. In addition, decreased activity of antioxidant enzymes also contributes Figure 5. ROS generation in mitochondria. Under physiological conditions, the free en-ergy of electron transfer from NADH and FADH2 to O2 is coupled to ATP synthesis.
Due to leakage from the mitochondrial electron transport chain, only a small amount of is produced, which is converted by MnSOD to H2O2. In ischemia, the inhibition of complex IV increases ROS production mainly by complex III. K.C., Krebs cycle; C I–V,mitochondrial complexes.
to elevated ROS levels in pathological conditions. Both prooxidant and antioxi-dant enzyme systems have an important role in the development of endothelialdysfunction in I/R injury and nitrate tolerance.
ROS sources in ischemia/reperfusion During ischemia and reperfusion, increases in various ROS, such as O − as well as reactive nitrogen species, e.g. ONOO− can be detected in tissues (Liand Jackson 2002). These reactive oxygen and nitrogen species are derived fromdiverse cellular and molecular sources in vessels. Both endothelial cells and circu-lating phagocytes produce ROS during I/R (Al-Mehdi et al. 1998; Kaminski et al. 2002). While endothelial cells produce O − continuously, neutrophils become more important with longer periods of reperfusion as they adhere to the endothe-lium and become activated (Fabian and Kent 1999). The most important source ofROS in phagocytic cells is the NADPH oxidase, which catalyzes the reduction of molecular oxygen to O − 2 . This radical in turn gives rise to other reactive oxygen metabolites, which can diffuse into endothelial cells and injure them. Endothelial cells themselves also produce ROS. For example, in cerebral arteries O − tion is increased 1–4 h after reperfusion. Subcellular microscopical examination shows that O − 2 is localized in cytosolic vesicles (Kim et al. 2002). Various enzymes are potential sources of ROS in the vessel wall. The involvement of each of theseoxidases to I/R injury will be presented in more detail in the following sections.
The reaction of XO with hypoxanthine and molecular oxygen to produce a burstof oxygen radicals was implicated in reperfusion models more than 20 years ago(Parks et al. 1983). Xanthine dehydrogenase, which uses NAD+ as a substrate,is converted under ischemic conditions to XO, which uses oxygen as an electronacceptor (substrate). Due to ATP consumption during hypoxia, hypoxanthine andxanthine are accumulated in the tissue, which upon reoxygenation are metabolized by XO and yield O − and H2O2 (Granger 1988) (Fig. 4). In the vessel wall, this conversion and increased activity of XO occurs in the endothelial layer during I/R.
Some of this enzyme also circulates in the blood and can bind to endothelial cells.
The activity of XO is increased for example in reoxygenated human umbilical veins 2 h after the hypoxic period (termed late reoxygenation) (Sohn et al. 2003).
Inhibition of this enzyme has protective effect in some models of I/R. Oxypurinolsimilarly to SOD decreases brain cortical endothelial cell damage caused by 10-hanoxia and 4-h reoxygenation, as measured by lactate dehydrogenase efflux intothe culture medium (Beetsch et al. 1998). Another XO inhibitor, allopurinol, alsorestores venodilation responses to ACh in rats (Flynn et al. 1999).
However, I/R injury also occurs when XO activity is low or absent (Muxfeldt and Schaper 1987). This is the case in patients undergoing coronary bypass surgery.
At the end of the ischemic period, and also 30 min following clamp removal, theratio of reduced/oxidized glutathione (GSH/GSSG) in the plasma is decreased, ROS in Ischemia/Reperfusion and Nitrate Tolerance indicating increased oxidative stress. Levels of hypoxanthine were increased in theplasma collected from the coronary sinus. Nevertheless, xanthine and uric acidlevels were found to be low, suggesting low XO activity (Carlucci et al. 2002).
Thus, the role of XO in I/R injury is variable. This oxidase seems to be im- portant for example in human umbilical vein reperfusion (Sohn et al. 2003), inmaintaining optimal mesenteric artery microcirculation upon resuscitation fromhemorrhagic shock (Flynn et al. 1999) and in the in vitro model of cerebral en-dothelial cell reoxygenation (Beetsch et al. 1998). On the other hand, there areseveral reports, where the contribution of XO is not critical for ROS generationin I/R. This suggests involvement of other enzyme systems, as discussed in thefollowing sections.
Mitochondrial oxidase Absence of the final electron acceptor increases ROS production in the mitochon-dria by complexes I–III (Fig. 5), of which complex III seems to be the most impor-tant (Pearlstein et al. 2002; Waypa and Schumacker 2002). Increased ROS levels ob-served in endothelial cells during hypoxia could be due to inhibition of cytochrome oxidase (complex IV) activity and increased O − 2 production by the ubisemiquinone at complex III (Pearlstein et al. 2002). O − and H2O2 released to the cytoplasm through anion channels and by diffusion, respectively, can act as intracellular signal-ing messengers linking tissue hypoxia to the subsequent responses. Mitochondria,by means of ROS production have been suggested to function as oxygen sensors inpulmonary artery endothelial cells and myocytes. Although the exact mechanismis still being debated, ROS derived from the mitochondria are thought to medi-ate vasoconstriction in hypoxic pulmonary arteries, thereby decreasing blood flowthrough hypoventilated alveoli (Waypa and Schumacker 2002).
In contrast to these signaling roles, ROS produced by the mitochondria during hypoxia/reoxygenation have harmful effects. Increased endothelial cell permeabil-ity is related to hypoxia-induced oxidative stress (Pearlstein et al. 2002). Exposure to hypoxia for 6 h followed by a 45-min reoxygenation period increased O − eration in bovine brain endothelial cells. Excess O − production was prevented by blocking the first two complexes of the mitochondrial electron transport chain, sug-gesting that mitochondria are the main sources of ROS formed during reoxygena-tion (Kimura et al. 2000). A negative correlation between the number of perfusedpostischemic sinusoids and the mitochondrial redox state also indicates an impor-tant role of mitochondria in hepatic microcirculation (Glanemann et al. 2003).
Decreased activity of antioxidant mitochondrial enzymes, such as MnSOD, further increases ROS release during hypoxia. Cardiac I/R significantly reducedmitochondrial SOD and glutathione peroxidase activities, so that enzymatic degra- dation of O − and H2O2 was compromised (Shlafer et al. 1987). There are thus two mechanisms, inhibited electron transfer through cytochromes and decreasedantioxidant enzymatic activity, leading to excessive mitochondrial ROS productionduring I/R.
Nitric oxide synthase In the absence of its substrate and/or cofactor NOS was shown to produce O − H2O2 (Vasquez-Vivar et al. 1998). Administration of the substrate L-Arg decreasedcoronary vascular resistance, increased blood flow and thereby reduced the extentof myocardial I/R injury (Agullo et al. 1999; Pernow and Wang 1999). Infusion of .NO donors had a similar effect on the ischemic myocardium, indicating that main- tenance of NO release is an important factor in protecting tissues from reperfusioninjury. In an in vitro model of vascular hypoxia and reoxygenation, incubation ofhypoxic endothelial cells with L-Arg decreased ferryl hemoglobin formation in themedium, which is a measure of oxidative stress (D'Agnillo et al. 2000). A recentstudy showed that L-Arg may provide protection against I/R injury in transplan-tations as well as in routine cardiac surgery. This was demonstrated in a model ofrat heart transplantation, where polymers of L-Arg improved coronary flow andreduced myocardial oxidative stress (Kown et al. 2003).
.NO synthesis also depends on the availability of H4B, a NOS cofactor modu- lated by the redox state of the cell. H4B is decreased in several pathophysiologicalconditions, including coronary artery disease and I/R. Deficiency of H4B seems toaccelerate endothelial dysfunction and myocardial I/R injury, while pretreatmentwith this cofactor reduced functional and metabolic abnormalities (Yamashiro etal. 2003).
In contrast to short ischemia, eNOS upregulation and increased NO formation was detected in endothelial cells exposed for 8 h to mild hypoxia (Sohn et al. 2003).
Long-term hypoxic conditions thus may lead to increased NO levels by activationof antioxidant enzymes and NOS upregulation.
Hypoxia alone or followed by reoxygenation also increased O − in endothelial cells (D'Agnillo et al. 2000; Kimura et al. 2000). As soon as it is produced, O − acts as a scavenger of NO to which it combines to form ONOO−.
This radical is more potent in oxidizing H 4B than O2 and uncouples NOS (Laursen et al. 2001). Therefore, O − production by eNOS appears to be a self-induced mechanism triggered by interaction with a primary O − source. An important candidate for such interaction is the non-phagocytic NAD(P)H oxidase.
The role of the vascular NAD(P)H oxidase in I/R injury can be studied with thehelp of inhibitors, such as diphenyleneiodonium (DPI), apocynin, as well as a natu-ral proline- and arginine-rich antibactetrial peptide (PR-39). While DPI inhibits allflavoproteins, the latter two are more specific since they prevent oxidase assemblyby binding to the cytosolic subunit p47phox. For example, apocynin concentration- dependently inhibited NADH-stimulated O − 2 production in rat aorta, thus increas- ing NO bioavailability and vasorelaxation (Hamilton et al. 2002).
Endothelial cells exposed to 24-h hypoxia showed an almost 3-fold increase in H2O2 production immediately upon reperfusion, which was not reversed by in-hibitors of XO, cyclooxygenase, NOS, CYP450 or mitochondrial electron transport ROS in Ischemia/Reperfusion and Nitrate Tolerance (Zulueta et al. 1995). In contrast, DPI, and/or the protein kinase C (PKC) in-hibitor calphostin, reduced H2O2 release in whole endothelial cells as well as ina membrane fraction (Zulueta et al. 1995). These results suggest that a PKC-dependent flavoprotein, such as NAD(P)H oxidase, is involved in hypoxia-induced In isolated sheep lungs, 30 min of ischemia followed by 180 min of reperfu- sion increased vascular permeability, which was prevented by preincubation withapocynin (Dodd and Pearse 2000). High concentrations (0.3 and 3 mmol/l) of thisNAD(P)H oxidase inhibitor increased pulmonary artery pressure observed in I/Rspecimens after 30-min reperfusion. In the same model, apocynin or DPI adminis- tration decreased O − production in the blood (Dodd and Pearse 2000), possibly by inhibiting both the neutrophil and vascular NAD(P)H oxidase.
In rat liver, I/R obtained by hepatic artery and portal vein clamping induced a biphasic increase in ROS production (Ozaki et al. 2000). The early oxidative burstin this tissue occurred within 5 min of reperfusion in the absence of neutrophils,suggesting that ROS were produced by the ischemic tissue itself. This increase insuperoxide production was inhibited by SOD and dominant-negative rac1, while gp91phox deficiency had no effect on O − production. Therefore a rac1 containing oxidase, which is different from the neutrophil oxidase, such as one of the vascular homologues, appears to be responsible for the early increase in O − hepatic tissue during I/R (Ozaki et al. 2000).
The production of ROS in vascular smooth muscle cells also seems to re- quire the small GTP-binding protein rac1. Primary cultures of rat vascular smoothmuscle cells showed a 10-fold increase in ROS production after 16 h/5 min hy-poxia/reoxygenation, which was reversed by rac1 inhibition using dominant-negati-ve adenovirus transduction (Kim et al. 1998). Similarly, ROS production, detectedby DCF, was inhibited when the cells were preincubated with N-acetylcysteine,catalase or DPI (Kim et al. 1998). Although this method of ROS measurementdetects predominantly H2O2, results from electron spin resonance measurements showed that rac1 increased O − generation, which is subsequently dismutated to H2O2 (Irani et al. 1997). Thus, rac1 as a component of the NAD(P)H oxidase seems to contribute to increased O − 2 production in vascular smooth muscle during ROS production is similarly increased in a model of lung ischemia, called – because of maintained ventilation – "oxygenated ischemia". Blocking lung perfu-sion for 1 h led to a 7-fold increase in DCF fluorescence indicating H2O2 gen-eration (Al-Mehdi et al. 1998). Tissue infiltration by phagocytes was low in thismodel, and fluorescence was localized predominantly to endothelial cells. PR-39dose dependently inhibited ROS generation and tissue lipid peroxidation. Thus anon-phagocytic NAD(P)H oxidase appears to be responsible for ROS production.
Multiple cellular sources of ROS may account for their increased generation during I/R. XO was identified as the first enzyme producing excess O − studies demonstrated the important contribution of the mitochondrial respiratorychain, NAD(P)H oxidase and eNOS to ROS-induced reperfusion injury. A low activity of antioxidant enzymes, such as GSH peroxidase, GSH reductase, SOD,and catalase was also observed after prolonged (24–48 h) endothelial cell hypoxia(Plateel et al. 1995). Overall, increased ROS production by oxidases and decreasedantioxidant capacity lead to injury in I/R.
Maintaining the balance between NO and ROS is important to protect blood vessels from I/R-induced injury. NO prevents neutrophil infiltration, has anti-platelet activating factor properties, preserves vascular permeability and endothe- lial function. Treatments increasing NO bioavailability, for example L-Arg, H4B, or simply NO donors, have great therapeutic potential in conditions associatedwith I/R. However, long-term nitrate administration can also lead to increasedROS formation, as further elaborated.
ROS sources in nitrate tolerance In nitrate tolerant arteries, the production of O − and its metabolite ONOO− are increased. The main source of O − appears to be the endothelium (Munzel and Harrison 1997), although, as shown by dihydroethidium staining, other compo- nents of the vessel wall also produce O − 2 (Schulz et al. 2002). Positive staining for nitrotyrosine indicates that ONOO− formation is also increased throughout thevessel wall (Mihm et al. 1999). Moreover, platelets are other potential sources of in nitrate tolerance (McVeigh et al. 2002).
Two enzymes are important sources of increased ROS production in nitrate tolerance. Firstly, uncoupled endothelial NOS and secondly, the vascular NAD(P)H oxidase, which releases low concentrations of O − into the cytoplasm and further alters the physiological function of NOS.
Nitric oxide synthase Recent studies suggest that prolonged NTG treatment induces a dysfunctional stateof NOS (Munzel et al. 2000). In healthy vessels, ACh induces vasodilation medi- ated by NO release, which is blocked by NOS inhibitors. For example, the L-Argantagonists N-monomethyl-L-Arg (L-NMMA), Nω-nitro-L-arginine (L-NNA) and L-nitroarginine methyl ester (L-NAME) decrease vasorelaxation and increase O − production in healthy large arteries (for references see Table 1). In contrast, whenapplied to nitrate-tolerant vessels NOS inhibitors improved responses to ACh, sug- gesting that NOS was uncoupled, thus producing the vasoconstrictor O − than NO (Gori et al. 2001). Similar results were obtained in cultured endothelialcells exposed to NTG (Kaesemeyer et al. 2000). Vascular responses to L-NAMEwere, however, restored when the animals were pretreated with low doses of pravas- tatin or atorvastatin, most likely due to their O − lowering effect (Fontaine et al.
Both O − 2 and ONOO− can oxidize H4B to H2B, and thereby uncouple eNOS (Landmesser et al. 2003). SOD administration decreases tissue superoxide levelsand improves relaxation (Munzel et al. 1995). Similarly, increased ROS produc-tion in nitrate-tolerant vessels can be reversed by supplementation with L-Arg, Table 1. Effect of nitric oxide synthase (NOS) inhibitors on O − production and endothelium-dependent vasorelaxation Untreated vessels Nitrate-tolerant vessels ↑(Guzik et al. 2002) ↓(Ishibashi et al. 2001) ↑(Gori et al. 2001) ↑(Boger et al. 2000) ↓(Gori et al. 2001) ↓(Heitzer et al. 2000) ↑(Oelze et al. 2000) ↓(Yada et al. 2003 ↓(Munzel et al. 2000) ↑(Gruhn et al. 2001) ↑(Munzel et al. 2000) ↓(Matoba et al. 2000) ↑(Mollnau et al. 2002) ↑(Laursen et al. 2001) ↓(Guzik et al. 2002) ↓(Kaesemeyer et al. 2000) EC ↑(Nakae et al. 2003) ↑(Muzaffar et al. 2003) ↓(Ruiz et al. 1997) ↑(Fontaine et al. 2003) statins ↓(Yamashita et al. 2000) ↓(Imaoka et al. 1999) ↓(Fontaine et al. 2003) statins EC, endothelial cells.
H4B, or antioxidant folates. Depleted intracellular stores of L-Arg are thought tocontribute to abnormal NOS activity in nitrate-tolerant patients, as suggested bythe fact that supplementation with L-Arg improves NOS dysfunction caused by nitrates (Parker et al. 2002). In vivo administration of H 4B also reduced O2 pro- duction (Vasquez-Vivar et al. 1998) and improved NO availability (Tiefenbacher2001). Similarly, H4B or folic acid can prevent endothelial dysfunction induced bycontinuous nitroglycerin treatment in healthy volunteers (Gori et al. 2001; Goriet al. 2003; Leopold and Loscalzo 2003). Several mechanisms have been suggested to explain the protective function of folates. First, the O − scavenging capability of folates could prevent NOS uncoupling. Second, folates may stabilize H4B andimprove its regeneration from H2B. Third, they could affect eNOS directly and increase the production of NO, rather than of O − (Verhaar et al. 2002).
The function of NOS depends on other ROS producing enzyme systems of the vessel wall, which by providing O − and ONOO− may oxidize H4B to H2B thereby inducing NOS uncoupling. The vascular NAD(P)H oxidase, for example, is suggested to play such role by generating low amounts of O − 2 , the "kindling radical" (Landmesser et al. 2003).
Increased O − production in aortic rings of nitrate-tolerant rabbits can be nor- malized by DPI, suggesting that O − is generated by a flavoprotein (Munzel et al.
1995). Specific inhibitors of several vascular oxidases (including flavoproteins, e.g.
NOS, XO, mitochondrial oxidase and CYP450), however, failed to affect O − duction. In contrast, addition of enzyme substrates NADH or NADPH to nitrate- tolerant rat aorta increased O − 2 -derived chemiluminescence (Fontaine et al. 2003).
The lipid-lowering statins showed beneficial effect on endothelial function in pa- tients receiving long-term nitrate treatment (Inoue et al. 2003). Statins lower O − production in nitrate tolerant vessels without increasing eNOS abundance, indi- cating that they improve vasorelaxation by decreasing O − 2 production rather than by directly increasing NO synthesis (Fontaine et al. 2003). Indeed, atorvastatininhibited the expression and activation of NAD(P)H oxidase (Wassmann et al.
2002). Responses to vasodilators and O − 2 lowering properties of statins were found impaired in the presence of NADH or NADPH (Fontaine et al. 2003). On balance,these data indicate that an NAD(P)H oxidase is activated in nitrate tolerance.
The mechanism by which NAD(P)H oxidase becomes activated may involve neurohumoral pathways (Munzel and Harrison 1997; Mollnau et al. 2002). Theiractivation is due to the fall in blood pressure caused by vasodilation upon nitrateintake. Increased plasma renin activity in nitrate-tolerant patients indicates reninangiotensin system (RAS) activation (Munzel et al. 1996). Angiotensin II is knownto increase NAD(P)H oxidase activity (Lassegue et al. 2001), and this activationseems to require PKC (Mollnau et al. 2002). In nitrate tolerance, PKC inhibitorsprevented the increased vasoconstriction to phenylephrine and improved relaxationto NTG (Zierhut and Ball 1996). Conversely, the PKC activator phorbol ester ROS in Ischemia/Reperfusion and Nitrate Tolerance 12,13-dibutyrate (PDBu) induced significantly increased vasoconstriction in NTGtolerant compared to control arteries (Munzel and Harrison 1997).
Nonintermittent nitrate administration was accompanied by abnormal platelet activation, resulting in their increased O − release (McVeigh et al. 2002). Ad- dition of NADH increased, whereas DPI abolished O − production. Oxypurinol, rotenone and indomethacine had no effect on platelet NADH oxidoreductase activ-ity (McVeigh et al. 2002). These results indicate that a platelet oxidase similar to the vascular NAD(P)H oxidase may be an additional source of O − nitrate treatment.
Thus NAD(P)H oxidases contained in the vessel wall and in platelets seem to be important contributors to elevated ROS production that accompanies nitrate tolerance. The product of these enzymes, O − 2 as well as ONOO−, uncouple eNOS 4B, which in turn leads to increased O2 production. Therefore, interaction of these enzymes is involved in development of nitrate tolerance.
Summary and therapeutic implication Increased ROS production complicates clinical conditions associated with I/R andalso the continuous nitrate administration used for the treatment of recurrent is-chemic heart attacks. The first sign of vessel impairment under these circumstancesis endothelial dysfunction. ROS are known to play an important role in its devel-opment.
Several enzymes in the vessel wall are involved in O − overproduction in these conditions. Even small increases in the release of ROS, such as O − ONOO−, may have an impact on endothelial physiology as they can uncoupleeNOS (Landmesser and Harrison 2001).
Antioxidant vitamins or folates, due to their low toxicity and minimal side effects, may be useful tools in preventing I/R injury and nitrate tolerance, sincethey reverse endothelial dysfunction (Verhaar et al. 2002; Erbs et al. 2003). SODhas also a beneficial effect in vascular pathologies accompanied by increased ROSproduction. However, other studies reported an inconsistent outcome of antioxidantsupplementation and the native SOD has been found unsuitable for clinical use.
Recent data suggest that the production and metabolism of ROS is compart- mentalized within the cell (Lassegue and Clempus 2003). One of the reasons whyexogenous antioxidants are not consistently effective in restoring endothelial func-tion in I/R or nitrate tolerance could be their low ability to penetrate cellularmembranes. Newly synthetized SOD mimetics are proposed to overcome this limi-tation, since they are small molecules and cross membranes easily (Salvemini andCuzzocrea 2003).
However, it is important to note that ROS have a dual function in the vascula- ture. This may be another reason that systemic delivery of antioxidant enzymes isnot always optimal. Although ROS are often considered toxic, they also have phys-iological roles in the endothelium. H2O2 for example is a potential candidate forEDHF in human mesenteric (Matoba et al. 2002) and coronary circulation (Yada et al. 2003). Besides the role of O − and H2O2 on vascular relaxation, ROS also have signaling functions (Griendling et al. 2000a), they can for example stimulateendothelial cell migration and angiogenesis (Ushio-Fukai et al. 2002). Thus, ROScan act as messengers during coronary collateral development in the ischemic my-ocardium, as shown with frequently repeated brief occlusions and reperfusion ofthe artery (Gu et al. 2003).
Targeted delivery of antioxidant drugs is an interesting approach to face the harmful effects of ROS. With cell specific antibodies (immunotargeting) antioxi-dant enzymes can be delivered to the sites of increased ROS production. Since theendothelium is an important source of ROS during ischemia and also in nitrate tol-erant vessels, endothelial cell targeting can be used to prevent vascular dysfunction(Kozower et al. 2003). Another way of specific targeting is to direct pharmacologi-cal agents to specific enzymes (for example to inhibit oxidases responsible for ROSoverproduction). Recognizing the exact enzymatic sources is therefore importantfor designing specific therapies.
Acknowledgements. I am grateful to Dr. Bernard Lass egue for his comments and helpful suggestions upon reading the manuscript. This work was supported by grants: APVT 20-02802 and VEGA 2/2052/22.
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