Vascular Consequences of Endothelial Nitric Oxide Synthase Uncoupling for the Activity and Expression of the Soluble Guanylyl Cyclase and th
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动脉硬化血栓血管生物学 2005年第8期
From II Medizinische Klinik (T.M., A.D., A.M.), Mainz Kardiologie und Angiologie Mainz, Germany; and Fachbereich Biologie (V.U.), Universit?t Konstanz, Germany.
Correspondence to Thomas Münzel, MD, Professor of Internal Medicine, II Medizinische Klinik und Poliklinik Johannes Gutenberg-Universit?t Mainz, Langenbeckstrasse 1, D-55131 Mainz, Germany. E-mail tmuenzel@uni-mainz.de
Series Editor: Kathy K. Griendling
Redox Mechanisms in Blood Vessels
ATVB in Focus
Previous Brief Reviews in this Series:
?Mueller CFH, Laude K, McNally JS, Harrison DG. Redox mechanisms in blood vessels. 2005;25:274–278.
?Gutterman DD, Miura H, Liu Y. Redox modulation of vascular tone: focus of potassium channel mechanisms of dilation. 2005;25:671–678.
?Nicholls SJ, Hazen SL. Myeloperoxidase and cardiovascular disease. 2005;25:1102–1111.
?Leopold JA, Loscalzo J. Oxidative enzymopathies and vascular disease. 2005;25:1332–1340.
Abstract
Endothelial dysfunction in the setting of cardiovascular risk factors, such as hypercholesterolemia, hypertension, diabetes mellitus, chronic smoking, as well as in the setting of heart failure, has been shown to be at least partly dependent on the production of reactive oxygen species (ROS), such as the superoxide radical, and the subsequent decrease in vascular bioavailability of nitric oxide (NO). Superoxide-producing enzymes involved in increased oxidative stress within vascular tissue include the NAD(P)H oxidase, the xanthine oxidase, and mitochondrial superoxide-producing enzymes. Superoxide produced by the NADPH oxidase may react with NO released by endothelial nitric oxide synthase (eNOS), thereby generating peroxynitrite. Peroxynitrite in turn has been shown to uncouple eNOS, thereby switching an antiatherosclerotic NO-producing enzyme to an enzyme that may initiate or even accelerate the atherosclerotic process by producing superoxide. Increased oxidative stress in the vasculature, however, is not restricted to the endothelium and has also been demonstrated to occur within the smooth muscle cell layer in the setting of hypercholesterolemia, diabetes mellitus, hypertension, congestive heart failure, and nitrate tolerance. Increased superoxide production by the endothelial and/or smooth muscle cells has important consequences with respect to signaling by the soluble guanylyl cyclase (sGC) and the cGMP-dependent protein kinase I (cGK-I), the activity and expression of which has been shown to be regulated in a redox-sensitive fashion. The present review summarizes current concepts concerning eNOS uncoupling and also focuses on the consequences for downstream signaling with respect to activity and expression of the sGC and cGK-I in various diseases.
Key Words: endothelium ? vasodilation ? nitric oxide ? endothelial NO-synthase ? oxidative stress
Introduction
Traditionally, the role of the endothelium was thought primarily to be that of a selective barrier to the diffusion of macromolecules from the vessel lumen to the interstitial space. During the past 20 years, numerous additional roles for the endothelium have been defined such as regulation of vascular tone, modulation of inflammation, promotion, and inhibition of vascular growth and modulation of platelet aggregation and coagulation. Endothelial dysfunction is a characteristic feature of patients with coronary atherosclerosis and more recent studies indicate that it may predict long-term atherosclerotic disease progression as well as cardiovascular event rate.1 Although the mechanisms underlying endothelial dysfunction may be multifactorial, there is a growing body of evidence that increased production of reactive oxygen species (ROS) may contribute considerably to this phenomenon. ROS production has been demonstrated to occur in the endothelial cell layer, but also within the media and adventitia, all of which may impair nitric oxide (NO) signaling within vascular tissue to endothelium-dependent, but also endothelium-independent, vasodilators. More recent experimental, but also clinical, studies point to a crucial role of endothelial nitric oxide synthase (eNOS) as a superoxide-producing enzyme in the setting of atherosclerosis, hypertension, congestive heart failure, and also nitrate tolerance. Figure 1 summarizes our observations that in all these pathophysiological settings vascular oxidative stress, as visualized by dihydroethidine-dependent fluorescence, is present within the endothelium, but also the smooth muscle cell layer and the adventitia, all of which plays an important role for the development of endothelial and/or vascular dysfunction. This review briefly addresses mechanisms underlying eNOS uncoupling and focuses on the consequences with respect to the activity and/or expression of the NO target, the soluble guanylyl cyclase (sGC), and the cGMP-dependent protein kinase I (cGK-I) present in vascular smooth muscle.
Figure 1. Detection of vascular superoxide formation by the fluorescence dye dihydroethidine in aortic tissue sections from different oxidative stress animal models and in the mammary artery from nitroglycerin (NTG)-treated patients. CHF indicates congestive heart failure; hypertension: angiotensin (AT II)-infused rats vs sham-treated Wistar rats; atherosclerosis: hyperlipidemic Watanabe rabbits (WHHL) vs New Zealand White rabbits (NZWR); diabetes: streptozotocin (STZ)-treated rats vs sham-treated Wistar rats; nitrate tolerance: NTG-infused patients vs patients without NTG treatment.
The L-Arginine/NO/cGMP Pathway in Vascular Tissue
The endothelium, a single-layered continuous cell sheet lining the luminal vessel wall, separates the intravascular (blood) from the interstitial compartment and the vascular smooth muscle. Based on cell count (6x1013), mass (1.5 kg), and surface area (1000 m2), the endothelium is an autonomous organ. Though for a long time regarded as a passive barrier for blood cells and macrosolutes, this view completely changed with the advent of endothelial autacoids like prostacyclin2 and NO,3 as well as with the discovery of integrins and other surface signals.4 It is now evident that the endothelium is at the cross-bridges of communication between blood and tissue cells and actively controls this process and the function of surrounding cells by a plethora of signaling routes. One of the prominent communication lines is established by the so-called L-arginine-NO-cyclic GMP pathway.5 This signaling cascade starts with eNOS (NOSIII), which generates NO and L-citrulline from L-arginine and O2 in response to receptor-dependent agonists (bradykinin, acetylcholine, ATP) and physicochemical stimuli (shear, stretch).6 Reducing equivalents are provided by NADPH, and electrons are passed via a flavin chain to the catalytic center, the enzymes heme iron (Figure 2, upper). The first step of the normal NOS reaction is a classical mono-oxygenation, which consumes 1 mol of NADPH and oxygen. Molecular oxygen bound to the iron is activated and split to accomplish a hydroxylation of the substrate, the guanidino-nitrogen of L-arginine, forming NG-hydroxy-L-arginine. The second step is an atypical mono-oxygenation, which consumes 1 mol O2 and 0.5 mol NADPH. It is a 3-electron oxidation of NG-hydroxy-L-arginine to afford the final products NO and L-citrulline. To guarantee this reaction path, the enzyme has to be homodimeric and the cofactor tetrahydrobiopterin must be present (Figure 2). For details, the reader is referred to excellent reviews on this topic.7,8
Figure 2. Scheme depicting electron flow in coupled vs uncoupled eNOS. Electron flow starts from NADPH to flavins FAD and FMN of the reductase domain, which delivers the electrons to the iron of the heme (oxygenase domain) and to the BH3· radical generated as an intermediate in the catalytic cycle. BH4 seems to be essential to donate an electron and proton to versatile intermediates in the reaction cycle of L-arginine/O2 to L-citrullin/NO. Calmodulin (CAM) controls electron flow in eNOS. Zinc ions (Zn) bound to NOS are required for dimer formation and stability. Monomeric eNOS or BH4/L-arginine–deficient eNOS is uncoupled and produces O2– (for explanation see text).
NO diffuses to the adjacent smooth muscle where it interacts with different receptor molecules, of which the sGC is the best characterized and presumably most important one with regard to control of vessel tone and smooth muscle proliferation. The catalytically active holoenzyme exists as an obligate heterodimer consisting of 1 and 1 ? subunit and a noncovalently bound ferrous heme.9 The most abundant isoform in mammalian tissues is the 1 (73 to 78 kDa)/?1 (70 kDa) heterodimer. Significant protein expression of 2 other homologues subunits has only been detected in the human placenta (2)10,11 and kidney (?2).12 At the genomic level, a dominant-negative splice variant of 2 classified as 2i has been detected.13 The interspecies homology of the individual subunits is high, whereas the intersubunit homology is lower.14 In contrast to 1 and ?1, the biological significance of 2, 2I, and ?2 is still obscure. The 2 subunit has recently been shown to be linked to cerebral maturation and sensory pathway refinement during postnatal development.15
A unifying concept of the molecular requisites for sGC activation has been put forth.16,17 Activation by NO requires sGC heme iron to be in the ferrous (II) state. On NO binding, the iron is moved slightly out of the porphyrin plane, thereby releasing a distal histidine (His105 of the ?1 subunit) from iron coordination.18 This triggers subsequent intramolecular re-arrangements influencing the catalytic center, resulting in an up to several-hundred-fold increase in cGMP formation. Depending on the cell type, cyclic GMP then elicits different biological responses, either by inhibition (cAMP-metabolizing phosphodiesterases) or by activation (cGMP-activated protein kinases, cGK, and cGMP-gated cation channels) of effector proteins.19 A comprehensive overview on cGK-I downstream signaling is provided.19 The mammalian cGK family consists of cGK-I, a splice variant cGK-I?, and cGK-II, which is encoded by a separate gene.19 Both cGK-I isoforms, not cGK-II, are expressed in vascular cells. In rat aortic smooth muscle cells, activated cGK-I increases the open probability of Ca2+-activated K+(BK) channels, thereby inducing a hyperpolarization of the smooth muscle cells and inhibition of agonist-induced Ca2+ influx. In addition, activated cGK-I? phosphorylates the inositol triphosphate receptor-associated G-kinase substrate, thereby inhibiting agonist-induced Ca2+ release and smooth muscle contraction. Another cGK-I substrate found in many cell types is the 46/50 kDa vasodilator-stimulated phosphoprotein. cGK-I phosphorylates vasodilator-stimulated phosphoprotein specifically at serine 239, and this reaction can be exploited as a biochemical monitor for the integrity and activity of the NO-cGMP pathway,20 as discussed. Phosphorylation and activation of cGMP-hydrolyzing phosphodiesterase 5 by cGK-I is a major mechanism to cease cGMP signaling.19,21
Oxidative Stress and Endothelial Dysfunction
The endothelium-derived relaxing factor, previously identified as NO3 or a closely related compound,22 has potent antiatherosclerotic properties. NO released from endothelial cells works in concert with prostacyclin to inhibit platelet aggregation,23 it inhibits the attachment of neutrophils to endothelial cells and the expression of adhesion molecules. NO in high concentrations inhibits the proliferation of smooth muscle cells.24 Therefore, under all conditions in which an absolute or relative NO deficit is encountered, the process of atherosclerosis is being initiated or accelerated. The half-life of NO and therefore its biological activity is decisively determined by oxygen-derived free radicals such as superoxide.25 Superoxide rapidly reacts with NO to form the highly reactive intermediate peroxynitrite.26 The rapid bimolecular reaction between NO and superoxide yielding peroxynitrite (rate constant: 5 to 10x109 M–1s–1) is 3- to 4-times faster than the dismutation of superoxide by the superoxide dismutase. Therefore, peroxynitrite formation represents a major potential pathway of NO reactivity pending on the rates of tissue superoxide production. Peroxynitrite in high concentrations is cytotoxic and may cause oxidative damage to proteins, lipids, and DNA.26 Recent studies also indicate that peroxynitrite may have deleterious effects on activity and function of the prostacyclin synthase27 and the eNOS.28 Other ROS such as the dismutation product of superoxide, hydrogen peroxide, and the hypochlorous acid released by activated neutrophils, are not free radicals, but have a powerful oxidizing capacity, which will further contribute to oxidative stress within vascular tissue.
Endothelial Dysfunction and Cardiovascular Risk Factors
It is well known that in the presence of cardiovascular risk factors endothelial dysfunction is frequently encountered. This has been shown for chronic smokers, patients with increased low-density lipoprotein levels, for patients with diabetes types I and II, for hypertensive patients, and for patients with metabolic syndrome. There are several potential abnormalities, which could account for reductions in endothelium-dependent vascular relaxation including changes in the activity and/or expression of the eNOS, decreased sensitivity of vascular smooth muscle cells to NO, or increased degradation of NO via its interaction with ROS such as superoxide. The NO degradation concept is the most attractive one because in the presence of cardiovascular risk factors, endothelial dysfunction is established and even more importantly it is markedly improved by the acute administration of the antioxidant vitamin C.29–32 Superoxide and/or peroxynitrite could also act further downstream by oxidative inactivation of sGC as well as activation of cGK-I.
eNOS Uncoupling Contributes to Endothelial Dysfunction
In most situations in which endothelial dysfunction caused by increased oxidative stress is encountered, the expression of the eNOS has been shown to be paradoxically increased rather than decreased.33–36 The mechanisms underlying increased expression of eNOS are likely to be secondary to increased endothelial levels of hydrogen peroxide, which has been shown to increase the expression at the transcriptional and translational level.37 The demonstration of endothelial dysfunction in the presence of increased expression of eNOS indicates that the capacity of the enzyme to produce NO may be limited. Very intriguing are observations that the eNOS itself can be a superoxide source, thereby causing endothelial dysfunction. It has become clear from studies with the purified enzyme that eNOS may become "uncoupled," eg, in the absence of the NOS substrate L-arginine or the cofactor tetrahydrobiopterin (BH4). In such uncoupled state, electrons normally flowing from the reductase domain of one subunit to the oxygenase domain of the other subunit are diverted to molecular oxygen rather than to L-arginine,38,39 resulting in production of superoxide rather than NO (Figure 2). That eNOS-derived superoxide/ROS formation bears consequences opposite to "normal" NOS function was first shown in U937 cells transfected with wild-type eNOS, which induced increased transcription of tumor necrosis factor- in an NOS inhibitor (L-NMA)-insensitive and superoxide-dismutase–sensitive fashion.40 We discuss several possibilities how eNOS uncoupling may occur.
eNOS Uncoupling Caused by Increased Peroxynitrite-Mediated BH4 Oxidation
For proper function of NOS, BH4 seems to be essential in several ways. BH4 stabilizes the NOS dimer, facilitates its formation, and protects NOS against proteolysis.41,42 It also increases the affinity of NOS for L-arginine and affects the spin state of the heme iron, the heme redox potential, and the oxygen binding. Most importantly, however, BH4 plays a decisive role for oxygen activation and the time-critical delivery of one electron and proton to the Fe(II)–O2 intermediate, which converts to an iron-oxo species and releases H2O in the catalytic cycle of NOS.43 BH4 provides the second electron in the first mono-oxygenation reaction that hydroxylates L-arginine to N-hydroxy-arginine. Rapid kinetics analysis by freeze-quench EPR revealed the transient formation of a BH4+· cation radical during this reaction, which rapidly splits off a proton to form a BH3· radical. The BH3· radical is reduced by one electron and proton delivered by the reductase domain to BH4, which participates in a second oxygen activation step leading to the final products NO and L-citrulline. In the absence of BH4, the Fe(II)–O2 intermediate decays under formation of superoxide and Fe(III). Dihydrobiopterin (BH2) and other derivatives such as sepiapterin can bind to NOS but cannot support NO formation.43 Therefore, limited availability of BH4 for NOS will inevitably result in increased superoxide formation at the expense of NO formation, ie, it will uncouple NOS.
What are the mechanisms leading to BH4 depletion? In vitro studies proposed that native low-density lipoprotein44 and even more pronounced oxidized low-density lipoprotein45 are able to stimulate endothelial superoxide production and that this phenomenon is inhibited by the NOS inhibitor L-NAME, pointing to a specific role of eNOS in superoxide production. Hypercholesterolemia also has been shown to increase vascular formation of superoxide via activation of the NAD(P)H oxidase46 and/or xanthine oxidase.47 Superoxide derived from both enzyme sources may lead to increased formation of peroxynitrite.33,48 Peroxynitrite in turn rapidly oxidizes the active NOS cofactor BH4 to cofactor inactive molecules such as BH2.33,49 These concepts, however, also imply that the uncoupling of eNOS would invariably require a priming event such as superoxide produced by the NAD(P)H oxidase and/or the xanthine oxidase (so-called kindling radicals) leading via increased formation of peroxynitrite eNOS to produce superoxide (bonfire radical).
eNOS Uncoupling Caused by Peroxynitrite-Mediated Oxidation of the Zinc–Thiolate Complex
Another interesting concept concerning eNOS uncoupling was provided by Zou et al. These authors showed that the exposure of the isolated enzyme to peroxynitrite leads to a disruption of the zinc–thiolate cluster resulting in an uncoupling of the enzyme.28 The authors also demonstrated that a similar phenomenon occurred when endothelial cells were exposed to high concentrations of glucose. Additional experiments revealed that BH4 was oxidized at concentrations being 10- to 100-fold higher than those needed to disrupt the zinc–thiolate complex. Based on these findings, the authors suggested that the principal mechanism of uncoupling is the oxidation of the zinc–thiolate center and the subsequent release of Zn2+ ions rather than BH4 oxidation.28
eNOS Uncoupling Caused by L-Arginine Deficiency or Increased Production of Asymmetrical Methyl Arginine
Several recently published studies demonstrate that increased concentrations of asymmetrical methyl arginines (ADMA) in cultured endothelial cells or in patients with endothelial dysfunction are associated with increased ROS production in supernatants, rodents, or human plasma.50–52 The question is whether increased ROS production is the reason for increased ADMA levels or whether increased production of ADMA actually contributes to the oxidative stress burden of the vasculature via uncoupling of eNOS. Interestingly, the activity of methylating enzymes such as the S-adenosylmethionine–dependent protein arginine methyltransferases (PRMTs) (type I)51 responsible for the ADMA synthesis or the activity of ADMA hydrolyzing enzymes such as DDAH50 is redox-sensitive. Thus, oxidative stress in the vasculature should always stimulate ADMA production and/or inhibit ADMA degradation in concentrations that significantly inhibit eNOS activity or even uncouple the enzyme, which would further increase superoxide production in a positive feedback fashion.53
Which Enzyme Produces the Kindling Radical for Increased Peroxynitrite Formation Ultimately Leading to eNOS Uncoupling?
Role for NAD(P)H Oxidase
The NAD(P)H oxidase is a superoxide-producing enzyme that has been first characterized in neutrophils.54 We know that a similar enzyme exists also in endothelial and smooth muscle cells, as well as in the adventitia. The activity of the enzyme in endothelial as well as smooth muscle cells is increased on stimulation with angiotensin II.55 The stimulatory effects of angiotensin II on the activity of this enzyme would suggest that in the presence of an activated renin angiotensin system (local or circulating), vascular dysfunction caused by increased vascular superoxide production is likely to be expected. Experimental hypercholesterolemia has been shown to be associated with an activation of the NAD(P)H oxidase46 and there is a close association with endothelial dysfunction and clinical risk factors and the activity of this enzyme in human saphenous veins in patients with coronary artery disease.56 In atherosclerotic arteries there is evidence for increased expression of the NAD(P)H oxidase subunit gp91phox and nox4, all of which may contribute to increased oxidative stress within vascular tissue.57
Interestingly, there is a growing body of evidence that the local renin angiotensin system is activated in the setting of hypercholesterolemia. In patients, ACE activity and therefore local angiotensin II concentrations are increased in atherosclerotic plaques,58,59 and inflammatory cells are capable of producing large amounts of angiotensin II. Increased angiotensin II concentrations along with increased levels of superoxide have been shown in the shoulder region of atherosclerotic plaques.60 In vessels from hypercholesterolemic animals46 as well as in platelets from hypercholesterolemic patients,61 there is an increase in the expression of the angiotensin II receptor subtype AT1. Thus, both experimental and clinical studies have provided evidence for stimulation of the renin angiotensin system in atherosclerosis and simultaneously for an activation of the NAD(P)H oxidase in the arterial wall. Similar evidence for an activation of this enzyme in the vasculature has been provided from experimental animal models of different forms of hypertension such as angiotensin II infusion62,63 and in SHR,64 as well as in different forms of diabetes mellitus.35
The proof of concept that superoxide produced by the NADPH oxidase may indeed trigger eNOS uncoupling was provided by David Harrison’s group in the experimental animal model of DOCA-salt hypertension. With these studies, the authors showed that superoxide induced by DOCA-salt treatment caused increased vascular superoxide production, which was significantly reduced by an inhibitor of eNOS such as L-NAME. Treatment of p47phox knockout animals with DOCA-salt caused markedly reduced levels of oxidative stress and abolished superoxide effects of NOS inhibition compatible with a prevention of eNOS uncoupling.65
Role for Xanthine Oxidase
Xanthine oxidoreductase catalyzes the sequential hydroxylation of hypoxanthine to yield xanthine and uric acid. The enzyme can exist in 2 forms that differ primarily in their oxidizing substrate specificity. The dehydrogenase form preferentially uses NAD+ as an electron acceptor but is also able to donate electrons to molecular oxygen. By proteolytic breakdown as well as thiol oxidation, xanthine dehydrogenase from mammalian sources can be converted to the oxidase form that readily donates electrons to molecular oxygen, thereby producing superoxide and hydrogen peroxide but does not reduce NAD+. Oxypurinol, an inhibitor of xanthine oxidoreductase, has been shown to reduce superoxide production and to improve endothelium-dependent vascular relaxations to acetylcholine in vessels from hyperlipidemic animals.47 This suggests an increase in the expression or activity of xanthine oxidase in early hypercholesterolemia. The mechanisms underlying such a phenomenon remain unclear; however, it has been demonstrated that certain cytokines can stimulate the expression of xanthine oxidase by the endothelium. An alternative mechanism may be that increased cholesterol levels trigger the release of xanthine oxidase (eg, from the liver) into the circulation where it binds to endothelial glycosaminoglycans.66 Human studies concerning the efficacy of xanthine oxidase inhibition on endothelial dysfunction are somewhat discrepant. Although Panza et al showed that endothelial dysfunction in hypercholesterolemic patients and hypertensive diabetic subjects is improved by acute inhibition of xanthine oxidase with oxypurinol and allopurinol,67,68 other groups failed to show similar efficacy69 for allopurinol. Its role in mediating increased oxidative stress in the setting of hypertension is not quite clear. Oxypurinol has blood pressure-lowering effects comparable to heparin-binding SOD in SHR70 but fails to demonstrate a positive effect on endothelial dysfunction in hypertensive patients.67
How Can We Assess eNOS Uncoupling?
It is important to note that eNOS-mediated superoxide production—by the isolated enzyme or in vascular tissue—is inhibited by NG-nitro-L-arginine (L-NNA) and its methylester NG-nitro-L-arginine methylester (L-NAME), because both substances antagonize the transfer of electrons to either L-arginine or oxygen. In contrast, L-NMMA has been previously shown to stimulate rather than to inhibit superoxide production by the isolated enzyme because of partial uncoupling of NADPH oxidation (electron transfer to oxygen). This has been shown for inducible NOS71 and for neuronal NOS.72 Similar phenomena may have to be expected when isolated enzymes are exposed to the structurally similar ADMA, which will prevent the oxidation of L-arginine and therefore reduce NO production or, as mentioned, will stimulate superoxide production by competing for the same binding site of the enzyme.
The L-NNA–sensitive and L-NAME–sensitive inhibition of superoxide formation is a convenient method to detect uncoupling of NOS in vascular tissues. Depending on the detection system used, addition of these NOS inhibitors to vascular cells and tissues will decrease the superoxide-derived signal, such as lucigenin-dependent (5 μmol/L) luminescence or the dihydroethidine fluorescence.73,74 The Harrison group showed that depletion of endogenous BH4 in mouse aorta by addition of exogenous peroxynitrite increased superoxide formation, and that addition of L-NAME, removal of the endothelium, or genetic knockout of eNOS prevented this radical response.33–36
Modulation of the Activity and Expression of the sGC and cGK-I In Vitro by ROS and Effects of Oxidative Stress on the Activity and Expression of the sGC and cGK-I In Vivo
See http://atvb.ahajournals.org for details.
Summary and Conclusions
Taken together, there is mounting evidence that endothelial dysfunction of the coronary or peripheral circulation has important prognostic implications for future cardiovascular events. Although the mechanisms underlying endothelial dysfunction are likely multifactorial (Figure 4), it is important to note that increased production of oxygen-derived free radicals by an uncoupled eNOS markedly contributes to this phenomenon. Increased superoxide production is not restricted to the endothelium but also involves the smooth muscle cell layer, where reactive oxygen species affect NO/cGMP signaling by altering the expression of sGC and by inhibiting the activity of the sGC and cGK-I.
Figure 4. Schematic representation of the role of reactive oxygen species in causing endothelial dysfunction. Under normal conditions, nitric oxide (NO) synthesized by endothelial nitric oxide synthase (NOSIII) stimulates soluble guanylyl cyclase (sGC), increasing cGMP, thus stimulating cGMP-dependent protein kinase I (cGK-I) and eliciting vasorelaxation. cGK-I also phosphorylates vasodilator-stimulated phosphoprotein, which can be taken as a biochemical marker of cGK-I activity. This pathway can, however, be inhibited at several sites. Angiotensin II, hypertension, hypercholesterolemia, chronic smoking, nitrate tolerance, and diabetes mellitus stimulate superoxide (O2) production within endothelial and smooth muscle cells and in the adventitia, which may inactivate NO and inhibit sGC directly, thereby diminishing cGK-I activity. Peroxynitrite (ONOO–) may also inhibit sGC directly and it uncouples NOSIII by oxidizing Zn–thiolate complexes within NOSIII, and/or by oxidizing the NOS cofactor tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2). This concept, however, also means that uncoupling of NOSIII would always require a priming event such as superoxide produced by the NAD(P)H oxidase and/or xanthine oxidase. Angiotensin II and nitroglycerin (NTG) also increase catabolism of cGMP by phosphodiesterase 1A1 (PDE1A1), which is usually accomplished by phosphodiesterase 5.
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Vergnani L, Hatrik S, Ricci F, Passaro A, Manzoli N, Zuliani G, Brovkovych V, Fellin R, Malinski T. Effect of native and oxidized low-density lipoprotein on endothelial nitric oxide and superoxide production : key role of L-arginine availability. Circulation. 2000; 101: 1261–1266.
Warnholtz A, Nickenig G, Schulz E, Macharzina R, Brasen JH, Skatchkov M, Heitzer T, Stasch JP, Griendling KK, Harrison DG, Bohm M, Meinertz T, Munzel T. Increased NADH-oxidase-mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the renin-angiotensin system. Circulation. 1999; 99: 2027–2033.
Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest. 1993; 91: 2546–2551.
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Lin KY, Ito A, Asagami T, Tsao PS, Adimoolam S, Kimoto M, Tsuji H, Reaven GM, Cooke JP. Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase. Circulation. 2002; 106: 987–992.
Boger RH, Sydow K, Borlak J, Thum T, Lenzen H, Schubert B, Tsikas D, Bode-Boger SM. LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of S-adenosylmethionine-dependent methyltransferases. Circ Res. 2000; 87: 99–105.
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Sydow K, Munzel T. ADMA and oxidative stress. Atheroscler Suppl. 2003; 4: 41–51.
Bastian NR, Hibbs JB Jr. Assembly and regulation of NADPH oxidase and nitric oxide synthase. Curr Opin Immunol. 1994; 6: 131–139.
Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.
Guzik TJ, West NE, Black E, McDonald D, Ratnatunga C, Pillai R, Channon KM. Vascular superoxide production by NAD(P)H oxidase: association with endothelial dysfunction and clinical risk factors. Circ Res. 2000; 86: E85–E90.
Sorescu D, Weiss D, Lassegue B, Clempus RE, Szocs K, Sorescu GP, Valppu L, Quinn MT, Lambeth JD, Vega JD, Taylor WR, Griendling KK. Superoxide production and expression of nox family proteins in human atherosclerosis. Circulation. 2002; 105: 1429–1435.
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Schieffer B, Schieffer E, Hilfiker-Kleiner D, Hilfiker A, Kovanen PT, Kaartinen M, Nussberger J, Harringer W, Drexler H. Expression of angiotensin II and interleukin 6 in human coronary atherosclerotic plaques: potential implications for inflammation and plaque instability. Circulation. 2000; 101: 1372–1378.
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Fukui T, Ishizaka N, Rajagopalan S, Laursen JB, Capers Qt, Taylor WR, Harrison DG, de Leon H, Wilcox JN, Griendling KK. p22phox mRNA expression and NADPH oxidase activity are increased in aortas from hypertensive rats. Circ Res. 1997; 80: 45–51.
Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK, Harrison DG. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone. J Clin Invest. 1996; 97: 1916–1923.
Morawietz H, Weber M, Rueckschloss U, Lauer N, Hacker A, Kojda G. Upregulation of vascular NAD(P)H oxidase subunit gp91phox and impairment of the nitric oxide signal transduction pathway in hypertension. Biochem Biophys Res Commun. 2001; 285: 1130–1135.
Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE, Harrison DG. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest. 2003; 111: 1201–1209.
White CR, Darley-Usmar V, Berrington WR, McAdams M, Gore JZ, Thompson JA, Parks DA, Tarpey MM, Freeman BA. Circulating plasma xanthine oxidase contributes to vascular dysfunction in hypercholesterolemic rabbits. Proc Natl Acad Sci U S A. 1996; 93: 8745–8749.
Cardillo C, Kilcoyne CM, Cannon RO,3rd, Quyyumi AA, Panza JA. Xanthine oxidase inhibition with oxypurinol improves endothelial vasodilator function in hypercholesterolemic but not in hypertensive patients. Hypertension. 1997; 30: 57–63.
Butler R, Morris AD, Belch JJ, Hill A, Struthers AD. Allopurinol normalizes endothelial dysfunction in type 2 diabetics with mild hypertension. Hypertension. 2000; 35: 746–751.
O’Driscoll JG, Green DJ, Rankin JM, Taylor RR. Nitric oxide-dependent endothelial function is unaffected by allopurinol in hypercholesterolaemic subjects. Clin Exp Pharmacol Physiol. 1999; 26: 779–783.
Nakazono K, Watanabe N, Matsuno K, Sasaki J, Sato T, Inoue M. Does superoxide underlie the pathogenesis of hypertension? Proc Natl Acad Sci U S A. 1991; 88: 10045–10048.
Olken NM, Marletta MA. NG-methyl-L-arginine functions as an alternate substrate and mechanism-based inhibitor of nitric oxide synthase. Biochemistry. 1993; 32: 9677–9685.
Pou S, Keaton L, Surichamorn W, Rosen GM. Mechanism of superoxide generation by neuronal nitric-oxide synthase. J Biol Chem. 1999; 274: 9573–9580.
Mulsch A, Oelze M, Kloss S, Mollnau H, Topfer A, Smolenski A, Walter U, Stasch JP, Warnholtz A, Hink U, Meinertz T, Munzel T. Effects of in vivo nitroglycerin treatment on activity and expression of the guanylyl cyclase and cGMP-dependent protein kinase and their downstream target vasodilator-stimulated phosphoprotein in aorta. Circulation. 2001; 103: 2188–2194.
Miller FJ Jr, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998; 82: 1298–1305.(Thomas Münzel; Andreas Da)
Correspondence to Thomas Münzel, MD, Professor of Internal Medicine, II Medizinische Klinik und Poliklinik Johannes Gutenberg-Universit?t Mainz, Langenbeckstrasse 1, D-55131 Mainz, Germany. E-mail tmuenzel@uni-mainz.de
Series Editor: Kathy K. Griendling
Redox Mechanisms in Blood Vessels
ATVB in Focus
Previous Brief Reviews in this Series:
?Mueller CFH, Laude K, McNally JS, Harrison DG. Redox mechanisms in blood vessels. 2005;25:274–278.
?Gutterman DD, Miura H, Liu Y. Redox modulation of vascular tone: focus of potassium channel mechanisms of dilation. 2005;25:671–678.
?Nicholls SJ, Hazen SL. Myeloperoxidase and cardiovascular disease. 2005;25:1102–1111.
?Leopold JA, Loscalzo J. Oxidative enzymopathies and vascular disease. 2005;25:1332–1340.
Abstract
Endothelial dysfunction in the setting of cardiovascular risk factors, such as hypercholesterolemia, hypertension, diabetes mellitus, chronic smoking, as well as in the setting of heart failure, has been shown to be at least partly dependent on the production of reactive oxygen species (ROS), such as the superoxide radical, and the subsequent decrease in vascular bioavailability of nitric oxide (NO). Superoxide-producing enzymes involved in increased oxidative stress within vascular tissue include the NAD(P)H oxidase, the xanthine oxidase, and mitochondrial superoxide-producing enzymes. Superoxide produced by the NADPH oxidase may react with NO released by endothelial nitric oxide synthase (eNOS), thereby generating peroxynitrite. Peroxynitrite in turn has been shown to uncouple eNOS, thereby switching an antiatherosclerotic NO-producing enzyme to an enzyme that may initiate or even accelerate the atherosclerotic process by producing superoxide. Increased oxidative stress in the vasculature, however, is not restricted to the endothelium and has also been demonstrated to occur within the smooth muscle cell layer in the setting of hypercholesterolemia, diabetes mellitus, hypertension, congestive heart failure, and nitrate tolerance. Increased superoxide production by the endothelial and/or smooth muscle cells has important consequences with respect to signaling by the soluble guanylyl cyclase (sGC) and the cGMP-dependent protein kinase I (cGK-I), the activity and expression of which has been shown to be regulated in a redox-sensitive fashion. The present review summarizes current concepts concerning eNOS uncoupling and also focuses on the consequences for downstream signaling with respect to activity and expression of the sGC and cGK-I in various diseases.
Key Words: endothelium ? vasodilation ? nitric oxide ? endothelial NO-synthase ? oxidative stress
Introduction
Traditionally, the role of the endothelium was thought primarily to be that of a selective barrier to the diffusion of macromolecules from the vessel lumen to the interstitial space. During the past 20 years, numerous additional roles for the endothelium have been defined such as regulation of vascular tone, modulation of inflammation, promotion, and inhibition of vascular growth and modulation of platelet aggregation and coagulation. Endothelial dysfunction is a characteristic feature of patients with coronary atherosclerosis and more recent studies indicate that it may predict long-term atherosclerotic disease progression as well as cardiovascular event rate.1 Although the mechanisms underlying endothelial dysfunction may be multifactorial, there is a growing body of evidence that increased production of reactive oxygen species (ROS) may contribute considerably to this phenomenon. ROS production has been demonstrated to occur in the endothelial cell layer, but also within the media and adventitia, all of which may impair nitric oxide (NO) signaling within vascular tissue to endothelium-dependent, but also endothelium-independent, vasodilators. More recent experimental, but also clinical, studies point to a crucial role of endothelial nitric oxide synthase (eNOS) as a superoxide-producing enzyme in the setting of atherosclerosis, hypertension, congestive heart failure, and also nitrate tolerance. Figure 1 summarizes our observations that in all these pathophysiological settings vascular oxidative stress, as visualized by dihydroethidine-dependent fluorescence, is present within the endothelium, but also the smooth muscle cell layer and the adventitia, all of which plays an important role for the development of endothelial and/or vascular dysfunction. This review briefly addresses mechanisms underlying eNOS uncoupling and focuses on the consequences with respect to the activity and/or expression of the NO target, the soluble guanylyl cyclase (sGC), and the cGMP-dependent protein kinase I (cGK-I) present in vascular smooth muscle.
Figure 1. Detection of vascular superoxide formation by the fluorescence dye dihydroethidine in aortic tissue sections from different oxidative stress animal models and in the mammary artery from nitroglycerin (NTG)-treated patients. CHF indicates congestive heart failure; hypertension: angiotensin (AT II)-infused rats vs sham-treated Wistar rats; atherosclerosis: hyperlipidemic Watanabe rabbits (WHHL) vs New Zealand White rabbits (NZWR); diabetes: streptozotocin (STZ)-treated rats vs sham-treated Wistar rats; nitrate tolerance: NTG-infused patients vs patients without NTG treatment.
The L-Arginine/NO/cGMP Pathway in Vascular Tissue
The endothelium, a single-layered continuous cell sheet lining the luminal vessel wall, separates the intravascular (blood) from the interstitial compartment and the vascular smooth muscle. Based on cell count (6x1013), mass (1.5 kg), and surface area (1000 m2), the endothelium is an autonomous organ. Though for a long time regarded as a passive barrier for blood cells and macrosolutes, this view completely changed with the advent of endothelial autacoids like prostacyclin2 and NO,3 as well as with the discovery of integrins and other surface signals.4 It is now evident that the endothelium is at the cross-bridges of communication between blood and tissue cells and actively controls this process and the function of surrounding cells by a plethora of signaling routes. One of the prominent communication lines is established by the so-called L-arginine-NO-cyclic GMP pathway.5 This signaling cascade starts with eNOS (NOSIII), which generates NO and L-citrulline from L-arginine and O2 in response to receptor-dependent agonists (bradykinin, acetylcholine, ATP) and physicochemical stimuli (shear, stretch).6 Reducing equivalents are provided by NADPH, and electrons are passed via a flavin chain to the catalytic center, the enzymes heme iron (Figure 2, upper). The first step of the normal NOS reaction is a classical mono-oxygenation, which consumes 1 mol of NADPH and oxygen. Molecular oxygen bound to the iron is activated and split to accomplish a hydroxylation of the substrate, the guanidino-nitrogen of L-arginine, forming NG-hydroxy-L-arginine. The second step is an atypical mono-oxygenation, which consumes 1 mol O2 and 0.5 mol NADPH. It is a 3-electron oxidation of NG-hydroxy-L-arginine to afford the final products NO and L-citrulline. To guarantee this reaction path, the enzyme has to be homodimeric and the cofactor tetrahydrobiopterin must be present (Figure 2). For details, the reader is referred to excellent reviews on this topic.7,8
Figure 2. Scheme depicting electron flow in coupled vs uncoupled eNOS. Electron flow starts from NADPH to flavins FAD and FMN of the reductase domain, which delivers the electrons to the iron of the heme (oxygenase domain) and to the BH3· radical generated as an intermediate in the catalytic cycle. BH4 seems to be essential to donate an electron and proton to versatile intermediates in the reaction cycle of L-arginine/O2 to L-citrullin/NO. Calmodulin (CAM) controls electron flow in eNOS. Zinc ions (Zn) bound to NOS are required for dimer formation and stability. Monomeric eNOS or BH4/L-arginine–deficient eNOS is uncoupled and produces O2– (for explanation see text).
NO diffuses to the adjacent smooth muscle where it interacts with different receptor molecules, of which the sGC is the best characterized and presumably most important one with regard to control of vessel tone and smooth muscle proliferation. The catalytically active holoenzyme exists as an obligate heterodimer consisting of 1 and 1 ? subunit and a noncovalently bound ferrous heme.9 The most abundant isoform in mammalian tissues is the 1 (73 to 78 kDa)/?1 (70 kDa) heterodimer. Significant protein expression of 2 other homologues subunits has only been detected in the human placenta (2)10,11 and kidney (?2).12 At the genomic level, a dominant-negative splice variant of 2 classified as 2i has been detected.13 The interspecies homology of the individual subunits is high, whereas the intersubunit homology is lower.14 In contrast to 1 and ?1, the biological significance of 2, 2I, and ?2 is still obscure. The 2 subunit has recently been shown to be linked to cerebral maturation and sensory pathway refinement during postnatal development.15
A unifying concept of the molecular requisites for sGC activation has been put forth.16,17 Activation by NO requires sGC heme iron to be in the ferrous (II) state. On NO binding, the iron is moved slightly out of the porphyrin plane, thereby releasing a distal histidine (His105 of the ?1 subunit) from iron coordination.18 This triggers subsequent intramolecular re-arrangements influencing the catalytic center, resulting in an up to several-hundred-fold increase in cGMP formation. Depending on the cell type, cyclic GMP then elicits different biological responses, either by inhibition (cAMP-metabolizing phosphodiesterases) or by activation (cGMP-activated protein kinases, cGK, and cGMP-gated cation channels) of effector proteins.19 A comprehensive overview on cGK-I downstream signaling is provided.19 The mammalian cGK family consists of cGK-I, a splice variant cGK-I?, and cGK-II, which is encoded by a separate gene.19 Both cGK-I isoforms, not cGK-II, are expressed in vascular cells. In rat aortic smooth muscle cells, activated cGK-I increases the open probability of Ca2+-activated K+(BK) channels, thereby inducing a hyperpolarization of the smooth muscle cells and inhibition of agonist-induced Ca2+ influx. In addition, activated cGK-I? phosphorylates the inositol triphosphate receptor-associated G-kinase substrate, thereby inhibiting agonist-induced Ca2+ release and smooth muscle contraction. Another cGK-I substrate found in many cell types is the 46/50 kDa vasodilator-stimulated phosphoprotein. cGK-I phosphorylates vasodilator-stimulated phosphoprotein specifically at serine 239, and this reaction can be exploited as a biochemical monitor for the integrity and activity of the NO-cGMP pathway,20 as discussed. Phosphorylation and activation of cGMP-hydrolyzing phosphodiesterase 5 by cGK-I is a major mechanism to cease cGMP signaling.19,21
Oxidative Stress and Endothelial Dysfunction
The endothelium-derived relaxing factor, previously identified as NO3 or a closely related compound,22 has potent antiatherosclerotic properties. NO released from endothelial cells works in concert with prostacyclin to inhibit platelet aggregation,23 it inhibits the attachment of neutrophils to endothelial cells and the expression of adhesion molecules. NO in high concentrations inhibits the proliferation of smooth muscle cells.24 Therefore, under all conditions in which an absolute or relative NO deficit is encountered, the process of atherosclerosis is being initiated or accelerated. The half-life of NO and therefore its biological activity is decisively determined by oxygen-derived free radicals such as superoxide.25 Superoxide rapidly reacts with NO to form the highly reactive intermediate peroxynitrite.26 The rapid bimolecular reaction between NO and superoxide yielding peroxynitrite (rate constant: 5 to 10x109 M–1s–1) is 3- to 4-times faster than the dismutation of superoxide by the superoxide dismutase. Therefore, peroxynitrite formation represents a major potential pathway of NO reactivity pending on the rates of tissue superoxide production. Peroxynitrite in high concentrations is cytotoxic and may cause oxidative damage to proteins, lipids, and DNA.26 Recent studies also indicate that peroxynitrite may have deleterious effects on activity and function of the prostacyclin synthase27 and the eNOS.28 Other ROS such as the dismutation product of superoxide, hydrogen peroxide, and the hypochlorous acid released by activated neutrophils, are not free radicals, but have a powerful oxidizing capacity, which will further contribute to oxidative stress within vascular tissue.
Endothelial Dysfunction and Cardiovascular Risk Factors
It is well known that in the presence of cardiovascular risk factors endothelial dysfunction is frequently encountered. This has been shown for chronic smokers, patients with increased low-density lipoprotein levels, for patients with diabetes types I and II, for hypertensive patients, and for patients with metabolic syndrome. There are several potential abnormalities, which could account for reductions in endothelium-dependent vascular relaxation including changes in the activity and/or expression of the eNOS, decreased sensitivity of vascular smooth muscle cells to NO, or increased degradation of NO via its interaction with ROS such as superoxide. The NO degradation concept is the most attractive one because in the presence of cardiovascular risk factors, endothelial dysfunction is established and even more importantly it is markedly improved by the acute administration of the antioxidant vitamin C.29–32 Superoxide and/or peroxynitrite could also act further downstream by oxidative inactivation of sGC as well as activation of cGK-I.
eNOS Uncoupling Contributes to Endothelial Dysfunction
In most situations in which endothelial dysfunction caused by increased oxidative stress is encountered, the expression of the eNOS has been shown to be paradoxically increased rather than decreased.33–36 The mechanisms underlying increased expression of eNOS are likely to be secondary to increased endothelial levels of hydrogen peroxide, which has been shown to increase the expression at the transcriptional and translational level.37 The demonstration of endothelial dysfunction in the presence of increased expression of eNOS indicates that the capacity of the enzyme to produce NO may be limited. Very intriguing are observations that the eNOS itself can be a superoxide source, thereby causing endothelial dysfunction. It has become clear from studies with the purified enzyme that eNOS may become "uncoupled," eg, in the absence of the NOS substrate L-arginine or the cofactor tetrahydrobiopterin (BH4). In such uncoupled state, electrons normally flowing from the reductase domain of one subunit to the oxygenase domain of the other subunit are diverted to molecular oxygen rather than to L-arginine,38,39 resulting in production of superoxide rather than NO (Figure 2). That eNOS-derived superoxide/ROS formation bears consequences opposite to "normal" NOS function was first shown in U937 cells transfected with wild-type eNOS, which induced increased transcription of tumor necrosis factor- in an NOS inhibitor (L-NMA)-insensitive and superoxide-dismutase–sensitive fashion.40 We discuss several possibilities how eNOS uncoupling may occur.
eNOS Uncoupling Caused by Increased Peroxynitrite-Mediated BH4 Oxidation
For proper function of NOS, BH4 seems to be essential in several ways. BH4 stabilizes the NOS dimer, facilitates its formation, and protects NOS against proteolysis.41,42 It also increases the affinity of NOS for L-arginine and affects the spin state of the heme iron, the heme redox potential, and the oxygen binding. Most importantly, however, BH4 plays a decisive role for oxygen activation and the time-critical delivery of one electron and proton to the Fe(II)–O2 intermediate, which converts to an iron-oxo species and releases H2O in the catalytic cycle of NOS.43 BH4 provides the second electron in the first mono-oxygenation reaction that hydroxylates L-arginine to N-hydroxy-arginine. Rapid kinetics analysis by freeze-quench EPR revealed the transient formation of a BH4+· cation radical during this reaction, which rapidly splits off a proton to form a BH3· radical. The BH3· radical is reduced by one electron and proton delivered by the reductase domain to BH4, which participates in a second oxygen activation step leading to the final products NO and L-citrulline. In the absence of BH4, the Fe(II)–O2 intermediate decays under formation of superoxide and Fe(III). Dihydrobiopterin (BH2) and other derivatives such as sepiapterin can bind to NOS but cannot support NO formation.43 Therefore, limited availability of BH4 for NOS will inevitably result in increased superoxide formation at the expense of NO formation, ie, it will uncouple NOS.
What are the mechanisms leading to BH4 depletion? In vitro studies proposed that native low-density lipoprotein44 and even more pronounced oxidized low-density lipoprotein45 are able to stimulate endothelial superoxide production and that this phenomenon is inhibited by the NOS inhibitor L-NAME, pointing to a specific role of eNOS in superoxide production. Hypercholesterolemia also has been shown to increase vascular formation of superoxide via activation of the NAD(P)H oxidase46 and/or xanthine oxidase.47 Superoxide derived from both enzyme sources may lead to increased formation of peroxynitrite.33,48 Peroxynitrite in turn rapidly oxidizes the active NOS cofactor BH4 to cofactor inactive molecules such as BH2.33,49 These concepts, however, also imply that the uncoupling of eNOS would invariably require a priming event such as superoxide produced by the NAD(P)H oxidase and/or the xanthine oxidase (so-called kindling radicals) leading via increased formation of peroxynitrite eNOS to produce superoxide (bonfire radical).
eNOS Uncoupling Caused by Peroxynitrite-Mediated Oxidation of the Zinc–Thiolate Complex
Another interesting concept concerning eNOS uncoupling was provided by Zou et al. These authors showed that the exposure of the isolated enzyme to peroxynitrite leads to a disruption of the zinc–thiolate cluster resulting in an uncoupling of the enzyme.28 The authors also demonstrated that a similar phenomenon occurred when endothelial cells were exposed to high concentrations of glucose. Additional experiments revealed that BH4 was oxidized at concentrations being 10- to 100-fold higher than those needed to disrupt the zinc–thiolate complex. Based on these findings, the authors suggested that the principal mechanism of uncoupling is the oxidation of the zinc–thiolate center and the subsequent release of Zn2+ ions rather than BH4 oxidation.28
eNOS Uncoupling Caused by L-Arginine Deficiency or Increased Production of Asymmetrical Methyl Arginine
Several recently published studies demonstrate that increased concentrations of asymmetrical methyl arginines (ADMA) in cultured endothelial cells or in patients with endothelial dysfunction are associated with increased ROS production in supernatants, rodents, or human plasma.50–52 The question is whether increased ROS production is the reason for increased ADMA levels or whether increased production of ADMA actually contributes to the oxidative stress burden of the vasculature via uncoupling of eNOS. Interestingly, the activity of methylating enzymes such as the S-adenosylmethionine–dependent protein arginine methyltransferases (PRMTs) (type I)51 responsible for the ADMA synthesis or the activity of ADMA hydrolyzing enzymes such as DDAH50 is redox-sensitive. Thus, oxidative stress in the vasculature should always stimulate ADMA production and/or inhibit ADMA degradation in concentrations that significantly inhibit eNOS activity or even uncouple the enzyme, which would further increase superoxide production in a positive feedback fashion.53
Which Enzyme Produces the Kindling Radical for Increased Peroxynitrite Formation Ultimately Leading to eNOS Uncoupling?
Role for NAD(P)H Oxidase
The NAD(P)H oxidase is a superoxide-producing enzyme that has been first characterized in neutrophils.54 We know that a similar enzyme exists also in endothelial and smooth muscle cells, as well as in the adventitia. The activity of the enzyme in endothelial as well as smooth muscle cells is increased on stimulation with angiotensin II.55 The stimulatory effects of angiotensin II on the activity of this enzyme would suggest that in the presence of an activated renin angiotensin system (local or circulating), vascular dysfunction caused by increased vascular superoxide production is likely to be expected. Experimental hypercholesterolemia has been shown to be associated with an activation of the NAD(P)H oxidase46 and there is a close association with endothelial dysfunction and clinical risk factors and the activity of this enzyme in human saphenous veins in patients with coronary artery disease.56 In atherosclerotic arteries there is evidence for increased expression of the NAD(P)H oxidase subunit gp91phox and nox4, all of which may contribute to increased oxidative stress within vascular tissue.57
Interestingly, there is a growing body of evidence that the local renin angiotensin system is activated in the setting of hypercholesterolemia. In patients, ACE activity and therefore local angiotensin II concentrations are increased in atherosclerotic plaques,58,59 and inflammatory cells are capable of producing large amounts of angiotensin II. Increased angiotensin II concentrations along with increased levels of superoxide have been shown in the shoulder region of atherosclerotic plaques.60 In vessels from hypercholesterolemic animals46 as well as in platelets from hypercholesterolemic patients,61 there is an increase in the expression of the angiotensin II receptor subtype AT1. Thus, both experimental and clinical studies have provided evidence for stimulation of the renin angiotensin system in atherosclerosis and simultaneously for an activation of the NAD(P)H oxidase in the arterial wall. Similar evidence for an activation of this enzyme in the vasculature has been provided from experimental animal models of different forms of hypertension such as angiotensin II infusion62,63 and in SHR,64 as well as in different forms of diabetes mellitus.35
The proof of concept that superoxide produced by the NADPH oxidase may indeed trigger eNOS uncoupling was provided by David Harrison’s group in the experimental animal model of DOCA-salt hypertension. With these studies, the authors showed that superoxide induced by DOCA-salt treatment caused increased vascular superoxide production, which was significantly reduced by an inhibitor of eNOS such as L-NAME. Treatment of p47phox knockout animals with DOCA-salt caused markedly reduced levels of oxidative stress and abolished superoxide effects of NOS inhibition compatible with a prevention of eNOS uncoupling.65
Role for Xanthine Oxidase
Xanthine oxidoreductase catalyzes the sequential hydroxylation of hypoxanthine to yield xanthine and uric acid. The enzyme can exist in 2 forms that differ primarily in their oxidizing substrate specificity. The dehydrogenase form preferentially uses NAD+ as an electron acceptor but is also able to donate electrons to molecular oxygen. By proteolytic breakdown as well as thiol oxidation, xanthine dehydrogenase from mammalian sources can be converted to the oxidase form that readily donates electrons to molecular oxygen, thereby producing superoxide and hydrogen peroxide but does not reduce NAD+. Oxypurinol, an inhibitor of xanthine oxidoreductase, has been shown to reduce superoxide production and to improve endothelium-dependent vascular relaxations to acetylcholine in vessels from hyperlipidemic animals.47 This suggests an increase in the expression or activity of xanthine oxidase in early hypercholesterolemia. The mechanisms underlying such a phenomenon remain unclear; however, it has been demonstrated that certain cytokines can stimulate the expression of xanthine oxidase by the endothelium. An alternative mechanism may be that increased cholesterol levels trigger the release of xanthine oxidase (eg, from the liver) into the circulation where it binds to endothelial glycosaminoglycans.66 Human studies concerning the efficacy of xanthine oxidase inhibition on endothelial dysfunction are somewhat discrepant. Although Panza et al showed that endothelial dysfunction in hypercholesterolemic patients and hypertensive diabetic subjects is improved by acute inhibition of xanthine oxidase with oxypurinol and allopurinol,67,68 other groups failed to show similar efficacy69 for allopurinol. Its role in mediating increased oxidative stress in the setting of hypertension is not quite clear. Oxypurinol has blood pressure-lowering effects comparable to heparin-binding SOD in SHR70 but fails to demonstrate a positive effect on endothelial dysfunction in hypertensive patients.67
How Can We Assess eNOS Uncoupling?
It is important to note that eNOS-mediated superoxide production—by the isolated enzyme or in vascular tissue—is inhibited by NG-nitro-L-arginine (L-NNA) and its methylester NG-nitro-L-arginine methylester (L-NAME), because both substances antagonize the transfer of electrons to either L-arginine or oxygen. In contrast, L-NMMA has been previously shown to stimulate rather than to inhibit superoxide production by the isolated enzyme because of partial uncoupling of NADPH oxidation (electron transfer to oxygen). This has been shown for inducible NOS71 and for neuronal NOS.72 Similar phenomena may have to be expected when isolated enzymes are exposed to the structurally similar ADMA, which will prevent the oxidation of L-arginine and therefore reduce NO production or, as mentioned, will stimulate superoxide production by competing for the same binding site of the enzyme.
The L-NNA–sensitive and L-NAME–sensitive inhibition of superoxide formation is a convenient method to detect uncoupling of NOS in vascular tissues. Depending on the detection system used, addition of these NOS inhibitors to vascular cells and tissues will decrease the superoxide-derived signal, such as lucigenin-dependent (5 μmol/L) luminescence or the dihydroethidine fluorescence.73,74 The Harrison group showed that depletion of endogenous BH4 in mouse aorta by addition of exogenous peroxynitrite increased superoxide formation, and that addition of L-NAME, removal of the endothelium, or genetic knockout of eNOS prevented this radical response.33–36
Modulation of the Activity and Expression of the sGC and cGK-I In Vitro by ROS and Effects of Oxidative Stress on the Activity and Expression of the sGC and cGK-I In Vivo
See http://atvb.ahajournals.org for details.
Summary and Conclusions
Taken together, there is mounting evidence that endothelial dysfunction of the coronary or peripheral circulation has important prognostic implications for future cardiovascular events. Although the mechanisms underlying endothelial dysfunction are likely multifactorial (Figure 4), it is important to note that increased production of oxygen-derived free radicals by an uncoupled eNOS markedly contributes to this phenomenon. Increased superoxide production is not restricted to the endothelium but also involves the smooth muscle cell layer, where reactive oxygen species affect NO/cGMP signaling by altering the expression of sGC and by inhibiting the activity of the sGC and cGK-I.
Figure 4. Schematic representation of the role of reactive oxygen species in causing endothelial dysfunction. Under normal conditions, nitric oxide (NO) synthesized by endothelial nitric oxide synthase (NOSIII) stimulates soluble guanylyl cyclase (sGC), increasing cGMP, thus stimulating cGMP-dependent protein kinase I (cGK-I) and eliciting vasorelaxation. cGK-I also phosphorylates vasodilator-stimulated phosphoprotein, which can be taken as a biochemical marker of cGK-I activity. This pathway can, however, be inhibited at several sites. Angiotensin II, hypertension, hypercholesterolemia, chronic smoking, nitrate tolerance, and diabetes mellitus stimulate superoxide (O2) production within endothelial and smooth muscle cells and in the adventitia, which may inactivate NO and inhibit sGC directly, thereby diminishing cGK-I activity. Peroxynitrite (ONOO–) may also inhibit sGC directly and it uncouples NOSIII by oxidizing Zn–thiolate complexes within NOSIII, and/or by oxidizing the NOS cofactor tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2). This concept, however, also means that uncoupling of NOSIII would always require a priming event such as superoxide produced by the NAD(P)H oxidase and/or xanthine oxidase. Angiotensin II and nitroglycerin (NTG) also increase catabolism of cGMP by phosphodiesterase 1A1 (PDE1A1), which is usually accomplished by phosphodiesterase 5.
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