Inducible nitric oxide synthase and cardiovascular disease

The simple gas nitric oxide (NO) has a diverse array of actions in numerous physiological and pathophysiological processes in the cardiovascular system. Three distinct NO synthase (NOS) isoforms, each encoded for by separate genes, have now been identified [1–4]. nNOS (or ‘neuronal’ NOS, NOS1) and eNOS (or ‘endothelial’ NOS, NOS3) are constitutive, Ca2+-regulated isoforms expressed not only in nervous tissue and endothelium respectively, but also in several other cell types. iNOS (or NOS2) can be expressed in almost any cell type upon appropriate stimulation. All NOS isoforms can be transcriptionally and post-transcriptionally regulated [5]. The generation of NO requires l-arginine, O2, NADPH, and tetrahydrobiopterin (BH4). In situations where there is l-arginine and/or BH4 deficiency, all the NOSs can generate superoxide as well as NO [6]. Appreciation of the role of NO in the cardiovascular system dates back to 1980 and the seminal report by Furchgott & Zawadzki of a labile endothelium-derived relaxing factor (EDRF) responsible for acetylcholine-induced vasodilatation [7]. In 1987, Salvador Moncada and colleagues [8] as well as Ignarro and co-workers [9] independently demonstrated that NO accounted for the biological activity of EDRF. Around the same time, it was shown that NO was involved in macrophage-induced cytotoxicity [10,11]. Endothelial NO synthesis was Ca2+-regulated and produced small amounts of NO, whereas the macrophage enzyme was Ca2+-independent and generated much larger amounts of NO. Furthermore, the macrophage enzyme was detectable only after exposure to cytokines or endotoxin (lipopolysaccharide, LPS), ie, was inducible. In 1989, Patrick Vallance and colleagues had used local intra-arterial infusion of a specific NOS inhibitor, NG-monomethyl l-arginine (l-NMMA), into the forearm to show that endogenous NO contributed to resting arteriolar vasodilator ‘tone’ and mediated acetylcholine-induced vasodilatation in humans [12]. In their Cardiovascular Research paper published that year, they reported that endogenous NO did not affect basal venous tone in normal humans, but did mediate both acetylcholine and bradykinin-induced venodilatation [13]. These were the first studies of the effects of endogenous NO on cardiovascular function in normal humans in vivo. Wright and colleagues in their paper published in 1992 reported the effects of l-NMMA on endotoxic shock in anaesthetised rabbits [14]. A Ca2+-independent inducible NOS (iNOS) had been implicated in endotoxin- or cytokine-induced vascular hyporesponsiveness and shock, and inhibition of NO synthesis had been suggested to reverse hypotension in this condition [15–19]. Wright et al. [14] reported that intravenous l-NMMA markedly exacerbated endotoxin-induced hypotension and mortality over the 3 h timespan of their experiment, but that co-treatment with an NO donor ameliorated these effects. They concluded that inhibition of constitutive NOS was deleterious, but that selective inhibition of iNOS might be beneficial. They also speculated that iNOS expression in the heart might account for myocardial depression in endotoxic shock. Subsequent studies by these and other workers led to the hypothesis that iNOS expression was deleterious not only with respect to hypotension and vascular hyporeactivity in endotoxic shock, but also in relation to myocardial dysfunction in this condition and others where the enzyme was expressed (e.g. dilated cardiomyopathy) (e.g. [20–23]). In the present update, we focus on the role of iNOS in endotoxic shock and other cardiovascular disorders. NO is now known to be a potent, locally acting vasodilator that has a central role in the regulation of vascular smooth muscle tone. In addition, it inhibits leukocyte and platelet adhesion to the endothelium, leukocyte activation and platelet aggregation, and endothelial permeability [6,24]. It thus optimises blood flow regulation in the microcirculation. In the heart, endothelium-derived NO modulates myocardial relaxation and diastolic function and the Frank-Starling response [25], and reduces oxygen consumption independent of effects on contractile function [26]. NO generated within cardiac myocytes by eNOS and possibly also nNOS may modulate excitation-contraction coupling via effects on sarcolemmal Ca2+ channels and sarcoplasmic reticular function [27,28]. It can also modulate heart rate and β-adrenergic inotropic responses [27]. NO has vascular and myocardial anti-proliferative potential, and can act as a bifunctional regulator of cell apoptosis [27,28]. NO-triggered downstream signal transduction may be cGMP-dependent or -independent [6,24]. The latter usually involves direct reactions of NO with amino, thiol, or diazo groups in proteins, and with haem and Fe2+ or sulphur centres. Under conditions where both NO and superoxide are generated, a diffusion-limited, essentially irreversible reaction between these molecules leads to the formation of peroxynitrite [29]. Low levels of peroxynitrite can be beneficial via stimulation of guanylyl cyclase, but higher levels generate highly reactive hydroxyl-like species that induce toxic effects secondary to protein oxidation [29]. A reduction in endothelial NO production or bioavailability contributes to ‘endothelial dysfunction’, which is a feature of many cardiovascular pathologies – e.g., hypertension, hypercholesterolaemia, atherosclerosis, diabetes, heart failure [30]. Endothelial dysfunction contributes to disease pathophysiology and, in at least some cases, may even have a primary pathogenetic role. In conditions such as ischaemia-reperfusion, the generation of peroxynitrite from reaction between superoxide and eNOS-derived NO may have damaging effects. The induction of iNOS in cardiovascular tissues has been suggested to be involved in the pathophysiology of several disorders. Upon exposure to cytokines or LPS, iNOS can be expressed in most cardiovascular tissues, e.g., vascular smooth muscle, endothelial cells, and cardiac myocytes [20,31–35]. However, in many in vivo settings (and especially in the heart), a major proportion of iNOS expression and activity may in fact be in infiltrating inflammatory cells (e.g. [36]). Induction of iNOS mRNA is inhibited by glucocorticoids (e.g. dexamethasone), transforming growth factor β(TGFβ), and osteopontin – a multifunctional extracellular matrix phosphoprotein that may itself be induced by cytokines [37]. On the other hand, iNOS expression is augmented by cAMP, angiotensin II, vasopressin and adrenomedullin. Cytokine-induced expression of iNOS is often accompanied by co-induction of GTP cyclohydrolase I, which regulates BH4 production [31], and cationic amino acid transporters, which regulate l-arginine transport [38]; co-induction of these proteins may be required for optimal NO production by iNOS. Numerous studies have shown that iNOS induction in vessels in vitro leads to vasodilatation and vascular hyporeactivity to vasoconstrictors. Many studies have also documented myocardial dysfunction after iNOS induction. In some studies, this was manifest as a decrease in baseline myocardial contractile function [21,34,35,39–42], whereas others reported just a reduction in β-adrenergic inotropic responsiveness [22,31,43]. iNOS-induced myocyte apoptosis and death have also been documented [44,45]. It should be noted that cytokine-induced changes in vascular or myocardial function may involve several NO-independent pathways. In addition, cytokines can activate pre-existing eNOS, so that NO-mediated dysfunction may not necessarily be due to induction of iNOS [46,47]. In vivo expression of iNOS mRNA and protein in cardiac and/or vascular tissues has been demonstrated in several conditions, e.g., endotoxic shock, cardiac allograft rejection, dilated cardiomyopathy, myocarditis, and heart failure (see below). A role of iNOS in the vascular hyporeactivity of endotoxic shock has been suggested by the results of many studies that have employed pharmacological inhibitors of NOS (both non-selective and iNOS-selective) in vivo. However, in the case of cardiac iNOS expression, investigation of functional consequences in vivo has been very limited. Nevertheless, based on the detection of iNOS and on the effects of cytokine-induced iNOS expression in vitro, it has been widely speculated that intra-cardiac iNOS expression leads to contractile depression and other harmful effects. In determining the relationship between evidence of iNOS expression and potential functional consequences, several factors need to borne in mind. First, the potential involvement of iNOS should ideally be based on the direct detection of iNOS mRNA, protein and activity; the use of glucocorticoid-inhibitable responses or Ca2+-independent NOS activity alone as markers of iNOS can be misleading. Second, the level of expression of iNOS mRNA or protein does not necessarily reflect functional activity, which may be impaired because of substrate or co-factor deficiency, or even be dysfunctional with the production of superoxide [6,48]. In this regard, the results of in vitro biochemical NOS activity assays performed in the presence of non-limiting concentrations of substrate and co-factors do not necessarily reflect the true activity of the enzyme in vivo. Third, the temporal and spatial expression/activity of iNOS may vary according to disease stage and severity. For example, the time course of iNOS expression and activity in endotoxic shock does not always correlate with functional vascular dilatation [49]. Fourth, cytokine-independent pathways and cytokine-dependent but NOS-independent pathways may be involved in many conditions. Finally, only relatively recently has it been appreciated that iNOS may have beneficial as well as harmful effects on cardiovascular function. A beneficial role of iNOS is not surprising given that its induction by cytokines usually occurs as a component of a host defence response. iNOS expression in macrophages has anti-viral [50] and anti-bacterial effects [51]. Other beneficial actions include cytoprotection, decreased leukocyte adhesion, anti-platelet activity, reduced vascular permeability, anti-oxidant activity, and improved cardiac diastolic function. On the other hand, the widespread expression of iNOS, particularly in non-inflammatory cells, may be harmful. As discussed above, this is especially likely in situations of concurrent oxidative stress, when high levels of peroxynitrite may be formed [29]. In the heart, high concentrations of peroxynitrite are reported to potently inhibit both contractility and respiration [52], reduce cardiac efficiency [53], and promote Ca2+ overload [54]. Septic shock resulting from gram negative bacterial infection is a major cause of morbidity and mortality worldwide. It is a systemic inflammatory process triggered by LPS and other microbial products [55,56]. Multiple signal transduction cascades are activated in the condition, including the production of cytokines (TNFα, IL-1, IL-2, interferon γ, leukaemia inhibitory factor), platelet activating factor, endothelin, kinins, eicosanoids, reactive oxygen species, and adhesion molecules, and the activation of the coagulation and complement pathways. Cardinal clinical features of the syndrome are hypotension, vascular hyporeactivity to vasoconstrictors, intrinsic myocardial depression (independent of changes in cardiac loading), blood flow maldistribution to organs, impaired oxygen extraction, and eventually multi-organ failure. It has been suggested that the induction of iNOS in multiple tissues is a central, even obligatory, component of the pathophysiology of evolving septic shock and of the presumed final common pathway leading to death. Overproduction of NO could, at least in theory, account for many of the clinical features described above since it (a) is a potent vasodilator and is involved in blood flow regulation, (b) can depress myocardial function, (c) can impair cellular respiration, and (d) is generated in large amounts in septic shock. Indeed, NOS inhibitors have been documented to improve or reverse hypotension in experimental models of septic shock (e.g. [17,57,58]) and in preliminary small clinical studies [59]. However, in many studies NOS inhibitors worsened overall outcome (e.g. [14]). Earlier studies, such as that by Wright et al. [14], suggested that the inhibition of eNOS was the reason for these deleterious effects, and that selective iNOS inhibition alone may be the preferred therapeutic option [21]. However, as discussed above, iNOS itself may also have beneficial effects. Indeed, some studies have shown that organ dysfunction in endotoxic shock is not prevented by selective inhibition of iNOS [60]. Recent studies of iNOS knockout mice have provided significant new information about the role of iNOS in endotoxic shock. As expected, LPS does not induce iNOS in these mice [61–63]. MacMicking et al. reported that hypotension and mortality in response to low-dose LPS were significantly lower in anaesthetized iNOS knockout mice [61]. However, in conscious mice injected higher doses of LPS, iNOS knockout animals were not protected against tissue damage. Furthermore, in iNOS knockout mice treated with LPS after priming with Propionobacterium, there was no difference in mortality compared to wild type mice [61]. Laubach et al. [62] independently generated iNOS-knockout mice, and found no difference in mortality between wild-type and knockout animals administered either low-dose or high-dose LPS. These investigators also reported that female knockout mice had a higher LPS-induced mortality than wild-type female mice [64]. A third independent group [63] reported that iNOS knockout animals were more resistant to LPS-induced death. This group also found that LPS-induced hypotension in conscious, instrumented knockout mice was significantly reduced [58]. Despite the somewhat contradictory nature of these data, it is clear that while a role for iNOS-derived NO in the vascular hyporeactivity of septic shock is supported by some studies in iNOS knockout mice [58,61,65], iNOS induction is not obligatory for LPS-induced shock and death [61,62,64]. At least part of the reason for this may be that iNOS has some beneficial effects in septic shock. In addition to the beneficial effects discussed earlier, in iNOS knockout mice given LPS, it has recently been reported that leukocyte adhesion and rolling in the microcirculation are markedly increased [66]; thus, iNOS-derived NO may be especially important for microvascular integrity and function. The results of iNOS knockout and other studies indicate that NO-independent pathways (either instead of or in parallel to iNOS) may contribute significantly to many aspects of septic shock pathophysiology. In normal humans, Vallance and colleagues addressed this question by developing a technique of local instillation of endotoxin into dorsal hand veins [67]. They found that endotoxin-induced venous hyporesponsiveness was glucocorticoid-inhibitable but not mediated by NO [67]. In experimental endotoxic shock in rats, widespread vascular dilatation was observed at a time when iNOS activity had returned to normal [49]. Haem oxygenase-1 (HO-1), an enzyme that generates carbon monoxide (CO) in the process of haem catabolism, has recently been found to be markedly induced in systemic vessels and other tissues (e.g., liver, lung, heart) in experimental endotoxic shock [68–70]. CO generated either by a constitutive haem oxygenase-2 (HO-2) or by HO-1 activates guanylyl cyclase and is capable of causing vasodilatation [71,72]. In rat endotoxic shock, an inhibitor of HO, zinc protoporphyrin IX, was found to abrogate endotoxin-induced hypotension [69]. Consistent with these findings, an NO-independent activation of guanylyl cyclase in the vasculature was reported in rat endotoxic shock [73]. Interestingly, it has been suggested that HO-1 may be inducible by NO [74]. As is the case with iNOS, it is likely that HO-1 induction may have beneficial (e.g., antioxidant) as well as deleterious effects [75]. The relative roles of iNOS, HO-1, and other induced proteins in septic shock remain to be worked out in detail. Intrinsic myocardial dysfunction plays a major part in the morbidity and mortality of septic shock [55]. iNOS mRNA is expressed within <3 h in inflammatory cells, cardiac and vascular smooth muscle in experimental endotoxic shock while Ca2+-independent NOS activity at about h after LPS in iNOS protein expression has been demonstrated in tissue of of In et al. published a paper that iNOS expression in cardiac myocytes contributed to myocardial depression in septic shock. These reported that the basal contractile function of myocytes h after LPS in was by exposure to a NOS inhibitor, and was prevented by with However, no direct evidence of iNOS expression or activity was provided [21]. studies have to this In a performed in an et al. not Ca2+-independent NOS activity in and found that in vitro cardiac depression was not by NOS et al. found no of non-selective NOS inhibitors or inhibitors on or myocyte function or h after LPS in A of of NOS inhibitors on in vitro cardiac function in has been reported by several groups In vivo cardiac depression by in was also suggested to be independent of NO The clear from the above studies is that iNOS induction is not an obligatory in the of intrinsic myocardial depression in or endotoxic shock. Indeed, it is known that LPS results in the expression of many proteins in the heart, e.g., decreased expression of eNOS and and increased expression of and HO-1 there is evidence a role for iNOS in the vascular hyporeactivity and hypotension of septic shock, it is clear that (a) iNOS is not involved in all aspects of septic shock myocardial (b) even where iNOS is other parallel pathways may that important when iNOS is and (c) iNOS may beneficial effects (e.g., anti-bacterial and It that NOS inhibition alone to be an in the of septic shock. of endotoxic shock may instead the of more than final common as well as the use of In experimental myocarditis, intra-cardiac iNOS expression to have beneficial effects, because of its anti-viral activity and its expression in infiltrating inflammatory cells of intravenous non-selective NOS inhibitors in experimental myocarditis, either or increased cardiac and mortality On the other hand, it has been suggested that cardiac iNOS induction may be harmful in myocarditis, because of its widespread expression in cardiac myocytes and as well as macrophages Indeed, in experimental rat models of myocarditis, an inhibitor, cardiac and and improved However, a of in knockout and wild type mice that iNOS expression was not for of disease the knockout mice to induce iNOS and had no the and of were studies have demonstrated the presence of Ca2+-independent NOS activity or of iNOS mRNA and protein in the cardiac tissue of with or heart failure. iNOS expression does not to be specific for dilated as was suggested but may be with heart failure evidence for iNOS protein expression in cardiac myocytes of was found by et al. and et al. However, et al. in a of from with dilated or cardiomyopathy, found that iNOS mRNA and protein were to vascular endothelial and smooth muscle cells, with some myocyte expression only in a of et al. found that Ca2+-independent NOS activity in with the of infiltrating macrophages but not with iNOS protein expression in cardiac et al. iNOS mRNA expression in only of and even in these only at a The functional role of cardiac iNOS expression in heart failure evidence to a deleterious is In with dilated cardiomyopathy, l-NMMA had no significant effects on baseline contractility but did the response to or intravenous This was with a of of endogenous NO on basal function, but an inhibition of β-adrenergic inotropic response. However, no of eNOS or iNOS expression/activity was in this and it NOS was responsible for the observed effects. et al. found no of l-NMMA on baseline of muscle from with high iNOS However, these investigators reported that high iNOS mRNA expression was with myocardial relaxation and a reduced inotropic response to with the response of normal was In et al. reported that l-NMMA had no either on baseline function or the response to in cardiac myocytes from et al. recently reported a in which of contractile function in dilated were with iNOS and eNOS mRNA expression, by in A was found between and the expression level of eNOS or iNOS mRNA, and these suggested that this involved a beneficial of NO on diastolic function. evidence iNOS in the pathophysiology of cardiac iNOS induction was documented in cardiac microvascular endothelial cells, and inflammatory cells in cardiac in This was by of myocytes and macrophages of protein and by apoptosis of macrophages and endothelial cells iNOS expression, and apoptosis were found in cardiac In a rat the inhibitor, features of not as well as with The of cardiac myocytes from rat heart also be augmented by In cardiac in the first after an was reported between the presence of iNOS mRNA in and and diastolic dysfunction by iNOS has also been implicated in dysfunction increased microvascular of rat cardiac in rats, iNOS is expressed in the which – leading to that it may be involved in this process However, iNOS expression in vessels may in fact be based on the that was in iNOS knockout mice Recent studies that the induction of HO-1 may contribute to against [75]. in about NO have been in the since the by Vallance and colleagues and Wright et al. [14] that this to were In in the of the NOSs have led to the of new experimental for out the physiological and pathophysiological roles of NO, e.g., the use of clinical investigation has also and many of the from experimental studies have been (or in As as iNOS is it is now clear that NO from this may have beneficial as well as deleterious effects. This is not surprising given that the induction of iNOS is an well host defence response. The between beneficial and deleterious effects may be particularly by the spatial and temporal of iNOS expression, and the presence or of concurrent oxidative respect to endotoxic shock, iNOS plays a significant role in the vascular hyporeactivity of this condition, but is not the only factor responsible for this Furthermore, some aspects of the pathophysiology of endotoxic shock (e.g., intrinsic myocardial may to a large be while iNOS-derived NO also has beneficial effects (e.g., on The use of or non-selective NOS inhibitors may well in of this the other cardiovascular in which iNOS expression has been the evidence for a role for iNOS is in it to be and in allograft it to be The role of iNOS in the myocardial dysfunction of heart failure to be the of Cardiovascular at