Beauty and the Beast. The nitric oxide paradox in systemic sclerosis{dagger}

M. Matucci Cerinic and M. B. Kahaleh1

Department of Medicine, Division of Rheumatology, University of Florence, Italy and
1 Department of Medicine, Division of Rheumatology, Medical College of Ohio, Toledo, OH, USA

Vascular endothelial dysfunction is a central event in the pathogenesis of a variety of human diseases, including systemic sepsis, ischaemia–reperfusion injury, adult respiratory distress syndrome, atherosclerosis and diffuse systemic inflammatory disorders.

In systemic sclerosis (SSc), the microvascular bed is the target of an immune–inflammatory injury that leads to dysregulation of vascular tone control and results in progressive disorganization of the vascular architecture, leading to vascular obliteration and diminished blood flow to the organs involved [1]. On the cellular level, endothelial dysfunction is characterized by a shift in the endothelial functional profile towards an inflammatory and vasospastic functional potential [2]. Raynaud's phenomenon (RP) is the most recognizable clinical sign that reflects this dysfunction. Several pathological mechanisms have been proposed as potential causes of RP [3]. The endothelial hypothesis suggests reduced production of the endothelium-derived vasodilatory mediators [prostacyclin, nitric oxide (NO)] and increased endothelial vasoconstrictive signals (endothelin) in the pathogenesis of RP [46]. However, while endothelin levels have unanimously been reported to be elevated, the exact status of NO production in SSc is confusing as both increased and decreased circulating total nitrate levels and production in SSc have been reported [610] (Table 1Go). Because the physiological and pathological functions of NO are diverse and often contradictory, in SSc the effects of NO may be seen as paradoxical—it may have a positive as well as a negative effect according to the perspective from which its involvement in and contribution to some pivotal physiological mechanisms is considered. We will focus here on the potentially positive and negative effects of NO on the pathogenesis of SSc.


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TABLE 1.  Levels of NO detected in the serum, urine, exhaled air and monocytes of patients with SSc

 
NO is biosynthesized from L-arginine and the recycling of L-citrulline [11]. Mammalian systems contain three well-characterized isoforms of nitric oxide synthase: neuronal (nNOS, also called NOS1), inducible (iNOS or NOS2) and endothelial (eNOS or NOS3). The endothelial form is expressed constitutively by the vascular endothelium, where it is regulated by shear-stress forces, endothelin, neuropeptides and certain cytokines. iNOS is expressed in the endothelium, smooth muscle cells, fibroblasts, macrophages and many other cell types, and is regulated chiefly by inflammatory mediators and cytokines [11]. The importance of eNOS in the maintenance of vasorelaxation, the prevention of vasospasm and platelet activation can be understood readily in a disease such as SSc, in which vasoconstriction is one of the main pathological processes that may influence disease pathogenesis and promote the development of complications.

Beauty: the protective role of nitric oxide. The NO generated constitutively by the endothelium plays a critical role in the control of vascular tone and the regulation of blood pressure and acts as an antithrombotic and cytoprotective agent [12]. Indeed, NO has been shown to inhibit adhesion molecule expression, platelet adhesion and aggregation and smooth muscle cell proliferation [12]. Its release is strictly dependent on the presence and integrity of the vascular endothelium [12]. Derangement of the endothelium in pathological states leads to impairment of constitutive NO synthesis and release, as in hyperlipidaemia, hypertension, diabetic vasculopathy and cigarette smoking [12]. The impact of reduced constitutive NO release on the vascular beds and surrounding tissue can be extensive. Dysregulated control of vascular tone and increased adhesiveness and thickening of the vascular wall may result from this deficiency. This sequence of pathological events may apply to SSc, in which injury of the vascular endothelium leads to vasospasm, platelet activation and intimal proliferation. Thus, deficient NO release in the steady state may play a pivotal role in the pathogenesis of these events. In SSc, failure of endothelium-dependent vasodilatation in response to substance P and other mediators has been shown in the acral circulation, the kidneys and the lungs [1315], suggesting failure of NO production at the endothelial level. This view is strengthened by a recent report showing deficient NO release from SSc vascular endothelium in vitro [16]. Decreased basal NO and shear stress-induced NO release by SSc microvascular endothelial cells in vitro has also been described [17]. Moreover, reduced endothelial NO synthase mRNA and protein levels were described in microvascular endothelial cells derived from SSc patients [18]. These findings suggest a key change in the endothelial phenotype that may result from or may predispose to the disease. This conclusion is reinforced by a similar defect in uninvolved microvascular endothelial cells, suggesting a generalized endothelial dysfunction in SSc [16]. Impaired basal NO production may contribute greatly to the development of SSc vascular disease by enhancing vasospasm, platelet aggregation, the up-regulation of endothelial and leucocyte adhesion molecules [selectins, ß-integrins and ICAM-1 (intercellular cell adhesion molecule 1)] and increasing vascular wall thickness. The utility of NO is also stressed in joint involvement, where, in an experimental model, it has been demonstrated that iNOS is useful in protecting from exacerbation of antigen-induced arthritis [19]. Indeed, the influence of deficient endothelial NO release may extend beyond the control of vascular tone and platelet activation. It is well known that the mechanism of reperfusion injury and the formation of free radicals is a pivotal event in SSc [20]. For this reason, the basal release of NO is considered to be a potent chemical barrier that protects the vascular endothelium from oxidation injury [21, 22]. This conclusion is based on the fact that the oxygen-derived free-radical superoxide anion, plays an important role in the pathogenesis not only of SSc but also of many other diseases. Recent demonstrations of the inactivation of by NO suggest that NO acts as a useful endogenous free-radical scavenger. This hypothesis was tested recently in an in vitro system by analysing the effect of NO on production. NO depressed the rate of reduction of cytochrome c by released from polymorphonuclear neutrophils or generated from the oxidation of hypoxanthine by xanthine oxidase [23]. These observations indicate that NO can be regarded as a scavenger of the superoxide anion and suggest that NO may provide a chemical barrier to cytotoxic free radicals. Moreover, NO donors inhibit lipid peroxidation in a concentration-dependent manner [22] and suppress the programmed death of endothelial cells [24, 25]. These findings suggest that NO may have a considerable protective effect on cellular viability and can act as an antioxidant protecting cells from oxidant-induced damage and preventing endothelial apoptosis. Thus, in SSc, a vicious cycle of oxidation injury [20], loss of endothelial adaptation and consequent functional alteration [26] may enhance and/or follow deficiency in the basal protective effect of NO against oxidation injury. This may suggest that NO supplementation in SSc patients could be a useful tool for controlling endothelial derangement following reperfusion injury.

The Beast: the harmful role of nitric oxide. Increased production of NO by the inducible form of NO synthase in SSc is suggested by increased expression of iNOS in lesional skin [7, 27], endothelial cells, fibroblasts, mononuclear cells infiltrating SSc skin [27], and alveolar macrophages [28], and by increased production of NO by peripheral blood mononuclear cells when stimulated with interleukin 1ß [29, 30]. The most interesting data concerning NO in the affected tissue have been provided by a UK group, who have shown that a metabolic switch occurs in cutaneous endothelial cells affected by the disease. They describe elegantly how the progression of the disease changes the NO synthase from the endothelial form (eNOS) to the inducible form (iNOS) [27]. Indeed, while the damage is occurring in the skin the cells express nitrotyrosine, which indicates active free-radical injury [27]. Moreover, the increased total circulating amount of nitrate that has been reported in SSc [610] and its correlation with markers of endothelial damage and disease activity [7] suggest that, despite the possible reduction in constitutive endothelial NO production, this increased total NO production may be accounted for mainly by iNOS. In this inflammatory milieu, free radicals produced by inflammatory cells may react with NO produced by iNOS to form peroxynitrate, a potent oxidant that mediates cellular and tissue injury in various pathological conditions [20]. The detrimental effects of peroxynitrate are due to its reactivity with a vast range of molecules, including amino acids, lipids, nucleic bases and antioxidants in oxidation- and nitration-related processes. This reactivity induces modification of the structure and function of target molecules, including carbohydrate and lipid biomolecules, leading to tissue dysfunction and injury. Moreover, the breakage of single-strand DNA and activation of the nuclear enzyme poly(ADP-ribose) synthetase may lead to ineffective cellular repair and contribute to cell damage and death [31]. This effect is particularly prominent in the hypoxic environment, a common finding in SSc [20]. This sequence of events may play a pivotal role in the pathogenesis of tissue damage in the ischaemia–reperfusion environment, as in RP, in which oxygen radicals and NO production may overwhelm the cellular defences, leading to oxidative vascular damage and endothelial apoptosis [26]. Evidence of increased endothelial apoptosis has been detected in SSc [32]. The switch from eNOS to iNOS when free radical production is increased as disease progresses may contribute to the transformation of NO from a protector to a noxious enhancer of inflammatory injury to cells, and therefore of endothelial apoptosis [21, 33]. Thus, low NO concentrations contribute to endothelial cell survival and high NO levels induce the apoptosis of endothelial cell [34]. Indeed, the noxious role of iNOS may also be supported by the evidence from animal experiments that genetic deficiency of iNOS reduces atherosclerosis and lowers plasma lipid peroxides in apolipoprotein E knockout mice [35].

It is interesting to note that transforming growth factor ß(TGF-ß), in a surprisingly low dose range, has the capacity to reduce oxidative injury caused by transient ischaemia [36]. This finding raises the intriguing possibility that excessive TGF-ß production in SSc and the resulting tissue fibrosis may be due to amplification of the protective role of TGF-ß.

The evidence for a pathogenic role of NO may thus suggest a therapeutic strategy, which could be implemented in a variety of ways, in order to reduce the increased levels of NO and to counteract its contribution to the formation of pro-oxidant species.

Any assessment of the role of NO in human disease must take into account the dual role of NO. The measurement of total nitrate in serum or urine does not seem sufficient to assess the exact contribution of this vital mediator to the pathogenesis of human disease. We believe that it is essential to identify the functional activity of eNOS and iNOS in order to understand the role of NO in disease pathogenesis. We should recognize that NO plays two roles in the disease state, one protective and the other injurious. The final outcome of the toxic and tissue-protective activities of NO depends on several factors, including the sites and relative concentrations of individual reactive species and their diffusion distances. An excellent example of the utility of the understanding of the variation in the level of NO according to different clinical conditions is its measurement in exhaled air. In SSc, it has been demonstrated that exhaled NO is increased during interstitial involvement [3740], even in conditions of subclinical inflammation [41], but that decreased levels are found when pulmonary hypertension is present [37, 40]. Thus, an increased level of exhaled NO identifies interstitial involvement while a decreased level is associated with severe vascular involvement. Therefore, the level of circulating NO should be investigated in different phases of the disease in order to understand its role and to design a sensible approach to therapy—whether NO supplementation/enhancement or neutralization—that can be used to treat the patient at a particular stage of disease. This might allow a rational and better-tailored approach without risking exacerbation of endothelial derangement.

In SSc, endothelial injury seems to reduce eNOS and increase iNOS activity, resulting in a vasoconstricting and proinflammatory environment in association with tissue damage. Another aspect that needs to be addressed in the future is eNOS polymorphism, in order to identify patients at risk of early vascular disease [42]. Clearly, more work is needed to understand the mechanisms involved in patients in whom the protective function of NO is deficient. Furthermore, new strategies to block the toxic effects of NO or enhance its beneficial effects may open a new avenue in the management of endothelial injury and vascular insufficiency in SSc.

The authors wish to acknowledge the Scleroderma Foundation (USA) and APAI (Associazione Patologie Autoimmuni) (Italy) for their continued support.

Notes

{dagger}This work was inspired by and is dedicated to Dr C. LeRoy. He patiently taught us the passion and the tools in the study and care of patients with systemic sclerosis. Back

Correspondence to: M. Matucci Cerinic, Department of Medicine, Section of Rheumatology, Villa Monna Tessa, Viale Pieraccini 18, 50139 Firenze, Italy. Back

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