Oxidative stress in the pathogenesis of systemic scleroderma: An overview

Abstract Systemic sclerosis (SSc) is a rare disorder of the connective tissue characterized by fibrosis of the skin, skeletal muscles and visceral organs. Additional manifestations include activation of the immune system and vascular injury. SSc causes disability and death as the result of end‐stage organ failure. Two clinical subsets of the SSc are accepted: limited cutaneous SSc (lc‐SSc) and diffuse cutaneous SSc (dc‐SSc). At present, the aetiology and pathogenesis of SSc remain obscure, and consequently, disease outcome is unpredictable. Numerous studies suggest that reactive oxidizing species (ROS) play an important role in the pathogenesis of scleroderma. Over the years, several reports have supported this hypothesis for both lc‐SSc and dc‐SSc, although the specific role of oxidative stress in the pathogenesis of vascular injury and fibrosis remains to be clarified. The aim of the present review was to report and comment the recent findings regarding the involvement and role of oxidative stress in SSc pathogenesis. Biomarkers proving the link between ROS and the main pathological features of SSc have been summarized.

of the SSc process via oestrogen receptor interactions. 4 The role of hormones in disease manifestation is also evident when considering SSc-associated pulmonary arterial hypertension. 5 Moreover, autoantibodies to oestrogen receptors have been found to correlate with disease activity in scleroderma patients. 6 Although pulmonary arterial hypertension occurs more frequently in females, males with this complication show an increased frequency of other serious SSc disease manifestations, such as scleroderma renal crisis, diffuse cutaneous disease, interstitial lung disease and an increased risk of mortality. 5 Genetic and environmental factors have been suggested to be involved in the aetiology of this disease, but the exact mechanisms involved in SSc pathogenesis are not well understood.
Several years ago Murrel 7 linked, for the first time, the pathogenesis of SSc to the occurring of oxidative stress. Several reports 8,9 have supported this hypothesis for both lc-SSc and dc-SSc, although  is a stable marker specific to omega-3 and omega-6 fatty acids peroxidation. In a case-control study, an inverse relationship was found between high plasmatic MDA levels and disease duration. 22 Riccieri et al 23 found that the increased hydroperoxide levels measured in plasma of SSc patients correlated with the capillaroscopy semiquantitative rating scale score (predictive of novel future severe organ involvement in SSc), and with the rating system for avascular areas.
These authors also reported that the levels of carbonyl groups, biomarkers of protein oxidation, inversely correlated with modified Rodnan's skin score, a standard outcome measure for skin disease in SSc, and were lower in patients with pulmonary fibrosis. shock protein 70 (Hsp70) levels. 28 Increased levels of Hsp70 in SSc patients have been associated with pulmonary fibrosis, skin sclerosis, renal vascular damage, oxidative stress and inflammation. 28 Oxidative post-transcriptional protein modifications, for example, nitrated proteins, known to be a marker of • NO-derived oxidants, 29 and advanced oxidation protein products (AOPP) have been measured in SSc plasma and skin. 30 Interestingly, AOPP in turn seems to be able to stimulate ROS formation in cell targets. Indeed, serum containing high AOPP levels isolated from SSc patients has been found to stimulate endothelial cells and fibroblasts from healthy donors to generate ROS involved in vascular or fibrotic complications. 30 Interestingly, AOPP generated by different oxidation patterns can induce the selective triggering of cells to produce H 2 O 2 or • NO, suggesting that these oxidation products may be involved in the generation of different types of ROS in SSc patients. 30 Moreover, urinary levels of both 8-hydroxy-2 0 deoxyguanosine (the main validated biomarker of endogenous oxidative damage to DNA) and F2-isoprostane (a product of ROS-mediated arachidonic acid peroxidation) were found to be higher in SSc patients than in controls. 31 VONA ET AL.

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The multivariate analysis indicated a relevant association with a fibrotic phenotype. In fact, 8-hydroxy-2 0 deoxyguanosine levels were significantly associated with the presence of pulmonary fibrosis, decreased forced vital capacity and decreased alveolar volume suggesting a potential predictive value of this biomarker. 31 Biomarkers of oxidative stress detected in SSc are listed in Table 1.
The total antioxidant capacity (TAC), which accounts for the ability of tissues to counteract oxidative stress, has been reported to be decreased in plasma of SSc patients. 14

| ROS SOURCES IN SSC
Ischaemia reperfusion events, clinically manifested in SSc patients as RP, have been considered the most likely contributors to the abnormal oxidative stress observed in this disorder. 8

ROS production in
SSc tissues could also be triggered by the interaction of cytokines or growth factors with their specific receptors, such as interleukin-6 (IL-6), IL-3, tumour necrosis factor a, angiotensin II, platelet-derived growth factor (PDGF), transforming growth factor b1 (TGF-b1), nerve growth factor, fibroblast growth factor, and granulocytemacrophage colony-stimulating factor. 3,8 ROS may be generated inside the vascular lumen by peripheral blood cells, or within the vessel wall by monocytes, endothelial cells, erythrocytes and adventitial fibroblasts in response to one or more noxious agents (Figure 1). 38 Cells involved in ROS production are reported below.

| Red blood cells
Because of their high iron concentration (~20 mmol/L), red blood cells (RBCs) can be considered as an "iron mine" but, paradoxically, they also represent one of the major components of blood antioxidant capacity and one of the cells with higher resistance to oxidative F I G U R E 1 Sources of reactive oxidizing species (ROS) in SSc. In SSc, ROS may be generated inside the vascular lumen by activated monocytes and macrophages, erythrocytes, endothelial cells and fibroblasts. In addition to ROS, activated endothelial cells release adhesion molecules, which can trigger an inflammatory cascade, elevating the plasma levels of pro-inflammatory cytokines in patients stress. 48 Crossing inflamed areas, RBCs can help to detoxify ROS, thus rescuing or partially "protecting" cells (eg, endothelial cells). A completely different situation may arise when RBCs cross a tissue where an intense production of ROS occurs. Under these conditions, RBCs may accumulate oxidative damage, and act as pro-oxidant "bullets" capable of modifying the behaviour and fate of endothelial cells. 49 It has been demonstrated that the systemic oxidative imbalance occurring in SSc patients induces changes in RBCs, including cytoskeleton oxidative denaturation and derangement, and loss of lipid asymmetry. 6 These changes can result in the alteration of RBC adhesive properties, aggregability and deformability, and correlate with disease severity. In agreement, high MDA and • NO levels have been measured in RBCs of SSc patients. 19  Bax is a pro-apoptotic member of the Bcl-2 family. 51 Moreover, it has been demonstrated that both cellular Abelson (c-Abl) and TGF-b1 serve as apoptosis suppressors in human dermal fibroblasts. c-Abl is known as a TGF-b1-modulating molecule in fibrosis. TGF-b1 is an important cytokine that induces fibroblast differentiation into myofibroblasts and is considered to be the major factor towards chronic fibrosis. SSc patients express elevated TGF-b1 levels in the early lesions, but not in established fibrotic tissue. 52 Kim and collaborators demonstrated that TGF-b1 induced the activation of Akt in normal and RA synovial fibroblasts, and that TGF-b1 exerted its anti-apoptotic effect, in part, through the PI3 kinase/Akt pathway. 53

| AN TIOXIDAN T THERAPY FOR SSC
A reduced concentration of classical antioxidants, such as antioxidant vitamins (ascorbic acid, a-tocopherol, and b-carotene) and minerals (zinc, selenium), has been found in RP and SSc. This antioxidant potential deficiency increases the propensity to oxidative stress, favouring the development of injury mediated by ROS generation.
Currently, molecules as N-acetyl-l-cysteine (NAC), antioxidant vitamins and polyphenols may be useful in the supportive therapy of SSc and RP. NAC acts as a precursor for the substrate (l-cysteine) in the synthesis of hepatic glutathione (GSH), and replenishes GSH in deficient cells. It influences protein thiols, supports glutathione synthesis and generates free sulfhydryl groups. In many studies, NAC has shown a beneficial influence on SSc, diminishing cellular ROS in fibroblast and replenishing free cellular thiols. 54 Vitamin E is a potent intracellular antioxidant 55 and protects the polyunsaturated fatty acids present in membrane phospholipids and in plasma lipoproteins.
Moreover, its administration considerably increases cell-mediated and humoural immune functions in human. 56  There are several possible explanations for these poor results, for example, (i) the short duration of therapy. It is possible that in order to be effective, the antioxidant therapy has to be given early in the SSc disease process, before the onset of irreversible tissue damage; (ii) the malabsorption syndrome resulting from increased collagen deposition in the intestines or bacterial overgrowth in SSc patients; and (iii) an altered renal clearance of the aqueous phase antioxidants (such as ascorbic acid) and its increased excretion in SSc patients.
Although recent findings supporting the efficacy of antioxidant therapy are very encouraging, [54][55][56]58 further studies are necessary to define the therapeutic potential of the analysed antioxidants in SSc.
Anyway, notwithstanding substantial advancements, the morbidity and mortality in SSc are still high and can largely be attributed to a delay in diagnosis. In fact, at the time of diagnosis, SSc is often well established with significant irreversible tissue and organ damage. An early and accurate diagnosis of SSc and the use of autoantibody testing embedded in evidence-based clinical care pathways will help improve SSc-associated clinical outcomes and healthcare expenditures.

| CONCLUSION S
This review comments on the recent findings regarding the involvement and the role of oxidative stress in SSc pathogenesis.
Biomarkers proving the link between ROS and the main pathological features of SSc could be of interest in the development of more appropriate diagnostics and therapeutic strategies. In fact, the pathogenesis of SSc appears as complex and involves a complex framework of "actors" such as endothelial cells, epithelial cells, fibroblasts and immunological mediators, resulting in dysregulated vascular remodelling and, ultimately, vasculopathy. In addition, taking into account the significant gender disparity in the occurrence of the disease as well as the well-known differences in ROS generation by XX and XY cells, including vessel cells, 60 the relevance of the redox state as a gender-associated determinant in the disease onset and progression cannot be ruled out.