Silvia Svegliati

Reactive oxygen species are required for maintenance and differentiation of primary lung fibroblasts in idiopathic pulmonary fibrosis.

Background Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal illness whose pathogenesis remains poorly understood. Recent evidence suggests oxidative stress as a key player in the establishment/progression of lung fibrosis in animal models and possibly in human IPF. The aim of the present study was to characterize the cellular phenotype of fibroblasts derived from IPF patients and identify underlying molecular mechanisms. Methodology/Principal Findings We first analyzed the baseline differentiation features and growth ability of primary lung fibroblasts derived from 7 histology proven IPF patients and 4 control subjects at different culture passages. Then, we focused on the redox state and related molecular pathways of IPF fibroblasts and investigated the impact of oxidative stress in the establishment of the IPF phenotype. IPF fibroblasts were differentiated into alpha-smooth muscle actin (SMA)-positive myofibroblasts, displayed a pro-fibrotic phenotype as expressing type-I collagen, and proliferated lower than controls cells. The IPF phenotype was inducible upon oxidative stress in control cells and was sensitive to ROS scavenging. IPF fibroblasts also contained large excess of reactive oxygen species (ROS) due to the activation of an NADPH oxidase-like system, displayed higher levels of tyrosine phosphorylated proteins and were more resistant to oxidative-stress induced cell death. Interestingly, the IPF traits disappeared with time in culture, indicating a transient effect of the initial trigger. Conclusions/Significance Robust expression of α-SMA and type-I collagen, high and uniformly-distributed ROS levels, resistance to oxidative-stress induced cell death and constitutive activation of tyrosine kinase(s) signalling are distinctive features of the IPF phenotype. We suggest that this phenotype can be used as a model to identify the initial trigger of IPF.

Oxidative stress and the pathogenesis of scleroderma : the Murrell's hypothesis revisited

Systemic sclerosis (SSc, scleroderma) is a devastating, immune-mediated, multisystem disorder characterized by microvasculature damage, circulating autoantibodies, and fibroblast activation, leading to massive fibrosis of skin, vessels, muscles, and visceral organs. Scleroderma causes disability and death as the result of end-stage organ failure. At present, no specific diagnostic nor therapeutic tools are available to handle the disease. In spite of significant effort, the etiology and pathogenesis of SSc remain obscure and, consequently, the disease outcome is unpredictable. Several years ago, Murrell suggested a unifying hypothesis linking the pathogenesis of scleroderma to the generation of a large excess of reactive oxygen species. This hypothesis has been substantiated by several reports indicating the presence of an abnormal redox state in patients with scleroderma. This review will summarize the available evidence supporting the link between free radicals and the main pathological features of scleroderma.

Long-term outcome and prospective validation of NIH response criteria in 39 patients receiving imatinib for steroid-refractory chronic GVHD

Forty adults aged 28 to 73 years were entered into a prospective trial of imatinib for the treatment of steroid-refractory chronic graft-versus-host disease (SR-cGVHD). After 6 months, intention-to-treat (ITT) analysis of 39 patients who received the drug, regardless of the duration of treatment, revealed 14 partial responses (PR), 4 minor responses (MR) with relevant steroid sparing (46%) according to Couriel criteria, and 20 ≥ PR (51.3%), as per the National Institutes of Health (NIH) criteria and NIH severity score changes. The best responses were seen in the lungs, gut, and skin (35%, 50%, and 32%, respectively). After a median follow-up of 40 months, 28 patients were alive, with a 3-year overall survival (OS) and event-free survival of 72% and 46%, respectively. The 3-year OS was 94% for patients responding at 6 months and 58% for nonresponders according to NIH response, suggesting that these criteria represent a reliable tool for predicting OS after second-line treatment. Monitoring of anti-platelet-derived growth factor receptor (PDGF-R) antibodies showed a significant decrease in PDGF-R stimulatory activity in 7 responders, whereas it remained high in 4 nonresponders. This study confirms the efficacy of imatinib against SR-cGVHD and suggests that the response at 6 months significantly predicts long-term survival.

New Insights into the Role of Oxidative Stress in Scleroderma Fibrosis

Systemic sclerosis (Scleroderma – SSc) is a connective tissue disorder of unknown aetiology characterized by extensive fibrosis of the skin and visceral organs, by vascular abnormalities and immunological manifestations. Recent evidence suggest that the cellular redox state may play a significant role in the progression of scleroderma fibrosis. Mechanisms involved include an autoamplification circuit linking ROS, Ras and ERK 1-2 which in turn amplifies and maintains the autocrine loop made up by cytokines, growth factors and their cognate receptors. This review summarizes the recent progress on the role of oxidative stress in the pathophysiology of scleroderma and disorders characterised by organ fibrosis

HaRas activates the NADPH oxidase complex in human neuroblastoma cells via extracellular signal-regulated kinase 1/2 pathway

In this study we have investigated the effects of the small GTP‐binding‐protein Ras on the redox signalling of the human neuroblastoma cell line, SK‐N‐BE stably transfected with HaRas(Val12). The levels of reactive oxygen species (ROS) and superoxide anions were significantly higher in HaRas(Val12) expressing (SK‐HaRas) cells than in control cells. The treatment of cells with 4‐(2‐aminoethyl) benzenesulfonylfluoride, a specific inhibitor of the membrane superoxide generating system NADPH oxidase, suppressed the rise in ROS and significantly reduced superoxide levels produced by SK‐HaRas cells. Moreover, HaRas(Val12) induced the translocation of the cytosolic components of the NADPH oxidase complex p67phox and Rac to the plasma membrane. These effects depended on the mitogen‐activated protein kinase kinase/extracellular signal‐regulated kinase (MEK/ERK1/2) pathway, as the specific MEK inhibitor, PD98059, prevented HaRas‐mediated increase in ROS and superoxide anions. In contrast, the specific phosphoinositide 3‐kinase (PI3K) inhibitors LY294002 and wortmannin were unable to reverse the effects of HaRas(Val12). Moreover, cholinergic stimulation of neuroblastoma cells by carbachol, which activated endogenous Ras/ERK1/2, induced a significant increase in ROS levels and elicited membrane translocation of p67phox and Rac. ROS generation induced by carbachol required the activation of ERK1/2 and PI3K. Hence, these data indicate that HaRas‐induced ERK1/2 signalling selectively activates NADPH oxidase system in neuroblastoma cells.

Oxidative DNA damage induces the ATM-mediated transcriptional suppression of the Wnt inhibitor WIF-1 in systemic sclerosis and fibrosis

DNA damage not only promotes a mitotic checkpoint response but also activates a pathway that can produce fibrotic disease. Connecting DNA Damage to Fibrotic Skin Thickening Systemic sclerosis (SSc) is an autoimmune disease characterized by the accumulation of collagen in the connective tissue, a process known as fibrosis. Fibrosis affects skin and internal organs and is frequently associated with activation of the Wnt signaling pathway. Svegliati et al. found loss of the Wnt inhibitor WIF-1 in skin biopsies from SSc patients compared with healthy skin. Antibodies in serum from SSc patients or oxidative DNA-damaging agents triggered a transcriptional suppression pathway that suppressed WIF-1 expression and promoted collagen production in normal fibroblasts. In fibroblasts cultured from SSc patients, inhibiting this pathway restored WIF-1 expression and reduced collagen production; in a mouse model of fibrosis, inhibiting this pathway prevented fibrotic skin thickening. Systemic sclerosis (SSc) is an autoimmune disease characterized by extensive visceral organ and skin fibrosis. SSc patients have increased production of autoreactive antibodies and Wnt signaling activity. We found that expression of the gene encoding Wnt inhibitor factor 1 (WIF-1) was decreased in fibroblasts from SSc patient biopsies. WIF-1 deficiency in SSc patient cells correlated with increased abundance of the Wnt effector β-catenin and the production of collagen. Knocking down WIF-1 in normal fibroblasts increased Wnt signaling and collagen production. WIF-1 loss and DNA damage were induced in normal fibroblasts by either SSc patient immunoglobulins or oxidative DNA-damaging agents, such as ultraviolet light, hydrogen peroxide, or bleomycin. The DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM) mediated WIF-1 silencing through the phosphorylation of the transcription factor c-Jun, which in turn activated the expression of the gene encoding activating transcription factor 3 (ATF3). ATF3 and c-Jun were recruited together with histone deacetylase 3 (HDAC3) to the WIF-1 promoter and inhibited WIF-1 expression. Preventing the accumulation of reactive oxygen species or inhibiting the activation of ATM, c-Jun, or HDACs restored WIF-1 expression in cultured SSc patient cells. Trichostatin A, an HDAC inhibitor, prevented WIF-1 loss, β-catenin induction, and collagen accumulation in an experimental fibrosis model. Our findings suggest that oxidative DNA damage induced by SSc autoreactive antibodies enables Wnt activation that contributes to fibrosis.

Epitope Specificity Determines Pathogenicity and Detectability of Anti–Platelet-Derived Growth Factor Receptor α Autoantibodies in Systemic Sclerosis

To identify the epitopes recognized by autoantibodies targeting platelet‐derived growth factor receptor α (PDGFRα) in systemic sclerosis (SSc) and develop novel assays for detection of serum anti‐PDGFRα autoantibodies.

Autoantibodies against the platelet-derived growth factor receptor in scleroderma: comment on the articles by Classen et al and Loizos et al.

patients is unclear. Drs. Zeidler and Rihl further suggest that some cases of PsA, which is another SpA, might also be caused by chlamydial infection. We find it interesting indeed that they recently detected C trachomatis in synovial fluid from patients with PsA. Not only are the clinical features of PsA and reactive arthritis indistinct, but palmoplantar pustular psoriasis and keratoderma blenorrhagicum cannot be differentiated grossly or histologically (2). Finally, we stress the fact that 14 (88%) of the 16 subjects in our study whose synovial tissue was positive for chlamydiae by PCR analysis had an asymptomatic initial infection. This can and certainly does make classification of the specific type of SpA less clinically apparent. Taken together, data from our study and the important observations cited by Zeidler and Rihl help to support the hypothesis initially proposed by Dr. Denys Ford that one agent may cause different clinical syndromes, and that one syndrome may be attributable to many infectious agents (3). Indeed, we discuss precisely those ideas in terms of recent data in a manuscript that is now in preparation (4). We agree entirely that a concerted effort needs to be undertaken to precisely define the role of infectious agents in all types of SpA.

Agonistic Anti-PDGF Receptor Autoantibodies from Patients with Systemic Sclerosis Impact Human Pulmonary Artery Smooth Muscle Cells Function In Vitro

One of the earliest events in the pathogenesis of systemic sclerosis (SSc) is microvasculature damage with intimal hyperplasia and accumulation of cells expressing PDGF receptor. Stimulatory autoantibodies targeting PDGF receptor have been detected in SSc patients and demonstrated to induce fibrosis in vivo and convert in vitro normal fibroblasts into SSc-like cells. Since there is no evidence of the role of anti-PDGF receptor autoantibodies in the pathogenesis of SSc vascular lesions, we investigated the biologic effect of agonistic anti-PDGF receptor autoantibodies from SSc patients on human pulmonary artery smooth muscle cells and the signaling pathways involved. The synthetic (proliferation, migration, and type I collagen gene α1 chain expression) and contractile (smooth muscle-myosin heavy chain and smooth muscle-calponin expression) profiles of human pulmonary artery smooth muscle cells were assessed in vitro after incubation with SSc anti-PDGF receptors stimulatory autoantibodies. The role of reactive oxygen species, NOX isoforms, and mammalian target of rapamycin (mTOR) was investigated. Human pulmonary artery smooth muscle cells acquired a synthetic phenotype characterized by higher growth rate, migratory activity, gene expression of type I collagen α1 chain, and less expression of markers characteristic of the contractile phenotype such as smooth muscle-myosin heavy chain and smooth muscle-calponin when stimulated with PDGF and autoantibodies against PDGF receptor, but not with normal IgG. This phenotypic profile is mediated by increased generation of reactive oxygen species and expression of NOX4 and mTORC1. Our data indicate that agonistic anti-PDGF receptor autoantibodies may contribute to the pathogenesis of SSc intimal hyperplasia.

NADPH oxidase, oxidative stress and fibrosis in systemic sclerosis

ABSTRACT Systemic sclerosis (SSc) is an autoimmune disease characterized by damage of small vessels, immune abnormalities and exaggerated production of extracellular matrix. The etiology of the disease is unknown and the pathogenesis ill defined. However, there is consistent evidence that oxidative stress contributes to the establishment and progression of the disease. This review examines the most relevant research regarding the involvement of free radicals and of nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidases; NOX) in the pathogenesis of systemic sclerosis. Graphical abstract Figure. No caption available. HighlightsOxidative stress may play a role in the molecular pathogenesis of fibrosis and in the development of systemic sclerosis.NADPH oxidases are involved in ROS generation in systemic sclerosis and other fibrotic disorders.Further work is needed to develop new specific NOX inhibitors for the treatment of several fibrotic disorders.