Fibrosis--a lethal component of systemic sclerosis.

REVIEWS Fibrosis—a lethal component of systemic sclerosis Yuen Yee Ho, David Lagares, Andrew M. Tager and Mohit Kapoor Abstract | Fibrosis is a pathological process characterized by excessive accumulation of connective tissue components in an organ or tissue. Fibrosis is produced by deregulated wound healing in response to chronic tissue injury or chronic inflammation, the hallmarks of rheumatic diseases. Progressive fibrosis, which distorts tissue architecture and results in progressive loss of organ function, is now recognized to be one of the major causes of morbidity and mortality in individuals with one of the most lethal rheumatic disease, systemic sclerosis (SSc). In this Review, we discuss the pathological role of fibrosis in SSc. We discuss the involvement of endothelium and pericyte activation, aberrant immune responses, endoplasmic reticulum stress and chronic tissue injury in the initiation of fibrosis in SSc. We then discuss fibroblast activation and myofibroblast differentiation that occurs in response to these initiating processes and is responsible for excessive accumulation of extracellular matrix. Finally, we discuss the chemical and mechanical signals that drive fibroblast activation and myofibroblast differentiation, which could serve as targets for new therapies for fibrosis in SSc. Ho, Y. Y. et al. Nat. Rev. Rheumatol. advance online publication 22 April 2014; doi:10.1038/nrrheum.2014.53

Introduction

Shriners Hospital for Children, Division of Surgical Research, McGill University, 1529 Cedar Avenue, Montreal, QC H3G1A6, Canada (Y.Y.H.). Pulmonary and Critical Care Unit and Centre for Immunology and Inflammatory Diseases, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA (D.L., A.M.T.). The Toronto Western Research Institute, Division of Orthopaedics, Toronto Western Hospital, The University Health Network, 60 Leonard Avenue, Toronto, ON M5T 2S8, Canada (M.K.). Correspondence to: M.K. mkapoor@ uhnresearch.ca

Systemic sclerosis (SSc) is a complex autoimmune dis ease of unknown aetiology. The disease has a variable clinical course, but, in general, SSc is characterized by microvascular dysfunction, immune abnormalities, chronic inflammation, and interstitial and perivascular fibrosis in the skin and internal organs. Patients with SSc can have severe disabilities, such as functional impair ment of the hands owing to digital ulcers, 1 and inter nal organ dysfunction, which often leads to premature death.2 In this Review, we discuss the triggers and cellu lar, molecular and mechanical mediators of tissue fibrosis in SSc, the rheumatic disease with the highest case-based mortality.3 We also discuss the potential to target these triggers and mediators of SSc with antifibrotic therapies.

Fibrosis in SSc Development of fibrosis Fibrosis is a common outcome of diseases characterized by chronic and prolonged tissue injury and/or inflam mation, such as rheumatic diseases. Fibrogenesis is a multistage process, now increasingly seen as the result of deregulated tissue repair responses, in which aber rantly sustained production of cytokines, growth factors, and angiogenic factors tilt tissue homeostasis towards interstitial hyperplasia and excessive accumulation of extracellular matrix (ECM). Excessive deposition of ECM components—including collagens, hyaluronic acid, fibronectin and proteoglycans—leads to progres sive tissue remodelling that can ultimately destroy tissue Competing interests The authors declare no competing interests.

architecture and cause loss of organ function. Once initi ated, fibrosis can escalate through multiple feed-forward amplification loops that are generated as a consequence of tissue damage, increased matrix stiffness, hypoxia, oxidative stress and accumulation of damage-associated molecular patterns (DAMPs), all of which can promote further fibroblast activation and myofibroblast dif ferentiation. Thus, primary vascular or immune events can cause persistent fibroblast activation and progres sive injury through ‘vicious’ cycles of profibrotic events.4 Few therapies that target fibrotic processes have been developed to date, and therapies that are able to reverse existing fibrotic lesions are not yet available. Disturbances of both the vascular and immune sys tems are thought to contribute to the development of SSc. Endothelial alterations often occur early in the establishment of the disease, and are followed by oblit eration of blood vessels that progressively causes tissue ischaemia and a cascade of stimulatory changes culmi nating in tissue fibrosis (Figure 1). ‘Leaky’ endothelium is believed to activate cells such as fibroblasts, macro phages, T cells, monocytes and mast cells to aberrantly secrete cytokines, growth factors and chemokines. These mediators drive inflammation, which leads to further activation of fibroblasts and recruitment of progenitor and/or stem cells from the bone marrow and circula tion (Figure 1). Strong evidence exists for the involve ment of both innate and adaptive arms of the immune system in the initiation of SSc pathogenesis. Prolonged inflammation and the presence of local and systemic profibrotic factors facilitate the transdifferentiation of resident fibroblasts and recruited cells such as pericytes,

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REVIEWS Key points ■■ Fibrosis is characterized by excessive accumulation of connective tissue components in organs or tissues, which is caused by deregulated wound-healing processes in response to chronic tissue injury and/or inflammation ■■ Chronic tissue injury and inflammation—hallmarks of rheumatic diseases —are crucial in activating the tissue repair mechanisms that result in fibrosis in systemic sclerosis (SSc) ■■ Fibrosis in SSc is typically characterized by prolonged and/or exaggerated activation of fibroblasts, a key feature of which is the differentiation of fibroblasts into myofibroblasts ■■ Endoplasmic reticulum stress has been hypothesized to contribute to the initiation of fibrotic tissue remodelling in rheumatic diseases, and mechanical cues have a crucial role in determining fibroblast activation in fibrosis ■■ Mechanotransduction, the capability of cells to transform mechanical cues into biochemical signals, has also been implicated in fibrotic mechanisms ■■ Therapies targeting either persistent fibroblast activation (such as aberrant immune responses), or the chemical and mechanical stimuli that drive myofibroblast differentiation, could have the potential to alleviate the symptoms of SSc

fibrocytes, endothelial and endothelial progenitor cells into myofibroblasts, which are responsible for the deposition of large quantities of ECM components. 5 Progressive replacement of tissue architecture by ECM components such as collagen and fibronectin results in functional impairment of affected organs. Almost all patients with SSc have skin fibrosis despite the hetero geneity of the disease. Skin manifestations are often associated with distinct patterns of organ involvement, disease severity and survival. Internal organ dysfunction in SSc manifests as pulmonary fibrosis and pulmonary hypertension, renal crisis, gastrointestinal dysmotil ity and malabsorption, and impaired cardiac function. Decline in lung function is the most common cause of death in patients with SSc.3

Skin fibrosis in SSc Tightened and thickened skin is the clinical hallmark of SSc. Two well-recognized clinical subsets are conven tionally used to describe patients with SSc on the basis of the extent of skin involvement: diffuse cutaneous SSc (dcSSc), which involves the forearms, arms, face, trunks and lower extremities; and limited cutaneous SSc (lcSSc), which is characterized by thickening of skin of the face, lower distal extremities, fingers and hands.6 Skin lesions in patients with early SSc are infiltrated with inflammatory cells, comprising mostly of T cells and monocytes, suggesting that innate and cell-mediated immunity have a crucial role in the early establishment of SSc. Increased accumulations of collagen I, III, V and VII are observed in the reticular dermis of SSc lesional skin.7 Human and animal studies have also demonstrated downregulation of microRNA let7a,8 microRNA‑29a9 and microRNA‑15010 in association with upregulation of COL1A1 mRNA and protein expression. Elevated levels of enzymes that catalyse post-translational col lagen modifications, such as lysyl hydroxylase and lysyl oxidase,11 have been reported. In the lower half of the dermis, dermal collagen bundles are replaced by a compact, wax-like, intensely fibrotic matrix. The

deposition of hyaluronan within the epidermis and dermis is also reported to be highly increased in SSc, most notably around blood vessels.12 In patients with SSc, telocytes (a distinct stromal cell population of human dermis) are severely damaged and progressively disappear from skin lesions.13 Telocyte loss could contribute to altered skin homeostasis13 and 3D organization of the ECM in SSc skin,13 as well as impaired skin regeneration14 and diminished functional stem cell niches.13,14 The decrease in numbers of telo cytes might also contribute to the abnormal activation of fibroblasts and mast cells in SSc skin as telocytes are involved in intercellular signalling that could influence the transcriptional activity of neighbouring cells.15,16 Genome-wide expression profiling of SSc skin has highlighted the molecular heterogeneity of SSc. Micro array analysis revealed that expression of genes involved in cell proliferation, inflammation, fibrosis and innate immunity are all found to be substantially increased in SSc skin biopsy samples compared with samples from healthy individuals, whereas downregulation of genes associated with fatty acids and lipid biosynthesis has been reported, which could explain the notable loss of subcu taneous fat observed in patients with SSc.17 Perturba tions in genes associated with transforming growth factor (TGF)‑β and Wnt signalling are most prominent in fibroblasts cultures from SSc skin biopsy samples.18

Triggers of fibrosis Endothelium and pericyte activation Injury to vascular endothelial cells (Figure 1) followed by disrupted or inappropriate repair processes are pos sibly the earliest and primary events in the pathogenesis of SSc. Dysregulation in vascular remodelling (charac terized by fibrotic intimal hyperplasia) has been shown to occur in all layers of the vessel wall in samples from patients with SSc.19,20 On a cellular level, patients with SSc exhibit upregulation of vasoconstrictive, thrombo genic, mitogenic and proinflammatory factors, and downregulation of vasodilatory, antithrombogenic and antimitogenic factors.21 In SSc, consistent evidence suggests that microvascu lar endothelium activation and damage are omnipresent and precede vascular remodelling. Thrombospondin 1, an antiangiogenic factor that blocks endothelial cell proliferation and induces their apoptosis, is released at high concentrations in SSc endothelial cells, epidermal keratinocytes and dermal connective tissue cells (sam ples from patients with SSc).22 Expression of adhesion molecules (such as vascular cell adhesion protein 123 and intercellular cell adhesion molecule 1 24), cytok ines and chemokines were upregulated in activated endothe lial cells, which promotes perivascular inflammatory infiltrates, such as macrophages and leucocytes, through the endothelium.23,24 This process causes chronic inflam mation in SSc, leading to further endothelial cell damage that facilitates the process of capillary breakdown. Further downstream events include the dysregulation of vascular tone control, and progressive disorganiza tion of the vascular architecture can trigger an excessive

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REVIEWS Exposure to toxins or irritants, autoantibodies, persistent infections, etc.

Autoimmunity B cells

Tissue damage

Innate immune activation Mast cell, macrophage, NK cell, DC, neutrophil, basophil, eosinophil IFN-γ, TNF, IL-1β, IL-6, TGF-β

Adaptive immune activation TREG cell TH1 cell TH2 cell TGF-β IFN-γ, IL-4, IL-2 IL-10, IL-13 Inflammation

Fibrosis

TH2>TH1

Plasma cells

Autoantibodies

Excessive ECM synthesis, collagen cross-linking and contraction

Increased tissue pressure, hypoxia, stress

Myofibroblast (α-SMA)

Activated fibroblast Contractile phenotype, reduced apoptosis

Pericyte Endothelial and/or epithelial cell

Fibroblast Recruitment and differentiation

Bone marrow MSC, fibrocyte

Figure 1 | Fibrogenesis in SSc. This schematic diagram shows the integration of cellular and immunological processes leading to the initiation and persistence of fibrosis in SSc. Fibrosis is initiated by tissue injury induced by genetic mutation and/or defects or environmental triggers, which induces autoimmunity and autoantibody production, which, in some cases, could worsen tissue injury and activate fibroblasts. Tissue injury also activates innate immune cells (such as resident macrophages or neutrophils) and the secretion of innate immune cytokines by these cells, leading to inflammation. The adaptive immune response is also activated and TH1 cells are largely responsible for the secretion of inflammatory cytokines and growth factors, whereas TH2 cells are predominantly profibrotic. Inflammation and autoimmunity activate fibroblasts and result in the transdifferentiation of fibroblasts to myofibroblasts. These cell populations in affected tissue expand as a result of the recruitment of their progenitors and the transdifferentiation of MSCs, epithelial cells and endothelial cells. The activation of fibroblasts and the differentiation of myofibroblasts are hallmarks of fibrosis. Activated myofibroblasts synthesize and deposit ECM components, leading to ECM accumulation, increased collagen crosslinking, contraction and fibrosis. Fibrotic tissues exhibit increased tissue pressure and hypoxia, which further activate resident fibroblasts and escalate fibrotic mechanisms. Abbreviations: DC, dendritic cell; ECM, extracellular matrix; MSC, mesenchymal stem cell; NK cell, natural killer cell; SSc, systemic sclerosis; TGF‑β, transforming growth factor β; TH cell, helper T cell; TREG cell, regulatory T cell.

accumulation of ECM components, which leads to the formation of a permanent fibrotic scar.21 Abnormal characteristics of microvascular pericytes —cells that have important functional roles in the regulation of vascular development, maturation and remodelling—have been observed early in the disease course in SSc. In SSc fibrotic lesions, pericytes showed markers of activation such as platelet-derived growth factor receptor β and high-molecular-weight melanomaassociated antigen.25 Studies suggest that endothelial cells and pericytes both undergo changes in the early establish ment of SSc.5 Additionally, pericytes can take a smooth- muscle-like phenotype by transdifferentiation into myofibroblasts and synthesize ECM components, which are then deposited perivascularly 26 (Figure 1). In a mouse model of kidney fibrosis, pericytes and fibroblasts were the main sources of collagen production.27 Activation of microvascular pericytes and the endot helium can therefore initiate vasculopathy and fibrosis in SSc, and could serve as important early targets in SSc antifibrotic therapy. Current molecular targets of SSc endothelium

dysregulation are endothelin‑1, 5-hydroxytryptamine, platelet-derived growth factor (PDGF) signalling and vascular endothelial growth factor (Box 1).

Innate and adaptive immunity and autoantibodies In SSc, damage to the endothelium is thought to drive chronic inflammation and wound-healing responses. Regardless of the initiating stimulus, however, chronic tissue injury results in the release of DAMPs that acti vate innate immune responses controlled by monocytederived cells. Innate immune activation can secondarily drive adaptive immune responses, which can further amplify inflammation in these diseases. Mitigating innate and/or adaptive immune responses, by reducing chronic inflammation, might therefore be able to lessen fibrosis in SSc. Monocyte-derived cells of the innate immune system (such as antigen-presenting dendritic cells [DCs]), also promote primary T‑cell and B‑cell responses, linking innate and adaptive immunity.28–30 In the innate immune compartment, the DCs are known to be the most potent



REVIEWS Box 1 | Antifibrotic targets and pharmacological agents ■■ ■■ ■■ ■■ ■■ ■■ ■■ ■■ ■■

■■ ■■ ■■ ■■ ■■ ■■ ■■ ■■ ■■ ■■ ■■ ■■ ■■ ■■

ET‑1: bosentan; ambrisentan 5-hydroxytryptamine: terguride PDGF: imatinib mesylate; sunitinib*; sorafenib* VEGF: cediranib; bevacizumab; axitinib TLR: TLR inhibitors (E5564, TAK‑242) IL‑4: cromoglicic acid IL‑6 receptor: tocilizumab IL‑13: humanized monoclonal antibody TGF‑β expression and activation: pirfenidone; αvβ6 antibody; ATI and ATII receptor blockers; ACE inhibitor; CAT‑192 (anti-TGF‑β1 monoclonal AB); caveolin scaffolding domain TGF‑β receptor I: SM305 SMAD3: SIS3 FAK: PF‑562271 Notch: DAPT Wnt: thiazolidinediones; paricalcitol; pyrvinium; DICKKOPF-related protein 1 PPARγ: rosiglitazone; triterpenoid CDDO Hsp90: 17-dimethylaminoethylamino‑17demethoxygeldanamycin Autophagy: rapamycin LOXL2: GS-6624 (formerly AB0024) ROCK: fasudil YAP: verteporfin MKL1: CCG‑1423 LPA1–LPA3: SAR100842 LPA1: BMS‑986202

*Also used in anti-VEGF therapy. Abbreviations: ACE, angiotensinconverting enzyme; CDDO, 2-cyano-3,12-dioxoolean-1,9-dien28-oic acid; ET‑1, endothelin‑1; FAK, focal adhesion kinase; Hsp90, heat shock protein 90; LOXL2, Lysyl oxidase-like 2; LPA, lysophosphatidic acid; MKL1, MKL/myocardin-like protein 1; PDGF, platelet-derived growth factor; PPARγ, peroxisome proliferator-activated receptor‑γ; ROCK, Rho-associated protein kinase; SMAD3, mothers against decapentaplegic homolog 3; TGF‑β, transforming growth factor‑β; TLR, Toll-like receptor; VEGF, vascular endothelial growth factor; YAP, Yes-associated protein.

activators and modulators of the adaptive immune system.28–30 DCs reside in peripheral tissues and possess a variety of pattern-recognition receptors, including Toll-like receptors (TLRs).31 Activation of DCs is medi ated by signalling pathways activated via the binding of microbial products to TLRs and leads to the induction of various soluble and membrane-bound proteins that are critical for the induction of several CD4+ and CD8+ T‑cell effector functions. TLR activation also leads to the production of interferon by DCs and to B‑cell matu ration.32 As such, innate immunity might regulate key aspects of autoimmunity. How TLR stimulation leads to fibrosis is not com pletely understood. Studies have suggested that TLR activation might directly stimulate fibroblast conver sion to myofibroblasts through TLR333 or TLR9.34 Farina et al.35 demonstrated that the TLR3 agonist poly(I:C), activated dermal fibrosis in vitro and in vivo by upregu lating interferon-responsive and TGF‑β-responsive gene expression. Circulating TLR4 agonist was detected in the serum of patients with SSc, but not in serum of healthy individuals or patients with primary Raynaud phenomenon.36 The nature of this putative TLR4 ligand remains to be elucidated. However, TLR4 agonist

activity in SSc serum has been demonstrated in studies of anti-fibroblast antibodies.37 This study also showed that expression of CC‑motif chemokine 2 (also known as monocyte chemoattractant protein‑1) by fibroblasts (induced by antifibroblast antibodies) is diminished in Tlr4-deficient mouse embryonic fibroblasts in vitro,37 suggesting a role for TLR4 in angiogenesis and fibrosis in SSc. TH1–TH 2 balance in SSc In many studies, the immune-cell infiltrates in the skin of patients with SSc are shown to consist primarily of CD3+ T cells, with CD4+ T cells predominating over CD8+ T cells.38,39 Macrophages are the dominant cell type in the skin of some patients with SSc.40 B cells, plasma cells and natural killer cells have also been detected. Increased numbers of mast cells, particularly degran ulated ones, were also detected in the skin of patients with SSc in the early stages of the disease.41 CD4+ helper T (TH) cells consist of two subgroups, type 1 (TH1) and type 2 (TH2) cells, which are charac terized by distinct cytokine secretion patterns. TH1 cells promote cell-mediated immunity by secreting IFN‑γ, IL‑1, IL‑2, IL‑6, oncostatin‑M and TNF; whereas TH2 cells elicit humoral immune responses by producing IL‑4, IL‑5, IL‑10 and IL‑13. TH2 cytokines have been shown to stimulate the synthesis of collagen by human fibroblasts,42,43 but TH1 cytokines such as IL‑1 and TNF suppress collagen production by fibroblasts in vitro.43 Studies demonstrated that cytokine balance is generally inclined to the TH2 phenotype rather than a TH1 pheno type in patients with SSc (Figure 1), favouring tissue fibrosis and antibody production. Increased serum levels of T H2 cytokines such as IL‑4 and IL‑13 have been reported in patients with SSc compared with healthy controls.44 Both IL‑4 and IL‑13 have been shown to stimulate the transcription of the human α2 type I collagen gene (COL1A2) in dermal fibroblasts.45,46 IL‑4 has been shown to promote fibro blast proliferation and synthesis of ECM proteins such as collagen45 and tenascin,47 and to stimulate the produc tion of TGF‑β48 and other cytokines such as IL‑6,49 as well as contribute to mononuclear cell infiltration by upregulating the expression of adhesion molecules by endothelial cells.50 The production of IL‑4 by CD8+ T cells in the lung is associated with an increased decline in pulmonary function in patients with SSc.51 Most individuals with SSc have elevated serum levels of the TH2 cytokine IL‑13, which positively correlate with the number of nailfold capillaroscopic findings (haemorrhages, sludging of blood, larger total loop and arterial diameters) compared with healthy individuals and patients with SSc who have normal IL-13 levels as controls.52 IL‑13 could contribute to fibrosis via the induction of TGF‑β production by macrophages, and has been demonstrated to induce fibrosis through TGF‑βindependent mechanisms, such as stimulating fibroblast proliferation and collagen production through the acti vation of innate immunity.53,54 Notably, the elevated levels of IL‑13 in serum of patients with SSc, were produced by



REVIEWS cytotoxic CD8+ T cells, which correlated with the extent of skin fibrosis.55 TH2 cytokines also enhance immunoglobulin produc tion by B cells. Activated B cells are known to pro duce IL‑6, which induces T H2-dominant immune responses.56,57 IL‑6 is long established as a proinflam matory cytokine that has a potential effect on tissue fibro sis,58 so increased IL‑6 production by activated B cells might directly promote tissue fibrosis in SSc. IL‑6 levels are reported to be upregulated in the serum, endothe lium and fibroblasts of patients with SSc, and is prevalent in the serum of patients with SSc who have pulmonary fibrosis.59 IL‑6 can also induce collagen production by normal dermal fibroblasts42 and promote differentiation of dermal fibroblasts into myofibroblasts.60 Currently, tocilizumab (Box 1), which binds the IL‑6 receptor, is licenced for treatment of active, moderate-to-severe rheu matoid arthritis and systemic juvenile idiopathic arthritis. In a study that involved 15 patients, tocilizumab was also shown to benefit patients with SSc who have refractory arthritis61 and a small case study involving two patients with SSc reported improvement in dermal fibrosis and unchanged lung fibrosis in both patients with tocilizumab treatment.62 However, these are observational trials and the drug has not been approved in the USA for SSc or arthritis associated with SSc. A subcutaneous form of tocilizumab and other promising IL‑6-blocking agents are also currently undergoing phase III clinical trials.63 Autoantibodies Studies have revealed that B cells have critical roles in a variety of systemic autoimmune diseases, including SSc. The chronic hyperactivity of B cells in SSc leads to the production of autoantibodies (Figure 1), some of which can play active functional parts in SSc. The generation of autoantibodies can also be caused by the increased occur rence or abnormal distribution of potential autoantigenic peptides and by molecular mimicry.64 The presence of autoantibodies against ubiquitous nuclear components is the hallmark of autoimmune dys regulation in SSc. Antinuclear antibodies, such as topoi somerase, RNA polymerase III and centromere proteins, are detected in >95% of patients with SSc.65 To date, it is not known whether antinuclear antibodies have any pathological role in SSc, but they seem to be markers of clinical, genetic and possibly aetiological patient subsets. Other autoantibody groups, however, might have a puta tive pathological role in the SSc, including those targeting endothelial cells as well as those against PDGF receptors66 and fibroblasts.67 PDGF-receptor-specific autoantibodies could induce the production of ECM components by fibroblasts through Ha–Ras–ERK1/2 pathways, followed by formation of vasculotoxic reactive oxygen species, the production of type I collagen and the activating con version of resting fibroblasts to myofibroblasts.66 Antifibrilin‑1 antibodies, shown to activate fibroblasts and stimulate the release of TGF‑β, are detectable in more than half of patients with SSc.68 Antibodies directed against matrix metalloproteinase (MMP)-1 and MMP‑3 are present in most patients with SSc.69 These antibodies

can prevent the degradation of excessive collagen and cause an accumulation of ECM.

Endoplasmic reticulum stress Endoplasmic reticulum (ER) stress has been hypothe sized to contribute to the initiation of fibrotic tissue remodelling in multiple diseases, including rheumatic diseases.70,71 The ER is the intracellular organelle respon sible for the proper folding and secretion of proteins; ER stress is an imbalance between the load of proteins enter ing the ER and its folding capacity. Under physiologi cal conditions, chaperone proteins assist in folding of nascent proteins, thereby preventing aggregation of pro teins in the ER. Binding immunoglobulin protein (BiP, also known as HSPA5) is one such protein that maintains transmembrane sensor protein PKR-like endoplasmic reticulum kinase (also known as eukaryotic translation initiation factor 2‑α kinase 3), cyclic AMP-dependent transcription factor ATF‑6 alpha (also known as acti vating transcription factor 6) and inositol-requiring enzyme 1 in their inactivate state.72 With protein accumu lation in the ER, these transmembrane proteins dissociate from BiP and become activated, leading to a network of signalling pathways collectively known as the adaptive unfolded protein response (UPR), which enables cells to adapt to ER stress by reducing the influx of nascent proteins into the ER, increasing the capacity of the ER to fold incoming proteins, and increasing ER‑associated protein degradation of proteins not properly folded 73 (Figure 2). If the adaptive UPR is unable to resolve sus tained ER stress, however, other signalling pathways induce a terminal UPR that leads to apoptosis to elimi nate damaged cells (Figure 2). The persistent presence of ER stress can, therefore, increase cell death in injured tissues, induction of epithelial–mesenchymal transition (EMT) and promote fibrotic remodelling instead of the restoration of normal tissue architecture.70,71 Peripheral blood mononuclear cells from patients with lcSSc and pulmonary arterial hypertension have shown increased expression of ER stress and UPR genes.74 Autophagy and ER stress If ER stress exceeds the capacity of the adaptive UPR to relieve it, a lysosome-dependent degradation process known as autophagy is activated.75 Although better known for its ability to generate amino acids and energy required for cell survival during periods of nutrient deprivation or hypoxia, autophagy, involving the self-degradation of intracellular components in lysosomes, can also eliminate aberrant and/or misfolded proteins.76 Increased levels of autophagy can also promote the survival of fibroblasts during the development of dermal fibrosis in SSc, by shifting their energy metabolism to fuel acquisition of a fibrotic phenotype. This prodermal fibrosis effect of increased levels of autophagy was suggested following observations in calveolin‑1-deficient mice, whose skin exhibits many of the same characteristics as skin from patients with SSc, and whose dermal fibroblast demon strate an increase in autophagy and a shift towards aerobic glycolysis.77 Increased autophagy can also consequently



REVIEWS Genetic factors, viruses, oxidative stress, metabolic factors, environmental triggers Injury Epithelial cell or fibroblast

ER stress

Prolonged and excessive ER stress

Adaptive UPR

Terminal UPR

Influx of nascent proteins to ER Capacity of ER to fold incoming proteins ER-associated protein degradation

Chaperone proteins e.g. BiP and HSP90, and ATFs (ATF4, ATF6)

EMT, apoptosis, autophagy, inflammation

Loss of epithelial cells and activation of fibroblasts

Homeostasis maintained

Fibrosis

Figure 2 | ER stress and the development of fibrosis. Genetic factors, viruses, oxidative stress, metabolic factors and environmental triggers can injure fibroblasts and epithelial cells, resulting in ER stress. To maintain homeostasis, a cascade of pathways collectively known as the adaptive UPR are activated to adapt ER stress by reducing the influx of nascent proteins into the ER, increasing the capacity of the ER to fold incoming proteins, and increasing ER‑associated protein degradation of proteins not properly folded. In the event of prolonged and excessive ER stress, terminal UPR is activated with notable increase in ER‑stress-associated chaperone proteins, such as BiP and HSP90, and ATFs, such as ATF4 and ATF6. Terminal UPR leads to EMT, apoptosis, autophagy and inflammation, all of which would lead to the loss of epithelial cells and activation of fibroblasts, and ultimately the development of fibrosis. Abbreviations: ATFs, activating transcription factors; BiP, binding immunoglobulin protein; EMT, epithelial–mesenchymal transition; ER, endoplasmic reticulum; HSP, heat shock protein; UPR, unfolded protein response.

promote fibrosis in SSc through multiple mechanisms enhancing fibroblast survival and persistence.77 Heat shock protein 90 ER stress studies have uncovered the role of heat shock protein 90 (Hsp90) in fibrosis. Hsp90 is a chaper one protein that maintains the structural integrity and proper regulation of a subset of cytosolic proteins;78 for example, in the folding and conformational stabiliza tion of TGF‑β receptors (TβRI and TβRII).79 Hsp90 is upregulated in patients with SSc and mouse models of skin fibrosis and is required for effective TGF‑β signal ling.80 Increased TGF‑β signalling is observed to stimu late Hsp90 expression in mouse and human SSc skin fibroblasts. The increased Hsp90 expression renders the cells more susceptible to TGF‑β by stabilizing the conformational folding of TβRI and TβRII and down stream intracellular fibrotic mediators. 80 Pharmaco logical inhibition of Hsp90 with the selective Hsp90 inhibitor 17-DMAG (17-dimethylaminoethylamino– 17-d emethoxygeldanamycin) prevented this TGF‑β

and Hsp90 endogenous amplification loop and attenu ated TGF‑β mediated fibrosis, suggesting Hsp90 as an important candidate in antifibrotic therapy.80 17-DMAG has also been shown to downregulate keratinocyte dif ferentiation in the epidermis, possibly by upregulating Smad2/3 phosphorylation, which induces cell-cycle arrest in keratinocytes.81 These observations are also demon strated in renal fibrosis, in which the inhibition of Hsp90 with 17-DMAG has been shown to decrease the expres sion of TGF‑β-induced ECM, such as α‑SMA, fibronectin and collagen I in HK2 human proximal tubular epithelial cells.82 The phosphorylation of SMAD2, Akt, GSK‑3β and ERK was also demonstrated to be inhibited in a time-dependent manner via a mechanism dependent on proteasome-mediated degradation of TβRII.82

Fibroblasts and myofibroblasts Fibrosis in SSc is typically characterized by prolonged and/or exaggerated activation of fibroblasts. Fibroblasts isolated from affected tissues of patients with SSc have shown stable changes in phenotype, including increased proliferation, migration, invasion of ECM and resistance to Fas-mediated apoptosis.83 The hallmark of fibrosis is the differentiation of fibro blasts into myofibroblasts (Figure 1). Myofibroblasts are specialized fibroblasts that acquired characteristics of smooth muscle cells, including the expression of α‑SMA in stress fibres within their cytoplasm.84 Myofibroblasts have increased capacity to synthesize collagen and other ECM components. Additionally, myofibroblasts are a major source of TGF‑β, though only upon co-expression of the extra domain A variant of fibronectin and in the presence of biomechanical tension.26,85 In contrast with physiological wound healing, in which myofibroblasts are present only transiently within granulation tissue before being removed by apoptosis (a crucial step in wound resolution), myofibroblasts persist in the aber rant wound-healing process that leads to fibrogenesis, causing ECM contracture and scarring.86 The source of activated fibroblasts in repairing wounds and fibrotic lesions has been controversial. Although much evidence points to an important role for the activation and proliferation of resident fibro blasts,87 the recruitment of fibroblast-like cells from the circulation and/or bone marrow, and the differentiation of other cells, such as epithelial cells, into mesenchymal cells could also contribute. In experimental models of pulmonary fibrosis, bone-marrow-derived fibrocytes have been shown to be recruited into injured or fibrotic tissues in response to chemokine cues,88,89 where they can differentiate into fibroblasts or myofibroblasts. Epithelial cells have been hypothesized to trans differentiate into fibroblasts and myofibroblasts, that is, undergo EMT, in response to TGF‑β and other growth factors and/or cytokines during the development of fibrosis. SSc tissues are chronically hypoxic,90 which can also promote EMT. Of note, lineage tracing experiments have failed to demonstrate evidence of EMT in kidney, liver and lung fibrosis in general.91–93 In addition to epi thelial cells, endothelial cells have also been hypothesized



REVIEWS to transdifferentiate into fibroblasts through endothelial– mesenchymal transition.94 Finally, lineage tracing experi ments have provided convincing evidence that pericytes are an important source of myofibroblasts during renal fibrogenesis,91 and these cells might also be a source of myofibroblasts in SSc.26

Soluble mediators and signalling TGF‑β TGF‑β, the most critical regulator of physiological wound healing and pathological fibrosis, is overexpressed in all fibrotic tissues and it induces collagen production in cultured fibroblasts, regardless of their origin.95 TGF‑β production correlates with the progression of fibrotic dis eases and TGF‑β inhibition has been shown to reduce fibrotic processes in many experimental models. Studies suggested that TGF‑β activity depends on its cellular source. TGF‑β from macrophages shows profibrotic properties whereas TGF‑β from CD4+ regulatory T (TREG) cells is anti-inflammatory and antifibrotic.96 Patients with SSc97 have impaired TREG-cell function, which could con tribute to aberrant TGF‑β activation. An anti-TGF‑β anti body CAT‑192 (Box 1) for SSc was reported as ineffective and associated with serious adverse effects in a clinical trial.98 Although TGF‑β is unequivocally a prominent stimulus and regulator of fibrosis, other triggers and contributors have also been identified. These include lysophosphatidic acid (LPA), peroxisome proliferatoractivated receptor-γ (PPARγ), FAK/src kinase, Notch signalling and Wnt signalling pathways. Lysophosphatidic acid Lysophosphatidic acid (LPA) is a bioactive lipid that has been implicated in the development of pathological fibrosis in multiple organs, including the lung, kidney, peritoneum and liver.99–103 Among rheumatic diseases, it has been implicated in the pathogenesis of both rheuma toid arthritis104,105 and SSc.106,107 LPA seems to be gener ated predominantly through the lysopholipase D activity of the enzyme autotaxin,108 and mediates diverse cellular responses through interactions with specific G proteincoupled receptors, of which at least six have been defini tively identified and designated LPA1‑6.109 LPA1-deficient mice have also been demonstrated to be dramatically protected from dermal fibrosis produced in the bleo mycin mouse model of SSc.110 In humans, the expression of both autotaxin and LPA1 is greatly elevated in injured compared with normal healthy skin111 and elevated levels of a particular species of LPA, arachidonoyl-LPA, have been noted to be increased in the serum of patients with SSc.112 A phase II trial of a dual LPA1–LPA3 antagonist (SAR100842) (Box 1) in SSc is ongoing.113 PPARγ PPARγ is a nuclear receptor initially identified in adipose tissue, with key roles in the regulation of adipogenesis, insulin sensitivity and energy homeostasis.114 PPARγ has gained attention in the past 15 years as a cell intrinsic anti-inflammatory and antifibrotic factor and is increas ingly implicated in both physiological and pathological

matrix remodelling. In normal fibroblasts, activation of PPAR‑γ by either natural (15d-prostaglandin J2)115 or synthetic (rosiglitazone116 and triterpenoid CDDO117) ligands (Box 1) resulted in abrogation of TGF‑β– induced collagen production, and Smad3-dependent and p300-dependent transcriptional responses.115 In addition, PPARγ ligands induce phosphate and tensin homologue, 118 a negative regulator of myofibroblast activation and collagen synthesis. The expression of PPARγ is reduced in lesional skin and lung biopsy samples from patients with SSc, and the suppression of PPARγ involves the TβRI and canonical Smad signal ling pathway.119 Fibroblast-specific deletion of PPARγ results in enhanced susceptibility to bleomycin-induced skin fibrosis and enhanced sensitivity to TGF‑β in mouse models.120 These observations indicate that PPARγ is a potent endogenous antagonist of TGF‑β signalling and further suggest that PPARγ has a physiological role in preventing excessive fibrosis.

The Notch signalling pathway Notch signalling, first discovered in Drosophila, is essen tial for cell-fate decision and development, and has been shown to mediate EMT and myofibroblast differen tiation.121 Studies showed that Notch is upregulated and persistently activated in human SSc skin fibroblasts.122 Normal skin fibroblasts stimulated by Jag‑1 (a Notch ligand) exhibit elevated collagen gene expression and the transdifferentiation of these cells to myofibroblasts in vitro.122 Pharmacological inhibition of Notch signal ling using DAPT reduces the expression of profibrotic cytokines, and observations suggested that targeting Notch signalling might be effective in preventing fibro sis in early inflammatory and later stage of SSc, when inflammation has been resolved.122 DAPT also induces regression of established fibrosis in an SSc experimen tal model, suggesting that Notch signalling might be a promising antifibrotic therapeutic target.123 The Wnt signalling pathway Canonical Wnt signalling has an important role in embryonic development involving the binding of LRP5– LRP6 and Frizzled receptors, the activation and nuclear translocation of β‑catenin to activate downstream target genes. Genome-wide expression profiling reported upregulated Wnt ligands and β‑catenin target genes in SSc.18 In vitro stimulation of normal fibroblast with Wnt ligands results in elevated expression of collagen and ECM matrix genes, myofibroblast differentiation and cell migration.124 Wnt signalling has been shown to induce skin fibrosis and subcutaneous lipoatrophy in mice.125 Inhibiting the signalling pathway with currently available drugs such as thiazolidinediones,126 paricalcitol,127 pyr vinium128 and DKK1129 could therefore be a promising antifibrotic approach (Box 1).

Mechanical forces Matrix stiffness In addition to signals from soluble mediators, the physi cal properties of the microenvironment direct normal



REVIEWS Fibroblast

Collagen type I Profibrotic soluble factors (TGF-β, ET-1, CTGF, LPA) Proto-myofibroblast

LOXL2

Targeting matrix crosslinking LOXL2 blocking antibody (GS-6624) Increased matrix stiffness

Mechanical tension

LOXL2 Myofibroblast

Matrix stiffness

LOXL2

ECM FAK G-Actin YAP/ TAZ

ROCK

P Rho

MKL1 Targeting mechanotransduction FAK inhibitor (PF-562271) ROCK inhibitor (fasudil) MKL1 (CCG-1423) YAP (verteporfin)

F-actin YAP/ TAZ TEAD

MKL1 SRF

Fibrosis Collagen α-SMA CTGF Survival Bcl-2

Integrin LOXL2 Myofibroblast apoptosis

Figure 3 | Targeting matrix stiffness and mechanotransduction in fibrosis. Fibroblast activation follows a serial sequence of events involving the formation of proto-myofibroblasts by the action of soluble growth factors. Fully activated fibroblasts, myofibroblasts, requires a stiff environment. Increased LOXL2 expression by proto-myofibroblasts promotes matrix stiffening, which further promote myofibroblast formation. Therapeutically, inhibiting myofibroblast mechanotransduction or targeting matrix crosslinking with blocking antibodies against LOXL2 ameliorates fibrogenesis in vivo. Abbreviation: Bcl-2, B-cell lymphoma 2; CTGF, connective tissue growth factor; ET-1, endothelin 1; FAK, focal adhesion kinase; LOXL2, lysyl oxidase-like 2; LPA, lysophosphatidic acid; MKL1, MKL/myocardin-like protein 1; ROCK, Rho-associated protein kinase; α-SMA, α-smooth muscle actin; SRF, serum response factor; TAZ, WW domain-containing transcription regulator protein 1; TEAD, TEA domain; TGF-β, transforming growth factor β; YAP, Yes-associated protein.

development and adult organ homeostasis. Given the essential roles of mechanical forces in the func tions of most, if not all, tissues, it is not surprising that deregulation of tensional homeostasis has been asso ciated with human diseases, including fibrotic condi tions. Abnormalities in the mechanical characteristics of both the cells and the ECM of fibrotic tissues have been described, and both sets of abnormalities might contribute to progression of fibrosis. Whereas no mor phological differences were detected between dermal fibroblasts isolated from patients with SSc and healthy individuals as controls, the fibroblasts from SSc lesional skin exhibit a reduced elastic constant, that is, were softer than control fibroblasts.130 Fibroblasts are exquisitely sen sitive to changes in the mechanical microenvironment, such as stretch or matrix stiffness,131 and softer cells might be even more sensitive to activation by mechanical

stimuli.130 Compelling evidence now shows that mecha nical cues have a crucial role in driving fibroblast acti vation in fibrosis. Importantly, it has been shown that matrix stiffness by itself can induce myofibroblast acti vation and collagen deposition. An increased collagen deposition and crosslinking in turn further increases matrix stiffness, this process creates a ‘vicious cycle’ of increased stiffness, increased myofibroblast activation and increased collagen deposition in fibroproliferation (Figure 3). The fact that matrix stiffening promotes fibrogenesis independently of soluble profibrotic factors such as TGF‑β potentially has tremendous implications for understanding the mechanisms of fibrosis progres sion.132,133 Thus, understanding the factors that contrib ute to matrix stiffening, as well as how fibroblasts sense and integrate mechanical cues, will open new therapeutic avenues for the treatment of fibroproliferative diseases.



REVIEWS Lysyl oxidase like‑2 The molecular determinants for augmented matrix rigidity in fibrosis remain to be fully understood. Work by several groups points to aberrant collagen crosslink ing as an important driver of matrix stiffness. The five member family of lysyl oxidase (LOX) enzymes, con sisting of LOX itself and lysyl oxidase-like 1–4, control tissue stiffness and elasticity by catalysing the cova lent crosslinking of both collagen and elastin fibres.134 The expression and activity of LOX family enzymes is tightly regulated during development and normal tissue homeostasis, whereas different members of this family have been shown to be upregulated in various fibrotic diseases.135 Studies have demonstrated upregulation of LOX mRNA136 and protein expression in SSc dermal fibroblasts; LOX expression is also increased in whole lesional skin from patients with SSc compared with their nonlesional skin or the skin of healthy individuals.136 Research findings have also implicated overexpression of lysyl oxidase-like 2 (LOXL2) in the development of human lung and liver fibrosis, suggesting that this par ticular enzymatic regulator of matrix stiffness also has the potential to be a therapeutic target for the treatment of pathological fibrosis 133 (Figure 3). A monoclonal antibody blocking LOXL2 function (AB0023) has been shown to mitigate lung and liver fibrosis in mouse models, 133 and a humanized version, GS-6624 (for merly AB0024) (Box 1), entered into a clinical trial to evaluate its safety and efficacy in patients with idiopathic pulmonary fibrosis.137 Mechanotransduction The mechanisms responsible for force-induced fibro blast activation remain to be fully elucidated, but include both altered mechanotransduction and augmented growth factor signalling. Mechanotransduction is the capability of cells to transform mechanical cues into bio chemical signals, and this capability determines cellular responses to mechanical forces. Mechanotransduction occurs at sites of focal adhesions, which are plasma membrane structures that serve as a nexus between the ECM and the contractile actin cytoskeleton.138 Integrins are cell surface receptors within these focal adhesions that are responsible for ECM binding and the activation of mechanotransduction pathways in fibroblasts. Focal adhesion kinase Integrin binding to the ECM results in conformational and organizational changes of some of the proteins within focal adhesions, including focal adhesion kinase (FAK). FAK has been shown to be an important mediator in mechanosensing by both responding to matrix stiff ness and regulating cell tension via the actin cytoskeleton. In SSc fibroblasts, FAK is constitutively phosphorylated, and this activated state is dependent on integrin β1.139 Pharmacological FAK inhibition abrogates profibrotic gene expression in human SSc lesional fibroblasts.139 In mice, FAK has been also shown to mediate tissue stress in a hypertrophic scar-like model of skin scarring.140 Mechanistically, FAK activation in fibroblasts links

mechanical stress to increased inflammation and sub sequent fibrosis seen in this hypertrophic scar model.140 In humans, FAK activation has also been observed to be upregulated in patients with idiopathic pulmonary fibrosis or SSc, suggesting that FAK has the potential to be a novel therapeutic target in human fibrotic diseases. In this regard, several orally available ATP-competitive FAK inhibitors have been developed and are in human clinical trials. PF‑562271 (Box 1, Figure 3) is one such inhibitor that has been demonstrated to inhibit FAK phosphorylation and prevent bleomycin-induced lung fibrosis in mice.141,142

Rho–ROCK The small GTPase Rho is involved in biomechani cal sensing and cell contraction. Rho effectors such as Rho-associated protein kinase 1 and 2 (ROCK1 and ROCK2) mediate Rho responses by phosphorylating downstream mediators, including myosin phosphatase target subunit 1 (also known as protein phosphatase 1 regulatory subunit 12A). Increased ROCK activity has been described in SSc and idiopathic pulmonary fibro sis fibroblasts, and ROCK inhibition reduces collagen expression in both of these cells in vitro.143,144 In vivo, pharmacological ROCK inhibition with fasudil, a smallmolecule nonselective ROCK inhibitor (Box 1), miti gates pathological fibrosis in rodents in multiple organs, including the lung, peritoneum and heart.145–147 Studies have shown that fasudil abrogates the ability of matrix stiffness to promote myofibroblast survival, resulting in selective apoptosis of these cells148 (Figure 3). Fasudil has been approved for the treatment of cerebral vasospasm in Japan, and could offer a novel approach for effectively treating fibrosis in humans (Box 1).

Transcription factors Transcription factors that control matrix stiffness-induced myofibroblast function are beginning to be identified. RhoA–ROCK-dependent cytoskeleton rearrangement can release several transcriptional activators that are nor mally sequestered in the cytosol, which then translocate into the nucleus to drive the expression of profibrotic gene programmes.

MKL1 MKL-myocardin-like protein 1 (MKL1) is a member of the myocardin family of transcriptional co-activators of serum response factor (SRF). The ability of MKL1 to direct transcription induced by SRF is regulated at the level of MKL1 subcellular localization. In the nucleus, MKL1– SRF complexes bind to CArG box sequences in serum response elements in the promoters of a contractile gene programme, such as α‑smooth muscle actin.149 Activa tion of these genes promotes a smooth-muscle-cell-like contractile phenotype in nonmuscle cells, such as myo fibroblasts. Pharmacological blockade of Mkl1-binding to Srf with CCG‑1423 (Box 1, Figure 3) diminished perito neal fibrosis in mice.150 Genetic deletion of Mkl1 limits myofibroblast function and the development of cardiac fibrosis in mice,151 as well as pulmonary fibrosis.152



REVIEWS YAP–TAZ The transcriptional co-activators YAP (Yes-associated protein) and TAZ (WW domain-containing transcrip tion regulator protein 1, also known as transcriptional co-a ctivator with PDZ-binding motif ) are unique mechanosensors first described in mammary epithelial cells.153 As with MKL1, YAP and TAZ nuclear localiza tion and transcriptional activation are driven by RhoA– ROCK-dependent cytoskeleton rearrangement. 154 Although located in the cytoplasm, YAP–TAZ have also been shown to limit TGF‑β signalling, further suggest ing that inhibiting YAP–TAZ nuclear translocation might offer a new therapeutic approach for fibrosis.155 A small molecule named verteporfin (Box 1) was identified in a chemical screen for YAP inhibitors, and has been shown to limit liver tumorogenesis in vivo.156

Conclusions Fibrosis causes much of the morbidity and mortality associated with SSc and can therefore be regarded as a lethal component of the disease. Persistent fibroblast activation and increased myofibroblast differentiation lead to excessive ECM deposition, distorting tissue architecture, diminishing organ function and, ultimately, leading to organ failure. Therapies that target either the causes of persistent fibroblast activation, such as aber rant immune responses and/or chronic ER stress, or the chemical and mechanical stimuli that drive myofibro blast differentiation, would therefore have great thera peutic potential in treating SSc. However, as described in this Review, multiple classes of immune and inflam matory cells, along with fibroblasts, activate a plethora 1.

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of molecular pathways that contribute to the initiation and progression of fibrosis in SSc. These molecular pathways are complex and interdependent, and some might be redundant, suggesting that successful treat ment of fibrosis in SSc could require simultaneous targeting of several pathways. An intergrated therapeu tic approach of concurrently targeting inflammatory cytokines, fibrotic mediators (transcriptional, chemical and mechanical), cell recruitment and cell-fate changes might be needed for antifibrotic therapy to succeed in SSc and other fibrotic diseases. In fact, efforts to develop multiple-candidate targeted therapeutics for SSc are now underway, and are at different stages of clinical advance ment.157 Such approaches are grounds for maintaining an optimistic outlook for the development of SSc treat ments that hopefully will be able to abate autoimmunity and inflammatory damage, restore vascular homeostasis, resolve existing scar tissues and prevent loss of, and even restore, specific organ function. Review criteria This Review was prepared by a thorough search of the PubMed and ClinicalTrials.gov databases for pertinent literature on the role of fibrosis in systemic sclerosis, rheumatoid arthritis and osteoarthritis. The following keywords were used alone or in combination: “fibrosis”, “fibroblasts”, “myofibroblast”, “systemic sclerosis”, “scleroderma”, “cytokines”, “inflammation”, “animal models of fibrosis”, and “knockout mice”. The search was restricted to English-language, full-length articles only. The search criteria were not restricted by date.

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Genetics of systemic sclerosis.

Treatment of systemic sclerosis.

Treatment of systemic sclerosis.

Lethal micromelic facial bones sclerosis dysplasia.

[Systemic sclerosis - an update].

Microalbuminuria in systemic sclerosis.

Epigenetics and systemic sclerosis.

Systemic sclerosis and pregnancy.

Systemic sclerosis (scleroderma). Introduction.

Diagnostic criteria of systemic sclerosis.

Gastrointestinal Manifestations of Systemic Sclerosis.

The prognosis of systemic sclerosis.

The immunopathology of systemic sclerosis.

Cardiovascular disease in systemic sclerosis.

Cardiac manifestations in systemic sclerosis.

Kidney involvement in systemic sclerosis.

Skin imaging in systemic sclerosis.

Parental influence on systemic sclerosis.

Penicillamine therapy in systemic sclerosis.

Systemic sclerosis in old age.

Progressive systemic sclerosis and polymyositis.

Thyroid function in systemic sclerosis.

Update on juvenile systemic sclerosis.

Endothelial dysfunction in systemic sclerosis.