• fibrosis;
  • immune dysfunction;
  • new treatments;
  • scleroderma;
  • vasculopathy


  1. Top of page
  2. Abstract
  3. Introduction
  4. References

Systemic sclerosis (SSc) is an autoimmune disorder with clinical manifestations resulting from immune activation, fibrosis development and damage of small blood vessels. Although there have been no established treatments for SSc, lots of new treatments targeting organ and pathogenesis are in the process of development. Transforming growth factor (TGF)-β is a major cytokine involved in the pathogenesis of fibrosis in SSc. The blockade of cell surface molecules capable of activating latent TGF-β, blockade of ligand by the pan-isoform-specific antibody, soluble TGF-β receptors and a recombinant latency associated peptide, as well as inhibitors for ALK5 and Smad3 are the potential strategies to abolish the pathological activation of TGF-β signaling in SSc fibroblasts. Besides TGF-β, connective tissue growth factor (CTGF)/CCN2, platelet-derived growth factor (PDGF) and endothelin-1 are the candidates for the new therapeutic targets. As for immune dysfunction in SSc, i.v. immunoglobulin infusion, stem cell transplantation and B-cell depletion are potential new therapies under or awaiting a randomized, double-blind, placebo-controlled trial, although their efficacies are still controversial. Phosphodiesterase-5 inhibitors, endothelin receptor antagonists and inhibitors for serotonin signaling are the new therapeutic targets for Raynaud’s phenomenon, digital ulceration and pulmonary arterial hypertension in SSc. Imatinib mesylate may be a novel new therapy for fibrosis and vasculopathy in SSc because it reverses the expression levels of Fli1, which is a transcription factor downregulated in SSc through an epigenetic mechanism and is likely to be involved in the development of fibrosis and vasculopathy in this disease. Potential therapeutic targets other than those described above are also reviewed.


  1. Top of page
  2. Abstract
  3. Introduction
  4. References

Systemic sclerosis (SSc) is an autoimmune disease characterized by small vessel vasculopathy and fibrosis of skin and internal organs. Clinical manifestations include thickening of the skin, Raynaud’s phenomenon, pulmonary artery hypertension (PAH), pulmonary fibrosis (PF) and involvement of other visceral organs. Although there have been no established treatments for SSc, lots of new treatments targeting organs and pathogenesis are in the process of development. In this article, novel new strategies for the treatment of SSc are reviewed.

New therapeutic targets for fibrosis in SSc

Mechanism of fibrosis in SSc

The fibrosis of the skin and internal organs in SSc is believed to be caused by the transition of quiescent fibroblasts to activated myofibroblasts, which characteristically overproduce dermal fibrillar collagen (type I, III, V), collagen-modifying enzymes and other extracellular matrix (ECM) components. This activation is likely to be caused by inflammation, autoimmune attack and vascular damage in vivo, which are also characteristics of SSc pathogenesis. However, cultured SSc fibroblasts, which are free from such microenvironmental factors, continue to produce excessive amounts of ECM proteins,1,2 suggesting that once activated, these cells establish a constitutive self-activation system. One of the major cytokines involved in this process is transforming growth factor (TGF)-β1.3 A growing body of evidence demonstrates that SSc fibroblasts resemble the cells exposed to exogenous TGF-β1 and appear to be constitutively activated by the stimulation of autocrine TGF-β.4–7 TGF-β is normally secreted as a latent complex, which is required to be activated in extracellular regions before binding its receptors and exerting its biological effects. In dermal fibroblasts, several membrane proteins, including integrin αVβ5 and thrombospondin 1(TSP1), catalyze the activation of latent TGF-β in the local microenvironment.8–12 Besides TGF-β, other cytokines, such as connective tissue growth factor (CTGF)/CCN2, platelet-derived growth factor (PDGF) and endothelin-1 (ET-1), have been reported to be involved in the activation of SSc fibroblasts.

Targets for blockade of TGF-β signaling

Cell surface molecules catalyzing latent TGF-β activation

One of the novel strategies for therapeutic disruption of TGF-β is the blockade of the activation of matrix-bound latent TGF-β. Two cell surface molecules expressed on dermal fibroblasts, integrin αVβ5 and TSP1, are the promising candidates for this intervention therapy (Fig. 1, Table 1). Integrin αVβ5 is a member of αV containing integrins, which recognize the RGV motif. Because latency-associated peptide (LAP) has the RGV motif, αV containing integrins function as a receptor for the latent form of TGF-β, a complex of active TGF-β and LAP. Integrin αVβ5 is expressed on the cell surface of dermal fibroblasts and plays a central role in the myofibroblast contraction-mediated TGF-β1 activation.13 Dermal fibroblasts constitutively expressing β5 integrin at high levels differentiate into myofibroblasts through the activation of endogenous latent TGF-β by integrin αVβ5.10 Importantly, integrin αVβ5 is expressed at high levels on cultured SSc fibroblasts (in vitro) and on activated fibroblasts in lesional skin of diffuse cutaneous SSc (dcSSc) in its early stage (in vivo).11 Furthermore, blockade of this integrin by functionally blocking antibody or antisense oligonucleotide results in the decrease of Smad3 phosphorylation levels and type I collagen expression in SSc fibroblasts, indicating that the disruption of this integrin partially attenuates the constitutive activation of TGF-β signaling in SSc fibroblasts.9 TSP1 is a molecule catalyzing latent TGF-β activation as well as integrin αVβ5 and is also expressed at high levels in cultured SSc fibroblasts (in vitro) and in activated fibroblasts in lesional skin of SSc (in vivo). Blockade of TSP1 partially abolishes the autocrine TGF-β signaling in SSc fibroblasts, indicating the involvement of TSP1 in the process of autocrine TGF-β signaling.12 Therefore, these two molecules are the promising targets for the treatment of fibrosis in SSc. Given that β5-knockout mice develop, grow and reproduce normally and show no abnormality in wound healing or susceptibility to adenovirus infection, which are major biological processes in which αVβ5 participates,14 most roles of αVβ5 can be compensated for by other αVβ5-independent pathways. This functional redundancy in αVβ5 makes pharmacological interference with αVβ5 function a promising approach to the treatment of SSc.


Figure 1.  Future targets for the inhibition of pathological transforming growth factor (TGF)-β signaling in systemic sclerosis (SSc). Integrin αVβ6, αVβ5 and thrombospondin-1, the cell surface molecules capable of activating latent form of TGF-β, are the potential therapeutic targets, which can inhibit the pathological TGF-β signaling without affecting the physiological TGF-β signaling. The blockade of ligand by the pan-isoform-specific antibody, soluble TGF-β receptors and a recombinant latency associated peptide, inhibitors for ALK5 and Smad3 and forced expression of Smad7 are the potential strategies to abolish the pathological activation of TGF-β–Smad signaling in SSc fibroblasts. Inhibition of c-Abl tyrosine kinase activity is another promising therapeutic strategy in SSc.

Download figure to PowerPoint

Table 1.   Future targets for the inhibition of pathological transforming growth factor (TGF)-β signaling in systemic sclerosis (SSc)
Cell surface molecules capable of activating latent TGF-βNeutrizing antibody against integrin αVβ6, integrin αVβ5 and thrombospondin-1
Active TGF-βNeutralizing antibody against active TGF-β, soluble TGF-β receptors that block all active TGF-β ligand, and a recombinantlatency-associated peptide (LAP)
ALK5SB-431542, SB-525334 Forced expression of Smad7
c-AblImatinib mesylate, dasatinib, nilotinib

Integrin αVβ6 is another αV containing integrin capable of catalyzing the activation of latent TGF-β in the local microenvironment. This integrin is mainly expressed on epithelial cells, but not at all in fibroblasts (Fig. 1). Although mice with targeted deletion of αvβ6 integrin develop spontaneous lung inflammation, the animals are protected from bleomycin-induced fibrosis.15 Blockade of integrin αVβ6 by functionally blocking antibody abolishes latent TGF-β activation and prevents the development of pulmonary fibrosis induced by intratracheal bleomycin or radiation.16,17 Theoretically, targeting of integrin αVβ6 can selectively diminish the pathologically important signaling, the αVβ6-dependent activation of latent TGF-β, without inducing any undesirable effect on normal TGF-β signaling indispensable for normal biological activities. Based on the results of animal models, integrin αVβ6 can be a new therapeutic target for PF in SSc.

Blockade of the ligand

Several recent studies have demonstrated that blocking TGF-β ligand may abrogate fibrosis in a number of mouse models.18 A recombinant human antibody against active TGF-β1 (CAT-192) was developed and a phase I/II trial of repeated doses of this medicine for early-stage dcSSc was performed.19 Unexpectedly, the study did not show any evidence of efficacy for CAT-192. Given that effective prevention of fibrosis has generally required the blockade of multiple isoforms of TGF-β in animal models of fibrosis,20,21 another therapeutic intervention capable of blocking all active TGF-β ligand is required to confirm if this type of treatment is effective for SSc. Thus, the pan-isoform-specific antibody, soluble TGF-β receptors that block all active TGF-β ligand and a recombinant LAP are the next candidates to achieve the complete blockade of TGF-β ligands.

Blockade of TGF-β receptor activity and Smad intracellular signal transduction

Small-molecule inhibitors, such as SB-431542 and SB-525334, bind to the adenosine triphosphate-binding domain of the serine/threonine kinase TGF-β receptor type I (TβRI, ALK5) and prevent ligand-induced Smad2/3 phosphorylation by ALK5 and consequent fibrotic responses in vitro.22,23 These inhibitors also improve experimental fibrosis in the kidneys, liver, blood vessels and lungs, and prevent diabetic nephropathy in the db/db mouse model.24–28 As well as ALK5 inhibitors, specific inhibitor of Smad3 (SIS3) attenuates the effects of TGF-β1, collagen type I production and transdifferentiation to myofibroblasts.29 Blockade of Smad2/3 activation is another strategy for SSc treatment. Inhibition of phosphatidyl-inositol-3 (PI3) kinase by LY294002 partially decreased TGF-β-dependent activation of Smad3, while completely diminishing constitutive activation of Smad3 in SSc fibroblasts,30 suggesting that the PI3 kinase pathway is a possible target for the treatment of SSc. Paclitaxel, a cancer drug that works by stabilizing the microtubules, attenuated constitutive Smad2 activation in skin grafts of SSc patients xenotransplanted onto severe combined immunodeficiency (SCID) mice.31 Although these compounds have not been tested in patients, they may represent future therapies against SSc. Induction of Smad7, an endogenous Smad inhibitor, is another strategy to disrupt Smad2/3 signaling. Indeed, gene transfer of inhibitory Smad7 reversed pulmonary, renal and peritoneal fibrosis in animal models.32–35

Blockade of non-canonical intracellular signal transduction

Although the Smad pathway constitutes a major mode of TGF-β signaling, recent evidence suggests that alternative non-Smad pathways are activated in parallel with Smad signaling and also mediate TGF-β responses.36 The non-Smad pathways include mitogen-activated protein kinases (MAPK),37–39 protein kinase C (PKC),40,41 the PI3 kinase pathway and its downstream target Akt,42 and non-receptor kinases c-Abl and c-Src.41,43 There is also evidence that some of these pathways, including extracellular signal-regulated kinase (ERK)1/2, Akt, c-Abl and PKC-δ, play a role in pathological fibrosis,44 but the specific mechanisms for any of these signaling molecules in the fibrotic process remain to be elucidated. Our latest studies identified a new non-canonical pathway, “c-Abl–PKC-δ–Fli1 pathway”, and demonstrated that this pathway is targeted by imatinib mesylate. These data are summarized in the forth section of this article.

Targeting of cytokines other than TGF-β 


Endothelin-1 is normally produced by endothelial cells but is also expressed by various cell types including fibroblasts.45,46In vitro, ET-1 induces fibroblasts to synthesize and contract ECM,47,48 acting synergistically with TGF-β.49,50 Serum ET-1 levels are elevated in SSc patients and correlate with the severity of skin involvement and PF.51,52 Consistently, ET-1 is produced in elevated amounts by SSc fibroblasts,53 suggesting that ET-1 plays an important part in the fibrosing process in SSc. Importantly, blockade of ET receptors, ETA and ETB, with bosentan significantly reduces α-smooth muscle actin (α-SMA), CCN2 and type I collagen overexpression and ECM contraction by SSc fibroblasts, while not affecting the basal activity of normal fibroblasts.47,54 Bosentan is widely used to treat PAH and to prevent the development of new digital ulcers in SSc patients,55,56 indicating the good tolerance for ETA/B receptor antagonism in those patients. Although the efficacy of bosentan for fibrotic lesions is still unknown, the blockade of ET receptors is likely to be of clinical benefit in alleviating the persistently activated fibroblasts in SSc as well as the profibrotic responses to TGF-β.


Connective tissue growth factor/CCN2, a member of the CCN family of proteins, is a cystein-rich matricellular protein and may operate in tandem with or downstream of TGF-β in the fibrotic pathway as well as ET-1. CCN2 is not normally expressed in dermal fibroblasts under physiological conditions. TGF-β is the most potent inducer of CCN2 identified so far and in SSc fibroblasts, where TGF-β signaling is highly activated, CCN2 is constitutively overexpressed. Given that CCN2 promotes ECM deposition and fibroblast adhesion and proliferation, the overexpression of CCN2 in fibrotic lesions appears to contribute to the development of SSc phenotype. In animal models, although TGF-β or CCN2 alone produce only a transient fibrotic response, these cytokines act together to promote sustained fibrosis. Therefore, the constitutive overexpression of CCN2 in SSc fibroblasts, which is already activated by autocrine TGF-β stimulation, would be expected to contribute to persistent fibrosis in this disease. Collectively, these observations suggest that CCN2 is a novel molecular target for therapeutic intervention in SSc.57–60 This notion is supported by the observation that antisense oligonucleotides against CCN2 alleviate fibrosis in animal models.61–63


Platelet-derived growth factor has been implicated in the pathogenesis of SSc as well as TGF-β, ET-1 and CCN264 because serum levels of PDGF are increased in patients with SSc and PDGF stimulation results in the increase of type I collagen production in normal dermal fibroblasts. Furthermore, the activation status of ERK1/2, downstream effectors of PDGF receptor signaling, is constitutively elevated in SSc lung and skin fibroblasts.65–67 A novel recent finding is that sera from patients with SSc possess autoantibodies activating PDGF receptor signaling in normal dermal fibroblasts,68 which may contribute to the upregulation of collagen synthesis in SSc fibroblasts in vivo. Collectively, these findings suggest that PDGF can be a therapeutic target for SSc. The inhibitors of PDGF receptor tyrosine kinase (imatinib mesylate, dasatinib and nilotinib), which also inhibits TGF-β signaling through the blockade of c-abl, exert antifibrotic effects in vitro and in vivo, as seen in animal studies and in a limited number of unselected cases refractory to other treatments. The therapeutic potential of PDGF receptor tyrosine kinase inhibitors for SSc is discussed in the fourth section of this article.

Type I interferon

Recent studies demonstrated that SSc-specific autoantibodies, such as anti-topoisomerase-I antibody and anti-nucleolar antibody, interact with nuclear proteins derived from apoptotic cells and this immune complex interacts with Toll-like receptor on plasmacytoid dendritic cells and subsequently induces the production of type I interferon (IFN-α/β).69 Widespread use of INF-α and -β for the treatment of chronic hepatitis C and multiple sclerosis demonstrated that SSc and SSc-like disorder can be caused by the administration of type I IFN.70–75 Because IFN-α suppresses the expression of type I collagen in normal dermal fibroblasts in vitro,76,77 a clinical trial of IFN-α was performed. Disappointingly, INF-α did not improve outcome at 1 year in patients with dcSSc in a randomized, double-blind, placebo-controlled trial.78,79 Another important observation in this study was that the administration of IFN-α exacerbated lung function of SSc patients. Taken together, these clinical and laboratory observations suggest that IFN-α is involved in the fibrotic process of SSc and blockade of IFN-α can be a new strategy for the treatment of SSc.

New therapeutic targets for immune dysfunction in SSc

Mechanism of immunological disturbances

Autoreactive immune system activation has also been implicated in the pathogenesis of SSc, yet the exact mechanisms responsible for this process are not fully understood. Increasing evidence demonstrated the disturbances of B-cell homeostasis and the abnormal activation of T cells in this disorder. Elevated serum levels of growth factors (TGF-β, CTGF/CCN2, vascular endothelial growth factor [VEGF], fibroblast growth factor [FGF]), interleukins (IL-2, 4, 6, 10, 13), chemokines and other cytokines (monocyte chemotactic protein-1 [MCP-1], IL-8, thymus and activation-regulated chemokine [TARC], fractalkine and tumor necrosis factor-α [TNF-α]) have also been revealed in patients with SSc.

In SSc patients, the clonal expansion of T cells has been detected in the circulation and the affected organs,80 such as skin and lung, suggesting the activation of T cells in response to an unknown antigen. Reflecting the T-cell activation, serum levels of soluble IL-2 receptor are elevated in SSc patients and correlates with the severity of dermal and pulmonary fibrotic lesions.81 Cytokines produced by activated T lymphocytes are involved in the regulation of ECM deposition by fibroblasts.82 Stimulated naive T cells differentiate into memory/effector T cells that are classified into T-helper cell (Th)1 and Th2 subsets based on their cytokine production profiles.83 Th1 cells secrete mainly IFN-γ and IL-2, whereas Th2 cells predominantly release IL-4, IL-5, IL-6, IL-10 and IL-13.83 It has been suggested that Th1 cytokines generally decrease ECM deposition, whereas Th2 cytokines increase it.82 Thus, cytokines produced by activated T cells may regulate fibrosis associated with SSc. Consistently, SSc patients who experience a shift in serum cytokine profile from a Th2 to a Th1 orientation, also experience improvements in skin fibrosis.84

As well as the abnormal activation of T cells, disturbances in B-cell homeostasis appear to be involved in the process of fibrosis and autoantibody production in SSc. An excellent study by Sato et al.85 demonstrated that CD19 expression on B cells were increased in SSc patients approximately 20%, which is comparable to the value leading to the production of autoantibody in a mouse model. Also, the upregulated expression of CD80 and CD86 on the memory B cells of SSc patients suggests that these cells are chronically activated.86 Comparison of the cell type composition and gene expression profiles in skin biopsies from SSc patients with normal individuals has revealed clear differences, including infiltration of B lymphocytes into affected skin.87 In a tight skin mouse, a novel experimental animal model of SSc, experimental B-cell depletion suppresses the development of skin fibrosis, autoantibody production and hypergammaglobulinemia in newborn mice.88 Imbalance of mRNA expression profiles of Th1 and Th2 cytokines was also improved in treated mice. Collectively, these experimental data suggest a pathological role of B cells in skin fibrosis. However, this intervention was inefficacious in mice with existing fibrosis, suggesting that imbalances in B-cell homeostasis may only be important during disease onset.88

The development of autoantibody is a prominent feature of collagen disease including SSc. Autoantibodies in SSc are classified into two groups: (i) autoantibodies directed against nuclear antigens (e.g. topoisomerase-I, centromere, RNA polymerase I/III); and (ii) autoantibodies with putative pathogenic roles. Recent studies demonstrated that anti-nuclear antibodies specific to SSc, such as anti-topoisomerase-I antibody and anti-nucleolar antibodies, react with nuclear antigens derived from apoptotic cells (e.g. apoptosis of endothelial cells by autoantibodies) and these immune complexes induce the production of IFN-α from circulating plasmacytoid dendritic cells.69 Because IFN-α can induce maturation of immature plasmacytoid dendritic cells and development of autoreactive T cells and B cells, these anti-nuclear antibodies may be involved in the process of autoimmune activation in SSc. The other group of autoantibodies with putative pathogenic roles includes anti-endothelial cell antibodies, which are estimated to occur in 44–84% of SSc patients and may induce apoptosis of endothelial cells.89–92 Nuclear antigens derived form apoptotic endothelial cells may lead to the activation of autoimmune reaction as described above. Anti-fibrillin-1 antibodies are detectable in more than 50% of SSc patients and can activate fibroblasts and stimulate release of TGF-β. Antibodies to matrix metalloproteinase (MMP)-1 and -3 may also occur in a high proportion of patients, preventing the degradation of excessive collagen. A putative pathogenic autoantibody to PDGF receptor (PDGFR) has been recognized in SSc patients, and implicated in collagen gene overexpression by fibroblasts.68 Thus, altered B-cell function may be a key link between autoimmunity and fibrosis. Therapeutic interventions restoring immunological homeostasis may be another promising strategy for the treatment of SSc.

Corticosteroid and immunosuppressants

Although few controlled clinical studies have been performed for medicines with anti-inflammatory and immunosuppressive properties, such as corticosteroid, methotrexate, cyclosporine A, azathioprine and mycophenolate mofetil, these medicines are widely used for the treatment of SSc patients, especially for those with a severe form and in early stage. A recent study from the German Network for Systemic Scleroderma demonstrated that 41.3% and 35.8% of all registered SSc patients were treated with corticosteroids and immunosuppressants, respectively. Among SSc patients with overlap syndrome or with diffuse cutaneous involvement, a much higher proportion was treated with these reagents.93

Corticosteroids are still a mainstay of treatment in SSc as well as most autoimmune rheumatic diseases. The use of corticosteroids in SSc is still controversial mainly because high-dose prednisone therapy has been proved to be associated with an increased risk of developing renal crisis in a retrospective case–control study.94, 95 There have been so far no well-controlled studies on the use of corticosteroids in SSc. Similarly, large well-controlled prospective clinical trials are lacking for most of the drugs with immunosuppressive or antifibrotic properties that have been widely used in the treatment of SSc. Although the small number of controlled studies failed to demonstrate a significant benefit of immunosuppressants, recent randomized controlled trials showed a modest benefit of cyclophosphamide and methotrexate for SSc in its early and active stage.96,97 The combination therapy of corticosteroid and cyclophosphamide for pulmonary fibrosis in SSc has been well-studied. One study demonstrated that predonisone (10 mg/day) and i.v. cyclophosphamide (IVCY) (750 mg/m2, monthly, 12 times) were effective for the suppression of active alveolitis in SSc patients.98 Another study, however, showed that deterioration in DLco (carbon monoxide diffusing capacity) occurred in the majority of SSc patients treated with predonisone and IVCY upon discontinuation of therapy even if the treatment initially stabilized lung disease.99 Furthermore, another one showed limited efficacy of IVCY and prednisone for alveolitis in SSc patients.100 Thus, the effect of this combination therapy for pulmonary disease in SSc patients is still controversial. The most important thing at this moment is to establish the treatment recommendations for SSc, which address the use of corticosteroids and immunosuppressive agents with respect to disease subsets, organ involvement and disease activity.

Intravenous immune globulin

Intravenous immune globulin (IVIG) is a potent immunomodulating agent whose efficacy has been demonstrated in various immune-mediated disorders, including idiopathic thrombocytopenia, immune neutropenia, autoimmune hemolytic anemia, pure red cell aplasia, Guillain–Barré syndrome, myasthenia gravis, Kawasaki disease, dermatomyositis and polymyositis. The efficacy of IVIG for the treatment of SSc has also been reported by a small number of open-label, uncontrolled studies101–103 as well as experiments on the tight-skin mouse. IVIG treatment (2 g/kg) on the tight-skin mouse resulted in a significant decrease in cutaneous collagen deposition compared to controls along with a reduction in type I collagen gene expression. Furthermore, the secretion of profibrotic cytokines, such as TGF-β1 and IL-4, by splenocytes of IVIG-treated mice in vitro was decreased.104 These data supported a role for IVIG in preventing fibrosis through a cytokine-mediated mechanism. Moreover, IVIG also downregulated the expression of chemokines such as MCP-1, macrophage colony-stimulating factor (M-CSF) and granulocyte–macrophage colony stimulating factor (GM-CSF), which contribute to inflammation and fibrosis in SSc.105 We previously demonstrated that myofibroblastic phenotype of SSc fibroblasts were reversed after the treatment of IVIG, which appeared to be at least in part attributed to the normalization of the expression levels of TGF-β receptors in SSc fibroblasts.101 To date, several mechanisms have been proposed for the immunomodulatory effect of IVIG:106 (i) inhibition of complement activities; (ii) neutralization of autoantibodies by anti-idiotypic antibodies; (iii) modulation of lymphocyte functions and cytokine synthesis; and (iv) blockade of Fcγ receptors on phagocytic cells. Our observation suggests that several of these mechanisms may work together in the initiation and maintenance of fibroblast activation in SSc. To further validate the efficacy of IVIG for SSc, a large randomized controlled study is currently under way in Japan.

Stem cell transplantation

Given that SSc has an autoimmune-related pathogenesis, particularly in its early stage, immunoablation by high-dose immunosuppressive therapy accompanied with autologous hemapoietic stem cell transplantation (HSCT) is a plausible potential therapy. The EBMT and EULAR database showed that approximately 70% of patients with autoimmune diseases treated with HSCT initially responded well, with durable remission/stabilization seen more frequently in multiple sclerosis and SSc than in rheumatoid arthritis and systemic lupus erythematosus.107 The rate of transplant-related mortality in SSc patients was acceptable. Based on these results, the European multicenter, prospective, randomized Autologous Stem cell Transplantation International Scleroderma (ASTIS) trial is currently underway, which compares immunoablation and autologous HSCT versus monthly IVCY therapy in patients with early dcSSc at high risk of mortality.108

B-cell depletion by biologics

Given that experimental B-cell depletion suppresses the development of skin fibrosis, autoantibody production and hypergammaglobulinemia in animal model of SSc,88 B-cell depletion is another potential therapeutic strategy for SSc. A small clinical trial recently performed demonstrated that rituximab, a monoclonal antibody for CD20, depletes peripheral and dermal B cells but did not provide any clear benefit in SSc skin involvement.109 However, the safety of the medication and the potential efficacy related to B-cell infiltration of other organs such as the lungs suggest that rituximab might be worth further studying for SSc-associated PF, perhaps in combination with cyclophosphamide.

New therapeutic targets for vasculopathy in SSc

Mechanism of SSc vasculopathy

Lesional skin of SSc patients often shows microvascular damage preceding dermal fibrosis, which is characterized by a decrease in the number of capillaries and the development of avascular areas mainly caused by apoptosis of endothelial cells.110 Intimal proliferation and the replacement of type IV collagen with proteoglycans in vascular basement membranes are commonly seen, which often result in vessel occlusion. In addition, a perivascular infiltration mainly consisting of activated T and B cells has been described at early stage. Anti-endothelial cell antibodies, which are found in approximately 40–50% of SSc patients, are likely to cause an antibody-dependent cellular cytotoxicity against endothelial cells as well as cytotoxic CD8+ T cells. Additional factors, including ischemia, reperfusion injury, superoxide radicals derived from platelets and neutrophils, cytokines and chemokines, further contribute to the development of the endothelial dysfunction in SSc. Especially, ET-1, a potent vasoconstrictor with a mitogenic action on fibroblasts and vascular smooth muscle cells, is elevated in plasma of SSc patients and believed to play an important role in the development of SSc vasculopathy. Nitric oxide (NO) and NO synthase (NOS) are decreased in endothelial cells of patients with SSc. In addition, an important role in the pathogenesis of SSc is attributed to platelet activation and to the coagulation and fibrinolysis mechanisms, whose dysregulation is thought to occur secondarily to endothelial dysfunction.111 Finally, angiotensin is also considered as an important contributor to the development of vascular manifestations in SSc through induction of fibrosis as well as vasoconstricton.112 All these pathways thus far have been used as therapeutic targets.

Another new therapeutic target for SSc vasculopathy is a transcription factor Fli1. A recent report demonstrated that Fli1 is a key transcription factor regulating endothelial cell behavior in the process of angiogenesis.113 Furthermore, we recently demonstrated that endothelial cell-specific Fli1 depletion results in the development of histopathological features of SSc vasculopathy in animal models (Asano et al., unpubl. data, 2009). Collectively, these results suggest that transcription factor Fli1 can be a potent candidate for the new therapeutic target in SSc.

Here, the three new therapeutic targets for Raynaud’s phenomenon, digital ulcers and PAH in SSc are described and the details regarding the potential therapy targeting transcription factor Fli1 is described in the fourth section of this article.

Phosphodiesterase-5 inhibitors

Phosphodiesterase-5 inhibitors, such as sildenafil, vardenafil and tadalafil, prevent the degradation of cyclic guanosine monophosphate (cGMP) in pulmonary vascular smooth muscle cells, resulting in relaxation of pulmonary arteries. Sildenafil has been proven effective for the treatment of PAH associated with connective tissue diseases, including SSc.114, 115 In addition, sildenafil is effective for reducing the frequency, severity and duration of Raynaud’s phenomenon in SSc patients.116, 117 The new derivatives, such as vardenafil and tadalafil, are currently undergoing evaluation for their use in PAH.118–120

Endothelin receptor antagonists

Endothelin receptor antagonists thus far consist of three drugs, bosentan, sitaxsentan and ambrisentan. Bosentan is dual endothelin receptor antagonist and the others selectively block the type A endothelin receptor. Bosentan is already widely used for the treatment of primary and secondary PAH associated with connective tissue diseases, including SSc. Although bosentan did not improve the 6-min walk distance or the pulmonary function tests in SSc patients with PAH, it significantly reduced the mortality in those patients.121–124 The impact of bosentan in SSc has been evaluated also in terms of its effect on digital ulcerations and Raynaud’s phenomenon. Although bosentan failed to improve the healing of ulcers, it prevented the development of new digital ulcerations and induced an improvement in the functionality of the hand and a diminution of pain in treated SSc patients.56 Selective ETA receptor antagonists, sitaxsentan and ambrisentan, have also been proven to be useful for the treatment of primary PAH.125,126 Both of these drugs are currently being evaluated in patients with SSc. The role of endothelin receptor antagonists in the management of SSc goes beyond the reversal of the potent vasoconstrictive effects of endothelin. Given that ET-1 can upregulate the expression of intercellular adhesion molecule-1 on the cell surface of fibroblasts127 and act as a downstream mediator of TGF-β, blockade of ET-1 is likely to inhibit the profibrotic phenotype of SSc fibroblasts (already described above).

Inhibitors for serotonin signaling

Recent findings implicate serotonin in the pathogenesis of PAH through the vasoconstriction and the stimulation of pulmonary vascular smooth muscle cell proliferation. Importantly, pulmonary microvascular endothelial cells derived from patients with primary PAH produce increased levels of serotonin in vitro, theoretically causing hyperplasia of pulmonary artery smooth muscle cells.128 Furthermore, selective 5HT transporter inhibitors, citalopram and fluoxetine, prevent the development of PAH in animal models.129 Selective serotonin reuptake inhibitors (SSRI) are other potent candidates for the treatment of PAH. A retrospective cohort study demonstrated that 15% of PAH patients used SSRI during the study period and that the risk of PAH-related death in these patients was lower although not statistically significant.130 Taken together, these experimental and clinical data suggest that 5HT transporter inhibitors and SSRI are new potent drugs for the treatment of PAH in SSc.

A genetic factor of SSc, transcription factor Fli1

Roles of Fli1 in endothelial cells, immune cells and fibroblasts

Fli1 is a member of the Ets family of transcription factors characterized by the presence of the evolutionary conserved DNA-binding (ETS) domain, which recognizes the purine-rich GGA/T core sequence. Based on comparative gene expression profile analysis of various human cell lines, Fli1 is expressed at high levels in both endothelial and hematopoietic cells.131Fli1 knockout mice are embryonic lethal at E11.5 due to severe hemorrhage in the central nervous system, indicating the importance of Fli1 in the development of vasculature.132Fli1 has been believed to be involved in the mechanism responsible for the autoimmune activation in systemic lupus erythematosus (SLE). The expression levels of Fli1 are increased in peripheral blood mononuclear cells (PBMC) derived from patients with SLE.133 H-2Kk-Fli1 transgenic mice, which overexpress Fli1 especially at the highest levels in the thymus and spleen, develop a high incidence of progressive immunological renal disease and ultimately die of renal failure caused by tubulointerstitial nephritis and immune-complex glomerulonephritis.134 MRL/lpr mice, the model animal of SLE, develop lupus nephritis. However, the prevalence of lupus nephritis is significantly decreased in MRL/lpr mice with heterozygous knockout of Fli1.135 Collectively, aberrant expression of Fli1 may lead to the activation of the autoimmune system. Although Fli1 is present in a relatively limited amount in dermal fibroblasts, recent studies have shown that Fli1 plays a pivotal role in the regulation of ECM genes, including type I collagen and tenascin-C,40,136–139 ECM-degrading enzyme MMP-1140 and the multifunctional matricellular factor CCN2.141 Thus, Fli1 plays pivotal roles in endothelial cells, immune cells and fibroblasts, which are the three main cell groups involved in the pathogenesis of SSc.

Transcriptional activity of Fli1 is regulated by TGF-β through phosphorylation-acetylation cascade in dermal fibroblasts

Recent studies provide strong evidence for a critical role of Fli1 in the repression of collagen type I genes and inhibition of the TGF-β-induced profibrotic gene program.136,141–146 TGF-β abrogates the function of this repressor through the sequential post-translational modifications that involves phosphorylation and acetylation (Fig. 2).142,144 Upon TGF-β stimulation, PKC-δ is activated and translocated into the nucleus. In the nucleus, PKC-δ directly interacts with Fli1 on the promoter region of type I collagen gene and phosphorylates Fli1 at threonine 312, which in turn increases the affinity of Fli1 with PCAF. Then, PCAF acetylates Fli1 at lysine 380 and acetylated Fli1 dissociates from the promoter of type I collagen gene due to conformational change and is targeted for degradation through a proteasomal pathway, resulting in a decreased steady-state level of Fli1 protein and a de-repression of collagen gene transcription. Our recent study demonstrated that TGF-β-dependent activation of PKC-δ is mediated by c-Abl tyrosine kinase, which has been implicated in the non-canonical pathway of TGF-β signaling.43, 147, 148 A novel non-canonical pathway of TGF-β signaling “c-Abl – PKC-δ–Fli1” would be a potent therapeutic target in SSc (the details are described below).


Figure 2.  “c-Abl – PKC-δ–Fli1” is one of the non-canonical pathways of transforming growth factor (TGF)-β signaling. c-Abl and protein kinase C (PKC)-δ are sequentially activated by TGF-β stimulation. Active PKC-δ translocates into the nucleus, directly interacts with Fli1 on the promoter region of type I collagen gene and phosphorylates Fli1 at threonine 312, which in turn increases the affinity of Fli1 with PCAF. Then, PCAF acetylates Fli1 at lysine 380 and acetylated Fli1 dissociates from the promoter of type I collagen gene due to conformational change and is targeted for degradation through a proteaosomal pathway, resulting in a decreased steady-state level of Fli1 protein and a de-repression of collagen gene transcription. Imatinib mesylate inhibits c-Abl tyrosine kinase activity and consequently blocks non-canonical TGF-β signaling. Imatinib mesylate also inhibits platelet-derived growth factor (PDGF) signaling pathway, which may be activated in systemic sclerosis fibroblasts by the stimulation mediated by the circulating PDGF receptor antibodies.

Download figure to PowerPoint

Role of Fli1 in the mechanism responsible for skin fibrosis in SSc

Aberrant expression of Fli1 has been implicated in the pathogenesis of cutaneous fibrosis in SSc.138,149 In the clinically involved skin of SSc patients with early active disease, Fli1-positive fibroblasts are either absent or only occasionally seen, while the majority of fibroblasts in healthy control skin express Fli1 at a relatively high level. Importantly, there is an inverse correlation between the expression of Fli1 and type I collagen in dermal fibroblasts in lesional and healthy skin.138 An animal model demonstrated that Fli1 functions as a transcriptional repressor of fibrillar collagen genes in vivo.143 Expression levels of the fibrillar collagens (type I, III, V) are markedly elevated in the skin of mice with the homozygous deletion of the C-terminal domain of the Fli1 gene (Fli1ΔCTA). Elevated expression of fibrillar collagens is maintained in fibroblasts cultured from the skin of these mice, indicating that the absence of functional Fli1 in fibroblasts is sufficient to alleviate repression of collagen genes both in vivo and in vitro. An excellent study by Wang et al.149 demonstrated that the downregulation of Fli1 through epigenetic mechanisms may mediate the fibrotic manifestations of SSc based on the following data: (i) the treatment of epigenetic inhibitors normalizes collagen expression in cultured SSc fibroblasts; (ii) the augmented collagen synthesis in SSc fibroblasts is linked to epigenetic repression of the collagen suppressor gene Fli1; and (iii) heavy methylation of the CpG islands and the increased acetylation of histone H3 and H4 in the promoter region of the Fli1 gene are detected in SSc fibroblasts and skin biopsy specimens. Collectively, these experimental data strongly suggest that Fli1 deficiency due to an epigenetic mechanism is involved in the mechanism responsible for the constitutive activation of SSc fibroblasts.

Role of Fli1 in the mechanism responsible for SSc vasculopathy

Although Fli1 is expressed at high levels in the microvasculature of healthy skin, the role of Fli1 in the vasculature has not been fully characterized. Recent studies of zebrafish and Xenopus embryos, however, have shown that Fli1 functions as a master regulator of the transcriptional network driving blood and endothelial cell lineages.113 Consistently, mice with targeted deletion of the Fli1 gene die at E11.5 due to cranial and spinal hemorrhages.132 In contrast to normal human skin, the levels of Fli1 are greatly reduced in endothelial and peri-endothelial cells in SSc skin, suggesting that the absence of Fli1 may be involved in the mechanism responsible for vasculopathy in SSc.138

Despite intensive studies, the causes of endothelial cell dysfunction in SSc have not been well understood. The absence of an animal model that recapitulates the major features of SSc vasculopathy has hindered progress in this area. Because of the early lethality of Fli1 null mice we recently generated mice with a conditional deletion of Fli1 in endothelial cells, and we showed that Fli1 is a critical regulator of vascular homeostasis in the skin. Importantly, vascular defects observed in SSc vasculature, such as stenosis of arterioles and dilation of capillaries, are also reproduced in these mice, supporting the notion that conditional Fli1 deficient mice may represent a useful model to investigate the molecular mechanisms involved in SSc vasculopathy (Asano et al., unpubl. data, 2009).

Imatinib mesylate reverses the expression levels of Fli1 and suppresses the production of type I collagen in SSc fibroblasts

Imatinib mesylate, a selective protein tyrosine kinase inhibitor against c-Abl, as well as PDGF receptor and c-kit, is now widely used for the treatment of several diseases. Imatinib has been shown to block the induction of c-Abl tyrosine kinase activity and fibrotic gene responses elicited by TGF-β, and normalized collagen overproduction in cultured SSc fibroblasts.146 Until recently, the anti-TGF-β effects of imatinib are thought to be associated with blockade of the activation of Smad1 and early growth response protein 1.146, 150 However, our latest study identified another novel mechanism by which imatinib blocks the TGF-β-dependent upregulation of type I collagen gene. As mentioned above, upon TGF-β stimulation c-Abl and PKC-δ are sequentially activated and Fli1 consequently dissociates from the promoters of type I collagen genes through phosphorylation-acetylation cascade. Because imatinib inhibits tyrosine kinase activity of c-Abl and subsequently serine/threonine kinase activity of PKC-δ, imatinib increases the binding activity of Fli1 to type I collagen promoter through dephosphorylation and deacetylation of Fli1, resulting in the suppression of type I collagen gene expression (Fig. 2). Indeed, addition of imatinib to cell cultures increases the binding of Fli1 to the promoter of type I collagen gene and subsequently suppresses the expression of type I collagen protein in SSc fibroblasts (Bujor et al., unpubl. data, 2009).

Imatinib mesylate is a novel potent medicine for the treatment of SSc

Numerous reports suggest that imatinib is effective in ameliorating skin fibrosis in some patients with SSc, and in fibrosing conditions, such as chronic graft-versus-host disease, nephrogenic systemic fibrosis and localized scleroderma.151–156 In addition, imatinib has been shown to ameliorate PAH, a frequent complication of limited cutaneous SSc, in animal models of PAH, individual case reports and a small phase II clinical trial.157–160 These clinical findings are plausible because imatinib can reverse the expression levels of Fli1 in fibroblasts and endothelial cells of SSc, where Fli1 is downregulated through an epigenetic mechanism. In terms of the pathogenesis of SSc, Fli1 is one of the permissive genetic background factors,149 whose aberrant expression may result in the activation of fibroblasts and endothelial cells and in the dysfunction of immune cells. Because imatinib blocks PDGF receptor tyrosine kinase as well as c-Abl tyrosine kinase, the reversal of Fli1 gene expression is likely to be one of the mechanisms by which imatinib improves clinical symptoms of SSc (Fig. 3). Thus, protein tyrosine kinase inhibitors are promising candidates for the treatment of SSc and dasatinib, another novel tyrosine kinase inhibitor that is active against multiple members of the Src family of kinases as well as c-Abl and the PDGF receptor, is now in early-stage clinical trials for SSc.


Figure 3.  Pathogenesis of systemic sclerosis (SSc) and the significance of imatinib mesylate for the treatment of SSc. Fli1 is downregulated in SSc fibroblasts through an epigenetic mechanism, suggesting that Fli1 is one of the genetic backgrounds. Imatinib mesylate reverses the expression levels of Fli1 in SSc, potentially resulting in the normalization of fibroblasts, endothelial cells and immune cells.

Download figure to PowerPoint


  1. Top of page
  2. Abstract
  3. Introduction
  4. References