Curr Rheumatol Rep (2014) 16:411 DOI 10.1007/s11926-014-0411-1
SCLERODERMA (J VARGA, SECTION EDITOR)
Recent Advances in Understanding the Pathogenesis of Scleroderma-Interstitial Lung Disease Tanjina Akter & Richard M. Silver & Galina S. Bogatkevich
Published online: 13 February 2014 # Springer Science+Business Media New York 2014
Abstract Systemic sclerosis (scleroderma, SSc) is a heterogeneous autoimmune connective tissue disease of unknown etiology. Interstitial lung disease (ILD) is a frequent complication, and a significant contributor to morbidity and mortality among SSc patients. SSc-ILD most commonly occurs within 10 years of diagnosis, and may be seen in patients with either the limited or diffuse cutaneous subset of SSc. SSc-ILD is a multifaceted disease process in which different factors and pathways are involved. Aberrant function of a variety of lung cells, cytokines, growth factors, peptides, and bioactive proteins, in combination with genetic and epigenetic regulators, have crucial functions in the pathogenesis of this disease. Here we present our view on recent advances regarding the pathogenesis of SSc-ILD. Keywords Systemic sclerosis (SSc) . Scleroderma . Pathogenesis . Interstitial lung disease (ILD) . Fibroblast, TGF-β . Thrombin . Wnt/β-catenin pathway . Genetics . Epigenetics
This article is part of the Topical Collection on Scleroderma T. Akter (*) : G. S. Bogatkevich Division of Rheumatology and Immunology, Department of Medicine, Medical University of South Carolina, 114 Doughty Street, Charleston, SC 29425, USA e-mail: [email protected] G. S. Bogatkevich e-mail: [email protected] R. M. Silver Division of Rheumatology and Immunology, Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, Charleston, SC 29425, USA e-mail: [email protected]
Introduction Systemic sclerosis (SSc) is a connective tissue disease characterized by vascular alterations, limited or widespread dermal and visceral organ fibrosis, and immunological dysregulation associated with specific autoantibodies [1]. Persistent overproduction of collagen and deposition of connective tissue is the main characteristic of this disease [2], and activation of collagen-producing cells is greatly increased in SSc patients [3, 4]. Systemic sclerosis often affects multiple systems, including the skin and visceral organs of the body. Pulmonary involvement is seen in 70 % to 90 % of cases and is now the leading cause of mortality. Approximately 40 % of SSc-ILD patients will die within 10 years of diagnosis [4, 5], so a better understanding of the pathogenesis of this complication is of paramount importance. The most common initiator of the pathogenesis of SSc-ILD is a persistent injury to lung cells, for example alveolar epithelial and endothelial cells, in which the presence of pro-fibrotic stimuli induces the differentiation of lung fibroblasts to a myofibroblast phenotype. Increased release of profibrotic cytokines, growth factors, peptides, and bioactive proteins results in stronger signaling, leading to the additional recruitment of myofibroblasts, accumulation of extracellular matrix, and scarring [6•, 7]. The principal consequence of this pathobiology is apoptosis of the alveolar epithelial cells, and accumulation and activation of fibroblasts from different origins [6•, 8]. Systemic sclerosis is characterized by alterations to both humoral and cellular immunity, and a variety of diseasespecific autoantibodies against self-antigens have been described in SSc patients. Autoimmune diseases, including SSc, are believed to be complex polygenic conditions, with predisposition and progression determined by multiple
411, Page 2 of 9
susceptibility genes interacting with epigenetic and environmental factors [9, 10]. Extensive supporting evidence for the involvement of genetics has recently been found, and several genes involved in immune regulation have been proposed as risk factors for the development of SSc. Involvement of several loci and microRNA has already been postulated for pulmonary fibrosis [11]. Alternatively, another line of research has provided evidence that pulmonary fibrosis in SSc is a result of an innate immune response driven by persistent microbial infection [12]. Excessive oxidative stress in SSc [13], and specifically in lung fibrosis [14], may be another important factor in pathogenesis. The objective of this review is to discuss the genetic susceptibility, cellular damage, and aberrant signaling pathways involved in the pathogenesis of SSc-ILD.
Cells Involved in the Pathogenesis of SSc-ILD Recurrent alveolar epithelial cell (AEC) injury, and an aberrant healing process accompanied by fibroproliferation with myofibroblast persistence, impaired hyperplasia, and increased retardation and/or apoptosis of AEC, is central to the pathogenesis of ILD [15]. The pathophysiological alteration of injured alveolar epithelial cell is reflected in the high-level secretion of glycoproteins, for example surfactant protein D (SP-D) and KL6, in the blood of SSc-ILD patients [16]. One of the most profound changes in epithelial cell morphology and biology is epithelial mesenchymal transition (EMT), which has been observed in scleroderma [8]. EMT is a biochemical process in which the epithelial cell loses its polarity and develops mesenchymal properties. These cells develop enhanced migratory capacity, invasiveness, increased resistance to apoptosis, and greatly increased production of extracellular matrix (ECM) components [17]. EMT has been observed both in SSc-ILD patients and in the bleomycin-induced mouse model of ILD [5]. The concept of transformation of epithelial cells to mesenchymal cells has been strongly criticized and challenged by other researchers, who observed that neither pericytes nor type-1 or type-2 AEC follow EMT pathways, but that instead a heterogeneous cell population is involved in this process [18]. More studies are required, both on animal models and on human fibrotic disease, for better understanding of this process. Little is known about the crucial mediators that determine whether AEC will undergo EMT or apoptosis during fibrosis. Human AEC in culture have a matrix-dependent preference, with AEC-II cells in the presence of fibronectin and/or fibrin in culture medium following EMT pathways. In the presence of transforming growth factor-β (TGF-β), these cells lose the epithelial cell marker pro-surfactant protein C (pro-SPC), lose apical–basal polarity, and express mesenchymal cadherin (Ncadherin). In contrast, AEC in the presence of laminin and/or
Curr Rheumatol Rep (2014) 16:411
collagen in cell culture medium undergo apoptosis rather than EMT [19]. The other major cell type involved in the pathogenesis of lung fibrosis is the fibroblast. Fibroblasts have heterogeneous phenotypic features, a vigorous replication pattern, and a central function in ECM formation [15, 20]. Activated lung fibroblasts can originate from resident lung fibroblasts, from circulating bone-marrow-derived fibrocytes, and from the differentiation of other cell types including epithelial cells, endothelial cells, and pericytes [4, 20]. Recruitment and activation of lung fibroblasts are induced by persistent lung injury, and an unsuccessful repair process summons more cells to the site of injury [21]. In cellular homeostatic conditions these recruited cells are cleared up by apoptosis. But in the case of lung fibrosis, fibroblasts do not undergo programmed cell death. This avoidance of apoptosis by fibroblasts is enabled by several proteins. One of these proteins, focal adhesion kinase (FAK), provides viability to fibroblasts through the PI3K-Akt-mediated pathway, thus helping them avoid apoptosis [22]. Fibroblasts are capable of different responses depending on the type of stimuli they receive, which may also facilitate their survival. For example, in the presence of IL-1β these cells express the pro-apoptotic protein calcium-insensitive nitric oxide synthase (iNOS), which produces nitric oxide and leads to cell death. However, in the presence of TGF-β the expression of iNOS is reduced or completely inhibited [23]. Additionally, fibroblasts isolated from SSc-ILD patients respond differently from normal lung fibroblasts. Thus, after stimulation with IL-6, healthy fibroblasts express Bcl-2-associated X-protein (BAX), a pro-apoptotic protein that leads to Fas-induced cell death, whereas scleroderma lung fibroblasts express B-cell lymphoma-2 (Bcl-2), which is an anti-apoptotic protein that confers apoptosis resistance [24]. Another important cause of the apoptosis resistance of lung fibroblasts is X-linked inhibitor of apoptosis protein (XIAP), which regulates Fas-mediated apoptosis. XIAP is over-expressed in fibroblastic foci in IPF, and inhibition or silencing of XIAP causes a loss of resistance to apoptosis in IPF fibroblasts [25]. Expression of Fas is also regulated by histone modification on the Fas promoter region. Histone deacetylation and histone 3 lysine 9 trimethylation are significantly higher both in human lung fibrosis and in the bleomycin mouse model, with over-expression of histone deacetylase (HDAC)-2 and HDAC-4. These down-regulate expression of Fas, and inhibit Fas-mediated apoptosis. In the presence of an HDAC inhibitor, Fas expression increases and cells recover susceptibility to Fas-mediated apoptosis [26]. Fibroblasts can also escape apoptosis by controlling the cell cycle. Human diploid fibroblasts enter a stable growth arrest near the end of their life; in this stage they accumulate pro-apoptotic ceramide but become resistant to ceramide-induced apoptosis [27].
Curr Rheumatol Rep (2014) 16:411
TGF-β Pathway The most widely studied mediator of SSc-ILD is TGF-β, which is released by injured lung cells. There are several proposed mechanisms, both interrelated and independent, underlying the pathogenesis of SSc-ILD. TGF-β leads to inhibition of AEC proliferation, expression of metalloproteinase-7 (MMP-7), induction of myofibroblasts, and accumulation of ECM proteins [4, 6•, 8, 28, 29]. In SSc-ILD, TGF-β has multiple functions in cell signaling, binding with its cellsurface heterodimeric receptor to activate canonical or noncanonical pathways. In the canonical pathway TGF-β causes phosphorylation of Smad2/3, which subsequently binds with Smad4. Smad4 acts as a transcription activator, leading to the expression of extracellular matrix proteins, e.g., collagen, plasminogen activator inhibitor-1, and connective-tissue growth factor (CTGF, CCN2) [8]. TGF-β-induced cell signaling initiated via a non-canonical pathway is mediated by specific regulatory proteins, including MAPK, PAR6 and RhoA, which subsequently affect cytoskeleton rearrangement, cell adhesion, and cell motility in pulmonary fibrosis [8]. Because TGF-β has a central function in development and progression of lung fibrosis, blocking of TGF-β could be a potential therapeutic strategy. Conditional knockout of TGF-β receptor type II (TβRII) attenuated lung fibrosis via regulating the phosphorylation of Smad-2 and Smad-3 in AECs; such knockout mice are resistant to bleomycin-induced lung fibrosis and have an increased survival rate [4]. As well as regulating the gene expression or the cell signaling, TGF-β also has a crucial function in regulation of immune response. Injured tissue releases TGF-β, which signals for the recruitment of immune cells, including macrophages, to the site of injury, which in turn release more TGF-β, exacerbating the fibrosis [30]. TGF-β also has a major function in the development, maturation, and proliferation of lymphocytes, mainly T-cells and B-cells, and in secretion of cytokines. It suppresses the expression of IL-2 and inhibits IL-2-dependent lymphocyte proliferation [31]. Additionally, TGF-β induces the pro-inflammatory cytokine IL-10, disrupting the balance between helper and regulatory T cells, and suppresses anti-fibrotic cytokine interferon-γ (INF-γ) [28, 29].
Thrombin Pathway Thrombin is a key mediator of the coagulation cascade and is a serine protease with pluripotent cellular effects, including induction of cytokines and regulation of cell-type-specific apoptosis in lung fibrosis [32, 33••]. Our laboratory and others have revealed greatly increased levels of thrombin in bronchoalveolar lavage fluid (BALF) from scleroderma patients with lung fibrosis and other fibrosing lung disease [34, 35]. It
Page 3 of 9, 411
has been revealed that BALF from normal subjects has a low level of thrombin activity, whereas BALF from SSc patients has up to 100-fold higher thrombin activity [34]. Elevated levels of thrombin activity have also been observed in bleomycin-induced pulmonary fibrosis in mice [36]. Thrombin is mitogenic for lung fibroblasts [34, 37, 38] and enhances the proliferative effect of fibrinogen on fibroblasts [39]. Thrombin is also a potent inducer of fibrogenic cytokines, chemokines, and growth factors, for example TGF-β [40], CTGF [41, 42], platelet-derived growth factor-AA (PDGF-AA) [34], chemokine ligand 2 (CCL2) [32], and ECM proteins, for example collagen, fibronectin, and tenascin, in a variety of cells, including lung fibroblasts [43–45]. Most cellular responses to thrombin are mediated via the G-protein-coupled receptor PAR-1, which is significantly increased in patients with SSc-ILD, notably in lung parenchyma associated with inflammatory and fibroproliferative foci [37, 46]. Elevated expression of PAR-1 has been also observed in patients with IPF and in a murine model of bleomycininduced lung fibrosis [36, 47]. Studies from our laboratory revealed that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype that is resistant to apoptosis, although thrombin also is capable of inducing apoptosis of AEC via a PAR-1-receptor-dependent pathway [33••, 37, 38]. Inhibition of thrombin by a direct inhibitor, dabigatran, has substantial antifibrotic effects both in vitro and in vivo [48, 49]. Dabigatran inhibits thrombin-induced differentiation of normal lung fibroblasts to the myofibroblast phenotype in vitro, and it decreases CTGF, α-SMA, and collagen type I in scleroderma lung fibroblasts [48]. Moreover, dabigatran significantly reduces thrombin-induced apoptosis of AEC [48]. Oral administration of dabigatran etexilate attenuates the development of bleomycin-induced pulmonary fibrosis in mice. Additionally, treatment with dabigatran etexilate reduces collagen, CTGF, and α-SMA expression in mice with bleomycin-induced lung fibrosis, whereas it has no effect on basal levels of these proteins [49].
Wnt/β-catenin Pathway SSc is characterized by widespread vascular injury and ischemia. In SSc-ILD, vascular injury leads to apoptosis of AEC type-1 cells and activation and proliferation of AEC type-2 (AT2) cells to repair the damaged lung, with concurrent activation of other pathways and fibroblast accumulation culminating in fibrosis [5]. AT2 cells may also be involved in fibrosis in other ways. The phenotype and gene-expression profile of AT2 cells from fibrotic lung tissue indicate an increased proliferation capacity and enhanced expression of proliferation-related genes [50]. Activation of the Wnt/βcatenin pathway has been observed in the fibroblastic foci of
411, Page 4 of 9
IPF patients, but not of patients with normal and/or pneumonia-related pulmonary disease [51]. The Wnt pathway consists of a large number of highly conserved growth factors, whose receptor-mediated activity leads to the accumulation of cytoplasmic β-catenin; this then translocates to the nucleus to regulate target gene expression in association with Akt and/or through T-cell-specific transcription factor and/or lymphoid-enhancer-binding factor1 family transcription factors [8, 52]. This pathway may also have a function in epithelial cell regeneration at bronchiolar–alveolar junctions and in epithelial–mesenchymal transitions, leading to the irreversible remodeling of pulmonary tissue [51]. High levels of nuclear β-catenin are observed in IPF fibroblastic foci, and activated Akt has been observed in fibroblasts from SSc-ILD patients [51, 53]. Further analysis reveals that expression of WNT1-inducible signaling protein-1 (WISP1), a product of the WNT target gene, is highly upregulated in the AT2 cells in hyperplastic areas of fibrotic lung. WISP1 is a member of the CCN family, consisting of four conserved cysteine-rich modular domains, acting through binding of integrin receptor, and affecting cellular functions including mitosis, adhesion, and migration. WISP1 works in an autocrine fashion to induce the release of cytokines, including SPP1, MMP-7, MMP-9, and PAI1, from alveolar epithelial cells [5, 50]. MMP-7 levels are elevated in SSc-ILD and are over-expressed in AT2 cells in other types of pulmonary fibrosis [50, 54]. Treatment with WISP1 in AT2 primary cell culture leads to an increase in cell proliferation and EMT, with profibrotic marker-gene expression. Co-localization of WISP1 with pro-SPC has been detected in both the bleomycin mouse model and in IPF [50]. Evidence for catenin-based Wnt signaling has been detected in fibrotic skin in SSc, and in a mouse model Wnt is related to collagen gene expression [55]. Wnt signaling is a very common mechanism in cancer, e.g., breast cancer, melanoma, prostate cancer, lung cancer, and several other cancer types, and has been regarded as a potential therapeutic target. Blocking Wnt signaling inhibits prostatic bud formation in prostate cancer [56]. In the bleomycin mouse model it has been revealed that inhibition of WISP1, by antiWISP1 antibody or siRNA, leads to a decrease in AT2-cell proliferation. Repetitive orotracheal instillation of antiWISP1 antibody in the bleomycin mouse model is associated with a significant decrease in collagen deposition, and with improved lung function and survival [50].
Genetic Associations Genetic studies have revealed that the incidence of SSc is 1.5–1.7 % in families with a history of scleroderma, compared with 0.026 % in the general population. SSc is not inherited in a Mendelian fashion, but a positive family
Curr Rheumatol Rep (2014) 16:411
history significantly increases the relative risk: by 15 to 19-fold in siblings and by 13 to 15-fold in first-degree relatives [57]. Although females are more susceptible to SSc [58], the severity of SSc-ILD in male patients is greater than in females [3]. Genetics studies suggest that SSc-ILD is correlated with the X-chromosome, which might in turn suggest that, because males have a single X-chromosome, any mutation(s) in the genes of this chromosome are not restored in males, leaving them more vulnerable to ILD. Racial differences observed among African-American and Caucasian SSc patients suggest African Americans have a genetic predisposition to SSc. Studies conducted in different locations in the US have consistently observed that African-American SSc patients have higher morbidity and mortality as compared with Caucasians [59, 60••, 61–63]. African Americans with SSc have a higher incidence of the anti-Scl-70 autoantibody [62], and express more TGF-β, IL-6, and other profibrotic factors [60••], as compared with Caucasian SSc patients. In contrast, expression of antifibrotic proteins, including hepatocyte growth factor (HGF) and PPAR-γ, is significantly lower in African-American SSc patients than in Caucasian SSc patients [61, 64•]. Extensive gene-based investigation is required to discover the underlying causes of this racial disparity.
HLA-dependent Genes The main interest in genetic-based studies related to SSc has been focused on genes of the major histocompatibility complex-class II (MHC-II). Demographic studies reveal variation in the expression pattern of MHC genes; for the most part, subtypes of MHC-II, DP, DQ, and DR have been revealed to be associated with SSc [65–67]. These different classes of allele are associated with a variety of diseasespecific autoantibodies, including anti-centromere, antitopoisomerase I, and other antinuclear antibodies [66]. The proposed effect of these genetic correlations has been criticized, because of high variation in different geographic and ethnic-based studies. A study in Mexico revealed that DR5 (DRB1*1104) affects SSc susceptibility [65]. In Japan, another study revealed association of SSc with “Transporter associated with antigen processing 1 and 2” (TAP 1 and 2) alleles [68]. DPB1*1301 and DRB1*15 affect pulmonary fibrosis in South-African and Korean patients. In Caucasians, MHCclass I subtypes (HLA-B*62 and HLA-Cw*602) are predominant in patients with lung fibrosis. Genetic susceptibility associated with HLA-DRB1*15 was observed in a Korean population and in French women with dcSSc. The presence of HLA-DRB1*11/07 haplotypes is associated with pulmonary fibrosis in French women, and in Spanish and Italian patients [69]. Two other alleles are recognized to be associated with
Curr Rheumatol Rep (2014) 16:411
Page 5 of 9, 411
Fig. 1 Cells and pathways involved in pathogenesis of lung fibrosis. Fibrosis is caused by a combination of several factors, including genetics and epigenetics cross-linked with heterogeneous cellular responses. The initiation of fibrosis is caused by injury to the alveolar cells, inducing activation of immune cells and release of thrombin, TGF-β, Wnt/β-catenin, and other fibrogenic proteins. These events stimulate the fibroblasts, recruiting them to the site of injury and resulting in the generation of myofibroblasts leading to tissue fibrosis
SSc. One of them, HLA-DRw11, seems to have a major function in determining the severity of SSc, and another one,
an HLA-DQ gene, is associated with anti-topoisomerase-I antibody development [70].
Table 1 Cells and pathways involved in SSc-ILD Cell involved in fibrosis AECs
Fibroblast
Main pathways TGF-β
Fate Apoptosis or retardation EMT Release of SP-D and KL-6 Apoptosis resistance Signal for cell migration, persistent lung injury, fibrosis Intermediates involved Smad-dependent signaling molecules Smad-independent signaling molecule (MAPK, PAR6, RhoA)
Thrombin
PAR1 activation
Wnt/β-catenin
β-catenin
Ref. [15] [8] [16] [23] [21]
Biological effects Gene expression regulation, ECM formation EMT Cell signaling Lymphocyte development, proliferation, and maturation, and cytokine release Induction of cytokines and ECM protein Increase fibroblast proliferation. Signal for apoptosis in AECs but not in fibroblast Regulate target gene expression Hyperplasia in AEC-II Release of cytokines
Ref. [6•] [8] [30, 31] [28] [32] [33••] [55] [5] [50]
411, Page 6 of 9
Curr Rheumatol Rep (2014) 16:411
Table 2 Pathways targeted therapeutic strategies in lung fibrosis Pathways of therapeutic Therapeutic strategies and rationales interest TGF-β
Thrombin
Wnt/β-catenin
Biological effects
Lung-epithelium-specific deletion of TGF-β receptor II (TβRII)
Deletion of TβRII regulates the phosphorylation of Smad 2 and Smad 3, and diminishes bleomycininduced lung fibrosis Direct thrombin inhibition: oral administration In in-vitro study, dabigatran etexilate reduces thrombinof thrombin inhibitor dabigatran etexilate induced apoptosis in AECs Oral uptake of dabigatran reduces bleomycin-induced fibrosis in mouse model Blockage of downstream target-gene product: orotracheal WISP1 inhibition attenuates bleomycin-induced fibrosis instillation of anti-WISP1 antibody and SiRNA to silence and regulates AEC type 2 cell proliferation WISP1, a downstream effector protein of this pathway
Non-HLA Genes Approximately 30 genes and gene regions have been identified as scleroderma-susceptible loci, most of which are related to immune function. Some of them may perform their function via their gene product, and others by polymorphisms [64•]. Up-regulation of CTGF/CCN2 gene expression was observed in primary fibroblast cells cultured from human SSc skin. A polymorphism in the promoter region of the CTGF (rs698698) gene has been detected in SSc patients in the UK and in Japan. Another SNP in the CTGF gene (rs9399005), related to the structure of CTGF mRNA and present in both systemic and localized (morphea) scleroderma skin, is believed to affect CTGF regulation. Serotonin is another potentially important mediator contributing to the vasoconstriction and fibrosis seen in SSc. A polymorphism was detected in the exonic region (C+1354T) of the serotonin-receptor gene (5-HT2A) [64•]. CD226, a member of the immunoglobulin superfamily, has a function in activation of T cells and natural killer cells. Three CD226 SNPs (rs763361, rs3479968, and rs727088) have been detected and suggested to affect predisposition to SSc-ILD [33••]. IRF5 and MMP-12 polymorphism are also believed to be associated with SSc-ILD [64•]. Other non-HLA genes believed to be involved in SSc include CSK; this codes for C-Src tyrosine kinase, which is known to be involved in myofibroblast differentiation and fibrosis. IRAK1 codes for interleukin (IL)-1-receptor-associated kinase-1. This gene product regulates different functions, in association with the NF-КB, and affects T-cell receptor signaling via toll-likereceptor activation and induction of interferon α and γ [64•]. The homozygous genotype of this gene is frequently observed in cases of diffuse cutaneous SSc [71]. STAT4 is a transcription factor for intercellular signal transduction, is activated by cytokines (TGF-β) and growth factors, and works in association with the TBX21 gene. TBX21 codes for a transcription factor that regulates the balance between T helper1 and T helper2 cells and alters cytokine balance. As a result, immune dysregulation, with over expression of IL-6, IL-2, IL-4, Il-5, Il-13, and TNF-α, has been observed in TBX21rs 11650354-
Ref.
[4]
[47] [48] [49]
CC genotypic SSc patients. Other genes associated with SSc are the B-cell scaffold protein with ankyrin repeats 1, tyrosine protein kinase or B lymphocyte kinase, T-cell surface glycoprotein CD3 zeta chain or T cell receptor zeta chain, tumor necrosis factor α-induced protein3, FAS, interleukin-23 receptor, allograft inflammatory factor-1, FCG receptor, and tumor necrosis factor ligand superfamily member 4 [71].
Epigenetics DNA methyltransferase expression is significantly higher in fibroblasts, leading to methylation in the CpG of nitric oxide synthase. This enzyme has an important function in platelet aggregation and leukocyte–endothelial cell adhesion [69]. CpG island methylation is also detected in the collagen transcription suppression factor Fli1, which is down regulated in SSc, and in turn may lead to high expression of collagen in SSc skin, lung, and other organs [72]. Other important transcription regulators are microRNA (miRNA), small noncoding RNA that have a function in post-transcriptional modification. In the human genome 924 miRNA sequences have been detected, and 24 miRNA have been detected in SSc skin samples, nine being up regulated and 15 being down regulated [69]. It has been revealed that approximately 10 % of miRNA is significantly altered in IPF patients. The up-regulated miRNA belong to the family of mir-155 and mir-21, and the down-regulated miRNA belong to the family of let-7, mir-29, mir-30, and miR-17–92 clusters. Mir-21 regulates the expression of inhibitory Smad 7 in IPF [11, 73, 74].
Conclusions The pathogenesis of SSc-ILD involves complex mechanisms that are not completely understood. The combined involvement of heterogeneous cell populations, with multiple signaling cascades, in association with immunological dysfunction, makes pathogenesis complex. Figure 1 illustrates that in the
Curr Rheumatol Rep (2014) 16:411
developmental stage of lung fibrosis, structural cell injury occurs; this induces innate immune responses and activation of TGF-β and thrombin. Part of the innate response is the recruitment of different immune cells: both lymphocytes and macrophages. Genetic and epigenetic factors combine to bring about lung injury. TGF-β and thrombin have versatile effects on pathogenesis of lung fibrosis by activating the fibroblasts, inducing apoptosis resistance, and regulating the expression of pro-fibrotic proteins and cytokines. Wnt/β-catenin pathways are activated in both AEC and fibroblast cells as a part of the normal wound-healing process, but persist in the fibroblastic foci at later stages of disease. The combination of all these events culminates in the persistent fibrosis characteristic of SSc-ILD. In this article we emphasized the involvement of structural cells, including AEC and fibroblasts, in SSc-ILD pathogenesis. We also reviewed the effect of individual profibrotic signaling cascades, including TGF-β, thrombin, and the Wnt/β-catenin pathway, which are summarized in Table 1, and potential therapeutic targets depending on these pathways, shown in Table 2. Additionally, we discussed the association of pathogenesis with genetic and epigenetic factors. Still, there are other mechanisms involved in pathogenesis that remain to be determined. SSc-ILD is a disease without effective treatment and accounts for significant mortality among SSc patients, and a better understanding of pathogenesis is critical for the development of novel and effective therapy.
Page 7 of 9, 411 4.
5.
6.•
7.
8.
9.
10.
11. 12. 13.
14.
15. 16.
Acknowledgment This work was in part supported by P60AR062755. Compliance with Ethics Guidelines
17.
Conflict of Interest Tanjina Akter declares that she has no conflict of interest. Richard M. Silver has received grant support from the NIAMS/ NIH and the Scleroderma Foundation. Galina S. Bogatkevich has received grant support from the NIH.
18.
Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.
19.
20. 21.
References 22.
Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance
23.
24. 1.
2. 3.
Marut W, Kavian N, Servettaz A, et al. Amelioration of systemic fibrosis in mice by angiotensin II receptor blockade. Arthritis Rheum. 2013;65:1367–77. Jimenez SA, Hitraya E, Varga J. Pathogenesis of scleroderma. Collagen. Rheum Dis Clin N Am. 1996;22:647–74. Silver RM, Wells AU. Histopathology and bronchoalveolar lavage. Rheumatology. 2008;47:62–4.
25.
26.
Li M, Krishnaveni MS, Li C, et al. Epithelium-specific deletion of TGF-β receptor type II protects mice from bleomycin-induced pulmonary fibrosis. J Clin Invest. 2014;121(1):277–87. Homer RJ, Herzog EL. Recent advances in pulmonary fibrosis: implications for scleroderma. Curr Opin Rheumatol. 2010;22: 683–9. Wei J, Bhattacharyya S, Tourtellotte WG, Varga J. Fibrosis in systemic sclerosis: emerging concepts and implications for targeted therapy. Autoimmun Rev. 2011;10:267–75. This summarizes the molecular and cellular pathways leading to SSc-fibrosis. Gilbane AJ, Denton CP, Holmes AM. Scleroderma pathogenesis: a pivotal role for fibroblasts as effector cells. Arthritis Res Ther. 2013;15:215. Willis BC, Borok Z. TGF-b-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol. 2007;293:525–34. Hamaguchi Y. Autoantibody profiles in systemic sclerosis: predictive value for clinical evaluation and prognosis. J Dermatol. 2010;37:42–53. Carmona FD, Cénit MC, Diaz-Gallo LM, Broen JC, et al. New insight on the Xq28 association with systemic sclerosis. Ann Rheum Dis. 2013;72:2032–8. Pandit KV, Milosevic J, Kaminski N. MicroRNAs in idiopathic pulmonary fibrosis. Transl Res. 2011;157:191–9. Meneghin A, Hogaboam CM. Infectious disease, the innate immune response, and fibrosis. J Clin Invest. 2007;117:530–8. Gabrielli A, Svegliati S, Moroncini G, et al. Oxidative stress and the pathogenesis of scleroderma: the Murrell’s hypothesis revisited. Semin Immunopathol. 2008;30:329–37. Gabrielli A, Svegliati S, Moroncini G, Amico D. New insights into the role of oxidative stress in scleroderma fibrosis. Open Rheumatol J. 2012;6:87–95. Chambers RC. The role of coagulation cascade proteases in lung repair and fibrosis. Eur Respir J. 2003;22:33–5. Hant FN, Ludwicka-Bradley A, Wang HJ, et al. Surfactant protein D and KL-6 as serum biomarkers of interstitial lung disease in patients with scleroderma. J Rheumatol. 2009;36:773–80. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009;119:1420–8. Rocka JR, Barkauskasb CE, Cronceb MJ, et al. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proc Natl Acad Sci U S A. 2011;108:1475–83. Kim KK, Kugler MC, Wolters PJ, et al. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A. 2006;103:13180–5. Hashimoto N, Jin H, Liu T, et al. Bone marrow–derived progenitor cells in pulmonary fibrosis. J Clin Invest. 2004;113:243–52. Noble PW. Epithelial fibroblast triggering and interactions in pulmonary fibrosis. Eur Respir Rev. 2008;17:123–9. Horowitz JC, Rogers DS, Sharma V, et al. Combinatorial activation of FAK and AKT by transforming growth factor-β1 confers an anoikis-resistant phenotype to myofibroblasts. Cell Signal. 2007;19:761–71. Zhang H-Y, Phan SH. Inhibition of myofibroblasts apoptosis by transforming growth factor β1. Am J Respir Cell Mol Biol. 1999;21:658–65. Moodley YP, Misso NL, Scaffidi AK, et al. Inverse effects of interleukin-6 on apoptosis of fibroblasts from pulmonary fibrosis and normal lungs. Am J Respir Cell Mol Biol. 2003;29:490–8. Ajayi IO, Sisson TH, Higgins PD, et al. X-linked inhibitor of apoptosis regulates lung fibroblast resistance to Fas-mediated apoptosis. Am J Respir Cell Mol Biol. 2013;49:86–95. Huang SK, Scruggs AM, Donaghy J, et al. Histone modifications are responsible for decreased Fas expression and apoptosis
411, Page 8 of 9
27.
28.
29.
30.
31. 32.
33.••
34.
35. 36.
37.
38.
39.
40.
41.
42.
43.
resistance in fibrotic lung fibroblasts. Cell Death Dis. 2013;4:621. doi:10.1038/cddis.2013.146. Hampel B, Malisan F, Niederegger H, et al. Differential regulation of apoptotic cell death in senescent human cells. Exp Gerontol. 2004;39:1713–21. Kitani A, Fuss I, Nakamura K, et al. Transforming growth factor (TGF)-β1–producing regulatory T cells induce Smad-mediated interleukin 10 secretion that facilitates coordinated immunoregulatory activity and amelioration of TGF-β1–mediated fibrosis. J Exp Med. 2003;198:1179–88. Sime PJ, O’Reilly KMA. Fibrosis of the lung and other tissues: new concepts in pathogenesis and treatment. Clin Immunol. 2001;99: 308–19. Khalil N, Bereznay O, Sporn M, Greenberg AH. Macrophage production of transforming growth factor beta and fibroblast collagen synthesis in chronic pulmonary inflammation. J Exp Med. 1989;70:727–37. Letterio JJ, Roberts AB. Regulation of immune responses by TGFβ. Annu Rev Immunol. 1998;16:137–61. Deng X, Mercer PF, Scotton CJ, Gilchrist A, Chambers RC. Thrombin induces fibroblast CCL2/JE production and release via coupling of PAR1 to Galphaq and cooperation between ERK1/2 and Rho kinase signaling pathways. Mol Biol Cell. 2008;19:2520–33. Bogatkevich GS, Highland KB, Akter T, et al. Inhibition of thrombin as a novel strategy in the treatment of scleroderma-associated interstitial lung disease. Edited by Radstake T. InTech. Croatia 2012:131–48. This paper provides evidence for a promising therapeutic strategy: use of thrombin inhibitor in the treatment of murine lung fibrosis. Ohba T, McDonald JK, Silver RM, et al. Scleroderma bronchoalveolar lavage fluid contains thrombin, a mediator of human lung fibroblast proliferation via induction of platelet-derived growth factor alpha-receptor. Am J Respir Cell Mol Biol. 1994;10:405–12. Hernández-Rodríguez NA, Cambrey AD, Harrison NK, et al. Role of thrombin in pulmonary fibrosis. Lancet. 1995;346:1071–3. Howell DCJ, Johns RH, Lasky JA, et al. Absence of proteaseactivated receptor-1 signaling affords protection from bleomycininduced lung inflammation and fibrosis. Am J Pathol. 2005;166: 1353–65. Bogatkevich GS, Gustilo E, Oates J, et al. Distinct PKC isoforms mediate cell survival and DNA synthesis in thrombin-induced myofibroblasts. Am J Physiol Lung Cell Mol Physiol. 2005;288: 190–201. Bogatkevich GS, Tourkina E, Silver RM, Ludwicka-Bradley A. Thrombin differentiates normal lung fibroblast to a myofibroblast phenotype via proteolytically activated receptor-1 and protein kinase C-dependent pathway. J Biol Chem. 2001;276:45184–92. Gray AJ, Bishop JE, Reeves JT, Laurent GJ. A alpha and B beta chains of fibrinogen stimulate proliferation of human fibroblasts. J Cell Sci. 1993;104:409–13. Bachhuber BG, Sarembock IJ, Gimple LW, Owens GK. alphaThrombin induces transforming growth factor-beta1 mRNA and protein in cultured vascular smooth muscle cells via a proteolytically activated receptor. J Vasc Res. 1997;34:41–8. Chambers RC, Leoni P, Blanc-Brude OP, Wembridge DE, Laurent GJ. Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease-activated receptor1. J Biol Chem. 2000;275:35584–91. Bogatkevich GS, Ludwicka-Bradley A, Nietert PJ, Silver RM. Scleroderma lung fibroblasts: contractility and connective tissue growth factor. Edited by Gabbiani G, Chaponnier C, Desmouliere A. Texas: Landes. Bioscience 2006:25–31. Tourkina E, Hoffman S, Fenton JW, Lipsitz S, et al. Depletion of protein kinase Cε in normal and scleroderma lung fibroblasts has opposite effects on tenascin expression. Arthritis Rheum. 2001;44: 1370–81.
Curr Rheumatol Rep (2014) 16:411 44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58. 59.
60.••
61.
62.
Chambers RC, Dabbagh K, McAnulty RJ, et al. Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1. Biochem J. 1998;333:121–7. Armstrong MT, Fenton 2nd JW, Andersen TT, Armstrong PB. Thrombin stimulation of matrix fibronectin. J Cell Physiol. 1996;166:112–20. Howell DC, Laurent GJ, Chambers RC. Role of thrombin and its major cellular receptor, protease-activated receptor-1, in pulmonary fibrosis. Biochem Soc Trans. 2002;30:211–6. Chambers RC. Procoagulant signalling mechanisms in lung inflammation and fibrosis: novel opportunities for pharmacological intervention? Br J Pharmacol. 2008;153:367–78. Bogatkevich GS, Ludwicka-Bradley A, Silver RM. Dabigatran, a direct thrombin inhibitor, blocks differentiation of normal fibroblasts to a myofibroblast phenotype and demonstrates anti-fibrotic effects on scleroderma lung fibroblasts. Arthritis Rheum. 2009;60: 3455–64. Bogatkevich GS, Ludwicka-Bradley A, Nietert PJ, et al. Antiinflammatory and antifibrotic effects of the oral direct thrombin inhibitor dabigatran etexilate in a murine model of interstitial lung disease. Arthritis Rheum. 2011;63:1416–25. Königshoff M, Kramer M, Balsara N, Wilhelm J, et al. WNT1inducible signaling protein-1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis. J Clin Invest. 2009;119:772–87. Chilosi M, Poletti V, Zamò A, et al. Aberrant Wnt/beta-catenin pathway activation in idiopathic pulmonary fibrosis. Am J Pathol. 2003;162:1495–502. Gordon MD, Nusse R. Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem. 2006;281:22429–33. Shi-Wen X, Chen Y, Denton CP, et al. Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/ phosphoinositide 3-kinase/Akt-dependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts. Mol Biol Cell. 2004;15:2707–19. Moinzadeh P, Krieg T, Hellmich M, et al. Elevated MMP-7 levels in patients with systemic sclerosis: correlation with pulmonary involvement. Exp Dermatol. 2011;20:770–3. Wei J, Melichian D, Komura K, et al. Canonical Wnt signaling induces skin fibrosis and subcutaneous lipoatrophy, a novel mouse model for scleroderma. Arthritis Rheum. 2011;63: 1707–17. Schneider AJ, Vezina CM, Richard E, et al. TCDD inhibition of canonical Wnt signaling disrupts prostatic bud formation in mouse urogenital sinus. Toxicol Sci. 2013;133:42–53. Arnett FC, Cho M, Chatterjee S, et al. Familial occurrence frequencies and relative risks for systemic sclerosis (scleroderma) in three United States cohorts. Arthritis Rheum. 2001;44:1359–62. Haustein U-F. Systemic sclerosis - an update. Lab Med. 2011;42: 562–72. Laing TJ, Gillespie BW, Toth MB, et al. Racial differences in scleroderma among women in Michigan. Arthritis Rheum. 1997;40:734–42. Silver RM, Bogatkevich G, Tourkina E, et al. Racial differences between blacks and whites with systemic sclerosis. Curr Opin Rheumatol. 2012;24:642–8. This article provides evidence of correlation between race-dependent predisposition to fibrosis and biological variable factors. Bogatkevich GS, Ludwicka-Bradley A, Highland KB, et al. Impairment of the antifibrotic effect of hepatocyte growth factor in lung fibroblasts from African Americans: possible role in systemic sclerosis. Arthritis Rheum. 2007;56:2432–42. Gelber AC, Manno RL, Shah AA, et al. Race and association with disease manifestations and mortality in scleroderma:
Curr Rheumatol Rep (2014) 16:411
63.
64.•
65.
66. 67.
a 20-year experience at the Johns Hopkins Scleroderma Center and review of the literature. Medicine (Baltimore). 2013;92: 191–205. Steen V, Domsic RT, Lucas M, et al. A clinical and serologic comparison of African American and Caucasian patients with systemic sclerosis. Arthritis Rheum. 2012;64: 2986–94. Bogatkevich GS, Highland KB, Akter T, Silver RM. The PPARγ agonist rosiglitazone is antifibrotic for scleroderma lung fibroblasts: mechanisms of action and differential racial effects. Pulm Med. 2012;2012:545172. doi:10.1155/2012/545172. This article provides functional evidence of race-based transcriptional factor over-activation and promising treatment for SSc-ILD on that basis. Vargas-Alarcón G, Granados J, Ibañez de Kasep G, et al. Association of HLA-DR5 (DR11) with systemic sclerosis (scleroderma) in Mexican patients. Clin Exp Rheumatol. 1995;13:11–6. Arnett FC. HLA and autoimmunity in scleroderma (systemic sclerosis). Int Rev Immunol. 1995;12:107–28. Reveille JD. Molecular genetics of systemic sclerosis. Curr Opin Rheumatol. 1993;5:753–9.
Page 9 of 9, 411 68.
Takeuchi F, Kuwata S, Nakano K, et al. Association of TAP1 and TAP2 with systemic sclerosis in Japanese. Clin Exp Rheumatol. 1996;14:513–21. 69. Luo Y, Wang Y, Wang Q, Xiao R, Lu Q. Systemic sclerosis: genetics and epigenetics. J Autoimmun. 2013;41:161–7. 70. Morel PA, Chang HJ, Wilson JW, et al. Severe systemic sclerosis with anti-topoisomerase I antibodies is associated with an HLADRw11 allele. Hum Immunol. 1994;40:101–10. 71. Romano E, Manetti M, Guiducci S, et al. The genetics of systemic sclerosis: an update. Clin Exp Rheumatol. 2011;29:75–86. 72. Wang Y, Fan PS, Kahaleh B. Association between enhanced type I collagen expression and epigenetic repression of the FLI1 gene in scleroderma fibroblasts. Arthritis Rheum. 2006;54:2271–9. 73. Makino K, Jinnin M, Hirano A, et al. The downregulation of microRNA let-7a contributes to the excessive expression of type I collagen in systemic and localized scleroderma. Immunology. 2013;190:3905–15. 74. Liu G, Friggeri A, Yang Y, et al. miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis. J Exp Med. 2010;207:1589–97.
SCLERODERMA (J VARGA, SECTION EDITOR)
Recent Advances in Understanding the Pathogenesis of Scleroderma-Interstitial Lung Disease Tanjina Akter & Richard M. Silver & Galina S. Bogatkevich
Published online: 13 February 2014 # Springer Science+Business Media New York 2014
Abstract Systemic sclerosis (scleroderma, SSc) is a heterogeneous autoimmune connective tissue disease of unknown etiology. Interstitial lung disease (ILD) is a frequent complication, and a significant contributor to morbidity and mortality among SSc patients. SSc-ILD most commonly occurs within 10 years of diagnosis, and may be seen in patients with either the limited or diffuse cutaneous subset of SSc. SSc-ILD is a multifaceted disease process in which different factors and pathways are involved. Aberrant function of a variety of lung cells, cytokines, growth factors, peptides, and bioactive proteins, in combination with genetic and epigenetic regulators, have crucial functions in the pathogenesis of this disease. Here we present our view on recent advances regarding the pathogenesis of SSc-ILD. Keywords Systemic sclerosis (SSc) . Scleroderma . Pathogenesis . Interstitial lung disease (ILD) . Fibroblast, TGF-β . Thrombin . Wnt/β-catenin pathway . Genetics . Epigenetics
This article is part of the Topical Collection on Scleroderma T. Akter (*) : G. S. Bogatkevich Division of Rheumatology and Immunology, Department of Medicine, Medical University of South Carolina, 114 Doughty Street, Charleston, SC 29425, USA e-mail: [email protected] G. S. Bogatkevich e-mail: [email protected] R. M. Silver Division of Rheumatology and Immunology, Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, Charleston, SC 29425, USA e-mail: [email protected]
Introduction Systemic sclerosis (SSc) is a connective tissue disease characterized by vascular alterations, limited or widespread dermal and visceral organ fibrosis, and immunological dysregulation associated with specific autoantibodies [1]. Persistent overproduction of collagen and deposition of connective tissue is the main characteristic of this disease [2], and activation of collagen-producing cells is greatly increased in SSc patients [3, 4]. Systemic sclerosis often affects multiple systems, including the skin and visceral organs of the body. Pulmonary involvement is seen in 70 % to 90 % of cases and is now the leading cause of mortality. Approximately 40 % of SSc-ILD patients will die within 10 years of diagnosis [4, 5], so a better understanding of the pathogenesis of this complication is of paramount importance. The most common initiator of the pathogenesis of SSc-ILD is a persistent injury to lung cells, for example alveolar epithelial and endothelial cells, in which the presence of pro-fibrotic stimuli induces the differentiation of lung fibroblasts to a myofibroblast phenotype. Increased release of profibrotic cytokines, growth factors, peptides, and bioactive proteins results in stronger signaling, leading to the additional recruitment of myofibroblasts, accumulation of extracellular matrix, and scarring [6•, 7]. The principal consequence of this pathobiology is apoptosis of the alveolar epithelial cells, and accumulation and activation of fibroblasts from different origins [6•, 8]. Systemic sclerosis is characterized by alterations to both humoral and cellular immunity, and a variety of diseasespecific autoantibodies against self-antigens have been described in SSc patients. Autoimmune diseases, including SSc, are believed to be complex polygenic conditions, with predisposition and progression determined by multiple
411, Page 2 of 9
susceptibility genes interacting with epigenetic and environmental factors [9, 10]. Extensive supporting evidence for the involvement of genetics has recently been found, and several genes involved in immune regulation have been proposed as risk factors for the development of SSc. Involvement of several loci and microRNA has already been postulated for pulmonary fibrosis [11]. Alternatively, another line of research has provided evidence that pulmonary fibrosis in SSc is a result of an innate immune response driven by persistent microbial infection [12]. Excessive oxidative stress in SSc [13], and specifically in lung fibrosis [14], may be another important factor in pathogenesis. The objective of this review is to discuss the genetic susceptibility, cellular damage, and aberrant signaling pathways involved in the pathogenesis of SSc-ILD.
Cells Involved in the Pathogenesis of SSc-ILD Recurrent alveolar epithelial cell (AEC) injury, and an aberrant healing process accompanied by fibroproliferation with myofibroblast persistence, impaired hyperplasia, and increased retardation and/or apoptosis of AEC, is central to the pathogenesis of ILD [15]. The pathophysiological alteration of injured alveolar epithelial cell is reflected in the high-level secretion of glycoproteins, for example surfactant protein D (SP-D) and KL6, in the blood of SSc-ILD patients [16]. One of the most profound changes in epithelial cell morphology and biology is epithelial mesenchymal transition (EMT), which has been observed in scleroderma [8]. EMT is a biochemical process in which the epithelial cell loses its polarity and develops mesenchymal properties. These cells develop enhanced migratory capacity, invasiveness, increased resistance to apoptosis, and greatly increased production of extracellular matrix (ECM) components [17]. EMT has been observed both in SSc-ILD patients and in the bleomycin-induced mouse model of ILD [5]. The concept of transformation of epithelial cells to mesenchymal cells has been strongly criticized and challenged by other researchers, who observed that neither pericytes nor type-1 or type-2 AEC follow EMT pathways, but that instead a heterogeneous cell population is involved in this process [18]. More studies are required, both on animal models and on human fibrotic disease, for better understanding of this process. Little is known about the crucial mediators that determine whether AEC will undergo EMT or apoptosis during fibrosis. Human AEC in culture have a matrix-dependent preference, with AEC-II cells in the presence of fibronectin and/or fibrin in culture medium following EMT pathways. In the presence of transforming growth factor-β (TGF-β), these cells lose the epithelial cell marker pro-surfactant protein C (pro-SPC), lose apical–basal polarity, and express mesenchymal cadherin (Ncadherin). In contrast, AEC in the presence of laminin and/or
Curr Rheumatol Rep (2014) 16:411
collagen in cell culture medium undergo apoptosis rather than EMT [19]. The other major cell type involved in the pathogenesis of lung fibrosis is the fibroblast. Fibroblasts have heterogeneous phenotypic features, a vigorous replication pattern, and a central function in ECM formation [15, 20]. Activated lung fibroblasts can originate from resident lung fibroblasts, from circulating bone-marrow-derived fibrocytes, and from the differentiation of other cell types including epithelial cells, endothelial cells, and pericytes [4, 20]. Recruitment and activation of lung fibroblasts are induced by persistent lung injury, and an unsuccessful repair process summons more cells to the site of injury [21]. In cellular homeostatic conditions these recruited cells are cleared up by apoptosis. But in the case of lung fibrosis, fibroblasts do not undergo programmed cell death. This avoidance of apoptosis by fibroblasts is enabled by several proteins. One of these proteins, focal adhesion kinase (FAK), provides viability to fibroblasts through the PI3K-Akt-mediated pathway, thus helping them avoid apoptosis [22]. Fibroblasts are capable of different responses depending on the type of stimuli they receive, which may also facilitate their survival. For example, in the presence of IL-1β these cells express the pro-apoptotic protein calcium-insensitive nitric oxide synthase (iNOS), which produces nitric oxide and leads to cell death. However, in the presence of TGF-β the expression of iNOS is reduced or completely inhibited [23]. Additionally, fibroblasts isolated from SSc-ILD patients respond differently from normal lung fibroblasts. Thus, after stimulation with IL-6, healthy fibroblasts express Bcl-2-associated X-protein (BAX), a pro-apoptotic protein that leads to Fas-induced cell death, whereas scleroderma lung fibroblasts express B-cell lymphoma-2 (Bcl-2), which is an anti-apoptotic protein that confers apoptosis resistance [24]. Another important cause of the apoptosis resistance of lung fibroblasts is X-linked inhibitor of apoptosis protein (XIAP), which regulates Fas-mediated apoptosis. XIAP is over-expressed in fibroblastic foci in IPF, and inhibition or silencing of XIAP causes a loss of resistance to apoptosis in IPF fibroblasts [25]. Expression of Fas is also regulated by histone modification on the Fas promoter region. Histone deacetylation and histone 3 lysine 9 trimethylation are significantly higher both in human lung fibrosis and in the bleomycin mouse model, with over-expression of histone deacetylase (HDAC)-2 and HDAC-4. These down-regulate expression of Fas, and inhibit Fas-mediated apoptosis. In the presence of an HDAC inhibitor, Fas expression increases and cells recover susceptibility to Fas-mediated apoptosis [26]. Fibroblasts can also escape apoptosis by controlling the cell cycle. Human diploid fibroblasts enter a stable growth arrest near the end of their life; in this stage they accumulate pro-apoptotic ceramide but become resistant to ceramide-induced apoptosis [27].
Curr Rheumatol Rep (2014) 16:411
TGF-β Pathway The most widely studied mediator of SSc-ILD is TGF-β, which is released by injured lung cells. There are several proposed mechanisms, both interrelated and independent, underlying the pathogenesis of SSc-ILD. TGF-β leads to inhibition of AEC proliferation, expression of metalloproteinase-7 (MMP-7), induction of myofibroblasts, and accumulation of ECM proteins [4, 6•, 8, 28, 29]. In SSc-ILD, TGF-β has multiple functions in cell signaling, binding with its cellsurface heterodimeric receptor to activate canonical or noncanonical pathways. In the canonical pathway TGF-β causes phosphorylation of Smad2/3, which subsequently binds with Smad4. Smad4 acts as a transcription activator, leading to the expression of extracellular matrix proteins, e.g., collagen, plasminogen activator inhibitor-1, and connective-tissue growth factor (CTGF, CCN2) [8]. TGF-β-induced cell signaling initiated via a non-canonical pathway is mediated by specific regulatory proteins, including MAPK, PAR6 and RhoA, which subsequently affect cytoskeleton rearrangement, cell adhesion, and cell motility in pulmonary fibrosis [8]. Because TGF-β has a central function in development and progression of lung fibrosis, blocking of TGF-β could be a potential therapeutic strategy. Conditional knockout of TGF-β receptor type II (TβRII) attenuated lung fibrosis via regulating the phosphorylation of Smad-2 and Smad-3 in AECs; such knockout mice are resistant to bleomycin-induced lung fibrosis and have an increased survival rate [4]. As well as regulating the gene expression or the cell signaling, TGF-β also has a crucial function in regulation of immune response. Injured tissue releases TGF-β, which signals for the recruitment of immune cells, including macrophages, to the site of injury, which in turn release more TGF-β, exacerbating the fibrosis [30]. TGF-β also has a major function in the development, maturation, and proliferation of lymphocytes, mainly T-cells and B-cells, and in secretion of cytokines. It suppresses the expression of IL-2 and inhibits IL-2-dependent lymphocyte proliferation [31]. Additionally, TGF-β induces the pro-inflammatory cytokine IL-10, disrupting the balance between helper and regulatory T cells, and suppresses anti-fibrotic cytokine interferon-γ (INF-γ) [28, 29].
Thrombin Pathway Thrombin is a key mediator of the coagulation cascade and is a serine protease with pluripotent cellular effects, including induction of cytokines and regulation of cell-type-specific apoptosis in lung fibrosis [32, 33••]. Our laboratory and others have revealed greatly increased levels of thrombin in bronchoalveolar lavage fluid (BALF) from scleroderma patients with lung fibrosis and other fibrosing lung disease [34, 35]. It
Page 3 of 9, 411
has been revealed that BALF from normal subjects has a low level of thrombin activity, whereas BALF from SSc patients has up to 100-fold higher thrombin activity [34]. Elevated levels of thrombin activity have also been observed in bleomycin-induced pulmonary fibrosis in mice [36]. Thrombin is mitogenic for lung fibroblasts [34, 37, 38] and enhances the proliferative effect of fibrinogen on fibroblasts [39]. Thrombin is also a potent inducer of fibrogenic cytokines, chemokines, and growth factors, for example TGF-β [40], CTGF [41, 42], platelet-derived growth factor-AA (PDGF-AA) [34], chemokine ligand 2 (CCL2) [32], and ECM proteins, for example collagen, fibronectin, and tenascin, in a variety of cells, including lung fibroblasts [43–45]. Most cellular responses to thrombin are mediated via the G-protein-coupled receptor PAR-1, which is significantly increased in patients with SSc-ILD, notably in lung parenchyma associated with inflammatory and fibroproliferative foci [37, 46]. Elevated expression of PAR-1 has been also observed in patients with IPF and in a murine model of bleomycininduced lung fibrosis [36, 47]. Studies from our laboratory revealed that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype that is resistant to apoptosis, although thrombin also is capable of inducing apoptosis of AEC via a PAR-1-receptor-dependent pathway [33••, 37, 38]. Inhibition of thrombin by a direct inhibitor, dabigatran, has substantial antifibrotic effects both in vitro and in vivo [48, 49]. Dabigatran inhibits thrombin-induced differentiation of normal lung fibroblasts to the myofibroblast phenotype in vitro, and it decreases CTGF, α-SMA, and collagen type I in scleroderma lung fibroblasts [48]. Moreover, dabigatran significantly reduces thrombin-induced apoptosis of AEC [48]. Oral administration of dabigatran etexilate attenuates the development of bleomycin-induced pulmonary fibrosis in mice. Additionally, treatment with dabigatran etexilate reduces collagen, CTGF, and α-SMA expression in mice with bleomycin-induced lung fibrosis, whereas it has no effect on basal levels of these proteins [49].
Wnt/β-catenin Pathway SSc is characterized by widespread vascular injury and ischemia. In SSc-ILD, vascular injury leads to apoptosis of AEC type-1 cells and activation and proliferation of AEC type-2 (AT2) cells to repair the damaged lung, with concurrent activation of other pathways and fibroblast accumulation culminating in fibrosis [5]. AT2 cells may also be involved in fibrosis in other ways. The phenotype and gene-expression profile of AT2 cells from fibrotic lung tissue indicate an increased proliferation capacity and enhanced expression of proliferation-related genes [50]. Activation of the Wnt/βcatenin pathway has been observed in the fibroblastic foci of
411, Page 4 of 9
IPF patients, but not of patients with normal and/or pneumonia-related pulmonary disease [51]. The Wnt pathway consists of a large number of highly conserved growth factors, whose receptor-mediated activity leads to the accumulation of cytoplasmic β-catenin; this then translocates to the nucleus to regulate target gene expression in association with Akt and/or through T-cell-specific transcription factor and/or lymphoid-enhancer-binding factor1 family transcription factors [8, 52]. This pathway may also have a function in epithelial cell regeneration at bronchiolar–alveolar junctions and in epithelial–mesenchymal transitions, leading to the irreversible remodeling of pulmonary tissue [51]. High levels of nuclear β-catenin are observed in IPF fibroblastic foci, and activated Akt has been observed in fibroblasts from SSc-ILD patients [51, 53]. Further analysis reveals that expression of WNT1-inducible signaling protein-1 (WISP1), a product of the WNT target gene, is highly upregulated in the AT2 cells in hyperplastic areas of fibrotic lung. WISP1 is a member of the CCN family, consisting of four conserved cysteine-rich modular domains, acting through binding of integrin receptor, and affecting cellular functions including mitosis, adhesion, and migration. WISP1 works in an autocrine fashion to induce the release of cytokines, including SPP1, MMP-7, MMP-9, and PAI1, from alveolar epithelial cells [5, 50]. MMP-7 levels are elevated in SSc-ILD and are over-expressed in AT2 cells in other types of pulmonary fibrosis [50, 54]. Treatment with WISP1 in AT2 primary cell culture leads to an increase in cell proliferation and EMT, with profibrotic marker-gene expression. Co-localization of WISP1 with pro-SPC has been detected in both the bleomycin mouse model and in IPF [50]. Evidence for catenin-based Wnt signaling has been detected in fibrotic skin in SSc, and in a mouse model Wnt is related to collagen gene expression [55]. Wnt signaling is a very common mechanism in cancer, e.g., breast cancer, melanoma, prostate cancer, lung cancer, and several other cancer types, and has been regarded as a potential therapeutic target. Blocking Wnt signaling inhibits prostatic bud formation in prostate cancer [56]. In the bleomycin mouse model it has been revealed that inhibition of WISP1, by antiWISP1 antibody or siRNA, leads to a decrease in AT2-cell proliferation. Repetitive orotracheal instillation of antiWISP1 antibody in the bleomycin mouse model is associated with a significant decrease in collagen deposition, and with improved lung function and survival [50].
Genetic Associations Genetic studies have revealed that the incidence of SSc is 1.5–1.7 % in families with a history of scleroderma, compared with 0.026 % in the general population. SSc is not inherited in a Mendelian fashion, but a positive family
Curr Rheumatol Rep (2014) 16:411
history significantly increases the relative risk: by 15 to 19-fold in siblings and by 13 to 15-fold in first-degree relatives [57]. Although females are more susceptible to SSc [58], the severity of SSc-ILD in male patients is greater than in females [3]. Genetics studies suggest that SSc-ILD is correlated with the X-chromosome, which might in turn suggest that, because males have a single X-chromosome, any mutation(s) in the genes of this chromosome are not restored in males, leaving them more vulnerable to ILD. Racial differences observed among African-American and Caucasian SSc patients suggest African Americans have a genetic predisposition to SSc. Studies conducted in different locations in the US have consistently observed that African-American SSc patients have higher morbidity and mortality as compared with Caucasians [59, 60••, 61–63]. African Americans with SSc have a higher incidence of the anti-Scl-70 autoantibody [62], and express more TGF-β, IL-6, and other profibrotic factors [60••], as compared with Caucasian SSc patients. In contrast, expression of antifibrotic proteins, including hepatocyte growth factor (HGF) and PPAR-γ, is significantly lower in African-American SSc patients than in Caucasian SSc patients [61, 64•]. Extensive gene-based investigation is required to discover the underlying causes of this racial disparity.
HLA-dependent Genes The main interest in genetic-based studies related to SSc has been focused on genes of the major histocompatibility complex-class II (MHC-II). Demographic studies reveal variation in the expression pattern of MHC genes; for the most part, subtypes of MHC-II, DP, DQ, and DR have been revealed to be associated with SSc [65–67]. These different classes of allele are associated with a variety of diseasespecific autoantibodies, including anti-centromere, antitopoisomerase I, and other antinuclear antibodies [66]. The proposed effect of these genetic correlations has been criticized, because of high variation in different geographic and ethnic-based studies. A study in Mexico revealed that DR5 (DRB1*1104) affects SSc susceptibility [65]. In Japan, another study revealed association of SSc with “Transporter associated with antigen processing 1 and 2” (TAP 1 and 2) alleles [68]. DPB1*1301 and DRB1*15 affect pulmonary fibrosis in South-African and Korean patients. In Caucasians, MHCclass I subtypes (HLA-B*62 and HLA-Cw*602) are predominant in patients with lung fibrosis. Genetic susceptibility associated with HLA-DRB1*15 was observed in a Korean population and in French women with dcSSc. The presence of HLA-DRB1*11/07 haplotypes is associated with pulmonary fibrosis in French women, and in Spanish and Italian patients [69]. Two other alleles are recognized to be associated with
Curr Rheumatol Rep (2014) 16:411
Page 5 of 9, 411
Fig. 1 Cells and pathways involved in pathogenesis of lung fibrosis. Fibrosis is caused by a combination of several factors, including genetics and epigenetics cross-linked with heterogeneous cellular responses. The initiation of fibrosis is caused by injury to the alveolar cells, inducing activation of immune cells and release of thrombin, TGF-β, Wnt/β-catenin, and other fibrogenic proteins. These events stimulate the fibroblasts, recruiting them to the site of injury and resulting in the generation of myofibroblasts leading to tissue fibrosis
SSc. One of them, HLA-DRw11, seems to have a major function in determining the severity of SSc, and another one,
an HLA-DQ gene, is associated with anti-topoisomerase-I antibody development [70].
Table 1 Cells and pathways involved in SSc-ILD Cell involved in fibrosis AECs
Fibroblast
Main pathways TGF-β
Fate Apoptosis or retardation EMT Release of SP-D and KL-6 Apoptosis resistance Signal for cell migration, persistent lung injury, fibrosis Intermediates involved Smad-dependent signaling molecules Smad-independent signaling molecule (MAPK, PAR6, RhoA)
Thrombin
PAR1 activation
Wnt/β-catenin
β-catenin
Ref. [15] [8] [16] [23] [21]
Biological effects Gene expression regulation, ECM formation EMT Cell signaling Lymphocyte development, proliferation, and maturation, and cytokine release Induction of cytokines and ECM protein Increase fibroblast proliferation. Signal for apoptosis in AECs but not in fibroblast Regulate target gene expression Hyperplasia in AEC-II Release of cytokines
Ref. [6•] [8] [30, 31] [28] [32] [33••] [55] [5] [50]
411, Page 6 of 9
Curr Rheumatol Rep (2014) 16:411
Table 2 Pathways targeted therapeutic strategies in lung fibrosis Pathways of therapeutic Therapeutic strategies and rationales interest TGF-β
Thrombin
Wnt/β-catenin
Biological effects
Lung-epithelium-specific deletion of TGF-β receptor II (TβRII)
Deletion of TβRII regulates the phosphorylation of Smad 2 and Smad 3, and diminishes bleomycininduced lung fibrosis Direct thrombin inhibition: oral administration In in-vitro study, dabigatran etexilate reduces thrombinof thrombin inhibitor dabigatran etexilate induced apoptosis in AECs Oral uptake of dabigatran reduces bleomycin-induced fibrosis in mouse model Blockage of downstream target-gene product: orotracheal WISP1 inhibition attenuates bleomycin-induced fibrosis instillation of anti-WISP1 antibody and SiRNA to silence and regulates AEC type 2 cell proliferation WISP1, a downstream effector protein of this pathway
Non-HLA Genes Approximately 30 genes and gene regions have been identified as scleroderma-susceptible loci, most of which are related to immune function. Some of them may perform their function via their gene product, and others by polymorphisms [64•]. Up-regulation of CTGF/CCN2 gene expression was observed in primary fibroblast cells cultured from human SSc skin. A polymorphism in the promoter region of the CTGF (rs698698) gene has been detected in SSc patients in the UK and in Japan. Another SNP in the CTGF gene (rs9399005), related to the structure of CTGF mRNA and present in both systemic and localized (morphea) scleroderma skin, is believed to affect CTGF regulation. Serotonin is another potentially important mediator contributing to the vasoconstriction and fibrosis seen in SSc. A polymorphism was detected in the exonic region (C+1354T) of the serotonin-receptor gene (5-HT2A) [64•]. CD226, a member of the immunoglobulin superfamily, has a function in activation of T cells and natural killer cells. Three CD226 SNPs (rs763361, rs3479968, and rs727088) have been detected and suggested to affect predisposition to SSc-ILD [33••]. IRF5 and MMP-12 polymorphism are also believed to be associated with SSc-ILD [64•]. Other non-HLA genes believed to be involved in SSc include CSK; this codes for C-Src tyrosine kinase, which is known to be involved in myofibroblast differentiation and fibrosis. IRAK1 codes for interleukin (IL)-1-receptor-associated kinase-1. This gene product regulates different functions, in association with the NF-КB, and affects T-cell receptor signaling via toll-likereceptor activation and induction of interferon α and γ [64•]. The homozygous genotype of this gene is frequently observed in cases of diffuse cutaneous SSc [71]. STAT4 is a transcription factor for intercellular signal transduction, is activated by cytokines (TGF-β) and growth factors, and works in association with the TBX21 gene. TBX21 codes for a transcription factor that regulates the balance between T helper1 and T helper2 cells and alters cytokine balance. As a result, immune dysregulation, with over expression of IL-6, IL-2, IL-4, Il-5, Il-13, and TNF-α, has been observed in TBX21rs 11650354-
Ref.
[4]
[47] [48] [49]
CC genotypic SSc patients. Other genes associated with SSc are the B-cell scaffold protein with ankyrin repeats 1, tyrosine protein kinase or B lymphocyte kinase, T-cell surface glycoprotein CD3 zeta chain or T cell receptor zeta chain, tumor necrosis factor α-induced protein3, FAS, interleukin-23 receptor, allograft inflammatory factor-1, FCG receptor, and tumor necrosis factor ligand superfamily member 4 [71].
Epigenetics DNA methyltransferase expression is significantly higher in fibroblasts, leading to methylation in the CpG of nitric oxide synthase. This enzyme has an important function in platelet aggregation and leukocyte–endothelial cell adhesion [69]. CpG island methylation is also detected in the collagen transcription suppression factor Fli1, which is down regulated in SSc, and in turn may lead to high expression of collagen in SSc skin, lung, and other organs [72]. Other important transcription regulators are microRNA (miRNA), small noncoding RNA that have a function in post-transcriptional modification. In the human genome 924 miRNA sequences have been detected, and 24 miRNA have been detected in SSc skin samples, nine being up regulated and 15 being down regulated [69]. It has been revealed that approximately 10 % of miRNA is significantly altered in IPF patients. The up-regulated miRNA belong to the family of mir-155 and mir-21, and the down-regulated miRNA belong to the family of let-7, mir-29, mir-30, and miR-17–92 clusters. Mir-21 regulates the expression of inhibitory Smad 7 in IPF [11, 73, 74].
Conclusions The pathogenesis of SSc-ILD involves complex mechanisms that are not completely understood. The combined involvement of heterogeneous cell populations, with multiple signaling cascades, in association with immunological dysfunction, makes pathogenesis complex. Figure 1 illustrates that in the
Curr Rheumatol Rep (2014) 16:411
developmental stage of lung fibrosis, structural cell injury occurs; this induces innate immune responses and activation of TGF-β and thrombin. Part of the innate response is the recruitment of different immune cells: both lymphocytes and macrophages. Genetic and epigenetic factors combine to bring about lung injury. TGF-β and thrombin have versatile effects on pathogenesis of lung fibrosis by activating the fibroblasts, inducing apoptosis resistance, and regulating the expression of pro-fibrotic proteins and cytokines. Wnt/β-catenin pathways are activated in both AEC and fibroblast cells as a part of the normal wound-healing process, but persist in the fibroblastic foci at later stages of disease. The combination of all these events culminates in the persistent fibrosis characteristic of SSc-ILD. In this article we emphasized the involvement of structural cells, including AEC and fibroblasts, in SSc-ILD pathogenesis. We also reviewed the effect of individual profibrotic signaling cascades, including TGF-β, thrombin, and the Wnt/β-catenin pathway, which are summarized in Table 1, and potential therapeutic targets depending on these pathways, shown in Table 2. Additionally, we discussed the association of pathogenesis with genetic and epigenetic factors. Still, there are other mechanisms involved in pathogenesis that remain to be determined. SSc-ILD is a disease without effective treatment and accounts for significant mortality among SSc patients, and a better understanding of pathogenesis is critical for the development of novel and effective therapy.
Page 7 of 9, 411 4.
5.
6.•
7.
8.
9.
10.
11. 12. 13.
14.
15. 16.
Acknowledgment This work was in part supported by P60AR062755. Compliance with Ethics Guidelines
17.
Conflict of Interest Tanjina Akter declares that she has no conflict of interest. Richard M. Silver has received grant support from the NIAMS/ NIH and the Scleroderma Foundation. Galina S. Bogatkevich has received grant support from the NIH.
18.
Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.
19.
20. 21.
References 22.
Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance
23.
24. 1.
2. 3.
Marut W, Kavian N, Servettaz A, et al. Amelioration of systemic fibrosis in mice by angiotensin II receptor blockade. Arthritis Rheum. 2013;65:1367–77. Jimenez SA, Hitraya E, Varga J. Pathogenesis of scleroderma. Collagen. Rheum Dis Clin N Am. 1996;22:647–74. Silver RM, Wells AU. Histopathology and bronchoalveolar lavage. Rheumatology. 2008;47:62–4.
25.
26.
Li M, Krishnaveni MS, Li C, et al. Epithelium-specific deletion of TGF-β receptor type II protects mice from bleomycin-induced pulmonary fibrosis. J Clin Invest. 2014;121(1):277–87. Homer RJ, Herzog EL. Recent advances in pulmonary fibrosis: implications for scleroderma. Curr Opin Rheumatol. 2010;22: 683–9. Wei J, Bhattacharyya S, Tourtellotte WG, Varga J. Fibrosis in systemic sclerosis: emerging concepts and implications for targeted therapy. Autoimmun Rev. 2011;10:267–75. This summarizes the molecular and cellular pathways leading to SSc-fibrosis. Gilbane AJ, Denton CP, Holmes AM. Scleroderma pathogenesis: a pivotal role for fibroblasts as effector cells. Arthritis Res Ther. 2013;15:215. Willis BC, Borok Z. TGF-b-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol. 2007;293:525–34. Hamaguchi Y. Autoantibody profiles in systemic sclerosis: predictive value for clinical evaluation and prognosis. J Dermatol. 2010;37:42–53. Carmona FD, Cénit MC, Diaz-Gallo LM, Broen JC, et al. New insight on the Xq28 association with systemic sclerosis. Ann Rheum Dis. 2013;72:2032–8. Pandit KV, Milosevic J, Kaminski N. MicroRNAs in idiopathic pulmonary fibrosis. Transl Res. 2011;157:191–9. Meneghin A, Hogaboam CM. Infectious disease, the innate immune response, and fibrosis. J Clin Invest. 2007;117:530–8. Gabrielli A, Svegliati S, Moroncini G, et al. Oxidative stress and the pathogenesis of scleroderma: the Murrell’s hypothesis revisited. Semin Immunopathol. 2008;30:329–37. Gabrielli A, Svegliati S, Moroncini G, Amico D. New insights into the role of oxidative stress in scleroderma fibrosis. Open Rheumatol J. 2012;6:87–95. Chambers RC. The role of coagulation cascade proteases in lung repair and fibrosis. Eur Respir J. 2003;22:33–5. Hant FN, Ludwicka-Bradley A, Wang HJ, et al. Surfactant protein D and KL-6 as serum biomarkers of interstitial lung disease in patients with scleroderma. J Rheumatol. 2009;36:773–80. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009;119:1420–8. Rocka JR, Barkauskasb CE, Cronceb MJ, et al. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proc Natl Acad Sci U S A. 2011;108:1475–83. Kim KK, Kugler MC, Wolters PJ, et al. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A. 2006;103:13180–5. Hashimoto N, Jin H, Liu T, et al. Bone marrow–derived progenitor cells in pulmonary fibrosis. J Clin Invest. 2004;113:243–52. Noble PW. Epithelial fibroblast triggering and interactions in pulmonary fibrosis. Eur Respir Rev. 2008;17:123–9. Horowitz JC, Rogers DS, Sharma V, et al. Combinatorial activation of FAK and AKT by transforming growth factor-β1 confers an anoikis-resistant phenotype to myofibroblasts. Cell Signal. 2007;19:761–71. Zhang H-Y, Phan SH. Inhibition of myofibroblasts apoptosis by transforming growth factor β1. Am J Respir Cell Mol Biol. 1999;21:658–65. Moodley YP, Misso NL, Scaffidi AK, et al. Inverse effects of interleukin-6 on apoptosis of fibroblasts from pulmonary fibrosis and normal lungs. Am J Respir Cell Mol Biol. 2003;29:490–8. Ajayi IO, Sisson TH, Higgins PD, et al. X-linked inhibitor of apoptosis regulates lung fibroblast resistance to Fas-mediated apoptosis. Am J Respir Cell Mol Biol. 2013;49:86–95. Huang SK, Scruggs AM, Donaghy J, et al. Histone modifications are responsible for decreased Fas expression and apoptosis
411, Page 8 of 9
27.
28.
29.
30.
31. 32.
33.••
34.
35. 36.
37.
38.
39.
40.
41.
42.
43.
resistance in fibrotic lung fibroblasts. Cell Death Dis. 2013;4:621. doi:10.1038/cddis.2013.146. Hampel B, Malisan F, Niederegger H, et al. Differential regulation of apoptotic cell death in senescent human cells. Exp Gerontol. 2004;39:1713–21. Kitani A, Fuss I, Nakamura K, et al. Transforming growth factor (TGF)-β1–producing regulatory T cells induce Smad-mediated interleukin 10 secretion that facilitates coordinated immunoregulatory activity and amelioration of TGF-β1–mediated fibrosis. J Exp Med. 2003;198:1179–88. Sime PJ, O’Reilly KMA. Fibrosis of the lung and other tissues: new concepts in pathogenesis and treatment. Clin Immunol. 2001;99: 308–19. Khalil N, Bereznay O, Sporn M, Greenberg AH. Macrophage production of transforming growth factor beta and fibroblast collagen synthesis in chronic pulmonary inflammation. J Exp Med. 1989;70:727–37. Letterio JJ, Roberts AB. Regulation of immune responses by TGFβ. Annu Rev Immunol. 1998;16:137–61. Deng X, Mercer PF, Scotton CJ, Gilchrist A, Chambers RC. Thrombin induces fibroblast CCL2/JE production and release via coupling of PAR1 to Galphaq and cooperation between ERK1/2 and Rho kinase signaling pathways. Mol Biol Cell. 2008;19:2520–33. Bogatkevich GS, Highland KB, Akter T, et al. Inhibition of thrombin as a novel strategy in the treatment of scleroderma-associated interstitial lung disease. Edited by Radstake T. InTech. Croatia 2012:131–48. This paper provides evidence for a promising therapeutic strategy: use of thrombin inhibitor in the treatment of murine lung fibrosis. Ohba T, McDonald JK, Silver RM, et al. Scleroderma bronchoalveolar lavage fluid contains thrombin, a mediator of human lung fibroblast proliferation via induction of platelet-derived growth factor alpha-receptor. Am J Respir Cell Mol Biol. 1994;10:405–12. Hernández-Rodríguez NA, Cambrey AD, Harrison NK, et al. Role of thrombin in pulmonary fibrosis. Lancet. 1995;346:1071–3. Howell DCJ, Johns RH, Lasky JA, et al. Absence of proteaseactivated receptor-1 signaling affords protection from bleomycininduced lung inflammation and fibrosis. Am J Pathol. 2005;166: 1353–65. Bogatkevich GS, Gustilo E, Oates J, et al. Distinct PKC isoforms mediate cell survival and DNA synthesis in thrombin-induced myofibroblasts. Am J Physiol Lung Cell Mol Physiol. 2005;288: 190–201. Bogatkevich GS, Tourkina E, Silver RM, Ludwicka-Bradley A. Thrombin differentiates normal lung fibroblast to a myofibroblast phenotype via proteolytically activated receptor-1 and protein kinase C-dependent pathway. J Biol Chem. 2001;276:45184–92. Gray AJ, Bishop JE, Reeves JT, Laurent GJ. A alpha and B beta chains of fibrinogen stimulate proliferation of human fibroblasts. J Cell Sci. 1993;104:409–13. Bachhuber BG, Sarembock IJ, Gimple LW, Owens GK. alphaThrombin induces transforming growth factor-beta1 mRNA and protein in cultured vascular smooth muscle cells via a proteolytically activated receptor. J Vasc Res. 1997;34:41–8. Chambers RC, Leoni P, Blanc-Brude OP, Wembridge DE, Laurent GJ. Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease-activated receptor1. J Biol Chem. 2000;275:35584–91. Bogatkevich GS, Ludwicka-Bradley A, Nietert PJ, Silver RM. Scleroderma lung fibroblasts: contractility and connective tissue growth factor. Edited by Gabbiani G, Chaponnier C, Desmouliere A. Texas: Landes. Bioscience 2006:25–31. Tourkina E, Hoffman S, Fenton JW, Lipsitz S, et al. Depletion of protein kinase Cε in normal and scleroderma lung fibroblasts has opposite effects on tenascin expression. Arthritis Rheum. 2001;44: 1370–81.
Curr Rheumatol Rep (2014) 16:411 44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58. 59.
60.••
61.
62.
Chambers RC, Dabbagh K, McAnulty RJ, et al. Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1. Biochem J. 1998;333:121–7. Armstrong MT, Fenton 2nd JW, Andersen TT, Armstrong PB. Thrombin stimulation of matrix fibronectin. J Cell Physiol. 1996;166:112–20. Howell DC, Laurent GJ, Chambers RC. Role of thrombin and its major cellular receptor, protease-activated receptor-1, in pulmonary fibrosis. Biochem Soc Trans. 2002;30:211–6. Chambers RC. Procoagulant signalling mechanisms in lung inflammation and fibrosis: novel opportunities for pharmacological intervention? Br J Pharmacol. 2008;153:367–78. Bogatkevich GS, Ludwicka-Bradley A, Silver RM. Dabigatran, a direct thrombin inhibitor, blocks differentiation of normal fibroblasts to a myofibroblast phenotype and demonstrates anti-fibrotic effects on scleroderma lung fibroblasts. Arthritis Rheum. 2009;60: 3455–64. Bogatkevich GS, Ludwicka-Bradley A, Nietert PJ, et al. Antiinflammatory and antifibrotic effects of the oral direct thrombin inhibitor dabigatran etexilate in a murine model of interstitial lung disease. Arthritis Rheum. 2011;63:1416–25. Königshoff M, Kramer M, Balsara N, Wilhelm J, et al. WNT1inducible signaling protein-1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis. J Clin Invest. 2009;119:772–87. Chilosi M, Poletti V, Zamò A, et al. Aberrant Wnt/beta-catenin pathway activation in idiopathic pulmonary fibrosis. Am J Pathol. 2003;162:1495–502. Gordon MD, Nusse R. Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem. 2006;281:22429–33. Shi-Wen X, Chen Y, Denton CP, et al. Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/ phosphoinositide 3-kinase/Akt-dependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts. Mol Biol Cell. 2004;15:2707–19. Moinzadeh P, Krieg T, Hellmich M, et al. Elevated MMP-7 levels in patients with systemic sclerosis: correlation with pulmonary involvement. Exp Dermatol. 2011;20:770–3. Wei J, Melichian D, Komura K, et al. Canonical Wnt signaling induces skin fibrosis and subcutaneous lipoatrophy, a novel mouse model for scleroderma. Arthritis Rheum. 2011;63: 1707–17. Schneider AJ, Vezina CM, Richard E, et al. TCDD inhibition of canonical Wnt signaling disrupts prostatic bud formation in mouse urogenital sinus. Toxicol Sci. 2013;133:42–53. Arnett FC, Cho M, Chatterjee S, et al. Familial occurrence frequencies and relative risks for systemic sclerosis (scleroderma) in three United States cohorts. Arthritis Rheum. 2001;44:1359–62. Haustein U-F. Systemic sclerosis - an update. Lab Med. 2011;42: 562–72. Laing TJ, Gillespie BW, Toth MB, et al. Racial differences in scleroderma among women in Michigan. Arthritis Rheum. 1997;40:734–42. Silver RM, Bogatkevich G, Tourkina E, et al. Racial differences between blacks and whites with systemic sclerosis. Curr Opin Rheumatol. 2012;24:642–8. This article provides evidence of correlation between race-dependent predisposition to fibrosis and biological variable factors. Bogatkevich GS, Ludwicka-Bradley A, Highland KB, et al. Impairment of the antifibrotic effect of hepatocyte growth factor in lung fibroblasts from African Americans: possible role in systemic sclerosis. Arthritis Rheum. 2007;56:2432–42. Gelber AC, Manno RL, Shah AA, et al. Race and association with disease manifestations and mortality in scleroderma:
Curr Rheumatol Rep (2014) 16:411
63.
64.•
65.
66. 67.
a 20-year experience at the Johns Hopkins Scleroderma Center and review of the literature. Medicine (Baltimore). 2013;92: 191–205. Steen V, Domsic RT, Lucas M, et al. A clinical and serologic comparison of African American and Caucasian patients with systemic sclerosis. Arthritis Rheum. 2012;64: 2986–94. Bogatkevich GS, Highland KB, Akter T, Silver RM. The PPARγ agonist rosiglitazone is antifibrotic for scleroderma lung fibroblasts: mechanisms of action and differential racial effects. Pulm Med. 2012;2012:545172. doi:10.1155/2012/545172. This article provides functional evidence of race-based transcriptional factor over-activation and promising treatment for SSc-ILD on that basis. Vargas-Alarcón G, Granados J, Ibañez de Kasep G, et al. Association of HLA-DR5 (DR11) with systemic sclerosis (scleroderma) in Mexican patients. Clin Exp Rheumatol. 1995;13:11–6. Arnett FC. HLA and autoimmunity in scleroderma (systemic sclerosis). Int Rev Immunol. 1995;12:107–28. Reveille JD. Molecular genetics of systemic sclerosis. Curr Opin Rheumatol. 1993;5:753–9.
Page 9 of 9, 411 68.
Takeuchi F, Kuwata S, Nakano K, et al. Association of TAP1 and TAP2 with systemic sclerosis in Japanese. Clin Exp Rheumatol. 1996;14:513–21. 69. Luo Y, Wang Y, Wang Q, Xiao R, Lu Q. Systemic sclerosis: genetics and epigenetics. J Autoimmun. 2013;41:161–7. 70. Morel PA, Chang HJ, Wilson JW, et al. Severe systemic sclerosis with anti-topoisomerase I antibodies is associated with an HLADRw11 allele. Hum Immunol. 1994;40:101–10. 71. Romano E, Manetti M, Guiducci S, et al. The genetics of systemic sclerosis: an update. Clin Exp Rheumatol. 2011;29:75–86. 72. Wang Y, Fan PS, Kahaleh B. Association between enhanced type I collagen expression and epigenetic repression of the FLI1 gene in scleroderma fibroblasts. Arthritis Rheum. 2006;54:2271–9. 73. Makino K, Jinnin M, Hirano A, et al. The downregulation of microRNA let-7a contributes to the excessive expression of type I collagen in systemic and localized scleroderma. Immunology. 2013;190:3905–15. 74. Liu G, Friggeri A, Yang Y, et al. miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis. J Exp Med. 2010;207:1589–97.
