Scleroderma pathogenesis: a pivotal role for fibroblasts as effector cells

Scleroderma (systemic sclerosis; SSc) is characterised by fibrosis of the skin and internal organs in the context of autoimmunity and vascular perturbation. Overproduction of extracellular matrix components and loss of specialised epithelial structures are analogous to the process of scar formation after tissue injury. Fibroblasts are the resident cells of connective tissue that become activated at sites of damage and are likely to be important effector cells in SSc. Differentiation into myofibroblasts is a hallmark process, although the mechanisms and cellular origins of this important fibroblastic cell are still unclear. This article reviews fibroblast biology in the context of SSc and highlights the potentially important place of fibroblast effector cells in fibrosis. Moreover, the heterogeneity of fibroblast properties, multiplicity of regulatory pathways and diversity of origin for myofibroblasts may underpin clinical diversity in SSc, and provide novel avenues for targeted therapy.

progresses more rapidly and aff ects the trunk and extremities [2]. Studies suggest that the extent and pattern of change in skin sclerosis, especially in dcSSc, refl ects the severity and frequency of signifi cant internal organ complications and impacts on survival and other important long-term disease outcomes. However, the relationship is complex and underscores the clinical heterogeneity of SSc [3]. Auto-antibodies are important diagnostic tools that also provide information about clinical risks of specifi c complications, such as lung fi brosis or SSc renal crisis [4]. Some reports support a functional role of anti-nuclear antibodies (ANAs) in the pathological development of SSc, including recent data suggesting antibodies against vascular receptors specifi c for endothelin or angiotensin II may associate with more progressive forms of SSc [3]. Intriguingly the agonist eff ects of auto-anti-platelet-derived growth factor receptor antibodies in modulating fi broblast intracellular signalling have been reported [5], although these studies have not been consistently repeated [6].
Th e pathophysiology of SSc includes vascular injury and infl ammation, and culminates in fi brosis. Th e disruption of the aff ected tissue's architecture due to fi brosis is orchestrated by the fi broblasts' excessive synthesis and deposition of extracellular matrix (ECM) proteins, including collagen type I [7]. Central to the development and progression of fi brosis is the activation of resident fi broblasts. Fibrosis, like wound healing, is instigated by fi broblast activation, proliferation and migration of these cells into the site of trauma and deposition of matrix proteins such as fi bronectin and collagen [8]. In wounds, the activated fi broblasts or myo fi broblasts are lost, although the mechanism(s) by which these cells are cleared from the site of trauma remain contentious and may include apoptosis as well as de-activation. In fi brotic pathologies like SSc, however, these cells persist and promote a pro-fi brotic micro environ ment rich in ECM and growth factors, such as fi broblast growth factor (FGF) and connective tissue growth factor (CTGF; CCN2).

Fibroblast biology and scleroderma
Th e connective tissue confers a structural scaff old that facilitates organ function. Composed of ECM, the most Abstract Scleroderma (systemic sclerosis; SSc) is characterised by fi brosis of the skin and internal organs in the context of autoimmunity and vascular perturbation. Overproduction of extracellular matrix components and loss of specialised epithelial structures are analogous to the process of scar formation after tissue injury. Fibroblasts are the resident cells of connective tissue that become activated at sites of damage and are likely to be important eff ector cells in SSc. Diff erentiation into myofi broblasts is a hallmark process, although the mechanisms and cellular origins of this important fi broblastic cell are still unclear. This article reviews fi broblast biology in the context of SSc and highlights the potentially important place of fi broblast eff ector cells in fi brosis. Moreover, the heterogeneity of fi broblast properties, multiplicity of regulatory pathways and diversity of origin for myofi broblasts may underpin clinical diversity in SSc, and provide novel avenues for targeted therapy. common cell found in the connective tissues are spindleshaped cells termed 'fi broblasts' . Th ese cells, which express vimentin but not desmin or alpha smooth muscle actin (α-SMA), are found in the majority of organs and are essential for connective tissue homeostasis [5]. An imbalance in the deposition of ECM proteins, including collagen type I and III, leads to the pathological changes observed in SSc. Fibroblasts are highly active cells and each cell synthesises approximately 3.5 million pro-collagen molecules per day [6]. Fibroblasts regulate matrix turnover through the expres sion of matrix metalloproteinases (MMPs), which degrade ECM, and their inhibitors, tissue inhibitors of metallo proteinases (TIMPs). Consistent with increased ECM deposition in SSc patients, serum levels of TIMPs in dcSSc and limited cutaneous SSc are signifi cantly raised compared to healthy controls. Th is supports the hypothesis that fi broblast-regulated matrix accumulation occurs through an imbalance in turnover of the ECM and this plays a pivotal role in SSc [9].
Fibroblasts are the key contributors to fi brosis in patients with SSc. In healthy individuals fi broblasts are protected from stress by the surrounding ECM, but during connective tissue diseases the damaged fi broblasts are no longer protected, causing the fi broblasts to attach to the ECM [10]. Upon tissue injury, fi broblasts migrate towards the wound and due to the presence of growth factors released by immune and blood cells diff erentiate into secretory myofi broblasts that are involved in repair during wound healing.

Myofi broblasts
In response to tissue injury, mesenchymal cells of fi broblastic lineage accumulate at the wound site and deposit and remodel new ECM and contract the wound site. Th e main fi broblastic cells responsible for this process are termed myofi broblasts and exhibit specifi c markers and phenotypic properties that are suited to this role. Normally as wounds repair and resolve myofi broblasts are lost from the site of injury, whereas in fi brotic pathologies such as SSc they remain [11,12]. Th e persistence and accumulation of a large number of myofi broblasts in connective tissues is responsible for the exaggerated and uncontrolled production of ECM during the development and progression of fi brotic pathologies such as SSc [5,12]. Myofi broblasts can arise from resident fi broblasts in a process termed fi broblast to myofi broblast transition. Unlike fi broblasts, myofi broblasts express the contractile protein α-SMA [5]. More recently, it has become recognised that myofi broblasts can arise from diff erent cellular sources, including pericytes and smooth muscle and epithelial cells (Figure 1), via a number of biological processes that we address elsewhere in this review.
Histological analysis of SSc skin has shown an abundance of myofi broblasts involved in lesional skin and fi brotic areas of the visceral organs from SSc patients [5,13]. Consistent with the presence of myofi broblasts in SSc and wounding, gene expression profi ling studies demonstrate a number of genes diff erentially regulated in wound healing fi broblasts and those derived from fi brotic regions of SSc patients [14]. Comprehensive transcriptional analysis of skin biopsies has demonstrated systematic diff erences in the gene expression profi le of dermal fi broblasts from SSc patients into subsets including infl ammatory and transforming growth factor (TGF)-β gene signatures [15,16]. Th e diff erent gene expression profi les exhibited by fi broblasts from SSc patients may refl ect the diverse origins of the cells that contribute to the formation of myofi broblasts. It remains unclear if SSc fi broblasts arising from these diverse cellular pools will respond to similar therapeutic interventions, and future studies will be needed to explore the relevance to the pathological development of fi brosis in SSc patients.

Positional identity of fi broblasts -relevance to SSc
Fibroblasts isolated from diff erent tissues display similar morphology but exhibit diverse functional properties. For example, the capacity of fi broblasts from diff erent anatomical sites to migrate or express extracellular matrix proteins varies [17]. Th ese diff erences are con sistent with the wide variety of biological and physical environments these cells are found in. Th e 'positional memory' of fi broblasts was elegantly highlighted by Chang and colleagues [18] in gene expression profi ling studies of fi broblasts from a variety of anatomical sites in the adult and foetus. A striking feature in this study was the distinct and characteristic transcriptional patterns displayed by fi broblasts, including genes associated with lipid metabolism, the TGFβ and Wnt cell signaling pathways, and fate determination [18]. Th is study highlighted the context-dependent activity of fi broblasts to generate appropriate extracellular microenvironments. For example, foetal lung and skin fi broblasts expressed high levels of the basement membrane protein type IV collagen in the skin and lung alveoli. In contrast, skin but not lung fi broblasts expressed signifi cant levels of type I and V collagen, which confers the tensile strength in the dermis [18]. Consistent with the diff erent anatomical environ mental requirements of organs, fi broblasts exhibit distinct immune-modulatory eff ects on leuko cytes, including recruitment [17]. Th e mechanism(s) by which fi broblasts acquire their positional identity remain unclear; however, it is likely epigenetic mechanisms play a signifi cant role. It remains to be investigated if SSc fi broblasts lose their positional identity and this in turn promotes the development of a pro-fi brotic environment. It is plausible that location-specifi c signatures for fi bro blasts and other cell types explain the diverse patterns of fi brosis between and within diff erent subsets of SSc.

Origin of fi broblasts in SSc: resident cells, trans-diff erentiation, and circulating fi brocytes
Th e origin of activated fi broblasts or myofi broblasts in fi brotic tissues was until comparatively recently believed to result from the expansion and activation of resident fi broblasts. However, recent studies have highlighted the potential for other tissue-resident and blood-borne cells to contribute to the pool of myofi broblasts (Figure 1) that arise in fi brotic tissues [19][20][21]. A number of tissueresident cells, including endothelial, epithelial and smooth muscle cells, can diff erentiate into fi broblastic-like cells [19,20,22,23]. In addition to tissue-resident cells, blood-borne cells such as fi brocytes also contribute to the heterogeneity of these myofi broblasts [24].
Th e diff erentiation of resident cells has been proposed as an important mechanism contributing to the development of tissue fi brosis in SSc (Figure 1). For example, endothelial to mesenchymal transition (EndoMT) was thought to be a rare phenomenon confi ned to embryonic development; however, Arciniegas and colleagues [20] elegantly demonstrated the capacity of adult endothelial cells to lose vascular markers such as E-cadherin and acquire myofi broblast markers, including α-SMA and type I collagen. Consistent with a putative pathological role of EndoMT in SSc, lung capillary endothelial cells can contribute to the pool of myofi broblasts/fi broblasts present in the bleomycin model of pulmonary SSc [19]. Like EndoMT, epithelial cells in a process termed epithelial to mesenchymal transition (EMT) are induced by TGF-β to take on fi broblast-like features [19].
EMT has been linked to cellular diff erentiation and tumour invasion for a number of years [14]. More recently, EMT has become strongly associated with renal and pulmonary fi brosis in pre-clinical models [25]; however, the contribution of EMT in the development of fi brotic pathologies, including SSc, remains contentious. During EMT, epithelial cells down-regulate epithelial markers such as E-cadherin and acquire mesenchymal/ myofi broblast markers, including α-SMA [5,21]. EMT, like EndoMT, is likely to lead to a signifi cant loss of the functional capacity of these cells to act as biological barriers and contribute further to the development of fi brosis. Th e importance of barrier loss in the development and progression of SSc, as well as the therapeutic benefi t of restoring barrier function, remain to be explored. EMT can be induced by a number of secreted factors, including TGF-β, CTGF and FGF-2, all of which have been implicated in SSc [26]. Studies from our own group have further highlighted a cellular link between microvascular damage and fi brosis via pericyte transdiff erentiating into myofi broblasts (pericyte to mesenchymal transition (PeMT)) [27,28]. Th e presence of activated pericytes in dcSSc skin and the capability of these cells to transition into myofi broblasts when activated further support their likely contribution to dermal fi brosis in SSc. Interestingly, elegant genetic ablation studies targeting ADAM12 support a role of PeMT during tissue injury. Th ese studies demonstrated that the loss of perivascular ADAM12-positive cells led to a marked loss of pro-fi brotic collagen-producing cells during injury. Work from our own group has shown ADAM12 to be elevated in lung fi broblasts from SSc patients, supporting the possible contribution of PeMT in the development of SSc [29].
In addition to local precursors, circulating cells are also able to contribute to the myofi broblasts that populate fi brotic tissues (Figure 1). Fibrocytes were initially described in the early 1990s as blood-borne collagenproducing cells with antigen-presenting capability [30]. Since then, they have been associated with a broad range of fi brosing disorders, including SSc, sickle cell lung disease, asthma, pulmonary hypertension and atherosclerosis [31][32][33][34][35]. Although the cell surface markers that identify fi brocytes remains ambiguous, it is widely accepted that these cells share immune and mesenchymal cell surface markers [36] and migrate to sites of tissue injury [37]. Th e pre-clinical bleomycin insult model, which serves as a model of SSc fi brosis, exhibits enhanced fi brocyte recruitment in the dermis and lung, supporting the notion that fi brocytes play a key role in SSc [25,32]. Previous studies have demonstrated that mice dosed with adenosine A 2A antagonists were protected from develop ing bleomycin-induced lung fi brosis [38]. Th e use of these A 2A antagonists also halted lung fi brocyte recruit ment, suggesting that these receptors must be involved in fi brocyte recruitment and supporting the contribution of fi brocytes in the development of pulmonary fi brosis [32]. Consistent with fi brocyte recruitment to sites of tissue injury, these cells express a number of chemokine receptors, including chemokine receptor type 4 (CXCR4). Analysis of SSc patients demonstrated the presence of CXCR4 + /collagen type I + cells only in SSc interstitial lung disease patients. Further, the expression of CXCR4 and its ligand, stromal cell-derived factor 1 (CXCL12), was also highly upregulated in SSc lung compared to healthy controls [27]. Th e SSc lungs that overexpress CXCR4 also lack caveolin 1, and show enhanced monocyte migration compared to controls. In the bleomycin-induced fi brosis model, the use of caveolin scaff olding domain (CSD) diminishes fi brocyte accumu lation in the lung and may represent a novel therapy in SSc [34].
Th e cellular origin of the mesenchymal cells that contri bute to the excessive accumulation of ECM and loss of tissue architecture in SSc fi brosis remains unclear. Indeed, the reported contributions through the cellular processes that give rise to these cells, including expansion of resident tissue fi broblasts, EMT, fi broblast to myofi broblast transition and accumulation of bone marrowderived and circulating fi brocytes, may vary in an organspecifi c manner (Figure 1). Future studies will be required to assess the relative contribution and therapeutic relevance in SSc.

Fibroblast-dependent dysregulated connective tissue repair -unifi cation of the pathogenic pathway in SSc
Th ere are a number of established pre-clinical models of SSc and these continue to be the models of choice in SSc research (Table 1). Established models include the bleomycin-induced fi brotic model and tight skin mouse (tsk). More recently, transgenic models (Table 1) have facilitated research into fi broblastic mechanisms that drive fi brosis. An example of such a transgenic model is the TβRI CA ; Cre-ER transgenic mouse expressing constitutively active TβRI in fi broblasts. Postnatal expression of this activated receptor in mice leads to many of the clinical, histological and biochemical features seen in SSc patients. In the TβRI CA ; Cre-ER mouse, fi brosis of the dermis, thinner epidermis, loss of hair follicles and fi brotic thickening of small blood vessels in the lung and kidney are all evident [39]. Further, fi bro blasts isolated from the skin exhibit increased expression of downstream TGF-β target genes and resemble that seen in SSc patients. Th ese fi broblasts also showed increased expression of plasminogen activator, elevated Smad2/3 phosphorylation and enhanced myofi broblast diff erentiation [39]. Th is model of SSc is an example of the advantage of transgenic models and would be suitable for therapies aimed at alleviating enhanced TGF-β signalling in SSc and fi brosis.
Another example of a transgenic mouse model is the TβRIIΔk-fi b model. Th is model possesses a kinase defi cient TβRII gene in fi broblasts leading to receptor expression-dependent balanced upregulation of TGF-β signalling. Th e mechanism of this model is validated in the human disease Loeys-Dietz syndrome where patients have mutations in the TβRII gene leading to increased expression of both collagen and CTGF, as well as increased pSmad2 in the nuclei, which indicates increased TGF-β signalling [40]. Th ere is an increase in latent TGF-β in the ECM and they all have a fi brotic phenotype with dermal fi brosis. Twenty-fi ve percent of these mice spon taneously develop lung fi brosis, which again recapitu lates the disease of SSc. Th e intratracheal administration of bleomycin to these transgenic mice resulted in an increase in fi broblast proliferation, an increase in myo fi bro blasts and an increase in type II alveolar epithelial cell apoptosis [41]. In the TβRIIΔk-fi b model the transgenic fi broblasts are shown to proliferate more rapidly than wild-type cells, and exhibit an increase in TGF-β markers, including CTGF [42]. Interestingly, the mice also develop vasculopathy, which is a key feature in SSc that can lead to important clinical complications, includ ing SSc renal crisis and pulmonary arterial hypertension [43]. Th e development of diverse relevant pathology together with altered tissue injury response in multiple cell types provides strong support for a central role of fi broblasts in determining susceptibility to organ-based complications in SSc. Th is is consistent with a working hypothesis of 'fi broblast-dependent dysregulated connec tive tissue repair' as a unifying feature of SSc. As well as providing insight into potential pathogenic mechanisms in human disease, the studies outlined above also support fi broblastic cells as logical targets for disease-modifying therapy in SSc.
Recent studies by Wei and colleagues [44] demonstrated elevated Wnt-10b expression in the skin of SSc patients. Consistent with a role for the wnt signalling path way in fi brosis, this study demonstrated that Wnt-10b overexpression in transgenic animals led to enhanced Wnt signalling, skin thickening and expression of pro-fi brotic genes, including those encoding type I collagen and CTGF. Interestingly, these animals did not exhibit activation of the canonical TGF-β signalling pathway involving Smad2, suggesting the fi brogenic eff ects of this model are not directly mediated through induction of endogenous TGF-β [44]. However, the interplay between TGF-β and Wnt-regulated pathways may be complex as other studies in human fi broblast cultures have suggested that TGF-β signalling can activate canonical Wnt signalling and a broader potential role for morphogens in SSc pathogenesis is possible, as discussed below [45]. Collectively, the transgenic models of SSc highlighted in Table 1 suggest that alterations in a number of pathways can ultimately lead to the develop ment of SSclike tissue fi brosis. Understanding the con tri bution of these pathways in promoting the forma tion of activated fi broblasts in SSc from diff erent cellular pools that give rise to activated fi broblasts remains a key issue and will be of signifi cant importance for future therapies.

Shifting the paradigm -novel fi broblast-activated signalling pathways
Recent integration of novel in vitro and in vivo approaches have led to the emergence of a number of novel molecules and signalling pathways in SSc, which has helped to elucidate the complex cellular and molecular mechanisms implicated in this fi brotic disease. For example, the family of transcription factor activator protein 1 (AP-1) has also been shown to be activated in SSc; recent studies have shown members of this family of transcription factors to be up-regulated in SSc, including c-jun and c-fos in skin and dermal fi broblasts of SSc [46], and Fra-2 [47]. Further studies have implicated the hedgehog morphogen, fi rst discovered in the 1980s [48]. Sonic hedgehog (SHH), one of the three hedgehog proteins, was shown to be elevated in skin fi broblasts, endothelial cells and keratinocytes in SSc patients. It was also shown to induce myofi broblast diff erentiation, suggesting that excessive activation of this pathway in SSc promotes a pro-fi brotic cascade [49]. Inhibition of the hedgehog pathway exhibited potent anti-fi brotic eff ects in a model of SSc [50], suggesting that targeting this pathway could provide a novel therapeutic strategy in SSc. Th e Wnt signalling pathway has also become an area of interest in SSc and recent genome studies have shown an increase in expression of the Wnt receptor FZD2 and decreased expression of the Wnt antagonists DKK2 and WIF1 in skin from dcSSc patients. Activation of this pathway also stimulated an increase in fi broblast migration and proliferation, suggesting it plays an important role in SSc [51].

Conclusion
Signifi cant advances in recent years have led to an appreciation that cells from a variety of origins can assume fi broblastic characteristics under pathological situations [19][20][21][22]. Th e default plasticity of cells to become fi broblasts and thus contribute to the reservoir of disease-causing myofi broblasts in SSc may in part explain the apparent heterogeneity in the disease. Th e capacity of SSc fi broblasts to promote the recruitment and diff erentiation of these cells to become pathological is actively being investigated. A key question, however, is whether SSc fi broblasts derived from diff erent cellular origins aff ect the progression of fi brosis in SSc or leads to diff erences in the disease mediators that future therapeutics will seek to target. Future studies to defi ne the interplay of fi broblast populations derived from diff erent cellular origins in SSc will be crucial in our understanding of how best to develop and treat SSc. In conclusion, emerging data further support a key role for pathways that are central to normal growth and development in regulating abnormal fi broblast properties in acquired disease and fi brosis. Th ese pathways, of the fi broblasts themselves, may ultimately provide therapeutic strategies for SSc that have a more direct antifi brotic eff ect than current treatments that target vasculo pathy or the immune system.