NIH Public Access
`Author Manuscript
`J Pathol. Author manuscript; available in PMC 2009 June 9.
`Published in final edited form as:
`J Pathol. 2008 January ; 214(2): 199–210. doi:10.1002/path.2277.
`Cellular and molecular mechanisms of fibrosis
`TA Wynn*
`Immunopathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and
`Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
`Abstract
`Fibrosis is defined by the overgrowth, hardening, and/or scarring of various tissues and is attributed
`to excess deposition of extracellular matrix components including collagen. Fibrosis is the end result
`of chronic inflammatory reactions induced by a variety of stimuli including persistent infections,
`autoimmune reactions, allergic responses, chemical insults, radiation, and tissue injury. Although
`current treatments for fibrotic diseases such as idiopathic pulmonary fibrosis, liver cirrhosis, systemic
`sclerosis, progressive kidney disease, and cardiovascular fibrosis typically target the inflammatory
`response, there is accumulating evidence that the mechanisms driving fibrogenesis are distinct from
`those regulating inflammation. In fact, some studies have suggested that ongoing inflammation is
`needed to reverse established and progressive fibrosis. The key cellular mediator of fibrosis is the
`myofibroblast, which when activated serves as the primary collagen-producing cell. Myofibroblasts
`are generated from a variety of sources including resident mesenchymal cells, epithelial and
`endothelial cells in processes termed epithelial/endothelial-mesenchymal (EMT/EndMT) transition,
`as well as from circulating fibroblast-like cells called fibrocytes that are derived from bone-marrow
`stem cells. Myofibroblasts are activated by a variety of mechanisms, including paracrine signals
`derived from lymphocytes and macrophages, autocrine factors secreted by myofibroblasts, and
`pathogen-associated molecular patterns (PAMPS) produced by pathogenic organisms that interact
`with pattern recognition receptors (i.e. TLRs) on fibroblasts. Cytokines (IL-13, IL-21, TGF-(cid:533)1),
`chemokines (MCP-1, MIP-1(cid:533)), angiogenic factors (VEGF), growth factors (PDGF), peroxisome
`proliferator-activated receptors (PPARs), acute phase proteins (SAP), caspases, and components of
`the renin–angiotensin–aldosterone system (ANG II) have been identified as important regulators of
`fibrosis and are being investigated as potential targets of antifibrotic drugs. This review explores our
`current understanding of the cellular and molecular mechanisms of fibrogenesis.
`Keywords
`fibrosis; myofibroblast; collagen; wound healing; liver; lung
`IntroductionIn contrast to acute inflammatory reactions, which are characterized by rapidly resolving
`vascular changes, oedema and neutrophilic inflammation, fibrosis typically results from
`chronic inflammation — defined as an immune response that persists for several months and
`in which inflammation, tissue remodelling and repair processes occur simultaneously. Despite
`having distinct aetiological and clinical manifestations, most chronic fibrotic disorders have
`*Correspondence to: TA Wynn, Immunopathogenesis, Section, Laboratory of Parasitic Diseases, National Institute of Allergy and
`Infectious Diseases, National Institutes of Health, 50 South Drive, Room 6154, MSC 8003, Bethesda, MD, 20892, USA. E-mail: E-mail:
`twynn@niaid.nih.gov.
`No conflicts of interest were declared.
`This article is a US government work and is in the public domain in the USA.
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`in common a persistent irritant that sustains the production of growth factors, proteolytic
`enzymes, angiogenic factors and fibrogenic cytokines, which stimulate the deposition of
`connective tissue elements that progressively remodel and destroy normal tissue architecture
`[1–3].Damage to tissues can result from various stimuli, including infections, autoimmune reactions,
`toxins, radiation and mechanical injury. The repair process typically involves two distinct
`phases: a regenerative phase, in which injured cells are replaced by cells of the same type,
`leaving no lasting evidence of damage; and a phase known as fibroplasia or fibrosis, in which
`connective tissues replaces normal parenchymal tissue. Although initially beneficial, the repair
`process becomes pathogenic when it is not controlled appropriately, resulting in substantial
`deposition of ECM components in which normal tissue is replaced with permanent scar tissue
`[4]. In some diseases, such as idiopathic pulmonary fibrosis, liver cirrhosis, cardiovascular
`fibrosis, systemic sclerosis and nephritis, extensive tissue remodelling and fibrosis can
`ultimately lead to organ failure and death (Table 1).
`Wound healing versus fibrosis
`When epithelial and/or endothelial cells are damaged, they release inflammatory mediators
`that initiate an anti-fibrinolytic coagulation cascade [5], which triggers blood-clot formation
`and formation of a provisional ECM. Platelets are exposed to ECM components, triggering
`aggregation, clot formation and haemostasis. Platelet degranulation also promotes vasodilation
`and increased blood vessel permeability, while myofibroblasts (activated collagen secreting,
`(cid:302)-SMA+ fibroblasts) and epithelial and/or endothelial cells produce MMPs, which disrupt the
`basement membrane, allowing inflammatory cells to be easily recruited to the site of injury.
`Growth factors, cytokines and chemokines are also produced, which stimulates the
`proliferation and recruitment of leukocytes across the provisional ECM. Some of the early
`responders include macrophages and neutrophils, which eliminate tissue debris, dead cells and
`any invading organisms. They also produce cytokines and chemokines, which are mitogenic
`and chemotactic for endothelial cells, which begin to surround the injured site. They also help
`form new blood vessels as epithelial/endothelial cells migrate towards the centre of the wound.
`During this period, lymphocytes and other cells become activated and begin secreting
`profibrotic cytokines and growth factors, such as TGF(cid:533), IL-13 and PDGF [6–8], which further
`activate the macrophages and fibroblasts. Activated fibroblasts transform into (cid:302)-SMA-
`expressing myofibroblasts as they migrate along the fibrin lattice into the wound. Following
`activation, the myofibroblasts promote wound contraction, the process in which the edges of
`the wound migrate towards the centre. Finally, epithelial and/or endothelial cells divide and
`migrate over the basal layers to regenerate the damaged tissue, which completes the wound-
`healing process. However, chronic inflammation and repair can trigger an excessive
`accumulation of ECM components, which leads to the formation of a permanent fibrotic scar.
`Collagen turnover and ECM remodelling is regulated by various MMPs and their inhibitors,
`which include the tissue inhibitors of metalloproteinases (TIMPs). Shifts in synthesis versus
`catabolism of the ECM regulate the net increase or decrease of collagen within the wound
`[9]. Fibrosis occurs when the synthesis of new collagen by myofibroblasts exceeds the rate at
`which it is degraded, such that the total amount of collagen increases over time.
`The cellular origins of myofibroblasts
`Local tissue myofibroblasts were originally believed to be the primary producers of ECM
`components following injury [5]; however, it is now thought that fibroblasts can be derived
`from multiple sources [10]. In addition to resident mesenchymal cells, myofibroblasts are
`derived from epithelial cells in a process termed epithelial–mesenchymal transition (EMT)
`[10–12]. More recently, it was suggested that a similar process occurs with endothelial cells,
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`termed endothelial–mesenchymal transition (EndMT) [13]. Bucala and colleagues also
`identified a unique circulating fibroblast-like cell derived from bone marrow stem cells [14].
`These blood-borne mesenchymal stem cell progenitors have a fibroblast/myofibroblast-like
`phenotype (they express CD34, CD45 and type I collagen) and are now commonly called
`fibrocytes [15–18]. Finally, in some tissues, resident fibroblasts are not the only source of
`myofibroblasts. For example, in liver fibrosis the resident hepatic stellate cell (HSC) appears
`to be the primary source of myofibroblasts, although bone-marrow-derived cells can also
`contribute [18,19]. Because it is now thought that fibrocytes and EMT-derived myofibroblasts
`participate with resident mesenchymal cells in the reparative process, there has been growing
`interest in dissecting the role of the various myofibroblast subpopulations in fibroproliferative
`disease [20]. Because bone marrow-derived fibrocytes must find their way to sites of tissue
`injury to participate in wound healing and fibrosis, there has been a great deal of interest in
`understanding the role of chemokines and acute phase proteins, such as serum amyloid P (SAP),
`in the development and recruitment of myofibroblasts [20–22]. Because fibrocytes and EMT-
`derived myofibroblasts produce a variety of factors that are involved in the fibrotic process
`[10], interrupting their development, recruitment and/or activation could provide a unique
`therapeutic approach to treat a variety of fibrotic diseases.
`Innate and adaptive immune mechanisms regulate myofibroblast activity
`Many fibrotic disorders are thought to have an infectious aetiology, with bacteria, viruses, fungi
`and multicellular parasites driving chronic inflammation and the development of fibrosis. It
`was recently suggested that conserved pathogen-associated molecular patterns (PAMPs) found
`on these organisms help maintain myofibroblasts at a heightened state of activation [23].
`Bacteria living in the gut can also contribute to the activation of myofibroblasts [24]. PAMPs
`are pathogen byproducts, such as lipoproteins, bacterial DNA and double-stranded RNA,
`which are recognized by pattern recognition receptors (PRRs) found on a wide variety of cells,
`including fibroblasts [25]. The interaction between PAMPs and PRRs serves as a first line of
`defence during infection and activates numerous proinflammatory cytokine and chemokine
`responses. In addition, because fibroblasts express a variety of PRRs, including Toll-like
`receptors (TLRs), Toll ligands can directly activate fibroblasts and promote their differentiation
`into collagen-producing myofibroblasts [23,24,26]. Thus, inhibiting TLR signalling might
`represent a novel approach to treat fibrotic disease.
`Nevertheless, pathogenic organisms are not responsible for all fibrotic disorders. Therefore,
`additional mechanisms must also participate in the activation of myofibroblasts. For example,
`in the case of systemic sclerosis (SSc), fibroblasts obtained from lesional skin or fibrotic lungs
`have a constitutively activated myofibroblast-like phenotype, characterized by enhanced ECM
`synthesis, constitutive secretion of cytokines and chemokines and increased expression of cell
`surface receptors [27–29]. Because most of the characteristics of fibroblasts from patients with
`SSc are reproduced in normal human fibroblasts following stimulation with TGF(cid:533), it is thought
`that the SSc fibroblast phenotype is maintained by an autocrine TGF(cid:533) signal. However,
`TGF(cid:533)/SMAD3-independent mechanisms have also been proposed [30,31], including a role for
`viruses such as CMV, which stimulate the production of auto-antibodies and connective tissue
`growth factor (CTGF), both of which are known to participate in the activation of
`myofibroblasts [28,32]. Epigenetic changes may also contribute to the persistent activation of
`myofibroblasts [33]. B cells have also been implicated, either by producing autoanti-bodies or
`by secreting IL-6, a well-known fibroblast growth factor [34]. Still other studies have argued
`that Th2-type cytokines derived from a variety of cellular sources are critically involved in the
`mechanism of fibrosis [35–38]. Therefore, paracrine signals derived from activated
`lymphocytes, autocrine factors produced by fibroblasts, as well as molecules derived from
`pathogenic organisms can cooperate to initiate and maintain myofibroblast activation.
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`Chemokines regulate fibrogenesis by controlling myofibroblast recruitment
`Chemokines are leukocyte chemoattractants that cooperate with profibrotic cytokines in the
`development of fibrosis by recruiting myofibroblasts, macrophages and other key effector cells
`to sites of tissue injury. Although a large number of chemokine signalling pathways are
`involved in the mechanism of fibrogenesis, the CC- and CXC-chemokine receptor families
`have consistently exhibited important regulatory roles. Specifically, CCL3 (macrophage
`inflammatory protein 1(cid:302)) and CC-chemokines such as CCL2 (monocyte chemoattractant
`protein-1), which are chemotactic for mononuclear phagocytes, were identified as profibrotic
`mediators. Macrophages and epithelial cells are believed to be the key sources of CCL3, and
`studies in the bleomycin model of pulmonary fibrosis showed that anti-CCL3 antibodies could
`significantly reduce the development of fibrosis [39,40]. Similar results were obtained when
`CCL2 was neutralized, suggesting that a variety of CC-chemokines are involved [41,42].
`Subsequent studies with CC-chemokine receptor 1 (CCR1)- and CCR2-deficient mice
`produced similar results, confirming critical roles for CCL3/CCL2-mediated signalling
`pathways in fibrogenesis [43–47]. Interestingly, in several of these blocking studies, the
`absence of fibrosis was associated with decreased IL-4/IL-13 expression [44,48], suggesting
`a direct link between CC-chemokine activity and the production of profibrotic cytokines such
`as IL-13. IL-13 is a potent inducer of several CC-chemokines, including CCL3, CCL4
`(MIP-1(cid:533)), CCL20 (MIP-3(cid:302)), CCL2, CCL11, CCL22 (macrophage-derived chemokine) and
`CCL6 (C10), among others, suggesting that a positive feedback mechanism exists between
`IL-13 and the CC-chemokine family [49,50]. As seen with anti-CCL3 and anti-CCL2 antibody
`treatment, antibodies to CCL6 significantly attenuated lung remodelling responses in IL-13-
`transgenic mice [50] as well as in mice challenged with bleomycin [49], indicating non-
`redundant roles for a variety of CC-chemokines in the pathogenesis of fibrosis. In mice, CXC
`chemokine receptor 4 (CXCR4), CC chemokine receptor 7 (CCR7) and CCR2 have also been
`shown to regulate the recruitment of fibrocytes to the lung [20,21]. Thus, interrupting specific
`chemokine signalling pathways could have a significant impact on the treatment of a variety
`of fibroproliferative diseases.
`Th1 and Th2 cells differentially regulate organ fibrosis
`Chronic inflammatory reactions are typically characterized by a large infiltrate of mononuclear
`cells, including macrophages, lymphocytes, eosinophils and plasma cells. Lymphocytes are
`mobilized to sites of injury and become activated following contact with various antigens,
`which stimulate the production of lymphokines that further activate macrophages and other
`local inflammatory cells. Thus, there is significant activation of the adaptive immune response
`in many chronic inflammatory diseases. Although inflammation typically precedes the
`development of fibrosis, results from a variety of experimental models suggest that fibrosis is
`not always characterized by persistent inflammation, implying that the mechanisms regulating
`fibrosis are to a certain extend distinct from those controlling inflammation. Findings from our
`own studies of schistosomiasis-induced liver fibrosis support this theory [35]. In this model,
`fibrosis develops progressively in response to schistosome eggs that are deposited in the liver,
`which induce a chronic granulomatous response. As in many other experimental models of
`fibrosis, CD4+ T cells play a prominent role in the progression of the disease. Studies conducted
`with multiple cytokine-deficient mice have demonstrated that liver fibrosis is strongly linked
`with the development of a CD4+ Th2 cell response (involving IL-4, IL-5, IL-13 and IL-21)
`[51–55].
`Several experimental models of fibrosis in addition to our own have also documented potent
`antifibrotic activities for the Th1-associated cytokines IFN(cid:534) and IL-12. In schistosomiasis,
`while treatment with IFN(cid:534) or IL-12 has no effect on the establishment of infection, collagen
`deposition associated with chronic granuloma formation is substantially decreased [51].
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`Similar results have been obtained in models of pulmonary, liver and kidney fibrosis [56–
`59]. These findings suggest that it might be possible to develop an antifibrosis vaccine based
`on immune deviation [51,60], in which the profibrotic effects of the Th2 response are switched
`off in favour of an antifibrotic Th1 response. Indeed, similar approaches have been proposed
`for individuals suffering from allergic airway inflammation [61], which is also driven by Th2-
`type responses. Studies investigating the gene expression patterns of fibrotic tissues found that
`markedly different gene expression profiles are induced during Th1 and Th2 polarized
`responses [62,63]. As might be expected, a large number of IFN(cid:534)-induced genes are
`upregulated in the tissues of mice exhibiting Th1-polarized responses, with no evidence of
`significant activation of the fibrosis-associated genes in this setting [62–64]. Instead, two major
`groups of genes were identified in Th1-polarized mice: those associated with the acute-phase
`reaction and apoptosis (cell death), findings which may explain the extensive tissue damage
`that is commonly observed when Th1 responses continue unchecked [65]. By contrast, several
`genes known to be involved in the mechanisms of wound healing and fibrosis were upregulated
`in animals exhibiting Th2-polarized inflammation [62,63]. The regulation and function of a
`few of the genes, including procollagens I, III and VI, arginase-1 [66], lysyl oxidase [67,68],
`matrix metalloproteinase-2 (MMP-2) [69,70], MMP-9 [71,72] and tissue inhibitor of matrix
`metalloproteinase-1 (TIMP-1) [73,74], have been investigated in some detail. Several
`additional Th2-linked genes [62,63], including haem oxygenase, procollagen III, secreted
`phosphoprotein 1, procollagen V, reticulocalbin and fibrillin 1 have also been reported in the
`fibrotic lungs of bleomycin-treated mice [75] and in CCl4-stimulated rat hepatic stellate cells
`(collagen-producing cells in the liver) [76], providing further evidence that fibrosis is often
`associated with the development of Th2-type responses.
`Unique roles for the Th2 cytokines IL-4, IL-5, IL-13 and IL-21 in fibrosis
`The Th2 cytokines IL-4, IL-5, IL-13 and IL-21 each have distinct roles in the regulation of
`tissue remodelling and fibrosis. IL-4 is found at increased levels in the bronchoalveolar lavage
`fluids of patients with idiopathic pulmonary fibrosis (IPF) [77], in the pulmonary interstitium
`of individuals with cryptogenic fibrosing alveolitis [78] and in peripheral blood mononuclear
`cells (PBMCs) of those suffering from periportal fibrosis [79]. Development of post-irradiation
`fibrosis is also associated with increased production of IL-4 [80]. Although the extent to which
`IL-4 participates in fibrosis varies in different diseases, it has long been considered a potent
`profibrotic mediator. In fact, studies have suggested that IL-4 is nearly twice as effective as
`TGF(cid:533) [81], another potent profibrotic cytokine that has been extensively studied [82].
`Receptors for IL-4 are found on many mouse [83] and human fibroblast subtypes [84] and in
`vitro studies showed the synthesis of the extracellular matrix proteins, types I and III collagen
`and fibronectin, following IL-4 stimulation. One of the first in vivo reports to investigate the
`contribution of IL-4 was a study of schistosomiasis in mice, in which neutralizing antibodies
`to IL-4 were shown to significantly reduce the development of hepatic fibrosis [52]. Inhibitors
`of IL-4 were also found to reduce dermal fibrosis in a chronic skin graft rejection model and
`in a mouse model of scleroderma [85,86].
`IL-13 shares many functional activities with IL-4 because both cytokines exploit the same
`IL-4R(cid:302)/Stat6 signalling pathways [87]. However, with the development of IL-13 transgenic
`and knockout mice [88,89], as well as IL-13 antagonists [53,90], unique and non-redundant
`roles for IL-13 and IL-4 have been revealed in numerous models. When IL-4 and IL-13 were
`inhibited independently, IL-13 was identified as the dominant effector cytokine of fibrosis in
`several experimental models of fibrosis [38,53,91–94]. In schistosomiasis, although the egg-
`induced inflammatory response was unaffected by IL-13 blockade, collagen deposition
`decreased by more than 85% [53,95], despite continued and undiminished production of IL-4
`[53,96]. Related studies have also shown a dominant role for IL-13 in the pathogenesis of
`pulmonary fibrosis. Over-expression of IL-13 in the lung triggered significant subepithelial
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`airway fibrosis in mice in the absence of any additional inflammatory stimulus [89], while
`treatment with anti-IL-13 antibody markedly reduced collagen deposition in the lungs of
`animals challenged with A. fumigatus conidia [91] or bleomycin [49]. In contrast, transgenic
`mice that over-expressed IL-4 displayed little evidence of subepithelial airway fibrosis, despite
`developing an intense inflammatory response in the lung [97]. Interestingly, two recent studies
`suggested that IL-13-regulated responses [98], including lung fibrosis [99], could develop in
`the absence of IL-4R(cid:302) or Stat6-mediated signalling, suggesting that IL-13 can exploit an
`additional signalling mechanism that is distinct from the IL-4R(cid:302)/Stat6-signalling pathway.
`Indeed, a recent report suggested that TGF(cid:533)1-driven pulmonary fibrosis might in some cases
`be dependent on IL-13-mediated signalling through the IL-13R(cid:302)2 chain [100], which was
`originally thought to operate exclusively as a decoy receptor for IL-13 and as an inhibitor of
`fibrosis [53,101].
`IL-5 and eosinophils have also been shown to regulate tissue fibrogenesis. The differentiation,
`activation and recruitment of eosinophils is highly dependent on IL-5, and eosinophils are an
`important source of fibrogenic cytokines, including TGF(cid:533)1 and IL-13. IL-5 and tissue
`eosinophils have been observed in a variety of diseases, including skin allograft rejection and
`pulmonary fibrosis [86,102,103]. However, studies with neutralizing anti-IL-5 antibodies and
`IL-5 knockout mice have often yielded conflicting results [104]. Early experiments with
`neutralizing anti-IL-5 monoclonal antibodies showed no reduction in liver fibrosis following
`S. mansoni infection, even though tissue eosinophil responses were markedly reduced [105].
`Although negative findings were also reported in some of the skin and lung fibrosis models
`[105,106], other studies observed significant reductions in fibrosis when IL-5 activity was
`neutralized [86,107–110]. A recent study demonstrated that although excessive amounts of
`IL-5 can exacerbate bleomycin-induced fibrosis, IL-5(cid:237)/(cid:237) mice showed no impairment in
`fibrosis [111], suggesting that IL-5 and/or eosinophils act as amplifiers rather than as direct
`mediators of fibrosis. In mice deficient in IL-5 and/or CCL11 (eotaxin), tissue eosinophilia
`was abolished and the ability of CD4+ Th2 cells to produce the profibrotic cytokine IL-13 was
`significantly impaired [112]. Eosinophils were also found to be an important source of IL-13
`in the schistosomiasis-induced model of liver fibrosis [55]. IL-5 and eosinophils can also
`regulate the TGF(cid:533) response in the lungs of mice [109,113]. Thus, one of the key roles of IL-5
`and eosinophils may be to facilitate production of important profibrotic cytokines like IL-13
`and/or TGF(cid:533), which function as the key mediators of fibrosis.
`Finally, similar to IL-5 [55], IL-21/IL-21R signalling was recently shown to promote fibrosis
`by facilitating the development of the CD4+ Th2 response [54]. IL-21R-signalling was also
`critical for Th2-cell survival and for the migration Th2 cells to the peripheral tissues [114]. In
`addition to supporting the development of Th2 responses, IL-21 also increased IL-4 and IL-13
`receptor expression on macrophages [54], which enhances the development of alternatively
`activated macrophages that are believed to be important regulators of fibrosis [66,115].
`Distinct and overlapping roles for TGF(cid:533) and Th2-type cytokines in fibrosis
`TGF(cid:533) has been the most intensively studied regulator of the ECM and has been linked with
`the development of fibrosis in a number of diseases [116–119]. There are three isotypes of
`TGF(cid:533) in mammals, TGF(cid:533)1, -2 and -3, all exhibiting similar biological activity [120]. Although
`a variety of cell types produce and respond to TGF(cid:533) [82], tissue fibrosis is primarily attributed
`to the TGF(cid:533)1 isoform, with circulating monocytes and tissue macrophages being the
`predominant cellular sources. In macrophages, the primary level of control is not in the
`regulation of TGF(cid:533)1 mRNA expression, but in the regulation of both the secretion and
`activation of latent TGF(cid:533)1. TGF(cid:533)1 is stored inside the cell as a disulphide-bonded homodimer,
`non-covalently bound to a latency-associated protein (LAP), which keeps TGF(cid:533) inactive.
`Binding of the cytokine to its receptors requires dissociation of the LAP, a process that is
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`catalysed by several agents, including cathepsins, plasmin, calpain, thrombospondin, integrin-
`(cid:302)v(cid:533)6 and matrix metalloproteinases [82,120,121], many of which have become potential
`targets of antifibrotic drugs. Once activated, TGF(cid:533) signals through transmembrane receptors
`that trigger signalling intermediates known as Smad proteins, which modulate transcription of
`important target genes, including procollagen I and III [122]. Dermal fibrosis following
`irradiation [123] and renal interstitial fibrosis induced by unilateral ureteral obstruction [116]
`are both reduced in Smad3-deficient mice, confirming an important role for the TGF(cid:533)
`signalling pathway. Macrophage-derived TGF(cid:533)1 is thought to promote fibrosis by directly
`activating resident mesenchymal cells including epithelial cells, which differentiate into
`collagen-producing myofibroblasts via EMT. Interestingly, a recent paper showed that the loss
`of TGF(cid:533) signalling in fibroblasts triggers intraepithelial neoplasia, suggesting that TGF(cid:533)1
`signalling critically regulates the activity of fibroblasts as well as the oncogenic potential of
`neighbouring epithelial cells [124]. In the bleomycin model of fibrosis, alveolar macrophages
`are thought to produce nearly all of the active TGF(cid:533) that promotes pulmonary fibrosis [125].
`Nevertheless, Smad3/TGF(cid:533)1-independent mechanisms of fibrosis have also been
`demonstrated in the lung and other tissues [30,126,127], suggesting that profibrotic mediators
`such as IL-4, IL-5, IL-13 and IL-21 can act separately from the TGF(cid:533)/Smad-signalling pathway
`to stimulate collagen deposition.
`There is also evidence that Th2 cytokines cooperate with TGF(cid:533) to induce fibrosis. IL-13
`induces the production of latent TGF(cid:533)1 in macrophages and can also serve as an indirect
`activator of TGF(cid:533) by upregulating expression of proteins that cleave the LAP [128,129].
`Indeed, IL-13 is a potent stimulator of both MMP and cathepsin-based proteolytic pathways
`that activate TGF(cid:533) [74,129]. Thus, the significant tissue remodelling associated with polarized
`Th2 responses may involve a pathway wherein IL-13-expressing CD4+ Th2 cells trigger
`macrophage production of TGF(cid:533)1, which then serves as the major stimulus for fibroblast
`activation and collagen deposition [100,128,130]. In support of this hypothesis, when TGF(cid:533)1
`activity was neutralized in the lungs of IL-13-transgenic mice, development of subepithelial
`fibrosis was significantly reduced [128]. However, related studies observed enhanced
`pulmonary pathology when the TGF(cid:533)/Smad signalling pathway was blocked [131,132],
`suggesting that TGF(cid:533) suppresses rather than induces tissue remodelling in some settings. The
`source of TGF(cid:533)1 appears to be critical, since macrophage-derived TGF(cid:533)1 is often profibrotic
`[128], while T cell-derived TGF(cid:533)1 appears to play a suppressive role [133]. Some studies
`investigating the mechanisms of IL-13-driven fibrosis also reported no reduction in fibrosis in
`MMP-9-, Smad3- and TGF(cid:533)1-deficient mice, suggesting that IL-13 can operate independently
`from TGF(cid:533)1 [30]. This may explain the unexpected failure of Smad/TGF(cid:533) inhibitors in some
`blocking studies [126,127]. Thus, it remains unclear to what extent IL-13 must act through
`TGF(cid:533)1 to trigger fibrosis. Given that numerous antifibrotic therapies are focused on inhibiting
`the TGF(cid:533)1 signalling pathway [82,134], it will be important to determine whether the collagen-
`inducing activity of IL-13 is dependent on TGF(cid:533)1 or whether IL-13 and other profibrotic
`mediators [135] can also operate independently, as has been suggested in some studies [30,
`53,135].
`Vascular changes often accompany the development of fibrosis
`In addition to fibroproliferation and deposition of ECM components, the pathogenesis of IPF,
`systemic sclerosis (SSc), liver fibrosis and many other fibrotic diseases, including many fibrotic
`diseases of the eye, are characterized by substantial vascular remodelling, which often occurs
`prior to the development of fibrosis. In the case of systemic sclerosis, vascular changes are a
`prominent and early manifestation of the disease, with impaired angiogenesis leading to the
`progressive disappearance of blood vessels [28,29]. It has been suggested that reduced numbers
`of circulating bone marrow-derived CD34+ endothelial progenitor cells, as well as their
`impaired differentiation into mature endothelial cells, might be contributing to the early
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`vascular defects in SSc [136]. In contrast to SSc, where fibrosis is associated with the loss of
`blood vessels, fibrosis and traction retinal detachments associated with advanced diabetic
`retinopathy (DR) are characterized by uncontrolled vascular proliferation [137]. Indeed, the
`common pathway for many fibrotic eye diseases, including age-related macular degeneration
`(ARMD) [138], is injury to the cornea and/or retina, which results in inflammatory changes,
`tissue oedema, hypoxia-driven neovascularization and ultimately fibrosis. Once new blood
`vessels begin to grow in the eye, they are prone to haemorrhage, leading to further activation
`of the wound-healing response, and ultimately development of severe fibrosis [139]. Therefore,
`prevention of the primary vascular abnormality has been the most promising therapeutic
`strategy for many diseases of the eye. Because various members of the CXC-chemokine family
`exhibit potent angiogenic or angiostatic activity [140], targeting the CXC-chemokine family
`might offer a unique approach to regulate angiogenesis and fibrosis.
`Angiotensin II plays a critical role in fibrosis
`Although all major components of the renin–angiotensin–aldosterone system exhibit
`profibrotic activity, ANG II appears to be the dominant hormone responsible for cardiac
`fibrosis in hypertensive heart disease [141]. ANG II also plays an important role in the
`development of renal and hepatic fibrosis [142]. ANG II, produ