Systemic sclerosis: a prototypic multisystem fibrotic disorder (original) (raw)
Overview: molecular and cellular determinants of the ECM. Fibrosis is the most characteristic pathological hallmark of SSc, and it is especially prominent in the diffuse cutaneous form of the disease (Figure 2; ref. 57). Progressive replacement of tissue architecture by collagen-rich ECM results in functional impairment of affected organs. The fibrotic process is most prominent in the skin, lungs, gastrointestinal tract, heart, tendons and ligaments, and endocrine glands; widespread perivascular fibrosis also occurs. Fibrotic damage to these affected organs accounts for much of the morbidity and mortality associated with SSc. The ECM consists of a cellular compartment of resident and infiltrating cells and a connective tissue compartment composed of collagens, proteoglycans, fibrillins, and adhesion molecules (58). The ECM also functions as a reservoir for TGF-β, CTGF, and other growth factors and matricellular proteins that, together with the connective tissue compartment, control mesenchymal cell differentiation, function, and survival. Excessive connective tissue accumulation is due to overproduction by fibroblasts and related mesenchymal cells activated by soluble factors in an autocrine and/or paracrine manner, or by cell-cell or cell-ECM interactions. Impaired ECM degradation and turnover and expansion of the pool of mesenchymal cells in lesional tissues further contribute to ECM accumulation (58).
Skin inflammation and fibrosis in SSc. (A) In early diffuse cutaneous SSc, moderate fibrosis in the upper dermis and at the dermal-epidermal junction is accompanied by evidence of keratinocyte hypertrophy with a flattening of the epidermis, leading to loss of reticular structure and decreased length of rete pegs (fingerlike structures that project up from the dermis and down from the epidermis, increasing the area of contact between the layers of the skin). In addition, inflammatory infiltrates are found in the dermis and near the dermal-epidermal junction, predominantly around small blood vessels. (B) Early-stage diffuse disease showing profound dermal inflammation characterized by perivascular mononuclear cellular infiltrate composed of monocytes and activated lymphocytes, with perivascular fibrosis and loss of pericytes and vessel integrity. (C) In established fibrosis, collagen accumulation leads to dermal thickening and the deposition of dense and closely packed collagen fibers throughout the dermis, with the loss of the microvasculature and dermal structures and the dermis-subcutaneous adipose tissue interface. All images are stained with H&E and photographed using a Zeiss Axioscope confocal microscope under light field. Original magnification, ×100 (A and C); ×200 (B).
The most abundant ECM components are collagens, a family of proteins that consists of over two dozen structural proteins with critical roles in organ development, growth, and differentiation. The genes encoding the various collagen proteins harbor conserved regulatory elements that are specifically recognized by DNA-binding transcription factors. The complement of generalized and tissue-specific transcription factors implicated in regulating the expression of the genes encoding collagen proteins includes SP1, SMAD3, ETS1, early growth response 1, and CCAAT-binding factor, which mediate stimulation, and SP3, CCAAT/enhancer binding protein, Y box-binding protein 1, c-KROX, and FLI-1, which suppress transcription (59). These transcription factors not only interact with one another but also with cofactors and chromatin-modifying enzymes such as p300/CREB-binding protein (p300/CBP), p300/CBP–associated factor, and histone deacetylases that are recruited to target genes by DNA-bound transcription factors. The relative levels, intracellular activities, and interactions among transcription factors and cofactors are controlled by extracellular cues, and alterations in these contribute to persistent fibroblast activation in SSc (60).
Cellular determinants of fibrosis: fibroblasts, myofibroblasts, pericytes, and transdifferentiation. Fibroblasts are key effectors of the fibrotic process. Soluble mediators generated in the local cellular microenvironment by platelets, endothelial cells, epithelial cells, and inflammatory cells provide cues that induce fibroblasts to secrete collagens and other ECM macromolecules to adhere to, contract, organize, and remodel connective tissue; to secrete and activate growth factors and cytokines; and to undergo transdifferentiation into contractile myofibroblasts (61). Together, these biosynthetic, contractile, and adhesive functions enable fibroblasts to mediate effective wound healing. Under normal circumstances, the fibroblast repair program is self limited, but pathological fibrotic responses are characterized by sustained and amplified fibroblast activation. Inappropriate fibroblast activation is the fundamental pathogenetic alteration underlying fibrosis in SSc. Recent DNA microarray studies have revealed that fibroblasts from different anatomic locations differ markedly in their gene expression patterns and show site-specific variations in their transcriptional profiles that seem to be related to their location within the body (62). Moreover, these variations in gene expression are retained and might be considered the “positional memory” of these fibroblasts (62). It will be important to investigate how alterations in fibroblast positional memory might contribute to inappropriate fibroblast activation and fibrosis.
The tissue pool of biosynthetically activated mesenchymal cells contributing to ECM accumulation and remodeling is expanded in fibrosis by proliferation and migration of resident fibroblasts, by in situ transdifferentiation of other cell types, and by the influx of mesenchymal progenitor cells from the circulation (Figure 1; ref. 1). Myofibroblasts are specialized fibroblasts that show features of smooth muscle cell differentiation, having prominent cytoplasmic stress fibers with α-SMA. Myofibroblasts synthesize collagens and other ECM components and are a major source of TGF-β during the fibrotic response (61). In normal wound healing, myofibroblasts can be detected transiently in the granulation tissue; their removal by apoptosis is a crucial step in wound resolution (63). By contrast, myofibroblasts persist in pathological fibrogenesis, resulting in excessively contracted ECM characteristic of chronic scar (63). Myofibroblast transdifferentiation from normal fibroblasts can be induced by TGF-β and requires coexpression of the extra domain A (ED-A) variant form of fibronectin. The presence of α-SMA+ myofibroblasts expressing ED-A fibronectin and THY-1 is strongly associated with SSc but is absent from normal skin (64, 65). Pericytes are smooth muscle–like structural cells that are found normally in the walls of small blood vessels and have key roles in microvascular integrity and function (66). In early-stage SSc, pericytes are activated and express receptors for PDGF and THY-1, all of which are features of wound healing (65). Under certain conditions, epithelial cells can undergo transformation to fibroblasts (67). Epithelial-mesenchymal transition (EMT) has a vital role during embryonic development. The process of EMT can be induced by TGF-β in cultured alveolar epithelial cells and suppressed by bone morphogenetic protein-7. Pathological EMT has been documented in cancer as well as in renal and pulmonary fibrosis. To date, the role of EMT in the pathogenesis of tissue fibrosis in SSc has not been examined.
The BM can serve as a source of fibroblast progenitors. Circulating BM-derived mesenchymal progenitors continuously replenish the resident fibroblast population as part of normal tissue homeostasis and might have a role in fibrosis (61). Fibrocytes are CD34+ BM-derived precursors normally present in small numbers in the peripheral blood (68). Fibrocytes synthesize collagen and express CD14 (a monocyte marker) as well as chemokine receptors CC chemokine receptor 3 (CCR3), CCR5, and CXC chemokine receptor 4 (CXCR4). The cell surface expression of chemokine receptors allows fibrocytes to respond to chemokine gradients and to traffic to and accumulate in specific tissues. Stromal cell–derived factor-1 (SDF-1, also known as CXCL12) is an important chemokine regulator of progenitor trafficking, and its expression, as well as that of its receptor, CXCR4, is elevated in lesional skin in early SSc (69). The pathogenic role for fibrocyte trafficking into lesional tissue has been established in animal models using neutralizing antibodies and in mice genetically deficient in CXCR4 (70). Other studies have identified multipotent monocyte-derived mesenchymal progenitors in peripheral blood (71). The roles of pericytes, fibrocytes, and other monocyte-derived circulating fibroblast progenitors in the pathogenesis of fibrosis in SSc remain to be fully elucidated.
Molecular determinants of fibrosis: TGF-β. The expression of ECM genes is normally tightly regulated by paracrine and/or autocrine actions of soluble mediators as well as by cell-cell contact, hypoxia, and contact with the surrounding ECM (59). Of the multiple cytokines implicated in SSc, TGF-β is considered to be the master regulator of both physiological (wound healing and tissue repair) and pathological (scar) fibrogenesis (60). In addition, TGF-β has essential roles in normal tissue repair, angiogenesis, immune regulation, cell proliferation, and cell differentiation (72). TGF-β is secreted by platelets, monocytes/macrophages, T cells, and fibroblasts. Most cell types express specific cell surface receptors for TGF-β and secrete TGF-β as a latent complex that is sequestered in the ECM, in part by an interaction with fibrillin-1 (73). Activation of latent TGF-β to its biologically active form capable of inducing cellular responses can be mediated by integrins, thrombospondins, THY-1, or plasmin (72).
The responses elicited by TGF-β are context dependent and specific for target cell lineage. In mesenchymal cells, TGF-β functions as a potent fibrogenic stimulus by enhancing collagen synthesis, proliferation, migration, adhesion, and transdifferentiation into myofibroblasts (72). Binding of TGF-β to the TGFβRII triggers an intracellular signal transduction cascade that leads to the induction of target genes (74). The evolutionarily conserved canonical TGF-β signal transduction pathway involves phosphorylation of TGFβRI (also known as ALK5), a transmembrane serine-threonine kinase that in turn phosphorylates SMAD proteins (Figure 3). Ligand-induced phosphorylation of SMAD2 and SMAD3 allows them to form heterocomplexes with SMAD4 and translocate from the cytoplasm into the nucleus, where they recognize and bind to a _cis_-acting DNA sequence (CAGAC) that defines the consensus SMAD-binding element (SBE). Upon SBE binding, activated SMAD proteins recruit transcriptional cofactors to the DNA, resulting in gene transcription. The conserved SBE is found in the promoters of many TGF-β–inducible genes, including type I collagen, plasminogen activator inhibitor-1, α-SMA, and CTGF. Ligand-induced signal transduction through the SMAD proteins is tightly controlled by endogenous inhibitors such as SMAD7. Deregulated expression or function of activating and inhibitory SMAD proteins and their cofactors has been documented in SSc fibroblasts (1, 58, 60) and might contribute to the initiation or propagation of the abnormal fibrogenic response (Table 2).
Profibrotic signaling by TGF-β through SMAD-dependent pathways. The ECM serves as a reservoir for latent TGF-β, which is maintained in an inactive form by latent TGF-β binding proteins (LTBPs). Upon activation, TGF-β binds to its cell surface receptors and triggers SMAD-mediated intracellular signal transduction. Activated SMAD proteins accumulate in the nucleus and bind to conserved SBE regulatory elements in target genes, recruit coactivators and chromatin-modifying enzymes such as p300/CBP to the DNA, and induce mRNA synthesis and cellular responses. Inhibitory SMAD7 blocks ligand-induced SMAD protein phosphorylation and shuts down SMAD-mediated signaling.
Intracellular signaling molecules and pathways deregulated in SSc fibroblasts
Although the SMAD pathway is considered to be the central conduit for signals from the TGF-β receptors, emerging evidence highlights the importance of non-SMAD pathways (75). Non-SMAD molecules activated by TGF-β include protein kinases (the MAPKs p38 and JNK, focal adhesion kinase [FAK], and TGF-β activated kinase 1), lipid kinases (such as PI3K and its downstream target, AKT), and the calcium-dependent phosphatase calcineurin (Figure 4). Recent studies indicate novel roles for the tyrosine kinase c-ABL (76) and early growth response 1 (77) in mediating the stimulation of profibrotic responses induced by TGF-β in fibroblasts. These novel non-SMAD pathways interact with each other and with SMAD proteins in complex, cell lineage–specific signaling networks.
TGF-β signaling through non-SMAD pathways. Receptor activation by TGF-β can cause activation of non-SMAD pathways involved in regulating cell proliferation, cytoskeletal rearrangement, ECM synthesis, and apoptosis. Activation of intracellular protein and lipid kinase cascades results in activation of DNA-binding transcription factors and regulation of gene expression. These signal transdution pathways might converge on or interact with the canonical SMAD pathway or operate completely independent of SMAD pathways. AP-1, acivator protein 1; EGR-1, early growth response 1; PAK2, p21-activated kinase 2; ROCK, Rho-associated, coiled-coil containing protein kinase 1; TAB1/2, TAK1-binding protein 1/2; TAK1, TGF-β activated kinase 1.
Molecular effectors of fibrosis: cytokines, growth factors, and chemokines. In addition to TGF-β, an expanding list of cytokines, growth factors, and chemokines that regulate mesenchymal cell function has been found to be overexpressed or abnormally regulated in patients with SSc. These soluble factors contribute to the pathogenesis of fibrosis and represent attractive potential targets for therapy. CTGF is a cysteine-rich modular protein belonging to the Cyr61/CTGF/NOV (CCN) family of matricellular growth factors, all of which function as adaptor molecules connecting the cell surface to the ECM (78). The CCN proteins have adhesive abilities that control cell attachment and migration, modulate the activities of TGF-β and other cytokines, and regulate cell differentiation, proliferation, apoptosis, and ECM synthesis (78). Although expressed at only very low levels in normal tissues, expression of CTGF is induced by TGF-β as well as ET-1 and hypoxia (78). The levels of CTGF are markedly elevated in lesional tissues from patients with SSc (79) and in mouse models of scleroderma (19). Because many activities of CTGF parallel those induced by TGF-β, it has been suggested that at least some TGF-β responses are mediated through endogenous CTGF (78). The identity of the fibroblast receptor for CTGF and the mechanism of action underlying profibrotic responses elicited by this matricellular protein are still incompletely characterized. PDGF, which is produced by macrophages, endothelial cells, and fibroblasts in addition to platelets, is a potent mitogen and chemoattractant for fibroblasts and can induce them to synthesize collagen, fibronectin, and proteoglycans and to secrete TGF-β1, monocyte chemoattractant protein 1 (MCP-1), and IL-6. The expression of PDGF and its receptors is elevated on SSc fibroblasts and in lesional tissues (80) and in bronchoalveolar lavage fluid of SSc patients (81). Autoantibodies specific for the PDGF receptor can be detected in serum from patients with SSc (51). These antibodies have been shown to induce activation of normal fibroblasts in vitro through PDGF receptor–mediated activation of the intracellular Ha-Ras–ERK1/2 cascade and generation of ROS (51).
The cytokines IL-4 and IL-13 have major roles in the pathogenesis of Th2 cell–mediated diseases such as asthma and in the pathogenesis of fibrotic disorders (40). In normal fibroblasts, IL-4 stimulates proliferation, chemotaxis, and collagen synthesis and enhances the production of TGF-β and CTGF (82). Levels of IL-4 are elevated in the sera of patients with SSc (83), and the number of IL-4–producing T cells is increased in their peripheral blood (84, 85). Expression of IL-4 protein and mRNA is markedly elevated in SSc lesional skin and cultured fibroblasts (86). The profibrotic activities of IL-13 involve both direct fibroblast activation and indirect mechanisms due to stimulation of TGF-β (87, 88). Although levels of IL-13 are elevated in the serum of patients with SSc, its role in inducing and maintaining tissue fibrosis in SSc remains to be determined. Chemokines have a broad range of cellular targets and biological activities and are increasingly implicated in fibrosis (60). In vitro, MCP-1 stimulates collagen production directly or indirectly through the induction of endogenous TGF-β and IL-4 (89). Levels of MCP-1 and of multiple other chemokines are elevated in the serum and bronchoalveolar lavage fluid from patients with SSc and correlate with the severity of fibrosis (90, 91). The expression of MCP-1 and MCP-3 and of their receptors is markedly elevated in lesional skin and cultured fibroblasts from patients with SSc, particularly in early-stage disease (92, 93).
Endogenous mechanisms for attenuating the fibrotic response. To avoid excessive scarring following injury, redundant biological mechanisms have evolved to suppress ECM synthesis and fibroblast proliferation and differentiation and thereby slow the fibrotic process. Appropriate temporospatial utilization of these mechanisms allows repair of damaged tissue without the development of fibrosis. The prototypic Th1 cytokine IFN-γ suppresses the expression of the genes encoding the collagen proteins and abrogates the stimulatory effects of TGF-β (94, 95). IFN-γ is also a potent inhibitor of fibroblast proliferation, ECM contraction, and fibroblast transdifferentiation to myofibroblasts (60). It has been shown that IFN-γ suppresses fibrotic responses in vivo and in vitro (96) and therefore might have a physiological function as an endogenous natural antifibrotic mediator. Although some studies suggest that IL-10 (alone or in combination with IFN-γ) also functions as a repressor of the fibrotic process (97), the role of IL-10 in SSc remains to be clarified.
Fibroblasts are equipped with endogenous molecules that can suppress the stimulation of ECM gene expression. For example, by blocking SMAD-mediated TGF-β signal transduction, SMAD7 limits the intensity and duration of the TGF-β response and prevents prolonged stimulation of collagen synthesis by normal fibroblasts (98). Impaired SMAD7 expression or function has been demonstrated in SSc fibroblasts (99). Additional molecules that function as intrinsic repressors of basal or inducible collagen synthesis include the transcription factors SP3, FLI-1, p53, RAS, and the nuclear hormone receptor PPARγ (100–103). Diminished expression or function of these endogenous inhibitors, or their impaired responsiveness to extracellular ligands, could contribute to excessive and sustained ECM upregulation in fibrosis.
SSc fibroblasts: autonomously activated effector cells in fibrosis. Over 3 decades ago, E.C. LeRoy demonstrated that fibroblasts explanted from lesional skin or the fibrotic lungs of patients with SSc displayed an abnormal activated phenotype that persisted for several passages in vitro (104). This seminal observation focused attention on the pivotal role of the fibroblast in the pathogenesis of fibrosis and spawned extensive research into the underlying mechanisms (105). Persistent fibroblast activation in the absence of the fibrotic tissue milieu, confirmed more recently by DNA microarray studies (52, 106, 107), indicates autonomous, signal-independent alterations in cell function. The SSc phenotype is characterized by enhanced ECM synthesis, constitutive secretion of cytokines and chemokines, and increased expression of cell surface receptors for fibrogenic signaling mediators (60). Moreover, SSc fibroblasts show evidence of myofibroblast transdifferentiation, due in part to constitutive activation of FAK (108). Elevated expression of the prosurvival factors BCL2 and AKT in fibroblasts from patients with SSc might account for their relative resistance to apoptosis (109–111). Because most of the SSc fibroblast characteristics can be induced in normal fibroblasts by incubation with TGF-β, it has been proposed that the SSc phenotype is due to autocrine TGF-β signaling (60). The levels of receptors for TGF-β are elevated on SSc fibroblasts, permitting them to mount a robust response to endogenously produced TGF-β or to subthreshold levels of exogenous TGF-β in their environment (112, 113). Furthermore, thrombospondin and αvβ3 integrins, both of which can mediate latent TGF-β activation, are elevated on SSc fibroblasts (114, 115). Consistent with the autocrine TGF-β hypothesis, SSc fibroblasts show evidence of ligand-independent intracellular TGF-β signaling, with elevated expression and nuclear accumulation of activated SMAD3 (99, 116) and its constitutive interaction with the p300/CBP coactivator (117, 118). Other studies have demonstrated defective expression of function of endogenous suppressors of TGF-β signaling such as FLI-1 and SMAD7 (27, 98, 119), and of PPARγ (J. Varga and M. Whitfield, unpublished observations).
However, fibroblast activation mediated by an autocrine TGF-β–SMAD signaling pathway cannot fully account for all of the phenotypic hallmarks of SSc fibroblasts, such as constitutive production of CTGF or ET-1 (120). Furthermore, small molecule inhibitors of TGFβRI-dependent SMAD signaling fail to fully normalize SSc fibroblasts in vitro (113, 121, 122). These recent observations therefore imply that SMAD-independent TGF-β signaling mechanisms, TGF-β–independent activation events, or both also have a role in inducing or maintaining the SSc phenotype. The autonomous phenotype of SSc fibroblasts might also result from altered integrin signaling from the surrounding ECM (123). Recent evidence suggests that epigenetic regulation in SSc fibroblasts might contribute to their persistent dysfunction (119). For example, the gene encoding FLI-1, an important negative regulator of collagen synthesis, seems to be constitutively silenced in SSc fibroblasts through a mechanism that involves DNA methylation or chromatin histone deacetylation (119). Silencing the expression of this negative regulator is associated with increased collagen synthesis (119).