Volume 9 Supplement 2
Fibrosis in connective tissue disease: the role of the myofibroblast and fibroblast-epithelial cell interactions
© BioMed Central Ltd 2007
Published: 15 August 2007
Fibrosis, characterized by excessive extracellular matrix accumulation, is a common feature of many connective tissue diseases, notably scleroderma (systemic sclerosis). Experimental studies suggest that a complex network of intercellular interactions involving endothelial cells, epithelial cells, fibroblasts and immune cells, using an array of molecular mediators, drives the pathogenic events that lead to fibrosis. Transforming growth factor-β and endothelin-1, which are part of a cytokine hierarchy with connective tissue growth factor, are key mediators of fibrogenesis and are primarily responsible for the differentiation of fibroblasts toward a myofibroblast phenotype. The tight skin mouse (Tsk-1) model of cutaneous fibrosis suggests that numerous other genes may also be important.
Fibrosis, the result of excess synthesis and deposition of collagen, is a feature of many connective tissue diseases (CTDs) and is the hallmark of scleroderma (systemic sclerosis [SSc]). In this rare, progressive and life-threatening autoimmune disease, fibrosis affects not only the skin but also internal organs such as lungs and kidneys, leading to organ dysfunction and failure . In SSc, fibrosis is the final stage in a series of pathological events that begins with vascular dysfunction, manifesting as altered vascular tone, endothelial activation and oxidative stress, followed by immunological activation and leucocyte-mediated extra-vascular inflammation . A complex network of intercellular interactions that involves a diverse range of molecules, including growth factors, cytokines, chemokines and endothelin, is believed to drive the pathological events that ultimately result in uncontrolled connective tissue fibrosis. In this review, which describes results from in vitro systems and animal models of cutaneous and visceral fibrosis, we explore cellular and molecular events associated with the initiation and maintenance of fibrosis in CTDs.
Initiators of fibrosis in connective tissue diseases
These findings suggest that induction of ET-1 by TGF-β requires an activation step, perhaps dependent on inflammatory status or other cellular signals. Interestingly, blocking the effects of TGF-β with a soluble TGF-β receptor (STR) antagonist leads to inhibition of ET-1 production by both injured stellate and normal endothelial cells in vivo (Figure 3b) . The concept of a cytokine hierarchy, in which ET-1 is induced by TGF-β, is also supported by data from experimental studies that showed that ET-1 does not increase TGF-β gene expression or increase TGF-β protein production in vascular smooth muscle cells .
As mentioned above, CTGF is a common target for both TGF-β and ET-1. Recent studies showed that ET-1 regulates CTGF independently of TGF-β and that, in turn, CTGF mediates ET-1 induced ECM accumulation. In an experimental study in which vascular smooth muscle cells were treated with 8 to 10 mol/l ET-1, there was a marked increase in type 1 collagen expression compared with control cells. However, when the treated cells were exposed to a CTGF antisense oligonucleotide that blocks the action of CTGF, type 1 collagen expression was markedly suppressed. Similar results were reported for levels of fibronectin, the production of which is also influenced by both ET-1 and CTGF .
From the available evidence it seems clear that TGF-β can induce ET-1, although its induction is variable and may be dependent on other signals such as inflammation. Both TGF-β and ET-1 can induce CTGF, with ET-1 acting independently of TGF-β. CTGF, a cytokine that stimulates fibroblast growth and upregulation of collagen and fibronectin in vitro , is a difficult molecule to study experimentally, but there is evidence to suggest it mediates certain effects of TGF-β and ET-1 on ECM production [12, 13]. More complex interactions between TGF-β and ET-1 are likely to depend on cross-regulation of receptors and activators.
Models of the profibrotic microenvironment
Models of organ fibrosis
The in vivo effects of ET-1 on cardiac, renal and pulmonary tissue have been studied in a number of animal models of fibrosis. Experimental work on salt-induced hypertension in rodents (deoxycorticosterone acetate-salt hypertensive rats), for example, found a marked increase in fibrosis in the myocardium of affected animals compared with controls. However, when animals were given treatment with a selective endothelin-1 receptor subtype A antagonist, effective endothelin blockade resulted in no collagen deposition in cardiac tissue . Earlier work on ET-1 transgenic mice, created by the transfer of the human ET-1 gene locus into the germline of mice, resulted in a pathological phenotype characterized by glomerulosclerosis, interstitial fibrosis and renal cysts without hypertension. In this model, pronounced renal fibrosis was associated with an age-dependent decrease in glomerular filtration that culminated in fatal kidney disease . The severe ET-1-induced renal disease seen in this model of fibrosis is consistent with evidence from patients that an activated renal endothelin system is associated with glomerular and interstitial injury . Other work in a transgenic mouse model has shown ET-1 over-expression to lead to the recruitment of inflammatory cells in respiratory tissues and subsequent development of progressive pulmonary fibrosis .
Models of skin fibrosis
More recently, a tight-skin mouse (Tsk-1) model has been used to enhance understanding of the underlying pathology of skin fibrosis in SSc. Tsk-1 mice develop tightening of the skin to the underlying tissues, due to a mutation in the gene encoding fibrillin 1. Fibrillin is a large ECM structural protein and the major component of microfibrils, found with or without associated elastin . The primary role of fibrillin is to form the scaffolding for elastic fibres. In Tsk-1 mice, fibrillin appears to alter fibrillin matrix structure, with a notable. increase in fibrillin in the superficial fascia of these animals. Tsk-1 mice exhibit increased elastogenesis in the superficial fascia (gain-of-function) and a loss of the dense elastic fibre band normally found at the interface between the intradermal muscle and deep connective tissue (loss-of-function).
The Tsk-1 mouse clearly provides a good model for evaluating how matrix regulates its own composition. Although there is evidence from this model to suggest that Tsk fibrillin regulates gene expression through altered interactions with other matrix proteins, the identities of these mediators remain to be elucidated.
A further model is the bleomycin-induced model of dermal fibrosis, in which bleomycin has been found to increase expression of ECM proteins, potentially via the mediation of TGF-β and CTGF/CCN2 . This model has been widely used to evaluate potential antifibrotic drugs. For example, using this model, the protein kinase inhibitor imatinib mesylate (a selective, dual inhibitor of TGF-β and platelet-derived growth factor) was shown to have potent antifibrotic effects , although perhaps not in later stages of fibrosis . Peptide inhibitors of TGF-β1 applied topically are also showing some promise . Other agents targeting other components of the fibrotic process have similarly demonstrated attenuation of bleomycin-induced skin sclerosis in mice .
Fibroblast to myofibroblast differentiation
In comparison with normal skin, biopsies of skin from SSc patients show a significant increase in myofibroblasts, particularly in the deep dermis. These cells, which are characterized by the presence of stress fibres containing α-smooth muscle actin (SMA), play a pivotal role in ECM deposition and wound contraction during the normal wound healing process . A strong correlation has also been observed between myofibroblast intensity in the skin of SSc patients and the Rodnan skin score, which reflects disease throughout the body. Thus, cutaneous changes in SSc patients may reflect what is happening systemically, even though the disease may not be clinically evident in large parts of the body.
There has been much speculation on the origin of activated fibroblasts in the skin of SSc patients and what influences their differentiation toward a myofibroblast phenotype. There is evidence that activated fibroblasts may arise from local conversion of dermal fibroblasts by soluble factors, a finding that is consistent with our CTGF staining data. It is also possible that activated fibroblasts are recruited from circulating or resting mesenchymal precursor cells in the tissue, or induced by cell-cell contacts, cytokines or other chemokines, or selected as an active subpopulation by one or other mechanism. Alternatively, they may be generated by clonal selection. As is discussed in greater detail below, in wound healing the differentiation of fibroblasts toward a myofibroblast phenotype is governed by several factors, including the proximity of fibroblasts to keratinocytes, the presence of endogenous TGF-β, and direct cell-cell contact between fibroblasts and keratinocytes to allow initial TGF-β activation.
Orchestrating fibrosis: interactions between endothelial and other cells
In the initial stages of any fibrotic condition, and certainly in SSc, there appears to be significant crosstalk between different cell types, including endothelial and mesenchymal cells. In vitro studies carried out during different stages of wound healing have shed light on the general activation mechanisms that are involved in fibrosis and on the intercellular crosstalk that influences cell behaviour. In the very early stages of wound healing, fibroblast activation is induced by epithelial cells in response to injury. In the skin, regulation during this very early phase of wound healing is due to direct contact between keratinocytes and the underlying fibroblasts, or to the release of cytokines from one cell to another, or, indeed, a combination of both. Interaction of fibroblasts and keratinocytes has also been found to modulate levels of two key enzymes that are involved in wound healing and remodelling: matrix metalloproteinase-2 and -9 . This interaction may be critical for optimal healing quality at a later stage in the wound healing process.
Selected genes induced by keratinocyte-fibroblast interactions
Type of gene
Extracellular matrix (ECM)
Hyaluronan synthase 2
Monocyte chemoattractant protein (MCP)-1
Latent TGF-β-binding protein (LTBP)1
Connective tissue growth factor (CTGF)
Heparin-binding epithelial growth factor (HB-EGF)
Granulocyte-colony stimulating factor (G-CSF)
α-Smooth muscle actin (α-SMA)
Vasodilator-stimulated phosphoprotein (VASP)
Myosin light chain kinase (MLCK)
Results from these experimental studies indicate that highly complex epithelial-mesenchymal crosstalk is involved in controlling the extent to which fibroblastic cells are induced in the underlying mesenchyma and in their subsequent differentiation to myofibroblasts.
As the experimental studies described here illustrate, myofibroblasts play a central role in mediating tissue fibrosis, which is the major clinical manifestation of CTDs such as SSc. TGF-β, ET-1, and CTGF are among the most important molecular mediators of fibrogenesis, with TGF-β and ET-1 intimately involved in the differentiation of fibroblasts toward a myofibroblast phenotype, characterized by the presence of stress fibres containing α-SMA. Cell culture experiments show that direct cell-cell contact between fibroblasts and keratinocytes is important for TGF-β activation and initiation of myofibroblast formation. Data from animal models of cardiac, renal, pulmonary and cutaneous fibrosis confirm the importance of these profibrotic mediators in fibrogenesis, while also highlighting other molecular entities that may be important as well. The Tsk-1 model of cutaneous fibrosis, for example, illustrates the importance of Tsk fibrillin and how it regulates gene expression through altered interactions with other matrix proteins.
connective tissue disease
connective tissue growth factor
smooth muscle actin
transforming growth factor
The authors would like to acknowledge medical writing support funded by an educational grant from Actelion Pharmaceuticals Ltd.
This article is part of Arthritis Research & Therapy Volume 9 Supplement 2: Advances in systemic sclerosis and related fibrotic and vascular conditions, and is based on presentations made at a symposium entitled Advances in systemic sclerosis and connective tissue disease, sponsored by Actelion Pharmaceuticals Ltd, held in Athens, Greece in April 2006. The full contents of the supplement are available online at http://arthritis-research.com/supplements/9/S2. This supplement has been supported by an educational grant from Actelion Pharmaceuticals Ltd.
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