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The recognition of the role of the myofibroblast in granulation tissue contraction and connective tissue remodeling during fibrocontractive diseases has allowed a theoretical and practical progress in the understanding of these pathologies. The observation that TGF-β is the key cytokine in myofibroblast differentiation, correlated with its role in collagen synthesis promotion, shows a coordinated mechanism in connective tissue remodeling. Recent work has furnished new knowledge concerning the molecular mechanisms of tension production by the myofibroblast and indicated that the N-terminal peptide of α-smooth muscle actin exerts an inhibitory action on myofibroblast contraction. Moreover the multiple derivation, both local and from circulating cells, of the myofibroblast begins to be understood. These data point to the myofibroblast as a major regulator of connective tissue remodeling and in turn of epithelial organization.

Mechanical forces are central to the control of 3D spatial organisation in connective tissue remodelling, repair and scarring. How this operates is increasingly seen as the next major research focus in this area. In contrast to mechanics at the tissue-scale, cell-level mechanics (or cytomechanics) is dominated by the cell-matrix-material interplay. The matrix completely modifies incoming, external mechanical cues whilst fibroblasts generate their own local forces to monitor and remodel that same matrix. By understanding these cell-material dynamics it is becoming clear how musculo-skeletal cells predictably adapt their responses to the perceived mechanical environment. Elements such as shape-change, orientation, matrix synthesis, migration can be explained relatively simply in terms of stress-shielding, matrix anisotropy, force vectors, rate of strain etc. In turn these translate into control of 3D tissue structure. (i) Anisotropy in fibrous collagen seems to be particularly important in modulating cytomechanical cues and so in controlling 3D remodelling. It is likely, then that the disorganised structure of scars resists subsequent remodelling because that same structural disorganisation obscures normal cytomechanical signaling. (ii) Reinterpretation of the role of cell force-generation suggests that this is necessary for (a) testing substrate material properties and (b) to produce incremental changes in tissue dimensions during remodelling. By understanding this interplay of cell-level forces with extracellular material properties (i.e., cytomechanics) new interpretations of connective tissue control and dysfunction are possible. Because of the predictable nature of (cyto) mechanics, these mechanisms are more direct and less complex than their biological counterparts.

Although mammalian connective tissues have a number of secondary functions (metabolic, depot, pseudo-regulatory), it is axiomatic that their PRIMARY role is structural and supportive. However, this mechanical function operates at both the macro and the cellular scales (micro-nano). Carriage of load at the macro, or tissue scale is familiar and relatively simple for us to understand in terms of the mechanical properties of the extra-cellular matrix (ECM) material. We are naturally familiar with the normal adaptation of structural tissues, their growth/ adaptation and reshaping to habitual use, aging and disease. We take for granted their repair and even fibrosis after injury and their ability to withstand the habitual mechanical loads placed on them. Our neuro-muscular systems are exquisitely tuned to use and to protect them. Yet to understand how these dynamics operate (and fail) we must look to the resident cells, which create and manipulate the ECM mechanical properties. This involves understanding the mechanical environment down at the cellular level; a much more tricky task. This world of cytomechanics is very unfamiliar and holds many surprises for the unwary biologist. Two examples, illustrating this topsy-turvy world are (i) the very limited DIRECT effects of gravity on cells, yet (ii) their exaggerated sensitivity to fluid viscosity.

It is assumed as a fixed point here that understanding the mechanisms of growth, adaptation and restoration of ECM material properties must be the focus of research in our field. Control of ‘material properties’ (understood rather better outside the biological disciplines) is dominated at least as much by micro-architecture and polymer distribution as by composition and density. The assumption of this author is that it is essential to identify the PRIMARY environmental cues used by tissue fibroblasts and the way in which they might operate, or fail. Since fibroblasts live in a sea of load-bearing polymer aggregate with the role of maintaining mechanical function, it seems inevitable that some of the most potent PRIMARY environmental cues will be mechanical. This chapter explores current concepts and knowledge of how cells monitor, respond to (and potentially misinterpret) local, micro-scale mechanical forces.

The role of myofibroblasts in various fibrotic disorders is currently well established. These smooth-muscle-like fibroblasts promote deposition of ECM proteins and contractility of lung parenchyma. The present studies were performed to characterize the contractile activity of SSc lung fibroblasts. Previously, we demonstrated that the early stages of interstitial lung disease of SSc are characterized by a prominence of cells prossessing a myofibroblast phenotype. A major feature of such myofibroblasts is contractility, explained by an over-expression of α-smooth muscle actin. Here, we demonstrate for the first time that the contractility of SSc lung fibroblasts dependends on expression of CTGF as well, and that the VWC domain is primarily responsible for the contractile activity of CTGF in human lung fibroblasts. Future studies are required to identify the mechanisms by which CTGF stimulates collagen gel contraction.

Fibroblasts are a heterogeneous population of structural cells whose primary function is the production of extracellular matrix for normal tissue maintenance and repair. However, fibroblasts provide much more than structural support as they synthesize and respond to many different cytokines and lipid mediators and are intimately involved in the processes of inflammation. It is now appreciated that fibroblasts exhibit phenotypic heterogeneity, differing not only between organ systems, but also within a given anatomical site. Subtypes of fibroblasts can be identified by the expression of markers such as Thy-1, a cell surface glycoprotein of unknown function. Initial characterization of fibroblasts as Thy-1 or Thy-1 can be performed by immunofluorescence or flow cytometry. They can be sorted according to their expression of Thy-1 by fluorescence-activated cell sorting (FACS), cloning and/or magnetic beading, yielding greater than 99% purity. Fibroblasts that are separated into Thy-1 and Thy-1 subsets exhibit differences in their morphological, immunological and proliferative responses and ability to differentiate into a-smooth muscle actin-expressing myofibroblasts and adipocyte-like lipofibroblasts, key cells for wound healing and fibrotic disorders. The identification of Thy-1 as a surface marker by which to separate fibroblast subtypes has yielded vital insight into diseases such as scarring and wound healing and highlights the concept of fibroblast heterogeneity. Future research into fibroblast subsets may lead to the tissue-specific treatment of disease such as idiopathic pulmonary fibrosis and Graves’ ophthalmopathy.

Asthma is characterized by functional and structural alterations of the bronchial epithelium, chronic airway inflammation and remodeling of the normal bronchial architecture. Bronchial myofibroblasts are thought to play a crucial role in the pathogenesis of subepithelial fibrosis, a prominent aspect of the remodeling process. The results of the studies reviewed in this report indicate that circulating fibrocytes contribute to the bronchial myofibroblast population and may be responsible for the excessive collagen deposition below the epithelial basement membrane in asthma. More information on the mechanisms regulating the migration and differentiation of these cells in the asthmatic airways may help identify novel targets for therapeutic intervention.

Most of the present knowledge on the pathomechanism of renal fibrosis is based on experimental studies with laboratory animals. Today, a variety of genetic and inducible animal models that mimic primary causes of human disease, such as diabetes mellitus, glomerulonephritis or lupus erythematodes are available. However, only few of these models progress consistently to interstitial fibrosis in the kidney involving interestitial fiberosis, tubular atrophy and glomerulosclerosis, common features of renal fibrogenesis. In this chapter, three different mouse models of human kidney disease are described highlighting their utility to study pathways leading to renal fibrosis.

The most common diseases that cause end stage renal failure (ESRF) differ significantly in their underlying primary pathomechanisms. Glomerulonephritis due to primary glom-erular inflammation, metabolic diseases such as diabetes mellitus, cystic nephropathies such as polycystic kidney disease, interstitial nephritis due to primary interstitial inflammation, and vasculopathies are among the leading causes of end stage renal failure. Despite the diversity of primary patho-mechanisms associated with these different kidney diseases, they all lead to an indistinguishable scarred/fibrotic kidney. The observation that chronic renal failure seems to possess common patterns independent of the underlying disease, has resulted in the speculation for the existence of a common pathogenic pathway leading to ESRF associated with fibrosis.

Peritoneal dialysis (PD) is an alternative to hemodialysis for the treatment of end-stage renal disease and is based on the use of the peritoneum as a semi-permeable membrane for water and solutes. Peritoneal membrane fibrosis (or sclerosis) is one of the most frequent complications of PD that includes a wide spectrum of peritoneal structural changes, ranging from mild inflammation to severe sclerosing peritonitis and encapsulating-sclerosing peritonitis. In parallel with fibrosis, the peritoneum shows a progressive increase of capillary number (angiogenesis) and vasculopathy, which are involved in increased small solute transport across the peritoneal membrane and ultrafiltration failure. Local production of vascular endothelial growth factor (VEGF) during PD appears to play a central role in the processes leading to peritoneal angiogenesis and functional decline. The most important factors of the PD solutions responsible of peritoneal deterioration are glucose and glucose degradation products, which stimulate transforming growth factor-β (TGF-β) and VEGF production by mesothelial cells (MC). TGF-β is a potent pro-fibrotic factor and inducer of epithelial-mesenchymal transition (EMT) of the MC.

This review discusses the mechanism implicated in peritoneal structural alteration and points to EMT of MC as protagonist and starter of peritoneal membrane injury through the increase of submesothelial fibroblast population. Possible mechanisms of regulation and new targets for inhibition of EMT or its deleterious effects are proposed.

While some progress has been made recently in identifying potential candidate genes that may be important in pathogenesis of pulmonary fibrosis, the list certainly is not complete. Using DNA microarray analysis to analyze the lung gene expression profiles in a rat model of pulmonary fibrosis has revealed over 600 genes that were ≥ 2-fold up or down-regulated. The highest up-regulated gene is identified as FIZZ1 (Found in Inflammatory Zone), and found to be expressed primarily by lung epithelial cells and not in fibroblasts. Further analysis shows that FIZZ1 stimulated α-smooth muscle actin and type I collagen expression independently of transforming growth factor-β, suggesting its potential as an inducer of myofibroblast differentiation. IL-4 and IL-13 are found to induce FIZZ1 expression in type II alveolar epithelial cells via a STAT6 mediated mechanism, and interestingly IL-4/IL-13 or STAT6 deficient mice shows reduced lung FIZZ1 expression and fibrosis. Thus the potential role of FIZZ1 in fibrosis represents an example of epithelial-mesenchymal crosstalk of significance to the genesis of myofibroblasts in lung fibrosis.

Cancer cell invasion necessitates the participation of host cells. One of the cell types that stimulates invasion of colon and other cancer cells is the myofibroblast, as evidenced from the histology of cancer and from coculture experiments. Cancer cells produce transforming growth factor-β (TGF-β) and TGF-β converts fibroblasts into pro-invasive myofibroblasts. In the in vitro system with human cancer cell lines and freshly isolated stromal cells, the pro-invasive activity of myofibroblasts is due to the combined action of Hepatocyte growth factor/scatter factor (HGF/SF) and tenascin-C, two molecules known to promote invasion in clinical tumors and their experimental surrogates. The myofibroblasts are themselves invasive and this activity is stimulated by TGF-β. N-cadherin is implicated in the invasion response of myofibroblasts. The question now is which of the multiple factors present in the tumor ecosystem is responsible for the pro-invasive switch that turns a benign tumor into a malignant one.

Hepatic stellate cells are perisinusoidal fibroblasts that transdifferentiate into myofibroblasts in response to paracrine factors released from cancer cells and cancer-activated endothelial cells. Tumor-associated myofibroblasts exhibit contractility, proliferation, production of extracellular matrix molecules and metalloproteases. They secrete soluble factors inducing proinflammatory and immune suppressant effects. Myofibroblasts are present in avascular micrometastasis prior to endothelial cell recruitment, and act as supporting stroma for tumor neoangiogenesis. In replacement-type cancer growth, the reticular arrangement of tumor-associated myofibroblasts provides a sinusoidal-type angiogenic pattern. In pushing-type cancer growth, fibrous tract-forming myofibroblasts support a portal-type angiogenic pattern. Additionally, tumor-activated myofibroblasts support cancer development via paracrine release of tumor invasion and proliferation-stimulating factors. In summary, this information suggests that the ability of cancer cells to activate hepatic stellate cells and to collaborate with myofibroblasts along the metastatic process may represent a key phenotypic property of liver-colonizing cancer cells. On the other hand, experimental anti-tumor and anti-angiogenic agents have inhibited intrametastatic recruitment and proangiogenic activities of hepatic myofibroblasts, suggesting that targeting tumorigenic effects of these cells may contribute to hepatic metastasis inhibition.