Role of growth factors in the pathogenesis of tissue fibrosis in systemic sclerosis.

The most severe clinical and pathologic manifestations of systemic sclerosis (SSc) are the result of a fibrotic process characterized by the excessive and often progressive deposition of collagen and other connective tissue macromolecules in skin and numerous internal organs. The mechanisms involved in the initiation and progression of the remarkable fibrotic process in SSc remain largely unknown. Extensive recent studies have indicated that a variety of polypeptide growth factors play a crucial role in this process. The most commonly implicated growth factors include transforming growth factor beta (TGF-β), connective tissue growth factor (CTGF), platelet derived growth factor (PDGF), and vascular endothelial growth factor (VEGF). Here, the experimental evidence supporting the participation of various growth factors in the pathogenesis of the fibrotic process in SSc and the molecular mechanisms involved will be reviewed

Strategies for anti-fibrotic therapies.

The fibrotic diseases encompass a wide spectrum of entities including such multisystemic diseases as systemic sclerosis, nephrogenic systemic fibrosis and sclerodermatous graft versus host disease, as well as organ-specific disorders such as pulmonary, liver, and kidney fibrosis. Collectively, given the wide variety of affected organs, the chronic nature of the fibrotic processes, and the large number of individuals suffering their devastating effects, these diseases pose one of the most serious health problems in current medicine and a serious economic burden to society. Despite these considerations there is currently no accepted effective treatment. However, remarkable progress has been achieved in the elucidation of their pathogenesis including the identification of the critical role of myofibroblasts and the determination of molecular mechanisms that result in the transcriptional activation of the genes responsible for the fibrotic process. Here we review the origin of the myofibroblast and discuss the crucial regulatory pathways involving multiple growth factors and cytokines that participate in the pathogenesis of the fibrotic process. Potentially effective therapeutic strategies based upon this new information are considered in detail and the major challenges that remain and their possible solutions are presented. It is expected that translational efforts devoted to convert this new knowledge into novel and effective anti-fibrotic drugs will be forthcoming in the near future. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease

Antifibrotic Effects of Vibratory Stimulation

Extracellular matrix (ECM) is a dynamic and complex environment characterized by biophysical, mechanical and biochemical properties specific for each tissue. Cells constantly experience dynamic mechanical loadings that include compression, shear, tension, hydrostatic pressure, and interstitial fluid flow. Through the process of mechano-chemical conversion, mechanical stimulation activates intracellular biochemical signaling that affects many aspects of cell behavior including cell proliferation and differentiation, as well as ECM deposition and organization during development, wound healing, and pathological diseases. Despite significant advances in understanding the dynamic relationship between mechanical forces and matrix remodeling, many of the unique mechanisms and associated responses to various physical stimuli remain to be elucidated. Fibrosis is a complex disease predominantly characterized by excessive and abnormal fibrous ECM deposition that leads to the failure of various organs: lung, liver, kidney and skin. During the normal wound healing process, injured tissue progresses through phases of hemostasis, acute inflammation, granulation tissue/fibroproliferative, matrix formation, and remodeling. Collectively, the fibro-proliferative stage terminates with the restoration of ECM homeostasis and the disappearance of myofibroblasts, probably through apoptosis. However, the chronic presence of diverse injuries, commonly involving the abnormal persistence of several profibrotic cytokines results in sustained myofibroblast activation, excessive ECM deposition, scar formation, and organ failure. Specifically, transforming growth factor-β (TGF-β) is a master switch that activates critical downstream molecules in the progression of fibrotic disease. Although various strategies designed to interfere with TGF-β expression, receptor binding, and signal transduction have been studied, a clinically safe and effective therapy has not yet been developed. The superficial layer of the lamina propria (SLLP) in the human vocal folds experiences a unique mechanical microenvironment of high frequency vibration during voice production. The presence of macrophages/myofibroblasts in the SLLP of healthy patients suggests that the mechanical stresses imposed during routine speech result in repetitive microtrauma, which is generally repaired without permanent alterations in vocal fold matrix composition or vocal quality. In addition, mechanical forces have recently been shown to alter the fibrotic phenotype in fibrotic fibroblasts. Therefore, the objective of this research is to understand the mechanisms regulating fibroblast matrix metabolism in the SLLP and investigate the potential of vibratory stimulation for treatment of fibrotic diseases. First, we characterized the transcriptional and translational changes of human dermal fibroblasts in response to vibratory stimulation and demonstrated that vibratory stimulation led to the down-regulation of the TGF-β signaling through reduced expression of TGF-β receptors and Smad signal transduction molecules and increased expression of SMAD7, ubiquitin ligases, and SIK1 and SKIL, transcriptional repressors responsible for signaling inhibition. Second, we then investigated the effects of variable vibratory regimes defined by varying frequency, amplitude, and duration on the expression of ECM-related transcripts in human dermal fibroblast and found significant dose-dependent and temporal changes in mRNA expression levels of HA-related molecules and profibrotic cytokines, while type I and III collagen expression was consistently down-regulated across a broad range of parameters. Finally, we tested the potential therapeutic efficacy of vibration for reversing the fibrotic phenotype in scleroderma-derived fibroblasts. These studies showed that vibratory stimulation significantly reduced the mRNA levels of sclerotic pathogenic targets and collagen synthesis and accumulation. These studies, therefore, suggest that vibration can be used as a clinical mechanotherapy for a wide range of fibrotic diseases such as systemic sclerosis and idiopathic pulmonary fibrosis

The role of the epidermis in pathogenesis of systemic sclerosis

Studies into the pathogenesis of systemic sclerosis (SSc) skin fibrosis to date have concentrated on dermal changes in the disease. Little attention has been paid to the epidermis in SSc. Epithelial-fibroblast interactions are believed to regulate wound healing and contribute to a number of fibrotic diseases. Recent proteomic data from our laboratory reveals altered keratinocyte (KC) specific proteins in SSc skin consistent with a wound healing phenotype of the disease epidermis. I therefore studied SSc KCs focusing on differentiation and KC-fibroblast interaction. I found that KC maturation is altered in SSc with abnormal persistence of cytokeratins 1, 10 and 14 into suprabasal layers. Cytokeratins 6 and 16, induced in wound healing KCs, were shown to be expressed in SSc epidermis. In addition, IL-1, a pivotal cytokine involved in KC and fibroblast events post epidermal injury, and its downstream signalling phosphoproteins p38 and JNK were elevated in SSc epidermis. I went on to study the effect of SSc epidermis on normal human fibroblasts. I found that SSc epidermis promoted fibroblast activation in an ET-1, TGF-β, and IL-1 dependent fashion. I suggest a double paracrine loop initiated by KC-derived IL-1 as a mechanism for epidermal-dermal co-activation in the disease, similar to that previously demonstrated for wound healing. There is a need for developing antifibrotic agents targeting epithelium-derived factors and their signalling pathways. I went on to study normal epidermal wound healing. A paradox during epithelial repair is that KCs proliferate despite a TGF-β dominated environment, which is known to be anti-proliferative. Our laboratory previously showed that prostanoids antagonise TGF-β-dependent events in human cells. The induction of prostanoids following injury could transiently free KCs from the anti-proliferative effects of TGF-β. I test this hypothesis by confirming transient induction of epidermal COX II and PGE2 following injury. I also show that PGE2 antagonises the anti-proliferative and pro-migratory effects of TGF-β on KCs. My work supports a model where induction of epidermal wound edge COX II leads to antagonism of TGF-β and allows KCs to proliferate prior to migration over the wound

Role of TGFbRII in myeloid cell mediated regenerative processes and fibroplasia

Tissue repair and fibrosis are controlled by the interaction of different cell lineages, their soluble factors and matrix signals. Recently, macrophages have been found to be crucial for proper tissue repair. In particular, the role of Transforming growth factor-β1 (TGF-β1) has been extensively studied during tissue repair and fibrosis. Fibrosis is characterized by excessive production and deposition of extracellular matrix, as well as immune cell infiltration. Macrophages are one of the main sources of TGF-β1. So far, studies on the mechanisms of tissue repair and fibrosis have mainly focused on macrophages or TGF-β1 individually. However, the specific function of TGF-β1 on macrophages in tissue repair and fibrosis still needs to be elucidated. To understand the macrophage specific role of TGFβ1-TGFβRII signaling in tissue repair and fibrosis, we generated a mouse model, which lacks TGFβRII in myeloid cells (TGFβRIIfl/fl/LysMCre). We observed that during mechanical tissue injury TGFβRII signaling in macrophages contributes to wound contraction, possibly by cross—talk between macrophages and fibroblasts. The attenuated wound contraction was accompanied by impaired myofibroblast differentiation and collagen deposition. However, the loss of TGFβRII signaling in macrophages did not lead to reduced expression of TGF- β1, which we proposed as one of the primary mechanisms in wound tissue underlying reduced myofibroblast formation observed in TGFβRIIfl/fl/LysMCre mice. Generation of cutaneous fibrosis by bleomycin injection for two and four weeks resulted in reduced fibrosis in TGFβRIIfl/fl/LysMCre mice, compared to control mice. The mechanisms leading to this phenotype were associated with reduced infiltration of immune cells, reduced deposition of collagen and diminished production of inflammatory mediators such as IL-1β, TNF-α and osteopontin-1 at the early stage of fibrosis formation. At the later stage, the expression of inflammatory mediators in TGFβRIIfl/fl/LysMCre mice was not altered compared to control mice, possibly due to compensatory mechanisms. Our data leads to the hypothesis that the reduced fibrosis is caused by the reduced expression of inflammatory mediators and accumulation of immune cells at the early stage of fibrosis in TGFβRIIfl/fl/LysMCre mice. Our results provide new insights into the crucial role of macrophage specific TGFβRII signaling in tissue repair and fibrosis

Activation of JNK Signaling Mediates Connective Tissue Growth Factor Expression and Scar Formation in Corneal Wound Healing

Connective Tissue Growth Factor (CTGF) and Transforming growth factor-β1 (TGF-β1) are key growth factors in regulating corneal scarring. Although CTGF was induced by TGF-β1 and mediated many of fibroproliferative effects of TGF-β1, the signaling pathway for CTGF production in corneal scarring remains to be clarified. In the present study, we firstly investigated the effects of c-Jun N-terminal kinase (JNK) on CTGF expression induce by TGF-β1 in Telomerase-immortalized human cornea stroma fibroblasts (THSF). Then, we created penetrating corneal wound model and determined the effect of JNK in the pathogenesis of corneal scarring. TGF-β1 activated MAPK pathways in THSF cells. JNK inhibitor significantly inhibited CTGF, fibronectin and collagen I expression induced by TGF-β1 in THSF. In corneal wound healing, the JNK inhibitor significantly inhibited CTGF expression, markedly improved the architecture of corneal stroma and reduced corneal scar formation, but did not have a measurable impact on corneal wound healing in vivo. Our results indicate that JNK mediates the expression of CTGF and corneal scarring in corneal wound healing, and might be considered as specific targets of drug therapy for corneal scarring

The role of IL-17 in the pathogenesis of systemic sclerosis

IL-17A is a pro-inflammatory cytokine secreted mainly by Th17 cells. It plays a critical role in the host defense system and is linked to various autoimmune diseases. The dual role of IL-17A in systemic sclerosis (SSc) pathogenesis is quite intriguing: on one hand IL-17A promotes pro-inflammatory cytokine synthesis and enhances immune reactions and on the other it decreases fibrotic responses and abrogates fibrosis. IL-17D belongs to the IL-17 family and could possibly regulate inflammation and autoimmune diseases including SSc. Potential mechanisms behind IL-17 family members in SSc pathogenesis is therefore worthy of investigation. Functional fibroblast assays were conducted and showed in the presence of IL-17A there was no change in cell morphology, viability or migration in dermal fibroblasts. RT-PCR was used to assess mRNA levels of key fibroblast molecules in response to IL-17A. Although most remained unaltered, IL17A was shown to inhibit expression of fibrboblast activation protein (FAP) in SSc at 50ng/mL (p=0.0144). Protein analysis using Western Blotting to examine fibroblast cell signalling pathways was employed to examine the impact of IL- 17A on the p38 MAPK pathway, Erk1/2 MAPK pathway and STAT3/gp130 pathways in fibroblasts. Measuring the culture medium by ELISA revealed that IL-17A did not induce IL-6 secretion in SSc fibroblasts. Exploring a well-defined clinical cohort of SSc patients, correlations between IL-17 family members IL- 17A and IL-17D and other cytokines and clinical parametres were examined. Although no differences were noted in circulating IL-17A and IL-17D between SSc and healthy controls, important clinical correlations between IL-17 subtypes and clinical parametres were observed. Interestingly IL-17A was shown to be associated with digital ulcers (IL-17A in patients with digital ulcers was significantly higher than patients without digital ulcers, p=0.0349). IL-17A was positively correlated with the level of erythrocyte sedimentation rate ( r=0.7188, p<0.0001, in the SSc-Pulmonary Arterial Hypertension group) and type-B natriuretic peptide (r=0.5013, p=0.0091, in the SSc-only group), which is consistent with the inflammatory aspect of IL17A. IL-10 was correlated with IL-17A in SSc-PAH patients (r=0.4506, p=0.0238). IL-17D was age-related in SSc (IL-17D in patients aged 40-60 was significantly lower than in patients aged over 60, p=0.018). IL-17D was related to the presence of lung fibrosis in CT scans of SSc patients (IL-17D in patients with no fibrosis in CT scans was significantly higher than in patients with mild fibrosis, p=0.0175). The level of IL-17A and IL-17D between healthy donors and SSc patients was compared and no significant change was detected. Haemoglobin (r=-0.4281, p=0.0259, in the SSc-only group) and mean corpuscular volume (r=-0.4599, p=0.0313, in the SSc-PAH group) were inversely correlated with IL-17D while MRSS was positively correlated with IL-17D in SSc (r=0.6934, p=0.001, in the SSc-only group). In LcSSc patients, Hb (r=0.-0.485, p=0.0001) was inversely correlated with IL-17D, while BNP (r=0.347, p=0.013) and MRSS (r=0.48, p=0.001) were positively correlated with IL-17D. This study shows that fibroblasts are able to respond to IL-17A by altering the expression of fibrogenic markers and that two IL-17 family members (IL-17A and IL-17D) exhibit important associations within defined SSc disease subsets. The precise role of the IL-17 family in SSc pathogenesis is of significant interest and studies reported here present opportunities for further explorations as potential therapy strategies for SSc

Fibrosis: a role for vitamin D

Chronic inflammation leads to fibrosis and eventually organ failure. Fibrosis is defined as a wound-healing response that has gone awry. It is featured by excessive production, deposition, and accumulation of extracellular matrix components. The key mediator cells of fibrotic disorders are the myofibroblasts, derived from different precursor cells. Myofibroblasts are responsible of stiff ECM, a hallmark of fibrosis. It is mandatory understanding the molecular pathways contributing to develop the fibrotic tissue to discovery anti-fibrotic therapies. Vitamin D, the precursor of seco-steroid hormone, appears to have anti-fibrotic properties. Vitamin D deficiency may contribute to development of different fibrotic disorders in several organs. It counteracts the pro-fibrotic signals, such as TGF-β1, through several biochemical mechanisms. Counteracting TGF-β1, Vitamin D inhibits myofibroblasts activation and ECM deposition

A Role of Myocardin Related Transcription Factor-A (MRTF-A) in Scleroderma Related Fibrosis.

In scleroderma (systemic sclerosis, SSc), persistent activation of myofibroblast leads to severe skin and organ fibrosis resistant to therapy. Increased mechanical stiffness in the involved fibrotic tissues is a hallmark clinical feature and a cause of disabling symptoms. Myocardin Related Transcription Factor-A (MRTF-A) is a transcriptional co-activator that is sequestered in the cytoplasm and translocates to the nucleus under mechanical stress or growth factor stimulation. Our objective was to determine if MRTF-A is activated in the disease microenvironment to produce more extracellular matrix in progressive SSc. Immunohistochemistry studies demonstrate that nuclear translocation of MRTF-A in scleroderma tissues occurs in keratinocytes, endothelial cells, infiltrating inflammatory cells, and dermal fibroblasts, consistent with enhanced signaling in multiple cell lineages exposed to the stiff extracellular matrix. Inhibition of MRTF-A nuclear translocation or knockdown of MRTF-A synthesis abolishes the SSc myofibroblast enhanced basal contractility and synthesis of type I collagen and inhibits the matricellular profibrotic protein, connective tissue growth factor (CCN2/CTGF). In MRTF-A null mice, basal skin and lung stiffness was abnormally reduced and associated with altered fibrillar collagen. MRTF-A has a role in SSc fibrosis acting as a central regulator linking mechanical cues to adverse remodeling of the extracellular matrix