Myofibroblast Biology Research Articles

Canadian Contributions in Fibroblast Biology

Fibroblasts are stromal cells found in virtually every tissue and organ of the body. For many years, these cells were often considered to be secondary in functional importance to parenchymal cells. Over the past 2 decades, focused research into the roles of fibroblasts has revealed important roles for these cells in the homeostasis of healthy tissue, and has demonstrated that activation of fibroblasts to myofibroblasts is a key step in disease initiation and progression in many tissues, with fibrosis now recognized as not only an outcome of disease, but also a central contributor to tissue dysfunction, particularly in the heart and lungs. With a growing understanding of both fibroblast and myofibroblast heterogeneity, and the deciphering of the humoral and mechanical cues that impact the phenotype of these cells, fibroblast biology is rapidly becoming a major focus in biomedical research. In this review, we provide an overview of fibroblast and myofibroblast biology, particularly in the heart, and including a discussion of pathophysiological processes such as fibrosis and scarring. We then discuss the central role of Canadian researchers in moving this field forwards, particularly in cardiac fibrosis, and highlight some of the major contributions of these individuals to our understanding of fibroblast and myofibroblast biology in health and disease.

ELTD1 Activation Induces an Endothelial-EMT Transition to a Myofibroblast Phenotype.

ELTD1 is expressed in endothelial and vascular smooth muscle cells and has a role in angiogenesis. It has been classified as an adhesion GPCR, but as yet, no ligand has been identified and its function remains unknown. To establish its role, ELTD1 was overexpressed in endothelial cells. Expression and consequently ligand independent activation of ELTD1 results in endothelial-mesenchymal transistion (EndMT) with a loss of cell-cell contact, formation of stress fibres and mature focal adhesions and an increased expression of smooth muscle actin. The effect was pro-angiogenic, increasing Matrigel network formation and endothelial sprouting. RNA-Seq analysis after the cells had undergone EndMT revealed large increases in chemokines and cytokines involved in regulating immune response. Gene set enrichment analysis of the data identified a number of pathways involved in myofibroblast biology suggesting that the endothelial cells had undergone a type II EMT. This type of EMT is involved in wound repair and is closely associated with inflammation implicating ELTD1 in these processes.

Fibrometabolism-An emerging therapeutic frontier in pulmonary fibrosis.

Fibrosis is the final pathological outcome and major cause of morbidity and mortality in many common and chronic inflammatory, immune-mediated, and metabolic diseases. Despite the growing incidence of fibrotic diseases and extensive research efforts, there remains a lack of effective therapies that improve survival. The application of omics technologies has revolutionized our approach to identifying previously unknown therapeutic targets and potential disease biomarkers. The application of metabolomics, in particular, has improved our understanding of disease pathomechanisms and garnered a wave of scientific interest in the role of metabolism in the biology of myofibroblasts, the key effector cells of the fibrogenic response. Emerging evidence suggests that alterations in metabolism not only are a feature of but also may play an influential role in the pathogenesis of fibrosis, most notably in idiopathic pulmonary fibrosis (IPF), the most rapidly progressive and fatal of all fibrotic conditions. This review will detail the role of key metabolic pathways, their alterations in myofibroblasts, and the potential this new knowledge offers for the development of antifibrotic therapeutic strategies.

Engineered microenvironment for the study of myofibroblast mechanobiology.

Myofibroblasts are mechanosensitive cells and a variety of their behaviours including differentiation, migration, force production and biosynthesis are regulated by the surrounding microenvironment. Engineered cell culture models have been developed to examine the effect of microenvironmental factors such as the substrate stiffness, the topography and strain of the extracellular matrix (ECM) and the shear stress on myofibroblast biology. These engineered models provide well-mimicked, pathophysiologically relevant experimental conditions that are superior to those enabled by the conventional two-dimensional (2D) culture models. In this perspective, we will review the recent advances in the development of engineered cell culture models for myofibroblasts and outline the findings on the myofibroblast mechanobiology under various microenvironmental conditions. These studies have demonstrated the power and utility of the engineered models for the study of microenvironment-regulated cellular behaviours. The findings derived using these models contribute to a greater understanding of how myofibroblast behaviour is regulated in tissue repair and pathological scar formation.

Myofibroblast Functions in Tissue Repair and Fibrosis: An Introduction.

Our understanding of myofibroblast biology has advanced over the past two decades, and the seemingly antagonistic roles of these cells in both normal tissue repair and fibrotic diseases are better reconciled. An age-related loss of cellular plasticity that results in impaired capacity for de-differentiation and apoptosis susceptibility may predispose individuals to non-resolving and progressive fibrotic disorders involving diverse organ systems.

Animal and Human Models of Tissue Repair and Fibrosis: An Introduction.

Reductionist cell culture systems are not only convenient but essential to understand molecular mechanisms of myofibroblast activation and action in carefully controlled conditions. However, tissue myofibroblasts do not act in isolation and the complexity of tissue repair and fibrosis in humans cannot be captured even by the most elaborate culture models. Over the past five decades, numerous animal models have been developed to study different aspects of myofibroblast biology and interactions with other cells and extracellular matrix. The underlying principles can be broadly classified into: (1) organ injury by trauma such as prototypical full thickness skin wounds or burns; (2) mechanical challenges, such as pressure overload of the heart by ligature of the aorta or the pulmonary vein; (3) toxic injury, such as administration of bleomycin to lungs and carbon tetrachloride to the liver; (4) organ infection with viruses, bacteria, and parasites, such as nematode infections of liver; (5) cytokine and inflammatory models, including local delivery or viral overexpression of active transforming growth factor beta; (6) "lifestyle" and metabolic models such as high-fat diet; and (7) various genetic models. We will briefly summarize the most widely used mouse models used to study myofibroblasts in tissue repair and fibrosis as well as genetic tools for manipulating myofibroblast repair functions in vivo.

A Rodent Model of Hypertrophic Scarring: Splinting of Rat Wounds.

Human hypertrophic scars are the result of imperfect healing of skin, which is particularly evident from the scars developing after severe burns. In contrast, mouse and rat full-thickness skin wounds heal normally without forming visible scar tissue, which reduces the suitability of rodent models for the study of skin scarring. We here provide a simple procedure to splint the edges of full-thickness rodent skin with a sutured plastic frame that prevents wound closure by granulation tissue contraction. The resulting mechanical tension in the wound bed and the lack of neo-epithelium amplify myofibroblast formation and generate hypertrophic features, not unlike those of human skin. In addition to producing scar tissue, the splint provides a reservoir that can be used for the delivery of cellular and acellular wound treatment regimen. Despite being simple and almost historical, wound splinting is a robust and reliable model to study myofibroblast biology.

Extracellular matrix remodeling associated with bleomycin-induced lung injury supports pericyte-to-myofibroblast transition

Of the many origins of pulmonary myofibroblasts, microvascular pericytes are a known source. Prior literature has established the ability of pericytes to transition into myofibroblasts, but provide limited insight into molecular cues that drive this process during lung injury repair and fibrosis. Fibronectin and RGD-binding integrins have long been considered pro-fibrotic factors in myofibroblast biology, and here we test the hypothesis that these known myofibroblast cues coordinate pericyte-to-myofibroblast transitions. Specifically, we hypothesized that αvβ3 integrin engagement on fibronectin induces pericyte transition into myofibroblastic phenotypes in the murine bleomycin lung injury model. Myosin Heavy Chain 11 (Myh11)-CreERT2 lineage tracing in transgenic mice allows identification of cells of pericyte origin and provides a robust tool for isolating pericytes from tissues for further evaluation. We used this murine model to track and characterize pericyte behaviors during tissue repair. The majority of Myh11 lineage-positive cells are positive for the pericyte surface markers, PDGFRβ (55%) and CD146 (69%), and display typical pericyte morphology with spatial apposition to microvascular networks. After intratracheal bleomycin treatment of mice, Myh11 lineage-positive cells showed significantly increased contractile and secretory markers, as well as αv integrin expression. According to RNASeq measurements, many disease and tissue-remodeling genesets were upregulated in Myh11 lineage-positive cells in response to bleomycin-induced lung injury. In vitro, blocking αvβ3 binding through cycloRGDfK prevented expression of the myofibroblastic marker αSMA relative to controls. In response to RGD-containing provisional matrix proteins present in lung injury, pericytes may alter their integrin profile.

The myofibroblast at a glance.

In 1971, Gabbiani and co-workers discovered and characterized the "modification of fibroblasts into cells which are capable of an active spasm" (contraction) in rat wound granulation tissue and, accordingly, named these cells 'myofibroblasts'. Now, myofibroblasts are not only recognized for their physiological role in tissue repair but also as cells that are key in promoting the development of fibrosis in all organs. In this Cell Science at a Glance and the accompanying poster, we provide an overview of the current understanding of central aspects of myofibroblast biology, such as their definition, activation from different precursors, the involved signaling pathways and most widely used models to study their function. Myofibroblasts will be placed into context with their extracellular matrix and with other cell types communicating in the fibrotic environment. Furthermore, the challenges and strategies to target myofibroblasts in anti-fibrotic therapies are summarized to emphasize their crucial role in disease progression.

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

Proteomic profile of TGF-β1 treated lung fibroblasts identifies novel markers of activated fibroblasts in the silica exposed rat lung

We performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) on control and TGF-β1-exposed rat lung fibroblasts to identify proteins differentially expressed between cell populations. A total of 196 proteins were found to be differentially expressed in response to TGF-β1 treatment. Guided by these results, we next determined whether similar changes in protein expression were detectable in the rat lung after chronic exposure to silica dust. Of the five proteins selected for further analysis, we found that levels of all proteins were markedly increased in the silica-exposed rat lung, including the proteins for the very low density lipoprotein receptor (VLDLR) and the transmembrane (type I) heparin sulfate proteoglycan called syndecan 2 (SDC2). Because VLDLR and SDC2 have not, to our knowledge, been previously linked to the pathobiology of silicosis, we next examined whether knockdown of either gene altered responses to TGF-β1 in MRC-5 lung fibroblasts. Interestingly, we found knockdown of either VLDLR or SDC2 dramatically reduced collagen production to TGF-β1, suggesting that both proteins might play a novel role in myofibroblast biology and pathogenesis of silica-induced pulmonary fibrosis. In summary, our findings suggest that performing LC-MS/MS on TGF-β1 stimulated lung fibroblasts can uncover novel molecular targets of activated myofibroblasts in silica-exposed lung.

Homing in on the hepatic scar: recent advances in cell-specific targeting of liver fibrosis.

Despite the high prevalence of liver disease globally, there are currently no approved anti-fibrotic therapies to treat patients with liver fibrosis. A major goal in anti-fibrotic therapy is the development of drug delivery systems that allow direct targeting of the major pro-scarring cell populations within the liver (hepatic myofibroblasts) whilst not perturbing the homeostatic functions of other mesenchymal cell types present within both the liver and other organ systems. In this review we will outline some of the recent advances in our understanding of myofibroblast biology, discussing both the origin of myofibroblasts and possible myofibroblast fates during hepatic fibrosis progression and resolution. We will then discuss the various strategies currently being employed to increase the precision with which we deliver potential anti-fibrotic therapies to patients with liver fibrosis.

PDGF-Mediated Regulation of Liver Fibrosis

The activation of mesenchymal cells to matrix secreting myofibroblasts is central to the initiation and development of liver fibrosis. Understanding the mechanisms that govern this process is a vital step in the rational development of new anti-fibrotic therapies. Platelet-derived growth factors (PDGFs) and their cognate receptors play a key role in hepatic stellate cell and liver myofibroblast biology. In this review, we outline the major hepatic cellular sources of PDGFs and their modes of action during liver fibrogenesis. Furthermore, we examine how the PDGF pathway may represent a tractable anti-fibrotic target, and how the PDGF receptors may be harnessed to allow cell-specific delivery of therapeutics to help treat patients with liver fibrosis.

The IκB kinase inhibitor ACHP strongly attenuates TGFβ1-induced myofibroblast formation and collagen synthesis.

Excessive accumulation of a collagen-rich extracellular matrix (ECM) by myofibroblasts is a characteristic feature of fibrosis, a pathological state leading to serious organ dysfunction. Transforming growth factor beta1 (TGFβ1) is a strong inducer of myofibroblast formation and subsequent collagen production. Currently, there are no remedies for the treatment of fibrosis. Activation of the nuclear factor kappa B (NF-κB) pathway by phosphorylating IκB with the enzyme IκB kinase (IKK) plays a major role in the induction of fibrosis. ACHP {2-Amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(4-piperidinyl)-3 pyridinecarbonitrile}, a selective inhibitor of IKK, prohibits the activation of the NF-κB pathway. It is not known whether ACHP has potential anti-fibrotic properties. Using adult human dermal and lung fibroblasts we have investigated whether ACHP has the ability to inhibit the TGFβ1-induced transition of fibroblasts into myofibroblasts and its excessive synthesis of ECM. The presence of ACHP strongly suppressed the induction of the myofibroblast markers alpha-smooth muscle actin (αSMA) and SM22α, as well as the deposition of the ECM components collagen type I and fibronectin. Furthermore, post-treatment with ACHP partly reversed the expression of αSMA and collagen type I production. Finally, ACHP suppressed the expression of the three collagen-modifying enzymes lysyl hydroxylase (PLOD1, PLOD2 and PLOD3) in dermal fibroblasts, but did not do so in lung fibroblasts. We conclude that the IKK inhibitor ACHP has potent antifibrotic properties, and that the NF-κB pathway plays an important role in myofibroblast biology.

MIOFIBROBLASTOS, CÉLULAS RELEVANTES NA SAÚDE E NA DOENÇA: REVISÕES DE LITERATURA

Recently, a large number of studies about myofibroblasts have been published. These cellular elements were primarily described for over 40 years and their ultrastructural characteristics, as well as its role in various physiological and pathological conditions continue stirring interest in health area. This paper aims to describe the cellular and molecular biology of myofibroblasts, and contextualize scientific findings of these cells recorded in articles over the past 40 years since its initial discovery.

Dynamic Action of Myofibroblasts in Skin and Bone Marrow Transplantation

Skin is highly accessible and evaluable organ, which accelerate the understanding of novel medical innovation in association with organ transplantation, engineering, and wound healing, as well as the stage-specific adaptability of transplanted Bone Marrow (BM) cells. In skin transplantation biology, multi-stage/-angle damages occur in both grafted donor and perilesional host skin, and need to be repaired properly for the engraftment and later maintenance of the local homeostasis and characteristic skin architecture, such as stratified squamoid epithelium and dermal components. These local events are more unlikely to be regulated by the host immunity, because the donor (allogenic) skin engraftment mostly accomplishes onto the immunocompromised or immunosuppressive animals. Accumulating evidences have emerged the importance of alpha-Smooth Muscle Actin (SMA)-positive myofibroblasts, via stage- and cell type specific contribution of TGF-beta, PDGF, ET-1, CCN-2 signaling pathways and/or mastocyte-derived mediators (e.g. histamine and tryptase), for the functional reorganization of the grafted skin. Moreover, particular cell lineages from BM cells have been shown to harbor the differentiation capacity into multiple skin cell phenotypes, including epidermal keratinocytes, and dermal endothelial cells and pericytes, under controlled by chemokines or cytokines, but the trans-differentiation into alpha-SMA+ myofibroblasts is possibly reversed by inactivation of MEK/ERK signal cascade. We review the recent update of the myofibroblast biology in association with the reconstitution of the engrafted skin, and also work on translating this attractive action into the application of BM transplantation medicine in genetic skin diseases.

Myofibroblast biology and interactions with the environment

AbstractNormal tissue repair includes a number of overlapping phases. After injury, there is usually an early inflammatory step. Next, as granulation tissue forms, fibroblasts invade the wound and begin to replace the provisional matrix with a more mature wound matrix. As the granulation tissue phase proceeds, fibroblasts acquire a new phenotype with prominent microfilament bundles. These typical myofibroblasts (MFs) have been shown to develop a smooth muscle‐like phenotype, and are responsible for wound contraction. Lastly, in the resolution phase of healing, there is considerable loss of cell types including MFs, by apoptosis. Inappropriate delay of apoptosis, and thus increased survival of MFs activated during the healing process, may be a factor which leads to pathological situations and excessive scarring. In most situations, MFs derive from local connective tissue cells but it has now become clear that other cells can contribute to MF production. Both MF differentiation and apoptosis are dependent on cytokines, mechanical stress and more generally on cell‐cell and/or cell‐matrix interactions. The control of the MF activity represents an important target to promote healing and to limit excessive scarring.

Regulation and Relevance of Myofibroblast Responses in Idiopathic Pulmonary Fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, incurable lung disease of unknown etiology with only limited treatment options. Current paradigms of disease pathogenesis feature recurrent or prolonged epithelial injury and an ensuing inflammatory response that culminates in the appearance of activated myofibroblasts. These cells are believed central to the excessive deposition of extracellular matrix that eventually obliterates the alveolar space to cause respiratory failure. Because the factors driving the accumulation of myofibroblasts remain poorly understood, effective therapies remain elusive. This review focuses on recent understanding of myofibroblasts including their seemingly uncontrolled proliferation and survival, their controversial origin in pathological IPF tissues, and the local biochemical and biomechanical matrix factors that drive their behavior. In addition, novel antifibrotics under development for the treatment of lung disease will be discussed. As our understanding of fibroblast and myofibroblast biology regulation expands, these cells may prove to be effective therapeutic targets.

AB0238 Effects of endothelin A/B receptor antagonist (bosentan) on alpha-smooth muscle actin (α-SMA) and extracellular matrix protein synthesis in primary cultures of systemic sclerosis skin fibroblasts

BackgroundIn systemic sclerosis (SSc) myofibroblasts are cellular mediator of fibrosis by the overproduction of extracellular matrix (ECM) proteins such as collagens and fibronectin (FN) (1, 2). Myofibroblasts, as activated fibroblasts...

Voltage-gated Na⁺ channels: novel players in fibroblast-to-myofibroblast transition with a potential role in atrial arrhythmogenesis?

Fibrosis entails the accumulation of excess extracellular matrix (ECM) components such as collagen. Excessive ECM deposition distorts normal tissue architecture, compromises cardiac pump function and disrupts cytoarchitecture of electrically coupled cardiomyocytes, predisposing to arrhythmias. Various triggers can promote cardiac fibrosis. Regardless of the initiating event, cardiac fibroblasts generally first proliferate and then differentiate into ECM-secreting myofibroblasts, the key mediators of fibrotic-tissue remodelling. Atria are more susceptible to fibrotic stimuli than ventricles, and fibrosis is important in atrial fibrillation (AF; Yue et al. 2011). Atrial-fibrotic remodelling causes conduction abnormalities and may promote electrical fibroblast–cardiomyocyte interactions that favour AF. While it is known that ECM-secreting myofibroblasts are central to atrial fibrosis formation, the precise regulatory mechanisms of fibroblast-to-myofibroblast transitions remain unclear (Wakili et al. 2011). A better understanding of the molecular biology underlying AF-promoting fibrosis may help to identify novel drugs for effective AF treatment (Dobrev et al. 2012). Cardiac fibroblasts express ion channels (Du et al. 2010; Li et al. 2009) but their functional role is poorly understood. Recent studies have provided evidence that Ca2+-permeable melastatin type-7 transient-receptor potential (TRPM7) channels regulate human atrial fibroblast/myofibroblast function, potentially contri-buting to AF pathophysiology (Du et al. 2010). Ca2+-permeable TRP canonical-3 (TRPC3) channels control atrial fibroblast proliferation and differentiation by regulating the Ca2+ influx that activates ERK signalling. TRPC3-channel expression is increased in human AF, probably contributing to fibroblast-to-myofibroblast transition (Harada et al. 2011). Thus, TRPM7 and TRPC3 channels and the related Ca2+ influx may play important roles in AF-promoting fibrotic remodelling. However, whether voltage-gated ion channels also contribute to the activation of atrial myofibroblasts is unknown. In a recent issue of The Journal of Physiology, Chatelier et al. (2012) demonstrate a unique de novo functional expression of Na+ currents in human atrial myofibroblasts. Human atrial fibroblasts did not show specific labelling for α-smooth muscle actin (α-SMA), an established marker of myofibroblasts, before 7 days in culture, but a strong staining after 12 days, indicating that the differentiation of fibroblasts into myofibroblasts occurred between 7 and 12 days of culture. The same time course was observed for expression of the Na+-channel subunit Nav1.5, with INa being recordable in every cell after 12 days in culture. RT-qPCR, immunofluorescence and pharmacological experiments showed that INa is predominantly conducted by Nav1.5. The most common method to identify myofibroblasts is immunostaining for α-SMC. One important merit of the work of Chatelier et al. (2012), which is the first to show INa in human atrial myofibroblasts, is the identification of INa as a potential marker of human atrial myofibroblasts, which should foster the electrophysiological separation of fibroblasts and myofibroblasts in diseased human atria. They also add INa to the growing list of ion channels potentially controlling the transition of fibroblasts to ECM-secreting myofibroblasts. Ca2+-handling abnormalities are critical determinants of atrial cardiomyocyte dysfunction in AF (Voigt et al. 2012), and abnormal Ca2+ entry is suggested to be essential for fibroblast proliferation, migration and differentiation into myofibroblasts in diseased hearts (Yue et al. 2011). Fibroblasts lack voltage-gated Ca2+ channels and Ca2+ influx is likely to occur predominantly via TRPM7 and TRPC3 channels. In addition, reverse-mode (Ca2+ influx) Na+–Ca2+ exchanger (NCX) may also participate in Ca2+ signalling in myofibroblasts. Myofibroblast INa had a greater window current than cardiomyocyte INa, which should produce a larger persistent Na+ entry, enhancing Ca2+ influx through NCX that may contribute to cell proliferation. However, whether INa participates in the regulation of Ca2+ fluxes in myofibroblasts needs exploration. Taken together, the work of Chatelier et al. (2012) opens new avenues in the understanding of voltage-gated ion channels in the molecular biology of myofibroblasts in the human atrium. Their study raises several important questions that need addressing in subsequent work. What is the source (endothelial cells?) of myofibroblasts de novo expressing Na+ channels? Do Na+ channels promote fibroblasts differentiation into myofibroblasts? Is ion-channel composition of fibroblasts and myofibroblasts different? Do Na+ channels accelerate myofibroblast proliferation, migration and secretion properties? Is the inhibition of Na+ channels in myofibroblasts an exploitable therapeutic strategy for AF? Answers to these questions should provide important insights into the multifunctional role of ion channels in cardiac myofibroblasts and their precise role in structural remodelling in AF. The authors’ work is supported by the European Network for Translational Research in Atrial Fibrillation, the Atrial Fibrillation Competence Network and German Center for Cardiovascular Research, the Deutsche Forschungsgemeinschaft, and by Fondation Leducq (European–North American Atrial Fibrillation Research Alliance).