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Scientific Reports Dec 2019Asthma is the most common chronic lung disease in children and young adults worldwide. Airway remodelling (including increased fibroblasts and myofibroblasts in airway...
Asthma is the most common chronic lung disease in children and young adults worldwide. Airway remodelling (including increased fibroblasts and myofibroblasts in airway walls due to chronic inflammation) differentiates asthmatic from non-asthmatic airways. The increase in airway fibroblasts and myofibroblasts occurs via epithelial to mesenchymal transition (EMT) where epithelial cells lose their tight junctions and are transdifferentiated to mesenchymal cells, with further increases in myofibroblasts occurring via fibroblast-myofibroblast transition (FMT). Transforming growth factor (TGF)-β is the central EMT- and FMT-inducing cytokine. In this study, we have used next generation sequencing to delineate the changes in the transcriptome induced by TGF-β treatment of WI-38 airway fibroblasts in both the short term and after differentiation into myofibroblasts, to gain an understanding of the contribution of TGF-β induced transdifferentiation to the asthmatic phenotype. The data obtained from RNAseq analysis was confirmed by quantitative PCR (qPCR) and protein expression investigated by western blotting. As expected, we found that genes coding for intermediates in the TGF-β signalling pathways (SMADs) were differentially expressed after TGF-β treatment, SMAD2 being upregulated and SMAD3 being downregulated as expected. Further, genes involved in cytoskeletal pathways (FN1, LAMA, ITGB1) were upregulated in myofibroblasts compared to fibroblasts. Importantly, genes that were previously shown to be changed in asthmatic lungs (ADAMTS1, DSP, TIMPs, MMPs) were similarly differentially expressed in myofibroblasts, strongly suggesting that TGF-β mediated differentiation of fibroblasts to myofibroblasts may underlie important changes in the asthmatic airway. We also identified new intermediates of signalling pathways (PKB, PTEN) that are changed in myofibroblasts compared to fibroblasts. We have found a significant number of genes that are altered after TGF-β induced transdifferentiation of WI-38 fibroblasts into myofibroblasts, many of which were expected or predicted. We also identified novel genes and pathways that were affected after TGF-β treatment, suggesting additional pathways are activated during the transition between fibroblasts and myofibroblasts and may contribute to the asthma phenotype.
Topics: Biomarkers; Cell Differentiation; Cells, Cultured; Fibroblasts; Gene Expression Profiling; Humans; Myofibroblasts; Signal Transduction; Transcriptome; Transforming Growth Factor beta
PubMed: 31889146
DOI: 10.1038/s41598-019-56955-1 -
Journal of Neuroinflammation Nov 2020Macular fibrosis causes irreparable vision loss in neovascular age-related macular degeneration (nAMD) even with anti-vascular endothelial growth factor (VEGF) therapy....
BACKGROUND
Macular fibrosis causes irreparable vision loss in neovascular age-related macular degeneration (nAMD) even with anti-vascular endothelial growth factor (VEGF) therapy. Inflammation is known to play an important role in macular fibrosis although the underlying mechanism remains poorly defined. The aim of this study was to understand how infiltrating macrophages and complement proteins may contribute to macular fibrosis.
METHODS
Subretinal fibrosis was induced in C57BL/6J mice using the two-stage laser protocol developed by our group. The eyes were collected at 10, 20, 30 and 40 days after the second laser and processed for immunohistochemistry for infiltrating macrophages (F4/80 and Iba-1), complement components (C3a and C3aR) and fibrovascular lesions (collagen-1, Isolectin B4 and α-SMA). Human retinal sections with macular fibrosis were also used in the study. Bone marrow-derived macrophages (BMDMs) from C57BL/6J mice were treated with recombinant C3a, C5a or TGF-β for 48 and 96 h. qPCR, Western blot and immunohistochemistry were used to examine the expression of myofibroblast markers. The involvement of C3a-C3aR pathway in macrophage to myofibroblast transition (MMT) and subretinal fibrosis was further investigated using a C3aR antagonist (C3aRA) and a C3a blocking antibody in vitro and in vivo.
RESULTS
Approximately 20~30% of F4/80 (or Iba-1) infiltrating macrophages co-expressed α-SMA in subretinal fibrotic lesions both in human nAMD eyes and in the mouse model. TGF-β and C3a, but not C5a treatment, significantly upregulated expression of α-SMA, fibronectin and collagen-1 in BMDMs. C3a-induced upregulation of α-SMA, fibronectin and collagen-1 in BMDMs was prevented by C3aRA treatment. In the two-stage laser model of induced subretinal fibrosis, treatment with C3a blocking antibody but not C3aRA significantly reduced vascular leakage and Isolectin B4 lesions. The treatment did not significantly alter collagen-1 fibrotic lesions.
CONCLUSIONS
MMT plays a role in macular fibrosis secondary to nAMD. MMT can be induced by TGF-β and C3a but not C5a. Further research is required to fully understand the role of MMT in macular fibrosis. Macrophage to myofibroblast transition (MMT) contributes to subretinal fibrosis. Subretinal fibrosis lesions contain various cell types, including macrophages and myofibroblasts, and are fibrovascular. Myofibroblasts are key cells driving pathogenic fibrosis, and they do so by producing excessive amount of extracellular matrix proteins. We have found that infiltrating macrophages can transdifferentiate into myofibroblasts, a phenomenon termed macrophage to myofibroblast transition (MMT) in macular fibrosis. In addition to TGF-β1, C3a generated during complement activation in CNV can also induce MMT contributing to macular fibrosis. RPE = retinal pigment epithelium. BM = Bruch's membrane. MMT = macrophage to myofibroblast transition. TGFB = transforming growth factor β. a-SMA = alpha smooth muscle actin. C3a = complement C3a.
Topics: Animals; Cells, Cultured; Complement C3a; Female; Fibrosis; Humans; Macrophages; Macular Degeneration; Male; Mice; Mice, Inbred C57BL; Myofibroblasts; Neovascularization, Pathologic; Retina
PubMed: 33239022
DOI: 10.1186/s12974-020-02033-7 -
Scientific Reports Aug 2022Fibro-adipogenic progenitors (FAPs) are essential in supporting regeneration in skeletal muscle, but in muscle pathologies FAPs the are main source of excess...
Fibro-adipogenic progenitors (FAPs) are essential in supporting regeneration in skeletal muscle, but in muscle pathologies FAPs the are main source of excess extracellular matrix (ECM) resulting in fibrosis. Fibrotic ECM has altered mechanical and architectural properties, but the feedback onto FAPs of stiffness or ECM properties is largely unknown. In this study, FAPs' sensitivity to their ECM substrate was assessed using collagen coated polyacrylamide to control substrate stiffness and collagen hydrogels to engineer concentration, crosslinking, fibril size, and alignment. FAPs on substrates of fibrotic stiffnesses had increased myofibroblast activation, depicted by αSMA expression, compared to substrates mimicking healthy muscle, which correlated strongly YAP nuclear localization. Surprisingly, fibrosis associated collagen crosslinking and larger fibril size inhibited myofibroblast activation, which was independent of YAP localization. Additionally, collagen crosslinking and larger fibril diameters were associated with decreased remodeling of the collagenous substrate as measured by second harmonic generation imaging. Inhibition of YAP activity through verteporfin reduced myofibroblast activation on stiff substrates but not substrates with altered architecture. This study is the first to demonstrate that fibrotic muscle stiffness can elicit FAP activation to myofibroblasts through YAP signaling. However, fibrotic collagen architecture actually inhibits myofibroblast activation through a YAP independent mechanism. These data expand knowledge of FAPs sensitivity to ECM and illuminate targets to block FAP's from driving progression of muscle fibrosis.
Topics: Adipogenesis; Cell Differentiation; Collagen; Extracellular Matrix; Fibrosis; Humans; Muscle, Skeletal; Myofibroblasts
PubMed: 35945422
DOI: 10.1038/s41598-022-17852-2 -
Ear, Nose, & Throat Journal Sep 2021
Topics: Adolescent; Child; Female; Head and Neck Neoplasms; Humans; Male; Medical Illustration; Myofibroblasts; Neoplasms, Muscle Tissue; Young Adult
PubMed: 31760795
DOI: 10.1177/0145561319890165 -
Histopathology Jun 2018Pathological differential diagnoses of pleuroparenchymal fibroelastosis (PPFE) include usual interstitial pneumonia (UIP) and pulmonary apical cap (PAC); however, there...
Pathological differential diagnoses of pleuroparenchymal fibroelastosis (PPFE) include usual interstitial pneumonia (UIP) and pulmonary apical cap (PAC); however, there are no specific immunostaining makers to distinguish between these diseases. We performed immunohistochemistry using several pleural mesothelial cell-related markers, including cytokeratin-5/6, CAM5.2, WT-1, calretinin, desmin and podoplanin, for PPFE (n = 4), UIP (n = 10) and PAC (n = 3) lung sections. Among the examined markers, in PPFE and PAC lungs podoplanin commonly showed positivity for spindle cells both in thickened pleura and subpleural fibroelastosis lesions; these cells were also stained with α-smooth muscle actin, a marker of myofibroblasts. However, even in elastic fibre-rich cases, UIP lungs did not show such podoplanin-positive myofibroblasts in pleura/subpleura and fibroblastic foci. These findings were also verified using immunofluorescence. By contrast, immunohistochemically as well as morphologically, the difference between PPFE and PAC was not apparent. The presence of podoplanin-positive myofibroblasts could be a pathological hallmark of PPFE, suggesting a pathogenic process distinct from UIP but common to PAC.
Topics: Aged; Databases, Factual; Diagnosis, Differential; Elastic Tissue; Female; Humans; Idiopathic Pulmonary Fibrosis; Lung Diseases, Interstitial; Male; Middle Aged; Myofibroblasts; Pleura
PubMed: 29468722
DOI: 10.1111/his.13494 -
Experimental Cell Research Aug 2011Myofibroblasts participate in tissue repair processes in diverse mammalian organ systems. The deactivation of myofibroblasts is critical for termination of the...
Myofibroblasts participate in tissue repair processes in diverse mammalian organ systems. The deactivation of myofibroblasts is critical for termination of the reparative response and restoration of tissue structure and function. The current paradigm on normal tissue repair is the apoptotic clearance of terminally differentiated myofibroblasts; while, the accumulation of activated myofibroblasts is associated with progressive human fibrotic disorders. The capacity of myofibroblasts to undergo de-differentiation as a potential mechanism for myofibroblast deactivation has not been examined. In this report, we have uncovered a role for MyoD in the induction of myofibroblast differentiation by transforming growth factor-β1 (TGF-β1). Myofibroblasts demonstrate the capacity for de-differentiation and proliferation by modulation of endogenous levels of MyoD. We propose a model of reciprocal signaling between TGF-β1/ALK5/MyoD and mitogen(s)/ERK-MAPK/CDKs that regulate myofibroblast differentiation and de-differentiation, respectively. Our studies provide the first evidence for MyoD in controlling myofibroblast activation and deactivation. Restricted capacity for de-differentiation of myofibroblasts may underlie the progressive nature of recalcitrant human fibrotic disorders.
Topics: Cell Differentiation; Cell Proliferation; Cells, Cultured; Humans; MyoD Protein; Myofibroblasts; Transforming Growth Factor beta1
PubMed: 21440539
DOI: 10.1016/j.yexcr.2011.03.016 -
The Journal of Clinical Investigation Jan 2018Fibrosis is the excessive accumulation of extracellular matrix that often occurs as a wound healing response to repeated or chronic tissue injury, and may lead to the... (Review)
Review
Fibrosis is the excessive accumulation of extracellular matrix that often occurs as a wound healing response to repeated or chronic tissue injury, and may lead to the disruption of organ architecture and loss of function. Although fibrosis was previously thought to be irreversible, recent evidence indicates that certain circumstances permit the resolution of fibrosis when the underlying causes of injury are eradicated. The mechanism of fibrosis resolution encompasses degradation of the fibrotic extracellular matrix as well as elimination of fibrogenic myofibroblasts through their adaptation of various cell fates, including apoptosis, senescence, dedifferentiation, and reprogramming. In this Review, we discuss the present knowledge and gaps in our understanding of how matrix degradation is regulated and how myofibroblast cell fates can be manipulated, areas that may identify potential therapeutic approaches for fibrosis.
Topics: Animals; Apoptosis; Cell Dedifferentiation; Cellular Reprogramming; Cellular Senescence; Extracellular Matrix; Fibrosis; Humans; Myofibroblasts
PubMed: 29293097
DOI: 10.1172/JCI93563 -
Biomedical Physics & Engineering Express Jul 2022Cardiac arrhythmias represent about 50% of the cardiovascular diseases which are the first cause of mortality in the world. Implantable medical devices play a major role...
Cardiac arrhythmias represent about 50% of the cardiovascular diseases which are the first cause of mortality in the world. Implantable medical devices play a major role for treating these arrhythmias. Nevertheless the leads induce an unwanted biological phenomenon called fibrosis. This phenomenon begins at a cellular level and is effective at a macroscopic scale causing tissue remodelling with a local modification of the active cardiac tissue. Fibrosis mechanism is complex but at the cellular level, it mainly consists in cardiac fibroblasts activation and differentiation into myofibroblasts. We developed a simplifiedmodel of cardiac fibrosis, with human cardiac fibroblasts whom differentiation into myofibroblasts was promoted with TGF-1. Our study addresses an unreported impedance-based method for real-time monitoring ofcardiac fibrosis. The objective was to study whether the differentiation of cardiac fibroblasts in myofibroblasts had a specific signature on the cell index, an impedance-based feature measured by the xCELLigence system. Primary human cardiac fibroblasts were cultured along 6 days, with or without laminin coating, to study the role of this adhesion protein in cultures long-term maintenance. The cultures were characterized in the presence or absence of TGF-1 and we obtained a significant cell index signature specific to the human cardiac fibroblasts differentiation.
Topics: Cells, Cultured; Electric Impedance; Fibroblasts; Fibrosis; Humans; Myofibroblasts; Transforming Growth Factor beta1
PubMed: 34243179
DOI: 10.1088/2057-1976/ac12e1 -
Cell Calcium Mar 2020During development, disease or in response to changes in local environmental and/or nutrient supply, cellular metabolism is substantially remodeled. Reduced... (Review)
Review
During development, disease or in response to changes in local environmental and/or nutrient supply, cellular metabolism is substantially remodeled. Reduced mitochondrial Ca uptake was recently reported to induce metabolic remodeling, which through stimulating alterations in the epigenome causes changes in gene expression associated with fibroblast to myofibroblast differentiation.
Topics: Animals; Calcium; Epigenome; Fibrosis; Humans; Mitochondria; Models, Biological; Myofibroblasts
PubMed: 31911235
DOI: 10.1016/j.ceca.2019.102155 -
Cells Jun 2022Fibrosis results from defective wound healing processes often seen after chronic injury and/or inflammation in a range of organs. Progressive fibrotic events may lead to... (Review)
Review
Fibrosis results from defective wound healing processes often seen after chronic injury and/or inflammation in a range of organs. Progressive fibrotic events may lead to permanent organ damage/failure. The hallmark of fibrosis is the excessive accumulation of extracellular matrix (ECM), mostly produced by pathological myofibroblasts and myofibroblast-like cells. The Hippo signaling pathway is an evolutionarily conserved kinase cascade, which has been described well for its crucial role in cell proliferation, apoptosis, cell fate decisions, and stem cell self-renewal during development, homeostasis, and tissue regeneration. Recent investigations in clinical and pre-clinical models has shown that the Hippo signaling pathway is linked to the pathophysiology of fibrotic diseases in many organs including the lung, heart, liver, kidney, and skin. In this review, we have summarized recent evidences related to the contribution of the Hippo signaling pathway in the development of organ fibrosis. A better understanding of this pathway will guide us to dissect the pathophysiology of fibrotic disorders and develop effective tissue repair therapies.
Topics: Fibrosis; Hippo Signaling Pathway; Humans; Myofibroblasts; Protein Serine-Threonine Kinases; Signal Transduction
PubMed: 35805148
DOI: 10.3390/cells11132065