Myofibroblast Phenotype Research Articles

Excess Transforming Growth Factor-α Changed the Cell Properties of Corneal Epithelium and Stroma.

PurposeThis study is to investigate the corneal anomaly caused by excess transforming growth factor-α (TGF-α) during mouse development.MethodsBitransgenic KeraRT/TGF-α mice, generated via cross-mating tetO-TGF-α and KeraRT mice, were induced to overexpress TGF-α by doxycycline commencing at embryonic day 0 or postnatal day 0 to different developmental stages. Bitransgenic mice with doxycycline induction were defined as TGF-αECK mice (TGF-α excess expression by corneal keratocytes). Mouse eyes were examined by hematoxylin and eosin staining, immunofluorescent staining and transmission electron microscopy. Protein and RNA from mouse cornea were subjected to western blotting and real-time quantitative polymerase chain reaction.ResultsIn TGF-αECK mice, TGF-α overexpression resulted in corneal opacity. Excess TGF-α initially caused corneal epithelial hyperplasia and subsequent epithelium degeneration as the mouse developed, which was accompanied by gradually diminished K12 expression from the periphery of corneal epithelium and increased K13 expression toward the corneal center. Interestingly, K14 was detected in all layers of corneal epithelium of TGF-αECK mice, whereas it was limited at basal layer of controls. Transmission electron microscopy showed desmosome loss between corneal epithelial cells of TGF-αECK mice. In TGF-αECK mice, keratocan expression was abolished; α-SMA expression was increased while expression of Col1a1, Col1a2, and Col5a1 was diminished. Cell proliferation increased in the corneal epithelium and stroma, but not in the endothelium of TGF-αECK mice.ConclusionsExcess TGF-α had detrimental effects on corneal morphogenesis during mouse development in that it changed the cell fate of corneal epithelial cells to assume conjunctival phenotypic expression of K13, and keratocytes to myofibroblast phenotype.

Differential impact of paired patient-derived BPH and normal adjacent stromal cells on benign prostatic epithelial cell growth in 3D culture.

Benign prostatic hyperplasia (BPH) is an age-related disease characterized by nonmalignant abnormal growth of the prostate, which is also frequently associated with lower urinary tract symptoms. The prostate with BPH exhibits enhanced growth not only in the epithelium but also in the stroma, and stromal-epithelial interactions are thought to play an important role in BPH pathogenesis. However, our understanding of the mechanisms of stromal-epithelial interactions in the development and progression of BPH is very limited. Matched pairs of glandular BPH and normal adjacent prostate specimens were obtained from BPH patients undergoing simple prostatectomy for symptomatic BPH. Tissues were divided further into fresh specimens for culture of primary prostatic stromal cells, and specimens were embedded in paraffin for immunohistochemical analyses. Proliferation assays, immunohistochemistry, and immunoblotting were used to characterize the primary prostate stromal cells and tissue sections. Coculture of the primary stromal cells with benign human prostate epithelial cell lines BHPrE1 or BPH-1 was performed in three-dimensional (3D) Matrigel to determine the impact of primary stromal cells derived from BPH on epithelial proliferation. The effect of stromal-conditioned medium (CM) on BHPrE1 and BPH-1 cell growth was tested in 3D Matrigel as well. BPH stromal cells expressed less smooth muscle actin and calponin and increased vimentin, exhibiting a more fibroblast and myofibroblast phenotype compared with normal adjacent stromal cells both in culture and in corresponding paraffin sections. Epithelial spheroids formed in 3D cocultures with primary BPH stromal cells were larger than those formed in coculture with primary normal stromal cells. Furthermore, CM from BPH stromal cells stimulated epithelial cell growth while CM from normal primary stromal cells did not in 3D culture. These findings suggest that the stromal cells in BPH tissues are different from normal adjacent stromal cells and could promote epithelial cell proliferation, potentially contributing to the development and progression of BPH.

Human Adipocyte Conditioned Medium Promotes In Vitro Fibroblast Conversion to Myofibroblasts

Adipocytes and adipose tissue derived cells have been investigated for their potential to contribute to the wound healing process. However, the details of how these cells interact with other essential cell types, such as myofibroblasts/fibroblasts, remain unclear. Using a novel in-vitro 3D human adipocyte/pre-adipocyte spheroid model, we investigated whether adipocytes and their precursors (pre-adipocytes) secrete factors that affect human dermal fibroblast behavior. We found that both adipocyte and pre-adipocyte conditioned medium induced the migration of fibroblasts, but only adipocyte conditioned medium induced fibroblast differentiation into a highly contractile, collagen producing myofibroblast phenotype. Furthermore, adipocyte mediated myofibroblast induction occurred through a TGF-β independent mechanism. Our findings contribute to a better understanding on the involvement of adipose tissue in wound healing, and may help to uncover and develop fat-related wound healing treatments.

Statistical Mechanics of Non-Muscle Myosin IIA in Human Bone Marrow-Derived Mesenchymal Stromal Cells Seeded in a Collagen Scaffold: A Thermodynamic Near-Equilibrium Linear System Modified by the Tripeptide Arg-Gly-Asp (RGD).

Mesenchymal stromal cells (MSCs) were obtained from human bone marrow and amplified in cultures supplemented with human platelet lysate. Once semi-confluent, cells were seeded in solid collagen scaffolds that were rapidly colonized by the cells generating a 3D cell scaffold. Here, they acquired a myofibroblast phenotype and when exposed to appropriate chemical stimulus, developed tension and cell shortening, similar to those of striated and smooth muscle cells. Myofibroblasts contained a molecular motor—the non-muscle myosin type IIA (NMMIIA) whose crossbridge (CB) kinetics are dramatically slow compared with striated and smooth muscle myosins. Huxley’s equations were used to determine the molecular mechanical properties of NMMIIA. Thank to the great number of NMMIIA molecules, we determined the statistical mechanics (SM) of MSCs, using the grand canonical ensemble which made it possible to calculate various thermodynamic entities such as the chemical affinity, statistical entropy, internal energy, thermodynamic flow, thermodynamic force, and entropy production rate. The linear relationship observed between the thermodynamic force and the thermodynamic flow allowed to establish that MSC-laden in collagen scaffolds were in a near-equilibrium stationary state (affinity ≪ RT), MSCs were also seeded in solid collagen scaffolds functionalized with the tripeptide Arg-Gly-Asp (RGD). This induced major changes in NMMIIA SM particularly by increasing the rate of entropy production. In conclusion, collagen scaffolds laden with MSCs can be viewed as a non-muscle contractile bioengineered tissue operating in a near-equilibrium linear regime, whose SM could be substantially modified by the RGD peptide.

AB0168 NINTEDANIB (TYROSINE-KINASE INHIBITOR) INHIBITS THE TRANSITION OF CIRCULATING FIBROCYTES ISOLATED FROM SYSTEMIC SCLEROSIS PATIENTS INTO MYOFIBROBLASTS: AN IN VITROSTUDY

Background:Systemic sclerosis (SSc) is a chronic connective disease characterized by microvascular alterations, dysregulated immune response and fibrosis [1,2]. Myofibroblasts are alpha-smooth muscle actin (alphaSMA)+cells and play a crucial role in fibrosis, through the excessive synthesis and deposition of extracellular matrix (ECM) proteins, in particular fibronectin (FN) and type I collagen (COL1) [3]. Despite myofibroblasts primarily derive from resident fibroblasts transition and differentiation, another important source is represented by circulating fibrocytes [4]. Nintedanib is a tyrosine kinase inhibitor approved for the treatment of idiopathic pulmonary fibrosis that interferes with the signalling pathways involved in the pathogenesis of fibrosis [5].Objectives:To investigate the possible effects of nintedanib in contrasting the ability of cultured mature fibrocytes from SSc patients to differentiate into profibrotic myofibroblasts.Methods:Circulating fibrocytes were obtained from peripheral blood mononuclear cells isolated from 5 limited cutaneous SSc patients (mean age 68 +/- 10 years) and then plated on FN-coated tissue culture dishes in growth medium (DMEM at 20% of fetal bovine serum, 1% of penicillin-streptomycin and 1% L-glutamine), to allow the adhesion of fibrocyte precursors. Adherent cells were maintained in growth medium for 8 days in order to allow their differentiation into fibrocytes. Differentiated fibrocytes were treated with nintedanib at the concentrations of 100nM and 1000nM for 3 and 24 hours (hrs) or maintained in growth medium without any treatment. The differentiation of fibrocytes into myofibroblasts was determined evaluating the gene expression of alphaSMA, fibroblast specific protein-1 (S100A4) COL1, FN and CXCR4 by quantitative real-time polymerase chain reaction, and the protein synthesis of alphaSMA, COL1 and FN by western blotting.Results:Nintedanib inhibited alphaSMA and S100A4 gene expression already at the concentration of 100nM in cultured fibrocytes and after 3 hrs of treatment, when compared with untreated cells. Furthermore, both concentrations of nintedanib (100nM and 1000nM) reduced the gene expression of COL1 and FN, whereas only 100nM downregulated the CXCR4 gene expression. At protein level, nintedanib 100nM and 1000nM reduced the synthesis of alphaSMA and COL1 after 24 hrs of treatment, whereas FN synthesis was reduced only by the nintedanib concentration of 1000nM.Conclusion:The preliminary results show that nintedanib may inhibit thein vitrotransition of SSc fibrocytes into myofibroblasts and their profibrotic activity, through the reduction of specific myofibroblast phenotype markers and ECM protein production. The results seem to suggest fibrocytes as further possible target of the antifibrotic action of nintedanib in SSc.

A novel pancreatic tumour and stellate cell 3D co-culture spheroid model

BackgroundPancreatic ductal adenocarcinoma is a devastating disease with poor outcome, generally characterized by an excessive stroma component. The purpose of this study was to develop a simple and reproducible in vitro 3D-assay employing the main constituents of pancreatic ductal adenocarcinoma, namely pancreatic stellate and cancer cells.MethodA spheroid assay, directly co-culturing human pancreatic stellate cells with human pancreatic tumour cells in 3D was established and characterized by electron microscopy, immunohistochemistry and real-time RT-PCR. In order to facilitate the cell type-specific crosstalk analysis by real-time RT-PCR, we developed a novel in vitro 3D co-culture model, where the participating cell types were from different species, human and mouse, respectively. Using species-specific PCR primers, we were able to investigate the crosstalk between stromal and cancer cells without previous cell separation and sorting.ResultsWe found clear evidence for mutual influence, such as increased proliferation and a shift towards a more mesenchymal phenotype in cancer cells and an activation of pancreatic stellate cells towards the myofibroblast phenotype. Using a heterospecies approach, which we coined virtual sorting, confirmed the findings we made initially in the human-human spheroids.ConclusionsWe developed and characterized different easy to set up 3D models to investigate the crosstalk between cancer and stroma cells for pancreatic cancer.

Platelet-Rich Plasma Modulates Gap Junction Functionality and Connexin 43 and 26 Expression During TGF-β1-Induced Fibroblast to Myofibroblast Transition: Clues for Counteracting Fibrosis.

Skeletal muscle repair/regeneration may benefit by Platelet-Rich Plasma (PRP) treatment owing to PRP pro-myogenic and anti-fibrotic effects. However, PRP anti-fibrotic action remains controversial. Here, we extended our previous researches on the inhibitory effects of PRP on in vitro transforming growth factor (TGF)-β1-induced differentiation of fibroblasts into myofibroblasts, the effector cells of fibrosis, focusing on gap junction (GJ) intercellular communication. The myofibroblastic phenotype was evaluated by cell shape analysis, confocal fluorescence microscopy and Western blotting analyses of α-smooth muscle actin and type-1 collagen expression, and electrophysiological recordings of resting membrane potential, resistance, and capacitance. PRP negatively regulated myofibroblast differentiation by modifying all the assessed parameters. Notably, myofibroblast pairs showed an increase of voltage-dependent GJ functionality paralleled by connexin (Cx) 43 expression increase. TGF-β1-treated cells, when exposed to a GJ blocker, or silenced for Cx43 expression, failed to differentiate towards myofibroblasts. Although a minority, myofibroblast pairs also showed not-voltage-dependent GJ currents and coherently Cx26 expression. PRP abolished the TGF-β1-induced voltage-dependent GJ current appearance while preventing Cx43 increase and promoting Cx26 expression. This study adds insights into molecular and functional mechanisms regulating fibroblast-myofibroblast transition and supports the anti-fibrotic potential of PRP, demonstrating the ability of this product to hamper myofibroblast generation targeting GJs.

LINC00312/YBX1 Axis Regulates Myofibroblast Activities in Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) has been recognized as a precancerous disorder in the oral cavity. Great effort has been made to inhibit the malignant progression of OSF over the past decades, but the cure of this fibrosis disease has not been discovered. In the present study, we found that a long noncoding RNA, LINC00312, was upregulated in OSF tissues, and positively associated with several fibrosis factors, such as α-SMA, type I collagen, and fibronectin. As such, we sought to investigate the role of LINC00312 in OSF progression and identify its interacting factor that mediated oral fibrogenesis. Our results showed that the inhibition of LINC00312 downregulated the myofibroblast activities, including collagen gel contractility, transwell migration, and wound healing, as well as the gene expression of myofibroblast markers. We verified that YBX1 was a downstream factor of LINC00312 and revealed that the downregulation of YBX1 repressed the gene expression of α-SMA and p-Smad2 along with the reduced myofibroblast phenotypes. Most importantly, we demonstrated that the LINC00312-induced myofibroblast activities were reverted by the knockdown of YBX1, suggesting that the LINC00312-mediated myofibroblast transdifferentiation was through YBX1. Collectively, our findings revealed that the LINC00312/ YBX1 axis may serve as a target for the development of therapies against OSF.

Hedgehog Signaling Demarcates a Niche of Fibrogenic Peribiliary Mesenchymal Cells

In response to tissue injury, stromal cells secrete extracellular matrix (ECM) components that remodel the tissue and lead to fibrosis. Parenchymal stellate cells are the primary contributors to fibrosis in models of hepatocellular and cholestatic injury. The liver comprises different, heterogenous compartments; stromal cells within those compartments might have unique identities and regional functions. The portal tract contains the bile duct, which is surrounded by stromal cells often called portal fibroblasts. We investigated the contributions of these cells to hepatic injury. We performed studies with Gli1:CreERT2;Rosa26:lox-STOP-lox-tdTomato mice. Mice underwent bile duct ligation or were fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine to induce cholestatic injury or were given carbon tetrachloride to induce liver fibrosis. Liver tissues were collected and analyzed by histology and immunofluorescence, and mesenchymal cells were isolated. We performed lineage tracing experiments to determine the fates of peribiliary mesenchymal cells (PMCs) that surround the bile duct after cholestatic and hepatocellular injury. We used cell sorting combined with RNA sequencing to isolate stellate cells and PMCs, and we identified determinants of cell identity within each population. Liver tissues were obtained from patients with primary sclerosing cholangitis, alcoholic liver disease, or nonalcoholic steatohepatitis or individuals without disease and were analyzed by quantitative reverse transcription polymerase chain reaction. Gli1 was a marker of mesenchymal cells that surround the biliary tree but not epithelial cells of the canals of Hering. Lineage-traced Gli1+ PMCs proliferated and acquired a myofibroblast phenotype after cholestatic injury; Gli1+ PMCs were found only surrounding the main duct of a portal tract but not the epithelial cells of the ductular reaction, which were instead encased by stellate cells. Compared with stellate cells, Gli1+ PMCs expressed a different subset of genes, including genes that are markers of active hedgehog signaling, Osr1 (encodes a transcription factor), and ECM-related genes. Loss of hedgehog signaling reduced expression of Osr1 and PMC-specific ECM genes. Liver tissues from patients with liver disease had increased expression of genes that define PMC identity compared with control liver tissues. In lineage-tracing studies of mice, we found that Gli1+ PMCs are a subset of stromal cells characterized by active hedgehog signaling that proliferate, acquire a myofibroblast phenotype, and surround the biliary tree in response to cholestatic injury.

The Regulation of Fibroblast to Myofibroblast Activation by Zinc Finger E‐Box Binding Homeobox 1 and 2

After a myocardial infarction (MI), a dense collagen scar is formed that initially prevents the heart from rupturing. However, after the scar is formed, collagen deposits accumulate and the extracellular matrix (ECM) is remodeled in the viable areas of the heart, distal from the site of injury, which causes the entire organ to vstiffen in a process called cardiac fibrosis (CF). CF often contributes to the development of heart failure (HF) which is a major clinical problem in Canada. Cardiac fibroblasts (FBs) provide structural support and remodel the ECM in the healthy heart, but after an MI, FBs become activated, proliferate, and adopt a myofibroblast (MF) phenotype. MFs can continue to produce ECM long after they become activated which leads to CF and ultimately HF. Two transcription factors that have been identified as potential contributors to CF are zinc finger E‐box binding homeobox 1 and 2 (ZEB1/2). ZEB1 and ZEB2 are essential for epithelial‐mesenchymal transition (EMT) and, previous studies in our lab have shown that ZEB2 is also directly involved with FB activation. Based on other research we hypothesize that ZEB1 may also regulate FB activation and be functionally redundant with ZEB2. Our initial results show that ZEB1 protein and RNA levels both increase as FBs convert to MFs. By comparing samples at varying time points we have increased our understanding of the FB to MF activation process. This study show that ZEB1 may be involved in the fibrotic process and further analysis will determine the extent of its involvement.Support or Funding InformationSupported by a grant from NSERC and from the St. Boniface Hospital Foundation Research without Borders initiative.

MP06-15 MYOFIBROBLASTS CYTOKINES SECRETED BY THE PROGRESSION OF BENIGN PROSTATIC HYPERPLASIA PROMOTE MONOCYTE RECRUITMENT AND INDUCE POLARIZATION OF THP1 TO M2-TYPE MACROPHAGES

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (MP06)1 Apr 2020MP06-15 MYOFIBROBLASTS CYTOKINES SECRETED BY THE PROGRESSION OF BENIGN PROSTATIC HYPERPLASIA PROMOTE MONOCYTE RECRUITMENT AND INDUCE POLARIZATION OF THP1 TO M2-TYPE MACROPHAGES Muqiu Zhang* Muqiu Zhang*Muqiu Zhang* More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000820.015AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Introduction and Objective: Our previous studies, stroma–epithelium ratios and collagen deposition were prevalent in rapidly-progressed BPH patients (age ≤50 years old) . Meanwhile, we also detected that increased M2 macrophage infiltration in rapidly-progressed BPH patients, M2 macrophage induces myofibroblast phenotype during the progression of benign prostatic hyperplasia. The present study mainly investigates if myofibroblasts cytokines secreted by the progression of benign prostatic hyperplasia may promote monocyte recruitment and induce polarization of THP1 to M2-type macrophages. METHODS: Methods: We separated the human primary prostate fibroblast (PrPF) cells from the early progressed BPH (PrPF-early), age-matched prostate (PrPF-control), and elderly BPH tissues (PrPF-old). In vitro, we uesd flow cytometry to detect CD163 and CD206 (M2-type macrophages’ markers ) expression of THP-1 cells after co-culture 4days with three groups of human primary prostate fibroblast, respectively. Quantitative PCR was used to identify cytokines secreted by three groups of human primary prostate fibroblast. RESULTS: Results: THP-1 was recruited by human primary prostate fibroblast of rapidly-progressed BPH than in the other two groups. Significant increase in the percentage of M2 macrophages after co-culture with PrPF-early. Cytokines MCP-1 and IL-6 were increased in primary prostate fibroblast of rapidly-progressed BPH. Primary prostate fibroblast of rapidly-progressed BPH could recruit THP-1 and induce polarization of THP1 to M2-type macrophages. CONCLUSIONS: Conclusion: The early-progressed BPH fibroblasts recruit THP-1 and induce polarization of THP1 to M2-type macrophages. Source of Funding: no © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e56-e57 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Muqiu Zhang* More articles by this author Expand All Advertisement PDF downloadLoading ...

Fibroblast-specific Stat1 deletion enhances the myofibroblast phenotype during tissue repair.

Signal transducer and activator of transcription 1 (Stat1) is a ubiquitously expressed latent transcription factor that is activated by many cytokines and growth factors. Global Stat1 knockout mice are prone to chemical-induced lung and liver fibrosis, suggesting roles for Stat1 in tissue repair. However, the importance of Stat1 in fibroblast-mediated and vascular smooth muscle cell (VSMC)-mediated injury response has not been directly evaluated in vivo. Here, we focused on two models of tissue repair in conditional Stat1 knockout mice: excisional skin wounding in mice with Stat1 deletion in dermal fibroblasts, and carotid artery ligation in mice with global Stat1 deletion or deletion specific to VSMCs. In the skin model, dermal wounds closed at a similar rate in mice with fibroblast Stat1 deletion and controls, but collagen and α-smooth muscle actin (αSMA) expression were increased in the mutant granulation tissue. Cultured Stat1 -/- and Stat1 +/- dermal fibroblasts exhibited similar αSMA+ stress fiber assembly, collagen gel contraction, proliferation, migration, and growth factor-induced gene expression. In the artery ligation model, there was a significant increase in fibroblast-driven perivascular fibrosis when Stat1 was deleted globally. However, VSMC-driven remodeling and neointima formation were unchanged when Stat1 was deleted specifically in VSMCs. These results suggest an in vivo role for Stat1 as a suppressor of fibroblast mediated, but not VSMC mediated, injury responses, and a suppressor of the myofibroblast phenotype.

Long non-coding RNA HOTAIR drives EZH2-dependent myofibroblast activation in systemic sclerosis through miRNA 34a-dependent activation of NOTCH

BackgroundSystemic sclerosis (SSc) is characterised by autoimmune activation, tissue and vascular fibrosis in the skin and internal organs. Tissue fibrosis is driven by myofibroblasts, that are known to maintain their...

SUN-160 Drp1-mediated mitochondrial fission promotes renal fibroblast activation and fibrogenesis

Mitochondria are extremely dynamic organelles, constantly undergoing antagonistic processes of fission and fusion. Multiple mitochondrial membrane GTPases regulate mitochondrial dynamics. Among them, Dynamin-related protein 1 (Drp1) executes fission by recruiting to the mitochondria outer membrane to drive scission. Drp1 activity can be reversibly modified by two critical phosphorylation sites. Phosphorylation of Drp1 at serine 616 (p-Drp1S616) promotes Drp1 activity. Conversely, phosphorylation of serine 637 (p-Drp1S637) represses its activity. Besides controlling morphology, mitochondrial dynamics play crucial roles in bioenergetics, metabolism, cell transformation, proliferation, and apoptosis. The imbalance of fission and fusion dynamics resulting in excessive mitochondrial fragmentation has been implicated in many chronic illnesses. Excessive mitochondrial fission acts as a pro-proliferative marker in lung fibrosis, its potential role in renal fibroblast activation and fibrogenesis has never been investigated. Mitochondrial dynamic features of fibroblasts were examined in fibrotic and nonfibrotic kidney. The potential role of Drp1-mediated mitochondrial fission in renal fibroblasts activation, proliferation and fibrogenesis using in vivo, ex vivo, and in vitro strategies. We showed more pronounced fragmented mitochondria in fibrotic than in non-fibrotic renal fibroblasts in humans(Figure 1A) and mice(Figure 1B), along with the increased phosphorylation of Drp1 at serine 616. Pharmacological inhibition of mitochondrial fission by mdivi-1(a specific inhibitor of Mdivi-1) attenuated myofibroblast accumulation and interstitial fibrosis in the obstructive kidneys of mice(Figure 1C and 1D). Moreover, ex vivo mdivi-1 treatment reversed mitochondrial fission and differentiated phenotype in primary myofibroblast-derived from fibrotic kidney. In vitro study, by using a rat fibroblast cell line(NRK-49F), Drp1 knockdown by using siRNA suppressed TGF-β1-elicited mitochondrial fragmentation, mtROS elevation and glycolytic shift, which promoted apoptosis and inhibited myofibroblast differentiation and proliferation in NRK-49F cells(Figure 1E and 1F). Further, phosphorylation of Drp1 at serine 616 (p-Drp1S616) and acetylation of H3K27(H3K27ac) was upregulated in fibrotic kidneys and TGF-β1-treated NRK-49F cells. Overexpression of inactive mutant of Drp1(Drp1S616A) rather than wild-type Drp1 rescued TGF-β1-induced myofibroblastic phenotype, along with reduced p-Drp1S616 and H3K27ac in Drp1-deleted NRK-49F cells. TGF-β1 treatment increased the enrichment of H3K27ac at the promoters of α-SMA and PCNA, which was hampered by overexpressed Drp1S616A but not wild-type Drp1 in Drp1-deficient cells.(For the reason that only one images can be uploaded, the rest of the data are not shown in the figure) Our results imply that mitochondrial dynamics is crucial for fibroblasts activation and proliferation, modulating Drp1-mediated mitochondrial fission in fibroblast may serve as a therapeutic target for retarding renal fibrogenesis.

The Shh/Gli signaling cascade regulates myofibroblastic activation of lung-resident mesenchymal stem cells via the modulation of Wnt10a expression during pulmonary fibrogenesis

Lung-resident mesenchymal stem cells (LR-MSCs) are important regulators of lung repair and regeneration, and evidence suggests that this cell population also plays a vital role in fibrosis. Crosstalk between sonic hedgehog (Shh) signaling and wingless/integrated (Wnt) has been demonstrated in idiopathic pulmonary fibrosis (IPF). However, the underlying correlation between LR-MSCs and the Shh-Wnt signaling cascade remains poorly understood. Here, we identified Wnt10a as a key factor in pulmonary fibrosis. Using a bleomycin mouse model, we found that highly expressed Wnt10a was secreted by LR-MSCs undergoing myofibroblastic differentiation. LR-MSCs with myofibroblast characteristics isolated from fibrotic lungs exhibited increased Shh pathway activity, suggesting their role as Shh targets. In vitro, LR-MSCs responded to stimulation by recombinant Shh, acquiring a myofibroblast phenotype. We further demonstrated that the Shh/glioblastoma (Gli) system machinery regulated LR-MSC-to-myofibroblast transition and pulmonary fibrosis via manipulation of Wnt/β-catenin signaling. Accordingly, inhibition of the Shh-Wnt signaling cascade prevented LR-MSC transformation into myofibroblasts and ameliorated pulmonary fibrotic lesions. Moreover, induction of Wnt10a expression and activation of Shh/Gli signaling were confirmed in human pulmonary fibrosis. In summary, this study linking the Shh-Wnt signaling cascade with LR-MSC fibrogenic activity furthered the current understanding of pulmonary fibrosis pathogenesis and might provide a new perspective in the development of treatment strategies for IPF.

The ER Unfolded Protein Response Effector, ATF6, Reduces Cardiac Fibrosis and Decreases Activation of Cardiac Fibroblasts.

Activating transcription factor-6 α (ATF6) is one of the three main sensors and effectors of the endoplasmic reticulum (ER) stress response and, as such, it is critical for protecting the heart and other tissues from a variety of environmental insults and disease states. In the heart, ATF6 has been shown to protect cardiac myocytes. However, its roles in other cell types in the heart are unknown. Here we show that ATF6 decreases the activation of cardiac fibroblasts in response to the cytokine, transforming growth factor β (TGFβ), which can induce fibroblast trans-differentiation into a myofibroblast phenotype through signaling via the TGFβ–Smad pathway. ATF6 activation suppressed fibroblast contraction and the induction of α smooth muscle actin (αSMA). Conversely, fibroblasts were hyperactivated when ATF6 was silenced or deleted. ATF6 thus represents a novel inhibitor of the TGFβ–Smad axis of cardiac fibroblast activation.

Biological dermal templates with native collagen scaffolds provide guiding ridges for invading cells and may promote structured dermal wound healing.

Dermal substitutes are of major importance in treating full thickness skin defects. They come in a variety of materials manufactured into various forms, such as films, hydrocolloids, hydrogels, sponges, membranes, and electrospun micro- and nanofibers. Bioactive dermal substitutes act in wound healing either by delivery of bioactive compounds or by being constructed from materials having endogenous activity. The healing success rate is highly determined by cellular and physiological processes at the host-biomaterial interface during crucial wound healing steps. Hence, it is important to design appropriate wound treatment strategies with the ability to work actively with tissues and cells to enhance healing. Therefore, in this study, we investigated biological dermal templates and their potential to stimulate natural cell adherence, guidance, and morphology. The most pronounced effect was observed in biomaterials with the highest content of native collagen networks. Cell attachment and proliferation were significantly enhanced on native collagen scaffolds. Cell morphology was more asymmetrical on such scaffolds, resembling native in vivo structures. Importantly, considerably lower expression of myofibroblast phenotype was observed on native collagen scaffolds. Our data suggest that this treatment strategy might be beneficial for the wound environment, with the potential to promote improved tissue regeneration and reduce abnormal scar formation.

Lack of Thy1 defines a pathogenic fraction of cardiac fibroblasts in heart failure

In response to heart injury, inflammation, or mechanical overload, quiescent cardiac fibroblasts (CFs) can become activated myofibroblasts leading to pathological matrix remodeling and decline in cardiac function. Specific targeting of fibroblasts may thus enable new therapeutic strategies to delay or reverse the progression of heart failure and cardiac fibrosis. However, it remains unknown if all CFs are equally responsive to specific pathological insults and if there exist sub-populations of resident fibroblasts in the heart that have distinctive pathogenic phenotypes. Here, we show that in response to transverse aortic constriction (TAC)-induced heart failure, previously uncharacterized Thy1neg (Thy1-/MEFSK4+/CD45-/CD31-) fraction of mouse ventricular fibroblasts became more abundant and attained a more activated, pro-fibrotic myofibroblast phenotype compared to Thy1Pos fraction. In a tissue-engineered 3D co-culture model of healthy cardiomyocytes and freshly isolated CFs, Thy1neg CFs from TAC hearts significantly decreased cardiomyocyte contractile function and calcium transient amplitude, and increased extracellular collagen deposition yielding a profibrotic heart tissue phenotype. In vivo, mice with global knockout of Thy1 developed more severe cardiac dysfunction and fibrosis in response to TAC-induced heart failure than wild-type mice. Taken together, our studies identify cardiac myofibroblasts lacking Thy1 as a pathogenic CF fraction in cardiac fibrosis and suggest important roles of Thy1 in pathophysiology of heart failure.

Could cathepsin-k be a driver of the myofibroblastic differentiation observed in dermatofibroma, atypical fibroxanthoma and pleomorphic dermal sarcoma?

Dermatofibroma (BFH), atypical fibroxanthoma (AFX) and dermal pleomorphic sarcoma (DPS) are skin-based soft-tissue neoplasms of uncertain lineage. They are classified as “fibrohistiocytic” neoplasms, even if the World Health Organization stated that this term connotes a polymorphic group of lesions that histologically resemble fibroblasts and histiocytes. It is well-known that this group of lesions shows a “fibro-histiocytic-dendritic” and/or a “myofibroblastic” phenotype, even within the same lesion. We studied the expression of cathepsin-k in 34 cases (25 BFH, 5 AFX, 4 DPS) with a broad panel of antibodies. 20 cases (5 dermatofibrosarcoma protuberans, 5 melanomas, 5 basal cell carcinomas, 5 squamous cell carcinomas) were chosen as controls. Although our results need to be validated, they support a myofibroblastic and/or partial myofibroblastic (“proto-myofibroblastic”) phenotype and the lineage-plasticity of these neoplasms, highlighting the potential role of cathepsin-k in myofibroblastic trans-differentiation. Cathepsin-k proved to be an additional immunoistochemical marker potentially useful in the diagnostic algorithm.

The Pathway from Radiation to Fibrosis: LET Dependence

It is well-known that Radiation-induced fibrosis (RIF) is a late event occurring months to years after the initial radiation exposure. Fibrotic lesions have been shown to manifest in many tissues including the skin, heart, lung, liver and kidney. Fibrosis occurs due to abnormal accumulation of extracellular matrix (ECM) proteins that result in loss of normal tissue and organ function. The cell type involved in RIF is myofibroblasts, which do not undergo apoptosis after healing but instead continue to accumulate, producing excessive amounts of ECM proteins, thereby damaging the tissues and organs. Reactive oxygen species, generated in response to radiation, is one signal that helps maintain the myofibroblast phenotype. In this review, we discuss molecular mechanisms leading to this late radiation event, known biomarkers for prediction, preclinical animal models of radiation-induced toxicity and current clinical trials designed for mitigation and treatment of radiation-induced fibrosis. We also discuss other physical properties such as linear energy transfer (LET) than the ones used in the clinics today which may have the potential to change our understanding on this inevitable pathway from radiation treatment to organ fibrosis.