scholarly journals FAK Inhibition Attenuates Corneal Fibroblast Differentiation In Vitro

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1682
Author(s):  
Vincent Yeung ◽  
Sriniwas Sriram ◽  
Jennifer A. Tran ◽  
Xiaoqing Guo ◽  
Audrey E. K. Hutcheon ◽  
...  
Keyword(s):  
Wound Healing ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Myofibroblast Differentiation ◽  
Corneal Scarring ◽  
Corneal Fibroblast ◽  
Corneal Fibroblasts ◽  
Corneal Wound ◽  
Tgf Β1

Corneal fibrosis (or scarring) occurs in response to ocular trauma or infection, and by reducing corneal transparency, it can lead to visual impairment and blindness. Studies highlight important roles for transforming growth factor (TGF)-β1 and -β3 as modulators in corneal wound healing and fibrosis, leading to increased extracellular matrix (ECM) components and expression of α-smooth muscle actin (αSMA), a myofibroblast marker. In this study, human corneal fibroblasts (hCF) were cultured as a monolayer culture (2D) or on poly-transwell membranes to generate corneal stromal constructs (3D) that were treated with TGF-β1, TGF-β3, or TGF-β1 + FAK inhibitor (FAKi). Results show that hCF 3D constructs treated with TGF-β1 or TGF-β3 impart distinct effects on genes involved in wound healing and fibrosis—ITGAV, ITGB1, SRC and ACTA2. Notably, in the 3D construct model, TGF-β1 enhanced αSMA and focal adhesion kinase (FAK) protein expression, whereas TGF-β3 did not. In addition, in both the hCF 2D cell and 3D construct models, we found that TGF-β1 + FAKi attenuated TGF-β1-mediated myofibroblast differentiation, as shown by abrogated αSMA expression. This study concludes that FAK signaling is important for the onset of TGF-β1-mediated myofibroblast differentiation, and FAK inhibition may provide a novel beneficial therapeutic avenue to reduce corneal scarring.

scholarly journals Cub domain-containing protein 1 negatively regulates TGF-β signaling and myofibroblast differentiation

2018 ◽  
Vol 314 (5) ◽  
pp. L695-L707 ◽  
Author(s):  
Nina Noskovičová ◽  
Katharina Heinzelmann ◽  
Gerald Burgstaller ◽  
Jürgen Behr ◽  
Oliver Eickelberg
Keyword(s):  
Cell Surface ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Proteome Analysis ◽  
Lung Fibroblasts ◽  
Myofibroblast Differentiation ◽  
Dependent Manner ◽  
Cub Domain ◽  
Tgf Β1

Fibroblasts are thought to be the prime cell type for producing and secreting extracellular matrix (ECM) proteins in the connective tissue. The profibrotic cytokine transforming growth factor-β1 (TGF-β1) activates and transdifferentiates fibroblasts into α-smooth muscle actin (α-SMA)-expressing myofibroblasts, which exhibit increased ECM secretion, in particular collagens. Little information, however, exists about cell-surface molecules on fibroblasts that mediate this transdifferentiation process. We recently identified, using unbiased cell-surface proteome analysis, Cub domain-containing protein 1 (CDCP1) to be strongly downregulated by TGF-β1. CDCP1 is a transmembrane glycoprotein, the expression and role of which has not been investigated in lung fibroblasts to date. Here, we characterized, in detail, the effect of TGF-β1 on CDCP1 expression and function, using immunofluorescence, FACS, immunoblotting, and siRNA-mediated knockdown of CDCP1. CDCP1 is present on interstitial fibroblasts, but not myofibroblasts, in the normal and idiopathic pulmonary fibrosis lung. In vitro, TGF-β1 decreased CDCP1 expression in a time-dependent manner by impacting mRNA and protein levels. Knockdown of CDCP1 enhanced a TGF-β1-mediated cell adhesion of fibroblasts. Importantly, CDCP1-depleted cells displayed an enhanced expression of profibrotic markers, such as collagen V or α-SMA, which was found to be independent of TGF-β1. Our data show, for the very first time that loss of CDCP1 contributes to fibroblast to myofibroblast differentiation via a potential negative feedback loop between CDCP1 expression and TGF-β1 stimulation.

scholarly journals Glucosamine impedes transforming growth factor β1-mediated corneal fibroblast differentiation by targeting Krüppel-like factor 4

2019 ◽  
Vol 26 (1) ◽  
Author(s):  
Ying-Jen Chen ◽  
Shih-Ming Huang ◽  
Ming-Cheng Tai ◽  
Jiann-Torng Chen ◽  
Chang-Min Liang
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Disease Model ◽  
Immunoblot Analysis ◽  
Transforming Growth Factor Β1 ◽  
Myofibroblast Differentiation ◽  
Alpha Smooth Muscle Actin ◽  
Corneal Fibroblasts ◽  
Tgf Β1

Abstract Background Transforming growth factor (TGF) family members play important roles in the regulation of corneal integrity, and the pathogenesis of corneal fibrosis. Currently, there are no effective agents targeting TGF-β signaling to diminish corneal fibrosis. Glucosamine (GlcN), which is widely used in the treatment of osteoarthritis, abrogates the morphologic effects of TGF-β2 on retinal pigmented epithelial cells in a mouse disease model. Here, we sought to determine whether GlcN would exert beneficial effects against TGF-β1-induced corneal fibrosis. Methods In human corneal fibroblasts (HCFs) treated with GlcN, the expression of Krüppel-like factor 4 (KLF4) and its downstream signaling effects were determined in the presence and absence of TGF-β1 using immunoblot analysis. We further explored GlcN inhibition of fibroblast-to-myofibroblast differentiation via KLF4 siRNA. The effect of cycloheximide on KLF4 protein levels with or without GlcN administration was assessed to determine whether GlcN affects the stability of the KLF4 protein. Results In HCFs, GlcN induced the expression of KLF4, which regulated the maturation and maintenance of the ocular surface. GlcN partially suppressed the TGF-β1-induced expression of alpha-smooth muscle actin (α-SMA) and reduced the collagen contraction capacity in HCFs, suggesting a decrease in fibroblast-to-myofibroblast differentiation. This effect appeared to be mediated through suppression of Smad2 phosphorylation and ERK-dependent signaling. The levels of KLF4 mRNA were increased by GlcN and decreased by TGF-β1 and the TGF-β1-induced α-SMA mRNA expression was upregulated when the KLF4 gene was silenced. GlcN also appeared to stabilize the KLF4 protein, reducing its turnover in corneal fibroblasts. Conclusion These findings shed light on a novel mechanism by which GlcN suppresses TGF-β1-induced fibroblast-to-myofibroblast differentiation through the upregulation of KLF4 expression. Current strategies for treating corneal fibrosis were not effective. Elevating KLF4 levels through the use of GlcN might provide an effective alternative to alleviate the development and progression of corneal fibrosis.

Acceleration of Palatal Wound Healing in Smad3-deficient Mice

2009 ◽  
Vol 88 (8) ◽  
pp. 757-761 ◽  
Author(s):  
K. Jinno ◽  
T. Takahashi ◽  
K. Tsuchida ◽  
E. Tanaka ◽  
K. Moriyama
Keyword(s):  
Wound Healing ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Monocyte Chemoattractant Protein 1 ◽  
Inflammatory Protein ◽  
Complex Process ◽  
Dermal Cells ◽  
Macrophage Inflammatory Protein ◽  
Tgf Β1

Wound healing is a well-orchestrated complex process leading to the repair of injured tissues. It is suggested that transforming growth factor (TGF)-β/Smad3 signaling is involved in wound healing. The purpose of this study was to investigate the role of TGF-β/Smad3 signaling in palatal wound healing in Smad3-deficient (Smad3−/−) mice. Histological examination showed that wound closure was accelerated by the proliferation of epithelium and dermal cells in Smad3−/− mice compared with wild-type (WT) mice. Macrophage/monocyte infiltration at wounded regions in Smad3−/− mice was decreased in parallel with the diminished production of TGF-β1, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1α compared with WT mice. Fibrocytes, expressing hematopoietic surface marker and fibroblast products, were recruited and produced α-smooth-muscle actin in WT mice, but were not observed in Smad3−/− mice. These results suggest that TGF-β/Smad3 signaling may play an important role in the regulation of palatal wound healing.

scholarly journals Platelet-Rich Plasma and Bone Marrow-Derived Mesenchymal Stromal Cells Prevent TGF-β1-Induced Myofibroblast Generation but Are Not Synergistic when Combined: Morphological in vitro Analysis

2018 ◽  
Vol 206 (6) ◽  
pp. 283-295 ◽  
Author(s):  
Flaminia Chellini ◽  
Alessia Tani ◽  
Larissa Vallone ◽  
Daniele Nosi ◽  
Paola Pavan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transforming Growth Factor ◽  
Synergistic Effects ◽  
Platelet Rich Plasma ◽  
Therapeutic Option ◽  
Smooth Muscle Actin ◽  
Combined Treatment ◽  
Biologically Active ◽  
Tgf Β1

The persistence of activated myofibroblasts is a hallmark of fibrosis of many organs. Thus, the modulation of the generation/functionality of these cells may represent a strategical anti-fibrotic therapeutic option. Bone marrow-derived mesenchymal stromal cell (MSC)-based therapy has shown promising clues, but some criticisms still limit the clinical use of these cells, including the need to avoid xenogeneic compound contamination for ex vivo cell amplification and the identification of appropriate growth factors acting as a pre-conditioning agent and/or cell delivery vehicle during transplantation, thus enabling the improvement of cell survival in the host tissue microenvironment. Many studies have demonstrated the ability of platelet-rich plasma (PRP), a source of many biologically active molecules, to positively influence MSC proliferation, survival, and functionality, as well as its anti-fibrotic potential. Here we investigated the effects of PRP, murine and human bone marrow-derived MSCs, and of the combined treatment PRP/MSCs on in vitro differentiation of murine NIH/3T3 and human HDFα fibroblasts to myofibroblasts induced by transforming growth factor (TGF)-β1, a well-known pro-fibrotic agent. The myofibroblastic phenotype was evaluated morphologically (cell shape and actin cytoskeleton assembly) and immunocytochemically (vinculin-rich focal adhesion clustering, α-smooth muscle actin and type-1 collagen expression). We found that PRP and MSCs, both as single treatments and in combination, were able to prevent the TGF-β1-induced fibroblast-myofibroblast transition. Unexpectedly, the combination PRP/MSCs had no synergistic effects. In conclusion, within the limitations related to an in vitro experimentation, our study may contribute to providing an experimental background for supporting the anti-fibrotic potential of the combination PRP/MSCs which, once translated “from bench to bedside,” could potentially offer advantages over the single treatments.

Expression of Transforming Growth Factor β1, β2, and β3 in Chronic, Cancer-Associated, Obstructive Pancreatitis

2006 ◽  
Vol 130 (3) ◽  
pp. 356-361 ◽  
Author(s):  
Yuki Fukumura ◽  
Toshio Kumasaka ◽  
Keiko Mitani ◽  
Kanae Karita ◽  
Koichi Suda
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Early Stage ◽  
Smooth Muscle Actin ◽  
Receptor Type ◽  
Type Ii ◽  
Soluble Receptor ◽  
Obstructive Pancreatitis ◽  
Tgf Β1

Abstract Context.—Myofibroblasts are considered to play central roles in pancreatic fibrosis. The potent fibrogenic capacities of transforming growth factor βs (TGF-βs) have been emphasized in vitro and in animal studies. However, the roles of TGF-βs in human chronic pancreatitis have not been fully clarified. Objective.—To investigate whether expressions of TGF-βs are related to myofibroblast distribution in chronic, cancer-associated, obstructive pancreatitis (COP). Design.—Histopathologic studies using hematoxylin-eosin and Elastica-Masson trichrome and immunohistochemical studies using antibodies against α-smooth muscle actin (SMA); CD68; TGF-β1, -β2, and -β3; and TGF-β soluble receptor type II were performed in 19 COP cases and 6 controls. By classifying COP tissues into 3 fibrosis phases by the amount of collagen deposits, immunoreactivities for TGF-βs, histopathologic changes, and myofibroblast distribution were examined for each fibrosis phase. Results.—Six cases were categorized in the early stage of fibrosis, 8 in the intermediate stage, and 5 in the advanced stage. Immunoreactivities for all 3 isoforms of TGF-β were observed in occasional myofibroblasts. In the early and intermediate stages, TGF-β1–expressing macrophages and neutrophils were distributed in the midst of myofibroblasts. TGF-β2 and TGF-β3 expressions were observed in ductal structures, sometimes even in sites where no or few myofibroblasts were seen. TGF-β soluble receptor type II was immunoreactive for myofibroblasts, endothelium, and ductal structures. Conclusions.—All 3 isoforms of TGF-βs may contribute to fibrosis in COP. Macrophages and neutrophils may be sources of fibrogenic TGF-β1. Infiltration of these cells appears to play an important role in the progression of COP fibrosis.

scholarly journals Celastrol-Induced Suppression of the MiR-21/ERK Signalling Pathway Attenuates Cardiac Fibrosis and Dysfunction

2016 ◽  
Vol 38 (5) ◽  
pp. 1928-1938 ◽  
Author(s):  
Mian Cheng ◽  
Gang Wu ◽  
Yue Song ◽  
Lin Wang ◽  
Ling Tu ◽  
...  
Keyword(s):  
Myocardial Fibrosis ◽  
Transforming Growth Factor Beta ◽  
Cardiac Dysfunction ◽  
Cardiac Fibrosis ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Signalling Pathway ◽  
Tgf Β1

Backgroud: Myocardial fibrosis results in myocardial remodelling and dysfunction. Celastrol, a traditional oriental medicine, has been suggested to have cardioprotective effects. However, its underlying mechanism is unknown. This study investigated the ability of celastrol to prevent cardiac fibrosis and dysfunction and explored the underlying mechanisms. Methods: Animal and cell models of cardiac fibrosis were used in this study. Myocardial fibrosis was induced by transverse aortic constriction (TAC) in mice. Cardiac hypertrophy and fibrosis were evaluated based on histological and biochemical measurements. Cardiac function was evaluated by echocardiography. The levels of transforming growth factor beta 1 (TGF-β1), extracellular signal regulated kinases 1/2 (ERK1/2) signalling were measured using Western blotting, while the expression of miR-21was analyzed by real-time qRT-PCR in vitro and in vivo. In vitro studies, cultured cardiac fibroblasts (CFs) were treated with TGF-β1 and transfected with microRNA-21(miR21). Results: Celastrol treatment reduced the increased collagen deposition and down-regulated α-smooth muscle actin (α-SMA), atrial natriuretic peptide (ANP), brain natriuretic peptides (BNP), beta-myosin heavy chain (β-MHC), miR-21 and p-ERK/ERK. Cardiac dysfunction was significantly attenuated by celastrol treatment in the TAC mice model. Celastrol treatment reduced myocardial fibroblast viability and collagen content and down-regulated α-SMA in cultured CFs in vitro. Celastrol also inhibited the miR-21/ERK signalling pathway. Celastrol attenuated miR-21 up-regulation by TGF-β1 and decreased elevated p-ERK/ERK levels in CFs transfected with miR-21. Conclusion: MiR-21/ERK signalling could be a potential therapeutic pathway for the prevention of myocardial fibrosis. Celastrol ameliorates myocardial fibrosis and cardiac dysfunction, these probably related to miR-21/ERK signaling pathways in vitro and in vivo.

scholarly journals Andrographolide Ameliorates Liver Fibrosis in Mice: Involvement of TLR4/NF-κB and TGF-β1/Smad2 Signaling Pathways

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Liteng Lin ◽  
Rui Li ◽  
Mingyue Cai ◽  
Jingjun Huang ◽  
Wensou Huang ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
History Of ◽  
Matrix Accumulation ◽  
Tgf Β1 ◽  
In Vitro Treatment ◽  
In Vitro Data ◽  
Sirius Red

Liver fibrosis is characterized by activated hepatic stellate cells (HSC) and extracellular matrix accumulation. Blocking the activation of HSC and the inflammation response are two major effective therapeutic strategies for liver fibrosis. In addition to the long history of using andrographolide (Andro) for inflammatory disorders, we aimed at elucidating the pharmacological effects and potential mechanism of Andro on liver fibrosis. In this study, liver fibrosis was induced by carbon tetrachloride (CCl4) and the mice were intraperitoneally injected with Andro for 6 weeks. HSC cell line (LX-2) and primary HSC were also treated with Andro in vitro. Treatment of CCl4-induced mice with Andro decreased the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), Sirius red staining as well as the expression of α smooth muscle actin (α-SMA) and transforming growth factor- (TGF-) β1. Furthermore, the expression of Toll-like receptor (TLR)4 and NF-κB p50 was also inhibited by Andro. Additionally, in vitro data confirmed that Andro treatment not only attenuated the expression of profibrotic and proinflammatory factors but also blocked the TGF-β1/Smad2 and TLR4/NF-κB p50 pathways. These results demonstrate that Andro prevents liver inflammation and fibrosis, which is in correlation with the inhibition of the TGF-β1/Smad2 and TLR4/NF-κB p50 pathways, highlighting Andro as a potential therapeutic strategy for liver fibrosis.

Epithelial-derived TGF-β2 modulates basal and wound-healing subepithelial matrix homeostasis

2006 ◽  
Vol 291 (6) ◽  
pp. L1277-L1285 ◽  
Author(s):  
H. Garrett R. Thompson ◽  
Justin D. Mih ◽  
Tatiana B. Krasieva ◽  
Bruce J. Tromberg ◽  
Steven C. George
Keyword(s):  
Extracellular Matrix ◽  
Wound Healing ◽  
Second Harmonic Generation ◽  
Harmonic Generation ◽  
Transforming Growth Factor ◽  
Second Harmonic ◽  
Smooth Muscle Actin ◽  
Critical Role ◽  
The Matrix

The epithelium influences the mesenchyme during dynamic processes such as embryogenesis, wound healing, fibrosis, and carcinogenesis. Since transforming growth factor-β (TGF-β) modulates these processes, we hypothesized that epithelial-derived TGF-β also plays a critical role in maintaining the extracellular matrix at basal conditions. We utilized an in vitro model of the epithelial-mesenchymal trophic unit in the human airways to determine the role of epithelial-derived TGF-β in modulating the extracellular matrix under basal and wound-healing conditions. When differentiated at an air-liquid interface, the human bronchial epithelium produces active TGF-β2 at a concentration of 50–70 pg/ml, whereas TGF-β1 is undetectable. TGF-β2 increases two- to threefold following scrape injury in a dose-dependent fashion and significantly enhances both α-smooth muscle actin expression in the underlying collagen-embedded fibroblasts and secretion of tenascin-C into the matrix. Multiphoton microscopy demonstrates substantially enhanced second harmonic generation from fibrillar collagen in the matrix. Pretreatment of the matrix with either sirolimus (2.5 nM) or paclitaxel (10 nM) abolishes the increases in both TGF-β2 and second harmonic generation in response to epithelial injury. In the absence of the epithelium, exogenous active TGF-β2 (0–400 pg/ml) produces a biphasic response in the second harmonic signal with a minimum occurring at the epithelial-derived basal level. We conclude that epithelial-derived TGF-β2 is secreted in response to injury, significantly alters the bulk optical properties of the extracellular matrix, and its tight regulation may be required for normal collagen homeostasis.

Fibroblast fate regulation by time dependent TGF-β1 and IL-10 stimulation in biomimetic 3D matrices

2017 ◽  
Vol 5 (9) ◽  
pp. 1858-1867 ◽  
Author(s):  
Jiranuwat Sapudom ◽  
Xiancheng Wu ◽  
Marina Chkolnikov ◽  
Michael Ansorge ◽  
Ulf Anderegg ◽  
...  
Keyword(s):  
Wound Healing ◽  
Time Dependent ◽  
Myofibroblast Differentiation ◽  
Late Stages ◽  
Tgf Β1

Myofibroblast differentiation and de-differentiation at early and late stages of wound healing is modelled in 3D biomimetic matrices based on collagen and fibronectin in vitro.

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