scholarly journals Abolishing Myofibroblast Arrhythmogeneicity by Pharmacological Ablation of α-Smooth Muscle Actin Containing Stress Fibers

2011 ◽  
Vol 109 (10) ◽  
pp. 1120-1131 ◽  
Author(s):  
Christian Rosker ◽  
Nicolò Salvarani ◽  
Stephan Schmutz ◽  
Teddy Grand ◽  
Stephan Rohr
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Actin ◽  
Stress Fibers ◽  
Muscle Actin

scholarly journals α-Smooth Muscle Actin Is Crucial for Focal Adhesion Maturation in Myofibroblasts

2003 ◽  
Vol 14 (6) ◽  
pp. 2508-2519 ◽  
Author(s):  
Boris Hinz ◽  
Vera Dugina ◽  
Christoph Ballestrem ◽  
Bernhard Wehrle-Haller ◽  
Christine Chaponnier
Keyword(s):  
Smooth Muscle ◽  
Contractile Activity ◽  
Smooth Muscle Actin ◽  
Focal Adhesions ◽  
Serum Levels ◽  
Stress Fibers ◽  
Muscle Actin ◽  
Cytoplasmic Actin ◽  
Β3 Integrin ◽  
Two Phases

Cultured myofibroblasts are characterized by stress fibers, containing α-smooth muscle actin (α-SMA) and by supermature focal adhesions (FAs), which are larger than FAs of α-SMA–negative fibroblasts. We have investigated the role of α-SMA for myofibroblast adhesion and FA maturation. Inverted centrifugation reveals two phases of initial myofibroblast attachment: during the first 2 h of plating microfilament bundles contain essentially cytoplasmic actin and myofibroblast adhesion is similar to that of α-SMA–negative fibroblasts. Then, myofibroblasts incorporate α-SMA in stress fibers, develop mature FAs and their adhesion capacity is significantly increased. When α-SMA expression is induced in 5 d culture by TGFβ or low serum levels, fibroblast adhesion is further increased correlating with a “supermaturation” of FAs. Treatment of myofibroblasts with α-SMA fusion peptide (SMA-FP), which inhibits α-SMA–mediated contractile activity, reduces their adhesion to the level of α-SMA negative fibroblasts. With the use of flexible micropatterned substrates and EGFP-constructs we show that SMA-FP application leads to a decrease of myofibroblast contraction, shortly followed by disassembly of paxillin- and β3 integrin-containing FAs; α5 integrin distribution is not affected. FRAP of β3 integrin-EGFP demonstrates an increase of FA protein turnover following SMA-FP treatment. We conclude that the formation and stability of supermature FAs depends on a high α-SMA–mediated contractile activity of myofibroblast stress fibers.

Stable incorporation of α-smooth muscle actin into stress fibers is dependent on specific tropomyosin isoforms

Cytoskeleton ◽  
2015 ◽  
Vol 72 (6) ◽  
pp. 257-267 ◽  
Author(s):  
Marco Prunotto ◽  
Maurizio Bruschi ◽  
Peter Gunning ◽  
Giulio Gabbiani ◽  
Franziska Weibel ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Actin ◽  
Stress Fibers ◽  
Muscle Actin ◽  
Tropomyosin Isoforms ◽  
Stable Incorporation

scholarly journals Focal adhesion size controls tension-dependent recruitment of α-smooth muscle actin to stress fibers

2006 ◽  
Vol 172 (2) ◽  
pp. 259-268 ◽  
Author(s):  
Jérôme M. Goffin ◽  
Philippe Pittet ◽  
Gabor Csucs ◽  
Jost W. Lussi ◽  
Jean-Jacques Meister ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Focal Adhesion ◽  
Stress Resistance ◽  
Smooth Muscle Actin ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Myofibroblast Differentiation ◽  
Muscle Actin ◽  
High Tension ◽  
Tension Generation

Expression of α-smooth muscle actin (α-SMA) renders fibroblasts highly contractile and hallmarks myofibroblast differentiation. We identify α-SMA as a mechanosensitive protein that is recruited to stress fibers under high tension. Generation of this threshold tension requires the anchoring of stress fibers at sites of 8–30-μm-long “supermature” focal adhesions (suFAs), which exert a stress approximately fourfold higher (∼12 nN/μm2) on micropatterned deformable substrates than 2–6-μm-long classical FAs. Inhibition of suFA formation by growing myofibroblasts on substrates with a compliance of ≤11 kPa and on rigid micropatterns of 6-μm-long classical FA islets confines α-SMA to the cytosol. Reincorporation of α-SMA into stress fibers is established by stretching 6-μm-long classical FAs to 8.1-μm-long suFA islets on extendable membranes; the same stretch producing 5.4-μm-long classical FAs from initially 4-μm-long islets is without effect. We propose that the different molecular composition and higher phosphorylation of FAs on supermature islets, compared with FAs on classical islets, accounts for higher stress resistance.

scholarly journals Inhibition of TGF-β2-induced migration and epithelial-mesenchymal transition in ARPE-19 by sulforaphane

2021 ◽  
Vol 14 (7) ◽  
pp. 973-980
Author(s):  
Yan-Bing Huang ◽  
◽  
Hui Zheng ◽  
Xiu-Xia Yang ◽  
Cheng-Cheng Yang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Transforming Growth Factor ◽  
Epithelial Mesenchymal Transition ◽  
Smooth Muscle Actin ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Actin Stress Fiber ◽  
Muscle Actin ◽  
Mesenchymal Transition ◽  
Actin Stress Fibers

AIM: To investigate the effects of sulforaphane (SFN) on transforming growth factor (TGF)-β2 stimulated migration and epithelial-mesenchymal transition (EMT) in ARPE-19 cells. METHODS: ARPE-19 cells were cultured in the presence or absence of SFN or TGF-β2. SFN toxicity was assessed by performing a lactate dehydrogenase assay (LDH) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays, and cell migration was evaluated by Transwell migration assay. Actin stress fiber formation in ARPE-19 cells was determined using immunofluorescence analysis. Immunoblotting analysis was used to determine fibronectin and α-smooth muscle actin expressions along with the degree of Smad and Akt phosphorylation. RESULTS: SFN inhibited ARPE-19 migration. Additionally, SFN attenuated TGF-β2-induced appearance of actin stress fibers as well as fibronectin and α-smooth muscle actin expressions in these cells. SFN also hindered the TGF-β2-stimulated phosphorylation of Smad2, Smad3, and Akt. SFN showed no cytotoxicity towards ARPE-19 cells. CONCLUSION: SFN inhibits TGF-β2-stimulated migration and EMT in ARPE-19 cells, probably by preventing the establishment of actin stress fibers and Akt and Smad2/3 signaling.

scholarly journals Titanium Substratum Roughness as a Determinant of Human Gingival Fibroblast Fibronectin and α-Smooth Muscle Actin Expression

Materials ◽  
2021 ◽  
Vol 14 (21) ◽  
pp. 6447
Author(s):  
Hong Li ◽  
Chengyu Guo ◽  
Yuchen Zhou ◽  
Hao Sun ◽  
Robin Hong ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Dental Implants ◽  
Smooth Muscle Actin ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Human Gingival Fibroblast ◽  
Gingival Fibroblast ◽  
Tissue Formation ◽  
Muscle Actin ◽  
Protein Levels

The most appropriate surface treatment to enhance gingival connective tissue formation on the abutment of dental implants remains undefined, with healing associated with a scar-like response. We have previously shown that topographies with an arithmetic average of the absolute profile height deviations (Ra) = 4.0 induces an anti-fibrotic phenotype in human gingival fibroblasts (HGFs) by causing nascent adhesion formation. With bacterial colonization considerations, we hypothesized that a lower Ra could be identified that would alter adhesion stability and promote a matrix remodeling phenotype. Focal adhesions (FAs) area decreased with increasing roughness, although no differences in cell attachment or proliferation were observed. Alpha smooth muscle actin (α-SMA) protein levels were significantly reduced on Ra = 3.0 and 4.0 vs. 0.1 (p < 0.05), with incorporation of α-SMA into stress fibers most prominent on Ra = 0.1. Fibronectin protein levels were reduced on 3.0 and 4.0 vs. 0.1 (p < 0.05), and Ra = 1.5 and deeper significantly altered fibronectin deposition. Addition of exogenous TGF-β3 increased HGF adhesion size on 0.1 surfaces, but not on any other topography. We conclude that Ra = 1.5 is sufficient to reduce adhesion size and inhibit α-SMA incorporation into stress fibers in HGFs, but 3.0 is required in the presence of exogenous TGF-β3. Our findings have implications for inhibiting fibrotic tissue formation surrounding percutaneous devices such as dental implants.

scholarly journals Focal adhesion features during myofibroblastic differentiation are controlled by intracellular and extracellular factors

2001 ◽  
Vol 114 (18) ◽  
pp. 3285-3296 ◽  
Author(s):  
Vera Dugina ◽  
Lionel Fontao ◽  
Christine Chaponnier ◽  
Jury Vasiliev ◽  
Giulio Gabbiani
Keyword(s):  
Smooth Muscle ◽  
Focal Adhesion ◽  
Smooth Muscle Actin ◽  
Three Dimensional ◽  
Focal Adhesions ◽  
Laser Scanning Microscopy ◽  
Stress Fibers ◽  
Muscle Actin ◽  
Cultured Fibroblasts ◽  
Actin Expression

Transforming growth factor β (TGFβ), the most established promoter of myofibroblast differentiation, induces ED-A cellular fibronectin and α-smooth muscle actin expression in fibroblastic cells in vivo and in vitro. ED-A fibronectin exerts a permissive action for α-smooth muscle actin expression. A morphological continuity (called fibronexus), a specialized form of focal adhesion, has been described between actin stress fibers that contain α-smooth muscle actin, and extracellular fibronectin, which contains the ED-A portion, in both cultured fibroblasts and granulation tissue myofibroblasts. We have studied the development of these focal adhesions in TGFβ-treated fibroblasts using confocal laser scanning microscopy, three-dimensional image reconstruction and western blots using antibodies against focal adhesion proteins. The increase in ED-A fibronectin expression induced by TGFβ was accompanied by bundling of ED-A fibronectin fibers and their association with the terminal portion of α-smooth muscle actin-positive stress fibers. In parallel, the focal adhesion size was importantly increased, and tensin and FAK were neoexpressed in focal adhesions; moreover, vinculin and paxillin were recruited from the cytoplasmic pool into focal adhesions. We have evaluated morphometrically the length and area of focal adhesions. In addition, we have evaluated biochemically their content of associated proteins and of α-smooth muscle actin after TGFβ stimulation and on this basis suggest a new focal adhesion classification, that is, immature, mature and supermature.When TGFβ-induced α-smooth muscle actin expression was blocked by soluble recombinant ED-A fibronectin, we observed that the fragment was localised into the fibronectin network at the level of focal adhesions and that focal adhesion supermaturation was inhibited. The same effect was also exerted by the ED-A fibronectin antibody IST-9. In addition, the antagonists of actin-myosin contractility BDM and ML-7 provoked the dispersion of focal adhesions and the decrease of α-smooth muscle actin content in stress fibers of pulmonary fibroblasts, which constitutively show large focal adhesions and numerous stress fibers that contain α-smooth muscle actin. These inhibitors also decreased the incorporation of recombinant ED-A into fibronectin network. Our data indicate that a three-dimensional transcellular structure containing both ED-A fibronectin and α-smooth muscle actin plays an important role in the establishment and modulation of the myofibroblastic phenotype. The organisation of this structure is regulated by intracellularly and extracellularly originated forces.

scholarly journals A deep learning approach to identify and segment alpha-smooth muscle actin stress fiber positive cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander Hillsley ◽  
Javier E. Santos ◽  
Adrianne M. Rosales
Keyword(s):  
Smooth Muscle ◽  
Deep Learning ◽  
Cardiac Fibroblasts ◽  
Smooth Muscle Actin ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Actin Stress Fiber ◽  
Muscle Actin ◽  
Alpha Smooth Muscle Actin

AbstractCardiac fibrosis is a pathological process characterized by excessive tissue deposition, matrix remodeling, and tissue stiffening, which eventually leads to organ failure. On a cellular level, the development of fibrosis is associated with the activation of cardiac fibroblasts into myofibroblasts, a highly contractile and secretory phenotype. Myofibroblasts are commonly identified in vitro by the de novo assembly of alpha-smooth muscle actin stress fibers; however, there are few methods to automate stress fiber identification, which can lead to subjectivity and tedium in the process. To address this limitation, we present a computer vision model to classify and segment cells containing alpha-smooth muscle actin stress fibers into 2 classes (α-SMA SF+ and α-SMA SF-), with a high degree of accuracy (cell accuracy: 77%, F1 score 0.79). The model combines standard image processing methods with deep learning techniques to achieve semantic segmentation of the different cell phenotypes. We apply this model to cardiac fibroblasts cultured on hyaluronic acid-based hydrogels of various moduli to induce alpha-smooth muscle actin stress fiber formation. The model successfully predicts the same trends in stress fiber identification as obtained with a manual analysis. Taken together, this work demonstrates a process to automate stress fiber identification in in vitro fibrotic models, thereby increasing reproducibility in fibroblast phenotypic characterization.

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