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  • 1
    Electronic Resource
    Electronic Resource
    Masters and Servants of the Force: Focal Adhesion Size Controls Recruitment of α-Smooth Muscle Actin to Stress Fibers (2005)
    Oxford, UK; Malden, USA : Blackwell Science Inc
    Wound repair and regeneration 13 (2005), S. 0 
    Details
    ISSN: 1524-475X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Mechanical stress is a crucial factor for modulating fibroblasts into differentiated myofibroblasts, which are characterized by expression of α-smooth muscle actin (α-SMA). Incorporation of α-SMA into stress fibers generates high contractile activity and drives the formation of “supermature” focal adhesions (FA) that are considerably longer (6–30 μm) compared with “classical” FAs (2–6 μm) of α-SMA-negative fibroblasts. We here show that in turn, supermature FAs control myofibroblast differentiation by communicating the level of extracellular matrix stress to the cytoskeleton. Culture on compliant silicone substrates reduces the size of supermature FAs to that of classical FAs and leads to a concomitant decrease of α-SMA expression. Incorporation of α-SMA into stress fibers requires the formation of FAs longer than 6 μm as demonstrated by plating myofibroblasts on arrays of adhesive islets with dimensions ranging from 1.5 × 2–20 μm and spacing between 2–6 μm, which are created on rigid culture surfaces by means of microcontact printing (μCP). Stretching 6 μm islets on flexible silicone membranes to 8 μm length induces stress fiber formation of α-SMA-EGFP transfected fibroblasts; this was not achieved by applying the same stretch (30%) to cells initially grown on 4 μm islets. By analyzing local deformations created in deformable micropatterned substrates by paxillin-EGFP transfected myofibroblasts, we determined a linear relationship between the size of supermature FAs and local force exertion; hence we determined the minimal tension at individual supermature FAs required for α-SMA recruitment.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1067-1927.2005.130117m.x
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  • 2
    Electronic Resource
    Electronic Resource
    About the Level of Stress Between Myofibroblasts (2005)
    Oxford, UK; Malden, USA : Blackwell Science Inc
    Wound repair and regeneration 13 (2005), S. 0 
    Details
    ISSN: 1524-475X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Fibroblast to myofibroblast modulation is characterized by expression of α-smooth muscle actin (α-SMA) and represents a crucial step in granulation tissue contraction during wound healing. We have recently demonstrated that myofibroblasts develop cadherin-type cell-cell adherens junctions (AJs), which join stress fibers over the cell membrane. Cadherin expression changes from N-cadherin to OB-cadherin upon TGF-β induced myofibroblast differentiation in culture and coincides with the enlargement of AJs. We here show that myofibroblast AJs exhibit a higher mechanical resistance compared with that of α-SMA-negative fibroblasts by measuring the binding strength between two cells. (1) By separating two suspended cells placed in contact with laser tweezers, we revealed two types of Ca2+-dependent adhesion bonds with different strength. The first bond exhibited a breaking force of 5.5 ± 1.5 nN and existed in both, fibroblasts and myofibroblasts. The strength of the second bond differed between the two cells, breaking at 18.2 ± 0.8 nN in fibroblasts and at 23.0 ± 1.5 nN in myofibroblasts. Measuring the adhesion force between two plated cells by atomic force microscopy after short contact grossly confirmed these observations. Since no cyoskeletal reinforcement was involved in these experimental conditions, we suggest that these differences represent the different cadherins involved in (myo)fibroblast adhesion. (2) Subjecting suspended cells that were attached to plated cells to hydrodynamic forces in a flow chamber demonstrated ∼18% higher adhesion of myofibroblasts over fibroblasts at shear forces of 4 Nm−2. This higher adhesion was reduced to the level of fibroblast attachment by a peptide that inhibits α-SMA-mediated contraction, indicating AJ reinforcement by α-SMA-positive stress fibers. To conclude, high cell-cell adhesion of myofibroblasts appears to be determined by their specific cadherin pattern and by α-SMA-mediated mechanical reinforcement of AJs.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1067-1927.2005.130117u.x
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    Paper (German National Licenses)
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  • 3
    Electronic Resource
    Electronic Resource
    Mechanical Stress Induces TGFβ1 Activation in Myofibroblast Culture (2005)
    Oxford, UK; Malden, USA : Blackwell Science Inc
    Wound repair and regeneration 13 (2005), S. 0 
    Details
    ISSN: 1524-475X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Differentiation of fibroblasts into contractile, α-smooth muscle actin (α-SMA) expressing myofibroblasts depends on the action of the cytokine TGFβ in conjunction with mechanical tension. The goal of this study was to assess whether mechanical stress may play a role in activating TGFβ. Previous studies have shown that myofibroblasts secrete TGFβ1 as a large latent complex, consisting of the latency associated protein (LAP), the latent TGFβ1 binding protein (LTBP-1) and TGFβ1. LTBP-1 targets the small latent complex of LAP and TGFβ1 to the extracellular matrix (ECM), providing a stock of latent TGFβ1. To reveal the mechanisms triggering the release of active TGFβ1 from LAP and LTBP-1, myofibroblasts were cultured on flexible silicone membranes for 3d before being subjected to unique 5% uniaxial stretch. After stress application we observed the course of TGFβ1 activation over 24 h by quantifying luciferase synthesis under the control of the TGFβ-inducible PAI-1 promoter. TGFβ activation showed two distinct peaks 1 h and 6 h after stretch. TGFβ1 mRNA levels were increased after 6 h as assessed by semi-quantitative RT-PCR. To further determine if rapidly activated TGFβ1 was released from ECM stores, we stretched myofibroblast-derived ECM after cells have been removed by desoxycholate or EDTA treatment. Finally, we assessed the importance of the actin cytoskeleton in TGFβ1 activation by stretching Triton-X-100- and cytochalasin-D-treated myofibroblasts. Our results suggest that mechanical activation of TGFβ in myofibroblast culture requires cell activity and a functional cytoskeleton.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1067-1927.2005.130117ac.x
    Library Location Call Number Volume/Issue/Year Availability
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    Paper (German National Licenses)
    Fulltext
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