| 講演要旨： |
Fibroblast-to-myofibroblast modulation represents a crucial step in the contraction of wound granulation tissue and in the production of connective tissue deformations typical of fibrocontractive diseases. Myofibroblast differentiation depends on the presence of mechanical stress, of specific growth factors and matrix components and is hallmarked by the neo-expression of α smooth muscle actin (α SMA) in cytoplasmic stress fibers, which confers a high contractile activity to fibroblastic cells. Cell-matrix adhesions are the central checkpoints in transmitting the high contractile activity of myofibroblasts to the ECM and in controlling myofibroblast differentiation by sensing the level of matrix stress. In contrast to normal dermal fibroblasts, tissue myofibroblasts develop complex adhesion structures with the ECM that are called 'fibronexus' or 'supermature' focal adhesion (FA). Investigating the formation, molecular structure and function of these key structures is important to understand the mechanisms of granulation tissue evolution and to develop anti-fibrosis therapies.
Supermature FAs of cultured myofibroblasts are considerably longer (6 30 μm) compared with 'classical' FAs (2 6 μm) of α-SMA-negative fibroblasts. To investigate the role of α SMA in FA maturation, myofibroblasts were treated with the α SMA fusion peptide (SMA-FP), an agent that specifically inhibits α SMA-mediated contractile activity. The use of flexible micro-patterned substrates and EGFP-tagged focal adhesion proteins demonstrated that SMA-FP first decreased myofibroblast contraction, shortly followed by the disassembly of supermature FAs, which finally reduced myofibroblast adhesion to the level of α SMA negative fibroblasts.
In turn, supermature FAs control myofibroblast differentiation by communicating the level of matrix stress to the cytoskeleton. Culture on compliant silicone substrates reduced the size of supermature FAs to that of classical FAs and lead to a concomitant decrease of α SMA expression. Incorporation of α SMA into stress fibers required the formation of FAs >6 μm (Figure) as demonstrated by plating myofibroblasts on arrays of adhesive islets with lengths ranging from 2-20 μm, created by means of microcontact printing (μCP). Stretching 6 μm islets on flexible silicone membranes to 8 μm length induced incorporation of α SMA into stress fibers; this was not achieved by applying the same stretch (35%) to cells initially grown on 4 μm islets. Finally, by analyzing local deformations created in deformable micropatterned substrates by paxillin-EGFP transfected myofibroblasts, we determined a close relationship between the size of supermature FAs and local force exertion; hence we were able to determine the minimal tension at individual supermature FAs required for α SMA recruitment.
|
