May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Cell Motility and Extracellular Matrix Remodelling During Cell Mediated Collagen Contraction - The Effect of Matrix Metalloproteinase Modulation
Author Affiliations & Notes
  • A. Ahir
    Div Cell Biology, Inst Ophthal/Univ College London, London, United Kingdom
  • S.E. Moss
    Div Cell Biology, Inst Ophthal/Univ College London, London, United Kingdom
  • P.T. Khaw
    Wound Healing Research Unit, Inst Ophthal/Univ College London, London, United Kingdom
  • M. Bailly
    Wound Healing Research Unit, Inst Ophthal/Univ College London, London, United Kingdom
  • Footnotes
    Commercial Relationships  A. Ahir, None; S.E. Moss, None; P.T. Khaw, None; M. Bailly, None.
  • Footnotes
    Support  MRC Component Grant 57188
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 2102. doi:
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      A. Ahir, S.E. Moss, P.T. Khaw, M. Bailly; Cell Motility and Extracellular Matrix Remodelling During Cell Mediated Collagen Contraction - The Effect of Matrix Metalloproteinase Modulation . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2102.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose:Wound healing processes are critical for the successful outcome of many types of surgery and cell mediated collagen contraction and remodelling are critical components of scarring. We aim to clarify mechanisms of cell mediated matrix remodelling and investigate the effect of matrix metalloproteinase (MMP) modulation. Methods:Primary human sceral fibroblasts were used in monolayers and 3D Collagen Type I gels. Cells were GFP transfected to follow motility, and 1um diameter fluorescent beads were embedded in gels to track gel movement. Gels were relaxed, or stressed for 24hrs before relaxing (pre-stressed), in the presence and absence of a broad spectrum MMP inhibitor (Ilomastat). Whole gels were photographed to record contraction over 7 days and collected at time intervals after casting for analysis by immunolabelling, confocal reflection microscopy and 4D imaging. Results:Rate of contraction was always greater in pre-stressed gels compared to relaxed ones. By 24hrs, pre-stressed gels had contracted over 50% compared to 30% for relaxed ones. By day 7 all gels had contracted by 70%. In concurrent experiments, F-actin stress fibres were found in 12% of cells by 6hrs of casting gels and maintaining stress, and increased to 45% by 72hrs. By contrast, cells in relaxed gels had a rounded morphology and no stress fibres (6% by 72hrs), although gel contraction did occur. Time lapse imaging illustrated transient protrusions extending from cells in relaxed gels only. Confocal reflection microscopy illustrated the transient protrusions in relaxed gels pulling collagen fibrils towards the cell mass. With prolonged incubation, 'holes' appeared in the matrix around cells in both relaxed and pre-stressed gels. With longer incubation times (96hrs), pre-stressed gels showed evidence of collagen fibril alignment . Incubation with Ilomastat prevented contraction of gels (less than 10% by 7 days), and completely blocked the appearance of 'holes' in the gels. Conclusions:We illustrate for the first time live imaging of collagen fibril organisation in response to fibroblast motility. Our results of stress fibre formation and pre-stressed gel contraction support previous reports that suggest cell migration mediates contraction. Ilomastat block of matrix degradation by MMPs may indicate that cells first digest the matrix and then migrate into the space. However, for relaxed gels our results suggest an alternative mechanism, with no stress fibre formation and transient cellular protrusions playing a role in contraction.

Keywords: wound healing • extracellular matrix • imaging/image analysis: non-clinical 
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