(2007) The effects of growth factors and mechanical tension on ocular fibroblasts in wound healing and scarring. Doctoral thesis , University of London.

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Abstract

Processes of wound healing or scarring cause many blinding eye conditions and can limit the success of surgical eye treatments. The mechanisms underlying matrix remodelling and contraction by connective tissue cells (fibroblasts) are not fully understood. Two main theories dominate the field: firstly, that fibroblasts in a wound begin to express alpha smooth muscle actin and transdifferentiate into myofibroblasts with enhanced contractile properties, and secondly that fibroblast migration might underly matrix condensation. The purpose of this work was to develop novel imaging techniques to investigate the mechanisms of tissue contraction and force generation using live cell and matrix imaging during fibroblast-mediated collagen gel contraction, and to determine the role played by growth factors and mechanical tension in that process. We have shown that fibroblasts from different parts of the eye display marked differences in macroscopic matrix contraction profiles, and that four factors determine the early matrix contraction profile: 1) cell size. 2) intrinsic cellular force. 3) growth factor-mediated actomyosin-based dynamic cell protrusive activity and 4) net pericellular matrix displacement. Intrinsic cellular force and dynamic activity appear to be independent unique characteristics of each cell type and might serve as predictors of matrix contraction. In addition, in corneal fibroblasts, we have shown that the recently identified PIXil rab5 RN I re-pathway mediates a specific type of cell protrusive activity leading to matrix contraction. Specific inhibitors of actomyosin- and rab5'RNTre-pathways are likely candidates to prevent corneal haze formation after surgery, trauma or inflammation, finally, we explored novel models of contraction involving real tissue by developing techniques to maintain and microscopically study ocular surface tissue ex vivo. We achieved successful concomitant live imaging of resident cells and extra-cellular matrix behaviour at high resolution in both conjunctival and corneal stroma by combining optimized strategies for tissue maintenance, fluorescent cell labeling, and confocal laser and/or two-photon microscopy. In summary, this thesis presents novel pathways of matrix contraction and imaging techniques to study these events in vitro and ex vivo.

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