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Abstract

After organ injury, a complex sequence of events is triggered to heal the tissue and to form a scar. Myofibroblasts, specialized contractile and secretory fibroblasts, are one of the many cell types that participate in the remodeling process. Myofibroblasts are responsible for tissue regeneration and tissue contraction, facilitating wound closure. However, uncontrolled occurrence of the myofibroblastic phenotype leads to pathological excessive extracellular matrix (ECM) secretion and aberrant tissue contraction, characteristic of severe diseases such as fibrosis or hypertrophic scars. Transforming growth factor β1 (TGFβ1) is the major cytokine that induces fibroblast-to-myofibroblast differentiation. In the myofibroblast, TGFβ1 is secreted as a latent complex (LLC) deposited in the surrounding ECM and must be liberated from ECM-stores to be active. Several mechanisms activate TGFβ1 most of which involve LLC proteolysis. Recently, myofibroblasts have been demonstrated to closely interact with the LLC by expressing specific integrins. In the central part of my thesis, I raise and test the hypothesis that myofibroblasts are able to liberate TGFβ1 from ECM-stores by directly exerting traction forces on LLC, independently from proteolysis. Using myofibroblast contraction agonists and antagonists, I demonstrate that levels of active TGFβ1 are directly correlated with the contractile state of the cell. Moreover, contraction of TX-100-extracted myofibroblast cytoskeleton with ATP directly increases the amounts of active TGFβ1, eliminating the possible contribution of proteases to the activation process. Using a culture system that I have specifically designed, I demonstrate that stretching myofibroblast cultures instantaneously liberates TGFβ1 from ECM-stores. Blocking the function of αvβ5, αvβ3 and a yet undetermined β1 integrin abolishes traction-mediated TGFβ activation suggesting that those integrins participate in the transduction of cellular forces to the LLC. Finally, using compliant culture substrates with tunable stiffness, I demonstrate that the traction-mediated activation of TGFβ1 is limited to a mechano-challenging, that is to say resistant, environment. I report a novel mechanism for TGFβ1 activation and highlight a new feature for integrins in the biology of this growth factor. At the levels of the cytoskeleton, the integrins and the ECM, therapeutic approaches can be envisaged to specifically inhibit TGFβ1 activation in the myofibroblast, suppressing the development of myofibroblast-related diseases.

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