Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. David O'Gorman

Abstract

Abnormal scarring is a type of benign fibrosis of the skin that can restrict mobility, dexterity and quality of life. There are few, if any, truly effective treatment options for these conditions. Hypertrophic scarring is a common form of abnormal scarring characterized by increased fibroblast proliferation and differentiation of apoptosis-resistant and hyper-contractile myofibroblasts that promote excessive deposition and contracture of the extracellular matrix (ECM). Periostin, a secreted ECM protein, is transiently expressed during normal cutaneous wound repair, but is abnormally abundant and persistent in abnormal scars and other benign fibroses that display enhanced fibroblast proliferation and myofibroblast differentiation. The objectives of this study were to elucidate the effects of periostin on fibroblast proliferation, myofibroblast differentiation and myofibroblast persistence in an in vitro human fibroblast model of hypertrophic scarring. Primary fibroblasts derived from patients with hypertrophic scars (HTS) or normal skin (NS) were cultured in two- and three-dimensional collagen cultures to more closely mimic their in vivo microenvironment. A series of in vitro techniques, including adenoviral transduction, WST-1 assays, stressed fibroblast populated collagen lattices (sFPCLs), western immunoblotting and immunofluorescence confocal microscopy were employed to assess periostin effects on fibroblast proliferation and myofibroblast differentiation under these culture conditions. Periostin treatment was shown to enhance HTS fibroblast proliferation in compliant 2D collagen cultures through Akt and Rho kinase dependent pathways. When subjected to isometric tension in sFPCLs, periostin enhanced myofibroblast differentiation, as evidenced by increases in collagen contraction, alpha smooth muscle actin (αSMA) and the formation of supermature focal adhesions. In contrast, no discernible effects of periostin treatment were evident in NS fibroblasts. Periostin signalling maintained αSMA levels in HTS fibroblasts in an environment of decreasing ECM tension, and this was correlated with changes in focal adhesion kinase, cofilin and myosin light chain activities. These studies are the first to identify periostin as a mediator of excessive proliferation, myofibroblast differentiation and persistence in human fibroblasts derived from hypertrophic scars. The findings reported here suggest that novel therapeutic interventions designed to deplete periostin levels in the dermis of hypertrophic scars may have utility for attenuating fibroblast proliferation and depleting myofibroblast populations, and thereby enhance scar resolution.


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Biochemistry Commons

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