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A5795 - Structure and Composition of Lung Extracellular Matrix Dictate Fibroblast Fate
Author Block: C. B. Upagupta, S. Sato, P. Bellaye, C. Shimbori, T. Yanagihara, M. R. Kolb; Medicine, McMaster University, Firestone Institute for Respiratory Health, Hamilton, ON, Canada.
Rational: Idiopathic pulmonary fibrosis (IPF) is a chronic disease, characterized by progressive scarring of lung tissue. IPF prognosis is poor, with most patients succumbing to the illness at a rate comparable to aggressive cancers. The causes of IPF remain elusive, and there is no curative therapy. The key component contributing to the development and progression of IPF is the accumulation of lung myofibroblasts leading to excess deposition of extracellular matrix (ECM) proteins. The effect of the ECM properties (composition and stiffness) on the phenotype and regulation of pulmonary fibroblasts is yet to be fully elucidated. However, there is evidence suggesting increased deposition of abnormal ECM may promote differentiation of fibroblasts into myofibroblasts; therefore, progressing the disease in an exponential manner. Hypothesis: The mechanical transduction pathway in response to increased ECM stiffness, along with the ECM composition might play a major role in fibroblast fate and myofibroblast differentiation.Methods: We first investigated the effect of stiffness by culturing primary rat fibroblasts on different stiffness plates (1, 50, 107 kPa), and the role of ECM composition by culturing in media supplemented with ECM homogenate. Changes within the cells were measured through mRNA expression of pro-fibrotic markers (Acta2, Col1a1). Next we utilized a decellularization protocol to remove all the cells within rat lungs, leaving behind just the ECM to holistically investigate the 3-dimensional (3D) role of the ECM. Pulmonary fibrosis was induced in rats using an adenovirus vector transcribing active TGF-β1 (AdTGF-β) and control (AdDL). The effect of ECM on fibroblast fate was investigated by recellularizing stiff and normal decellularized lungs with primary rat fibroblasts. The role of the mechanical transduction pathway within these ECMs was also investigated using a ROCK inhibitor. Results: Both increased stiffness and the fibrotic ECM homogenate had a clear impact on cell phenotype and differentiation, as seen through increases in pro-fibrotic markers. Furthermore, the stiff 3D ECM also resulted in an increase in α-SMA expression of fibroblasts, that correlates with the level of fibrosis within the lungs. However, administration of the ROCK inhibitor within our ex-vivo model notably attenuates this fibrotic ECM driven myofibroblast differentiation. Conclusion: These findings suggest that both ECM composition and stiffness play a major role in myofibroblast differentiation. Therefore, targeting one or both these properties may serve as a potential avenue for the therapy of IPF and other fibrosis diseases.