Substrate stiffness regulates extracellular matrix deposition by alveolar epithelial cells

Aim: The aim of the study was to address whether a stiff substrate, a model for pulmonary fibrosis, is responsible for inducing changes in the phenotype of alveolar epithelial cells (AEC) in the lung, including their deposition and organization of extracellular matrix (ECM) proteins. Methods: Freshly isolated lung AEC from male Sprague Dawley rats were seeded onto polyacrylamide gel substrates of varying stiffness and analyzed for expression and organization of adhesion, cytoskeletal, differentiation, and ECM components by Western immunoblotting and confocal immunofluorescence microscopy. Results: We observed that substrate stiffness influences cell morphology and the organization of focal adhesions and the actin cytoskeleton. Surprisingly, however, we found that substrate stiffness has no influence on the differentiation of type II into type I AEC, nor does increased substrate stiffness lead to an epithelial-mesenchymal transition. In contrast, our data indicate that substrate stiffness regulates the expression of the alpha 3 laminin subunit by AEC and the organization of both fibronectin and laminin in their ECM. Conclusions: An increase in substrate stiffness leads to enhanced laminin and fibronectin assembly into fibrils, which likely contributes to the disease phenotype in the fibrotic lung.