Lung Parenchymal Tissue Stiffness in Fibrosis and Cellular Responses to Substrate Stiffness

Abstract

Stiffening of the lung parenchyma is one of the cardinal features of progressive pulmonary fibrosis, giving rise to a heterogeneous microenvironment with continuous gradients in stiffness. To define the local mechanical environment of the lung fibroblast, we directly measured the local elastic modulus and elasticity gradients of tissue strips from bleomycin treated mouse lung at the cellular scale using AFM force mapping technique. Elastographs revealed a heterogeneous spatial distribution of tissue stiffness with shear moduli ranging from 0.01 to 20 kPa in both normal and fibrotic lung. Fibrotic lung presented incremental stiffness and spatial heterogeneity. The median shear modulus shifted from 0.1 kPa in normal lung to 3 kPa in fibrotic lung. We then prepared a model system using 2D collagen-coated polyacrylamide substrates with stiffness gradients ranging from 0.1 to 50 kPa to study the role that local stiffness play in focusing fibroblast growth and activation. Human lung fibroblasts growing on stiffness gradients exhibited increased spatial density as substrate stiffness increased, suggesting that proliferation and/or migration responses on stiffness bias fibroblast accumulation to the stiffest region. Immunostaining indicated that stiffness increases focal adhesion size, alpha-smooth muscle actin expression, procollagen I expression, as well as the fibrogenic effect of TGF-beta1 on lung fibroblast. Together these results demonstrate that fibroblast accumulation and activation are biased to areas of increased substrate stiffness, and more importantly, that regional mechanical factors could underpin fibrotic progression in the lung through positive feedback loops of fibroblast recruitment and activation.