Matrices of Physiologic Stiffness Potently Inactivate Idiopathic Pulmonary Fibrosis Fibroblasts

Abstract

Fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) have been shown to differ from normal lung fibroblasts in functional behaviors that contribute to the pathogenesis of IPF, including the expression of contractile proteins and proliferation, but how such behaviors vary in matrices with stiffness matched to normal and fibrotic lung tissue remains unknown. Here, we tested whether pathologic changes in matrix stiffness control IPF and normal lung tissue-derived fibroblast functions, and compared the relative efficacy of mechanical cues to an antifibrotic lipid mediator, prostaglandin E 2 (PGE 2 ). Fibroblasts were grown on collagen I-coated glass or hydrogel substrates of discrete stiffnesses, spanning the range of normal and fibrotic lung tissue. Traction microscopy was used to quantify contractile function. The CyQuant Cell Proliferation Assay (Invitrogen, Carlsbad, CA) was used to assess changes in cell number, and PGE 2 concentrations were measured by ELISA. We confirmed differences in proliferation and PGE 2 synthesis between IPF and normal tissue-derived fibroblasts on rigid substrates. However, IPF fibroblasts remained highly responsive to changes in matrix stiffness, and both proliferative and contractile differences between IPF and normal fibroblasts were ablated on physiologically soft matrices. We also confirmed the relative resistance of IPF fibroblasts to PGE 2 , while demonstrating that decreases in matrix stiffness and the inhibition of Rho kinase both potently attenuate contractile function in IPF-derived fibroblasts. We conclude that pathologic changes in the mechanical environment control important IPF fibroblast functions. Understanding how mechanical cues control fibroblast function may offer new opportunities for targeting these cells, even when they are resistant to antifibrotic pharmacological agents or biological mediators.Keywords: pulmonary fibrosis; lung; extracellular matrix; fibroblast contractility Idiopathic pulmonary fibrosis (IPF) is a devastating, progressive fibrosing disease with no proven pharmacological therapy (1). The fibroblast is the end effector cell of fibrosis, and fibroblasts increase in number and activation status during IPF. Scattered aggregates of proliferating fibroblasts are consistently observed in fibrotic lungs (2, 3), and the progression of IPF is accompanied by an excessive activation of lung fibroblasts to a synthetic and contractile myofibroblast phenotype responsible for the deposition, contraction, and remodeling of the lung's extracellular matrix (ECM) (4, 5).To understand the pathogenic mechanisms at work in IPF, much effort has been devoted to identifying functional differences in IPF-derived fibroblasts compared with fibroblasts isolated from normal lung tissue. These efforts have demonstrated, among a host of changes, that IPF fibroblasts are more contractile (6), express higher concentrations of a-smooth muscle actin (a-SMA) (7, 8), Type I collagen (8-10), and tissue inhibitors of metalloproteinase (11) than do normal lung tissue-d...

Lung Extracellular Matrix and Fibroblast Function

“…Indeed, recent data suggest that ECM protein translation in IPF is positively influenced by the IPF ECM, even in fibroblasts derived from normal donors (32). In line with these findings, a study now demonstrates that ECM within a physiologic range of stiffness is capable of reversing the activated myofibroblast phenotype (33), lending further credence to the idea that targeting ECM stiffness may be an appropriate therapeutic approach in fibrotic disorders. However, no studies to date have uncoupled pathologic ECM stiffness from pathologic ECM composition, raising the prospect that ECM composition, or the combination of pathologic ECM composition and stiffness, drives a feedforward loop in fibrotic lung disease.…”

Matrix, Mesenchyme, and Mechanotransduction

“…In support of the pathological relevance of this pathway, mice genetically deficient in MRTF-A are protected from bleomycin-induced lung fibrosis, and targeting of Rho kinase upstream of actin polymerization and MRTF-A effectively attenuates bleomycin-induced fibrosis in both preventive and therapeutic dosing regimens (32). Importantly, fibroblasts isolated from patients with IPF remain responsive to inactivation of this signaling pathway (32) and overall remain largely responsive to the matrix mechanical environment (33), suggesting that targeting the matrix mechanical environment or its downstream signaling pathways may be an effective strategy for interrupting profibrotic cellular activation.…”

Prostaglandin E2 Inhibits α-Smooth Muscle Actin Transcription during Myofibroblast Differentiation via Distinct Mechanisms of Modulation of Serum Response Factor and Myocardin-related Transcription Factor-A

“…Key features in the pathogenesis of fibrotic disorders such as IPF include expansion of the population of fibroblasts, their differentiation into myofibroblasts that express contractile proteins such as ␣-smooth muscle actin (␣-SMA), and the excessive production of extracellular matrix proteins such as collagen that compose the tissue scar (2,3). Myofibroblasts play a critical role in fibrotic tissue remodeling (4) because of the following: 1) their relative resistance to apoptosis (5, 6); 2) their robust capacity for extracellular matrix protein generation (7), and 3) their contribution to tissue contraction and hence stiffness (8,9). Transforming growth factor-␤1 (TGF-␤1) is widely implicated in the pathogenesis of fibrotic diseases (10) and is the best studied inducer of fibroblast differentiation into myofibroblasts.…”