Inhibition of mechanosensitive signaling in myofibroblasts ameliorates experimental pulmonary fibrosis
Yong Zhou, … , Karen Bernard, Victor J. Thannickal
Yong Zhou, … , Karen Bernard, Victor J. Thannickal
Published February 22, 2013
Citation Information: J Clin Invest. 2013;123(3):1096-1108. https://doi.org/10.1172/JCI66700.
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Research Article

Inhibition of mechanosensitive signaling in myofibroblasts ameliorates experimental pulmonary fibrosis

Abstract

Matrix stiffening and myofibroblast resistance to apoptosis are cardinal features of chronic fibrotic diseases involving diverse organ systems. The interactions between altered tissue biomechanics and cellular signaling that sustain progressive fibrosis are not well defined. In this study, we used ex vivo and in vivo approaches to define a mechanotransduction pathway involving Rho/Rho kinase (Rho/ROCK), actin cytoskeletal remodeling, and a mechanosensitive transcription factor, megakaryoblastic leukemia 1 (MKL1), that coordinately regulate myofibroblast differentiation and survival. Both in an experimental mouse model of lung fibrosis and in human subjects with idiopathic pulmonary fibrosis (IPF), we observed activation of the Rho/ROCK pathway, enhanced actin cytoskeletal polymerization, and MKL1 cytoplasmic-nuclear shuttling. Pharmacologic disruption of this mechanotransduction pathway with the ROCK inhibitor fasudil induced myofibroblast apoptosis through a mechanism involving downregulation of BCL-2 and activation of the intrinsic mitochondrial apoptotic pathway. Treatment with fasudil during the postinflammatory fibrotic phase of lung injury or genetic ablation of Mkl1 protected mice from experimental lung fibrosis. These studies indicate that targeting mechanosensitive signaling in myofibroblasts to trigger the intrinsic apoptosis pathway may be an effective approach for treatment of fibrotic disorders.

Authors

Yong Zhou, Xiangwei Huang, Louise Hecker, Deepali Kurundkar, Ashish Kurundkar, Hui Liu, Tong-Huan Jin, Leena Desai, Karen Bernard, Victor J. Thannickal

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Figure 1

Fasudil induces myofibroblast apoptosis both in vitro and in vivo, while normal lung fibroblasts are not susceptible to fasudil-induced cell death.

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Fasudil induces myofibroblast apoptosis both in vitro and in vivo, while...
(A) Myofibroblasts (myoFBs) isolated from lungs of patients with IPF (n = 8) and fibroblasts of non-IPF control subjects (failed donors; n = 6) were cultured in the presence or absence of 25 μM fasudil (Fasu) for 24–48 hours. Control fibroblasts (Ctrl FBs) were treated with 4 ng/ml TGF-β1 (Tβ1) and/or fasudil. Cells were double stained for TUNEL (green) and α-SMA (red); nuclei were stained with DAPI (blue). Confocal fluorescent images were overlaid to show apoptotic myofibroblasts. Scale bars: 20 μM. (B) Apoptotic cells (i.e., TUNEL positive) were quantified and expressed as the percentage of total cells. At least 300 cells were counted per condition. **P < 0.01, ***P < 0.001 vs. PBS; ##P < 0.01 vs. TGF-β1. (C–E) Frozen lung tissue sections from mice treated with saline (Sal) or with bleomycin (Bleo) in combination with PBS or fasudil were stained for TUNEL, α-SMA, and nuclei as in A. Higher-magnification views of the boxed regions in the bleomycin plus PBS and bleomycin plus fasudil images are shown in D and E, respectively (enlarged ×4- to ×5-fold). Scale bars: 50 μM.