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Vimentin intermediate filament assembly regulates fibroblast invasion in fibrogenic lung injury
Ranu Surolia, … , Victor J. Thannickal, Veena B. Antony
Ranu Surolia, … , Victor J. Thannickal, Veena B. Antony
Published April 4, 2019
Citation Information: JCI Insight. 2019;4(7):e123253. https://doi.org/10.1172/jci.insight.123253.
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Research Article Pulmonology

Vimentin intermediate filament assembly regulates fibroblast invasion in fibrogenic lung injury

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Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive disease, with a median survival of 3–5 years following diagnosis. Lung remodeling by invasive fibroblasts is a hallmark of IPF. In this study, we demonstrate that inhibition of vimentin intermediate filaments (VimIFs) decreases the invasiveness of IPF fibroblasts and confers protection against fibrosis in a murine model of experimental lung injury. Increased expression and organization of VimIFs contribute to the invasive property of IPF fibroblasts in connection with deficient cellular autophagy. Blocking VimIF assembly by pharmacologic and genetic means also increases autophagic clearance of collagen type I. Furthermore, inhibition of expression of collagen type I by siRNA decreased invasiveness of fibroblasts. In a bleomycin injury model, enhancing autophagy in fibroblasts by an inhibitor of VimIF assembly, withaferin A (WFA), protected from fibrotic lung injury. Additionally, in 3D lung organoids, or pulmospheres, from patients with IPF, WFA reduced the invasiveness of lung fibroblasts in the majority of subjects tested. These studies provide insights into the functional role of vimentin, which regulates autophagy and restricts the invasiveness of lung fibroblasts.

Authors

Ranu Surolia, Fu Jun Li, Zheng Wang, Huashi Li, Kevin Dsouza, Vinoy Thomas, Sergey Mirov, Dolores Pérez-Sala, Mohammad Athar, Victor J. Thannickal, Veena B. Antony

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Figure 7

Induction of autophagy by inhibition of VimIF assembly decreases invasiveness.

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Induction of autophagy by inhibition of VimIF assembly decreases invasiv...
Stable transfection with mEmerald-vimentin construct (WT) and GFP-vimentin-C328S construct (C328S) in Vim–/– mouse fibroblasts. (A) Fluorescence images showing arrangement of vimentin in WT and C328S fibroblasts. Scale bars: 50 μm. (B) Immunoblot analysis for LC3B and vimentin in transfected fibroblasts after 6 hours of serum starvation. β-Actin served as loading control. Densitometry data are representative of 3 experiments. (C) Immunofluorescence staining of pulmospheres using WT and C328 fibroblasts for LC3B (red) and green fluorescence–tagged vimentin expression after 6 hours of serum starvation. Scale bars: 100 μm. (D) Fluorescence images of IPF pulmospheres containing mEmerald-vimentin–transfected IPF fibroblasts treated with vehicle (control) or WFA. Scale bars: 250 μm. (E) Representative image of pulmospheres treated with vehicle or WFA. Graph shows calculated zone of invasion percentage from 6 IPF lung pulmospheres from controls not treated and treated with WFA. Each dot represents the mean value of calculated zone of invasion from 5 pulmospheres for each IPF subject. Data are expressed as mean ± SEM. ***P = 0.002, compared with vehicle-treated pulmospheres. (F) Transmission electron microscopy images of vehicle- and WFA-treated pulmospheres. Scale bars: 2 μm; magnified images, 500 nm. (G) Calculation formula for determination of fold change ratio for zone of invasion (ZOI) area. Median values of ZOI ≥1 represent pulmospheres nonresponsive to the treatment with WFA, and median values of ZOI <1 represent pulmospheres responsive to WFA treatment. (H) Invasiveness of pulmospheres from an individual patient not treated and treated with WFA. Box-and-whisker plots for IPF patient pulmospheres treated with WFA. Whiskers show maximum to minimum values. Lines in boxes represent median values.

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