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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 3

Inhibition of VimIF assembly induces autophagy in IPF fibroblasts.

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Inhibition of VimIF assembly induces autophagy in IPF fibroblasts.
(A) R...
(A) Representative electron photomicrograph and quantitative graph showing increased number of autophagic vacuoles in vehicle and WFA-treated IPF fibroblasts. Data are presented as mean ± SEM. Three cells per IPF patient were analyzed for the quantification of autophagic vacuoles. Each dot represents an individual patient. **P < 0.01, as compared with vehicle-treated fibroblasts. Scale bars: 10 μm. (B) Human fibroblasts were treated with vehicle or WFA, and laser confocal microscopy was performed to detect LC3B by indirect immunofluorescence. Nuclei were costained with DAPI (blue). Scale bars: 20 μm. Graph represents the quantification of LC3BII puncta in WFA-treated fibroblasts. ***P < 0.001, as compared with vehicle-treated fibroblasts. Data are mean ± SD (n = 5 patients). (C) Immunoblot analysis for vimentin, LC3B, and beclin 1 of cell lysates from WFA-treated IPF fibroblasts. Asterisk indicates vimentin 56-kDa band. Each lane represents an individual subject. β-Actin served as loading control. Densitometry for (D) vimentin (band at 56 kDa), (E) beclin 1, and (F) LC3BII/LC3B I ratio normalized to β-actin for the Western blot. *P < 0.05, compared with vehicle-treated fibroblasts. (G) Immunoblot analysis for LC3B of cell lysates from IPF fibroblasts treated with chloroquine (CQ) and cotreated with WFA/CQ. β-Actin served as loading control. Data are representative of 3 experiments. The graph represents densitometry analysis for LC3BII/LC3B I ratio normalized to β-actin for the immunoblot blot analysis of LC3B. ***P < 0.001, compared with vehicle-treated fibroblasts. (H) Representative images of cells expressing the RFP-GFP-LC3B construct followed by the treatment with vehicle alone (control) or CQ, WFA, and WFA/CQ cotreatment for 12 hours. CQ-treated cells served as a positive control. Nuclei were costained with DAPI (blue). Natural-pH LC3B-positive autophagosome (green fluorescence) and acidic-pH LC3B-positive autophagolysosome (red fluorescence) were detected respectively using a fluorescence microscope. Scale bars: 20 μm

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