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A2654 - Loss of Autophagic Homeostasis Regulates the Invasive Capacity of Vimentin Rich Fibroblasts in 3D Lung Pulmospheres from Patients with Idiopathic Pulmonary Fibrosis
Author Block: R. Surolia1, F. Li2, H. Li1, Z. Wang1, H. Kim3, V. J. Thannickal4, V. B. Antony2; 1Univ of Alabama, Birmingham, AL, United States, 2Univ of Alabama at Birmingham, Birmingham, AL, United States, 3Department of Radiology, Univ of Alabama, Birmingham, AL, United States, 4Univ of Alabama At Birmingham, Birmingham, AL, United States.
Remodeling of the lung architecture characterized by a pathogenic accumulation of invasive fibroblasts is a hallmark of IPF. The mechanisms of increased invasiveness of fibroblasts and myofibroblasts in IPF are not yet fully elucidated. Importantly, autophagy is impaired in fibroblasts from patients with IPF. We hypothesized that vimentin regulates both autophagy and invasiveness of IPF fibroblasts. IPF patient lung biopsy derived 3D pulmospheres were used to study the effect of aberrant autophagy on vimentin expression and invasiveness. RT- PCR, western blots and immunostaining were used to delineate the relationship between Vimentin intermediate filament (Vim IF) and the autophagic marker Beclin1. Withaferin A, pharmaceutical inhibitor of Vim IF assembly, was used to demonstrate the effect of Vim IF breakdown on the invasiveness and autophagy of lung fibroblasts. In in vivo experiments using a bleomycin treated mouse model of lung fibrosis, mice were treated with Withaferin A for every alternate day post bleomycin administration till 21 days. Mice were sacrificed on day 21 and the extent of fibrosis was assessed with histology of lung sections and micro CT scans. The level of autophagic markers was assessed by Westerns blots of mice lung lysates and immunohistochemical staining for LC3B. Pulmospheres from IPF patient lungs demonstrated increased levels of vimentin and invasiveness as compared to pulmospheres form normal lung. The pulmospheres from IPF patient tissue had decreased autophagic markers as compared to pulmospheres from control lung tissue. Immunoprecipitation studies demonstrated that Vim IF bind to Beclin 1. Vim IF-Beclin1 conjugation deprives cellular autophagic machinery of Beclin1 that results in suppression of autophagy in IPF fibroblasts. Withaferin A treatments breakdown Vim IF assembly and induces autophagy. In vivo studies in bleomycin exposed mice demonstrated that Withaferin A treatment increased autophagy in lungs, and these mice were protected from fibrotic lung injury. We conclude that vimentin regulates invasiveness of fibroblasts through altering autophagic homeostasis in fibrotic lung disease