Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

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Abstract

Transforming growth factor- β ₁ (TGF- β ₁) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF- β ₁ may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF- β ₁-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF- β ₁ to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF- β ₁ promoted collagen type I α2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF- β ₁ in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3 β indicated the localization of punctate LC3 β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF- β ₁-induced autophagy is required for the fibrogenic response in hATMyofbs.

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Most cited references 54

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In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

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Autophagy is a metabolic process that degrades and recycles intracellular organelles and proteins with many connections to human disease and physiology. We studied the role of autophagy during hepatic stellate cell (HSC) activation, a key event in liver fibrogenesis. Analysis of the autophagic flux during in vitro activation of primary mouse HSCs was performed using a DsRed-GFP-LC3B encoding plasmid. The effect of autophagy inhibition by bafilomycin A1 on the in vitro activation process of human and mouse HSCs was examined by measuring proliferation, presence of activation markers by RT-qPCR, immunofluorescence, and Western blotting. Analysis of lipid droplet and microtubule-associated protein light chain 3 beta (LC3B) colocalization in the presence of PDGF-BB was investigated by immunocytochemistry. A significant increased autophagic flux was observed during culture induced mouse HSC activation. Treatment of mouse HSCs and human HSCs with autophagy inhibitor bafilomycin A1 results in a significant decreased proliferation and expression of activation markers. In addition, lipid droplets and LC3B colocalization was increased after PDGF-BB treatment in quiescent HSCs. During HSC activation, autophagic flux is increased. The demonstration of partly inhibition of in vitro HSC activation after treatment with an autophagy inhibitor unveils a potential new therapeutic strategy for liver fibrosis. Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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Author and article information

Journal

Journal ID (nlm-ta): Cell Death Dis

Journal ID (iso-abbrev): Cell Death Dis

Title: Cell Death & Disease

Publisher: Nature Publishing Group

ISSN (Electronic): 2041-4889

Publication date (Print): March 2015

Publication date (Electronic): 19 March 2015

Publication date PMC-release: 1 March 2015

Volume: 6

Issue: 3

Page: e1696

Affiliations

[1 ]Department of Physiology, Manitoba Institute of Child Health , Winnipeg, Manitoba, Canada

[2 ]Biology of Breathing Group, Manitoba Institute of Child Health , Winnipeg, Manitoba, Canada

[3 ]Department of Physiology and Institute of Cardiovascular Sciences, St. Boniface Research Centre, University of Manitoba , Winnipeg, Manitoba, Canada

[4 ]Department of Human Anatomy and Cell Science, University of Manitoba , Winnipeg, Manitoba, Canada

[5 ]Department of Internal Medicine, Manitoba Institute of Child Health , Winnipeg, Manitoba, Canada

[6 ]Cardiac Sciences Program, St. Boniface General Hospital , Winnipeg, Manitoba, Canada

Author notes

[* ]Department of Physiology, Molecular Cardiology Laboratory, Institute of Cardiovascular Sciences, R3038 St. Boniface General Hospital Research Centre , 351 Tache Avenue, Winnipeg, Manitoba, Canada R2H 2A6; Tel: +204 235 3419; Fax: +204 233 6723. E-mail: idixon@ 123456sbrc.ca

Article

Publisher Item ID: cddis201536

DOI: 10.1038/cddis.2015.36

PMC ID: 4385916

PubMed ID: 25789971

SO-VID: bebf4201-2ed9-464c-98f0-ac20f8d7e2ba

License:

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International Licence. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons licence, users will need to obtain permission from the licence holder to reproduce the material. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0

History

Date received : 13 May 2014

Date revision received : 09 January 2015

Date accepted : 16 January 2015

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