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Autophagy mediates tumor necrosis factor-α-induced phenotype switching in vascular smooth muscle A7r5 cell line
Vascular smooth muscle cells (VSMC) dedifferentiation from a contractile to a synthetic phenotype contributes to atherosclerosis. Atherosclerotic tissue has a chronic inflammatory component with high levels of tumor necrosis factor-α (TNF-α). VSMC of atheromatous plaques have increased autophagy, a...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947899/ https://www.ncbi.nlm.nih.gov/pubmed/29750813 http://dx.doi.org/10.1371/journal.pone.0197210 |
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author | García-Miguel, Marina Riquelme, Jaime A. Norambuena-Soto, Ignacio Morales, Pablo E. Sanhueza-Olivares, Fernanda Nuñez-Soto, Constanza Mondaca-Ruff, David Cancino-Arenas, Nicole San Martín, Alejandra Chiong, Mario |
author_facet | García-Miguel, Marina Riquelme, Jaime A. Norambuena-Soto, Ignacio Morales, Pablo E. Sanhueza-Olivares, Fernanda Nuñez-Soto, Constanza Mondaca-Ruff, David Cancino-Arenas, Nicole San Martín, Alejandra Chiong, Mario |
author_sort | García-Miguel, Marina |
collection | PubMed |
description | Vascular smooth muscle cells (VSMC) dedifferentiation from a contractile to a synthetic phenotype contributes to atherosclerosis. Atherosclerotic tissue has a chronic inflammatory component with high levels of tumor necrosis factor-α (TNF-α). VSMC of atheromatous plaques have increased autophagy, a mechanism responsible for protein and intracellular organelle degradation. The aim of this study was to evaluate whether TNF-α induces phenotype switching of VSMCs and whether this effect depends on autophagy. Rat aortic Vascular smooth A7r5 cell line was used as a model to examine the phenotype switching and autophagy. These cells were stimulated with TNF-α 100 ng/mL. Autophagy was determined by measuring LC3-II and p62 protein levels. Autophagy was inhibited using chloroquine and siRNA Beclin1. Cell dedifferentiation was evaluated by measuring the expression of contractile proteins α-SMA and SM22, extracellular matrix protein osteopontin and type I collagen levels. Cell proliferation was measured by [(3)H]-thymidine incorporation and MTT assay, and migration was evaluated by wound healing and transwell assays. Expression of IL-1β, IL-6 and IL-10 was assessed by ELISA. TNF-α induced autophagy as determined by increased LC3-II (1.91±0.21, p<0.001) and decreased p62 (0.86±0.02, p<0.05) when compared to control. Additionally, TNF-α decreased α-SMA (0.74±0.12, p<0.05) and SM22 (0.54±0.01, p<0.01) protein levels. Consequently, TNF-α induced migration (1.25±0.05, p<0.05), proliferation (2.33±0.24, p<0.05), and the secretion of IL-6 (258±53, p<0.01), type I collagen (3.09±0.85, p<0.01) and osteopontin (2.32±0.46, p<0.01). Inhibition of autophagy prevented all the TNF-α-induced phenotypic changes. TNF-α induces phenotype switching in A7r5 cell line by a mechanism that required autophagy. Therefore, autophagy may be a potential therapeutic target for the treatment of atherosclerosis. |
format | Online Article Text |
id | pubmed-5947899 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-59478992018-05-25 Autophagy mediates tumor necrosis factor-α-induced phenotype switching in vascular smooth muscle A7r5 cell line García-Miguel, Marina Riquelme, Jaime A. Norambuena-Soto, Ignacio Morales, Pablo E. Sanhueza-Olivares, Fernanda Nuñez-Soto, Constanza Mondaca-Ruff, David Cancino-Arenas, Nicole San Martín, Alejandra Chiong, Mario PLoS One Research Article Vascular smooth muscle cells (VSMC) dedifferentiation from a contractile to a synthetic phenotype contributes to atherosclerosis. Atherosclerotic tissue has a chronic inflammatory component with high levels of tumor necrosis factor-α (TNF-α). VSMC of atheromatous plaques have increased autophagy, a mechanism responsible for protein and intracellular organelle degradation. The aim of this study was to evaluate whether TNF-α induces phenotype switching of VSMCs and whether this effect depends on autophagy. Rat aortic Vascular smooth A7r5 cell line was used as a model to examine the phenotype switching and autophagy. These cells were stimulated with TNF-α 100 ng/mL. Autophagy was determined by measuring LC3-II and p62 protein levels. Autophagy was inhibited using chloroquine and siRNA Beclin1. Cell dedifferentiation was evaluated by measuring the expression of contractile proteins α-SMA and SM22, extracellular matrix protein osteopontin and type I collagen levels. Cell proliferation was measured by [(3)H]-thymidine incorporation and MTT assay, and migration was evaluated by wound healing and transwell assays. Expression of IL-1β, IL-6 and IL-10 was assessed by ELISA. TNF-α induced autophagy as determined by increased LC3-II (1.91±0.21, p<0.001) and decreased p62 (0.86±0.02, p<0.05) when compared to control. Additionally, TNF-α decreased α-SMA (0.74±0.12, p<0.05) and SM22 (0.54±0.01, p<0.01) protein levels. Consequently, TNF-α induced migration (1.25±0.05, p<0.05), proliferation (2.33±0.24, p<0.05), and the secretion of IL-6 (258±53, p<0.01), type I collagen (3.09±0.85, p<0.01) and osteopontin (2.32±0.46, p<0.01). Inhibition of autophagy prevented all the TNF-α-induced phenotypic changes. TNF-α induces phenotype switching in A7r5 cell line by a mechanism that required autophagy. Therefore, autophagy may be a potential therapeutic target for the treatment of atherosclerosis. Public Library of Science 2018-05-11 /pmc/articles/PMC5947899/ /pubmed/29750813 http://dx.doi.org/10.1371/journal.pone.0197210 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 (https://creativecommons.org/publicdomain/zero/1.0/) public domain dedication. |
spellingShingle | Research Article García-Miguel, Marina Riquelme, Jaime A. Norambuena-Soto, Ignacio Morales, Pablo E. Sanhueza-Olivares, Fernanda Nuñez-Soto, Constanza Mondaca-Ruff, David Cancino-Arenas, Nicole San Martín, Alejandra Chiong, Mario Autophagy mediates tumor necrosis factor-α-induced phenotype switching in vascular smooth muscle A7r5 cell line |
title | Autophagy mediates tumor necrosis factor-α-induced phenotype switching in vascular smooth muscle A7r5 cell line |
title_full | Autophagy mediates tumor necrosis factor-α-induced phenotype switching in vascular smooth muscle A7r5 cell line |
title_fullStr | Autophagy mediates tumor necrosis factor-α-induced phenotype switching in vascular smooth muscle A7r5 cell line |
title_full_unstemmed | Autophagy mediates tumor necrosis factor-α-induced phenotype switching in vascular smooth muscle A7r5 cell line |
title_short | Autophagy mediates tumor necrosis factor-α-induced phenotype switching in vascular smooth muscle A7r5 cell line |
title_sort | autophagy mediates tumor necrosis factor-α-induced phenotype switching in vascular smooth muscle a7r5 cell line |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947899/ https://www.ncbi.nlm.nih.gov/pubmed/29750813 http://dx.doi.org/10.1371/journal.pone.0197210 |
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