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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...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
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.
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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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