Artículo de revista
Autophagy mediates tumor necrosis factor-alpha-induced phenotype switching in vascular smooth muscle A7r5 cell line
Fecha
2018Registro en:
PLoS one 13(5): e0197210, May 2018.
10.1371/journal.pone.0197210
Autor
García Miguel, Marina
Riquelme, Jaime A.
Norambuena Soto, Ignacio
Morales, Pablo E.
Sanhueza Olivares, Fernanda
Núñez Soto, Constanza Belén
Mondaca Ruff, David
Cancino Arenas, Nicole
San Martín, Alejandra
Chiong Lay, Mario
Institución
Resumen
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-alpha (TNF-alpha). 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-alpha 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-alpha 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 alpha-SMA and SM22, extracellular matrix protein osteopontin and type I collagen levels. Cell proliferation was measured by [H-3]-thymidine incorporation and MTT assay, and migration was evaluated by wound healing and transwell assays. Expression of IL-1 beta, IL-6 and IL-10 was assessed by ELISA. TNF-alpha 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-alpha decreased alpha-SMA (0.74 +/- 0.12, p<0.05) and SM22 (0.54 +/- 0.01, p<0.01) protein levels. Consequently, TNF-alpha 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-alpha-induced phenotypic changes. TNF-alpha 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.