Autophagy mediates tumor necrosis factor-alpha-induced phenotype switching in vascular smooth muscle A7r5 cell line

Abstract

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.