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

• Published in: PloS one Vol. 13; no. 5; p. e0197210
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 11.05.2018; Public Library of Science (PLoS)
• Subjects: Animals; Aorta; Arteriosclerosis; Atherosclerosis; Atherosclerosis - metabolism; Atherosclerosis - pathology; Autophagy; Biocompatibility; Biology and Life Sciences; Biomedical materials; Cancer; Cell death; Cell growth; Cell Line; Cell proliferation; Chloroquine; Chronic illnesses; Collagen; Collagen (type I); Cytokines; Disease; Enzyme-linked immunosorbent assay; Extracellular matrix; Hypertension; Immunology; Inflammation; Interleukin 10; Interleukin 6; Interleukin-10 - metabolism; Interleukin-1beta - metabolism; Interleukin-6 - metabolism; Kinases; Matrix protein; Medicine and Health Sciences; Metabolism; Muscle contraction; Muscle, Smooth, Vascular - metabolism; Muscle, Smooth, Vascular - pathology; Muscles; Myocytes, Smooth Muscle - metabolism; Myocytes, Smooth Muscle - pathology; Necrosis; Osteopontin; p62 Protein; Phagocytosis; Phenotypes; Physical Sciences; Physiology; Plaques; Proteins; Rats; Research and analysis methods; siRNA; Smooth muscle; Switching; Therapeutic applications; Thymidine; Tumor Necrosis Factor-alpha - metabolism; Tumor necrosis factor-TNF; Tumor necrosis factor-α; Tumors; Wound healing; Chile
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0197210

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 [3H]-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.