Autophagy mediates tumor necrosis factor-α-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-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.

Activation of TFEB ameliorates dedifferentiation of arterial smooth muscle cells and neointima formation in mice with high-fat diet

“…Moreover, augmented autophagy was found to inhibit SMC migration 18,19 . On the contrary, defective autophagy due to impaired autophagy induction contributes to uncontrolled cell growth or migration 20–22 , which promotes neointimal lesions in the arterial wall as well as atherosclerotic development 22–24 . In addition to impaired autophagy induction, the defective autophagy pathway is also associated or caused by lysosome dysfunction or injury 7,25 .…”

NPS2390, a selective calcium-sensing receptor antagonist controls the phenotypic modulation of hypoxic human pulmonary arterial smooth muscle cells by regulating autophagy

“…[4] Moreover, inhibition of autophagy by drugs can inhibit PDGF on synthetic phenotype. [5,6] Autophagy may participate in the process of phenotypic modulation of VSMCs. Furthermore, the molecular mechanism of autophagy is complicated and related to several distinct signaling pathways.…”

PI3Kγ promotes vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis via a SOX9-dependent mechanism

“…Likewise, the pathogenesis of transplant arteriosclerosis involves the interaction between immune and nonimmune factors, which provokes endothelial injury and vascular inflammation, resulting in VSMC phenotypic modulation that plays a central role in the initiation and perpetuation of neointimal formation in graft vessels [ 3 , 7 , 10 , 14 ]. Specifically, the pleiotropic cytokine TNF-α has also been shown to be a critical modulator of VSMC phenotypic modulation in the progression of neointimal formation [ [15] , [16] , [17] ]. However, the molecular mechanisms underlying VSMCs phenotypic modulation has not been fully understood.…”