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Autophagy mediates tumor necrosis factor-?-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-? (TNF-?). 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-? 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-? 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 ?-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-1?, IL-6 and IL-10 was assessed by ELISA. TNF-? 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-? decreased ?-SMA (0.74±0.12, p<0.05) and SM22 (0.54±0.01, p<0.01) protein levels. Consequently, TNF-? 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-?-induced phenotypic changes. TNF-? 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.

SUBMITTER: Garcia-Miguel M

PROVIDER: S-EPMC5947899 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

García-Miguel Marina M Riquelme Jaime A JA Norambuena-Soto Ignacio I Morales Pablo E PE Sanhueza-Olivares Fernanda F Nuñez-Soto Constanza C Mondaca-Ruff David D Cancino-Arenas Nicole N San Martín Alejandra A Chiong Mario M

PloS one 20180511 5

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 a ...[more]

PMID: 29750813

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Project description:The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox'd Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival.

Dataset Information

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

Publications

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

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