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.

Fig 1. TNF-α activates autophagy in A7r5 cells through an-IKKα dependent pathway.

(A) Western blot analysis of Beclin1, p62, LC3-II and GAPDH in A7r5 cells stimulated with 0, 1, 10 and 100 ng/mL of TNF-α (n = 4; *p<0.05 vs 0 ng/mL TNF-α). (B) Western blot analysis of Beclin1, p62, LC3-II and GAPDH in A7r5 cells incubated with TNF-α (100 ng/mL) at 0, 3, 16, 24 and 48 h (n = 4; *p<0.05, **p<0.01 vs 0 h). (C) Western blot analysis of p62, LC3-II and GAPDH in A7r5 cells treated with TNF-α (100 ng/mL) for 24 h and co-administered with or without chloroquine (CQ, 20 μmol/L) during the last 4 h of stimulus (n = 3; **p<0.01, ***p<0.001 vs control. (D) Visualization of autophagic vesicles by confocal microscopy in A7r5 cells transduced with adenovirus overexpressing LC3-GFP at MOI = 150 for 24 h and incubated with TNF-α (100 ng/mL) for 24 h and co-administered with or without CQ (20 μmol/L) during the last 4 h of TNF-α stimulus (n = 3). (E) Western blot analysis of LC3-II in A7r5 cells pre-treated with or without BAY-11-7082 (1 and 10 μmol/L) for 30 min, followed by incubation with TNF-α (100 ng/mL) for 24 h (n = 3; *p<0.05 vs control). Data are expressed as mean ± SEM and analyzed by one-way ANOVA followed Dunnett post-test (A, B and C) and paired t test comparing each condition to its control (E).

Fig 2. TNF-α induces dedifferentiation of A7r5 cells by an autophagy-dependent pathway.

(A) Western blot analysis of α-SMA and SM22 (n = 3–4; *p<0.05, **p<0.01 vs control) or (B) collagen type I and osteopontin (n = 3–4; **p<0.01 vs control) in A7r5 cells incubated with TNF-α (100 ng/mL) for 48 h in the presence or absence of siScramble and siBeclin1. GAPDH was used as loading control. (C) Visualization of actin filaments in A7r5 cells stained with rhodamine-phalloidin after treatment with TNF-α (100 ng/mL) for 48 h in the presence or absence of chloroquine (CQ, 5 μmol/L) during the last 24 h of TNF-α stimulus. Lower panel represent a fluorescence intensity profile of the lines depicted on the images. Data are expressed as mean ± SEM and analyzed by two-way ANOVA, followed by Holm Sidak post-test.

Fig 3. TNF-α requires autophagy to induce migration in A7r5 cells.

(A) Assessment of migration by the wound healing and transwell assays in A7r5 cells stimulated with TNF-α (100 ng/mL) for 24 h in the presence or absence of chloroquine (CQ, 20 μmol/L) during the last 4 h of TNF-α stimulus (n = 4; **p<0.01 vs control) or (C) siScramble and siBeclin1 for 24 h (n = 4; **p<0.01 vs control). Migration was visualized using a phase contrast microscope (upper panels of A and C). The results of the wound healing and transwell assays were quantified by measuring wound width and the number of cells that migrated through the Boyden chamber, respectively (lower panels of A and C). (B) Zymography analysis of matrix metalloproteinase 9 (MMP-9) in A7r5 cells stimulated with TNF-α (100 ng/mL) for 24 h (n = 3; *p<0.05 vs control). Data are expressed as mean ± SEM and analyzed by one-way ANOVA, followed by Holm Sidak (A and C) and Dunnett (B) post-tests.

Fig 4. TNF-α induces proliferation of A7r5 cells through an autophagy-dependent mechanism.

(A) Determination of proliferation by the MTT assay and (B) incorporation of [³H]-thymidine in A7r5 cells treated with TNF-α (100 ng/mL) for 24 h in the presence or absence of chloroquine (CQ, 20 μmol/L) during the last 4 h of TNF-α stimulus (n = 4; **p<0.01 vs control) or (C and D) siScramble and siBeclin1 (n = 4; *p<0.05, **p<0.01 vs control). Data are expressed as mean ± SEM and analyzed by one-way ANOVA, followed by Holm Sidak (A and C) and Dunnett (B and D) post-tests.

Fig 5. Autophagy mediates TNF-α-induced secretion of IL-6 in A7r5 cells.

(A) Determination of IL-6 mRNA using RT-qPCR in A7r5 cells treated with TNF-α (100 ng/mL) for 30 min, 1 and 6 h (n = 6; *p<0.05, **p<0.01 vs 0 h). (B) Determination of IL-6 using ELISA assay in A7r5 cells treated with TNF-α (100 ng/mL) for 24 and 48 h (n = 4; **p<0.01, ***p<0.001 vs 0 h) or (C) co-administered with or without chloroquine (CQ, 20 μmol/L) during the last 4 h of the 24 h stimulus with TNF-α (n = 4; ***p<0.001 vs control). Data are expressed as mean ± SEM and analyzed by one-way ANOVA, followed by Dunnett and Tukey post-tests.