Metformin Attenuates Hyperglycaemia-Stimulated Pro-Fibrotic Gene Expression in Adventitial Fibroblasts via Inhibition of Discoidin Domain Receptor 2

Figures

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  Figure 1. Metformin prevents HG-induced increase in Fibronectin and Collagen type I protein expression in vascular adventitial fibroblasts. Cells were pre-treated with metformin hydrochloride (10 µM) for 45 min prior to HG supplementation. (A) Protein was isolated after 12 h of HG and subjected to western blot analysis for detection of Fibronectin, with β-Actin as loading control. (B) Protein was isolated after 12 h of HG and subjected to western blot analysis for detection of Collagen type I, with β-Actin as loading control. Data are expressed as mean ± SEM (standard error of mean). The groups were analysed by one-way ANOVA (p ≤ 0.05) and Tukey’s multiple comparisons (p ≤ 0.05). Significant comparisons along with their p values are depicted in the Figure. The data are representative of three independent experiments, n = 3.

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  Figure 2. Metformin prevents HG-induced increase in DDR2 expression in vascular adventitial fibroblasts. Cells were pre-treated with metformin hydrochloride (10 µM), 45 min prior to HG supplementation. (A) DDR2 mRNA levels were determined by Taqman real-time PCR at 6 h post-HG treatment, with β-Actin as loading control. (B) Protein was isolated after 12 h of HG treatment and subjected to western blot analysis for detection of DDR2, with β-Actin as loading control. Data are expressed as mean ± SEM (standard error of mean). The groups were analysed by one-way ANOVA (p ≤ 0.05) and Tukey’s multiple comparisons (p ≤ 0.05). Significant comparisons along with their p values are depicted in the Figure. The data are representative of three independent experiments, n = 3.

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  Figure 3. DDR2 knockdown prevents HG-induced increase in Fibronectin expression in vascular adventitial fibroblasts. Cells were transiently transfected with DDR2 siRNA (5 pmol) (indicated by (+) sign) or scrambled siRNA (5 pmol) (indicated by (−) sign). Following exposure of the transfected cells to HG for 12 h, Fibronectin protein expression was examined by western blot analysis and normalized to β-Actin. 3,3′-diaminobenzidine (DAB) reagent was used for detection of β-Actin, and brightness/contrast was adjusted uniformly to match with other blots. Data are expressed as mean ± SEM (standard error of mean). The groups were analysed by one-way ANOVA (p ≤ 0.05) and Tukey’s multiple comparisons (p ≤ 0.05). Significant comparisons along with their p values are depicted in the Figure. The data are representative of three independent experiments, n = 3.

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  Figure 4. Metformin prevents HG-induced increase in TGF-β1 mRNA and protein expression in vascular adventitial fibroblasts. Cells were pre-treated with metformin hydrochloride (10 µM), 45 min prior to HG supplementation. (A) TGF-β1 mRNA levels were determined by Taqman real-time PCR at 6 h post-HG treatment, with β-Actin as loading control. (B) Protein was isolated after 12 h of HG and subjected to western blot analysis for detection of TGF-β1, with β-Actin as loading control. Data are expressed as mean ± SEM (standard error of mean). The groups were analysed by one-way ANOVA (p ≤ 0.05) and Tukey’s multiple comparisons (p ≤ 0.05). Significant comparisons along with their p values are depicted in the Figure. The data are representative of three independent experiments, n = 3.

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  Figure 5. DDR2 acts downstream of TGF-β1 to regulate Fibronectin expression. (A) Cells were transiently transfected with TGF-β1 siRNA 1 and 2 (5 pmol) (indicated by (+) sign) or scrambled siRNA (5 pmol) (indicated by (−) sign) prior to treatment with HG for 12 h. Protein was isolated and subjected to western blot analysis for detection of Fibronectin, with β-Actin as loading control. Validation of TGF-β1 knockdown is also shown. (B) Sub-confluent, quiescent cultures of vascular adventitial fibroblasts in M199 were pre-treated with SMAD2 inhibitor, PD169316, or SMAD3 inhibitor, SIS3, for 1 h and subsequently with HG for 12 h. Following treatment with HG, protein was isolated and subjected to western blot analysis for detection of Fibronectin, with β-Actin as loading control. 3,3′-diaminobenzidine (DAB) reagent was used for detection of β-Actin and brightness/contrast was adjusted uniformly to match with other blots. (C) Cells were co-transfected with DDR2 cDNA plasmid (1µg) (indicated by (+) sign) or empty plasmid (1µg) (indicated by (−) sign) and TGF-β1 siRNA 1 and 2 (5 pmol) (indicated by (+) sign) or scrambled siRNA (5 pmol) (indicated by (−) sign) (lipofectamine 2000-complexed) for 8 h…

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  Figure 6. Schematic depiction of plausible mechanisms underlying the protective effect of metformin against hyperglycaemia-induced vascular fibrosis. Metformin inhibits TGF-β1/SMAD2/3 signalling to inhibit DDR2-dependent pro-fibrotic gene expression in HG-stimulated vascular adventitial fibroblasts.