PRMT5 critically mediates TMAO-induced inflammatory response in vascular smooth muscle cells

Figures

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  Fig. 1. TMAO upregulates PRMT5 and VCAM-1 expression in rat VSMC. A VSMC were treated with different concentrations of TMAO (100–2000 μM) for 24 h. Subsequently, cell viability was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and quantitative analysis of cell viability. B VSMC were treated with TMAO (600 μM) for indicated periods (4–48 h), and cell viability was detected using MTT assay and quantitative analysis of cell viability. C VSMC were treated for indicated concentrations of TMAO for 24 h. Representative blots and quantitative analysis of VCAM-1 and PRMT5. The protein expression level was normalized to β-actin. D VSMC were treated with TMAO (600 μM) for indicated periods. Representative blots and quantitative analysis of VCAM-1 and PRMT5. The protein expression level was normalized to β-actin. Data shown are means ± SD, n = 6 per group from three independent experiments. **P < 0.01, ***P < 0.001 using a one-way ANOVA followed by Tukey’s post hoc test.

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  Fig. 2. PRMT5 is responsible for TMAO-induced VCAM-1 expression. VSMC were transfected with PRMT5 siRNA (siPRMT5) or PRMT5 cDNA (PRMT5 OE), then stimulated with TMAO (600 μM) for 24 h. A, B Representative blots and quantitative analysis of VCAM-1 and PRMT5. The protein expression level was normalized to β-actin. C Representative images obtained by fluorescence microscope show cell adhesion. Data shown are means ± SD, n = 6 per group from three independent experiments. ***P < 0.001 using a one-way ANOVA followed by Tukey’s post hoc test.

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  Fig. 3. PRMT5-mediated NF-κB p65 methylation is involved in TMAO-stimulated VCAM-1 expression in VSMC. A VSMC were pretreated with BAY 11-7085 for 4 h and then stimulated with TMAO (600 μM) for 24 h. Representative blots and quantitative analysis of VCAM-1. The protein expression level was normalized to β-actin. B VSMC were treated with TMAO (600 μM) for indicated periods. Co-immunoprecipitation analysis of the interaction between PRMT5 and p65. C VSMC were treated with TMAO (600 μM) for 24 h. Representative blots of arginine methylation of p65. Immunoprecipitated SYM10 was blotted with an antibody against p65. D Representative blots of arginine methylation of p65. Immunoprecipitated SYM10 was blotted with an antibody against p65 in VSMC transfected with PRMT5 siRNA (siPRMT5) or PRMT5 cDNA plasmid (PRMT5 OE) followed by TMAO (600 μM) stimulation. E Flag-tagged p65 wide-type (p65WT) or R30A mutant (p65R30A) plasmid was transfected to VSMC, and then treated with TMAO (600 μM). Representative blots of arginine methylation of p65. Immunoprecipitated SYM10 was blotted with an antibody against p65. F Flag-tagged p65 wide-type (p65WT) or R30Amutant (p65R30A) plasmid was transfected to VSMC, and…

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  Fig. 4. PRMT5 contributes to TMAO induced vascular inflammation in vivo. SMC-PRMT5-KO or SMC-Cre mice were subjected to wire-induced carotid artery injury. TMAO (86 μmol) or saline was administered daily by intraperitoneal injection for 7 days. A Representative photomicrographs for VCAM-1 in carotid arteries at 7 days after wire injury. Scale bar, 50 μm. B Representative photomicrographs for CD68 in carotid arteries at 7 days after wire injury. Scale bar, 50 μm. C Representative blots and quantitative analysis of VCAM-1 and PRMT5. The protein expression level was normalized to β-actin. D Representative blots of arginine methylation of p65. Immunoprecipitated SYM10 was blotted with an antibody against p65. Data shown are means ± SD, n = 6 per group from three independent experiments. ***P < 0.001 using a one-way ANOVA followed by Tukey’s post hoc test.

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  Fig. 5. NOX4 knockdown decreases H2O2 production in VSMC treated with TMAO. A Representative blots and quantitative data of Nox1, Nox2 and Nox4 protein expression in TMAO (600 μM) treated VSMC at indicated periods. The protein expression level was normalized to β-actin. B, C VSMC were treated with scrambled siRNA, Nox1 or Nox4 siRNA, and then treated with TMAO (600 μM). B H2O2 and C O2•− levels were evaluated by DHE and Amplex Red probe, respectively. Data shown are means ± SD, n = 6 per group from three independent experiments. **P < 0.01, ***P < 0.001 using a one-way ANOVA followed by Tukey’s post hoc test.

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  Fig. 6. Nox4 knockdown downregulates PRMT5 and VCAM-1 expression. A VSMC were pretreated with N-acetyl-cysteine (NAC) and then stimulated with TMAO (600 μM) for 24 h. Representative blots and quantitative analysis of VCAM-1 and PRMT5. The protein expression level was normalized to β-actin. H2O2 stimulation served as positive control. B–D VSMC were treated with scrambled siRNA (siScr) or Nox4 siRNA (siNOX4) and then treated with TMAO (600 μM). B Representative blots and quantitative analysis of Nox4 and PRMT5. The protein expression level was normalized to β-actin. C Representative blots and quantitative data of VCAM-1 protein expression. The protein expression level was normalized to β-actin. H2O2 stimulation served as positive control. D Representative images obtained by fluorescence microscope show cell adhesion. Data shown are means ± SD, n = 6 per group from three independent experiments. **P < 0.01, ***P < 0.001 using a one-way ANOVA followed by Tukey’s post hoc test.

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  Fig. 7. NF-YA is responsible for TMAO-induced PRMT5 expression. A VSMC were treated with TMAO (600 μM) for indicated periods. Representative blots and quantitative analysis of NF-YA. The protein expression level was normalized to β-actin. B VSMC were transfected with scrambled siRNA (siScr) or Nox4 siRNA (siNox4) and then stimulated with TMAO (600 μM) for 24 h. Representative blots and quantitative analysis of NF-YA. The protein expression level was normalized to β-actin. C VSMC were transfected with NF-YA siRNA (siNF-YA) or siScr and then stimulated with TMAO (600 μM) for 24 h. Representative blots and quantitative analysis of NF-YA and PRMT5. The protein expression level was normalized to β-actin. Data shown are means ± SD, n = 6 per group from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 using a one-way ANOVA followed by Tukey’s post hoc test.

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  Fig. 8. PRMT5 promotes TMAO-induced VCAM-1 expression via methylation of NF-κB p65 in VSMC. H2O2 generated from the TMAO-stimulated Nox4 upregulation can promote PRMT5 expression and increase methylation of NF-κB p65 on R30, leading to the induction of VCAM-1 and macrophage adhesion.