Myostatin Inhibits Vascular Smooth Muscle Cell Proliferation and Local 14q32 microRNA Expression, But Not Systemic Inflammation or Restenosis

Figures

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  Figure 1. (A–F): Immunocytochemical staining of VSMCs. (A–C)—VSMCs treated with 10 nM myostatin showed uptake of myostatin in the cell, concentrating mostly in the nucleus. (A)—Hoechst only, (B)—Myostatin only, (C)—Merged image. (D–F)—VSMCs not treated with myostatin showed no endogenous myostatin presence in the cells at all. (D)—Hoechst only, (E)—Myostatin only, (F)—Merged image. Picture taken with 63× magnification.

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  Figure 2. (A–C): microRNAs in myostatin treated VSMCs relative to untreated control (100%). (A)—miR-433-3p expression in VSMCs treated with 10 nM and 20 nM myostatin only showed a downregulation at 10 nM treatment (p = 0.04). (B)—miR-494-3p expression in VSMCs treated with 10 and 20 nM myostatin was downregulated at 20 nM treatment (p = 0.03) and showed a trend towards downregulation at 10 nM myostatin (p = 0.06). (C)—miR-495-3p expression in VSMCs treated with 10 and 20 nM myostatin was decreased with more than 80% by both treatment concentrations (p = 0.0098 and p = 0.0088, respectively). Mean expression is shown and error bar represents SEM (n = 3). One-sample t-test performed with 100% of control expression as hypothetical value. * p < 0.05, ** p < 0.01.

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  Figure 3. (A–C): PCNA expression, cell migration and cell viability in myostatin treated VSMCs relative to non-myostatin treated control (100%). (A)—PCNA mRNA expression in myostatin treated VSMCs was downregulated at 10 nM myostatin, but not at 20 nM myostatin, n = 3. (B)—Scratch wound healing in myostatin treated VSMCs was not changed, n = 3. (C)—Cell viability in myostatin treated VSMCs remained unchanged, n = 4. Mean expression is shown and error bars represent SEMs. One-sample t-test performed with 100% of Negative Control expression as control group.

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  Figure 4. (A–F): Quantification of restenosis in cuffed femoral arteries in different treatment groups. (A)—Media area was similar in different treatment groups. (B)—Lumen stenosis did not differ between treatment groups. (C)—Intima/Media ratio was similar in different treatment groups. (D)—Lumen area in different treatment groups was equal. (E)—Neointima area in different treatment groups did not differ. (B–E): Quantification of not occluded arteries. Mean expression is shown and error bar represents SEM. n = 7 in control group, n = 4 in 0.1 µg/day, n = 4 in 0.2 µg/day, n = 4 in 0.4 µg/day. Student’s t-test, performed between myostatin treated groups and control group with α = 0.05, did not show any significant differences. (F)—representative examples of cuffed femoral arteries in different treatment groups. LEE = Lamina Elastica Externa, LEI = Lamina Elastica Interna, NI = Neointima, M = Media.

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  Figure 5. (A–D): Immunofluorescent stainings in different treatment groups. (A)—Myostatin-Hoechst immunofluorescent double staining. Representative mouse of every treatment group is shown and zoom-in of femoral artery shows presence of myostatin in all treated groups, but not in the control group. (B)—Ki-67-αSMA-Hoechst immunofluorescent triple staining in cuffed femoral arteries of different treatment groups shows few proliferating cells, especially not in the αSMA area. (C)—BrdU-Myostatin-Hoechst immunofluorescent triple staining in cuffed femoral arteries of different treatment groups shows again little proliferating cells and those cells are not double stained with myostatin. (D)—Hoechst-MAC3 immunofluorescent double staining of cuffed femoral arteries showed macrophages in all groups in both the intimal and medial layers, but no differences were found.

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  Figure 6. PCNA mRNA expression in femoral arteries of myostatin treated mice. PCNA mRNA expression is significantly lower in myostatin treated groups at all dosages compared to the control group. Mean expression is shown and error bar represents SEM (n = 8 for 0.0, 0.1 and 0.4 µg/day myostatin group, n = 6 for baseline and 0.2 µg/day group). Student’s t-test performed between treated groups and control group with α = 0.05. * p < 0.05, ** p < 0.01.

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  Figure 7. (A–D): Effect of myostatin on macrophages. (A–D)—TNFα in supernatant of bone marrow macrophages stimulated with myostatin. (A,B)—in vivo treated mice with 0.0 vs. 0.4µg myostatin/day and not treated in vitro with myostatin (A) or in vitro treated with 10 nM myostatin (B) did not show significant differences. (C,D)—in vivo not treated (C) or treated (D) and in vitro not treated or treated with myostatin did not show significant differences. Mean expression is shown as average of duplicate measurements of pooled cells and error bar represents SEM. Student’s t-test did not show significant differences.

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  Figure 8. (A–F): microRNA expression in femoral arteries and macrophages. (A)—miR-433-3p expression in femoral arteries of 0.1, 0.2, 0.4 µg/day of myostatin relative to control mice (100%) showed significant downregulation of miR-433-3p at 0.4 µg/day (p = 0.003). (B)—miR-494-3p expression in femoral arteries of 0.1, 0.2, 0.4 µg/day of myostatin relative to control mice (100%) showed significant downregulation of miR-494-3p at 0.1 (p = 0.001) and 0.4 µg/day (p < 0.0001). (C)—miR-495-3p expression in femoral arteries of 0.1, 0.2, 0.4 µg/day of myostatin relative to control mice (100%) showed significant downregulation of miR-495-3p at 0.2µg/day (p = 0.0003). For (A–C) Mean expression is shown and error bars represent SEM (n = 8 for 0.0, 0.1 and 0.4 treatment groups, n = 6 for 0.2 treatment group). One-sample t-test performed with 100% expression of control group. * p < 0.05, ** p < 0.01, **** p < 0.0001. (D)—miR-433-3p expression did not show any differences between groups. (E)—miR-494-3p expression did not show any differences between groups. (F)—miR-495-3p expression did not show any differences. For (D–E) bone marrow monocytes matured into macrophages from in vivo treated mice with 0.0…