Soluble Interleukin-6 Receptor Regulates Interleukin-6-Dependent Vascular Remodeling in Long-Distance Runners

Figures

• 

  FIGURE 1. Plasma interleukin-6 (IL-6) levels in sedentary subjects and athletes with high (HT) and low (LT) training loads. HT-group: ≥100 km by week, n = 22; LT group: <100 km by week, n = 22; and a healthy and inactive physically group (control, CT; n = 21). (A) Basal venous blood samples were obtained 1 week prior to the marathon. After performing an outlier analysis using the ROUT method (Q = 10%), two values in the LT group (corresponding to 11.3 and 18.8) and 1 value in the HT group (corresponding to 23.5) were discarded. Data were analyzed by one-way ANOVA followed by post hoc Holm-Sidak’s multiple comparisons test. (B) Post-exercise venous blood samples were obtained at the finish line within 5 min of the end of the marathon. Different letters indicate significant differences from different groups at the same time. Data were analyzed by two-way ANOVA followed by post hoc Sidak test; a vs. b p < 0.0001. (C) Changes in plasma IL-6 levels. After performing an outlier analysis using the ROUT method (Q = 10%), a single value in the HT group (corresponding to 316) was discarded. Data were analyzed by Student t-test. Plasma IL-6 levels were determined using an ELISA kit. All data are…

• 

  FIGURE 2. Plasma soluble interleukin-6 receptor (sIL-6R) levels in sedentary subjects and athletes with high (HT) and low (LT) training loads. HT-group: ≥100 km by week, n = 22; LT group: <100 km by week, n = 22; and a healthy and inactive physically group (control, CT; n = 21). (A) Basal venous blood samples were obtained 1 week prior to the marathon. Data were analyzed by one-way ANOVA followed by post hoc Holm-Sidak’s multiple comparisons test. (B) Post-exercise venous blood samples were obtained at the finish line within 5 min of the end of the marathon. Different letters indicate significant differences from different groups at the same time. Data were analyzed by two-way ANOVA followed by post hoc Sidak test; a vs. b p < 0.05. (C) Change in plasma sIL-6R levels. Data were analyzed by Student t-test. *p < 0.05 vs. LT group. Plasma sIL-6R levels were determined using an ELISA kit. (D) IL-6/sIL-6R ratios were calculated. Different letters indicate significant differences from different groups at the same time. Data were analyzed by two-way ANOVA followed by post hoc Sidak test; c vs. d p < 0.0001. (E) spg130 was determined in the same plasma samples obtained prior to and immediately…

• 

  FIGURE 3. Aortic diameter in in sedentary subjects and athletes with high and low training loads. HT-group: ≥ 100 km by week, n = 19; LT group: <100 km by week, n = 20; and a healthy and inactive physically group (control, CT; n = 14). (A) Ascending aortic and (B) aortic root diameters were determined by transthoracic echocardiography and normalized by body surface. Upper panels are representative images of the echocardiography. Lower panels are the quantifications. Data are mean ± SD. Data were analyzed by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *p < 0.05 vs. sedentary group. Correlation between (C) ascending aortic or (D) aortic root diameters with plasma IL-6 levels, were evaluated by Pearson’s analysis. Plasma IL-6 levels, determined immediately after running the marathon, were multiplied by training load -considered as kilometers ran per week-, and years of training (□) HT group: (○) LT group.

• 

  FIGURE 4. Interleukin-6 (IL-6) induces A7r5 vascular smooth muscle cell migration. (A) A7r5 VSMCs were treated with 0, 100, 200, and 300 ng/mL IL-6, and cell migration was assessed by wound healing assay. (B,C) A7r5 VSMCs were treated with 300 ng/mL IL-6 or 20 ng/mL PDGF-BB, and cell migration was assessed by wound healing assay (B) or Transwell assay (C). Wound healing (%) after 24 h and migrated cells per field in Transwell chamber after 4 h were measured using ImageJ software (n = 5). (D–F) A7r5 VSMCs were treated with 300 ng/mL IL-6 or 20 ng/mL PDGF-BB for 24 h. α-Smooth muscle actin (α-SMA) (D), SM22 (E), and calponin (F) levels were determined by Western blot (n = 5). β-tubulin was used as a loading control. Upper panels are representative images. Lower panels are measurements. Data represent mean ± SEM and were analyzed using one-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *p < 0.05, ***p < 0.001, ****p < 0.0001 vs. control; ##p < 0.01 vs. IL-6.

• 

  FIGURE 5. Soluble interleukin-6 receptor (sIL-6R) inhibits interleukin-6 (IL-6)-induced A7r5 vascular smooth muscle cell migration. (A) A7r5 VSMCs were treated with 300 ng/mL IL-6, 300 ng/mL sIL-6R, or 300 ng/mL IL-6 + 300 ng/mL sIL-6R. (B) A7r5 VSMCs were treated with 20 ng/mL PDGF-BB, 300 ng/mL sIL-6R, or 20 ng/mL PDGF-BB + 300 ng/mL sIL-6R. Wound healing (%) after 24 h was measured using ImageJ software (n = 5). (C) A7r5 VSMCs were treated with 300 ng/mL sIL-6R for 1 h. Phosphorylated focal adhesion kinase (p-FAK) and total FAK were determined by Western blotting. GAPDH was used as a loading control. (D–I) A7r5 VSMCs were treated with 300 ng/mL IL-6, 300 ng/mL sIL-6R, or 300 ng/mL IL-6 + 300 ng/mL sIL-6R for 24 h. α-smooth muscle actin (α-SMA) (D), calponin levels (E), SM22 (F), collagen type I (G), and cyclin D1 (H) were determined by Western blot. GAPDH and β-tubulin were used as a loading control. Upper panels are representative images. Lower panels are measurements (n = 4). (I) Cell proliferation was measured using the MTT assay (n = 4). Data represent mean ± SEM and were analyzed using one-way ANOVA followed by Holm-Sidak’s multiple comparisons test in panels (C–E); and two-way…