RhoBTB1 reverses established arterial stiffness in angiotensin II–induced hypertension by promoting actin depolymerization

Figures

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  Figure 1. Mouse model and protocol. (A) S-RhoBTB1 mice expressing a Tx-inducible, smooth muscle–specific Rhobtb1 transgene were obtained by breeding ISM-Cre mice, which express Tx-inducible, smooth muscle–specific Cre-recombinase, and RhoBTB1IND mice, which express a LoxP-STOP-LoxP-Rhobtb1-tdTomato transgene. Cre-recombinase catalyzes the removal of the LoxP-STOP-LoxP cassette, inducing expression of Rhobtb1 and tdTomato. (B) ISM-Cre mice and S-RhoBTB1 mice received either vehicle or Ang II treatment via osmotic minipumps for 5 weeks. All animals received 5 consecutive daily Tx injections 2 weeks after pump implantation. BP was continuously measured by radiotelemetry from 1 week before pump implantation until the end of the protocol. PWV was measured before pump implantation, 2 weeks after pump implantation, and at 0, 1, and 2 weeks after the completion of Tx injections. Tissues were harvested at the end of the protocol. (C) Representative Western blot detecting tdTomato and GAPDH in aorta (n = 2–3 biological replicates). Mice that did not receive Tx received the corn oil vehicle. Positions of actual size markers are shown. (D) Relative Rhobtb1 mRNA levels in aorta from the indicated…

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  Figure 2. Arterial stiffness. (A and B) PWV was measured at baseline (BL) before Ang II treatment, 2 weeks after Ang II pump implantation (week 3), and 0, 1, and 2 weeks (weeks 4–6) after completion of Tx injections (A); n = 20–27, as indicated in dot plots in B. (B) Individual PWV recordings at each time point from A are shown. (C and D) Aortic compliance was evaluated by the pressure-diameter (C) and stress-strain relationships (D). Sample numbers are as follows: ISM-CRE vehicle, n = 6; S-RhoBTB1 vehicle, n = 9; ISM-Cre Ang II, n = 6; S-RhoBTB1 Ang II, n = 9.All data are presented as a mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons were used for PWV analysis. Two-way ANOVA with repeated measurements were used for pressure-diameter and stress-strain relationships. *P < 0.05 vs. ISM-Cre, vehicle; #P < 0.05 vs. ISM-Cre, Ang II.

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  Figure 3. Blood pressure. BP was continuously measured by radiotelemetry (sample numbers are as follows: ISM-CRE vehicle, n = 6; S-RhoBTB1 vehicle, n = 5; ISM-Cre Ang II, n = 8; S-RhoBTB1 Ang II, n = 12.). (A–C) Systolic BP (SBP, A), mean arterial BP (MAP, B), and diastolic BP (DBP, C) during the entire protocol are presented as weekly averages. (D–E)Hourly SBP (D), MAP (E), and DBP (F) were analyzed during the last week of the protocol. All data are presented as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons were used for data analysis at each time point. *P < 0.05 vs. ISM-Cre, vehicle.

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  Figure 4. Vascular reactivity. (A–C) Vascular reactivity was studied using wire myography on carotid arteries (sample numbers are as follows: ISM-CRE vehicle, n = 6; S-RhoBTB1 vehicle, n = 6; ISM-Cre Ang II, n = 9; S-RhoBTB1 Ang II, n = 11); and (D–F) mesenteric arteries (sample numbers are as follows: ISM-CRE vehicle, n = 6; S-RhoBTB1 vehicle, n = 9; ISM-Cre Ang II, n = 5; S-RhoBTB1 Ang II, n = 9.) (A and D). ACh-mediated vasodilation was used to evaluate endothelial-dependent vascular relaxation. (D) One mesenteric artery sample was excluded because its reading fell out of the cutoff value predetermined for quality control. (B and E) Sodium nitroprusside–mediated (SNP-mediated) vasodilation was used to evaluate endothelium-independent vascular relaxation. (C and F) Serotonin-mediated (5-HT–mediated) vasoconstriction was measured. All data are presented as a mean ± SEM. *P < 0.05 vs. ISM-Cre, vehicle. Two-way ANOVA with repeated measurements were used for statical analysis. The Emax values for each biological replicate are provided in Supplemental Figure 2. Emax, maximum effect.

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  Figure 5. PCA and vascular SMC differentiation markers. (A) PCA of global gene expression in the RNA-Seq data set (n = 10). (B) Transactivation profiles of vascular smooth muscle differentiation markers in each sample.

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  Figure 6. Vascular SMC remodeling. (A) Masson’s trichrome staining of aorta cross-sections from indicated animals 2 weeks after the completion of Tx injections (n = 5–9, as indicated in the dot plots in B. Scale bar: 100 μm. (B) Wall area, lumen area, and wall area to lumen area ratio from Masson’s trichrome staining were used to evaluate vascular smooth muscle remodeling. All data are presented as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons were used for data analysis. *P < 0.05 vs. ISM-Cre, vehicle.

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  Figure 7. Extracellular matrix. (A) Transactivation profiles of classical ECM components contributing to arterial stiffness such as collagen, elastin, matrix metalloproteinase, tissue inhibitors of metalloproteinases, Lox, Tgm2, and Tgfb1 (n = 10). (B and C) Collagen deposition was assessed by the area of collagen in Masson’s trichrome staining (B) and hydroxyproline assay (C). n = 8–11, as indicated in the dot plots. (D) Elastin contents were measured in aortic samples, using biochemical assays (n = 8–11, as indicated in the dot plot). All data are presented as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons were used for data analysis. *P < 0.05 vs. ISM-Cre, vehicle.

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  Figure 8. Actin polymerization. (A and B) F-actin and G-actin were separated by ultracentrifugation and assessed by Western blot (with 6–10 biological replicates, as indicated in the dot plot in B). The last lane was loaded with purified actin as a positive control. Molecular weight markers are as indicated in kilodaltons. (B) Densitometry was used to quantify F-actin and G-actin content in the Western blot. Actin polymerization was indicated by the ratio of F-actin to G-actin (n = 6–10, as indicated in the dot plot). One sample from the ISM-Cre vehicle group and another from the S-RhoBTB1 vehicle group were identified as outliers and were excluded using the Grubb’s outlier test. (C) Actin polymerization was confirmed using fluorescence staining in aorta (n = 4–7, as indicated in the dot plot in D). F-actin was stained with Alexa Fluor 594–phalloidin (red, upper panels) and G-actin was stained by Alexa Fluor 488–DNase I (green, lower panels). Scale bar: 100 μm. (D) MFI of the same volume of aortic samples was measured under the same optical configuration using confocal microscopy. Ratio of F-actin to G-actin was used to assess action polymerization. All data are presented as mean ± SEM (n…

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  Figure 9. Cofilin and VASP. (A) Aortic P-cofilin and cofilin levels were detected by Western blotting (n = 6–10, as indicated in the dot plots). (B) P-VASP and total VASP levels were assessed by Western blot in aortic samples (n = 6–10, as indicated in the dot plots). These Western blots were performed with 6–10 biological replicates in 3 (cofilin) or 4 (VASP) blots for each probe. Measurements of all samples were reflected in the corresponding Western blot analysis panel. Molecular weight markers are as indicated in kilodaltons. All data are presented as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons were used for data analysis. *P < 0.05 vs. ISM-Cre, vehicle. #P < 0.05 vs. ISM-Cre, Ang II. NS, not significantly different from vehicle-treated samples.