High Salt Intake Worsens Aortic Dissection in Mice

Figures

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  Figure 1.. Mouse model of aortic dissection (AD). The mouse AD model was created by continuous infusion of β-aminopropionitrile (BAPN) and Ang II (angiotensin II) for 14 d. A, Representative macroscopic and histological images from hematoxylin-eosin (HE) and Elastica van Gieson (EVG) staining taken before (normal) and after BAPN+Ang II (B+A) treatment. Scale bar=500 μm. B, The incidence of AD for the indicated doses of B+A (1000 ng/kg per min). C, The incidence of AD by 150 mg/kg per d BAPN and 1000 ng/kg per min Ang II. The numbers of mice in each group are shown in parentheses. D, Volcano plots comparing the control (Cont; without any challenge) and B+A AD models, and human aortas with a normal tricuspid aortic valve (TAV) and congenitally abnormal bicuspid aortic valve (BAV). The blue-shaded area and the red-shaded area indicate the genes with significant (P<0.05) suppression (fold change, <0.5) and induction (fold change, >2), respectively.

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  Figure 2.. Effect of high-salt intake on an aortic dissection (AD) model in WT (wild type) or IL-17KO (IL [interleukin]-17A knockout) mice. A, Representative macroscopic images for the WT and IL-17KO mice given normal drinking water or high-salt water (1% NaCl, high-salt challenge). B, The AD lesion lengths (mm). Data are from 16 mice in each experimental group. C, The plasma concentration of IL-17A (pg/mL) and Il17a mRNA expression levels in the AD model. D, The mRNA levels of Il17ra, Il17rc, Rorc, and Tcrg determined by quantitative reverse transcription polymerase chain reaction before and 1 d after starting the high-salt challenge and β-aminopropionitrile+angiotensin II (B+A) treatment. The mRNA expression levels were normalized for Actab and are shown relative to the control without any experimental interventions. The number of mice in each group is shown in parentheses. The means (red lines)±SEs (blue lines) are shown. *P<0.05, **P<0.01, ***P<0.001. N.S. indicates not significant.

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  Figure 3.. Effect of high-salt challenge on NFκB (nuclear factor-kappa B) and the transcriptome. A, Representative immunoblots and quantitative analyses for activated (phosphorylated) NFκB (P-NFκB), total NFκB, and β-actin, which served as the internal loading control. WT (wild type) and IL-17KO (IL [interleukin]-17A knockout) mice were given normal (normal salt) or 1% NaCl (high salt). An identical sample (C) was loaded on both electrophoresis gels, for WT samples and IL-17KO samples, so that the relative expression levels could be determined. The means (red lines)±SEs (blue lines) are shown. B, Volcano plots comparing the normal and high-salt conditions in WT mice without (WT Cont) or with β-aminopropionitrile (BAPN)+Ang II (angiotensin II; WT B+A) and IL-17KO mice without (IL-17KO Cont) or with BAPN+Ang II (IL-17KO B+A). The blue-shaded area and the red-shaded area indicate the genes with significant (P<0.05) suppression (fold change, <0.5) and induction (fold change, >2), respectively.

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  Figure 4.. Expression of ECM (extracellular matrix) genes in the thoracic aorta. A, A volcano plot of the effect of Il17a deletion (IL-17KO [IL (interleukin)-17A knockout]) comparing WT (wild type) and IL-17KO mouse aorta without any challenge. The blue-shaded area and the red-shaded area indicate the genes with significant (P<0.05) suppression (fold change, <0.5) and induction (fold change, >2), respectively. B, Heat map of the hierarchical clustering analysis for genes with the annotation of extracellular matrix. After log conversion of the signal value, the distance from the median is shown in green, black, and red when lower, intermediate, and higher than those of other samples, respectively, within a given gene. The samples are from WT or IL-17KO mice treated with (H) or without (N) high-salt challenge and with or without β-aminopropionitrile+angiotensin II (B+A) challenge. None of the samples had aortic dissection.

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  Figure 5.. Histological and biomechanical analyses of aortic walls. A–D, Aortic samples were stained for collagen fibers by Picrosirius Red and observed by bright-field illumination (A and B) or polarized light illumination (C and D). Representative photomicrographs are shown with the luminal side of the aortic section facing up (A and C). Quantitative analysis of Picrosirius Red staining is shown for bright-field (B) and polarized light illumination (D) as the mean (red lines)±SE (blue lines). *P<0.05, ***P<0.001. E and F, Elastic lamellae were analyzed by Elastica van Gieson (EVG) staining (E) and their area shown relative to the medial area (F). Scale bars=50 μm. G and H, Biomechanical analysis of the excised aortic rings. G, Force-displacement curves are shown for the descending aorta from WT (wild type; black lines) and IL-17KO (IL [interleukin]-17A knockout) mice (red lines). The average force-displacement curve corresponds to the dotted rectangle in the plot of the individual traces. H, The force/displacement ratios, as an indicator of aortic wall stiffness, and the peak force (mN) for the breakage of aortas are shown. Data represent the mean (red lines)±SE (blue lines) of 21…

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  Figure 6.. TGFβ (transforming growth factor-beta) signaling in the aortic walls. A, Representative immunoblots for total and activated (phosphorylated) Smad2 and Smad3, total Smad4, and Smad7, as well as β-actin, which served as the internal loading control. WT (wild type) and IL-17KO (IL [interleukin]-17A knockout) mice were given normal (normal salt) or 1% NaCl (high salt). C, An identical sample was loaded on both electrophoresis gels, for WT samples and IL-17KO samples, so the relative expression levels could be determined. B, Quantitative analyses for P-Smad2, total Smad2, P-Smad3, total Smad3, Smad4, and Smad7. *P<0.05, ***P<0.001. Data are shown for individual samples with the mean (red lines)±SE (blue lines) of 8 independent observations for each experimental group. C–E, TGFβ signaling at the single-cell level. Representative immunofluorescence images for 5-μm paraffin-embedded sections (C), the results of imaging cytometry as scattergrams (D), and histograms of SMA (smooth muscle α-actin) and P-Smad2 (E) are shown for sections of proximal descending aorta from WT or IL-17KO mice and for the negative control samples (NegCont) in which primary antibodies were omitted. Eight mice were…

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  Figure 7.. Cross talk between the IL (interleukin)-17 and TGFβ (transforming growth factor-beta) pathways in aortic smooth muscle cells (SMCs) in culture. A, Representative immunoblots for the time course of activated (phosphorylated) Jnk (P-Jnk) after IL-17A stimulation in aortic SMCs in culture. B, Quantitative analysis of P-Jnk. *P<0.05. C, Representative immunoblots for activated (phosphorylated) Smad2 (P-Smad2), P-Smad3, total Smad2, and total Smad3 with and without IL-17 pretreatment and TGFβ1 stimulation. GAPDH served as an internal loading control (Cont). D, Quantitative analyses of the immunoblots in C are shown as the mean (red lines)±SE (blue lines). ***P<0.001.

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  Figure 8.. Smooth muscle cell (SMC) differentiation status in the aorta. Aortic samples from WT (wild type) and IL-17KO (IL [interleukin]-17A knockout) mice given normal (N) or high (H) salt and β-aminopropionitrile+angiotensin II (B+A) were immunoblotted for SMC differentiation markers, synthetic phenotype markers SMemb and vimentin, and contractile phenotype markers SM2 and calponin-1. β-actin served as the internal loading control. A, Representative images and (B) the quantitative analysis are shown. The means (red lines)±SEs (blue lines) are shown for 8 independent experiments for each group. *P<0.05, **P<0.01. N.S. indicates not significant.

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  Figure 9.. Proinflammatory signaling in the aorta. A, Representative images from immunoblotting for P-Stat3 and P-Jnk. B, Quantitative analysis. β-actin served as an internal loading control. WT (wild type) and IL-17KO (IL [interleukin]-17A knockout) mice were treated with (H) and without (N) high salt and β-aminopropionitrile+angiotensin II (B+A). The means (red lines)±SEs (blue lines) are presented for 8 independent experiments in each group. *P<0.05, **P<0.01. N.S. indicates not significant.