MRTF-A promotes angiotensin II-induced inflammatory response and aortic dissection in mice

Figures

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  Fig 1. AD model by AngII administration. (A) Representative images are shown before (Control) and 4 days after starting AngII administration. Bar 1 mm. (B) Systolic blood pressure during the observational period. (C) Representative histological images with elastica van Gieson (EVG) and hematoxylin & eosin (H&E) staining 4 days after starting AngII administration. Bar 0.2 mm. (D) Evans blue-perfused aortae from WT mice 4 days after AngII infusion. Black arrow (left image): hematoma. Green line (upper and lower right images)s: area of ascending aorta. Red line (upper right image): area of hematoma. Arrowheads: Evans blue-stained tears in bright field images and red fluorescence in a dark field image.

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  Fig 2. Protein expression and role of MRTF-A in AD model. (A) Ascending aortic sections were stained for MRTF-A, SMA and DAPI in WT mice 4 days after starting AngII administration. Bar 0.1 mm. (B) Immunofluorescence staining for MRTF-A and SMA with nuclear DAPI staining before, 1 day and 4 days after starting AngII infusion in WT ascending aorta. Bar 0.1 mm.(C) Immunoblot analysis for MRTF-A in the aortic tissue before, 1 day and 4 days after starting AngII infusion in WT ascending aorta. Representative immunoblot images and quantitative analysis are shown. Gapdh served as an internal loading control. (D) Representative images of aortas from WT and MRTF-A-KO mice AngII administered for 4 days. (E) Area ratio of hematomas and tears in the ascending aorta from WT or MRTF-A-KO mice. Red and blue bars indicate the medians and interquartile ranges, respectively. The numbers in parenthesis indicate the number of biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001.

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  Fig 3. Transcriptome analysis of ascending aorta. (A) The result of hierarchical clustering analysis is shown for AngII- and MRTF-A-regulated genes by the heat map. The samples are from WT or MRTF-A-KO mice with or without AngII infusion for 1 day. The numbers in parenthesis indicate the number of biological replicates. Each gene is color-coded by red and green colors for the induction and the suppression, respectively, from the median. Comparison 1 (C1); without (Control) and with AngII in WT, Comparison 2 (C2); Control and AngII in MRTF-A-KO, Comparison 3 (C3); WT and MRTF-A-KO in Control, Comparison 4 (C4); WT and MRTF-A-KO in AngII. Comparison 2 was not used for further analysis. (B, C) Diagrams indicate number of genes with significant expression changes in each comparison as indicated in panel A. The numbers of upregulated (up) and downregulated (down) genes are shown.

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  Fig 4. Inflammatory response in mouse AD model. (A) mRNA expressions were examined by qRT-PCR for Il6, Tnf and Ccl2, and normalized by Actb. The samples were obtained from WT or MRTF-A-KO mice with or without AngII administration for 1 day. (B, C) Protein expressions were examined by western blotting for pStat3, Stat3 and Gapdh. Representative images (B) and quantitative analysis (C) of western blotting are shown. The samples were ascending aortae from WT or MRTF-A-KO mice with or without AngII administration for 1 day. The numbers in parenthesis indicate the number of biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, N.S. not significant. (D) Macroscopic and histological images are shown with EVG staining and Cd45 immunohistochemical staining along with the isotype control. Dotted line in the macroscopic image demarcates the aortic tissue. Aortic samples were obtained at day 4 of AngII infusion. Black and white arrowheads indicate the intimal tear and Cd45-positive staining, respectively. Bar 0.1 mm.

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  Fig 5. Apoptosis in mouse AD model. (A) Representative histological images are shown with TUNEL and EVG staining. The samples are from WT or MRTF-A-KO mice before (Control) and 1–4 days after starting AngII administration. White arrowheads indicate TUNEL-positive cells. Black arrowheads indicate the tear. (B) Immunofluorescence staining for Cd31, SMA, TUNEL and DAPI on ascending aortae are shown from WT mice 1 day after AngII administration. White arrowheads indicate TUNEL-positive nuclei. Bars 0.1 mm.

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  Fig 6. Myc and apoptosis in mouse AD tissue. (A, B) Protein expressions are shown by western blotting for Myc and Gapdh. Representative images (A) and quantitative analysis (B) are shown using ascending aortae from WT or MRTF-A-KO mice with or without AngII administration for 1 day. The numbers in parenthesis indicate the number of biological replicates. * P < 0.05, *** P < 0.001 compared with WT control. (C) Fluorescence staining for Myc, TUNEL, and DAPI on ascending aorta are shown from WT mice 1 day after AngII administration. (D) Fluorescence stainings are shown for Il6 and TUNEL in mouse aorta 1 day after starting AngII administration. Staining with rabbit IgG isotype control (IC) is also shown. White rectangles correspond to the magnified images at the bottom of the panels. Bar 0.1 mm.

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  Fig 7. Pharmacological inhibitor of MRTF-A suppressed aortic dissection. Hematomas and tears in the ascending aorta from WT mice with vehicle (DMSO) or CCG-203971 treatment 4 days after starting AngII infusion. Representative images (A) and quantitative analysis for the hematoma and the tear (B) are shown. Bar 1 mm. Red and blue bars indicate the medians and interquartile ranges, respectively. (C) mRNA expressions were examined by qRT-PCR for Il6, Tnf and Ccl2, and normalized by Actb. The samples were obtained from WT with AngII administration for 1 day with or without CCG-203971 treatment. The numbers in parenthesis indicate the number of biological replicates. * P < 0.05, *** P < 0.001. N.S. not significant. (D) TUNEL and DAPI staining of aortic samples with AngII administration for 1 day with or without CCG-203971 treatment. Bar 0.1 mm.