SMAD3 contributes to ascending aortic dilatation independent of transforming growth factor-beta in bicuspid and unicuspid aortic valve disease

Figures

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  Figure 1. Transforming growth factor-beta (TGFß) ligand concentration correlates to ascending aortic diameter in tricuspid (TAV), but not bicuspid (BAV) or unicuspid (UAV) aortic valve patients. (A) Graph depicting the TGFß1 ligand concentration in non-dilated (ND) aortas from individuals with a TAV, BAV or UAV. (B) Fluorescent micrographs (top; blue = DAPI, red = TGFß1) and corresponding graph (bottom) depicting the TGFß1 concentration in the aortic media from individuals with ND aortas and either a TAV, BAV or UAV. (C) Graphs depicting the relationship between TGFß ligand concentration (whole aorta: top, media: bottom) and maximal ascending aortic diameter in TAV (blue) and BAV/UAV (pink) aortas. (D) Graphs depicting the TGFß1 ligand concentration (whole aorta: top, media: bottom) in ND and D aortas from individuals with a TAV, BAV or UAV. *P < 0.05. IHC = immunohistochemistry. Scalebar = 20 µm.

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  Figure 2. SMAD2 phosphorylation is increased in non-dilated bicuspid (BAV) and unicuspid (UAV) aortic valve aortas, and increases with dilatation in tricuspid aortic valve (TAV) aortas. (A) Fluorescent micrographs of ascending aortic cross-sections immunolabeled for phosphorylated SMAD2 (green points inside nuclei, indicated by white arrows) and counter-stained with DAPI (red). (B) Graph depicting the percentage of phosphorylated SMAD2 (pSMAD2)-positive smooth muscle cells in non-dilated (ND) aortas from individuals with a TAV, BAV or UAV. (C) Graph depicting the relationship between pSMAD2 and maximal ascending aortic diameter in TAV (blue) and BAV/UAV (pink) aortas. (D) Graphs depicting the percentage of pSMAD2-positive SMCs in ND and D aortas from individuals with a TAV, BAV or UAV. *P < 0.05. Scalebar = 10 µm.

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  Figure 3. SMAD3 phosphorylation is increased in non-dilated bicuspid (BAV) and unicuspid (UAV) aortic valve aortas, and increases with dilatation in tricuspid aortic valve (TAV) and BAV/UAV aortas. (A) Fluorescent micrographs of ascending aortic cross-sections immunolabeled for phosphorylated SMAD3 (green points inside nuclei, indicated by white arrows) and counter-stained with DAPI (red). (B) Graph depicting the percentage of phosphorylated SMAD3 (pSMAD3)-positive smooth muscle cells in non-dilated (ND) aortas from individuals with a TAV, BAV or UAV. (C) Graph depicting the relationship between pSMAD3 and maximal ascending aortic diameter in TAV (blue) and BAV/UAV (pink) aortas. (D) Graphs depicting the percentage of pSMAD3-positive SMCs in ND and D aortas from individuals with a TAV, BAV or UAV. *P < 0.05. Scalebar = 10 µm.

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  Figure 4. Phosphorylation of SMAD2 and SMAD3 is dependent upon TGFß1 ligand concentration in TAV, but not BAV/UAV aortas. (A) Graphs depicting the relationship between TGFß1 ligand concentration (whole aorta: top, medial layer: bottom) and phosphorylated SMAD2 in TAV (blue) and BAV/UAV (pink) aortas. (B) Graphs depicting the relationship between TGFß1 ligand concentration (whole aorta: top, medial layer: bottom) and phosphorylated SMAD3 in TAV (blue) and BAV/UAV (pink) aortas. (C) Graph depicting the relationship between phosphorylated SMAD2 and phosphorylated SMAD3 in TAV (blue) and BAV/UAV (pink) aortas.

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  Figure 5. Elastin degradation is dependent upon transforming growth factor-beta ligand in tricuspid (TAV), but not bicuspid (BAV) or unicuspid (UAV) aortic aneurysms. (A) Photomicrographs of ascending aortic cross-sections labeled with Movat’s pentachrome, depicting elastin (black filaments) breaks and degradation in BAV aortas. Blue stain represents mucoid extracellular matrix deposition. (B) Graph depicting elastin breaks in non-dilated (ND) aortas from individuals with a TAV, BAV or UAV. (C) Graph depicting the relationship between elastin breaks and maximal ascending aortic diameter in TAV (blue) and BAV/UAV (pink) aortas. (D) Graphs depicting the relationships between TGFß1 ligand concentration (whole aorta: left, medial layer: right) and elastin breaks in TAV (blue) and BAV/UAV (pink) aortas. (E) Graphs depicting the TGFß1 ligand concentration in ascending aortas with mild elastin loss (EL) and severe EL for TAV (left) and BAV/UAV (right) aortas. *P < 0.05. Scalebar = 50 µm. Fluores. = fluorescence.

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  Figure 6. Aortic remodeling is dependent upon phosphorylation of SMAD3, but not SMAD2 in bicuspid (BAV) and unicuspid (UAV) aortic valve-associated ascending aortic aneurysms. (A) Graph depicting the relationship between elastin breaks and phosphorylated SMAD2 (pSMAD2) in TAV (blue) and BAV/UAV (pink) aortas. (B) Graph depicting the relationship between elastin breaks and phosphorylated SMAD3 (pSMAD3) in TAV (blue) and BAV/UAV (pink) aortas. (C–E) Graphs depicting the level of pSMAD2 (dark grey) and pSMAD3 (light grey) in ascending aortas with mild or severe elastin loss (EL; C), fibrosis (D) and mucoid extracellular matrix accumulation (MEMA; E) in TAV (left) and BAV/UAV (right) aortas. *P < 0.05.