A Functional Variant of SMAD4 Enhances Thoracic Aortic Aneurysm and Dissection Risk through Promoting Smooth Muscle Cell Apoptosis and Proteoglycan Degradation

Figures

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  Fig. 1. Representative MALDI-TOF MS spectra of rs12455792 and linkage disequilibrium (LD) analysis of the tagger SNPs in SMAD4 gene. (A) Genotypes of rs12455792 are determined by plotting peak intensity (y-axis) against mass (Da) (x-axis). (a) MALDI-TOF MS spectrum of a single peak at 8949.0 Da indicates homozygous genotype of CC. (b) MALDI-TOF MS spectrum of a single peak at 8932.5 Da indicates homozygous genotype of TT. (c) MALDI-TOF MS spectrum of 2 peaks at 8932.5 Da and 8949.0 Da represents heterozygous genotype of CT. (B) Pairwise LD among 5 tagger SNPs of SMAD4. The r2 value in each diamond indicates the pairwise correlation between SNPs. The color scale ranges from white to black reflects lower to higher r2 values.

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  Fig. 2. rs12455792 affected SMAD4 gene expression. (A) In silico prediction of rs12455792 impact on RNA folding structures. The structures corresponding to rs12455792C (a) or T allele (c). Mountain plots related to rs12455792C (b) or T allele (d). y-axis: minimum free energy or entropy. x-axis: sequence position. (B) Reporter plasmids construction. The schematic diagram of pGL3-basic vector (a) and pGL3-SMAD4-TRS reporter (b). (C) Representative graph of relative luciferase activity of cells transfected with pGL-3 basic vector or pGL3-SMAD4-TRS reporter. Six replicates for each group and the experiment was repeated at least three times. *P < 0.05. (D) Western blot analysis for SMAD4 expression in aorta tissues from patients with different genotypes (a) and corresponding quantitative analysis (b).

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  Fig. 3. SMAD4 inhibited Versican and Decorin degradation via ADAMTS-4. Western blot analysis and quantitative analysis for Versican (A) or Decorin (B) degradation products in aortic tissues of patients. *P < 0.05, ns: not significant. (C) Western blot analysis for ADAMTS-1/4 protein levels in SMAD4-silenced cells or not. (D) Versican degradation products detection in medium of cells with different treatment.

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  Fig. 4. SMAD4 inhibited the apoptosis of HASMCs. (A) Movat's pentachrome staining of normal aortic tissues or tissues from TAAD patients with different genotype. In each group, Scale bar = 50 μm (left)/100 μm (right). (B) Representative scatterplots of flow cytometry for apoptosis analysis. (a) FSC/SSC scatterplot for cells gating. Annexin V-FITC/PI scatterplots of parental (b), siRNA-NC (c), siRNA-SMAD4 (d) group. Quantitative analysis of SMCs (C) and fibers (D) (n = 5). *P < 0.05. (E) Percentages of early or late-stage apoptotic cells in different groups.*P < 0.05. All experiments were performed in triplicates. Data was presented as mean ± SD.

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  Fig. 5. Schematic diagram for potential roles of the SMAD4 variant in pathological progression of TAAD. Variant T allele of rs12455792 might inhibit SMAD4 expression by affecting the affinity between transcription factors and 5′UTR of this gene, resulting in MSCs apoptosis and Versican degradation, and further facilitate the progress of TAAD.