The Role of Wnt/β-Catenin Pathway Mediators in Aortic Valve Stenosis

Figures

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  FIGURE 1. Stenotic aortic valves have greater mRNA expression of Wnt-signaling mediators compared to healthy valves. Increased mRNA expression was found for Wnt-signaling mediators DVL. AVS, aortic valve stenosis. The data are represented as mean ± SEM. Significance level was set at P < 0.05. *P < 0.05, **P < 0.01 compared to control groups; n = 31 for AVS samples and n = 5 for normal samples. Student’s t-test was used for statistical analysis.

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  FIGURE 2. Stenotic aortic valves have greater immunostaining of Wnt/β-catenin mediators compared to healthy valves. Low (A) DVL2, (B) GSK-3β, (C) SFRP2, (D) β-catenin immunoreactivity observed in histologically normal leaflet (arrows indicate weak immunostaining in native cells). Prevalent immunostaining of (E) αSMA in histologically normal leaflets. Co-localization of (F) DVL2, (G) GSK-3β, (H) SFRP2 (I), β-catenin immunoreactivity with macrophages around areas of calcification (arrows), indicated by (J) CD68 staining. Co-localization of (K) DVL2, (L) GSK-3β, (M) SFRP2 (N), β-catenin immunoreactivity in valve interstitial cells in the thickened fibrosa (arrows), indicated by (O) αSMA staining. *Calcified foci; v, ventricular side of the leaflet. Arrows point to regions of interest.

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  FIGURE 3. Stenotic aortic valves have greater protein expression of Wnt-signaling mediators compared to healthy valves. (A) Representative Western blot of DVL2, GSK-3β, p-GSK-3β, and of β-catenin protein expression in AVS patients compared to healthy patients. Densitometry analysis of DVL2 (B), GSK-3β, p-GSK-3β (C), and of β-catenin (D) protein expression. The data are represented as mean ± SEM. Significance level was set at P < 0.05. *P < 0.05 compared to control groups; n = 6 for AVS samples and n = 3 for normal samples. Students t-test was used for statistical analysis. AVS, aortic valve stenosis.

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  FIGURE 4. Proteomic profiling of HAVICs treated with CHIR99021. (A) Representative images of CV staining in HAVICs treated with CHIR99021 in DMEM or OSM. (B) Quantification of CV staining. The data are represented as mean ± SEM. Significance level was set at P < 0.05. *P < 0.05, **P < 0.01 compared to the specified group; n = 6 HAVIC lines were used for each group. One-way ANOVA followed by Tukey’s post hoc multiple comparisons test was used for statistical analysis. (C) mRNA expression of Wnt ligands after treatment with CHIR99021. The data are represented as mean ± SEM. Significance level was set at P < 0.05. *P < 0.05, **P < 0.01 compared to OSM groups; n = 3 HAVIC lines were used for each group. Students t-test was used for statistical analysis. (D) Venn diagram representing the number of differentially expressed proteins in HAVICs treated with CHIR99021 for 3 weeks in OSM compared to cells incubated in OSM alone. Fischer’s exact t-test was used for statistical analysis. (E) Network graphic of the highly enriched gene ontology biological process organophosphate metabolic processes. HAVICs, human aortic valve interstitial cells; CV, crystal violet; OSM, osteogenic media.

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  FIGURE 5. Fetuin A attenuates calcification in HAVICs and is associated with inactivation of Wnt signaling genes. (A) Representative images of endogenous Fetuin A in HAVICs localized to the golgi apparatus (G), endoplasmic reticulum (ER), and vesicles (V). Arrows point to regions of interest. Scale bar: 20 μm. (B) Representative Alizarin Red S staining images of HAVICs treated with different concentrations of Fetuin A. (C) Quantification of calcium deposition using acid extraction following Alizarin Red S stain of HAVIC relative to Crystal Violet stain. The data are represented as mean ± SEM. Significance level was set at P < 0.05. *P < 0.05, **P < 0.01 compared to the specified group; n = 6 HAVIC lines were used for each group. One-way ANOVA followed by Tukey’s post hoc multiple comparisons test was used for statistical analysis. (D) mRNA expression of osteogenic and Wnt signaling genes after treatment with Fetuin A. The data are represented as mean ± SEM. Significance level was set at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control groups; n = 6 HAVIC lines were used for each group. Students t-test was used for statistical analysis. (E) Representative Western blot of…