Differential BMP Signaling Mediates the Interplay Between Genetics and Leaflet Numbers in Aortic Valve Calcification

Figure 1. DCBLD2 Is Down-Regulated in Human CAVD, and Dcbld2 Deletion Leads to Isolated BAV and Valvular Remodeling in a Subset of Mice (A) Immunofluorescent staining of DCBLD2 (red) and CD31 (green) in normal human aortic valve leaflets (upper panels) and aortic valve leaflets with advanced CAVD (lower panels). Nuclei are stained blue with DAPI. (B) Western blot analysis (upper panel) and quantification (lower panel) of DCBLD2 expression in normal human aortic valve leaflets and aortic valve leaflets with advanced CAVD. ∗∗∗P < 0.001 (2-tailed t-test). (C) Immunohistochemical staining of DCBLD2 in WT (upper panels) and Dcbld2−/−(lower panels) murine aortic valves. (D) Photographic images of 9- to 12-month-old WT (left), Dcbld2−/− TAV (middle), and Dcbld2−/− BAV (right) aortic valves. Individual leaflets are marked with asterisks. (E) Prevalence of TAV and BAV in Dcbld2−/− mice. (F-H) LV ejection fraction (F), LV mass (G), and LV outflow tract velocity (H) assessed by echocardiography in WT, Dcbld2−/− TAV, and Dcbld2−/− BAV mice. ∗P < 0.05 (Kruskal-Wallis test with Dunn’s multiple comparisons). (I) Hematoxylin and eosin staining of WT (left), Dcbld2−/− tricuspid (middle), and Dcbld2−/−…

Figure 2. Dcbld2 Deficiency Promotes Valvular Fibrosis, Calcification, and Stenosis (A and B) Immunofluorescent staining (A) and quantification (B) of collagen type I (COL1, red) in WT aortic valve (left), Dcbld2−/− TAV (middle), and Dcbld2−/− BAV (right) leaflets. ∗∗P < 0.01 (1-way analysis of variance with Tukey’s multiple comparisons). (C and D) Alizarin red staining (C) and quantification (D) of valvular calcification in WT (left), Dcbld2−/− TAV (middle), and Dcbld2−/− BAV (right) mice. ∗P < 0.05, ∗∗P < 0.01 (Kruskal-Wallis test with Dunn’s multiple comparisons). (E and F) Aortic valve leaflet separation (E) and peak transvalvular velocity (F) in WT, Dcbld2−/− TAV, and Dcbld2−/− BAV mice assessed by echocardiography. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (1-way analysis of variance with Tukey’s multiple comparisons for E, and Kruskal-Wallis test with Dunn's multiple comparisons for F). Abbreviations as in Figure 1.

Figure 3. DCBLD2 Regulates BMP2 Expression and Signaling in VICs (A and B) Western blot analysis (A) and quantification (B) of BMP2 expression in normal human aortic valve leaflets and aortic valve leaflets with advanced CAVD. ∗P < 0.05 (Mann-Whitney U test). (C) Immunofluorescent staining of BMP2 (red) and CD31 (green) in normal human aortic valve leaflets (upper panel) and aortic valve leaflets with advanced CAVD (lower panel). Nuclei are stained blue with DAPI. (D-F) RT-PCR analysis of Bmp2 mRNA (D), and Western blot analysis of s-BMP2 protein expression (E) and its quantification (F) in pVIC transfected with scrambled or DCBLD2 siRNAs. ∗P < 0.05 (paired Student's t-test). (G and H) Western blot analysis (G) and its quantification (H) of SMAD1/5/9 phosphorylation in pVIC treated with porcine BMP2 (50 ng/mL) for the indicated time points. ∗P < 0.05 (paired Student's t-test). (I and J) Representative images (I) and quantification (J) of Alizarin red staining of pVIC transfected with scrambled or DCBLD2 siRNAs, or DCBLD2 siRNA with recombinant human Noggin. n = 3. ∗P < 0.05, ∗∗P < 0.01 (1-way analysis of variance with Tukey’s multiple comparisons). The Figure is representative of 2…

Figure 4. Endothelial DCBLD2 Regulates Endothelial BMP2 Expression and VIC Calcification (A-C) RT-PCR analysis of Bmp2 mRNA (A), and Western blot analysis of c-BMP2 protein expression (B) and its quantification (C) in WT and Dcbld2−/− EC. ∗P < 0.05 (2-tailed t-test), ∗∗P < 0.01 (Mann-Whitney U test). (D-F) RT-PCR analysis of Bmp2 mRNA (D) and Western blot analysis of s-BMP2 protein (E) expression and its quantification (F) in porcine valvular endothelial cells transfected with scrambled or DCBLD2 siRNAs. ∗∗P < 0.01 (paired Student's t-test). (G) Quantification of calcified nodule formation in pVIC alone and pVIC co-cultured with WT or Dcbld2−/− EC. n = 3. ∗∗P < 0.01, ∗∗∗P < 0.001 (1-way analysis of variance with Tukey’s multiple comparisons). c-BMP2 = cytosolic BMP2; EC = endothelial cell; other abbreviations as in Figures 1 and 3.

Figure 5. Differential BMP2 Signaling Underlies the Difference in Valvular Calcification Between Dcbld2−/− BAV and TAV (A)Bmp2 mRNA expression in WT and Dcbld2−/− aortic valve tissues. ∗∗P < 0.01 (Mann-Whitney U test). (B and C) Examples of phosphorylated SMAD1/5/9 immunofluorescent staining in red (B) and quantification (C) in WT aortic valve, Dcbld2−/− TAV and Dcbld2−/− BAV leaflets. Nuclei are stained blue with DAPI. ∗∗∗P < 0.001 (1-way analysis of variance with Tukey’s multiple comparisons). (D) Osteocalcin mRNA expression in WT, Dcbld2−/− TAV, and Dcbld2−/− BAV. ∗P < 0.05, ∗∗P < 0.01 (Kruskal-Wallis test with Dunn's multiple comparisons). (E and F) Western blot analysis (E) and quantification (F) of phosphorylated SMAD1/5/9 in pVIC transfected with scrambled or DCBLD2 siRNA in the presence or absence of LDN-193189 (0.5 μmol/L). ∗P < 0.05, ∗∗∗P < 0.001 (1-way analysis of variance with Tukey’s multiple comparisons). (G and H) Representative examples of phosphorylated SMAD1/5/9 immunofluorescent staining in red (G) and quantification (H) in Dcbld2−/− BAV mice and Dcbld2−/− BAV mice treated with LDN-193189 (6 mg/kg). Nuclei are stained blue with DAPI. ∗∗∗P < 0.001 (2-tailed t-test). (I)…