Reduced Proteolytic Cleavage of von Willebrand Factor Leads to Aortic Valve Stenosis and Load-Dependent Ventricular Remodeling

Figures

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  Figure 1. Characterization of the Murine Models at Age 30 Weeks (A to D) Mean ± SEM plasma lipid concentrations including total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides. (E and F) Mean ± SEM systolic and diastolic awake blood pressure (BP) measurements. (G and I) Mean ± SEM signal intensity on contrast ultrasound molecular imaging signal from the aortic root for endothelial-associated vWF by microbubble targeting of the A1 binding domain, platelet adhesion by targeting GPIbα, and control microbubble contrast agent. P values are adjusted for multiple comparisons. Examples of contrast ultrasound molecular imaging of the aortic root from all 3 murine groups are illustrated in Supplemental Figure 1. ∗P < 0.05; ∗∗P < 0.001 (adjusted). HDL = high-density lipoprotein; LDL = low-density lipoprotein; vWF = von Willebrand factor; WSD = Western-style diet; WT = wild type.

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  Figure 2. Systemic Arterial Hemodynamics at Age 50 Weeks Mean ± SEM values are shown for (A) systolic BP, (B) diastolic BP, (C) aortic distensibility, and (D) arterial elastance. P values are adjusted for multiple comparisons. ∗P < 0.01; ∗∗P < 0.001 (adjusted). Abbreviations as in Figure 1.

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  Figure 3. Echocardiographic Data on Aortic Valve Hemodynamics at Age 50 Weeks (A) Examples of pulsed-wave Doppler at the aortic valve with angle-correction alignment and corresponding spectral Doppler measurements from each of the murine strains (note different velocity scales for spectral Doppler recordings). Graphs depict mean ± SEM) 2-dimenisonal ultrasound and Doppler-derived (B) peak aortic gradient, (C) mean aortic gradient, (D) aortic valve resistance, and (E) valvulo-arterial impedance (Zva). P values are adjusted for multiple comparisons.∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 (adjusted). Abbreviations as in Figure 1.

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  Figure 4. Aortic Valve Histology at Age 50 Weeks (A) Examples of aortic root histology at the level of the valve for wild-type mouse, LDLR−/− mouse on WSD, and 2 different LDLR−/−AD13−/− mice on WSD at age 50 weeks illustrating Masson’s trichrome at low and high magnification (top 2 rows), Mac-2 (galectin-3) immunofluorescence with positive staining in the green fluorescent range (third row), and picrosirius red staining visualized under polarized light (bottom row). Quantitative microscopy data (mean ± SEM) are provided for measurement of (B) plaque area, (C) average valve thickness, (D) valve leaflet area with positive Mac-2 staining, and (E) valve leaflet area with positive picrosirius staining. P values are adjusted for multiple comparisons. Alizarin red staining of the leaflets from LDLR−/−AD13−/− mice showing (F) dense focal calcific nodules in the body of the leaflet; and (G) low-intensity staining of individual VICs (low magnification image of the leaflets shown, with inset illustrating high magnification of the area denoted by the dashed lines). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 (adjusted). Abbreviations as in Figure 1.

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  Figure 5. Valve Endothelial Cell-Associated vWF Examples of aortic valve immunofluorescence for vWF and CD31 at the level of the aortic valve at age 50 weeks from (A) a wild-type mouse, (B) an LDLR−/− mouse on WSD, and (C) an LDLR−/−AD13−/− mouse on WSD. Sequential panels from left to right represent incremental increases in magnification from the regions outlined by the dashed lines. Counterstaining for nuclei was performed with 4′,6-diamidino-2-phenylindole (DAPI). Examples of single immunofluorescent staining for vWF that were used for quantitative assessment are illustrated in Supplemental Figure 2. Abbreviations as in Figure 1.

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  Figure 6. Platelet Adhesion Patterns at Age 50 Weeks (A to D) Examples of aortic valve dual immunofluorescence for vWF and platelet CD41 at age 50 weeks from (A) a wild-type mouse, (B) an LDLR−/− mouse on WSD, and (C and D) 2 separate LDLR−/−AD13−/− mice on WSD. Sequential panels from left to right represent incremental increases in magnification from the regions outlined by the dashed lines. Red arrow in C indicates evidence for a long vWF multimer on the valve surface with platelets at the distal end (yellow staining indicating dual CD41 and vWF staining). (E) Two examples of aortic valve dual immunofluorescence at high magnification for CD31 and platelet CD41 at age 50 weeks from LDLR−/−AD13−/− mice on WSD. Arrows indicate the evidence for platelet staining for particles that appear <1 μm in size. (F and G) Transmission electron microscopy showing evidence of extracellular microvesicles (arrows) directly attached to the valvular endothelial cells. The structure denoted by the arrowhead in G may represent either microvesicle with contents but is also consistent with intermediate secretory pods that have been described after Weibel-Palade body (WPB) cell membrane fusion. Graphs depict…

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  Figure 7. Valve Interstitial Cell Myofibroblastic and Osteogenic Transformation (A and B) Examples of aortic valve immunofluorescence for (A) α-smooth muscle actin (αSMA) and (B) matrix metalloproteinase (MMP)-9 from each murine strain. High-magnification images of the area defined by dashed lines are provided below each image. Additional examples of αSMA staining are shown in Supplemental Figure 3. Graphs depict mean ± SEM immunofluorescent-positive area MMP-9 expressed as (C) total area and (D) proportion of total leaflet area. P values are corrected for multiple comparisons. (D) In situ hybridization for osteopontin mRNA expression (red staining) illustrating positive staining for aortic plaque in both LDLR−/− mice on WSD and LDLR−/−AD13−/− mice on WSD, but leaflet staining (arrows) only in the latter. Examples of dual immunofluorescent staining for: (E) pSMAD2 and platelet CD41, and (F) β-catenin and platelet CD41 from w LDLR−/−AD13−/− mice on WSD, with high-magnification images shown to the right. Additional examples from all 3 strains are shown in Supplemental Figure 4. ∗P < 0.01; ∗∗P < 0.001 (adjusted). Abbreviations as in Figure 1.

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  Figure 8. Echocardiographic Data on LV function and Morphology at Age 50 Weeks Graphs depict mean ± SEM. (A) Left ventricular (LV) mass, (B) LV mass indexed to body mass, (C) left ventricular end-diastolic volume index (LVEDVi), (D) left ventricular ejection fraction (LVEF); (E) stroke volume, (F) stroke volume index (SVi), (H) single-plane longitudinal strain (GLS), (I) single-plane longitudinal strain rate (SR), (J) single-plane circumferential strain (GCS), (K) single-plane circumferential SR, and (L) cardiac work index. (G) Representative images of the LV in the parasternal long- and short-axis (top) used for many of the measurements, and of circumferential strain curves (bottom) for a 6-segment model in the short-axis. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 (adjusted).