Temporal Progression of Aortic Valve Pathogenesis in a Mouse Model of Osteogenesis Imperfecta

Figures

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  Figure 1. Aortic valve cusps from oim/oim mice thicken over time. Movat’s Pentachrome stain of aortic valve (AoV) regions from wildtype (WT) (A,C,E) and oim/oim (B,D,F) mice at 3 (A,B) (n = 3 for each genotype), 6 (C,D) (n = 3 for each genotype), and 12 (E,F) (n = 4 WT, n = 5 oim/oim) months. Arrows indicate AoV cusps. Elastic fibers (black); collagen fibers (yellow); mucin/proteoglycan (bright blue); fibrin (bright red); and muscle (red). (G) Quantitation of AoV cusp area based on 2D tissue sections as indicated. (H) The number of DAPI-positive nuclei normalized to AoV cusp area for each genotype and time point. Individual data points are plotted and represent biological replicates. * p < 0.05 ** p < 0.01, **** p < 0.0001 based on non-parametric 1-way ANOVA analysis. Note increased AoV cusp area in oim/oim mice at 12 months of age compared to 3 months.

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  Figure 2. Cardiac function of oim/oim mice deteriorates by 12 months of age. (A,B) Movat’s pentachrome staining of whole heart sections from 12 month old wildtype (WT) (A) and oim/oim (B) mice. N = 9 for WT, n = 10 for oim/oim. Echocardiography findings of WT and oim/oim mice at 12 months of age including ejection fraction (C), stroke volume (D), fractional shortening (E), aortic valve (AoV) mean gradient (F) and AoV mean velocity (G). Individual data plots from each mouse are shown. * p < 0.05 based on non-parametric t-test analysis. (H,I) Representation of color doppler imaging from WT and oim/oim mice. Note retrograde blood flow in oim/oim mice (arrow and arrowhead) indicative of insufficiency observed in 5/10 oim/oim mice and 0/10 WT mice. Ao, aorta. LV, left ventricle.

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  Figure 3. The ECM proteome is altered in oim/oim mice at 3 months of age. The ECM proteome is altered in oim/oim mice at 3 months of age. Individual AoV ring isolates (containing annulus ring and cusps) from n = 3 wildtype and oim/oim mice at 3 months of age were decellularized, enriched for the ECM, and subjected to liquid chromatography tandem mass spectrometry analysis. (A) Principal component analysis of all decellularized samples largely showed proteomic homology amongst genotypes, with biological variance observed for one biological replicate in each cohort. (B) Significantly altered proteins in oim/oim mice compared to wildtype controls (abundance ratio (sample/control) log2 fold change > 1 and p-value < 0.05. (C) Significantly altered proteins from (B) filtered for an association with the “ECM” based on gene ontology classification terms. (D) Volcano plot of differentially expressed proteins in AoVs from 3-month-old oim/oim mice versus wildtype (green, downregulated; red, upregulated). Fmod, fibromodulin; Itih1, inter-alpha-trypsin inhibitor heavy chain H2; Mgp, matrix gla protein; Vcan, versican.

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  Figure 4. Gene ontology analysis of the altered ECM proteome in decellularized AoVs from oim/oim mice at 3 months of age. Gene ontology was performed using DAVID analysis on differentially expressed proteins as shown in Figure 3C (A–C) and those filtered for ECM association, as shown in Figure 3D (D–F). Outputs are reported as significantly affected GO classification terms related to biological processes (A,D), cellular compartments (B,E) and molecular functions (C,F). Dark yellow indicates p-value 0.05, light yellow indicates p-value < 0.01.

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  Figure 5. Proteoglycan abundance is altered in oim/oim mice from 3 months of age. Immunohistochemistry to detect versican (Vcan) (red, (A,B)), chondroitin sulfate proteoglycan (CSPG) (green, (D–I)), and fibromodulin (Fmod) (red, (K,L)) expression in aortic valve (AoV) regions from wildtype (WT) (A,D,F,H,K) and oim/oim (B,E,G,I,L) mice. Vcan and Fmod are shown at 3 months of age; CSPG at 3 (D,E), 6 (F,G), and 12 (H,I) months (n = 3 for each genotype and time point). DAPI (blue) indicate nuclei. (C,J,M) Corrected total cell fluorescence (CTCF) of Vcan (C) and CSPG (J) Fmod (M) in AoV regions normalized to area. * p < 0.05 based on non-parametric 1-way ANOVA analysis.

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  Figure 6. Collagen homeostasis is altered in aortic valve cusps during early stages of valve disease in 3 month old oim/oim mice. Immunocytochemistry to detect Col1a1 (red, (A,B)), Collagen Hybridization Peptide (CHP) (green) and Col6a5 (red, (K,L)) in aortic valve (AoV) regions from wildtype (WT) (A,D,F,H,K) and oim/oim (B,E,G,I,L) mice. Col1a1 and Col6a5 are shown at 3 months of age; CHP at 3 (D,E), 6 (F,G), and 12 (H,I) months. n = 3 for each genotype at each time point. Arrows in L indicate redistribution of CHP reactivity at the tip of the AoV cusp. DAPI (blue) indicate nuclei. (C,J,M) Corrected total cell fluorescence (CTCF) of Col1a1 (C), CHP (J), and Col6a5 (M) normalized to area. * p < 0.05 based on non-parametric 1-way ANOVA analysis.

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  Figure 7. Summary of AoV valvuopathy in oim/oim mice. The temporal progression of AoV disease in oim/oim mice is initiated by mutations in Col1a2, which is hypothesized to lead to alterations in biochemical signals that are required to maintain physiological ECM homeostasis. As a result, secondary changes in the ECM proteome occur during early stages (3 months) of disease in oim/oim mice that, over time, cause structural deterioration (from 6 months onwards) and significant dysfunction by late stages (12 months).