Aortic Stenosis Hyalectan Remodeling Revealed by Proteomics and Glycoproteomics

Figures

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  Figure 1.. Proteomics of extracellular matrix (ECM) and ECM-associated proteins in human aortic valves (AVs). A, Representative images illustrating the analyzed sample groups. B, Immunoblots validate the efficiency of the ECM extraction protocol. Intracellular proteins (such as GAPDH and vimentin), as well as ECM cleavage products like the neoepitope NITEGE392 of aggrecan (generated by ADAMTS [a disintegrin and metalloproteinase with thrombospondin motifs]-specific cleavage), are detected in the NaCl fraction, while decorin, versican, and the aggrecan core protein are predominantly found in the GuHCl fraction. C, The circular heatmap shows hierarchical clustering of significantly altered ECM proteins in the soluble (left, NaCl extract) and core (right, GuHCl extract) matrisome fractions. The outer ring compares noncalcified calcific aortic valve disease (CAVD) samples (n=17) to controls (n=29), while the inner ring compares calcified to noncalcified CAVD samples (n=17). Red and blue indicate up- and downregulated protein changes relative to median levels, respectively (see color scale). For CAVD noncalcified vs control, statistical significance was calculated using the limma package with…

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  Figure 2.. Glycoproteomic analysis of calcific aortic valve disease (CAVD). A, The glycoproteomics workflow employed advanced mass spectrometry fragmentation techniques for glycopeptide analysis: HCD-alt-EThcD alternates between Higher-energy Collisional Dissociation (HCD) and Electron Transfer/Higher-energy Collisional Dissociation (EThcD). HCD provides detailed peptide sequencing; EThcD preserves glycosidic bonds, revealing glycan masses and glycosylation sites. B, Heatmap of significantly altered proteoglycan glycopeptides in the core matrisome. Glycopeptides from glycosites with >3 glycoforms were labeled. Detailed information on glycopeptides and glycoform information can be found in Tables S5 and S6. Significance was determined using the limma package (eBayes adjustment; P<0.05, Benjamini-Hochberg correction; fold changes [FC] >1.5) and Student paired t test for noncalcified vs calcified regions in CAVD (P<0.05, Benjamini-Hochberg correction; FC >1.5). C, Gene expression of versican (VCAN), aggrecan (ACAN), hyaluronan and proteoglycan link protein 1 (HAPLN1) in control, noncalcified, and calcified CAVD. Significance was calculated using the limma package for CAVD vs controls and…

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  Figure 3.. Versican (VCAN)-to-aggrecan (ACAN) transition during calcification development in human aortic valves (AVs). A, Scaled ACAN/VCAN ratios in control AVs, noncalcified and calcified calcific aortic valve disease (CAVD) regions, determined by proteomics analysis Statistical significance was calculated using the limma package (age and sex adjustment and Benjamini Hochberg correction) or paired Student t test for noncalcified vs calcified regions (**P<0.01; Benjamini-Hochberg correction). Data are shown as mean±SEM. B, Volcano plot showing a significant positive association between the ACAN/VCAN ratio and AV pressure gradient. Ratios were based on scaled protein abundances from calcified AV regions. Multivariable relative risk regression analysis was performed after adjustment for age and bicuspid valve covariates to examine the association between extracellular matrix protein changes and AV pressure gradients. C, Immunoperoxidase images showing staining for VCAN (noncleaved) and ACAN (noncleaved) in human control and calcified AVs. All sections were counterstained with hematoxylin to visualize nuclei.

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  Figure 4.. Valve interstitial cells (VIC) subpopulation marker analysis in human and mouse single-cell RNA-seq. A, Heatmap showing normalized protein abundance of selected cell markers. For calcific aortic valve disease (CAVD) noncalcified vs controls (*P<0.05) and CAVD calcified vs controls (#P<0.05) in the NaCl fraction, significance was calculated using the limma package for CAVD vs controls with Benjamini-Hochberg correction for P value. B, Immunoblot validation of selected proteins among control, CAVD noncalcified, and CAVD calcified groups. Corresponding densitometry quantification in the NaCl fraction can be found in Figure S4B. C, Gene expression analysis of human VICs cultured in control, TGFβ1, osteogenic, or chondrogenic media shows a strong increase of VCAN, particularly under TGFβ1 treatment with myofibroblast marker periostin (POSTN), and a strong induction of ACAN, particularly under chondrogenic conditions with chondrogenic/osteogenic biomarker SPP1. Statistical significance across different time points was assessed using 1-way ANOVA followed by Tukey post hoc test, *P<0.05, and **P<0.01. Data are shown as mean±SEM. D, Left: Uniform Manifold Approximation and Projection…

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  Figure 5.. Adamts5Δcat/ApoE−/− mice exhibit an aortic stenosis (AS) phenotype. A, Adamts5Δcat mice lack ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs) catalytic activity, hindering hyalectan cleavage. Reduced levels of ADAMTS-mediated aggrecan (NVTEGE373) and versican (DPEAAE441) fragments confirmed impaired hyalectan processing in their aortic valves (AVs). B, Representative images of dissected tricuspid/bicuspid AV from 12-week-old Adamts5Δcat and Adamts5Δcat/ApoE−/− mice; arrowheads indicate commissures between AV leaflets. The percentage of mice with different genotypes displaying bicuspid AVs can be found in Figure S6A. C, Representative aortic cusp separation distance and (D) mean blood velocity in 12-week-old mice and corresponding ultrasound images. Four-group data analysis can be found in Figure S6C and S6D. E, Percentage of 12-week-old and 52-week-old mice showing AS and aortic regurgitation (AR). F, Representative Oil Red O-stained images of aortic roots from 52-week-old mice. Their corresponding AV thickness and lipid deposition quantification can be found in Figure S6E. Adamts5Δcat/ApoE−/− abbreviated as TS5Δcat/ApoE−/−.

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  Figure 6.. Proteomics analysis of aortic valves (AVs) from Adamts5Δcat/ApoE−/− mice. A, Scatter plots highlighting common changes between 12-week male mice (ApoE−/−, n=8; Adamts5Δcat/ApoE−/−, n=8) and 52-week male mice (ApoE−/−, n=4; Adamts5Δcat/ApoE−/−, n=4). Red dots represent proteins with consistent change directions across both comparisons and significantly altered in at least one comparison. Each point represents an individual protein, and the solid blue line represents the linear regression fit. The gray shaded area indicates the 95% CI of the regression line. B, Immunoblots for PTX3 (pentraxin 3), HAPLN1 (hyaluronan and proteoglycan link protein 1) and the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs)-specific cleavage product of Vcan, identified by the versican DPEAAE441 neoepitope. Comparisons between Adamts5Δcat/ ApoE−/− (abbreviated as TS5Δcat/ApoE−/− in figures) vs ApoE−/− mice are shown. C, Gene expression levels of HA synthases (Has1, Has2) in ApoE−/−, and Adamts5Δcat/ApoE−/− mice at 12 weeks. Statistical significance was calculated using an unpaired Student t test. *P<0.05. Data are presented as means±SEM. D, Representative immunofluorescence images…