Transcriptomic Signatures of Calcific Aortic Valve Stenosis Severity in Human Tricuspid and Bicuspid Aortic Valves

Figures

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  Figure 1. UMAP Plot Depicting Samples Relationships Based on Gene Expression (A) Uniform Manifold Approximation and Projection (UMAP) according to CAVS severity level. (B) UMAP according to aortic valve fibrocalcific remodeling score.

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  Figure 2. Differential Gene Expression Analysis for Calcific Aortic Valve Stenosis (A) Volcano plot of the differentially expressed genes (DEGs). 2,951 genes were differentially expressed between very severe or severe calcific aortic valve stenosis (n = 348) and mild or no calcific aortic valve stenosis (n = 86). 1,254 genes were down-regulated (blue) and 1,697 genes were up-regulated (red). The top ten most differentially expressed genes are labeled. (B) Heatmap of the top 100 most differentially expressed genes. AVR = aortic valve replacement; BAV = bicuspid aortic valve; TAV = tricuspid aortic valve.

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  Figure 3. Gene Modules Derived From the Expression Gradient Analysis (A) Cluster dendrogram of 266 genes exhibiting a gradient of expression with a fold-change threshold of 1.5. Colors represent coexpression modules. (B) Enriched terms for the coexpressed modules derived from weighted correlation network analysis. The gene ratio is the number of genes in the set associated with an ontology term divided by the total number of genes in the set. P values are calculated based on the cumulative hypergeometric distribution. (C) Network nodes represent proteins, and edges represent protein-protein associations (ie, proteins jointly contribute to a shared function without necessarily binding to each other). (D) Gene expression profile grouped by disease severity levels for 3 central genes among the 266 exhibiting an expression gradient. Box plots represent median with variance. Lower and upper hinges represent the 25th and 75th percentile, respectively. Whiskers represent the variability outside the upper and lower quartiles and bold dots represent outliers.

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  Figure 4. WGCNA of the Total Genes in the Data Set (A) Cluster dendrogram of the 25,390 genes included in the data set. Colors represent coexpression modules. (B) Enriched terms for the coexpressed modules. The gene ratio is the number of genes in the set associated with an ontology term divided by the total number of genes in the set. P values are calculated based on the cumulative hypergeometric distribution.

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  Figure 5. Differential Gene Expression Analysis in Calcified vs Noncalcified Tissues (A) Volcano plot of the DEGs showing up-regulated genes in red and down-regulated genes in blue. The top 10 most significantly differentially expressed genes are labeled. (B) Venn diagram showing common genes between calcification and gradient analyses. (C) Enriched terms for the differentially expressed genes according to calcification level. (D) Enriched terms for genes common to calcification and gradient analyses. The gene ratio is the number of genes in the set associated with an ontology term divided by the total number of genes in the set. P values are calculated based on the cumulative hypergeometric distribution.

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  Figure 6. Differences in Expression Profile Between Bicuspid Aortic Valve and Tricuspid Aortic Valve Tissues (A) Volcano plot of the differentially expressed genes between bicuspid aortic valve and tricuspid aortic valve tissues with severe forms of calcific aortic valve stenosis, up-regulated genes are in red and down-regulated genes are in blue. (B) Enriched terms for the 17 genes associated with disease severity only in bicuspid aortic valve. The gene ratio is the number of genes in the set associated with an ontology term divided by the total number of genes in the set. P values are calculated based on the cumulative hypergeometric distribution. (C) Gene expression profile in bicuspid aortic valve samples grouped by disease severity levels for 3 genes among the 17 genes associated with disease severity only in bicuspid aortic valve. Box plots represent median with variance. Lower and upper hinges represent the 25th and 75th percentile, respectively. Whiskers represent the variability outside the upper and lower quartiles and bold dots represent outliers.

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  Figure 7. Differences in Expression Profile in Individuals With Early-Onset Calcific Aortic Valve Stenosis and a Tricuspid Valve (A) Volcano plot of the differentially expressed genes between tricuspid aortic valve samples age 65 years or younger and those older than 65 years with severe forms of calcific aortic valve stenosis. Up-regulated genes are in red and down-regulated genes are in blue. (B) Protein-protein interaction network of the most dysregulated genes (fold change >2.8). Network nodes represent proteins, and edges represent protein-protein associations (ie, proteins jointly contribute to a shared function without necessarily binding to each other). (C) Heatmap of the top 100 most significantly differentially expressed genes. (D) Gene expression profile of tricuspid aortic valve samples grouped by disease severity levels for 4 genes among the 240 differentially expressed genes. Very severe or severe (VSS) samples are divided into early-onset cluster, age 65 years or younger, and age older than 65 years. Box plots represent median with variance. Lower and upper hinges represent the 25th and 75th percentiles, respectively. Whiskers represent the variability outside the upper and…

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  Figure 8. Immune Cell Type Proportions According to Calcific Aortic Valve Stenosis Severity and Valve Morphology (A) Immune cell type proportions according to calcific aortic valve stenosis severity. (B) Immune cell type proportions according to valve morphology in tissues with severe forms of calcific aortic valve stenosis. Proportions were derived from deconvolution of transcript counts using CIBERSORTx and the LM22 data set, which is a validated leukocyte gene signature matrix. Box plots represent median with variance. Lower and upper hinges represent the 25th and 75th percentiles, respectively. Whiskers represent the variability outside the upper and lower quartiles and bold dots represent outliers. P values were obtained from a t-test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. NS = not significant.