Multi-omics of in vitro aortic valve calcification

FIGURE 1. Comparison of proteomic and transcriptomic profiles of human valve interstitial cells (VIC) from patients with or without calcific aortic valve disease (CAVD) in standard cultivation (control) and in osteogenic medium (differentiated). (A) Venn diagram shows the number of unique gene products identified by shotgun proteomics and RNA-seq transcriptomics. (b-c) Partial least squares-discriminant analysis (PLS-DA) score plots based on transcriptomic (B) and proteomic (C) data. Yellow triangles—differentiated VIC from patients with CAVD, green crosses—differentiated VIC from healthy donors, gray pluses—control VIC from healthy donors, blue circles—control VIC from patients with CAVD.

FIGURE 2. Differentially expressed genes involved in osteogenic differentiation of human valve interstitial cells (VIC) from healthy and calcified valves were identified by transcriptomics and proteomics analysis at early and late time points respectively. (A) Volcano plot of RNA-seq analysis of control VIC and 48 h after induction of osteogenic differentiation. Log2 fold change—level of change in expression-Log10P—logarithm of P-value. Dotted lines cut off transcripts with P-value < 0.05 and Log2 fold change > |1|. (B) Volcano plot of proteomics analysis of control VIC and 10 days after induction of osteogenic differentiation Log2 fold change—level of change in expression-Log10P—logarithm of P-value. Dotted lines cut off transcripts with P-value < 0.05 and Log2 fold change > |1|. (C) Venn diagram representing overlapping of DEGs with Log2 fold change > |2| between control and differentiated VIC founded in proteomic and transcriptomic data. (D) Scatter plot of comparison of Log2Fold Change of genes identified in both proteomic and transcriptomic data. The dotted line separates the border of Log2 fold change > |1|. (E) Bar chart representing the number of DEGs with Log2 fold change > |2|…

FIGURE 3. Pathway enrichment analysis of upregulated genes involved in osteogenic differentiation of human aortic valve interstitial cells (VIC) by KEGG: biological processes database. Abscissa axis represents biological processes associated with VIC osteogenic differentiation in early timepoint by RNA-seq transcriptomics data (A) or later timepoint by shotgun proteomics data (B); ordinate axis represents number of identified differentially expressed transcripts (A) or proteins (B) enriched in corresponding biological process. Asterisk marked KEGG (*): biological processes enriched in both proteomics and transcriptomics data.

FIGURE 4. Pathway enrichment analysis of upregulated genes involved in osteogenic differentiation of human aortic valve interstitial cells (VIC) by KEGG: biological processes database. Abscissa axis represents biological processes associated with VIC osteogenic differentiation in early timepoint by RNA-seq transcriptomics data (A) or later timepoint by shotgun proteomics data (B); ordinate axis represents number of identified differentially expressed transcripts (A) or proteins (B) enriched in corresponding biological process. Asterisk marked KEGG (*): biological processes enriched in both proteomics and transcriptomics data.

FIGURE 5. Evaluation of the expression levels of the most responsive genes in comparison of undifferentiated and differentiated human valve interstitial cells (VIC) from healthy donors and patients with CAVD using qPCR.

FIGURE 6. Effect of ZBTB16 (PLZF) on human valve interstitial cells (VIC) osteogenic differentiation. (A) Results of Alizarine red stain of VIC cultured in standard medium (undif control), osteogenic medium (dif control), osteogenic medium with transduction by an empty vector (dif TRC), osteogenic medium with transduction by construction for ZBTB16 overexpression (dif ZBTB). (B) Results of qPCR of Zbtb16, Runx2, and Col1a in VIC cultured in standard medium (undif control), osteogenic medium (dif control), osteogenic medium with transduction by construction for ZBTB16 overexpression (dif ZBTB). *p-value < 0.05. (C) Clusterization by sparse partial least square discriminant analysis (sPLS-DA). (D) Volcano plot demonstrating results of analysis of differential expression by limma between VIC in osteogenic conditions and VIC in osteogenic conditions with ZBTB16 overexpression. Log2 fold change—level of change in expression -Log10P—logarithm of adjusted p–value. Dotted lines cut off transcripts with adjusted p-value < 0.05 and Log2 fold change > |1|.