Fig 2: Identification and validation of differentially expressed genes involved in integrin signaling pathway in porcine aortic valve interstitial cells (pAVICs) exposed to nitric oxide (NO) donor or S-nitrosoglutathione (GSNO). (A) Heatmap represents Z-scored expression of 32 differentially expressed genes involved in the integrin signaling pathway in individual cells from control, NO donor, and GSNO treated pAVICs. (B) Diagram representing the protein product of differentially regulated integrin pathway genes, which were S-nitrosylated in valve interstitial cells (VICs). The circle inside represents S-nitrosylated proteins enriched after NO donor treatment. (C) Transcriptional expression of 11 representative genes from the integrin signaling pathway was estimated in control, NO donor, and GSNO-treated pAVICs by quantitative real time polymerase chain reaction (RT-PCR). (D) Immunofluorescence images represent the decreased expression of ITGA8 (red) with NO donor and GSNO when compared to untreated control pAVICs, co-stained with nuclear DAPI (blue). Scale bar: 20 µm. (E) The graph shows the quantification of fluorescence intensity of ITGA8 expression in control, NO donor, and GSNO treated pAVICs normalized to the number of cells. *Indicates p = 0.05.

Fig 3: Increased expression of ITGA8 and vinculin (VCL) in calcified mouse aortic valve. (A) Violin plots demonstrate the expression of ITGA8 and VCL in porcine aortic valve interstitial cells (pAVICs) and mouse P30 aortic valve cells. (B) Immunofluorescence staining of aortic valve sections demonstrates the increase of ITGA8 (Integrin α8; red) expression in calcific conditions (Usp9xfl/Y; Tie2-Cre+) compared to (Usp9xfl/Y; Tie2-Cre–) controls (n = 2) and co-stained with nuclear DAPI (blue). Arrows indicate the representative area of expression of ITGA8. Yellow boxes indicate the magnified area of the tissue sections shown right of each image. Scale bar: 100 μm. (C) Immunohistochemistry of aortic valve sections demonstrate increased expression of VCL in (Usp9xfl/Y; Tie2-Cre+) compared to (Usp9xfl/Y; Tie2-Cre–) controls (n = 2). Negative control with no primary antibody is shown in the first column. Boxes indicate the magnified area of tissue sections shown below each image. Scale bar: 100 μm.