Proteomic Architecture of Valvular Extracellular Matrix

Figures

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  Figure 1. Illustration of ECM Pipeline Extraction From Human Aortic Valve Tissue (A) Macroscopic analysis of calcified valve tissue. (B) Dissection of fibrotic stage tissue. (C) Dissection of calcified stage tissue. (D) Diagram to compare proteome between noncalcified, low-calcified, and highly calcified tissue from the same aortic valve. (E) ECM extraction process. (F) Alizarin Red staining of noncalcified valve versus (G) staining of calcified valve with calcium nodes highlighted with yellow rectangles the corresponding dispersive x-ray energy graphs showing the calcium peak (circled) are below each section. Proteins were isolated from the calcified (yellow rectangle) and noncalcified (green) stages of the same valve tissue. Masson trichrome staining of (H) a calcified valve and (I) a control noncalcified valve. The high-calcium samples were taken from the area indicated by the black highlighted box (I) and the low-calcium samples from the area indicated by the red highlighted box. ECM = extracellular matrix; LC-MS/MS = liquid chromatography tandem mass spectrometry.

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  Figure 2. Representation of the Top 20 Significant Proteins in the 3 Disease Stages Core matrisome proteins are highlighted in red. (A) Significant ECM proteins between highly calcified (HIGH) and control valve (VAHN) tissues. (B) Significant ECM proteins between low-calcified (LOW) and control valve tissues. (C) Significant ECM proteins between high- and low-calcified tissues from the same valve are labeled with asterisks. (D) Venn diagram showing the comparison of the proteomes in the 3 different stages. ECM = extracellular matrix; VAHN = normal human aortic valve.

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  Figure 3. Heat Map Representation of ECM Proteins in the 3 Different Stages (A) Comparing highly calcified tissue (VAHC-HIGH) to control valve (VAHN) tissue. (B) Low calcified tissue (VAHC-LOW) compared with VAHN. (C) Low calcified stage compared with high calcified stage tissue in the same valve. Abbreviations as in Figure 2.

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  Figure 4. Network Analysis Surrounding ECM Proteins (A) Network of significant proteins when comparing high calcified to control valve tissues (VAHN). (B) The network between low-calcified tissue and control valves. Orange circle outline = core matrisome protein, yellow circle outline = matrisome-associated protein, green dot = up-regulated, red dot = down-regulated. Abbreviations as in Figure 2.

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  Figure 5. IF Staining of Selected Proteins Immunofluorescence (IF) staining (left) and quantification (right) show the up-regulation of osteopontin, versican, and fibronectin, and down-regulation of TUBA4 in calcified (VAHC) versus noncalcified (VAHN) valves (∗p < 0.05, ∗∗p < 0.01).

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  Figure 6. Characterization of Human Aortic Valve Cells (A) Isolated human valve cells are positive for vimentin and αSMA as shown with both immunofluorescence and Western blotting. Boxed areas (left) are shown at higher magnification on the right. (B) Treatment of cells with phosphate-based calcifying medium enhanced the expression of α-SMA. (C) Diagram showing the process followed to screen extracellular matrix expression during valve calcification. CTL = control.

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  Figure 7. Human Valve Cell Calcification In Vitro (A) Human valve interstitial cells (hVICs) were treated with phosphate-based calcifying medium (CM) for 7 days. Calcium nodes were stained with Alizarin Red and observed with a polarized light microscope. The dashed-line boxes indicate the regions shown at higher magnification in the solid-line boxes. (B) The quantification of calcium was performed with Arsenazo III comparing control (CTL) with cells treated with CM. Scanning electron microscopy analyses shows (C) noncalcified cells and (D) calcified nodes in phosphate-treated cells. Boxed areas (left) are shown at higher magnification on the right. Calcium nodes are highlighted with an artificial green color. Calcium nodes were revealed with energy dispersive x-ray to show their hydroxyapatite nature. Calcium nodes were revealed with energy dispersive x-ray, in black eclipse, to show their hydroxyapatite nature (C, D). ∗∗∗p < 0.001.

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  Figure 8. ECM Expression During Valve Cell Calcification In Vitro (A) Venn diagram showing cellular and secreted proteins during calcification. (B) Top 20 secreted proteins expressed, with comparison of their expression at 0, 3, and 7 days of calcifying medium treatment. ECM proteins are labeled in red. (C) Venn diagram showing cellular protein expression at 0, 3, and 7 days. ECM = extracellular matrix.