Unveiling the Angiogenic Potential and Functional Decline of Valve Interstitial Cells During Calcific Aortic Valve Stenosis Progression

Figures

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  FIGURE 1. Isolation process of VICs from calcified (C‐VICs) and noncalcified (NC‐VICs) area of the aortic valve. Noncalcified and calcified areas shown are representative of the complete cohort.

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  FIGURE 2. Characterisation of NC‐VICs and C‐VICs. (A) Morphology. Phase‐contrast image of VICs isolated from NC‐VICs and C‐VICs in culture at different magnifications (×100, ×200, ×400). (B) CD90 and CD31 expression in acoustic cytometry by NC‐VICs and C‐VICs (n = 6) (C) Col1A1, Fn1, THY 1, CD44, CDH2, S100A4, TWIST1, VIM, NT5E, ENTPD1, ADORA2B, P2RY2, P2RY12, S1PR2 mRNA expression by MSCs, C‐VICs and NC‐VICs (n = 3, mean ± SEM). ns, p > 0.05.

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  FIGURE 3. Functional characterisation of NC‐VICs and VICs. (A) Osteogenic differentiation of MSC, VICs isolated from NC‐VICs and C‐VICs revealed by Alizarin Red staining. Adipogenic differentiation of MSCs revealed by the presence of lipid drops on phase‐contrast image (scale 400 μm). Quantification of osteogenic differentiation of MSCs, NC‐VICs and C‐VICs (OD 405 nm, n = 5 to 7) (B) Cumulative population doubling of MSC, NC‐VICs and C‐VICs (n = 4). C. Maximum cumulative population doubling of MSC, NC‐VICs and C‐VICs (n = 4). ns, p > 0.05, *p < 0.05 and, **p < 0.01.

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  FIGURE 4. Blood vessels infiltration evaluated by haematoxylin and eosin (H&E) staining. (A) Matrigel plug of ECFCs+NC‐VICs at lower (1,25× left pannel) and higher magnification (10× central, 20× right panel) (B) Matrigel plug of ECFCs+C‐VICs at lower and higher magnification (same order) (C) Matrigel plug of ECFCs+MSCs at lower and higher magnification (same order). Arrows indicate functional vessels containing erythrocytes. (D) Vessels number quantification (n = 10 matrigel plugs for each group) (E) Diameter quantification of n = 10 Matrigel plugs for each group (A–C). **p < 0.01 and ***p < 0.001. (F) Matrigel plug section of ECFCs+NC‐VICs, ECFCs+C‐VICs, ECFCs+MSCs (from left to right). CD31 stain (red) was performed to identify endothelial cells while perivascular cells are stained by αSMA (green). (G) Quantification of αSMA stain in n = 10 different Matrigel plugs for the 3 different groups mentioned (H) 3D reconstruction of the confocal images proposed in (F).

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  FIGURE 5. Co‐culture of ECFCs + NC‐VICs, ECFCs + C‐VICs (A) Co‐culture of ECFCs+NC‐VICs and ECFCs+C‐VICs (400× magnification, zoom), ECFCs are positive for von Willebrand factor (vWF) stain (red) and NC‐VICs or C‐VICs are stained for calponin (green) (B) Quantification of calponin stain in n = 3 different co‐culture in duplicate (C) Quantification of nuclei in co‐culture of ECFCs+NC‐VICs and ECFCs+C‐VICs stained by calponin (D) Co‐culture of ECFCs+NC‐VICs and ECFCs+C‐VICs (400× magnification, zoom), ECFCs are positive for vWF stain (red) and NC‐VICs or C‐VICs are stained for αSMA (green). (E) Quantification of αSMA stain in n = 3 different co‐culture in duplicate (F) Quantification of nuclei in co‐culture of ECFCs+NC‐VICs and ECFCs+C‐VICs stained by αSMA. *p < 0.05 and ***p < 0.001.