Ultrastructural and Immunohistochemical Detection of Hydroxyapatite Nucleating Role by rRNA and Nuclear Chromatin Derivatives in Aortic Valve Calcification: In Vitro and In Vivo Pro-Calcific Animal Models and Actual Calcific Disease in Humans

Figures

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  Figure 1. Immunocytochemical detection of rRNA in 15-day-long bovine aortic valve interstitial cell (bAVIC) cultures. (A) Immunopositivity of untreated bAVICs. (B) Immunopositivity of mineralizing bAVICs as well as vesicular cell debris (arrowheads) and calcific nodule areas (cn; inset). (C) Alizarin red calcium staining of mineralizing bAVICs and calcific nodules (cn; inset). Bar: 1 mm.

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  Figure 2. Ultrastructure of bAVICs from 15-day-long pro-calcific cultures. (A–C) Enlargement of RER cisternae (arrowheads) and RER membrane lysis, with ribosome release and merging with the nearby phthalocyanine-positive material (single asterisk: less condensed PPM; double asterisk: more condensed PPM) derived from ongoing membranous organelle degeneration. (D) Immunogold labelling of rRNA with gold particles (white arrowheads) decorating the intracellular PPM (asterisk) and a cross-sectioned degenerating RER cisterna (black arrowhead). (E) Cell-derived byproducts showing a mix of PPM (asterisk) and more electrondense phthalocyanine-positive layers (PPLs; counterposed arrowheads). (F1,F2) Immunogold labelling of rRNA showing gold particles within PPM (asterisks) and PPLs (counterposed arrowheads) at the level of vesicular cell debris. Bar: 0.5 μm (A–C,E); 0.25 μm (D,F1,F2).

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  Figure 3. Immunohistochemical detection of rRNA in porcine aortic valve leaflets (pAVLs) after 7-day-long implantation in rat subcutis. (A) Immunopositivity of residual endothelial cells and mineralizing pAVICs as well as cell debris (arrowhead) in the tunica fibrosa. (B) Paraserial section showing positivity to von Kossa staining of mineralizing pAVICs and cell debris (arrowhead). Bar: 0.25 mm.

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  Figure 4. RER ultrastructure in AVICs populating 7-day-long-implanted pAVLs. (A) Enlarged RER cisternae (circle) in a mineralizing cell showing electrondense PPM and disappearance of nuclear envelope (counterposed arrowheads; N: nucleus). (B) Magnification of the cytoplasm area encircled in (A) showing release of ribosomes from dissolving RER membranes and melting with the nearby PPM (arrowheads). Bar: 0.5 μm.

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  Figure 5. Immunohistochemical detection of rRNA in human stenotic aortic valve leaflets (hAVLs). (A,B) Immunopositivity of hAVICs, including those bordering calcific nodules (cn; arrowheads), and cell debris (arrows). Bar: 0.25 mm.

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  Figure 6. Ultrastructure of AVICs populating stenotic hAVLs. (A) Ribosomes (black/white arrowheads) embedded within PPM/PPL in a mineralizing hAVIC. (B,C) Immunogold labelling of rRNA with gold particles decorating peripheral PPLs. (D) Immunogold labelling of rRNA showing gold particles within PPM/PPLs in a hAVIC undergone advanced calcific degeneration. (E) Immunogold labelling of rRNA showing gold particles onto a PPL lining a vesicle-shaped cell byproduct. Inset: magnification of the PPL-squared region. Bar: 0.25 μm (A–E); 0.1 μm ((E) inset.

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  Figure 7. Post-embedding von Kossa silver staining of ribosomes in mineralizing AVICs. (A) Silver particle precipitation onto free ribosomes and those associated with RER membranes in a bAVIC at early calcification stages. (B–D2) Silver particle precipitation onto ribosomes associated with membranes lining enlarged RER cisternae or released nearby in hAVICs affected by ongoing calcific degeneration. Bar: 0.25 μm.

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  Figure 8. Post-embedding von Kossa staining of nuclear chromatin in mineralizing AVICs. Major silver particle precipitation onto nuclear heterochromatin (rectangular boxes) in mineralizing AVICs from (A) 28-day-long subdermally implanted pAVLs, with the largest silver particles being distributed along peripheral PPLs (counterposed arrowheads), (B) stenotic hAVLs, and (C) pro-calcific cultures. Bar: 0.25 μm.