Calcium-Dependent Cytosolic Phospholipase A2α as Key Factor in Calcification of Subdermally Implanted Aortic Valve Leaflets

Figures

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  Figure 1. Histological sections of porcine aortic valve leaflets in the native state (nAVL) or subdermally implanted into rat subcutis for 2 to 28 days (iAVL 2d, iAVL 7d, iAVL 14d, and iAVL 28d) after the immunohistochemical detection of cytosolic phospholipase A2α (cPLA2α; left column) matched against para-serial sections subjected to von Kossa silver reaction (vK) for calcium binding site identification (right column). (A–D) Negligible positivity for cPLA2α showed by both nAVL (A) and iAVL 2d (C) vs. negativity for vK showed by both nAVL (B) and iAVL 2d (D). (E–J) Time-dependent increase in cPLA2α-positive valvular cells in iAVL 7d (E), iAVL 14d (G), and iAVL 28d (I) vs. parallel increase in vK-positive ones (F,H,J). Bar: 0.5 mm.

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  Figure 2. Comparison between para-serial histological sections of an aortic valve leaflet implanted into rat subcutis for 7 days (7d) after immunohistochemical detection of (A) vimentin, (B) cytosolic phospholipase A2α (cPLA2α), and (C) α-smooth muscle actin (ASMA). Co-localization in superimposable areas of cPLA2α-positive valvular cells (B) with both vimentin-positive (A) and ASMA-positive (C) ones. Bar: 0.25 mm.

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  Figure 3. Thin sections of aortic valve leaflets implanted into rat subcutis for 2 days (2d; left column) vs. those implanted for 14 days (14d; right column). (A,B) Pre-embedding reaction with phthalocyanine cuprolinic blue (CB) showing CB-reactive dense bodies (arrows) in a valvular cell at early degeneration stage vs. appearance of a CB-positive, membrane-derived pericellular layer (counterposed arrows) embedding outside collagen fibrils (double arrows) and elastin fibers (e) at late degeneration stage. (C,D) CB-positive dense bodies (arrows in C) showing negligible positivity to additional post-embedding von Kossa reaction (vK) vs. a CB-positive pericellular layer exhibiting large silver particle superimposition after vK (D). (E,F) Immunogold labelling of cytosolic phospholipase A2α (cPLA2α) showing selective decoration of mitochondrial membranes in a valvular cell at early degeneration stage (E) vs. large decoration of a pericellular layer at late degeneration stage (F). Bar: 1 μm (A,B); 0.5 μm (C,E,F); 0.25 μm (D).

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  Figure 4. Histological section of an aortic valve leaflet implanted into rat subcutis for 14 days (iAVL 14d) vs. another implanted for 28 days (iAVL 28d) after immunohistochemical detection of interleukin-6 (IL-6). (A) Positivity of valvular endothelial cells and host’s cells of the peri-implant connective tissue (p.i.c.t.). (B) Positivity of valvular subendothelial cells and host’s cells of the peri-implant connective tissue (p.i.c.t.). Bar: 0.25 mm.

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  Figure 5. Immunohistochemical detection of osteopontin (OPN) in aortic valve leaflets implanted into rat subcutis for 28 days (28d). (A) Histological section showing positive valvular cells exhibiting marked reactivity at their edges (counterposed arrows). (B) Thin section subjected to immunogold labelling showing a membrane-derived pericellular layer decorated by gold particles. Bar: 0.25 mm (A); 0.25 μm (B).

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  Figure 6. Histological section of an aortic valve leaflet implanted into the rat subcutis for 28 days (iAVL 28d) after immunohistochemical detection of fetuin-A. (A) Positivity of blood plasma of vessels irrorating the peri-implant connective tissue (p.i.c.t.), with no positivity showed by valvular cells. (B) Magnification of the immunopositive vessels in the framed area in (A). Bar: 0.5 mm (A); 0.1 mm (B).

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  Figure 7. Immunohistochemical detection of annexin-V (Anx-V) in aortic valve leaflets implanted into rat subcutis for 2 days (2d; line above) vs. 14 days (14d; line below). (A) Histological section showing immunopositivity for sporadic valvular cells. (B) Thin section subjected to immunogold labelling showing mitochondrial membranes decorated by gold particles in a valvular cell at early degeneration stage. (C) Histological section showing increased numbers of immunopositive valvular cells, with marked reactivity at their edges (counterposed arrows). (D) Thin section subjected to immunogold labelling showing a membrane-derived pericellular layer decorated by gold particles in a valvular cell at late degeneration stage. Bar: 0.25 mm (A,C); 0.25 μm (B,D).