Ultrastructural and immunohistochemical detection of hydroxyapatite nucleating role by rRNA and nuclear chromatin derivatives in pro-calcific aortic valve interstitial cell cultures and ex vivo mineralized valve leaflets

In calcified aortic valve leaflets, major hydroxyapatite (HA) nucleators seem to consist in the so-called “calcium-phospholipid-phosphate complexes” made of acidic phospholipids derived from degenerating membranous components of mineralizing cells [1]. Early evidence of the latter as main pro-calcific foci was provided by the silver reaction introduced in 1901 by Julius von Kossa (vKr), based on replacement of phosphate- or carbonate-bounded calcium ions with silver ions and subsequent reduction to metallic silver [2]. The vKr still represents the method of choice for calcium-binding site visualization in light microscopy; however, later introduction of procedural adjustments, consisting in the re-embedding of vK-reacted semithin sections, enabled the application of this silver staining also in the electron microscopy field [3]. In our previous studies on in vivo and in vitro experimentally induced aortic valve interstitial cell (AVIC) calcification [4,5], re-embedding of vK-reacted semithin sections obtained from calcified samples after pre-embedding copper phthalocyanine reaction revealed major HA nucleational sites to be acid-phospholipid-rich peripheral layers (PPLs) edging mineralized AVICs and PPL-derived vesicular byproducts. Here, the same combined procedure was employed to assess whether ribosomal RNA (rRNA) and nuclear chromatin contribute to AVIC mineralization, extending the analysis also to human valve leaflets affected by actual calcific stenosis. For both experimental and pathological conditions, at early calcification stages silver particles were found to superimpose to free and membrane-bounded ribosomes. Subsequent melting of the latter with intracellular phthalocyanine-positive material and pericellular PPLs was observed, with these pro-calcific substrates being decorated by gold particles after immunogold labelling of rRNA. Silver particle deposition onto nuclear chromatin was also detectable, in absence of features of apoptotic or oncotic cell death. In conclusion, the use of vKr adapted to electron microscopy enabled the identification of rRNA and nuclear chromatin as additional sites of HA nucleation during AVIC mineralization, providing more information on valve cell pro-calcific death.