Calcification onset in aortic valves has been largely debated, being increasingly relevant the pro-calcific role of cell-membrane-derived acidic phospholipids as part of the so called “calcium-phospholipid-phosphate complexes” [1]. Our previous studies on in vivo experimentally and pathologically calcified aortic valve leaflets as well as aortic valve interstitial cell (AVIC) pro-calcific cultures strengthened the cell-mediated triggering of valve calcification. Indeed, the degenerative process was found to depend on overall cytomembrane dissolution with lipid release and appearance of a phthalocyanine-positive acidic material (PPM) stuffing cell cytoplasm, followed by its spreading towards cell edges with formation of peripheral layers (PPLs) acting as major hydroxyapatite (HA) nucleators [2-4]. Here, additional involvement of anionic nucleic acids in valve mineralization has been investigated. Ultrastructural analyses of mineralizing cultured AVICs as well as those populating in vivo calcified valve leaflets showed ribosome detachment from degenerating rough endoplasmic reticulum cisternae and their embedding within both intracellular PPM and forming peripheral PPLs. Using antibodies against ribosomal RNA (rRNA), immunohistochemical analyses actually showed positivity at level of PPM/PPL material characterizing mineralized AVICs and cell-derived vesicular byproducts. Moreover, immunogold labelling reactions revealed gold particles to decorate PPM/PPLs, supporting the evidence that rRNA derived from ribosome degeneration contributes to the formation of such pro-calcific acidic material. Interestingly, nuclear DNA resulted as an additional HA nucleational site in mineralizing AVICs, as revealed by silver particle deposition onto nuclear chromatin after post-embedding von Kossa reactions for selective calcium-binding site visualization. In conclusion, the present results indicate that nucleic acids may contribute to AVIC mineralization both in vivo and in vitro, with their acidic nature promoting PPM/PPL capacity of HA nucleation. The obtained results are also consistent with previous finding that complete removal of nucleic acids during decellularization of native heart valves is of paramount importance to attain durable, calcification-free valve biosubstitutes [5].
