Introduction. In spite of the concept that physiological, pathological and experimental calcification processes are very similar to each other, accumulating data indicate specific differences to exist. A distinct process of cell degeneration/mineralization occurs in aortic valves subjected to calcification subdermal models. This was found to be characterized by acidic lipid clustering at the surface of cells and matrix-vesicle-like bodies, that acts as major apatite nucleators (Ortolani F et al: Connect Tissue Res 43:44-55, 2002; Ortolani F et al: Histochem J 34: 41-50, 2002; Ortolani F et al: Histol Histopathol 18: 1131-40, 2003) in association with calcium-binding protein Annexin-V (Anx-V). Here the possibility was investigated that these degenerative processes also occur in pathological valve calcification. Material and Methods. After surgical replacement with valve prostheses in patients affected by aortic valve stenosis, samples were excised from the explanted, calcified valves and subjected to pre-embedding reaction with 0.05% Cuprolinic Blue in phosphate solutions containing 2.5% glutaraldehyde plus 0.05M MgCl2, pH 4,8 (GA-CB). Semithin sections of GA-CB-reacted samples underwent von-Kossa-silver-staining and re-embedded to achieve reacted ultrathin sections (GA-CB-S). Histological sections underwent von-Kossa-silver-staining (S). Cryosections underwent reactions for alkaline phosphatase activity (AP) and immunohistochemical reactions for Anx-V. LR-White-thin sections underwent immunogold reactions for Anx-V. Polyclonal AB anti-Anx-V R88 was used (courtesy of Klaus von der Mark). Results. Light microscopy showed colocalized positivity between GA-CB and S reactions for several cell groups, whereas non-colocalized AP-reactivity appeared for several others. At the ultrastructural level, degenerative features were observed which were closely superimposable to those in the subdermal model. Clustering of acid lipids was observed around cell debris, matrix-vesicle-like bodies and elastic fibers as well as colocalization between GA-CB and GA-CB-S reactivity. Immunogold labelling showed Anx-V to be closely associated with the accumulating lipidic material. Conclusions. In spite of the heterogenous patterns observed and undependently from eziopathogenesis, these results suggest that common mechanisms are shared by the different forms of this pathological calcification and experimental one, including initial hypoxia-induced up-regulation (Denko N et al: Clin Cancer Res 6: 480-7, 2000) of Anx-V and subsquent protein translocation due to its avidity for the exudating lipids resulting from concurrent cell phospholipidosis.
