Co-existence of regulated and mechanistic processes in stenotic aortic valve calcification as in the subdermal model

In vascular tissue ectopic calcification, cells regulate calcium nucleation by sinthesizing mineralization-competent matrix, releasing matrix vesicles and/or by undergoing necrosis, thereby accumulating degeneration products. Of these, using a calcification subdermal model acidic lipids were found to cluster at the surface of cells and arise matrix-vesicle-like bodies, acting as major apatite nucleators, in association with undetermined calcium-binding proteins (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). Since cell additional roles may consist in actively regulated processes controlling the expression of intracellular mineralization-related proteins, this study investigated whether calcium-binding proteins Annexin-V (Anx-V) and alkaline phosphatase (AP) could be detected in subdermal model and/or pathological calcification. Two lots of samples excised from (A) porcine aortic valve leaflets (PAVs) retrieved after 1-day- to 4-week-long implantation in rat subcutis, and (B) human aortic valve leaflets (HAVs) explanted from patients affected by valve calcific stenosis were subjected to pre-embedding reactions with 0.05% Cuprolinic Blue in phosphate solutions containing 2.5% glutaraldehyde plus 0.05M MgCl2, pH 4,8 (GA-CB) and processed for TEM analysis. Some 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). Cryo-sections underwent reactions for AP activity 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). Within the first implantation week, all cells exhibited symptoms of active reaction to initial hypoxia, such as extrusion of multiple lamellipodies and accumulation of dense bodies, while only transient, negligible AP activity was observed. Starting from 1-week long sub-cutis implantation, co-localization appeared of GA-CB reactions and GA-CB-S reactions at the edges of cells, matrix-vesicle-like bodies, and then around elastic fibers, confirming calcium nucleation to be primed by peculiar cell degeneration, with acidic lipids undergoing dissociation, acidification, peripherical clustering and subsequent outside spreading. In addition, immunogold labelling showed Anx-V to be closely associated with the accumulating lipid moieties. In HAVs, heterogeneous patterns of calcification were observed at different places, including irregular distribution of sharp positivity for AP reactions. However, at the ultrastructural level, a lot of degenerative features mimicked those in PAVs, concerning initial reactivity of cells, co-localization between GA-CB and GA-CB-S reactions, and immunoreactivity for Anx-V, being the only difference the presence of cell debris edged by proteolipidic layers, instead of entire cells. These data show that several of the different mechanisms which take place in various forms of valve calcific stenosis are closely comparable with those occurring in subdermal-model-induced calcification, including a regulated process, where initial hypoxia might induce Anx-V up-regulation (Denko N et al: Clin Cancer Res 6: 480-7, 2000) followed by a mechanistic process, where this protein is subjected to translocation because of its avidity for the exudating acidic lipids arised from concurrent cell degeneration.