Involvement of endogenous annexin-V as co-factor in aortic valve calcification as revealed by immunogold labelling

Introduction - To date, the mineralization processes incurring in soft tissues are still debated. Previously, it was found that modified Glutaraldehyde-Cuprolinic-blue reactions (GA-CB) promote demineralization of calcified aortic valves visualizing cells and matrix vesicles outlined by peculiar, CB-reactive layers which closely correlated with initial mineralization loci (1,2). Anionic nature and differential chemical/enzymatic extractivity suggested these structures to be formed by acidic phospholipids . In addition, specific pre-embedding reactions with Glutaraldehyde-Malachite-green (GA-MG) confirmed this assumption (3). Material and Methods - After 1d-, and 1-, 2-, and 4-week long implantations in rat subcutis, porcine aortic valve leaflets were subjected to: (A) pre-embedding GA-CB reaction and (B) GA-MG reaction (0.05% Cuprolinic Blue or 0.1% Malachite green in buffer solutions, respectively; pH 4,8). (C) Semithin sections of GA-CB-reacted samples underwent post-embedding von Kossa silver staining and re-embedded, so achieving GA-CB-S-reacted thin sections. (D) Histological sections underwent von Kossa silver-staining. (E) Cryosections underwent immunohistochemical reaction for Annexin V, and (F) LR-White-thin sections underwent immunogold reaction. Polyclonal AB anti-Annexin-V R88 was used. Results - Co-localization was confirmed between GA-CB-, GA-MG-, and GA-CB-S-reactivities. von Kossa reaction on histological sections revealed calcification to start at cell level after 2-day-long subdermal implantation involving more and more cells and then spreading from cells toward the extracellular matrix progressively, after longer implantation periods. On cryostatic sections, Annexin-V-reactivity appeared to co-localize with silver precipitation. On thin sections, immunogold-labelling showed initial reactivity for Annexin V to occur (i) within the cells, at level of mitochondrial membranes and other cytomembranes, firstly, and then (ii) at cell edges, also after cytomembrane disappearance. Later (iii) Annexin-V-spreading took place toward the spaces interposed between the iuxta-cellular collagen fibrils. Conclusions - These data show that a process of cell degeneration/mineralization occurs in this calcification model, in which acidic phospholipids accumulate at cell surface acting as major apatite nucleators with co-involvement of associated calcium-binding proteins. Of these, Annexin V has been identified as intracellular product that may represent a co-factor in calcification. The underlying process might be shared by calcifying bioprosthetic valves and/or soft tissues affected by pathological mineralization. References - (1) Ortolani F. et al., Connect. Tissue Res. 43: 44-55, 2002. (2) Ortolani F. et al., Histochem. J. 34: 41-50, 2002. (3) Ortolani F. et al., Histol. Histopathol. 18, 1131-1140, 2003