Acidic lipid clustering at the surface of cells and matrix vesicles and associated calcium-binding protein Annexin-V (Anx-V) have been found to act as major apatite nucleators in aortic valves subjected to experimental calcification (Ortolani F et al: Connect Tissue Res 43: 44-55, 2002 - Histochem J 34: 41-50, 2002 - Histol Histopathol 18: 1131-40, 2003). Here, possibility was investigated that these degenerative processes also occur in pathological valve calcification. After explantation, human calcified valves affected by aortic stenosis were subjected to reaction with 0.05% Cuprolinic Blue + 2.5% glutaraldehyde + 0.05M MgCl2 in phosphate solutions, pH 4,8 (GA-CB). Semithin sections of GA-CB-reacted samples underwent von-Kossa-silver-staining and re-embedded to achieve reacted thin sections (GA-CB-S). Histological sections underwent von-Kossa-silver-staining (S). Cryosections underwent immunohistochemical reactions for Anx-V. LR-White-thin sections underwent immunogold reactions for Anx-V. Ployclonal AB anti-Anx-V R88 was used (courtesy of Klaus von der Mark). As for experimental valve calcification, it was observed clustering of acid lipids around cell debris, matrix vesicles and elastic fibers, colocalization between GA-CB and GA-CB-S reactivity, and immunogold labelling for Anx-V which was closely associated with the accumulating lipidic material. These results suggest common mechanisms to be shared by this pathological calcification and experimental one, including initial hypoxia-induced up-regulation of Anx-V (Denko N et al: Clin Cancer Res 6: 480-7, 2000) and subsequent translocation due to protein avidity for the exudating lipids.
