Ultrastructural and immunohistochemical evidentiation of lipid and annexin-V involvement in subdermal calcification of aortic valve leaflets

Introduction: To date, mineralization mechanism occurring in heart valves is not well understood, even if common steps have been shown to exist between calcification occurring in physiological, pathological or experimental conditions. A major problem for characterizing the structural changes occurring in calcifying tissues consists in tissue masking by mineral or extraction of tissue components from sample, when decalcifying agents are used. Modified glutaraldehyde-Cuprolinic-blue reactions (GA-CB) enabled us to demineralize calcified aortic valves with concurrent binding of CB to anionic material which was layered around cells and matrix vesicles, just where major apatite precipitation is observable in undecalcified samples. Their anionic nature and differential chemical/enzymatic extractivity suggested that these CB-reactive layers may be formed by acidic phospholipids. Further investigation has been carried out to confirm this datum and to localize possible lipid-associated calcium-binding proteins. Methods: After 6-week implantation in rat subcutis, porcine aortic valve leaflets were subjected to (a) pre-embedding GA-CB reaction (0.05% Cuprolinic Blue in phosphate solution buffered at pH 4,8), and (b) GA-MG reaction (0.1% Malachite green in solution buffered at pH 4,8). Semithin sections of GA-CB-reacted samples underwent von Kossa silver reaction and re-embedded to achieve GA-CB-S-reacted thin sections. Cryosections and LRW-thin sections underwent immunohistochemical reaction for Annexin-V localization. Results: After GA-CB-S-reactions, major silver precipitation occurred within the CB-reactive layers outlining cells and matrix vesicles in selective manner. In addition, GA-MG-reactivity closely co-localized with GA-CB-S-reactivity. The same distribution was also found for annexin-V. Conclusions: These data show that in the subdermal calcification model, valve mineralization is primed by acidic phospholipids accumulated at cell and matrix vesicle surfaces, which will act as major apatite nucleators in association with calcium-binding proteins. This is someway comparable with calcium-acidic-phospholipid-phosphate complexes and their interactions, as reported for physiological calcification.