In situ detection of apoptosis as promotor of aortic valve calcification in the subdermal model

In previous ultrastructural research on subdermally implanted aortic valve leaflets (AVLs), we described a distinct pattern of cell degeneration, in which acidic lipids give raise to peculiar, pericellular layers acting as initial nucleators of apatite crystals, presumably in association with calcium-binding protein Annexin-V, as revealed by immunogold labelling. Here, we used conventional electron microscopy and a TUNEL assay (ApopTag-Peroxidase) to assess (i) which changes are induced on cells, starting from the usual pre-implantation treatment (fixation in 0.6% glutaraldehyde, nitrogen atmosphere), and (ii) which type of cell death may precede this calcific degeneration. Samples were excised from (i) native AVLs, (ii) AVLs undergone pre-implantation treatment alone (pre-T-AVLs), and (iii) AVLs subdermally implanted for 2 days, 2 weeks, and 6 weeks (SI-AVLs). Ultrastructural features exhibited by pre-T-AVLs and 2-week-SI-AVLs were consistent with early apoptosis occurrence and TUNEL-reactivity resulted for pre-T-AVLs and all SI-AVLs. These data indicate that hypoxic/anoxic conditions due to glutaraldehyde-dependent toxic effects and concurrent oxygen-lacking environment prime uncomplete, apoptotic cell death, which represents the initial event triggering this peculiar cell degeneration and subsequent apatite precipitation. More proper comparison is now allowed between the experimental calcific process occurring in the subdermal model and physiological or pathological processes.