Apoptosis involvement in the initial degenerative cascade triggering aortic valve calcification in the subdermal model

Introduction - In previous research on subdermally implanted aortic valve leaflets (AVLs) (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), a distinct pattern of cell degeneration was found to be characterized by acidic lipid layering at cell edges and subsequent generation of matrix-vesicle-like bodies, as revealed by modified reactions with phthalocyanines, and acting as initial apatite crystal nucleators, as revealed by combined phthalocyanine-silver reactions, in association with calcium-binding protein Annexin-V (Anx-V), as revealed by immunogold labelling. Material and methods - Samples were excised from (i) native aortic valve leaflets from pigs (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). pre-T consisted in fixation in 0.625% glutaraldehyde (GA)) in 10 mM borate buffer, pH 7.4, containing 0.9% (w/v) NaCl for 6 hours, under nitrogen atmosphere, and subsequent storage in 0,2% GA for 2 days. Implantations were performed into abdominal pouches of 3-week-old male Sprague-Dawley rats. For apoptosis detection, all samples underwent TUNEL assay using Apoptag Plus Peroxidase "in situ" detection kit (Chemicon Int.) and counterstaining with methyl green. Ultrastructural features were analyzed after conventional processing of for electron microscopy of other samples from each lot. Results - TUNEL assays showed initial positivity for pre-T-AVLs at endothelium level topically and for sporadical interstitial cell; reactivity involved most interstitial cells in 2d-SI-AVLs and, with increasing course, in 2w-SI- and 6w-SI-AVLs, co-localizing with calcification foci. Electron microscopy showed negligible cell injury for endothelial cells and hyperactivity signs for most interstitial cells for pre-T-AVLs, initial cell degeneration signs in 2d-SI-AVLs and progressive, peculiar cell death, as previously reported for 2w- and 6w-SI-AVLs. Conclusions - 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.