Immunohistochemical localization of osteopontin and in-situ detection of interstitial cell apoptosis in aortic valve leaflets subjected to in vivo experimental calcification

A distinct pattern of cell degeneration has been described for porcine calcifying aortic valve leaflets (AVLs) after xenogenic subdermal implantation (Ortolani et al., 2002; Histol Histopathol 2003; Histol Histopathol, in press). It this situation degraded acidic lipids cluster at cell surfaces in association with calcium-binding protein Annexin-V, acting as primary nucleators of apatite. As reported, ectopic mineralization could depend on the expression of proteins involved in regulating normal osteogenesis instead of a mechanistic one. Of these, osteopontin (OPN) is an acidic phosphoprotein also present at high levels in calcified vascular tissues and plays an inhibiting role on mineralization process. In the present investigation, immunohistochemical reactions and immunogold labelling (mouse antimouse OPN monoclonal Ab, santa Cruz) showed that actually OPN is present in AVLs after the usual preimplantation treatment with 0.6% glutaraldehyde, and subdermal implantations for 2 days, 2 weeks, and 6 weeks. Additionally, TUNEL assay (ApopTag-Peroxidase-Chemicon Int.) and ultrastructural evidences suggested that incomplete apoptosis takes place, anticipating the degradation cascade as previously reported to occur. These data support the concept that subdermal model dependent hypoxic/anoxic conditions affect AVL cells inducing both active reactions; the transient expression of osteogenesis-involved proteins, and cell death. The latter primes the onset of the mechanistic phenomena culminating in apatite salt crystallization.