Fig. 5.

Osteopontin (OSTP) positive staining was observed in the collagen rich fibrosa layer and near the ventricularis surface in the normal tissues (a). In the CAVD tissues (b), positive OSTP staining was found in the calcification sites and in regions immediately adjacent to calcified nodules forming a boundary surrounding the calcific nodule. Occasionally, OSTP staining was seen associated with cell clusters in a metaplasia zone and possibly next to the early calcification sites. Osteonectin (SPARC) staining was more diffuse and loosely scattered in the normal tissues (c). The endothelial lining was also positive for SPARC. However, intense but patchy SPARC staining was observed in calcification site and in the regions interspersed with cells near calcific nodule (d). Osteocalcin (OSTCN) staining was diffusely distributed in the normal tissue (e). In the CAVD tissue (f), OSTCN-positive staining was observed near the calcific area and associated with fibrous structures of ECM. Occasional, OSTCN was associated with cells in a stratified banding fashion. Osteoprotegerin (OPG/TR11B) was diffusely distributed in the normal tissue (g). OPG was closely associated with calcified areas in CAVD tissues (h), and the staining appeared more intensely in the ventricularis. RANK (TNR11) was faintly distributed throughout the normal tissue (i). RANK staining in CAVD tissues appeared patchy near the calcified areas (j). Negative control stains without primary antibodies are provided. Positive staining is shown by DAB chromogen in brown color. Scale bar =100μm.