Identification of specific calcium-binding noncollagenous proteins associated with glutaraldehyde-preserved bovine pericardium in the rat subdermal model.

Abstract

Calcification of glutaraldehyde-preserved bioprosthetic heart valves (BHVs) results in their clinical failure. The mechanism of this pathologic calcification is not well defined. Since serum proteins are known to be taken up in mineralized tissue, we hypothesized that serum proteins derived from several calcium-binding noncollagenous proteins (NCPs) of bone and teeth also may be associated with pathologically mineralized BHVs. Using a rat subdermal model of BHV calcification, glutaraldehyde-preserved bovine pericardium (GPBP) was implanted for 1, 3, 14, and 60 days, and then subjected to an extraction procedure designed to isolate only NCPs tightly bound to the mineral phase. Gel electrophoresis and Coomassie Brilliant Blue staining demonstrated that these proteins became associated with GPBP over time, paralleling reported calcium uptake by the tissue. Stains-All staining demonstrated a marked accumulation of highly acidic, phosphorylated NCPs associated with 60-day GPBP extracts. Some of these proteins were detected in rat serum but were absent from extracts of GPBP incubated in rat serum in vitro. Western blotting with antibodies to three NCPs found in bone and teeth-bone acidic glycoprotein 75 (BAG 75), osteopontin, and SPARC-demonstrated that these NCPs were tightly bound to the mineral phase of calcified GPBP. A fourth NCP, bone sialoprotein II (BSP II) was barely detectable. Thus each identified NCP showed a different pattern of GPBP association relative to mineral deposition, suggesting unique roles for each in pathologic calcification. SPARC increased within 3 days of GPBP implantation but decreased by 2 weeks. BAG 75 and osteopontin uptake was detected in the initial mineral deposits and increased mineralization proceeded. BSP II never increased significantly over the entire-period. Further studies, which should include immunohistochemistry, will be important for delineating the source, location, and function of these three NCPs and for identifying others that also may be involved in this pathological process. Most important, the new insights into the mechanism of pathologic calcification described here present exciting opportunities for novel approaches to BHV calcification prevention.