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.

Biological Calcification

“…Several findings do, however, point to a cellular function. Gura et al (1997) found non-collagenous proteins tightly bound to the mineral phase in subdermal implants of glutaraldehyde-preserved bovine pericardium with marked accumulation of highly acidic, phosphorylated proteins; they reported that ON increased within three days after implantation but its duration decreased by two weeks, and that BAG-75 and OPN uptake could be detected in initial inorganic deposits and increased with calcification. Levy et al (1980) have detected OC in three calcified porcine xenograft valves and in a calcified homograft valve.…”

Effects of periodate and chondroitin 4-sulfate on proteoglycan stabilization of ostrich pericardium. Inhibition of calcification in subcutaneous implants in rats

“…PGs and GAGs were extracted in 4 m guanidine HCl in sodium acetate, pH 5.8, containing protease inhibitors: 1 mm p-hydroxymercuribenzoate, 1 mm phenylmethylsulfonyl fluoride, 1 mg/ml soybean trypsin inhibitor and 10 mm EDTA [22]. After dialysis (MWCO 12,000) at 4 C for 72 h against distilled water, the PG/GAGs content of the extracted samples was determined with a Blyscan assay kit (Biocolor Ltd., Belfast, North Ireland) and expressed as micrograms per milligram of wet weight tissue.…”

Composition of calcifications in children with juvenile dermatomyositis: Association with chronic cutaneous inflammation

“…Pathologic calcifications in soft tissue, similar to those present in calcified heart valves (1), are a complication of several connective tissue diseases, increasing morbidity and mortality (2, 3). In contrast to adults with dermatomyositis (DM), in whom calcification is relatively uncommon, it is estimated that 20–40% of children with juvenile DM have calcific deposits (3).…”