Calcification is a biological process absolutely essential to the survival of all vertebrate organisms, including man. However, the unwanted deposition of mineral in vascular walls and valves contributes significantly to pathogenesis in arteriosclerosis. Despite its critical importance, the mechanism of calcification is not well understood. The long-term objective of the proposed research is to elucidate the mechanism of endochondral calcification. This calcifying system is not only critical to bone development, but also appears to be closely analogous to ectopic mineral deposition. The proposed research will explore the role of matrix vesicles (MV) structures now widely accepted as initiators of both normal calcification in cartilage and early bone formation, in the mechanism of mineral deposition. Attention will be focused on 1) the metabolism of Ca and Pi, and the characterization of very early mineral forms in MV during the induction of mineral formation, 2) characterization of key MV proteins involved in this process, 3) exploration of the relationship between MV and collagen in mineral deposition, and 4) utilization of specific inhibitors to elucidate the sequence of events in MV mineral deposition. Special attention will be directed towards: a) characterization of constitutive MV proteins [the newly-discovered lipid-dependent Ca2+-binding proteins, the water-soluble peripheral proteins, and the collagen-binding proteins] and their relationship to the induction of mineral formation by MV, b) characterization of mineral precursors formed during early stages of MV mineralization, and c) elucidating the effect of the electrolyte environment on MV mineralization. Epiphyseal growth plate cartilage from rapidly growing chickens will be used to provide an abundant source of actively calcifying material for isolation of cells and MV. Experimental methods will include: Tissue fractionation, cell culture, assays of MV 45Ca- and 32Pi-metabolism, protein purification (extraction, chromatography, electrophoresis) and characterization using amino acid analysis, peptide mapping and sequencing, immunology (polyclonal and monoclonal antibodies, Western blots) and molecular biology (mRNA isolation, Northern blots, cDNA library, cDNA cloning and sequencing). Spectroscopy (FTIR, NMR, UV), electron microscopy and x-ray diffraction will be used to characterize MV mineral phases.
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