Proteoglycans (PGs) are complex molecules containing a core protein to which are attached one or more negatively charged glycosaminoglycans. They give articular cartilage its ability to undergo rapid reversible deformation. They exist as multiple populations with different structures and functional properties. Aggrecan, the major population, undergoes structural changes with age, reflecting in part a change in chondrocyte expression but also alterations taking place in the matrix with time. The human articular cartilage matrix becomes enriched with age in nonfunctional fragments which may weaken the matrix and thus predispose the tissue to progressive degeneration. Recent advances in our understanding of these age-related changes have been made possible by the development of culture systems in which the human cells remain phenotypically stable for long periods of time. In the alginate bead system, PGs laid down in different pools can be extracted without disrupting the conformation of native PG aggregates. Sensitive immunoassays to quantify substructures and levels of aggrecan-derived fragments in body fluids are making it possible to ask questions that could not have been addressed before. The goal of this proposal is to take advantage of these tools in studies aimed at further elucidating changes in the metabolism of human articular cartilage PGs with aging. First, phenotypically stable normal articular chondrocytes from humans of different ages will be cultured in alginate beads to further study age-related differences in the response of these cells to IGF-I and IL-1. Studies will examine age-related differences in the synthesis and turnover of different PG populations which accumulate in two pools representing PGs located in the vicinity of the cell and in the regions of the matrix more distant from the cells. Experiments will be performed to study age-related differences of changes in the expression of these PG populations (CS-rich and CS-poor aggrecan, decorin, KS-containing fibromodulin, type IX collagen) and of hyaluronate (HA), the composition of specific populations and the kinetics of incorporation and turnover of each population into the two pools. Attempts will be made to study the incorporation and turnover of aggrecan within stable aggregates of two distinct sizes. Bovine chondrocytes and agarose cultures will be used in some studies to develop new approaches and for comparative purposes. Small samples of human cartilage will be analyzed to correlate age-related changes in the composition of PGs in the matrix with the information obtained in culture. Second, the cultured human chondrocytes will be used to study age-related differences in the conversion of aggrecan molecules to smaller products such as the HA-binding region fragment which accumulates with age in normal human articular cartilage. Experiments will be devised to examine what proportion of the fragments in the two pools are present in aggregates and can bind to HA. Third, studies will be performed to help test the hypothesis that the level of KS epitope in body fluids in adult life provides a measure of the concentration of total KS present. This will be achieved by measuring and comparing concentrations of KS epitope (using a well-characterized ELISA) and total KS (determined by HPLC analysis of disaccharides generated after treatment of samples with keratanase II) in body fluids and aggrecan from normal adults of different ages.
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