PROTEOGLYCAN STRUCTURE, METABOLISM AND ROLE IN TENDON

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The long-term goal of this project is to understand the structure, metabolism, regulation, and roles of one category of extracellular matrix constituent in tendon - the proteoglycans. Tendon generally contains only a small amount of the small proteoglycan decorin. However, an aggrecan- rich fibrocartilage develops where the bovine deep flexor tendon passes under bone and experiences compression as well as tension in situ. In vitro experiments support the hypothesis that mechanical loading leads to increased synthesis and accumulation of large proteoglycans, generating a tissue with the added compressive stiffness needed for its particular mechanical environment. Experiments are proposed to investigate the long- term and short-term responses of cells in tendon to the imposition of specific mechanical loads . (1) A new device will be constructed to load discrete regions of a fetal bovine tendon in vitro with normal tensile stress, normal compressive stress, or shear stress, for time periods ranging from minutes to days. The effects of different loading environments on the amount and type of proteoglycan synthesized, its structure and turnover, as well as on mRNA expression and localization for several matrix constituents, will be determined. Synthesis and activation of TGF-Beta as a result of loading will be measured. (2) Confocal microscopy will be used to visualize immediate cellular responses to tissue loading, such as changes in (Ca++] or altered organization of the cytoskeleton. The activity of tyrosine phosphorylation or tyrosine phosphatase will be assessed after different loading regimens. (3) The role of extracellular matrix organization and specific matrix components in regulation the cellular response to tissue loading will be determined using enzymes to alter matrix integrity and drugs to alter cytoskeletal organization. This integrated experimental plan will, for the first time, allow correlation of distinct measured mechanical loads with relevant cellular responses in one tissue. Fibrocartilage provides compressive stiffness and a gliding surface in precisely the location where such properties are useful to the tendon. The inappropriate development of fibrocartilage leads to pathology associated with repetitive motion syndromes, altered healing properties, and rheumatic disease. The studies of this proposal will increase understanding of tissue-level responses that allow soft connective tissues to appropriately modulate their composition to meet a changing mechanical environment.