The retinal pigment epithelium (RPE) plays a number of vital roles in maintaining visual cell function; one of these is the metabolism of extracellular matrix (ECM) components. The studies outlined in this proposal will answer specific questions concerning the mechanisms involved in the degradation of one of the components of the ECMs surrounding the RPE: glycosaminoglycans (GAGs). The proposed studies will identify and characterize GAG degradative pathways in both the intracellular and extracellular compartments of the RPE in vitro. The experimental studies utilize RPE cultures initiated from defined regions of the eye to measure the turnover of GAGs within individual compartments of the RPE cell layer (intracellular compartment, trypsin-soluble glycocalyx, basal extracellular matrix). Exogenously applied compounds as well as inherited enzyme deficiencies will be used to experimentally modulate (stimulate, inhibit) in a selective manner either intracellular or extracellular GAG turnover. Biosynthetically radiolabeled basement membrane preparation will be used to determine whether ECM components are reinternalized during degradation or remain extracellular, and what factors affect this process. Radiolabeled basement membrane preparations also will be used to distinguish degradation by cell surface enzymes (e.g. requiring direct cell contact) from that of secreted enzymes. These studies will provide valuable information concerning the normal functioning of the RPE, and its interactions with extracellular matrices. In addition, these studies may provide insights into aging or disease related changes in the RPE and/or the ECMs surrounding the RPE.
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