A thickening in basement membranes (or epithelial-associated extracellular matrix (ECM)) is a common feature of a number of disease states. For example, ECM thickening has been observed 1) associated with the renal tubules and glomeruli (GBM) of patients with polycystic kidney disease (PKD), 2) associated with the seminiferous tubules in male patients with certain forms of impaired fertility, 3) associated with the GBM of patients with Alport syndrome, and 4) in patients with diabetes mellitus. Progressive secondary complications of these diseases have been tied to abnormalities associated with this ECM thickening. Although an explanation as to the mechanisms by which abnormal ECM thickening occurs is not currently available, it is evident that the normal biosynthesis and maintenance of ECM is an important aspect of healthy differentiated tissues. As indicated, the cellular and molecular basis for alterations in ECM formation is not understood, but may be explained in terms of problems in the balance of ECM component synthesis, polymerization, and turnover. These processes all involve cell/ECM interactions to some degree and imply that ECM formation is normally tightly controlled by both cellular and extracellular processes. Experimental approaches to evaluate the mechanisms which govern normal ECM formation and the mechanisms by which abnormally thickened ECM occurs have been hampered by a lack of in vitro and in vivo cellular models. In order to approach these problems we have developed an in vivo cellular model in which epithelial-associated ECM formation can be experimentally induced in a short time frame for subsequent analysis of the cellular mechanisms involved in the process. In addition, methods have been developed to trigger the formation of abnormally thickened ECM so that both the normal and abnormal process can be directly compared and evaluated. The model we have developed is simply comprised of an epithelial bilayer with an intervening ECM. In addition, we have determined that this in vivo model responds to hyperglycemic conditions by thickening its ECM as observed in various pathological conditions. As opposed to vertebrate animal models currently available however, the cell system we utilize develops an ECM within 24-96 hr and doubles the thickness of this ECM within this same time frame when exposed to elevated levels of glucose. This model was developed using the Cnidarian, Hydra vulgaris. The proposed project will utilize this in vivo model to analyze normal ECM formation and determine if abnormal thickening of ECM results from 1) cellular abnormalities in the synthesis and accumulation of ECM components, 2) abnormalities in the extracellular assembly of ECM components., and/or 3) abnormalities in the degradation of ECM components. A combination of morphological, biochemical, and molecular approaches will be utilized to test these hypotheses. This project will provide basic information on the cellular mechanisms of ECM formation under normal conditions and under conditions in which ECM thickening occurs.
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