Collagen stabilizes the structure of most organs. The applicants have demonstrated that fibrils are initially assembled as discrete fibril segments. These segments are incorporated into discontinuous fibers within developing extracellular matrices. As development proceeds, a regulated maturation of segments occurs. This involves the transformation of intermediates (segments) within the immature tissue into longer and, in most tissues, larger diameter fibrils (tendon); into longer fibrils with no change in diameter (cornea); or perhaps to persistence of segments (rapid remodeling). Relative to this application, it is hypothesized that segments undergo a post-depositional fusion, followed by molecular rearrangements that give rise to longer fibrils of the mature tissue. This hypothesis predicts changes in segment structure as fibrillogenesis proceeds from assembly of segments to fibril growth. The applicants' model of fibril growth predicts a stabilization and destabilization of segments at specific times in development. They hypothesize that this process involves temporal and spatial changes in components associated with the segment surface. They suggest that their data implicates the fibril-associated proteoglycans and collagens. In this application, they will study the growth of fibrils from preformed intermediates in two contrasting tissues, the tendon and cornea. Specifically, they will characterize the structure of segments during and after the period of rapid fibril growth. They also will determine whether a correlation exists between candidate macromolecules and expression/interaction at specific stages of fibril growth using morphological, biochemical and molecular genetic approaches. As candidates are substantiated, their roles in the regulation of fibril growth will be examined. Regulation via decorin and fibromodulin will be studied initially; the applicants' data indicating a decrease in decorin during the predicted period of destabilization, and an increase in fibromodulin during the period of matrix stabilization. The temporal expression of these candidate molecules will be altered during development. Retroviral constructs will be prepared to reduce (antisense or dominant-negative), or prolong expression of the putative regulatory molecules. The effects will be analyzed, in vitro, using a collagen gel model system populated by infected cells. The effects on tendon development in situ also will be analyzed. It is suggested that an understanding of the mechanisms regulating fibril formation, growth, matrix assembly and the development of tissue-specific architecture will lead to the understanding of development, growth, repair and pathobiology, as well as manipulations of inherited disorders.
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