The cornea uses extracellular matrix (ECM) molecules, in particular, collagens, to set up an architecture that serves two crucial functions: refraction and transmission of light. Our overall objective is to understand how the cornea regulates the synthesis, assembly, and differential expression of a specific set of collagens to achieve this architecture. Collagen fibrils in the corneal stroma are thin, uniform diameter structures, evenly spaced apart at discreet distances, and arranged in orthogonal layers. We hypothesize that fibrillar collagen XXIV and FACIT collagen XIV contribute to regulating the fibril diameters, and that FACIT collagen XII regulates the spacing of the fibrils in the corneal stroma. To address these hypotheses we propose in Aim 1 to determine what changes occur in the cornea of a transgenic mouse that secretes reduced quantities of type XII collagen. Our preliminary data indicate that collagen fibrils are spaced much closer together in the transgenic cornea. The fibril diameters, fibril spacing and biomechanical strength of transgenic mouse corneas will be measured and compared to wild type mice. We will also assess how other matrix molecules are affected by a paucity of collagen XII and what are the ophthalmologic consequences of making a cornea with reduced levels of collagen XII. It is possible that these mice represent an animal model for the human corneal disease, keratoconus.
In Aim 2, we will determine whether collagen XII is a proteoglycan in the cornea, as well as a collagen, similar to its structure in cartilage. This is of interest in the cornea because proteoglycans have been implicated in correct collagen fibril spacing, and mice with the known corneal proteoglycan genes knocked out have not shown significant alterations in corneal fibril spacing. If collagen XII is a proteoglycan, we will measure what percentage of the total proteoglycan it represents.
In Aim 3 we will demonstrate whether newly identified fibrillar collagen type XXIV and FACIT collagen XIV are fibril diameter regulators in the developing chick cornea. The amount of collagen XXIV and XIV secreted in the cornea will be reduced by injecting into the cornea in ovo virus encoding truncated versions of each collagen. Truncated and normal chains will combine and be degraded intracellularly, reducing the amount of secreted collagen XIV and XXIV. The alterations in fibril architecture resulting from the dominant negative phenotype will be evaluated by transmission electron microscopy. ? ?
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