Graduation Year

2014

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Medicine

Major Professor

David E. Birk

Keywords

Collagen, Collagen XII, Collagen XIV, Cornea, Endothelium, FACIT Collagen

Abstract

Purpose: Corneal transparency depends upon the precise organization of corneal stromal extracellular matrix and corneal endothelial function. Stromal structure and extracellular matrix organization is responsible for proper refraction of light into the eye. The corneal endothelium is responsible for pumping excess fluid out of the cornea, effectively maintaining corneal hydration and thickness. Corneal transplantation is the current form of treatment for corneal endothelial and stromal dystrophies. The mechanisms controlling stromal collagen fibril packing and organization into orthogonal layers as well as maturation of the endothelium into a fully functioning cellular layer are unknown. Collagens XII and XIV, fibril associated collagens with interrupted triple helices (FACIT), have been implicated in matrix-matrix interactions regulating structure, cell behavior, and cell-matrix interactions. The overall aim is to determine the role of collagens XII and XIV in fibril assembly, fibril packing, lamellar assembly, stromal organization, corneal thickness, and endothelial maturation. The general hypothesis is that collagens XII and XIV regulate cornea stromal matrix development and structure, endothelial development, and corneal function. This dissertation assesses three specific hypotheses: 1) Collagen XIV regulates lateral fibril growth and fibril packing through fibrillar surface interactions; 2) Collagen XII regulates fibril packing, lamellar assembly, stromal organization, corneal thickness, and therefore, corneal function; and 3) FACIT collagens in the specialized posterior stroma regulate the acquisition of function in the corneal endothelium.

Materials and Methods: The temporal and spatial expression patterns of collagens XII and XIV were determined in the murine cornea using quantitative PCR, semi-quantitative immuno-blots and immuno-localization approaches. To determine the regulatory roles of collagens XII and XIV in stromal and endothelial development, mouse models null for collagens XII or XIV were

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utilized. This was coupled with ultrastructural and morphometric analyses of fibril assembly, fibril packing, lamellar organization, and endothelial maturation. The roles of collagens XII and XIV in corneal structure were determined using measurements of corneal thickness at postnatal day (P) 30 and P60.

Results: Collagen XIV had a dynamic expression pattern in wild type (WT) corneal development. Corneas at P4 expressed the highest amount of collagen XIV with a sharp reduction by P10. Collagen XIV localized in the full thickness of the stroma at P4 and P14. At P30 and P90 there was less immuno-reactivity for collagen XIV in the WT stroma. The collagen XIV null stromas contained larger diameter fibrils when compared to P30 WT stromas. The null stromas also exhibited irregular spacing of fibrils. In the absence of collagen XIV there was an abnormal increase in corneal thickness. Unlike collagen XIV, collagen XII localized homogenously throughout the WT corneal stroma from P4 to P90. Collagen XII content was relatively constant in the cornea from P4 to P90. The collagen XII P30 null stromas contained areas of increased fibril density and disruption of lamellar organization. Corneal thickness increased in the absence of collagen XII at P60. Corneas deficient in Col12a1-/- and/or Col14a1-/- exhibited a delay in maturation. The null corneal endothelia retained vacuoles seen only in the immature WT P4 cornea. The P30 Col12a1-/- and Col14a1-/- endothelia had patchy localization of ZO-1 similar to that of an immature endothelium. There was an abnormal increase in thickness at P30 in the absence of collagens XII and XIV suggesting an increase in stromal hydration.

Conclusions: Collagen XIV regulates fibril assembly, and regular fibril packing in early stromal development. Collagen XII regulates fibril packing, lamellar assembly, stromal organization, and influences the keratocyte network. Both collagens XII and XIV regulate endothelial maturation and acquisition of function through interactions between the stroma and underlying endothelium. Understanding the mechanisms behind stromal organization and endothelial maturation will improve treatment of stromal and endothelial dystrophies, as well as other diseases that involve extracellular matrix-cell interactions mediated by FACIT collagens.

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