The mature, functioning cornea results from the synthesis and assembly of several collagenous matrices, such as Bowman's membrane, the stroma, and Descemet's membrane. Transparency depends on the small uniform diameter and regular spacing of collagen fibrils in the stroma, and is acquired by compacting the stroma and coalescing the collagen fibril bundles. This compaction is accompanied by an increase in the fibril-associated collagen, type XIV. This collagen has recently been shown to facilitate the movement of fibrils in hydrated collagen gels in vitro, and therefore in vivo it may have a similar function in facilitating compaction of the stroma. Our studies suggest that all tissues express type XIV collagen mRNA (e.g., corneal epithelium and stroma), but not all translate the mRNA (e.g., the protein is not expressed by corneal epithelium). To examine how transcription is regulated by the corneal epithelium (a non-expressor of the protein), and stroma (an expressor of protein) we will measure transcriptional activity using gene transfections with CAT constructs containing the type XIV collagen gene promoter. We will also examine how translational regulation is conferred by isolating and analyzing polysomes (intracellular structures containing translatable mRNAs) and monosomes (structures with non-translatable mRNAs) from corneal epithelia and stromas for the presence of type XIV collagen mRNAs. The expression of corneal proteoglycans varies during development. We have found 4 splice variants of type XIV collagen that may bind these proteoglycans with different affinities. We will test this using affinity coelectrophoresis. We will also use quantitative PCR to determine which variants are prevalent at different developmental stages, especially around the time that transparency is acquired. To assess the functional role of type XIV collagen in compaction, early corneas will be infected in ovo with a retroviral construct that synthesizes a truncated type XIV collagen polypeptide. This will create a dominant negative phenotype for the collagen XIV molecule, which should interfere with its normal function. Lastly, we have found that another fibril-associated collagen, type XII, is prevalent in the interfacial matrices of the cornea, where Bowman's membrane and Descemet's membrane adjoin the stroma. Immunohistochemistry and gene structural analysis suggest that in cornea a new isoform of the molecule exists, one which may provide structural stability to these matrices. We will use a PCR based strategy to obtain a cDNA for the corneal type XII isoform, to elucidate its structure.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Visual Sciences A Study Section (VISA)
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Fisher, Richard S
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Rutgers University
Schools of Pharmacy
New Brunswick
United States
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Gordon, Marion K; DeSantis-Rodrigues, Andrea; Hahn, Rita et al. (2016) The molecules in the corneal basement membrane zone affected by mustard exposure suggest potential therapies. Ann N Y Acad Sci 1378:158-165
DeSantis-Rodrigues, Andrea; Chang, Yoke-Chen; Hahn, Rita A et al. (2016) ADAM17 Inhibitors Attenuate Corneal Epithelial Detachment Induced by Mustard Exposure. Invest Ophthalmol Vis Sci 57:1687-98
Chang, Yoke-Chen; Wang, James D; Hahn, Rita A et al. (2014) Therapeutic potential of a non-steroidal bifunctional anti-inflammatory and anti-cholinergic agent against skin injury induced by sulfur mustard. Toxicol Appl Pharmacol 280:236-44
Chang, Yoke-Chen; Wang, James D; Svoboda, Kathy K et al. (2013) Sulfur mustard induces an endoplasmic reticulum stress response in the mouse ear vesicant model. Toxicol Appl Pharmacol 268:178-87
Zheng, Ruijin; Po, Iris; Mishin, Vladimir et al. (2013) The generation of 4-hydroxynonenal, an electrophilic lipid peroxidation end product, in rabbit cornea organ cultures treated with UVB light and nitrogen mustard. Toxicol Appl Pharmacol 272:345-55
Chao, Ming Wei; Po, Iris P; Laumbach, Robert J et al. (2012) DEP induction of ROS in capillary-like endothelial tubes leads to VEGF-A expression. Toxicology 297:34-46
Black, Adrienne T; Gordon, Marion K; Heck, Diane E et al. (2011) UVB light regulates expression of antioxidants and inflammatory mediators in human corneal epithelial cells. Biochem Pharmacol 81:873-80
Chao, Ming-Wei; Kozlosky, John; Po, Iris P et al. (2011) Diesel exhaust particle exposure causes redistribution of endothelial tube VE-cadherin. Toxicology 279:73-84
Gordon, Marion K; Desantis, Andrea; Deshmukh, Manjeet et al. (2010) Doxycycline hydrogels as a potential therapy for ocular vesicant injury. J Ocul Pharmacol Ther 26:407-19
Gordon, Marion K; Hahn, Rita A (2010) Collagens. Cell Tissue Res 339:247-57

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