Gene expression profiles in the trabecular meshwork and other tissues of the eye angle (iris, ciliary body, walls of Schlemm’s canal) are not very well described. This information is critical for a better understanding of the functions of these tissues and for identification of new genes associated with glaucoma. A cDNA library from the human trabecular meshwork has been constructed (in collaboration with Dr. Vincent Raymond, Laval University Medical Center, Canada). This human trabecular meshwork cDNA library is the first cDNA library obtained from isolated trabecular meshwork. Two thousand cDNAs were randomly sequenced from the 5’ end. Sequences with a length of at least 100 bp could be organized into 1027 groups. Two hundred twenty seven of these contained at least two clones. About 20% of the sequences corresponded to ESTs, novel or uncharacterized sequences. Several genes implicated in glaucoma (MYOC/TIGR, PITX2, CYP1B1b) were identified among the sequenced clones. cDNAs encoding MYOC/TIGR formed the third most abundant group of clones (13 cDNAs) in the library. Such high abundance of the MYOC/TIGR cDNA has never been reported in any characterized library and is consistent with its important role in the trabecular meshwork. Homeodomain-containing transcripton factor PITX2 was present 3 times among 2000 analyzed clones. Mutations in PITX2 gene are associated with Rieger syndrome and may lead to glaucoma. Potential binding sites for this transcription factor were identified in the proximal MYOC/TIGR promoters. Wild-type but not mutated forms of PITX2 were able to stimulate MYOC/TIGR promoter activity 10 fold in co-transfection experiments (in collaboration with Dr. M. Walter, University of Alberta, Canada). Gel-shift experiments indicated that PITX2 is able to interact with the MYOC/TIGR promoter. Together, these data connect two genes implicated in glaucoma for the first time. Transgenic mice overexpressing Myoc/Tigr in the eye (in collaboration with Dr. S. John, The Jackson Laboratory) and Myoc/Tigr knockout mice (in collaboration with Dr. R. Johnson, Anderson Cancer Center, Houston) were generated. A BAC clone containing at least 20 kb of the 5’- and 3’ flanking sequences of the mouse Myoc/Tigr gene was used to produce transgenic mice. Twenty transgenic lines were obtained. They gave variable levels of Myoc/Tigr gene expression in the eye. The best over-expressing line gave an 8-fold increase of Myoc/Tigr mRNA in the eye. However, it did not lead to detectable changes in eye morphology. Although the Myoc/Tigr mRNA and protein were absent in the eyes of Myoc/Tigr null mice as judged by northern and western blot analysis, the eye morphology and intraocular pressure were normal after one year of observations. We conclude that the increased level of wild-type Myoc/Tigr protein or its complete absence are not deleterious and do not lead to pronounced changes in intraocular pressure. Therefore, most glaucoma-causing mutations in the human MYOC/TIGR gene are probably gain-of-function mutations. A novel gene encoding olfactomedin-related protein was identified and named optimedin. The expression patterns of optimedin and Myoc/Tigr overlap and both proteins show similar intra- and extracellular localization. We demonstrated that optimedin and Myoc/Tigr are able to interact. These observations place optimedin as the first protein partner interacting with MYOC/TIGR and provide a new avenue for exploring MYOC/TIGR and glaucoma. Two rat models of glaucoma were used to study molecular changes taking place in the retina after experimental elevation of intraocular pressure (IOP). Elevation of IOP was achieved by a cauterization of episcleral veins or by an injection of concentrated saline solutions into the episcleral vein (in collaboration with Dr. J. Morrison, Casey Eye Institute). Affimetrix arrays were used to study changes in gene expression in the retina after high IOP induction. Several genes changing their expression have been identified. The role of these genes in the glaucoma progression is now analyzed.
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