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Jaclyn Bermudez, Hannah C. Webber, Bartley Brown, Terry Braun, Abbot F Clark, Weiming Mao; Differential gene expression between trabecular meshwork cells of glucocorticoid responder and non-responder bovine eyes. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4703.
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© ARVO (1962-2015); The Authors (2016-present)
Glucocorticoid (GC) induced glaucoma (GIG) is a secondary glaucoma that results in ocular hypertension (OHT) and may lead to blindness. The trabecular meshwork (TM) is the primary site of injury that causes elevated aqueous humor outflow resistance and increased intraocular pressure (IOP), the primary risk factor for glaucoma. About 40% of the population develops GIG after GC treatment. However, the molecular mechanisms responsible for GC-mediated increased outflow resistance and elevated IOP in GIG are unknown. Here, we compared the genes that are differentially expressed in bovine responder and non-responder TM cells.
Paired bovine eyes were used for testing GC responsiveness and bovine TM (BTM) cell strain establishment. The anterior segment of one eye was perfusion-cultured with 100uM dexamethasone (DEX). The contralateral eye was used to establish BTM cell strains without prior perfusion. Eyes with an IOP increase greater than 2.82 mmHg were defined as GC-responders and the others as non-responders. We treated 3 responder and 3 non-responder BTM cell strains with either 0.1% ethanol (EtOH) as a vehicle control or 100uM DEX for 7 days. Proteins were extracted for western immunoblotting, and RNA was extracted for RNA sequencing (RNAseq) analysis. Gene expression was compared between responder and non-responder cells after comparisons between treatments. The UMD3.1 genome assembly of bovine was used for mapping and annotation. Tophat2 was used for mapping, Cuffquant for quantitation, and Cuffnorm and Cuffdiff for normalization and differential expression analyses. QPCR was used to validate differential RNA expression identified from sequencing data.
Western immunoblotting showed DEX-induced fibronectin expression in responder cells. RNAseq showed 328 genes differentially expressed in the presence of DEX in responders and non-responder cells, 607 genes in the presence of DEX in responder cells, and 94 genes with DEX in non-responder BTM cells. 27 pathways were found to be closely associated with DEX responsiveness. The RNAseq data were consistent with qPCR confirmation studies.
The gene expression profile is different between GC-responder and non-responder TM cells. The differentially expressed genes and associated pathways may play an important role in GC-induced IOP elevation, resulting in GIG.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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