Browsing by Subject "Glucocorticoids / pharmacology"
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Item A Comparison of Gene Expression Profiles between Glucocorticoid Responder and Non-Responder Bovine Trabecular Meshwork Cells Using RNA Sequencing(PLOS, 2017-01-09) Bermudez, Jaclyn Y.; Webber, Hannah C.; Brown, Bartley; Braun, Terry A.; Clark, Abbot F.; Mao, WeimingThe most common ocular side effect of glucocorticoid (GC) therapy is GC-induced ocular hypertension (OHT) and GC-induced glaucoma (GIG). GC-induced OHT occurs in about 40% of the general population, while the other 60% are resistant. This study aims to determine the genes and pathways involved in differential GC responsiveness in the trabecular meshwork (TM). Using paired bovine eyes, one eye was perfusion-cultured with 100nM dexamethasone (DEX), while the fellow eye was used to establish a bovine TM (BTM) cell strain. Based on maximum IOP change in the perfused eye, the BTM cell strain was identified as a DEX-responder or non-responder strain. Three responder and three non-responder BTM cell strains were cultured, treated with 0.1% ethanol or 100nM DEX for 7 days. RNA and proteins were extracted for RNA sequencing (RNAseq), qPCR, and Western immunoblotting (WB), respectively. Data were analyzed using the human and bovine genome databases as well as Tophat2 software. Genes were grouped and compared using Student's t-test. We found that DEX induced fibronectin expression in responder BTM cells but not in non-responder cells using WB. RNAseq showed between 93 and 606 differentially expressed genes in different expression groups between responder and non-responder BTM cells. The data generated by RNAseq were validated using qPCR. Pathway analyses showed 35 pathways associated with differentially expressed genes. These genes and pathways may play important roles in GC-induced OHT and will help us to better understand differential ocular responsiveness to GCs.Item BMP and Activin Membrane Bound Inhibitor Regulates the Extracellular Matrix in the Trabecular Meshwork(ARVO Journals, 2018-04) Hernandez, Humberto; Millar, J. Cameron; Curry, Stacy M.; Clark, Abbot F.; McDowell, Colleen M.Purpose: The trabecular meshwork (TM) has an important role in the regulation of aqueous humor outflow and IOP. Regulation of the extracellular matrix (ECM) by TGFbeta2 has been studied extensively. Bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) has been shown to inhibit or modulate TGFbeta2 signaling. We investigate the role of TGFbeta2 and BAMBI in the regulation of TM ECM and ocular hypertension. Methods: Mouse TM (MTM) cells were isolated from B6;129S1-Bambitm1Jian/J flox mice, characterized for TGFbeta2 and dexamethasone (DEX)-induced expression of fibronectin, collagen-1, collagen-4, laminin, alpha-smooth muscle actin, cross-linked actin networks (CLANs) formation, and DEX-induced myocilin (MYOC) expression. MTM cells were transduced with Ad5.GFP to identify transduction efficiency. MTM cells and mouse eyes were transduced with Ad5.Null, Ad5.Cre, Ad5.TGFbeta2, or Ad5.TGFbeta2 + Ad5.Cre to evaluate the effect on ECM production, IOP, and outflow facility. Results: MTM cells express TM markers and respond to DEX and TGFbeta2. Ad5.GFP at 100 MOI had the highest transduction efficiency. Bambi knockdown by Ad5.Cre and Ad5.TGFbeta2 increased fibronectin, collagen-1, and collagen-4 in TM cells in culture and tissue. Ad5.Cre, Ad5.TGFbeta2, and Ad5.TGFbeta2 + Ad5.Cre each significantly induced ocular hypertension and lowered aqueous humor outflow facility in transduced eyes. Conclusions: We show for the first time to our knowledge that knockdown of Bambi alters ECM expression in cultured cells and mouse TM, reduces outflow facility, and causes ocular hypertension. These data provide a novel insight into the development of glaucomatous TM damage and identify BAMBI as an important regulator of TM ECM and ocular hypertension.Item Glucocorticoid receptor GRbeta regulates glucocorticoid-induced ocular hypertension in mice(Springer Nature, 2018-01-16) Patel, Gaurang C.; Liu, Yang; Millar, J. Cameron; Clark, Abbot F.Prolonged glucocorticoid (GC) therapy can cause GC-induced ocular hypertension (OHT), which if left untreated progresses to iatrogenic glaucoma and permanent vision loss. The alternatively spliced isoform of glucocorticoid receptor GRbeta acts as dominant negative regulator of GR activity, and it has been shown that overexpressing GRbeta in trabecular meshwork (TM) cells inhibits GC-induced glaucomatous damage in TM cells. The purpose of this study was to use viral vectors to selectively overexpress the GRbeta isoform in the TM of mouse eyes treated with GCs, to precisely dissect the role of GRbeta in regulating steroid responsiveness. We show that overexpression of GRbeta inhibits GC effects on MTM cells in vitro and GC-induced OHT in mouse eyes in vivo. Ad5 mediated GRbeta overexpression reduced the GC induction of fibronectin, collagen 1, and myocilin in TM of mouse eyes both in vitro and in vivo. GRbeta also reversed DEX-Ac induced IOP elevation, which correlated with increased conventional aqueous humor outflow facility. Thus, GRbeta overexpression reduces effects caused by GCs and makes cells more resistant to GC treatment. In conclusion, our current work provides the first evidence of the in vivo physiological role of GRbeta in regulating GC-OHT and GC-mediated gene expression in the TM.