Effects of dexamethasone for primary trabecular meshwork cell gene expression
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ABSTRACT: To clarify the effects of dexamethasone treatment for primary trabecular meshwork cell gene expression, which may relates to the pathophysiology of glucocorticoid-induced glaucoma Three lots (lot #2584, 3423 and 4973) of primary culture human trabecular meshwork (TM) cells were purchased from ScienCell Research Laboratories (Carlsbad, CA). The TM cells were treated with and without 100nM dexamethasone (DEX) for 14 days. Genomewide gene expression analysis was carried out using Agilent 8X60K array.
Project description:To identify genes whose expressions in primary human trabecular meshwork (TM) cell cultures are affected by the transcription factorfoxc1 and to identify genes that may have roles in glaucoma. Expression profiles derived using microarrays were compared between TM control cells and cells treated with foxc1 siRNAs. TM1 and TM2 cells were cultured in 6 well plates were transfected with either of two FOXC1 specific siRNAs or scrambled siRNA (as control). The scrambled siRNA treatment was performed in duplicate for each TM. Forty eight hours after exposure to siRNAs, cells were harvested and RNA was extracted. Considering the two siRNAs for FOXC1 transcription factor, the two scrambled siRNA treatments, and the two TM primary cultures, we performed 8 array hybridizations.
Project description:Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, a leading cause of blindness worldwide. Regulation of aqueous humor drainage through the TM, however, is poorly understood. The role of RhoA GTPase-mediated contractile activity, cell adhesive interactions, and gene expression in regulation of aqueous humor outflow was investigated using adenoviral vector-driven expression of constitutively active RhoA (RhoAV14). Organ cultured anterior segments from porcine eyes expressing RhoAV14 exhibited significant reduction of aqueous humor outflow. Cultured TM cells expressing RhoAV14 revealed strong contractile cell morphology, increased actin stress fibers and focal adhesions, along with increased levels of phosphorylated myosin II, and collagen IV, fibronectin and laminin. cDNA microarray analysis of RNA extracted from RhoAV14 expressing human TM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins, cytokines, integrins, cytoskeletal proteins and signaling proteins. Conversely, various ECM proteins stimulated robust increases in phosphorylation of myosin II, paxillin and focal adhesion kinase, and activated Rho GTPase and actin stress fiber formation in TM cells, indicating a potential regulatory feedback interaction between ECM-induced mechanical strain and Rho GTPase-induced isometric tension in TM cells. Collectively, these data demonstrate that sustained activation of Rho GTPase signaling in the aqueous humor outflow pathway increases resistance to aqueous humor outflow through the trabecular pathway by influencing the contractile force, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells. Keywords: Gene Expression Two condition experiment: Human trabecular mesh work cells infected with Adenivirus expressing GFP Vs Adenovirus expressing GFP and constitutively active RhoAV14
Project description:Trabecular meshwork (TM) tissue is subjected to constant mechanical stress due to the ocular pulse created by the cardiac cycle. This induces alterations in membrane lipids and associated cell-cell adhesion and cell-extracellular matrix (ECM) interactions triggering intracellular signaling responses to counter mechanical insults. A loss of such response can lead to elevated intraocular pressure (IOP), a major risk factor for primary open-angle glaucoma. The purpose of this study was to understand the response of TM under mechanical stretch using a multi-omics approach. We performed an unbiased mRNA sequencing for changes in transcripts, mass spectrometry (MS)-based quantitative proteomics for protein changes, and multiple reaction monitoring (MRM) profiling based MS and HPLC-based MS to identify lipid changes. With the help of the multi-omics data analysis on human TM cells under mechanical stress, we provide evidence for the regulation of TM actin cytoskeleton and ECM interactions at the mRNA and protein levels, which can further modulate the TM lipids contents effectively regulating the mechanical properties of TM membrane. Overall, in this study, we propose the mechanistic interplay of macromolecules to bring about a concerted cellular response in TM cells to achieve mechanotransduction and IOP regulation
Project description:Glucocorticoids with different chemical structures but similar glucocorticoid receptor potency regulate subsets of common and unique genes in human trabecular meshwork cells. Gene expression changes of human trabecular meshwork cells, TM 86 and TM 93, due to treatment with dexamethasone (Dex), fluocinolone acetonide (FA), and triamcinolone acetonide (TA).
Project description:The normal gene expression profiles of the tissues in the eye are a valuable resource for considering genes likely to be involved with disease processes. This is based on the assumption that transcript abundances in healthy tissue are correlated to the continued health of that tissue. Expression values were compared with publically available EST and RNA-sequencing resources. The estimated gene and exon level abundances are available online on the Ocular Tissue Database. Ten different tissues were obtained from 6 different individuals and RNA was pooled. The tissues included: retina, optic nerve head (ONH), optic nerve (ON), ciliary body (CB), trabecular meshwork (TM), sclera, lens, cornea, choroid/RPE and iris.