Publications -- Abe Clark
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/31655
This collection is limited to articles published under the terms of a creative commons license or other open access publishing agreement since 2016. It is not intended as a complete list of the author's works.
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Browsing Publications -- Abe Clark by Author "Bermudez, Jaclyn Y."
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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 HDAC Inhibitor-Mediated Epigenetic Regulation of Glaucoma-Associated TGFbeta2 in the Trabecular Meshwork(ARVO Journals, 2016-07-01) Bermudez, Jaclyn Y.; Webber, Hannah C.; Patel, Gaurang C.; Liu, Xiangyang; Cheng, Yi-Qiang; Clark, Abbot F.; Mao, WeimingPURPOSE: Elevated intraocular pressure (IOP) in primary open-angle glaucoma (POAG) results from glaucomatous damage to the trabecular meshwork (TM). The glaucoma-associated factor TGFbeta2 is increased in aqueous humor and TM of POAG patients. We hypothesize that histone acetylation has a role in dysregulated TGFbeta2 expression. METHODS: Protein acetylation was compared between nonglaucomatous TM (NTM) and glaucomatous TM (GTM) cells using Western immunoblotting (WB). Nonglaucomatous TM cells were treated with 10 nM thailandepsin-A (TDP-A), a potent histone deacetylase inhibitor for 4 days. Total and nuclear proteins, RNA, and nuclear protein-DNA complexes were harvested for WB, quantitative PCR (qPCR), and chromatin immunoprecipitation (ChIP) assays, respectively. Paired bovine eyes were perfused with TDP-A versus DMSO, or TDP-A versus TDP-A plus the TGFbeta pathway inhibitor LY364947 for 5 to 9 days. Intraocular pressure, TM, and perfusate proteins were compared. RESULTS: We found increased acetylated histone 3 and total protein acetylation in the GTM cells and TDP-A treated NTM cells. Chromatin immunoprecipitation assays showed that TDP-A induced histone hyperacetylation associated with the TGFbeta2 promoter. This change of acetylation significantly increased TGFbeta2 mRNA and protein expression in NTM cells. In perfusion-cultured bovine eyes, TDP-A increased TGFbeta2 in the perfusate as well as elevated IOP. Histologic and immunofluorescent analyses showed increased extracellular matrix and cytoskeletal proteins in the TM of TDP-A treated bovine eyes. Cotreatment with the TGFbeta pathway inhibitor LY364947 blocked TDP-A-induced ocular hypertension. CONCLUSIONS: Our results suggest that histone acetylation has an important role in increased expression of the glaucoma-associated factor TGFbeta2. Histone hyperacetylation may be the initiator of glaucomatous damage to the TM.Item TGFbeta2 Induces the Formation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells Through the Smad and Non-Smad Dependent Pathways(ARVO Journals, 2017-02) Montecchi-Palmer, Michela; Bermudez, Jaclyn Y.; Webber, Hannah C.; Patel, Gaurang C.; Clark, Abbot F.; Mao, WeimingPurpose: Increased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor beta2 (TGFbeta2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFbeta2 signaling pathways in CLAN formation. Methods: Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGFbeta2, with or without the inhibitors of TGFbeta receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFbeta2 plus inhibitors for 10 days or pretreated with TGFbeta2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4',6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett's post hoc test. Results: TGFbeta2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFbeta receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFbeta receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption. Conclusions: TGFbeta2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers.Item The Role of Wnt/beta-Catenin Signaling and K-Cadherin in the Regulation of Intraocular Pressure(ARVO Journals, 2018-03) Webber, Hannah C.; Bermudez, Jaclyn Y.; Millar, J. Cameron; Mao, Weiming; Clark, Abbot F.Purpose: Wnt/beta-catenin signaling in the trabecular meshwork (TM) is required for maintaining normal intraocular pressure (IOP), although the mechanism(s) behind this are unknown. We hypothesize that Wnt/beta-catenin signaling regulates IOP via beta-catenin's effects on cadherin junctions. Methods: Nonglaucomatous primary human TM (NTM) cells were treated with or without 100 ng/ml Wnt3a, 1 mug/ml sFRP1, or both for 4 to 48 hours. Cells were immunostained for beta-catenin, total cadherins, or cadherin isoforms. Membrane proteins or whole-cell lysates were isolated for Western immunoblotting and probed for cadherin isoforms. RNA was extracted for cDNA synthesis and qPCR analysis of cadherin expression. Some NTM cells were cultured on electric plates for cell impedance assays. Ad5.CMV recombinant adenoviruses encoding K-cadherin, and/or sFRP1 were injected into eyes of 4- to 6-month-old female BALB/cJ mice (n = 8-10). Conscious IOPs were assessed for 35 days. Results: Upon Wnt3a treatment, total cadherin expression increased and beta-catenin accumulated at the TM cell membrane and on processes formed between TM cells. qPCR showed that Wnt3a significantly increased K-cadherin expression in NTM cells (P < 0.01, n = 3), and Western immunoblotting showed that Wnt3a increased K-cadherin in NTM cells, which was inhibited by the addition of sFRP1. Cell impedance assays showed that Wnt3a treatment increased transcellular resistance and anti-K-cadherin siRNA decreased transcellular resistance (P < 0.001, n = 4-6). Our in vivo study showed that K-cadherin significantly decreased sFRP1-induced ocular hypertension (P < 0.05, n = 6). Western immunoblotting also showed that K-cadherin alleviated sFRP1-induced beta-catenin decrease in mouse anterior segments. Conclusions: Our results suggest that cadherins play important roles in the regulation of TM homeostasis and IOP via the Wnt/beta-catenin pathway.