Browsing by Subject "Trabecular Meshwork / drug effects"
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Item ATF4 leads to glaucoma by promoting protein synthesis and ER client protein load(Springer Nature, 2020-11-05) Kasetti, Ramesh B.; Patel, Pinkal D.; Maddineni, Prabhavathi; Patil, Shruti; Kiehlbauch, Charles; Millar, J. Cameron; Searby, Charles C.; Raghunathan, Vijaykrishna; Sheffield, Val C.; Zode, Gulab S.The underlying pathological mechanisms of glaucomatous trabecular meshwork (TM) damage and elevation of intraocular pressure (IOP) are poorly understood. Here, we report that the chronic endoplasmic reticulum (ER) stress-induced ATF4-CHOP-GADD34 pathway is activated in TM of human and mouse glaucoma. Expression of ATF4 in TM promotes aberrant protein synthesis and ER client protein load, leading to TM dysfunction and cell death. These events lead to IOP elevation and glaucomatous neurodegeneration. ATF4 interacts with CHOP and this interaction is essential for IOP elevation. Notably, genetic depletion or pharmacological inhibition of ATF4-CHOP-GADD34 pathway prevents TM cell death and rescues mouse models of glaucoma by reducing protein synthesis and ER client protein load in TM cells. Importantly, glaucomatous TM cells exhibit significantly increased protein synthesis along with induction of ATF4-CHOP-GADD34 pathway. These studies indicate a pathological role of ATF4-CHOP-GADD34 pathway in glaucoma and provide a possible treatment for glaucoma by targeting this pathway.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.Item ID1 and ID3 are Negative Regulators of TGFbeta2-Induced Ocular Hypertension and Compromised Aqueous Humor Outflow Facility in Mice(ARVO Journals, 2021-05-03) Mody, Avani A.; Millar, J. Cameron; Clark, Abbot F.Purpose: In POAG, elevated IOP remains the major risk factor in irreversible vision loss. Increased TGFbeta2 expression in POAG aqueous humor and in the trabecular meshwork (TM) amplifies extracellular matrix (ECM) deposition and reduces ECM turnover in the TM, leading to a decreased aqueous humor (AH) outflow facility and increased IOP. Inhibitor of DNA binding proteins (ID1 and ID3) inhibit TGFbeta2-induced fibronectin and PAI-1 production in TM cells. We examined the effects of ID1 and ID3 gene expression on TGFbeta2-induced ocular hypertension and decreased AH outflow facility in living mouse eyes. Methods: IOP and AH outflow facility changes were determined using a mouse model of Ad5-hTGFbeta2C226S/C288S-induced ocular hypertension. The physiological function of ID1 and ID3 genes were evaluated using Ad5 viral vectors to enhance or knockdown ID1/ID3 gene expression in the TM of BALB/cJ mice. IOP was measured in conscious mice using a Tonolab impact tonometer. AH outflow facilities were determined by constant flow infusion in live mice. Results: Over-expressing ID1 and ID3 significantly blocked TGFbeta2-induced ocular hypertension (P < 0.0001). Although AH outflow facility was significantly decreased in TGFbeta2-transduced eyes (P < 0.04), normal outflow facility was preserved in eyes injected concurrently with ID1 or ID3 along with TGFbeta2. Knockdown of ID1 or ID3 expression exacerbated TGFbeta2-induced ocular hypertension. Conclusions: Increased expression of ID1 and ID3 suppressed both TGFbeta2-elevated IOP and decreased AH outflow facility. ID1 and/or ID3 proteins thus may show promise as future candidates as IOP-lowering targets in POAG.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.