Browsing by Subject "Blotting, Western"
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Item Crosstalk Between Transforming Growth Factor Beta-2 and Toll-Like Receptor 4 in the Trabecular Meshwork(ARVO Journals, 2022-03) Hernandez, Humberto; Medina-Ortiz, Wanda E.; Luan, Tomi; Clark, Abbot F.; McDowell, Colleen M.Purpose: The trabecular meshwork (TM) is involved in the outflow of aqueous humor and intraocular pressure (IOP) regulation. Regulation of the extracellular matrix (ECM) by TGFbeta2 signaling pathways in the TM has been extensively studied. Recent evidence has implicated toll-like receptor 4 (TLR4) in the regulation of ECM and fibrogenesis in liver, kidney, lung, and skin. Here, we investigated the role of TGFbeta2-TLR4 signaling crosstalk in the regulation of the ECM in the TM and ocular hypertension. Methods: Cross sections of human donor eyes, primary human TM cells in culture, and dissected mouse TM rings were used to determine Tlr4 expression in the TM. Trabecular meshwork cells in culture were treated with TGFbeta2 (5 ng/mL), TLR4 inhibitor (TAK-242, 15 muM), and a TLR4 ligand (cellular fibronectin isoform [cFN]-EDA). A/J (n = 13), AKR/J (n = 7), BALBc/J (n = 8), C3H/HeJ (n = 20), and C3H/HeOuJ (n = 10) mice were injected intravitreally with adenovirus 5 (Ad5).hTGFbeta2c226s/c228s in one eye, with the uninjected contralateral eye serving as a control. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Results: Toll-like receptor 4 is expressed in the human and mouse TM. Inhibition of TLR4 signaling in the presence of TGFbeta2 decreases fibronectin expression. Activation of TLR4 by cFN-EDA in the presence of TGFbeta2 further increases fibronectin, laminin, and collagen-1 expression, and TLR4 signaling inhibition blocks this effect. Ad5.hTGFbeta2c226s/c228s induces ocular hypertension in wild-type mice but has no effect in Tlr4 mutant (C3H/HeJ) mice. Conclusions: These studies identify TGFbeta2-TLR4 crosstalk as a novel pathway involved in ECM regulation in the TM and ocular hypertension. These data further explain the complex mechanisms involved in the development of glaucomatous TM damage.Item Expression of Mutant Myocilin Induces Abnormal Intracellular Accumulation of Selected Extracellular Matrix Proteins in the Trabecular Meshwork(Association for Research in Vision and Ophthalmology, 2016-11-01) Kasetti, Ramesh B.; Phan, Tien N.; Millar, J. Cameron; Zode, Gulab S.PURPOSE: Abnormal accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) is associated with decreased aqueous humor outflow facility and IOP elevation in POAG. Previously, we have developed a transgenic mouse model of POAG (Tg-MYOCY437H) by expressing human mutant myocilin (MYOC), a known genetic cause of POAG. The purpose of this study is to examine whether expression of mutant myocilin leads to reduced outflow facility and abnormal ECM accumulation in Tg-MYOCY437H mice and in cultured human TM cells. METHODS: Conscious IOP was measured at various ages of Tg-MYOCY437H mice using a rebound tonometer. Outflow facility was measured in 10-month-old Tg-MYOCY437H mice. Selected ECM proteins were examined in human TM-3 cells stably expressing mutant myocilin and primary human TM cells (n = 4) as well as in the TM of Tg-MYOCY437H mice by real-time PCR, Western blotting, and immunostaining. Furthermore, TM cells expressing WT or mutant myocilin were treated with 5 mM sodium 4-phenylbutyrate (PBA), and ECM proteins were examined by Western blot and immunostaining. RESULTS: Starting from 3 months of age, Tg-MYOCY437H mice exhibited significant IOP elevation compared with wild-type (WT) littermates. Outflow facility was significantly reduced in Tg-MYOCY437H mice (0.0195 mul/min/mm Hg in Tg-MYOCY437H vs. 0.0332 mul/min/mm Hg in WT littermates). Increased accumulation of fibronectin, elastin, and collagen type IV and I was observed in the TM of Tg-MYOCY437H mice compared with WT littermates. Furthermore, increased ECM proteins were also associated with induction of endoplasmic reticulum (ER) stress markers, GRP78 and CHOP in the TM of Tg-MYOCY437H mice. Human TM-3 cells stably expressing DsRed-tagged Y437H mutant MYOC exhibited inhibition of myocilin secretion and its intracellular accumulation compared with TM cells expressing WT MYOC. Expression of mutant MYOC in TM-3 cells or human primary TM cells induced ER stress and also increased intracellular protein levels of fibronectin, elastin, laminin, and collagen IV and I. In addition, TM-3 cells expressing mutant myocilin exhibited reduced active forms of matrix metalloproteinase (MMP)-2 and MMP-9 in conditioned medium compared with TM-3 cells expressing WT myocilin. Interestingly, both intracellularly accumulated fibronectin and collagen I colocalized with mutant myocilin and also with ER marker KDEL further suggesting intracellular accumulation of these proteins in the ER of TM cells. Furthermore, reduction of ER stress via PBA decreased selected ECM proteins in primary TM cells. CONCLUSIONS: These studies demonstrate that mutant myocilin induces abnormal ECM accumulation in the ER of TM cells, which may be responsible for reduced outflow facility and IOP elevation in myocilin-associated glaucoma.Item Glucocorticoid Receptor Transactivation Is Required for Glucocorticoid-Induced Ocular Hypertension and Glaucoma(ARVO Journals, 2019-05) Patel, Gaurang C.; Millar, J. Cameron; Clark, Abbot F.Purpose: Glucocorticoid (GC)-induced ocular hypertension (GC-OHT) is a serious side effect of prolonged GC therapy that can lead to glaucoma and permanent vision loss. GCs cause a plethora of changes in the trabecular meshwork (TM), an ocular tissue that regulates intraocular pressure (IOP). GCs act through the glucocorticoid receptor (GR), and the GR regulates transcription both through transactivation and transrepression. Many of the anti-inflammatory properties of GCs are mediated by GR transrepression, while GR transactivation largely accounts for GC metabolic effects and side effects of GC therapy. There is no evidence showing which of the two mechanisms plays a role in GC-OHT. Methods: GRdim transgenic mice (which have active transrepression and impaired transactivation) and wild-type (WT) C57BL/6J mice received weekly periocular dexamethasone acetate (DEX-Ac) injections. IOP, outflow facilities, and biochemical changes to the TM were determined. Results: GRdim mice did not develop GC-OHT after continued DEX treatment, while WT mice had significantly increased IOP and decreased outflow facilities. Both TM tissue in eyes of DEX-treated GRdim mice and cultured TM cells isolated from GRdim mice had reduced or no change in the expression of fibronectin, myocilin, collagen type I, and alpha-smooth muscle actin (alpha-SMA). GRdim mouse TM (MTM) cells also had a significant reduction in DEX-induced cytoskeletal changes, which was clearly seen in WT MTM cells. Conclusions: We provide the first evidence for the role of GR transactivation in regulating GC-mediated gene expression in the TM and in the development of GC-OHT. This discovery suggests a novel therapeutic approach for treating ocular inflammation without causing GC-OHT and glaucoma.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.