Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21683
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Browsing Eye / Vision by Author "Clark, Abbot"
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Item Application of the CRISPR interference method in regulating TGFβ2 in the trabecular meshwork(2017-03-14) Nguyen, Nghia; Webber, Hannah; Bermudez, Jaclyn; Clark, Abbot; Mao, Weiming; Goan, DanielIntroduction: Glaucoma is an eye disease that damages the optic nerve and leads to gradual loss of vision. Glaucoma is the second leading cause of blindness globally. The trabecular meshwork (TM) is an ocular tissue responsible for controlling and drainage the aqueous humor which is a fluid that fills the eye. In primary open angle glaucoma (POAG), the most frequent type of glaucoma, there is a dysfunction within the TM that decreases the outflow of aqueous humor and elevates intraocular pressure (IOP). Transforming growth factor beta 2 (TGFβ2) is a protein that controls cell growth, differentiation, proliferation, and apoptosis. Many studies have shown that elevated TGFβ2 induces glaucoma phenotypes in the eye, including elevated IOP. Therefore, lowering TGFβ2 levels in the TM is a potential therapeutic strategy for treating glaucomatous changes in the TM as well as lowering IOP. Since our published study showed that elevated TGFβ2 is likely due to histone hyperacetylation, the purpose of this study was to determine whether the novel CRISPR interference technology, which is able to deacetylate histones in a gene-specific manner, is suitable for the manipulation of TGFβ2 levels in the TM. Methods: Four sets of oligos were designed close to the transcriptional start site of the TGFβ2 gene using the online CRISPR sgRNA design tool (http://crispr.mit.edu/) for the construction of sgRNAs. These oligos were sub-cloned into the target sgRNA expression vector (Addgene). The dCas9-KRAB expression vector was purchased from Addgene. The sgRNA expression vector and dCas9-KRAB vector were co-transfected in transformed human TM cells (GTM3). Four days after transfection, we isolated mRNA and protein for quantitative PCR (qPCR) and Western immunoblotting analyses. Results: The expression of dCas9-KRAB and/or sgRNA did not show toxicity to GTM3 cells. qPCR analysis showed that the 2 two-vector system dramatically repressed the level of TGFβ2 in GTM3 cells. Conclusions: The CRISPR/dCAS9 interference method is effective in lowering the level of TGFβ2 in the HTM. Further studies are required to determine the specificity and suitability of this technology in other genes and primary human TM cells.Item C1q induction and glial activation following optic nerve injury(2017-03-14) Liu, Yang; Clark, Abbot; Allums, ElliottPurpose: Complement protein 1 subunit q (C1q) is a component of the C1 complex of the classical pathway of complement activation. It plays a role in synaptic development and pruning of central nervous system, as well as in the pathogenesis of various neurodegenerative diseases. In this study, we characterized C1q expression in C57BL/6J mice in an optic nerve crush (ONC) model of neurodegeneration. We also examined glial activation to determine possible sources of the increased C1q expression. Methods: Acute injury was induced in adult C57BL/6J mice by intraorbital ONC performed approximately 1 mm posterior to the optic nerve head with self-closing forceps for four seconds. C1q expression and glial activation (GFAP) was determined at 3 and 7 days post ONC by immunohistochemistry (IHC) as well as Western Blotting. Results: C1q expression increased in the crush site in the optic nerve, the inner plexiform layer (IPL) and the outer plexiform layer (OPL) of the retina 3 days after ONC. C1q expression further increased 7 days after ONC in the crush site, IPL, OPL, as well as the ganglion cell layer (GCL). Optic nerve injury increased glial fibrillary acidic protein (GFAP) expression in the GCL layer, extending through the retinal layers, 7 days post ONC and ED1 expression in the crush site 3 and 7 days following ONC. Conclusions: This study shows that C1q may play a role in neurodegeneration and could have potential as a therapeutic target. Glial cells may be responsible for the increased expression in C1q following ONC.Item Crosstalk Between Transforming Growth Factor Beta-2 and Toll-Like Receptor 4 in the Trabecular Meshwork(2017-03-14) Medina-Ortiz, Wanda E.; Curry, Stacy; Luan, Tomi; Clark, Abbot; McDowell, Colleen; Hernandez, HumbertoPurpose: The trabecular meshwork (TM) plays an important role in the regulation of aqueous humor outflow and intraocular pressure (IOP). Regulation of the ECM by TGFβ2 in the TM and toll-like receptor 4 (TLR4) in fibrogenesis has been extensively studied. Here, we investigate the role of TGFβ2-TLR4 signaling crosstalk and BMP/activin membrane-bound inhibitor (BAMBI) in the regulation of the TM ECM and ocular hypertension. Methods: TLR4 expression was evaluated in cross-sections of human donor eyes, primary human TM cells, and dissected mouse TM rings. TM cells were treated with TGFβ2 (5ng/ml), TLR4 inhibitor (TAK-242, 15mM), and/or TLR4 ligand (cFN-EDA, 10mg/mL). A/J (n=13), AKR/J (n=7), BALBc/J (n=8), C3H/HeJ (n=20), and C3H/HeOuJ (n=10) were injected intravitreally with Ad5.hTGFβ2. Further, B6;129S1-Bambitm1Jian/J mice were injected intravitreally with either Ad5.TGFβ2 (n=10), Ad5.Cre (n=9), or Ad5.TGFβ2 + Ad5.Cre (n=10). The uninjected contralateral eyes served as controls. Mouse TM (MTM) cells were isolated from B6;129S1-Bambitm1Jian/J mice using magnetic beads and transduced with Ad5.TGFβ2 or Ad5.Cre in cell culture. Results: TLR4 is expressed in the human and mouse TM. Inhibition of TLR4 signaling in the presence of TGFβ2 decreases fibronectin expression. Activation of TLR4 by cFN-EDA in the presence of TGFβ2 further increases fibronectin, laminin, and collagen-1 expression, and TLR4 signaling inhibition blocks this effect. Ad5.hTGFβ2 induces ocular hypertension in wild-type mice but has no effect in Tlr4 mutant (C3H/HeJ) mice. Ad5.Cre, Ad5.TGFβ2, or Ad5.TGFβ2 + Ad5.Cre each induced ocular hypertension significantly throughout the time course compared to uninjected control eyes. Bambi knockdown by Ad5.Cre leads to increased fibronectin expression in MTM cells. Conclusions: Here we show a TGFβ2-TLR4 crosstalk pathway that we hypothesize is regulated by TGFβ2 negative regulator BAMBI. Conditional knockdown of BAMBI in the TM with Ad5.Cre induces fibronectin expression, reduces aqueous humor outflow facility and causes ocular hypertension. These data provide a novel pathway involved in the development of glaucomatous TM damage and provide potential new targets to lower IOP.Item Effects of Wnt/beta-catenin and SMAD/TGF-beta crosstalk on cadherins in the trabecular meshwork(2017-03-14) Mao, Weiming; Clark, Abbot; Webber, HannahPurpose: We have shown cross-inhibition of the primary open angle glaucoma (POAG)-associated Wnt/beta-catenin and SMAD/TGFb signaling pathways in trabecular meshwork (TM) but the downstream effects of this crosstalk are unknown. Wnt transcription factor and cadherin accessory protein, beta-catenin, plays a role in cadherins junction stability. We studied the role of Wnt/beta-catenin and SMAD/TGFb signaling on cadherins junctions in the TM and TM cell adhesion. Methods: Confluent primary TM (NTM) cells (donated from Alcon) were treated for 2 or 24 hours with or without 100ng/mL Wnt3a or 5ng/mL TGFb2 and their membrane-bound protein, conditioned media, and total protein were isolated for western immunoblotting. Samples were probed for fibronectin (FN), PAI-1, p-Smad3, beta-catenin, GAPDH, Pan-, K-, or OB-cadherin. Some NTM cells were grown to 80% confluence then transfected with 0.3nM or 0.5nM K-cadherin, OB-cadherin, or non-targeting siRNA. Forty-eight or 72 hours after transfection, cells were harvested for western immunoblotting or immunofluorescence. NTM cells were plated for the Acea iCelligence system to determine cellular impedance with data collected every 30 minutes to establish baseline. After 24 hours, culture medium was replaced with transfection mixes as described. Cell impedance was continuously measured for an additional 96 hours. Bright field images of the cells were taken. Results: Wnt3a treatment resulted in increased K-cadherin expression. Co-treatment of Wnt3a and TGFb2 decreased the expression of K-cadherin. Three days after transfection with 0.5nM K-cadherin siRNA, decreased K-cadherin expression was observed in both whole cell lysate and membrane-bound fractions. Transfection with K-cadherin siRNA decreased NTM cellular impedance compared to non-targeting siRNA control. Conclusions: Crosstalk between Wnt/beta-catenin and SMAD/TGFb signaling pathways in the TM may regulate the expression of cadherins as well as NTM cell adhesion.Item Glucocorticoid receptor GRβ regulates glucocorticoid-induced ocular hypertension and glaucoma in mice(2017-03-14) Liu, Yang; Millar, J. Cameron; Clark, Abbot; Patel, GaurangPurpose: Glucocorticoid (GC) induced ocular hypertension (OHT) is a serious side effect of prolonged GC therapy and if left untreated it can lead to iatrogenic glaucoma and permanent vision loss. The Alternatively spliced isoform of glucocorticoid receptor GRβ acts as a dominant negative regulator of GC activity. Our previous studies have shown that GRβ regulates GC responsiveness and that overexpressing GRβ in trabecular meshwork (TM) cells inhibits GC-induced and glaucomatous damage in TM cells. The purpose of this study was to determine whether increased expression of GRβ can reverse GC-induced OHT in mice. Methods: Mouse trabecular meshwork cells (MTM) were transduced with Ad5.null or Ad5.hGRβ expression vectors at MOI-50. After 24 hours MTM cells were treated with dexamethasone (DEX) or vehicle control (0.1% ethanol). To generate GC-OHT, C57BL/6J mice received weekly bilateral periocular (administrated through conjunctival fornix) injections of dexamethasone acetate (DEX-Ac, 200ug/eye). Several weeks after DEX-Ac administration, mouse eyes were injected intravitreally with Ad5.null or Ad5.hGRβ expression vectors (3x107 pfu/eye) to transduce the TM. Nighttime intraocular pressure (IOP) was measured using a TonoLab rebound tonometer, and outflow facilities were measured in living mice using our constant flow infusion technique. Fibronectin and collagen I expression were evaluated using immunoblotting of mouse anterior segment tissues. The unpaired Student’s t-test (2-tailed) and One-way ANOVA were used for statistical analysis. Results: DEX treatment of MTM cells increased fibronection expression, whereas transduction of MTM cells with Ad5.hGRβ maintained fibronectin expression at control levels as shown by immunocytochemistry. DEX-Ac significantly increased IOP from days 3-44 (n=23, p Conclusion: Overexpression of GRβ in the TM of mouse eyes reversed GC-OHT. GRβ gene therapy may be a useful therapeutic approach to treat GC-OHT and glaucoma.Item Inhibition of Transforming Growth Factor-β2 Signaling Prevents ECM Remodeling, Endoplasmic Reticulum Stress and Ocular Hypertension in Steroid-induced Glaucoma(2017-03-14) Maddineni, Prabhavathi; Patel, Pinkal; Millar, Cameron; Phan, Tien; Searby, Charles; Clark, Abbot; Sheffield, Val; Zode, Gulab; Kasetti, RameshPurpose: Ocular hypertension is a serious side effect of glucocorticoid (GC) therapy. Abnormal accumulation of extracellular matrix (ECM) and chronic endoplasmic reticulum (ER) stress in the trabecular meshwork (TM) is associated with GC-induced ocular hypertension. In the present study, we examined the role of TGFβ2 signaling in dexamethasone (Dex)-induced ECM remodeling, ER stress and ocular hypertension. Methods: Conscious IOP and outflow facility was measured in C57 mice treated with vehicle or Dex eye drops up to 7-weeks. TGFβ2 & fibronectin levels in the aqueous humor (AH) were analyzed by Western blotting. Effect of inhibition of TGFβ signaling on Dex-induced ER stress and ECM accumulation was examined by Western blot, immunostaining & Smad reporter assay in TM cells treated with or without TGFβ signaling inhibitors (SIS3 and LY364947). To further examine the role of TGF-β2 signaling, IOP, ECM and ER stress was examined in WT or Smad3-/- mice treated with Dex. Results: Dexamethasone (Dex) mediated reduction of outflow facility and IOP elevation is associated with increased abnormal extracellular matrix (ECM) accumulation in the TM, inducing ER stress. Biochemical analysis of the aqueous humor samples from Dex-treated eyes revealed significantly increased bioactive form of transforming growth factor-β2 (TGF-β2), a major regulator of ECM in the TM. Dex treatment increased both precursor and bioactive form of TGF-β2 in the conditioned medium and activated TGF-β2-induced Smad signaling pathway in primary human TM cells as evident from increased phosphorylation of Smad3 and increased Smad luciferase activity. Inhibition of TGF-β2 signaling significantly reduced Dex-induced abnormal intracellular ECM accumulation and ER stress in human TM cells. Smad3-/- mice, which are required for TGF-β2 signaling, protected from Dex-induced ocular hypertension, ER stress and abnormal ECM accumulation further indicating the role of TGF-β2 signaling in GC-induced glaucoma. Interestingly, knock out of ER stress-induced transcriptional factors, ATF4 and CHOP prevented activation of TGF-β2 signaling and also reduced intracellular ECM accumulation in the TM, thus preventing Dex-induced ER stress and IOP elevation. Conclusions: These studies indicate that Dex-induced TGF-β2 signaling is responsible for ECM remodeling, ER stress and elevation of IOP in GC-induced glaucoma.Item Tissue Transglutaminase Mediated Ocular Hypertension and Effects of a Small Molecule Crosslinking Modulator(2017-03-14) Millar, Cameron; Clark, Abbot; Raychaudhuri, UrmimalaPurpose: Transforming Growth Factor-β2 (TGF-β2) induces expression of the crosslinking enzyme tissue transglutaminase (TGM2) in human trabecular meshwork (TM) cells. We studied (1) whether TGM2 overexpression in the TM can increase intraocular pressure (IOP) and decrease aqueous humor (AH) outflow facility (C) in mice and (2) whether a small molecule TGM2 inhibitor can decrease ECM crosslinking in-vitro. Methods: 2μl of the expression vector Ad5.CMV.TGM2 (1-50 × 106 pfu) was injected intravitreally (OS) in BALBc/J (n=18) or C57BL/6J mice (n=9), while contralateral eyes served as uninjected controls. Daytime conscious IOPs were measured (Tonolab) twice/week. C was measured following IOP elevation in BALBc/J (n=6) and C57BL/6J (n=3) mice. In vitro, primary human glaucoma TM cells (GTM 125, GTM 60 A and GTM 46) were treated with a small molecule TGM2 inhibitor (5nM). Results: Ad5.CMV.TGM2 injection significantly elevated IOP, where BALBc/J showed maximum IOP at Day 19, [15.86 mmHg (injected) vs. 10.7 mmHg (control), (p Conclusions: TGM2 overexpression in the mouse TM significantly elevates IOP and decreases the AH outflow facility. A TGM2 inhibitor decreased crosslinking in TM primary cells. In future, we will study the role of TGM2 and the TGM2 small molecule inhibitor in TGFβ2 induced ocular hypertension.Item Transforming Growth Factor β2 Regulates the Expression of microRNAs (miRNAs) in Human Optic Nerve Head Cells(2017-03-14) Tovar-Vidales, Tara; Clark, Abbot; Lopez, NavitaPurpose: microRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that epigenetically regulate post-transcriptional gene expression. miRNAs are known to modulate cellular functions such as extracellular matrix (ECM) turnover. There is evidence that dysregulation of miRNA expression has a role in the pathogenesis of fibrotic diseases including glaucoma. Glaucoma is the leading cause of irreversible blindness and is associated with fibrotic changes to the optic nerve head (ONH), the initial site of glaucomatous damage to the retina and optic nerve. Our previous study showed that expression of the pro-fibrotic cytokine TGFβ2 is elevated in the ONH of glaucoma eyes compared to age-matched normal eye. However, there currently is little knowledge regarding the roles of miRNAs in the ONH. The purpose of this study was to determine if there are differences in expression of pro-fibrotic and anti-fibrotic miRNAs in normal ONH cells treated with or without TGFβ2. Methods: Primary human ONH cell strains derived from normal donor eyes were grown to 100% confluency. ONH cells were treated with 5ng/ml TGFβ2 or with control medium for 24hrs. RNA was isolated and cDNA synthesis performed for miRNA qPCR arrays to compare expression levels of pro-fibrotic and anti-fibrotic miRNAs in normal human ONH cells treated with or without TGFβ2. Results: Normal ONH cells exposed to TGFβ2 showed that several anti-fibrotic miRNAs were downregulated (hsa-miR-107, hsa-miR-132-3p, hsa-miR-141-3p hsa-miR-18a-5p, hsa-miR-194-5p, hsa-miR-204-5p) compared to control cells. In contrast, only one pro-fibrotic miRNA was upregulated (hsa-miR-34a-5p) in ONH cells treated with TGFβ2 compared to control. The most prominent targets of these miRNAs include connective tissue growth factor (CTGF), gremlin 2 and lysyl oxidase-like 3 (LOX-L3). Conclusions: Our results suggest that miRNAs expressed by ONH cells may be regulated by TGFβ2. These miRNAs may target CTGF, crosslinking enzymes and BMP antagonists to modify the ECM in the ONH.