Eye / Vision

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Glucocorticoid receptor GRβ regulates glucocorticoid-induced ocular hypertension and glaucoma in mice
(2017-03-14) Liu, Yang; Millar, J. Cameron; Clark, Abbot; Patel, Gaurang
Purpose: 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.
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, Ramesh
Purpose: 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.
Tissue Transglutaminase Mediated Ocular Hypertension and Effects of a Small Molecule Crosslinking Modulator
(2017-03-14) Millar, Cameron; Clark, Abbot; Raychaudhuri, Urmimala
Purpose: 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.
Crosstalk Between TGFβ2 and TLR-4 Signaling Pathways in the Glaucomatous Trabecular Meshwork
(2017-03-14) Hernandez, Humberto; Curry, Stacy; McDowell, Colleen; Roberts, Amanda
Purpose: Glaucoma is a heterogeneous group of optic neuropathies that increases extracellular matrix (ECM) proteins in the trabecular meshwork (TM) and leads to thinning of the retinal nerve fiber layer and vision loss. TM regulates aqueous humor outflow and intraocular pressure (IOP). In this study, we employ experimental cell culture methods to determine whether the crosstalk between transforming growth factor beta 2 (TGFβ2) and toll-like receptor 4 (TLR4) signaling pathways regulate ECM production. We hypothesize that TGFβ2-TLR4 crosstalk is a pathway that assist TLR4 ligands to augment TGFβ2 signaling and regulate ECM production in the TM. Methods: Transformed human TM (GTM3) and primary human TM (HTM) cells were grown to confluency. Cells were pre-treated with TAK-242 for 2 hours followed by treatment of TGFβ2 for 24 hours (RNA) or 48 hours (protein). To activate TLR4, HTM cells were plated on dishes pre-coated with cellular fibronectin (cFN) containing the FN-EDA isoform in the presence or absence of TGFβ2. ECM expression was assessed by western immunoblotting and quantified by densitometry. Results: These experiments revealed a TGFβ2-TLR4 signaling crosstalk that regulates ECM production. TGFβ2 treatment in GTM3 cells enhanced fibronectin (FN) and collagen-1 mRNA expression and FN protein expression in the condition medium and cell lysate, while TAK-242 significantly blocked this effect. Activating TLR4 using cFN-EDA alone increased FN expression in HTM3 cells. TGFβ2 with cFN-EDA treatment significantly enhanced FN expression compared to TGFβ2 alone. TLR4 inhibitor TAK-242 significantly blocked TGFβ2 and cFN-EDA induced ECM changes. For future studies, I will use low molecular weight hyaluronan (LMWH) as a TLR4 activator. I expect the LMWH to enhance the effect of TGFβ2 induced ECM production. Conclusions: These studies identified TGFβ2-TLR-4 crosstalk as a novel pathway involved in ECM regulation in the TM. These data provide novel targets to further explore the molecular mechanism involved in glaucomatous TM development.
Protective effect of ID protein on TGFβ2 induced fibrosis in human trabecular meshwork cells: implication for developing a glaucoma therapy. (2017)
(2017-03-14) Millar, J. Cameron; Wordinger, Robert J.; Clark, Abbot F.; Mody, Avani
Purpose: Primary open angle glaucoma (POAG) is one of the most prevalent forms of glaucoma. Elevated transforming growth factor β2 (TGFβ2) expression in the trabecular meshwork (TM) causes increased deposition of extracellular matrix (ECM) and prevents ECM turnover by increasing expression of plasminogen activator inhibitor-I (PAI-1), leading to elevated intraocular pressure (IOP) in POAG patients. In fibrotic pulmonary diseases, bone morphogenetic proteins (BMPs) induce inhibitor of DNA binding proteins (ID1, ID3), which are transcription regulators known to suppress bHLH promoter activity, thereby inhibiting TGFβ induced ECM production. However, in TM cells the underlying mechanism for BMP4 inhibition of TGFβ2-induced fibrosis remains undetermined. Our study will determine whether ID1and ID3 proteins are downstream targets of BMP4, which attenuates TGFβ2 induction of ECM proteins in cultured human TM cells. Methods: Primary human TM (HTM) cells were treated with BMP4 for 0-48hr, and ID1 and ID3 mRNA and protein expression was determined by Q-PCR and western immunoblotting. HTM cells were treated with a BMPR inhibitor to confirm that BMP4 signaling is necessary for induction of ID1 and ID3 protein expression. GTM3 cells were transfected with ID1 or ID3 vectors to determine their inhibitory effects on TGFβ2 induced fibronectin and PAI-1 protein expression. Ad5-CMV-hId1 and Ad5-CMV-hID3 viral vectors along with Ad5-CMV-hTGFb2C226S/C288S were injected intravitreally to observe IOP changes in female BALB/cJ mice. Results: BMP4 significantly induced early expression of ID1 and ID3 mRNA (p Conclusions: BMP4 induced ID1 and ID3 suppresses TGFβ2 induced fibronectin and PAI-1. This makes ID1 and/or ID3 strong candidates for developing disease-modifying IOP lowering therapies.
C1q induction and glial activation following optic nerve injury
(2017-03-14) Liu, Yang; Clark, Abbot; Allums, Elliott
Purpose: 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.
Endothelin Mediated Changes in Gene Expression Determined by RNA-sequencing of the Translatome in Primary Retinal Ganglion Cells.
(2017-03-14) Krishnamoorthy, Raghu; Stankowska, Dorota; Chaphalkar, Renuka M.
Purpose: Endothelin treatment has been shown to produce increased cell death in primary RGCs, however the underlying changes in gene expression are not completely understood. The purpose of the study was to assess endothelin-mediated changes in mRNA expression that occur at the translational level. Methods: Primary RGCs were isolated from post-natal day 5 rat pups by immunopanning with an antibody to Thy1.1. RGCs obtained were allowed to attach and maintained for 7 days for neurite outgrowth to occur. The RGCs were either untreated or treated with endothelin-1 (100 nM) for 24 h in trophic factor-free medium. Following brief incubation with cycloheximide to inhibit protein synthesis, total polysomes were isolated by magnesium precipitation and polysomal RNA was extracted using the Trizol reagent. Libraries for RNA-Seq were prepared with KAPA Stranded RNA-Seq Kit. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on Illumina Hiseq3000/4000 for a pair end 150 run. The reads were first mapped to the latest UCSC transcript set using STAR version 2.4.1d and the gene expression level was quantified to annotation model (Partek E/M). Differentially expressed genes were identified using differential gene expression (GSA) algorithm in Partek. Genes showing altered expression with p Results: Analysis of gene ontology of the changes in gene expression revealed a significant increase in expression of several mitochondrial genes including mitochondrial intermediate peptidase (MIPEP) (3.3-fold), cytochrome c oxidase assembly factor (SURF1) (8.7-fold) and Apolipoprotein O Like (APOOL) (7.5-fold). On the other hand, a decrease in expression of mitochondrial genes cytochrome c oxidase subunit 4I2 (8-fold), cytochrome c oxidase 6B2 (8-fold), and DNA polymerase gamma 2 (POLG2) (7-fold) was observed. Conclusions: Analysis of the translatome offers a glimpse into de novo protein synthesis which is an important manifestation of changes in gene expression. Endothelin treatment produced changes in several key regulators of mitochondrial metabolism and bioenergetics which could be indicative of their involvement in neurodegeneration in glaucoma.
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, Navita
Purpose: 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.
A Small Chemical Chaperone, Sodium 4 Phenylbutyrate Inhibits TGFβ2-Induced ECM Remodeling in Human TM Cells
(2017-03-14) Kasetti, Ramesh; Phan, Tien; Maddineni, Prabhavathi; Zode, Gulab; Patel, Pinkal
Purpose: The pathological mechanisms underlying increased outflow resistance at the trabecular meshwork (TM) that is responsible for elevating intraocular pressure (IOP) have not been fully delineated. We have previously shown that progressive accumulation of unfolded proteins and endoplasmic reticulum (ER) stress play an important role in the pathophysiology of glaucomatous TM damage in myocilin-associated POAG. However, it is not understood whether other glaucoma factors lead to similar pathological ER stress in the TM, leading to IOP elevation. Transforming growth factor β2 (TGFβ2) is known to induce abnormal extracellular matrix (ECM) deposition in the TM cells, which may be responsible for IOP elevation. Here, we examined whether TGFβ2 induces ER stress and whether reducing ER stress via a small chemical chaperone, sodium 4-phenylbutyrate (PBA) reduces TGFβ2–induced ECM remodeling. Methods: Human GTM-3 cells or primary TM cells (n=2) were treated with TGFβ2 (5ng/ml) with or without 5mM PBA for 48 hours. Total cellular lysates, conditioned medium, and fixed cells were examined for ECM and ER stress markers by Western blotting and immunostaining. We also used RT-PCR to demonstrate XBP1 splicing in response to TGFβ2 treatment. Results: TGFβ2 increased synthesis and deposition of ECM proteins in GTM-3 and primary TM cells. TGFβ2 induced ER stress as evident from increased ER chaperones and splicing of XBP-1. Treatment of TM cells with TGFβ2 and PBA demonstrated reduced synthesis and deposition of ECM and ER stress markers as evident from reduced fibronectin, GRP78, GRP94 and CHOP. Conclusions: Our studies suggest that TGFβ2 induces abnormal ECM accumulation and ER stress, and PBA may reduce TGFβ2-induced IOP elevation by decreasing abnormal ECM accumulation and reducing ER stress.
Role of Glutaredoxin 2 (Grx2) in protecting the retina from light-induced damage
(2017-03-14) Xavier, Christy; Wu, Hongli; Liu, Xiaobin
Purpose: Glutaredoxin 2 (Grx2), a glutathione-dependent oxidoreductase, is known to repair oxidative damage of protein thiol groups and also serve as electron donors for ribonucleotide reductase. Grx2 is highly expressed in tissues with high energy demand like heart, brain, liver, and kidney. Our previous study has shown that Grx2 is highly expressed in the neural retina where its physiological functions remain completely unknown. In this study, we evaluated the role of Grx2 in protecting the retina from light-induced damage by using Grx2 gene knockout (KO) mice as a model. Methods: Wild type (WT) and Grx2 KO mice were exposed to white light at 12,000 lux for 1 hour after dark adaptation. The retinal damage was evaluated by the electroretinogram (ERG) recording, spectral domain optical coherence tomography (SD-OCT) measurement, and fundus examination. Hematoxylin and Eosin (H&E) staining was used to analyze the morphological changes in the retina. To better understand the molecular basis of how Grx2 protects the retina from light induced damage, we performed the whole transcriptome shotgun sequencing (RNA-seq) to analyze the full transcriptome of the retinal tissue in light-exposed Grx2 KO mice. The gene network was explored using DESeq2 pathway analysis software. The selected genes of interest were further confirmed by real-time PCR and Western Blot. Results: Light-exposed Grx2 KO mice showed severe loss of both a- and b-wave amplitudes and the outer nuclear layer (ONL) in the Grx2 deficient mice was significantly thinner compared to that of light-exposed WT mice. We identified thousands of genes with statistical significant expression changes and classified them into cellular processes and molecular pathways. Interestingly, assessment of gene expression profile indicated that several nuclear factor erythroid 2 (Nrf2) regulated antioxidant genes including SOD1, NQO1, and catalase were dysregulated in Grx2 KO mice, which indicated that Grx2 may be a novel regulator of the Nrf2 defense pathway. Conclusions: Our results suggest that Grx2 may protect the retina from light-induced retinal degeneration. The protective effects of Grx2 in the retina may be explained at least in part by its ability to control the Nrf2 signaling pathway.
An Antiapoptotic Peptide for Neuroprotection in Glaucoma
(2017-03-14) Krishnamoorthy, Raghu R.; Sampathkumar, Sruthi; Nagaraj, Ram; Stankowska, Dorota L.
Purpose: Axonal degeneration and death of retinal ganglion cells (RGC) are primary contributors to vision loss in glaucoma. The purpose of this study was to determine if intraperitoneal administration of the core peptide derived from small heat shock protein αB-crystallin (ABCP) could inhibit RGC death in animal models of glaucoma. Materials and Methods: Brown Norway rats were retrogradely labeled (to detect RGCs) using Fluoro-gold and IOP was elevated (150 mmHg/days) in one eye using the Morrison’s method, while the contralateral eye served as control. The rats were intraperitoneally injected with 10μg of ABCP (n=3 animals per group) three times per week for five weeks. Surviving RGCs were counted in retinal flat mounts. In another model of ischemia reperfusion (I/R) injury, C57BL/6 mice were subjected to IOP elevation of 120 mmHg for 30 min, followed by rapid reperfusion. Intraperitoneal ABCP injections were given 3h before and immediately after the procedure and then once daily post I/R injury for 14 days. RGC apoptosis was assessed using a TUNEL assay (n=2 animals per group). Results: Intraperitoneal injections of ABCP significantly (p Conclusions: Intraperitoneally administered ABCP peptide was able to significantly attenuate RGC death in two animal models of glaucoma. These findings suggest that ABCP has the potential to be developed as a neuroprotective agent in glaucoma.
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, Daniel
Introduction: 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.
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, Humberto
Purpose: 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.
Neuroprotective effects of an endothelin receptor antagonist in a rat model of ocular hypertension.
(2017-03-14) McGrady, Nolan; Krishnamoorthy, Raghu; Jefferies, Hayden
Purpose: Endothelin 1 is elevated in both patients and in animal models of glaucoma and has been shown to contribute to neurodegeneration. Since endothelin 1 acts through two receptors, endothelin A receptor and endothelin B receptor, the purpose of this study is to determine if antagonism of both endothelin receptors by Macitentan can promote neuroprotection during intraocular pressure (IOP) elevation in rats. Methods: IOP was elevated in the left eye of adult male retired breeder Brown Norway rats using Morrison’s model of ocular hypertension (injection of hypertonic saline through episcleral veins) and maintained for 4 weeks. Contralateral eyes served as the corresponding contralateral controls. Rats were then separated into treated and untreated groups, with the treated group receiving Macitentan (10 mg/kg) 3 times per week in the diet (administered in gel packs). After one month of treatment, rats were sacrificed and retinal flat mounts were prepared for both IOP elevated and contralateral eyes of both the treated and untreated rats. The flat mounts were immunostained for the retinal ganglion cell-specific marker Brn3a, imaged in a confocal microscope and masked cell counts were performed. Results: IOP elevation in untreated rats showed a dramatic decline in retinal ganglion cell counts compared to the contralateral controls, whereas rats treated with Macitentan showed significant preservation of retinal ganglion cell numbers. Conclusion: Currently, therapies for the treatment of glaucoma are solely focused on the reduction of IOP. The study described herewith demonstrates the ability of a dual endothelin receptor antagonist, Macitentan, to promote retinal ganglion cell survival independent of IOP. Due to its IOP independent action, this therapy could be developed for neuroprotection either independent or concurrent with available glaucoma therapies.
Endothelin receptors are targets for neuroprotection in a rat model of glaucoma
(2017-03-14) Stankowska, Dorotoa; Rendon, Caitlin; Krishnamoorthy, Raghu; McGrady, Nolan
Purpose: The endothelin system has been shown to play a causative role in the neurodegenerative effects seen in animal models of glaucoma. However, the underlying mechanisms are not completely understood. The goal of this study was to investigate the interaction between the ETA and ETB receptors and the involvement of the MAP kinase pathways in endothelin mediated cell death and determine if the dual ETA/ETB receptor antagonist, macitentan could attenuate neurodegenerative changes following IOP elevation in Brown Norway rats. Methods: Cultured transformed 661W cells were transiently transfected with a plasmid DNA encoding the ETA receptor and treated with ET-1 or ET-3 for 24 h. Phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) was determined by immunoblotting. Wild type and ETB-deficient Wistar-Kyoto rats were subjected to IOP elevation by the Morrison’s method and maintained for 2 weeks. Retinal sections obtained from the rats were subjected to immunohistochemical analysis of ERK1/2 and JNK and their phosphorylation. Brown Norway rats subjected to IOP elevation in one eye were either untreated or treated with macitentan (10 mg/kg body wt) for 1 month. Retinal flatmounts obtained from these rats were used to determine RGC counts following staining with the RBPMS antibody. Results: Cell culture experiments showed an appreciable upregulation of pERK1/2, while pJNK levels were not appreciably altered, following overexpression of the ETA receptor in 661W cells. ETB-deficient rats showed increased immunostaining for pERK1/2 in the nerve fiber layer (NFL), ganglion cell layer (GCL) and inner plexiform layer (IPL), compared to wild type rats. Following IOP elevation, ERK1/2 phosphorylation was greatly reduced in wild type rats, while ETB-deficient rats showed better preservation of pERK1/2 levels. Conversely, immunostaining for pJNK in wild type rats was increased in the NFL following IOP elevation, but was attenuated in ETB-deficient rats. Rats fed with macitentan displayed increased RGC survival by 25 to 42% following IOP elevation, compared to untreated rats. Conclusions: There is a substantial body of evidence for the pro-survival role of ERKs, and the pro-death role of JNKs. The current study points to an involvement of ERK1/2 and JNK signaling with endothelin receptor expression following IOP elevation. Blocking both ETA and ETB receptors has neuroprotective effects on RGCs during ocular hypertension.
Overexpression of ATF-4 in trabecular meshwork causes elevation of intra ocular pressure and reduction of outflow facility in a CHOP dependent manner
(2017-03-14) Kasetti, Ramesh; Zode, Gulab; Maddineni, Prabhavathi
Purpose: Primary open-angle glaucoma (POAG) has been primarily associated with reduced aqueous humor outflow facility through trabecular meshwork (TM) and elevated intraocular pressure (IOP). Studies based on both human as well as mice models revealed that chronic endoplasmic reticulum (ER) stress in TM is one of the causative factors responsible for TM dysfunction and ocular hypertension. The purpose of this study is to examine whether forced expression of UPR downstream pro apoptotic molecules ATF4 and CHOP leads to reduced outflow facility and IOP elevation in normal C57 mice. Methods: Ad5.ATF-4/Ad5.CHOP/Ad5.empty virus (pfu=2x107) were injected intravitreally into C57BL/6J or CHOP KO (CHOP-/-) mice. Conscious IOP of both the eyes was monitored once in a week until 7 weeks using rebound tonometer. Outflow facility was measured by constant-flow infusion technique. Also, we examined the expression levels of ATF-4 in the TM of age-matched normal and POAG donors by immunohistochemistry. Results: Forced expression of ATF-4 but not CHOP caused significant IOP elevation (23.97 mmHg in Ad5.ATF4 v/s 14.6 mmHg in Ad5.null mice) and reduced outflow facility (0.022µL/min/mmHg in Ad5.ATF4 v/s 0.04 in Ad5.null mice) in C57BL/6J mice. Elevation of IOP in C57BL/6J was prominent from 3 weeks post injection and sustained until 7 weeks. Interestingly Ad5.ATF-4 did not elevate IOP (17.7 mmHg) in CHOP-/- mice, indicating that ATF-4 interaction with CHOP is the prerequisite for ATF-4-induced IOP elevation. Also, ER stress-induced pro death marker, ATF-4 was significantly increased in human post-mortem glaucomatous TM tissues compared to normal TM tissues. Expression of ATF4 in primary TM cells induced oxidative and ER stress and also upregulated pro-apoptotic markers. Conclusions: This data indicates that chronic ER marker ATF4 is increased in the glaucomatous TM tissues, which may be associated with TM dysfunction, reduction of outflow facility and IOP elevation via induction of ER and oxidative stress. Furthermore, interaction of ATF-4 and CHOP is essential to carry out downstream signal transduction pathways.
Effects of Wnt/beta-catenin and SMAD/TGF-beta crosstalk on cadherins in the trabecular meshwork
(2017-03-14) Mao, Weiming; Clark, Abbot; Webber, Hannah
Purpose: 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.
Multifunctional small molecule TLR4 antagonist for treating ocular neovascularization
(2017-03-14) Panda, Santosh; Cai, Jiyang; Stankowska, Dorota; Ufret-Vincenty, Rafael; Acharya, Suchismita
Purpose: The multifactorial pathological challenge of ocular neovascularization is difficult to address so far only by anti-VEGF therapy. We have tested our hypothesis that, a novel class of natural product derived compound with toll like receptor 4 (TLR4) antagonist activity can ameliorate the hyper-inflammation produced by macrophage/macroglia over activation as well as decrease choroidal neovascularization (CNV) size in mice. Methods: Inhibition of cytokines: Mouse bone marrow derived macrophages were treated with high mobility group box1 (HMGB1, 100 ng/mL), an endogenous TLR4 ligand for 8 hours, with or without 100 mg/mL of test compounds. The mRNA levels of TNF-a, iNOS were measured by real-time RT-PCR, and normalized to the control cells. Inhibition of VEGF production: ARPE-19 cells were treated either with 100ng/mL or without HMGB1 along with the compounds (50μg/mL) for 24 h. Supernatants were collected and assayed using human VEGF ELISA kit according to manufacturer’s instructions. Experiments were repeated three times and one way ANOVA was used for statistical analysis. Inhibition of CNV: Laser CNV was induced in C57BL/6 mice (male, 10-12 weeks, n = 5). Each eye received 4 laser burns. The compounds (200 μg/mL) or BSS (vehicle) were administered by IP injection once before and once daily up to 10 days following laser injury. Fundus fluorescein angiography and optical coherence tomography was used to visualize the CNV lesions. RPE/choroid/sclera flat mounts were prepared and stained with both FITC conjugated isolectin B4 and anti-ICAM-2 antibody to quantitatively measure the size of CNV lesion. Results: Compound treatment significantly (p Conclusions: Our results are consistent with our hypothesis that this novel class of compounds will decrease ocular inflammation and neovascularization. Further structure optimization of the lead compound and TLR4 dependent and independent mechanistic investigation are underway.

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