Eye / Vision

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21735


Recent Submissions

Now showing 1 - 14 of 14
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    BMP4 Induced ID Protein Protects TM From Glaucomatous Effects of TGFβ-2
    (2015-03) Mody, Avani A.; Wordinger, Robert J.; Clark, Abbot F.
    Purpose: Elevated levels of pro-fibrotic growth factor transforming growth factor b2 (TGFb2) are have been reported in the aqueous humor and trabecular meshwork (TM) of primary open angle glaucoma (POAG) patients. TGFb2 treatment results in accumulation of extracellular matrix (ECM) molecules in the TM, which are associated with increased outflow resistance. Expression of TGFb2 in rodent eyes and ex-vivo anterior segment perfusion models elevate intraocular pressure, suggesting that TGFb2 plays a role in the development and progression of glaucoma. Interestingly, bone morphogenetic protein 4 (BMP4) inhibits ECM proteins that are up-regulated in TM cells by TGFb2.The purpose of our study is to determine the mechanism by which BMP4 inhibits the TGFb2 induction of ECM proteins in the TM. Prominent downstream targets of the BMP4 pathway are inhibitor of DNA binding proteins (IDs). There are four family members of IDs (ID1-4). In this study, we determine the expression of IDs in TM cells and determine the role of BMP4 induced ID1 and ID3 in regulating TGFb2 induction of ECM proteins Methods: Time and dose dependent BMP4 induction of ID1 and ID3 were studied in cultured human TM cells by Q-PCR and western blot analysis. GTM3 cells were transfected with pCMV-ID1 and pCMV-ID3 plasmids to determine their effect on TGFb2 induced ECM proteins (Fibronectin, PAI-1) by western blot analysis. Results: BMP4 (10ng/ml) significantly induced ID1 and ID3 mRNA and protein expression, starting 30 minutes after treatment. Conclusions: BMP4 induced ID1 and ID3 expression in TM cells, and ID1/ID3 suppressed the profibrotic ECM effects of TGFb2. Therefore, the BMP4 suppression of TGFb2 effects in TM cells may be mediated by ID1 and ID3. Further this study suggests ID1 and ID3 to be potential therapeutic targets for POAG.
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    Retina ganglion cell subtype specific cell death following optic nerve crush in mice
    (2015-03) Daniel, Steffi; Huberman, Andrew; Clark, Abbot F.; McDowell, Colleen M.
    Purpose: Glaucoma is an optic neuropathy that causes cupping of the optic disc, retina ganglion cell (RGC) loss, and characteristic visual field defects. Published literature suggests differential RGC susceptibility to damage. However, the mechanisms by which the optic nerve and RGCs become more susceptible to injury and damage are largely unknown. We investigated individual RGC subtypes’ susceptibility to damage after optic nerve crush (ONC). Methods: We utilized two mouse strains that selectively express GFP in individual RGC subtypes: CB2-GFP strain (selectively expresses GFP in transient OFF-alpha RGCs) and TRHR-GFP strain (selectively expresses GFP in On-Off direction selective RGCs). ONC was performed unilaterally, with the contralateral eye serving as a control. RGC subtype specific damage was evaluated at 0, 1, 3, 7, and 14 days post ONC. RGC damage was assessed by immunofluorescence of labeled retinal flat mounts using the GFP biomarker for the specific RGC subtypes and NeuN for total RGCs. Results: Throughout the 14 day time course, GFP positive RGCs in the CB2-GFP strain died faster than the GFP positive RGCs in the TRHR-GFP strain, with similar rates of total RGC death in each strain. The half-life (T1/2) of GFP positive cells in the TRHR-GFP strain was T1/2=7.11 days with total RGC death T1/2=9.65 days. The half-life of GFP positive cells in the CB2-GFP strain was T1/2=4.19 days with total RGC death T1/2=10.77 days. There was a significant difference in percent cell survival of each individual RGC subtype at 3 days (TRHR-GFP, 61.3 +/- 7.4%; CB2-GFP, 38.2 +/- 14.3%; n=4, p=0.029), 7 days (TRHR-GFP, 63.1 +/- 26.4%; CB2-GFP, 22.2 +/- 5.3%; n=4-5, p=0.011) and 10 days (TRHR-GFP, 38.0 +/- 2.9%; CB2-GFP, 3.5 +/- 3.5%; n=4-5, p<0.001) post-crush. There was no significant difference in percent cell survival of each individual RGC subtype at 1 day (TRHR-GFP, 76.2 +/- 20.9%; CB2-GFP, 70.3 +/- 10.0%; n=4-5, p=0.621) and 14 days (TRHR-GFP, 5.0 +/- 6.5%; CB2-GFP, 2.6 +/- 3.3%; n=3-4, p=0.556) post-crush. Conclusions: These studies demonstrate differences in individual RGC subtype susceptibilities to ONC. These data provide valuable information to develop new targets to slow and/or prevent the progression of RGC damage and new methods to detect early damage in diseases such as glaucoma.
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    Contribution of the Endothelin Receptor A to Neurodegeneration in a Rat Model of Ocular Hypertension
    (2015-03) McGrady, Nolan R.; Minton, Alena Z.; Krishnamoorthy, Raghu R.
    Purpose: The endothelin system of peptides and their receptors, primarily the ETB receptor, has been shown to have a neurodegenerative role in glaucoma. The purpose of this study was to examine alterations in ETA receptor expression within the retina following IOP elevation by the Morrison’s model of ocular hypertension in Brown Norway rats. Methods: IOP was elevated in the left eye of adult male retired breeder Brown Norway rats using the Morrison model of ocular hypertension (by injection of hypertonic saline through episcleral veins) while the contralateral eye served as the control. Rats were maintained for either two or four weeks following IOP elevation and sacrificed. Retina sections were obtained from both control and IOP-elevated eyes and analyzed for changes in ETA receptor expression by immunohistochemistry. ETA receptor immunostaining was co-localized with β-III-Tubulin, which is selectively expressed in retinal ganglion cells. In separate experiments a live/dead assay was performed using calcein AM and ethidium homodimer on stable 661W clones overexpressing the ETA receptor to determine the effect an increase in ETA receptors could have on cell viability. Results: After two weeks of IOP elevation rat eyes showed an increase in immunostaining for ETA receptors in multiple retinal layers. The most prominent increase in ETA receptor expression was observed in the outer plexiform layer and a moderate increase was seen in the ganglion cell layer and inner plexiform layer. Following four weeks of IOP elevation an increase in ETA receptor expression was observed primarily in the outer plexiform layer compared to that in the corresponding contralateral eyes. A modest increase in the ganglion cell layer was also observed. Cell culture studies showed that cells overexpressing the ETA receptor had a greater number of dead or dying cells compared to the empty vector expressing cells. Conclusion: Elevated intraocular pressure results in a appreciable change in ETA receptor expression. Overexpression of the ETA receptor results in a decrease in cell viability in cultured 661W cells. Further work is needed to understand the precise role of the ETA receptor in neurodegeneration during ocular hypertension.
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    A Study of Consistency of Dexamethasone Responsiveness between Paired Bovine Eyes
    (2015-03) Hickman, Colton; Bermudez, Jaclyn Y.; Mao, Weiming; Clark, Abbot F.
    Purpose: Glucocorticoid (GC) therapy can lead to elevated intraocular pressure (IOP) and GC-induced glaucoma. IOP elevation is also a major risk factor for the development and progression of primary open angle glaucoma (POAG). Glaucomatous and GC-induced IOP elevation is due to increased aqueous outflow resistance in the trabecular meshwork (TM). Because the pathological findings and clinical presentations of the two types of glaucoma are similar, GC-induce ocular hypertension is often used as a model to study POAG. Although the bovine eye perfusion culture model has been established, the consistency of paired bovine eyes to GC treatment has not been determined. Therefore, this study is to determine if Dexamethasone (DEX) changes IOP similarly in paired bovine eyes. Methods: Fresh bovine eyes were obtained from local abattoir, transferred to the lab and carefully dissected. The vitreous, uveal tract, retina, retinal pigment epithelium, and lens were removed. The remaining anterior segment, which contained the cornea, sclera, and TM, was mounted and sealed on a custom-made acrylic dish with an O-ring. Perfusion medium was infused by a syringe pump at a constant infusion rate of 5 μL/min. After IOP was stable, bovine eyes were perfused with medium containing 0.1% dexamethasone for 6-7 days. Bovine eyes with IOP elevation of more than 2.82mmHg was defined as a responder eye according to our published results. Results: Of the seven pairs of bovine eyes tested, one pair of eyes were DEX responders and the other six pairs were non-responders. The responder pair had IOP elevation of greater than 2.82mm Hg in both eyes. The other six pairs of non-responder eyes showed IOP change between -0.6 and 1.7 mm Hg. Conclusion: Our study showed that the DEX-responsiveness in paired bovine eyes are highly consistent. As suggested by early studies, it is very likely that induced IOP elevation and glaucoma are highly associated with genetic background. These results further validate the use of paired bovine eyes in glaucoma research. Due to the small samples size, further experiments are required. We will also try to determine possible genetic components such as the ratio between GC receptor isoforms GRα and GRβ in the TM cells.
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    Histone Acetylation as an Epigenetic Regulator of Glaucoma-Associated Growth Factors in the Trabecular Meshwork
    (2015-03) Bermudez, Jaclyn Y.; Webber, Hannah; Cheng, Yi-Qiang; Clark, Abbot F.; Mao, Weiming
    Glaucoma is a leading cause of blindness in the U.S. and worldwide. This disease leads to progressive, irreversible damage to the optic nerve and visual function. The primary risk factor of primary open angle glaucoma (POAG), the major type of glaucoma, is elevated intraocular pressure (IOP). IOP elevation in glaucoma patients is due to glaucomatous insults to the trabecular meshwork (TM) and compromised TM function, which increase aqueous humor outflow resistance. In the glaucomatous TM (GTM), there is excessive extracellular matrix (ECM) protein deposition. Many studies have suggested that cell signaling pathways, such as the transforming growth factor beta (TGF-β) and Wnt signaling pathways, play key roles in TM homeostasis. The growth factors that are associated with these pathways, including TGFβ2 and sFRP1, are increased in the GTM cells compared to normal TM cells. Little is known about the role of epigenetics in regulating glaucoma-associated growth factors in the TM. We hypothesize that histone acetylation is responsible for the increased expression of glaucoma associated factors in the TM. Primary human TM cell cultures were treated with 10nM Thailandepsin (TDP-A), a histone deacetylase inhibitor (HDACi), or 1% DMSO as vehicle control for 3 - 4 days. TM cells were a kind gift from Novartis. RNA was extracted for qPCR to compare gene expression. We also treated paired perfusion cultured bovine anterior segments with DMSO or TDP-A for 7 to 10 days. Additionally, we treated paired perfused anterior segments with TDP-A or TDP-A plus sFRP-1 or TGFβ receptor 1 inhibitors. The IOPs of the bovine eyes were continuously monitored. Data were analyzed by using Student’s t-test. P values less than 0.05 were considered significant. We found that TDP-A elevated the expression of sFRP-1 and TGFβ2 in TM cell cultures. Our bovine eye perfusion culture study also showed that TDP-A treatment increased the expression of sFRP-1 and TGFβ2 as well as significantly elevated IOP (n=9, p less than 0.05). Furthermore, use of sFRP-1 or TGFβ receptor 1 inhibitors decreased IOP. Histone acetylation may play an important role in the dysregulation of growth factors in the GTM. This mechanism provides a unique opportunity to elucidate the etiology of POAG. Also, TDP-A is a potent HDACi that can be used as a powerful research tool in glaucoma research.
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    Transforming Growth Factor-β2 Regulated Expression of Bone Morphogenetic Protein 1 (BMP-1), Tissue Transglutaminase (TGM2), Lysyl Oxidase (LOX), Procollagen C-endopeptidase Enhancer 1 (PCOLCE1), and Periostin (POSTN) in Human Optic Nerve Head Cells
    (2015-03) Tovar, Tara; Clark, Abbot F.; Wordinger, Robert J.
    Purpose: Transforming growth factor-β2 (TGF-β2) increases the expression of BMP1 (an enzyme responsible for the cleavage and maturation of ECM proteins) as well as TGM2 and LOX enzymatic activity to promote cross-linking. TGF-β2 has been implicated in glaucoma damage to the optic nerve head (ONH). Other factors associated with the ECM remodeling include POSTN and PCOLCE1. The purpose of this study was to determine (a) whether human ONH cells express BMP1, TGM2, LOX, POSTN and PCOLCE1, (b) whether expression of BMP1, TGM2, LOX, POSTN and PCOLCE1 are regulated by TGF-β2, and (c) if TGF-β2 regulates TGM2 and LOX activity. Methods: Primary human ONH cells were obtained from Alcon Laboratories (Fort Worth, Texas). Human ONH cells were isolated and subjected to qPCR (n=3) and Western immunoblotting (WB; n=6) for BMP1, TGM2, LOX, POSTN and PCOLCE1. qPCR was used to determine whether expression of BMP1, TGM2, LOX, POSTN and PCOLCE1 in ONH cells are regulated by TGF-β2 (5ng/ml). WB results were used to determine the effects of TGF-β2 on BMP1, TGM2, LOX, POSTN and PCOLCE1 protein expression in cell lysates of ONH cells treated for 48 hours. TGM2 and LOX activity assays were used to determine differences between non-treated and TGF-β2 treated in ONH cells. Results: Human ONH cells expressed mRNA and protein for BMP1, TGM2, LOX, POSTN and PCOLCE1. Exogenous TGF-β2 statistically increased BMP1, TGM2, LOX, POSTN mRNA expression at 24 hours compared to their controls. TGF-β2 statistically decreased PCOLCE1 mRNA expression compared to their controls at 24hours. WB analysis showed induced BMP1, TGM2, and POSTN levels in cell lysates after TGF-β2 treatment compared to controls. However, WB analysis showed that TGF-β2 decreased PCOLCE1 levels in cell lysates. TGF-β2 increased both TGM2 and LOX enzyme activity in the ONH. Conclusions: BMP1, TGM2, LOX, POSTN and PCOLCE1 are expressed in human ONH cells. These molecules may be involved in the normal function of the ONH. Altered expression of BMP1, TGM2, LOX, POSTN and PCOLCE1 by TGF-β2 may lead to functional changes and ECM remodeling within the ONH.
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    Crosstalk of transforming growth factor beta-2 and toll-like receptor 4 in the trabecular meshwork
    (2015-03) Hernandez, Humberto; Medina-Ortiz, Wanda E.; Clark, Abbot F.; McDowell, Colleen M.
    Purpose: Glaucoma is characterized by progressive optic neuropathy that is associated with elevated intraocular pressure (IOP) and extracellular matrix (ECM) remodeling. The trabecular meshwork (TM) is involved in the outflow of aqueous humor and IOP regulation. Glaucomatous eyes show elevated levels of transforming growth factor-β 2 (TGF-β2) and its signaling pathways in the ECM of the TM have been extensively studied. Recent evidence has implicated toll-like receptor 4 (TLR4) in the regulation of ECM and fibrogenesis in the liver, kidney, lung and skin. Based on the potential for shared signaling pathways, we hypothesize that endogenous TLR4 ligands activate TLR4 and augment TGF-β2 signaling sensitivity by downregulating BAMBI, leading to increase ECM production in the TM and increase IOP. Methods: Cross-sections of human donor eyes and dissected mouse TM rings were used to determine TLR4 expression in the TM. Primary human TM cells were used to test for the expression of BAMBI. To study the role of TGF-β2 and TLR4 crosstalk in the expression of ECM proteins, four primary human TM cell strains were treated with a selective TLR4 inhibitor (TAK-242, 15µM), TGFβ2 (5ng/ml), and a TLR4 ligand (Fibronectin-EDA isoform). In-vivo studies were carried out to examine the induction of ocular hypertension in wild-type (A/J, AKR/J, BALBc/J, and C3H/HeOuJ) or Tlr4 mutant strains of mice (C3H/HeJ) by intravitreally injecting Ad5.hTGFβ2226/228 (2.5x107 pfu) in one eye while the contralateral uninjected eye was used as negative controls. Results: Our studies reveal the expression of TLR4 in the human and mouse TM. BAMBI is expressed in human TM cells and its expression is significantly decreased in the presence of TGF-β2. Inhibition of TLR4 in the presence of TGF-β2 decreases fibronectin and collagen-1 expression. Activation of TLR4 in the presence of TGF-β2 increases fibronectin and collagen-1 expression and TLR4 inhibition blocks this effect. Our in-vivo studies show that Ad5.hTGF-β2 induces ocular hypertension in wild-type mice but has no effect in Tlr4 mutant mice. Conclusions: These studies identify TGFβ2 – TLR4 crosstalk as a novel pathway involved in ECM regulation in the TM and ocular hypertension. These data provide potential new targets to lower IOP and further explain the mechanisms involved in the development of glaucomatous TM damage.
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    Wnt induction of SMAD/TGFβ signaling in the Trabecular Meshwork
    (2015-03) Webber, Hannah C.; Clark, Abbot F.; Mao, Weiming
    Purpose: Primary Open Angle Glaucoma is a progressive, irreversibly blinding disease, the leading risk factor of which is increased intraocular pressure (IOP) thought to be due to an inherent pathological change in the trabecular meshwork (TM) tissue. Canonical Wnt signaling genes are expressed in the TM, the primary site for regulation of aqueous humor outflow and therefore IOP. Canonical Wnt signaling activation has been found to regulate IOP, but the mechanism behind this phenomenon remains unknown. Extracellular matrix deposition in the TM caused by increased activation of the TGF-β pathway by the TGF-β2 ligand has been associated with increased IOP and with primary open angle glaucoma. In other cell types and diseases, evidence exists for crosstalk between the TGF-β and Wnt signaling pathways. Our study aims to pinpoint the affect of Wnt signaling on the glaucoma-associated TGF-β pathway in the TM. Methods: Lentivirus-based luciferase assays were conducted in normal trabecular meshwork (NTM) and glaucomatous trabecular meshwork (GTM) cells by using TGF-β/SMAD or TCF/LEF (Wnt) signaling reporter vectors. Trabecular meshwork cells were all kind gifts from Novartis. Cell were treated with or without 100ng/ml Wnt3a or 5ng/ml TGF-β2. In some experiments, siRNAs were also used to knock down smads in NTM cells. Western immunoblotting was performed on nuclear and cytosolic fractions of NTM and GTM cells with corresponding primary as well as secondary antibodies. Results: In NTM cells, Wnt3a treatment increased TGFβ/SMAD pathway reporter activity (n=5 p less than 0.05) but TGF-β2 did not affect and even slightly decreased TCF/LEF (Wnt) signaling activity, although this decrease was not statistically significant (n=5, P greater than 0.05). SiRNA knockdown of SMAD pathway mediator smad3 decreased Wnt3a-induced SMAD/TGFβ signaling activity (n=6 p less than 0.05) in NTM cells. However, nuclear fractions of NTM and GTM cells showed translocation of smad4 (co-smad) into the nucleus upon Wnt3a treatment but not smad2 or smad3. Nuclear fractions also showed translocation of β-catenin by TGFβ2 treatment. Conclusions: The Wnt pathway ligand Wnt3a is able to activate SMAD/TGFβ transcriptional activity in TM cells, but not vice versa. This activation seems to involve translocation of only smad4. We hypothesize that a protein complex consisting of β-catenin and smad4 can form in the TM. By selectively recruiting other smad proteins into the complex during SMAD activation, the SMAD/TGFβ pathway can be differentially regulated. Defining how Wnt and SMAD signaling pathways crosstalk in the TM is imperative in defining the role of Wnt signaling in IOP regulation, and could lead to discovery of a therapeutic target for regulation of TGF-β pathway and therefore regulation of POAG.
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    Characterization of Novel Sigma-1 Receptor (σ1-r) Ligands
    (2015-03) Broyles, Heather V.; Park, Yong H.; Li, Linya; Ellis, Dorette Z.
    Purpose: Sigma-1 receptors (σ-1rs) are non-opioid ligand receptors that are associated with the endoplasmic reticulum. Upon agonist stimulation during stress, these receptors have the ability to translocate to the plasma membrane. σ-1rs are known to mediate ion channels such as L-type Voltage Gated Calcium Channels (VGCCs), thus facilitating neuroprotective effects in neurons such as retinal ganglion cells (RGCs). The purpose of this study was to determine if novel σ-1r agonist (PB190) and novel σ-1r antagonist (PB212) in purified RGCs have similar actions to that of known σ-1r agonists and antagonists, such as Pentazocine and BD1063, respectively. Methods: Purification and the culture of RGCs were performed by a double immunopanning technique using an antibody to Thy1.1 from P3-P7 Sprague-Dawley rats. RGCs were cultured for 5-7 days in vitro prior to experiments. Purified RGCs were incubated for 30 minutes with 1µM treatments of either Pentazocine, PB190, BD1063, PB212, or PB212 + PB190 combined. FURA-2 AM fluorescent dye was used to determine calcium concentration within RGCs. Immunoblot and immunocytochemistry were used to determine purity of RGCs and expression of σ-1r. Results: Immunocytochemistry determined 98% purity of isolated RGC cultures following 7 days in vitro. σ-1r expression was identified in purified RGCs through immunocytochemistry and immunoblot (MW~26 kDa). Intracellular calcium concentration [Ca2+]i was determined in RGCs where control (no treatment) group calcium levels were equal to 265 ± 104 nM. Pentazocine [Ca2+]i levels were equal to 115 ± 13 nM. PB190 [Ca2+]i levels were equal to 102 ± 9 nM. BD1063 [Ca2+]i levels were equal to 119 ± 13 nM. PB212 [Ca2+]i levels were equal to 118 ± 14 nM. PB190 and PB 212 co-treatment [Ca2+]i levels were equal 135 ± 15 nM. At the concentrations used (1µM), all of these σ-1r ligands significantly (Ca2+]i levels compared to control group. Conclusion: All σ-1r ligands utilized in this study modulated basal intracellular calcium levels. The new compounds PB190 and PB212 have similar effects in decreasing basal calcium levels as those seen in the known compounds Pentazocine and BD1063. Activation or inhibiting σ-1r could be modulating calcium channels involved in homeostasis such as the VGCCs. Future studies are needed to determine if these new ligands are more efficacious than that of known σ-1r ligands, and what other calcium channels they may modulate.
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    Mechanisms Underlying AMPA-Mediated Excitotoxicity of Retinal Ganglion Cells Under Hypoxic Conditions
    (2015-03) Park, Yong H.; Broyles, Heather V.; McGrady, Nolan R.; Yorio, Thomas
    Purpose: Excessive AMPA receptor (AMPAR) stimulation has been implicated in producing excitotoxicity in many neurodegenerative diseases such as glaucoma. The purpose of this study was to investigate if AMPAR desensitization attenuates excitotoxicity in purified retinal ganglion cells (RGCs) under normoxic and hypoxic conditions. Methods: Purified RGCs were treated with AMPAR agonists (100µM s-AMPA (desensitizing), 100µM kainic acid (non-desensitizing)), an AMPAR modulator (100µM cyclothiazide), AMPAR antagonist (50µM CFM-2), and kainate receptor antagonist (50µM UBP301) for 72h in RGC defined medium. To determine if excitotoxicity occurs following hypoxic injury, RGCs were subjected to oxygen-glucose deprivation (OGD) for 4h, followed by s-AMPA treatment under OGD for an additional 4h. Live-Dead Assays were carried out to assess cell viability. AMPA receptor mediated calcium influxes in RGCs were determined by imaging with fura-2 AM following 4h either normoxic-glucose-free or OGD treatments. Results: Significantly enhanced viability was found in RGCs treated with 100µM s-AMPA (84 ± 1% viable) compared to vehicle (0.1% DMSO) group (71 ± 4% viable) alone (ps-AMPA in combination with cyclothiazide or kainic acid significantly reduced cell viability to 50 ± 3% and 54 ± 2%, respectively (ps-AMPA (22 ± 5% viable) following OGD injury compared to those under normoxic-glucose-free conditions (67 ± 4% viable). Additionally, no significant decrease in RGC survival was observed when OGD was carried out for 8h in the presence of s-AMPA in the cell culture medium (85 ± 2% viable). Increased calcium influx was observed when RGCs were maintained in OGD (1085 ± 97 nM) compared to control (764 ± 72 nM). However, there was no difference between treatments of normoxic-glucose-free and RGC medium with RGC defined medium. Conclusions: Desensitization of AMPAR is a key determinant of s-AMPA-mediated excitotoxicity, whereby blocking the desensitization of AMPAR induces cell death. Future studies will determine if AMPAR subunits with greater ion current influx possibly mediate increased sensitivity to excitotoxicity following injury.
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    Role of Extracellular Matrix Crosslinking Enzymes in the Trabecular Meshwork
    (2015-03) Raychaudhuri, Urmimala; Vidales, Tara Tovar; Wordinger, Robert J.; Clark, Abbot F.
    Purpose: Transforming Growth Factor - β2 (TGFβ2) increases deposition of extracellular matrix (ECM) in the trabecular meshwork (TM), which could be responsible for increased aqueous humor (AH) outflow resistance in primary open angle glaucoma (POAG). TGFβ2 induces expression of extracellular matrix (ECM) crosslinking enzymes tissue transglutaminase (TGM2), Lysyl oxidase (LOX) and Lysyl-oxidase like 2 (LOXL2) in the TM. These enzymes covalently crosslink ECM proteins leading to resistance to ECM degradation and turnover. In POAG, there is increased expression of TGFβ2, which increases TGM2, LOX and LOXL2 expression. Increased expression and crosslinking activity of these enzymes may enhance ECM deposition in the outflow pathway. This could lead to increased AH outflow resistance and elevated IOP. To determine whether these crosslinking enzymes play a role in regulating IOP, we developed and validated TGM2, LOX and LOXL2 expression vectors in-vitro. Viral vectors expressing these enzymes will be used in ex-vivo and in-vivo models to study the effect of their overexpression on IOP and AH outflow resistance. Materials and methods: Transformed glaucomatous TM (GTM-3) and primary TM cells were transfected with plasmids expressing TGM2, LOX and LOXL2. Conditioned medium was collected, and overexpression was determined using western immunoblots. TGM2 activity was assessed by exposing cells to biotin-cadaverine followed by incubation with AlexaFluor 488 streptavidin-conjugate followed by fluorescence microscopy. LOX and LOXL2 enzyme activity was evaluated by western blots of the substrate tropoelastin in transfected cells with or without the LOX inhibitor b -aminoproprionitrile (BAPN) for 48 hours. Results: TM cells transfected with TGM2, LOX and LOXL2 significantly overexpressed the enzymes. The TGM2 activity assay demonstrated increased crosslinking activity in cells transfected with TGM2 expression plasmid. LOX and LOXL2 activity was increased in cells transfected with LOX and LOXL2 expression vectors as exhibited by increased expression of the LOX substrate tropoelastin in western blotting. Conclusions: Our results indicate that TGM2, LOX and LOXL2 expression vectors significantly over-express these proteins and also show increased enzyme activity. In conclusion, these plasmid constructs will be packaged into adenovirus expression vectors and tested for effects on AH outflow and IOP in ex-vivo and in-vivo models.
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    Phosphoproteomic changes in the retina following optic nerve crush
    (2015-03) Liu, Yang; Clark, Abbot F.; Pang, Iok-Hou
    Purpose: Phosphorylation is a major type of protein post-translational modification. The identification and characterization of protein phosphorylation changes in disease models is an effective strategy to delineate the underlying disease mechanisms. In this study, we evaluated the phosphoproteomic changes in the retina induced by optic nerve crush (ONC) in the mouse. Methods: Intraorbital ONC was performed in adult C57BL6/J mice. Retinas were collected at 0, 6, and 12 h following optic nerve injury. Retinal proteins labeled with CyDye-C2 were subjected to 2D-PAGE. 2D gel phosphoprotein immunostaining was performed, followed by in-gel image analysis. Proteins with significant changes in phosphorylation in retinas of the injured eyes compared to the control eyes were spot-picked, tryptic digested, and peptide fragments were analyzed by MALDI-TOF (MS) and TOF/TOF (tandem MS/MS). Identified proteins were validated by Western blotting and immunofluorescence staining. Results: Intraorbital ONC increased phosphorylation of many retinal proteins. Among them, 96 were spot-picked and identified. An initial DAVID analysis showed that these proteins fall into several specific biological themes, such as apoptosis, survival, and regeneration of neurons, as well as glial activation. One of the identified phosphoproteins, PEA-15, has been confirmed by Western blot analysis; ONC increased phosphorylation of this protein without affecting its expression level. Immunofluorescence staining using phospho-PEA-15-specific antibody demonstrated that increased phosphorylated PEA-15 co-localized with GFAP, a marker for Müller cells and astroglia in the retina. Conclusions: This study provides new insights into mechanisms of retinal ganglion cell degeneration after optic nerve injury, as well as central nervous system (CNS) neurodegeneration, since the retina is an extension of the CNS. These new insights will lead to novel therapeutic targets for retinal and CNS neurodegeneration.
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    In Vivo Detection and Modulation of Reactive Oxygen Species in a Mouse Model of Retinal Ischemia/Reperfusion
    (2015-03) Silverman, Sean; Kim, Byung-Jin; Clark, Abbot F.; Pang, Iok-Hou
    Purpose: Ischemia results in deprival of oxygen and metabolic substrates, energy depletion, and ultimately cell death. As a result, there is a significant and detrimental increase in free radical formation, mediators of oxidative stress. Our study aims to establish a novel method for noninvasive in vivo detection of reactive oxygen species (ROS). Methods: Retinal ischemia/reperfusion (I/R) was induced in left eyes of C57BL/6J mice. They were cannulated in their anterior chamber, and intraocular pressure (IOP) was raised to 120 mmHg for 60 minutes. Right eyes served as internal controls. Detection of ROS was conducted by a chemiluminescent compound L-012. At indicated days after I/R, L-012 (75mg/kg) was injected intraperitoneally. Pupils were dilated using phenylephrine HCl 2.5% and mice were placed in the small animal In Vivo Imaging System Lumina XR 15 minutes after L-012 administration. In some studies, ROS scavenger TEMPOL (100mg/kg) or NADPH oxidase inhibitor apocynin (50mg/kg) was injected 30 minutes prior to L-012 treatment. At day 14, eyes were harvested and paraffin embedded for H&E staining. Retinal morphological changes were evaluated. All measurements were conducted in Living Image software (Caliper Life Sciences) and statistical analysis was performed using SigmaPlot (Systat). Results: L-012 chemilluminescent signals were successfully detected in the I/R-injured eyes following systemic L-012 administration. Over a 14-day time course, only 24 and 48 hours post I/R were statistically significant (p<0.05) signals detected for greater than 1 hour after L-012 injection. No toxicity or gross inflammation was observed throughout the eye. Treatment with both TEMPOL and apocynin caused a statistically significant (p<0.01) reduction of L-012 radiance at both 24 and 48-hour time points. Conclusions: Our studies indicate retinal I/R causes a transient and significant induction in ROS production. L-012 appears to be a reliable and nontoxic tool for noninvasive detection of ROS in mice. Furthermore, we showed TEMPOL and apocynin successfully reduce chemilluminescent signal through the removal of excess ROS. Previous detection of ROS was possible only in post mortem samples; however, our method does not require euthanasia of animals fulfilling a largely unmet need in the study of oxidative stress.
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    Novel Strategy for RPE Protection: Glutaredoxin-Targeting Natural Products
    (2015-03) Liu, Xiaobin; Xavier, Christy; Jann, Jamieson; Giordano, Dante; Wu, Hongli
    Purpose: Oxidative stress-induced retinal pigment epithelial (RPE) cell damage is known as an important factor in the pathogenesis of retinopathies, such as age-related macular degeneration (AMD). In our previous study, we identified glutaredoxin 1 (Grx1), a thiol-disulfide oxidoreductase, as a new cytoprotective enzyme in RPE cells. In this study, we searched for small molecule Grx1 inducers from natural products to protect RPE cells from oxidative damage. Methods: Five natural antioxidant phenolics, including Salvianolic acid A (Sal A), Salvianolic acid B (Sal B), total salvianolic acid, curcumin, and epigallocatechin gallate (EGCG) were screened for their Grx1-inducing activities and cytoprotective effects in primary human RPE cells. Grx1 expression was examined by Western blot. Cell viability was evaluated with the WST8 assay. The level of protein glutathionylation (PSSG) was measured by using anti-PSSG antibody. Results: Among all the tested compounds, Sal B was found to be the most potent Grx1 inducer, which upregulated Grx1 by ~3 fold at 50 µM after 24 h. In both a time and dose-dependent manner, Sal B protected cells from H2O2-induced cell viability loss. Sal B also reduced annexin V positive cells, decreased Bax/Bcl-2 ratio, prevented caspase-3 cleavage, and inhibited ROS production. Additionally, H2O2-induced PSSG accumulation was markedly decreased by Sal B treatment. Moreover, knockdown of Grx1 by siRNA significantly reduced the cytoprotective effects of Sal B. Conclusions: Sal B protects primary human RPE cells from oxidative stress-induced damage by upregulating Grx1. Naturally occurring Grx1 inducers may be used as new therapeutic strategies to treat oxidative stress-related retinopathies like AMD.