Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21711
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Browsing Eye / Vision by Author "Clark, Abbot"
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Item Effect of BAMBI expression on intraocular pressure and aqueous humor outflow facility in mice(2016-03-23) Millar, Cameron; Clark, Abbot; McDowell, Colleen; Hernandez, HumbertoPurpose: Elevated intraocular pressure (IOP) is an important risk factor in the development of glaucoma. TGFβ2 is well known to be involved in regulating both the extracellular matrix in the trabecular meshwork (TM) as well as ocular hypertension. BAMBI (BMP and activin membrane-bound inhibitor), a TGF-β pseudoreceptor, has been shown to be a negative regulator of TGF-β2. However, the role of BAMBI in regulating IOP is unknown. We investigated whether knockdown of BAMBI results in ocular hypertension in mice due to uninhibited TGFβ2 signaling. Methods: B6;129S1-Bambitm1Jian/J mice were injected intravitreally with 2.5x107 pfu of either Ad5.TGFβ2 (n=10), Ad5.Cre (n=9), or Ad5.TGFβ2 + Ad5.Cre (n=10), in one eye of each animal. The contralateral uninjected eyes were used as negative controls. IOP was measured using a TonoLab rebound tonometer. Aqueous humor outflow facility was assessed using a constant flow infusion method. Student’s t-test was used to compare between vector-treated and control uninjected eyes. Results: Injection with either Ad5.Cre, Ad5.TGFβ2, or Ad5.TGFβ2 + Ad5.Cre each induced ocular hypertension starting at day 7 post-injection and maintained significant IOP elevation throughout the 56 day time course compared to uninjected control eyes (p Conclusions: Here we show for the first time that conditional knockdown of BAMBI in the TM with Ad5.Cre induces ocular hypertension by reducing aqueous humor outflow facility. These data further explain the mechanisms involved in the development of glaucomatous TM damage and provide potential new targets to lower IOP.Item Epigenetic regulation of TGFβ2 in the pathogenesis of glaucoma(2016-03-23) Webber, Hannah; Liu, Xiangyang; Cheng, Yi-Qiang; Clark, Abbot; Mao, Weiming; Bermudez, Jaclyn Y.Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The primary risk factor for the development and progression of this optic neuropathy is increased intraocular pressure (IOP) caused by glaucomatous damage to the trabecular meshwork (TM). The glaucoma-associated factor, transforming growth factor beta 2 (TGFβ2) is increased in the TM of POAG patients. TGFβ2 elevates IOP in perfusion cultured human eyes and in rodents. We hypothesize that histone acetylation plays a role in dysregulated TGFβ2 expression. To test our hypothesis, we treated primary non-glaucomatous human TM (NTM) cells as well as perfusion cultured bovine eyes with 10 nM thailandepsin-A (TDP-A), a potent histone deacetylase inhibitor. We found that TDP-A increased protein acetylation in the TM using Western immunoblotting. Chromatin immunoprecipitation showed that TDP-A induced histone hyperacetylation associated with the TGFβ2 promoter. This change of acetylation significantly increased TGFβ2 expression in NTM cells as shown by quantitative PCR (n=6, pItem GRα and GRβ expression levels in trabecular meshwork determines steroid responsiveness upon glucocorticoid treatment(2016-03-23) Zode, Gulab; Mao, Weiming; Clark, Abbot; Patel, GaurangPurpose Glucocorticoid (GC) induced ocular hypertension (OHT) is a serious side effect of prolonged GC therapy with patients showing elevated intraocular pressure (IOP). Two major isoforms of glucocorticoid receptor (GRα and GRβ) regulate GCs sensitivity and specificity in various tissues. GRβ acts as a dominant negative regulator of GC activities and has been shown to regulate GC responsiveness in trabecular meshwork (TM). We evaluated GRα and GRβ expression levels in two mouse strains and studied how expression levels regulate GC and GC-induced OHT. Methods TM cells from C57BL/6J and BALB/cJ mice strains were isolated and characterized. RNA was isolated from TM cells and evaluated for GRα and GRβ expression levels using quantitative (Q)-PCR. To study how both TM cell lines respond to Dexamethasone (DEX) they were treated with DEX (100nM) and myocilin (MYOC) expression in TM cells was determined by Q-PCR analysis. Three month old C57BL/6J and BALB/cJ mice strains were used to evaluate changes in IOP upon DEX treatment. Mice were peri-ocularly injected with DEX-Acetate (100ug/eye) in both eyes. Conscious IOP measurements were taken using a TonoLab tonometer. Two-tailed Student’s t-test and One-way ANOVA were used for statistical analysis. Results MTM cells from both strains (C57BL/6J and BALB/cJ) expressed TM markers, including collagen IV, laminin and α-smooth muscle actin. GRα expression levels between both strains were similar. TM cells from BALB/cJ mice expressed significantly higher levels of GRβ compared to TM cells from C57BL/6J. When TM cells were treated with 100nM DEX, TM cells from C57BL/6J showed induction of myocilin expression compared to untreated controls whereas; TM cells from BALB/cJ did not show myocilin induction. IOP measurements upon DEX-Acetate treatment showed significant IOP elevation in C57BL/6J mice (ΔIOP of 3.5mmHg, p Conclusions In mouse, GRα and GRβ expression levels determines GC responsiveness. Higher GRβ expression levels leads to GC resistance. The current findings provide an important foundation for comparisons of GRα and GRβ expression levels in the TM among different strains. Also, manipulating GRα to GRβ expression levels holds a promise for desensitizing cells and tissues to GCs effects.Item Perfusion-cultured bovine anterior segments as a model for studying TGFB2-induced ocular hypertension and glaucoma(2016-03-23) Bermudez, Jaclyn; Clark, Abbot; Mao, Weiming; Wileman, JustinPurpose: Primary open angle glaucoma (POA) is a leading cause of blindness worldwide. The most important risk factor is elevated intraocular pressure (IOP), which is due to glaucomatous damage to the trabecular meshwork (TM). Damaged TM does not function properly and creates an obstruction to aqueous humor outflow, and therefore elevates IOP. Transforming growth factor beta 2 (TGFβ2) is elevated in about 50% POAG patients, and is known to increase IOP in several experimental models. The purpose of this study was to determine if TGFβ2 induces IOP elevation in perfusion cultured bovine eyes, which are an important glaucoma research model. Methods: Fresh bovine eyes were obtained, transferred to the lab and carefully dissected. Vitreous humor, uvea, retina, retinal pigment epithelium, and lens were removed. The remaining anterior segment tissue, which contained the sclera, cornea, and TM, was mounted and sealed on a custom-made plexiglass dish with an O-ring using four screws. Perfusion medium was infused by a syringe pump at a constant infusion rate of 5 ul/min. After baseline IOP was established, bovine eyes were perfused with or without 10ng/ml TGFB2 for up to 7 days. IOP was recorded by a pressure transducer and a computerized system. Changes in IOP were calculated by subtracting baseline IOP from IOP post treatment. Conditioned medium was collected for Western immunoblotting (WB). Results: TGFβ2 increased IOP in treated bovine eyes by about 4.5 mmHg while the fellow control eye did not show significant changes in IOP. WB showed that fibronectin, a TGFβ2-inducible protein, was increased in perfusate collected from the TGFβ2 treated eyes. Conclusion: Our study showed that TGFβ2 is able to induce ocular hypertension in perfusion cultured bovine eyes. This will provide researchers a useful model to study POAG.Item Tissue transglutaminase causes intraocular pressure elevation in mice(2016-03-23) Millar, Cameron; McDowell, Colleen; Clark, Abbot; Raychaudhuri, UrmimalaPurpose: The profibrotic cytokine TGF-β2 increases expression of the crosslinking enzyme tissue transglutaminase (TGM2). In the trabecular meshwork (TM), excessive crosslinking of ECM proteins mediated by TGM2 could increase extracellular matrix (ECM) protein deposition, thereby decreasing the aqueous humor outflow facility. We hypothesize that increased expression of TGM2 increased ECM crosslinking in TM cells, and increases aqueous humor outflow resistance leading to elevated intraocular pressure (IOP) in mice. Methods: MTM cells were grown to confluency and transduced with Ad5.TGM2 (MOI of 75). On Day 5, MTM cells were fixed with 4% PFA for immunocytochemistry (ICC). Ad5.TGM2 (1.28 - 106 pfu in 2ml) was injected intravitreally into the left eye of female BALBc/J retired breeder mice (n = 18). The uninjected (right) eye served as a control. Daytime conscious IOP measurements were taken twice a week using a TonoLab rebound tonometer for approximately 3 weeks. Aqueous humor outflow facilities (C) was studied on day 23 (n = 6) using our published constant flow infusion method. Results: In cultured MTM cells, treatment with Ad5.TGM2 increased immunostaining of ε-(γ-glutamyl)lysine (GGEL) bonds, demonstrating increased TGM2 crosslinking activity after treatment with Ad5.TGM2. In BALBc/J mice, injection of Ad5.TGM2 significantly increased IOP from day 14 to 22, with the maximum difference elevation at Day 19, (15.86 +/- 1.06 mmHg (injected) versus 10.7 +/- 0.48 mmHg (control) (p Conclusion: Increased expression of TGM2 in mouse TM cells increases the ECM cross-linking activity of TGM2. Increased expression of TGM2 in the TM of the living mouse increases aqueous outflow resistance and elevates IOP. In the future, we will study whether TGM2 is responsible for TGF-β2 induced ocular hypertension.