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1.
Exp Eye Res ; 188: 107763, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31421135

RESUMO

Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness, and individuals with ocular hypertension are at risk to develop POAG. Currently, the only modifiable risk factor for glaucoma progression is lowering of intraocular pressure (IOP). A novel mechanism for lowering IOP involves activation of the type B natriuretic peptide receptor (NPR-B), the naturally occurring agonist of which is C-type natriuretic peptide (CNP). Being a cyclic peptide of 22 amino acids, CNP does not readily penetrate the cornea and its ocular hypotensive effect requires intraocular injection. TAK-639 is a synthetic, cornea-permeable, 9-amino acid CNP analog has been studied for the treatment of ocular hypertension and POAG. We assessed TAK-639 in a receptor binding profile and the effects of TAK-639 on NPR-B-mediated cyclic GMP production in cultured transformed human trabecular meshwork (TM) cells (GTM-3). We also evaluated the effects of topical ocular administration of TAK-639 on mouse IOP and aqueous humor dynamics. Among 89 non-natriuretic peptide receptors, transporters, and channels evaluated, TAK-639 at 10 µM displaced ligand binding by more than 50% to only two receptors: the type 2 angiotensin receptor (IC50 = 8.2 µM) and the cholecystokinin A receptor (IC50 = 25.8 µM). In vitro, TAK-639 selectively activates NPR-B (EC50 = 61 ±â€¯11 nM; GTM-3 cells) relative to NPR-A (EC50 = 2179 ±â€¯670 nM; 293T cells). In vivo, TAK-639 lowered mouse IOP by three mechanisms: increase in aqueous humor outflow facility (C), reduction in the aqueous humor formation rate (Fin), and reduction in episcleral venous pressure (Pe). The maximum mean IOP decreases from baseline were -12.1%, -21.0%, and -36.1% for 0.1%, 0.3%, and 0.6% doses of TAK-639, respectively. Maximum IOP-lowering effect was seen at 2 h, and the duration of action was >6 h. With TAK-639 0.6%, at 2 h post-dose, aqueous outflow facility (C) increased by 155.8%, Fin decreased by 41.0%, the uveoscleral outflow rate (Fu) decreased by 52.6%, and Pe decreased by 31.5% (all p < 0.05). No ocular adverse effects were observed. TAK-639 is an efficacious IOP-lowering agent, with a unique combination of mechanisms of action on both aqueous formation and aqueous outflow facility. Further study of this mechanism of treatment may optimize pharmacologic outcomes and provide disease management in patients with POAG and ocular hypertension.

2.
Invest Ophthalmol Vis Sci ; 60(6): 1967-1978, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31050723

RESUMO

Purpose: Glucocorticoid (GC)-induced ocular hypertension (GC-OHT) is a serious side effect of prolonged GC therapy that can lead to glaucoma and permanent vision loss. GCs cause a plethora of changes in the trabecular meshwork (TM), an ocular tissue that regulates intraocular pressure (IOP). GCs act through the glucocorticoid receptor (GR), and the GR regulates transcription both through transactivation and transrepression. Many of the anti-inflammatory properties of GCs are mediated by GR transrepression, while GR transactivation largely accounts for GC metabolic effects and side effects of GC therapy. There is no evidence showing which of the two mechanisms plays a role in GC-OHT. Methods: GRdim transgenic mice (which have active transrepression and impaired transactivation) and wild-type (WT) C57BL/6J mice received weekly periocular dexamethasone acetate (DEX-Ac) injections. IOP, outflow facilities, and biochemical changes to the TM were determined. Results: GRdim mice did not develop GC-OHT after continued DEX treatment, while WT mice had significantly increased IOP and decreased outflow facilities. Both TM tissue in eyes of DEX-treated GRdim mice and cultured TM cells isolated from GRdim mice had reduced or no change in the expression of fibronectin, myocilin, collagen type I, and α-smooth muscle actin (α-SMA). GRdim mouse TM (MTM) cells also had a significant reduction in DEX-induced cytoskeletal changes, which was clearly seen in WT MTM cells. Conclusions: We provide the first evidence for the role of GR transactivation in regulating GC-mediated gene expression in the TM and in the development of GC-OHT. This discovery suggests a novel therapeutic approach for treating ocular inflammation without causing GC-OHT and glaucoma.

3.
Invest Ophthalmol Vis Sci ; 59(5): 2154-2166, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29801150

RESUMO

Purpose: The trabecular meshwork (TM) has an important role in the regulation of aqueous humor outflow and IOP. Regulation of the extracellular matrix (ECM) by TGFß2 has been studied extensively. Bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) has been shown to inhibit or modulate TGFß2 signaling. We investigate the role of TGFß2 and BAMBI in the regulation of TM ECM and ocular hypertension. Methods: Mouse TM (MTM) cells were isolated from B6;129S1-Bambitm1Jian/J flox mice, characterized for TGFß2 and dexamethasone (DEX)-induced expression of fibronectin, collagen-1, collagen-4, laminin, α-smooth muscle actin, cross-linked actin networks (CLANs) formation, and DEX-induced myocilin (MYOC) expression. MTM cells were transduced with Ad5.GFP to identify transduction efficiency. MTM cells and mouse eyes were transduced with Ad5.Null, Ad5.Cre, Ad5.TGFß2, or Ad5.TGFß2 + Ad5.Cre to evaluate the effect on ECM production, IOP, and outflow facility. Results: MTM cells express TM markers and respond to DEX and TGFß2. Ad5.GFP at 100 MOI had the highest transduction efficiency. Bambi knockdown by Ad5.Cre and Ad5.TGFß2 increased fibronectin, collagen-1, and collagen-4 in TM cells in culture and tissue. Ad5.Cre, Ad5.TGFß2, and Ad5.TGFß2 + Ad5.Cre each significantly induced ocular hypertension and lowered aqueous humor outflow facility in transduced eyes. Conclusions: We show for the first time to our knowledge that knockdown of Bambi alters ECM expression in cultured cells and mouse TM, reduces outflow facility, and causes ocular hypertension. These data provide a novel insight into the development of glaucomatous TM damage and identify BAMBI as an important regulator of TM ECM and ocular hypertension.

4.
Invest Ophthalmol Vis Sci ; 59(3): 1454-1466, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29625468

RESUMO

Purpose: Wnt/ß-catenin signaling in the trabecular meshwork (TM) is required for maintaining normal intraocular pressure (IOP), although the mechanism(s) behind this are unknown. We hypothesize that Wnt/ß-catenin signaling regulates IOP via ß-catenin's effects on cadherin junctions. Methods: Nonglaucomatous primary human TM (NTM) cells were treated with or without 100 ng/ml Wnt3a, 1 µg/ml sFRP1, or both for 4 to 48 hours. Cells were immunostained for ß-catenin, total cadherins, or cadherin isoforms. Membrane proteins or whole-cell lysates were isolated for Western immunoblotting and probed for cadherin isoforms. RNA was extracted for cDNA synthesis and qPCR analysis of cadherin expression. Some NTM cells were cultured on electric plates for cell impedance assays. Ad5.CMV recombinant adenoviruses encoding K-cadherin, and/or sFRP1 were injected into eyes of 4- to 6-month-old female BALB/cJ mice (n = 8-10). Conscious IOPs were assessed for 35 days. Results: Upon Wnt3a treatment, total cadherin expression increased and ß-catenin accumulated at the TM cell membrane and on processes formed between TM cells. qPCR showed that Wnt3a significantly increased K-cadherin expression in NTM cells (P < 0.01, n = 3), and Western immunoblotting showed that Wnt3a increased K-cadherin in NTM cells, which was inhibited by the addition of sFRP1. Cell impedance assays showed that Wnt3a treatment increased transcellular resistance and anti-K-cadherin siRNA decreased transcellular resistance (P < 0.001, n = 4-6). Our in vivo study showed that K-cadherin significantly decreased sFRP1-induced ocular hypertension (P < 0.05, n = 6). Western immunoblotting also showed that K-cadherin alleviated sFRP1-induced ß-catenin decrease in mouse anterior segments. Conclusions: Our results suggest that cadherins play important roles in the regulation of TM homeostasis and IOP via the Wnt/ß-catenin pathway.


Assuntos
Caderinas/fisiologia , Pressão Intraocular/fisiologia , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/metabolismo , Via de Sinalização Wnt/fisiologia , Proteína Wnt3A/farmacologia , beta Catenina/fisiologia , Animais , Western Blotting , Caderinas/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Isoformas de Proteínas/metabolismo , Transdução de Sinais/fisiologia , beta Catenina/metabolismo
5.
Exp Eye Res ; 171: 106-110, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29535003

RESUMO

Glaucoma is a vision threatening optic neuropathy that affects millions of people worldwide. In primary open angle, increased intraocular pressure (IOP) is the main risk factor for the development of this disease. Studies investigating the causes and mechanisms of increased IOP show fibrotic changes in the trabecular meshwork (TM) that are different from those of age-matched controls. Tissue transglutaminase (TGM2), an extracellular matrix (ECM) crosslinking enzyme, covalently crosslinks ECM proteins and causes excessive ECM protein deposition in the TM that could cause increased IOP. Previous literature reports increased expression of TGM2 in glaucomatous eyes compared to controls. We recently have shown that overexpression of TGM2 causes increased ECM crosslinking in the TM, increases IOP, and decreases aqueous humor (AH) outflow facility in mouse eyes. Therefore, we wanted to study the effect of TGM2 knockout (KO) on IOP in TGM2 floxed mice. Ad5.Cre transduction caused partial KO of TGM2, which decreased TGM2 expression in the TM region of mouse eyes. TGM2 KO significantly decreased IOP by itself and also in TGFß2 induced ocular hypertensive mice. TGM2 KO also restores the outflow facility in TGFß2 transduced eyes. Overall, TGM2 KO rescued the TGFß2-induced ocular hypertensive phenotype. Thus, TGM2 may offer potential as a new therapeutic target for glaucoma.

6.
Sci Rep ; 8(1): 862, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29339763

RESUMO

Prolonged glucocorticoid (GC) therapy can cause GC-induced ocular hypertension (OHT), which if left untreated progresses to iatrogenic glaucoma and permanent vision loss. The alternatively spliced isoform of glucocorticoid receptor GRß acts as dominant negative regulator of GR activity, and it has been shown that overexpressing GRß in trabecular meshwork (TM) cells inhibits GC-induced glaucomatous damage in TM cells. The purpose of this study was to use viral vectors to selectively overexpress the GRß isoform in the TM of mouse eyes treated with GCs, to precisely dissect the role of GRß in regulating steroid responsiveness. We show that overexpression of GRß inhibits GC effects on MTM cells in vitro and GC-induced OHT in mouse eyes in vivo. Ad5 mediated GRß overexpression reduced the GC induction of fibronectin, collagen 1, and myocilin in TM of mouse eyes both in vitro and in vivo. GRß also reversed DEX-Ac induced IOP elevation, which correlated with increased conventional aqueous humor outflow facility. Thus, GRß overexpression reduces effects caused by GCs and makes cells more resistant to GC treatment. In conclusion, our current work provides the first evidence of the in vivo physiological role of GRß in regulating GC-OHT and GC-mediated gene expression in the TM.


Assuntos
Glucocorticoides/farmacologia , Pressão Intraocular/efeitos dos fármacos , Hipertensão Ocular/etiologia , Receptores de Glucocorticoides/metabolismo , Animais , Colágeno Tipo I/metabolismo , Proteínas do Citoesqueleto/metabolismo , Dexametasona/farmacologia , Proteínas do Olho/metabolismo , Feminino , Fibronectinas/metabolismo , Vetores Genéticos/metabolismo , Glicoproteínas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hipertensão Ocular/metabolismo , Hipertensão Ocular/patologia , Receptores de Glucocorticoides/genética , Malha Trabecular/citologia , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/metabolismo
7.
Methods Mol Biol ; 1695: 109-120, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29190023

RESUMO

Mice and rats are being increasingly used in glaucoma research and much useful data have been generated from them. One aspect of using these animals for this purpose involves assessment of aqueous humor dynamics. Several techniques have been described in the literature for the determination of one or more of these parameters in rodents, in both living animals and eyes perfused ex vivo. Here, we describe the practical details for a technique for the determination of all principal parameters of aqueous humor dynamics (intraocular pressure (IOP), aqueous humor formation rate (Fin), uveoscleral outflow rate (Fu), aqueous outflow facility (C), and episcleral venous pressure (Pe)) in the living rat and mouse eye, in a single experimental session.


Assuntos
Humor Aquoso/fisiologia , Retina/fisiologia , Animais , Pressão Intraocular , Camundongos , Ratos , Tonometria Ocular
8.
Invest Ophthalmol Vis Sci ; 58(14): 6197-6211, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29222550

RESUMO

Purpose: Tissue transglutaminase (TGM2) is elevated in glaucomatous trabecular meshwork (TM) tissues. We investigated whether increased expression of TGM2 increases extracellular matrix crosslinking in the TM, thereby increasing aqueous humor outflow resistance and elevating intraocular pressure (IOP) in mouse eyes. Methods: GTM3, primary human GTM 125-05, and cultured mouse TM cells were transduced with adenovirus serotype 5 expressing human transglutaminase 2 (Ad5.TGM2; multiplicity of infection [MOI]-75) and fixed for immunocytochemistry. To test the effect on IOP in living eyes, Ad5.TGM2 was injected intravitreally into one eye of BALB/cJ (n = 18) or C57BL/6J mice (n = 9). The uninjected contralateral eye and Ad5.GFP served as controls. Daytime conscious IOPs were measured twice per week. Aqueous outflow facility (C) was measured by constant flow infusion on completion of IOP measurements. Immunohistochemistry was performed on BALB/cJ mouse eyes to study TGM2 expression and activity. Results: The treatment of cultured TM cells with Ad5.TGM2 increased immunostaining of N-ε(γ-glutamyl) lysine crosslinks. Ad5.TGM2 injection significantly increased IOP in BALB/cJ (15.86 mm Hg [injected] vs. 10.70 mm Hg [control]) and in C57BL/6J mice (17.09 mm Hg [injected] vs. 12.01 mm Hg [control]). Mean aqueous outflow facility in the injected eyes of BALB/cJ (0.013 µL/min/mm Hg) and C57BL/6J mice (0.012 µL/min/mm Hg) was significantly lower than in the uninjected control eyes (BALB/cJ, 0.021 µL/min/mm Hg; C57BL/6J, 0.019 µL/min/mm Hg). The Ad5.TGM2 transduction of mouse eyes increased TGM2 expression in the TM region and increased N-ε(γ-glutamyl) lysine crosslinks. Conclusions: The increased expression of TGM2 in the TM increases N-ε(γ-glutamyl) lysine crosslinking in the TM, increases aqueous outflow resistance, and elevates IOP in mice. TGM2 may be at least partially responsible for ocular hypertension in POAG.


Assuntos
Humor Aquoso/enzimologia , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Glaucoma de Ângulo Aberto/genética , Pressão Intraocular , RNA/genética , Malha Trabecular/enzimologia , Transglutaminases/genética , Animais , Western Blotting , Células Cultivadas , Proteínas de Ligação ao GTP/biossíntese , Glaucoma de Ângulo Aberto/enzimologia , Glaucoma de Ângulo Aberto/patologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Malha Trabecular/patologia , Transglutaminases/biossíntese
9.
Sci Rep ; 7(1): 14951, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29097767

RESUMO

Increased synthesis and deposition of extracellular matrix (ECM) proteins in the trabecular meshwork (TM) is associated with TM dysfunction and intraocular pressure (IOP) elevation in glaucoma. However, it is not understood how ECM accumulation leads to TM dysfunction and IOP elevation. Using a mouse model of glucocorticoid (GC)-induced glaucoma, primary human TM cells and human post-mortem TM tissues, we show that increased ECM accumulation leads to endoplasmic reticulum (ER) stress in the TM. The potent GC, dexamethasone (Dex) increased the secretory protein load of ECM proteins in the ER of TM cells, inducing ER stress. Reduction of fibronectin, a major regulator of ECM structure, prevented ER stress in Dex-treated TM cells. Overexpression of fibronectin via treatment with cellular fibronectin also induced chronic ER stress in primary human TM cells. Primary human TM cells grown on ECM derived from Dex-treated TM cells induced ER stress markers. TM cells were more prone to ER stress from ECM accumulation compared to other ocular cell types. Moreover, increased co-localization of ECM proteins with ER stress markers was observed in human post-mortem glaucomatous TM tissues. These data indicate that ER stress is associated with increased ECM accumulation in mouse and human glaucomatous TM tissues.

10.
Invest Ophthalmol Vis Sci ; 58(13): 5731-5742, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29114841

RESUMO

Purpose: Cromakalim prodrug 1 (CKLP1) is a water-soluble ATP-sensitive potassium channel opener that has shown ocular hypotensive properties in ex vivo and in vivo experimental models. To determine its mechanism of action, we assessed the effect of CKLP1 on aqueous humor dynamics and in combination therapy with existing ocular hypotensive agents. Methods: Outflow facility was assessed in C57BL/6 mice by ex vivo eye perfusions and by in vivo constant flow infusion following CKLP1 treatment. Human anterior segments with no trabecular meshwork were evaluated for effect on pressure following CKLP1 treatment. CKLP1 alone and in combination with latanoprost, timolol, and Rho kinase inhibitor Y27632 were evaluated for effect on intraocular pressure in C57BL/6 mice and Dutch-belted pigmented rabbits. Results: CKLP1 lowered episcleral venous pressure (control: 8.9 ± 0.1 mm Hg versus treated: 6.2 ± 0.1 mm Hg, P < 0.0001) but had no detectable effect on outflow facility, aqueous humor flow rate, or uveoscleral outflow. Treatment with CKLP1 in human anterior segments without the trabecular meshwork resulted in a 50% ± 9% decrease in pressure, suggesting an effect on the distal portion of the conventional outflow pathway. CKLP1 worked additively with latanoprost, timolol, and Y27632 to lower IOP, presumably owing to combined effects on different aspects of aqueous humor dynamics. Conclusions: CKLP1 lowered intraocular pressure by reducing episcleral venous pressure and lowering distal outflow resistance in the conventional outflow pathway. Owing to this unique mechanism of action, CKLP1 works in an additive manner to lower intraocular pressure with latanoprost, timolol, and Rho kinase inhibitor Y27632.


Assuntos
Anti-Hipertensivos/uso terapêutico , Humor Aquoso/fisiologia , Cromakalim/uso terapêutico , Pressão Intraocular/efeitos dos fármacos , Pró-Fármacos/uso terapêutico , Amidas/uso terapêutico , Animais , Segmento Anterior do Olho/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Humanos , Latanoprosta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Soluções Oftálmicas , Prostaglandinas F Sintéticas/uso terapêutico , Piridinas/uso terapêutico , Coelhos , Esclera/irrigação sanguínea , Timolol/uso terapêutico , Tonometria Ocular , Pressão Venosa/efeitos dos fármacos
11.
Exp Eye Res ; 164: 95-108, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28822760

RESUMO

Mice are now routinely utilized in studies of aqueous humor outflow dynamics. In particular, conventional aqueous outflow facility (C) is routinely measured via perfusion of the aqueous chamber by a number of laboratories. However, in mouse eyes perfused ex-vivo, values for C are variable depending upon whether the perfusate is introduced into the posterior chamber (PC) versus the anterior chamber (AC). Perfusion via the AC leads to posterior bowing of the iris, and traction on the iris root/scleral spur, which may increase C. Perfusion via the PC does not yield this effect. But the equivalent situation in living mice has not been investigated. We sought to determine whether AC versus PC perfusion of the living mouse eye may lead to different values for C. All experiments were conducted in C57BL/6J mice (all ♀) between the ages of 20 and 30 weeks. Mice were divided into groups of 3-4 animals each. In all groups, both eyes were perfused. C was measured in groups 1 and 2 by constant flow infusion (from a 50 µL microsyringe) via needle placement in the AC, and in the PC, respectively. To investigate the effect of ciliary muscle (CM) tone on C, groups 3 and 4 were perfused live via the AC or PC with tropicamide (muscarinic receptor antagonist) added to the perfusate at a concentration of 100 µM. To investigate immediate effect of euthanasia, groups 5 and 6 were perfused 15-30 min after death via the AC or PC. To investigate the effect of CM tone on C immediately following euthanasia, groups 7 and 8 were perfused 15-30 min after death via the AC or PC with tropicamide added to the perfusate at a concentration of 100 µM. C in Groups 1 (AC perfusion) and 2 (PC perfusion) was computed to be 19.5 ± 0.8 versus 21.0 ± 2.1 nL/min/mmHg, respectively (mean ± SEM, p > 0.4, not significantly different). In live animals in which tropicamide was present in the perfusate, C in Group 3 (AC perfusion) was significantly greater than C in Group 4 (PC perfusion) (22.0 ± 4.0 versus 14.0 ± 2.0 nL/min/mmHg, respectively, p = 0.0021). In animals immediately following death, C in groups 5 (AC perfusion) and 6 (PC perfusion) was computed to be 21.2 ± 2.0 versus 22.8 ± 1.4 nL/min/mmHg, respectively (mean ± SEM, p = 0.1196, not significantly different). In dead animals in which tropicamide was present in the perfusate, C in group 7 (AC perfusion) was greater than C in group 8 (PC perfusion) (20.6 ± 1.4 versus 14.2 ± 2.6 nL/min/mmHg, respectively, p < 0.0001). C in eyes in situ in living mice or euthanized animals within 15-30 min post mortem is not significantly different when measured via AC perfusion or PC perfusion. In eyes of live or freshly euthanized mice, C is greater when measured via AC versus PC perfusion when tropicamide (a mydriatic and cycloplegic agent) is present in the perfusate.


Assuntos
Câmara Anterior/fisiologia , Humor Aquoso/fisiologia , Pressão Intraocular/fisiologia , Segmento Posterior do Olho/fisiologia , Animais , Câmara Anterior/efeitos dos fármacos , Câmara Anterior/metabolismo , Humor Aquoso/metabolismo , Modelos Animais de Doenças , Feminino , Pressão Intraocular/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Antagonistas Muscarínicos/farmacologia , Segmento Posterior do Olho/efeitos dos fármacos , Segmento Posterior do Olho/metabolismo , Malha Trabecular/metabolismo , Tropicamida/farmacologia
12.
Am J Pathol ; 187(4): 713-723, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28167045

RESUMO

Glucocorticoid (GC)-induced ocular hypertension (OHT) is a serious adverse effect of prolonged GC therapy that can lead to iatrogenic glaucoma and permanent vision loss. An appropriate mouse model can help us understand precise molecular mechanisms and etiology of GC-induced OHT. We therefore developed a novel, simple, and reproducible mouse model of GC-induced OHT. GC-induced myocilin expression in the trabecular meshwork (TM) has been suggested to play an important role in GC-induced OHT. We further determined whether myocilin contributes to GC-OHT. C57BL/6J mice received weekly periocular conjunctival fornix injections of a dexamethasone-21-acetate (DEX-Ac) formulation. Intraocular pressure (IOP) elevation was relatively rapid and significant, and correlated with reduced conventional outflow facility. Nighttime IOPs were higher in ocular hypertensive eyes compared to daytime IOPs. DEX-Ac treatment led to increased expression of fibronectin, collagen I, and α-smooth muscle actin in the TM in mouse eyes. No changes in body weight indicated no systemic toxicity associated with DEX-Ac treatment. Wild-type mice showed increased myocilin expression in the TM on DEX-Ac treatment. Both wild-type and Myoc-/- mice had equivalent and significantly elevated IOP with DEX-Ac treatment every week. In conclusion, our mouse model mimics many aspects of GC-induced OHT in humans, and we further demonstrate that myocilin does not play a major role in DEX-induced OHT in mice.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Dexametasona/análogos & derivados , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Hipertensão Ocular/induzido quimicamente , Anestesia , Animais , Peso Corporal/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Dexametasona/administração & dosagem , Dexametasona/efeitos adversos , Vias de Administração de Medicamentos , Esquema de Medicação , Feminino , Fibronectinas/metabolismo , Injeções , Injeções Intraoculares , Pressão Intraocular , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hipertensão Ocular/fisiopatologia , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/patologia
13.
Invest Ophthalmol Vis Sci ; 57(14): 6058-6069, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27820874

RESUMO

Purpose: Abnormal accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) is associated with decreased aqueous humor outflow facility and IOP elevation in POAG. Previously, we have developed a transgenic mouse model of POAG (Tg-MYOCY437H) by expressing human mutant myocilin (MYOC), a known genetic cause of POAG. The purpose of this study is to examine whether expression of mutant myocilin leads to reduced outflow facility and abnormal ECM accumulation in Tg-MYOCY437H mice and in cultured human TM cells. Methods: Conscious IOP was measured at various ages of Tg-MYOCY437H mice using a rebound tonometer. Outflow facility was measured in 10-month-old Tg-MYOCY437H mice. Selected ECM proteins were examined in human TM-3 cells stably expressing mutant myocilin and primary human TM cells (n = 4) as well as in the TM of Tg-MYOCY437H mice by real-time PCR, Western blotting, and immunostaining. Furthermore, TM cells expressing WT or mutant myocilin were treated with 5 mM sodium 4-phenylbutyrate (PBA), and ECM proteins were examined by Western blot and immunostaining. Results: Starting from 3 months of age, Tg-MYOCY437H mice exhibited significant IOP elevation compared with wild-type (WT) littermates. Outflow facility was significantly reduced in Tg-MYOCY437H mice (0.0195 µl/min/mm Hg in Tg-MYOCY437H vs. 0.0332 µl/min/mm Hg in WT littermates). Increased accumulation of fibronectin, elastin, and collagen type IV and I was observed in the TM of Tg-MYOCY437H mice compared with WT littermates. Furthermore, increased ECM proteins were also associated with induction of endoplasmic reticulum (ER) stress markers, GRP78 and CHOP in the TM of Tg-MYOCY437H mice. Human TM-3 cells stably expressing DsRed-tagged Y437H mutant MYOC exhibited inhibition of myocilin secretion and its intracellular accumulation compared with TM cells expressing WT MYOC. Expression of mutant MYOC in TM-3 cells or human primary TM cells induced ER stress and also increased intracellular protein levels of fibronectin, elastin, laminin, and collagen IV and I. In addition, TM-3 cells expressing mutant myocilin exhibited reduced active forms of matrix metalloproteinase (MMP)-2 and MMP-9 in conditioned medium compared with TM-3 cells expressing WT myocilin. Interestingly, both intracellularly accumulated fibronectin and collagen I colocalized with mutant myocilin and also with ER marker KDEL further suggesting intracellular accumulation of these proteins in the ER of TM cells. Furthermore, reduction of ER stress via PBA decreased selected ECM proteins in primary TM cells. Conclusions: These studies demonstrate that mutant myocilin induces abnormal ECM accumulation in the ER of TM cells, which may be responsible for reduced outflow facility and IOP elevation in myocilin-associated glaucoma.


Assuntos
Proteínas do Citoesqueleto/genética , DNA/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Olho/genética , Regulação da Expressão Gênica , Glaucoma de Ângulo Aberto/genética , Glicoproteínas/genética , Mutação , Malha Trabecular/metabolismo , Animais , Humor Aquoso/metabolismo , Western Blotting , Células Cultivadas , Proteínas do Citoesqueleto/biossíntese , Análise Mutacional de DNA , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Proteínas da Matriz Extracelular/genética , Proteínas do Olho/biossíntese , Glaucoma de Ângulo Aberto/metabolismo , Glaucoma de Ângulo Aberto/fisiopatologia , Glicoproteínas/biossíntese , Humanos , Pressão Intraocular , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Malha Trabecular/patologia
14.
Invest Ophthalmol Vis Sci ; 56(10): 5764-76, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26325415

RESUMO

PURPOSE: We evaluated differences in aqueous humor dynamics (AHD) among several mouse strains within younger and older age groups. METHODS: Albino (A/J, BALB/cJ) and pigmented (C3H/HeJ, C57-BL/6J) mice (young [2½-4½ months] and aged [10-12 months]) were studied. Intraocular pressure (IOP) was measured. In cannulated eyes, episcleral venous pressure (Pe) was assessed (blood reflux). Other AHD parameters (outflow facility [C], aqueous humor formation rate [Fin]) were assessed (constant flow infusion). Uveoscleral outflow rate (Fu) was obtained by calculation (Fu(calc)) using the modified Goldmann equation, and in additional eyes (for comparison), by FITC-dextran perfusion (Fu(FITC-dex)). RESULTS: Intraocular pressure was higher in pigmented strains, but did not exhibit age-dependence, except in the C57-BL/6J strain. Fu(calc) decreased with age in BALB/cJ (↓83.3%), C3H/HeJ (↓78.0%), and C57-BL/6J (↓85.0%) strains. In the A/J strain, Fu(calc) decreased with age (↓70.0%), but not significantly. Fin decreased with age in the C3H/HeJ (↓53.6%) strain. In C57-BL/6J and A/J strains, Fin decreased with age, but not significantly. C in the BALB/cJ strain increased with age (↑62.5%). In C3H/HeJ and C57-BL/6J strains, C increased with age, but not significantly. Episcleral venous pressure ranged from 6.0 to 6.6 mm Hg (albino strains) to 8.5 to 8.9 mm Hg (pigmented strains). Pe was not age dependent, but was higher in pigmented animals. CONCLUSIONS: In mouse, Fu and Fin diminish with age. C tends to increase as animals progress to middle life. There are strain differences in Fu, IOP, C, Fin, and Pe. The current findings provide an important foundation for comparisons among different strains in different study reports.


Assuntos
Envelhecimento/fisiologia , Humor Aquoso/fisiologia , Fatores Etários , Animais , Feminino , Pressão Intraocular/fisiologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos , Especificidade da Espécie , Tonometria Ocular , Pressão Venosa/fisiologia
15.
Exp Eye Res ; 141: 33-41, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26025608

RESUMO

Rodents are increasingly being used as glaucoma models to study ocular hypertension, optic neuropathy, and retinopathy. A number of different techniques are used to elevate intraocular pressure in rodent eyes by artificially obstructing the aqueous outflow pathway. Another successful technique to induce ocular hypertension is to transduce the trabecular meshwork of rodent eyes with viral vectors expressing glaucoma associated transgenes to provide more relevant models of glaucomatous damage to the trabecular meshwork. This technique has been used to validate newly discovered glaucoma pathogenesis pathways as well as to develop rodent models of primary open angle glaucoma. Ocular hypertension has successfully been induced by adenovirus 5 mediated delivery of mutant MYOC, bioactivated TGFß2, SFRP1, DKK1, GREM1, and CD44. Advantages of this approach are: selective tropism for the trabecular meshwork, the ability to use numerous mouse strains, and the relatively rapid onset of IOP elevation. Disadvantages include mild-to-moderate ocular inflammation induced by the Ad5 vector and sometimes transient transgene expression. Current efforts are focused at discovering less immunogenic viral vectors that have tropism for the trabecular meshwork and drive sufficient transgene expression to induce ocular hypertension. This viral vector approach allows rapid proof of concept studies to study glaucomatous damage to the trabecular meshwork without the expensive and time-consuming generation of transgenic mouse lines.


Assuntos
Glaucoma , Pressão Intraocular/fisiologia , Malha Trabecular/metabolismo , Vírus/genética , Animais , Modelos Animais de Doenças , Vetores Genéticos , Glaucoma/genética , Glaucoma/metabolismo , Glaucoma/fisiopatologia , Camundongos Transgênicos , Malha Trabecular/virologia , Transgenes
16.
Exp Eye Res ; 141: 74-90, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25933714

RESUMO

Glaucoma is a leading cause of blindness, which is treatable but currently incurable. Numerous animal models therefore have both been and continue to be utilized in the study of numerous aspects of this condition. One important facet associated with the use of such models is the ability to accurately and reproducibly measure (by cannulation) or estimate (by tonometry) intraocular pressure (IOP). At this juncture there are several different approaches to IOP measurement in different experimental animal species, and the list continues to grow. We feel therefore that a review of this subject matter is timely and should prove useful to others who wish to perform similar measurements. The general principles underlying various types of tonometric and non-tonometric techniques for non-continuous determination of IOP are considered. There follows discussion of specific details as to how these techniques are applied to experimental animal species involved in the research of this disease. Specific comments regarding anesthesia, circadian rhythm, and animal handling are also included, especially in the case of rodents. Brief consideration is also given to possible future developments.


Assuntos
Glaucoma/diagnóstico , Pressão Intraocular/fisiologia , Tonometria Ocular/métodos , Animais , Animais de Laboratório , Modelos Animais de Doenças , Glaucoma/fisiopatologia
18.
Invest Ophthalmol Vis Sci ; 53(11): 7043-51, 2012 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-22956608

RESUMO

PURPOSE: We previously discovered elevated levels of secreted frizzled-related protein 1 (sFRP1), the Wnt signaling pathway inhibitor, in the glaucomatous trabecular meshwork (GTM), and found that key canonical Wnt signaling pathway genes are expressed in the trabecular meshwork (TM). The purpose of our study was to determine whether a functional canonical Wnt signaling pathway exists in the human TM (HTM). METHODS: Western immunoblotting and/or immunofluorescent microscopy were used to study ß-catenin translocation as well as the actin cytoskeleton in transformed and primary HTM cells. A TCF/LEF luciferase assay was used to study functional canonical Wnt signaling, which was confirmed further by WNT3a-induced expression of a pathway target gene, AXIN2, via quantitative PCR. Intravitreal injection of an Ad5 adenovirus expressing Dickkopf-related protein-1 (DKK1) was used to study the in vivo effect of canonical Wnt signaling on IOP in mice. RESULTS: WNT3a induced ß-catenin translocation in the HTM, which was blocked by co-treatment with sFRP1. Similarly, WNT3a enhanced luciferase levels in TCF/LEF luciferase assays, which also were blocked by sFRP1. Furthermore, AXIN2 expression was elevated significantly by WNT3a. However, neither WNT3a nor sFRP1 affected actin cytoskeleton organization, which theoretically could be regulated by noncanonical Wnt signaling in HTM cells. Exogenous DKK1, a specific inhibitor for the canonical Wnt signaling pathway, or sFRP1 elevated mouse IOP to equivalent levels. CONCLUSIONS: There is a canonical Wnt signaling pathway in the TM, and this canonical Wnt pathway, but not the noncanonical Wnt signaling pathway, regulates IOP.


Assuntos
Malha Trabecular/metabolismo , Via de Sinalização Wnt/fisiologia , Actinas/metabolismo , Adenoviridae/genética , Proteína Axina/genética , Western Blotting , Células Cultivadas , Densitometria , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Pressão Intraocular/fisiologia , Injeções Intravítreas , Microscopia de Fluorescência , Transporte Proteico/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Malha Trabecular/efeitos dos fármacos , Proteína Wnt3A/farmacologia , beta Catenina/metabolismo
19.
Exp Eye Res ; 100: 65-72, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22575566

RESUMO

Elevated intraocular pressure (IOP) is a causative risk factor for the development and progression of glaucoma. Glaucomatous mutations in myocilin (MYOC) damage the trabecular meshwork and elevate IOP in humans and in mice. Animal models of glaucoma are important to discover and better understand molecular pathogenic pathways and to test new glaucoma therapeutics. Although a number of different animal models of glaucoma have been developed and characterized, there are no true models of human primary open angle glaucoma (POAG). The overall goal of this work is to develop the first inducible mouse model of POAG using a human POAG relevant transgene (i.e. mutant MYOC) expression in mouse eyes to elevate IOP and cause pressure-induced damage to the optic nerve. Four mouse strains (A/J, BALB/cJ, C57BL/6J, and C3H/HeJ) were used in this study. Ad5.MYOC.Y437H (5 × 10(7) pfu) was injected intravitreally into one eye, with the uninjected contralateral eye serving as the control eye. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Optic nerve damage was determined by scoring PPD stained optic nerve cross sections. Retinal ganglion cell and superior colliculus damage was assessed by Nissl stain cell counts. Intravitreal administration of viral vector Ad5.MYOC.Y437H caused a prolonged, reproducible, and statistically significant IOP elevation in BALB/cJ, A/J, and C57BL/6J mice. IOPs increased to approximately 25 mm Hg for 8 weeks (p < 0.0001). In contrast, the C3H/HeJ mouse strain was resistant to Ad5.MYOC.Y437H induced IOP elevation for the 8-week time period. IOPs were stable (12-15 mm Hg) in the uninjected control eyes. We also determined whether there were any strain differences in pressure-induced optic nerve damage. Even though IOP was similarly elevated in three of the strains tested (BALB/cJ, C57BL/6J, and A/J) only the A/J strain had considerable and significant optic nerve damage at the end of 8 weeks with optic nerve damage score of 2.64 ± 0.19 (n = 18, p < 0.001) in the injected eye. There was no statistical difference in retinal ganglion cell death or superior colliculus damage at the 8-week time point in any of the strains tested. These results demonstrate strain dependent responses to Ad5.MYOC.Y437H-induced ocular hypertension and pressure-induced optic nerve damage.


Assuntos
Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Proteínas do Olho/genética , Glaucoma de Ângulo Aberto/genética , Glicoproteínas/genética , Doenças do Nervo Óptico/genética , Adenoviridae/genética , Animais , Feminino , Vetores Genéticos , Glaucoma de Ângulo Aberto/metabolismo , Glaucoma de Ângulo Aberto/patologia , Técnicas Imunoenzimáticas , Pressão Intraocular , Injeções Intravítreas , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Hipertensão Ocular/genética , Hipertensão Ocular/metabolismo , Hipertensão Ocular/patologia , Doenças do Nervo Óptico/metabolismo , Doenças do Nervo Óptico/patologia , Especificidade da Espécie , Tonometria Ocular , Transgenes
20.
Invest Ophthalmol Vis Sci ; 52(2): 685-94, 2011 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-20861483

RESUMO

PURPOSE: To characterize a technique that concurrently assesses all aqueous humor hydrodynamic parameters in mouse eyes. METHODS: Mouse outflow facility (C) was determined by multiple flow-rate infusion and episcleral venous pressure (Pe) measured by manometry. The animals were then euthanatized, eliminating aqueous formation rate (Fin) and Pe. C was determined again (C(dead)) while uveoscleral outflow (Fu(dead)) and Fin were deduced. To assess whether Fu(dead) would remain the same as Fu(live), the animals were perfused with FITC-dextran and Fu determined. The effects of IOP-lowering drugs on the parameters of aqueous hydrodynamics were also evaluated. RESULTS: Under the conditions tested, Fu(live) (0.012 ± 0.003 µL/min) was not different from Fu(dead) (0.015 ± 0.003 µL/min; P = 0.472). In anesthetized mice, IOP = 11.4 ± 0.2 mm Hg (mean ± SEM, n = 8), C = 0.018 ± 0.0006 µL/min/mm Hg, Pe = 5.4 ± 0.2 mm Hg, Fin = 0.14 ± 0.0007 µL/min, and Fu = 0.029 ± 0.005 µL/min. C(dead) was not different from C (P = 0.317). Latanoprost reduced IOP by increasing C by 0.009 ± 0.0003 µL/min/mm Hg (P < 0.001), without affecting Fin or Fu. Betaxolol reduced Fin by 0.075 ± 0.021 µL/min (P = 0.009). Brimonidine increased C by 0.005 ± 0.0005 µL/min/mm Hg (P < 0.001) and Fu by 0.013 ± 0.003 µL/min (P = 0.007). CONCLUSIONS: In this study, a unique technique was developed to concurrently assess IOP, C, Pe, Fin, and Fu in the mouse eye. This experimental approach should be useful to evaluate effects of pharmacologic agents or genetic manipulations on aqueous humor dynamics in mice and other animal models.


Assuntos
Anti-Hipertensivos/administração & dosagem , Humor Aquoso/metabolismo , Pressão Intraocular/fisiologia , Esclera/metabolismo , Malha Trabecular/metabolismo , Úvea/metabolismo , Animais , Betaxolol/administração & dosagem , Tartarato de Brimonidina , Corantes , Dextranos , Fluoresceína-5-Isotiocianato/análogos & derivados , Pressão Intraocular/efeitos dos fármacos , Latanoprosta , Masculino , Manometria , Camundongos , Camundongos Endogâmicos BALB C , Prostaglandinas F Sintéticas/administração & dosagem , Quinoxalinas/administração & dosagem , Tonometria Ocular , Pressão Venosa
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