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1.
Sci Rep ; 8(1): 15472, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30341380

RESUMO

We investigated the effect of a new Rho kinase inhibitor, SB772077B (SB77) on aqueous outflow facility (OF) in human eyes using human organ-cultured anterior segment (HOCAS). IOP was monitored for 24 h post-treatment with either SB77 (0.1/10/50 µM) or vehicle after a stable baseline pressure. The hydrodynamic pattern of aqueous outflow was analysed by labelling outflow pathway with red fluorescent microspheres. The effect of SB77 on cell morphology, actin stress fibers, focal adhesions, ECM, status of RhoA activation and myosin light chain phosphorylation (p-MLC) were evaluated and compared with Y27632, by immunostaining using primary human trabecular meshwork (HTM) cells. Following 24 h treatment, SB77 increased OF by 16% at 0.1 µM (N = 6), 29% at 10 µM (N = 8; p = 0.018) and 39% at 50 µM (N = 8; p = 0.004) in human eyes. There was an overall increase in tracer quantity and in area along inner wall of Schlemm's canal. Treatment with SB77 showed no evidence of cytotoxicity and caused a significant reduction in the expression of fibrotic markers compared to Y27632. The present findings indicate that SB77 treatment was effective in enhancing OF and reducing fibrotic markers in an ex vivo model. Thus SB77 may be a potential clinical candidate for the management of glaucoma.


Assuntos
Humor Aquoso/metabolismo , Inibidores Enzimáticos/metabolismo , Olho/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Humanos , Hidrodinâmica , Modelos Biológicos , Técnicas de Cultura de Órgãos
2.
Invest Ophthalmol Vis Sci ; 59(1): 298-310, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29340644

RESUMO

Purpose: To determine if proteasome inhibition using MG132 increased the efficiency of FIV vector-mediated transduction in human trabecular meshwork (TM)-1 cells and monkey organ-cultured anterior segments (MOCAS). Methods: TM-1 cells were pretreated for 1 hour with 0.5% dimethyl sulfoxide (DMSO; vehicle control) or 5 to 50 µM MG132 and transduced with FIV.GFP (green fluorescent protein)- or FIV.mCherry-expressing vector at a multiplicity of transduction (MOT) of 20. At 24 hours, cells were fixed and stained with antibodies for GFP, and positive cells were counted, manually or by fluorescence-activated cell sorting (FACS). Cells transduced with FIV.GFP particles alone were used as controls. The effect of 20 µM MG132 treatment on high- and low-dose (2 × 107 and 0.8 × 107 transducing units [TU], respectively) FIV.GFP transduction with or without MG132 was also evaluated in MOCAS using fluorescence microscopy. Vector genome equivalents in cells and tissues were quantified by quantitative (q)PCR on DNA. Results: In the MG132 treatment groups, there was a significant dose-dependent increase in the percentage of transduced cells at all concentrations tested. Vector genome equivalents were also increased in TM-1 cells treated with MG132. Increased FIV.GFP expression in the TM was also observed in MOCAS treated with 20 µM MG132 and the high dose of vector. Vector genome equivalents were also significantly increased in the MOCAS tissues. Increased transduction was not seen with the low dose of virus. Conclusions: Proteasome inhibition increased the transduction efficiency of FIV particles in TM-1 cells and MOCAS and may be a useful adjunct for delivery of therapeutic genes to the TM by lentiviral vectors.


Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Vetores Genéticos , Vírus da Imunodeficiência Felina/genética , Leupeptinas/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Malha Trabecular/metabolismo , Transdução Genética , Animais , Segmento Anterior do Olho/metabolismo , Células Cultivadas , Citometria de Fluxo , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/genética , Humanos , Macaca mulatta , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase em Tempo Real , Transfecção
3.
PLoS One ; 11(3): e0151644, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27011234

RESUMO

BACKGROUND AND PURPOSE: The intraocular pressure (IOP)-lowering and side effects in response to different prostaglandin F2α analogues can be variable, but, the underlying basis for this difference remains unknown. This study investigated the differential changes of cellular proteins relevant to IOP-lowering effects of latanoprost and bimatoprost. METHODS: The human T lymphoblast (MOLT-3) cell line and immortalized human trabecular meshwork (iHTM) cells were studied by quantitative PCR and by immunofluorescence after treatment with either latanoprost or bimatoprost. New Zealand white rabbit eyes were treated topically with each agent and, following euthanasia, anterior segment tissues were studied with immunostaining. RESULTS: In cultured MOLT-3 cells, mRNA expression of both c-fos and matrix metalloproteinase 9 increased significantly in response to each agent. In addition, there was little change in tissue inhibitor of metalloproteinase (TIMP)-3 mRNA, but a significant decrease in TIMP-4. Fibronectin mRNA in MOLT-3 cells was down-regulated with bimatoprost, but was up-regulated with latanoprost. Immunofluorescence analysis of iHTM cells showed that intracellular fibronectin was significantly decreased by bimatoprost, but was increased by latanoprost. Both latanoprost and bimatoprost increased mRNA expression of NF-кB p65 and decreased that of IкBα. Aquaporin-1 mRNA expression was significantly down-regulated by bimatoprost. Immunostaining also revealed a significant decrease of aquaporin-1 in the ciliary epithelium of New Zealand white rabbits after bimatoprost treatment. CONCLUSIONS: Similarities in protein expression produced by latanoprost and bimatoprost in vitro may be relevant to the mechanism for their IOP-lowering effects in vivo. Differences in fibronectin expression and in aquaporin-1 expression in response to each agent may contribute to variability in the IOP-lowering efficacy in some studies.


Assuntos
Anti-Hipertensivos/farmacologia , Bimatoprost/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Pressão Intraocular/efeitos dos fármacos , Prostaglandinas F Sintéticas/farmacologia , Animais , Aquaporina 1/genética , Linhagem Celular , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Latanoprosta , NF-kappa B/genética , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , Coelhos , Transdução de Sinais/efeitos dos fármacos
4.
Invest Ophthalmol Vis Sci ; 56(5): 3075-83, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-26024091

RESUMO

PURPOSE: The cellular mechanisms linking elevated IOP with glaucomatous damage remain unresolved. Mechanical strains and short-term increases in IOP can trigger ATP release from retinal neurons and astrocytes, but the response to chronic IOP elevation is unknown. As excess extracellular ATP can increase inflammation and damage neurons, we asked if sustained IOP elevation was associated with a sustained increase in extracellular ATP in the posterior eye. METHODS: No ideal animal model of chronic glaucoma exists, so three different models were used. Tg-Myoc(Y437H) mice were examined at 40 weeks, while IOP was elevated in rats following injection of hypertonic saline into episcleral veins and in cynomolgus monkeys by laser photocoagulation of the trabecular meshwork. The ATP levels were measured using the luciferin-luciferase assay while levels of NTPDase1 were assessed using qPCR, immunoblots, and immunohistochemistry. RESULTS: The ATP levels were elevated in the vitreal humor of rats, mice, and primates after a sustained period of IOP elevation. The ecto-ATPase NTPDase1 was elevated in optic nerve head astrocytes exposed to extracellular ATP for an extended period. NTPDase1 was also elevated in the retinal tissue of rats, mice, and primates, and in the optic nerve of rats, with chronic elevation in IOP. CONCLUSIONS: A sustained elevation in extracellular ATP, and upregulation of NTPDase1, occurs in the posterior eye of rat, mouse, and primate models of chronic glaucoma. This suggests the elevation in extracellular ATP may be sustained in chronic glaucoma, and implies a role for altered purinergic signaling in the disease.


Assuntos
Trifosfato de Adenosina/metabolismo , Antígenos CD/genética , Apirase/genética , Modelos Animais de Doenças , Glaucoma/metabolismo , Pressão Intraocular/fisiologia , Segmento Posterior do Olho/metabolismo , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Contagem de Células , Doença Crônica , Feminino , Immunoblotting , Imuno-Histoquímica , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Ratos Endogâmicos BN , Reação em Cadeia da Polimerase em Tempo Real , Células Ganglionares da Retina/patologia , Transdução de Sinais
5.
Invest Ophthalmol Vis Sci ; 56(4): 2431-8, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25783606

RESUMO

PURPOSE: Purified Clostridium botulinum exoenzyme C3 transferase (C3) effects on the actin cytoskeleton in human trabecular meshwork cells (HTM) and on the outflow facility response in monkey organ-cultured anterior segments (MOCAS) were determined in the presence or absence of viral vectors. METHODS: Human adenovirus type 5 (AdV) and feline immunodeficiency virus (FIV) vectors were produced using kits. Cell soluble purified C3 (C3cs) was purchased commercially. Recombinant C3 (C3rec) cDNA was overexpressed in Escherichia coli and purified. The HTM cells were incubated with up to 10 µg/mL C3cs or with 5 µg of C3rec and/or viral vector (multiplicity of infection [MOI] = 25). Cells then were fixed and stained for actin. Outflow facility in MOCAS was measured at baseline, 4 hours, 24 hours, and 3 to 4 days following bolus injection of AdV (1.6 × 107 transducing units) and/or 2.5 µg C3rec. RESULTS: The HTM cells treated for 4 hours with C3cs (all doses) or for 24 hours with C3rec developed a rounded morphology and lost stress fibers. Cells transduced with vectors alone showed no changes at any time point. Cells exposed to C3rec and cotransduced with either viral vector showed significant disruption of the actin cytoskeleton within 4 hours after exposure, which persisted at 24 hours. In MOCAS, the AdV vector alone had no effect on outflow facility, but enhanced the response to C3rec at 4 hours. CONCLUSIONS: Coadministration of viral vectors enhances the ability of C3 transferase to disrupt actin stress fiber formation in HTM cells and increase outflow facility in MOCAS. Viral vectors potentially could be used to increase the bioavailability of proteins for cells that are difficult to transfect.


Assuntos
Actinas/metabolismo , Adenovírus Humanos/genética , Humor Aquoso/metabolismo , Complemento C3/farmacologia , Vetores Genéticos/farmacologia , Vírus da Imunodeficiência Felina/genética , Transferases/metabolismo , Animais , Humor Aquoso/virologia , Gatos , Células Cultivadas , Modelos Animais de Doenças , Seguimentos , Haplorrinos , Humanos , Técnicas de Cultura de Órgãos
6.
Invest Ophthalmol Vis Sci ; 55(11): 7499-507, 2014 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-25324280

RESUMO

PURPOSE: To identify stem cells in the chamber angle of the monkey eye by detection of 5-bromo-2'-deoxyuridine (BrdU) long-term retention. METHODS: Four cynomolgus monkeys were treated with BrdU via subcutaneous pumps for 4 weeks. The eyes of two animals were processed immediately thereafter (group 1) while in the other animals, BrdU treatment was discontinued for 4 weeks to allow identification of cells with long-term BrdU retention (group 2). The number of BrdU-positive nuclei was quantified, and the cells were characterized by immunohistochemistry and transmission electron microscopy (TEM). RESULTS: The number of BrdU-positive cells was higher at Schwalbe's line covering the peripheral end of Descemet's membrane than in Schlemm's canal (SC) endothelium, trabecular meshwork (TM), and scleral spur (SS). Labeling with BrdU in SC, TM, and SS was less intense and the number of labeled cells was smaller in group 2 than in group 1. In contrast, in cells of Schwalbe's line the intensity of BrdU staining and the number of BrdU-positive cells was similar when group 1 and 2 monkeys were compared with each other, indicating long-term BrdU retention. Cells that were BrdU-positive in Schwalbe's line region stained for the stem cell marker OCT4. Details of a stem cell niche in Schwalbe's line region were identified by TEM. CONCLUSIONS: We provide evidence for a niche in the Schwalbe's line region harboring cells with long-term BrdU retention and OCT4 immunoreactivity. The cells likely constitute a population of adult stem cells with the capability to compensate for the loss of TM and/or corneal endothelial cells.


Assuntos
Células-Tronco Adultas/ultraestrutura , Câmara Anterior/citologia , Bromodesoxiuridina , Células-Tronco Adultas/metabolismo , Animais , Câmara Anterior/metabolismo , Bromodesoxiuridina/farmacocinética , Macaca fascicularis , Macaca mulatta , Microscopia Eletrônica de Transmissão , Reprodutibilidade dos Testes , Tomografia de Coerência Óptica
7.
Curr Eye Res ; 39(9): 859-70, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24559478

RESUMO

Currently, the most effective outflow drugs approved for clinical use are prostaglandin F2α analogues, but these require daily topical self-dosing and have various intraocular, ocular surface and extraocular side effects. Lentiviral vector-mediated delivery of the prostaglandin F synthase (PGFS) gene, resulting in long-term reduction of intraocular pressure (IOP), may eliminate off-target tissue effects and the need for daily topical PGF2α self-administration. Lentiviral vector-mediated delivery of the PGFS gene to the anterior segment has been achieved in cats and non-human primates. Although these results are encouraging, our studies have identified a number of challenges that need to be overcome for prostaglandin gene therapy to be translated into the clinic. Using examples from our work in non-human primates, where we were able to achieve a significant reduction in IOP (2 mm Hg) for 5 months after delivery of the cDNA for bovine PGF synthase, we identify and discuss these issues and consider several possible solutions.


Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Hidroxiprostaglandina Desidrogenases/genética , Lentivirus/genética , Animais , Segmento Anterior do Olho/metabolismo , Terapia Genética , Glaucoma/terapia , Pressão Intraocular , Macaca
8.
Invest Ophthalmol Vis Sci ; 54(7): 5103-10, 2013 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-23800771

RESUMO

PURPOSE: To determine the effect of the nitric oxide donor, sodium nitroprusside (SNP), and the nitric oxide synthase (NOS) inhibitor, L-nitro-arginine-methylester (L-NAME), on IOP, mean arterial pressure (MAP), pupil diameter (PD), refraction (Rfx), aqueous humor formation (AHF), and outflow facility (OF) in monkeys. METHODS: Monkeys were treated with single or multiple topical treatments of 500 µg SNP or L-NAME to one eye. IOP was determined by Goldmann applanation tonometry, PD with vernier calipers in room light, Rfx by Hartinger coincidence refractometry, AHF by fluorophotometry, and MAP with a blood pressure monitor. OF was determined by two-level constant pressure perfusion following anterior chamber exchange. RESULTS: Following four topical treatments with 500 µg SNP, 30 minutes apart, IOP was significantly decreased from 2 to 6 hours compared with the contralateral control with the maximum IOP reduction of 20% at 3 hours (P < 0.001). PD, Rfx, and AHF were unchanged. Effects on MAP were variable. OF after SNP exchange was significantly increased by 77% (P < 0.05) at 10(-3) M. Topical L-NAME had no effect on IOP, PD, Rfx, or MAP. CONCLUSIONS: Enhancement of nitric oxide concentration at targeted tissues in the anterior segment may be a useful approach for IOP reduction for glaucoma therapy. Additional studies are warranted before conclusions can be made regarding the effect of NOS inhibition on ocular physiology in nonhuman primates.


Assuntos
Segmento Anterior do Olho/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Macaca fascicularis/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/fisiologia , Nitroprussiato/farmacologia , Análise de Variância , Animais , Segmento Anterior do Olho/fisiologia , Humor Aquoso/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Frequência Cardíaca/efeitos dos fármacos , Pressão Intraocular/efeitos dos fármacos , Óxido Nítrico/antagonistas & inibidores , Pupila/efeitos dos fármacos , Refração Ocular/efeitos dos fármacos
9.
Invest Ophthalmol Vis Sci ; 53(4): 2368-76, 2012 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-22427549

RESUMO

Purpose. The scanning laser polarimetry with variable corneal compensation (GDx VCC) methodology was established and verified in monkeys with experimental glaucoma (ExpG). Terminal GDx parameters were correlated with axon counts and electrophysiologic measures. The effects of memantine on these parameters were investigated. Methods. ExpG was induced in monkeys and intraocular pressure monitored weekly. Some monkeys received memantine in their diet before and after ExpG induction (1-10 months). GDx VCC scans, stereophotographs, and multifocal visual evoked potential (mfVEP) data were collected at baseline and every 6 to 8 weeks until euthanasia. Optic nerves were prepared for axon counting and other morphologic analysis. Results. There was no difference in IOP elevation exposure between memantine-treated and no-memantine-treated monkeys. The percentage of the optic nerve area composed of connective tissue septa was significantly greater in ExpG eyes than in Fellow eyes. There was a strong positive correlation between axon counts and terminal GDx parameter measures. Animals not receiving memantine exhibited significantly lower mfVEP amplitudes in ExpG eyes compared with the ipsilateral baseline or the final value in the Fellow eye. ExpG eyes from memantine-treated animals had higher overall mean amplitudes that were not significantly different relative to the ipsilateral baseline and final amplitudes in the Fellow eye. Conclusions. The authors' studies confirm that GDx VCC can be utilized in monkey ExpG studies to detect early retinal structural changes and that these changes are highly correlated with optic nerve axon counts. These structural changes may or may not lead to central functional changes as shown by the mfVEP in response to investigational therapies.


Assuntos
Axônios/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glaucoma/metabolismo , Pressão Intraocular/efeitos dos fármacos , Memantina/farmacologia , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Potenciais Evocados Visuais/efeitos dos fármacos , Feminino , Macaca fascicularis , Masculino , Nervo Óptico/metabolismo , Fotografação/métodos , Polarimetria de Varredura a Laser/métodos
10.
Arch Biochem Biophys ; 518(1): 53-60, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22198282

RESUMO

Ocular hypertension is the greatest known risk factor for glaucoma that affects an estimated 70 million people worldwide. Lowering intraocular pressure (IOP) remains the mainstay of therapy in the management of glaucoma. By means of microarray analysis, we have discovered that 1α,25-dihydroxyvitamin D(3) (1α,25-(OH)(2)D(3)) regulates genes that are known to be involved in the determination of intraocular pressure (IOP). Topical administration of 1α,25-(OH)(2)D(3) or its analog, 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D(3) (2MD), markedly reduces IOP in non-human primates. The reduction in IOP is not the result of reduced aqueous humor formation, while a 35% increase in aqueous humor drainage by 1α,25-(OH)(2)D(3) was found but this increase did not achieve significance. Nevertheless, our results suggest that 1α,25-(OH)(2)D(3), or an analog thereof, may present a new approach to the treatment of glaucoma.


Assuntos
Calcitriol/análogos & derivados , Calcitriol/farmacologia , Pressão Intraocular/efeitos dos fármacos , Administração Tópica , Animais , Humor Aquoso/efeitos dos fármacos , Humor Aquoso/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Calcitriol/administração & dosagem , Calcitriol/química , Cálcio/sangue , Olho/irrigação sanguínea , Olho/efeitos dos fármacos , Olho/metabolismo , Feminino , Macaca fascicularis , Masculino , Camundongos , Ratos , Transcriptoma/efeitos dos fármacos
11.
Invest Ophthalmol Vis Sci ; 52(9): 6820-4, 2011 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-21757583

RESUMO

PURPOSE: The effect of total volume perfused on outflow resistance (the reciprocal of outflow facility) and the effect of age on the rate of change in resistance as a function of total volume were determined in rhesus and cynomolgus monkeys. METHODS: Outflow facility was measured under general anesthesia by two-level constant pressure perfusion in one eye of 22 rhesus and 17 cynomolgus monkeys (ranging in age, respectively, from 4 to 25 and from 3 to 12 years). Total volume perfused was calculated from data obtained during the perfusion. RESULTS: Resistance decreased in both cynomolgus and rhesus monkeys as total volume perfused increased (-0.085 ± 0.021 and -0.022 ± 0.011 mm Hg/µL/min/µL(tot); P = 0.001 and P = 0.047, respectively). Rate of change in resistance significantly increased in cynomolgus monkeys as total volume perfused increased (0.0018 ± 0.0.0007 mm Hg/µL/min/µL(tot), P = 0.033); however, this was not the case in rhesus monkeys. After accounting for total volume perfused, the rate of change in resistance significantly decreased with increasing age in rhesus monkeys (-0.0068 ± 0.0026 [mm Hg/µL/min]/µL(tot)/y, P = 0.017). There was no significant difference in rate of change in resistance with age, after accounting for total volume, in the cynomolgus monkeys. CONCLUSIONS: The present study supports previous findings indicating that total washout is largely dependent on perfusion volume. However, in populations with old/elderly animals, such as our rhesus group, we found that age does play a significant role in rate of change in resistance, and may be an even more important factor to consider in the rate of resistance change than volume perfused in aged animals.


Assuntos
Envelhecimento/fisiologia , Câmara Anterior/fisiologia , Humor Aquoso/fisiologia , Animais , Pressão Intraocular , Macaca fascicularis , Macaca mulatta , Reologia/métodos
12.
Exp Eye Res ; 93(3): 321-7, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21147103

RESUMO

The effects of various nitric oxide compounds and their inhibitors on monkey ciliary muscle contraction in vitro were investigated in both the longitudinal and circular vectors. The responses to nitric oxide compounds in carbachol precontracted ciliary muscle consisted of an initial relaxation often followed by recovery to near carbachol precontracted levels while the compound was still present. Sodium nitroprusside produced the greatest relaxation responses (nearly 100% relaxation in both vectors at 10(-3) M). The highest concentrations of isosorbide dinitrate (10(-4) M) and L-arginine (10(-3) M) produced relaxation responses of approximately 50% in both vectors. 8-Bromo cyclic GMP produced the smallest relaxation responses (25-35%). Nitric oxide synthase inhibition enhanced carbachol contraction up to 20% in the longitudinal but not the circular vector. Phosphodiesterase inhibition did not further enhance the relaxation response to L-arginine. Guanylate cyclase inhibition partially attenuated the relaxation response to sodium nitroprusside. Nitric oxide generating compounds were effective in relaxing precontracted monkey ciliary muscle in vitro. Endogenous production of nitric oxide is likely involved in the regulation of the contractile response in monkey ciliary muscle. Nitric oxide generating compounds may have potential value in therapeutic areas where modulation of ciliary muscle tension is desirable.


Assuntos
Corpo Ciliar/fisiologia , Músculo Liso/fisiologia , Doadores de Óxido Nítrico/farmacologia , Animais , Carbacol/farmacologia , Corpo Ciliar/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Contração Isométrica/efeitos dos fármacos , Macaca fascicularis , Macaca mulatta , Masculino , Relaxamento Muscular/fisiologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores
13.
Exp Eye Res ; 91(4): 486-90, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20620138

RESUMO

Sodium orthovanadate (Na(3)VO(4)) is reported to reduce IOP by affecting aqueous formation, but whether it also affects outflow facility (OF) is unclear. We tested the effect of Na(3)VO(4) on OF and intraocular pressure (IOP) in live cynomolgus monkeys, and on actin and cell adhesion organization in cultured human trabecular meshwork (HTM) cells. Total OF (n = 12) was measured by 2-level constant pressure perfusion of the monkey anterior chamber (AC) before and after exchange with 1 mM Na(3)VO(4) or vehicle in opposite eyes. Topical 1% Na(3)VO(4) or vehicle only was given twice daily (each 2 × 20 µL drops) for 4 days to opposite eyes (n = 8), and Goldmann IOP was measured before and hourly after treatment for 6 h on Days 1 and 4. Filamentous actin and vinculin-containing cell adhesions were examined by epifluorescence microscopy after the cells had been incubated with 1 mM Na(3)VO(4) for 24 h. A) In monkeys, Na(3)VO(4) increased OF by 29.3 ± 8.8% (mean ± s.e.m.) over the perfusion interval when adjusted for baseline and contralateral eye washout (p = 0.01; n = 12). B) Day 1 baseline IOP was 16.2 ± 1.5 mmHg in treated eyes and 15.9 ± 1.3 mmHg in the contralateral control eyes. Following treatment on Day 1, IOP was no different (p > 0.05) between treated eyes and control eyes at any time-point or compared to baseline. Day 4 mean IOP averaged over hours 2-6 was 13.5 ± 0.8 mmHg in treated eyes and 16.1 ± 0.2 mmHg in control eyes. Treated eye IOP was lower than its Day 4 baseline (p < 0.005), lower than control eyes for the same Day 4 interval (p = 0.009), and lower than the Day 1 baseline (p = 0.0000). Control eye IOP on Day 4 was not significantly different from baseline on Day 1. C) Incubation of HTM cells with 1 mM Na(3)VO(4) for 24 h caused a loss of actin stress fibers and vinculin-containing adhesions. Cell retraction and separation was also observed in vanadate-treated cultures. Reformation of actin stress fibers, vinculin-containing adhesions and confluent monolayers occurred within 24 h after Na(3)VO(4)-containing culture medium was replaced with Na(3)VO(4)-free medium. Ocular administration of Na(3)VO(4) to live monkeys significantly increases OF and reduces IOP. Na(3)VO(4) reversibly disrupts actin and cell adhesion organization and causes retraction and separation of cultured HTM cells. Na(3)VO(4) increases pressure-dependent outflow in live monkeys. Altered actin architecture in the TM may play a part in this increased OF.


Assuntos
Humor Aquoso/metabolismo , Pressão Intraocular/efeitos dos fármacos , Malha Trabecular/efeitos dos fármacos , Vanadatos/farmacologia , Actinas/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Macaca fascicularis , Masculino , Microscopia de Fluorescência , Malha Trabecular/metabolismo , Vanadatos/administração & dosagem , Vinculina/metabolismo
14.
Exp Cell Res ; 316(9): 1500-12, 2010 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-20302860

RESUMO

In the trabecular meshwork (TM) of the eye, regulation of tissue contractility by the PPRARI sequence within the Heparin II (HepII) domain of fibronectin is believed to control the movement of aqueous humor and dictate the level of intraocular pressure. This study shows that the HepII domain utilizes activated alpha4beta1 integrin and collagen to mediate a co-signaling pathway that down-regulates contractility in TM cells. siRNA silencing of alpha4beta1 integrin blocked the actin disrupting effects of both PPRARI and the HepII domain. The down-regulation of the actin cytoskeleton and contractility did not involve syndecan-4 or other heparan sulfate proteoglycans (HSPGs) since siRNA silencing of syndecan-4 expression or heparitinase removal of cell surface HSPGs did not prevent the HepII-mediated disruption of the actin cytoskeleton. HepII-mediated disruption of the cytoskeleton depended upon the presence of collagen in the extracellular matrix, and cell binding studies indicated that HepII signaling involved cross-talk between alpha4beta1 and alpha1/alpha2beta1 integrins. This is the first time that the PPRARI sequence in the HepII domain has been shown to serve as a physiological alpha4beta1 ligand, suggesting that alpha4beta1 integrin may be a key regulator of tissue contractility.


Assuntos
Colágeno/metabolismo , Fibronectinas/metabolismo , Heparina/metabolismo , Integrina alfa4beta1/metabolismo , Pressão Intraocular , Transdução de Sinais , Malha Trabecular/metabolismo , Actinas/metabolismo , Animais , Western Blotting , Adesão Celular , Células Cultivadas , Citoesqueleto/metabolismo , Regulação para Baixo , Citometria de Fluxo , Imunofluorescência , Humanos , Integrina alfa4beta1/antagonistas & inibidores , Integrina alfa4beta1/genética , Macaca mulatta , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Malha Trabecular/citologia
15.
Invest Ophthalmol Vis Sci ; 51(4): 2060-6, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19933177

RESUMO

Purpose. To determine the effects of COCH transgene expression on cultured human trabecular meshwork (HTM) cell morphology and on outflow facility (OF) in monkey organ cultured anterior segments (MOCAS). Methods. An adenoviral (Ad) vector expressing both cochlin (COCH) and green fluorescent protein (GFP) (AdCOCHGFP) or GFP alone (AdGFP) was used to transduce cultured HTM cells (multiplicity of transduction, 2.8 and 28). COCH transgene expression in transduced HTM cells and the culture medium was verified by Western blot analysis and immunofluorescence detection 5 days after transduction. MOCAS were used to test the effect of Ad vectors (2.8 x 10(10) viral particles per segment) on OF. The morphology of transduced MOCAS was evaluated by light microscopy. Results. Western blot analysis showed a viral vector dose-dependent expression of cochlin in transduced cells and the culture medium. There was no notable morphologic change in transduced cells. In MOCAS, cochlin expression was detectable in the medium by 3 days after transduction. A 35% decrease in OF in AdCOCHGFP-transduced MOCAS was detected after 3 days, decreasing by 76% after 12 days when compared to control segments injected with AdGFP. Anterior segment pressure (ASP) more than doubled (P < 0.05) in segments injected with AdCOCHGFP at 12 days after transduction. Light microscopy revealed normal angle structures in transduced segments. Conclusions. Ad vector delivery of the COCH transgene resulted in cochlin expression in HTM cells and MOCAS. Cochlin expression was effective in decreasing OF and increasing ASP in MOCAS, suggesting possible involvement of cochlin in IOP elevation in vivo. COCH gene delivery has potential for use in developing a glaucoma model.


Assuntos
Humor Aquoso/metabolismo , Expressão Gênica/fisiologia , Proteínas/genética , Malha Trabecular/metabolismo , Transgenes/fisiologia , Adenoviridae/genética , Adolescente , Adulto , Animais , Segmento Anterior do Olho , Western Blotting , Células Cultivadas , Proteínas da Matriz Extracelular , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Humanos , Macaca fascicularis , Macaca mulatta , Masculino , Técnicas de Cultura de Órgãos , Transfecção
16.
Surv Ophthalmol ; 54(4): 472-86, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19539835

RESUMO

In a chronic disease such as glaucoma, a therapy that provides a long lasting local effect with minimal systemic side effects, while circumventing the issue of patient compliance, is very attractive. The field of gene therapy is growing rapidly and ocular applications are expanding. Our understanding of the molecular pathogenesis of glaucoma is leading to greater specificity in ocular tissue targeting. Improvements in gene delivery techniques, refinement of vector construction methods, and development of better animal models combine to bring this potential therapy closer to reality.


Assuntos
Terapia Genética/métodos , Glaucoma/terapia , Doenças do Nervo Óptico/terapia , Animais , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Terapia Genética/tendências , Humanos , Pressão Intraocular
17.
Hum Gene Ther ; 20(3): 191-200, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19301472

RESUMO

We injected lentiviral vectors into the eyes of live nonhuman primates to assess potential for glaucoma gene therapy. Anterior chambers of five cynomolgus monkeys were injected with green fluorescent protein (GFP)-encoding feline immunodeficiency viral vectors. The monkeys were monitored for in vivo transgene expression and clinical parameters. Their eyes were harvested 2-15 months postinjection for tissue analyses. All seven eyes injected with 1.0-2.0 x 10(8) transducing units (TU) showed substantial GFP fluorescence in the trabecular meshwork (TM), which was observable even by goniophotographic monitoring for up to 15 months. Only the lowest dose (0.03 x 10(8) TU) failed to result in TM fluorescence detectable in vivo, and five of the eight vector-injected eyes continued to display substantial GFP expression when enucleated eyes were examined at 2, 7, or 15 months postinjection. Some transduced cells were also detected in the iris and ciliary body. Mild, transient postinjection inflammatory responses exceeding that induced by a control saline injection were observed, but vectors did not raise intraocular pressure and were well tolerated. The results demonstrate the first lentiviral vector transduction of the nonhuman primate aqueous humor outflow pathway and support application of the system to human glaucoma gene therapy.


Assuntos
Humor Aquoso/metabolismo , Vetores Genéticos/biossíntese , Vírus da Imunodeficiência Felina/genética , Transgenes/fisiologia , Animais , Humor Aquoso/citologia , Corpo Ciliar/citologia , Corpo Ciliar/metabolismo , Feminino , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Glaucoma de Ângulo Aberto/terapia , Proteínas de Fluorescência Verde , Humanos , Injeções , Iris/citologia , Iris/metabolismo , Macaca fascicularis , Masculino , Proteínas Recombinantes/biossíntese , Fatores de Tempo , Malha Trabecular/citologia , Malha Trabecular/metabolismo
18.
Trans Am Ophthalmol Soc ; 107: 146-58, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20126491

RESUMO

PURPOSE: To determine the effects of the advanced glycation end product (AGE) cross-link breaker alagebrium on intraocular pressure (IOP), accommodation (ACC), outflow facility (OF), anterior segment morphology, and ocular AGE and receptors for AGE (RAGE) in older rhesus monkeys. METHODS: Six rhesus monkeys (aged 19 to 20 years) received 3 or 4 intracameral and intravitreal (final concentration, 1 mM) injections of alagebrium to one eye over 2.5 to 3 weeks and vehicle to the opposite eye. ACC and OF responses to intramuscular or intravenous pilocarpine were measured at baseline and at 1 to 2 weeks and 2, 4, and 6 months postinjection. IOP was measured prior to all injections, ACC, and OF measurements. Monkeys were euthanized 3 to 6 months after the last injection, the eyes were enucleated, and anterior and posterior segments were examined by electron microscopy or immunohistochemistry. RESULTS: No significant differences were found in ACC or IOP at any time point after alagebrium treatment. Baseline OF was higher (37.0 +/- 6.0%; P < or = .005) in alagebrium-treated vs control eyes at 6 months postinjection. In 3 monkeys, alagebrium-treated eyes, compared to control eyes, showed greater focal plaque formation, similar to that seen in primary open-angle glaucoma, in the juxtacanalicular meshwork/inner wall of Schlemm's canal. No changes in anterior segment AGE or RAGE were detectable. However, some areas of the retina and optic nerve head exhibited decreased AGE and increased RAGE immunostaining. CONCLUSIONS: Intraocular injection of AGE cross-link breakers is an unlikely approach for glaucoma therapy. However, it may generate a model for further study of glaucomatous-like plaque formation. Immunohistochemical changes in the posterior segment in response to alagebrium warrant further functional studies.


Assuntos
Segmento Anterior do Olho/efeitos dos fármacos , Segmento Anterior do Olho/fisiologia , Olho/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Receptores Imunológicos/metabolismo , Tiazóis/farmacologia , Acomodação Ocular/efeitos dos fármacos , Animais , Segmento Anterior do Olho/ultraestrutura , Olho/efeitos dos fármacos , Olho/patologia , Feminino , Glaucoma de Ângulo Aberto/patologia , Imuno-Histoquímica , Injeções , Pressão Intraocular/efeitos dos fármacos , Macaca mulatta , Microscopia Eletrônica , Disco Óptico/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Refração Ocular/efeitos dos fármacos , Retina/metabolismo , Tiazóis/administração & dosagem , Corpo Vítreo
19.
Exp Eye Res ; 88(4): 713-7, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18793636

RESUMO

Abnormally high resistance to aqueous humor drainage via the trabecular meshwork and Schlemm's canal is highly correlated with the development of primary open-angle glaucoma. Contractility of the actomyosin system in the trabecular cells or inner wall endothelium of Schlemm's canal is an important factor in the regulation of outflow resistance. Cytoskeletal agents, affecting F-actin integrity or actomyosin contractility, or gene therapies, employing overexpression of caldesmon or Rho-A inhibition, can decrease outflow resistance in the drainage pathway. In this review, we discuss the mechanisms underlying these and similar effects on trabecular outflow resistance in living animals and/or in cultured ocular anterior segments from enucleated animal or human eyes.


Assuntos
Actomiosina/fisiologia , Malha Trabecular/fisiologia , Animais , Proteínas de Ligação a Calmodulina/farmacologia , Glaucoma de Ângulo Aberto/fisiopatologia , Humanos , Pressão Intraocular/efeitos dos fármacos , Pressão Intraocular/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/ultraestrutura , Moduladores de Tubulina/farmacologia
20.
Invest Ophthalmol Vis Sci ; 50(1): 235-41, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18757505

RESUMO

PURPOSE: To determine the active site in the Heparin II (HepII) domain of fibronectin that regulates outflow facility in cultured anterior segments and disrupts the actin cytoskeleton in transformed human trabecular meshwork (TM-1) cells. METHODS: Outflow facility was determined by two-level, constant-pressure perfusion in cultured anterior segments of rhesus and cynomolgus monkey eyes. One segment from each pair was exchanged with either the HepII domain or an integrin/syndecan binding peptide (IDAPS or PPRARI) from the HepII domain. To assay changes in the actin cytoskeleton, TM-1 cells were incubated for 24 hours with or without the HepII domain, PPRARI, or IDAPS. Changes were monitored with phase and immunofluorescence microscopy. RESULTS: HepII domain (100 microg/mL) and PPRARI (500 microg/mL) increased outflow facility by 31% +/- 13% (n = 9, P < 0.05) and 24% +/- 9% (n = 8, P < 0.05), respectively in cultured anterior segments after an overnight infusion. Perfusion with IDAPS (500 microg/mL) had no effect on outflow facility. In TM-1 cultures, 250 microg/mL of the HepII domain or 4 mg/mL of PPRARI disrupted the assembly of actin filaments. A lower concentration of PPRARI (2 mg/mL) disrupted the actin cytoskeleton when used in combination with a nondisrupting concentration of the HepII domain (30-60 microg/mL). In contrast, IDAPS did not disrupt the actin cytoskeleton under any condition tested. CONCLUSIONS: The active site in the HepII domain that regulates outflow facility in cultured anterior segments and disrupts the actin cytoskeleton in TM-1 cells is the syndecan/integrin binding sequence, PPRARI.


Assuntos
Humor Aquoso/metabolismo , Domínio Catalítico , Fibronectinas/química , Heparina/química , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Malha Trabecular/efeitos dos fármacos , Actinas/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Humanos , Integrinas/química , Macaca fascicularis , Macaca mulatta , Microscopia de Fluorescência , Técnicas de Cultura de Órgãos , Sindecanas/química , Malha Trabecular/metabolismo
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