RESUMO
Frank-Ter Haar syndrome (FTHS), also known as Ter Haar syndrome, is an autosomal-recessive disorder characterized by skeletal, cardiovascular, and eye abnormalities, such as increased intraocular pressure, prominent eyes, and hypertelorism. We have conducted homozygosity mapping on patients representing 12 FTHS families. A locus on chromosome 5q35.1 was identified for which patients from nine families shared homozygosity. For one family, a homozygous deletion mapped exactly to the smallest region of overlapping homozygosity, which contains a single gene, SH3PXD2B. This gene encodes the TKS4 protein, a phox homology (PX) and Src homology 3 (SH3) domain-containing adaptor protein and Src substrate. This protein was recently shown to be involved in the formation of actin-rich membrane protrusions called podosomes or invadopodia, which coordinate pericellular proteolysis with cell migration. Mice lacking Tks4 also showed pronounced skeletal, eye, and cardiac abnormalities and phenocopied the majority of the defects associated with FTHS. These findings establish a role for TKS4 in FTHS and embryonic development. Mutation analysis revealed five different homozygous mutations in SH3PXD2B in seven FTHS families. No SH3PXD2B mutations were detected in six other FTHS families, demonstrating the genetic heterogeneity of this condition. Interestingly however, dermal fibroblasts from one of the individuals without an SH3PXD2B mutation nevertheless expressed lower levels of the TKS4 protein, suggesting a common mechanism underlying disease causation.
Assuntos
Anormalidades Múltiplas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Anormalidades do Olho/complicações , Cardiopatias Congênitas/complicações , Anormalidades Musculoesqueléticas/complicações , Mutação/genética , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Pré-Escolar , Mapeamento Cromossômico , Anormalidades do Olho/genética , Feminino , Inativação Gênica , Cardiopatias Congênitas/genética , Homozigoto , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Anormalidades Musculoesqueléticas/genética , Proteínas de Transferência de Fosfolipídeos/química , SíndromeRESUMO
BACKGROUND: The loss of RGCs expressing Thy-1 after optic nerve injury has an initial phase of rapid decline followed by a longer phase with slower reduction rate. This study used longitudinal retinal imaging of mice expressing cyan fluorescent protein under control of the Thy-1 promoter (Thy1-CFP mice) to determine how the α2-adrenergic agonist brimonidine influences loss of Thy1 promoter activation. METHODS: Baseline images of the fluorescent retinal neurons in 30 Thy1-CFP mice were obtained using a modified confocal scanning laser ophthalmoscope. Next, brimonidine (100 ug/kg, IP) was administered either one time immediately after optic nerve crush, or immediately after optic nerve crush and then every 2 days for four weeks. A control group received a single saline injection immediately after optic nerve crush. All animals were imaged weekly for four weeks after optic nerve crush. Loss of fluorescent retinal neurons within specific retinal areas was determined by counting. RESULTS: At one week after optic nerve crush, the proportion of fluorescent retinal neurons retaining fluorescence was 44±7% of baseline in control mice, 51±6% after one brimonidine treatment, and 55±6% after brimonidine treatment every other day (P<0.05 for both brimonidine treatment groups compared to the control group). Subsequently, the number of fluorescent retinal neurons in the group that received one treatment differed insignificantly from the control group. In contrast, the number of fluorescent retinal neurons in the group that received repeated brimonidine treatments was greater than the control group by 28% at two weeks after crush and by 32% at three weeks after crush (P<0.05 at both time points). Rate analysis showed that brimonidine slowed the initial rate of fluorescent cell decline in the animals that received multiple treatments (P<0.05). Differences in the rate of loss among the treatment groups were insignificant after the second week. CONCLUSION: Repeated brimonidine treatments protect against loss of fluorescence within fluorescent retinal neurons of Thy1-CFP mice after optic nerve crush. As most of fluorescent retinal neurons in this system are RGCs, these findings indicate that repeated brimonidine treatments may protect RGC health following optic nerve crush.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/uso terapêutico , Compressão Nervosa , Traumatismos do Nervo Óptico/tratamento farmacológico , Regiões Promotoras Genéticas , Substâncias Protetoras/uso terapêutico , Quinoxalinas/uso terapêutico , Antígenos Thy-1/fisiologia , Análise de Variância , Animais , Tartarato de Brimonidina , Contagem de Células , Modelos Animais de Doenças , Feminino , Estudos Longitudinais , Masculino , Camundongos , Microscopia de Fluorescência , Traumatismos do Nervo Óptico/genética , Traumatismos do Nervo Óptico/patologia , Células Ganglionares da Retina/efeitos dos fármacosRESUMO
Many ocular pathologies, including retinopathy of prematurity (ROP), diabetic retinopathy, and age-related macular degeneration, result in vision loss because of aberrant neoangiogenesis. A common feature of these conditions is the presence of hypoxic areas and overexpression of the proangiogenic vascular endothelial growth factor (VEGF). The prevailing current treatment, laser ablation of the retina, is destructive and only partially effective. Preventive and less destructive therapies are much more desirable. Here, we show that mice lacking c-Jun N-terminal kinase 1 (JNK1) exhibit reduced pathological angiogenesis and lower levels of retinal VEGF production in a murine model of ROP. We found that hypoxia induces JNK activation and regulates VEGF expression by enhancing the binding of phospho-c-Jun to the VEGF promoter. Intravitreal injection of a specific JNK inhibitor decreases retinal VEGF expression and reduces pathological retinal neovascularization without obvious side effects. These results strongly suggest that JNK1 plays a key role in retinal neoangiogenesis and that it represents a new pharmacological target for treatment of diseases where excessive neoangiogenesis is the underlying pathology.
Assuntos
Hipóxia/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Doenças Retinianas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Ativação Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Hipóxia/genética , Hipóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Proteína Quinase 8 Ativada por Mitógeno/genética , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Inibidores de Proteínas Quinases/farmacologia , Doenças Retinianas/genética , Doenças Retinianas/patologia , Transcrição Gênica/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
PURPOSE: The goal of this study is to determine whether increased optic atrophy type 1 (OPA1) expression protects against retinal ganglion cell (RGC) death in glaucomatous DBA/2J mice. METHODS: Intraocular pressure in DBA/2J mice was measured, and pre-glaucomatous DBA/2J mice eyes were transfected with recombinant adeno-associated virus serotype 2 (AAV2) constructs including AAV2-wild type (WT) mOPA1 for two months. Increased OPA1 expression was confirmed by western blotting and RGC survival was assessed by retrograde labeling with FluoroGold. In addition, apoptotic cell death and mitochondrial structure were determined in AAV2-WT mOPA1-transfected differentiated RGC-5 cells exposed to elevated hydrostatic pressure (30 mmHg) for three days. RESULTS: WT AAV2-mOPA1 transfection significantly increased 90 kDa and 80 kDa OPA1 isoforms in the retina of glaucomatous DBA/2J mice. OPA1 immunoreactivity was increased in the inner nuclear layer, inner plexiform layer, and ganglion cell layer in nine month-old glaucomatous DBA/2J mice transfected with AAV2-WT mOPA1. Overexpression of OPA1 significantly increased RGC survival at two months after AAV2-WT mOPA1 transfection, and decreased activation of both astroglia and microglia in the retina of glaucomatous DBA/2J mice. Also, overexpression of OPA1 in differentiated RGC-5 cells resulted in less apoptotic cell death and blocked mitochondrial fission following elevated hydrostatic pressure. CONCLUSIONS: OPA1 can directly modulate RGC survival, and increasing OPA1 expression may protect against RGC death in glaucomatous optic neuropathy.
Assuntos
Citoproteção , GTP Fosfo-Hidrolases/metabolismo , Glaucoma/enzimologia , Glaucoma/patologia , Células Ganglionares da Retina/enzimologia , Animais , Apoptose , Astrócitos/enzimologia , Astrócitos/patologia , Sobrevivência Celular , Dependovirus/metabolismo , Modelos Animais de Doenças , Glaucoma/fisiopatologia , Proteínas de Fluorescência Verde/metabolismo , Pressão Intraocular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/enzimologia , Microglia/patologia , Mitocôndrias/metabolismo , Células Ganglionares da Retina/patologia , TransfecçãoRESUMO
PURPOSE: This study was conducted to determine whether elevated hydrostatic pressure alters mitochondrial structure, triggers release of the dynamin-related guanosine triphosphatase (GTPase) optic atrophy type 1 (OPA1) or cytochrome C from mitochondria, alters OPA1 gene expression, and can directly induce apoptotic cell death in cultured retinal ganglion cell (RGC)-5 cells. METHODS: Differentiated RGC-5 cells were exposed to 30 mmHg for three days in a pressurized incubator. As a control, differentiated RGC-5 cell cultures were incubated simultaneously in a conventional incubator. Live RGC-5 cells were then labeled with MitoTracker Red and mitochondrial morphology was assessed by fluorescence microscopy. Mitochondrial structural changes were also assessed by electron microscopy and three-dimensional (3D) electron microscope tomography. OPA1 mRNA was measured by Taqman quantitative PCR. The cellular distribution of OPA1 protein and cytochrome C was assessed by immunocytochemistry and western blot. Caspase-3 activation was examined by western blot. Apoptotic cell death was evaluated by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. RESULTS: Mitochondrial fission, characterized by the conversion of tubular fused mitochondria into isolated small organelles, was triggered after three days exposure to elevated hydrostatic pressure. Electron microscopy confirmed the fission and noted no changes to mitochondrial architecture, nor outer membrane rupture. Electron microscope tomography showed that elevated pressure depleted mitochondrial cristae content by fourfold. Elevated hydrostatic pressure increased OPA1 gene expression by 35+/-14% on day 2, but reduced expression by 36+/-4% on day 3. Total OPA1 protein content was not changed on day 2 or 3. However, pressure treatment induced release of OPA1 and cytochrome C from mitochondria to the cytoplasm. Elevated pressure also activated caspase-3 and induced apoptotic cell death. CONCLUSIONS: Elevated hydrostatic pressure triggered mitochondrial changes including mitochondrial fission and abnormal cristae depletion, alteration of OPA1 gene expression, and release of OPA1 and cytochrome C into the cytoplasm before the onset of apoptotic cell death in differentiated RGC-5 cells. These results suggest that sustained moderate pressure elevation may directly damage RGC integrity by injuring mitochondria.
Assuntos
Apoptose , Diferenciação Celular , Citocromos c/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/enzimologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Linhagem Celular , GTP Fosfo-Hidrolases/genética , Regulação da Expressão Gênica , Pressão Hidrostática , Imuno-Histoquímica , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Células Ganglionares da Retina/ultraestrutura , Antígenos Thy-1/metabolismoRESUMO
PURPOSE: To investigate the effect on circadian variation of intraocular pressure (IOP) of eliminating the alpha2A-, alpha2B-, or the alpha2C-adrenergic receptor subtypes in the mouse. METHODS: A microneedle method was used to measure IOP in knockout mice lacking the alpha2A-, alpha2B-, or the alpha2C-receptor (alpha2A-R(-/-), alpha2B-R(-/-), alpha2C-R(-/-)), in wild-type mice of the alpha2B knockout strain (alpha2B-R(+/+)), and in the background strain mice, C57BL/6. All mice were maintained in a 12-hour light-dark cycle commencing at 0600 hours. IOP was measured at 0900 and 2100 hours in the five groups: C57BL/6 (n = 8), alpha2A-R(-/-) (n = 10), alpha2B-R(-/-) (n = 8), alpha2B-R(+/+) (n = 8), and alpha2C-R(-/-) (n = 10). In parallel experiments, eyes from the alpha2A-R(-/-), alpha2B-R(-/-), alpha2C-R(-/-), and C57BL/6 mice were embedded in epoxy resin, and semithin sections were stained with toluidine blue. RESULTS: IOP at 0900 hours in B6, alpha2A-R(-/-), alpha2B-R(-/-), alpha2B-R(+/+), and alpha2C-R(-/-) mice was 17.1 +/- 1.8, 17.7 +/- 1.4, 17.1 +/- 2.1, 17.6 +/- 1.3, and 17.3 +/- 0.9 mm Hg, respectively (mean +/- SD). IOP at 2100 hours in the same eyes was 19.6 +/- 1.9, 19.2 +/- 2.2, 20.5 +/- 1.5, 19.7 +/- 0.8, and 21.3 +/- 2.7 mm Hg, respectively. There was no significant difference among these genotypes in IOP measured at either time point (P > 0.05, ANOVA). Within each genotype, IOP at 2100 hours was significantly higher than IOP at 0900 hours (C57BL/6, alpha2B-R(-/-), alpha2B-R(+/+), and alpha2C-R(-/-): P < 0.01; alpha2A-R(-/-): P < 0.05, paired t-test). Differences in the diurnal IOP change among the different genotypes were insignificant (P > 0.05, ANOVA). Histopathologic assessment found minimal differences in the structural organization of the anterior segment among the alpha2A-R(-/-), alpha2B-R(-/-),alpha2C-R(-/-), or C57BL/6 mice. CONCLUSIONS: These results indicate that IOP magnitude and circadian variation are minimally altered by the absence of the alpha2A-, alpha2B-, or alpha2C-receptor subtypes in transgenic mice.
Assuntos
Ritmo Circadiano/fisiologia , Pressão Intraocular/fisiologia , Receptores Adrenérgicos alfa 2/fisiologia , Animais , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Tonometria OcularRESUMO
PURPOSE: Glutamate receptor activation-induced excitotoxicity has been hypothesized to cause retinal ganglion cell (RGC) death in glaucoma and to link mitochondrial dysfunction in both acute and chronic neurodegenerative disorders. However, the relationships among elevated intraocular pressure (IOP), glutamate receptor-mediated excitotoxicity, and mitochondrial dysfunction in glaucoma remains unknown. The goal of this study was to determine whether the N- methyl D-aspartate (NMDA) glutamate receptor antagonist MK801 can block optic atrophy 1 (OPA1) release and subsequent apoptotic cell death, as well as whether acute IOP elevation triggers OPA1 release and alters OPA1 gene and protein expression in the rat retina after ischemia. METHODS: Sprague Dawley rats received injections of MK801 (10 mg/kg) or vehicle and then transient retinal ischemia was induced by acute IOP elevation. Following subcellular fractionation, changes in cytoplasmic and mitochondrial OPA1 were assessed by western blot analysis. Also, the expression of OPA1 mRNA was measured by Taqman qPCR, the distribution of OPA1 protein was assessed by immunohistochemistry, and apoptotic cell death was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. RESULTS: The ~65 and 90 kDa isoforms of OPA1 were increased in the cytosol in the rat retina at 6 h and at 12 h, but only the 90 kDa isoform of OPA1 was decreased at 12 h after ischemia induced by acute IOP elevation. This suggests that ischemic insult induced OPA1 release from the mitochondria in retinas. Pretreatment with MK801 blocked this effect and significantly increased OPA1 immunoreactivity in the inner retinal layers, as well as OPA1 gene expression and total protein expression in retinas at 12 h after ischemia. Further, pretreatment with MK801 prevented apoptotic cell death in retinas at 12 h after ischemia. Following acute IOP elevation, Bcl-2 mRNA expression in retinas was decreased at 3 h and 6 h but increased at 12 h and 24 h. In contrast, Bax mRNA expression in these retinas was increased in the first 12 h and then plateaued. Moreover, pretreatment with MK801 increased Bcl-2 mRNA expression, but did not alter the course of Bax mRNA expression. CONCLUSIONS: These results indicate that OPA1 release from mitochondria triggered by acute IOP elevation is inhibited by blockade of glutamate receptor activation. Because this effect was accompanied by increases of Bcl-2 expression, no changes of Bax expression, and blockade of apoptosis, these findings indicate that glutamate receptor activation following acute IOP elevation may lead to a distinct mitochondria-mediated cell death pathway in ischemic retina. These results support further studies to determine whether ischemia-induced OPA1 release may be an important component of the biochemical cascade leading to pressure-related ischemic damage in glaucomatous retina.
Assuntos
Apoptose , GTP Fosfo-Hidrolases/metabolismo , Isquemia/metabolismo , Isquemia/patologia , Receptores de Glutamato/metabolismo , Retina/metabolismo , Retina/patologia , Animais , Apoptose/efeitos dos fármacos , Maleato de Dizocilpina/farmacologia , GTP Fosfo-Hidrolases/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Pressão Intraocular/efeitos dos fármacos , Isquemia/fisiopatologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismoRESUMO
Current methods for in vivo retinal ganglion cells (RGCs) imaging involve either retrograde or intravitreal injection of chemical or biological tracers, which are invasive and may require repeated injection for serial long-term assessment. We have developed a confocal scanning laser ophthalmoscope technique (blue-light CSLO or bCSLO) to image retinal ganglion cells (RGCs) in mice expressing cyan fluorescent protein under the control of a Thy-1 promoter. Fluorescent spots corresponding to CFP-expressing retinal ganglion cells were discernable with the bCSLO. 96.1+/-2.6% of CFP expressing cells also were retrograde labeled with DiI indicating the bCSLO imaged fluorescent spots are RGCs. The imaging of Thy-1 promoter-driven CFP expression in these mice could serve as a sensitive indicator to reflect the integrity of RGCs, and provides a non-invasive method for longitudinal study of the mechanism of RGC degeneration and the effect of neuroprotective agents.
Assuntos
Microscopia Confocal/métodos , Oftalmoscopia/métodos , Células Ganglionares da Retina/ultraestrutura , Animais , Corantes Fluorescentes , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Reprodutibilidade dos TestesRESUMO
PURPOSE: It is not known whether the prostaglandin FP receptor plays an important role in endogenous 24-hour regulation of intraocular pressure. The purpose of this study was to compare 24-hour intraocular pressure (IOP) in FP receptor-knockout mice with that of wild-type mice that have normal FP receptor expression. METHODS: The 24-hour IOP profile was determined by rebound tonometry in FP-knockout and wild-type mice. Peak and trough IOP was then measured by microneedle cannulation of the anterior chamber in homozygous (FP(-/-); n = 8), heterozygous (FP(+/-); n = 14), and C57BL/6 background strain mice (FP(+/+); n = 11). To confirm any differences in baseline IOP between genotypes, midafternoon IOP was also measured in a larger, separate group of FP(-/-) mice (n = 20), FP(+/-) mice (n = 49), and FP(+/+) (n = 23) wild-type littermates. RESULTS: Trough IOPs were measured between 10 AM and 12 PM, peak IOPs were measured between 8 and 10 PM. For FP(+/+), FP(+/-), and FP(-/-) mice trough IOP was 16.2, 15.3, and 15.1 mm Hg and peak IOPs were 18.2, 18.4, and 17.7 mm Hg, respectively. There was no significant difference among genotypes for mean peak or mean trough IOP or for peak-trough difference in IOP among genotypes (P > 0.05, ANOVA). In addition, there was no significant difference in midafternoon IOP between genotypes in a larger population (n = 92) of FP-knockout and wild-type mice. CONCLUSIONS: An intact FP receptor does not appear to be critical for normal 24-hour IOP regulation in the mouse eye.
Assuntos
Ritmo Circadiano/fisiologia , Pressão Intraocular/fisiologia , Receptores de Prostaglandina/fisiologia , Animais , Feminino , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tonometria OcularRESUMO
PURPOSE: Previous studies have shown that increased trans-scleral permeability after exposure to certain prostaglandins is associated with increased intrascleral matrix metalloproteinases (MMPs). The present study was undertaken to determine whether these MMPs could directly alter transscleral permeability. METHODS: Freshly enucleated mouse eyes were incubated with human MMP-1, -2, and -14 for 4 hours at 37 degrees C. The eyes then were incubated with 10 or 70 kDa dextran-tetramethylrhodamine-lysine for 16 to 32 minutes at 37 degrees C. Two methods of analysis were used. In the first, quickly isolated retinas were homogenized and centrifuged. Fluorescence in the supernatants was determined by microspectrofluorimetry. In the second, the eyes were fixed in 4% paraformaldehyde, and frozen sections were prepared. After the identity of the sections was masked, the intensity of fluorescence in anterior, middle, and posterior regions of the outer retina and inner retina was scored with a 7-point grading scheme. RESULTS: The concentration of 10-kDa fluorescent dextran was 5.14 +/- 1.61 microg/mL (mean +/- SD, n = 33) in the control retinal supernatants, and 6.37 +/- 2.67 microg/mL (n = 40) in the retinal supernatants from the MMP-treated eyes. This increase was statistically significant (P < 0.02, t-test). The structural organization of the retina and other ocular tissues was maintained in all experimental conditions. Histologic scoring of fluorescence found significantly increased dextran in the outer retina of eyes treated with MMPs for 32 minutes (the score of control eyes was 2.5 +/- 0.4 and of MMP-treated eyes was 3.5 +/- 0.1, mean +/- SD; P = 0.02, n = 3). Analysis by region found greater scores in the third of the retina nearest to the optic nerve head. CONCLUSIONS: These results show that MMP-1, -2, and -14 can directly increase transscleral permeability and support the view that the increased MMP-1 and -2 observed after topical PG treatment could contribute to increased uveoscleral outflow.
Assuntos
Biotina/análogos & derivados , Dextranos/metabolismo , Metaloproteinases da Matriz/farmacologia , Retina/metabolismo , Rodaminas/metabolismo , Esclera/metabolismo , Animais , Biotina/metabolismo , Camundongos , Permeabilidade/efeitos dos fármacos , Espectrometria de FluorescênciaRESUMO
PURPOSE: Mitochondrial fission is a cellular response to stress that has an important role in neuronal cell death in neurodegenerative diseases. The purpose of this study was to determine whether elevated hydrostatic pressure induces mitochondrial fission and dysfunction in cultured retinal ganglion cells. METHODS: RGC-5 cells were differentiated with succinyl concanavalin A (50 microg/mL) and transferred to a pressurized incubator in which 30 mm Hg of pressure was applied for 1, 2, or 3 days. As a control, differentiated cells from an identical passage were incubated simultaneously in a conventional incubator at each of the time points. Live RGC-5 cells were then labeled with a red fluorescent mitochondrial dye and mitochondrial morphology was assessed by fluorescence microscopy and electron microscopy. After elevated hydrostatic pressure, the cellular adenosine triphosphate (ATP) levels were also measured by a luciferase-based assay. RESULTS: Mitochondrial fission, characterized by the conversion of tubular fused mitochondria into isolated small organelles, was triggered in >74.3% +/- 1.9% of mitochondria at 3 days after elevated hydrostatic pressure. Only 4.7% +/- 1.4% of nonpressurized control cells displayed mitochondrial fission after 3 days. Electron microscopy showed that elevated hydrostatic pressure for 3 days induced abnormal cristae depletion and decreased the length of the mitochondria. On elevation of hydrostatic pressure, the fission-linked protein, Drp-1 was translocated from the cytosol to the mitochondria. Elevated hydrostatic pressure also resulted in a significant, time-dependent reduction of cellular ATP. CONCLUSIONS: Elevated hydrostatic pressure triggered mitochondrial fission, abnormal cristae depletion, Drp-1 translocation, and cellular ATP reduction in differentiated RGC-5 cells. Increased understanding of the molecular mechanisms that regulate the cellular response to elevated pressure including mitochondrial fission may provide new therapeutic targets for protecting RGCs from elevated hydrostatic pressure.
Assuntos
Trifosfato de Adenosina/metabolismo , Pressão Hidrostática , Mitocôndrias/patologia , Células Ganglionares da Retina/patologia , Animais , Apoptose , Western Blotting , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Citosol/metabolismo , Dinaminas/metabolismo , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Ratos , Células Ganglionares da Retina/metabolismoRESUMO
PURPOSE: To determine whether oxidative adduct formation or heme oxygenase-1 (HO-1) expression are altered in retinal ganglion cell (RGC) cultures exposed to elevated hydrostatic pressure and in a mouse model of glaucoma. METHODS: Cultured RGC-5 cells were subjected to 0, 30, 60, or 100 mm Hg hydrostatic pressure for 2 hours, and the cells were harvested. Parallel experiments examined the recovery from this stress, the effect of direct 4-hydroxy-2-nonenal (HNE) treatment, and the effect of pretreatment with resveratrol or quercetin. Mice were anesthetized and intraocular pressure was increased to 30, 60, or 100 mm Hg for 1 hour; then the retinas were harvested. HNE adduct formation and HO-1 expression were assessed by immunocytochemistry and immunoblotting. RESULTS: Increases of HNE-protein adducts (up to 5-fold) and HO-1 expression (up to 2.5 fold) in pressure-treated RGC-5 cells were dose dependent. During recovery experiments, HNE-protein adducts continued to increase for up to 10 hours; in contrast, HO-1 expression decreased immediately. HNE, at a concentration as low as 5 muM, led to neurotoxicity in RGC-5 cells. HNE adducts and HO-1 expression increased in the mouse retina and optic nerve after acute IOP elevation up to 5.5-fold and 2-fold, respectively. Antioxidant treatment reduced the oxidative stress level in pressure-treated RGC-5 cells. CONCLUSIONS: This study demonstrates that oxidative stress is an early event in hydrostatic pressure/IOP-induced neuronal damage. These findings support the view that oxidative damage contributes early to glaucomatous optic neuropathy.
Assuntos
Glaucoma/etiologia , Pressão Hidrostática/efeitos adversos , Doenças do Nervo Óptico/etiologia , Estresse Oxidativo , Doenças Retinianas/etiologia , Células Ganglionares da Retina/patologia , Aldeídos/farmacologia , Animais , Apoptose , Western Blotting , Linhagem Celular Transformada , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Fluoresceínas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Glaucoma/metabolismo , Glaucoma/patologia , Heme Oxigenase-1/metabolismo , Pressão Intraocular , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Proteínas Nucleares , Doenças do Nervo Óptico/metabolismo , Doenças do Nervo Óptico/patologia , Ratos , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismoRESUMO
PURPOSE: To determine the effect of bimatoprost on intraocular pressure in the prostaglandin FP receptor knockout mouse. METHODS: The IOP response to a single 1.2-microg (4 microL) dose of bimatoprost was measured in the treated and untreated fellow eyes of homozygote (FP+/+, n = 9) and heterozygote (FP+/-, n = 10) FP-knockout mice, as well as in wild-type C57BL/6 mice (FP+/+, n = 20). Serial IOP measurements were also performed after topical bimatoprost in a separate generation of homozygous FP-knockout mice and wild-type littermate control animals (n = 4 per group). Aqueous humor protein concentrations were measured to establish the state of the blood-aqueous barrier. Tissue, aqueous humor and vitreous concentrations of bimatoprost, latanoprost, and their C-1 free acids were determined by liquid chromatography and tandem mass spectrometry. RESULTS: A significant reduction in IOP was observed in the bimatoprost-treated eye of wild-type mice at 2 hours, with a mean difference and 95% confidence interval (CI) of the difference in means of -1.33 mm Hg (-0.81 to -1.84). Bimatoprost did not lead to a significant reduction in IOP in either the heterozygous knockout -0.36 mm Hg (-0.82 to +0.09) or homozygous FP-knockout mice 0.25 mm Hg (-0.38 to +0.89). The lack of an IOP response in the FP-knockout mice was not a consequence of blood-aqueous barrier breakdown, as there was no significant difference in aqueous humor protein concentration between treated and fellow eyes. Tissue and aqueous humor concentrations of bimatoprost, latanoprost, and their C-1 free acids indicate that latanoprost, but not bimatoprost, is hydrolyzed in the mouse eye after topical administration. CONCLUSIONS: An intact FP receptor gene is critical to the IOP response to bimatoprost in the mouse eye.
Assuntos
Anti-Hipertensivos/farmacologia , Pressão Intraocular/efeitos dos fármacos , Lipídeos/farmacologia , Receptores de Prostaglandina/fisiologia , Amidas , Animais , Anti-Hipertensivos/farmacocinética , Humor Aquoso/metabolismo , Bimatoprost , Barreira Hematoaquosa/fisiologia , Cloprostenol/análogos & derivados , Proteínas do Olho/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Lipídeos/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Soluções Oftálmicas/farmacocinética , Soluções Oftálmicas/farmacologia , Receptores de Prostaglandina/deficiência , Tonometria OcularRESUMO
Experimental models have enhanced our understanding of the biology of glaucoma. Moreover, they have enabled the testing of potential therapies prior to the initiation of human trials. Each have advantages and limitations. In vitro cell and organ culture offer direct cellular accessibility and microenvironmental control, as well as efficient comparison between many experimental conditions or potential therapeutic compounds. However, they generally have less relevance to clinical glaucoma than in vivo models. Rat models allow moderate sized investigations of intact biological systems that have greater relevance to glaucoma than in vitro experiments, but less than primate experiments. Mouse models are similar to rat models but have the advantage of investigating mutant and transgenic strains mimicking specific aspects of glaucoma that are not available in other model systems. Primate models of glaucoma generally are the most relevant to human glaucoma but must be limited in scope because of availability and the high cost of experimentation.
Assuntos
Glaucoma , Modelos Biológicos , Animais , Pesquisa Biomédica , Técnicas de Cultura de Células , Modelos Animais de Doenças , Humanos , Técnicas de Cultura de Órgãos , Doadores de TecidosRESUMO
Exploitation of the mouse as a model for glaucoma has been advanced by the development of methods to measure mouse intraocular pressure (IOP), identification of mutant mouse strains in which IOP spontaneously increases, and the development of treatments to induce elevated IOP. These developments enable investigations that directly test the influence of specific gene product alterations on the progression of glaucoma. Moreover, new transgenic mouse models have been produced with genetic mutations that parallel human gene mutations that have been linked to the onset of glaucoma. These new mouse models and technologies have potential for uncovering the biological basis of glaucoma as well as for evaluating new treatments.
Assuntos
Modelos Animais de Doenças , Pressão Intraocular , Hipertensão Ocular/complicações , Animais , Camundongos , Camundongos Transgênicos , Disco Óptico/patologia , Doenças do Nervo Óptico/etiologiaRESUMO
Long-term dietary supplementation with resveratrol protects against cardiovascular disease, osteoporesis, and metabolic decline. This study determined how long-term dietary resveratrol treatment protects against retinal ganglion cell (RGC) dendrite loss after optic nerve injury and alters the resolution of the unfolded protein response. Associated changes in markers of endoplasmic reticulum stress in RGCs also were investigated. Young-adult Thy1-yellow fluorescent protein (YFP) and C57BL/6 mice received either control diet or diet containing resveratrol for approximately 1 year. Both groups then received optic nerve crush (ONC). Fluorescent RGC dendrites in the Thy1-YFP mice were imaged weekly for 4 weeks after ONC. There was progressive loss of dendrite length in all RGC types within the mice that received control diet. Resveratrol delayed loss of dendrite complexity and complete dendrite loss for most RGC types. However, there were variations in the rate of retraction among different RGC types. Three weeks after ONC, cytoplasmic binding immunoglobulin protein (BiP) suppression observed in control diet ganglion cell layer neurons was reversed in mice that received resveratrol, nuclear C/EBP homologous protein (CHOP) was near baseline in control diet eyes but was moderately increased by resveratrol; and increased nuclear X-box-binding protein-1 (XBP-1) observed in control diet eyes was reduced in eyes that received resveratrol to the same level as in control diet uncrushed eyes. These results indicate that protection of dendrites by resveratrol after ONC differs among RGC types and suggest that alterations in long-term expression of binding immunoglobulin protein, CHOP, and XBP-1 may contribute to the resveratrol-mediated protection of RGC dendrites after ONC.
Assuntos
Dendritos/metabolismo , Dendritos/patologia , Suplementos Nutricionais , Traumatismos do Nervo Óptico/patologia , Desdobramento de Proteína/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Estilbenos/administração & dosagem , Estilbenos/farmacologia , Animais , Proteínas de Ligação a DNA/metabolismo , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Transcrição de Fator Regulador X , Resveratrol , Células Ganglionares da Retina/citologia , Fatores de Tempo , Fator de Transcrição CHOP/metabolismo , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
PURPOSE: To determine whether brimonidine protects against the retraction and loss of retinal ganglion cell (RGC) dendrites after optic nerve crush (ONC). METHODS: Fluorescent RGCs of mice expressing yellow fluorescent protein (YFP) under the control of the Thy-1 promoter (Thy1-YFP mice) were imaged in vivo and assigned to one of six groups according to dendrite structure. The mice then received brimonidine every other day starting 2 days before, or 2 or 6 days after, unilateral ONC. Control animals received vehicle every other day starting 2 days before ONC. Control animals received vehicle every other day starting 2 days before ONC. Total dendrite length, dendrite branching complexity, and the time until complete loss of dendrites were assessed weekly for 4 weeks. RESULTS: Overall, brimonidine treatment significantly slowed the complete loss of RGC dendrites and significantly slowed the reduction of total dendrite length and branching complexity. Separate analysis of each RGC group showed brimonidine significantly delayed the time until complete loss of dendrites in four of the RGC groups. These delays generally were similar when treatment started either 2 days before or 2 days after ONC, but were smaller or absent when treatment started 6 days after ONC Protection against loss of total dendrite length and loss of branching complexity was observed in three of the RGC groups. In two of these RGC groups, protective effects persisted until the end of the study. CONCLUSIONS: Brimonidine protects many RGC types against dendrite retraction, loss of branching complexity, and complete loss of dendrites following ONC. However, the pattern and magnitude of this protection differs substantially among different RGC types. These results indicate that requirements for RGC-protective therapies following optic nerve injury may differ among RGC types.
Assuntos
Citoproteção/efeitos dos fármacos , Dendritos/efeitos dos fármacos , Compressão Nervosa , Traumatismos do Nervo Óptico/patologia , Quinoxalinas/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Tartarato de Brimonidina , Dendritos/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Microscopia de Fluorescência , Células Ganglionares da Retina/patologiaRESUMO
There are several unorthodox features, which distinguish the non-redundant and unique novel matrix metalloproteinase-26 (MMP-26) (an enzyme that has recently evolved and does not exist in rodents but is present in humans) from other members of the MMP superfamily. This report describes our recent efforts to gain a better understanding of the mechanisms which restrict expression of MMP-26 to certain cell/tissue types. We examined transcriptional regulation of the human MMP-26 gene in normal and malignant cells. The AP-1 and Tcf-4 sites of the MMP-26 promoter appear most potent in regulating the expression of the MMP-26-luciferase chimera in HEK293 embryonic kidney and MCF7 breast carcinoma cells. Key regulators of the Wnt pathway (beta-catenin and lymphoid enhancer-binding factor/T-cell factor with which beta-catenin associates) enhanced the transcriptional activity of MMP-26 suggesting that the MMP-26 gene is a likely target of the Wnt pathway. Immunostaining, gene arrays and reverse-transcriptase polymerase chain reaction (RT-PCR) confirm the presence of MMP-26 in normal cells, including the apical epithelial conjunctiva cells of the human eye, as well as in malignant cells of epithelial origin. MMP-26 predominantly accumulates in its proenzyme form in the intracellular milieu of the transfected breast carcinoma MCF7 cells. This study brings us a step forward towards a better understanding of the unconventional role, regulation and functions of epithelial cell MMP-26 in physiological conditions and in neoplasms.
Assuntos
Carcinoma/enzimologia , Proteínas do Citoesqueleto/metabolismo , Metaloproteinases da Matriz/genética , Transativadores/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/enzimologia , Regulação Enzimológica da Expressão Gênica , Genes Reporter , Humanos , Imunoquímica , Luciferases/análise , Fator 1 de Ligação ao Facilitador Linfoide , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz Secretadas , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Esclera/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta Catenina , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
PURPOSE: This study was undertaken to assess directly whether there is uveoscleral outflow in the mouse eye by monitoring the movement of intracamerally injected fluorescent dextran. METHODS: After anesthesia, NIH Swiss mice received intracameral injection of 1.5 microL of 0.2 pg/microL 70-kDa dextran conjugated to tetramethyl-rhodamine and to lysine. After survival times of 10, 20, 60, and 120 minutes, the experiments were terminated by transcardial perfusion with 2% paraformaldehyde. The eyes were enucleated and embedded in paraffin, and sections were prepared. These sections were then analyzed by fluorescence microscopy. RESULTS: Fluorescent tracer in the eyes of animals that survived for 10 minutes was prominent in the iris root and ciliary processes and was of moderate intensity in the adjacent sclera. Moderate intensity fluorescence also was observed in the trabecular meshwork and adjacent cornea. At 20 minutes, intense fluorescence was observed in the ciliary processes and the ciliary muscle. This fluorescence in the ciliary muscle extended from the posterior edge of the ciliary muscle's tail into the anterior choroid. At 60 minutes, the fluorescence in the choroid extended to the equator and adjacent sclera. The intensity of the fluorescence within the ciliary processes of these eyes was substantially reduced when compared with the 20-minute-survival eyes. At 120 minutes, label was observed only within trabecular meshwork and Schlemm's canal. CONCLUSIONS: These results indicate that at least a portion of aqueous outflow in the mouse eye is through the uveoscleral outflow pathway.
Assuntos
Humor Aquoso/metabolismo , Corantes Fluorescentes , Esclera/metabolismo , Úvea/metabolismo , Animais , Câmara Anterior/metabolismo , Dextranos , Injeções , Camundongos , Microscopia de Fluorescência , RodaminasRESUMO
PURPOSE: The present study was undertaken to determine whether treatment of ciliary muscle cells with the prostaglandin (PG) analogue latanoprost acid alters transcription of mRNA for matrix metalloproteinase (MMP)-1, -2, -3, and -9. METHODS: Human ciliary smooth muscle cell cultures were grown to confluence and treated for 24 hours with medium supplemented with latanoprost acid or vehicle. Total RNA was then isolated, and the expression of mRNAs for MMP-1, -2, -3, and -9 were determined using Taqman and energy-transfer real-time PCR analyses. All results were normalized according to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in each sample. RESULTS: Specificity and linearity of each real-time PCR assay were confirmed by electrophoresis and serial dilution analysis of oligonucleotides containing the amplicon sequence. Addition of latanoprost acid for 24 hours increased expression of MMP-1 by 3- to 13-fold in three of five primary ciliary muscle lines. Addition of 8, 40, and 200 nM latanoprost acid for 24 hours increased MMP-1 mRNA in a dose-dependent manner. Analysis of cultures exposed to 200 nM latanoprost acid for 4, 6, 12, or 24 hours revealed an increase and then a decline of MMP-1 mRNA, with peak expression at 6 to 12 hours after initiation of treatment. Parallel assessments of RNA from ciliary muscle cultures exposed to latanoprost acid for 24 hours revealed increased MMP-1, -3, and -9 mRNAs and reduced MMP-2 mRNA, when compared with RNA from vehicle-treated cultures. CONCLUSIONS: Latanoprost acid induced a dose-dependent increase of MMP-1, -3, and -9 gene transcription in cultured human ciliary smooth muscle cells. These results are consistent with increased MMPs contributing to the increased uveoscleral outflow facility observed after topical latanoprost.