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1.
Cell ; 139(2): 299-311, 2009 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-19837033

RESUMEN

Mutations in the genes encoding the Wnt receptor Frizzled-4 (FZD4), coreceptor LRP5, or the ligand Norrin disrupt retinal vascular development and cause ophthalmic diseases. Although Norrin is structurally unrelated to Wnts, it binds FZD4 and activates the canonical Wnt pathway. Here we show that the tetraspanin Tspan12 is expressed in the retinal vasculature, and loss of Tspan12 phenocopies defects seen in Fzd4, Lrp5, and Norrin mutant mice. In addition, Tspan12 genetically interacts with Norrin or Lrp5. Overexpressed TSPAN12 associates with the Norrin-receptor complex and significantly increases Norrin/beta-catenin but not Wnt/beta-catenin signaling, whereas Tspan12 siRNA abolishes transcriptional responses to Norrin but not Wnt3A in retinal endothelial cells. Signaling defects caused by Norrin or FZD4 mutations that are predicted to impair receptor multimerization are rescued by overexpression of TSPAN12. Our data indicate that Norrin multimers and TSPAN12 cooperatively promote multimerization of FZD4 and its associated proteins to elicit physiological levels of signaling.


Asunto(s)
Receptores Frizzled/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Retina/embriología , Transducción de Señal , beta Catenina/metabolismo , Animales , Diterpenos , Células Endoteliales/metabolismo , Receptores Frizzled/genética , Humanos , Ratones , Receptores Acoplados a Proteínas G/genética , Tetraspaninas , beta Catenina/genética
2.
Ophthalmology ; 128(2): 248-255, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-32777229

RESUMEN

PURPOSE: There is an urgent need for treatments that prevent or delay development to advanced age-related macular degeneration (AMD). Drugs already on the market for other conditions could affect progression to neovascular AMD (nAMD). If identified, these drugs could provide insights for drug development targets. The objective of this study was to use a novel data mining method that can simultaneously evaluate thousands of correlated hypotheses, while adjusting for multiple testing, to screen for associations between drugs and delayed progression to nAMD. DESIGN: We applied a nested case-control study to administrative insurance claims data to identify cases with nAMD and risk-set sampled controls that were 1:4 variable ratio matched on age, gender, and recent healthcare use. PARTICIPANTS: The study population included cases with nAMD and risk set matched controls. METHODS: We used a tree-based scanning method to evaluate associations between hierarchical classifications of drugs that patients were exposed to within 6 months, 7 to 24 months, or ever before their index date. The index date was the date of first nAMD diagnosis in cases. Risk-set sampled controls were assigned the same index date as the case to which they were matched. The study was implemented using Medicare data from New Jersey and Pennsylvania, and national data from IBM MarketScan Research Database. We set an a priori threshold for statistical alerting at P ≤ 0.01 and focused on associations with large magnitude (relative risks ≥ 2.0). MAIN OUTCOME MEASURES: Progression to nAMD. RESULTS: Of approximately 4000 generic drugs and drug classes evaluated, the method detected 19 distinct drug exposures with statistically significant, large relative risks indicating that cases were less frequently exposed than controls. These included (1) drugs with prior evidence for a causal relationship (e.g., megestrol); (2) drugs without prior evidence for a causal relationship, but potentially worth further exploration (e.g., donepezil, epoetin alfa); (3) drugs with alternative biologic explanations for the association (e.g., sevelamer); and (4) drugs that may have resulted in statistical alerts due to their correlation with drugs that alerted for other reasons. CONCLUSIONS: This exploratory drug-screening study identified several potential targets for follow-up studies to further evaluate and determine if they may prevent or delay progression to advanced AMD.


Asunto(s)
Neovascularización Coroidal/diagnóstico , Evaluación Preclínica de Medicamentos/métodos , Medicamentos Genéricos/uso terapéutico , Degeneración Macular Húmeda/diagnóstico , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Neovascularización Coroidal/prevención & control , Minería de Datos , Progresión de la Enfermedad , Reposicionamiento de Medicamentos/métodos , Femenino , Humanos , Revisión de Utilización de Seguros , Masculino , Medicare/estadística & datos numéricos , Estados Unidos , Degeneración Macular Húmeda/prevención & control
3.
J Biol Chem ; 293(52): 20137-20156, 2018 12 28.
Artículo en Inglés | MEDLINE | ID: mdl-30389787

RESUMEN

Myocilin (MYOC) was discovered more than 20 years ago and is the gene whose mutations are most commonly observed in individuals with glaucoma. Despite extensive research efforts, the function of WT MYOC has remained elusive, and how mutant MYOC is linked to glaucoma is unclear. Mutant MYOC is believed to be misfolded within the endoplasmic reticulum, and under normal physiological conditions misfolded MYOC should be retro-translocated to the cytoplasm for degradation. To better understand mutant MYOC pathology, we CRISPR-engineered a rat to have a MYOC Y435H substitution that is the equivalent of the pathological human MYOC Y437H mutation. Using this engineered animal model, we discovered that the chaperone αB-crystallin (CRYAB) is a MYOC-binding partner and that co-expression of these two proteins increases protein aggregates. Our results suggest that the misfolded mutant MYOC aggregates with cytoplasmic CRYAB and thereby compromises protein clearance mechanisms in trabecular meshwork cells, and this process represents the primary mode of mutant MYOC pathology. We propose a model by which mutant MYOC causes glaucoma, and we propose that therapeutic treatment of patients having a MYOC mutation may focus on disrupting the MYOC-CRYAB complexes.


Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas del Ojo/metabolismo , Glaucoma/metabolismo , Glicoproteínas/metabolismo , Mutación Missense , Malla Trabecular/metabolismo , Cadena B de alfa-Cristalina/metabolismo , Sustitución de Aminoácidos , Animales , Cristalinas/genética , Cristalinas/metabolismo , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Femenino , Glaucoma/genética , Glaucoma/patología , Glicoproteínas/genética , Humanos , Masculino , Ratones Mutantes , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica , Ratas Sprague-Dawley , Malla Trabecular/patología , Cadena B de alfa-Cristalina/genética
4.
BMC Med Genet ; 17(1): 47, 2016 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-27439461

RESUMEN

BACKGROUND: Macular corneal dystrophy (MCD) is a rare autosomal recessive disorder that is characterized by progressive corneal opacity that starts in early childhood and ultimately progresses to blindness in early adulthood. The aim of this study was to identify the cause of MCD in a black South African family with two affected sisters. METHODS: A multigenerational South African Sotho-speaking family with type I MCD was studied using whole exome sequencing. Variant filtering to identify the MCD-causal mutation included the disease inheritance pattern, variant minor allele frequency and potential functional impact. RESULTS: Ophthalmologic evaluation of the cases revealed a typical MCD phenotype and none of the other family members were affected. An average of 127 713 variants per individual was identified following exome sequencing and approximately 1.2 % were not present in any of the investigated public databases. Variant filtering identified a homozygous E71Q mutation in CHST6, a known MCD-causing gene encoding corneal N-acetyl glucosamine-6-O-sulfotransferase. This E71Q mutation results in a non-conservative amino acid change in a highly conserved functional domain of the human CHST6 that is essential for enzyme activity. CONCLUSION: We identified a novel E71Q mutation in CHST6 as the MCD-causal mutation in a black South African family with type I MCD. This is the first description of MCD in a black Sub-Saharan African family and therefore contributes valuable insights into the genetic aetiology of this disease, while improving genetic counselling for this and potentially other MCD families.


Asunto(s)
Distrofias Hereditarias de la Córnea/genética , Mutación , Sulfotransferasas/genética , Adulto , Córnea/patología , Distrofias Hereditarias de la Córnea/patología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Humanos , Masculino , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple , Sudáfrica , Carbohidrato Sulfotransferasas
5.
Exp Eye Res ; 146: 269-275, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-27060376

RESUMEN

Increased histone deacetylase (HDAC) activity and the resulting dysregulation of protein acetylation is an integral event in retinal degenerations associated with ischemia and ocular hypertension. This study investigates the role of preconditioning on the process of acetylation in ischemic retinal injury. Rat eyes were unilaterally subjected to retinal injury by 45 min of acute ischemia, and retinal neuroprotection induced by 5 min of an ischemic preconditioning (IPC) event. HDAC activity was evaluated by a fluorometric enzymatic assay with selective isoform inhibitors. Retinal localization of acetylated histone-H3 was determined by immunohistochemistry on retina cross sections. Cleaved caspase-3 level was evaluated by Western blots. Electroretinogram (ERG) analyses were used to assess differences in retinal function seven days following ischemic injury. In control eyes, analysis of HDAC isoforms demonstrated that HDAC1/2 accounted for 28.4 ± 1.6%, HDAC3 for 42.4 ± 1.5% and HDAC6 activity 27.3 ± 3.5% of total activity. Following ischemia, total Class-I HDAC activity increased by 21.2 ± 6.2%, and this increase resulted solely from a rise in HDAC1/2 activity. No change in HDAC3 activity was measured. Activity of Class-II HDACs and HDAC8 was negligible. IPC stimulus prior to ischemic injury also suppressed the rise in Class-I HDAC activity, cleaved caspase-3 levels, and increased acetylated histone-H3 in the retina. In control animals 7 days post ischemia, ERG a- and b-wave amplitudes were significantly reduced by 34.9 ± 3.1% and 42.4 ± 6.3%, respectively. In rats receiving an IPC stimulus, the ischemia-induced decline in ERG a- and b-wave amplitudes was blocked. Although multiple HDACs were detected in the retina, these studies provide evidence that hypoacetylation associated with ischemic injury results from the selective rise in HDAC1/2 activity and that neuroprotection induced by IPC is mediated in part by suppressing HDAC activity.


Asunto(s)
Histona Desacetilasas/metabolismo , Precondicionamiento Isquémico , Neuroprotección/fisiología , Retina/metabolismo , Acetilación , Análisis de Varianza , Animales , Western Blotting , Caspasa 3/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Histonas/metabolismo , Inmunohistoquímica , Masculino , Ratas
6.
Invest Ophthalmol Vis Sci ; 62(7): 26, 2021 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-34160562

RESUMEN

Purpose: Dysregulation of the alternative complement pathway is a major pathogenic mechanism in age-related macular degeneration. We investigated whether locally synthesized complement components contribute to AMD by profiling complement expression in postmortem eyes with and without AMD. Methods: AMD severity grade 1 to 4 was determined by analysis of postmortem acquired fundus images and hematoxylin and eosin stained histological sections. TaqMan (donor eyes n = 39) and RNAscope/in situ hybridization (n = 10) were performed to detect complement mRNA. Meso scale discovery assay and Western blot (n = 31) were used to measure complement protein levels. Results: The levels of complement mRNA and protein expression were approximately 15- to 100-fold (P < 0.0001-0.001) higher in macular retinal pigment epithelium (RPE)/choroid tissue than in neural retina, regardless of AMD grade status. Complement mRNA and protein levels were modestly elevated in vitreous and the macular neural retina in eyes with geographic atrophy (GA), but not in eyes with early or intermediate AMD, compared to normal eyes. Alternative and classical pathway complement mRNAs (C3, CFB, CFH, CFI, C1QA) identified by RNAscope were conspicuous in areas of atrophy; in those areas C3 mRNA was observed in a subset of IBA1+ microglia or macrophages. Conclusions: We verified that RPE/choroid contains most ocular complement; thus RPE/choroid rather than the neural retina or vitreous is likely to be the key site for complement inhibition to treat GA or earlier stage of the disease. Outer retinal local production of complement mRNAs along with evidence of increased complement activation is a feature of GA.


Asunto(s)
Coroides , Activación de Complemento , Proteínas del Sistema Complemento/genética , Degeneración Macular , Retina , Epitelio Pigmentado de la Retina , Anciano , Autopsia/métodos , Coroides/metabolismo , Coroides/patología , Vía Alternativa del Complemento , Femenino , Perfilación de la Expresión Génica/métodos , Atrofia Geográfica/patología , Humanos , Degeneración Macular/metabolismo , Degeneración Macular/patología , Masculino , ARN Mensajero/análisis , Retina/metabolismo , Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología
7.
J Ocul Pharmacol Ther ; 36(5): 269-281, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32176566

RESUMEN

Purpose: To identify new targets and compounds involved in mediating cellular contractility or relaxation in trabecular meshwork (TM) cells and test their efficacy in an ex vivo model measuring outflow facility. Methods: A low-molecular weight compound library composed of 3,957 compounds was screened for cytoskeletal changes using the Acea xCelligence impedance platform in immortalized human NTM5 TM cells. Hits were confirmed by 8-point concentration response and were subsequently evaluated for impedance changes in 2 primary human TM strains, as well as cross-reactivity in bovine primary cells. A recently described bovine whole eye perfusion system was used to evaluate effects of compounds on aqueous outflow facility. Results: The primary screen conducted was robust, with Z' values >0.5. Fifty-two compounds were identified in the primary screen and confirmed to have concentration-dependent effects on impedance in NTM5 cells. Of these, 9 compounds representing distinct drug classes were confirmed to modulate impedance in both human primary TM cells and bovine cells. One of these compounds, wortmannin, an inhibitor of phosphoinositide 3-kinase, increased outflow facility by 11%. Conclusions: A robust phenotypic assay was developed that enabled identification of contractility modulators in immortalized TM cells. The screening hits were translatable to primary TM cells and modulated outflow facility in an ex vivo perfusion assay.


Asunto(s)
Impedancia Eléctrica/efectos adversos , Glaucoma/tratamiento farmacológico , Ensayos Analíticos de Alto Rendimiento/métodos , Presión Intraocular/efectos de los fármacos , Malla Trabecular/efectos de los fármacos , Wortmanina/farmacología , Anciano de 80 o más Años , Animales , Bovinos , Citoesqueleto/efectos de los fármacos , Glaucoma/fisiopatología , Humanos , Presión Intraocular/fisiología , Contracción Muscular/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3/administración & dosificación , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Malla Trabecular/citología , Malla Trabecular/metabolismo , Malla Trabecular/fisiología , Wortmanina/administración & dosificación
8.
Cancer Res ; 67(8): 3871-7, 2007 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-17413002

RESUMEN

The sonic hedgehog (Shh) pathway is activated in approximately 30% of human medulloblastoma resulting in increased expression of downstream target genes. In about half of these cases, this has been shown to be a consequence of mutations in regulatory genes within the pathway, including Ptc1, Smo, and Sufu. However, for some tumors, no mutations have been detected in known pathway genes. This suggests that either mutations in other genes promote tumorigenesis or that epigenetic alterations increase pathway activity in these tumors. Here, we report that 3% to 4% of mice lacking either one or both functional copies of Cxcr6 develop medulloblastoma. Although CXCR6 is not known to be involved in Shh signaling, tumors derived from Cxcr6 mutant mice expressed Shh pathway target genes including Gli1, Gli2, Ptc2, and Sfrp1, indicating elevated pathway activity. Interestingly, the level of Ptc1 expression was decreased in tumor cells although two normal copies of Ptc1 were retained. This implies that reduced CXCR6 function leads to suppression of Ptc1 thereby increasing Smoothened function and promoting tumorigenesis. We used a direct transplant model to test the sensitivity of medulloblastoma arising in Cxcr6 mutant mice to a small-molecule inhibitor of Smoothened (HhAntag). We found that transplanted tumors were dramatically inhibited in mice treated for only 4 days with HhAntag. These findings suggest that HhAntag may be effective against tumors lacking mutations in known Shh pathway genes.


Asunto(s)
Meduloblastoma/genética , Receptores de Quimiocina/genética , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Femenino , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Proteínas Hedgehog/metabolismo , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Receptores Patched , Receptor Patched-1 , Receptores CXCR , Receptores CXCR6 , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Receptores de Quimiocina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened
9.
Clin Pharmacol Ther ; 106(4): 874-883, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31038730

RESUMEN

We sought to refine understanding about associations identified in prior studies between angiotensin-II receptor blockers, metformin, selective serotonin reuptake inhibitors, fibric-acid derivatives, or calcium channel blockers and progression to glaucoma filtration surgery for open-angle glaucoma (OAG). We used new-initiator, active-comparator cohort designs to investigate these drugs in two data sources. We adjusted for confounders using stabilized inverse-probability-of-treatment weights and evaluated results using "intention-to-treat" and "as-treated" follow-up approaches. In both data sources, Kaplan-Meier curves showed trends for more rapid progression to glaucoma filtration surgery in patients taking calcium channel blockers compared with thiazides with as-treated (MarketScan P = 0.15; Medicare P = 0.03) and intention-to-treat follow-up (MarketScan P < 0.01; Medicare P = 0.10). There was suggestion of delayed progression for selective serotonin reuptake inhibitor compared with tricyclic antidepressants in Medicare, which was not observed in MarketScan. Our study provided support for a relationship between calcium channel blockers and OAG progression but not for other investigated drugs.


Asunto(s)
Bloqueadores de los Canales de Calcio , Progresión de la Enfermedad , Glaucoma de Ángulo Abierto/fisiopatología , Anciano , Antidepresivos/efectos adversos , Antidepresivos/uso terapéutico , Antihipertensivos/efectos adversos , Antihipertensivos/uso terapéutico , Bloqueadores de los Canales de Calcio/efectos adversos , Bloqueadores de los Canales de Calcio/uso terapéutico , Factores de Confusión Epidemiológicos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/prevención & control , Femenino , Glaucoma de Ángulo Abierto/epidemiología , Humanos , Hipoglucemiantes/efectos adversos , Hipoglucemiantes/uso terapéutico , Estimación de Kaplan-Meier , Masculino , Medicare/estadística & datos numéricos , Medición de Riesgo/métodos , Estados Unidos
10.
PLoS One ; 13(11): e0201683, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30496180

RESUMEN

Pluripotent stem cells (PSCs) edited with genetic reporters are useful tools for differentiation analysis and for isolation of specific cell populations for study. Reporter integration into the genome is now commonly achieved by targeted DNA nuclease-enhanced homology directed repair (HDR). However, human PSCs are known to have a low frequency of gene knock-in (KI) by HDR, making reporter line generation an arduous process. Here, we report a methodology for scarless KI of large fluorescent reporter genes into PSCs by transient selection with puromycin or zeocin. With this method, we can perform targeted KI of a single reporter gene with up to 65% efficiency, as well as simultaneous KI of two reporter genes into different loci with up to 11% efficiency. Additionally, we demonstrate that this method also works in mouse PSCs.


Asunto(s)
Técnicas de Sustitución del Gen/métodos , Células Madre Pluripotentes/metabolismo , Animales , Línea Celular , Genes Reporteros , Humanos , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Ratones , Células Madre Pluripotentes/citología , Transgenes
11.
Sci Rep ; 8(1): 14339, 2018 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-30254279

RESUMEN

The knockout (KO) of the adiponectin receptor 1 (AdipoR1) gene causes retinal degeneration. Here we report that ADIPOR1 protein is primarily found in the eye and brain with little expression in other tissues. Further analysis of AdipoR1 KO mice revealed that these animals exhibit early visual system abnormalities and are depleted of RHODOPSIN prior to pronounced photoreceptor death. A KO of AdipoR1 post-development either in photoreceptors or the retinal pigment epithelium (RPE) resulted in decreased expression of retinal proteins, establishing a role for ADIPOR1 in supporting vision in adulthood. Subsequent analysis of the Mfrprd6 mouse retina demonstrated that these mice are lacking ADIPOR1 in their RPE layer alone, suggesting that loss of ADIPOR1 drives retinal degeneration in this model. Moreover, we found elevated levels of IRBP in both the AdipoR1 KO and the Mfrprd6 models. The spatial distribution of IRBP was also abnormal. This dysregulation of IRBP hypothesizes a role for ADIPOR1 in retinoid metabolism.


Asunto(s)
Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Receptores de Adiponectina/deficiencia , Receptores de Adiponectina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Visión Ocular , Animales , Proteínas del Ojo/metabolismo , Humanos , Ratones , Receptores de Adiponectina/genética , Retinoides/metabolismo , Proteínas de Unión al Retinol/metabolismo
12.
Invest Ophthalmol Vis Sci ; 59(5): 1704-1716, 2018 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-29610853

RESUMEN

Purpose: The nitric oxide/soluble guanylate cyclase/protein kinase G (NO/sGC/PKG) is known to be involved in the regulation of intraocular pressure (IOP) and may be dysregulated in glaucoma. The purpose is to demonstrate that the sGC activator MGV354 lowers IOP in a monkey model of glaucoma and could be considered as a possible new clinical drug candidate. Methods: Changes to cGMP were assessed in primary human trabecular meshwork (hNTM) cells and binding studies were conducted using human sGC full-length protein. Ocular safety tolerability, exposure, and efficacy studies were conducted in rabbit and monkey models following topical ocular dosing of MGV354. Results: sGC was highly expressed in the human and cynomolgus monkey outflow pathways. MGV354 had a 7-fold greater Bmax to oxidized sGC compared to that of reduced sGC and generated an 8- to 10-fold greater cGMP compared to that of a reduced condition in hTM cells. A single topical ocular dose with MGV354 caused a significant dose-dependent reduction of 20% to 40% (versus vehicle), lasting up to 6 hours in pigmented rabbits and 24 hours postdose in a cynomolgus monkey model of glaucoma. The MGV354-induced IOP lowering was sustained up to 7 days following once-daily dosing in a monkey model of glaucoma and was greater in magnitude compared to Travatan (travoprost)-induced IOP reduction. Mild to moderate ocular hyperemia was the main adverse effect noted. Conclusions: MGV354 represents a novel class of sGC activators that can lower IOP in preclinical models of glaucoma. The potential for sGC activators to be used as effective IOP-lowering drugs in glaucoma patients could be further determined in clinical studies.


Asunto(s)
Antihipertensivos/farmacología , Activadores de Enzimas/farmacología , Glaucoma/tratamiento farmacológico , Presión Intraocular/efectos de los fármacos , Piperidinas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Guanilil Ciclasa Soluble/metabolismo , Administración Oftálmica , Animales , Antihipertensivos/administración & dosificación , Células Cultivadas , GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Activadores de Enzimas/administración & dosificación , Glaucoma/fisiopatología , Humanos , Inmunohistoquímica , Macaca fascicularis , Hipotensión Ocular/tratamiento farmacológico , Soluciones Oftálmicas , Piperidinas/administración & dosificación , Pirazoles/administración & dosificación , Piridinas/administración & dosificación , Conejos , Malla Trabecular/metabolismo
13.
Invest Ophthalmol Vis Sci ; 48(11): 5221-8, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17962477

RESUMEN

PURPOSE: Mucolipidosis II and III (ML II; ML III) are lysosomal storage diseases characterized by a deficiency in GlcNAc-1-phosphotransferase. Patients with ML III have retinal disease, but in cases of the more clinically severe ML II, human ophthalmic studies are limited. In this study, retinal function and overall disease were assessed in mice lacking GNPTAB, the gene mutated in patients with ML II. METHODS: Mice deficient in GNPTAB were generated from Omnibank, a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones as part of a large-scale effort to knock out, phenotypically screen, and thereby validate pharmaceutically tractable genes for drug development. Routine diagnostics, expression analysis, histopathology, and ERG analyses were performed on mice lacking GNPTAB. In addition, measurements of serum lysosomal enzymes were performed. RESULTS: Severe retinal degeneration was observed in mice deficient in GNPTAB. Heterozygous mice were phenotypically normal and in situ hybridization showed expression across the neural retina. Compared to wild-type mice, the GNPTAB homozygous mice were smaller, had elevated levels of serum lysosomal enzymes, exhibited cartilage defects, and had cytoplasmic alterations in secretory cells of several exocrine glands. CONCLUSIONS: Mice deficient in GNPTAB exhibited severe retinal degeneration. Additional features observed in patients with ML II, a lysosomal storage disease, are also present in these mice. Understanding underlying mechanisms of this gene in the eye will increase its therapeutic potential for the treatment of retinal diseases.


Asunto(s)
Glándulas Exocrinas/patología , Trastornos del Crecimiento/enzimología , Mucolipidosis/enzimología , Degeneración Retiniana/enzimología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/fisiología , Animales , Catepsina D/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Genotipo , Glicósido Hidrolasas/sangre , Trastornos del Crecimiento/sangre , Trastornos del Crecimiento/fisiopatología , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mucolipidosis/sangre , Mucolipidosis/fisiopatología , Fotograbar , Retina/fisiopatología , Degeneración Retiniana/sangre , Degeneración Retiniana/fisiopatología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
14.
Invest Ophthalmol Vis Sci ; 58(7): 2991-3003, 2017 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-28605810

RESUMEN

Purpose: To discover novel therapies that lower IOP by increasing aqueous humor outflow facility, ex vivo ocular perfusion systems provide a valuable tool. However, currently available designs are limited by their throughput. Here we report the development of a compact, scalable perfusion system with improved throughput and its validation using bovine and porcine eyes. Methods: At a fixed IOP of 6 mm Hg, flow rate was measured by flow sensors. We validated the system by measuring the outflow responses to Y-39983 (a Rho kinase inhibitor), endothelin-1 (ET-1), ambrisentan (an antagonist for endothelin receptor A [ETA]), sphigosine-1-phosphate (S1P), JTE-013 (antagonist for S1P receptor 2 [S1P2]), S-nitroso-N-acetylpenicillamine (SNAP, a nitric oxide [NO] donor), and 3-Morpholino-sydnonimine (SIN-1, another NO donor). Results: The instrument design enabled simultaneous measurements of 20 eyes with a footprint of 1 m2. Relative to vehicle control, Y-39983 increased outflow by up to 31% in calf eyes. On the contrary, ET-1 decreased outflow by up to 79%, a response that could be blocked by pretreatment with ambrisentan, indicating a role for ETA receptors. Interestingly, the effect of ET-1 was also inhibited by up to 70% to 80% by pretreatment with NO donors, SNAP and SIN-1. In addition to testing in calf eyes, similar effects of ET-1 and ambrisentan were observed in adult bovine and porcine eyes. Conclusions: The compact eye perfusion platform provides an opportunity to efficiently identify compounds that influence outflow facility and may lead to the discovery of new glaucoma therapies.


Asunto(s)
Humor Acuoso/metabolismo , Glaucoma/fisiopatología , Presión Intraocular/fisiología , Perfusión/instrumentación , Piridinas/farmacología , Malla Trabecular/metabolismo , Animales , Humor Acuoso/efectos de los fármacos , Bovinos , Diseño Asistido por Computadora , Modelos Animales de Enfermedad , Endotelina-1/farmacología , Diseño de Equipo , Glaucoma/metabolismo , Glaucoma/terapia , Pirazoles/farmacología , Porcinos , Malla Trabecular/efectos de los fármacos
15.
Eur J Pharmacol ; 787: 47-56, 2016 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-26948313

RESUMEN

Intraocular pressure (IOP) lowering drugs that are approved for the treatment of glaucoma and ocular hypertension have limited activity on increasing aqueous humor movement through the trabecular meshwork and Schlemm's canal (TM/SC). The TM/SC complex is considered the conventional outflow pathway and is a primary site of increased resistance to aqueous humor outflow in glaucoma. Novel mechanisms that enhance conventional outflow have shown promise in IOP reduction via modulation of several pathways including Rho kinase, nitric oxide/soluble guanylate cyclase/cGMP, adenosine A1, prostaglandin EP4/cAMP, and potassium channels. The clinical translatability of these pharmacological modulators based on pre-clinical efficacy models is currently being explored. In addition, identification of pathways from GWAS and other studies involving transgenic rodent models with elevated/reduced IOP phenotypes have begun to yield additional insights into IOP regulation and serve as a source for the next generation of IOP lowering targets. Lastly, improvements in drug delivery technologies to enable sustained IOP reduction are also discussed.


Asunto(s)
Presión Intraocular/efectos de los fármacos , Terapia Molecular Dirigida/métodos , Animales , Descubrimiento de Drogas , Humanos
16.
Elife ; 52016 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-27823583

RESUMEN

The tumor microenvironment is a critical modulator of carcinogenesis; however, in many tumor types, the influence of the stroma during preneoplastic stages is unknown. Here we explored the relationship between pre-tumor cells and their surrounding stroma in malignant progression of the cerebellar tumor medulloblastoma (MB). We show that activation of the vascular regulatory signalling axis mediated by Norrin (an atypical Wnt)/Frizzled4 (Fzd4) inhibits MB initiation in the Ptch+/- mouse model. Loss of Norrin/Fzd4-mediated signalling in endothelial cells, either genetically or by short-term blockade, increases the frequency of pre-tumor lesions and creates a tumor-permissive microenvironment at the earliest, preneoplastic stages of MB. This pro-tumor stroma, characterized by angiogenic remodelling, is associated with an accelerated transition from preneoplasia to malignancy. These data expose a stromal component that regulates the earliest stages of tumorigenesis in the cerebellum, and a novel role for the Norrin/Fzd4 axis as an endogenous anti-tumor signal in the preneoplastic niche.


Asunto(s)
Carcinogénesis , Proteínas del Ojo/metabolismo , Receptores Frizzled/metabolismo , Meduloblastoma/fisiopatología , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Ratones
17.
Nat Commun ; 6: 6228, 2015 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-25736573

RESUMEN

The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover therapies for blindness. Here we report the discovery of adiponectin receptor 1 (AdipoR1) as a regulator of these cells' functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1(-/-) mice. We also find decreased photoreceptor-specific phosphatidylcholine containing very long-chain polyunsaturated fatty acids and severely attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1(-/-) mice. RPE-rich eyecup cultures from AdipoR1(-/-) reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity.


Asunto(s)
Ácidos Docosahexaenoicos/metabolismo , Células Fotorreceptoras de Vertebrados/fisiología , Receptores de Adiponectina/metabolismo , Epitelio Pigmentado de la Retina/fisiología , Animales , Electrorretinografía , Hibridación in Situ , Ratones , Ratones Noqueados , Fosfatidilcolinas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Epitelio Pigmentado de la Retina/metabolismo
18.
ACS Med Chem Lett ; 6(1): 84-8, 2015 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-25589936

RESUMEN

The structure of LX7101, a dual LIM-kinase and ROCK inhibitor for the treatment of ocular hypertension and associated glaucoma, is disclosed. Previously reported LIM kinase inhibitors suffered from poor aqueous stability due to solvolysis of the central urea. Replacement of the urea with a hindered amide resulted in aqueous stable compounds, and addition of solubilizing groups resulted in a set of compounds with good properties for topical dosing in the eye and good efficacy in a mouse model of ocular hypertension. LX7101 was selected as a clinical candidate from this group based on superior efficacy in lowering intraocular pressure and a good safety profile. LX7101 completed IND enabling studies and was tested in a Phase 1 clinical trial in glaucoma patients, where it showed efficacy in lowering intraocular pressure.

19.
Invest Ophthalmol Vis Sci ; 45(8): 2767-77, 2004 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-15277503

RESUMEN

PURPOSE: Semaphorin 4A (Sema4A) is a member of the transmembrane class 4 family of semaphorins. It has recently been shown to participate in cell-cell communication in the immune system. High levels of sema4A are also present in brain and eye, but its function in the central nervous system has not been studied. To investigate the function of Sema4A, we generated mice deficient in this transmembrane signaling molecule. METHODS: An embryonic stem (ES) cell clone with a retroviral gene-trap insertion in the sema4A gene was used to generate mice lacking this transmembrane semaphorin. Fundus photography, fluorescein angiography, and electroretinography were used to evaluate retinal anatomy and physiology in mice lacking Sema4A. Electron microscopy and immunohistochemistry with cell-type-specific markers were used to characterize retinal development. In situ hybridization with sema4A-specific riboprobes was used to localize expression of this gene in the developing and adult eye. RESULTS: Fundus photography performed at 14 weeks of age revealed severe retinal degeneration, attenuated retinal vessels, and depigmentation in mice lacking Sema4A. At this age, the outer nuclear layer was reduced to a single row of photoreceptor cells, and the outer plexiform layer was thin and disorganized. Disruption of Sema4A also compromised the physiological function of both rod and cone photoreceptors. Developmental studies in Sema4A-deficient mice revealed abnormal morphology of photoreceptor outer segments during the time at which they establish contacts with apical microvilli of the retinal pigment epithelium (RPE). Sema4A is expressed in the inner retina and RPE during the time at which photoreceptor outer segments elongate. CONCLUSIONS: These findings identify a previously unknown function of Sema4A in the developing visual system and provide a useful model for understanding cell-cell interactions that occur between photoreceptors and the RPE.


Asunto(s)
Células Fotorreceptoras de Vertebrados/fisiología , Degeneración Retiniana/fisiopatología , Semaforinas/fisiología , Animales , Comunicación Celular , Electrorretinografía , Angiografía con Fluoresceína , Técnica del Anticuerpo Fluorescente Indirecta , Eliminación de Gen , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuroglía/metabolismo , Neuroglía/patología , Células Fotorreceptoras de Vertebrados/ultraestructura , Epitelio Pigmentado Ocular/metabolismo , Epitelio Pigmentado Ocular/patología , Degeneración Retiniana/metabolismo , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Vasos Retinianos/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semaforinas/deficiencia
20.
Brain Res Mol Brain Res ; 115(2): 121-9, 2003 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-12877983

RESUMEN

The Reelin signaling pathway controls neuronal positioning during mammalian brain development by binding to the very low density lipoprotein receptor and apolipoprotein E receptor-2, and signaling through the intracellular adapter protein Disabled-1 (Dab1). To identify new components in the Reelin signaling pathway, we used a yeast two-hybrid screen to select Dab1-interacting proteins. Here, we report the characterization of a new mouse Dab1-interacting protein that is orthologous to rat Dab2IP, a Ras-GTPase activating protein previously shown to bind to Dab2/DOC. The interaction of Dab1 and Dab2IP was confirmed in biochemical assays and by co-immunoprecipitation from brain lysates. The site of interaction between Dab1 and Dab2IP was narrowed to the Dab1-PTB domain and the NPxY motif in Dab2IP. The deduced amino acid sequence of mouse Dab2IP encompasses 1,208 residues containing several protein interaction motifs as well as a Ras-like GAP-related domain. Northern blot analysis revealed at least two isoforms of Dab2IP mRNA in the brain, both of which exhibited increased expression during development. In situ hybridization analyses indicated that Dab2IP mRNA is diffusely expressed throughout the developing and the adult brain. Using a polyclonal antiserum specific for Dab2IP, we observed protein expression in the soma and processes of neurons in a variety of brain structures, including the developing cerebral cortex. Our findings suggest that Dab2IP may function as a downstream effector in the Reelin signaling pathway that influences Ras signaling during brain development.


Asunto(s)
Encéfalo/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Elementos sin Sentido (Genética)/metabolismo , Northern Blotting , Western Blotting , Encéfalo/citología , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Calbindinas , Embrión de Mamíferos , Proteínas Activadoras de GTPasa/clasificación , Proteínas Activadoras de GTPasa/inmunología , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Pruebas de Precipitina/métodos , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteína Reelina , Proteína G de Unión al Calcio S100/metabolismo , Técnicas del Sistema de Dos Híbridos , Levaduras
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