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PURPOSE: To investigate the functional role that the zinc e-box binding homeobox 1 (ZEB1) gene, which underlies the genetic basis of posterior polymorphous corneal dystrophy 3 (PPCD3), plays in corneal endothelial cell proliferation, apoptosis, migration, and barrier function. METHODS: A human corneal endothelial cell line (HCEnC-21T) was transfected with siRNA targeting ZEB1 mRNA. Cell proliferation, apoptosis, migration, and barrier assays were performed: Cell proliferation was assessed with cell counting using a hemocytometer; cell apoptosis, induced by either ultraviolet C (UVC) radiation or doxorubicin treatment, was quantified by measuring cleaved caspase 3 (cCASP3) protein levels; and cell migration and barrier function were monitored with electric cell-substrate impedance sensing (ECIS). RESULTS: ZEB1 knockdown in HCEnC-21T cells transfected with siRNA targeting ZEB1 did not result in a significant difference in cell proliferation when compared with control. Although knockdown of ZEB1 in HCEnC-21T cells sensitized the cells to UV-induced apoptosis, ZEB1 knockdown did not alter the cells' susceptibility to doxorubicin-induced apoptosis, as measured with cCASP3 protein levels, compared with controls. Similarly, no difference was observed in cell migration following ZEB1 knockdown. However, cell barrier function increased significantly following ZEB1 knockdown. CONCLUSIONS: The corneal endothelium in PPCD3 is characterized by morphologic, anatomic, and molecular features that are more consistent with an epithelial-like rather than an endothelial-like phenotype. Although these characteristics have been well documented, we demonstrate for the first time that susceptibility to UV-induced apoptosis and cell barrier function are significantly altered in the setting of reduced ZEB1. The significance of an altered cellular response to apoptotic stimuli and increased cell barrier function in the pathobiology of PPCD remains to be fully elucidated.
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Distrofias Hereditarias de la Córnea/fisiopatología , Endotelio Corneal/fisiología , Regulación de la Expresión Génica/fisiología , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Antibióticos Antineoplásicos/toxicidad , Apoptosis/fisiología , Western Blotting , Caspasa 3/metabolismo , Línea Celular , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Doxorrubicina/toxicidad , Impedancia Eléctrica , Endotelio Corneal/efectos de la radiación , Técnicas de Silenciamiento del Gen , Humanos , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Transfección , Rayos Ultravioleta/efectos adversosRESUMEN
Congenital hereditary endothelial dystrophy (CHED), Harboyan syndrome (CHED with progressive sensorineural deafness), and potentially a subset of individuals with late-onset Fuchs' endothelial corneal dystrophy are caused by mutations in the SLC4A11 gene that results in corneal endothelial cell abnormalities. Originally classified as a borate transporter, the function of SLC4A11 as a transport protein remains poorly understood. Elucidating the transport function(s) of SLC4A11 is needed to better understand how its loss results in the aforementioned posterior corneal dystrophic disease processes. Quantitative PCR experiments demonstrated that, of the three known human NH2-terminal variants, SLC4A11-C is the major transcript expressed in human corneal endothelium. We studied the expression pattern of the three variants in mammalian HEK-293 cells and demonstrated that the SLC4A11-B and SLC4A11-C variants are plasma membrane proteins, whereas SLC4A11-A is localized intracellularly. SLC4A11-B and SLC4A11-C were shown to be multifunctional ion transporters capable of transporting H+ equivalents in both a Na+-independent and Na+-coupled mode. In both transport modes, SLC4A11-C H+ flux was significantly greater than SLC4A11-B. In the presence of ammonia, SLC4A11-B and SLC4A11-C generated inward currents that were comparable in magnitude. Chimera SLC4A11-C-NH2-terminus-SLC4A11-B experiments demonstrated that the SLC4A11-C NH2-terminus functions as an autoactivating domain, enhancing Na+-independent and Na+-coupled H+ flux without significantly affecting the electrogenic NH3-H(n)+ cotransport mode. All three modes of transport were significantly impaired in the presence of the CHED causing p.R109H (SLC4A11-C numbering) mutation. These complex ion transport properties need to be addressed in the context of corneal endothelial disease processes caused by mutations in SLC4A11.
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Proteínas de Transporte de Anión/genética , Antiportadores/genética , Variación Genética/genética , Transporte Iónico/genética , Línea Celular , Membrana Celular/genética , Distrofias Hereditarias de la Córnea/genética , Endotelio Corneal/metabolismo , Células HEK293 , Pérdida Auditiva Sensorineural/genética , Humanos , Proteínas de la Membrana/genética , Persona de Mediana Edad , Mutación/genéticaRESUMEN
PURPOSE: To report potentially pathogenic mutations in the keratin 3 (KRT3) and keratin 12 (KRT12) genes in two individuals with clinically diagnosed Meesmann corneal dystrophy (MECD). METHODS: Slit-lamp examination was performed on the probands and available family members to identify characteristic features of MECD. After informed consent was obtained, saliva samples were obtained as a source of genomic DNA, and screening of KRT3 and KRT12 was performed. Potentially pathogenic variants were screened for in 200 control chromosomes. PolyPhen-2, SIFT, and PANTHER were used to predict the functional impact of identified variants. Short tandem repeat genotyping was performed to confirm paternity. RESULTS: Slit-lamp examination of the first proband demonstrated bilateral, diffusely distributed, clear epithelial microcysts, consistent with MECD. Screening of KRT3 revealed a heterozygous missense variant in exon 1, c.250C>T (p.(Arg84Trp)), which has a minor allele frequency of 0.0076 and was not identified in 200 control chromosomes. In silico analysis with PolyPhen-2 and PANTHER predicted the variant to be damaging to protein function; however, SIFT analysis predicted tolerance of the variant. The second proband demonstrated bilateral, diffusely distributed epithelial opacities that appeared gray-white on direct illumination and translucent on retroillumination. Neither parent demonstrated corneal opacities. Screening of KRT12 revealed a novel heterozygous insertion/deletion variant in exon 6, c.1288_1293delinsAGCCCT (p.(Arg430_Arg431delinsSerPro)). This variant was not present in either of the proband's parents or in 200 control chromosomes and was predicted to be damaging by PolyPhen-2, PANTHER, and SIFT. Haplotype analysis confirmed paternity of the second proband, indicating that the variant arose de novo. CONCLUSIONS: We present a novel KRT12 mutation, representing the first de novo mutation and the first indel in KRT12 associated with MECD. In addition, we report a variant of uncertain significance in KRT3 in an individual with MECD. Although the potential pathogenicity of this variant is unknown, it is the first variant affecting the head domain of K3 to be reported in an individual with MECD and suggests that disease-causing variants associated with MECD may not be restricted to primary sequence alterations of either the helix-initiation or helix-termination motifs of K3 and K12.
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Distrofia Corneal Epitelial Juvenil de Meesmann/genética , Queratina-12/genética , Queratina-3/genética , Mutación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Niño , Distrofia Corneal Epitelial Juvenil de Meesmann/patología , Análisis Mutacional de ADN , Femenino , Heterocigoto , Humanos , Mutación INDEL , Queratina-12/química , Queratina-3/química , Masculino , Persona de Mediana Edad , Mutación Missense , Linaje , Polimorfismo de Nucleótido SimpleRESUMEN
PURPOSE: To report the identification of a novel frameshift mutation and copy number variation (CNV) in PIKFYVE in two probands with fleck corneal dystrophy (FCD). METHODS: Slit-lamp examination was performed to identify characteristic features of FCD. After genomic DNA was collected, PCR amplification and automated sequencing of all 41 exons of PIKFYVE was performed. Using genomic DNA, quantitative PCR (qPCR) was performed to detect CNVs within PIKFYVE. RESULTS: In the first FCD proband, numerous panstromal punctate opacities were observed in each of the proband's corneas, consistent with the diagnosis of FCD. Screening of PIKFYVE demonstrated a novel heterozygous frameshift mutation in exon 19, c.3151dupA, which is predicted to encode for a truncated PIKFYVE protein, p.(Asp1052Argfs*18). This variant was identified in an affected sister but not in the proband's unaffected mother or brother or 200 control chromosomes. The second FCD proband presented with bilateral, discrete, punctate, grayish-white stromal opacities. Exonic screening of PIKFYVE revealed no causative variant. However, CNV analysis demonstrated the hemizygous deletion of exons 15 and 16. CONCLUSIONS: We report a novel heterozygous frameshift mutation (c.3151dupA) and a CNV in PIKFYVE, representing the first CNV and the fifth frameshift mutation associated with FCD.
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Secuencia de Bases , Distrofias Hereditarias de la Córnea/genética , Variaciones en el Número de Copia de ADN , Mutación del Sistema de Lectura , Fosfatidilinositol 3-Quinasas/genética , Eliminación de Secuencia , Adulto , Córnea/metabolismo , Córnea/patología , Distrofias Hereditarias de la Córnea/diagnóstico , Distrofias Hereditarias de la Córnea/patología , Análisis Mutacional de ADN , Exones , Femenino , Expresión Génica , Heterocigoto , Humanos , Datos de Secuencia MolecularRESUMEN
PURPOSE: To report the identification of five novel nonsense mutations in the zinc finger E-box binding homeobox 1 (ZEB1) gene and exclusion of promoter region mutations in individuals without ZEB1 coding region mutations in posterior polymorphous corneal dystrophy (PPCD). METHODS: Slit-lamp examination and DNA collection were performed for individuals diagnosed with PPCD and, when available, affected and unaffected family members. Genomic DNA prepared from peripheral blood leukocytes and buccal epithelial cells underwent PCR amplification and automated sequencing of the ZEB1 gene and 1 kb 5' of ZEB1, presumably containing the ZEB1 promoter region. RESULTS: Thirteen unrelated individuals with PPCD were identified, and genomic DNA was collected from each individual. ZEB1 mutations were identified in six of the 13 probands, five of which were novel: p.(Gly150Alafs*36; spontaneous), p.(His230Argfs*7), p.(Ser638Cysfs*5), p.(Glu1039Glyfs*6), and p.(Gln884Argfs*37). Screening of the ZEB1 promoter region in 31 probands with PPCD without a ZEB1 coding region mutation identified only two known single nucleotide polymorphisms (SNPs) whose frequency in the affected probands did not differ significantly from that in the general population. CONCLUSIONS: We report five novel frame-shift mutations, one confirmed as spontaneous, in the ZEB1 gene associated with PPCD, bringing the total number of reported pathogenic mutations to 24, and the percentage of PPCD associated with ZEB1 mutations to 32%. The absence of ZEB1 promoter region mutations in probands without a ZEB1 coding region mutation indicates that other genetic loci, such as the PPCD1 locus, are involved in the pathogenesis of PPCD.
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Codón sin Sentido/genética , Distrofias Hereditarias de la Córnea/genética , Proteínas de Homeodominio/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Emparejamiento Base/genética , Secuencia de Bases , Análisis Mutacional de ADN , Exones/genética , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Linaje , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Background Silver possesses cytotoxic properties against many microorganisms and is regularly used in wound care. Current evidence supporting the use of one type of silver-containing wound dressing (SCWD) is insufficient. Materials and methods To examine the ability of selected SCWDs to inhibit the growth of two strains of bacteria (Escherichia coli and Staphylococcus aureus) commonly found in wounds, an in vitro wound model was used. Bacteria were applied to the surface of nutrient agar, and a piece of each SCWD was applied to the bacteria. The plates were incubated at 37°C overnight. The zone of inhibition (ZI) around each SCWD was measured in cm2. Results The mean ZI for Acticoat Flex-3 on E. coli was 1.59 ± 0.15 cm2, which was significantly greater than that observed for Aquacel Ag (p<0.001), Mepilex Ag (p<0.0001), Mepitel Ag (p<0.001), Optifoam (p<0.0001), and Tegaderm Alginate Ag (p<0.01), but statistically indistinguishable from Maxorb II Ag. The mean ZI on S. aureus was 1.21 ± 0.16 cm2, which was greater than Aquacel Ag (p<0.05), Mepilex (p<0.0001), Optifoam (p<0.0001), and Tegaderm Alginate Ag (p<0.05), but statistically indistinguishable from Maxorb II Ag or Mepitel Ag. Conclusion Of the SCWDs tested, Acticoat Flex-3 demonstrated the most robust antimicrobial effect. Herein, we show that Acticoat Flex-3 may provide the most wound protection against bacterial infection. In conclusion, these data provide clinicians with additional independent evidence to inform their clinical practice on the use of specific wound dressings.
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Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an extracellular protein and endogenous regulator of matrix metalloproteinases (MMPs) secreted by astrocytes in response to CNS myelin injury. We have previously reported that adult TIMP-1 knock-out (KO) mice exhibit poor myelin repair following demyelinating injury. This observation led us to hypothesize a role for TIMP-1 in oligodendrogenesis and CNS myelination. Herein, we demonstrate that compact myelin formation is significantly delayed in TIMP-1 KO mice, a situation that coincided with dramatically reduced numbers of white matter astrocytes in the developing CNS. Analysis of differentiation in CNS progenitor cells (neurosphere) cultures from TIMP-1 KO mice revealed a specific deficit of NG2(+) oligodendrocyte progenitor cells. Application of recombinant murine TIMP-1 (rmTIMP-1) to TIMP-1 KO neurosphere cultures evoked a dose-dependent increase in NG2(+) cell numbers, while treatment with GM6001, a potent broad-spectrum MMP inhibitor did not. Similarly, administration of rmTIMP-1 to A2B5(+) immunopanned oligodendrocyte progenitors significantly increased the number of differentiated O1(+) oligodendrocytes, while antisera to TIMP-1 reduced oligodendrocyte numbers. We also determined that A2B5(+) oligodendrocyte progenitors grown in conditioned media derived from TIMP-1 KO primary glial cultures resulted in reduced differentiation of mature O1(+) oligodendrocytes. Finally, we report that addition of rmTIMP-1 to primary glial cultures resulted in a dose-dependent proliferative response of astrocytes. Together, these findings describe a previously uncharacterized role for TIMP-1 in the regulation of oligodendrocytes and astrocytes during development and provide a novel function for TIMP-1 on myelination in the developing CNS.
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Astrocitos/metabolismo , Diferenciación Celular/fisiología , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Análisis de Varianza , Animales , Astrocitos/citología , Células Cultivadas , Técnicas de Cocultivo , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Electrónica , Oligodendroglía/citología , Inhibidor Tisular de Metaloproteinasa-1/genéticaRESUMEN
Enteroviruses, including coxsackieviruses, exhibit significant tropism for the central nervous system, and these viruses are commonly associated with viral meningitis and encephalitis. Previously, we described the ability of coxsackievirus B3 (CVB3) to infect proliferating neuronal progenitor cells located in the neonatal subventricular zone and persist in the adult murine central nervous system (CNS). Here, we demonstrate that cultured murine neurospheres, which comprise neural stem cells and their progeny at different stages of development, were highly susceptible to CVB3 infection. Neurospheres, or neural progenitor and stem cells (NPSCs), isolated from neonatal C57BL/6 mice, supported high levels of infectious virus production and high viral protein expression levels following infection with a recombinant CVB3 expressing enhanced green fluorescent protein (eGFP) protein. Similarly, NPSCs isolated from neonatal actin-promoter-GFP transgenic mice (actin-GFP NPSCs) were highly susceptible to infection with a recombinant CVB3 expressing DsRed (Discosoma sp. red fluorescent protein). Both nestin-positive and NG2(+) progenitor cells within neurospheres were shown to preferentially express high levels of viral protein as soon as 24 h postinfection (p.i.). By day 3 p.i., viral protein expression and viral titers increased dramatically in NPSCs with resultant cytopathic effects (CPE) and eventual cell death. In contrast, reduced viral replication, lower levels of CPE, and diminished viral protein expression levels were observed in NPSCs differentiated for 5 or 16 days in the presence of fetal bovine serum (FBS). Despite the presence of CPE and high levels of cell death following early CVB3 infection, surviving neurospheres were readily observed and continued to express detectable levels of viral protein as long as 37 days after initial infection. Also, CVB3 infection of actin-GFP NPSCs increased the percentage of cells expressing neuronal class III ß-tubulin following their differentiation in the presence of FBS. These results suggest that neural stem cells may be preferentially targeted by CVB3 and that neurogenic regions of the CNS may support persistent viral replication in the surviving host. In addition, normal progenitor cell differentiation may be altered in the host following infection.
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Diferenciación Celular , Enterovirus Humano B/fisiología , Enterovirus Humano B/patogenicidad , Células-Madre Neurales/virología , Animales , Células Cultivadas , Efecto Citopatogénico Viral , Enterovirus Humano B/genética , Enterovirus Humano B/ultraestructura , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Células-Madre Neurales/citología , Células-Madre Neurales/ultraestructura , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral , Proteína Fluorescente RojaRESUMEN
IL-6 is crucial for the induction of many murine models of autoimmunity including experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. To establish the role of site-specific production of IL-6 in autoimmunity, we examined myelin oligodendrocyte glycoprotein immunization-induced EAE in transgenic mice (GFAP-IL6) with IL-6 production restricted to the cerebellum. Myelin oligodendrocyte glycoprotein-immunized (Mi-) GFAP-IL6 mice developed severe ataxia but no physical signs of spinal cord involvement, which was in sharp contrast to Mi-wild type (WT) animals that developed classical EAE with ascending paralysis. Immune pathology and demyelination were nearly absent from the spinal cord, but significantly increased in the cerebellum of Mi-GFAP-IL6 mice. Tissue damage in the cerebellum in the Mi-GFAP-IL6 mice was accompanied by increased total numbers of infiltrating leukocytes and increased proportions of both neutrophils and B-cells. With the exception of IL-17 mRNA, which was elevated in both control immunized and Mi-GFAP-IL6 cerebellum, the level of other cytokine and chemokine mRNAs were comparable with Mi-WT cerebellum whereas significantly higher levels of IFN-gamma and TNF-alpha mRNA were found in Mi-WT spinal cord. Thus, site-specific production of IL-6 in the cerebellum redirects trafficking away from the normally preferred antigenic site the spinal cord and acts as a leukocyte "sink" that markedly enhances the inflammatory cell accumulation and disease. The mechanisms underlying this process likely include the induction of specific chemokines, activation of microglia, and activation and loss of integrity of the blood-brain barrier present in the cerebellum of the GFAP-IL6 mice before the induction of EAE.
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Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/patología , Inflamación/etiología , Interleucina-6/biosíntesis , Animales , Autoinmunidad , Sistema Nervioso Central/metabolismo , Cerebelo/patología , Quimiotaxis , Citocinas/análisis , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/fisiopatología , Interleucina-6/inmunología , Ratones , Ratones Transgénicos , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/efectos adversos , Glicoproteína Mielina-Oligodendrócito , ParálisisRESUMEN
BACKGROUND: Concomitant corneal ectasia and posterior lamellar corneal opacification is rare, and the genetic relationship between these two conditions is unclear. We report the genetic and clinical characterization of this phenotype in three unrelated individuals. MATERIALS AND METHODS: One previously reported affected individual and two unreported, unrelated, affected individuals were recruited for the study. Subjects and unaffected relatives underwent slit lamp examination, refraction, and multi-modal imaging. Saliva samples were obtained from two of the three affected individuals, from which DNA was extracted. Sanger sequencing was performed to identify mutations in genes associated with posterior amorphous corneal dystrophy (PACD), brittle cornea syndrome (BCS), and posterior polymorphous corneal dystrophy (PPCD), while copy number variation (CNV) analysis was used to identify CNV in the PACD locus. RESULTS: Affected individuals demonstrated bilateral corneal steepening, stromal thinning and lamellar posterior corneal opacification. Corneal topography and tomography revealed conical or globular corneal steepening and decreased thickness. Anterior segment optical coherence tomography demonstrated hyperreflectivity of the posterior stroma in each of the affected individuals. Genetic testing did not detect a heterozygous deletion involving the PACD locus on chromosome 12 or a pathogenic mutation in the genes associated with BCS or PPCD. CONCLUSIONS: Corneal ectasia may be associated with posterior lamellar stromal opacification that appears consistent with PACD. However, genetic testing for PACD as well as BCS and PPCD in affected individuals fails to reveal pathogenic deletions or mutations, indicating that other genetic factors are involved.
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Córnea/patología , Opacidad de la Córnea/diagnóstico , Sustancia Propia/patología , Queratocono/diagnóstico , Distrofias Hereditarias de la Córnea/diagnóstico , Distrofias Hereditarias de la Córnea/genética , Topografía de la Córnea , Variaciones en el Número de Copia de ADN , Dilatación Patológica , Anomalías del Ojo/diagnóstico , Anomalías del Ojo/genética , Femenino , Humanos , Recién Nacido , Inestabilidad de la Articulación/congénito , Inestabilidad de la Articulación/diagnóstico , Inestabilidad de la Articulación/genética , Masculino , Mutación , Anomalías Cutáneas/diagnóstico , Anomalías Cutáneas/genética , Microscopía con Lámpara de Hendidura , Adulto JovenRESUMEN
PURPOSE: To investigate the presence of pre-Descemet corneal dystrophy (PDCD) in association with X-linked ichthyosis (XLI) in an 11-year-old boy using multimodal imaging and genetic analysis. METHODS: Corneal opacities were examined and imaged with slit-lamp biomicroscopy, anterior segment optical coherence tomography, noncontact specular microscopy, and in vivo confocal microscopy. Cytogenomic array analysis was performed using genomic DNA isolated from the patient. RESULTS: Corneal opacities characteristic of PDCD located in the posterior corneal stroma just anterior to Descemet membrane were identified by slit-lamp biomicroscopy. A pre-Descemet hyper-reflective line, consistent with these opacities, was seen with anterior segment optical coherence tomography. Scheimpflug tomography revealed a bimodal peak light scattering. In vivo confocal microscopy findings were unremarkable. Copy number analysis identified a 4389 kbp hemizygous deletion on the X chromosome (chr. X: 6,540,898-8,167,604), resulting in the deletion of 4 genes, including the known locus of XLI, the STS gene. CONCLUSIONS: This report demonstrates that PDCD-associated XLI may present in children and that the diagnosis may be confirmed through multimodal imaging in conjunction with genetic analysis.
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Distrofias Hereditarias de la Córnea/diagnóstico , Ictiosis Ligada al Cromosoma X/diagnóstico , Microscopía Confocal/métodos , Imagen Multimodal , Microscopía con Lámpara de Hendidura/métodos , Esteril-Sulfatasa/genética , Tomografía de Coherencia Óptica/métodos , Niño , Distrofias Hereditarias de la Córnea/genética , Sustancia Propia/patología , ADN/genética , Lámina Limitante Posterior/patología , Humanos , Ictiosis Ligada al Cromosoma X/genética , Masculino , Esteril-Sulfatasa/metabolismoRESUMEN
The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided a source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. However, concerns over cell identity remain, and a comprehensive characterization of the cultured CEnC across serial passages has not been performed. To this end, we compared two established CEnC culture methods by assessing the transcriptomic changes that occur during in vitro expansion. In confluent monolayers, low mitogenic culture conditions preserved corneal endothelial cell state identity better than culture in high mitogenic conditions. Expansion by continuous passaging induced replicative cell senescence. Transcriptomic analysis of the senescent phenotype identified a cell senescence signature distinct for CEnC. We identified activation of both classic and new cell signaling pathways that may be targeted to prevent senescence, a significant barrier to realizing the potential clinical utility of in vitro expansion.
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Técnicas de Cultivo de Célula/métodos , Endotelio Corneal/citología , Adolescente , Adulto , Movimiento Celular , Proliferación Celular , Senescencia Celular , Niño , Preescolar , Biología Computacional , Trasplante de Córnea , Femenino , Humanos , Masculino , Fenotipo , Transducción de Señal , Transcriptoma , Adulto JovenRESUMEN
The zinc finger e-box binding homeobox 1 (ZEB1) transcription factor is a master regulator of the epithelial to mesenchymal transition (EMT), and of the reverse mesenchymal to epithelial transition (MET) processes. ZEB1 plays an integral role in mediating cell state transitions during cell lineage specification, wound healing and disease. EMT/MET are characterized by distinct changes in molecular and cellular phenotype that are generally context-independent. Posterior polymorphous corneal dystrophy (PPCD), associated with ZEB1 insufficiency, provides a new biological context in which to understand and evaluate the classic EMT/MET paradigm. PPCD is characterized by a cadherin-switch and transition to an epithelial-like transcriptomic and cellular phenotype, which we study in a cell-based model of PPCD generated using CRISPR-Cas9-mediated ZEB1 knockout in corneal endothelial cells (CEnCs). Transcriptomic and functional studies support the hypothesis that CEnC undergo a MET-like transition in PPCD, termed endothelial to epithelial transition (EnET), and lead to the conclusion that EnET may be considered a corollary to the classic EMT/MET paradigm.
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Endotelio Corneal/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Cadherinas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Córnea/metabolismo , Distrofias Hereditarias de la Córnea/genética , Distrofias Hereditarias de la Córnea/metabolismo , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Humanos , Factores de Transcripción/metabolismo , Transcriptoma , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
Increased matrix metalloproteinase (MMP) proteolytic activity contributes to the pathogenesis of many neuroinflammatory and neurodegenerative conditions in the CNS. To fully understand this process, it is important to define the MMP expression profile of specific cell types, including the CNS-resident cells astrocytes and microglia. While previous studies have characterized astrocyte MMP expression by using mixed glial cultures, these results are likely complicated by the presence of contaminating microglia within these cultures. In the current study, we sought to clarify this complexity, by taking a novel approach to prepare pure astrocyte cultures entirely devoid of microglia, by promoting neural stem cell (NSC) differentiation into astrocytes. The MMP expression profile of mixed glial cultures, neurosphere-derived astrocytes, and pure microglia was characterized by RNase protection assay. This revealed that MMP gene expression is largely cell-type specific. Astrocytes constitutively expressed MMP-11, MMP-14, and MMP-2 and showed induction of MMP-3 in response to IL-1beta but did not respond to lipopolysaccharide (LPS). In contrast, microglia constitutively expressed high levels of MMP-12 and showed strong induction of MMP-9 and MMP-14 in response to LPS. Gelatin zymography confirmed that LPS and TNF-alpha induced strong expression of MMP-9 in microglia but not astrocytes. In summary, these studies demonstrate that neurosphere-derived astrocytes represent an attractive alternative system in which to study astrocyte behavior in vitro. Using this system, we have shown that astrocytes and microglia express distinct sets of MMP genes and that microglia, not astrocytes, are the major source of MMP-9 in response to LPS or TNF-alpha.
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Astrocitos/enzimología , Perfilación de la Expresión Génica/métodos , Regulación Enzimológica de la Expresión Génica/fisiología , Metaloproteinasas de la Matriz/biosíntesis , Microglía/enzimología , Animales , Animales Recién Nacidos , Astrocitos/citología , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Metaloproteinasas de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Microglía/citologíaRESUMEN
PURPOSE: To present the clinical and cytogenetic features of a previously unreported family with posterior amorphous corneal dystrophy (PACD) associated with a heterozygous deletion of the small leucine-rich proteoglycan (SRLP) genes on chromosome 12. METHODS: Clinical characterization was performed using slit lamp biomicroscopic and optical coherence tomography (OCT) imaging. Genomic DNA was collected from affected and unaffected family members, and a cytogenomic array was used to identify copy number variations (CNV) present in the PACD locus. RESULTS: Three members of a Guatemalan family presented with clinical characteristics consistent with PACD: bilateral posterior stromal lamellar opacification, decreased corneal curvature, and iridocorneal adhesions. OCT imaging demonstrated decreased corneal thickness and hyperreflectivity of the posterior third of the corneal stroma. CNV analysis confirmed the presumed clinical diagnosis of PACD by revealing a 0.304 Mb heterozygous deletion in the PACD locus on chromosome 12 that included the four SLRP genes (KERA, LUM, DCN, and EPYC) deleted in each of the PACD families in which CNV analysis has been reported. CONCLUSIONS: This is the first report of the OCT appearance of PACD and the second confirmation of a heterozygous deletion of chromosome 12q21.33 as the cause of PACD, highlighting the utility of array-based cytogenomics to confirm the suspected clinical diagnosis of PACD. As the smallest previously reported pathogenic deletion was 0.701 Mb, the 0.304-Mb deletion we report is the smallest identified to date and reduces the size of the PACD locus to 0.275 Mb.
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Cromosomas Humanos Par 12/genética , Distrofias Hereditarias de la Córnea/genética , Variaciones en el Número de Copia de ADN , Eliminación de Secuencia , Proteoglicanos Pequeños Ricos en Leucina/genética , Adolescente , Preescolar , Distrofias Hereditarias de la Córnea/diagnóstico por imagen , Topografía de la Córnea , Femenino , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Mutación , Linaje , Microscopía con Lámpara de Hendidura , Tomografía de Coherencia ÓpticaRESUMEN
The inoculation of MOG peptides into C57BL/6 mice induces CD4(+) and CD8(+) T cells, and recent work has shown that adoptive transfer of the latter population, after extensive in vitro stimulation, can cause EAE in naïve recipient mice. Herein, we have evaluated the incidence and severity of EAE, and the induction of CD4(+) and CD8(+) T cells, following MOG peptide inoculation of wt mice and of LMP-2KO mice that lack an intact immunoproteasome, a cytoplasmic organelle that is induced by chronic inflammation and that may be important for the presentation of MHC class I epitopes to CD8(+) T cells. We report that EAE, evaluated by both clinical and histological criteria, is similar in LMP-2KO mice and wildtype C57B/6 mice (wt) in response to immunization with MOG peptides MOG(35-55) and MOG(40-54), suggesting that the immunoproteasome does not play a key role in the development of demyelinating disease. Furthermore, and consistent with previous reports, peptide-specific CD8(+) T cells were barely detectable in the CNS of peptide-immunized mice, although peptide-specific CD4(+) T cells were abundant. Therefore, we used a new technique to look for autoreactive CD8(+) T cells in MOG peptide-immunized mice, and we report the identification of CD4(+) and CD8(+) T cells that, as late as 19 days after peptide injection, are actively producing IFNgamma in vivo, in response to in vivo antigen contact.
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Cisteína Endopeptidasas/deficiencia , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/inmunología , Glicoproteína Asociada a Mielina , Fragmentos de Péptidos , Linfocitos T/efectos de los fármacos , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Citometría de Flujo , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de la Mielina , Glicoproteína Mielina-Oligodendrócito , Factores de TiempoRESUMEN
Purpose: To investigate the molecular basis of posterior polymorphous corneal dystrophy (PPCD) by examining the PPCD transcriptome and the effect of decreased ZEB1 expression on corneal endothelial cell (CEnC) gene expression. Methods: Next-generation RNA sequencing (RNA-seq) analyses of corneal endothelium from two PPCD-affected individuals (one with PPCD3 and one of unknown genetic cause) compared with two age-matched controls, and primary human CEnC (pHCEnC) transfected with siRNA-mediated ZEB1 knockdown. The expression of selected differentially expressed genes was validated by quantitative polymerase chain reaction (qPCR) and/or assessed by in situ hybridization in the corneal endothelium of four independent cases of PPCD (one with PPCD3 and three of unknown genetic cause). Results: Expression of 16% and 46% of the 104 protein-coding genes specific to ex vivo corneal endothelium was lost in the endothelium of two individuals with PPCD. Thirty-two genes associated with ZEB1 and 3 genes (BMP4, CCND1, ZEB1) associated with OVOL2 were differentially expressed in the same direction in both individuals with PPCD. Immunohistochemistry staining and RNA-seq analyses demonstrated variable expression of type IV collagens in PPCD corneas. Decreasing ZEB1 expression in pHCEnC altered expression of 711 protein-coding genes, many of which are associated with canonical pathways regulating various cellular processes. Conclusions: Identification of the altered transcriptome in PPCD and in a cell-based model of PPCD provided insight into the molecular alterations characterizing PPCD. Further study of the differentially expressed genes associated with ZEB1 and OVOL2 is expected to identify candidate genes for individuals with PPCD and without a ZEB1 or OVOL2 mutation.
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Colágeno/metabolismo , Distrofias Hereditarias de la Córnea/genética , Transcriptoma , Adolescente , Adulto , Estudios de Casos y Controles , Colágeno Tipo IV/metabolismo , Distrofias Hereditarias de la Córnea/metabolismo , Células Endoteliales/metabolismo , Endotelio Corneal/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Reacción en Cadena de la Polimerasa , Adulto Joven , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
BACKGROUND/AIMS: To report the clinical, histopathological and genetic features of a variant of lattice corneal dystrophy (LCD) associated with two pathogenic mutations in the transforming growth factor-B-induced (TGFBI) gene. METHODS: Clinical characterisation was performed by slit lamp examination and in vivo confocal microscopic imaging (IVCM). Histopathological characterisation was performed with light microscopic examination of an excised corneal button and a peripheral blood samples were collected for TGFBI screening. RESULTS: A 42-year-old woman presented with progressive photophobia and decreased visual acuity in both eyes. Slit lamp examination demonstrated punctate and linear branching opacities in the mid and posterior corneal stroma, corresponding to hyper-reflective opacities noted on IVCM and amyloid deposition noted on histopathological examination of an excised corneal button. TGFBI screening revealed two previously reported heterozygous missense mutations: c.337G>A (p.(Val113Ile)) in exon 4 and c.1673T>C (p.(Leu558Pro)) in exon 12. Screening of an affected sibling with a similar phenotype revealed that she was also heterozygous for both mutations, while screening of another sibling with punctate but not linear stromal opacities revealed that she was heterozygous for only the p.(Leu558Pro) mutation. CONCLUSIONS: The p.(Val113Ile) mutation results in an alteration of the atypical LCD phenotype associated with the p.(Leu558Pro) mutation. This represents only the second report of the alteration of the phenotype of a TGFBI dystrophy by a second, non-homozygous pathogenic mutation, and thus provides insight into the phenotype-genotype correlation of the TGFBI dystrophies.
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Córnea/patología , Distrofias Hereditarias de la Córnea/diagnóstico , Distrofias Hereditarias de la Córnea/genética , Microscopía Confocal , Mutación , Fotofobia/fisiopatología , Adulto , Distrofias Hereditarias de la Córnea/fisiopatología , Progresión de la Enfermedad , Femenino , Estudios de Asociación Genética , Humanos , Linaje , Fenotipo , Fotofobia/etiología , Fotofobia/genéticaRESUMEN
PURPOSE: To identify the genetic basis of posterior polymorphous corneal dystrophy (PPCD) in families mapped to the PPCD1 locus and in affected individuals without ZEB1 coding region mutations. METHODS: The promoter, 5' UTR, and coding regions of OVOL2 was screened in the PPCD family in which linkage analysis established the PPCD1 locus and in 26 PPCD probands who did not harbor a ZEB1 mutation. Copy number variation (CNV) analysis in the PPCD1 and PPCD3 intervals was performed on DNA samples from eight probands using aCGH. Luciferase reporter assays were performed in human corneal endothelial cells to determine the impact of the identified potentially pathogenic variants on OVOL2 promoter activity. RESULTS: OVOL2 mutation analysis in the first PPCD1-linked family demonstrated segregation of the c.-307T>C variant with the affected phenotype. In the other 26 probands screened, one heterozygous coding region variant and five promoter region heterozygous variants were identified, though none are likely pathogenic based on allele frequency. Array CGH in the PPCD1 and PPCD3 loci excluded the presence of CNV involving either OVOL2 or ZEB1, respectively. The c.-307T>C variant demonstrated increased promoter activity in corneal endothelial cells when compared to the wild-type sequence as has been demonstrated previously in another cell type. CONCLUSIONS: Previously identified as the cause of PPCD1, the OVOL2 promoter variant c.-307T>C was herein identified in the original family that established the PPCD1 locus. However, the failure to identify presumed pathogenic coding or non-coding OVOL2 or ZEB1 variants, or CNV involving the PPCD1 and PPCD3 loci in 26 other PPCD probands suggests that other genetic loci may be involved in the pathogenesis of PPCD.
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Distrofias Hereditarias de la Córnea/genética , Mutación/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Regiones no Traducidas 5'/genética , Línea Celular , Segregación Cromosómica/genética , Cromosomas Humanos Par 20/genética , Variaciones en el Número de Copia de ADN/genética , Familia , Femenino , Sitios Genéticos , Humanos , Masculino , Linaje , Reproducibilidad de los Resultados , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
PURPOSE: To describe 2 unrelated families with multiple members demonstrating a less commonly recognized vortex pattern of corneal deposits confirmed to be granular corneal dystrophy type 1 (GCD1) after identification of the p.(Arg555Trp) mutation in the transforming growth factor ß-induced gene (TGFBI). METHODS: A slit-lamp examination was performed on individuals from 2 families, one of Mexican descent and a second of Italian descent. After DNA extraction from affected individuals and their unaffected relatives, TGFBI screening was performed. RESULTS: Eight of 20 individuals in the Mexican family and 20 of 55 in the Italian family demonstrated corneal stromal opacities. Seven of the 8 affected individuals in the Mexican family and 4 of the 20 affected individuals in the Italian family demonstrated a phenotype characterized by a "sea fan" or vortex pattern of superficial stromal corneal deposits originating from the inferior aspect of the cornea. Screening of TGFBI in both families revealed a heterozygous missense mutation [p.(Arg555Trp)] in exon 12, confirming the diagnosis of GCD1. CONCLUSIONS: Our findings demonstrate that GCD1 may present with a vortex pattern of anterior stromal deposits. Although this pattern of dystrophic deposits is not recognized by clinicians as a typical phenotype of GCD1, it is consistent with the production of the majority of the TGFBI protein by the corneal epithelium.