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1.
Mol Vis ; 25: 415-426, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31523119

RESUMEN

Purpose: Maintenance of a transparent corneal stroma is imperative for proper vision. The corneal stroma is composed of primarily collagen fibrils, small leucine-rich proteoglycans (SLRPs), as well as sparsely distributed cells called keratocytes. The lattice arrangement and spacing of the collagen fibrils that allows for transparency may be disrupted due to genetic mutations and injuries. The purpose of this study is to examine the therapeutic efficacy of human umbilical cord mesenchymal stem/stromal cells (UMSCs) in treating congenital and acquired corneal opacity associated with the loss of collagen V. Methods: Experimental mice, i.e., wild-type, Col5a1f/f and Kera-Cre/Col5a1f/f (Col5a1∆st/∆st , collagen V null in the corneal stroma) mice in a C57BL/6J genetic background, were subjected to a lamellar keratectomy, and treated with or without UMSC (104 cells/cornea) transplantation via an intrastromal injection or a fibrin plug. In vivo Heidelberg retinal tomograph (HRT II) confocal microscopy, second harmonic generated (SHG) confocal microscopy, histology, and immunofluorescence microscopy were used to assess the corneal transparency of the regenerated corneas. Results: Col5a1∆st/∆st mice display a cloudy cornea phenotype that is ameliorated following intrastromal transplantation of UMSCs. Loss of collagen V in Col5a1∆st/∆st corneas augments the formation of cornea scarring following the keratectomy. UMSC transplantation with a fibrin plug improves the healing of injured corneas and regeneration of transparent corneas, as determined with in vivo HRT II confocal microscopy. Second harmonic confocal microscopy revealed the improved collagen fibril lamellar architecture in the UMSC-transplanted cornea in comparison to the control keratectomized corneas. Conclusions: UMSC transplantation was successful in recovering some corneal transparency in injured corneas of wild-type, Col5a1f/f and Col5a1∆st/∆st mice. The production of collagen V by transplanted UMSCs may account for the regeneration of corneal transparency, as exemplified by better collagen fiber organization, as revealed with SHG signals.


Asunto(s)
Opacidad de la Córnea/congénito , Opacidad de la Córnea/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Animales , Colágeno Tipo V/metabolismo , Opacidad de la Córnea/patología , Sustancia Propia/patología , Colágenos Fibrilares/metabolismo , Humanos , Ratones Endogámicos C57BL , Resultado del Tratamiento , Cordón Umbilical/citología
2.
PLoS One ; 14(3): e0212569, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30840655

RESUMEN

PURPOSE: Pseudoexfoliation (PEX) syndrome is an age-related systemic disease with ocular manifestations. The development of animal models is critical in order to elucidate the cause of the disease and to test potential treatment regimens. The purpose of this study is to report phenotypes found in mouse eyes injected with Adenovirus coding Wnt5a. Some of the phenotypes resemble those found in PEX patients while others are different. METHODS: Recombinant Adenovirus coding Wnt5a or green fluorescent protein (GFP) were injected into mouse eyes. Two months after the injection, eyes were examined for PEX phenotypes using slit lamp, fluorescence stereomicroscope, histological staining, immunostaining and transmission electron microscope. RESULT: Certain ocular features of PEX syndrome were found in mouse eyes injected with recombinant Adenovirus coding Wnt5a. These features include accumulation of exfoliation-like extracellular material on surfaces of anterior segment structures and its dispersion in the anterior chamber, saw-tooth appearance and disrupted basement membrane of the posterior iris pigment epithelium, iris stromal atrophy and disorganized ciliary zonules. Ultrastructure analysis of the exfoliation material revealed that the microfibril structure found in this model was different from those of PEX patients. CONCLUSION: These features, resembling signs of ocular PEX syndrome in patients, suggest that new information obtained from this study will be helpful for developing better mouse models for PEX syndrome.


Asunto(s)
Síndrome de Exfoliación , Cristalino , Epitelio Pigmentado de la Retina , Proteína Wnt-5a , Animales , Modelos Animales de Enfermedad , Síndrome de Exfoliación/genética , Síndrome de Exfoliación/metabolismo , Síndrome de Exfoliación/patología , Femenino , Humanos , Cristalino/metabolismo , Cristalino/ultraestructura , Masculino , Ratones , Ratones Transgénicos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/ultraestructura , Proteína Wnt-5a/biosíntesis , Proteína Wnt-5a/genética
3.
Bio Protoc ; 8(10)2018 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-29951567

RESUMEN

Stem cells are widely used for numerous clinical applications including limbal stem cell deficiency. Stem cell derived from the bulge region of the hair follicle have the ability to differentiate into a variety of cell types including interfollicular epidermis, hair follicle structures, sebaceous glands and corneal epithelial cells when provided the appropriate cues. Hair follicle stem cells are being studied as a valuable source of autologous stem cells to treat disease. The protocol described below details the isolation and expansion of these cells for eventual clinical application. We used a dual-reporter mouse model to visualize both isolation and eventual differentiation of these cells in a limbal stem cell-deficient mouse model.

4.
Bio Protoc ; 8(10): e2849, 2018 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-34285966

RESUMEN

The goal of this protocol is to establish a procedure for cultivating stem cells on a fibrin carrier to allow for eventual transplantation to the eye. The ability to transfer stem cells to a patient is critical for treatment for a variety of disorders and wound repair. We took hair follicle stem cells from the vibrissae of transgenic mice expressing a dual reporter gene under the control of the Tet-on system and the keratin 12 promoter (Meyer- Blazejewska et al., 2011 ). A clonal growth assay was performed to enrich for stem cells. Once holoclones formed they were transferred onto a fibrin carrier and cultivated to obtain a confluent epithelial cell layer. Limbal stem cell deficient (LSCD) mice were used as the transplant recipient in order to test for successful grafting and eventual differentiation into a corneal epithelial phenotype.

5.
Invest Ophthalmol Vis Sci ; 58(11): 4800-4808, 2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-28973326

RESUMEN

Purpose: We created a novel inducible mouse line Keratocan-rtTA (KeraRT) that allows specific genetic modification in corneal keratocytes and tenocytes during development and in adults. Methods: A gene-targeting vector (Kera- IRES2-rtTA3) was constructed and inserted right after the termination codon of the mouse Kera allele via gene targeting techniques. The resulting KeraRT mouse was crossed to tet-O-Hist1H2B-EGFP (TH2B-EGFP) to obtain KeraRT/TH2B-EGFP compound transgenic mice, in which cells expressing Kera are labeled with green fluorescence protein (GFP) by doxycycline (Dox) induction. The expression patterns of GFP and endogenous Kera were examined in KeraRT/TH2B-EGFP. Moreover, KeraRT was bred with tet-O-TGF-α to generate a double transgenic mouse, KeraRT/tet-O-TGF-α, to overexpress TGF-α in corneal keratocytes upon Dox induction. Results: Strong GFP-labeled cells were detected in corneal stroma, limbs, and tail when KeraRT/TH2B-EGFP mice were fed Dox chow. There was no GFP in any single transgenic KeraRT or TH2B-EGFP mouse. Histological analysis showed that GFP in the cornea was limited to stromal keratocytes of KeraRT/TH2B-EGFP, which is consistent with Kera expression. Induction of GFP occurred in 24 hours and reached a plateau by 7 days after Dox induction. GFP could be detected 3-months after induction of KeraRT/TH2B-EGFP. Ectopic expression of TGF-α in corneal keratocytes caused hyperplasia in the corneal epithelium and stroma. Conclusions: The novel Dox inducible KeraRT driver mouse line is a useful genetic tool for gene manipulation and elucidating gene functions in corneal stroma and tendons of limbs and tail during embryonic development, homeostasis and pathogenesis.


Asunto(s)
Sustancia Propia/metabolismo , Técnicas de Sustitución del Gen , Ratones Transgénicos/genética , Proteoglicanos/genética , Tendones/metabolismo , Animales , Queratocitos de la Córnea/metabolismo , Modelos Animales de Enfermedad , Epitelio Corneal/metabolismo , Técnicas de Inactivación de Genes , Vectores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Proteoglicanos/metabolismo , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo
6.
Exp Eye Res ; 163: 58-63, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28950938

RESUMEN

The epidermal growth factor receptor (EGFR) signaling has a pivotal role in the regulation of morphogenesis during development and maintenance of homeostasis in adult eyelid and its adnexa. Studies have demonstrated that during eyelid morphogenesis the EGFR signaling pathway is responsible for keratinocyte and mesenchymal cell proliferation and migration at the eyelid tip. For meibomian gland morphogenesis, EGFR signaling activation stimulates meibomian gland epithelial cell proliferation. EGFR signaling pathway functions through multiple downstream signals such as ERK, Rho/ROCK and integrin and is regulated by a variety of upstream signals including Adam17, GPR48 and FGFR signaling. Herein we review the literature that describe the role of EGFR and its related signaling pathways in eyelid and meibomian gland morphogenesis.


Asunto(s)
Receptores ErbB/fisiología , Párpados/embriología , Glándulas Tarsales/embriología , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Células Epiteliales/fisiología , Párpados/fisiología , Humanos , Glándulas Tarsales/fisiología , Transducción de Señal/fisiología
7.
Ocul Surf ; 15(4): 704-712, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28284825

RESUMEN

BACKGROUND AND PURPOSE: Meibomian glands (MGs) play an important role in the maintenance of ocular surface health, but the mechanisms of their development are still poorly understood. The MGs arise from the epithelium at the junction of eyelid fusion, raising the possibility that defective eyelid fusion disturbs the formation of MGs. METHODS: We examined, histologically and functionally, the development of MGs in mice with either normal or defective eyelid fusion, displaying eye-closed at birth (ECB) or eye-open at birth (EOB) phenotypes, respectively. RESULTS: The Meibomian anlage was detected in the epithelium at the eyelid fusion junction immediately after birth at postnatal day 0 (PD0), and it extended into the eyelid stroma at PD1 and started to branch and produce meibum at PD7 in the ECB mice. In contrast, few if any MG structures were detectable in the EOB mice in the early postnatal periods. The Meibomian gland ductile system was seen aligned along the eyelid margin in the adult ECB mice, but was absent or scarce in that of the EOB mice. While MG abnormalities were found in all EOB mice, the severity varied and corresponded to the position and the size of eye opening but not the genetic defects of the mice. CONCLUSION: Proper Meibomian gland formation and development require eyelid closure and fusion.


Asunto(s)
Glándulas Tarsales , Animales , Enfermedades de los Párpados , Ratones , Morfogénesis
8.
Mol Vis ; 22: 168-76, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26957900

RESUMEN

PURPOSE: To identify the lineage that contributes to the morphogenesis of the meibomian gland. METHODS: To examine which cell lineage gives rise to the meibomian gland, the expression of Pax6 as well as that of various cytokeratin markers, including keratin 14 (Krt14), Krt15, Krt4, and Krt10, was examined with immunofluorescent staining of C57BL/6J mouse eyelids from P2 to P11 pups and adult mice. RESULTS: Pax6 was localized to the cytoplasm within the acinar region of the meibomian glands during morphogenesis but was absent in the fully developed gland. Keratin 14 was expressed throughout the gland at all stages whereas keratin 15 was absent at all stages. Keratin 4, a marker of mucosal lineage, was present throughout the gland and was colocalized with keratin 10 (epidermal lineage marker) in the developing duct at P4. This colocalization region decreased as the gland developed becoming restricted to the central duct near the opening to the acini in the fully developed gland. CONCLUSIONS: We identified a unique cell lineage that expresses markers characteristic of mucosal and epidermal epithelia during meibomian gland morphogenesis. This unique group of cells was located in the central duct with a concentration near the ductule orifice. The expression of these cells reduced during meibomian gland morphogenesis and may play a role in the development and homeostasis of the gland.


Asunto(s)
Linaje de la Célula/fisiología , Párpados/crecimiento & desarrollo , Glándulas Tarsales/crecimiento & desarrollo , Morfogénesis/fisiología , Animales , Biomarcadores/metabolismo , Proteínas del Ojo/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas de Homeodominio/metabolismo , Queratina-10/metabolismo , Queratina-4/metabolismo , Glándulas Tarsales/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo
9.
Development ; 142(19): 3383-93, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-26443636

RESUMEN

The development of organs with an epithelial parenchyma relies on reciprocal mesenchymal-epithelial communication. Mouse corneal epithelium stratification is the consequence of a coordinated developmental process based on mesenchymal-epithelial interactions. The molecular mechanism underlying these interactions remains unclear. The Wnt/ß-catenin signaling pathway is involved in fundamental aspects of development through the regulation of various growth factors. Here, we show that conditional ablation of either ß-catenin (Ctnnb1(cKO)) or co-receptors Lrp5/6 (Lrp5/6(cKO)) in corneal stromal cells results in precocious stratification of the corneal epithelium. By contrast, ectopic expression of a murine Ctnnb1 gain-of-function mutant (Ctnnb1(cGOF)) retards corneal epithelium stratification. We also discovered that Bmp4 is upregulated in the absence of ß-catenin in keratocytes, which further triggers ERK1/2 (Mapk3/1) and Smad1/5 phosphorylation and enhances transcription factor p63 (Trp63) expression in mouse corneal basal epithelial cells and in a human corneal epithelial cell line (HTCE). Interestingly, mouse neonates given a subconjunctival BMP4 injection displayed a phenotype resembling that of Ctnnb1(cKO). Conditional ablation of Bmp4 eradicates the phenotype produced in Ctnnb1(cKO) mice. Furthermore, ChIP and promoter-luciferase assays show that ß-catenin binds to and suppresses Bmp4 promoter activity. These data support the concept that cross-talk between the Wnt/ß-catenin/Bmp4 axis (in the stromal mesenchyme) and Bmp4/p63 signaling (in the epithelium) plays a pivotal role in epithelial stratification during corneal morphogenesis.


Asunto(s)
Proteína Morfogenética Ósea 4/antagonistas & inhibidores , Epitelio Corneal/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Morfogénesis/fisiología , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Animales , Western Blotting , Proteína Morfogenética Ósea 4/administración & dosificación , Inmunoprecipitación de Cromatina , Doxiciclina , Fluorescencia , Galactósidos , Técnicas Histológicas , Inmunohistoquímica , Indoles , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/deficiencia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/deficiencia , Luciferasas , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Fosfoproteínas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transactivadores/metabolismo
10.
Dev Biol ; 406(2): 147-57, 2015 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-26363126

RESUMEN

Transforming growth factor alpha (TGFα) belongs to the epidermal growth factor (EGF) family and is known to play an important role during eyelid morphogenesis. In this study, we showed that ectopic expression of TGFα in the stroma of Kera-rtTA/tet-O-TGFα bitransgenic mice results in precocious eye opening, abnormal morphogenesis of the meibomian gland, tendon and tarsal plate malformation and epithelium hyperplasia. TGFα did not change proliferation and differentiation of meibocytes, but promoted proliferation and inhibited differentiation of the tarsal plate tenocytes. These results suggest that proper formation of the tendon and tarsal plate in the mouse eyelid is required for normal morphogenesis of the meibomian gland.


Asunto(s)
Párpados/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Glándulas Tarsales/anomalías , Morfogénesis/fisiología , Factor de Crecimiento Transformador alfa/metabolismo , Animales , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Cartilla de ADN/genética , Regulación del Desarrollo de la Expresión Génica/genética , Técnicas Histológicas , Inmunohistoquímica , Glándulas Tarsales/embriología , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Tendones/citología , Tendones/embriología , Factor de Crecimiento Transformador alfa/genética
11.
Nature ; 511(7509): 353-7, 2014 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-25030174

RESUMEN

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs), and LSC deficiency is a major cause of blindness worldwide. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts, a gene allowing for prospective LSC enrichment has not been identified so far. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5) marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs in mice and p63α-positive LSCs in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Limbo de la Córnea/citología , Limbo de la Córnea/fisiología , Regeneración , Células Madre/metabolismo , Cicatrización de Heridas , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/deficiencia , Animales , Apoptosis , Biomarcadores/metabolismo , Diferenciación Celular , Proliferación Celular , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Trasplante de Células Madre , Células Madre/citología , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo
12.
PLoS One ; 8(12): e82730, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24367547

RESUMEN

Lumican (Lum), a small leucine-rich proteoglycan (SLRP) family member, has multiple matricellular functions both as an extracellular matrix component and as a matrikine regulating cell proliferation, gene expression and wound healing. To date, no cell surface receptor has been identified to mediate the matrikine functions of Lum. This study aimed to identify a perspective receptor that mediates Lum effects on promoting wound healing. Transforming growth factor-ß receptor 1 (ALK5) was identified as a potential Lum-interacting protein through in silico molecular docking and molecular dynamics. This finding was verified by biochemical pull-down assays. Moreover, the Lum function on wound healing was abrogated by an ALK5-specific chemical inhibitor as well as by ALK5 shRNAi. Finally, we demonstrated that eukaryote-specific post-translational modifications are not required for the wound healing activity of Lum, as recombinant GST-Lum fusion proteins purified from E. coli and a chemically synthesized LumC13 peptide (the last C-terminal 13 amino acids of Lum) have similar effects on wound healing in vitro and in vivo.


Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Sulfato de Queratano/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Adulto , Anciano , Animales , Western Blotting , Línea Celular , Células Cultivadas , Proteoglicanos Tipo Condroitín Sulfato/genética , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Sulfato de Queratano/genética , Lumican , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Simulación de Dinámica Molecular , Proteínas Serina-Treonina Quinasas/genética , Proteoglicanos/genética , Proteoglicanos/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiología
13.
Invest Ophthalmol Vis Sci ; 54(10): 6502-9, 2013 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-23963164

RESUMEN

PURPOSE: Dexamethasone (DEX) regulates aqueous humor outflow by inducing a reorganization of the cytoskeleton to form cross-linked actin networks (CLANs) in trabecular meshwork (TM) cells. Rho-associated protein kinase (ROCK) has been demonstrated to have an important role in this process, but the upstream components leading to its activation remain elusive. The purpose of the study is to demonstrate that noncanonical Wnt signaling mediates the DEX-induced CLAN formation in TM cells. METHODS: The TM cells were treated with 100 nM DEX in low serum medium for over 7 days. The medium was changed every 3 days. The cells were harvested and subjected to molecular analysis for the expression of Wnt ligands. Stress fiber structures were revealed by Phalloidin staining. Lentivirus-based shRNA against noncanonical Wnt receptor (Ror2) was used to determine the role of noncanonical Wnt signaling in DEX-induced CLAN formation. RESULTS: The DEX induced stress fiber rearrangement in TM cells. A noncanonical Wnt ligand (Wnt5a) was upregulated by DEX as demonstrated by Wnt ligand degenerate PCR, real-time quantitative PCR (qRT-PCR), and Western blotting. Knocking-down Ror2, the receptor of noncanonical Wnt signaling, abolished the effects of DEX on the TM cells. CONCLUSIONS: Our data suggest that DEX induces the upregulation of noncanonical Wnt ligand Wnt5a. Recombinant WNT5a protein induces CLAN formation through the noncanonical Wnt receptor ROR2/RhoA/ROCK signaling axis. Given the similarities between DEX-induced ocular hypertension and primary open-angle glaucoma, our results provide a mechanism of action for applying ROCK inhibitor to treat primary open-angle glaucoma.


Asunto(s)
Actinas/metabolismo , ADN/genética , Dexametasona/farmacología , Glaucoma de Ángulo Abierto/genética , Proteínas Proto-Oncogénicas/genética , Malla Trabecular/metabolismo , Regulación hacia Arriba , Proteínas Wnt/genética , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patología , Células Cultivadas , Glaucoma de Ángulo Abierto/tratamiento farmacológico , Glaucoma de Ángulo Abierto/metabolismo , Glucocorticoides/farmacología , Humanos , Proteínas Proto-Oncogénicas/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/biosíntesis , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Malla Trabecular/citología , Malla Trabecular/efectos de los fármacos , Proteínas Wnt/biosíntesis , Proteína Wnt-5a
14.
Invest Ophthalmol Vis Sci ; 52(11): 8208-15, 2011 Oct 17.
Artículo en Inglés | MEDLINE | ID: mdl-21917935

RESUMEN

PURPOSE: The aim of this study was to elucidate the mechanisms governing epithelial cell migration and proliferation during wound healing. METHODS: The authors used wound healing of mouse corneal epithelium to examine the role TGF-ß signaling plays during the healing process. To achieve this goal, they used transgenic mice in which the TGF-ß receptor type II (Tbr2) was conditionally ablated from the corneal epithelium. Epithelium debridement wounds were made, followed by the assessment of cell migration, proliferation, and immunostaining of various signaling pathway components. RESULTS: The authors showed that in the absence of TGF-ß signaling corneal epithelial wound healing is delayed by 48 hours; this corresponds to a delay in p38MAPK activation. Despite the delayed p38MAPK activation, ATF2, a substrate of p38MAPK, is still phosphorylated, leading to the suppression of cell proliferation at the leading edge of the wound. These data provide evidence that in the absence of TGF-ß signaling, the suppression of cell proliferation during the early stages of wound healing is maintained through the JNK activation of ATF2. CONCLUSIONS; Together the data presented here demonstrate the importance of the TGF-ß and MAPK signaling pathways in corneal epithelial wound healing.


Asunto(s)
Comunicación Celular/fisiología , Epitelio Corneal/fisiología , Lesiones Oculares/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Cicatrización de Heridas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Factor de Transcripción Activador 2/metabolismo , Animales , Movimiento Celular , Proliferación Celular , Desbridamiento , Epitelio Corneal/lesiones , Lesiones Oculares/patología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Masculino , Ratones , Ratones Transgénicos , Fosforilación , Heridas no Penetrantes/metabolismo , Heridas no Penetrantes/patología
15.
Graefes Arch Clin Exp Ophthalmol ; 249(12): 1795-803, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21847577

RESUMEN

PURPOSES: To determine the relationship between immunohistochemical reactivity to osteopontin, vimentin, keratin 8/18, LZTS1, and beta-catenin and clinical and histopathological prognostic factors for metastasis and death in archival specimens of primary uveal melanomas, and the prognostic value of the evaluated study variables for death from metastasis. METHODS: Retrospective analysis of clinical records and formalin-fixed, paraffin-embedded slides of primary uveal melanomas treated by enucleation during May 1 1999, through June 30 2009. Immunofluorescent staining of each tumor was assessed on newly prepared histologic slides after the application of antibodies directed against five biomarkers associated with unfavorable prognosis in uveal melanoma. RESULTS: After exclusions, our study group consisted of 82 cases. Immunofluorescence was observed in 40.2% of specimens evaluated for keratin, 50.0% evaluated for osteopontin, 26.8% evaluated for ß-catenin, 65.9% evaluated for vimentin, and 70.7% evaluated for LZTS1. Through available follow-up, 27 patients (32.9%) were dead of confirmed or suspected metastatic uveal melanoma. None of the patients whose tumor exhibited strong immunoreactivity to ß-catenin died of metastasis. In contrast, patients whose tumor exhibited immunoreactivity of any intensity to LZTS1 were more likely to develop metastasis. In multivariate Cox proportional hazards modeling, a composite variable that took into account the immunostaining for both ß-catenin and LZTS1 had a statistically significant relationship with patient's survival time. CONCLUSIONS: Our study suggests that conventional clinical and histopathological prognostic factors, and immunoexpression of ß-catenin and LZTS1 combined may allow better prognostication of metastasis than clinical and histomorphological factors alone.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Coroides/mortalidad , Neoplasias de la Coroides/patología , Cuerpo Ciliar/patología , Melanoma/mortalidad , Melanoma/secundario , Proteínas de Neoplasias/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Coroides/metabolismo , Cuerpo Ciliar/metabolismo , Proteínas de Unión al ADN/metabolismo , Enucleación del Ojo , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Queratina-18/metabolismo , Queratina-8/metabolismo , Masculino , Melanoma/metabolismo , Persona de Mediana Edad , Osteopontina/metabolismo , Pronóstico , Estudios Retrospectivos , Proteínas Supresoras de Tumor/metabolismo , Vimentina/metabolismo , beta Catenina/metabolismo
16.
Stem Cells ; 29(1): 57-66, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20957740

RESUMEN

Limbal stem cell deficiency (LSCD) leads to severe ocular surface abnormalities that can result in the loss of vision. The most successful therapy currently being used is transplantation of limbal epithelial cell sheets cultivated from a limbal biopsy obtained from the patient's healthy, contralateral eye or cadaveric tissue. In this study, we investigated the therapeutic potential of murine vibrissae hair follicle bulge-derived stem cells (HFSCs) as an autologous stem cell (SC) source for ocular surface reconstruction in patients bilaterally affected by LSCD. This study is an expansion of our previously published work showing transdifferentiation of HFSCs into cells of a corneal epithelial phenotype in an in vitro system. In this study, we used a transgenic mouse model, K12(rtTA/rtTA) /tetO-cre/ROSA(mTmG) , which allows for HFSCs to change color, from red to green, once differentiation to corneal epithelial cells occurs and Krt12, the corneal epithelial-specific differentiation marker, is expressed. HFSCs were isolated from transgenic mice, amplified by clonal expansion on a 3T3 feeder layer, and transplanted on a fibrin carrier to the eye of LSCD wild-type mice (n = 31). The HFSC transplant was able to reconstruct the ocular surface in 80% of the transplanted animals; differentiating into cells with a corneal epithelial phenotype, expressing Krt12, and repopulating the corneal SC pool while suppressing vascularization and conjunctival ingrowth. These data highlight the therapeutic properties of using HFSC to treat LSCD in a mouse model while demonstrating a strong translational potential and points to the niche as a key factor for determining stem cell differentiation.


Asunto(s)
Enfermedades de la Córnea/cirugía , Epitelio Corneal/citología , Anomalías del Ojo/cirugía , Folículo Piloso/trasplante , Limbo de la Córnea/anomalías , Limbo de la Córnea/cirugía , Trasplante de Células Madre , Células Madre/fisiología , Animales , Transdiferenciación Celular , Células Cultivadas , Folículo Piloso/citología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vibrisas/citología , Vibrisas/trasplante
17.
J Cell Sci ; 123(Pt 17): 2987-95, 2010 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-20699360

RESUMEN

An important aspect of wound healing is the recruitment of neutrophils to the site of infection or tissue injury. Lumican, an extracellular matrix component belonging to the small leucine rich proteoglycan (SLRP) family, is one of the major keratan sulfate proteoglycans (KSPGs) within the corneal stroma. Increasing evidence indicates that lumican can serve as a regulatory molecule for several cellular processes, including cell proliferation and migration. In the present study, we addressed the role of lumican in the process of extravasation of polymorphonuclear leukocytes (PMNs) during the early inflammatory phase present in the healing of the corneal epithelium following debridement. We used Lum(-/-) mice and a novel transgenic mouse, Lum(-/-),Kera-Lum, which expresses lumican only in the corneal stroma, to assess the role of lumican in PMN extravasation into injured corneas. Our results showed that PMNs did not readily invade injured corneas of Lum(-/-) mice and this defect was rescued by the expression of lumican in the corneas of Lum(-/-),Kera-Lum mice. The presence of lumican in situ facilitates PMN infiltration into the peritoneal cavity in casein-induced inflammation. Our findings are consistent with the notion that in addition to regulating the collagen fibril architecture, lumican acts to aid neutrophil recruitment and invasion following corneal damage and inflammation.


Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/inmunología , Enfermedades de la Córnea/inmunología , Lesiones de la Cornea , Lesiones Oculares/inmunología , Sulfato de Queratano/inmunología , Neutrófilos/inmunología , Animales , Córnea/inmunología , Córnea/metabolismo , Córnea/patología , Enfermedades de la Córnea/metabolismo , Enfermedades de la Córnea/patología , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Lesiones Oculares/metabolismo , Lesiones Oculares/patología , Citometría de Flujo , Humanos , Inmunohistoquímica , Lumican , Ratones , Lavado Peritoneal , Cicatrización de Heridas/inmunología
18.
Invest Ophthalmol Vis Sci ; 51(9): 4562-8, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20393120

RESUMEN

PURPOSE: The purpose of this study was to characterize a Krt12-Cre knock-in mouse line for corneal epithelium-specific gene ablation and to analyze the allelic selection of the keratin 12 (Krt12) gene during corneal type-epithelium differentiation. METHODS: Knock-in mice were generated by gene targeting. The authors examined the expression patterns of several reporter genes in the corneas of bitransgenic Krt12cre/+/ROSA(EGFP), Krt12Cre/+/ZEG, and Krt12Cre/+/ZAP mouse lines. Krt12 and cre recombinase (Cre) immunostaining was performed. Corneal epithelial cells from bitransgenic Krt12Cre/+/ROSA(EGFP) mice were examined by fluorescence-activated cell sorter. RESULTS: Mosaic and spiral expression patterns of EGFP were observed in young and adult bitransgenic Krt12Cre/+/ZEG mice, respectively. Immunostaining revealed that Cre- cells were also Krt12 negative in the corneal epithelia of Krt12Cre/-/ZAP mice. Using FACS analysis, 60% to 70% of the corneal epithelial cells from Krt12Cre/+/ROSAEGFP mice were EGFP positive, whereas 20% to 30% were negative. RT-PCR revealed that EGFP+ cells express both Krt12Cre and Krt12+ alleles, whereas EGFP- cells express only Krt12+. In the Krt12Cre/- cornea, the number of epithelial cells expressing Cre is the same as that found in Krt12Cre/Cre, which can be explained by the fragility of corneal epithelial cells that did not produce Krt12 because the Krt12Cre allele was not transcribed. CONCLUSIONS: These observations are consistent with the notion that clonal limbal stem cells randomly activate Krt12 alleles in the process of terminal differentiation. The authors suggest that this selection is advantageous for retaining epithelial cells expressing the Krt12+ allele and that it allows tolerance to structural mutations of Krt12.


Asunto(s)
Células Epiteliales/citología , Queratina-12/genética , Limbo de la Córnea/citología , Limbo de la Córnea/fisiología , Células Madre/citología , Alelos , Animales , Diferenciación Celular/fisiología , Células Epiteliales/fisiología , Epitelio Corneal/citología , Epitelio Corneal/fisiología , Expresión Génica/fisiología , Técnicas de Sustitución del Gen , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Operón Lac , Ratones , Ratones Transgénicos , Mosaicismo , Células Madre/fisiología
19.
J Cell Sci ; 123(Pt 8): 1285-94, 2010 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-20332116

RESUMEN

Beta-catenin signaling has been shown to play a fundamental role in embryonic development and tumorigenesis. In this study, we investigated the role of beta-catenin (Ctnnb1) in corneal homeostasis and tumorigenesis. Conditional expression of a murine Ctnnb1 gain-of-function mutation alone caused corneal neoplasia and neovascularization, resembling human ocular surface squamous neoplasia (OSSN). These corneas displayed an upregulation of cell proliferative markers (PCNA and p63), while presenting downregulation of both the Pax-6 transcription factor and the corneal differentiation marker cytokeratin 12. In addition, the expression of limbal-type keratin 15 ectopically extended to cornea, but the pattern of conjunctival keratin 4 and epidermal keratin 10 were unchanged. Moreover, epithelial E-cadherin and laminins decreased concomitantly with elevated levels of MMP-7. We also noticed a dramatic upregulation of pro-angiogenic factors (Vegf-A, Vegfr1) and angiopoietins in these corneas. Interestingly, all human OSSN specimens examined revealed nuclear beta-catenin immunoreactivity. Taken together, these results argue that beta-catenin activation is a crucial step during OSSN pathogenesis. Thus, inhibition of beta-catenin might be beneficial for treating this disease.


Asunto(s)
Transformación Celular Neoplásica/patología , Córnea/metabolismo , Córnea/patología , Proteínas Mutantes/metabolismo , beta Catenina/metabolismo , Envejecimiento/patología , Animales , Membrana Basal/enzimología , Membrana Basal/patología , Cadherinas/metabolismo , Diferenciación Celular , Membrana Celular/metabolismo , Membrana Celular/patología , Núcleo Celular/metabolismo , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Regulación hacia Abajo , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Neoplasias del Ojo/metabolismo , Neoplasias del Ojo/patología , Humanos , Hiperplasia , Metaloproteinasa 7 de la Matriz/metabolismo , Ratones , Modelos Biológicos , Neoplasias de Células Escamosas/metabolismo , Neoplasias de Células Escamosas/patología , Transporte de Proteínas , Regulación hacia Arriba
20.
Biochem Biophys Res Commun ; 362(4): 940-5, 2007 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-17765873

RESUMEN

MicroRNAs are known to regulate the expression of many mRNAs by binding to complementary target sequences at the 3'UTRs. Because of such properties, miRNAs may regulate tissue-specific mRNAs as a cell undergoes transdifferentiation during regeneration. We have tested this hypothesis during lens and hair cell regeneration in newts using microarray analysis. We found that distinct sets of miRNAs are associated with lens and hair cell regeneration. Members of the let-7 family are expressed in both events and they are regulated in a similar fashion. All the let-7 members are down regulated during the initiation of regeneration, which is characterized by dedifferentiation of terminally differentiated cells. This is the first report to correlate expression of miRNAs as novel regulators of vertebrate regeneration, alluding to a novel mechanism whereby transdifferentiation occurs.


Asunto(s)
Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Internas/metabolismo , Cristalino/citología , Cristalino/metabolismo , Proteoma/metabolismo , Regeneración/fisiología , Salamandridae/metabolismo , Animales , Diferenciación Celular/fisiología , Regulación de la Expresión Génica/fisiología , Cristalino/crecimiento & desarrollo , MicroARNs/genética , Morfogénesis/fisiología , Salamandridae/anatomía & histología , Salamandridae/crecimiento & desarrollo
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