RESUMO
Corneal nerve fibers serving sensory, reflex, and neurotrophic functions sustain corneal homeostasis and transparency to promote normal visual function. It is not known whether corneal epithelium is also important for the corneal innervation. Herein, we generated a compound transgenic mouse strain, K14rtTA;tetO-Cre (TC);Shp2flox/flox, in which Shp2 was conditionally knocked out from K14-positive cells including corneal epithelium (Shp2K14ce-cko) upon doxycycline (dox) administration. Our data reveal that Shp2K14ce-cko caused corneal denervation. More specifically, corneal epithelium thickness and corneal sensitivity reduced dramatically in Shp2K14ce-cko mice. In addition, corneal epithelial wound healing after debridement was delayed substantially in the mutant mice. These defects manifested in Shp2K14ce-cko mice resemble the symptoms of human neurotrophic keratopathy. Our in vitro study shows that neurite outgrowth of the mouse primary trigeminal ganglion cells (TGCs) was inhibited when as cocultured with mouse corneal epithelial cells (TKE2) transfected by Shp2-, Mek1/2-, or ∆Np63-targeted siRNA but not by Akt1/2-targeted siRNA. Furthermore, ∆Np63 RNA interference downregulated Ngf expression in TKE2 cells. Cotransfection experiments reveal that Shp2 tightly monitored ΔNp63 protein levels in HEK293 and TKE2 cells. Taken together, our data suggest that the Shp2-mediated MAPK pathway regulated ΔNp63, which in turn positively regulated Ngf in epithelium to promote corneal innervation and epithelial homeostasis.
Assuntos
Córnea , Sistema de Sinalização das MAP Quinases , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Cicatrização , Animais , Córnea/inervação , Córnea/metabolismo , Córnea/fisiologia , Lesões da Córnea/metabolismo , Epitélio Corneano/metabolismo , Homeostase/genética , Homeostase/fisiologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Transgênicos , Neuritos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
In order to understand the pathobiology of neurotrophic keratopathy, we established a mouse model by coagulating the first branch of the trigeminal nerve (V1 nerve). In our model, the sensory nerve in the central cornea disappeared and remaining fibers were sparse in the peripheral limbal region. Impaired corneal epithelial healing in the mouse model was associated with suppression of both cell proliferation and expression of stem cell markers in peripheral/limbal epithelium as well as a reduction of transient receptor potential vanilloid 4 (TRPV4) expression in tissue. TRPV4 gene knockout also suppressed epithelial repair in mouse cornea, although it did not seem to directly modulate migration of epithelium. In a co-culture experiment, TRPV4-introduced KO trigeminal ganglion upregulated nerve growth factor (NGF) in cultured corneal epithelial cells, but ganglion with a control vector did not. TRPV4 gene introduction into a damaged V1 nerve rescues the impairment of epithelial healing in association with partial recovery of the stem/progenitor cell markers and upregulation of cell proliferation and of NGF expression in the peripheral/limbal epithelium. Gene transfer of TRPV4 did not accelerate the regeneration of nerve fibers. Sensory nerve TRPV4 is critical to maintain stemness of peripheral/limbal basal cells, and is one of the major mechanisms of homeostasis maintenance of corneal epithelium.
Assuntos
Epitélio Corneano , Células-Tronco , Canais de Cátion TRPV/metabolismo , Nervo Trigêmeo/metabolismo , Cicatrização/fisiologia , Animais , Células Cultivadas , Epitélio Corneano/citologia , Epitélio Corneano/lesões , Epitélio Corneano/metabolismo , Técnicas de Inativação de Genes , Camundongos , Células-Tronco/citologia , Células-Tronco/metabolismo , Canais de Cátion TRPV/genética , Nervo Trigêmeo/químicaRESUMO
Corneal neovascularization and inflammatory fibrosis induced by severe injury or infection leads to tissue opacification and even blindness. Transient receptor potential (TRP) channel subtypes contribute to mediating these maladaptive responses through their interactions with other receptors. TRPV1 is one of the contributing channel isoforms inducing neovascularization in an alkali burn mouse wound healing model. VEGF-A upregulation contributes to neovascularization through interaction with its cognate receptors (VEGFR). Since the TRP isoform in this tissue, TRPA1, is also involved, we determined here if one of the pathways mediating neovascularization and immune cell infiltration involve an interaction between VEGFR and TRPA1 in a cauterization corneal mouse wound healing model. Localization of TRPA1 and endothelial cell (EC) CD31 immunostaining pattern intensity determined if TRPA1 expression was EC delimited during cauterization induced angiogenesis. Quantitative RT-PCR evaluated the effects of the absence of TRPA1 function on VEGF-A and TGF-ß1 mRNA expression during this process. Macrophage infiltration increased based on rises in F4/80 antigen immunoreactivity. TRPA1 immunostaining was absent on CD31-immunostained EC cells undergoing neovascularization, but it was present on other cell type(s) adhering to EC in vivo. Absence of TRPA1 expression suppressed both stromal neovascularization and inhibited macrophage infiltration. Similarly, the increases occurring in both VEGF-A and TGF-ß1 mRNA expression levels in WT tissue were blunted in the TRPA1-/- counterpart. On the other hand, in the macrophages their levels were invariant and their infiltration was inhibited. To determine if promotion by TRPA1 of angiogenesis was dependent on its expression on other unidentified cell types, the effects were compared of pharmacological manipulation of TRPA1 activity on EC proliferation tube formation and migration. In the presence and absence of a fibroblast containing feeder layer. Neither VEGF-induced increases in human vascular endothelial cell (HUVEC) proliferation nor migration were changed by a TRPA1 antagonist HC-030031 in the absence of a feeder layer. However, on a fibroblast feeder layer this antagonist suppressed HUVEC tube formation. In conclusion, during corneal wound healing transactivation by VEGFR of TRPA1 contributes to mediating neovascularization and macrophage infiltration. Such crosstalk is possible because of close proximity between VEGFR delimited expression on EC and TRPA1 expression restricted to cell types adhering to EC.
Assuntos
Neovascularização da Córnea/fisiopatologia , Substância Própria/patologia , Canal de Cátion TRPA1/deficiência , Animais , Neovascularização da Córnea/metabolismo , Substância Própria/metabolismo , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Canal de Cátion TRPA1/antagonistas & inibidores , Canal de Cátion TRPA1/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/fisiologiaRESUMO
Ectodysplasin A (Eda), a member of the tumour necrosis factor superfamily, plays an important role in ectodermal organ development. An EDA mutation underlies the most common of ectodermal dysplasias, that is X-linked hypohidrotic ectodermal dysplasia (XLHED) in humans. Even though it lacks a developmental function, the role of Eda during the postnatal stage remains elusive. In this study, we found tight junctional proteins ZO-1 and claudin-1 expression is largely reduced in epidermal, corneal and lung epithelia in Eda mutant Tabby mice at different postnatal ages. These declines are associated with tail ulceration, corneal pannus formation and lung infection. Furthermore, topical application of recombinant Eda protein markedly mitigated corneal barrier dysfunction. Using cultures of a human corneal epithelial cell line and Tabby mouse skin tissue explants, Eda up-regulated expression of ZO-1 and claudin-1 through activation of the sonic hedgehog signalling pathway. We conclude that EDA gene expression contributes to the maintenance of epithelial barrier function. Such insight may help efforts to identify novel strategies for improving management of XLHED disease manifestations in a clinical setting.
Assuntos
Ectodisplasinas/metabolismo , Epitélio/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Animais , Infecções Bacterianas/patologia , Córnea/microbiologia , Córnea/patologia , Humanos , Inflamação/patologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/farmacologia , Pele/patologia , Proteínas de Junções Íntimas/metabolismoRESUMO
We generated cornea-specific plakoglobin (Jup; junctional plakoglobin) knockout mice in order to investigate the function of plakoglobin on the maintenance of the homeostasis of corneal epithelium in mice. Cornea epithelium-specific conditional knockouts (JupCEΔ/CEΔ) (cKO) were obtained by breeding keratin12-Cre (Krt12-Cre) mice to Jup-floxed (Jupf/f) mice. Light and transmission electron microscopic and immunohistochemical analyses were carried out to determine consequence of the loss of plakoglobin on maintaining corneal epithelium integrity under mechanical stress, e.g., brushing and wound healing. Immunohistochemistry analysis demonstrated that, although Jup ablation did not affect BrdU incorporation, basal cell-like cells labeled for keratin 14 were ectopically present in the supra-basal layer in mutant corneal epithelium, suggestive of altered cell differentiation. Plakoglobin-deficient epithelium exhibits increased fragility against mechanical intervention when compared to wild-type controls under identical treatment. Closure of an epithelial defect was significantly delayed in JupCEΔ/CEΔ epithelium. Our findings indicate that the lack of plakoglobin significantly affects corneal epithelium differentiation, as well as its structural integrity. Plakoglobin is essential to the maintenance of the structure of the corneal epithelium and its wound healing.
Assuntos
Epitélio Corneano/fisiologia , Cicatrização , gama Catenina/fisiologia , Animais , Lesões da Córnea , Epitélio Corneano/ultraestrutura , Camundongos TransgênicosRESUMO
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.
Assuntos
Proteína Morfogenética Óssea 4/antagonistas & inibidores , Epitélio Corneano/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Morfogênese/fisiologia , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Animais , Western Blotting , Proteína Morfogenética Óssea 4/administração & dosagem , Imunoprecipitação da Cromatina , Doxiciclina , Fluorescência , Galactosídeos , Técnicas Histológicas , Imuno-Histoquímica , Indóis , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/deficiência , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/deficiência , Luciferases , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Fosfoproteínas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transativadores/metabolismoRESUMO
The present study attempts to elucidate the role of TRPV1 cation channel receptor on primary repair in an incision-wounded mouse cornea in vivo. Previous study revealed that blocking TRPV1 suppressed myofibroblast formation and expression of transforming growth factor ß1 (TGFß1) in cultured keratocytes or ocular fibroblasts. Male C57BL/6 (wild-type; WT) mice and male C57BL/6 Trpv1-null (KO) mice incurred a full-thickness incision injury (1.8 mm in length, limbus to limbus) in the central cornea of one eye with a surgical blade under general and topical anesthesia. The injury was not sutured. On days 0, 5, and 10, the eyes were enucleated, processed for histology, immunohistochemistry, and real-time RT-PCR gene expression analysis to evaluate the effects of the loss of TRPV1 on primary healing. Electron microscopy observation was also performed to know the effect of the loss of TRPV1 on ultrastructure of keratocytes. The results showed that the loss of Trpv1 gene delayed closure of corneal stromal incision with hindered myofibroblast transdifferentiation along with declines in the expression of collagen Ia1 and TGFß1. Inflammatory cell infiltration was not affected by the loss of TRPV1. Ultrastructurally endoplasmic reticulum of TRPV1-null keratocytes was more extensively dilated as compared with WT keratocytes, suggesting an impairment of protein secretion by TRPV1-gene knockout. These results indicate that injury-related TRPV1 signal is involved in healing of stromal incision injury in a mouse cornea by selectively stimulating TGFß-induced granulation tissue formation.
Assuntos
Lesões da Córnea/patologia , Canais de Cátion TRPV/deficiência , Cicatrização , Animais , Córnea/patologia , Córnea/ultraestrutura , Lesões da Córnea/metabolismo , Inflamação/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miofibroblastos/patologia , Canais de Cátion TRPV/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
The ocular surface epithelia, including the stratified but non-keratinized corneal, limbal and conjunctival epithelium, in concert with the epidermal keratinized eyelid epithelium, function together to maintain eye health and vision. Abnormalities in cellular proliferation or differentiation in any of these surface epithelia are central in the pathogenesis of many ocular surface disorders. Goblet cells are important secretory cell components of various epithelia, including the conjunctiva; however, mechanisms that regulate goblet cell differentiation in the conjunctiva are not well understood. Herein, we report that conditional deletion of transforming growth factor ß receptor II (Tgfbr2) in keratin 14-positive stratified epithelia causes ocular surface epithelial hyperplasia and conjunctival goblet cell expansion that invaginates into the subconjunctival stroma in the mouse eye. We found that, in the absence of an external phenotype, the ocular surface epithelium develops properly, but young mice displayed conjunctival goblet cell expansion, demonstrating that TGFß signaling is required for normal restriction of goblet cells within the conjunctiva. We observed increased expression of SAM-pointed domain containing ETS transcription factor (SPDEF) in stratified conjunctival epithelial cells in Tgfbr2 cKO mice, suggesting that TGFß restricted goblet cell differentiation directly by repressing Spdef transcription. Gain of function of Spdef in keratin 14-positive epithelia resulted in the ectopic formation of goblet cells in the eyelid and peripheral cornea in adult mice. We found that Smad3 bound two distinct sites on the Spdef promoter and that treatment of keratin 14-positive cells with TGFß inhibited SPDEF activation, thereby identifying a novel mechanistic role for TGFß in regulating goblet cell differentiation.
Assuntos
Diferenciação Celular/fisiologia , Túnica Conjuntiva/crescimento & desenvolvimento , Células Caliciformes/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Imunoprecipitação da Cromatina , Túnica Conjuntiva/citologia , Citometria de Fluxo , Técnicas Histológicas , Queratina-14/metabolismo , Microdissecção e Captura a Laser , Luciferases , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteína Smad3/metabolismoRESUMO
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.
Assuntos
Pálpebras/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glândulas Tarsais/anormalidades , Morfogênese/fisiologia , Fator de Crescimento Transformador alfa/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Primers do DNA/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas Histológicas , Imuno-Histoquímica , Glândulas Tarsais/embriologia , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Tendões/citologia , Tendões/embriologia , Fator de Crescimento Transformador alfa/genéticaRESUMO
Conjunctival goblet cells primarily synthesize mucins to lubricate the ocular surface, which is essential for normal vision. Notch signaling has been known to associate with goblet cell differentiation in intestinal and respiratory tracts, but its function in ocular surface has yet to be fully characterized. Herein, we demonstrate that conditional inhibition of canonical Notch signaling by expressing dominant negative mastermind-like 1 (dnMaml1) in ocular surface epithelia resulted in complete suppression of goblet cell differentiation during and subsequent to development. When compared with the ocular surface of wild-type mice (OS(Wt)), expression of dnMaml1 at the ocular surface (OS(dnMaml1)) caused conjunctival epithelial hyperplasia, aberrant desquamation, failure of Mucin 5ac (Muc5ac) synthesis, subconjunctival inflammation and epidermal metaplasia in cornea. In addition, conditional deletion of Notch1 from the ocular surface epithelia partially recapitulated OS(dnMaml1) phenotypes. We have demonstrated that N1-ICD (Notch1 intracellular domain) transactivated the mouse Krüppel-like factor 4 (Klf) promoter and that Klf4 directly bound to and significantly potentiated the Muc5ac promoter. By contrast, OS(dnMaml1) dampened Klf4 and Klf5 expression, and diminished Muc5ac synthesis. Collectively, these findings indicated that Maml-mediated Notch signaling plays a pivotal role in the initiation and maintenance of goblet cell differentiation for normal ocular surface morphogenesis and homeostasis through regulation of Klf4 and Klf5.
Assuntos
Túnica Conjuntiva/metabolismo , Epitélio Corneano/patologia , Receptor Notch1/metabolismo , Transdução de Sinais , Ativação Transcricional , Animais , Diferenciação Celular , Proliferação de Células , Túnica Conjuntiva/embriologia , Túnica Conjuntiva/patologia , Córnea/embriologia , Córnea/metabolismo , Córnea/patologia , Epitélio Corneano/embriologia , Epitélio Corneano/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Hiperplasia/genética , Hiperplasia/patologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/patologia , Metaplasia/metabolismo , Metaplasia/patologia , Camundongos , Camundongos Transgênicos , Mucina-5AC/genética , Mucina-5AC/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Receptor Notch1/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Intrauterine infection (chorioamnionitis) aggravates neonatal hypoxic-ischemic (HI) brain injury, but the mechanisms linking systemic inflammation to the CNS damage remain uncertain. Here we report evidence for brain influx of T-helper 17 (TH17)-like lymphocytes to coordinate neuroinflammatory responses in lipopolysaccharide (LPS)-sensitized HI injury in neonates. We found that both infants with histological chorioamnionitis and rat pups challenged by LPS/HI have elevated expression of the interleukin-23 (IL-23) receptor, a marker of early TH17 lymphocytes, in the peripheral blood mononuclear cells. Post-LPS/HI administration of FTY720 (fingolimod), a sphingosine-1-phosphate receptor agonist that blocks lymphocyte trafficking, mitigated the influx of leukocytes through the choroid plexus and acute induction of nuclear factor-κB signaling in the brain. Subsequently, the FTY720 treatment led to attenuated blood-brain barrier damage, fewer cluster of differentiation 4-positive, IL-17A-positive T-cells in the brain, less proinflammatory cytokine, and better preservation of growth and white matter functions. The FTY720 treatment also provided dose-dependent reduction of brain atrophy, rescuing >90% of LPS/HI-induced brain tissue loss. Interestingly, FTY720 neither opposed pure-HI brain injury nor directly inhibited microglia in both in vivo and in vitro models, highlighting its unique mechanism against inflammation-sensitized HI injury. Together, these results suggest that the dual hit of systemic inflammation and neonatal HI injury triggers early onset of the TH17/IL-17-mediated immunity, which causes severe brain destruction but responds remarkably to the therapeutic blockade of lymphocyte trafficking.
Assuntos
Movimento Celular/efeitos dos fármacos , Hipóxia-Isquemia Encefálica/patologia , Hipóxia-Isquemia Encefálica/prevenção & controle , Inflamação/prevenção & controle , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Propilenoglicóis/farmacologia , Esfingosina/análogos & derivados , Animais , Animais Recém-Nascidos , Atrofia/tratamento farmacológico , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Corioamnionite/tratamento farmacológico , Corioamnionite/metabolismo , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Feminino , Cloridrato de Fingolimode , Humanos , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Recém-Nascido , Lipopolissacarídeos , Linfócitos/citologia , NF-kappa B/metabolismo , Gravidez , Propilenoglicóis/uso terapêutico , Ratos , Receptores de Interleucina/metabolismo , Esfingosina/farmacologia , Esfingosina/uso terapêutico , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Substância Branca/efeitos dos fármacosRESUMO
PURPOSE: To examine the developmental pathobiology of the eyelid and the cornea caused by epithelial ß-catenin gain-of-function (gof) during mouse embryogenesis. METHODS: Compound mutant mice (Ctnnb1(GOFOSE) , gof of ß-catenin in the epidermis and the ocular surface epithelium) were generated by time-mating keratin 5-promoter-Cre recombinase (Krt5-Cre) and Ctnnb1(fE3/WT) (floxed exon 3 of Ctnnb1) mice. Eyes obtained from wild-type (WT) and mutant embryos at various gestation stages until E18.5 were examined with histology and immunohistochemistry. The ultrastructure of the ocular tissues of the E18.5 embryos was also examined. RESULTS: Expression of the gof-ß-catenin mutant protein in the epidermis severely impaired eyelid morphogenesis at E15.5, E17.5, and E18.5. The mutant stroma exhibited impaired keratocyte differentiation with accelerated cell proliferation and reduction in the accumulation of collagen type I. The mutant embryos also showed hyperproliferative nodules in the ocular surface epithelia with anomaly of cornea-type epithelial differentiation and the absence of the epithelial basement membrane. CONCLUSIONS: Expression of the gof-ß-catenin mutant protein in basal epithelial cells disrupts eyelid and cornea morphogenesis during mouse embryonic development due to the perturbation of cell proliferation and differentiation of the epithelium and the neural crest-derived mesenchyme.
Assuntos
Córnea/embriologia , Córnea/metabolismo , Pálpebras/embriologia , Pálpebras/metabolismo , Mutação , beta Catenina/genética , beta Catenina/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Córnea/citologia , Transição Epitelial-Mesenquimal , Epitélio/embriologia , Epitélio/metabolismo , Pálpebras/citologia , Feminino , Idade Gestacional , Queratina-5/genética , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Morfogênese/genética , Gravidez , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
This review summarizes a recent finding regarding the intrinsic canonical Notch signaling pathway in regulating normal ocular surface morphogenesis and its role in the pathogenesis of goblet cell deficiency-associated keratoconjunctivitis sicca (KCS, or dry eye). Specifically, we used novel transgenic mice to investigate the mechanism of how the Notch1 activation may serve as the upstream control of expression of transcription factors Krüppel-like factors 4 or 5 (Klf4 or Klf5) which in turn controls goblet cell differentiation and activates mucin 5/ac synthesis during ocular surface morphogenesis.
Assuntos
Síndromes do Olho Seco/fisiopatologia , Células Caliciformes/fisiologia , Receptor Notch1/fisiologia , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular/fisiologia , Congressos como Assunto , Túnica Conjuntiva/citologia , Proteínas de Ligação a DNA/metabolismo , Síndromes do Olho Seco/metabolismo , Células Caliciformes/citologia , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Fatores de Transcrição/metabolismoRESUMO
PURPOSE: RNA nanoparticles derived from the three-way junction (3WJ) of the pRNA of bacteriophage phi29 DNA packaging motor were previously found to be thermodynamically stable. As the nanoparticles could have potential in ocular drug delivery, the objectives in the present study were to investigate the distribution of pRNA nanoparticles after subconjunctival injection and examine the feasibility to deliver the nanoparticles to the cells of cornea and retina. METHODS: Alexa647-labeled pRNA nanoparticles (pRNA-3WJ and pRNA-X) and double-stranded RNA (dsRNA) were administered via subconjunctival injection in mice. Alexa647 dye was a control. Topical administration was performed for comparison. Ocular clearance of pRNA nanoparticles and dsRNA after the injection was assessed using whole-body fluorescence imaging of the eyes. The numbers of cells in the ocular tissues with nanoparticle cell internalization were determined in fluorescence microscopy of dissected eye tissues. RESULTS: After subconjunctival injection, pRNA nanoparticles and dsRNA were observed to distribute into the eyes and cleared through the lymph. pRNA-3WJ, pRNA-X, and dsRNA were found in the cells of the conjunctiva, cornea, and sclera, but only pRNA-X was in the cells of the retina. Topical administration was not effective in delivering the nanoparticles to the eye. CONCLUSIONS: The pRNA nanoparticles were delivered to the cells in the eye via subconjunctival injection, and cell internalization was achieved in the cornea with pRNA-3WJ and pRNA-X and in the retina with pRNA-X. Only the X-shape pRNA-X could enter the retina.
Assuntos
Túnica Conjuntiva/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Olho/efeitos dos fármacos , Olho/metabolismo , Nanopartículas/administração & dosagem , RNA/administração & dosagem , Proteínas Virais/administração & dosagem , Administração Tópica , Animais , Túnica Conjuntiva/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/metabolismo , RNA/metabolismo , Proteínas Virais/metabolismoRESUMO
Cell polarity during eye development determines the normal retinal lamination and differentiation of photoreceptor cells in the retina. In vertebrates, blood vessel epicardial substance (Bves) is known to play an important role in the formation and maintenance of the tight junctions essential for epithelial cell polarity. In the current study, we generated a transgenic zebrafish Bves (zbves) promoter-EGFP zebrafish line to investigate the expression pattern of Bves in the retina and to study the role of zbves in retinal lamination. Immunostaining with different specific antibodies from retinal cells and transmission electron microscopy were used to identify the morphological defects in normal and Bves knockdown zebrafish. In normal zebrafish, Bves is located at the apical junctions of embryonic retinal neuroepithelia during retinogenesis; later, it is strongly expressed around inner plexiform layer (IPL) and retinal pigment epithelium (RPE). In contrast, a loss of normal retinal lamination and cellular polarity was found with undifferentiated photoreceptor cells in Bves knockdown zebrafish. Herein, our results indicated that disruption of Bves will result in a loss of normal retinal lamination.
Assuntos
Retina/citologia , Vasos Retinianos/metabolismo , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Sequência de Bases , Polaridade Celular , Primers do DNARESUMO
(1) Background: Our previous data indicated that disturbance of the Transforming Growth Factor beta (TGFB) signaling pathway via its Type-2 Receptor (TGFBR2) can cause a Corneal Ectasia (CE)-like phenotype. The purpose of this study is to elucidate whether the SMAD4-dependent signaling pathway is involved in the TGFBR2-related CE-like pathogenesis. (2) Methods: Smad4 was designed to be conditionally knocked out from keratocytes. Novel triple transgenic mice, KerartTA; Tet-O-Cre; Smad4flox/flox (Smad4kera-cko), were administered with doxycycline (Dox). Optical Coherence Tomography (OCT) was performed to examine Central Corneal Thickness (CCT), Corneal Radius, Anterior Chamber and CE-like phenotype and compared to the littermate Control group (Smad4Ctrl). (3) Results: The OCT revealed normal cornea in the Smad4Ctrl and a CE-like phenotype in the Smad4kera-cko cornea, in which the overall CCT in Smad4kera-cko was thinner than that of Smad4Ctrl at P42 (n = 6, p < 0.0001) and showed no significant difference when compared to that in Tgfbr2kera-cko. Furthermore, the measurements of the Anterior Chamber and Corneal Radius indicated a substantial ectatic cornea in the Smad4kera-cko compared to Smad4Ctrl. The H&E staining of Smad4kera-cko mimics the finding in the Tgfbr2kera-cko. The positive immunostaining of cornea-specific marker K12 indicating the cell fate of cornea epithelium remained unchanged in Smad4kera-cko and the Proliferating Cell Nuclear Antigen (PCNA) immunostaining further indicated an enhanced proliferation in the Smad4kera-cko. Both immunostainings recapitulated the finding in Tgfbr2kera-cko. The Masson's Trichrome staining revealed decreased collagen formation in the corneal stroma from both Smad4kera-cko and Tgfbr2kera-cko. The collagen type 1 (Col1a1) immunostaining further confirmed the reduction in collagen type 1 formation in Smad4kera-cko. (4) Conclusions: The aforementioned phenotypes in the Smad4kera-cko strain indicated that the SMAD4-dependent signaling pathway is involved in the pathogenesis of the CE-like phenotype observed in Tgfbr2kera-cko.
Assuntos
Doenças da Córnea , Camundongos , Animais , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Doenças da Córnea/patologia , Camundongos Transgênicos , Transdução de Sinais , Fenótipo , ColágenoRESUMO
Bves is widely observed in the cell junction of the skin, epicardium, intestine, and cornea of both developmental embryos and mature adults. However, it is not clear how Bves confers its role in intercellular adhesion. Here, we identified the zebrafish bves (zBves) and found that the epidermal barrier function could be disrupted after knockdown of Bves, and these zBves morphants were sensitive to osmotic stress. A loss of zBves would affect the partitioning defective protein (PAR) junctional complex identified by the rescue experiment with tjp-2/ZO-2 or the PAR complex (par-3, par-6, and prkci/atypical (a)PKC) mRNAs, in which the survival rate of embryos increased 11, 24, 25, and 28%, respectively, after injection with junctional components; the tjp-2 and aPKC mRNA-rescued embryos also had 24 and 45% decreases in the defective rate. Immunofluorescent studies demonstrated that the aggregation of aPKC around the cell junctions had disintegrated in zBves morphants. However, the expression and assembly of zBves were not influenced by aPKC-MO. These results indicate that a loss of zBves affects the proteins involved in the pathway of the PAR junctional complex, especially aPKC, and both aPKC and Bves are indispensable to claudin expression.
Assuntos
Claudinas/biossíntese , Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Junções Íntimas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Proteína da Zônula de Oclusão-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Claudinas/genética , Embrião não Mamífero/citologia , Isoenzimas/genética , Proteína Quinase C/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Junções Íntimas/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteína da Zônula de Oclusão-2/genéticaRESUMO
Notch signaling is pivotal for the morphogenesis and homeostasis of many tissues. We found that aberrant Notch activation in mouse neural-crest-derived periocular mesenchymal cells (POMCs), which contribute to the formation of corneal and eyelid stroma, results in blepharophimosis. Compound transgenic mice overexpressing the Notch1 intracellular domain (N1-ICD) in POMCs (POMC(N1-ICD)) showed relatively minor effects on the cornea, but increased cell apoptosis and decreased cell proliferation during eyelid morphogenesis. Eyelid closure at E15.5 and eyelid formation at birth were incomplete. In further analyses, overexpression of N1-ICD impaired eyelid levator smooth muscle formation by downregulating the transcription factor FoxL2. This is similar to the effect of haploinsufficiency of FOXL2 in humans, which results in type II BPES (blepharophimosis, ptosis and epicanthus inversus syndrome). In vitro studies showed that FoxL2 expression is augmented by a low dose of N1-ICD but was downregulated by a high dose, depending on the extent of Hes-1 and Hey-1 activation. Moreover, transfection of CMV-FoxL2 enhanced α-SMA promoter activity. These data strongly imply that a physiologically low level of Notch1 is crucial for proper FoxL2 expression in POMCs, which is, in turn, essential for Müeller muscle formation and normal eyelid development.
Assuntos
Blefarofimose/metabolismo , Blefarofimose/patologia , Pálpebras/metabolismo , Pálpebras/patologia , Fatores de Transcrição Forkhead/metabolismo , Músculo Liso/metabolismo , Receptor Notch1/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Blefarofimose/genética , Western Blotting , Proliferação de Células , Imunoprecipitação da Cromatina , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/genética , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Receptor Notch1/genéticaRESUMO
PURPOSE: We hypothesized that Transforming growth factor beta receptor 2 (Tgfbr2) deletion in keratocyte (Tgfbr2kera-cko), the corneal stroma cell, can result in corneal thinning and generate a potential model for Cornea Ectasia (CE). METHODS: Corneal thickness of Tgfbr2kera-cko and Tgfbr2Ctrl was examined with Optical Coherence Tomography (OCT) at post-natal (P) days 42 and 70, respectively. Histological H&E staining, transmission electron micrograph (TEM), and immunofluorescence staining (IFS) were harnessed to examine corneal cell morphology, proliferation, differentiation, and collagen fibrils. RESULTS: Slit-Lamp revealed that corneas were transparent in both Tgfbr2kera-cko and Tgfbr2Ctrl, however, Tgfbr2kera-cko cornea was 33.5% and 42.9% thinner as compared with those of Tgfbr2Ctrl at P42 and P70, respectively. H&E and semithin section staining with toluidine blue-O confirmed that Tgfbr2kera-cko cornea has a thinner stroma. In contrast, the epithelium in Tgfbr2kera-cko was substantially thicker. The cell proliferation marker Ki67 expression level increased â¼9% in Tgfbr2kera-cko corneal epithelium as compared with that in Tgfbr2Ctrl, however, the Krt14 and Krt12 expression pattern was not obviously changed in Tgfbr2kera-cko corneal epithelium. It was noticed that Col1a1 expression was substantially reduced in Tgfbr2kera-cko as compared with that in Tgfbr2Ctrl. TEM showed that keratocytes were unhealthy and stromal collagen fibril density was significantly reduced in Tgfbr2kera-cko as compared with that in Tgfbr2Ctrl cornea. Moreover, mechanical eye-rubbing on Tgfbr2kera-cko resulted in corneal hydrops and edema. CONCLUSION: Tgfbr2 in keratocytes is indispensable for the corneal stroma at postnatal homeostasis. The cornea phenotype manifested in these Tgfbr2kera-cko mice resembles corneal ectasia disease in humans.
Assuntos
Córnea , Doenças da Córnea , Receptor do Fator de Crescimento Transformador beta Tipo II , Animais , Humanos , Camundongos , Colágeno , Córnea/patologia , Doenças da Córnea/patologia , Substância Própria , Dilatação Patológica/metabolismo , Dilatação Patológica/patologia , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismoRESUMO
INTRODUCTION: This two-part study aimed to investigate the therapeutic potential of topical spironolactone in ocular graft-versus-host disease (oGVHD). While off-label use of topical spironolactone has been described in dry eye, its efficacy in managing signs and symptoms of oGVHD remains unstudied. Preclinically, we tested the hypothesis that spironolactone induces corneal lipid synthesis in a mouse model. Clinically, we assessed patient response to spironolactone with a retrospective observational design. METHODS: Both immortalized and primary human corneal epithelial cells were stained with oil red O after 9 days of treatment with spironolactone. C57BL/6 mice were dosed thrice daily with one drop in each eye for 18 days. Corneal tissue was stained with oil red O and BODIPY™. Twenty eyes with oGVHD, as defined by the International Chronic oGVHD Consensus Group, were studied. Corneal fluorescein staining, lid margin vascularity, meibomian gland obstruction, meibum turbidity, zone A posterior lid margin vascularity, and oGVHD diagnostic criteria severity grading were compared in a pre-post study. Follow-up times ranged from 7 to 21 weeks, with a median time of 12 weeks. Statistical analysis was done with STATA 17 by fitting data to a non-parametric model. RESULTS: In vitro results showed an increased number and density of oil red O staining granules in the treatment group versus control in both primary and immortalized human corneal epithelium. In vivo, results showed translation to the mouse model with increased corneal epithelial BODIPY™ signal compared to untreated control. oGVHD patients had improved lid margin vascularity (p = 0.046), corneal fluorescein staining (p = 0.021), and International oGVHD Consensus Group severity scores (p = 0.011) after treatment with topical spironolactone. Minimal adverse effects were noted, the most common being mild stinging lasting less than a minute after instillation. CONCLUSION: The improved severity scores, lid margin inflammation, and corneal fluorescein staining after weeks of treatment support the rationale that topical spironolactone may benefit oGVHD. The observed lipid production by the corneal epithelium is thought to contribute to this protective effect against ocular surface erosive disease in oGVHD. A mineralocorticoid receptor antagonist, spironolactone may offer therapeutic benefits in oGVHD while avoiding undesirable side effects of topical or systemic glucocorticoids.