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1.
Toxicol Lett ; 322: 1-11, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31884112

RESUMO

Chloropicrin (CP), a warfare agent now majorly used as a soil pesticide, is a strong irritating and lacrimating compound with devastating toxic effects. To elucidate the mechanism of its ocular toxicity, toxic effects of CP (0-100 µM) were studied in primary human corneal epithelial (HCE) cells. CP exposure resulted in reduced HCE cell viability and increased apoptotic cell death with an up-regulation of cleaved caspase-3 and poly ADP ribose polymerase indicating their contribution in CP-induced apoptotic cell death. Following CP exposure, cells exhibited increased expression of heme oxygenase-1, and phosphorylation of H2A.X and p53 as well as 4-hydroxynonenal adduct formation, suggesting oxidative stress, DNA damage and lipid peroxidation. CP also caused increases in mitogen activated protein kinase-c-Jun N-terminal kinase and inflammatory mediator cyclooxygenase-2. Proteomic analysis revealed an increase in the carbonylation of 179 proteins and enrichment of pathways (including proteasome pathway and catabolic process) in HCE cells following CP exposure. CP-induced oxidative stress and lipid peroxidation can enhance protein carbonylation, prompting alterations in corneal epithelial proteins as well as perturbing signaling pathways resulting in toxic effects. Pathways and major processes identified following CP exposure could be lead-hit targets for further biochemical and molecular characterization as well as therapeutic intervention.


Assuntos
Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Epitélio Anterior/efeitos dos fármacos , Hidrocarbonetos Clorados/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Praguicidas/toxicidade , Carbonilação Proteica/efeitos dos fármacos , Caspase 3/metabolismo , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Dano ao DNA , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Epitélio Anterior/metabolismo , Epitélio Anterior/patologia , Heme Oxigenase-1/metabolismo , Histonas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Peroxidação de Lipídeos , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
2.
Invest Ophthalmol Vis Sci ; 60(12): 3854-3862, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31529118

RESUMO

Purpose: Subconjunctival injection of antagomir-21 attenuates the progression of corneal neovascularization. We examined the underlying mechanism by investigating the regulation of microRNA (miR)-21 expression and the involvement of miR-21 in the homeostasis of corneal epithelial cells. Methods: Corneal epithelial cells were cultured with TGF-ß1 and/or under hypoxia conditions. miR-21 expression was measured by quantitative PCR. The direct targets of miR-21 were validated by the 3'-UTR luciferase reporter assay. Alterations of proangiogenic signaling and the epithelial-mesenchymal transition (EMT) phenotype after miR-21/Sprouty2 (SPRY2) knockdown were examined by Western blotting. The effect of conditioned medium on angiogenesis was assessed using the tube formation assay. Wound healing was evaluated by the migration and scratch assays. Results: TGF-ß1 or hypoxia upregulated miR-21, and miR-21 silencing abolished TGF-ß1/hypoxia-induced hypoxia inducible factor (HIF)-1α and VEGF expression. miR-21 inhibited SPRY2 by directly targeting its 3'-UTR. Simultaneous silencing of miR-21 and SPRY2 significantly upregulated p-ERK, HIF-1α, and VEGF and promoted angiogenesis. Induction of miR-21 or inhibition of SPRY2 reduced the levels of cytokeratin (CK)-3 and CK-12 and promoted EMT. Transwell and wound healing assays indicated that miR-21 promoted cell migration. Conclusions: TGF-ß1 or hypoxia induced miR-21 and inhibited SPRY2, thereby enhancing proangiogenic signaling, suppressing the epithelial phenotype, and promoting wound healing in corneal epithelial cells.


Assuntos
Epitélio Anterior/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Membrana/fisiologia , MicroRNAs/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/fisiologia , Animais , Western Blotting , Movimento Celular/fisiologia , Células Epiteliais/citologia , Transição Epitelial-Mesenquimal , Epitélio Anterior/efeitos dos fármacos , Hipóxia/metabolismo , Queratina-12/metabolismo , Queratina-3/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Fator de Crescimento Transformador beta1/farmacologia
3.
Invest Ophthalmol Vis Sci ; 60(12): 3776-3785, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31503282

RESUMO

Purpose: To investigate the therapeutic effects of targeting signal transducer and activator of transcription-3 (STAT3) activation on the ocular surface damage of dry eye in mice. Methods: Adult Balb/C and C57BL/6 mice with benzalkonium chloride (BAC) treatment, lacrimal gland excision, and meibomian gland dysfunction were used as dry eye models. The levels of phosphorylated STAT3 (p-STAT3) were detected with immunofluorescence staining and Western blotting. STAT3 inhibition was performed by topical application of STAT3 inhibitor S3I-201. Corneal epithelial barrier function, tear production, and conjunctival goblet cell density were quantified with fluorescein sodium staining, phenol red cotton test, and histochemical staining. The expressions of matrix metalloproteinase (MMP)-3/9, TUNEL, and inflammation cytokines were assessed with immunofluorescence staining, qPCR, and ELISA assays. The therapeutic effect of S3I-201 was further compared with the Janus kinase inhibitor tofacitinib and ruxolitinib. Results: Elevated levels of nuclear p-STAT3 were detected in the corneal and conjunctival epithelium of three dry eye models. Topical application of S3I-201 improved corneal epithelial barrier function, increased tear production and conjunctival goblet cell density in BAC-induced dry eye mice. Moreover, S3I-201 decreased the expression of MMP-3/9, suppressed the apoptosis of corneal and conjunctival epithelial cells, and reduced the levels of IL-1ß, IL-6, IL-17A, and IFN-γ. Compared with tofacitinib and ruxolitinib, the STAT3 inhibitor S3I-201 showed superior improvement of tear production and inflammatory cytokine expression in lacrimal gland. Conclusions: Elevated STAT3 activation is involved in the pathogenesis of dry eye, while targeting STAT3 effectively alleviates BAC-induced ocular surface damage.


Assuntos
Modelos Animais de Doenças , Síndromes do Olho Seco/tratamento farmacológico , Proteínas Inibidoras de STAT Ativados/uso terapêutico , Fator de Transcrição STAT3/antagonistas & inibidores , Administração Oftálmica , Animais , Western Blotting , Túnica Conjuntiva/metabolismo , Citocinas/metabolismo , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Ensaio de Imunoadsorção Enzimática , Epitélio/metabolismo , Epitélio Anterior/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Células Caliciformes/patologia , Marcação In Situ das Extremidades Cortadas , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Soluções Oftálmicas , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição STAT3/metabolismo , Lágrimas/fisiologia
4.
Invest Ophthalmol Vis Sci ; 60(13): 4074-4083, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31561249

RESUMO

Purpose: We have observed noticably weak epithelial attachment in vitamin D receptor knockout mice (VDR KO) undergoing epithelial debridement. We hypothesized that VDR KO negatively affects corneal epithelial cell desmosomes and/or hemidesmosomes. Methods: Transcript levels of desmosome and hemidesmosome proteins in VDR KO corneas were assessed by qPCR. Western blotting and immunochemistry were used to detect proteins in cultured cells exposed to 1,25(OH)2D3 and 24R,25(OH)2D3. Results: VDR KO resulted in decreased corneal desmosomal desmoglein 1 (DSG1) and desmocollin 2 (DSC2) mRNA, and hemidesmosomal plectin mRNA. DSG1 and plectin protein expression were reduced in VDR KO corneas. DSG1 protein expression increased in VDR wild types (VDR WT) and VDR KO mouse primary epithelial cells (MPCEC) treated with 1,25(OH)2D3 and 24R,25(OH)2D3. 24R,25(OH)2D3 treatment resulted in increased plectin and integrin ß4 levels in VDR WT MPCEC, and decreased levels in VDR KO MPCEC. Treatment of human corneal epithelial cells (HCEC) with 1,25(OH)2D3 and 24R,25(OH)2D3 resulted in increased DSC2 and DSG1 protein expression. Plectin and integrin ß4 were only increased in 24R,25(OH)2D3 treated HCEC. Conclusions: VDR KO results in reduced desmosomal and hemidesmosomal mRNA and protein levels. 1,25(OH)2D3 and 24R,25(OH)2D3 increased DSG1 protein in all cells tested. For hemidesmosome proteins, 24R,25(OH)2D3 increased plectin and integrin ß4 protein expression in VDR WT and HCEC, with decreased expression in VDR KO MPCEC. Thus, vitamin D3 is involved in desmosome and hemidesmosome junction formation/regulation, and their decreased expression likely contributes to the loosely adherent corneal epithelium in VDR KO mice. Our data indicate the presence of a VDR-independent pathway.


Assuntos
Desmossomos/metabolismo , Células Epiteliais/metabolismo , Epitélio Anterior/metabolismo , Hemidesmossomos/efeitos dos fármacos , Vitamina D/fisiologia , Vitaminas/fisiologia , Animais , Desmocolinas/metabolismo , Desmogleína 1/metabolismo , Células Epiteliais/efeitos dos fármacos , Epitélio Anterior/efeitos dos fármacos , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Receptores de Calcitriol/deficiência , Vitamina D/farmacocinética
5.
Invest Ophthalmol Vis Sci ; 60(12): 3718-3726, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31479112

RESUMO

Purpose: To evaluate the effects of human amniotic membrane-derived fibroblast (AMF) cell supernatant (AMF-sup) on corneal epithelium. Methods: The phenotype of AMF cells was analyzed by flow cytometry using cell-surface markers. AMF cells were also induced to form osteoblasts and neural cells, and cell phenotypes were observed by staining and RT-PCR. Cultivated human corneal limbal epithelial sheets generated using AMF-sup were analyzed using immunohistochemistry and colony-forming efficiency, and the wound healing of epithelial defects was observed using a tissue-punch method. The effects of instillation of each supernatant in a rabbit corneal epithelial wound healing model were compared. Results: Mesenchymal stem cell (CD29, CD44, CD73, and CD90) and neural crest (CD49d and CD56) markers were expressed on the AMF cell surface. Following induction of differentiation, isolated AMF cells showed characteristics of osteoblasts and neural cells. Application of AMF-sup resulted in maintenance of the limbal epithelial phenotype and immature state, and significantly promoted wound healing in cultivated human corneal limbal epithelial sheets (P < 0.05) and rabbit corneal epithelium (P < 0.05) compared with the control. Conclusions: These data suggest that AMF cells have multi-differentiation potential, and that AMF-sup is effective in maintaining the limbal epithelial phenotype and promoting corneal epithelial wound healing, which may be of value in ocular surface reconstruction.


Assuntos
Âmnio/citologia , Fatores Biológicos/farmacologia , Lesões da Córnea/tratamento farmacológico , Epitélio Anterior/efeitos dos fármacos , Fibroblastos/fisiologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Epitélio Anterior/metabolismo , Epitélio Anterior/patologia , Feminino , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Limbo da Córnea/citologia , Camundongos , Células NIH 3T3 , Fenótipo , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cicatrização/fisiologia
6.
ACS Appl Mater Interfaces ; 11(41): 37491-37501, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31532610

RESUMO

Microbial keratitis is a serious sight threatening infection affecting approximately two million individuals worldwide annually. While antibiotic eye drops remain the gold standard treatment for these infections, the significant problems associated with eye drop drug delivery and the alarming rise in antimicrobial resistance has meant that there is an urgent need to develop alternative treatments. In this work, a nitric oxide releasing contact lens gel displaying broad spectrum antimicrobial activity against two of the most common causative pathogens of microbial keratitis is described. The contact lens gel is composed of poly-ε-lysine (pεK) functionalized with nitric oxide (NO) releasing diazeniumdiolate moieties which enables the controlled and sustained release of bactericidal concentrations of NO at physiological pH over a period of 15 h. Diazeniumdiolate functionalization was confirmed by Fourier transform infrared (FTIR), and the concentration of NO released from the gels was determined by chemiluminescence. The bactericidal efficacy of the gels against Pseudomonas aeruginosa and Staphylococcus aureus was ascertained, and between 1 and 4 log reductions in bacterial populations were observed over 24 h. Additional cell cytotoxicity studies with human corneal epithelial cells (hCE-T) also demonstrated that the contact lens gels were not cytotoxic, suggesting that the developed technology could be a viable alternative treatment for microbial  keratitis.


Assuntos
Anti-Infecciosos , Lentes de Contato , Ceratite/tratamento farmacológico , Óxido Nítrico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/crescimento & desenvolvimento , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/crescimento & desenvolvimento , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Epitélio Anterior/metabolismo , Epitélio Anterior/microbiologia , Epitélio Anterior/patologia , Humanos , Teste de Materiais , Óxido Nítrico/química , Óxido Nítrico/farmacologia
7.
Invest Ophthalmol Vis Sci ; 60(10): 3538-3546, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31415077

RESUMO

Purpose: To determine if trigeminal innervations of the corneal epithelium maintains its integrity and homeostasis through controlling the nicotinamide adenine dinucleotide (NAD) content of this tissue. Methods: Corneal denervation of C57BL/6 mice was induced by squeezing the nerve bundles that derive from the trigeminal ganglion and was confirmed by whole-mount corneal nerve staining and the sensation test. The apoptosis of the corneal epithelium was examined by TUNEL assay and annexin V/propidium iodide staining. NAD biosynthesis-related enzymes were analyzed by quantitative PCR, immunofluorescence staining, and Western blotting. FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), exogenous nicotinamide mononucleotide (NMN), and NAD+ were used to evaluate the effect of NAD+ on the apoptosis of cultured corneal epithelial cells and epithelial detachment in denervated mice. Protein expression that related to apoptosis and phosphorylation were analyzed by Western blotting. Results: The denervated mice showed spontaneous corneal epithelial detachment and cell apoptosis accompanied with impaired epithelial NAD+ contents due to low levels of NAMPT. Similarly, inhibition of NAMPT recapitulated epithelial detachment as in denervated mice and induced apoptosis in cultured corneal epithelial cells. The replenishment of NMN or NAD+ partially slowed down corneal nerve fiber degeneration, reduced the epithelial defect in denervated mice, and improved apoptosis induction in FK866-treated cells by restoring the activation levels of SIRT1, AKT, and CREB. Conclusions: Corneal denervation lowered epithelial NAD+ contents through reducing the expression of NAMPT and caused cell apoptosis and epithelial defects, suggesting that corneal innervations contribute to epithelial homeostasis by regulating NAD+ biosynthesis.


Assuntos
Apoptose , Córnea/inervação , Denervação , Epitélio Anterior/patologia , NAD/metabolismo , Nervo Trigêmeo/fisiologia , Animais , Anexina A5/metabolismo , Western Blotting , Proteína de Ligação a CREB/metabolismo , Córnea/metabolismo , Doenças da Córnea/diagnóstico , Doenças da Córnea/metabolismo , Epitélio Anterior/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sirtuína 1/metabolismo , Doenças do Nervo Trigêmeo/diagnóstico , Doenças do Nervo Trigêmeo/metabolismo , Proteína X Associada a bcl-2/metabolismo
8.
Cornea ; 38 Suppl 1: S34-S41, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31403532

RESUMO

In its early stages, an embryo polarizes to form cell subpopulations that subsequently produce specific organ cell types. These cell subpopulations are defined by transcription factors (TFs) that activate or repress specific genes. Although an embryo comprises thousands of TFs, surprisingly few are needed to determine the fate of a given cell. The ectoderm divides into the neuroectoderm and surface ectoderm, the latter of which gives rise to epidermal keratinocytes and corneal epithelial cells (CECs). Meanwhile, neuroectoderm cells give rise to other parts of the eye such as the corneal endothelium and retina. To investigate the regulatory role of TFs in CECs, we overexpressed the "core TFs" (PAX6, OVOL2, and KLF4) in human fibroblasts and found that the cells adopted a CEC-like quality. OVOL2 overexpression was even able to directly induce cells with a neuroectoderm fate toward a surface ectoderm fate, designated "direct reprogramming." Conversely, suppression of OVOL2 or PAX6 expression induced CECs to show qualities consistent with neural lineage cells or epidermal keratinocytes, respectively. This suggests that these core TFs can maintain the CEC phenotype through reciprocal gene regulation. Direct reprogramming has important implications for cell therapies. The potential benefits of cells derived by direct reprogramming compared with induced pluripotent stem cells include the fact that it requires less time than reprogramming a cell back to the pluripotent state and then to another cell type. Further understanding of the reciprocally repressive mechanism of action for core TFs could lead to alternative treatments for regenerative medicine not requiring cell transplantation.


Assuntos
Epitélio Anterior/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Fator de Transcrição PAX6/genética , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Linhagem da Célula , Epitélio Anterior/citologia , Redes Reguladoras de Genes , Humanos , Fatores de Transcrição Kruppel-Like/biossíntese , Fator de Transcrição PAX6/biossíntese , Fatores de Transcrição/biossíntese
9.
Exp Eye Res ; 187: 107776, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31465769

RESUMO

Vitamin D is a fat-soluble prohormone that can be activated both systemically and within individual tissues. Our lab has previously demonstrated that the corneal epithelium can activate vitamin D and that the vitamin D metabolites 1,25(OH)2D3 and 24R,25(OH)2D3 can affect corneal epithelial migration, proliferation, and tight and gap junction function. These vitamin D-derived metabolites signal through the vitamin D receptor (VDR). The purpose of this study was to specifically determine the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell gap junction proteins. Connexin (Cx) 26, 30 and 43 protein expression was detected in a human corneal epithelial cell line (HCEC), wild type and vitamin D receptor knockout (VDR-/-) mouse corneas, and cultured mouse primary epithelial cells (MPCEC). In vitro gap junction function was assessed using the scrape loading/dye transfer assay. HCEC and MPCEC were treated with 1,25(OH)2D3 or 24R,25(OH)2D3. Western blotting was used to detect gap junction proteins. Vitamin D3 effects on epithelial intracellular Ca++ (Ca++i) were determined using the dye Cal-520. Cx26 and Cx43 protein levels were significantly increased in HCEC and MPCEC treated with both 1,25(OH)2D3 and 24R,25(OH)2D3. Cx30 and Cx43 protein levels were also significantly increased in VDR-/- MPCEC. In vitro gap junction connectivity was significanlty enhanced in HCEC and MPCEC cultured with 24R,25(OH)2D3 and 1,25(OH)2D3. Ca++i was not affected by 1,25(OH)2D3 or 24R,25(OH)2D3 in HCEC or MPCEC. We conclude that both 1,25(OH)2D3 and 24R,25(OH)2D3 are positive regulators of connexin proteins and gap junction communication in the corneal epithelium. These vitamin D metabolites appear to signal through both VDR-dependent and -independent pathways. The effects of vitamin D on corneal epithelial gap junctions do not seem to be dependent on Ca++i.


Assuntos
24,25-Di-Hidroxivitamina D 3/farmacologia , Calcitriol/farmacologia , Conexinas/metabolismo , Epitélio Anterior/efeitos dos fármacos , Receptores de Calcitriol/fisiologia , Animais , Western Blotting , Cálcio/metabolismo , Linhagem Celular , Conexina 30/metabolismo , Conexina 43/metabolismo , Epitélio Anterior/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais
10.
Invest Ophthalmol Vis Sci ; 60(10): 3570-3583, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31419300

RESUMO

Purpose: Single-cell RNA-sequencing (scRNA-seq) was used to interrogate the relatively rare stem (SC) and early transit amplifying (TA) cell populations in limbal/corneal epithelia from wild-type and autophagy-compromised mice. Methods: We conducted scRNA-seq on ocular anterior segmental tissue from wild-type and beclin 1-deficient (beclin1+/-) mice, using a 10X Gemomics pipeline. Cell populations were distinguished by t-distributed stochastic neighbor embedding. Seurat analysis was conducted to compare gene expression profiles between these two groups of mice. Differential protein expression patterns were validated by immunofluorescence staining and immunoblotting. Results: Unbiased clustering detected 10 distinct populations: three clusters of mesenchymal and seven clusters of epithelial cells, based on their unique molecular signatures. A discrete group of mesenchymal cells expressed genes associated with corneal stromal SCs. We identified three limbal/corneal epithelial cell subpopulations designated as stem/early TA, mature TA, and differentiated corneal epithelial cells. Thioredoxin-interacting protein and PDZ-binding kinase (PBK) were identified as novel regulators of stem/early TA cell quiescence. PBK arrested corneal epithelial cells in G2/M phase of the cell cycle. Beclin1+/- mice displayed a decrease in proliferation-associated (Ki67, Lrig1) and stress-response (H2ax) genes. The most increased gene in beclin1+/- mice was transcription factor ATF3, which negatively regulates limbal epithelial cell proliferation. Conclusions: Establishment of a comprehensive atlas of genes expressed by stromal and epithelial cells from limbus and cornea forms the foundation for unraveling regulatory networks among these distinct tissues. Similarly, scRNA-seq profiling of the anterior segmental epithelia from wild-type and autophagy-deficient mice provides new insights into how autophagy influences proliferation in these tissues.


Assuntos
Autofagia/fisiologia , Epitélio Anterior/citologia , Limbo da Córnea/citologia , Células-Tronco Mesenquimais/citologia , RNA/genética , Transcriptoma/genética , Animais , Proteína Beclina-1/fisiologia , Biomarcadores/metabolismo , Contagem de Células , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Epitélio Anterior/metabolismo , Feminino , Imuno-Histoquímica , Limbo da Córnea/metabolismo , Glicoproteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA
11.
Invest Ophthalmol Vis Sci ; 60(8): 2836-2847, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31266058

RESUMO

Purpose: To assess how DNA damage-inducible transcript 4 (DDIT4) and autophagic flux are altered in dry eye disease and reveal the underlying mechanisms. Methods: C57BL/6 mice were exposed to desiccating stress (subcutaneous scopolamine [0.5 mg/0.2 mL] 3 times a day, humidity < 30%) for 7 days. Primary human corneal epithelial cells and cells from a human corneal epithelial cell line were cultured under hyperosmolarity. Western blot assays and immunofluorescence staining were used to measure changes in protein expression. mRNA expression was analyzed by RT-PCR and quantitative real-time PCR. Autophagosomes were observed through electron microscopy. Cellular reactive oxygen species and mitochondrial function were detected with 2',7'-dichlorodihydrofluorescein diacetate and mitochondrial membrane potential assays. Cell Counting Kit-8 and lactate dehydrogenase assays were used to measure cell death. Apoptosis was analyzed by Annexin V-PI flow cytometry. Results: Increased expression of microtubule-associated protein 1 light chain 3 (LC3-II), sequestosome 1 (SQSTM1), and DDIT4 were observed in corneal epithelial cells in in vitro and mice models of dry eye. The electron microscopy revealed large autophagic vacuoles with poorly degraded materials in human corneal epithelial cells under hyperosmolarity. In addition, we found that DDIT4 knockdown significantly suppressed the expression of LC3-II and SQSTM1 by disrupting reactive oxygen species release and restoring mitochondrial function under hyperosmolarity. Moreover, the ablation of DDIT4 effectively preserved cell viability and inhibited apoptosis. Conclusions: Excessive reactive oxygen species release through DDIT4 induction can lead to impaired autophagy and decreased cell viability in dry eye disease.


Assuntos
Autofagia , Síndromes do Olho Seco/metabolismo , Estresse Oxidativo/fisiologia , Fatores de Transcrição/metabolismo , Animais , Anexina A5/metabolismo , Apoptose/fisiologia , Autofagia/fisiologia , Western Blotting , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Epitélio Anterior/metabolismo , Epitélio Anterior/patologia , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/metabolismo
12.
Graefes Arch Clin Exp Ophthalmol ; 257(9): 1915-1924, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31321523

RESUMO

PURPOSE: In vivo microenvironments are critical to tissue homeostasis and wound healing, and the cornea is regulated by a specific microenvironment complex that consists of cell-cell interactions, air-liquid interfaces, and fluid flow stimulation. In this study, we aimed to clarify the effects of and the correlations among these three component factors on the cell kinetics of corneal epithelial cells. METHODS: Human corneal epithelial-transformed (HCE-T) cells were cocultured with either primary rat corneal fibroblasts or NIH 3T3 fibroblasts. We employed a double-dish culture method to create an air-liquid interface and a gyratory shaker to create fluid flow stimulation. Morphometric and protein expression analyses were performed for the HCE-T cells. RESULTS: Both the primary rat fibroblasts and the NIH 3T3 cells promoted HCE-T cell proliferation, and the presence of fluid flow synergistically enhanced this effect and inhibited the apoptosis of HCE-T cells. Moreover, fluid flow enhanced the emergence of myofibroblasts when cocultured with primary rat fibroblasts or NIH 3T3 cells. Extracellular signal-regulated kinase and p38 signaling were regulated either synergistically or independently by both fluid flow and cellular interaction between the HCE-T and NIH 3T3 cells. CONCLUSION: The cell-cell interaction and fluid flow stimulation in the air-liquid interface synergistically or independently regulated the behavior of HCE-T cells. Fluid flow accelerated the phenotypic change from corneal fibroblasts and NIH 3T3 cells to myofibroblasts. Elucidation of the multicomponent interplay in this microenvironment will be critical to the homeostasis and regeneration of the cornea and other ocular tissues.


Assuntos
Lesões da Córnea/metabolismo , Epitélio Anterior/metabolismo , Células-Tronco Mesenquimais/citologia , Cicatrização/fisiologia , Animais , Western Blotting , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Lesões da Córnea/patologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Epitélio Anterior/patologia , Homeostase , Humanos , Imuno-Histoquímica , Ratos , Ratos Wistar , Transdução de Sinais
13.
PLoS Pathog ; 15(6): e1007825, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31220184

RESUMO

Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S. marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection.


Assuntos
Toxinas Bacterianas/metabolismo , Células Epiteliais/metabolismo , Epitélio Anterior/metabolismo , Infecções por Proteus/metabolismo , Proteus/metabolismo , Infecções por Serratia/metabolismo , Serratia marcescens/metabolismo , Animais , Toxinas Bacterianas/genética , Morte Celular , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Epitélio Anterior/microbiologia , Epitélio Anterior/patologia , Humanos , Camundongos , Perforina/genética , Perforina/metabolismo , Proteus/genética , Infecções por Proteus/genética , Infecções por Proteus/microbiologia , Infecções por Proteus/patologia , Células RAW 264.7 , Infecções por Serratia/genética , Infecções por Serratia/microbiologia , Infecções por Serratia/patologia , Serratia marcescens/genética , Suínos , Sistemas de Secreção Tipo V/genética , Sistemas de Secreção Tipo V/metabolismo
14.
Cutan Ocul Toxicol ; 38(4): 375-383, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31223032

RESUMO

Purpose: A comet assay is one of the genotoxicity methods for evaluating the potential of chemicals to induce DNA strand breaks. To investigate the usefulness of comet assays for evaluating the genotoxic potential of ophthalmic solutions, a three-dimensional (3D) reconstructed human corneal epithelial model (3D corneal model) was exposed to conditions mimicking topical ocular instillation administration. Methods: The 3D corneal model was exposed to acridine orange, ethidium bromide, hydrogen peroxide, 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat), 4-nitroquinoline 1-oxide (4-NQO), acrylamide and methyl methanesulfonate (MMS). To mimic the ocular surface condition to which ophthalmic solutions are administered, the exposure time was set to 1 minute. Likewise, human corneal epithelial (HCE-T) cells, as monolayer cultured cells, were exposed to the same chemicals, for comparison. Results: In the 3D corneal model, the amount of DNA fragments was statistically significantly increased in cells treated with each of the test chemicals except acrylamide. In HCE-T cells, the amount of DNA fragments was statistically significantly increased in acridine orange-, ethidium bromide-, hydrogen peroxide-, 4-NQO- and MMS-treated cells but not in paraquat- or acrylamide-treated cells. In the 3D corneal model, the lowest concentrations at which we observed DNA damage were about 100 times higher than the concentrations in HCE-T cells. Since the 3D corneal model is morphologically similar to human corneal tissue, form a multilayer and having tight junctions, it may be that the test chemicals only permeated about 1% into the 3D corneal model. Conclusion: These results suggest that the comet assay using 3D cell culture models may reflect in vivo conditions better than do monolayer cultured cells, and that the comet assay may be useful for the evaluation of genotoxic potential of topical ophthalmic solution.


Assuntos
Ensaio Cometa/métodos , Epitélio Anterior/efeitos dos fármacos , Soluções Oftálmicas/toxicidade , 4-Nitroquinolina-1-Óxido/toxicidade , Laranja Acridina/toxicidade , Acrilamida/toxicidade , Administração Oftálmica , Linhagem Celular , Córnea , Dano ao DNA , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Epitélio Anterior/citologia , Epitélio Anterior/metabolismo , Etídio/toxicidade , Humanos , Peróxido de Hidrogênio/toxicidade , Técnicas In Vitro , Metanossulfonato de Metila/toxicidade , Paraquat/toxicidade , Quinolonas/toxicidade
15.
Exp Eye Res ; 185: 107681, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31150636

RESUMO

Microenvironmental factors regulate stem cell fate. Fibronectin (FN), a key extracellular matrix component of the microenvironment, has been linked to various stem cell behaviors. However, how FN controls self-renewal, proliferation, and homeostasis of limbal stem cells remains unclear. Our study investigated the roles of FN in the self-renewal of rabbit limbal epithelial stem cells (rLESCs) by assessing rLESC proliferation and stemness in the presence and absence of FN. We further examined the effect of FN on non-canonical Wnt signaling during rLESC proliferation by evaluating the expression of cell cycle regulators. We found that rLESC proliferation increased after FN treatment and that 12.5 µg/cm2 FN maintained rLESC stemness. FN facilitated rLESC self-renewal by promoting Wnt11 and Fzd7 interaction. Furthermore, FN modulated cell cycle regulators to enhance rLESC proliferation via the upregulation of ROCK1 and ROCK2. Our study provides new insights into the mechanism through which FN regulates the self-renewal of rLESCs; specifically, this occurs via stimulation of the Wnt11/Fzd7/ROCK non-canonical Wnt pathway. The roles of FN in the self-renewal of limbal epithelial stem cells should be further investigated for the potential treatment of limbal deficiency.


Assuntos
Epitélio Anterior/efeitos dos fármacos , Fibronectinas/farmacologia , Receptores Frizzled/metabolismo , Limbo da Córnea/citologia , Células-Tronco/efeitos dos fármacos , Proteínas Wnt/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Western Blotting , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Epitélio Anterior/metabolismo , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Técnicas de Silenciamento de Genes , Masculino , RNA Interferente Pequeno/genética , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Células-Tronco/metabolismo , Via de Sinalização Wnt/fisiologia
16.
Exp Eye Res ; 185: 107664, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31085182

RESUMO

HSV-1 infection in corneal epithelium initiates the process of herpes simplex keratitis. We investigated the dynamic change of the host proteins in corneal epithelial cells infected with HSV-1 to understand the virus-host interaction. iTRAQ coupled with LC-MS/MS was applied to quantitatively analyze the protein profiles in HSV-1 infected corneal epithelial cells at 6 and 24 h post-infection (hpi), and the results were validated by multiple reaction monitoring (MRM). We also performed bioinformatic analysis to investigate the potentially important signal pathways and protein interaction networks in the host response to HSV-1 infection. We identified 292 proteins were up-regulated and 168 proteins were down-regulated at 6 hpi, while 132 proteins were up-regulated and 89 proteins were down-regulated at 24 hpi, which were validated by MRM analysis. We found the most enriched GO terms were translational initiation, cytosol, poly(A) RNA binding, mRNA splicing via spliceosome and extracellular exosome for the dysregulated proteins. KEGG pathway analysis revealed significant changes in metabolism pathway characterized by decreased tricarboxylic acid cycle activity and increased glycolysis. Proteins interaction network analysis indicated several proteins including P4HB, ACLY, HSP90AA1 and EIF4A3, might be critical proteins in the host-virus response. Our study for the first time analyzed the protein profile of HSV-1 infected primary corneal epithelial cells by quantitative proteomics. These findings help to better understand the host-virus interaction and the pathogenesis of herpes simplex keratitis.


Assuntos
Epitélio Anterior/virologia , Herpesvirus Humano 1/fisiologia , Western Blotting , Linhagem Celular , Cromatografia Líquida , Biologia Computacional , Regulação para Baixo , Epitélio Anterior/metabolismo , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica/fisiologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Proteômica , RNA Mensageiro/metabolismo , Transdução de Sinais , Espectrometria de Massas em Tandem , Regulação para Cima
17.
Exp Eye Res ; 182: 167-174, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30930125

RESUMO

Alzheimer's disease (AD) primarily affects the brain and is the most common form of dementia worldwide. Despite more than a century of research, there are still no early biomarkers for AD. It has been reported that AD affects the eye, which is more accessible for imaging than the brain; however, links with the cornea have not been evaluated. To investigate whether the cornea could be used to identify possible diagnostic indicators of AD, we analyzed the proteolytic processing and isoforms of amyloid precursor protein (APP) and evaluated the expression of AD-related genes and proteins in corneal fibroblasts from wild-type (WT) corneas and corneas from patients with granular corneal dystrophy type 2 (GCD2), which is related to amyloid formation in the cornea. Reverse transcription polymerase chain reaction (RT-PCR) analysis was used to assess the expression of AD-related genes, i.e., APP, ADAM10, BACE1, BACE2, PSEN1, NCSTN, IDE, and NEP. RT-PCR and DNA sequencing analysis demonstrated that isoforms of APP770 and APP751, but not APP695, were expressed in corneal fibroblasts. Moreover, the mRNA ratio of APP770/APP751 isoforms was approximately 4:1. Western blot analysis also demonstrated the expression of a disintegrin and metalloprotease domain-containing protein 10 (ADAM10), beta-site APP-cleaving enzyme 1 (BACE1), nicastrin, insulin degradation enzyme, and neprilysin in corneal fibroblasts. Among these targets, the levels of immature ADAM10 and BACE1 protein were significantly increased in GCD2 cells. The expression levels of APP, ADAM10, BACE1, and transforming growth factor-beta-induced protein (TGFBIp) were also detected by western blot in human corneal epithelium. We also investigated the effects of inhibition of the autophagy-lysosomal and ubiquitin-proteasomal proteolytic systems (UPS) on APP processing and metabolism. These pathway inhibitors accumulated APP, α-carboxy-terminal fragments (CTFs), ß-CTFs, and the C-terminal APP intracellular domain (AICD) in corneal fibroblasts. Analysis of microRNAs (miRNAs) revealed that miR-9 and miR-181a negatively coregulated BACE1 and TGFBIp, which was directly associated with the pathogenesis of AD and GCD2, respectively. Immunohistochemical analysis indicated that APP and BACE1 were distributed in corneal stroma cells, epithelial cells, and the retinal layer in mice. Collectively, we propose that the cornea, which is the transparent outermost layer of the eye and thus offers easy accessibility, could be used as a potential biomarker for AD diagnosis and progression.


Assuntos
Doença de Alzheimer/complicações , Precursor de Proteína beta-Amiloide/genética , Distrofias Hereditárias da Córnea/genética , Epitélio Anterior/metabolismo , Regulação da Expressão Gênica , RNA/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/biossíntese , Animais , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Ensaio de Imunoadsorção Enzimática , Epitélio Anterior/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Imuno-Histoquímica , Camundongos
18.
Exp Eye Res ; 184: 107-125, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30981716

RESUMO

Corneal Epithelial Stem Cells (CESCs) and their proliferative progeny, the Transit Amplifying Cells (TACs), are responsible for maintaining the integrity and transparency of the cornea. These stem cells (SCs) are widely used in corneal transplants and ocular surface reconstruction. Molecular markers are essential to identify, isolate and enrich for these cells, yet no definitive CESC marker has been established. An extensive literature survey shows variability in the expression of putative CESC markers among vertebrates; being attributed to species-specific variations, or other differences in developmental stages of these animals, approaches used in these studies and marker specificity. Here, we expanded the search for CESC markers using the amphibian model Xenopus laevis. In previous studies we found that long-term label retaining cells (suggestive of CESCs and TACs) are present throughout the larval basal corneal epithelium. In adult frogs, these cells become concentrated in the peripheral cornea (limbal region). Here, we used immunofluorescence to characterize the expression of nine proteins in the corneas of both Xenopus larvae and adults (post-metamorphic). We found that localization of some markers change between larval and adult stages. Markers such as p63, Keratin 19, and ß1-integrin are restricted to basal corneal epithelial cells of the larvae. After metamorphosis their expression is found in basal and intermediate layer cells of the adult frog corneal epithelium. Another protein, Pax6 was expressed in the larval corneas, but surprisingly it was not detected in the adult corneal epithelium. For the first time we report that Tcf7l2 can be used as a marker to differentiate cornea vs. skin in frogs. Tcf7l2 is present only in the frog skin, which differs from reports indicating that the protein is expressed in the human cornea. Furthermore, we identified the transition between the inner, and the outer surface of the adult frog eyelid as a key boundary in terms of marker expression. Although these markers are useful to identify different regions and cellular layers of the frog corneal epithelium, none is unique to CESCs or TACs. Our results confirm that there is no single conserved CESC marker in vertebrates. This molecular characterization of the Xenopus cornea facilitates its use as a vertebrate model to understand the functions of key proteins in corneal homeostasis and wound repair.


Assuntos
Biomarcadores/metabolismo , Epitélio Anterior/metabolismo , Proteínas do Olho/metabolismo , Larva/metabolismo , Xenopus laevis/metabolismo , Animais , Western Blotting , Immunoblotting , Metamorfose Biológica , Microscopia de Fluorescência , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo
19.
Int Immunopharmacol ; 71: 372-381, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30952101

RESUMO

PURPOSE: To explore the effects of netrin-1 on inflammation in Aspergillus fumigatus-infected mouse corneas and on proliferation and migration in human corneal epithelial cells (HCECs). METHODS: Netrin-1 and the receptor A2BAR were detected in normal and infected corneas from C57BL/6 mice and RAW 264.7 cells. The mice were injected subconjunctivally with recombinant netrin-1. The severity of the disease was determined by clinical scores, photography with a slit lamp, RT-PCR, western blotting, myeloperoxidase (MPO) assays and immunofluorescence staining of polymorphonuclear neutrophilic leukocytes (PMNs). The effects of netrin-1 on RAW 264.7 cells in vitro were determined by RT-PCR. The role of A2BAR was demonstrated in vivo by detecting the expression of IL-1ß, TNF-α, and IL-10 in corneas pretreated subconjunctivally with an A2BAR antagonist (PSB1115). RAW 264.7 cells were stimulated with Aspergillus fumigatus (A. fumigates) and netrin-1 with or without PSB1115 pretreatment. A cell counting kit-8 (CCK-8) assay was used to evaluate cell proliferation ability, and cell migration ability was determined by cell scratch experiments with HCECs. RESULTS: Netrin-1 expression decreased slightly after A. fumigatus infection and then increased to its peak. A2BAR expression increased at 1 day post infection (p.i.), with a subsequent decline. Compared to the PBS control, exogenous netrin-1 attenuated the inflammatory response, PMN infiltration, and expression of the proinflammatory factors IL-1ß and TNF-α, while IL-10 expression was up-regulated. In RAW 264.7 cells, recombinant netrin-1 obviously inhibited the mRNA expression of IL-1ß and TNF-α and promoted the mRNA expression of the anti-inflammatory cytokine IL-10. Pretreatment with PSB1115 resulted in disease aggravation and higher levels of the proinflammatory factors IL-1ß and TNF-α both in vivo and in vitro. And the effect of netrin-1 on inflammatory factors was abolished by PSB1115. Moreover, compared to the control treatment, exogenous netrin-1 significantly facilitated the proliferation and migration of HCECs. CONCLUSIONS: Netrin-1 attenuates inflammation in C57BL/6 mice infected with A. fumigatus, and it may play this role via the receptor A2BAR. Additionally, netrin-1 can promote the proliferation and migration of HCECs.


Assuntos
Aspergilose/metabolismo , Aspergillus fumigatus/fisiologia , Epitélio Anterior/metabolismo , Netrina-1/metabolismo , Receptor A2B de Adenosina/metabolismo , Antagonistas do Receptor A2 de Adenosina/administração & dosagem , Animais , Movimento Celular , Proliferação de Células , Citocinas/genética , Citocinas/metabolismo , Epitélio Anterior/patologia , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Netrina-1/administração & dosagem , Netrina-1/genética , Células RAW 264.7 , Proteínas Recombinantes/administração & dosagem , Xantinas/administração & dosagem
20.
Cornea ; 38(7): 905-913, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30969262

RESUMO

PURPOSE: We previously showed that cannabinoid-related GPR18 receptors are present in the murine corneal epithelium, but their function remains unknown. The related CB1 receptors regulate corneal healing, possibly via chemotaxis. We therefore examined a potential role for GPR18 in corneal epithelial chemotaxis and wound healing. METHODS: We examined GPR18 messenger RNA (mRNA) and protein expression in the cornea. We additionally examined GPR18 action in cultured bovine corneal epithelial cells (bCECs) using Boyden and tracking assays, as well as proliferation and signaling. Finally, we examined wound closure in murine corneal explants. RESULTS: GPR18 mRNA was upregulated with injury in the mouse cornea. GPR18 protein was present in basal epithelial cells of the mouse and cow and redistributed to the wound site upon injury. GPR18 ligand N-arachidonoylglycine induced bCEC chemotaxis. The endocannabinoid arachidonoylethanolamine also induced chemotaxis via fatty acid amide hydrolase-mediated metabolism to N-arachidonoylglycine. GPR18 receptor activation additionally induced bCEC proliferation. In an explant model, the GPR18 antagonist O-1918 slowed corneal epithelial cell migration and the rate of corneal wound closure. CONCLUSIONS: Corneal GPR18 activation induced both chemotaxis and proliferation in corneal epithelial cells in vitro and impacted wound healing. GPR18 may contribute to the maintenance of corneal integrity.


Assuntos
Proliferação de Células/fisiologia , Quimiotaxia/fisiologia , Lesões da Córnea/metabolismo , Epitélio Anterior/metabolismo , Receptores Acoplados a Proteínas-G/fisiologia , Cicatrização/fisiologia , Animais , Bovinos , Movimento Celular/fisiologia , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Regulação para Cima
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