RESUMO
Purpose: Corneal neovascularization (CNV) impairs corneal transparency and visual acuity. The study aims to deepen our understanding of the molecules involved in CNV induced by alkali burns, facilitate a better grasp of CNV mechanisms, and uncover potential therapeutic targets. Methods: Eighty-four mice were selected for establishing CNV models via alkali burns. On days 3, 7, and 14 after the burns, corneal observations and histological investigations were conducted. An integrated analysis of RNA sequencing (RNA-seq)-based transcriptomics and label-free quantitative proteomics was performed in both normal and burned corneas. Bioinformatics approaches, encompassing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, were applied to discern differentially expressed genes (DEGs) and crucial signaling pathways. Four potentially CNV-related genes were validated using quantitative real-time PCR (qRT-PCR) and Western blot. Results: Significant CNV was observed on the seventh day. Forty-one genes were differentially expressed in neovascularized corneas, with 15 upregulated and 26 downregulated at both mRNA and protein levels. Bioinformatics analysis revealed that these DEGs participated in diverse biological processes, encompassing retinol and retinoic acid metabolism, neutrophil chemotaxis, and actin filament assembly, along with significant enrichment pathways like cytochrome P450, tyrosine, and phenylalanine metabolism. The upregulation of lymphocyte cytosolic protein 1 (LCP1) and cysteine and glycine-rich protein 2 (CSRP2) genes and the downregulation of transglutaminase 2 (TGM2) and transforming growth factor-beta-induced (TGFBI) genes were confirmed. Conclusions: We analyzed gene expression differences in mouse corneas 7 days after alkali burns, finding 41 genes with altered expression. The exact role of these genes in CNV is not fully understood, but exploring angiogenesis-related molecules offers potential for CNV treatment or prevention.
Assuntos
Queimaduras Químicas , Neovascularização da Córnea , Animais , Camundongos , Neovascularização da Córnea/genética , Queimaduras Químicas/genética , Proteômica , Neovascularização Patológica , Perfilação da Expressão Gênica , Modelos Animais de DoençasRESUMO
INTRODUCTION: Study of inflammatory cytokines in patients with caustic gastrointestinal tract injury is sketchy. This study investigated the cytokine profiling of patients with caustic substance ingestion, and analyzed the differences between patients with severe and mild injury. METHODS: This prospective, cross-sectional study enrolled 22 patients admitted to Chang Gung Memorial Hospital between March and October 2018. All patients underwent esophagogastroduodenoscopy in 24 hours. Patients were categorized into two subgroups, as mild (<2b, n = 11) or severe (≥2b, n = 11) group. RESULTS: The neutrophil count was higher in severe than mild group (P = 0.032). Patients in mild and severe groups exhibited significantly higher circulating inflammatory cytokines than healthy control, including interleukin (IL)-2, IL-5, IL-8, IL-9, IL-12, IL-13, interferon-gamma inducible protein-10, macrophage inflammatory protein-1 beta, regulated upon activation, normal T cell expressed and presumably secreted and tumor necrosis factor-alpha. Furthermore, the levels of IL-2 and tumor necrosis factor-alpha were significantly higher in patients with severe group than mild group. Although there was no difference in cumulative survival between both groups (P = 0.147), the severe group received more operations (P = 0.035) and suffered more gastrointestinal complications (P = 0.035) than mild group. CONCLUSION: Caustic substance ingestion produces mucosal damages and leads to excessive neutrophils and inflammatory cytokines in peripheral blood.
Assuntos
Citocinas/análise , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/lesões , Traumatismos Abdominais , Adulto , Idoso , Idoso de 80 Anos ou mais , Queimaduras Químicas/genética , Queimaduras Químicas/imunologia , Cáusticos/toxicidade , Estudos Transversais , Citocinas/sangue , Feminino , Humanos , Interleucinas/análise , Interleucinas/sangue , Contagem de Leucócitos/métodos , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Estudos Prospectivos , Taiwan , Traumatismos Torácicos , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/sangueRESUMO
Leucine-rich α-2-glycoprotein-1 (LRG1) is a novel profibrotic factor that modulates transforming growth factor-ß (TGF-ß) signaling. However, its role in the corneal fibrotic response remains unknown. In the present study, we found that the LRG1 level increased in alkali-burned mouse corneas. In the LRG1-treated alkali-burned corneas, there were higher fibrogenic protein expression and neutrophil infiltration. LRG1 promoted neutrophil chemotaxis and CXCL-1 secretion. Conversely, LRG1-specific siRNA reduced fibrogenic protein expression and neutrophil infiltration in the alkali-burned corneas. The clearance of neutrophils effectively attenuated the LRG1-enhanced corneal fibrotic response, whereas the presence of neutrophils enhanced the effect of LRG1 on the fibrotic response in cultured TKE2 cells. In addition, the topical application of LRG1 elevated interleukin-6 (IL-6) and p-Stat3 levels in the corneal epithelium and in isolated neutrophils. The clearance of neutrophils inhibited the expression of p-Stat3 and IL-6 promoted by LRG1 in alkali-burned corneas. Moreover, neutrophils significantly increased the production of IL-6 and p-Stat3 promoted by LRG1 in TKE2 cells. Furthermore, the inhibition of Stat3 signaling by S3I-201 decreased neutrophil infiltration and alleviated the LRG1-enhanced corneal fibrotic response in the alkali-burned corneas. S3I-201 also reduced LRG1 or neutrophil-induced fibrotic response in TKE2 cells. In conclusion, LRG1 promotes the corneal fibrotic response by stimulating neutrophil infiltration via the modulation of the IL-6/Stat3 signaling pathway. Therefore, LRG1 could be targeted as a promising therapeutic strategy for patients with corneal fibrosis.
Assuntos
Queimaduras Químicas/genética , Quimiotaxia/efeitos dos fármacos , Queimaduras Oculares/genética , Glicoproteínas/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Álcalis , Ácidos Aminossalicílicos/farmacologia , Animais , Benzenossulfonatos/farmacologia , Queimaduras Químicas/tratamento farmacológico , Queimaduras Químicas/metabolismo , Queimaduras Químicas/patologia , Linhagem Celular , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/tratamento farmacológico , Queimaduras Oculares/patologia , Fibrose/prevenção & controle , Regulação da Expressão Gênica , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Purpose: Corneal alkali burns (CABs) are a common clinical ocular disease, presenting a poor prognosis. Although some long noncoding RNAs (lncRNAs) reportedly play a key role in epigenetic regulation associated with CABs, studies regarding the lncRNA signature in CABs remain rare and elusive. Methods: A CAB model was established in C57BL/6J mice and profiling of lncRNA expressions was performed by RNA-Seq. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to predicate the related pathological pathways and candidate genes. RT-qPCR was used to verify the expression pattern of lncRNAs and related mRNAs, both in vitro and in vivo. Data were statistically analyzed by GraphPad Prism version 6.0. Results: In all, 4436 aberrantly expressed lncRNAs were identified in CAB mice when compared with control mice. In the top 13 aberrantly expressed lncRNAs, Bc037156 and 4930511E03Rik were confirmed as the most significantly altered lncRNAs. Pathway analysis revealed that mitogen-activated protein kinase (MAPK) signaling pathway was most enriched. Following 4930511E03Rik siRNA treated, Srgn, IL-1ß and Cxcr2 were significant upregulated in corneal epithelial cells, corneal keratocytes, and bone marrow dendritic cells, with NaOH treatment. Moreover, after Bc037156 siRNA treated, expression levels of IL-1ß and Srgn were significantly downregulated in the three cell lines. Conclusions: Our study suggests that Bc037156 and 4930511E03Rik may be involved in inflammation, immune response, and neovascularization by regulating Srgn, IL-1ß, and Cxcr2 expression after CAB. These candidate lncRNAs and mRNAs may be the potential targets for the treatment strategy of the alkali injured cornea.
Assuntos
Queimaduras Químicas/genética , Lesões da Córnea/genética , Epigenômica/métodos , Queimaduras Oculares/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Transcriptoma/genética , Álcalis/toxicidade , Animais , Queimaduras Químicas/metabolismo , Queimaduras Químicas/patologia , Lesões da Córnea/induzido quimicamente , Lesões da Córnea/metabolismo , Modelos Animais de Doenças , Queimaduras Oculares/metabolismo , Queimaduras Oculares/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismoRESUMO
Alkali burn to the cornea is one of the most intractable injuries to the eye due to the opacity resulting from neovascularization (NV) and fibrosis. Numerous studies have focused on studying the effect of drugs on alkali-induced corneal injury in mouse, but fewer on the involvement of alkali-induced DNA methylation and the PI3K/AKT/mTOR signaling pathway in the mechanism of alkali-induced corneal injury. Thus, the aim of this study was to determine the involvement of DNA methyltransferase 3 B-madiated DNA methylation and PI3K/AKT/mTOR signaling modulation in the mechanism of alkali-induced corneal injury in a mouse model. To this end, we used bisulfite sequencing polymerase chain reaction and Western blot analysis, to study the effects of 5-aza-2'-deoxycytidine and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, which inhibit methyltransferase and PI3K respectively, on DNA methylation and expression of downstream effectors of PI3K related to corneal NV, including TSC1 and mTOR genes. The results showed that, after an intraperitoneal injection of rapamycin (2 mg/kg/day) for seven days, the alkali-induced opacity and NV were remarkably decreased mainly by suppressing the infiltration of immune cells into injured corneas, angiogenesis, VEGF expression and myofibroblasts differentiation; as well as by promoting corneal cell proliferation and PI3K/AKT/mTOR signaling. More significantly, these findings showed that epigenetic regulatory mechanisms by DNA methylation played a key role in corneal NV, including in corneal alkali burn-induced methylation modification and rapamycin-induced DNA demethylation which involved the regulation of the PI3K/AKT/mTOR signaling pathway at the protein level. The precise findings of morphological improvement and regulatory mechanisms are helpful to guide the use of rapamycin in the treatment of corneal angiogenesis induced by alkaline-burn.
Assuntos
Queimaduras Químicas/prevenção & controle , Lesões da Córnea/prevenção & controle , Queimaduras Oculares/induzido quimicamente , Imunossupressores/uso terapêutico , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/genética , Proteína 1 do Complexo Esclerose Tuberosa/genética , Actinas/genética , Animais , Western Blotting , Queimaduras Químicas/genética , Queimaduras Químicas/patologia , Cromonas/farmacologia , Lesões da Córnea/genética , Lesões da Córnea/patologia , Metilação de DNA , Modelos Animais de Doenças , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Hidróxido de Sódio/toxicidade , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Purpose: This study investigated the role of S100 calcium binding protein A4 (S100A4) in corneal wound healing and the underlying mechanism of the S100A4-mediated PI3K/Akt/mammalian target of rapamycin (mTOR) pathway. Methods: The rabbit corneal alkali burn model was established in vivo. S100A4 expression, wound healing, inflammation, and autophagy in rabbit cornea after alkali burn were detected. The NaOH-treated rabbit corneal stromal cells (rCSCs) were transfected with overexpressed S100A4 or silencing S100A4 to examine the effect of S100A4 on corneal wound healing in vitro. The effect of S100A4 on cell viability, proliferation, migration, invasion, fibrosis, and autophagy of rCSCs after alkali burn was analyzed. Then the functional rescue experiments were carried out. The PI3K inhibitor, LY294002, was used to elucidate the PI3K/Akt/mTOR signaling pathway in rCSCs. Results: S100A4 silencing promoted rabbit corneal wound healing by inhibiting fibrosis and inflammation and promoting autophagy in alkali-burned cornea, corresponding to increased levels of LC3, Beclin 1, and Atg4B but lowered α-smooth muscle actin, TNF-É, and p62 levels. Moreover, silencing S100A4 inhibited proliferation, migration, invasion, and fibrosis of NaOH-treated rCSCs and promoted the differentiation of rCSCs into corneal cells and the autophagy of damaged rCSCs. The inhibitory role of S100A4 in wound healing was achieved via activation of the PI3K/Akt/mTOR pathway. Conclusions: S100A4 silencing confers a promising effect on wound healing of alkali-burned cornea by blocking the PI3K/Akt/mTOR pathway, supporting the advancement of corneal gene therapies for wound healing.
Assuntos
Queimaduras Químicas/genética , Lesões da Córnea/genética , Queimaduras Oculares/genética , Regulação da Expressão Gênica , Fosfatidilinositol 3-Quinases/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100/genética , Serina-Treonina Quinases TOR/metabolismo , Álcalis/toxicidade , Animais , Autofagia , Queimaduras Químicas/metabolismo , Queimaduras Químicas/patologia , Lesões da Córnea/induzido quimicamente , Lesões da Córnea/metabolismo , Modelos Animais de Doenças , Queimaduras Oculares/metabolismo , Queimaduras Oculares/patologia , Feminino , Masculino , Coelhos , Proteína A4 de Ligação a Cálcio da Família S100/biossíntese , Transdução de Sinais , Cicatrização/genéticaRESUMO
Wound healing differs significantly between men and women in a tissue-dependent manner. Dermal wounds heal faster in women whereas mucosal wounds heal faster in men. However, the effect of sex as a variable in corneal wound healing is largely unknown. The primary objective of this study was to test whether sex is a biological variable in corneal wound healing activated by the trauma or injury using an established in vivo rabbit model with male and female New Zealand White rabbits. Corneal wounds in rabbits were produced by a single topical alkali (0.5N Sodium hydroxide) application. Serial slit-lamp, stereo biomicroscopy, and applanation tonometry evaluated corneal opacity, anterior segment ocular health, and intraocular pressure (IOP), respectively, at various times during the study. Fourteen days after alkali-wound, corneal tissues were collected after humane euthanasia to examine cellular and molecular wound healing parameters. Quantitative PCR (qPCR) and immunofluorescence were used to quantify changes in the extracellular modeling protein levels of alpha-smooth muscle actin (α-SMA), Fibronectin (FN), Collagen-I (Col-I), and Transforming growth factor beta 1 (TGFß1) involved in corneal healing. Hematoxylin and Eosin (H&E) staining was used to study histopathological changes in morphology and TUNEL assay to evaluate levels of apoptotic cell death. Male and female rabbits showed no significant differences in corneal opacity (Fantes score) or intraocular pressure (IOP) values (9.5⯱â¯0.5â¯mm Hg) in live animals. Likewise, no statistically significant sex-based differences in the mRNA levels of α-SMA (maleâ¯=â¯5.95⯱â¯0.21 fold vs. femaleâ¯=â¯5.32⯱â¯0.043), FN (maleâ¯=â¯3.02⯱â¯0.24 fold vs. femaleâ¯=â¯3.23⯱â¯0.27), Col-I (maleâ¯=â¯3.12⯱â¯0.37 fold vs. femaleâ¯=â¯3.31⯱â¯0.24), TGFß1 (maleâ¯=â¯1.65⯱â¯0.06 fold vs. femaleâ¯=â¯1.59⯱â¯0.053); and protein levels of α-SMA (maleâ¯=â¯74.16⯱â¯4.6 vs. femaleâ¯=â¯71.58⯱â¯7.1), FN (maleâ¯=â¯60.11⯱â¯4.6 vs. femaleâ¯=â¯57.41⯱â¯8.3), Col-I (maleâ¯=â¯84.11⯱â¯2.8 vs. femaleâ¯=â¯84.55⯱â¯3.6), TGFß1 (maleâ¯=â¯11.61⯱â¯2.8 vs. femaleâ¯=â¯9.5⯱â¯3.04) were observed. Furthermore, H&E and TUNEL analyses found no statistically significant differences in cellular structures and apoptosis, respectively, in male vs. female corneas. Consistent with earlier reports, wounded corneas showed significantly increased levels of these parameters compared to the unwounded corneas. Our data suggest that sex is not a major biological variable during active early stages of corneal wound healing in rabbits in vivo.
Assuntos
Queimaduras Químicas/fisiopatologia , Lesões da Córnea/fisiopatologia , Queimaduras Oculares/induzido quimicamente , Fatores Sexuais , Cicatrização/fisiologia , Actinas/genética , Animais , Queimaduras Químicas/genética , Colágeno Tipo I/genética , Lesões da Córnea/genética , Queimaduras Oculares/genética , Queimaduras Oculares/fisiopatologia , Fibronectinas/genética , Imunofluorescência , Marcação In Situ das Extremidades Cortadas , RNA Mensageiro/genética , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Hidróxido de Sódio/toxicidade , Fator de Crescimento Transformador beta1/genéticaRESUMO
Neprilysin (NEP), an ectoenzyme that modulates inflammation by degrading neuropeptides, was recently identified in the human corneal epithelium. The cornea expresses many NEP substrates, but the function of NEP in homeostatic maintenance and wound healing of the cornea is unknown. We therefore investigated the role of this enzyme under naive and injured conditions using NEP-deficient (NEP-/-) and wild type (WT) control mice. In vivo ocular surface imaging and histological analysis of corneal tissue showed no differences in limbal vasculature or corneal anatomy between naive NEP-/- and WT mice. Histological examination revealed increased corneal innervation in NEP-/- mice. In an alkali burn model of corneal injury, corneal wound healing was significantly accelerated in NEP-/- mice compared to WT controls 3 days after injury. Daily intraperitoneal administration of the NEP inhibitor thiorphan also accelerated corneal wound healing after alkali injury in WT mice. Collectively, our data identify a previously unknown role of NEP in the cornea, in which pharmacologic inhibition of its activity may provide a novel therapeutic option for patients with corneal injury.
Assuntos
Queimaduras Químicas/tratamento farmacológico , Lesões da Córnea/tratamento farmacológico , Neprilisina/antagonistas & inibidores , Inibidores de Proteases/uso terapêutico , Tiorfano/uso terapêutico , Cicatrização/efeitos dos fármacos , Animais , Queimaduras Químicas/genética , Queimaduras Químicas/patologia , Córnea/efeitos dos fármacos , Córnea/metabolismo , Córnea/patologia , Lesões da Córnea/genética , Lesões da Córnea/patologia , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neprilisina/genéticaRESUMO
The aim of the present study was to investigate the effect of microRNA 146a (miR146a) on promoting the repair of corneal alkali burn with bone marrow mesenchymal stem cells (MSCs). A total of 24 SpragueDawley female rats were divided into a normal group (Control), a normal MSC treatment group (Normal MSCs), an miR146a knockout MSC treatment group (miR146alow MSCs) and an miR146a highexpression MSC treatment group (miR146ahigh MSCs) according to the random number table. Quantitative polymerase chain reaction was used to evaluate the expression levels of miR146a. MTT assay was performed to measure the cell viability of mesenchymal stem cells (MSCs) and apoptosis was measured by flow cytometry. The expression levels of p65 nuclear factor (NF)κB, proliferating cell nuclear antigen (PCNA) and Fas proteins were analyzed by western blotting. MSCs were tested for the secretion levels of vascular endothelial growth factor (VEGF), CD45, interferon (IFN)γ and interleukin (IL)10 by ELISA. The miR146ahigh MSCs improved cell viability of MSCs and inhibited apoptosis of MSCs following alkali burn. miR146ahigh MSCs decreased the expression levels of p65NFκB and PCNA, and enhanced the expression level of Fas. Furthermore, miR146ahigh MSCs improved the cornea opacity and enhanced the inhibition of neovascularization in the rats following alkali burn. miR146ahigh MSCs inhibit the expression of VEGF, CD45, IFNγ, while enhanced the expression of IL10. Therefore, miR146a promotes the repair of corneal alkali burn in rats treated with MSCs.
Assuntos
Queimaduras Químicas/terapia , Queimaduras Oculares/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Regulação para Cima , Álcalis/efeitos adversos , Animais , Apoptose , Queimaduras Químicas/genética , Queimaduras Químicas/patologia , Sobrevivência Celular , Células Cultivadas , Córnea/patologia , Neovascularização da Córnea/genética , Neovascularização da Córnea/patologia , Neovascularização da Córnea/terapia , Modelos Animais de Doenças , Queimaduras Oculares/genética , Queimaduras Oculares/patologia , Feminino , Técnicas de Inativação de Genes , Células-Tronco Mesenquimais/citologia , Ratos , Ratos Sprague-DawleyRESUMO
Exposure to mustard gas can lead to variations in the proteome of corneal epithelium cells and after a latency period produces delayed symptoms in the eyes of chemical victims. Hence, a comparative proteome analysis was conducted between the corneal epithelial cells of chemical victims from Iran-Iraq war (1980-1988) and healthy donors. To this end, corneal epithelium samples from victims and healthy individuals were collected, and the proteome of these samples were prepared for two-dimensional electrophoresis and the analysis of spots by statistical software. Selected spots were further analyzed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Twenty four proteins were identified of which eighteen proteins showed downregulation while six proteins were upregulated in the victims in comparison to the normal individuals. Also, six protein spots were confirmed by western-blot analysis. In conclusion, all the twenty-four identified proteins are involved in pathways which their up- or down-regulation leads to the accumulation of undesired substrates, cell death and apoptosis. Bioinformatics' tools indicated that these identified proteins were involved in various metabolic processes, DNA damage response, immune response and etc. The present study provides a suitable platform for further clinical studies.
Assuntos
Queimaduras Químicas/genética , Substâncias para a Guerra Química/toxicidade , Epitélio Corneano/metabolismo , Proteínas do Olho/genética , Gás de Mostarda/toxicidade , Proteoma/genética , Adulto , Queimaduras Químicas/etiologia , Queimaduras Químicas/metabolismo , Queimaduras Químicas/patologia , Eletroforese em Gel Bidimensional , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/patologia , Proteínas do Olho/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Guerra do Iraque 2003-2011 , Masculino , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Mapeamento de Interação de Proteínas , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
PURPOSE: To characterize the molecular, clinical, and histopathological profiles in the rat cornea after alkali injury over a 21-day period. METHODS: Alkali injury was induced in one eye of male Lewis rats. Corneal opacity and corneal neovascularization were assessed daily. Real-time qRT-PCR analysis and immunohistochemical staining were conducted to examine inflammation, neovascularization, and fibrosis. RESULTS: We found that within 2 hours of chemical exposure, corneal opacification rapidly developed with an acute increase in various cytokine expressions, while several cytokines demonstrated a secondary peak by day 7. Early neutrophil infiltration peaked at day 1 post-injury while macrophage infiltration peaked at day 7. Throughout the time course of the study, corneal opacity persisted and neovascularization, lymphangiogenesis, and fibrosis progressed. CONCLUSIONS: This study highlights the molecular, clinical, and histopathological changes throughout the progression of alkali injury in the rat cornea. These profiles will assist in the development of new strategies and therapies for ocular alkali injury.
Assuntos
Queimaduras Químicas/patologia , Neovascularização da Córnea/patologia , Opacidade da Córnea/patologia , Modelos Animais de Doenças , Queimaduras Oculares/induzido quimicamente , Animais , Queimaduras Químicas/genética , Queimaduras Químicas/imunologia , Córnea/efeitos dos fármacos , Córnea/patologia , Neovascularização da Córnea/induzido quimicamente , Neovascularização da Córnea/genética , Neovascularização da Córnea/imunologia , Opacidade da Córnea/induzido quimicamente , Opacidade da Córnea/genética , Opacidade da Córnea/imunologia , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Queimaduras Oculares/genética , Queimaduras Oculares/imunologia , Fibrose , Expressão Gênica/fisiologia , Inflamação/patologia , Linfangiogênese/imunologia , Macrófagos/imunologia , Masculino , Neutrófilos/imunologia , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase em Tempo Real , Hidróxido de Sódio , CicatrizaçãoRESUMO
OBJECTIVE: The purpose of this study is to explore the inhibitory effects of S100A4 gene silencing on alkali burn-induced corneal neovascularization (CNV) in rabbit models. METHODS: Sixty-five rabbits were used to establish alkali-induced CNV models. After the operation, rabbits were given daily antibiotic eye drops and an eye ointment to prevent infection. The models were assigned to either an S100A4 siRNA or an empty vector group. Thirty rabbits were selected as the normal control group. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the mRNA expression of S100A4, vascular endothelial growth factor (VEGF), and tumor necrosis factor-α (TNF-α) in corneal tissues. Immunohistochemistry was used to detect the protein expression of VEGF in corneal tissues, and an enzyme-linked immunosorbent (ELISA) assay was used to detect the protein expression of VEGF and TNF-α in the aqueous humor. RESULTS: The qRT-PCR results showed that S100A4 mRNA expression was lower in the S100A4 siRNA group than in the empty vector group at 1, 3, 7, 14, and 28 days after an alkali burn. When compared with the empty vector group, the expression of VEGF and TNF-α mRNA was downregulated in the S100A4 siRNA group. The immunohistochemistry results revealed that VEGF protein expression was downregulated in the S100A4 siRNA group when compared to the empty vector group at 1, 3, 7, 14, and 28 days after an alkali burn. The ELISA results suggest that VEGF and TNF-α protein expression is downregulated in the S100A4 siRNA group in comparison to the empty vector group at 1, 3, 7, 14, and 28 days after an alkali burn. CONCLUSIONS: These findings indicate that S100A4 gene silencing can inhibit alkali burn-induced CNV in rabbits.
Assuntos
Queimaduras Químicas/genética , Queimaduras Químicas/patologia , Neovascularização da Córnea/induzido quimicamente , Neovascularização da Córnea/genética , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/genética , Inativação Gênica , Proteína A4 de Ligação a Cálcio da Família S100/genética , Álcalis , Animais , Humor Aquoso/metabolismo , Córnea/metabolismo , Córnea/patologia , Neovascularização da Córnea/patologia , Queimaduras Oculares/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The aim of this study was to examine whether mesenchymal stem cells (MSCs) and/or corneal limbal epithelial stem cells (LSCs) influence restoration of an antioxidant protective mechanism in the corneal epithelium and renewal of corneal optical properties changed after alkali burns. The injured rabbit corneas (with 0.25 N NaOH) were untreated or treated with nanofiber scaffolds free of stem cells, with nanofiber scaffolds seeded with bone marrow MSCs (BM-MSCs), with adipose tissue MSCs (Ad-MSCs), or with LSCs. On day 15 following the injury, after BM-MSCs or LSCs nanofiber treatment (less after Ad-MSCs treatment) the expression of antioxidant enzymes was restored in the regenerated corneal epithelium and the expressions of matrix metalloproteinase 9 (MMP9), inducible nitric oxide synthase (iNOS), α-smooth muscle actin (α-SMA), transforming growth factor-ß1 (TGF-ß1), and vascular endothelial factor (VEGF) were low. The central corneal thickness (taken as an index of corneal hydration) increased after the injury and returned to levels before the injury. In injured untreated corneas the epithelium was absent and numerous cells revealed the expressions of iNOS, MMP9, α-SMA, TGF-ß1, and VEGF. In conclusion, stem cell treatment accelerated regeneration of the corneal epithelium, restored the antioxidant protective mechanism, and renewed corneal optical properties.
Assuntos
Antioxidantes/uso terapêutico , Queimaduras Químicas/terapia , Epitélio Corneano/patologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Substâncias Protetoras/uso terapêutico , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Álcalis , Animais , Queimaduras Químicas/enzimologia , Queimaduras Químicas/genética , Queimaduras Químicas/patologia , Diferenciação Celular/efeitos dos fármacos , Opacidade da Córnea/complicações , Opacidade da Córnea/terapia , Paquimetria Corneana , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Limbo da Córnea/citologia , Metaloproteinase 9 da Matriz/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Substâncias Protetoras/farmacologia , Coelhos , Superóxido Dismutase/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: Impaired healing of corneal injuries can result in ulceration and complete loss of vision, especially in the elderly. Such patients frequently also exhibit vitamin D insufficiency. 1,25-dihydroxyvitamin D3 is the active vitamin D metabolite. As it affects cell proliferation and inflammation, we herein aimed at elucidating its influence on corneal wound healing after alkali burn by using in vitro and ex vivo techniques. METHODS: mRNA abundance in human corneal epithelial cells in response to vitamin D3 was determined by RT-PCR. Corneal re-epithelialization after alkaline burn was analyzed using enucleated mouse eyes and fluorescein staining. RESULTS: Human corneal epithelial cells (HCEC) expressed the vitamin D receptor (VDR) and retinoid x receptor (RXR) and were responsive to 1,25- dihydroxyvitamin D3, as shown by induction of the 1,25- dihydroxyvitamin D3 responsive gene cyp-24A1 and slightly reduced abundance of IL-6 mRNA. However, no effect on cell vitality and migration was observed. In contrast, re-epithelialization of mouse corneas ex vivo was dose dependently inhibited by 1,25- dihydroxyvitamin D3. CONCLUSIONS: These data indicate that topically applied 1,25- dihydroxyvitamin D3 does not seem to be suitable for therapy of corneal lesions.
Assuntos
Queimaduras Químicas/tratamento farmacológico , Calcitriol/farmacologia , Doenças da Córnea/tratamento farmacológico , Queimaduras Oculares/induzido quimicamente , Vitaminas/farmacologia , Cicatrização/efeitos dos fármacos , Administração Tópica , Animais , Queimaduras Químicas/genética , Queimaduras Químicas/metabolismo , Calcitriol/administração & dosagem , Linhagem Celular , Doenças da Córnea/genética , Doenças da Córnea/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reepitelização/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Hidróxido de Sódio , Vitamina D3 24-Hidroxilase/genética , Vitamina D3 24-Hidroxilase/metabolismo , Vitaminas/administração & dosagemRESUMO
PURPOSE: To evaluate the effects of dry eye on ocular surface protease activity and sight threatening corneal complications following ocular surface chemical injury. METHODS: C57BL/6 mice were subjected to unilateral alkali burn (AB) with or without concomitant dry eye for 2 or 5 days. Mice were observed daily for appearance of corneal perforation. Whole corneas were harvested and lysed for RNA extraction. Quantitative real-time PCR was performed to measure expression of inflammation cytokines, matrix metalloproteinases (MMP). Matrix metalloproteinase-9 activity, gelatinase activity, and myeloperoxidase (MPO) activity were evaluated in corneal lysates. Presence of infiltrating neutrophils was evaluated by immunohistochemistry and flow cytometry. RESULTS: Eyes subjected to the combined model of AB and dry eye (CM) had 20% sterile corneal perforation rate as soon as 1 day after the initial injury, which increased to 35% by 5 days, delayed wound closure and increased corneal opacity. Increased levels of IL-1ß, -6, and MMPs-1, -3, -8, -9, and -13, and chemokine (C-X-C motif) ligand 1 (CSCL1) transcripts were found after 2 days in CM compared with AB corneas. Increased MMP-1, -3, -9, and -13 immunoreactivity and gelatinolytic activity were seen in CM corneas compared with AB. Increased neutrophil infiltration and MPO activity was noted in the CM group compared with AB 2 days post injury. CONCLUSIONS: Desiccating stress worsens outcome of ocular AB, creating a cytokine and protease storm with greater neutrophil infiltration, increasing the risk of corneal perforation.
Assuntos
Queimaduras Químicas/genética , Queimaduras Oculares/genética , Regulação da Expressão Gênica , Metaloproteinase 9 da Matriz/genética , Estresse Oxidativo , RNA/genética , Cicatrização , Álcalis/toxicidade , Animais , Queimaduras Químicas/enzimologia , Queimaduras Químicas/patologia , Modelos Animais de Doenças , Progressão da Doença , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/enzimologia , Queimaduras Oculares/patologia , Citometria de Fluxo , Metaloproteinase 9 da Matriz/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo RealRESUMO
PURPOSE: Chemical burn in cornea may cause permanent visual problem or complete blindness. In the present study, we investigated the role of microRNA 206 (miR-206) in relieving chemical burn in mouse cornea. METHOD: An alkali burn model was established in C57BL/6 mice to induce chemical corneal injury. Within 72 hours, the transient inflammatory responses in alkali-treated corneas were measured by opacity and corneal neovascularization (CNV) levels, and the gene expression profile of miR-206 was measured by quantitative real-time PCR (qPCR). Inhibitory oligonucleotides of miR-206, miR-206-I, were intrastromally injected into alkali-burned corneas. The possible protective effects of down-regulating miR-206 were assessed by both in vivo measurements of inflammatory responses and in vitro histochemical examinations of corneal epithelium sections. The possible binding of miR-206 on its molecular target, connexin43 (Cx43), was assessed by luciferase reporter (LR) and western blot (WB) assays. Cx43 was silenced by siRNA to examine its effect on regulating miR-206 modulation in alkali-burned cornea. RESULTS: Opacity and CNV levels, along with gene expression of miR-206, were all transiently elevated within 72 hours of alkali-burned mouse cornea. Intrastromal injection of miR-206-I into alkali-burned cornea down-regulated miR-206 and ameliorated inflammatory responses both in vivo and in vitro. LR and WB assays confirmed that Cx43 was directly targeted by miR-206 in mouse cornea. Genetic silencing of Cx43 reversed the protective effect of miR-206 down-regulation in alkali-burned cornea. CONCLUSION: miR-206, associated with Cx43, is a novel molecular modulator in alkali burn in mouse cornea.
Assuntos
Queimaduras Químicas/terapia , Conexina 43/metabolismo , Córnea/metabolismo , Lesões da Córnea/terapia , Neovascularização da Córnea/terapia , MicroRNAs/metabolismo , Oligonucleotídeos/administração & dosagem , Hidróxido de Sódio , Animais , Queimaduras Químicas/genética , Queimaduras Químicas/metabolismo , Queimaduras Químicas/patologia , Conexina 43/genética , Córnea/irrigação sanguínea , Córnea/patologia , Lesões da Córnea/induzido quimicamente , Lesões da Córnea/genética , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Neovascularização da Córnea/induzido quimicamente , Neovascularização da Córnea/genética , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Opacidade da Córnea/induzido quimicamente , Opacidade da Córnea/genética , Opacidade da Córnea/metabolismo , Opacidade da Córnea/patologia , Opacidade da Córnea/prevenção & controle , Modelos Animais de Doenças , Regulação para Baixo , Células HEK293 , Humanos , Injeções Intraoculares , Ceratite/induzido quimicamente , Ceratite/genética , Ceratite/metabolismo , Ceratite/patologia , Ceratite/prevenção & controle , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Transfecção , CicatrizaçãoRESUMO
PURPOSE: In this study, we aimed to assess whether the expression pattern of galectins is altered in Pseudomonas aeruginosa-infected and chemically burned mouse corneas. METHODS: Galectin (Gal) fingerprinting of normal, P. aeruginosa-infected, and silver nitrate-cauterized corneas was performed by Western blotting, immunofluorescence staining, and qRT-PCR. RESULTS: In normal corneas, Gal-1 was distributed mainly in the stroma, Gal-3 was localized mainly in epithelium, and Gal-7, -8, and -9 were detected in both corneal epithelium and stroma. Expression levels of the five galectins were drastically altered under pathological conditions. In both infected and cauterized corneas, overall Gal-3 expression was downregulated, whereas overall Gal-8 and -9 were upregulated. Changes in the expression level of Gal-7, -8, and -9 were distinct in the epithelium of infected and cauterized corneas. Expression of these three galectins was upregulated in corneal epithelium of infected corneas but not in cauterized corneas. Consistent with the changes in protein expression: (1) Gal-7, -8, and -9 mRNA expression was upregulated in cauterized corneas, and (2) Gal-3 mRNA was downregulated and Gal-9 mRNA expression was upregulated in infected corneas. CONCLUSIONS: Our data demonstrate differential regulation of various members of the galectin family in the course of corneal infection and neovascularization. The emerging functionality of the sugar code of cell surface receptors via endogenous galectins reflect to the pertinent roles of the five tested galectins in the diseases of cornea.
Assuntos
Queimaduras Químicas/genética , Córnea/metabolismo , Lesões da Córnea/genética , Infecções Oculares Bacterianas/genética , Galectinas/genética , Pseudomonas aeruginosa/isolamento & purificação , RNA Mensageiro/genética , Animais , Western Blotting , Queimaduras Químicas/metabolismo , Queimaduras Químicas/patologia , Córnea/patologia , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Impressões Digitais de DNA/métodos , Infecções Oculares Bacterianas/metabolismo , Infecções Oculares Bacterianas/microbiologia , Galectinas/metabolismo , Camundongos , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecção dos Ferimentos/genética , Infecção dos Ferimentos/metabolismo , Infecção dos Ferimentos/microbiologiaRESUMO
BACKGROUND: Lymphangiogenesis is one of the major causes of corneal graft rejection. Among the lymphangiogenic factors, vascular endothelial growth factor (VEGF)-C and -D are considered to be the most potent. Both bind to VEGF receptor 3 (VEGFR3) to activate Prospero homeobox 1 (Prox1), a transcription factor essential for the development and maintenance of lymphatic vasculature. MicroRNAs (miRNAs) bind to the 3' untranslated regions (3' UTRs) of target genes in a sequence-specific manner and suppress gene expression. In the current study, we searched for miRNAs that target the pro-lymphangiogenic factor Prox1. RESULTS: Among the miRNAs predicted by the bioinformatic analysis to seed match with the 3' UTR of Prox-1, we chose 3 (miR-466, miR-4305, and miR-4795-5p) for further investigation. Both the miR-466 and miR-4305 mimics, but not the miR-4795-5p mimic, significantly reduced the luciferase activity of the Prox-1 3' UTR reporter vector. In primary lymphatic endothelial cells (HDLEC), miR-466 mimic transfection suppressed Prox1 mRNA and protein expression, while miR-4305 mimic transfection did not. Experiments using mutated reporter constructs of the two possible seed match sites on the 3' UTR of Prox1 suggested that the target site 2 directly bound miR-466. HDLEC transfected with the miR-466 mimic suppressed tube formation as compared to the scrambled control. Furthermore, HDLEC transfected with a miR-466 inhibitor showed enhanced tube formation as compared to control inhibitor transfected cells, and this inhibitory effect was counteracted by Prox1 siRNA. The miR-466 mimic reduced angiogenesis and lymphangiogenesis resulting in clearer corneas in an cornea injury rat model compared to the scrambled control. CONCLUSIONS: Our data suggest that miR-446 may have a protective effect on transplanted corneas by suppressing Prox1 expression at the post-transcriptional level. The results of the current study may provide insights into the mechanisms of lymphangiogenesis resulting from corneal graft rejection and alkali-burn injuries, as well as into the development of new treatments for lymphangiogenic eye diseases.
Assuntos
Queimaduras Químicas/genética , Lesões da Córnea/genética , Queimaduras Oculares/genética , Proteínas de Homeodomínio/genética , Linfangiogênese , MicroRNAs/genética , Proteínas Supressoras de Tumor/genética , Álcalis/toxicidade , Animais , Queimaduras Químicas/etiologia , Lesões da Córnea/induzido quimicamente , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Queimaduras Oculares/induzido quimicamente , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , MicroRNAs/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Supressoras de Tumor/metabolismoRESUMO
Sulfur mustard (SM) is a strong bifunctional alkylating agent that produces severe tissue injuries characterized by erythema, edema, subepidermal blisters and a delayed inflammatory response after cutaneous exposure. However, despite its long history, SM remains a threat because of the lack of effective medical countermeasures as the molecular mechanisms of these events remain unclear. This limited number of therapeutic options results in part of an absence of appropriate animal models. We propose here to use SKH-1 hairless mouse as the appropriate model for the design of therapeutic strategies against SM-induced skin toxicity. In the present study particular emphasis was placed on histopathological changes associated with inflammatory responses after topical exposure of dorsal skin to three different doses of SM (0.6, 6 and 60mg/kg) corresponding to a superficial, a second-degree and a third-degree burn. Firstly, clinical evaluation of SM-induced skin lesions using non invasive bioengineering methods showed that erythema and impairment of skin barrier increased in a dose-dependent manner. Histological evaluation of skin sections exposed to SM revealed that the time to onset and the severity of symptoms including disorganization of epidermal basal cells, number of pyknotic nuclei, activation of mast cells and neutrophils dermal invasion were dose-dependent. These histopathological changes were associated with a dose- and time-dependent increase in expression of specific mRNA for inflammatory mediators such as interleukins (IL1ß and IL6), tumor necrosis factor (TNF)-α, cycloxygenase-2 (COX-2), macrophage inflammatory proteins (MIP-1α, MIP-2 and MIP-1αR) and keratinocyte chemoattractant (KC also called CXCL1) as well as adhesion molecules (L-selectin and vascular cell adhesion molecule (VCAM)) and growth factor (granulocyte colony-stimulating factor (Csf3)). A dose-dependent increase was also noted after SM exposure for mRNA of matrix metalloproteinases (MMP9) and laminin-γ2 which are associated with SM-induced blisters formation. Taken together, our results show that SM-induced skin histopathological changes related to inflammation is similar in SKH-1 hairless mice and humans. SKH-1 mouse is thus a reliable animal model for investigating the SM-induced skin toxicity and to develop efficient treatment against SM-induced inflammatory skin lesions.