RESUMEN
We investigated the effects of loss of tenascin C on the healing of the stroma using incision-injured mice corneas. Tenascin C was upregulated in the stroma following incision injury to the cornea. Wild-type (WT) and tenascin C-null (knockout (KO)) mice on a C57BL/6 background were used. Cell culture experiments were also conducted to determine the effects of the lack of tenascin C on fibrogenic gene expression in ocular fibroblasts. Histology, immunohistochemistry and real-time reverse transcription PCR were employed to evaluate the healing process in the stroma. The difference in the incidence of wound closure was statistically analyzed in hematoxylin and eosin-stained samples between WT and KO mice in addition to qualitative observation. Healing of incision injury in corneal stroma was delayed, with less appearance of myofibroblasts, less invasion of macrophages and reduction in expression of collagen Iα1, fibronectin and transforming growth factor ß1 (TGFß1) in KO mice compared with WT mice. In vitro experiments showed that the loss of tenascin C counteracted TGFß1 acceleration of mRNA expression of TGFß1, and of collagen Iα1 and of myofibroblast conversion in ocular fibroblasts. These results indicate that tenascin C modulates wound healing-related fibrogenic gene expression in ocular fibroblasts and is required for primary healing of the corneal stroma.
Asunto(s)
Lesiones de la Cornea , Sustancia Propia/fisiopatología , Regulación de la Expresión Génica/genética , Tenascina/deficiencia , Cicatrización de Heridas/fisiología , Análisis de Varianza , Animales , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Fibronectinas/metabolismo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
The extracellular matrix components osteopontin and tenascin-C are ligands of α9 integrin, and both play roles in corneal wound fibrosis and neovascularization. It has been shown that loss of osteopontin impairs closure of incisional wounds in the mouse cornea. Detailed analyses suggest that the loss of osteopontin reduces macrophage invasion and myofibroblast differentiation in the healing stroma in association with suppression of fibrogenic gene expression in response to injury. Cultured ocular fibroblasts derived from knockout mice showed an impairment of activation of p38 MAPK and Smad3 upon exposure to transforming growth factor ß1. The loss of tenascin-C delays stromal healing in association with suppression of fibrogenic gene expression and macrophage invasion. With regard to neovascularization, the loss of either osteopontin or tenascin-C suppressed the growth of new blood vessels from the limbal region toward the central cornea in response to corneal cauterization in mice. Gene expression analysis further showed that lack of osteopontin or tenascin-C resulted in inhibition of angiogenic and proinflammatory gene expression. In conclusion, osteopontin or tenascin-C, α9 integrin ligands, play an important role in stromal healing (or fibrosis) and neovascularization in mouse cornea.
Asunto(s)
Sustancia Propia/metabolismo , Proteínas del Ojo/fisiología , Osteopontina/fisiología , Tenascina/fisiología , Cicatrización de Heridas/fisiología , Animales , Humanos , Cadenas alfa de Integrinas/fisiologíaRESUMEN
We examined whether absence or blocking of transient receptor potential vanilloid subtype 1 (TRPV1) affects the level of inflammation and fibrosis/scarring during healing of injured tissue using an alkali burn model of cornea in mice. A cornea burn was produced with 1 N NaOH instilled into one eye of TRPV1-/- (KO) (n = 88) or TRPV1+/+ (n = 94) mice. Examinations of the corneal surface and eye globe size suggested that the loss of TRPV1 suppressed inflammation and fibrosis/scarring after alkali burn, and this was confirmed by histology, IHC, and gene expression analysis. The loss of TRPV1 inhibited inflammatory cell invasion and myofibroblast generation in association with reduction of expression of proinflammatory and profibrogenic components. Experiments of bone marrow transplantation between either genotype of mice showed that KO corneal tissue resident cells, but not KO bone marrow-derived cells, are responsible for KO-type wound healing with reduced inflammation and fibrosis. The absence of TRPV1 attenuated expression of transforming growth factor ß 1 (TGFß1) and other proinflammatory gene expression in cultured ocular fibroblasts, but did not affect TGFß1 expression in macrophages. Loss of TRPV1 inhibited myofibroblast transdifferentiation in cultured fibroblasts. Systemic TRPV1 antagonists reproduced the KO type of healing. In conclusion, absence or blocking of TRPV1 suppressed inflammation and fibrosis/scarring during healing of alkali-burned mouse cornea. TRPV1 is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing.
Asunto(s)
Quemaduras Oculares/patología , Inflamación/metabolismo , Inflamación/patología , Canales Catiónicos TRPV/metabolismo , Álcalis , Animales , Células Cultivadas , Técnicas de Cocultivo , Córnea/metabolismo , Córnea/patología , Quemaduras Oculares/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Regulación de la Expresión Génica , Inmunohistoquímica , Inflamación/genética , Macrófagos/metabolismo , Macrófagos/patología , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/genética , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Cicatrización de Heridas/genéticaRESUMEN
PURPOSE: To examine the effects of a histone deacetylase inhibitor, Trichostatin A (TSA), on the behavior of macrophages and subconjunctival fibroblasts in vitro and on ocular surface inflammation and scarring in vivo using an alkali burn wound healing model. METHODS: Effects of TSA on expression of inflammation-related growth factors or collagen I were examined by real-time RT-PCR or immunoassay in mouse macrophages or human subconjunctival fibroblasts. Effects of TSA on trans forming growth factor ß (TGFß)/Smad signaling were evaluated with western blotting and/or immunocytochemistry. Alkali-burn injuries on the eyes of mice were performed with three µl of 0.5 N NaOH under general and topical anesthesia. TSA (600 µg/Kg daily) or vehicle was administered to animals via intraperitoneal (i.p.) injection. Histology and real-time RT-PCR investigations evaluated the effects of TSA on the healing process of the cornea. RESULTS: TSA inhibited TGFß 1 and vascular endothelial growth factor (VEGF) expression in macrophages, and TGFß1 and collagen I in ocular fibroblasts. It elevated the expression of 5'-TG-3'-interacting factor (TGIF) and Smad7 in fibroblasts and blocked nuclear translocation of phospho-Smad2. Real-time PCR and immunocytochemistry studies showed that systemic administration of TSA suppressed the inflammation and fibrotic response in the stroma and accelerated epithelial healing in the alkali-burned mouse cornea. CONCLUSIONS: Systemic administration of TSA reduces inflammatory and fibrotic responses in the alkali-burned mouse ocular surface in vivo. The mechanisms of action involve attenuation of Smad signal in mesenchymal cells and reduction in the activation and recruitment of macrophages. TSA has the potential to treat corneal scarring in vivo.
Asunto(s)
Oftalmopatías/tratamiento farmacológico , Oftalmopatías/patología , Ácidos Hidroxámicos/uso terapéutico , Inflamación/complicaciones , Inflamación/tratamiento farmacológico , Animales , Quemaduras Químicas/complicaciones , Quemaduras Químicas/tratamiento farmacológico , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Conjuntiva/patología , Citocinas/metabolismo , Oftalmopatías/complicaciones , Oftalmopatías/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ácidos Hidroxámicos/farmacología , Inflamación/genética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Neovascularización Patológica/complicaciones , Neovascularización Patológica/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
PURPOSE: To investigate the role of tenascin-C in epithelial-mesenchymal transition (EMT) of the lens epithelium during wound healing in mice. Tenascin-C is a component of the extracellular matrix in patients having post-operative capsular opacification. METHODS: The crystalline lens was injured by needle puncture in tenascin-C null (KO, n=56) and wild-type (WT, n=56) mice in a C57BL/6 background. The animals were killed at day 2, 5, or 10 post-injury. Immunohistochemistry was employed to detect alpha-smooth muscle actin (alphaSMA), a marker of EMT, collagen type I, transforming growth factor beta1 (TGFbeta1), phospho-Smad2, phospho-adducin, and phospho-myosin light chain 9 (MLC9). The expression levels of phospho-adducin and phospho-MLC9 were used as markers for the activation of protein kinase C and Rho kinase, respectively. RESULTS: The expression of tenascin-C was upregulated in WT lens epithelial cells adjacent to the capsular break at day 5. The results showed that injury-induced EMT of the mouse lens epithelium, as evaluated by histology and the expression patterns of alphaSMA and fibronectin, was attenuated in the absence of tenascin-C. Upregulation of TGFbeta1 expression in the epithelium was also inhibited, and loss of tenascin-C attenuated the phosphorylation of Smad2 and adducin in epithelial cells adjacent to the capsular break. The expression of phospho-adducin was suppressed, while the expression level of phospho-MLC9 was unchanged, in the healing epithelium in the absence of tenascin C. CONCLUSIONS: Tenascin-C is required for injury-induced EMT in the mouse lens epithelium. The mechanism behind this might involve impaired activation of cytoplasmic signaling cascades; i.e., TGFbeta/Smad and protein kinase C-adducing signaling, in the absence of tenascin-C.
Asunto(s)
Epitelio/lesiones , Epitelio/patología , Cristalino/lesiones , Cristalino/patología , Mesodermo/patología , Tenascina/deficiencia , Animales , Proteínas de Unión a Calmodulina/metabolismo , Epitelio/metabolismo , Fibronectinas/metabolismo , Inmunohistoquímica , Cristalino/metabolismo , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteína Smad2/metabolismo , Tenascina/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Maintenance of the transparency and regular shape of the cornea are essential to the normal vision, whereas opacification of the tissue impairs vision. Fibrogenic reaction leading to scarring in an injured cornea is characterized by appearance of myofibroblasts, the key player of the fibrogenic reaction, and excess accumulation of fibrous extracellular matrix. Inflammatory/fibrogenic growth factors/cytokines produced by inflammatory cells play a pivotal role in fibrogenic response. Signaling systems involved in myofibroblast formation and fibrogenesis are activated by various growth factors, i.e., transforming growth factor beta or others. Modulation of transforming growth factor beta signal transduction molecules, e.g., Smad and mitogen-activated protein kinases, by gene transfer and other technology provides a new concept of prevention/treatment of unfavorable fibrogenesis in the cornea.
Asunto(s)
Córnea/fisiología , Córnea/fisiopatología , Enfermedades de la Córnea/prevención & control , Enfermedades de la Córnea/terapia , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Transdiferenciación Celular , Córnea/patología , Enfermedades de la Córnea/patología , Enfermedades de la Córnea/fisiopatología , Endotelio Corneal/patología , Fibrosis , Técnicas de Transferencia de Gen , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Miofibroblastos/patología , Proteínas Smad/genética , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Cicatrización de HeridasRESUMEN
PURPOSE: We determined if the immunohistochemical expression pattern of transient receptor potential vanilloid (TRPV) family members and TRP ankyrin 1 (TRPA1) differs among a healthy conjunctival epithelium and diseased epithelia. MATERIALS AND METHODS: Subjects include a normal conjunctival epithelium, pterygium epithelium, epithelial dysplasia or carcinoma in situ. RESULTS: TRPV1, TRPV4 or TRPA1 was detected in both the cytoplasm and nuclei, or in either the nuclei or cytoplasm, of these different epithelial layers, respectively. There was no difference in the expression pattern of these three TRP isoforms. On the other hand, the expression patterns of TRPV2 and TRPV3 differed dramatically among these different subjects. TRPV2 was observed in the basal layer epithelium of a normal conjunctiva and pterygium. Its pattern was scattered in this region, although TRPV2 was absent throughout most of the dysplastic epithelium. TRPV2 was detected only in some of the suprabasal epithelial cells of a carcinoma in situ. TRPV3 was faintly detected in the cytoplasm of all the cell layers and also in the nuclei of some of the basal cells in a normal conjunctiva and in the pterygia epithelium, while in situ it was uniformly expressed in all of the dysplasia and carcinoma nuclei in all epithelial cell layers. CONCLUSION: These results suggest that TRPV2 and TRPV3 expression pattern analysis might be potential diagnostic markers of ocular surface epithelial disorders.
RESUMEN
Fibrotic diseases, e.g., cutaneous and corneal scarring, keloids, and liver and lung fibrosis, etc., are characterized by appearance of myofibroblasts, the key player of the fibrogenic reaction, and excess accumulation of extracellular matrix with resultant tissue contraction and impaired functions. Inflammatory/fibrogenic growth factors/cytokines produced by injured tissues play a pivotal role in fibrotic tissue formation. Ocular tissues are also susceptible to fibrotic diseases. In this article, the pathogenesis of such fibrotic disorders in the eye, i.e., scarring in the cornea and conjunctiva, post-cataract surgery fibrosis of the lens capsule and proliferative vitreoretinopathy are reviewed. Focus is put on the roles of myofibroblast and signals activated by the fibrogenic cytokine, transforming growth factor beta. Modulation of signal transduction molecules, e.g., Smad and mitogen-activated protein kinases, by gene transfer and other technology is beneficial and can be an important treatment regiment to overcome (prevent or treat) these diseases.
Asunto(s)
Conjuntiva/patología , Córnea/patología , Oftalmopatías/terapia , Terapia Genética/métodos , Cápsula del Cristalino/patología , Retina/patología , Animales , Oftalmopatías/patología , Fibroblastos/patología , Fibrosis/terapia , Técnicas de Transferencia de Gen , Humanos , Proteínas Quinasas Activadas por Mitógenos/genética , Piel/patología , Proteínas Smad/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
PURPOSE: Injury-mediated corneal epithelial wound healing in vivo is mediated through different cell signaling pathways depending on whether or not the basement membrane is removed. Given this dependence, we ascertained if c-jun N-terminal kinase (JNK/SAPK) mitogen-activated protein kinase (MAPK) cell signaling mediates this response in vivo and in vitro, irrespective of the presence or absence of the basement membrane. Furthermore, in vitro the relative contribution was determined by the JNK/SAPK pathway to that of its p38 and ERK MAPK counterparts in mediating injury-induced increases in cell migration. METHODS: Corneal epithelial debridement was performed in C57BL/6 mice and their organ-cultured eyes without removal of the basement membrane. In rabbits, following basement membrane removal by keratectomy, fluorescein-staining monitored reepithelialization was performed as in the mice. Immunohistochemistry evaluated changes in JNK phosphorylation status and localization. JNK inhibitor I and its inactive analogue determined if JNK signaling activation contributes to wound healing. BrdU staining assessed cell proliferation. A scratch wound assay of healing rates in SV40-immortalized human corneal epithelial cell line (HCEC) evaluated the relative contributions by p38 and ERK and JNK MAPK signaling activation to wound healing. A TUNEL assay probed for apoptosis after wound closure of HCEC. MTT assay evaluated corneal epithelial viability. RESULTS: Two hours following mice corneal epithelial debridement, phospho-JNK was transiently upregulated in the nucleus, whereas total JNK was constitutively expressed. JNK inhibitor I suppressed epithelial spreading in organ-cultured mouse eyes and rabbit corneal blocks, irrespective of the presence or absence of basement membrane. No proliferation was detected at the wound edges. In HCEC, a p38 (SB203580) and a JNK pathway inhibitor (JNK inhibitor I) inhibited migration rates more than U0126-induced ERK, whereas the JNK inhibitor I inactive analogue had no effect. JNK pathway inhibition wound closure in this region was not associated with either any TUNEL or BrdU-positive cells. Cell viability was unaffected by any of these MAPK inhibitors. CONCLUSION: JNK/SAPK pathway activation stimulates wound healing in vitro and in vivo, irrespective of the presence or absence of the basement membrane. Therefore, studies on how wound closure is elicited in HCEC are relevant for identifying how MAPK signaling mediates this response in vivo and in organ-cultured eyes. This realization suggests that the JNK signaling system has a role in vivo that is intermediate to those of ERK and p38 in mediating increases in cell migration.
Asunto(s)
Movimiento Celular/fisiología , Epitelio Corneal/lesiones , Epitelio Corneal/fisiología , Lesiones Oculares/fisiopatología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Transducción de Señal/fisiología , Animales , Apoptosis , Membrana Basal , Desbridamiento , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Técnicas para Inmunoenzimas , Etiquetado Corte-Fin in Situ , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Fosforilación , Conejos , Regulación hacia Arriba , Cicatrización de Heridas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
PURPOSE: To investigate the effects of loss of osteopontin (OPN) in the healing of the injured cornea in mice. Cell culture study was also conducted to clarify the effects of OPN in fibroblast behaviors. METHODS: Ocular fibroblasts from wild-type (WT) and OPN-null (KO) mice were used to study the role of OPN on cell behavior. The effect of the lack of OPN on corneal wound healing was evaluated in mice. RESULTS: In cell culture, OPN is involved in cell adhesion and in the migration of ocular fibroblasts. Adhesion of the corneal epithelial cell line was not affected by exogenous OPN. OPN was upregulated in a healing, injured mouse cornea. Loss of OPN did not affect epithelial healing after simple epithelial debridement. Healing of an incision injury in cornea was delayed, with less appearance of myofibroblasts and transforming growth factor beta1 expression in a KO mouse than in a WT mouse. The absence of OPN promoted tissue destruction after an alkali burn, resulting in a higher incidence of corneal perforation in KO mice than in WT mice. CONCLUSIONS: OPN modulates wound healing-related fibroblast behavior and is required to restore the physiological structure of the cornea after wound healing.
Asunto(s)
Sustancia Propia/lesiones , Glicoproteínas/fisiología , Cicatrización de Heridas/fisiología , Animales , Quemaduras Químicas/metabolismo , Adhesión Celular/efectos de los fármacos , Técnicas de Cultivo de Célula , Movimiento Celular/efectos de los fármacos , Desbridamiento , Modelos Animales de Enfermedad , Quemaduras Oculares/inducido químicamente , Fibroblastos/efectos de los fármacos , Glicoproteínas/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Hidróxido de Sodio , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
PURPOSE: To understand the role of TGF-beta related signals in the repair of a corneal endothelium defect and also to evaluate the therapeutic effect of Smad7 gene transfer on injury induced fibrosis of the corneal endothelium in rats. METHODS: (1) Japanese albino rabbits (n=108) were used. Blocks of central cornea (4 x 4 mm) were prepared. After partially scraping the endothelium to produce a defect, the blocks were organ cultured for 24 h in the presence of either exogenous growth factors, transforming growth factor beta (TGF-beta)-neutralizing antibody, or inhibitors of each TGF-beta related signal. Endothelium repair was assayed under light microscopy. (2) Adult Wistar rats (n=62) were then used. Smad7 expressing adenoviral vector (Smad7-Ad) or non-functioning control vector (Cre-Ad) was administered to the anterior chamber of an eye. The cornea was burned with topical 1 N NaOH (10 microl) three days later. After specific intervals, the eye was histologically observed. RESULTS: (1) The endothelial layer that elongated toward the defect lacked proliferation after 24 h in organ culture. Endogenous TGF-beta was required for endothelium defect repair. Inhibition of p38 and Erk but not c-Jun NH(2)-terminal kinase (JNK) and ALK5 signal (Smad) retarded such cell spreading. (2) Adenoviral Smad7 overexpression suppressed fibrogenic reaction of the endothelium of an alkali-burned cornea as evaluated by immunohistochemistry for phospho-Smad2, collagen I, and alpha-smooth muscle actin, a marker for endothelial-mesenchymal transition (EnMT), and by electron microscopy. CONCLUSIONS: Inhibition of Smad and JNK signals do not affect corneal endothelium defect repair. Inhibition of Smad suppresses fibrogenic reaction via EnMT of corneal endothelium in vivo.
Asunto(s)
Endotelio Corneal/efectos de los fármacos , Endotelio Corneal/patología , Transducción de Señal/efectos de los fármacos , Proteínas Smad/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Adenoviridae/genética , Animales , Anticuerpos/farmacología , Quemaduras/patología , Movimiento Celular/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Endotelio Corneal/ultraestructura , Fibrosis , Vectores Genéticos/genética , Integrasas/metabolismo , Ratones , Pruebas de Neutralización , Técnicas de Cultivo de Órganos , Conejos , Ratas , Ratas Wistar , Proteínas Smad/metabolismo , Transducción Genética , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Factor de Crecimiento Transformador beta2/farmacología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
PURPOSE: We examined the role of connective tissue growth factor (CTGF) in transforming growth factor beta1 (TGFbeta1)-related behavior in cultured human subconjunctival fibroblasts (SCFs), protein production, mRNA expression of CTGF and type I collagen alpha1 chain (colIA1), and cell proliferation and migration. TGFbeta1 is the major factor involved in bleb scarring following filtration surgery. METHODS: An antisense deoxynucleotide (antisense) (5 microM) for CTGF mRNA was used to block endogenous CTGF expression. Effects of antisense on extracellular matrix (ECM) production and immunolocalization, mRNA expression, and cell proliferation and migration were examined in human SCF cultures with or without TGFbeta1 (5 ng/ml). Cell migration was examined in an in vitro wound model of monolayer fibroblast cultures. RESULTS: CTGF antisense reduced mRNA expression of CTGF and colIA1 and production of the ECM components type I collagen, and fibronectin much more markedly in cells treated with TGFbeta1 compared with control fibroblasts, and it inhibited the proliferation of cultured SCFs to 71.9% of that of controls after 13 days of culture. CTGF antisense also delayed defect closure in monolayer cell sheets. In the culture, the defect was closed by TGFbeta1 by 36 h, whereas 7.0% of the defect remained at 48 h in the antisense-treated culture. CONCLUSIONS: These findings indicate that CTGF is involved in ECM production in SCFs activated by exogenous TGFbeta1 in vitro. Inhibition of CTGF expression may be effective in preventing undesirable scar formation during healing following filtration surgery.
Asunto(s)
Colágeno Tipo I/metabolismo , Conjuntiva/efectos de los fármacos , Fibronectinas/metabolismo , Proteínas Inmediatas-Precoces/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Movimiento Celular/fisiología , Proliferación Celular , Células Cultivadas , Niño , Preescolar , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Conjuntiva/citología , Conjuntiva/metabolismo , Factor de Crecimiento del Tejido Conjuntivo , ADN sin Sentido/farmacología , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Oligonucleótidos Antisentido/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
PURPOSE: To determine whether a Chinese herbal medicine component, emodin, suppresses inflammatory/fibrogenic reaction in cultured subconjunctival fibroblasts and reduces injury-induced increases in ocular surface inflammation in mice. METHODS: Effects of emodin were measured in human subconjunctival fibroblasts on proliferation and migration with colorimetry and scratch wound assay, respectively. Neovascularization was evaluated using an endothelial cell-fibroblast coculture model. Proinflammatory mediator and extracellular matrix component gene and protein expression was characterized with real-time reverse transcription-polymerase chain reaction, enzyme immunoassay, and immunocytochemistry, respectively. Western blotting and immunohistochemistry evaluated the activation of nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK). In a mouse corneal alkali-burn model, the effects of emodin on ocular surface inflammation and fibrosis were evaluated. RESULTS: Emodin suppressed tumor necrosis factor alpha (TNF-alpha)-induced fibroblast migration and fibronectin deposition in vitro. VEGF induced neovascularization but did not affect cell proliferation and collagen type 1 production. Monocyte/macrophage-chemoattractant protein-1 gene and protein expression declined. Emodin inhibited TNF-alpha-induced NF-kappaB p65 and JNK activation but did not affect transforming growth factor beta1-induced Smad2/3 signaling. In vivo, emodin inhibited proinflammatory and fibrogenic reactions. CONCLUSIONS: Emodin suppressed in vitro TNF-alpha-induced stimulation of proinflammatory reaction. In a mouse ocular alkali burn model, this herbal component lessened inflammation and scarring. Additional studies are warranted to evaluate the therapeutic potential of emodin in lessening ocular tissue inflammation and resultant fibrosis after injury.
Asunto(s)
Conjuntiva/efectos de los fármacos , Conjuntivitis Alérgica/prevención & control , Medicamentos Herbarios Chinos/farmacología , Emodina/farmacología , Animales , Western Blotting , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Técnicas de Cocultivo , Colágeno Tipo I/metabolismo , Conjuntiva/citología , Conjuntiva/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
PURPOSE: To evaluate the role of tumor necrosis factor alpha (TNFalpha) in regulation of corneal epithelial cell migration. METHODS: Cell culture of immortalized corneal epithelial cell line was employed to examine the role of transforming growth factor beta1 (TGFbeta1) and TNFalpha on cell migration and cell signaling. Healing of central epithelial defect was also observed in organ culture in the presence or absence of neutralizing antibody against either TNFalpha or TGFbeta1. RESULTS: In cell cultures of corneal epithelial cell line, adding TNFalpha suppresses activation of p38 signal and cell migration, but not Smad2 activation, upon TGFbeta1 exposure. In an organ culture system, healing of an epithelial defect was promoted by the loss of TNFalpha. A neutralizing antibody against TNFalpha also promoted closure of an epithelial defect of organ cultured WT mouse corneas. Anti-TGFbeta neutralizing antibody reversed facilitation of epithelial healing in KO corneas in organ culture. CONCLUSIONS: TNFalpha inhibits migration of corneal epithelial cells.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Epitelio Corneal/citología , Epitelio Corneal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Línea Celular , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Desbridamiento , Epitelio Corneal/enzimología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: To determine the effects of Smad7 gene transfer in the prevention of fibrogenic responses by the retinal pigment epithelium, a major cause of proliferative vitreoretinopathy after retinal detachment, in mice. METHODS: Retinal detachment-induced proliferative vitreoretinopathy in a mouse model. Forty-eight eyes received either an adenoviral gene transfer of Smad7 or Cre recombinase gene only. The eyes were histologically analyzed. A retinal pigment epithelial cell line, ARPE-19, was used to determine whether Smad7 gene transfection suppresses the fibrogenic response to transforming growth factor (TGF) beta2 exposure. RESULTS: The Smad7 gene transfer inhibited TGF-beta2/Smad signaling in ARPE-19 cells and expression of collagen type I and TGF-beta1 but had no effect on their basal levels. In vivo Smad7 overexpression resulted in suppression of Smad2/3 signals and of the fibrogenic response to epithelial-mesenchymal transition by the retinal pigment epithelium. CONCLUSION: Smad7 gene transfer suppresses fibrogenic responses to TGF-beta2 by retinal pigment epithelial cells in vitro and in vivo. Clinical Relevance Smad7 gene transfer might be a new strategy to prevent and treat proliferative vitreoretinopathy.
Asunto(s)
Modelos Animales de Enfermedad , Expresión Génica/fisiología , Epitelio Pigmentado Ocular/metabolismo , Proteína smad7/genética , Factor de Crecimiento Transformador beta/farmacología , Vitreorretinopatía Proliferativa/prevención & control , Adenoviridae/genética , Animales , Western Blotting , Línea Celular , Colágeno Tipo I/metabolismo , Fibronectinas/metabolismo , Fibrosis , Técnica del Anticuerpo Fluorescente Indirecta , Vectores Genéticos , Ratones , Epitelio Pigmentado Ocular/efectos de los fármacos , Epitelio Pigmentado Ocular/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transfección , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia Arriba , Vitreorretinopatía Proliferativa/metabolismo , Vitreorretinopatía Proliferativa/patologíaRESUMEN
PURPOSE: To determine in a lens epithelial cell line, alpha-TN4, whether genipin, an intestinal metabolite component of the herbal medicine inchin-ko-to, suppresses profibrogenic myofibroblast generation and upregulation of fibrogenic cytokines and to evaluate the potential benefit of the medicine in preventing posterior capsule opacification (PCO). SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: In this study, alpha-TN4 cell proliferation, migration, and expression of alpha-smooth muscle actin (alpha-SMA), the hallmark of myofibroblast generation, were assayed with a colorimetric assay, scratch wound assay, immunohistochemistry, and Western blot analysis. Gene expression of transforming growth factor-beta1 (TGF-beta1) and connective tissue growth factor (CTGF) was characterized with real-time reverse transcription-polymerase chain reaction. In addition, p38 mitogen-activated protein kinase (p 38 MAPK), extracellular signal-regulated kinase (ERK) limb, and Smad signalings were evaluated by Western blotting and immunohistochemistry. Cytotoxicity of genipin was evaluated using a commercial colorimetric assay kit for nuclear matrix protein 41/7 (NMP41/7) in culture medium. RESULTS: Genipin suppressed cell proliferation and migration in association with inhibition of Smad and p38 MAPK phosphorylation, although ERK signaling was enhanced. Genipin suppressed mRNA expression of TGF-beta1 and CTGF. Cytoplasmic fiber formation declined based on less intense alpha-SMA immunocytochemical staining. However, alpha-SMA protein expression was actually not altered. This negative result suggests that genipin attenuated formation of alpha-SMA-containing cytoskeleton. Treatment of the cells with genipin for 48 hours did not increase the release of NMP41/7 to the medium, indicating this compound is not cytotoxic. CONCLUSION: Because genipin suppressed alpha-TN4 lens cell fibrogenic behaviors, it may be of therapeutic value in preventing PCO.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Células Epiteliales/efectos de los fármacos , Cristalino/citología , Piranos/farmacología , Actinas/metabolismo , Antígenos Nucleares/metabolismo , Western Blotting , Proteínas de Ciclo Celular , Línea Celular , Factor de Crecimiento del Tejido Conjuntivo , Medicamentos Herbarios Chinos/toxicidad , Células Epiteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Proteínas Inmediatas-Precoces/genética , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/genética , Glicósidos Iridoides , Iridoides , Proteínas Asociadas a Matriz Nuclear/metabolismo , Piranos/toxicidad , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1 , Regulación hacia Arriba , Cicatrización de Heridas/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Background. Epithelial dysplasia is categorized as conjunctival/corneal intraepithelial neoplasia which is a precancerous lesion. The lesion is usually developed at the limbal region and grows towards central cornea in association with neovascularization into the lesion. Here, we report a case of isolated nonvascularized corneal epithelial dysplasia surrounded by normal corneal epithelium with immune histochemical finding of ocular surface tissues cytokeratins, for example, keratin 13 and keratin 12. Case Presentation. A 76-year-old man consulted us for visual disturbance with localized opacification of the corneal epithelium in his left eye. His visual acuity was 20/20 and 20/200 in his right and left eye, respectively. Slit lamp examination showed a whitish plaque-like lesion at the center of his left corneal epithelium. No vascular invasion to the lesion was found. The lesion was surgically removed and subjected to histopathological examination and diagnosed as epithelial dysplasia. Amyloidosis was excluded by direct fast scarlet 4BS (DFS) staining. Immunohistochemistry showed that the dysplastic epithelial cells express keratin 13 and vimentin, but not keratin 12, indicating that the neoplastic epithelial cells lacked corneal-type epithelium differentiation. Conclusions. The lesion was diagnosed as nonvascularized epithelial dysplasia of ocular surface. Etiology of the lesion is not known.
RESUMEN
The authors report two cases of congenital retrobulbar cyst or large peripapillary staphyloma with alcoholic mothers. In the first case, a 53-day-old newborn was referred for evaluation of microphthalmia in her left eye. Ophthalmic examination showed microcornea, microphthalmia, and an abnormally enlarged optic disc with an excavation in her left eye. Imaging revealed the presence of a cystic lesion in her left orbit that seemed to be communicated with the vitreous cavity. She did not have chromosomal abnormality. In the second case, a large peripapillary staphyloma was detected in the right eye of a 7-month-old newborn. The mother of each patient was an alcoholic. Retrobulbar cystic lesion or peripapillary staphyloma could be considered to be a series of abnormalities caused by the degree of the failure of the optic cup. Effects of each mother's alcohol intake on the development of the eye, especially the closure of the optic cup, is to be further investigated.
Asunto(s)
Quistes , Oftalmopatías , Disco Óptico/anomalías , Consumo de Bebidas Alcohólicas/efectos adversos , Femenino , Humanos , Lactante , Embarazo , Efectos Tardíos de la Exposición PrenatalRESUMEN
PURPOSE: This study investigated the effects of loss of tenascin C (TNC) in the development of neovascularization in a corneal stroma in mice. Cell culture study was also conducted to clarify the roles of TNC in the expression of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)ß1 in fibroblasts and macrophages. METHODS: Ocular fibroblasts and macrophages from wild-type (WT) and TNC-null (KO) mice were used to study the role of TNC in the expression of VEGF and TGFß1. The effects of the absence of TNC on angiogenic gene expression, inflammatory cell invasion, and cornea neovascularization in the corneal stroma were then evaluated after cauterization of the center of the cornea in mice. Histologic, immunohistochemical, and mRNA expression analyses were performed. RESULTS: Absence of TNC suppressed expression of VEGF and counteracted upregulation of TGFß1 by exogenous TGFß1 in ocular fibroblast culture. Such effects of the absence of TNC were not observed in cultured macrophages. Absence of TNC attenuated expression of both VEGF and TGFß1 mRNA as well as neovascularization into the stroma after cauterization at the center of the cornea in mice. Absence of TNC suppressed macrophages, but not neutrophils, invading the cauterized cornea. CONCLUSIONS: TNC is involved in angiogenic gene expression in ocular fibroblasts in vitro and in vivo and is required for macrophage invasion and neovascularization of injured corneal stroma.
Asunto(s)
Neovascularización de la Córnea/metabolismo , Sustancia Propia/irrigación sanguínea , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/fisiología , Tenascina/fisiología , Factor de Crecimiento Transformador beta1/genética , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Células Cultivadas , Neovascularización de la Córnea/patología , Fibroblastos/metabolismo , Técnicas para Inmunoenzimas , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Neurotropic keratopathy is one of the major refractory corneal disorders, which was first recognized in 1824. This category of diseases is caused by damage to the trigeminal nerve and the consequent loss of corneal sensation. It leads to various types of corneal disorder, including superficial keratopathy, persistent epithelial defects and corneal ulcers. In the present article, we review the pathobiology and prevention/treatment of neurotrophic corneal diseases. The goals of treatment of neurotrophic keratopathy are to prevent the progression of corneal damage to promote epithelial healing in case that the original damage in the trigeminal nerve or its braches is difficult. The therapy should be prompt and based on the clinical stage of the disease. Although at present, there are no accurate treatment methods for neurotrophic keratopathy, several effective treatments have been reported. Signals derived from TRP channels are involved in homeostasis of tissues by controlling cell behaviors, i. e., gene expression for inflammation, cell migration, or cell survival/cell death. Targeting TRP channel in the cornea is expected to contribute to the development of a new treatment method for neurotrophic keratopathy. Further study and clinical trial are need to establish this new treatment strategy.