RESUMO
We investigated the effects of lacking TNFα on the development and regression of Argon-laser-induced choroidal neovascularization (CNV) in mice. We lasered ocular fundus for induction of CNV in both wild-type (WT) and TNFα-null (KO) mice. Fluorescence angiography was performed to examine the size of CNV lesions. Gene expression pattern of wound healing-related components was examined. The effects of exogenous TNFα on apoptosis of human retinal microvascular endothelial cells (HRMECs) and on the tube-like structure of the cells were investigated in vitro. The results showed that Argon-laser irradiation-induced CNV was significantly larger in KO mice than WT mice on Day 21, but not at other timepoints. Lacking TNFα increased neutrophil population in the lesion. The distribution of cleaved caspase3-labelled apoptotic cells was more frequently observed in the laser-irradiated tissue in a WT mouse as compared with a KO mouse. Exogenous TNFα induced apoptosis of HRMECs and accelerated regression of tube-like structure of HRMECs in cell culture. Taken together, TNFα gene knockout delays the regression of laser-induced CNV in mice. The mechanism underlying the phenotype might include the augmentation of neutrophil population in the treated tissue and attenuation of vascular endothelial cell apoptosis.
Assuntos
Neovascularização de Coroide , Animais , Argônio , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Humanos , Lasers , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Necrose Tumoral alfaRESUMO
Purpose: We examined the effects of travoprost on cell proliferation-related signals and E-cadherin expression in vitro and in situ in order to obtain evidence to support the hypothesis that topical travoprost impairs the integrity of the corneal epithelium.Methods: A human corneal epithelial cell culture was treated with travoprost (0.4 mg/ml) and/or PD168393 (an EGF receptor inhibitor, 10 µM). The culture was then processed for cell proliferation, an mRNA expression analysis of epidermal growth factor (EGF) and E-cadherin, and protein expression analysis of E-cadherin by immunocytochemistry and Western blotting. The eyes of C57/BL6 mice were incubated in serum-free medium plus travoprost (0.4 mg/ml) and/or PD168393 (10 µM). After being cultured for 24 h, the expression patterns of phospho-EGFR, phospho-ERK, E-cadherin, and Ki67 were immunohistochemically examined in paraffin sections.Results: The addition of travoprost up-regulated EGF mRNA expression and cell proliferation in the corneal epithelial cell culture, and this was cancelled by the addition of PD168393. This FP agonist also decreased E-cadherin expression levels in the cell-cell contact zone, and this was cancelled by the addition of PD168393. In the organ culture, the addition of travoprost to the medium up-regulated the expression of phospho-EGFR and phospho-ERK as well as cell proliferation, and down-regulated the expression of E-cadherin in the corneal epithelium, particularly in basal cells, whereas PD168393 reversed these effects.Conclusions: Travoprost activates epithelial cell proliferation by up-regulating an EGF-related signal in association with the suppression of E-cadherin localization in the cell-cell contact zone. Modulation of the EGF signal may be a strategy to minimize the negative impact of this mitogen on reformation of corneal barrier function during epithelial renewal.
Assuntos
Anti-Hipertensivos/farmacologia , Caderinas/genética , Dinoprosta , Fator de Crescimento Epidérmico/genética , Células Epiteliais/efeitos dos fármacos , Quinazolinas/farmacologia , Travoprost/farmacologia , Animais , Caderinas/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Córnea/citologia , Células Epiteliais/metabolismo , Receptores ErbB/antagonistas & inibidores , Glaucoma , Humanos , Camundongos Endogâmicos C57BLRESUMO
In humans suffering from pulmonary disease and a mouse model, transient receptor potential vanilloid 4 (TRPV4) channel activation contributes to fibrosis. As a corneal alkali burn induces the same response, we determined if such an effect is also attributable to TRPV4 activation in mice. Accordingly, we determined if the alkali burn wound healing responses in wild-type (WT) mice are different than those in their TRPV4-null (KO) counterpart. Stromal opacification due to fibrosis in KO (n = 128) mice was markedly reduced after 20 days relative to that in WT (n = 157) mice. Immunohistochemistry revealed that increases in polymorphonuclear leukocytes and macrophage infiltration declined in KO mice. Semi-quantitative real time RT-PCR of ocular KO fibroblast cultures identified increases in proinflammatory and monocyte chemoattractant protein-1 chemoattractant gene expression after injury. Biomarker gene expression of fibrosis, collagen1a1 and α-smooth muscle actin were attenuated along with macrophage release of interleukin-6 whereas transforming growth factor ß, release was unchanged. Tail vein reciprocal bone marrow transplantation between WT and KO chimera mouse models mice showed that reduced scarring and inflammation in KO mice are due to loss of TRPV4 expression on both corneal resident immune cells, fibroblasts and infiltrating polymorphonuclear leukocytes and macrophages. Intraperitoneal TRPV4 receptor antagonist injection of HC-067047 (10 mg/kg, daily) into WT mice reproduced the KO-phenotype. Taken together, alkali-induced TRPV4 activation contributes to inducing fibrosis and inflammation since corneal transparency recovery was markedly improved in KO mice.
Assuntos
Álcalis/farmacologia , Córnea/patologia , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/patologia , Técnicas de Inativação de Genes , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genética , Actinas/genética , Animais , Córnea/efeitos dos fármacos , Opacidade da Córnea/complicações , Queimaduras Oculares/complicações , Queimaduras Oculares/genética , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/patologia , Interleucina-6/genética , Camundongos , Canais de Cátion TRPV/metabolismo , Fator de Crescimento Transformador beta/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
We evaluated the effects of the loss of Smad3 on the development of experimental argon laser-induced choroidal neovascularization (CNV) in mice. An in vitro angiogenesis model was also used to examine the role of transforming growth factor-ß1 (TGFß1)/Smad3 signaling in vessel-like tube formation by human umbilical vein endothelial cells (HUVECs). CNV was induced in eyes of 8-12-week-old B6.129-background Smad3-deficient (KO) mice (n=47) and wild-type (WT) mice (n=47) by argon laser irradiation. Results showed that the size of the CNV induced was significantly smaller in KO mice as compared with WT mice at day 14 as revealed by high-resolution angiography with fluorescein isothiocyanate-dextran. Immunohistochemistry and real-time reverse transcription-polymerase chain reaction of RNA extracted from laser-irradiated choroidal tissues were conducted on specimens at specific timepoints. Invasion of macrophages (F4/80+), but not neutrophils (myeloperoxidase+), and appearance of myofibroblasts (α-smooth muscle actin+) were suppressed in laser-irradiated KO tissues. mRNA expression of inflammation-related factors, that is, vascular endothelial growth factor (VEGF), macrophage-chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and TGFß1 in choroidal tissues was suppressed by the loss of Smad3. We then examined the effects of adding a Smad3 inhibitor, SIS3, or an ALK5 inhibitor, SB431542, on tube formation promoted by TGFß1 or VEGF in HUVECs cocultured with fibroblast feeder. Further addition of SIS3 or SB431542 augmented vessel-like tube formation by HUVECs in the presence of TGFß1 or VEGF. In conclusion, lack of Smad3 attenuated the growth of laser-induced CNV with suppression of inflammation by macrophages in mice. Because blocking TGFß1/Smad3 signal stimulated the activity of angiogenesis of HUVECs in vitro, the reduction of CNV in vivo in KO mice is attributed to a decrease in growth factor levels in the tissue by the loss of Smad3.
Assuntos
Neovascularização de Coroide/prevenção & controle , Macrófagos/patologia , Proteína Smad3/deficiência , Animais , Neovascularização de Coroide/etiologia , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana , Humanos , Mediadores da Inflamação/metabolismo , Isoquinolinas/farmacologia , Lasers de Gás/efeitos adversos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Piridinas/farmacologia , Pirróis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/genética , Fator de Crescimento Transformador beta1RESUMO
Corneal scarring/fibrosis disturbs normal transparency and curvature of the tissue and thus impairs vision. The lesion is characterized by appearance of myofibroblasts, the key player of the fibrogenic reaction, and excess accumulation of extracellular matrix. Inflammatory/fibrogenic growth factors or cytokines expressed in inflammatory cells that infiltrate into injured tissues play a pivotal role in fibrotic tissue formation. In this article the pathogenesis of fibrosis/scarring in the corneal stroma is reviewed focusing on the roles of myofibroblast, the key player in corneal stromal wound healing and fibrosis, and cytoplasmic signals activated by the fibrogenic cytokine, transforming growth factor ß (TGFß). Although it is established that TGFß/Smad signal is essential to the process of keratocyte-myofibroblast transformation in a healing corneal stroma post-injury. This article emphasizes the involvement of non-TGFß molecular mechanisms in modulating Smad signal. We focus on the roles of matricellular proteins, i.e., osteopontin and tenascin C, and as cellular components, the roles of transient receptor potential (TRP) cation channel receptors are discussed. Our intent is to draw attention to the possibility of signal transduction cascade modulation (e.g., Smad signal and mitogen-activated protein kinases, by gene transfer and other related technology) as being beneficial in a clinical setting to reduce or even prevent corneal stromal tissue fibrosis/scarring and inflammation.
Assuntos
Doenças da Córnea/fisiopatologia , Substância Própria/fisiopatologia , Miofibroblastos/fisiologia , Transdução de Sinais/fisiologia , Proteínas Smad/fisiologia , Cicatrização/fisiologia , Diferenciação Celular/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Humanos , Macrófagos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Conjunctival and subconjunctival fibrogenesis and inflammation are sight compromising side effects that can occur subsequent to glaucoma filtration surgery. Despite initial declines in intraocular pressure resulting from increasing aqueous outflow, one of the activated responses includes marshalling of proinflammatory and pro-fibrogenic cytokine mediator entrance into the aqueous through a sclerostomy window and their release by local cells, as well as infiltrating activated immune cells. These changes induce dysregulated inflammation, edema and extracellular matrix remodeling, which occlude outflow facility. A number of therapeutic approaches are being taken to offset declines in outflow facility since the current procedure of inhibiting fibrosis with either mitomycin C (MMC) or 5-fluorouracil (5-FU) injection is nonselective. One of them entails developing a new strategy for reducing fibrosis induced by wound healing responses including myofibroblast transdifferentiation and extracellular matrix remodeling in tissue surrounding surgically created shunts. The success of this endeavor is predicated on having a good understanding of conjunctival wound healing pathobiology. In this review, we discuss the roles of inappropriately activated growth factor and cytokine receptor linked signaling cascades inducing conjunctival fibrosis/scarring during post-glaucoma surgery wound healing. Such insight may identify drug targets for blocking fibrogenic signaling and excessive fibrosis which reduces rises in outflow facility resulting from glaucoma filtration surgery.
Assuntos
Cirurgia Filtrante/efeitos adversos , Glaucoma/cirurgia , Cicatrização/fisiologia , Doenças da Túnica Conjuntiva/fisiopatologia , Citocinas/metabolismo , Fibrose/fisiopatologia , Glaucoma/fisiopatologia , Humanos , Pressão Intraocular/fisiologia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/metabolismo , Complicações Pós-Operatórias/fisiopatologia , Trabeculectomia/métodosRESUMO
Corneal photorefractive surgery is currently performed by ablation of corneal stroma under the stromal flap. A stromal flap is created using a femtosecond (FS) laser or mechanical microkeratome, although the FS laser procedure is considered safer and more accurate. This review assesses and compares the use of FS laser versus mechanical microkeratome ablation for corneal stromal characteristics mainly examined by histology and cellular biological responses. Supporting data from our studies, using corneas of enucleated porcine eye globes, are included in this review. Histological analysis and experimental studies of cellular/tissue responses to FS laser irradiation should be further investigated, and the equipment used to perform these techniques should be improved.
Assuntos
Substância Própria/ultraestrutura , Cirurgia da Córnea a Laser , Lasers de Excimer/uso terapêutico , Retalhos Cirúrgicos/patologia , Animais , Humanos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Suínos , CicatrizaçãoRESUMO
PURPOSE: To examine the role of conjunctival Muc16 in the homeostasis of the ocular surface epithelium and stroma using Muc16-null knockout (KO) mice. METHODS: We used KO mice (n = 58) and C57/BL6 (WT) mice (n = 58). Histology and immunohistochemistry were employed to analyze the phenotypes in the ocular surface epithelium. The expression of phospho-Stat3, AP-1 components, interleukin 6 (IL-6), and tumor necrosis factor-α (TNFα) in the cornea and conjunctiva was examined. The shape of the nuclei of corneal epithelial cells was examined to evaluate intraepithelial cell differentiation. Epithelial cell proliferation was studied using bromo-deoxyuridine labeling. Finally, the wound healing of a round defect (2-mm diameter) in the corneal epithelium was measured. The keratocyte phenotype and macrophage invasion in the stroma were evaluated after epithelial repair. RESULTS: The loss of Muc16 activated Stat3 signal, affected JunB signal, and upregulated the expression of IL-6 in the conjunctiva. Basal-like cells were observed in the suprabasal layer of the corneal epithelium with an increase in proliferation. The loss of Muc16 accelerated the wound healing of the corneal epithelium. The incidence of myofibroblast appearance and macrophage invasion were more marked in KO stroma than in WT stroma after epithelial repair. CONCLUSIONS: The loss of Muc16 in the conjunctiva affected the homeostasis of the corneal epithelium and stroma. The mechanism might include the upregulation of the inflammatory signaling cascade (i.e., Stat3 signal, and IL-6 expression in the KO conjunctiva). Current data provides insight into the research of the pathophysiology of dry eye syndrome.
Assuntos
Antígeno Ca-125/genética , Túnica Conjuntiva/metabolismo , Substância Própria/metabolismo , Epitélio Corneano/metabolismo , Traumatismos Oculares/genética , Regulação da Expressão Gênica , Proteínas de Membrana/genética , RNA/genética , Animais , Apoptose , Antígeno Ca-125/biossíntese , Túnica Conjuntiva/patologia , Substância Própria/lesões , Substância Própria/patologia , Modelos Animais de Doenças , Epitélio Corneano/lesões , Epitélio Corneano/patologia , Traumatismos Oculares/metabolismo , Traumatismos Oculares/patologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Cicatrização/genéticaRESUMO
Limbal stem cell deficiency (LSCD) leads to severe ocular surface abnormalities that can result in the loss of vision. The most successful therapy currently being used is transplantation of limbal epithelial cell sheets cultivated from a limbal biopsy obtained from the patient's healthy, contralateral eye or cadaveric tissue. In this study, we investigated the therapeutic potential of murine vibrissae hair follicle bulge-derived stem cells (HFSCs) as an autologous stem cell (SC) source for ocular surface reconstruction in patients bilaterally affected by LSCD. This study is an expansion of our previously published work showing transdifferentiation of HFSCs into cells of a corneal epithelial phenotype in an in vitro system. In this study, we used a transgenic mouse model, K12(rtTA/rtTA) /tetO-cre/ROSA(mTmG) , which allows for HFSCs to change color, from red to green, once differentiation to corneal epithelial cells occurs and Krt12, the corneal epithelial-specific differentiation marker, is expressed. HFSCs were isolated from transgenic mice, amplified by clonal expansion on a 3T3 feeder layer, and transplanted on a fibrin carrier to the eye of LSCD wild-type mice (n = 31). The HFSC transplant was able to reconstruct the ocular surface in 80% of the transplanted animals; differentiating into cells with a corneal epithelial phenotype, expressing Krt12, and repopulating the corneal SC pool while suppressing vascularization and conjunctival ingrowth. These data highlight the therapeutic properties of using HFSC to treat LSCD in a mouse model while demonstrating a strong translational potential and points to the niche as a key factor for determining stem cell differentiation.
Assuntos
Doenças da Córnea/cirurgia , Epitélio Corneano/citologia , Anormalidades do Olho/cirurgia , Folículo Piloso/transplante , Limbo da Córnea/anormalidades , Limbo da Córnea/cirurgia , Transplante de Células-Tronco , Células-Tronco/fisiologia , Animais , Transdiferenciação Celular , Células Cultivadas , Folículo Piloso/citologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vibrissas/citologia , Vibrissas/transplanteRESUMO
The anterior segment of the eye ball, i. e., cornea and conjunctiva, serves as the barrier to the external stimuli. Cornea is transparent and is a "window"of the light sense, while conjunctiva covers the sclera, the main part of the eyeshell. Fibrosis/scarring in cornea potentially impairs vision by the reduction of its transparency and the alteration of the regular curvature. Fibrotic reaction in conjunctiva is also of a clinical importance because inflammatory fibrosis in this tissue affects the physiology of the cornea and also of a problem post-eye surgery. In this review we discuss on the topic that is quite critical in vision. Although various growth factors are considered to be involved in, focus was put on the roles of transforming growth factorß (TGFß).
Assuntos
Segmento Anterior do Olho/patologia , Cicatriz/patologia , Oftalmopatias/patologia , Animais , Segmento Anterior do Olho/imunologia , Segmento Anterior do Olho/metabolismo , Segmento Anterior do Olho/fisiopatologia , Cicatriz/imunologia , Cicatriz/metabolismo , Cicatriz/fisiopatologia , Cicatriz/terapia , Oftalmopatias/imunologia , Oftalmopatias/metabolismo , Oftalmopatias/fisiopatologia , Oftalmopatias/terapia , Fibrose , Humanos , Mediadores da Inflamação/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Visão OcularRESUMO
PURPOSE: To investigate the effects of loss of osteopontin (OPN) in the development of neovascularization in corneal stroma in mice. Cell culture study was also conducted to clarify the effects of OPN in transforming growth factor (TGF) beta1-driven cell signaling and expression of vascular endothelial growth factor (VEGF). METHODS: Ocular fibroblasts from wild-type and OPN-null mice were used to study the role of OPN in TGFbeta1 signal and VEGF expression. The effect of the absence of OPN on corneal neovascularization was evaluated in mice. RESULTS: In ocular fibroblast culture, loss of OPN attenuated TGFbeta1 signals (Smad3 and p38) and reduced expression of VEGF. Loss of OPN attenuated neovascularization in corneal stroma in mice. CONCLUSIONS: OPN is involved in VEGF expression in cultured fibroblasts and is required for neovascularization in corneal stroma in vivo.
Assuntos
Neovascularização da Córnea/metabolismo , Osteopontina/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Córnea/citologia , Neovascularização da Córnea/etiologia , Neovascularização da Córnea/prevenção & controle , Substância Própria/irrigação sanguínea , Fibroblastos/citologia , Fibroblastos/metabolismo , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteopontina/farmacologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Regulação para CimaRESUMO
Transforming growth factor b (TGF beta) is believed to be the most important ligand in the pathogenesis of fibrotic diseases in the eye. Such ocular fibrotic diseases include scarring in the cornea and conjunctiva, fibrosis in the corneal endothelium, post-cataract surgery fibrosis of the lens capsule, excess scarring the tissue around the extraocular muscles in the strabismus surgery and proliferative vitreoretinopathy. In the proliferative stage of diabetic retinopathy, fibrogenic reaction causes tractional retinal detachment in association with contraction of the tissue. A myofibroblast, the major cellular component in the fibrotic lesions, is derived from both mesenchymal cells (in cornea and conjunctiva) and epithelial cell types (lens or retinal pigment epithelium or corneal endothelium) through epithelial-mesenchymal transition (EMT). The myofibroblasts cause excess accumulation of fibrogenic extracellular matrix with resultant tissue contraction and impaired functions. Although various cytokine signaling pathways are involved in the fibrogenic reaction in tissues, TGF beta/Smad signal is the critical one. Blocking Smad signal by chemical or natural inhibitors or anti-Smad gene introduction effectively suppress fibrogenic reaction; inhibition of both fibroblast-myofibroblast conversion or EMT. Such strategies can be clinically tested.
Assuntos
Oftalmopatias/fisiopatologia , Fator de Crescimento Transformador beta/fisiologia , Células Epiteliais/patologia , Oftalmopatias/patologia , Fibrose , Humanos , Mesoderma/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
PURPOSE: To examine the effects of introduction of the adenoviral peroxisome proliferator-activated receptor (PPAR)-gamma gene on postinjury conjunctival scarring in mice. Its effects on fibrogenic reaction of cultured human subconjunctival fibroblasts (hSCFs) were also evaluated. METHODS: The effects of PPARgamma gene introduction on expression of type I collagen, fibronectin, and connective tissue growth factor (CTGF) in hSCFs were examined. A circumferential incision was made in the equatorial conjunctiva of the right eye of generally anesthetized adult C57BL/6 mice (n = 72). PPARgamma cDNA-expressing adenoviral vector was topically applied; the control eye received nonfunctioning adenoviral vector. At 2, 5, 7, and 14 days (each, n = 18), the eyes were processed for histologic or immunohistochemical examination to evaluate tissue scarring. Expression of type I collagen and growth factors was evaluated by real-time reverse transcription-polymerase chain reaction in 32 eyes from control and treatment groups. RESULTS: PPARgamma overexpression suppressed type I collagen, fibronectin, and CTGF in cultured hSCFs at the mRNA or protein level. In vivo experiments showed that PPARgamma gene introduction suppressed monocyte/macrophage invasion, generation of myofibroblasts, and mRNA upregulation of cytokines/growth factors and collagen Ialpha2 chain (Col 1A2) in healing conjunctiva. CONCLUSIONS: PPARgamma gene transfer suppresses the fibrogenic reaction in hSCFs as well as the injury-induced scarring of conjunctival tissue in mice, suggesting the effectiveness of this strategy in preventing excess scarring after filtration surgery. The mechanism may include suppression of activation of fibroblasts and reduction of macrophage invasion.
Assuntos
Adenoviridae/genética , Túnica Conjuntiva/metabolismo , Doenças da Túnica Conjuntiva/prevenção & controle , Regulação da Expressão Gênica/fisiologia , PPAR gama/genética , Actinas/metabolismo , Animais , Antígenos de Diferenciação , Células Cultivadas , Colágeno Tipo I/genética , Túnica Conjuntiva/lesões , Fator de Crescimento do Tecido Conjuntivo/genética , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Fibronectinas/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , CicatrizaçãoRESUMO
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.
Assuntos
Endotélio Corneano/efeitos dos fármacos , Endotélio Corneano/patologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/antagonistas & inibidores , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Adenoviridae/genética , Animais , Anticorpos/farmacologia , Queimaduras/patologia , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Endotélio Corneano/ultraestrutura , Fibrose , Vetores Genéticos/genética , Integrases/metabolismo , Camundongos , Testes de Neutralização , Técnicas de Cultura de Órgãos , Coelhos , Ratos , Ratos Wistar , Proteínas Smad/metabolismo , Transdução Genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta2/farmacologia , Cicatrização/efeitos dos fármacosRESUMO
Fibrotic diseases are characterized by the appearance of myofibroblasts, the key cell type involved in the fibrogenic reaction, and by excess accumulation of extracellular matrix with resultant tissue contraction and impaired function. Myofiborblasts are generated by fibroblast-myofibrobalst conversion, and in certain tissues through epithelial-mesenchymal transition (EMT), a process through which an epithelial cell changes its phenotype to become more like a mesenchymal cell. Although inflammatory/fibrogenic growth factors/cytokines produced by injured tissues orchestrate the process of EMT, transforming growth factor beta (TGFbeta) is believed to play a central role in the process. Unlike fibrotic lesions in kidney or other tissues where myofibroblasts are generated from both fibroblasts and epithelial cells, fibrotic lesions in the eye crystalline lens are derived only from lens epithelial cells without contamination of fibroblast-derived myofibroblasts. Thus, this tissue is suitable to investigate detailed mechanisms of EMT and subsequent tissue fibrosis. EMT in retinal pigment epithelium is involved in the development of another ocular fibrotic disease, proliferative vitreoretinopathy, a fibrosis in the retina. EMT-related signal transduction cascades, i. e., TGFbeta/Smad, are a target to prevent or treat unfavorable ocular tissue fibrosis, e. g., fibrotic diseases in the crystalline lens or retina, as well as possibly in other organs.
Assuntos
Células Epiteliais/efeitos dos fármacos , Oftalmopatias/patologia , Oftalmopatias/prevenção & controle , Mesoderma/efeitos dos fármacos , Animais , Matriz Extracelular/patologia , Traumatismos Oculares/tratamento farmacológico , Traumatismos Oculares/patologia , Fibrose , Terapia Genética , Humanos , Procedimentos Cirúrgicos Oftalmológicos , Transdução de Sinais/fisiologia , Proteína Smad3/fisiologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/fisiologiaRESUMO
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.
Assuntos
Colágeno Tipo I/metabolismo , Túnica Conjuntiva/efeitos dos fármacos , Fibronectinas/metabolismo , Proteínas Imediatamente Precoces/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Criança , Pré-Escolar , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Túnica Conjuntiva/citologia , Túnica Conjuntiva/metabolismo , Fator de Crescimento do Tecido Conjuntivo , DNA Antissenso/farmacologia , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
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.
Assuntos
Túnica Conjuntiva/patologia , Córnea/patologia , Oftalmopatias/terapia , Terapia Genética/métodos , Cápsula do Cristalino/patologia , Retina/patologia , Animais , Oftalmopatias/patologia , Fibroblastos/patologia , Fibrose/terapia , Técnicas de Transferência de Genes , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Pele/patologia , Proteínas Smad/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
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.
Assuntos
Túnica Conjuntiva/efeitos dos fármacos , Conjuntivite Alérgica/prevenção & controle , Medicamentos de Ervas Chinesas/farmacologia , Emodina/farmacologia , Animais , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Técnicas de Cocultura , Colágeno Tipo I/metabolismo , Túnica Conjuntiva/citologia , Túnica Conjuntiva/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/metabolismoRESUMO
PURPOSE: To examine the effects of blocking p38 mitogen-activated protein kinase (MAPK) on post-injury conjunctival scarring in mice. Its effects on the behaviors of cultured subconjunctival fibroblasts were also investigated. METHODS: An in vivo study was conducted using an adenoviral vector carrying a dominant-negative (DN)-p38MAPK gene. A circumferential incision was made in the equatorial conjunctiva by scissors in the right eye of generally anesthetized adult C57BL/6 mice. DN-p38MAPK-expressing adenoviral vector was topically applied. The left control eye received non-functioning adenoviral vector. At 2, 5, and 7 days (each, n=22) the eyes were processed for histological or immunohistochemical examination to evaluate the tissue scarring. The expressions of type-I collagen and growth factors were evaluated by real time-reverse transcriptase-polymerase chain reaction. The effects of p38MAPK inhibitor on the proliferation, migration, and fibrogenic gene/protein expression of cultured human fibroblasts were also studied. RESULTS: The in vivo DN-p38MAPK gene introduction blocked the phospho-p38 expression with reduction of myofibroblast generation and suppression of mRNA expression of connective tissue growth factor (CTGF) and monocyte/macrophage chemoattractant protein-1 (MCP-1) in the mouse-injured conjunctiva. Blocking p38MAPK signal in the fibroblasts by a chemical inhibitor counteracted TGFbeta1's enhancement of expressions of type-I collagen, fibronectin, and CTGF. It also retarded cell migration, but cell proliferation was unchanged. CONCLUSIONS: Inhibiting p38MAPK signal impairs the fibrogenic reaction induced by the subconjunctival fibroblasts in vivo and in vitro, suggesting its potential effectiveness in preventing excessive scarring following glaucoma filtering surgery.
Assuntos
Cicatriz/prevenção & controle , Túnica Conjuntiva/enzimologia , Túnica Conjuntiva/lesões , Cicatrização , Ferimentos Penetrantes/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Adenoviridae/genética , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno/genética , Colágeno Tipo I , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/patologia , Fator de Crescimento do Tecido Conjuntivo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibrose/metabolismo , Expressão Gênica , Técnicas de Transferência de Genes , Genes Dominantes , Vetores Genéticos , Humanos , Imidazóis/farmacologia , Proteínas Imediatamente Precoces/genética , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Piridinas/farmacologia , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Cicatrização/efeitos dos fármacos , Ferimentos Penetrantes/metabolismo , Ferimentos Penetrantes/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
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.