RESUMO
The aim of this study was to better understand the role of apoptosis in a retinal ischaemia-reperfusion injury model and to determine whether sildenafil citrate treatment can prevent retinal cell apoptosis. Thirty-six rats were divided into a control group (n = 6) and two experimentally induced ischaemia-reperfusion groups (7 and 21 days; n = 15 per group). The induced ischaemia-reperfusion groups were treated with sildenafil for 7 and 21 days (n = 10 per group), and 10 animals were treated with a placebo for the same period (n = 5 per group). Paracentesis of the anterior chamber was performed with a 30-G needle attached to a saline solution (0.9%) bag positioned at a height of 150 cm above the eye for 60 min. Intraocular pressure was measured by rebound tonometer (TonoVet® ). The eyes were analysed by histology and morphometry, and by immunohistochemistry and qRT-PCR for expression of Caspase-7, Caspase-6, Caspase-9, Tnf-r2, Fas-l, Bcl-2 and Bax. Sildenafil-treated animals showed lower levels of histopathological changes (inflammatory, cellular and tissue) than their placebo-treated counterparts at both 7 and 21 days. The retinal ganglion cell layer (RGC) was preserved in the sildenafil groups (SG), with a cell count closer to control than in the placebo groups (PG). Caspase-7 expression was significantly higher in both treated groups at 7 days compared to controls. Gene expression levels in both treatment groups differed from the controls, but in SG Bax and Caspase-6 expression levels were similar to control animals. These results suggest that the main mechanism of retinal cell death in this model is apoptosis, as there is an increase in pro-apoptotic factors and decrease in the anti-apoptotic ones. Also, sildenafil seems to protect the retinal ganglion cell layer from apoptosis. Cell survival was evident in the histological and morphometric analyses, and sildenafil treatment had a protective effect in the apoptosis pathways, with gene expression levels in SG similar to the controls.
Assuntos
Traumatismo por Reperfusão/prevenção & controle , Doenças Retinianas/prevenção & controle , Vasos Retinianos/patologia , Citrato de Sildenafila/uso terapêutico , Vasodilatadores/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Pressão Intraocular/efeitos dos fármacos , Masculino , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/patologia , Ratos Endogâmicos Lew , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Doenças Retinianas/fisiopatologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologiaRESUMO
The pancreatic and duodenal homeobox factor 1 (Pdx1) protein is the most pivotal transcription factor in the development of islet ß cells. This study investigated the role of Pdx1 and its mechanism in differentiating induced pluripotent stem cells (iPSCs) into islet ß cells. iPSCs derived from human skin fibroblasts were cultured in vitro and directionally induced to differentiate for 20 days. The expression of insulin-related genes was then detected by RT-PCR, and the expression of several differentiation-related transcription factors was assessed both before and after the differentiation process. Lastly, the specific promoter regions where Pdx1 binds were detected by ChIP. The insulin-related genes, MafA, insulin, Glut2, Nkx6.1, GCK, and Tcf1, showed increased expression during differentiation, and nearly peaked on the 20th day. Similarly, the expression of transcription factors, Pdx1, Ngn3, and Pax6 showed enhanced expression during differentiation as compared with that of the control group. ChIP experiments confirmed that Pdx1 activates the expression of the downstream transcription factors, Ngn3 and Pax6, by combined with the promoter regions of insulin (Insulin-P), Ngn3 (Ngn3-P), and Pax6 (Pax6-P). In conclusion, Pdx1 activates downstream transcription factors Ngn3 and Pax6, and may be one of the mechanisms that promote differentiation of iPSCs into islet ß cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Proteínas do Olho/biossíntese , Proteínas de Homeodomínio/biossíntese , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Fatores de Transcrição Box Pareados/biossíntese , Proteínas Repressoras/biossíntese , Transativadores/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Imunoprecipitação da Cromatina , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas do Olho/genética , Genes Homeobox , Proteínas de Homeodomínio/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Insulina/genética , Células Secretoras de Insulina/citologia , Proteínas do Tecido Nervoso/genética , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genética , Transativadores/genética , Ativação TranscricionalRESUMO
BACKGROUND: Cryotherapy is a no invasive technique that uses intense cold to freeze and destroy cancer tissues. There are no descriptions of its effects over the expression of vascular endothelial growth factor and pigment epithelium-derived factor. METHODS: Experimental study in cryogenic spot were applied in the right sclera of twelve pigs for ten minutes. Other 3 pigs were used as normal controls. Animals were sacrificed at 7, 14 and 21 and the tissues of choriodes and retina were dissected in areas of approximately 1 cm2 surrounding cryogenic spots. Expression levels of vascular endothelial growth factor and pigment epithelium-derived factor were determined analyzed using polymerase chain reaction coupled to reverse-transcription. RESULTS: Vascular endothelial growth factor was significantly downregulated (24%, p< 0.05) seven days post-treatment meanwhile pigment epithelium-derived factor levels increased 44.8% (p< 0.05) as compared to normal controls (untreated). Both vascular endothelial growth factor and pigment epithelium-derived factor levels remain the same until day 14 but returned to basal expression at day 21. DISCUSSION: This work expose the relation of cryotherapy with the expression of two factors related to angiogenesis. RESULTS showed significant changes on the expression of vascular endothelial growth factor and pigment epithelium-derived factor illustrating that both proteins are regulated in response to cryogenic treatment in relatively short periods (21 days).
Antecedentes: la crioterapia es una técnica no invasiva que usa frio intenso para congelar y destruir los tejidos cancerosos. Sus efectos en la expresión del factor de crecimiento del endotelio vascular y el factor derivado del epitelio pigmentado no se han descrito. Material y métodos: estudio experimental en modelos experimentales de crioterapia. En la esclera del ojo derecho de 12 cerdos se aplicó un punto de congelamiento durante 10 segundos. Se usaron 3 cerdos como controles normales. Los animales se sacrificaron a los 7, 14 y 21 días y el tejido de coroides y retina se seccionó en áreas de aproximadamente 1 cm2 circundantes al punto de congelamiento. Los niveles de expresión del factor de crecimiento del endotelio vascular y factor derivado del epitelio pigmentado se determinaron y analizaron por reacción en cadena de la polimerasa acoplada a reverso-transcripción. Resultados: los niveles de factor de crecimiento del endotelio vascular disminuyeron significativamente (24%, p < 0.05) a los 7 días postratamiento, mientras que la expresión del factor derivado del epitelio pigmentado aumentó 44.8% (p< 0.05) en comparación con los niveles de las muestras normales. Los niveles de expresión se mantuvieron hasta el día 14 y regresaron a valores basales en el día 21. Conclusiones: este trabajo expone la relación entre la crioterapia y la expresión de dos factores angiogénicos. Los resultados muestran cambios significativos en la expresión del factor de crecimiento del endotelio vascular y factor derivado del epitelio pigmentado, y evidencian que ambas proteínas son reguladas en respuesta al tratamiento criogénico en periodos relativamente cortos (21 días).
Assuntos
Neovascularização de Coroide/genética , Crioterapia , Proteínas do Olho/biossíntese , Regulação da Expressão Gênica , Fatores de Crescimento Neural/biossíntese , Neovascularização Retiniana/genética , Esclera/metabolismo , Serpinas/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Apoptose , Corioide/metabolismo , Neovascularização de Coroide/etiologia , Endotélio Vascular/metabolismo , Proteínas do Olho/genética , Fatores de Crescimento Neural/genética , Retina/metabolismo , Neovascularização Retiniana/etiologia , Serpinas/genética , Sus scrofa , Suínos , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The cause of keratoconus is unknown. However, an earlier report demonstrated magnesium deficiency in keratoconus patients, and suggested that magnesium deficiency could pathologically affect the mechanisms of the cornea. Experimental and clinical papers concerning a possible relationship between keratoconus and magnesium deficiency were reviewed. These studies have demonstrated molecular and cellular alterations specific to the keratoconic cornea, including: thinning and fragmentation of membranes, degenerated cells and collagen fibres, swelling of the mitochondria, and biochemical abnormalities in protein synthesis. Similar alterations have reportedly been induced by magnesium deficiency. This review suggests a possible relationship between the specific keratoconic disorders and the alteration induced by magnesium deficiency at the intracellular and extracellular levels. Although the etiology of keratoconus is still unknown, this paper may give some new ideas for further experimental and clinical studies on the etiology of keratoconus.
Assuntos
Ceratocone/etiologia , Deficiência de Magnésio/complicações , Apoptose , Proteínas do Olho/biossíntese , Humanos , Ceratocone/metabolismo , Ceratocone/patologia , Deficiência de Magnésio/metabolismo , Deficiência de Magnésio/patologiaRESUMO
To test for a role for the cellular prion protein (PrP(c)) in cell death, we used a PrP(c)-binding peptide. Retinal explants from neonatal rats or mice were kept in vitro for 24 h, and anisomycin (ANI) was used to induce apoptosis. The peptide activated both cAMP/protein kinase A (PKA) and Erk pathways, and partially prevented cell death induced by ANI in explants from wild-type rodents, but not from PrP(c)-null mice. Neuroprotection was abolished by treatment with phosphatidylinositol-specific phospholipase C, with human peptide 106-126, with certain antibodies to PrP(c) or with a PKA inhibitor, but not with a MEK/Erk inhibitor. In contrast, antibodies to PrP(c) that increased cAMP also induced neuroprotection. Thus, engagement of PrP(c) transduces neuroprotective signals through a cAMP/PKA-dependent pathway. PrP(c) may function as a trophic receptor, the activation of which leads to a neuroprotective state.
Assuntos
Anisomicina/farmacologia , Apoptose/efeitos dos fármacos , AMP Cíclico/análogos & derivados , AMP Cíclico/fisiologia , Proteínas do Olho/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas PrPC/metabolismo , Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Anisomicina/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Apoptose/fisiologia , Colforsina/farmacologia , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/biossíntese , Proteínas do Olho/imunologia , Flavonoides/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Soros Imunes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Fosforilação , Proteínas PrPC/química , Processamento de Proteína Pós-Traducional , Ratos , Ratos Endogâmicos , Retina/metabolismo , Tionucleotídeos/farmacologia , Fosfolipases Tipo C/farmacologiaRESUMO
In the present study we have characterized the postnatal (PN) development of the retina in the Brazilian opossum, Monodelphis domestica. Monodelphis, a small, pouchless marsupial, undergoes a protracted period of postnatal development. Using bromodeoxyuridine immunohistochemistry, we have investigated postnatal neurogenesis of the retina. In addition, we have examined the differentiation of the retina by using antibodies directed against the presynaptic terminal-associated proteins synaptotagmin, Rab3A, synaptophysin and synaptosomal-associated protein-25 (SNAP-25), and have characterized their spatial and temporal distribution during postnatal development. This study is the first systematic comparison of the developmental expression of multiple presynaptic terminal-associated proteins in relation to retinal neurogenesis and differentiation. At birth (1PN), the Monodelphis retina was relatively undifferentiated morphologically and birthdating analysis revealed mitotically active cells throughout the retina. The 8PN retina was organized into two cellular layers: an outer region of mitotically active neuroepithelial cells and an inner region of postmitotic cells. The inner plexiform layer formed between 5PN and 10PN, and exhibited unique patterns of immunoreactivity with the antibodies used in this analysis. By 25PN the retina was well laminated, and synaptotagmin-, Rab3A-, synaptophysin- and SNAP-25-like immunoreactivities exhibited distinct and specific patterns within the plexiform layers, although they had not yet achieved their mature, adult patterns. These results indicate that each of these proteins exhibits developmentally regulated changes in its cellular localization, and therefore may play important roles during morphogenesis and synaptogenesis of the vertebrate retina.
Assuntos
Proteínas de Ligação ao Cálcio , Proteínas do Olho/biossíntese , Proteínas de Membrana , Proteínas do Tecido Nervoso/biossíntese , Gambás/metabolismo , Terminações Pré-Sinápticas/metabolismo , Retina/metabolismo , Animais , Bromodesoxiuridina , Diferenciação Celular/fisiologia , Proteínas de Ligação ao GTP/biossíntese , Imuno-Histoquímica , Glicoproteínas de Membrana/biossíntese , Gambás/crescimento & desenvolvimento , Retina/citologia , Retina/crescimento & desenvolvimento , Sinaptofisina/biossíntese , Proteína 25 Associada a Sinaptossoma , Sinaptotagminas , Proteínas rab3 de Ligação ao GTPRESUMO
The contribution of the ciliary body to the origin of vitreous proteins was investigated in rabbits by incubating explants of this eye component under novel conditions. At the end of incubations for up to 21 h, the tissues were processed histologically and were shown to be in an excellent state of morphological preservation. When radioactive amino acids and fucose were added to the culture medium, protein and glycoprotein synthesis and secretion were detected using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) plus fluorography. The origin of these secretory products was traced by autoradiography to the ciliary epithelium. When samples of vitreous bodies - injected intravitreally with the same radioactive precursors - were run beside samples of the tissue culture media, comigration of at least 8 radioactively labelled bands including the one previously identified as transferrin was detected. This indicates that some vitreous proteins may be secreted by the ciliary body and that cultures of explants of ciliary body-iris are useful tools for studies on vitreous protein secretion.
Assuntos
Corpo Ciliar/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Aminoácidos/metabolismo , Animais , Autorradiografia , Corpo Ciliar/efeitos dos fármacos , Meios de Cultura , Cicloeximida/farmacologia , Eletroforese em Gel de Poliacrilamida , Proteínas do Olho/biossíntese , Fucose/metabolismo , Glicoproteínas/biossíntese , Iris/efeitos dos fármacos , Iris/metabolismo , Masculino , Técnicas de Cultura de Órgãos , Epitélio Pigmentado Ocular/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , CoelhosRESUMO
Intermediate filaments of epithelial cells generally consist of specific combinations of keratins. However, cultured epithelial cells from certain tissues and some epithelial tumors have been shown also to express vimentin. In the present study, the expression of vimentin by epithelial cells in healing corneal wounds (partial thickness penetrating wounds) and in tissue culture was analyzed. Both immunohistochemical and immunotransblot analyses indicated that although vimentin was not detected in the normal rabbit corneal epithelium in vivo, cultured rabbit corneal epithelial cells co-express keratins and vimentin. At 1 day post-wounding, vimentin was not detectable in the epithelial cells that had covered the denuded stroma. However, at 2 days postwounding, the epithelium at the base of the epithelial plug immunoreacted with both anti-vimentin and antikeratin monoclonal antibodies. Immunotransblot analyses of the extracts of the epithelial plugs confirmed the presence of vimentin (Mr = 58k). The 58k band was not detected in the extract of normal rabbit corneal epithelium. At day/5, vimentin was no longer detectable in the epithelium. This study demonstrated that corneal epithelial cells transiently co-express vimentin and keratins in vivo during wound healing and in tissue culture. The time-course of the transient expression of vimentin suggests that the vimentin expression in the epithelial cells during healing is not linked to cell proliferation or to the centripetal migration of the epithelium during early stages (first 24 h) of healing, but may be linked to cell-matrix interactions or the migration of basal cells in the upward direction at the following stage of healing.
Assuntos
Lesões da Córnea , Proteínas do Olho/biossíntese , Filamentos Intermediários/metabolismo , Vimentina/biossíntese , Cicatrização , Animais , Córnea/metabolismo , Epitélio/metabolismo , Imunofluorescência , Regulação da Expressão Gênica , Queratinas/biossíntese , CoelhosRESUMO
L-[3H]fucose was injected either intravitreally or intra-aqueously into adult rabbits which were killed at several time points after injection. SDS-polyacrylamide gel electrophoresis and fluorography of iris extracts revealed that most of the proteins are glycoproteins containing fucose residues. Autoradiography of semi-thin histologic sections demonstrated that glycoprotein synthesis was most prominent in the epithelium of the iris, while little protein synthesis was evident in the stroma of the iris. The results of these experiments indicated that the glycoproteins of the iris undergo renewal. The protein band pattern of the iris extracts was very similar to that of extracts of the ciliary body. The high-molecular-weight cartilage matrix glycoprotein (CMGP), an intrinsic component of the ciliary body, vitreous, and aqueous humor, was detected by immunohistologic studies only in the stroma of the iris. The results of immunohistochemical analyses of the eyes of young rabbits (1-21 days old), in addition to the autoradiographic findings, strongly suggest that CMGP is not an intrinsic glycoprotein of the iris stroma, at least in this species.