RESUMO
Retinal neovascularization (RNV) is an important pathological feature of vitreoretinopathy that can lead to severe vision loss. The purpose of this study was to identify the role of ephrin-A5 (Efna5) in RNV and to explore its mechanism. The expression pattern and biological significance of Efna5 were investigated in a mouse model of oxygen-induced retinopathy (OIR). The expression of Efna5 and downstream signaling pathway members was determined by RT-PCR, immunofluorescence, immunohistochemistry, and western blot analyses. shRNA was used to knockdown Efna5 in the retina of the OIR mouse model. Retinal flat mounts were performed to evaluate the impact of Efna5 silencing on the RNV process. We found that the Efna5 was greatly upregulated in the retina of OIR mice. Elevated Efna5 mainly colocalized with the retinal vessels and endothelial cells. We then showed that knockdown of Efna5 in OIR mouse retinas using lentivirus-mediated shRNA markedly decreased the expression of Efna5 and reduced the retinal neovascularization and avascular retina area. We further showed hypoxia stimulation dramatically increased both total and phosphorylation levels of ERK1/2 and the phosphorylation levels of Akt in OIR mice. More importantly, knockdown of Efna5 could inhibit the p-Akt and p-ERK signaling pathways. Our results suggested that Efna5 may regulate the RNV. This study suggests that Efna5 was significantly upregulated in the retina of OIR mice and closely involved in the pathological retinal angiogenesis.
Assuntos
Efrina-A5/metabolismo , Neovascularização Retiniana/etiologia , Retinopatia da Prematuridade/etiologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Efrina-A5/antagonistas & inibidores , Efrina-A5/genética , Técnicas de Silenciamento de Genes , Injeções Intravítreas , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Retinopatia da Prematuridade/genética , Retinopatia da Prematuridade/metabolismo , Regulação para CimaRESUMO
Glioblastoma (GBM) is the most common and most aggressive intrinsic brain tumour in adults. Integrated transcriptomic and epigenomic analyses of glioblastoma initiating cells (GIC) in a mouse model uncovered a novel epigenetic regulation of EfnA5. In this model, Bmi1 enhances H3K27me3 at the EfnA5 locus and reinforces repression of selected target genes in a cellular context-dependent fashion. EfnA5 mediates Bmi1-dependent proliferation and invasion in vitro and tumour formation in an allograft model. Importantly, we show that this novel Polycomb feed-forward loop is also active in human GIC and we provide pre-clinical evidence of druggability of the EFNA5 signalling pathway in GBM xenografts overexpressing Bmi1.
Assuntos
Efrina-A5/metabolismo , Glioblastoma/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Animais , Anti-Hipertensivos/farmacologia , Proliferação de Células , Doxazossina/farmacologia , Sistemas de Liberação de Medicamentos , Efrina-A5/antagonistas & inibidores , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Histonas/metabolismo , Humanos , Lisina/metabolismo , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Células-Tronco Neurais/metabolismo , Neurogênese , Complexo Repressor Polycomb 1/genética , Células Tumorais CultivadasRESUMO
Recent findings suggest that ephrinA5 (Efna5) has a novel role in female mouse fertility, in addition to its well-defined role as a neurogenesis factor. Nevertheless, its physiological roles in ovarian granulosa cells (GC) have not been determined. In this study, mouse GC were cultured and transfected with ephrin A5 siRNA and negative control to determine the effects of Efna5 on GC apoptosis, proliferation, cell cycle progression, and related signaling pathways. To understand the mode signaling, the mRNA expression levels of Efna5 receptors (Eph receptor A5, Eph receptor A3, Eph receptor A8, and Eph receptor B2) were examined. Both mRNA and protein expressions of apoptosis-related factors (Bax, Bcl-2, Caspase 8, Caspase 3, and Tnfα) and a proliferation marker, Pcna, were investigated. Additionally, the role of Efna5 on paracrine oocyte-secreted factors and steroidogenesis hormones were also explored. Efna5 silencing suppressed GC apoptosis by downregulating Bax and upregulating Bcl-2 in a Caspase 8-dependent manner. Efna5 knockdown promoted GC proliferation via p-Akt and p-ERK pathway activation. The inhibition of Efna5 enhanced BMH15 and estradiol expression, but suppressed GDF9, while progesterone level remained unaltered. These results demonstrated that Efna5 is a pro-apoptotic agent in GC and plays important role in folliculogenesis by mediating apoptosis, proliferation, and steroidogenesis in female mouse. Therefore Efna5 might be potential therapeutic target for female fertility disorders.
Assuntos
Efrina-A5/genética , Estradiol/metabolismo , Fertilidade/genética , Células da Granulosa/metabolismo , Progesterona/metabolismo , Transdução de Sinais/genética , Animais , Apoptose/genética , Caspase 3/genética , Caspase 3/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Ciclo Celular/genética , Proliferação de Células , Efrina-A3/genética , Efrina-A3/metabolismo , Efrina-A5/antagonistas & inibidores , Efrina-A5/metabolismo , Efrina-B2/genética , Efrina-B2/metabolismo , Feminino , Regulação da Expressão Gênica , Células da Granulosa/citologia , Camundongos , Cultura Primária de Células , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Stroke causes loss of neurological function. Recovery after stroke is facilitated by forced use of the affected limb and is associated with sprouting of new connections, a process that is sharply confined in the adult brain. We show that ephrin-A5 is induced in reactive astrocytes in periinfarct cortex and is an inhibitor of axonal sprouting and motor recovery in stroke. Blockade of ephrin-A5 signaling using a unique tissue delivery system induces the formation of a new pattern of axonal projections in motor, premotor, and prefrontal circuits and mediates recovery after stroke in the mouse through these new projections. Combined blockade of ephrin-A5 and forced use of the affected limb promote new and surprisingly widespread axonal projections within the entire cortical hemisphere ipsilateral to the stroke. These data indicate that stroke activates a newly described membrane-bound astrocyte growth inhibitor to limit neuroplasticity, activity-dependent axonal sprouting, and recovery in the adult.
Assuntos
Axônios/metabolismo , Efrina-A5/metabolismo , Plasticidade Neuronal/fisiologia , Recuperação de Função Fisiológica/fisiologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Axônios/patologia , Comportamento Animal , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Efrina-A5/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/fisiologia , Rede Nervosa/fisiopatologia , Fosforilação , Transdução de Sinais , Coloração e RotulagemRESUMO
The calcium-stimulated adenylate cyclase 1 (AC1) has been shown to be required for the refinement of the retinotopic map, but the mechanisms involved are not known. To investigate this question, we devised a retinotectal coculture preparation that reproduces the gradual acquisition of topographic specificity along the rostrocaudal axis of the superior colliculus (SC). Temporal retinal axons invade the entire SC at 4 d in vitro (DIV) and eliminate exuberant branches caudally by 12 DIV. Temporal and nasal axons form branches preferentially in the rostral or caudal SC, respectively. Retinal explants from AC1-deficient mice, AC1(brl/brl), maintain exuberant branches and lose the regional selectivity of branching when confronted with wild-type (WT) SC. Conversely, WT retinas correctly target AC1(brl/brl) collicular explants. The effects of AC1 loss of function in the retina are mimicked by the blockade of ephrin-A5 signaling in WT cocultures. Video microscopic analyses show that AC1(brl/brl) axons have modified responses to ephrin-A5: the collapse of the growth cones occurs, but the rearward movement of the axon is arrested. Our results demonstrate a presynaptic, cell autonomous role of AC1 in the retina and further indicate that AC1 is necessary to enact a retraction response of the retinal axons to ephrin-A5 during the refinement of the retinotopic map.