RESUMEN
Activation of the ERK mitogen-activated protein kinase pathway has been implicated in pro-survival and cellular protective mechanisms, so that chronic ERK activation may be a useful therapeutic strategy. Here, we further explored the consequences of prolonged ERK activation following expression of constitutively active form of MEK, MEK-EE, in cardiac myocytes. We confirmed that chronic MEK-EE overexpression halved myocyte death following glucose deprivation, but surprisingly this was not associated with preserved intracellular ATP levels. Whilst activities of a number of antioxidant enzymes were not altered upon MEK-EE expression, paradoxically Cu/Zn superoxide dismutase activity was almost halved upon MEK-EE expression. When we then exposed myocytes to the superoxide generator menadione, we observed significantly higher death of MEK-EE expressing myocytes. Pre-incubation with U0126 inhibited menadione-induced death. Our results are the first to show that MEK-ERK signalling can act to increase or decrease cell survival, the outcome depending on the form of stress stimulus encountered.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/fisiología , Miocitos Cardíacos/citología , Adenosina Trifosfato/metabolismo , Animales , Antioxidantes/farmacología , Butadienos/farmacología , Supervivencia Celular/fisiología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Glucosa/deficiencia , Glucosa/metabolismo , Ácido Glutámico/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Nitrilos/farmacología , Mutación Puntual , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Superóxido Dismutasa/metabolismo , Factores de Tiempo , Vitamina K 3/farmacologíaRESUMEN
Successful clinical transplantation of whole skeletal muscles can be limited by impaired muscle revascularization and regeneration. The aim of this study was to enhance the revascularization (and hence speed of regeneration) of transplanted whole muscles by transducing muscles with the vascular endothelial growth factor (VEGF) gene before transplantation, using a recombinant adeno-associated virus (rAAV). The rAAV encoding VEGF and green fluorescent protein (GFP) (rAAV.VEGF.GFP) was injected into the tibialis anterior muscles of adult BALB/c mice. One month after injection whole muscle autotransplantation was performed. Muscles were sampled 7 days after autografting. GFP expression was examined as an indicator of persistent transgene expression after grafting, and immunohistochemistry was used to identify VEGF, blood vessels, and newly formed myotubes. After grafting, GFP expression persisted only in a few surviving myofibers in the periphery of rAAV.VEGF.GFP-pretreated muscles, although abundant VEGF expression was seen in myogenic cells in all grafted muscles. Quantitative analysis demonstrated that, although only small numbers of rAAV.VEGF.GFP-transduced myofibers were present, whole muscle grafts preinjected with rAAV.VEGF.GFP were significantly more vascular than saline-injected and uninjected control muscle grafts. Furthermore, rAAV.VEGF.GFP-injected whole muscle transplants were further advanced in terms of regeneration (myotube formation) compared with the uninjected control muscle transplants. This study clearly shows that rAAV-mediated VEGF expression persists only in myofibers that survive the necrosis induced by muscle transplantation; however, this amount of VEGF results in significantly increased revascularization and regeneration of whole muscle transplants.
Asunto(s)
Factores de Crecimiento Endotelial/genética , Terapia Genética , Péptidos y Proteínas de Señalización Intercelular/genética , Linfocinas/genética , Músculo Esquelético/fisiología , Neovascularización Fisiológica/fisiología , Regeneración/fisiología , Animales , Dependovirus , Factores de Crecimiento Endotelial/metabolismo , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Linfocinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Músculo Esquelético/trasplante , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
This work aims to investigate the effect of compromised lysosomal enzyme activity on the accumulation of photoreceptor-derived debris in the retinal pigment epithelial (RPE) cells and to examine if this accelerated debris accumulation can induce retinal abnormalities similar to those observed in aged individuals. A mutated, enzymatically inactive form of cathepsin D (CatD), generated by site-directed mutagenesis was used to produce stable cell lines and transgenic mice. There was a strong increase in enzymatically inactive CatD protein production in the mutated CatD DNA transfected D407 cells (D407MCD). The presence of the inactive CatD has been linked to an impairment in bovine rod outer segment (BROS) digestion and was confirmed by a statistically significant increase of undigested residual BROS in the medium of D407MCD when compared to the control vector-transfected D407 cells (t-test, P < or = 0.016, P < or = 0.003) or untransfected D407 cells (t-test, P < or = 0.008, P < or = 0.003). The impairment was also confirmed in vivo by demonstration of BROS-derived debris accumulation in the RPE cell layer of transgenic mice. These results demonstrated that the mutated and inactive CatD form could lead to impairment of photoreceptor outer segments (POS) proteolysis. It is proposed that this initial impairment of POS proteolysis may result in the accumulation of CatD-opsin-like complexes in the pigment epithelium, which further compromises RPE cell functions and thus causes the changes observed in aging humans.