RESUMEN
OBJECTIVE: To investigate early events leading to microvascular cell loss in diabetic retinopathy. RESEARCH DESIGN AND METHODS: FOXO1 was tested in vivo by DNA binding activity and by nuclear translocation in microvascular cells in retinal trypsin digests. In vivo studies were undertaken in STZ-induced diabetic rats and Zucker diabetic fatty rats using the tumor necrosis factor (TNF)-specific blocker, pegsunercept, or by inhibiting FOXO1 with RNAi. Microvascular cell apoptosis, formation of pericyte ghosts, and acellular capillaries were measured. Upstream and downstream effects of high-glucose-induced FOXO1 were tested on rat microvascular endothelial cells (RMECs) by small-interfering RNA (siRNA) in vitro. RESULTS: DNA binding or nuclear translocation of FOXO1, which was reduced by TNF inhibition, was elevated in type 1 and type 2 diabetic retinas. Diabetes stimulated microvascular cell apoptosis; pericyte ghost and acellular capillary development was inhibited by FOXO1 siRNA. High glucose in vitro decreased FOXO1 phosphorylation and DNA binding activity and decreased Akt phosphorylation in RMECs. High-glucose-stimulated FOXO1 DNA binding activity was mediated through TNF-alpha and formation of reactive oxygen species (ROS), while inhibitors of TNF and ROS and FOXO1 siRNA reduced high-glucose-enhanced RMEC apoptosis. The caspase-3/7 activity and capacity of high glucose to increase mRNA levels of several genes that regulate RMEC activation and apoptosis were knocked down by FOXO1 siRNA. CONCLUSIONS: FOXO1 plays an important role in rat retinal microvascular cell loss in type 1 and type 2 diabetic rats and can be linked to the effect of high glucose on FOXO1 activation.
Asunto(s)
Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/patología , Endotelio Vascular/patología , Factores de Transcripción Forkhead/genética , Proteínas del Tejido Nervioso/genética , Vasos Retinianos/patología , Animales , Apoptosis , Núcleo Celular/fisiología , Factores de Transcripción Forkhead/fisiología , Hiperglucemia/genética , Inmunohistoquímica , Microcirculación , Proteínas del Tejido Nervioso/fisiología , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Ratas Zucker , TransfecciónRESUMEN
Retinal microvascular cell loss plays a critical role in the pathogenesis of diabetic retinopathy. To examine this further, type 1 streptozotocin-induced diabetic rats and type 2 Zucker diabetic fatty rats were treated by intravitreal injection of the tumor necrosis factor-specific inhibitor pegsunercept, and the impact was measured by analysis of retinal trypsin digests. For type 2 diabetic rats, the number of endothelial cells and pericytes positive for diabetes-enhanced activated caspase-3 decreased by 81% and 86%, respectively, when treated with pegsunercept (P < 0.05). Similarly, the number of diabetes-enhanced terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive endothelial cells and pericytes decreased by 81% and 67% respectively when treated with pegsunercept (P < 0.05). Diabetes-increased activated caspase-3- and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive microvascular cell numbers were both reduced by 81% and 80%, respectively, in pegsunercept-treated type 1 diabetic rats (P < 0.05). Inhibition of tumor necrosis factor reduced type 1 diabetes-enhanced pericyte ghost formation by 87% and the number of type 2 diabetes-enhanced pericyte ghosts by 62% (P < 0.05). Similarly, increased acellular capillary formation caused by type 1 and type 2 diabetes was reduced by 68% and 67%, respectively, when treated with pegsunercept (P < 0.05). These results demonstrate a previously unrecognized role of tumor necrosis factor-alpha in promoting the early pathogenesis of diabetic retinopathy leading to loss of retinal microvascular cells and demonstrate the potential therapeutic benefit of modulating its activity.
