FOXO1 Plays an Important Role in Enhanced Microvascular Cell Apoptosis and Microvascular Cell Loss in Type 1 and Type 2 Diabetic Rats

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 ST7-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 I and type 2 diabetic rats and can be linked to the effect of high glucose on FOXO1 activation. Diabetes 58:917-925, 2009