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OBJECTIVE - To investigate early events leading to microvascular cell loss in diabetic retinopathy.
RESEARCH DESIGN AND METHODS- FOXOl 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 FOXOl with RNAi. Microvascular cell apoptosis, formation of pericyte ghosts, and acellular capillaries were measured. Upstream and downstream effects of high-glucoseinduced FOXOl were tested on rat microvascular endothelial cells (RMECs) by small-interfering RNA (siRNA) in vitro.
RESULTS- DNA binding or nuclear translocation of FOXOl, 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 FOXOl siRNA. High glucose in vitro decreased FOXOl phosphorylation and DNA binding activity and decreased Akt phosphorylation in RMECs. High-glucose-stimulated FOXOl DNA binding activity was mediated through TNF-α and formation of reactive oxygen species (ROS), while inhibitors of TNF and ROS and FOXOl 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 FOXOl siRNA
CONCLUSIONS- FOXOl 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 FOXOl activation. Diabetes 58:917-925, 2009
Diabetic retinopathy, the leading cause of vision loss in occupational-age adults (1,2), is charac- terized by early vascular lesions, including ap- optosis of microvascular cells, formation of pericyte ghosts, and the development of acellular capillar- ies before the onset of clinical complications (3,4). The formation of acellular capillaries eventually leads to hyp- oxia, setting the stage for proliferative diabetic retinopathy that ultimately results in impaired vision (5-8).
The loss of critical microvascular cells in the early stages of this complication are not well understood. To investigate this issue, we examined in type 1 and type 2 diabetic rats the role of the transcription factor FOXOl, a forkhead transcription factor that regulates cell death, inhibits cell cycle progression, and modulates differentiation in various cell types (9-11)....