We report for the first time that diabetes increases FOXO1 mRNA levels and DNA binding activity in the retina. In addition, both type 1 and type 2 diabetic models exhibited increased FOXO1 nuclear translocation in retinal microvascular cells. These increases occurred in a time frame when increased inflammation, caspase-3–positive cells, and enhanced apoptosis are present (8–26 weeks). Moreover, TNF-mediated diabetes enhanced FOXO1 mRNA levels, DNA binding, and nuclear translocation. Thus, we propose that diabetes increases TNF-α, which enhances FOXO1 mRNA levels, nuclear translocation, and DNA binding in retinas of type 1 and type 2 diabetic rats.
The functional significance of FOXO1 was tested by RNAi. Intravitreal injection of FOXO1 siRNA significantly reduced FOXO1 mRNA levels and FOXO1 nuclear translocation, while control-scrambled siRNA had no effect. The effect of FOXO1 knockdown by siRNA was evident 6 weeks after the second injection, indicating that silencing is effective for at least 6 weeks. In type 1 diabetic rats, FOXO1 siRNA significantly decreased microvascular cell apoptosis, the formation of pericyte ghosts, and the development of acellular capillaries. Similar results were found in type 2 diabetic rats. FOXO1 siRNA significantly reduced cleaved caspase-3– and TUNEL-positive pericytes and endothelial cells. This represents the first demonstration that FOXO1 plays a critical role in diabetes-induced apoptosis and retinal microvascular cell loss.
In retinal microvascular endothelial cells, high glucose was shown to increase FOXO1 mRNA and protein levels. In addition, high glucose reduced FOXO1 phosphorylation, consistent with increased DNA binding activity, since FOXO1 is deactivated by phosphorylation. High glucose did not affect total Akt levels but did significantly decrease the active phosphorylated isoforms of Akt. When Akt was inhibited there was a further decrease in FOXO1 phosphorylation, indicating that high glucose levels could modulate FOXO1 through its effect on Akt. In addition, hypoinsulinemia or insulin resistance associated with diabetes could potentially contribute to decreased phosphorylation of Akt in vivo and contribute to enhanced FOXO1 activity (25
As discussed above, TNF-mediated diabetes enhanced FOXO1 activation both in vitro and in vivo. Furthermore, TNF stimulated apoptosis that was reduced by FOXO1 siRNA. Similarly, ROS inhibitor reduced high-glucose–stimulated FOXO1 DNA binding activity and high-glucose–induced apoptosis. Also, high-glucose–stimulated apoptosis was inhibited by FOXO1 siRNA. Thus, several pathways by which high glucose increased FOXO1 were tested. In each case, the factor was shown to mediate high-glucose–stimulated apoptosis of retinal endothelial cells. The results obtained in vitro and in vivo are strikingly similar and indicate that hyperglycemia per se could stimulate apoptosis of microvascular cells through a mechanism not involving other cell types, although it cannot be ruled out that other cell types or leukostasis in the retina may play an important role in the apoptotic process (26
In vitro mRNA profiling suggests that FOXO1 mediates high-glucose–induced mRNA expression of genes that modulate endothelial cell activation and apoptosis and the basal expression of genes that affect angiogenesis, which were not altered by high-glucose conditions. A surprising finding was that FOXO1 siRNA reduced mRNA levels of several genes that regulate proinflammatory and procoagulant responses. Thus, FOXO1 could induce expression of genes that participate in the very early events that affect diabetic retinal microvascular endothelial cells, many of which are known to be increased by diabetes (28
). This is significant since it has not previously been recognized that FOXO1 could modulate proinflammatory gene expression. However, it is consistent with activation of FOXO1 during the early inflammatory phase of diabetic retinopathy. In addition, FOXO1 siRNA modulated a high percentage of proapoptotic genes, consistent with its known proapoptotic function (20
). This is supported by data reported here that FOXO1 siRNA inhibited high-glucose–stimulated caspase-3 activity and apoptosis in microvascular endothelial cells in vitro. Furthermore, inhibition of caspase-3 blocked high-glucose–stimulated apoptosis, consistent with FOXO1-regulated caspase-3 mRNA levels and caspase-3 activity. Last, mRNA expression of genes associated with angiogenesis were also enhanced by high glucose and/or reduced by FOXO1 siRNA. This is consistent with a report that FOXO1 overexpression induces genes that regulate angiogenesis and vascular remodeling (31
). Thus, high glucose and FOXO1 had a dramatic effect on genes that regulate a relatively diverse array of genes that modulate endothelial cell behavior, several of which are thought to contribute to diabetic retinopathy (30
). FOXO1 can affect gene expression without directly binding to DNA (32
). For example, FOXO1 can regulate transcription by enhancing the activity of CCAAT/enhancer binding protein α or by modulating the DNA binding and transcriptional activity of PAX3 (33
Few studies have examined the role of transcription factors in early diabetic retinopathy, and a limitation of our studies is that it is unknown if these findings pertain to humans with diabetes. Findings from the short-term application of FOXO1 RNAi point to the possibility of using long-term inhibition of FOXO1 by shRNA in diabetic retinopathy. Moreover, FOXO1 may potentially play an important role in other diabetes complications, where locally enhanced inflammation and apoptosis are associated with pathologic changes. In summary, studies presented here demonstrate a mechanistic link between diabetes, enhanced TNF-α levels in the retina, increased FOXO1 nuclear translocation, and microvascular cell apoptosis. This provides a basis for understanding how chronic low-grade inflammation may play a role in early diabetic retinopathy.