Diabetes Enhanced FOXO1 Activation Induces the Loss of Retinal Microvascular Cells in Diabetic Retinopathy

Abstract

Purpose:: The death and subsequent disappearance of retinal microvascular cells is accelerated in diabetic retinopathy and is recognized as an important early change in the diabetic retina. Our goal was to investigate the activation of the pro-apoptotic transcription factor FOXO1 in the diabetic retina and to examine its functional role in promoting microvascular cell death.

Methods:: FOXO1 activation was measured in nuclear protein extracts from the retina of type 2 diabetic ZDF rats and matched normoglycemic controls by ActiveMotiff assay and in retinal trypsin digests by nuclear translocation using confocal laser scanning microscopy. The contribution of FOXO1 to microvascular cell death was measured in vivo by intravitreal injection of FOXO1 siRNA compared to scrambled siRNA. Retinal trypsin digests were then examined by the TUNEL assay for apoptosis and by immunofluorescence for activated caspase-3. Rats were euthanized after they had been diabetic for six months. Significance was determined by analysis of variance with Scheffe's post-hoc for comparisons between multiple groups at the P<0.05 level.

Results:: Diabetes caused a three fold increase (P<0.05) in activation of FOXO1 assessed by ActiveMotif assay and more than three fold (P<0.05) nuclear translocation in RTDs by confocal microscopy. In vitro transfection of retinal microvascular endothelial cells with FOXO1 siRNA effectively silenced FOXO1 and reduced apoptosis by 60% (P<0.05) compared to scrambled siRNA. Intravitreal injection of FOXO1 siRNA resulted in an 69% reduction of apoptosis of retinal pericytes and 74% of microvascular endothelial cells in vivo compared to scrambled siRNA (P<0.05). TNF was elevated more than three fold in the retina of ZDF diabetic rats and when TNF was specifically inhibited by intravitreal injection of pegsunercept in diabetic rats FOXO1 elevation was reduced 64% percent (P<0.05). Inhibition of TNF also reduced pericyte and microvascular endothelial cell apoptosis and acellular capillary development in the retina 64% to 80% (P<0.05).

Conclusions:: Studies presented here point to strategies that may prevent apoptosis in humans. Thus, silencing FOXO1 or inhibiting its activation could have therapeutic usefulness in treating diabetic retinopathy or other diabetic microvascular complications that involve enhanced apoptosis.

Keywords: diabetes • transcription factors • apoptosis/cell death