Abstract
Purpose: :
An early step in diabetic retinopathy is the accelerated death of microvascular cells. We determined whether TNF dysregulation significantly contributed to enhanced apoptosis and whether it a mechanism involved diabetes–enhanced activation of the pro–apoptotic transcription factor, FOXO1.
Methods: :
A TNF–specific inhibitor, etanercept was used to investigate the role of TNF in microvascular cell apoptosis in streptozotocin induced diabetic rats. Apoptosis was measured in retinal trypsin digests by the TUNEL assay. In vivo retinal specimens were assessed for activation of transcription factors NFΚB and FOXO1 by EMSA and caspase–3 activity by fluorimetric assay. Apoptosis of primary cultures of retinal pericytes and endothelial cells was assessed in vitro. Apoptosis was measured by histone–associated cytoplasmic DNA and FOXO1 activation by EMSA and ELISA. The role of FOXO1 in TNF induced microvascular cell apoptosis was functionally examined by silencing with siRNA.
Results: :
In diabetic rats there was a five fold increase in microvascular cell apoptosis in the retina, which was blocked by etanercept (P<0.05). Diabetes–enhanced activation of NFΚB and FOXO1 was also reversed by the TNF–specific antagonist. In vitro studies demonstrated that TNF stimulated apoptosis of retinal pericytes and endothelial cells were largely dependent upon FOXO1. By use of specific kinase inhibitors, TNF stimulated FOXO1 activation and microvascular cell apoptosis was shown to significantly dependent on p38 and JNK MAP kinases (P<0.05). Specific Akt and NFΚB inhibitors enhanced apoptosis suggesting that they activate pro–survival signals in retinal pericytes.
Conclusions: :
These data indicate that TNF plays a significant role in diabetes–enhanced apoptosis of retinal microvascular cells and that the loss of these cells involves TNF induced activation of the MAP kinase pathway and the transcription factor FOXO1.
Keywords: diabetic retinopathy • apoptosis/cell death • vascular cells