Abstract
Purpose: :
Endothelial cell death is a hallmark of diabetic retinopathy and inflammation plays a pivotal role in driving vascular injury. We recently discovered that endoplasmic reticulum (ER) stress is a key player in diabetes-induced retinal inflammation. However, the signaling pathways that link these critical pathogenic processes remain unclear. Here, we investigated the role of activating transcription factor 4 (ATF4), a major downstream effector of ER stress, in diabetic retinopathy.
Methods: :
Rat retinal Müller (rMC-1) and endothelial (TRi-BRB) cells were used in in vitro studies. High glucose (25 mM) and TNF-α (10 ng/ml) were used as diabetic insults. ATF4 activity was manipulated by adenoviruses expressing wildtype (WT) or a dominant negative mutant (DN) of ATF4. For in vivo experiments, ATF4 knockout (KO) and WT mice were induced diabetes by streptozotocin. Inflammatory gene expression was determined real-time RT-PCR, Western blot analysis and immunohistochemistry. Inflammatory signaling pathways mediated by JNK and STAT3 were analyzed. Apoptosis was assessed by TUNEL staining and caspase activation.
Results: :
In cultured rMC-1 and TRi-BRB cells, high glucose treatment induced a significant increase in nuclear ATF4 level, which was abolished by ER stress inhibitors 4-PBA and TUDCA, suggesting that ATF4 induction by hyperglycemia is driven by ER stress. Blockade of ATF activity by Ad-ATF4DN significantly attenuates high glucose-induced ICAM-1, TNF-α and VEGF expression. Conversely, over-expression of ATF4 results in inflammatory gene up-regulation, which was abolished by inhibition of STAT3 and JNK activation. In addition, over-expression of ATF4 induces the pro-apoptotic gene CHOP expression, caspase 3 activation and cell apoptosis in TRi-BRB cells, while ATF4 inhibition attenuates TNF-α-induced endothelial cell apoptosis. Consistently, intravitreal injection of Ad-ATF4 induces a remarkable increase in retinal inflammation in non-diabetic mice. In diabetic animals, suppressing ATF4 function by Ad-ATF4DN or genetic depletion of ATF4 using ATF4 KO mice markedly decreased retinal inflammation, ameliorated retinal cell apoptosis and mitigates retinal vascular permeability.
Conclusions: :
ATF4 activated by ER stress in diabetes up-regulates retinal inflammatory gene expression and mediates endothelial cell apoptosis, and therefore contributes to the pathogenesis of diabetic retinopathy.
Keywords: inflammation • apoptosis/cell death • diabetic retinopathy