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Sarah X. Zhang, Yimin Zhong, Yanming Chen, Tim Townes, Joshua J. Wang; Activating Transcription Factor 4 is A Novel Pathogenic Gene in Retinal Inflammation and Endothelial Cell Death in Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2011;52(14):996.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
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