Abstract
Purpose: :
We previously reported that endoplasmic reticulum (ER) stress is a key player in diabetic retinal inflammation and vascular leakage. However, the signaling pathways that link these critical pathogenic processes remain unclear. Activating transcription factor-4 (ATF4) is a transcriptional factor induced by various cellular stresses. The objective of this study is to delineate the role of ATF4 in vascular endothelial inflammation and vascular leakage in retinal diseases such as diabetic retinopathy and uveitis.
Methods: :
Immortalized retinal endothelial (TRi-BRB) and primary brain endothelial cells were exposed to high glucose (25 mM), TNF-α (10 ng/ml), or LPS (250 ng/ml) to induce ER stress and inflammation. ATF4 activity was manipulated by adenoviruses expressing wildtype (WT) or a dominant negative mutant (DN) of ATF4. Mouse models of diabetes and uveitis were induced by streptozotocin and LPS, respectively, in heterozygous ATF4 knockout (KO) and wild type (WT) mice. Inflammatory gene expression was determined by real-time RT-PCR, Western blot analysis, immunohistochemistry, and ELISA. Inflammatory signaling pathways, including JNK, STAT3, and NF-ΚB, were analyzed.
Results: :
ER stress and ATF4 expression were increased by high glucose, TNF-α and LPS in retinal and brain endothelial cells. Inhibiting ATF4 activity by Ad-ATF4DN, or loss of ATF4 gene significantly attenuated high glucose- or LPS-induced upregulation of ICAM-1, TNF-α and VEGF, and reduced secretion of MCP-1. Conversely, over-expressing ATF4 was sufficient to induce expression of inflammatory genes in cultured endothelial cells, and in mouse retinas after intravitreal injection of Ad-ATF4. This was in part through activation of the JNK and STAT3 pathways. In mouse models of diabetes and uveitis, depletion of ATF4 gene significantly prevented increased expression of retinal adhesion molecules (ICAM-1) and inflammatory cytokines (MCP-1, IL-1β and VEGF), and mitigated retinal vascular permeability.
Conclusions: :
Our results indicate that ATF4 is a key regulator of ER stress-associated inflammation in vascular endothelial cells. Inhibition of ATF4 activation may provide a new therapeutic target in inflammatory retinal diseases, such as diabetic retinopathy and uveitis.
Keywords: inflammation • stress response • edema