Activation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2.

Recent studies have revealed a role of endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) in the regulation of RPE cell activity and survival. Herein, we examined the mechanisms by which the UPR modulates apoptotic signaling in human RPE cells challenged with cigarette smoking extract (CSE). Our results show that CSE exposure induced a dose- and time-dependent increase in ER stress markers, enhanced reactive oxygen species (ROS), mitochondrial fragmentation, and apoptosis of RPE cells. These changes were prevented by the anti-oxidant NAC or chemical chaperone TMAO, suggesting a close interaction between oxidative and ER stress in CSE-induced apoptosis. To decipher the role of the UPR, overexpression or down-regulation of XBP1 and CHOP genes was manipulated by adenovirus or siRNA. Overexpressing XBP1 protected against CSE-induced apoptosis by reducing CHOP, p-p38, and caspase-3 activation. In contrast, XBP1 knockdown sensitized the cells to CSE-induced apoptosis, which is likely through a CHOP-independent pathway. Surprisingly, knockdown of CHOP reduced p-eIF2α and Nrf2 resulting in a marked increase in caspase-3 activation and apoptosis. Furthermore, Nrf2 inhibition increased ER stress and exacerbated cell apoptosis, while Nrf2 overexpression reduced CHOP and protected RPE cells. Our data suggest that although CHOP may function as a pro-apoptotic gene during ER stress, it is also required for Nrf2 up-regulation and RPE cell survival. In addition, enhancing Nrf2 and XBP1 activity may help reduce oxidative and ER stress and protect RPE cells from cigarette smoke-induced damage.

Myofibroblasts in macular fibrosis secondary to neovascular age-related macular degeneration - the potential sources and molecular cues for their recruitment and activation.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in developed countries. Neovascular AMD (nAMD) accounts for 90% of AMD-related vision loss. Although intravitreal injection of VEGF inhibitors can improve vision in nAMD, approximately 1/3 of patients do not benefit from the therapy due to macular fibrosis. The molecular mechanism underlying the transition of the neovascular lesion to a fibrovascular phenotype remains unknown. Here we discussed the clinical features and risk factors of macular fibrosis secondary to nAMD. Myofibroblasts are key cells in fibrosis development. However, fibroblasts do not exist in the macula. Potential sources of myofibroblast precursors, the molecular cues in the macular microenvironment that recruit them and the pathways that control their differentiation and activation in macular fibrosis were also discussed. Furthermore, we highlighted the challenges in macular fibrosis research and the urgent need for better animal models for mechanistic and therapeutic studies.

The Role of IRE-XBP1 Pathway in Regulation of Retinal Pigment Epithelium Tight Junctions.

PURPOSE: The retinal pigment epithelium (RPE) tight junctions play a pivotal role in maintaining the homeostatic environment of the neural retina. Herein, we investigated the role of X-box binding protein 1 (XBP1), an endoplasmic reticulum (ER) stress-responsive transcription factor, in regulation of RPE tight junctions. METHODS: Human RPE cell line (ARPE-19) and primary primate RPE cells were used for in vitro experiments and RPE-specific XBP1 knockout (KO) mice were used for in vivo study. Endoplasmic reticulum stress was induced by a sublethal dose of thapsigargin or tunicamycin. XBP1 activation was manipulated by IRE inhibitor 4µ8C, which suppresses XBP1 mRNA splicing. The integrity of tight junctions and the involvement of calcium-dependent RhoA/Rho kinase pathway were examined. RESULTS: Induction of ER stress by thapsigargin, but not tunicamycin, disrupted RPE tight junctions in ARPE-19 cells. Inhibition of XBP1 activation by 4µ8C resulted in a remarkable downregulation of tight junction proteins (ZO-1 and occludin) and defects in tight junction formation in the presence or absence of ER stress inducers. Overexpression of active XBP1 partially reversed 4µ8C-induced anomalies in tight junctions. Mechanistically, XBP1 inhibition resulted in increased intracellular Ca2+ concentration, upregulation of RhoA expression, redistribution of F-actin, and tight junction damage, which was attenuated by Rho kinase inhibitor Y27632. In vivo, deletion of XBP1 in the RPE resulted in defective RPE tight junctions accompanied by increased VEGF expression. CONCLUSIONS: Taken together, these results suggest a protective role of XBP1 in maintaining RPE tight junctions possibly through regulation of calcium-dependent RhoA/Rho kinase signaling and actin cytoskeletal reorganization.

The unfolded protein response in retinal vascular diseases: implications and therapeutic potential beyond protein folding.

Angiogenesis is a complex, step-wise process of new vessel formation that is involved in both normal embryonic development as well as postnatal pathological processes, such as cancer, cardiovascular disease, and diabetes. Aberrant blood vessel growth, also known as neovascularization, in the retina and the choroid is a major cause of vision loss in severe eye diseases, such as diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, and central and branch retinal vein occlusion. Yet, retinal neovascularization is causally and dynamically associated with vasodegeneration, ischemia, and vascular remodeling in retinal tissues. Understanding the mechanisms of retinal neovascularization is an urgent unmet need for developing new treatments for these devastating diseases. Accumulating evidence suggests a vital role for the unfolded protein response (UPR) in regulation of angiogenesis, in part through coordinating the secretion of pro-angiogenic growth factors, such as VEGF, and modulating endothelial cell survival and activity. Herein, we summarize current research in the context of endoplasmic reticulum (ER) stress and UPR signaling in retinal angiogenesis and vascular remodeling, highlighting potential implications of targeting these stress response pathways in the prevention and treatment of retinal vascular diseases that result in visual deficits and blindness.