Our findings suggest that strategies targeting NOX1 have the potential to be effective treatments for a range of ischemic retinopathies.

Abstract-Neovascularization is a hallmark feature of retinopathy of prematurity and diabetic retinopathy. Type 1 angiotensin receptor blockade reduces neovascularization in experimental retinopathy of prematurity, known as oxygen-induced retinopathy (OIR). We investigated in OIR whether inhibiting aldosterone with the aldosterone synthase inhibitor FAD286 reduced neovascularization as effectively as angiotensin receptor blockade (valsartan). OIR was induced in neonatal Sprague-Dawley rats, and they were treated with FAD286 (30 mg/kg per day), valsartan (10 mg/kg per day), or FAD286ϩvalsartan. The cellular sources of aldosterone synthase, the mineralocorticoid receptor, and 11␤-hydroxysteroid dehydrogenase 2 were evaluated in retinal cells involved in neovascularization (primary endothelial cells, pericytes, microglia, ganglion cells, and glia). In OIR, FAD286 reduced neovascularization and neovascular tufts by 89% and 67%, respectively, and normalized the increase in vascular endothelial growth factor mRNA (1.74-fold) and protein (4.74-fold) and was as effective as valsartan and FAD286ϩvalsartan. In retina, aldosterone synthase mRNA was reduced with FAD286 but not valsartan. Aldosterone synthase was detected in microglia, ganglion cells, and glia, whereas mineralocorticoid receptor and 11␤-hydroxysteroid dehydrogenase 2 were present in all of the cell types studied. Given the location of aldosterone synthase in microglia and their contribution to retinal inflammation and neovascularization in OIR, the effects of FAD286 on microglial density were studied. The increase in microglial density (ionized calcium binding adaptor protein 1 immunolabeling) in OIR was reduced with all of the treatments. In OIR, FAD286 reduced the increase in mRNA for tumor necrosis factor-␣, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and monocyte chemoattractant molecule 1. These findings indicate that aldosterone inhibition may be a potential treatment for retinal neovascularization.

Activation-induced cell death (AICD) plays a critical role in immune homeostasis and tolerance. In T-cell-dependent humoral responses, AICD of B cells is initiated by Fas ligand (FasL) on T cells, stimulating the Fas receptor on B cells. In contrast, T-cell-independent B cell responses involve innate-type B lymphocytes, such as marginal zone (MZ) B cells, and little is known about the mechanisms that control AICD during innate B cell responses to Toll-like receptor (TLR) activation. Here, we show that MZ B cells undergo AICD in response to TLR4 activation in vivo. The transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI) receptor and TLR4 cooperate to upregulate expression of both FasL and Fas on MZ B cells and also to repress inhibitors of Fas-induced apoptosis signaling. These findings demonstrate an unappreciated role for TACI and its ligands in the regulation of AICD during T-cell-independent B cell responses.

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Neovascular retinopathies are major causes of vision loss; yet treatments to prevent the condition are inadequate. The role of regulatory T cells in neovascular retinopathy is unknown. Here we show that in retinopathy regulatory T cells are transiently increased in lymphoid organs and the retina, but decline when neovascularization is established. The decline is prevented following regulatory T cells expansion with an IL-2/anti-IL-2 mAb complex or the adoptive transfer of regulatory T cells. Further, both approaches reduce vasculopathy (vaso-obliteration, neovascularization, vascular leakage) and alter the activation of Tmem119+ retinal microglia. Our in vitro studies complement these findings, showing that retinal microglia co-cultured with regulatory T cells exhibit a reduction in co-stimulatory molecules and pro-inflammatory mediators that is attenuated by CTLA-4 blockade. Collectively, we demonstrate that regulatory T cells are recruited to the retina and, when expanded in number, repair the vasculature. Manipulation of regulatory T cell numbers is a previously unrecognized, and promising avenue for therapies to prevent blinding neovascular retinopathies.

Our findings indicate that an RORγ/IL-17A axis influences VEGF production and neovascular retinopathy by mechanisms involving neuroglia. Inhibition of RORγ and IL-17A may have potential for the improved treatment of neovascular retinopathies.

BackgroundInflammation and the excess production of reactive oxygen species (ROS) contribute significantly to the pathogenesis of ischemic retinopathies such as diabetic retinopathy and retinopathy of prematurity. We hypothesized that GKT137831, a dual inhibitor of NADPH oxidases (NOX) 1 and NOX4, reduces inflammation in the ischemic retina by dampening the pro-inflammatory phenotype of retinal immune cells as well as macroglial Müller cells and neurons.MethodsIschemic retinopathy was induced in Sprague-Dawley rats by exposure to 80 % O2 cycled with 21 % O2 for 3 h per day from postnatal day (P) 0 to P11, followed by room air (P12 to P18). GKT137831 was administered P12 to P18 (60 mg/kg, subcutaneous) and comparisons were to room air controls. Retinal inflammation was examined by measuring leukocyte adherence to the retinal vasculature, ionized calcium-binding adaptor protein-1-positive microglia/macrophages, and the mRNA and protein levels of key inflammatory factors involved in retinal disease. Damage to Müller cells was evaluated by quantitating glial fibrillary acidic protein-positive cells and vascular leakage with an albumin ELISA. To verify the anti-inflammatory actions of GKT137831 on glia and neurons involved in ischemic retinopathy, primary cultures of rat retinal microglia, Müller cells, and ganglion cells were exposed to the in vitro counterpart of ischemia, hypoxia (0.5 %), and treated with GKT137831 for up to 72 h. ROS levels were evaluated with dihydroethidium and the protein and gene expression of inflammatory factors with quantitative PCR, ELISA, and a protein cytokine array.ResultsIn the ischemic retina, GKT137831 reduced the increased leukocyte adherence to the vasculature, the pro-inflammatory phenotype of microglia and macroglia, the increased gene and protein expression of vascular endothelial growth factor, monocyte chemoattractant protein-1, and leukocyte adhesion molecules as well as vascular leakage. In all cultured cell types, GKT137831 reduced the hypoxia-induced increase in ROS levels and protein expression of various inflammatory mediators.ConclusionsNOX1/4 enzyme inhibition with GKT137831 has potent anti-inflammatory effects in the retina, indicating its potential as a treatment for a variety of vision-threatening retinopathies.

Citation: Deliyanti D, Alrashdi SF, Tan SM, et al. Nrf2 activation is a potential therapeutic approach to attenuate diabetic retinopathy. Invest Ophthalmol Vis Sci. 2018;59:815-825. https:// doi.org/10.1167/iovs.17-22920 PURPOSE. Oxidative stress is a causal factor in the development of diabetic retinopathy; however, clinically relevant strategies to treat the disease by augmenting antioxidant defense mechanisms have not been fully explored. We hypothesized that boosting nuclear factor erythroid-2-related factor 2 (Nrf2) antioxidant capacity with the novel Nrf2 activator dh404, would protect the retina in diabetes including vision-threatening breakdown of the bloodretinal barrier (BRB) and associated damage to macroglial Müller cells.METHODS. Sprague-Dawley rats were randomized to become diabetic or nondiabetic and administered dh404 by gavage for 10 weeks. Complementary in vitro studies were performed in cultured Müller cells exposed to hyperglycemia. RESULTS.In diabetes, dh404 prevented vascular leakage into the retina and vitreous cavity as well as upregulation of the vascular permeability and angiogenic factors, VEGF, and angiopoietin-2, and inflammatory mediators, including TNF-a and IL-6. Müller cells, which maintain BRB integrity and become gliotic in diabetes with increased immunolabeling for glial fibrillary acidic protein, were protected by dh404. In diabetes, dh404 bolstered the antioxidant capacity of the retina with an increase in hemeoxygenase-1, nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) quinine oxidoreductase-1, and Nrf2. Further, dh404 attenuated the diabetes-induced increase in oxidative stress as measured by dihydroethidium and 8-oxo-2 0 -deoxyguanosine (8-OHdG) immunolabeling as well as NADPH oxidase isoform expression. Studies in Müller cells supported these findings with dh404 attenuating the hyperglycemia-induced increase in vascular permeability, angiogenic and inflammatory mediators, and oxidative stress.CONCLUSIONS. Our data demonstrate the ability of dh404 to protect the retina against diabetesinduced damage and potentially prevent vision loss.