Nuclear factor-erythroid 2 related factor 2 (Nrf2)-mediated signaling plays a central role in maintaining cellular redox homeostasis of hepatic cells. Carbon monoxide releasing molecule-A1 (CORM-A1) has been reported to stimulate up-regulation and nuclear translocation of Nrf2 in hepatocytes. However, the role of CORM-A1 in improving lipid metabolism, antioxidant signaling and mitochondrial functions in nonalcoholic steatohepatitis (NASH) is unknown. In this study, we report that CORM-A1 prevents hepatic steatosis in high fat high fructose (HFHF) diet fed C57BL/6J mice, used as model of NASH. The beneficial effects of CORM-A1 in HFHF fed mice was associated with improved lipid homeostasis, Nrf2 activation, upregulation of antioxidant responsive (ARE) genes and increased ATP production. As, mitochondria are intracellular source of reactive oxygen species (ROS) and important sites of lipid metabolism, we further investigated the mechanisms of action of CORM-A1-mediated improvement in mitochondrial function in palmitic acid (PA) treated HepG2 cells. Cellular oxidative stress and cell viability were found to be improved in PA + CORM-A1 treated cells via Nrf2 translocation and activation of cytoprotective genes. Furthermore, in PA treated cells, CORM-A1 improved mitochondrial oxidative stress, membrane potential and rescued mitochondrial biogenesis thru upregulation of Drp1, TFAM, PGC-1α and NRF-1 genes. CORM-A1 treatment improved cellular status by lowering glycolytic respiration and maximizing OCR. Improvement in mitochondrial respiration and increment in ATP production in PA + CORM-A1 treated cells further corroborate our findings. In summary, our data demonstrate for the first time that CORM-A1 ameliorates tissue damage in steatotic liver via Nrf2 activation and improved mitochondrial function, thus, suggesting the anti-NASH potential of CORM-A1.

Diabetic retinopathy (DR) is the most common cause of blindness in people under the age of 65. Unfortunately, the current screening process for DR restricts the population that can be evaluated and the disease goes undetected until irreversible damage occurs. Herein, we aimed to evaluate homocysteine (Hcy) as a biomarker for DR screening. Hcy levels were measured by enzyme-linked immuno sorbent assay (ELISA) and immunolocalization methods in the serum, vitreous and retina of diabetic patients as well as in serum and retina of different animal models of DM representing type 1 diabetes (streptozotocin (STZ) mice, Akita mice and STZ rats) and db/db mice which exhibit features of human type 2 diabetes. Our results revealed increased Hcy levels in the serum, vitreous and retina of diabetic patients and experimental animal models of diabetes. Moreover, optical coherence tomography (OCT) and fluorescein angiography (FA) were used to evaluate the retinal changes in mice eyes after Hcy-intravitreal injection into normal wild-type (WT) and diabetic (STZ) mice. Hcy induced changes in mice retina which were aggravated under diabetic conditions. In conclusion, our data reported Hcy as a strong candidate for use as a biomarker in DR screening. Targeting the clearance of Hcy could also be a future therapeutic target for DR.

Stress-associated premature senescence (SAPS) is involved in retinal microvascular injury and diabetic retinopathy. We have investigated the role and mode of action of miR-34a in retinal endothelial cells senescence in response to glucidic stress. Human retinal microvascular endothelial cells (HuREC) were exposed to glucidic stress (high glucose (HG) = 25 mM d-glucose) and compared to cells exposed to normal glucose (NG = 5 mM) or the osmotic control l-glucose (LG = 25 mM). HG stimulation of HuREC increased the expression of miR-34a and induced cellular senescence. HG also increased the expression of p16ink4a and p21waf1, while decreasing the histone deacetylase SIRT1. These effects were associated with diminished mitochondrial function and loss of mitochondrial biogenesis factors (i.e., PGC-1α, NRF1, and TFAM). Transfection of the cells with miR-34a inhibitor (IB) halted HG-induced mitochondrial dysfunction and up-regulation of senescence-associated markers, whereas miR-34a mimic promoted cellular senescence and mitochondrial dysfunction. Moreover, HG lowered levels of the mitochondrial antioxidants TrxR2 and SOD2, an effect blunted by miR-34a IB, and promoted by miR-34a mimic. 3’-UTR (3’-untranslated region) reporter assay of both genes validated TrxR2 as a direct target of miR-34a, but not SOD2. Our results show that miR-34a is a key player of HG-induced SAPS in retinal endothelial cells via multiple pathways involved in mitochondrial function and biogenesis.

Oxidative damage has been identified as a major causative factor in degenerative diseases of the retina; retinal pigment epithelial (RPE) cells are at high risk. Hence, identifying novel strategies for increasing the antioxidant capacity of RPE cells, the purpose of this study, is important. Specifically, we evaluated the influence of selenium in the form of selenomethionine (Se-Met) in cultured RPE cells on system xc- expression and functional activity and on cellular levels of glutathione, a major cellular antioxidant. ARPE-19 and mouse RPE cells were cultured with and without selenomethionine (Se-Met), the principal form of selenium in the diet. Promoter activity assay, uptake assay, RT-PCR, northern and western blots, and immunofluorescence were used to analyze the expression of xc-, Nrf2, and its target genes. Se-Met activated Nrf2 and induced the expression and function of xc- in RPE. Other target genes of Nrf2 were also induced. System xc- consists of two subunits, and Se-Met induced the subunit responsible for transport activity (SLC7A11). Selenocysteine also induced xc- but with less potency. The effect of Se-met on xc- was associated with an increase in maximal velocity and an increase in substrate affinity. Se-Met increased the cellular levels of glutathione in the control, an oxidatively stressed RPE. The Se-Met effect was selective; under identical conditions, taurine transport was not affected and Na+-coupled glutamate transport was inhibited. This study demonstrates that Se-Met enhances the antioxidant capacity of RPE by inducing the transporter xc- with a consequent increase in glutathione.

Significance: The studies described here are relevant to the cure of diabetic retinopathy, a leading cause of blindness with currently limited therapeutic options. Here we provided evidence showing that agonists of growth hormone-releasing hormone (GHRH) can significantly diminish retinal neurovascular injury characterizing the early stages of diabetic retinopathy through antioxidant and anti-inflammatory effects. The results of the presented studies provide information on the potential therapeutic effects of GHRH agonists and shed light on the role of hypothalamic hormones in retinal physiology and their effect on visual disorders. In addition, our findings suggest protective effects of GHRH analogs in other disease conditions affecting retinal neuronal cells and, possibly, other nonretinal neurons.

Nicotinamide adenine dinucleotide (NAD⁺) plays an important role in various key biological processes including energy metabolism, DNA repair, and gene expression. Accumulating clinical and experimental evidence highlights an age-dependent decline in NAD⁺ levels and its association with the development and progression of several age-related diseases. This supports the establishment of NAD⁺ as a critical regulator of aging and longevity and, relatedly, a promising therapeutic target to counter adverse events associated with the normal process of aging and/or the development and progression of age-related disease. Relative to the above, the metabolism of NAD⁺ has been the subject of numerous investigations in various cells, tissues, and organ systems; however, interestingly, studies of NAD⁺ metabolism in the retina and its relevance to the regulation of visual health and function are comparatively few. This is surprising given the critical causative impact of mitochondrial oxidative damage and bioenergetic crises on the development and progression of degenerative disease of the retina. Hence, the role of NAD⁺ in this tissue, normally and aging and/or disease, should not be ignored. Herein, we discuss important findings in the field of NAD⁺ metabolism, with particular emphasis on the importance of the NAD⁺ biosynthesizing enzyme NAMPT, the related metabolism of NAD⁺ in the retina, and the consequences of NAMPT and NAD⁺ deficiency or depletion in this tissue in aging and disease. We discuss also the implications of potential therapeutic strategies that augment NAD⁺ levels on the preservation of retinal health and function in the above conditions. The overarching goal of this review is to emphasize the importance of NAD⁺ metabolism in normal, aging, and/or diseased retina and, by so doing, highlight the necessity of additional clinical studies dedicated to evaluating the therapeutic utility of strategies that enhance NAD⁺ levels in improving vision.