4-hydroxy-2-nonenal and Macular-Degeneration

Appropriate diet is essential for the regulation of age-related macular degeneration (AMD). In particular the type of dietary polyunsaturated fatty acids (PUFA) and poor antioxidant status including carotenoid levels concomitantly contribute to AMD risk. Build-up of oxidative stress in AMD induces PUFA oxidation, and a mix of lipid oxidation products (LOPs) are generated. However, LOPs are not comprehensively evaluated in AMD. LOPs are considered biomarkers of oxidative stress but also contributes to inflammatory response. In this cross-sectional case-control study, plasma omega-6/omega-3 PUFA ratios and antioxidant status (glutathione, superoxide dismutase and catalase), and plasma and urinary LOPs (41 types) were determined to evaluate its odds-ratio in the risk of developing exudative AMD (n = 99) compared to age-gender-matched healthy controls (n = 198) in adults with Chinese diet. The odds ratio of developing exudative AMD increased with LOPs from omega-6 PUFA and decreased from those of omega-3 PUFA. These observations were associated with a high plasma omega-6/omega-3 PUFA ratio and low carotenoid levels. In short, poor PUFA and antioxidant status increased the production of omega-6 PUFA LOPs such as dihomo-isoprostane and dihomo-isofuran, and lowered omega-3 PUFA LOPs such as neuroprostanes due to the high omega-6/omega-3 PUFA ratios; they were also correlated to the risk of AMD development. These findings indicate the generation of specific LOPs is associated with the development of exudative AMD.

Topics: 3-Hydroxyacyl CoA Dehydrogenases; Acetyl-CoA C-Acyltransferase; Aged; Aldehydes; Antioxidants; Biomarkers; Carbon-Carbon Double Bond Isomerases; Carotenoids; Diet; Enoyl-CoA Hydratase; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Female; Humans; Isoprostanes; Lipid Peroxidation; Macular Degeneration; Male; Middle Aged; Neuroprostanes; Oxidation-Reduction; Oxidative Stress; Racemases and Epimerases; Risk Factors

The discovery that genetic abnormalities in complement factor H (FH) are associated with an increased risk for age-related macular degeneration (AMD), the most common cause of blindness among the elderly, raised hope of new treatments for this vision-threatening disease. Nonetheless, over a decade after the identification of this important association, how innate immunity contributes to AMD remains unresolved. Pentraxin 3 (PTX3), an essential component of the innate immunity system that plays a non-redundant role in controlling inflammation, regulates complement by interacting with complement components. Here, we show that PTX3 is induced by oxidative stress, a known cause of AMD, in the retinal pigmented epithelium (RPE). PTX3 deficiency in vitro and in vivo magnified complement activation induced by oxidative stress, leading to increased C3a, FB, and C3d, but not C5b-9 complex formation. Increased C3a levels, resulting from PTX3 deficiency, raised the levels of Il1b mRNA and secretion of activated interleukin (IL)-1β by interacting with C3aR. Importantly, PTX3 deficiency augmented NLRP3 inflammasome activation, resulting in enhanced IL-1β, but not IL-18, production by the RPE. Thus, in the presence of PTX3 deficiency, the complement and inflammasome pathways worked in concert to produce IL-1β in sufficient abundance to, importantly, result in macrophages accumulating in the choroid. These results demonstrate that PTX3 acts as an essential brake for complement and inflammasome activation by regulating the abundance of FH in the RPE, and provide critical insights into the complex interplay between oxidative stress and innate immunity in the early stages of AMD development. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: Aldehydes; Animals; C-Reactive Protein; Cells, Cultured; Choroid; Complement Activation; Complement C3a; Complement Factor H; Cysteine Proteinase Inhibitors; Humans; Immunity, Innate; Inflammasomes; Interleukin-1beta; Macrophages; Macular Degeneration; Mice, Inbred C57BL; Nerve Tissue Proteins; Oxidative Stress; Retinal Pigment Epithelium

Age-related macular degeneration (AMD) affects the retina and is the most common cause of blindness in elderly persons in developed countries. The retina is constantly subjected to oxidative stress; to avoid the effects of oxidative stress, retinal pigment epithelial (RPE) cells possess potent anti-oxidant systems. Disruption of these systems leads to dysfunction of RPE cells, which then accelerates the development of AMD. Here, we investigated the role of thioredoxins (TRXs), scavengers of intracellular reactive oxygen species, by assessing the effect of TRX overexpression on cell viability, morphology, NF-κB expression, and mitochondrial membrane potential, in RPE cells. TRX-overexpressing cell lines were generated by infection of an established human RPE cell line (ARPE) with adeno-associated virus vectors encoding either TRX1 or TRX2. We showed that overexpression of TRXs reduced cell death caused by 4-hydroxynonenal (4-HNE)-induced oxidative stress; TRX2 was more effective than TRX1 in promoting cell survival. 4-HNE caused perinuclear NF-κB accumulation, which was absent in TRX-overexpressing cells. Moreover, overexpression of TRXs prevented depolarization of mitochondrial membranes; again, TRX2 was more effective than TRX1 in maintaining the membrane potential. The difference in the protective effects of these TRXs against oxidative stress may be due to their expression profile. TRX2 was expressed in the mitochondria, while TRX1 was expressed in the cytoplasm. Thus, TRX2 may directly protect mitochondria by preventing depolarization. These results demonstrate that TRXs are potent antioxidant proteins in RPE cells and their direct effect on mitochondria may be a key to prevent oxidative stress.

Topics: Aldehydes; Antioxidants; Cell Death; Cell Line; Cell Survival; Humans; Macular Degeneration; Membrane Potential, Mitochondrial; NF-kappa B; Oxidative Stress; Reactive Oxygen Species; Retina; Retinal Pigment Epithelium; Thioredoxins

In the aging human eye, oxidative damage and accumulation of pro-oxidant lysosomal lipofuscin cause functional decline of the retinal pigment epithelium (RPE), which contributes to age-related macular degeneration. In mice with an RPE-specific phagocytosis defect due to lack of αvβ5 integrin receptors, RPE accumulation of lipofuscin suggests that the age-related blindness we previously described in this model may also result from oxidative stress. Cellular and molecular targets of oxidative stress in the eye remain poorly understood. Here we identify actin among 4-hydroxynonenal (HNE) adducts formed specifically in β5(-/-) RPE but not in neural retina with age. HNE modification directly correlated with loss of resistance of actin to detergent extraction, suggesting cytoskeletal damage in aging RPE. Dietary enrichment with natural antioxidants, grapes or marigold extract containing macular pigments lutein/zeaxanthin, was sufficient to prevent HNE-adduct formation, actin solubility, lipofuscin accumulation, and age-related cone and rod photoreceptor dysfunction in β5(-/-) mice. Acute generation of HNE adducts directly destabilized actin but not tubulin cytoskeletal elements of RPE cells. These findings identify destabilization of the actin cytoskeleton as a consequence of a physiological, sublethal oxidative burden of RPE cells in vivo that is associated with age-related blindness and that can be prevented by consuming an antioxidant-rich diet.

Topics: Actin Cytoskeleton; Aldehydes; Animals; Antioxidants; Apoptosis; Blindness; Cells, Cultured; Dietary Supplements; Electroretinography; Lipofuscin; Macular Degeneration; Male; Mice; Mice, 129 Strain; Mice, Knockout; Oxidation-Reduction; Photoreceptor Cells, Vertebrate; Phytotherapy; Plant Preparations; Protein Carbonylation; Protein Stability; Receptors, Vitronectin; Retina; Retinal Pigment Epithelium; Vitis

Amyloid β peptides (Aβ) have been implicated in the pathogenesis of age-related macular degeneration (ARMD) and glaucoma. In this study, retinas of mice overexpressing Aβ (Tg) were compared to those of wild-type mice (Wt) and analyzed for oxidative stress parameters. We observed a progressive decrease in all retinal cell layers, which was significantly greater in Tg mice at 14 months and culminated in loss of the outer retina at 18 months of age. We also observed higher levels of reactive oxygen species, glial fibrillary acidic protein, and hydroperoxide in Tg versus Wt mice (14 months). These effects were associated with phosphorylation/activation of the apoptosis signal kinase 1 and the p38 mitogen-activated kinase. Western blotting analysis revealed progressive increases in the levels of thioredoxin 1 and thioredoxin inhibitory protein in Tg compared to Wt mice. No changes were observed in the levels of thioredoxin reductase 1 (TrxR1); however, measurements of TrxR1 activity showed a 42.7±8% reduction in Tg mice versus Wt at 14 months of age. Our data suggest that Aβ-mediated retinal neurotoxicity involves impairment of the thioredoxin system and enhanced oxidative stress, potentially implicating this mechanism in the pathogenesis of ARMD and glaucoma.

Topics: Aldehydes; Amyloid; Amyloid beta-Peptides; Animals; Apoptosis; Carrier Proteins; Caspase 3; Enzyme Activation; Gene Expression; Glial Fibrillary Acidic Protein; Humans; Lipid Peroxidation; Macular Degeneration; MAP Kinase Kinase Kinase 5; Mice; Mice, Transgenic; Oxidation-Reduction; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Reactive Oxygen Species; Retina; Retinal Ganglion Cells; Thioredoxin Reductase 1; Thioredoxins

Oxidative stress and inflammation are known to be associated with age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells play the principal role in the immune defense of macula, and their dysfunction is a crucial event leading to clinically relevant changes seen in AMD. In the present study, we have examined the ability of oxidative stress to activate inflammasome signaling in the human ARPE-19 cells by adding the lipid peroxidation end product 4-hydroxynonenal (HNE) to cell cultures pre-treated or not treated with the endotoxin, LPS. Our results indicate that LPS and HNE significantly increased the production of IL-6 and IL-18, respectively. LPS treatment preceding HNE induced an even greater increase in the production of IL-18 than HNE alone. In addition to IL-18, HNE significantly increased the production of IL-1β. The productions of IL-1β and IL-18 were reduced in the cell cultures pre-treated with the Caspase-1 inhibitor. PCR analysis revealed that HNE induced an over 5-fold increase in the amount of NLRP3 mRNA compared to control cells; LPS had no effect. In conclusion, our present data suggest that oxidative stress can activate NLRP3 inflammasomes in RPE cells which occupy center stage in the pathogenesis of AMD.

Topics: Aldehydes; Carrier Proteins; Cell Line; Gene Expression Regulation; Humans; Inflammasomes; Interleukin-18; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Macular Degeneration; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress; Signal Transduction

Protein modifications resulting from reactive aldehydes are thought to be involved in the pathogenesis of various degenerative diseases. Aged cynomolgus monkey (Macaca fascicularis) spontaneously develop drusen in the macula, consistent with the phenotype observed in early-stage age-related macular degeneration (AMD), indicating that this animal is an optimum model for AMD. In retinal sections from three monkeys with macular degeneration, regardless of their size, drusen were consistently positive with immunohistochemical labeling against protein modifications by 4-hydroxynonenal and 4-hydroxyhexenal, end products of non-enzymatic oxidation of n-6 and n-3 polyunsaturated fatty acids, respectively. Positive labeling for both modifications was observed in the ganglion cell layer, the inner nuclear layer, the outer nuclear layer, and the retinal pigment epithelium. However, no consistent differences in location or intensity of the labeling were observed between monkeys with normal macula and macular degeneration. The results suggest a possible association between drusen formation and protein modifications by aldehydes in the pathogenesis of AMD.

Topics: Aldehydes; Animals; Disease Models, Animal; Eye Proteins; Female; Macaca fascicularis; Macular Degeneration; Retinal Drusen

Oxidative damage to proteins, lipids, and DNA has been suggested to be a mechanism for age-related macular degeneration (AMD). The retina is particularly susceptible to lipid peroxidation due to high concentrations of easily oxidized polyunsaturated fatty acids in the presence of abundant oxygen. One of the most toxic products of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), can modify and inactivate proteins. The hypothesis was that 4-HNE-modified proteins would accumulate and serve as a marker for progressive stages of AMD.. Proteins containing HNE adducts were identified in both the macular and peripheral regions during four progressive stages of AMD. The proteins were resolved by two-dimensional (2-D) gel electrophoresis before detection of HNE-adducted proteins. Modified proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The total content of HNE adducts was compared using a slot blot immunoassay. One-dimensional Western blot analysis was used to measure levels of proteins involved in HNE detoxification.. Nineteen proteins that were consistently modified regardless of stage of AMD or retinal region were identified. These proteins are involved in two main functions: energy production and stress response. No change in total HNE-adducted protein was observed between regions or stages. Modest increases in content of proteins involved in HNE detoxification were observed.. Consistently modified proteins indicate preferred protein targets for oxidation by HNE. HNE-modified proteins were not different between regions or stages, suggesting that pathways for detoxification of HNE or removal of damaged proteins are adequate. Consistent levels of HNE-modified proteins suggest that HNE is not a sensitive retinal biomarker for AMD.

Topics: Adult; Aged; Aged, 80 and over; Aging; Aldehydes; Animals; Biomarkers; Blotting, Western; Disease Progression; Electrophoresis, Gel, Two-Dimensional; Female; Humans; Lipid Peroxidation; Macular Degeneration; Male; Middle Aged; Oxidative Stress; Rats; Rats, Inbred F344; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Pathological features of age-related macular degeneration such as the formation of extracellular deposits and neovascularization are frequently viewed as outcomes of compromising processes within retinal pigment epithelial cells, but the initiating circumstances are poorly understood. Here we tested the hypothesis that photooxidation events initiated by A2E, a blue light-excitable aging fluorophore of the retinal pigment epithelium, can set the stage for altered cellular signaling and changes in the expression of genes that can impact the extracellular milieu. Proteins modified by lipid peroxidation products (4-hydroxynonenal; malondialdhyde) and advanced glycation end products were detected at sites of blue light irradiation both in association with the cultured A2E-laden retinal pigment epithelial cells and within the fibronectin substrate on which the cells were grown. RAGE, the cell surface receptor that transduces the effects of advanced glycation end products, was also upregulated, and RAGE expression co-localized with the deposition of advanced glycation end products. Blue light triggered alterations in gene expression was also evidenced by elevations in both transcripts and protein for vascular endothelial growth factor, a potent angiogenic and permeability-enhancing factor. These findings indicate that cell associated and extracellular modification of proteins by lipid peroxidation products and advanced glycation end products together with increased expression of RAGE and vascular endothelial growth factor may be induced consequent to blue light illumination of A2E-burdened retinal pigment epithelial cells. Thus, photooxidative events that are not an immediate threat to retinal pigment epithelial cell viability may nevertheless elicit sustained perturbation that could ultimately alter neighboring tissues and impact retinal pigment epithelial cell function.

Topics: Aldehydes; Cells, Cultured; Epithelial Cells; Eye Proteins; Gene Expression; Glycation End Products, Advanced; Growth Inhibitors; Humans; Immunoblotting; Immunohistochemistry; Light; Lipid Peroxidation; Macular Degeneration; Malondialdehyde; Pigment Epithelium of Eye; Pyridinium Compounds; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Retinoids; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation; Vascular Endothelial Growth Factors