Chapter 22 Mechanisms of Oxidative Stress in Retinal Injury
Oxidative stress has been implicated in the development and progression of retinal diseases. This chapter will focus on three forms of retinal pathology that are related to oxidative stress: age-related macular degeneration (AMD), diabetic retinopathy, and inherited retinal degenerations. We will discuss specific mechanisms of oxidative stress that affect the retinal pigment epithelium (RPE), retinal vasculature, photoreceptors, and mitochondria. We will also consider the evidence linking oxidative stress and inflammation in the pathogenesis of retinal disease. Finally, we will explore potential therapies targeting oxidative stress in the retina.
Reactive oxygen species (ROS) are a major source of retinal oxidative stress. These highly reactive particles include free radicals, peroxides, and singlet oxygen. Free radicals, such as the hydroxyl radical (OH•), hydroperoxyl radicals (HO2•), superoxide anion (O2–•), and lipid peroxyl radicals, are strong oxidizing agents with an unpaired electron in the outer shell. Peroxides (e.g., hydrogen peroxide (H2O2), lipid peroxides) and singlet oxygen (1O2) have a full complement of electrons in an unstable state.1
Under physiologic conditions, the body produces ROS through normal metabolic processes, such as glycolysis and the Krebs cycle. Aging and disease may disturb the balance between ROS generation and clearance, resulting in oxidative damage to macromolecules.2 The majority of endogenous ROS are produced by mitochondria through the electron transport chain, which converts 2–3% of all utilized oxygen into ROS.3 Stimuli such as aging, inflammation, irradiation, air pollutants, and cigarette smoke lead to increased ROS, and thus increased cellular oxidative injury.1,4,5
The body’s defense against increasing oxidative stress consists of molecules with antioxidant capacity, including vitamins C and E, carotenoids, and other free radical scavengers. Most ROS are eliminated immediately by antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase, and catalase. For example, the superoxide anion, produced by the mitochondria during the electron transport stage of cellular respiration, is converted to the less noxious hydrogen peroxide molecule (H2O2) by SOD.1 Smaller antioxidant molecules (e.g., vitamin C (ascorbate), vitamin E (tocopherol), and carotenoids) act on free radicals directly, reducing ROS such as the hydroxyl radical.
As most free radical chain reactions are prevented by free radical-scavenging molecules, it appears that free radicals are often not the direct cause of oxidative damage but rather act to initiate further oxidative damage by nonradical oxidants. The redox hypothesis describes radical-free oxidative stress in which disrupted thiol redox circuits interfere with regulation of cellular redox status, affecting cell signaling and physiological regulation. Redox-sensitive thiols include the amino acid cysteine (Cys), the Cys-derived disulfide cystine (CySS), the Cys-containing tripeptide glutathione (GSH), and glutathione disulfide (GSSG). Sulfur redox couples act as “on/off” switches regulating gene expression and protein function. Because the Cys/CySS and GSH/GSSG couples are not in equilibrium, it is plausible that abnormal levels of nonradical oxidants could be sufficient to disrupt normal function.6
The retina is particularly vulnerable to oxidative stress because of its high oxygen consumption and high proportion of polyunsaturated fatty acids (PUFAs).1,2 High light exposure and phagocytosis in the RPE contribute to the oxidative burden of the retina. The turnover rate of photoreceptors is high, with these cells shedding about 10% of their outer-segment discs each day. The disc membranes, in particular the PUFAs, are subject to peroxidation, which is highly damaging to the RPE. Two carotenoids, lutein and zeaxanthin, comprise the macular pigment that protects against ROS in the retina. Lutein and zeaxanthin can quench the reactive singlet oxygen and form an optical filter that blocks highly damaging blue light from reaching the photoreceptors.1
Oxidative stress contributes to diseases of the retina, including AMD, diabetic retinopathy, and inherited retinal degenerations (Table 22.1). To set the stage for a detailed discussion of the underlying mechanisms of oxidative damage to the retina, we will discuss the pathology and evidence for a role of oxidative stress in each of these diseases.
Light exposure associated with increased generation of ROS and AGEs52,53 and increased risk of AMD1,2
|Inherited retinal degenerations|
AMD, age-related macular degeneration; RPE, retinal pigment epithelium; ROS, reactive oxygen species; AGE, advanced glycation end-product; RAGE, receptor of AGEs; VEGF, vascular endothelial growth factor; t-BHP, tert-butyl hydroperoxide; NF-κB, nuclear factor-κB; GSH, glutathione; ERGs, electroretinograms; GST, glutathione-S-transferase; GSHPx, glutathione peroxidase.
AMD, the leading cause of irreversible vision loss in older individuals in the western world, is a complex disease influenced by factors such as genetics, demographics, and environmental exposure. Approximately 1.5% of individuals in the USA over the age of 40 (about 1.75 million people) develop the sight-threatening advanced stages of the disease, and this number is projected to approach 3 million by 2020.7
AMD can be divided into an early, typically asymptomatic form, and a late form that often results in severe central vision loss. In the early stages of AMD, whitish-yellow waste deposits called drusen accumulate in the macula, usually localized between the RPE and Bruch’s membrane. These deposits may be small and discrete (hard drusen) or larger and more confluent (soft drusen). Drusen may also be present between the photoreceptors and RPE or within the photoreceptor cell layer (reticular drusen).8,9 Histologically, drusen consist of numerous proteins (e.g., complement, immunoglobulins, amyloid-β) and lipids (e.g., phospholipids, cholesterol, apolipoproteins).10,11 Early dry AMD may develop into advanced dry or advanced wet AMD. Advanced dry AMD (geographic atrophy) is marked by cell death in the choriocapillaris and overlying RPE. Advanced wet AMD is characterized by choroidal neovascularization, in which abnormal choroidal vessels extend into the subretinal space.
Risk factors for AMD include older age, high body mass index, and high light exposure.12,13 Smoking, the strongest environmental risk factor for AMD, has been linked to disease onset and progression in multiple large, epidemiologic studies.14–19 Additionally, high dietary intake of carotenoids and antioxidant supplementation have been linked with lower risk of AMD.20,21
Multiple lines of evidence point to the role of oxidative stress in the pathogenesis of AMD. Several AMD risk factors, particularly aging, smoking, and light exposure, have been linked to increased levels of oxidative stress.1 Additionally, proteome analysis of human donor eyes revealed oxidative modifications to proteins and DNA in Bruch’s membrane, drusen, and RPE.23 The higher prevalence of these oxidative changes in AMD patients versus controls23 suggests an increased oxidative state in the retinas of AMD patients.
Impairment of the retinal antioxidant defense system may also play a role in AMD, as some studies have reported decreased macular pigment in AMD patients.24–26 Significantly lower levels of macular pigment (lutein and zeaxanthin) were found by direct measurement in AMD versus control eyes at autopsy.24 Additionally, dietary supplementation with antioxidants (vitamin C, vitamin E, and β-carotene) and zinc was shown to decrease the risk of progression to advanced AMD in the Age-Related Eye Disease Study (AREDS).22 In past observational studies, high dietary intake of lutein/zeaxanthin and omega-3 fatty acids has corresponded to reduced prevalence and incidence of AMD.27 These compounds are now under investigation for the treatment of AMD in the AREDS2 clinical trial.
Diabetic retinopathy, a common microvascular complication of both type 1 and type 2 diabetes mellitus (DM), is the leading cause of vision loss in working-age adults.28 In the USA, approximately 26 million people are living with diabetes, and the number of diabetic adults with retinopathy is projected to triple to 16 million by the year 2050.29 The majority of DM (90–95%) is represented by type 2 diabetes, which involves impaired insulin action at target tissues and impaired insulin release. In contrast, the primary cause of type 1 diabetes is the autoimmune destruction of pancreatic β cells.
Clinically, diabetic retinopathy is defined as the presence of specific retinal microvascular signs in an individual with DM. The nonproliferative form is characterized by microaneurysms, hemorrhages, hard exudates, cotton-wool spots, venous dilation and beading, and intraretinal microvascular abnormalities. The more severe proliferative form is characterized by retinal neovascularization, which can lead to vitreous hemorrhage and tractional retinal detachment. Intraretinal macular edema, which may be present in either form of retinopathy, can also lead to severe vision loss.
Interestingly, retinal capillary cells in diabetics undergo accelerated apoptosis that precedes the detection of any of the histopathological changes characteristic of diabetic retinopathy.30 Although the exact mechanism for this accelerated apoptosis remains uncertain, studies have pointed to the involvement of oxidative stress-activated caspases and nuclear factor-κB (NF-κB) in retinal cell death.31,32 Additional experiments revealed that inhibition of superoxide accumulation in diabetes prevents this early retinal cell death.33,34
Oxidative stress appears to be a major risk factor for the onset and progression of diabetes. Many of the common DM risk factors, including obesity and age, foster an oxidative environment that may alter insulin sensitivity by increasing insulin resistance or impairing glucose tolerance. Hyperglycemia, a common result of both types of diabetes, contributes in turn to the progression and maintenance of an overall oxidative environment.35
Hyperglycemia may contribute to oxidative stress by generating ROS directly or disrupting the cellular redox balance. This hyperglycemia-induced oxidative stress can occur via several well-studied mechanisms, including increased polyol pathway flux, increased intracellular formation of advanced glycation end products (AGEs), activation of protein kinase C (PKC), and disturbance of the hexosamine biosynthesis pathway. The overproduction of superoxide by the mitochondrial transport chain has been proposed as a possible root cause for these metabolic changes.36
The polyol pathway leads to the reduction of glucose to sorbitol via aldose reductase in an nicotinamide adenine dinucleotide phosphate (NADPH)-dependent manner. Sorbitol is then oxidized to form fructose by sorbitol dehydrogenase. The polyol pathway consumes NADPH, an essential cofactor for regenerating the intracellular antioxidant GSH. By decreasing the amount of free GSH, the polyol pathway may increase the susceptibility of cells to oxidative injury.36
AGEs are modifications of proteins or lipids that become nonenzymatically glycated and oxidized after contact with aldose sugars. Early glycation and oxidation result in the formation of Schiff bases and Amadori products. Further glycation of proteins and lipids causes molecular rearrangements that lead to the generation of AGEs.37 AGEs appear to damage cells by modifying intracellular proteins, such as transcription factors, and altering extracellular proteins and extracellular matrix molecules. These modified proteins can then bind and activate receptor of AGEs (RAGE), resulting in the upregulation of the transcription factor NF-κB and the production of inflammatory cytokines and growth factors.36
The activation of PKC leads to multiple characteristics of diabetic retinopathy, including increased vascular permeability, endothelial cell proliferation and apoptosis, and neovascularization.38 Hyperglycemia-induced ROS inhibit the key glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), leading to an increase in glycolytic pathway precursors. Increased levels of triose phosphate upregulate diacylglycerol, the primary activator of PKC.38,39 Activated PKC can induce expression of vascular endothelial growth factor (VEGF),40 a major effector of blood flow and vessel permeability and a primary stimulator of neovascularization. PKC also leads to the synthesis of NF-κB, a proinflammatory transcription factor, and transforming growth factor-β1 (TGF-β), which can contribute to the occlusion of capillaries seen in diabetic retinopathy.36,38
The hexosamine biosynthesis pathway may also mediate some of the toxic effects of hyperglycemia. The inhibition of GADPH by ROS causes diversion of glycolytic metabolites to the hexosamine pathway, producing uridine diphosphate N-acetylglucosamine. N-acetylglucosamine is added to serine and threonine residues of transcription factors, often resulting in pathological changes in gene expression. Examples include increased expression of TGF-β and plasminogen activator inhibitor-1.36
Hyperglycemia-induced alterations in each of these DM-associated pathways can be linked to oxidative stress. Toward that end, Brownlee has proposed a “unifying mechanism” that hypothesizes that all of these pathway changes are a result of mitochondrial superoxide overproduction in the endothelial cells of both large and small blood vessels.41 In this model, diabetic hyperglycemia causes increased production of ROS by mitochondria. Greater concentration of ROS in the body induces nuclear DNA strand breaks. This DNA damage activates the DNA repair enzyme poly(ADP-ribose) polymerase, which reduces GADPH activity by the addition of ADP ribose polymers. Decreased GAPDH activity results in a backup of the glycolytic pathway and activation of the polyol, AGE, PKC, and hexosamine pathways, discussed above.36
Inherited retinal degenerations are a group of disorders characterized by primary and progressive loss of photoreceptor cells, leading to irreversible vision loss. Over 170 genes and greater than 200 loci have been associated with inherited retinal degenerations (www.sph.uth.tmc.edu/retnet). The proteins encoded by these genes are diverse in function, including not only proteins required for phototransduction but also structural proteins, RNA splicing factors, intracellular trafficking molecules, and proteins involved in the phagocytosis and regulation of intracellular pH.42
The most common form of these diseases, retinitis pigmentosa (RP), which has an estimated prevalence of around 1 in 4000,43 involves a primary insult to rods that initiates rod cell death followed by cone cell death (rod–cone dystrophy). Other forms of inherited retinal degeneration include cone dystrophy, cone–rod dystrophy, and macular dystrophy. The most common inherited macular dystrophy is Stargardt macular dystrophy, an autosomal recessive disease with a carrier frequency of approximately 1 in 50.44
The first symptom of RP is typically night blindness, followed by progressive loss of the peripheral visual field. Fundoscopic exam classically demonstrates a pale optic nerve, attenuated retinal vessels, and bone spicule pigmentation in the retinal midperiphery. The widespread retinal degeneration leads to diminution or abolishment of the a-waves and b-waves on electroretinograms (ERGs). A variety of molecular defects has been identified in individuals with RP, including mutations in genes encoding rhodopsin, peripherin, and cGMP phosphodiesterase. These genes are typically rod-specific, but some also code for more general proteins, such as structural proteins in cilia and their associated transport molecules.
The unifying feature in animal models and human cases of RP seems to be the apoptotic cell death of rod photoreceptors, although the mechanisms that lead to photoreceptor cell death in RP remain poorly defined. While most mutations in RP specifically affect rods, both rods and cones die. Several mechanisms have been proposed for the dependence of cone survival on the presence of functioning rods. One theory is that rods produce a survival factor that is required by cones and that modulates the thioredoxin antioxidant defense system in the retina.45 Oxidative stress or alterations in the cellular redox state have also been proposed by several groups as a common mediator of photoreceptor cell death.46 Several lines of evidence support this hypothesis, including the induction of apoptosis by oxidants such as H2O2 and the inhibition of apoptosis by antioxidants.
Specific mechanisms of oxidative damage to the retina are unique to the affected retinal cell types. Morphology and function of the RPE, retinal vasculature, and photoreceptors can be severely impaired by the increased oxidative stress associated with disease. Growing evidence suggests that the mitochondria of retinal cells contribute to oxidative stress-related pathology and that inflammatory processes are closely tied to pathologic oxidative stress in the retina.
Oxidative stress can lead to the accumulation of debris and ultimately cell death in the RPE, as manifest in the drusen and geographic atrophy of AMD. Abnormally high concentrations of oxidized products may also interfere with the ability of RPE cells to regulate key angiogenic factors, resulting in retinal neovascularization.
Oxidative damage to the RPE can be induced by oxidizing agents or light. Under normal conditions, phagocytosis of photoreceptor outer segments by the RPE generates ROS via the NADPH oxidase system, leading to increased intracellular H2O2 and increased catalase activity.47 The phosphoinositide 3-kinase (PI3K)-Akt pathway may protect RPE cells from such oxidative stress. The oxidant H2O2 was demonstrated to induce PI3K-dependent Akt activation in cultured RPE cells. Akt activation can enhance RPE survival by phosphorylating and thus inactivating multiple proapoptotic factors.48 Cultured human RPE cells exhibited signs of senescence when treated with the oxidizing agents tert-butyl hydroperoxide (t-BHP)49 or H2O2,50 suggesting that oxidative stress may contribute to the RPE senescence seen in AMD. Oxidative stress induced by hypoxia/reoxygenation in cultured human RPE cells led to the accumulation of extracellular matrix and may be linked to the thickening of Bruch’s membrane seen in early AMD.51 Additionally, animal and laboratory studies have shown that blue light damages the RPE through ROS generation.52 RPE cells treated with subapoptotic levels of ultraviolet light showed increases in both ROS and AGEs.53
The AGE-induced activation of RAGE may play a key role in RPE apoptosis. In human donor eyes, RAGE were found to colocalize with AGE deposits and macular disease in AMD retinas, while normal retinas displayed little or no immunolabeling for either AGE or RAGE. This study also showed that AGEs stimulate RAGE-mediated activation in cultured RPE cells in a dose-dependent fashion.54 RAGE increased secretion of VEGF in cultured RPE cells, suggesting that AGE-mediated activation of the RAGE axis in RPE cells could contribute to neovascular macular disease.55
Further evidence supports the idea that oxidative stress in the RPE has a significant influence on VEGF regulation. Treatment of cultured human RPE cells with the oxidant dl-buthionine-(S,R)-sulfoximine caused a significant decrease in intracellular GSH and GSH/GSSG ratios. This change in thiol redox status was associated with increased VEGF-A secretion as well as significant induction of VEGF receptors VEGFR-1 and VEGFR-2.56 Both VEGF-A and VEGF-C were upregulated in cultured RPE cells treated with t-BHP, with secretion higher to the apical side than to the basal side,57 suggesting that VEGF may directly affect the photoreceptors. VEGF secretion in RPE cells is regulated, in part, by the mitogen-activated protein kinases, including c-Jun-activated kinase, p38, and Erk. Studies in cultured RPE cells demonstrated that constitutive VEGF secretion is regulated by p38, while oxidative stress-induced VEGF secretion is regulated by both p38 and Erk.58 Finally, it appears that autocrine VEGF-A can enhance RPE cell survival under oxidative stress, utilizing the autocrine VEGF-A/VEGFR-2/PI3K/Akt pathway.59
Increased oxidative stress can lead to alterations in both the retinal and choroidal vasculature. In diabetes, hyperglycemia-induced ROS may link elevated glucose and multiple metabolic abnormalities associated with the development of retinopathy.41 Retinal capillary cell cultures containing endothelial cells and pericytes incubated in high-glucose medium exhibited increased oxidative stress markers. In these cells, increased caspase-3 and apoptosis were seen after 5 days in culture.32
Oxidative stress has been correlated with increased production of the angiogenic growth factor and vascular permeability factor VEGF and may be involved in the upregulation of endothelial cell VEGF expression seen in diabetes.60 It appears that increased VEGF expression in vascular endothelial cells may result from peroxynitrite, a highly reactive oxidant formed by the combination of superoxide anion with nitric oxide.61 Peroxynitrite mediates a variety of biological processes including inhibition of key metabolic enzymes, lipid peroxidation, nitration of protein tyrosine residues, and oxidation of thiol pools.62
A hyperglycemic environment may promote the damaging effects of VEGF observed in diabetes. Interestingly, VEGF has been shown to be a potent survival factor for endothelial cells in vitro and in vivo.63 Despite its neuroprotective properties, the increased levels of VEGF seen in diabetic retinas64 are associated with increased retinal endothelial cell death. Cultured endothelial cells subjected to serum withdrawal-induced apoptosis were protected by VEGF in normal glucose but not in high-glucose or peroxynitrite media.62 Further experiments determined that high-glucose treatment blocks the prosurvival effect of VEGF and causes accelerated endothelial cell apoptosis via the action of peroxynitrite.62
Oxidative stress can also affect retinal endothelial cells’ ability to transport glucose into the cell. Exposure of endothelial cells to sustained oxidative stress in the form of H2O2 resulted in decreased glucose transport activity due to increased internalization of GLUT1, the most common retinal glucose transporter.65 H2O2-induced oxidative stress was also found to increase the rate of GLUT1 internalization by a proteasome-dependent mechanism involving inactivation of Akt.
Oxidative stress may have direct and indirect effects on the choroidal endothelial cells (CEC), and thus the choroidal neovascularization seen in AMD. Treatment of cultured RPE and CEC with the oxidant t-BHP resulted in decreased viability and increased proliferation of both cell types.66 RPE cells exposed to t-BHP for 24 hours were found to release basic fibroblast growth factor, a prominent proangiogenic factor. This finding suggests that oxidative stress may stimulate choroidal neovascularization via RPE-mediated growth factor release. Interestingly, CECs exposed to oxidative stress-induced AGEs in culture demonstrated increased proliferation as well as upregulation of VEGF, suggesting that AGEs may also promote choroidal neovascularization.67
The process of retinal degeneration disrupts the physiologic balance of oxidants and antioxidants in the retinal spaces. As a source of ROS, photoreceptors contribute to the oxidative burden of retina, particularly in a pathologic oxidative environment. The vitality of rods and cones is directly affected by the redox status of surrounding tissue.
Photoreceptor cell loss or a reduction in energy-demanding activities like phototransduction can lead to elevated tissue oxygen concentrations because choroidal blood vessels are not autoregulated by local oxygen levels. Accordingly, increases in outer retinal oxygen concentrations have been confirmed in multiple animal models.68–70 The inner retinal arterioles, in contrast, do autoregulate, leading to their attenuation in the presence of high oxygen concentrations.71 This oxygen imbalance induced by photoreceptor dysfunction is manifest by the thinned retinal vessels seen clinically in RP.
Alterations in the cellular redox state appear to mediate photoreceptor cell death. An in vitro model of photoreceptor apoptosis demonstrated an early and sustained increase in intracellular ROS accompanied by a rapid depletion of intracellular GSH.46 Evidence suggests that multiple oxidative stress-related mechanisms underlie the programmed cell death of photoreceptors. Classically, caspase-dependent apoptotic pathways have been described.72,73 More recently, it has been shown that programmed cell death can also be accomplished through caspase-independent apoptosis,74–76 as well as via autophagy.77 In a photoreceptor cell line treated with the nitric oxide donor sodium nitroprusside, cytosolic calcium levels increased during photoreceptor apoptosis, leading to activation of caspases as well as the calcium-dependent proteases calpains.78 Inhibitors of both caspases78 and calpains,79 applied independently to the same photoreceptor cell line, failed to prevent apoptosis. These results indicate that, in addition to the classic caspase-dependent pathways of apoptosis, calpain activity may be crucial for apoptosis.
Calpains may lead to retinal oxidative damage by impairing DNA repair mechanisms. Studies in retinal degeneration (rd1) mice, which have a mutation in exon 7 of the beta subunit of the rod photoreceptor PDE6 gene, have demonstrated downregulation in defensive mechanisms against oxidative stress (e.g., glutathione-S-transferase and glutathione peroxidase), potentially allowing an increase in intracellular ROS.80,81 A major product of ROS reactions in the DNA is the highly mutagenic oxidated nucleotide 8-oxo-guanosine (8-oxoG). Mismatches of 8-oxoG with adenine or cytosine are corrected by the specific DNA repair enzyme 8-oxoG DNA glycosylase (OGG1).82 OGG1 is regulated by cAMP response element-binding protein (CREB), and OGG1 and CREB have both been found to be downregulated in the rd1 retina. Since OGG1 can be inactivated by calpains, an increase in calpain activity could severely compromise a cell’s ability to repair oxidative stress-induced DNA damage.
Cones appear to be sensitive to retinal oxygen concentration. In multiple animal models of RP, increased levels of oxygen in the retina result in progressive oxidative damage to cones.71,83,84 Treatment of rd1–/– mice with a cocktail of antioxidants downregulated markers of oxidative damage in cones, reduced cone cell loss, and preserved cone ERGs. These results suggest that oxidative damage is important in cone death regardless of the underlying mechanism that kills rods.85 Most recently, studies in rd1–/– mice showed that NADPH oxidase plays a critical role in generation of the oxidative stress that leads to cone death in RP.85
Initial experiments investigating the progressive death of cones after rod cell death showed that grafting of normal photoreceptors (97% rods) into the eye of the rodless rd1–/– mouse before the cones degenerated had a positive effect on the host cones, even at some distance from the transplant.86 Further studies identified a protein that allowed for cone survival, termed rod-derived cone viability factor (RdCVF).45 RdCVF, encoded by the exon 1 of the nucleoredoxin-like 1 (Nxnl1) gene, corresponds to a truncated thioredoxin (TRX)-like protein. Because TRX proteins are key to maintaining a reducing intracellular environment,87 disruption of TRXs can lead to conditions of increased oxidative stress. Nxnl1 knockout mice have been shown to be sensitive to oxidative stress, suggesting that RdCVF is part of an endogenous redox-based signaling pathway involved in retinal maintenance.87