Figure 14.1 Diagrammatic representation of detoxification of ROS superoxide anion. In the presence of SOD, superoxide anion is immediately transformed into hydrogen peroxide. Three enzymes catalyze decomposition of hydrogen peroxide: catalase (CAT), Gpx, and Prx. Reduced glutathione (GSH) and Trx (Trx_Red) provide electrons for Gpx and Prx, respectively. Using NADPH as the final electron donor, oxidized glutathione (GSSG) and Trx are reduced back by GR and TR.

Nonenzymatic compounds are also key components of the antioxidant network. The tripeptide GSH is one of several highly important small compounds. It contains a free sulfhydryl (SH) group, which is highly reactive with ROS. In RPE cells, the intercellular concentration of GSH is in the millimolar range and provides a first-line buffer system against free radical attack. GSH also serves as a cofactor in the detoxification of ROS by antioxidant enzymes. Several other small molecule antioxidants, including vitamin C, vitamin E, and zinc, are also of particular importance in protecting the retina from oxidative stress. They are discussed in more detail later in the chapter.

The antioxidant systems in RPE cells are highly efficient and highly error resistant under normal physiologic conditions. However, the balance between the generation of ROS and their clearance by the antioxidant systems can be disturbed by exogenous factors, such as pollutants and tobacco use, or physiologic conditions, such as aging and concomitant systemic disease. The retina is prone to oxidative stress for several reasons. First, the rate of oxygen consumption in the retina is greater than in any other tissue of the human body (25). This high-energy utilization is necessarily accompanied by high ROS production. Second, the retina is continually subjected to intense focal light exposure. In the presence of photosensitizers or chromophores (visual pigments and lipofuscin), photo-oxidation occurs resulting in the production of ROS, which includes singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals (26). Third, RPE cells can function as phagocytes, phagocytizing and degrading shed photoreceptor outer segments (POS). The phagocytosis of POS stimulates respiratory burst (rapid release of ROS), which likely occurs by utilizing plasma membrane NADPH oxidase (27–29). Moreover, the outer segments are extremely abundant in polyunsaturated fatty acids (PUFAs), which readily undergo lipid peroxidation. The resultant oxidized intermediates are long-lived and covalently modify various kinds of molecules including proteins, thereby impairing the stability and functions of their targets. Thus, lipid peroxidation initiates a chain reaction, which amplifies the initial number of free radicals. Two major products of lipid peroxidation, carboxyethylpyrrole (CEP) and malondialdehyde (MDA), have been recently linked to AMD and are discussed in more detail later in the chapter.

As the retina ages, it becomes more susceptible to oxidative injury due partly to an age-related decline in the antioxidant capacity. For example, a reduction in catalase activity in aging RPE was demonstrated by both biochemical and immunohistochemical analyses of human donor eyes (30,31). Systemically, the plasma GSH pool has an age-dependent decrease in total level as well as an oxidation in the redox status (32,33). Meanwhile, age-related increases in chromophores like lipofuscin cause increases in pro-oxidants that further stress and sometimes surpass the antioxidant system capacity.

Lipofuscin

Lipofuscin, also known as aging pigment, is an autofluorescent and electron-dense material that accumulates with age in postmitotic cells, including the RPE (34). It has a brown-yellow color and is enzymatically nondegradable. The rate of lipofuscin accumulation shows a negative correlation with longevity in animals. Thus, the accumulation of lipofuscin is considered a hallmark of aging (35,36). An age-dependent buildup of lipofuscin occurs in human RPE cells; at the age of 90, lipofuscin occupies about 19% of the RPE cell volume, whereas in the first 10 years of life, it occupies only 1% (37). The increased lipofuscin in the RPE causes an increase in autofluorescence of the fundus and precedes the development of geographic atrophy. Thus, high autofluorescence of the fundus can be a useful clinical biomarker that can assist in monitoring the progression of atrophic AMD (37–39) (Fig. 14.2).

The composition of lipofuscin is highly heterogeneous and includes oxidized proteins and lipids. Two degradation pathways, autophagy and phagocytosis, contribute to lipofuscin accumulation in the lysosomes. In the RPE, the role of phagocytosis has been well established by multilayered evidence. On the molecular level, the best-characterized fluorophore of the RPE lipofuscin, N-retinylidene-N-retinylethanolamine (A2E), derives from unique components of phagocytized POS (40,41). On the cellular level, long-term exposure of POS leads to formation of lipofuscin-like autofluorescent granules in cultured RPE cells (42). On the whole animal level, RCS rats (which have early photoreceptor degeneration) show less lipofuscin accumulation with age in the RPE (43). Compared to phagocytosis, the role of autophagy in RPE lipofuscin formation is less defined. However, results from both in vivo and in vitro studies indicate that RPE lipofuscin is not produced solely from phagocytosis. Studies with long-term cultured RPE cells show that autofluorescent inclusions resembling lipofuscin granules accumulate in the absence of outer segments or the A2E (44). Mice lacking alphaVbeta5 integrin are defective in phagocytosis of POS; nonetheless, lipofuscin accumulation with age is still apparent in the RPE of these mice (45). We recently developed a murine model of AMD using nuclear factor erythroid 2–related factor (Nrf2)–deficient mice. In this model, increased presence of autophagy intermediate structures was detected in the aging RPE, indicating dysregulated autophagy (46). Lipofuscin-associated fluorescence was more pronounced in aging RPE from knockout mice than in an age-matched wild-type control. More conclusively, components originating from autophagy have been identified in human RPE lipofuscin using proteomic approaches (47,48). Thus, like in other postmitotic tissues, autophagy appears to be involved in lipofuscinogenesis in the RPE (49).

Lipofuscin augments oxidative stress by increasing ROS generation and weakening antioxidant defense. Lipofuscin granules are photosensitizers that produce ROS in response to light exposure (50–53). It has recently been reported that photo-oxidation products of the major lipofuscin fluorophore A2E may contribute to drusen formation in human AMD (54). They may also directly damage the intracellular antioxidant defense. In fact, RPE cells fed with the aging pigment have decreased activity of SOD and catalase (55). Accumulated lipofuscin compromises cellular functions including lysosome-mediated degradative pathways, which are responsible for removal of damaged proteins and organelles (55–58). Altogether, lipofuscin causes considerable oxidative damage in aging RPE by increasing ROS production and impairing removal of ROS.

ASSOCIATION BETWEEN AMD AND OXIDATIVE STRESS: EVIDENCE BASED ON EPIDEMIOLOGIC AND CLINICAL ASSOCIATION STUDIES

Population-based studies have established a strong correlation between oxidative stress and AMD. Risk factors of AMD, such as aging, smoking, and sunlight exposure, are closely linked to oxidative stress (59–66). Markers of oxidative damage are detected both locally and systemically in patients with AMD. Moreover, antioxidant supplementation is an effective intervention in delaying the progression of AMD (67). Together, epidemiologic and clinical studies clearly demonstrate an association between oxidative stress and AMD.

Oxidation Markers in the AMD

Oxidative stress can be evaluated by various markers. In the past, we used plasma thiol/disulfide redox status and/or the concentration of thiol metabolites as a systemic indicator of oxidative stress. We showed that oxidation of plasma GSH and cysteine pools is associated with AMD risk factors including aging, smoking, and CFH polymorphism (32,62,68,69). Our recent studies further showed that plasma level of oxidized cysteine is sensitive to the supplements recommended by the Age-Related Eye Disease Study (AREDS). Dietary antioxidants and zinc indeed reverse the tendency of oxidation of the plasma cysteine in patients with AMD (70,71). Therefore, monitoring the redox status of plasma cysteine may provide a tool for predicting the effectiveness of antioxidant intervention in AMD patients.

The retina is rich in PUFA and susceptible to lipid peroxidation, and therefore, lipid peroxidation has been widely studied in the context of AMD. A relationship between dietary intake of PUFA and AMD is proposed by several published studies. In human eyes with AMD, an increased staining of oxidized phospholipids is prominent in the outer segments and the RPE (72). Presence of lipid modification in lipofuscin from human RPE is confirmed by proteomic analysis (48). Drusen, the earliest clinical manifestation of AMD, contains adducts of CEP and MDA, both of which are products of lipid peroxidation (73,74). The increased level of those two adducts is observed not only in the RPE/Bruch’s membrane/choroid complex but also in the plasma of patients with AMD (74–76). Recent studies on those two lipid peroxidation markers have provided critical information in elucidating functional roles for oxidative stress in AMD.

CEP adducts are derived from oxidation of docosahexaenoic acid (DHA), a fatty acid component of the membrane. DHA is an essential PUFA with highest abundance in the retina and accounts for approximately 50% of the fatty acids in POS membrane (77). Oxidative cleavage of DHA gives rise to 4-hydroxy-7-oxohept-5-enoic acid, and the latter covalently modifies proteins to form CEP adducts (78). Immunocytochemical studies showed that in the eye, CEP adducts mainly locate in the POS and RPE (76). CEP modifications have been identified in drusen of human AMD eyes. In addition, human AMD eyes have more CEP adducts in RPE/Bruch’s membrane/choroid complex than do age-matched controls (73). Systemically, AMD human plasma has increased levels of CEP adducts and anti-CEP autoantibodies compared with age-matched non-AMD control human plasma (76,79). CEP adducts are not only an oxidative damage marker associated with AMD. A recent study by Hollyfield et al. (80) showed that systemic administration of CEP adducts can initiate dry AMD-like phenotype in animal models. Thus, these studies convincingly suggest that the CEP adducts contribute to the AMD pathology of the outer retina.

MDA is a commonly used lipid peroxidation marker. In contrast to CEP (which is uniquely generated from DHA), MDA is the decomposition product from oxidation of PUFAs that contain more than two methylene-interrupted double bonds (81). Plasma samples from patients with AMD have higher levels of MDA than that of a control cohort (82,83). The negative regulator of the complement system, CFH, was recently found to specifically bind to the plasma MDA adducts, but not to the other oxidative modifications tested, such as CEP and 4-hydroxynonenal (74). CFH binds to MDA adducts via its two short consensus repeats (SCRs), SCR7 or SCR20, and neutralizes proinflammatory effects of MDA adducts. More strikingly, the AMD risk allele of CFH Y402H, which has an amino acid substitution in the region of SCR7, has impaired binding ability to MDA modifications and decreased protection. Together, using both clinical and experimental approaches, this elaborate study provides the first clue to solve the mystery on functional roles of the genetic risk factor CFH in AMD.

CAUSAL ROLES OF OXIDATIVE STRESS IN AMD: EVIDENCE FROM ANIMAL MODELS

Despite decades of efforts and the strong clinical association between oxidative stress and AMD, mechanisms of how oxidative injury leads to RPE degeneration and the link with chronic inflammation remain elusive. Severe oxidative stress induces apoptosis, but loss of RPE cells occurs mainly in the late stages of AMD. Cellular and molecular mechanisms of the early RPE degeneration remain largely unknown. In recent years, compelling evidence has been obtained from animal models, in which oxidative stress is introduced either by increasing exogenous pro-oxidant level, such as injection of CEP, or by compromising endogenous antioxidant systems such as deletion of the stress response gene Nrf2.

Superoxide Dismutase Knockout Mice

Mammals have three isoforms of SOD: the cytoplasmic Cu/Zn-SOD (SOD1), the mitochondrial MnSOD (SOD2), and the extracellular Cu/Zn-SOD (SOD3). All three require catalytic metals (Cu or Mn) for their activities (16). SODs play significant roles in oxidative defense, which is evident by increased oxidative damage in mice deficient of the cytoplasmic SOD1 or the mitochondrial SOD2. In fact, mice that completely lack the SOD1 have accelerated aging and shortened lifespan, and SOD2 knockout mice are lethal (84,85).

SODs are highly expressed in the retina (86), and manipulation of SODs in mice generates phenotypes related to human AMD. Mice deficient of the cytoplasmic SOD1 are the first animal model that demonstrated the causative roles of oxidative stress in pathogenesis of AMD (87). The SOD1 knockout mice showed AMD-like pathology in the outer retina (deposition of inflammatory proteins in the RPE/choroid complex, thickened Bruch’s membrane, and sub-RPE deposition) and developed drusen after 9 months. In young knockouts, constant light exposure at an intensity similar to outdoor sunlight caused drusen formation within a few weeks. After 10 months, about 10% of the mice developed choroidal neovascularization (CNV) and subretinal fluid leakage during fluorescein angiography. SOD2 is critical in defense against mitochondrial oxidative stress. Sod2−/− mice have dilated cardiomyopathy and die within weeks after birth (85). Retinal toxicity is evident even in these short-lived mutant mice, which show a progressive thinning of retina mainly involving the inner retinal and photoreceptor layers (88). RPE-specific knockdown of SOD2 leads to phenotypic changes related to AMD, but mainly in the RPE/Bruch’s membrane. Those changes include accumulation of oxidized proteins in the RPE/choroid complex, vacuolization and degeneration of the RPE, thickening of Bruch’s membrane, and increased levels of autofluorescence and A2E (89).

While the SOD models proved the concept that oxidative injury can lead to RPE degeneration and AMD-like pathology, they had certain limitations. SOD1 is a universal enzyme that directly scavenges superoxide anions and provides the first line of defense against oxidative stress. Lack of SOD1 resulted in severe damage to the neural retina with degeneration of the retina and the RPE occurring at almost the same time (90). Significant changes in the thickness of outer nuclear layer were noted as early as 30 weeks. This is in contrast to the clinical observation in AMD patients who typically retain their retinal function until late stages of the disease.

Mice Deficient of Nuclear Factor Erythroid 2–Related Factor

Nrf2 is a transcription factor controlling the cellular antioxidant and detoxification responses. Under basal conditions, Nrf2 is targeted for degradation by its inhibitor protein, Keap1, and remains at low to undetectable level in nontransformed cells (91,92). Upon conditions of oxidative stress or exposure to electrophilic compounds, Nrf2 dissociates from Keap1 and translocates into the nucleus in which it up-regulates genes containing a cis-acting antioxidant response element (ARE) in their promoter region (93). Genes under the transcriptional control of Nrf2 encode various enzymes, such as Trx1, Prx, glutamate–cysteine ligase, GSH S-transferase, heme oxygenase, NAD(P)H–quinone reductase, and glutamate–cysteine exchanger, which are essential for detoxification of xenobiotics and endogenous reactive intermediates (93,94).

Nrf2 knockout mice have normal embryonic development and comparable basal level of antioxidant status to the wild-type animals at young age (94,95). Accelerated aging is evident in Nrf2 null mice. The life expectancy is about 20 months, which is the life expectancy of only 60% of wild-type mice with the same genetic background (96). Nrf2 knockout mice have increased sensitivity to a variety of pharmacologic and environmental toxicants, and the manifestation depends on tissues and stimuli (94,97,98). Certain stimuli can induce ocular pathology in Nrf2-deficient mice. Hyperoxia results in more severe retinal vaso-obliteration in neonatal Nrf2 knockout mice compared with wild-type controls (99). Nrf2 null mice are more sensitive to ischemia/reperfusion-induced retinal toxicity. In response to ischemia/reperfusion, knockout mice have increased proinflammatory mediators, leukocyte infiltration, loss of ganglion cells, and retinal capillary degeneration (100). Chronic exposure to cigarette smoking induces more oxidative stress in Nrf2-deficient mice as evidenced by increased staining of 8-hydroxydeoxyguanosine in the RPE. Accordingly, Nrf2−/− mice displayed abnormal RPE basal infoldings and vacuoles and thickening of Bruch’s membrane (98).

We recently reported that Nrf2−/− mice developed age-related RPE and choroidal degeneration resembling cardinal features of human AMD, including RPE degeneration, Bruch’s membrane thickening, and spontaneous CNV (46). Between 8 and 11 months, drusen-like deposits emerged in fundus of Nrf2 KO knockout mice. With aging, atrophic RPE lesions occurred, and some of these lesions would eventually develop into sites of CNV. Knockout mice have moderate decrease in retinal function, as measured by ERG. Drusen formation, RPE atrophy, and CNV are also confirmed by histologic studies. Using histologic and electron microscopy, we further showed increased thickness of Bruch’s membrane and RPE vacuolation. Basal laminar and basal linear deposits were found exclusively in Nrf2−/− mice. Accumulation of lipofuscin granules was detected by fluorescence and electron microscopy. Immunostaining of eye sections revealed increased deposition of proteins that related to innate immunity (i.e., C3d, vitronectin, and serum amyloid P) and a marker of oxidative injury (nitrotyrosine) between the RPE and Bruch’s membrane in Nrf2−/− mice. The same proteins have been found in drusen and Bruch’s membrane of human AMD eyes (73,101).

Dry AMD-Like Pathology in Mice Immunized with CEP-Conjugated Albumin

CEP adducts originate from lipid peroxidation and have increased levels in patients with AMD both locally and systemically. Mice immunized with CEP-modified mouse serum albumin (CEP-MSA) have some phenotypic changes resembling AMD (80). Pathologic changes are apparent in the RPE, including vacuolation, hyperpigmentation, hypopigmentation, and atrophy. Effects of CEP-MSA immunization are long-lasting and progressive. Twelve months after the immunization, changes in the RPE persist and further extend to Bruch’s membrane. Sub-RPE elevation and basal laminar deposits occur, and there is significant increase in thickness of Bruch’s membrane. Deposition of complement-related proteins on Bruch’s membrane is also evident although no spontaneous CNV develops in the whole time frame. Thus, CEP-immunized mice provide a model of the atrophic form of AMD.

Findings from the CEP model suggest that a systemic challenge with immunogens can cause localized responses in the outer retina, and highlight the contributions of systemic factors such as complement protein and humoral or cell-mediated immunity to AMD. This is consistent with our previous findings that showed a more oxidized environment in plasma of AMD patients. These data support the use of antioxidant compounds to alleviate oxidative stress throughout the entire body.

Retinal and RPE Degeneration in Mouse Models of Iron Overload

Iron overload is clinically associated with AMD. Compared to normal donors, human AMD eyes have increased iron within the RPE (60,102). Although iron acts as essential cofactor of enzymes, ferrous iron is a potent source of ROS through the Fenton reaction. To mimic the condition of iron overload, Dunaief et al. generated double knockout mice (DKO) with both Cp and Heph deficiency (103). Cp and Heph are both multicopper ferroxidases that facilitate iron export from cells. Consequently, reduced activity of Cp and Heph in the DKO results in an age-dependent iron accumulation in the retina with the highest level in the RPE/choroid (104). The mice eventually develop retinal degeneration, which shares many features with AMD including photoreceptor degeneration, lipofuscin accumulation, RPE hyperplasia, RPE atrophy, sub-RPE deposits, and CNV (103,104). Infiltration of macrophages and deposition of complement components are also evident in the mice. Since iron overload has definite clinical associations with AMD, iron-chelating compounds may have a role in the development of novel therapeutic agents (105).

HOW OXIDATIVE STRESS CONTRIBUTES TO AMD: POTENTIAL MECHANISMS

Although clinical and laboratory studies indicate functional roles of oxidative stress in AMD, underlying mechanisms remain undefined. Available data suggested that oxidative stress could contribute to the pathophysiology of AMD in multiple ways, both locally and systemically.

Interaction Between Oxidative Stress and Inflammation in AMD

AMD involves inflammation, which is supported by the presence of complement proteins in drusen (73,106), association of multiple complement gene polymorphisms with AMD (1–4), and accumulation of inflammatory and immune response proteins in the Bruch’s membrane/choroid complex of AMD eyes (107). Available evidence suggests that oxidative stress may favor a proinflammatory environment and serve either as an initiating insult or as an amplifying adjuvant of the inflammatory responses in the pathogenesis of AMD. Oxidative stress and inflammation can also be involved in feed-forward loops to greatly accelerate the disease progression. Similar to observations in many other chronic human diseases, the interactive roles of oxidative stress and inflammation have been clearly demonstrated in the pathogenesis of AMD (108–111).

Oxidatively modified molecules may serve as an initial signal for innate immunity. Studies from recent years have shown that oxidized phospholipids modify endogenous molecules and generate immunogenic and proinflammatory oxidation-specific epitopes (OSE). The pathophysiologic significance of OSE has been established in several chronic diseases (112). CEP and MDA adducts are major OSE associated with AMD. The proinflammatory and immunogenic features of those epitopes are demonstrated by the following: (a) Intravitreal injections of MDA-BSA in mice trigger IL-8 production in the RPE (74), and (b) mice immunized with CEP-BSA have C3 deposition in the RPE/Bruch’s membrane and develop some AMD-resembling phenotypes (80). Interestingly, proteins encoded from normal and risk alleles of CFH show different reactivity to MDA adducts. Plasma CFH can bind to MDA-related OSE and inhibit their proinflammatory effects, while the AMD risk allele of CFH (Y402H) has impaired ability of binding MDA adducts and decreased efficiency in neutralizing their proinflammatory effects (74).

Oxidative stress may foster a proinflammatory environment in the eye by other means as well. Transcription factors responsible for up-regulating the inflammatory response, such as nuclear factor kappa B (NFκB), can be activated by oxidative stress (113–115). RPE cells are known to produce chemokines and cytokines and are involved in local immune responses. Oxidative stress can change the profile of cytokines and chemokines secreted by the RPE. Phagocytosis of oxidized POS induces IL-8 and MCP-1 production and decreases CFH production from RPE cells (116,117). In the presence of complement sufficient medium, hydrogen peroxide can reduce the surface expression of complement inhibitors DAF and CD59, decrease the surface inhibition mediated by CFH, and lead to complement activation on the surface of RPE cells (118). Oxidative damage induced by bisretinoid pigments of RPE lipofuscin has similar effects on complement activation, as evidenced by the accumulation of C3 split product iC3b (119,120). A direct molecular link between oxidative stress and CFH expression in the RPE has been demonstrated by Wu et al. (121). They identified an FOXO3 binding site in the promoter region of CFH and showed that the binding of the repressor FOXO3 to CFH promoter is subject to regulation by oxidative stress.

Oxidative Stress and Choroidal Neovascularization

Oxidative stress may generate a proangiogenic environment in the retina and contribute to the development of exudative AMD. In vitro cell culture studies have shown that oxidative stress stimulates VEGF production from the RPE (122,123). Furthermore, CEP adducts induce VEGF secretion and promote angiogenesis in vivo (124,125). About 10% of Sod1 knockout mice develop spontaneous CNV (126). Sod1 knockout mice crossed with transgenic mice expressing VEGF under the rhodopsin promoter developed significantly increased neovascularization in the subretinal space. Sod1 null mice also showed increased retinal neovascularization when subjected to oxygen-induced ischemia (127,128). These results suggest that when choroidal and retinal neovascularization begin to develop, oxidative stress potentiates VEGF production (from the RPE and other types of cells), thereby promoting the proliferation of the neovascularization.

Other Mechanisms

Oxidative stress may be involved in AMD pathology independent of inflammation. For example, DICER1 is a ribonuclease required for maturation of small interference RNA and microRNA (129). Recently, it has been attributed to the pathology of atrophic AMD (130,131). Expression of DICER1 is susceptible to oxidants, and therefore, direct down-regulation of DICER1 can be one possible mechanism mediating the contribution of oxidative stress to AMD. A common feature shared by mouse models of AMD involving oxidative stress is RPE pathology, including atrophy. Oxidative stress may induce RPE cell death and lead to atrophic AMD.

Intervention Targeting Oxidative Stress

Currently, antioxidant and zinc supplementation is the only treatment proven effective to impede the development and progression of AMD except for antiangiogenic pharmacotherapy that solely targets the exudative form of AMD. The therapeutic potential of antioxidant supplementation was supported by several clinical studies including the AREDS I. The multicentered, randomized AREDS I showed that high intake of the supplemental antioxidants (vitamin C, vitamin E, and beta-carotene) and zinc decreased the risk of progression from intermediate AMD to advanced AMD by 25% (132). Modifying antioxidant status can actually reduce the genetic risk as indicated by the Rotterdam Study (133). However, protection achieved by dietary antioxidant supplementation is modest, and the effectiveness remains controversial. The controversy may result from the lack of sufficient statistical power in detecting the phenotypic changes of the chronic disease and low potency of the current antioxidant regimen. More potent antioxidants with less long-term toxicities prove to be more effective. Furthermore, nonenzymatic antioxidants act in a synergistic fashion as is seen among vitamin C, vitamin E, and GSH, which greatly complicates the task of formulating an antioxidant supplement balanced to achieve optimal efficacy. Alternatively, reagents boosting the endogenous antioxidant network may offer a new direction to improve therapeutic outcomes.

Vitamin C (Ascorbate)

Vitamin C, the most effective water-soluble antioxidant found in the blood, may be essential for protection against diseases that involve oxidative stress (134). Vitamin C exists at high levels in the retina and is very efficient in removing hydrogen peroxide (135). Pretreatment with vitamin C protects against light toxicity in rats (67). AREDS I showed that vitamin C, together with vitamin E, beta-carotene, and zinc, reduced the risk for progression of AMD. However, vitamin C alone does not appear sufficient to provide adequate protection against oxidative stress (136).

Vitamin E

Vitamin E comprises a class of tocopherol compounds of which alpha-tocopherol is the most common and potent form (25). They are lipid-soluble and membrane-bound antioxidants considered as a primary nonenzymatic defense against lipid peroxidation. The RPE and the outer segments of the rods in the retina are rich in vitamin E, which is important in protecting the retina from oxidative injury (137,138). Typically, vitamin E acts as a chain-breaking antioxidant, which prevents the propagation of free radical damage to lipids in biologic membranes. To achieve maximum efficiency, vitamin E requires the presence of ascorbate and GSH (139). Deficiency of vitamin E causes retinal degeneration in monkeys (140,141). Supplementation of vitamin E protects outer segments from oxidative damage (67,132,142,143). Similar to vitamin C, there is no strong evidence that supports the efficacy of vitamin E when used alone, but it is included in the AREDS supplement formula, which has been proven effective (142,144).

Carotenoids

Carotenoids are tetraterpenoid organic pigments naturally occurring in photosynthetic organisms especially plants. They can be divided into two classes: non–oxygen-containing carotenes, such as beta-carotene, and oxygen-containing xanthophylls, such as lutein and zeaxanthin. Dietary analyses by the Eye Disease Case–Control Study (EDCCS) group suggest carotenoids have protective effects in AMD (145,146). In this study, an inverse relationship between carotenoid supplementation and risk for exudative AMD was demonstrated. Lutein, zeaxanthin, and beta-carotene were the major species (of those examined) that accounted for the protective effect of the carotenoids against oxidative stress.

Beta-carotene is a precursor to vitamin A. It is highly abundant in the blood but normally is present only in trace amounts in the retina (67). Beta-carotene acts as general scavenger to a broad spectrum of ROS, including superoxide radicals, lipid peroxidation intermediates, and Fenton-generated radicals. However, the role of beta-carotene in AMD is inconsistent. The AREDS supplementation regimen contained 15 mg of beta-carotene and resulted in the aforementioned 25% risk reduction in progression to advanced AMD (147). The risk reduction effect is confirmed by the Rotterdam Study (148), but challenged by the Blue Mountain Eye Study, in which an increased risk of developing neovascular AMD was associated with increased intake of beta-carotene (146,149).

The xanthophyll carotenoids, lutein and zeaxanthin, are the major components of macular pigment (150). These membrane-localized carotenoids function as lipid antioxidants by two means: (a) as light filters and (b) as ROS quenchers. Lutein and zeaxanthin efficiently absorb blue light that passes through the anterior segment and decrease the short-wavelength light exposure of the macula (151,152). The high content of double bonds in the tetraterpenoid backbone enables these carotenoids to easily supply electrons to quench ROS and prevent further damage to lipids. Thus, they prevent lipid peroxidation and stabilize the membrane.

As discussed above, carotenoid intake from food was negatively associated with the risk of exudative AMD in the EDCCS. Several other clinical studies have evaluated the effects of the macular carotenoids. However, the results are still controversial due to the limited sample size, the relatively short lengths of follow-up, and outcome measures concerns (137). There currently is a large-scale longitudinal clinical trial, AREDS 2, that is designed to evaluate the effects of supplemental lutein, zeaxanthin, and omega-3 on the progression to advanced AMD (http://clinicaltrials.gov/ct2/show/NCT00345176) that will be invaluable in answering the question about the benefit of carotenoids in the treatment of AMD.

Glutathione

GSH is a major component of the endogenous antioxidant system that protects cells from oxidative stress. As briefly mentioned above, the nonenzymatic antioxidants vitamin C and vitamin E require GSH for maximal activity. Vitamin C (ascorbate) scavenges hydrogen peroxide forming oxidized dehydroascorbate whose reduction depends on GSH. Vitamin C further supports antioxidant function of vitamin E by reducing the radical form of vitamin E (138). Thus, by fulfilling the redox cycle of vitamin C and vitamin E, GSH plays a critical role in the maintenance of the eye mediated by these antioxidants (153,154). In addition, GSH serves both as a direct antioxidant and as an important substrate for the enzymatic antioxidant systems such as GSH peroxidase (155,156). Deficiency of GSH leads to tissue damage in animals and even early mortality in newborn rats and guinea pigs (157). In the context of the eye, GSH protects photoreceptor cell membranes from lipid peroxidation (158,159). Furthermore, compounds inducing GSH synthesis protect against oxidative injury in cultured RPE (160).

Zinc

Zinc is a trace element present in high concentrations in eye tissues, particularly the retina and choroid (161,162). It participates in the antioxidant defense in both direct and indirect ways (163–165). Zinc protects susceptible protein SH groups from oxidative modification by binding directly to these groups; by binding to adjacent sites, thus creating steric hindrance to the SHs; and by binding to distant sites and causing a conformational change in the protein’s tertiary structure to sequester the SHs. Zinc can also antagonize redox-active transition metal catalysts in the Fenton reaction such as copper and iron, thereby inhibiting the production of ROS. Zinc is indispensable for activity of Cu/Zn-SOD, the first-line enzymatic antioxidant defense. Moreover, zinc can induce enzymes that are critical for the antioxidant network, such as catalase, metallothioneins (MTs), and Nrf2 (67,148). Zinc deficiency results in increased lipid peroxidation in subcellular membranes of the liver.

The beneficial effects of zinc with regard to the progression of AMD have been demonstrated by both large-scale randomized controlled clinical trials and population-based studies (67). In AREDS, zinc, either alone or with antioxidants, significantly reduced the risk of progression from an intermediate stage to an advanced stage of AMD (164). Of particular note was the observation that both zinc alone and zinc with antioxidant supplementation had much greater efficacy than was found with supplementation of the three antioxidants, vitamin C, vitamin E, and beta-carotene. The potency of zinc may be attributed to effects other than directly scavenging ROS. Our study using in vitro cultured RPE cells showed that zinc activates the Nrf2-dependent antioxidant system in the RPE, which is the major activation site in the eye. We also found that feeding mice a high-zinc diet increased Nrf2 activity (unpublished data) (46). Increasing cellular GSH synthesis is one of the downstream events of Nrf2. Accordingly, higher levels of GSH were found in the retina and RPE from the mice fed a high-zinc diet than were found in tissues from the mice fed a normal diet. Nrf2 is a master regulator of cellular antioxidant and detoxification systems. Mice deficient of Nrf2 have ocular pathology resembling human AMD, and enhancing transcriptional activity of Nrf2 can protect against oxidative injury to the RPE in this model (166). Thus, our data from both in vitro and in vivo studies indicate that Nrf2 is a potential node that mediates the protective effect of zinc in AMD.

Nrf2 Inducers

Besides zinc, several structurally different Nrf2 inducers have been tested for their protective effects against oxidative retinal injury. The majority of these inducers are naturally occurring compounds, such as sulforaphane and curcumin, or from compounds biochemically derived from them, such as triterpenoid analogs. Sulforaphane [(-)- 1-isothiocyanato-(4R)-(methylsulfinyl)butane] is a potent Nrf2 inducer existing in vegetables such as broccoli and Brussels sprouts (167,168). It activates an array of detoxification responses dependent on Nrf2. Protection against oxidative and proto-oxidative damage is evident in RPE cells pretreated with sulforaphane (169). In vivo study in rodent models of retinal injury has also confirmed the protection conferred by sulforaphane. Using a light damage model, Yodoi et al. showed that sulforaphane is effective in protecting photoreceptors and the RPE from oxidative injury and the protective signals emanating from Nrf2-mediated pathways (170). Sulforaphane delayed photoreceptor cell death in tubby mouse, a model of Usher syndrome, which exhibits progressive photoreceptor degeneration shortly after birth (171). Compared to vehicle-treated animals, sulforaphane-treated tub/tub mice showed increased thickness of outer nuclear layer and improved visual function as detected by ERG recording.

A new class of Nrf2 inducers, triterpenoid derivatives, has been investigated recently. These compounds have strong potencies, capable of inducing phase 2 enzymes and protecting against oxidative stress at subnanomolar concentrations (172). One of the most potent synthetic triterpenoid analogs, 2-cyano-3,12-dioxooleanan-1,9(11)-dien-28-oci acid (CDDO) (172

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