Chapter 18

Oxidative Stress and Systemic Changes

in Age-Related Macular Degeneration

Milam A. Brantley Jr., Melissa P. Osborn, Jiyang Cai, and Paul Sternberg Jr.

Abstract Several lines of evidence point to a systemic role for oxidative stress in age-related macular degeneration (AMD). Age and smoking are associated with increasing levels of systemic oxidative stress and oral antioxidant supplements have been shown to slow the progression of the disease. In addition, plasma levels of reactive oxygen species and lipid peroxidation products have been closely associated with AMD, and decreased antioxidant enzyme activity has been reported in AMD patients. Polymorphisms in mitochondrial DNA (mtDNA) and in genes coding for antioxidant enzymes have also been linked to AMD. Oxidative stress and inßammatory mediators have been shown to play a role in AMD, although it is unclear whether inßammation is aggravated by oxidative stress or vice versa. As the interaction between inßammation and oxidative stress may be critical to development and progression of AMD, a combination therapy that reduces systemic changes in redox status and controls local inßammation may be able to prevent or at least slow the development of sight-threatening late-stage disease.

18.1Introduction

Age-related macular degeneration (AMD), the leading cause of irreversible vision loss in older individuals in the Western world, is a complex disease inßuenced by factors such as genetics, demographics, and environmental exposures. 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 [1]. As AMD prevalence increases dramatically with age, people over 85 are ten times more likely to experience advanced AMD than those aged 70Ð74 [2].

M.A. Brantley Jr. ¥ M.P. Osborn ¥ J. Cai ¥ P. Sternberg Jr. (*)

Vanderbilt Eye Institute, Vanderbilt University, 2311 Pierce Avenue, Nashville, TN 37232, USA e-mail: paul.sternberg@vanderbilt.edu

in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-61779-606-7_18, © Springer Science+Business Media, LLC 2012

AMD can be divided into an early form, in which patients usually do not have symptoms, and a late form, which may result in severe central vision loss. The hallmark of early AMD is the presence of drusenÑwhitish-yellow deposits typically localized between the retinal pigment epithelium (RPE) and BruchÕs membrane. These deposits may be small and discrete (hard drusen) or larger and more conßuent (soft drusen). Drusen may also be present between the photoreceptors and RPE or within the photoreceptor cell layer (reticular drusen) [3, 4]. Histologically, drusen consist of numerous proteins (e.g., complement, immunoglobulins, amyloid-b) and lipids (e.g., phospholipids, cholesterol, apolipoproteins) [5, 6].

The presence of soft and/or reticular drusen increases the risk of progressing to a more advanced form of the disease. In the Rotterdam Eye Study, the 5-year progression rate to late AMD in patients with high-risk drusen was 28% [7]. While there are no direct interventions for early AMD, vitamin supplementation has been shown to protect against AMD progression. The Age-Related Eye Disease Study (AREDS), a multicenter, randomized clinical trial sponsored by the National Eye Institute, demonstrated that daily intake of supplemental antioxidants (b-carotene, vitamin C, vitamin E) and zinc reduced the risk of progression to advanced AMD by 25% over 5 years in high-risk early AMD patients [8].

Sight-threatening late AMD can be divided into ÒdryÓ and ÒwetÓ forms. In advanced dry AMD, extensive loss of the choriocapillaris and overlying RPE results in regions of retinal geographic atrophy (GA). A patient with GA often experiences gaps in an image or missing letters in a line of text. Currently, no effective treatment to slow GA progression is available. In wet AMD (neovascular, or exudative AMD), which is responsible for 90% of AMD-related vision loss [9], abnormal choroidal vessels extend into the subretinal space in a process known as choroidal neovascularization (CNV). Leakage of blood or serous ßuid from these vessels can lead to a detachment of the neurosensory retina from the underlying RPE, which may cause straight lines to appear distorted. Extensive scarring or additional hemorrhages may occur within weeks to months of the initial detachment, often resulting in permanent vision loss.

The current standard of care for neovascular AMD consists of intravitreal injections of drugs that target vascular endothelial growth factor (VEGF). The anti-VEGF antibodies ranibizumab and bevacizumab inhibit the interaction between VEGF and its receptors, thus mitigating the angiogenic and permeability-enhancing effects of VEGF. In the pivotal clinical trials MARINA and ANCHOR, monthly injections of ranibizumab improved or maintained vision over 2 years in over 90% of treated individuals [10, 11]. In fact, 35Ð40% of MARINA and ANCHOR patients demonstrated signiÞcant improvement in visual acuity, making ranibizumab the Þrst treatment of neovascular AMD to reverse vision loss in some patients.

A variety of factorsÑdemographic, environmental, and geneticÑcontribute to the risk of developing AMD and advancing to late stages of the disease. Studies have demonstrated that older age, higher body mass index (BMI), and greater light exposure correspond to higher prevalence of AMD [12, 13]. Smoking, the strongest environmental risk factor for AMD, has been linked to AMD onset and progression in multiple large, epidemiologic studies [14Ð19]. Importantly, smoking cessation was shown to reduce the risk for dry AMD, suggesting that smoking is a modiÞable

risk factor for AMD [20]. Additionally, high dietary intake of carotenoids and antioxidant supplementation have been linked with lower risk of AMD [8, 21, 22].

The hereditary component of AMD is supported by several lines of evidence, including familial and twin studies [23Ð25]. Recent population-based studies demonstrated that a single nucleotide polymorphism (SNP) in the complement factor H (CFH) gene is strongly linked with AMD [26Ð29]. As the primary regulator of the alternative arm of the complement cascade, CFH plays a critical role in innate immunity and inßammatory response. In these seminal studies, individuals with one risk allele for this SNP had signiÞcantly increased risk of AMD (odds ratios [ORs] ranging from 2.5 to 4.6), and two risk alleles conferred correspondingly higher risk (ORs ranging from 3.3 to 7.4). Multiple reports have conÞrmed this association in different populations [30Ð34]. The signiÞcant inßuence of the complement pathway on macular degeneration was further substantiated when polymorphisms in genes coding for complement factor B/C2, C3, complement factor I, and CFH-related proteins 1 and 3 were also shown to inßuence AMD susceptibility [35Ð40]. Recently, an association has been reported between AMD and the SERPING1 gene, which encodes the C1 inhibitor, a regulator of the classic complement pathway [41, 42].

A second locus, encompassing the ARMS2 (Age-related maculopathy susceptibility 2) and HTRA1 (HtrA serine peptidase 1) genes on chromosome 10q26, has also been consistently associated with AMD [43Ð46]. It has proven difÞcult to determine whether ARMS2 or HTRA1 is responsible for the association with AMD because they are very close to each other on the chromosome and are in strong linkage disequilibrium. Interestingly, ARMS2 was reported to localize to the mitochondrial outer membrane, suggesting the involvement of a mitochondrial pathway in AMD [47]. This Þnding, however, was not conÞrmed in a subsequent study [48].

Polymorphisms in numerous other genes may exert smaller effects on AMD susceptibility than do the major contributors CFH and ARMS2/HTRA1. Two recent genome-wide association studies (GWAS) showed that the hepatic lipase (LIPC) and tissue inhibitor of metalloprotease 3 (TIMP3) genes may inßuence AMD risk [49, 50]. LIPC, a critical enzyme in high-density lipoprotein (HDL) cholesterol metabolism, has been localized to the retina [49], and LIPC variants have been speciÞcally associated with advanced AMD [51]. A mutation in the TIMP3 gene, involved in degradation of the extracellular matrix, causes SorsbyÕs fundus dystrophy [52]. This early-onset macular degenerative disease, which typically presents before the age of 40, shares clinical features with AMD [53]. The link between TIMP3 polymorphisms and AMD remains to be conÞrmed by further genetic association studies.

While a wide range of risk factors for AMD have been identiÞed, the molecular cause of this complex disease remains unknown. Several lines of evidence implicate oxidative stress in the pathophysiology of AMD [54], as well as in numerous other chronic diseases, including heart disease, diabetes, and neurodegenerative disorders [55, 56]. Although the body produces reactive oxygen species (ROS) as by-products of normal metabolic processes (e.g., glycolysis and the Krebs cycle), aging and disease may disturb the balance between ROS generation and clearance, resulting in oxidative damage to macromolecules [57].

ROS are highly reactive atoms, ions, or molecules that contain oxygen. They 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 [58]. The majority of endogenous ROS are produced by mitochondria through the electron transport chain, which converts 2Ð3% of all utilized oxygen into ROS [59]. Stimuli such as aging, inßammation, irradiation, air pollutants, and cigarette smoke lead to increased ROS [58, 60, 61].

Most ROS are eliminated immediately by antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GSHPx), 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 [58]. Smaller antioxidant molecules (e.g., vitamin C [ascorbate], vitamin E [tocopherol], and carotenoids) function as direct radical scavengers, reducing ROS such as the hydroxyl radical.

The retina is particularly susceptible to oxidative stress because of its high oxygen consumption, its high proportion of polyunsaturated fatty acids (PUFAs), and its exposure to visible light [57, 58]. The unique phagocytic function of the RPE provides an additional oxidative burden. 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. The 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 Þlter that blocks highly damaging blue light from reaching the photoreceptors.

Retinal oxidation has been investigated in the context of AMD. Oxidative modiÞcations to proteins and DNA have been detected in BruchÕs membrane, drusen, retina, and RPE [62]. The higher prevalence of these oxidative changes in AMD patients vs. controls [62] suggests an increased oxidative state in the retina of AMD patients. Also, impairment of the retinaÕs antioxidant defense system may play a role in AMD, as some studies have reported decreased levels of macular pigment in AMD patients [63Ð65]. It remains unclear whether AMD is strictly a disease of the retina/RPE complex or if damage to these structures is a local manifestation of a truly systemic disease. Two of the primary risk factors for AMD, smoking and aging, correspond to increased levels of free radicals and thus oxidative stress throughout the body [60, 61]. High dietary intake of antioxidants has been linked with reduced risk of AMD [21, 66], and oral vitamin supplementation has been shown to slow AMD progression [8], further suggesting a systemic component to the disease. Additionally, modiÞed levels of plasma biomarkers of oxidative stress, as well as antioxidant enzymes, have been found in AMD patients [54]. Studies of blood complement protein levels demonstrated increased systemic complement activation in AMD patients [67, 68]. Furthermore, patients with membranoproliferative glomerulonephritis type II (MPGN II) and systemic complement activation develop retinal