Oxidative damage and age-related macular degeneration

Abstract

This article provides current information on the potential role of oxidation in relation to age-related macular degeneration (AMD). The emphasis is placed on the generation of oxidants and free radicals and the protective effects of antioxidants in the outer retina, with specific emphasis on the photoreceptor cells, the retinal pigment epithelium and the choriocapillaris. The starting points include a discussion and a definition of what radicals are, their endogenous sources, how they react, and what damage they may cause. The photoreceptor/pigment epithelium complex is exposed to sunlight, is bathed in a near-arterial level of oxygen, and membranes in this complex contain high concentrations of polyunsaturated fatty acids, all considered to be potential factors leading to oxidative damage. Actions of antioxidants such as glutathione, vitamin C, superoxide dismutase, catalase, vitamin E and the carotenoids are discussed in terms of their mechanisms of preventing oxidative damage. The phototoxicity of lipofuscin, a group of complex autofluorescent lipid/protein aggregates that accumulate in the retinal pigment epithelium, is described and evidence is presented suggesting that intracellular lipofuscin is toxic to these cells, thus supporting a role for lipofuscin in aging and AMD. The theory that AMD is primarily due to a photosensitizing injury to the choriocapillaris is evaluated. Results are presented showing that when protoporphyric mice are exposed to blue light there is an induction in the synthesis of Type IV collagen synthesis by the choriocapillary endothelium, which leads to a thickened Bruch's membrane and to the appearance of sub-retinal pigment epithelial fibrillogranular deposits, which are similar to basal laminar deposits. The hypothesis that AMD may result from oxidative injury to the retinal pigment epithelium is further evaluated in experiments designed to test the protective effects of glutathione in preventing damage to cultured human pigment epithelial cells exposed to an oxidant. Experiments designed to increase the concentration of glutathione in pigment epithelial cells using dimethylfumarate, a monofunctional inducer, are described in relation to the ability of these cells to survive an oxidative challenge. While all these models provide undisputed evidence of oxidative damage to the retinal pigment epithelium and the choriocapillaris that is both light- and oxygen-dependent, it nevertheless is still unclear at this time what the precise linkage is between oxidation-induced events and the onset and progression of AMD.

Figure 1

Schematic sequence of damaging reactions. The sequence begins with either an oxidation, a photoxidation or a photosensitization event and ends with a damaging reaction resulting in a disease. It is proposed that oxidants and free radicals produced as a result of these initial events cause lipid peroxidation, the oxidation of critical bonds in proteins and DNA strand breaks. Cells possess an armory of protectants, including antioxidants and enzymes, that serve to quench the oxidants and free radicals, thereby minimizing the damage and the need for repair and replacement. In the case of light-dependent damage to ocular tissues, such as the retina in AMD, dark sunglasses will reduce exposure to sunlight.

Figure 2

Biochemistry of oxygen derived metabolites. The basic elements and reactions by which oxygen is converted to a variety of oxidants and free radicals are shown. Major oxygen derived metabolites include superoxide anion, hydrogen peroxide, hydroxyl radical and singlet oxygen. Also shown is the approximate half-life (t_1/2) for each metabolite.

Figure 3

Cellular sources of oxygen radicals. Listed above are a number of enzyme catalyzed reactions and activities in cells which are known to produce oxidants and oxygen free radicals. Particularly relevant to the photoreceptor cell/RPE complex are the four activities highlighted in red.

Figure 4

Basic properties and structure of vitamin E. Keys facts regarding vitamin E and its role as a scavenger of free radicals; the structure of vitamin E. * A group of eight fat-soluble compounds * α-tocopherol is the biologically most active form * Absorbed into lymphatics from the intestines * Protects lipids from peroxidative damage * A chain-breaking antioxidant that reacts with, ·O₂^{− 1}O₂, peroxyl (ROO·), and alkoxyl (RO·) radicals * Major lipid-soluble antioxidant protecting membranes and lipoproteins from injury * Vitamin E· (radical form) is reduced back to Vitamin E by Vitamin C

Figure 5

Basic properties and structures of carotenoids. Basic properties of the macular carotenoids lutein and zeaxanthin; the structures of the macular carotenoids are shown with other carotenoids for comparison. * Absorb blue light, protective against short wavelength visible light * Quench singlet oxygen * Quench triplet state of photosensitizers * Inhibit autoxidation of lipids * β-carotene, an effective antioxidant at low P O₂; assume same for macular carotenoids * Undergo autoxidation * Small amount of oxidation products detected in normal human and monkey retinas

Figure 6

Histogram demonstrating the phototoxicity of lipofuscin. Lipofuscin-fed RPE cells were either maintained in the dark (black bars) or exposed to light (gold bars): A. blue light (400–550 nm); B. amber light (550–800 nm) for up to 48 h. Cell viability was determined using the MTT assay [47]. Vertical bars represent standard error of the mean.

Figure 7

Choriocapillaris in a mouse model of protoporphyria. In the mouse model of protoporphyria with approximately a 10-fold increase in protoporphyrin IX and exposure to blue light (380–430 nm, 14 μW/cm²), a time and light dependent increase in choriocapillary and subretinal pigment epithelial basal laminar-like deposits are demonstrated (see arrows).

Figure 8

Normal choriocapillaris in mouse. In dark controls, no increase of the choriocapillary basement membrane was noted.

Figure 9

Sub-retinal pigment epithelial deposits in a mouse model of protoporphyria. Protoporphyric mouse model exposed to blue light demonstrates sub-retinal pigment epithelium fibrillogranular deposit (white arrow) with fibrils measuring up to 16 nm with a periodicity of 13 nm.

Figure 10

Type IV collagen staining of choriocapillaris in a mouse model of protoporphyria. Intense Type IV collagen specific staining of basement membrane of the choriocapillaris especially notable around chroiocapillary endothelial fenestrations.

Figure 11

Enhancement of collagen synthesis following photosensitization. Collagen synthesis was increased with light and PP IX exposure when compared to cells exposed to PP IX and dark (p=0.0004), controls in light (p=0.003) and controls in dark (p=0.003; Figure 10). The use of a blue light filter eliminated radiation below 450 nm. Total energy delivered remained the same by increasing intensity irradiation of longer wavelengths.

Figure 12

Shift in glutathione redox status with aging, AMD, and diabetes. Calculated redox potential (E_h) for GSH pool in blood plasma of younger and older controls and patients with AMD or diabetes. Values for E_h were calculated as E_h = E_o + RT/ZF ln ([GSSG]/(2[GSH])) where E_o was taken as 0.24 V [61], R is the universal gas constant, T is the absolute temperature, Z is the valence, and F is the Faraday constant.

Figure 13

Dimethylfumarate protects cultured RPE cells from oxidative injury. A 24 h pretreatment with 0.1 mM dimethylfumarate (DMF) protects cultured human RPE cells from oxidative injury associated with a toxic dose of tertiary butyl hydroperoxide (tBHP). Viability of cells was determined by measuring the extent of leakage of lactic acid dehydrogenase from the cells into the incubation media.