Iron Induced Oxidative Damage As a Potential Factor in Age-Related Macular Degeneration: The Cogan Lecture

Abstract

Iron is a potent generator of oxidative damage whose levels increase with age, potentially exacerbating age-related diseases. Several lines of evidence suggest that iron accumulation may be a factor in age-related macular degeneration (AMD). AMD retinas have more iron within the photoreceptors, RPE, and drusen than do age-matched control retinas. Accelerated AMD-like maculopathy develops in patients with retinal iron overload from the hereditary disease aceruloplasminemia. Mice with retinal iron overload resulting from knockout of ceruloplasmin and its homologue hephaestin exhibit retinal degeneration with some features of AMD, including subretinal neovascularization, accumulation of RPE lipofuscin and sub-RPE deposits, and RPE/photoreceptor death. Increased understanding of the mechanisms of retinal iron homeostasis may help in the development of therapies to prevent iron overload. For example, herein it is shown that one regulator of systemic iron homeostasis, HFE, is expressed in the RPE. Thus, patients with the common disease hereditary hemochromatosis, which is often caused by an HFE mutation, may have retinal iron overload predisposing to AMD. Preliminary data suggest that iron chelation can reduce RPE iron overload in mice and protect them from degeneration, suggesting that iron-binding drugs may one day prove useful in reducing RPE oxidative stress and decreasing the risk of AMD progression.

The pathogenesis of age-related macular degeneration (AMD) is not well understood, but is thought to involve oxidative stress ¹ and inflammation. ^{2 3 4 5 6} Because iron overload has been implicated in the age-related neurodegenerations Alzheimer’s disease and Parkinson’s disease (reviewed in Ref. ⁷ ) and is a potent generator of oxidative stress through the Fenton reaction, we have explored the possibility that iron contributes to AMD. 

Iron is absorbed in the intestine, but very little iron is excreted, leading to an increase in tissue iron levels with age. Consistent with this, retinal iron levels are higher in maculas from post mortem donors older 65 than in those younger than 65 (Hahn and Dunaief, unpublished, 2006). This iron accumulation is potentially toxic. 

Although iron is important in normal retinal function as an essential metabolic component of both heme- and non-heme-containing proteins including RPE65, ⁸ if dysregulated, it can cause damaging oxidative stress. ⁹ Ferrous iron (Fe⁺²) reacts with H₂O₂ in the Fenton reaction to produce the highly reactive hydroxyl radical, which can damage proteins, lipids, and nucleic acids. To protect cells against this damage, a set of iron-binding and transport proteins (iron-handling proteins) transfers iron from the gut to the plasma and, ultimately, to cells including retinal neurons and RPE cells. ¹⁰ In the brain, there is considerable iron flux, with iron crossing the blood-brain barrier through capillary endothelial cells and through the choroid plexus into the cerebral spinal fluid (CSF; reviewed in Ref. ¹¹ ). Dysfunction of iron-handling proteins leads to disease in both the brain and retina. ^{12 13}  

Several lines of evidence indicate the necessity for tight retinal iron regulation: (1) An iron foreign body lodged in one part of the eye can result in a generalized photoreceptor and RPE degeneration throughout the retina, after diffusion of iron through the vitreous and retina. Similarly, injection of ferrous iron into the vitreous of an experimental animal causes photoreceptor degeneration. ^{14 15 16} (2) In models of subretinal hemorrhage, iron released from red blood cells is believed to participate in photoreceptor degeneration, ¹⁷ and iron chelation can ameliorate the degeneration (Youssef TA et al., IOVS 2002;43;ARVO E-Abstract 3000). (3) Royal College of Surgeons (RCS) rats, with deficient phagocytosis of photoreceptor outer segments, have increased RPE and photoreceptor iron compared with control animals, which may contribute to their retinal degeneration. ¹⁸ (4) Patients with hereditary diseases causing retinal iron overload, aceruloplasminemia, Friedreich’s ataxia, and pantothenate kinase-associated neurodegeneration ^{19 20 21 22} have retinal degeneration. (5) There are higher RPE and photoreceptor iron levels in AMD retinas than in age-matched control retinas, ^{23 24} suggesting that iron-mediated RPE toxicity may contribute to retinal degeneration in AMD. Levels of the iron carrier protein transferrin are also increased in AMD retinas. ²⁵  

Unique characteristics of the retina render it particularly dependent on tight iron regulation. The retina is constantly exposed to photo-oxidative stress, which could be exacerbated by even normal levels of labile iron. Indeed, treatment of rats with the iron chelator deferoxamine ameliorates photic injury. ²⁶ Photic injury upregulates ceruloplasmin (Cp), a ferroxidase that may protect the retina from photo-oxidative stress by oxidizing toxic ferrous iron (Fe⁺²) to the more inert ferric form (Fe⁺³). ²⁷ Photoreceptors require iron, in part as an essential cofactor in membrane biogenesis to replace their shed outer segments ²⁸ and as a cofactor for the enzyme guanylate cyclase, which produces cGMP, an essential component of the phototransduction cascade. The highest levels of retinal iron are in the choroid, RPE, and photoreceptors. ²⁹ The outer segments contain iron and the RPE, which phagocytoses shed outer segments, is subject to increases in iron through this ingestion. 

The Multicopper Ferroxidases Ceruloplasmin and Hephaestin

Mutation of the ferroxidase Cp leads to RPE iron accumulation. Patients with the disease aceruloplasminemia, a hereditary deficiency of Cp, ^{30 31} begin to show retinal degeneration in the fourth or fifth decade. The degeneration is characterized by drusen-like deposits and RPE atrophy (Fig. 1) . ^{19 22} Although histology in human aceruloplasminemia has not been published, mice lacking both Cp and its homologue Heph have an accelerated retinal iron overload (Fig. 2)and age-dependent retinal degeneration consisting of photoreceptor death, RPE hypertrophy with lipofuscin accumulation, sub-RPE deposits, and subretinal neovascularization (Fig. 3) . ³²  

Cp is a copper-binding glycoprotein found mainly in plasma but also present in several other tissues, including retina and brain. ³³ Two forms of Cp are produced by alternative splicing, and both are present in the retina. ²⁷ One form is secreted and the other is anchored to the cell surface. As a ferroxidase, Cp functions as an antioxidant by converting hydroxyl-radical producing ferrous iron (Fe⁺²) to the less toxic ferric form (Fe⁺³). ³⁴ Cp−/− neural cells have increased susceptibility to oxidative stress, ³⁵ confirming Cp’s antioxidant function. Cp also facilitates iron transport by oxidizing iron to the ferric form; only the ferric form can bind transferrin, the major extracellular iron transporter. Cp may facilitate iron loading onto transferrin in the retina, as transferrin is made by RPE and photoreceptors and is bound by photoreceptors and other retinal cells. ^{29 36}  

Cp levels are elevated by several ocular injuries and stresses, most likely to convert iron to the less toxic ferrous form. These conditions include the rat retina after optic nerve crush, ³⁷ the mouse retina after light-induced damage, ^{27 38} the glaucomatous human and monkey retinas, ^{39 40} the diabetic rat retina, ⁴¹ and the aqueous and vitreous of the inflamed rabbit eye. ⁴²  

Hereditary Hemochromatosis and HFE

Hereditary (primary) hemochromatosis is a genetic disorder in which the body absorbs an excess of dietary iron. The majority of cases are the result of a mutated HFE gene product. HFE protein binds to β2-microglobulin ⁴³ and acts as a regulator of iron absorption by decreasing the affinity of the transferrin receptor for transferrin. ^{44 45} HFE mutations are also associated with reduced levels of the iron regulatory hormone hepcidin, which plays an important role in the regulation of intestinal iron absorption. ⁴⁶ In approximately 60% to 90% of cases, a missense mutation occurs causing tyrosine substitution for cysteine (C282Y), resulting in dysregulated transferrin-mediated iron uptake in the gut. The excess iron is deposited in the parenchymal cells within organs throughout the body including the liver, pancreas, joints, and heart. Though present at birth, the genetic defect does not manifest signs and symptoms until later in life, usually between the ages of 30 and 50 in men, and greater than 50 in women. Women often present after menopause when iron loss through menstruation and pregnancy ceases. 

Clinical symptoms include weakness, fatigue, arthralgias, arthritis, intermittent abdominal pain, loss of libido, and impotence. Physical examination may show skin hyperpigmentation, hepatomegaly, evidence of heart failure, testicular atrophy, elevated liver enzymes, hyperglycemia, low testosterone levels, and hypothyroidism. 

Roth and Foos ⁴⁷ reported histopathology of a small series of patients with hereditary hemochromatosis showing iron deposits in the peripapillary retinal pigment epithelium, ciliary epithelium, and the sclera. Several of these patients had drusen. 

In a post mortem retina (courtesy of W. R. Green, Wilmer Eye Institute, Baltimore, MD) of a 60-year-old man who died of complications of hereditary hemochromatosis, Perls’ stain detected iron in drusen (Richa and Dunaief, unpublished, 2006). It is possible that iron toxicity to the RPE contributed to the formation of these drusen and may cause ongoing RPE damage. The potential association between hereditary hemochromatosis, or carrier state for a mutation in the HFE gene that may moderately increase dietary iron absorption, and AMD is worthy of investigation. 

Retinal iron homeostasis is most likely influenced by HFE, as the HFE protein is present in the RPE, photoreceptor inner segments and Müller cells (Fig. 4) . This labeling pattern is specific, as control specimens with other species-matched antibodies show dissimilar labeling patterns and omission of the primary antibody eliminates the label. 

Potential Therapeutics

In most diseases involving iron overload, the primary goal is to reduce the amount of iron in the body. It has been achieved by serial phlebotomy, limitation of dietary iron, and, in some cases, iron chelation. Several reports suggest that iron chelation may help in the treatment of several neurologic diseases such as Alzheimer’s disease, Parkinson’s disease, ⁴⁸ Huntington’s disease, and Friedreich’s ataxia. ⁴⁹ Thus, it is plausible that iron chelation can be useful in retinal disease associated with iron overload. 

From an ophthalmologic perspective however, it would be premature to consider iron chelation with deferoxamine (DFO) for AMD. Iron chelation with DFO has occasionally been associated with retinal toxicity. ^{50 51 52 53} The mechanism of DFO toxicity is unknown, but it may result from retinal iron deficiency if doses are too high. If DFO were one day tested as a prevention for advanced AMD, the dose would have to be carefully titrated, along with careful monitoring for retinal toxicity. 

Additional chelating agents are currently being investigated. Chemical compounds bearing an 8-hydroxyoxyquinoline moiety show high affinity for iron, free radical scavenging capability, and in vitro neuroprotective activity. ⁴⁸ One such agent protected mice from the Parkinson’s-like disease caused by the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). ⁵⁴  

Supplementation with the dibasic amino acid l-arginine and its precursor l-citrulline may warrant further investigation in the treatment of AMD. ⁵⁵ These amino acids have been shown in vitro to protect against iron-induced glycoprotein insolubility, a proposed mechanism contributing to drusen formation. It is suggested that normal function of iron transport within the retina and RPE relies on proper endocytosis and lysosomal function and that glycoprotein insolubility may act as a potential derailment in this process. 

Future Investigations

Until the effect of dietary iron on retinal iron levels is understood, I suggest that patients with retinal disease avoid taking oral iron supplements or eating a diet high in red meat unless there is medical indication for the supplements such as iron deficiency anemia. Iron chelation may one day prove useful in prevention of advanced AMD. As an initial step, the retinal toxicity profile of several iron chelators will be tested in mice. Their efficacy in reducing retinal iron overload and preventing retinal degeneration in the Cp/Heph mutant mice will be investigated. 

Since a number of observations suggest that iron is important in retinal degeneration (Table 1) , the prevalence of iron overload in retinal degeneration and the mechanisms controlling retinal iron homeostasis will be the subjects of ongoing investigation. It is possible that polymorphisms in genes affecting retinal iron homeostasis, including Cp, Heph, ferroportin, transferrin, ferritin, HFE, or hepcidin can affect the risk of AMD. 

 Supported by Research to Prevent Blindness (William and Mary Greve Scholar award to JLD and an unrestricted award), National Eye Institute Grant EY015240, the International Retina Research Foundation, the F. M. Kirby Foundation, the Steinbach Foundation, a gift from Alma Cohen, and the Paul and Evanina Bell Mackall Foundation Trust.

 Disclosure: J.L. Dunaief, None

 

Figure 1.

 

Fundus photographs and fluorescein angiographic results from patient with aceruloplasminemia. OD (top) and OS (bottom). Fundus photographs in panels (A) and (E) were obtained at age 47, while the remainder of the images including fluorescein angiograms were obtained at age 56. Comparison of (A) and (B) shows that white oval and reticular subretinal lesions developed between 1995 to 2004. Macular depigmentation corresponds to the RPE transmission defect in the fluorescein angiogram early (C) and late (D) phases. In OS, the macular lesions spread into the more peripheral macula from 1995 (E) to 2004 (F). Fluorescein angiography of the left eye reveals in the mid (G) and late (H) phases a macular window defect consistent with RPE atrophy. Whitish subretinal peripapillary lesions are also present (F). Reprinted from Ophthalmology, 112, Dunaief JL, Richa C, Franks EP, et al., Macular degeneration in a patient with aceruloplasminemia, a disease associated with retinal iron overload, 1062–1065, © 2005, with permission from the American Academy of Ophthalmology.

Figure 2.

 

Adult (6 month) Cp−/−Heph−/Y RPE cells accumulate iron. Prussian Blue Perls’ stain reveals punctate blue iron label only in the RPE. Other areas of the retina as well as wild-type retinas had no Perls’ label (not shown). Section was counterstained with hematoxylin/eosin. Reprinted from Hahn P, Qian Y, Dentchev T, et al. Disruption of ceruloplasmin and hephaestin in mice causes retinal iron overload and retinal degeneration with features of age-related macular degeneration. Prod Natl Acad Sci U S A. 2004;101:13850–13855. © 2004 National Academy of Sciences, USA.

Figure 3.

 

Nine month old Cp−/−Heph−/Y mice have retinal degeneration. In an area of hypertrophic, hyperplastic (area demarcated by arrowheads) RPE cells, a necrotic RPE cell also observed by electron microscopy (diamond inset) is present. Within the area of RPE hyperplasia, there is local photoreceptor thinning and subretinal neovascularization (red *) visible as small vessels containing erythrocytes (rectangular inset). The hyperplastic RPE have formed a localized cyst (Cy). The degree of RPE hypertrophy can be appreciated by comparing the hypertrophic cells on the left of the image to the normal size RPE on the right. Reprinted from Hahn P, Qian Y, Dentchev T, et al. Disruption of ceruloplasmin and hephaestin in mice causes retinal iron overload and retinal degeneration with features of age-related macular degeneration. Prod Natl Acad Sci USA. 2004;101:13850–13855. © 2004 National Academy of Sciences, USA.

Figure 4.

 

Fluorescence photomicrograph of BALB/c mouse retina immunolabeled with anti-HFE antibody. Anti-HFE immunoreactivity (red) is present in the RPE, inner segments, and Müller cell end feet. Nuclei are labeled with 4′,6′-diamino-2-phenylindole (DAPI; blue). Scale bar, 50 μm.

Table 1.

 

Summary of Iron’s Importance in Retinal Degeneration

1. Iron, even at normal concentrations, is a potent generator of oxidative stress.

2. Retinal iron levels are high and increase with age and in AMD.

3. Intravitreal iron injection causes retinal degeneration.

4. Iron chelation protects rabbit retina from subretinal blood (Trese M et al. IOVS 2002;43:ARVO E-Abstract 4017).

5. Inherited defects in iron metabolism cause retinopathy.

6. Aceruloplasminemia (hereditary retinal iron overload) causes maculopathy.

7. Cp/Heph-knockout mice have retinal degeneration with increased oxidative stress, RPE lipofuscin, sub-RPE deposits, sub-RPE neovascularization, and death of RPE and photoreceptors.

8. Iron chelation protects rat retinas from light damage 26 and RPE cells from oxidant-induced death (Lukinova, Amado, and Dunaief, unpublished, 2006).

I am profoundly grateful to my mentors, chronologically: Daniel Albert, Stephen Goff, Don Zack, Morton Goldberg, Ann Milam, Craig Thompson, and Jean Bennett. Stuart Fine has been my medical retina mentor and exceptionally supportive chairman. Paul Hahn and Tzvete Dentchev did much of the described work in my lab. Jared Iacovelli performed the HFE immunofluorescence experiment described herein. Leah Harris provided the Cp/Heph mice and has been a steadfast collaborator. I also thank for their constant support my mother Leah Dunaief and my wife Rachel Dunaief.