Pathogenetic Mechanisms in Age-Related Macular Degeneration

Published on 08/03/2015 by admin

Filed under Opthalmology

Last modified 22/04/2025

Print this page

rate 1 star rate 2 star rate 3 star rate 4 star rate 5 star
Your rating: none, Average: 0 (0 votes)

This article have been viewed 1557 times

Chapter 64 Pathogenetic Mechanisms in Age-Related Macular Degeneration

Introduction

Age-related macular degeneration (AMD) can be divided into early and late stages. In early disease visual acuity is good and in the fundus focal deposits are seen in Bruch’s membrane, called drusen. The distribution and size of drusen varies from one patient to another, although their attributes are highly concordant between eyes of an individual. There may also be pigmentary changes at the level of the retinal pigment epithelium (RPE).

The three forms of late AMD cause loss of central vision. In most communities the most common is choroidal neovascularization (CNV), in which blood vessels grow inwards into or through Bruch’s membrane. Detachment of the retinal pigment epithelium (PED), in which there is accumulation of fluid between the RPE and Bruch’s membrane, is relatively uncommon. In geographic atrophy (GA) there is well-defined loss of RPE and photoreceptor cells.

It is generally considered that GA is the default pathway of the disease process and that CNV occurs as a reactive event during the evolution of change. The treatment of CNV is well established and is described elsewhere in this book. There is no well-recognized treatment whereby the disease mechanisms during transition from early AMD to GA can be modified.

The structures involved in the disease process are the photoreceptor cells in the outer retina, the retinal pigment epithelium (RPE), Bruch’s membrane and the capillary bed in the inner choroid (choriocapillaris). In AMD changes occur in all these tissues throughout the eye, although they are most marked at the macula that subserves central vision in which there is a high density of cones. The changes in each of these tissues represent a potential target for treatment based on the current understanding of the relevant pathogenic mechanisms. In this chapter the logic of the various therapeutic approaches will be discussed.

Structural changes

Choroid

In the young, the choroidal capillary bed is formed of a sinusoidal complex in which the capillary bed is fenestrated and lacks tight junctions. It is believed that the nature of the choriocapillaris is determined largely by the constitutive expression of vascular endothelial growth factor (VEGF) outwards toward the choroid by the RPE.15 In one morphometric study it was found that the density of the choriocapillaris is decreased with age in eyes without AMD6 and choroidal casts have shown that the capillary bed may become tubular.7 With advanced AMD, loss or narrowing of the choriocapillaris occurs.811

A clue as to possible clinical detection of change in the choriocapillaris came from studies of Sorsby fundus dystrophy, a monogenic disorder characterized by major thickening of Bruch’s membrane and a prolonged choroidal filling phase on fluorescein angiography.12 It was thought that the diffusely thickened Bruch’s membrane represented a barrier to diffusion of VEGF towards the choroid resulting in changes in the capillary bed to a tubular state such that acquisition of fluorescence of the inner choroid is irregular and delayed.13,14 This angiographic sign has also been identified in patients with AMD.15 It is not known whether this sign indicates only change in circulation, or if slow egress of dye through the fenestrae and diffusion through tissues also contributes to this angiographic abnormality. The potential significance of this clinical sign has been established by demonstrating discrete areas of scotopic threshold elevation of up to 3.4 log units and slow dark adaptation which corresponded closely to regions of choroidal perfusion abnormality.16,17 Loss of photopic function was less marked. Subsequent studies have also shown that the recovery from bleaching is prolonged18 and the functional loss has an impact on daily tasks.19

Bruch’s membrane

A direct relationship between aging and thickness of Bruch’s membrane has been established both by electron and light microscopy,20,21 but in one study the correlation coefficients (R2)were only 0.57 and 0.32, respectively, with great variation in the elderly.22 Thus about half of the change in thickness must be explained by factors other than age, such as genetic or environmental influences.

Several studies on the nature of the deposits have been undertaken. Consequent upon discussion of the pathogenesis of PEDs it was hypothesized that reduction of the hydraulic conductivity of Bruch’s membrane would hamper movement of water towards the choroid thus causing it to accumulate in the sub-RPE space.23 This demands that Bruch’s membrane contain a high lipid content that would increase the resistance of fluid flow. A series of investigations followed to test this hypothesis and support was derived from both histopathological, biochemical, biophysical, and clinical observations. A study of frozen tissue undertaken using histochemical staining on human eyes with an age range between 1 and 95 years showed accumulation of lipids with age that varied greatly both in the quantity and form of lipids in the elderly.24 Some eyes stained for neutral lipids alone, some stained predominantly for phospholipids, and others stained equally for both neutral lipids and phospholipids. To confirm these conclusions, material extracted by universal lipid solvents from tissue of eye-bank fresh eyes was analyzed by thin layer and gas chromatography.25,26 After separation, the chemical species were identified by mass spectroscopy which included fatty acids, cholesterol, triglycerides, and phospholipids. This study confirmed the conclusion that the quantity of total lipid in Bruch’s membrane increases with age. Little or no lipid was extracted from specimens from donors younger than 50 years of age. In specimens from donors older than 50 years, the increase was exponential. Eyes from donors over the age of 60 years showed wide variation of total lipid extracted from donors of similar age, and that the ratios of phospholipids to neutral fats was different from one specimen to another. The ratio of neutral lipids to phospholipids did not correlate with the total quantity of lipid. The finding that the major lipid species were phospholipids and fatty acids rather than cholesterol and cholesterol esters, and that only 50% of the phospholipids was phosphatidylcholine led to the conclusion that the lipids were of a cellular (presumably RPE), rather than plasma origin.26 Curcio, using different extraction methods, reported that cholesterol and cholesterol esters were the major lipids rather than phospholipids. As in the previous study, however, it was concluded that the lipids were of RPE origin on the basis of the nature of the cholesterol.27 Unlike atheroma, there was little free cholesterol.

Finally, measurements of hydraulic conductivity of Bruch’s membrane showed that it becomes reduced with age,28,29 and after the age of 50 years there is a close direct linear relationship between resistance of fluid flow and lipid content.

Clinical observations were sought to support the concept that the biochemical content as well as thickness of Bruch’s membrane influenced subsequent clinical behavior. It was hypothesized that drusen that are hyperfluorescent on fluorescein angiography must be hydrophilic allowing free diffusion of water-soluble sodium fluorescein into the abnormal deposit and that there would be binding of dye to polar molecules. In contrast, if the drusen were hypofluorescent it would imply that they are hydrophobic due to the presence of neutral lipids. This conclusion was supported by histological observations in which it was shown that in vitro binding of sodium fluorescein correlated well with the biochemical contents of drusen as shown by histochemistry.30 Drusen rich in neutral lipids did not bind fluorescein whereas those with little lipid content bound fluorescein strongly.

It would be predicted that the highest resistance to water flow in Bruch’s membrane would be found in eyes destined to suffer tears of the detached RPE in which there is sufficient tangential stress in the detached tissues to cause them to rupture. The determination that a tear in one eye implied high risk of a similar event occurring in the fellow eye31 provided the opportunity to test the concept. A comparison was made of the drusen in the fellow eye of a tear with those of the fellow eye of one with visual loss due to subretinal neovascularization. It was shown that the drusen were larger, more confluent, and less fluorescent on angiography in the former group than in the latter.32 Thus, there is good reason to believe that thickening and lipid accumulation in Bruch’s membrane would hamper movement of metabolites and water between the RPE and choroid.

There is considerable lipid trafficking through Bruch’s membrane and lipids are believed to accumulate as they fail to pass freely through a thickened Bruch’s membrane. This demands that Bruch’s membrane becomes thicker as a prerequisite for this lipid accumulation. Analysis has been undertaken of proteins in aging Bruch’s membrane since this is likely to initiate the thickening. Recent studies have shown that several of the proteins associated with the immune system such as C3, C5b-C9 and CFH are present in high quantity in Bruch’s membrane in AMD.33 These observations serve to underline the potential significance of a disordered immune system to AMD. However, unlike inflammation elsewhere there is no infiltration by inflammatory cells. Beta-amyloid has also been identified.34 In the inner part of Bruch’s membrane there are high levels of vitronectin.35,36 The origin of the proteins is in doubt given that there is RPE expression of some of the constituents although a major contribution may come from plasma. The state of the proteins is unknown but circumstantial evidence suggests that they are oligomerized37 and that this may be generated by high levels of zinc or other metallic ions. In Bruch’s membrane the levels of zinc are very high.38 The levels of bioavailable zinc are many times that necessary to cause oligomerization of CFH in vitro37 and the high risk variant of CFH would predictably oligomerize more readily than the low-risk variant. Thus the proteins may not have the biological properties of the monomers.

Further insight into the possible mechanisms of accumulation of material in Bruch’s membrane was derived from observation in the CFH−/− mouse.39 It is acknowledged that a gene knockout is not necessarily homologous with a polymorphism, that the immune system in mouse is dissimilar from human and mouse does not have a macula. However, if reduction of CFH activity is important, as has been concluded from genetic studies, the observations may help in understanding AMD. In this mouse there is thickening of the renal glomerulus basement membrane, but surprisingly, Bruch’s membrane was thinner than in age-matched mice. This implies that dysregulation of the immune system alone may not explain thickening Bruch’s membrane and that the presence of the CFH protein is important to the process.

The retinal pigment epithelium

Accumulation of residual bodies that fluoresce can be used as an index of age change in the RPE. A quadratic relationship exists between age, and both autofluorescence and residual body quantity as measured by autofluorescence imaging as seen by light microscopy and electron microscopy, respectively.22 The slowing of accumulation in the elderly was not surprising since the population of photoreceptors decreases in late life.42 The relationship between age and autofluorescence, however, is not close, with an adjusted R2 of only 0.45, and for residual bodies the R2 was 0.50,22 reflecting the wide variation in the eyes from elderly donors. Thus 50% of the variation in either autofluorescence or residual bodies is not explained by aging, the suspicion being that genetic or environmental factors would play a role in determining the variance. Most surprising was the relationship between autofluorescence and residual body volume. The relationship was direct, which would have been expected since it is from the residual bodies that the autofluorescence is derived. However, the R2 was only 0.26. In retrospect, the variation between specimens should not have been surprising since only a small proportion of the material in residual bodies fluoresces, and this proportion may be influenced by circumstances such as vitamin A content in diet.43 If rodents are given a diet low in vitamin A the residual bodies do not fluoresce. In littermates given a diet high in vitamin A the residual content of the RPE is similar but they fluoresce brightly. From this observation it might be concluded that those with high autofluorescence levels had a diet high in vitamin A.

The clinical relevance of these finding is underlined by the ability now to image RPE autofluorescence in vivo through the efforts of Fitzke and von Rückmann.44 This is achieved using a confocal scanning laser ophthalmoscope with an excitation wavelength of 488 nm generated by an argon laser. Emission is recorded above 500 nm by inserting a barrier filter. The evidence that the signal originates from lipofuscin in the RPE is derived from the work of Delori and coworkers.45 It has been shown that in early AMD the distribution of autofluorescence varies from one patient to another. In about half of cases of early AMD autofluorescence is homogeneous, whereas in the remainder diffusely irregular or focally increased autofluorescence is seen.46 Drusen do not appear to explain the differences, since apart from serogranular drusen at the fovea, drusen in AMD do not autofluoresce significantly. It has been shown that in bilateral early AMD the pattern is symmetrical, implying that the autofluorescence characteristics reflect the form of disease in an individual that may be determined by the genetic or environmental influences. In patients with unilateral visual loss from AMD, focal increased autofluorescence in the good eye is associated with GA in the other eye, and predicts the development of GA in the good eye. This impression was reinforced by the observation that a high level of autofluorescence is found around the perimeter of GA, and that this area becomes atrophic within one year,47 whereas cases without marginal hyperautofluorescence tend not to have progression of their GA.

The underlying molecular mechanisms by which changes in the RPE result in the development of GA have been subject to debate. It has been argued that the cytoplasmic volume occupied by the residual bodies may interfere with cell metabolism.48 It has been shown that lipofuscin is a free radical generator and that may cause cell damage.49 In addition, there is evidence for toxic effects of individual lipofuscin compounds. A2-E, a Schiff-base product of retinaldehyde and ethanolamine, has surfactant-like properties on biomembranes that have been shown in one study to increase intralysosomal pH by inhibition of the ATP-dependent lysosomal proton pump that in turn would inhibit activity of lysosomal hydrolases.50 Furthermore, A2-E has been shown to cause leakage of lysosomes in vitro.51 Release of lysosomal content may cause further RPE cell dysfunction and cell death. Another study failed to confirm a rise in lysosomal pH, possibly because lower quantities of lipofuscin were used, but it did show that lipid degradation was reduced.52 Thus, both studies imply that lipofuscin reduces the activity of phagolysosomal enzymes.

The possible consequences of reduced RPE lysosomal degradation have been investigated in vivo. Interference with degradation of lysosomes was achieved in 11-week-old Sprague–Dawley rats by injection of 5 µl of a lysosomal protease inhibitor, E-64 (2.22 µM) intravitreally.53 A single injection of E-64 caused a transient accumulation of phagolysosome-like inclusion bodies in the RPE. Furthermore, 2 or 3 injections on alternate days caused progressive accumulation of these inclusions associated with changes in intracellular organelles such as loss of smooth endoplasmic reticulum and RPE cell conformation. This was accompanied by shortening and loss of photoreceptor outer segments without prior dysmorphic changes, photoreceptor loss, reduction of fenestrae in the choroidal capillaries, and invasion of Bruch’s membrane by fibroblasts and pericytes. Intravitreal injection of vehicle for comparison induced no structural changes.

It was considered likely that the changes in the RPE reflected reduced metabolism of lipids and reduction of basolateral VEGF expression caused the loss of fenestrae. The shortening and loss of the outer segments was thought to be due to impaired morphogenesis of disc membranes rather than a direct effect of E-64 on photoreceptor cells, because there was no vesiculation of disk membranes. The shortening of the OS could be explained by the lack of available lipids due to the inability of the RPE to break down the contents of the phagosome. The findings imply greater dependence upon the availability of products of phagosomal degradation for OS renewal than was previously considered, and that acquisition of plasma-derived material is insufficient to sustain this process fully. The ability of the RPE to recycle lipids has been well illustrated.54,55 The observed changes in rats are similar in many respects to age changes in RPE, photoreceptor, and choroid in humans, although there are major differences between such an acute experiment, and the consequences of life-long metabolic activity, and species differences between rat and human.

Thus, both experimental evidence and clinical observations illustrate potential pathogenetic mechanisms of geographic atrophy and explain the association of geographic atrophy with focal increased autofluorescence if the latter is witness to the inability to recycle phagosomal contents.

Another potential intriguing consequence of the presence of lipofuscin is the demonstration that photodegradation products of the fluorophore induce the complement cascade that may be relevant to Bruch’s membrane thickening.56

Measurement of visual function over areas of increased autofluorescence showed loss of scotopic function that was much greater than photopic, and that the loss was as great as 3.5 log units.57 The question as to whether the loss is due to cell loss or cell dysfunction was not addressed.

Outer retina

Relative to other structures there is less information on physical changes in the neuroretina in AMD. From early histological studies it was concluded that photoreceptor cell loss occurs progressively in early AMD although it was thought that this may occur as a consequence of RPE dysfunction.59,60 Clinical studies have served to support this conclusion that photoreceptor loss occurs early. Two representative papers describe ocular coherence tomography in patients with geographic atrophy.61,62 Areas of the fundus beyond the edge of atrophy in which the retina appeared normal by ophthalmoscopy apart from drusen were imaged to determine the thickness of the photoreceptor layer. In some subjects there was an abrupt change from the lack of photoreceptor cells in the area of atrophy, to a normal thickness outer nuclear layer. In the majority, however, there was evidence of major photoreceptor loss for a considerable distance beyond the edge. As a consequence of these observations it appears to be likely that the functional losses in early AMD are due in part or completely to cell loss rather than cell dysfunction. The magnitude of functional loss implies major photoreceptor loss in early AMD.

Observations in the CFH knockout mouse may also be relevant to cell loss in AMD.39 It had been shown that visual function was reduced when compared with age-matched mice despite the lack of expected Bruch’s membrane thickening. In these mice the photoreceptor outer segments were dysmorphic and there was increased C3 expression in the outer retina. The relevance of C3 to outer segment morphology or of the genetic risk factors to presence of C3 are unknown. As a result of these observations it was concluded that the consequences of the high-risk CFH polymorphism may not be restricted to its influence upon Bruch’s membrane. Of possible relevance is that RPE expression of CFH in vitro appears to be apical into the outer retina rather than through the basolateral domain into the choroid.63 These observations raise the possibility that there is a role played by the immune system in photoreceptor physiology, and that disturbance of these may cause demise of photoreceptor cells that is unrelated to changes in Bruch’s membrane or the RPE.

Therapeutic implications

Preservation of the photoreceptor cells might be best achieved by improving RPE function by reduction of accumulation of lipofuscin that may be achieved through manipulation of A2E formation in the photoreceptor outer segment. Restriction of vitamin A availability presumably would achieve this end. Neuroprotective agents might be of benefit as has been hypothesized in the treatment of glaucoma.64 Similarly, such an approach might slow or prevent photoreceptor cell loss and this has been attempted with the use of a slow release device that uses cells embedded in a matrix that express CNTF.65 There is early indication that this may be successful in the management of retinitis pigmentosa.66

This chapter is by no means exhaustive and additional factors have been considered that are not tissue-specific, such as free radical damage and mitochondrial dysfunction.67,68 There is a large body of circumstantial evidence to support each although neither is proven. If mitochondrial dysfunction were important it would affect photoreceptors more than other cells since they require large quantities of oxygen to maintain the dark current.

Conclusion

In age-related macular degeneration changes occur in the choroid, Bruch’s membrane, the RPE, and outer retina. Both genetic and environmental influences have been identified as conferring risk of disease and presumably initiate disease through their cumulative effect. However, there is doubt as to tissues primarily affected by these factors. It is most likely that the balance of changes in the various tissues would vary from one case to another given that many factors confer risk and that they vary from one case to another. Thus in some individuals Bruch’s membrane thickening may be the most threatening change whereas it may be the RPE alterations in others. It is also possible that photoreceptor loss may not be due to dysfunction of either the RPE or Bruch’s membrane in some cases but is a consequence of an abnormality limited to the outer retina. If correct, therapy directed to one aspect of the disorder should be reserved to those in whom the specific tissue is the primary threat to photoreceptor survival. To achieve this goal it would be necessary to have knowledge of changes to the various tissues involved. Hints or even proof might come from genetic research.

The photoreceptor population could be assessed using imaging such as optical coherence tomography, or visualization of the photoreceptor cells with adaptive optics. The same objective could be achieved using psychophysics means such as visual field recording, microperimetry or fine matrix mapping. Assessment of the concentration of bleachable rhodopsin using reflectometry might be the most effective method of determining the population of viable photoreceptor cells.69,70 Whatever testing device is used it would be important to test the scotopic as well as the photopic system. The health of the RPE might be assessed by measuring absolute levels of autofluorescence. There is some doubt as to whether or not the thickness of Bruch’s membrane could be measured in vivo, but it is likely that the state of the choroid is determined by access of VEGF expressed outward by the RPE. Thus choroidal thickness and choriocapillaris flow would be influenced by the biophysical properties of Bruch’s membrane. Both these have been measured recently although variation in normal subjects has yet to be fully established. Characterization of disease in this way would be important in selecting patients for a specific therapeutic approach and in monitoring the response to treatment. It is true that the tissues are metabolically interdependent and modulation of age change in one tissue may have secondary benefits on its neighbors.

References

1 Korte GE, Repucci V, Henkind P. RPE destruction causes choriocapilary atrophy. Invest Ophthalmol Vis Sci. 1984;25:1135–1145.

2 Blaauwgeers HG, Holtkamp GM, Rutten H, et al. Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris. Evidence for a trophic paracrine relation. Am J Pathol. 1999;155:421–428.

3 Kannan R, Zhang N, Sreekumar PG, et al. Stimulation of apical and basolateral VEGF-A and VEGF-C secretion by oxidative stress in polarized retinal pigment epithelial cells. Mol Vis. 2006;12:1649–1659.

4 Saint-Geniez M, Kurihara T, Sekiyama E, et al. An essential role for RPE-derived soluble VEGF in the maintenance of the choriocapillaris. Proc Natl Acad Sci U S A. 2009;106:18751–18756.

5 McLeod DS, Grebe R, Bhutto I, et al. Relationship between RPE and choriocapillaris in age-related macular degeneration. Invest Ophthalmol Vis Sci. 2009;50:4982–4991.

6 Ramrattan RS, van der Schaft TL, Mooy CM, et al. Morphometric analysis of Bruch’s membrane, the choriocapillaris, and the choroid in aging. Invest Ophthalmol Vis Sci. 1994;35:2857–2864.

7 Olver J. Quoted by AC Bird in: Therapeutic targets in age-related macular disease. J Clin Invest. 2010;120:3033–3041.

8 Hogan MJ. Macular diseases, pathogenesis: electron microscopy of Bruch’s membrane. Trans Am Acad Ophthalmol. 1965;69:683–690.

9 Green WR, Key SN. Senile macular degeneration: a histopathological study. Trans Am Ophthalmol Soc. 1977;75:180–250.

10 Sarks SH. Changes in the region of the choriocapillaris in aging and degeneration. 23rd Concilium Ophthalmol, Kyoto; 1978 p. 228–38.

11 Tso MOM. Pathogenetic factors of aging macular degeneration. Ophthalmology. 1985;92:628–635.

12 Polkinghorne PJ, Capon MR, Berninger TA, et al. Sorsby’s fundus dystrophy: a clinical study. Ophthalmology. 1989;96:1763–1768.

13 Meves H. Die pathologisch-anatomischen gefassveranderungen des Auges bei der beningen und malingen Nephrosklerose. Graefes Arch Ophthalmol. 1948;168:287.

14 Friedman E, Smith TR, Kuwabara T, et al. Choroidal vascular patterns in hypertension. Arch Ophthalmol. 1964;71:842.

15 Pauleikhoff D, Chen JC, Chisholm IH, et al. Choroidal perfusion abnormalities in age related macular disease. Am J Ophthalmol. 1990;109:211–217.

16 Chen JC, Fitzke FW, Pauleikhoff D, et al. Functional loss in age-related Bruch’s membrane change with choroidal perfusion defect. Invest Ophthalmol Vis Sci. 1992;33:334–340.

17 Steinmetz RL, Haimovici R, Jubb C, et al. Symptomatic abnormalities of dark adaptation in patients with age-related Bruch’s membrane change. Br J Ophthalmol. 1993;77:549–554.

18 Owsley C, Jackson GR, White M, et al. Delays in rod-mediated dark adaptation in early age-related maculopathy. Ophthalmology. 2001;108:1196–1202.

19 Scilley K, Jackson GR, Cideciyan AV, et al. Early age-related maculopathy and self-reported visual difficulty in daily life. Ophthalmology. 2002;109:1235–1242.

20 Green WR, Key SN. Senile macular degeneration: a histopathological study. Trans Am Ophthalmol Soc. 1977;75:180–250.

21 Ramrattan RS, van der Schaft TL, Mooy CM, et al. Morphometric analysis of Bruch’s membrane, the choriocapillaris, and the choroid in aging. Invest Ophthalmol Vis Sci. 1994;35:2857–2864.

22 Okubo A, Rosa RH, Bunce KV, et al. The relationships between age changes in retinal pigment epithelium and Bruch’s membrane. Invest Ophthalmol Vis Sci. 1999;40:443–449.

23 Bird AC, Marshall J. Retinal pigment epithelial detachments in the elderly. Trans Ophthalmol Soc UK. 1986;105:674–682.

24 Pauleikhoff D, Harper CA, Marshall J, et al. Aging changes in Bruch’s membrane: a histochemical and morphological study. Ophthalmology. 1990;97:171–178.

25 Holz FG, Sheraidah G, Pauleikhoff D, et al. Analysis of lipid deposits extracted from macular and peripheral Bruch’s membrane. Arch Ophthalmol. 1994;112:402–406.

26 Sheraidah G, Steinmetz R, Maguire J, et al. Correlation between lipids extracted from Bruch’s membrane and age. Ophthalmology. 1993;100:47–51.

27 Li CM, Chung BH, Presley JB, et al. Lipoprotein-like particles and cholesteryl esters in human Bruch’s membrane: initial characterization. Invest Ophthalmol Vis Sci. 2005;46:2576–2586.

28 Moore DJ, Hussain AA, Marshall J. Age related variation in the hydraulic conductivity of Bruch’s membrane. Invest Ophthalmol Vis Sci. 1995;36:1290–1297.

29 Starita C, Hussain AA, Patmore A, et al. Localization of the site of major resistance to fluid transport in Bruch’s membrane. Invest Ophthalmol Vis Sci. 1997;38:762–767.

30 Pauleikhoff D, Zuels S, Sheraidah G, et al. Correlation between biochemical composition and fluorescein binding of deposits in Bruch’s membrane. Ophthalmology. 1992;99:1548–1553.

31 Chuang EL, Bird AC. Bilaterality of tears of the retinal pigment epithelium. Br J Ophthalmol. 1988;72:918–920.

32 Chuang EL, Bird AC. The pathogenesis of tears of the retinal pigment epithelium. Am J Ophthalmol. 1988;105:185–190.

33 Hageman GS, Luthert PJ, Victor Chong NH, et al. An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch’s membrane interface in aging and age-related macular degeneration. Prog Retin Eye Res. 2001;20:705–732.

34 Yoshida T, Ohno-Matsui K, Ichinose S, et al. The potential role of amyloid beta in the pathogenesis of age-related macular degeneration. J Clin Invest. 2005;115:2793–2800.

35 Hageman GS, Mullins RF, Russell SR, et al. Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells. FASEB J. 1999;13:477–484.

36 Wasmuth S, Lueck K, Baehler H, et al. Increased vitronectin production by complement-stimulated human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 2009;50:5304–5309.

37 Nan R, Gor J, Lengyel I, et al. Uncontrolled zinc- and copper-induced oligomerisation of the human complement regulator factor H and its possible implications for function and disease. J Mol Biol. 2008;384:1341–1352.

38 Lengyel I, Flinn JM, Peto T, et al. High concentration of zinc in sub-retinal pigment epithelial deposits. Exp Eye Res. 2007;84:772–780.

39 Coffey PJ, Gias C, McDermott CJ, et al. Complement factor H deficiency in aged mice causes retinal abnormalities and visual dysfunction. Proc Natl Acad Sci U S A. 2007;104:16651–16656.

40 Lovell MA. A potential role for alterations of zinc and zinc transport proteins in the progression of Alzheimer’s disease. J Alzheimers Dis. 2009;16:471–483.

41 Li W, Chen S, Ma M, et al. Complement 5b-9 complex-induced alterations in human RPE cell physiology. Med Sci Monit. 2010;16:17–23.

42 Curcio CA, Medeiros NE, Millican CL. Photoreceptor loss in age-related macular degeneration. Invest Ophthalmol Vis Sci. 1996;37:1236–1249.

43 Katz ML, Norberg M. Influence of dietary vitamin A on autofluorescence of leupeptin-induced inclusions in the retinal pigment epithelium. Exp Eye Res. 1992;54:239–246.

44 von Rückmann A, Fitzke FW, Bird AC. Distribution of fundus autofluorescence with a scanning laser ophthalmoscope. Br J Ophthalmol. 1995;79:407–412.

45 Delori FC, Dorey CK, Staurenghi G, et al. In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelial lipofuscin characteristics. Invest Ophthalmol Vis Sci. 1995;36:718–729.

46 Lois N, Coco R, Hopkins J, et al. Fundus autofluorescence in patients with age-related macular degeneration and high risk characteristics. Am J Ophthalmol. 2002;133:341–349.

47 Holz FG, Bellman C, Staudt S, et al. Fundus autofluorescence and development of geographic atrophy in age-related macular degeneration. Invest Ophthalmol Vis Sci. 2001;42:1051–1056.

48 Feeney-Burns L, Eldred GE. The fate of the phagosome: conversion to “age-pigment” and impact in human retinal pigment epithelium. Trans Ophthalmol Soc UK. 1984;103:416–421.

49 Rozanowska M, Korytowski W, Rozanowski B, et al. Photoreactivity of aged human RPE melanosomes: a comparison with lipofuscin. Invest Ophthalmol Vis Sci. 2002;43:2088–2096.

50 Holz FG, Schutt F, Kopitz J, et al. Inhibition of lysosomal degradative functions in RPE cells by a retinoid component of lipofuscin. Invest Ophthalmol Vis Sci. 1999;40:737–743.

51 Schutt F, Bergmann M, Holz FG, et al. Isolation of intact lysosomes from human RPE cells and effects of A2-E on the integrity of the lysosomal and other cellular membranes. Graefes Arch Clin Exp Ophthalmol. 2002;240:983–988.

52 Lakkaraju A, Finnemann SC, Rodriguez-Boulan E. The lipofuscin fluorophore A2E perturbs cholesterol metabolism in retinal pigment epithelial cells. Proc Natl Acad Sci U S A. 2007;104:11026–11031.

53 Okubo A, Sameshima M, Unoki K, et al. Ultrastructural changes associated with accumulation of inclusion bodies in rat retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2000;41:4305–4312.

54 Wiegand RD, Koutz CA, Stinson AM, et al. Conservation of docosahexaenoic acid in rod outer segments of rat retina during n-3 and n-6 fatty acid deficiency. J Neurochem. 1991;57:1690–1699.

55 Stinson AM, Wiegand RD, Anderson RE. Recycling of docosahexaenoic acid in rat retinas during n-3 fatty acid deficiency. J Lipid Res. 1991;32:2009–2017.

56 Zhou J, Kim SR, Westlund BS, et al. Complement activation by bisretinoid constituents of RPE lipofuscin. Invest Ophthalmol Vis Sci. 2009;50:1392–1399.

57 Scholl HPN, Bellmann C, Dandekar SS, et al. Photopic and scotopic fine matrix mapping of retinal areas of increased fundus autofluorescence in patients with age related macular degeneration. Invest Ophthalmol Vis Sci. 2004;45:574–583.

58 Radu RA, Mata NL, Nusinowitz S, et al. Treatment with isotretinoin inhibits lipofuscin accumulation in a mouse model of recessive Stargardt’s macular degeneration. Proc Natl Acad Sci U S A. 2003;100:4742–4747.

59 Hogan MJ. Role of the retinal pigment epithelium in macular disease. Trans Am Acad Ophthalmol Otolaryngol. 1972;76:64–80.

60 Sarks SH. Ageing and degeneration in the macular region: a clinico-pathological study. Br J Ophthalmol. 1976;60:324–341.

61 Wolf-Schnurrbusch UEK, Enzmann V, Brinkmann CK, et al. Morphological changes in patients with geographic atrophy assessed with a novel spectral OCT-SLO combination. Invest Ophthalmol Vis Sci. 2008;49:3095–3099.

62 Fleckenstein M, Issa PC, Helb HM, et al. High-resolution spectral domain-OCT imaging in geographic atrophy associated with age-related macular degeneration. Invest Ophthalmol Vis Sci. 2008;49:4137–4144.

63 Kim YH, He S, Kase S, et al. Regulated secretion of complement factor H by RPE and its role in RPE migration. Graefes Arch Clin Exp Ophthalmol. 2009;247:651–659.

64 Fu QL, Li X, Yip HK, et al. Combined effect of brain-derived neurotrophic factor and LINGO-1 fusion protein on long-term survival of retinal ganglion cells in chronic glaucoma. Neuroscience. 2009;162:375–382.

65 Zhang K, Hopkins JJ, Heier JS, et al. Ciliary neurotrophic factor delivered by encapsulated cell intraocular implants for treatment of geographic atrophy in age-related macular degeneration. Proc Natl Acad Sci U S A. 2011;108:6241–6245.

66 Talcott KE, Ratnam K, Sundquist SM, et al. Longitudinal study of cone photoreceptors during retinal degeneration and in response to ciliary neurotrophic factor treatment. Invest Ophthalmol Vis Sci. 2011;52:2219–2226.

67 Beatty S, Koh H, Phil M, et al. The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv Ophthalmol. 2000;45:115–134.

68 Brennan LA, Kantorow M. Mitochondrial function and redox control in the aging eye: role of MsrA and other repair systems in cataract and macular degenerations. Exp Eye Res. 2009;88:195–203.

69 Kemp CM, Jacobson SG, Faulkner DJ. Two types of visual dysfunction in autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci. 1988;29:1235–1241.

70 Chuang EL, Sharp DM, Fitzke FW, et al. Retinal dysfunction in central serous retinopathy. Eye. 1987;1:20–25.