Chapter 64 Pathogenetic Mechanisms in Age-Related Macular Degeneration
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.
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.1–5 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.8–11
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
It is most likely that modification of the choroid in AMD is a response to alteration of neighboring tissues rather representing an intrinsic change, although the consequent reduction of metabolic supply to the outer retina may play a contributory role in the generating of disease. However, the possibility that choroidal changes occur independently of changes in other tissues cannot be excluded. This is not seen as a good target for therapy currently.
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.
If thickening of Bruch’s membrane and impedance of metabolic exchange and fluid movement is important to the pathogenesis of AMD, several therapeutic approaches might be considered. Reduction of the availability of the constituent proteins may slow the disease process, such as might be achieved with the chronic use of anti-inflammatory agents. Once thickening is established, mobilizing lipids or breaking down the oligomers may be achieved with the use of antibodies or possibly zinc buffers, as has been suggested in Alzheimer disease.40 There might be potential risks in rapid generation of monomers.41 Alternatively, the lipids might be mobilized. All these approaches would increase hydraulic conductivity and improve metabolic exchange between the RPE and choriocapillaris. In addition, it may induce an increase in choroidal circulation and in the density of fenestrae.
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.
If increased lipofuscin is important to genesis of GA, it would argue against dietary supplementation with vitamin A. Efforts are now underway to reduce the accumulation of lipofuscin therapeutically by restricting the availability of vitamin A to the retina. Initial results have shown the potential benefits of this approach in the ABCA4 knockout mouse.58 Agents that increase lysosomal activity or lower phagolysosomal pH might also be effective. On theoretical grounds light restriction might also be helpful.