Development and Progression of AMD
Maria Luz Cachulo, MD
Coimbra University Hospital - Coimbra, Portugal
Age-related macular degeneration (AMD) remains the leading cause of irreversible vision loss in the developed world among individuals older than 50 years(1,2,3).
Patients with intermediate to large soft/confluent drusen with or without hyper or hypopigmentation areas in the macula and no neovascular membrane or geographic atrophy are considered to have early age related maculopathy (ARM).
Geographic atrophy and wet age related degeneration (AMD) are more advanced forms of AMD that are more often associated with vision disability.
Although the treatment of AMD has evolved to include laser photocoagulation, photodynamic therapy, surgical macular translocation and antiangiogenic agents, treatment options for advanced AMD are limited.
Furthermore, the early form of ARM, albeit less devastating than the wet form, has even fewer viable treatment options. AMD is characterized by ageing changes at photoreceptors, retinal pigment epithelium (RPE), Bruch’s membrane and choroid(4,5,6).
Such ageing changes are considered to play a major role in development and progression of AMD. AMD is a bilateral disease and approximately 10 to 20 % of patients with early ARM will develop the wet form of AMD.
Whereas patients with early ARM in both eyes are at increased risk of developing either geographic atrophy or wet AMD, once wet AMD develops in one eye there is a higher risk of subsequent development of choroidal neovascularization (CNV) in the second eye(7,8).
Several funduscopic findings have been associated with increased risk of development of CNV in fellow eyes of patients with unilateral neovascular AMD.
Various reports have emphasized the devastating effects of CNV in the visual function of these patients(7,9,10). It is crucial to understand the natural history of the development of CNV or the process underlying the conversion from early ARM to late AMD.
A better understanding of the involved pathophysiologic process or the identification of biomarkers for the conversion would enhance our ability to diagnose and treat the wet AMD, to develop better therapies and eventually to prevent vision loss associated with the disease(11).
This review summarizes the various biomarkers of AMD and analyzes whether or not they may, one day, be exploited to determine risks of disease onset, measure progression of disease or even assess the effects of treatment of AMD.
Potential biomarkers are important to identify since some might be utilized to reflect the disease state of a particular patient and to individualize therapy.
Although studies have yielded promising results for nutrient and inflammatory biomarkers, these results have been inconsistent.
At present, the best available marker of AMD risk is single nucleotide polymorphisms (SNPs).
SNPs in complement factor H (CFH) and PLEKHA1/ARMS2/HtrA1 capture a substantial fraction of AMD risk and permit the identification of individuals at high risk of developing AMD.
Patients with AMD in the first eye are known to have high risk of bilateral involvement. In prospective studies in white populations, the annual rate of fellow eye involvement was reported to be around 6% to 9%(9,12,13).
The characterization of early ARM phenotypes is challenging. By combining different imaging modalities of the macula and correlating this information, we are better able to determine the presence of functional macular alterations in the fellow eye of patients with this disease.
To identify morphological and/or functional early markers of CNV development in fellow eyes of patients with exudative AMD in the other eye, our group performed a single center, prospective, observational, longitudinal two year study, enrolling patients with neovascular AMD in one eye (the non-study eye) and early ARM in the fellow eye (study eye) at risk for the development of CNV.
It was possible to identify a sequence of alterations at the chorioretinal interface during the development of CNV and progression of early ARM to neovascular AMD using different imaging methods simultaneously and at regular intervals to characterize markers or predictors of conversion to CNV.
It was also possible to correlate the evolution of the identified alterations with the development of CNV and to demonstrate the reliability and relative value of different clinical methodologies used to identify AMD disease progression.
AMD is a complex disease caused by the combination of genetic predisposition and environmental factors.
The prevalence of AMD increases with age.
The adverse effect of smoking is well established.
Genetic predisposition has been demonstrated by familial aggregation studies and twin studies. Using genome linkage scan and association studies, multiple potentially causative genes have been identified.
The chromosomes most commonly implicated are 1q25-31 and 10q26.
In particular, variants in the gene for the complement factor H (CFH) and the genes PLEKHA1/LOC387715/HTRA1, Factor B (BF) and complement component 2 (C2) have been implicated as major risk or protective factors for the development of AMD.
There have been some advances in the treatment of this condition; however, a complete cure remains remote but hopeful.
Understanding the causative environmental and genetic interactions will facilitate the development of future preventive methods and treatments.
AMD-associated SNPs may eventually serve to identify at-risk individuals and separate AMD patients into homogenous groups for preventive and therapeutic studies.
These genetic biomarkers also serve as powerful tools in the elucidation of the underlying etiology of AMD.
We limit our discussion predominantly to those SNPs that are consistently associated with AMD in multiple case–control studies and, thus, have the strongest potential to serve as genetic biomarkers.
In early 2005, four groups reported independently that common variants in the gene encoding CFH confer major susceptibility to AMD(14,15,16,17).
A year later, at-risk and protective haplotypes were identified in two other genes encoding complement proteins, BF and C2(18).
These markers include:
• Complement factor H (Chromosome 1q32, Entrez Gene ID 3075) Polymorphisms in the complement factor H gene (CFH) are associated with a significantly increased risk for the development of age-related macular degeneration (AMD). The most documented risk-conferring single-nucleotide polymorphism results in a tyrosine-to-histidine substitution at position 402 (Y402H) of the CFH protein.
• Complement factor B (chromosome 6p21.3, Entrez Gene ID 629)
• Complement Component 2 (chromosome 6p21.3, Entrez Gene ID 717)
• PLEKHA1/ARMS2/HtrA1 (chromosome 10q26, Entrez Gene ID 387715/5654/59338)
• Excision repair cross-complementing rodent repair deficiency, complementation group 6 (chromosome 10q11.23, Entrez Gene ID 2074)
• VEGF (chromosome 6p12, Entrez Gene ID 7422)
Various immunological molecules and inflammatory mediators have been identified at the site of AMD lesions(19).
Pro inflammatory cytokines are released from immune cells during an inflammatory response. These cytokines mediate inflammatory effects.
Elevated levels of AMD biomarkers, e.g., markers of systemic inflammation: C-Reactive Protein (CRP), Interleukin 6 (IL-6), Tumor Necrosis Factor alpha-Receptor II (TNF α-RII), Intercellular Adhesion Molecule (IcAM), Vascular Cell Adhesion Molecule (VCAM ); lipid biomarkers: Apolipoprotein B (ApLP B) or Lipoprotein (a) (Lp-a); homocysteine (Hc); and fibrinogen (Fbg), are predictive of development and progression of AMD.
CRP may be involved in the pathogenesis of AMD through chronic inflammation leading to oxidative damage, endothelial dysfunction, drusen development or the degeneration of Bruch’s membrane(20).
CRP may also have a direct role in AMD development through its ability to induce complement activation.
Several studies performed by Seddon and colleagues(21,22) showed an association between CRP levels and AMD and elevated CRP levels may serve as a marker for AMD progression.
However, Hogg and colleagues found no significant association between CRP plasma levels and AMD or AMD progression in both case–control and prospective studies(23).
IL-6 is a marker for systemic inflammation, such as acute pancreatitis, chronic arthritis and geriatric syndromes.
Seddon and colleagues found a correlation between the level of IL-6 and chances of AMD progression(22).
This study shows that elevated IL-6 levels may serve as a marker for progression of AMD.
However, Klein and colleagues found no significant association between IL-6 plasma levels and AMD or AMD progression(24).
Fibrinogen is an established biomarker of acute and chronic inflammation(25,26).
Lip and colleagues found elevated levels of plasma fibrinogen in AMD cases compared with controls(25).
A case–control analysis from the large Blue Mountains Eye Study in Australia detected significantly elevated plasma fibrinogen levels in AMD patients compared with controls
(p < 0.05)(27).
In another study using patients recruited from the Muenster Aging and Retina Study population in Münster (Germany), no association between plasma fibrinogen levels and AMD was showed(28).
Vascular endothelial growth factor
There is strong evidence suggesting that VEGF is a good candidate AMD biomarker.
VEGF is also elevated in RPE cells of AMD patients and in the AMD patient post-mortem eyes(25).
This evidence points to a role for elevated VEGF levels in AMD.
A study by Lip found increased plasma VEGF levels in 78 AMD patients compared with 25 age-matched controls (p = 0.0196)(25) with no significant difference found between dry and wet AMD cases in a comparison of plasma VEGF values(25).
Tsai and colleagues found increased plasma VEGF levels in 77 AMD patients compared with 42 controls (p < 0.001)(30) and significantly higher plasma VEGF levels in wet AMD patients compared with dry AMD patients (p < 0.05)(30).
These results suggest that high VEGF levels may play a role in predisposing individuals to neovascular AMD.
Additional studies must be undertaken to establish its role as a biomarker for this disease.
Functional multimodal imaging of the macula
Multimodal imaging of the macula provides improved visualization of the macular alterations seen in early ARM.
• Color Fundus Imaging
• Fluorescein Angiography
• Indocianine Green Angiography
• Fundus Autofluorescence
• Optical Coherence Tomography
• Retinal Leakage Analyzer
Color fundus imaging
Grading of stereoscopic color photographs collected during 5 years of follow-up in more than 3000 AREDS participants were used to develop a detailed grading scale(29).
A simplified scale was developed based on the presence or absence of 2 features characteristic of AMD that were easily identified clinically (drusen size and pigment abnormalities) and highly associated with the development of advanced AMD, especially when the status of both eyes was considered.
Color fundus imaging and AREDS photographic grading helps us to make an AMD severity scale that would provide clinically useful risk categories for the development of advanced AMD in persons with earlier stages of AMD. Defining risk factors and counting them provides a convenient way to define risk categories.
These categories may be useful in discussing with patients their risk of progression to vision-threatening AMD and in developing inclusion criteria for clinical studies of AMD.
The simplest scheme counts the presence of at least 1 large drusen (diameter greater than or equal to that of a large vein at the disc margin) and the presence of any pigment abnormality as 1 risk factor each and sums their presence across both eyes when both are free of advanced AMD.
One risk factor is assigned to patients who have no large drusen in either eye but intermediate-sized drusen (diameter ≥ one half that of a large drusen) in both eyes (Table 1).
The 5-year risk of advanced AMD using this scale increases in the approximate sequence of: 0 factors, 0.5%; 1 factor, 3%; 2 factors, 12%; 3 factors, 25%; and 4 factors, 50%.
This scale may be useful clinically, either with ophthalmoscopy or slit lamp biomicroscopy, or in less optimal photographs using less complex grading procedures than those used in AREDS.
For clinical purposes, as the number of risk factors increases from 0 to 4, the 5-year risk of advanced AMD in at least one eye increases in the easily remembered approximate sequence of 0.5%, 3%, 12%, 25%, and 50%.
Extensive drusen area, as seen on fundus photograph, is the greatest risk factor for the progression of AMD(31).
When examiners are asked to mentally aggregate the amount of drusen occupying a given macular subfield(32), as in the International System, where drusen areas were estimated to within 10% to 25% or 25% to 50%, and so on(33), these semi-quantitative estimates prove difficult for human observers.
Clearly, there is a need to implement more precise techniques to improve the quality of data being gathered in clinical trials and epidemiological studies.
Fluorescein angiography is the standard examination for diagnosis and classification of conversion from early ARM to exudative AMD.
Indocyanine green angiography
In AMD, digital indocyanine green (ICG) angiography is a technique that may enable improved imaging of occult CNV(34).
Hot spots are observed frequently in retinal angiomatous proliferation (RAP), polypoidal choroidal vasculopathy and focal occult CNV(35).
ICG angiography may demonstrate neovascular connections between the choroid and retina, in the form of anastomoses.
The presence of late ICG hot-spots may be an important biomarker in the development of neovascular membrane.
They appeared not only when a RAP developed but in other forms of CNV as well.
The hot spots may appear not only when conversion occurs but even before conversion suggesting that this finding may be associated to future conversion in some subtypes of neovascular AMD.
Fundus autofluorescence (FAF) imaging is a non-invasive method for examination and follow-up patients with macular disease that supplies additional information to that obtained using fundus photography and fluorescence angiography(36).
It is based on the detection of fundus autofluorescence, which is associated primarily with the lipofuscin content of retinal pigment epithelium(36).
Areas of increased FAF may correlate to areas of hyperpigmentation, yellowish soft drusen, or normal fundus appearance. However some drusen are related to areas of decreased FAF.
Areas covered with reticular drusen usually show a reticular FAF pattern with small areas of decreased FAF surrounded by normal FAF.
The areas of hypopigmentation on fundus photographs may be associated with decreased FAF suggestive of absence of retinal pigment epithelium or degeneration.
There are also FAF images with no or minimal changes in patients with funduscopically visible drusen, and vice versa(36). Smith et al(37) have retrospectively studied the relationship between reticular patterns of autofluorescence and choroidal neovascularization.
They showed that reticular hyperfluorescence appears to be a marker for reticular pseudodrusen, which are known to be frequently associated with CNV development.
Other studies have suggested a higher risk of CNV development in eyes showing a patchy pattern of autofluorescence(38,39).
In our series, among the eyes which developed CNV in study eye during follow-up no clear pattern of association could be established to their predominant FAF pattern at baseline.
In this study the presence of a pattern of minimal change in fundus autofluorescence at baseline in eyes with neovascular AMD in the fellow eye appears to indicate a lower risk of developing CNV at two years.
Eyes with fewer abnormalities in FAF pattern at baseline appear to have a slower progression to neovascular AMD in patients with unilateral neovascular AMD.
The relatively small population studied makes necessary further studies with a more prolonged follow-up in order to clarify the relationship between fundus autofluorescence and the risk for progression to neovascular AMD, which was not apparent in this two-year study.
Fundus autofluorescence imaging can be even more effective in evaluating ARM progression when it is combined with Optical Coherence Tomography, as with Spectralis HRA+OCT (Heidelberg Engineering).
With the simultaneous recordings of high-resolution OCT, it is possible to evaluate corresponding morphological substrates like underlying microstructural changes in the retina and the retinal pigment epithelium(40,41).
Stratus OCT showed increased retinal thickness from intraretinal fluid accumulation in every eye that developed CNV.
This increase in fluid accumulation, however, could not be identified before conversion, in any of the eyes.
OCT findings confirm that this method is a good indicator of the presence of active CNV and therefore can be used reliably to monitor CNV treatments(42).
Retinal leakage analyser
The retinal leakage analyzer showed leakage of fluorescein into the vitreous associated with the area of CNV, confirming the observations of Merin et al(43), using vitreous fluorometry.
Leakage location correlated well with the CNV site.
Furthermore, it showed, before conversion, sites of abnormal breakdown of the blood-retinal barrier in 13 of the 17 eyes (76%) that converted to exudative AMD.
An alteration of the blood-retinal barrier appears, in this study, to be a particularly promising predictor of conversion from dry to wet AMD.
There were also sites of alteration of the blood-retinal barrier in 23% of the study eyes that did not convert during the two-year period of the study.
It is now of particular interest to follow closely the future evolution of these eyes to see if these eyes with early ARM and localized alterations of the blood-retinal barrier demonstrated by the retinal leakage analyzer are the first to develop CNV.
We believe that by examining the natural history of eyes at high risk of converting from early ARM to exudative AMD designing a different imaging methodologies of these will help us to obtain a better understanding of what happens on the retina and in the chorio-retinal interface.
Our current research is focusing on understanding the alteration in the blood-retinal barrier at the RPE level.
Other studies are looking at photoreceptor function to determine how much of it is altered and damaged before, after, or simultaneously with disease.
We believe that studies such as these can help us to understand the natural history of age-related maculopathy.