Macular Degeneration 13 Contact Hours

ABSTRACT

Macular degeneration is the most common cause of severe vision loss in the elderly in the developed world and is the cause of one third of all forms of untreatable loss of vision. It is an age related painless and irreversible degenerating condition of the eyes associated with the damage and ultimate death of photoreceptors. In most cases the patient’s central vision is lost but the peripheral vision remains intact. It is estimated that 9 million older Americans have one form or another of macular degeneration and that about 1.75 million suffer from an advanced stage of the condition. This article discusses the two presentations of macular degeneration and guides health care professionals on how to manage patients that present with this condition.

LEARNING OBJECTIVES

1. Discuss the epidemiology of Macular Degeneration 2. Discuss the effects of macular degeneration on the patient 3. Review the two types of macular degeneration 4. List and review the causes of macular degeneration 5. Identify the factors that increase a patients risk of developing macular degeneration 6. List common drugs that may contribute to the risk of macular degeneration 7. Describe the pathophysiology of macular degeneration 8. Identify the signs and symptoms of macular degeneration 9. Screen and identify patients that require further investigation by an ophthalmologist 10. Review the findings of an ophthalmoscope indicative of macular degeneration 11. Review the prognosis of the condition 12. List foods that are useful for the prevention of macular degeneration 13. Identify supplements and herbals that may slow disease progression 14. Review pharmacological agents for the management of macular degeneration 15. Explore treatment options available to patients 16. Recognize lifestyle measures that can be taken to prevent macular degeneration or slow its

progress 17. Review vision rehabilitation techniques 18. Identify aims of treatment and develop an individualized treatment plan for the patient 19. Evaluate points for discussion with the family or caregivers 20. Summarize the findings of the age related eye disease study (AREDS)

Introduction

Macular degeneration is the leading cause of irreversible visual impairment in the elderly [1]. It is a

degenerative disease of the central part of the retina, known as the macula leading to a loss of central

vision which is essential for most of daily activities [2]. The condition is associated with a loss of visual

acuity due to degeneration of the choriocapillaris, retinal pigment epithelium (RPE), and photoreceptors

usually beginning with Drusen and pigmentary changes in the Bruch’s membrane [1,3].

Macular degeneration may develop from the early stage to the late stage which occurs in the non-

neovascular (non-exudative or dry) form or neovascular (exudative or wet) form, usually beginning with

the former and progressing to the latter [3]. It is important to note that each eye can be at a different

stage of degeneration; however, the development of an advanced stage of the condition in one eye

accelerates the neovascularization in the fellow eye [1].

There is increasing evidence to show that inflammation and the immune system play a vital role in the

development of the disease alongside smoking, hypertension, obesity and poor dietary habits [4].

The results from the Age-Related Eye Disease Study demonstrated that supplementation with high dose

anti-oxidants, and zinc were useful in patients with an intermediate form of dry AMD in one or both

eyes or with an advance form of dry AMD in vision loss due to AMD in one eye only [4]. The current

therapy for the exudative form of AMD includes intravitreal injections of endothelial growth factor

inhibitors [4]. While there is no cure for AMD, good nutrition, supplementation and improved physical

activity have been shown to slow down disease progression [1].

Epidemiology

Macular degeneration affects 30-50million people worldwide and is the leading cause of irreversible

blindness in developed countries in people aged 50 years or above [1,2]. Over 1.75 million persons were

reported with AMD in the year 2000 and it is thought that the incidence will increase to almost 3 million

in 2020 [2].

The prevalence of AMD has been shown to increase exponentially with every decade after 50 years [4].

In many Western countries the prevalence of AMD in individuals above the age of 55 is 1.6% and

increases with age to about 13% in persons above the age of 84 [2].

The loss of central visual acuity leads to reduction of activities of daily living as well as mobility and

increased the risk of falls, fractures and depression in the elderly [5].

Types of macular degeneration

The disease begins as an early stage disease and progresses through an intermediate stage into an

advanced stage associated with the loss of vision. Early stages of AMD are characterized by a macula

that has yellowish colored subretinal deposits called Drusen and/or increased pigment. Patients with

early AMD have stable visual acuity for many years and loss of vision is gradual [5]. The Age Related Eye

Disease Study (AREDS) used the following stages of AMD as follows:

Category Name Description

1 No AMD No or few small drusen:<63 µm in diameter

2 Early AMD A combination of multiple small drusen, few intermediate drusen (63 to 124 µm in diameter), or RPE abnormalities.

3 Intermediate Extensive intermediate drusen, at least one large drusen (125 µm in diameter), or geographic atrophy not involving the center of the fovea.

4 Advanced Characterized by geographic atrophy or neovascular maculopathy

Source: [6]

In its advanced stages, AMD causes a significant loss of vision and occurs in one of two morphological

types: dry AMD or wet AMD.

Dry AMD

The dry form of AMD is also known as non-exudative, non-neovascular or atrophic AMD. This is the

commonest form of AMD, seen in about 90% of cases [7]. Vision loss in dry AMD is low, gradual and

usually associated with moderate visual impairment as well as functional limitations including

fluctuating vision, difficulty reading, and limited vision at night or under conditions of reduced

illumination. The macula shows areas of depigmentation upon examination [5]. The lesions may remain

asymptomatic for a long time and the development of atrophy that leads to geographic atrophy may

also be delayed in dry AMD [7].

Geographic atrophy (GA) is an advanced stage of dry AMD in which the fovea is often initially spared

from degeneration [8]. It is characterized by loss of photoreceptors, RPE and the choriocapillaris within

the macula. Geographic atrophy can manifest as single or multiple areas measuring 175µm or more or

RPE loss or depigmentation with associated choriocapillaris atrophy [3]. More often than not GA

appears as a small, singular parafoveal lesion while fewer lesions may appear subfoveal or multifocal at

initial detection.[9] Since the RPE is essential in the functioning and survival of retinal photoreceptors,

geographic atrophy is associated with visual atrophy and the moderate to severe loss of vision [7,9].

On optical coherence tomography (OCT), lesions of GA, are seen as a loss of the corresponding retinal

layers. Fundus autoflourescence show these as a loss of normal autoflourescence [3].

This form of macular degeneration progresses slowly over time and is responsible for 20% of cases of

legal blindness associated with macular degeneration [7, 9]. Currently there is no effective treatment for

geographic atrophy but a number of potential therapies are under investigation [7].

GA lesions have been classified into 4 primary phenotypes by the Fundus Autoflourescence in Age-

Related Macular Degeneration study group:

focal: single or individual small spots of increased fundus autoflourescence at the margin of the

atrophic patch

banded: continuous stippled-band or ring-shaped zone of increased FAF surrounding the entire

atrophic area

patchy: large patchy areas of increased FAF outside the atrophic area

diffuse: increased FAF at the margin of the atrophic area and beyond [3].

Wet AMD

Wet AMD is also referred to as exudative or neovascular AMD. It is less common than the dry form,

accounting for about 10% of all cases of macular degeneration [7]. However, its presence usually

indicates a more advanced stage of the disease and it is associated with rapid distortion and a sudden

loss of central vision over a period of weeks to months [2]. A number of studies have demonstrated that

eyes of patients with wet AMD have two times the expected prevalence of vitreomacular adhesion and

are less likely to have a posterior vitreous detachment [12]. Fluid and exudate may accumulate

underneath the retina in patients with neovascular AMD resulting in severe macular edema [5, 7]. If left

untreated, the neovascular membrane forms a big scar in the macular area resulting in a sudden

decrease in central vision [7]. Wet AMD present as one of the following:

Serous and/or hemorrhagic detachment of the sensory retina or RPE

Retinal hard exudates (a secondary phenomenon resulting from chronic leakage from any

source)

Subretinal and sub-RPE fibrovascular proliferation

Disciform scar

Choroidal neovascularization [6].

Choroidal neovascularization (CNV) is an advanced stage of wet AMD. It can be further subdivided in

two types:

subretinal (type 1) or

sub-retinal pigment epithelium (type 2) space [3]

CNV can lead to the development of polypoidal choroidal vasculopathy (PCV) which is a condition that is

associated with grape like clusters of the new vessels [3].

The condition progresses from drusen to the development of choroidal neovascularization (CNV)

whereby the choriocapillaris cross the Bruch’s Membrane and spread laterally within the planes of these

lesions [12]. The location of the neovascular lesion is described based on its proximity to the center of

the fovea avascular zone (FAZ) and can be divided as follows:

Extrafoveal lesions are found ≥ 200 μm and < 2500 μm from the center of the FAZ

Juxtafoveal lesions are those that occur between 1–199 μm from the center of the FAZ

Subfoveal lesions are under the center of the FAZ [1].

Etiology

The etiology of AMD is multifactorial and involves an interplay of genetic, environmental, metabolic and

functional factors including aging, family history, smoking, high blood pressure, obesity,

hypercholesterolemia and arteriosclerosis [4,13]. As the name suggests, the underlying cause of macular

degeneration is simply the deterioration of the central portion of the retina known as the macula. This

results in the loss of central vision, while the peripheral vision remains intact [5]. Central vision is the

part of vision that is required for identifying letters, numbers, facial features, border surfaces, angles

and colors, reading, driving, watching television, and many other activities that require “high-definition”

vision [5, 14]. Since the patients peripheral vision is not affected, an individual with AMD does not

normally require canes or guide dogs.

Risk Factors for Macular Degeneration

While numerous risk factors for age related macular degeneration have been identified, their

association to condition is variable and the evidence for some is poor. In most reviews, age, smoking,

family history and cataract surgery seem to be the obvious risk factors [15]. Risk factors range from

demographic, top nutritional, lifestyle, medical, environmental and ocular features [15]. As new and

relatively effective treatments become available, the importance of early identification of patients with

risk factors becomes even more prominent [15]. Furthermore, developing a reliable risk assessment

model has become of greater importance than ever [16]. Such risk score models would assist in

targeting high risk individuals for lifestyle changes with the aim of reducing the risk of AMP progression,

allowing a differential diagnosis of AMD and its subtypes, identifying patients that can be included in

clinical trials and selecting therapies [17].

An ideal AMD risk assessment model should include the identification of those individuals with early

AMD who are at the highest risk of progression to the advanced stage of the condition leading to

blindness. Furthermore, it should be able to predict when progression may occur. The model would take

into accounts patient demographics, environmental factors, phenotypic risk factors and genetic factors.

Recently, a number of attempts at developing a risk assessment model and validating have been made.

One model demonstrated good performance on measures of discrimination, calibration and overall

performance [16]. A more recent model has been proposed to predict the progression to advanced

stages of AMD in 2 independent cohorts [17]. Another model specifically assessed a patient’s risk of

developing geographic atrophy, the first study of its kind [18].

Individuals identified as high risk patients should be advised to seek prompt medical advice if they

develop visual symptoms of distortion or reduced vision [19].

Older age

Old age has been identified as the biggest risk factor for AMD [12, 15, 20]. The prevalence of AMD

increases with age and has been known to increase exponentially with every decade after 50 years of

age [4, 5, 21]. The melanin pigment that protects the retina from radiation diminishes with age [22].

Many of the pathological processes that are associated with AMD are a normal process of aging.

Smoking

Cigarette smoking is one of the biggest preventable factors of AMD. It has been shown to increase a

patient’s risk of developing AMD by up to three times that in non-smokers [2, 5, 21]. Individuals who

smoke one pack a day or more have a higher risk of developing AMD than non-smokers or individuals

that have given up smoking more than 10 years ago. The AREDS showed that smoking was linked to

three out of five stages of macular degeneration.

A number of possible mechanisms for this effect have been proposed. One school of thought is that

smoking diminishes the melanin pigment that protects the retina from radiation.

Other studies have evaluated the role of nicotine from cigarette smoke in the angiogenesis, one of the

underlying pathological mechanisms in AMD [23]. In this regard, nicotine imitates the effect of other

angiogenic growth factors by promoting endothelial cell migration, proliferation, survival, tube

formation and nitric oxide(NO) production in vitro. This effect has been demonstrated at the average

tissue and plasma concentrations of even light smokers. The nicotine has been shown to stimulate

nicotinic acetylcholine receptors (nAChRs), primarily the α7 homomeric type, on endothelial cells to

induce angiogenic processes. Furthermore, studies have shown that these receptors have a synergistic

effect with angiogenic growth factor receptors at both the phosphoproteomic and genomic levels [23].

A number of studies have supported the hypothesis that endothelial nAChRs may promote choroidal

neovascularization as well as retinal edema, both of which are features of AMD [23].

Furthermore, smoking has been identified as a modifier for genetic risk of AMD. The genes that are

involved in the pathology of AMD are discussed later in this paper. One study, while not achieving

associations with statistical significance, was able to identify novel regions with potential gene-smoking

interactions at the major risk loci [24].

Additionally, cigarette smoke is a risk factor for atherosclerosis, which is in turn a recognized risk factor

for the development of AMD.

Family History

Most studies have consistently related family history to an increased risk of AMD, an association that

may be explained through various genetic findings [12, 23]. Only one systemic review performed on two

case controlled studies was not able to demonstrate an association between family history and the risk

of developing AMD [15]. The two major loci associated with AMD are Iq32 and 10q36 [3]. One of the

most widely studied and recognized genetic association is with the rs1061170 variant(Y402H) of the

complement factor H gene, CFH. This variant has been shown to lead to affect the alternative pathway

of complement regulation and increases a patient’s risk of developing AMD [19]. The second high risk

locus ARMS2 has been particularly found to contribute significantly to the development of the disease

[25].

One study examined the risk of developing AMD on the basis of family history using two approaches.

The first part of the study was a case control study of reported family history, while the second part

examined the siblings of patients with AMD. All data was adjusted for age, smoking habits, history and

genotype. The study concluded that a patient with a first degree relative suffering from AMD is at a

significantly higher risk of developing the condition [26].

Female Gender

The evidence for the female gender as a factor is contradictory. While a recent meta-analysis showed no

difference in the risk of developing AMD between two genders, other studies have demonstrated that

females are indeed at a higher risk of developing this condition [15]. One study in particular showed that

the incidence of AMD is three times greater in post-menopausal women than in men of a similar age

[22]. Furthermore it is specifically the exudative form of the disease, which is also the more severe form

that is more prevalent in women than in men [5]. Interestingly, there was one study that showed male

gender to be a higher risk factor for the development of AMD [20].

A systemic review of population based studies was conducted to gain a better understanding of the

demographic distribution of patients with AMD. Studies of patients with geographic atrophy,

neovascular AMD and late AMD were used dating as far back as 1950. There was some evidence to

suggest that women were at a higher risk of developing AMD compared to men, particular the

neovascular type of the condition [27].

Obesity

While overweight and obesity are linked to an increased risk of late AMD the association is not as strong

as that with age [12, 24].

Sun exposure

To date epidemiologists have not agreed on whether high exposure to sunlight increases a patient’s risk

of developing AMD. A systemic review conducted to analyze the data available shows that individuals

with higher amounts of sunlight exposure are at a statistically significant increased risk of AMD [28]. One

author concludes that patients who spend more than 5 hours a day in the sun are twice as likely to have

AMD as those with less than 2 hours a day of sun exposure [22].

Atherosclerosis

AMD may share some features with atherosclerotic plaques and in fact atherosclerosis may present as a

risk factor for the development of AMD. The lipoprotein deposition in the vessel walls of atherosclerosis

has largely influenced the thinking on how lipids contribute to AMD [12]. The details of this association

and the effect of lipids in AMD are discussed under the pathophysiology of this condition.

A number of studies have been conducted to examine the relationship between various cardiovascular

diseases to the increased risk of AMD [29, 30].

The Beaver Dam Studies were conducted to examine the relationship of atherosclerosis to the 10-year

cumulative incidence of AMD [29]. Specifically, the role of intima-media thickness, plaque in carotid

artery, angina, myocardial infarction and stroke were measured against an outcome of AMD. The study

found that the intima media thickness and plaques of the carotid artery has a weak association with the

development of AMD, one that was independent of systemic and genetic risk factors. No association

was found with angina, myocardial infarction, and stroke. The study was not able to determine whether

the carotid intima media thickness is a risk indicator of processes affecting Bruch's membrane and the

retinal pigment epithelium, or in fact a measure of atherosclerosis affecting susceptibility to AMD [29].

When examining specifically the effect of a myocardial infarction on the risk of developing early AMD,

researchers found a significantly higher occurrence of the condition in patients that had experienced a

myocardial infarction [31].

In the reverse scenario, conflicting evidence has been demonstrated regarding the risk of cardiovascular

disease in patients with AMR. One study conducted to examine the effect of AMD on the incidence of

stroke demonstrated that persons with AMD are at a higher risk of both cerebral infarction and

intracerebral hemorrhage. It is postulated that this may be due to the sharing of common

pathophysiological pathways between AMD and stroke [32]. On the contrary a study published a few

months earlier was not able to associate AMD with an increased risk of coronary heart disease or

cardiovascular disease [33].

Hypertension

There is a small amount of research hinting that hypertension is a presenting risk factor for AMD. In one

study that examined the association of AMD with long term average blood pressure parameters

including the pulse pressure, researchers linked hypertension to AMD.

After adjusting for sex, age, educational level, smoking habits, body mass index, plasma HDL and LDL

cholesterol, genetic polymorphisms in CFH Y402H, APOE2, APOE4, and ARMS2 A69S the study

demonstrated that high PP may increase a patients risk of developing AMD [34].

Diabetes

When the relationship between the ten year incidence of age related macular degeneration and a

patients diabetic retinopathy classification was measured, it was found that elderly patients with

nonproliferative diabetic retinopathy (NPDR), and proliferative diabetic retinopathy (PDR) seemed to be

at a higher risk of AMD compared to those without diabetes mellitus or diabetic retinopathy [35].

Lung Function

The correlation between lung function and disease with the development of AMD has demonstrated

conflicting information to date. While some studies have reported a loose correlation, others have

demonstrated no correlation. The Atherosclerosis Risk in Communities Study (ARIC) examined the cross-

sectional relationship between lung disease or function and early AMD in a large population-based

sample of white and African Americans in the United States using a combination of spirometry and

questionnaires. The Wisconsin grading protocol was used to grade AMD photographs from the fundus.

This study concluded that patients with poor lung function or lung diseases such as asthma were not at a

higher risk of developing AMD [36].

Infectious agents

There are very few and small studies that investigate the effect of infectious agents on the development

of AMD. In fact, there are no recent papers that discuss any link between the two.

Polypharmacy

Aspirin is one of the most widely used medications worldwide and commonly prescribed for the

secondary prevention of myocardial infarction, stroke and other recurrent cardiovascular conditions.

One study found that regular use of aspirin increased the risk of AMD, particularly the exudative form

[37]. This was further investigated in a study that examined the relationship between the regular use of

aspirin and the 15 year incidence of AMD, particularly the neovascular type. From this study, and

correcting for cardiovascular disease and smoking, the research team concluded that regular aspirin use

doubled a patient’s risk of developing AMD over a period of 15 years [38].

Alcohol

While smoking is a known risk factor for AMD, little is known about the association of alcohol

consumption with AMD [39]. In fact, it is postulated to have both positive and negative effects on the

development of AMD. An Australian based study was set up to determine whether there was in fact

such an association and what it entailed. The alcohol consumption of participants was determined using

a structure interview and AMD was assessed using digital macula photographs. After adjusting for age,

sex, smoking habits, country of birth, education level, physical activity and energy intake from food, the

study showed that patients who consumed more than 20g of alcohol a day had a 20% higher chance of

developing early AMD than those who did not consume any alcohol. The authors concluded that there

was a modest association between the consumption of alcohol and the risk of developing AMD [39].

An earlier meta-analysis had already shown that the consumption of more than 30g of alcohol per day

on a regular basis increased the risk of early AMD by 47-67% [40]. Other studies suggested that the

quantity as well as the type of alcohol consumed affect the risk of AMD differently [39].

Ethnicity

AMD seems to be more prevalent in non-Hispanic whites than in blacks or Mexican Americans [5]. There

is one study, based in Oklahoma, demonstrating the prevalence of AMD in an Indian population. AMD

was measured using retinal photographs and the condition was graded by the University of Wisconsin

Ocular Epidemiology Reading Center using the Wisconsin Age-Related Maculopathy Grading system.

Only photographs that were considered gradable were considered for the study. The study, a first of its

type specific for Oklahoma individuals, revealed that this population has a relatively high prevalence of

AMD as compared to other ethnic groups. However, the authors propose further research to confirm

these findings [41].

Iris Color

A darker iris color seems to have a protective effect in the development of AMD, however the difference

between different iris colors does not seem to be significant [15].

Features such as age, current smoking status, cataract surgery and family history are all risk factors that

are easily identified during a patient evaluation. Other significant factors that may not be so obvious

such as BMI, hypertension, cardiovascular disease and diabetes are easily identifiable through simple

testing.

Hypothyroidism

A small body of literature has shown a possible relationship between AMD and hypothyroidism [42].

One study conducted on patients over the age of 50 used self- reported data on thyroid conditions as

well as AMD. The data were analyzed to determine whether there was indeed any correlation between

the two conditions. The results showed that hypothyroidism may be a risk factor for AMD, although the

researchers were not able to establish a clear connection. Further studies are still warranted in this area

[42].

C-Reactive Protein

A number of studies have demonstrated that elevated C-reactive protein levels are associated with a

high risk of AMD. The mechanism behind this association is yet to be discovered [43].

Pathophysiology

The retina is part of the central nervous system that captures and converts light energy into an

electrochemical signal for transmission to the brain through the optic nerve [12]. It contains more than

50 different cell types that can be divided into at least 5 cell classes. Each of these cells perform unique

functions that ultimately provide the visual centers in the brain information to form and perceive visual

images [12].

A high metabolic rate and complex membrane structure is required for the retina to undertake its role

[44]. In performing its role, it is continuously exposed to a photo-oxidative environment [44]. The outer

segments of the photoreceptors are enriched in the highly photosensitive docosahexaenoic acid and

other polyunsaturated fatty acids that are relatively easily oxidized. In the retina are mechanisms that

allow the uptake of lipids and metabolize them to adapt to this photo-oxidative environment [44]. In

particular it takes up circulating LDL to nourish all the cellular layers with lipids. Furthermore it is able to

generate cholesterol molecules [44].

The macula is a specialized structure of retina that is responsible for exceptional color vision and visual

acuity. It is the most sensitive area of the ocular fundus that is rich in cone photoreceptors and ganglion

cells [14]. AMD is a disorder that affects the macula and hence a patients visual acuity.

The pathophysiology of AMD has been studied using molecular dissections of histopathologic specimens

and genetic linkage analyses [1]. While genetic studies indicate an inflammatory component, clinical

trials point towards oxidative stress, and physiological studies indicate a role of ischemia and hypoxia

[45, 46]. It is possible that all or more than one of these mechanisms contribute to the disease at

different stages. Understanding them is important in developing drugs to target the different pathways

and hence stop the loss of vision.

The disease involves 4 distinct layers of the outer retina: the photoreceptors, retinal pigment epithelium

(RPE), the Bruch’s membrane, and the choriocapillaris [47]. The hallmarks of the condition are the

presence of Drusen and the inner collagenous layer of the Bruchs membrane [7]. Degenerative changes

have been observed in the photoreceptor layer (rod and cones), the retinal pigment epithelium (RPE)

and choriocapillaris [8, 45].

Photoreceptors

Photoreceptors, found in the posterior retina, are neurons that transduce light, converting light into a

signal that can be transmitted through the neuron. Photoreceptors can be classified into rods or cones

depending upon their shape and distribution across the retina. Furthermore, they have variable

sensitivity, with rods being able to be triggered by a very small number of photons. Brighter light usually

triggers the signal in the cones. Cones cells can be further divided into the red, green and blue,

depending upon the wavelength of light that they respond to [8].

The density of photoreceptors varies considerably across the 1,000-mm2 retina. The fovea that is about

0.8mm in diameter has a cone density approaching 200,000/mm2. The parafovea is 2–4 mm away from

the foveal center and has a similar density of rods. Surrounding the fovea is a pile-up of inner retinal

neurons [12].

Photoreceptors consume the highest oxygen per gram of tissue than any other body cell and are

therefore densely packed with mitochondria. The aging retina has been seen to accumulate

mitochondrial DNA deletions and cytochrome c oxidase-deficient cones, primarily in the fovea, a process

that is thought to contribute to degeneration of the macula in aging [8].

Retinal Pigment Epithelium (RPE)

The RPE is a layer of nurse cells that functions to maintain the health of photoreceptors [47]. Over 100

million rod and cone photoreceptors are located at the outer surface of the retinal sheet and supported

by the RPE [12]. On a daily basis, the retinal photoreceptors are exposed to light stimuli, oxygen, and

lipid peroxidation products derived from the photoreceptor subjecting them to oxidative stress [48-53].

In the normal healthy eye, the photoreceptors and underlying pigment epithelium are supplied with

essential nutrients to combat this oxidative stress and maintain good vision. Free radicals from light and

oxygen attack the rod and cone cell membranes. Before any disease process can be initiatied, the RPE

cells need to be polarized [8].

Specifically, the RPE, a polarized monolayer provides phagocytic activity towards the photoreceptor

outer segment tips, Vitamin A metabolism, maintenance of retinal attachment and coordination of

cytokine mediated immune protection [12, 14, 54]. The functions of the RPE can be broken down as

follows:

Provides nutrition to the outer segments of the photoreceptors

Forms an outer blood-retinal barrier that prevent diffusion and transport of material from the

choroid

Phagocytosis of outer segments thereby maintaining the environment of the retina [8].

Secretes variety of growth factors: fibroblast growth factors (FGF-1, FGF-2, and FGF-5) and transforming

growth factor-β (TGF-β) (insulin-like growth factor-I (IGF-I), ciliary neurotrophic factor (CNTF), platelet-

derived growth factor (PDGF) ,VEGF, lens epithelium-derived growth factor (LEDGF), members of the

interleukin family, and pigment epithelium-derived factor (PEDF) to help build and sustain the choroid

and photoreceptors.

Bruch’s Membrane

The Bruch’s membrane is the innermost 2-4µm layer of the choroid subadjacent to the RPE. It is a

connective tissue that lies between the RPE and the dense capilliary bed known as the choriocapillaris

that supplies it with nutrients.[8, 12]. It lies flat along one side of the dense A5-layer vessel wall that

underlies the RPE, the Bruch’s Membrane is an elastin- and collagen-rich extracellular matrix (ECM) that

acts as a molecular sieve [47]. The membrane has both a biochemical and a physical role and can be

divided into:

the most anterior layer is the basal lamina of the RPE that is 0.14–0.15 μm thick

the inner collagenous layer that is approximately 1.4 μm in diameter

a central elastin layer that is porous

an outer collagenous layer about 0.7 μm in diameter

the most posterior layer that is the basement membrane of the endothelium of the

choriocapillaris [12]

The Bruch’s membrane has been implicated in the pathology of conditions such as choroidal

neovascularization (CNV) and has a number of functions including:

controlling the diffusion of nutrients and waste products between the choroid and RPE including

minerals, antioxidants, trace elements and various components of serum that are essential for

the normal vision by the photoreceptors [12, 55].The diffusion properties of the membrane are

dependent upon various factors including the hydrostatic pressure on both sides of the

membrane, the concentration of the diffusing molecules. Furthermore the structure and

composition of the membrane can play a large role in the diffusion properties, and these are in

turn affected by the age, genetics, environmental factors, retinal location, drugs and disease

act as a physical support allowing the adhesion of RPE cells and surface for migration

promoting the healing of wounds

act as a physical barrier [8]

Choriocapillaris

The retina is supplied by two vascular systems:

the intrinsic retinal circulation supplies the inner retinal layers,

the choriocapillaries that supplies the photoreceptors and the RPE [12].

The choriocapillaris is 200-300µm thick and is found lying posterior to the Bruchs Membrane. It has the

highest blood flow per unit volume in the body, 7 times greater in the macula than the periphery [12]. It

is highly perforated particularly on the retinal side, an indication that it is involved in secretion and/or

filtration processes. Furthermore, VEGF receptor-1 and -2 (VEGFR-1 and -R2) have been shown to be

expressed on the retinal side of the vasculature [8]. A unique feature of the choriocapillaris is that it

constitutively express intracellular adhesion molecule-1 (ICAM-1), a molecule that is responsible for firm

adherence to endothelial cells by leukocytes with CD11b/CD18 on their surface, like macrophages and

neutrophils. The RPE are thought to extract nutrients from the choriocapillaris and transport them to

the photoreceptors, and remove waste products from the photoreceptors [8].

The pathological changes seen in AMD are discussed below based upon the part of the macula that they

occur in.

Changes in the photoreceptors

While it is thought that the first changes that occur in AMD may be a dysfunction in rod photoreceptor,

the earliest detectable changes in the macula are the formation of drusen and pigmentary changes in

the macula [3]. It has been shown that the loss of rods exceeds the loss of cones in patients with AMD

[8]. The eyes of patients with AMD have been seen to contain apoptotic photoreceptors, RPE, and inner

nuclear layer cells. It has been proposed that the Fas/Fas ligand system has a role to play in

photoreceptor apoptosis in AMD. Moreover an association between the loss of photoreceptors and loss

of RPE has been documented. The loss of photoreceptors in wet AMD is associated with the formation

of a scar, and the presence of RPE on the scar has been shown to decrease the severity of AMD.

Furthermore, thicker scars lead to a greater degeneration of photoreceptors [8].

Changes to the RPE

Various changes to the RPE are observed in AMD, many as a result of increased oxidative stress. These

include:

changes in pigmentation and the reduction of melanosomes,

reduction in the cell density of RPE. This may result from apoptosis, which is caused by

accumulation of toxic substances

increase in the number of lipofuscin granules [8].

Photodegradation of the RPE is also thought to contribute to the AMD, although the exact mechanism

has not yet been determined [56].

Changes in the Bruch’s Membrane

The Bruch’s membrane is a dynamic layer whose continuous turnover is mediated by matrix

metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) through couples

synthesis and degradation [3, 55]. RPE and choroidal endothelial cells release MMPs 1, 2,3 and 9 as well

as TIMPs 1, 2 and 3 into the Bruch’s membrane [55]. Furthermore high molecular weight species of

MMPs including termed HMW1&2 made up of polymers MMPs 2 and 9, as well as large macromolecular

weight complex (LMMC), made of HMW1, 2, pro-MMP 9 and smaller quantities of pro-MMP 2 have

been identified in human Bruch’s Membrane [57, 58]. Even though there are processes in place to

rejuvenate the Bruch’s membrane, the process of aging deteriorates it. As such it is unable to fulfill its

role in transporting fluids, amino acids and larger molecular complexes [57]. This in turn means that

debris accumulates in the membrane leading to a disruption in the homeostasis of the overlying

photoreceptor layer [55].

The thickness of the Bruch’s Membrane is seen to increase as an individual ages due to the

accumulation of undefined subtances in the collagenous areas and intercapillary regions [8].

Furthermore, the Bruch’s membrane harbors normal and abnormal extracellular matrix material,

increases in advanced glycolation/lipoxidation end products (AGEs and ALEs), accumulates lipid rich

debris and inter molecular fibril cross links. AGEs and ALEs are known potent inhibitors of MMPs, while

the intermolecular fibril cross links reduce the susceptibility of the collagen molecule to proteolytic

action [55]. Accumulation of the high molecular weight MMP species is thought to isolate monomeric

species, thereby eliminating them from the activation process [55]. The end result of all these changes is

a reduction in the turnover of the ECM and the Bruch’s Membrane [55]. These changes seem to be

greater in the Bruch’s membrane on patients with AMD. Of note in these patients is the reduced

hydraulic conductivity and diffusional capacity for amino acids and carrier sized molecules [55]. These

patients were shown to have increased levels of TIMP-3 inhibitor and pentosidine AGEs, and reduced

active MMPs 2 and 9 leading to the suggestion that abnormalities in the MMP system play a role in the

development of the AMD.

Additionally, there is the accumulation of the lipid rich basal laminal deposits (BlamD) and basal linear

deposits (BlinD). Basal deposits are diffusely distributed lesions associated with AMD that differ in size,

composition and significance. It is postulated that the lipids deposit as a result of the RPE failing to

process cellular debris associated with outer segment turnover [1,12]. Lipoproteins found in the Bruch’s

Membrane contain high amounts of fatty acid linoleate and low amounts of docosahexanoate. They also

contain apolipoproteins B, A-I and E. Yet again these lesions in AMD seem to share certain mechanisms

with atherosclerotic plaque formation, with the exception that lipoproteins retained in the Bruch’s

membrane are of intraocular origin whereas those retained in large arteries are derived from plasma

LDL [47].

BlinD is a thin lipid rich layer of about 0.4–2 μm that occurs between the basement membrane of the

RPE and the inner collagenous layer of the Bruch’s Membrane [12]. Since BlinD has a similar composition

to drusen and is located in the same plane, researchers believe that they may be an alternate form of

drusen [3, 12]. Both BlinD and soft drusen have been shown to be much more highly enriched in UC as

compared to membranes of surrounding cells [12].

BlamD on the other hand is more fibrous and associated with aging and AMD [3]. These deposits are

found between the RPE and its basement membrane in the form of small pockets resembling basement

membrane. Further investigation reveals that they are composed of material that resembles the

basement membrane and contain laminin, fibronectin, and type IV and VI collagen. In the eyes of

patients with AMD however, they appear as a thick continuous layer of up to 15 μm. These deposits are

more heterogeneous, containing vitronectin, MMP-7, TIMP-3, C3, and C5b-9 as well as EC and UC.

Contrary to prior belief the elastin fibers in this ECM do in fact turn over and in the process may

degenerate. Since the eyes of patients with AMD display larger gaps in macular elastin layer integrity

compared with controls, it is postulated that degeneration of elastin may contribute to the thickening of

the Bruch’s membrane in AMD [1, 3].

Both the thickening of the Bruch’s membrane and the presence of drusen may result in the formation of

hydrophobic barriers to impede the passes of fluid and nutrients between the choroid and outer retina

resulting in relative ischemia [1]. Oxygen diffuses from the choriocapillaries through the Bruch’s

membrane and retinal pigment epithelium towards the avascular outer retina. Here it is used up in

various processes in the photoreceptors [46]

Changes in the choroid and choriocapillaris

The choroidal thickness has been shown to decrease in patients with AMD although the exact effect of

this is yet to be identified [8].

Drusen

Drusen are focal deposits of extracellular debris that typically form between the basal lamina of the RPE

and the inner collagenous layer of the Bruch’s membrane. They are generally round in shape with a

yellowish color. These lesions are considered to be the hallmarks of the AMD and are characteristic of

the aging eye and age related maculopathy. They can be sequestered and can be detected through

various assays [47].

Drusen are classified into hard or soft depending upon their borders and the level of risk that they

confer to the advancement of the disease [12]. Soft drusen that are the most common type of drusen in

the macula have a high membrane coil content and pose a higher risk of development of AMD [12,47].

They are slightly larger than hard drusen and do not have well-defined margins [7]. Hard drusen tend to

be smaller and well defined. The distinct features of the drusen may give an indication of the stage of

AMD [47]. Furthermore, the content of drusen is important in understanding how the lesions that are

specific to ADM form. The molecules trapped in drusen have varying roles including the processing of

extracellular enzymes, the stigmata of formative processes eg: the extrusion or secretion of cellular

materials and cellular invasion [47].

When they were first identified, it was thought they were composed entirely of lipids [7]. Further

investigation revealed that they contained carbohydrates as well [7]. Presently they are known to

comprise lipids, carbohydrates, zinc and at least 129 different proteins excluding extracellular matrix

[47]. It has been found that drusen are composed of 40% of the lipids EC and PC cumulatively [47]. The

discovery of this information has been useful in determining contributory pathways as well as allowing

construction of improved in vivo and in vitro model druse systems [47]. Druse proteins involved in

inflammation and innate immunity include amyloid-β, immunoglobulin light chains, factor X, C3, and late

stage activated complement components such as the C5b-9 complex, are of particular interest in the

development of AMD [12, 47]. Additionally, various studies have isolated ubiquitin, integrins,

lipoproteins, tissue inhibitor of metalloproteinase 3, advanced glycation end products and major

histocompatibility complex (MHC) class II antigens [7]. Organelles, cellular and basal lamina fragments,

lipofuscin and melanin are common cellular components found in drusen that are derived from RPE [7].

More recently, human leukocyte antigen and differentiation antigens were detected leading to the

conclusion that druse may not only be the result of waste material that cannot be eliminated but also

the by- product of local active processes that involve inflammation, the complement system and local

and systemic immune related mechanisms [7]. In fact some of these components may also be revealed

in the non-ocular pathological conditions that are based on inflammation and complement such as

atherosclerosis [7]. Amyloid beta, a key component in drusen, is a waste product that accumulates in

the central nervous system during the process of aging and in age related disease such as Alzheimer’s

disease [7, 59]. Interestingly, in addition to accumulating along the Bruch’s membrane, amyloid beta is

also observed in the photoreceptor outer segment [59]. On this basis, it can be proposed that

inflammation is a central pathological basis for all these age related conditions [7,59]

All drusen, whether hard or soft, contain the two chemical forms of cholesterol, unesterified cholesterol,

(UC) and cholesterol that is esterified into long chain fatty acids (EC) [12]. Neutral and polar lipids have

been identified in drusen using sudanophilic dyes, filipin or polarizing microscopy [12]. The abnormal

metabolism of lipids has been assosciated with AMD [60]. It has been shown that the export of

intracellular cholesterol is impaired with decreased ABCA1 expression in aged macrophages, a process

that is regulated by liver X receptor and microRNA-33, leading to higher levels of free cholesterol within

senescent macrophages. An increase in free intracellular lipids results in the polarization of older

macrophages to an abnormal state that promotes vascular proliferation including angiogenesis.

Angiogenesis is an underlying mechanism of common age related disorders such as atherosclerosis,

cancer and neovascular AMD [61]

Drusen are thought to form when lipoprotein particles are trapped in a hydrophilic matrix followed by

the fusion of lipid particles and pooling. It has also been shown that RPE-secreted lipoproteins are not

the only source of lipids in the drusen formation due to the differing compositions. Specifically, the

composition of drusen seems to have a lower UC and higher fatty acid content than the RPE-secreted

lipoproteins. It is possible that the lipoproteins found in drusen serve as a stimulus for complement

mediated activation, as does in the wall of the atherosclerotic artery [47].

It is interesting to note that EC and UC also accumulate in normal Bruch’s membrane throughout an

individual’s adult life [47]. This accumulation is a result of the storage of lipoprotein particles that are

60-80nm in diameter and that contain large amounts of EC, UC, phosphatidylcholine (PC) and

apoliprotein B [47]. It is postulated that the RPE secrete the lipoprotein that accumulates in Bruch’s

membrane although some lipoproteins may be of hepatic or intestinal origin [47].

Oxidative Stress

In macular degeneration, the retinal pigment epithelium (RPE), cannot keep up with the removal of lipid

debris and is unable to respond effectively to the increased oxidative stress [62]. Furthermore, the

concentration of photo-oxidative reactive species may increase and that of α-tocopherol, an important

antioxidant, may decrease. The combined effect of these actions is believed to contribute to the

pathogenesis of AMD.

This synergistic effect has been reiterated in a recent study evaluating the role of oxidative stress in

AMD [63]. The researchers also found that patients with AMD had an increase total thiol level and a

raised PON1 actitivity.

Additionally, it has been proposed that the accumulation of advanced-glycation end products (AGEs) in

RPE and the Bruch’s Membrane contribute to oxidative stress. AGEs have also been isolated from

Drusen [8].

Hypoxia

Choroidal blood flow is disturbed in the development of AMD, possibly resulting in hypoxia and impaired

energy metabolism in the elderly. Furthermore, other features in AMD, such as confluent drusen, serous

or hemorrhagic retinal detachment, retinal edema and vitreoretinal adhesion may also impair the

delivery of oxygen and nutrients from the choroid to the retina [46]. Drusen, retinal elevation, tissue

edema and cystoid spaces may achieve this effect by increasing the diffusion distance, while

vitreoretinal adhesion may limit the transvitreal delivery of oxygen to the retina and cytokine clearance

[46].

These proposals have mostly been based on studies on choroidal perfusion, which are not the only

contributor to retinal hypoxia found in AMD eyes. Most cases of choroidal neovascularization in AMD

have been localized close to areas with poor choroidal perfusion on indocyanine green angiography.

Additionally, choroidal blood flow has been studied using laser Doppler, Doppler Imaging and pulsatile

ocular blood flow by various research groups over the years. More recently, choroidal histology

specimens of human eyes with the exudative form of AME showed choriocapillaris dropout adjacent to

active choroidal neovascularization. It is postulated that the dropout regions were hypoxic [46].

Drusen, particularly the thick confluent type that deposit under the retinal pigment and a thickened

Bruch’s membrane both increase the distance between the retinal cells and the choriocapillaris. Since

the oxygen tension inside the drusen is lower than the outer surface, the oxygen and other essential

nutrients must cross the drusen down a concentration gradient to get to the retina. This process seems

to come into play more in confluent drusen than in hard small drusen in which the oxygen can normally

diffuse between the lesions, a suggestion that might also explain why confluent drusen have a greater

association with neovascularization than hard drusen [46].

Similarly, the deposition of lipids in the Bruch’s membrane may restrict the diffusion of oxygen by

increasing the diffusion distance rather than a lower diffusion coefficient. This could possibly lead to

hypoxia in the retinal pigment epithelium (RPE) and subsequent production of VEGF. Previous studies

have shown that RPE cells exposed to lower levels of oxygen tend to produce more VEGF than when

exposed to normal atmospheric levels [46].

The partial pressure of oxygen in the RPE of a normal eye is very high due to its close proximity to the

choriocapillaris, thus decreases to almost 0mm Hg in the inner portion of the photoreceptors. However,

if the distance from the choriocapillaris to the photoreceptors is increased through processes such as

retinal detachments, the tissues may experience hypoxia [46].

The situation could be worsened by a resulting vicious cycle in which a detachment that increases

hypoxia leading to the production of VEGF in the retina. Increased VEGFs increase vascular permeability

and stimulate the growth of new vessels leading to increased fluid accumulation under the retina. Water

accumulation in retinal edema also increases the distance between the capillaries and the oxygen

consuming photoreceptors, increasing the diffusion distance and worsening the hypoxia [46].

Other studies have found that vitreomacular adhesion is associated with choroidal neovascularization

exudative AMD. Furthermore, hypoxia-inducible factor (HIF), HIF-1a and HIF-2a have been detected in

the endothelium and macrophages of the choroidal neovascular membranes in AMD [46]. Under

hypoxic conditions, the HIF transcription factors activate the expression of the VEGF gene, increasing the

formation of VEGFs that induce angiogenesis. HIF has been shown to cause apoptosis, possibly leading

to geographic atrophy [46].

Age and Lipid Deposition

While the Bruch’s membrane undergoes neutral lipid deposition during the process of aging, the

presence of BlinD and drusen are the hallmarks of AMD. Lipid accumulation in AMD is probably different

[1].

An analysis using three histochemical stains, oil red O, Bromine Sudan Black B and Bromine-Acetone-

Sudan Black B performed to identify lipids in eyes from donors between the ages of 1-95 years showed

that oil red O-binding material was localized exclusively to BrM, whereas two other dyes labeled cells

throughout the choroid in addition to BrM [12].

Oil red O-binding neutral lipid is a major age related deposition in the Bruch’s membrane, sclera, cornea,

and intima of large arteries [64]. In the intima, the oil red O-positive material is made up of small (60–

200 nm) extracellular droplets that are higher in EC relative to UC [12]. It is known that EC in the sclera,

cornea and arterial intima is derived from LDL translocated from plasma into connective tissues. The

smaller LDL particles are trapped by the extracellular matrix, the LDL protein and/or phospholipid

components are degraded and the remaining lipid components are fused. Whether it is this process that

manifests in the eyes or there is a process unique to the retina that results in the deposition of lipids,

remains to be established [12]

EC has been identified as the primary lipoprotein accumulating in the Bruch’s membrane with age,

through various light microscopic histochemistry, physical chemistry, ultrastructure, and lipid profiling of

tissues and isolated lipoproteins [12]. Furthermore, it has been shown the EC is localized to the Bruch’s

membrane and not found in the choroid. This leads to the investigation on how age-related BrM lipid

accumulation occurs.

The lipoprotein droplets in the RPE are much larger (1–2 μm), and they have less EC (105) and more

retinyl ester than the particles found in the Bruch’s membrane [12]. Additionally, since few RPE cells

have droplets in any one eye, the release of droplets is not a probable mechanism for the large and

universal accumulation of lipids in the Bruch membrane with age. This led to the postulation that EC are

released from a healthy cell within the core of an apoB-containing lipoprotein [12].

Biochemical, histochemical and ultrastructural studies have revealed that RPE in fact secretes

apolipoprotein (apoB)-lipoprotein particles of unusual composition into Bruch’s membrane [12]. Both

mRNA and protein for the large subunit of microsomal triglyceride transfer protein (MTP) of the apoB

system has been isolated in the human RPE [12]. It is suggested that the RPE has the capability of

secreting lipoprotein particles since both apoB and MTP are expressed on it. Furthermore, it expresses

mRNA for acylcholesterol acyltransferase-2 (ACAT-2), a cholesterol esterifying enzyme that is associated

with the production of lipoproteins [12]. The RPE secretes a large lipoprotein with an EC rich neutral

lipid core from its basolateral side into the Bruch’s membrane for eventual clearance into plasma [12]

These apo-B lipoproteins have been shown to resemble plasma apo-B lipoproteins in many ways [12].

They accumulate in the Bruch’s membrane as the eye ages and result in the formation of a lipid wall,

impeding the RPE as well as the functioning of the photoreceptors [12]. This process is very similar to

atherosclerosis whereby apo-B becomes trapped in the arteries. In both cases, the resulting conditions

are pathological adaptive responses to the deposition of lipids [3].

Oxysterols

Oxysterols have been recently identified as contributing factors to a number of pathological conditions

[65]. Their formation and role in the retina and pathological conditions such as age related macular

degeneration is yet to be established. 7-ketocholesterol (7KCh) has so far emerged as the most

abundant oxysterol in the retina [65]. It is thought to trigger inflammatory and cell death pathways in a

number of cells including those of the retina [65]

7KCh is the oxidized form of cholesterol that is found in oxidized LDL. It is associated with

atherosclerotic plaques and has been implicated in foam cell formation in macrophages as well as

proinflammatory and cell death pathways [65]. The end result of all these processes is the formation of

atheromatous plaques. This oxysterol has been found to be highly toxic to cultured RPE cells and is

thought to be responsible for most of the cytotoxicity resulting from oxidized LDL.

Cholesterol is oxidized by both enzymatic and non-enzymatic pathways. The latter leads the formation

of 7KCh as well as 7-hydroxycholesterol (7HCh), andepimeric 5, 6-epoxycholesterol (5, 6-epoxCh) [65].

Circulating cholesterol is taken up by the RPE in the form of lipoprotein particles. Circulating LDL is taken

up via LDL receptors found in the basal layer of the RPE whereas in the neural retina, LDL and VLDL are

taken up by VLDL receptors through the vascular endothelial cells. A number of possible mechanisms for

the metabolism of 7KCh have been proposed [65].

Various studies have linked 7KCh to the development of AMD. The formation and accumulation of 7KCh

is likely to affect the choriocapillaris and RPE, leading to loss of their functions, induction of VEGF,

weakening of Bruch’s membrane and causing AMD. Further research is still required in this area [65].

Vascular Resistance

Increased vascular resistance may compromise metabolic exchange across the RPE leading to

accumulation of lipoproteins in the Bruch’s membrane and hence AMD. A number of factors have been

shown to contribute to increased vascular resistance in the choriocapillaris [3].

Infiltration by lipids in the sclera and other ocular tissues leads to increased resistance in the choroidal

vessels, thereby diminishing the ability of the choriocapillaris to clear lipoproteins from the RPE and

Bruch’s membrane [3].

Vascular resistance is further increased by the presence of hyperopia, an established risk factor for

developing AMD. Moreover, the density of choriocapillaris increases in both wet and dry AMD, leading

to an increased resistance and impaired flow [3].

Inflammation and Immune Response

As discerned earlier, inflammatory pathways have a central role in both AMD as well as other age

related pathological conditions such as atherosclerosis [3, 7, 66]

Generation of inflammatory related molecules in the Bruch’s membrane, recruitment of macrophages

and dendritic cells, complement activation and microglial activation in the macular area are some of the

immunological responses detected so far [45, 67]. Genetic variation in the complement pathway and

environmental factors may exert their effects as risk factors in the development of AMD through such

inflammatory pathways [3].

Indirectly, inflammatory responses can upregulate vascular endothelial growth factor (VEGF) and other

proangiogenic factors that are essential for chronic neovascularization [3].

Other findings that link inflammatory and immune responses to AMD include age related changes in

retinal microglial activation that cause lipofuscin accumulation and, infectious agents that may

potentially trigger inflammatory responses. [3]

Drusen have been shown to be sites of immune complex formation and that they are generated as an

inflammatory or complement mediated response in AMD. In addition to demonstrating

immunoreactivity to certain immunoglobulins, molecules that are involved in the cellular and humoral

immune responses as well as the complement pathway have been found in drusen. Furthermore, the

Bruch’s membrane, and the choroidal vessels also contain molecules involved in the complement

pathway. It is postulated that RPE cells injured by light damage, oxidative stress, lipofuscin accumulation

etc, recruit and activate dendritic cells from the choroid. The dendritic cells elicit an inflammatory or

complement mediated response to generate drusen. It is interesting to note that there is no mention of

lipid deposition in this model. [3]

Environmental and genetic factors can also trigger the inflammatory response leading to disease

progression [7]. A number of potential mechanisms have been proposed to explain the pathogenic

mechanism of AMD in the eye [7]. These are discussed below in turn.

Immune privilege

The eye as well as the brain, testes, placenta and fetus are immune privileged sites [7]. In all tissues, the

synthesis of complement proteins normally occurs in hepatic tissue from where they are distributed.

The eye, particularly the retina, however demonstrates extra hepatic synthesis of small proteins that are

involved in the complement system [7]. This immune privilege allows it to provide protection on one

hand but may predispose it to the development of autoimmune diseases. Such a vital structure,

inflammation and immune responses in the eye could lead to serious damage [7].

The entry of immune cells into the eye is blocked by various features in the eye such as the lack of

intraocular lymphatic drainage, presence of lens and cornea and the presence of endothelial barriers

such as the occludent intercellular junctions. Therefore antigens have to be insulated inside the eye

itself [7]. Furthermore, the eye structure expresses several anti-inflammatory and immunosuppressive

molecules that contribute to the immune and inflammatory response [7]. Sympathetic innervation and

the secretion of neuropeptides adds to this phenomenon [7].

In this way, the eye maintains homeostasis preventing any damage that could result in the death of RPE

cells and the consequent development of pathological ocular conditions such as AMD.

Specifically, the ocular pigment epithelial cells produce immune regulator soluble and cell surface

molecule. These have two fold activity; not only do they express those components of the immune

system that trigger an immune cascade and form proinflammatory mediators, but they also work to

prevent damage to the retina by blocking the inflammatory response. In particular they express toll like

receptors, complement components and MHC molecules which cause an inflammatory response. To

modulate this response, they also release immunosuppressive mediators and induce regulatory T cells

[7, 68].

Autoimmunity

There is a growing body of evidence to show that autoimmunity plays an important role in the

pathogenesis of AMD. Sera from monkeys and humans affected with both early and advanced stages of

AMD were shown to contain anti-retinal antibodies [7]. AMD is associated with RPE degeneration that

leads to the loss of production of immunosuppressive and anti-inflammatory mediators and disruption

of the blood-retinal barrier, affecting the homeostatic balance of the immune privilege [7]. This leaves

the eye open to the entry of immune cells that do not recognize ocular antigens as self and proliferate

to produce autoantibodies. Whether this is a primary or a secondary response to disease progression

remains unclear since the autoantibodies are seen in patients with both early and advanced stages of

disease progression [7].

It is also postulated that autoimmunity may be linked to AMD through oxidative stress [7]. Oxidative

stress is known to play a key role in the development of AMD retinal damage as shown by a number of

studies [7, 69].

Chronic Local inflammation

Vitronectin is a common component of three pathological and possibly related conditions: AMD,

atherosclerosis or elastosis. Vitronectin production is upregulated when RPE cells are stimulated with

complement. It is found in drusen in high levels and shared components have also been found in

amyloid deposits and glomerular dense deposits disease. Similarly, amyloid beta is found in drusen as

well as the plaques that characterize Alzheimer’s disease. In all these diseases, elements that belong to

the pro-inflammatory and immune system such as acute phase proteins and complement are

accumulated [70-72]. It is possible that the accumulation of this debris triggers a local inflammatory

response that activates the immune response.

Amyloid-beta promotes RPE cells to enter senescence leading to the secretion of increased amounts of

IL-8 and MMP-9. MMP-9 degrades the tight junction proteins and barrier dysfunction in the senescent

cells and promotes IL-8 to a more active form [73].

In the long term, the organism ends up causing further damage to the retina while adapting to this

inflammation [7].

Parainflammation

It has been shown that the cells of the innate immune system are able to trigger an immune response

upon recognition of stressed cells or tissues in order to restore homeostasis. The degree of this process

known as para-inflammation is determined by the level and the source of stress. Para-inflammation

drives the recently discovered concept of inflammaging [7].

Various types of adaptation and maintenance of tissue/cell homeostasis through inflammatory

pathways have been identified. Under basal conditions of development, growth and aging, apoptosis

operates to maintain homeostasis. During infection or damage conditions, the tissues or cells react and

repair. If the cells fluctuate between a state of homeostasis and infection, then para-inflammation,

subinflammation, low level inflammation, sterile inflammation, physiological inflammation, or

inflammaging all come into play [7].

Para-inflammation has an important role to play in maintaining homeostasis and monitoring tissue

malfunction. In AMD and other age related disorders, oxidized lipids, proteins and DNA may form as a

result of increased oxidative stress. In such a situation, para-inflammation is triggered as a mechanism

to repair and remodel the tissue through various processes including microglial, macrophage and

complement activation [7]. The inflammatory responses involved in AMD and aging are similar leading

to the suggestion that some individuals develop AMD as a normal part of aging whereas others do not.

In line with this it is thought that the inflammatory responses that lead to AMD are probably not

controlled as well and hence result in a pathological condition. This is complicated further by unhealthy

lifestyles and genetics [7].

Advanced Glycation Endproducts (AGEs)

AGEs accumulate as a natural part of the aging process and have been associated with age related

diseases such as Alzheimer’s disease, osteoarthritis, atherosclerosis and AMD. These molecules deposit

in Drusen and in the Bruch’s membrane of the eye. Various studies have been conducted to determine

the role of these products in the development of AMD. While some researchers have found that they

promote oxidative stress, others conclude that AGEs play a role in the accumulation of apoptosis and

lipofuscin. A more recent proposal is that they activate complement and chronic inflammation and are

implicated in para-inflammation [74]. One study conducted to investigate this proposal found that while

all anti-inflammatory cytokines including IL10, IL1ra and IL9 were all overexpressed, some pro-

inflammatory cytokines such as IL4, IL15 and IFN-γ were overexpressed, and others includingIL8, MCP1,

IP10 were underexpressed following stimulation by AGEs [74]. This led to the discovery that the RPE

exists in a para inflammatory state under the influence of AGEs [74]. Furthermore it was seen that the

cells contained higher levels of mRNA that codes for chemokine, CXCL11, and viperin, all of which have a

role in the inflammatory response. The authors of this study concluded that AGEs exert their effect in

AMD through inflammatory pathways [74].

Microglia

Microglia are resident immune cells found in the CNS where they have a role in both innate and

adaptive immunity. Interestingly microglia are found in the amyloid plaques of Alzheimer’s disease and

have been associated with Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis,

suggesting a role for these in different neurological disease [7].

Microglia activation is central to all kinds of neuroinflammation with the release of inflammatory

mediators and phagocytosis [7]. The role of microglia has been recognized in a number of retinal

degenerative diseases including AMD, retinitis pigmentosa and, late onset retinal degeneration [7].

Microglia have been shown to have two primary roles. In normal cells they have been shown to release

neuroprotective and anti-inflammatory factors under physiological condition. In CNS damage, they

remove waste materials and degenerated cells to halt further injury [7]. If however, the microglia trigger

a cycle of persistent activation and recruitment, they may be harmful. It is thought that all these actions

described above are replicated by the microglia found in the eyes. In the normal retina, microglia are

inactive and restricted to the inner retinal layers [7]. Studies on the eyes of patients with AMD have

revealed that microglia are active and spread to the outer nuclear layers where they probably remove

cell debris [7]. However, in the process, they may result in further degeneration as they do in

Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis [7].

Furthermore microglia may also promote the development of AMD through the impaired expression of

chemokine motif (C-X3-C) receptor 1 (CX3CR1). CX3CR1 is a protein that is sometimes referred to as the

fractalkine receptor or G-protein coupled receptor 13[7]. It binds the chemokine motif (C-X3-C) ligand

(CX3CL1) that is involved in the adhesion and migration of leukocytes necessary for the immune

response. CX3CR1 expressed in microglia on the brain and retina control the reaction to a inflammatory

stimuli in these organs. It has been demonstrated that a deficiency of CX3CR1 actually results in

neurotoxicity by dysregulating the microglial response. Furthermore, CX3CR1 knockout mice

demonstrate subretinal accumulation of microglia resulting in an increase of photoreceptor

phagocytosis and toxicity [7]. Additionally, intracellular lipids transform the microglial cells into foamy

cells that resemble drusen and may in fact lead to the formation of drusen [7].

Researchers have shown that microglia can be activated by endogenous photoreceptor proteins through

TLR4, aggravating the death of RPE cells. It is postulated that this leads to a number of degenerative

retinal disorders including AMD [75].

Autophagy and Heterophagy

Autophagy is the name given to the process that degrades cytoplasmic material and organelles. It is a

tightly regulated lysosomal clearance process used to remove large unwanted structures such as

ascytoplasmic material (ubiquitinated macromolecules), organelles (damaged mitohochondria,

endoplasmic reticulum, peroxisomes), and internalized pathogens [76,77]

To date three different routes of autophagy have been identified:

The chaperone mediated route that is, associated with the transport of proteins with a specific

sequence signal from the cytoplasm into the lysosome through the lysosomal membrane

Microautophagy that is said to occur when the lysosomal membrane itself sequesters a portion

of the cytoplasm by a process similar to the pinching off of phagosomes from the plasma

membrane

Macroautophagy which occurs through the formation of an autophagosome: a double

membrane bound vesicle that contains cytoplasm and/or organelles [7,78].

The process is stimulated by the formation of phagophores in response to stress stimuli such as

oxidative stress. Phagophores are isolation membranes that are formed at the phagophore assembly

site [79, 80]. In normal cells phagophores elongate and engulf a portion of the cytosol and form

autophagosomes which invaginate the material that is to be degraded and package it [14, 78, 81]. The

mature autophagosomes fuse with primary lysosomes allowing the lyososomal enzymes to degrade

their contents. In this way autophagy reduces the toxicity of the protein aggregates, a process that is

thought to prevent age related diseases [82]. In aged post mitotic RPE cells (as with other cells), the

process of autophagy may fail at some point leading to the accumulation of aggregate-prone proteins,

cellular degeneration increased lipofuscinogenesis, RPE damage and finally cell death causing AMD

[14,78,81-88].

Heterophagy is another process through which the RPE cells prevent pathological conditions such as

AMD. This is the diurnal phagocytosis of the photoreceptor outer segment (POS) fragments by RPE cells

[14]. In a process similar to that seen in autophagy, autophagosomes and phagosomes form, mature and

fuse with lysosomes [89]. The process is complex and involved the interplay of POS binding, membrane

invagination and the circadian rhythm. Focal Adhesion Kinase (FAK) and Mertyrosine kinase (MerTK),

activated by the αvβ5 receptors regulate the process of POS heterophagy. Milk fat globule- EGF8 (MFG-

E8), a ligand of αvβ5 integrin regulates the circadian rhythm of phagocytosis. It has been shown that

reduction in αvβ5, occurs during the aging process, leads to an accumulation of lysosomal lipofuscin

[14].

Impairment in lysosomal function

Lysosomal clearance and lysosomal enzymes form an important part of autophagy and heterophagy,

both processes that prevent AMD. It is therefore safe to assume that animpairment in lysosomal

function will contribute to the development of AMD. To date various mechanisms that disturb lysosomal

clearance have been identified [14, 82, 90].

A variant of an inhibitor of the cysteine proteinases, cystating C has been associated with the

advancement of AMD. This molecule, known as variant B cystatin C is thought to exert effect by

inhibiting proteolytic regulator secretion, mistargeted signaling and inappropriate cell proteins [91].

Lysosomes do not have mechanisms for clearing these proteins leading to the degeneration of RPE and

subsequent development of AMD [14].

One of the lysosomal hydrolases involved in autophagy and heterophagy are known as the cathepsins

and include the cysteine cathepsins and aspartic protease cathepsin D [14, 92]. So far 11 cathepsins

have been characterized in humans: cathepsins B, C, F, H, K, L, O, S, V, W, and X. Studies have revealed

that they all share the same core structure and are all monomers with a 30 kDa molecular weight with

the exception of Cathepsin C that exists in a tetrameric form [92]. While most cathepsins behave as

endopeptidases, a few have exopeptidase properties [92]. The RPE has been shown to contain

cathepsins A, D, E and S, whereby cathepsin D is involved in the breakdown of rod outer segments and

rhodopsin into glycopeptides. From experiments on cathepsin D knockout mice, it has been

demonstrated that dysfunction of cathepsins may contribute to the development of AMD [14].

Inflammasone:

Inflammasome is a component of the innate immune system that has been implicated in AMD through

its modulatory effect on the maturation and secretion of pro-inflammatory cytokines IL-1 and IL-18 [14].

Inflammasone monitors cell stress through pattern recognition receptors that are able to recognize

molecular conformations such as the pathogen associated molecular patterns and damage associated

molecular patterns [3]. These pattern recognition receptors include Toll-like receptors (TLRs), Nod-like

receptors (NLRs), the C-like leptin receptors and virus specific RIG-I like receptors [93].

One of the inflammasomes found in human RPE cells is the NLR pyrin domain containing-3 (NLRP3) [87,

94, 95]. It has been shown to be activated by a broad spectrum of activators infection, injury, other

inflammatory disorders and endogenous aggregates including uric acid crystals, amyloid polypeptides in

diabetes.

The activation of the NLRP3 inflammasome required 2 signals. The first one follows from the activation

of NFκβ or MAPK pathways that are initiated by the ligand binding of TLR or NOD receptor [14]. A

second signal results in the oligomerization of NLRP3 through its nucleotide-binding domain (NBD).

Oxidative stress has been seen to play a large role in this activation mechanism.

More recently a single and double stranded endogenous nucleic acid, AluRNA was shown to activate

NLRP3. It is thought that AluRNA exerts its effects through the inflammatory cytokine interleukin IL-8

resulting in RPE degeneration and the advancement of GA. Another study has demonstrated that drusen

activated NLRP3 inflammasome and IL-I8 expression in cultured monocytes [3].

Complement

Complement plays a pivotal role in the pathogenesis of AMD as has been identified by a number of

studies although the exact mechanisms behind this process are to be determined [3,96]. The

complement system is made up of several plasma proteins with enzymatic actions. These are activated

by a proteolytic cascade and have different roles including pathogen opsonization, recruitment of

inflammatory cells, removal of waste material or direct cell lysis.

The complement system is made up of 3 pathways:

The classic pathway that is activated by antibodies

The alternative pathway that is directly triggered by the surface components of different

pathogens and lectin

The mediated pathway that is activated by serum proteins that have the ability to bind

encapsulated bacteria.

All these pathways eventually cause the activation of the central component C3 and cleave it into C3a

and C3b fragments [71, 97, 98]. C3b in turn splits C5 into C5a and C5b. C5b binds to component 6 and

catalyzes the addition of component 7, 8 and 9 to form the C5b-9 membrane attack complex (MAC) [3].

The MAC creates pores through the lipid bilayer facilitating cellular lysis and clearance [3].

Complement proteins are not only synthesized in the hepatocytes but also in extra hepatic tissues

including the neural retina, RPE and choroid. Furthermore, the accumulation of lipofuscin components

and apolipoproteins has been shown to activate the alternative complement pathway [3].

Many components of the complement system have been found in the drusen, RPE, the Bruch’s

membrane, basal deposits, retina and choroidal capillaries [3]. These include C1q, mannose binding

lectin, complement factor B (CFB), complement factor I (CFI), complement factor H (CFH), C3 and

fragments of C3, late stage complement components such as C5-9, the C5b-9 (membrane attack

complex, MAC), various regulatory proteins such as membrane cofactor protein, complement receptor

1, vitronectin, and clusterin, and activators such as amyloid beta, C-reactive protein, and

immunoglobulin. These components demonstrate low levels of activity in a normal functioning eye

where they provide defense against pathogens and confer immune privileges.

Furthermore, multiple genetic studies based on polymorphisms related to complement genes have

confirmed the link between AMD and the complement system. Some of these genes include

complement factor H (CFH), complement factor H –related (CFHR) 1 and 3, CFB, C2, C3 and CFI.

CFH is a serum glycoprotein that plays a vital role in the alternative pathway facilitating the

differentiation of self from non-self. It works by preventing the rapid breakdown of the unstable C3a-

C3b thioester bond in any aqueous environment and protects normal cells from intrinsic complement

activation [99]. The detection of a specific CFH gene variant that is linked to the risk of AMD has led to

the notion that the alternative pathway has the greatest role in the development of AMD. Mutations in

CFH lead to alterations in structure and eventually function resulting in excessive complement activation

and hence immune mediated damage [7].

A recent study has shown that all-trans-retinal (atRal) sensitizes RPE cells to an attack by the alternative

complement pathway [100]. Another study has shown that impaired complement regulators may result

in AMD through impaired regulation of complement. Particularly, the complement regulatory protein

CD59 was studied. Researchers found that the levels of this protein were increased in early AMD but

decreased in the RPE overlying drusen in patients with advanced AMD[100].

Amyloid Beta and Apolipoprotein E Accumulation

Amyloid beta (Aβ) is a peptide that is found in both senile plaques as well as drusen. It is thought to have

a role in inflammatory events that contribute to RPE dysfunction [3]. The effects of the deposition of

amyloid beta in drusen can be broken down as follows:

Blocking of CFI and hence blocking the inactivation of C3b- this leads to a continuous low grade

activation of the complement system with subsequent chronic inflammation

Increase the production of monocyte chemo attractant protein-1 (MCP-1), interleukin-1 beta (IL-

1b) and tumor necrosis factor-alpha (TNF-a) from macrophages and microglia which activate

CFB in RPE cells [7].

It has been postulated that amyloid beta may induce the secretion of MMPs that disrupt the integrity of

RPE. MMP-9 in particular secretes TJ proteins such as zonula occludens-1, occludin and F-actin that are

an important factor in the breakdown of the barrier [101].

Neprilysin is an amyloid beta degrading peptidase. The development of typical AMD associated with the

subretinal accumulation of amyloid beta has been demonstrated in mice [7].

Apolipoprotein E, another major component of drusen plays a role in normal lipid catabolism and

transport [3]. Light microscopy, ultrastructural studies, lipid histochemistry, assay of isolated

lipoproteins, and gene expression analysis have all contributed to the postulation that apolipoproteins

contribute to the accumulation of esterified cholesterol-rich lipids deposited in the Bruch’s Membrane

[12]. Furthermore, it has been proposed that the retention of a sub-endothelial apolipoprotein B

lipoprotein may be a contributing factor in the formation of AMD lesions [12].

It is interesting to note that different variants of the APOE gene have been implicated in Alzheimer’s

disease and AMD although they seem to have opposite effects in the two conditions. The E4 allele is a

risk factor for Alzheimer’s, but not for AMD, and the E2 allele is a risk factor for Alzheimer’s but not for

AMD.

Other potential mediators of inflammation and AMD

Many other mediators of inflammation have been linked to AMD. These include pro-inflammatory

cytokines IL-1, IL-6, and TNF-a that are released by the choroid and IL-6 and IL-8 released by the RPE

cells [7].

TNF-α is a pleotropic inflammatory cytokine that plays a number of roles in cellular activites and

physiological functions including cell proliferation, survival, differentiation and apoptosis [102, 103].

Increased levels of TNF-α have been associated with the death of various retinal neurons causing retinal

neurodegenerative disorders [102]. By acting as an upstream regulator, this cytokine has indirect toxicity

in retinal neurons [102].

It has been seen to be expressed in the choroidal neovascularizations of age-related macular

degeneration eyes where it is known to disrupt barrier function and cause angiogenesis [103].

With regard to gene studies, one research group was able to find that a patient with AMD had a single

nucleotide polymorphism (SNP) in TNFRSF10A: one of the genes that regulate the function of one of

members of the TNF superfamily [103]. Another study demonstrated that a polymorphism in the tumor

necrosis factor-α -1031 T/C gene may be associated with wet AMD [104].

Seeing that TNF-α is a key mediator in the inflammatory pathways associated with AMD, regulating the

TNF-α signaling pathway becomes an attractive target for the treatment of this disorder [102].

Other studies have focused on the effects of factor Xa and thrombin in AMD. These lead to the

production of a variety of cytokines, chemokines and growth factors by RPE cells [105]. It has been

shown that factor Xa and thrombin can cause inflammation and fibrosis intravitreally leading to

disorders such as AMD [105].

RPE cells also produce VEGF and monocyte chemoattractant protein-1(MCP-1) that serve as a

chemoattractant for macrophages [7]. The precise role of macrophages has not yet been fully elucidated

although there are a number of proposals [7]. Free radicals, pro-apoptic factors and matrix

metalloproteinases have all been seen to activate macrophages [7].

Lipofuscin accumulation

In the normal eye, light sensitive photoreceptor outer segments are renewed constantly [3]. RPEs

phagocytose photoreceptor outer segment discs for lysosomal degradation [3].

The by-products of this phagocytic process are known to accumulate contributing to the age relating

changes in the Bruch’s membrane and the RPE [3]. One of these products is a granular pigment known

as lipofuscin[3]. Accumulation of lipofuscin in the RPE leads to increased fundus autoflourescence which

eventually results in GA [3].

Lipofuscin is composed of a number of flourophores including A2E. A2E is a pyridium bis-retinoid that

accounts for the fluorescent properties of lipofuscin. It is made up of 2 vitamin A aldehyde molecules

and 1 ethanolamine molecule and is the basis of the clinical fundus autoflourescence imaging [3]. A2E is

toxic to the RPE cells in a number of ways:

In inhibits the degradative functions of lysosomes and the breakdown of cholesterol in the RPE

It has photosensitizing properties leading to blue light induced apoptosis

It induces the complement system[3].

Angiogenesis

Angiogenesis involves an interplay of different highly complex biochemical and cellular processes [106].

It requires sequential receptor activation by several growth factors including fibroblast growth factors

(FGF) both acidic and basic, transforming growth factor (TGF) – α and TGF-β, hepatocyte growth factor,

tumor necrosis factor-α, angiogenin, interleukin-8 (IL-8) and angiopoietins (Ang). There are four

angiopoietins: Ang-1 and Ang-4, activate the endothelial receptor tyrosine kinase Tie2 receptor, whereas

Ang-2 and Ang-3 inhibit Ang-1-induced Tie2 phosphorylation [107].

Physiological angiogenesis is vital for many processes in the human body including growth,

development, maintenance and repair [106]. Conversely, pathological angiogenesis, that often occurs in

response to tissue hypoxia or inflammation leads to the growth of tumors and destruction of normal

tissue [106].

Angiogenesis plays a greater role in the progression of AMD rather than the early stages and initial

development [3]. A characteristic of neovascular AMD is vascular growth and leakage occurring in the

sub RPE, subretinal space and neural retina [3].

The process of angiogenesis in CNV involves the proliferation of endothelial cells and their migration

along the Bruch’s membrane. This is followed by the formation of endothelial tubes that are leaky and

tortous and may extend into the subretinal space. This is followed by vessel maturation[3]. Discontinuity

of the Bruch’s membrane is essential to the development of CNV and this can occur from exogenous

factors such as mechanical disruption or endogenous factors such as proteolysis or complement attack.

Vascular endothelial growth factor (VEGF) has been shown to have a central role to play in angiogenesis

[106]. VEGF is a potent endothelial cell mitogen and vascular permeability factor that promotes the

migration and proliferation of the endothelial cells [3]. Angiogenesis requires vascular endothelial

growth factor (VEGF) during embryonic growth and development and postnatal blockage of VEGF has

been shown to result in stunted growth, renal failure leading to increased mortality, and impaired organ

development [106]. In juveniles the result is ovarian failure and stunted growth [106]. In adults infused

with VEGF, the microvascular perfusion is stimulated in patients with severe coronary artery disease and

ischemic limbs [106]. While this effect may be lifesaving as a response to myocardial ischemia, it may be

pathological when It results in hyperpermeability in the eyes and other organs [106, 108].

VEGF is not a single molecule but a combination of several isomers that break up into 5 subgroups

namely: VEGFA, VEGFB, VEGFC, VEGFD and placental growth factor [106]. VEGFA is the primary isomer

involved in the regulation of both physiological and pathological angiogenesis [106]. Further

investigation has shown that out of the six different human isoforms of VEGFA, VEGFA165, is the most

important isoform in the process of angiogenesis. Strategies to alter the process of angiogenesis have

been developed based on the information on these isoforms and their relative importance[106].

VEGF in the blood activates 3 membrane spanning tyrosine kinases: VEGFR-1, VEGFR-2, VEGFR-3 leading

to a biochemical cascade[106, 108]. Specifically, stimulation of VEGFR-1 results in the release of tissue

specific growth factors, recruitment of endothelial progenitors and induction of matrix

metalloproteinases (MMPs) [109]. MMPs contribute to the breakdown of the ECM and increase VEGF

expression and secretion from RPE cells via a feedback mechanism [3]. The binding of VEGF165 to the

extracellular domains of VEGFR2 results in dimerization which leads to the activation of intracellular

tyrosine kinase moieties. These trigger various biochemical cascades in the cell that produces the

mitogenic, angiogenic, permeability enhancing and anti-apoptic effects of VEGF [109, 110]. They:

upregulate the production of MMPs dissolving the intercellular matrix

cleave sequestered VEGF-A isomers amplifying the effect of VEGF

phosphorylate intercellular proteins to release tight junction and

Promote mitosis and the recruitment of endothelial cell progenitors to produce new vascular

endothelial cells [110]

In areas of highest VEGF concentrations, pre-existing vessels are seen to give rise to vascular buds that

are immature and leak blood. They elongate into tubules and become fully competent when they are

covered by a sheath of pericytes to form mature vascular loops [110].

Since pericytes require platelet derived growth factor (PDGF) for proliferation, migration, and survival,

interrupting the process of PDGF may prevent vessel maturation and prolong the interval during which

the neovascular complex responds to anti-VEGF therapy [110].

Since VEGFR-1 has a higher affinity for VEGF than VEGFR-2, drugs targeting VEGF pathways use VEGFR-1

binding sequences [106]. In the eye, VEGF is produced by retinal pigment epithelial cells, neurons, glial

cells, endothelial cells, ganglion cells, Muller cells, and smooth muscle cells[106]. Its synthesis is

triggered by tissue hypoxia and it mainly targets vascular endothelial cells in the retina[106].

Additionally, VEGF-messenger RNA synthesis can be upregulated by molecules associated with

intraocular inflammatory conditions such as epidermal growth factor, insulin-like growth factors, TGF-α,

TGF-β, keratinocyte growth factor, insulin like growth factor, platelet derived growth factor, fibroblast

growth factor, interleukin-1α, interleukin-6 and protein kinase C[106].

The detection of VEGF in neovacular membranes has been the primary driver for the development of

anti-VEGF therapies. The use of anti-VEGF agents in AMD is discussed later in this article.

Cell Death Pathways

All processes in advanced AMD pathways eventually lead to the death of photoreceptor cells. This is the

ultimate cause of vision loss, both through apoptosis and necrotosis [3].

Excessive levels of light induce apoptosis in photoreceptors, although the exact mechanism of how this

happens is unknown [111]. It has been shown that endoplasmic reticulum stress may play a pivotal role

in light exposure induced retinal damage through the activation of the C/EBP-homologous protein

(CHOP) pathway [111].

Cells that are in the phase of apoptosis are rounded and shrunken with a condensed and fragmented

nuclear DNA. Before they lyse and result in inflammation, they are engulfed by white blood cells [3].

Genetics

A number of studies have linked genetics to AMD even though studying the genetics of AMD remains a

challenge for a number of reasons [3]. Siblings are ideal controls when performing the studies. However,

since AMD appears late in life, in many cases the siblings of patients may no longer be alive [3].The

condition can exist in 2 different types and both types may coexist in the same patient, making it

difficult to identify what gene defect is associated with what subtype. Furthermore, the phenotypic

characteristics of AMD have not been clearly identified [3]. Since environmental factors seem to interact

with genetic factors, it is difficult to isolate the effect of a gene polymorphism.

Nevertheless, work in this area continues and linkage and association studies have identified several

genes that may have a role in the development of AMD. 2 major loci seem to be the most replicated and

emerge as the most important in AMD: Iq32 and 10q36[3, 112].

The Iq32 locus contains several genes that are essential to immune function and the inflammatory

response [3]. These include CFH genes, CFHR genes and F13B genes. The 10q26.13 locus has genes for

PLEKHAI, ARMS2 and HTRA1.

The five genes encoding CFH related (CHFR) proteins, CFHR1-5 genes are found at the Iq32-Iq31.1 loci

[3,21]. A number of studies have demonstrated the association of Y402H polymorphism in the CFH gene

and AMD [21]. There is evidence that dysregulation of CFH may result in the pathogenic features of

AMD[3,21]. While polymorphism of this gene has been repeatedly linked to AMD via the inflammatory

pathway, a more recent proposal is that it may also have a role to play in with vascular factors that

contribute to the pathology of AMD [21].

F13B forms part of the RCA gene cluster that encodes the non catalytic B-subunit of coagulation factor

XIII (FXIII) [3]. The role of FXIII is to regulate platelet adhesion and specifically stabilizes fibrin clots and

ECMs[3]. FXIII is a tetrameric complex of 2A and 2B, which dissociates into the A and B dimers in the

presence of Ca2+ ions. The FXIII-A portion behaves as a catalyst in the coagulation cascade.

The F13B and CFH/CFHR lie in close proximity to another, encode structurally similar proteins and act on

systems that are closely related (complement and coagulation)[3].

The 10q26.13 has genes for PLEKHAI, ARMS2 and HTRA1

PLEKHAI encodes for the tandem pleckstrin homology domain-containing protein 1 (TAPP1) that

regulates B-cell activation and autoantibody production. This finding has led to the postulation that

PLEKHAI may have an immunomodulatory role in the pathogenesis of AMD[3].

ARMS2 codes for protein of unknown function, though the ARMS2 gene product has been seen to bind

several ECM proteins and has been found in multiple tissues including the retina. It is thought that the

risk of AMD is modified by ARMS2 variants through mitochondrial pathways and possibly oxidative

stress[3].

HTRA1 expression is seen to increase in patients with AMD and single nucleotide polymorphisms in

HTRA1 have a strong association with the onset of AMD [113]. It encodes for high temperature

requirement A (HtrA) proteins that may alter the intergrity of connective tissue in the retina by

interaction with ECM components [3]. The elastic properties of the HTRA1 protein may be the cause of

the Bruch membrane and choroidal lesions that are seen in mice with HTRA1 overexpression [3]. One

study has shown that it is 2 common variants of the HTRA1 found within the insulin like growth

factorIGF-1 binding domain that contribute the increased risk of AMD [114]. Another study has shown

that the induction of HtrA1 by oxidative stress leads to the premature death of cells and senescence

through p38 MAPK [113].

Many other loci have been associated with AMD although their contribution is yet to be established

fully. These can be grouped into those associated with immune function and inflammation, extracellular

matrix and cell adhesion, lipid/protein metabolism and transport, angiogenesis, cellular stress and

toxicity [3].

A recent study has identified seven new loci near the genes COL8A1-FILIP1L, IER3-DDR1, SLC16A8,

TGFBR1, RAD51B, ADAMTS9 and B3GALTL that may possibly be associated with AMD. Further research

is required to identify and establish their roles [115]. Two genome-wide association studies have

discovered a variation in several genes including LIPC (hepatic lipase), ABCA1 (ATP-binding cassette,

subfamily A member 1) and TIMP3 (encoding metalloproteinaseinhibitor 3), among others [24].

Inflammation and Immunity

Inflammation and immune function pathways in AMD are associated with genes from Iq32 locus, the

PLEKHAI from the 10q26 locus as well as a few others are discussed next.

Chimeric CFH transgenic mice have been shown to develop typical features of AMD [116].

Various studies and meta-analysis have demonstrated a reduced risk of AMD in patients with C2/CFB

polymorphisms at the 6p21.3 locus [3]. A study conducted to investigate the link between the C2-CFB-

RDBP-SKIV2L region and the development of AMD showed that SKIV2L has a significant role to play in

the development of neovascular AMD but not in polypoidal choroidal vasculopathy (PCV). This effect

was seen to be independent of CFH and HTRA1 [117]. Another study conducted on the Han Chinese

population showed that the rs429608 genetic variant in the SKIV2L gene was significantly associated

with AMD [118].

Other gene polymorphisms that have some data to link them to AMD through inflammatory pathways

include:

C3/CFD at the 19p13.3 – p13.2 loci,

CFI at the 4q25 loci

SERPING1 found at 11q12-q13.1

RORA found at 15q22.

CX3CR1 gene found at 3p21.3[3].

CCR3 gene polymorphism may lead to chronic inflammation in the complement system resulting in

AMD. A recent study has shown that there are two CCR3 single nucleotide polymorphisms that could be

responsible and are involved in the development of AMD [119].

Extracellular Matrix and Cell Adhesion

In addition to F13B and ARMS2/HTRA1, there are a number of other genes that affect the structure of

the ECM in AMD. Furthermore since the Bruch’s membrane is an ECM composed of elastin and collagen,

any polymorphisms in the genes that encode for ECM may result in pathological changes in the Bruch’s

membrane at various stages of AMD[3].

FBLN5 located at 14q32.1 encodes for Fibulin 5, an ECM protein that promotes elastic fiber

assembly and maturation.

HMCN1 or FBLN6 found on loci 1q25.3 – q31.1. Hemicentin (or fibulin 6) is the elastin associated

component of the ECM.

ROBO1 located at 3p12; the gene product is an immunoglobulin that is down-regulated in

patients with neovascular AMD [3].

CST3 located at 20p11.21 encodes cystatin C, an abundant extracellular inhibitor of cysteine

proteases that is involved in ECM remodeling in retinal degeneration.

COL8A1 and COL10A1 that encode the α-chains of the type VIII and type X collagens.

MMP-9 and TIMP-3 encode the MMPs and TIMPs

Lipid Transport and Metabolism

Lipid accumulation is central to AMD, and it is very likely that genetics plays an important role in this

process alongside other factors such as aging. Several genes have been linked to lipid transport and

metabolism in AMD [3].

ABCA1(9q31.1) and ABCA4(1p22.1-p21): expressed in the retina and RPE, encodes a cholesterol

efflux pump in the cellular lipid removal pathway

ABCA4 is primarily expressed in the retina. It encodes the retina specific ATP-binding cassette

transporter (ABCR) which clears all-trans-retinal and is associated with the buildup of A2E and

lipofuscin

CETP (16q21) which encodes for cholesteryl transfer protein (CETP), a protein that forms a part

of the retinas lipid transport system.

CYP24A1 (20q13) that encodes a mitochondrial enzyme of the cytochrome P450 family involved

in drug metabolism, lipid synthesis, and anti-inflammatory effects.

ELOVL4(6q14) that encodes ELOVL fatty acid elongase 4

FADS1-3(11q12.2-q13.1), which encode 3 members of the fatty acid desaturase family

LIPC (11q12) which codes hepatic lipase,

LPL (8p22) which encodes lipoprotein lipase

LPR6 (12p13.2) that encodes low density lipoprotein receptor related protein 6.

Angiogenesis

The genes associated with angiogenesis include:

SERPINF (17p13.3) encodes PEDF, an endogenous inhibitor of angiogenesis [3]

VEGFA (6p12) that encodes VEGF[3]

VLDLR (9p24): very low density lipoprotein receptor that has an important role in the

metabolism of triglycerides and very low density lipoproteins; however, rather than affecting

AMD by altering lipid metabolism, VLDLR seems to increase the risk of AMD by resulting in

angiogenesis[3].

CC chemokine receptor 3 (CCR3) that has recently shown to contribute to choroidal

neovascularization. It is a receptor for eotaxin and is found on many cells including white blood

cells as well as mast cells and eosinophils that are involved in allergic reactions such as

angiogenesis [119].

Cellular Stress and Toxicity

The extent of tissue damage in AMD is dependent upon how the cell responds to some level of cellular

stress or toxicity [3]. Cellular responses to stress can be divided into those that are protective and those

that are destructive [3]. Activation of detoxification or survival pathways for example constitutes

protective pathways [3]. On the other hand activation of cell death pathways such as apoptosis or

necrosis is destructive [3].

Many genes associated with AMD influence the cellular stress responses through various processes

including inflammation, ECM function, lipid metabolism and angiogenesis [3]. For example complement

and the coagulation cascade are both proteolytic cascades that serve as cellular responses to injury.

HTRA1 enzymes for example are a family of proteases that are involved in regulated proteolysis, a form

of cellular stress response [3]. ECM components including ACE and ARMS2 have a part to play in

oxidative stress and lipid transporters that are encoded by ABCA4 and APOE[3].

It is very likely that genes that encode for receptors have an even greater role to play in the stress

response of the cell since they provide the basis for interaction and signaling of the external

environment [3]. Such genes include CX3CR1, ROBO1, RORA, TLR3, TLR4 and VLDLR[3].

Toll like receptors (TLRs) are essential receptors of the innate immune system where they behave as first

responders for protection against viral and bacterial pathogens. Several TLRs have been shown to have a

role in regulating cell death and survival in non pathogen injuries such as stroke and oxidative stress. A

study conducted to examine the effect of TLR3 in regulating cell survival during oxidative stress

demonstrated that the TLR3 signaling was activated in the presence of oxidative stress. The result is a

protective effect on RPE cells leading to increased cell viability through a central mechanism of signal

transducer and activator of transcription 3 (STAT3) which was a central mechanism in this effect [120].

Based on these findings the authors of this study propose that dysregulation of TLR3 activity may a

factor in the development of AMD.

Other genes that have been linked to the increased risk of AMD through cellular stress response include:

GSTM1 (1p13.3), GSTP1 (11q13), and GSTT1( 22q11.23): these encode for glutathione S-

transferases that is involved in xenobiotic metabolism and detoxification of peroxidized lipids [3]

SOD2 (6q25.3) superoxidase dismutase 2: encodes and antioxidant enzyme that maintains

reactive oxygen species including hydrogen peroxide [3]

TF (3q22.1): transferring proteins that manage free iron levels in the blood and may be altered

in ocular diseases including AMD and retinal degenerations [3]

paraoxonase 1 (PON1) L55M and Q192R single nucleotide polymorphism: the MM and QQ

genotypes have been found to be associated with a decreased risk of AMD following a study

that adjusted for age, gender, and the prevalence of smoking, hypertension, diabetes, and

hypercholesterolemia [121].

Microglia

Microglia is the name given to immune cells found in the central nervous system. They are thought to

undergo changes in their gene expression patterns during the aging process leading to pathogenic

phenotypes that are responsible for age related neurodegenerative disorders [122]. One study has

demonstrated that molecular pathways including those that are responsible for immune function and

regulation, angiogenesis, and neurotrophin signaling undergo age related changes. From the findings the

authors concluded that this is possibly due to the senescent microglia altering their constitutive support

functions and regulation of neuroinflammation and neurodegeneration in the CNS [122].

Phagocytosis

P2X7 has been identified as a scavenger receptor that mediates the phagocytosis of apoptic cells.

Furthermore it has been shown that the P2X4Tyr315Cys variant is two times more frequent in patients

with AMD. Additionally the two minor alleles, Tyr315Cys in the P2RX4 gene and Gly150Arg in the P2RX7

gene are overexpressed in patients with AMD [123].

Diagnosis

Since the onset of the condition is so gradual and often goes unnoticed for a long time, routine dilated

eye examinations are recommended [22]. Furthermore, it is useful for primary care physicians to know

their patient profiles and be able to identify high risk patients. The diagnosis is usually established by an

ophthalmologist.

Signs and Symptoms

Patients complain of acute vision loss, metamorphopshia or blurred vision, scotomas or chronic

distortion of vision [5, 119]. Early disease is associated with very gradual loss of vision. Late disease can

lead to a significant loss of vision over a relatively short time

Many patients with AMD will also present with depressive symptoms. Recent insights into the

information the pathways by which impaired vision leads to depression strengthen the association

between the 2 conditions. It may be important to evaluate an AMD patient for depression and treat

both conditions simultaneously [124].

Often patients that have AMD associated with a macular scotoma will have difficulty writing legibly. It is

postulated that the reason for this is the close proximity of the retinal locations of the pen tip and the

fPRL or fovea [125].

It has been shown that about 13% of patients with AMD present with a condition known as the Charles-

Bonnet Syndrome. This is a condition related to mentally healthy patients with loss of vision and

complex visual hallucinations. The hallucinations are well defined, organized and clear images over

which the subject has very little or no control over [126]. Charles Bonnet Syndrome is a benign condition

that may regress in many cases as the visual cortex adapts to the loss of vision [127].

AMD can be graded into one of several categories including:

without druse,

several minute drusen and no RPE changes,

retinal RPE alterations but no drusen,

both small drusen and RPE changes,

several large and intermediate size drusen ,

RPE detachment,

geographic atrophy

choroidal neovascular membrane with disciform scarring [119].

Drusen are usually the first clinical sign, but not the first detectable change in an eye with AMD. Patients

may complain of impaired dark adaptation when moving from bright to dimmer environments although

central visual acuity remains the same. For this reason, testing dark adaptation may be useful in patients

to detect early stages of AMD [3].

Drusen are described clinically by their size and contour [3]. Small drusen are those that are less

than<63µm. Small, hard drusen are generally few and tend to distribute at the retinal periphery. They

are commonly associated with aging, seen in the eyes of healthy young and middle aged adults and are

usually not indicative of AMD [3, 7]. Hard drusen however tend to coalesce into larger. Small hard

drusen increase incidence of large soft drusen as well as RPE abnormalities thereby increasing the

possibility of the disease progressing to a more advanced stage [7, 47]. It is interesting to note that

“hard” drusen have also been isolates in conditions such as long standing serious retinal detachment or

hereditary macular dystrophies, although the characteristics of those drusen seem to differ slightly [7].

Soft drusen that are usually larger are often more numerous and tend to localize in the macular area.

They are an early sign of AMD and a high risk factor for the development of AMD [7]. Whether they

advance the disease or are simply a sign of things to come remains to be determined [7].

The presence of intermediate drusen that are 63-125µm and large drusen that are >125µm in the

macular region is an indication of AMD [3].

Cuticular or basal laminar drusen are small, uniformly-sized, and yellow. These do not seem to be

connected to AMD [3]. Reticular pseudodrusen are a network of yellow, oval or roundish lesions with a

diameter of 125-250µm [3]. They are best seen with a red-free imaging or infrared wavelengths of the

scanning laser opthalmoscope but are not hyperflourescent on fluorescein angiography [3]. Subretinal

drusenoid deposits or reticular pseudodrusen are associated with AMD and thought to be a sign of

possible progression to advance AMD [3].

Focal hyper-pigmentation of the RPE is seen as focal areas of grey or black pigment

Disease progression is monitored through clinical examination of macula as well as the use of disease

classification systems [3]. A number of investigations have been conducted to formulate an ideal

classification system for AMD. One study attempted to achieve this using a modified Delphi process

[128]. The researchers concluded that the following classification system was useful in predicting the

progression and risk of AMD:

no visible drusen or pigmentary abnormalities should be considered to have no signs of AMD

presence of small drusen (<63 μm), also termed drupelets, should be considered to have normal

aging changes with no clinically relevant increased risk of late AMD developing

the presence of medium drusen (≥63-<125 μm), but without pigmentary abnormalities thought

to be related to AMD, should be considered to have early AMD

the presence of large drusen or with pigmentary abnormalities associated with at least medium

drusen should be considered to have intermediate AMD.

Individuals with lesions associated with neovascular AMD or geographic atrophy should be considered

to have late AMD [128].

Home monitoring

Amsler Grid

The Amlser grid is a 4 x 4 inch checkerboard square that has proven to be an effective tool for

monitoring progression of the condition [5]. Patient is given a copy of the grid to use regularly at home.

The patient is instructed to hold the grid up straight and stand 35cm back from the grid. They close one

eye and focus the open eye on the center dot in the grid and repeat with the other eye. The patient

should be directed to perform this test daily at home and if the patient detects line distortions or

scotomas for more than one ot two days, he or she should contact a health care practitioner[5].

Unfortunately, the Amsler Grid has shown to have poor accuracy as a screening test[5, 129].

Mobile Handheld Devices

More recently, novel computing systems using mobile handheld devices have been tested to monitor

the retinal visual function of patients with AMD. One particular study investigated the usefulness of the

Health Management Tool (HMT), one such computing system, in neovascular AMD patients taking

ranibizumab [130]. The researchers found that elderly patients were willing to and were capable of

complying with the use of mobile handheld devices for the self monitoring of retinal visual function on a

daily basis [130].

Screening tests

Various screening tests may be used by an ophthalmologist to establish a diagnosis. These include

dilated funduscopic examination and various forms of retinal imaging. In advanced cases, referral to a

retinal specialist may be required.

Visual Acuity Tests

Visual acuity tests include near, distant, pinhole or reading. A recent study showed that reading acuity

might be a more sensitive measure for vision decrease than distance visual acuity in patients with

macular diseases such as AMD. Furthermore, the authors found that reading acuity was useful in

assessing intravitreal therapy efficacy[131]. Overall however, visual acuity screening tests seem to have

low accuracy when compared with a full ophthalmologic examination for identifying the presence of

AMD [129]. The Snellen Eye Chart is widely used to screen for visual acuity [129]. The patient is asked to

stand 20 feet from the chart and read letters. Inability to read letters on or below the 20/40 line with

their best-corrected vision (using glasses) indicates the need for further evaluation. It is interesting to

note however that there are no studies to evaluate its accuracy against a clinically relevant reference

standard [129].

Funduscopic examination

Digital color retinal fundus images are used to screen for many eye diseases including AMD [63].

Color fundus photographs are useful in finding landmarks such as drusen evaluating retinal

detachments. Furthermore, when used alongside angiography, they are useful in determining the

etiology of blocked fluorescence. Funduscopic examination also provides a reliable baseline for patients

with advanced non-neovascular AMD and monitoring the effectiveness of therapy in treated patients

[6].

Optical Coherence tomography

Optical Coherence Tomography (OCT) is a non invasive imaging technique that provides high resolution,

cross sectional images of the retina, retinal nerve fiber layer and the optic nerve head, that cannot be

obtained with any other imaging technology presently available [6,132]. It is a useful technique in any

patient with AMD with complaints of visual acuity and is valuable in assessing the correlation between

drusen and choroidal thickness, both of which are a good measure of visual acuity [1, 132, 133].

Furthermore it can be used to determine the presence of subretinal fluid [6], one of the earliest

manifestations of neovascularization [6]. Following the structural changes in the retina accurately

provides a good platform for evaluating the response of the eye to therapy. This technique allows the

differential diagnosis of AMD, ruling out cases of visual acuity loss that may be the result of other

conditions such as subtle epiretinal membrane or vitreomacular traction [1]. OCT provides an insight to

the etiology of metamorphosia and visual acuity loss through a detailed evaluation of the RPE and

photoreceptor layer.

It is possible that advances in OCT such as spectral domain OCT, longer-wavelength OCT systems

including the swept-source technology, along with Doppler OCT and en-face imaging may allow

increased resolution of the retina [6,132]. One study performed to assess drusenoid retinal pigment

epithelial detachments (DPED) secondary to age-related macular degeneration (AMD) utilized spectral

domain OCT. The researchers found this technique is able to detect slight changes in the area and

volume of drusenoid retinal pigment epithelial detachment [134]. They concluded that it was valuable in

identifying the natural history of disease progression and to monitor eyes with AMD [134].

Another study demonstrated that distinct morphological alterations visible on SD OCT imaging in the

eyes caused by GA are associated with faster rates of enlargement, increase lesion size and multifocal

patches of atrophy [135].

One research group wanted to determine whether OCT is a useful tool in predictive outcomes at 12

months in eyes with neovascular AMD in patients treated with intravitreal ranibizumab and whether

baseline OCT features can predict a change in visual acuity from baseline to 12 months. They conducted

a retrospective, observational study and collected data from cross sectional images of the macular using

the Spectralis OCT (HRA+OCT Heidelberg Engineering) from 94 eyes of patients undergoing intravitreal

ranibizumab therapy for neovascular AMD. It was shown that an intact ellipsoid zone and the external

limiting membrane in the subfoveal area at baseline were the only 2 independent good prognostic

indicators of final visual acuity at 12 months. The group found that none of the morphological features

at baseline were suitable predictors of visual acuity by 12 months. Putting all the findings of the study

together, the authors concluded that the integrity of the outer retinal layers was an essential

component in determining final visual acuity at 12 months in eyes of patients using intravitreal

ranibizumab for neovascular AMD.

Fluorescein angiography

Flourescein angiography is indicated to confirm neovascularization in patients that complain of new

metamorphosia or unexplained blurred vision. It may also be useful when a clinical examination reveals

an elevation of the RPE or retina, subretinal blood, hard exudate or subretinal fibrosis. All this

information helps determine whether treatment is indicated, what therapeutic intervention may be

suitable, whether the eye is eligible for laser treatment, and allow the recording of a baseline from

which treatment effectiveness and disease progression can be monitored [5,6].

Flourescein angiography should be performed by a physician who is aware of the potential risks

associated with the procedure including death [6]. Any medical facility offering this procedure should

have in place an emergency care plan should medical complications arise [6]. The results should be

interpreted by an individual experienced in managing patients with neovascular AMD as soon as

possible since extrafoveal or juxtafoveal lesions can quickly spread causing irreversible damage and

render any therapeutic modality ineffective [6].

Using the pattern of fluorescence from the angiogram, the neovascular lesion may be categorized as

either:

Classic: whereby there is bright uniform, early hyperfluorescence exhibiting leakage in the late

phase and obscuring the boundaries.

Occult: that is associated with either

fibrovascular PED which is seen as an area of irregular elevation of the RPE. This area is

neither bright nor discrete, rather with a stippled hyperfluorescence present in the

midphase of the angiogram and leakage or staining by the late phase.

late leakage from an undetermined source mostly seen as a speckled hyperfluorescence

with dye pooled in the subretinal space in the late phase. The source is undetermined as

it does not correspond to classic CNV or fibrovascular PED in the early or the midphase

of the angiogram [1].

When reviewing an angiogram, the presence of hemorrhage, blocked fluorescence that is not due to a

hemorrhage, or serous detachment of the RPE should also be reviewed [1].

Recently the Age-Related Macular Degeneration Detection of Onset of new Choroidal

neovascularization Study (AMD DOC Study) showed that fluorescein angiography is currently the best

diagnostic method available for the detection of new onset CNV [137].

Furthermore, preliminary data from a small sample study have shown that computer aided methods to

segment CNV lesions provide similar results to those with manual segmentation and in fact may reduce

the burden of manual segmentation as well as reduce inter- and intra-observer variability [138].

Indocyanine Green (ICG) Angiography

More recently indocyanine green (ICG) has been used in angiography. This dye allows better delineation

of the choiroidal circulation over fluorescein angiography. ICG angiography has therefore found a role in

the detection of areas of occult CNV. It may also be useful in evaluating pigment epithelial detachment,

poorly defined CNV, and lesions such as retinal angiomatous proliferation or polypoidal choroidal

vasculopathy. In fact it is thought the PCV may be mistaken for CNV, without the use of ICG angiography

particularly in patients of African or Asian descent. [6].

The interpretation of CNV lesions from ICG angiography leads to their categorization into one of the

following 3 classes:

focal hot spots

plaques

a combination of focal hot spots and plaques [1]

Fundus autoflourescence

Fundus autoflourescence (FAF) is a screening test that is based upon the detection of pigments [9]. The

retina is known to exhibit an inherent autofloursecence that can clinically be imaged using

ophthalmoscopy, spectroflourometry and adaptive optics [139]. This emission derives from the complex

mixture of bisretinoid pigments that accumulate in the RPE cells as lipofuscin as has been demonstrated

by the spectral features, spatial distribution, age-dependent intensities and disease-related

characteristics of fundus autofluorescence [139].

Fundus autoflourescence detects the pigments lipofuscin and melanin using short wave FAF (SW-FAF)

and near infrared FAF (NIR-FAF) respectively [9]. Short wave FAF (SW-FAF) uses excitation 488nm

employed with a confocal scanning laser ophthalmoscope (cSLO) and emission >500nm. This is an

imaging method that allows in vivo topographic mapping of lipofuscin distribution. Near infrared FAF

(NIR-FAF) has slightly higher excitation and emission values of 787nm and>800nm respectively. This

technique is useful for the imaging of the distribution of melanin, a flourophore of the RPE cells and

choroid- useful in quantifying GA that is secondary to AMD [9].

The lipofuscin related pigments of the retina have been shown to display a range of absorbance maxima

in the visible spectrum including 430 nm (all-trans-retinal dimer), 439 nm (A2E), 449 nm (A2PE), 426 nm

(isoA2E), 490 nm (A2- dihydropyridine-phosphatidylethanolamine, A2-DHP-PE) and 510 nm (all-trans-

retinal dimer-phosphatidylethanolamine and all-trans-retinal dimer-ethanolamine) [139].

The compounds are formed as a result of the light capturing function of the retina. Therefore individuals

that lack the 11-cis- and all-trans-retinal chromophores of visual pigment, such as patients with early

onset retinal dystrophy associated with mutation in the RPE65, there is not formation of lipofuscin and

consequently fundus autoflourescence is not observed [139].

Fundus autoflourescence is useful in a number of retinal conditions including geographic atrophy

whereby the absence of RPE shows up as a severely reduced FAF signal when atrophic areas of GA are

assessed [1]. Localized areas of enhanced fundus autofluorescence may be observed as an enhanced

signal at the margin of geographic atrophy. It may be predictive of visual acuity outcomes in neovascular

AMD, although this role is yet to be established [1].

When comparing the two wavelengths, a recent study has shown the SW-FAF underestimates GA when

compared to NIR-FAF at baseline. It was found the GA always seemed larger on NIR-FAF than SW-FAF

images. This is possibly because the normal hypo-autoflourescence in SW-FAF due to the foveal

pigments is difficult to distinguish from the hypo-autoflourescence caused by the GA [9].

Ultrasonography

Ultrasonography may be indicated in a few cases for the diagnosis of neovascular AMD. If the media is

obscured by a CNV break through hemorrhage, B-scan ultrasonography may be required as a clear view

of the fundus through an ophthalmoscope is blocked. If the lesion simulates a tumor, the acoustic

properties of the eye may provide an useful insight into the condition [1]

When comparing the measurements of drusen from manual segmentation of color fundus photographs

to those obtained from spectral domain OCT images, it was found that the two techniques only provided

a fair agreement. The authors proposed that both approaches should be used as they provide

complimentary information about the drusen [140].

With regard to the detection of new onset choroidal neovascularization, one study demonstrated that

OCT has a greater specificity than the use of the Amsler Grid [137].

Assessing the severity

Static and flicker perimetry have been tested to assess the severity of AMD recently. One study has

shown that these techniques are similarly affected across the spectrum of AMD severity and seem to be

valid techniques for assessing retinal sensitivity in AMD between the time that drusen are >125µm and

late AMD [141].

Differential Diagnosis

While making a differential diagnosis of AMD, it is useful to note that a patient suffering from AMD is

typically aging and may have drusen in the affected eye as well as the fellow eye. The differential

diagnosis of dry AMD includes a number of other conditions that affect the RPE and choriocapillaris

including:

Pattern dystrophy

Stargardt’s disease

Best’s disease

Angioid streaks

Adult Vitelliform Dystrophy

Similarly CNV, may be caused by other conditions such as:

Angioid Streaks

Best’s disease

Choroidal osteoma

Fundus flavumaculatus

Idiopathic causes

Multifocal choroditis

Optic disc drusen

Pathological myopia

Pattern dystrophies

Photocoagulation

Sarcoidosis

Serpiginous or geographic choroiditis

Toxoplasmic retinochoroiditis

Traumatic choroidal rupture

Polypoidal choroidal vasculopathy [1].

Prognosis and History

The risk of progression to an more advanced stage depends upon a number of factors including the

characteristics of the macula, the status of the fellow eye and the patients genetic makeup [1].

One study group attempted to develop a severity scale that is able to estimate a patient’s 5 year risk of

developing a more advanced form of AMD. The results of this study are summarized in the table below

[1].

Finding Risk of advanced AMD in 5 years

dry AMD in both eyes but no large drusen or pigment changes in either eye 0.4–3.1%

dry AMD in both eyes with a large drusen or pigment change appears in both eyes

11.8%

large drusen and pigment changes in both eyes 47.3%

One study showed that an individual suffering from systemic arterial hypertension with either subfoveal

or juxtafoveal CNV lesions in one eye and multiple drusen with focal RPE hyperpigmentation has a 87%

risk of developing CNV in the fellow eye over a 5 year period.

As a general observation, CNV associated with AMD has a poor prognosis [1].

Early AMD

Patients with early AMD have a central visual acuity similar to that of patients with normal maculae. The

AREDs study showed that patients with early AMD were low risk patients that had a 1.3% chance of

advancing to category 4 over 5 years in either eye [6].

Intermediate AMD

The risk of progression from category 3 to 4 over 5 years has been shown to be around 18% in the

AREDS. The risk is lower (6.3%) for patients with large drusen in one eye, but 26% in patients with

bilateral large drusen [6].

Advanced AMD

The Beaver Dam Eye study showed that approximately 22% of the fellow eyes of patients with advanced

AMD developed either geographic atrophy or neovascular changes over 5 years [142].

Occult CNV

Studies performed on patients with occult CNV demonstrated a poor visual outcome over 5 years. The

results of the Verteporfin in Photodynamic Therapy (VIP) Study showed that at 12 months, 73% of eyes

experienced a loss of vision from the baseline, out of which the vision loss was severe in 32% of cases

[143]. Further along at 24 months, the loss of vision from baseline increased to 79% and the proportion

of cases with severe vision loss increased to 43%. The mean loss of visual acuity from baseline was four

lines (20 letters) and five lines (25 letters) at 12 and 24 months, respectively [143].

Classic CNV

In the Macular Photocoagulation Study Group (MPS), untreated patients with classic CNV showed a

reduction of visual acuity of 1.9 lines at 3 months and 4.4 lines at 24 months. At 3 months, 11% of eyes

demonstrated severe visual loss and at 24 months, 37% of eyes demonstrated severe visual loss [144].

Another study conducted later, the Treatment of Age-related Macular Degeneration with Photodynamic

Therapy (TAP) study showed a similar trend. The researchers found a mean loss of 2 lines at 3 months,

3.5 lines at 12 months, and 3.9 lines at 24 months. Furthermore, 36% of eyes had severe visual loss at 24

months [145, 146].

The Age-Related Eye Disease Study (AREDS) group attempted to formulate a clinical scale that can be

used to define risk categories for the development of advanced AMD. It utilizes a grading system

whereby each eye is assigned one risk factor for the presence of one or more large drusen (125 µm in

diameter) and one risk factor for any pigment abnormality [6]. Patients that have intermediate drusen

but no large drusen in both eyes are assigned one risk factor. The presence of advanced AMD in one eye

is counted as two risk factors [6]. If the eyes have large drusen and RPE hypo/hyperpigmentary changes,

they are counted as four risk factors [6]. The risk factors are added up for both eyes giving a 5-step scale,

0-4. The approximate 5-year risk of developing advanced AMD in at least one eye increases according to

this scale by the following percentages:

Zero factors: 0.5%

One factor : 3%

Two factors: 12%

Three factors: 25%;

Four factors: 50%. [6]

Some studies on the effect of AMD in the development of other conditions have also been conducted. It

has been shown that diabetes mellitus and end-stage renal disease are more prevalent in patients with

nAMD than in those with PCV. Specific systemic conditions might be associated with the development of

nAMD [147].

Management

Due to the aging population, the impact of macular degeneration will only become greater in the future.

This gives rise to a need for preventive strategies as well as effective treatment options

The approach used in the management of AMD is focused on identifying and attacking the disease in its

early stages, slowing down progression and loss of vision [3]. Various lifestyle changes and dietary

constituents have been established as having a beneficial effect on preventing the disease and halting

progression [3]. The current focus in research is on targeting complement pathways, disturbing the light

cycle and intervening in the actions of inflammasome and other inflammatory mediators [3].

Foods for sight preservation

Numerous studies have shown that nutrition plays an important role in AMD; patients who consume

diets high in fruits and vegetables particularly green leafy vegetables have been seen to have a lower

risk of wet AMD

Cold water fish

Cold water, deep dwelling fish such as sardines, cod and mackerel are an excellent source of

docosahexanoic acid (DHA). Algae eating fish also contain high amounts of DHA. DHA is vital in providing

structural support to the cell membranes including that of photoreceptors. It has been recommended

for dry eyes, treatment of macular degeneration and preservation of sight [22]. Studies have shown that

individuals consuming fish less than once a month are two times more likely to develop macular

degeneration than those that consume fish at least once a week. Moreover, it has been demonstrated

that patients with a high level of dietary cholesterol have a 2.7 time greater chance of developing AMD

than patients that consumed low levels of dietary cholesterol.

There is evidence to suggest that omega-3 dietary fatty acids, that contain DHA, inhibit angiogenesis,

although the exact mechanism by which this occurs is yet to be understood. One study has shown that it

is the epoxydocosapentaenoic acids (EDPs), which are lipid mediators produced by cytochrome P450

epoxygenases from omega-3 fatty acid docosahexaenoic acid, which have an inhibitory effect on VEGF-

induced angiogenesis in vivo. Furthermore EDPS were shown to suppress endothelial cell migration and

protease production in vitro through a pathway that utilizes VEGF receptors 2. On the other hand

derivatives of omega – arachidonic acid were shown to increase angiogenesis [148]

Another study assessed the link between late AMD and plasma n3polyunsaturated fatty acids (PUFA).

Researchers performed non-mydriatic color retinal photographs at all examinations and spectral domain

optical coherence tomography at follow-up and adjusted for age, gender, smoking, education, physical

activity, plasma HDL-cholesterol, plasma triglycerides, CFH Y402H, apoE4, and ARMS2 A69S

polymorphisms, and follow-up time. It was observed that patients with a comparatively higher plasma

total n-3 PUFA had a lower risk of late AMD. The authors propose that further research is required in

this area to determine the extent and mechanism of this effect [149].

Green leafy vegetables

Green leafy vegetables such as spinach and kale are rich in caroteinoids, particularly lutein and xanthine.

The role of lutein and xanthine is discussed later in this article [22].

Eggs

Rich in cysteine, sulfur, lecithin, amino acids and lutein; sulfur and sulfur compounds have a protective

role towards the lens of the eye and prevent cataract formation [22].

Garlic and Onions

Garlic, onions, shallots and capers are rich in sulfur. Sulfur is required in the production of glutathione

that functions as an important antioxidant for the body including the lens of the eye [22].

Soy

Soy is low in fat and high in protein. It contains essential fatty acids, phytoestrogen, Vitamin E as well as

natural anti-inflammatory agents [22].

Yellow Fruits and Vegetables

Yellow fruits and vegetables such as carrots and sweet potatoes contain Vitamin A, C and E, beta-

carotein and lutein. They are particularly important in daytime vision [22].

Blueberries and Grapes

Blueberries and grapes contain anthocyanins that improve night vision. Dark adaptation can be

improved within 30 minutes with a cupful of blueberries, huckleberry jam or a 100-mg bilberry

supplement [22].

Wine

In addition to its cardio-protective role, red wine contains many important nutrients that protect vision

[22].

Nuts

Grains such a flaxseed, contain a high amount of omega-3 fatty acids that help lower serum cholesterol

and stabilize cell membranes [22].

Extra virgin olive oil

Extra virgin olive oil is a monounsaturated fat; diets high in saturated fats increase the risk of AMD

whereas those high in unsaturated fats decrease the risk; butter and margarine should therefore be

substituted by extra virgin olive oil or other unsaturated fats [22].

Supplements

Lutein and Zeaxanthin

Zeaxanthin and lutein are both xanophylls that compose macular pigment and have been identified as

essential components for eye health through epidemiological, clinical and interventional studies [3,

150]. Their accumulation in the macular pigment is dependant upon dietary intake and serum levels and

they are also found in the iris, choroid and the lens [22].

Lutein and zeaxanthin, an isomer of lutein, are caroteinoid pigments that impart a yellow-orange color

to various common goods such as cantaloupe, pasta, corn, carrots, orange/yellow peppers, fish, salmon

and eggs. Lutein is a yellow pigment that protects the macula from sun damage and from blue light [22].

In fact it is the yellow color of the retina that the name macula lutea derives from [22]. Furthermore,

they improve visual acuity and eliminate harmful reactive oxidative species [22].

Lutein and zeaxanthin have been linked to a reduced risk of AMD [22,150]. They are currently under

investigation as part of the AREDS2, were excluded from the original AREDS as they were not easily

formulated for study [3].

They are both dietary nutrients obtained from plants either directly or indirectly and concentrated in the

retina [3]. These carotenoids are found in a variety of foods including apples, broccoli, brussel sprouts,

celery, collard greens, corn, cucumber, egg yolk, grapes, green beans, honeydew melon, kale, kiwi,

mango, orange pepper, orange juice, peas, pumpkin, scallions, spinach, squash and zucchini [5].

One study has been conducted recently to examine the relationship between macular pigment optical

density (MPOD) and high-contrast visual acuity (HC-VA) and low-contrast visual acuity (LC-VA) in eyes

with early age-related macular degeneration (AMD). The study concluded that low levels of macular

pigment lead to reduced visual function in both healthy eyes and eyes with early AMD [151].

It is proposed that healthy individuals should take up to 6mg of lutein a day while those with AMD

require at least 10mg of lutein a day for a number of months to allow the nutrient to concentrate in the

macula [22].

Vitamin A, beta-carotene and other carotenoids

Vitamin A protects the retinal cell membrane from light damage [22]. It is required to provide adequate

levels of rhodopsin for optimal rod function [22]. A deficiency in Vitamin A has been associated with

keratomalacia, xerophthlamia and visual impairment [22].

The value of beta carotene in the management of AMD is inconclusive [22]. Its use has been associated

with increased yellowing of the skin and an increased risk of developing lung cancer in current smokers

or smokers who stopped within the last year [6].

Vitamin C

Vitamin C (ascorbic acid) is known to recycle tocopherol retinal tissue and to reduce the loss of

rhodopsin and photoreceptor cell nuclei that occurs upon exposure to light. Additionally it rejuvenates

vitamin E and cell membrane related enzymes.

One study of patients with AMD found that the individuals with AMD had a lower intake of tocopherol,

magnesium, zinc, pyridoxine and folic acid [22]. Tocopherols protect against lipid peroxidation in the cell

membrane. Furthermore, d-alpha tocopherol has been shown to have a large protective role in the

management of macular degeneration. The current recommendations for the intake of Vitamin C for the

prevention of AMD are at least 60 mg once daily [22].

Alpha lipoic acid

Alpha lipoid acid regenerates the reduced form of Vitamin C and in this way has a beneficial effect in

AMD that is very similar to that of Vitamin C. In addition to recycling tocopherol in retinal tissue, it

serves as a nerve stabilizer that reduces insulin resistance [6].

B-vitamins

A deficiency of pyridoxine has been observed in two studies of AMD patients. Out of these, one study

also demonstrates a deficiency in folate. Another study showed that patients who had taken folate,

pyridoxine and cyanocobalamin supplements for 7.3 years had a lower rate of development of AMD [6].

A more recent trial has concluded that patients with elevated serum levels of homocysteine and clinical

deficiencies in folate and Vitamin B12 have a higher risk incident of AMD. These findings strengthen the

suggestion that Vitamin B12 and folate reduce the risk of AMD [152].

Vitamin D

Recently a number of studies have demonstrated the beneficial effect of Vitamin D supplementation in

reducing the prevalence of early AMD [153]. Vitamin D is a circulating steroid hormone that is classically

associated with bone mineralization and the regulation of calcium ions and phosphorous ions [154]. It is

synthesized in the human body from the conversion of 7-dehydrocholesterol by UV rays absorbed by the

skin. Various dietary sources and supplement also provide the body with Vitamin D. Vitamin D is

activated biologically in the liver through metabolism by liver enzymes into a form that can interact with

Vitamin D receptors (VDR).

The first study that linked Vitamin D to AMD was published in 1994 [155]. This study found that serum

Vitamin D levels were inversely associated with early AMD but not advanced AMD [155].

The CAREDS study group in particular targeted early AMD in postmenopausal women. Specifically, the

relationship between the serum 25-hydroxyvitamin D (25(OH)D) concentrations and the incidence of

early AMD was assessed. After adjusting for the major non genetic risk factors of AMD, the study

concluded that the Vitamin D status of a female has a significant impact on her odds of early AMD [153].

It is thought that Vitamin D modulates the immune system and hence prevents pathological conditions

that are associated with the inflammatory process [153]. The vitamin D receptor (VDR) is expressed on

cells of the human immune system including the vertebrate retinal tissue. They have been isolated from

cultured human retinal endothelial cells. Here they interact with circulating 25(OH)2D. The 25(OH)2D

acts to suppress pro-inflammatory cytokines. Furthermore, since Vitamin D has an inhibitory effect on

angiogenesis, it is thought that it may have a role in preventing the progression of AMD from the early

stages to more advanced neovascular conditions. The CARED study was not able to establish this with

the data collected. Further research is still required to establish the findings of the CAREDS study as well

as the possible effects of Vitamin D on the disease progression [153].

Around about the same time, a systems based analysis of the role of vitamin D on the pathogenesis of

AMD was conducted. This study revealed a protective association between UV radiation and

neovascular AMD after adjusting for smoking and polymorphisms in CFH and HTRA1. In conclusion, the

authors proposed further evaluation of the hypothesis [154].

Interestingly another study conducted to examine the same hypothesis revealed no connection between

the risk of AMD and patients Vitamin D levels [156]. The study had been set up to confirm the findings of

previous studies that in fact did find an inverse relationship between Vitamin D levels and AMD [156].

Glutathione

Glutathione is a tripeptide that has a protective role in human RPE cells that underlie the macula.

Glutathione is abundant in avocados, asparagus, garlic, watermelon and cruciferous vegetables [22]. It is

manufactured in the liver after ingestion of the appropriate amino acids and sulfur containing foods. A

water soluble compound, this peptide serves as an antioxidant and regenerator of vitamin E,

carotenoids and intracellular enzymes.

As it is broken down in the stomach rapidly, many patients may require supplementation. Some

recommendations for supplementation are:

600mg N-acetylcysteine twice daily

1000mg methysulfonylmethane once daily

200mg S-adenosylmethionine (SAMe)

250mg alpha-lipoic acid twice daily [22]

Bioflavonoids

There are not many studies that demonstrate the usefulness of bioflavonoids in AMD. One study

demonstrated that a preparation of eucalyptus and citrus bioflavonoids contributes to the protection of

bovine and rat retina from induced lipid peroxidation [22].

Amino acids

Taurine, a non bound amino acid has been shown to stabilize biological membranes that protect rod

outer segments, support cardiovascular function and modulate nerve transmission. A deficiency in

taurine has been linked to retinal degeneration and supplementation has led to the stabilization of

retinal changes [22].

Arginine, another amino acid is an important regulator of ocular perfusion. It has been shown to

increase retinal blood flow when administered intravenously [22].

Minerals

Zinc and Copper

High concentrations of zinc are found in the retina, RPE and choroid. This mineral serves as a cofactor

for many retinal enzymes including superoxide dismutase, catalase, carbonic anhydrase, retinol

dehydrogenase and protein phosphorylase [22]. It also discharges vitamin A from the liver [22]. A

supplement containing 100mg/day of zinc was found to slow the progression of AMD significantly [22].

Another study demonstrated an inverse relationship between zinc consumption and drusen formation

[22]. Zinc has been associated with an increased risk of hospitalizations due to genitourinary disorders

and copper deficiency anemia [6].

Copper is required for the synthesis of superoxide dismutase, a retinal enzyme [22]. The levels of copper

in the body are adversely affected by prolonged levels of zinc in excess of 30mg, requiring consequent

copper supplementation.

An ideal supplement that balances the zinc and copper levels in the body is one that contains 15-30mg

of zinc and 2mg of copper [22].

Magnesium

Magnesium is required for the nerve conduction and dilation of blood vessels. In this way it maintains

blood flow to the eye and brain in older persons with macular degeneration [22]. The recommended

doses for supplementation ares 400-500mg at bedtime.

Selenium

Selenium is a cofactor for vitamin E and glutathione enzymes. Patients with low selenium levels in the

serum and smoking have been shown to be at a high risk of developing AMD. The maximum

recommended dose for selenium supplementation is 200mcg/day [22].

DHA

The role of DHA has been discussed earlier in this article. It is recommended that DHA supplementation

should be maintained between 500-1000mg/day [22].

Herbal Supplements

A number of herbal supplements have been shown to reduce a patient’s risk of developing AMD.

Ginkgo

Ginkgo increases cerebral blood flow including blood flow to the retina resulting in an excitatory effect.

Ginkgo supplements should be administered with care to patients taking blood thinners as this herb has

a blood thinning effect [22].

Sage

Sage also works to improve the blood circulation although it has a calming rather than excitatory effect.

The current recommended dose is 1g taken twice daily although more studies are required to establish

its effects [22].

Bilberry

Bilberry improves the effectiveness of rhodopsin which is necessary for night vision. Its effect on AMD is

unclear although it is sometimes prescribed or added to eye care supplements [22].

Milk thistle

Silymarin, extract from milk thistle, useful in promoting liver function. Since fat soluble vitamins,

glutathione and B vitamins are stored in the liver, silymarin, an important supporter of liver function

ensures the effective storage and utilization of these vitamins. The usual dose is 150mg two to three

times daily. An alternative source of silymarin is 200mg SAMe twice daily [22].

Thermal Laser Photocoagulation.

Thermal laser photocoagulation (TLP) was the choice of treatment for many years in the management of

patients with wet AMD. In this procedure, the laser is directed towards the CNV to destroy it. This

procedure has however been associated with a high percentage of recurrences after treatment [157].

Laser photocoagulation therapy has been shown to increase the risk of short term visual acuity loss of 6

or more lines compared to observation. However, over a period of 2 years, it was shown to be superior

to observation [129].

Thermal laser photocoagulation surgery has been associated with severe vision loss following treatment,

the rupture of the Bruch’s membrane with subretinal or vitreous hemorrhage and, tears in the RPE [6].

Pharmacological Therapy

Various strategies have been used and proposed in the past including laser photocoagulation for

neovascular AMD, submacular surgery, external beam irradiation, proton beam irradiation, focal

radiation, intravitreal steroids for their antiangiogenic properties and, transpupillary thermotherapy.

These did not demonstrate efficacy, and were associated with severe adverse effects or were surpassed

by more recent therapies.

There is insufficient evidence to support the use of statins in the management of AMD for both

prevention of or delaying the onset [3].

The prognosis for patients with neovascular AMD has improved significantly with the development of

verteprofin photodynamic therapy, and antiangiogenic therapy namely intravitreal pegaptanib sodium,

intravitreal bevacizumab and intravitral ranibizumab [158].

Photodynamic therapy

Takes advantage of certain unique properties of subretinal neovascular vessels and is based on the fact

that neovascular tissue differs from normal blood vessels when it comes to retaining dye. This

therapeutic modality utilizes a combination of drugs and laser therapy in which a verteporfin

photosensitive compound that localizes to the target tissue is injected into a peripheral vein and excited

with laser light [3].

Verteporfin is activated by light of a specific wavelength. Activated verteporfin forms free radicals that

close down the leaky subretinal vessel leading to cellular injury [3, 5] and was approved by the FDA in

2000, PDT was the first pharmacological treatment for neovascular AMD [3, 158].

Patients using PDT should be advised to avoid exposure to the sun and other sources of bright light.

Some patients may complain of injection site problems, photosensitivity and infusion related back pain

during the first year of therapy [5].

One study concluded that both TLP and PDT are equally ineffective for the treatment of recurrent CNV

in patients with AMD [157]. This lack of effectiveness prompted researchers to develop newer therapies.

Antiangiogenic Therapy

Based on the angiogenic roles of VEGF in neovacscularization, VEGF inhibition has become one of the

targets of successful therapies for neovascular AMD. The introduction of anti-VEGF agents was a turning

point in the management of neovascular AMD. It has been shown that visual improvements of between

+6.9 letters to +11.3 letters can be achieved using bevacizumab (Avastin®, Genentech, S. San Francisco,

CA, USA/Roche, Basel, Switzerland), ranibizumab (Lucentis®, Genentech, S. San Francisco, CA,

USA/Roche, Basel, Switzerland), and aflibercept (Eylea®, Regeneron, Tarrytown, NY, USA) intravitreally in

patients with CNV [110].

Pegaptanib sodium, an RNA binding anti-VEGF165aptamer was the first antiangiogenic tested [159]. It

was made available in 2004 and resulted in improved vision with repeated injections [3,106]. Patients

given intravitreal injections every 6 weeks experience a decrease in the average 1 year vision loss due to

AMD from loss of 15 letter to the loss of 7 letters [3,106]. These results were shown to be similar to

those obtained from the use of Visudyne (verteporfin photodynamic therapy) [3,46].

The safety of pegaptanib has been demonstrated in clinical studies for over four years and examined at

doses up to ten times greater than clinical doses. All these have demonstrated systemic and ocular

safety [159].

More recently the use of pegaptanib as maintenance therapy for patients with neovascular age related

macular degeneration in patients that experienced a clinical improvement with induction therapy has

been tested.

The use of this agent has declined since the development of more potent drugs ranibizumab and

bevacizumab, that are both derived from the same monoclonal antibody precursor [106, 160]. Even

then one study showed that maintenance therapy with pegaptanib is a useful alternative to prolonged

non-selective VEGF inhibition using agents (ranibizumab and bevacizumab discussed below) [106].

Unlike pegaptanib, ranibizumab is seen to bind to and inhibit the biological activity of all active forms of

VEGF-A [161].

Bevacizumab is a full length murine derived anti-VEGF antibody that has been approved for treatment of

advanced adenocarcinoma of the colon. In 2004 it was tested intravitreally for use in AMD although

concerns over systemic adverse effects resulted in halting of initial studies for its use in this respect

[106]. Furthermore, scientists did not expect the full length antibody (148kDa) to cross the retina and

therefore abandoned the idea. However in 2005, groups demonstrated its efficacy in clinical trials [3].

Following these findings, bevacizumab has been widely used by retina surgeons as an off-label indication

[159]. The average vision improvement was shown to increase from 20/235 to 20/172 [162].

The next step was to determine the lowest effective dose and least frequent administration schedule.

One study was conducted with the aim of distinguishing the effect of 1.25mg to 2.5mg intravitreal

bevacizumab in the treatment of inflammatory choroidal neovascularization at 24 months [162]. The

researchers found that these doses both demonstrated stability and improvement in best corrected

visual acuity (BCVA), optical coherence tomography and fluorescein angiogram [162]. The same research

group further tested the difference between using 1.25mg and 2.5mg in patients with subfoveal

choroidal neovascularization secondary to AMD. The effect, measured as a change in BCVA and optical

coherence tomography, was positive. However the difference between the use of intravitreal

bevacizumab at doses of 1.25mg or 2.5mg was not statistically significant [162].

Ranibizumab is an anti-VEGF antibody fragment that binds all VEGF isoforms and has also demonstrated

promising results [3, 106, 159]. It is formed by cleaving bevacizumab into a smaller Fab fragment that is

48kDa [106]. The use of ranibizumab in the management of neovascular AMD was approved in 2006 by

the FDA [158].

The minimally classic occult trial in the anti-VEGF antibody ranibizumab in the treatment of neovascular

age related macular degeneration (MARINA) study found that patients gained more letters of vision than

placebo injections [106].

The Anti-VEGF Antibody for the treatment of predominantly classis choroidal neovascularization in age-

related macular degeneration (ANCHOR) study showed a greater improvement in vision loss than

photodynamic therapy (10.7 letters vs 9.8 letters) [106, 163].

The long term effects of intravitreal ranibizumab on the retinal arteriolar diameter in patients with

neovascular AMD were examined recently on ten eyes of 10 patients. The patients had not been

previously treated for neovascular AMD and were given three-monthly injections of ranibizumab

intravitreally during the trial. Based on visual acuity and OCT findings, the patients were retreated if

required and the diameter of the retinal arterioles was measured. The researchers found that

intravitreal ranibizuman induced sustained retinal arteriolar vasoconstriction in eyes with neovascular

AMD [164].

Another study examined the effect of ranibizumab on vascularized pigment epithelial detachments

based upon the different subtypes of lesions. The outcomes were measured using BCVA and changes in

the height of pigment epithelial detachment. The researchers found that ranibizumab was effective at

stabilizing vision in patients with pigment epithelial detachments. However, it demonstrated better

tolerability in patients with serous pigment epithelial detachments [165].

In a trial performed to assess the effectiveness of ranibizumab by measuring the reduction in the height

of pigment epithelial detachments (PED), patients with PEDs associated with naïve neovascular AMD,

visual acuity of >20/200, and height of PED >150 μm on OCT were given 3 injections of ranibizumab in

monthly intervals as upload therapy. A review was performed at 14 weeks after the initial treatment to

assess the height of PED, presence of intraretinal or subretinal fluid, intraretinal cysts, macular volume,

retinal thickness, presence of foveal depression, presence of hemorrhage, and visual acuity. It was found

that during the uploading phase of intravitreal ranibizumab 15% of the cases showed a complete

resolution of PED [166]

Intravitreal ranibizumab has been shown to be equally effective against both types of wet AMD: typical

neovascular AMD (tAMD) and PCV. One study measured the best-corrected visual acuity and central

retinal thickness in patients who had received three consecutive intravitreal ranibizumab treatments

(0.5 mg) every month in all types of wet AMD. Both phenotypes showed a significant improvement in

the central retinal thickness during the 12 months following initial intravitreal ranibizumab treatments,

but the improvement in best-corrected visual acuity was higher in patients with typical neovascular

AMD that those with PCV [167].

Since VEGF is only blocked temporarily, ranibizumab has to be administered monthly over a period of

approximately 2 years [168]. Despite these necessary monthly visits, patient adherence remains high.

Upon interviewing patients it was found that a decrease in vision upon examination was a greater

reason for concern than a monthly intravitreal injection [169].

A recent report reviewed the effectiveness and safety of combination therapies in the management of

AMD. The study revealed that the photodynamic therapy in combination with antivascular endothelial

growth factor and steroids is useful as second line therapy in patients that do not respond to first line

monotherapy with anti-VEGF agents. Furthermore, it is a useful option in patients that find monthly

injections a great burden. The use of radiation and antiplatelet-derived growth factor is still under

investigation but studies so far have demonstrated positive results. The authors concluded that using

therapies which target other pathways in addition to anti-VEGF agents may increase visual improvement

and offer more convenient dosing regimens [170].

In an attempt to reduce the number of intravitreal injections required for effective management of

neovascular AMD, one group studied the effect of adding PDT to ranibizumab therapy [168]. They found

that although there was a significant reduction in the number of required intravitreal injections with the

addition of PDT therapy, the functional outcomes in the combined therapy group were worse. This is

therefore not a useful option to reduce the number of intravitreal injections.

Another group investigated the safety and efficacy of bevacizumab and a steroid (triamcinolone

acetonide) for CNV unresponsive to anti-VEGF monotherapy in CNV secondary to AMD. Patients with

CNV due to AMD were treated with a combination of 1mg of intravitreal bevacizumab and 4mg of

triamcinolone. The patients eyes were assessed at 7 days, 1, 3, 6, 9, and 12 months for BCVA, fluorescein

angiography, indocyanine green angiography, and OCT. Retreatment with intravitreal bevacizumab or

combination therapy was considered at the clinicians discretion at every follow up visit pro re nata. It

was found that there was an overall improvement in the BCVA from 0 to 12 months. It was interesting to

note that the patients had experienced a loss of BCVA observed 6 months prior to combination therapy

even though they were on regular anti-VEGF therapy. Furthermore, the central retinal thickness

decreased as observed on OCT measurements. The authors concluded that using a combination of

intravitreal bevacizumab and triamcinolone acetonide was safe and effective in managing CNV in

patients that do not responsd to anti-VEGF monotherapy. In fact, this combination seemed to reverse

the loss of patient vision [171].

A Similar trial, known as the LuceDex trial, was conducted to compare the effectiveness of ranibizumab

monotherapy to a combination of intravitreal ranibizumab and dexamethasone in the treatment of

neovascular AMD. The study was able to demonstrate a possible benefit of adding dexamethasone to

ranibizumab in the management of neovascular AMD [172]. The authors propose that further research

is required to establish the benefit of adding dexamethasone to ranibizumab [172].

Ranibizumab has also been tested in submacular hemorrhage secondary to wet AMD [173]. A

retrospective study was conducted on patients with either neovascular AMD or PCV and presenting

with fovea-involving submacular hemorrhage ≥ 4 disk areas in size, of <10 days of duration. Patients

were treated with a single 0.05-mL intravitreal injection of 50 μg alteplase, 0.3 mL of 100% C3F8, and

facedown positioning for 1 week. Those that presented with newly diagnosed AMD received 3

consecutive monthly intravitreal injections of 0.5 mg ranibizumab, followed by monthly retreatment as

needed. It was shown that ranibizumab or photodynamic therapy combined with tissue plasminogen

activator and C3F8 was useful in clearing submacular hemorrhage and improving visual acuity [173].

The EXCITE trial was set up to demonstrate the efficacy and safety of a 0.3mg quarterly and 0.5mg

quarterly ranibizumab treatment regimen versus a 0.3mg monthly dosing regimen in neovascular AMD

[174]. The researchers measured the mean change on BCVA measured by early treatment diabetic

retinopathy study like charts, the central retinal thickness from baseline through 12 months and the

incidence of adverse effects. At three months, all treatment groups maintained BCVA, whereas at 12

months, the monthly regimen showed superiority over the 3 month group in terms of BCVA gain [174].

The SUSTAIN trial examined to evaluate the safety and efficacy of individualized ranibizumab treatment

in patients with neovascular AMD [175]. The study conducted over 12 months, was performed on

ranibizumab-naïve patients who were given three monthly injections of ranibizumab (0.3 mg)

followed by pro re nata (PRN) retreatment for 9 months based on pre-specified retreatment criteria. The

researchers measured the frequency of adverse effects, monthly change in the BCVA and central retinal

thickness from baseline, the time to the first retreatment and the total number of the treatments

required. The safety profile of ranibizumab was similar to previous findings substantiating earlier studies

and patients on a less than monthly retreatment schedule were exposed to a similar risk as those on a

monthly schedule in terms of adverse effects. The most frequently encountered adverse effects were

reduced visual acuity, retinal hemorrhage, conjunctival hemorrhage and increased intraocular pressure.

A low average number of re-treatments achieved efficacy outcomes [175]. Based upon the visual acuity

and optical coherence assessment, the visual acuity of the patients reached a maximum after the first 3

monthly injections and decreased slightly over the next few months with an as needed schedule.

Following this it was maintained at the same level for the remainder of the study [175].

The results of the SECURE study aimed to establish the long term safety of ranibizumab 0.5mg in

neovascular AMD were published recently [176].The study lasted 24 months and was set up as an open

label, multicenter, phase IV extension to the EXCITE and SUSTAIN trials [176].. Ranibizumab 0.5mg was

administered if a patient experienced a loss in BCVA of >5 Early Treatment Diabetic Retinopathy Study

letters measured against high visual acuity value obtained in SECURE, EXCITE and SUSTAIN studies. The

incidence of ocular and non-ocular adverse events and serious adverse events, mean change in BCVA

from baseline over time and the number of injections were all recorded [176]. The results demonstrated

that the ranibizumab administered as per a visual acuity guided flexible dosing regimen at the

investigators discretion was well tolerated over 2 years and no new safety signals were identified in

patients that continued treatment for up to 3 years. However, patients lost BCVA from the SECURE

study baseline, which the authors attributed to disease progression and possible undertreatment [176].

Recently the safety and efficacy of intravitreal bevacizumab for the treatment of eyes with neovascular

AMD with baseline visual acuity better than 70 letters (Snellen equivalent better than 20/40) was

examined [177]. The researchers divided the 90 patients into 3 groups based upon their baseline visual

acuity: Group 1 (better than 70 letters), Group 2 (70 to 61 letters), and Group 3 (60 to 51 letters). The

primary outcome measures were the best-corrected visual acuity and central retinal thickness using OCT

was used to guide intravitreal bevacizumab therapy in an as needed regimen. Both BCVA and central

retinal thickness were measured at baseline and at each follow-up visit. It was found that the use of

intravitreal bevacizumab for patients with wet AMD and baseline visual acuity better than 70 letters was

safe and able to maintain this vision for 12 months [177].

A study conducted in Australia examines the use of an inject-and-extend regimen for the management

of AMD. This prospective multicenter non randomized trial demonstrated the efficacy and safety of the

inject-and-extend ranibizumab regimen. The authors proposed that further investigation is required to

determine the usefulness of this regimen with existing regimens in terms of efficacy and economic

viability [178].

Bevacizumab quickly became more popular than ranibizumab and its wide off label use is due to its low

cost [158]. A single dose of ranibizumab costs 40 times more than bevacizumab [179]. Currently,

bevacizumanb is the most widely used drug in the United States for the management of neovascular

AMD even though there is no large scale clinical trials to support its use [179].

Genetics and the response to bevacizumab and ranibizumab

While some researchers continue to identify single nucleotide polymorphisms that are associated with

AMD, other study groups have focused on the effect of genetics in their response to therapeutic

modalities including anti-angiogenic therapy. One study assessed the effect of single-nucleotide

polymorphisms of interleukin 8, vascular endothelial growth factor, erythropoietin, complement factor

H, complement component C3, and LOC387715 genes on the patient response to bevacizumab

treatment in exudative AMD [180]. The retrospective trial used data from patient clinical records,

smoking history, OCT and angiographies to determine patient response. Patients were divided into

responders, partial responders, or non-responders based on the disappearance of intra- or subretinal

fluid as observed with OCT after 3 initial treatment visits and a median time of 3.5 months. It was found

the A allele and the homozygous AA genotype of the interleukin 8 -251A/T reduced the clinical response

to bevacizumab therapy to the extent that patients did not respond completely. Patients with an

interleukin 8 promoter polymorphism -251A/T were shown to leave persisting fluid on OCT [180].

In another study, the link between seventeen single nucleotide polymorphisms (SNPs) in known AMD

risk-associated genes and the outcome of anti-vascular endothelial growth factor (VEGF) treatment in

neovascular AMD was investigated [181]. Specifically the genes tested were including CFH (rs800292,

rs3766404, rs1061170, rs2274700 and rs393955), HTRA1 (rs11200638), CFHR1-5 (rs10922153,

rs16840639, rs6667243, and rs1853883), LOC387715/ARMS2 (rs3793917 and rs10490924), C3

(rs2230199 and rs1047286), C2 (rs547154), CFB (rs641153) and F13B (rs6003). Patients were treated

with 3 monthly injections of either ranibizumab or bevacizumab injections followed by 9 months of "as

required" injections based on clinician’s decision at each follow-up visit according to retreatment

criteria. The effect of therapy on the mean change in visual acuity was used as the primary outcome

measure. It was found that there was a strong association between the HTRA1 promoter SNP

(rs11200638) and A69S at LOC387715/ARMS2 and a poorer visual outcome for ranibizumab or

bevacizumab treatment in neovascular AMD [181].

Another study focused on the association between the response to ranibizumab and VEGF-A gene SNPs,

rs699947 (-2578A/C) and rs1570360 (-1154G/A) in wet AMD. Patients were treated with a loading phase

of 3 monthly injections of 0.5mg ranibizumab injections given intravitreally. The patient’s visual acuity

was measured before and after the treatment. It was found that patients carrying the VEGF-A -2578C

allele demonstrated a significantly higher response to treatment, whereas those carrying the VEGF-A -

2578AA did not show an early functional response to ranibizumab. The author concludes that the VEGF-

A -2578A/C single nucleotide polymorphism may be an important allele in determining a patient’s

response to the early functional response to ranibizumab [182].

Comparison of Bevacizumab and Ranibizumab

Given that bevacizumab is so much cheaper than ranibizumab, researchers have been interested in

finding out whether one of the drug entities is more efficacious than another. A number of trials have

been carried out in this regard.

The Comparison of Age-related Macular Degeneration Treatments Trial (CATT), the Alternative

Treatments to Inhibit VEGF in Age Related Choroidal Neovascularization Trial and other trials have

demonstrated similar results with ranibizumab and bevacizumab [3].

In the CATT, patients with neovascular AMD were either administered intravitreal ranibizumab or

bevacizumab either on a monthly or an as needed with evaluation schedule. A mean change in the visual

acuity after one year with a non-inferiority limit of 5 letters on the eye chart was the primary outcome

measured [179]. The as needed regimen was widely used in bevacizumab since the intraocular safety of

bevacizumab and the duration of its therapeutic effect were unknown. Similarly an as needed regimen

for ranibizumab is often employed. In the first year of the follow up, both drugs showed equivalent

effects on visual acuity at all tested points [179]. The outcomes measured were the mean number of

letters gained, the proportion of patients in whom visual acuity was maintained and the proportion of

those who had a gain of at least 15 letters [179].

When testing the effectiveness of a less-than-monthly regimen with both drugs, excellent results for

visual acuity were obtained, with a mean gain of 5.9 letters with bevacizumab given as needed and 6.8

letters with ranibizumab [179].

The trial also examined the effect of the drugs on the presence of fluid in or under the retina [179]. It

was shown that both drugs results in a substantial and immediate reduction in the amount of fluid in or

under the retina [179]. While the first year CATT study examined disease activity by means of time

domain optical coherence tomography, the second year of the ongoing trial extends to assess whether

high resolution spectrum domain OCT results in increased detection of fluid and subsequent treatment

[179].

Pharmacogenetic studies in the CATT showed no association between genotype and treatment response

[3]. In one study to determine whether VEGF polymorphisms affected the outcome of anti-VEGF

therapy, patients administered bevacizumab intravitreally and VEGF polymorphisms were recorded.

Genotyping for the VEGF polymorphisms rs1413711, rs3025039, rs2010963, rs833061, rs699947,

rs3024997, and rs1005230 was performed and the mean change in visual acuity at the end of the

treatment was measured. It was found that VEGF polymorphisms did not contribute to the success of

anti-VEGF treatments significantly [183].

A slightly different approach was used by one study that examined the effect of intravitreal injection of

bevacizumab, ranibizumab or pegaptanib on the level of vascular endothelial growth factor (VEGF) in

the plasma of patients with diabetic macular edema and those with exudative AMD [184]. Out of the 30

patients suffering from AMD, 10 received 1.25 mg bevacizumab, 10 0.5 mg ranibizumab and 10 0.3 mg

pegaptanib. ELISA technique was used to measure the concentration of VEGF before the injections, after

7 days and after one month. It was shown that while bevacizumab reduced the VEGF levels in the blood

plasma, ranibizumab and pegaptanib has no such significant effect [184]. It is proposed that this effect

may be useful in decreased number of retreatments required following initial therapy [184].

Safety of anti-VEGF therapies

Intravitreal anti-VEGF agents have generally been found to be safe. The systemic use of bevacizumab in

cancer chemotherapy has been associated with an increase in the incidence of hypertension, bleeding

and thromboembolic events [159]. It is likely that systemic exposure to the drug is much lower when

administered intravitreally for the management of AMD since the exposure is likely to be diminished by

the higher volume of distribution [159]. Therefore, the risk of developing adverse effects is very likely

lower than those recorded with its use in cancer chemotherapy. Furthermore, the safety concerns are

probably greater in patients that are already at a high risk of developing hypertension, stroke and

cardiovascular disease [159].

A study to examine the ocular and systemic side effects of bevacizumab and ranibizumab when

administered intravitreally in patients with AMD showed that the bevacizumab was associated more

commonly with side effects than ranibizumab. The research group found that while serious adverse

effects associated with these two agents were relatively rare, acute intraocular inflammation was seen

to occur more frequently in patients receiving bevacizumab [185]. The authors propose further

investigation of these findings particularly for the long term use of these agents as the present study

was not able to establish this [185].

Similarly, a systemic review conducted on the safety of using anti-VEGFs agents for over 52 weeks

concluded that further research is required on the effects of these agents when administered for over 2

years [186]. The authors recognize that while there is a relatively low risk of side effects with the use of

these agents on the short term, long term effects are unrecognized and may be deleterious [186].

One study compared the use of anti-VEGF therapy with either photodynamic therapy or pegaptanib use

[160]. Patients receiving intravenous pegaptanib, bevacizumab or ranibizumab were set up against a

control group of patients receiving photodynamic therapy [160]. The primary outcomes measured were

the incident of myocardial infarction, bleeding and incident stroke. After adjustments for baseline

characteristics, and comorbid conditions were made, the study found no significant differences in the

hazard of mortality or myocardial infarction between bevacizumab use and the other therapies

examined [160]. Furthermore there was no statistically significant relationship between the treatment

group and a risk of bleeding events or stroke, although ranibizumab demonstrated the lowest risk of

side effects [160]. The study concluded that the use of bevacizumab or ranibizumab does not increase

the risk of mortality, myocardial infarction, bleeding or stroke, when compared to photodynamic

therapy or pegaptanib use [160].

A similar study compared the risk of gastrointestinal bleeding and thromboembolic events between anti-

VEGF therapy and photodynamic therapy as well as a non-treated community sample [187]. Data were

obtained from hospital and death records of patients that had been treated with anti-VEGF therapy

(bevacizumab or ranibizumab) or photodynamic therapy. The study found that the 12 month rate of

myocardial infarction was higher in patients that had received anti-VEGF therapy [187]. No statistically

significant difference was observed between the bevacizumab and ranibizumab patients. Furthermore,

the number of injections of anti-VEGF did not seem to increase the patients 12 month risk of myocardial

infarction [187]. The risk of stroke and gastrointestinal bleeding did not vary between the groups. It was

concluded that although cardiovascular side effects were rare in patients administered anti-VEGF

intravitreally, these patients were more likely to experience a fatal or non-fatal MI [187]. The authors

could not confirm whether this was due to the underlying AMD or the anti-VEGF agent [187].

A systemic review of articles on the clinical safety of ranibizumab in age-related macular degeneration

was conducted on data from 1984 onwards [188]. The MARINA study concluded that every injection of

ranibizumab was associated with a 0.05% risk of endophthalmitis, whereas the ANCHOR study showed a

<0.1% rate of the same [188, 189]. Transient increases in intraocular pressure were noted and

incidences of intraocular inflammation were not serious in most cases. With regard to cardiovascular

events, the MARINA and PIER studies revealed 4.6% and 0% incidences of systemic arterial

thromboembolic events in patients receiving ranibizumab and 3.8% and 0% rates in patients receiving

placebo respectively [188, 190]. The SAILOR study showed an insignificant but higher rate of

cerebrovascular stroke with 0.5mg ranibizumab compared to 0.3mg ranibizumab [188]. The review

concluded that there was no increased risk of stroke with ranibizumab therapy and that repeated

intravitreal ranibizumab was well tolerated over a period of 2 years of treatment [188].

A retrospective study conducted to compare the incidence of arterial thromboembolic events between

patients treated with bevacizumab and those treated with ranibizumab found that bevacizumab raised

the risk of arterial thromboembolic events to a greater extent than ranibizumab [191]. The arterial

thromboembolic events examined included stroke, myocardial infarction, angina pectoris,

peripheral thromboembolic disease, transient ischemic attack, sudden death and lethal stroke. The

authors concluded that the new arterial thromboembolic events could not be attributed exclusively to

the anti-VEGFs since the elderly population present with a number of cardiovascular risk factors. They

proposed that the findings be further investigated and confirmed with clinical trials [191].

In summary, the main adverse events to look out for with ranibizumab are endophthalmitis, retinal

detachment, and traumatic injury to the lens as per the ANCHOR and MARINA studies. Other adverse

events that have been reported include conjunctival hemorrhage, eye pain, vitreous floaters, increased

intraocular pressure within 60 minutes of injection, and intraocular inflammation [189, 190].

Bevacizumab was reported to cause bacterial endophthalmitis tractional retinal detachments, uveitis,

rhegmatogenous retinal detachment, and vitreous hemorrhage [6]

Early studies on pegaptanib have shown that it may be associated with endophthalmitis, traumatic

injury to the lens, retinal detachment, anaphylaxis/anaphylactoid reactions including angioedema, pain

in the eye, the presence of vitreous floaters, punctate keratitis, vitreous opacities, cataract, anterior

chamber inflammation, visual disturbance, eye discharge, and corneal edema [192].

Aflibercept, a newer anti-VEGF agent, is a fully human recombinant fusion protein composed of the

second immunoglobulin binding domain of the VEGFR1 and the third immunoglobulin binding domain of

the VEGFR2 receptors [161].

The agent was developed by Regeneron and Bayer and was approved by the FDA in Nov 2011. It forms

part of Regeneron’s “Trap” products which catch, hold and block cytokines. The intravitreal injection is

known as VEGF Trap-Eye.

Aflibercept binds all isoforms of VEGF as well as placental growth factors [193]. Placental growth factor

is present in human CNV membranes, and has been shown to contribute to the development of CNV in

animal models [161]. Furthermore, aflibercept has a binding affinity for VEGF that is fairly higher than

that of ranibizumab or, bevacizumab. For this reason, ablifercept may provide effective blocking of VEGF

even at low dosage concentrations and result in a longer duration of action and increased dosing

intervals [161].

Phase I studies demonstrated a dose dependent decrease in retinal thickness. The administration of

intravitreal injections of 2mg and 4mg aflibercept showed that half the patients did not suffer from

retinal leakage and were able to maintain vision gains at 12 weeks after a single dose injection [161].

The phase II trial is referred to as the clinical evaluation of anti-angiogenesis in the retina study (CLEAR-

IT) trial [194]. This examined the outcomes and vision, injection frequency and safety of ablifercept in

two parts: during the 12-week fixed dosing period and then the as needed 52- week period in patients

with exudative AMD [194, 195]. Patients with subfoveal choroidal neovascularization secondary to wet

AMD were given either 0.5 mg or 2 mg every 4 weeks or 0.5, 2, or 4 mg every 12 weeks during the fixed-

dosing period (weeks 1-12). For the rest of the 52 weeks they were on an as needed regimen and

following monthly evaluation were treated with their assigned dose if required. The primary outcome

measure was the change in choroidal thickness. The secondary outcomes measured were the mean

change in best corrected visual accuracy, the proportion of patients with 15-letter loss or gain and

safety. In the second part of the trial the time to first PRN injection and reinjection frequency were also

measured [194, 195].

During the first 12 weeks, it was demonstrated that repeated monthly intravitreal administration of

ablifercept significantly reduced retinal thickness and improved visual acuity. Furthermore the regimen

was well tolerated by the patients [195].

At 52 weeks it was found that an as needed schedule of aflibercept was able to maintain the outcomes

gained in the 12 week fixed dosing period and that the frequency of reinjections was low. Patients

tolerated repeated dosing well through the 52 week period [194].

Two parallel Phase 3 studies have now been conducted on aflibercept [196]. The VEGF Trap-Eye:

Investigation of Efficacy and Safety in wet Age-Related Macular Degeneration (VIEW 1) study was based

in North America whereas the VIEW 2 study included patients from Europe, Asia Pacific, Japan, and Latin

America [196]. During the initial 12 months of both studies, patients were treated with EYLEA, 0.5

milligram (mg) every four weeks, 2mg every four weeks, and 2mg every eight weeks (following three

initial monthly injections), compared to ranibizumab 0.5mg every four weeks. During months 12-24, the

patients followed the same regimen as the first 12 months and were evaluated monthly to establish the

need for retreatment [196].

The year 1 results of the VIEW 1 trial showed that monthly injections of 2 mg aflibercept led to greater

vision gains than ranibizumab (10.9 letters vs. 8.1 letters; p<0.05) whereas no statistically significant

difference was seen in the VIEW 2 trial (7.6 letters vs. 9.4 letters (p≥0.05) [197]. Based upon this data, it

is postulated that the treatment burden may be decreased with the use of 2mg aflibercept every 8

weeks as compared to ranibizumab administered every 4 weeks following a 3 month loading dose [198].

The year 2 results showed that fewer injections of aflibercept were required in the 2mg every eight

weeks group than the ranibizumab group (4.2 vs. 4.7) [196].

Both aflibercept and ranibizumab were considered generally safe with the incidence of adverse effects

being the same across all treatment groups. The most common ocular adverse effects noted were

conjunctival hemorrhage, eye pain, retinal hemorrhage, and reduced visual acuity. The study group also

identified falls, pneumonia, myocardial infarction and atrial fibrillation as the most commonly reported

adverse effects, occurring in more than 1% of the study population [196].

To add to the results of the first year study, fewer injections of aflibercept are required for the same

effect as ranibizumab. Aflibercept comes in at a slightly lower price than ranibizumab. This combined

with the need for fewer injections may translate into substantial savings for patients as well as reduced

treatment burden [161].

Another study was performed to investigate very high- and very low-dose intravitreal aflibercept in

patients with neovascular age-related macular degeneration [199]. Patients with lesions ≤12 disc areas,

≥50% active CNV, and BCVA≤20/40 were divided into two groups. One group was treated with 0.15mg

aflibercept and the other with 4 mg. The change in retinal thickness from baseline was measured after 8

weeks as a primary outcome measure. The change of best visual acuity from baseline was the secondary

outcome measure. It was shown that high dose aflibercept (4mg) when compared to low dose therapy

(0.15mg) resulted in a significant reduction in foveal thickening at weeks 4 (34.2 vs 13.3, P=0.0065) and

6 (23.8 vs. 5.9, P=0.0380) and improved the best visual acuity at week 6. The 4mg dosing regimen was

associated with a considerably reduced need for retreatments after week 8 and similar adverse effect

profile as that of 0.15mg aflibercept [199].

Further research was conducted on the effects of aflibercept in patients non responsive to bevacizumab

and ranibizumab therapy.

A retrospective analysis was conducted in this regard to assess the efficacy of 2.0mg aflibercept

administered intravitreally to patients with treatment resistant neovascular AMD [200]. The study group

was patients with persistent subretinal and/or intraretinal fluid despite previous treatments with

intravitreal ranibizumab (0.5 mg) [200]. The eyes were treated with intravitreal aflibercept (2.0 mg) and

assessed after 3 consecutive injections and after 6 months of treatment. The change in visual acuity,

central foveal thickness, height and diameter of the pigment epithelial detachment on OCT were used as

outcome measures. The study found that the intravitreal aflibercept significantly improved visual and

anatomical outcomes in eyes with persistent subfoveal fluid despite previous treatment with

ranibizumab [200].

Another study examined the short term efficacy of aflibercept use in the treatment of neovascular AMD

associated with retinal pigment epithelial detachment that is refractory or develops tachyphylaxis to

bevacizumab and ranibizumab [201]. The review was a retrospective review conducted using the

medical records of patients. Three eyes of three female patients aged 49, 55 and 65 met the inclusion

criteria for the study [201]. It was found that large serous pigment epithelial detachment and subretinal

fluid that were previously unresolved with intravitreal bevacizumab and/or ranibizumab therapy were

completely treated with aflibercept injections over a 3-month period [201]. Furthermore all three eyes

demonstrated an improvement in visual acuity. The authors concluded that aflibercept may have a

potential role to play in the management of serous PED in neovascular AMD patients if bevacizumab and

ranibizumab fail. Larger case controlled studies are proposed to substantiate these findings [201].

Another anti-VEGF, axitinib, has more recently been tested for the management of CNV in patients with

AMD [202]. Axitinib is a potent and selective inhibitor of VEGF-1, 2 and 3 and is approved in the US for

the management of patients with advanced renal cell carcinoma after failure of one prior systemic

therapy [202]. For this purpose, it is taken 5 mg twice daily, taken with or without food, up to a

maximum dose of 10mg twice daily [202].

One study examined the effects of this molecule in C57BL/6 mice in which experimental CNV lesions

were induced by laser photocoagulation [203]. The mice were treated with 5 mg/kg/day axitinib or

vehicle for two weeks beginning on either day 1 or day 7 after induction. Two weeks after induction the

mice were tested for CNV using choroidal flat mounts perfused with fluorescein-labeled dextran [203].

The CNV lesions were quantified using immunofluorescence staining with isolectin IB4. The results

showed that axitinib inhibited the progression of CNV in animal models [203].

In addition to integrating the use of aflibercept into the AMD management schedule and developing

more efficacious anti-VEGFs, research groups are working on developing other anti-VEGF therapies.

One of the therapeutic agents being explored is the anti-VEGF molecule sFLT01 [204]. This novel soluble

chimeric molecule is delivered by intravitreal injection of an AAV2 vector and provides persistent

expression [204]. AAV vectors are valuable tools in gene therapy as they are non pathogenic, present

with minimal toxicity and immunogenicity, transduce non dividing cells and have a potential for long

term expression. AAV20-sFLTO1 results in a transgene product that has a high binding affinity for VEGF.

This molecule is a dimer that is composed of the domain 2 of Flt-1 (VEGF-R1) and the human

immunoglobulin G1 (IgG1) heavy chain Fc fragment. It has demonstrated efficacy in a murine model or

retinal vascularization in previous studies [204-207]. The most recent study conducted on the molecule

examines the transduction and efficacy of intravitreal administration of AAV2-sFLT01 in a non-human

primate with CNV. The results of this preliminary study show that the intravitreal administration of

AAV2-sFLT01 may be effective long-term in the management of CNV [204].

Recently, Opthotech presented the results of their phase II study examining the results of combining the

anti-platelet derived growth factor (PDGF) aptamer E10030 (Fovista) with ranibizumab. Fovista

specifically targets PDGF-B, the subunit of PDGF that regulates cells associated with the walls of newly

formed small blood vessels known as neovascular pericytes [208]. Both in vitro and in vivo, Fovistas

shown to bind to PDGF-B with high specificity and affinity, and inhibit its functions.

The 6 month trial demonstrated promising results in phase II, showed that vision improved with

combination therapy by 60% over monotherapy with ranibizumab (+10.6 letters vs. +6.5 letters at 24

weeks) [208]. The mean visual acuity differences increased and the choroidal thickness decreased

between the 2 groups as the trial progressed [110]. While these results are encouraging, they need to be

confirmed with phase III trials, which are currently underway. As explained earlier, it is proposed that

anti-PDGF agents may increase the time that neovascular complexes respond to anti-VEGF therapy.

Furthermore anti-PDGFs are thought to lead to the contraction and dissolution of the CNV membrane,

possibly through the death of pericytes and the loss of endothelial cells [110].

RetinoStat(®) is an equine infectious anemia virus based lentiviral gene therapy vector developed by

Oxford Biomedica (Oxford, United Kingdom) [209]. It expresses endostatin, an angiostatic protein, as

well as angiostatin, and is delivered by a subretinal injection for the treatment of exudative AMD. The

rationale behind the use of angiogenic inhibitors is based on the fact that collagen XVIII / endostatin is a

component of the Bruch’s membrane [209]. There is evidence to demonstate that this may be impaired

in AMD causing abnormalities in the elastic fibres and a fractured Bruch’s membrane [3]. A trial to assess

the safety and biodistribution of this molecule for AMD was carried out over 6 months in rhesus

macaques and Dutch belted rabbits. It was demonstrated that with viral delivery of the molecule

intravitreally, the level of human endostatin and angiostatin proteins peaked at around one month of

dosing in the eyes of the rabbits and remained elevated for the rest of remainder of the study [209].

Overall, RetinoStat was well tolerated and concentrated in the retina [209]. Short term effects included

mild to moderate ocular inflammation which resolved within a month of dosing. Since the viral delivery

of the molecules has been effective in animal models, further clinical trials are currently underway [209].

ISONEP is the trade name for the Sonepcizumab/LT 1009 molecule that is an humanized monoclonal

antibody against a bioactive lipid, sphingosine 1-phosphate. Sphingosine 1-phosphate is a target for the

treatment of AMD as it has been shown to modulate angiogenesis, inflammation, and fibrosis. Currently,

the molecule is undergoing two phase I trials: One to assess the efficacy and Safety Study of ISONEP with

and without anti-VEGF therapy to treat AMD, and another to study the safety of ISONEP in the

management of AMD [210, 211]. A third study, designed to evaluate the safety and potential efficacy of

ISONEP following one, two or three injections of ISONEP, as needed, for the treatment of pigment

epithelial detachment in patients with exudative AMD or PCV, was terminated in July 2012 by the

sponsor. It is claimed that safety concerns were not the reason for the early termination of this trial

[212].

Some researchers have focused on targeting other cytokines, including tyrosine kinases. However, while

these agents are potent and have a broad spectrum of activity, their use is limited by their high rate of

adverse effects [3].

Pazopanib is a potent multitargeted receptor tyrosine kinase inhibitor of several receptor tyrosine

kinases, including VEGF receptor 1 (VEGFR1), VEGFR2, VEGFR3, PDGF receptor-α(PDGFRα), PDGFRβ and

KIT [213]. It has been shown to inhibit VEGF-induced VEGFR2 phosphorylation, tumour angiogenesis and

the growth of human tumour xenografts in studies in mice and is approved in the US for the

management of advanced renal cell carcinoma [213]. Currently it is under investigation by

GlaxoSmithKline (Brentford, Middlesex, United Kingdom).

Recently the effects of ranibizumab, sorafenib, and pazopanib on the light-induced overexpression of

PDGF and VEGF- A, the vascular endothelial growth factor A receptors 1 and 2, and neuropilin 1 and 2

were investigated [214]. As discussed earlier cumulative light exposure, growth factors and growth

factor receptor signaling all have a substantial impact on the development of AMD. Human RPE cells

were exposed to white light and then treated with either 0.125 mg/mL ranibizumab, 1 μg/mL sorafenib,

or 1 μg/mL pazopanib. Reverse transcription-polymerase chain reactions, immunohistochemistry, and

enzyme-linked immunosorbent assays were used to assess the viability of the cells, the expression of

VEGF receptors 1 and 2 and neuropilin 1 and 2 as well as their mRNA, and finally the secretion of VEGF

and PDGF. It was found the sorafenib and pazopanib treatment groups demonstrated a reduced

expression of VEGFR-1 and 2 and neuropilin1 [214]. Sorafenib further reduced neuropilin 2. Additionally,

these two agents reduced light induced over expression and secretion of VEGF and PDGF considerably

[214].

Another study was conducted to establish the mechanisms by which this drug inhibits angiogenesis and

experimental choroidal neovascularization (CNV) [215]. It was found that its effectiveness in the

inhibition of angiogenesis and the prevention of CNV was largely due to the down-regulation of VEGF

release in the retina as well as the impaired VEGF-induced signaling and chemotaxis [215]. The authors

propose that a convenient topical dosing regimen of pazopanib might be a useful product for the

management of ocular neovascular diseases such as neovascular AMD [215].

Phase I trials of pazopanib have shown a positive effect on visual acuity whereas phase II trials are

underway. Furthermore, there seemed to be an apparent allele dosage effect for the TT genotype (vs

the CC or the CT genotype) of the CFH gene [216].

Therapies in the pipeline

Matrix metalloproteinases

Since abnormalities in the MMP have been shown to contribute to the development of AMD, it seems

logical to target these pathways as a viable therapeutic option to treat eyes in the early stages of AMD.

Increased hydraulic conductivity in aged Bruch’s membrane has been shown in the past, following

cultivation with MMP2 and 9 isolated from cultured human cells. A more recent study has been

inititated to assess the potential use of selective retina therapy to induce the release for activated

MMPs from RPE cells as a means of rectifying the pathological alterations of the Bruch’s membrane that

are associated with AMD [55]. Selective retina therapy is a micropulse laser technique that only targets

the RPE and not the neurosensory retina. In this way it avoids the development of microscotoma [55].

The heat is confined to the interior of a given RPE cell, ensuring that the overlying photoreceptors and

the underlying Bruchs membrane with the choroid are not affected adversely. The findings of this study

showed that following selective retina therapy, MMP2s are released on the basal side of the RPE as a

part of the wound healing process. These have a potential to slow degenerative macular aging processes

before irreversible functional loss has occurred [55].

One of the metalloproteins that has been researched in methallothionen(MT), is a low molecular weight

cysteine rich protein [217]. The four isoforms of MT found in mammals have a variety of roles including

the detoxification of heavy metals, the regulation of essential metal homeostasis and protection against

oxidative stress [217]. It is thought that the role of MT in the development of AMD may be through

oxidative stress or zinc toxicity pathway [217].

Complement

The complement pathway seems to be a possible target in the management of AMD even though its

role and timing is not completely established. Currently, data from genetic and histologic studies is being

used to develop drugs that target the complement system.

Potentia pharmaceuticals completed Phase I trials to assess the safety of intravitreal POT-4 therapy in

patients with neovascular AMD [218]. POT-4, a derivative of the cyclic peptide compstatin, is a

complement factor C3 inhibitor [218]. It works by blocking the complement activation cascade which

may otherwise lead to local inflammation, tissue damage and the upregulation of angiogenic factors

such as VEGF in the eye. At the end of the phase I trials it was proposed that POT-4 holds the potential

to be effective against both dry and wet AMD [218]. Following this Alcon Laboratories took over further

testing of this entity under the name of APL-1. APL-1 is currently in Phase II clinical trials for age-related

macular degeneration [218].

Eculizumab is a human monoclonal antibody that binds to the complement protein C5 with high affinity

[219]. It has an inhibitory role blocking the cleavage of C5 to C5a and C5b and therefore preventing the

generation of the MAC. The potential of the parent chain from which eculizumab has been derived to

cause a pro-inflammatory response is reduced by replacing the heavy chain constant region with human

IgG [219]. This process of humanization also increases the drugs half-life. The drug is administered

intravenously as a weekly dose during the loading phase followed by two weekly maintenance doses

over a complete six month period. Two studies have been carried out on eculizumab so far: in 2 studies

as a treatment for patients with AMD.

The Complement Inhibition of Systemic Eculizumab for the treatment of Non-exudative AMD

(COMPLETE) trial was a prospective, randomized, Phase II single center placebo controlled trial

conducted in three parts [220, 221]. The aim of this study was to assess the effect of eculizumab in

patients with drusen secondary to AMD. The high dose group was given 900mg of eculizumab weekly for

four weeks followed by 1200mg every 2 weeks for 6 months [220, 221]. The standard dose group was

given 600mg weekly for 4 weeks, followed by 900mg every two weeks for 6 months. The placebo group

was given saline intravenously in the same schedule. The results of the trial showed no reduction in

drusen volume in either study or any delay in slowing the progression of GA [220, 221]. The only benefit

noted was that none of the patients in the treatment groups developed wet AMD from dry AMD, but 2

patients in the placebo group did [220, 221].. It is possible that GA is not dependent upon complement

activation. Even though the inhibition of complement did not affect drusen volume, it was observed that

the growth of drusen is dependant upon the number of complement at risk alleles expressed by the

individual [220, 221]. Based upon this the authors proposed that the delivery method used may have

been insufficient to inhibit complement [220, 221]. Other possible explanations presented for the failure

of the trial were that the duration of the trial was too short or that the patient group was too small [220,

221].

Genetech are investigating an anti-factor D antibody, FCFD4514S, for the treatment of dry AMD. Factor

D mediates the cleavage of Factor B and in this way modulates complement activation rather than

shutting it down completely. A phase I study was conducted to establish the safety, tolerability,

pharmacokinetics, and immunogenicity of an intravitreal (ITV) injection of FCFD4514S in patients with

GA [222]. This study has been extended to investigate the effects of the long term use of FCFD4514S in

patients with GA [222]. Phase II trials are currently underway to determine the safety, tolerability, and

evidence of activity of FCFD4514S intravitreal injections administered monthly or every other month in

patients with geographic atrophy [222].

Taligen therapeutics is developing TA106, a Fab fragment of a monoclonal antibody that is a

complement factor B inhibitor [222, 223]. This product is still in its preclinical stages of development as

it is being developed for inflammatory diseases that have alternative complement pathway pathological

bases. The formulation is being developed for localized delivery [223].

Two peptidomimetic C5a receptor antagonists are also in preclinical assessment for AMD.

EvaluatePharma is developing the molecule JSM-7717 and Jerini AG is developing JPE-1375. The basis of

these molecules is that they competitively bind to the C5a receptor and neutralize interaction. They are

therefore thought to have the potential to suppress the inflammatory response without having an

adverse effect on complement related immunity [222].

CR2-fH is a synthetic recombinant form of CFH that has been shown to reduce angiogenesis and prevent

CNV in mice with AMD. This drug is currently undergoing preclinical experiments [222].

As discussed earlier, mutation in the gene that encodes SERPING1, a C1-inhibitor (C1INH), may

contribute to the development of AMD. C1NH is currently used in the US for the treatment of hereditary

angioneurotic edema. It has not been tested in AMD yet [3].

Ophthotech is developing ananti c5 aptamer, ARC 1905 for the management of both wet and dry AMD

[224]. It has been well tolerated and has shown some improvement in visual acuity after 8 weeks of

treatment. Phase I trials are not complete for this molecule.

Aurin tricarboxylic acid (ATA) is an orally effective agent that prevents the formation of MAC. It is seen

to block two stages of the alternative complement pathway and may be useful at reducing AMD [225].

Another approach being investigated used a light emitting diode to avert excessive para-inflammation. A

lack of complement factor H has been shown to activate complement and confer para-inflammation

leading to AMD and other retinal disorders [226]. The toll like receptors, TLR-2 and TLR-4 have

specifically been implicated in the abnormal complement activation, due to CFH polymorphisms and

macrophage migration and infiltration has been linked to TLR-4 activity [226]. The mitochondrial inner

membrane in the aged retina selectively absorbs 670 and 830 nm light, which may influence the

efficiency of mitochondria. Furthermore irradiation between 660–680 nm has been shown to increase

electron transfer in Cytochrome C oxidase, leading to augmented ATP synthesis in vitro. It has been

shown that 670 nm light exposure over a one week period reduced the numbers of microglia and

activated macrophages significantly. In previous studies 670 nm LED has been shown to alleviate para-

inflammation in the normal aged outer retina. A recent study has shown that this light regulates the

innate immune response in the neural retina in a mouse model of AMD [226].

Inflammatory and Immune Response

Fas Ligand (FasL) and tumor necrotic factor related apoptosis inducing ligand (TRAIL), apoptic inducer

factors for cells expressing the correspondent receptor are all implicated in retinal immune privilege [7].

FasL is widely expressed in ocular tissues and is thought to form a barrier against inflammatory

mediators and neovascular endothelial cells. In one experiment, mice that were laser treated to induce

CNV demonstrated a regression of neovascularization when injected with FasL [7, 227].

Alprostadil is a prostaglandin E1 that is currently being tested in a prospective randomized multicenter

study for its use in the management of dry AMD. Patients were given an infusion of 60µg alprostadil or

placebo given over 3 months. The mean difference in best visual acuity from baseline was used as a

outcome measure. The study showed that alprostadil caused a positive effect in vision until the end of

the follow up [228].

Inflammasome

A couple of studies to examine usefulness of targeting inflammasone have also been conducted recently

based on the findings of two independent academic teams. Both these research groups discovered that

inflammasome reduces retinal damage in mouse models, one focusing on the management of dry AMD

and the other of wet AMD [229].

The first uses the MYD88 inhibitor to target inflammasone mediated MYD88 activation for dry AMD

treatment. It is known that the reduction of RNase DICER1 leads to an accumulation of AluRNA in the

RPE of patients with GA [230]. The research group demonstrates that a reduction in DICER1 levels or

increased exposure to Alu RNA activates the NACHT, LRR and PYD domains-containing protein 3 (NLRP3)

inflammasome, stimulating MYD88 signaling. This signaling pathway is independent of TLR and occurs

via IL18 in the RPE [95]. Furthermore, the pharmacological or genetic inhibition of the inflammasome

components NLRP3, Pycard, Caspase-1 as well as MyD88, or IL18 was found to prevent the breakdown

of the RPE caused by the deficiency of DICER1 or increased AluRNA exposure. This, combined with the

finding that the RPE of patients with GA contains greater than normal amounts of NLRP3, PYCARD, and

IL18 and evidence of increased Caspase-1 and MyD88 activation, leads to the proposal that MyD88

inhibitors may be a viable option for prevention or treatment of GA.

The second study focused on the protective role of NLRP3 in wet AMD through the induction of IL-18 by

drusen components. The study showed that drusen activates the NLRP3 inflammasome. This leads to

the secretion of the interleukins, interleukin-1b (IL-1b) and interleukin-8 (IL-8). Moreover, drusen also

contain C1Q which is also known to activate the NLRP3 inflammasome through a lysosomal acidification

and cathepsin B-dependent mechanism. These findings demonstrate a clear involvement of NLRP3 and

IL-18 in the progression of AMD [94].

Light Cycle

The light cycle is believed to result in the accumulation of toxic A2E in the photoreceptors in patients

with AMD. Interfering with this light cycle has been proposed as another possible approach to the

management of AMD.

Fenretinide is an oral retinoid antagonist that interrupts the normal visual cycle and thus reduce the

toxic waste buildup that is part of that cycle [231]. A phase 2b trial of the compound Fenretinide by

ReVision Therapeutics demonstrated that this compound slows the expansion of the area of GA in the

eye and delays the progression to neovascular AMD [231]. Retinol derived toxins in the eye may

exacerbate lesion growth and promote the development of GA. Fenretinide has been shown to limit the

delivery of retinol to the eye by reducing the serum levels of retinol binding protein (RBP), decelerating

the accumulation of retinol derived toxins in the retina and slowing the growth of lesions. Fenretinide is

also thought to carry anti-inflammatory and anti-angiogenic properties that delay progression to wet

AMD [232]. The study tested the use of 100mg of Fenretinide daily administered orally. It was noted

however that patients complained of diminished dark adaptation, a feature that may be use limiting in

the development of Fenretinide in AMD [232].

ACU-4429, under development by Acucela, Seattle, Washington, USA, is a small molecule visual cycle

modulator that inhibits the isomerase complex and has been shown to prevent the accumulation of A2E

in mouse models of retinal degeneration [233]. It has completed a phase I trial in AMD and a phase II

trial is currently underway. The phase I trial examined the tolerability, pharmacokinetics,

pharmacodynamics, and safety of a single, orally administered dose of ACU-4429 in healthy subjects.

The subjects were administered a single dose of ACU-4429 in escalating doses from 2mg to 75. Full field

electroretinograms were recorded both before and after exposure to full field bleaching light.

Pharmacokinetics samples were drawn at predetermined intervals and adverse events, vital signs,

clinical laboratory assays, electrocardiograms as well as other ophthalmologic examinations were

conducted to monitor safety. The oral administration of ACU-4429 was shown to inhibit the B-wave of

the electroretinograms, an effect that was dose dependant. Furthermore, safety was demonstrated in

oral doses of up to 75mg given daily [233]. Adverse effects were also dose dependant and at the most

included dyschromatopsia and alteration in dark adaptation [233]

Amyloid β peptides, identified in drusen, have been implicated in the pathology of AMD as discussed

earlier. Drawing upon this theory, one research group investigated the role of anti-amyloid therapy in

the protection of RPE damage and the loss of vision in patients with AMD. Based upon the findings of

this trial, the authors proposed that there is a potential in pursuing anti-amyloid therapy in AMD [234].

Two drugs based upon this theory are currently being tested: Glatiramer acetate and RN6G

(bapeneuzimab).

Pfizer is investigating bapineuzimab, an antiamyloid monoclonal antibody that is used for Alzheimer’s

disease. This molecule is currently undergoing 2 clinical trials investigating its role in the management of

patients with dry AMD using single intravenous doses ranging from 0.3 to 30 mg/kg [3].

Glatiramer acetate is currently used for its immunomodulatory properties in the management of

multiple sclerosis and has been proposed to exert its effects in a number of ways including suppressing T

cells, downregulating inflammatory cytokines, reducing amyloid β-induced retinal microglial cytotoxicity,

and promoting the formation of a neuroprotective phenotype of microglia [235]. In the past, glatiramer

acetate has been shown to reduce the dursen area. More recently this effect has been observed to a

significant extent in patients with dry AMD. There is therefore a possibility that this molecule has a

potential role in the management of dry AMD [236].

Neuroprotection

Although the pathogenic mechanisms leading to GA have not been fully identified, it is thought that

oxidative stress plays an important role in the development of this condition [237]. Antioxidants that do

not classify as vitamins and minerals are being investigated as neuroprotective agents to prevent GA.

These may have a role in both preventing toxicity in the retina as well as blocking cell death pathways

[237].

Piperidine nitroxides such as Tempol have the ability to react directly with free radicals and prevent

their oxidative properties [237]. Tempol-H is one such compound that protects the RPE cells from

damage by free radicals and the photoreceptors from acute light induced damage. This has led to the

investigation of the compound OH-551 in the treatment of GA [237]. This disubstituted hydroxylamine

compound is converted to Tempol-H, exerting its anti-oxidant effects in the retina. A phase II trial was

carried out to investigate the safety and preliminary efficacy of OT-551 [237]. The medication was

administered topically in a concentration of 0.45% three times daily for 2 years. The OT-551 was well

tolerated by participants, with the absence of any serious adverse effects [237]. While there was a slight

benefit in maintaining visual acuity, there was no significant effect on changes in the area of GA,

contrast sensitivity, microperimetry measurements, and total drusen area from baseline [237]. It was

concluded that at the current concentration and mode of delivery, OT-551 did not have a great benefit

in the management of GA [237].

Ciliary neurotrophic factor (CNTF) is a neurotrophic factor that has been shown to retards the loss of

photoreceptors during the breakdown of the retina. It is a member of the IL-6 family of cytokines and

mediates its activities through a heterotrimeric complex that contains CNTF receptor alpha (CTNF-α),

gp130, and LIF receptor beta (LIFRβ). The mechanism by which CNTF protects the photoreceptors is not

fully understood [238]. Delivering the CNTF to the retina was a major challenge until NT-501, an

encapsulated cell technology (ECT) polymer implant containing human RPE cells, genetically modified to

secrete CTNF, was developed [239]. ECT implants do not store the drug, but actually produce it and

deliver it in situ. The NT-501 implant is 6mm long and 1mm in diameter. It is inserted into the vitreous

from where it releases CNTF in two different output rates for 12 months or longer: 5ng/day and

20ng/day [235].

A phase 2 study was conducted to assess the safety profile of NT-501 in patients with GA, evaluate the

effect of CNTF on structure and function, and determining the dose and primary end point for future

studies. The researchers found that both the implant and technique were well tolerated and the CNTF

delivered via the implant seemed to slow the progression of vision loss in patients with GA, particularly

those with 20/63 or better vision at baseline [238].

Thioredoxins (TRXs) exert their anti-oxidant effect by scavenging intracellular reactive oxygen species.

One trial investigated the effect of TRX overexpression on cell viability, morphology, NF-κB expression,

and mitochondrial membrane potential, in RPE cells [240]. To generate TRX over expressing cell lines, an

established human RPE cell line was infected with adeno-associated virus vectors encoding either TRX1

or TRX2. While both TRXs reduced cell death caused by 4-hydroxynonenal (4-HNE)-induced oxidative

stress, TRX2 seemed to have a greater benefit in promoting cell survival. Additionally, TRX2 seemed

more effective at maintaining the membrane potential than TRX1. The authors proposed that the

difference in effect between the two TRXs was possibly due to the fact that the TRX2 was expressed in

the mitochondria, while TRX1 was expressed in the cytoplasm. As anti-oxidants, TRXs may therefore be

useful in preventing oxidative stress and hence AMD [240].

A research group at Alcon Research Ltd conducted a trial to evaluate the efficacy of the 5-HT1A agonist,

AL-8309B, in protecting the retina from severe blue light–induced photo-oxidative damage in mice

[241]. 5-HT1A receptors have been implicated in processes such as the control of sleep, feeding, and

anxiety. Blue light oxidizes A2E in human ARPE-19 cells leading to the activation of the alternate

complement pathway and the formation of C3a [241]. Retinas damaged by blue light demonstrate

complement activation and the formation of carboxyethylpyrrole (CEP)-adduct, a product that is found

in the retinas of AMD patients. The researchers found that 1.75% AL-8309A administered topically to the

eye was an effective neuroprotectant. The molecule is currently undergoing further trials to establish its

effectiveness in humans [241].

Brimonidine, an α-2 adrenoceptor agonist currently used to reduce intraocular pressure in patients with

glaucoma is also being investigated for its neuroprotective properties in AMD [235]. Through various

studies in the past, brimonidine has been shown to have a protective role in ganglion cells, bipolar cells

and photoreceptors [235]. Brimonidine exerts its effects in a number of ways including increasing the

expression of basic fibroblast growth factor mRNA thereby delaying apoptosis, increasing the expression

of proteins that regulate mitochondrial membrane permeability, and suppressing the accumulation of

glutamate which causes neuronal cell death [235].

A 2 year phase II clinical trial to evaluate the brimonidine implant in patients with GA is currently

underway [235]. It utilizes a sustained release biodegradable implant that is injected via a 22-gauge

needle to deliver brimonidine. Patients are treated with either 200µg brimonidine, 400 μg brimonidine

or sham injections [235, 242].

A number of investigations that have not yet reached clinical trials are also underway. One group is

working on the finding that when caspase pathways are blocked, receptor interacting protein (RIP)

kinases promote necrosis and overcome apoptosis inhibition [243]. Programmed necrosis seems to be

just as important in promoting cell death as apoptosis and it has been shown that the

concomittant inhibition of RIP kinases and caspases is essential for effective neuroprotection [243]. This

study group is using a necrostatin to inhibit necrosis and caspase inhibition to delay apoptosis [244].

Stem Cells

Stem cell therapy focuses on replacing the retinal pigment epithelium (RPE), a monolayer of cells vital to

photoreceptor cell health [245]. Stem cells are a promising approach for the treatment of AMD as has

been demonstrated by recent studies. Cell transplant therapies do exist but due to the risk of an

inadequate supply of donor cells their therapeutic success has been limited [246]. Furthermore

transplanting intact sheets and suspension of primary RPE cells has not been entirely effective in terms

of graft survival or vision improvement [247]. Stem cells may also be used to deliver therapeutic drugs

such as cytokines and growth factors into the retina.

Some researchers have focused on developing techniques for successful cell transplantation. One group

has developed a biodegradable electrospun (ES) scaffold designed to direct the growth of retinal

ganglion cell (RGC) axons radially, a process that imitates the orientation of axons in the retina. It was

shown that RGCs on the ES scaffold followed a radial pattern of the host retinal nerve fibers when

transplanted onto retinal explants. Furthermore, the RGCs transplanted directly grew axons in no

particular pattern. The use of this scaffold was recommended in cell transplant therapies [248].

A similar technique using a scaffolding system was tested by another research group whereby

conjunctiva mesenchymal stem cells (CJMSCs) were seeded onto poly-L-lactic acid (PLLA) nanofibrous

scaffolds and were induced to differentiate toward photoreceptor cell lineages [249]. This study

concluded that nanofibrous scaffolds were a suitable cell carrier for retinal tissue engineering. The

authors proposed that CJMSCs might have a potential role in the regeneration of retinal cells.

However, due to the limitations of cell transplantation, other groups have tried to develop better

models of replacing degenerating retinal cells.

In one trial, it was shown that a combination of trabecular meshwork mesenchymal stem cells

(TMMSCs) and basement membrane support from the amniotic membrane is a possibly viable source of

cells for subretinal transplantation in regenerative therapy in AMD [249]. Due to problems such as the

complicated surgical procedures for extraction, restricted accessibility of pluripotent retinal stem cells

and the formation of cell orientation in rosettes have all restricted the use of mature photoreceptors,

retinal progenitor cells (RPC), retinal sheets and RPE for cell transplantation to the subretinal space. This

has led to the discovery of alternative sources including mesenchymal stem cells [249].

Another research group focused on the use of human embryonic stem cells (HESCs). HESCs are able to

self-renew indefinitely while maintaining a stable undifferentiated state [245]. They are isolated post

fertilization from the inner cell mass of the human blastocyte and can be stored as pluripotent cells with

mitotic inactivated mouse embryonic fibroblasts (MEFs). Preliminary trials for HESCs have shown that

they do not demonstrate hyperproliferation, tumorigenicity, ectopic tissue formation, or apparent

rejection after 4 months [247]. In one study carried out on mice it is demonstrated that retinal stem cells

isolated from the retina of adults may produce function photoreceptor cells that can potentially restore

the loss of vision as a result of the loss of photoreceptors [250].

Controlling the differentiation of human pluripotent cells using defined protocols has been shown to

convert 80% of the pluripotent cells to an RPE phenotype in as little as 14 days [251]. Another study

group defines a protocol for the generation of RPEs from HESCs that is simple and scalable [252].

One study has focused on the use of renewable sources of cells following the identification of factors

that proliferate RPE cells in vitro [246]. In this trial, the small molecule WS3 is used to reversibly

proliferate primary RPE cells that have been isolated from both fetal and adult human donors. Upon

withdrawal of WS3, the RPE cells mature and differentiate [246].

Retinal prosthesis

Retinal prosthesis studies are currently underway with the aim of restoring some form of vision to

patients with AMD and other retinal degenerative disorders [253]. Most of the studies stimulate the

inner retinal cells electrically through electrodes that are implanted on or near the retina. The activation

of spatially distinct population of cells in the retina is essential for the patient to gain pattern vision, and

therefore the electrical stimulation needs to be localized [253]. Putting all these factors together, one

study group developed a hexagonal return (hexapolar) configuration in place of the traditionally used

monopolar or bipolar return configurations. The researchers found that this configuration was a more

effective method for electrically stimulating spatially distinct populations of cells in retinal tissue than

the monopolar or bipolar configuration.

Drug Delivery

Another area of research is the development of improved drug delivery systems to increase the

concentrations of medications in the retina and reduce systemic side effects.

Two studies using nanoparticle systems to deliver drugs are summarized next. These are being

developed in an attempt to overcome monthly intraocular injections that are widely used yet invasive

techniques. Nanoparticle strategies are less invasive, offer less risk to the patient and provide longer

term angiogenesis and fibrosis [254]

In one trial, it was demonstrated that single intravenous injection of targeted biodegradable

nanoparticles carrying the recombinant Flt23k intraceptor plasmid homes to neovascular lesions in the

retina and decreased CNV lesions in primate and murine AMD eyes. Furthermore it was found that

subretinal fibrosis was suppressed and no ocular or systemic toxicity relating to the nanoparticle therapy

was observed. These findings substantiated the proposal that a nanoparticle-based platform was a

viable option for targeted, vitreous-sparing, extended-release, nonviral gene therapy [254].

In a similar study, folate-functionalized poly(ethylene glycol)-b-polycaprolactone (folate-PEG-b-PCL)

were synthesized for assembling into nanoparticles of ~130nm and used to encapsulate triamcinolone

acetonide. The nanoparticles were programmed to release triamcinolone acetonide over a period of

4weeks at pH 5.5 and 8weeks at pH 7.4. Their uptake by RPE cells was assessed. It was found that not

only did the triamcinolone acetonide demonstrate reduced cytotoxicity when delivered via the

nanoparticles, but the cellular uptake was significantly higher than particles without folate modification

[255].

Thermal gels have also been utilized in drug delivery for AMD. Reversible thermal gels undergo gelation

as the temperature increases. The polymer, poly(ethylene glycol)-poly(serinol hexamethylene urethane)

(ESHU) forms a physical gel at 37 °C and has demonstrated positive compatibility with ocular cells. It is

used to provide the sustained release of the anti-VEGF agent, bevacizumab. In this formulation,

bevacizumab is released over 17 weeks in vitro properties that can be altered by changing the

concentration of the drug or the ESHU [256].

Radiation Therapy

Radiation therapies are under consideration to reduce the risk and the burden of monthly intravitreal

injections of anti-VEGF agents. For example, one of the therapies that has shown promising results is the

stereotactic low-voltage x-ray irradiation system, Oraya. Phase I studies have shown a beneficial effect

[257].

Lifestyle Interventions

Stress Management

Hormonal imbalance and depression, that can not be associated with stress, contribute to poor retinal

health. Depression leads to food habituation, overeating and abnormal sleep patterns [22]

Limiting Alcohol Intake

While excess alcohol results in cardiovascular and liver disorders, it has been shown that one or two

glasses of red wine daily may in fact reduce the risk of AMD by 40-50%. Red wine is thought to exert this

beneficial effect by improving retinal and choroidal blood flow, a benefit not observed with white wine

which lacks the bioflavonoids that confer the protective properties to red wine [22].

Smoking Cessation

Smoking cessation should be encouraged not only to reduce the progression of the condition but also to

improve response to therapies such as laser photocoagulation.

Using sunglasses, hats and visors

Phototoxicity is a known causative factor in macular degeneration. All individuals should be encouraged

to wear sunglasses with light protection, hats and visors from childhood onwards. Individuals that work

outdoors, frequently use tanning beds, or have dilated pupils are at a higher risk of developing macular

degeneration later in life. Microscope light during cataract surgery can have a similar effect as prolonged

exposure to the sun [22].

Effects of medications

There are a variety of medications that directly affect the health of the RPE including phenothiazine,

hydroxychloroquine and ethambutol [22]. Some medicines may indirectly induce AMD by altering

digestion or liver function [22]. The liver is essential in filtering nutrients and toxins, storing fat soluble

vitamins, activating the B vitamins and manufacturing glutathione [22].

Maintaining hydration

Water hydrates the body, flushes the liver and kidneys and decreases appetite. All these factors

indirectly improve ocular health [22].

Regular exercise

Exercise improves cardiovascular health and, stimulates the cardiovascular system. Furthermore it

decreases intraocular pressure and improves ocular blood flow [22].

Breathing techniques

Various breathing techniques and meditation relax the mind, strengthen the diaphragm and improve

blood flow to the eye [22].

Estrogen

It has been shown that post-menopausal women taking HRT have a lower incidence of macular

degeneration particularly the exudative form [22].

Sleep behavior

Sleep is an essential component of ocular health and is vital for the restoration of photoreceptors. It is

during sleep that the eye can restore photoreceptor integrity and replenish nutrients consumed during

the day.

Vision rehabilitation

Vision rehabilitation is an important therapy for patients with any loss of vision. Patients should be

referred to a qualified vision rehabilitation specialist to learn strategies on how to maximize their

remaining vision [3].

The various strategies employed include near vision devices, distant-vision devices and non-optical aids

[5].

Examples of near vision devices include direct illumination using a gooseneck or adjustable lamp to

sharpen images, magnifiers both hand held and table attachable, lit magnifiers, high power reading

glasses, telescope focused for near vision and electronic reading tables [3, 5].

Telescopes mounted on glasses or held in the hand, protective filters and sun lenses to reduce glare are

all devices that can be used to improve distant vision [5].

A number of non-optical aids are also available to the patient. Reading stands may be suggested to allow

reading material to be suitably positioned to facilitate reading. Clear sheets of colored acetate filters

enhance contrast. Dials with large lettering may be attached to televisions, stoves etc. Large print

publications available at libraries may be chosen over small print. Bold line writing paper allows the

patient to write freely. Stencils specially cut out of cheque books, envelopes etc may help with writing

payee information, addresses etc [5].

Since patients are at a greater risk of slipping and falls in the bathroom various strategies for improving

the patients functioning in the bathroom have also been proposed [5]. These include the use of

contrasting colors for the cup, soap and soap dish or using a wall mounted soap dispenser. Installing a

wall mounted mirror with an extension arm will allow for closer viewing while shaving, applying makeup

etc [5]. Keeping all bathroom cabinets closed and organizing the contents allows for the right product to

be retrieved easily [5]. Using different shaped containers for different products reduces the chance of

confusing the bottles. Marking the position of the hot and cold faucet handles on the wall or sink to the

patients liking helps the patient to select the same temperature setting each time. Similarly, marking the

bathtub at the desired water level with a black tape will ensure it does not overflow [5].

Recently CentraSight Implatable Miniature Telescope prosthesis has been approved and is used in

conjunction with cataract surgery [3].

Educating Families

It is very important to educate families on the condition as part of the treatment program. This will

improve therapeutic outcomes and decrease the challenges faced by the patient due to the support

provided by the family.

Follow up Testing After Treatment

The American Academy of Ophthlamology Retinal Panel recommends that all patients with AMD who

have been treated with ranibizumab, bevacizumab, pegaptanib sodium injection, verteporfin PDT or

thermal laser photocoagulation surgery should be examined at regular intervals [6]. Biomicroscopy of

the fundus, OCT, fluorescein angiography and fundus photography will all provide signs of exudation, if

any [6]. These recommendations provide guidance on the intervals for follow up visits:

Patients treated with bevacizumab and ranibizumab should have a follow up examination

approximately 4 weeks after treatment

Patients treated with pegaptanib should be advised to have a follow up examination at 6 weeks

following treatment.

Patients that have received verteporfin PDT treatment for subfoveal CNV should have follow-up

examinations and fluorescein angiograms at least every 3 months for up to 2 years [6]

In patients in the stability phase of ranibizumab therapy, a trial was conducted to assess changes in

visual acuity if the follow up intervals are decreased. It was shown that patients whose follow up visit

intervals were reduced from approximately 8 weeks to 4 weeks demonstrated improved visual acuity

and reduced severe vision loss [258]. Furthermore, the study found that the stability phase was an

appropriate time to evaluate the outcome and effectiveness of therapy.

The American Academy of Ophthalmology Retina Panel recommends that when taking a follow up

history, health care professionals should make a note of any symptoms including decreased vision and

metamorphopsia, any changes in medication or supplements taken by the patient, any changes in

medical and ocular history and any changes in social history particularly smoking habits [6].

Age Related Eye Disease Study (AREDS)

The Age-Related Eye Disease Study (AREDS) was one of the largest clinical trials designed to learn about

the natural history and risk factors of AMD and cataract and to evaluate the effect of high doses of

Vitamin C, Vitamin E, beta-carotene and zinc on the progression of these conditions. The trial was

sponsored by the National Eye Institute and upon completion was followed by the AREDS-2 trials that

tested several changes to the formulation.

The initial AREDS was an 11-center double-masked clinical trial which tested high-dose supplementation

with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss

[259]. There were a total of 4,757 participants, ranging between the ages of 55-80 years of age. These

patients were chosen on the basis that they had extensive small drusen, intermediate drusen, large

drusen, noncentral geographic atrophy, or pigment abnormalities in 1 or both eyes, or advanced AMD or

vision loss due to AMD in 1 eye. Additionally, they had to have at least 1 eye at BCVA of 20/32 or better.

In this way the study was able to establish the effects of the supplements in patients with early,

intermediate or advanced forms of AMD.

The participants were randomly assigned a dosing regimen of daily oral tablets containing one of the

following:

Antioxidants alone: 500mg Vitamin C, 400IU of Vitamin E and 15mg of beta carotene or,

Zinc alone with copper added to prevent deficiency of copper :80mg of zinc in the form of zinc

oxide and 2mg of copper as cupric oxide, or

Antioxidants plus zinc or

Placebo [259]

All participants were required to take two tablets in the morning and two in the evening with or after

meals to prevent stomach irritation by zinc [259].

At baseline the participant’s height, weight, blood pressure, manifest refraction, best-corrected visual

acuity, and intraocular pressure were measured together with stereoscopic fundus photographs [259].

Other participant details recorded included the individuals demographic information, smoking habits

and history, history of sunlight exposure, medical history, history of specific prescription drug

medication, nonprescription medication, vitamin and mineral use [259].

At each follow up examination, conducted every 6 months, slitlamp biomicroscopy and ophthalmoscopy

were performed to assess visual acuity. Additionally, general physical and ophthalmic examinations wer

conducted annually [259]. Two years after the randomization, stereoscopic fundus photographs of the

macula were taken on an annual basis [259].

The researchers measured the progression to or treatment of AMD using fundus photography as well as

at least moderate drusen (≥ 15 letters visual acuity loss from baseline) [259]. Estimated pill counts were

performed for the patients and it was seen that most patients took 75% or more of the assigned

medications and adherence was balanced by treatment [259].

The effect of the formulations on the 1117 category 1 patients, that had few drusen, could not be

assessed in this study as only 5 of them developed AMD [259]. Additionally, the category 2 patients,

those who had extensive small drusen, pigment abnormalities, or at least 1 intermediate size druse,

demonstrated too few advanced AMD events to be assessed further for the purpose of this study [259].

The study group found that the highest benefit of using zinc alone or in combination with antioxidants

was observed in patients with extensive intermediate drusen, large drusen, or noncentral GA in 1 or

both eyes (category 3), or advanced AMD or visual acuity <20/32 attributable to AMD in 1 eye (category

4) [259]. In these categories, the treatments were shown to reduce the risk of progression to advanced

AMD, with the zinc and antioxidant regimen showing a greater benefit than the zinc alone or antioxidant

alone group[259]. The placebo group patients were at the highest risk of developing advanced AMD

[259].

With regard to safety, there was a relatively low incidence of reported side effects and upon further

investigation of these effects, no statistical correlation was established between the adverse effects and

the formulations [259].

The long term effects of the AREDS formulation of high dose antioxidants and zinc supplements on the

progression of AMD was examined in a follow up trial. 3549 of the surviving participants of the original

AREDS enrolled in the follow up study [260]. The study group found that five years after the clinical trial

had ended, only patients with neovascular AMD benefited from the AREDS formulation. Those with GA

did not show any improvement with the continued use of this combination of supplements [260].

Furthermore it was observed that no adverse effects were associated with the AREDS formulation and

that patients assigned to zinc containing formulations had in fact a reduced mortality. The study

strengthened the findings of the initial recommendations that Category 3 and 4 participants should

consider the AREDS formulation to delay disease progression [260].

In 2006, the AREDS research group initiated a second study in an attempt to improve the AREDS

formulation, the AREDS-2 trial [261]. From observational data it had been seen that other nutritional

supplements may be beneficial in reducing the risk of progression of AMD to advanced stages. In the

AREDS-2 the aim of the study group was to evaluate the efficacy and safety of the antioxidants, lutein

plus zeaxanthin (L+Z), which are in the same family as beta-carotene, and/or ω-3 long-chain

polyunsaturated fatty acid (LCPUFA) supplementation in reducing the risk of developing advanced AMD

[261]. They also assessed the effect of the reduction of zinc and the omission of β-carotene, which had

been shown to increase the risk of lung cancer in smokers from the previous study, from

original AREDS formulation [261]. The multicenter, phase III, randomized, controlled clinical trial was

conducted on patients between the ages of 50 to 85 years, who suffered from bilateral intermediate

AMD or advanced AMD in 1 eye [261].

Once again, patients were randomly assigned one of the following therapies that appeared the same for

purposes of masking,

10mg lutein and 2mg zeaxanthin

1000mg omega-3 fatty acids containing ω-3 LCPUFAs (650mg eicosapentaenoic acid + 350mg

docosahexaenoic acid)

lutein/zeaxanthin and omega-3 fatty acids

placebo [261]

The participants were required to take these medications daily for a total of 5 years. Additionally, the

participants were offered a secondary randomization of a slightly modified AREDS formulation:

Original AREDS formulation: 500mg Vitamin C, 400IU of Vitamin E, 15mg of beta carotene, 80mg

of zinc and 2mg of copper, (original AREDS formulation) or

Lower the levels of zinc: 500mg Vitamin C, 400IU of Vitamin E, 15mg of beta carotene, 25mg of

zinc and 2mg of copper or

Omit beta-carotene: 500mg Vitamin C, 400IU of Vitamin E, 80mg of zinc and 2mg of copper, or

Lower the levels of zinc and omit beta-carotene: 500mg Vitamin C, 400IU of Vitamin E, 25mg of

zinc and 2mg of copper [261]

Patient progression to advanced AMD established by the centralized grading of the annual fundus

photographs [261].

Interestingly, the findings did not show any additional benefit of daily supplementation with lutein +

zeaxanthin, DHA + EPA, or lutein + zeaxanthin and DHA + EPA in addition to the original AREDS

formulation on progression to advanced AMD or changes in visual acuity. On the other hand, no harmful

effects of adding DHA + EPA or lutein + zeaxanthin were noted [262]. The research group proposed that

apart from a true lack of efficacy, the null results may be due to other factors such as inadequate dosing,

inadequate duration of treatment, or both [262].

However, data obtained from the secondary randomization showed a possible benefit of using lutein

and zeaxanthin in delaying the progression of AMD to the advanced form when administered without

beta-carotene [262]. The research group proposed that further research is required in this area.

Furthermore, lutein and zeaxanthin did not demonstrate an increased risk of lung cancers in smokers, as

was previously observed with beta-carotene [262].

There was no statistically significant difference between the population taking low dose zinc and that

taking high dose zinc [262]. The study was therefore not able to provide a clear recommendation on the

levels of zinc that are required to delay advancement of the condition [262].

Fellow Eye

In many patients while AMD presents as a unilateral disease, the risk of the fellow eye developing AMD

remains high. The American Academy of Ophthalmology Retina Panel recommends reducing this risk by

taking AREDS supplements, a measure that has been shown to be effective [6]. Furthermore, the panel

recommends that patients monitor their vision regularly and visit an ophthalmologist periodically even

in the absence of symptoms [6]. In the case of any new significant visual symptoms, prompt medical

attention should be sought. Patients that have been identified as high risk patients require more

frequent examinations than those at a lower risk [6].

A recent cohort study of patients enrolled in a multicenter, randomized clinical trial was conducted to

assess whether the drug, drug dosing regimen, as well as patient risk factors affected the incidence of

CNV in the fellow eyes of patients treated for CNV with either ranibizumab or bevacizumab [6]. The

patients found that over 2 years, there was no statistically significant difference between ranibizumab

and bevacizumab in the incidence of CNV in the fellow eye [6]. Furthermore, the genotype of 4 single

nucleotide polymorphisms (SNPs): rs1061170 (CFH), rs10490924 (ARMS2), rs11200638 (HTRA1), and

rs2230199 (C3) did not affect the incidence of CNV in the fellow eye [6].

Conclusion

Age Related Macular Degeneration remains the highest causes of blindness in the Western world. The

etiology of this condition is largely unknown and is thought to involve an interplay of a number of

modifiable and non-modifiable risk factors. As the underlying pathological mechanisms of the

degenerative retinal disorder are discovered, therapies targeted at the involved pathways are being

investigated. The management of neovascular AMD is hopeful with the availability of anti-angiogenic

therapies, both established ones as well as upcoming ones. There is however, no therapy currently

available for the management of geographic atrophy although scientists are developing molecules that

have so far shown some efficacy and safety in phase I and II clinical trials.

Additionally, the identification of single nucleotide polymorphisms, challenging as it has been, has led to

the development of gene therapies that are all still in the early stages of clinical trials. Different research

groups are targeting genes that affect different parts of the pathological processes identified so far.

Lastly, the National Institute of Health has funded a large clinical trial examining the effects of

nutritional supplements in slowing down the progression of AMD. A formulation has been developed

using the first part of the trial. The results of the second part of the trial have recently been published

paving the way for future investigation.

In summary therefore, despite the available therapeutic modalities available in the market, the

condition continues to prevail at a high rate and there is a lot of room for more efficacious, more potent

and safer therapies. Health care practitioners are advised to watch this space for new and upcoming

therapies.

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Macular Degeneration Final examination questions

Choose True or False for questions 1 through 10 and mark your answers online at Nursing.EliteCME.com.

1. In the year 2000, over 1.75 million individuals were reported to have AMD.

True False

2. Individuals with early AMD have drusen that are : <63 µm in diameter.

True False

3. High exposure to sunlight is thought to increase a patients’ risk of developing AMD.

True False

4. AGEs and ALEs both work to inhibit MMPs.

True False

5. Microautophagy is that type of autophagy that occurs through the formation of an

autophagosome: a double membrane bound vesicle that contains cytoplasm and/or organelles.

True False

6. Iq32 is the only major gene locus seen to be associated with AMD.

True False

7. Advanced AMD has a poor prognosis and is associated with severe vision loss.

True False

8. Vertoporfin photodynamic therapy is the first pharmacological therapy to become available for

the management of AMD, using a combination of a photosensitive compound and laser light.

True False

9. Fenretinide is a molecule, currently under development for the management of AMD, that is

thought to work by targeting the inflammasome system.

True False

10. The original AREDS demonstrated the increased risk of lung cancer in smokers taking beta-

carotene supplements.

True False