SURVEY OF OPHTHALMOLOGY VOLUME 48 • NUMBER 3 • MAY–JUNE 2003

MAJOR REVIEW

Age-Related Macular Degeneration: Etiology,

Pathogenesis, and Therapeutic Strategies
Jayakrishna Ambati, MD,1,2 Balamurali K. Ambati, MD,2,3 Sonia H. Yoo, MD,2,4
Sean Ianchulev, MD,2 and Anthony P. Adamis, MD2,5,6

1
Ocular Angiogenesis Laboratory, Department of Ophthalmology, University of Kentucky, Lexington, Kentucky;
2
Massachusetts Eye & Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts;
3
Department of Ophthalmology, Medical College of Georgia, Augusta, Georgia; 4Bascom Palmer Eye Institute,
5
University of Miami School of Medicine, Miami, Florida; Children’s Hospital, Harvard Medical School, Boston,
Massachusetts; and 6Eyetech Research Center, Woburn, Massachusetts, USA

Abstract. Age-related macular degeneration is the principal cause of registered legal blindness among
those aged over 65 in the United States, western Europe, Australia, and Japan. Despite intensive
research, the precise etiology of molecular events that underlie age-related macular degeneration is
poorly understood. However, investigations on parallel fronts are addressing this prevalent public
health problem. Sophisticated biochemical and biophysical techniques have refined our understanding
of the pathobiology of drusen, geographic atrophy, and retinal pigment epithelial detachments.
Epidemiological identification of risk factors has facilitated an intelligent search for underlying
mechanisms and fueled clinical investigation of behavior modification. Gene searches have not only
brought us to the cusp of identifying the culpable gene loci in age-related macular degeneration, but
also localized genes responsible for other macular dystrophies. Recent and ongoing investigations,
often cued by tumor biology, have revealed an important role for various growth factors, particularly
in the neovascular form of the condition. Transgenic and knockout studies have provided important
mechanistic insights into the development of choroidal neovascularization, the principal cause of vision
loss in age-related macular degeneration. This in turn has culminated in preclinical and clinical trials
of directed molecular interventions. (Surv Opthalmol 48:257–293, 2003. 쑖 2003 by Elsevier Inc. All
rights reserved.)

Key words. age-related macular degeneration • choroidal neovascularization •

drusen • macular dystrophies

Age-related macular degeneration (AMD), already
the leading cause of irreversible blindness among the
elderly in industrialized nations,109,240,305 promises
to exact an even greater toll with the imminent
demographic right-shift. While our understanding of
molecular events presaging AMD has grown in the
last decade, its pathogenesis remains puzzling, and
consequently our therapeutic arsenal remains lim-
ited. Nevertheless, the burgeoning development of
directed molecular interventions (monoclonal anti-
bodies, aptamers, ribozymes, gene transfer therapy)
combined with the development of targeted delivery
modalities offers hope in the battle against this
blinding affliction. The abbreviations used through-
out the review along with their definitions are listed
in Table 1.

257
쑖 2003 by Elsevier Inc. 0039-6257/03/$–see front matter
All rights reserved. doi:10.1016/S0039-6257(03)00030-4
258 Surv Ophthalmol 48 (3) May–June 2003
I. Morphologic Forms in AMD and
Their Natural History
A. DRUSEN
The clinical hallmark of AMD is the appearance
of drusen,144 localized deposits lying between the
basement membrane of the retinal pigment epi-
thelium (RPE) and Bruch’s membrane. In the ear-
liest stage, they may be visible ophthalmoscopically
as semi-translucent punctate dots in retroillumina-
tion. As the overlying RPE thins, drusen are more
obvious as yellow-white deposits; they may regress,
leaving atrophy in their wake. Clinically, drusen are
classified morphologically either as hard or soft. Hard
drusen are pinpoint yellow-white lesions, typically less
than 63 µm in diameter, that appear as window
defects on fluorescein angiography. In small num-
bers, they are not considered risk factors for the de-
velopment of AMD,392 but numerous hard drusen
are an independent risk factor for visual loss from
AMD.278 Soft drusen generally are larger and have
indistinct edges with a tendency to become conflu-
ent. With increasing age, drusen can become calcified
or filled with cholesterol, appearing crystalline or
polychromatic. Typically drusen are clustered in the
central macula.
Sarks and colleagues have presented a refined clas-
sification of drusen incorporating not only clinical
characteristics, but also temporal evolution, histopa-
thology, and angiographic findings.390 Apart from
hard drusen, they have identified drusen that derive
from clusters of hard drusen (hard or soft cluster-
derived drusen), membranous drusen, and regressing
drusen.
TABLE 1
Abbreviations
ABCR ATP-binding cassette transporter
AGE advanced glycation products
AMD age-related macular degeneration
Ang1/2 angiopoietin-1/2
BlamD basal laminar deposits
BlinD basal linear deposits
CNV choroidal neovascularization
ECM extracellular matrix
eNOS endothelial nitric oxide synthase
FA fluorescein angiography
FGF2 basic fibroblast growth factor
ICAM-1 intercellular adhesion molecule-1
ICGA indocyanine green angiography
MMP matrix metalloproteinase
MPS Macular Photocoagulation Study
PEDF pigment epithelium derived factor
RAP retinal angiomatous proliferation
ROI reactive oxygen intermediates
RPE retinal pigment epithelium
TAP treatment of AMD with
photodynamic therapy
VEGF vascular endothelial growth factor
AMBATI ET AL
These drusen subtypes exhibit characteristic angio-
graphic findings.33 Both hard drusen and hard clus-
ter-derived drusen (63–250 µm) appear as window
defects (early hyperfluorescence that fades in late
phase) on fluorescein angiography (FA) and hyper-
fluoresce during indocyanine green angiography
(ICGA). Soft cluster-derived drusen, however, exhibit
weak and delayed hyperfluorescence on FA and are
hypofluorescent during ICGA. Membranous drusen
(63–250 µm) show very faint delayed hyperfluores-
cence on FA but are not detectable on ICGA. Re-
gressing drusen demonstrate hyperfluorescence on
FA and early ICGA hyperfluorescence. Although
drusen alone do not account for significant loss of
visual acuity in AMD, they can lead to deficits in
macular function such as color contrast sensitivity,
central visual field sensitivity, and spatiotemporal sen-
sitivity.125,297,298,440,454,479 Instead, detachments and
loss of the overlying RPE and retina seem to be the
principal cause of central visual acuity loss. A prospec-
tive evaluation of patients with drusen in the fellow
eyes of unilateral exudative AMD suggests that the
risk of developing choroidal neovascularization
(CNV) in the second eye peaks at 4 years, with
an increasing incidence of geographic atrophy there-
after.394 The annual incidence of developing CNV
ranges from 5–14%. Greater size, confluence, and
greater number of drusen were associated with in-
creasing rates of CNV. The risk of legal blindness
in the fellow eye of patients with unilateral visual
loss from CNV is approximately 12% over 5 years.278
In patients with bilateral drusen and good vision
in both eyes, the annual incidence of new atrophic or
exudative lesions is approximately 8% over 3 years.205
These patients have a cumulative risk of 14.5% of
developing CNV over 4 years.426 In this group conflu-
ence of drusen, focal RPE hyperpigmentation or atro-
phy, and delayed choroidal filling were strong risk
factors. Interestingly, spontaneous resolution of
large confluent drusen, hyperpigmentation, or non-
geographic RPE atrophy was observed in a signifi-
cant proportion of patients in a 5-year follow up of
the Maryland Waterman study.57
Four independent risk factors for the development
of CNV in fellow eyes have been established from
multivariate analyses in the Macular Photocoagula-
tion Study (MPS).59,278 The presence of five or more
drusen seems to be the strongest predictor, with a
relative risk of 2.1. The second in order is focal RPE
hyperpigmentation with a relative risk of 2.0, fol-
lowed by systemic hypertension and the finding of
one or more large drusen. Of 127 fellow eyes free
of neovascular AMD at study entry in patients with
extrafoveal CNV, 26% developed CNV over 5 years.59
Similarly, MPS subfoveal and juxtafoveal studies fol-
lowed 670 fellow eyes free of neovascular AMD at
AGE-RELATED MACULAR DEGENERATION
study entry, 35% of which developed CNV by the end
of the 5-year period.278
Extramacular drusen also are a risk factor for the
development of AMD.265 Reticular pseudodrusen
refer to a yellowish interlacing network 125 µm to
250 µm in width, usually appearing along the super-
otemporal arcade and corresponding to choroidal
venous attenuation, that do not fluoresce on fluores-
cein or indocyanine green angiography.34 They are
an important risk factor for the development of CNV
(66%) in one or both eyes.
B. GEOGRAPHIC ATROPHY
Geographic atrophy refers to confluent areas (de-
fined as 175 µm in minimum diameter) of RPE cell
death accompanied by overlying photoreceptor
atrophy.156 Often it develops in the parafoveal
region, sparing the fovea until late in the dis-
ease,145,156,280,389,396 and is bilateral in more than half
of patients.156,364,389 Geographic atrophy can develop
following fading of drusen, in an area of RPE attenua-
tion, following flattening of a RPE detachment, or
involution of CNV. The visible atrophy is usually
accompanied by atrophic underlying choriocapil-
laris,294,370 and large areas of atrophy reveal underly-
ing choroidal blood vessels seen ophthalmoscopically
or angiographically.
Areas of geographic atrophy are characterized by
diffuse, irregular patches of increased autofluores-
cence, which precede the development and enlarge-
ment of geographic atrophy.201 This suggests that
accumulation of RPE lipofuscin, the dominant
fundus fluorophore, may be associated with the
pathogenesis of geographic atrophy.
Historically, patients with geographic atrophy were
not considered to be at risk for developing CNV.
However, Green et al showed a 34% incidence of
geographic atrophy in a histopathologic study of pa-
tients with CNV.155 Geographic atrophy and CNV
both tend to develop in patients with large drusen
and pigmentary changes.204,239 Patients with bilateral
atrophy can develop CNV at a rate of 2–4% over
two-year period.453 Similarly, CNV can conceal an
atrophic process, thus obscuring the distinction.
Geographic atrophy leads to gradual progression
of visual loss, most likely because photoreceptors
overlying areas of RPE atrophy are metabolically de-
pendent on RPE cells. Marked apoptosis of not only
the outer nuclear layer (primarily rods), but also
the inner nuclear layer of the retina, is found near
clusters of RPE atrophy.101 However, at least in some
patients, overlying photoreceptors may be viable as
demonstrated by the striking improvement in vision
after macular translocation surgery.44 Even before
visual acuity drops significantly, there is consider-
able visual impairment due to scotomas near fixation.
259
Geographic atrophy leads to a reduction in dark-
adapted function, with a profound drop in acuity and
retinal sensitivity in dim environment.454–456 Of eyes
with visual acuity better than 20/50, fully half lose
three or more lines of vision over 2 years, and a
quarter lose 6 or more lines.456 Geographic atrophy
is responsible for 20% of the legal blindness from
AMD.115
C. CHOROIDAL NEOVASCULARIZATION
Choroidal neovascularization (CNV) refers to the
growth of new blood vessels from the choroid. In
AMD, these vessels may remain beneath the RPE, or
breach the RPE and enter the subretinal space. The
earliest sign of CNV often is subretinal or sub-RPE
hemorrhage. Appearing as a greenish gray subretinal
tissue, CNV characteristically leaks fluorescein on an-
giography. However, subretinal blood or lipid exu-
dates, also signs of CNV, may block the angiographic
hyperfluorescence. CNV may not exhibit fluorescein
leakage if it has undergone involution or is enveloped
by RPE proliferation. Repeated leakage of blood,
serum, and lipid can stimulate fibroglial organization
leading to a cicatricial (disciform) scar.
The fluorescein angiographic leakage patterns of
CNV are classified either as classic or occult (Fig. 1).
The former refers to discrete areas that hyperfluore-
sce early in the study and continue to exhibit progres-
sive leakage with increasing intensity and extent.
Occult CNV refers either to a fibrovascular RPE de-
tachment with irregular elevation of the RPE with
stippled hyperfluorescence and late leakage, or to
late leakage of undetermined origin.
Data from the Macular Photocoagulation Study
(MPS) Group suggest that the natural history of CNV
is bleak. Over 5 years, 62% of eyes with extrafoveal
CNV275 and 65% of eyes with juxtafoveal CNV276
suffer severe visual loss (loss of 6 or more lines of
visual acuity). Of eyes with subfoveal CNV, those with
poor initial vision have a 25–35% probability of severe
visual loss, while those with relatively good initial
vision have a 55–65% probability of severe visual
loss.277 Although the MPS data demonstrated the
benefit of thermal laser photocoagulation for extrafo-
veal and juxtafoveal CNV, as well as a select group
of subfoveal CNV,275–277 since most CNV membranes
are occult,56,120 the vast majority (87%) of patients
with newly diagnosed exudative AMD do not meet
MPS criteria for laser photocoagulation.127 Similarly,
while ocular photodynamic therapy with verteporfin
is beneficial to patients with predominantly classic
CNV and some with occult CNV,483,484 most patients
do not experience visual improvement. Moreover,
only a small fraction (18%) of patients with CNV are
eligible for this therapy.48
260 Surv Ophthalmol 48 (3) May–June 2003
Retinal angiomatous proliferation (RAP), also
called deep retinal vascular anomalous complex or
retinochoroidal anastomosis, has been recognized
as a peculiar form of neovascular AMD.185,186,250,425,516
RAP refers to new vessel development extending
outward from the neurosensory retina, sometimes
anastomosing with the choroidal circulation.256 Inter-
mediate and late stages of RAP are angiographically
imaged better with ICG than fluorescein.250 The pres-
ence of intraretinal hemorrhages, telangiectasia, and
microaneurysms can aid in the diagnosis of this
entity, especially in the early stage.
As many as 20–30% of occult CNV with focal hot
spots on ICG angiography may fall into this cate-
gory.250,425 Typically, RAP commences as intraretinal
capillary proliferation, later extending into the su-
bretinal space, and finally can terminate with frank
CNV.516 This last stage sometimes is associated with
a vascularized RPE detachment. These arise often
adjacent to areas of focal hyperpigmentation,185
and are particularly resistant to thermal laser photo-
coagulation185,186,250,425 or photodynamic therapy.382
D. RPE DETACHMENT
Soft drusen can coalesce to form overlying serous
detachments of the RPE (RPED). Thus the angio-
graphic distinction between soft drusen and small
RPE serous detachments is arbitrary. Studies using
strict angiographic criteria of uniform fluorescence to
define a serous detachment of the RPE have reported
AMBATI ET AL
Fig. 1. A: Early and late phase FA
of classic CNV. Early phase shows a
discrete area of RPE/choroidal leak-
age, that increases both in intensity
and area in the late phase. B: Early and
late phase FA of occult CNV. Early
phase shows no choroidal leakage,
but late phase shows marked leak-
age at the level of the RPE/choroid
of unknown origin.
that one-third to one-half of patients over 50 years
old with RPE detachments developed CNV and
severe visual loss within several years from the onset
of the detachment.67,107 Large and highly elevated
RPEDs can induce tangential stress and lead to a
rupture (RPE tear). Hypofluorescent RPEDs are
more likely to progress to a RPE tear, probably
because of the increased lipid content in Bruch’s
membrane. RPEDs can cause visual loss in patients
with AMD.302 Visual loss can also follow spontane-
ous or laser-induced flattening of the RPED, probably
due to metabolic failure of the RPE.
II. Pathology
AMD is a degenerative disease that affects the outer
neural retina, RPE, Bruch’s membrane, and the cho-
roid. The locus of the primary insult remains in dis-
pute. The hallmarks of the disease are diffuse and
focal thickening of Bruch’s membrane (drusen) to-
gether with the development of hypo- and hyper-
pigmented areas of RPE, the former signifying areas
of RPE cell loss.154–156
A. NONEXUDATIVE AMD
Histopathological studies of the choroid disclose
a relative decrease in vessel density and a narrowing
of vessel lumens. This phase of the disease is often
referred to as dry AMD. In contrast, wet or neovascu-
lar AMD is characterized by the proliferation of
AGE-RELATED MACULAR DEGENERATION
blood vessels from the choriocapillaris, through
Bruch’s membrane, and into and around the RPE
and photoreceptor layers. Vision loss occurs through
the accumulation of exudate and hemorrhage, and
the secondary cell death and reactive gliosis it
precipitates.
The pathological abnormalities in AMD are most
pronounced in the area centralis, particularly so in
the central macula. At various stages, the RPE, photo-
receptors, and the choroidal vasculature can be in-
volved. The earliest pathological changes are the
appearance of basal laminar deposits (BlamD) and
basal linear deposits (BlinD).155,156 BlamD consist of
membrano-granular material and foci of wide spaced
collagen between the plasma membrane and basal
lamina of the RPE. BlinD consist of vesicular mate-
rial located in the inner collagenous zone of Bruch’s
membrane. There is an increased amount of BlamD
in eyes with AMD,155 whereas BlinD apparently is a
specific marker for AMD.86 Although these early basal
deposits can affect vision and exhibit a faint pattern
of staining in the late phases of the fluorescein angio-
gram, they may escape ophthalmoscopic evaluation.
Immunohistochemical analyses have shown vari-
ous molecular constituents, such as apolipoproteins
B and E, different immunoglobulins, factor X, amy-
loid P component, complement C5 and C5b-9 termi-
nal complexes, fibrinogen, vitronectin, and others,
to be present in all phenotypes of hard and soft
drusen.29,171,172,218,269,284,323 It is interesting that many
of these constituents, including a number of acute
phase reactants, are participants in the humoral and
cellular immunity. Such recent observations suggest
a role for immunological and inflammatory processes
in drusen pathogenesis. Preliminary studies have
characterized the presence of morphologically dis-
tinct choroidal cells, which have been identified
as dendritic, antigen-presenting cells by marker anal-
yses, lending further weight to the presumptive role
of immunological processes in AMD pathogenesis.170
Later in the disease, soft drusen (defined as being
larger than 63 µm in diameter) may appear. Most
often, soft drusen represent an accumulation of
BlinD or localized detachment of BlamD.58,155
B. EXUDATIVE AMD
The development of CNV is the hallmark of exuda-
tive AMD. The choroid gives birth to CNV, which then
extends through a defect in Bruch’s membrane and
into a plane between BlamD and Bruch’s mem-
brane.155 CNV also can extend through the RPE into
the subretinal space. CNV arises as a capillary-like
structure, usually with multiple points of origin, and
evolves into arterioles and venules.155,156,398 CNV can
cause serous or sanguineous detachment of the RPE
261
or retina, pigment modeling, RPE tears, and lipid
exudation. Consistent with the predilection of CNV
for the central macula, there is a thinning of the
elastic and outer collagenous laminae of Bruch’s
membrane in the foveal region in eyes with
AMD.169 Whereas Bruch’s membrane is 145 nm thick
in the periphery, at the fovea it decreases to 55 nm.
The porosity of its elastic lamina increases from
nearly 0% in the periphery (17 mm from the fovea)
to approximately 35% at the fovea.169
Histological examination of eyes with CNV or disci-
form scars has revealed macrophages adjacent to thin
areas and ruptures in Bruch’s membrane.228,351,490
The histopathologic features of surgically excised
CNV in patients with AMD, ocular histoplasmosis syn-
drome, myopia, and idiopathic and pattern dystrophy
suggest that CNV represent a stereotypic, nonspecific
response, regardless of underlying disease.124,159 The
most prominent cellular components of the mem-
branes in decreasing order of frequency were RPE,
vascular endothelium, fibrocytes, macrophages, and
photoreceptors. The most prominent extracellular
components in the membranes were collagen, fibrin,
basal laminar deposit, and fragments of Bruch’s
membrane.159
The disciform scar represents an end-stage. It is
usually vascularized, almost invariably from the cho-
roidal circulation but sometimes with retinal contri-
bution as well, and can have both subretinal and
sub-RPE components.154,155 The degree of RPE and
photoreceptor degeneration is proportional to the
diameter and thickness of the disciform scar. A tear
of the RPE can accompany a minority of disciform
scars. Detachments of the RPE or the neurosensory
retina are found in 10% of autopsied AMD eyes, and
can have serous or sanguineous components.155
C. GEOGRAPHIC ATROPHY
Geographic atrophy of the RPE and overlying pho-
toreceptor layer is present in 37% of AMD eyes at
autopsy.155 Contrary to common belief, this dry form
of AMD is associated with CNV or disciform scarring
in 34% of eyes with geographic atrophy. Drusen of
all types accompany geographic atrophy. Geographic
atrophy is characterized by absence of the RPE, lead-
ing to loss of photoreceptors.156 The choriocapillaris
also may be absent.
Any of the stages in the overlapping morphological
spectrum of AMD can witness changes in the RPE,
including attenuation, atrophy, hypertrophy, hyper-
plasia, and pigment clumping in the subretinal
space.58,155
III. Risk Factors and Relation to Etiology
Vast epidemiological studies have shed light on var-
ious factors associated with an altered risk of AMD.
262 Surv Ophthalmol 48 (3) May–June 2003 AMBATI ET AL

Apart from aiding early detection and treatment,
these risk factors may also provide insight into the
etiology of the disease. Table 2 provides a list on
ongoing experimental trials for AMD.
A. DEMOGRAPHIC FACTORS
The incidence, prevalence, and progression of all
forms of AMD increase with advancing age.243,307,427
In experimental models, older animals are more
susceptible to induction of CNV.108 Female sex is
associated with a slightly greater prevalence of AMD
in many studies; however, the longer life span of and
greater health-care utilization by women confound
the issue. A meta-analysis of population based studies
reveals that women have only a slightly higher preva-
lence of the disease and that this risk is confined
mainly to those over 75 years of age.110 Pooled data
from the Beaver Dam Eye Study, Blue Mountains Eye
Study, and the Rotterdam Study revealed no sex dif-
ferences in AMD risk.427 However, recent analyses
from the Blue Mountains Eye Study suggest that the
5-year incidence of neovascular AMD among women
is double that of men (1.2% vs. 0.6%).307 In addi-
tion, experimental models suggest that female an-
imals are more susceptible to induction of CNV, with
higher expression of kinase insert domain receptor
(KDR), a receptor for vascular endothelial growth
factor (VEGF), a potent angiogenic cytokine.464 A
mechanistic basis is suggested by retinal capillary en-
dothelial cells upregulation of VEGF synthesis and
KDR expression in response to 17β-estradiol.460
All forms of AMD are more prevalent in the white
population than more darkly pigmented
races,131,241,358 leading to the belief that melanin may
be protective against development of CNV. There is
some suggestion that pigmented animals are less
likely to develop neovascularization.383 The presence
of melanin also seems to protect against formation of
RPE lipofuscin, a marker of cellular senescence and
promoter of oxidative damage.225,450 Although some
reports have found an association between light-col-
ored irides and AMD,212,306,502 data pooled from large
studies conducted in three continents reported no
association between iris color and AMD.427 Studies
of monozygotic twins showed markedly similar inci-
dences of AMD changes151,179,234,296 which, while sug-
gestive of a genetic component, seem to militate
against an overriding role of pigmentation because
macular pigment optical density varies greatly
between such twins.178 A recent study found no differ-
ences in macular or melanin pigment densities be-
tween eyes with and without early AMD.45 In sum,
the data seem to suggest that racial differences in
AMD prevalence may be due to factors other than
pigmentation.
B. CARDIOVASCULAR FACTORS
Vascular risk factors have been hypothesized as
important pathogenetic factors for the development
of AMD. Cigarette smoking is third only to older age
and family history as a significant risk factor for AMD.
Smoking has been associated with an increased risk

TABLE 2
Ongoing Experimental Trials for AMD
Application Translation state
Exudative AMD
VEGF rhuFab (VEGF antibody fragment) Vitreous injections Phase II
EYE001 (VEGF aptamer) Vitreous injections Phase II/III
Anecortave acetate Sub-Tenon injection Phase II
Envision (Fluocinolone acetonide) Vitreous implant Phase I/II
TTT4CNV (Transpupillary Thermotherapy for CNV) Laser application Phase III
AMDRATS (AMD Radiation Therapy Study) External beam Phase I
AdPEDF (Adenoviral vector coding for PEDF) Vitreous injections Phase I
Squalamine Intravenous Phase I/II
Combretastatin Intravenous Phase I/II
SST (Submacular Surgery Trials) Surgery Phase III
Early retreatment of photodynamic therapy Laser application Phase II
Identification and treatment of feeder vessels Laser application Phase I
Limited macular translocation versus photodynamic therapy trial Surgery/Laser Phase I/II
Celecoxib with PDT in AMD Oral/Laser Phase II
Nonexudative AMD
Genistein Oral Phase I
Lutein for AMD Oral Phase II
PTAMD (Prophylactic Treatment of AMD) Laser application Phase III
CNVPT (CNV Prevention Trial) Laser application Phase III
MIRA-1 (Multi-Center Investigation of Rheophoresis for AMD) Systemic Phase I
AGE-RELATED MACULAR DEGENERATION
of developing AMD in most population-based stud-
ies.6,94,242,292,308,427,495 Both prior and current smok-
ers are at increased risk for AMD,212,428,470 with these
individuals developing AMD 5 to 10 years before
non-smokers, respectively.308 This may be due to
smoking’s effects on antioxidant metabolism and
choroidal blood flow. Risk ratios of 2.0 or more for
neovascular AMD associated with smoking have been
described.76,407 Male smokers may be at higher risk
of developing nonexudative AMD than female
smokers.308 However female smokers may be at
particular risk for progression to advanced AMD (3.5-
fold).81 These authors found that men who contin-
ued or ceased smoking and past female smokers had
a small suggestion of increased risk that was not statis-
tically significant. Smokers also are at increased risk
for large or extensive drusen and geographic
atrophy.6,308 Cigarette smoking is associated with in-
creasing rates of recurrent CNV after laser photoco-
agulation.274 The finding that nicotine stimulates
neovascularization by inducing endothelial cell pro-
liferation and accelerating fibrovascular growth189
provides another mechanistic basis for these observa-
tions. Nicotine appears to increase severity of choroi-
dal neovascularization in a murine model of laser
injury.451
The purported link between atherosclerosis and
AMD,132,133,494 spurred in part by the similarity
between the molecular composition of drusen and
atherosclerotic deposits,323 finds no tenable epidemi-
ological support.211,236,237 However, the association
between systemic hypertension and neovascular
AMD is more persuasive.6,211,438 The visual benefits
of laser photocoagulation of CNV do not extend to
those with systemic hypertension,276 suggesting an
etiologic relationship to angiogenesis. The role of
angiotensin II, a culprit in systemic hypertension, in
potentiating retinal neovascularization in models of
retinopathy of prematurity270,315 and diabetic reti-
nopathy148 is intriguing and suggestive of a similar
role in CNV. That angiotensin II induces the expres-
sion of VEGF and angiopoietin 2,136,343 growth fac-
tors involved in angiogenesis (see below), provides
further mechanistic evidence for its putative role.
There is preliminary evidence that blockade of an
angiotensin II receptor partially inhibits experi-
mental CNV in a rat model.196
C. LIGHT EXPOSURE
Excessive exposure to light can damage the retina.
Photooxidative damage, mediated by reactive oxygen
intermediates (ROI), has been implicated as a culprit
in the development of AMD (see below). Clinical
study of this important factor is hindered by the diffi-
culty of quantifying light exposure over a lifetime.
263
Accordingly, there have been conflicting reports on
the association of ultraviolet or visible light with
AMD.53,83,88,212,306,466,470,504 The association between
cataract surgery and development of signs of late
AMD244 may suggest a detrimental effect of increased
light exposure. One study suggests that the use of
hats and sunglasses may be slightly protective.83 The
prevailing mechanistic hypothesis is that photoactiva-
tion of protoporphyrin may lead to ROI in the outer
retina and choroid.152 In turn, this may lead to lipid
peroxidation of the outer segments of photorecep-
tor cells.
D. DIETARY AND MEDICATION FACTORS
The interest in dietary connections is spurred by
the fact that it is modifiable. A prospective study of
participants in the Nurses’ Health Study and the
Health Professionals Follow-up Study, together cull-
ing more than 70,000 men and women, revealed that
total fat intake was positively associated with risk of
AMD.72 In particular, high linolenic acid intake was
associated with a 49% increased risk of AMD, and
high docosahexaenoic acid was associated with a 30%
reduced risk of AMD. Higher dietary consumption
of fish was associated with a lower risk of AMD.72,429 A
case-control study of patients with neovascular AMD
reported that higher intake of linoleic acid and vege-
table, monounsaturated, and polyunsaturated fats
was associated with a greater risk for advanced AMD,
and that high intake of ω-3 fatty acids and fish were
inversely associated with risk for AMD when intake of
linoleic acid was low.406 However, the Third National
Health and Nutrition Examination Survey did not
find any association between dietary fat or fish intake
and AMD.194
Higher energy-adjusted intake of cholesterol was
associated with a higher risk of AMD.288,429 Analysis
of body-mass index has revealed that obese individ-
uals are at higher risk for dry and neovascular
AMD,6,397,430 while extremely lean individuals are at
higher risk for dry AMD.397
A prospective study among female nurses and male
health professionals did not disclose any protective
effect of alcohol consumption.71 In fact, among
women who consumed more than 30 g of alcohol
per day, there was a modest increased risk of AMD.
There appears to be no association between coffee
or caffeine intake and AMD.480
The high levels of certain anti-oxidant vitamins and
minerals in the retina, and the concentration of ca-
rotenoids in the macula have led to the speculation
that micronutrient supplementation may confer
protection from AMD. The initial reports on the
correlation between high serum carotenoids and
lower risk of AMD111,470 could not establish causality.
264 Surv Ophthalmol 48 (3) May–June 2003
Very low levels of lycopene, the most abundant
serum carotenoid, but not the macular pigment ca-
rotenoids (lutein and zeaxanthin), are associated
with AMD.287
Although there was an inverse association between
carotenoid, vitamin E, or zinc intake and early
AMD,493 and a pilot randomized trial suggested a
beneficial role for oral zinc supplementation,324 two
large prospective studies, which together enrolled
more than 100,000 men and women, found that
moderate zinc intake, via food or supplements, did
not protect persons without AMD from developing
the condition over an 8–10-year period.73 A recent
prospective randomized trial of vitamin E supple-
mentation revealed no differences in the rate of
development of early or late stages of AMD.467
However, data from the Age-Related Eye Disease
Study (AREDS) suggest that supplementation with
very high doses of zinc, vitamin C, vitamin E, and β-
carotene provide a modest protective effect on pro-
gression to advanced neovascular AMD in patients
with extensive drusen or fellow eyes with neovascular
AMD.7 This benefit did not extend to patients without
AMD or with few drusen. However, the recommenda-
tions of this study have been questioned on various
grounds.1,23,408 Although higher blood levels of vita-
min E may be protective against AMD,503 there is no
convincing evidence to support its supplementa-
tion,467,493,503 and these differences may be due en-
tirely to coexisting lower serum lipid levels.287
One of the reasons for inconclusive evidence re-
garding dietary supplements is that the aforemen-
tioned observational studies cannot adequately
control for the numerous differences that surely
exist between people with diets rich in micronutri-
ents and those that do not. Ongoing experimental
trials such as the L-glutathione augmentation trial for
geographic atrophy31 are likely to yield more solid
evidence in this regard. Meanwhile, absent convinc-
ing evidence, patients should be cautioned that nutri-
ent supplements are not necessarily innocuous. For
instance, among male smokers, β-carotene supple-
mentation increases the risk of lung cancer and mor-
tality,340,469 whereas multivitamin supplementation
increases overall cancer mortality.498 High serum
levels of zinc are associated with Alzheimer’s dis-
ease,386 promote amyloid fibril aggregation,62 worsen
outcomes after acute stroke,346 may cause RPE cell
apoptosis,509 and exacerbate diabetes mellitus.85,374
Higher rates of hospitalizations for genitourinary
dysfunction in males taking the high dose micronutri-
ent supplementation were observed in AREDS.7
Data from the Physicians’ Health Study I seems to
rule out any large beneficial effect of low-dose aspirin
use in AMD.75 Although the Beaver Dam Eye Study
uncovered no association between medication use
AMBATI ET AL
and early AMD,238 the use of angiotensin-converting
enzyme inhibitors or cholesterol-lowering medica-
tions was associated with an increased risk of AMD
in an Australian cohort.292 A smaller cross-sectional
study in the United Kingdom reports that elderly
users of statins have one-eleventh the risk of devel-
oping AMD compared to non-users.174 This is striking
because the discovery that some statins actually pro-
mote angiogenesis251 seems to be at odds with this
finding. Perhaps the cholesterol-lowering effect of
these drugs, which may result in decreased BlinD
in Bruch’s membrane,98 or their anti-inflammatory
effect436 may be salutary in this condition. However,
these preliminary findings in a very small cohort
should be interpreted with much caution.492
IV. Animal Models
There are no animal models that faithfully repli-
cate all features of clinical AMD. This, in large part,
has hampered the delineation of etiology and the
development of a desirable prophylactic or curative
treatment. However, spontaneous and iatrogenic
conditions have shed some light.
A. SPONTANEOUS AGING
Primates and other animals develop drusenoid
changes in the retina with aging, and these underly-
ing processes have been studied as a possible model
for the pathogenesis of human AMD.
The prevalence of age-related macular changes in
primates appears to be similar to humans. Rhesus
monkeys develop drusen as they age, although
slightly accelerated compared to humans. Other
changes include pigment mottling, hypopigmented
spots, and dull or absent foveal reflex. A statistically
significant correlation has been established between
the absence of foveal reflex and the presence of pig-
ment mottling/hypopigmentation of macula.312
Among a closed colony of rhesus monkeys, drusen
were observed in 58% of the monkeys and 47% of
their eyes.208 The prevalence and severity of drusen
were linearly related to increasing age and signifi-
cantly higher in females and specific maternal lin-
eages. Although frank CNV was not identified, late
hyperfluorescence consistent with degenerative scar-
ring and atrophy was seen in some eyes.
Aging primate eyes with AMD manifest RPE histo-
pathologic changes very similar to those observed in
human eyes. Heterogeneous membranous, granular
and cellular deposition is seen in the collagenous
zones of Bruch’s membrane alongside dense bodies
in Bruch’s membrane and RPE cytoplasm.487 How-
ever, these deposits are dissimilar to human AMD
AGE-RELATED MACULAR DEGENERATION
drusen both in ultrastructural morphology and bio-
chemical composition.198,322
One of the strains of the senescence-accelerated
mouse, SAM P8, exhibits some features of AMD. It has
thickened Bruch’s membrane, lipoidal degeneration
and atrophy of the RPE, mild choriocapillaris atro-
phy, and abnormal deposits in the inner Bruch’s
membrane akin to BlamD seen in clinical AMD.282
Perhaps most significant, some SAM P8 mice
have abnormal intra-Bruch’s neovascularization. Al-
though frank CNV is not present, this model may
provide some insight into the early events of CNV
pathogenesis.
B. TRANSGENIC MODELS
Targeted modification of the genome is a powerful
tool in examining the role of individual genes and
proteins that may play a role in a disease process.
The multi-pronged strategy of studying deletional
mutants, transgenic and knock-in animals permits
the parsing of individual genes and their potential
contribution. Campochiaro and colleagues created
transgenic mice that overexpress VEGF in the photo-
receptors under control of the rhodopsin
promoter.336,476 In one line of these mice that had
sustained overexpression of VEGF, new vessels origi-
nated from the deep retinal capillary bed and ex-
tended through the photoreceptor layer into the
subretinal space. Although retinal neovascularization
was observed, CNV was not. However, among another
line of these mice which had transient high-level
VEGF expression accompanied by photoreceptor de-
generation (with subsequent loss of VEGF), CNV
developed during the transient period of ectopic
VEGF production. These data suggest that healthy
photoreceptors, and perhaps a healthy Bruch’s mem-
brane, prevent CNV formation despite high levels
of VEGF.
D’Amato and colleagues created transgenic mice
that overexpress VEGF in the RPE under control
of the RPE65 promoter.403 The eyes of these mice
revealed intrachoroidal CNV that did not pen-
etrate through an intact Bruch’s membrane and RPE
cell layer. Overexpression of VEGF by the RPE in rats,
achieved by subretinal injection of a recombinant
adenovirus vector encoding VEGF, led to the tran-
sient (weeks) development of frank CNV that
breached Bruch’s membrane to result in subretinal
new vessels that leak fluorescein like clinical
CNV.35,439 Similar use of an adeno-associated virus
vector encoding VEGF led to sustained development
of CNV in rats.497
The development of frank CNV in the viral
models may be due, in part, to iatrogenic breaks in
Bruch’s membrane induced by subretinal injection of
265
virus or to the inflammation and macrophage recruit-
ment induced by adenoviruses.36 Differential species
susceptibility may also be at play as the rat Bruch’s
membrane is easier to fracture with laser photocoagu-
lation. The absence of CNV when photoreceptors
overexpress VEGF and the absence of retinal neo-
vascularization when the RPE overexpresses VEGF
demonstrates a striking localization of paracrine
effect. Taken together, these data support a two-hit
hypothesis, indicating that both VEGF overexpres-
sion and a defect in Bruch’s membrane are required
for bone fide CNV.
C. LASER INJURY
Ryan and colleagues described a laser injury model
of CNV in primates,387 which has provided valuable
information about the natural history of CNV, the
role of the RPE in re-establishing the blood retinal
barrier, and its role in the scarring process. Laser
burns applied to the fundus resulted in CNV only if
Bruch’s membrane was disrupted, as evidenced by
the appearance of a bubble during the procedure.
Interestingly, while 40% of macular laser lesions in-
duced CNV that leaked on fluorescein angiography,
fewer than 3% of extra-macular lesions induced CNV.
All lesions exhibiting fluorescein leakage con-
tained CNV upon histopathological examina-
tion.300,301 However, 80% of non-leaking lesions also
contained histopathological CNV. The difference in
leakage was due to the presence (or absence) of
subretinal fluid overlying the CNV. In this model, all
lesions eventually ceased fluorescein leakage, but not
because of a reduction in or disappearance of CNV.
Ultrastructural investigation revealed that both leak-
ing and non-leaking vessels had fenestrations similar
to native choriocapillaris, but that RPE proliferation
resulted in envelopment of CNV leading to cessa-
tion of leakage.299,302 That RPE may resorb or prevent
accumulation of subretinal fluid has also been sub-
stantiated in clinicopathological studies of human
CNV.
Although the laser-induced model of CNV may
involve processes not relevant to AMD, it captures
many of the important features of the human condi-
tion. Laser photocoagulation that disrupts Bruch’s
membrane can induce CNV in humans.122 Migration
of choroidal vascular endothelial cells and newly
formed vessels into the subretinal space through de-
fects in Bruch’s membrane, accumulation of subreti-
nal fluid, congregation of leukocytes adjacent to
arborizing neovascular tufts,157,328,334,353,363 and fi-
brovascular scarring are shared features of the exper-
imental model and CNV secondary to AMD. In both
cases, the newly formed CNV contains fenestrated
endothelial cells and permeable inter-endothelial
266 Surv Ophthalmol 48 (3) May–June 2003
junctions.299,393,477 Leakage of fluorescein from these
immature new vessels into the subretinal space in
the animal model appears angiographically similar to
the leakage of classic CNV in AMD.299 Macrophages
have been found in close proximity to thinned and
perforated areas of Bruch’s membrane,228,490 and
participate in the digestion of the outer collagenous
zone of Bruch’s membrane,490 both of which facili-
tate the subretinal entry of CNV.
There are also similarities in growth factor profiles
between laser-induced CNV and CNV in AMD. Immu-
nostaining has demonstrated the presence of VEGF
in RPE and leukocytes,124,213,253,271,344,418,496 VEGF
receptors in RPE and endothelial cells,496 basic
fibroblast growth factor (FGF2) in RPE cells,
choriocapillaris endothelial cells, and in macro-
phages,26,124,331,376 transforming growth factor-β
(TGF-β) in RPE, fibroblasts, and choriocapillaris en-
dothelium,26,246,332,376 tumor necrosis factor-α (TNF-
α) expression in leukocytes,334 and involvement of
Fas and Fas-ligand197,222 in both conditions.
The laser injury model has been used to investigate
the efficacy of various drugs, although the high cost
associated with primates limits widespread use. Rat
models are more feasible for investigating therapeu-
tic efficacy, for example, via vitreous implants,379 and
have provided information about the spatial and tem-
poral expression of growth factors and cellular adhe-
sion molecules during the development of CNV.521 A
recent model of CNV in mice possesses the additional
advantage of being able to parse the effects of manip-
ulating specific genes on CNV development.477 It may
also permit study regression of CNV because these
lesions continue to leak on fluorescein angiography
for several months.257
V. Theories of Etiology
A. GENETICS
Much evidence points to a familial component of
AMD. Twin-concordance, linkage studies, and bio-
molecular investigations implicating genetic factors
are compelling. Genetic analyses of AMD have been
hindered by the late onset of the disease, which re-
sults in limited study pedigrees, as well as the inter-
twined nature of multiple genetic influences acting
in concert with myriad environmental provocations.
Furthermore, the clinical classification of AMD may
not lend itself to genetic analyses: clinical heteroge-
neity may not necessarily correlate with genetically
different forms of AMD. For example, one study
reported a significant odds ratio of 3.1 for exudative
AMD in siblings, while the odds ratio of 1.5 for geo-
graphic atrophy was not statistically significant, sug-
gesting varying relative genetic contributions for dif-
ferent subtypes of AMD.405
AMBATI ET AL
Nonetheless, familial aggregation of AMD has
been demonstrated. First-degree relatives of cases are
three times more likely to develop exudative AMD
than controls.233 This study also estimated that the
proportion of end-stage AMD in the population at-
tributed to genetic factors was more than 20%. Twin
studies suggest greater than 90% concordance in
monozygotic twins.151,234,296 Unlike these studies
that used selected cases, a population-based study
revealed an overall concordance of 37% in mono-
zygotic twins versus 19% for dizygotic twins for early
AMD.179 This study estimated the overall inheritabil-
ity of early AMD at 45%, with particularly high inher-
itability for 20 or more hard drusen (81%), large
soft drusen (⬎125 µm) (57%), and pigmentary
changes (46%).
Sibling analyses demonstrate that 55% to 57% of
the variability in AMD could be attributed to a single
gene segregation.191 It is possible that a single gene
is responsible for susceptibility, although many others
could influence phenotype. In one large family with
10 AMD patients (predominantly with the dry pheno-
type) spanning three generations, linkage to
chromosome 1q25-q31 has been reported.235 A
genome-wide scanning approach confirmed the pres-
ence of a susceptibility locus at 1q31 and identified
another such region at 17q25.501
A statistically significant association of manganese
superoxide dismutase gene polymorphism with AMD
has been reported.231 This is intriguing because of
the role of this xenobiotic-metabolizing enzyme in
oxidative stress, and highlights the interaction be-
tween environment and genetics in AMD. An allelic
variant of the CST3 gene, which codes for cystatin C,
an inhibitor of the cathepsin enzymes that function in
the RPE to process rod outer segments, was associated
with exudative AMD, particularly in men, in a
German population-based study.523 A specific Alu
insert polymorphism, absent in the majority of the
population, in the angiotensin converting enzyme
(ACE) gene, which apart from its role in blood pres-
sure regulation also plays a role in cell proliferation
and death, seems to confer protection from the dry
(atrophic) form of AMD.175
The apolipoprotein E ε4 allele is associated with a
57% reduction in the risk of exudative AMD, while
the ε2 allele is associated with a 50% increased
risk.232,432 This finding assumes added interest in view
of the presence of apolipoprotein E immunoreactiv-
ity in drusen and BlamD.323 Interestingly, apolipo-
protein E deficient mice exhibit accumulation of
particles similar to BlinD98 and BlamD245 at an earlier
age and have more membrane-bounded material in
Bruch’s membrane than control mice.
The search for candidate genes also naturally fo-
cuses on the hereditary retinal dystrophies with phe-
notypic similarity to AMD. The gene in Stargardt
AGE-RELATED MACULAR DEGENERATION
disease, characterized by progressive atrophy of the
RPE and overlying photoreceptors in the macular
area, is caused by mutations in the ABCR gene at
chromosome 1p21, a rod-specific ATP-binding cas-
sette transporter.17
Allikmets et al suggest that ABCR is a susceptibility
gene for AMD based on the finding that a greater
percentage of non-neovascular AMD patients had
missense mutations in the exons of this locus com-
pared to controls.16 However, this interpretation has
been contested for not including certain sequence
variations that occurred in controls, the inclusion of
which would have nullified the significance of the
differences.92 It has also been suggested that some of
these mutations are more likely to polymorphic vari-
ants than disease-causing mutations. Furthermore,
the original study of Allikmets has been assailed for
not using the same heteroduplex analysis and single-
strand conformational polymorphism analysis strat-
egies for screening for mutations in controls as in
AMD patients.
Subsequent studies demonstrated no significant
differences between the large number of sequence
variations in controls and AMD patients.140,433 How-
ever, Allikmets et al have recently reported that pa-
tients with AMD who have relatives with Stargardt
disease have mutations in their ABCR gene.14 They
have also identified two sequence changes, G1961E
and D2177NE, that are found much more frequently
in AMD patients than in controls. However, these
missense mutations are rare in both populations, and
even this observation has been recently chal-
lenged.167 A recent multicenter study500 and a
German study377 reported that there was no signifi-
cant difference in the proportion of any genetic
variants of ABCR between patients with and without
AMD. Establishing causality of highly polymorphic
genes such as ABCR in a prevalent disease like AMD
will be difficult.
Linkage studies have indicated that the genes asso-
ciated with Doyne honeycomb retinal dystrophy
(EFEMP1),444 Best disease (dystrophin),15 and
Sorsby fundus dystrophy (TIMP-3),93 all hereditary
dystrophies bearing phenotypic resemblance to
AMD, are not associated with AMD. Mutations in the
peripherin/RDS gene, which are involved in myriad
retinal degenerations of the RP family, also have not
been found to play a role in AMD.416 Recently, how-
ever, EFEMP1 has been found to accumulate beneath
RPE cells overlying drusen in AMD.289
B. OXIDATIVE STRESS
For greater detail, the reader is referred an excel-
lent review of the role of oxidative stress in AMD.40
Many age-related pathologies have been attributed
267
to cumulative oxidative damage caused by reactive
oxygen intermediates (ROI) such as hydrogen perox-
ide (H2O2), singlet oxygen (1O2), superoxide anion
(O2⫺), and hydroxyl radical (OH⫺). ROI arise as
byproducts of cellular metabolism or photochemical
reactions. Cell membranes are prime targets for ROI-
induced damage as they contain polyunsaturated
fatty acids, whose double bonds are a source of elec-
trons, which in turn produces lipid peroxyl radicals
that perpetuate the electron appropriation.
The retina is particularly susceptible to oxidative
damage because of the following reasons:
• High O2 tension11
• Tremendous exposure to irradiation in view of
its raison d’etre
• High proportion of polyunsaturated fatty acids
in the photoreceptor outer segments39,445
• Numerous chromophores (lipofuscin, melanin,
rhodopsin, cytochrome c oxidase) in the retina
and RPE95,141,385
• RPE phagocytosis of photoreceptor disks gener-
ates ROI465
In humans, the macula contains less amounts of
docosahexaenoic acid, the principal retinal polyun-
saturated fatty acid,445 than the periphery,491 and is
more susceptible to lipid peroxidation, particularly
with age.91 This counter-intuitive finding (decreased
polyunsaturated fatty acid content leads to de-
creased chance of lipid peroxidation) actually may
be secondary to locally increased lipid peroxidation
in the macula.91 It also may be an adaptive response
of the macula supported by the increased density of
cones, which contain less of this fatty acid than
rods.381 A diet deficient in linolenic acid protects
rats from phototoxicity,63,341 echoing the results of a
clinical trial.72 A diet deficient in docosahexaenoic
acid was protective in rats,63 but this is contrast to
human data.72
Lipofuscin refers to a diverse group of autofluores-
cent lipid and protein aggregates that accumulate in
RPE cells. There is considerable evidence linking
the formation of lipofuscin to autooxidative tissue
damage suggesting that lipofuscin is, at least in part, a
product of autooxidation. It is believed that impaired
phagocytosis of photoreceptor outer segments by the
RPE results in lipofuscin formation.227 The recent
identification of recoverin, an outer segment specific
protein, in lipofuscin supports this hypothesis.401 In
the RPE, lipofuscin accumulation increases with age
and is concentrated in the macula, maximally in
the parafoveal ring where rods are densest.96,507
Lipofuscin is purported to disrupt RPE function
by mechanical distortion of cellular architecture and
potentiating phototoxicity. Lipofuscin accumulation
greatly reduces RPE phagocytic capacity.448 RPE cells
268 Surv Ophthalmol 48 (3) May–June 2003
fed lipofuscin granules and exposed to short wave-
length visible light (390–550 nm) undergo lipid per-
oxidation, suffer structural disintegrity, marked
decrease in activity of catalase, superoxide dismutase,
and CatD, reduction of glutathione levels, and ulti-
mately cell death.89,385,415 These photooxidative alter-
ations are more pronounced upon exposure to
blue light.505
N-retinyl-N-retinylidene ethanolamine (A2E), a li-
pofuscin component,106 induces apoptosis in RPE
by specifically targeting the mitochondrial enzyme
cytochrome oxidase.459 A2E compromises RPE cell
function by inhibiting lysosomal degradation of pho-
toreceptor phospholipid,118,202 disrupting mem-
brane integrity, and via phototoxicity400 by initiating
blue light-induced apoptosis of RPE cells.437
RPE dysfunction mediated by ROI has been sug-
gested as a possible cause of AMD. The phagocytosis of
photoreceptor outer segments by RPE cells generates
extracellular H2O2, an oxidative stress, and upregu-
lates the expression of the antioxidants catalase and
metallothionein, presumably as a compensatory
mechanism.465 H2O2 treatment of RPE cells results in
marked mitochondrial DNA damage.37 This damage
results in decreased mitochondrial redox function.
Exposure of RPE cells to concentrations of H2O2
that cause in vitro mitochondrial DNA damage also
promotes apoptosis, as measured by caspase-3 prote-
ase activity.216 H2O2 upregulates the pro-apoptotic
proteins p53 and p21, and downregulates bcl-2, an
anti-apoptotic protein. In a model of oxidative injury,
t-butylhydroperoxide induced apoptosis of RPE cells
by caspase activation via mitochondrial damage.64
Consequently, RPE mitochondrial DNA appears
susceptible to oxidative stress, and thus may serve
as a catalyst in the initial events leading to RPE dys-
function in vivo. With age, damage to mitochondrial
DNA may accumulate in RPE cells, and render them
more susceptible to apoptosis.
Because exposure to continuous bright visible
(green) light leads to selective degeneration of rod
photoreceptor cells in rats,329 it has been suggested
that long-term exposure to sunlight may predispose
to the development of AMD, perhaps via photosensi-
tization reactions. It has been suggested that proto-
porphyrin IX, a hemoglobin precursor, may act as an
endogenous photosensitizer.153 Supportive of this,
protoporphyric mice develop basal laminar-like de-
posits beneath the RPE and in Bruch’s membrane and
the choriocapillaris, as well as thickening of Bruch’s
membrane and attenuation of the choriocapillaris,
similar to AMD patients.152 The greater number of
melanosomes in the RPE of darkly pigmented eyes
would be expected to protect the choriocapillaris
from such photosensitization, which is consistent
AMBATI ET AL
with the decreased incidence of AMD in darkly pig-
mented individuals. Although it is clear that light
exposure and oxygen radicals can damage the RPE
and choriocapillaris, there still is no definitive causal
linkage between photooxidative damage and AMD.
Among elderly (ⱖ20 years old) rhesus macaques,
macular drusen were observed in 47%.339 Higher
plasma levels of thiobarbituric acid reactive sub-
stances, a biomarker of oxidative stress, were mea-
sured in animals with greater than 10 drusen
compared to animals without drusen, suggesting a
role for oxidative lipid damage in their formation.
Among cynomolgus monkeys with early onset AMD,
increased albumin, decreased 3-phosphate dehydro-
genase, catalase and gluthathione peroxidase activi-
ties were found in the affected retinas.325,326 These
changes are compatible increased oxidative stress.
Fourfold lower zinc concentrations were found in
affected retinas, but plasma concentrations were not
significantly different. Similarly, the synthesis of
metallothionein, a free radical scavenger whose ex-
pression is induced by transcription factors requir-
ing zinc, was suppressed in affected retinas. This also
points towards an increased peroxidation process
in the development of macular degenerative
changes.325,326
Because of the importance of antioxidants for scav-
enging free radicals, many investigators have at-
tempted to discern whether dietary antioxidants and
mineral cofactors confer protection from AMD. Vita-
min C supplementation inhibited light induced pho-
toreceptor damage in rats.267,342 Monkeys placed on
vitamin E deficient diets developed focal disruption
of photoreceptor outer segments and accumulated
more lipofuscin in their RPE cells than animals with
sufficient vitamin E diets.188 The macular pigments
lutein and zeaxanthin quench singlet oxygen105,138
and filter damaging blue light.123,395 They, in addi-
tion to vitamin E and the carotenoid lycopene,
reduce the formation of lipofuscin in rabbit and
bovine RPE cells exposed to hyperoxia and photore-
ceptor outer segments.449
The presence of advanced glycation products
(AGE), chemically modified lipids, nucleic acids, and
proteins, which are elevated in AMD,214 leads to the
upregulation of genes that promote RPE aging.207
ROI are generated in parallel with AGE either di-
rectly or through AGE-RAGE (receptor for AGE)
interaction.515 AGE also increase VEGF expression
in RPE,272 and can induce angiogenesis.337
Differential expression analysis reveals that the ex-
pression of HSC70, a constitutively produced heat
shock protein functioning as a chaperone in cellular
protein processing, markedly decreases in human
and rhesus monkey retina from youth to old age.49
AGE-RELATED MACULAR DEGENERATION
Decreased HSC70 expression during aging may com-
promise the stress response mechanism of the meta-
bolically active retina, and increase susceptibility to
stress-induced apoptosis.165 The tyrosine kinase in-
hibitor genistein, a component of soy products, po-
tently inhibits endothelial cell proliferation and
angiogenesis,121 possibly through reduced expres-
sion of hsp70, a heat shock protein.522 Intravi-
treous injection of genistein inhibits choriocapillaris
regeneration without apparent effect on RPE wound
healing.283 Oral administration of genistein moder-
ately inhibits laser-induced CNV in mice.255 The
recent finding that genistein is capable of producing
thymic and immune abnormalities raises a cautionary
flag however.518
Oxidative stress also may promote neovasculariza-
tion. H2O2 induces a dose-dependent reduction in
the expression of pigment epithelial derived factor
(PEDF), an endogenous anti-angiogenic mole-
cule.335 ROI also upregulate VEGF in RPE.252 Oxida-
tive stress leads to deposition of extracellular matrix
along Bruch’s membrane and increased levels of the
angiogenic factor FGF2 in RPE cells.321
C. HYDRODYNAMIC CHANGES
Bruch’s membrane is situated strategically between
the nutrient fount of the choriocapillaris and the
metabolically active RPE, which subserves photo-
receptor homeostasis. As such, mechanical or func-
tional injury to this two-way conduit for nutrients
and cellular breakdown products would compromise
retinal function. With age Bruch’s membrane under-
goes numerous changes that impede normal fil-
tration.
The progressive increase in lipid content in
Bruch’s membrane throughout life is exaggerated
in the macula compared to the periphery.203 These
neutral fat deposits differ from atheromas as they
are derived from a cellular origin and not the periph-
eral blood, for they contain low levels of cholesterol
ester and phosphotidylcholine, the major plasma
lipids.38,203,419 It is believed that progressive accumu-
lation of extracellular material containing lipid in
Bruch’s membrane may influence the development
of AMD by altering its diffusion characteristics.
The age-related exponential decline in hydraulic
conductivity of Bruch’s membrane is more marked in
the macula, and inversely related to its total lipid
content.119,314 The impedance to diffusion through
Bruch’s membrane compromises metabolic ex-
change between the choroid and retina,119 and dis-
rupts photoreceptor function.54 Increased levels of
AGE214 and RAGE177 are found in AMD. The pres-
ence of AGE decreases the hydraulic conductivity of
Bruch’s membrane.209
269
With age, Bruch’s membrane thickness increases
(135% over 10 decades370), thus increasing the diffu-
sional path length. The accompanying 45% age-re-
lated decline in choriocapillaris density may further
impair diffusion by reducing clearance from Bruch’s
membrane.370 A direct relationship between Bruch’s
membrane thickness and RPE autofluorescence, a
marker of lipofuscin accumulation, has been demon-
strated in aged eyes.338 Over 9 decades the permeabil-
ity of Bruch’s membrane to serum proteins decreases
by 90%.313
The solubility of collagen in Bruch’s membrane
declines by 50–60% over 9 decades, and may contrib-
ute to debris accumulation in Bruch’s membrane.223
With age, the glycosaminoglycan composition of
Bruch’s membrane changes, increasing in size and
in the total fraction of heparan sulfate, alterations
that may compromise Bruch’s membrane filtration
function.195 Heparan sulfate also binds VEGF165, the
predominant RPE VEGF isoform, and may contrib-
ute to choriocapillaris loss through the reduction of
VEGF, an endothelial cell survival factor.18
It is believed that the origin of the abnormal
deposits in Bruch’s membrane is from the RPE.
Highly metabolic photoreceptors shed their outer
segments that are then phagocytosed by the RPE and
degraded by lysosomal enzymes. It is possible that
the material discharged into Bruch’s membrane is
abnormal as a consequence of incomplete phagolyso-
somal degradation, and in particular may contain
large molecules and membrane complexes, obstruct-
ing diffusion. The activity of certain lysosomal en-
zymes is decreased within human RPE cells in aged
eyes55,78 and various degradative enzymes have been
identified with different regional distributions.55
This abnormal material may collect as discrete
deposits in the inner portion of Bruch’s membrane
between the basement membrane of the RPE and
the inner collagenous layer (drusen). Additionally,
diffuse thickening of the inner part of Bruch’s mem-
brane (linear deposits) is seen with age. The similarity
of staining pattern between debris accumulated in a
diffuse and discrete manner implies a similar origin
of linear deposits and drusen.
The accumulation of breakdown products derived
from the RPE is believed to play a major role in the
induction of RPE detachments and new vessel growth
under Bruch’s membrane. Large confluent hypofluo-
rescent, hydrophobic drusen predispose to RPE de-
tachments, whereas hyperfluorescent, hydrophilic
drusen predispose to CNV.77,350,399 This observation
is in consonance with the suggestion that sub-RPE
fluid is derived from the RPE,50 wherein increased
hydrophobicity of Bruch’s membrane would increase
resistance to water flow from the RPE to the choroid,
leading to RPE detachments. Conversely in the latter
270 Surv Ophthalmol 48 (3) May–June 2003 AMBATI ET AL

instance, greater exposure to serum derived angio- senescence. Successive passage of cultured RPE cells
genic factors has been speculated to promote CNV. results in diminished PEDF production, suggesting
an age-related decline,481 although this is not univer-
D. RPE SENESCENCE sally observed.291
Senescence refers to the loss of cellular growth
potential. This replicative failure is a form of viable E. HEMODYNAMIC CHANGES
cell cycle arrest characterized by the inability (or
There is evidence that the choroidal blood flow is
refusal) to proliferate in the face of mitogenic stimuli.
impaired in patients with AMD; however, the exact
There is a discrete alteration in the morphology and
nature of the impairment has not been elucidated.
physiology of these cells, reflecting altered gene ex-
The fluorescein angiographic appearance of choroi-
pression. Cellular senescence is observed in a wide
dal perfusion is modified in some patients with
variety of human cell types, and has been accused of
AMD.349 A prolonged choroidal filling phase during
contributing to the development of AMD. Markers
initial dye transit is observed in roughly a quarter of
of senescence, such as altered gene expression and
eyes with age-related changes at the level of Bruch’s
shortening of chromosomal telomeres, have been
membrane, and is suggested to correlate with diffuse
identified in cultured RPE cells exposed to AGE,
thickening of Bruch’s membrane. These observations
which form in Bruch’s membrane with age,180 and
have led to speculation that an abnormality of the
oxidative stress. RPE cells appear to undergo senes-
choriocapillaris reduces diffusion into the intravascu-
cence earlier than other cell types such as fibro-
lar space of waste material derived from the RPE,
blasts.373 With increasing age, foveal RPE cells
with waste material accumulating in the outer part
selectively lose their hexagonal shape and decrease
of Bruch’s membrane. A clinical correlate has been
in density.99,499 Such cells harbor the potential to
demonstrated by way of elevated scotopic thresholds
wreak havoc and may contribute to age-related pa-
and slow dark adaptation,173,442 especially in eyes with
thologies.
prolonged choroidal filling.70
Alteration of β-galactosidase activity is a marker
Computer-assisted image analysis of video ICG an-
of RPE senescence.290,304 Using this technique, an
giography revealed that the age-related decrease in
accumulation of senescent RPE cells has been dem-
the foveal choriocapillaris blood flow is further at-
onstrated in older monkey eyes.290,304 There is an
tenuated in AMD patients.160,161 Laser Doppler flow-
age-related, progressive accumulation of lipofuscin,
metry demonstrated that the decrease in choroidal
lysosomal storage material, in RPE cells. RPE lyso-
blood flow is mainly due to decreased choroidal
somes digest photoreceptor outer segments continu-
blood volume,namely, the density and diameter of
ously. A decrease in lysosomal activity accompanies
the choriocapillaris are attenuated,161 correlating
aging.506,512 One of these enzymes, cathepsin D
with histopathological findings.391 In AMD eyes,
(CatD), is most important for opsin proteolysis.368
there are areas of watershed or focal hypofluo-
A transgenic mouse that expressed an enzymatically
rescence in the macula; interestingly, CNV developed
inactive form of CatD exhibited bloated RPE cells
in these areas.384
filled with autofluorescent debris, basal laminar and
Friedman advanced a hemodynamic model of the
linear deposits, and progressive photoreceptor de-
pathogenesis of AMD, which proposed that the disor-
generation.369 The association of a gene mutation
der is caused by decreased compliance of the sclera
in cystatin C, an inhibitor of cathepsins, with AMD
and choroidal vessels leading to increased resistance
is intruiging.523
in choroidal blood flow.132,133 He postulates that li-
In AMD, there are increased advanced glycation
poidal infiltration of the sclera and Bruch’s mem-
endproducts (AGE) and their reactive intermediates
brane lead to decreased compliance and increased
in Bruch’s membrane. Treatment with AGE induces a
resistance of the sclera and choroidal vessels. This
range of pathophysiological changes in RPE cells,
in turn leads to decreased choroidal perfusion and
including downregulation of CatD mRNA and pro-
impaired RPE transport. Decreased choroidal perfu-
tein expression.293
sion results in the formation of drusen, RPE atrophy,
Exposed to near-ultraviolet irradiation suppresses
and lipoidal infiltration of Bruch’s membrane, while
RPE proliferation, decreases cellular melanin levels,
increased intravascular pressure is suggested to lead
and increases lipofuscin accumulation.266 These phe-
to RPE detachments and CNV.
nomena are substantially eliminated in anaerobic con-
ditions. Both the near-ultraviolet-irradiated RPE cells
and aged RPE cells express markedly less PEDF and F. CNV AND ANGIOGENESIS
tissue inhibitor of metalloproteinase-3 (TIMP-3). The The development of new capillaries from preex-
former is a known neurotrophic factor.41 These find- isting networks, angiogenesis, is integral to embry-
ings suggest that phototoxicity may contribute to RPE onic development, somatic growth, and tissue repair
AGE-RELATED MACULAR DEGENERATION
(Fig. 2). However, it also can prove harmful by feed-
ing tumor growth or by destroying normal ocular
architecture via aberrant neovascularization. An as-
semblage of molecular players orchestrates the com-
plex process of vessel formation, and their
identification is largely the serendipitous conse-
quence of oncology research.
1. VEGF
Vascular endothelial growth factor (VEGF) was ini-
tially discovered as a peptide secreted by tumor cells
that increased vascular permeability.410 Later it was
rediscovered as an angiogenic growth factor with
high specificity for vascular endothelial cells.226,263
VEGF is likely to be a pivotal angiogenic factor be-
cause of these important characteristics:
• It has a high degree of selectivity to endothe-
lial cells
• Reciprocal oxygen regulation: hypoxia drives
VEGF synthesis422 and hyperoxia inhib-
its it360,365,513
• VEGF can diffuse to its target because it contains
a signal sequence for extracellular secretion
• Multiple components of angiogenesis (endothe-
lial cell proliferation, survival, and migration)
as well as vascular hyperpermeability can be ef-
fected by VEGF
VEGF, which is so crucial to embryonic vascular
development that its absence is lethal,65 is a potent
and selective mitogen of vascular endothelial cells.
The secretion of this diffusible protein is upregulated
by hypoxia. VEGF induces expression of urokinase-
type and tissue-type plasminogen activators, as well
as that of metalloproteinase interstitial collagenase.
This co-induction promotes degradation of the local
extracellular matrix and facilitates endothelial cell
migration. It also helps mediate vascular hyperper-
meability, and the resulting leakage of plasma pro-
teins has been postulated to serve a substrate for
endothelial cell growth. These features, combined
271
with its target selectivity, make it an attractive target
for anti-angiogenic therapy.
A collection of studies unraveled the role of VEGF
in ocular neovascularization. VEGF has been shown
to be necessary8 and sufficient478 for the development
of retinal and iris neovascularization4,21,114,303 in ex-
perimental models. It has also been implicated in the
development of retinal and iris neovascularization
in ischemic diseases such as diabetic retinopathy,3
retinal vein occlusion,485 retinopathy of prematu-
rity,520 and neovascular glaucoma.485 Evidence has
now emerged for a putative role in CNV as well.
VEGF is overexpressed in the RPE of autopsy eyes
with AMD and in transdifferentiated RPE cells of
surgically excised CNV membranes (Fig. 3A).124,271
Intravitreous injections of VEGF induce proliferation
of choroidal endothelial cells in non-human pri-
mates.478 Repetitive intravitreous injection of rhu-
FabV2, an active fragment of a recombinant
humanized monoclonal VEGF antibody, inhibits the
development of laser-induced CNV in a primate
model.249 Adenoviral transfection of a VEGF gene into
the RPE of rats led to development of CNV that
subsequently breached Bruch’s membrane to result in
subretinal new vessels that leak fluorescein like clini-
cal CNV (Fig. 3B).35,439 A murine model of VEGF
overexpression in RPE cells exhibits increased cho-
roidal vascular density but these new vessels do not
penetrate Bruch’s membrane.403 Adenoviral transfec-
tion of a soluble VEGF receptor into the RPE of rats
inhibited the development of laser-induced
CNV.206 Intravitreous injection of an oligonucleotide
targeted to the VEGF sequence inhibited laser-in-
duced CNV in rats,143 but neither intravitreous or
subretinal administration of this agent reduced
laser-induced CNV in monkeys.417
VEGF is constitutively produced by RPE cells,5,420
and may be involved in paracrine signaling between
the RPE and choriocapillaris. It has been dem-
onstrated that VEGF can induce endothelial fenestra-
tions.378 These findings are consistent with a trophic
Fig. 2. Schematic diagram of angio-
genesis in choroidal neovasculariza-
tion.
272 Surv Ophthalmol 48 (3) May–June 2003
Fig. 3. A: In situ hybridization of surgically excised CNV from patient with AMD demonstrating VEGF mRNA pre-
dominantly localized to RPE cells. B: Light micrograph at 4 weeks after subretinal injection of Ad. VEGF showing
development of CNV (white arrow) with migration and flattening of the RPE (dark arrowheads). (Reprinted from Baffi
et al35 with permission of Investigative Ophthalmology and Visual Science.)
maintenance of the choriocapillaris by the RPE via
VEGF, which is a known survival factor.18 RPE secre-
tion of VEGF is polarized, that is, under normoxic
conditions, basal (towards Bruch’s membrane) secre-
tion of VEGF is 2- to 7-fold higher than apical (toward
photoreceptors) secretion.51 This disparity is exag-
gerated under hypoxic conditions. Furthermore,
VEGF receptors are preferentially localized to the
inner choriocapillaris endothelium. The presence of
VEGF receptors on RPE cells162 suggests an autocrine
function as well. Interestingly, many of the factors
implicated in AMD, namely, AGE, ROI, and hypoxia,
are all potent stimuli of VEGF gene expression in
RPE.252,272,421
The near-absence of retinal neovascularization in
AMD, combined with the finding of intraretinal, but
not choroidal, neovascularization in a murine model
of VEGF overexpression by photoreceptors,336 dem-
onstrates the importance of directionality. The in-
creased thickness and hydrophobicity of Bruch’s
membrane may prevent VEGF from reaching the
choriocapillaris, causing it to atrophy, which, in turn,
would reduce the clearance of debris from Bruch’s
membrane. This feed-forward cascade, together with
attendant hypoxia and Bruch’s degradation, would
induce VEGF, promoting CNV.
2. FGF2
VEGF is not the only growth factor implicated in
CNV formation. Basic fibroblast growth factor
(FGF2) is present within RPE cells in surgically re-
moved CNV membranes,26,124,253,271 and FGF2 mRNA
is upregulated in RPE, choroidal vascular endothelial
cells, and fibroblasts in laser-induced CNV lesions in
rats.331,519 Sustained release of FGF2 by subretinal
pellets induces CNV in minipigs431 and rabbits.230
However, targeted disruption of the FGF2 gene did
AMBATI ET AL
not inhibit the development of laser-induced CNV
in mice, suggesting that it is not necessary for the
development of CNV.477
The role of FGF2 in CNV was further clarified by
using low-intensity laser photocoagulation that dis-
rupts photoreceptors and RPE cells, releasing their
intracellular contents and high-intensity laser photo-
coagulation that breaks Bruch’s membrane and
damaged the choriocapillaris.514 Low-intensity photo-
coagulation results in CNV among mice whose
photoreceptors overexpress FGF2 but not in wild-
type mice. High-intensity photocoagulation leads to
a greater area of CNV in these transgenic mice than
among wildtypes. Cumulatively, these data suggest
that retinal overexpression of FGF2 is angiogenic
only in the setting of cellular injury that overcomes
physiological mechanisms that sequester it.
3. Angiopoietins
The angiopoietin (Ang) family of molecules are,
in addition to VEGF, the only known molecules with
almost exclusive specificity for endothelial cells. Ang1
induces endothelial cells to recruit periendothelial
support cells and associate with the extracellular
matrix and mesenchyme, promoting vascular integ-
rity. It has an indispensable role in vascular develop-
ment as its absence leads to embryonic lethality.457
Ang1 is expressed in normal human vessels, implying
a role for maintenance of the adult vasculature as
well. Interestingly, whereas overexpression of VEGF
leads to the development of numerous leaky vessels,
Ang1 overexpression results in an increased density
and caliber of non-leaky vessels.458,475
Ang2 is a natural antagonist of Ang1 that binds to
the same endothelial cell receptor, Tie2.281 Overex-
pression of Ang2 leads to angiogenic deficits reminis-
cent of the Ang1-deficient state. In humans, Ang2
AGE-RELATED MACULAR DEGENERATION
is expressed only at sites of vascular remodeling.281
Endothelial cell chemotaxis toward Ang1 is blocked
by Ang2.508 Unlike VEGF, neither Ang1 nor Ang2
induces endothelial cell proliferation.508 Both mole-
cules modulate VEGF-induced neovascularization. In
a corneal micropocket assay, Ang1 promotes matura-
tion of the immature vessels induced by VEGF,
whereas Ang2 augments neovascularization incited
by VEGF.
Both hypoxia and VEGF upregulate the expression
of Ang2 but not Ang1 in endothelial cells.333 In a
murine model of ischemia-induced retinal neovascu-
larization, Ang2 was upregulated in the ischemic
layers of the retina as well as the new vessels, sug-
gesting that it facilitates hypoxia and VEGF induced
neovascularization by destabilizing existing vascu-
lature.
Histological examination of CNV in AMD and
other diseases disclosed that Ang1 and Ang2 were
present.344 Ang2 is expressed in highly vascular areas
of the CNV membrane, paralleling the distribution of
VEGF. The Tie2 receptor is expressed in the vascular
structures as well as the RPE and in fibroblast-like
cells. Ang1 and Ang2 were also expressed by the mac-
rophages in the CNV membrane. Interestingly, VEGF
selectively upregulates Ang1 synthesis and secretion
by RPE cells.182 Adenoviral-mediated systemic gene
delivery of the soluble Tie2 receptor, which acts as
an Ang antagonist, markedly inhibits the develop-
ment of laser-induced CNV in mice.181 Ang1 may
have a potential therapeutic use as it suppresses
VEGF-induced leukostasis and inflammation by in-
hibiting expression of ICAM-1, vascular cell adhe-
sion molecule-1 (VCAM-1), and E-selectin.229
4. PEDF
Pigment epithelium-derived factor (PEDF) is a gly-
coprotein produced in abundance by the RPE.481 This
neurotrophic growth factor for photoreceptors also
inhibits angiogenesis and proliferation of several cell
types. This endogenous molecule has also attracted
interest because of its regulation by oxygen reciprocal
to that of VEGF. Corneal and vitreous body avascular-
ity have been attributed to their high levels of PEDF.90
Its anti-angiogenic action may stem from endothelial
cell apoptosis.443
Systemic delivery of recombinant PEDF inhibited
ischemia-induced retinopathy in mice,443 and VEGF-
induced retinal vascular leakage in diabetic rats. Ade-
noviral transfection of PEDF into RPE cells partially
inhibited CNV in a murine model of laser injury, as
well as retinal neovascularization in transgenic mice
whose photoreceptors overexpress VEGF.317 Adeno-
associated viral transfection of PEDF inhibited laser-
induced CNV in mice.319 Recent evidence suggests
273
that PEDF may cause regression of established
CNV.318 Subretinal transplanation of autologous iris
pigment epithelial cells expressing PEDF inhibited
laser-induced CNV in rats.409 In the rat model of
ischemia-induced retinopathy, an increased VEGF/
PEDF ratio correlated with the presence of retinal
neovascularization.142 Similar imbalances may be
present in CNV.
Situated at the crucial junction between Bruch’s
membrane and the neurosensory retina, the RPE
has long been speculated to play a pivotal role in the
modulation of CNV. Although the discovery of RPE
production of VEGF suggested a causal role for RPE in
the development of CNV, the finding that it also
produces PEDF suggests that it can play a dual role.
The RPE appears to have a paradoxical relation-
ship with the choriocapillaris. In the normal eye, the
RPE very likely serves a trophic maintenance func-
tion. The preferential localization of choriocapillaris
fenestrations on the side facing the RPE is consistent
with this hypothesis.51 In addition, absence of the
RPE is known to lead to choriocapillaris atrophy in
geographic atrophy or after surgical debridement.
The RPE also appears to be involved in the devel-
opment of CNV. However, RPE cells have been ob-
served to arrest CNV growth by encapsulation.302 It
has been postulated that laser photocoagulation may
stimulate proliferation of RPE cells to envelop CNV
and stop leakage.
In vitro, there is a decreased production of PEDF by
senescent RPE cells,481 although this is not a universal
finding.291 The declining vitreous concentration of
PEDF with increasing age is more marked in patients
with AMD.200 It is not clear whether this is due to
decreased synthesis or increased proteolytic activity
in the vitreous.510
In view of its potent anti-angiogenic activity in dif-
ferent models, it is puzzling that endogenous PEDF
does not prevent the development of CNV. Perhaps
the small amounts constitutively produced are over-
whelmed by angiogenic factors. Or it may be that
the apically polarized secretion of PEDF42 is spatially
disadvantaged by the basally polarized secretion of
VEGF from RPE.51 The pooling of PEDF in the inter-
photoreceptor matrix may explain the lack of inva-
sion of CNV into the neurosensory retina.511 Recent
work has demonstrated that while PEDF inhibits the
growth of endothelial cells exposes to FGF2, it pro-
motes proliferation of endothelial cells exposed to
high VEGF levels,210 providing a sobering note of
caution. The paradoxical effects of PEDF were under-
lined by the recent finding that whereas low levels
of PEDF inhibit laser induced CNV in mice, high
levels of PEDF actually increase CNV development.113
274 Surv Ophthalmol 48 (3) May–June 2003
5. Nitric Oxide
Nitric oxide (NO), which is involved in regulating
systemic blood pressure, also plays a crucial role in
VEGF-induced angiogenesis and hyperpermeability.
VEGF induces the upregulation of endothelial nitric
oxide synthase (eNOS) and the subsequent release of
NO, which is produced by the action of eNOS, from
endothelial cells;347,486 in turn endogenous NO en-
hances VEGF synthesis.100 Pharmacological blockade
or genetic disruption of eNOS inhibits VEGF-in-
duced angiogenesis and hyperpermeability.137 eNOS
deficiency or blockade reduces oxygen-induced
retinal vasoobliteration and vitreous neovasculari-
zation.61
NO appears to induce angiogenesis by promoting
endothelial cell migration and differentiation into
capillaries by upregulating the αvβ3 integrin on en-
dothelial cells,262 a critical mediator of cell-matrix
adhesion and migration. NO also reduces extracellu-
lar matrix adhesion, permitting their migration.
In contrast to eNOS, inducible NOS (iNOS) can
inhibit angiogenesis by downregulating the VEGF re-
ceptor.412 In a chemical injury model of corneal neo-
vascularization, iNOS expression was associated with
reduced angiogenesis.411 In a murine model of
ischemic retinopathy, iNOS inhibits VEGF-driven
neovascularization.412 Preliminary studies suggest
that NO may play a role in CNV. Human CNV mem-
branes express high levels of iNOS in RPE cells and
macrophages.187 In mice with laser-induced rupture
of Bruch’s membrane, CNV was inhibited in the
iNOS deficient mice but not the eNOS deficient
animals.30
6. Extracellular Matrix
Invasion and migration of endothelial cells
through the extracellular matrix (ECM) during angi-
ogenesis are orchestrated by the integrin family of
cell adhesion molecules. Various integrins facilitate
migration by interacting with adhesion proteins in
the ECM, such as collagen, fibronectin, fibrinogen,
laminin, vitronectin, and von Willebrand factor. This
process is potentiated by the secretion of proteolytic
enzymes matrix metalloproteinases (MMPs) and
modulated by tissue inhibitors of metalloproteinases
(TIMPs). Endothelial cell proliferation occurs in an
anchorage dependent manner, mediated by the inte-
grin family of adhesion receptors.
FGF2 and tumor necrosis factor-alpha (TNF-α)
induce αvβ3 expression, while VEGF and trans-
forming growth factor-alpha (TGF-α) induce αvβ5
expression on developing blood vessels.60,129 Expres-
sion of the αvβ3130 and perhaps αvβ5375 integrins is
found in CNV of patients with AMD. A cyclic peptide
AMBATI ET AL
antagonist of the αv integrins inhibited retinal neo-
vascularization in a murine model without disrupting
established vessels.176,273 A monoclonal antibody to
αvβ3 integrin conjugated to a mitomycin C-dextran
inhibited experimental CNV in a rat model.221
MMPs are a family of enzymes associated with re-
modeling of the ECM in neovascular processes. Sev-
eral MMPs are expressed in CNV membranes in
patients with AMD,219,441 and VEGF can induce MMP
production.488 MMP-2 localizes to the areas of new
vascular proliferation, whereas MMP-9 expression is
greatest at the margins of the CNV membranes,441
suggesting a synergistic enzymatic effect. MMP-7,
which has been implicated in tumor progression,2 is
expressed in Bruch’s membrane of CNV mem-
branes.219 In AMD patients, there is increased gene
expression of TIMP-3 and its protein product in
Bruch’s membrane compared to age-matched
controls.32,112,220 Similar elevations of MMP-9 also are
found. MMP-9 expression is upregulated in the laser
injury model of CNV, and MMP-9 deficient mice have
decreased rates of laser-induced CNV.259
TIMP-3, produced by the RPE, is an ECM compo-
nent of Bruch’s membrane. TIMP-3 inhibits che-
motaxis of vascular endothelial cells toward VEGF
and FGF2, inhibits collagen gel invasion and capillary
morphogenesis in vitro, and inhibits FGF2-induced
angiogenesis in the chick allantoic membrane assay
in vivo.27 During normal aging, TIMP-3 content in
Bruch’s membrane of the macula shows a significant
increase. TIMP-3 content in AMD eyes was elevated
relative to that of age-matched normal eyes. Higher
levels of TIMP-3 may contribute to the thickening of
Bruch’s membrane observed in AMD.
TIMP-3 inhibits endothelial cell motility.27 Virally
mediated overexpression of the TIMP-3 gene by the
RPE inhibited the development of laser-induced CNV
in rats.463 In this model, MMP-2 mRNA expression
markedly increased after laser photocoagulation and
mainly localized to macrophage-like and RPE-like
cells invading the choroid, subretinal space and inner
retina.254 Accordingly, MMP-2 deficient mice have
decreased rates of laser-induced CNV.47
While increased amounts of MMP-2 in subretinal
space of AMD patients361 and of MMP-2 and MMP-
9 in Bruch’s membrane with increasing age have
been described,163 the detected enzymes were almost
entirely inactive forms. Thus, attributing a direct
pathogenic role is simplistic. Accumulation of inac-
tive MMPs is more likely a consequence of remodel-
ing of Bruch’s membrane rather than causal.
Increased deposition of collagen and other ECM
components may induce synthesis of MMPs; however,
increased concentration of TIMPs or decreased po-
rosity of Bruch’s membrane may prevent activation
AGE-RELATED MACULAR DEGENERATION
of MMPs and limit their diffusion. The relevant ques-
tion of whether activation of MMPs contributes to
CNV remains unanswered.
Although Prinomastat (formerly AG3340), a selec-
tive oral inhibitor of MMP-2, -9, -13 and -14, inhibited
tumor growth and angiogenesis in a variety of preclin-
ical models, including cancer of colon, breast, lung,
and in melanoma and glioma models,414 it was inef-
fective in patients with AMD.52 Moreover it caused
severe and intolerable side effects.
Disturbed interaction of RPE cells with their ECM
could play a role in the development of AMD. RPE cell
adhesion to vitronectin is mediated by αvβ5 and to
fibronectin via α5β1. Soluble vitronectin, fibrinogen,
and fibronectin stimulate RPE cells to release VEGF, a
process potentiated by thrombospondin 1 (TSP1).321
TSP1 is produced by RPE cells in a manner related
to their proliferative state66,309 and may play a role
in modulating CNV. It also is produced by platelets
and monocytes.215,402 TSP1-induces VEGF secretion
that is dependent on binding to αvβ5 and α5β1 inte-
grins. These data suggest the potential modulatory
role of VEGF release from RPE by ECM and αvβ5 and
α5β1 integrins.321
In seeming contradiction, TSP-1 also inhibits endo-
thelial cell proliferation, migration, and angiogen-
esis.150 Platelet-derived TSP-1 inhibits angiogenesis
in a rat model of ROP.413 In bovine retinal microcapil-
lary endothelial cells, VEGF induces a biphasic re-
sponse of TSP-1 expression; an initial downregulation
followed by marked upregulation. VEGF-induced
endothelial cell proliferation is inhibited by exoge-
nous TSP-1 and potentiated by anti-TSP-1 antibody.
Thus in ischemic retina, VEGF-mediated TSP-1 in-
duction in ischemia-induced angiogenesis may be a
negative feedback mechanism.461
G. SUBCLINICAL INFLAMMATION
Although intraocular inflammation is not clinically
apparent in AMD, multiple lines of evidence support
an influential role for inflammation in this condition.
In fact, a role for inflammation in neovascular AMD
was conjectured more than a century ago.345 Several
early histopathologists identified foci of inflamma-
tory cells in autopsy eyes with AMD.183,190,348
Strangely, this school of thought was not revived until
decades later, perhaps because of the concurrent
development of powerful molecular biological tools
that permitted more precise delineation of the in-
flammatory component.
The cores of drusen are derived from choroi-
dal dendritic cells, immune antigen-presenting
cells.170,171 The presence of HLA-DR, immunoglobu-
lin light chains, vitronectin, and terminal comple-
ment complexes in drusen suggest that immune
275
mediated processes are involved in their biogenesis
of drusen (Fig. 4).82,172,218,323 Histochemical staining
of drusen and BlamD in Bruch’s membrane show
accumulation of neutral fats and phospholipids with
age. It has been suggested that focal concentration of
these materials may produce a powerful chemotactic
stimulus for leukocytes, possibly acting via a comple-
ment cascade.28,217,228 Macrophages appear to prefer-
entially engulf the wide-banded collagen of BlamD
in patients with AMD.228,269,351,490
The presence of circulating anti-retinal autoanti-
bodies in the sera of patients with AMD, which may
be merely an associated or reactive response, never-
theless lends further credence to the inflammatory
paradigm.69,164,354
The presence of leukocytes in histological studies
of CNV has elicited interest in their role in the devel-
opment of neovascular AMD. Leukocytes, macro-
phages in particular, have been implicated in the
pathogenesis of AMD158,228,353,490 as their spatiotem-
poral distribution correlates with arborizing CNV in
humans (Fig. 5)157 and in animal models.328
In eyes with established CNV, Bruch’s membrane
is attenuated for some distance around the break.490
Beneath these thinned areas are regions of deeper
choroidal infiltration comprising of macrophages,
lymphocytes, fibroblasts, and myofibroblasts.490 Be-
cause activated macrophages secrete proteolytic en-
zymes such as collagenase and elastase, which can
erode thinned Bruch’s membrane, and facilitate mi-
gration of choroidal capillaries, their presence sug-
gests an active role rather than a passive non-specific
inflammation. In situ hybridization studies have re-
vealed high levels of monocyte chemoattractant pro-
tein-1 (MCP-1) in RPE cells of AMD eyes, which may
account for the presence of macrophages in
CNV.158,435 Under flow conditions, MCP-1 can induce
leukocyte adhesion to vascular endothelium.147
There is growing evidence that leukocyte mediated
angiogenesis involves the interaction of cellular
adhesion molecules and VEGF.43,295 VEGF induces
the expression of intercellular adhesion molecule-1
(ICAM-1) on tumor and retinal vascular endothe-
lium, and regulates leukocyte adhesion to endo-
thelial cells.310,367 ICAM-1 blockade decreases
VEGF-induced leukostasis in the retina310,311 and an-
giogenesis in the cornea.43,320 These systems are inter-
twined as leukocytes, which possess receptors for and
migrate in response to VEGF,80 can also produce
and release VEGF.184,462 The modulation of angioten-
sin-II-induced leukocyte adhesion by reactive
oxygen intermediates and nitric oxide is further evi-
dence of this complexity.19,20
We recently reported that the development of
laser-induced CNV is markedly diminished in mice
276 Surv Ophthalmol 48 (3) May–June 2003
Fig. 4. A: Fluorescence light micrograph depicting drusen (asterisk) immunoreactivity to anti-Complement 5 antibody
(green). RPE autofluorescence is yellow. (Reprinted from Mullins et al322 with permission of FASEB J.) B: Light micrographs
of drusen core (arrow) and a portion of the connected cell body on the choroidal side of the elastic lamina (arrowhead)
depicting immunoreactivity to anti-HLA-DR antibody. (Reprinted from Hageman et al169 with permission of Progress in
Retinal and Eye Research.)
genetically deficient in ICAM-1 or its cognate recep-
tor on leukocytes, CD18.388 These data suggest a
causal role for leukocyte adhesion to vascular endo-
thelium in the development of experimental CNV.
Combined with the immunolocalization of ICAM-
1192,330 and MCP-1158 in human CNV, and the pres-
ence of leukocytes concentrated around newly
formed CNV,157 the evidence for leukocyte involve-
ment in the development of CNV is compelling
(Fig. 3).
VI. Therapeutic Prospects
Although an array of risk factors has been epidemi-
ologically related to AMD, their role in the progres-
sion of the disease remains equivocal. Recent
studies have helped unravel some of the molecular
processes involved, but their intricate interplay and
complexity make the understanding of AMD still
quite elusive. Still, with a greater understanding of
the etiology of AMD have come therapeutic strategies
that have moved beyond the limited approach of
thermal laser photocoagulation. Photodynamic ther-
apy represents such a milestone in new options, but it
too has restricted benefit. More directed and rational
therapies aimed at the causal molecular mechanisms
have been advanced. A review of the most recent
treatment options and preventive measures are dis-
cussed below in terms of their ability to halt and
reverse the progression of AMD.
A. ANTI-ANGIOGENIC THERAPY
Therapy aimed at the angiogenic processes under-
lying CNV possesses the unique advantage of ad-
dressing the most destructive feature of AMD.
Sustained and effective anti-angiogenic therapy
AMBATI ET AL
would not only halt and reverse CNV, but also allow
freedom from recurrences, and prevent the develop-
ment of neovascularization. A modular approach fo-
cused on single molecules may show substantial
efficacy, especially if master control switches like
VEGF are targeted. However it is possible that multi-
ply-redundant neovascular pathways employed by the
highly vascular choroid, whose vascular biology may
be different than that of the cornea or retina, demand
more comprehensive strategies. Finally, a parallel
neuroprotective approach may prove highly useful,
especially if it can spare the neurosensory retina from
the harsh microenvironment resulting from CNV.
1. Steroids
Steroids’ broad-spectrum suppression of inflam-
mation often translates into anti-angiogenic activity.
Laser-induced CNV in rats was inhibited by systemic
delivery of dexamethasone104 or intravitreous in-
jection of triamcinolone acetonide.79 An uncon-
trolled case series suggested that a single intravitreous
injection of triamcinolone acetonide stabilized vision
in patients with subfoveal recurrence of CNV after
laser photocoagulation of extrafoveal CNV.371 Ste-
roid inhibition of CNV is likely related to broad in-
hibition of cytokines and pleiotropic effects. In
particular, triamcinolone downregulates ICAM-1 ex-
pression on a human epithelial cell line and decreases
its paracellular permeability.355 Prednisolone inhib-
its human RPE cell proliferation induced by platelet
derived growth factor or FGF2.224 Interestingly, these
authors found that VEGF does not induce RPE cell
proliferation. Because RPE cell proliferation may be a
salutary phenomenon in enveloping CNV to prevent
subretinal fluid leakage and possibly induce regres-
sion,302 it is possible that this and other steroids may
AGE-RELATED MACULAR DEGENERATION 277

Fig. 5. A: Histologic two-dimensional reconstruction demonstrating macrophages in spatial proximity to choroidal

neovascularization. The shaded area indicates an area of intact RPE, and the unshaded area corresponds to RPE defect/
disciform scar. (Reprinted from Grossniklaus et al157 with permission of Archives of Ophthalmology.) B: Immunohistochemistry
for CD68 reveals numerous macrophages located in the RPE monolayer, the stroma, and around the neovascular vessels
(V) in a CNV membrane (C) caused by AMD. (Reprinted from Oh et al333 with permission of Investigative Ophthalmology
and Visual Science.) C: Histology of eye of patient with dry AMD showing leukostasis in the congested choroid beneath
hyaline druse compromising predominantly monocytes. (Reprinted from Penfold et al352 with permission of Progress in
Retinal and Eye Research.) D: Electron micrograph of eye of patient with CNV showing accumulation of leukocytes adherent
to abluminal surface of a new subretinal blood vessel. (Reprinted from Penfold et al352 with permission of Progress in
Retinal and Eye Research.) E: Choriocapillaris of transgenic mouse overexpressing VEGF in the RPE showing with abundant
adherent leukocytes (arrows). (Reprinted from Schwesinger et al403 with permission of American Journal of Pathology.)

have an adverse impact on preexisting CNV. Use of
corticosteroids must also recognize potential adversit-
ies such as the induction of RPE dysfunction.362 A
multicenter clinical trial of an intravitreous implant
containing fluocinolone acetonide in isolation or in
conjunction with photodynamic therapy is underway.
Angiostatic steroids have anti-angiogenic activity
independent of glucocorticoid action.84 Anecorta-
veacetate, the most widely studied of this class, pos-
sesses significant anti-angiogenic activity in several
neovascular models, including rabbit corneal neovas-
cularization, hypoxic retinalneovascularization in
rats and kittens, and murine uveal melanoma.356 Be-
cause anecortave acetate is rapidly deacetylated it
achieves ocular penetration even upon topical appli-
cation. Its angiostatic activity is reported tobe related,
in part, to upregulation of plasminogen activator in-
hibitor expression inthe retinas of rats in a model
of retinopathy of prematurity.356 Interestingly, mice
deficient in plasminogen activator inhibitor 1 (PAI-
1) have a decreased incidence of laser-induced CNV
compared to their wild-type brethren.260 When PAI-
1 expression was restored in the PAI-1 deficient mice,
the wild-type pattern of CNV was restored. These ob-
servations demonstrate the pro-angiogenic activity of
PAI-1 and confirm the perplexing and often paradox-
ical nature of the angiogenic pathways.
Results from a phase IB study suggest that sub-
Tenon juxtascleral injection of anecortave acetate
may stabilize vision in patients with predominantly
classic subfoveal CNV for up to 6 months.424 No statis-
tically significant difference was conferred by using
anecortave acetate in conjunction with Visudyne (No-
vartis Ophthalmics). A pivotal phase III clinical trial
is underway.
2. Targeted Molecular Therapy
VEGF is an attractive target in anti-CNV therapy
because of its role outlined previously. Intravitreous
injection of an oligonucleotide targeted to the VEGF
sequence inhibited laser-induced CNV.143 Following
the evidence that intravitreous injections of rhu-
FabV2 (the active fragment of a humanized mono-
clonal antibody to VEGF) inhibits development of
laser-induced CNV in cynomolgus monkeys,249 a
phase I study of this agent (in or without combination
278 Surv Ophthalmol 48 (3) May–June 2003
with photodynamic therapy with Visudyne) was
conducted. Preliminary evidence from this study sug-
gest that monthly intravitreous injections of this anti-
body fragment improved vision in 26% of eyes at
some point during the 3-month study.193 A phase III
trial is being planned.
Another exciting advance is the development of
the VEGF aptamer, a synthetic RNA compound spe-
cifically designed to bind to extracellular VEGF.
Phase IB results suggest that monthly intravitreous
injections of this aptamer stabilized vision in 83% of
patients with subfoveal CNV and improved vision by
3 or more lines in 26% of eyes.423 When combined
with Visudyne, vision improvement by 3 or more
lines was seen in 60%, and the fraction of eyes
requiring retreatment with Visudyne was 40% com-
pared to 93% in the original TAP trial. A phase III
trial is underway, in isolation or in conjunction
with Visudyne.471
A potentially promising strategy is immunoconju-
gation—coupling an antibody directed against an an-
tigen found selectively or preferentially in areas of
CNV with a cellular toxin. Exploiting the presence
of CD105 (endoglin) in CNV membranes, immuno-
conjugates of anti-CD105 monoclonal antibody and
dextran binding mitomycin C were found to inhibit
the proliferation of human umbilical vein endothe-
lial cells.517 Similarly, a monoclonal antibody to αvβ3
integrin coupled to mitomycin C-dextran was shown
to inhibit laser-induced CNV in rats.221
In this gene therapy era, subretinal injection of
adenoviral or adeno-associated viral vectors has been
used to transform the RPE into a factory for sustained
local delivery of a drug or a gene in experimental
models of CNV. Delivered in such fashion, angios-
tatin,257 the soluble flt-1 receptor, which acts as a
VEGF scavenger,206,258 and the tissue inhibitor of met-
alloproteinases-3 (TIMP-3) gene inhibited the devel-
opment of laser-induced CNV in rats,463 and PEDF
did so in mice.317,319 Systemic delivery of adenoviral
vectors expressing secretable endostatin316 or the
soluble Tie2 receptor, which acts as an Ang antago-
nist,181 inhibited the development of laser-induced
CNV in mice.181 A phase I study of intravitreous injec-
tion of an adenovirus encoding PEDF has com-
menced in patients with neovascular AMD.372
Gene delivery, whether through viral vectors,
naked plasmids, or antisense oligonucleotides, must
be delivered in a targeted fashion and one approach
may be the transscleral route,25 which may be consid-
ered for sustained delivery of protein-based drugs as
well. Preliminary evidence suggests that subconjunc-
tival injection of adenoviruses coding for PEDF146
or soluble flt-1 (a VEGF scavenger)97 inhibits laser-
induced CNV by using the episcleral tissue as a factory
to secrete these anti-angiogenic molecules, which
AMBATI ET AL
then can cross the sclera into the choroid and retina,
by virtue of their molecular weights (46 kD for PEDF
and 75 kD for soluble flt-1)22,24 and effect their desir-
able actions.
B. SURGICAL APPROACHES
Surgical excision of subfoveal CNV has shown sub-
stantial benefit in patients with conditions other than
AMD, such as myopia and ocular histoplasmosis
syndrome.473 The unimpressive results in AMD have
been attributed to the entanglement of RPE with
the CNV complex resulting in the almost obligatory
removal of both structures, which also leads to loss
of underlying choriocapillaris.468 The ongoing Sub-
macular Surgery Trials reported that the outcome
of surgical excision of recurrent subfoveal CNV in
patients with prior thermal laser photocoagulation of
juxtafoveal or extrafoveal CNV was no better than
laser photocoagulation, either in visual acuity or
in quality of life measures.446,447 It is possible that
coupling this procedure with transplantation of
RPE or iris pigment epithelium may improve
outcomes.12,13,261,474
Rotation of the neurosensory retina to reposition
it with respect to the underlying RPE has achieved
marked short-term visual improvement in some pa-
tients.9,10,103,134,135,149,264,327,359,404,482 Unfortunately,
the results of this procedure are unpredictable and
the long-term prognosis is unclear. Moreover, several
cases have been plagued by the development of in-
tractable proliferative vitreoretinopathy, leading to
total retinal detachment and loss of all vision. Fur-
ther technical refinements and better definition of
surgical candidates may expand the scope of this
approach.
C. OTHER APPROACHES
As ionizing radiation preferentially damages mi-
totic tissue, this modality has been advocated for the
treatment of choroidal neovascularization. Unfortu-
nately, this area has been cluttered by conflicting
reports, disparate radiation doses, type of radiation,
and dose fractions. Some studies have reported bene-
ficial outcomes46,68,117,285 whereas others failed to
demonstrate efficacy.247,366,434,489 In addition, al-
though no severe side effects have been reported
after radiation therapy for AMD, a subgroup of pa-
tients may experience extensive growth of CNV after
radiation, causing greater functional damage than
occurs spontaneously.168 A randomized, clinical trial
(Age-Related Macular Degeneration Radiotherapy
Trial) to assess the efficacy of external beam radiation
versus observation for the treatment of new and re-
current subfoveal CMV in AMD is underway.286
AGE-RELATED MACULAR DEGENERATION 279

Pilot trials that suggested morphologic resolution efficacy. The closure of the Fellow Eye arms of the
or visual improvement following laser photocoagula- CNVPT and PTAMD laser to drusen trials due to
tion to drusen116,126,268 spurred the Choroidal Neo- increased CNV in treated eyes is a reminder that non-
vascularization Prevention Trial (CNVPT), which intervention sometimes may be preferable.
applied subthreshold argon laser burns to the
macula. Although the CNVPT demonstrated laser-
induced drusen reduction associated with improved VII. Challenges
visual acuity and contrast sensitivity at 1 year,199 en-
AMD promises to remain a major threat to vision
rollment in the fellow-eye arm of the trial (patients
for decades to come. Current management options
with fellow eyes with exudative AMD) was suspended
represent a modest advance, but do not greatly alter
because of higher rates of CNV (15% vs. 3%) in these
the unrelenting course and prognosis of the disease
laser-treated eyes.74 Another multicenter random-
itself. New advances in the understanding of the mo-
ized prospective trial, the Prophylactic Treatment of
lecular mechanisms underpinning photoreceptor
AMD Trial (PTAMD) using diode laser photocoagu-
apoptosis, geographic atrophy, CNV, and disciform
lation, demonstrated no visual benefit,380 with treated
scarring hopefully will unlock the door to more tar-
fellow eyes experiencing higher rates of CNV (18%
geted and effective therapeutic modalities. There are
vs. 10%) and worse vision.128 The bilateral arms of
several milestones that must be achieved on the road
both trials are continuing.
to satisfactory outcomes in AMD. The absence of an
The resolution of drusen following laser photoco-
animal model that faithfully recapitulates every phase
agulation may be due to the recruitment of
of the AMD hampers study of its pathogenesis as well
phagocytes from the choriocapillaris.102 This is associ-
as therapeutic advances. Filling this lacuna would be
ated with an increase in protrusion of choriocapillary
a boon to research in this arena. A further under-
endothelial processes into Bruch’s membrane, which
standing of the factors triggering neovascularization
may represent an early event in angiogenesis,166 per-
and permeability is another major challenge. Because
haps explaining the higher rates of CNV in eyes of
angiogenesis mechanisms are generally conserved
patients already predisposed to exudative AMD.
throughout the body, this area of research is where the
greatest near-term progress is likely to be made. By
D. FAILED STRATEGIES elucidating the molecular mechanisms of AMD
pathogenesis, sure to be stimulated by insights gar-
Numerous drugs that evinced promise in pilot
nered from genomic and proteomic investigations,
studies have not survived wider examination. One
ophthalmologists and visual scientists will eventually
such disappointment was interferon-α, which showed
find the keys to preserving vision in the face of AMD.
potential efficacy in an unrandomized study.139 How-
The desire to bring potential treatments to the
ever, a randomized dose-escalation clinical trial dem-
fore, however, must be tempered by circumspection.
onstrated no benefit in progression of CNV and was
For example, the highly variable and fluctuating
associated with worse visual outcome than placebo
course taken by visual acuity in AMD patients452 dic-
at the highest drug dosage.357
tates caution in interpreting the results of clinical
Another clinical failure was thalidomide, the infa-
trials that are of short duration, non-randomized,
mous teratogen that in recent years has recaptured
or rely on historical controls for comparison. The
interest in oncology due to its potent anti-angiogenic
recognition that modular approaches to this dis-
properties. Thalidomide inhibits corneal angiogen-
ease, whether megadose micronutrient supple-
esis in a rabbit micropocket model,87,248 and
ments or antagonism of angiogenic factors, disregard
human RPE cell proliferation induced by platelet
the very likely complex and multifactorial underlying
derived growth factor or FGF2.224 However, the clini-
mechanisms is essential to solving the Rubik’s cube
cal trial of thalidomide in subfoveal CNV was ham-
of AMD.
pered by the high dropout rate of older AMD subjects
who were unable to tolerate the side effects.279 Im-
portantly, no significant anti-angiogenic effect was
found even in the small group of tolerant patients. VIII. Method of Literature Search
The Prinomastat metalloproteinase inhibitor trial References for this review were generated by a com-
also was stopped for similar reasons. prehensive literature search of the electronic
The failure of tin ethyl etiopurpurin (Purlytin) PubMed database (1966–2003) and of the Associa-
photodynamic therapy in subfoveal CNV472 despite tion for Research in Vision and Ophthalmology ab-
promising phase I/II data and the success of vertepor- stracts (1996–2002). Additional articles, unavailable
fin (Visudyne) suggests that peculiarities of individ- on electronic archives, were selected from a review of
ual drugs in this class may dictate their clinical the bibliographies of the articles generated from the
280 Surv Ophthalmol 48 (3) May–June 2003
above search. The literature search was not limited to
the English language; foreign language publications
were translated to English. To ensure that this review
is as up-to-date as possible, current issues of American
Journal of Ophthalmology, Archives of Ophthalmology, Brit-
ish Journal of Ophthalmology, Current Eye Research, Experi-
mental Eye Research, Eye, Graefes Archive for Clinical and
Experimental Ophthalmology, Investigative Ophthalmology
and Visual Science, Nature, Nature Medicine, Ophthalmol-
ogy, Progress in Retinal and Eye Research, Retina, Science,
and Survey of Ophthalmology were regularly reviewed
during the period of preparation. The following key
words and combinations of these words were used in
compiling the search: age-related macular degenera-
tion, angiogenesis, angiopoietin, angiotensin, animal
model, Bruch’s membrane, cell adhesion molecules, choroid,
choroidal neovascularization, choriocapillaris, drusen, ex-
tracellular matrix, FGF, gene therapy, geographic atrophy,
inflammation, integrin, lipofuscin, MMP, neovasculariza-
tion, nitric oxide, oxidative stress, PEDF, photooxidation,
radiation, retina, retinal pigment epithelium, risk factor,
senescence, surgery, TIMP, transgenic, transplant, TSP-1,
and VEGF.
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AGE-RELATED MACULAR DEGENERATION 293

F. CNV and angiogenesis VI. Therapeutic prospects

1. VEGF A. Anti-angiogenic therapy
2. FGF2 1. Steroids
3. Angiopoietins 2. Targeted molecular therapy
4. PEDF B. Surgical approaches
5. Nitric oxide C. Failed strategies
6. Extracellular matrix VII. Challenges
G. Subclinical inflammation VIII. Method of Literature Search