Anatomy and Physiology Section 1
Retinal and Choroidal Vasculature:
Retinal Oxygenation
Maria B. Grant, Gerard A. Lutty
INTRODUCTION
Oxygen is necessary for the existence of mammals because it is
required to generate adenosine triphosphate (ATP) oxidatively.
Although the partial pressure of oxygen (pO2) is 149 mmHg (21%
of atmospheric oxygen) at sea level, arterial oxygen content is as
low as 75–100 mmHg (10–14%) and tissue pO2 is much lower. The
oxygen level in inner segments of photoreceptors (mitochondria-
rich) after dark adaptation is between 0 and 5 mmHg (0.7%) but
up to 20 mmHg in the light. Inner retinal oxygen is normally
20 mmHg, so normoxia depends on the area of retina and dark/
light state.1,2 Hypoxia is an oxygen level below normoxia while
hyperoxia is achieved by inhaling high levels of oxygen as in the
isolette of the neonatal intensive care unit.
The retina is one of the most metabolically active tissues in the
body. It has two unique zones of oxygenation.1 The inner retina
is supplied with oxygen by the retinal vasculature. The retinal
vasculature is autoregulated because it is responsive to changes
in systemic oxygen levels, keeping the inner retina at a relatively
constant level. If the retinal vasculature is compromised, as in
ischemic retinopathies, the retina becomes hypoxic in that area.
The outer retina is supplied solely by the choroidal vasculature.
Unlike retina, choroidal vessels are not autoregulated, so sys-
temic levels of oxygen control the level of oxygen in choroid.
Supply of oxygen to choroid is diminished by stenosis of the
ophthalmic artery, which is the branch off the internal carotid
that is most likely to be stenosed because it is a right-angle
branch.
Comparison of retinal and
choroidal vasculatures
Although less than 300 µm apart in distance, the retinal and
choroidal vasculatures are vastly different in many attributes in
addition to autoregulation. The initial retinal vasculature in
human starts forming around 14 weeks’ gestation (WG) by vas-
culogenesis, development by differentiation and assembly of
vascular precursors, angioblasts.3–5 The deep capillary network
forms after 20 WG by angiogenesis, development by migration,
and proliferation of endothelial cells from existing blood vessels.
The driving force for vascular development is physiological
hypoxia; metabolic requirements of developing neurons are only
met by stimulating the development of a retinal vasculature.6,7
It is mostly a bilayered system, a superficial network, and a deep
capillary plexus; however, there are multiple layers of capillaries
in the peripapillary region, the radial peripapillary capillaries.
There is only one layer of blood vessels in the periphery at
the ora serrata where the retina thins to 100 µm. The retinal
Chapter
18 
vasculature is supplied with blood directly by the central retinal
artery in humans. The retinal vasculature has a traditional end-
arterial hierarchy: arteries branch to arterioles, which supply a
capillary network that is drained by venules, and then veins
remove the blood from the retina (Fig. 18.1). The retinal vascu-
lature forms the inner blood–retinal barrier (BRB), restricting
passage of molecules that do not have receptors or transporters
on the luminal surface of the endothelial cells. Capillaries have
a lumen diameter of 3.5–6 µm, permitting passage of red blood
cells only after deformation of their disc shape. The retinal capil-
laries and venules have perivascular pericytes and the retina has
the highest endothelial cell-to-pericyte ratio in the body, 1 : 1.8
The choroidal vasculature forms well before the retinal vessels
(6–9 WG), although its maturation is only completed after
20 WG. It develops by hemovasculogenesis, formation of blood
cells and blood vessel cells from a common progenitor, the
hemangioblast.9 The choroidal vasculature provides oxygen and
nutrients to the photoreceptors. The capillary system, the chorio-
capillaris, lies directly under the Bruch’s membrane, while inter-
mediate and large blood vessels of the system lie posterior to the
capillaries. The short and long ciliary arteries supply blood to
the choroidal vasculature while 4–6 vortex veins remove blood
from this vast system. Unlike the retina, the hierarchy in choroid
is lobular, similar to kidney glomeruli (Fig. 18.1). The lobules
change in shape, vascular density, and size depending upon area
and the location of feeding arterioles and draining venules also
varies by geographic location of the lobule.10 The capillaries are
broad and flat, having luminal diameters ranging from 10 to
38 µm in diameter. Another major difference from retina is that
the capillaries are fenestrated, allowing the passage of small
molecules and solutes through these 60–70-nm pores. The cho-
riocapillaris is sided in that the majority of the fenestrations are
on the retinal side as well as all three types of vascular endothe-
lial growth factor (VEGF) receptors.11 Pericytes, however, are
mostly on the scleral side of these capillaries. Control of vascular
tone in choroid may be accomplished by mast cells, which lie
abluminal to arteries and arterioles, or choroidal ganglion cells.12
HISTORY OF RETINAL ISCHEMIA
Ischemia is the restriction in oxygen supply without considering
actual levels of oxygen. Michaelson13,14 and Wise15 hypothesized
that areas of vascular loss in retina must be hypoxic because the
high metabolic rate requires a continuous supply of oxygen.
They observed that neovascularization always formed adjacent
to these nonperfused areas and, therefore, an angiogenic factor
must be produced by the hypoxic retina. They hypothesized
that this factor X must be hypoxia-inducible and diffusible.

434
Section 1
A D
Anatomy and Physiology
Basic Science and Translation to Therapy
p stained by alkaline phosphatase (APase)
e hierarchy. (E) When sectioned, the
c choriocapillaris (c) shows the broad flat
B E
p apparent and, at higher magnification (inset),
e
C F
Subsequently, oxygen was measured directly in retinas of several
species and it demonstrated that nonperfused areas were indeed
hypoxic.1,2 It was not until 1989 that factor X was discovered,
purified, and characterized as vascular endothelial growth factor
(VEGF).16 This factor was first shown to be responsible for the
increased vascular permeability seen in some retinopathies.17
NORMOXIA
The studies of Wangsa-Wirawan and Linsenmeier1 and Yu and
Cringle2 using oxygen electrodes directly assessed oxygen levels
from choroid to vitreous in various species. The oxygen tension
is around 70 mmHg in choroid and plummets to zero at the
inner segments in the dark (Fig. 18.2). Inner retina is around
10–20 mmHg. There are regional variations in oxygen concentra-
tion within the retina. Yu et al.18 showed that oxygen consump-
tion in outer retina is highest in the parafoveal region while inner
retinal oxygen in the fovea (approximately 5 mmHg) reflected
the lack of a retinal vasculature and the predominantly choroidal
source of oxygen.
HYPEROXIA
Life in utero is hypoxic, so when a child is born prematurely, the
normoxic environment is actually hyperoxic. Prematurely born
Fig. 18.1 Comparison of retinal (A-C) and
choroidal (D-F) vasculatures. (A) The retinal
vasculature, stained here with adenosine
diphosphatase (ADPase) enzyme
histochemistry, has an end-arterial hierarchy:
arteries (arrow) have a capillary-free zone,
and arterioles and then capillaries, which
are drained by venules, and then veins
(arrowhead). (B) A capillary in the deep
capillary network has a pericyte and
endothelial cell. (C) Transmission electron
microscopy (TEM) of a retinal capillary shows
a pericyte (p) within the shared basement
membrane (arrow) with an endothelial cell
(e). (Courtesy of DB Archer, TA Gardiner,
and AW Stitt.) (D) The choriocapillaris,
enzyme histochemistry, has a lobular
endothelial cell APase activity in
c
lumens of the choriocapillaris which are
positioned under retinal pigment epithelial
cells (top) and Bruch’s membrane between
them. (F) With TEM the thin processes of the
choriocapillaris endothelial cells (arrow) are
the fenestrations in these endothelial cells
are visible.
children are placed in 40% oxygen, making their tissue further
hyperoxic, which yields vaso-obliteration (endothelial cells die
and pericytes and progenitors survive).19 The only direct mea-
surements of oxygen in a model of retinopathy of prematurity
(ROP) were performed by Ernest and Goldstick.20 They found in
kitten after 80–90% O2 that preretinal pO2 over avascular retina
was close to zero but was normal over vascularized retina. Vaso-
obliteration from hyperoxia does not occur in the choroid of
humans and dogs19 but does occur when rats are exposed to
hyperoxia.21 Loss of vasculature in vaso-obliteration makes the
retina hypoxic when the child is returned to room air. Exposure
of the adult vasculature to hyperoxia causes constriction but not
vaso-obliteration. During hyperoxia breathing (100% oxygen),
the inner retinal pO2 remains unchanged due to autoregulation
while the choroidal pO2 rises to 250 mmHg in cat22 and 220 mmHg
in the minipig, due to a lack of metabolic control of the choroidal
vasculature.23
HYPOXIA
Complex homeostatic mechanisms are designed to maintain
O2 concentration in each cell within a narrow range. While
O2 consumption increases with the metabolic activity of the
organism, exposure to O2 must be limited due to the potentially

80
60
Oxygen tension, mmHg
40
20
110 100 90 80 70 60 50
Retinal depth, %
damaging effects of reactive oxygen species (ROS). Hypoxia,
the state of low oxygen concentration, promotes the formation
of blood vessels and is important for the formation of a vascular
system in embryos.24 Disease occurs when the retina and choroid
are deprived of adequate oxygen supply; this can also be
described as a mismatch of oxygen supply versus demand at
the cellular level within ocular tissues.
The blood O2-carrying capacity is maintained by the O2-
regulated production of erythropoietin (EPO), which stimulates
the proliferation and survival of red blood cell progenitors.
Semenza and coworkers25,26 performed seminal studies to iden-
tify hypoxia-inducible factor-1 (HIF-1). HIF-1 orchestrates a
pleiotropic adaptive response to hypoxia by inducing the expres-
sion of more than 100 genes encoding glycolytic enzymes and
glucose transporters (thereby facilitating the glycolytic switch in
energy metabolism typically observed under hypoxic condi-
tions), matrix metalloproteinases, and angiogenic, mitogenic,
and survival factors, including EPO.27,28 Other molecules upreg-
ulated by HIF-1 that have profound effects on vasculature
include 5’ nucleotidase, an enzyme that is the major source of
the potent vasodilator adenosine in the body, and VEGF. HIFs
are vital to development and, in mammals, deletion of the HIF-1
genes results in perinatal death. HIF-1 is expressed in all cell
types and functions as a master regulator of oxygen homeostasis
by playing critical roles in embryonic development and post­
natal physiology.
Hypoxia-inducible factor
HIF is a highly conserved transcriptional complex which is a
heterodimer composed of an alpha and a beta subunit. HIF-1
belongs to the PER-ARNT-SIM (PAS) subfamily of the basic
helix–loop–helix (bHLH) family of transcription factors. The
alpha and beta subunit both contain an N-terminus bHLH
domain for DNA binding, a central region with PAS domain,
Fig. 18.2 Oxygen profile measured with
microelectrodes in cat retina during light
and dark adaptations. The schematic
at the top shows where, anatomically, 435
the measurements were taken from
choriocapillaris (left, retinal depth = 100)
to the internal limiting membrane (right,
Chapter 18
retinal depth = 0). (Reproduced with
Light permission from Wangsa-Wirawan ND,
Linsenmeier RA. Retinal oxygen. Arch
Dark
Ophthalmol 2003;121:547–55.)
Retinal and Choroidal Vasculature:
40 30 20 10 0
which facilitates heterodimerization, and a C-terminus, which
recruits transcriptional coregulatory proteins.
The activity of HIF depends on the intracellular levels of its
inducible alpha subunit. In the presence of oxygen, HIF-1α is
hydroxylated on two critical proline residues (Pro402 and Pro564)
in the so-called oxygen-dependent degradation domain. Three
prolyl hydroxylases have been identified in mammalian cells
and use O2 as a substrate to generate 4-hydroxyproline at resi-
dues 402 and/or 564 of HIF-1α. The hydroxylation reaction
also requires 2-oxoglutarate (α-ketoglutarate) as a substrate and
generates succinate as a side product. These prolyl hydroxylases
have a high Km for O2 that is slightly above atmospheric con-
centration; thus O2 is rate-limiting for enzymatic activity under
physiological conditions and any change in cellular O2 concen-
tration is directly transduced into changes in the rate of HIF-1α
hydroxylation.29
Factor-inhibiting HIF-1 (FIH-1), which was identified in a
yeast two-hybrid screen as a protein that interacts with, and
inhibits the activity of the HIF-1α transactivation domain,30
functions as asparaginyl hydroxylase.31 As in the case of the
prolyl hydroxylases, FIH-1 appears to use O2 and 2-oxoglutarate,32
although it has a Km for O2 that is three times lower than
the prolyl hydroxylases.33 Hydroxylation provides a mechanism
for regulating protein–protein interactions, similar to the effect
of phosphorylation and other posttranslational modifications.
However, this hydroxylation occurs in an O2-dependent manner,
thus establishing a direct link between cellular oxygenation and
HIF-1 activity. Following HIF-1α hydroxylation, the protein
becomes targeted for ubiquitination by an E3 ligase complex
(including the von Hippel–Lindau (VHL) tumor suppressor
protein) and subsequent proteasomal degradation.
Under hypoxic conditions, the HIF prolyl-hydroxylases are
inhibited, because these HIF prolyl-hydroxylases utilize oxygen
as a cosubstrate. Hypoxia results in an increase in succinate,

Hyperoxia
436 OH OH
Prolyl E3 Ligase
hydroxylase
complex
Hif-1α Hif-1α Hif-1α
Section 1
Hypoxia
Angiogenic growth factors
Hif-1β
Angiogenic survival factors
(ARNT)
Anatomy and Physiology
Basic Science and Translation to Therapy
Cytoskeletal proteins
Proapoptotic proteins
Glucose transporters
Glycolytic enzymes
Nucleus Hif-1β
(ARNT)
Transcription
Hif-1α
Hif-1β
(ARNT)
due to inhibition of the electron transport chain in the mito-
chondria, which serves to inhibit further HIF prolyl-hydroxylase
activity. When stabilized by hypoxic conditions, HIF increases
the expression of critical genes that promote survival in low-
oxygen conditions, including glycolytic enzymes, which allow
ATP synthesis in an oxygen-independent manner. HIF activates
the transcription of genes encoding secreted signaling mole-
cules, including angiogenic growth factors and survival factors,
cell surface receptors, extracellular matrix proteins and modify-
ing enzymes, transcription factors, cytoskeletal proteins, pro-
apoptotic proteins, and glucose transporters and glycolytic
enzymes (Fig. 18.3).29
HIF-induced VEGF, stromal-derived factor-1 (SDF-1), and
EPO promote neovascularization. HIF-1 acts by binding to HIF-
responsive elements in promoters that contain the sequence
NCGTG, which is present in the promoters for VEGF, SDF-1,
EPO, and many other genes. In addition to hypoxia, other factors
such as nuclear factor κB (NF-κB) modulate HIF-1α expression
in the presence of normal oxygen pressure. Thus, conditions
such as tissue inflammation can lead to local HIF-1α expres-
sion.34 HIF-1 DNA-binding activity and target gene expression
are induced in cells exposed not only to hypoxia but also to the
iron chelator desferrioxamine or to cobalt chloride.35
A structurally and functionally related protein to HIF-1α, des-
ignated HIF-2α, is the product of the EPAS1 gene. HIF-2α can
also heterodimerize with HIF-1ß.36 HIF-1α:HIF-1ß and HIF-
2α:HIF-1ß heterodimers have overlapping yet distinct target
gene specificities.37 HIF-2α, unlike HIF-1α, is not expressed in all
cell types and HIF-2α can be inactivated by cytoplasmic seques-
tration. This “compartmentalization” of oxygen-sensitive signal-
ing components also influences the hypoxic response.38,39
HIF deficiency and its resultant pathology
O2 delivery to cells of the developing embryo becomes limited
by diffusion such that establishment of a functioning circulatory
Fig. 18.3 Hypoxia-inducible factor 1 (Hif-1α)
in hypoxia and hyperoxia.
Ub
Degradation
system is required for embryonic survival by embryonic day 9
(E9) in the mouse. In wild-type mouse embryos, HIF-1α expres-
sion increases dramatically between E8.5 and E9.5, whereas
embryos that lack HIF-1α expression die between E9.5 and E10.5
and show cardiac malformations, vascular regression, and
massive cell death.40 Complete HIF-2α deficiency is also associ-
ated with embryonic lethality41 and because the embryos survive
longer than HIF-1a–/– mice, effects on multiple organ systems can
be demonstrated.42
Complete HIF-1α deficiency results in developmental defects;
however, partial HIF-1α deficiency is sufficient to result in
impaired responses to physiological stimuli. A particularly dra-
matic example is the loss of O2 sensing in the carotid body of
HIF-1a+/– mice.43 Although the carotid bodies are anatomically
and histologically normal and depolarize normally in response
to cyanide application, they show essentially no response to
hypoxia. Thus partial HIF-1α deficiency in the carotid body
results in a complete loss of the ability to sense and/or respond
to changes in the arterial PO2 by stimulation of the central
nervous system cardiorespiratory centers. The HIF-1 target
genes that are critical for O2 sensing and/or efferent responses
by the carotid body have not been identified.
Mice with HIF-1α conditionally knocked out using PAX6-Cre
have delayed development of the outer retinal plexus but not
the superficial or deep plexus.44 However, when HIF-1α was
knocked down only in Müller cells using a Cre-LOX system, and
the animals were made diabetic with streptozotocin, vascular
permeability in retina was reduced and leukostasis and overpro-
duction of VEGF and intercellular adhesion molecule (ICAM)-1
were attenuated in adult mice.45
Another dramatic phenotype is the complete inability of HIF-
1α–/– myeloid cells (granulocytes and macrophages) to respond
to inflammatory stimuli.46 Myeloid cells are dependent on gly-
colysis for ATP generation, perhaps reflecting the hypoxic
microenvironment that is often associated with inflammation
and infection. HIF-1α deficiency results in ATP deficiency, which
impairs critical myeloid cell functions such as aggregation,
motility, invasion, and bacterial killing. HIF-1 also plays
critical roles in B-lymphocyte development47 and T-lymphocyte
activation.48
HIF-activated genes relevant to
physiological and pathological
ocular angiogenesis
The paragraphs above provide a brief summary of the critical
role of HIF-1α in oxygen sensing, development, and physiology.
HIF-1α plays an equally important role in disease pathophysiol-
ogy, including retinal diseases. As a result, there is considerable
interest in HIF-1 α as a therapeutic target.49 In cardiovascular
diseases, increased HIF-1α activity induced as a result of HIF-1α
gene therapy,50 small-molecule inhibitors of prolyl hydroxylase
activity,51 or inhibitors of HIF-1α–VHL interaction52 may provide
a means to stimulate neovascularization in ischemic tissue. In
contrast, small-molecule inhibitors of HIF-1 activity may be
useful as antiangiogenic agents. However, because HIF-1α func-
tions as a global regulator of oxygen homeostasis, it may not be
a useful therapeutic target if the treatment results in unintended
and undesirable side-effects.
An alternative therapeutic approach that may be particularly
relevant to the treatment of ocular pathology is to focus on
modulation of HIF-1α target genes. However, the protein prod-
ucts of these target genes must also be delivered in a precise and
perfectly timed manner. EPO is an oxygen-regulated hormone
stimulating erythrocyte production and is critical for retinal
angiogenesis. Increasing EPO expression in phase 1 of the
murine ROP model (postnatal days 7–12) is protective and
results in less neovascularization during phase 2 (postnatal days
12–17).53 In contrast, EPO mRNA expression levels in retina are
highly elevated during the hypoxia-induced proliferation phase
of retinopathy (phase 2) and inhibition of retinal EPO mRNA
expression with RNA interference results in suppressed retinal
neovascularization.53
The best-known gene activated by HIF-1α is VEGF, first iden-
tified as a potent promoter of vascular permeability17 and endo-
thelial cell proliferation.15 VEGF has become known as a master
regulator of angiogenesis.54 Tight control of physiologic VEGF
levels is required for proper embryological development.55
Although it was initially thought that the postembryonic role of
VEGF was restricted to a few processes, it is now quite clear that
VEGF acts as a pluripotent growth factor essential for a wide
variety of physiological processes,56 including maintenance of
the adult microvasculature,57 neuronal survival,58 and trophic
maintenance of ocular tissues.
VEGF in health and in ocular disease
VEGF is produced by many cell types in the retina, including
retinal pigment epithelium (RPE),59 vascular endothelial cells,60
pericytes,60 retinal neurons,61 Müller cells,61 and astrocytes,62 sug-
gesting that VEGF has important functions in ocular homeosta-
sis. RPE-secreted VEGF plays an important role in maintaining
the choriocapillaris.11,63,64 VEGF secretion by retinal cells and the
RPE is stimulated in response to hypoxia.60 VEGF administration
protects retinal neurons from apoptosis.65 Moreover, chronic
VEGF inhibition can lead to a significant loss of retinal ganglion
cells in normal adult animals.65
While VEGF is critical to maintaining normal ocular func-
tion, overproduction of VEGF is deleterious. Elevated levels
of VEGF have been strongly implicated in the pathogenesis 437
of ocular neovascular diseases such as neovascular age-related
macular degeneration (NV in AMD)66 and proliferative diabetic
Chapter 18
retinopathy67 as well as diabetic macular edema.68 Elevated
VEGF levels are observed in central and branch retinal vein
occlusion (CRVO and BRVO),69 neovascular glaucoma,70 and
ROP.71 Blocking VEGF action is now an established strategy
for the treatment of NV in AMD, with two agents (the RNA
aptamer pegaptanib sodium72 and the humanized murine
monoclonal antibody antigen-binding fragment ranibizumab73)
Retinal and Choroidal Vasculature:
having received regulatory approval for the intravitreal treat-
ment of NV in AMD.
While current standard of care for NV in AMD uses intravitreal
delivery of an anti-VEGF agent on a repeated basis, this routine
clinical practice does not address all the nuances of VEGF biology.
The biology of the VEGF proteins is extremely complex. The
VEGF family members are part of a superfamily of cysteine
knot proteins and include VEGF-B, -C, -D, and placental growth
factor (PlGF). Alternative splicing events for VEGFA give rise to
at least 14 subtypes of VEGF, namely, VEGF111, VEGF121, VEGF121b,
VEGF145, VEGF145b, VEGF148, VEGF162, VEGF165, VEGF165b, VEGF183,
VEGF183b, VEGF189, VEGF189b,74 and VEGF206.75,76 Following the
discovery of the antiangiogenic isoform of VEGF, VEGF165b, and
its associated family of isoforms, a further layer of complexity
was added to understanding the regulation of VEGF.
All VEGF isoforms are essential regulators of angiogenesis
and vascular permeability. VEGFs elicit their intracellular activi-
ties via the activation of two receptor tyrosine kinases (RTKs):
VEGFR-1 and -2. VEGFR-1, a high-affinity fms-like tyrosine
kinase-1,77 and VEGFR-2, a kinase insert domain-containing
receptor,78 are transmembrane glycoproteins consisting of a
seven-tandem immunoglobulin-like domains, which serves as
the extracellular ligand-binding region, a single-transmembrane
domain, and a cytoplasmic domain consisting of two tyrosine
kinase catalytic domains. Moreover, it has also been reported
that a family of cell surface glycoproteins, particularly
neuropilin-1, act as isoform-specific coreceptors for VEGF-A.79
The VEGF family and their respective receptors are outlined in
Fig. 18.4.
Ligand binding to the extracellular domain of VEGFR-2 results
in a maximal increase of kinase activity following the induction
of receptor dimerization and subsequent phosphorylation of
tyrosine residues on the intracellular domain of the receptor.80
This event is crucial for the recruitment of additional signaling
molecules that contain Src homology 2 or phosphotyrosine
binding domains, which mediate further downstream signaling
cascades.81 The association of RTKs with coreceptors, such as
NP-1, in the case of VEGFR-2:VEGF165 signaling/interaction,
can enhance the functional signal transduction and facilitate
diverse cellular responses.80 VEGFR1/R2 signaling activates
RAS, raf1, MEK1, and ERK1/ERK2 and stimulates PI3K/AKT/
PKCz/MAPK pathways to mediate proliferation, migration, and
cell survival (Fig. 18.4).
VEGFR-2 is the major mediator of angiogenic signaling in
endothelial cells and is required for de novo vessel formation,
vasculogenesis, and for angiogenesis, the formation of vessels
from pre-existing vasculature.82 The pathways leading to VEGFR
internalization and the role of receptor degradation in VEGF
signaling remain controversial and differ for VEGFR-1 and -2.
 VEGFRs generate signal output at the plasma membrane and on
their way to degradation through endocytic vesicles,83 whereas,
438 in unstimulated cells, VEGFR-2 is predominantly located in recy-
cling endosomes identified by Rab4 and/or Rab5.84 VEGFR inter-
nalization is clathrin-mediated and transport is further directed
by the endosomal sorting complex required for transport pro-
Section 1
including the nucleus,88 where receptors encounter distinct sig-
naling molecules.89
In addition to EPO and VEGF, SDF-1 is hypoxia-regulated and
ischemic tissues express high levels of SDF-1 to recruit repara-
tive cells to the injured region. SDF-1 activation of either CXCR-4
or CXCR-7 results in stimulation of the Ras/Raf/Mek/ERK

teins.85 VEGFR signaling is also regulated by ubiquitination, not
only of the receptor itself, but also of receptor-associated signal-
ing molecules.86 Specific VEGFR trafficking regulates biological
output, as shown for arterial morphogenesis, for example.87
The molecular basis for ligand specificity of VEGFR signaling
is poorly understood. It is well accepted however that VEGF
Anatomy and Physiology
Basic Science and Translation to Therapy
pathway and the PI3K/Akt pathway to promote endothelial cell
proliferation and neovascularization. Thus ligand–receptor
interaction of VEGF to VEGF-R1 and VEGF-R2 and SDF-1 to
CXCR4/CXCR-7 and their subsequent internalization sets in
motion the cascade of cellular effects of VEGF and SDF-1
(Fig. 18.4). The VEGF and SDF-1 signaling pathways appear

receptors can associate with distinct coreceptors such as neuro-
pilins, integrins, semaphorins, or heparan sulfate glycosamino-
glycans, and engage distinct signaling molecules giving rise to
specific signal output. Ligand-specific signaling may also result
from receptor trafficking to specific cellular compartments,
to be intimately connected with HIF-1 activation (Fig. 18.3), as
the promoters of each of these factors contain a HIF response
element. Because hypoxic tissue releases SDF-1 and VEGF,
varying O2 concentrations would have an effect on expression of
the receptors for these factors.

VEGF-A
VEGF-B VEGF-E
Insulin
PIGF
IGF-1

IGF-2 SDF-1

VEGF-R1 VEGF-R2 IGF-1R CXCR4/CXCR7

Signaling by
intracellular RAS PI3K/Akt PLCg
translocation

RAF1 PKCz PKC

MEK1 MAPK

ERK1

ERK2

Fig. 18.4 Vascular endothelial growth factor (VEGF) family and its receptors. PlGF, placental growth factor; IGF, insulin-like growth factor;
SDF, stromal-derived factor.
Bone marrow-derived progenitor cells
(BMPC) and vascular repair
In conditions like diabetic retinopathy and ROP, areas of retinal
vasodegeneration occur and lead to retinal ischemia which in
turn induces the expression of hypoxia-regulated angiogenic
factors. Typically, BMPCs robustly respond to these factors,
including VEGF and SDF-1.90
Importantly, all hypoxia-regulated angiogenic factors are
modulators of bone marrow-derived stem cells. Specifically,
hematopoietic stem cells and other BMPCs reside in the bone
marrow and are mobilized into the peripheral circulation by
increased levels of EPO, SDF-1, and VEGF released by the isch-
emic tissue. Hematopoietic stem cell/BMPC mobilization occurs
in response to vascular injury throughout the body and, when
functioning properly, leads to revascularization of injured
areas.91 In healthy individuals, bone marrow-derived CD34+
endothelial progenitor cells as well as other BMPC successfully
orchestrate the reparative process. These BMPC demonstrate a
limited ability to differentiate directly into endothelial cells and
form components of new blood vessels by vasculogenesis, but
show marked ability to provide paracrine support for the resi-
dent vasculature. This paracrine support facilitates resident
endothelial cell recovery.
Disease-associated BMPC dysfunction
In diabetes, for example, BMPC are dramatically altered and
cannot facilitate the repair process. Diabetic individuals have
fewer circulating CD34+ cells and an increased number of
inflammatory BMPC such as CD14+ cells than nondiabetics.92–94
This diabetes-related bone marrow dysfunction is closely linked
to the impaired healing response experienced by many diabetic
patients and to the vasodegenerative aspect of diabetic macro-
and microvascular complications.94–96 Diabetes-induced BMPC
defects occur in part due to uncoupling of nitric oxide synthases,
enhanced NADPH oxidase activity, and increased generation of
ROS such as superoxide and peroxynitrite (ONOO-)97 within
BMPC. While stem and progenitor cells are deemed more resis-
tant to oxidative stress,98 the highly oxidative diabetic milieu has
a clearly detrimental effect on the function of these cells.99 Pro-
longed oxidant exposure reduces reparative function100 by
impairing antioxidant defense enzymes. Previously, we and
others have shown that diabetic CD34+ cells exhibit decreased
migration and adhesion activities in vitro, and consequently
reduce recruitment to areas of injury.96 In addition to oxidative
stress, other key mechanisms implicated in diabetes-induced
BMPC dysfunction include a reduction of cathepsin L activity101
and an upregulation of thrombospondin-1.100,102 While these
functional defects are profound, strategies that successfully
reverse BMPC defects in diabetics have included: (1) enhance-
ment of angiogenic stimulus by increasing BMPC mobilization
using granulocyte colony-stimulating factor and targeting
SDF-1103; (2) use of nitric oxide donors to correct migration and
promote cell deformability104; (3) enhancing cell interactions
with substrate proteins to increase attachment to basement
membranes105; (4) reducing high levels of endogenous trans-
forming growth factor-β to normal levels106; and (5) treatment
with rosiglitazone107 or atorvastatin.108
In addition to CD34+ cells, other populations of bone
marrow cells may attempt vascular repair, such as CD14+ cells,
discussed above, which can, under select circumstances, form
endothelial-like cells109; however, the blood vessels formed by
the endothelial cells of CD14+ origin eventually generate patho-
logical blood vessels with increased permeability and contribute 439
to the pathology of diabetic retinopathy.
We and others have been particularly interested in a novel
Chapter 18
factor expressed in increased concentrations in hypoxic tissue,
insulin-like growth factor-binding protein 3 (IGFBP-3). While
the standard IGF-dependent actions of the family of IGF-binding
proteins have been well described, recently several IGF-
independent actions have been discovered for IGFBPs, including
IGFBP-3. These IGF-1 independent actions have been charac­
terized as regulating cell fate and apoptosis.110–113 The role of
Retinal and Choroidal Vasculature:
IGFBP-3 in the control of cell growth remains an area under
intense study, since IGFBP-3 may enhance or suppress cell
growth, depending on specific conditions.112,114 In the retina,
multiple forms of IGFBP (2–5) are secreted by retinal endothelial
cells.113 IGFBP-3 has been shown to enhance cell proliferation
in retinal endothelial cells and to decrease the formation of
neovascular tufts in a murine model of oxygen-induced reti-
nopathy.115,116 These studies suggest that IGFBP-3 may be
vascular-protective in the retina. Recently, we also have dem-
onstrated that IGFBP-3 is neuroprotective in the retina and
reduces injury-induced retinal inflammation.117
Key factors that modulate VEGF function
in the retina
The retina is one of the last organs to become vascularized in the
fetus. The vasculogenic part of the process of human retinal
vascularization begins in about gestational week 14 and appears
to depend on a physiological hypoxia7,118 brought about by an
increase in metabolic demand within the developing retina.3–5
This physiological hypoxia induces the local release of VEGF
which, together with IGF-1, regulates angiogenesis and therefore
normal vascularization of the retina.118,119 Retinal vascularization
is complete by 36–40 WG.
ROP is a two-phase disease in which the phases are mirror
images; the controlling growth factors are deficient in phase 1
and in excess in phase 2. Phase 2 involves uncontrolled prolifera-
tive growth of retinal blood vessels in response to hypoxia. The
therapeutic intention would be to prevent the first phase of ces-
sation of vessel and neural retinal development and then the
second destructive phase would be prevented. This has been
successfully performed using exogenous EPO, VEGF, and
IGFBP-3 during phase 1 to prevent phase 2. These two phases,
seen in premature infants, can be duplicated in animal models
of ROP. Hyperoxia causes vaso-obliteration and cessation of
normal retinal blood vessel development, which mimics phase
1 of ROP. IGF-1 levels rise in the third trimester of pregnancy
but not in the preterm infant. The low IGF-1 in the preterm infant
is due to the infant no longer being exposed to the support of
the maternal environment, including maternal sources of IGF-1.
IGF-1 levels rise slowly after preterm birth, as these babies are
unable to produce adequate IGF-1 compared to term infants.120
In these premature infants, IGF-1 is further reduced by poor
nutrition,121 acidosis, hypothyroxinemia, and sepsis.122 IGF-1
appears important for retinal and brain growth.119 Low levels of
IGF-1 appear to play an important role in the early cessation of
retinal growth that precipitates ROP.
IGF-1 mediates its effects through activation of the IGF-1 recep-
tor (IGF-1R) (Fig. 18.3). IGF-1R is a receptor tyrosine kinase and
 is well established as a key regulator of cell growth and survival
with activation of Ras-ERK pathway, the PI3K/Akt pathway and
440 PKC (Fig. 18.4). Insulin and IGF-2 can also signal using the IGF-R.
There is also a growing body of data to support a role for the
structurally and functionally related insulin receptor (IR) in cell
survival, even though its major function has been to modulate
Section 1
metabolism. Bidirectional cross-talk between IGF-1R and IR is
observed, where specific inhibition of either receptor confers a
compensatory increase in activity for the reciprocal receptor.
Although fluctuating oxygen has long been associated with
the development of ROP, oxygen-regulated factors like VEGF
appear to be directly modulated by IGF-1. IGF-1 is a key growth
Anatomy and Physiology
Basic Science and Translation to Therapy
factor in early retinal development. IGF-1 controls maximum
VEGF activation of the Akt endothelial cell survival pathway
(Fig. 18.3). Thus loss of IGF-1 leads to loss of VEGF signaling
and retinal vaso-obliteration. This vaso-obliteration leads to
phase 2 of ROP which is the proliferative phase. At this point,
suppression of IGF-1 and VEGF can reduce neovascularization.
Thus IGF-1 is critical to normal retinal vascular development
and a lack of IGF-1 in the early neonatal period is associated with
lack of vascular growth and with subsequent proliferative ROP.
In IGF-1-null mice, the retinal blood vessels grow more slowly
than in those of normal mice, a pattern very similar to that seen
in premature babies with ROP.
ADULT RETINAL HYPOXIA AND ETIOLOGY
Diabetic retinopathy
Much like ROP, diabetic retinopathy has a vaso-obliteration
phase that leads to a proliferative phase. The vaso-obliteration
is not due to hyperoxia but rather to vaso-occlusion and vaso­
degeneration of the microvasculature, setting up the ischemic
environment that leads to the vasoproliferative end-stage condi-
tion. Clinically the early pathology has been classified as non-
proliferative (microaneurysms, exudates, leakage, capillary
nonperfusion) resulting in hypoxia and the end-stage pathology
as proliferative (preretinal neovascularization). The purely vaso-
degenerative, nonproliferative form of the disease is by far the
most common and represents a disease of the neurovascular
unit, resulting in dysfunction and eventual death of several of
the key cells that maintain the BRB: pericytes, vascular endothe-
lial cells, Müller glia and neurons. Kohner and Henkind ele-
gantly demonstrated that, in diabetic individuals, areas of
nonperfusion on fluorescein angiography are associated with
acellular capillaries in trypsin digests.123 Direct measurement of
oxygen in diabetic cat retinas demonstrated that even small
aneurysms can result in a decrease in retinal interstitial oxygen.124
There are many mechanisms implicated in the pathogenesis
of diabetic retinopathy but one that has gained considerable
attention in the last decade is inflammation. The environment
of hyperglycemia, abnormal lipids, increased oxidative stress,
elevated serum and tissue advanced glycation endproducts
(AGE)/receptor for AGE, increased serum/tissue cytokines,
elevated blood pressure, and endoplasmic reticulum stress are
the likely initiators of inflammation.125 Pathways of inflamma-
tion converge with pathways of endothelial dysfunction and
coagulation to accelerate the pathogenesis of this disease.126
Initially nonspecific indicators of inflammation such as white-
cell count and fibrinogen were found to be predictive of incident
diabetes.127 Subsequently plasminogen activator inhibitor-1
(PAI-1), C-reactive protein, and fibrinogen were shown to be
independent predictors.128 These observations are supported by
several other prospective studies, in which tissue plasminogen
activator, another marker of reduced fibrinolysis,129 and von
Willebrand factor, a marker of endothelial injury, were predic-
tive.130 In one clinical study of type 2 diabetics, adhesion mol-
ecules were higher in subjects with retinopathy than those
without,131 and in another population-based cohort, composite
scores of both inflammatory and endothelial function markers
were strongly associated with the presence of diabetic retinopa-
thy.132 Similarly, E-selectin values were found to be increased
in a group with type 1 diabetes and retinopathy.133 Adiponectin
was increased in the advanced stages of retinopathy.134 These
results should be interpreted cautiously, however, as serum
markers are not necessarily indicators of tissue events. However,
P-selectin, ICAM-1, and polymorphonuclear leukocyte numbers
are all elevated in human diabetic retina.135 Experimental work
in diabetic rat retinas later demonstrated that inflammatory
cytokine-mediated leukostasis occurs early in diabetic retina
and neutralizing ICAM-1 and CD-18 prevents it.136–138 From these
studies, it appears that inflammation-mediated EC injury and
vaso-occlusion may cause nonperfusion and subsequrent
hypoxia in diabetic retina.139,140
The hypothesis that inflammation is critical to the develop-
ment of diabetic retinopathy arose from initial reports that dia-
betic patients taking salicylates to treat rheumatoid arthritis had
a lower-than-expected incidence of diabetic retinopathy.141 The
subsequent decades demonstrated an increase in inflammatory
markers and growth factors in the diabetic vitreous and retina.
Recently microarray analyses substantiated a marked inflamma-
tory response in the retinas of diabetic rodents.142 Confirmation
of the importance of inflammatory factors and growth factors is
supported by additional rodent studies that show that blocking
these factors prevents the development of lesions characteristic
of the retinopathy in animals. Specific inflammatory molecules
that have been shown to contribute to structural or functional
alterations that are characteristic of the retinopathy include
NF-κβ143; inducible nitric oxide synthase143; cytochrome c
oxidase143; ICAM140; 5-lipoxygenase144; interleukin-1β145; tumor
necrosis factor (TNF)-α146; and VEGF.67,147 Inflammation can
intensify the generation of AGEs that are produced in response
to hyperglycemia and increased oxidative stress.
Retinal vein occlusion (RVO)
Occlusion of large retinal blood vessels is a common occurrence,
which results in retinal hypoxia. RVO is the second most
common sight-threatening retinal vascular disorder after dia-
betic retinopathy.148 RVO represents an obstruction of the retinal
venous system that involves either the central retinal vein or
a branch retinal vein. RVO is typically due to external compres-
sion or disease of the vein wall, such as is seen in vasculitis.149
Central retinal artery occlusion (CRAO) results in sudden, cata-
strophic visual loss and branch retinal arteriolar occlusion
(BRAO) causes sudden segmental visual loss and may recur to
involve other branch retinal arterioles. CRAO studies have
shown that the ischemic retinal whitish opacity and swelling
of CRAO are essentially located in the perifoveolar region of
the macula. Oxygen supply and nutrition from the choroidal
vascular bed to the thinner peripheral retina help in its much
longer survival and the maintenance of peripheral visual fields.
The diagnosis is clinical and based on the observation of the
ocular fundus: venous dilatation and tortuosity, flame-shaped
retinal hemorrhages, retinal edema and cotton-wool exudates
affecting all the retinal sectors (in CRVO) or the sector of the
retina drained by the affected vein in BRVO. Open-angle glau-
coma is the most frequent local alteration predisposing to RVO
as it compromises venous outflow by increasing intraocular
pressure. Raised intraocular pressure causes external compres-
sion of the central retinal vein as it passes through the lamina
cribrosa, resulting in turbulent blood flow distal to the compres-
sion leading to thrombus formation.
The natural history of RVO is highly variable; in some cases
the retinal findings progressively disappear and there is a
good visual outcome, while in other cases severe complications
like ocular neovascularization (proliferative retinopathy), vitre-
ous hemorrhage, neovascular glaucoma, and macular edema
develop. Using retinal oximetry in BRVO, venular saturation
was found to be highly variable between patients (12–93%)150;
this was attributed to variable severity of the disease, recana-
lization, degree of occlusion, collateral vasculature, or tissue
atrophy. The majority of patients with CRVO have signs of
macular edema at presentation whereas only 5–15% of eyes
with BRVO develop macular edema over the first year. Venous
collateral channels represent tortuous vessels that develop
locally, mainly around the optic disc, and are usually associ-
ated with a long-standing vascular obstruction. Vitreous hem-
orrhage develops in 10% of eyes with CRVO within 9 months
of presentation and in about 40% of eyes with BRVO.148 If
there is restoration of circulation in the central retinal artery,
the retinal capillaries in the central, thickest part of the macular
region do not refill because of compression by the surround-
ing swollen superficial retinal tissue, resulting in the “no-reflow
phenomenon,”151and consequently in permanent ganglion cell
death in the nonperfused retina; the area of central retinal
capillary nonfilling may vary from eye to eye depending upon
the severity of retinal swelling in the macular region. This
results in the variable size of the permanent central scotoma.
In considering the outcome of CRAO, it is important to con-
sider retinal tolerance time to acute retinal ischemia. The chance
of recovery of vision only exists as long as the retina has
reversible ischemic damage. The retina suffers no detectable
damage with CRAO of up to 97 minutes, but after that, the
longer the CRAO, the more extensive the irreversible ischemic
retinal damage.
It is generally believed that the CRAO is always either embolic
or thrombotic in origin. Embolism is far more common than
thrombosis,152 as was pointed out almost a century ago by
Coats.153,154 An inflammatory etiology is also postulated in RVO
as RVO is associated with immunological diseases in young
patients. To support this contention, patients can experience
prompt resolution of symptoms with the use of periocular ste-
roids.155 Giant cell arteritis is an important and well-known cause
of CRAO, and is an ophthalmic emergency because of the high
risk of bilateral visual loss, which is preventable.
Elevated levels of PAI-1, lipoprotein(a), and hyperhomocyste-
inemia, and low circulating levels of folic acid, vitamin B12
and vitamin B6 have been implicated in the pathogenesis of
this disease.156 While the pathogenesis is complex and largely
unknown, medical treatment includes identification and correc-
tion of vascular risk factors. The use of fluorescein fundus angi-
ography before (showing occlusion of the central retinal artery)
and immediately after thrombolysis may show improvement in
and/or restoration of retinal circulation and retinal function.
It is also important to consider that fibrinolytic agents can dis-
solve only platelet fibrin emboli.157 Retinal emboli are made of
74% cholesterol, 10.5% calcific material, and only 15.5% of plate- 441
let fibrin. Fibrinolytic agents cannot dissolve cholesterol or calci-
fied material. Therefore, there is no scientific rationale for the use
Chapter 18
of fibrinolytic agents in at least 85% of CRAO cases. For the
remaining 15%, if the diagnosis is made within 15 days from
onset of clinical manifestations, low-molecular-weight heparins
at anticoagulant doses are typically used 10–15 days followed by
half dose for a total of 90 days.158 No data are available on the
possible role of antithrombotic/antiplatelet strategies in the
long-term prevention of recurrent RVO159; however, they are
Retinal and Choroidal Vasculature:
routinely prescribed to most patients with diabetes, hyperten-
sion, or arterial disease. Of Virchow’s three classical factors that
play a role in thrombogenesis – stasis, vessel wall damage, and
hypercoagulability – the first two have long been reported in
patients with RVO, whereas the third has not been sufficiently
investigated until recently.
Sickle-cell disease (SCD)
The pathological processes involved in SCD can affect virtually
every vascular bed including the retina and in its advanced
stages has the potential to cause blindness.160 Classification of
ophthalmic manifestations of SCD in the retina is based on the
presence or absence of vascular proliferation, which is the most
important precursor of blinding complications and precedes
development of a vitreous hemorrhage or retinal detachment.
Ischemia occurs due to obstruction of capillaries with sickle red
blood cells and subsequent thrombus formation. The subsequent
scenario for disease pathogenesis is similar to that for ROP,
diabetic retinopathy, and RVO, described above, except that
there is no retinal leakage of retinal blood vessels only from
preretinal neovascularization, even though there is loss of
peripheral vasculature and elevation of VEGF.161 Neovascular-
ization that occurs can lead to fibrosis that can then result in
retinal detachment.
Ocular ischemic syndrome (OIS)
OIS occurs at a mean age of 65 years and is rare before the
age of 50. Men are affected twice as often as women,162 reflect-
ing their higher incidence of atherosclerotic disease; however,
no racial predilection exists. Bilateral involvement may occur
in up to 22% of cases.163,164 Sturrock and Mueller estimated 7.5
cases per million persons every year,165 but this is likely an
underestimation as OIS can be easily misdiagnosed. Kearns166
reported that, of patients with occlusion of the internal carotid
artery undergoing surgical anastomosis between the superficial
temporal artery and the middle cerebral artery, 18% presented
with OIS. Up to 29% of patients with a symptomatic carotid
artery occlusion manifest retinal vascular changes that are
usually asymptomatic; however, 1.5% of them progress per year
to symptomatic OIS.167 OIS develops especially in patients with
poor collateral circulation between the internal and external
carotid arterial systems. Insufficient collateral vascular flow in
OIS patients explains the frequent association with cerebral
infarctions and the poor neurologic outcomes.168 Degree of inter-
nal carotid artery stenosis, presence of collateral vessels, and
compensation by collaterals are important in assessing OIS
disease severity. Also if the OIS is bilateral or there are associ-
ated systemic vascular diseases, this tends to worsen the
prognosis.
 GA: Nonatrophic GA: Atrophic Wet AMD

442
Section 1

A D G
Anatomy and Physiology
Basic Science and Translation to Therapy

B E H

c c c
C F I

Fig. 18.5 The choroid and retinal pigment epithelium (RPE) in geographic atrophy (A-F) and wet (neovascular) age-related macular degeneration
(AMD) (G-I). (A) Alkaline phosphatase (APase)-stained choroidal blood vessels in a nonatrophic region of geographic atrophy (GA) choroid.
(B) When sectioned, this area has normal-appearing RPE cells (arrowhead) and viable choriocapillaris lumens filled with serum APase.
(C) Higher magnification shows the intimate relationship between RPE, Bruch’s membrane, and choriocapillaris (c). (D) The atrophic region of
this GA choroid has no RPE and an attenuated choriocapillaris with narrow lumens. (E) Cross-sections of this area demonstrate the APase
reaction product in a few remaining choriocapillaris lumens and the endothelial cells of artery (bottom). (F) At higher magnification it is apparent
that the APase– capillaries (c) are only collagenous tubes between intercapillary septa. (G) In a flat choroid preparation of a wet AMD subject,
a large fan-shaped choroidal neovascular formation is present and the RPE monolayer to the left appears normal. (H) A section of this
subject immediately in advance of the choroidal neovascularization demonstrates viable hypertrophic RPE (arrowhead) over an attenuated
choriocapillaris. (I) At higher magnification, the remnants of atrophic choriocapillaris lumens (c) are present between intercapillary septa and
a single APase+ lumen remains, yet RPE are still present.

Retinal detachment center of our understanding of ocular angiogenesis. Retinal

Retinal detachment causes the sensory retina to be distant from
choriocapillaris, thus reducing its oxygen supply and resulting
in photoreceptor degeneration. Linsenmeier and Padnick-
Silver169 demonstrated in cat that retinal detachment resulted in
a significant decrease in outer retinal oxygen, having a serious
metabolic effect on photoreceptors. In subsequent work, Linsen-
meier demonstrated that hyperoxia may have clinical benefit
after retinal detachment because it normalized photoreceptor
oxygen consumption and prevented photoreceptor dysfunc-
tion.170 Furthermore, hyperoxia prevents proliferation and reac-
tivity of retinal Müller cells in the detached feline retina, limiting
retinal injury.171
Consequences of retinal ischemia
In 1971, Judah Folkman reported in the New England Journal
of Medicine that all cancer tumors are angiogenesis-dependent.172
If a tumor could be stopped from growing its own blood
supply, he surmised, it would wither and die. Though his
hypothesis was initially disregarded by most experts in the
field, Folkman persisted with his research. After more than a
decade, his theory became widely accepted and is now at the
neovascularization is defined as a state where new pathologic
vessels originate from the existing retinal veins and extend
along the inner surface of the retina.
Vascular permeability
Growth factors such as VEGF have been implicated in both
retinal neovascularization and vascular hyperpermeability.173
Antibodies to VEGF improve visual function in patients with
diabetic macular edema.174 In experimental diabetes and in
VEGF-induced permeability, alterations of the tight junction (TJ)
complex of microvascular endothelial cells alters the BRB.175
A role of classical PKC isoforms (cPKCs) but especially PKCβ
in regulating VEGF-induced vascular permeability is well
accepted.176 VEGF activation of PKCβ leads to phosphorylation
and reorganization of the TJ complex, increasing vessel wall
permeability.177 VEGF increases the phosphorylation of the
TJ protein, occludin, at multiple sites,178 including Ser490.179
Phosphorylation at Ser490 allows subsequent ubiquitination
and endocytosis of occludin and fosters breaks in the TJ.180
Despite this well-supported mechanism, the PKCβ inhibitor
ruboxistaurin failed to achieve Food and Drug Administration
approval for diabetic retinopathy. cPKC inhibition also failed to
prevent TNF-α-induced permeability,181 a proinflammatory
cytokine also implicated in diabetic retinopathy. Thus targeting
permeability in the retina continues to be a difficult clinical
problem.
ADULT CHOROIDAL ISCHEMIA
The choroidal vasculature is an expansive vascular plexus that
handles 90% of the blood from the ophthalmic artery. It was
often assumed that the choriocapillaris was so vast that genera-
tion of choroidal ischemia rarely occurred. However, there are
several diseases in which the loss of choroidal vasculature is
extensive, resulting in apparent choroidal ischemia.
Diabetic choroidopathy was first described by Hidiyat and
Fine.182 The use of alkaline phosphatase enzyme histochemistry
on human choroid permitted quantification of vascular loss in
choroid. Viable vessels were positive for alkaline phosphatase
while acellular, dysfunctional capillaries lacked it, and choroidal
neovascularization had the greatest activity.10 This technique
demonstrated that there was four times greater loss in chorio-
capillaris in diabetic subjects than in aged control subjects.183
Loss of alkaline phosphatase activity was associated with the
presence of polymorphonuclear leukocytes.184 Areas of chorio-
capillaris loss were also associated with choroidal neovascular-
ization and Bruch’s membrane deposits. A consequence of
choriocapillaris dropout is loss of outer retinal oxygenation. This
may be the reason for loss of blue cones and other cones in
diabetic retina when there is no retinopathy present.185,186
Decreased choroidal blood flow has been measured in
AMD.187–190 Using alkaline phosphatase activity and quantifica-
tion of RPE in AMD, the loss of choriocapillaris has been dem-
onstrated in exudative or wet AMD as well as geographic
atrophy (Fig. 18.5). RPE loss occurs first in geographic atrophy
and then choriocapillaris degeneration occurs. Although some
capillaries survive in the area of RPE atrophy, a 50% loss of
vasculature occurs and the surviving capillaries are highly con-
stricted.191,192 This undoubtedly contributes to photoreceptor loss
in the area of degeneration. Capillary loss occurs in exudative
AMD as well but in the presence of a complete RPE monolayer
(Fig. 18.5). This suggests that the RPE in this area are hypoxic
and VEGF production may be increased, causing choroidal neo-
vascularization. Large-vessel stenosis is also often observed in
AMD choroid, suggesting that the choriocapillaris blood supply
may be limited as well. Unfortunately, we have no way of mea-
suring oxygen directly in the choroid so we can only assume
ischemia occurs when there is loss of viable choriocapillaris and
stenosis of large and intermediate choroidal blood vessel.
CONCLUSIONS
Both too little and too much oxygen can be damaging to the
retina. Retina cannot function properly nor survive without the
support of two vasculatures: retinal and choroidal. If either vas-
culature is dysfunctional, retinal hypoxia occurs and HIF-1α
becomes stable and induces expression of key factors to assist
in retinal cell survival. Reduced retinal oxygenation causes
impaired neuroretinal activity in young healthy persons.193 One
key factor upregulated by HIF-1α is VEGF, which stimulates
new angiogenesis and increased vascular permeability. These
pathological events can be controlled by destroying ischemic
retina (photocoagulation), inhibiting VEGF, or reducing levels of
HIF-1α.
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