DIABETES/METABOLISM RESEARCH AND REVIEWS REVIEW ARTICLE

Diabetes Metab Res Rev 2003; 19: 442–455.

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/dmrr.415

Vascular endothelial growth factor and diabetic

retinopathy: pathophysiological mechanisms
and treatment perspectives

Ruth B. Caldwell1,2,4 * Summary

Manuela Bartoli1,5
M. Ali Behzadian1,3 Retinal neovascularization and macular edema are central features of
Azza E. B. El-Remessy1,3 diabetic retinopathy, the major cause of blindness in the developed world.
Current treatments are limited in their efficacy and are associated with
Mohamed Al-Shabrawey1
significant adverse effects. Characterization of the molecular and cellular
Daniel H. Platt1 processes involved in vascular growth and permeability has led to the
R. William Caldwell3 recognition that the angiogenic growth factor and vascular permeability
1
factor vascular endothelial growth factor (VEGF) plays a pivotal role in the
Vascular Biology Center, The
retinal microvascular complications of diabetes. Therefore, VEGF represents
Medical College of Georgia Augusta,
an exciting target for therapeutic intervention in diabetic retinopathy. This
GA 30912
review highlights the current understanding of the mechanisms that regulate
2
Department of Cellular Biology and VEGF gene expression and mediate its biological effects and how these
Anatomy, The Medical College of
processes may become altered during diabetes. The cellular and molecular
Georgia Augusta, GA 30912
alterations that characterize experimental models of diabetes are considered
3
Department of Pharmacology and in relation to the influence of high glucose-mediated oxidative stress on VEGF
Toxicology, The Medical College of expression and on the mechanisms of VEGF’s actions under hyperglycemic
Georgia Augusta, GA 30912
induction. Finally, potential therapeutic strategies for preventing VEGF
4
Department of Ophthalmology, The overexpression or blocking its pathological effects in the diabetic retina
Medical College of Georgia Augusta, are considered. Copyright  2003 John Wiley & Sons, Ltd.
GA 30912
5
Department of Pathology, The Keywords diabetic retinopathy; retina; vascular endothelial growth factor;
Medical College of Georgia Augusta, angiogenesis; vascular permeability; reactive oxygen species
GA 30912

*Correspondence to:
Ruth B. Caldwell, Vascular Biology
Center, Department of Cellular
Biology and Anatomy, Department
of Ophthalmology, The Medical
College of Georgia, Augusta, GA
30912. E-mail: rcaldwel@mcg.edu
Received: 23 April 2003
Revised: 12 August 2003
Accepted: 18 August 2003
Introduction
Clinical features of diabetic retinopathy
Diabetic retinopathy is the most common complication of diabetes and a
leading cause of blindness in the western world, affecting approximately
three-fourths of diabetic patients within 15 years after onset of the disease
[1–3]. Although the best way to avoid visual loss is to initiate treatment
before symptoms develop, many diabetic patients are referred for treatment
only after visual complications have already occurred. Hyperglycemia is
known to be the primary pathogenic factor in the development of diabetes
complications [4,5], but the mechanisms by which elevated blood glucose
causes tissue injury and disease progression in the retina are not yet clear.
The clinical progression of diabetic retinopathy follows a pattern
characteristic of ischemic retinopathy (for review, see [6,7]). The disease
begins with biochemical and cellular alterations that are not clinically
evident. Vascular alterations during this period include increased adhesion
of leukocytes to the vessel wall, alterations in blood flow, death of retinal

Copyright  2003 John Wiley & Sons, Ltd.

VEGF and Diabetic Retinopathy
pericytes (perivascular contractile cells), basement mem-
brane thickening and subtle increases in vascular perme-
ability. Blockage of the retinal capillaries causes localized
hypoxia, which is thought to lead to increased tissue pro-
duction of angiogenic factors. This growth factor release
is likely to be involved in the permeability increase
and may contribute to the pathological vascular growth
that is seen later. With time, endothelial cells lining
the microvessels are lost, leading to capillary closure
and formation of acellular, nonperfused vessels. As the
disease progresses, obvious alterations in the vascular
structure can be seen upon ophthalmoscope examination.
These include microaneurysms, dot/blot hemorrhages,
cotton-wool spots, venous beading and vascular loops. At
this stage, the blood vessels become prominently leaky,
allowing blood and fluid to accumulate in the retinal tis-
sue, forming exudate deposits. The retinal edema causes
reduced vision if the macula is affected, but is other-
wise asymptomatic. The condition of macular edema
is also referred to as nonproliferative retinopathy. In
many patients, the retinopathy progresses to proliferative
retinopathy, which is characterized by the growth of new
blood vessels on the surface of the retina. The walls of
the new blood vessels are weak and may break, allow-
ing blood to leak out. This can cloud the vitreous and
compromise vision. In more advanced stages of prolifera-
tive retinopathy, newly formed fibro-vascular tissue grows
from the retinal surface into the vitreous cavity. This can
cause retinal detachment and will result in blindness if
untreated. Proliferative retinopathy typically develops in
patients with type 1 diabetes, whereas nonproliferative
retinopathy with macular edema is more common in
patients with type 2 diabetes.
Current therapy for diabetic
retinopathy
Many therapies for diabetic retinopathy have been eval-
uated clinically (for review, see [8]). Laser photocoag-
ulation has long been the recommended treatment for
patients with advanced proliferative diabetic retinopathy
or clinically significant diabetic macular edema. While
this treatment is usually effective, the procedure destroys
neural tissue and can decrease peripheral vision, impair
night vision and change color perception. Moreover, in
some patients the retinopathy continues to progress in
spite of appropriate treatment. Therefore, there is a great
need for the development of new noninvasive therapies
to prevent and treat diabetic retinal vascular disease.
The Early Treatment Diabetic Retinopathy Study
evaluated aspirin treatment and found no improvement
of visual acuity in patients with macular edema and
no change in the subsequent development of severe
preproliferative diabetic retinopathy. Clinical trials of
aldose reductase inhibitors also showed no significant
effect on the development or progression of diabetic
retinopathy. Small-scale studies showed that blocking the
action of growth hormone using a synthetic somatostatin
Copyright  2003 John Wiley & Sons, Ltd.
443
analog had beneficial effects at near toxic doses, whereas,
a receptor blocker was without effect.
Epidemiologic observations have suggested that the risk
and/or progression of diabetic retinopathy is increased by
hypertension. Studies in which strict control of blood
pressure was obtained using an angiotensin converting
enzyme (ACE) inhibitor or a beta adrenergic blocker
found reductions in the progression of retinopathy and
risk of deterioration in visual acuity [9]. Because the
previous studies were not specifically designed to study
retinopathy, a new study is underway to see whether
blockade of the renin-angiotensin system with the AT1-
receptor blocker candesartan can prevent the incidence
and progression of retinopathy.
Another promising therapy in clinical trials is the use
of oral inhibitors of the beta isoform of protein kinase
C (PKCβ). Studies in a variety of experimental models
in vivo and in vitro have demonstrated the involvement of
PKCβ in the very early ocular complications of diabetes
(for review, see [10]). These observations have led to the
initiation of clinical trials testing the ability of the specific
PKCβ inhibitor LY333531 to slow or halt the development
of proliferative diabetic retinopathy and reduce or reverse
the progression of diabetic macular edema (for review,
see [8,11]).
A therapeutic strategy for treating diabetic retinopathy
just beginning to be tested in clinical trials involves
the use of agents that directly inhibit angiogenesis.
Until recently, the major emphasis in clinical trials
of antiangiogenic therapies has been on determining
their ability to inhibit the growth of vascularized
tumors [12,13]. However, several agents found to be
effective in controlling tumor growth have now been
evaluated for their efficacy in the treatment of ocular
neovascularization associated with age-related macular
degeneration [14,15]. The results may have implications
for other kinds of pathological neovascularization,
including diabetic retinopathy. Interferon alpha-2a, which
had been shown to be antiangiogenic in animal and
in vitro models, was found to be ineffective in halting
the progression of neovascular macular degeneration
in a double-blind, placebo-controlled clinical trial [16].
Thalidomide is another promising antiangiogenic drug
that has been tested for patients with age-related macular
degeneration in phase 3 clinical trials, but the results
are not yet known. To date, the most promising results
for treating age-related macular degeneration have been
obtained with antiangiogenic reagents targeting vascular
endothelial growth factor (VEGF) [8]. The discussion that
follows focuses on VEGF, its mechanisms of actions under
hyperglycemic induction, its role in diabetic retinopathy
and potential strategies for blocking its adverse affects on
the retinal vasculature during diabetes.
Hyperglycemia in diabetic retinopathy
As outlined above, type 1 and type 2 diabetes differ
in manifestation of retinal complications. However, the
Diabetes Metab Res Rev 2003; 19: 442–455.
444
microvascular alterations in both conditions have the
same pathophysiological basis in that the occurrence
and progression of retinopathy is closely correlated with
the degree of hyperglycemia. Clinical trials have shown
that intensive blood glucose control can delay the onset
and reduce the severity of the retinal complications in
patients with either type 1 or type 2 diabetes [4,5].
In other words, the initial stages of retinopathy that
are collectively related to endothelial dysfunction (i.e.
leukocyte adhesion, blood flow alterations, capillary
closure and formation of leaky vessels) are most probably
due to the effects of elevated blood glucose concentration
on microvascular tissues.
Numerous studies in various models indicate that
hyperglycemia in vivo or high glucose exposure in vitro
induces the formation of angiogenic factors (for review,
see [17,18]). While multiple growth factors and cytokines
have been implicated in retinal neovascular diseases,
VEGF has been identified as a primary mediator of the
vascular alterations in diabetic retinopathy (for review,
see [15,19]). Expression of VEGF is increased by hypoxia,
oxidative stress, high glucose and inflammatory reactions.
Furthermore, retinal endothelial cells have large numbers
of high affinity, high sensitivity VEGF receptors which
have been found to be increased in diabetes [20,21].
Oxidative stress in diabetic retinopathy
The molecular mechanisms of hyperglycemia-induced
endothelial dysfunction are not fully understood. How-
ever, multiple biochemical pathways associated with
hyperglycemia/diabetes-induced vascular injury can
increase the production of reactive oxygen species. These
include glucose auto-oxidation, the polyol pathway and
formation of advanced glycation end products. Moreover,
all of these mechanisms seem to reflect a hyperglycemia-
induced process initiated by superoxide overproduction
by the mitochondrial electron-transport chain (for review,
see [22]). Recent studies showing that diabetes-induced
retinal vascular dysfunction can be prevented by inhibitors
of reactive oxygen species provide further support for the
role of oxidative stress in diabetic retinopathy [23,24].
Oxidative stress has been correlated with the increased
production of VEGF under in vitro conditions and is
thought to be involved in the upregulation of VEGF
expression during diabetes [5,25–27]. Studies showing
that diabetes-induced increases in both retinal VEGF
concentrations and in lipid peroxidation in streptozotocin-
diabetic rats can be prevented by antioxidant treatment
[28] provide strong support for this hypothesis.
The mechanisms by which oxidative stress contributes
to diabetic vascular dysfunction and VEGF overexpression
are complex. It has been shown that inhibitors of
nitric oxide synthase prevent diabetes-induced vascular
dysfunction in rats [24,29,30], indicating that formation
of reactive nitrogen species plays a role in the
pathology. Recently, attention has been focused on the
role of peroxynitrite in diabetic vascular dysfunction.
Copyright  2003 John Wiley & Sons, Ltd.
R. B. Caldwell et al.
Peroxynitrite is a highly reactive oxidant formed by the
combination of nitric oxide with superoxide. Research
demonstrating the presence of the peroxynitrite marker
nitrotyrosine in placental vessels and plasma of diabetic
patients and retinas of diabetic rats suggests a role for
peroxynitrite in the development of diabetic complications
[30–32]. A working model to explain the potential
interactions between oxidative and nitrative stress in
the development of diabetic vascular disease is shown
in Figure 1. This model is based on studies of cultured
endothelial cells showing that high glucose-induced
superoxide formation leads to an increase in intracellular
calcium, which not only causes activation of endothelial
nitric oxide synthase (NOS3) to form nitric oxide but also
causes NOS3 ‘uncoupling’, leading to further increases
in superoxide formation [33–36]. Superoxide combines
rapidly with nitric oxide to form peroxynitrite, thereby
causing deactivation of nitric oxide. Data showing a
reduction in nitric oxide bioavailability and vasodilatory
function in endothelial cells exposed to high glucose
provide further support for this concept [34,35].
Peroxynitrite initiates a variety of pathological pro-
cesses including inhibition of key metabolic enzymes
by nitration of protein tyrosine residues, lipid perox-
idation and reduction of cellular antioxidant defenses
by oxidation of thiol pools and induction of DNA
strand breaks, leading to apoptosis [37–39]. Peroxynitrite
can also oxidize specific cellular components, including
the nitric oxide synthase cofactor tetrahydrobiopterin
and may also affect the L-arginine transporter [40,41].
Reduction in availability of tetrahydrobiopterin and/or
L-arginine uncouples nitric oxide synthase, leading to fur-
ther increases in superoxide and peroxynitrite. This cycle
can be interrupted by scavenging superoxide or perox-
ynitrite or inhibiting nitric oxide synthase. Studies in
animal models of diabetes and cultured endothelial cells
exposed to high glucose have demonstrated that increases
in peroxynitrite formation are directly correlated with dia-
betes and high glucose-induced VEGF expression and with
increases in retinal vascular permeability and reduced
endothelial cell survival [30,36,42,43].
Overview of VEGF actions and its
potential role in diabetic retinopathy
VEGF, a multifunctional vasoactive
factor
VEGF is a member of a large family of angiogenic
growth factors. There are six known members of the
VEGF family: VEGF-A (commonly referred to as VEGF),
placental growth factor, VEGF-B, VEGF-C, VEGF-D and
VEGF-E, for review, see [44]).
VEGF was first discovered as vascular permeability
factor, but subsequently was recognized as an angiogenic
factor and as a specific mitogen for vascular endothelial
cells (for review, see [45]). VEGF’s mitogenic activity
Diabetes Metab Res Rev 2003; 19: 442–455.
VEGF and Diabetic Retinopathy
Electr
on
c Oxidase
Tr n
ha
ans
i
port
Ca 2+ i
NOS3
L-Arginine
Tetrahydrobiopterin
Figure 1. Mechanisms of high glucose-induced formation of reactive oxygen and nitrogen species, including superoxide (O2 − ),
nitric oxide (NO) and peroxynitrite (ONOO-)
is demonstrated in vascular and lymphatic endothelial
cells, but is not seen at appreciable levels in other
cell types. VEGF also functions as a vasodilator,
a promoter of endothelial cell migration and an
antiapoptotic, endothelial cell survival factor [46–48].
Studies done in vitro have demonstrated VEGF mRNA
and protein expression by numerous retinal cell types,
including retinal-pigmented epithelial cells, pericytes,
astrocytes, Muller glia and endothelial cells [49–52].
VEGF expression in cultured cells is substantially
increased by hypoxia [49,52,53] and inflammatory
mediators [54–56], all of which are increased by
oxidative stress. In vitro studies also indicate that
high glucose-mediated oxidative stress increases the
autocrine expression of VEGF within retinal microvascular
endothelial cells [57].
Role of VEGF in diabetic retinopathy
A number of clinical studies have shown a strong
correlation between increases in intraocular VEGF
concentration and the development of proliferative
diabetic retinopathy (for review, see [15]). VEGF may
also have a key role in the pathogenesis of diabetic
macular edema due to its action in increasing vascular
permeability. Studies in streptozotocin diabetic rats
have shown that retinal vascular alterations similar to
background diabetic retinopathy in patients are associated
with increases in retinal VEGF levels and increased
expression of VEGFR2 [20,21]. Inhibition of VEGF can
Copyright  2003 John Wiley & Sons, Ltd.
445
High Glucose
NAD(P)H
RAGE
Aldose
reductase
PKC
O2−
Uncoupling
ONOO −
NO
prevent the diabetes-induced permeability, indicating a
direct role for VEGF in this pathology [58].
Because of the lack of rodent models for proliferative
diabetic retinopathy, considerable research has been done
in neonatal animals in which retinal neovascularization
is induced by hyperoxia exposure. Using these models
it has been shown that neovascularization is closely
associated with increases in VEGF production and that
the pathological vascular growth can be prevented by
treatment with selective VEGF inhibitors [49,59–63].
Regulation of VEGF gene expression
Altered VEGF expression has profound pathological
consequences. Remarkably, the disruption of a single
VEGF allele results in embryonic lethality due to
severe vascular abnormalities, thus indicating that
adequate VEGF expression levels are critical for vascular
development [64]. Conversely, the need for tight control
of VEGF expression is evident in the pathological
consequences of VEGF overexpression during ischemic
retinopathy (for review, see [15,19]).
Tissue hypoxia upregulates VEGF by increasing both
the expression and stability of VEGF mRNA [65].
Hypoxic transcriptional regulation of VEGF is mediated
at least in part by the action of the heterodimeric
complex, hypoxia-inducible factor-1 [66]. Hypoxia-
inducible factor-1 is composed of the constitutively
produced hypoxia-inducible factor-1β subunit and the
inducible component hypoxia-inducible factor-1α [67].
Diabetes Metab Res Rev 2003; 19: 442–455.
446
Hypoxia-inducible factor-1α complexes with hypoxia-
inducible factor-1β and is translocated to the nucleus
where it binds the hypoxia response elements to promote
the expression of VEGF, erythropoietin, and other genes
whose products are involved in oxygen transport and
glucose metabolism [68]. The role of hypoxia-inducible
factor-1α in diabetic retinopathy is not yet clear, but
it has been implicated in VEGF overexpression and
hyperpermeability of the blood-retinal barrier in diabetic
animals treated with intensive insulin therapy during early
diabetes [69].
Of particular interest in relation to VEGF overexpression
during diabetic retinopathy are the effects of TGF-β, TNF-
α, IGF-1, interleukins, advanced glycation end products
and reactive oxygen species, all of which are upregulated
in diabetes [70–76]. The influence of these factors on
transcriptional events leading to VEGF overexpression
is still not completely understood. However, oxidative
stress has been found to increase VEGF expression by
a mechanism involving signal transducer and activator
of transcription factor 3 (STAT3) [77]. STAT proteins
are latent cytoplasmic transcription factors that regulate
gene expression induced by cytokines, interferons and
growth factors (for review, see [78–80]). STAT3 binding
sites have been identified in the VEGF promoter, and
mutations of these regions can block VEGF mRNA
transcription [81]. STAT3 has been shown to play a
pivotal role in tumor and heart angiogenesis by inducing
VEGF expression. STAT3 is activated by reactive oxygen
species as well as by inflammatory mediators, and its
constitutive activation has been shown to correlate with
increased rates of VEGF expression and angiogenesis in
in vivo and in vitro [81–85]. It has been shown that VEGF
selectively induces STAT3 activation and VEGF autocrine
expression in retinal microvascular endothelial cells,
whereas macrovascular endothelial cells from the aorta
are unaffected [86,87]. Studies now in progress indicate
that STAT3 also plays a fundamental role in inducing
VEGF expression in retinal endothelial cells exposed to
oxidative stress involving peroxynitrite formation [88].
VEGF receptors
Two high-affinity VEGF receptors, VEGFR1 (Flt-1 or
fms-like tyrosine kinase) and VEGFR2 (Flk-1/KDR or
fetal liver kinase) have been identified in vascular
endothelial cells [89–92]. These receptors are similar in
structure – both are class III receptor tyrosine kinases with
seven immunoglobulin-like loops in their extracellular
domain and a kinase insert region in the intracellular
domain – but they differ in their biological activities.
VEGFR1 displays a higher ligand affinity than VEGFR2
[89,92]. However, studies with cultured endothelial cells
indicate that lack of VEGFR1 has little effect on VEGF
function, whereas lack of VEGFR2 prevents VEGF-induced
cellular responses (for review, see [45]).
Studies with knockout mice revealed that absence of
either VEGFR1 or VEGFR2 results in embryonic lethality
Copyright  2003 John Wiley & Sons, Ltd.
R. B. Caldwell et al.
due to the failure to develop a normal vasculature
[93,94]. VEGFR2 null mice exhibit altered angioblast
differentiation, indicating VEGFR2’s involvement in
endothelial cell lineage determination. By contrast,
VEGFR1 null mice develop abnormal vascular-like
structures in which the lumen is filled with endothelial
cells, indicating a role for VEGFR1 as negative modulator
of vascular formation [95]. Overexpression of a short
soluble form of VEGFR1 results in negative regulatory
effects. Finally, recent evidence has implicated VEGFR1
in cell migration and in the induction of stem cell
recruitment and vasculogenesis in various models of
ischemic retinopathy [96,97], suggesting a role for
VEGFR1 in postnatal vasculogenesis. Involvement of
VEGFR1 in diabetic retinopathy remains to be determined.
VEGFR2 plays a prominent role in mediating VEGF’s
angiogenic actions. Upon VEGF binding, VEGFR2 under-
goes dimerization and transphosphorylation followed by
a complex cascade of signaling events leading to multiple
cell responses, including vasodilation, increased vascular
permeability and increases in endothelial cell migra-
tion, proliferation and survival (Figure 2, for review, see
[98]). In diabetic retinas, VEGFR2 expression is increased
and the intracellular signaling events associated with its
activation play a significant role in the onset and/or pro-
gression of retinopathy [20,21,99]. Thus, VEGFR2 and its
downstream mediators represent important therapeutic
targets.
Recent studies have indicated that VEGF activation
of VEGFR2 involves formation of superoxide anion
in endothelial cells through a mechanism involving
activation of NAD(P)H oxidase. This effect has been
shown to be required for VEGF’s mitogenic properties
[76], indicating that superoxide can serve as an
intracellular signaling mediator of angiogenesis. In
addition, this result implies that VEGF stimulation of
VEGFR2 is itself a potential source of oxidative stress in
diabetic retinopathy.
Mechanisms of VEGF-induced increases
in vascular permeability
Early breakdown of the blood-retinal barrier in diabetes
at the level of the retinal vascular endothelium has been
well established by analyses in both diabetic patients and
experimental animals (for review, see [100]).
The permeability defect has been shown to correlate
with increases in the expression of VEGF [58,101–103].
Recent studies in streptozotocin diabetic rats have shown
that the initial breakdown of the blood-retinal barrier
is associated with increases in expression of the both
endothelial and neuronal nitric oxide synthase as well
as with increases in VEGF expression [30,103,104].
Moreover a VEGF-neutralizing receptor construct has
been shown to prevent the diabetes-induced increases in
expression of VEGF and endothelial nitric oxide synthase
and the barrier breakdown [104], underlining a potential
role for VEGF in the early vascular dysfunction.
Diabetes Metab Res Rev 2003; 19: 442–455.
VEGF and Diabetic Retinopathy
The specific mechanism of the VEGF-induced bar-
rier dysfunction has been controversial, but studies of
VEGF’s permeability-inducing actions in vitro have shown
that VEGF induces a transient and reversible hyper-
permeability in cultured endothelial cells that persists
for only 30 min, which is associated with the for-
mation of fenestrae, a transcellular permeability route
[105]. In vitro studies with cultured retinal microvascular
endothelial cells have shown that this immediate perme-
ability increase occurs by a mechanism of transcytosis
in caveolin-coated vesicles requiring activation of nitric
oxide synthase and nitric oxide formation [106].
It was subsequently found that the rapid and transient
permeability increase is followed by a sustained increase
in paracellular permeability starting 4 to 6 h after VEGF
treatment [107]. Since VEGF induces urokinase receptor
(uPAR) expression [108], which may initiate plasmin
formation, it appears likely that the VEGF-induced
permeability results from disruption of endothelial
cell-to-cell attachments by uPAR-mediated initiation
of proteolytic activities. This would generate leaky
vessels and allow endothelial cells to penetrate their
underlying basement membrane, proliferate and migrate,
setting the stage for retinal neovascularization. These
in vitro results are consistent with results of studies in
streptozotocin-induced diabetic rats showing increased
vascular permeability associated with reductions in
levels of the tight junction protein occludin [100,109].
Subsequent work has shown that this diabetes-induced
increase in vascular permeability is associated with
overexpression of VEGF and uPAR and with oxidative
stress as evidenced by increased formation of nitric oxide,
superoxide and peroxynitrite [30]. Moreover, each of
these alterations could be prevented by inhibiting nitric
Src
PI3K
PlCγ
PKB/Akt
IP3 DAG
Survival NOS Ca ++ PKC
NO
Cell proliferation/permeability
Vasodilation
Angiogenesis
Figure 2. Signaling pathways activated by VEGF
Copyright  2003 John Wiley & Sons, Ltd.
447
oxide synthase or scavenging peroxynitrite, indicating a
role for nitric oxide synthase and peroxynitrite formation
in the pathology.
The identification of mediators that regulate VEGF
effects on blood/tissue barrier breakdown may have sig-
nificant clinical implications. In vitro analyses of the sig-
naling pathways by which VEGF stimulates the autocrine
production of nitric oxide and prostacyclin by vascular
endothelial cells indicate that VEGF activation of VEGFR2
involves the formation of protein complex between
VEGFR2 and c-Src and that c-Src activation is required
for VEGF induction of nitric oxide and prostacyclin for-
mation [110] (Figure 2). Further studies have shown that
VEGF induces uPAR gene expression in cultured retinal
endothelial cells by inducing transcriptional activation
of β-catenin and that the delayed paracellular phase of
the VEGF-induced permeability increase correlates with
uPA/uPAR activation [107]. These findings may shed new
light on the mechanism of VEGF-induced barrier dysfunc-
tion. Further definition of the VEGF-induced signaling
pathways leading to these events should aid in the devel-
opment of more specific and efficient therapies for the
treatment of diabetic macular edema and prevention of
proliferative retinopathy.
VEGF’s angiogenic actions
VEGF’s functions in stimulating endothelial cell migration
and proliferation during angiogenesis have been well
established (for review, see [45,111]) and the role of
these functions in proliferative diabetic retinopathy is
also clear. However, there is increasing evidence that
neovascularization is not only due to angiogenesis but
may also involve the process of vasculogenesis in which
VEGF
VEGFR2
P P
P
P β - catenin
FAK
STAT1 uPAR
&
STAT3 Paxilin
MAPK Permeability
Gene
PGI2
expression Cytoskeletal
rearrangement
Cell migration
Diabetes Metab Res Rev 2003; 19: 442–455.
448
circulating bone marrow-derived endothelial progenitor
cells are recruited into the neovascularization process
[112]. Relatively little is known yet about the effects
of diabetes on vasculogenesis and the recruitment of
endothelial precursor cells. However, recent studies
indicate that vasculogenesis is impaired in a mouse model
of diabetes [113]. Studies of human endothelial precursor
cells isolated from diabetic patients showed impairments
in their rate of proliferation, adhesion to endothelium
and incorporation into vascular structures, suggesting
that alteration in endothelial precursor cell function may
account for impaired growth of the peripheral vasculature
during diabetes [114]. The effects of diabetes on
endothelial precursor cell recruitment and vasculogenesis
in retina have not yet been studied. However, recent
studies in a mouse model of ischemic retinopathy have
shown that bone marrow–derived hematopoietic stem
cells participate in retinal neovascularization [96]. Studies
have also shown that these cells can be used to rescue
and stabilize the degenerating vasculature in a mouse
model of retinal degeneration or to target and inhibit
new vessel formation using a cell-based gene therapy in a
mouse model of retinopathy of prematurity [115]. Thus,
it is possible that alteration in endothelial precursor cell
function could be involved in the capillary dropout stage
of diabetic retinopathy. Further study is needed to directly
test this hypothesis. Unfortunately, however, research in
this area will be seriously limited until good models for
diabetic retinopathy become available.
VEGF autocrine induction of VEGF
expression in activated endothelial
cells
VEGF expression has been described mainly as a
paracrine event. However, the development of new
capillary networks during neovascularization may require
the existence of autocrine growth stimulation. VEGF’s
autocrine ability to stimulate its own production in
the microvascular endothelium has been described in
hypoxia, brain tumors, when the cell-to-cell junctions
are disrupted, or during in vitro angiogenesis induced
by advanced glycation end products [84,116]. The role
of VEGF autocrine expression in diabetic retinopathy
is not yet known, but high glucose and peroxynitrite
both appear to induce VEGF autocrine expression in
cultured retinal endothelial cells [57,88]. Diabetes has
also been found to induce the production of inflammatory
mediators via the cyclooxygenase pathway in the rat retina
[117,118]. Products of arachidonic acid metabolism
via cyclooxygenase (prostaglandins, prostacyclin and
thromboxane) are known to play a role in ocular
inflammation and are able to induce VEGF expression
in vitro [54–56]. VEGF itself has been shown to induce
prostacyclin formation in cultured endothelial cells and
ICAM-1 expression under both in vivo and in vitro
conditions, resulting in amplification of the inflammatory
process [110,119]. It appears likely that the effects of
Copyright  2003 John Wiley & Sons, Ltd.
R. B. Caldwell et al.
diabetes in causing pathological overgrowth of the retinal
microvasculature are due in part to VEGF’s autocrine
actions in triggering VEGF overexpression in retinal
microvascular endothelial cells. Parallel actions of high
glucose/oxidative stress in inhibiting the production of
proteins that normally inhibit VEGF expression may
enhance this autocrine process. This hypothesis is
supported by studies showing that decreased endostatin
levels are correlated with increased VEGF levels in
patients with proliferative diabetic retinopathy [120]
and that increased intraocular expression of endostatin
reduces VEGF-induced retinal vascular permeability and
inhibits retinal neovascularization in transgenic mice with
overexpression of VEGF in the retina [121].
VEGF’s prosurvival actions
VEGF’s function as a survival factor for endothelial cells
has been clearly documented (for review, see [15]).
Paradoxically, even though levels of both VEGF and
VEGFR2 are substantially increased in diabetic retinas,
VEGF’s prosurvival function is compromised as shown by
the fact that retinal capillary dropout occurs at the same
time that expression of VEGF and VEGFR2 is increased
[15,122]. Thus, alteration in VEGF protein expression
does not fully explain disease progression in diabetic
retinopathy. The VEGF prosurvival signaling pathway in
endothelial cells may also be altered during diabetes.
VEGF activation of VEGFR2 is known to result
in the transduction of antiapoptotic signals via the
phosphatidylinositol 3
-kinase/Akt signaling pathway
[123,124]. However, VEGF also stimulates activation of
the stress-activated serine/threonine protein kinase, p38
mitogen activated protein kinase [125] – an important
modulator of the proapoptotic process in various cell
types, including endothelial cells. Blockade of VEGF-
mediated activation of phosphatidylinositol 3
-kinase or
Akt signaling has been shown to result in increased
apoptosis due to potentiation of p38 mitogen activated
protein kinase activation [126]. Studies in retinal
endothelial cells cultured in high glucose medium
or treated with peroxynitrite have shown a similar
phenomenon of accelerated apoptosis even in the
presence of exogenous VEGF [42,127]. This pro-apoptotic
effect is associated with increases in phosphorylation
of P38 mitogen activated protein kinase, decreases
in Akt-kinase activity and tyrosine nitration of the
P85 and P110 subunits of phoshatidylinositol 3
-kinase,
suggesting a key role for peroxynitrite in high glucose
effects impairing endothelial cell survival [42]. Previous
work in vitro has shown that the p85 regulatory
subunit of phoshatidylinositol 3
-kinase is a target for
peroxynitrite-induced protein nitration on tyrosine and
that tyrosine nitration of p85 blocks its interaction
with the p110 catalytic subunit [128], indicating that
peroxynitrite can alter cell survival responses mediated by
phoshatidylinositol 3
-kinase. Observations of increased
tyrosine nitration in retinal proteins of diabetic rats
Diabetes Metab Res Rev 2003; 19: 442–455.
VEGF and Diabetic Retinopathy
suggest that peroxynitrite formation is likely to play an
important role in mediating the suppressive effects of
diabetes on endothelial cell survival [30,36]. Peroxynitrite
may be ultimately responsible for the pathophysiology
previously attributed to nitric oxide alone [129,130].
Therapeutic strategies for preventing
VEGF’s pathological effects
Inhibitors of VEGF and VEGFRs
VEGF and its receptors are good targets for ther-
apeutic intervention in diabetic retinopathy because
(a) VEGF receptors are highly specific and (b) they are
expressed in increased numbers during pathological vas-
cular growth. Several different strategies have been found
effective in preventing retinal neovascularization in mod-
els of ischemic retinopathy, including soluble VEGFR1,
anti-VEGF-neutralizing antibodies, soluble VEGF-receptor
chimeric proteins, antisense oligonucleotides, and inhibi-
tion of a VEGF-specific protein kinase [49,63,131,132].
Two VEGF-directed therapies are currently under eval-
uation in clinical trials for use in the treatment of
angiogenesis in ocular diseases. EYE001 (macugen) is a
VEGF antisense pegylated RNA aptamer (oligonucleotide
that acts like a high-affinity anti-VEGF antibody) that is
now being tested for use in treating neovascularization in
age-related macular degeneration and diabetic retinopa-
thy [132]. Another therapy now being evaluated for use
in macular degeneration involves the use of a monoclonal
antibody fragment directed against VEGF [133]. Both of
these agents are administered by intravitreous injection.
In addition to the above approaches, extensive clinical
research is being done to evaluate the effectiveness
of VEGF inhibitors for preventing tumor angiogenesis.
Phase II/III clinical trials are now in progress testing a
recombinant human anti-VEGF antibody directed against
free VEGF [44,134,135]. This antibody functions by
binding to VEGF and blocking its ability to bind to
its receptor. Another antibody directed against VEGFR2
is now in Phase I trials for treatment of cancer [44].
This monoclonal antibody has been shown to block both
VEGFR-2-mediated permeability and tumor angiogenesis
in mice [136].
An alternative approach has been the use of soluble
VEGF receptors as a decoy to sequester VEGF and
reduce its bioavailablity. Recent studies using either
adenovirus-mediated overexpression of soluble VEGFR1
[137] or liposomes carrying plasmids expressing soluble
VEGFR1 [138] showed decreased tumor angiogenesis
and increased tumor apoptosis. Similarly, studies done
in rats that had been implanted with tumor cells
infected with dominant negative expression of VEGFR-
2 showed impaired angiogenesis and reduced tumor
vascular density [139].
Another technique that is now in Phase I/II clinical
trials for cancer therapy involves the use of ribozymes
Copyright  2003 John Wiley & Sons, Ltd.
449
targeting VEGF mRNA. Ribozymes are naturally occurring
or engineered RNAs with enzymatic activity for degrading
mRNA. The ribozyme, angiozyme, specifically targets
and cleaves VEGF receptor mRNA and has been
shown to inhibit angiogenesis in animal tumor models
[140].
In addition to the above approaches, a number of
small molecules have been synthesized that inhibit
VEGF receptor tyrosine kinase activity. SU5416 inhibits
VEGFR2 and VEGFR1 intracellular signaling and has
had promising clinical results in inhibiting tumor growth
[141]. It also has some activity against platelet-derived
growth factor receptor, the VEGF receptor Flt-4 and c-
kit [142]. ZD4190 is a potent inhibitor of VEGFR1 and
VEGFR2 kinase activity, and VEGF-stimulated endothelial
cell proliferation in vitro. It has also been shown to
inhibit VEGF-induced angiogenesis and to have antitumor
activity in mice [143].
Inhibitors of VEGF expression
Knowledge of the transcriptional events associated with
VEGF expression in diabetic retinopathy is limited.
However, investigators at the National Cancer Institute
have identified a number of small molecule inhibitors
of HIF-1 activity and VEGF expression that are being
developed as inhibitors of tumor angiogenesis [144].
These molecules may also be useful for treatment of
diabetic retinopathy.
A number of inhibitors of hypoxia-inducible factor-
1 have been shown to negatively modulate VEGF
expression. This is the case of the immunosuppressant
rapamycin that has been shown to possess an antian-
giogenic activity by inhibiting VEGF expression [145].
Several studies addressing rapamycin effects on VEGF
expression have shown that it is a negative modula-
tor of the phosphatidylinositol 3
-kinase/Akt-dependent
mTOR (mammalian target of rapamycin) activation. This
leads to a reduction in the transactivating function of
hypoxia-inducible factor-1 [146–148]. Recent studies
conducted with cultured retinal endothelial cells indicated
that the antioxidant N-(2-mercapto-2-methylpropionyl)-
L-cysteine (bucillamine) inhibits VEGF expression by
inhibiting hypoxia-inducible factor-1 transcriptional activ-
ity [149]. Another study in streptozotocin-induced dia-
betic rats showed that bucillamine-blocked diabetes-
induced increases in VEGF expression and vascular per-
meability [150].
Less information is available regarding approaches to
inhibit STAT3 activation of VEGF expression. Because
of the overexpression of VEGF and the angiogenic
requirements for tumor growth, drug discovery pro-
grams are actively searching for peptide inhibitors of
STAT3 [151]. One such inhibitor has been identi-
fied and will soon be available for laboratory research
[152].
Diabetes Metab Res Rev 2003; 19: 442–455.
450
HMG-CoA reductase inhibitors (statins)
Recent studies have indicated that statins possess remark-
able vasoprotective effects in a wide variety of pathologi-
cal conditions including diabetes [153]. Statins’ protective
actions are exerted mainly on the microvasculature and
appear to be independent of their lipid lowering proper-
ties and rather to be the result of their antiinflammatory
and antioxidant properties as well as their actions as
inducers and agonists of nitric oxide synthase. Statins’
pleiotropic effects are due in part to their molecular action
as inhibitors of the HMG-CoA reductase, which catalyzes
the synthesis of L-mevalonate. L-mevalonate is a precursor
of cholesterol and isoprenoid intermediates such as farne-
syl pyrophosphate and geranyl–geranyl pyrophosphate.
Isoprenoids are important lipid moieties needed for the
posttranslational modification of a variety of small G pro-
teins (Ras-like proteins) such as Rho and Rac-1 [154].
Statin-mediated inhibition of Rho leads to upregulation
of endothelial nitric oxide synthase (NOS3) activation
of the phosphatidylinositol 3
-kinase/Akt pathway and
increased nitric oxide production and statin-induced inhi-
bition of Rac-1 results in blockade of NAD(P)H oxidase
activity [155]. Statins have also been shown to down-
regulate VEGF expression induced by advanced glycation
end products and to inhibit angiogenesis associated with
tumors [156–158]. These contrasting angiogenic and
angiostatic properties have been explained by in vitro
studies showing a biphasic, dose-dependent effect of
statins [158,159]. However, several studies in experimen-
tal animals and in patients indicate that statins suppress
pathological angiogenesis at clinically relevant doses that
have been shown to induce angiogenesis in models of
acute ischemia [160,161].
Finally, statins have been shown to inhibit angiotensin-
induced activation of STAT3 in vitro [160]. If this process
has a role in VEGF overexpression and the associated
alterations in vascular function and growth during
diabetes, statin therapy may be useful in treating diabetic
retinopathy. Anecdotal evidence indicates that lipid
exudation associated with breakdown of the blood-retinal
barrier is reduced in diabetic patients treated with statins
[162]. However, the number of patients studied was
small. Randomized clinical trials are needed to evaluate
the efficacy of statins as therapy for diabetic retinopathy.
Because the statins are well known drugs whose efficacy,
side effects and toxicity have been well characterized in
more than ten years of clinical use, studies determining
their efficacy in preventing retinal neovascularization and
diabetic retinopathy will be important.
Cyclooxygenase-2 inhibitors
Retinas of diabetic rats as well as diabetic patients exhibit
inflammation associated alterations, such as vasodilata-
tion, vascular leakage, platelet aggregation and leukocyte
adhesion [163]. High glucose treatment of cultured retinal
endothelial cells has been shown to induce the production
Copyright  2003 John Wiley & Sons, Ltd.
R. B. Caldwell et al.
of prostenoid mediators of inflammation via the cyclooxy-
genase pathway [131,164]. High-dose aspirin (nonselec-
tive inhibitor of cyclooxygenase-1 and cyclooxygenase-2)
has been reported to reduce the incidence of diabetic
retinopathy in patients [165] and to prevent the devel-
opment of retinal vascular changes in diabetic dogs
[166]. Because retinal expression of cyclooxygenase-2,
but not cyclooxygenase-1 is elevated during diabetes, it
is considered likely that cyclooxygenase-2 activity may
be responsible for the aspirin effect [167]. Studies in
the streptozotocin-induced diabetic rat model showing
that cyclooxygenase-2 inhibitor treatment inhibits reti-
nal VEGF expression and vascular leakage support this
hypothesis [117]. Further work is needed to determine
the effects of selective inhibitors of cyclooxygenase-2 in
preventing diabetic retinopathy in patients.
Antioxidants
The role of oxidative stress in diabetic vascular
dysfunction and VEGF overexpression is well established.
Production of superoxide anion can result from various
mechanisms active in diabetes, including activation
of the mitochondrial electron-transport chain, glucose
auto-oxidation, protein glycation, increased glucose flux
through the polyol pathway, and prostanoid production
[22,168]. In this regard, normalization of glucose-
stimulated superoxide production has been found to block
at least three independent pathways of hyperglycemia-
induced vascular damage [22]. Studies showing that
VEGF itself induces formation of superoxide anion in
cultured endothelial cells and that superoxide anion
formation is required for VEGF’s angiogenic actions
[76] suggest that superoxide inhibitors/scavengers may
also improve diabetic retinopathy by inhibiting the
VEGF intracellular signaling pathway. Furthermore,
antioxidants such as vitamin E have been shown to prevent
some of the vascular dysfunctions associated with diabetes
in animal models [169]. In one study of diabetic patients
who had minimal or no retinopathy, treatment with high-
dose vitamin E for four months was found to significantly
reverse diabetes-induced alterations in retinal blood flow
[170].
Another potential target for antioxidant therapy
is peroxynitrite. Diabetes- and high-glucose-induced
vascular dysfunction has long been thought to involve
the inactivation of endothelium-derived nitric oxide by its
combination with superoxide anion to form peroxynitrite.
This concept has been supported by research showing
nitrotyrosine formation in the plasma of diabetic patients
[32] and in blood vessels and retinas from diabetic
rats [30,171–173]. Moreover, studies in diabetic rats
have also shown that increases in retinal nitrotyrosine
levels and lipid peroxidation are correlated with VEGF
overexpression and breakdown of the blood-retinal
barrier and that all of these alterations can be prevented
by treatment with the peroxynitrite scavenger uric acid
or the nitric oxide synthase inhibitor L-NAME [30].
Diabetes Metab Res Rev 2003; 19: 442–455.
VEGF and Diabetic Retinopathy
Preclinical studies in the mouse model for oxygen-
induced retinopathy provide further support for the role
of peroxynitrite in retinal neovascular disease. These
studies have shown that increased peroxynitrite formation
is associated with retinal capillary dropout and that
NOS3 deletion or treatment with a nitric oxide synthase
inhibitor diminishes VEGF expression and inhibits retinal
neovascularization [174,175].
Conclusion
For many years, diabetic retinopathy has been the major
cause of new adult blindness in the industrialized world.
Recent improvements in the care and management
of diabetic patients have decreased the incidence and
severity of diabetes complications in the retina as
well as other organ systems and advances in the
use of laser photocoagulation therapy have improved
the visual outcome for many of the patients that
do develop diabetic retinopathy. Nevertheless, many
patients still develop diabetic retinopathy in spite of
good diabetes control. Moreover, laser therapy maintains
vision rather than recovering lost vision and can
itself lead to additional visual loss. Therefore, there
is a great need for new therapies that are safe and
effective for preventing and treating diabetes retinal
complications.
A number of promising therapies are currently under
investigation in clinical trials, including inhibitors of
angiotensin converting enzyme activity, angiotensin
receptor blockers and inhibitors of the protein kinase
C beta isoform. Recently, a wealth of clinical and
experimental evidence has accumulated to demonstrate
that overexpression of VEGF has a key role in diabetes-
induced retinal vascular dysfunction. Therefore, the
development of agents that directly target VEGF and
its receptors is an area of active clinical research.
New therapeutic strategies not yet in clinical trials
but supported by promising preclinical data involve
the use of pharmacological approaches to block VEGF
overexpression or prevent its adverse effects on vascular
permeability and growth. These include various inhibitors
of VEGF transcriptional activation, the statin HMG-
CoA reductase inhibitors, cyclooxygenase-2 inhibitors and
antioxidant therapies.
A new area that is just beginning to be considered
for potential use in the treatment of diabetes-induced
vascular disease involves the use of endothelial progenitor
cells. While the specific role of endothelial progenitor
cells in diabetic retinopathy is not yet clear, the
results of initial studies in other models of ischemic
retinopathy have supported their role in pathological
angiogenesis and suggested a potential use as tools for
drug delivery in the treatment of retinal vascular disease.
Further investigation is needed to establish the role
of endothelial progenitor cells in ischemic retinopathy
and to determine their involvement in diabetes retinal
complications.
Copyright  2003 John Wiley & Sons, Ltd.
451
Acknowledgements
NIH-R01-EY04618, NIH-R01-EY11766, American Heart Associ-
ation, and unrestricted research award to the Department of
Ophthalmology from Research to Prevent Blindness
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