angiogenesis and antiangiogenesis in the neonate relevance to retinopathy of prematurity

8/10/2019 Angiogenesis and Antiangiogenesis in the Neonate Relevance to Retinopathy of Prematurity

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Angiogenesis and Antiangiogenesisin the Neonate: Relevance to Retinopathyof PrematurityAshima Madan, MD*

Objectives After completing this article, readers should be able to:1. Delineate the major stimulus for growth of retinal blood vessels by angiogenesis in

utero.

2. Describe the pathogenesis of retinal neovascularization in preterm infants.

3. List the angiogenic factors that regulate growth of new blood vessels.

4. List potential antiangiogenesis therapies.

IntroductionThe molecular mechanisms underlying new blood vessel growth currently are being

investigated in several laboratories. Angiogenesis or neovascularization has been impli-

cated in various unrelated disease processes, such as retinopathy of prematurity (ROP),diabetic retinopathy, choroidal neovascularization, macular degeneration, and tumor

angiogenesis. On the other hand, growth of new blood vessels is desired and beneficial in

wound healing and myocardial and limb ischemia. All of these unrelated conditions likely

share several common mechanisms in the final pathway that culminates in angiogenesis.

Identification of a number of growth factors along with the ability to manipulate the

mouse embryo genetically has resulted in increased understanding of the molecular

mechanisms regulating angiogenesis. This review summarizes current knowledge of the

factors regulating new blood vessel growth and its relevance to retinal neovascularization

in preterm infants.

Vascularization of the Retina and Retinopathy of PrematurityThe outer layers of the retina are supplied by the choroid plexus, which lies between theoutermost layer of the retina and the retinal pigment epithelium (RPE). The inner layers

are supplied by a superficial plexus beneath the inner limiting membrane and a deep plexus

in the inner nuclear layer (Figs 1 and 2). Vascularization of the retina in utero, where

arterial oxygen tension is less than 30 mm Hg, occurs by a process of vasculogenesis and

angiogenesis. Vasculogenesis, the de novo formation of capillaries from endothelial cells

that have differentiated from mesodermal precursors at approximately 16 weeks gestation

in the posterior region around the optic disc, is seen only during embryonic development.

Angiogenesis, the formation of blood vessels from existing blood vessels, begins around

25 weeks gestation in the foveal region and is responsible for increasing vascular density of

the superficial plexus and formation of the deep plexus.

Newly formed blood vessels spread across the surface of the retina following the central

peripheral gradient of retinal ganglion cell maturation toward the peripheral retina. Bloodvessels initially develop in the superficial plexus, followed by growth radially outward into

the deep plexus only as far as the junction between the inner nuclear and peripheral layers

(Fig. 2). Physiologic hypoxia, created by the increased metabolic demands of the fetal

retina, leads to the release of vascular endothelial growth factor (VEGF) from neural

cellsthe Muller cells in the inner nuclear layer and the astrocytes in the ganglion cell

layer. VEGF acts in a paracrine manner and is the major stimulus for the growth of retinal

blood vessels by angiogenesis in utero.

Several lines of evidence indicate that vasculogenesis in the human retina is independent

of metabolic demand and hypoxia-induced VEGF expression. First, VEGF expression has

not been detected in the human retina before 20 weeks gestation. Second, neuronal

*Associate Professor of Pediatrics, Stanford University School of Medicine, Stanford, CA.

Article ophthalmology

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maturation is greatest around 15 to 18 weeksgestation

in the perifoveal region, which is avascular at this stage of

gestation. Third, although VEGF knockout mice form

vessels by vasculogenesis, such vessels are very abnormal.

Although there is considerable variation in the time

course of retinal vascularization among infants, approxi-mately 70% of the retina is vascularized at 27 weeks

gestation; complete vascularization occurs by 36 weeks

gestation on the nasal side and by 40 weeks gestation on

the temporal side. The susceptibility of preterm infants of

a younger gestational age to retinopathy compared with

more mature infants is due to the presence of a larger

region of avascular retina. The question of why the retina

exclusively is affected by administered oxygen has per-

plexed scientists. It has been hypothesized to be due to

the unique relationship between the retinal and choroi-

dal blood vessels as well as the unusual properties of the

choroidal vasculature.The pathogenesis of retinal neovascularization in pre-

term infants occurs in two phases. In the first phase,

hyperoxia produces vasoconstriction and vaso-

obliteration of developing retinal blood vessels. This

phase likely represents an exaggerated natural protective

response to a surplus of oxygen. The second phase is

initiated with concomitant development of the photore-

ceptor retinal layer with advancing postnatal age and the

resulting increase in oxygen requirements. Photorecep-

tors have a high metabolic demand because they need to

resynthesize photosensitive pigment continuously, con-

stantly rebuild their cilia, and maintain membrane mech-

anisms for neural signaling. It is interesting to note that

retinal consumption of oxygen is much higher in the dark

than in the light. Retinal hypoxia, created by the inability

of the vaso-obliterated blood vessels to meet the meta-

bolic demands of the retina, results in the release of

angiogenic growth factors and subsequent revasculariza-tion. However, this exuberant blood vessel growth is

pathologic; the blood vessels tend to be leaky and friable,

resulting in hemorrhage and exudation followed byfi-

broplasia and ultimately retinal detachment and loss of

vision. Although it has been suggested that the fibropla-

sia may be caused by either invasion of the vitreous by

mesenchymal precursors or the effect of fibroblast

growth factor, the cause offibroplasia in ROP remains

unknown.

Angiogenesis

Angiogenesis is a complex process that begins with mi-gration of inflammatory cells toward the hypoxic or

inflammatory signal and the release of angiogenic factors

(Table 1). Endothelial cells release proteolytic enzymes

such as matrix metalloproteinases that disrupt the vascu-

lar basement membrane and extracellular matrix. Vascu-

lar adhesion molecules enhance adhesion of the endothe-

lial cells to the extracellular matrix. The endothelial cells

then migrate in the form of a solid column called the

vascular sprout toward the angiogenic stimulus. By pro-

ducing proteolytic activity, the advancing cell column

migrates through the extracellular matrix. Behind the

advancing region, cells begin to differentiate, adhere to

Table 1.Angiogenic Factors Regulating Growth of New Blood Vessels

Angiogenic Factors Function

Vascular endothelial growth factor (VEGF) Endothelial cell (EC)-specific mitogen, prevents apoptosisBasic fibroblast growth factor, hepatocyte growth

factor (HGF), tumor necrosis factor, nuclearfactor-kappaB, interleukin-8

Increase proliferation of endothelial cells, potentiate effect ofVEGF (HGF)

Placental growth factor-1 Selective agonist for Flt-1Hypoxia-inducible factor-1 alpha Transcription factor, upregulates several angiogenic factors in

response to hypoxiaMetalloproteinases Proteases that degrade endothelial cell matrix (ECM)Alpha v beta 3 and alpha v beta 5 integrins Vascular adhesion molecules mediating binding of EC to ECME-Selectin Mediates cell-to-cell contactPlatelet-derived growth factor Recruits pericytesTransforming growth factor-beta Stabilizes blood vessels by induction of pericyte

differentiation, basement membrane productionFlk-1, Flt-1, neuropilin VEGF tyrosine kinase (TR) receptors, initiate signaltransduction cascade

Tie1 and Tie2 TR receptors for angiopoietinsAngiopoietins Ligands for Tie2, remodelling and stabilization of vessels

ophthalmology angiogenesis

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one another, form a new basement membrane, and de-

velop a lumen to form a new capillary. Anastomosis of

adjacent sprouting vessels establishes the vascular supply

to the region. Formation of the early vascular network is

followed by several maturation steps involving attraction

of smooth muscle cells and pericytes around the endo-

thelium as well as vascular remodeling involving changes

in lumen diameter and vessel wall thickness to accommo-

date the needs of the local tissue.

Angiogenesis is a staged process that is tightly con-

trolled by a balance between angiogenic and angiostatic

factors. The endothelial cell growth factor family consists

of the vascular endothelial growth factor and angiopoi-

etin groups. All factors in this complex pathway must

function in coordination to form functional vessels.

Vascular Endothelial Growth FactorVEGF is the key growth factor driving ocular blood

vessel growth. Both Michaelson and Ashton had pro-

posed in the early 1950s that an angiogenic factor was

released from the retina. However, it was not until the

1980s that VEGF was identified as the key molecule in

angiogenesis. VEGF-A exists in at least four different

isoformsVEGF 121, VEGF 165, VEGF 189, and

VEGF 206 that are produced by alternate splicing of

the VEGF mRNA. VEGF binds to tyrosine kinase cell

surface receptors, VEGF receptor-1 or Flt-1, and VEGFreceptor-2 or Flk-1. Neuropilin-1, a neuronal cell recep-

tor that mediates neuronal guidance, has been identified

as a coreceptor for VEGF and is critically involved in

vascular development.

The distribution of Flt-1 and Flk-1 receptors differs

markedly during normal retinal development, and the

receptors have different roles in endothelial cell prolifer-

ation and differentiation. In situ hybridization and im-

munohistochemistry experiments in mice that are 5 days

postpartum have shown that the Flt-1 protein colocalizes

with retinal vessels, with Flk-1 detectable only in the

neural retina. These studies showed a 60-fold inductionof Flt-1 from postnatal day 3 to day 26, but no significant

change in Flk-1 expression. Mice deficient in Flt-1 and

Flk-1 died in utero between postnatal days 8.5 and 9.5.

Flt-1 knockout mice developed disordered vascularity

due to overgrowth of endothelial cells; mice that had

targeted disruption of the Flk-1 gene failed to develop a

vasculature and had very few endothelial cells.

VEGF gene expression is regulated by oxygen ten-

sion. Expression increases in response to hypoxia and is

downregulated by hyperoxia. The mechanism of

hypoxia-inducible expression is mediated at least partially

via hypoxia-inducible factor-1 alpha (HIF-1-alpha), a

transcription factor that transactivates several hypoxia-

inducible genes.

Several lines of evidence indicate that VEGF is the key

molecule involved in development of normal blood ves-

sels as well as in retinopathy. First, knockout mice that

have disruption of even one allele of the VEGF gene have

impaired blood vessel formation. On the other hand,

transgenic mice that have overexpression of VEGF in the

photoreceptor layer develop intraretinal and subretinal

neovascularization. Increased expression of VEGF

mRNA is seen anterior to the developing blood vessels

on postnatal day 7 in the normal mouse retina (retinal

vascularization in mice occurs after birth and is complete

by 14 days). Second, VEGF levels are increased in the

vitreous of patients who have diabetic retinopathy and

other neovascular disorders of the eye as well as in a

primate model of iris neovascularization. In situ hybrid-

ization experiments have localized the site of production

of VEGF during the process of neovascularization to the

inner nuclear layer of the retina. The attenuation of

blood vessels that is seen in the first phase of hyperoxia-

induced retinal vessel loss can be inhibited by intravitreal

administration of exogenous VEGF in animal models.

Conversely, the increase in VEGF expression and neo-

vascularization seen in the second phase of retinopathy

can be inhibited in the mouse model by use of antisense

oligonucleotides, receptor binding chimeric proteins,and monoclonal antibodies. Thus, VEGF is not just a

mitogen; it also acts as an endothelial cell survival factor

by preventing apoptosis of endothelial cells. This survival

effect appears to be developmentally regulated. VEGF is

not required for the maintenance of blood vessels in the

adult vasculature.

Current knowledge suggests that under normoxic

conditions, VEGF is produced at a maintenance level

adequate to support existing blood vessels. During hyp-

oxia, VEGF levels increase above this level to induce

growth of new blood vessels. Under hyperoxic condi-

tions, VEGF levels decrease below the maintenance leveland lead to attenuation of existing blood vessels.

Angiogenic FactorsOther angiogenic factors, such asfibroblast growth fac-

tor (FGF), transforming growth factors (TGF) alpha and

beta, hepatocyte growth factor, tumor necrosis factor-

alpha, and interleukin-8 (IL-8), also are involved in

angiogenesis. Although basic fibroblast growth factor

(bFGF, or FGF2) previously was implicated in ocular

neovascularization, FGF2-deficient mice developed the

same degree of neovascularization as control animals in

the oxygen-induced mouse model of retinopathy. IL-8




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levels are increased in the vitreous fluid of patients who

have proliferative diabetic retinopathy. Nuclear factor-

kappaB (NF-kappaB) and a rat homolog of IL-8 are

increased in endothelial and glial cells in oxygen-induced

retinopathy. Knockout mice lacking the hypoxia-

inducible factor-2 alpha gene (HIF-2 alpha) do not

develop neovascularization when exposed to the

hyperoxia/normoxia paradigm used to generate mice

that have retinopathy. These mice have decreased levels

of erythropoietin (Epo), indicating a role for Epo in

experimental ROP.

ProteolysisBinding of VEGF to the receptor is followed by degra-

dation of the endothelial cell basement membrane by

proteases that degrade collagen and other extracellular

matrix components and disrupt the basement membrane

barrier, thus enabling endothelial cells to migrate from

the vessels and proliferate. Expression of protease genes

is induced by cytokines and angiogenic growth factors

such as bFGF and VEGF-A. The matrix metalloprotein-

ases (MMP) are a family of proteases that selectively

degrade extracellular matrix. They consist of secreted

and membrane-associated endopeptidases that act on

various substrates. The endothelial sprout must traverse

several types of extracellular matrixes during the process

of growth: first the basement membrane, then thefibrinogen-rich provisional matrix created by leakiness of

the surrounding vessels, and finally the collagen and

fibronectin-rich intersitial matrix. MMP activity helps in

this process. The most convincing evidence for the role

of MMP in angiogenesis is that use of MMP inhibitors

inhibits in vivo and in vitro angiogenic responses. Also,

MMP-deficient mice have a delayed angiogenic response

during development. Although there is consensus that

MMP activity clearly is implicated in angiogenesis, their

precise role in this process and the details of their inter-

action with other endothelial cell functions (see endothe-

lial cell adhesion) is not yet defined completely. Someevidence suggests that MMPs may help to produce an-

giogenesis inhibitors such as angiostatin. Thus, MMP

activity possibly has a dual role (facilitating angiogenesis

and releasing angiogenesis inhibitors) in new blood ves-

sel growth.

Endothelial Cell Adhesion and MigrationVascular cell adhesion molecules such as integrins alpha v

beta 3 and alpha v beta 5 are critical to the formation and

maintenance of the newly formed blood vessel. The

integrins are a family of heterodimeric transmembrane

receptors consisting of an alpha and beta subunit that

each recognizes a unique set of extracellular matrix li-

gands. They assist in the migration of endothelial cells by

mediating binding of the endothelial cell to the extracel-

lular matrix. Integrin-mediated adhesion initiates several

intracellular signaling events that regulate cell survival

and division. Inhibition of either matrix hydrolysis or

integrin/ligand interaction can inhibit normal and

pathologic retinal blood vessel formation. Integrin alpha

v beta 3 is a receptor for a wide variety of extracellular

ligands and is expressed at high levels on activated endo-

thelial cells. Studies suggest that alpha v beta 3 can bind

MMP-2 and localize the active form of this enzyme on

the surface of angiogenic vessels, thus enabling the en-

dothelial cells to degrade the extracellular matrix during

invasion. However, the relationship between endothelial

cells and the extracellular matrix is complex, and further

studies are needed to elucidate the signaling pathway

induced by interaction between vascular adhesion mole-

cules and endothelial cells.

It has been difficult to reconcile the results of studies

using monoclonal antibodies to block alpha v beta 3 or

alpha v beta 5 integrins with the results of knockout mice

experiments with integrin-deficient mice. Inhibition of

integrin function with monoclonal antibodies prevented

angiogenesis in animal models. Conversely, in knockout

mice experiments, beta 3-negative mice contained nor-

mal brain and intestinal blood vessels, suggesting thatbeta 3 is not essential for angiogenesis. Only 20% of alpha

v integrin knockout mice survive to term, but these

animals die within a few hours due to brain and intestinal

blood vessel abnormalities and hemorrhages, indicating

that the alpha v unit is important for angiogenesis. One

possibility is that other adhesion molecules in the knock-

out mouse model,such as alpha v beta 5,may takeover in

the absence of alpha v integrins. However, this has not

been confirmed. Another possibility is that monoclonal

antibodies also directly inhibit the effect of other inte-

grins.

Migration and proliferation of cells is followed byformation of tubes that have a patent lumen. E-selectin,

a glycoprotein that mediates endothelial cell-cell con-

tacts, is important for lumen formation.

Remodeling and Stabilization of BloodVesselsThe final steps in the process involve remodeling and

stabilization of the blood vessels by recruitment of mes-

enchymal cells and differentiation to pericytes. Platelet-

derived growth factor (PDGF), which exists in three

isoforms, plays a role in pericyte recruitment. PDGF-B-

and PDGF receptor-deficient mice do not have pericytes




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in their blood vessels and develop microaneurysms, thus

suggesting that endothelial cells of the vascular sprouts

are unable to recruit local mural cells in the absence of

PDGF. Transforming growth factor-beta (TGF-beta) is

one of a large family of growth factors that is implicated

in cellular growth and differentiation. TGF-beta-

deficient embryos have delayed-to-absent vasculogenesis

as well as inadequate contact between endothelial and

mesothelial cells. It is speculated that TGF-beta acts at

multiple steps to stabilize blood vessel formation via

several actions that include inhibition of endothelial cell

proliferation and migration as well as induction of peri-

cyte differentiation. TGF also can alter the integrin pro-

files and stimulate basement membrane production.

Angiopoietins and Tyrosine Kinase ReceptorsIn addition to the receptors for VEGF, the tyrosine

kinase immunoglobulin and epidermal growth factor-

like extracellular domains (Tie) receptors, Tie1 and Tie2,

which are expressed by endothelial cells, are another

group of tyrosine kinase receptors that play a critical role

in embryonic angiogenesis. Both Tie1 and Tie2 are

required for structural integrity of endothelial cells; mice

deficient in Tie1 die during the perinatal period due to

respiratory difficulties. Tie2-deficient mouse embryos do

not survive beyond embryogenesis day 9.5 to 10.5 and

have prominent abnormalities in the vasculature.The angiopoietins are ligands for Tie2. Angiopoietin-1

(Ang-1) is associated with developing blood vessels. This

activating ligand induces tyrosine phosphorylation of

Tie2 in endothelial cells, sprouting of capillaries, and

survival of endothelial cells. Disruption of the Ang-1

gene in mouse embryos produces a phenotype that is very

similar to Tie2-deficient mice. These animals develop a

vasculature but have disordered remodeling of blood

vessels and lack periendothelial supporting cells. In dia-

betic retinopathy, it is possible that Ang-1 could be used

in the future as an agent to prevents leakiness of blood

vessels. Ang-2, a natural antagonist of Ang-1, is a desta-bilizing factor that promotes differentiation of endothe-

lial cells by blocking Ang-1 activation of Tie2. In normal

mice, retinal vascularization occurs postnatally in con-

junction with regression of the hyaloid vessels encasing

the lens. Ang-2-deficient mice do not develop retinal

vascularization or regression of hyaloid vessels, thus sup-

porting the role of Ang-2 as a destabilizing agent. Ang-2

mRNA expression is increased in the inner nuclear layer

and ganglion cell layer in the oxygen-induced mouse

model of retinopathy, indicating that it may play a role in

ROP.

Tie1 signal transduction has been difficult to study

because no ligand has been identified for this receptor. It

is speculated that Tie1 may be involved in ligand-

independent signaling or may form a heterodimer with

Tie2 and modulate Tie2 signaling.

AntiangiogenesisResearch in the early 1970s showed that formation of

new blood vessels was important for tumor growth and

progression. Since then, antiangiogenic therapy aimed at

prevention of endothelial cell migration and induction of

apoptosis has been tried as a treatment for cancer. Several

early trials showed no benefit. However, an increased

understanding of the angiogenic processes and factors

involved has led to newer strategies that target either:

1) proangiogenic factors, their receptors, or downstream

signaling molecules; 2) increased expression of endoge-

nous inhibitors or administration of exogenous inhibi-

tors; or 3) the new blood vessels directly. One of the

challenges is the designing of appropriate assays to mon-

itor the effect of the antiangiogenic agents.

Several inhibitors of tumor angiogenesis have been

described (Table 2). Some are naturally occurring mole-

cules, such as angiostatin, endostatin, platelet factor-4,

thrombospondin-1, interferon-alpha, troponin I, and

tissue inhibitors of metalloproteinases (TIMP).

Antiangiogenic therapy has been extended to diseases

causing intraocular neovascularization, particularly dia-betic retinopathy. Exogenous inhibitors have been tested

most commonly in the mouse model of retinopathy.

Studies in animals have shown that alpha v integrin

antagonist peptides and antibodies produce a reduction

in retinal neovascularization as well as inhibition of an-

giogenesis in tumors and in arthritis. The humanized

Table 2.Endogenous Inhibitors ofAngiogenesis

StatinsAngiostatin (inhibits EC proliferation and migration

by binding ATPase and annexin II on endothelialcells)

Endostatin (inhibits EC proliferation by binding tointegrin)

Platelet factor-4 (blocks binding of FGF to EC) Thrombospondin (modulates EC proliferation and

migration) Interferon-alpha (downregulates action of VEGF) TIMP family (inhibition of metalloproteinases)

EC endothelial cell, FGF fibroblast growth factor, VEGF vascular endothelial growth factor, TIMP tissue inhibitors ofmetalloproteinases




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form of this antibody is being tested in clinical trials.

Retinal neovascularization was inhibited in transgenic

mice expressing a growth hormone antagonist gene, and

the degree of inhibition was proportional to serum levels

of growth hormone as well as insulinlike growth factor-1

(IGF-1). This inhibition was decreased by administration

of exogenous IGF-1, suggesting a potential therapy for

intraocular neovascularization. Transgenic mice deficient

for the endothelial nitric oxide synthase (eNOS) gene

have decreased hyperoxia-induced attenuation of the

retinal vasculature compared with normal controls,

which suggests a possible therapeutic role for inhibitors

of eNOS activity in the future. Angiotensin II also has

been shown to play a possible role in induction of neo-

vascularization in the oxygen-induced mouse model of

retinopathy. Administration of perindopril, an

angiotensin-converting enzyme inhibitor, to mice de-

creased the number of endothelial cells in the retinas of

treated versus untreated animals.

Increased understanding of endothelial cell physiol-

ogy and identification of inhibitors has led to an exten-

sion of antiangiogenesis research from the bench to the

bedside. This exciting, novel approach is being tested in

several clinical trials for diabetic retinopathy that is in

phase II /III. Some of the antiangiogenic strategies that

are undergoing testing in clinical trials for ocular disease

include VEGF-A inhibitors such as anti-VEGF aptamer,humanized anti-VEGF Fab monoclonal antibody (rhu-

Fab), thalidomide, angiostatic steroids, protein kinase

C-beta inhibitor (LY333531), growth hormone antago-

nists such as long-acting somatostatin, and integrin an-

tagonists. Treatment of ocular angiogenesis depends on

adequate transfer of the drug to the back of the eye,

where pathologic neovascularization typically occurs.

This can be achieved by using eye drops or intravitreal

injections. Gene transfer to the eye using a safe viral

vector that constitutively expresses an antiangiogenic

protein or by using a liposome expression vector is an-

other promising technology that is being tested in clini-cal trials in patients who have myocardial ischemia or

limb ischemia.

Possible Role of Antiangiogenesis in ROPTo date, there are no trials of antiangiogenic agents in

ROP, and several unique features of ROP must be ad-

dressed before they can be undertaken. First, the blood

vessels in ROP are immature, and unlike mature blood

vessels, they are VEGF-dependent. Second, unlike in

neovascularizing diseases affecting adults, a large part of

the peripheral retina in infants is avascular. Therefore,

antiangiogenic agents must be selective for abnormal

vessels. Third, because neovascularization typically oc-

curs at34 to36 weeks gestation, therapy can be targeted

to a particular duration. With VEGF levels decreased in

the first phase of ROP and increased in the second phase,

two different strategies potentially can be used in this

population. The first is administration of VEGF or a

VEGF receptor agonist to rescue the vessels during the

hyperoxic phase. The second is to antagonize selectively

VEGF or downstream signaling molecules in abnormal

vessels seen in the second phase of retinopathy. One of

the concerns with the first approach is that VEGF also

can stimulate abnormal vessel proliferation and increased

permeability in ROP. Recent work by Shih and associates

has shown that selective use of placental growth factor I

(PGFI), a selective agonist for VEGF receptor-1, pro-

tects the retinal vessels from hyperoxia-induced obliter-

ation without any concomitant increase in retinal neovas-

cularization during normal retinal vascular development.

A similar vasoprotective effect of PGFI was shown in

oxygen-damaged retinal vessels. The exact mechanism

underlying this selective effect on pathologic angiogen-

esis while sparing normal blood vessel growth is un-

known. The signal transduction pathway resulting in

vasoprotection by VEGF or PGFI is yet to be identified.

Phosphorylation of the survival kinase Akt1 and inhibi-

tion of apoptosis along with increased expression of

antiapoptotic genes has been shown to be important.

Use of Supplemental Oxygen for Preventionof Threshold ROPPhelps and colleagues employed the kitten model of

oxygen-induced retinopathy to show that use of supple-

mental oxygen (28%) in animals recovering from expo-

sure to 80% oxygen decreased the degree of neovascular-

ization compared with animals recovering in normoxia.

This effect possibly is mediated by a decrease in VEGF

levels. The Supplemental Therapeutic Oxygen for Pre-

threshold ROP (STOP-ROP) was a randomized trial

conducted to test this hypothesis. Infants who had pre-threshold ROP and were unable to maintain an oxygen

saturation of 94% in room air were randomized to either

the control (O2saturations of 88% to 94%) or the treat-

ment group (O2saturations of 96% to 99%). No statisti-

cally significant difference was noted in the rate of con-

version to threshold ROP between the two groups,

indicating the lack of an adverse effect of oxygen in active

prethreshold ROP. However, infants in the supplemen-

tal group had a much higher incidence of adverse pulmo-

nary events, such as pneumonia and exacerbation of

pulmonary disease. Although the current data do not

support the use of supplemental oxygen for prethreshold




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ROP, it may have a role in selected cases of ROP. A

subgroup analysis suggested a benefit among infants who

did not have plus disease. In addition, infants who were

excluded from the study by virtue of maintaining O2saturations less than 94% in room air (ie, those who had

less severe pulmonary disease) had a much lower inci-

dence of progression to threshold ROP than those en-

rolled in the STOP-ROP study. Further studies are re-

quired to define more clearly the effect of oxygen on

retinal neovascularization in preterm infants.

ConclusionSeveral exciting new developments have expanded un-

derstanding of the mechanisms underlying angiogenesis.Continued identification and characterization of molec-

ular factors that regulate blood vessel growth hold con-

siderable promise for the future of antiangiogenesis as a

therapeutic approach for ROP.

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NeoReviews Quiz

8. Vascularization of the retina during fetal development includes vasculogenesis (the de novo formation ofcapillaries from endothelial cells) followed by angiogenesis (the formation of blood vessels from existingblood vessels). Vascular endothelial growth factor (VEGF) plays a key role in retinal vascularization, bothduring normal development and in the pathogenesis of retinopathy of prematurity. Of the following, themost accurate statement regarding retinal vascularization is that:

A. Angiogenesis begins around 16 weeks of gestational age.B. Consumption of oxygen is much higher in the retina in the dark than in the light.C. Vascularization of the retina is complete by 36 weeks of gestational age.D. Vasculogenesis is dependent on hypoxia-induced VEGF.E. VEGF is produced by endothelial cells in the retina.

9. Angiogenesis is a complex process that involves several angiogenic factors that have specific putativeroles. Of the following, the angiogenic factor mostinvolved in the recruitment of pericytes in remodelingand stabilization of blood vessels is:

A. Fibroblast growth factor.B. Interleukin-8.C. Platelet-derived growth factor.

D. Tumor necrosis factor-alpha.E. Vascular endothelial growth factor.

10. Retinopathy of prematurity is characterized by two phases: an initial phase of vasoconstriction and vaso-obliteration of developing retinal blood vessels and a subsequent phase of hypoxia-induced abnormalneovascularization. An angiogenic factor that protects against both vaso-obliteration andneovascularization would be useful in the treatment of retinopathy of prematurity. Of the following, theangiogenic factor that has the mostpromise as a potential treatment of retinopathy of prematurity is:

A. Fibroblast growth factor.B. Placental growth factor.C. Platelet-derived growth factor.D. Transforming growth factor.E. Vascular endothelial growth factor.



angiogenesis and antiangiogenesis in the neonate relevance to retinopathy of prematurity

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