g#- 'Ns,?i NIH Public Access
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`Pub Ii shed in final edited fonn as:
`ProgRetinEyeRes. 2008 July~ 27(4): 331-371. doi:10.1016/j.preteyeres.2008.05.001.
`
`Vascular Endothelial Growth Factor in Eye Disease
`
`J.S. Penna, A. Madanb, R.B. Caldwellc, M. Bartolic, R.W. Caldwellc, and M.E. Hartnettd
`avanderbilt University School of Medicine, Nashville, TN
`
`bStanford University School of Medicine, Palo Alto, CA
`
`cMedical College of Georgia, Augusta , GA
`
`dUniversity of North Carolina School of Medicine, Chapel Hill, NC
`
`Abstract
`Collectjvely, angiogenic ocular conditions represent the leading cause of irreversible vision loss in
`developed countries. In the U.S., for example, retinopathy of prematurity, diabetjc retinopathy and
`age-related macular degeneration are the principal causes of blindness in the infant, working age
`and elderly populations, respectively. Evidence suggests that vascular endothelial growth factor
`(VEGF), a 40 k:Da dimeric glycoprotein, promotes angiogenesis in each of these conditions,
`making it a highly significant therapeutic target. However, VEGF is pleiotropic, affecting a broad
`spectrum of endothelial, neuronal and glial behaviors, and confounding the validity of anti-VEGF
`strategies, particularly under chronic disease conditions. In fact, among other functions VEGF can
`influence cell proliferation, cell mjgration, proteolysis, cell survival and vessel penneability in a
`wide variety of biological contexts. This article will describe the roles played by VEGF in the
`pathogenesis of retinopathy of prematurity, diabetic retinopathy and age-related macular
`degeneration. The potential disadvantages of inhibiting VEGF will be discussed, as will the
`rationales for targeting other VEGF-related modulators of angiogenesis.
`
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`Keywords
`retina; angiogenesis; vascular endothelial growth factor; age-related macular degeneration;
`diabetic retinopathy; retinopathy of prematurity
`
`1. Introduction
`1.1 Vascular Endothelial Growth Factor (VEGF)
`Vascular endothelial growth factor (VEGF), a dimeric glycoprotein of approximately 40
`kDa, is a potent, endothelial cell mitogen tJ1at stimulates proliferation, migration and tube
`fonnation leading to angiogenic growth of new blood vessels. It is essential for angiogenesis
`during development; the deletion of a single allele arrests angiogenesis and causes
`embryonic 1.etliality (Ferrara et al., 1996). In mammals, the VEGF family consists of seven
`members: VEGF-A (typically, and hereafter, referred to as VEGF), VEGF-B, VEGF-C,
`VEGF-D, VEGF-E, VEGF-F and PlGF (placental growtl1 factor) (Fig. IA). Alternative
`
`© 2008 Elsevier Ltd. All rights reserved.
`Corresponding auihor: John S. Penn, Ph.D., Vanderbilt Eye Institute, 8016 Medical Center East, North Tower, Vanderbilt University
`School of Medicine, Nashville, 1N 37232-8808.
`Publisbet·'s Disclaimer: 111is is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our
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`splicing results in several VEGF variants. In humans, these include the relatively abundant
`VEGF121, VEGF16s, VEGF189 and VEGF206, and several less abundant forms (Fig. lB).
`The solubility of these splice variants (collectively referred to as VEGFxxx) is dependent on
`heparin binding affinity. VEGF206 and VEGF 189 bind very tightly to heparin and, thus,
`remain sequestered in the e>..1racellular matrix. VEGF J6S binds heparin with less affinity, but
`also can be associated with the matrix, and VEGF 121 lacks heparin-binding capacity,
`rendering it highly soluble. Investigations in mice genetically engineered to express less than
`the full complement of splice variants confinn that the relative solubility of VEGF splice
`variants strongly affects their specific bioactivities (Taka11ash.i and Shibuya, 2005).
`Moreover, plasmin and various metalloproteinases can cleave VEGF l6S, resulting in an N(cid:173)
`terminal 113-amino acid peptide that is non-heparin-binding, but retains its bioactivity (Keyt
`et al., 1996; Ferrara et al., 2003). Our understanding of the relative expression of the
`different VEGF isoforms tmder normal or pathological conditions and the molecular
`regulators of VEGF alternative splicing is relatively limited.
`
`Recently, discovery of the so called " VEGFxX,'Cb isoforms" has sparked new interest in the
`molecular events iliat regulate VEGF expression (for review, see Ladomery et al., 2007).
`The VEGFxx.,'<b isofonus share approxin1ately 94-98% homolo!,ry with the corresponding
`VEGF>,,'XX isoforms and have the same length. However, due to alterations in the C(cid:173)
`terminus they bind to VEGF receptors, but do not fully activate tllem and act as " dominant
`negative splice variants" (Bates et al., 2002). Studies showing that VEGF l6Sb is
`downregulated in angiogenic tissues (Ladomery et al., 2007) suggest a primary role for this
`isofonn in controlling VEGF activity in health and disease. Administrntion of VEGF 165b has
`been shown to inhibit retinal angiogenesis in the mouse model of oxygen-induced
`retinopathy (Konopatskaya et al., 2006).
`
`1.2 VEGF Receptors
`
`VEGFR-1/Flt-l (ftns-like tyrosine kinase) and VEGFR-2/KDR/Flk-l (kinase insert domain(cid:173)
`containing receptor/fetal liver kinase), along witl1 structurally related receptors, Flt-3/Flk-2
`and VEGFR-3/Flt-4, belong to the receptor tyrosine kinase family (Fig. IA) (Hanks and
`Quinn, 1991; Blume-Jensen and Hunter, 2001). VEGFR-1 and -2 are primarily involved in
`angiogenesis, (Yancopoulos et al., 2000) whereas Flt-3 and Flt-4 are involved in
`hematopoiesis and ly mphangiogenesis (Jussila and Alitalo, 2000). The VEGFRs contain an
`approximately 750-amino-acid-residue e>..tracellular domain, which is organized into seven
`i1mnunoglobulin-like folds. Adjacent to the e>..tracellular domain is a single tnmsmembrane
`region, followed by aju>..1an1e1nbrane domain, a split tyrosine-kinase domain that is
`interrupted by a 70-amino-acid kinase insert, and a C-tenninal tail.
`
`VEGF receptor activation requires dimerization. Guided by the binding properties of tl1e
`ligands, VEGFRs fonn both homodimers and heterodimers (Rahimi, 2006). The signal
`transduction properties of the VEG FR heterodimers, compared with homodimers, remain to
`be fully elucidated. Dime1ization of VEGFR is accompanied by activation of receptor kinase
`activity, leading to autophosphorylation. Site-directed mutagenesis studies have
`demonstrated tliat the Tyrl214 residue, located in the crubo>..-y tenninus of VEGFR-2, is
`required for the ligand-dependent autophosphorylation of tl1e receptor and its ability to
`activate signaling proteins. Signal trdilSduction is propagated when activated VEGF
`receptors phosphorylate SH2 domain-containing protein substrates.
`
`In addition to VEGFRs, VEGF serves as a ligand to anotller family of receptors, the
`neuropilins. Neuropilins are 120- to 130-kDa non-tyrosine kinase receptors that mediate
`critical functions in tumor cells and the nervous and vascular systems. In endotl1elial cells,
`neuropilins serve as receptors for the class 3 semaphorins and co-receptors for VEGF family
`members. The role ofNeuropilin-1 (NRP-1) in mediating VEGF activity is now being
`
`ProgRelii1 Eye Res. Author manuscript; availabl e in PMC 2013 .lune 14.
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`elucidated. VEGF signaling through NRP-1 stimulates endothelial cell migration and
`adhesion. The addition of an anti-NRP-1 antibody suppressed the mitogenic effects of
`VEGF 165 on bovine retinal endothelial cells (RECs) (Oh et al., 2002). In another il1 vitro
`model, the VEGF-dependent differentiation of a subset of lmman bone marrow-derived cells
`into vascular precursors, and subsequent prolifenition of these cells, required the activation
`of a VEGFR-2/NRP-l-dependent signaling pathway (Fons et al., 2004). Finally, VEGF
`promotion of the synthesis and release of prostacyclin (PGI2), an important mediator of
`angiogenesis, is thought to be mediated via NRP-1 binding (Neagoe et al., 2005). The
`angiogenic effects regulated tlrrough VEGF binding to NRP-2 are less well chanicterized
`and appear to be modulated differently from tl1e effects controlled by NRP-1. For example,
`VEGF selectively up-regulates NRP-1, but not NRP-2, on endoilielial cells (Oh et al., 2002).
`
`BIAcore analysis has shown NRP-1 to intenict wiili VEGFR-1, greatly reducing its binding
`affinity for VEGF 165 (Fuh et al., 2000). Co-culture systems of endotl1elial cells and breast
`carcinoma cells indicate that NRP-1 significantly enhances VEGF l6S binding to VEGFR-2
`(Soker et al., 2002). In aortic endotl1elial cells, NRP-2 interacted with VEGFR-1, but less is
`known at present about how this influences VEGF bioactivity (Gluzman-Poltordk et al ,
`2001). Finally, using multiple in vitJv systems, NRP-2 was shown to intenict witl1
`VEGFR-3, leading to lymphangiogenic activity, but no internction was seen between NRP-2
`and VEGFR-2 (Karp~men et al., 2006).
`
`1.3 VEGF Signaling
`
`Few SH2 domain-containing proteins have been shown to interact directly with VEGFR-2.
`Phospholipase C-y (PLC-y) binds to phosphorylated Tyrll75 (Tyrll73 in tl1e mouse), and
`mediates the activation of tl1e mitogen-activated protein kinase (MAPK) cascade, leading to
`proliferation of endotl1elial cells (Takal1ashi et al, 2001) (Fig. 2). PLCy activates protein
`kinase C via tl1e production of diacylglycerol and increased concentmtions of int:r'dcellular
`calcium. A Tyr l l 73Phe mutation of VEGFR-2 causes embryonic lethality due to vascular
`defects, mimicking tl1e defects of VEGFR-r 1- mice (Sakurai et al., 2005). These data
`demonstrate an essential function of the Tyr 1173 residue du.ring vascular development.
`
`In addition to PLCy, the adaptor molecule, Shb, also binds to phosphory lated Tyr 117 5.
`VEGF-induced migmtion and PI3K activation is inhibited by small interfering RNA
`(siRNA)-mediated knockdown of Shb in endotl1elial cells (Holmqvist et al., 2004). The
`serine/threonine kinase, Akt, is activated downstream of PI3K and mediates endothelial cell
`survival (Fujio and Walsh, 1999). Akt also regulates nitric oxide (NO) production by direct
`phosphorylation and activation of endothelial NO synthase (eNOS). Finally, phosphorylated
`Tyrl 175 is known to interact witl1 Sek (lgarashi et al., 1998; Sakai et al., 2000), an adaptor
`molecule that binds Grb2, and participates in MAPK signaling in tl1e epidennal growth
`factor pathway (Thelemann et al., 2005).
`
`Another important phosphorylation site in VEGFR-2 is Tyr95 l (Tyr949 in the mouse), a
`binding site for the signaling adaptor VRAP (VEGF receptor-associated protein)
`(Matsumoto et al., 2005). The phosphorylated Tyr951-VRAP pathway has been shown to
`regulate endothelial cell migration (Matsumoto et al., 2005; Zeng et al , 2001). Reduced
`microvessel density and tumor growtll in vRAP-/- mice confirm an essential function for
`iliis residue in endothelial cells of tl1e angiogenic phenotype (Matsumoto et al., 2005).
`VEGF induces the fonnation of a complex between VRAP and Src (Matsumoto et al.,
`2005), indicating that VRAP might regulate Src activation and its signaling downstream of
`VEGFR-2.
`
`Mice that express a Tyrl212Phe (corresponding to the human Tyrl214) VEGFR-2 mutant
`are viable and fertile (Sakurai et al., 2005). However, phosphorylation ofTyrl212/I214 has
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`ProgRelii1 Eye Res. Author manuscript; available in PMC 2013 .lune 14.
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`been implicated in VEGF-induced actin remodeling through the sequential activation of
`CDC42 and p38 MAPK (Lamalice et al., 2004). lnhibition of p38 MAPK augments VEGF(cid:173)
`induced angiogenesis in the chicken chorioallantoic membmne (CAM) assay (lssbmcker et
`al., 2003 ; Matsumoto et al., 2002), without an accompanying increase in vascular
`permeability (lssbrucker et al., 2003). Moreover, p38 MAPK induces phosphorylation of
`heat-shock protein-27 (HSP27), a molecular chaperone that positively re!,'lllates VEGF(cid:173)
`induced actin reorganization and migration (McMullen et al., 2005; Rousseau et al., 1997).
`
`The existence of multiple ligands and receptors provides initial diversity to VEGF
`bioactivity. Groupings of receptor homo- and heterodimers, activated by both common and
`specific ligands, further augment the diversity of VEGF si!,•naling. A final level of diversity
`is provided by activation of distinct signaling intermediates downstream of each VEGF
`receptor. The combination of these features yields an elaborate signaling network capable of
`regulating the extremely complex angiogenic cascade.
`
`1.4 Regulation of VEGF Expression
`During hypoxia, VEGF gene expression increases via seveml different mechanisms (Dor et
`al., 2001). These mechanisms include increased transcription, rnRNA stability, and protein
`translation using an internal ribosomal entry site, as well as increased expression of ox.-ygen
`regulated protein 150, a chaperone required for intracellular tnmsport of proteins from the
`endoplasmic reticulum to the Golgi apparatus prior to secretion (Chen and Shyu, 1995;
`Forsythe et al., 1996; Levy et al., 1996; Levy et al., 1998; Ozawa et al., 2001).
`
`The increase in VEGF trc:lllscription is largely mediated via hypoxia inducible factor-1
`(HIF-1) (Fig. 3). HIF-1 is a heterodirneric transcription factor composed of two subunits -
`the constitutively produced HIF-1[3 subunit and the inducible component, HIF-la. (Wang
`and Semenza, 1995). Under normoxic conditions, HIF-la. is inactivated and targeted for
`proteasomal degradation by hydrox.-ylation, whereas under hypoxic conditions the specific
`hydrox.-ylases are inhibited, resulting in the rescue ofHIF-la. from degmdation (Schofield
`and Ratcliffe, 2004). When this occurs, HIF-la. complexes with HIF-1[3, translocates into
`the nucleus and binds to a specific sequence in the 5' flanking region of the VEGF gene, the
`hypoxia responsive element (HRE) (Ikeda et al., 1995; Laughner et al., 2001; Shima et al.,
`1996; Wenger, 2002; Forsythe et al., 1996). The in1portance of interaction between HIF-la.
`and the VEGF promoter has been confinned in studies of HIF-la. - /- mouse embryonic stem
`cells, in which basal expression of VEGF mRNA remains low in response to hypoxia
`(Canueliet et al., 1998; Iyer et al., 1998).
`
`Two additional isoforms of HIF, known as HIF-2a. and HIF-3a., have been identified by
`screening for proteins that associate with HIF-1[3 (Ratcliffe, 2007). HIF-2a. appears to be
`closely related to HIF-la. and can promote HRE-dependent gene transcription. While
`structurally and fimctionally sintilar, HIF-la. and HIF-2a. appear to exert different biological
`functions, as demonstrated in studies using knockout mice (Hu et al., 2003). For example,
`while HIF-la. antagonizes c-Myc function, inhibiting renal cell carcinoma (RCC) growth,
`HIF-2a. promotes cell cycle progression in hypoxic RCC and many other cell lines (Gordan
`et al., 2007). Interestingly, the most distantly related isofonn, HIF-3a., appears to antagonize
`HRE-dependent gene expression, suggesting a possible negative influence on hypoxia(cid:173)
`induced gene expression. Additional study is needed to detennine if HIF-2a. or HIF-3a. is
`involved in the regulation of retinal VEGF ex.'Pression.
`
`Clearly, post-transcriptional events are also important in the regulation ofVEGF production
`in tl1e diseased retina, as underscored by tl1e correlation of polymorphisms within the 5' -
`untranslated region (UTR) of t11e VEGF gene with the occurrence of age-related macular
`degeneration (AMD). The 3'UTR and t11e 5'UTR of the VEGF gene are important sites of
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`ProgRelii1 Eye Res. Author manuscript; availabl e in PMC 201 3 .lune 14.
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`regulation controlling mRNA stability and the rate of translation through the internal
`ribosomal entry site (IRES) (for review, see Yoo et al., 2006).
`
`Evidence for the increase in VEGF mRNA stability in response to hypoxia comes from i11
`v1t1v mRNA degradation assays that have led to the identification of adenylate/uridylate-rich
`elements (AREs) in the 3 1 UTR of VEGF mRNA. YEGF mRNA is extremely labile in
`nom10xic conditions, with a half-life of less than 1 hour, as compared with the average half(cid:173)
`life of 10 to 12 hours for eukaryotic mRNA. During hypoxia, the half-life of VEGF mRNA
`increases by two to three-fold (Levy et al., 1996) due to a stabilizing effect of HuR, a 36
`kDa RNA-binding protein, which binds with high affinity to AREs in the 3 '-UTR of VEGF
`rnRNA, protecting it from degradation by endonucleases (Brennan and Steitz, 2001 ;
`Robinow et al., 1988).
`
`Post-transcriptional regulation can also occur in tJ1e in 51 -UTR of VEGF 1nRNA. This
`region contains multiple IRES. These are specific sites of attachment to the ribosomal
`machinery, which provide sites for initiation of translation alternative to the classical 5' cap(cid:173)
`and elF-dependent translational system (for review, see van der Velden and Thomas, 1999).
`Several IRES have been identified in the 5' -UTR of YEGF mRNA, and these provide
`alternative sites of translational control ofVEGF e>.'J)ression (Bomes et al , 2004). Notably,
`evidence suggests that IRES sites in the VEGF 5' -UTR can potentially control tl1e
`generation of alternatively spliced VEGF (Bornes et al., 2004; Huez et al., 2001 ).
`
`AnotJ1er regulatory mechanism consists of increased production of oxygen regulated protein
`150 (ORPI50) in response to hypoxia. Studies using human macrophages transfected with
`adenovirus coding for ORPl 50 showed that overexpression of ORP150 resulted in increased
`VEGF secretion in hypoxia. Evidence suggests that under hypoxic conditions, ORP150
`functions as a molecular chaperone to facilitate YEGF protein transport and secretion
`(Ozawa et al., 2001). VEGF is not only regulated by hypoxia.
`
`VEGF function is also affected by insulin like growtl1 factor 1 (IGF-1) which plays an
`important role in retinal vasculariz.ation. Several lines of evidence, including in vitro studies,
`support the notion tJ1at IGF-1 is critical for vessel development (King et al, 1985; Grant et
`al., 1993). Pretenn infants with reduced serum levels ofTGF-1 have a higher incidence of
`development of retinopathy (Hellstrom et al., 2003). Mice null for the IGF-1 gene have
`retarded retinal vascular growth, compared to wild type controls (Hellstrom et al., 2001).
`However, the action ofIGF-1 is not mediated by decreasing VEGF expression, as the
`amount ofVEGF mRNA is si1nilar in knock out and wild type control mice; instead IGF-1
`acts by decreasing YEGF activation of tl1e Akt signaling pathway. Both MAPK and A.kt
`pathways have been shown to be necessary for endothelial cell survival (Smith et al., 1999).
`
`1.5 Retinal Expression of VEGF and VEG FR
`
`The influence of VEGF in retinal diseases is profound. It has been implicated in a large
`number of retinal diseases and conditions including, but not limited to, highly prevalent
`conditions like AMD and diabetic retinopathy; less conunon disorders such as retinopathy of
`prematurity, sickle cell retinopathy and retinal vascular occlusion; and as a non-causal, but
`important, secondary influence in neovascular glaucoma (Bock et al., 2007) and inl1erited
`retinal dystrophies (Penn et al., 2000). Collectively, these conditions, all of which have
`critical angiogenic components, account for the vast majority of irreversible vision loss in
`developed countries.
`
`At least five retinal cell types have the capacity to produce and secrete YEGF. These include
`the retinal pigmented epithelium (RPE) (Miller et al., 1997), astrocytes (Stone et al., 1995),
`Mi.iller cells (Robbins et al., 1997), vascular endothelium (Aiello et al., 1995) and ganglion
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`ProgRelii1 Eye Res. Author manuscript; available in PMC 2013 .lune 14.
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`cells (Ida et al., 2003). These cells differ widely in their responses to hypoxia; i11 vitro
`studies show that Miiller cells and astrocytes generally produce the !,>reatest amounts of
`VEGF under hypoxic conditions (Morrison et al., 2007; Aiello et al., 1995; Hata et al.,
`1995). To date, the relative capacity of these cells to produce specific splice variants remains
`unclear, as do the patterns of splice variant production throughout retinal development and
`aging.
`
`The distinct roles of the different VEGF splice variants in retinal vascular development is
`being explored, however, in mice expressing only a single variant (Stalmans et al., 2002).
`Vascular development was nonnal in the retinas of mice expressing only VEGF !64
`( VEGF1641164), indicating that this variant is sufficient for directing nonnal vascular growth
`and remodeling. In contrast, retinas of VEGF1201120 mice exhibited severe vascular defects,
`displaying retarded venous and severely flawed arterial development. VEGF1881188 mice had
`normal development of retinal veins but little or no arterial !,>rowth.
`
`Evidence for the expression patterns and roles of VEGFR in retinal tissues comes from a
`variety of species and experimental venues. In the human retina VEGFR-l and -2 can be
`e:x-pressed by neural, glial and vascular elements. In adults, expression is generally restricted
`to the inner nuclear layer (Miiller cells and amacrine cells), the ganglion cell layer, and the
`relinal vasculature (Stitt et al. , 1998). However, during retinal neurogenesis VEGFR-2 is
`also expressed by neural progenitor cells (Hashimoto et al. , 2006). Notably, neural cell
`VEGFR-2 can be activated by VEGF iI1 vitro (Yang et al., 1996). In cultured retinal
`pericytes VEGFR-1 , but not -2, is expressed (Takagi et al. , 1996), whereas in cultured RPE
`cells, both receptors are expressed and are induced by oxidative stress (Sreekumar at al.,
`2006). In the mouse, ganglion cells express both receptors, but only VEGFR-2 is increased
`by intraocular inoculation with herpesvirus (Vinores et al , 2001). Studies in newborn mice
`using the VEGFR-specific kinase inhibitor, SU5416, indicate that Millier cell survival or
`proliferation during retinal development is VEGFR- and MAPK-dependent (Robinson et al.,
`200 l). In a study of patients with diabetic retinopathy, VEGFR-1 expression dominated in
`nonnal retina, but was not increased in the diabetic retina, while VEGFR-2 levels were
`increased, particularly in the vascular elements (Smith et al., 1999). Finally, VEGFR-1 and
`-2 are found on uterine smooth muscle cells in vivo. When these cells are cultured in vitro,
`VEGFR-1 can be phosphorylated and is capable of inducing smooth muscle cell
`proliferation (Brown et al., 1997). To date, neither VEGFR-1 nor -2 has been identified in
`retinal smooth muscle cells.
`
`This article will review the role of VEGF in angiogenesis related to three blinding
`conditions: retinopathy of prematurity, diabetic retinopathy and age-related macular
`degeneration. These conditions constitute Lhe leading causes of irreversible vision loss in
`infants, and working age and elderly Americans, respectively. That VEGF is believed to
`play a causal role in all three of these disorders underscores its p rofound impact in eye
`disease. VEGF antagonists have already proven their value in tumor angiogenesis and
`cho.roidal neovascularization, and new VEGF antagonists are being tested pre-clinically and
`clinically for other ocular indications. Only with a more complete understanding of VEGF
`and its retinal and choroidal activities can we hope to develop better strategies to prevent,
`retard or repair the damage caused by ocular neovascularization.
`
`2. Retinopathy of Prematurity
`
`Retinopathy of prematurity (ROP), a neovascularizing disease affecting preterm infants, is
`one of the most common causes of childhood blindness in the world. Recent estimates
`indicate that each year in the United States, 68% of the approximately 10,000 babies born
`with a birth weight of less than 1250 grams will develop ROP. Thirty six percent of these
`
`ProgRelii1 Eye Res. Author manuscript; available in PMC 2013 .lune 14.
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`infants will progress to severe ROP, a condition that can lead to retinal detaclunent and
`blindness. The incidence of the disease is highly correlated with the gestational age of the
`infant at birth. That is, the more immature the infant, the higher the likelihood of developing
`severe ROP (CRYO-ROP Cooperative Group, 1988; ETROP Cooperative Group, 2003).
`ROP is caused by perturbation of the process of normal vascular development of the retina.
`Its correlation with gestational age stems from the fact that the retina is one of the last
`organs to be vascularized in the human fetus. In very premature infants, the retina is nearly
`avascular at birth. The process of retinal vascular development nonnally occurs in the
`hypoxic uterine environment, but it must occur in a relatively hyperoxic e>.'tra-uterine
`environment in these infants. For reasons defined in the following paragraphs, this leads to
`arrested growth of retinal blood vessels, followed by their unregtllated growth into the
`vitreous cavity, with potentially catastrophic consequences.
`
`2.1 Pathogenesis of ROP
`
`The pathogenesis of ROP is hypothesized to consist of two distinct phases (Madan and
`Penn, 2003). In the initial phase, nonnal retinal vascular growth is retarded. This occurs as a
`consequence, primarily, of exposure to extra-uterine hyperoxia aggravated by therapeutic
`oxygen delivery, but it may also be due to other noxious stimuli, and/or to the premature
`withdrawal of certain maternally derived factors at tJ1e time of birt11. Relative retinal hypoxia
`results from the increasing metabolic demands of the developing neural retina that are unmet
`secondal)1 to the attenuation of blood vessels. This leads to the second phase ofROP,
`consisting of the release of VEGF and other angiogenic factors, producing excessive growth
`of abnonnal leaky blood vessels into the vitreous, followed by vitreous hemorrhage and
`tractional retinal detachment. This second phase is probably encouraged by the weaning of
`infants from oxygen therapy, but removal from therapy is not required for the neovascular
`response to occur. Understanding the process of nonnal retinal vascularization can provide
`important clues regarding the molecular mechanisms underlying the pathogenesis of the two
`phases ofROP.
`
`2.2 Development of the Retinal Vasculature
`The process of nonnal vascularization has been extensively examined in the kitten, mouse
`and rat retinas (Ashton et al., 1957; Ashton, 1961; Ashton, 1970; Blanks and Johnson, 1983;
`Connolly et al., 1988; Chan-Ling et al., 1990; Smith et al, 1994; Stone et al., 1995; DorrelJ
`and Friedlander, 2006). Several studies have also examined the process in the human fetal
`relina (Michaelson, 1948: Nilausen, 1958; Cogan, 1963; Ashton, 1970; Nishimura and
`Taniguchi, 1982; Penfold et al.. 1990; Gariano et al.. 1994: Hughes et al., 2000).
`Development of the retinal vasculature follows a common pattern. in all species (Dorre!J and
`Friedlander, 2006), but there are some dissimilarities as well (Ashton, 1968; Gariano et al. ,
`1994). For example, in humans the process occurs primarily during the latter half of
`gestation, whereas in rodents the process is completed in the first two weeks after birth
`(Michaelson et al., 1954; Ashton, 1970; Stone et aJ., 1995; Engem1an and Meyer, 1965).
`Generally, the completion of retinal vascular development is coincident witJ1 eye opening in
`mammalian species.
`
`Retinal tissue is provided witl1 oxygen and nutrients by the adjacent choriocapillary plexus,
`which supplies the avascular photoreceptor layers, and by the superficial and deep capillary
`plexuses within the retina, supplying its inner layers (Yu and Cringle, 2001). The superficial
`plexus lies immediately beneath the inner limiting membrane, while t11e deep plexus
`penneates the inner nuclear layer. The endothelial cells lining tl1e vessels in tJ1e retinal
`capillary plexuses fonn tight junctions and provide an important part of the blood-retinal
`barrier, while the choroidal vessels are fenestrated, lacking this barrier (Raviola, 1977;
`Campochiaro, 2000). At the choroid/retina interface, barrier function is provided by tight
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`ProgRelii1 Eye Res. Author manuscript; availabl e in PMC 2013 .lune 14.
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`Regeneron Exhibit 1055.007
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`Page 8
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`junctions between adjacent retinal pi1:,11nent epithelial cells. Additionally, prior to the
`fonnation of the retinal vasculature, a trnnsient network of vessels called the hyaloid
`vascular system forms to nourish the immature lens. The vessels of the hyaloid ex1:end from
`their source at the optic nerve head to the posterior surface of the lens, where they bifurcate
`to fonn the dense capillary arbor known as the ttmica vasculosa lentis. This network
`regresses during mid-gestation in the human and two to three weeks post-natally in the
`mouse and rat, coinciding with the period of retinal vascular development (Gogat et al.,
`2004; Fruttiger, 2007).
`
`2.2.1 The Superficial Plexus- Retinal astrocytes I:,'llide the formation of the retinal
`vasculature in manunals. Inuuediately prior to the development of the vascular plex'Us, a
`network of astrocytes rndiates from the optic nerve head across the surface of the imma11.1re
`retina in a central to peripheral pattern. This network forms the scaffold upon which the
`primary vascular plex11s is fom1ed (Ling and Stone, 1988; Fmttiger et al., 1996). Astrocytes
`express VEGF, which promotes endothelial cell proliferation and migration within this
`superficial plane (Stone et al., 1995). The formation of this primary vascular plexus begins
`in the region around the optic disc at the base of the hyaloid artery. A network of capillaries
`spreads across the developing neural layer along the inner surface of the retina towards the
`periphery. Because of its location, this vessel network is often called the superficial plexus.
`In rodents, the development of the superficial ple,ms begins at birth, while in humans the
`process begins at about 16 weeks gestation (Provis, 200 l ; Saint-Geniez and D ' Amore, 2004;
`Dorrell and Friedlander, 2006).
`
`VEGF expression in the retina is closely linked to retinal vascular development. On post(cid:173)
`natal day zero (PO), intense VEGF expression is noted in the innennost nerve fiber layer in
`the mouse retina. From PO - P7, expression increases and is i:,rreater at the leading edge of
`blood vessel growth (Gariano et al., 2006). As blood vessel growth proceeds, there is a
`grndual decrease in VEGF expression (Fig. 4). Th