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`Published in final edited form as:
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`Frog Rem? Etc Res. 2008 July ‘_. 27(4): 331—37]. doi:10.l0l6ij.pretcyercs.2008.05.00l.
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`Vascular Endothelial Growth Factor in Eye Disease
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`J.S. Penna, A. Madanb. R.B. Caldwellc. M. Bartolic, R.W. Caldwell“, and ME. Hartnettd
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`a\l'anderbilt University School of Medicine, Nashville, TN
`
`blStanford University School of Medicine, Palo Alto, CA
`
`l=Medical College of Georgia, Augusta, GA
`
`dUniversity of North Carolina School of Medicine, Chapel Hill, NC
`
`Abstract
`
`Collectively. angiogenic oeular conditions represent the leading cause of irreversible vision loss in
`developed countries. In the U.S.. for example. retinopathy of prematurity. diabetic retinopathy and
`age-related macular degeneration are the principal causes of blindness in lite infant. working age
`and elderly populations. respectively. Evidence suggests that vascular endothelial growth factor
`(VEGF). a 40 kDa dimcric glycoprotcin. promotes angiogenesis in each of these conditions,
`making it a highly significant therapeutic target. However. VEGF is plciotropic, 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 migration. proteolysis. cell survival and vessel permeability in a
`wide variety of biological contexts. This article will describe the roles played by VEGF in the
`pathogenesis of retinopathy of prematurity. diabetic rctinopathy and a ge-relatcd macular
`degeneration. The potential disadvantages of inhibiting VEGF will be discussed, as will the
`rationales for targeting other VEGF-related modulators of angiogenesis.
`
`Keywords
`
`retina: angiogenesis: vascular endothelial growth factor; age-related macular degeneration:
`diabetic rctinopathy; rctinopathy of prematurity
`
`1. Introduction
`
`1.1 Vascular Endothelial Growth Factor (VEGF)
`
`Vascular endothelial growth factor (VEGF). a dimcric glycoprotein of approximately 40
`kDa. is a potent- endothelial cell mitogen that stimulates proliferation, migration and tube
`formation leading to angiogenic gr0w1h of new blood vessels. It is essential for angiogenesis
`during development; the deletion of a single allele arrests angiogenesis and causes
`embryonic lethality (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 growth factor) (Fig. IA). Alternative
`
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`it} 2008 Elsevier Ltd. .-'\ll rights reserved.
`Corresponding author: .iohn S. Penn. Pli.D,, Vanderbilt Eye Institute. 8016 Medical Center East, North Tower. Vanderbilt University
`School ol‘ Medicine. Nashville. TN 3T232—8808.
`
`Publisher's Disclaimer: This is a l-‘Dl-‘ file oi‘an unedited manuscript that has been accepted for publication. As a service to our
`customers we are providing this early version of the manuscript. 'llie manuscript will undergo copyediting, typesetting. and review of
`the resulting proof before it is published in its filial citable form. Please note that during the production process errors may be
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`splicing results in several VEGF variants. In humans. these include the relatively abundant
`VEGFlgl. VEGF165. VEGF 189 and VEGF206. and several less abundant forms (Fig. 1B).
`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 extracellular matrix. VEGF165 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 confirm that the relative solubility of VEGF splice
`variants strongly affects their specific bioactivities {Takahashi and Shibuya. 2005).
`Moreover. plasrnin and various rnetalloproteinases can cleave VEGF165. resulting in an N-
`terminal lI3-arnino 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 isofonns under normal or pathological oorrditions and the molecular
`regulators of VEGF alternative splicing is relatively limited.
`
`Recently. discovery of the so called “VEGFXXXIJ isofonns” has sparked new interest in the
`molecular events that regulate VEGF expression (for review. see Ladomery et al.. 2007).
`The VEGFXXXb isofomrs share approximately 94—98% homology with the corresponding
`VEGFxxx isofonns and have the same length. However. due to alterations in the C-
`terminus they bind to VEGF receptors. but do not fully activate them and act as "dominant
`negative splice variants” {Bates et al.. 2002). Studies showng that VEGF165b is
`dowruegulated in angiogenic tissues (Ladomery et al.. 2007) suggest a primary role for this
`isoforrn in controlling VEGF activity in health and disease. Administration 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-lr'Flt-l (fms-like tyrosine kinase) and VEGFR-Zr’KDRIFIk-l (kinase insert domain-
`containing receptori’fetal liver kinase), along with structurally related receptors. Flt-3fFlk-2
`and VEGFR-3lFlt-4. belong to the receptor tyrosine kinase family (Fig. 1A) (Hanks and
`QuimL 1991: Blume-Jensen and Hunter. 2001). VEGFR-I and -2 are primarily involved in
`angiogenesis. (Yancopoulos et al.. 2000) whereas Flt-3 and Flt-4 are involved in
`hematopoiesis and lymphangiogenesis (Jussila and Alitalo. 2000). The VEGFRs contain an
`approximately 750-anuno-acid-residue extracellular domain. which is organized into seven
`irmnunoglobulin—like folds. Adjacent to the extracellular domain is a single trausrnernbrarre
`region. followed by a juxtamembrane domain, a split tyrosine-kinase domain that is
`interrupted by a TO-amino-acid kinase insert, and a C-tenninal tail.
`
`VEGF receptor activation requires dimerization. Guided by the binding properties of the
`ligands, VEGFRs forrrr both lrornodimers and lreterodirners (Rahimi. 2006). The signal
`transduction properties of the VEGFR heterodimers. compared with hornodirners. remain to
`be fully elucidated. Dimerization of VEGFR is accompanied by activation of receptor kinase
`activity. leading to autoplrosphorylation. Site-directed mutagenesis studies have
`demonstrated that the Tyrl2 l4 residue. located in the carboxy terminus of VEGFR-2. is
`required for the ligand-dependent autophosphorylation of the receptor and its ability to
`activate signaling proteins. Signal transduction is propagated when activated VEGF
`receptors plrosphorylate SH2 domain-containing protein substrates.
`
`In addition to VEGFRs. VEGF serves as a ligand to another family of receptors. the
`neuropilins. Neuropilins are 120- to I30-kDa non-tyrosine kinase receptors that mediate
`critical functions in tumor cells and the nervous and vascular systems. In errdotlrelial cells.
`neuropilins serve as receptors for the class 3 semaphorirrs and co-receptors for VEGF family
`members. The role of Neuropilin—I (NRP-I) in mediating VEGF activity is now being
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`elucidated. VEGF signaling through NRP-l stimulates endothelial cell migration and
`adhesion. The addition of an anti-NRP-l antibody suppressed the mitogenic effects of
`VEGF165 on bovine retinal endothelial cells (RECs) (011 et al.. 2002). In another in Vitro
`model. the VEGF-dependent differentiation of a subset of human bone marrow-derived cells
`into vascular precursors. and subsequent proliferation of these cells. required the activation
`of a VEGFR-2l'NRP-l -dependent signaling pathway (Fons et al.. 2004). Finally. VEGF
`promotion of the synthesis and release of prostacyclin (P612). an important mediator of
`angiogenesis. is thought to be mediated via NRP-l binding (Neagoe et al.. 2005). The
`angiogenic effects regulated tlu‘ough VEGF binding to NRP-2 are less well characterized
`and appear to be modulated differently from the effects controlled by NRP-l. For example.
`VEGF selectively up-regulates NRP-l, but not NRP-2, on endothelial cells (Oh et al.. 2002).
`
`BlAcore analysis has shown NRP-l to interact with VEGFR-l. greatly reducing its binding
`affinity for VEGF165 (Fuh et al.. 2000). Co-culture systems of endothelial cells and breast
`carcinoma cells indicate that NRP-l significantly enhances VEGF165 binding to VEGFR—2
`(Soker et al.. 2002). In aortic endothelial cells, NRP-2 interacted with VEGFR-l. but less is
`known at present about how this influences VEGF bioactivity (Gluzman-Poltorak et al..
`2001). Finally. using multiple in who systems. NRP-2 was shown to interact with
`VEGFR-3. leading to lymphartgiogenic activity, but no interaction was seen between NRP-2
`and VEGFR-2 (Karpanen et al.. 2006).
`
`1.3 VEGF Signaling
`
`Few SH2 domain-containing proteins have been shown to interact diIECtly with VEGFR-2.
`Phospholipase C -*y (PLC‘y) binds to phosphorylated Tyrl 175 (Tyrl 173 in the mouse). and
`mediates the activation of the mitogen—activated protein kinase (MAPK) cascade. leading to
`proliferation of endothelial cells (Takahashi et al.. 2001) (Fig. 2). PLCy activates protein
`kinase C via the production of diacylglycerol and increased concentrations of intracellular
`calcium. A Tyrl 173Phe mutation of VEGFR-2 causes embryonic lethality due to vascular
`defects. mimicking the defects of VEGFR-2 " mice (Sakurai et al.. 2005). These data
`demonstrate an essential function of the Tyr1173 residue during vascular development.
`
`In addition to PLCT. the adaptor molecule. Shb, also binds to phosphorylated Tyrl 175.
`VEGF-induced migration and PI3K activation is inhibited by small interfering RNA
`(siRNA)—mediated knockdown of Shb in endothelial cells (Holmqvist et al.. 2004). The
`Serinefthreonine kinase. Akt. is activated downstream of PI3K and mediates endothelial cell
`
`survival (Fuj io and Walsh. 1999). Akt also regulates nitric oxide (NO) production by direct
`phosphorylation and activation of endothelial NO synthase (eNOS). Finally. phosphorylated
`Tyr1175 is known to interact with Sck (Igarashi et al.. 1998; Sakai et al.. 2000). an adaptor
`molecule that binds Grb2, and participates in MAPK signaling in the epidermal growth
`factor pathway (Thelemann et al.. 2005).
`
`Another important phosphorylation site in VEGFR—2 is Tyr951 (Tyr‘949 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 growth in VRAPT— mice confirm an essential function for
`this residue in endothelial cells of the angiogenic phenotype (Matsumoto et al.. 2005).
`VEGF induces the formation 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 2] Tyr] 2 I 2Phe (corresponding to the human Tyr1214) VEGFR-Z mutant
`are viable and fertile (Sakurai et al.. 2005). However. phosphorylation of Tyr1212s’1214 has
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`been implicated in VEGF-induced actin remodeling through the sequential activation of
`CDC42 and p38 MAPK (Lamalice et al.. 2004). Inhibition of p38 MAPK augments VEGF—
`induced angiogenesis in the chicken chorioallantoic membrane (CAM) assay (Issbmcker et
`al.. 2003; Matsurnoto 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 regulates VEGF-
`induced actin reorganization and migration (Mthrllen etal.. 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 signaling. 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 several different mechanisms (Dor et
`al.. 2001). These mechanisms include increased transcription. mRNA stability. and protein
`translation using an internal ribosornal entry site. as well as increased expression of oxygen
`regulated protein 150. a chaperone required for intracellular transport of proteins from the
`endoplasmic reticulum to the Golgi apparatus prior to secretion (Chen and Slryu. 1995;
`Forsythe etal., 1996; Levy et al.. 1996‘, Levy et al.. 1998; Ozawa et al.. 2001).
`
`The increase in VEGF transcription is largely mediated via hypoxia inducible factor-l
`(HLF-l) (Fig. 3). HLF-l is a heterodimeric transcription factor composed of two subunits —
`the constitutively produced HlF-lfi subunit and the inducible component. HIF-lo. (Wang
`and Semenza. 1995). Under normoxic conditions. HlF- la. is inactivated and targeted for
`proteasoma] degradation by hydroxylation. whereas under hypoxic conditions the specific
`hydroxylases are inhibited. resulting in the rescue of HIP-la. from degradation (Schofield
`and Ratcliffe. 2004). When this occurs. HlF-lo. complexes with HLF- l|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) (lkeda et al.. 1995. Laughner et al.. 2001: Shirna et al..
`1996; Wenger. 2002; Forsythe et al.. 1996). The importance of interaction between HIF-la.
`and the VEGF promoter has been confirmed in studies of HIP-107"" mouse embryonic stem
`cells. in which basal expression of VEGF mRNA remains low in response to hypoxia
`(Canneliet et al.. 1998; Iyer et al.. 1998).
`
`Two additional isoforms of HIF, known as HIP-2o. and HIP-3a.. have been identified by
`screening for proteins that associate with HIP-[[3 (Ratcliffe. 2007). HIP-2o. appears to be
`closely related to HlF- lo. and can promote HRE-dependent gene transcription. While
`structurally and functionally similar. HIP-lo. and HLF-2o. appear to exert different biological
`functions. as demonstrated in studies using knockout mice (Hu et al.. 2003). For example.
`while HlF- lo. antagonizes c—Myc function. inhibiting renal cell carcinoma {RCC) growth.
`HlF-2o. promotes cell cycle progression in hypoxic RC C and many other cell lines (Gordan
`et al.. 2007). Interestingly. the most distantly related isoform, HIP-3a. appears to antagonize
`HRE-dependerrt gene expression. suggesting a possible negative influence on hypoxia-
`induced gene expression. Additional study is needed to determine if HIP-2o. or HLF-3u. is
`involved in the regulation of retinal VEGF expression.
`
`Clearly. post-transcriptional events are also important in the regulation of VEGF production
`in the diseased retina, as underscored by the correlation of polymorphisms within the 5'-
`untranslated region (UTR) of the VEGF gene with the occurrence of age-related Inacular
`degeneration (AMD). The 3’ UTR and the 5'UTR of the VEGF gene are important sites of
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`regulation controlling lnRN A 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 lnRNA stability in response to hypoxia comes from in
`who mRNA degradation assays that have led to the identification of adenylateturidylate-rich
`elements (ARES) in the 3 ’ UTR of VEGF lnRNA. VEGF mRNA is extremely labile in
`normoxic conditions. with a half-life of less than 1 hour. as compared with the average half-
`life of 10 to 12 hours for eukaryotic mRNA. During hypoxia the half-life of VEGF mRNA
`increases by two to tluee-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
`mRNA. protecting it from degradation by endonucleases (Brennan and Steitz. 2001;
`Robinow et al.. 1988).
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`Post—transcriptional regulation can also occur in the in 5’-UTR of VEGF mRNA. This
`region contains multiple fRES. These are specific sites of attachment to the ribosornal
`machinery, which provide sites for initiation of translation alternative to the classical 5' cap-
`and elF-dependent translational system (for review. see van der Velden and Thomas, I999).
`Several IRES have been identified in the 5' -UTR of VEGF mRNA, and these provide
`alternative sites of translational control of VEGF expression (Bornes et al.. 2004). Notably.
`evidence suggests that IRES sites in the VEGF 5’-UTR can potentially control the
`generation of alternatively spliced VEGF (Bornes et al.. 2004: Huez et al.. 2001).
`
`Another regulatory mechanism consists of increased production of oxygen regulated protein
`150 (ORP150) in response to hypoxia. Studies using human macrophages transfected with
`adenovirus coding for ORP150 showed that overexpression of ORP150 resulted in increased
`VEGF secretion in hypoxia. Evidence suggests that under hypoxic conditions, ORPI 50
`functions as a molecular chaperone to facilitate VEGF protein transpon and secretion
`(Oyawa et al., 2001). VEGF is not only regulated by hypoxia.
`
`VEGF function is also affected by insulin like growth factor 1 (IGF- l) which plays an
`important role in retinal vaSCulariyation. Several lines of evidence, including in i-‘frm studies.
`suppon the notion that lGF-l is critical for vessel development (King et al._. 1985; Grant et
`al., 1993). Preterm infants with reduced serum levels of IGF-I have a higher incidence of
`development of retinopathy (Hellstrom et al., 2003). Mice null for the lGF-l gene have
`retarded retinal vaswlar growth. compared to wild type controls (Hellstrorn et al., 2001 ).
`However. the action of lGF-l is not mediated by decreasing VEGF expression. as the
`amount of VEGF mRNA is similar in knock out and wild type control mice: instead lGF-l
`acts by decreasing VEGF activation of the Akt signaling pathway. Both MAPK and Akt
`pathways have been shown to be necessary for endothelial cell survival (Smith et al._. 1999).
`
`1.5 Retinal Expression of VEGF and VEGFR
`
`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 common 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 inherited
`retinal dy strophies (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 VEGF. These include
`the retinal pigmented epithelium (RPE) (Miller et al., 1997), astrocytes (Stone et al._. 1995),
`Muller cells (Robbins et al., 1997), vaSCular endotltelium (Aiello et al._. 1995) and ganglion
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`cells (Ida et 31., 2003). These cells differ widely in their responses to hypoxia; r}: rr'tro
`studies show that Mtiller cells and astrocytes generally produce the greatest 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 tluoughout 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 normal in the retinas of mice expressing only VEGFIM
`( VEGFJM’IM), indicating that this variant is sufficient for directing normal vascular growth
`and remodeling. In contrast, retinas of VEGFJM'?” mice exhibited severe vascular defects.
`displaying retarded venous and severely flawed arterial development. VEGF15’&"1881nice had
`normal development of retinal veins but little or no arterial growth.
`
`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
`expressed by neural, glial and vascular elements. In adults, expression is generally restricted
`to the inner nuclear laycr (Muller cells and amacrinc cells), the ganglion cell layer, and the
`retinal vasculaturc (Stilt et al.. 1998). However. during retinal neurogcnesis VEGFR-Z is
`also expressed by neural progenitor cells (I—Iashimoto et at. 2006). Notably, neural cell
`VEGFR-2 can be activated by VEGF in Vitro (Yang et al., 1996). In cultured retinal
`pericytcs VEGFR-l, but not -2, is expressed (Takagi et al., 1996), whereas in cultured RPE
`cells, both receptors are expressed and are induced by oxidativc stress (Sreekumar at al.,
`2006). In the mouse, ganglion cells express both receptors, but only VEGFR-2 is increased
`by intraocular inoculation with hcrpesvinis {Vinores et al,, 2001). Studies in newborn mice
`using the VEGFR-spccitic kinasc inhibitor, SU5416, indicate that Muller 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-I expression dominated in
`normal retina, but was not increased in the diabetic retina, while VEGFR-2 levels were
`increased, pattiCularly in the vaSCular elements (Smith ct at, 1999). Finally, VEGFR-I and
`-2 are found on uterine smooth muscle cells in viva. When these cells are cultured in wire,
`VEGFR-I can be phosphorylated and is capable of inducing smooth muscle cell
`proliferation (Brown et al,, 1997). To date, neither VEGFR-l nor -2 has been identified in
`retinal smooth muscle cells.
`
`This article will review the role of VEGF in angiogencsis related to three blinding
`conditions: retinopathy of prematurity, diabetic retinopathy and age-related macular
`degeneration. These conditions constitute the 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 profound impact in eye
`disease. VEGF antagonists have already proven their value in tumor angiogencsis and
`choroidal neovascularization, and new VEGF antagonists are being tested pro-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 neovasculariyation.
`
`2. Retinopathy of Prematurity
`
`Retinopathy of prematurity (ROP). a neovascularizing disease affecting preterm infants. is
`one of the most conunon 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 grants will develop ROP. Thirty six percent of these
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`infants will progress to severe ROP, a condition that can lead to retinal detachment 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 normally occurs in the
`hypoxic uterine environment, but it must occur in a relatively hyperoxic extra-uterine
`environment in these infants. For reasons defined in the following paragraphs, this leads to
`arrested growth of retinal blood vessels. followed by their unregulated growth into the
`vitreous cavity, with potentially catastrophic consequences.
`
`2.1 Pathogenesis of ROP
`
`The pathogenesis of ROP is hypothesired to consist of two distinct phases (Madan and
`Penn, 2003). In the initial phase, normal 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, andfor to the premature
`withdrawal of certain maternally derived factors at the time of birth. Relative retinal hypoxia
`results from the increasing metabolic demands of the developing neural retina that are unmet
`secondary to the attenuation of blood vessels. This leads to the second phase of ROP,
`consisting of the release of VEGF and other angiogenic factors, producing excessive growth
`of abnormal 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 occm. Understanding the process of normal retinal vasculariration can provide
`important clues regarding the molecular mechanisms underlying the pathogenesis of the two
`phases of ROP.
`
`2.2 Development of the Retinal Vasculature
`
`The process of normal vascularization has been extensively examined in the kitten, mouse
`and rat retinas {Ashton et al., 1953’; Ashton, 1961; Ashton, 1970; Blanks and Johnson. 1983 ;
`Connolly et al., 1988‘, Chan-Ling et al., 1990. Smith et al., I994; Stone et al., I995; Derrell
`and Fricdlander, 2006). Several studies have also examined the process in the human fetal
`retina (Michaelson. 1948; Nilauscn. I958; Cogan. 1963; Ashton, 1970; Nishimura and
`Taniguchi. 19821Penfold et al.. 1990: Gariano et al.. 1994: Hughes et al., 2000).
`Development of the retinal vasculature follows a common pattern in all species (Donell and
`Friedlander, 2006), but there are some dissimilarities as well (Ashton, I968; 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, I970; Stone et al., 19951Engennan and Meyer, I965).
`Generally, the completion of retinal vaswlar development is coincident with eye opening in
`mammalian species.
`
`Retinal tissue is provided with 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, 200 l ). The superficial
`plexus lies immediately beneath the inner limiting membrane. while the deep plexus
`permeates the inner nuclear layer. The endothelial cells lining the vessels in the retinal
`capillary plexuses form tight junctions and previde an important part of the blood-retinal
`barrier, while the choroidal vessels are fenestrated, lacking this barrier (Raviola, 197?;
`Campochiaro, 2000). At the choroidfretina interface, barrier function is provided by tight
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`ng Refill Eye Res. Author manuscript; available in PMC 2013 June 14.
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`Regeneron Exhibit 1055.007
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`junctions between adjacent retinal pigment epithelial cells. Additionally, prior to the
`formation of the retinal vasculature. a transient network of vessels called the hyaloid
`vascular system forms to nourish the immature lens. The vessels of the hyaloid extend from
`their source at the optic nerve head to the posterior surface of the lens, where they bifurcate
`to form the dense capillary arbor known as the tunica 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 guide the formation of the retinal
`vasculature in manmlals. Inuuediately prior to the development of the vascular plexus, a
`network of astrocytes radiates from the optic nerve head across the surface of the immature
`retina in a central to peripheral pattern. This network forms the scaffold upon which the
`primary vascular plexus is formed (Ling and Stone, 1988: Fnrttiger et al..1996). Astrocytcs
`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.
`1n rodents. the development of the superficial plexus begins at birth. while in humans the
`process begins at about 16 weeks gestation (Provis, 2001; Saint-Geniez and D’Amore, 2004;
`Dorrell and Friedlander. 2006).
`
`VEGF expression in the retina is closely linked to retina] vascular development. On post-
`natal day zero (P0), intense VEGF expression is noted in the innennost nerve fiber layer in
`the mouse retina. From P0 - P7. expression increases and is greater at the leading edge of
`blood vessel growth (Gariano et al.. 2006). As blood vessel growth proceeds. there is a
`gradual decrease in VEGF expression (Fig. 4). The pattern suggests a strong correlation
`between the presence or absence of physiologically patent blood vessels and the level of
`local VEGF expression.
`
`Studies indicate that development of the retinal vasculature may occur by both
`vasculogenesis, dc mm formation of blood vessels from mesodermal precursor cells
`(angioblasts). and angiogenesis, sprouting of new vessels from existing blood vessels
`(Ashton. 1966: Ashton, 1970'. Flower et al._. 1985'. McLeod et al., 1987; Kretzer and Hittner.
`1988; Chan-Ling etal.. 1990; Jiang et al., 1995‘. Risau and Flamme. 1995; Risau. 1997'.
`Hughes et al., 2000). "Spindle-cells”, so called for their shape. have been identified within
`the inner plexifonn layer of the immature mammalian retina (Flower et al._. 1985', McLeod et
`al., 1987; Stone and Dreher, 1987', Watanabe and Raff, 1988', Hughes et al., 2000; Taomoto
`et al., 2000). These cells are purported to coalesce to fonn vascular cords, which
`subsequently form the patent vessels that constitute the superficial vascular plexus. By a
`process that is not yet completely understood. the vessels then undergo remodeling, leading
`to the development of mature arteries. veins and capillaries. The identification of vascular
`precursor cells in the retinas of various species supports the notion that vasculogenesis
`participates in the formation of the primary plexus (Chan Ling et al., 2004). However. some
`evidence argues in favor of angiogenesis as being the mechanism d