Article
`
`Ocular Angiogenesis: The Science Behind the Symptoms
`With a variety of factors contributing to neovascularization, many therapies
`are being developed
`
`January 1, 2011
`
`PEER REVIEWED
`
`Ocular Angiogenesis: The Science Behind the Symptoms
`
`With a variety of factors contributing to neovascularization, many therapies
`are being developed.
`
`Matthew Dombrow, MD · Ron A. Adelman, MD, MPH, FACS
`
`With most things in nature, a balance must be struck between two opposing forces, each necessary for a
`functional end product. Enormously complex interactions between production, degradation and reformation are
`necessary for any living organism. The eye, being one of the most complex and evolved organs, must also adhere
`to this delicate balance. Angiogenesis and antiangiogenesis are one of these many pairings that must achieve
`balance. In his groundbreaking 1971 article, Judah Folkman introduced the idea of a “diffusible message” released
`from solid tumor cells to invoke a robust capillary-sprouting response, more robust than ordinary wound healing or
`inflammation, to help support its cancerous growth. In the same light, Folkman introduced the concept of
`1
`antiangiogenesis.
`
`A distinction between vasculogenesis and angiogenesis bears mention. Vasculogenesis is blood-vessel formation
`via endothelial progenitor cells and hemangioblast differentiation, while angiogenesis is the formation of new
`capillaries from pre-existing blood vessels. Angiogenesis requires endothelial cell migration, proliferation, survival,
`vessel maturation, vessel-wall remodeling and degradation of the extracellular matrix. It is a normal part not only of
`development but also of healing. Endothelial cell number is normally stable without significant proliferation, likely
`due to a balance between proangiogenic factors (ie, vascular endothelial growth factor) and antiangiogenic
`(angiostasis) factors (ie, pigment epithelium–derived factor). When there is an imbalance between these two,
`pathology ensues.
`
`Ocular angiogenesis is a major cause of much ocular disease and blindness. It is a significant contributing factor in
`diabetic retinopathy, exudative AMD, corneal graft rejection, corneal neovascularization, retinopathy of prematurity,
`retinal vein occlusion, neovascular glaucoma and sickle cell retinopathy. In this article, different forms of ocular
`angiogenesis, their mediators and implications for treatment will be reviewed.
`
`RETINAL AND CHOROIDAL NEOVASCULARIZATION
`Retinal Neovascularization
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`Proliferative diabetic retinopathy (Figures 1-3), RVO and sickle cell proliferative vitreoretinopathy are the most
`common forms of retinal neovascularization. Other causes include familial exudative vitreoretinopathy, sarcoidosis,
`pars planitis, radiation retinopathy, ocular ischemic syndrome and Eales disease. A combination of angiogenesis
`and vasculogenesis occurs during retinal neovascularization. Normally, minimal endothelial cell proliferation occurs
`in the retina. “Hypoxia is believed to be the initial stimulus that causes an upregulation of growth factors, integrins
`2
`and proteinases, which result in endothelial cell proliferation and migration.” Hypoxia upregulates VEGF mRNA in
`3
`retinal endothelial cells, RPE cells, pericytes, Müller cells and ganglion cells.
`4
`
`Figure 1. Severe proliferative diabetic retinopathy with trac-tional retinal detachment.
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`Figures 2 and 3. Early phase fluorescein angiography of leaking neovascular membranes and hemorrhage
`in patients with PDR.
`
`Choroidal Neovascularization
`
`Despite alterations in choroidal blood flow in AMD, it may not be enough to produce significant hypoxia to induce
`CNV.
` Like retinal neovascularization, CNV is a combination of angiogenesis and vasculogenesis. CNV is most
`3,5,6
`7
`commonly seen in wet AMD (Figures 4 and 5). Other common causes are high myopia, choroidal rupture, angioid
`streaks, ocular histoplasmosis syndrome, multifocal choroiditis, punctate inner choroidopathy and iatrogenic
`causes (intense laser photocoagulation). Wet AMD is the leading cause of severe vision loss in the elderly in the
`United States. Oxidative damage and inflammation (and less so hypoxia) are thought to tip the pro- and
`8
`antiangiogenic-factor balance in favor of angiogenesis.
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`Figures 4 and 5. Fundus photography and mid-phase fluorescein angiography of an active choroidal
`neovascular membrane in a patient with exudative age-related macular degeneration.
`
`INFLAMMATION AND COMPLEMENT
`
`In experimental models, a robust immune response is seen quickly after choroidal neovascular membrane (CNVM)
`is induced by laser. Within 72 hours, there is an influx of neutrophils, macrophages, natural killer cells, microglial
`cells and edema. Macrophages are found near ruptured or thin areas in Bruch's membrane and have been
`9
`isolated from CNVM removed via submacular surgery.
` Activated macrophages secrete collagenase and
`10
`elastase, which may erode Bruch's membrane, allowing for vascular mobilization. Recently, inhibition of vascular
`adhesion protein-1 decreased macrophage accumulation in CNV lesions and reduced CNV size and expression
`11
`of other inflammatory molecules including TNF-α, ICAM-1 and MCP-1.
`11
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`Drusen are likely the byproducts of RPE cells with some protein components, possibly arising from the choroid. It
`is still not truly known if drusen are epiphenomena of AMD, an active player in the inflammatory cascade, or a
`passive player via its physical barrier and disruption of transport across Bruch's membrane.
` Shen was able to
`12
`produce a CNV membrane by subretinal injection of Matrigel (BD Biosciences, Sparks, MD), a soluble basement
`membrane preparation mimicking drusen formation.
` In the past decade, noting similarities between drusen found
`13
`in AMD and drusen found in patients with membranoproliferative glomerulonephritis type 2, Hageman and
`colleagues formed the basis of complement system dysfunction and its role in CNVM formation.
` Specifically,
`14-16
`variants of complement factor H (CFH) and alterations in the alternative complement pathway have been
`genetically linked to AMD.
` A myriad of targets of the complement sys tem exist and several phase 1 and 2
`14-17
`trials are ongoing. Potential agents work by either replacing a defective component (ie, CFH with the Y402H
`mutation) or by blocking a complement pathway (C3 and C5 inhibitors).
`18
`
`VEGF
`
`Vascular endothelial growth factor was initially named vascular permeability factor, as it originally was isolated in
`tumor ascites fluid from guinea pigs.
` VEGF is 50,000 times more potent as a vasodilator than histamine.
` It
`19
`20
`includes a family of growth factors — VEGF-A, VEGF-B, VEGF-C, VEGF-D and PlGF (placental growth factor).
`The major mediator of tumor angiogenesis is VEGF-A. Despite a very high affinity between VEGF and VEGF
`receptor 1 (VEGFR-1), most transduction occurs with VEGFR-2. VEGFR-1 may be a “decoy” receptor, preventing
`VEGF binding to VEGFR-2.
` Thus, the interaction of VEGF and VEGFR-2 is crucial.
`3,21
`
`Vascular endothelial growth factor is expressed in most types of human cancers and is highly selective for
`endothelial cells.
` As adapted from Hicklin, VEGF is involved in: (1) endothelial cell proliferation via activation of
`10,22
`mitogen-activated protein kinases; (2) endothelial cell permeability via opening of endothelial fenestrations and cell
`junctions; (3) tumor-cell invasion via induction of metallo-proteinases (MMPs) and urokinase plasminogen
`activators (UPA), hence promoting extracellular matrix degradation; (4) migration through activation of FAK, p38
`and nitric oxide; (5) survival of new endothelial cells by inhibiting apoptosis; and (6) activation and stabilization of
`the vascular network.
`23
`
`Induction of VEGF results from hypoxia via hypoxia-inducible factor 1 (HIF-1), low pH, inflammatory cytokines (IL-
`6), growth factors (basic fibroblast growth factor), sex hormones (androgens and estrogens), chemokines,
`oncogene activation and decreased activity of tumor suppressor gene activity.
` Numerous studies have shown
`22,23
`increased aqueous and vitreous levels of VEGF and VEGFR-1 in a variety of ocular proliferative conditions.
`24 31
`Surgically removed CNVMs from AMD patients show increased VEGF expression in fibroblasts and RPE cells.
`32 34
`
`But is VEGF itself sufficient enough to produce retinal and choroidal neovascularization? VEGF is found in the
`Bruch's membrane–choriocapillaris complex of normal healthy donor eyes.
` Some animal models had difficulty
`10,35
`producing both retinal and choroidal neovascularization. Ozaki implanted intravitreal, sustained-release VEGF
`pellets that induced retinal neovascularization in rabbits but not in primates.
` Tolentino showed that intravitreal
`36
`injections of VEGF in monkey eyes produced capillary nonperfusion and vessel dilation, but preretinal
`neovascularization in only the peripheral retina and not posterior pole.
` Transgenic mice in which rhodopsin
`37
`promoter was coupled to a VEGF gene, hence producing increased intraretinal VEGF, did form intraretinal and
`subretinal neovascularization. However, as Campochiaro has demonstrated, when increased VEGF levels are
`combined with photoreceptor degeneration, CNVM may form. Thus, healthy photoreceptors may somehow prevent
`the choriocapillaris from responding to elevated VEGF levels.
`38,39
`
`Basic FGF
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`Fibroblast growth factor (FGF) is a heparin-binding growth factor with a high affinity for heparin sulfate
`proteoglycans. FGF and associated receptor tyrosine kinases (RTKs) are expressed in virtually all cells —
`overexpression plays a role in many cancers.
` A recent question is whether it is truly involved in retinal and
`40
`choroidal neovascularization. Studies have shown increased levels of FGF and FGF-like peptides in ischemic
`retinopathy and laser-induced CNV. Following FGF-impregnated microsphere injection into the subretinal space,
`AMD-like neovascular membranes formed in rabbit eyes.
` However, other studies suggest that FGF is only
`3,41-43
`angiogenic when it accompanies other cell injury, thus unmasking “control mechanisms that sequester FGF2.”
`3,44
`Newer evidence may show that FGF may not, at least alone, be a factor in ocular angiogenesis. FGF2-deficient
`transgenic mice developed the same amount of retinal and choroidal neovascularization as in wild-type mice.
`FGF2 retinal overexpression did not produce retinal neovascularization in mice.
`45,46
`
`IGF-1
`
`The role of insulin-like growth factor (IGF)-1 in angiogenesis was seen as early as 1953, when Poulsen noted
`regression of neovascularization in proliferative diabetic retino- pathy patients after pituitary infarction.
` Pituitary
`47
`ablation was even a treatment for PDR at one time. IGF-1 is elevated in the serum and vitreous in PDR.
`48,49
`Elevated growth hormone/IGF-1 has been implicated in retinal neovascularization in mouse models independent of
`VEGF levels.
` More recently, Hellstrom studied the relationship between IGF-1 and VEGF in retinopathy of
`50
`prematurity. Knock-out IGF-1 mice's normal retinal vascular development was arrested despite adequate VEGF
`levels. Infants with higher levels of IGF-1 had more normal vasculature development. Hellstrom proposed that
`infants with a low level of IGF-1 develop an avascular retina, and in conjunction with subsequent elevated VEGF
`levels, proliferative ROP ensues.
`51
`
`MMP/UPA
`
`Extracellular proteinases are vital for the migration of endothelial cells through the extracellular matrix. Urokinase
`plasminogen activator and the matrix metalloproteinase (MMP) family are primarily involved in angiogenesis.
`MMPs are upregulated in angiogenic lesions and “their inhibition or genetic ablation diminishes angiogenic
`switching, tumor size and growth.”
`3,11,52-54
`
`Prinomostat (AG3340), an MMP inhibitor, was shown to inhibit CNVM growth and leakage when injected
`intravitreally in an animal model, but early human trials studying use for cancers were terminated. It is currently
`being studied for possible ocular use.
` Intravitreal injection of A6, a urokinase system inhibitor, showed promise
`55,56
`in successful treatment of monkey CNVM — up to a 76% reduction in CNVM size was safely observed.
`57
`
`IL-8 AND TNF-α
`
`Interleukin-8 is also thought to play a role in the inflammatory component of ocular neovascularization. It is a
`cytokine that is a chemotactic factor for neutrophils and lymphocytes. IL-8 levels are significantly elevated in the
`vitreous of patients with proliferative neovascularization, including PDR, RVO and Eales disease.
` It is also
`31,58
`thought to aid in the inflammatory component of angiogenesis as shown in rat cornea studies.
` Elner showed that
`59
`stimulated RPE cells synthesized IL-8, thereby possibly augmenting the inflammatory cascade in angiogenesis.
`60
`
` CNV membranes
`Elevated levels of TNF-α have been isolated from fibrovascular membranes in PDR eyes,
`61
`62
`and animal models of hypoxia-induced retinal neovascularization.
` TNF-α antagonists have been successful in
`63
`treating T-cell–mediated noninfectious uveitis and juvenile idiopathic arthritis, but its role in ocular angiogenesis
`has not been well established.
`
`PEDF
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`Pigment epithelium–derived factor (PEDF) is believed to be the most potent angiogenesis inhibitor. Hypoxia has
`been shown to downregulate PEDF, thus permitting neovascularization.
` Vitreous samples from PDR and RVO
`64,65
`patients showed lower PEDF levels than in controls, and PDR eyes treated with PRP showed the ability to
`replenish their levels of PEDF.
` Systemic and intravitreal PEDF administration decreases retinal
`31,66
`neovascularization in a hypoxia-induced neovascularization mouse model.
` Mori showed decreased CNV using
`67,68
`an adenoviral vector transfer of PEDF.
` A phase 1 trial of an adenoviral vector delivered intravitreal PEDF
`69-71
`(AdPEDF) completed in 2006 showed stabilization of CNVM in wet AMD patients for up to 12 months.
`72
`
`TGF-β
`
`Transforming growth factor–β's exact role remains somewhat more elusive in angiogenesis. It is believed to be
`both pro- and antiangiogenic, depending on its concentration and surrounding environment. Low levels lead to
`angiogenesis by upregulating angiogenic factors and proteases. High levels stabilize basement membrane
`formation, recruit smooth muscle cells, and inhibit endothelial cell growth.
` Ogata showed TGF-β levels remained
`73
`elevated in photocoagulated RPE cells, while angiogenic factors, including VEGF and IL-8, were reduced.
` In the
`74
`context of cancer and tumor angiogenesis, Bernebeu describes TGF-β's “dual and paradoxical role”; first it acts as
`a tumor suppressor in a premalignant stage, and then its role becomes “exploited” by active tumor cells to aid in its
`own growth. Tumor cells can become resistant to TGF-β via mutating TGF-β receptors, thus reacting as if in a low-
`level TGF-β environment.
`75
`
`Uveal Melanoma
`
`Uveal melanoma cells are one of the few cell types that use a process coined “molecular mimicry” to enhance their
`microcirculation. Folberg and Maniotis found de novo vascular channels that were completely devoid of endothelial
`cells in aggressive melanoma cells.
` This suggested tumor cell ability to form new circulatory channels
`76
`independent of angiogenesis, hence discovering a potential new way to diagnose and treat tumor growth. In
`addition to this novel concept, angiogenesis also plays a role in uveal melanoma's growth. In 1979, Folkman
`implanted aqueous humor aspirated from eyes with uveal melanoma, eyes with retinoblastoma and control eyes
`into the chorioallantoic membrane of chick embryos. The majority of tumor-laden samples produced an angiogenic
`response in the chick embryo, while only one sample from a control eye did (this patient later developed
`lymphocytic leukemia).
`77
`
`Recent reports of VEGF expression in uveal melanoma have been highly variable. Generally, elevated VEGF
`levels have been seen in samples with a necrotic tumor component but do not appear to be related to tumor size
`or presence of metastases.
` This year, bevacizumab (Avastin, Genentech) and gene transfer of PEDF (an
`78-80
`angiogenesis suppressor) decreased uveal melanoma growth and hepatic micrometastases in a mouse
`model.
`81,82
`
`DISCUSSION
`
`In addition to the above factors, several other growth factors, cytokines and environmental conditions are involved
`in the normal and abnormal cascade of angiogenesis. As with any disease process, it is vital to understand the
`pathophysiology in order to better target treatments. With regard to the eye, preventing angiogenesis is
`multifactorial and complex. Despite our lack of full understanding of these diseases (eg, AMD), we have been able
`to halt or at least slow the progression of angiogenesis.
`
`Laser photocoagulation has been the mainstay of treatment for retinal neovascularization. It has been used for
`CNV and then was replaced with photodynamic therapy, which in turn has been largely replaced by anti-VEGF
`injections. Currently, anti-VEGF treatment has taken center stage due to the primary role VEGF has in ocular
`neovascularization. In the past decade, tremendous advancements have been made in the treatment of wet AMD.
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`Newer anti-VEGF agents are under investigation, such as aflibercept (VEGF Trap-Eye, Regeneron). It has a
`longer duration of action compared to ranibizumab (Lucentis, Genentech) and thus may allow for a less frequent
`dosing schedule. There are also clinical trials evaluating small-interfering RNA and combination therapy with
`brachytherapy to address VEGF-induced angiogenesis.
`
`Additionally, other targets of angiogenesis are under investigation, such as receptor tyrosine kinase inhibitors to
`target VEGF and PDGF receptors, complement modulators, MMP and UPA inhibitors, and even doxycycline.
`Recently, Cox showed that oral doxycycline, through its anti-inflammatory properties and MMP-suppression
`effects, reduced angiogenesis in CNVM and pterygium mouse models.
` For now, anti-VEGF agents are the
`52
`mainstay for the treatment of choroidal angiogenesis. For retinal angiogenesis, such as in PDR, laser
`photocoagulation and/or anti-VEGF agents may be used.
`
`CONCLUSIONS
`
`A delicate balance must be struck between angiogenesis and its counterpart, angiostasis. Retinal
`neovascularization and CNV may ensue when this balance goes awry. Retinal neovascularization is usually
`initiated in hypoxic states (eg, PDR), whereas CNV is usually a result of aging, chronic inflammation and oxidative
`damage (eg, AMD). VEGF has been strongly implicated in both, but other contributing factors include complement,
`fibroblast growth factor, insulin-like growth factor 1, matrix metalloproteinase, urokinase, interleukin-8, tumor
`necrosis factor-α and pigment epithelium–derived growth factor.
`
`Due to its enormous complexity, there are many pathways to target when treating ocular neovascularization.
`Although not a very elegant treatment, laser photocoagulation has been a successful treatment for retinal
`neovascularization. A bit more refined, targeting VEGF and its resulting molecular cascade of events has shown
`great hope in the treatment of both retinal and choroidal neovascularization. Potentially longer-lasting treatments
`that target more specific pathways may come about in the treatment of ocular neovascularization, thus reducing
`angiogenic-related eye disease and blindness. RP
`
`REFERENCES
`
`1. Folkman J. Tumor Angiogenesis: therapeutic implications. N Engl J Med. 1971;285:1182-1186.
`2. Engerman RL, Pfaffenbach D, Davis MD. Cell turnover of capillaries. Lab Invest. 1967;17:738-43.
`3. Das A, McGuire PG. Retinal and choroidal angiogenesis: pathophysiology and strategies for inhibition. Prog
`Retin Eye Res. 2003;22:721-748.
`4. Aiello L P, Northrup JM, Keyt BA, Takagi H, Iwamoto MA. Hypoxic regulation of vascular endothelial growth
`factor in retinal cells. Arch Ophthalmol. 1995;113:1538-1544.
`5. Grunwald JE, Hariprasad SM, DuPont J, et al. Foveolar choroidal blood flow in age-related macular
`degeneration. Invest Ophthalmol Vis Sci. 1998; 39:385-390.
`6. Campochiaro PA. Retinal and choroidal neovascularization. J Cell Physiol. 2000; 184:301-310.
`7. Sengupta N, Caballero S, Mames RN, Butler JM, Scott EW, Grant MB. The role of adult bone marrow-derived
`stem cells in cho roidal neovascualrization. Invest Ophthalmol Vis Sci. 2003;44:4908-4913.
`8. Age-related macular degeneration: what you should know. National Eye Institute Web site.
`http://www.nei.nih.gov/health/maculardegen/nei_wysk_amd.PDF. Accessed October 31, 2010.
`9. Nusenblatt RB, Ferris F 3rd.. Age-related macular degeneration and the immune response; implications for
`therapy. Am J Ophthalmol. 2007; 144:618-626.
`10. Bressler SB. Introduction: Understanding the role of angiogenesis and antiangiogenic agents in age-related
`macular degeneration. Ophthalmology. 2009; 116(Suppl):S1-S7.
`11. Noda K, She H, Nakazawa T, et al. Vascular adhesion protein-1 blockade suppresses choroidal
`neovascualrization. FASEB J. 2008;22:2928-2935.
`12. Nozaki M, She H, Nakazawa T, et al. Drusen complement components c3a and c5a promote choroidal
`
`Samsung et al. v. Regeneron IPR2023-00884
`Regeneron Pharmaceuticals, Inc. Exhibit 2113 Page 8
`
`

`

`neovascularization. Proc Natl Acad Sci U S A. 2006; 103:2328-2333.
`13. Shen D et al. Exacerbation of retinal degeneration induced by subretinal injection of Matrigel in CCL2/MCP-1
`deficient mice. Paper presented at: Annual meeting of the Association of Research for Vision and Ophthalmology;
`April 30-May 4, 2005; Fort Lauderdale, FL.
`14. Gehrs KM, Jackson JR, Brown EN, Allikmets R, Hageman GS. Complement, age-related macular
`degeneration and a vision of the future. Arch Ophthalmol. 2010;128:349-358.
`15. Hageman GS, Luthert PJ, Victor Chong NH, Johnson LV, Anderson DH, Mullins RF. An integrated hypothesis
`that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch's membrane in ter face in
`age-related macular degeneration. Prog Retin Eye Res. 2001;20:705-732.
`16. Anderson DH, Mullins R F, Hageman GS, Johnson LV. A role for local inflammation in the formation of drusen
`in the aging eye. Am J Ophthalmol. 2002; 134:411-431.
`17. Haines JL, Hauser MA, Schmidt S, et al. Complement factor H variant increases the risk of age-related
`macular degeneration. Science. 2005; 308:419-421.
`18. Zarbin MA, Rosenfeld PJ. Pathway-based therapies for age-related macular degeneration: an integrated
`survey of emerging treatment alternatives. Retina. 2010;30:1350-1367.
`19. Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF. Tumor cells secrete a vascular
`permeability factor that promotes accumulation of ascites fluid. Science. 1983;219:983-985.
`20. Dvorak HF. Vascular permeability factor/vascular endothelial growth factor: a critical cytokine in tumor
`angiogenesis and a potential target for diagnosis and therapy. J Clin Oncol. 2002;20:4368-4380.
`21. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9:669-676.
`22. Kerbel RS. Tumor angiogenesis. N Engl J Med. 2008;358:2039-2049.
`23. Hicklin DJ, Ellis LM. Role of vascular endothelial growth factor pathway in tumor growth and angiogenesis. J
`Clin Oncol. 2005;23:1011-1027.
`24. Adamis AP, Miller JW, Bernal MT, et al. Increased vascular endothelial growth factor levels in the vitreous of
`eyes with proliferative diabetic retinopathy. Am J Ophthalmol. 1994;118:445-450.
`25. Aiello L P, Avery RL, Arrigg PG, et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic
`retinopathy and other retinal disorders. N Engl J Med. 1994;331:1480-1487.
`26. Malecaze F, Clamens S, Simorre-Pinatel V, et al. Detection of vascular endothelial growth factor messenger
`RNA and vascular endothelial growth factor-like activity in proliferative diabetic retinopathy. Arch Ophthalmol.
`1994;112:1476-1482.
`27. Pe'er J, Folberg R, Itin A, Gnessin H, Hemo I, Keshet E. Upregulated expression of vascular endothelial growth
`factor in proliferative diabetic retinopathy. Br J Ophthalmol. 1996;80:241-245.
`28. Ambati J, Chalam KV, Chawla DK, et al. Elevated gamma-aminobutyric acid, glutamate, and vascular
`endothelial growth factor levels in the vitreous of patients with proliferative diabetic retinopathy. Arch Ophthalmol.
`1997; 115:1161-1166.
`29. Hattenbach LO, Allers A, Gümbel HO, Scharrer I, Koch FH. Vitreous concentrations of TPA and plasminogen
`activator inhibitor are associated with VEGF in proliferative diabetic vitreoretinoapthy. Retina. 1999;19:383-389.
`30. Matsunaga N, Chikaraishi Y, Izuta H, et al. Role of soluble vascular endothelial growth factor-1 in the vitreous
`in proliferative diabetic retinopathy. Ophthalmology. 2008;115:1916-1922.
`31. Murugeswari P, Shukla D, Rajendran A, Kim R, Namperumalsamy P, Muthukkaruppan V. Proinflammatory
`cytokines and angiogenic and anti-angiogenic factors in vitreous of patients with proliferative retinopathy and eales’
`disease. Retina. 2008;28:817-824.
`32. Amin R, Puklin JE, Frank RN. Growth factor localization in choroidal neovascualr membranes of age-related
`macular degeneration. Invest Ophthalmol Vis Sci. 1994;35:3178-3188.
`33. Frank RN, Amin RH, Eliott D, Puklin JE, Abrams GW. Basic fibroblast growth factor and vascular endothelial
`growth factor are present in epiretinal and choroidal neovascualr membranes. Am J Ophthalmol. 1996;122:393-
`403.
`34. Lopez PF, Sippy BD, Lambert HM, Thach AB, Hinton DR. Transdifferentiated retinal pigment epithelial cells are
`immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration-
`
`Samsung et al. v. Regeneron IPR2023-00884
`Regeneron Pharmaceuticals, Inc. Exhibit 2113 Page 9
`
`

`

`related choroidal neovascular membranes. Invest Ophthalmol Vis Sci. 1996;37:855-868.
`35. Bhutto IA, McLeod DS, Hasegawa T, Kim S Y, Merges C, Tong P, Lutty GA. Pigment epithelium-derived factor
`(PEDF) and vascular endo thelial growth factor (VEGF) in aged human choroids and eyes with age-related
`macular degeneration. Exp Eye Res. 2006;82:99-110.
`36. Ozaki H, Hayashi H, Vinores SA, Moromizato Y, Campochiaro PA, Oshima K. Intravitreal sustained release of
`VEGF causes retinal neovascularization in rabbits and breakdown of the blood-retinal barrier in rabbits and
`primates. Exp Eye Res. 1997;64:505-517.
`37. Tolentino MJ, McLeod DS, Taomoto M, Otsuji T, Adamis AP, Lutty GA. Pathologic features of vascular
`endothelial growth factor-induced retinopathy in the nonhuman primate. Am J Ophthalmol. 2002;133:373-385.
`38. Yamada H, Yamada E, Kwak N, et al. Cell injury unmasks a latent proangiogenic phenotype in mice with
`increased expression of FGF2 in the retina. J Cell Physiol. 2000;185:135-142.
`39. Campochiaro PA. Retinal and choroidal neovascularization. J Cell Physiol. 2000;184:301-310.
`40. Cook KM, Figg WD. Angiogenesis inhibitors: Current strategies and future prospects. CA Cancer J Clin.
`2010;60:222-243.
`41. Zhang NL, Samadani EE, Frank RN. Mitogenesis and retinal pigment epithelial cell antigen expression in the
`rat after krypton laser photocoagulation. Invest Ophthalmol Vis Sci. 1993;34:2412-2424.
`42. Kimura H, Spee C, Sakamoto T, et al. Cellular response in subretinal neovascularization induced by bFGF
`impregnated microspheres. Invest Ophthalmol Vis Sci. 1999;40:524-528.
`43. Sivalingham A, Kenney J, Brown GC, Benson WE, Donoso L. Basic fibroblast growth factor levels in the
`vitreous of patients with proliferative diabetic retinopathy. Arch Ophthalmol. 1990;108:869-872.
`44. Yamada H, Yamada E, Kwak N, et al. Cell injury unmasks a latent proangiogenic phenotype in mice with
`increased expression of FGF2 in the retina. J Cell Physiol. 2000;185:135-142.
`45. Ozaki H, Okamoto N, Ortega S, et al. Basic fibroblast gowth factor is neither necessary nor sufficient for the
`development of retinal neovascularization. Am J Pathol. 1998;153:757-765.
`46. Tobe T et al. Targeted disruption of the FGF2 gene doe not prevent choroidal neovascularization in a murine
`model. Am J Pathol. 1998 Nov;153(5):1641-6.
`47. Poulsen JE. Recovery from retinopathy in a case of diabetes with Simmonds’ disease. Diabetes. 1953 Jan-
`Feb;2(1);7-12.
`48. Merimee TJ et al. Insulin-like growth factors. Studies in diabetics with and without retinopathy. N Engl J Med.
`1983 Sep 1;309(9):527-30
`49. Grant M et al. Insulin-like growth factors in vitreous. Studies in control and diabetic subjects with
`neovascularization. Diabetes. 1986 Apr;35(4);416-20.
`50. Smith LE, Kopchick JJ, Chen W, et al. Essential role of growth hormone in ischemia-induced retinal
`neovascularization. Science. 1997;276;1706-1709.
`51. Hellstrom A, Perruzzi C, Ju M, et al. Low IGF-1 suppresses VEGF-survival signaling in retinal endothelial cells:
`direct correlation with clinical retionopathy of prematurity. Proc Natl Acad Sci U S A. 2001;98;5804-5808.
`52. Cox CA, Amaral J, Salloum R, et al. Doxycycline's effect on ocular angiogenesis: an in vivo analysis.
`Ophthalmology. 2010;117:1782-1791.
`53. Plantner JJ, Smine A, Quinn TAl. Increase in interphotoreceptor matrix gelatinase A (MMP-2) associated with
`age-related macular degeneration. Exp Eye Res 1998;67:637-645.
`54. Bergers G, Brekken R, McMahon G, et al. Matrix metalloproteinase-9 triggers the angiogenic switch during
`carcinogenesis. Nat Cell Biol. 2000;2:737-744.
`55. El Bradey M, Cheng L, Bartsch DU, et al. Preventive versus treatment effect of Ag3340, a potent matrix
`metalloproteinase inhibitor in a rat model of choroidal neovascularization. J Ocular Pharm Ther. June;20:217-236.
`56. Blodi BA. AG3340 Study Group. Effects of prinomastat (AG3340), an angiogenesis inhibitor, in patients with
`subfoveal choroidal neovascularization associated with age-related macular degeneration. Invest Ophthalmol Vis
`Sci. 2001 42:S311.
`57. Koh HJ, Freeman WR, Azen S P, et al. Effect of a novel octapeptide urokinase fragment, A6, on experimental
`choroidal neovascularization in the monkey. Retina. 2006;26:202-209.
`
`Samsung et al. v. Regeneron IPR2023-00884
`Regeneron Pharmaceuticals, Inc. Exhibit 2113 Page 10
`
`

`

`58. Yoshida A, Yoshida S, Khalil AK, Ishibashi T, Inomata H. Role of NF-kappaB-mediated interleukin-8 expression
`in intraocular neovascularization. Invest Ophthalmol Vis Sci. 1998;39:1097-1106.
`59. Koch AE, Polverini PJ, Kunkel SL, et al. Interleukin-8 as a macrophage-derived mediator of angiogenesis.
`Science. 1992;258:1798-1801.
`60. Elner VM, Strieter RM, Elner SG, Baggiolini M, Lindley I, Kunkel SL. Neutrophil chemotactic factor (IL-8) gene
`expression by cytokine-treated retinal pigment epithelial cells. Am J Pathol. 1990;136:745-750.
`61. Limb GA, Chignell AH, Green W, LeRoy F, Dumonde DC. Distribution of TNF alpha and its reactive vascular
`adhesion molecules in fibrovascular membranes of proliferative diabetic retinopathy. Br J Ophthalmol.
`1996;80:168-173.
`62. Oh H, Takagi H, Takagi C, et al. The potential angiogenic role of macrophages in the formation of choroidal
`neovascular membranes. Invest Ohpthalmol Vis Sci. 1999;40:1891-1898.
`63. Majka S, McGuire PG, Das A. Regulation of matrix metalloproteinase expression by tumor necrosis factor in a
`murine model of retinal neovascularization. Invest Ophthalmol Vis Sci. 2002;43:260-266.
`64. Lange J, Yafai Y, Reichenbach A, Wiedemann P, Eichler W. Regulation of pigment epithelium-derived factor
`production and release by retinal glial (Muller) cells under hypoxia. Invest Ophthalmol Vis Sci. 2008;49:5161-5167.
`65. Dawson DW, Volpert OV, Gillis P, et al. Pigment epithelium-derived factor: a potent inhibitor of angiogenesis.
`Science. 1999;285:245-248.
`66. Spranger J, Osterhoff M, Reimann M, et al. Loss of antiangiogenic pigment epithelium-derived factor in
`patients with angiogenic eye disease. Diabetes. 2001;50:2641-2645.
`67. Stellmach V, Crawford SE, Zhou W, Bouck N. Prevention of ischemia-induced retinopathy by the natural ocular
`antiangiogenic agent pigment epithelium-derived factor. Proc Natl Acad Sci U S A. 2001;98:2593-2597.
`68. Duh EJ, Yang HS, Suzuma I, et al. Pigment epithelium-derived factor suppresses ischemia-induced retinal
`