Idiopathic subfoveal choroidal neovascular membrane in a 21-month-old child: Ultrastructural features and implication for membranogenesis

Idiopathic subfoveal choroidal neovascular membrane in a 21-month-old child: Ultrastructural features and implication for membranogenesis Anthony B. Daniels, MD, MSc,a,b Frederick A. Jakobiec, MD, DSc,a,b,c Corey B. Westerfeld, MD,a,b,d Akira Hagiwara, MD,a,b,d,e Norman Michaud, MS,a,b,c and Shizuo Mukai, MDa,b,d PURPOSE

To report the clinical and pathologic features of an idiopathic choroidal neovascular membrane (CNVM) in a 21-month-old child and to discuss the unique findings of infantile CNVM in the context of understanding the mechanism of membrane formation.

METHODS

The CNVM was removed by submacular surgery. Light and electron microscopic tissue analyses were used to elucidate the structure and constituents of the CNVM. Postoperative vision was 20/60 at 10 months without evidence of recurrence. Endothelium-lined vascular channels were observed within a membrane composed entirely of retinal pigment epithelial (RPE) cells with an associated fibrocollagenous and amorphous matrix. No inflammatory cells were identified. The RPE cells toward the inner (photoreceptor) side of the membrane exhibited a less-differentiated appearance, having lost their polarity and most of their cytoplasmic melanin granules. They secreted more prominent fibrils and mucopolysaccharides. Fenestrated endothelial cells surrounded by pericytes were present between the proliferating RPE cells.

RESULTS

CONCLUSIONS

Submacular surgery can be beneficial for idiopathic CNVM in pediatric patients, even at this early age. Proliferating RPE cells in the current membrane appear to be capable of performing all of the necessary functions associated with membranogenesis, even in the absence of inflammatory cells. ( J AAPOS 2010;14:244-250)

S

ubfoveal choroidal neovascular membrane (CNVM) is rarely found in patients younger than 18 years of age.1,2 In this age group, it is most often associated with chorioretinal inflammatory conditions; on pathologic examination, an inflammatory cell infiltrate is often present in the membrane.3,4 Other causes of pediatric CNVM, including developmental abnormalities, dystrophies, and trauma, have been described, yet many case series also include the category of idiopathic CNVM (Table 1).3-12 Inflammatory CNVM, such as that found in the presumed ocular histoplasmosis syndrome, is not uncommon in children.3,10 In contrast, idiopathic CNVM is extremely Author affiliations: aDepartment of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts; bHarvard Medical School, Boston, Massachusetts; cDavid G. Cogan Laboratory of Ophthalmic Pathology, and dRetina Service, Massachusetts Eye and Ear Infirmary, Boston; and eDepartment of Ophthalmology and Visual Science, Chiba University Graduate School of Medicine, Chiba, Japan This study was funded by gifts to the Mukai Fund, Massachusetts Eye and Ear Infirmary. A portion of this work was presented at the Third Meeting of the Association of Pediatric Retina Surgeons, Duck Key, Florida, January 2009. Submitted November 3, 2009. Revision accepted January 15, 2010. Published online April 23, 2010. Reprint requests: Shizuo Mukai, MD, Retina Service, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114-3096 (email: shizuo_mukai@meei. harvard.edu). Copyright Ó 2010 by the American Association for Pediatric Ophthalmology and Strabismus. 1091-8531/2010/$36.00 1 0 doi:10.1016/j.jaapos.2010.01.010

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rare, especially in young children, with only 2 cases reported in the first decade of life.5,6 Current management options for pediatric subfoveal CNVM include observation, laser photocoagulation, photodynamic therapy, intravitreal injection of bevacizumab, and submacular surgery. Surgical removal of subfoveal CNVM appears to be promising in all types of pediatric CNVM, especially in advanced cases that are chronic, where the starting visual acuity is poor (\20/200), and in cases in which there is significant neurosensory detachment.3,5,10 The visual outcome in idiopathic cases has been generally good, as summarized in Table 1. On pathologic analysis, pediatric CNVM usually contains an admixture of retinal pigment epithelium (RPE), fibrocytes, collagen, vascular endothelium, and lymphocytes, whereas basal laminar deposits commonly seen in adult CNVM are absent.3 In the CNVM associated with age-related macular degeneration, the cellular constituents of the membranes and the signaling pathways leading to their development are being elucidated.13-17 It is not known what types of cells and signaling pathways are responsible for membranogenesis in children, especially in the absence of an inflammatory provocation or condition. In this report, we describe the youngest patient to date (21 months) with an idiopathic CNVM, which was successfully managed by surgical removal of the CNVM. Light microscopic evaluation of one-micron Epon sections and electron microscopic analysis revealed a membrane with

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Table 1. Idiopathic choroidal neovascularization in children in the English-language literature Author a

Daniels et al (2010) Jain et al (2002)5 Cakir et al (2009)6 Wilson and Mazur (1988)7 Giovannini et al (2007)8 Sears et al (1999)3 Mimouni et al (2003)9 Uemura and Thomas (2000)10 Giansanti et al (2005)11 Goshorn et al (1995)4 Gomi et al (2007)12

Age

Treatment

21 mo 6 yr 8 yr 10 yr 10 yr 14 yr 11 yr 11 yr 11 yr 13 yr 12 yr 14 yr 16 yr 13 yr 15 yr 16 yr 16 yr 16 yr

Surgical removal Surgical removal Intravitreal bevacizumab Laser photocoagulation PDT 1 intravenous steroid PDT Surgical removal PDT PDT PDT Surgical removal Surgical removal Surgical removal PDT PDT None None TA, Intravit. bevacizumab

Initial VA

Final VA

CF CF 20/400 20/40 20/200 20/50 20/400 NR NR NR 20/300 20/200 20/300 20/80 20/200 20/25 20/80 20/100

20/60 20/30 20/50 20/30 20/200 20/100 20/125 NR NR NR 20/30 20/20 20/30 20/63 20/125 20/20 20/25 20/33

VA, visual acuity; CF, counting fingers; PDT, photodynamic therapy; NR, not reported; TA, posterior sub-Tenon’s triamcinolone acetonide injection. Current study.

a

a unique cellular pattern devoid of inflammatory cells and consisting entirely of proliferating RPE cells and neovascular units. These observations suggests that an inflammation-independent mechanism involving the RPE can play a key role in the formation of idiopathic CNVM in children.

Patients and Methods Visual acuity was measured preoperatively by the use of Allencards and postoperatively by Snellen chart with single letters, both according to standard practice. For surgical removal of the CNVM, after standard 3-port pars plana vitrectomy, a 32-gauge subretinal cannula was used to create a retinotomy temporal to the membrane in the neurosensory detachment, to inject balanced-salt solution into the subretinal space around the membrane, and to gently mobilize the CNVM. Posterior hyaloid was not surgically detached.18 Subretinal forceps were inserted through the retinotomy into the subretinal space to grasp the edge of the membrane and to deliver it in toto through the retinotomy. No hemorrhage was encountered. Subsequent air-fluid exchange was performed. The excised membrane was placed in a mixture of 2.5% glutaraldehyde and 2% formaldehyde fixative buffered in 0.1 M cacodylate followed by 2% osmium fixation in an aqueous solution. One-micron Epon sections were prepared and stained with toluidine blue for light microscopic evaluation and ultrathin sections were prepared for ultrastructural analysis. The institutional review board determined that approval was not required for this study. Informed consent was obtained for all procedures, and all work reported herein is compliant with the Health Insurance Privacy and Accountability Act.

Case Report A 21-month-old boy with no significant past medical history was noted to have right esotropia for 5 months. Exam-

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ination by a pediatric ophthalmologist and a retina specialist diagnosed an elevated central macular lesion in his right eye. Serologic testing for Toxocara canis, Toxoplasma gondii, Histoplasma capsulatum, cytomegalovirus, herpes simplex virus types 1 and 2, and rubella were all negative. Examination of his left eye was completely normal, and there was no family history of any macular dystrophies, any familial syndromes, or any other eye disease. The patient was referred to the Retina Service of the Massachusetts Eye and Ear Infirmary for evaluation of the macular lesion. In addition to the aforementioned history, he was a full-term product of an uneventful pregnancy, with no other ocular history, including no history of trauma. Retrospective examination of his family photographs revealed leukocoria by flash photography as far back as age 13 months, 8 months before our evaluation. The examination revealed visual acuity of counting fingers in the right eye and 10/30 by Allen cards in the left. A constant right esotropia was present. Intraocular pressure and anterior segment examination were normal in each eye. Dilated fundus examination revealed a yellowish white, mildly elevated subretinal lesion in the central macula, part of which was under the fovea. It was surrounded by a neurosensory detachment greater than twice the area of the CNVM (Figure 1A). The remainder of the retinal examination, including peripheral retinal examination with scleral depression, was normal, as was the findings from the examination of the left eye. Fluorescein angiography demonstrated hyperfluorescence of the lesion with late leakage of the dye (Figure 1B) and pooling of fluorescein in the area of the neurosensory detachment (Figure 1C). The remainder of the fluorescein angiogram, including peripheral sweeps with the 130 lens with the RetCam II system (Clarity Medical Systems, Pleasanton, CA), was normal. The diagnosis of subfoveal CNVM was made. Fluorescein angiography of the left eye was

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FIG 1. A, Preoperative 80 RetCam II fundus photograph. B and C, Fluorescein angiogram at 22 s and 10 min 47 s, respectively, demonstrating a subfoveal, type 2 choroidal neovascular membrane surrounded by a neurosensory detachment. D, Postoperative 80 RetCam II fundus photograph. E and F, Fluorescein angiogram at 23 s and 7 min 46 s, revealing no recurrence of the membrane and absorption of the subretinal fluid. Retinal pigment epithelial changes corresponding to the areas of previous choroidal neovascular membrane and serous retinal detachment are present.

normal. The CNVM was thought to be idiopathic with no clinical evidence of inflammation or trauma. Surgical removal of the CNVM was recommended and was carried out without complication as described in the Methods section. On examination under anesthesia 1 month postoperatively, there was total resorption of the subretinal fluid, with no recurrence of the CNVM. Retinal pigment epithelial changes were present corresponding to the areas of previous CNVM and neurosensory detachment (Figure 1D). No leakage or pooling of fluorescein was seen by angiography (Figures 1E and 1F). After removal of the CNVM, the patient was treated with occlusion therapy, and his visual acuity improved to 20/60 by the 10-month postoperative visit. He had complete office-based dilated fundoscopic examinations at the 3-month, 5-month, and 10-month visits,

and there was no evidence of recurrence. Visual acuity of the left eye was 20/20. Light and Electron Microscopic Findings The excised CNVM measured around 1.5  1.0 mm and was evaluated with light microscopy and transmission electron microscopy with the use of standard techniques as described in the Methods section. Low-power examination of the plastic sections disclosed a moderately cellular membrane with more pigmented cells present at the base (ie, the surface next to Bruch’s membrane) than at the apex next to the photoreceptors (Figure 2A). Orientation of the excised membrane was possible because fragments of photoreceptor outer segments were found adjacent to its apical surface. Both the pigmented and less-pigmented

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cells were elongated rather than cuboidal or columnar in shape, and they were separated from each other by a delicate, loosely fibrillar matrix. Scattered small blood vessels with obvious lumens were present, but inflammatory cells were absent (Figures 2B and 2C). The more intensely pigmented epithelial cells at the base possessed large round fully melaninized granules that were evenly dispersed throughout the cytoplasm and that did not obscure the nucleus or exhibit coarse clumping (Figure 2C). No macrophages were detected. Transmission electron microscopy revealed at the base of the membrane a remnant of cuboidal RPE cells. A continuous basement membrane was discovered along the straight plasmalemmal surface abutting the new stroma in lieu of apical villi. Some elongated cells demonstrated subplasmalemmal thin actin-type filaments possessing fusiform densities (Figure 3A). In occasional regions the RPE cells adopted a polyhedral shape and contained within their cytoplasms parallel stacks of rough endoplasmic reticulum and myriad mitochondria but no phagolysosomes. Basement membrane material was present on the outer aspect of the cells facing the stroma but not between the apposed cells (Figure 3B). The microcirculatory elements consisted of endothelial cells with fenestrae (attenuated foci of endothelial cytoplasm consisting of a membranous diaphragm) encircled on the outside by multiple interrupted layers of pericytes with scattered thin filaments (Figure 3C). Reduplicated basement membrane material enveloped both the endothelial cells and pericytes. No inflammatory cells or fibrocytes were observed in any of the ultrathin sections of the membrane that were examined. Rare RPE cells manifested an intracytoplasmic pseudolumen and cytoplasmic lipofuscin but true intracellular lumens were absent (Figure 3D).

Discussion

FIG 2. Light microscopy of the excised choroidal neovascular membrane. A, Multilaminar subretinal membrane is composed predominantly of elongated plump pigmented cells on the bottom (next to Bruch’s membrane), which become less pigmented toward the top (next to photoreceptors; 1-mm Epon section, toluidine blue, original magnification 200). B, Scattered small blood vessels (arrows) are present at all levels of the membrane (1-mm Epon section, toluidine blue, original magnification 400). C, Gradual transition of more

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We describe a 21-month-old patient with idiopathic CNVM. By history, the CNVM was present even as early as age 13 months. There was no inflammatory, infectious, developmental, or traumatic predisposition, and ophthalmoscopy and fluorescein angiography of the other eye was normal. The CNVM was subfoveal and subretinal (type 2 membrane). This child is by far the youngest reported with an idiopathic CNVM. The next youngest was reported in a 6-year-old patients, and although the CNVM was removed surgically it was not studied pathologically.5 Surgical removal of the CNVM was recommended because of the following: (1) there was a risk of amblyopia; heavily pigmented cells below into lightly pigmented cells above (1-mm Epon sections, toluidine blue, original magnification 400). No lumens are observed. Microvessels are also clearly seen in this field (arrows).

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FIG 3. Transmission electron micrographs of the excised choroidal neovascular membrane. A, Cuboidal cells on the bottom are detached from Bruch’s membrane (original magnification 4600). There are no apical villi. Linear basement membrane (arrows) separates the plasmalemma from the fibrillar matrix. The elongated cell above also exhibits a coating of basement membrane material (arrows) and contains thin cytoplasmic actin-type filaments with densities beneath the cell membrane (crossed arrow). Note that the melanin granules are singly dispersed. B, A cluster of mostly depigmented epithelial cells elaborates basement membrane when abutting the stroma (arrow; original magnification, 4600). Abundant and often parallel stacks of rough-surfaced endoplasmic reticulum (ER) are present along with many mitochondria (M). C, A capillary unit is composed of fenestrated endothelium (E) surrounded by pericytes (P) (original magnification, 3400). Reduplicated basement membrane investing both cell types is prominent (arrows). Amorphous and fibrillar material constitutes the surrounding stroma (S). The epithelial cells (Ep) in the vicinity of the capillaries lack melanin granules but are rich in mitochondria (M). Note the clear-cut basal lamina elaborated by these cells (crossed arrows). No inflammatory cells are present. D, Retinal pigment epithelium cell with rare melanin granules (arrows) displays an intracytoplasmic pseudolumen (PL) and lipofuscin (LF; original magnification, 7900). Collagen fibers (CF) are prominent in the extracellular space. N, nucleus of RPE cell.

(2) the CNVM appeared partially organized with a subfoveal edge; (3) the neurosensory detachment was greater than twice the area of the CNVM; and (4) the chronicity as evidenced by the duration of leukocoria and esotropia. Previous studies (footnotes 1, 3, 10) of submacular surgery in pediatric CNVM have reported good visual outcome for membranes of various etiologies, with 92% of patients having an improvement of vision (footnote 1). However, some eyes never attained vision better than 20/400, and there have been reported recurrences.3,10 The 6 reported cases of surgical removal of the idiopathic CNVM in children, including the presently described case, have resulted in significant visual improvement from preoperative visual acuities ranging from 20/200 to

counting fingers to postoperative visual acuities ranging from 20/20 to 20/125 (Table 1).3,5,10 The 2 reported cases of surgically excised pediatric idiopathic CNVMs that recurred did so within the first month after surgical removal.10 If one includes all pediatric CNVMs, regardless of etiology, one still finds that all recurrences occurred within 6 weeks, except for a single case of posttraumatic CNVM that recurred at 6 months.10 There are no late recurrences that we could find in the literature, and even in that last case, repeated surgery at 9 months yielded a final visual acuity of 20/30. Standard surgical techniques for submacular surgery were used in our case, although because of a strongly adherent posterior hyaloid, no attempt was made to detach it.18 Postoperative visual acuity in our patient

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Volume 14 Number 3 / June 2010 continues to improve, measuring 20/60 at 10 months after surgery. Goshorn and colleagues4 have reported spontaneous regression of pediatric CNVM, but visual outcome was usually poor when the starting visual acuity was less than 20/200. We thought that a wait-and-see approach with the hope of spontaneous resolution was not the best option in this case, with an actively leaking CNVM with a neurosensory detachment greater than twice the area of the CNVM in an infant at risk for deprivation amblyopia and having poor initial visual acuity and evidence for chronicity. Other treatment modalities, including laser photocoagulation7 and photodynamic therapy with verteporforin,8,9,11 have been used with some success in cases of pediatric CNVM. Macular laser photocoagulation is possible under anesthesia in a child this young, usually with the indirect-ophthalmoscope laser-delivery system. One would expect a central scotoma after treating a submacular CNVM even if there were significant neurosensory detachment over the CNVM. In terms of photodynamic therapy, we do not know of any center that is set up to do this in a 21-month-old infant, and we also do not know whether there is systemic toxicity from verteporforin in such a young child. Agents against vascular endothelial growth factor (VEGF) have yet to be systematically evaluated for pediatric CNVM, although some centers are starting to do this.6,12,19 Although we considered using intravitreal bevacizumab in this eye, we believed that the partially organized appearance of the membrane and its subfoveal location would limit visual recovery because, even with resolution of the neurosensory detachment and regression of the CNVM, a subfoveal organized residual scar would occur. There is also the critical question of potential systemic side effect of intravitreal anti-VEGF agents in young children.19 Unlike most CNVMs in children that are inflammatory in nature,3,4 the histopathologic and ultrastructural analyses demonstrated the membrane in our case to be strictly noninflammatory, consisting entirely of proliferating RPE cells, among which capillary channels were insinuated. Light and electron microscopic examination confirmed the absence of an inflammatory-cell component. Histopathologic studies of CNVM encountered in children and adults have featured prominent inflammation.3 Grossniklaus and Green13 described the cellular constituency of CNVM as a spectrum, with minimal inflammation at one end and a conspicuous inflammatory component at the other. Our case appears to define the noninflammatory extreme of this spectrum. In our patient’s subfoveal membrane, the endothelial cells of the neovascular units manifested fenestrae in keeping with their choroidal origin. The pericytes possessed variable numbers of cytoplasmic thin, actin-type filaments, lacking fusiform densities, were ensheathed by basement membranes and were in an intimate circumferential relationship to the endothelial cells. The RPE cells in the outer

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portion closest to the native RPE position (ie, resting on Bruch’s membrane) displayed an ultrastructural appearance closest to that of normal RPE, including a cuboidal shape and dense melaninization. The RPE cells located toward the inner zone of the membrane were elongated, exhibited oval or round nuclei, contained scattered subplasmalemmal cytoplasmic actin filaments subserving migration, and became progressively less pigmented toward the apex. This zonal pattern, the relative hypocellularity of the membrane caused by matrix accumulation elaborated by the RPE cells, and the absence of adenomatous units strongly militate against an RPE neoplasm, which furthermore has never been reported in such a young patient.20 The innermost cells near the photoreceptors produced more collagen fibrils and amorphous mucopolysaccharides in the absence of fibrocytes but retained the capacity for basement membrane production. Fibrocytes have been found in a majority of CNVMs previously studied.3 The absence of fibrocytes is another unique feature of the CNVM described here. The RPE is thought to play an important role in CNVM formation, but evidence exists that the inflammatory cells embedded within the membrane express the matrix metalloproteinases necessary to digest through tissue planes21 and that are also responsible for producing the fibrin scaffold on which the CNVM grows.22 In these models, the RPE cells have a regulatory role, first by recruiting macrophages that produce the membrane matrix itself,22 and subsequently by reining in this activity by elaborating tissue inhibitors of metalloproteinases.21 In the CNVM from our patient, however, because there were no inflammatory cells, the RPE cells appear to have initiated vasogenesis rather than simply serving a regulatory role. It is possible that inflammatory cells were present at an earlier stage of CNVM formation, but we did not find any cellular debris as evidence for this. There are studies demonstrating in vitro that VEGFsignaling by the RPE plays a role in the normal development of the vasculature in the human retina23 and choriocapillaris.24,25 Although we have no evidence for or against the role of such VEGF-mediated signaling in the formation of the CNVM in our patient, the fact that only RPE cells were present among the vascular channels in this membrane strongly suggests that these cells are the likely source of the angiogenic signaling molecules, and this finding is consistent with reports demonstrating that the RPE cells can synthesize VEGF in the formation of CNVM.26 The pathogenesis of submacular CNVMs, especially those found in children, remains incompletely understood. Although inflammatory cells appear to play an important role in many of the cases, the RPE cells are the most commonly seen cells of the CNVM.3 Our case shows that CNVM can exist strictly without an inflammatory component suggesting that inflammatory cells are not necessary for CNVM formation. Further studies are needed to elucidate the pathways involved in the formation of CNVM in children. Finally, the choice of treatment modalities for

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subfoveal CNVM in children remains case-dependent. We have shown that surgical removal can be effective even in an infant, although the future role of the anti-VEGF agents in the management of pediatric CNVM is yet to be determined.

Literature Search MEDLINE was searched via PubMed, using combinations of the following terms: idiopathic choroidal neovascular (82 articles returned) OR idiopathic choroidal neovascularization (193 articles returned) OR idiopathic choroidal neovascularisation (same 193 articles returned). No limits were set on publication date or language. All articles returned by this search were reviewed, as well as pertinent references gleaned from these articles. All articles in foreign languages had English abstracts in MEDLINE, and where the possibility persisted that a child younger than 21 months might be included in the article (eg, the article did not specify the youngest person in the age range), the original article was reviewed in the original language with native-speaking colleagues where necessary. References 1. Sivaprasad S, Moore AT. Choroidal neovascularization in children. Br J Ophthalmol 2008;92:451-4. 2. Spaide RF. Choroidal neovascularization in younger patients. Curr Opin Ophthalmol 1999;10:177-81. 3. Sears J, Capone A Jr, Aaberg T Sr, et al. Surgical management of subfoveal neovascularization in children. Ophthalmology 1999;106: 920-4. 4. Goshorn EB, Hoover DL, Eller AW, et al. Subretinal neovascularization in children and adolescents. J Pediatr Ophthalmol Strabismus 1995;32:178-82. 5. Jain K, Shafiq AE, Devenyi RG. Surgical outcome for removal of subfoveal choroidal neovascular membranes in children. Retina 2002;22: 412-7. 6. Cakir M, Cekic O, Yilmaz OF. Intravitreal bevacizumab for idiopathic choroidal neovascularization. J AAPOS 2009;13:296-8. 7. Wilson ME, Mazur DO. Choroidal neovascularization in children: Report of five cases and literature review. J Pediatr Ophthalmol Strabismus 1988;25:23-9. 8. Giovannini A, Neri P, Mercanti L, Brue C. Photodynamic treatment versus photodynamic treatment associated with systemic steroids for idiopathic choroidal neovascularization. Br J Ophthalmol 2007;91: 620-3. 9. Mimouni KF, Bressler SB, Bressler NM. Photodynamic therapy with Verteporfin for subfoveal choroidal neovascularization in children. Am J Ophthalmol 2003;135:900-902.

Volume 14 Number 3 / June 2010 10. Uemura A, Thomas MA. Visual outcome after surgical removal of choroidal neovascularization in pediatric patients. Arch Ophthalmol 2000;118:1373-8. 11. Giansanti F, Virgili G, Varano M, et al. Photodynamic therapy for choroidal neovascularization in pediatric patients. Retina 2005;25: 590-96. 12. Gomi F, Nishida K, Oshima Y, et al. Intravitreal bevacizumab for idiopathic choroidal neovascularization after previous injection with posterior subtenon triamcinolone. Am J Ophthalmol 2007;143: 507-10. 13. Grossniklaus HE, Green WR. Choroidal neovascularization. Am J Ophthalmol 2004;137:496-503. 14. Thomas JW, Grossniklaus HE, Lambert HM, et al. Ultrastructural features of surgically excised idiopathic subfoveal neovascular membranes. Retina 1993;13:93-8. 15. Grossniklaus HE, Hutchinson AK, Capone A Jr, et al. Clinicopathologic features of surgically excised choroidal neovascular membranes. Ophthalmology 1994;101:1099-111. 16. Lopez PF, Sippy BD, Lambert HM, et al. Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration. Invest Ophthalmol Vis Sci 1996;37:855-68. 17. Kvanta A, Algvere PV, Berglin L, Seregard S. Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor. Invest Ophthalmol Vis Sci 1996;37: 1929-34. 18. Lit ES, Kim RY, D’Amico DJ. Surgical removal of subfoveal choroidal neovascularization without removal of posterior hyaloid: A consecutive case series in younger patients. Retina 2001;21:317-23. 19. Avery RA. Extrapolating anti-vascular endothelial growth factor therapy into pediatric ophthalmology: Promise and concern. J AAPOS 2009;13:329-31. 20. Shields JA, Shields CL, Gunduz K, Eagle RC Jr. Neoplasms of the retinal pigment epithelium: The 1998 Albert Ruedemann, Sr, Memorial Lecture, Part 2. Arch Ophthalmol 1999;117:601-8. 21. Steen B, Sejersen S, Berglin L, et al. Matrix metalloproteinases and metalloproteinase inhibitors in choroidal neovascular membranes. Invest Ophthalmol Vis Sci 1998;39:2194-200. 22. Grossniklaus HE, Ling JX, Wallace TM, et al. Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization. Mol Vis 2002;8:119-26. 23. Gogat K, Le Gat L, Van Den Berghe L, et al. VEGF and KDR gene expression during human embryonic and fetal eye development. Inv Ophthalmol Vis Sci 2004;45:7-14. 24. Sakamato T, Sakamato H, Murphy TL, et al. Vessel formation by choroidal endothelial cells in vitro is modulated by retinal pigment epithelial cells. Arch Ophthalmol 1995;113:512-20. 25. Marneros AG, Fan J, Yokoyama Y, et al. Vascular endothelial growth factor expression in the retinal pigment epithelium is essential of choriocapillaris development and visual function. Am J Pathol 2005;167: 1451-9. 26. Spilsbury K, Garrett KL, Shen W-Y, et al. Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization. Am J Pathol 2000;157:135-44.

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