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Arch Ophthalmol. 2004;122:650-652.

The development of fibrous tissue in patients with occult choroidal neovascularization (CNV) associated with age-related macular degeneration (AMD) is one of the major determinants of vision loss. Usually this process is slow with the development of disciform scarring that exceeds 50% of the lesion occurring in only 20% of eyes by 12 months.¹ The typical time course is between 30 and 100 months of follow-up.² Interestingly, in the absence of subretinal blood or classic CNV, no eyes with occult CNV developed more than 50% disciform scarring in a 9- to 12-month follow-up.³

In the present case, we describe clinicopathologic findings in a patient with AMD who developed progressive subretinal fibrovascular proliferation associated with occult CNV.

Report of a Case
A 76-year-old man had a 3-month history of vision loss and a visual acuity of 20/60 OD as well as turbid subretinal fluid in that eye (Figure 1A). The fellow eye demonstrated a visual acuity of 20/30 and the retinal examination findings in this eye showed drusen and pigmentary changes of the retinal pigment epithelium (RPE). On stereofluorescein angiography, leakage of undetermined origin at the level of the RPE consistent with a 4-disc area of occult CNV was identified in the right eye (Figure 1B and C). The right eye underwent a rapid and progressive fibrosis over the ensuing 6 months (Figure 1D-F) with visual acuity declining to 20/400. The patient underwent vitrectomy with removal of the subretinal neovascular fibrotic scar.

View larger version (63K): [in this window] [in a new window] Figure 1. Fundus photographs and early and late frames of the fluorescein angiogram of the patient at baseline (A-C) and at 6 months (D-F).

The surgical specimen measured 4 x 3.5 x 1.2 mm. Microscopic analysis of the neovascular complex demonstrated a well-demarcated lesion with a 2-component fibrous scar (Figure 2A). The overall thickness of the lesion was approximately 1200 µm with the subretinal and intra-Bruch's membrane components measuring 1100 and 100 µm, respectively. Fibroblastlike cells were evident throughout the extensive matrix of collagen. Many microvascular channels were prominent in the region external to the RPE and within the outer subretinal area. The innermost region of fibrosis was relatively acellular and contained few vascular structures.

View larger version (95K): [in this window] [in a new window] Figure 2. A, Hematoxylin-eosin staining revealed extensive fibrous tissue internal to Bruch's membrane with fibroblastlike cells evident (arrows). Many small vascular channels were detected (asterisks). The inner layer of Bruch's membrane was intact (small arrowheads). The retinal pigment epithelial (RPE) layer was attenuated with focal disruption and loss of pigment granules into surrounding tissues (large arrowheads). B, The subretinal pigment epithelial (subRPE) tissue was markedly hypercellular confirmed by DAPI staining of the cell nuclei. Scale bar = 50 µm.

DAPI staining for cellular nuclei demonstrated striking hypercellularity in the subRPE component of the scar (Figure 2B). Many cells in this area were positive for CD68⁺, a macrophage marker (Figure 3A). The neovascular structures stained positively for CD34⁺, a marker for microvascular endothelium and hematopoetic stem cells (Figure 4A), and for von Willebrand factor (vWF) (Figure 4B), a marker for differentiated endothelial cells. Numerous individual cells identified as CD34⁺ cells were also located throughout the scar. Within the complex, multiple individual isolated cells were CD34⁺ but vWF negative, indicating possible immature endothelial cells (Figure 4A and B). Tissue stains for vascular endothelial growth factor (VEGF) and pigment epithelium–derived factor were negative but strongly positive for platelet-derived growth factor (PDGF) B within neovascular endothelial cells (Figure 3B). The VEGF from the subretinal fluid was below the detectable levels by enyme-linked immunosorbent assay (Quantikine Human VEGF Immunoassay; R&D Systems, Inc, Minneapolis, Minn).

View larger version (74K): [in this window] [in a new window] Figure 3. A, Immunoperoxidase staining for CD68+ demonstrates extensive staining (arrows) within the subretinal pigment epithelial (subRPE) and subretinal space. B, Cy3 conjugated fluorescent immunolabeling (red) for platelet-derived growth factor B exhibiting positive staining within multiple vascular channels (asterisks) both in the subRPE and subretinal components of the lesion. Scale bar = 50 µm.

View larger version (42K): [in this window] [in a new window] Figure 4. Cy3 conjugated fluorescent immunolabeling (red) of CD34+ (Becton Dickinson, San Jose, Calif) (A) and von Willebrand factor (vWF)–positive (Becton Dickinson) (B) cells found both adjacent to vascular structures (asterisks) and as individual cells (arrows) in the both the subretinal pigment epithelial (subRPE) and subretinal components of the lesion. Throughout the lesion more cells were CD34+ positive (A) than were vWF positive (B). Scale bar = 50 µm.

Comment
This patient demonstrated rapid fibrosis of an occult fibrous neovascular membrane. The histopathology of the present lesion contained microvascular channels, a prominent fibrotic response, hypercellularity of the tissue comprising both inflammatory and endothelial cells, and expression of PDFG B within vascular channels.

A hypercellular response was most prominent in that portion of the scar adjacent to the RPE. Part of the cellular responses appeared to be a prominent inflammatory component consisting of CD68⁺ macrophages. This finding suggests that these cells may participate in the evolution of fibrous neovascular membranes and support the inflammatory paradigm of CNV in AMD. The origin of vascular elements contributing to the subretinal fibrovascular membrane in AMD remains uncertain. In the present case, endothelial cells located within the microvascular channels were strongly positive for CD34⁺ and vWF. Many individual cells isolated within the fibrous matrix of this membrane were CD34⁺ but vWF negative. In addition, many endothelial marker positive cells were identified as isolated, individual cells rather than embedded in a neovascular channel. These findings, in this case, suggest that a cellular rather than a vascular invasion may have played a role in the pathogenesis of the neovascular structures. In addition, while vWF staining is specific to endothelial cells, CD34⁺ also serves as a marker for hematopoetic stem cells. Recently, work with stem cells has highlighted the capacity of these cells to differentiate into vascular elements both in vitro and in vivo under the influence of VEGF and PDGF.⁴ Whether this process occurs in the pathogenesis of CNV remains speculative.

Consistent with a previous report on cytokine production in CNV undergoing involution,⁵ this case exhibited a lack of VEGF expression both by immunohistochemistry and quantitative enzyme-linked immunosorbent assay. However, prominent PDGF staining was detected within the vascular channels. In summary, this surgically excised fibrovascular membrane demonstrates additional findings that add to our knowledge of the molecular biology of neovascular AMD.

None of the authors has a proprietary interest in any test or product described within this article.

AUTHOR INFORMATION

Karl Csaky, MD, PhD
Bethesda, Md

Judit Baffi, MD, PhD
Bethesda, Md

Chi-Chao Chan, MD
Bethesda, Md

Gordon A. Byrnes, MD
Bethesda, Md

Corresponding author and reprints: Karl Csaky, MD, PhD, Bldg 10, Room 10N119, NEI/NIH, 9000 Rockville Pike, Bethesda, MD 20895-1857 (e-mail: kcsaky{at}helix.nih.gov).

REFERENCES
1. Bressler NM, Frost LA, Bressler SB, Murphy RP, Fine SL. Natural course of poorly defined choroidal neovascularization associated with macular degeneration. Arch Ophthalmol. 1988;106:1537-1542. FREE FULL TEXT 2. Soubrane G, Coscas G, Francais C, Koenig F. Occult subretinal new vessels in age-related macular degeneration: natural history and early laser treatment. Ophthalmology. 1990;97:649-657. ISI | PUBMED 3. Stevens TS, Bressler NM, Maguire MG, et al. Occult choroidal neovascularization in age-related macular degeneration: a natural history study. Arch Ophthalmol. 1997;115:345-350. FREE FULL TEXT 4. Yamashita J, Itoh H, Hirashima M, et al. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature. 2000;408:92-96. FULL TEXT | PUBMED 5. Grossniklaus HE, Ling JX, Wallace TM, et al. Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization. Mol Vis. 2002;8:119-126. ISI | PUBMED

SECTION EDITOR: W. RICHARD GREEN, MD

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