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(Investigative Ophthalmology and Visual Science. 2006;47:1642-1645.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0779
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The Presence of AC133-Positive Cells Suggests a Possible Role of Endothelial Progenitor Cells in the Formation of Choroidal Neovascularization

Carl M. Sheridan,1 Deborah Rice,1 Paul S. Hiscott,1 David Wong,1,2 and David L. Kent1,3

1From the Department of Ophthalmology, School of Clinical Sciences, University of Liverpool, Liverpool, United Kingdom; 2St. Paul’s Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom; and the 3Eye Service, Aut Even Hospital, Kilkenny, Ireland.


   Abstract
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PURPOSE. Recent evidence suggests that vasculogenesis as well as angiogenesis occurs throughout the body during neovascularization. The recruitment of circulating stem cells is a key feature of vasculogenesis. The purpose of the present study was to determine whether markers of endothelial progenitor cells (EPCs) are present in choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD).

METHODS. Surgically excised CNV (n = 9) membranes from patients with AMD were probed with immunohistochemical techniques using the following monoclonal antibodies: AC133 a putative marker of EPCs and hematopoietic stem cells (HSCs); the endothelial cells markers CD31, CD34, and von Willebrand factor (vWF); and cytokeratins and CD68, markers for retinal pigment epithelium (RPE) and macrophages, respectively. After secondary antibody amplification, reactions were visualized with fast red substrate.

RESULTS. Six of nine specimens demonstrated cells positive for AC133 that were all found within predominantly cellular regions of the specimens. In the avascular fibrous stromal core of all specimens, the predominant cells were RPE cells and macrophages. The peripheral component of all CNV membranes was highly vascular and showed varying immunoreactivity for all endothelial markers. The greatest immunoreactivity for endothelial markers was observed with CD34 and vWF and least for CD31.

CONCLUSIONS. These findings support animal studies that vasculogenesis, in addition to angiogenesis, may contribute to the neovascularization that occurs in AMD.

Angiogenesis may be defined as the formation of new blood vessels from pre-existent vasculature and, until recently, was considered the sole mechanism by which neovascularization occurs in postnatal life. However, modification of this concept is now needed, because endothelial progenitors cells (EPCs) or angioblasts derived from bone marrow stem cells have been identified in the adult and have been shown to participate in blood vessel formation in both physiological and pathologic states.1 2 3 4 5 6 7 Hence, it now appears that vasculogenesis, the formation of blood and blood vessels from the in situ differentiation of pluripotent progenitor cells or hemangioblasts, which gives rise to the circulatory system in the embryo, also plays a role in new blood vessel formation postnatally—so-called postnatal vasculogenesis. Furthermore, recent evidence from bone marrow transplantation experiments, suggesting that stem cells (HSCs) can be found in several nonhematopoietic tissues in the body, raises the possibility that these cells may be capable of actual tissue regeneration in addition to vasculogenesis.8 9 10 11 12 13

The process of submacular angiogenesis seen in association with a variety of chorioretinal disorders is termed choroidal neovascularization (CNV). It is most commonly encountered in age-related macular degeneration (AMD), the commonest cause of irreversible vision loss in the Western world.14 15 The purpose of the present study was to investigate whether adult bone-marrow–derived stem cells are present in CNV secondary to AMD.


   Methods
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The methods conformed to the Declaration of Helsinki for research involving human subjects and have Liverpool Research Ethics Committee approval. Nine surgically excised subfoveal CNV membranes secondary to AMD were obtained during pars plana microsurgery. Normal human eyes were obtained from the local eye bank (Royal Liverpool University Hospital). All specimens (globes and membranes) were fixed in formalin and embedded in wax. After the blocks were cooled, 5-µm sections were cut with a rotary microtome (model A532; Shandon, Sewickley, PA). Sections were placed on 3% aminopropylethoxysilane-coated (APES; Sigma-Aldrich, Poole, UK) glass slides before they were dewaxed in a 60°C oven for 20 minutes. They were then transferred to xylene for 5 minutes (one change) and rehydrated through 5-minute immersions in descending ratios of alcohol (99%–95%; one change of each) to distilled water before being either stained histologically (with hematoxylin and eosin) or immunohistochemically.

Immunoreactivity for monoclonal antibody (AC133-1; Miltenyi Biotec, Cologne, Germany) against a human antigen recently identified as a marker of both EPCs and HSCs and absent on mature endothelium was compared to that for cell markers specific for fully differentiated vascular endothelial cells: von Willebrand factor (vWF), CD34 and CD31 (all 1:100; Dako, High Wycombe, UK), macrophages (CD68; KP1, 1:100; Dako), and RPE (panel cytokeratins/cytokeratin 18 clone CY90 (CK18, 1:10; Sigma-Aldrich). Briefly, sections were rehydrated by washing in TBS for 10 minutes. Nonspecific binding was blocked by exposure to 5% normal goat serum (NGS; Sigma-Aldrich) in TBS (vol/vol) for 20 minutes in a humidity chamber. The blocking solution was then drained off, and the primary antibody was added overnight at +4°C at the manufacturer’s recommended dilution. After three 5-minute washes with Tris-buffered saline (TBS) and according to the manufacturer’s instructions, secondary amplification (Envision; Dako) of alkaline phosphatase (AP)-labeled polymer conjugates to affinity-purified goat anti-mouse immunoglobulins was performed with a 30-minute incubation at room temperature. After a subsequent washing in TBS (three times, 5 minutes each), immunoreactivity was visualized with fast red chromogen for 5 minutes and then sections were mounted under a glass coverslip, using mounting medium (Immunomount; Euro-Path Ltd., UK). The cell type that exhibited positive immunostaining was determined by morphologic and cytologic criteria as well as immunostaining. In negative control experiments, preimmune serum replaced the specific antibodies. All specimens were sequentially viewed in their entirety under a x40 objective and each field of view was scored as either a fibrotic or cellular region (>50%) of the membrane. The number of cells per field were scored as 0 (–); 1 to 3 (+); 4 to 10 (++), or >10 (+++) cells per field.


   Results
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Morphology of CNV Membranes
Histochemical evaluation revealed a stereotypical morphology for all nine specimens of CNV consisting of a relatively avascular and paucicellular fibrous stromal core and a peripheral cell-rich outer zone. The membranes contained cells, arranged in layers or foci, with a variable amount of extracellular fibrous tissue. Eight of the nine CNV-AMD specimens displayed a predominantly cellular histology (>50% of total membrane; see Table 1 ).


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  		TABLE 1. Summary of CNV’s Cellular Distribution

 
Immunohistochemistry of CNV Membranes
Confirmation of the mature differentiated nature of the endothelial cells making up new blood vessels was demonstrated by the positivity of these cells to known EC markers (Fig. 1) . Endothelial cell markers CD31, CD34, and vWF were positive in all nine CNV membranes studied and were found in both fibrotic (Figs. 1A 1B 1C) and cellular (Figs. 1D 1E 1F) regions of the membranes. Positive immunostaining was observed for all three markers in large blood vessels, small capillaries, and apparent single cells. The range of immunopositive staining ranged from 1% to >50% for the endothelial cell markers throughout each membrane (Fig. 1) . Six of the nine CNV membranes studied contained cells that were immunoreactive for markers against AC133 (Fig. 2) . No positive reaction for AC133 cells was observed in any of the fibrotic regions of the membranes studied. Cellular regions of six membranes demonstrated less than 0.1% positivity with AC133 and were observed as sparse individual cells (Figs. 2A 2D) . None of these positive cells were incorporated within large blood vessels or small capillaries. Areas showing AC133 positivity were further analyzed in sequential sections stained with markers to endothelial cells and cytokeratins revealed the presence of CD34-positive cells within this area (>1%–10%) with some CD34 cells appearing as isolated cells (Fig. 2B) . Cytokeratin immunostaining was negative within this area.


Figure 1
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  		FIGURE 1. Photomicrographs of a fibrotic (A–C) and cellular (D–F) CNV membrane. The fibrous membrane consisted of a fibrous core containing vessels that were immunoreactive for the endothelial cell marker CD34 (A; arrows). Surrounding the fibrovascular tissue were cytokeratin-positive RPE cells (B; arrows; cytokeratin 18). The specimen was negative for the antibody directed against AC133 (C). The cellular region of a CNV membrane showed immunoreactivity for cytokeratin-positive cells (D) throughout the membrane. The membrane also consisted of blood vessels, which were immunoreactive for the endothelial cell markers CD34 (E; arrows) and vWF (F). Endothelial cell immunoreactivity was seen in large mature blood vessels (open arrows) and isolated cells or small capillaries (filled arrows).

 

Figure 2
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  		FIGURE 2. A cellular region within a CNV membrane has an isolated cell that was immunoreactive for AC133 (A; arrow). The sequential sections showed immunoreactivity for CD34 (B) and mouse IgG negative control (C). A high-magnification photomicrograph demonstrated an AC133+ cell (arrow) within the CNV (D). (E) Photomicrograph of a normal eye in the region of the ciliary processes which demonstrated a single cell (arrow) showing positivity for AC133 (A). AC133 positivity was not observed in any other ocular tissues studied (n = 12).

 
Immunohistochemistry on three whole globes demonstrated no immunoreactivity in the retina or choroid to AC133 antibody (n = 6). Only 1 positive cell was seen in all sections (n = 12) of normal globes studied. This positive cell was observed adjacent to a small blood vessel of the ciliary body (see Fig. 2E ).


   Discussion
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There is now strong evidence to support the concept that cells resident in the bone marrow can enter the circulation and participate in neovascularization and regeneration in adult species.1 2 3 4 5 7 8 9 10 11 12 In keeping with this evidence, the results of the present study suggest that vasculogenesis may contribute to the clinical entity we term neovascular AMD, supporting a potential role for these cells, not just in blood vessel formation, but also in the reparative response known to occur in CNV.16 Although they have the ability to differentiate into mature endothelial cells, they can be separated from mature circulating endothelial cells on the basis that they do not express characteristic mature EC markers.17 18 19 Nevertheless, both precursor and mature endothelial cells can express markers specific to endothelial cells.20 21 22 23 24 Isolating a specific marker for EPCs is complicated by the fact that cell markers such as VEGFR1 (flt-1), CD34, platelet endothelial cell adhesion molecule (PECAM; CD31), and vWF can also be shared by HSCs.25 In addition, HSCs and EPCs also share the cell surface marker AC133.26 27 Expression of AC133 is rapidly downregulated as both EPCs and HSCs differentiate, so much so that CD34-positive cells that express AC133 appear to be true indicators of cells representative of either EPCs or HSCs.26 27 28

In our study, AC133-positive cells were sparsely present in six of nine specimens. We were unable to identify any AC133-positive cells that had been incorporated into blood vessels in the specimens, which is probably due to the rapid downregulation of AC133 during differentiation.26 27 28 In addition, the single time point of analysis (i.e., membrane removal and fixation) makes it more difficult to observe colocalization. Nevertheless, the positive labeling for AC133 suggests that these cells may be HSCs or EPCs. The paucity of cells may be a reflection of the disease itself, or it could reflect the duration of the neovascular process in that all patients had CNV diagnosed several months before surgery, more that sufficient time to allow precursors to differentiate into mature endothelial cells. Perhaps earlier surgery would yield greater AC133-positive cell numbers consistent with earlier disease. Alternatively, the few cells present may reflect the sensitivity of the monoclonal antibodies (mAbs) used in the present study. It may also be a possibility that the recruitment of stem cells is an initial event in the pathobiology of CNV and is not a feature of disease progression. Based on the findings in studies of experimental CNV, this is certainly a distinct possibility.29 30

Because vasculogenesis is being increasingly recognized as a component of both physiologic and pathologic neovascularization in the adult, it is perhaps not surprising that the AC133-positive cells identified raises the possibility of vasculogenesis in CNV. However, it is intriguing to speculate that these cells are not only contributing to neovascularization through EPCs but, if HSCs are truly present, these cells could give rise to cells that have true regenerative abilities. HSCs are thought to retain a high degree of plasticity, which could commit them to a role as regenerative precursors in nonhematopoietic tissues after injury.3 8 9 11 12 13 31 32 33 At the cellular level, CNV has all the hallmarks of a stereotypical tissue repair response.34 35 36 37 The findings of the present study in conjunction with those in other studies suggest that the clinical entity CNV is in fact a true wound-healing response of which neovascularization is just a single component.3 5 Even in experimental models of CNV, Bruch’s membrane is damaged with laser to initiate a wound-healing response of which neovascularization is just a single component.29 30 38 Indeed, the presence of HSCs may not only contribute to the neovascularization necessary for this reparative response, but also may supply a regenerative component to the RPE/Bruch’s membrane complex. If this were the case and bearing in mind that systemic stem cell input may contribute to the development of CNV, then it may be timely to consider therapies other than antiangiogenesis, which prevent healing, and consider treatments that promote repair and regeneration.39 In this context, the therapy would involve modifying the disciform scarring response in an attempt to prevent destruction of or enhance the rescue of RPE and photoreceptors. Ultimately, it is hoped that the treatment of AMD will be preventative. Until such time, modifying the natural history of CNV remains a realistic expectation of treatment. The demonstration that AC133 positive cells are present in neovascular AMD suggests not only that postnatal vasculogenesis is a component of CNV but also may provide a novel avenue of therapeutic exploitation to enhance recovery and even regeneration of functioning tissue after the development of CNV.


   Footnotes
 
Supported by the Dunhill Medical Trust; Fight for Sight; the R&D Support Fund of the Royal Liverpool and Broadgreen University Hospitals, and St. Paul’s Foundation for the Prevention of Blindness.

Submitted for publication June 21, 2005; revised October 4, 2005; accepted January 30, 2006.

Disclosure: C. Sheridan, None; D. Rice, None; P. Hiscott, None; D. Wong, None; D. Kent, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Carl M. Sheridan, Ophthalmology, School of Clinical Sciences, University of Liverpool, Daulby Street, Liverpool L69 3GA, UK; c.sheridan{at}liverpool.ac.uk.


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