|Molecular Vision 2005;
Received 8 March 2005 | Accepted 8 July 2005 | Published 14 July 2005
Glycoconjugates of choroidal neovascular membranes in age-related macular degeneration
Robert F. Mullins,
Michael A. Grassi,
Jessica M. Skeie
Center for Macular Degeneration, Department of Ophthalmology and Visual Science, The University of Iowa Carver College of Medicine, Iowa City, IA
Correspondence to: Robert F Mullins, 4135E MERF, 375 Newton Road, Iowa City, IA, 52242; Phone: (319) 335-8222; FAX: (319) 335-6641; email: firstname.lastname@example.org
Purpose: Choroidal neovascularization (CNV) is a complication of multiple eye diseases, including age-related macular degeneration, that usually results in irreversible vision loss. It is characterized by proliferation and growth of choroidal blood vessels through Bruch's membrane into the subpigment epithelial and/or subretinal space. The purpose of this study was to characterize the carbohydrate groups associated with CNV by lectin histochemistry.
Methods: Frozen sections from three human eyes with CNV (two fixed eyes and one unfixed eye) were prepared. Sections containing choroidal neovascular membranes were incubated with a battery of biotinylated lectins directed against a number of distinct oligosaccharide moieties. Lectin labeling of the vessels in CNV was visualized with avidin-Texas red.
Results: Several carbohydrate groups were preferentially associated with the vascular elements in CNV. Glycoconjugates that react with lectins derived from wheat germ, soybean, and hairy vetch seed (sWGA, SBA, and VVA, respectively) all showed reactivity with CNV vessels that was higher than the labeling of the surrounding matrix. SBA and sWGA also reacted with CNV vessels at low concentrations at which normal retinal and choroidal vessels were largely unlabeled.
Conclusions: Choroidal neovascular membranes possess a distinct set of carbohydrate moieties. These data may be valuable in understanding endothelial cell biology in CNV.
A final common pathway and source of vision loss for many diseases is choroidal neovascularization (CNV) and its subsequent disciform scarring . In this condition, the choroidal vasculature penetrates Bruch's membrane, resulting in injury to the retinal pigment epithelium and photoreceptor cells of the neural retina. Over forty conditions have been associated with CNV . Of these age related macular degeneration (AMD) is the most prevalent.
AMD is the most common cause of severe visual loss in the developed world, impairing more than 10 million people in the United States alone . Approximately one in three people over the age of 75 are affected to some degree . Moreover, the US Census Bureau predicts that the number of people in this age group will increase by 60-80% in the next 25 years, making the prevalence of blindness from AMD greater than glaucoma and diabetic retinopathy combined. The vast majority of patients with AMD who suffer from legal blindness are affected with the neovascular form of the condition. It is estimated that 90% of eyes with severe vision loss due to AMD have CNV , underscoring the fact that, although it affects only a fraction of all AMD patients, CNV is the major cause of vision loss in this disease.
The overall visual prognosis for CNV remains dismal. Severe visual loss occurs in over 60% of CNV cases over a five year period [6-8]. In general, the currently available therapeutic modalities are only able to decrease the extent to which vision is lost and are incapable of restoring vision [9-12].
The identification of distinct markers that differentially label components of CNV could yield important insights into the pathogenesis of this condition. The resultant clinical benefit of molecularly targeted treatment of CNV would be significant. In this study, we performed a lectin histochemical assay of choroidal neovascular membranes (CNVMs) from three donors to determine whether a specific carbohydrate composition is associated with the neovascular complex.
Lectin histochemistry is a morphological technique that takes advantage of the carbohydrate binding characteristics of plant, animal and fungal proteins . Different cell types, and cells under different environmental influences, alter their surface carbohydrate composition, and these alterations may be detected histologically or biochemically. This approach has been utilized on eyes with early AMD, to determine compositional characteristics of drusen and basal laminar deposits [14-16].
In this study, we examined the carbohydrate moieties present in CNV and compared the labeling pattern of CNV with normal retinal and choroidal blood vessels. We found that a number of carbohydrate moieties were present on the vascular elements of CNVMs and disciform scars, including those recognized by soybean agglutinin (SBA), Vicia villosa agglutinin (VVA),Ulex europaeus agglutinin I (UEA-I), and succinyl wheat germ agglutinin (sWGA). SBA and sWGA were found to recognize the vascular elements of CNVMs at concentrations that failed to show strong labeling of normal vessels of the retina and choroid.
Aged donor eyes from three individuals with CNVMs were received from the Iowa Lions Eye Bank (Iowa City, IA). Eyecups or macular punches were either fixed (Cases 1 and 2) or were embedded unfixed (Case 3), as described below. Eyes were fixed by immersion for 2 h in 4% paraformaldehyde solution diluted in 10 mM phosphate buffered saline (PBS), pH 7.4. Eyes were fixed within 5 h of death. Maculae were washed in PBS and were then infiltrated and embedded in sucrose solution . The maculae from these eyes were serially sectioned on a Microm HM505E cryostat and employed in the lectin histochemical study. Labeling patterns of the CNVMs in these eyes were compared with the patterns in normal retinal and choroidal endothelial cells (ECs) in these same eyes.
In order to determine whether the sucrose infiltration and embedment affected the pattern of labeling, a third eye with subpigment epithelial CNV (Case 3) was embedded unfixed in optimal cutting temperature medium (OCT; Ted Pella; Redding, CA) within 12 h of death. Frozen sections from this donor were evaluated with the same battery of lectins.
Patterns of lectin labeling in two eyes with choroidal neovascularization were evaluated. The panel of biotinylated lectins and their specificities are shown in Table 1. All lectins were obtained from Vector Laboratories (Burlingame, CA). Lectin labeling was performed essentially as described previously  except that biotinylated lectins were utilized, followed by detection with avidin-Texas red (Vector Labs). The divalent cations MgCl2 and CaCl2 (1 mg/mL each) were included in all steps of the labeling protocol. Sections were blocked for 15 min in PBS with 1 mg/mL bovine serum albumin (Sigma, St. Louis, MO). Sections were then incubated in the biotinylated lectin diluted 1:50 to 1:400 in PBS (with albumin, MgCl2 and CaCl2) for 30 min, followed by three 5-min washes, and incubation in avidin-Texas red (25 μg/mL) and the nuclear counterstain DAPI (4'-6-diamidino-2-phenylindole) for 30 min. Following three 5-min washes in PBS, sections were coverslipped in Aquamount (Lerner Laboratories, Pittsburgh, PA).
Additional sections were stained using conventional hematoxylin/eosin staining (H&E), periodic acid-Schiff (PAS), or Masson's trichrome (MT). PAS and MT staining were performed at the University of Iowa F. C. Blodi Ocular Pathology Laboratory (Iowa City, IA).
Sections were photographed on an Olympus BX41 microscope with a fluorescence attachment and filter sets for fluorescein, rhodamine, and DAPI. In order to discriminate between autofluorescence and Texas red labeling (particularly in areas of RPE lipofuscin, which is highly autofluorescent), all photographed fields were imaged in red, green and blue channels.
Eyes were also labeled with antibodies directed against type IV collagen (Chemicon rabbit polyclonal antibody, Temecula, CA) to visualize vascular basal laminae and with NBT/BCIP (Vector Laboratories) in order to visualize the endogenous alkaline phosphatase activity of endothelial cells . Immunohistochemical labeling was performed as described previously .
The three eyes with neovascular membranes and disciform scars showed features typical of those described in the literature for CNVMs associated with AMD [20-24], reviewed in .
In Case 1, a large, compact subfoveal scar was noted that was eosinophilic and stained blue on MT stain, suggesting a significant amount of collagen (Figure 1A). The scar contained fibrovascular material that was present on both sides of a detached layer of basal laminar deposit with a discontinuous RPE layer on its inner surface. Islands of RPE were noted within the scar, as were rare vessels (Figure 1A). Photoreceptor degeneration was noted above the scar, and several rosettes were present in the overlying retina. At the temporal edge of the scar, a flat layer of active blood vessels was noted lying between the outer aspect of Bruch's membrane and the degenerated RPE (Figure 1B,C).
In Case 2, a two-layered CNVM was present. The retina overlying the CNVM exhibited severe cystic changes at the level of the inner nuclear layer (Figure 2A). In addition, the outer nuclear layer and photoreceptor outer segments were extremely attenuated. A detached layer of meandering basal laminar deposit (BlamD) was also noted, with a layer of loose proteinaceous material between the outer layers of Bruch's membrane and the layer of BlamD  (Figure 2B). PAS-reactive material in the BlamD and the residual Bruch's membrane was observed, with active blood vessels and matrix elements occupying this space (Figure 2B). Case 3, an unfixed eye with a comparatively long death-preservation time, showed similar characteristics, except that the CNVM was located only in the subpigment epithelial space, between a layer of BlamD and Bruch's membrane (Figure 2C).
The labeling patterns of eleven lectins were determined in the retinal blood vessels, choroidal blood vessels, the glycoconjugates within the scar, and the vascular components of the neovascular membrane. The observed patterns are described in Table 1 and in Figure 3, Figure 4, Figure 5, and Figure 6.
Several lectins showed intense labeling of matrix elements within the CNVM and/or disciform scar. The material within the scar was labeled with PNA (in Case 2 only; Figure 2A) and PSL (in Cases 1 and 2). A sheet of matrix in the subretinal space, most likely corresponding to the basal lamina of dystrophic RPE cells, was labeled with SBA, PNA, and VVA (Figure 3B). When used at higher concentrations (20 μg/mL), sWGA labeled basal laminar deposits (Figure 3C). At this concentration, sWGA also labeled normal retinal and choroidal vasculature. PNA also labeled photoreceptor rosettes, which were observed in the overlying retina, as described previously (Figure 3A) .
Vessels in the CNVM were labeled with UEA-I (which labeled all vessels in the eyes studied, Figure 4A,B). Reactivity of the CNVM vessels was also noted with VVA (Figure 5A), PSL, SBA (Figure 5B,C), and sWGA (Figure 5D). These probes also showed variable labeling of vessels in normal retina and/or choroid. SBA was notably higher in CNV vessels in Case 2 than in normal retinal and choroidal vessels, and exhibited positive reactivity of these vessels at relatively low concentrations (2.5 μg/mL) that did not label other structures in the eye. Similarly, sWGA labeled choroidal neovessels at concentrations at which other vascular beds were unlabeled or very weakly labeled. Similar results were obtained between sucrose embedded (Cases 1 and 2) and unfixed (Case 3) CNVMs (Table 1).
We also sought to evaluate the glycoconjugates associated with a large feeder vessel breaching Bruch's membrane in Case 2 (Figure 6A). Two lectins found to react with subpigment epithelial vessels in CNV were utilized in dual labeling experiments with an antibody directed against type IV collagen. Both SBA (Figure 6B) and sWGA (Figure 6C) reacted with components of this large vessel. The SBA-reactive glycoconjugates appeared to be external to the inner layer of ECs, whereas sWGA appeared to localize to the EC surface internal to the layer of collagen IV (Figure 6C).
Endothelial cells express a broad spectrum of glycoconjugates that differ between species, between types of vessels, and between ECs in healthy and diseased states. Changes in the distribution of EC carbohydrate surface molecules have been described in several pathologic conditions, including experimental abdominal cryptorchidism , normal and dystrophic umbilical cord vasculature , and experimentally-injured brain microvascular ECs .
In addition, several studies have also shown that ECs in angiogenic and migrating states may express a different set of cell surface carbohydrate moieties than quiescent ECs. Studies on chicken chorioallantoic membrane ECs during normal vascular development  and on migrating cultured aortic ECs following experimental debridement  have shown that the lectin-binding profile of ECs changes during growth and migration. Glycoconjugates containing N-acetyl-glucosamine (GlcNAc) were found to be upregulated in both cases. Interestingly, vessels in choroidal neovascular membranes were also found to react intensely with sWGA, a marker for GlcNAc-containing oligosaccharides. Whether these reactive carbohydrates are components of glycoproteins or glycolipids can not be readily determined by lectin histochemistry, and evaluating the biochemical nature of these glycoconjugates will be an interesting extension of these studies.
Although relatively little is understood about the biochemistry of CNV ECs, recent studies have begun to explore the expression of EC markers in human and animal CNVMs. Immunolabeling of CNVMs has shown that endoglin/CD105 is associated with CNV ECs [32,33], and adhesion molecules such as ICAM-1/CD54 and E-selectin are present in human  and experimental  choroidal neovascularization. Endothelial cells in a mouse model of CNV have been shown to react with Lycopersicon esculentum agglutinin , a lectin from tomato with affinity for GlcNAc oligomers. In our hands, this lectin did label rosettes and CNVMs in Case 2, but did not show strong specificity for CNV over normal vessels or other matrix elements (data not shown). Lectin reactivities of blood vessels differ significantly among mammalian species , and it will be interesting to determine the similarities in the glycoconjugate profiles of mouse experimental CNV and human CNV.
Although the function of altered EC oligosaccharide chains in CNV remains unclear, there are several steps in the process of CNV formation in which glycosylation is likely to play a role. Angiogenesis is regulated in part by the interactions of matrix metalloproteinases (MMPs) and their substrates . Mutations in TIMP3, a regulator of metalloproteinases, result in a form of macular dystrophy that can be complicated by CNV [39,40]. Recently it has been shown that some members of the MMP family of enzymes modulate their interactions based upon their glycosylation state . Moreover, endothelial cell activation molecules such as ICAM-1, which is required for CNV formation in the murine model of CNV  and which is localized to CNVMs in human eyes , have functions that similarly rely on a specific state of glycosylation .
In the current study we describe the localization of a battery of lectins to CNVMs in human eyes. There are a number of limitations in the current study. The patterns of lectin labeling were evaluated in only a small number of eyes. In addition, the eyes used in this study had disciform scars, an end stage cicatricial response in CNV. It is possible that eyes with earlier staged CNV might reveal a different set of carbohydrate moieties. Whether abnormal vessels assayed at various stages of CNV maturation would demonstrate different glycoconjugate profiles is an interesting and open question. We did find, however, that SBA (a lectin from the soybean Glycine max) and sWGA (succinylated wheat germ agglutinin) label CNV endothelial cells. Significantly, these lectins reacted with the abnormal microvasculature in CNV at concentrations at which labeling of normal retinal and choroidal vasculature was absent or markedly reduced, which suggests that CNV ECs may possess specific glycoconjugates. The identification of the glycoconjugate profile of the abnormal vessels in CNV may be useful in understanding the pathophysiology of CNV.
Supported in part by RO3 EY14563 (RFM), a Carver Trust Medical Research Initiative Grant (RFM); and the Heed Foundation (MAG). The authors wish to thank Elizabeth Malone and Marissa Olvera for technical assistance, and Dr. Nasreen Syed and Christy Ballard for their assistance with special staining techniques, and the Iowa Lions Eye Bank for procuring the human donor eyes used in this study. A patent application based on this work has been filed.
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