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(Investigative Ophthalmology and Visual Science. 1999;40:1855-1859.)
© 1999 by The Association for Research in Vision and Ophthalmology, Inc.

N{varepsilon}(Carboxymethyl)Lysin and the AGE Receptor RAGE Colocalize in Age-Related Macular Degeneration

Hans-Peter Hammes1, Hans Hoerauf2, Alex Alt1, Erwin Schleicher3, Jes Thorn Clausen4, Reinhard G. Bretzel1 and Horst Laqua2

1 From the Third Medical Department of Internal Medicine, Justus–Liebig-University, Giessen, Germany; the 2 Eye Clinic, Medical University Lübeck, Germany; the 3 Fourth Medical Department, Eberhard–Karl-University, Tübingen, Germany; and 4 BioSciences, Novo Nordisc A/L, Bagsvaerd, Denmark.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate whether glycoxidation products and the receptor for advanced glycation end products (RAGE) are present and colocalize in subfoveal membranes of patients with age-related macular degeneration (ARMD).

METHODS. Surgically removed subfoveal fibrovascular membranes from 12 patients, 11 related to ARMD and 1 to an idiopathic membrane, were analyzed for the presence of the glycoxidation product N{varepsilon}-(carboxymethyl)lysin (CML), one of the receptors for advanced glycation end products, RAGE, and the activation of NFkB, using immunohistochemistry.

RESULTS. CML-like immunoreactivity was found in all ARMD specimens examined adjacent or colocalized with RAGE, but not in the idiopathic membrane. RAGE immunoreactive material was found in CD68-positive cells and in the fibrous matrix. CD68-positive cells and surrounding areas stained for p50, the activated form of NFkB.

CONCLUSIONS. These results indicate that glycoxidation products are present in subretinal membranes of patients with ARMD. The concomitant expression of RAGE in these membranes and the finding of activated NFkB is suggestive of an implication of glycoxidation product formation in the pathogenesis of the disease.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Age-related macular degeneration (ARMD) is the main cause of legal blindness in the elderly population.1 Advanced stages of ARMD develop into two variants, one of which involves sprouting of new blood vessels from the choriocapillaris into the subretinal space.2 Despite detailed knowledge about the natural course of choroidal neovascularizations, the underlying cause remains poorly understood.

The aging process is associated with increased formation and deposition of chemically modified proteins, lipids, and nuclear acids called advanced glycation end products (AGE; for review, see Ref. 3) . In parallel with AGE, reactive oxygen intermediates are produced, either directly through the glycation process or indirectly as the consequence of the interaction of cellular AGE receptors with their respective ligands. Some products form by sequential glycation and oxidation (glycoxidation products), such as N{varepsilon}-(carboxymethyl) lysine (CML), which is not only a glycoxidation product, but can also form from the peroxidation of lipoproteins.

Glucose-modified bovine serum albumin (BSA ) containing CML has been shown to induce the expression of growth factors in vitro, thus linking the CML and other AGE with the formation of neovascularizations in aged patients. We speculated that CML could be involved in the formation of subretinal neovascular membranes, and we investigated whether CML is present in subretinal membranes of patients with ARMD and studied the presence and localization relative to CML of one of the AGE receptors, RAGE, in these membranes.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
For the human research, the tenets of the Declaration of Helsinki were followed, and informed consent was obtained.

Materials
Twelve subfoveal neovascular membranes, 11 excised from patients with age-related macular degeneration (aged 72–81 years, 10 with occult and 1 with a classic subfoveal neovascular membrane) and 1 excised from a patient with idiopathic neovascular membrane (female, age 17) were examined after removal during a three-port pars plana vitrectomy. Membranes were immediately placed in Bouin fixative for 12 hours at room temperature and subsequently embedded in paraffin. Serial sections were cut at 5 µm. One donor eye was obtained from a 42-year old male donor without diabetes or any known ocular disease. After fixation in Bouin fixative for 12 hours, the retina was similarly processed for immunohistochemistry.

Immunohistochemistry
CML.
Paraffin-embedded subretinal membrane sections were immunostained using a standard alkaline phosphatase–anti-alkaline phosphatase protocol and a monoclonal anti-CML antibody (6D12) whose specificity has been described previously.4 Histochemical detection was performed using newfuchsin chromogen substrate (Dako; Carpinteria, CA) as chromogen and hemalaun as a counterstain. For control sections, the primary antibody was omitted or replaced by a nonimmune mouse IgG. Additional sections were stained using a polyclonal antibody recognizing protein-bound CML, which was characterized previously, and the avidin-biotin, complex peroxidase staining method (ABC; Vector, Burlingame, CA) as described.5

RAGE.
RAGE immunohistochemistry was performed using a peroxidase–antiperoxidase protocol and a polyclonal antibody raised against the extracellular part of the RAGE (truncated RAGE). The gene sequence for the extracellular part of the human RAGE described by Schmidt et al.6 was obtained from Gene Bank. cDNA was cloned from a human lung cDNA library by standard reverse transcription–polymerase chain reaction procedures, inserted into the vector pBac, and expressed in the baculovirus system. New Zealand White rabbits were injected subcutaneously with 50 µg of the truncated RAGE emulsified in Freund’s complete adjuvant, followed by three booster injections with 50 µg truncated RAGE emulsified in Freund’s incomplete adjuvant every 2 weeks. Ten days after the last injection. the animals were bled, and sera were isolated. The antibody was characterized by western blot analysis using membranes from Hek 293 cells transfected with RAGE and retinal extracts of diabetic and nondiabetic rats. A single band was observed in retinal extracts from normal and diabetic rats at Mr of approximately 39 kDa, and bands with Mr between 45 and 52 kDa were observed in samples from transfected cell membranes (because of different posttranslational glycosylation of RAGE in Hek cells), whereas no bands were detected in nontransfected cells. As controls, the antibody was preadsorbed with excess soluble RAGE or omitted. Both cases resulted in the disappearance of the band (not shown). Antibody binding was visualized by 3,3'-diaminobenzidine (0.06%), and sections were counterstained with hemalaun.

Semiquantitative Grading of CML and RAGE Immunoreactivity
Staining intensity of the monoclonal CML antibody was evaluated using a four-step system (0, unreactive; +, mildly positive; ++, moderately positive; and +++, strongly positive). The grading was performed for areas with high cellularity and for matrix-rich areas of the membranes by two observers unaware of the samples’ identity (HPH, AA).

NFkB p50
To identify the activated transcription factor NFkB p50 in subretinal membranes, sections were incubated with an affinity-purified rabbit polyclonal antibody (2 µg/ml) raised against amino acids 350 to 363 mapping within the nuclear location signal region of human NFkB 50 (Santa Cruz Biotechnology, Santa Cruz, CA), and detection was performed using the indirect immunoperoxidase method (ABC Kit, Vector).

CD68
For the identification of human macrophages, sections were immunostained with an anti-human CD68 monoclonal antibody (4 µg/ml; Dako) and the alkaline phosphatase detection system as described.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Subretinal neovascular membranes consisted of areas with fibrovascular and fibrous tissue, focal RPE deposits, depositions of amorphous material, and focal cell infiltrations (Fig. 1 A).



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  		Figure 1. Immunolocalization of CML-modified proteins in subfoveal neovascular membranes from patients with ARMD. (A) Negative control of a membrane showing fibrovascular (white arrow) and fibroproliferative (black arrow) segments, and areas of foamlike deposits (white arrowhead). (B) Section adjacent to (A) stained with the polyclonal CML antibody. Note the strong immunolabeling of fibrovascular and fibroproliferative areas and near the foamlike deposits. Gray arrow indicates area used for colocalization studies shown in Figure 2 . CML-positive deposits in (C). Areas of cellular infiltrations in (D) matrix, (E) in fibrillous deposits, and (F) amorphous deposits. (G) CML staining of the retina. (H) RAGE staining of the retina. (C, D, E, F, G) Monoclonal CML antibody (6D12). Original magnification, (A, B) x6.3; (C, D, E, F, G) x25.

 
CML-immunoreactive material was found in all membranes examined and was heterogeneously distributed within the membranes, including fibrovascular and fibrous elements, foamlike deposits, and near RPE inclusions (Fig. 1B) .

Using the monoclonal antibody, CML was detected in the vicinity of vascular structures (Fig. 1C) , in perinuclear cell compartments, in the extracellular matrix (Fig. 1D) , in foamlike areas (Fig. 1E) , and in amorphous material (Fig. 1F) . In the retina of a patient without known ocular diseases, no CML reactivity was discernible (Fig. 1G) , but RAGE was moderately present in the inner limiting membrane and the inner plexiform and nuclear layers (Fig. 1H) .

To study the possible colocalization of CML with RAGE, we used defined fibrovascular areas (Figs. 1A 1B and Figs. 2 A, 2B, 2C, 2D, 2E). When compared with the immunolabeling of CML (Fig. 2A) , RAGE colocalized in fibrovascular areas of the membranes and near RPE deposits (Fig. 2B) . These findings were consistent for all samples examined (for quantitation see Table 1 ). The distribution of CML- and RAGE-immunoreactivity indicate a colocalization of both in neovascular membranes of patients with ARMD.



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  		Figure 2. Accumulation of CML in areas of subfoveal membranes colocalizing with CD68+ cells expressing RAGE and activated NFkB p50. (A) Magnification x25 of the area indicated in Figures 1A and 1B . (gray arrow), stained with the polyclonal CML antibody. (B) Adjacent section stained with the polyclonal RAGE antibody. (C) Adjacent section stained with the monoclonal CD68 antibody, labeling macrophages. (D) Adjacent section stained with the polyclonal antibody against NFkB p50. (E) Negative control of (A), incubated with nonimmune IgG. Black arrows indicate corresponding areas.

 

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  		Table 1. Semiquantitative Evaluation of CML Immunohistochemistry in Subfoveal Neovascular Membranes of Patients with ARMD (Cases 1–11) and in One Patient (Case 12) with Idiopathic Subfoveal Neovascular Membrane

 
Macrophages are one cell type expressing RAGE, indicated by CD68 immunostaining in areas of RAGE immunostaining (Figs. 2B 2C) .

Next, we hypothesized that cells expressing RAGE would show functional consequences induced by binding of AGE-type ligands to RAGE. It was found that the activated form of NFkB, NFkB p50, was widely expressed by cells in these membranes, indicating the functional role of RAGE in the subretinal membranes of ARMD patients (Figs. 2D) .

In contrast to these findings, and to demonstrate that ARMD is different from other diseases associated with subretinal neovascularizations, we used the same histochemical parameters to study the subretinal membrane from a 17-year-old girl with idiopathic subretinal neovascular membrane. This membrane was negative for CML and RAGE (Figs. 3 A, 3B).



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  		Figure 3. Idiopathic subfoveal membrane of a 17-year-old patient. (A) CML immunostaining. (B) RAGE immunostaining. (C) Negative control. Note the absence of immunolabeling in (A) and (B). Original magnification, x25.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our findings indicate that CML-like immunoreactive material is present in membranes of patients with ARMD, RAGE is associated with CML-immunoreactive material within membranes, RAGE is expressed by CD68-positive cells in this tissue, and NFkB p50 is present as an indication of functional activation.

CML is involved in the aging process, because it accumulates in long-lived matrix structures such as skin collagen of normal people.3 Although CML neither forms crosslinks nor induces free radicals, it is regarded as a biomarker of oxidant stress in the respective tissues. CML is absent from tissues of young age and is not found in retinae from control subjects, at least not with the CML antibody used in the present study. The presence of CML in subfoveal neovascular membranes is surprising, because the material was not deposited long enough to assume CML accumulation with age. It is more likely, that CML accumulation, or an underlying process leading to it, is involved in the pathogenesis of subretinal neovascularization.

CML is colocalized in the subretinal membranes with RAGE. On binding to AGE, RAGE mediates a variety of cellular responses, including increased intracellular oxidant stress and chemotaxis. Preliminary data indicate that CML-modified proteins, as recognized by the antibodies used in our study, are ligands for RAGE, activating intracellular signaling pathways and altering gene expression.7 Moreover, reactive oxygen intermediates themselves can stimulate RAGE expression because reactive oxygen intermediates induce NFkB, and the RAGE promoter has two NFkB-binding sites.8

Subretinal neovascularization associated with ARMD can be explained, at least in part, by the activity of factors stimulating the growth of both vascular and matrix components. Among others, basic fibroblast growth factor and vascular endothelial growth factor (VEGF) have been found in subfoveal fibrovascular membranes.9 Whereas the exact biochemical stimulus for basic fibroblast growth factor is unknown, the major stimulus for VEGF expression is hypoxia.10 VEGF stimulation in ARMD may be the result of increased oxidative stress, and the formation of glycoxidation products may be a contributing factor. Both reactive oxygen intermediates and AGE are capable of inducing VEGF expression in retinal cells, especially in RPE,11 and AGEs by themselves are angiogenic.12

The scenario of CML colocalized with RAGE and inflammatory cells present in neovascular membranes suggests that this system may be involved in the initiation and/or propagation of ARMD. Supporting evidence for our observations comes from a recent study showing that CML is present in soft drusen (preceding choroidal neovascular membranes) and in nearby RPE cells, whereas in control eyes, no CML was found.13 The role of RAGE was not investigated.

Taken together, the evidence in this study suggests that CML-like immunoreactivity is present in subfoveal neovascular membranes of patients with ARMD and that they colocalize with one of the AGE receptors, RAGE. Because current therapeutic options are limited, further studies are needed to determine the implication of these findings and their functional consequences in the pathogenesis of ARMD. More detailed studies extending this and other preliminary observations, are warranted.


   Acknowledgements
 
The authors thank Dres Henrik Vissing and Poul Baad Rasmussen, Novo Nordisc, for cloning and production of the AGE-receptor; Seikoh Horiuchi for his kind gift of the 6D12 monoclonal CML-antibody; and Kerstin Schneider for help with the manuscript.


   Footnotes
 
Reprint requests: Hans-Peter Hammes, Third Medical Department, Justus–Liebig-University, Rodthohl 6, 35392 Giessen, Germany.

Supported by the German Research Council (HA 1755/3-1).

Submitted for publication August 14, 1998; revised January 27, 1999; accepted February 24, 1999.

Proprietary interest category: N.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Klein, R, Klein, BEK, Linton, KLP (1992) Prevalence of age-related maculopathy The Beaver Dam Eye Study. Ophthalmology. 99,933-943[Medline][Order article via Infotrieve]
  2. Bird, AC, Bressler, NM, Bressler, SB, et al (1995) An international classification and grading system for age-related maculopathy and age-related macular degeneration Surv Ophthalmol 39,367-374[Medline][Order article via Infotrieve]
  3. Baynes, JW, Thorpe, SR (1999) Perspectives in diabetes Role of oxidative stress in diabetic complications. A new perspective on an old paradigm. Diabetes. 48,1-9[Abstract]
  4. Ikeda, K, Higashi, T, Sano, H, et al (1996) , Ne-(carboxymethyl)lysine protein adduct is a major immunological epitope in proteins modified with advanced glycation end products of the Maillard reaction Biochemistry 35,8075-8083[Medline][Order article via Infotrieve]
  5. Schleicher, ED, Wagner, E, Nerlich, AG (1997) Increased Accumulation of the Glycoxidation Product Ne-(carboxymethyl)lysine in Human Tissues in Diabetes and Aging J Clin Invest 99,457-468[Medline][Order article via Infotrieve]
  6. Schmidt, AM, Vianna, M, Gerlach, M, et al (1992) Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface J Biol Chem 267,14987-14997[Abstract/Free Full Text]
  7. Fu, C, Pischetsrieder, M, Hofmann, M, Yan, SF, Stern, D, Schmidt, AM (1998) Carboxymethyl-lysine advanced glycation endproduct modifications of proteins are ligands for RAGE that activate cell signalling pathways (abstract) Circulation 98,1651[Abstract/Free Full Text]
  8. Li, J, Schmidt, AM (1997) Characterization and functional analysis of the promoter of RAGE, the receptor for advanced glycation end products J Biol Chem 272,16498-16506[Abstract/Free Full Text]
  9. Frank, RN, Amin, RH, Eliott, D, Puklin, JE, Abrams, GW (1996) Basic fibroblast growth factor and vascular endothelial growth factor are present in epiretinal and choroidal neovascular membranes Am J Ophthalmol 122,393-403[Medline][Order article via Infotrieve]
  10. . Pe‘er J,Shweiki, D, Itin, A, Hemo, I, Gnessin, H, Keshet, E (1995) Hypoxia-induced expression of vascular endothelial growth factor by retinal cells is a common factor in neovascularizing ocular diseases. Lab Invest. 72,638-645[Medline][Order article via Infotrieve]
  11. Kuroki, M, Voest, EE, Amano, S, et al (1996) Reactive oxygen intermediates increase vascular endothelial growth factor expression in vitro and in vivo J Clin Invest 98,1667-1675[Medline][Order article via Infotrieve]
  12. Lu, M, Kuroki, M, Amano, S, et al (1998) Advanced glycation end products increase retinal vascular endothelial growth factor expression J Clin Invest 101,1219-1224[Medline][Order article via Infotrieve]
  13. Ishibashi, T, Murata, T, Hangai, M, et al (1998) Advanced glycation end products in age-related macular degeneration Arch Ophthalmol 116,1629-1632[Abstract/Free Full Text]



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