 |
|
Human Retinal Pigment Epithelium Cell Changes and Expression of  B-CrystallinA Biomarker for Retinal Pigment Epithelium Cell Change in Age-Related Macular Degeneration
Soma De, PhD;
David M. Rabin, BS;
Enrique Salero, PhD;
Patricia L. Lederman, BS;
Sally Temple, PhD;
Jeffrey H. Stern, PhD, MD
Arch Ophthalmol. 2007;125(5):641-645.
ABSTRACT
| |
Objective To examine changes in the retinal pigment epithelium (RPE) in eyes with age-related macular degeneration (AMD) and specifically to characterize  B-crystallin expression in RPE cells as a biomarker in this disease.
Methods Maculae from human patients diagnosed as having AMD or from age-matched control eyes were isolated, cryosectioned, and analyzed immunohistochemically for B-crystallin and for cell type–specific markers.
Results In eyes with dry and wet AMD, B-crystallin was heterogeneously expressed by a subpopulation of RPE cells in the macular region (frequently in cells adjacent to drusen) and in areas of RPE hypertrophy associated with wet AMD. In contrast, B-crystallin was not detected at significant levels in control RPE.
Conclusion Accompanying the formation of drusen in early-stage and late-stage AMD, RPE cells undergo change to express B-crystallin.
Clinical Relevance The detection of B-crystallin in the RPE of patients with early and advanced AMD implicates this as an AMD biomarker. Sporadic expression of B-crystallin by RPE cells localized adjacent to drusen in early AMD indicates that changes in the gene expression of RPE cells accompany early stages of the disease and introduces novel potential targets for AMD therapy.
INTRODUCTION
Age-related macular degeneration (AMD) is a progressive degeneration of photoreceptors and underlying retinal pigment epithelium (RPE) cells in the macula region of the retina. It is a highly prevalent disease and a major cause of blindness in the Western world.1-2 Early morphological changes include thickening of the Bruch's membrane, the basement membrane formed by the RPE and underlying choroidal blood vessels.3 Drusen, pale excrescences of variable size, and other deposits accumulate below the RPE on Bruch's membrane; clinical and histopathologic investigations have shown that these extracellular deposits are the hallmark of early AMD.4 As AMD advances, areas of geographic atrophy (GA) of the RPE can cause visual loss, or choroidal neovascularization can occur to cause wet, or exudative, AMD with accompanying central visual loss.
Drusen contain various lipids, polysaccharides, and glycosaminoglycans.4-5 The identity of the cells that generate these components is unclear. Direct proteomic analysis of drusen isolated by microdissection from donor eyes with and without AMD has identified a set of proteins that are more prevalent in drusen from patients with AMD.6-7 Crystallins are the most abundant proteins found in drusen from donors diagnosed as having AMD.
Crystallins, a group of small heat shock proteins, form oligomeric complexes and function as molecular chaperones protecting other proteins from denaturation and destabilization.8 They are induced by accumulation of abnormally folded proteins resulting from stress.9 Elevated expression of small heat shock proteins has been implicated in the pathogenesis of neurodegenerative disorders, including Alzheimer's disease.10-11 Cell culture studies12-13 demonstrated that B-crystallin might protect cells from apoptosis. In cultured RPE, induction of B-crystallin occurs when human RPE cells are stressed by heat shock or oxidant-mediated injury, which are stresses implicated in the pathogenesis of AMD.14 In addition, intense light exposure increases expression of B-crystallin in photoreceptors and RPE in rats, which has been suggested to protect cells from light damage.15 Given these observations, we sought to study whether human RPE cells change to express B-crystallin in AMD.
METHODS
SOURCE OF TISSUE
Human ocular tissues were obtained from The Eye-Bank for Sight Restoration, Inc, New York, NY, and the National Disease Research Interchange, Philadelphia, Pa. Eyes with early, midstage, and advanced AMD and age-matched control eyes were used. Eyes with early AMD had not received a diagnosis of AMD by an ophthalmologist but had some hard macular drusen and possibly small soft drusen observed by histologic analysis and were from patients who likely did not experience AMD-related visual loss. Eyes with midstage AMD were from patients diagnosed as having AMD who had moderate numbers of hard and soft macular drusen but not GA. Eyes with advanced AMD were from patients diagnosed as having AMD by an ophthalmologist and contained choroidal neovascularization or numerous hard or soft drusen, usually accompanied by scarring or GA and visual loss. Age-matched eyes that were from patients not having a diagnosis of AMD served as control specimens for this study. Twenty-three eyes were used (11 controls and 12 with AMD). Among the 11 controls, 9 eyes (5 with and 4 without nonmacular drusen) were from patients older than 60 years, while 2 eyes (without nonmacular drusen) were from patients younger than 60 years. Among the 12 eyes with AMD, 3 eyes were classified as having early AMD, 7 eyes as having midstage AMD, and 3 eyes as having advanced AMD (1 with the wet form and 2 with the dry form).
The Table summarizes the findings; 1 eye from each eye classification group is described hereinafter. Eye 1 from a 75-year-old man with early dry AMD had several hard drusen and a few soft or diffuse drusen, with minimal RPE and photoreceptor loss. Eye 2 from a 91-year-old man with advanced dry AMD had GA, some RPE loss, and numerous various-sized drusen. Eye 3 from a 74-year-old man with advanced wet AMD had an exudative lesion observed in macular sections. Eye 4 from an 82-year-old man with midstage dry AMD had several hard and soft drusen.
TISSUE PROCESSING AND HISTOLOGIC ANALYSIS
Eyes were procured within 12 hours of death and were fixed in a 10% formalin solution. An incision was made 3 mm posterior to the limbus, and the cornea, iris, lens, and vitreous were removed. A block of tissue approximately 6 to 8 mm2 containing the macula was excised, rinsed in phosphate-buffered saline (PBS) for 2 hours, and then cryoprotected in 30% sucrose in PBS. The tissue was embedded in optimal-cutting temperature compound (Tissue-Tek; Sakura Finetek USA, Inc, Torrance, Calif) and stored at –80°C. Fourteen-micrometer-thick cryostat sections were collected for immunohistochemistry.
IMMUNOHISTOCHEMICAL ANALYSIS
Sections were rinsed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes, and then treated with potassium permanganate for 30 minutes at room temperature to bleach the RPE autofluorescence, followed by treatment with oxalic acid until colorless.16-17 After washing with PBS, sections were incubated for 30 minutes in hydrogen peroxide in methanol, blocked for 30 minutes in normal serum at room temperature, and incubated with primary antibody overnight at 4°C. Primary antibodies A-crystallin and B-crystallin (1:2500; Stressgen, San Diego, Calif) and RPE65 (1:200) and fluorescent secondary antibodies (1:800; Jackson Immunoresearch Laboratories, West Grove, Pa, or Molecular Probes, Eugene, Ore) were used. Nuclei were stained using 4,6-diamidino-2-phenylindole (DAPI) (1 µg/µL; Sigma-Aldrich Co, St Louis, Mo) for 5 to 10 minutes. Phase and fluorescent images were acquired using an inverted microscope (Axiovert 200) and a digital camera (AxioCam MRm) with AxioVision version 4.5 software (all from Carl Zeiss, Thornburg, NY).
RESULTS
Here we demonstrate that B-crystallin is expressed at elevated levels in the RPE from patients with AMD. Expression of B-crystallin was examined using immunohistochemistry on cryostat sections obtained from 23 human eyes with different stages of AMD progression compared with aged controls (Table). The B-crystallin protein was not detected in any of the RPE of age-matched controls, but was seen in the underlying choroidal layer (Table and Figure 1). The A-crystallin was present throughout many retinal layers, as described previously.17
|
|
|
|
Figure 1. Retinal pigment epithelium (RPE) from healthy human eyes does not express B-crystallin, as shown in photomicrographs of healthy human retinas labeled with anti–B-crystallin (red) and anti–A-crystallin (green). Nuclei are stained with 4,6-diamidino-2-phenylindole (DAPI) (blue). Higher-power views of C and G are shown in D and H, respectively, and demonstrate a lack of B-crystallin staining in the RPE. Br indicates Bruch's membrane; Chr, choroidal neovascularization; GCL, ganglion cell layer; INL, inner nuclear layer; and ONL, outer nuclear layer. Original magnification x10 (A-C), x20 (E-G), x40 (D), and x60 (H). The bar in G indicates 50 µm; the bar in H, 25 µm.
|
|
|
In contrast, B-crystallin was found in RPE cells in all eyes with AMD examined, revealing a strong correlation between B-crystallin expression and AMD (Table and Figures 2, 3, and 4). There were fewer B-crystallin–positive cells in eyes with early AMD (exemplified by eye 1 with early dry AMD), and they tended to be restricted to the macular region (Figure 2A-D). In contrast, B-crystallin–positive cells were more numerous throughout the macula and extended into the periphery in the RPE of eyes with more advanced dry AMD (exemplified by eye 2 with advanced dry AMD) (Figure 2E-P). A similar trend was observed using ABC labeling, a different visualization method for staining (data not shown).
|
|
|
|
Figure 2. The B-crystallin is expressed in retinal pigment epithelium (RPE) cells in eyes with early and advanced age-related macular degeneration (AMD). A-E, Eye 1. The B-crystallin staining is seen in cells of the RPE layer, frequently near drusen in the eye with early dry AMD. Shown is a patch of highly labeled cells in the macular region. F-T, Eye 2. The B-crystallin is more widespread throughout the RPE of eyes with more advanced dry AMD and is frequently seen in RPE cells lying above drusen. Br indicates Bruch's membrane; Dr, druse; and ONL, outer nuclear layer. Original magnification x20 (A-D), x32 (F-I and K-N), x40 (E and P-S), and x60 (J, O, and T). The bar in S indicates 50 µm; the bar in T, 25 µm.
|
|
|
|
|
|
|
Figure 4. Eye 3. The B-crystallin is expressed in retinal pigment epithelium (RPE) cells of eyes with wet AMD, as demonstrated in hypertrophic RPE cells near the site of choroidal neovascularization in the macular region of eye 3 with advanced wet AMD. The RPE cells were identified by RPE65 staining. Br indicates Bruch's membrane; DS, disciform scarring; and ONL, outer nuclear layer. Original magnification x32. The bar in L indicates 50 µm.
|
|
|
We compared the incidence of B-crystallin with that of A-crystallin by double-immunostaining. The A-crystallin was found in all the retinal layers in eyes with AMD and sometimes in the RPE layer (Figures 1, 2, and 4). Strong and distinct expression of A-crystallin was observed in the Bruch's membrane in the regions of GA in eye 2 with advanced dry AMD (data not shown).
The B-crystallin was encountered frequently in RPE cells that were associated with drusen, but it was also detected in RPE cells in which no adjacent drusen were visible, as shown in Figure 3. In early AMD, B-crystallin–positive RPE cells were found more frequently in direct contact with drusen than not (P<.001, t test), but this association is lost as AMD advances and B-crystallin expression becomes more widespread in the RPE.
EXPRESSION OF B-CRYSTALLIN IN EYES WITH WET AMD
In the eyes with wet AMD that we examined (exemplified by eye 3 with advanced wet AMD in the Table and in Figure 4), significant B-crystallin expression was observed. Expression was strong in hypertrophic RPE cells and near choroidal neovascularization. Staining with the RPE-specific marker RPE65 showed colocalization of RPE65 and B-crystallin in a subpopulation of cells in this layer. The B-crystallin was also seen in the ganglion cell layer, inner nuclear layer, outer nuclear layer, and choroidal connective tissue.
B-CRYSTALLIN–POSITIVE RPE CELLS WERE NOT UNDERGOING CELL DEATH
Because B-crystallin expression has been shown to be protective against cell death and because apoptosis accompanies AMD,18 we investigated whether these B-crystallin–positive RPE cells in eyes with AMD were undergoing apoptosis. In double-labeling experiments, RPE cells that were TUNEL (terminal deoxynucleotidyl transferase–mediated biotin-deoxyuridine 5-triphosphate nick-end labeling) positive were not B-crystallin positive, and RPE cells that were B-crystallin positive were not TUNEL labeled (data not shown).
B-CRYSTALLIN IS EXPRESSED IN A SUBPOPULATION OF CULTURED RPE CELLS
To confirm that B-crystallin can be expressed by RPE cells, we isolated primary RPE cells from 57- to 90-year-old cadaveric eyes, cultured these for up to 3 weeks, and stained the cultures for RPE markers and B-crystallin expression. A subpopulation of RPE cells expressed B-crystallin, and the incidence of positive cells was 80% at 4 days (data not shown). Hence, this biomarker is normally expressed in a subpopulation of RPE cells in culture, allowing studies of mechanisms of induction and suppression.
COMMENT
It is important to understand the mechanisms involved in early AMD to develop novel treatments. Here we show that the stress-response protein B-crystallin is a reliable biomarker of AMD-affected RPE cells from early through late stages of the disease. This indicates that individual RPE cells undergo phenotypic alteration in AMD and that the change includes expression of B-crystallin.
We found that B-crystallin is heterogeneously expressed in RPE cells in eyes with drusen, but we did not detect it in the RPE from healthy aged eyes. In eyes with dry AMD, B-crystallin expression occurred more frequently in RPE cells apposing drusen and occurred less frequently in RPE cells not adjacent to visible drusen. Hence, there is a clear association between B-crystallin expression and drusen formation in the early stages of AMD. The function of B-crystallin in AMD is unknown; however, it may help protect stressed RPE cells from cell death.
In conclusion, the detection of B-crystallin in the RPE throughout AMD progression opens up new avenues for exploring the changes in RPE cells that accompany AMD. The RPE cells change expression in an individual cell-by-cell manner rather than as a generalized process diffusely affecting the entire RPE layer. This leads us to hypothesize that the mechanism of AMD includes the activation of cell-intrinsic programs to alter individual RPE cells. Further studies of such changes in RPE cell phenotype will aid in our understanding of the mechanisms of drusen formation and, ultimately, the pathogenesis of AMD.
AUTHOR INFORMATION
Correspondence: Sally Temple, PhD, Center for Neuropharmacology and Neuroscience, Albany Medical College, 42 New Scotland Ave, Albany, NY 12208 (temples{at}mail.amc.edu).
Submitted for Publication: August 15, 2006; final revision received October 19, 2006; accepted October 22, 2006.
Author Contributions: Dr Temple had full access to all of the data in this study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Financial Disclosure: None reported.
Funding/Support: This study was supported by the Ruth and Milton Steinbach Foundation, New York. All human research tissue was obtained with funding to Dr Stern.
Role of the Sponsor: The Ruth and Milton Steinbach Foundation approved the concept of the study but was not directly involved in its design or conduct, in the handling of the data, or in the preparation of the manuscript.
Acknowledgment: We thank T. Michael Redmond, PhD, for his generous donation of the RPE65 antibody.
Author Affiliations: Center for Neuropharmacology and Neuroscience (Drs De, Salero, and Temple and Mr Rabin and Ms Lederman) and Department of Ophthalmology (Dr Stern), Albany Medical College, Albany, NY.
REFERENCES
| |
1. Ambati J, Ambati BK, Yoo SH, Ianchulev S, Adamis AP. Age-related macular degeneration: etiology, pathogenesis, and therapeutic strategies. Surv Ophthalmol. 2003;48:257-293.
FULL TEXT
|
ISI
| PUBMED
2. Congdon N, O’Colmain B, Klaver CC; et al, Eye Diseases Prevalence Research Group. Causes and prevalence of visual impairment among adults in the United States. Arch Ophthalmol. 2004;122:477-485.
FREE FULL TEXT
3. Green WR, Key SN III. Senile macular degeneration: a histopathologic study. Trans Am Ophthalmol Soc. 1977;75:180-254.
PUBMED
4. Abdelsalam A, Del Priore L, Zarbin MA. Drusen in age-related macular degeneration: pathogenesis, natural course, and laser photocoagulation–induced regression. Surv Ophthalmol. 1999;44:1-29.
FULL TEXT
|
ISI
| PUBMED
5. Hageman GS, Luthert PJ, Victor Chong NH, Johnson LV, Anderson DH, Mullins RF. An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE–Bruch's membrane interface in aging and age-related macular degeneration. Prog Retin Eye Res. 2001;20:705-732.
FULL TEXT
|
ISI
| PUBMED
6. Crabb JW, Miyagi M, Gu X; et al. Drusen proteome analysis: an approach to the etiology of age-related macular degeneration. Proc Natl Acad Sci U S A. 2002;99:14682-14687.
FREE FULL TEXT
7. Hollyfield JG, Salomon RG, Crabb JW. Proteomic approaches to understanding age-related macular degeneration. Adv Exp Med Biol. 2003;533:83-89.
ISI
| PUBMED
8. Graw J. The crystallins: genes, proteins and diseases. Biol Chem. 1997;378:1331-1348.
ISI
| PUBMED
9. Richter-Landsberg C, Goldbaum O. Stress proteins in neural cells: functional roles in health and disease. Cell Mol Life Sci. 2003;60:337-349.
FULL TEXT
|
ISI
| PUBMED
10. Shinohara H, Inaguma Y, Goto S, Inagaki T, Kato K. B crystallin and HSP28 are enhanced in the cerebral cortex of patients with Alzheimer's disease. J Neurol Sci. 1993;119:203-208.
FULL TEXT
|
ISI
| PUBMED
11. van Rijk AF, Bloemendal H. Alpha-B-crystallin in neuropathology. Ophthalmologica. 2000;214:7-12.
FULL TEXT
|
ISI
| PUBMED
12. Andley UP, Song Z, Wawrousek EF, Fleming TP, Bassnett S. Differential protective activity of A- and B-crystallin in lens epithelial cells. J Biol Chem. 2000;275:36823-36831.
FREE FULL TEXT
13. Mehlen P, Schulze-Osthoff K, Arrigo AP. Small stress proteins as novel regulators of apoptosis: heat shock protein 27 blocks Fas/APO-1– and staurosporine-induced cell death. J Biol Chem. 1996;271:16510-16514.
FREE FULL TEXT
14. Alge CS, Priglinger SG, Neubauer AS; et al. Retinal pigment epithelium is protected against apoptosis by B-crystallin. Invest Ophthalmol Vis Sci. 2002;43:3575-3582.
FREE FULL TEXT
15. Sakaguchi H, Miyagi M, Shadrach KG, Rayborn ME, Crabb JW, Hollyfield JG. Clusterin is present in drusen in age-related macular degeneration. Exp Eye Res. 2002;74:547-549.
FULL TEXT
|
ISI
| PUBMED
16. Proulx S, Guerin SL, Salesse C. Effect of quiescence on integrin 5β1 expression in human retinal pigment epithelium. Mol Vis. 2003;9:473-481.
ISI
| PUBMED
17. Nakata K, Crabb JW, Hollyfield JG. Crystallin distribution in Bruch's membrane–choroid complex from AMD and age-matched donor eyes. Exp Eye Res. 2005;80:821-826.
FULL TEXT
|
ISI
| PUBMED
18. Dunaief JL, Dentchev T, Ying GS, Milam AH. The role of apoptosis in age-related macular degeneration. Arch Ophthalmol. 2002;120:1435-1442.
FREE FULL TEXT
|
