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From The Eye Institute, The Cleveland Clinic Foundation, Ohio.
Abstract
PURPOSE. To assess the distribution, content, and function of tissue inhibitor of metalloproteinases (TIMP)-3 during aging in normal eyes for comparison with the levels observed in eyes with age-related macular degeneration (AMD).
METHODS. Donor tissues analyzed included 36 normal eyes (14–96 years old) and 15 AMD eyes (74–98 years old). A tissue strip including the fovea was used for immunohistochemistry. Western blot analysis was performed on extracts of the retinal pigment epithelium (RPE)–choroid complex from the posterior part of each eye. Immunoreactivity of TIMP-3 bands in each western blot was densitometrically quantitated. The inhibitory function of TIMP-3 was evaluated with reverse zymography.
RESULTS. TIMP-3 was present uniformly across Bruch’s membrane in the normal samples. In samples from donors more than 50 years of age, immunostaining was intense. TIMP-3 content ranged from 92 to 1061 ng/cm2 and increased with age (r = 0.66). In AMD eyes, TIMP-3 distribution in Bruch’s membrane was abundant in areas of continuous soft drusen but absent in areas below RPE atrophy. TIMP-3 levels in AMD eyes were significantly higher than in age-matched normal eyes (577 versus 877 ng/cm2; P = 0.009). Inhibitory activity correlated well with TIMP-3 content (r = 0.82) and was also significantly higher in AMD eyes than in age-matched normal eyes (P < 0.001).
CONCLUSIONS. During normal aging, TIMP-3 content in Bruch’s membrane of the macula shows a significant increase. TIMP-3 content in AMD eyes was elevated relative to that of age-matched normal eyes. Higher levels of TIMP-3 may contribute to the thickening of Bruch’s membrane observed in AMD.
Matrix metalloproteinases (MMPs) and the tissue inhibitors of matrix metalloproteinases (TIMPs) play important roles in regulating the turnover of the extracellular matrix (ECM). MMPs constitute a family of secreted enzymes, currently with more than 20 members, that are involved in degrading components of the ECM in the normal course of matrix turnover and renewal.1 MMPs are also implicated during the initial stages of neovascularization, in which they are thought to be required, along with other proteases, for degradation of components of the capillary basement membrane as a prerequisite for new vessel outgrowth.2 The TIMPs, which are represented by four distinct gene products, are thought to suppress excessive degradation of ECM and may play an important functional role in limiting neovascularization.3 4 5 6 7 8 9 10
TIMP-3 is unique in that once secreted, it binds to component(s) of the ECM, whereas other TIMPs do not.11 In the outer eye wall, immunohistochemical studies indicate that TIMP-3 is present in normal Bruch’s membrane,12 13 and in situ hybridization studies indicate that the RPE is a major site of TIMP-3 gene expression.14 15 One role of TIMP-3 in Bruch’s membrane may be as a potent local inhibitor of MMP activity, regulating the rate of Bruch’s membrane turnover, as well as limiting choroidal neovascularization.
Sorsby’s fundus dystrophy is an early-onset, inherited form of macular degeneration, characterized by thickening of Bruch’s membrane and submacular neovascularization, which are also features of AMD. Mutations in the gene coding for TIMP-3 have been found in families with Sorsby’s fundus dystrophy.16 Immunohistochemical studies of a donor eye from a Sorsby’s patient showed extensive TIMP-3 accumulation in the thickened Bruch’s membrane.17 These observations led us to evaluate TIMP-3 content and distribution in Bruch’s membrane of AMD donor eyes, which are known to accumulate drusen and exhibit abnormal thickening of this layer. Although no mutations in the coding region or the regulatory elements of the TIMP-3 gene have been discovered in patients with AMD to date,18 19 excess TIMP-3 within the ECM could prevent normal matrix remodeling and could be causally involved in the increased thickening of Bruch’s membrane that occurs in AMD.
Because of the importance of Bruch’s membrane permeability in the trafficking of metabolites between choroid and RPE20 and the known early alterations in this lamina in AMD,21 it is important to understand the relationship of TIMP-3 in normal aging and AMD. In this study we follow the age-related changes in TIMP-3 distribution, content, and inhibitory activity during normal aging and compare these levels with those present in age-matched AMD donor eyes.
Methods
Donor Tissues
Thirty-two eyes from normal human donors (Table 1)
and 15 eyes from AMD donors (Table 2)
were used. Normal eyes were obtained through the Cleveland Eye Bank ,
Ohio, and the National Disease Research Interchange (Philadelphia, PA).
They were enucleated between 1.5 and 7 hours after death and preserved
at 4°C for 1 to 14 hours. Immediately after arrival at our laboratory
or National Disease Research Interchange, they were frozen in liquid
nitrogen and stored at -80°C until sample preparation. AMD donor
eyes were obtained through the Eye Donor Program of the Foundation
Fighting Blindness (Hunt Valley, MD). They were enucleated between 1.5
and 8 hours after death and retained at 4°C for 0.5 to 11.5 hours
before freezing in liquid nitrogen before storage at -80°C.
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For biochemical samples, the retina, any adhering vitreous, and sclera were removed from the remaining 10 x 10-mm aerial expanse of tissue and the RPE–Bruch’s membrane–choroid complex was retained for extraction. Each sample was weighed and then homogenized in 350 µl of extraction buffer, consisting of 500 mM Tris-HCl (pH 7.6), 200 mM NaCl, 1% Triton X-100, and protease inhibitors (400 µg/ml EDTA, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 100 µg/ml phenylmethylsulfonyl fluoride). After incubation for 15 minutes on ice followed by centrifugation at 13,000 rpm for 30 minutes at 4°C, the supernatant was collected and stored at -70°C. Protein concentration of each sample was measured using bicinchoninic acid (BCA; Pierce, Rockford, IL) and bovine serum albumin (BSA) as a reference standard.
TIMP-3 content was evaluated as the amount per area (10 x 10 mm2). Wet tissue weight and protein content of each sample are presented in Tables 1 and 2 .
Immunohistochemistry
The 2 x 10-mm tissue strip including the fovea was embedded
in optimum cutting temperature (OCT) compound (Tissue-Tek, Sakura
Finetek, Torrance, CA) and frozen in liquid nitrogen for
immunocytochemistry in the absence of fixation. Indirect
immunohistochemistry was performed on 8-µm-thick cryosections. After
quenching endogenous peroxidase activity with 0.3% hydrogen peroxide
and blocking nonspecific antibody binding with 5% BSA in PBS
containing 0.3% Triton X-100, the sections were incubated overnight at
4°C with a mouse monoclonal anti-human TIMP-3 antibody (either Clone
136 -13H4, provided by Suneel Apte or 136-17B12, provided by Kazushi
Iwata, [Fuji Chemical Industries, Toyama, Japan]). The primary
antibody was used at 1:1000 dilution. Biotinylated horse anti-mouse IgG
(1:200 dilution; Vector, Burlingame, CA) was used as a secondary
antibody followed by the avidin–biotin complex method (Elite
ABC, Vector). Immunoreactivity was resolved with
horseradish peroxidase-aminoethylcarbazole, which produces a magenta
reaction product. Sections were counterstained with hematoxylin.
Control sections were prepared in an identical manner with the
exception that nonimmune mouse IgG was used as the primary antibody.
Microscopic Evaluation
Because no detailed clinical history was available on any of the
donor eyes classified as AMD, it was necessary to evaluate the status
of the disease in the AMD donor tissues based on the histopathology of the tissue samples. For classification of the AMD
disease status, we assessed the following criteria, also used in the
recently published ARMD Grading System22
: hard drusen
status: (-) no hard drusen, (+) 1 to 3 hard drusen in the 10-mm
section, (++) 4 or more hard drusen in the 10-mm section; soft drusen
status: (-) no soft drusen, (+) patchy soft drusen present but not
continuous, (++) continuous soft drusen extending more than 0.5 mm; the
presence and length of choroidal neovascular membranes; and RPE atrophy
measured in the full 10-mm length of the section. We could not evaluate
basal laminar or linear deposits, because these features cannot be
distinguished within drusen with light microscopy on unfixed tissue
samples. The listed features are presented for each AMD donor eye in
Table 2 .
Western Blot Analysis
Immediately before electrophoresis, 3.5 µl of sample buffer (4x
NuPage sample buffer, Novex, San Diego, CA) containing dithiothreitol
(final concentration, 50 mM) was added to 6.5 µl of the protein
extract. The 4x sample buffer (pH 8.5) consisted of 1.17 M sucrose,
563 mM Tris base, 423 mM Tris-HCl, 278 mM sodium dodecyl sulfate (SDS),
2.05 mM EDTA, 0.88 mM Coomassie Blue R250 and 0.70 mM phenol red. After
heating for 10 minutes at 70°C, 10-µl volumes of each sample were
loaded into gel slots for electrophoresis on 10% gels (Bis-Tris;
Novex) with 2-(N-morpholino) ethane sulfonic acid SDS
running buffer (NuPage MES-SDS; Novex). Recombinant human TIMP-3
protein (10 ng) was included in a separate lane as a positive control.
Proteins were transferred from the gel to polyvinylidene difluoride
membranes using blotting apparatus (XCell II; Novex). After transfer,
membranes were incubated with blocking solution (2% BSA in
Tris-buffered saline) for 1 hour. The monoclonal anti-human TIMP-3
antibody (Clone 136-17B12) conjugated to horseradish peroxidase was
applied at 1:1000 dilution and incubated overnight at 4°C. After
rinsing, immunoreactivity was displayed with the chemiluminescent
method (ECL, Amersham, Arlington Heights, IL) and captured on
radiographic film during a 10- to 15-second exposure. The
immunoreactivity signal was digitized on a Scanwizard
(Microtek, Redondo Beach, CA) flatbed scanner. The intensity of
immunoreactivity was quantitated from the digitized images using
commercial software (Digital Science 1D; Eastman Kodak, Rochester, NY)
on a personal computer (Power Macintosh, Apple Computer, Cupertino,
CA). The densitometric intensity was converted to amount of protein
(nanograms per sample) by comparing the intensity of immunoreactivity
with that of varying amounts of recombinant TIMP-3 standard.
Protein Isolation and N-Terminal Sequence Analysis
In addition to recognizing the TIMP-3 bands at 24 and 27 kDa, the
monoclonal antibody also interacted with two additional bands at 37 and
40 kDa. To determine whether these were aggregates of TIMP-3 or
unrelated proteins, we excised the higher molecular weight bands from
polyvinylidene difluoride membrane after electrotransfer from a
separate gel and analyzed the bands by Edman microsequencing using a
(Procise 492; PE Biosystems, Foster City, CA) protein sequencer
in the Molecular Biology Core Laboratory, Case Western Reserve
University (Cleveland, OH).
Reverse Zymography
TIMP-3 functional activity in each sample was established with
reverse zymography using protease–substrate gel electrophoresis, as
described previously.11
In brief, the extracted protein
from the RPE–choroid complex was diluted to 75 times and activated by
adding calcium chloride at a final concentration of 10 mM. After mixing
with 4x loading buffer (40 mM Tris, 8% SDS, 40% glycerol, and 0.01%
bromphenol blue), 20 µl of the sample (without heating or reducing
agents) was separated by electrophoresis in a matrix consisting of
0.1% SDS, 12% polyacrylamide gel containing MMP-2, MMP-9, and 0.1%
gelatin (from a reverse zymography kit which also includes TIMP-3
standards provided by Dylan R. Edwards, The University of Calgary,
Alberta, Canada). To remove SDS, the gel was rinsed overnight in the
following buffer (50 mM Tris [pH 7.5], 5 mM
CaCl2, and 25 mg/ml Triton X-100). The gel was
then placed in an incubation buffer (50 mM Tris [pH 7.5 ] and 5 mM
CaCl2) to allow MMPs to degrade gelatin for 24
hours at 37°C. Coomassie blue stains only the protein (gelatin) in
the region where MMP activity has been inhibited. Intensity of the
Coomassie blue–stained gelatin was quantitated as described earlier.
The values obtained were compared with the intensity of Coomassie blue
staining of the gelatin in lanes with known concentrations of TIMP-3
standards.
Statistical Analysis
Western blot analysis and reverse zymography were repeated three
times on each sample. The results are expressed as the mean ± 1
SD.
Diversity of tissue weight and protein concentration of samples were analyzed with unpaired Student’s t-test. Correlations between TIMP-3 content and age or inhibitory activity were determined with Pearson’s correlation coefficient. Differences in TIMP-3 content and inhibitory activity between AMD and age-matched normal tissue were analyzed with the Mann–Whitney test.
Results
TIMP-3 Distribution and Content in Normal Eyes
Table 1
presents a summary of all the data and vital information
from each normal eye used in this analysis.
Distribution.
TIMP-3 immunoreactivity was present in Bruch’s membrane in each normal
donor tissue used and was distributed across the full thickness of this
membrane. Although immunoreactivity was uniform in each donor sample,
in general, eyes from younger donors showed less intense immunostaining
than eyes from older donors. In addition to TIMP-3 immunolocalization
in Bruch’s membrane, samples from 80- and 90-year-old donors commonly
exhibited intense staining in the capillary bed matrix. TIMP-3
immunoreactivity was not evident in the neurosensory retina, the
choroid, or the sclera. In the control tissues where nonimmune mouse
IgG was substituted for the monoclonal IgG, Bruch’s membrane showed no
immunoreactivity. Examples of TIMP-3 immunolocalization from donors at
a variety of representative ages are presented in Figure 1
.
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The 40-kDa immunoreactive band yielded the N-terminal sequence of LAIPALAQEQPQGQ, which shows less than 40% homology to any known protein. Although the identity of this 40-kDa protein was not established, the sequence obtained did not identify this protein as an aggregate of TIMP-3.
The immunoreactivity present in the 24- and 27-kDa western blot bands was variable among different samples. Notably, the relative amounts of immunoreactivity were found to be age dependent, with less intense signals present in samples from young donors than from older donors. Densitometric measurements of specific TIMP-3 immunoreactivity in the 24- and 27-kDa bands suggest that the TIMP-3 content ranged from a low of 92 ng/cm2 in a sample from a 14-year-old donor of normal eyes to 1061 ng/cm2 in a sample from a 90-year-old donor of normal eyes (Table 1) . A direct relationship between TIMP-3 content and age (Fig. 3) appears to be significant in all the normal tissue analyzed (correlation coefficient, r = 0.66; n = 32).
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TIMP-3 Distribution and Content in AMD Eyes
Table 2
presents the summary of all the data and vital information
from each AMD eye used in this analysis.
Distribution.
Representative examples of several features characteristic of AMD eyes
are shown in Figure 4
. Each AMD eye used contained soft drusen, with 7 of the 15 samples
having a continuous expanse of soft drusen for the full 10-mm length of
the sample section examined. Whenever soft drusen were observed,
whether isolated or continuous, each was intensely immunoreactive with
the TIMP-3 antibody (Figs. 1
4A)
. Hard drusen, when present, were also
strongly immunoreactive. Most of the AMD donor tissues (13 of 15)
contained areas of RPE atrophy, which involved from 0.5 to 8.5 mm of
the 10-mm section length examined. In Bruch’s membrane below the
expanses of RPE atrophy, TIMP-3 immunoreactivity was either not evident
or barely detectable (Fig. 4C)
. Choroidal neovascularization was
present in 8 of the 15 AMD donor eyes. RPE atrophy, where little or no
TIMP-3 immunoreactivity was present, as described earlier, was always
noted below the areas of choroidal neovascularization (Fig. 4)
. In
areas where the RPE had proliferated around a choroidal neovascular
membrane or a fibroblastic scar, TIMP-3 immunoreactivity was observed
surrounding the hyperplastic RPE (Fig. 4C)
. The immunoreactivity is
subtle, however, and shows irregular distribution, possibly because of
a loss of function and polarity of the proliferated RPE. In regions of
transition between areas of RPE atrophy and a normal RPE, TIMP-3
immunoreactivity decreases but is still present in soft drusen and
below the central elastin layer of Bruch’s membrane (Fig. 4B)
.
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The amount of functional TIMP-3 present in Bruch’s membrane below the macula of the normal human retina appears to be age dependent. Sections from the fovea of the donors from the second and third decade of life were weakly immunoreactive with anti-TIMP-3 antibody. This correlated well with lower amounts of immunoreactivity observed in western blot analysis. Additionally, TIMP-3 distribution changed in the 9th and 10th decade samples, with extension of immunoreactivity from Bruch’s membrane into the matrix surrounding the choriocapillaris. Quantitative analysis showed that TIMP-3 content and function in the macula increased with age with a significant correlation coefficient (r = 0.66 and 0.67, respectively). Proteins that increase or decrease with age can be referred to as senescence-related proteins and are candidate molecules for age-related diseases.23 24 Accordingly, we propose that TIMP-3 is a senescence-related protein.
Immunostaining of TIMP-3 was conspicuous in extensive accumulation of drusen, which is characteristic of AMD eyes. Quantitative analysis showed that TIMP-3 levels were significantly elevated in the macula of AMD eyes compared with normal eyes. AMD eyes, however, showed a nonuniform distribution, with virtually no TIMP-3 immunoreactivity below the area of RPE atrophy and abundant immunoreactivity in the area outside the atrophic areas where the RPE was present, which is consistent with the observation in Sorsby’s fundus dystrophy and retinitis pigmentosa.17 This indicates that TIMP-3 distribution in AMD eyes with RPE atrophy is nonuniform. To determine the TIMP-3 content below areas where the RPE remains, we assumed that the area of RPE atrophy was circular and that the diameter was represented by the length of atrophy measured in the tissue section. We then calculated the area of RPE atrophy and subtracted that area from 10 x 10 mm2. With this adjusted denominator, it was apparent that the TIMP-3 content below areas where RPE is present is approximately two times higher in AMD eyes than in age-matched normal eyes (Fig. 5) .
The monoclonal antibody used in this study recognized recombinant TIMP-3 in western blot analysis exhibiting characteristic 27- and 24-kDa bands. Although two higher molecular weight bands (approximately 37 and 40 kDa) appeared in the blots of most tissue samples, N-terminal amino acid sequence analysis revealed that these proteins were unrelated to TIMP-3. Reverse zymography also showed that these higher molecular weight components have no inhibitory activity. The 37-kDa nonspecific band was found to be GAPDH, a ubiquitous intracellular enzyme, and the purified protein showed cross-reactivity with the TIMP-3 antibody but with low affinity. In immunohistochemistry, however, no intracellular staining was observed. The presence of cross-reactivity in the western blot analysis and the absence of intracellular staining in immunohistochemistry probably resulted from differences in protein content. Coomassie staining of the transferred membrane showed a large amount of 37-kDa protein in the samples. The total amount of the protein in the tissue extract was large, but the concentration in each cell in the tissue sections was not sufficient to show nonspecific immunoreactivity.
Although neither structural mutations nor mutations in regulatory regions of the TIMP-3 gene are thought to be a cause of AMD,18 19 it is possible that a mutation in a gene regulating TIMP-3 translation causes TIMP-3 elevation or that TIMP-3 change occurs downstream of other disorders—for example, changes in the ECM-binding partner. Our immunohistochemical results show that soft drusen were markedly stained with anti-TIMP-3 antibody in AMD eyes. This may imply that an elevated TIMP-3 level in the macula of AMD resulted from TIMP-3 accumulation in soft drusen. Drusen are thought to be composed of incompletely degraded debris that is exocytosed by RPE cells. Various hereditary25 26 27 or nonhereditary factors such as protein alterations,27 oxidative stress,29 30 31 or disorders of hydrolytic enzymes32 33 may participate, along with aging, in accelerating the accumulation of this debris. Those insufficiently digested materials may possess a domain with an affinity for TIMP-3 binding. Because TIMP-3 broadly inhibits MMPs, drusen with excess TIMP-3 may retard Bruch’s membrane renewal. This may result in the thickening of Bruch’s membrane, reducing Bruch’s membrane permeability in the trafficking of metabolites and nutrients between the choroid and RPE, ultimately resulting in RPE and photoreceptor atrophy.
In areas where choroidal neovascularization was observed, the RPE was absent, and virtually no TIMP-3 immunoreactivity was evident in the subjacent Bruch’s membrane. There are two possible sequences to these changes: atrophy of the RPE leads to a decrease in TIMP-3, which is permissive to neovascularization; or choroidal neovascularization causes RPE atrophy, which is followed by TIMP-3 loss. Although current results do not indicate which occurs first, the antiangiogenic activity of TIMP-39 10 makes the former a more likely scenario. We speculate that, in some AMD eyes, Bruch’s membrane thickening causes an atrophy of RPE, and TIMP-3 levels decrease in the area of RPE atrophy, which is permissive for choroidal neovascularization.
We conclude that the TIMP-3 content in Bruch’s membrane in the macula increases during normal aging and that TIMP-3 content is elevated beyond normal levels in the macular region of AMD eyes. This suggests that TIMP-3 may be one of the key molecules causally involved in Bruch’s membrane thickening during normal aging and in AMD. Further studies are needed to identify the TIMP-3 binding partner(s) in Bruch’s membrane and drusen, to determine whether elevated TIMP-3 levels are caused by increased accumulation or increased synthesis of this inhibitor, and to establish whether TIMP-3 elevation is causally involved in the thickening of Bruch’s membrane, which is associated with age-related macular degeneration.
Acknowledgements
The authors thank Joan Fisher of The Foundation Fighting Blindness for her help in securing the AMD donor eyes used in the analysis; Jill Spitzer of the Cleveland Eye Bank for help in securing the normal eyes less than 70 years of age; and the National Disease Research Interchange for obtaining the normal eyes more than 70 years of age; Suneel Apte, Bela Anand–Apte, John W. Crabb, and Dylan R. Edwards for valuable discussions and technical advice during the course of this study; and Mary E. Rayborn for help in proofreading the manuscript.
Footnotes
Supported by The Foundation Fighting Blindness and the Retina Research Foundation.
Submitted for publication January 20, 1999; revised April 9, 1999; accepted April 20, 1999.
Proprietary interest category: N.
Corresponding author: Motohiro Kamei, The Eye Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue FFB-33, Cleveland, OH 44195. E-mail: kameim@cesmtp.ccf.org
References
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N. H. Sachdev, N. Di Girolamo, P. J. McCluskey, A. V. Jennings, R. McGuinness, D. Wakefield, and M. T. Coroneo Lens Dislocation in Marfan Syndrome: Potential Role of Matrix Metalloproteinases in Fibrillin Degradation Arch Ophthalmol, June 1, 2002; 120(6): 833 - 835. [Full Text] [PDF]
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J. H. Qi, Q. Ebrahem, K. Yeow, D. R. Edwards, P. L. Fox, and B. Anand-Apte Expression of Sorsby's Fundus Dystrophy Mutations in Human Retinal Pigment Epithelial Cells Reduces Matrix Metalloproteinase Inhibition and May Promote Angiogenesis J. Biol. Chem., April 12, 2002; 277(16): 13394 - 13400. [Abstract] [Full Text] [PDF]
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S. G. Jacobson, A. V. Cideciyan, J. Bennett, R. M. Kingsley, V. C. Sheffield, and E. M. Stone Novel Mutation in the TIMP3 Gene Causes Sorsby Fundus Dystrophy Arch Ophthalmol, March 1, 2002; 120(3): 376 - 379. [Abstract] [Full Text] [PDF]
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M. A Majid, V. A Smith, D. L Easty, A. H Baker, and A. C Newby Adenovirus mediated gene delivery of tissue inhibitor of metalloproteinases-3 induces death in retinal pigment epithelial cells Br. J. Ophthalmol., January 1, 2002; 86(1): 97 - 101. [Abstract] [Full Text] [PDF]
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 V. LAMBERT, C. MUNAUT, A. NOEL, F. FRANKENNE, K. BAJOU, R. GERARD, P. CARMELIET, M. P. DEFRESNE, J.-M. FOIDART, and J.-M. RAKIC Influence of plasminogen activator inhibitor type 1 on choroidal neovascularization FASEB J, April 1, 2001; 15(6): 1021 - 1027. [Abstract] [Full Text] [PDF]
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S. Majka, P. McGuire, S. Colombo, and A. Das The Balance between Proteinases and Inhibitors in a Murine Model of Proliferative Retinopathy Invest. Ophthalmol. Vis. Sci., January 1, 2001; 42(1): 210 - 215. [Abstract] [Full Text]
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S. L. Bernstein, A. M.-H. Liu, B. C. Hansen, and R. I. Somiari Heat Shock Cognate-70 Gene Expression Declines during Normal Aging of the Primate Retina Invest. Ophthalmol. Vis. Sci., September 1, 2000; 41(10): 2857 - 2862. [Abstract] [Full Text]
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C S SETHI, T A BAILEY, P J LUTHERT, and N H V CHONG Matrix metalloproteinase biology applied to vitreoretinal disorders Br. J. Ophthalmol., June 1, 2000; 84(6): 654 - 666. [Full Text]
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N. H. V. Chong, R. A. Alexander, T. Gin, A. C. Bird, and P. J. Luthert TIMP-3, Collagen, and Elastin Immunohistochemistry and Histopathology of Sorsby’s Fundus Dystrophy Invest. Ophthalmol. Vis. Sci., March 1, 2000; 41(3): 898 - 902. [Abstract] [Full Text]
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, normal samples; 
,
AMD samples; 
, adjusted AMD samples.
