(Investigative Ophthalmology and Visual Science. 2001;42:1626-1630.)
© 2001
by The Association for Research in Vision and Ophthalmology, Inc.
Age and Topographic Variation of Insulin-like Growth Factor–Binding Protein 2 in the Human RPE
Noritake Miyamura1,
Kazuaki Mishima1,
Shigeru Honda1,
Amy E. Aotaki-Keen1,2,
Lawrence S. Morse1,
James T. Handa1 and
Leonard M. Hjelmeland1,2
1 From the Departments of Ophthalmology and
2 Molecular and Cellular Biology, University of California, Davis.
 |
Abstract
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PURPOSE. Previous studies have shown that insulin-like growth factor–binding
protein (IGFBP)-2 is markedly upregulated in senescent RPE cells in
vitro, and might therefore be a marker of senescent cells in vivo. This
study was conducted to determine whether IGFBP-2 expression in human
RPE cells from the macula and periphery varies with age in vivo.
METHODS. Paraformaldehyde (4%)-fixed and optimal cutting temperature (OCT)
compound–embedded human eyes from 17 patients were cryosectioned and
subjected to high-sensitivity digoxigenin (DIG)-labeled cRNA in situ
hybridization to determine the expression of IGFBP-2. Complementary
immunohistochemistry experiments using a polyclonal anti-IGFBP-2
antibody were performed to confirm IGFBP-2 protein expression.
Specimens were examined by light microscopy, and images were captured
with a digital camera. The total numbers of RPE cells and IGFBP-2 mRNA
expression–positive RPE cells were counted for each section, and the
ratio of labeled RPE cells to total RPE cells counted was calculated
for both macular and peripheral regions of each donor.
RESULTS. IGFBP-2 mRNA expression was detected in the ganglion cell layer, inner
and outer nuclear layers, and inner segments of photoreceptor cells in
all 17 eyes. In 16 of 17 eyes, IGFBP-2 mRNA expression was detected in
the RPE. In 11, the ratio of labeled cells to total RPE cells counted
per section in the macula was 1.2 times greater than the ratio in the
periphery (P = 0.008). The ratio of labeled RPE
cells in the macula decreased with age (P =
0.0064). Immunohistochemistry studies for IGFBP-2 confirmed the
expression pattern found by in situ hybridization.
CONCLUSIONS. There is a topographical and age-related change in IGFBP-2 expression
in RPE cells from human donor eyes. This distribution is likely not to
represent senescent RPE cells in vivo.
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Introduction
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Age-related macular degeneration (ARMD), a leading cause of
blindness in western societies, is a progressive degeneration of the
photoreceptors and their underlying retinal pigment epithelium (RPE) in
the macular region of the retina. Although influences from genetic to
environmental have been implicated in the pathogenesis of ARMD,
chronological age is the primary determinant of disease onset. A series
of studies have suggested that RPE cells have a phenotype that varies
as a function of topography and/or age. For example, Burke and
Soref1
showed that RPE cells from the macula of the human
eye or the area centralis of the bovine eye have more limited
replicative life-spans in vitro than do cells from more peripheral
regions of the posterior pole. Flood et al.2
observed that
the growth rate and numbers of nondividing cells in primary cultures of
human RPE were directly correlated with the chronological age of the
donor eye.
Our laboratory has recently investigated the phenomenon of senescence
in RPE cells as it might pertain to the pathogenesis of
ARMD.3
4
We first demonstrated that the combined
senescence-associated ß-galactosidase (SABG)/bromo-deoxyuridine
incorporation assay identified senescent RPE cells in
vitro.5
Mishima et al.,6
adapting this same
method for use in the posterior pole of the primate eye, showed
selective SABG labeling in a group of RPE cells in a 29-year-old, but
not a 2-year-old, rhesus macaque eye. Because measurement of SABG
activity is dependent on the postmortem and fixation times, this
technique is unfortunately not feasible in human donor eyes. Recently,
we found that IGFBP-2 mRNA expression is dramatically upregulated in
human senescent RPE cells in vitro.7
Similarly, Shelton et
al.8
reported marked upregulation of IGFBP-2 in senescent
RPE 340 cells by microarray analysis. In our attempt to identify a
marker of senescence in vivo, we hypothesized that IGFBP-2 is
preferentially upregulated in macular RPE cells with age in vivo.
Herein, we report topographic differences of IGFBP-2 expression in RPE
cells as well as an age-related decline in the macula from human donor
eyes.
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Methods
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Tissue Processing
Fresh globes from 10 male and 7 female donors, ranging in age
from 27 to 83 years at the time of death, were obtained from the Sierra
Tissue Eye Bank (Sacramento, CA) within 21 hours of death. Table 1
outlines the age, sex, postmortem time, and systemic and
ophthalmic disease histories of each donor. Inspection with a
dissecting microscope did not reveal any obvious posterior segment
disease. The globes were fixed in phosphate-buffered saline (PBS; pH
7.4) containing 4% paraformaldehyde at 4°C. Using microscopic
guidance, the anterior segment was removed, and the posterior segment
was divided into five sections (approximately 6 x 6 mm): nasal
(nasal side of disc), superior (superior to arcade vessels), inferior
(inferior to arcade vessels), macular (centered around the foveola),
and temporal region (temporally outside of macula to equator). The
tissue was cryoprotected using the technique of Barthel and
Raymond9
and Mishima et al.6
All tissue
blocks were stored at -80°C until used. Cryosections (10 µm) were
cut with a cryotome (Bright Instrument Co., Huntingdon, UK), mounted on
coated glass slides (Vectabond; Vector Laboratories, Burlingame, CA),
and air dried at room temperature for 4 hours.
In Situ Hybridization
The cDNA containing plasmids for human IGFBP-2 and human opsin
were kindly provided by Shunichi Shimasaki and Jeremy Nathans,
respectively. The IGFBP-2 cDNA was 446 bp and inserted into a cloning
vector (pBluescript II SK(+); Stratagene, La Jolla, CA). Opsin cDNA was
inserted into the cloning vector (pBluescript II SK(+); Stratagene)
after digestion with EcoRI (Roche, Indianapolis, IN) and
ApaI (Roche) to 450 bp. The antisense and sense digoxigenin
(DIG)-labeled RNA riboprobes were synthesized according to the labeling
kit protocol (Roche). The length and integrity of the synthesized
riboprobes were quantified by gel electrophoresis. The concentrations
were estimated by the dot-spot test using DIG-labeled control RNA.
In situ hybridization histochemistry was performed according to
Braissant and Wahli,10
with slight modifications. After
postfixation in 4% paraformaldehyde-PBS for 10 minutes, sections were
immersed in 0.25% acetic anhydride for 10 minutes and 5x SSC for 15
minutes. Prehybridization was performed at 57°C for 2 hours in the
hybridization mixture (50% formamide, 5x SSC, and 40 µg/ml salmon
sperm DNA). After denaturing the probes for 5 minutes at 80°C,
hybridization was performed at 57°C for 40 hours with a cover
(Parafilm; American Can Company, Greenwich, CT) in a chamber saturated
with the hybridization mixture in a hybridization oven (Fisher
Scientific, Los Angeles, CA). Sections were washed and equilibrated in
buffer 1 (100 mM Tris, 150 mM NaCl, and 50 mM
MgCl2 [pH 7.5]) for 5 minutes and incubated in
alkaline phosphatase-coupled anti-DIG antibody (Roche) diluted 1:5000
in buffer 2 (buffer 1 with 0.5% DIG blocking reagent added) at room
temperature for 2 hours. The sections were equilibrated in buffer 3
(100 mM Tris, 100 mM NaCl, and 50 mM MgCl2 [pH
9.5]) for 5 minutes and color developed at room temperature in buffer
3 containing 5-bromo-4-chloro-3-indoyl phosphate 4-toluidine salt
(BCIP)-nitroblue tetrazolium chloride (NBT; Roche) overnight. Staining
was stopped by TE buffer (10 mM Tris, 1 mM EDTA [pH 8.0]) for
15 minutes. Nonspecific background staining was removed in 95% ETOH
for 1 hour. Selected sections were bleached with potassium
permanganate, as previously described.6
Sections were
counterstained with nuclear fast red (Vector Laboratories), dehydrated,
and mounted.
Immunohistochemistry
Eleven eyes (cases 1, 2–5, 9, 10, 12, 14–17) were investigated
by immunohistochemistry using a previously published
technique.11
After blocking with 3% goat serum and 3%
blocking reagent (Blotto; Santa Cruz Biotechnology, Santa, Cruz, CA),
sections were incubated at 4°C overnight with 1:1000 anti-bovine
IGFBP-2 rabbit polyclonal antibody (Upstate Biotechnology Inc., Lake
Placid, NY). Detection was performed using an ABC staining kit (Vector
Laboratories). Control sections were incubated with 1:1000 normal
rabbit immunoglobulin fraction (Dako, Carpinteria, CA) instead of the
primary antibody. Some sections were bleached with potassium
permanganate, as described earlier.6
Sections were
counterstained with nuclear fast red (Vector Laboratories), dehydrated,
and mounted.
Data Analysis
Specimens were observed under a light microscope (BH-2; Olympus
Optical Co., Ltd., Tokyo, Japan) with a charge-coupled device camera
(ProgRes 3012; Kontron Elektronik GmbH, Eching, Germany). Digitalized
images were captured through the digital camera plug-in directly to
graphic software (Photoshop 5.0; Adobe, Mountain View, CA). The number
of total RPE cells and in situ hybridization–labeled RPE cells were
counted. Each section contained at least 300 RPE cells. The ratio of
labeled RPE cells to total RPE cells counted per section was calculated
for both macular and peripheral regions in each eye. Statistical
significance was determined using the Wilcoxon signed-rank test for the
ratio of labeled macular cell versus the ratio of labeled peripheral
cells. Spearman’s rank correlation was used to determine the influence
of age on each ratio. P < 0.01 was considered
significant.
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Results
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In Situ Hybridization for IGFBP-2
IGFBP-2 mRNA expression was detected in the ganglion cell layer,
inner and outer nuclear layers, and inner aspects of photoreceptor
cells in all 17 globes (Fig. 1A)
. No labeling was detected with the IGFBP-2 sense probe, whereas opsin
mRNA expression was detected in the outer nuclear layer and inner
segment of photoreceptor cells (Figs. 1B
1C)
. Fifteen of 16 eyes
(except in case 11) showed IGFBP-2 mRNA labeling in the RPE. The RPE in
general showed nonuniform labeling characterized by clumps of labeled
cells adjacent to unlabeled cells. In 11 eyes, the macular RPE showed a
1.2-fold greater ratio of labeled cells to total RPE cells counted per
section compared with those counted in the periphery (P = 0.008, Wilcoxon signed-rank test). The number of IGFBP-2–labeled RPE
cells decreased with age in the macula, whereas the number of
IGFBP-2–labeled RPE cells in the periphery did not change (Fig. 2)
. The IGFBP-2–labeled RPE cell ratio in the macula decreased with age
(Fig. 3A
, P = 0.0064, Spearman’s rank correlation). In contrast,
the IGFBP-2–labeled RPE cell ratio in the periphery did not change
with age (Fig. 3B)
. These results suggest that the IGFBP-2 expression
in macular RPE cells was preferentially decreased with age compared
with those in the periphery.
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Figure 1. In situ hybridization using anti-sense IGFBP-2 (A), sense
IGFBP-2 (B), and anti-sense opsin (C) mRNA probes
in the macula of a 65-year-old eye (case 9). The DIG-labeled in situ
hybridization reaction with BCIP/NBT appears blue-purple.
(A) IGFBP-2 expression was detected in the ganglion cell
layer, inner and outer nuclear layers, and inner photoreceptor
segments. Arrowheads: RPE cells with IGBP-2 labeling.
(B) No labeling was seen with the sense probe.
(C) Opsin mRNA expression was detected only in the outer
nuclear layer and inner segment of photoreceptor cells ( ). Scale
bar, 50 µm.
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Figure 2. Labeling of IGFBP-2 mRNA by in situ hybridization in RPE cells by
region and age. RPE sections from a 27-year-old (case 1) macula
(A) and periphery (B), a 63-year-old (case 6)
macula (C) and periphery (D), and an 83-year-old
(case 17) macula (E) and periphery (F). The
DIG-labeled in situ hybridization reaction with BCIP/NBT appears
blue-purple. More IGFBP-2–labeled cells
(arrowheads) were seen in the macula than the periphery at
all ages. The number of IGFBP-2–labeled RPE cells decreased with age
in the macula, whereas the number of IGFBP-2–labeled cells in
periphery remained unchanged. Scale bar, 50 µm.
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Figure 3. Graph of the ratio of labeled to total cells counted as a function of
age in the macula (A) and periphery (B). The
ratio of labeled cells in the macula decreases with age (A;
P = 0.0064 Spearman’s rank correlation). The ratio of
labeled cells in the periphery showed no age-related change
(B).
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Immunohistochemical Staining of IGFBP-2
Immunohistochemical experiments were performed to determine
whether there was a corresponding decrease in IGFBP-2 protein in the
macula in 11 eyes. IGFBP-2 protein was located in the ganglion cells,
inner segment of photoreceptors (data not shown), RPE, and choroid.
Sections treated with rabbit IgG showed no staining (data not shown).
In the eyes that showed immunohistochemical staining for IGFBP-2
(n = 7), all eyes exhibited more RPE cells labeled with
IGFBP-2 in the macula than the periphery (Fig. 4)
. This differential immunostaining of IGFBP-2 between the macula and
the periphery was more obvious in young eyes.
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Figure 4. Immunohistochemical staining of IGFBP-2 in RPE cells by region and age.
The sections from a 27-year-old (case 1) macula (A) and
periphery (B), a 63-year-old (case 6) macula (C)
and periphery (D), and an 83-year-old (case 17) macula
(E) and periphery (F). The immunohistochemical
staining with BCIP-NBT appears blue-purple. More RPE cells
were labeled for IGFBP-2 (arrowheads) in the macula than in
the periphery in the young and middle-aged specimens. More
IGFBP-2–labeled macular RPE cells were visualized in the 27-year-old
than in the 63-year-old eye, whereas the immunoreactivity in the
peripheral RPE was the same for both ages. The RPE in the oldest eye
did not show immunoreactivity in either the macula or periphery. Scale
bar, 50 µm.
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Discussion
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In this study, we showed a topographic and age-related variation
in IGFBP-2 mRNA expression in human RPE cells by determining the number
of labeled cells and calculating the ratio of IGFBP-2–labeled RPE
cells to the total number of cells counted per section. We identified
two important associations between IGFBP-2 expression and human RPE
cells in vivo: First, more cells expressed IGFBP-2 in the macula than
the periphery at all ages in our collection, and second, the number of
labeled cells decreased with age in the macula but remained unchanged
in the periphery. Our immunohistochemical experiments supported these
findings.
To quantify topographic and age-related changes in IGFBP-2 expression,
we developed a method to count RPE cells. This method, by using
the ratio of labeled cells to total cells counted per section,
quantifies the expression differences that were qualitatively seen when
examining the sections. An advantage of using the ratio of labeled
cells to total cells is that the confounding influences, such as an
age-related decline in cell density, are excluded. We also considered
the effect of variable postmortem and tissue processing times on the
overall patterns of IGFBP-2 expression. One effect of prolonged
postmortem and processing times may be the loss of both IGFBP-2 mRNA
and protein. This effect would make processing time a significant
variable in our results. To exclude the effect of postmortem and
processing times, we examined specific correlations between the ratio
of labeled cells in the macula versus the ratio of labeled cells in the
periphery for individual eyes (i.e., the ratio of the two labeling
ratios). This analysis yielded the same results concerning topographic
and age-related variations in IGFBP-2 expression (data not shown). To
further exclude the effect of postmortem and processing times, we
incubated freshly enucleated rhesus macaque eyes at room temperature
from 0 to 24 hours before fixation. We found no difference in RPE cell
IGFBP-2 mRNA labeling in pattern and extent.
Our studies add to the topographical and age-dependent morphologic
changes to the RPE that have been reported in the
literature,1
12
13
14
15
16
and suggest that the phenotype of the
RPE differs depending on its location and age. For example, Liles et
al.16
found that catalase activity was higher and show a
greater decrease with age in the macula than in the periphery in normal
eyes, whereas superoxide dismutase (SOD) activity does not change.
Furthermore, Burke and Soref1
demonstrated that RPE cells
from the macula of the human eye or the area centralis of the bovine
eye have more limited replicative life-spans in vitro than do cells
from more peripheral regions of the posterior pole. In a separate
study, the same laboratory showed that cytochrome oxidase activity in
bovine and human RPE cells was lower in area centralis-macular cells at
all donor ages and that the highest activities were from the oldest
donors.15
Finally, this group observed phenotypic
variations in the staining of tight-junction complexes of RPE cells in
situ, noting a chimeric pattern reminiscent of our current
observations.17
The rationale for studying IGFBP-2 came from recent in vitro work from
our laboratory and others who showed marked upregulation of IGFBP-2
with replicative senescence using quantitative Northern and microarray
analyses, respectively.7
8
Our data contradict our
hypothesis that IGFBP-2 may be a useful marker of senescence in RPE
cells in vivo, because we found decreased IGFBP-2 expression with age.
The reason for this in vivo and in vitro difference in expression of
IGFBP-2 is unresolved, but our results highlight the potential
deficiencies of in vitro culture systems.18
19
Previously published studies indicate that the expression of IGFBP-2
appears to be affected by oxidative stress and chronological
age.20
21
22
23
Arnold et al.20
showed that
IGFBP-2 progressively decreases with age in human cerebrospinal fluid,
and speculate that IGFBP-2 is an important regulator of neuronal
function, especially during aging. Tham et al.24
showed
that IGF-II and IGFBP-2 were elevated in the cerebrospinal fluid of
patients with Alzheimer disease compared with normal subjects. Although
the exact role of IGFBP-2 in the RPE is unclear at this time, these
findings offer the intriguing possibility that IGFBP-2 helps to
maintain the RPE in a differentiated state in health, and during aging
or exposure to critical levels of oxidative stress, IGFBP-2 expression
could decline and promote the loss of RPE differentiation. Further
investigations are needed to determine IGFBP-2–specific functions in
the RPE cell during health and aging.
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Footnotes
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Supported by Grants EY06473 (LMH) and EY00344 (JTH) from the National Institutes of Health, a Research to Prevent Blindness Manpower Award (JTH), and a University of California Davis Health Science Award (JTH, LSM).
Submitted for publication December 7, 2000; revised February 9, 2001; accepted February 23, 2001.
Commercial relationships policy: N.
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: Leonard M. Hjelmeland, Vitreoretinal Research Laboratory, School of Medicine University of California, One Shields Avenue, Davis, CA 95616-8794. lmhjelmeland{at}ucdavis.edu
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Top
Abstract
Introduction
