Molecular Vision : 13:2310-2319 <http://www.molvis.org/molvis/v13/a261>
Received 11 July 2006 | Accepted 12 December 2007 | Published 21 December 2007
© 2007 Molecular Vision
Bruch’s membrane aging decreases phagocytosis of outer segments
by retinal pigment epithelium
Kai Sun,1 Hui Cai,1 Tongalp H. Tezel,2 David Paik,1 Elizabeth R. Gaillard,3 Lucian V. Del Priore1
1Department
of Ophthalmology, Harkness Eye Institute, Columbia University, New York, NY;; 2Kentucky Lions Eye Center,
University of Louisville, Louisville, KY;; 3Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL.
Purpose: We have shown previously that aging of human Bruch’s membrane affects the attachment, survival and gene
expression profile of the overlying retinal pigment epithelium (RPE). Herein we determine the effects of Bruch’s
membrane aging on RPE phagocytosis of rod outer segments.
Methods: Explants of human Bruch’s membrane were prepared from cadaver donor eyes (aged 9–81years) within 48 h
of death, and 6 mm punches were embedded with the basal lamina in a 96-well plate. Approximately 50,000 ARPE-19
cells per well were seeded onto the explant surface and cultured for two weeks until they reached confluence. In addition,
ARPE-19 were also seeded onto RPE-derived extracellular matrix (RPE-ECM) that was unmodified or modified by
nonenzymatic nitration. Bovine rod outer segments were purified by sucrose gradient centrifugation, labeled with 10 ug/
ml fluorescein isothiocyanate, and added to ARPE-19 cultured on Bruch’s membrane or RPE-ECM for 24 h. Phagocytic
activity was quantified by flow cytometry of harvested cells.
Results: The ability of RPE to phagocytose rod outer segments decreased as a function of aging of Bruch’s membrane;
mean phagocytotic activity of ARPE-19 on younger Bruch’s membrane was significantly higher than on older Bruch’s
membrane (129.7 ± 34.8 versus 67.4 ± 4.2 arbitrary units, respectively; p<0.01). Nitrite treatment of RPE-ECM decreased
rod outer segment phagocytosis compared to untreated RPE-ECM and mimicked the effects of aging of human Bruch’s
membrane.
Conclusions: Aging of human Bruch’s membrane decreases rod outer segment phagocytosis by ARPE-19. This effect
can be mimicked by nonenzymatic nitration of extracellular matrix in vitro. Our observations may have implications for
understanding the role of aging changes within Bruch’s membrane on pathogenesis of age-related macular degeneration
and other disorders.
In the normal human eye, the retina pigment epithelium
(RPE) forms a hexagonal cell monolayer that lines Bruch’s
membrane internally and separates the neural retina from the
choriocapillaris [1]. The RPE is responsible for maintaining
the integrity of the neural retina, choriocapillaris, and Bruch’s
membrane [1]. The apical RPE processes surround the distal
tip of the outer segments [1]. The RPE performs several
crucial functions important for maintaining the outer retina,
including phagocytosis of the distal tips of outer segments,
recycling of visual pigment, and transferring nutrients from
the choriocapillaris to the neural retina [2-4]. In addition, the
RPE is responsible for maintaining the integrity of the
choriocapillaris, as surgical RPE removal or pharmacological
RPE damage by intravitreal injection of ornithine or iodate
leads to secondary choriocapillaris atrophy [5,6]. The
integrity of the RPE monolayer is maintained if there is proper
attachment of the RPE to one another and to Bruch’s
membrane [7]. The attachment between the RPE and inner
aspects of Bruch’s membrane are mediated by integrin
receptors on the RPE and ligands within the basal lamina that
Correspondence: Lucian V. Del Priore, MD, PhD, Department of
Ophthalmology, Harkness Eye Institute of Columbia University, 635
West 165th Street, New York, NY, 10032; Phone: 212-305-2923;
Fax: 212-342-1724; email: ldelpriore@yahoo.com.
include laminin, fibronectin, and collagen type IV [8]; cellcell attachment is mediated by cadherins present on the lateral
RPE cell border [8].
Age-related macular degeneration (AMD) is
characterized by cellular changes in the RPE, choriocapillaris,
and outer retina and by structural changes within Bruch’s
membrane that include diffuse thickening, accumulation of
drusen, basal laminar, and basal linear deposits, collagen
cross-linking in the inner and outer collagen layer,
calcification, and fragmentation of the elastin layer and
lipidization [9-13]. Cellular changes in advanced AMD
include atrophy of the RPE, choriocapillaris, and outer retina
in nonexudative AMD and the development of choroidal
neovascularization in exudative AMD [14-18]. The
connection between the ultrastructural changes observed in
Bruch’s membrane with age and cellular changes that develop
in AMD is not known. However, it is known that the structural
changes within Bruch’s membrane precede cellular changes
in the RPE by one or two decades [9,10,12,19] and that agerelated changes within Bruch’s membrane can induce changes
in the attachment, survival, proliferation, and gene expression
profile of the overlying RPE [20-24]. The chronology of these
changes suggests that changes in Bruch’s membrane may
induce changes in the behavior of the overlying RPE.
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In the human eye in vivo, the RPE and Bruch’s membrane
must by necessity age together, thus making it impossible to
separate the effects of Bruch’s membrane aging from the
effects of cellular aging. Studying the effects of aged Bruch’s
membrane in the human eye in vivo creates a chicken-versusthe-egg (which comes first) dilemma since cellular aging of
the RPE may initiate aging of Bruch’s membrane which in
turn may accelerate RPE aging [25]. In the current study we
used a system previously developed in our laboratory to study
the effects of age-related changes within Bruch’s membrane
on the behavior of human RPE [20-22]. In essence, RPE from
tissue culture were seeded onto acellular human Bruch’s
membrane explants harvested from donors of different ages.
After repopulation of the surface, RPE monolayers were then
fed purified labeled outer segments to determine the effects
of Bruch’s membrane aging on RPE phagocytic ability. This
experimental design allowed us to separate the effects of
substrate aging from the effects of RPE aging. Our results
suggest that age-related changes in Bruch’s membrane can
decrease the phagocytosis ability of the RPE, and have
important implications for understanding age-related changes
in RPE function observed in AMD.
METHODS
Preparation of immortalized ARPE-19: Immortalized human
RPE (ARPE-19), obtained from American Type Culture
Collection (Manassas, VA), were cultured and propagated in
Dulbecco's Modified Eagle Medium (DMEM; Gibco, Grand
Island, NY) containing 10% fetal bovine serum (FBS),
100 IU/ml penicillin G, 100 ug/ml streptomycin, 100 ug/ml
gentamicin, and 2.5 ug/ml Amphotericin B (Sigma, St. Louis,
MO). The cells were incubated in a humidified atmosphere of
5% CO2 and 95% air at 37 °C, and the culture medium was
changed three times per week. Approximately 50,000 cells
were plated into 96-well plates (Nalge Nunc International,
Rochester, NY) and allowed to reach confluence (typically
two weeks). Eight wells of the culture were immunostained
with mouse antihuman ZO-1 antibody using fluorescence
conjugated secondary goat antimouse antibody (Invitrogen
Corp, Carlsbad, CA).
Isolation of Bruch’s membrane explants: Bruch’s membrane
explants were obtained from young (aged 9–46 years) and
older (aged 74–81 years) donors (Table 1) obtained through
the National Disease Research Interchange (Philadelphia,
PA). All donor eyes were enucleated within 10 h and shipped
in a sterile container at 4 °C and received in our laboratory
within 48 h of death. Two Bruch’s membrane explants were
harvested from each donor with the basal lamina layer of
Bruch’s membrane on the apical surface [20-22]. The
posterior pole of each eyecup was inspected visually with
direct and retroillumination under a dissecting microscope,
and globes were discarded if there were any evidence of
subretinal bleeding, extensive drusen, or irregular
pigmentation of the macular RPE. The eyes were placed in
carbon dioxide-free media (Gibco), and an incision was made
through the sclera 3 mm posterior to the limbus and extended
circumferentially. Four radial incisions were then made
through the sclera, and the sclera was peeled away. The
anterior segment and vitreous were removed and discarded.
The choroid-Bruch’s membrane-RPE complex was removed
after trimming its attachment to the optic nerve. Native RPE
were removed by bathing the explant with 0.02 M ammonium
hydroxide in a 60-mm polystyrene Petri dish (Falcon; Becton
Dickinson, Lincoln Park, NJ) for 20 min at room temperature
followed by washing three times in phosphate buffered saline
(PBS). The explant was then floated in carbon dioxide-free
media over an unlaminated, hydrophobic 125–175 micronthick polytetrafluoroethylene membrane (Millipore, Bedford,
MA) with 0.5 micron pores with the basal lamina of the RPE
facing the membrane. The curled edges were flattened from
the choroidal side with fine forceps without touching Bruch’s
membrane. Next, 4% agarose (Sigma) was poured onto the
Bruch’s membrane-choroid complex from the choroidal side,
and the tissue was kept at 4 °C for 2–3 min to allow the agarose
to solidify. The polytetrafluoroethylene membrane was
peeled off. Next, 6 mm circular buttons were trephined on a
Teflon sheet from peripheral Bruch’s membrane and placed
on 4% agarose at 37 °C in untreated polystyrene wells of a 96well plate (Corning Costar Corp., Cambridge, MA). Allowing
the agarose to solidify within 2–3 min at room temperature
enabled the Bruch’s membrane explant to stabilize. The
acellular explants were rinsed gently with PBS three times for
5 min before they were sterilized with 20,000 rad of gamma
radiation sterilized and stored at 4 °C.
Isolation of bovine rod outer segments: Fresh bovine eyes
were transported on ice from a local slaughterhouse to the
laboratory. The eyes were washed once with Hanks’ basal salt
solution (HBSS; Gibco), which also contained 100 U/ml
penicillin and 0.1 mg/ml streptomycin. The anterior half of
the eye was removed and discarded. The vitreous was
removed, and the retina was allowed to gently float off the
TABLE 1.
Tissue ID
Death To
Enucleation
Time (hours)
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
Sample 11
4
2.5
8
3
3
7
4
6.5
8
5.3
4
Enucleation To
Bruch's
Membrane
Extraction Time
(hours)
21
27.5
46
38
25
10
41
18
32
31
28
Age
(year-old)
Cause of Death
9
14
16
20
41
46
74
75
76
80
81
motor vehicle accident
head injury
head injury
motor vehicle accident
head injury
head injury
breast cancer
cardiopulmonary Arrest
coronary artery disease
cardiopulmonary Arrest
leukemia
All donor eyes were enucleated within 10 h and shipped in a
sterile container at 4 °C and received in our laboratory within
48 h of death.
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RPE by pipetting HBSS into the subretinal space. The retina
was removed after cutting at the optic nerve. It was then put
into a glass tube on ice that contained isolation medium
composed of 20% sucrose, 20 mM Tris-Cl, 2 mM MgCl2, and
130 mM NaCl (ph 7.2), and it was homogenized. One
milliliter of the retinal homogenate was layered on top of a
10%–60% linear sucrose gradient and centrifuged at 141,000
g for 2 h at 4 °C in a Beckman SW-40 rotor (Beckman Coulter,
Inc., Fullerton, CA). Rod outer segments (ROS) sedimented
at between 27-50% sucrose gradient. Bands containing rod
outer segments were collected and diluted with HBSS. The
rod outer segments were pelleted by centrifugation at 7700 g
for 10 min at 4 °C, suspended in the buffered salt solution, and
again pelleted at 800 g for 10 min [26,27]. Purified ROS were
stored at 4 °C in Dulbecco’s modified Eagles medium
(DMEM) containing 17% sucrose, 100 ug /ml streptomycin,
and 100 U/ml penicillin. The rod outer segments were used
within 4 weeks. Prior to use, they were centrifuged at 9,000 g
for 10 min to remove storage medium [28].
Fluorescent labeling of isolated bovine rod outer segments:
Isolated rod outer segments were pelleted in HBSS, suspended
in serum-free culture medium (DMEM) and transferred to a
1.5 ml microcentrifuge tube, where it was stained with
fluorescein isothiocyanate (FITC; Sigma) using a
modification of the method of Boyle and McLaughlin [29]. A
2 mg/ml stock solution of FITC in 0.1 mol/L sodium
bicarbonate at pH 9.0–9.5 was prepared under dim red light,
filter-sterilized, and stored in aliquots at –20 °C. The FITC
stock was added to the suspended rod outer segments (final
concentration 10 ug/ml) and incubated for 1 h at room
temperature in the dark with gentle stirring. The FITC-stained
rod outer segments were pelleted for 4 min at 7,000 rpm in a
microcentrifuge, suspended in growth medium, counted, and
diluted to a final concentration of 1.0 × 107/ml using a Coulter
Z1 cell counter (Beckman Coulter). Microscopic examination
of the labeled rod outer segments showed intact outer
segments after labeling (data not shown).
Incubation of cultured retinal pigment epithelium cells with
fluorescein isothiocyanate-labeled rod outer segments: RPE
cells were fed FITC-labeled outer segments two weeks after
the RPE were seeded onto Bruch’s membrane explants. For
this purpose, confluent RPE cultures were overlaid with
FITC-rod outer segments (107/ml in DMEM with 20% fetal
bovine serum) and incubated at 37 °C for 24 h. After 24 h the
cells were rinsed with DMEM with 20% FBS to remove FITClabeled ROS from the medium. Eight cultures were fixed at
this point using 2% paraformaldehyde in 0.1 M phosphate
buffer at pH 7.4 for subsequent imaging.
Measurement of retinal pigment epithelium cell area:
Visualization of the RPE border was facilitated by labeling
fixed cells with Vybrant Cell-labeling Solution Di-I
(Molecular Probes, Eugene, OR) before imaging. Briefly, two
weeks after cells were cultured on Bruch’s membrane, they
were labeled with Vybrant CM-Di-I in DMEM with 10% FBS
for 20 min at 37 °C and washed three times with cell culture
medium. The size of RPE cells were measured using Image J
software (NIH, Bethesda, MD) at 20x objective.
Flow cytometry: The fluorescence (excitation=488 nm,
emission=530 nm) of 10,000 of unfixed cells/well was then
assayed immediately on a FACSan (Becton Dickinson
Immunocytometry System, San Jose, CA) using a live gate to
exclude cell fragments, rod outer segments particles, and other
unwanted debris. A logarithmic scale of relative fluorescent
intensity was used, and rod outer segments phagocytosis was
calculated by subtracting the geometric mean
autofluorescence of control cells from the geometric mean
fluorescence of cells challenged with FITC-labeled rod outer
segments. Cellular autofluorescence was determined for each
population by analyzing cells from wells with no added rod
outer segments. Following rod outer segments challenge,
unbound and cell membrane surface-bound ROS were
removed by washing the wells three times with PBS, treated
for 10 min with 0.25% trypsin containing 1 mM edetic acid
[30], pelleted, washed again with PBS, and suspended in 500
ul of PBS with 10 mM glucose at pH 8.0.
DNA microarray to measure mRNA level of MerTK:
Affymetrix DNA microarray was performed as follows [24].
Briefly, ARPE19 were seeded onto 10 independent Bruch’s
membrane explants (five young donors age 31–47 years and
five older donors aged 71–81 years). ARPE-19 cells were
seeded onto the explants surface as described in the previous
section. Total RNA isolation and microarray target
(U133plus2, Affymetrix, Santa Clara, CA) hybridization,
washing, staining, and scanning probe arrays were done
following a standard Affymetrix GeneChip Expression
Analysis Manual. Gene expression analyses, including global
normalization and scaling, were performed using the
Affymetrix GCOS Manager software [24].
Preparation of nitrite-treated extracellular matrix: To
prepare tissue culture wells coated with RPE- extracellular
matrix (ECM), we plated ARPE-19 into 96-well plates and
allowed them to grow to confluence. RPE cells were removed
by adding 20 mM ammonium hydroxide buffer for 20 min,
followed by washing with PBS three times, and drying to
obtain 96 well plates coated with RPE-derived ECM. Sodium
nitrite-treated ECM was prepared by adding 100 mM sodium
nitrite in pH 4 acetate buffer for one week. Wells were then
washed at least four times with PBS, further incubated with
PBS for 4 h, and then washed at least two additional times to
completely remove nitrite [31].
RESULTS
Two weeks after seeding onto Bruch’s membrane explants
ARPE-19 formed a hexagonal monolayer with prominent
expression of ZO-1 at the cell border. As Figure 1 shows,
ARPE-19 appeared similar after culturing onto a younger (20
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years old) donor Bruch’s membrane versus an older (80 years
old) donor Bruch’s membrane. As evident in Figure 2,
ingestion of the outer segment material was more prominent
in ARPE-19 seeded onto younger versus older Bruch’s
membrane. Phase contrast microscopy did not show apparent
morphological differences between ARPE-19 seeded onto
younger (Figure 2A) versus older (Figure 2B) Bruch’s
membrane. Fluorescence microscopy of ARPE-19 seeded
onto a 16-year-old Bruch’s membrane showed intense
labeling of ingested outer segments (2C, solid arrow), whereas
cells seeded onto an 81-year-old Bruch’s membrane had less
intense labeling after feeding fluorescent-labeled outer
segments (2D, solid arrow). Flow cytometry of harvested
ARPE-19 revealed that the population of cells containing
labeled outer segments was higher in RPE cultured on young
Bruch’s membrane (Figure 3, top panel) compared to
ARPE-19 cultured on older Bruch’s membrane (Figure 3,
bottom panel).
The average fluorescence intensity per cell, which is a
measure of the phagocytosis ability of ARPE-19, increases as
a function of incubation time (Figure 4) up to 24 hours after
feeding of outer segments. This observation is consistent with
the study reported elsewhere [32]. The average fluorescent
intensity per cell decreased as the age of human Bruch’s
membrane increased (Figure 5), indicating that aging of
human Bruch’s membrane decreases the ability of the RPE to
ingest outer segment material.
We also determined the effects of Bruch’s membrane
aging on the expression levels of MerTK, which is a receptor
tyrosine kinase involved in phagocytosis of outer segments by
RPE. Our results demonstrate that the expression level of
MerTK was affected by the age of Bruch’s membrane; the
expression level of MerTK mRNA was lower in RPE cultured
on older Bruch's membrane than on younger Bruch’s
membrane (61.53 ± 9.2 versus 100 ± 11.2 arbitrary units,
respectively; p=0.026, Figure 6).
Figure 1. ZO-1 immune staining of ARPE-19 cells cultured to
confluence on Bruch’s membrane Fluorescent microscopy shows
ZO-1 immune staining of ARPE-19 cells cultured for two weeks until
they reached confluence on (A) a 20-year-old Bruch’s membrane and
(B) an 80-year-old Bruch’s membrane. Note the hexagonal shape of
the ARPE-19 monolayer, with no apparent morphological difference
between ARPE-19 cultured on younger versus older Bruch’s
membrane.
We then compared the effects of nonenzymatic nitration
of the substrate on the phagocytosis ability of ARPE-19. The
ingestion of labeled rod outer segments increased with rod
outer segment concentration on both unmodified and nitritemodified RPE derived ECM (Figure 7) with the plateau value
higher on unmodified ECM. At all rod outer segment
concentrations tested, the phagocytosis of outer segments was
higher on unmodified ECM versus nitrite-modified ECM
(Figure 7).
We then compared the effects of Bruch’s membrane
aging and nonenzymatic nitration of RPE-ECM on ARPE-19
phagocytosis (Figure 8). Using the Student t-test, we found
the average fluorescent intensity per cell on younger Bruch’s
membrane (129.7 ± 34.8 arbitrary units) was higher than on
older Bruch’s membrane (67.4 ± 4.2 arbitrary units,
respectively; p<0.01). This may imply that aging decreases
ARPE-phagocytosis by about 50% compared to the baseline
value on young Bruch’s membrane. Interestingly, the mean
fluorescence intensity of ARPE19 cells (reflecting amount of
ingested FITC-labeled ROS) is reduced by about 50% when
cultured on nonenzymatic nitration of RPE derived ECM
(126.7 ± 12.8 versus 60.8 ± 8.2 arbitrary units for unmodified
versus nitrite-treated RPE-ECM, respectively; student t-test p
< 0.01, Figure 8).
Figure 2. Binding and uptake of fluorescein isothiocyanate -rod outer
segment particles by ARPE-19 are decreased when cultured on older
Bruch’s membrane A: Phase contrast microscopic image shows
ARPE-19 two- weeks after seeding onto a 16-year-old donor Bruch’s
membrane. B: Phase contrast image shows ARPE-19 two weeks after
seeding onto an 81-year-old donor Bruch’s membrane. C:
Fluorescence image of ARPE-19 on a 16-year- old donor Bruch’s
membrane fed with labeled rod-outer-segments shows cells border
(broken arrows) and intense labeling of ingested outer segments
(solid arrow). D: Fluorescence image of ARPE-19 on an 81-year-old
donor Bruch’s membrane fed with labeled rod-outer-segments shows
cells border (broken arrows) and less labeling of ingested outer
segments (solid arrow) (compare C to D).
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We then determined the average cell area on aged versus
young Bruch’s membrane, and unmodified and nitrite–treated
RPE-ECM, to ensure that the effects we observed were not
due to a simple difference in surface area between the cells.
The mean area of RPE cells on younger Bruch’s membrane
was similar to the cell area on older Bruch’s membrane
(Figure 9). Culturing the cells on harvested ECM led to a
larger surface area compared to RPE cultured on human
Bruch’s membrane; however there was no difference between
RPE cultured on untreated versus nitrite-treated extracellular
matrix (Figure 9).
DISCUSSION
AMD is a complex disorder characterized by cellular changes
in the RPE, choriocapillaris, and outer retina and by structural
changes within Bruch’s membrane that include diffuse
thickening, accumulation of basal laminar and basal linear
deposits, collagen cross-linking within the inner and outer
collagen layers, and elastin fragmentation [12,13]. Cellular
changes that occur in AMD include RPE and choriocapillaris
atrophy, photoreceptor atrophy, and the development of
choroidal neovascularization [9-13]. In the aging eye, the
Figure 3. Representative flow cytometry data Counts to right of
arrow represent population of ARPE-19 containing fluorescein
isothiocyanate-labeled rod-outer- segments. The area under the
curve to the right of the arrow is greater in retinal pigment epithelium
cultured on younger Bruch’s membrane (top panel, from a 14-yearold donor) than on older Bruch’s membrane (bottom panel, from a
76-year-old donor).
connection between aging changes in the basement membrane
and aging changes within cells is not known. However, it is
known that the aging changes within Bruch’s membrane occur
several decades before cellular changes, thus suggesting that
Bruch’s membrane dysfunction can induce changes in the
adjacent cells.
We have previously developed a system for isolating the
effects of basement membrane aging from the effects of
cellular aging from the effects of cellular aging. We have this
system to show that RPE attachment and proliferation
decrease and apoptosis rate increases when cultured on aged
Bruch’s membrane [20-22]; the gene expression profile of
human RPE is also altered by aging of Bruch’s membrane
[24]. Herein we demonstrate that aging of Bruch’s membrane
has a direct effect on an important physiologic function of
human RPE, namely, phagocytosis of outer segments. To our
knowledge, this is the first direct evidence that aging of human
Bruch’s membrane leads to a diminution in RPE phagocytic
ability.
Phagocytosis is an important physiologic process
performed by the RPE, which is one of the most active
phagocytic epitheliums in the human body. In vivo,
approximately 3%–5% of the distal tips of photoreceptor outer
segments are shed daily, and proper RPE phagocytosis is
necessary to maintain the health and integrity of the neural
retina and choriocapillaris [2,4,33]. In vivo, impairment of
RPE phagocytosis would lead to improper handling of
photoreceptor outer segments with accumulation of material
Figure 4. Phagocytosis of ARPE-19 as a function of incubation time
ARPE-19 were cultured for two weeks to confluence on a human
Bruch’s membrane explant harvested from a 14-year-old human
donor. Average fluorescein isothiocyanate-labeled rod-outersegments (FITC-ROS) intensity per cell increased linearly with
incubation time. Each data point is the average of three experiments
with duplicate wells. Background autofluorescence was subtracted.
Error bar represents standard deviation.
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within the RPE and deposition of abnormal material within
Bruch’s membrane. Lipofuscin accumulation has been
associated with age-related macular degeneration [34,35].
Improper handling of ingested photoreceptor material may
create a positive feedback loop in which aging changes in
Bruch’s membrane may reduce RPE phagocytosis, leading to
further accumulation of material within Bruch’s membrane,
further impairing phagocytosis. Interestingly, a decrease in
Figure 5. Phagocytosis ability of retinal pigment epithelium as a
function of Bruch’s membrane age The average fluorescent intensity
per cell decreased as the age of human Bruch’s membrane increased.
Each data point is the average of three experiments with duplicate
wells. The error bar represents standard deviation.
Figure 6. Relative mRNA of c-mer proto-oncogene tyrosine kinase
in ARPE19 cells cultured on younger or older Bruch’s membrane
measured with Affymetrix DNA microarray (probe ID 40648_at).
Data represent average of five replicates with each experimental
group. The differences were statistically significant (p= 0.026). Error
bar represents standard deviation.
phagocytic ability may be associated with an increase in RPE
melanogenesis, and clinically, RPE hyperpigmentation is a
risk factor for the development of AMD. As human age,
alterations in melanogenesis occur in the epidermis
throughout the human body [36].
At the moment, we do not know which molecular change
or changes in Bruch’s membrane are responsible for the agedependent decline in RPE phagocytosis ability we have
observed. However it is tempting to speculate that nitrite
modification of the substrate may be responsible for this effect
since nitrite modification of ECM decreases the phagocytosis
ability of the RPE to an extent comparable to Bruch’s
membrane aging. We have shown previously that nitrite
modification decreases the ability of RPE to attach to
extracellular matrix, inhibits cell proliferation, and increases
cell apoptosis and necrosis [37]. Nitrite-mediated collagen
damage leads to collagen cross-linking in vitro and may
underlie the changes that we have seen. There is an age-related
linear decline in collagen solubility, which reflects an increase
in cross-linking, with increasing patient age [38]. In vivo,
pathological collagen cross-linking is nonenzymatic [39], and
cross-linking agents include nonreducing sugars, reactive
oxygen species, and reactive nitrogen species. Pentosidine,
which results from nonenzymatic glycation, increases with
increasing age in human Bruch’s membrane [40,41]. Nitritemediated nonenzymatic collagen cross-linking, shown in
vitro [31] and likely to occur in vivo, and has been associated
with chronic inflammation and cigarette smoking [42,43],
which are associated with the development of AMD [44,45].
We cannot exclude the possibility that other changes are
responsible for the effect of Bruch’s membrane aging on RPE
phagocytosis, since the proteomics of aging Bruch’s
membrane are not understood completely. In addition to
Figure 7. Comparison of the phagocytosis ability of retinal pigment
epithelium cells cultured onto untreated retinal pigment epitheliumderived extracellular matrix versus nitrite-treated retinal pigment
epithelium-derived extracellular matrix. ARPE-19 ingestion of
labeled rod outer segments (ROS) is higher on untreated versus
nitrite-treated retinal pigment epithelium-derived extracellular
matrix (RPE-ECM). Each data point is the average of three
experiments with duplicate wells. Background autofluorescence was
subtracted.
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nitrite modification, numerous other changes occur within
Bruch’s membrane with age including posttranslational
modification of existing proteins, deposition of abnormal
proteins and lipids, topographic reorganization of protein
structure, and changes in the presentation of ligand binding
sites necessary for cell attachment [12,13].
Our results suggest that expression of the gene encoding
for MerTK, a tyrosine kinase known to be involved in RPE
phagocytosis, decreases with aging of human Bruch’s
membrane. Several studies have demonstrated that MerTK
plays an important role in phagocytosis of outer segments by
the RPE. The Royal College of Surgeons rat (RCS rat) is a
well characterized animal model of retinal degeneration that
arises from a defect in phagocytosis, with accumulation of
debris in the subretinal space leading to progressive loss of
photoreceptors [46]. The defect results from a mutation in the
Mertk gene, which is normally expressed in the RPE [47]. In
the RCS rat, a small deletion of DNA disrupts the gene
encoding the receptor tyrosine kinase Mertk [46]. Mice
homozygous for a targeted disruption of a homologous gene
manifest a retinal dystrophy phenotype similar to RCS rats
[48]. Tissue culture studies suggest that the RPE is the site of
action of Mertk, and Mertk protein plays a key role in RPE
phagocytosis. Mertk protein is absent from RCS but not wild-
type cultured RPE cells. Delivery of rat Mertk to cultured RCS
RPE cells by means of a recombinant adenovirus restored the
cells to complete phagocytic competency [49]. Mutation of
the human ortholog, MERTK, were detected in 3 of 328 DNA
samples from patients with various retinal dystrophies
diagnosed with retinitis pigmentosa, thus demonstrating that
a defect in RPE phagocytosis can lead to human retinal disease
[50]. A MERTK missense mutation in humans can lead to a
severe cone-rod dystrophy with childhood onset when in
compound heterozygous form with a R722X allele [51]. The
similarity in phenotypes between the two rodent models, and
the detected presence of a similar phenotype in humans with
mutations in the homologous gene, suggests that alterations
in phagocytosis can lead to retinal degeneration in both
humans and rodents [48]. Correction of this phagocytic defect
with gene therapy can prolong photoreceptor cell survival in
the RCS rat [47,52,53].
In summary, we have shown that aging of human Bruch’s
membrane leads to a decline in the phagocytic ability of RPE
cells, and that the magnitude of this effect is comparable to
the effects of nonenzymatic nitration of RPE-derived
extracellular matrix. Currently, AMD is thought of as a
cellular disease in which pathological alterations in the RPE,
outer retina, or possibly macrophages lead to secondary
Figure 8. Comparison of effects of Bruch’s membrane aging and
nonenzymatic nitration of retinal pigment epithelium-derived
extracellular matrix (RPE-ECM) on ARPE-19 phagocytosis.
Average fluorescein isothiocyanate-labeled rod-outer- segments
(FITC-ROS) intensity per cell on younger than 50-year-old donor
Bruch’s membrane was higher than on older than 70 year-old donor
Bruch’s membrane (129.7 ± 34.8 S.D. versus 67.4 ± 4.2 arbitrary
units; * = p < 0.01 with student t-test). Mean fluorescence of RPE
after ingesting FITC-ROS is higher for cells seeded onto untreated
ECM compared to nitrite-treated ECM (126.7 ± 12.8 S.D. versus 60.8
± 8.2 arbitrary units; * = p < 0.01 with student t-test). Note that the
magnitude of 50% reduction effect is the same for aging of human
Bruch’s membrane and nonenzymatic nitration of RPE-ECM.
Figure 9. Area of RPE two weeks after seeding onto different
substrates Cells cultured on older Bruch’s membrane (age > 70) were
slightly larger than cells cultured on younger Bruch's membrane (age
< 50), but the difference was not statistically significant. Cells
cultured onto unmodified retinal pigment epithelium-derived
extracellular matrix (RPE-ECM) were larger than cells seeded onto
younger or older Bruch’s membrane; cell size was not changed by
nitrite treatment of RPE-ECM. Asterisk (*) indicates p<0.01 for
unmodified or modified RPE-ECM versus younger or older Bruch’s
membrane.
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© 2007 Molecular Vision
change in Bruch’s membrane; our own studies offer a different
paradigm to interpret aging changes in this disease. In our
model, age-related changes in human Bruch’s membrane, that
may include but are not limited to nonenzymatic nitration,
could lead to extensive disruption of RPE function, including
RPE phagocytosis with accumulation of material beneath the
RPE. Interestingly, a clinical trial is currently under way to
determine if systemic dialysis can be used to alter Bruch’s
membrane in age-related macular degeneration [54].
Preliminary studies suggest that soft drusen may resolve and
cell function may improve after this procedure, which may
improve the function of the RPE, photoreceptors, and outer
retina [54]. Additional studies are required to determine the
ultimate effects of dialysis and the use of other techniques to
refurbish human Bruch’s membrane on the function of the
RPE and other cells [55].
ACKNOWLEDGMENTS
This study was supported by the Robert L. Burch III Fund, the
Macula Society, the Foundation Fighting Blindness, the
Hickey’s Family Fund, and unrestricted funds from Research
to Prevent Blindness. Dr. Tezel is the recipient of an RPB
Career Development Award.
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The print version of this article was created on 21–December–2007. This reflects all typographical corrections and errata to the
article through that date. Details of any changes may be found in the online version of the article.
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