Female Gender, Estrogen Loss, and Sub-RPE Deposit
Formation in Aged Mice
Scott W. Cousins,1 Maria E. Marin-Castaño,1,2 Diego G. Espinosa-Heidmann,1,2
Anastassia Alexandridou,1 Liliane Striker,3 and Sharon Elliot 3
PURPOSE. Estrogen status influences the incidence and severity
of many diseases in women. Because women with early menopause appear at risk for worse ARMD, estrogen deficiency may
also contribute to the onset or severity of ARMD in women. It
has been observed that aged male C57BL/6 mice fed a high-fat
diet and briefly exposed to blue-green light exhibit development of significant sub-RPE deposits and mild Bruch’s membrane (BrM) thickening. This model was used in an attempt to
delineate the role of gender and estrogen status in this model.
METHODS. C57BL/6 male and female mice of 9 or 16 months
were fed a high-fat diet for 4.5 months. Several groups of
9-month-old female mice underwent estrogen depletion by
ovariectomy, with or without supplementation with exogenous 17␤-estradiol. After 4 weeks of a high-fat diet, the eyes
were exposed to seven 5-second doses of nonphototoxic levels
of blue-green light over 2 weeks. Three and a half months after
cessation of blue light treatment, transmission electron microscopy was performed to assess severity of deposits, BrM
changes, and choriocapillaris endothelial morphology. In some
mice, gelatin zymography and Western blot analyses were
performed on protein extracts of freshly isolated RPE to determine the effect of estrogen on matrix metalloproteinase
(MMP)-2 activity in the RPE.
RESULTS. Both male and female 16-month-old mice showed
qualitatively similar basal laminar deposit morphology, but the
severity of thickness, continuity, and content was significantly
greater in female mice. Aged female mice also demonstrated a
trend toward more severe endothelial changes and increased
BrM thickening compared with age-matched male mice. Ovariectomized middle-aged mice showed more severe deposits
than sham-surgery control animals. However, ovariectomized
mice that received high-dose estrogen supplementation also
showed significant deposits, although they had thinner BrMs
than did the estrogen-deficient mice. Loss of RPE MMP-2 activity correlated with deposit severity, with estrogen-deficient
mice expressing less MMP-2 than ovary-intact control mice.
CONCLUSIONS. Female gender in aged mice and estrogen deficiency in middle-aged mice appears to increase the severity of
sub-RPE deposit formation. Estrogen deficiency may increase
From the 1Department of Ophthalmology, Bascom Palmer Eye
Institute, Miami Florida; and the 3Vascular Biology Institute, University
of Miami School of Medicine, Miami, Florida.
2
Joint 2nd authorship.
Presented at the annual meetings of the Association for Research
in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2001 and
May 2002.
Supported by National Eye Institute Grant EYAI13318.
Submitted for publication March 21, 2002; revised August 13 and
September 30, 2002; accepted October 10, 2002.
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: Scott W. Cousins, Bascom Palmer Eye
Institute, William L. McKnight Vision Research Center, 1638 NW 10th
Avenue, Miami, FL 33136; scousins@med.miami.edu.
Investigative Ophthalmology & Visual Science, March 2003, Vol. 44, No. 3
Copyright © Association for Research in Vision and Ophthalmology
susceptibility to formation of sub-RPE deposits by dysregulating turnover of BrM, contributing to collagenous thickening
and endothelial changes. Estrogen supplementation at the dosages used in this study does not appear to protect against
formation of sub-RPE deposits. (Invest Ophthalmol Vis Sci.
2003;44:1221–1229) DOI:10.1167/iovs.02-0285
A
ge-related macular degeneration (ARMD) is the most common cause of lost central vision in the elderly.1 The histopathology of early ARMD demonstrates accumulation of specific lipid-rich deposits under the retinal pigmented epithelium
(RPE). During aging, deposits, called basal laminar deposits or
BLDs, initially accumulate between the RPE and its basement
membrane, but progression into ARMD requires additional
formation of deposits within Bruch’s membrane (BrM, called
basal linear deposit and drusen).1,2 The pathogenesis of deposits in ARMD is unknown, but the process is influenced by
genetic, systemic health, and environmental risk factors.
The incidence and severity of many diseases have been
associated with the reduction of estrogen in women.3– 6 Although information is limited, several observational studies
have suggested that estrogens may serve to protect women
from the development or progression of ARMD. Early menopause may be associated with the development of ARMD in
women,7,8 implying that estrogen deficiency may play a role in
the pathogenesis of ARMD.9 Nonetheless, these few observational studies have provided the basis for a large clinical trial to
evaluate estrogen-replacement therapy to prevent the onset or
progression of ARMD.
Among their many actions, estrogens regulate the expression of genes important for extracellular matrix (ECM) turnover, including collagen and matrix metalloproteinases
(MMPs).10,11 For example, estrogens have been shown to increase both MMP-9 mRNA and activity in glomerular mesangial
cells,12,13 and to increase MMP-2 activity and protein expression in human granulosa lutein cells.10 The effects of estrogen
on the retina or RPE are unknown. Estrogen-mediated regulation of genes, which are expressed in RPE and are important
for matrix turnover, may provide a pathogenic mechanism to
explain the link between estrogen status and ARMD. In this
regard, MMP-2 may be important since it preferentially degrades extracellular matrix components such as types IV and I
collagen.14 –16 Dysregulation in the relative production of
MMP-2 and collagen may lead to net deposition in BrM and
contribute to formation of sub-RPE deposits.
We have recently shown that normal human RPE contains
functionally active estrogen receptors (ERs) of both subtypes
and that those and MMP-2 are regulated by estrogens.17 A dose
dependency was observed, with physiologic levels of estrogen
causing upregulation (compared with RPE without estrogen)
but superphysiologic concentrations revealed a paradoxical
absence of effect. These data suggest that loss of estrogen
and/or estrogen function is associated with downregulation of
expression of MMP-2 and its activity in the RPE.
In this study, we sought to determine whether gender and
estrogen status affect formation of deposits and RPE expression
of MMP-2 in vivo. We have recently developed a mouse model
1221
1222
Cousins et al.
for formation of sub-RPE deposits based on feeding C57BL/6
mice a diet high in polyunsaturated fats followed by exposure
to blue light.18 Briefly, the results indicated that in aged mice
(16 months) but not in younger mice, moderately severe subRPE deposits developed, consistent with BLD. Significant thickening and other changes occurred in BrM, but without definite
drusen, and a variable degree of endothelial injury was present
in the choriocapillaris without definite neovascularization. In
this prior analysis of sub-RPE deposits and mild thickening of
BrM, we did not analyze differences between male and female
mice. We performed a study to better delineate the role of age,
gender, and estrogen depletion or supplementation in this
model.
METHODS
Mice
C57BL/6 male or female mice of ages 9 or 16 months were purchased
from the National Institute of Aging (Bethesda, MD). C57BL/6 were
chosen because, compared with other strains of mice, they exhibit
development of plasma hyperlipidemia when fed a high-fat diet.19 The
guidelines of the ARVO Statement for the Use and of Animals in
Ophthalmic and Vision Research were followed, and the Division of
Veterinary Resources approved all experiments.
Experimental Model for Sub-RPE Deposits
In our recently developed model of formation of sub-RPE deposits
based on feeding mice a high-fat diet followed by exposure to bluegreen light,18 experimental mice were switched from a regular diet
(Diet 5001; PMI Nutrition International Test Diet, Richmond, IN) to
high-fat chow (Diet 5015; PMI Nutrition International Test Diet) at the
beginning of the experiments to allow tissue distribution of dietary
lipid. The high-fat diet more than doubled the relative percentage of
calories from fat but maintained the same number of calories per gram
of food (by reducing the percentage of calories from carbohydrates).
Dietary fat was increased from 11% of total calories to 25% of total
calories, equally divided between increased saturated and unsaturated
fat. The diets contained the following constituents: 64% carbohydrates,
30% protein, 6.5% total fat, 3.6% unsaturated fat, 1.2% linoleic acid, and
0.05 IU/g vitamin E for the regular chow versus 55% carbohydrates,
18% protein, 26% total fat, 8.2% unsaturated fat, 2.5% linoleic acid, and
0.04 IU/g vitamin E for the high-fat chow.
In the right eye, a brief, repetitive exposure to nonphototoxic
levels of argon laser 488 nm blue-green light (Model 910A Argon Laser;
Coherent, Palo Alto, CA) was added to induce transient production of
RPE oxidant,20 –24 4 weeks after starting the high-fat chow. After
dilation of eyes with 0.5% tropicamide and 2.5% phenylephrine and
with anesthesia induced in the animal by intramuscular injection of 0.1
mL of a cocktail of xylazine (8.5 mg/mL), acepromazine (1.5 mg/mL),
and ketamine (42 mg/mL), seven 5-second exposures to 20 mJ (approximately one third the threshold intensity to produce a clinical
lesion) of argon laser 488-nm blue-green light was administered 2 to 3
days apart over a 2-week period. Pilot experiments demonstrated that
60 to 80 mJ was required to produce a distinct retinal burn clinically
visible as a light gray lesion, and that 120 mJ was needed to produce an
intense white burn with BrM injury detectable by histologic examination 24 hours later. The delivery system used a probe producing a
200-␮m spot, and the energy intensity was calibrated before each
application by a photometer (210 Power Meter; Coherent) held at a
standardized distance from the probe. Retinal illumination required a
specially designed biconcave contact lens (focal distance, ⫺6.5 mm at
670 nm; 153.8 dioptric power) to neutralize the optical power of the
natural lens and to enlarge the retinal spot size. When threshold injury
(i.e., visible retinal burns requiring 60 –100 mJ) were applied to characterize the actual area of retinal illumination, the retinal area of direct
burn occurred in the superotemporal retina and appeared to be approximately 700 to 1000 ␮m, or approximately three to six disc areas,
IOVS, March 2003, Vol. 44, No. 3
accounting for approximately 5% to 8% of the retinal surface area.
Runoff (spread from the edge of the burn) and significant scattering
through the delivery system both occur. Thus, actual amount of energy
delivered to the retina is impossible to calculate, and retinal illumination is uneven over its surface. All mice were maintained on 12-hour
light– dark cycles with ambient light maintained by fluorescent lighting, and cages were kept on middle shelves of the cage rack. The
high-fat chow diet was continued for three additional months, after the
2-week exposure to blue-green light. Mice were then killed and the
eyes immediately removed for transmission electron microscopy
(TEM) or recovery of RPE for MMP zymography and Western blot
analysis.
Experimental Protocol
Studies were divided into three protocols:
1. To evaluate age-related gender differences, the deposit model
described earlier was used in male and female mice aged 9
months (n ⫽ 5 of each gender) or 16 months (n ⫽ 5 male, 6
female). The nonexposed left eye was not evaluated in this
study.
2. To evaluate the effect of estrogen status in middle-aged mice,
9-month-old female mice were divided into three groups: ovary
intact (controls, n ⫽ 6), ovariectomy plus implantation of an
empty pellet (n ⫽ 5), or ovariectomy followed by subcutaneous
implantation of a sustained release pellet containing a 90-day
supply of 17␤-estradiol (1.7 mg/pellet; (n ⫽ 7). Ovariectomies
were performed with animals under general anesthesia in sterile
conditions with a 1-cm laparotomy incision. The peritoneal
cavity was entered, the ovaries identified, retracted, and excised
bilaterally. The incision was closed with 6-0 interrupted sutures
(Vicryl; Ethicon, Piscataway, NJ). Two weeks after ovariectomy,
subcutaneous implantation of the empty or estrogen-containing
pellet was performed, and the high-fat diet was initiated. The
deposit model described previously was performed. Ninety
days after implantation, the original pellet was removed and
replaced with a fresh implant to ensure constant estrogen for
135 days of the experiment. The drug delivery pellet is a
commercially available polylactide-containing polymer with
well-defined pharmacokinetic and drug-release characteristics
(Innovative Research of America, Sarasota, FL). The nonexposed left eye was not evaluated in this study.
3. To evaluate the effect of 17␤-estradiol on MMP-2 expression and
activity in RPE, five additional groups of 9-month-old mice were
evaluated: ovary intact (n ⫽ 7); ovariectomy (n ⫽ 7), ovariectomy plus high-dose (1.7 mg/pellet; n ⫽ 7), moderate-dose
(0.36 mg/pellet; n ⫽ 7), or low-dose (0.18 mg/pellet; n ⫽ 7)
estradiol implant (NE-121, Innovative Research of America).
Surgery was conducted as explained previously. The deposit
model was performed exposing both eyes to the blue-green
light. Six weeks after the high-fat chow was started, the eyes
were removed for analysis of MMP-2 in RPE. This time point was
chosen to evaluate the physiologic effect of estrogen at the
midpoint of deposit formation.
RPE Isolation
After enucleation, the eyes were rinsed with 10% gentamicin for
sterilization and twice with PBS (1⫻). The eyes were then placed in a
dish containing PBS (1⫻) and with the aid of a dissecting microscope,
were opened by a circumferential incision at the ora serrata. The
anterior segment was removed, and the vitreous-retina was separated
from the RPE and choroid eyecup with a round-tipped disposable blade
(K20-1504; Katena Products, Inc., Denville, NJ) and Tennant forceps
(K5-5230; Katena Products, Inc.). The remaining eyecup was incubated with Dulbecco’s modified Eagle’s medium (DMEM)/F12 (1:1
vol/vol) supplemented with 10% fetal bovine serum (FBS) at 37°C for
10 minutes. Then, using a Barraquer spatula (K3-2310; Katena Products, Inc.) for blunt dissection and scraping, the RPE monolayer was
Estrogen and Experimental BLD
IOVS, March 2003, Vol. 44, No. 3
dissected from BrM and choroid. The RPE was aspirated with a micropipette and transferred into individual tubes (Eppendorf, Fremont,
CA) containing 250 ␮L cold lysis buffer or (1⫻) Earle’s balanced salt
solution (EBSS) and homogenized on ice with a pestle. RPE samples of
left and right eyes of individual mice from each group were pooled and
stored at – 80°C until protein extraction and analysis.
An aliquot from the isolated RPE cells were stained with mouse
anti-cytokeratin-18 antibody (Sigma, St. Louis, MO) and with mouse
anti-endothelial cell (CD146) monoclonal antibody (Chemicon International, Inc., Temecula, CA) to ensure that there was no contamination with endothelial cells.
Western Blot Analysis
Dissected RPE monolayer was homogenized with a pestle in lysis
buffer and centrifuged for 30 minutes at 15,000g at 4°C. Supernatant
was collected, and protein concentration was determined by bicinchoninic acid (BCA) protein assay.12 All samples were then diluted in
Laemmli buffer and boiled. Twenty micrograms of the samples was
loaded on a 10% polyacrylamide gel. Prestained markers were used to
estimate molecular weight. Electrotransfer to nitrocellulose was performed by electroelution. Immunoblot analysis was performed with
MMP-2 antibody (mAb 13405; Chemicon), and immunoreactive bands
were determined by exposing the nitrocellulose blots to a chemiluminescence solution and exposure to autoradiographic film (Hyperfilm
ECL; Amersham Pharmacia Biotech, Piscataway, NJ). Confluent cell
layers were washed with PBS (1⫻) and collected in the presence of
lysis buffer. RPE tissue and cell homogenates were centrifuged for 30
minutes at 15,000g at 4°C. Supernatant was collected, and protein
concentration was determined by BCA protein assay. All samples were
then diluted in Laemmli buffer and boiled. Ten milligrams of samples
for ER type a and MMP-2 or 40 mg for ER type b were loaded on a 10%
polyacrylamide gel. Prestained markers were used to estimate molecular weight. Electrotransfer to nitrocellulose was performed by electroelution. Immunoblot analysis was performed with each anti-ERa,
-ERb, and -MMP-2 antibody (H-184 and N-19; Santa Cruz Biotechnology,
Santa Cruz, CA; and mAb 13405; Chemicon), and immunoreactive
bands were determined by exposing the nitrocellulose blots to a
chemiluminescence solution and exposing to autoradiographic film
(Hyperfilm ECL; Amersham Pharmacia Biotech).
MMP-2 Activity
The tissue homogenate supernatants were collected, and protein concentration was determined. MMP-2 activity was assessed with 10%
zymogram gels (Invitrogen Corp., Carlsbad, CA), as described previously.25 Briefly, 20 ␮g of proteins from RPE tissue were used. The
medium was diluted to normalize for protein quantity before the
addition of 5⫻ Laemmli buffer under nonreducing conditions. After
electrophoresis, gels were washed for 1 hour in 2.5% Triton X-100 and
incubated 24 hours in 50 mM Tris buffer. The gels were stained with
Coomassie Blue and air dried. Densitometry, using NIH image ver. 1.6
(available by ftp from zippy.nimh.nih.gov/or from http://rsb.info.nih.
gov/nih-image; developed by Wayne Rasband, National Institutes of
Health, Bethesda, MD) was used to analyze relative MMP-2 activity.
Each zymography assay was repeated at least three times.
Serum Levels of 17␤-Estradiol
Serum (50 ␮L) was removed by cardiac puncture, and 17␤-estradiol
was measured in triplicate for each group by a competitive enzyme
immunoassay kit (Active Estradiol EIA kit DSL-10-4300; Diagnostic
Systems Laboratories, Inc., Webster, TX).
Histology and TEM
Mice were killed by anesthetic overdose and perfused with saline
followed by a mixture of 3% glutaraldehyde and 2% paraformaldehyde.
Eyes were immediately enucleated and the corneas removed and fixed
in 3% glutaraldehyde and 2% paraformaldehyde in PBS (0.1 M, pH 7.3)
1223
overnight. The lens was removed, and the posterior segment (retina,
choroid, and sclera) was quadrisected to contain the perioptic nerve
portion at the apex and the ciliary body at the base. The superotemporal quadrant of retina, choroid, and sclera was submitted for electron
microscopic sectioning. The tissue was fixed in 1% osmium tetroxide
for 1 hour, rinsed in PBS, dehydrated in EtOH, and then imbedded in
Spur’s resin. Thick and ultrathin sections (0.7-1.0 ␮m) were cut on a
microtome (MT-2; Porter Blum, Hatfield, PA). Thick sections were
stained with toluidine blue and examined by light microscopy. Ultrathin sections were stained with 4% uranyl acetate and lead citrate and
examined with a transmission electron microscope (model CX-100;
JEOL, Tokyo, Japan).
Semiquantitative Grading System
For each specimen, a single cross-section was examined, and lowpower transmission electron micrographs were made of the entire
section from perioptic to ciliary body portion (usually approximately
10 micrographs). Then, two to four representative high-power micrographs were made from each low-power section, by an individual
unaware of the experimental conditions, and were used for semiquantitative scoring. The high-power micrographs were graded by two
independent examiners for the presence and severity of BLD. A severity score of 0 to 15 points was determined for each section by summation of the median scores of all the micrographs from a section on
each of five different categories of abnormalities (from 0 to 3 points for
each): continuity of BLD; maximal thickness of BLD; nature of deposit
content (homogeneous, banded structures, membranous debris, granular material); presence of BrM abnormalities; and assessment of other
choriocapillaris endothelial damage or invasion. BrM thickness was
also directly measured in three different standardized locations in each
image and then averaged to provide a mean score for that micrograph.
The mean of 10 high-power micrographs was used to assign and
average BrM thickness for an individual specimen.
Groups were compared by determining the mean ⫾ SD, and the
t-test was used for statistical analysis of the differences. In addition, the
frequency of BLD was determined using two different criteria. “Any
BLD” was defined as the presence of any discrete focal nodule of
homogenous material of intermediate electron density between the
RPE cell membrane and BrM in at least one micrograph within a
section. “Moderate BLD” was defined as the presence, in at least three
micrographs, of the following: continuous BLD extending under two
or more cells, deposit thickness equal to or greater than 20% of RPE
cell cross-sectional thickness, or the presence of any banded structures
within the BLD. Differences in the relative frequency were tested using
␹2 analysis.
Data from statistical analysis of zymography, Western blot analysis,
and estradiol levels are expressed as percentage of control (ovary
intact) or as arbitrary densitometry units. One-way analysis of variance
and the Dunnett multiple-comparison post hoc test or Student’s t-test
were performed for the statistical analysis.
RESULTS
Gender Differences in Deposit Formation
in Aged Mice
Recently, we have shown that 16-month-old mice, fed a
high-fat diet and exposed to blue light, develop morphologic
changes consistent with early ARMD, including sub-RPE
deposits, thickening of BrM, and morphologic changes in
the choriocapillaris. However, young (2-month-old) mice
did not exhibit the anatomic changes and 9-month-old developed infrequent but mild changes. We sought to determine the contribution of gender to the morphologic
changes by comparing male and female aged mice. Lipid
level determination confirmed previous results in which
middle-aged and aged C57BL/6 mice fed high-fat chows
developed a 1.4- to 2-fold elevated plasma triglycerides and
1224
Cousins et al.
IOVS, March 2003, Vol. 44, No. 3
FIGURE 1. TEM of the outer retina
and choroid from 16-month-old fatfed female or male mice exposed to
blue-green light. Left: specimen
from an aged female mouse revealed moderately thick sub-RPE
deposits (under white dotted line)
containing many banded structures
(white asterisk) consistent with
BLDs. BrM revealed marked collagenous thickening and scattered debris (black asterisk). Invasion by
several cellular processes (black arrows) apparently emanating from
the choriocapillaris (CC) was also
evident. The endothelium revealed
some loss of fenestrations with
thickened basement membrane.
Right: specimen from an aged male
mouse revealed continuous, moderately thick sub-RPE deposits (under white dotted line) containing
banded structures (white asterisk)
similar to those observed in the female. However, BrM was only
mildly and irregularly thickened.
The endothelial morphology appears slightly abnormal, with mild reduplication of the basement membrane, but evidence of severe injury or invasion was not observed.
Magnification, ⫻25,000.
cholesterol compared with age-matched mice fed regular
diets.18
Sixteen-month-old female and male mice exhibited qualitatively similar morphologic changes internal to the RPE
basement membrane (Fig. 1). Mice of neither gender
showed significant photoreceptor degeneration or morphologic changes in the RPE indicative of severe degeneration
or injury. However, TEM revealed that mice of both genders
had moderately severe basal laminar deposits. In general,
deposits in both genders were multifocal and moderately
thick, often containing oval-shaped banded structures (i.e.,
fibrous long-spaced collagen) and other debris embedded in
a homogenous material of moderate electron density. These
findings are typical of BLD observed in humans.1,2 Table 1
shows that semiquantitative scoring suggested that the deposits were more severe in female than in male mice (10.6 ⫾
2.4 in females versus 7.4 ⫾ 1.8 in males), indicating a
modest but significant increase in continuity, thickness, and
the nature of the deposit content.
Qualitative and quantitative differences between male and
female mice were also observed within BrM and the choriocapillaris. In general, BrM of male mice demonstrated moderate collagenous thickening with accumulation of minimal
amorphous debris (Fig. 1, right). The choriocapillaris endothelium revealed normal morphology or showed mild hypertrophy. Compared with male mice, female mice often demonstrated pronounced collagenous thickening of BrM with
accumulation of various kinds of debris and deposits (Fig. 1
left, asterisk). In approximately half the specimens, prominent cellular processes were identified, suggesting cellular
invasion from the choriocapillaris (possibly representing
endothelial cells; Fig. 1, left, arrows; Fig. 2 right, arrowheads). The choriocapillaris endothelium often demonstrated
morphologic changes suggestive of chronic injury, especially
loss of fenestrations and hypertrophy when compared with
normal aged female mice consuming a regular diet and not
exposed to blue-green light (Fig. 2, left). However, no definite neovascularization or vascular lumen formation was
identified within BrM. Measurement of BrM demonstrated increased thickness, although very high variability among specimens from aged females prevented statistical significance (Table 1). Neither male nor female 9-month-old mice had
moderate or severe deposits, BrM thickening, or endothelial
changes, and therefore the differences were not quantitatively
analyzed.
TABLE 1. Effect of Gender on sub-RPE Deposits in Aged Mice
Group
n
Frequency of
Any BLD
(%)
16 mo male
16 mo female
5
6
100
100
Frequency of
Moderate BLD
(%)
Mean Severity
Score
(15 max.)
Mean BrM
Thickness
Frequency of
Endothelial Invasion
(%)
100
100
7.4 ⫾ 1.8
10.6 ⫾ 2.4*
15.6 ⫾ 3.3
30.0 ⫾ 21.0†
0
50‡
Any BLD, presence of any homogeneous deposit (focal or continuous) between the RPE cell membrane and its basement membrane in at least
one (of five) transmission electron micrographs from an individual specimen (i.e., one micrograph with a score of at least 1 point). Moderate BLD,
presence of continuous deposits underlying several cells, or presence of banded structures (fiber long-spaced collagen), or focal deposit thickness
more than one quarter the cell height in at least three TEMs from an individual specimen (i.e., three micrographs with a minimum score of three
or more).
* P ⫽ 0.045.
† P ⫽ NS.
‡ P ⫽ 0.02.
Estrogen and Experimental BLD
IOVS, March 2003, Vol. 44, No. 3
1225
FIGURE 2. TEM of the outer retina
and choroid of 16-month-old female
mice. Left: specimen from an aged
female mouse consuming regular
chow and not exposed to blue light
revealed normal RPE without deposits and minimally thickened BrM. The
choriocapillaris (CC) appeared normal. Right: specimen from an aged
female mouse fed high-fat chow and
exposed to blue-green light revealed
moderately thick sub-RPE deposits
(under white dotted line) containing
banded structures (white arrow).
There was irregular thickening of
BrM. The endothelium revealed
thickening (white asterisk) with
some loss of fenestrations (black arrows) and reduplication of the basement membrane (black asterisks). Invasion by several cellular processes (black arrowheads), apparently
emanating from the choriocapillaris, was also evident. Abbreviations as in Figure 1. Magnification, ⫻25,000.
Effect of Estrogen Depletion and
Supplementation on Deposit Severity
To better determine the role of estrogen depletion as a mechanism for the gender differences in aged mice, we compared
the effect of ovariectomy on severity of deposits in 9-month-old
female mice. Reduction of estrogen is among the most important hormonal deficiencies induced by ovariectomy. We confirmed that ovariectomy caused a reduction in plasma estradiol
(Table 2). Compared with aged-matched females (97 pg/mL)
ovariectomized females demonstrated approximately 30 pg/
mL. Male mice had approximately 47.5 pg/mL, resulting from
metabolism of normal testosterone and other sex hormones.
We examined the subretinal morphologic changes in the
ovariectomized mice. As expected, sham-surgery control animals had minimal changes in RPE morphology (Fig. 3, top left).
Minor, focal homogenous nodular deposits under the RPE were
observed, and minimal changes in BrM or choriocapillaris were
present. In contrast, ovariectomy induced significant morphologic changes (Fig. 3, top right). Moderately severe BLD were
observed with typical morphologic features including banded
structures and other heterogeneous debris. Also, moderate
collagenous thickening of BrM was present in most eyes, although intra-BrM debris or cellular invasion was not observed.
However, significant morphologic choriocapillaris changes
were often apparent, especially hypertrophy and loss of fenestration (Fig. 3, bottom left). These qualitative differences were
TABLE 2. Effect of Estrogen Depletion or Supplementation on Blood
Levels of 17␤-Estradiol
Group
9-month male
9-month female (Ovary intact)
18.5-month female (ovary intact but
postmenopausal)
9-month female (ovariectomized)
9-month female (ovariectomized ⫹
supplemental estrogen [1.7 mg/pellet])
9-month female (ovariectomized ⫹
supplemental estrogen [0.36
mg/pellet])
9-month female (ovariectomized ⫹
supplemental estrogen [0.18
mg/pellet])
Plasma 17␤-Estradiol
Concentration (pg/ml)
47.5 ⫾ 1.9
97 ⫾ 3.4
36.1 ⫾ 0.2
30.8 ⫾ 0.8
545 ⫾ 5.3
175 ⫾ 2.9
82 ⫾ 1.4
Data are expressed as mean picograms per milliliter ⫾ SEM. n ⫽
7 for all groups except 9-month male, where n ⫽ 5.
reflected in quantitative measures of BLD severity score and
BrM thickness (Table 3).
Because estrogen depletion was associated with more severe sub-RPE deposits, we investigated the ability of estrogen
replacement therapy with subcutaneous implantation of pellets containing a sustained release formulation of estradiol.
Three different dosages were evaluated in preliminary experiments. As expected, pellets containing different estradiol dosages were associated with predictable plasma levels 6 weeks
after implantation (Table 2). Ovariectomized mice implanted
with the 1.7-mg/pellet showed blood estradiol levels of 545 ⫾
5.3 pg/mL, whereas mice implanted with the 0.18-mg/pellet
dose showed blood estradiol at the physiological level of 82 ⫾
1.4 pg/mL.
Based on the presumption that a higher concentration of
estradiol would provide better regulation of MMP-2 in the RPE
and vascular protective effects, we evaluated the capacity of a
1.7-mg dose in preventing sub-RPE deposits. Mice receiving
implantation of high-dose pellets unexpectedly showed development of BLDs that were only slightly less severe than those
in ovariectomized mice without estrogen (4.9 ⫾ 3.6 in estrogen-supplemented mice versus 5.6 ⫾ 3.2 in estrogen-depleted
mice; Fig. 3, bottom right). In general, the qualitative features
were identical. However, BrM appeared thinner in estrogensupplemented mice (12.7 ⫾ 7.5 in supplemented mice versus
20.7 ⫾ 4.1 in ovariectomized mice; Table 3).
Effect of Estrogen Depletion and
Supplementation in RPE Expression of MMP-2
Estrogen replacement may have failed to protect against formation of deposits after ovariectomy for at least two reasons:
First, estrogen replacement alone may not be sufficient, because other ovarian hormones contribute to protection from
deposit accumulation. Second, estrogen may demonstrate a
paradoxical bimodal dose effect on RPE production of molecules that may influence the turnover of the RPE basement
membrane and other extracellular matrix molecules. Previously, we have shown that superphysiologic concentrations of
estradiol failed to increase production of MMP-2 in cultured
human RPE compared with the two- to threefold increase in
activity induced by physiologic concentrations.17 We sought to
determine whether a bimodal response in the regulation of
MMP-2 activity was evident in RPE of ovariectomized mice,
with and without estrogen replacement.
RPE were isolated from eyecups of mouse eyes in five
groups of 9-month-old female mice: ovary intact, after ovariectomy, or after estrogen replacement with low-, moderate-, or
1226
Cousins et al.
IOVS, March 2003, Vol. 44, No. 3
FIGURE 3. TEM of the outer retina
and choroid from fat-fed, blue-green
light– exposed 9-month-old female
mice undergoing sham surgery
(ovary-intact), ovariectomy alone, or
ovariectomy with estrogen replacement. Top left: specimen from a middle-aged sham-surgery (ovary-intact)
female mouse revealed minimal subRPE deposits and minimally thickened BrM. The choriocapillaris (CC)
appeared normal. Top right: specimen from a middle-aged ovariectomized mouse reveals moderately
thick continuous BLD (under white
dotted line) with banded structure
(white asterisk), irregular thickening
of BrM, and normal choriocapillaris.
Marked thickening and reduplication
of the basement membrane was evident (black asterisks). Bottom left:
another specimen from an ovariectomized mouse revealed nodular BLDs
and irregularly thickened BrM. The
endothelium appeared hypertrophied (white asterisk) with irregular
protrusions into BrM, loss of fenestrations (open white arrowheads), and
reduplication of the basement membrane (black asterisk). No invasive
processes were observed. Bottom
right: specimen from an ovariectomized mouse with estrogen replacement revealed moderately thick BLD
(under white dotted line) of severity
similar to that in the ovariectomized
mice with banded structures (white
asterisk). However, BrM appeared to
be normally compact and the endothelium was normal. Magnification,
⫻25,000.
high-dose estrogen-containing pellets for 6 weeks. Protein was
extracted and gelatin zymography for MMP-2 activity and Western blot for detection of latent MMP-2 protein was performed.
Female 9-month mice demonstrated significant MMP-2 activity
and expression of latent protein (Figs. 4, 5). By comparison,
ovariectomized mice demonstrated 70% loss of MMP-2 activity.
TABLE 3. Effect of Estrogen Depletion or Supplementation on Deposit Severity in 9-Month-Old Mice
Group
n
Frequency of
Any BLD
(%)
Ovary intact
Ovariectomized
Supplemental estrogen
6
5
7
100
100
71
Frequency of
“Moderate” BLD
(%)
Mean Severity Score
(15 max.)
Mean Bruch’s
Membrane Thickness
0
60*
71†
1.8 ⫾ 1.3
5.6 ⫾ 3.2‡
4.9 ⫾ 3.6§
16.5 ⫾ 3.4
20.7 ⫾ 4.1㛳
12.7 ⫾ 2.5¶
Frequencies of BLD are as described in Table 1.
* P ⫽ 0.02 versus ovary intact (␹2 test).
† P ⫽ 0.008 versus ovary intact (␹2 test).
‡ P ⫽ 0.023 versus ovary intact.
§ P ⫽ 0.07 versus ovary intact; P ⫽ NS versus ovariectomy.
㛳 P ⫽ ns versus ovary intact.
¶ P ⫽ 0.039 versus ovary intact; P ⫽ 0.002 versus ovariectomy.
IOVS, March 2003, Vol. 44, No. 3
FIGURE 4. RPE-derived MMP-2 activity. Protein was extracted from
RPE of seven pairs of eyes in each group and the left and right eye
pooled from groups of mice treated by sham ovariectomy (ovary
intact), ovariectomy (O) without estrogen, or ovariectomy with 17␤estradiol replacement: high (1.7 mg/pellet), moderate (0.36 mg/pellet), or low (0.18 mg/pellet) dose. Top: gelatin zymogram from a
representative experiment. Bottom: data are the average of results in
seven pairs of eyes. **Significant difference (P ⬍ 0.01) compared with
the sham-surgery control.
A striking dose effect on activity of MMP-2 was observed in the
estrogen-supplemented groups. Mice receiving the high-dose
supplement demonstrated low MMP-2 activity with similar levels as observed in the ovariectomized mice without supplement. However, mice receiving the lowest-dose implants
(reproducing physiologic blood estrogen concentration) demonstrated MMP-2 levels similar to the ovary-intact mice.
DISCUSSION
Estrogens are a large family of chemically related steroid compounds produced mainly by the ovary but can also result from
testosterone metabolism and extragonadal production. 17␤Estradiol is the main estrogen produced by the ovary. Since the
discovery of ERs in nearly every tissue, estrogen has been
considered to be a powerful regulator of many biological processes. In particular, estrogen has been implicated as a regulatory factor in many chronic degenerative and vascular diseases,
including Alzheimer’s disease,26 atherosclerosis, 27,28 and renal
glomerulosclerosis.12 A large body of literature of retrospective
and observational research has suggested that estrogen status
influences progression of disease: Female gender and late onset
of menopause are beneficial, estrogen depletion by menopause
or oophorectomy is associated with increased risk of disease
progression, and hormone replacement therapy (HRT) seems
to be protective.
ARMD is characterized by the accumulation of various lipidrich deposits and progressive thickening in BrM.29 Several
observational studies have suggested that estrogens protect
women from the development of ARMD,3 and estrogen replacement therapy is currently being studied in a large clinical
trial for the prevention of ARMD. However, our knowledge
regarding the actions of estrogens in RPE, a crucial target tissue
in the progression of ARMD is limited.
In these experiments, we evaluated the effect of age, gender, and estrogen status in the regulation of severity of sub-RPE
Estrogen and Experimental BLD
1227
deposits and in RPE expression of MMP-2. These studies confirm our previous findings that older age renders mice susceptible to high-fat diet-induced formation of deposits, especially
in response to blue-green laser light. The changes include
sub-RPE deposits (i.e., BLD), thickening of BrM and endothelial
invasion. In the current study, old (16 months) female mice
had more severe sub-RPE deposits and endothelial changes to
the choriocapillaris than did age-matched male mice. Certainly
other factors associated with ovariectomy and postmenopause,
other than estrogen depletion, could contribute to the formation of BLD.
We also showed that estrogen deficiency may increase the
susceptibility of middle-aged (9 months) female mice to formation of sub-RPE deposits, because ovariectomy was associated
with more severe collagenous thickening, accumulation of
deposits, and endothelial changes than were observed in ovaryintact control animals. High-dose estrogen replacement did not
prevent the onset of formation of deposits, although less BrM
thickening was observed. The explanation for this lack of
protection against formation of deposits may be that estrogen
exerts a bimodal dose response on RPE MMP-2 activity and
protein expression, as we observed in vitro in cultured human
RPE cells. Ovariectomy to cause subphysiologic blood estrogen
levels or high-dose replacement to induce superphysiologic
estrogen concentration was associated with diminished activity of MMP-2 in the RPE. Normal estrogen levels in 9-month-old
mice or replacement with exogenous estradiol to reproduce
physiologic blood estrogen concentrations, correlated with
higher RPE MMP-2 activity. Thus, loss of MMP-2 activity in the
RPE correlated with development of deposits, suggesting that
regulation of the extracellular matrix by MMP-2 may play a role
in the pathogenesis of deposits.
In the mouse model, choriocapillaris endothelial changes,
including hypertrophy, loss of fenestration, reduplication of
the endothelial basement membrane, and possible cellular invasion into BrM, were an important feature in some aged and
ovariectomized female mice. Abnormal regulation of ECM turnover by “injured” endothelial cells in the absence of estrogen is
possibly another component in this model. In a preliminary
experiment not shown, the choroidal MMP-2 response to estrogen depletion and replacement appeared to be similar to
FIGURE 5. RPE-derived MMP-2 protein. Protein was extracted from
RPE as described in Figure 4. Top: Western blot from a representative
experiment. Bottom: data are the average of results in seven pairs of
eyes. **Significant difference (P ⬍ 0.01) compared with the shamsurgery control.
1228
Cousins et al.
that observed in the RPE. Nevertheless, the choroidal specimens processed for this study contained a wide range of
heterogeneous cell types in addition to the choriocapillaris
endothelial cells, including pericytes, macrophages, melanocytes, and others. Thus, much more thorough analysis of regulation of choroidal ECM is required to draw any meaningful
conclusions.
17␤-Estradiol can influence cell biology in several important
areas, especially as gene regulators through activation of two
nuclear receptors and as antioxidants.26,30 –33 Extracellular estrogen directly permeates cell membranes to enter the cytosol.
Estrogen mediates its actions through two nuclear receptors
ER␣ and ER␤, which on binding their ligand in the cytoplasm,
translocate into the nucleus, becoming transcription factors to
modulate gene expression.26,34,35 ERs can influence AP-1– dependent, NF-␬␤– dependent, and other transcriptional regulation.30,31,33 Although ERs generally inhibit transcriptional
functions, many cell-specific differences have been noted, and
each cell type must be independently evaluated. Additional
complexity is introduced by injury-dependent and concentration-dependent changes in expression of ER, as well as interactions between ER␣ and ER␤, all of which can lead to positive
or negative feedback that profoundly alters estrogen activity.26,36,37
In general, ER activation usually prevents dysregulated production of matrix molecule expression in response to injury or
hormonal stimulation (e.g., in vascular smooth muscle cells
within the glomerulus or coronary artery), but many cellspecific exceptions have been noted. In several tissues, estrogens have been shown to modulate the expression and activity
of molecules important for turnover of ECM, including MMP2.8,10,11,38 MMP-2 has type IV collagenolytic activity but also
cleaves type I, V, VII, and XI collagens as well as laminin.
Because many of these molecules are part of BrM39 – 41 and
BLD,42 altered production or activity of MMP-2 may influence
accumulation of deposits, collagenous thickening, and biochemical function of BrM. Estrogen-mediated regulation of RPE
genes important for matrix turnover may be a potential mechanism by which estrogens exert their protective effects against
ARMD. Estrogens can also function as antioxidants,27 a property not investigated in this study.
Two recent prospective studies evaluating the putative protective benefit of HRT in coronary artery disease in women
have raised the issue of an “estrogen paradox.”43,44 Estrogen
depletion has been confirmed to increase risk for cardiovascular events, but estrogen replacement therapy with conventional modalities (sustained levels of high physiologic concentrations rather than the natural cyclic, fluctuating levels) was
not found to be protective.43– 46 Prospective studies are currently ongoing in other diseases as well. However, at least in
coronary artery disease, the paradox presented is that estrogen
depletion is harmful, but conventional replacement is not necessarily beneficial to those women predisposed to cardiac
disease.46 Our results may reflect on this paradox by suggesting
a strong concentration-dependent difference in the regulation
of MMP expression: Physiologic concentrations of estrogen,
but not sub- or superphysiologic levels, upregulated MMP-2
activity (and probably many other molecules). Studies are underway to confirm and extend these observations in vitro.17
Also, we intend to perform in vivo studies to evaluate different
doses of estrogen and their impact on RPE and choriocapillaris
ECM production.
We expect that these and similar studies will assist in testing
a hypothetical model for early formation of deposits in ARMD.
We propose that initial oxidant injury to the RPE causes the
formation of extruded RPE cell membrane blebs, which in
association with downregulation of MMP-2, accumulate between the RPE and its basement membrane as part of BLDs.
IOVS, March 2003, Vol. 44, No. 3
Estrogen reduction may promote this process by loss of estrogen signaling through the ERs, which may lead to downregulation of MMP-2, a major gelatinase responsible for degradation
of type IV collagen, to cause thickening of the RPE basement
membrane as well as collagenous thickening of BrM. In support of this model, we have observed that cultured human RPE,
exposed to mild, sublethal oxidant injury, develop cell membrane blebs and downregulation of MMP-2 activity.47,48 Similar
mechanisms may be responsible for choriocapillaris endothelial injury as well. Studies are under way to confirm these
observations in vitro and in vivo.
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