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From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan.
Abstract
PURPOSE. Estrogen is known to promote angiogenesis in gonads. The presence of estrogen receptors in the vascular endothelium of organs other than gonads has been reported. The goal of this study was to determine whether estrogen promotes the proliferation of retinal microvascular endothelial cells and to explore the mechanism of it.
METHODS. DNA was quantitated using primary cultures of bovine retinal endothelial cells that were incubated with different doses of 17 ß-estradiol (E2), VEGF, or both. The changes in expression level of VEGF and VEGF receptor-2 (VEGFR2) were measured using northern blot analysis after treatment with E2. The presence of estrogen receptors in the endothelial cells was studied by immunohistochemistry and western blot analysis.
RESULTS. 17 ß-Estradiol (E2) increased the DNA level in bovine retinal capillary endothelial cells (BRECs) by 177% at 1 nM (P < 0.05) and 150% at 10 nM (P < 0.05) by comparison with unstimulated BREC. One hundred nanomole tamoxifen completely blocked the E2-induced DNA synthesis in BRECs. Ten nanomole E2 augmented vascular endothelial growth factor (VEGF)–induced DNA synthesis in BRECs significantly (160%, P < 0.01). Ten nanomole E2 also increased VEGF mRNA expression, which peaked after 24 hours (6.7 times, P < 0.05), and VEGF receptor-2 (VEGFR2) mRNA expression, which peaked after 9 hours (2.4 times, P < 0.05). The mRNA expression level of VEGFR2 peaked with 10 nM E2 (P < 0.05) and that of VEGF reached maximum with 1 nM E2 (15 times, P < 0.001). VEGFR2 and VEGF proteins increased in parallel with their mRNA levels. Immunocytochemistry showed estrogen receptor expression in BRECs, and western blot analysis indicated the presence of a 67-kDa protein that was compatible with the estrogen receptor.
CONCLUSIONS. These findings suggest that E2 may stimulate BREC growth by the receptor-mediated pathway and that E2 may augment the VEGF-dependent angiogenesis partly through the upregulation of VEGFR2.
Estrogen is a member of steroid hormone well known for its classic roles in the homeostasis of sex steroid hormones and the maintenance of gonad function.1 Recently, the existence of the estrogen receptor in the vascular endothelium of organs other than gonads has been reported, and its role in both physiological and pathologic conditions has begun to interest many investigators, who have found that estrogen exerts its effect on nitric oxide synthase2 and lipid metabolism3 ; affects the release of prostaglandins and leukotrienes, thereby inhibiting the chemotaxis of white blood cells4 5 ; and inhibits leukocyte infiltration by reducing intercellular adhesion molecules.6 Among the many roles of estrogen, one of the most important is its angiogenic property, principally in the gonadal organs; estrogen is involved in the physiological condition of menstruation and in some pathologic situations, such as in endometriosis.7
Diabetic retinopathy is characterized by proliferative neovascularization of the retina and is one of the leading causes of visual loss in adults in the United States.8 In female patients, the diabetic retinopathy often deteriorates during pregnancy.9 10 11 The serum level of estrogen during pregnancy is raised nearly 1000 times that of nonpregnancy. These suggest that estrogen may have some effect on vascular proliferation, but to our knowledge there has been no report on the presence of estrogen receptors or effects of estrogen on ocular vasculature. Thus, in the study described herein, we investigated the effects of 17 ß-estradiol (E2), the major functional derivative of estrogen, on retinal endothelial cell proliferation using bovine retinal endothelial cells (BRECs). Furthermore, because elevated levels of vascular endothelial growth factor (VEGF) in patients with diabetic retinopathy seem to play an important role in neovascularization,12 13 14 we also explored if estrogen could have some synergistic effect with VEGF on BREC proliferation.
Materials and Methods
Materials
VEGF was purchased from Genzyme (Cambridge, MA); plasma-derived
horse serum was obtained from Wheaton (Pipersville, PA);
32P-dCTP and 35S-methionine
were obtained from Amersham (Buckinghamshire, UK); 17 ß-estradiol,
tamoxifen, fibronectin, sodium pyrophosphate, sodium fluoride, sodium
orthovanadate, aprotinin, leupeptin, and phenylmethylsulfonyl fluoride
were obtained from Sigma (St. Louis, MO); and protein A-Sepharose beads
were from Pharmacia Biotech (Uppsala, Sweden). For immunocytochemistry,
mouse monoclonal anti-estrogen receptor antibody was obtained from ABR
Inc. (Golden, CO); for western blot analysis, mouse monoclonal
anti-estrogen receptor antibody was purchased from NeoMarkers (Fremont,
CA); and for immunoprecipitation analysis, rabbit polyclonal anti-human
VEGF antibody and VEGF receptor-2 (VEGFR2) antibody were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Human VEGFR2 cDNA was a
generous gift from Lloyd P. Aiello and George L. King (Joslin Diabetes
Center, Boston, MA).
Cell Culture
Primary cultures of BRECs were isolated from freshly isolated calf
eyes obtained from a local abattoir by homogenization and a series of
filtration steps, as described by King et al.15
Primary
BRECs were grown on fibronectin-coated dishes (Iwaki Glass, Tokyo,
Japan) containing phenol red–free Dulbecco’s modified Eagle’s medium
(DMEM, Sigma) with 5.5 mM glucose, charcoal/dextran-treated 10%
plasma–derived horse serum, 50 mg/l heparin, and 50 U/l endothelial
cell growth factor (Boehringer Mannheim, Indianapolis, IN). The cells
were cultured in 5% CO2 at 37°C; medium was
changed every 3 days. Endothelial cell homogeneity was confirmed by
positive immunostaining for anti–factor VIII antibodies analyzed by
confocal microscopy. Cells were plated at a density of 2 x
104 cells/cm2 and passaged
when confluent (approximately 1 x 105
cells/cm2). The medium was again changed every 3
days, and cells from passages 3 through 10 were used for the
experiments.
DNA Content Assay in Retinal Endothelial Cells
BRECs were seeded sparsely (approximately 2500 cells/well) on
24-well culture plates (Iwaki Glass, Tokyo, Japan) and incubated for 24
hours with phenol red–free DMEM containing charcoal-filtered 10% calf
serum (GIBCO Laboratories, Grand Island, NY). The medium was changed to
each of the experimental conditioned media the following day, as
follows: (1) to study the effect of estrogen on BREC proliferation, the
medium were conditioned with 1 pM to 100 nM E2 in
either the presence or absence of VEGF, added at a concentration of 0.6
nM, which has been reported to induce the maximum proliferative effect
on BRECs16
; (2) to observe augmented VEGF-dependent cell
proliferation by E2, VEGF was added at 0.0006 to
0.6 nM concentrations to 10 nM E2; (3) to study
the effect of estrogen receptor antagonist, a 10-fold excess of
tamoxifen was added to the medium 3 hours before stimulation with
E2. In each of these experiments, the cells were
incubated in the experimental medium at 37°C for 4 days and lysed in
0.1% sodium dodecyl sulfate; DNA content was measured by means of
Hoechst-33258 dye and a fluorometer (model TKO-100; Hoefer Scientific
Instruments, San Francisco, CA). It has previously been shown that
total cellular DNA content measured in this manner correlates closely
with actual cell number, as determined by hemocytometer counting of
trypsinized cells.16
RNA Extraction
The medium was decanted, and the cells were lysed directly in the
culture dishes with 600 µl guanidinium thiocyanate. RNA was extracted
by adding 240 µl of chloroform and shaking for 10 seconds, then
cooling at 4°C for 5 minutes. The suspension was centrifuged at
15,000 rpm for 15 minutes at 4°C, and the aqueous phase transferred
to a new tube. The RNA was precipitated by adding 600 µl isopropanol
and incubated on ice for 15 minutes at 4°C. The RNA pellets were
washed once with 75% ethanol, dried, resuspended in 20 µl diethyl
pyrocarbonate (DEPC)–treated water, and incubated for 10 minutes at
60°C. RNA purity was determined by the ratio of optical density (OD)
measured at 260 and 280 nm
(OD260/OD280), and RNA
quantity was estimated at OD260.
Northern Blot Analysis
Northern blot analysis was performed on 15 µg total RNA after
1% agarose–2 M formaldehyde gel electrophoresis and subsequent
capillary transfer to Biodyne nylon membranes (Pall BioSupport, East
Hills, NY) and ultraviolet crosslinking using a FUNA-UV-LINKER (model
FS-1500; Funakoshi, Tokyo, Japan). Radioactive probes were generated
using an Amersham Megaprime labeling kit and
32P-dCTP. Blots were prehybridized, hybridized,
and washed 4 times in 0.5 x SSC, 5% sodium dodecyl sulfate
(SDS), at 65°C for 1 hour in a rotating hybridization oven (TAITEC,
Koshigaya, Japan). All signals were analyzed using a densitometer
(model BAS-2000 II; Fuji Photograph Film, Tokyo, Japan), and the
samples of each lane were normalized using the 36B4 cDNA probe.
Immunoprecipitation
Confluent BRECs were serum-deprived for 24 hours and then
treated with 10 nM E2 or vehicle for 9 hours for
VEGFR2 protein evaluation or for 24 hours for VEGF protein evaluation.
Cells were then incubated with 100 µCi/ml of
35S-methionine in methionine-free DMEM for 4
hours, washed 3 times with cold phosphate-buffered saline (PBS), and
then solubilized in 1 ml of lysis buffer containing 1% Triton X-100,
50 mM HEPES, 10 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium
fluoride, 1 mM sodium orthovanadate, 1 µg/ml aprotinin, 1 µg/ml
leupeptin, and 2 mM phenylmethylsulfonyl fluoride. After centrifugation
at 12,000 rpm for 10 minutes, equal amounts of protein from each sample
were preabsorbed with protein A–Sepharose beads and reacted with an
excess amount of rabbit anti-human VEGFR2 antibody or rabbit anti-human
VEGF antibody; immunoprecipitation was then done with protein
A–Sepharose beads. The resin was washed 5 times with lysis buffer and
boiled for 5 minutes in nonreducing SDS–polyacrylamide gel
electrophoresis sample buffer. Each sample was electrophoresed on a
7.5% SDS–polyacrylamide gel, and all signals were analyzed with a
densitometer (model BAS-2000 II; Fuji Photograph Film).
Immunocytochemistry
BRECs were seeded sparsely (approximately 2500 cells/well)
on 4-well chamber slides (Nalge Nunc International, Naperville, IL) and
incubated for 4 days with phenol red–free DMEM containing
charcoal-filtered 10% calf serum. The medium was then discarded, and
the BRECs fixed in acetone for 10 minutes at 4°C. After a 10-minute
wash with PBS, slides were incubated in a humidified chamber for 2
hours with primary antibody (1:50, mouse monoclonal anti-estrogen
receptor antibody, ABR Inc. and then rinsed for 10 minutes with PBS at
room temperature. Slides were next incubated for 1 hour with secondary
antibody (goat anti-mouse IgG antibody; 1:200), and then incubated with
streptavidine for 2 hours at room temperature. A confocal laser
scanning microscope (model LSM 410 invert Laser Scan Microscope; Zeiss,
Oberkochen, Germany) was used for the immunocytochemical analysis.
Digitized images were captured by computer and stored on an optical
disc for subsequent display. Photographic images were printed with a 35
mm film printer.
Western Blot Analysis
Cells were washed 3 times with cold PBS and then solubilized in
100 µl lysis buffer (1% Triton X-100, 50 mM HEPES, 10 mM EDTA, 10 mM
sodium pyrophosphate, 100 mM sodium fluoride, 1 mM sodium
orthovanadate, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 2 mM
phenylmethylsulfonyl fluoride). After centrifugation at 12,000 rpm for
10 minutes, 30 µg protein was subjected to 7.5% SDS gel
electrophoresis and transferred electrically to a nitrocellulose
membrane (Schleicher & Schuell, Keene, NH). The membrane was then
soaked in the blocking buffer (PBS containing 0.1% Tween-20 and 3%
bovine serum albumin) for 1 hour at room temperature and incubated with
anti-estrogen receptor antibody (1:100) for 1.5 hours. The blots were
washed with PBS containing Tween-20, and the signals detected by an ECL
western blot analysis system (Amersham).
Statistical Analysis
All determinations were performed in triplicate, and all
experiments were repeated at least three times. Results are expressed
as the mean ± SEM, unless otherwise indicated. Statistical
analysis used Student’s t-test or ANOVA to compare
quantitative data populations with normal distributions and equal
variance. Data were analyzed using the Mann–Whitney rank sum test or
the Kruskal–Wallis test for populations with nonnormal distributions
or unequal variance. P < 0.05 was considered
statistically significant.
Results
Effect of E2 on DNA Synthesis in BRECs
The effect of exogenous E2 on DNA synthesis
in BRECs is shown in Figure 1
. BREC DNA synthesis was increased by the addition of exogenous
E2; the maximal stimulatory effect was achieved
with between 1 nM (177% ± 78.1%, P < 0.05) and 10
nM (150% ± 20.8%, P < 0.05). At concentrations
above 10 nM, increased cell growth was not apparent, and its effect was
eliminated entirely by a 10-fold excess dose of tamoxifen, an estrogen
receptor antagonist (Fig. 1)
.
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In the present study, we first investigated the possible role of estrogen in the angiogenic response of retinal endothelial cells, especially in direct relation to the presence of VEGF. We studied DNA content in the endothelial cells to estimate BREC proliferation.16 The enhancement of DNA synthesis in BRECs by E2 suggests that E2 may promote proliferation of BRECs. Furthermore, this E2-induced increase in DNA synthesis was inhibited completely by pretreatment with tamoxifen. Tamoxifen has been shown to influence cell behavior differently according to the concentration used; it inhibits protein kinase C at concentrations greater than 100 µM17 and inhibits lung cancer and human melanoma cell growth by a non-estrogen receptor–specific mechanism at concentrations of 1 to 10 µM18 19 ; at concentrations below 1 µM, tamoxifen works as an estrogen receptor antagonist.20 21 The concentration used in our experiments (100 nM of tamoxifen) is thus low enough to act as an estrogen receptor antagonist, and our results support the hypothesis that the estrogen-dependent increase in DNA synthesis in BRECs is an estrogen receptor–mediated event. We also confirmed the presence of estrogen receptors in cultured retinal endothelium by both immunocytochemistry and western blot analysis. In our experiments, peak response in DNA synthesis in BRECs was achieved with between 1 and 10 nM of E2. The physiological level of plasma estrogen ranges from picomolar to less than 100 nanomolar concentrations, the latter observed during pregnancy.22 These findings suggest that estrogen receptors may be present in retinal vascular endothelium and that estrogen may actually promote receptor-mediated endothelial proliferation in the eye in vivo, especially during pregnancy when plasma estrogen levels reach nanomolar ranges. In our DNA content assay, we found that DNA synthesis decreased with E2 at concentrations of 100 nM or more. A similar decline in proliferative response was observed in a number of reports.23 24 25 26 This may suggest that E2 may promote cell proliferation at a limited range of its concentration in vivo as well.
In angiogenesis, a variety of growth factors are released that affect
one another, and estrogen itself has been reported to have some
features that could contribute to angiogenesis; in breast carcinoma
cell lines, estrogen makes cells more responsive to the proliferative
effect of insulinlike growth factors, basic fibroblast growth factor
(b-FGF), transforming growth factor-
, and epidermal growth
factor27
; estrogen may also facilitate the release of
angiogenic factors such as b-FGF by increasing 27-kDa heat shock
protein28
; and in the uterus, estrogen is reported to
increase VEGF expression, which may accompany angiogenesis during
menstruation.7
29
30
These indicate that factors other
than VEGF could also contribute to estrogen-induced endothelial
proliferation; however, considering the importance of VEGF in ocular
diseases such as proliferative diabetic retinopathy,12
13
we concentrated on the effect of estrogen in relation to VEGF. As
presented in Figure 2B
, 10 nM of E2 not only
augmented BREC proliferation at each concentration of VEGF that we used
but it also increased the sensitivity of BRECs to VEGF. These results
clearly show the effect of E2 on endothelial
cells in direct relation to VEGF.
We next studied how estrogen enhanced the effect of VEGF. VEGF has two functional high affinity tyrosine kinase receptors, fms-like tyrosine kinase (Flt, VEGFR1) and a kinase insert domain–containing receptor (KDR, VEGFR2).31 32 In vitro studies have shown that VEGFR1 is expressed in both endothelial cells and pericytes, whereas VEGFR2 is expressed in microvascular endothelial cells.33 34 In diabetic retinopathy, pericytes disappear during the early stages,35 and it has been suggested that VEGFR2 plays a major role in VEGF-dependent angiogenesis.36 In our experiments, a significant increase in VEGFR2 in BRECs was observed 6 hours after treatment with E2. This prompt response suggests that E2 may directly induce VEGFR2 mRNA. To our knowledge, this is the first quantitative report that VEGFR2 mRNA is increased by E2 in vitro. This could be supported by a previous report that estrogen enhanced the immunohistochemical expression of VEGFR2 in the vascular endothelial cells of the rat pituitary.37 This may partly explain why E2 continues to augment cell proliferation even with maximal doses of VEGF. VEGFR2 was increased significantly with E2 at a concentration of 10 nM, which is compatible with the findings that E2 enhancement of BREC proliferation in the presence of VEGF was most marked at this concentration. Additionally, we also studied the effect of estrogen on VEGF expression in BRECs, because the upregulation of VEGF mRNAs by estrogen has been shown by several investigators.7 29 The increase in VEGF mRNAs in BRECs by estrogen may suggest that the effect of VEGF could also be enhanced by an autocrine manner. The expression of VEGF in endothelial cells, however, has never been documented in vivo, and the significance of the upregulation of the gene in BREC is still to be investigated.
Considering that estrogen is constitutively present in the circulatory system in a rather wide range of concentrations, varying according to the time during the menstruation cycle or to the months of pregnancy, it will not be easy to implicate it in a specific pathologic event. Yet the consistency in the proliferative response of BRECs and the increased expression of VEGFR2 by E2 at 10 nM indicate that the physiological level of estrogen such as that observed during pregnancy could enhance VEGF-induced proliferation of endothelial cells partly by increasing expression of VEGFR2. This may imply that during pregnancy VEGF may be effectively used for vascular construction in the fetus but could worsen angiogenic ocular diseases such as diabetic retinopathy. In conclusion, further understanding of the roles of estrogen in neovascularization at the cellular and molecular levels may contribute to our therapeutic and preventive strategies to ocular neovascularization.
Acknowledgements
Financial support for this work was provided by Grant-in-Aid for Scientific Research A2-10307042 and C-09671793 from the Ministry of Education, Science, and Culture of the Japanese Government.
Footnotes
Reprint requests: Michiko Mandai, Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto 606, Japan.
Submitted for publication November 2, 1998; revised March 12, 1999; accepted April 21, 1999.
Proprietary interest category: N.
References
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