|Molecular Vision 2004;
Received 3 August 2004 | Accepted 1 December 2004 | Published 6 December 2004
The role of estrogen and estrogen receptorβ in choroidal neovascularization
Mai Tanemura,1 Noriko Miyamoto,1
Michiko Mandai,2 Hiroshi
Kamizuru,1 Sotaro Ooto,1 Tsutomu Yasukawa,1 Masayo
Takahashi,2 Yoshihito Honda1
1Department of Ophthalmology and Visual Science, Kyoto University Graduate School of Medicine, Kyoto, Japan; 2Translational Research Center, Kyoto University Hospital, Kyoto, Japan
Correspondence to: Michiko Mandai, 54 Yoshida-kawahara-cho Sakyo, Kyoto, Japan; Phone: (81) 75-751-4717; FAX: (81) 75-751-4731; email: email@example.com
Purpose: Choroidal neovascularization (CNV) under the age of 50 is more often observed in women than in men. The effects of 17β-estradiol (E2) on choroidal neovascularization development were investigated both in animal models and cultured cells to see if estrogen receptors (ERs) are involved in the process.
Methods: CNV was induced by fundus laser photocoagulation in adult male and female rats. The degree of CNV development was scored and compared between them. Gene expression levels of VEGF receptor 2 (VEGFR2) and ERs after photocoagulation were compared between genders using real time PCR. CNV formation and the gene expressions were also examined in ovariectomized females with and without E2 treatment. The roles of ERs were studied by overexpressing them in human umbilical vein endothelial cells (HUVECs). The localization of estrogen receptorβ (ERβ) and VEGFR2 in CNV were studied immunohistochemically.
Results: CNV scores were significantly higher in females than in males 14 and 21 days after photocoagulation (<0.05). VEGFR2 and ERβ gene levels were increased more in females than in males on day 7 (3.4 fold compared to 1.8 fold) and on day 3 (5.8 fold compared to 2.3 fold) after photocoagulation, respectively. Both ERβ and VEGFR2 gene expressions were additively enhanced by photocoagulation and E2 treatment in ovariectomized females. E2 significantly enhanced VEGFR2 gene expression and cell proliferation in HUVECs overexpressing ERβ. ERβ and VEGFR2 are well co-localized in CNV tissue.
Conclusions: Estrogen may promote CNV development by increasing VEGFR2 gene expression via ERβ.
Estrogen is classically a sex steroid hormone that maintains gonad function. Recently, numerous other effects of estrogen on organs other than the gonads have been reported including the cardiovascular and nervous systems, bone integrity  and carcinogenesis . In these reports, the importance of estrogen is often related to gender difference in susceptibility to certain diseases. Similarly, in ocular diseases, we previously reported a possible role of estrogen in uveitis, which may support the etiological background of gender difference in certain uveitis . We also observed that 17β-estradiol (E2) played an important role in the regulation and modulation of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2) mRNA, and subsequent endothelial cell proliferation [4,5]. This led us to question a possible role of E2 in ocular angiogenesis.
Choroidal neovascularization (CNV) is one of the leading causes of visual loss in developed countries. It has different features according to the age group of the patients who develop the disease. In patients under the age of 50, the causes of CNV include myopic degeneration, inflammation, trauma, angioid streaks, and a number of other causes grouped as idiopathic . These CNVs often develop at the site of the impaired Bruch's membrane, and in this age group, women are more susceptible to CNV development [6,7]. In our study, 75% of patients with myopic CNV and 65% of patients with idiopathic CNV diagnosed at our clinic were women (unpublished data). On the other hand, in patients who are aged 50 and over, age related macular degeneration (AMD) is the leading cause of CNV, which is more prevalent among female patients in the United States  but more often observed in male patients in Japan [9,10]. These etiologies may mean that there is less gender differential in the occurrence of CNV over the age of 50, though females are more commonly susceptible to CNV under the age of 50. This gender differential among younger people may implicate the relevance of estrogen at least in simpler angiogenic changes including, for instance, an injury evoked process. Recently, the presence of estrogen receptor has been reported in human myopic CNV tissue, which also suggests that estrogen may play some role in the pathology of CNV .
There exist two estrogen receptor subtypes: estrogen receptor α and β (ERα, ERβ). The distribution and the role of each receptor differ according to the organs [1,12]. In ocular tissues, both types of receptors are expressed in the posterior segment but the role of each receptor type has not yet been explored enough [13,14]. In this study, we investigated the effects of E2 on CNV development after laser injury and assessed the possibility of involvement of ERs in the process of CNV formation.
Mature Brown-Norway rats (6 to 9 weeks) weighing between 130 and 150 g were used. The animals were handled in accordance with institutional guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
The rats were anesthetized with sodium pentobarbital (37.5 mg/kg) given intraperitoneally and their pupils were dilated with 1% tropicamide eye drops. Laser photocoagulation was performed to induce CNV under the following conditions: power 250 mW; wave length 514 nm; duration 0.05 s; and spot size 50 μm. To evaluate the extent of CNV development, 8 laser burns were delivered around the optic disc of each eye. For the quantitative study of gene expression, 100 laser burns were applied to each eye.
Evaluation of the development of CNV
Fluorescein angiograms (FAG) were taken on day 7, 14, and 21 after laser photocoagulation to evaluate CNV development and its activity. Each rat was injected with 0.5 ml of 10% fluorescein sodium (Fluorescite; Alcon, Fortworth, TX) intraperitoneally and fundus angiogram photographs were taken at early and late phases. The formation of CNV was evaluated according to the size and the presence or absence of dye leakage. The guideline for the CNV scoring was as follows: no leakage (score 0); minimum leakage or a staining of tissue with no leakage (score 1); small but evident leakage less than 1/4 disc area (indicating small active CNV; score 2); large evident leakage (large active CNV; score 3). A typical photograph of each score is shown in Figure 1. Overall photo-intensity was normalized by retinal capillary fluoro-intensity and each of eight photocoagulated lesions was graded into one of the four scores. Three independent experienced ophthalmologists judged the scores in a masked manner and the scores among the three judges were consistent. The average of the sum of the scores of all eight spots in each eye was calculated to determine the "CNV score of the eye".
Extraction of mRNA and cDNA synthesis
To evaluate gene expression levels of VEGFR2, ERα and ERβ in the CNV induced ocular tissues, the total cellular RNA was isolated from the rat retinal tissue (including retinal pigment epithelium (RPE) and CNV tissues extending into the retina) using the acid guanidinium thiocyanate-phenol-chloroform extraction method. Each sample (5 μg) was used to make cDNA using a first strand cDNA synthesis kit. (Pharmacia-Biotech, Uppsala, Sweden) A detailed description of the protocol has been published previously [4,15,16].
Real time PCR
To obtain relative changes in the levels of the target gene expression, real time PCR was carried out using SYBR green PCR master mix (Applied Biosystems Japan, Tokyo, Japan). Amplification and detection were performed using an ABI Prism 7700 system (Applied Biosystems Japan, Tokyo, Japan) according to the manufacturer's instructions. The PCR cycling program consisted of 40 two step cycles of 15 s at 95 °C and 60 s at 60 °C. Each specimen was amplified in duplicate. In order to avoid contamination from background signals, all the target bands were confirmed as appearing as a single band on agarose gels each time after quantification. Primers used in this experiment are shown in Table 1. Each experiment was repeated three times to confirm reproducibility. Nucleotide sequencing confirmed that the PCR products were derived from the target cDNA. The samples were quantified, compared with the standard curve created from serial dilution of the highest concentrated sample and the data was normalized by GAPDH message level.
Ovariectomy and E2 administration
Female rats were ovariectomized and allowed a 7 day washout period before laser photocoagulation. E2 was administrated daily thereafter to the rats in E2 treated groups. E2 (10 μg; Sigma, St. Louis, MO) was dissolved in ethanol, diluted in 0.2 ml of PBS, and injected intraperitoneally once a day for the described period following laser photocoagulation. Serum E2 concentration was monitored using Estradiol EIA kit (Cayman Chemical, Ann Arbor, MI) and was confirmed to be in or above the range of normal female rats throughout the 24 h interval period between the injections.
Human umbilical vein endothelial cells (HUVECs) were purchased from TOYOBO (Osaka, Japan) and were cultured in 6 well plates in charcoal stripped phenol free endothelial cell growth medium (TOYOBO). Cells were plated at a density of 1x105 cells/well and passaged when confluent. Cells from passage 3 through 7 were used for the experiments.
Vectors and transfection
hER/pSG5 and pSG5/pERβf1 were kindly provided by Professor Pierre Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire, University Louis Pasteur, Strasbourg, France). The control mock vector was made by removing the ERβ coding sequence from pSG5/pERβf1 at the two BamHI sites. The plasmid was transfected using Fugene6 (Roche, Mannheim, Germany) according to the manufacturer's instructions. In brief, 2 μg DNA mixed with 6 μl Fugene6 in 100 μl Hanks' balanced salt solution (Invitrogen, Tokyo, Japan) was added to each well of the 6 well plates. After 5 h of incubation, the medium was changed to fresh endothelial cell growth medium.
E2 treatment and isolation of total RNA and cDNA synthesis from HUVECs
On the day following the transfection, the medium was replaced with fresh endothelial cell growth medium with E2 at a final concentration of 0, 0.01, 0.1, or 1 nM. The total RNA was isolated in each well 24 h later by using RNeasy Mini Kit (Qiagen, Valencia, CA), and 5 μg of each sample was made into cDNA with a first strand cDNA synthesis kit (Pharmacia-Biotech, Uppsala, Sweden). The gene expression of VEGFR2 and ERβ was analyzed by real time PCR.
Cell proliferation analysis
Cell proliferation was assessed by BrdU uptake using the cell proliferation ELISA kit (colorimetric, Roche, Penzberg, Germany) according to manufacturer's instructions. HUVECs overexpressing ERα, ERβ or mock were cultured in 96 well plates (3000 cells/well) with 0, 0.01, 0.1, or 1 nM E2 for 24 h and labeled with BrdU(10 μM) in the last 6 h. BrdU incorporated in newly synthesized cellular DNA was labeled with peroxidase conjugated anti-BrdU antibody and substrate, and measured as an absorbance value at 370 nm by an ELISA reader (spectraMAX GeminiEM; Molecular Devices, Osaka, Japan). Results are shown as a ratio to the control (mock transfected without E2 treatment) absorbance value. Eight wells were examined for each condition and the experiments were repeated 5 times.
The localization of VEGFR2 and ERβ protein was evaluated by immunohistochemistry on frozen sections of female rat eyes 3 days after laser photocoagulation using a confocal laser scanning microscope. (Carl Zeiss, Oberkochen, Germany). For double detection, images were superimposed using a morphometric analysis program. Sections were incubated with anti-ERβ rabbit IgG (1:200) and anti-VEGFR2 mouse IgG (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. Control sections were incubated with normal rabbit serum and the isotype control of mouse IgG (DAKO, Glostrup, Denmark 1:50). The secondary antibodies were FITC conjugated goat anti-rabbit IgG and TRITC conjugated rabbit anti-mouse IgG. (DAKO, Glostrup, Denmark 1:30) To reduce nonspecific binding of anti-mouse secondary antibody, rat serum was added to the anti-mouse secondary antibody at a dilution of 1:10.
Results are expressed as mean±standard error of the mean. Since the normality assumptions were met in all of our data by Kolmogorov-Smirnov test, we used parametric statistical analysis. The difference between two groups was analyzed using unpaired t-tests. Statistical analysis among multiple groups was performed using one way ANOVA after the normality of the data was tested. Post hoc comparison was made with the Fisher's protected least significant difference test. The α level was set to 0.05.
Gender differences in the development of CNV
As shown in Figure 2, the CNV score was highest on day 14 in both genders, and tended to be higher in females at any time point. On day 14 and 21, the difference between the two genders was statistically significant (p<0.05).
Time course of gene expression of VEGFR2 and ERβ after laser photocoagulation
The gene expression of VEGFR2, ERα, and ERβ were examined at different time points in rat retinae that received 100 shots of laser photocoagulation using real time PCR. The fold increases to the expression levels in untreated eyes were evaluated in males and females.
As shown in Figure 3A, VEGFR2 gene expression increased at each time point in both genders. The fold increase of VEGFR2 expression in females was highest on day 7, and the difference between males (1.8±0.1 fold) and females (3.4±0.4 fold) at this time point was statistically significant (p<0.01).
Gene expression of ERβ increased most on day 3 after laser photocoagulation in both genders (Figure 3B) and the increase was significantly higher in females (5.8±1.0 fold) than in males (2.3±0.5 fold; p<0.05).
There was no significant change in the gene expression levels of ERα at any time point after laser photocoagulation in either of the genders (data not shown).
Effect of E2 on CNV formation in ovariectomized rats
In our preliminary study, E2 enhanced laser induced CNV development and VEGFR2 expression levels in male rats (data not shown; preliminary study), and in order to better analyze the specific effect of E2 on CNV formation, we used ovariectomized female rats in comparison with regular female rats for further experiments. The CNV scores at 14 and 21 days after laser photocoagulation are shown in Figure 4. The scores were statistically higher in ovariectomized females with E2 treatment than those without E2 treatment both on day 14 (p<0.05) and 21 (p<0.01).
Effect of E2 on gene expression of VEGFR2 and ERβ in ovariectomized rats
Since the expression of VEGFR2 was at its maximum in female rats on day 7 after laser photocoagulation, we investigated the effect of E2 on VEGFR2 gene expression in ovariectomized female rats on that day. Ovariectomized rats received photocoagulation and/or 7 days of E2 treatment before they were sacrificed.
In ovariectomized rats, VEGFR2 gene expression tended to decrease in comparison with intact females (0.74±0.22 fold), and E2 treatment or laser photocoagulation tended to increase VEGFR2 gene expression, though these were not statistically significant. Laser photocoagulation with E2 treatment significantly increased VEGFR2 gene expression levels to 2.5±0.37 fold (p<0.01; Figure 5A).
As for ERβ, ovariectomy itself did not change the ERβ expression level. Either laser photocoagulation or E2 treatment alone enhanced ERβ gene expression in ovariectomized rats to 1.8±0.21 fold (p<0.01) and 1.8±0.10 fold (p<0.01), respectively. Simultaneous treatment of E2 and photocoagulation further increased ERβgene expression to 2.5±0.19 fold (p<0.01; Figure 5B).
The effect of ERs and E2 on cell proliferation
The effect of 2 subtypes of ERs on endothelial cell proliferation was tested using HUVEC culture. Cell proliferation evaluated by BrdU uptake was significantly increased in HUVECs overexpressing ERβ with 0.1 nM E2 treatment (p<0.01; Figure 6). The cells transfected ERα or mock vector did not respond to different E2 concentrations.
The effect of ERs and E2 on VEGFR2 gene expression in HUVECs
ERα, ERβ, and mock were each transfected to HUVECs and VEGFR2 gene expression levels were evaluated after 24 h incubation with or without 0.1 nM E2. Without E2 treatment all groups showed the same level of VEGFR2 expression. The physiological concentration of E2 (0.1 nM) significantly increased VEGFR2 gene expression only in HUVECs overexpressing ERβ(p<0.01; Figure 7A).
ERβ expression levels had no significant difference among each preparation (data not shown), but we also normalized the VEGFR2 expression levels by ERβ expression levels to study a dose dependency of VEGFR2 expression of E2 concentration (Figure 7B). Treatment of 0.1 nM E2 again significantly increased VEGFR2 expression (2.4±0.61 fold, p<0.05) but 1 nM E2 did not have any effect on the VEGFR2 message levels.
Localization of ERβ and VEGFR2 in CNV tissue
In immunohistochemistry, VEGFR2 and ERβ were most detected at the site of the CNV area in our rat model. Also, ERβ and VEGFR2 immunoreactivity were well co-localized at CNV (Figure 8). No significant staining was observed with the control antibodies.
Etiologically, in their reproductive years, more women suffer from CNV than men, whereas in later years, the gender differential becomes less consistent [6-10,17,18]. In this study CNV scores were higher in female rats than in males of a reproductive age, or E2 treated ovariectomized rats than those without E2 treatment. These results were compatible with human etiology, indicating an important role of estrogen on ocular angiogenesis. This angiogenic effect of estrogen was a consistent observation with previous reports in which estrogen had a similar effect on basic FGF induced in vivo angiogenesis using Matrigel plugs [19,20]. These reports showed that basic FGF induced angiogenesis was reduced in estrogen receptor knockout mice or ovariectomized mice, and angiogenesis was restored by estrogen replacement in the latter. Since we previously observed that estrogen enhanced retinal vascular endothelial cell proliferation and VEGFR2 expression in these cells in vitro, we presently focused on the possibility that estrogen may also enhance ocular angiogenesis in vivo by modulating VEGFR2.
VEGF is one of the most potent growth factors that contribute to angiogenesis, which is also known to play an important role in the development of CNV [21-28]. VEGF has two functional receptors, VEGFR1 (Flt) and VEGFR2 (KDR) [29,30]. In vitro studies have shown that VEGFR1 is expressed in both endothelial cells and pericytes, whereas VEGFR2 is expressed in microvascular endothelial cells [31,32]. It has been suggested that VEGFR2 plays a major role in VEGF dependent angiogenesis . In the present study, VEGFR2 mRNA expression increased following laser photocoagulation in both genders, suggesting that VEGFR2 may play a significant role in the formation of CNV in our model. Furthermore, in correlation with CNV scores, the fold increase in VEGFR2 expression after laser photocoagulation was greater in females. We had previously observed that E2 increased VEGFR2 mRNA expression as soon as 6 h after administration in bovine retinal capillary endothelial cells . These results suggested some direct effect of E2 on VEGFR2 in the development of CNV formation.
ER has two isoforms, α and β, both of which are reported to exist in many tissues other than the gonads. The distribution of each ER subtype differs according to the organs. For example, ERα is dominantly expressed in testis, pituitary, uterus, kidney, and adrenal gland etc. On the other hand, various neural tissues (e.g., cerebellum, brain stem, spinal cord) express ERβ dominantly . Each receptor subtype seems to have a specific role in each tissue, for example, ERα affects protection against brain injury , or in the developing brain ERβ influences migration and neuron survival .
In the present study, the expression levels of ERβ significantly increased after laser photocoagulation especially in females, while expression levels of ERα did not change in either of the genders.
Interestingly ERβ gene expression significantly increased 3 days after laser photocoagulation followed by a delayed increase in VEGFR2 expression on day 7. This time course delay may imply that ERβ induction may subsequently lead to VEGFR2 induction. Furthermore, the expression level of ERβ increased with E2 treatment alone in ovariectomized females, and the simultaneous treatment of E2 with laser photocoagulation further enhanced ERβ gene expression.
Lindner reported that ERβ gene expression increased in rat endothelial cells of the aorta after vascular injury . In the process of CNV formation in humans, it is known that injury to the posterior tissue, including Bruch's membrane, is an important trigger for CNV formation in pathologic myopia, trauma, and angioid streaks . In the present study, ERβ induction may have been caused by injury to the posterior eye (including Bruch's membrane) by laser photocoagulation, and the induction of ERβ seems to be further enhanced in the presence of E2.
The reports on effects of E2 on the induction of ERs, however, are not consistent. In rat retinal pigment epithelium cells, estrogen induces both ERα and ERβ . In omental adipose tissue, E2 increases ERα but decreases ERβ . In prepubertal rat uteri, the estrogen analog genistein decreases ERα but not ERβ . In the immature mouse uteri, E2 downregulates ERβ synthesis but upregulates ERα synthesis . The effects of ERs on cell proliferation are different according to organs or cells . But as for endothelial cells it is generally regarded that E2 enhances cell proliferation both in vitro and in vivo [20,41-45] and in some cells E2 may have different effects on cell proliferation according to its concentration [42,43].
Thus, we studied the role of ERs in the vascular cell proliferation using HUVEC culture. As shown in our results, the expression of VEGFR2 and cell proliferation were significantly enhanced in ERβ overexpressing cells in the presence of E2 up to 0.1 nM, but no such results were obtained in ERα or mock transfected cells. These results suggest that E2 may play an important role via ERβ in inducing VEGFR2, and in subsequent endothelial cell proliferation.
Our immunohistochemical study also suggested the importance of ERβ. A previous study showed that in the posterior part of the normal human eye, ERβ is expressed mainly in the ganglion cell layer in the retina and choroidal structures . In our study, both ERβ and VEGFR2 were highly expressed in the CNV areas. The co-localization of these two proteins may also support the idea that ERβ may be induced in the vascular endothelial cells at the site of photocoagulation and may mediate the induction of VEGFR2 and promote CNV development.
Robinson and co-workers reported that 2-methoxyestradiol, an E2 metabolite, may inhibit CNV formation . In this report CNV were induced without destruction of Bruch's membrane and E2 concentration seemed higher than the physiological level. In our HUVEC cell culture, cell proliferation was enhanced with 0.1 nM of E2 but not with higher concentration. Similar results were observed in our previous study in bovine retinal endothelial cell culture . One possibility is that E2 may have different effects on vascular endothelial cell proliferation according to its concentrations. This tendency was also reported in some other cells [42,43].
Taken together, we propose that injury to the posterior part of the eye could trigger an induction of ERβ in the vascular endothelial cells in the early stage of CNV formation, which may further enhance the development of CNV in the presence of E2. This might also partially explain why females of a reproductive age are more susceptible to CNV than males.
We thank Professor Pierre Chambon (University Louis Pasteur, Strasbourg, France) for the human ER expression vectors, and we thank Dr. Satoshi Kashii MD, PhD for his constant support.
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