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Endocrinology Vol. 144, No. 1 326-334
Copyright © 2003 by The Endocrine Society

ARTICLE

Soluble Vascular Endothelial Growth Factor Receptor 1 Inhibits Edema and Epithelial Proliferation Induced by 17ß-Estradiol in the Mouse Uterus

Julie M. Hastings, Diana R. Licence, Graham J. Burton, D. Stephen Charnock-Jones1 and Stephen K. Smith1

Reproductive Molecular Research Group, Department of Pathology, University of Cambridge (J.M.H., D.R.L., D.S.C.-J., S.K.S.), Cambridge, United Kingdom CB2 1QP; Department of Anatomy, University of Cambridge (G.J.B.), Cambridge, United Kingdom CB2 3DY; and Department of Obstetrics and Gynecology, Rosie Hospital (D.S.C.-J., S.K.S.), Cambridge, United Kingdom CB2 2QH

Address all correspondence and requests for reprints to: Dr. Stephen Charnock-Jones, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom CB2 1QP. E-mail: dscj1{at}cam.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The uterine response to 17ß-estradiol (E2) includes increased water retention, enhanced vascular permeability, DNA and RNA synthesis, and increased cellular mitosis. We have used the natural antagonist of vascular endothelial growth factor A (VEGF-A), sflt-1 (soluble form of flt-1), to determine whether the edematous and proliferative effects of E2 in the uterus are mediated by VEGF-A. Female BALB/c mice were ovariectomized and treated with E2 (10 µg/kg) in the absence or presence of sflt-1 (0.8 and 4.0 mg/kg) for 24 h. E2 induced increases in uterine mass from 25.3 to 36.8 mg, in total cross-sectional uterine area from 771 to 1133 µm2, in cross-sectional endometrial area from 268 to 569 µm2, and in the mitotic index of lumenal epithelial cells from 0% to 53%. Antagonism with sflt-1 reduced the E2-induced increases in total uterine area to 779 µm2, endometrial area to 398 µm2 and the mitotic index of lumenal epithelial cells to 25%, but the E2-induced increase in uterine mass was not significantly reduced. From these data we conclude that the edematous response and proliferation of lumenal epithelial cells in the murine uterus are mediated in part through VEGF-A. These data suggest that sflt-1 could be a useful anti-VEGF-A agent and may be effective in modifying uterine biology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS ARE molecules that increase the size and weight of the uterus (1). The early phases of this response include increased water retention, enhanced vascular permeability, prostaglandin release, and increased glucose metabolism (2). Several agents have been suggested as mediators of this response, including histamine and the prostaglandins (3, 4, 5, 6, 7, 8).

An inducer of vascular permeability that is 1000 times more potent than histamine was isolated from a guinea pig cell line and named vascular permeability factor (9). Subsequent molecular cloning revealed that vascular permeability factor was encoded by the same gene as vascular endothelial growth factor A (VEGF-A) (10, 11, 12). VEGF-A was identified in medium conditioned by normal bovine pituitary folliculo-stellate cells and shown to be a potent, specific, and direct-acting endothelial cell mitogen and motogen (13, 14).

VEGF-A mRNA and protein are present in endometrium (15, 16, 17, 18, 19). VEGF mRNA and protein are present in glandular and lumenal epithelium and stroma in the proliferative phase of the human menstrual cycle (15, 17). After ovulation, VEGF-A is found predominantly in the epithelial compartment, its synthesis in the stroma being reduced, suggesting that steroid hormones may play a role in its regulation (15, 18). Shifren et al. (17) found a small rise in the levels of VEGF-A mRNA in the luteal phase of the cycle. However, progesterone did not increase secreted VEGF-A in cultured endometrial explants (20). In a more recent study increased mRNA and protein levels of VEGF189 were seen in decidualized stromal cells in the late luteal phase in vivo and in vitro when isolated stromal cells were treated with E2 and progesterone (21).

In the mouse uterus, VEGF-A expression is greater in proestrus and estrus than in diestrus (19). VEGF-A mRNA was described in the epithelium in proestrus and in the stroma in estrus; however, VEGF-A protein is restricted to the epithelial compartment during both proestrus and estrus (19). VEGF-A mRNA is markedly increased in the uterus of ovariectomized rodents 2 h after treatment with E2, and this correlates with an increase in uterine mass (22). The VEGF-A gene contains two half-palandromic estrogen response elements in its promoter region, and E2 stimulates the production of VEGF-A in human and rodent uteri (15, 23).

VEGF-A-induced responses are mediated by two structurally related tyrosine kinase receptors, VEGF receptor type 1 (VEGF-R1) or flt-1 (fms-like tyrosine kinase) and VEGF-R2 or kinase domain region, and its murine homolog, fetal liver kinase-1, flk-1 (24, 25). Both flt-1 and flk-1 have seven immunoglobulin (Ig)-like extracellular domains, a single transmembrane domain, and a consensus tyrosine kinase cytoplasmic sequence that is interrupted by a kinase insert domain (26).

A cDNA sequence encoding an alternatively spliced form of flt-1 (sflt-1), lacking the seventh extracellular Ig domain, the transmembrane domain, and the cytoplasmic domain, was identified in a human umbilical vein endothelial cell cDNA library (27). Boocock et al. (28) identified an additional variant transcript of flt-1 in ovarian cancer cell lines that contained an in-frame termination codon in the fifth extracellular Ig domain of the gene. These sflt-1 molecules compete with full length flt-1 for binding to VEGF-A and inhibit VEGF-A-induced endothelial cell mitogenesis and motogenesis as well as vascular permeability (27, 28, 29, 30). sflt-1 also binds placenta growth factor; VEGF-B is known to bind flt-1 and therefore would be expected to bind sflt-1, although this has not to date been formally demonstrated (29, 31). However, both placenta growth factor and VEGF-B are known to be absent or poorly expressed in human endometrium, whereas VEGF-A is abundant.

Antagonism of VEGF-A with a soluble fusion protein of the first three extracellular domains of flt-1 and Fc-IgG, referred to as Flt-(1–3)-IgG, inhibited corpus luteal angiogenesis in a rat model of hormonally induced ovulation (32). This antagonism blocked the postovulatory increase in progesterone and subsequent maturation of the endometrium; thus, it was not possible in this experiment to investigate the direct effect of VEGF-A antagonism on the endometrium. However, a more recent study has suggested that the increased vascular permeability at the site of uterine implantation can be blocked by antagonists of VEGF-A (33).

In this study we sought to determine whether the E2-mediated increase in uterine size and uterine edema could be blocked by antagonism of VEGF-A. We also examined whether the effects of E2 on the proliferation of epithelial and stromal cells was affected by inhibition of VEGF-A.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female BALB/c mice (Harlan, Bicester, UK; 8 wk of age; 22–25 g) were maintained in standard housing. All procedures and care of the animals were performed in accordance with the regulations laid down by the United Kingdom Home Office. After a 10-d period of acclimatization, both ovaries were removed via a dorsal midline incision under inhalation anesthesia. After a recovery period of 3 wk, mice received 10 µg/kg E2 (Sigma, Poole, UK) in peanut oil sc in the absence or presence of sflt-1 (a gift from Metris Therapeutics, Slough, UK) in PBS ip (4 or 0.8 mg/kg). The sflt-1 protein used in this study was produced by transfection of CHO cells with a cDNA encoding the five-extracellular Ig domain construct described by Boocock et al. (28). Control mice were treated with equal volumes of oil and PBS. Twenty-three hours after treatment, mice received an ip injection of 3,5-bromodeoxyuridine (BrdU; Sigma) in PBS (40 mg/kg). One hour later, animals were killed under terminal anesthesia. The uteri were removed, trimmed of fat, and separated into two uterine horns, with excision of the cervix. The mass of each uterine horn was recorded. Each treatment group contained five and seven animals in two separate, but identical, experiments.

Morphometric analysis
The midlevel segment of the right uterine horn was placed in 4% glutaraldehyde (Sigma) for 24 h at room temperature before processing into 0.1 M HEPES (Life Technologies, Inc., Paisley, UK). The tissue was passed through graded alcohols and propylene oxide before embedding in Spur resin. The uterine horns were orientated such that transverse sections (1 µm) were mounted onto 3-aminopropyltriethoxysilane-coated microscope slides using a Reichert Ultracut E microtome (Shandon, Runcorn, Cheshire, UK) and were counterstained with methylene blue. The uterine area and volume fraction were measured on the same section of uterine horn.

Uterine area measurement
Total uterine and endometrial area measurements were performed with a Laborlux D microscope (Leitz, Rockleigh, NJ) at x4 magnification attached to the Video Interactive Digitizing System (Synoptics, Cambridge, UK) calibrated with a slide graticule. Three separate measurements were made by tracing around the on-screen image of the sections with a cursor. The outer uterine perimeter and the inner lumenal surface were traced: the area bounded by the latter was subtracted from that bounded by the former, and this value was taken as the total uterine cross-sectional area. Similarly, the interface between the myometrial and endometrial layers was also traced; the inner lumenal surface area was subtracted from this, and this value was taken as the endometrial area. Two independent investigators measured the total uterine and endometrial areas on two separate occasions.

Volume fraction measurement
Volume fraction measurements were performed using an Olympus Corp. BH.2 microscope (New Hyde Park, NY) at x60 magnification. Ten random fields of endometrium were selected, and a test grid of 56 points was superimposed on the sections. The numbers of test points falling on glandular epithelial cells, lumenal epithelial cells, endothelial cells, stromal cells, glandular lumenal spaces, uterine lumenal spaces, blood vessel lumenal spaces, and interstitial spaces were counted. The volume fraction occupied by each component was then calculated by expressing the number of points hitting that component as a percentage of the total number of test points applied.

Immunohistochemical double staining of endothelial and proliferating cells
Uterine tissue was placed in buffered formalin for 6 h at room temperature. Tissue was passed through graded alcohols and xylene and was embedded in paraffin wax in an orientation such that transverse sections (5 µm) could be mounted on Polysine slides (BDH, Poole, UK). Deparaffined and rehydrated sections were pretreated in citrate buffer (0.01 M, pH 6.0) in a pressure cooker under maximal pressure for 1 min and minimal pressure for 9 min, and sections were left to cool for 20 min. To identify those cells undergoing DNA synthesis, sections were incubated with anti-BrdU (Roche, Lewis, UK), rabbit antimouse Ig (DAKO Corp., Ely, UK), immune complexes of mouse alkaline phosphatase and anti-alkaline phosphatase (Sigma) and Fast Red (Sigma). To identify endothelial cells, sections were further incubated with biotinylated Bandiera simplicofolia agglutinin-1 (BS1; Sigma), streptavidin-horseradish peroxidase (Zymed Laboratories, Inc., San Francisco, CA), and 3,3-diaminobenzidine (Sigma). Ten fields of endometrium were selected in a random manner, and the number of points falling on BrdU+/BS1+, BrdU-/BS1+, BrdU+/BS1-, or BrdU-/BS1- cells within the lumenal epithelial and vascular compartments was counted at x60 magnification using an Olympus Corp. BH.2 microscope, as described above. The volume of proliferating cells was expressed as a percentage of the total volume of endothelial or epithelial cells.

Statistics
Nonparametric statistical analysis was performed on uterine mass and area measurements of control and E2-treated animals using the one-tailed Mann-Whitney U test; E2 animals and those treated with differing doses of sflt-1 were compared by Kruskal-Wallis one-way ANOVA. Volume fraction measurements and the level of proliferation (the level of BrdU incorporation and mitotic indexes) of control and E2-treated animals were statistically compared using Fisher’s exact test; E2- and sflt-1-treated animals were compared using Fisher’s exact test and Bonferroni test (for multiple analysis). Analysis was carried out using InStat (GraphPad Software, Inc., San Diego, CA). P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Uterine mass
E2 significantly increased the wet uterine mass of control mice from 25.3 mg [n = 7; 95% confidence limits (CL), 21.4–31.3 mg] to 36.8 mg (n = 7; CL, 27.7–59.0 mg; U statistic = 3.000; P = 0.0020; Fig. 1Go). The wet uterine mass of mice treated with 0.8 mg/kg sflt-1 was 37.5 mg (n = 5; 95% CL, 34.4–39.1 mg). The uterine mass of mice treated with 4 mg/kg sflt-1 was 34.1 mg (n = 7; 95% CL, 26.8–40.4 mg). Thus, neither low nor higher doses of sflt-1 altered the increase in uterine mass induced by E2.



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  		Figure 1. Uterine mass of ovariectomized mice. Ovariectomized BALB/c mice were treated with E2 in peanut oil (10 µg/kg) or E2 and sflt-1 (0.8 or 4.0 mg/kg). Control mice were treated with an equal volume of oil and PBS alone. Ovariectomized mice had a median wet uterine mass of 25.3 mg, and E2-treated mice had a mass of 36.8 mg (n = 7 in each group). This increase was statistically significant (P = 0.004, by one-tailed Mann-Whitney U test). Mice treated with E2 and 0.8 mg/kg sflt-1 (n = 5) or 4.0 mg/kg sflt-1 (n = 7) had median wet uterine masses of 37.50 and 34.10 mg, respectively. Data points represent the uterine mass of each animal from one of two identical, but separate, experiments; bars represent the median uterine mass for each group.

 
Tissue morphometry
The endometrium of control animals was compact. Glands were small and closed, with little evidence of secretory products (Fig. 2Go, A and B). The lumenal epithelium was unorganized, with a noncolumnar morphology and nuclei located at random positions in the cell. There was little, if any, interstitial space between the stromal cells. Treatment with E2 increased the size of endometrial epithelial and stromal cells (Fig. 2Go, C and D). Endometrial glands had an obvious lumen, many of which contained secretory products. Interstitial space appeared between stromal cells. Lumenal epithelial cells attained a columnar appearance, with basally located nuclei. Treatment with a low dose of sflt-1 (0.8 mg/kg) partially inhibited the E2-induced morphological changes (Fig. 2Go, E and F). Some glands showed signs of degeneration, whereas others remained healthy, but the stroma was expanded. The endometria of animals treated with a high dose of sflt-1 (4.0 mg/kg) and E2 largely contained degenerate glands with no lumen or secretory products visible. The interstitial space between stromal cells was compressed, and the lumenal epithelium had lost its columnar structure, with nuclei located randomly throughout the cells (Fig. 2Go, G and H).



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  		Figure 2. Morphometric comparison of murine endometrium in mice treated with E2 and sflt-1. Ovariectomized BALB/c mice were treated with E2 in peanut oil (10 µg/kg) or E2 and sflt-1 (0.8 or 4.0 mg/kg). Control mice were treated with an equal volume of oil and PBS alone. Uterine horns were harvested and processed into Spur resin as described in Materials and Methods. Transverse sections (1 µm) were counterstained with methylene blue. The endometrium of control animals (A and B) showed signs of degeneration, with unorganized lumenal epithelium (arrow), small closed glands (asterisks), and little interstitial space. Mice treated with E2 (C and D) had an expanded endometrium, with open glands (asterisks) and more interstitial space in the stroma. Treatment with sflt-1 [0.8 mg/kg sflt-1 (E and F) and 4.0 mg/kg sflt-1 (G and H)] resulted in the reversion of murine endometrium to a morphological phenotype similar to that of control animals. Magnification: A, C, E, and G, x10; B, D, F, and H, x63.

 
Total uterine area
The total uterine, cross-sectional area of control mice (771 µm2; n = 7; CL, 581-1017 µm2) was increased significantly by E2 (1135 µm2; n = 7; CL, 784-1734 µm2; U statistic, 9.000; P = 0.0265). The total uterine area of mice treated with 0.8 and 4.0 mg/kg sflt-1 was 1105 µm2 (n = 6; CL, 287-2830 µm2) and 779 µm2 (n = 7; CL, 667-1090 µm2), respectively (Fig. 3AGo). Therefore, sflt-1 reduced the E2-induced increase in total uterine area by 31% (Kruskal-Wallis statistic, 5.793; P < 0.001)



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  		Figure 3. Uterine and endometrial areas of ovariectomized mice. Ovariectomized BALB/c mice were treated with E2 in peanut oil (10 µg/kg) or E2 and sflt-1 in PBS (0.8 or 4.0 mg/kg). Control animals received an equal volume of oil and PBS alone. Total uterine (A) and endometrial (B) areas were measured as described in Materials and Methods. The total uterine areas of ovariectomized mice was increased by E2 from 771 to 1135 µm2 (P = 0.027, by one-tailed Mann-Whitney U test). Ovariectomized and E2-treated mice had endometrial areas of 268 and 589 µm2, respectively: this increase was significant (P = 0.002, by one-tailed Mann-Whitney U test). The E2-induced increases in total uterine and endometrial areas were reduced by sflt-1 (P < 0.001, by Kruskal-Wallis nonparametric ANOVA). Data points represent the area of each animal from one of two identical, but separate, experiments; bars represent the median area for each group.

 
Endometrial area
E2 doubled the cross-sectional endometrial area of control mice from 268 µm2 (n = 7; CL, 218–368 µm2) to 569 µm2 (n = 7; CL, 400–726 µm2; Fig. 3BGo). Mice treated with low (0.8 mg/kg) and high (4.0 mg/kg) doses of sflt-1 had a median endometrial area of 426 µm2 (n = 5; CL, 309-1720 µm2) and 398 µm2 (n = 5; 95% CL, 351–590 µm2), respectively (Fig. 3BGo). However, sflt-1 reduced the E2-induced increase in endometrial area by 30% (Kruskal-Wallis statistic, 11.262; P < 0.001).

Endometrial volume fraction
The volume fraction of epithelium, stroma, endothelium, and extracellular space was not changed by treatment of control mice with E2 and/or sflt-1. Stromal cells comprised the largest fraction (57–81%) of both treated and nontreated endometria (Fig. 4Go). The glandular and lumenal epithelium comprised 7–29% and 0–18%, respectively, of the endometrium. Endothelial cells comprised 2–8% of this endometrium. A small proportion of the volume of ovariectomized endometrium (<5%) represented spatial fractions, including extracellular space and glandular, uterine, and blood vessel lumens (Table 1Go). There was also no change in the total cellular (epithelium, endothelium, and stroma) or spatial fractions (glandular and uterine lumen, interstitial space, and blood vessel lumen) between the endometrium of treated and nontreated mice.



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  		Figure 4. Volume fraction of cellular compartments of the murine uterus. Ovariectomized BALB/c were treated with E2 in peanut oil (10 µg/kg) or E2 with sflt-1 in PBS [0.8 mg/kg (Low sflt-1) or 4.0 mg/kg (High sflt-1)] as described in Materials and Methods. Control animals received an equal volume of oil and PBS alone. The volume fractions of the cellular components of the endometrium were measured using a test grid of 56 points as described in Materials and Methods. The volume fraction is represented as a percentage of the total volume. The major constituents of endometrium were stroma and epithelium. Treatment of ovariectomized mice with E2 or E2 and sflt-1 did not induce a change in the endometrial volume fraction. Data points represent the volume fraction of each animal from one of two identical, but separate, experiments; bars represent the median volume fraction for each group.

 

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  		Table 1. The volume fraction of spatial compartments of the murine uterus

 
Measurement of cellular proliferation
The lectin BS1 and anti-BrdU antibody were used to visualize proliferating endothelial and epithelial cells as described in Materials and Methods (Fig. 5AGo). Few, if any, cells demonstrated incorporation of BrdU in control nuclei (Fig. 5CGo). E2 induced a significant increase in the number of glandular and lumenal epithelial cells demonstrating anti-BrdU immunoreactivity, the greatest increase being in the lumenal cells (Fig. 5EGo). Scattered cells throughout the stroma showed signs of DNA synthesis (Fig. 5EGo). The volume fraction of lumenal epithelial cells that have incorporated BrdU was increased from 0% (n = 4) to 53% (n = 4) by treatment with E2 (Fig. 6AGo). This increase was statistically significant (P < 0.0001, by Fisher’s exact test). sflt-1 partially inhibited this increase in the volume of proliferating epithelial cells (Fig. 5Go, G and I); the volume fractions of proliferating epithelial cells in mice treated with 0.8 and 4.0 mg/kg sflt-1 were 33% (n = 4) and 25% (n = 4), respectively (Fig. 6AGo). This inhibition was significant at higher doses of sflt-1 (P < 0.001, by Fisher’s exact and Bonferroni tests). To confirm the reliability of BrdU incorporation and detection, the number of lumenal epithelial cells containing mitotic figures was measured and represented as a percentage of the total number of lumenal epithelial cells (Fig. 5Go, D, F, H, and J). This mitotic index was significantly increased by E2 treatment from 0 (n = 5) to 2.6042% (n = 5; by P < 0.001, by Fisher’s exact test; Fig. 6BGo). The mitotic indexes of lumenal epithelial cells in animals treated with 0.8 and 4.0 mg/kg sflt-1 were 1.4614% (n = 5) and 1.3285% (n = 5), respectively (Fig. 6BGo). This inhibition was significant at higher doses of sflt-1 (P < 0.01, by Fisher’s exact and Bonferroni tests).



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  		Figure 5. Proliferative response of the endometrium of the murine uterus. BALB/c mice were ovariectomized and treated with an sc dose of either E2 in peanut oil (10 mg/kg) or E2 with sflt-1 in PBS (0.8 or 4.0 mg/kg). Control animals were treated with an equal volume of oil and PBS as described in Materials and Methods. One hour before death, animals received 40 mg/kg BrdU. Uterine horns were harvested and processed into paraffin wax for detection of BrdU and endothelial cells with anti-BrdU antibody and the lectin BS1 or into Spur resin for detection of mitotic figures. A, Proliferating endothelial cells (stained both red and brown; asterisk). B, Negative control slide where BrdU antibody and BS1 lectin have been omitted from the procedure. Little BrdU uptake (C) and no mitotic figures (D) could be detected in control animals. Treatment with E2 induced entry of the lumenal epithelial cells into the cell cycle with a significant increase in BrdU uptake (arrows in E) and the number of mitotic figures (arrows in F) in the lumenal epithelium. Low doses of sflt-1 (0.8 mg/kg) marginally inhibited the E2-induced increases in BrdU uptake (G) and mitotic figures (H). High doses of sflt-1 (4.0 mg/kg) significantly inhibited the E2-induced increases in BrdU uptake (I) and mitotic figures (J). Magnification: A and B, x40; C, E, G, and I, x10; D, F, H, and J, x63.

 


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  		Figure 6. BrdU uptake and mitotic index of lumenal epithelium of the murine uterus. Ovariectomized BALB/c were treated with E2 in peanut oil (10 µg/kg) or E2 with sflt-1 in PBS (0.8 or 4.0 mg/kg) as described in Materials and Methods. Control animals received an equal volume of oil and PBS alone. One hour before death, animals received 40 mg/kg BrdU. Cells undergoing DNA synthesis were visualized with antibodies against BrdU. The volume fraction of BrdU+ lumenal epithelial cells was measured using a test grid of 56 points and is represented here as a percentage of the total volume fraction of lumenal epithelial cells in A. The proportion of lumenal epithelial cells containing mitotic figures is represented as a percentage of the total number of lumenal epithelial cells in B. The level of epithelial cell proliferation was increased by treatment with E2 (P < 0.0001, by Fisher’s exact test). This increase could be inhibited by 4.0 mg/kg sflt-1 (P < 0.01, by Fisher’s exact and Bonferroni tests). Data points represent the volume fraction of each animal from one of two identical, but separate, experiments; bars represent the median volume fraction for each group.

 
The volume fractions of BrdU+/BS1+, BrdU-/BS1+, BrdU+/BS1-, and BrdU-/BS1- cells were calculated to determine the volume fraction of proliferating endothelial cells within the endometrium. This volume (<1%) was consistent for mice treated with or without E2 in the absence or presence of sflt-1. Thus, E2 treatment for 24 h did not increase the volume fraction of proliferating endothelial cells in the mouse uterus.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The response of the rodent uterus to E2 is complex and includes enhanced vascular permeability, increased cellular mitosis, and DNA and RNA synthesis. Our data agree with earlier studies that demonstrate the ability of E2 to induce edema in the rodent uterus (3, 4, 34). Simultaneous treatment with E2 and sflt-1 blocks the E2-induced increase in total uterine and endometrial cross-sectional area in a dose-dependent manner. The endometrium of mice treated with both E2 and sflt-1 has a morphological appearance similar to that of ovariectomized mice, with small, compact glands and stroma. The volume fraction of cells and spaces in the endometrium is not changed by treatment with E2 or E2 and sflt-1. These data suggest that all compartments of the endometrium increase in size uniformly 24 h after treatment with E2 and that the VEGF antagonist sflt-1 partially inhibits this edematous response. Over the time course of this study we did not show increased endothelial cell proliferation.

Taken together, these findings are consistent with those of Rabbani and Rogers (33), who showed that treatment with anti-VEGF-A antibody reduced the number of implantation sites. The level of endothelial cell proliferation in the implantation sites was not different in treated and untreated animals. Rabbani and Rogers (33) suggest that the reduction in implantation site number is due to the inhibition of vascular permeability and not an effect on proliferation of endothelial cells. More specifically, anti-VEGF-A monoclonal antibody inhibited capillary permeability in an adapted version of the Miles assay in rats (21). These researchers further show that under the influence of E2 and progesterone, VEGF189 protein and mRNA levels are increased in clusters of decidualized stromal cells during the mid to late secretory phase in the human menstrual cycle. In other organs the chimeric Ig construct of sflt-1-(1–3) reduced cerebral edema induced by VEGF-A (35).

The mechanism by which VEGF-A induces vascular permeability is unclear. Roberts and Palade (36) showed that VEGF-A increased the permeability of venular and capillary endothelium in the male rat cremaster muscle by opening intercellular, endothelial gap junctions and by the induction of fenestrae in nonfenestrated vessels. Treatment with VEGF-A induces a reorganization of cytoplasmic vesicles and vacuoles to form clusters of interconnected structures, called vesiculo-vacuolar organelles, that connect with the plasma membrane through the fenestrae or pores (36). The opening of interendothelial junctions occurs at lower concentrations of VEGF-A than those required for the induction of angiogenesis, suggesting that VEGF-A-induced vascular permeability can occur in the absence of angiogenesis (37). The structural changes induced by VEGF-A occur within 10 min of administration and can be blocked by anti-VEGF antibodies (36).

The effect of E2 on endothelial cell proliferation in the endometrium remains enigmatic. There are three stages of vessel growth in the human menstrual cycle: during menstruation to repair the vascular bed, under the influence of E2 during the proliferative phase, and under the influence of E2 and progesterone during the secretory phase (38). Although there are high levels of endothelial cell proliferation throughout the menstrual cycle, previous studies have failed to correlate these periods with endothelial cell proliferation or the levels of VEGF-A within the endometrium (39, 40, 41). Rogers (42) suggested that there may be a continuous remodeling of the endometrial vasculature throughout the menstrual cycle rather than endothelial cell growth. Such remodeling could involve relatively high levels of endothelial cell turnover, with high levels of endothelial cell proliferation and cell death. Mechanisms of VEGF-A-induced angiogenesis may include vascular sprouting, intussusception, or elongation (42). This vascular sprouting can occur without endothelial cell proliferation (43). However, a recent study by Heryanto and Rogers (44) demonstrated a significant increase in the percentage of proliferating endometrial endothelial cells in the uterus of mice treated with E2. Endometrial degeneration was induced by Heryanto and Rogers (44) in C57 BL/6J x CBA mice by ovariectomy and butan-2-ol administration, and animals were left to recover for 7 d; endometrial degeneration in our study was induced by ovariectomy of BALB/c mice, with a recovery period of 21 d. These differences may explain why our data and those reported by Heryanto and Rogers (44) show different results.

Although treatment with sflt-1 had no effect on endothelial cell proliferation, this treatment had a marked effect on lumenal epithelial cells. There is evidence for direct VEGF-A induction of epithelial cell proliferation in the fetal lung (45). Although there is no evidence to support the expression of VEGF-A receptors on adult uterine epithelial cells, their presence cannot be excluded. Therefore, it is possible that VEGF-A is directly stimulating epithelial cell proliferation, and that sflt-1 blocks this. An alternative explanation is that VEGF-A acts indirectly on epithelial cells. There is precedent for indirect stimulation of epithelial cell proliferation in the estrogen receptor knockout mouse (46). These researchers showed that the proliferative response of uterine epithelial cells to E2 was indirect. Uterine epithelial cells of estrogen receptor knockout or wild-type mice were cocultured with stromal cells from knockout or wild-type mice under the renal capsule of intact, adult mice; only when the estrogen receptor was present on the stromal cells did E2 induce the proliferation of epithelial cells. The researchers concluded that paracrine factors released by stromal cells are necessary for E2-induced uterine epithelial cell proliferation. However, estrogen receptor-positive epithelial cells respond directly to E2 to increase the production and release of many growth factors, including lactoferrin and VEGF-A (15, 23, 47). In the prostate of castrated rats treatment with testosterone induces vascular regeneration before epithelial proliferation (48). From these data Folkman (49) hypothesized that testosterone induced the production of VEGF-A by prostatic epithelium, and that this VEGF-A induced the production of epithelial growth factors by the endothelium. He suggested that the normal mass of an organ is tightly regulated by the vascular endothelium. Twenty-four hours after a single injection of E2, the endothelial cells of the rat uterus show signs of protein synthesis, including increased numbers of cytoplasmic granules, increased rough endoplasmic reticulum, and a well developed Golgi apparatus (4). Many factors secreted by endothelial cells induce the proliferation of epithelial cells, including fibroblast growth factor, platelet-derived growth factor, and IGF-I (50). Therefore, in our system E2 induces the release of VEGF-A from epithelial and stromal cells (22); this VEGF-A may then stimulate endothelial cells to produce growth factors that, in turn, stimulate epithelial cell proliferation. Blockade of VEGF-A with sflt-1 would therefore lead to a reduction in epithelial cell proliferation.

An alternative explanation depends on the increase in serum factors released into the tissue due to the increase in capillary permeability. Solid epithelial tumors lacking adequate access to a blood supply will grow only until passive diffusion can no longer provide adequate nutrients (51). Increased microvascular density in solid tumors is a poor prognostic indicator (52, 53, 54). Tumor vessels are leaky, which is thought to be a reflection of the increased levels of VEGF-A present in the tumor, and may provide easy access for tumor cells to circulating nutrients, including serum factors (55). Serum is a rich source of growth factors; it is well established that cells are functionally more active when cultured in vitro in the presence of serum (56, 57, 58). Likewise, by inducing vascular permeability in endometrial blood vessels with E2 we may enhance the availability of nutrients and serum factors to the uterine epithelium, thereby promoting their proliferation. Treatment with sflt-1 would reduce the level of epithelial cell proliferation by blocking vascular permeability and, therefore, nutrient and serum factor availability. It is likely that in our system sflt-1 inhibits the proliferation of lumenal epithelial cells by both of these mechanisms.

Antibodies against VEGF-A block VEGF-A-induced vascular permeability in the Miles assay, reduce the number of implantation sites in the rodent uterus, and reduce VEGF-A-induced cerebral edema (22, 35, 37). We now show that sflt-1, an antagonist of VEGF-A, reduces E2-induced edema and lumenal epithelial cell proliferation in the mouse uterus.

In conclusion, our data demonstrate that the E2-mediated increase in size of the rodent uterus is due in part to the actions of VEGF-A. The increase in uterine size is a consequence of increased edema, which is secondary to an increase in vascular permeability. These changes are necessary for the increase in lumenal epithelial cell proliferation. Potential applications of anti-VEGF agents, such as sflt-1, include the control of early stage solid tumors and vascular permeability-dependent conditions, such as endometriosis and implantation.


   Footnotes
 
This work was supported by the Medical Research Council (Program Grant G9623012 awarded to S.K.S. and D.S.C.-J. and Cooperative Grant awarded to S.K.S., D.S.C.-J., and G.J.B.).

1 D.S.C.-J. and S.K.S. contributed equally to this work. Back

Abbreviations: BrdU, 3,5-Bromodeoxyuridine; BS1, Bandiera simplicofolia agglutinin-1; CL, confidence limits; E2, 17ß-estradiol; Ig, immunoglobulin; sflt-1, spliced form of flt-1; VEGF-A, vascular endothelial growth factor A; VEGF-R1, vascular endothelial growth factor receptor type 1.

Received June 20, 2002.

Accepted for publication September 12, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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