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. 2008 Aug 7;149(12):6076–6083. doi: 10.1210/en.2008-0521

Vascular Endothelial Growth Factor Mediates the Estrogen-Induced Breakdown of Tight Junctions between and Increase in Proliferation of Microvessel Endothelial Cells in the Baboon Endometrium

Graham W Aberdeen 1, Stanley J Wiegand 1, Thomas W Bonagura Jr 1, Gerald J Pepe 1, Eugene D Albrecht 1
PMCID: PMC2613057  PMID: 18687775

Abstract

To assess whether there is a link between estrogen, vascular endothelial growth factor (VEGF), and early aspects of uterine angiogenesis, an acute temporal study was conducted in which ovariectomized baboons were pretreated with VEGF Trap, which sequesters endogenous VEGF, and administered estradiol at time 0 h. Serum estradiol levels approximated 500 pg/ml 4–6 h after estradiol administration. VEGF mRNA levels in endometrial glandular epithelial and stromal cells were increased to values 6 h after estradiol that were 3.74 ± 0.99-fold (mean ± se) and 5.70 ± 1.60-fold greater (P < 0.05), respectively, than at 0 h. Microvessel interendothelial cell tight junctions, which control paracellular permeability, were present in the endometrium at time 0 h, but not evident 6 h after estradiol administration. Thus, microvessel paracellular cleft width increased (P < 0.01, ANOVA) from 5.03 ± 0.22 nm at 0 h to 7.27 ± 0.48 nm 6 h after estrogen. In contrast, tight junctions remained intact, and paracellular cleft widths were unaltered in estradiol/VEGF Trap and vehicle-treated animals. Endometrial microvessel endothelial cell mitosis, i.e. percent Ki67+/Ki67− immunolabeled endothelial cells, increased (P < 0.05) from 2.9 ± 0.3% at 0 h to 21.4 ± 7.0% 6 h after estrogen treatment but was unchanged in estradiol/VEGF Trap and vehicle-treated animals. In summary, the estrogen-induced disruption of endometrial microvessel endothelial tight junctions and increase in endothelial cell proliferation were prevented by VEGF Trap. Therefore, we propose that VEGF mediates the estrogen-induced increase in microvessel permeability and endothelial cell proliferation as early steps in angiogenesis in the primate endometrium.

A NEW VASCULAR network develops via angiogenesis within the uterine endometrium during each menstrual cycle to support growth and differentiation for implantation. Ovarian estrogen has a pivotal role in establishing the endometrial vascular bed (for review, see Refs. 1,2,3), however, relatively little is known about the mechanisms by which estrogen regulates this fundamentally important process.

Increased microvessel permeability and endothelial cell proliferation are essential early events in angiogenesis. Enhanced vessel permeability results in the extravasation of proteins such as fibrin that serve as a substratum for endothelial cell migration and assembly into new microvessels (4,5). Tight junctions are fusions of the outer leaflets of plasma membranes between adjacent endothelial cells that act as intercellular seals by which microvessel paracellular permeability is controlled (6,7). Vascular endothelial growth factor (VEGF) has a central role in stimulating microvascular endothelial cell permeability (4), proliferation, migration, and assembly into capillary tubes (8). Moreover, estrogen is a potent stimulus of endometrial vascular permeability/edema (9,10,11).

We (12,13,14) and others (15) have shown that endometrial VEGF mRNA levels and the microvessel network within the baboon and rhesus monkey endometrium were decreased by ovariectomy and restored by chronic estrogen, but not progesterone, administration. Moreover, acute estradiol administration to ovariectomized baboons elevated endometrial VEGF mRNA expression within 2 h and disrupted the tight junctions between adjacent endometrial microvascular endothelial cells within 6 h (14). In addition, estradiol stimulated endothelial cell proliferation and microvessel density in the human (16), rhesus monkey (15), sheep (17), and mouse (18) endometrium. Therefore, we propose that the rapid estradiol-induced up-regulation of VEGF expression in the primate uterus mediates the well-established early action of estrogen on uterine microvascular permeability and endothelial cell proliferation. To assess this possibility in the present study, we determined the temporal relationship between a rapid surge in estrogen levels and expression of VEGF, microvessel tight junction morphology, and endothelial cell mitosis in the endometrium of baboons after administration of estradiol and VEGF Trap, a recombinant protein that selectively sequesters and, thus, suppresses the bioavailability of endogenous VEGF.

Materials and Methods

Animals

Adult female baboons (Papio anubis), originally obtained from the Southwest Foundation for Biomedical Research (San Antonio, TX), mean ± se age of 11.1 ± 1.3 yr (which was similar for each randomly assigned treatment group), exhibiting regular menstrual cycles and weighing 12–15 kg were used in this study. Baboons were housed individually in large primate cages in air-conditioned rooms, 12-h light, 12-h dark cycle, and received primate chow (Teklad-Harlan, St. Louis, MO) and fresh fruit twice daily and water ad libitum. Baboons were cared for and used strictly in accordance with U.S. Department of Agriculture regulations and the Guide for the Care and Use of Laboratory Animals prepared by the National Research Council (National Academy Press, 1996). The experimental protocols used in the present study were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.

Female baboons were anesthetized with isoflurane, bilaterally ovariectomized to remove the principal source of estrogen and progesterone, and then left for at least 60 d before being used for acute temporal study. During the 5 d immediately preceding the study, ovariectomized baboons were injected sc daily with the highly specific aromatase inhibitor letrozole (4,4′-[1,2,3-triazyol-1-yl-methylene]-bis-benzonitrite; Novartis Pharma AG, Basel, Switzerland) at a dosage of 0.5 mg/0.25 cc sesame oil to suppress potential aromatization in nonovarian sites. At −24 h of the temporal study, baboons received VEGF Trap (supplied by Regeneron Pharmaceuticals, Inc., Tarrytown, NY), a Chinese hamster ovary-derived soluble fusion protein consisting of portions of the ligand binding domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1 (19). VEGF Trap binds VEGF-A, VEGF-B, and placenta growth factor with high affinity, preventing binding of VEGF to its endogenous receptors. VEGF Trap (1 mg/kg body weight) or hFc protein was administered via an antecubital vein over a 2-min period in sterile buffer [5 mm phosphate, 5 mm citrate, 50 mm sodium chloride, 5% sucrose, and 0.1% polysorbate 20 (pH 6.0)]. At 0800 h on the following day, baboons were anesthetized with isoflurane, placed in a supine position on a 37 C warming pad on a surgical table, and administered 5% dextrose (25 ml/h) via a 21-gauge catheter (Intracath, 19 gauge, 24 in.; Becton Dickinson Vascular Access, Sandy, UT) inserted into a peripheral saphenous vein. A midline 5- to 6-cm abdominal incision was then made to expose the uterus for subsequent biopsy, and at time 0 h of the temporal study, VEGF Trap (n = 4) or hFc protein pretreated baboons were administered either estradiol [a bolus of 1.0 μg/kg body weight 17β-estradiol (Sigma-Aldrich Corp., St. Louis, MO) in 0.5 ml ethanol-normal saline delivered via a 23-gauge needle into an antecubital vein plus three SILASTIC brand implants (Dow Corning, Midland, MI) sc, 5-mm outside diameter, 6-cm length, containing 17β-estradiol] to elicit a rapid surge and sustained release of hormone (n = 10) or ethanol-saline-hFc vehicle (n = 8). Because the results for tight junction and paracellular cleft parameters were similar in baboons treated with estradiol and estradiol plus hFc protein, these data were combined for purposes of presentation. At 0 h, all animals also received an injection of Evans blue (2 mg/kg body weight, in 1.0 ml saline) via a 25-gauge needle into each internal iliac artery to assess vascular permeability.

Blood samples (2 ml) were obtained via the peripheral saphenous vein catheter periodically during the study period for determination of serum estradiol concentrations by RIA (20) and VEGF Trap levels by ELISA (21).

Single 5-mm diameter core biopsies (Acu-Punch; Acuderm, Inc., Ft. Lauderdale, FL) were obtained from the uterine fundus, alternating from anterior and posterior surfaces, extending transmurally from outer surface to lumen at 0, 2, 4, and 6 h. Gelatin sponge (Gelfoam; Pharmacia Corp. and Upjohn Co., Kalamazoo, MI) and single 5–0 chromic suture (Ethicon, Inc., Sommerville, NJ) were applied after each biopsy to close the excision. After the last endometrial sample was removed, the baboons were euthanized with sodium pentobarbital (Euthasol, 100 mg/kg body weight, iv; Virbac AH, Inc., Fort Worth, TX). The tissue samples were sectioned longitudinally into quarters, and one section was embedded in a Cryomold filled with optimal cutting temperature (OCT) medium (Sakura Finetek USA, Inc., Torrance, CA), frozen on dry ice, and stored at −80 C for subsequent VEGF mRNA analysis in glandular epithelial and stromal cells isolated by laser capture microdissection (LCM). In another quarter of uterine specimen, the myometrium was dissected free under a dissecting microscope, and the endometrium containing both functionalis and basalis zones was prepared for electron microscopical analysis of endothelial tight junctions and paracellular clefts. Another tissue section was fixed in 10% PBS buffered formalin for 24 h and embedded in paraffin for immunocytochemistry, whereas the endometrium from the last tissue quarter was dissected free, weighed, and extracted with formamide and analyzed for Evans blue content by spectrophotometry.

LCM of endometrial cells

Glandular epithelial and stromal cells were isolated from the endometrium by LCM as described previously (12). Briefly, serial 8-μm sections of the uterine biopsy were cut longitudinally (to include endometrium and myometrium) on a Jung Frigocut 2800E cryostat at −20 C (Leica Corp., Deerfield, IL) and mounted onto glass slides. Sections were immediately fixed in ethanol, stained with eosin, and washed in xylene. Slides were air-dried and transferred to a desiccator at room temperature, and an Arcturus PixCell II LCM system equipped with an Olympus Corp. microscope (Arcturus Engineering, Inc., Mountain View, CA) was then used to capture glandular (but not luminal) epithelial and stromal (but not observable blood vessels) cells randomly from both the basalis and functionalis zones of the endometrium. Optimal conditions for cell isolation included a laser power of 40 mW and duration of 1.5–2.5 msec, and laser spot size of 7.5 or 15 μm for glandular epithelium (depending on gland size) and 15 or 30 μm for stroma. Captured cells were then mixed with lysis buffer (Rneasy; QIAGEN, Inc., Valencia, CA), microcentrifuged, stored in lysate buffer overnight at −80 C, and RNA extracted within 72 h. We have previously shown (12,13) that a homogeneous population of cytokeratin-positive glandular epithelial and vimentin-positive stromal cells are isolated by LCM from the endometrium of the baboon uterus.

RT-PCR of VEGF mRNA

VEGF primers.

Oligonucleotide primers were designed using LightCycler probe design software (Roche Diagnostics Corp., Penzberg, Germany), based on the VEGF human gene sequence, and supplied by TIB MOLBIOL (Adelphia, NJ). The VEGF primers [primer 1: upstream, 5′-GCATTGGAGCCTTGCCTT-3′ (position 24–41); and primer 2: downstream, 5′-GCCTTGGTGAGGTTTGAT-3′ (position 342–325)] spanned exons 1–3 of the VEGF gene, and were upstream of the alternative splice site that generates the different 121, 145, 165, 189, and 206 isoforms of VEGF-A. Thus, a single 323-bp PCR product reflecting all VEGF-A isoforms was generated.

18S rRNA primers.

Oligonucleotide 18S rRNA primers were based on the human gene sequence (National Center for Biotechnology Information sequence database, accession no. M10098). LightCycler probe design was used to create the primers: primer 3, upstream, 5′-TCAAGAACGAAAGTCGGAGG-3′ (positions 1126–1145); and primer 4, downstream, 5′-GGACATCTAAGGGCATCACA-3′ (positions 1614–1595).

RT and real-time PCR.

Reverse transcription (RT) of total RNA from LCM isolates was performed according to the manufacturer’s directions (Invitrogen Corp., Carlsbad, CA). A 13-μl reaction volume containing 1 mm each of deoxy-ATP, deoxy-CTP, deoxy-GTP, and deoxy-TTP, 1× RT buffer, 250 ng random primers, and total RNA was incubated at 65 C for 5 min, followed by incubation on ice for 1 min. Reaction buffer, 200 U Superscript III RT (Invitrogen) and 40 U RNAguard (Amersham Pharmacia Biotech, Piscataway, NJ) were added to create a final 20-μl reaction volume that was incubated at 25 C for 5 min, followed by incubation at 50 C for 60 min. The RT reaction was terminated by heat inactivation of the enzyme at 70 C for 15 min and cooled to 4 C.

VEGF mRNA levels were quantified in a LightCycler real-time PCR unit (Roche Diagnostics) using the Fast Start DNA Master SYBR Green I Kit for the PCR. A 19 μl-reaction mix containing either VEGF- or 18S rRNA-specific primers was prepared according to the manufacturer’s protocol, and a 1-μl aliquot of RT reaction was added for a final volume of 20 μl. The reaction profile consisted of denaturation at 95 C for 8 min, 40 cycles of amplification (95 C for 5 sec, 52 C for 5 sec, and 72 C for 16 sec) and product formation measured and displayed in real time. Due to the limited amount of total RNA collected by LCM, the levels of 18 S rRNA were quantified by real-time PCR to serve as the reference gene. Efficiency corrected calibrator-normalized relative quantification was performed with analysis software (LightCycler version 4) using the concentration and efficiency of premade standard curves specific for each product to correct for differences in the efficiencies of target and reference genes, and an in-run calibrator to normalize quantification. We have confirmed that RNA obtained from LCM isolated cells was intact within the region spanned by the VEGF primers, and exhibited distinct 28S and 18S rRNA bands (12). Moreover, VEGF mRNA and 18 S rRNA analysis by real-time PCR confirmed similar melting peaks for VEGF mRNA from cells isolated by LCM from the baboon endometrium, and the calibrator and the absence of PCR product when template or RT was deleted from the reaction mix (data not shown).

Electron microscopical analysis of endothelial cell tight junctions and paracellular clefts

Microvessel endothelial cell tight junctions and paracellular clefts were analyzed by electron microscopy, essentially as previously described in our laboratory with modifications (14). In brief, endometrial biopsies were dissected into 1 mm3 pieces and fixed for 24 h in phosphate-buffered 4% formaldehyde/1% glutaraldehyde. After a wash with sucrose buffer, endometrial fragments were incubated in 1% osmium tetroxide for 1 h, dehydrated/rehydrated through a series of graded alcohols, and embedded in Epon resin (Poly/Bed 812; Polysciences, Inc., Warrington, PA). Thick sections (1 μm) were cut, counterstained with hematoxylin, and visualized at ×100 and ×200 magnifications to identify vascularized areas of endometrium. Thin sections (80 nm) of identified vascular areas were cut and placed onto copper grids, stained with uranyl acetate and lead citrate, and viewed with an electron microscope (Joel JEM-1200EX; Tokyo, Japan) under low-power resolution (3, 6, 10, and 12 K) for identification of intact microvessels.

The presence/absence of tight junctions and paracellular cleft widths between adjacent microvessel endothelial cells were determined on eight to 12 randomly chosen microvessels, i.e. comprised of only one to four intact endothelial cells, from each endometrial sample. Only junctional membranes that were sectioned perpendicularly and within the same section plane were used for analysis. Paracellular cleft tight junctions were defined as obvious bridging points at the narrowest point of membrane apposition between adjacent microvessel endothelial cells. A minimum of four to eight electron micrographs at 60 K was taken of each endometrial microvessel with identifiable paracellular cleft tight junctions as the focal point of each photomicrograph. Final electron photomicrograph magnification approximated ×178,200 when corrected for calibration and negative to print enlargement.

Paracellular cleft widths were measured manually with a digital slide rule (mm, decimal - hundredths) from the 60-K electron micrographs “blindly” by the same individual without prior knowledge of treatment group or sample time points. Final paracellular cleft width (nm) = width (mm) × conversion factor (mm to nm @ 60 K; 10.664)/negative to print magnification factor (2.71). The intraassay (i.e. between vessel, within animal) coefficient of variation for cleft width was less than 12.3%, indicating that endothelial cell morphometric analysis was consistent across different microvessels in the endometrium.

Microvascular permeability

The Evans blue indicator diffusion method (22) was used to assess endometrial vascular permeability in vivo in baboons. Evans blue irreversibly binds electrostatically to plasma albumin in a 10:1 molar ratio, and this complex extravasates into surrounding tissues whenever there is increased vascular permeability. Evans blue was extracted from endometrial tissue samples in formamide for 18 h at 4 C, and the extract centrifuged at 70,000 rpm for 45 min at 4 C. Endometrial supernatant (60 μl in duplicate) was diluted in formamide, absorbance at 620 and 740 nm measured spectrophotometrically, and concentration of dye in extracts calculated from a standard curve of Evans blue. The permeability index was calculated as Evans blue absorbance/tissue wet weight (μg). (Fisher Scientific, Pittsburgh, PA.)

Ki67 and von Willebrand factor immunocytochemistry

Paraffin blocks were cut (7 μm) and adjacent sections placed onto separate Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA), deparaffinized with xylene, rehydrated in graded concentrations of ethanol, and incubated for 10 min in 0.3% hydrogen peroxide for 10 min to block endogenous peroxide. Antigen retrieval involved boiling in 10 mm Na citrate for 5 min, cooling for 1 h, washing with potassium PBS (KPBS) for 5 min, followed by incubation with 5% normal goat serum in KPBS for 30 min to block nonspecific binding. Tissue sections were incubated overnight at 4 C with primary antibodies to Ki67 (monoclonal mouse antihuman, 1:250 dilution; DakoCytomation, Carpinteria, CA) and von Willebrand factor (rabbit antihuman, 1:1000 dilution; DakoCytomation). Sections were then incubated for 1 h at room temperature with biotinylated secondary Igs (Vector Laboratories, Burlingame, CA) for Ki67 [antimouse IgG (H+L), 1:200 dilution] and von Willebrand factor [antirabbit IgG (H+L), 1:200 dilution], washed twice in KPBS, immersed for 45 min in avidin-biotin-peroxidase complex (ABC Elite; Vector Laboratories), and developed using diaminobenzidine as the chromagen. Tissues were then counterstained for 30 sec with diluted (1:5) Harris hematoxylin, washed twice with running water, dehydrated with ethanol, washed with xylene (5 min), and coverslipped with Permount and allowed to dry overnight. Quality control for Ki67 and von Willebrand immunocytochemistry included substitution of nonimmune normal horse serum for the primary antibodies, utilization of isotype-matched secondary Ig IgM, and titration of primary antibodies to concentrations beyond detectable staining.

Endothelial cell proliferation

Adjacent endometrial tissue sections containing both functionalis and basalis regions were analyzed and quantified using image analysis to identify immuno-von Willebrand factor-positive and Ki67-positive endothelial cells. Only von Willebrand-positive staining endothelial cells were counted and analyzed from microvessels, i.e. capillaries, arterioles, and venules. Capillaries and venules were discriminated from each other under high-power light microscopy through the assessment of vessel lumen size and absence (capillaries) or presence (venules/arterioles) of pericytes fused with the basement membrane of endothelial cells. Capillaries consisted solely of a single layer of endothelial cells with a lumen size not much greater than the diameter of a red blood cell. Large vessels, e.g. having several layers of smooth muscle, and Ki67-positive glandular and luminal epithelial cells were excluded in the analysis of endothelial cell proliferation. The entire endometrium from a total of three to six representative pairs of adjacent tissue sections from each time point was evaluated “blindly” without prior knowledge of treatment groups or specified time points. The number of positive-staining cells counted per histological specimen ranged from two to 21 cells for Ki67 and 85–415 cells for von Willebrand factor. The percentage of Ki67-positive endothelial cells was calculated for each respective time point by the number of Ki67-positive endothelial cells divided by the number of von Willebrand factor-positive cells (i.e. total number of “microvessel” endothelial cells).

Statistical analysis

Baboons were randomly assigned to the treatment groups, and data expressed as the means ± se. The data displayed a normal gaussian distribution. Therefore, statistical analysis of the data was performed by ANOVA with post hoc comparisons of the means by Student-Newman-Keuls or Dunnett multiple comparison tests using statistical analysis software (SAS Institute Inc., Cary, NC).

Results

Serum estradiol and VEGF Trap levels

Serum estradiol levels were elevated to over 1500 pg/ml within 15 min estradiol treatment, then rapidly declined and plateaued after 4–6 h to approximately 500 pg/ml (Fig. 1), a level approximating that of the late proliferative phase of the normal baboon menstrual cycle (23). In baboons simultaneously treated with estradiol and VEGF Trap, the pattern and levels of serum estradiol were similar to those observed after estradiol treatment alone (Fig. 1). In contrast, serum estradiol remained at baseline levels, i.e. less than 10 pg/ml, in saline/hFc vehicle-treated animals.

Figure 1.

Figure 1

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Serum estradiol levels (means ± se) in ovariectomized baboons after administration of estradiol (bolus iv injection at 1 μg/kg body weight and sc insertion of three SILASTIC brand capsules containing estradiol) at time 0 h (n = 10), VEGF Trap (bolus iv injection at 1 mg/kg body weight) at −24 h plus estradiol at 0 h (n = 4), or saline/hFc vehicle at 0 h (n = 8).

Serum VEGF Trap levels reached 16.6 ± 3.6 and 15.2 ± 4.3 μg/ml 24 and 30 h (i.e. at times 0 and 6 h of temporal study), respectively, after VEGF Trap administration. Serum VEGF Trap levels then declined to 7.9 ± 1.1 μg/ml 72 h (i.e. +48 h of temporal study) after VEGF Trap treatment. Physiological parameters remained constant in baboons at 0 and 6 h of the acute experimental treatment period, including mean arterial blood pressure (71.7 ± 2.4 and 67.8 ± 9.1 mm, respectively) and blood oxygen saturation levels (95.2 ± 2.8 and 94.5 ± 0.7%, respectively).

VEGF mRNA

VEGF mRNA levels (mean ± se, corrected for 18 S rRNA) at time 0 h in glandular epithelial (0.40 ± 0.07) and stromal (0.29 ± 0.04) cells were rapidly increased within 2 h estradiol administration, reaching values at 6 h that were 3.74 ± 0.99-fold and 5.70 ± 1.60-fold greater (P < 0.05), respectively, than at time 0 h (Fig. 2). Endometrial VEGF mRNA levels also were increased (P < 0.02) in baboons 6 h after the administration of estradiol and VEGF Trap (data not shown), but not after administration of saline vehicle as shown in our previous study (14).

Figure 2.

Figure 2

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Endometrial VEGF mRNA levels (fold increase vs. 0 h, means ± se) determined by real-time RT-PCR in glandular epithelial and stromal cells isolated by LCM from ovariectomized baboons treated with estradiol as detailed in the legend of Fig. 1. *, Significantly different (P < 0.05, ANOVA and Dunnett test) vs. value at 0 h.

Microvessel tight junctions and paracellular clefts

Microvessel interendothelial cell tight junctions were present in endometrial tissue obtained at time 0 h from baboons untreated (Fig. 3A) or treated 24 h previously with VEGF Trap (Fig. 3C). However, 6 h after the administration of estradiol, tight junctions were either not observed or partially broken down between microvessel endothelial cells (Fig. 3B). Consequently, mean (±se) microvessel paracellular cleft width increased (P < 0.01) from 5.03 ± 0.20 nm at time 0 h, i.e. immediately before estradiol treatment, to 7.27 ± 0.48 nm 6 h after estradiol administration. In contrast, microvessel tight junctions remained intact (Fig. 3D), and microvessel paracellular width remained similar in value at times 0 and 6 h in estradiol/VEGF Trap-treated (4.85 ± 0.29 nm and 5.04 ± 0.30 nm, respectively) and saline/hFc vehicle-treated (4.91 ± 0.17 nm and 5.07 ± 0.19 nm, respectively) animals. Thus, the net increase in paracellular cleft width between 0 and 6 h was substantially greater (P < 0.001) in estradiol-treated (2.23 ± 0.54) than in estradiol/VEGF Trap (0.19 ± 0.12) or saline/hFc vehicle (0.13 ± 0.07) treated baboons (Fig. 4).

Figure 3.

Figure 3

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Electron photomicrographs illustrating endometrial tight junctions (arrowheads) and paracellular clefts between adjacent microvessel endothelial cells of baboons 0 h (A and C) and 6 h (B and D) after administration of estradiol (A and B) or estradiol plus VEGF Trap (C and D). Scale bar, 20 nm.

Figure 4.

Figure 4

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Net gain (means ± se) in paracellular cleft width (nm) between adjacent endometrial microvessel endothelial cells of ovariectomized baboons before (0 h) and 6 h after acute administration of estradiol (E2) (n = 10), estradiol plus VEGF Trap (n = 4), or saline/hFc vehicle (n = 8). *, Different at P < 0.001 (ANOVA and Newman-Keuls test) vs. other treatment groups.

Vascular permeability

Vascular permeability (i.e. Evans blue absorbance/tissue weight; mean ± se value for 2 and 6 h) in estradiol-treated baboons was 14.9 ± 8.0. In contrast, vascular permeability remained essentially at baseline levels in animals treated with both estradiol and VEGF Trap (1.9 ± 0.8) or saline/hFc vehicle (3.6 ± 1.5).

Microvessel endothelial cell proliferation

Ki67 immunolabeling (Fig. 5A) of von Willebrand factor positive (Fig. 5B) endometrial microvessel endothelial cells was negligible at 0 h in ovariectomized baboons. However, 6 h after the administration of estradiol, many of the endothelial cells lining blood vessel walls exhibited nuclear Ki67 immunostaining (Fig. 5C). In contrast, Ki67 immunostaining remained sparse throughout the endometrium of animals concomitantly treated with estradiol and VEGF Trap (Fig. 5E). The level of endometrial microvessel mitosis, expressed as the percentage of Ki67+/Ki67− cells (Fig. 6), was over 7-fold greater (P < 0.05) 6 h after estradiol administration (21.4 ± 7.0) than at 0 h (2.9 ± 0.3). In contrast, Ki67 immunolabeling of microvessel endothelial cells was similar at 0 and 6 h in estradiol/VEGF Trap (2.6 ± 0.3 and 3.8 ± 0.8, respectively) or saline/hFc vehicle (3.9 ± 1.5 and 5.0 ± 1.1, respectively) treated baboons (Fig. 6).

Figure 5.

Figure 5

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Ki67 and von Willebrand factor (vWF) immunocytochemical labeling of endometrial microvessel endothelial cells in baboons at 0 h (A and B) and 6 h after the administration of estradiol (E2) (C and D) or estradiol plus VEGF Trap (E and F). G and H, Isotype-matched secondary Ig IgM controls. Magnification, ×400.

Figure 6.

Figure 6

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Proliferation of endometrial microvessel endothelial cells, expressed as percentage of Ki67+/Ki67 − cells, in baboons 0, 2, and 6 h after administration of estradiol (E2) (n = 5), estradiol plus VEGF Trap (n = 3), or saline/hFc vehicle (n = 5). *, Different (P < 0.05, ANOVA and Newman-Keuls test) from time 0 h.

Discussion

The present study shows that endometrial microvessel tight junctions between endothelial cells were disrupted, vascular permeability was increased, and endothelial cell proliferation was elevated by acute administration of estradiol to ovariectomized baboons. Most significantly, the estrogen-induced breakdown of paracellular tight junctions and increase in permeability and endothelial cell mitosis were prevented by concomitant administration of estrogen and VEGF Trap, a receptor-based protein that sequesters and, thus, controls the bioavailability of endogenous VEGF. Therefore, we conclude that VEGF mediates the estrogen-induced increase in microvessel endothelial permeability and proliferation in the primate endometrium. We believe this is the first study to link estrogen, VEGF, and microvessel angiogenesis in the primate uterus. The results shown for VEGF in the baboon are consistent with in vivo studies that showed that VEGF antibody administration blocked estradiol-induced uterine edema in the rat (24), and VEGF receptor antagonists inhibited estradiol-induced endometrial endothelial cell proliferation in the mouse (18).

VEGF Trap has also been shown in vivo to block angiogenesis and follicular and luteal development in the marmoset and macaque ovary (21,25). Therefore, the latter and the present studies demonstrate the feasibility of using VEGF Trap in vivo in the nonhuman primate to investigate the mechanisms underlying the action of estrogen on angiogenesis in the uterus and ovary, one of the few sites other than during wound healing where angiogenesis occurs in the adult. Furthermore, we suggest that the results obtained in the baboon are extrapolatable to the human and indicate the translational value of this nonhuman primate model.

Although the present study was conducted acutely in ovariectomized baboons, we suggest that the VEGF-mediated estradiol-induced changes in microvessel endothelial cell function are applicable to the physiological regulation of endometrial angiogenesis during the normal menstrual cycle. Thus, chronic estradiol administration to ovariectomized rhesus monkeys (15) and baboons (13), which yielded serum levels that approximated the proliferative phase of the normal menstrual cycle, also elevated endometrial VEGF expression and vascular endothelial cell proliferation. Moreover, along with the normal increase in ovarian production and serum levels of estradiol, endometrial VEGF expression is elevated during the baboon (12,13) and human (26,27) menstrual cycle. Glandular epithelial cells are clearly a major source of VEGF in the endometrium as shown in the present and prior studies (12,15,26,28), whereas lymphocytes present in the endometrial stroma express (29) and, thus, are a possible source of the VEGF observed in stromal tissue isolated from estradiol-treated baboons. Both estradiol and VEGF are highly potent stimuli of endometrial edema and vascular permeability (8,9,11,30) and estradiol stimulated endometrial microvascular volume density in vivo in sheep (17). Moreover, conditioned medium from human endometrial glandular epithelial cells incubated with estradiol stimulated microvessel endothelial cell tube formation (31). Collectively, the results of the latter studies and those obtained with concurrent treatment of baboons with estradiol and VEGF Trap are consistent with the concept that VEGF modulates fundamentally important aspects of endometrial angiogenesis elicited by estrogen during the normal menstrual cycle.

The present study shows that an increase in endometrial endothelial cell immunoreactivity of Ki67, a protein present during and possibly important to progression through all active phases of the cell cycle (32), occurred 6 h after the administration of estradiol to ovariectomized baboons. Estrogen also induces a rapid increase within 2–4 h in expression of the cell cycle regulators cyclin A and D in the endometrium (33). Bromodeoxyuridine labeling of endometrial endothelial cells was also markedly up-regulated 24 h after administration of estradiol to ovariectomized mice (18), however, earlier times were not assessed. Although potential early effects of estrogen on endometrial endothelial cell proliferation apparently have also not been examined in other nonhuman primates, endometrial endothelial cell Ki67 immunolabeling was significantly increased in ovariectomized rhesus monkeys treated for several days with estradiol and/or progesterone to artificially induce proliferative and secretory phases of the menstrual cycle but not in hormone-deprived monkeys (15). Based on the results of the present and prior studies, it appears that Ki67, as well as the cell cycle regulator cyclins, provide a rapid index of estrogen-stimulated endothelial cell proliferation in the endometrium.

Microvessel tight junction assembly involves the expression, phosphorylation, interaction, and subcellular redistribution of key structural and signaling proteins, notably occludin, claudin, zonula occludens, and junctional adhesion molecules (6,34). As a result of the latter changes, the extracellular domains of occludin and claudin become organized into transmembrane loops or fibrils that form a tight junction paracellular seal between adjacent microvessel endothelial cells. In vitro studies with human brain microvessel endothelial cells have shown that VEGF suppressed expression, phosphorylation, interaction, and spatial organization of occludin and zonula occludens, which led to tight junction dissolution and increased microvessel endothelial cell permeability (34,35). Thus, it appears that occludin and zonula occludens are downstream effectors of the VEGF signaling pathway involved in microvessel permeability. Considering the latter aspects of tight junction biology and the results of the current study, we propose that the disintegration/loss of tight junctions between endometrial microvessel cells of estrogen-treated baboons resulted from VEGF-induced changes in the tight junction assembly proteins. The estradiol/VEGF Trap-treated baboon provides an excellent experimental model to assess this possibility in future study.

Vascular endothelial cells express estrogen receptor β (36,37,38) and, thus, have the capacity to respond directly to estrogen action (39). For example, estradiol promoted proliferation of (40), down-regulated expression of the tight junction protein occludin by, and increased paracellular permeability of human umbilical vein endothelial cells in culture (41). In contrast, conditioned medium from human endometrial glandular epithelial cells incubated with estradiol, but not estradiol added directly, stimulated human uterine microvessel tube formation/angiogenesis (31). Moreover, the present study clearly shows that estradiol enhanced endometrial glandular epithelial and stromal cell VEGF expression, and that the estrogen-induced loss of endometrial microvessel tight junctions and increase in microvessel endothelial cell proliferation were prevented by pretreatment of baboons with VEGF Trap, indicating that VEGF mediated these processes. Therefore, although we recognize the potential direct effects of estrogen on microvessel endothelial cell function, we propose based on our present and recent studies that estrogen indirectly promotes microvessel endothelial cell permeability and proliferation by up-regulating endometrial glandular epithelial and stromal VEGF formation.

VEGF also increases transendothelial cell transport, i.e. across rather than between cells, via caveoli and fenestrations (42,43,44,45), which in addition to paracellular tight junctions, potentially may control permeability and, thus, endothelial cell migration and angiogenesis. It is not known whether estrogen controls transendothelial as well as paracellular transfer in the endometrium. However, we propose based on the results of the current study that estrogen regulates intercellular tight junction breakdown and, thus, permeability as pivotal steps in vessel development in the uterus and that VEGF mediates this process.

In summary, estradiol disrupted tight junctions between microvessel endothelial cells, and increased vascular permeability and endothelial cell proliferation in the endometrium of the baboon, and pretreatment with VEGF Trap prevented these effects. Therefore, we conclude that VEGF mediates the estrogen-induced increase in microvessel endothelial cell permeability and proliferation as early steps in angiogenesis in the primate endometrium.

Acknowledgments

We thank Regeneron Pharmaceuticals, Inc., for the vascular endothelial growth factor Trap. The secretarial assistance of Wanda James with the manuscript is sincerely appreciated.

Footnotes

This work was supported by National Institute of Child Health and Human Development/National Institutes of Health through cooperative agreement U54 HD-36207 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 7, 2008

Abbreviations: LCM, Laser capture microdissection; RT, reverse transcription; VEGF, vascular endothelial growth factor.

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