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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 11 5803-5809
Copyright © 2004 by The Endocrine Society

Regulation of Placental Vascular Endothelial Growth/Permeability Factor Expression and Angiogenesis by Estrogen during Early Baboon Pregnancy

Eugene D. Albrecht, Victoria A. Robb and Gerald J. Pepe

Departments of Obstetrics, Gynecology, Reproductive Sciences, and Physiology, Center for Studies in Reproduction, University of Maryland School of Medicine (E.D.A., V.A.R.), Baltimore, Maryland 21201; and Department of Physiological Sciences, Eastern Virginia Medical School (G.J.P.), Norfolk, Virginia 23507

Address all correspondence and requests for reprints to: Dr. Eugene D. Albrecht, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Bressler Research Laboratories 11-019, 655 West Baltimore Street, Baltimore, Maryland 21201. E-mail: ealbrech{at}umaryland.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have recently shown that there was a developmental increase in placental trophoblast vascular endothelial growth/permeability factor (VEG/PF) expression and vascularization that closely paralleled maternal serum estrogen levels during advancing baboon gestation. The present study determined whether estrogen regulates these important aspects of primate development. VEG/PF mRNA levels were determined by competitive RT-PCR in isolated villous placental cells, and placental vascularization was assessed by image analysis. Placentas were obtained on d 60 of gestation (length of gestation is 184 d) from baboons in which estrogen levels on d 25–59 were increased by daily administration of aromatizable androstenedione or decreased by aromatase inhibitor CGS 20267. Androstenedione treatment increased maternal serum estradiol levels 3-fold (P < 0.01) and placental villous cytotrophoblast VEG/PF mRNA level to a value (mean ± SE, 26,836 ± 5,625 attomoles/µg total RNA) 2.5-fold greater (P < 0.05) than that in untreated animals (11,645 ± 1,746 attomoles/µg RNA). In contrast, administration of CGS 20267 decreased serum estradiol (P < 0.01) and placental cytotrophoblast mRNA (2,912 ± 693 attomoles/µg RNA; P < 0.05) levels by 75%, effects prevented by concomitant administration of CGS 20267 and estradiol. VEG/PF mRNA levels in inner villous cells were unaltered. Coinciding with the increase in placental VEG/PF expression, the percent vascularized area (3.46 ± 0.23) and vessel density (493 ± 34 vessels/mm2) of the villous placenta in untreated baboons on d 60 were increased (P < 0.01) in baboons in which estrogen levels were elevated by androstenedione treatment (6.54 ± 0.56 and 743 ± 27 vessels/mm2, respectively). It is concluded that estrogen has an important role in stimulating trophoblast VEG/PF expression and consequently villous placental angiogenesis to promote fetal growth and development in early primate pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A NEW VASCULAR network develops via angiogenesis within the villous placenta during human and nonhuman primate pregnancy to advance placental, and thus fetal, growth and development (see Refs.1 and 2 for reviews). Vascular endothelial growth/permeability factor (VEG/PF) is an endothelial cell-specific mitogen that promotes angiogenesis (see Refs.3 and 4 for reviews). VEG/PF (5, 6, 7, 8) and its fms-like tyrosine kinase (flt-1) and kinase-insert domain containing (flk-1) receptors (9, 10, 11) are expressed in the human villous placenta. However, relatively little is known about the regulation of VEG/PF expression and angiogenesis in the villous placenta during human pregnancy (12).

Estrogen stimulates VEG/PF mRNA expression and angiogenesis in the rat (13, 14, 15), sheep (16, 17), baboon (18, 19), and human (20, 21) uterus. Chronic estrogen treatment enhanced angiogenesis in normal, but not estrogen-receptor-null, mice (22). Collectively, these data suggest that estrogen has an important role in regulating angiogenesis in the uterus and that this process may be mediated via VEG/PF.

Using the baboon as a nonhuman primate model to study human fetal-placental development, we recently demonstrated that cytotrophoblasts were a major source of VEG/PF mRNA and protein in the baboon villous placenta (23). In addition, there was a developmental increase in cytotrophoblast VEG/PF mRNA levels and placental vascularization, which closely paralleled maternal serum estradiol concentrations during advancing baboon pregnancy. We propose, therefore, that estrogen has an important role in promoting VEG/PF expression and the new vascular system that develops within the placenta during primate pregnancy. To examine this possibility, VEG/PF mRNA levels and vascularization were determined within the villous placenta of baboons in which maternal serum estradiol levels were prematurely elevated or suppressed early in pregnancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female baboons (Papio anubis), originally obtained from the Southwest Foundation for Biomedical Research (San Antonio, TX) and weighing 13–15 kg, were used in this study. Baboons were individually housed in aluminum stainless-steel, large-primate cages and received 20% protein pellets (Primate Diet, Harlan, Madison, WI) and fresh fruit twice daily, vitamins daily, and water ad libitum. Females were paired with male baboons for 5 d at the anticipated time of ovulation, as estimated by menstrual cycle history and turgescence of external sex skin. Baboons were cared for and used strictly in accordance with USDA regulations and the NIH Guide for the Care and Use of Laboratory Animals. This experimental protocol was approved by the institutional animal care and use committee of University of Maryland School of Medicine.

At 1- to 2-d intervals throughout the study period, baboons were sedated with ketamine HCl (10 mg/kg body weight, im), and a maternal saphenous venous blood sample (2–4 ml) was collected. Serum estradiol levels were determined by RIA using an automated chemiluminescent immunoassay system (Immulite, Diagnostic Products Corp., Los Angeles, CA) as described previously (24).

To examine the effect of chronically elevating estrogen in early pregnancy, placentas were obtained via cesarean section on d 60 of gestation (length of gestation is 184 d) from halothane-anesthetized baboons untreated (n = 6) or treated daily on d 25–59 with androstenedione (30 mg/d, sc, in 1.0 ml sesame oil; n = 6). Androstenedione is readily taken up by and converted to estrogen within the primate placenta (25, 26), resulting in a physiological distribution of estrogen locally within the trophoblast.

To assess the importance of endogenous estrogen on angiogenesis, placentas were obtained on d 60 from baboons treated sc daily on d 25–59 with the highly specific aromatase inhibitor CGS 20267 [4,4'-[1,2,4-triazol-1-yl-methylene]-bis-benzonitrite; Letrozole (Novartis Pharma AG, Basel, Switzerland) starting at 0.1 mg and increasing to 1.0 mg/d by d 35; n = 5] or with CGS 20267 and estradiol benzoate (0.1 to 1.0 mg/d; n = 3).

Placental tissue processing

Several randomly selected sections of villous tissue were either frozen in liquid nitrogen for VEG/PF mRNA analysis or fixed in formalin and embedded in paraffin for immunocytochemistry and blood vessel quantification. From the remainder of the villous placenta, enriched fractions of cytotrophoblasts and cells of the inner villous core were obtained and used for VEG/PF mRNA quantification. Villous tissue was dispersed in Hanks’ balanced salt solution containing hyaluronidase, collagenase, and deoxyribonuclease, and cell fractions were isolated via 5–70% Percoll gradient centrifugation (1200 x g) as described previously (27). Kliman et al. (28) and our laboratory (Ref.27 and unpublished observations) have previously shown that enriched cytokeratin-positive cytotrophoblast and {alpha}-antichymotrypsin-positive inner villous cell fractions were obtained using the latter cell isolation methods. Although an enriched fraction of syncytiotrophoblast was also obtained, the amount of total RNA obtained in this cell fraction was insufficient to quantify VEG/PF mRNA by competitive RT-PCR in animals of the present study.

Competitive RT-PCR of VEG/PF mRNA

VEG/PF mRNA levels were quantified by competitive RT-PCR as established by Riedy et al. (29) and modified in our laboratory (23, 30). Villous tissue and isolated cells were homogenized in 4 M guanidine isothiocyanate, and total RNA was obtained via cesium chloride gradient centrifugation. Oligonucleotide primers were selected from the human VEG/PF cDNA sequence (31) and spanned exons 1, 2, and 3 (first base pair of initiating codon was designated 1). The following primers were used: primer 1, downstream, 5'-GGTGAGGTTTGATCCGCATAATCTGCGCATCAGGGGCACACAGGAT-3' (positions 336–311 and 243–224); primer 2, upstream, 5'-AATTTAATACGACTCACTATAGGGACTGCTGTCTTGGGTGCATTGG-3' [T7 polymerase sequence (underlined) and position 10–30]; primer 3, downstream, 5'-GGTTTGATCCGCATAATCTGC-3' (position 331–311); and primer 4, upstream, 5'-CTGCTGTCTTGGGTGCATTGG-3' (position 10–30). Primers 3 and 4 were upstream of the alternative splice site and thus generated a single 323-bp VEG/PF PCR product. The competitive reference standard (CRS) was synthesized from 150 ng cDNA template using primers 1 and 2 and the MEGAscript T7 in vitro transcription kit (Ambion, Inc., Austin, TX) and had a 67-bp deletion to differentiate it from wild-type target mRNA.

To quantify VEG/PF mRNA, a constant amount of total RNA (75–300 ng, depending on cell fraction) was added to an RT mixture containing 3-fold serial dilutions of the VEG/PF CRS (5400–200 attomoles). Upon completion of the RT, 5 µl of the RT mixture were added to a PCR mixture containing 20 pmol each of primers 3 and 4 and amplified for 26 sequential cycles. The presence of potential pseudogene or genomic DNA contamination was checked by omitting either the RT enzyme or RNA. The PCR products were fractionated in a 2% agarose gel containing ethidium bromide, visualized with a UV transilluminator, and photographed using type 665 positive/negative film (Polaroid Corp., Cambridge, MA). Negatives were analyzed by autoradiographic scanning using a model 620 Video Densitometer and 1-D Analyst software (Bio-Rad, Laboratories, Hercules, CA). The intensity of the amplified mRNA product was represented as the relative area under each sample band. The logarithm (log) of the ratio of VEG/PF CRS to VEG/PF target area was plotted as a function of the log concentration of VEG/PF CRS added to each PCR. The concentration of VEG/PF target mRNA was determined where the ratio of log of CRS and target area was equal to 0 (i.e. the equivalence point).

To confirm that there were no changes in a constitutively expressed cellular RNA, 18S rRNA levels were also quantified in placental cell fractions by RT-PCR using primers selected from the human 18S rRNA cDNA sequence (32). The PCR products were gel-fractionated, photographed, and scanned, and 18S rRNA levels were expressed in relative optical units.

Immunocytochemistry of VEG/PF and von Willebrand factor

The cellular localization of VEG/PF and von Willebrand factor protein was determined by immunocytochemistry as detailed previously (23). Briefly, paraffin blocks of placental villous tissue were serially sectioned at 4 µm, boiled in 0.01 M sodium citrate, pretreated with Protease (Biomeda, Foster City, CA) for 5 min at room temperature, incubated in H2O2 to inhibit endogenous peroxidase, and blocked with serum-free Protein Block (DakoCytomation, Carpinteria, CA). Tissues were incubated overnight at 4 C with polyclonal goat antibody to VEG/PF (AF-293-NA, 1:500 dilution; Santa Cruz Laboratories, Santa Cruz, CA) or rabbit antibody to von Willebrand factor (1:1500 dilution; DakoCytomation). Tissues were incubated 1 h with either biotinylated antigoat or antirabbit immunoglobulins (Vector Laboratories, Inc., Burlingame, CA) and an avidin-biotin-peroxidase complex (ABC Elite, Vector Laboratories, Inc.). Tissue sections were developed using diaminobenzidine for VEG/PF or diaminobenzidine and 2.5% nickel sulfate for von Willebrand factor and were counterstained with hematoxylin (VEG/PF) or eosin (von Willebrand factor). Negative controls for immunocytochemistry included preabsorption of the primary antibody with a 10-fold excess of human recombinant VEG/PF protein (Santa Cruz Biotechnology, Inc.), omission of the primary antibody, and substitution of goat/rabbit immunoglobulin G (DakoCytomation) for primary antibody.

Image analysis of placental vascularization

The level of placental vascularization was quantified by computer-assisted image analysis, as described previously by our laboratory (23). Quantification of blood vessels, i.e. arterioles, arteries, venules, and veins, in placental villous tissue was performed with an Eclipse E 1000M microscope (Nikon, Tokyo, Japan) attached to a color video camera (Dage-MTI, Michigan City, IN). Color images were digitized by a Power Macintosh G3 computer (Apple Computer, Cupertino, CA) and visualized on a high resolution monitor. von Willebrand factor-immunoreactive vessels were shaded in with a green pseudo-color, and the total villous area was circumscribed with a yellow pseudo-color, as illustrated in Fig. 1Go. Image analysis software (IP Lab Scientific Image Processing, Scanalytics, Fairfax, VA) was used for interactive manipulation of the image and data collection. Information on vessel number and area was imported into an Excel worksheet program.



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  		FIG. 1. Representative photomicrograph illustrating computerassisted image analysis of placental vascularization on d 60 of baboon pregnancy. von Willebrand-immunolabeled vessels were shaded in with green, and the total villous area was circumscribed with yellow for purposes of quantifying the level of placental villous vascularization.

 
The proportion of villous placenta comprised of blood vessels, i.e. percent vascularized area, was determined by dividing the total vessel area by the total villous area examined. The number of vessels per square millimeter of villous tissue, i.e. vessel density, and the size distribution of vessels were also determined. A mean (±SE) value for vessel counts on each placenta was determined on 20–40 different placental villous sections (two views per villous section by five sections per biopsy by two to four biopsies per placenta).

Statistical analysis

Data were expressed as the mean ± SE and were analyzed by two-way ANOVA with post hoc comparison of means by Newman-Keuls multiple comparison test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum estradiol levels and placental and fetal body weights

The administration of androstenedione increased the mean ± SE serum estradiol concentrations in maternal saphenous (0.58 ± 0.15 ng/ml) and uterine (1.60 ± 0.42 ng/ml) venous samples to values on d 60 that were approximately 3-fold greater (P < 0.01) than those in untreated baboons (0.20 ± 0.03 and 0.57 ± 0.07 ng/ml, respectively; Table 1Go). Within 1–2 d of CGS 20267 administration, serum estradiol declined to very low levels, and on d 60 of gestation, maternal peripheral saphenous (0.05 ± 0.01 ng/ml) and uterine (0.11 ± 0.02 ng/ml) vein serum estradiol concentrations were 20–25% (P < 0.001) of those in untreated animals (Table 1Go). Administration of CGS 20267 and estradiol restored serum estradiol to levels similar to those in untreated baboons.


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  		TABLE 1. Serum estradiol levels and placental and fetal body weights in baboons

 
As shown in Table 1Go, placental and fetal body weights on d 60 of gestation were not significantly altered by administration of androstenedione or CGS 20267 with or without estradiol.

Placental VEG/PF mRNA levels

Figure 2Go shows a representative quantitative analysis of VEG/PF mRNA levels by competitive RT-PCR in placental cytotrophoblasts obtained on d 60 from baboons untreated or treated with CGS 20267 on d 25–59 of gestation. The expected 323-bp VEG/PF target product and 256-bp VEG/PF CRS product generated by PCR are shown in Fig. 2AGo. No PCR product was detected when either RNA or RT enzyme was omitted from the reaction (data not shown). Correlation coefficients (r2) determined by linear regression of the slopes of the log of the CRS to target areas plotted as a function of increasing amounts of CRS were 0.96 and 0.99 for RNA obtained from CGS 20267-treated and untreated animals (Fig. 2BGo), respectively, indicating linear PCR amplification in each case. However, analysis of the equivalence points from each plot indicated that VEG/PF mRNA levels were lower in cytotrophoblasts from CGS 20267-treated compared with untreated animals.



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  		FIG. 2. Representative competitive RT-PCR of VEG/PF in baboon placental cytotrophoblasts. Total RNA (75 ng) from placental cytotrophoblasts obtained on d 60 of gestation from baboons untreated or treated daily on d 25–59 with CGS 20267 (as detailed in Table 1Go) was mixed with 3-fold serial dilutions of VEG/PF CRS. Samples were reverse transcribed and amplified for 26 cycles in the presence of specific primers. A, The 323-bp target product from total RNA and the 256-bp product from the CRS separated on a 2% agarose gel and stained with ethidium bromide. B, Intensities of the amplified products in A were analyzed by densitometry, and the log of the ratios of VEG/PF CRS and target areas in cytotrophoblasts from CGS 20267-treated ({circ}) and untreated (•) animals was plotted as a function of the quantity of CRS added to each PCR. Lines were constructed by linear regression analysis, and VEG/PF mRNA levels were determined from the equivalence points (i.e. intersection of the vertical with regression lines).

 
The mean levels of 18S rRNA (relative OD units) in inner villous cells, cytotrophoblasts, and whole villous tissue were similar on d 60 of gestation in untreated (1.60, 1.55, and 1.55, respectively), androstenedione-treated (1.75, 1.60, 1.60, respectively), and CGS 20267-treated (1.77, 1.70, and 1.60, respectively) baboons, indicating that RNA levels were not altered by experimental treatment.

Cytotrophoblasts were a major source of VEG/PF mRNA in the villous placenta on d 60 of gestation (Fig. 3Go), as we have previously shown throughout baboon pregnancy (23). Administration of androstenedione elevated baboon placental cytotrophoblast VEG/PF mRNA to levels on d 60 of gestation (26,836 ± 5,625 attomoles/µg total RNA), which were approximately 2.5-fold greater (P < 0.05) than those in untreated animals (11,645 ± 1,746 attomoles/µg total RNA; Fig. 3Go). Androstenedione administration also increased VEG/PF mRNA in whole villous tissue to levels (18,389 ± 2,256 attomoles/µg RNA) that were more than 5-fold greater (P < 0.001) than those in untreated controls (3,380 ± 594). However, VEG/PF mRNA levels in the inner villous cell fraction were not significantly different in untreated (3,462 ± 642) and androstenedione-treated (5,572 ± 1,022) baboons (Fig. 3Go).



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  		FIG. 3. VEG/PF mRNA levels in placental inner villous cells and cytotrophoblasts isolated by Percoll gradient centrifugation and whole villous tissue on d 60 of gestation from baboons untreated or treated daily on d 25–59 of gestation with androstenedione ({Delta}4A), CGS 20267, or CGS 20267 plus estradiol (E2) benzoate as detailed in Table 1Go. Total RNA was analyzed by competitive RT-PCR using primers upstream from the alternative splice site. Individual values represent the mean (±SE) of three to six baboons for each placental cell fraction and each treatment regimen (with the exception of inner villous cells obtained from baboons treated with CGS 20267 plus estrogen; n = 1). *, P < 0.05; **, P < 0.01 (vs. untreated control within respective tissue fraction, by ANOVA and Newman-Keuls multiple comparison test).

 
Suppression of placental estrogen formation and serum estrogen levels in baboons by administration of the aromatase inhibitor CGS 20267 decreased cytotrophoblast VEG/PF mRNA to a level (2,912 ± 693 attomoles/µg RNA) that was 25% (P < 0.05) of that observed in untreated controls (Fig. 3Go). Concomitant administration of CGS 20267 plus estradiol prevented this decline in cytotrophoblast VEG/PF mRNA level and increased expression to a value (26,509 ± 6,720 attomoles/µg RNA) greater (P < 0.01) than that in untreated animals and similar to that observed with androstenedione treatment. In contrast to the decline in cytotrophoblast VEG/PF mRNA, the whole villous tissue VEG/PF mRNA level was elevated (P < 0.05) approximately 3-fold by the administration of CGS 20267 (10,978 ± 2,339 attomoles/µg RNA), an effect that was reversed by CGS 20267 plus estradiol. VEG/PF mRNA levels in the inner villous cell fraction were not significantly altered by CGS 20267 (Fig. 3Go).

Placental VEG/PF protein expression

VEG/PF protein was localized by immunocytochemistry in relatively high levels within the cytoplasm of placental villous cytotrophoblasts on d 60 of baboon gestation (Fig. 4AGo). VEG/PF protein was also present, although in apparently lower levels, in the cytoplasm of the syncytiotrophoblast and cells of the inner villous core, including the vascular endothelium. The qualitative pattern of cellular VEG/PF protein localization in the villous placenta was not altered by androstenedione, CGS 20267, or CGS 20267 and estradiol treatment (not shown). There was little or no immunoreactivity for VEG/PF in the villous placenta when the primary antibody was preabsorbed with excess recombinant VEG/PF (Fig. 4BGo).



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  		FIG. 4. Representative photomicrograph of VEG/PF immunocytochemistry in the villous placenta on d 60 of gestation in an untreated baboon (A). B, VEG/PF immunocytochemistry when the primary VEG/PF antibody was preabsorbed with human recombinant VEG/PF. C, Cytotrophoblast cytoplasm/nucleus; S, syncytiotrophoblast nucleus; IVS, intervillous space. Scale bar, 40 µm.

 
Placental vascularization

The percent vascularized area of the villous placenta in untreated baboons on d 60 of pregnancy (3.46 ± 0.23; Fig. 5Go) was increased (P < 0.01) almost 2-fold by administration of androstenedione, to a level (6.54 ± 0.56) previously shown in untreated baboons on d 100, i.e. at midgestation (23). Consistent with results obtained for vascularized area, vessel density was also increased (P < 0.01) in baboons treated with androstenedione (743 ± 27 vessels/mm2 villous tissue) compared with that in untreated controls (493 ± 34 vessels/mm2; Fig. 6Go). In contrast, percent vascularized area (4.19 ± 0.56 and 5.12 ± 0.85) and placental vessel density (526 ± 64 and 609 ± 33 vessels/mm2) in baboons treated with CGS 20267 and CGS 20267 plus estradiol, respectively, were not significantly different from respective values in untreated baboons.



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  		FIG. 5. Percent vascularized area (mean ± SE) of the villous placenta determined by image analysis on d 60 of gestation in baboons untreated (n = 6) or treated on d 25–59 of gestation with androstenedione (n = 6), CGS 20267 (n = 5), or CGS 20267 plus estradiol benzoate (n = 3). *, P < 0.01 vs. untreated controls (by ANOVA and Newman-Keuls multiple comparison test).

 


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  		FIG. 6. Blood vessel density (mean ± SE) of the villous placenta determined by image analysis in the same baboons in which percent vascularized areas are shown in Fig. 5Go. *, P < 0.01 vs. untreated controls.

 
The majority of blood vessels in the villous placenta on d 60 of gestation were less than 200 µm2 in cross-sectional area in the baboons of this study (Fig. 7Go). The size distribution of blood vessels in the placenta, however, appeared relatively similar in untreated, androstenedione-treated, CGS 20267-treated, and CGS 20267- plus estradiol-treated animals.



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  		FIG. 7. Size distribution of blood vessels determined in placental villous tissue in the same baboons in which percent vascularized areas are shown in Fig. 5Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study showed that placental villous cytotrophoblast VEG/PF mRNA levels in early pregnancy were increased in baboons in which estrogen levels were elevated by administration of the estrogen precursor androstenedione and decreased when estrogen levels were suppressed by administration of the aromatase inhibitor CGS 20267. Moreover, the decline in placental cytotrophoblast VEG/PF expression in estrogen-suppressed baboons was prevented/reversed by simultaneous administration of aromatase inhibitor and estradiol. Our previous study showed that there was a developmental increase in placental cytotrophoblast VEG/PF mRNA levels in association with the rise in serum estradiol concentrations with advancing baboon pregnancy (23). Others have shown that estrogen stimulated VEG/PF mRNA expression in vivo in the rat (14, 15), sheep (17), and baboon (18, 19) uterus and in vitro in human endometrial cells (20, 33). Systematic deletions of the human VEG/PF promoter have led to identification of upstream sequences critical for estrogen receptor-activated VEG/PF gene transcription (34, 35), although this has not been performed for the baboon VEG/PF gene. Moreover, we and others have shown that estrogen receptor {alpha} and ß are expressed within villous trophoblasts of the human (36, 37) and baboon (38) placenta, thereby providing a mechanism for the paracrine/autocrine action of estrogen in this tissue. Collectively, the results of these previous related studies and the present findings are consistent with the concept that estrogen plays an important role in up-regulating VEG/PF expression by the villous trophoblast during early primate pregnancy.

The current study also showed that the increase in cytotrophoblast VEG/PF expression in androstenedione-treated baboons early in gestation was associated with an increase in placental villous vascularization to a level previously observed at midgestation when estrogen levels become elevated (23). Estrogen also stimulated vascularization of the uterus (16) and uterine and placental blood flow (39) in sheep. It is well established that VEG/PF has a central role in promoting angiogenesis by enhancing vascular endothelial cell proliferation, migration, and assembly into microvessels (4). Studies correlating placental VEG/PF and kinase-insert domain containing/flk-1 receptor expression with villous angiogenesis suggest that VEG/PF and its tyrosine kinase receptors are involved in establishing the capillary bed within the developing intermediate villi during the first half of human pregnancy (2, 40). Successful placentation and embryonic/fetal growth and development apparently are dependent upon optimal vascularization of the villous placenta (1, 2, 12). A deficiency in placental villous vascularization has been associated with spontaneous abortion and embryonic death (41). Thus, we propose that the up-regulation of villous cytotrophoblast VEG/PF in response to estrogen and the proximity of these cells to the inner villous mesenchyme provide a system for promoting vasculogenesis and blood flow within the placenta and, consequently, growth and development of the fetus during early primate pregnancy. Despite the increase in the level of placental vascularization in androstenedione-treated baboons, however, placental and fetal body weights were not significantly altered, suggesting that the fetal growth rate may be maximal at this interval of gestation.

Although placental blood vessel development was increased in baboons in which estrogen levels and cytotrophoblast VEG/PF expression were elevated by androstenedione administration, placental vascularization and placental and fetal body weights were unaltered in CGS 20267-treated baboons despite the decline in serum estrogen and cytotrophoblast VEG/PF mRNA levels. The underlying reason(s) for this apparent dichotomy is unknown; however, it is possible that the synthesis of other angiogenic factors, e.g. placental growth factor (42), may be enhanced to sustain angiogenesis when cytotrophoblast VEG/PF formation is decreased under the latter experimental conditions. Alternatively, or in addition, other cells of the villous placenta may become a significant source of VEG/PF with estrogen deprivation to maintain angiogenesis, perhaps secondary to induction of hypoxia, which has a well established role in stimulating VEG/PF expression in various tissues, including the placental trophoblast (43, 44). Indeed, in estrogendeprived baboons, whole villous tissue VEG/PF mRNA levels were increased, possibly in a compensatory fashion, by cells other than villous cytotrophoblasts or those inner villous cells isolated by the Percoll gradient centrifugation process employed in the present study. Additional studies are needed to elucidate the potential role of other peptide growth factors and different compartments of the placenta in promoting placental neovascularization in the absence of estrogen.

The increase in placental whole villous VEG/PF mRNA levels in androstenedione-treated baboons paralleled and presumably reflected the rise in VEG/PF mRNA expression by villous cytotrophoblasts, which comprise a significant part of the placenta in early pregnancy (45) and have previously been shown to be a significant source of VEG/PF in the human placenta (5, 7, 11, 46). It seems unlikely that the increase in whole villous VEG/PF mRNA reflected syncytiotrophoblast activity, because the latter cellular fraction expressed a relatively low level of VEG/PF in the baboon (23) and human (7, 11) placenta during the first third of pregnancy. Hofbauer macrophages and fibroblasts are also a source of VEG/PF in the inner villous core of the human and baboon placenta (5, 10, 11). However, inner villous cell VEG/PF mRNA levels were not altered in baboons in which estrogen levels were increased by androstenedione or decreased by CGS 20267 administration. Therefore, it appears that estrogen acts specifically on the villous cytotrophoblast cell fraction with respect to the formation of VEG/PF. Although VEG/PF protein levels were not quantified in the present study, both VEG/PF mRNA and protein were extensively expressed by villous cytotrophoblasts in early baboon pregnancy.

Androgens have been shown to up-regulate VEG/PF in other tissues (47); however, it seems unlikely that the stimulatory effects of androstenedione administration on placental VEG/PF expression and angiogenesis in baboons of the present study resulted from an androgenic effect of androstenedione. Thus, placental cytotrophoblast VEG/PF mRNA levels were decreased in baboons in which the aromatization of C19 steroid androgens and thus estrogen levels were suppressed and in which we (48) previously showed that androstenedione levels were increased by administration of aromatase inhibitor CGS 20267. Moreover, the decline in VEG/PF mRNA levels in estrogen-deprived baboons was prevented by simultaneous treatment with CGS 20267 and estradiol.

In summary, the results of the present study show that placental villous cytotrophoblast VEG/PF expression and vascularization were increased when estrogen levels were elevated by androstenedione administration during early baboon pregnancy. Moreover, placental cytotrophoblast VEG/PF expression was decreased when estrogen levels were suppressed by administration of an aromatase inhibitor, an effect prevented by concomitant treatment of baboons with aromatase inhibitor and estradiol. We conclude that estrogen has an important role in increasing trophoblast VEG/PF expression and consequently villous placental angiogenesis to promote fetal growth and development in early primate pregnancy.


   Acknowledgments
 
We gratefully acknowledge Novartis Pharma AG (Basel, Switzerland) for generously providing the aromatase inhibitor Letrozole for use in this study. We also sincerely appreciated the secretarial assistance of Mrs. Wanda James with the manuscript, and technical assistance of Ms. Donna Suresch with the VEG/PF immunocytochemistry.


   Footnotes
 
This work was supported by NIH Research Grant R01-HD-13294.

Abbreviations: CRS, Competitive reference standard; VEG/PF, vascular endothelial growth/permeability factor.

Received March 25, 2004.

Accepted July 27, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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