Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print July 21, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.030882
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/5/1694    most recent
biolreprod.104.030882v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Robb, V. A.
Right arrow Articles by Albrecht, E. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Robb, V. A.
Right arrow Articles by Albrecht, E. D.
Agricola
Right arrow Articles by Robb, V. A.
Right arrow Articles by Albrecht, E. D.
BIOLOGY OF REPRODUCTION 71, 1694–1698 (2004)
DOI: 10.1095/biolreprod.104.030882
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Acute Temporal Regulation of Placental Vascular Endothelial Growth/Permeability Factor Expression in Baboons by Estrogen1

Victoria A. Robb3, Gerald J. Pepe4, and Eugene D. Albrecht2,3

Department of Obstetrics, Gynecology and Reproductive Sciences,3 Center for Studies in Reproduction, University of Maryland School of Medicine, Baltimore, Maryland 21201 Department of Physiological Sciences,4 Eastern Virginia Medical School, Norfolk, Virginia 23507


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vascular endothelial growth/permeability factor (VEG/PF) has an established role in angiogenesis, however, the regulation of placental VEG/PF expression during primate pregnancy is incompletely understood. A temporal study was conducted in baboons to determine the effect of acute administration of estradiol on the expression of VEG/PF by cells of the villous placenta. VEG/PF mRNA levels were determined by reverse transcription-polymerase chain reaction in isolated placental cell fractions of baboons after acute i.v. and i.m. administration of estradiol. Within 2 h of estradiol treatment, VEG/PF mRNA (attomoles/ micrograms total RNA) increased within villous cytotrophoblasts to a level (mean ± SEM, 12 612 ± 2419) that was almost 2-fold greater (P < 0.05) than in untreated controls (6810 ± 1368). Cytotrophoblast VEG/PF mRNA levels remained elevated (P < 0.01) 6 h after estradiol treatment (15 006 ± 506), but were not different from controls 18 h after estradiol administration. VEG/ PF mRNA levels in whole villous tissue also were greater 6 h (12 667 ± 2284, P < 0.05) and 18 h (16 080 ± 3816, P < 0.01) after estradiol treatment than in untreated animals (3380 ± 594). In contrast, VEG/PF mRNA levels in cells of the inner villous core were not altered by estradiol treatment. Expression of both the VEG/PF121 and VEG/PF165 mRNA species appeared to increase in the placenta 6 h after estradiol treatment of baboons. We propose that estrogen regulates VEG/PF expression within the placenta in a cell-specific manner, providing a paracrine system to promote vascularization of the villous placenta during the first half of primate pregnancy.

estradiol, placenta, pregnancy, steroid hormones, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
With advancing primate pregnancy, an extensive vascular network develops within the villous placenta by in situ differentiation of fetal mesenchymal cells into blood vessels (i.e., vasculogenesis) and proliferation of existing vessels (i.e., angiogenesis). Relatively little is known, however, about the factors involved in regulating placental vascularization during primate pregnancy (see [1, 2] for reviews). Vascular endothelial growth/permeability factor (VEG/PF) has a well established role in angiogenesis [3], including the induction of microvascular permeability an early step, which appears critical to angiogenesis [4]. VEG/ PF is expressed as five isoforms, generated by alternate splicing of the mRNA to yield proteins of 121, 145, 165, 189, and 206 amino acids. In other tissues, chronic estrogen administration in vivo increased VEG/PF expression (e.g., in the rat [5], sheep [6], and baboon [7] uterus).

Using the baboon as a nonhuman experimental model, we recently showed that placental VEG/PF mRNA levels and the level of vascularization increased in association with the rise in serum estrogen levels that occur with advancing stages of pregnancy [8]. Moreover, chronically elevated serum estradiol levels early in baboon pregnancy increased placental villous trophoblast VEG/PF mRNA expression and neovascularization of the placenta (E.D. Albrecht, V.A. Robb, and G.J. Pepe, unpublished results). Therefore, we suggested that estrogen has an important role in establishing the new vascular system within the placenta during primate pregnancy and that VEG/PF mediates this process. However, because long-term administration of steroid hormones may result in cellular differentiation, it is not known whether or not the increase in placental VEG/ PF expression induced by estrogen represents direct effects on VEG/PF mRNA expression. To address this possibility, a temporal study was conducted in baboons to determine the effect of acute administration of estradiol on the expression of VEG/PF by isolated cell fractions of the villous placenta in early gestation when endogenous estradiol levels are low.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Female baboons (Papio anubis) weighing 13–18 kg were individually housed in large aluminum and stainless steel primate cages, and received high-protein monkey chow twice daily, fresh fruit, and vitamins daily, and water ad libitum. Females were paired with male baboons for 5 days at the anticipated time of ovulation as estimated by menstrual cycle history and turgescence of external sex skin. The first day of gestation was designated as 2 days preceding the day of detumescence.

To assess the temporal effect of acute estradiol administration on placental VEG/PF expression on Day 54 of gestation (length of gestation is 184 days), baboons were briefly sedated with ketamine HCl (10 mg/kg body weight, i.m.) and treated with 100 µg of 17ß-estradiol in 1.0 ml of 5% ethanol:saline administered as a bolus via an antecubital vein to achieve a rapid increase in blood estrogen levels and 500 µg estradiol benzoate i.m. in 0.5 ml of sesame oil to achieve a sustained increase in blood estrogen levels throughout the study period. Baboons were then anesthetized with halothane, and placentas were obtained via cesarean delivery after no treatment or 6 h after ethanol:saline vehicle treatment (results of n = 8 baboons combined, because results were similar in untreated and vehicle-treated animals), or 2 h (n = 6), 6 h (n = 6), or 18 h (n = 5) after estradiol treatment. Blood samples (2 ml) were obtained throughout the study period from a maternal saphenous vein, and serum estradiol levels were determined by RIA using an automated chemiluminescent immunoassay system (Immulite; Diagnostic Products Corp, Los Angeles, CA), as described previously [9].

Baboons were cared for and used strictly in accordance with U.S. Department of Agriculture regulations and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Publication 86-23, 1985). This experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.

Placental Tissue

Randomly selected sections (approximately 4 mm3 each) of villous tissue were excised from each placenta and stored frozen in liquid nitrogen for mRNA analysis. From the majority of the remaining placental villous tissue, enriched fractions of cytotrophoblasts and cells of the inner villous core were obtained as previously described by our laboratory [10] and by Kliman et al. [11] and used for quantification of VEG/PF mRNA levels by competitive reverse transcription-polymerase chain reaction (RT-PCR). Briefly, villous tissue was minced in Hanks balanced salt solution (HBSS; Life Technologies Inc., Gaithersburg, MD) and digested in HBSS containing 0.1% hyaluronidase (type I-S; Sigma Chemical, St. Louis, MO), 0.1% collagenase (Type H; Sigma Blend), and 0.01% deoxyribonuclease I (1680 Kunitz units/mg; Sigma). Enriched cell fractions were then isolated by 5%–70% Percoll (Pharmacia Fine Chemicals, Piscataway, NJ) gradient centrifugation at 1200 x g. Kliman et al. [11] and we (unpublished data) have previously shown that highly enriched cytokeratin-positive cytotrophoblast and {alpha}-antichymotrypsin-positive inner villous cell fractions were obtained from the placenta using the latter cell isolation procedure. Although an enriched fraction of syncytiotrophoblast was also obtained, sufficient RNA was not isolated for competitive RT-PCR analysis of VEG/ PF mRNA.

RT-PCR of VEG/PF

The mRNA levels for VEG/PF were quantified by the competitive RT-PCR assay established by Riedy et al. [12], as modified by our laboratory [13]. Placental cell fractions were homogenized in 4 M guanidine isothiocynate, layered over 5.7 M cesium chloride, and total RNA was pelleted by centrifugation.

Oligonucleotide primers were designed (Invitrogen Life Technologies, Carlsbad, CA) from selected regions within the human VEG/PF cDNA sequence [14]:

  1. Primer 1: downstream, 5'-GGTGAGGTTTGATCCGCATAATCTGCGCATCAGGGGCACACAGGAT-3' (position 336–311 linked to 243–224).
  2. Primer 2: upstream, 5'-AATTTAATACGACTCACTATAGGGACTGCTGTCTTGGGTGCATTGG-3' (position T7 polymerase sequence [underlined] linked to 10–30).
  3. Primer 3: downstream, 5'-GGTTTGATCCGCATAATCTGC-3' (position 331–311).
  4. Primer 4: upstream, 5'-CTGCTGTCTTGGGTGCATTGG-3' (position 10– 30).

Because primers 3 and 4 are upstream of the alternative splice site that generates the different isoforms of VEG/PF, a single 323-base pair (bp) PCR product was generated. A homologous competitive reference standard (CRS) was prepared using primers 1 and 2 and had a 67 bp deletion (length, 256 bp) compared with wild-type target mRNA. Total RNA (3.2 µg) from baboon placenta was reverse transcribed in a reaction mixture containing 1 mM deoxy(d)NTPs (Invitrogen), 1 mM dithiothreitol, 200 U Superscript RNase H-reverse transcriptase (RT; Invitrogen), 40 U RNAguard ribonuclease inhibitor (Pharmacia Biotech, Piscataway, NJ), 1x RT buffer, and 250 ng random primers (Invitrogen). After 60 min, the RT mixture was incubated at 70°C for 15 min and 5 µl of the RT mixture added to a PCR reaction mixture containing 0.2 mM dNTPs (Invitrogen), 1x PCR buffer, 1.25 U cloned Thermus aquaticus DNA polymerase (Amplitaq, Perkin-Elmer Corp/Cetus, Norwalk, CT) and 20 pmol each of primers 1 and 2. PCR was performed in a programmable thermal cycler (MJ Research Inc., Cambridge, MA) and samples were amplified in 25 sequential cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min, with a final extension at 72°C for 5 min. The PCR mixture was fractionated by electrophoresis in a 2% agarose gel and visualized with ethidium bromide. The amplified product containing a sequence for the T7 polymerase, as well as the designated deletion, was gel purified using a Qiaex DNA gel extraction kit (Qiagen, Inc., Valencia, CA) . The CRS was synthesized from 150 ng of cDNA template using the MEGAscript T7 in vitro transcription kit (Ambion, Inc., Austin, TX) and quantitated via UV absorption spectrophotometry at an optical density of 260 nm.

To quantify VEG/PF mRNA, a constant amount of total RNA (75–300 ng) was added to an RT mixture containing 3-fold serial dilutions of the VEG/PF CRS (5400–200 attomoles). Negative controls, in which either the RT enzyme or RNA was omitted from the RT reaction, were performed to test for any contaminating pseudogene or genomic DNA. Upon completion of the RT, 5 µl of the RT reaction was added to a PCR mixture containing 20 pmol each of primers 3 and 4 (26 cycles). 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 scanned using a model 620 Video Densitometer and 1-D Analyst II data analysis software (Bio-Rad Laboratories, Hercules, CA). The intensity of amplified product was represented as the relative area under each sample band. The logarithm (log) of the ratio of VEG/PF CRS area to VEG/PF target area was plotted as a function of the concentration of VEG/PF CRS added to each PCR reaction. The concentration of VEG/PF target mRNA was determined where the ratio of the log of CRS and target was equal to 0 (i.e., the equivalence point).

Northern Blot Analysis of VEG/PF

Expression of the mRNAs for the VEG/PF species were determined by Northern blot analysis as previously described in our laboratory [15]. Poly(A)+-enriched RNA was prepared by centrifugation of 500–750 µg total RNA from whole villous tissue over columns of oligo (deoxythymidine) cellulose (Pharmacia, Piscataway, NJ). Approximately 4 µg of poly(A)+-enriched RNA was denatured in 50% formamide, 2.2 M formaldehyde, and 20 mM 3-[N-morpholino] propane sulfonic acid (MOPS) pH 7.0, and size-fractioned by electrophoresis in 1.0% agarose gel containing 0.6 M formaldehyde and 20 mM MOPS pH 7.0. RNA was transferred overnight by capillary action in 10x saline-sodium citrate (SSC; 1.5 M NaC1-0.15 M sodium citrate-2H2O pH 7.0) onto a nylon membrane (Gene Screen; Dupont-New England Nuclear Corp, Boston, MA). RNA-containing membranes were UV cross-linked, baked in a vacuum oven at 80°C for 2 h, and prehybridized in buffer containing 50% formamide, 0.1% polyvinylpyrrolidone, 0.1% BSA, 0.1% Ficoll, 2.5x SSPE (0.375 M NaCl, 0.025 M NaH2PO4-H2O, 0.0025 M EDTA), 1.0% SDS, 10% dextran sulfate, and denatured salmon sperm DNA (100 µg/ml) for 24 h at 42°C prior to addition of probe. The cDNA for human VEG/PF was provided by the Collaborations Program of Genetech (South San Francisco, CA). The human cDNA for ß-actin (No. 65128) was obtained from American Type Culture Collection (Manassas, VA). The cDNAs were labeled with 50 µCi [{alpha}-32P] dCTP (3000 Ci/mmol, Amersham Corp., Arlington Heights, IL) to a specific activity of approximately 109 dpm/µg DNA using the Random-Primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN). Hybridization was performed in fresh buffer at 42°C for 21–24 h with approximately 106 cpm/ml [32P] cDNA probe. After hybridization, membranes were washed twice at room temperature for 10 min in 2x SSC, then at 65°C for 20 min in 2x SSC, 1% SDS, followed by room temperature wash in 0.1x SSC. Membranes were exposed to Kodak X-AR film (Eastman Kodak, Rochester, NY) at –80°C. After exposure, membranes were stripped before rehybridization. Intensities of blots were analyzed by densitometric autoradiographic scanning using a Model 620 Video Densitometer and 1-D Analyst II software (Bio-Rad). The relative intensities of the mRNA transcripts for VEG/PF were related to those of ß-actin to determine specific effects on expression.

Statistics

Data are expressed as the mean ± SEM. Placental cell VEG/PF mRNA levels in baboons were analyzed by two-way analysis of variance (ANOVA; 3 x 4 factorial design).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Serum Estradiol

Mean ± SEM maternal peripheral serum estradiol levels in baboons increased from 0.12 ± 0.02 ng/ml at Time 0 to a peak of 1.19 ± 0.52 ng/ml at 15 min after acute i.v. and i.m. injections of estradiol (Fig. 1). Serum estradiol then declined after 1 h to a plateau at which levels at 2 h (0.48 ± 0.02 ng/ml), 6 h (0.44 ± 0.16 ng/ml), and 18 h (0.55 ± 0.09 ng/ml) were approximately 4-fold greater than pretreatment values.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 1. Mean maternal peripheral serum estradiol levels in five baboons before and after a bolus injection at Time 0 of estradiol i.v. plus estradiol benzoate i.m. on Day 54 of gestation (term = 184 days)

VEG/PF mRNA

Figure 2 shows a representative quantitative analysis of VEG/PF mRNA levels by competitive RT-PCR in cytotrophoblasts obtained from baboons untreated and 2 h after treatment with a bolus of estradiol. The expected 323-bp VEG/PF target product and the 256-bp VEG/PF CRS product generated by PCR are shown in Figure 2A. No PCR product was detected when either RNA or RT enzyme were omitted from the reaction (data not shown). The slopes of the log of CRS to target areas plotted as a function of increasing amounts of CRS were similar for RNA obtained from untreated and estrogen-treated cytotrophoblasts (Fig. 2B), indicating no difference in amplification efficiency.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 2. Representative competitive RT-PCR of VEG/PF in baboon placental cytotrophoblasts. Total RNA from cytotrophoblasts was collected on Day 54 of gestation from baboons either untreated or treated with estradiol for 2 h as detailed in the legend to Figure 1. A) The 323-bp VEG/ PF target and 256-bp VEG/PF CRS shown separated in agarose gels. B) The products shown in (A) were analyzed by densitometry and the log of the ratios of VEG/PF CRS and target plotted as a function of CRS added to the reaction. VEG/PF mRNA levels were determined by linear regression of the products and the equivalence points (i.e., intersection of vertical and regression lines)

Cytotrophoblasts were the major source of VEG/PF mRNA in the villous placenta of untreated baboons on Day 54 of gestation (Fig. 3), as we have previously shown throughout pregnancy [8]. VEG/PF mRNA levels determined in cytotrophoblasts of baboons treated with ethanol: saline vehicle were similar to values in untreated control baboons (data not shown). Within 2 h of estradiol treatment, VEG/PF mRNA increased within the villous cytotrophoblast fraction to a level (mean ± SEM = 12 613 ± 2419 attomoles/µg total RNA) that was almost 2-fold greater (P < 0.05) than that in untreated controls (6810 ± 1368 attomoles/µg total RNA). Cytotrophoblast VEG/PF mRNA levels remained elevated (P < 0.01) 6 h after estrogen treatment (15 006 ± 506), but were not significantly different from control 18 h after estradiol administration. Corresponding to the findings in cytotrophoblasts, VEG/PF mRNA levels in whole villous tissue were greater 6 h (12 667 ± 2284 attomoles/µg total RNA, P < 0.05) and 18 h (16 080 ± 3816, P < 0.01) after estradiol treatment than in untreated controls (3380 ± 594). In contrast, VEG/ PF mRNA levels in cells of the inner villous core were not significantly changed at any of the time points after acute estrogen treatment (Fig. 3) compared to untreated baboons (3462 ± 642 attomoles/µg total RNA).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 3. VEG/PF mRNA levels in placental inner villous cells, cytotrophoblasts, and whole villous tissue obtained on Day 54 from baboons either untreated or 2, 6, or 18 h after estradiol administration. Individual values represent the means (± SEM) of three to six baboons for each placental cell fraction and each treatment group except value for inner villous cells at 2 h, for which n = 1. Values were different from untreated controls (within respective tissue fraction) at P < 0.05* or P < 0.01** (two-way ANOVA)

To confirm that there were no changes in a constitutively expressed cellular RNA, the levels of 18S rRNA in the various placental cell fractions were semiquantified by RT-PCR using specific 18S rRNA primers. The mean levels of 18S rRNA were similar on Day 54 of gestation in baboons untreated or treated with estradiol (data not shown), suggesting that total RNA levels were not affected by treatment.

To determine which isoform mRNA might be affected by estradiol treatment, we utilized Northern blot analysis. The mRNA for VEG/PF121 and VEG/PF165 were the predominate isoforms expressed in the baboon placenta on Day 54 of gestation (Fig. 4A). Expression of both the VEG/ PF121 and VEG/PF165 mRNA species appeared to increase in the placenta 6 h after estradiol treatment of baboons (Fig. 4B).



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 4. A) Northern blot analysis of mRNA expression of VEG/PF121 and VEG/PF165 isoforms in placental villous tissue obtained on Day 54 of gestation from baboons untreated or 6 h after injection of estradiol. B) Values (n = 3 untreated; n = 3 estradiol treated) are expressed as VEG/PF121 and VEG/PF165 corrected for ß-actin (arbitrary autoradiogram units)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study show that VEG/PF mRNA levels in placental villous cytotrophoblasts were rapidly elevated within 2 h of acute estradiol administration to baboons in early gestation. The increase in placental VEG/PF appeared to reflect up-regulation of the VEG/PF121 and VEG/PF165 species, which are the most abundantly expressed isomers, and which are believed to mediate early aspects of angiogenesis such as increased microvascular permeability [4]. We also reported recently that placental villous cytotrophoblast VEG/PF mRNA levels were increased with advancing baboon pregnancy, coinciding with the rise in serum estradiol levels [8] and early in baboon pregnancy by prematurely and chronically elevating estrogen levels (E.D. Albrecht, V.A. Robb, and G.J. Pepe, unpublished results). Moreover, placental VEG/PF mRNA levels were decreased by suppressing estrogen levels after administration of an aromatase inhibitor early in baboon gestation, an effect prevented or reversed by concomitant treatment with estradiol (E.D. Albrecht, V.A. Robb, and G.J. Pepe, unpublished results). Systematic deletions of the VEG/PF promoter have identified upstream sequences critical for estrogen receptor-activated gene transcription [16, 17]. Also, we and others have demonstrated that estrogen receptor {alpha} and ß are expressed within human [18, 19] and baboon [20] villous trophoblasts. Although VEG/PF protein levels were not quantified in the present study, both VEG/ PF mRNA and protein were expressed extensively by villous cytotrophoblasts in human [21–24] and baboon ([8] and E.D. Albrecht, V.A. Robb, and G.J. Pepe, unpublished results). Collectively, these observations are consistent with the concept that estrogen acts to up-regulate expression of VEG/PF by villous cytotrophoblasts during primate pregnancy. Therefore, estrogen appears to exert a central important regulatory effect on VEG/PF expression within the placenta, as previously demonstrated within the uterine endometrium of the rat [5], sheep [6], baboon [7, 25], and human [26]. It remains to be determined, however, whether the rapid stimulatory effect of estrogen on placental trophoblast VEG/PF mRNA levels shown in the present study reflected transcriptional events in VEG/PF synthesis.

The regulatory effect of estrogen on placental VEG/PF mRNA expression appeared to be specific for villous cytotrophoblasts, because VEG/PF mRNA levels within an enriched fraction of cells of the placental inner villous compartment were unaltered by acute administration of estradiol. Moreover, although we could not measure VEG/PF mRNA levels within placental syncytiotrophoblasts in baboons of the present study, we have shown previously that VEG/PF mRNA expression in syncytiotrophoblast was very low and not changed despite the rise in estrogen typical of advancing stages of baboon pregnancy [8]. In addition, cytotrophoblast VEG/PF mRNA levels were not altered by suppressing estrogen production by administration of aromatase inhibitor in the second half of baboon pregnancy (E.D. Albrecht, V.A. Robb, and G.J. Pepe; unpublished observations). Consequently, it appears that estrogen regulates VEG/PF expression within the placenta in a cell- and developmental-specific manner during primate pregnancy.

In addition to estrogen, other factors, notably oxygen levels [1, 27, 28], have an important role in regulating VEG/PF expression. Thus, placental VEG/PF expression is up-regulated by hypoxia [21, 28, 29] and down-regulated by hyperoxia [28]. Based on our recent report ([8] and E.D. Albrecht, V.A. Robb, and G.J. Pepe, unpublished results), the studies of others, and the present study, we propose as illustrated in Figure 5 that a paracrine system involving estrogen and other factors and different cell types exists to promote vascularization within the developing placenta. We postulate that estrogen produced by the syncytiotrophoblast [30], and possibly other factors such as oxygen and nitric oxide of trophoblast or stromal origin (or a combination of these) (reviewed in [2]), regulate in a paracrine manner the expression of VEG/PF by immediately adjacent villous cytotrophoblasts and possibly other cell components, which in turn, promotes angiogenesis within the fetal stromal compartment during primate pregnancy.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 5. Proposed model for the role of syncytiotrophoblast estrogen and other factors in regulating cytotrophoblast VEG/PF expression and consequently angiogenesis within the fetal stromal compartment of the villous placenta during primate pregnancy

In summary, VEG/PF mRNA levels in placental villous cytotrophoblasts were elevated rapidly within 2 h of acute estradiol administration to baboons in early pregnancy. We propose that estrogen regulates VEG/PF expression in a cell- and developmental-specific manner providing a paracrine system to promote vascularization of the villous placenta during the first half of primate pregnancy.


   ACKNOWLEDGMENTS
 
We appreciate the secretarial assistance of Mrs. Wanda James with the preparation of the manuscript and figures.


   FOOTNOTES
 
1 Suppported by National Institutes of Health Research Grant RO1 HD-13294. Back

2 Correspondence: Eugene D. Albrecht, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Bressler Research Laboratories Room 11-019, 655 West Baltimore Street, Baltimore, MD 21201. Fax: 410 706 5747; ealbrech{at}umaryland.edu Back

Received: 12 April 2004.

First decision: 28 April 2004.

Accepted: 9 July 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ahmed A, Dunk C, Ahmad S, Khaliq A. Regulation of placental vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) and soluble Flt-1 by oxygen—a review. Placenta 2000 21:Suppl AS16-S24
  2. Reynolds LP, Redmer DA. Angiogenesis in the placenta. Biol Reprod 2001 64:1033-1040[Abstract/Free Full Text]
  3. Ferarra N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997 18:4-25[Abstract/Free Full Text]
  4. Dvorak HF, Nagy JA, Feng D, Brown LF, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis. Curr Top Microbiol Immunol 1999 237:97-132[Medline]
  5. Cullinan-Bove K, Koos RD. Vascular endothelial growth factor/vascular permeability factor expression in the rat uterus: rapid stimulation by estrogen correlates with estrogen-induced increases in uterine capillary permeability and growth. Endocrinology 1993 133:829-837[Abstract/Free Full Text]
  6. Reynolds LP, Kirsch JD, Kraft KC, Redmer DA. Time-course of the uterine response to estradiol-17ß in ovariectomized ewes: expression of angiogenic factors. Biol Reprod 1998 59:613-620[Abstract/Free Full Text]
  7. Niklaus AL, Aberdeen GW, Babischkin JS, Pepe GJ, Albrecht ED. Effect of estrogen on vascular endothelial growth/permeability factor expression by glandular epithelial and stromal cells in the baboon endometrium. Biol Reprod 2003 68:1997-2004[Abstract/Free Full Text]
  8. Hildebrandt VA, Babischkin JS, Koos RD, Pepe GJ, Albrecht ED. Developmental regulation of vascular endothelial growth/permeability factor messenger ribonucleic acid levels in and vascularization of the villous placenta during baboon pregnancy. Endocrinology 2001 142:2050-2057[Abstract/Free Full Text]
  9. Albrecht ED, Aberdeen GW, Pepe GJ. The role of estrogen in the maintenance of primate pregnancy. Am J Obstet Gynecol 2000 182:432-438[CrossRef][Medline]
  10. Henson MC, Babischkin JS, Pepe GJ, Albrecht ED. Effect of the antiestrogen ethamoxytriphetol (MER-25) on placental low density lipoprotein uptake and degradation in baboons. Endocrinology 1988 122:2019-2026[Abstract/Free Full Text]
  11. Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss JF III. Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 1986 118:1567-1582[Abstract/Free Full Text]
  12. Riedy MC, Timm EA Jr, Stewart CC. Quantitative RT-PCR for measuring gene expression. Biotechniques 1995 18:70-76[Medline]
  13. Babischkin JS, Pepe GJ, Albrecht ED. Estrogen regulation of placental P-450 cholesterol side-chain cleavage enzyme messenger ribonucleic acid levels and activity during baboon pregnancy. Endocrinology 1997 138:452-459[Abstract/Free Full Text]
  14. Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, Fiddes JC, Abraham JA. The human gene for vascular endothelial growth factor. J Biol Chem 1991 266:11947-11954[Abstract/Free Full Text]
  15. Babischkin JS, Pepe GJ, Albrecht ED. Developmental expression of placental trophoblast P-450 cholesterol side-chain cleavage, adrenodoxin and {Delta}5-3ß-hydroxysteroid dehydrogenase/isomerase messenger ribonucleic acids during baboon pregnancy. Placenta 1996 17:595-602[CrossRef][Medline]
  16. Hyder SM, Stancel GM. Regulation of angiogenic growth factors in the female reproductive tract by estrogens and progestins. Mol Endocrinol 1999 13:806-811[Free Full Text]
  17. Mueller MD, Vigne JL, Minchenko A, Lebovic DI, Leitman DC, Taylor RN. Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors alpha and beta. Proc Natl Acad Sci U S A 2000 97:10972-10977[Abstract/Free Full Text]
  18. Billiar RB, Pepe GJ, Albrecht ED. Immunocytochemical identification of the oestrogen receptor in the nuclei of human placental syncytiotrophoblasts. Placenta 1997 18:365-370[CrossRef][Medline]
  19. Bukovsky A, Caudle MR, Cekanova M, Fernando RI, Wimalasena J, Foster JS, Henley DC, Elder RF. Placental expression of estrogen receptor beta and its hormone binding variant-comparison with estrogen receptor alpha and a role for estrogen receptors in asymmetric division and differentiation of estrogen-dependent cells. Reprod Biol Endocrinol 2003 1:36-56[CrossRef][Medline]
  20. Pepe GJ, Davies WA, Dong KW, Luo H, Albrecht ED. Cloning of the 11ß-hydroxysteroid dehydrogenase (11ß-HSD) gene in the baboon: effects of estradiol on promoter activity of 11ß-HSD-1 and -2 in placental JEG-3 cells. Biochim Biophys Acta 1999 144:101-110
  21. Sharkey AM, Charnock-Jones DS, Boocock CA, Brown KD, Smith SK. Expression of mRNA for vascular endothelial growth factor in human placenta. J Reprod Fertil 1993 99:609-615
  22. Jackson MR, Carney EW, Lye SJ, Knox Ritchie JW. Localization of two angiogenic growth factors (PDECGF and VEGF) in human placentae throughout gestation. Placenta 1994 15:341-353[Medline]
  23. Ahmed A, Li XF, Dunk C, Whittle MJ, Rushton DI, Rollason T. Colocalisation of vascular endothelial growth factor and its flt-1 receptor in human placenta. Growth Factors 1995 12:235-243[Medline]
  24. Clark DE, Smith SK, Sharkey AM, Charnock-Jones DS. Localization of VEGF and expression of its receptors flt and KDR in human placenta throughout pregnancy. Human Reprod 1996 11:1090-1098[Abstract/Free Full Text]
  25. Albrecht ED, Aberdeen GW, Niklaus AL, Babischkin JS, Suresch DL, Pepe GJ. Acute temporal regulation of vascular endothelial growth/ permeability factor expression and endothelial morphology in the baboon endometrium by ovarian steroids. J Clin Endocrinol Metab 2003 88:2844-2852[Abstract/Free Full Text]
  26. Shifren JL, Tseng JF, Zaloudek CJ, Ryan IP, Meng YG, Ferrara N, Jaffe RB, Taylor RN. Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab 1996 81:3112-3118[Abstract/Free Full Text]
  27. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992 359:843-845[CrossRef][Medline]
  28. Shore VH, Wang TH, Wang CL, Torry RJ, Caudle MR, Torry DS. Vascular endothelial growth factor, placenta growth factor and their receptors in isolated human trophoblast. Placenta 1997 18:657-665[CrossRef][Medline]
  29. Wheeler T, Elcock CL, Anthony FW. Angiogenesis in the placental environment. Placenta 1995 16:289-296[CrossRef][Medline]
  30. Albrecht ED, Pepe GJ. Placental steroid hormone biosynthesis in primate pregnancy. Endocr Rev 1990 11:124-150[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
T. W. Bonagura, G. J. Pepe, A. C. Enders, and E. D. Albrecht
Suppression of Extravillous Trophoblast Vascular Endothelial Growth Factor Expression and Uterine Spiral Artery Invasion by Estrogen during Early Baboon Pregnancy
Endocrinology, October 1, 2008; 149(10): 5078 - 5087.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
D. P. Hewitt, P. J. Mark, and B. J. Waddell
Glucocorticoids Prevent the Normal Increase in Placental Vascular Endothelial Growth Factor Expression and Placental Vascularity during Late Pregnancy in the Rat
Endocrinology, December 1, 2006; 147(12): 5568 - 5574.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/5/1694    most recent
biolreprod.104.030882v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Robb, V. A.
Right arrow Articles by Albrecht, E. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Robb, V. A.
Right arrow Articles by Albrecht, E. D.
Agricola
Right arrow Articles by Robb, V. A.
Right arrow Articles by Albrecht, E. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS