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Endocrinology Vol. 142, No. 5 2050-2057
Copyright © 2001 by The Endocrine Society

ARTICLES

Developmental Regulation of Vascular Endothelial Growth/Permeability Factor Messenger Ribonucleic Acid Levels in and Vascularization of the Villous Placenta During Baboon Pregnancy1

Victoria A. Hildebrandt, Jeffery S. Babischkin, Robert D. Koos, Gerald J. Pepe and Eugene D. Albrecht

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

Address all correspondence and requests for reprints to: Eugene D. Albrecht, Ph.D., 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
 
Vascular endothelial growth/permeability factor (VEG/PF) has an important role in angiogenesis; however, very little is known about the developmental regulation of VEG/PF and the vascular system within the placenta during human pregnancy. In the present study, therefore, a developmental approach was used in the baboon to determine the placental source of VEG/PF and its fms-like tyrosine kinase (flt-1) and kinase-insert domain containing (KDR/flk-1) receptors, and whether the rise in estrogen with advancing pregnancy was associated with a corresponding increase in placental VEG/PF expression and vascularization. VEG/PF messenger RNA (mRNA) levels were determined by competitive RT-PCR in villous cell fractions isolated by Percoll gradient centrifugation from placentas obtained on days 45 and 54 (very early), 60 (early), 100 (mid), and 165–170 (late) of baboon pregnancy (term = 184 days). Maternal peripheral serum estradiol increased from very low concentrations early in gestation (0.15–0.20 ng/ml) to an early surge of over 2.5 ng/ml on days 60–85, and peak levels of 4–6 ng/ml late in baboon pregnancy. VEG/PF mRNA was expressed in low level in the syncytiotrophoblast (<2,000 attomol/µg total RNA), and values in this fraction did not change significantly with advancing gestation. VEG/PF mRNA expression was slightly greater in the inner villous core cell fraction; however, levels decreased (P < 0.05) between early and late gestation. Cytotrophoblasts were a major source of VEG/PF mRNA and levels increased (P < 0.01) from 3,631 ± 844 attomol/µg total RNA on day 45 to 25,807 ± 5,873 attomol/µg total RNA on day 170. VEG/PF protein expression determined by immunocytochemistry was abundant in cytotrophoblasts and lower in the syncytiotrophoblast and inner villous core cells. The flt-1 and KDR/flk-1 receptors were expressed in the vascular endothelial cells of the baboon villous placenta. The percentage of villous placenta occupied by blood vessels and the number of vessels/mm2 villous tissue, determined by image analysis, progressively increased (P < 0.001; r = 0.97) from 3.4 ± 0.2% and 447 ± 29, respectively, on day 54 to 15.9 ± 0.9% and 1,375 ± 71, respectively, on day 170. In summary, the present study shows that villous cytotrophoblasts were a major source of VEG/PF mRNA and protein in the baboon villous placenta, and that cytotrophoblast VEG/PF mRNA levels and vascularization of the villous placenta closely paralleled the increase in estradiol concentrations of advancing pregnancy. These results are consistent with the concept that estrogen has an important role in establishing the new vascular system within the developing placenta during primate pregnancy and that VEG/PF mediates this process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING HUMAN and nonhuman primate pregnancy, the placenta undergoes extensive neovascularization to promote fetal growth and development. The vascular network within the placental villi develops by in situ differentiation of fetal mesenchymal cells, i.e. vasculogenesis, and proliferation of existing vessels, i.e. angiogenesis, a process which results from proteolysis of the basal lamina and proliferation and migration of endothelial cells. Vascular endothelial growth/permeability factor (VEG/PF) is a potent endothelial cell-specific mitogen that stimulates the formation of new blood vessels (Refs. 1, 2, 3 , for reviews) and vascular permeability (4, 5). VEG/PF is encoded from a single gene by differential exon splicing and expressed as 5 isoforms of 121, 145, 165, 189, and 206 amino acids (6 , for review), although the 121 and 165 isoforms seem to have the greatest angiogenic activity (7). Inactivation of the VEG/PF gene in transgenic mice results in significant defects in the vasculature of embryonic tissues and organs, as well as embryonic lethality (8). VEG/PF messenger RNA (mRNA) and protein are expressed by cytotrophoblasts, syncytiotrophoblast, and Hofbauer macrophage cells within the human villous placenta (9, 10, 11, 12, 13, 14). VEG/PF binds to two structurally related transmembrane tyrosine kinase receptors, fms-like tyrosine kinase (flt-1) and kinase-insert domain containing (KDR/flk-1) receptors (15, 16), which are expressed by vascular endothelial cells within placental villi (12, 17).

Although VEG/PF has an important role in angiogenesis, very little is known about the regulation of VEG/PF expression by the placenta during human pregnancy. Using the baboon as a primate model to study the endocrinology of human pregnancy, we have shown that estrogen has a central integrative role in modulating communication between the placenta and fetus critical for fetal development (18, 19). Estrogen up-regulates VEG/PF expression in the rat (20, 21), sheep (22), and human (23, 24) uterus. Estrogen also rapidly stimulates angiogenesis in the rat (25, 26) and sheep (27) uterus and enhances uteroplacental blood flow (28, 29, 30), in addition to regulating other aspects of cardiovascular function during pregnancy (30, 31). We propose, therefore, that one mechanism by which estrogen promotes placental and fetal development in the primate is to regulate expression of VEG/PF by, and consequently neovascularization of, the villous placenta.

In the present study, therefore, a developmental approach was used in the baboon to determine the placental cell source of VEG/PF and its flt-1 and KDR/flk-1 receptors, and whether the rise in levels of estrogen with advancing pregnancy was associated with a corresponding increase in placental VEG/PF expression and vascularization.


   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 aluminum stainless-steel large 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.

At 1- to 2-day intervals between days 20 and 170 of gestation (length of gestation is 184 days), pregnant baboons were briefly sedated with ketamine HCl (10 mg/kg body weight im) and a 2–4 ml blood sample was collected from a maternal peripheral saphenous vein. Serum estradiol was determined by RIA using an automated chemiluminescent immunoassay system (Immulite, Diagnostic Products Corp., Los Angeles, CA) as described previously (32).

Baboons were cared for and used strictly in accordance with USDA regulations and the NIH Guide for the Care and Use of Laboratory Animals (Publication No. 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 cell isolation
Placentas were obtained from baboons by cesarean section under halothane-nitrous oxide anesthesia on days 45 and 54 (very early), 60 (early), 100 (mid) or 165–170 (late) of gestation. Two to four randomly selected sections (approximately 4 mm3each) of villous tissue in areas lacking calcification or infarction were excised from each placenta and stored frozen in liquid nitrogen for RNA analysis. Two to four additional sections (4 mm3) of villous tissue were similarly collected and fixed overnight in 10% buffered formalin and embedded in paraffin for immunocytochemical analyses and vessel quantification.

From the majority of the remaining placental villous tissue, enriched fractions of cytotrophoblasts, syncytiotrophoblast, and cells of the inner villous core were obtained as previously described by our laboratory (33) and Kliman et al. (34) and used for quantification of VEG/PF mRNA levels by RT-PCR. Briefly, villous tissue was minced in HBSS (Life Technologies, Inc., Gaithersburg, MD), and digested in 100 ml calcium- and magnesium-free HBSS containing 0.025% soybean trypsin inhibitor (Type 1-S, Sigma, St. Louis, MO), 0.1% hyaluronidase (type I-S, Sigma), 0.1% collagenase (Type H, Sigma Blend), 0.01% deoxyribonuclease I (1680 Kunitz Units/mg, Sigma), 1.0% FBS (Life Technologies, Inc.), and 4 mM sodium bicarbonate (pH7.4) in a shaking water bath at 37 C for 40 min. Enriched cell fractions were then isolated by 5–70% Percoll (Pharmacia Fine Chemicals, Piscataway, NJ) gradient centrifugation at 1,200 x g. Kliman et al. (34) and we (unpublished data) have previously shown that highly enriched cytokeratin-positive cytotrophoblast, placental lactogen-positive syncytiotrophoblast, and {alpha}-antichymotrypsin-positive inner villous cell fractions were obtained from the human and baboon placenta using the latter cell isolation procedure.

RT-PCR of VEG/PF
The mRNA levels for VEG/PF were quantified by the competitive RT-PCR assay established by Riedy et al. (35), as modified by our laboratory (36). Placental cell fractions were homogenized in 4 M guanidine isothiocynate, 0.025 M sodium acetate (pH 6.0), and 0.83% 2ß-mercaptoethanol, extracted with chloroform: isoamyl alcohol (24, 1), layered over 5.7 M cesium chloride, and total RNA pelleted by centrifugation for 21 h at 174,000 x g at 20 C.

Oligonucleotide primers were selected from the human VEG/PF complementary DNA sequence (37), spanned exons 1, 2, and 3 (first bp of initiating codon is designated as 1), and were synthesized by Life Technologies, Inc.:

Primer 1: downstream, 5'-GGTGAGGTTTGATCCGCATAATCTGCGCATCAGGGGCACACAGGAT-3' (position 336–311 linked to 243–224); Primer 2: upstream, 5'-AATTTAATACGACTCACTATAGGGACTGCTGTCTTGGGTGCATTGG-3' (position T7 polymerase sequence [underlined] linked to 10–30); Primer 3: downstream, 5'-GGTTTGATCCGCATAATCTGC-3' (position 331–311); 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, these primers generated a single 323-bp PCR product. The competitive reference standard (CRS) was prepared by the methods of Riedy et al. (35) 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 at 42 C for 60 min in a 20 µl reaction mixture containing 1 mM each of deoxy(d)ATP, dCTP, dGTP, and dTTP (Life Technologies, Inc.), 1 mM dithiothreitol, 200 U Superscript RNase H-RT (Life Technologies, Inc.), 40 U RNAguard ribonuclease inhibitor (Pharmacia Biotech, Piscataway, NJ), 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 250 ng random primers (Life Technologies, Inc.). After 60 min, the RT mixture was incubated at 70 C for 15 min and cooled to 4 C and 5 µl of the RT mixture was added to a PCR mixture (45 µl) containing 0.2 mM each of dATP, dCTP, dGTP, dTTP (Life Technologies, Inc.), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 1.25 U cloned Thermus aquaticus DNA polymerase (Amplitaq, Perkin-Elmer 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. After the last cycle, samples were incubated for an additional 5 min at 72 C to allow for complete PCR product synthesis. A portion of the PCR mixture was fractionated by electrophoresis in a 2% agarose gel and visualized with ethidium bromide. The amplified product contained a sequence for the T7 polymerase, as well as the designated deletion, compared with the wild-type mRNA strand. A second round of PCR was performed to generate additional PCR product which was gel purified twice using a Qiaex DNA gel extraction kit (QIAGEN, Inc., Valencia, CA). The CRS was synthesized from 150 ng of complementary DNA template using the MEGAscript T7 in vitro transcription kit (Ambion, Inc., Austin, TX).

To quantify VEG/PF mRNA, a constant amount of total RNA was added to an RT mixture containing 3-fold serial dilutions of the VEG/PF CRS (5400–200 attomol). Upon completion of the RT, 5 µl of the RT mixture was added to a PCR mixture containing 20 pmol each of primers 3 and 4 (26 cycles). 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. 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 scanning using a model 620 Video Densitometer and 1-D Analyst II data analysis software (Bio-Rad Laboratories, Inc., Hercules, CA). The intensity of amplified mRNA product was represented as the relative area under each sample band. A correction factor (38) was used to account for the relative size difference between the CRS and the wild-type target mRNA: corrected relative area = VEG/PF CRS relative area x VEG/PF target mRNA length (323 bp)/CRS mRNA length (256 bp). 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. The concentration of VEG/PF target mRNA was determined where the ratio of CRS and target mRNA was equal to 1 (i.e. the equivalence point).

To identify which VEG/PF isoforms were expressed in the whole villous placenta, total RNA (1 µg) was reversed transcribed as described above and upon completion of the RT, 20 pmol each of VEG/PF primers which spanned the alternative splice site resulting in individual amplification of all VEG/PF isoforms (20), was added for the PCR (26 cycles). The PCR products were fractionated in a 2% agarose gel containing ethidium bromide, visualized with an UV transilluminator, and photographed.

To confirm that any developmental changes in VEG/PF mRNA expression were specific, steady-state levels of a constitutively expressed cellular RNA [18S ribosomal RNA (rRNA)] were also semiquantified in the various placental cell fractions by RT-PCR using specific 18S rRNA primers.

Immunohistochemistry of Factor VIII, VEG/PF, flt-1 and KDR/flk-1
Paraffin blocks of placental villous tissue were serially sectioned (4 µm), deparaffinized, rehydrated in graded concentrations of ethanol, and incubated in a dilute trypsin solution at 37 C for 15 min (flt-1 and KDR/flk-1) or boiled in 0.01 M Na citrate (pH 6.0) for 6 min (VEG/PF and human endothelial cell-specific von Willebrand-factor, Factor VIII). Tissues were then incubated in H2O2 for 15 min to inhibit endogenous peroxidase and blocked with 10% normal goat serum for 1 h. Tissues were incubated overnight at 4 C in a humidified chamber with polyclonal rabbit antibody to Factor VIII (1:1,500 dilution, DAKO Corp., Santa Barbara, CA), goat antibody to VEG/PF (AF-293-NA, 1:25 dilution, R & D Systems, Minneapolis, MN), or rabbit antibodies to flt-1 (C-17, 1:500) or KDR/flk-1 (C-1158, 1:250, both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Tissues were incubated 1 h (room temperature) with either biotinylated antigoat or antirabbit immunoglobulins (Vector Laboratories, Inc., Burlingame, CA), then 1 h with an avidin-biotin-peroxidase complex (ABC Elite, Vector Laboratories, Inc.). Sections were developed using diaminobenzidine (Sigma) with 2.5% nickel chloride as the chromagen to produce a black reaction product localizing Factor VIII or diaminobenzidine only for VEG/PF, flt-1 and KDR/flk-1. Tissue sections were lightly counterstained with eosin (Factor VIII) or Harris’ hematoxylin (others).

Negative controls included omission of the primary antibody, substitution of goat/rabbit immunoglobulin IgG (DAKO Corp.) for primary antibody, and/or preabsorption of the primary antibody with 10-fold excess of control peptide for flt-1 (Santa Cruz Biotechnology, Inc.) or a 10-fold excess of human recombinant VEG/PF protein (R & D Systems).

Quantification of placental vascularization by image analysis
Quantification of blood vessels, i.e. arterioles, arteries, venules, and veins, in placental villous tissue was accomplished using a Nikon Eclipse E 1000 M microscope (Nikon, Tokyo, Japan) coupled to a color video camera (3 CCD, Dage-MTI, MI City, IN) via a video tube. Color images were digitized by a Power Macintosh G3 computer and visualized on a high resolution monitor. An image-analysis software package (IP Lab Scientific Image Processing, Scanalytics, Fairfax, VA) was used for interactive manipulation of the image and data collection. The method of computer-aided vessel counting was based on the ability to shade-in Factor VIII immunoreactive vessels with a green pseudo-color and circumscribe total villous area examined with a yellow pseudo-color. The software quantified highlighted pixels into individual vessel areas and information regarding vessel number and area was imported into a worksheet program (Excel) and saved.

The percentage of the villous placenta occupied by blood vessels, i.e. percent vascularized area, was determined by dividing total vessel area by the total villous area examined. Number of vessels per mm2 of villous tissue and the size distribution of vessels were also determined. A mean (±SE) value for vessel counts on each placenta was determined on 20 to 40 different placental villous sections (2 views per villous section x 5 sections per biopsy x 2–4 biopsies per placenta).

Statistics
Data are expressed as the means ± SE. Statistical differences between groups were determined by ANOVA with posthoc comparisons of the means by Newman-Keuls multiple comparison test. Linear regression analysis was used to determine changes in placental vascularization with advancing gestation and to correlate placental VEG/PF mRNA and serum estradiol levels.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum estradiol
Maternal peripheral serum estradiol increased from very low concentrations early in gestation (0.15–0.20 ng/ml, Fig. 1Go) to approximately 0.35 ng/ml on days 53–59 of gestation. Estradiol then exhibited an early surge of over 2.5 ng/ml on days 60–85, a transient decline to approximately 1 ng/ml at midgestation, and a progressive increase thereafter reaching peak levels of 4–6 ng/ml late in gestation.



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  		Figure 1. Maternal peripheral serum estradiol concentrations in baboons between days 20 and 170 of gestation (length of gestation is 184 days). Each data point represents mean estradiol level of at least six animals (modified from Ref. 32 ).

 
RT-PCR of VEG/PF
Using RT-PCR and primers that spanned the alternative splice site, the mRNAs for VEG/PF 121 (434 bp), 165 (566 bp), and 189 (638 bp) were expressed in whole placental villous tissue (Fig. 2Go), whereas VEG/PF 145 (506 bp) was visible only after a second round of PCR (data not shown). The relative level of expression of different isoforms in whole villous tissue appeared similar at early, mid, and late baboon gestation. RT-PCR products were not observed when either RT enzyme or RNA were omitted from the reaction.



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  		Figure 2. RT-PCR analysis of VEG/PF mRNA isoforms in baboon whole placental villous tissue obtained at early (day 60), mid (day 100), and late (day 170) gestation. Total RNA (1 µg) was reversed transcribed and amplified for 26 cycles using VEG/PF primers that spanned the alternative splice site.

 
Because level of expression of the various VEG/PF isoforms in whole placental villous tissue appeared similar throughout gestation, placental VEG/PF mRNA levels in villous cell fractions were quantified by competitive RT-PCR using primers upstream from the alternative splice site which yielded a single 323-bp product. Figure 3Go shows a representative quantitative analysis of VEG/PF mRNA levels by competitive RT-PCR in cytotrophoblasts obtained at early and late baboon gestation. The expected 323-bp VEG/PF target product and the 256-bp VEG/PF CRS product generated by PCR are shown in Fig. 3AGo. No PCR product was detected when either RNA or RT enzyme was omitted from the reaction (data not shown). The slopes of the log of the CRS to target areas plotted as a function of increasing amounts of CRS were similar for RNA obtained from cytotrophoblasts at early and late gestation (Fig. 3BGo), indicating no difference in amplification efficiency. However, analysis of the equivalence points from each plot indicated that VEG/PF mRNA levels were greater in cytotrophoblasts in late than early gestation.



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  		Figure 3. Representative competitive RT-PCR of VEG/PF in baboon placental cytotrophoblasts. Total RNA (75 ng) from placental cytotrophoblasts obtained at early (day 60) and late (day 170) gestation 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 CRS separated on 2% agarose gels and stained with ethidium bromide. B, Intensities of amplified products in A were analyzed by densitometry, and the log of the ratios of VEG/PF CRS and target areas in cytotrophoblasts from early (•) and late ({circ}) gestation 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).

 
Figure 4Go illustrates the developmental pattern of VEG/PF mRNA expression in syncytiotrophoblast, inner villous core cells, whole villous tissue obtained from baboons on days 60–170 of gestation, and villous cytotrophoblasts obtained from baboons on days 45, 54, 60, 100, and 170 of gestation. VEG/PF mRNA appeared to be expressed in low levels in the syncytiotrophoblast (<2,000 attomol/µg total RNA) throughout gestation (Fig. 4Go), although the quality of RNA (i.e. ratio of distinct 28S and 18S rRNA bands after mini-gel electrophoresis) did not appear as good in syncytiotrophoblast when compared with the other placental cell types (data not shown). VEG/PF mRNA expression on day 60 appeared slightly greater in the inner villous cells than in syncytiotrophoblast, and decreased (P < 0.05, ANOVA) in this cell fraction from a mean (± SE) of 3,462 ± 642 attomol/µg total RNA on day 60 to 1,724 ± 287 attomol/µg RNA on day 170 of gestation. In contrast, VEG/PF mRNA levels in whole villous tissue approximated 4,000 attomol/µg RNA and did not change significantly with gestation (Fig. 4Go). Of the placental cell fractions examined, villous cytotrophoblasts were the major source of VEG/PF mRNA throughout baboon pregnancy. In cytotrophoblasts, VEG/PF mRNA increased from 3,631 ± 844 attomol/µg RNA on day 45 to 10,959 ± 2,308 attomol/µg RNA on day 60, then declined slightly at midgestation, before exhibiting a 2.5-fold increase (P < 0.01) on day 170 (25,807 ± 5,873 attomol/µg RNA). This developmental increase in cytotrophoblast VEG/PF mRNA levels paralleled and was significantly correlated with the maternal serum estradiol concentrations shown in Fig. 1Go (analysis by linear regression, r2 = 0.843, P < 0.03). In contrast, the levels for 18S rRNA were constant between days 45 and 170 of gestation in the syncytiotrophoblast (1.51 ± 0.12, overall mean ±SE, relative optical density units), inner villous core (1.74 ± 0.06), whole villous tissue (1.60 ± 0.03), and cytotrophoblast (1.71 ± 0.05) cell fractions.



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  		Figure 4. VEG/PF mRNA levels in syncytiotrophoblast, inner villous cells, and villous cytotrophoblasts isolated by Percoll gradient centrifugation and whole villous tissue, from baboons at very early (days 45 and 54, cytotrophoblasts only), early (day 60), mid (day 100) and late (day 170) gestation. Total RNA was analyzed by competitive RT-PCR using primers upstream from the alternative splice site. Individual values represent the means (±SE) of at least four baboons for each placental cell fraction and each stage of gestation.

 
Immunocytochemistry of VEG/PF, flt-1, and KDR/flk-1
VEG/PF protein was localized by immunocytochemistry in relatively high level within the cytoplasm of villous cytotrophoblasts (Fig. 5AGo). VEG/PF immunoreactivity was also observed in cytoplasm of the apical border of the syncytiotrophoblast and in vascular endothelial cells consistent with binding to VEG/PF receptors in these two locations, as well as in presumptive Hofbauer macrophage cells within the inner villous core.



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  		Figure 5. Representative photomicrographs of VEG/PF (A), flt-1 (B), and KDR/flk-1 (C) immunocytochemistry in the villous placenta on day 170 of baboon pregnancy. D, VEG/PF immunocytochemistry using primary VEG/PF antibody preabsorbed with excess human recombinant VEG/PF. C, Cytotrophoblast nucleus; S, syncytiotrophoblast nucleus; Ve, vascular endothelial cell. Magnification, x200. Inset in panel A shows a cytotrophoblast magnified x 400.

 
There was considerable immunocytochemical staining for flt-1 (Fig. 5BGo) and KDR/flk-1 (Fig. 5CGo) VEG/PF receptors in vascular endothelium of the baboon villous placenta. The villous syncytiotrophoblast also exhibited substantial immunoreactivity for flt-1, but not KDR/flk-1. When the primary antibody was preabsorbed with excess immunogen, there was little or no immunoreactivity for VEG/PF (Fig. 5DGo) or the flt-1 or KDR/flk-1 receptors (data not shown).

Placental vascularization
The percent vascularized area of the villous placenta increased (P < 0.001, linear regression, r = 0.97) from a relatively low level on days 54 (3.4 ± 0.2) and 60 (3.5 ± 0.2) of gestation to a maximum of 15.9 ± 0.9 on day 170 (Fig. 6Go). A similar pattern was found when vascularity was determined as the number of vessels/mm2 villous tissue (Fig. 7Go). Thus, vascular density increased (P < 0.01) from 447 ± 29 and 493 ± 34 on days 54 and 60, respectively, to 977 ± 101 on day 100 and exhibited a further increase (P < 0.001) to 1,375 ± 71 on day 170.



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  		Figure 6. Percent vascularized area, determined by image analysis as the ratio of total vessel area and total villous area examined, of placental villous tissue obtained from baboons at very early (day 54), early (day 60), mid (day 100), and late (day 170) gestation. Each data point represents the mean (±SE) determined on 20–40 different tissue sections examined for a single baboon placenta.

 


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  		Figure 7. Vascular density (vessels/mm2) determined in placental villous tissue of the same baboons on which data are shown in Fig. 6Go. Values (means ± SE) with different superscript letters are different (P < 0.01–0.001, ANOVA, Newman Keuls multiple comparison test) from each other. n = 6 placentas at each gestational stage.

 
Figure 8Go, which illustrates the size distribution of blood vessels in the villous placenta, indicates that at all stages of gestation the majority of the vessels were less than 200 µm2 in cross-sectional area. On days 54, 60, and 100 of gestation, the size distribution of vessels was similar. In contrast, on day 170 there were fewer (P < 0.01) blood vessels of less than 100 µm2 and more (P < 0.05–0.01) vessels of greater than 100 µm2 when compared with the earlier stages of gestation.



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  		Figure 8. Size distribution of blood vessels determined in placental villous tissue of the same baboons on which data are shown in Fig. 6Go. Values are the means (± SE) of six placentas examined at each stage of gestation. Different at P < 0.05* or 0.01** (ANOVA, Newman-Keuls multiple comparison test) for that respective vessel size/class compared with other stages of gestation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study show that villous cytotrophoblasts were a major source of VEG/PF mRNA and protein in the baboon villous placenta and that VEG/PF mRNA levels in cytotrophoblasts closely paralleled maternal serum estradiol concentrations throughout baboon pregnancy. Thus, the early surge in estrogen on day 60 and progressive increase in estrogen during the second half of baboon pregnancy were associated with corresponding increases in cytotrophoblast VEG/PF mRNA levels. We have shown that estrogen receptor {alpha} is expressed within baboon villous cytotrophoblasts (39). Estrogen rapidly stimulated VEG/PF mRNA expression in vivo in the rat (20, 21) and sheep (22) uterus and in a time-dependent manner in vitro in human endometrial cells (23, 24). Moreover, chronic estrogen treatment induced angiogenesis in the uterus of normal but not estrogen receptor null transgenic mice (26). Collectively, the results of these prior studies and the current study are consistent with the concept that estrogen regulates VEG/PF expression by the villous trophoblast during advancing primate pregnancy. Additional in vivo studies in which estrogen levels are experimentally altered during primate pregnancy, and/or in vitro studies using cultures of trophoblast cells, must be performed to definitively establish such a role for estrogen on placental VEG/PF expression.

The current study further shows that the developmental increase in estrogen and VEG/PF expression was associated with a progressive rise in placental vascularization. Considering the well established role of VEG/PF in angiogenesis and the results of the present study, it appears that estrogen has an important role in establishing the vascular system within the developing placenta during primate pregnancy and that VEG/PF mediates this process. Thus, the well established action of estrogen in enhancing uteroplacental blood flow (28, 29, 40) may reflect enhanced microvascularization/angiogenesis (27), as well as vasodilation. The increases in vascularization, vessel density, and proportion of larger blood vessels in the villous placenta during latter stages of baboon pregnancy are consistent with increased vascularization of the villous placenta during advancing human pregnancy (41).

The observation of high expression of VEG/PF mRNA and protein in villous cytotrophoblasts of the baboon placenta is consistent with the demonstration by in situ hybridization and immunocytochemistry of VEG/PF mRNA and protein in human placental cytotrophoblasts (10, 12, 13). VEG/PF mRNA expression by villous cytotrophoblasts isolated and assayed by RNase protection was also demonstrated in the human placenta (42). The present study used a very different experimental approach, however, to show for the first time that there was a developmental increase in cytotrophoblast VEG/PF mRNA expression with advancing primate pregnancy. The extensive expression of VEG/PF by villous cytotrophoblasts juxtaposed the inner villous mesenchyme would provide a system for promoting angiogenesis in the fetal compartment of the developing placenta, particularly in the first half of gestation.

Although VEG/PF protein was clearly localized by immunocytochemistry in the baboon syncytiotrophoblast, VEG/PF mRNA expression in the latter cell fraction was low and did not change throughout baboon pregnancy. Although this apparently low level of VEG/PF mRNA expression may in part reflect some RNA degradation in syncytiotrophoblast, mRNA levels for other regulatory components, e.g. P-450 cholesterol side-chain cleavage enzyme (43) and chorionic somatomammotropin (44), progressively increase with advancing baboon pregnancy in syncytiotrophoblast isolated and analyzed by the same procedures used in the present study. The latter indicates that methodological aspects of syncytiotrophoblast isolation cannot account for the absence of change in syncytiotrophoblast VEG/PF mRNA expression during baboon pregnancy.

The presence of VEG/PF within baboon syncytiotrophoblast is consistent with VEG/PF mRNA and protein expression, determined by in situ hybridization, RNase protection assay, and immunocytochemistry, in human placental syncytiotrophoblast (10, 12, 13, 42). In addition to expression by the syncytiotrophoblast, localization of VEG/PF protein in baboon syncytiotrophoblast may reflect binding to syncytiotrophoblast of VEG/PF produced by other cells, such as cytotrophoblasts. Indeed, VEG/PF flt-1 receptor was expressed in baboon placental syncytiotrophoblast, as shown previously in the human placenta (11, 12, 13). In addition, flt-1 and KDR/flk-1 proteins were expressed in inner villous vascular endothelium in both species (12, 13), as anticipated for the action of VEG/PF in promoting angiogenesis in this location. Thus, the qualitative patterns of cellular expression of VEG/PF and flt-1 and KDR/flk-1 receptors appear very similar in the baboon and human villous placenta. These observations point to the value of the baboon as a nonhuman primate model to study regulation of placental angiogenesis.

There is a progressive increase in cytotrophoblast and syncytiotrophoblast volumetric composition with advancing human (45) and baboon (46) gestation. However, as a result of morphological differentiation of cytotrophoblasts into syncytiotrophoblast, the ratio of syncytiotrophoblast: cytotrophoblast volumes is greatly increased in late baboon pregnancy (46). Consequently, even though syncytiotrophoblast VEG/PF mRNA expression remained relatively constant and low through gestation, with increase in mass the syncytiotrophoblast may also become an important source of VEG/PF for angiogenesis within the villous placenta in latter stages of primate pregnancy.

VEG/PF mRNA levels were moderately high in inner villous cells isolated from the baboon placenta, presumably reflecting expression shown by immunocytochemistry in presumptive Hofbauer macrophages and possibly fibroblasts. Stromal cells of the human villous placenta, particularly Hofbauer macrophages, also express VEG/PF mRNA and protein (9, 12, 13). The decline in VEG/PF mRNA levels in the inner villous cell fraction between early and late baboon pregnancy may have resulted from decreased numbers of Hofbauer cells and fibroblasts, which has been shown in the second half of baboon pregnancy (47). The decline in inner villous core combined with the increase in cytotrophoblast VEG/PF mRNA levels between early and late gestation presumably accounted for the lack of change with gestation in VEG/PF mRNA levels measured in whole villous placenta. These results point to the limitation of analyzing whole villous placental tissue when studying physiological processes such as angiogenesis in a complex heterogenous tissue such as the primate placenta.

The early surge in estradiol levels observed on day 60 of baboon pregnancy may have a role in accelerating placental angiogenesis and other aspects of fetal-placental development. A similar peak in estradiol seems to appear in the second trimester of human pregnancy (48). The factors contributing to this surge in estrogen remain to be determined. However, because placental syncytiotrophoblast volume progressively increases with advancing human (45) and baboon (46) gestation, and P-450 aromatase is not rate-limiting for placental estrogen biosynthesis (49), it is unlikely that the early surge in estrogen reflects a change in trophoblast mass or aromatase levels. Possibly, this rise in estrogen results from increased production of C19-steroid estrogen precursors, e.g. dehydroepiandrosterone and androstenedione, by the fetal and/or maternal adrenal glands.

Although serum estradiol and cytotrophoblast VEG/PF mRNA levels exhibited a transient decrease at midgestation, placental vascularization progressively increased through early, mid, and late baboon gestation. Therefore, factors in addition to estrogen may regulate placental neovascularization during primate pregnancy. Alternatively, because only VEG/PF mRNA was measured in the current study, trophoblast VEG/PF protein formation/levels may increase in a more linear fashion with advancing pregnancy to account for the constant rise in vascularization. Additional study is needed to determine these possibilities.

In summary, the present study shows that villous cytotrophoblasts were a major source of VEG/PF mRNA and protein in the baboon villous placenta. Moreover, cytotrophoblast VEG/PF mRNA levels and placental vascularization closely paralleled the increase in estradiol concentrations of advancing baboon pregnancy. These results are consistent with the concept that estrogen may have an important role in establishing the new vascular system within the developing placenta during primate pregnancy and that VEG/PF mediates this process.


   Acknowledgments
 
The authors sincerely appreciated the assistance of Dr. Graham W. Aberdeen with the experimental procedures in baboons and Ms. Donna L. Suresch with the immunocytochemistry. The secretarial assistance of Mrs. Wanda H. James with the manuscript is gratefully acknowledged.


   Footnotes
 
1 This work was supported by NIH Research Grant R-01-HD-13294 and NIH Grant U-54-HD-36207 as part of the Specialized Cooperative Centers Program in Reproduction Research. Back

Received September 8, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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