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Regular Articles |
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From the Departments of Stomatology,*Anatomy, Obstetrics, Gynecology, andReproductive Sciences, and PharmaceuticalChemistry,¶ University of California SanFrancisco, San Francisco, California; and the Molecular/Cancer BiologyLaboratory, Biomedicum Helsinki and LudwigInstitute for Cancer Research, University of Helsinki, Helsinki,Finland
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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1, an adhesion molecule highly expressed by endovascular
cytotrophoblasts, and increased apoptosis. In severe
preeclampsia and hemolysis, elevated liver enzymes, and
low platelets syndrome, immunolocalization on tissue sections
showed that cytotrophoblast VEGF-A and VEGFR-1 staining decreased;
staining for PlGF was unaffected. Cytotrophoblast secretion of the
soluble form of VEGFR-1 in vitro also increased.
Together, the results of this study showed that VEGF family
members regulate cytotrophoblast survival and that expression of
a subset of family members is dysregulated in severe forms of
preeclampsia.
) are
accomplished by the placenta’s specialized epithelial cells, termed
"cytotrophoblasts." In a subset of chorionic villi, these cells
form aggregates that attach the conceptus to the uterine wall.
Cytotrophoblasts that emanate from these sites invade the interstitium
of the decidua, the inner third of the myometrium, and uterine blood
vessels. Although there are many circumstances in which normal and
abnormal cells carry out interstitial invasion, several aspects of
cytotrophoblast vascular invasion are unique. During this process,
placental cells of fetal origin intravasate the superficial portions of
uterine vessels that lie near the implantation site. Initially these
vessels are plugged by cytotrophoblast aggregates. As invasion
proceeds, the placental cells form heterotypic interactions with, then
replace, the endothelium. They also intercalate within the muscular
tunica, a process that substantially increases the vessel diameter
while decreasing its resistance. In this manner uterine arterioles, and
to a lesser extent veins, are remodeled into unique hybrid vessels
composed of fetal and maternal cells. Once the lumina reform, uterine
blood flow is diverted to the intervillous space where nutrient, gas,
and waste exchange occur.
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6ß4 integrin, dramatically alter this repertoire as
they invade the uterus. In essence, they replace their epithelial-like
receptors with adhesion molecules typical of endothelial cells,
eg, vascular endothelial (VE)-cadherin, vascular cell adhesion
molecule-1, platelet-endothelial cell adhesion molecule-1, and Vß3
integrin. Other aspects of the cell surfaces of invasive
cytotrophoblasts also resemble vascular cells. For example, they
express urokinase plasminogen activator3
and the thrombin
receptor.4 The functional importance of this transformation program is highlighted by the fact that failures in executing portions of it are associated with a subset of pregnancy complications—for example, preeclampsia, the leading cause of maternal mortality in the Western world, which also increases perinatal mortality fivefold.5 The clinical diagnostic criteria of this syndrome include the new onset of hypertension and the appearance of proteinuria and edema during pregnancy, all of which could be explained by functional alterations in the maternal vascular endothelium.6 Preeclampsia and approximately half the cases of intrauterine growth restriction are associated with particular placental pathologies. The extent of interstitial invasion by cytotrophoblasts is variable, but frequently shallow, and endovascular invasion is consistently rudimentary, making it extremely difficult to find any maternal vessels that contain cytotrophoblasts.7,8 These anatomical defects suggested to us that in preeclampsia, cytotrophoblast differentiation along the invasive pathway is abnormal. Biopsies of the uterine wall of women with this syndrome showed that invasive cytotrophoblasts retain expression of adhesion receptors characteristic of stem cells and fail to turn on receptors that promote invasion and/or assumption of an endothelial phenotype.8 The concept that failed cytotrophoblast invasion and pseudovasculogenesis are linked to the maternal vascular pathology seems plausible, but has yet to be proved. Thus, the chain of events that links a defect in placentation to the maternal systemic disorder is under intense investigation.
We are very interested in understanding the molecular pathways that regulate cytotrophoblast pseudovasculogenesis, as well as the possible defects that occur in pregnancy complications such as preeclampsia. A vast array of physiological factors (eg, hypoxia, shear stress) and effector pathways (eg, transcription factors, growth factors, chemokines, cytokines, and protein fragments) governs blood vessel development and growth, either directly or indirectly.9 To focus the proposed investigation, we have been considering cytotrophoblast pseudovasculogenesis, which involves cells derived from the extraembryonic lineages, in the context of master regulatory pathways that govern de novo differentiation and assembly of blood vessels within the embryo. Gene deletion studies in mice have pointed to the particular importance of three families of ligands and their tyrosine kinase receptors—vascular endothelial growth factors and their receptors (VEGF/VEGFRs), angiopoietins and their Tie receptors (Ang/Tie), and ephrins and their Eph receptors (ephrin/Ephs)—in vasculogenesis and angiogenesis.10-13 The discrete phenotypes of the null animals suggest distinct roles for individual families, with VEGF family members and their receptors having important actions during the initial stages of vasculogenesis and angiogenesis, both during development and as a result of pathological processes.14,15
In accord with our hypothesis that cytotrophoblast pseudovasculogenesis co-opts a subset of the ligand-receptor interactions that govern conventional vasculogenesis, we studied the expression of VEGF family members and their receptors during this process in vivo. Immunolocalization experiments performed on tissue sections of the maternal-fetal interface showed that the expression of many of these ligands and receptors is modulated as cytotrophoblasts invade the uterus and its blood vessels in normal pregnancy. We also used an in vitro model of this process to understand the effects of interfering with interactions between VEGF and its receptors on cytotrophoblast differentiation/invasion and apoptosis. The results of these experiments, together with data showing that defects in VEGF and VEGFR expression exist in severe forms of preeclampsia, suggest that VEGF-VEGFR interactions play an important role in differentiation and survival of the unique cytotrophoblast subpopulation that invades the uterine wall, occupies the maternal vessels, and channels maternal blood to the placenta during pregnancy.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Antibodies That Recognize VEGFs and Placental Growth Factor (PlGF)
A mouse anti-VEGF-A monoclonal antibody (mAb) (M293; R & D Systems, Minneapolis, MN) and a goat anti-VEGF-C polyclonal antibody (pAb) (R&D Systems) were used at a dilution of 1:50 for immunolocalization analyses of paraffin-embedded tissues. A goat anti-PlGF-1, 2 pAb (R&D Systems) was used at a dilution of 1:25 for immunolocalization on tissues that were embedded in optimal cutting temperature (OCT) medium (Miles Scientific, Naperville, IL). A goat anti-VEGF-B pAb and a mouse anti-VEGF-D mAb, both from R&D Systems, were also used for immunolocalization analyses of OCT-embedded tissues at dilutions of 1:10 and 1:50, respectively. VEGF-A and PlGF levels in conditioned medium were measured separately using ELISA kits (R&D Systems). A mouse mAb (A4.6.1) that recognizes all isoforms of human VEGF-A (gift of Dr. N. Ferrara, Genentech, South San Francisco, CA) was used in immunoblotting experiments.
Antibodies That Recognize VEGF Receptors
The 190.11 anti-VEGFR-1 mouse mAb (diluted 1:400)16 and the rabbit N-931 anti-VEGFR-2 antibody (diluted 1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used for immunostaining. The 9D9 anti-VEGFR-3 mouse mAb (Molecular/Cancer Biology Laboratory, University of Helsinki, Finland) was used for immunostaining and immunoblotting (diluted 1:500). The C-17 rabbit anti-human VEGFR-1 antibody (diluted 1:1000, Santa Cruz Biotechnology Inc.) and the 2-10-1 anti-VEGFR-2 mAb (diluted 1:200; gift of Dr. K. Chwalisz, Scherring AG, Berlin, Germany) were used for immunoblotting. Soluble VEGFR-1 (sVEGFR-1) in conditioned medium was quantified with an ELISA kit that was developed using the 190.11 mAb.17
Antibodies That Recognize Adhesion Receptors
The TS2/7 mAb that specifically recognizes integrin 1/ß1
(Endogen, Woburn, MA) was used for immunostaining and
immunoprecipitation at a dilution of 1:50. mAbs BV6, BV9, and TEA
against VE-cadherin (gifts of Dr. Elisabetta Dejana, Mario Negri
Institute, Milan, Italy) were all used at a dilution of 1:10 for
immunostaining and immunoblotting.
Antibodies That Recognize Cytokeratin
The OV-TL 12/30 mAb against cytokeratin 7 (diluted 1:25; DAKO, Carpinteria, CA) was used for immunolocalization analyses of paraffin-embedded tissues. Rat anti-human cytokeratin mAb 7D3, produced in the Fisher laboratory, was used at a dilution of 1:50 for immunolocalization analyses of OCT-embedded tissues.18
Tissue Sources for Immunolocalization Experiments
Placentas were obtained from normal pregnant women and
patients with preeclampsia. We analyzed 19 control samples from
patients with no evidence of preeclampsia, gestational hypertension, or
a medical history that suggested an increased risk of developing
preeclampsia. Where applicable, the labor status is indicated with L
denoting women who experienced labor and NL denoting women who did not.
The gestational ages at which these samples were collected were as
follows: 6 weeks (n = 1), 10 weeks
(n = 1), 12 weeks (n =
1), 14 weeks (n = 1), 16 weeks
(n = 1), 17 weeks (n =
1), 19 weeks (n = 1), 20 weeks
(n = 1), 22 weeks (n =
1), 24 weeks (n = 1; NL), 26 weeks
(n = 2; NL), 32 weeks (n
= 1; NL), 36 weeks (n = 2; 1 L and 1 NL), 38
weeks (n = 3; L), and 40 weeks
(n = 1; NL). We analyzed 13 samples from
patients with preeclampsia diagnosed according to the classic criteria
originally recommended by Dr. Leon Chesley and modified by the National
Institutes of Health:19
no history of hypertension before
pregnancy; increase in diastolic pressure of 15 mm Hg or systolic
pressure of 30 mm Hg compared with blood pressure obtained before 20
weeks of gestation; proteinuria 
0.5 g/24 hours or 30 mg/dl (or 1+
on urine dipstick) in a catheterized specimen; hyperuricemia >5.5
mg/dl (or 1 SD greater than the normal mean value before term); return
to normal blood pressure and resolution of proteinuria by 12 weeks
postpartum. Severe preeclampsia (SPE) was diagnosed according to the
following criteria:20
systolic blood pressure 160 mm Hg
and/or diastolic pressure 110 mm Hg; proteinuria of 5 g in a
24-hour period or 3+ on urine dipstick; presence of cerebral or visual
disturbances. The criteria used to diagnose the syndrome of hemolysis,
elevated liver enzymes, and low platelets (HELLP) have been
published.21
Samples were collected for embedding in OCT
from women with the diagnoses indicated at the following weeks of
gestation: 26 weeks (n = 3; 1 SPE, 2 HELLP; NL),
28 weeks (n = 1 SPE; NL), 29 weeks
(n = 1 SPE; NL), 30 weeks
(n = 1 HELLP; L), 31 weeks
(n = 1 SPE; L), 32 weeks
(n = 1 SPE; L), 33 weeks
(n = 1 SPE; L), 35 weeks
(n = 1 SPE; L), 36 weeks
(n = 1 SPE; L), 38 weeks
(n = 2; 1 PE, 1 HELLP; L). Samples were
collected for embedding in paraffin from women with the diagnoses
indicated at the following weeks of gestation: 25 weeks
(n = 1 HELLP; NL), 29 weeks
(n = 1 HELLP; NL), 29 weeks
(n = 1 SPE; L), 30 weeks
(n = 1 HELLP; L), 31 weeks
(n = 1 SPE; L), 32 weeks
(n = 1 SPE; L), 33 weeks
(n = 1 SPE; L), 35 weeks
(n = 1 SPE; L), 36 weeks
(n = 1 SPE; L), 38 weeks
(n = 2; 1 PE, 1 HELLP; L).
Immunolocalization
Placental tissues were processed for double indirect immunolocalization as previously described.22,23 For detection of VEGF-B and VEGF-D and the VEGFRs, tissues were fixed in 3% paraformaldehyde for 30 minutes, washed three times in phosphate-buffered saline (PBS), infiltrated with 5 to 15% sucrose followed by OCT medium, and frozen in liquid nitrogen. Sections (5 µm) were prepared using a cryostat (Slee International, Tiverton, RI) and collected on charged and precleaned microscope slides (Fisher Scientific, Pittsburgh, PA). For detection of PlGF, tissues were directly embedded in OCT and frozen without previous fixation. Sections, prepared as described above, were fixed in cold acetone for 5 minutes before staining as follows. The sections were incubated in a mixture of anti-cytokeratin (to localize trophoblasts) and another primary antibody for 1 hour to overnight. Then the sections were rinsed, incubated with the appropriate species-specific secondary antibodies conjugated to rhodamine or fluorescein, washed three times in PBS for 10 minutes, and mounted with Vectashield medium (Vector, South San Francisco, CA). Samples were examined with a Zeiss Axiophot Epifluorescence microscope (Thornwood, NY) equipped with filters to selectively view the rhodamine and fluorescein fluorescence. For detection of VEGF-A, tissues were fixed in 10% neutral buffered formalin for 24 hours and embedded in paraffin. Sections (5 µm) were cut on a Leica microtome and stained as described by Zhang and colleagues.24 For detection of VEGF-C, sections cut from paraffin blocks were incubated with the primary antibody for 3 hours and washed in PBS (three times for 10 minutes). Bound antibody was detected by incubation with diaminobenzoate (Vector). The sections were examined using a Zeiss microscope. As a control for each experiment, the staining patterns of the primary and secondary antibodies alone were assessed.
For immunolocalization of antigens expressed by cultured cytotrophoblasts, isolated cells were plated on coverslips coated with Matrigel (Collaborative Biomedical Products, Bedford, MA) for various periods of time, then fixed in 3% paraformaldehyde for 5 minutes, and permeabilized with cold acetone or methanol for another 5 minutes. Samples were stained and analyzed as described above.
Cytotrophoblast Isolation and Culture
Cytotrophoblasts were isolated from chorionic villi of 7- to 24-week human placentas by routine procedures established in our laboratory.25 Briefly, the placentas were obtained immediately after elective pregnancy terminations. After a series of collagenase and trypsin digestions, cytotrophoblasts were separated from contaminating cell types on Percoll gradients. Purified cells were used immediately or cultured in serum-free Dulbecco’s modified Eagle’s medium-high glucose, with 2% Nutridoma (Boehringer Mannheim Biochemicals, Indianapolis, IN), on Matrigel-coated or human placental laminin-coated (Life Technologies, Inc., Rockville, MD) substrates for the times indicated.
RNA Isolation and Northern Blot Hybridization
Total RNA was extracted from purified first and second trimester cytotrophoblasts either immediately on isolation or after 12 hours in culture according to published methods.26 Blots were prepared as previously described.27 Before transfer, gels were stained with acridine orange to ensure integrity of the RNA samples and confirm equal loading. The probes were generated by random priming with [32P]CTP and the Klenow fragment of DNA polymerase I28 of the following fragments: bp 57 to 638 of the VEGF165 cDNA (GenBank accession number M32977), bp 1 to 362 of the VEGF-B167 cDNA (GenBank accession number U48801), bp 494 to 1661 of the VEGF-C cDNA (GenBank accession number X94216), the entire coding regions of the VEGF-D (1064 bp) gene (GenBank accession number AJ000185), bp 304 to 944 of the PlGF cDNA (GenBank accession number X54936), bp 706 to 2310 of the Flt1 (VEGFR-1) cDNA (GenBank accession number X51602), bp 6 to 715 of the KDR (VEGFR-2) cDNA (GenBank accession number L04947), and bp 1 to 595 of the Flt4 (VEGFR-3) cDNA (GenBank accession number X68203). Probes had a specific activity of 2 x 109 dpm/pg. The final posthybridization washes were conducted in 0.3x standard saline citrate (150 mmol/L NaCl, 15 mmol/L sodium citrate, pH 7.4) and 0.1% sodium dodecyl sulfate (SDS) at 65°C. Blots were stripped in 0.1x standard saline citrate with 0.5% SDS at 95°C.
ELISAs
Levels of VEGF-A, PlGF, and sVEGFR-1 in first (n = 5), second (n = 5), and third trimester (n = 5) cytotrophoblast-conditioned medium were assessed by ELISA. Patients with preeclampsia whose placentas were used as sources of cells had the diagnoses indicated at the following weeks of gestation: 26 weeks (n = 2; 1 SPE, 1 HELLP), 31 weeks (n = 1 SPE), 32 weeks (n = 1 SPE), and 36 weeks (n = 1 SPE). Equal numbers of cytotrophoblasts (1 x 106/ml) were cultured for 48 hours on Matrigel substrates as described above, and then medium was collected and centrifuged at 12,000 x g for 5 minutes. The ELISAs were performed according to the manufacturer’s instructions (VEGF-A and PlGF) or published methods (sVEGFR-117 ). The results were analyzed on a Vmax kinetic microplate reader (Molecular Devices Corporation, Sunnyvale, CA). The statistical significance of the data was analyzed by using an analysis of variance test.
Cell Extraction and Immunoblotting
Freshly isolated cytotrophoblasts or cytotrophoblasts cultured on either Matrigel-coated or human placental laminin-coated wells were washed twice with PBS and extracted with 200 µl of lysis buffer (50 mmol/L Tris buffer, pH 7.6, containing 1% Nonidet P-40, 0.1% SDS, 120 mmol/L NaCl, 100 µmol/L phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg Na2VCO3). Cell extracts were centrifuged at 12,000 x g for 5 minutes to remove insoluble materials. Samples containing equal amounts of protein were mixed with SDS sample buffer and separated by SDS-polyacrylamide gel electrophoresis under nonreducing conditions (VEGF-A and VEGFR-2) or reducing conditions (VEGFR-1 and VEGFR-3 and VE-cadherin). After the proteins were transferred to nitrocellulose, the membranes were incubated first with primary antibody, then with peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Labs, Inc., West Grove, PA), by using procedures standard in our laboratory.27 Immune complexes were visualized using enhanced chemiluminescence and Hyperfilm (Amersham Life Sciences-USB, Arlington Heights, IL). The entire experiment was done three times using different batches of cytotrophoblasts.
Function-Perturbing Fc Fusion Proteins
Fusion proteins consisting of the first three repeats of
VEGFR-1/Flt1 or VEGFR-3/Flt-4 fused to the Fc portion of IgG
[VEGFR-1(1-3)-Fc and VEGFR-3(1-3)-Fc] were produced in the Alitalo
laboratory.29,30
As a control, nonimmune IgG was added to
some of the wells. Initially, the activity of the fusion and control
proteins was tested at concentrations ranging from 500 ng/ml to 50
µg/ml. Thereafter, 15 µg/ml, the lowest protein concentration that
had maximum effects on cell morphology and integrin 1 expression,
was used routinely. The entire experiment was done three times using
different batches of cytotrophoblasts.
Cell-Surface Biotin Labeling and Immunoprecipitation
Cytotrophoblasts cultured on Matrigel-coated wells were washed
twice with PBS and then incubated for 90 minutes in a 0.1% solution of
freshly prepared biotin (Pierce, Rockford, IL) in ice-cold PBS. The
cells were washed three times, also in ice-cold PBS, then extracted
with 200 µl of the lysis buffer described above. Cell lysates were
centrifuged at 12,000 x g for 5 minutes. Samples of
equal volume that contained equal amounts of protein were mixed with
the TS2/7 mAb (1 µg/ml) and precipitated with protein A-Sepharose
CL-4B (Zymed Labs., South San Francisco, CA). The immunoprecipitates
were separated by SDS-polyacrylamide gel electrophoresis under reducing
conditions and transferred to nitrocellulose. The nitrocellulose
membranes were incubated with streptavidin-horseradish peroxidase, and
biotin-labeled integrin 1/ß1 was visualized by standard enhanced
chemiluminescence procedures.
Invasion Assay
Invasion assays were conducted as described previously.22,25 Briefly, isolated cytotrophoblasts (0.25 x 106) were plated on Transwell inserts (6.5 mm; Costar, Cambridge, MA) containing polycarbonate filters (pore size, 8 µm) that had been coated with Matrigel. Culture medium containing either a fusion protein [VEGFR-1(1-3)-Fc and VEGFR-3(1-3)-Fc] or control IgG was added. After 48 hours the cultures were stained with the 7D3 antibody, which specifically reacts with human cytokeratin, to visualize the cytotrophoblasts. The filters were cut from the supports and mounted, upper surface facing down, on slides. The number of cytokeratin-positive cells and cell processes on the lower surface of the filter was counted. Each experimental condition was tested in triplicate, and the entire assay was done seven times. Data were expressed as percentage of control. The statistical significance of the data was analyzed by Student’s t-test.
Terminal Deoxynucleotidyl Transferase (TdT)-Mediated dUTP Nick-End Labeling (TUNEL)
Cytotrophoblasts were plated on Matrigel- or human laminin-coated coverslips under the same experimental and control conditions as described above for the invasion assays. After 24 hours, the coverslips were fixed in 3% paraformaldehyde for 30 minutes, washed twice with PBS, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 4 minutes. Then the cells were incubated in the TUNEL reaction mixture (Boehringer Mannheim Biochemicals, Indianapolis, IN) for 1 hour at 37°C. The results were analyzed using a Zeiss Epifluorescence microscope. Data were expressed as the number of brightly staining cytotrophoblasts/total cytotrophoblasts per x40 field. Thirty randomly chosen fields were counted on each coverslip. Each experimental condition was tested in triplicate, and the entire experiment was done six times. The statistical significance of the data was analyzed by analysis of variance.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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First, we immunolocalized VEGF family members in tissue sections
of the maternal-fetal interface (diagrammed in Figure 1
). In first trimester chorionic villi,
cells and fetal vessels in villus stromal cores stained with
anti-VEGF-A (Figure 2B)
, as did a subset
of the syncytiotrophoblasts that are in contact with maternal blood at
the villous surface (data not shown). The majority of cytotrophoblast
stem cells, which are attached to the trophoblast basement membrane of
chorionic villi, and cytotrophoblasts in the proximal regions of cell
columns, which bridge the gap between the placenta and the uterine
wall, did not react with anti-VEGF-A (Figure 2B)
. Expression was
dramatically up-regulated on cytotrophoblasts in the distal regions of
columns. Within the superficial uterine wall, most cytotrophoblasts
also stained with anti-VEGF (Figure 2D)
, although some cells in the
deeper portions of the decidua and the superficial myometrium
showed no immunoreactivity. Particularly intense staining was observed
in association with the placental cells that occupied the lumina of
uterine vessels, although cytotrophoblasts in the wall often lacked
immunoreactivity (Figure 2F)
. In areas where the maternal endothelium
had not yet been replaced, the latter cells also reacted with
anti-VEGF-A. Neither the pattern of VEGF-A localization nor the
staining intensity changed during the first-to-second-trimester
interval analyzed, 6 to 22 weeks of gestation. At term, stromal cells
and blood vessels in the villous cores (data not shown) and a subset of
cytotrophoblasts in the uterine wall stained with anti-VEGF-A (see
Figure 12B
). Very weak anti-VEGF-B staining was detected, such that
there was no discernable pattern at any of the gestational ages
analyzed, ie, 6 weeks to term (data not shown). In contrast, very
strong anti-VEGF-C staining was observed in cytotrophoblast stem cells
and in the column region, particularly during the second trimester
interval (Figure 3, B and D)
.
Endovascular cytotrophoblasts also reacted with anti-VEGF-C in a
pattern that was similar to staining with anti-VEGF-A (data not shown).
This staining pattern did not change from 6 to 22 weeks of gestation,
but by term little or no staining was detected (Figure 3F)
. We failed
to detect expression of VEGF-D (data not shown).
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. Anti-PlGF reacted with
cytotrophoblasts in the distal regions of columns (Figure 4B)
and with
invasive placental cells in the interstitial (Figure 4D)
and
endovascular (Figure 4F)
uterine compartments. At term, fetal blood
vessels stained brightly as compared to cytotrophoblasts in the uterine
wall, which in some areas showed weak immunoreactivity and in others
failed to stain (data not shown).
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. Within the uterine wall,
cytotrophoblasts in the decidua and myometrium either failed to react
with anti-VEGFR-2 or stained weakly (data not shown). In contrast,
placental cells within the lumina of vessels stained brightly as
compared to cytotrophoblasts that had invaded the vessel wall and the
adjacent decidua (Figure 6B)
. Staining
for VEGFR-1 showed a different pattern. This antigen was detected in
association with syncytiotrophoblasts, but not the villous stroma,
cytotrophoblast stem cells, or sites of column initiation (Figure 5D)
.
Anti-VEGFR-1 reactivity was very specifically up-regulated within the
first three to four cell layers of columns, then maintained throughout
the rest of the column. Cytotrophoblasts in the decidua and the
myometrium stained brightly, but the endovascular subpopulation of
placental cells, in either vessel walls or lumina, did not react with
anti-VEGFR-1 (Figure 6D)
. VEGFR-3 displayed yet a third distinct
staining pattern. In chorionic villi, cell columns, decidua, and
myometrium the pattern of trophoblast immunoreactivity was nearly
identical to that of anti-VEGFR-1, except that syncytiotrophoblast
staining was relatively weaker (Figure 5F)
. In contrast to VEGFR-1,
staining for VEGFR-3 was strong on endovascular cytotrophoblasts
(Figure 6F)
. At term, staining for VEGFR-1 was comparable to that
observed in first and second trimester, whereas anti-VEGFR-2
and anti-VEGFR-3 immunoreactivity was down-regulated in all locations;
endovascular expression of the latter receptor was nearly absent (data
not shown). Finally, we observed no differences in VEGF ligand and
receptor expression between patients who experienced labor and those
who did not.
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Plating cytotrophoblast stem cells isolated from first and second
trimester placentas on complex matrix substrates such as Matrigel
promotes their differentiation along the pathway that leads to invasion
and pseudovasculogenesis, rather than fusion to form
syncytium.2,25
Here we investigated whether these culture
conditions supported expression of the same VEGF ligands and receptors
that were detected in the immunolocalization experiments performed on
tissue sections of the maternal-fetal interface. Northern blot
hybridization and immunoblot approaches were used to detect mRNA and
protein expression, respectively. Northern blot hybridization (Figure 7A)
showed that fibroblasts and
cytotrophoblasts isolated from first and second trimester placentas
primarily expressed VEGF-A and VEGF-C mRNAs; low levels of VEGF-B mRNA
were also detected, whereas VEGF-D was not. In contrast,
cytotrophoblasts, but not fibroblasts, expressed PlGF mRNA, and the
message levels were higher in the first than in the second trimester.
In cytotrophoblasts, VEGF-A, VEGF-C, and PlGF message levels did not
change with time in culture. Surprisingly, ELISA failed to detect
VEGF-A protein in the culture medium of first and second trimester as
well as term cytotrophoblasts plated on Matrigel (Figure 7B
, left), a
result that was confirmed by immunoblotting (data not shown). In these
experiments, VEGF release by the choriocarcinoma cell lines JAR, JEG,
and BeWo served as positive controls. If instead cytotrophoblasts were
cultured for 12 hours on a defined laminin substrate, substantial
amounts of VEGF-A, detected by immunoblotting, were secreted into the
medium (Figure 7C)
. The immunoreactive bands spanned a higher molecular
mass interval than the VEGF165 control,
suggesting the presence of higher molecular mass isoforms or
glycoforms. In contrast, cytotrophoblasts plated on both Matrigel and
laminin substrates released PlGF into the culture medium, as did the
choriocarcinoma cell lines (Figure 7B
, right).
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. Before plating, lysates prepared from
first trimester cytotrophoblasts contained two immunoreactive bands,
one that migrated slightly faster and one slightly slower than the
endothelial form of the protein. In contrast, in second trimester cell
lysates, only the more slowly moving band was detected. We did not
further investigate the reason for the differences among the samples in
the number and electrophoretic migration of the immunoreactive bands,
but the explanation likely includes the unusual
polylactosamine-containing carbohydrate structures that
cytotrophoblasts add to proteins, a process that is gestationally
regulated,31
and/or receptor
phosphorylation.14
|
. VEGFR-3 expression was much higher in
the first than in the second trimester, and again variations in the
pattern of immunoreactive bands were observed among samples (Figure 8)
. VEGF Family Members Promote Differentiation and Survival of Cytotrophoblasts
To understand the effects of VEGF family members, we plated
cytotrophoblast stem cells on Matrigel substrates in the presence of
fusion proteins that contained the ligand-binding domains of VEGFR-1
and VEGFR-3, receptors whose expression is up-regulated as
cytotrophoblasts differentiate in culture. We examined endpoints
indicative of changes in the cell’s adhesive and invasive
properties.22
After 12 hours in culture, cytotrophoblasts
plated under control versus experimental conditions showed
morphological differences. The control cells formed discrete aggregates
that were interconnected via cells and cell processes (Figure 9A)
. In contrast, cytotrophoblasts plated
in the presence of the VEGFR-1(1-3)-Fc fusion protein, which blocks
VEGF-A, PlGF, and VEGF-PlGF heterodimer binding to endogenous VEGFR-1,
flattened on the matrix substrate, and fewer interconnections were
evident (Figure 9D)
. The latter effect was enhanced when the cells were
plated in the presence of a VEGFR-3(1-3)-Fc fusion protein, which
blocks VEGF-C binding to endogenous VEGFR-3 (Figure 9G)
.
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1ß1 (Figure 9C)
.
Cytotrophoblasts plated in the presence of the VEGFR-1(1-3)-Fc fusion
protein showed wide variations in staining intensities; a few cells
stained brightly, many showed much weaker immunoreactivity, and some
did not react with the antibody (Figure 9F)
. Essentially the same
results were obtained when cytotrophoblasts were plated in the presence
of the VEGFR-3(1-3)-Fc fusion protein (Figure 9I)
. Immunoprecipitation
confirmed that control cells expressed higher levels of integrin
1ß1 than did cytotrophoblasts cultured in the presence of either
fusion protein (Figure 10A)
. In
contrast, inhibiting ligand binding to VEGFR-1 or VEGFR-3 did not
change the staining pattern for VE-cadherin, another cell adhesion
molecule whose expression is up-regulated during differentiation (data
not shown). In accord with this result, immunoblot analyses also failed
to detect any differences in VE-cadherin expression between the control
and experimental cells (Figure 10B)
.
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1
expression is highly correlated with a reduction in cytotrophoblast
invasiveness. We looked for the same correlation under the experimental
conditions tested here. As compared to control cultures, invasion was
reduced by 
50% when cytotrophoblasts maintained in medium that
contained either VEGFR-1(1-3)-Fc or VEGFR-3(1-3)-Fc reached the
undersides of the filters (Figure 11A)
.
During the course of these experiments, we also evaluated the
morphology of the cytotrophoblasts that remained on the upper surfaces
of the filters. The sharp increase in the number of cells with a
shrunken appearance suggested that exposure to either VEGFR-1(1-3)-Fc
or VEGFR-3(1-3)-Fc for 48 hours induced apoptosis, an idea that
was confirmed by using the TUNEL method to visualize cells undergoing
programmed cell death. Typically, 40 to 50% of the cells cultured
under the experimental conditions were labeled. This translated into an
approximately fivefold increase in cytotrophoblast apoptosis as
compared to control cells (Figure 11B)
.
|
In the final set of experiments, we investigated whether the
expression of VEGF family members is dysregulated in severe forms of
preeclampsia, serious complications that appear during the second half
of pregnancy. The associated pathological changes at the maternal-fetal
interface include shallow cytotrophoblast invasion and deficits in
pseudovasculogenesis.8
Here we compared, in control
pregnancies and those complicated by SPE and HELLP syndrome,
cytotrophoblast staining for the subset of VEGF ligands and receptors
that are expressed until term: VEGF-A and VEGFR-1/Flt-1. In control
pregnancy at 38 weeks of gestation, strong staining for VEGF-A was
detected on cytotrophoblasts that invaded both the interstitial and
vascular compartments of the uterine wall (Figure 12B)
. When the pregnancy was
complicated by severe forms of preeclampsia, cytotrophoblasts either
stained weakly with anti-VEGF-A (Figure 12D)
or, more often, failed to
demonstrate immunoreactivity (Figure 12F)
. As compared to control
tissue (Figure 13B)
, cytotrophoblast
VEGFR-1 expression was also diminished in preeclampsia, although the
range of staining intensities was greater than that observed for
VEGF-A. In some areas no antibody reactivity was observed (Figure 13D)
,
whereas in others weak staining was evident (Figure 13F)
. Finally,
recent reports from other investigators suggest that cytotrophoblasts
also secrete the soluble form of VEGFR-1 (sVEGFR-1), a potential
angiogenesis inhibitor.32,33
Accordingly, we measured
sVEGFR-1 levels in cytotrophoblast-conditioned medium. As compared to
control third trimester cells, those isolated from the placentas of
women with severe forms of preeclampsia released approximately twice
the amount of the soluble receptor (Figure 14)
; levels were also higher than those
detected in medium of either first or second trimester control
cytotrophoblasts.
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Interpreting these data in the context of existing information about the function of particular VEGF ligands and receptors gives some insight into the role they play in the unique differentiation pathway that forms the human maternal-fetal interface.14,34 With regard to receptors, deletion of the gene encoding VEGFR-2 has the severest effects; the mice die at embryonic day (E) 8.535 because differentiation of endothelial and hematopoietic cells is blocked.36,37 VEGFR-1 appears to act downstream of VEGFR-2. Null embryos, which also die at E 8.5, have an excess of endothelial cells that do not assemble into functional vessels.38 Unexpectedly, deletion of the receptor’s kinase domain does not interfere with angiogenesis—possible evidence of cooperative interactions with VEGFR-2.39 Deletion of VEGFR-3, which also stimulates growth of lymphatic vessels, causes death at E 9.5. In this case, vasculogenesis and angiogenesis appear to proceed normally, but large vessels are organized abnormally.40 Overexpression of soluble VEGFR-3 produces lymphedema.41
With regard to receptor ligands, threshold levels of VEGF interactions with its receptors (VEGFR-1 and VEGFR-2) are important, because deletion of only one copy of the VEGF gene causes death at E 11 to 12 because of abnormalities in both angiogenesis and blood-island formation.42,43 Recently, additional family members (VEGF-B, VEGF-C, VEGF-D, and PlGF) were discovered. Mice that lack VEGF-B, which binds to VEGFR-1, have impaired cardiac function.44 VEGF-C, a ligand for VEGFR-2 and VEGFR-3, has multiple actions. Mice engineered to overexpress this ligand in the skin have selective hyperplasia of the superficial lymphatic vasculature.45 But VEGF-C also induces angiogenesis.46 Human VEGF-D binds and activates VEGFR-2 and VEGFR-3, suggesting roles in angiogenesis and lymphangiogenesis.47 Finally, PlGF interactions with VEGFR-1 potentiates angiogenic responses to VEGF.48
Other investigators have studied the expression of VEGF ligands and receptors in the murine47 and human placentas.49 In the latter organ, Northern blot hybridization analyses of ligand expression shows that floating villi encode primarily VEGF-B and VEGF-C mRNA.50 In contrast, Clark and colleagues33 found, by in situ hybridization, mRNA for VEGF-A, but not its VEGF-B and VEGF-C forms, in the stroma of floating villi. The latter investigators also showed that mRNA for the VEGF family member PlGF was abundant in villous cytotrophoblasts, syncytium, and invasive cytotrophoblasts. Immunostaining localized both VEGF-A and PlGF protein to the endothelium of fetal blood vessels found within the cores of floating villi.50 VEGF-A and VEGF-C protein expression has also been reported in association with cytotrophoblasts within the uterine wall.51-53 With regard to VEGF receptors, invasive cytotrophoblasts appear to express VEGFR-1 mRNA and protein,51,52,54 whereas VEGFR-2 expression was detected in some studies53 but not others.52 Investigators in the latter study also suggested that the placenta produces and releases a soluble form of Flt-1 that could act as an antagonist.33 Finally, trophoblast expression of VEGFR-3 was recently described.53 The results of the work described here support some of these conclusions, eg, placental production of sVEGFR-1.32,33 Discrepancies are likely explained by the fact that we focused on a unique placental compartment—anchoring villi and the associated invasive cytotrophoblast population that invades the uterine wall, as well as blood vessels that traverse this region. Additionally, we studied this compartment throughout gestation, which revealed that cytotrophoblast expression of VEGF family members changes as pregnancy advances.
Our analyses of cytotrophoblast VEGF ligands and receptors suggested
that their expression is precisely regulated as these unusual cells
exit the mitotic cycle and undergo pseudovasculogenesis in
vivo. In turn, these intricate expression patterns allowed us to
generate hypotheses about the autocrine effects of VEGF on
cytotrophoblast differentiation and invasion. These processes, which
are key to placental development, mainly occur during the first half of
pregnancy. Accordingly, we were particularly interested in
ligand-receptor pairs that could function during this time period. For
example, the coordinated expression of VEGF-C and VEGFR-2 by early
gestation cytotrophoblast stem cells suggested that signals transmitted
through the latter receptor could affect important functions, eg,
proliferation. However, we were unable to test this hypothesis because
early gestation cytotrophoblasts rapidly down-regulated VEGFR-2
expression as they differentiated in vitro. The latter
observation is in accord with the results of our previous studies that
show cytotrophoblast differentiation, both in situ and in
culture, is associated with permanent withdrawal from the cell
cycle.55,56
Our previous studies also suggest that
invading cytotrophoblasts up-regulate the expression of molecules that
play functional roles in uterine invasion and
pseudovasculogenesis.1
Thus, the immunolocalization data
that result from the present study led us to theorize that PlGF,
VEGF-A, and VEGF-C interactions with VEGFR-1 are critical for invasion
and pseudovasculogenesis, as are VEGF-C signals transduced through
VEGFR-3. Our in vitro experiments showed this to be the
case, as the addition of fusion proteins that specifically interfered
with ligand binding to either receptor altered the cell’s ability to
express integrin 1, an adhesion molecule that is an accurate
barometer of the cell’s invasive capacity,22,23
a
correlation also demonstrated in the experiments described here.
Finally, signals transmitted through both receptors also play important
functions in cytotrophoblast survival, a finding that is in accord with
the central role played by VEGF signals in assembly and maintenance of
the vasculature. Thus, the reduced invasion we observed is almost
certainly a secondary phenomenon related to increased apoptosis.
Cytotrophoblast-derived VEGF family ligands are also likely to have paracrine effects on other populations of both fetal and maternal cells. In this regard it is interesting to note that PlGF and VEGF-C are more likely to play a role, because cytotrophoblast VEGF-A remained tightly associated with the extracellular matrix, a property of certain isoforms of the molecule (VEGF189, VEGF165, VEGF145) but not others (VEGF121).57 Although we did not study receptor expression by potential target cells, the intrinsic placental vasculature that forms within the stromal cores of chorionic villi could be influenced by factors produced in the trophoblast layers. This arrangement would coordinate trophoblast differentiation with that of the extraembryonic fetal vasculature, an important component of normal placental development. On the opposite side of the placenta, the maternal vasculature is another likely target. In this location cytotrophoblast-derived angiogenesis inhibitors, such as sVEGFR-1, could help cytotrophoblasts gain entrance to and occupy uterine vessels without triggering the cascade of events that normally leads to angiogenesis of the same population of vessels during the normal menstrual cycle.
The effects of severe forms of preeclampsia, conditions associated with failed pseudovasculogenesis and shallow invasion, on cytotrophoblast expression of VEGF family ligands and receptors also suggest the importance of particular molecules. In these cases the cells down-regulated expression of VEGF-A and VEGFR-1; sVEGFR-1 release increased. We speculate that the end result is an imbalance in the production of angiogenic factors at the maternal-fetal interface. Evidence that shows altered VEGF and PlGF levels in the blood of women with preeclampsia supports this hypothesis. For example, some investigators report that VEGF-A levels are elevated.58,59 In vitro studies suggest that this phenomenon could contribute to the maternal vascular defects associated with this syndrome.60 Whether the sources of enhanced levels of circulating VEGF-A include the maternal-fetal interface, where we found that cell-associated staining decreased, remains to be determined.
Since the origin of the preeclampsia syndrome is unknown, we have no
way of knowing how near our observations lie to the root causes of
these pregnancy complications. Studies from the laboratories of many
investigators summarized above suggest that an absence of the normal
repertoire of VEGF family members at the maternal-fetal interface could
result in the deficits in cytotrophoblast differentiation we observe in
preeclampsia. Data presented here offer further support for this link
as we were able to replicate important aspects of the phenotype of
cytotrophoblasts in preeclampsia by removing endogenous VEGF ligands
from the culture medium as the cells differentiated in
vitro: decreased integrin 1 expression8
(Figures 9 and 10)
and increased apoptosis62
(Figure 11)
.
Finally, VEGF family ligands and receptors are only a portion of the molecules with angiogenic and/or vasculogenic effects that are expressed by trophoblast cells. Thus we predict that other trophoblast-derived vasculogenic/angiogenic factors, such as angiopoietin ligands and their TIE receptors, as well as a broad array of fibroblast growth factor family members, will play important roles in cytotrophoblast pseudovasculogenesis and occupation of the maternal vessels. However, it is interesting to note that the placenta, which often co-opts pathways used in the development of other organs and tissues, can also use novel methods. Thus, we also suspect that these unusual processes could involve cytotrophoblast production of novel molecules with unique functions that are yet to be discovered.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants HL 64597 and HD 30367.
Accepted for publication January 10, 2002.
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