Professional Documents
Culture Documents
6, December 1997
Copynight ) American Society for Investigative Pathology
During human placental development, cytotrophoblast stem cells differentiate and invade the uterus.
Simultaneously, the cells modulate their expression
of several classes of stage-specific antigens that mark
transitions in the differentiation process and play a
role in either uterine invasion (integrin cell-extracellular matrix receptors and matrix metailoproteinase-9) or immune interactions (HLA-G). The pregnancy disease pre-eclampsia is associated with
shallow cytotrophoblast invasion. Previously we
showed, by immunofluorescence localization on placental tissue, that in pre-eclampsia invasive cytotrophoblasts fail to properly modulate their integrin repertoire. This finding suggests possible abnormalities
in the differentiation pathway that leads to uterine
invasion. Here we used a culture system that supports
this differentiation process to compare the differentiative and invasive potential of cytotrophoblasts obtained from control (n = 8, 22 to 38 weeks) and
pre-eclamptic (n = 9, 24 to 38 weeks) placentas. In
culture, the cells from pre-eclamptic placentas failed
to properly modulate al integrin and matrix metalloproteinase-9 expression at the protein and mRNA
levels. Their invasive potential was also greatly reduced. Likewise, the cells failed to up-regulate HLA-G
protein and mRNA expression. These results suggest
that defective cytotrophoblast differentiation/invasion can have significant consequences to the outcome of human pregnancy (ie, development of preeclampsia) and that, by the time delivery becomes
necessary, the defect is not reversed by removing the
cells from the maternal environment. (Am J Pathol
1997, 151:1809-1818)
1809
1810
Lim et al
We reasoned that pregnancy diseases in which cytotrophoblast invasion is abnormal at a microscopic level
could be associated with disruption of invasion at a molecular level, ie, result from an imbalance of the regulatory
mechanisms that govern invasion. Previous work suggested that pre-eclampsia could be an interesting example of such a disease. Histological studies of the maternal-fetal interface (eg, placental bed biopsies) show that,
in pre-eclampsia, interstitial cytotrophoblast invasion is
often shallow and endovascular invasion nearly absent.15-17 Pre-eclampsia is important because it occurs
frequently (7% of nulliparous patients) and has serious
consequences for both the mother (eg, high blood pres-.
sure, proteinuria, and edema) and the fetus (intrauterine
growth retardation). As a result, pre-eclampsia and hypertensive diseases of pregnancy are a leading cause of
maternal death18 and contribute significantly to premature deliveries in the United States.19
We began studying cytotrophoblast differentiation in
this syndrome by using immunolocalization techniques to
determine whether differentiating/invading cytotrophoblasts in tissue sections of the maternal-fetal interface
correctly switch their integrin repertoire. In normal pregnancies, cytotrophoblasts that are differentiating along
the invasive pathway first up-regulate the expression of
integrin a5/01, then al/f1.7 In pre-eclampsia, invasive
cytotrophoblasts do not completely switch their integrin
repertoire. The cells express integrin a5/f31 but not al/
11,17 thus displaying an adhesion phenotype our function-perturbation studies suggest could inhibit invasion.11
In addition, Redline20 recently showed that, in pregnancies complicated by pre-eclampsia, intermediate/invasive cytotrophoblasts have an increased proliferative index, as evidenced by increased staining for proliferating
cell nuclear antigen (PCNA) and reduced expression of
human placental lactogen, another antigen that is produced as a consequence of the differentiation process.
Together, these results suggest that, in pre-eclampsia,
cytotrophoblasts start to differentiate along the invasive
pathway but cannot complete this process. However,
studies performed on tissue sections do not allow us to
examine the differentiative potential of cytotrophoblasts
and to determine whether the observed defect is reversible.
In experiments described here, we used a culture
system that promotes cytotrophoblast stem cell differentiation along the invasive pathway to compare the differentiative potential of cells isolated from placentas of gestation-matched control and pre-eclamptic patients. The
results showed that, in pre-eclampsia, cytotrophoblast
integrin switching in vitro was altered in a pattern that
replicated the changes we previously described in vivo,
ie, in tissue sections of placental bed biopsies. Cytotrophoblast expression of al integrin protein and mRNA
was all but absent. Their ability to up-regulate MMP-9
production, as well as to invade ECM in vitro, was also
impaired. Likewise, their expression of HLA-G, a stagespecific antigen that probably plays an important role in
maternal-fetal immune interactions, dramatically decreased. Together, these data suggest that, in preeclampsia, cytotrophoblasts start to differentiate along
1811
Immunohistochemistry
Mouse monoclonal antibody to the human integrin al
subunit (IgG) was obtained from T-Cell Laboratory, Boston, MA. Rat monoclonal antibodies to human integrin
subunit a5 (BIIG2) and cytokeratin (7D3) were made in
this laboratory and described previously.7 Mouse monoclonal antibody to HLA-G (IgM) was also produced in this
laboratory.23 Species-specific monoclonal antibodies to
IgG and IgM were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Highly purified cytotrophoblasts were cultured at a
concentration of 2.5 x 105 cells/ml on Matrigel-coated
(Collaborative Research, Bedford, MA) 12-mm coverslips
in serum-free medium supplemented with 2% Nutridoma.
For time zero analysis, cytotrophoblasts were allowed to
adhere to the coverslips for 10 minutes at 370C. After 0,
12, 24, 36, and 48 hours of culture, the cells were washed
with phosphate-buffered saline (PBS) and fixed in 2.5%
paraformaldehyde at room temperature for 10 minutes.
Next, cells were permeabilized with 100% methanol at
-200C, and nonspecific reactivity was blocked by incubating them with 0.2% bovine serum albumin for 20 minutes. Primary antibody was added at 40C for 10 to 12
hours. Then the cells were washed in PBS and incubated
with species-specific secondary antibodies at room temperature for 1 hour. In all cases, the cells were double
stained with antibodies that detect cytokeratin as described previously.8 Cytokeratin-positive cells were
counted on an Axiophot microscope (Zeiss), and the
percentage of these cells that expressed integrin al or
a5 and HLA-G was calculated.
Immunoblotting
Conditioned medium was collected from control and preeclamptic cytotrophoblasts at 12-hour intervals for 3 days
RNA Extraction
RNA was extracted from cytotrophoblasts cultured at a
concentration of 1.0 x 106 cells/ml in serum-free medium
on Matrigel as described above. Total RNA was extracted according to published methods.25 Briefly, 5 x
106 cells per sample were homogenized in 500 ,ul of
guanidine buffer (4 mol/L guanidine isothiocyanate, 25
mol/L sodium citrate, 0.5% sarcosyl), followed by the
addition of 50 ,ul of 2 mol/L sodium acetate (pH 4.0), 500
,ul of water-saturated phenol and 100 ,ul of chloroform.
After centrifugation, the RNA was pelleted from the aqueous phase by the addition of 500 Al of isopropanol and
reprecipitated from a solution containing 10 mmol/L Tris
(pH 7.5), 1 mmol/L EDTA, and 0.5% SDS. The pellet was
then washed with cold (40C) 70% ethanol, vacuum dried,
and dissolved in sterile water. The concentration of RNA
was determined by measuring the absorbance at 260
nm.
Northern Blotting
Total RNA (8 ,ug) was separated by formaldehyde-agarose gel electrophoresis, transferred to Nytran membranes (Schleicher and Schuell, Keene, NH), and analyzed by Northern blot hybridization as described
previously.2627 The probe for human integrin al was
synthesized by random priming of full-length cDNA (the
gift of Dr. E. Marcantonio, Columbia University) using
[32P]CTP and the Klenow fragment of DNA polymerase 1.
The probe for MMP-9 was a 384-bp PCR product near the
5' end of the cDNA that has the least homology to MMP2,28 and the probe for HLA-G was synthesized by random priming of the 450-bp Pvull fragment from the 3'
untranslated region of HLA-G. Probes had a specific
activity of 2 x 109 dpm/,g. In all experiments, gels were
1812 Lim et al
AJP December 1997, Vol. 151, No. 6
Figure 1. Cytotrophoblast (CTB) expression of integrin al protein dramatically decreases in syndrome of hemolysis, elevated liver enzymes, and low platelets
(HELLP). Control CTBs isolated from the placenta of a patient who delivered at 27 weeks of gestation due to cervical incompetence were plated on Matrigel-coated
coverslips, cultured in serum-free medium for 48 hours and then stained with monoclonal antibodies against integrin a 1 (A) and cytokeratin (CK; B). CTBs isolated
from the placenta of a patient who delivered at 28 weeks of gestation due to HELLP syndrome were cultured under the same conditions for 48 hours, after which
their integrin al (C) and CK expression (D) was assessed. By 48 hours in culture many of the control CTBs stained with an antibody against the al integrin subunit
(A), whereas few of the CTBs isolated from the placenta of the patient with HELLP syndrome reacted with this antibody (C).
Invasion Assay
Invasion assays were performed using one of two methods previously described.29'30 In the first method, 2.5 x
105 cytotrophoblasts were cultured on Transwell inserts
(6.5 mm; Costar, Cambridge, MA) containing polycarbonate filters that had been coated with 10 ,l of Matrigel.
After 72 hours of culture, the cells were stained with
anti-cytokeratin antibody, and the number of cell projections on the bottom side of the filter was counted using a
fluorescence microscope as described.29 In the second
method, after the 72-hour culture period, cells were fixed
and processed for scanning electron microscopy.30 The
filters were removed from the inserts, critical-point dried,
sputter-coated with gold palladium, and then imaged in
an ETEC Autoscan Model U-1 scanning electron microscope. Invasion was quantified by counting the number
of cells and processes on the underside of the filter.
Statistical Analysis
All data were analyzed using analysis of variance with
Scheffe F-test for post hoc analysis (StatView, Abacus
Concepts, Berkeley, CA). A P value of <0.05 was considered significant.
Results
We compared the ability of cytotrophoblasts isolated
from normal and pre-eclamptic placentas to differentiate
along the invasive pathway by culturing these cells under
conditions that promote this process. First, we assayed
their expression of integrins and MMP-9, stage-specific
antigens that modulate their invasiveness. The integrins
of interest were the al and a5 subunits. By 48 hours in
culture, cells isolated from a control placenta (27 weeks)
reacted with an antibody that recognizes the al subunit
(Figure 1A). Cytotrophoblasts isolated from placentas
that were obtained from a pregnancy (28 weeks) complicated by pre-eclampsia all reacted with cytokeratin antibody (Figure 1D), but almost none reacted with the anti-al antibody (Figure 1C). In contrast, cells isolated from
both the control and pre-eclamptic placentas expressed
1813
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integrin ca5 (Figure 2, A and C) and reacted with anticytokeratin antibody (Figure 2, B and D).
To quantify these results, we determined the number of
cytokeratin-positive cells (ie, cytotrophoblasts) in both
groups that expressed integrin a1 immediately after isolation and after culture for 12, 24, 36, and 48 hours. The
results are shown in Figure 3. At time 0, an average of 8%
of control cytotrophoblasts (23 to 33 weeks) reacted with
the anti-cal integrin antibody, and by 48 hours this percentage increased to 36%. However, cells isolated from
pregnancies complicated by pre-eclampsia (24 to 32
weeks) demonstrated a marked decrease in their ability
to up-regulate integrin al expression in culture. Starting
at time 0, 1% of these cytotrophoblasts expressed integrin al, and after 48 hours, only 4% of the cells reacted
with the anti-ca1 integrin antibody. In contrast, a similar
percentage of cytotrophoblasts isolated from the placentas of normal and pre-eclamptic patients expressed integrin a5 in vitro at all times analyzed (Figure 4). Before
culture, approximately 53% of cells in both groups reacted with the anti-a5 antibody, and this increased to an
average of 80% after 48 hours.
Because our previous studies showed that MMP-9 expression is also required for cytotrophoblast invasiveness
in vitro, 0 we used Western blotting to compare the ability
of cytotrophoblasts in both groups to up-regulate expression of this proteinase in vitro (Figure 5). The amount of
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Hours in Culture
illi)a )1 pteit) ii) il C' -CcI,iilXipSia thltl)
Figure 3. iFcwXrt (CTBXs c'xpriss ii)iC'i.i(
ill
plejt'ltis (of
pa,ticilts witlh rtieclampsii (t = +, 24 to 32 XXeeks) ltr Comtr)ol pitienits (i
-+ 23 to 33 weeC'ks) WX C't' C Liltt'(iel (M) MItriiel-c(ilted C(Xeirlips insert)iOi-t te
ItICCliLllii1()0, 12, 24, 36, inC 48 h rtos.X'lic ceXlls wre fix\ed inCh staineC -Is
dcIsc Iil-t'Cd iti Mt,iteriIls inC Methti)Cds. CIn tite pe'cIttnlteagt(, CK-positiXve celXs
thit iIXO ex\ptreXseCl inltegi ii) cr1 XXsiXtCelee
iinc- BitrX sXhtXX the i-ne-in pctXe tlle tX\XO gIlOLIIC)iS It
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te'Xt Ili'e iilCie1- (o TIX iXis iIitCdCIli()llietIlc itiXs of pitien)ts XXitt
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1814
Lim et al
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Figure 4. Simnilar pcrcentagexs of C113s 'plre'ss
ietegililn a) protpein ill prieclampsipia ind in gestation-illtched colntrol piregnancies. 'th'le patients and
Iletliocds of anllx'iS iire decriiidil in Intlre 3, except that an antihiody to
iintc1Wi a ti toin aSXes the
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CTBs isolated froiii the placentas o)1 patiets xNxith pie-ecklillpsia to express
ilnteg'till nn inl vil/r is connsisient xx-tilt thilei exptession of this inte'grin i)i viltO
dItetlillilneCIdV LISinI(g tilhe Sxamllte alltihodIx to staill placental hed hiope
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The dlit'letncexheixxS
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As a first step toward understanding why cytotrophoblasts from placentas of pre-eclamptic patients fail to
correctly modulate integrin al and MMP-9 protein expression in vitro, we performed Northern hybridization to
compare levels of the corresponding mRNAs in these
cells with levels in gestation-matched control cytotrophoblasts. RNA was extracted from the cells at 0 hour, ie,
immediately after isolation, or after culture for 12 and 36
hours. Northern hybridization was performed using cDNA
probes that detect either integrin al mRNA (4.1 kb and
5.2 to 6.5 kb) or MMP-9 mRNA (2.8 kb). At 0 hour, integrin
l1 mRNA was detected in control 26-week cytotrophoblasts, and there was a small increase in message level
after the cells had been cultured for 12 hours (Figure 6A).
In contrast, cytotrophoblasts isolated from the 27-week
nl,qr-ant_
Pnrl_qmntirinvnracc2arl
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IILLI a-1
iUZ,3t U co
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mRNA at either time point that the band is not visible in
the photograph. After 36 hours in culture, there was a
very slight increase in ca1 mRNA expression by both
pre-eclamptic and control cells (data not shown). Analysis of cytotrophoblast integrin a1 mRNA levels in another
sample pair (24-week control/29-week pre-eclamptic)
gave essentially the same results.
A different blot of the same RNA samples shown in
Figure 6A was probed to detect MMP-9 mRNA (Figure
6B). In accord with our protein data, no message was
detected in control cells at 0 hour. By 12 hours MMP-9
mRNA was found, and levels continued to increase during the 36-hour culture period. In contrast, no MMP-9
mRNA was detected at either 0 or 12 hours in preeclamptic cytotrophoblasts. By 36 hours message was
present, but the levels, quantified by scanning densitometry, were approximately 15-fold less than those found in
control cells. These data suggest that the impaired ability
of cytotrophoblasts isolated from the placentas of preeclamptic patients to switch on expression of integrin al
and MMP-9 in culture is due, at least in part, to a dramatic
decrease in the levels of the corresponding mRNAs.
As these molecules mediate cytotrophoblast invasion
in vitro, we examined the ability of pre-eclamptic cytotrophoblasts to invade Matrigel substrates. We compared
the invasiveness of cells isolated from the placentas of
patients with pre-eclampsia who delivered at 29, 30, 27,
and 28 weeks to cells isolated from control patients who
delivered at 22, 24, 26, and 38 weeks. A graph summarizing these data is shown in Figure 7. In all cases, the
cells isolated from patients with pre-eclampsia were severalfold less invasive than the control cells. Overall, these
observations suggest that, in pre-eclampsia, the invasive
ability of cytotrophoblasts is dramatically decreased.
Finally, we also compared the ability of cytotrophoblasts in the two groups to modulate their expression of
stage-specific antigens that are likely to play a role in
immune interactions with maternal cells. In this case, the
antigen of interest was HLA-G, an unusual major histocompatibility complex class lb molecule expressed by
fully differentiated cytotrophoblasts that have invaded the
uterus."a Immediately after isolation, an average of 21%
of control preterm cytotrophoblasts expressed HLA-G,
A.
CON
Oh 12h
PE
Oh 12h
.., .
__ n_
,-.
.......-7-77"-
.I
........T
I;
- 28S
-28S
alto
MMP
-18S
-18S
Figure 6. CTB expression of integrin al and MMP-9 mRNA is reduced in pre-eclampsia. Total RNA was prepared from CTBs isolated from the placenta of a patient
with pre-eclampsia (PE; 27 weeks) and a gestationally matched control patient (CON; 26 weeks) and analyzed by Northern blotting as described in Materials and
Methods. Control CTBs expressed al integrin mRNA immediately after isolation (0 h), and the level increased only slightly after 12 hours of culture (A). In contrast,
CTBs isolated from the placenta of a pre-eclamptic patient expressed so little al mRNA at either 0 or 12 hours that the bands are not visible in the photograph.
CTB expression of mRNA for MMP-9 was also affected by pre-eclampsia (B). As compared with control cells, the onset of expression was delayed and the amount
detected was decreased.
Discussion
Our results suggest that cytotrophoblast differentiation
along the invasive pathway is abnormal in pre-eclampsia.
Specifically, the cells start to differentiate, as shown by
their up-regulation of integrin a5 expression. But their
ability to up-regulate expression of integrin al, MMP-9,
and HLA-G, stage-specific antigens that are associated
with later stages of the differentiation process, is impaired. We also show that this defect has functional consequences; cytotrophoblasts isolated from the placentas
of pre-eclamptic patients were much less invasive in vitro
0
Cytotrophoblast
Invasion
._A
._
en
m
100 -
0
0
80
7;
za
C)
0.
60*
control
PE
40
20 -
Hours in Culture
0
72 hrs
Hours
In
culture
Figure 7. Cytotrophoblast invasiveness is markedly reduced in pre-eclampsia. Invasion was quantified using the first assay method described in Materials and Methods. The data are expressed as percentage of control invasion.
The mean cell count (n = 4 separate experiments) of 639.3 + 191.6 (SEM)
was used as the 100% value. The absolute value for pre-eclamptic cells was
45.7 + 20.2. The difference between the two groups was statistically significant (P
0.037).
1816
Lim et al
-266
-168
HLA-GO
Figure 9. CTB expression of HLA-G mRNA is markedly reduced in preeclampsia. The blot shown in Figure 6A was stripped and rehybridized with
a cDNA probe that corresponds to a 450-bp region of the 3' untranslated
region of HLA-G. PE, pre-eclampsia; CON, control.
than gestation-matched control cells. These data (diagrammed in Figure 10) suggest that, in pre-eclampsia,
cytotrophoblasts differentiating along the invasive pathway cannot complete this process. As a result, they do
not express normal levels of molecules that are required
for invasion (eg, integrin ca1 and MMP-9), a factor that
could restrict invasion to the superficial portions of uterine
arterioles, the placental pathology most frequently associated with pre-eclampsia. Our finding that HLA-G expression is also impaired raises the interesting possibility
that interactions between the invasive subpopulation of
cytotrophoblasts and maternal immune cells change in
pre-eclampsia.
Understanding the end result of pre-eclampsia's effect
on cytotrophoblast differentiation/invasion is helping us to
identify the factors that are critical to normal cytotrophoblast invasion. To date we have identified two circum-
stances in which normal cytotrophoblasts exhibit elements of the pre-eclampsia phenotype. The first is when
first-trimester cytotrophoblasts are cultured in an environment of reduced oxygen tension. We hypothesized that
the lack of endovascular invasion could lead to hypoxia
at the maternal-fetal interface, initiating a feedback
mechanism in which a reduction in oxygen tension further
impairs cytotrophoblast differentiation/invasion. To test
this idea experimentally, we compared the differentiative
and invasive capacity of normal first-trimester cytotrophoblasts cultured either under standard tissue culture conditions (20% 02) or in a hypoxic environment (2% 02). We
previously showed that cytotrophoblasts cultured in 20%
02 undergo normal differentiation along the invasive
pathway, ie, switch their repertoire of stage-specific antigens, and rapidly degrade ECMs on which they are
plated. In hypoxia, the cells' integrin expression is altered
to the pattern observed in pre-eclampsia; ie, they express integrin a5 but not cr1. In addition, their ability to
invade ECM is markedly reduced.29 These results suggest that a reduction in oxygen tension at the maternalfetal interface can lead to abnormalities in cytotrophoblast differentiation/invasion that are similar to those
observed in pre-eclampsia.
The second circumstance in which normal cytotrophoblasts exhibit a pre-eclampsia phenotype is at term.
Starting with the earliest-gestation samples that are routinely available to us (6 weeks), we have noted a progressive decline in the cytotrophoblasts' differentiative and
invasive capacity as gestation progresses. With regard to
differentiation, cells isolated from term placentas have a
greatly reduced ability to up-regulate expression of integrins cr5 and al,11 MMP-9,8 and HLA-G23 in vitro. For
example, the percentage of normal 12- to 16-week cytotrophoblasts that react with antibody against integrin ar1
Normal Pregnancy
Preecfampsla
A. Morphology
FETUS
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Figure 10. Schema illustrating how pre-eclampsia changes cytotrophoblast invasion at the morphological and molecular levels. Comparison of the morphology
of the matemal-fetal interface in normal pregnancy (left, A) and in pre-eclampsia (right, A) shows that this syndrome is associated with abnormally shallow uterine
invasion and, in particular, with failure of CTB to penetrate the uterine arterioles. We theorize that these morphological abnormalities are the result of a defect
in CTB differentiation (B). Our data suggest that in pre-eclampsia, the cells start to differentiate along the invasive pathway (as shown by their ability to express
integrin a5) but do not complete this process (as shown by their reduced ability to express HLA-G, integrin arl, and MMP-9).
1817
Acknowledgments
We thank Ms. Evangeline Leash for editing the manuscript.
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