ample, gestational trophoblastic disease, which in- cludes a spectrum of pregnancy-related trophoblas- tic proliferative abnormalities like hydatidiform mole,.
149, A411zcrncanjourna1/ ol/ Patbologlol.Nol
4, OctobeIr 1996 Copt'rArgii C AoleZic;ica Society /r) Inv'cstiativi( latbholog,t
Abnormal Expression of Plasminogen Activator Inhibitors in Patients with Gestational Trophoblastic Disease
Amparo Estell6s,* Salvador Grancha,* Juan Gilabert,t Terri Thinnes,t Melitina Chirivella,§ Francisco Espaha,* Justo Aznar,11 and David J. Loskutofft ro0n1 the Centro ce lInvestigaci6onl* Cec1r/o .lcjltlrzal,t Serv icio de Anatomia Patolohica,5 and Dcpartancento dc Biopatologia Clnica/ HIoIspita/l Univirsitario La Fe, V'aliciCa, Spain, and the Department of I ascil/-a Biol/otj,, Mhe Scripps Researcb Ii.stitite, Lajolla. Ca/i/d rnia
We previously reported significantly elevated levels of plasminogen activator inhibitor type I (PAI-i) in plasma and placenta from pregnant women with severe pre-eclampsia, and preeclampsia is a frequent problem in molar pregnancies. As increases in PAI-I may contribute to the placental alterations that occur in preeclampsia, we have begun to investigate changes in PAI-I as well as PAI-2 and several other components of thefibrinolytic system in patients with trophoblastic disease. Significant increases in plasma PAI-I and decreases in plasma PAI-2 levels were observed in molar pregnancies when compared with the levels in normal pregnant women of similar gestational age. PAI-I antigen levels also were increased, and PAI-2 levels were decreased in placenta from women with molar pregnancies compared with placenta obtained by spontaneous abortion. Immunohistochemical analysis revealed strong positive and specific staining of PAI-I in trophoblastic epithelium in molar pregnancies and relatively weak staining of PAI-2. No association between the distribution of PAI-I and vitronectin was found, and no specific signalfor tissue type PA, urokinase type PA, tumor necrosis factor- a, or interleukin-I was detected. In situ hybridization revealed an increase in PAI-I but not PAI-2 mRNAs in placenta from molar pregnancies in comparison with placenta from abortions. These results demonstrate increased PAI-I protein and mRNA in trophoblas-
tic disease and suggest that localized elevated levels of PAI-I may contribute to the hemostatic problems associated with this disorder. (Am J Pathol 1996, 149:1229 -1239)
Plasminogen activators (PAs) are serine proteases that convert the zymogen plasminogen into the active serine protease plasmin, the primary enzyme responsible for the removal of fibrin deposits. The fibrinolytic system also plays an important role in many other processes including reproduction (eg, ovulation, blastocyst implantation, and pregnancy).1'2 The activity of the PAs is regulated by specific PA inhibitors (PAls). Type 1 PAI (PAI-1) appears to be the physiological inhibitor of both urokinase PA (u-PA) and tissue-type PA (t-PA) and may therefore be the primary regulator of plasminogen activation in vivo.3'- Although PAI-2 (the so-called placental-type PAI)6 inhibits u-PA, it is a relatively poor inhibitor of single-chain t-PA.7 Both PAI-1 and PAI-2 increase throughout normal pregnancy.8-10 Although dramatic changes in the expression of coagulation' 1,12 and fibrinolytic'-", 13,14 components occur during normal pregnancy, they are not life-threatening, and normal hemostatic balance, in general, is maintained. However, there are certain clinical situations in obstetrics that actually alter the coagulation/fibrinolysis equilibrium to such an extent that they may be life-threatening.6 -10, 13-18 For example, gestational trophoblastic disease, which includes a spectrum of pregnancy-related trophoblastic proliferative abnormalities like hydatidiform mole, chorioadenoma destruens, and choriocarcinois frequently associated with the dissemima,
'"
Supported in part by National Institutes of Health grant HL47819 (to D. J. Loskutoff) and in part by FIS grants 93/0380 and 96/1256 (to A. Estelles). Accepted for publication May 29, 1996. Address reprint requests to Dr. Amparo Estelles, Centro de Investigacion, Hospital Universitario La Fe, Avenida de Campanar 21, 46009 Valencia, Spain.
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nated intravascular coagulation syndrome,21'22 and an association between molar pregnancies that persist into the second trimester and pre-eclampsia has been reported.19'20 As pre-eclampsia is rarely seen before the 24th week, pre-eclampsia that develops before this time strongly suggests hydatidiform mole or extensive molar changes. Fatal eclampsia associated with molar pregnancy and pre-eclampsia and hydatidiform mole in early pregnancy have been reported.19'20 Histologically, hydatidiform moles are characterized by abnormalities of the chorionic villi consisting of varying degrees of trophoblastic proliferation and edema of villous stroma.20 The histological structure is characterized by hydropic degeneration and swelling of the villous stroma, the absence of blood vessels in the swollen villi, proliferation of the trophoblastic epithelium, and the absence of the fetus and amnion.20 There are often a large number of Hofbauer cells. The trophoblastic covering varies enormously from mole to mole, and occasionally the trophoblast degenerates or is enmeshed in fibrin.19'20 The spectrum of cells that are in the mole is transitional between the cytotrophoblast and syncytium.19'20 Although the incidence of hydatidiform moles is approximately 1 in 2000 pregnancies in the United States and Europe, it may be much higher in other countries. It is approximately 10 times more frequent in women older than 45 compared with those of ages 20 to 40. The risk of developing neoplasia from a complete mole is approximately 20%.19 Although alterations in plasma PAI-1 and PAI-2 occur in pregnant women who are prone to thrombotic disorders, the cause and origin of the altered level of expression of these molecules in these patients is still unclear. Immunohistochemical studies of placenta from normal pregnancies demonstrate that the villous trophoblast and syncytiotrophoblast both stain intensely with antibodies to PAI-2,23'24 whereas a weak reactivity is detected in the cells using antibodies to PAI-1.23 We have previously reported that plasma PAI-1 levels are significantly elevated in pregnant women with severe pre-eclampsia in comparison with normal pregnant women, whereas a concomitant decrease in plasma PAI-2 levels is observed in pregnancies complicated with intrauterine fetal growth retardation.9 15'18'25 We have also provided evidence that altered production of these two molecules in the syncytiotrophoblast is responsible, at least in part, for the changes in their plasma concentrations.25 The ultrastructural changes in the villous trophoblast in pre-eclampsia also occur in hydatidiform moles associated with pre-eclampsia.20 Little information is available about the biochemical
changes in placenta of pre-eclamptic patients26 or patients with trophoblastic disease, and the role of these alterations in the pathogenesis of the disease is not clear. In a recent study,27 plasma PAI-1 and PAI-2 levels were examined in two molar pregnancies. Although PAI-2 levels were below the level of detection, plasma PAI-1 levels were increased compared with normal pregnancies. Little is known about the factors that regulate these PAls in normal and molar placentas. In this regard, vitronectin (Vn) is a PAI-1-binding protein that can stabilize PAI-1 activity,28'29 and this adhesive protein has also been found in placental tissue.30'31 However, although PAI-1 and Vn antigens appear to co-localize in trophoblasts from normal placenta,31 their distribution in trophoblastic disease is unknown. The aim of the present study was to confirm and extend these preliminary studies of hydatidiform mole pregnancies by determining the plasma concentration and tissue distribution not only of PAI-1 and PAI-2 but also of Vn, t-PA, and u-PA. Our results demonstrate significant increases in plasma PAI-1 antigen and in placental PAI-1 antigen and mRNA in molar pregnancies compared with controls. In general, PAI-2 levels were decreased in these patients.
Materials and Methods Clinical Groups The hydatidiform mole group included 11 women in the first trimester of pregnancy (gestational age, 14.5 + 0.5 weeks of pregnancy). In each case, the diagnosis of hydatidiform mole was confirmed by histological examination. The plasma control group for this study consisted of 10 healthy normotensive women in the first trimester of pregnancy (gestational age, 14 + 1 weeks of pregnancy). The control group for placental extracts included 4 spontaneous abortions resulting from cervical incompetence in the first trimester of pregnancy (gestational age, 16.5 + 4.4 weeks of pregnancy). All of the women were from the Maternity Center of La Fe Hospital, Valencia, Spain, and informed consent was obtained before sample extraction from all of the patients and pregnant women.
Blood Samples Blood was collected into 3.8% sodium citrate (9:1, v/v, blood:anticoagulant) by venipuncture using the cubital vein. The citrated blood was centrifuged at 1500 x g
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for 15 minutes at 40C, and the resulting plasma was collected and stored at -6000 until used.
and high molecular weight u-PA. The interassay variability was 10%.
Collection of Tissue Samples and Extraction of Protein from Placenta
Assay for PAI- 1 and PAI-2 Antigen
Placental samples for this study were obtained immediately after delivery as previously described.25 Briefly, villous trophoblast tissue was carefully removed from the placenta, rinsed in cold phosphatebuffered saline (PBS) to remove blood, and processed for immunohistochemistry and in situ hybridization as previously described.25 In both cases, the sections were fixed overnight at 40C in 4% paraformaldehyde and embedded in paraffin blocks. Some specimens were flash-frozen in liquid nitrogen for extraction of protein. Proteins were extracted from the frozen placenta by cutting the tissue into small pieces and incubating approximately 1 g of it at 40C for 24 hours in 10 ml of 0.1 mol/L Tris/HCI, pH 7.5, 2 mol/L KSCN. The cellular debris was removed by centrifugation (1 0,000 x g for 30 minutes), and the supernatant was dialyzed against PBS. Total protein in the samples was determined by utilizing the BCA protein assay (Pierce, Rockford, IL).
PAI-1 and PAI-2 antigen levels in plasma and placenta samples were quantified by commercially available ELISAs (Tint Elize PAI-1 and PAI-2, Biopool). According to the manufacturer, the PAI-1 assay detects free and complexed forms of PAI-1 with the same efficiency. The PAI-2 assay detects both the high (60-kd) and low (48-kd) molecular weight forms of PAI-2.
Assay for Transforming Growth Factor (TGF)-,B Antigen TGF-f antigen levels in placenta samples were quantified by a commercially available immunoassay (Quantikine human TGF-,B1, R&D Systems, Minneapolis, MN). The assay has a sensitivity of 5.0 pg/ml. The intra- and interassay variability was 5 and 7%, respectively.
Antibodies for Immunohistochemistry Assay for PAI- 1 Activity The PAI-1 activity assay was performed as previously described using an amidolytic method.15 One unit of PAI is defined as the amount that inhibits 1 IU of single-chain t-PA in 15 minutes at room temperature under the conditions used. The intra- and interassay variability was 6 and 10%, respectively.
Assay for t-PA Antigen t-PA antigen was determined by using a commercially available enzyme-linked immunosorbent assay (ELISA; Imulyse t-PA, Biopool, Umea, Sweden). The assay detects free and complexed t-PA with similar efficiency. The intra- and interassay variability was 4 and 6%, respectively.
Assay for u-PA Antigen u-PA antigen was quantified by a commercially available ELISA (Tint Elize u-PA, Biopool). According to the manufacturer, the assay measures single-chain u-PA and the high molecular weight form of u-PA with approximately the same efficiency. The low molecular weight form of u-PA is measured with approximately 25% of the efficiency of single-chain u-PA
Antiserum to purified human PAI-1 was raised in New Zealand rabbits according to standard procedures, and the specific antibodies to PAI-1 were affinity purified using human PAI-1 bound to cyanogen-bromide-activated Sepharose 4B.32 Affinity-purified rabbit anti-human Vn,33 anti-human interleukin (IL)-1 (Endogen, Boston, MA), anti-human tumor necrosis factor (TNF)-a (Endogen), caprylic-acid-purified rabbit anti-human t-PA, and affinity-purified goat antihuman PAI-2 or goat anti-human u-PA (American Diagnostica, Greenwich, CT) were also used.
Immunohistochemistry Immunohistochemical staining was performed using the avidin-biotin-peroxidase method as previously described.25'34 Briefly, fixed and paraffin-embedded placental tissues were cut into 3- to 5-,um sections using a microtome, placed onto slides coated with polylysine, and air dried. The sections were deparaffinized in xylene, cleared in 95% ethanol, incubated in 3% hydrogen peroxide in methanol for 10 minutes, and rehydrated through graded ethanol. After washing with Tris-buffered saline (TBS), pH 7.4, the sections were treated with increasing concentrations of Triton X-100 (0.1 to 1%) in TBS for permeabilization. To unmask antigen immunoreactivity, the
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sections were incubated (370C for 5 minutes) with 0.23% (w/v) pepsin (2830 U/mg, Worthington Biochemical Corp., Freehold, NJ) in 0.01 N HCI. Tissue sections were subsequently incubated for 1 hour at 220C with 1% normal goat or rabbit serum in TBS to minimize nonspecific binding. The sections then were incubated with the affinity-purified rabbit or goat antibodies. In all experiments, preimmune rabbit IgGs or preimmune goat IgGs were used as control. After washing with 1% Triton/TBS (three times for 3 minutes each), a 1:100 dilution of the second antibody (biotinylated goat anti-rabbit IgG or rabbit anti-goat IgG; Jackson Immunoresearch Laboratories, West Grove, PA) was added. The tissue sections were washed again with 1% Triton/TBS (three times for 3 minutes each), incubated with streptavidin-peroxidase conjugate prepared according to the manufacturer's instructions for 10 minutes at room temperature, washed with 1% Triton/TBS (three times for 3 minutes each), and then treated with a freshly prepared aminoethylcarbazole chromogen containing 0.02% hydrogen peroxide at room temperature for 15 minutes. Finally, the sections were counterstained with Mayer's hematoxylin for 3 minutes at room temperature, rinsed well with tap water, and mounted on a GVA-mount (Zymed, San Francisco, CA).
In Situ Hybridization In situ hybridizations were carried out essentially as described.25'35 Briefly, hybridizations were performed by adding 600,000 cpm of 35S-labeled riboprobe in 20 ,ul of prehybridization buffer containing 2.5 mg/ml transfer RNA to the tissue sections. The slides were incubated at 55°C overnight, washed with 2X standard saline citrate (SSC), treated with RNAse A, washed again with 2X SSC, and finally washed at 600C for 2 hours in 0.1X SSC. All of the SSC solutions contained 10 mmol/L 2-mercaptoethanol and 1 mmol/L EDTA. To prepare sections for autoradiography, the slides were washed in 0.5X SSC without 2-mercaptoethanol, dehydrated in a graded alcohol series containing 0.3 mol/L NH4Ac, dried, coated with NTB2 emulsion, and exposed in the dark at 40C for 2, 4, and 12 weeks. Slides were developed, fixed, washed, and counterstained with hematoxylin and eosin. Parallel sections were analyzed using the sense probe as control, and no specific signal was detected after 12 weeks of exposure.
Statistical Analysis Levels of significance were determined by the Student t- and by the Mann-Whitney nonparametric U tests. Values
Sm
.......
*.
Figure 3. Immunohistochemistry and in situ hybridization of PAI-1 and Voi from molar placenta. Paraffin sectiotns of placentas from molair pregnancies ivere analyzed by inmmunohistochemical tecbniques for PAI-1 antigen (A and E) andjbr Vn (B and F). Positive staining is indicated by the brown color. Paraffin sectionts oJfplacentasfrom molar pregnianzcies were also analyzed by in situL hybridization (blue color) fbr PAI-1 mRNA (C antd G) and for Vn mnRNA (D and H). C anid G were exposedfor 2 wveeks, whereas D and H were exposed/for 12 uwee-ks. Original maicign/ificationi, X 400.
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centas of the different clinical groups. Prominent PAI-1 immunoreactivity was detected in the trophoblastic epithelium of molar placenta (Figures 1A and 2A) despite the immaturity of the placental villi and in contrast to the weak PAI-2 immunoreactivity (Figure 1C). These results are very similar to those previously reported by our group in severe pre-eclampsia with intrauterine fetal growth retardation.25 The positive PAI-1 immunoreactivity suggests, but does not prove, that PAI-1 is actually synthesized by the stained cells. To demonstrate PAI-1 gene expression in specific cells within placental tissue, we have used an in situ hybridization analysis. A strong positive signal for PAI-1 mRNA was detected in trophoblastic epithelium from molar pregnancies after only 2 weeks of exposure (Figures 1B and 2B). In contrast, a relatively weak signal for PAI-1 mRNA was observed in isolated placental villi from abortions (Figure 2D), and this signal was apparent only after prolonged (ie, 12 weeks) exposure. We previously reported an increase in PAI-1 mRNA in placenta from pre-eclamptic patients.25 To our knowledge, this is the first report of increased PAI-1 expression in molar placenta, and the factors regulating the production of PAI-1 in this disease therefore remain to be defined. A variety of compounds (eg, cytokines and growth factors) are capable of inducing the expression of PAI-1,4 and normal placentas are able to produce several different cytokines, hormones, and growth factors.3842 In this regard, human TNF was reported to stimulate gene transcription of PAI-1 and simultaneously suppress constitutive gene expression of t-PA in human fibrosarcoma cells.43 Preliminary experiments were performed to determine whether changes in the levels of some cytokines correlates with the expression of PAI-1 in molar pregnancies. Unfortunately, immunohistochemical analysis failed to reveal TNF-a or IL-1 immunoreactivity in trophoblastic epithelium in the molar placentas studied. More sensitive approaches will be necessary to relate changes in these cytokines to PAI-1 biosynthesis. In the present report, a slight increase in TGF-,B levels was observed in placental extracts from molar pregnancies in comparison with placental extracts from abortions. TGF-3 is known to induce the synthesis of PAI-1 and can decrease the induction of PAI-2.4445 Our results suggest that the increased PAI-1 levels obtained in molar pregnancies may be mediated, at least in part, by TGF-,B. However, more studies are required to delimit the role of this growth factor in the alterations of PAI levels observed in molar pregnancies.
It has been reported that Vn is a PAI-1-binding protein that can localize PAI-1 in tissues and stabilize its activity.2829'46 This adhesive glycoprotein has also been detected in placental tissue,3031 and in a recent report, data were presented to suggest that it co-localized with PAI-1 in trophoblasts of normal placenta.31 However, in the present study, immunohistochemical and in situ hybridization analysis (Figure 3) indicate that these molecules do not co-localize in placental tissue from molar pregnancies. In fact, their distribution patterns appear to be quite distinct. In conclusion, the results in this report demonstrate increased PAI-1 antigen in plasma and increased PAI-1 antigen and mRNA in tissue from trophoblastic disease, and these increases in PAI-1 are similar to those associated with pre-eclampsia. The parallelism between the two pathologies could indicate a common physiopathological mechanism. Our data are consistent with the possibility that the increase in PAI-1 can play a role in the origin of placental damage produced by both pathological conditions.
Acknowledgments We thank Encarnacibn Hervas, MaCarmen Martfn, Fuencisla Cuesta, Pilar Escamilla, and Araceli Serralbo for their technical assistance and Esther Olmos and Marcia McRae for their secretarial assistance.
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