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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 12 5995-6001
Copyright © 2003 by The Endocrine Society

Endothelial Cells and Peripheral Blood Mononuclear Cells Are a Potential Source of Extraplacental Activin A in Preeclampsia

D. S. Tannetta, S. Muttukrishna, N. P. Groome, C. W. G. Redman and I. L. Sargent

Nuffield Department of Obstetrics and Gynaecology (D.S.T., C.W.G.R., I.L.S.), University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom; Department of Obstetrics and Gynaecology (S.M.), Royal Free University College London Medical School, London WC1E 6HX, United Kingdom; and School of Biological and Molecular Sciences (N.P.G.), Oxford Brookes University, Headington, Oxford OX3 0BP, United Kingdom

Address all correspondence and requests for reprints to: Dr. Dionne Tannetta, Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington OX3 9DU, United Kingdom. E-mail: d.s.tannetta{at}obs-gyn.ox.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An excessive systemic inflammatory response, involving endothelial cells and leukocytes, underlies the maternal symptoms of preeclampsia. Activin A is raised in preeclampsia, suggesting a possible involvement in its pathophysiology. The placenta is the main source of activin A in normal pregnancy. We investigated whether peripheral blood mononuclear cells (PBMCs) and endothelium, activated by proinflammatory stimuli, were a potential source of activin A in preeclampsia. Both endotoxin and TNF{alpha} stimulated activin A secretion by PBMCs from nonpregnant, preeclamptic, and matched normal pregnant women (P < 0.05). Pregnancy increased the responsiveness of PBMCs to endotoxin (P < 0.05), whereas only the preeclamptic group were significantly more responsive to TNF{alpha} (P < 0.05). Human umbilical vein endothelial cells secreted activin A spontaneously and in response to TNF{alpha} (P < 0.05), but recombinant IL-1ß and IL-6 had no significant effect over the 72-h culture period. Inhibin A and follistatin were undetectable (<2 pg/ml and < 20 pg/ml, respectively) in PBMCs and human umbilical vein endothelial cell culture media. These data suggest that PBMCs and endothelium, activated by TNF{alpha}, could be extraplacental sources of activin A in preeclampsia. The pathological significance of increased activin A in preeclampsia is unknown, although it may have a role in the mechanisms underlying endothelium dysfunction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PREECLAMPSIA IS A pregnancy-specific disease, which is a major cause of maternal and fetal morbidity and mortality. Its etiology is as yet unknown. A major step in unraveling its pathogenesis came with the finding that generalized maternal endothelial cell dysfunction underlies the features of the maternal syndrome (1, 2). We have proposed that the endothelial dysfunction is part of a more generalized systemic inflammatory response that occurs in the maternal circulation in normal pregnancy but is exaggerated in preeclampsia where, in some aspects, it is as intense as that seen in sepsis (3, 4).

It is generally agreed that the agent(s) that provoke the inflammatory response in normal pregnancy and preeclampsia originate from the placenta (5). There are many placental products, which are raised in the circulation of preeclamptic women, that are candidates for this factor, including proinflammatory cytokines (6, 7, 8), syncytiotrophoblast microparticles (9, 10) and lipid peroxides (11). Another potential candidate is activin A.

Activin A is a glycoprotein hormone produced by many tissues (12, 13). During normal pregnancy, the fetoplacental unit is the main source of activin A, with the placenta producing the majority of the secreted activin A (13, 14). It belongs to the TGFß superfamily, a group of proteins whose members are involved in the control of cell proliferation and differentiation in many systems (15). Activin A activity is tightly controlled by a high affinity binding protein, follistatin, which regulates its availability and clearance (16, 17), and by the structurally related molecule, inhibin, which appears to function as an antagonist, competing with activin for binding to its receptors (18).

We have previously shown that high levels of activin A circulate during normal pregnancy. Maternal blood levels are raised but constant at around 2 ng/ml, compared with approximately 250 pg/ml in nonpregnant women, up to 26–28 wk, after which time there is a further rapid rise, reaching a peak at term of around 25 ng/ml (19). Activin A levels are further raised in the maternal circulation in preeclampsia, with levels as much as 9 times higher than those seen in gestational age-matched normal pregnant controls (20, 21). Longitudinal studies performed in this laboratory have also shown that activin A levels are raised before the manifestation of maternal symptoms in some women developing preeclampsia and may reflect the severity of the disease (22), suggesting a role in its pathogenesis, although its suitability as a predictive marker is questionable (22, 23, 24, 25).

A probable source of the increased secretion of activin A in preeclampsia is the placenta. The expression of both {alpha}- and ßA-subunit mRNA and proteins is up-regulated in placentas from preeclamptic pregnancies (26, 27, 28, 29, 30). The raised levels could be a consequence of the maternal inflammatory response. Secretion of activin A by cytotrophoblasts from normal placentas in vitro is stimulated by the proinflammatory cytokines TNF{alpha} and IL-1ß (31), the circulating levels and placental production of which are increased in preeclampsia (6, 7, 8, 32, 33). Alternative stimuli for the increased activin A production by placental and extraplacental sources could also be a yet unidentified placental factor(s).

Additional, uninvestigated sources of activin A in preeclampsia are activated monocytes and endothelial cells. Both monocytes and macrophages are known to secrete activin A in response to proinflammatory stimuli (34, 35, 36, 37, 38). Several types of endothelial cells, relevant to pregnancy, such as umbilical vein endothelial cells (39, 40) and vascular endothelial cells of myometrial blood vessels (41) and placental vessels (42) express ßA-subunit mRNA. Activation of endothelial cells, by exposure to medium conditioned by oxidized low-density lipoprotein-treated monocytes, also causes an up-regulation in ßA-subunit mRNA expression (43). To date, secretion of ßA-dimer, the biologically active form of activin A, has not been reported.

We therefore sought to determine whether peripheral blood mononuclear cells (PBMCs) and endothelial cells could be contributing to the raised levels of activin A in normal pregnancy and preeclampsia as a consequence of the systemic inflammatory response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

All materials were carefully sourced to guarantee minimal levels of endotoxin contamination. The medium used to culture PBMCs contained RPMI 1640 with L-glutamine purchased from Invitrogen (Paisley, Scotland), heat-inactivated fetal calf serum (FCS), purchased from Autogen Bioclear (Wiltshire, UK) and 100 mM sodium pyruvate solution, ß-mercaptoethanol, and penicillin (5000 U/ml) and streptomycin (5 mg/ml) solution purchased from Sigma-Aldrich Co. Ltd. (UK). Hanks’ balanced salt solution was purchased from Invitrogen, Histopaque-1077 was supplied by Sigma-Aldrich, and cell suspension 24-well plates was purchased from Sarstedt Ltd. (Leicester, UK). Human umbilical vein endothelial cell (HUVEC) culture medium reagents [M199 medium containing L-glutamine, endothelial cell growth supplement from bovine neural tissue, heparin and penicillin, and streptomycin and neomycin solution (5000 U, 5 mg, and 10 mg/ml, respectively, in saline)], collagenase type 1A, gelatin, PBS, and BSA were obtained from Sigma-Aldrich (Poole, Dorset, UK). Heat-inactivated FCS for HUVEC culture and Accutase were purchased from PAA Laboratories (Yeovil, Somerset, UK). Nunc tissue culture plastic ware was used for HUVEC culture (Fisher Scientific, Loughborough, UK). Endotoxin (lipopolysaccharide from Escherichia coli serotype 026:B6) was purchased from Sigma-Aldrich, and recombinant human TNF{alpha}, IL-6, and IL-1ß were purchased from R&D systems (Abingdon, UK). Hoechst 33258 (bis-benzimide) was purchased from Molecular Probes Inc. (Poortgebouw, The Netherlands) as a 10 mg/ml stock solution in water.

PBMC culture for determination of secreted activin A, inhibin A, and follistatin

Blood samples were collected from nonpregnant women (10); women suffering from preeclampsia (PE) (n = 10), and normal pregnant women (n = 10) matched to the PE group for maternal age (±4 yr), gestational age (±13 d), and parity (0, 1–3, 4+). The nonpregnant women used in this study were healthy, normally cycling women, between the ages of 20–35 yr, who were not taking any medication or on any form of hormonal contraceptive. Normal pregnant women were selected if they had no history of hypertension or chronic illness, a singleton pregnancy without known fetal abnormality, and natural conception. PE was defined as new hypertension (blood pressure > 140/90 mm Hg on two consecutive occasions) and new proteinuria (24 h secretion of >=500 mg), in the absence of urinary tract infection. This study was approved by the Central Oxford Research Ethics Committee, and informed consent was obtained from all individuals recruited onto the study.

PBMCs were isolated from 40 ml heparinized blood, using Histopaque-1077 separation. After isolation, PBMCs were washed in PBS, resuspended in culture medium, and counted. PBMC culture medium (CM) consisted of RPMI 1640 (batches with very low endotoxin levels were used) containing L-glutamine, supplemented with 1 mM sodium pyruvate, 2 x 10-5 M ß-mercaptoethanol, penicillin (50 U/ml), streptomycin (50 µg), and 10% (vol/vol) heat-inactivated FCS (the same batch, which contained a very low level of endotoxin, was used for all of the PBMC experiments). The cell suspension was diluted in culture medium to give 2 x 106 cells/ml. Cells were then plated out at a density of 2 x106 cells/ml/well into 24-well suspension plates. Treatments were added immediately after the cells were plated out, in a volume of 50 µl Hanks’ balanced salt solution. The treatments tested were endotoxin (0, 0.2, 1, 5, and 50 ng/ml) and TNF{alpha} (0, 1, 10, and 100 ng/ml). The PBMCs were cultured for 20 h at 37 C in 5% CO2 and 95% air, after which time the conditioned culture medium was collected, centrifuged to remove any cells, and the resultant supernatant stored at -20 C until subsequent analysis for activin A, inhibin A, and follistatin.

HUVEC isolation and routine culture

Umbilical cords were obtained, with consent, from uncomplicated normotensive term pregnancies delivered by cesarean section. HUVECs were isolated according to a previously published method (44). Cells were cultured in HUVEC CM (M199 containing L-glutamine supplemented with 10% (vol/vol) heat-inactivated FCS, endothelial cell growth supplement (30 µg/ml), heparin (90 µg/ml), and penicillin, streptomycin, and neomycin (50 U/ml, 50 µg, and 100 µg/ml, respectively). Several batches of FCS were screened by measuring the proliferation of HUVECs in the presence of increasing amounts of the FCS to be tested before a batch suitable for HUVEC culture was found. The batch of FCS considered suitable was then used throughout the experiments carried out on HUVECs. Isolated HUVECs were seeded into 25-cm2 flasks coated with 1% gelatin. Once confluent, the cells were washed with PBS and then detached using Accutase. The resultant pellet was resuspended with HUVEC CM and passaged into 75-cm2 and then 175-cm2 gelatin-coated flasks. Cells between passages 4 and 5 were used for all experiments.

HUVEC culture procedure for determination of secreted activin A, inhibin A, and follistatin

On d 1, HUVECs (50,000 cells/well) were plated out into 1% gelatin-coated 24-well plates in 1 ml HUVEC CM. The cells were left overnight at 37 C in 5% CO2 and 95% air to allow cells to adhere to the plate. On d 2, CM was removed and fresh HUVEC CM (1 ml/well) was added to each well along with treatments to be tested. The treatments tested were TNF{alpha} (0, 0.1, 1, 10, 100 ng/ml), IL-6 (0, 0.1, 1, 10, 100 ng/ml), and IL-1ß (0, 0.05, 0.5, 5, and 50 ng/ml). Treatments were tested in HUVEC CM rather than a basal CM because HUVECs could not be maintained satisfactorily in basal medium over the culture period. Culture medium was replaced every 24 h, on d 3, 4, and 5, and fresh treatments were added at this time. HUVEC-conditioned CM was collected at each time point, centrifuged to remove any cells, and the supernatant stored at -20 C for subsequent analysis of activin A, inhibin A, and follistatin. Adherent cells were kept for determination of DNA content in each well.

Quantitation of HUVEC DNA

Total DNA/well was quantified using a previously published method (45). This method measures the enhancement of Hoechst 33258 fluorescence when it binds to DNA. At the end of the 72-h culture period, conditioned medium was removed for analysis, and adherent cells were washed with PBS. Diluent (PBS + 1% BSA + 0.1% sodium azide; 250 µl/well) was added to all wells before the plates were stored at -40 C. Cell lysates were obtained by freeze/thaw cycles (two) followed by sonication (sonicating water bath for 10 min). Calf thymus DNA reconstituted in diluent was used to generate a standard curve, using a doubling dilution range of 0.156–10 µg/ml. Blank sample (diluent), standards, and cell lysates were then assayed in duplicate (50 µl/tube) following the previously published protocol (45).

Activin A ELISA

Total Activin A (follistatin bound + unbound) was measured using a two-site ELISA with high specificity for activin A, as described previously (46). Affinity-purified human activin A was used as the assay standard. One minor modification was made to the assay protocol for the analysis of HUVEC-conditioned CM, in that the activin A standard curve was diluted in HUVEC CM. The detection limit of the assay for purified human activin A was 50 pg/ml. Intra- and interassay coefficients of variation were 9% and 10%, respectively.

Inhibin A ELISA

Total inhibin A was measured using a two-site ELISA described previously (47). To measure inhibin A in HUVEC-conditioned CM, the inhibin A standard curve was diluted in HUVEC CM. The sensitivity of the assay was 2 pg/ml, and the intra- and interassay variations were 5.2% and 6.5%, respectively.

Follistatin ELISA

Follistatins (bound and unbound) were measured using a two-site ELISA described previously (48). The follistatin standard curve was generated by diluting the standards in HUVEC CM for the measurement of follistatin in HUVEC-conditioned CM. The sensitivity of the assay was 20 pg/ml. Intra- and interassay variations were 7% and 9%, respectively.

Statistical analysis

Activin A secretion by PBMCs is expressed as picograms per milliliter of neat culture medium. Data for activin A secretion by HUVECs are expressed as picograms per milliliter neat culture medium per microgram DNA to correct for differences in the numbers of cells per well because of treatment effect. All data for HUVEC experiments represent the mean ± SEM of four separate experiments. Kruskall-Wallis nonparametric test and Dunn’s multiple comparison post hoc test were performed to analyze the effect of treatment dose and the effect of subject group (for PBMC activin A secretion) and time in culture (for HUVEC activin A secretion). Statistical analyses were carried out using GraphPad Prism version 4.0 (GraphPad Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of proinflammatory stimuli on activin A secretion by PBMCs

No spontaneous release of activin A was detectable in any of the three groups, but a dose-dependent release was stimulated by increasing doses of endotoxin [1 ng/ml lipopolysaccharide nonpregnant (NP): P < 0.05; matched normal pregnant (MP): P < 0.01; and PE: P < 0.01] (Fig. 1Go). The levels of activin A secretion were significantly higher in the MP (P < 0.05) and PE groups (P < 0.05), compared with the NP group (Fig. 1Go). There was no significant difference between the levels of activin A secreted by the MP and PE groups.



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  		FIG. 1. Activin A secretion by PBMCs, collected from NP (A) (n = 10), MP (B) (n = 10), and PE (C) (n = 10) women and cultured over a 20-h period in the presence of increasing doses of endotoxin. All subjects were matched for age and parity. NP and PE subjects were additionally matched for gestational age. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 when compared with the untreated control values in the same treatment group. a, Significant difference (P < 0.05) when compared with the same treatment dose in the NP group.

 
The proinflammatory cytokine TNF{alpha} stimulated a dose-dependent increase in activin A secretion by PBMCs from all three groups (10 ng/ml; NP: P < 0.01; MP: P < 0.01; and PE: P < 0.0001) (Fig. 2Go). The PE group secreted significantly higher levels of activin A in response to TNF{alpha} than the NP group (P < 0.05). There was no significant difference between the levels of activin A secreted by the MP and PE groups in response to any of the doses of TNF{alpha} tested.



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  		FIG. 2. Activin A secretion by PBMCs, collected from NP (A) (n = 10), MP (B) (n = 10), and PE (C) (n = 10) women and cultured over a 20-h period in the presence of increasing doses of TNF{alpha}. All subjects were matched for age and parity. NP and PE subjects were additionally matched for gestational age. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 when compared with the untreated control values in the same treatment group. a, Significant difference (P < 0.05) when compared with the same treatment dose in the NP group.

 
Effect of proinflammatory stimuli on PBMC inhibin A and follistatin secretion

Inhibin A and follistatin levels, in culture medium from untreated PBMCs and PBMCs treated with increasing doses of TNF{alpha} and endotoxin were below the detection limit of the assay (2 pg/ml for inhibin A and 20 pg/ml for follistatin) (data not shown).

Effect of proinflammatory cytokines on HUVEC activin A secretion

Activin A was secreted spontaneously by HUVECs throughout the culture period (control well activin A concentrations (picograms per milliliter per microgram DNA per 24 h): 0–24 h = 654.85 ± 69.49; 24–48 h = 583.27 ± 57.16 and 48–72 h = 878.79 ± 136.49) (Fig. 3Go). Increasing doses of recombinant TNF{alpha} stimulated an increase in activin A secretion after 24 and 72 h in culture (0–24 h: P < 0.05; 48–72 h: P < 0.01) (Fig. 3AGo). Over time (between 48 and 72 h in culture), there was a significant increase in activin A secretion in response to 1 and 100 ng/ml TNF{alpha} (P < 0.05). Recombinant IL-1ß (0.05–50 ng/ml) tended to cause an increase in activin A secretion, but the effect did not reach significance (Fig. 3BGo). Recombinant IL-6 (0.1–100 ng/ml) had no effect on activin A secretion throughout the culture period (Fig. 3CGo).



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  		FIG. 3. Effect of increasing doses of TNF{alpha} (A), IL-1ß (B), and IL-6 (C) on HUVEC secretion of activin A after 24 h (closed triangle), 48 h (closed square), and 72 h (closed inverted triangle) in culture. Data presented are mean ± SEM of four experiments. *, P < 0.05 and **, P < 0.01 when compared with the untreated control values in the same period of culture. a, Significant difference (P < 0.05) when 48-h culture period was compared with the same treatment dose at 72 h in culture.

 
Effect of proinflammatory cytokines on HUVEC inhibin A and follistatin secretion

Inhibin A and follistatin levels, in culture medium from untreated HUVECs and HUVECs treated with increasing doses of TNF{alpha}, IL-6, and IL-1ß were below the detection limit of the assay (2 pg/ml for inhibin A and 20 pg/ml for follistatin) (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well established that the placenta is a source of activin A in normal pregnancy (49). There is also growing evidence that placental production of activin A is increased in pregnancies complicated with preeclampsia (26, 27, 28, 29, 30). Possible stimuli for the increase in placental activin A include oxidative stress and increased production of proinflammatory cytokines. Significantly higher circulating levels of activin A in pregnancies with poor placental perfusion resulting in babies with intrauterine growth retardation was recently reported (50), supporting the observation of increased fetoplacental production of activin A in a sheep model of fetoplacental hypoxia (51). We have previously shown that cytotrophoblasts in vitro secrete increased levels of activin A in response to the proinflammatory cytokines TNF{alpha} and IL-1ß (31), whose production is also increased in placentas from preeclamptic pregnancies (32, 33). Circulating levels of TNF{alpha} and IL-6 are also raised in preeclamptic women (6, 7, 8), and activin A levels are further increased in preeclamptic pregnancies complicated by babies with intrauterine growth retardation (52), suggesting that a maternal component of preeclampsia further increases the production of activin A. Even in normal pregnancy, in which we have shown that there is already activation of the maternal innate immune system (3), removal of the fetoplacental unit reduces activin A levels by only 50% (53). Given the relatively short half-life of activin A in the circulation (53), again this suggests that pregnancy stimulates the production of activin A by extraplacental sources. The experiments described here support the hypothesis that activin A could also be produced by maternal peripheral blood mononuclear cells and endothelium in women with PE.

We demonstrated that PBMCs from normal pregnant women produce significantly higher levels of activin A in response to proinflammatory stimuli than cells from nonpregnant women and that this is further increased in PE. We have previously shown that a marked systemic inflammatory response is established in the third trimester of normal pregnancy, characterized by increased expression of activation markers on the surface of monocytes, lymphocytes, and granulocytes, which is further increased in PE (3, 4). Because activated monocytes have been shown to secrete activin A, the present observation supports the hypothesis that in PE monocytes are stimulated to secrete activin A. There was no increase in spontaneous secretion of activin A by normal pregnant or preeclampsia PBMCs. This may be due to highly activated cells secreting activin A being lost from the circulation, either through margination, the process whereby circulating activated monocytes migrate into the tissues and mature into macrophages (54), or apoptosis, because higher levels of apoptotic microparticles have been found in the circulation of preeclamptic women (55).

The other major cell type involved in the systemic inflammatory response in PE is the endothelium. Because it was not possible to obtain endothelial cells from pregnant women, we studied the effects of proinflammatory cytokines on HUVECs in vitro and found that they secreted higher levels of activin A in response to TNF{alpha} and IL-1ß. Other studies also indicate that activated endothelial cells are a source of activin A. Exposure of endothelial cells to the conditioned medium from human monocytes treated with oxidized low-density lipoprotein up-regulates the expression of ßA-subunit (43), suggesting that secretion of activin A is stimulated when endothelial cells become activated. Because generalized vascular endothelial cell activation underlies the maternal symptoms of PE (1, 2), these observations strongly suggest that activated endothelial cells are an extraplacental source of activin A in PE.

In support of a systemic inflammatory response stimulating the increased production of activin A by immune cells, circulating activin A levels are raised in patients suffering from septicemia (our unpublished observation) in which there is also an intense intravascular inflammatory response. The observation of a stimulatory effect of TNF{alpha} on both PBMC and HUVEC activin A secretion is highly significant because TNF{alpha} is raised in the circulation of normal pregnant women and further raised in preeclamptic women (6, 7, 8). Moreover, several studies report circulating TNF{alpha} levels in septic patients (56, 57, 58), which are comparable with the levels reported in preeclamptic women (6, 7, 8). These levels are much lower than the concentrations tested in the present experiments (approximately 15 pg/ml vs. 1–100 ng/ml, respectively). The in vitro model of acute exposure of cells to a single high dose may not be a good representation of the in vivo situation, in which immune cells are exposed chronically and continuously to proinflammatory stimuli. Alternative stimuli for the increased activin A production by placental and extraplacental sources could be a yet-to-be-identified placental factor(s). However, a more likely explanation is the presence of several proinflammatory stimuli, which act concomitantly and perhaps synergistically on the maternal immune cells to provoke the exaggerated immune response to pregnancy seen in preeclamptic women.

In the present study, we were unable to detect inhibin A secretion by either activated PBMCs or endothelial cells, suggesting that the placenta is the only source of the increased circulating inhibin A in normal pregnancy and PE. Similarly, we were unable to detect the activin binding protein, follistatin, in conditioned medium from either resting or activated PBMCs or HUVECs. However, a previous study reported that endothelial cells do express follistatin mRNA at low levels (39). A possible reason for this discrepancy is that the ELISA used in the present study measures primarily the 288 form of follistatin, with 10% cross-reactivity with the 315 form (48). As yet, the forms of follistatin produced by endothelial cells have not been determined. It would appear that follistatin is unlikely to be a major product of the inflammatory response induced by pregnancy because there is no significant increase in circulating follistatin levels in preeclampsia, compared with normal pregnancy (59).

Although there have been many studies of activin A levels in pregnancy and preeclampsia, there has been little attempt to investigate its function. Of key relevance to this issue are studies showing that activin A plays a role in inflammatory responses, although its nature is far from clear because both proinflammatory (60, 61) and antiinflammatory effects (62, 63, 64) have been reported. The observation that activin A inhibits endothelial cell proliferation (39, 40) suggests a possible involvement in the endothelial cell dysfunction apparent in PE. In conclusion, the increased circulating activin A levels observed in PE may result from both placental and systemic sources, including inflammatory leukocytes and activated endothelium. However, the function of activin A in normal pregnancy is still not understood, and the significance of the changes induced by PE requires further elucidation.


   Acknowledgments
 
We thank Mrs. Hazel Colburn and Mrs. Carol Simms for blood sample collection and Dr. Zsuzsa Kertesz for technical advice.


   Footnotes
 
This work was supported by Tommy’s, The Baby Charity, Grant 54.

Abbreviations: CM, Culture medium; FCS, fetal calf serum; HUVEC, human umbilical vein endothelial cell; MP, matched normal pregnant; NP, nonpregnant; PBMC, peripheral blood mononuclear cell; PE, preeclampsia.

Received December 9, 2002.

Accepted August 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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