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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 9 3059-3062
Copyright © 1997 by The Endocrine Society

Original Studies

The Expression and Activity of Prostaglandin H Synthase-2 Is Enhanced in Trophoblast from Women with Preeclampsia1

Roger D. Johnson, Yoel Sadovsky, Carol Graham, Eyal Y. Anteby, Kenneth L. Polakoski, Xiaohua Huang and D. Michael Nelson

Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: D. Michael Nelson, M.D., Ph.D., Department of Obstetrics and Gynecology, 4911 Barnes Hospital Plaza, St. Louis, Missouri 63110-1094. NELSON DM{at}kids.wustl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Preeclampsia is associated with altered biosynthesis of vasoactive prostanoids in placental villi. The two isozymes of prostaglandin H synthase (PGHS) are essential for prostanoid synthesis. We tested the hypothesis that PGHS-2 expression is elevated in trophoblast from preeclamptic women, compared with trophoblast from healthy women. Using immunofluorescent staining, we demonstrated a higher PGHS-2 expression in villi from preeclampsia, compared with normal pregnancy. Cytotrophoblasts cultured from placentas of preeclamptic women expressed higher levels of PGHS-2 compared with cytotrophoblasts from normal placentas. This enhanced expression of PGHS-2 correlated with increased media levels of both thromboxane and prostaglandin E2, two products of PGHS activity. The increased prostanoid production by trophoblast from preeclamptic women was markedly reduced by NS-398, a specific inhibitor of PGHS-2. We conclude that both expression and activity of PGHS-2 are enhanced in trophoblasts from preeclamptic women compared with trophoblast from normal pregnancies. The increased production of prostanoids may contribute to the clinical syndrome of preeclampsia. Our data suggest that a selective inhibitor of PGHS-2 might provide a therapeutic alternative to prophylactic low-dose aspirin in modifying the prostanoid profile in preeclampsia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PREECLAMPSIA is a disease characterized by hypertension, proteinuria, edema, and platelet aggregation (1). Although the cause of preeclampsia is unknown, the disease is associated with vasoconstriction, partly attributed to an elevated thromboxane to prostacyclin ratio (2, 3). Thromboxane A2, an end product of prostaglandin H synthase (PGHS), causes vasoconstriction and platelet aggregation. One source of thromboxane is villous trophoblast (4, 5).

PGHS is a rate-limiting enzyme in prostanoid biosynthesis from arachidonic acid substrate (6). There are two isozymes with PGHS activity, each encoded by a separate gene. Most studies indicate that PGHS-1 is constitutively expressed, whereas PGHS-2 is inducible by a variety of endocrine, paracrine, and inflammatory mediators (6). Both PGHS isozymes are expressed in placental trophoblast (4). Whether or not the altered prostanoid production observed in preeclampsia is associated with changes in the differential expression or activity of PGHS isozymes in trophoblast is unclear. We tested the hypothesis that PGHS-2 expression in trophoblast from women with preeclampsia is enhanced, when compared with trophoblast from healthy women. We correlated trophoblast expression of PGHS-2 with prostanoid production, in the presence or absence of a selective PGHS-2 inhibitor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell isolation and culture

This study was approved by the Human Studies Committee of Washington University. Placentas were obtained immediately after a singleton delivery from either women with uncomplicated pregnancies or from women with preeclampsia. Villous cytotrophoblasts were isolated by the trypsin-DNase, Percoll gradient centrifugation method described by Kliman et al. (7) and characterized under our culture conditions (4, 8, 9). The use of freshly isolated vs. frozen cells for primary culture did not influence prostanoid production (unpublished data). Women with preeclampsia met the following criteria: nulliparity, no known or clinically evident antecedent renal or cardiovascular disease, blood pressure > 140/90 on two or more occasions separated by 6 h, proteinuria of > 300 mg/24 h or >= 2+ on dipstick of catheterized urine, hyperuricemia of > 5.5 mg/dl, and normal blood pressures without medications at the postpartum examination.

Triplicate trophoblast cultures for each paradigm were plated at 2.5 x 105 cells/cm2 in the presence of 10 µM NS-398 (Biomol, Plymouth Meeting, PA) or vehicle on 24-well plates. Cells were cultured in serum-free medium 199 (tissue culture facility, Washington University) containing 20 mM HEPES (Sigma, St. Louis, MO), and 2 mM L-glutamine (Sigma) in a 5% CO2 atmosphere, at 37 C. Cell viability, determined by trypan blue exclusion, exceeded 95%. Culture media were stored at -20 C until assayed for thromboxane B2 and prostaglandin E2.

Immunohistochemistry

Villous tissues from four healthy and five preeclamptic women were fixed for 2 h in 10% phosphate-buffered formalin and processed for paraffin embedding. Histochemical detection of PGHS-2 expression on five micron sections of tissue was done in duplicate as previously described (4), using a 1:100 (vol/vol) dilution of a polyclonal antibody (Cayman, Ann Arbor, MI) that recognized the C-terminal region of PGHS-2. Immunostaining intensity was visually scored as 0 (least fluorescent) to 6+ (most fluorescent) by two independent observers who were blinded to the clinical history. A preliminary evaluation of stained specimens established the reproducibility of the fluorescence score, with an interobserver and intraobserver variation <=1.

Northern and Western blot

Primary trophoblast were plated at 2 x 106 cells/cm2 on 10 cm2 dishes and harvested 24 h later. Poly-A messenger RNA (mRNA) was isolated by the guanidinium isothiocyanate/oligo (deoxythymidine)-cellulose chromatography (Pharmacia Biotech, Piscataway, NJ). The mRNA (3 µg/lane) was separated in denaturing 1% agarose gel containing 1.5% formaldehyde, then transferred onto a nylon membrane (Duralon-UV, Stratagene, La Jolla, CA). PGHS-2 and cyclophilin DNA probes were labeled with {alpha}32P by a random primer method, using an oligolabeling kit (Pharmacia Biotech). The labeled probes (1 x 106 cpm/ml) were hybridized to the blots in 50% formamide at 42 C overnight. Washing was performed twice at 37 C for 30 min in SSC (0.15 M sodium chloride and 0.075 M sodium citrate), which contained 0.1% SDS, and twice at 37 C for 30 min in 0.1 x SSC with 0.1% SDS, and exposed to Kodak XAR film (Eastman Kodak, Rochester, NY) for 24–96 h at -75 C. The mRNA loading was verified by cyclophilin, detected on the same blot.

For Western blot, cells were harvested by scraping in lysis buffer that contained 1% SDS, 10 mM dithiothreitol, 10 mM ethyleneglycol, bis-tetra-acetic acid (Sigma), 0.5 mM leupeptin, 1.0 mM pepstatin A, 10 mM phenylmethylsulfonyl fluoride (Sigma), 1 mM tosylphenyl-alanine chloromethyl ketone (Sigma) in 20 mM Tris, pH 8.2. Samples were sonicated (Kontes sonicator, Vineland, NJ) and boiled for 2 min. Protein samples (40 µg/lane) were electrophoresed on 10% SDS-polyacrylamide gels, then transferred to an Immoblon-P membrane (Millipore, Bedford, MA). The gels were stained with Coomassie brilliant blue R-250 (Sigma) to ensure equal loading. The membranes were exposed to anti-PGHS-2 goat polyclonal antibody (Santa Cruz, CA) at 1:100 for 1 h at room temperature, followed by a secondary antigoat antibody (Santa Cruz) for 1 h, then processed for chemiluminescence (Amersham, Arlington Heights, IL). Membranes were exposed to film (Kodak X-OMAT, Sigma) for 10 min.

RIA

Concentrations of prostaglandin E2 and thromboxane in media were determined for each placenta in triplicate cultures by specific RIA assays, following the manufacturer’s instructions (Perceptive Diagnostics, Cambridge, MA). Thromboxane A2 was estimated by measuring its stable metabolite, thromboxane B2. Levels were expressed as pg eicosanoid/mg cellular protein. The interassay and intraassay variation for prostaglandin E2 was 7.2% and 12.4%, and for thromboxane B2 5.8% and 11.2%, respectively.

Statistics

Statistical analyses for differences in immunohistochemical fluorescence were performed by the Mann-Whitney rank sum test. Differences of band density and differences of media prostanoid level were analyzed by Student’s t test, with a P <= 0.05 determined as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
An elevated level of total PGHS in many pathological conditions results from a higher expression of the inducible PGHS-2 isozyme (6). We initially used a semiquantitative, blinded analysis of the immunohistochemical staining for placental villous PGHS-2 to test the hypothesis that there is higher expression of PGHS-2 in villous trophoblast from preeclamptic women, compared with healthy women. We found that trophoblast from preeclamptic women (n = 5) exhibited more fluorescence when compared with the fluorescence of villi from healthy (n = 4) controls (P < 0.05; Fig. 1Go), indicating that the expression of PGHS-2 protein is elevated in trophoblast from preeclamptic women.



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  		Figure 1. Photomicrographs of intact villus, stained immunocytochemically for PGHS-2. A, An example of a typical villus from a normal placenta. A fluorescence score of 2 was assigned by two evaluators blinded to the source. B, An example of a villus from a preeclamptic pregnancy, that was assigned a score of 5. There was no staining in control sections when a nonspecific sera was used. C, Histogram of fluorescence scores. The fluorescence was quantified as described in Materials and Methods. Bar, 30 µm.

 
We next determined whether or not trophoblast isolated from villi showed enhanced PGHS-2 mRNA expression, compared with cultured trophoblast from uncomplicated pregnancies. We found a higher expression of PGHS-2 in three of five trophoblast cultures that were derived from placentas of preeclamptic women, compared with trophoblast (n = 4) from normal controls (Fig. 2Go). This trend, however, was not statistically significant (P = 0.1).



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  		Figure 2. Northern analysis of PGHS-2 mRNA in cultured human trophoblasts from three representative placentas from healthy women (lanes 1–3) and three representative trophoblast cultures from women with preeclampsia (lanes 4–6). Human tracheal endothelial cells (hTEC) provided a positive control for PGHS-2 mRNA, and COS-7 cells provided a negative control. Each lane was loaded with 3 µg poly A-selected mRNA.

 
Next, we used Western blotting with an antibody specific for PGHS-2 to confirm that the trend in mRNA level detected in trophoblast from preeclamptic pregnancies was translated to a higher level of PGHS-2 protein as shown in Fig. 3Go. Trophoblast from women with preeclampsia expressed more PGHS-2 protein compared with trophoblast from uncomplicated pregnancies (n = 3 for each group, P < 0.05). Two bands detected with the PGHS-2 specific antibody represent different glycosylation forms of PGHS-2 as previously described (6).



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  		Figure 3. Western blot analysis of PGHS-2 protein in cultured human trophoblasts from two representative healthy women and two representative women with preeclampsia. Each lane was loaded with 40 µg protein. Human fetal fibroblasts (hFF) were used as a positive control, and COS-7 cells served as a negative control.

 
The PGHS isozymes undergo suicide inactivation (6). Therefore, a higher protein expression may not correlate with a higher production of prostanoids. To determine if the increased PGHS-2 expression observed in placentas from preeclamptic women lead to enhanced production of prostanoids, we measured the media level of thromboxane B2 and prostaglandin E2, both prostanoids produced by trophoblast through the enzymatic action of PGHS. We found that trophoblast from placentas of women with preeclampsia produced significantly more (P < 0.02) thromboxane B2 and prostaglandin E2 than cells cultured from normal placentas (Fig. 4Go). These results confirm that the increased expression of PGHS in trophoblast leads to enhanced prostanoid production.



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  		Figure 4. Prostanoid production by human trophoblast from preeclamptic and normal pregnancies. Cells were cultured 24 h in serum-free media. Prostanoid levels were normalized to protein. Results are mean ± SD (n = 4 for each paradigm). P < 0.02 for prostaglandin E2 (PGE2) and for thromboxane B2 (TxB2), normal vs. preeclampsia.

 
The release of prostanoids by cultured trophoblast results from the combined enzymatic activity of PGHS-1 and PGHS-2 (6). To determine if the higher level of prostanoid in the culture media of trophoblast from preeclamptic pregnancy reflected the enzymatic action of PGHS-2, we cultured cells in the presence or absence of the selective PGHS-2 inhibitor, NS-398 (10). As shown in Fig. 5Go, NS-398 lowered the level of prostaglandin E2 by 2.1-fold and thromboxane B2 by 5.6-fold in media from normal trophoblasts. NS-398 caused a more pronounced diminution in the level of either prostaglandin E2 (6-fold) or thromboxane B2 (64-fold) from trophoblasts of preeclamptic women. The difference in the magnitude of PGHS-2 inhibition between normal and preeclamptic pregnancies was significant (P < 0.05), supporting the notion that there is a higher activity of PGHS-2 in trophoblasts from preeclamptic women, compared with healthy controls. As listed in the legends, the different culture conditions used in the inhibitor studies are responsible for the lower media prostanoid levels for cells illustrated in Fig. 5Go, compared with Fig. 4Go.



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  		Figure 5. Media levels of prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) from trophoblast of healthy women, or women with preeclampsia, cultured in the absence or presence of NS-398 (10 µM). Cells were allowed to attach 4 h in serum containing media, which was then replaced with serum free M199, containing NS-398 or vehicle. Cells were harvested for protein and media assayed for prostanoids at 24 h. Results are mean ± SD (n = 4 for each paradigm). The differences between cells exposed to vehicle vs. cells exposed to NS-398 was significant (P < 0.01) for each of the paradigms. The difference in the magnitude of PGHS-2 inhibition between healthy and preeclamptic women was significant (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data show that there is a higher expression of PGHS-2 in trophoblast from villi of preeclamptic women, compared with trophoblast in villi from normal pregnancy. Cytotrophoblasts from villi of preeclamptic pregnancies release abnormally high levels of two PGHS end products, thromboxane and prostaglandin E2. These findings suggest that PGHS-2, rather than PGHS-1, accounts for excess PGHS activity in preeclampsia, which may adversely affect feto-placental function. Selective inhibition of PGHS-2 by the isozyme specific inhibitor NS-398 limits the bulk of prostanoid release by trophoblast from preeclamptic pregnancy, supporting this conclusion.

Prostanoids play key roles in the human placenta, modifying vessel responsiveness to vasoactive agents such as endothelin-1 and angiotensin II (11). Thromboxane is a potent vasoconstrictor that alters placental blood flow, and excess thromboxane could thereby predispose to the fetal growth disturbances that often accompany preeclampsia (1, 3). Walsh (2) identified an imbalance of excess thromboxane and deficient prostacyclin production by villous tissues in preeclampsia. Trophoblast is one compartment that produces thromboxane (5, 12), and our study shows that the excess thromboxane from trophoblast in preeclampsia is secondary to PGHS-2 activity. Woodworth et al. (13) showed a higher expression of thromboxane synthase in villous tissues from preeclamptic women compared with normal pregnancies, which may explain the excess thromboxane produced by trophoblast in preeclampsia (2, 5). This possibility is not excluded by our study. However, higher PGHS expression clearly contributes to the abnormally high levels of prostanoids released because a second end-product of PGHS, prostaglandin E2, is not a product of thromboxane synthase and is also released by trophoblast at a higher level in preeclamptic, compared with normal pregnancy.

Aspirin is used for prophylaxis in some women at risk for preeclampsia (14). Its beneficial effect may be explained by irreversible inhibition of activity of both PGHS isozymes, resulting in overall decreased prostanoid production and a purported change in the ratio of vasodilating to vasoconstricting prostanoids (14). However, the success of aspirin in reducing the risk for preeclampsia has been recently questioned (15, 16). The higher trophoblast expression of PGHS-2 associated with higher thromboxane production in preeclampsia suggests that a selective inhibition of PGHS-2, may be an alternative way to inhibit thromboxane formation and beneficially influence the prostanoid balance in pregnancy.


   Acknowledgments
 
The authors thank Ms. Veronica Mulherin for her assistance in the preparation of this manuscript.


   Footnotes
 
1 This work was supported by a grant from the National Institutes of Health to D. Michael Nelson (R01-HD-29190). Back

Received February 25, 1997.

Revised June 10, 1997.

Accepted June 14, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sibai BM. 1996 Treatment of hypertension in pregnant women. N Engl J Med. 335:257–265.[Free Full Text]
  2. Walsh SW. 1985 Preeclampsia: an imbalance in placental prostacyclin and thromboxane production. Am J Obstet Gynecol. 152:335–340.[Medline]
  3. American College of Obstetrics, and Gynecology Technical Bulletin. 1996 Hypertension in pregnancy. 219 (January).
  4. Johnson RD, Walsh SW, Everson WV, Nelson DM. 1995 Differentiation and growth on a fibrin matrix modulate the cyclooxygenase expression and thromboxane production by cultured human placental trophoblasts. Prostaglandins Leukot Essent Fatty Acids. 52:21–27.[CrossRef][Medline]
  5. Ding ZQ, Rowe J, Sinosich MJ, Saunders DM, Gallery EDM. 1996 In-vitro secretion of prostanoids by placental villous cytotrophoblasts in pre-eclampsia. Placenta. 17:407–411.[CrossRef][Medline]
  6. Smith WL, DeWitt DL. 1996 Prostaglandin endoperoxide H synthases 1 and 2. Advance Immunol. 62:167–215.
  7. Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss III, JF. 1996 Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118:1567–1582.
  8. Church SL, Farmer DR, Nelson DM. 1992 Induction manganese superoxide dismutase in cultured human trophoblast during in vitro differentiation. Dev Biol. 149:177–184.[CrossRef][Medline]
  9. Farmer DR, Nelson DM. 1992 A fibrin matrix modulates the proliferation, hormone secretion and morphologic differentiation of cultured human placental trophoblast. Placenta. 13:163–177.[CrossRef][Medline]
  10. Endo T, Ogushi F, Sone S. 1996 LPS-dependent cyclooxygenase-2 induction in human monocytes is down-regulated by IL-13, but not by IFN-{gamma}. J Immunol. 156:2240–2246.[Abstract]
  11. Howarth SR, Vallance P, Wilson CA. 1995 Role of thromboxane A2 in the vasoconstrictor response to endothelin-1, angiotensin II and 5-hydroxytryptamine in human placental vessels. Placenta. 16:679–689.[CrossRef][Medline]
  12. Nelson DM, Walsh SW. 1989 Thromboxane and prostacyclin production by different compartments of the human placental villus. J Clin Endocrinol Metab. 68:676–683.[Abstract/Free Full Text]
  13. Woodworth SH, Li X, Lei ZM, et al. 1994 Eicosanoid biosynthetic enzymes in placental and decidual tissues from preeclamptic pregnancies: Increased expression of thromboxane-A2 synthase gene. J Clin Endocrinol Metab. 78:1225–1231.[Abstract]
  14. Hauth JC, Cunningham FG. 1995 Low-dose aspirin during pregnancy. In: Cunningham FG, MacDonald PC, Gant NS, Leveno KJ, Gilstrap LC, eds. Williams Obstetrics Supplement 14, August/September. Ortho Pharmaceutical Corporation, Raritan, New Jersey.
  15. Bower SJ, Harrington KF, Schuchter K, McGirr C, Campbell S. 1996 Prediction of pre-eclampsia by abnormal uterine Doppler ultrasound and modification by aspirin. Brit J Obstet Gynaecol. 103:625–629.[Medline]
  16. Caritis SN. 1997 Low dose aspirin does not prevent preeclampsia in high risk women. Am J Obstet Gynecol. 176:53 (Abstract).



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