Eicosanoid secretion by human endothelial cells exposed to normal pregnancy and preeclampsia plasma in vitro

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(2), 91-97 © HarcourtBrace& Co. Ltd 1998

Eicosanoid s e c r e t i o n by h u m a n e n d o t h e l i a l cells e x p o s e d to normal preg. n a n c y and p r e e c l a m p s i a p l a s m a ,n vitro C. J. M. de G¢oot, 1 J. T. Murai, ~ J-L. Vigne, 2 R. N. Taylor 2 ~Department of Obstetrics and Gynecology, University Hospital Leiden, PO Box 9600, 2300 RC Leiden, The Netherlands 2W. M. Keck Laboratory of Reproductive and Developmental Molecular Biology, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, School of Medicine M-1489, San Francisco, CA 94143-0132, USA

Eicosanoids play an important role in the pathogenesis of preeclampsia. The major eicosanoid metabolite reported to be secreted by endothelial cells, the vasodilator prostacyclin, is generally reduced in preeclampsia. By contrast, it was shown previously that prostacyclin secretion by cultured human umbilical vein endothelial (HUVE) cells is increased significantly after exposure to blood from preeclamptic women. In the current study, eicosanoid profiles in conditioned media from HUVE cells incubated with pregnancy plasma were analyzed by high-performance liquid chromatography, thin layer chromatography and quantitative radio- and enzyme immunoassays. More prostaglandin F2,, prostacyclin and 8-isoprostane were secreted after exposure to plasma from preeclamptic women than plasma from matched, normal pregnant patients. Predominant secretion of the vasoconstrictor prostaglandin F2, by HUVE cell cultures and a stimulatory effect of preeclampsia plasma on eicosanoid biosynthesis underscore the importance of bioactive lipids in the vasospasm associated with clinical preeclampsia. Summary

INTRODUCTION

The major pathophysiologic changes of preeclampsia: increased blood pressure, vascular permeability and activation of the coagulation cascade, suggest that endothelial cell dysfunction plays an important role in this disorder. 1,2 Evidence of this phenomenon has been detected as alterations in biochemical markers of endothelial cell function and increased lipid peroxidation in women with preeclampsia. 2 One biochemical marker that derives directly from the vascular endothelial cells is prostacyclin. Prostacyclin (PGI2) is reported to be the principal eicosanoid produced by human endothelial cells and is measured as its stable metabolite 6-keto-prostaglandin FI~.3 PGI2 has biological properties distinct from other eicosanoids, particularly its potent local vasodilatory and platelet anti-aggregatory activities and its marked chemical instability.4 Studies describing the concentrations of

this eicosanoid in plasma, serum, urine, amniotic fluid, cord blood and placental tissue generally indicate that the production of PGI2 is reduced in preeclampsia. 5 Paradoxically, however, cultured endothelial cells exposed to plasma or serum from women with preeclampsia produced significantly more PGI2 than cells exposed to plasma or serum from normal pregnant women. <7 To resolve this apparent discrepancy, we undertook a systematic analysis of eicosanoid production by cultured human umbilical vein endothelial (HUVE) cells after exposure to pregnancy plasma. High-performance liquid chromatography (HPLC) and thin layer chromatography (TLC) were used to establish eicosanoid profiles. Radioand enzyme immunoassays were performed to quantify specific prostanoid metabolites secreted into the culture media of HUVE cells exposed to plasma from normal pregnant women or women with preeclampsia. MATERIALS AND METHODS

Received 24 April 1997 Accepted 12 June 1997 Correspondence to: Robert N. Taylor, Tel. 0O 1 415 476 4556; Fax. 00 1 415 753 3271

Blood specimen collection, clinical assessment and definitions

After giving written informed consent, pregnant women 91

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with singleton gestations were recruited to participate in a protocol approved by the University of California, San Francisco Committee on Human Research. Plasma samples were collected during the late third trimester of pregnancy prior to the onset of labor and frozen at -70°C before use. The subjects were assigned as preeclamptic using criteria promulgated in a recent NIH consensus report on pregnancy research: 8 nulliparity, hypertension, proteinuria, hyperuricemia, and reversal of hypertension and proteinuria after pregnancy. Hypertension was defined as an increase of 30 mmHg systolic or 15 mmHg diastolic compared to values obtained before 20 weeks' gestation, or an absolute blood pressure _>140/90 mmHg (Korotkoff V, seated position). Proteinuria was designated as _ 2+ (100 mg/dl) on a voided or _> 1+ (30 mg/dl) on a catheterized specimen. Hyperuricemia was defined as _>5.5 mg/dl (_>1 standard deviation above the normal mean concentration at term)? Patients were matched for race, and as closely as possible for maternal age (+ 5 years), and duration of pregnancy (+ 4 weeks). Mean arterial blood pressure (MAP) was calculated from the average of blood pressure readings taken before 20 completed weeks' gestation and again at the time of admission to the delivery suite, before intravenous fluid or pharmacological (including anesthetic) therapy, according to the equation MAP = (diastolic + [systolic - diastolic]/3). Human umbilical vein endothelial (HUVE) cell cultures HUVE cells were obtained from umbilical cords of normal neonates as described previously. ~° Briefly, endothelial cells were isolated by collagenase digestion (1 mg/ml, 15 rain) and grown in commercially prepared culture medium (Clonetics, San Diego, CA, USA) at 37°C in 5% CO2. The medium was changed every other day. Primary cultures passaged three to five times were dispersed with 0.05% trypsin, plated in gelatin-coated 48-well dishes at l0 S cells/well and allowed to grow to confluent monolayers. HUVE cells were quiesced in serum-free medium for 24 h. The sernm-free media was replaced with 10% heparinized plasma from women with normal pregnancies or plasma from women with preeclampsia. After 24 h the supernatants were collected and stored in indomethacin (2.8 gM) at -20°C for up to 3 months. Eicosanoids secreted into the supernatants were measured as described below. The addition of 100 U/ml of heparin alone, the concentration used in these experiments, had no effect on eicosanoid production. The maintenance of endothelial cell phenotype was confirmed by immunocytochemical staining of representative cultures for yon Willebrand factor. 11 Total protein and DNA determinations of the HUVE cells revealed no differential effects of 10% normal pregnancy plasma vs 10% preeclampsia plasma or 10% fetal

calf serum containing media under the culture conditions, consistent with our previous results, 12 and thus were not confounding variables in these analyses. Measurements of eicosanoids: high-performance liquid chromatography (HPLC) To evaluate the general profile of eicosanoids secreted in vitro, HUVE cells were grown in media containing 3 nM [3H]arachidonic acid (New England Nuclear, Boston, MA, USA) for 24 h during the pretreatment quiescence period. After 24 h exposure to pregnancy plasma, the lipid fractions of the conditioned media were extracted twice with chloroform/methanol/acetic acid (180:20:1, by vol), dried under nitrogen gas at room temperature and resuspended in ethyl acetate. HPLC was performed using a Waters Model 204 liquid chromatograph with two pumps (a Model 6000A and Model 510) controlled by an automated gradient maker (Model 680) and a Waters Injector (Model U6K). For detection of the eluted compounds, UV absorbance at 254 nm was monitored using a Kratos Spectroflow (Model 15) device and radioactivity was detected using a Radiomatic detector (FLO/ONE-[3, Model A250). A 3.9 x 1 5 0 m m Nova-Pack C18 reverse phase HPLC column from Waters was equilibrated initially in 26% acetonitrile, pH 3.5. The samples were injected into the column and the retained compounds were eluted using a step gradient of 26-80% acetonitrile, pH 3.5.13 The total length of each run was 80 min with a flow rate of 1 ml/min. After each standard or sample the column was purged with 100% acetonitrile to remove any residual material. Under these conditions, the elution time for [3H]6-keto-prostaglandin (PG) FI~ (prostacyclin metabolite) was 10 min and for [3H]arachidonic acid was 70 min. Unlabeled eicosanoid standards, 6-keto-PGFl~, PGI2, PGE2, PGF2~, 8-isoprostane and 5-hydroxyeicosatetraenoic acid (HETE) had elution times of 10, 11, 28, 32, 32 and 64 min, respectively. Overall recovery ranged from 78-82% and did not differ among the standards. Data were expressed as radioactivity in each peak (counts per minute) as a percentage of the total counts per minute recovered during each separation. Measurements of eicosanoids: thin layer chromatography (TLC) Conditioned media samples from cultured HUVE cells were generated and extracted as described above. TLC plates (20 x 20 cm, PE Sil G/UV, Whatman Ltd, UK) were washed to remove contaminating impurities in a solvent system containing ethyl acetate:water:2,2,4 trimethylpentane:acetic acid (5.5:5.0:2.5:1.0, by VO1).14 Aliquots of the lipid extracts (20 ~1) were spotted onto the TLC plates with a mixture of prostaglandin standards as carriers

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(2), 91-97

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Preeclampsia and eicosanoid production in vitro

and internal controls. Separation of the prostaglandins was enhanced by repeating development in the solvents described above. Rf values of 0.36, 0.43 and 0.66 were noted for 6-keto-PGFl~, PGF2~and PGE2, respectively. TLC plates were developed using 10% phosphomolybdic acid in ethanol and dried in a vacuum oven at 80°C for 5 min. Lanes of the TLC plates were cut into pieces, dissolved in ethanol and scintillation cocktail, quantified in a [~ scintillation counter. Overall recovery after extraction was 81%. For each sample, recovered radioactivity was plotted against retention time, to show the distribution of radioactive products formed during incubation. Data are presented as radioactivity in each peak (counts per minute), with the quantity expressed as a percentage of the total counts per minute recovered from that lane. The sum of the percentages tended to be slightly less than 100 because scattered areas of radioactivity did not correspond to known prostaglandins. Measurements of eicosanoids: radioimmunoassays

PGI2 (measured as its stable metabolite, 6-keto-PGFl~), PGF2~ and PGE2 were measured independently in HUVE cell conditioned media by radioimmunoassays (RIAs) employing highly specific antisera (Advanced Magnetics, Cambridge, MA, USA). The cross-reactivities of each of the three RIAs for other eicosanoid metabolites were < 7%. The sensitivities of the assays were 1-2 pg/ml, with inter- and intraassay coefficients of variation < 11%. None of the prostaglandins were detected in unconditioned culture media. Prostaglandin concentrations were expressed as ng prostaglandin released/106 cells/24 h. Measurements of eicosanoids: enzyme immunoassays

Enzyme immunoassay (EIA) kits (Cayman Chemical, Ann Arbor, MI, USA) for PGI2 (6-keto-PGFl~), PGF2~ and 8-iso-

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prostane (8-epi-PGF2~) were used to verify and extend the results of the HPLC, TLC and RIA data. The cross-reactivities of each of the three EIAs for other eicosanoid metabolites were _<2%. The sensitivities of the assays were 10-25 pg/ml, with inter-and intraassay coefficients of variation _<9%. As noted above, prostanoid concentrations were expressed as ng prostaglandin released/10 ~ cells/24 h. Statistical analyses

The data are presented as means + standard error and were analyzed by unpaired, two-tailed Student's btests or Mann-Whitney tests. One-factor analysis of variance (ANOVA) was used to determine the predominant eicosanoid product in chromatographic profiles. Twofactor ANOVA with repeated measures was used to analyze differences among eicosanoid metabolite (PGI2, PGF2~ and 8-isoprostane) concentrations (dependent variables) and between normal m~d preeclamptic patients (independent variables). For all analyses, P = 0.05 was accepted as a threshold for significant differences. RESULTS

Demographic and clinical data from the two study groups are summarized in Table 1. The MAPs, levels of proteinuria, and serum uric acid concentrations at term differed significantly between the groups, as anticipated from the definition criteria. Ethnicity, maternal age, gestational age at delivery, blood pressures at < 20 weeks' gestation and hematocrit values at term did not differ between the two groups (P>_0.10 for all parameters). Analysis of eicosanoids secreted into conditioned media of HI.WE cells exposed to plasma from women with preeclampsia or normal pregnancies, resolved by HPLC, unexpectedly showed a predominant peak co-

Table 1 Demographic and clinical features of normal pregnant and preeclamptic subjects

Maternal age (years) Gestational age (weeks) at delivery Mean arterial pressure < 20 weeks (mmHg) Systolic pressure < 20 weeks (mmHg) Diastolic pressure < 20 weeks (mmHg) Mean arterial pressure at delivery (mmHg) Systolic pressure at delivery (mmHg) Diastolic pressure at delivery (mmHg) Serum uric acid at delivery (mg/dl) Proteinuria at delivery (mg/dl) Hematocdt at delivery (vol/vol %)

Normal subjects (n = 11)

Preeciamptic subjects (n= 11)

29.1 39.8 79.5 107.8 65.3 90.7 119,1 76.4 3.7 < 10 36.6

23.6±2.0 38.3±3.0 77.7±1.8 106.7±2.5 63.2±2.0 109.6±5.6" 151.1±9.2" 88.9±4.0" 7.0±0.3* >30 t 36.4±1.0

+_2.1 ± 0.6 ± 1.5 ± 3.5 _+ 1.7 + 3.6 ± 3.9 ± 3.6 ± 0.2 _+ 1.3

Values are the means +_+_SE. *P< 0.05 vs normal controls by unpaired Student's t-test. * P < 0,05 vs normal controls by Mann-Whitney test.

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elution time (min) Fig. Representative examples of high-performance liquid chromatography profiles of [3H]arachidonic acid metabolites. HUVE cells preincubated with incubated [3H]arachidonic acid were exposed to plasma from normal pregnant women (open symbols, dashed line) and plasma from women with preeclampsia (solid symbols, black line) for 24 h. Data are presented as the radioactivity recovered (cpm/106 HUVE cells) plotted against elution time, in minutes. Arrows at the top represent the elution times of eicosanoid standards (12 = 6-keto-PGG,~, E2 = PGE2, F2,, = PGF2~, HETE = 5-hydroxyeicosatetraenoic acid, AA = arachidonic acid).

migrating with the PGF2~ standard ( P < 0 . 0 5 , ANOVA, Fig.). Production of two major eicosanoids, PGF2~ and PGI~, was significantly greater in HUVE cells exposed to preeclampsia plasma than cells exposed to plasma from controls (P = 0.05, ANOVA with repeated measures, Fig.). The conversion of [~H]arachidonic acid to PGF2~was significantly greater in HUVE cells exposed to plasma from women with preeclampsia compared to plasma from normal pregnant controls (3.6 + 1.2O/ovs 0.4 _+ 0.1% recovered radioactivity/10~ HUVE cells/24h, P<0.05, n = 8). Consistent with our previous results, 6 more PGI2 also was produced by HUVE cells exposed to plasma from women with preeclampsia than plasma from normal pregnant women (0.2 + 0.1% vs 0.1 + 0.1% recovered radioactivity/106 HUVE cells/24 h, n = 8), although, these data did not reach a statistically significant level of difference. HUVE cells also converted [~H]arachidonic acid to small amounts of other eicosanoids including PGE2 and HETE after exposure to plasma from both groups (Fig.). While the overall metabolism of [3H]arachidonic acid

was greater in HUVE cells treated with plasma from women with preeclampsia (40.2 _+ 10.8% vs 21.8 + 5.4% converted radioactivity/106 HUVE cells/24 h, n = 8), this likewise did not reach statistical significance. TLC was used as an alternative technique to analyze the spectrum of eicosanoids produced by these cells after exposure to pregnancy plasma. PGF2~was again found to be the predominant eicosanoid produced in HUVE cells exposed to plasma from both pregnant patient groups. The conversion rates of [3H]arachidonic acid to PGF2~and PGI2 (converted radioactivity/106 HLTVEcells/24 h [n = 3]) were 4.9 + 4.3% and 0.6 + 0.4% in HUVE cells cultured in plasma from preeclamptic women and 0.8 + 0.4% and 0.4 + 0.1% in HUVE cells cultured in plasma from normal pregnant women, respectively. The combined data for the HPLC and TLC analyses are summarized in Table 2. For more precise analyses, RIAs and EIAs were performed to quantify production of the predominant prostanoids. We confirmed that cultured HUVE cells produced relatively more PGF2~than PGI2 after exposure to plasma from both pregnancy groups (P< 0.05, n = 11, ANOVA with repeated measures). HUVE cells exposed to plasma from preeclamptic women produced about fourfold more PGF2~and PGI2 than the same cells exposed to plasma from normal pregnant women (P<0.05, n = 11, ANOVA, Table 3). PGE2 production was at the sensitivity limit of our ILIA, confirming the HPLC findings that Table 2 Percent conversion of [3H]arachidonic acid to prostaglandin F2~,and 6-keto-prostaglandin FI~ by HUVE cells after exposure to plasma from women with normal pregnancies and preeclampsia. These data were obtained from HPLC and TLC determinations

[SH]Arachidonic acid conversion (%/106 HUVE cells/24 h)

HUVE cells exposed to 10% plasma from Normal Preeclamptic subjects subjects (n= 11) (n= 11)

PGF2,~ 6-Keto-PGFI~ PGE2

0.6 + 0.2 0.2 _+0.1 Not detected

4.4 _+2.6 0.4 _+0.3 Not detected

NB Prostaglandin F~ and 8-isoprostane could not be distinguished by these chromatographic methods.

Table 3 Prostaglandin F2~, 6-keto-prostaglandin FI,~ and 8isoprostane concentrations secreted by HUVE cells after exposure to plasma from women with normal pregnancies and preeclampsia. These data were obtained from radio- and enzyme immunoassays

HUVE cells exposed to 10% plasma from Prostanoid secretion Normal Preeclamptic (ng/106 H UVE subjects subjects cells/24 h) PGFa~ 6-Keto-PGFI~ 8-1soprostane

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(2), 91-97

48.0 -+ 12.6 (n = 11) 167.3 + 65.1 (n = 11 ) 9.9 _+2.8 (n= 11) 37.7_+ 11.1 (n= 11) 4.1 + 0.3 (n = 4) 7.2 + 0.7 (n = 4)

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Preoclampsia and eicosanoid production in vitro

this [3H]arachidonic acid metabolite is not secreted in appreciable amounts by HUVE cells cultured under these conditions. The newly described vasoconstrictor eicosanoid metabolite, 8-isoprostane (8-epi-PGF2~), can arise non-enzymatically via lipid peroxidation 15 and is a potential mediator of vasospasm in preeclampsia. We found that 8-isoprostane co-migrates with PGF2= under the HPLC conditions used in this report. Hence, EIAs were performed to determine the contribution of this compound to the major [3H]arachidonic acid metabolite peak observed by HPLC. The absolute concentrations of 8-isoprostane in HUVE cell conditioned media were low, but these followed the same trend as other prostanoids, being significantly increased in cells exposed to preeclampsia plasma (7.2 + 0.7 ng/10 ~HUVE cells/24 h) compared to cells incubated with plasma from normal pregnant women (4.1 + 0.3ng/10 e HUVE cells/24h, P<0.05, n = 4, Student's t-test, Table 2). The high ratios of PGF2~:8-isoprostane (23:1 and 12:1 in HUVE cells exposed to preeclampsia and normal pregnancy plasma, respectively) suggest that 8isoprostane is a relatively minor prostanoid product of these cells. DISCUSSION

Eicosanoids are believed to play an important role in the pathogenesis of preeclampsia and recently have been the target of multicentered aspirin trials to prevent this syndrome. I~
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arteries from women with preeclampsia vs normotensive pregnant controls) s-2s These contrary findings might be explained by differences in the clinical definition of preeclampsia 29 or in methodological techniques to quantify eicosanoid productionY In vivo measurements of plasma prostanoids are plagued by high rates of clearance and low circulating concentrations. Due to abundant platelet production of prostaglandins, acute effects of venipuncture itself can obscure underlying differences between normal and preeclamptic women. 3° A series of structurally unique PGF2-1ike compounds, the isoprostanes, have vasoconstrictor activity. These protanoids can be produced in vivo by a non-cyclooxygenase-dependent mechanism involving free radical catalyzed peroxidation of arachidonic acid. 15 A number of studies has shown these compounds to be accurate markers of lipid peroxidation and oxidative stress. 8Isoprostane can elicit vasoconstrictor effects by two independent mechanisms: activation of a thromboxane Aa-like receptor and stimulation of endothelin-1 secretion. 31 The results of the current study, to our knowledge the first in HUVE cells, indicate that 8-isoprostane is a relatively minor prostanoid product of these cells. Nevertheless, HUVE cells produced significantly more of this isoprostane after exposure to plasma from preeclamptic women than after exposure to plasma from normal pregnant patients. In vitro models have been used to study relative ratios of vasoconstrictor:vasodilator prostaglandin production. Ody et al32 noted important variations in eicosanoid production when endothelial cells were cultured under different conditions. Subconfluent piglet aorta endothelial cells secreted similar amounts of PGF2~ and PGI2 whereas confluent cultures preferentially produced PGI2. Variations in HUVE cell passage number, sources of media and culture conditions all can influence prostanoid secretion in vitroY In the current study we chose pregnancy plasma rather than serum to avoid the confounding effects of cellular products released into serum during blood coagulation? To verify our findings, four independent methods were used to evaluate the eicosanoid production. Metabolic labeling of prostanoids was accomplished by incorporating [aH]arachidonic acid into HUVE cell membrane lipid pools. HPLC and TLC were used to separate newly formed and secreted eicosanoids. Relatively small percentages of [3H]arachidonic acid were converted to prostanoid products, as described previously by Alhenc-Gelas et al. 34 Geling et aP 5 showed predominant production of PGFa~ in cultured HUVE cells by TLC, whereas Marcus et aP s reported that P G I 2 w a s t h e major [3H]arachidonic acid metabolic product. RIAs and EIAs allowed direct quantification of accumulated prostanoids, avoiding effects of exogenous arachidonic acid stimulation on newly formed eicosanoids.

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U n d e r t h e s e conditions, PGF;~ again was f o u n d to b e t h e p r e d o m i n a n t metabolite. Thus, p r e d o m i n a n c e of PGF2~ p r o d u c t i o n does n o t a p p e a r to be an artifact of excess a r a c h i d o n i c acid. Systematic e v a l u a t i o n of e n d o t h e l i a l cell e i c o s a n o i d profiles is i m p o r t a n t b e c a u s e v a s c u l a r t o n e d e p e n d s o n t h e relative ratio of eicosanoids r a t h e r t h a n t h e a b s o l u t e c o n c e n t r a t i o n of a single metabolite. W a l s h a n d colleagues h a v e e m p h a s i z e d t h e i m p o r t a n c e of t h e s e ratios in p l a c e n t a l p r o s t a n o i d biosynthesis. 36 A preferential s e c r e t i o n of PGF2~ vs PGI2 was n o t e d in e n d o t h e l i a l cells e x p o s e d to p r e g n a n c y plasma, particularly w h e n p r e e c l a m p s i a p l a s m a vs n o r m a l p r e g n a n c y p l a s m a was used. These d a t a i m p l y t h a t a n overall vasoconstrictor e n d o t h e l i a l r e s p o n s e is i n d u c e d b y circulating p r e e c l a m p s i a - a s s o c i a t e d factors. Q u a n t i t a t i v e differences in p r o s t a n o i d p r o d u c t i o n m i g h t b e e x p l a i n e d b y p h o s p h o l i p a s e A2 a c t i v a t i o n a n d i n c r e a s e d release of arachidonic acid from e n d o t h e l i a l cell m e m b r a n e p h o s p h o l i p i d s of w o m e n w i t h p r e e c l a m p s i a S In s u m m a r y , we f o u n d t h a t t h e v a s o c o n s t r i c t o r PGF2~ is p r e d o m i n a n t l y s e c r e t e d b y HLTVE cell c u l t u r e s e x p o s e d to p r e g n a n c y plasma. PGF2¢, PGI2 a n d 8-isoprostane p r o d u c tion b y c u l t u r e d HUVE cells e x p o s e d to p l a s m a from p r e e c l a m p t i c w o m e n was significantly g r e a t e r t h a n b y identical cells e x p o s e d to p l a s m a o b t a i n e d f r o m m a t c h e d , n o r m a l p r e g n a n t controls. These o b s e r v a t i o n s u n d e r s c o r e t h e i m p o r t a n c e of b i o a c t i v e lipid m e t a b o l i s m in pree c l a m p s i a a n d s u g g e s t t h a t e n d o t h e l i u m - d e r i v e d PGF2~ is one of several possible m e d i a t o r s of t h e v a s o s p a s m o b s e r v e d clinically in this s y n d r o m e .

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ACKNOWLEDGEMENTS We thank Jean Perry RN, MS and the members of the UCSF, Preeclampsia Clinical Data Core for accumulating patient samples and data. This is manuscript #26 from the UCSF Preclampsia Program Project supported by NIH grants P01-HD24180 and HD30367. CJMDG was supported by Ter Meu]en Fund, Royal Netherlands Academy of Arts and Sciences.

REFERENCES 1. Roberts J. M., Taylor R. N., Musci T. J., Rodgers G. M., Hubel C. ,4., McLaughlin M. K. Preeclampsia: an endothelial cell disorder. AmJObstetGyneco11989; 161: 1200-1204. 2. Friedman S..4., Taylor R. N., Roberts J. M. Pathophysiology of preeclampsia. Clin Perinato11991; 18: 661-682. 3. Spector ,4. ,4. Lipid and lipoprotein effects on endothelial eicosanoid formation. Semfn Thromb Hemost 1988; 14: 196-201. 4. Moncada S., Gryglewski R., Bunting S., Vane J. R. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 1976; 263: 663-665. 5. Friedman S. ,4. Preeclampsia: A review of the role of prostaglandins. Obstet C#necol 1988; 71: 122-137. 6. de Groot C. J. M., Davidge S. T., Friedman S. ,4., McLaughlin M. K., Roberts J. M., Taylor R. N. Plasma from preeclanaptic

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