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Endothelial cell proliferation is suppressed by plasma but not serum from women with preeclampsia
Endothelial cell proliferation is suppressed by plasma but not serum from women with preeclampsia Alexander I~ Smfirason, DPhil, Ian L. Sargent, PhD, and Christopher W.G. Redman, FRCP Oxford, United Kingdom OBJECTIVES" Evidence has been sought for a circulating factor derived from the placenta that suppresses endothelial cell proliferation and hence contributes to the maternal endothelial cell disturbances of preeclampsia. STUDY DESIGN- The effects of sera and Plasmas from women with proteinuric preeclampsia and from matched normal pregnant control women on endothelial cell proliferation were compared. The recovery of endothelial cell inhibitory activity from syncytiotrophoblast microvesicles added to male blood and prepared as plasma or serum was determined to investigate the possible placental origin of the inhibitory factor. RESULTS: Sera from women with preeclampsia did not inhibit endothelial cell proliferation. In contrast, plasma from preeclamptic women significantly suppressed endothelial cell growth at 20% dilution compared with controls, and suppression was more pronounced in severe preeclampsia. The inhibitory activity of syncytiotrophoblast microvesicles added to blood could not be recovered from serum, only from plasma, which may explain why there was no suppression with sera from preeclamptic women. CONCLUSIONS: These results confirm that there is a blood-borne endothelial cell suppressive factor in preeclampsia that may be derived from the placenta. (AM J OSSTE-rGYNECOL1996;174:787-93.)
Key words: Preeclampsia, endothelial cells, serum, plasma, syncytiotrophoblast microvillous membranes
Preeclampsia is a multisystem disease of p r e g n a n c y of u n k n o w n cause. T h e m a t e r n a l syndrome is characterized by increased b l o o d pressure, edema, proteinuria, and a b n o r m a l clotting, liver, and renal functions, all of which may result f r o m generalized vascular endothelial cell dysf u n c t i o n ) ' 2 T h e cause of this is unknown, but there is evidence for a circulating endothelial cell "toxic': factor in preeclampsia. Sera from preeclamptic w o m e n are rep o r t e d to be cytotoxic to endothelial cells, a' 4 and e n d o thelial cells c u l t u r e d in preeclampsia sera show an increased release of cellular fibronectin, ~ an increased triglyceride content, and a decreased prostacyclin release. 6 Preeclampsia is a placental disease. 7 So the " t o x i c " factor is most likely to originate f r o m the syncytiotrophoblast, which is a m u h i n u c l e a t e d syncytium with an extensive microvillous brush b o r d e r in contact with the m a t e r n a l blood. We have shown that the syncytiotrophoblast microvillous m e m b r a n e s o f n o r m a l placentas contain a factor(s)
From the Harris Birthright Pre-eclampsia Research Unit, Nuffield Department of Obstetrics and Gynaecology, University of Oxford. Supported by a grant from Birthrigtat. Received for publication August 30, 1994; revisedJanuary 8, 1995; acceptedJuly 11, 1995. Reprint requests:Alexander K- Smdrason, DPhil, Nuffield Department of Obstetrics and Gynaecolog);John Radcliffe Hospital, Oxford, United Kingdom, OX3 9DU. Copyright © 1996 by Mosby-Year Book, Inc. 0002-9378/96 $5.00+ 0 6/1/67723
that specifically inhibits endothelial cell proliferation and disrupts the continuity of established endothelial cell monolayers without causing cell death, s T h e same syncytiotrophoblast microvillous m e m b r a n e s preparations do not disturb cultures of o t h e r cell types ( a d h e r e n t or nonadherent). However, there is no difference in the activity of equivalent amounts of syncytiotrophoblas t microvillous m e m b r a n e s p r e p a r e d f r o m n o r m a l or preeclamptic placentas, s We have therefore c o n c l u d e d that if this factor is relevant to the maternal endothelial dysfunction of preeclampsia it is because it is shed into the maternal circulation in a b n o r m a l amounts. This is supported by our r e c e n t work 9' 1o c o n f i r m i n g previous reports n that syncytiotrophoblast are present in increased amounts in the uterine vein b l o o d of w o m e n with preeclampsia compared with similar samples f r o m n o r m a l p r e g n a n t women. It is n o t yet certain whether trophoblasts routinely e n t e r the peripheral circulation, ° probably most are trapped in the capillaries of the lungs. 1~ We propose that this deportation of whole trophoblast is simply the most visible aspect of a m o r e extensive process that includes the s h e d d i n g of microvillous fragments (containing the endothelial cell inhibitory- activity) that, unlike the intact trophoblast cells, would be carried t h r o u g h o u t the maternal circulation. This hypothesis is consistent with the observation that there are fewer microvilli on the surface of the placenta in preeclampsia and that they are structurally abnormal. ~s 787
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February 1996 AmJ ObstetGynecol
Sm&rason, Sargent, and Redman
Table I. Subjects in serum and plasma studies Serum study Preeclampsia (n = 2O)
Age (yr)* Nulliparoust Blood pressure in early pregnancy (ram Hg)* SBP DBP Maximum blood pressure (ram Hg) * SBP DBP Proteinuria (21+) at samplingt Proteinuria (>_1+) before delivery~Gestadonal age at sampling (days) * Gestational age at delivery (days) * Birth weight (gm)* Twinst
I
Plasma study
Pregnant controls (n = 20)
Preeclampsia (n = 20)
Pregnant controls (n = 20)
23.5 (17-32) 18 (90)
23.4 (18-31) 18 (90)
27.2 (18-38) 20 (100)
27.0 (18-36) 20 (100)
114 (80-150) 66 (40-88)
116 (95-130) 66 (45-80)
124 (110-150) 77 (70-85)
111 (80-130) 66 (30-80)
170 (140-200) 115 (100-130) 20 (100)
128 (110-150) 79 (70-95) 0 (0)
174 (145-210) 114 (90-140) 20 (100)
126 (110-145) 79 (66-100) 0 (0)
20 (100)
0 (0)
20 (100)
1 (5)
213 (165-254)
215 (173-259)
228 (201-261)
229 (206-264)
221 (168-254)
277 (243-291)
232 (203-261)
280 (252-293)
1342 (248-2845) 2 (10)
3248 (2312-4240) 2 (10)
1736 (797-3189) 0
3487 (2531-4476) 0
• SBP, Systolicblood pressure; DBP, diastolic blood pressure. *Mean and range. tNumber and percent.
To investigate this question further, we sought evidence for an endothelial cell inhibitory activity, initially in preeclampsia serum, and, having failed to detect it, subsequently in plasma. Experiments to determine the reason for the discrepancy between the activity of serum and plasma are presented.
Methods Subjects. The study groups were women with preeclampsia, matched control women with normal pregnancies, and n o n p r e g n a n t women. Women with preeclampsia had new hypertension (diastolic blood pressure consistently >_90 m m Hg with previously lower readings) and new proteinuria (more than one urine sample showing + on dipstick testing in the absence of urinary infection) that subsequently regressed after delivery. For each woman with preeclampsia a healthy pregn a n t control was found who was matched for age (_+4 years), parity (0, 1 to 3, >_4), and gestational age (+13 days) and not receiving any medication. Nonpregnant control women were hospital personnel of childbearing age (mean age 30.5 years, SD 5.7) but who otherwise cannot be regarded as matched controls. Further details of the subjects are shown in Table I. This study was approved by the Central Oxford Research Ethics Committee. Serum was prepared from peripheral vein blood that was centrifuged at 2500g for 10 minutes after clotting and kept frozen at -40 ° C. For the plasma study peripheral vein blood was collected into sterile plastic tubes containing sodium heparin (40 u n i t s / m l blood) and cen-
trifuged at 2500g for 25 minutes. The plasma was collected and frozen at -40 ° C. Previous experiments showed that concentrations of heparin between 10 and 320 u n i t s / m l blood made little difference to endothelial cell tritiated thymidine incorporation when grown in 20% plasma (data not shown). Isolation of human endothelial cells. H u m a n umbilical vein endothelial cells were isolated according to t h e method ofJaffe et al ~4 by use of 0.25 m g / m l collagenase type 1A (Sigma, St. Louis). The cells were cultured in RPMI 1640 medium containing 25 m m o l / L HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Gibco, Grand Island, N.Y.), 100 u n i t s / m l benzylpenicillin (Glaxo, Greenford, United Kingdom), 100 p g / m l streptomycin (Sigma), and 20% heat-inactivated fetal calf serum (Imperial, Andover, United Kingdom) in gelatin (Sigma)-coated tissue culture flasks (Nunc, Roskilde, Denmark), as previously described. 8 After 4 to 6 days in primary culture the cells were detached with trypsin-ethylenediaminetetraacetic acid (trypsin 1:250, 0.5 gm/L; ethylenediaminetetraacetic acid, 0.2 gin/L) (Sigma) and used in the assays described in the following text. Endothelial cell proliferation assay. To each gelatincoated well of a 96-well flat-bottomed microtiter plate (Nunc), 15 x 10~ endothelial cells were added in 200 pl of culture medium containing the specified concentration of test serum or plasma, which had been rapidly thawed at 37 ° C. After a 24-hour incubation at 37 ° C in 5% carbon dioxide in air, the cells were pulsed with 1 p,Ci per well (1 mCi = 37 MBq) of (methyl-SH)-thymidine (25
Volume 174, Number 2 Am J Obstet C.ynecol
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Fig. 1. Effect of peripheral vein serum from women with preeclampsia and matched pregnant controls on endothelial cell proliferation (tritiated thymidine incorporation). Mean and SD (n = 20 pairs).
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Fig. 2. Fate of syncytiotrophoblast microvillous membranes (STBM) added into blood. Syncytiotrophoblast microvillous membranes were added to fresh blood at 2.3 or 93 mg wet weight per milliliter blood. When 20% serum or plasma is tested, this corresponds to syncytiotrophoblast microvillous membranes concentrations of 170 and 1700 pg wet weight per well, respectively, if it is assumed that hematocrit is 45% and that syncytiotrophoblast microvillous membranes are retained in cell-fi-ee fraction after centrifugation. Suppression of endotheliaI cell proliferation (tritiated thymidine incorporation) in plasma (hatched bars) and serum (open bars) can be compared with standard curve for this syncytiotrophoblast microvillous membranes preparation (solid line) run at same time. Results are expressed as percentage of proliferation obtained in cultures containing serum or plasma in absence of seeded syncytiotrophoblast microviltous membranes. Median of five replicates.
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790
Sm&rason, Sargent, and Redman
February 1996 Am J Obstet Gynecol
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Statistical m e t h o d s . To minimize the effects of extreme outlying readings, the median rather than mean counts per minute of the five replicates was used when the thymidine incorporation index was calculated. For the same reason the n u m b e r of replicates was five rather than the more conventional three. Results for women with preeclampsia, matched pregnant controls, and n o n p r e g n a n t controls were compared with one-way analysis of variance followed by post hoc multiple comparisons with Bonferroni adjustment with the SPSS for Windows (SPSS, Chicago) statistical computer program. The Wilcoxon ranksum test was used to compare the results for women with low and normal platelets.
Results cesarean section at 39 weeks' gestation because of a previous cesarean section. Syncytiotrophoblast microvillous membranes were prepared as previously describedfl and the membranes were resuspended in sterile endotoxin7 free phosphate-buffered saline solution at two dilutions (25 and 250 mg wet weight/ml). One milliliter of each of these dilutions or of phosphate-buffered saline solution alone was mixed in plastic tubes with 10 ml aliquots of freshly drawn male blood, without (to give serum) or with 400 units of heparin sodium (to give plasma). Plasma was prepared immediately and serum, by centrifugation at 2500 gm for 25 minutes, after clotting. The samples were frozen at -20 ° C until required. The sera and plasma were tested in the endothelial cell proliferation assay at a 20% dilution. Known amounts of syncytiotrophoblast microvillous membranes (from the same placenta) were tested on the same batch of endothelial cells in 20% AB serum to produce a standard curve.
At all three serum concentrations tested (2%, 10%, 20%) there were no differences in the incorporation of tritiated thymidine by endothelial cells grown in sera from preeclamptic women compared with those from normal pregnant women (Fig. 1). Because these results were clearly contrary to the hypothesis, we tested whether the previously discovered endothelial cell suppressive activity of syncytiotrophoblast microvillous membranes could be recovered from serum or heparin plasma after syncytiotrophoblast microvillous membranes were added to unclotted blood. When tested in the endothelial cell proliferation assay, most of the expected syncytiotrophoblast microvillous membranes suppressive activity was recovered from the plasma (Fig. 2). The lower concentration (170 lag wet weight/well) caused 70% suppression (30% proliferation) when 86% was expected by comparison with the standard curve for syncytiotrophoblast microvillous membranes prepared
Sm&rason, Sargent, and Redman 791
Volume 174, Number 2 .~n J Obstet GynecoI
Table II. Preeclampsia cases in plasma study analyzed according to platelet count Platelet counts at sampling
Maturity at sampling (days) Maturity at delivery (days) Birth weight of infant (gm) Maximum blood pressure (mm Hg) SBP DBP Plasma aspartate aminotransferase (IU/L) Human umbilical vein endothelial cell proliferation in 2% plasma (%) Human umbilical vein endothelial cell proliferation in 20% plasma (%)
'Wormal"platelets (>150x 109/L) (n = 15)
Low platelets (<150× 109/L) (n = 5)
229 (201-261) 240 (203-261) 1632 (797-3189)
217 (210-225) 217 (210-225) 1443 (1055-1460)
170 115 27 104
(145-210) (95-140) (16-84) (89-133)
81 (55-113)
170 t10 579 126
(160-t80) (90-120) (29-1464)* (111-134)?
49 (39-95)f
Values are median and range. SBP, Systolicblood pressure; DBP, diastolic blood pressure. *p < 0.01, Wilcoxon rank-sum test. tP < 0.05, Wilcoxon rank-sum test.
from the same placenta. Some of the activity may have been lost in aggregates being pelleted on centrifugation. In contrast,' almost no suppressive activity was recovered from the serum. As a result of this experiment, it was clear that heparin plasma, not serum, should be tested if evidence for shedding of the syncytiotrophoblast microvillous membranes suppressive factor into the circulation in preeclampsia was to be sought. Hence a further set of clinical samples was collected from women with preeclampsia, matched pregnant controls, and, in addition, from n o n p r e g n a n t women, and the plasma was prepared as described above. There was no effect of preeclampsia plasma at 2% and 10%, but at a 20% dilution it significantly (p < 0.05) inhibited endothelial cell proliferation compared with both the normal pregnant and n o n p r e g n a n t controls (Fig. 3). At this dilution endothelial cell proliferation was also significantly reduced (p< 0.05) in normal pregnant plasma compared with n o n p r e g n a n t control. No morphologic changes were detected in the endothelial cell monolayers before harvesting at any of the plasma concentrations tested. The effects of sera or plasma from preeclamptic or normal pregnant women were expressed as proliferation index values relative to standard pools of sera or plasmas from n o n p r e g n a n t donors. The absolute values of thymidine incorporation depended on the concentration of serum or plasma tested (Fig. 4). There was a wide range of suppression caused by the different samples of plasma from preeclamptic women when tested at 20% (Fig. 5). All the cases were severe by conventional definitions by being proteinuric, and most of them had early-onset preeclampsia (Table I). However, we wondered whether there was a correlation with the severity of the disease. Because a low platelet count reflects more severe disease, the preeclampsia group was subdivided into those with lower and higher platelet
counts at the time of sampling. This necessitated choosing an arbitrary threshold of 150 x 109/L, which was chosen without knowledge of the experimental results. This yielded five cases below this limit, deemed to be more severe, and 15 above, classified as less severe. Preeclamptic women with lower platelets had significantly higher plasma aspartate aminotransferase activities, lower endothelial cell proliferation in 20% plasma, and higher proliferation in 2% plasma (Table II). There was no significant correlation between the proliferation index at 20% plasma and length of gestation or birth weight (data not shown).
Comment The starting point of this study came from the observatmns of others that sera from preeclamptic women damaged cultured h u m a n endothelial cells, as measured by increased release of chromium 51. 3' 4We assessed a different aspect of endothelial cell function--spontaneous proliferation--and were unable to show any effect of preeclampsia sera. We argued that if the sera were indeed toxic to endothelial cells then proliferation should be inhibited as well. That it was not suggests that in this context ~Cr release reflects alterations in cell membrane permeability but not cell death or damage sufficient to affect proliferation. Plasma from women with preeclampsia significantly inhibited endothelial cell growth in vitro compared with plasma from normal pregnant and n o n p r e g n a n t women. This is consistent with the hypothesis that more trophoblast-associated endothelial cell inhibitory activity is shed from the placenta into the maternal circulation in preeclampsia. Titration experiments showed that this inhibition was not merely a toxic effect of plasma (or the heparin anticoagulant) because high concentrations of nonpregnant plasma had no such effect. It is also unlikely to be the result of a lack of growth factors in preeclampsia
792 Sm&rason, Sargent, and Redman
because endothelial proliferation was not lower in 2% preeclampsia plasma compared with the other groups. No comparable inhibition was seen with preeclampsia sera, suggesting that the endothelial cell inhibitory activity is lost during the clotting process. In seeking an explanation f o r this difference, syncytiotrophoblast microvillous membrane preparation with known endothelial cell inhibitory activity was seeded into blood where it was found that the activity was recovered only from plasma, not from serum. The active factor in the syncytiotropboblast microvillous membranes may be destroyed by the clotting enzymes, or the syncytiotrophoblast microvillous membranes may simply become enmeshed in the clot. Loss of the syncytiotrophoblast microvillous m e m b r a n e s associated inhibitory activity in serum cannot be due to serum-associated blocking factors because the standard curve shown was carried out in serum. Thus, if the activity in the blood o f preeclamptic women is derived from syncytiotrophoblast microvillous fragments, this would account for the difference between the effects of serum and plasma in vitro. Compared with nonpregnant women, 20% plasma from pregnant normal control women also suppressed endothelial cell proliferation, although not to the same extent as preeclampsia plasma. This is compatible with the concept of a low-level release o f syncytiotrophoblast microvillous fragments in normal pregnancies that is exacerbated in preeclampsia. This could be comparable to trophoblast deportation, which can occur in normal pregnancy but is much greater in preeclampsia. 12 Blood levels of other factors known to suppress endothelial cell proliferation in vitro are raised in preeclampsia, including platelet factor W, 1~ a marker of platelet activation, 16 interleukin-6, and tumor necrosis factor-0L 17 Other factors that may disturb endothelial function but are not known to specifically suppress endothelial cell proliferation are also raised in preeclampsia. These inelude plasma neutrophil elastase, ~8' 19 lipid peroxides, 2° soluble fragments of fibrin(ogen), 21-2~ and antiendothelial cell antibodies. 24 However, all these factors have been shown to be present in preeclampsia serum and there are no experimental data suggesting that plasma levels are higher. These are therefore unlikely to be the cause of the inhibition of endothelial cell proliferation seen here. Because the serum and plasma samples in this study were not from the same individuals, it might be argued that the differences in activity reflect differences in the disease severity between the two patient groups. However, all patients had early-onset proteinuric preeclampsia, and the diagnostic criteria and the procedure for matching pregnant controls were identical for both groups. The apparent association between the most severe cases of preeclampsia and the highest suppression of endothelial cell proliferation in 20% plasma could have several possible explanations. There may be a greater release of the endothelial cell inhibitory activity from the hypoxic placenta in these patients, or the plasma endo-
February 1996 AmJ Obstet Gynecol
thelial cell suppressive activity may be due to a factor produced by the abnormal activation of other systems in preeclampsia, such as platelets, liver, or neutrophils. The finding that plasma from some patients with severe disease gave normal endothelial cell proliferation might indicate that the endothelial cell inhibitory activity is released from the placenta in surges, possibly connected to placental vascular accidents. It is interesting that the normal control pregnant woman whose plasma gave the lowest endothelial cell proliferation for that group had hypertension and a trace of proteinuria 7 weeks later and was delivered of a growthretarded baby. The significance of this observation cannot be developed except by a much larger prospective study. However, it raises the question whether the low measurement reflected the occult preeclamptic process. This study is consistent with the hypothesis that a factor is shed from the placenta in preeclampsia that causes the maternal endothelial dysfunction. Having established that there is an endothelial cell inhibitory factor on the syncytiotrophoblast microvillous membrane and that a similar activity is present in the plasma of preeclamptic women, it is now essential to determine whether the two factors are the sa~qe and if so whether the plasma factor is derived from the placenta. Only in this way will we estabfish the link between the placenta and the maternal syndrome.
REFERENCES
1. Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: an endothelial cell disorder. AuJ OBSXETG~qECOL1989;161:1200-4. 2. RobertsJM, Redman CWG. Pre-eclampsia: more than pregnancy-induced hypertension. Lancet 1993;341:1447-51. 3. Rodgers GM, Taylor RN, RobertsJM. Preeclampsia is associated with a serum factor cytotoxic to human endothelial cells. ANJ OBSTETG'mECOL1988;159:908-14. 4. Tsukimori K, Maeda H, Shingu M, Koyanagi T, Nobunaga M, Nakano H. The possible role of endothelial cells in hypertensive disorders during pregnancy. Obstet Gynecol 1992;80: 229-33. 5. Taylor RN, Casal DC, Jones LA, Varma M, Martin JN Jr, RobertsJM. Selective effects of preeclamptic sera on human endothelial cell procoagulant protein expression. AM J OBSTETG'mECOL1991;165:1705-10. 6. Lorentzen B, Endresen MJ, Hovig T, Haug E, Henriksen T. Sera from preeclamptic women increase the content of triglycerides and reduce the release of prostacyclin in cultured endothelial cells. Thromb Res 1991;63:363-72. 7. Redman CW. Current topic: pre-eclampsia and the placenta. Placenta 1991;12:301-8. 8. Smfirason AK, Sargent IL, Starkey PM, Redman CWG. The effect of placental syncytiotrophoblast microvillous membranes t-ore normal and pre-eclamptic women on the growth of endothelial ceils in vitro. Br J Obstet Gynaecol 1993;100:943-9. 9. Chua S, Wilkins T, Sargent I, Redman C. Trophoblast deportation in pre-eclamptic pregnancy. Br J Obstet Gynaecol 1991;98:973-9. 10. Sargent IL, Johansen M, Chua S, Redman CWG. Clinical experience: isolating trophoblast from maternal blood. Ann N YAcad Sci 1994;731:154-61. 11. Jaameri KEU, Koivuniemi AP, Carpen EO. Occurrence of trophoblasts in the blood of toxaemic patients. Gynaecologia 1965;160:315-20.
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12. Attwood HD, Park WW. Embolism to the lungs by" trophoblast. J Obstet Gynaecol Br Commonw 1961;64:611-7. 13. Jones CJ, Fox H. An ultrastructural and ultrahistochemical study of the human placenta in maternal pre-eclampsia. Placenta 1980;1:61-76. 14. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J Clin Invest 1973;52:2745-56. 15. Ballegeer VC, Spitz B, De Baene LA, Van Assche AF, Hidajat M, Criel AM. Platetet activation and vascular damage in gestational hypertension. AMJ OBSTETGYNECOL1992;166:62933. 16. Malone TE, Gray GS, PetroJ, et al. Inhibition of angiogenesis by recombinant human platelet factor-4 and related pepddes. Science 1990;247:77-9. 17. Vince G, Starkey P, Austgulen R, Kwiatkowski D, Redman C. Plasma concentrations of IL-6, TNF and TNF-R are increased in women with pre-eclampsia. BrJ Obstet Gynaecol 1995;102:20-5. 18. Greer IA, Haddad NG, Dawes J, Johnstone FD, Calder AA. Neutrophil activation in pregnancy-induced hypertension. BrJ Obstet Gynaecol 1989;96:978-82.
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19. Harlan JM. Neutrophil-mediated vascular injury. Acta Med Scand Suppl 1987;715:123-9. 20. Wang YP, Walsh SW, Guo JD, Zhang JY. Maternal levels of prostacyclin, thromboxane, vitamin E, and lipid peroxides throughout normal pregnancy. A_,aJ OBSTETGYNECOL1991; 165:1690-4. 21. Rowland FN, Donovan MJ, Picciano PT, Wilner GD, Kreutzer DL. Fibrin-mediated vascular injury: identification of fibrin peptides that mediate endothelial cell retraction. Am J Pathol 1984;117:418-28. 22. Dang CV, Bell ~gR, Kaiser D, Wong A. Disorganization of cultured vascular endothelial cell monolayers by fibrinogen fragment D. Science 1985;227:1487-90. 23. Proietti AB, Johnson MJ, Proietti FA, Repke JT, Bell WR. Assessment of fibrin(ogen) degradation products in preeclampsia using imrnunoblot, enzyme-linked immunosorbent assay, and latex-based agglutination. Obstet Gynecol 1991;77:696-700. 24. Rappaport VJ, Hirata G, Yap HK, Jordan SC. Antivascular endothelial cell antibodies in severe preeclanlpsia. AM J OBSTETC*YNECOL1990;162:138-46.
1-800-55-MOSBY This n u m b e r links you to the full text of articles published in over 25,000 journals, including all Mosby journals. MOSBY Document Express~, a rapid response information retrieval service, provides quick t u r n a r o u n d , 24-hour availability, and speedy delivery methods. For inquiries and pricing information, call our toll-free 24-hour o r d e r line: 1-800-55-MOSBY; outside the U n i t e d States: 415-259-5046; fax: 415-259-5019; E-mail: [email protected]. MOSBY Document ExDresga is offered in cooperation with Dynamic Information Corp.
Key words: Preeclampsia, endothelial cells, serum, plasma, syncytiotrophoblast microvillous membranes
Preeclampsia is a multisystem disease of p r e g n a n c y of u n k n o w n cause. T h e m a t e r n a l syndrome is characterized by increased b l o o d pressure, edema, proteinuria, and a b n o r m a l clotting, liver, and renal functions, all of which may result f r o m generalized vascular endothelial cell dysf u n c t i o n ) ' 2 T h e cause of this is unknown, but there is evidence for a circulating endothelial cell "toxic': factor in preeclampsia. Sera from preeclamptic w o m e n are rep o r t e d to be cytotoxic to endothelial cells, a' 4 and e n d o thelial cells c u l t u r e d in preeclampsia sera show an increased release of cellular fibronectin, ~ an increased triglyceride content, and a decreased prostacyclin release. 6 Preeclampsia is a placental disease. 7 So the " t o x i c " factor is most likely to originate f r o m the syncytiotrophoblast, which is a m u h i n u c l e a t e d syncytium with an extensive microvillous brush b o r d e r in contact with the m a t e r n a l blood. We have shown that the syncytiotrophoblast microvillous m e m b r a n e s o f n o r m a l placentas contain a factor(s)
From the Harris Birthright Pre-eclampsia Research Unit, Nuffield Department of Obstetrics and Gynaecology, University of Oxford. Supported by a grant from Birthrigtat. Received for publication August 30, 1994; revisedJanuary 8, 1995; acceptedJuly 11, 1995. Reprint requests:Alexander K- Smdrason, DPhil, Nuffield Department of Obstetrics and Gynaecolog);John Radcliffe Hospital, Oxford, United Kingdom, OX3 9DU. Copyright © 1996 by Mosby-Year Book, Inc. 0002-9378/96 $5.00+ 0 6/1/67723
that specifically inhibits endothelial cell proliferation and disrupts the continuity of established endothelial cell monolayers without causing cell death, s T h e same syncytiotrophoblast microvillous m e m b r a n e s preparations do not disturb cultures of o t h e r cell types ( a d h e r e n t or nonadherent). However, there is no difference in the activity of equivalent amounts of syncytiotrophoblas t microvillous m e m b r a n e s p r e p a r e d f r o m n o r m a l or preeclamptic placentas, s We have therefore c o n c l u d e d that if this factor is relevant to the maternal endothelial dysfunction of preeclampsia it is because it is shed into the maternal circulation in a b n o r m a l amounts. This is supported by our r e c e n t work 9' 1o c o n f i r m i n g previous reports n that syncytiotrophoblast are present in increased amounts in the uterine vein b l o o d of w o m e n with preeclampsia compared with similar samples f r o m n o r m a l p r e g n a n t women. It is n o t yet certain whether trophoblasts routinely e n t e r the peripheral circulation, ° probably most are trapped in the capillaries of the lungs. 1~ We propose that this deportation of whole trophoblast is simply the most visible aspect of a m o r e extensive process that includes the s h e d d i n g of microvillous fragments (containing the endothelial cell inhibitory- activity) that, unlike the intact trophoblast cells, would be carried t h r o u g h o u t the maternal circulation. This hypothesis is consistent with the observation that there are fewer microvilli on the surface of the placenta in preeclampsia and that they are structurally abnormal. ~s 787
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February 1996 AmJ ObstetGynecol
Sm&rason, Sargent, and Redman
Table I. Subjects in serum and plasma studies Serum study Preeclampsia (n = 2O)
Age (yr)* Nulliparoust Blood pressure in early pregnancy (ram Hg)* SBP DBP Maximum blood pressure (ram Hg) * SBP DBP Proteinuria (21+) at samplingt Proteinuria (>_1+) before delivery~Gestadonal age at sampling (days) * Gestational age at delivery (days) * Birth weight (gm)* Twinst
I
Plasma study
Pregnant controls (n = 20)
Preeclampsia (n = 20)
Pregnant controls (n = 20)
23.5 (17-32) 18 (90)
23.4 (18-31) 18 (90)
27.2 (18-38) 20 (100)
27.0 (18-36) 20 (100)
114 (80-150) 66 (40-88)
116 (95-130) 66 (45-80)
124 (110-150) 77 (70-85)
111 (80-130) 66 (30-80)
170 (140-200) 115 (100-130) 20 (100)
128 (110-150) 79 (70-95) 0 (0)
174 (145-210) 114 (90-140) 20 (100)
126 (110-145) 79 (66-100) 0 (0)
20 (100)
0 (0)
20 (100)
1 (5)
213 (165-254)
215 (173-259)
228 (201-261)
229 (206-264)
221 (168-254)
277 (243-291)
232 (203-261)
280 (252-293)
1342 (248-2845) 2 (10)
3248 (2312-4240) 2 (10)
1736 (797-3189) 0
3487 (2531-4476) 0
• SBP, Systolicblood pressure; DBP, diastolic blood pressure. *Mean and range. tNumber and percent.
To investigate this question further, we sought evidence for an endothelial cell inhibitory activity, initially in preeclampsia serum, and, having failed to detect it, subsequently in plasma. Experiments to determine the reason for the discrepancy between the activity of serum and plasma are presented.
Methods Subjects. The study groups were women with preeclampsia, matched control women with normal pregnancies, and n o n p r e g n a n t women. Women with preeclampsia had new hypertension (diastolic blood pressure consistently >_90 m m Hg with previously lower readings) and new proteinuria (more than one urine sample showing + on dipstick testing in the absence of urinary infection) that subsequently regressed after delivery. For each woman with preeclampsia a healthy pregn a n t control was found who was matched for age (_+4 years), parity (0, 1 to 3, >_4), and gestational age (+13 days) and not receiving any medication. Nonpregnant control women were hospital personnel of childbearing age (mean age 30.5 years, SD 5.7) but who otherwise cannot be regarded as matched controls. Further details of the subjects are shown in Table I. This study was approved by the Central Oxford Research Ethics Committee. Serum was prepared from peripheral vein blood that was centrifuged at 2500g for 10 minutes after clotting and kept frozen at -40 ° C. For the plasma study peripheral vein blood was collected into sterile plastic tubes containing sodium heparin (40 u n i t s / m l blood) and cen-
trifuged at 2500g for 25 minutes. The plasma was collected and frozen at -40 ° C. Previous experiments showed that concentrations of heparin between 10 and 320 u n i t s / m l blood made little difference to endothelial cell tritiated thymidine incorporation when grown in 20% plasma (data not shown). Isolation of human endothelial cells. H u m a n umbilical vein endothelial cells were isolated according to t h e method ofJaffe et al ~4 by use of 0.25 m g / m l collagenase type 1A (Sigma, St. Louis). The cells were cultured in RPMI 1640 medium containing 25 m m o l / L HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Gibco, Grand Island, N.Y.), 100 u n i t s / m l benzylpenicillin (Glaxo, Greenford, United Kingdom), 100 p g / m l streptomycin (Sigma), and 20% heat-inactivated fetal calf serum (Imperial, Andover, United Kingdom) in gelatin (Sigma)-coated tissue culture flasks (Nunc, Roskilde, Denmark), as previously described. 8 After 4 to 6 days in primary culture the cells were detached with trypsin-ethylenediaminetetraacetic acid (trypsin 1:250, 0.5 gm/L; ethylenediaminetetraacetic acid, 0.2 gin/L) (Sigma) and used in the assays described in the following text. Endothelial cell proliferation assay. To each gelatincoated well of a 96-well flat-bottomed microtiter plate (Nunc), 15 x 10~ endothelial cells were added in 200 pl of culture medium containing the specified concentration of test serum or plasma, which had been rapidly thawed at 37 ° C. After a 24-hour incubation at 37 ° C in 5% carbon dioxide in air, the cells were pulsed with 1 p,Ci per well (1 mCi = 37 MBq) of (methyl-SH)-thymidine (25
Volume 174, Number 2 Am J Obstet C.ynecol
Sm~rason, Sargent, and Redman
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C i / m m o l , A m e r s h a m , A I n e r s h a m , U n i t e d K i n g d o m ) for a f u r t h e r 18 hours. I m m e d i a t e l y b e f o r e h a r v e s t i n g t h e m o r p h o l o g i c features o f t h e e n d o t h e l i a l cell m o n o layer in e a c h c u l t u r e well were assessed by use o f a n i n v e r t e d p h a s e - c o n t r a s t m i c r o s c o p e . Because t h e e n d o thelial cells are a d h e r e n t , they were first r i n s e d with p h o s p h a t e - b u f f e r e d saline s o l u t i o n a n d t h e n d e t a c h e d with t r y p s i n - e t h y l e n e d i a m i n e t e t r a a c e t i c acid. T h e cells were t h e n h a r v e s t e d a n d t h e filters (Wallac, T u r k u ) c o u n t e d in a flat-bed scintillation c o u n t e r , 1205 Betaplate (Pharmacia-Wallac, Turku). T h e o p t i m a l n u m b e r o f cells p e r well a n d t h e t i m e o f a d d i n g tritiated t h y m i d i n e to t h e cultures to o b t a i n t h e m a x i m u m c o u n t s h a d previously b e e n d e t e r m i n e d , a n d t h e level o f t h y m i d i n e i n c o r p o r a t i o n was f o u n d to reflect t h e cell n u m b e r (data n o t shown). To s t a n d a r d i z e t h e assay b e t w e e n d i f f e r e n t plates a n d e n d o t h e l i a l cell p r e p a r a t i o n s , 10 replicates f r o m a c o n t r o l p o o l o f s e r u m or p l a s m a were i n c l u d e d o n e a c h plate. For the s e r u m study t h e c o n t r o l p o o l was o b t a i n e d f r o m 10 m a l e b l o o d g r o u p AB-positive d o n o r s a n d for t h e p l a s m a study f r o m 10 n o n p r e g n a n t w o m e n . A tritiated t h y m i d i n e i n c o r p o r a t i o n ( p r o l i f e r a t i o n ) i n d e x was t h e n c a l c u l a t e d for e a c h s a m p l e in r e l a t i o n to t h e c o n t r o l s e r u m o r p l a s m a p o o l o n t h e same plate, as follows: T r i t i a t e d t h y m i d i n e i n c o r p o r a t i o n (% o f c o n t r o l pool) = Sample (median counts/min) Control pool (mean counts/rain)
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Seeding of syncytiotrophoblast membranes into blood. A n e x p e r i m e n t was d e s i g n e d to e x p l o r e t h e fate o f syncyt i o t r o p h o b l a ~ t m e m b r a n e s a d d e d to blood. A p l a c e n t a was collected f r o m a h e a l t h y w o m a n h a v i n g a n elective
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Fig. 1. Effect of peripheral vein serum from women with preeclampsia and matched pregnant controls on endothelial cell proliferation (tritiated thymidine incorporation). Mean and SD (n = 20 pairs).
250
Fig. 2. Fate of syncytiotrophoblast microvillous membranes (STBM) added into blood. Syncytiotrophoblast microvillous membranes were added to fresh blood at 2.3 or 93 mg wet weight per milliliter blood. When 20% serum or plasma is tested, this corresponds to syncytiotrophoblast microvillous membranes concentrations of 170 and 1700 pg wet weight per well, respectively, if it is assumed that hematocrit is 45% and that syncytiotrophoblast microvillous membranes are retained in cell-fi-ee fraction after centrifugation. Suppression of endotheliaI cell proliferation (tritiated thymidine incorporation) in plasma (hatched bars) and serum (open bars) can be compared with standard curve for this syncytiotrophoblast microvillous membranes preparation (solid line) run at same time. Results are expressed as percentage of proliferation obtained in cultures containing serum or plasma in absence of seeded syncytiotrophoblast microviltous membranes. Median of five replicates.
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790
Sm&rason, Sargent, and Redman
February 1996 Am J Obstet Gynecol
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Statistical m e t h o d s . To minimize the effects of extreme outlying readings, the median rather than mean counts per minute of the five replicates was used when the thymidine incorporation index was calculated. For the same reason the n u m b e r of replicates was five rather than the more conventional three. Results for women with preeclampsia, matched pregnant controls, and n o n p r e g n a n t controls were compared with one-way analysis of variance followed by post hoc multiple comparisons with Bonferroni adjustment with the SPSS for Windows (SPSS, Chicago) statistical computer program. The Wilcoxon ranksum test was used to compare the results for women with low and normal platelets.
Results cesarean section at 39 weeks' gestation because of a previous cesarean section. Syncytiotrophoblast microvillous membranes were prepared as previously describedfl and the membranes were resuspended in sterile endotoxin7 free phosphate-buffered saline solution at two dilutions (25 and 250 mg wet weight/ml). One milliliter of each of these dilutions or of phosphate-buffered saline solution alone was mixed in plastic tubes with 10 ml aliquots of freshly drawn male blood, without (to give serum) or with 400 units of heparin sodium (to give plasma). Plasma was prepared immediately and serum, by centrifugation at 2500 gm for 25 minutes, after clotting. The samples were frozen at -20 ° C until required. The sera and plasma were tested in the endothelial cell proliferation assay at a 20% dilution. Known amounts of syncytiotrophoblast microvillous membranes (from the same placenta) were tested on the same batch of endothelial cells in 20% AB serum to produce a standard curve.
At all three serum concentrations tested (2%, 10%, 20%) there were no differences in the incorporation of tritiated thymidine by endothelial cells grown in sera from preeclamptic women compared with those from normal pregnant women (Fig. 1). Because these results were clearly contrary to the hypothesis, we tested whether the previously discovered endothelial cell suppressive activity of syncytiotrophoblast microvillous membranes could be recovered from serum or heparin plasma after syncytiotrophoblast microvillous membranes were added to unclotted blood. When tested in the endothelial cell proliferation assay, most of the expected syncytiotrophoblast microvillous membranes suppressive activity was recovered from the plasma (Fig. 2). The lower concentration (170 lag wet weight/well) caused 70% suppression (30% proliferation) when 86% was expected by comparison with the standard curve for syncytiotrophoblast microvillous membranes prepared
Sm&rason, Sargent, and Redman 791
Volume 174, Number 2 .~n J Obstet GynecoI
Table II. Preeclampsia cases in plasma study analyzed according to platelet count Platelet counts at sampling
Maturity at sampling (days) Maturity at delivery (days) Birth weight of infant (gm) Maximum blood pressure (mm Hg) SBP DBP Plasma aspartate aminotransferase (IU/L) Human umbilical vein endothelial cell proliferation in 2% plasma (%) Human umbilical vein endothelial cell proliferation in 20% plasma (%)
'Wormal"platelets (>150x 109/L) (n = 15)
Low platelets (<150× 109/L) (n = 5)
229 (201-261) 240 (203-261) 1632 (797-3189)
217 (210-225) 217 (210-225) 1443 (1055-1460)
170 115 27 104
(145-210) (95-140) (16-84) (89-133)
81 (55-113)
170 t10 579 126
(160-t80) (90-120) (29-1464)* (111-134)?
49 (39-95)f
Values are median and range. SBP, Systolicblood pressure; DBP, diastolic blood pressure. *p < 0.01, Wilcoxon rank-sum test. tP < 0.05, Wilcoxon rank-sum test.
from the same placenta. Some of the activity may have been lost in aggregates being pelleted on centrifugation. In contrast,' almost no suppressive activity was recovered from the serum. As a result of this experiment, it was clear that heparin plasma, not serum, should be tested if evidence for shedding of the syncytiotrophoblast microvillous membranes suppressive factor into the circulation in preeclampsia was to be sought. Hence a further set of clinical samples was collected from women with preeclampsia, matched pregnant controls, and, in addition, from n o n p r e g n a n t women, and the plasma was prepared as described above. There was no effect of preeclampsia plasma at 2% and 10%, but at a 20% dilution it significantly (p < 0.05) inhibited endothelial cell proliferation compared with both the normal pregnant and n o n p r e g n a n t controls (Fig. 3). At this dilution endothelial cell proliferation was also significantly reduced (p< 0.05) in normal pregnant plasma compared with n o n p r e g n a n t control. No morphologic changes were detected in the endothelial cell monolayers before harvesting at any of the plasma concentrations tested. The effects of sera or plasma from preeclamptic or normal pregnant women were expressed as proliferation index values relative to standard pools of sera or plasmas from n o n p r e g n a n t donors. The absolute values of thymidine incorporation depended on the concentration of serum or plasma tested (Fig. 4). There was a wide range of suppression caused by the different samples of plasma from preeclamptic women when tested at 20% (Fig. 5). All the cases were severe by conventional definitions by being proteinuric, and most of them had early-onset preeclampsia (Table I). However, we wondered whether there was a correlation with the severity of the disease. Because a low platelet count reflects more severe disease, the preeclampsia group was subdivided into those with lower and higher platelet
counts at the time of sampling. This necessitated choosing an arbitrary threshold of 150 x 109/L, which was chosen without knowledge of the experimental results. This yielded five cases below this limit, deemed to be more severe, and 15 above, classified as less severe. Preeclamptic women with lower platelets had significantly higher plasma aspartate aminotransferase activities, lower endothelial cell proliferation in 20% plasma, and higher proliferation in 2% plasma (Table II). There was no significant correlation between the proliferation index at 20% plasma and length of gestation or birth weight (data not shown).
Comment The starting point of this study came from the observatmns of others that sera from preeclamptic women damaged cultured h u m a n endothelial cells, as measured by increased release of chromium 51. 3' 4We assessed a different aspect of endothelial cell function--spontaneous proliferation--and were unable to show any effect of preeclampsia sera. We argued that if the sera were indeed toxic to endothelial cells then proliferation should be inhibited as well. That it was not suggests that in this context ~Cr release reflects alterations in cell membrane permeability but not cell death or damage sufficient to affect proliferation. Plasma from women with preeclampsia significantly inhibited endothelial cell growth in vitro compared with plasma from normal pregnant and n o n p r e g n a n t women. This is consistent with the hypothesis that more trophoblast-associated endothelial cell inhibitory activity is shed from the placenta into the maternal circulation in preeclampsia. Titration experiments showed that this inhibition was not merely a toxic effect of plasma (or the heparin anticoagulant) because high concentrations of nonpregnant plasma had no such effect. It is also unlikely to be the result of a lack of growth factors in preeclampsia
792 Sm&rason, Sargent, and Redman
because endothelial proliferation was not lower in 2% preeclampsia plasma compared with the other groups. No comparable inhibition was seen with preeclampsia sera, suggesting that the endothelial cell inhibitory activity is lost during the clotting process. In seeking an explanation f o r this difference, syncytiotrophoblast microvillous membrane preparation with known endothelial cell inhibitory activity was seeded into blood where it was found that the activity was recovered only from plasma, not from serum. The active factor in the syncytiotropboblast microvillous membranes may be destroyed by the clotting enzymes, or the syncytiotrophoblast microvillous membranes may simply become enmeshed in the clot. Loss of the syncytiotrophoblast microvillous m e m b r a n e s associated inhibitory activity in serum cannot be due to serum-associated blocking factors because the standard curve shown was carried out in serum. Thus, if the activity in the blood o f preeclamptic women is derived from syncytiotrophoblast microvillous fragments, this would account for the difference between the effects of serum and plasma in vitro. Compared with nonpregnant women, 20% plasma from pregnant normal control women also suppressed endothelial cell proliferation, although not to the same extent as preeclampsia plasma. This is compatible with the concept of a low-level release o f syncytiotrophoblast microvillous fragments in normal pregnancies that is exacerbated in preeclampsia. This could be comparable to trophoblast deportation, which can occur in normal pregnancy but is much greater in preeclampsia. 12 Blood levels of other factors known to suppress endothelial cell proliferation in vitro are raised in preeclampsia, including platelet factor W, 1~ a marker of platelet activation, 16 interleukin-6, and tumor necrosis factor-0L 17 Other factors that may disturb endothelial function but are not known to specifically suppress endothelial cell proliferation are also raised in preeclampsia. These inelude plasma neutrophil elastase, ~8' 19 lipid peroxides, 2° soluble fragments of fibrin(ogen), 21-2~ and antiendothelial cell antibodies. 24 However, all these factors have been shown to be present in preeclampsia serum and there are no experimental data suggesting that plasma levels are higher. These are therefore unlikely to be the cause of the inhibition of endothelial cell proliferation seen here. Because the serum and plasma samples in this study were not from the same individuals, it might be argued that the differences in activity reflect differences in the disease severity between the two patient groups. However, all patients had early-onset proteinuric preeclampsia, and the diagnostic criteria and the procedure for matching pregnant controls were identical for both groups. The apparent association between the most severe cases of preeclampsia and the highest suppression of endothelial cell proliferation in 20% plasma could have several possible explanations. There may be a greater release of the endothelial cell inhibitory activity from the hypoxic placenta in these patients, or the plasma endo-
February 1996 AmJ Obstet Gynecol
thelial cell suppressive activity may be due to a factor produced by the abnormal activation of other systems in preeclampsia, such as platelets, liver, or neutrophils. The finding that plasma from some patients with severe disease gave normal endothelial cell proliferation might indicate that the endothelial cell inhibitory activity is released from the placenta in surges, possibly connected to placental vascular accidents. It is interesting that the normal control pregnant woman whose plasma gave the lowest endothelial cell proliferation for that group had hypertension and a trace of proteinuria 7 weeks later and was delivered of a growthretarded baby. The significance of this observation cannot be developed except by a much larger prospective study. However, it raises the question whether the low measurement reflected the occult preeclamptic process. This study is consistent with the hypothesis that a factor is shed from the placenta in preeclampsia that causes the maternal endothelial dysfunction. Having established that there is an endothelial cell inhibitory factor on the syncytiotrophoblast microvillous membrane and that a similar activity is present in the plasma of preeclamptic women, it is now essential to determine whether the two factors are the sa~qe and if so whether the plasma factor is derived from the placenta. Only in this way will we estabfish the link between the placenta and the maternal syndrome.
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12. Attwood HD, Park WW. Embolism to the lungs by" trophoblast. J Obstet Gynaecol Br Commonw 1961;64:611-7. 13. Jones CJ, Fox H. An ultrastructural and ultrahistochemical study of the human placenta in maternal pre-eclampsia. Placenta 1980;1:61-76. 14. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J Clin Invest 1973;52:2745-56. 15. Ballegeer VC, Spitz B, De Baene LA, Van Assche AF, Hidajat M, Criel AM. Platetet activation and vascular damage in gestational hypertension. AMJ OBSTETGYNECOL1992;166:62933. 16. Malone TE, Gray GS, PetroJ, et al. Inhibition of angiogenesis by recombinant human platelet factor-4 and related pepddes. Science 1990;247:77-9. 17. Vince G, Starkey P, Austgulen R, Kwiatkowski D, Redman C. Plasma concentrations of IL-6, TNF and TNF-R are increased in women with pre-eclampsia. BrJ Obstet Gynaecol 1995;102:20-5. 18. Greer IA, Haddad NG, Dawes J, Johnstone FD, Calder AA. Neutrophil activation in pregnancy-induced hypertension. BrJ Obstet Gynaecol 1989;96:978-82.
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