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Potential role of endothelin-1 in normal and hypertensive pregnancies
Potential role of endothelin-l in normal and hypertensive pregnancies Dimitrios S. Mastrogiannis, MD, PhD, William F. O'Brien, MD, Judith Krammer, MD, and Ray Benoit, BS Tampa, Florida Endothelins are the most potent naturally occurring vasoconstrictors yet discovered. 80th normal and abnormal pregnancies are associated with significant changes in vascular smooth muscle; therefore the potential role of endothelin in pregnancy was investigated. Plasma immunoreactive endothelin-1 concentration was measured by radioimmunoassay in blood from women with normal pregnancy and preeclampsia and in cord blood from normal pregnancies. Endothelin-1 levels were elevated in pregnant women during labor when compared with levels in nonpregnant women and patients with normal pregnancies before labor. Preeclampsia in non laboring women before treatment was associated with higher endothelin values when compared with values in normal nonlaboring patients and women with preeclampsia after magnesium sulfate infusion. The umbilical venous concentration of endothelin was 10 times higher than normal pregnant levels and four times higher than levels in laboring patients. (AM J OBSTET GVNECOL 1991;165:1711-6.)
Key words: Endothelin, pregnancy, preeclampsia, labor, fetus The role of the endothelium in modulating cardiovascular homeostasis via regulation of the vascular smooth muscle tone has been recognized recently. Endothelium regulates the reactivity of the smooth muscles through production of both vasodilators and vasoconstrictors. I The endothelins are a group of vasoactive peptides first isolated from bovine aortic endothelial cells and are the most potent vasoconstrictors yet discovered. 2 Three distinct human endothelin genes have been identified. Endothelin-l is the only endothelin made by human endothelial cells. Endothelin-2 is produced in the kidney and endothelin-3 is mainly associated with neural tissue. 3 Endothelin appears to playa role in the pathogenesis of some forms of hypertension"~6 Additionally, endothelin concentration is elevated in patients with acute myocardial infarction or vasculitis and in patients with uremia who are undergoing hemodialysis.7~9 Preeclampsia, a hypertensive disorder unique to pregnancy, is characterized by generalized vasoconstriction that is due, in part, to increased sensitivity of
From the Division of Obstetrics / Maternal-Fetal Medicine, College of Medicine, University of South Florida. Supported in part by a Southern Medical Association Research Grant. Dr. Mastrogiannis is a recipient of a Southern Medical Association Research Grant Award. Presented in part at the Thirty-eighth Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 20-23,
1991.
Reprint requests: Dimitrios Mastrogiannis, MD, PhD, Temple University Hospital, Department of Obstetrics and Gynecology, Room 7-0PD, 3401 N. Broad St., Philadelphia, PA 19140.
6/6/33234
vascular smooth muscle to the effects of vasopressors. IO This is in contrast to normal pregnancy where the vascular bed is less sensitive to vasoconstrictors. Io Several lines of evidence implicate endothelial cell injury as a basic pathogenic mechanism in preeclampsia resulting in impaired synthesis of vasodilators and a possible increase in the production of vasoconstrictors. II Preliminary studies suggested that endothelin concentrations are elevated in women with preeclampsia, compared with controls. 12, 13 Both normal and hypertensive pregnancies are associated with significant changes in vascular smooth muscle tone; therefore the potential role of endothelin in various pregnancy conditions and pregnancy complications was investigated in this study. Material and methods One hundred twenty-eight plasma samples were prospectively collected from volunteers presenting for care at the University of South Florida and the Tampa General Hospital. Eighteen samples were collected from nonpregnant women of reproductive age without any medical or gynecologic complications. Forty-seven samples were collected from women with normal pregnancies at several gestational age intervals (11 samples from 6 to 12 weeks, 19 samples from 16 to 20 weeks, 9 samples from 26 to 33 weeks, and 8 samples from 35 to 42 weeks). Sixteen samples were collected from non laboring patients with preeclampsia before (n = 10) or after (n = 6) the institution of magnesium sulfate infusion. Sixteen samples were collected from patients in labor (eight patients with normal pregnancies and eight with preeclampsia). The collection of blood from hospital1711
1712 Mastrogiannis et al.
December 1991 Am J Obstet Gynecol
Table I. Comparison of normal, hypertensive, and umbilical venous groups (mean ± SD) Normal pregnancies, patients in labor (n = 8)
Age (yr) Gestational age at delivery (wk) Systolic pressure (mm Hg) Diastolic pressure (mm Hg) Hematocrit (%) Platelets (l03/ mm 3) Uric acid (mg/dl) Birth weight (gm) Vaginal delivery (no.) Cesarean section (no.)
21.9 ± 3.4 38.6 ± 4 126.2 ± 14 75 ± 5 34.3 ± 3.7 196 ± 44 4.1 ± 0.26 3135.1 ± 893.4 4 (50%) 4 (50%)
ized women was done on admission to the labor and delivery suite with the patients in the supine position and before any medications and thus reflects the first stage of labor in laboring women. Thirty-one samples were collected from the umbilical vein immediately after delivery in normal term pregnancies, before and after umbilical cord clamping. All patients gave written informed consent as approved by the University of South Florida's Institutional Review Board. Preeclampsia was defined according to the following criteria: (1) nulliparity, (2) blood pressure ~140/90 mm Hg measured on two occasions at least 6 hours apart, (3) proteinuria of ~30 mg/dl (defined as + 1) by dipstick on random catheterized specimen, (4) no known history of hypertension before pregnancy, and (5) resolution of hypertension and proteinuria in the postpartum period. All volunteers with normal pregnancies denied a history of hypertension or other medical complications. Patients with a history of illicit drug use or other medications were not included. Blood pressure measurements were obtained from patients in the sitting position after 15 minutes of rest, with phase IV Korotkoff sound used for the diastolic blood pressure. Obstetric and demographic data were recorded from the medical records. Blood samples were collected from each patient in the sitting position. Plasma was prepared from blood collected into chilled tubes containing 5 mg/ml ethylenediaminetetraacetic acid and 500 U I ml aprotinin. The plasma was separated by centrifugation and the samples were stored at - 80 C until assayed. On the day of the assay, 2 ml of thawed plasma was acidified with an equal volume 0.1 % trifluoroacetic acid and 2N hydrochloric acid (pH 3), extracted over a C18 column, and eluted with 60% acetonitrile in 0.1 % trifluoroacetic acid. Recoveries of 90% for both radiolabeled and unlabeled samples were observed. These extracts were evaporated to dryness under nitrogen and then assayed in duplicate with a specific radioimmunoassay (Peninsula Laboratories, Inc., Belmont, Calif.). Interassay and 0
Umbilical venous group (n = 31)
21.8 39.1 122.2 72.6 36.01 227
± 5.6
± ± ± ± ±
1.2 8.9 7.8 2.8 72
3355 ± 446 19 (61.3%) 12 (38.7%)
Patients with preeclampsia (n = 24)
21.5 ± 4.5 34.4 ± 4.6 160 ± 22 106 ± 13 34.5 ± 5.25 189 ± 68 6.5 ± 1.3 2245 ± 953 16 (66.7%) 8 (33.3%)
intraassay coefficient of variation for endothelin-l was 7%. Endothelin concentrations were corrected for midpoint displacement of each curve. We did not find differences in values from samples processed before or after freezing. Cross-reactivity with endothelin-2 and endothelin-3 was 7%. The person performing the radioimmunoassays was blinded as to the clinical condition of the patient. All data were expressed as mean ± SD and were analyzed by the SAS (Statistical Analysis Systems, Cary, N.C.) statistical package, with the general linear model procedure for unbalanced designs, Duncan's multiple range test, unpaired and paired Student t tests, and X2 tests where appropriate. A probability of p < 0.05 was considered significant. Results
The endothelin-l concentration in normal nonpregnant women of reproductive age (mean age 32.4 ± 4.7 years, range 30 to 39 years) was 1.8 ± 0.6 pg/ml. There was no significant association between the subject'S age and endothelin level concentrations for either nonpregnant or pregnant women and there was no significant difference in mean age between patients with normal pregnancies (28 ± 5 years) and nonpregnant controls. All patients in the antepartum group remained normotensive and were delivered of normal infants at term. In Table I obstetric and demographic data are presented for the normal labor, preeclampsia, and umbilical venous sample groups. Systolic and diastolic blood pressure, urinary protein concentration, and serum uric acid concentration were significantly higher (p < 0.001) in the patients with preeclampsia. Endothelin-l concentration in the nonpregnant and antepartum groups is shown in Fig. 1. We did not observe a difference in endothelin concentrations between pregnant and nonpregnant patients (by SAS General Linear Model procedure for unbalanced designs). A possible effect of gestational
Endothelin levels in pregnancy
Volume 165 Number 6, Part I
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Fig. 2. Effect of labor on maternal venous plasma endothelin-l in normal pregnancy and preeclampsia (Duncan's multiple range test).
age with increasing endothelin concentrations in the second and early third trimesters did not reach statistical significance (p = 0.06) . As noted in Fig. 2, levels of endothelin-I were significantly higher in laboring women than in nonlaboring women and all patients with preeclampsia had concentrations higher than those in non laboring women with normal pregnancies. The increase noted in women with preeclampsia was similar to that associated with labor (SAS, General Linear Model procedure). Women with preeclampsia (samples taken before magnesium sulfate infusion) had significantly elevated plasma endothelin concentrations when compared with samples obtained during magnesium sulfate infusion in nonpaired samples (p < 0.05) (Fig. 3). Similar results were found when paired samples were used (endothe-
lin level before magnesium sulfate, 6.6 ::!: 3.9 pg / ml; after magnesium sulfate, 4.75 ::!: 2.9 pg / ml (p < 0.02). Furthermore, endothelin was elevated in nonlaboring women with preeclampsia before treatment compared with patients with normal pregnancy (p < 0.001). Women with preeclampsia who labored had endothelin concentrations similar to those oflaboring patients with normal pregnancies (Duncan's multiple range test) (Fig. 3). All patients with preeclampsia had hematocrits that were similar to those of patients with normal pregnancies. In non paired samples the umbilical venous concentration of endothelin was 10 times higher than in normal pregnancies and four times higher than in laboring women (p < 0.001) (Fig. 4) . Similar data were found in paired samples from mothers in labo r and umbilical
1714
Mastrogiannis et al.
December 1991 Am J Obslcl Gynecol
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Fig. 3. Effect of magnesium sulfate and labor o n ma terna l venous plasma endothelin-l levels in women with preecla mpsia (unpaired Student t test) .
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Fig. 4. Maternal ve no us a nd umbilical venous endothelin-l levels before and after cord clamping (unpaired Stude nt t test).
vems at delivery (maternal endothelin-l 4.0 ± 3.26 pg / ml, umbilical vem endothelin-l 17.6 ± 10.9 pg/ ml). T he concentration of endothelin was not different before or after cord clamping in paired samples (Fig. 4). Furthermore, no difference in umbilical venous samples from vaginal deliveries (n = 19) compared with cesarean sections (n = 12) was found. Endothelin concentrations were not different in patients delivered by cesarean section and in those delivered vaginally. No correlation between endotheli n values (in the preeclampsia group) and level of maternal blood pressure or clinical severity was observed. Comment
Endothelin-l is the most active vasopressor substance yet discovered, with a potency 10 times that of angiotensin IU Other properties of endothelin include in-
d uction of uterine contractions, release of prostaglandins, and the release of endothelial-derived relaxing factors. 14 · 1fi It is now believed that endothelin-l binds to a specific membrane receptor leading to intracellular biochemical signals involving t he activation of phospholipase C, with a release of inositol phosphates and diacylglycerol and mobilization of intracellular calcium . The res ultant increase in intracellular calcium activates protein kinase C. '4. " Many substa nces, including thrombin, epine phrine, and the calcium ionophore A23 187, cause the slow release of immunoreactive endothelin-l from cultured endothelial cells. 2 In healthy humans plasma levels of endothelin-l measured by radioimmunoassay have been estimated to range from 0.26 to 5 pg I m\. 18 T h e concentration of endothelin-l is likely to be much highe r at the interface between the endothelium and smooth muscle. Therefore endothelin-l is more likely to act as a local autacoid
Volume 165 Number 6, Part 1
rather than as a systemic regulating hormone. 19 Paracrine effects of endothelin are very important, because many pharmacologic actions of endothelin-l are not substantiated naturally by the systemic concentrations found, which are much lower than those required for the actions observed in vitro.18 This is particularly true because the increased concentrations of endothelin-l are not necessarily related to the expected degree of vasoconstriction but are probably secondary effects of the simultaneous production of endothelial-relaxing factors and of vasodilatory prostaglandin release. IS It is also worth noting that the kidneys are 10 times more sensitive than other vascular regions to the vasoconstrictor effects of endothelin-1. 20 The potent vasoconstrictor action of endothelin-l, together with elevated plasma levels found in myocardial infarction, vasculitis, and essential or pulmonary hypertension, contributes to the hypothesis that endothelin-l may be involved in hypertensive states."s Its ability to stimulate the release of aldosterone and catecholamines from adrenal glands may contribute to hypertension, as would its ability to stimulate the release of renin. 20, 21 In laboratory animals the sensitivity of renal artery segments to endothelin-l was greater in rats with spontaneous hypertension than in normal animals. 22 Normal pregnancy state is characterized by generalized refractoriness to vasopressor substances such as angiotensin 11. 10 On the contrary, pregnancy-induced hypertension is characterized by widespread vasoconstriction. 10 The dominant view is that the vasoconstriction results mainly or wholly from an abnormal sensitivity of vascular smooth muscle to the vasoconstrictive effect of pressor substances. This study was concerned with endothelin-l levels found in normal and preeclamptic pregnancies during labor, as well as in umbilical vein blood. Endothelin levels were elevated in women with preeclampsia without labor before any institution of treatment. This finding confirms that of Taylor et al. 12 and Kamoi et al. 13 Furthermore, we showed that magnesium infusion lowers the level of endothelin as compared with the preinfusion level. Surprisingly, no difference in endothelin values was found in laboring patients with preeclampsia compared with laboring patients with normal pregnancies. We cannot explain at this time the high endothelin levels in normal pregnancies in the first stage of labor. It has recently been proposed, however, that endothelin-l and oxytocin modulate calcium through independent receptors, and endothelin, like oxytocin, is an important modulator of uterine contractibility with possible implications in the physiologic characteristics of normallabor.23 The mechanism of endothelin elevation in preeclampsia is unknown, but there are a few possible ex-
Endothelin levels in pregnancy
1715
planations. First, endothelin is basically a locally acting factor at the junction between endothelium and smooth muscle layer; therefore disruption and destruction of these anatomic boundaries can lead to a leak of endothelin from its local environment to the bloodstream with subsequently higher peripheral blood levels. Second, an abnormal production of endothelin by the affected endothelium might be a primary mechanism for the increase of endothelin locally and in the bloodstream. Third, increased production of endothelin from placental or fetal tissue in preeclampsia, or increased diffusion into the maternal circulation, might explain the elevated levels found in preeclampsia. Supporting the last hypothesis is evidence that the endothelin-l receptor was identified in the membranes from a human placenta. 24 Endothelin is found in the amniotic fluid and it is shown to be synthesized not only by endothelial cells derived from human umbilical vein but also by amnion cells. 25 Last, a defect of endothelin clearance could be a mechanism for the increased levels in preeclampsia. A different plasma volume of distribution in preeclampsia versus normal pregnancy would be an important factor. In our study the hematocrits did not differ significantly between the normotensive and preeclamptic groups. However, plasma albumin concentration may be a better indicator of plasma volume, independent of iron stores. Unfortunately, we did not test albumin concentrations in our patients. Pharmacologic concentrations of endothelin-l increase prostacyclin release from endothelial cells in vitro.15, 26 This property may counterbalance its vasoconstrictor effects in vivo and serve as a negative feedback. Given the observation that the prostacyclin levels are reduced, in preeclampsia, then an abnormality of endothelin-prostacyclin interaction could exist. We observed a lowering of endothelin values in patients during magnesium sulfate infusion. Recent in vitro studies have suggested that magnesium increases the prostacyclin production from endothelial cells, an observation that could not be supported in vivo as measured by systemic prostacyclin metabolite excretion in women who received magnesium sulfate infusion. 27 However, an increase in renal prostacyclin production was observed in patients in pre term labor after magnesium sulfate infusion. Endothelin has a major action on the glomerulus and is synthesized by kidney cells, so that a change in the prostacyclin environment in the kidney could play some role in addition to the vasodilatory effect with subsequent release of the stretch stimulus of the vessel caused by magnesium sulfate. A recent study" that also found increased levels of endothelin-l in patients with preeclampsia failed to find a difference in the second-trimester levels of this peptide in women in whom preeclampsia eventually developed. These data support the hypothesis that en-
1716
Mastrogiannis et al.
dothelin elevation in preeclampsia is probably a secondary effect of another mechanism that initiates the change in endothelium that is basic to this disease. The high concentration of endothelin-l in the umbilical venous circulation in our study supports the previous report by Usuki et al. 25 and cannot be adequately explained at this time. 24 2(; Our data do not support a role in the contraction of umbilical vessels after delivery, because we did not find increased levels over a course of time (from before to after cord clamping). However, other investigators have suggested that endothelin-l may contribute to fetal hemodynamic change, e.g. , closure of the umbilical vessels occurring at delivery.28 These investigators collected cord blood after the clamping of the umbilical cord. As mentioned earlier, endothelin was found to be synthesized by endothelial cells derived from umbilical cords and amnion cells,25 suggesting that endothelin might playa significant role in the regulation of uteroplacental circulation . Endothelin was reported to contribute to the closure of the ductus arteriosus in lambs, emphasizing the importance of this peptide in gestation. 29 Further study is needed to clarify this issue. REFERENCES I. Furchgou RF, Vanhoutte PM. Endothelium-derived re-
laxing and contracting factors. FASEB J 1989;3 :2007-18. 2. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascu lar endothelial cells. Nature 1988;332 :411-5. 3. ~ayler W~. End othelin: isoforms, binding sites and posSible Imphcauons m pathology. Trends Pharmacol Sci 1990; 11 :96-9. 4. Wu CC , Bohr DF. Role of endoth elium in the response to endothelin in hypertension. Hypertension 1990; 16:677-81. 5. Saito Y, Nakao K, Mukoyama M, Imura H. In creased plasma endothelin level in patients with essen tial hypertension. N Engl J Med 1990;332:205. 6. Davenport AP, Ashby MJ, Easton P, et al. A sensitive radioimmunoassay measuring endothelin-like immunoreacti~ity in human plas ma: comparison of levels in patients with essential hypertensIOn and normotensive control subjects. Clin Sci 1990;78:261 -4. 7. Kurihara H, Yoshizumi M, Sugiyama T, et al. The possible role of endothelin-l in the pathogenesis of coronary vasospasm . J Cardiovasc Pharmacol 1989; 13(suppl 5):SI32-7. 8. Kanno K, Hirata Y, Num ano F , et al. Endothelin -l and vasculitis. JAMA 1991;264:2868. 9. Koyama H, Nis hzawa Y, Morii H , Tabata T, Inoue T, Yamaji T. Plasma endoth elin levels in patients with uraemia. Lancet 1989;2:992-3. 10. Cu nnin gham FG, MacDonald PC, Gan t NF. Williams' obstetrics. 18th ed. Norwalk, Connecticut: Appleton & Lange, 1989:658-72. II. RobertsJ.M, Taylor RN , Musci 11, et al. Preeclampsia: an endotheli al cell disorder. AM J OBSTET GYI\ECOL 1989; 161: 1200-4.
December 1991
Am.J Obstet GY llecol
12. Taylor RN, Varma M, Teng NNH, Roberts JM. Women with preeclampsia have higher plasma endothelin levels than women with normal pregnancies. J Clin Endocrinol Metab 1990;7 1: 1675-7. 13. Kamoi K, Sudo N, Ishibashi M , Yamaji T. Plasma endothelin-I levels in patients with pregnancy-induced hyperte nsion. N EnglJ Med 1990;323: 1486-7. 14. Masaki T. The discovery, the present state , and the future pros pects of endothelin. .l Cardiovasc Pharmacol 1989;13(suppI5):SI-4. 15. Rae GA, Trybulec M, de Nucci G, Vane JR. EndothelinI releases eicosanoids from rabbit isolated perfused kidney and spleen. J Ca rdiovasc Pharmacol 1989; 13 (suppl 5):589-92. 16. War.ner TD, Mitchell .lA, de Nucci G, Vane JR. Endothelm-l and endothelin-3 release EDRF from isolated perfused artenal vessels of the rat and rabbit. J Cardiovasc Pharmacol 1989; 13(suppl 5):S85-8. . 17. Arai H, Hori S, Aramori I , Ohkubo H, Nakan ishi S. Clonin g and expression of a eDNA encoding an endothelin receptor. Nature 1990;348:730-2. 18. Vane JR, Anggard EE, Botting RM. Regulatory functions of the vascular endothelium. N Engl J Med 1990;323:2736. 19. MacCumber MW, Ross CA, Glaser BM, Snyder SH . Endothelin: visualization of mRNAs by in situ hybridization provides ev idence for local action . Proc Nat! Acad Sci USA 1989;86:7285-9. 20. ~ad r KF, M~rray JJ, Breyer MD, Takahashi K, Inagami r, Harns RC. Mesanglal cell, glomerular and renal vascular responses to endothelin in the rat kidney. J Clin Invest 1989;83:336-42. 21. Cozza EN , Gomez-Sanchez C E, Foecking MF, Chiou S. Endothelin binding to cultured calf ad renal zona glo merulosa cells and stimulation of aldosterone secretion. .I Clin Invest 1989;84 : 1032-5. 22. Watanabe TX, Kumagaye SI, Nishio H, Nakajima K, Kimura T, Sakakibara S. Effects of endoth elin-I and endothelin-3 on blood pressure in conscious hypertensive rats . .J Cardiovasc Pharmacol 1989; 13(suppl 5): S207-8. 23. Maher E, Bardeguez A, Gardner JP, e tal. Endothelin and oxytocin induced calcium signalin g in cultured human myometrial cells. J Clin Invest 1991 ;87: 1251-8 . 24 . ~akajo S: S U,?iura M, Snajdar RM , Boehm FH, In agami r. SolubilizatIOn and Identification of human placental endothehn receptor. Biochem Biophys Res Com mun 1989;164:205-11. 25. Usuk i S, Saitoh T, Sawamura T, et al. Increased maternal plasma concentrations of endothelin-l during labor pain or In d ehvenes and the existence of large amount of endothelin in amniotic fluid . Gy necol Endocrinol 1990 ;4:919. 26. Mitchell MD, Romero R.l, Lepera R, Rittenhouse L, Edwin SS. Actions of endothelin-I on prostaglandin production by gestational tissues. Prostaglandins 1990;40:627-35. 27. O'Brien WF, Williams MC, Benoit R, Sawai SK, Knuppel RA. The effect of magnesium sulfate infusion on systemic and renal prostacydin prod uction . 1990;40:529-38. 28. Nissel N, Hemsen A, Lunell N-O, Wolff K, Lundberg MJ Maternal and fetal levels of a novel polypeptide , endothelin: evide nce for release during pregnancy and delivery. Gynecol Obstet Invest 1990;30: 129-32. 29. Cocea?i F, Armstrong C, Kelsey L. Endothelin is a potent constnc tor of the lamb ductus arteriosus. Ca n J Physiol Pharmacol 1989;67:902-4.
Key words: Endothelin, pregnancy, preeclampsia, labor, fetus The role of the endothelium in modulating cardiovascular homeostasis via regulation of the vascular smooth muscle tone has been recognized recently. Endothelium regulates the reactivity of the smooth muscles through production of both vasodilators and vasoconstrictors. I The endothelins are a group of vasoactive peptides first isolated from bovine aortic endothelial cells and are the most potent vasoconstrictors yet discovered. 2 Three distinct human endothelin genes have been identified. Endothelin-l is the only endothelin made by human endothelial cells. Endothelin-2 is produced in the kidney and endothelin-3 is mainly associated with neural tissue. 3 Endothelin appears to playa role in the pathogenesis of some forms of hypertension"~6 Additionally, endothelin concentration is elevated in patients with acute myocardial infarction or vasculitis and in patients with uremia who are undergoing hemodialysis.7~9 Preeclampsia, a hypertensive disorder unique to pregnancy, is characterized by generalized vasoconstriction that is due, in part, to increased sensitivity of
From the Division of Obstetrics / Maternal-Fetal Medicine, College of Medicine, University of South Florida. Supported in part by a Southern Medical Association Research Grant. Dr. Mastrogiannis is a recipient of a Southern Medical Association Research Grant Award. Presented in part at the Thirty-eighth Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 20-23,
1991.
Reprint requests: Dimitrios Mastrogiannis, MD, PhD, Temple University Hospital, Department of Obstetrics and Gynecology, Room 7-0PD, 3401 N. Broad St., Philadelphia, PA 19140.
6/6/33234
vascular smooth muscle to the effects of vasopressors. IO This is in contrast to normal pregnancy where the vascular bed is less sensitive to vasoconstrictors. Io Several lines of evidence implicate endothelial cell injury as a basic pathogenic mechanism in preeclampsia resulting in impaired synthesis of vasodilators and a possible increase in the production of vasoconstrictors. II Preliminary studies suggested that endothelin concentrations are elevated in women with preeclampsia, compared with controls. 12, 13 Both normal and hypertensive pregnancies are associated with significant changes in vascular smooth muscle tone; therefore the potential role of endothelin in various pregnancy conditions and pregnancy complications was investigated in this study. Material and methods One hundred twenty-eight plasma samples were prospectively collected from volunteers presenting for care at the University of South Florida and the Tampa General Hospital. Eighteen samples were collected from nonpregnant women of reproductive age without any medical or gynecologic complications. Forty-seven samples were collected from women with normal pregnancies at several gestational age intervals (11 samples from 6 to 12 weeks, 19 samples from 16 to 20 weeks, 9 samples from 26 to 33 weeks, and 8 samples from 35 to 42 weeks). Sixteen samples were collected from non laboring patients with preeclampsia before (n = 10) or after (n = 6) the institution of magnesium sulfate infusion. Sixteen samples were collected from patients in labor (eight patients with normal pregnancies and eight with preeclampsia). The collection of blood from hospital1711
1712 Mastrogiannis et al.
December 1991 Am J Obstet Gynecol
Table I. Comparison of normal, hypertensive, and umbilical venous groups (mean ± SD) Normal pregnancies, patients in labor (n = 8)
Age (yr) Gestational age at delivery (wk) Systolic pressure (mm Hg) Diastolic pressure (mm Hg) Hematocrit (%) Platelets (l03/ mm 3) Uric acid (mg/dl) Birth weight (gm) Vaginal delivery (no.) Cesarean section (no.)
21.9 ± 3.4 38.6 ± 4 126.2 ± 14 75 ± 5 34.3 ± 3.7 196 ± 44 4.1 ± 0.26 3135.1 ± 893.4 4 (50%) 4 (50%)
ized women was done on admission to the labor and delivery suite with the patients in the supine position and before any medications and thus reflects the first stage of labor in laboring women. Thirty-one samples were collected from the umbilical vein immediately after delivery in normal term pregnancies, before and after umbilical cord clamping. All patients gave written informed consent as approved by the University of South Florida's Institutional Review Board. Preeclampsia was defined according to the following criteria: (1) nulliparity, (2) blood pressure ~140/90 mm Hg measured on two occasions at least 6 hours apart, (3) proteinuria of ~30 mg/dl (defined as + 1) by dipstick on random catheterized specimen, (4) no known history of hypertension before pregnancy, and (5) resolution of hypertension and proteinuria in the postpartum period. All volunteers with normal pregnancies denied a history of hypertension or other medical complications. Patients with a history of illicit drug use or other medications were not included. Blood pressure measurements were obtained from patients in the sitting position after 15 minutes of rest, with phase IV Korotkoff sound used for the diastolic blood pressure. Obstetric and demographic data were recorded from the medical records. Blood samples were collected from each patient in the sitting position. Plasma was prepared from blood collected into chilled tubes containing 5 mg/ml ethylenediaminetetraacetic acid and 500 U I ml aprotinin. The plasma was separated by centrifugation and the samples were stored at - 80 C until assayed. On the day of the assay, 2 ml of thawed plasma was acidified with an equal volume 0.1 % trifluoroacetic acid and 2N hydrochloric acid (pH 3), extracted over a C18 column, and eluted with 60% acetonitrile in 0.1 % trifluoroacetic acid. Recoveries of 90% for both radiolabeled and unlabeled samples were observed. These extracts were evaporated to dryness under nitrogen and then assayed in duplicate with a specific radioimmunoassay (Peninsula Laboratories, Inc., Belmont, Calif.). Interassay and 0
Umbilical venous group (n = 31)
21.8 39.1 122.2 72.6 36.01 227
± 5.6
± ± ± ± ±
1.2 8.9 7.8 2.8 72
3355 ± 446 19 (61.3%) 12 (38.7%)
Patients with preeclampsia (n = 24)
21.5 ± 4.5 34.4 ± 4.6 160 ± 22 106 ± 13 34.5 ± 5.25 189 ± 68 6.5 ± 1.3 2245 ± 953 16 (66.7%) 8 (33.3%)
intraassay coefficient of variation for endothelin-l was 7%. Endothelin concentrations were corrected for midpoint displacement of each curve. We did not find differences in values from samples processed before or after freezing. Cross-reactivity with endothelin-2 and endothelin-3 was 7%. The person performing the radioimmunoassays was blinded as to the clinical condition of the patient. All data were expressed as mean ± SD and were analyzed by the SAS (Statistical Analysis Systems, Cary, N.C.) statistical package, with the general linear model procedure for unbalanced designs, Duncan's multiple range test, unpaired and paired Student t tests, and X2 tests where appropriate. A probability of p < 0.05 was considered significant. Results
The endothelin-l concentration in normal nonpregnant women of reproductive age (mean age 32.4 ± 4.7 years, range 30 to 39 years) was 1.8 ± 0.6 pg/ml. There was no significant association between the subject'S age and endothelin level concentrations for either nonpregnant or pregnant women and there was no significant difference in mean age between patients with normal pregnancies (28 ± 5 years) and nonpregnant controls. All patients in the antepartum group remained normotensive and were delivered of normal infants at term. In Table I obstetric and demographic data are presented for the normal labor, preeclampsia, and umbilical venous sample groups. Systolic and diastolic blood pressure, urinary protein concentration, and serum uric acid concentration were significantly higher (p < 0.001) in the patients with preeclampsia. Endothelin-l concentration in the nonpregnant and antepartum groups is shown in Fig. 1. We did not observe a difference in endothelin concentrations between pregnant and nonpregnant patients (by SAS General Linear Model procedure for unbalanced designs). A possible effect of gestational
Endothelin levels in pregnancy
Volume 165 Number 6, Part I
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age with increasing endothelin concentrations in the second and early third trimesters did not reach statistical significance (p = 0.06) . As noted in Fig. 2, levels of endothelin-I were significantly higher in laboring women than in nonlaboring women and all patients with preeclampsia had concentrations higher than those in non laboring women with normal pregnancies. The increase noted in women with preeclampsia was similar to that associated with labor (SAS, General Linear Model procedure). Women with preeclampsia (samples taken before magnesium sulfate infusion) had significantly elevated plasma endothelin concentrations when compared with samples obtained during magnesium sulfate infusion in nonpaired samples (p < 0.05) (Fig. 3). Similar results were found when paired samples were used (endothe-
lin level before magnesium sulfate, 6.6 ::!: 3.9 pg / ml; after magnesium sulfate, 4.75 ::!: 2.9 pg / ml (p < 0.02). Furthermore, endothelin was elevated in nonlaboring women with preeclampsia before treatment compared with patients with normal pregnancy (p < 0.001). Women with preeclampsia who labored had endothelin concentrations similar to those oflaboring patients with normal pregnancies (Duncan's multiple range test) (Fig. 3). All patients with preeclampsia had hematocrits that were similar to those of patients with normal pregnancies. In non paired samples the umbilical venous concentration of endothelin was 10 times higher than in normal pregnancies and four times higher than in laboring women (p < 0.001) (Fig. 4) . Similar data were found in paired samples from mothers in labo r and umbilical
1714
Mastrogiannis et al.
December 1991 Am J Obslcl Gynecol
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vems at delivery (maternal endothelin-l 4.0 ± 3.26 pg / ml, umbilical vem endothelin-l 17.6 ± 10.9 pg/ ml). T he concentration of endothelin was not different before or after cord clamping in paired samples (Fig. 4). Furthermore, no difference in umbilical venous samples from vaginal deliveries (n = 19) compared with cesarean sections (n = 12) was found. Endothelin concentrations were not different in patients delivered by cesarean section and in those delivered vaginally. No correlation between endotheli n values (in the preeclampsia group) and level of maternal blood pressure or clinical severity was observed. Comment
Endothelin-l is the most active vasopressor substance yet discovered, with a potency 10 times that of angiotensin IU Other properties of endothelin include in-
d uction of uterine contractions, release of prostaglandins, and the release of endothelial-derived relaxing factors. 14 · 1fi It is now believed that endothelin-l binds to a specific membrane receptor leading to intracellular biochemical signals involving t he activation of phospholipase C, with a release of inositol phosphates and diacylglycerol and mobilization of intracellular calcium . The res ultant increase in intracellular calcium activates protein kinase C. '4. " Many substa nces, including thrombin, epine phrine, and the calcium ionophore A23 187, cause the slow release of immunoreactive endothelin-l from cultured endothelial cells. 2 In healthy humans plasma levels of endothelin-l measured by radioimmunoassay have been estimated to range from 0.26 to 5 pg I m\. 18 T h e concentration of endothelin-l is likely to be much highe r at the interface between the endothelium and smooth muscle. Therefore endothelin-l is more likely to act as a local autacoid
Volume 165 Number 6, Part 1
rather than as a systemic regulating hormone. 19 Paracrine effects of endothelin are very important, because many pharmacologic actions of endothelin-l are not substantiated naturally by the systemic concentrations found, which are much lower than those required for the actions observed in vitro.18 This is particularly true because the increased concentrations of endothelin-l are not necessarily related to the expected degree of vasoconstriction but are probably secondary effects of the simultaneous production of endothelial-relaxing factors and of vasodilatory prostaglandin release. IS It is also worth noting that the kidneys are 10 times more sensitive than other vascular regions to the vasoconstrictor effects of endothelin-1. 20 The potent vasoconstrictor action of endothelin-l, together with elevated plasma levels found in myocardial infarction, vasculitis, and essential or pulmonary hypertension, contributes to the hypothesis that endothelin-l may be involved in hypertensive states."s Its ability to stimulate the release of aldosterone and catecholamines from adrenal glands may contribute to hypertension, as would its ability to stimulate the release of renin. 20, 21 In laboratory animals the sensitivity of renal artery segments to endothelin-l was greater in rats with spontaneous hypertension than in normal animals. 22 Normal pregnancy state is characterized by generalized refractoriness to vasopressor substances such as angiotensin 11. 10 On the contrary, pregnancy-induced hypertension is characterized by widespread vasoconstriction. 10 The dominant view is that the vasoconstriction results mainly or wholly from an abnormal sensitivity of vascular smooth muscle to the vasoconstrictive effect of pressor substances. This study was concerned with endothelin-l levels found in normal and preeclamptic pregnancies during labor, as well as in umbilical vein blood. Endothelin levels were elevated in women with preeclampsia without labor before any institution of treatment. This finding confirms that of Taylor et al. 12 and Kamoi et al. 13 Furthermore, we showed that magnesium infusion lowers the level of endothelin as compared with the preinfusion level. Surprisingly, no difference in endothelin values was found in laboring patients with preeclampsia compared with laboring patients with normal pregnancies. We cannot explain at this time the high endothelin levels in normal pregnancies in the first stage of labor. It has recently been proposed, however, that endothelin-l and oxytocin modulate calcium through independent receptors, and endothelin, like oxytocin, is an important modulator of uterine contractibility with possible implications in the physiologic characteristics of normallabor.23 The mechanism of endothelin elevation in preeclampsia is unknown, but there are a few possible ex-
Endothelin levels in pregnancy
1715
planations. First, endothelin is basically a locally acting factor at the junction between endothelium and smooth muscle layer; therefore disruption and destruction of these anatomic boundaries can lead to a leak of endothelin from its local environment to the bloodstream with subsequently higher peripheral blood levels. Second, an abnormal production of endothelin by the affected endothelium might be a primary mechanism for the increase of endothelin locally and in the bloodstream. Third, increased production of endothelin from placental or fetal tissue in preeclampsia, or increased diffusion into the maternal circulation, might explain the elevated levels found in preeclampsia. Supporting the last hypothesis is evidence that the endothelin-l receptor was identified in the membranes from a human placenta. 24 Endothelin is found in the amniotic fluid and it is shown to be synthesized not only by endothelial cells derived from human umbilical vein but also by amnion cells. 25 Last, a defect of endothelin clearance could be a mechanism for the increased levels in preeclampsia. A different plasma volume of distribution in preeclampsia versus normal pregnancy would be an important factor. In our study the hematocrits did not differ significantly between the normotensive and preeclamptic groups. However, plasma albumin concentration may be a better indicator of plasma volume, independent of iron stores. Unfortunately, we did not test albumin concentrations in our patients. Pharmacologic concentrations of endothelin-l increase prostacyclin release from endothelial cells in vitro.15, 26 This property may counterbalance its vasoconstrictor effects in vivo and serve as a negative feedback. Given the observation that the prostacyclin levels are reduced, in preeclampsia, then an abnormality of endothelin-prostacyclin interaction could exist. We observed a lowering of endothelin values in patients during magnesium sulfate infusion. Recent in vitro studies have suggested that magnesium increases the prostacyclin production from endothelial cells, an observation that could not be supported in vivo as measured by systemic prostacyclin metabolite excretion in women who received magnesium sulfate infusion. 27 However, an increase in renal prostacyclin production was observed in patients in pre term labor after magnesium sulfate infusion. Endothelin has a major action on the glomerulus and is synthesized by kidney cells, so that a change in the prostacyclin environment in the kidney could play some role in addition to the vasodilatory effect with subsequent release of the stretch stimulus of the vessel caused by magnesium sulfate. A recent study" that also found increased levels of endothelin-l in patients with preeclampsia failed to find a difference in the second-trimester levels of this peptide in women in whom preeclampsia eventually developed. These data support the hypothesis that en-
1716
Mastrogiannis et al.
dothelin elevation in preeclampsia is probably a secondary effect of another mechanism that initiates the change in endothelium that is basic to this disease. The high concentration of endothelin-l in the umbilical venous circulation in our study supports the previous report by Usuki et al. 25 and cannot be adequately explained at this time. 24 2(; Our data do not support a role in the contraction of umbilical vessels after delivery, because we did not find increased levels over a course of time (from before to after cord clamping). However, other investigators have suggested that endothelin-l may contribute to fetal hemodynamic change, e.g. , closure of the umbilical vessels occurring at delivery.28 These investigators collected cord blood after the clamping of the umbilical cord. As mentioned earlier, endothelin was found to be synthesized by endothelial cells derived from umbilical cords and amnion cells,25 suggesting that endothelin might playa significant role in the regulation of uteroplacental circulation . Endothelin was reported to contribute to the closure of the ductus arteriosus in lambs, emphasizing the importance of this peptide in gestation. 29 Further study is needed to clarify this issue. REFERENCES I. Furchgou RF, Vanhoutte PM. Endothelium-derived re-
laxing and contracting factors. FASEB J 1989;3 :2007-18. 2. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascu lar endothelial cells. Nature 1988;332 :411-5. 3. ~ayler W~. End othelin: isoforms, binding sites and posSible Imphcauons m pathology. Trends Pharmacol Sci 1990; 11 :96-9. 4. Wu CC , Bohr DF. Role of endoth elium in the response to endothelin in hypertension. Hypertension 1990; 16:677-81. 5. Saito Y, Nakao K, Mukoyama M, Imura H. In creased plasma endothelin level in patients with essen tial hypertension. N Engl J Med 1990;332:205. 6. Davenport AP, Ashby MJ, Easton P, et al. A sensitive radioimmunoassay measuring endothelin-like immunoreacti~ity in human plas ma: comparison of levels in patients with essential hypertensIOn and normotensive control subjects. Clin Sci 1990;78:261 -4. 7. Kurihara H, Yoshizumi M, Sugiyama T, et al. The possible role of endothelin-l in the pathogenesis of coronary vasospasm . J Cardiovasc Pharmacol 1989; 13(suppl 5):SI32-7. 8. Kanno K, Hirata Y, Num ano F , et al. Endothelin -l and vasculitis. JAMA 1991;264:2868. 9. Koyama H, Nis hzawa Y, Morii H , Tabata T, Inoue T, Yamaji T. Plasma endoth elin levels in patients with uraemia. Lancet 1989;2:992-3. 10. Cu nnin gham FG, MacDonald PC, Gan t NF. Williams' obstetrics. 18th ed. Norwalk, Connecticut: Appleton & Lange, 1989:658-72. II. RobertsJ.M, Taylor RN , Musci 11, et al. Preeclampsia: an endotheli al cell disorder. AM J OBSTET GYI\ECOL 1989; 161: 1200-4.
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Am.J Obstet GY llecol
12. Taylor RN, Varma M, Teng NNH, Roberts JM. Women with preeclampsia have higher plasma endothelin levels than women with normal pregnancies. J Clin Endocrinol Metab 1990;7 1: 1675-7. 13. Kamoi K, Sudo N, Ishibashi M , Yamaji T. Plasma endothelin-I levels in patients with pregnancy-induced hyperte nsion. N EnglJ Med 1990;323: 1486-7. 14. Masaki T. The discovery, the present state , and the future pros pects of endothelin. .l Cardiovasc Pharmacol 1989;13(suppI5):SI-4. 15. Rae GA, Trybulec M, de Nucci G, Vane JR. EndothelinI releases eicosanoids from rabbit isolated perfused kidney and spleen. J Ca rdiovasc Pharmacol 1989; 13 (suppl 5):589-92. 16. War.ner TD, Mitchell .lA, de Nucci G, Vane JR. Endothelm-l and endothelin-3 release EDRF from isolated perfused artenal vessels of the rat and rabbit. J Cardiovasc Pharmacol 1989; 13(suppl 5):S85-8. . 17. Arai H, Hori S, Aramori I , Ohkubo H, Nakan ishi S. Clonin g and expression of a eDNA encoding an endothelin receptor. Nature 1990;348:730-2. 18. Vane JR, Anggard EE, Botting RM. Regulatory functions of the vascular endothelium. N Engl J Med 1990;323:2736. 19. MacCumber MW, Ross CA, Glaser BM, Snyder SH . Endothelin: visualization of mRNAs by in situ hybridization provides ev idence for local action . Proc Nat! Acad Sci USA 1989;86:7285-9. 20. ~ad r KF, M~rray JJ, Breyer MD, Takahashi K, Inagami r, Harns RC. Mesanglal cell, glomerular and renal vascular responses to endothelin in the rat kidney. J Clin Invest 1989;83:336-42. 21. Cozza EN , Gomez-Sanchez C E, Foecking MF, Chiou S. Endothelin binding to cultured calf ad renal zona glo merulosa cells and stimulation of aldosterone secretion. .I Clin Invest 1989;84 : 1032-5. 22. Watanabe TX, Kumagaye SI, Nishio H, Nakajima K, Kimura T, Sakakibara S. Effects of endoth elin-I and endothelin-3 on blood pressure in conscious hypertensive rats . .J Cardiovasc Pharmacol 1989; 13(suppl 5): S207-8. 23. Maher E, Bardeguez A, Gardner JP, e tal. Endothelin and oxytocin induced calcium signalin g in cultured human myometrial cells. J Clin Invest 1991 ;87: 1251-8 . 24 . ~akajo S: S U,?iura M, Snajdar RM , Boehm FH, In agami r. SolubilizatIOn and Identification of human placental endothehn receptor. Biochem Biophys Res Com mun 1989;164:205-11. 25. Usuk i S, Saitoh T, Sawamura T, et al. Increased maternal plasma concentrations of endothelin-l during labor pain or In d ehvenes and the existence of large amount of endothelin in amniotic fluid . Gy necol Endocrinol 1990 ;4:919. 26. Mitchell MD, Romero R.l, Lepera R, Rittenhouse L, Edwin SS. Actions of endothelin-I on prostaglandin production by gestational tissues. Prostaglandins 1990;40:627-35. 27. O'Brien WF, Williams MC, Benoit R, Sawai SK, Knuppel RA. The effect of magnesium sulfate infusion on systemic and renal prostacydin prod uction . 1990;40:529-38. 28. Nissel N, Hemsen A, Lunell N-O, Wolff K, Lundberg MJ Maternal and fetal levels of a novel polypeptide , endothelin: evide nce for release during pregnancy and delivery. Gynecol Obstet Invest 1990;30: 129-32. 29. Cocea?i F, Armstrong C, Kelsey L. Endothelin is a potent constnc tor of the lamb ductus arteriosus. Ca n J Physiol Pharmacol 1989;67:902-4.