European Journal of Obstetrics & Gynecology and Reproductive Biology 120 (2005) 33–38 www.elsevier.com/locate/ejogrb
Increased plasma levels of Urotensin-II in preeclampsia–eclampsia: a new mediator in pathogenesis? Ozcan Balat a,*, Fuat Aksoy a, Irfan Kutlar a, Mete Gurol Ugur a, Hakan Iyikosker b, Ayse Balat c, Ru¨ksen Anarat d a
Department of Obstetrics and Gynecology, Faculty of Medicine, Gaziantep University, P.T.T S¸ubesi, P.K: 34, 27310 Gaziantep, Turkey b Department of Obstetrics and Gynecology, State Hospital, Adıyaman, Turkey c Department of Pediatrics, Faculty of Medicine, Gaziantep University, Gaziantep, Turkey d Baskent University, Department of Biochemistry, Adana, Turkey Received 15 October 2003; received in revised form 23 July 2004; accepted 31 July 2004
Abstract Objective: To assess the possible role of human Urotensin-II (hU-II), a vasoactive peptide, in the pathophysiology of preeclampsia–eclampsia prospectively. Study design: Sixty subjects, 30 with a diagnosis of preeclampsia–eclampsia (group I) and 30 control subjects (group II), who had been admitted between January, 2002 and December, 2002, were taken into the study. Patients in group I had an increase in blood pressure after 28th week of gestation, without any history of hypertensive disease and/or preeclampsia or eclampsia. hU-II levels were assessed using a radioimmunoassay method. Results: No statistically significant difference in terms of age, gestational age, gravidity, abortion and parity was detected among groups (P > 0.05). Plasma hU-II levels in the preeclampsia–eclampsia and control groups were 10.11 5.94 pg/mL and 3.93 1.73 pg/mL, respectively. Difference between plasma hU-II levels of the two groups was found to be statistically significant (P < 0.00001). Also there was correlation between hU-II levels and mean arterial pressures in both groups (r = 0.73, P < 0.0001 and r = 0.72, P < 0.0001 for groups I and II, respectively). Conclusion: Results of our study strongly suggest an important role for hU-II in the pathophysiology of preeclampsia–eclampsia. Further studies concerning placenta and cord blood samples will more clearly elucidate the role of Urotensin-II in the pathogenesis of preeclampsia– eclampsia, and its feto-maternal effects. # 2004 Published by Elsevier Ireland Ltd. Keywords: Urotensin-II; Pregnancy; Preeclampsia; Eclampsia
1. Introduction The mechanisms responsible for the pathogenesis of preeclampsia have not yet been fully elucidated. A multifactorial etiology is a plausible explanation for today. Nowadays, endothelial dysfunction is frequently emphasized in the pathogenesis of preeclampsia [1–4]. Recent studies suggest the possible roles of cytokines, lipid peroxides and free oxygen radicals in the formation of endothelial dysfunction as potential mediators [5]. * Corresponding author. Tel.: +90 5374132105; fax: +90 3423357374. E-mail address:
[email protected] (O. Balat). 0301-2115/$ – see front matter # 2004 Published by Elsevier Ireland Ltd. doi:10.1016/j.ejogrb.2004.07.029
Urotensin is a peptide first characterized biologically by Bern and colleagues in 1967 and originally isolated from the urohypophysis of the goby (the urohypophysis being a neurosecretory organ found only in fishes) [6–8]. Human Urotensin II (hU- II) is a newly cloned, cyclic peptide of 11 aminoacids cleaved from a larger preprourotensin II (PP UII) precursor peptide of about 130 amino acids [7]. Messenger RNA encoding PP U-II has been demonstrated to be present in various tissues such as brain, pituitary, heart, kidney, adrenal gland, placenta and colonic mucosa [9]. hUII peptides are rapidly degraded by proteases in plasma. Also, hU-II molecules circulate in a bound form or in a larger precursor molecule, or both, that are not recognized
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by antibodies [10]. Following the development of radioimmunoassays for the detection of hU-II levels of 5 1 fmol/mL have been measured in plasma from healthy individuals. These levels are relatively low, suggesting that hU-II is not predominantly a circulating hormone. They are, however, comparable to normal circulating levels of endothelin-1 (ET-1), which acts principally in a paracrine/ autocrine manner and are consistent with hU-II functioning as an endothelium-derived mediator [8]. This peptide family has specific and important physiologic effects. Several studies revealed that this peptide family is involved in the (patho)physiological control of cardiovascular function. However, this neurohormone may target multiple organ systems and may serve several alternative physiological roles (osmoregulation, metabolic actions, etc.) [8–11]. hU-II is the most potent vasoconstrictor identified to date, nevertheless hU-II has an indirect vasodilator effect in addition to its direct vasoconstrictor effect [6–10]. It has been demonstrated that hU-II has a vasoconstrictor effect on smooth muscle cells of cardiac and arterial blood vessels through a G-protein coupled receptor named GPR14. Some authors suppose hU-II as an arterial selective spasmogen, however vasoconstrictor effects on veins are also reported [8]. The aim of this prospective study is to assess the role of Urotensin II, a vasoactive peptide, in the pathophysiology of disease in preeclamptic and eclamptic pregnants that have increased blood pressures after 28th week of gestation, who have no prior hypertensive disease and/or history of preeclampsia or eclampsia.
2. Material and methods Sixty subjects, 30 with a diagnosis of preeclampsia– eclampsia and 30 under the control group, who had been admitted between January, 2002 and December, 2002, were taken into the study. Patients were separated into two groups. Group I consisted of patients with preeclampsia–eclampsia, and group II (control group) consisted of normal, healthy pregnancies. The selection criteria of patients were: patients with no previous history of preeclampsia and/or eclampsia or a systemic disease like chronic hypertension, diabetes mellitus, chronic renal failure, thyroid function disorder, heart disease, etc., singleton pregnancies with a living fetus, and gestational ages of 28 weeks and above were included in the content of the study. The patients in group I had no history of antihypertensive medication. Detailed histories were obtained from all patients. Gestational age details about the timing when the samples were obtained and gestational age at delivery, and demographic data including age, gravidity, parity and abortion of all patients were recorded. Body mass indices of all patients were calculated. Also systemic physical examinations and pelvic examinations, complete blood
counts, routine urinary and biochemical analysis were all performed. We have separated preeclamptic patients according to their blood pressures and proteinuria levels. Mild preeclampsia is defined as blood pressure greater than 140/90 mmHg, and development of (>300 mg/L) proteinuria noted in clean-catch specimen. Severe preeclampsia is defined as blood pressure greater than 160 mmHg systolic or 110 mmHg diastolic on 2 occasions 6 h apart, and proteinuria exceeding 2 g in a 24 h period or 2–4+ on dipstick testing. Blood pressures of all patients were measured with sphingomanometry (Erka, Germany) after 15 min of bed rest. In order to assess daily protein excretion, 10 mL of midflow spot daily urinary sampling was obtained from each patient. Urinary samples were evaluated with diagnostic multistick method (Dipstick) (Combur 10 Test M, Miditron Junior II, Roche Diagnostics). The values obtained from evaluation of samples were presented as 30 mg/dL for 1(+) results, 100 mg/dL for 2(+) results, and 500 mg/dL for 3(+) results, according to the prospectus of the test. From all patients 10 mL blood samples were obtained and put in sterile vacuum tubes containing EDTA when admitted to the hospital. Tubes were gently shaken for the purpose of anticoagulation, and 0.6 TIU/mL of aprotinin was added to each tube. The tubes were then shaken gently again in order to inhibit proteolytic activity, and centrifuged at a speed of 1600 g/min for 15 min. Resultant plasma samples were kept in deep freeze at a temperature of 80 8C until the day of assessment procedure. Plasma samples were defrosted on the day of assessment, and were evaluated with radioimmunoassay (RIA) method by using an Urotensin-II (human) kit (Phoenix Pharmaceuticals Inc, Cat No: RK-071-05). Results were reported as mean standard deviation. Statistical evaluation of patient data in terms of age, gestational age, number of gravidity, abortion, parity, body mass index, mean arterial pressure and laboratory results was performed using Student’s t-test. Plasma levels of hU-II were statistically analyzed both within and between the groups by using Mann–Whitney U test. P values below 0.05 have been accepted to be significant. Also the correlation of mean arterial pressure to plasma hU-II values in both groups has been analyzed. Data was analyzed with the help of SPSS 9.0 (Statistical Package for Social Sciences) computer analysis program.
3. Results Mean age of the patients in groups I and II were 27.2 4.4 and 28.4 5.1, respectively. The difference between groups was not statistically significant (P > 0.05). Mean gestational age when the samples were obtained from the patients in groups I and II were 35.4 2.3 and 37.5 1.4 weeks, respectively (P > 0.05). Mean gestational age at delivery in groups I and II were 35.9 2.4 and 37.6 1.4
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Table 1 Comparison of the groups according to the age, gravidity, abortion and parity, and body mass index
Age Gravidity Abortion Parity Body mass index
Group I (n = 30)
Group II (n = 30)
P
27.2 4.4 2.7 1.8 0.3 0.6 1.4 1.3 28.7 3.2
28.4 5.1 2.5 1.5 0.3 0.5 1.2 1.1 27.9 3.1
>0.05 >0.05 >0.05 >0.05 >0.05
weeks, respectively. The difference between groups was not statistically significant (P > 0.05). Ten patients in group I and 7 in group II were nullipars, 33.3% and 23%, respectively (P > 0.05). Also, no statistically significant difference in terms of gravidity, abortion and parity, and body mass index was detected among groups (P > 0.05) (Table 1). The mean systolic blood pressures of the patients in groups I and II were 166 19 and 114 21 mmHg, respectively. The difference between groups was statistically significant (P < 0.05). Also the mean diastolic blood pressures of the patients in groups I and II were 111 12 and 72 7 mmHg, respectively. The difference between groups was statistically significant (P < 0.05). Also, in group I there was no statistically significant difference between the mean systolic and diastolic blood pressures between patients with eclampsia and preeclampsia, 168 20 and 165 19, 112 12 and 112 11 mmHg, respectively (P > 0.05). The corrected mean arterial pressure values of groups I and II are 126 12 and 83 7 mmHg, respectively. This difference was statistically significant (P < 0.0001). The urinary protein levels of patients in groups I and II were 346.75 124.19 and 24.57 6.31 mg/dL, respectively. The difference between groups was statistically significant (P < 0.001). Subgroup analysis of group I revealed no significant difference between the urinary protein levels of eclamptic and preeclamptic patients, 348.88 128.20 and 345 124.28 mg/dL, respectively (P > 0.05). Plasma hU-II levels of the patients are shown in Table 2 and Fig. 1. The mean urotensin-II levels of patients in groups I and II were 10.11 5.94 and 3.93 1.73 pg/mL, respectively (P < 0.00001). We have also found significant differences in plasma urotensin-II levels between eclampsia (median 9.50, minimum 6.00 – maximum 34.00), and
The key factor widely cited in preeclampsia is placental ischemia/hypoxia. According to this concept, inadequate trophoblast invasion and remodelling of the uterine spiral arteries during preeclampsia results in a reduction in uteroplacental perfusion [1–5]. This reduction in placental oxygenation is assumed to enhance the synthesis and release of vasoactive factors, which induce widespread injury of maternal vascular endothelium, and results in increased formation of endothelin, thromboxane, superoxide, increased vascular sensitivity to angiotensin II, and
Table 2 Plasma hU-II levels of the patients
Table 3 Plasma hU-II levels of the patients according to the severity of disease in group I
Plasma hU-II level (pg/mL)
Group I, median (minimum– maximum)
Group II, median (minimum– maximum)
P value
8.00 (6.00–34.00)
4.00 (0.20–6.50)
0.05). We have also determined a positive correlation between hU-II levels and mean arterial pressures in both groups (r = 0.73, P < 0.0001 and r = 0.72, P < 0.0001 for groups I and II, respectively) (Figs. 2 and 3).
4. Discussion
Mild preeclampsia Severe preeclampsia Eclampsia
Number of patients (n)
Plasma hU-II levels (pg/mL), median (minimum–maximum)
P value
8 13 9
7.25 (6.00–9.00) 8.00 (6.00–13.50) 9.50 (6.00–34.00)
>0.05
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Fig. 2. Correlation of mean arterial pressure to hU-II in group I.
Fig. 3. Correlation of mean arterial pressure to hU-II in group II.
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decreased formation of vasodilators such as nitric oxide and prostacyclin. These endothelial abnormalities, in turn, cause hypertension by impairing renal function and increasing total peripheral resistance [1–5]. Urotensin-II is a systemic and local vasoactive agent that has direct vasoconstrictor effect in arteries and veins [6–10]. Presence of Urotensin-II and its receptor GPR14 together in various tissues including kidney, and demonstration of its urinary excretion suggests autocrine and paracrine effects of this agent [10]. In healthy individuals, plasma levels of hU-II have been measured, and its presence in the circulation has been shown, although these levels are relatively low. This finding suggests that hU-II is not predominantly a circulating hormone. According to the results of the present studies, peripheral organs are thought to be the source of circulating hU-II [8,10]. A couple of studies have revealed that hU-II has a more potent vasoconstrictor effect than ET-1 on smooth muscle cells of arteries isolated from monkeys [8,12,13]. A study concerning systemic administration of hU-II in anesthetized monkeys demonstrated 300% increase in total peripheral resistance in a dose dependent manner [14]. Besides the direct vasoconstrictor effect of hU-II on smooth muscle cells, hU-II released from the endothelium indirectly contributes to vasodilation by enhancing release of endothelium derived NO (nitric oxide) and prostacyclin [8,12–16]. Inhibition of nitric oxide synthase enzyme by L-NAME and PGI2 synthesis by indometacine contributes to the vasoconstrictor effect of hU-II together with the enhanced vasoconstrictor effect of hU-II in endothelium denuded vessels which in turn leads to hypoxia, and consequent endothelial dysfunction [13]. To date, there are two studies in the literature concerning effects of systemic administration of hU-II in healthy individuals. Affolter et al. [16] observed no change in central and peripheral blood pressures of volunteers who have been given infusions of hU-II to reach up to plasma levels that are 100-fold of normal plasma values in a study designed to research hemodynamic effects of systemic hU-II infusion. They concluded that hU-II had no probable physiologic role in the short term regulation of vascular tone or blood pressure in humans. In a similar study, Wilkinson et al. [17] reported no local or systemic hemodynamic effect of high levels of hU-II in the circulation reached by hU-II infusion. During the trial, the study group has been administered 600 mg aspirin by oral route and infusion of L-NAME for inhibition of prostanoids and NO, and then no hemodynamic change due to hU-II infusion has been observed. No local or systemic change has been observed again, and they reported that hUII does not participate in regulation of vascular tone. Nevertheless, this result can be explained by the presence of ‘healthy’ endothelium that can preserve its normal functions in healthy individuals. In case of endothelial dysfunction,
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increase in hU-II secretion contributes to vasoconstriction together with release of vasodilator agents [8]. The tone of smooth muscles is provided by a mechanism of interaction of vasoactive agents in either an autocrine or paracrine manner. It is obvious that hU-II has an important contribution to this mechanism. In diseases with endothelial dysfunction like preeclampsia, both local and systemic changes in release and effects of vasoactive agents will lead to disequilibrium between these agents, therefore indicating a primary or secondary role in pathogenesis of disease. We could not find any study in the literature concerning Urotensin-II levels in preeclamptic patients. Our study is unique in literature in respect of demonstrating increased plasma levels of Urotensin-II, a vasoconstrictor agent that is 10-fold more potent than ET-1, in patients with preeclampsia–eclampsia. There is widespread endothelial injury in preeclamptic patients as in patients with hypertension. Some local and systemic changes take place as a result of these endothelial injuries. Besides many other contributing factors in the complex pathophysiological mechanism of preeclampsia, vasoactive peptides have been one of the major focuses of concerning studies. This preliminary study was planned to evaluate a possible role of hU-II. However, we cannot propose from the findings of our study that hU-II has a primary role in the pathophysiology of preeclampsia. It may also be a totally secondary change to raise the blood pressure in order to increase the uterine perfusion. Autocrine and paracrine effects of Urotensin-II, its reported high levels at circulation in cardiovascular disorders, and its interaction between vasoactive agents in the pathophysiology of preeclampsia like NO, prostaglandins, ET-1, and adrenomedullin may suggest a possible role of Urotensin-II in the pathogenesis of preeclampsia. Detection of higher maternal plasma hU-II levels in preeclampsia–eclampsia group may support this hypothesis. Consequent studies are needed to explain whether this difference between normotensive and hypertensive groups is a primary or secondary feature. Further studies concerning placenta and cord blood samples will sure more clearly elucidate the role of Urotensin-II in the pathogenesis of preeclampsia, and its maternal and fetal effects.
5. Condensation Increased plasma levels of Urotensin-II may play a role in the pathogenesis of preeclampsia–eclampsia.
Acknowledgement The authors thank Assist. Prof. Dr. Saime Sahinoz MD for her contribution to the statistical analysis of the manuscript.
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