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Angiogenin and vascular endothelial growth factor in pregnancies complicated by preeclampsia
International Journal of Gynecology and Obstetrics (2005) 88, 112 — 117
www.elsevier.com/locate/ijgo
CLINICAL ARTICLE
Angiogenin and vascular endothelial growth factor in pregnancies complicated by preeclampsia M. Shaarawya,*, F. Al-Sokkaryb, M. Shebaa, O. Wahbaa, H.O. Kandila, I. Abdel-Mohsena a
Department of Obstetrics and Gynecology, Faculty of Medicine, Cairo, Egypt Cairo and Al-Azhar Universities, Cairo, Egypt
b
Received 10 June 2004; received in revised form 1 October 2004; accepted 18 October 2004
KEYWORDS Angiogenin; Vascular endothelial growth factor; Preeclampsia
Abstract Objective: To investigate the role of the angiogenic factors, vascular endothelial growth factor (VEGF) and angiogenin in the pathophysiology of preeclampsia and how their concentrations correlate with the severity of the disease and fetal outcome. Patients and Methods: A prospective study was carried out on 71 pregnant patients with preeclampsia and 20 pregnant normotensive controls. Maternal serum levels of VEGF and angiogenin were determined in all cases by enzyme immunoassay. Assessment of fetal well-being using the Biophysical Profile Score (BPS), umbilical and uterine artery Doppler velocimetry, and infant birthweight were carried out. Results: Maternal serum VEGF and angiogenin levels were significantly increased in cases of mild and severe preeclampsia compared to controls. Their increase was positively correlated with elevated systolic and diastolic blood pressure, as well as poor BPS, abnormal Doppler velocimetry, and low birthweight. Conclusion: Elevated levels of both VEGF and angiogenin could confirm the existence of vascular reactivity and endothelial disturbance in preeclampsia. Measurement of these angiogenic factors in maternal serum may be a useful as biomarkers for the assessment of the severity of the disease and of fetal outcome. D 2004 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction * Corresponding author. 21 El-Khalifa El-Maamoun Street, Apt 701, Roxy Building, Heliopolis, Cairo, Egypt. Tel./fax: +20 2 623 2497. E-mail address: [email protected] (M. Shaarawy).
Preeclampsia continues to be one of the leading causes of maternal and perinatal morbidity and mortality. Although the exact pathophysiological process of preeclampsia still remains to be unrav-
0020-7292/$ - see front matter D 2004 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2004.10.005
Angiogenin and vascular endothelial growth factor in pregnancies complicated by preeclampsia elled, several mechanisms, varying from immune maladaptation to gene expression [1], have been proposed. Dysfunction of the vascular endothelium is now thought to be the primary event that explains the variable expression of this disorder. This view is based on morphologic evidence of glomerular endotheliosis and biochemical evidence of endothelial activation or damage including elevated levels of plasminogen activator inhibitor type 1, fibronectin and thrombomodulin [2]. In addition, altered endothelial production of such vasoactive substances as prostacyclin, nitric oxide, and endothelin has been implicated in the abnormal hemodynamics associated with the disorder [3]. This maternal endothelial dysfunction could be mediated by factors present in the maternal circulation as a result of impaired trophoblast invasion [4]. Angiogenic growth factors are thought to play a substantial role in placentation and can be strong endothelial cell activators [5]. It has been speculated that vascular endothelial growth factor (VEGF), by causing endothelial cell alteration, is involved in the natural history of preeclampsia [7,8]. The role of VEGF serum levels in pregnancy and preeclampsia, however, has been discussed controversially. Several studies have shown that VEGF concentrations are increased in the plasma or serum of women with preeclampsia [6—11], while others revealed decreased concentrations [12—14]. On the other hand Hefler et al. [15] reported no significant difference of serum VEGF levels between women with preeclampsia and healthy pregnant women. Hayashi et al. [16] suggested that regulatory mechanisms of angiogenin may exist during pregnancy. Maternal and fetal circulating angiogenin levels did not differ significantly in pregnancies with small-for-gestational age and appropriate-forgestational age infants [17]. Only two studies have shown that serum angiogenin concentration did not change significantly in preeclampsia when compared to that of controls [13,18]. This article shows for the first time how maternal serum angiogenin and VEGF concentrations change parallely in normal pregnancy and in pregnancies complicated by preeclampsia and how their concentrations correlate with maternal blood pressure, biophysical profile, umbilical uterine artery Doppler velocimetry, and fetal outcome in preeclampsia.
2. Patients and methods This study comprised 91 nulliparous pregnant women in the late third trimester (34—40 weeks
113
gestation) who attended the antenatal clinic of Cairo University Hospital between October 2002 and December 2003. They were divided into three groups. The first group included 20 normotensive healthy pregnant women who served as controls. The second group consisted of 55 women with mild preeclampsia. The third group consisted of 16 women with severe preeclampsia. This study was approved by Cairo University Hospital Review Board and informed consent for participation was obtained from each study participant. Preeclampsia was diagnosed clinically on the basis of elevated blood pressure, proteinuria, and edema [19]. Mild and severe preeclampsia were subgrouped according to the criteria of Cunningham et al. [19]. Mild preeclampsia was defined as an absolute systolic blood pressure of at least 140 mm Hg or an increase of at least 30 mm Hg systolic over baseline in the first 20 weeks of pregnancy and proteinuria (V300 mg per 24 h). The criteria for severe preeclampsia were blood pressure of at least 160 mm Hg or at least 100 mm Hg diastolic and proteinuria over 5 g per 24 h, in addition to oliguria (V400 ml in 24 h), cerebral or visual disturbances, epigastric pain, or pulmonary edema. Control women were matched with those with preeclampsia for race, maternal age, and gestational age at delivery and for time of blood sampling. Patients with history of chronic hypertension, diabetes, renal or cardiovascular disease were excluded. All controls delivered vaginally. Four of the 55 mildly preeclamptic women underwent elective cesarean because of cephalopelvic disproportion. Five women with severe preeclampsia had elective cesarean delivery, two because of failure to control the rise of blood pressure and developing signs of impending eclampsia and three because of previous abdominal deliveries. All patients and healthy controls underwent a complete real-time ultrasonographic examination, including confirmation of gestational age, assessment of fetal growth patterns, fetal biophysical profile score (BPS), in addition to umbilical and uterine artery Doppler velocimetry studies. These investigations were done on admission for both controls and patients. Four variables were tested using a real-time B-mode ultrasound scanner for the assessment of BPS. These parameters included fetal breathing movement, fetal movement, fetal tone, and fluid volume. Each variable was scored 2 if normal and 0 if abnormal, and the scores were combined to form the BPS score. A BPS of 6 or less was considered abnormal. A blood sample was taken from each participant before administration of any drug known to affect blood pressure and before any medical or surgical
114 Table 1
M. Shaarawy et al. Clinical characteristics of preeclamptic and normotensive pregnant women
Characteristics
Normotensive n=20
Mild preeclampsia n=55
Severe preeclampsia n=16
Age (year) Body mass index (kg/m2) Hemoglobin level (g/dl) Hematocrit (%) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Gestational age at sampling (weeks) Gestational age at delivery (weeks) Birth weight (kg) Abnormal BPS (b6) Abnormal Doppler velocimetry
24.6F0.7 25.2F0.5 12.4F0.12 36F1.5 115F6 76F4 36.8F1.2 39.8F0.4 3.6F0.16 0 0
26.1F0.8 27.1F1.2 12.2F0.14 37F0.8 145F4* 92F2* 36.2F0.8 39.4F0.5 3.2F0.18 11(20%) 0
25.4F0.9 26.4F0.6 11.8F0.35 35F1.1 170F8* 114F5* 36.5F1.1 37.1F0.6* 2.5F0.22* 9(56%)* 10(62%)
Data are given as meanFstandard error. * Pb0.001.
intervention. Blood samples were obtained on admission by clean venous puncture without stasis. Sera were separated by centrifugation and were kept at 80 8C until time of analyses. Serum VEGF concentration was determined by enzyme immunoassay. Reagent kits (Quantikine human VEGF) were purchased from R&D system (Minneapolis, MN). The intraassay coefficient of variation (three samples of known concentration assayed 20 times on one plate) was 4.1%. The interassay coefficient of variation (three samples of known concentration assayed in 20 separate assays) was 6.4%. The minimum detectable dose of VEGF was 5 pg/ml. Serum angiogenin concentration was determined by enzyme immunoassay. Reagent kits were also purchased from R&D system (Quantikine human angiogenin). The intra- and interassay coefficient of variation were 3.3% and 7.2%, respectively. The minimal detectable dose of angiogenin was 6 pg/ ml. Results were statistically evaluated using unpaired Student’s t-test. Pb0.05 was considered statistically significant.
3. Results Clinical and demographic data from normotensive and control women are summarized in Table 1. As anticipated from definition criteria, systolic and Table 2
diastolic pressures were significantly higher in the preeclamptic groups than in the normotensive pregnant group. Gestational age at delivery and infant birthweight were significantly lower in the severe preeclamptic group compared to the normotensive pregnant women. None of the infant birthweights in the normotensive pregnant were below the 10th percentile for gestational age (small for gestational age); in contrast, 21 of the infants in the preeclamptic groups were small for gestational age. Among severe preeclamptic women, there were four stillbirths. All controls had normal BPS, whereas 11 (20%) of mild preeclamptic and 9 (56%) of severe preeclamptic patients had abnormal BPS. Doppler velocimetry of the umbilical artery in cases of mild preeclampsia was normal as in the control group [systolic/diastolic (S/D) ratio b3, resistance index (RI) b0.58]. In cases of severe preeclampsia, 10 of 16 women had abnormal Doppler velocimetry results (S/D ratio N3.0, RI N0.58). Loss of diastolic flow in the umbilical artery occurred in four cases of severe preeclampsia. In this study, maternal serum VEGF levels in women with mild and severe preeclampsia were significantly higher than that of controls ( pb0.001; Table 2). Maternal serum angiogenin levels in women with mild and severe preeclampsia were also significantly elevated when compared to that of control ( pb0.001). The rise of
Serum angiogenin and VEGF levels in preeclamptic and normotensive pregnant women Number of cases
MeanFS.E.M.
Mean fold rise
P value
VEGF (pg/ml) Normotensive Mild preeclampsia Severe preeclampsia
20 55 16
184F18 396F35 608F111
— 2.15 3.30
b0.001 b0.001
Angiogenin (ng/ml) Normotensive Mild preeclampsia Severe preeclampsia
20 55 16
141F22 478F37 784F122
— 3.39 5.56
— b0.001 b0.001
Angiogenin and vascular endothelial growth factor in pregnancies complicated by preeclampsia
115
Table 4 Correlation between serum angiogenin, VEGF concentrations, and clinical parameters in preeclamptic women
Systolic blood pressure Diastolic blood pressure Biophysical profile Birth weight
Figure 1 Regression line equation and correlation coefficient brQ between serum angiogenin and VEGF.
angiogenin in preeclampsia is much higher than that of VEGF. There was a significant positive correlation between serum VEGF and serum angiogenin concentrations in preeclamptic patients (Fig. 1). Maternal serum VEGF and angiogenin levels in relation to the fetal biophysical profile score (BPS) in preeclamptic patients are depicted in Table 3. Serum VEGF and angiogenin levels in patients with abnormal BPS were significantly higher than the corresponding levels of cases with normal BPS. All preeclamptic women with abnormal BPS had abnormally elevated values of serum VEGF and serum angiogenin concentrations (more than the upper 95% confidence limit for controls; N220 pg/ml for VEGF and N185 ng/ml for angiogenin). There were significant associations between poor biophysical profile score and high VEGF and angiogenin concentration (Table 4). Serum VEGF and angiogenin levels in preeclamptic patients with abnormal umbilical and uterine artery Doppler velocimetry were significantly
Table 3 Serum VEGF and angiogenic levels in relation to biophysical profile score, Doppler velocimetry, and birthweight in preeclamptic patients Serum VEGF level (pg/ml) meanFS.E.
Serum angiogenin level (ng/ml) meanFS.E.
Biophysical profile Score Normal (57) 449F36 Abnormal (14) 956F88 P value b0.0005
352F30 885F86 b0.0005
Doppler velocimetry Normal (61) 451F34 Abnormal (10) 1087F71 P value b0.0005
370F29 952F122 b0.0005
Fetal birthweight Normal (50) Low (21) P value
295F20 859F77 b0.0005
360F24 1002F47 b0.0005
Serum angiogenin concentration
Serum VEGF concentration
r
r
p value
p value
0.32
b0.001
0.44
b0.001
0.34
b0.001
0.44
b0.001
0.28 0.27
b0.01 b0.01
0.30 0.29
b0.01 b0.01
higher than the corresponding levels in cases with normal Doppler velocimetry ( pb0.0005). All preeclamptic women with abnormal Doppler velocimetry had abnormally elevated values of serum VEGF and angiogenin concentrations. Correlation studies between serum concentration of VEGF and angiogenin and severity of preeclampsia as indicated by measurement of systolic and diastolic blood pressure revealed that both angiogenic factors were significantly positively correlated with both systolic and diastolic blood pressure ( pb0.001). Women with preeclampsia who delivered low birthweight infants had significantly higher serum VEGF and angiogenin levels than those who delivered infants with normal birthweight ( pb0.0005). All women who delivered low birthweight infants had serum VEGF and angiogenin values exceeding the corresponding 95% confidence limit of normal cases (Table 3). This study showed significant negative correlation between serum VEGF or serum angiogenin concentrations and infant birthweight in preeclamptic patients at pb0.01 (Table 4).
4. Discussion In this study, maternal serum vascular endothelial growth factor level was significantly higher in preeclamptic patients than that of controls ( pb0.001). These results are in agreement with several studies [6—11]. Baker et al. [6] suggested that VEGF has a role in the endothelial cell activation that occurs in the disease. Sharkey et al. [8] reported that VEGF may be involved in the maternal endothelial cell dysfunction associated with preeclampsia. An increase of VEGF, a potent regulator of microvascular permeability, may also contribute to the extravasation of plasma proteins and the subsequent development of proteinuria, a characteristic feature of preeclampsia. Furthermore, Bosio et al. [10] concluded that raised
116 plasma VEGF levels precede the clinical onset of preeclampsia and subsequently show a positive association with elevated peripheral resistance. It appears paradoxic that VEGF concentration should be increased in preeclampsia when endotheliumdependent dilatation is reduced [20]. It may be related to endothelial cell damage associated with the condition that has been shown to increase VEGF production, possible as a mechanism of endothelial repair [21]. One possibility is that VEGF originates from the placenta and its production is up-regulated in response to local ischemia. Hypoxia is known to up-regulate VEGF production, and areas of infarction are a feature of the placenta in preeclampsia [22]. VEGF induces matrix metalloproteinase production in endothelial cells which leads to endothelin cleavage to yield a potent vasoconstrictor [23]. The reduction of the vasodilator nitric oxide (NO) in preeclampsia may in part lead to vasospasm which produces hypoxia. The latter may be a cause of elevated VEGF in preeclampsia [24]. The hyperdynamic circulation that characterizes the latent phase of preeclampsia causes vascular shear stress, which in turn increases VEGF levels. Because VEGF normally acts as vasodilator, its increase may represent an unsuccessful vascular rescue response [10]. On the other hand, our results are contradictory to several studies [12—14], which demonstrated significant decrease of serum VEGF levels in preeclampsia. Levine et al. [25] reported that the antiangiogenic protein, total soluble fms-like tyrosine kinase 1 (sFlt-1), is significantly elevated in serum of women with preeclampsia. The increase of sFlt-1 is associated with a significant decrease of serum-free VEGF. They reported that sFLt-1 binds free placental growth favor (PIGF) and VEGF and might play in the pathogenesis of the disease. Moreover, Hefler et al. [15] reported that serum VEGF level did not significantly differ between women with preeclampsia and healthy pregnant women. At least four transcripts encoding mature monomeric VEGF containing 121,165,189, and 206 amino acid residues (VEGF121, VEGF165, VEGF189, VEGF206), each preceded by a 26-amino acid residue signal peptide, have been detected. VEGF121 and VEGF165 are diffusible proteins that are secreted into the medium. VEGF189 and VEGF206 have high affinity for heparin and mostly bound to heparin— containing proteoglycans in the extracellular matrix. The quantitative VEGF immunoassay used in this study is designed to measure both VEGF121 and VEGF165, the diffusible isoforms of VEGF. This methodological specificity may explain why several authors could not detect the rise of serum VEGF in preeclampsia.
M. Shaarawy et al. In this study, VEGF level could differentiate between preeclampsia and controls at a cutoff value of 312 pg/ml with a sensitivity of 94% and a specificity of 91%. Moreover, at cutoff value of 396 pg/ml, VEGF could discriminate mild from severe preeclampsia with a sensitivity of 74% and specificity of 82%. Maternal serum angiogenin levels in women with mild and severe preeclampsia were found to be significantly elevated when compared to that of controls ( pb0.001). A cutoff value of serum angiogenin of 478 ng/ml is clinically useful for differentiating preeclamptic patients from healthy controls with a sensitivity of 92% and a specificity of 88%. Moreover, a cutoff value of 784 ng/ml can discriminate mild from severe preeclampsia with a sensitivity of 76% and a specificity of 81%. However, two previous studies could not detect any significant changes of serum angiogenin between preeclamptic and normal pregnant women [14,19]. The present data show a significant positive correlation between serum VEGF and serum angiogenin concentrations in preeclamptic patients. Both angiogenic factors, angiogenin and VEGF, were significantly correlated with both systolic and diastolic blood pressure ( pb0.001). All preeclamptic patients with abnormal BPS or abnormal uterine artery Doppler velocimetry had abnormally elevated values of serum VEGF and angiogenin concentrations. Preeclamptic patients who delivered low birthweight infants had significantly higher serum VEGF and angiogenin levels than those who delivered infants with normal birthweight (Table 3). There was a significant negative correlation between serum VEGF or serum angiogenin concentration and infant birthweight in preeclamptic patients ( pb0.01). During normal pregnancy, serum VEGF has been found positivity correlated with birthweight in both the human and pig [26]. In placental and related chorioallantoic tissues, VEGF is up-regulated by hypoxia and down-regulated by hyperoxia [27]. In preeclampsia and intrauterine growth restriction (IUGR), fetoplacental blood flow is severely impaired, and transplacental gas exchange is poor, placing the fetus at risk of hypoxia and acidosis [12]. While several reports [8,28] indicated that plasma VEGF concentrations were significantly higher in women with preeclampsia delivering low birthweight infants, Tse et al. [29] reported no significant difference in the concentration and distribution of VEGF between placentae of normal and growth-retarded infants. Our results confirm the hypothesis of increased VEGF expression induced by placental hypoxia, which lead to an increase of fetoplacental flow impedance in preeclampsia as a leading cause of
Angiogenin and vascular endothelial growth factor in pregnancies complicated by preeclampsia IUGR or fetal stress at time of delivery. This study demonstrates a significant negative correlation between serum angiogenin and birthweight in preeclamptic patients. Several authors [13,18,30] reported significant differences between serum levels of angiogenin in severe preeclampsia and controls for fetal birthweight. In conclusion, elevated levels of both angiogenic factors, VEGF and angiogenin, could confirm the existence of vascular reactivity and endothelial disturbance in preeclampsia. Measurement of these angiogenic factors in maternal serum may be useful as biomarkers for the assessment of the severity of the disease and fetal outcome.
References [1] Lie RT, Rasmussen S, Burnborg H, Gjessing HK, Lie-Nielsen E, Igrens LM. Fetal and maternal contributions to risk of preeclampsia: population based study. Br Med J 1998;316: 1343 — 7. [2] Shaarawy M, El-Didy H. Thrombomodulin, plasminogen activator inhibitor type 1 and fibronectin as biomarkers of endothelial damage in preeclampsia and eclampsia. Int J Obstet Gynecol 1996;55:135 — 9. [3] Kraayenbrink AA, Decker GA, Van Kamp GJ, van Geijn HP. Endothelial vasoactive mediators in preeclampsia. Am J Obstet Gynecol 1993;169:160 — 5. [4] Pijnenborg R, Anthony J, Davey DA, Tiltman A, Vercruysse L, van Assche L. Placental bed spiral arteries in the hypertensive disorders of pregnancy. Br J Obstet Gynaecol 1991;98:648 — 55. [5] Vuoral P, Hatva E, Lymboussaki A, Kaipainen A, Joukov V, Persico MG, et al. Expression of vascular endothelial growth factor and placenta growth factor in human placenta. Biol Reprod 1997;56:489 — 94. [6] Baker PN, Krasnow J, Roberts JM, Yeo KT. Elevated serum levels of vascular endothelial growth factor in patients with preeclampsia. Obstet Gynecol 1995;86:815 — 21. [7] Hayman R, Brockelsby J, Kenny L, Baker P. Preeclampsia: the endothelium, circulating factor(s) and vascular endothelial growth factor. J Soc Gynecol Investig 1999; 6:3 — 10. [8] Sharkey AM, Cooper JC, Balmforth JR, McLaren J, Clark DE, Charnock-Jones DS, et al. Maternal plasma levels of vascular endothelial growth factor in normotensive pregnancies and in pregnancies complicated by preeclampsia. Eur J Clin Invest 1996;26:1182 — 5. [9] Hunter A, Aitkenhead M, Caldwell C, McCracken G, Wilson D, MiClure N. Serum levels of vascular endothelial growth factor in preeclamptic and normotensive pregnancy. Hypertension 2000;36(6):965 — 9. [10] Bosio PM, Wheeler T, Anthony F, Conroy R, O’herlihy C, McKenna P. Maternal plasma vascular endothelial growth factor concentrations in normal and hypertensive pregnancies and their relationship to peripheral vascular resistance. Am J Obstet Gynecol 2001;184(2):146 — 52. [11] Helske S, Vuorela P, Carpen O, Hornig C, Weich H, Halmesmaki E. Expression of vascular endothelial growth factor receptors 1, 2 and 3 in placentas from normal and complicated pregnancies. Mol Hum Reprod 2001;7(2):205 — 10.
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[12] Lyall F, Greer IA, Boswell F, Fleming R. Suppression of serum vascular endothelial growth factor immunoreactivity in normal pregnancy and preeclampsia. Br J Obstet Gynecol 1997;104:223 — 8. [13] Reuvekamp A, Velsing-Aarts FV, Poulina IE, Capello JJ, Duits AJ. Selective deficit of angiogenic growth factor characterises pregnancies complicated by preeclampsia. Br J Obstet Gynecol 1999;106(10):1019 — 22. [14] Livingston JC, Chin R, Haddad B, McKinney ET, Ahokas R, Sibai BM. Reductions of vascular endothelial growth factor and placental growth factor concentrations in severe preeclampsia. Am J Obstet Gynecol 2000;183(6):1554 — 7. [15] Hefler L, Obermair A, Husslein P, Kainz C, Tempfer C. Vascular endothelial growth factor serum levels in pregnancy and preeclampsia. Acta Obstet Gynecol Scand 2000;79:77 — 8. [16] Hayashi K, Yanagihara T, Hata T. Serum angiogenin levels during menstrual cycle and pregnancy. Gynecol Obstet Invest 2000;50(1):7 — 12. [17] Yamashiro C, Ohnishi Y, Kanenishi K, Hayashi K, Yanagihara T, Hata T. Maternal and fetal circulating angiogenin levels in pregnancies with small for gestational age and appropriate for gestational age infants. Gynecol Obstet Invest 2000; 49(3):156 — 9. [18] Kolben M, Bla ¨ser J, Ulm K, Schmitt M, Schneider KT, Tschesche H, et al. Angiogenin plasma levels during pregnancy. Am J Obstet Gynecol 1997;176:37 — 41. [19] Cunningham FG, McDonald PC, Gant NF, Leveno KS, Gilstrap J, editors. Appleton and Lang. Norwalk, Connecticut7 William’s Obstetrics; 1993. p. 763 — 818. [20] Knock G, Poston L. Bradykinin-mediated relaxation of isolated maternal resistance arteries in normal pregnancy and preeclampsia. Am J Obstet Gynecol 1996;765:1662 — 74. [21] Tsurumi Y, Murohara T, Krasinsk K, Chen D, Witzenbichler B, Kearney M, et al. Reciprocal relation between VEGF and NO in the regulation of endothelial integrity. Nat Med 1997; 3:879 — 86. [22] Taylor C, Stevens H, Anthony F. Influence of hypoxia on VEGF and chorionic gonadotrophin in the trophoblast-derived cell lines, JEG, JAR and BeWo. Placenta 1997;18:541 — 8. [23] Fenandez-Patron C, Radomski M, Davidge S. Vascular matrix metalloproteinase-2 cleaves big endothelin-1 yielding a novel vasoconstrictor. Crit Res 1999;85:906 — 11. [24] El-Salahy E, Ahmed M, El-Garieb A, Tawfik H. New scope in angiogenesis: role of VEGF, NO, lipid peroxidation, and vitamin E in the pathophysiology of preeclampsia among Egyptian females. Clin Biochem 2001;34:323 — 9. [25] Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, et al. Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 2004;350:672 — 83. [26] Vonnahme K, Ford S. Increases in VEGF during the second trimester of gestation are correlated with calf birth weight in beef heifers. Reprod Physiol 2002;66:69 — 74. [27] Shweiki D, Itin A, Soffer D, Keshet E. VEGF induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992;359:843 — 5. [28] Brockelsby J, Antony F, Johnsin IR, Baker P. The effect of VEGF on endothelial cells: a potential role in preeclampsia. Am J Obstet Gynecol 2000;82:176 — 83. [29] Tse J, Lao T, Chan C. Expression of VEGF in third trimester placentae is not increased in growth-restricted fetuses. J Soc Gynecol Investig 2001;8:77 — 82. [30] Yamashiro C, Ohnishi Y, Kanenish K, Hayashik K, Hata T. Maternal and fetal circulating angiogenin levels in pregnancies with small-for-gestational age and appropriate for gestational age infants. Gynecol Obstet Invest 2000;49: 156 — 64.
www.elsevier.com/locate/ijgo
CLINICAL ARTICLE
Angiogenin and vascular endothelial growth factor in pregnancies complicated by preeclampsia M. Shaarawya,*, F. Al-Sokkaryb, M. Shebaa, O. Wahbaa, H.O. Kandila, I. Abdel-Mohsena a
Department of Obstetrics and Gynecology, Faculty of Medicine, Cairo, Egypt Cairo and Al-Azhar Universities, Cairo, Egypt
b
Received 10 June 2004; received in revised form 1 October 2004; accepted 18 October 2004
KEYWORDS Angiogenin; Vascular endothelial growth factor; Preeclampsia
Abstract Objective: To investigate the role of the angiogenic factors, vascular endothelial growth factor (VEGF) and angiogenin in the pathophysiology of preeclampsia and how their concentrations correlate with the severity of the disease and fetal outcome. Patients and Methods: A prospective study was carried out on 71 pregnant patients with preeclampsia and 20 pregnant normotensive controls. Maternal serum levels of VEGF and angiogenin were determined in all cases by enzyme immunoassay. Assessment of fetal well-being using the Biophysical Profile Score (BPS), umbilical and uterine artery Doppler velocimetry, and infant birthweight were carried out. Results: Maternal serum VEGF and angiogenin levels were significantly increased in cases of mild and severe preeclampsia compared to controls. Their increase was positively correlated with elevated systolic and diastolic blood pressure, as well as poor BPS, abnormal Doppler velocimetry, and low birthweight. Conclusion: Elevated levels of both VEGF and angiogenin could confirm the existence of vascular reactivity and endothelial disturbance in preeclampsia. Measurement of these angiogenic factors in maternal serum may be a useful as biomarkers for the assessment of the severity of the disease and of fetal outcome. D 2004 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction * Corresponding author. 21 El-Khalifa El-Maamoun Street, Apt 701, Roxy Building, Heliopolis, Cairo, Egypt. Tel./fax: +20 2 623 2497. E-mail address: [email protected] (M. Shaarawy).
Preeclampsia continues to be one of the leading causes of maternal and perinatal morbidity and mortality. Although the exact pathophysiological process of preeclampsia still remains to be unrav-
0020-7292/$ - see front matter D 2004 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2004.10.005
Angiogenin and vascular endothelial growth factor in pregnancies complicated by preeclampsia elled, several mechanisms, varying from immune maladaptation to gene expression [1], have been proposed. Dysfunction of the vascular endothelium is now thought to be the primary event that explains the variable expression of this disorder. This view is based on morphologic evidence of glomerular endotheliosis and biochemical evidence of endothelial activation or damage including elevated levels of plasminogen activator inhibitor type 1, fibronectin and thrombomodulin [2]. In addition, altered endothelial production of such vasoactive substances as prostacyclin, nitric oxide, and endothelin has been implicated in the abnormal hemodynamics associated with the disorder [3]. This maternal endothelial dysfunction could be mediated by factors present in the maternal circulation as a result of impaired trophoblast invasion [4]. Angiogenic growth factors are thought to play a substantial role in placentation and can be strong endothelial cell activators [5]. It has been speculated that vascular endothelial growth factor (VEGF), by causing endothelial cell alteration, is involved in the natural history of preeclampsia [7,8]. The role of VEGF serum levels in pregnancy and preeclampsia, however, has been discussed controversially. Several studies have shown that VEGF concentrations are increased in the plasma or serum of women with preeclampsia [6—11], while others revealed decreased concentrations [12—14]. On the other hand Hefler et al. [15] reported no significant difference of serum VEGF levels between women with preeclampsia and healthy pregnant women. Hayashi et al. [16] suggested that regulatory mechanisms of angiogenin may exist during pregnancy. Maternal and fetal circulating angiogenin levels did not differ significantly in pregnancies with small-for-gestational age and appropriate-forgestational age infants [17]. Only two studies have shown that serum angiogenin concentration did not change significantly in preeclampsia when compared to that of controls [13,18]. This article shows for the first time how maternal serum angiogenin and VEGF concentrations change parallely in normal pregnancy and in pregnancies complicated by preeclampsia and how their concentrations correlate with maternal blood pressure, biophysical profile, umbilical uterine artery Doppler velocimetry, and fetal outcome in preeclampsia.
2. Patients and methods This study comprised 91 nulliparous pregnant women in the late third trimester (34—40 weeks
113
gestation) who attended the antenatal clinic of Cairo University Hospital between October 2002 and December 2003. They were divided into three groups. The first group included 20 normotensive healthy pregnant women who served as controls. The second group consisted of 55 women with mild preeclampsia. The third group consisted of 16 women with severe preeclampsia. This study was approved by Cairo University Hospital Review Board and informed consent for participation was obtained from each study participant. Preeclampsia was diagnosed clinically on the basis of elevated blood pressure, proteinuria, and edema [19]. Mild and severe preeclampsia were subgrouped according to the criteria of Cunningham et al. [19]. Mild preeclampsia was defined as an absolute systolic blood pressure of at least 140 mm Hg or an increase of at least 30 mm Hg systolic over baseline in the first 20 weeks of pregnancy and proteinuria (V300 mg per 24 h). The criteria for severe preeclampsia were blood pressure of at least 160 mm Hg or at least 100 mm Hg diastolic and proteinuria over 5 g per 24 h, in addition to oliguria (V400 ml in 24 h), cerebral or visual disturbances, epigastric pain, or pulmonary edema. Control women were matched with those with preeclampsia for race, maternal age, and gestational age at delivery and for time of blood sampling. Patients with history of chronic hypertension, diabetes, renal or cardiovascular disease were excluded. All controls delivered vaginally. Four of the 55 mildly preeclamptic women underwent elective cesarean because of cephalopelvic disproportion. Five women with severe preeclampsia had elective cesarean delivery, two because of failure to control the rise of blood pressure and developing signs of impending eclampsia and three because of previous abdominal deliveries. All patients and healthy controls underwent a complete real-time ultrasonographic examination, including confirmation of gestational age, assessment of fetal growth patterns, fetal biophysical profile score (BPS), in addition to umbilical and uterine artery Doppler velocimetry studies. These investigations were done on admission for both controls and patients. Four variables were tested using a real-time B-mode ultrasound scanner for the assessment of BPS. These parameters included fetal breathing movement, fetal movement, fetal tone, and fluid volume. Each variable was scored 2 if normal and 0 if abnormal, and the scores were combined to form the BPS score. A BPS of 6 or less was considered abnormal. A blood sample was taken from each participant before administration of any drug known to affect blood pressure and before any medical or surgical
114 Table 1
M. Shaarawy et al. Clinical characteristics of preeclamptic and normotensive pregnant women
Characteristics
Normotensive n=20
Mild preeclampsia n=55
Severe preeclampsia n=16
Age (year) Body mass index (kg/m2) Hemoglobin level (g/dl) Hematocrit (%) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Gestational age at sampling (weeks) Gestational age at delivery (weeks) Birth weight (kg) Abnormal BPS (b6) Abnormal Doppler velocimetry
24.6F0.7 25.2F0.5 12.4F0.12 36F1.5 115F6 76F4 36.8F1.2 39.8F0.4 3.6F0.16 0 0
26.1F0.8 27.1F1.2 12.2F0.14 37F0.8 145F4* 92F2* 36.2F0.8 39.4F0.5 3.2F0.18 11(20%) 0
25.4F0.9 26.4F0.6 11.8F0.35 35F1.1 170F8* 114F5* 36.5F1.1 37.1F0.6* 2.5F0.22* 9(56%)* 10(62%)
Data are given as meanFstandard error. * Pb0.001.
intervention. Blood samples were obtained on admission by clean venous puncture without stasis. Sera were separated by centrifugation and were kept at 80 8C until time of analyses. Serum VEGF concentration was determined by enzyme immunoassay. Reagent kits (Quantikine human VEGF) were purchased from R&D system (Minneapolis, MN). The intraassay coefficient of variation (three samples of known concentration assayed 20 times on one plate) was 4.1%. The interassay coefficient of variation (three samples of known concentration assayed in 20 separate assays) was 6.4%. The minimum detectable dose of VEGF was 5 pg/ml. Serum angiogenin concentration was determined by enzyme immunoassay. Reagent kits were also purchased from R&D system (Quantikine human angiogenin). The intra- and interassay coefficient of variation were 3.3% and 7.2%, respectively. The minimal detectable dose of angiogenin was 6 pg/ ml. Results were statistically evaluated using unpaired Student’s t-test. Pb0.05 was considered statistically significant.
3. Results Clinical and demographic data from normotensive and control women are summarized in Table 1. As anticipated from definition criteria, systolic and Table 2
diastolic pressures were significantly higher in the preeclamptic groups than in the normotensive pregnant group. Gestational age at delivery and infant birthweight were significantly lower in the severe preeclamptic group compared to the normotensive pregnant women. None of the infant birthweights in the normotensive pregnant were below the 10th percentile for gestational age (small for gestational age); in contrast, 21 of the infants in the preeclamptic groups were small for gestational age. Among severe preeclamptic women, there were four stillbirths. All controls had normal BPS, whereas 11 (20%) of mild preeclamptic and 9 (56%) of severe preeclamptic patients had abnormal BPS. Doppler velocimetry of the umbilical artery in cases of mild preeclampsia was normal as in the control group [systolic/diastolic (S/D) ratio b3, resistance index (RI) b0.58]. In cases of severe preeclampsia, 10 of 16 women had abnormal Doppler velocimetry results (S/D ratio N3.0, RI N0.58). Loss of diastolic flow in the umbilical artery occurred in four cases of severe preeclampsia. In this study, maternal serum VEGF levels in women with mild and severe preeclampsia were significantly higher than that of controls ( pb0.001; Table 2). Maternal serum angiogenin levels in women with mild and severe preeclampsia were also significantly elevated when compared to that of control ( pb0.001). The rise of
Serum angiogenin and VEGF levels in preeclamptic and normotensive pregnant women Number of cases
MeanFS.E.M.
Mean fold rise
P value
VEGF (pg/ml) Normotensive Mild preeclampsia Severe preeclampsia
20 55 16
184F18 396F35 608F111
— 2.15 3.30
b0.001 b0.001
Angiogenin (ng/ml) Normotensive Mild preeclampsia Severe preeclampsia
20 55 16
141F22 478F37 784F122
— 3.39 5.56
— b0.001 b0.001
Angiogenin and vascular endothelial growth factor in pregnancies complicated by preeclampsia
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Table 4 Correlation between serum angiogenin, VEGF concentrations, and clinical parameters in preeclamptic women
Systolic blood pressure Diastolic blood pressure Biophysical profile Birth weight
Figure 1 Regression line equation and correlation coefficient brQ between serum angiogenin and VEGF.
angiogenin in preeclampsia is much higher than that of VEGF. There was a significant positive correlation between serum VEGF and serum angiogenin concentrations in preeclamptic patients (Fig. 1). Maternal serum VEGF and angiogenin levels in relation to the fetal biophysical profile score (BPS) in preeclamptic patients are depicted in Table 3. Serum VEGF and angiogenin levels in patients with abnormal BPS were significantly higher than the corresponding levels of cases with normal BPS. All preeclamptic women with abnormal BPS had abnormally elevated values of serum VEGF and serum angiogenin concentrations (more than the upper 95% confidence limit for controls; N220 pg/ml for VEGF and N185 ng/ml for angiogenin). There were significant associations between poor biophysical profile score and high VEGF and angiogenin concentration (Table 4). Serum VEGF and angiogenin levels in preeclamptic patients with abnormal umbilical and uterine artery Doppler velocimetry were significantly
Table 3 Serum VEGF and angiogenic levels in relation to biophysical profile score, Doppler velocimetry, and birthweight in preeclamptic patients Serum VEGF level (pg/ml) meanFS.E.
Serum angiogenin level (ng/ml) meanFS.E.
Biophysical profile Score Normal (57) 449F36 Abnormal (14) 956F88 P value b0.0005
352F30 885F86 b0.0005
Doppler velocimetry Normal (61) 451F34 Abnormal (10) 1087F71 P value b0.0005
370F29 952F122 b0.0005
Fetal birthweight Normal (50) Low (21) P value
295F20 859F77 b0.0005
360F24 1002F47 b0.0005
Serum angiogenin concentration
Serum VEGF concentration
r
r
p value
p value
0.32
b0.001
0.44
b0.001
0.34
b0.001
0.44
b0.001
0.28 0.27
b0.01 b0.01
0.30 0.29
b0.01 b0.01
higher than the corresponding levels in cases with normal Doppler velocimetry ( pb0.0005). All preeclamptic women with abnormal Doppler velocimetry had abnormally elevated values of serum VEGF and angiogenin concentrations. Correlation studies between serum concentration of VEGF and angiogenin and severity of preeclampsia as indicated by measurement of systolic and diastolic blood pressure revealed that both angiogenic factors were significantly positively correlated with both systolic and diastolic blood pressure ( pb0.001). Women with preeclampsia who delivered low birthweight infants had significantly higher serum VEGF and angiogenin levels than those who delivered infants with normal birthweight ( pb0.0005). All women who delivered low birthweight infants had serum VEGF and angiogenin values exceeding the corresponding 95% confidence limit of normal cases (Table 3). This study showed significant negative correlation between serum VEGF or serum angiogenin concentrations and infant birthweight in preeclamptic patients at pb0.01 (Table 4).
4. Discussion In this study, maternal serum vascular endothelial growth factor level was significantly higher in preeclamptic patients than that of controls ( pb0.001). These results are in agreement with several studies [6—11]. Baker et al. [6] suggested that VEGF has a role in the endothelial cell activation that occurs in the disease. Sharkey et al. [8] reported that VEGF may be involved in the maternal endothelial cell dysfunction associated with preeclampsia. An increase of VEGF, a potent regulator of microvascular permeability, may also contribute to the extravasation of plasma proteins and the subsequent development of proteinuria, a characteristic feature of preeclampsia. Furthermore, Bosio et al. [10] concluded that raised
116 plasma VEGF levels precede the clinical onset of preeclampsia and subsequently show a positive association with elevated peripheral resistance. It appears paradoxic that VEGF concentration should be increased in preeclampsia when endotheliumdependent dilatation is reduced [20]. It may be related to endothelial cell damage associated with the condition that has been shown to increase VEGF production, possible as a mechanism of endothelial repair [21]. One possibility is that VEGF originates from the placenta and its production is up-regulated in response to local ischemia. Hypoxia is known to up-regulate VEGF production, and areas of infarction are a feature of the placenta in preeclampsia [22]. VEGF induces matrix metalloproteinase production in endothelial cells which leads to endothelin cleavage to yield a potent vasoconstrictor [23]. The reduction of the vasodilator nitric oxide (NO) in preeclampsia may in part lead to vasospasm which produces hypoxia. The latter may be a cause of elevated VEGF in preeclampsia [24]. The hyperdynamic circulation that characterizes the latent phase of preeclampsia causes vascular shear stress, which in turn increases VEGF levels. Because VEGF normally acts as vasodilator, its increase may represent an unsuccessful vascular rescue response [10]. On the other hand, our results are contradictory to several studies [12—14], which demonstrated significant decrease of serum VEGF levels in preeclampsia. Levine et al. [25] reported that the antiangiogenic protein, total soluble fms-like tyrosine kinase 1 (sFlt-1), is significantly elevated in serum of women with preeclampsia. The increase of sFlt-1 is associated with a significant decrease of serum-free VEGF. They reported that sFLt-1 binds free placental growth favor (PIGF) and VEGF and might play in the pathogenesis of the disease. Moreover, Hefler et al. [15] reported that serum VEGF level did not significantly differ between women with preeclampsia and healthy pregnant women. At least four transcripts encoding mature monomeric VEGF containing 121,165,189, and 206 amino acid residues (VEGF121, VEGF165, VEGF189, VEGF206), each preceded by a 26-amino acid residue signal peptide, have been detected. VEGF121 and VEGF165 are diffusible proteins that are secreted into the medium. VEGF189 and VEGF206 have high affinity for heparin and mostly bound to heparin— containing proteoglycans in the extracellular matrix. The quantitative VEGF immunoassay used in this study is designed to measure both VEGF121 and VEGF165, the diffusible isoforms of VEGF. This methodological specificity may explain why several authors could not detect the rise of serum VEGF in preeclampsia.
M. Shaarawy et al. In this study, VEGF level could differentiate between preeclampsia and controls at a cutoff value of 312 pg/ml with a sensitivity of 94% and a specificity of 91%. Moreover, at cutoff value of 396 pg/ml, VEGF could discriminate mild from severe preeclampsia with a sensitivity of 74% and specificity of 82%. Maternal serum angiogenin levels in women with mild and severe preeclampsia were found to be significantly elevated when compared to that of controls ( pb0.001). A cutoff value of serum angiogenin of 478 ng/ml is clinically useful for differentiating preeclamptic patients from healthy controls with a sensitivity of 92% and a specificity of 88%. Moreover, a cutoff value of 784 ng/ml can discriminate mild from severe preeclampsia with a sensitivity of 76% and a specificity of 81%. However, two previous studies could not detect any significant changes of serum angiogenin between preeclamptic and normal pregnant women [14,19]. The present data show a significant positive correlation between serum VEGF and serum angiogenin concentrations in preeclamptic patients. Both angiogenic factors, angiogenin and VEGF, were significantly correlated with both systolic and diastolic blood pressure ( pb0.001). All preeclamptic patients with abnormal BPS or abnormal uterine artery Doppler velocimetry had abnormally elevated values of serum VEGF and angiogenin concentrations. Preeclamptic patients who delivered low birthweight infants had significantly higher serum VEGF and angiogenin levels than those who delivered infants with normal birthweight (Table 3). There was a significant negative correlation between serum VEGF or serum angiogenin concentration and infant birthweight in preeclamptic patients ( pb0.01). During normal pregnancy, serum VEGF has been found positivity correlated with birthweight in both the human and pig [26]. In placental and related chorioallantoic tissues, VEGF is up-regulated by hypoxia and down-regulated by hyperoxia [27]. In preeclampsia and intrauterine growth restriction (IUGR), fetoplacental blood flow is severely impaired, and transplacental gas exchange is poor, placing the fetus at risk of hypoxia and acidosis [12]. While several reports [8,28] indicated that plasma VEGF concentrations were significantly higher in women with preeclampsia delivering low birthweight infants, Tse et al. [29] reported no significant difference in the concentration and distribution of VEGF between placentae of normal and growth-retarded infants. Our results confirm the hypothesis of increased VEGF expression induced by placental hypoxia, which lead to an increase of fetoplacental flow impedance in preeclampsia as a leading cause of
Angiogenin and vascular endothelial growth factor in pregnancies complicated by preeclampsia IUGR or fetal stress at time of delivery. This study demonstrates a significant negative correlation between serum angiogenin and birthweight in preeclamptic patients. Several authors [13,18,30] reported significant differences between serum levels of angiogenin in severe preeclampsia and controls for fetal birthweight. In conclusion, elevated levels of both angiogenic factors, VEGF and angiogenin, could confirm the existence of vascular reactivity and endothelial disturbance in preeclampsia. Measurement of these angiogenic factors in maternal serum may be useful as biomarkers for the assessment of the severity of the disease and fetal outcome.
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