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ORIGINAL RESEARCH |
From the Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Helsinki, Finland; Department of Gene Regulation and Differentiation, Division of Molecular Biotechnology, Braunschweig, Germany; and Molecular and Cancer Biology Laboratory, Haartman Institute, University of Helsinki, Finland.
Address reprint requests to: Erja Halmesmäki, MD, PhD Departments of Obstetrics and Gynecology Helsinki University Central Hospital PL 140 Helsinki, 00029 HYKS Finland E-mail: erja.halmesmaki{at}helsinki.fi
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Abstract |
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Methods: We assessed amniotic fluid from the second (n = 35, gestational weeks 14–19) and third (n = 29) trimesters of healthy women, and from the third trimesters of preeclamptic (n = 22) and diabetic women with (n = 11) or without preeclampsia (n = 34) and from women with fetal growth restriction (FGR) (n = 14) for soluble VEGF receptor-1 (VEGFR-1) by enzyme-linked immunosorbent assay.
Results: In early normal pregnancy, AF-soluble VEGFR-1 levels were higher (median 22 ng/mL, range 2.3–29.5 ng/mL) than in the third trimester (median 13 ng/mL, range 0.5–32 ng/mL; P < .05). In preeclamptic women during the third trimester, levels were higher (median 20 ng/mL, range 10.5–37 ng/mL; P < .05) than healthy controls. The lowest third-trimester levels were in diabetic women (median 11 ng/mL, range 0.5–27 ng/mL). In women with preeclampsia and diabetes, AF-soluble VEGFR-1 levels remained lower (median 13, range 6–32 ng/mL; P < .05) than in women with preeclampsia alone. Amniotic fluid levels of soluble VEGFR-1 in women with FGR (median 19.5 ng/mL, range 5–40 ng/mL) did not statistically differ from those of controls. The AF levels of soluble VEGFR-1 did not correlate with those of erythropoietin. Soluble VEGFR-1 was clearly detectable (median 14 ng/mL, range 9–22 ng/mL) in culture media from placental biopsies (n = 20).
Conclusion: Preeclampsia is associated with increased levels of soluble VEGFR-1, which are independent of erythropoietin, another hypoxia-inducible factor.
Pregnancies complicated by the multiorgan failure of preeclampsia are associated with poor placental growth and inadequate physiologic changes in the vasculature of the placental bed.1 Systemic symptoms of women were thought to be due to endothelial cell dysfunction.2 Another condition with reduced growth of placental vasculature is fetal growth restriction (FGR), the cause of which is most often unclear. Placentas from women with diabetes mellitus tend to grow larger than in uncomplicated pregnancies. A common factor for all those conditions is the constant danger to the fetus of hypoxia, which ultimately might lead to central nervous system (CNS) damage or even fetal death. Such fetal compromise can be assessed by amniotic fluid (AF) erythropoietin, the levels of which increase with fetal hypoxia.3
Similar oxygen-sensing mechanisms that regulate the expression of erythropoietin and vascular endothelial growth factor (VEGF) have been shown.4 Vascular endothelial growth factor, a vascular endothelial cell mitogen that also induces vascular permeability, is in the placenta and various fetal tissues.5,6 The VEGF receptor-1 (VEGFR-1) (or fms-like tyrosine kinase-1), is also upregulated by hypoxia, and a soluble form of the VEGFR-1 occurs in AF.5,7,8 Soluble VEGFR-1 might help regulate activity of VEGF in the AF compartment.
It is not known whether preeclampsia, diabetes mellitus, or FGR is associated with changes in AF levels of soluble VEGFR-1, but in placentas of preeclamptic women the expression of its ligand VEGF was reduced.9 However, results on the association of preeclampsia and circulating VEGF levels are contradictory.10,11 The role of VEGF in pregnancies complicated by diabetes mellitus has not been defined, but increased VEGF levels in the vitreous part of the eye have been reported in diabetic retinal neovascularization.12 The aim of the present study was to evaluate whether AF-soluble VEGFR-1 levels are altered in women with preeclampsia, FGR, or diabetes mellitus.
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Materials and Methods |
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Amniotic fluid samples from the third trimester were originally collected for assessment of fetal lung maturity from preeclamptic (24 samples from 22 women, gestational weeks 28–39) and diabetic (35 samples from 34 women, gestational weeks 27–39) women, from women with diabetes and preeclampsia (n = 11, gestational weeks 32–37), and from women whose pregnancies were complicated by FGR (n = 14, gestational weeks 27–36) without preeclampsia or other etiologic factor. Delivery was within 1–2 days of last amniocentesis. Diagnostic criteria were preeclampsia (blood pressure [BP] exceeding 140/90 mmHg on at least two consecutive measurements and proteinuria of at least 300 mg per 24-hour collection [before commencing medication, see below]); FGR (fetal weight at least two standard deviations [SDs] below average for gestational age); and diabetes (maternal insulin treatment).
Women with preeclampsia and growth-restricted fetuses routinely were given two 12-mg intramuscular injections of betamethasone (Celeston Chronodose; Schering Plough, Heist-op-Den-Berg, Belgium), each separated by 12 hours, to enhance maturing of fetal lungs. All the preeclamptic women were on antihypertensive therapy of labetalol hydrochloride (Albetol; Leiras, Turku, Finland) 400 – 800 mg/day or nifedipine (Adalat; Bayer AG, Leverkusen, Germany) 15– 60 mg/day. Diabetic mothers also were taking individualized insulin therapy (Protaphan 100 IU/mL, Actrapid 100 IU/mL; Novo Nordisk A/S, Bagsv
rd, Denmark).
Control samples of AF (n = 24) from women with uncomplicated pregnancies were collected by needle aspiration during elective cesareans for fetopelvic disproportion or breech presentation. Five other AF samples were collected from mothers (gestational weeks 25–30) with antibodies against Rhesus factor; amniocentesis was originally done for assessment of AF bilirubin levels. All bilirubin levels were within normal range, like all other laboratory and clinical findings. Thus, those samples were included in the control group (n = 29) to represent early third trimester. Clinical characteristics of study subjects are given in Table 1
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Amniotic fluid erythropoietin levels were measured by radioimmunoassay (RIA) (Epo-Trac; Incstar Corp., Stillwater, MN). The intra-assay and interassay coefficients of variation were 5.1–8.1% and 5.4–8.6%, respectively. Erythropoietin is used as a routine marker of fetal hypoxia in our hospital14 and is measured at the request of clinicians. Thus, erythropoietin values were available from samples of 23 diabetic and 14 preeclamptic subjects, six women with preeclampsia and diabetes, and six women with FGR. Samples from 11 healthy women were used for comparison.
Comparisons between study groups were done with the Mann-Whitney U test. Associations between the different parameters were investigated by Spearman rank correlation. In cases when more than one AF sample was analyzed from one woman, the value of the sample drawn closest to delivery was used to study associations between AF-soluble VEGFR-1 levels and clinical parameters. For statistical analysis, samples with soluble VEGFR-1 levels below the detection limit of the assay (1 ng/mL) were set to 0.5 ng/mL, ie, the median between zero and the lowest limit of detection. Results are expressed as median, range.
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Results |
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). In diabetic women the AF levels of soluble VEGFR-1 tended to be lower (median 11 ng/mL, range 0.5–27 ng/mL) than healthy controls, but the difference was not statistically significant (Figure 1). There was no association between soluble VEGFR-1 levels and severity of diabetes or maternal glucose balance, evaluated by the percentage of glycosylated hemoglobin (HbA1c) in maternal plasma (data not shown).
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). There was no correlation between AF-soluble VEGFR-1 levels and degree of maternal proteinuria, blood thrombocyte count, or serum alanine aminotransferase levels (data not shown). In diabetic women with superimposed preeclampsia, the median AF-soluble VEGFR-1 level was 13 ng/mL (range 6–32 ng/mL). Those levels were slightly higher than women with diabetes without superimposed preeclampsia, but clearly lower than women with preeclampsia and no other disorders (P < .05, Figure 1).
In pregnancies complicated by FGR the soluble VEGFR-1 levels in AF were slightly higher than healthy controls (median 19.5 ng/mL, range 5–40 ng/mL), but the difference was not statistically significant (Figure 1). The AF-soluble VEGFR-1 levels did not correlate with gestational age, fetal birth weight, placental weight, or erythropoietin levels in any of the groups (data not shown). Higher AF erythropoietin levels, compared with controls (median 5 U/L, range 4.2–10 U/L), were seen in AF from preeclamptic (median 13 U/L, range 5–28 U/L, P = .001) and diabetic (median 11 U/L, range 5–30 U/L, P < .05) women, those with diabetes and superimposed preeclampsia (median 14.5 U/L, range 6–75 U/L, P < .05), and those with FGR (median 8.4 U/L, range 7–26 U/L, P < .05), (Figure 2). Culture media from placenta biopsy specimens contained clearly detectable amounts of soluble VEGFR-1 (median 14 ng/mL, range 9–22 ng/mL, Figure 1).
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Discussion |
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We observed that in healthy pregnancies, AF levels of soluble VEGFR-1 were higher in the second trimester than at term. To find out whether that decline is gradual or rapid at some specific time point would require serial, ie, unethical, AF sampling of healthy pregnant women. The placenta was found to be a source of soluble VEGFR-1, although we cannot verify that placental soluble VEGFR-1 is secreted into AF, or that it would be the sole source of AF-soluble VEGFR-1. Sources such as fetal urine or membranes also are possible. It is unknown how AF VEGFR-1 concentration varies with changes in AF volume.
The effect of insulin on VEGF expression has not been investigated, but in vitro studies showed that high glucose levels stimulate VEGF expression by human vascular smooth muscle cells.18 Under hypoxic conditions, glioma cells also respond to hyperglycemia with increased VEGF production.19 In our diabetic subjects, AF-soluble VEGFR-1 levels tended to be lower than in controls, but we found no association between those levels and erythropoietin, severity of illness, or percentage of glycosylated hemoglobin, HbA1c, in maternal blood. Thus, at least in term pregnancy, AF-soluble VEGFR-1 levels did not clearly associate with diabetic states of pregnancy.
During the last trimester, AF-soluble VEGFR-1 levels in women with preeclampsia were higher than in controls or diabetic women. Owing to different gestational ages between groups, it cannot be concluded whether the difference was associated with preeclampsia or duration of pregnancy. In favor of the hypothesis that preeclampsia affects AF-soluble VEGFR-1 levels is that diabetic women with preeclampsia had higher levels than other diabetic women, and the groups delivered in the same gestational weeks. Preeclamptic placentas had reduced levels of trophoblastic mRNA levels of soluble VEGFR-1s ligand VEGF,9 and so we are interested in knowing how ligand and receptor interplay is affected by preeclampsia.
The clinical symptoms of preeclampsia, maternal edema, elevated BP, and proteinuria appear during the latter half of pregnancy. We have not studied first-trimester AF of women who subsequently develop preeclampsia, but we would be interested in knowing whether the changes in AF-soluble VEGFR-1 levels of preeclamptic women antedate clinical symptoms. We also studied AF samples from women with FGR. The reason for FGR often remains unknown, and is associated with restricted growth of the vascular placenta, which we hypothesized might be associated with changes in AF-soluble VEGFR-1 levels. No statistically significant differences between the FGR group and controls were seen. Due to the rarity of FGR, our sample was small, which might explain the lack of statistical significance and the tendency for the soluble VEGFR-1 levels of FGR group to be higher than those of healthy women.
Erythropoietin production, like VEGF, is known to increase in response to poor tissue oxygenation.3,20 Erythropoietin-induced endothelial cell proliferation can be inhibited by VEGF neutralizing antibodies,21 and erythropoietin and soluble VEGFR-1 are both expressed by placenta.20 The VEGFR-1 gene is upregulated by hypoxia and has a hypoxia-inducible-factor binding sequence.22 It is logical to assume a similar regulation between soluble VEGFR-1 and erythropoietin and VEGF genes. However, we did not see any association between those two parameters, although the erythropoietin concentrations in AF varied between groups, as in diabetic and hypertensive pregnancies.14 That suggests different regulatory mechanisms for soluble VEGFR-1 and erythropoietin production and secretion into AF.
Elevated levels of total soluble VEGFR-1 in AF associated with preeclampsia, and those levels were reduced with diabetes. Soluble VEGFR-1 is produced by the placenta, but its exact origin is unknown. Further studies are needed to find out whether AF or serum levels of soluble VEGFR-1 might be a tool for diagnosing preeclampsia.
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Footnotes |
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Received May 17, 1999. Received in revised form September 7, 1999. Accepted September 15, 1999.
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References |
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2. Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Pre-eclampsia: An endothelial cell disorder. Am J Obstet Gynecol 1989;161:1200–4.[Medline]
3. Caro J, Erslev AJ, Silver R, Miller O, Birgegard G. Erythropoietin production in response to anemia or hypoxia in the newborn rat. Blood 1982;60:984–8.
4. Goldberg MA, Schneider TJ. Similarities between the oxygensensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin. J Biol Chem 1994;269: 4355–9.
5. Ferrara N. Vascular endothelial growth factor: Molecular and biological aspects. In: Claesson-Welsh L, ed. Vascular growth factors and angiogenesis. Berlin: Springer-Verlag, 1999:1–30.
6. Vuorela 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.[Abstract]
7. Kendall RL, Thomas KA. Inhibition of vascular endothelial growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci U S A 1993;90:10705–9.
8. Banks RE, Forbes MA, Searles J, Pappin D, Canas B, Rahman D, et al. Evidence for the existence of a novel pregnancy-associated soluble variant of the vascular endothelial growth factor receptor, Flt-1. Mol Hum Reprod 1998;4:377–86.
9. Cooper JC, Sharkey AM, Charnock-Jones DS, Palmer CR, Smith SK. VEGF mRNA levels in placentae from pregnancies complicated by pre-eclampsia. Br J Obstet Gynaecol 1996;103:1191–6.[Medline]
10. Kupferminc MJ, Daniel Y, Englender T, Baram A, Many A, Jaffa AJ, et al. Vascular endothelial growth factor is increased in patients with preeclampsia. Am J Reprod Immunol 1997;38:302–6.
11. Lyall F, Greer IA, Boswell F, Fleming R. Suppression of vascular endothelial growth factor immunoreactivity in normal pregnancy and in pre-eclampsia. Br J Obstet Gynaecol 1997;104:223–8.[Medline]
12. Aiello LP, Avery RL, Arrigg PG, Keyt BA, Jampel HD, Shah ST, et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 1994;331:1480–7.
13. Hornig C, Behn T, Bartsch W, Yayon A, Weich HA. Detection and quantification of complexed and free soluble human vascular endothelial growth factor receptor-1 (sVEGFR-1) by ELISA. J Immunol Methods 1999;226:169–77.[Medline]
14. Teramo KA, Widness JA, Clemons GK, Voutilainen P, McKinlay S, Schwartz R. Amniotic fluid erythropoietin correlates with umbilical plasma erythropoietin in normal and abnormal pregnancy. Obstet Gynecol 1987;69:710–6.[Abstract]
15. Shibuya M, Ito N, Claesson-Welsh L. Structure and function of vascular endothelial growth factor receptor-1 and -2. In: Claesson-Welsh L, ed. Vascular growth factors and angiogenesis. Heidelberg, Germany: Springer-Verlag, 1999:59–84.
16. Olofsson B, Pajusola K, Kaipainen A, von Euler G, Joukov V, Saksela O, et al. Vascular endothelial growth factor B, a novel growth factor for endothelial cells. Proc Natl Acad Sci U S A 1996;93:2576–81.
17. Dumont DJ, Jussila L, Taipale J, Lymboussaki A, Mustonen T, Pajusola K, et al. Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science 1998;282:946–9.
18. Natarajan R, Bai W, Lanting L, Gonzales N, Nadler J. Effects of high glucose on vascular endothelial growth factor expression in vascular smooth muscle cells. Am J Physiol 1997;273:H2224–31.
19. Brooks SE, Gu X, Kaufmann PM, Marcus DM, Caldwell RB. Modulation of VEGF production by pH and glucose in retinal Müller cells. Curr Eye Res 1998;17:875–82.[Medline]
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