Molecular Human Reproduction, Vol. 7, No. 2, 205-210,
February 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Expression of vascular endothelial growth factor receptors 1, 2 and 3 in placentas from normal and complicated pregnancies
1 Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, PL 140, 00290 Helsinki, 2 Department of Pathology, Haartman Institute, University of Helsinki, 00014 Helsinki, Finland and 3 Department of Gene Regulation and Differentiation, Division of Molecular Biotechnology, Braunschweig, Germany
Abstract
Extensive angiogenesis and invasion of the maternal decidua by trophoblasts are essential for the development and function of the placenta. Vascular endothelial growth factors (VEGF), placenta growth factor (PlGF) and their receptors VEGFR-1/Flt-1, VEGFR-2/KDR and VEGFR-3/Flt4 have important roles in vasculogenesis and angiogenesis. We have studied the localization of these proteins by immunohistochemistry and Western blotting in the placenta and of PlGF in maternal serum, and their association with diabetes, pre-eclampsia, fetal growth restriction (FGR) and fetal alcohol syndrome (FAS). VEGFR-1 and VEGFR-3 were detected mainly in the syncytiotrophoblastic layer whereas VEGFR-2 was detected in the vascular endothelial cells of the placenta. VEGFR-1, but not the other receptors, showed increased expression in placental syncytiotrophoblasts from 50% of patients with severe pre-eclampsia and FGR when compared with normal placentas. PlGF was undetectable in 38 of 44 samples of amniotic fluid of mothers with normal and complicated pregnancies. However, maternal serum PlGF concentrations were significantly lower in pre-eclamptic patients and in those with FGR when compared to diabetic women or healthy controls. These results suggest that low maternal serum PlGF and increased placental expression of its receptor VEGFR-1 are associated with pre-eclampsia and FGR.
placental growth factor/placenta/pre-eclampsia/trophoblast/VEGF receptors
Introduction
A number of vascular endothelial growth factors (VEGF) have been shown to be expressed in the placenta (Vuorela et al., 1997
). The first identified VEGF is a 45 kDa disulphide-linked homodimeric glycoprotein shown to be angiogenic in vivo (Ferrara and Davis-Smyth, 1997). VEGF also induces vascular permeability (Dvorak et al., 1995), stimulates endothelial cell migration and growth (Dvorak et al., 1995; Ferrara and Davis-Smyth, 1997) and maintains the integrity of newly formed blood capillaries (Alon et al., 1995). In addition to VEGF, a closely related protein, placenta growth factor (PlGF), is abundantly expressed in the placenta (Maglione et al., 1991). Small amounts of mRNA of the other members of the VEGF family, VEGF-B and VEGF-C, have also been detected in the placenta (Klagsbrun and D'Amore, 1996; Vuorela et al., 1997).
Three receptors for the VEGF protein family have been identified. PlGF, VEGF and VEGF-B bind to and traduce signals via VEGFR-1/Flt-1 (de Vries et al., 1992), while both VEGF and VEGF-C bind to VEGFR-2/KDR (Terman et al., 1992; Millauer et al., 1994; Joukov et al., 1996). A specific receptor for VEGF-C is VEGFR-3/Flt4 which has been suggested to play a role in the regulation of angiogenesis and lymphangiogenesis (Enholm et al., 1998).
In normal pregnancies, fetal trophoblasts invade the maternal decidua and remodel the spiral arteries, converting them to low resistance vessels. In pregnancies complicated by pre-eclampsia, this trophoblastic cell invasion is inadequate, resulting in poor placental perfusion and fetal hypoxia (Lim et al., 1997; Zhou et al., 1998). Pre-eclampsia is characterized by hypertension, proteinuria, endothelial cell dysfunction and increased sensitivity to vasopressor agents.
Maternal diabetes mellitus predisposes the fetus to hyperglycaemia, macrosomia and hypoxia, all of which are associated with embryonic anomalies. The role of VEGF in placentas of diabetic women has not been studied, but increased intraocular VEGF concentrations have been shown to be associated with retinal oedema and neovascularization in diabetic patients (Aiello et al., 1994; Malacaze et al., 1994). Data are not available on the possible role of VEGF in pregnancies complicated by fetal growth restriction (FGR) or fetal alcohol syndrome (FAS).
The aim of our study was to determine whether there is any difference in the expression of VEGF receptors in the placentas of healthy, diabetic and pre-eclamptic women, and of women whose fetuses suffered from FGR and FAS. PlGF concentrations in healthy and pathological maternal serum and amniotic fluid were also studied since aberrant trophoblast production of PlGF, bound to VEGFR-1, may contribute to the vascular and placental pathologies noted in these obstetric complications (Torry et al., 1999).
Materials and methods
Sample collection
The study was conducted in accordance with the guidelines and approval given by the Ethical Committee of the Department of Obstetrics and Gynaecology of Helsinki University Hospital. Placental samples and amniotic fluid were collected from term pregnancies delivered by elective Caesarean section done for obstetric indications.
Placental tissues, from women whose pregnancies were complicated by pre-eclampsia (n = 8), FGR (n = 5), FAS (n = 2) and diabetes (n = 8) were compared with those obtained from normal healthy controls (n = 8). All these patients also gave amniotic fluid and blood samples for PlGF analyses. Including these, and in total, amniotic fluid PlGF from 13 healthy, 13 diabetic and 10 pre-eclamptic women and from eight mothers with FGR was investigated. Maternal serum was collected in total from 22 healthy, 18 diabetic and 18 pre-eclamptic women and from nine mothers with FGR within the last 24 h before delivery and stored at –20°C until analysis. Clinical details of the patients are presented in Table I.
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Pre-eclampsia was defined as systolic and diastolic blood pressures above 140 and 90 mmHg, respectively, in at least two consecutive measurements, and proteinuria (>300 mg per 24 h collection) before commencing medication. Group FGR included infants with birthweight below –2 SD for gestational age. FAS was identified according to the following criteria: fetal growth retardation, neurological abnormalities and characteristic facial features (Claren and Smith, 1978
). The diabetes mellitus group included women with insulin treatment for at least 10 years either with or without renal complications. Maternal side placental samples were collected from patients following informed consent, snap-frozen in liquid nitrogen and stored at –70°C for further investigation. The specimens were embedded in Tissue-Tek (OCT Compound 4583; Miles Inc. Diagnostics Division Elkhart, IN, USA), 5 µm sections were cut at –24°C, mounted onto silane-coated slides and stored at –70°C for up to 2 weeks.
Immunohistochemistry
Anti-VEGF antibody was an affinity-purified rabbit polyclonal antibody raised against a peptide corresponding to amino acids 1–191 of VEGF of human origin with a deletion from amino acids 142 to 185 (Santa Cruz Biotechnology, Santa Cruz, Canada). Monoclonal anti-VEGFR-1 antibody (bioreactor grade culture supernatant, clone FLT-19) and monoclonal anti-VEGFR-2 antibody (mouse ascites fluid, clone KDR-1) were also used (Simon et al., 1998). Monoclonal antibodies against VEGFR-3 have been described (Jussila et al., 1998). Rabbit polyclonal antibodies against human von Willebrand factor (vWF; Dakopatts, Klostrup, Denmark) were used to identify endothelial cells.
Sections were fixed in ice-cold acetone for 10 min, air-dried for 1 min and washed in phosphate-buffered saline (PBS) for 5 min. Endogenous peroxidase activity was quenched by incubation in methanol with 0.6% hydrogen peroxide for 10 min. After PBS washes (2x5 min) the tissues were treated for 30 min at room temperature with 2% normal swine serum (Dakopatts) for anti-VEGF stainings, and with 5% horse serum (Vectastain Elite mouse monoclonal ABC kit; Vector Laboratories, Burlingame, CA, USA) for the other stainings. The primary antibodies were then incubated on the slides for 2 h in a humid chamber at room temperature. Anti-VEGF antibodies were used at 2 g/ml, anti-VEGFR-1 and -VEGFR-2 at dilutions 1:200 and anti-VEGFR-3 at 2 µg/ml.
Excess antibody was washed away with PBS (3x5 min) and biotinylated swine antibody against rabbit IgG (1:200; Dakopatts) or biotinylated horse antibody against mouse IgG (1:200, Vectastain ABC Elite biotin-avidin system; Vector Laboratories), respectively, were then incubated on the slides for 2 h at room temperature to stain VEGF or the other antigens, respectively. Following PBS washes the sections were incubated in the ABC solution (Vectastain ABC Elite biotin-avidin system) for 60 min at room temperature and rinsed with PBS. Bound antibody conjugates were visualized using a solution of 3-amino-9-ethylcarbazole, 0.03% hydrogen peroxide, 14 mmol/l acetic acid, and 33 mmol/l sodium acetate. Counterstaining was carried out using Mayer's haematoxylin (Merck, Darmstadt, Germany) and the test slides were mounted with Aquamount improved mounting medium (BHD Laboratory Supplies, Poole, Dorset, UK).
In control stainings, the primary antibodies were replaced by PBS or non-immune rabbit and mouse serum in equal concentration as the primary antibodies. Blocking of the anti-VEGFR-3 was also carried out by incubating the antibodies with a 10-fold molar excess of the immunogen (Jussila et al., 1998).
All the slides stained by immunohistochemistry were evaluated by a consultant pathologist (O.C.), who was blinded on the clinical information. Staining intensity was scored from negative (-) to light (+), moderate (++) and strong (+++) staining.
Western Blotting
Preparation of lysates
Samples of placental tissue (0.5 g) were homogenized in liquid nitrogen and diluted in 3 ml of lysis buffer (20 mmol/l HEPES) (pH 7.4), 5 mmol/l EDTA, 5 mmol/l dithiothreitol, 1% glycerin, 1% Tween 20 (all Sigma, St Louis, MO, USA) including one protease inhibitor cocktail tablet (Boehringer Mannheim, Mannheim, Germany) per 10 ml buffer. After centrifugation at 20 000 g at 4°C for 5 min the supernatant was stored at –70°C until further use. An aliquot of 50 µl was used for determination of total protein concentration by BCA protein assay reagent (Pierce, Rockford, IL, USA).
Hybridization
A total of 30 µg of each sample was loaded onto a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoretic gel and separated under reducing conditions. Western blotting onto polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA) was performed for 20 min at 15 V in a semi-dry blotting chamber (Biorad, München, Germany). The membrane was saturated with 20% non-fat milk in TBS (20 mmol/l Tris–HCl, 150 mmol/l NaCl, pH 8.0) overnight at 4°C. The monoclonal antibody Flt-1 (Sigma, St Louis, MO, USA) was used diluted 1:1000 in TBS containing 10% non-fat milk for 1 h at room temperature. After three washes for 5 min with TBST (TBS, 0.1% Tween 20) the second goat anti-mouse horseradish peroxidase-conjugated antibody (Bethyl, Wolfenbüttel, Germany) was used at 1:20 000 in TBS containing 10% non-fat milk for 1 h at room temperature. After three washes for 5 min in TBST the membrane was incubated for 1 min in ECL Western-blotting detection reagent (Amersham, Little Chalfont, Buckinghamshire, UK), and exposed for 90 s to Kodak X-OMAT AR film (Kodak, Rochester, New York, USA).
Immunoassay of PlGF
Maternal serum and amniotic fluid PlGF was quantified by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Quantikine rhPlGF; R&D Systems Europe Ltd, Abingdon, UK). The linear range of the assay was from 16 to 2000 ng/l. The inter- and intra-assay coefficients of variation were 6.3 and 7.9% respectively.
Statistics
Comparisons between the groups were performed by the non-parametric Mann-Whitney U-test. Correlations between the different parameters were investigated by Spearman rank correlation.
Results
Distribution of VEGF in placenta
As described previously (Vuorela et al., 1997), the strongest VEGF-like immunoreactivity was located in the endothelial cells of the capillaries of the villi and larger vessels. No significant differences in intensity or localization of the staining were detected between the normal and pathological tissues examined (data not shown).
The distribution of VEGFR-1 in placenta
Staining of VEGFR-1 was detected in placental syncytiotrophoblasts, as well as in endothelial cells of placental villi from normal uncomplicated pregnancies (Figure 1A). A similar staining pattern was observed in placentas of diabetic patients, and stronger staining was detected in two of eight samples (Figure 1B). The staining intensity of the syncytiotrophoblasts from the eight pre-eclamptic women did not differ from that of normal pregnancies in four women (Figure 1C) but was significantly enhanced in the other four (Figure 1D). Some deterioration of normal placental structures was present in the FGR samples, but strong VEGFR-1 staining of syncytiotrophoblasts was also detected in two of five of these cases (Figure 1E). The disintegrated trophoblasts of the alcoholic patients with FAS showed only very weak staining of the syncytiotrophoblasts (Figure 1F). Interestingly, although the anti-VEGFR-1 monoclonal antibodies did not stain basement membranes in blood vessels or beneath the trophoblastic layer, the thick basement membrane beneath the amniotic epithelial cells gave weak staining (Figure 1G). Semiquantitative analysis of the stainings is shown in Table II.
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Distribution of VEGFR-2 and VEGFR-3
VEGFR-2 was localized almost exclusively to the endothelial cells of blood vessels of placental villi. No significant differences in the staining intensity were observed when pre-eclamptic and diabetic placentas were compared with those of normal pregnancies (Figure 2A-C
).
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The anti-VEGFR-3 antibodies strongly stained the syncytiotrophoblast layer of the villi (Figure 3A-C
). Staining was detected in the microvilli which cover the trophoblasts and enlarge their absorptive surface area. Here the staining showed a granular pattern. No staining was observed when primary antibody was blocked by incubating the anti-VEGFR-3 antibodies with a 10-fold molar excess of the antigen used in the immunizations (Figure 3D).
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Western blotting
Western blot hybridization showed that VEGFR-1 expression was increased in one of two placentas from pre-eclamptic women when compared to tissues from two healthy and two diabetic mothers (data not shown). These results were consistent with the immunohistochemical data from the same patients.
Immunoassay of PlGF
Maternal serum samples from pre-eclamptic women had significantly lower concentrations of PlGF (164 ± 169 pg/ml, mean ± SD) when compared to those from healthy controls (608 ± 307 pg/ml, P < 0.001) or diabetic women (690 ± 423 pg/ml, P < 0.001) whose concentrations did not differ from those of healthy subjects. PlGF concentrations in maternal serum were also lower (P < 0.01) in the FGR group (192 ± 151 pg/ml) when compared to healthy women (Figure 4).
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Patients with FAS were also included in the FGR group because of the rarity of this condition. Amniotic fluid PlGF concentrations were below the detection limit in all but one patient with FAS (80 pg/ml) and five healthy subjects (range 18-61 pg/ml).
Discussion
VEGF and its receptors are essential for embryonic vascular development as loss of even a single VEGF allele results in embryonic death (Carmeliet et al., 1996; Ferrara et al., 1996). VEGF and VEGFR-1 are expressed in the placenta throughout gestation (Ahmed et al., 1995; Clark et al., 1996) and the possible effect of VEGF on trophoblast invasion and motility is suggested to be mediated by functional receptors (Lash et al., 1999). A characteristic feature of VEGF regulation is the induction of its mRNA by tissue hypoxia and hypoglycaemia (Shweiki et al., 1992, 1995).
Increased VEGF concentrations have been detected in diabetic ocular fluid and VEGF is suggested to be essential for retinal and iris neovascularization, characteristic of diabetic proliferative retinopathy (Aiello et al., 1994; Malacaze et al., 1994). In addition to VEGF, increased PlGF expression has also been detected in proliferative diabetic retinopathy (Khaliq et al., 1998) and therefore the expression of PlGF in diabetic pregnancy was interesting to study. However, we were not able to show increased expression of VEGFR-1 in syncytiotrophoblasts in the majority of diabetic patients. It is possible that the influence of maternal diabetes on the expression of vascular endothelial growth factors is mostly local, concentrating in certain tissues. Individual variation in glucose balance and variable hyperglycaemia among the patients could explain some differences between our study subjects since advanced glycosylation end-products have been shown to increase retinal VEGF expression (Lu et al., 1998).
Here we have shown that VEGFR-1 expression is increased in the trophoblasts of placentas from pregnancies complicated by pre-eclampsia, but this was not observed in all cases. This may be explained by individual differences between the patients. The sample with strongest staining was taken from a patient of 26 weeks gestational age with blood pressure 220/120 mmHg and proteinuria of 11 g per 24 h. The other samples were taken from patients with milder disease at gestational weeks 30-33. Thus the staining may be associated with the severity of the disease and gestational week.
VEGFR-1 has not been shown to locate in the basement membranes of blood vessels or beneath the trophoblastic layer. However, we were able to detect VEGFR-1 in the thick basement membrane beneath the amniotic epithelial cells. Our findings may represent staining of the soluble form of the VEGFR-1 (sVEGFR-1), which is present in the amniotic fluid (Banks et al., 1998; Vuorela et al., 2000) secreted by the placenta, and is a potent VEGF antagonist regulating the action of VEGF in the placenta (Clark et al., 1998). VEGFR-1 binds VEGF, VEGF-B and PlGF. Hypoxic conditions reduce placental PlGF expression (Shore et al., 1997) and up-regulate VEGF expression (Gerber et al., 1997). The up-regulation of VEGFR-1 among some pre-eclamptic patients and their low serum concentrations of PlGF could therefore reflect hypoxia. Thus, the increased VEGFR-1 expression is probably not a specific change characteristic for pre-eclampsia, but is possibly associated with abnormal placental function and hypoxia.
Our group has previously shown that maternal serum and amniotic fluid contain PlGF but its concentrations in umbilical cord venous serum are below the detection limit. Earlier we analysed amniotic fluid PlGF concentrations from 11 patients and some values were below the detection limit, the mean (± SD) amniotic fluid PlGF concentration was 30 ± 18 ng/l (Vuorela-Vepsäläinen et al., 1999). Our new studies by ELISA also showed that amniotic fluid contained some PlGF (range 18-80 ng/l), and its concentrations were below the detection limit in all pre-eclamptic patients. Our new data seem more reliable since in this study we analysed 44 samples from different patient groups and did not count the mean value of the 38 samples with PlGF concentrations below the detection limit. Our finding of decreased PlGF concentrations in maternal serum of pre-eclamptic patients is supported by Reuvekamp et al. (1999). Furthermore, we have shown earlier that the amniotic fluid concentrations of sVEGFR-1, a splice variant of the VEGFR-1 gene (Kendall and Thomas, 1993), are elevated in pre-eclampsia, and that the placenta abundantly secretes sVEGFR-1 (Vuorela et al., 2000). As the PlGF ELISA only seems to detect the unbound PlGF (Hornig et al., 2000), our findings of increased sVEGFR-1, abundantly expressed by the pre-eclamptic syncytiotrophoblasts, may explain the low PlGF concentrations found in pre-eclamptic subjects.
In conclusion, we have preliminary findings of increased VEGFR-1 expression in syncytiotrophoblasts in pre-eclamptic patients and women with FGR as well as reduction of the special ligand for this receptor, PlGF, in maternal serum in these conditions. It seems that the members of the VEGF family and their receptors are involved in the pathogenesis of human pregnancy, complicated by placental dysfunction.
Acknowledgments
We wish to thank Prof. Kari Alitalo for great support. We also wish to thank Dr Lotta Jussila for providing the anti-VEGFR-3 antibody. This study was supported by the Finnish Foundation for Diabetes Research, the Jahnsson Foundation and The Clinical Research Fund of the Helsinki University Central Hospital.
Notes
4 To whom correspondence should be addressed at: POB 140, 00029 HUS, Finland. E-mail: erja.halmesmaki{at}hus.fi 
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E. Geva, D. G. Ginzinger, C. J. Zaloudek, D. H. Moore, A. Byrne, and R. B. Jaffe Human Placental Vascular Development: Vasculogenic and Angiogenic (Branching and Nonbranching) Transformation Is Regulated by Vascular Endothelial Growth Factor-A, Angiopoietin-1, and Angiopoietin-2 J. Clin. Endocrinol. Metab., September 1, 2002; 87(9): 4213 - 4224. [Abstract] [Full Text] [PDF]
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 Y. Li, M.-N. Wang, H. Li, K. D. King, R. Bassi, H. Sun, A. Santiago, A. T. Hooper, P. Bohlen, and D. J. Hicklin Active Immunization Against the Vascular Endothelial Growth Factor Receptor flk1 Inhibits Tumor Angiogenesis and Metastasis J. Exp. Med., June 17, 2002; 195(12): 1575 - 1584. [Abstract] [Full Text] [PDF]
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 C. Wulff, H. Wilson, S. E. Dickson, S. J. Wiegand, and H. M. Fraser Hemochorial Placentation in the Primate: Expression of Vascular Endothelial Growth Factor, Angiopoietins, and Their Receptors Throughout Pregnancy Biol Reprod, March 1, 2002; 66(3): 802 - 812. [Abstract] [Full Text] [PDF]
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G. Lash, A. MacPherson, D. Liu, D. Smith, S. Charnock-Jones, and P. Baker Abnormal fetal growth is not associated with altered chorionic villous expression of vascular endothelial growth factor mRNA Mol. Hum. Reprod., November 1, 2001; 7(11): 1093 - 1098. [Abstract] [Full Text] [PDF]
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