Vascular endothelial growth factor and placenta growth factor

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1To whom correspondence should be addressed at: Obstetrical Unit, Department of Obstetrics and Gynaecological ... Key words: Down's syndrome/PLGF/pregnancy/VEGF ..... To the best of our knowledge, this is the first study describing.

Molecular Human Reproduction Vol.7, No.8 pp. 765–770, 2001

Vascular endothelial growth factor and placenta growth factor concentrations in Down’s syndrome and control pregnancies F.Debieve1, A.Moiset, K.Thomas, S.Pampfer and C.Hubinont Department of Obstetrics and Gynaecological Endocrinology, Universite´ Catholique de Louvain, Brussels, Belgium 1To

whom correspondence should be addressed at: Obstetrical Unit, Department of Obstetrics and Gynaecological Endocrinology, Faculty of Medicine, OBST 5330, Avenue Em. Mounier 53, B-1200 Brussels, Belgium. E-mail: [email protected]

Vascular endothelial growth factor (VEGF) and placenta growth factor (PLGF) are considered to play important roles in angiogenesis and vascular permeability during placental development. Since trisomy 21 placentae show trophoblastic hypoplasia and hypovascularity, we investigated PLGF and VEGF synthesis in Down’s syndrome pregnancies. Maternal serum was collected from 102 euploid and 24 trisomy 21 pregnancies between 15 and 20 weeks gestation and tested for these two factors by enzyme-linked immunosorbent assays. Protein extracts from 15 normal and six trisomy 21 placentae were also tested. VEGF was not detected in maternal serum, while PLGF increased significantly with gestational age. Serum PLGF, transformed as a multiple of the gestational age median (MoM), in Down’s syndrome pregnancies was significantly lower than in euploid controls (mean 0.67 ⍨ 0.043 MoM versus 1.00 ⍨ 0.047 MoM, analysis of variance F ⍧ 11.605, P < 0.001). Both VEGF and PLGF were detected in placental protein extracts without variation according to gestational age. Down’s syndrome placentae had significantly less PLGF compared to normal placentae (Mann–Whitney, P < 0.05) but no difference was observed in placental VEGF content (Mann–Whitney, P ⍧ 0.94). Considering the biological properties of PLGF, this decrease may provide new insights into the mechanism(s) leading to the structural and functional anomalies described in trisomy 21 placentae. Key words: Down’s syndrome/PLGF/pregnancy/VEGF

Introduction Vascular endothelial growth factor (VEGF) is a 45 kDa disulphide-linked homodimeric glycoprotein. This growth factor is known to be an angiogenic agent promoting endothelial cell proliferation (Connolly et al., 1989) and vascular permeability (Keck et al., 1989; Ferrera and Davis-Smyth, 1997). As a result of alternative splicing, four transcripts encoding for 121, 165, 189 and 206 amino acid-long proteins have been described (Ferrera and Davis-Smyth, 1997). VEGF121 and VEGF165 are diffusible proteins, while VEGF189 and VEGF206 have a high affinity for heparin and are mostly bound to proteoglycans in the extracellular matrix. VEGF is widely expressed in many normal human tissues including activated macrophages, renal glomerular visceral epithelium, keratinocytes, hepatocytes, smooth muscle cells, uterine endometrium and corpus luteum (Ferrera and Davis-Smyth, 1997). During human pregnancy, VEGF expression has been detected mainly in the placental villous mesenchyme and at a lower concentration in trophoblast cells, as well as in several fetal © European Society of Human Reproduction and Embryology

tissues (Kaipainen et al., 1993). In-vitro studies on human placenta show that hypoxia up-regulates VEGF mRNA (Shore et al., 1997) and protein (Dunk and Ahmed, 1997), while hyperoxia causes a decrease in VEGF protein (Khaliq et al., 1999). The role of VEGF in human placenta is not well determined. However, VEGF knockout studies on mice have resulted in malformed vasculature followed by early embryonic death (Carmeliet et al., 1996; Ferrara et al., 1996). Placenta growth factor (PLGF) is a 50 kDa homodimeric glycoprotein sharing 53% sequence homology at the amino acid level with VEGF (Maglione et al., 1991; Hauser and Weich, 1993). Two isoforms resulting from alternative splicing are described: PLGF-1 (149 amino acids) and PLGF-2 (170 amino acids), and these are identical except for the insertion of a heparin-binding domain at the carboxyl end of PLGF-2 (Hauser et al., 1993). PLGF homodimers are themselves angiogenic (Ziche et al., 1997) and may enhance the activity of sub-optimal VEGF concentrations, increasing endothelial cell permeability and mitogenic activity (Park et al., 1994). 765

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Moreover, VEGF/PLGF heterodimers have a mitogenic activity on human umbilical vein endothelial cells (HUVEC) (DiSalvo et al., 1995). Expression of PLGF mRNA is mainly found in human placental trophoblast cells (Shore et al., 1997; Vuorela et al., 1997), but also in choriodecidua (Khaliq et al., 1999), HUVEC and in human choriocarcinoma cell lines (Hauser and Weich, 1993). Physiopathological studies on human placentae show that hypoxia down-regulates PLGF mRNA expression, whereas hyperoxia and fetal growth retardation have an up-regulating effect (Khaliq et al., 1999). Decreased PLGF concentrations are found in maternal serum from pregnancies complicated with pre-eclampsia (Torry et al., 1998). However, the exact PLGF role in placenta remains undetermined. Down’s syndrome is the most common chromosomal abnormality among newborns (Goshen, 1999). Trisomy 21 pregnancies are associated with hormonal changes in maternal serum concentrations, as used in clinical practice as screening parameters. The most commonly used serum markers are human chorionic gonadotrophin (HCG) concentration, which is increased, and unconjugated oestriol and α-fetoprotein (AFP) concentrations, which are low, between 15–20 weeks gestation. In recent years, additional markers have been investigated in order to increase the predictive value of the triple test; these include inhibin A, free βHCG and pregnancy-associated placental protein A (PAPP-A) (Ross and Elias, 1997). Despite the worldwide use of this screening method, almost no data regarding the biomolecular mechanisms of these hormonal changes are available, and data in the literature are controversial (Goshen, 1999). Moreover, none of these markers has been associated with the functional and structural anomalies related to the disease. Histopathological analysis of trisomy 21 placentae shows trophoblastic hypoplasia and villous hypovascularity from the first to the third trimester (Qureshi et al., 1997; Jauniaux and Hustin, 1998), although fetal growth retardation is mainly observed during the third trimester and occurs less frequently than in other chromosomal disorders (Chen et al., 1972; Kuhlmann et al., 1990; Qureshi et al., 1997). Moreover, a defect in cytotrophoblast differentiation into syncytium has been demonstrated in Down’s syndrome (Frendo et al., 2000). Considering the predicted roles of VEGF and PLGF and their possible effects on placental characteristics, the aim of the present study was to find a correlation between Down’s syndrome and these angiogenic factors. VEGF and PLGF concentrations in maternal serum and placental protein extracts were compared between Down’s syndrome pregnancies and euploid control pregnancies.

Materials and methods Sample populations Maternal serum samples, stored at –25°C, were selected from our prenatal diagnosis unit bank, upon approval of our local ethics committee. All serum samples were collected between 15–20 weeks gestation as part of a Down’s syndrome screening programme based on the triple test, or prior to an amniocentesis performed for maternal age (from 37 years) or maternal anxiety. Blood samples were collected by cubital vein puncture, clotted for 1 h at room temperature and centrifuged. The Down’s syndrome pregnancies were not identified

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Table I. Population characteristics of maternal serum samples from euploid and Down’s syndrome pregnancies

Number Maternal age (years)a Maternal weight (kg)a Smokers (%) Collecting period (years) aMedian

Euploid

Down’s syndrome

102 32.00 ⫾ 4.93 65.42 ⫾ 7.98 5 2

24 37.50 ⫾ 4.76 74.30 ⫾ 12.00 6 6

⫾ SD.

at the time of the sampling. Gestational age was determined by the last menstrual period or crown–rump length (CRL) measurement if different from the last menstrual period-derived gestational age by more than 7 days. The euploid population, attested by either a normal birth outcome or karyotype result, was based on 102 serum samples from different pregnancies (17 samples at each week of gestation from 15–20 weeks). All were singleton spontaneous pregnancies with a normal outcome, excluding pre-eclampsia, fetal growth retardation or insulin-dependent diabetes. Karyotype results were obtained in 25 pregnancies. The Down’s syndrome population was based on 24 serum samples, collected between 15–20 weeks gestation, from Down’s syndrome pregnancies, all attested later on by karyotyping. Characteristics of the study population are presented in Table I. The control placental tissue population comprised 15 samples from singleton euploid pregnancies divided into three groups according to gestational age: the first ranging from 6–15 weeks pregnancy (n ⫽ 5), the second from 20–30 weeks pregnancy (n ⫽ 5) and the third from 37–40 weeks pregnancy (n ⫽ 5). They were collected either after therapeutic abortion, or after pregnancy loss in the case of cervical incompetence, and at term upon approval of the local ethics committee. In the case of abortion, prostaglandin analogues were used, except for the first trimester placental samples obtained by mechanical evacuation. Abnormal karyotypes were excluded, and for the second and third trimester samples, pre-eclampsia or growth retardation were absent. The routine anatomopathological analyses were normal. Placental tissue was identified according to the external appearance and biopsies were taken in the softest, thickest portion of the placenta, away from calcification and fibrous areas. Placental tissue samples ranging from 15–35 weeks gestation (two samples at 15 weeks, one at 24 weeks, one at 32 weeks and two at 35 weeks gestation) were collected from Down’s syndrome pregnancies attested by karyotyping after therapeutic abortion or delivery (n ⫽ 6). All placental samples were collected in a 2 year period, immediately frozen and conserved in liquid nitrogen. Total proteins were isolated by a published method (Chomczynski and Sacchi, 1987), resuspended in phosphate-buffered saline–1% sodium dodecyl sulphate (SDS), and quantified with the Bradford method, using BSA as standard. Placental protein extracts were stored at –80°C. VEGF and PLGF assays VEGF and PLGF were independently tested with a specific enzymelinked immunosorbent assay (ELISA) according to the manufacturer’s protocol (Quantikine human VEGF and Quantikine human PLGF, R&D Systems). These assays used human recombinant VEGF165 and PLGF as standards. VEGF and PLGF assays had a sensitivity of 9 and 7 pg/ml respectively. The inter-plate and intra-plate coefficients of variation were all lower or equal to 10% for both assays. The VEGF and PLGF assays had 20 and 5% cross-reactivity respectively with the naturally occurring PLGF/VEGF heterodimer but no cross-reactivity

VEGF and PLGF in Down’s syndrome

Figure 1. Maternal serum placenta growth factor (PLGF) concentrations in euploid pregnancies in relation to gestational age, and linear regression.

between VEGF or PLGF and VEGF-C or VEGF-B has been described (Vuorela-Vepsa¨ la¨ inen et al., 1999). No cross-reactivity between VEGF and PLGF has been described (Quantikine rhVEGF and rhPLGF). Placental protein extracts were analysed for VEGF and PLGF with the same described ELISAs. The protein buffer used caused no interference with the assays. Statistical analysis The median serum concentration for each gestational age was determined by a linear regression in the euploid population to overcome the relatively small sample size. Euploid and trisomy 21 values were then converted into multiples of the corresponding regressed median. Linear regression was used to test a possible relationship between gestational age regressed MoM and maternal age, weight or sample storage time. A Kolmogorov–Smirnov test comparing actual and expected values assessed goodness of fit to gaussian distribution in both populations. In maternal serum values, comparison between euploid and Down’s syndrome populations was made using a one-way analysis of variance (ANOVA). In the case of heterogeneous distribution (ANOVA, P ⬍ 0.05), a post-hoc Scheffe´ ’s test was used to compare subgroups. In placental protein extract values, the relationship to gestational age was assessed by a linear regression, and comparison between euploid and Down’ syndrome populations was made using a Mann– Whitney U-test. Results were expressed as mean ⫾ SEM or median with interquartile range (IQR).

Results The maternal serum PLGF concentration in euploid pregnancies showed significant increase between 15–20 weeks pregnancy (Figure 1). The weekly observed values fitted a simple linear regression model described by the equation: 27.231⫻gestational age – 307.008 (R2 ⫽ 0.259, P ⬍ 0.001). This regression equation was used to determine the gestational age median. All serum PLGF values, in both euploid and Down’s syndrome groups, were then transformed as a multiple of the regressed median (MoM) at the same gestational age. In order to assess the normality of the distribution in both groups, a Kolmogorov–Smirnov normality test was used. The comparison between actual and expected values revealed no statistically significant difference in both Down’s syndrome

Figure 2. Box plot of placenta growth factor (PLGF) multiple of the regressed median (MoM) for gestational age in normal and trisomy 21 maternal serum between 15–20 weeks gestation. Horizontal lines represent successively the 10th, 25th, 50th, 75th and 90th percentiles. **P ⬍ 0.001, post-hoc Scheffe´ ’s test.

pregnancies group (P ⬎ 0.99) and euploid pregnancies group (P ⫽ 0.35). There was no evidence of association of serum PLGF with maternal age in both studied groups (R2 ⫽ 0.003, P ⫽ 0.62 for euploid group and R2 ⫽ 0.015, P ⫽ 0.56 for Down’s syndrome group). Maternal weight at the time of blood sampling was significantly lower in normal pregnancy than in Down’s syndrome pregnancy (ANOVA F ⫽ 9.308, P ⬍ 0.01 by Scheffe´ ’s test). Nevertheless, no association of serum PLGF with maternal weight was observed in both groups (R2 ⫽ 0.004, P ⫽ 0.73 for the euploid group and R2 ⫽ 0.008, P ⫽ 0.76 for the Down’s syndrome group). No significant difference in the smoker ratio between groups was observed. The collecting period, and consequently the storage time, was different between groups. Nevertheless, there was no association of serum PLGF concentrations with storage time in both studied groups (R2 ⫽ 0, P ⫽ 0.97 for the euploid group and R2 ⫽ 0.049, P ⫽ 0.30 for the Down’s syndrome group). A significant difference was observed between euploid and Down’s syndrome serum PLGF MoM (ANOVA F ⫽ 11.605, P ⬍ 0.001 by Scheffe´ ’s test) (Figure 2). Serum PLGF in the euploid pregnancies group showed a mean of 1.00 ⫾ 0.047 MoM and a median of 0.89 MoM (IQR ⫽ 0.64 MoM). In the Down’s syndrome pregnancies group, the serum PLGF mean was 0.67 ⫾ 0.043 MoM and the median 0.69 MoM (IQR ⫽ 0.30 MoM). No VEGF was detected in any maternal serum samples tested. Conversely, PLGF and VEGF were both detected in placental protein extracts (Figure 3). There was no difference in the storage time between groups. No variation according to gestational age was observed for PLGF (R2 ⫽ 0.058, P ⫽ 0.39) or VEGF (R2 ⫽ 0.096, P ⫽ 0.26). Moreover, there was no significant difference in the gestational age distribution between control and Down’s syndrome pregnancies (Kolmogorov–Smirnov test, P ⫽ 0.77). Comparison of placental protein extracts from Down’s syndrome pregnancies and euploid pregnancies showed a significant difference in PLGF content per total protein, with significantly less PLGF in Down’s syndrome placental extracts (Mann–Whitney U-test 767

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Figure 3. Placenta growth factor (PLGF) (A) and vascular endothelial growth factor (VEGF) (B) content in placental protein extracts from control (open circles) and trisomy 21 (filled circles) placentae related to gestational age. The straight lines represent the linear regression for control placentae.

P ⫽ 0.02). The VEGF content showed no significant variation between euploid and Down’s syndrome pregnancies (Mann– Whitney U-test P ⫽ 0.94).

Discussion To the best of our knowledge, this is the first study describing PLGF and VEGF changes in Down’s syndrome pregnancy in comparison with normal euploid pregnancy; but our results should be confirmed on a larger series of specimens. Even if Down’s syndrome is the most common chromosomal abnormality among newborns, its incidence is relatively low (about 1/700) (Goshen et al., 1999). It is consequently difficult to conduct a significant prospective study concerning a lowrisk population. This also explains the broad collecting period in the Down’s syndrome group in this retrospective study. Nevertheless, no significant influence of storage time on serum PLGF concentrations was observed. Moreover, there was no difference in storage time between Down’s syndrome and control placental protein extracts. The main difficulty in performing a study including human placentae is to obtain normal preterm placentae, at each week of gestation, in a sufficient number to carry out reliable statistical tests, with the assurance of a subsequent normal pregnancy. For evident ethical reasons, it was impossible to obtain completely normal preterm placentae. Thus, we used therapeutic abortion (e.g. mucoviscidosis, holoprosencephaly) and spontaneous pregnancy loss without fetal death in utero (cervical incompetence) with normal anatomopathological analysis of the placenta. No VEGF was detected in maternal serum by the sandwich two-antibody ELISA method used in this study. VEGF immunoreactivity has been detected by others during pregnancy using a direct competitive radioimmunoassay with a single polyclonal antibody (Anthony et al., 1997; Wheeler et al., 1999). It has been found that VEGF in maternal serum is linked to a pregnancy-specific protein which suppresses the VEGF immunoreactivity in the sandwich two-antibody ELISA used (Vuorela et al., 1997). This protein was not identified, and did not seem to correspond to α2-macroglobulin, pregnancy 768

zone protein, pregnancy associated plasma protein-A or the soluble form of the fms-like tyrosine kinase-1 (FLT-1) VEGF/ PLGF receptor (Vuorela-Vepsa¨ la¨ inen et al., 1999). However, Banks et al. have identified a pregnancy-associated soluble variant of the FLT-1 receptor in plasma from pregnant women and amniotic fluid, present as a multimeric structure (Banks et al., 1998). Moreover, Hornig et al. have shown that the soluble FLT-1 receptor could bind both VEGF and PLGF in amniotic fluid, but 10-fold less for PLGF than for VEGF (Hornig et al., 2000). However, no data are available concerning the biological activity of bound VEGF or PLGF. The VEGF expression is considerably lower than that of PLGF in trophoblast cells (Shore et al., 1997). Nevertheless, we found similar concentrations of PLGF and VEGF in euploid placental extracts. This finding supports the presence of a factor suppressing the VEGF immunoreactivity in maternal serum rather than a low production (Vuorela-Vepsa¨ la¨ inen et al., 1999). Nevertheless, no difference in placental VEGF content between Down’s syndrome and normal pregnancies was observed. This study shows that PLGF concentrations are significantly lower in Down’s syndrome pregnancies in both maternal serum and placental protein extracts. Considering the biological properties of PLGF, this decrease may provide new insights into the mechanism(s) leading to the structural and functional anomalies described in trisomy 21 placentae. Histopathological observations in trisomy 21 placentae from first (Jauniaux and Hustin, 1998), second (Qureshi et al., 1997; Kuhlmann et al., 1990) and third trimester (Rochelson et al., 1990) have shown villous abnormalities, including hypovascularity and trophoblast hypoplasia. Defective cell proliferation in trisomy 21 placentae has been excluded (Qureshi et al., 1997). PLGF is known to have an angiogenic activity (Ziche et al., 1997) and may enhance the VEGF mitogenic activity (Park et al., 1994). Moreover, VEGF/ PLGF heterodimers also have a mitogenic activity (DiSalvo et al., 1995). In addition, the presence of a functional FLT1 receptor on human trophoblast cells suggests a role in trophoblast invasion, differentiation, and metabolic activity (Shore et al., 1997). Consequently, the lower PLGF values in Down’s syndrome pregnancies found in this study could

VEGF and PLGF in Down’s syndrome

contribute to the histopathological anomalies in trisomy 21 placentae. Nevertheless, the trophoblast hypotrophy and the hypovascularity of the Down’s syndrome placenta have not been verified in this study, although anatomopathological observations have been obtained by many other groups (Khulmann et al., 1990; Rochelson et al., 1990; Qureshi et al., 1997; Jauniaux and Hustin, 1998). Further studies correlating the extent of the placental and serum PLGF decreases and the trophoblast and vascular development will be needed. In addition to the histopathological modifications, decrease in PLGF could also contribute to the physiopathological mechanisms leading to hormonal maternal serum changes associated with trisomy 21 pregnancy. An association between low maternal serum AFP concentrations and fetal Down’s syndrome has been proposed (Merkatz et al., 1984) and suggested as a method of screening for Down’s syndrome (Cuckle and Wald, 1984). Fetal synthesis and its diffusion through placenta and membranes determine the concentration of AFP in maternal serum (Los et al., 1985). Low AFP in newborn trisomy 21 babies after 35 weeks has been reported (Cuckle et al., 1986), but all other studies measuring fetal serum AFP during the second trimester by umbilical cord sampling have detected normal AFP concentrations in trisomy 21 fetuses (Nicolini et al., 1988; Scioscia et al., 1988; Seller, 1990). Low PLGF concentrations in Down’s syndrome pregnancies, associated with the PLGF vascular permeability properties (Park et al., 1994), could suggest a role for PLGF in decreasing the fetal AFP transfer rate to the maternal compartment rather than a decreased fetal synthesis. The same conclusions could be suspected concerning low unconjugated oestriol in Down’s syndrome pregnancies (Canick et al., 1987). The mechanism leading to high HCG concentrations in maternal serum from Down’s syndrome pregnancies is not well established. The placental HCG mRNA expression is not altered in trisomy 21 (Brizot et al., 1995). Recently, Frendo et al. have found a defect in cytotrophoblast differentiation into syncytiotrophoblast in Down’s syndrome (Frendo et al., 2000). This could reflect an immaturity of the trophoblastic tissue as suggested by others (Chard, 1991; Jauniaux et al., 2000). This immaturity could reflect or be caused by low PLGF in trisomy 21 placentae. One could argue that low PLGF concentrations in Down’s syndrome pregnancy are the consequence of growth retardation and smaller placentae. But intrauterine growth retardation (IUGR) is mainly observed during the third trimester and at a lower extent in trisomy 21 (Chen et al., 1972; Kuhlmann et al., 1990; Khaliq et al., 1999). Moreover, in-vitro studies on human placentae have shown that IUGR is associated with placental hyperoxia and increased PLGF expression (Khaliq et al., 1999). Thus, the low PLGF in trisomy 21 is apparently not associated with fetal growth restriction. On the other hand, hypoxia down-regulates PLGF mRNA and protein synthesis (Khaliq et al., 1999); and placental bed hypoxia is a possible mechanism involved in pre-eclampsia (Khong et al., 1986; Roberts et al., 1989) which is associated with reduced PLGF serum concentrations (Torry et al., 1998). Hypoxia could be therefore related to reduced PLGF concentrations observed in Down’s syndrome pregnancies.

In conclusion, we speculate that low PLGF concentration could influence vascular permeability of fetal placental vessels and thereby participate in the physiopathological mechanism leading to hormonal maternal serum changes associated with trisomy 21 pregnancy. We suggest that PLGF could therefore be a new maternal serum marker for Down’s syndrome screening, but further studies will be needed on a larger series to assess its usefulness in improving the sensitivity and specificity of the actual screening test.

Acknowledgements Placental and serum samples were collected with the kind help of Prof. P.Bernard from the obstetrics department. Karyotypes were obtained thanks to Prof. C.Verellen-Dumoulin and Dr M.Freund from the medical genetic centre. The authors are grateful to C.Brulet and A.M.Delait for their excellent technical assistance. Fre´ de´ ric Debie`ve is a Research Assistant from the ‘Fonds National de la Recherche Scientifique’ (F.N.R.S., Belgium) and research is supported by a grant from FNRS (grant 3.4501.97, FRSM).

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