Molecular Human Reproduction, Vol. 9, No. 7, 395-398,
July 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Modulation of placental vascular endothelial growth factor by leptin and hCG
Submitted on April 17, 2002; resubmitted on February 5, 2003. accepted on March 28, 2003
Department of Obstetrics and Gynaecology, Clinic of Infertility and Gynaecological Endocrinology–WHO Collaborating Centre in Human Reproduction, University Hospital of Geneva, 32 Boulevard de la Cluse, 1211 Geneva, Switzerland
1 To whom correspondence should be addressed at: Clinique de Stérilité et d’Endocrinologie Gynécologique, Hôpital Cantonal Universitaire de Genève, 32, Boulevard de la Cluse, 1211 Genève 14, Switzerland. e-mail: paul.bischof{at}hcuge.ch
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ABSTRACT |
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Vascular endothelial growth factor (VEGF) has been identified as an endothelium-specific mitogen and inducer of angiogenesis and endothelial cell survival. Leptin and hCG have also been suggested as possible regulators of angiogenesis in various models. In-vivo and in-vitro assays revealed that leptin has an angiogenic activity and that the vascular endothelium is a target for leptin. Thus, we hypothesized that products of cytotrophoblastic cells may play a role in placental angiogenesis and we therefore investigated the effects of leptin and hCG on cytotrophoblast VEGF secretion. We incubated cytotrophoblastic cells (CTB) with recombinant human leptin (rhLept) (0–4 pg/ml) or hCG (0–30 000 IU/ml) for 4 h. rhLept significantly stimulated hCG (P = 0.0045) and decreased VEGF release (P = 0.0008) by CTB in a concentration-dependent manner. On the other hand, increasing concentrations of hCG (0–30 000 IU/ml), induced a significant inhibition of leptin secretion (P = 0.0028) and a marked dose-dependent stimulation of VEGF165 secretion (P < 0.0001). We observed an increase of >1000-fold in basal trophoblastic VEGF secretion with physiological concentrations of hCG in vitro. An inhibitory effect of hCG on trophoblastic leptin secretion was also observed, suggesting that hCG might exert a possible negative feedback on trophoblastic release of leptin. We hypothesize that trophoblastic products such as hCG and leptin are probably involved in the control of VEGF secretion at the maternal–fetal interface.
Key words: angiogenesis/hCG/leptin/trophoblast/VEGF
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Introduction |
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Angiogenesis is generally a quiescent process in the healthy adult male with an extremely low turnover of endothelial cells. However, in the female, angiogenesis is highly regulated and turned on for brief periods of time in selected organs such as the ovary and the endometrium. Examples of such regulated angiogenesis include follicular growth, corpus luteum formation, endometrium differentiation and repair, and embryo implantation and development (Reynolds et al., 1992; Reynolds and Redmer, 1995). Among the list of angiogenic factors (Folkman and Klagsbrun, 1987), vascular endothelial growth factor (VEGF) was identified as an endothelium-specific mitogen and inducer of angiogenesis and endothelial cell survival (Plouet et al., 1989; Conn et al., 1990; Alon et al., 1995; Gerber et al., 1998).
Analysis of the DNA sequence of a variety of human VEGF clones indicates that VEGF may exist as one of four different isoforms as a result of alternative splicing. VEGF165 is the major VEGF isoform (Ferrara, 1999). In human placenta, VEGF expression has been analysed by in-situ hybridization and immunohistochemical studies (Ahmed et al., 2000). It is localized to the villous trophoblast, the decidua and macrophages of both fetal and maternal origin (Sharkey et al., 1993; Ahmed et al., 1995; Vuorela et al., 1997). Trophoblast cells also secrete VEGF in vitro (Shore et al., 1997; Ahmed et al., 2000). VEGF appears unique in its role as an angiogenic factor, in that the two known human receptors for VEGF, VEGFR-1 (formerly flt-1) and VEGFR-2 (formerly KDR) are expressed on endothelial cells (de Vries et al., 1992; Terman et al., 1992). Interestingly, human trophoblast cells, particularly the invasive extravillous cytotrophoblast cells (CTB) also express VEGF receptors (Charnock-Jones et al., 1994; Athanassiades et al., 1998). Both VEGF receptor forms are present in first trimester placental tissue (Ahmed et al., 1995; Vuckovic et al., 1996).
Leptin and hCG, both produced by human trophoblasts (Chardonnens et al., 1999), have also been suggested as possible regulators of angiogenesis in various models (Rizk et al., 1997; Henson and Castracane, 2000). Leptin was found to generate a growth signal via a tyrosine kinase-dependent pathway and promote angiogenic processes via activation of the leptin receptor (Ob-R) in endothelial cells (Park et al., 2001). In-vivo and in-vitro assays revealed that leptin has an angiogenic activity and that vascular endothelium is a target for leptin (Sierra-Honigmann et al., 1998). In contrast to the leptin-deficient ob/ob mice, where no vascular fenestrations are detected, capillary fenestrations are found in leptin-producing adipose tissue in lean mice. Thus, leptin plays a critical role in the maintenance and the regulation of vascular fenestrations in adipose tissue. Furthermore, leptin and fibroblast growth factor (FGF)-2 and VEGF synergistically stimulate angiogenesis (Cao et al., 2001).
Taking these observations together, we hypothesized that products of cytotrophoblastic cells may play a role in placental angiogenesis. We therefore investigated the effects of leptin and hCG on cytotrophoblast VEGF secretion.
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Materials and methods |
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CTB were isolated and purified as previously described (Bischof et al., 1991). Briefly, trophoblast tissue was obtained from legal abortions at 8–12 weeks of normal pregnancy (from the last menstrual period). The tissue was digested with trypsin and cytotrophoblastic cells separated from blood cells and syncytia on a discontinuous Percoll gradient and immunopurified by antibody-coated magnetic particles (anti-CD45; Dyna Beads, Switzerland) in order to eliminate contaminating leukocytes. After immunopurification the cell suspension represents 90–97% cytotrophoblastic cells, with a 3–7% contamination by fetal stromal cells. Lymphomyeloid cells are no longer present (Bischof et al., 1991). CTB viability was assessed by Trypan Blue (Sigma) exclusion and cells diluted to 106 cells/ml with culture medium. Recombinant human leptin (rhLept) was purchased from R&D Systems (Bühlmann Laboratories, Switzerland). hCG (Profasi) was purchased from Ares–Serono (Switzerland).
Culture conditions
CTB (100 µl, 106 cells/ml, >90% viability) were incubated in duplicates in the presence or absence of rhLept (0–4 pg/ml) or hCG (0–30 000 IU/ml). Incubation was performed in 12-well tissue culture plates (Costar, USA) under a 5% CO2 and 95% air atmosphere in a humid incubator at 37°C for 4 h. Culture medium was Dulbecco’s modified Eagle’s medium (Gibco, Switzerland) containing 2 mmol/l L-glutamine, 4.2 mmol/l magnesium sulphate, 25 mmol/l HEPES, 1% gentamycin, 1% amphotericin B, 100 µg/ml streptomycin and 100 IU/ml penicillin, in the absence of serum. The supernatants were collected after 4 h of incubation, aliquoted and stored at –20°C until assayed. Duplicate wells were run for each treatment condition and the experiments repeated four times with different CTB preparations.
Hormone and protein assays
VEGF165 was measured in the supernatants by an enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Germany) with a sensitivity of 5.0 pg/ml and intra- and inter-assay coefficient of variation (CV) <6.5 and <8.5% respectively. HCG was measured in the supernatants by a microparticle enzyme immunoassay (IMX; Abbot, USA) with a sensitivity of 0.1 mIU/ml and intra- and inter-assay coefficients of variation <4%. Leptin was measured in the supernatants by an ELISA (DRG diagnostics, Cis-Medipro, Switzerland) with a sensitivity of 0.2 ng/ml and an intra- and inter-assay CV of 4.3 and 8.6% respectively. According to the manufacturer, the leptin assay does not cross-react with human proteins such as proinsulin, C-peptide, glucagon or insulin-like growth factor-I.
Statistics
In order to normalize the distribution and minimize the inter-experimental variability, all results were expressed as a percentage of their respective controls, CTB in the absence of hCG or rhLept, and presented as mean ± SEM. Statistical evaluations were performed with a personal computer using the Statview Program (Abascus). Tests included one-way analysis of variance with Fisher’s test to compare the effects of different concentrations of rhLept or hCG. Regression analysis was performed to determine the dose dependency of rhLept or hCG effects. P < 0.05 was considered to be statistically significant.
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Results |
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Effects of leptin on hCG and VEGF secretion in human CTB
RhLept (0–4 µg/l) augmented hCG release by CTB in a concentration-dependent manner (P = 0.0045, Figure 1). Basal hCG release was 118 ± 12 IU/l. When compared to controls, hCG release was 115.7 ± 6.8% (P = 0.0436), 117.3 ± 6% (P = 0.0317), 127 ± 5.7% (P = 0.0058) and 130.5 ± 7.5% (P = 0.0034), after incubation with 0.5, 1, 2 and 4 pg/ml of rhLept respectively.
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RhLept (0–4 µg/l) decreased VEGF release by CTB in a concentration-dependent manner (P = 0.0008, Figure 2). Basal VEGF release was 7.0 ± 0.5 pg/ml. When compared with control, VEGF release was 94 ± 1.8% (P = 0.0013), 95.2 ± 1.2% (P = 0.0059) and 92 ± 1.9% (P = 0.0001) after incubation with 1, 2 and 4 pg/ml of rhLept respectively. However, this modest effect was still observed after incubation for 20 h (results not shown).
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Effects of hCG on leptin secretion in human CTB
Basal leptin secretion was 1.29 ± 0.17 ng/ml. When incubated for 4 h (n = 4) with increasing concentrations of hCG (0–30 000 IU/ml), a significant inhibition of leptin secretion was observed. When compared with controls, leptin secretion was 92.3 ± 2.9% (P = 0.0095), 91.3 ± 5.3% (P = 0.0042), 90.8 ± 2% (P = 0.0028) and 90.8 ± 5.2% (P = 0.0028) after incubation with 5000, 10 000, 20 000 and 30 000 IU/ml of hCG respectively (Figure 3). This effect was still observed after incubation for 20 h with removal of hCG at 4 h (results not shown).
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Effects of hCG on VEGF secretion in human CTB
CTB were incubated for 4 h with raising concentrations of hCG (0–30 000 IU/ml, n = 4 experiments). Basal VEGF secretion was 111.9 ± 0.1 pg/ml. There was a marked dose-dependent increase in VEGF165 secretion (P < 0.0001) in response to hCG reaching a maximum of 106 000 ± 14 500% of controls (P < 0.0001), when incubated with 30 000 IU/ml of hCG (Figure 4). When VEGF secretion was measured at 8 h after incubation with hCG, VEGF secretion was dose-dependently increased 10-fold above controls (P < 0.0001). At 24 h after incubation, we did not find any effect of hCG on trophoblastic VEGF secretion (results not shown).
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Discussion |
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An appropriate blood supply is essential for the development of the feto-placental unit. Maternal blood flow in the intervillous space increases 20-fold during pregnancy due to vasomotor changes of the distal intramyometrial portions of uteroplacental arteries and the transformation and dilatation of decidual segments. This results essentially from the invasive behaviour of cytotrophoblastic cells that invade and colonize the endometrial spiral arteries allowing for a loss of their elasticity (Fisher, 2000). The increased blood requirement is also met by intense angiogenic processes taking place in the fetal stroma of the placental villi and the maternal endometrium. Abnormal angiogenesis during early pregnancy may lead to fetal growth retardation and/or pre-eclampsia (Roberts, 1998). Although several angiogenic factors have been identified at the feto-maternal interface, the regulation of this process remains obscure (Folkman and Klagsbrun, 1987; Smith, 2000).
In the present study we observed an increase of >1000-fold in trophoblastic VEGF secretion in vitro when CTB were cultured with physiological concentrations of hCG. This observation is consistent with other studies where VEGF expression was induced by LH in the ovary (Geva and Jaffe 2000) and by hCG in human endometrium (Licht et al., 2001) and in the ovary (Lee et al., 1997). This indicates that hCG probably plays an important role in the control of endometrial and placental vascularization by paracrine (on the endometrium) as well as juxtacrine (on the trophoblast) mechanisms. Since the stimulatory effect of hCG on VEGF secretion is no longer observed at 24 h, it is tempting to speculate that hCG favours VEGF secretion rather than synthesis.
Since leptin stimulates hCG secretion through a GnRH-dependent pathway (Chardonnens et al., 1999) and increases the pulsatile release of hCG in first-trimester trophoblastic explants (Islami et al., 2003), we wondered if this peptide was able to directly or indirectly (through hCG) modify the expression of VEGF. Despite leptin stimulating hCG, it inhibited the release of VEGF. Although this effect was statistically significant and maintained up to 24 h of culture, it was modest compared with the effect of hCG on VEGF. The physiological significance of an inhibitory effect of leptin and a stimulatory effect of hCG on trophoblastic VEGF secretion is far from being understood particularly since in vivo hCG is produced by syncytiotrophoblast and not cytotrophoblast as in in-vitro experiments. Thus our results cannot easily be applied to an in-vivo situation.
It is also interesting to note that, in addition to the stimulatory effect of leptin on hCG secretion, we observed an inhibitory effect of hCG on trophoblastic leptin secretion. This effect seems to be maximal at 5000 IU/ml of hCG, possibly because of saturation of hCG receptors. This new observation suggests that hCG might exert a possible negative feedback on the trophoblastic release of leptin.
Our in-vitro observations are somewhat difficult to reconcile with in-vivo observations in patients with pre-eclampsia. Indeed, it has been reported that this pathology increases circulating VEGF (Sharkey et al., 1996) as well as leptin (Mise et al., 1998). Both of these increases can be mimicked in vitro by culturing trophoblastic cells under hypoxic conditions (Taylor at al., 1997; Grosfeld et al., 2001). In contrast, hCG is decreased under such hypoxic culture conditions (Esterman et al., 1996). Thus, if these in-vitro results are also true in vivo, then in pre-eclamptic pregnancies the stimulation of VEGF by hCG would be reduced. One possible explanation for this discrepancy is that hypoxia-induced VEGF release can be regarded as a compensatory mechanism (angiogenesis) that employs a different pathway from normoxic conditions. However, these in-vitro experiments were carried out with term placentas, where the interactions between hypoxia, hCG and VEGF could be different from first trimester trophoblast. During first trimester pregnancy, we would speculate that hypoxia induces hCG secretion, which stimulates trophoblastic VEGF release. Clearly the physiological interaction between hCG, leptin and VEGF under hypoxic conditions is a complex one and remains to be studied in an appropriate model. Our data support the idea that trophoblastic products such as hCG and leptin are probably involved in the control of VEGF secretion at the maternal–fetal interface.
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Acknowledgements |
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We thank Mrs Christine Wuillemin and Mrs Claire Gruffat for their skilful technical help. D.Islami is the recipient of a scholarship from the Novartis Research Foundation.
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