Background
Adverse gestational outcomes such as preeclampsia (PE) and intrauterine growth restriction (IUGR) are thought to be caused, at least in part, by deficiencies in processes critical to placental development, including extravillous trophoblast (EVT) invasion. EVT invade the maternal decidua and replace the endothelium of uterine spiral arteries, thus increasing vessel diameter to ensure adequate blood flow required for oxygen and nutrient delivery to the placenta [
1,
2]. In both human villous explants and primary trophoblast cultures, insulin-like growth factors I and II (IGF-I and IGF-II, respectively), stimulate trophoblast proliferation and EVT migration [
3,
4].
The bioavailability of IGF-I and -II is modulated by six insulin-like growth factor-binding proteins (IGFBPs) [
3,
5,
6], with the release of the IGFs generally achieved through proteolysis of the IGFBPs [
7]. In addition to reducing the availability of the IGFs, in some contexts IGFBPs increase the half-life of IGFs, concentrate them in particular regions and/or potentiate their effects [
8]. Furthermore, IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-5 all exert IGF-independent effects in a variety of cell models and tissues [
3,
6,
9]. For example, IGFBP-1 stimulates migration in an IGF-independent manner in HTR-8/SVneo cells, an immortalized trophoblast cell line commonly used to model EVT migration and invasion [
10]. In BeWo cells, an immortalized choriocarcinoma cell line commonly used to model the villous cytotrophoblast, IGFBP-3 inhibits proliferation in an IGF-independent manner [
11]. The proteolytic fragments of cleaved IGFBP-3 and −5 have also been shown to have effects in other systems [
12‐
14].
The pappalysins, pregnancy-associated plasma proteins-A and -A2 (PAPP-A and PAPP-A2, respectively), are two IGFBP proteases that have been consistently associated with a range of pregnancy complications. PAPP-A is a protease of IGFBP-4 and −5 that is produced by the placenta [
15] and circulates in the maternal blood at high levels during pregnancy. Abnormally low levels of PAPP-A in the first trimester have frequently been associated with increased risk of PE and IUGR [
16]. Pregnancy-associated plasma protein-A2 (PAPP-A2) shares 45% amino acid identity with PAPP-A, cleaves IGFBP-5 but not IGFBP-4, and is also produced by the syncytiotrophoblast and released into the maternal circulation during pregnancy [
17]. PAPP-A2 levels in the placenta and maternal circulation are higher at term in preeclamptic pregnancies and pregnancies with severe fetal growth restriction [
18‐
21], and PAPP-A2 levels are also elevated in the first-trimester maternal serum of pregnancies that subsequently develop preeclampsia [
22].
The mechanistic links between circulating pappalysin levels and pregnancy complications, and whether altered pappalysin expression plays a causal role in placental pathologies, remain unknown. However, whatever role the pappalysins play in placental development and physiology likely involves their IGFBP substrates, IGFBP-4 and −5. While IGFBP-1 is the most abundant IGFBP within the maternal decidua [
23] and is relatively well-studied, the roles of IGFBP-4 and −5 in placental development have received little attention.
The purpose of this study was to investigate whether the pappalysins might influence EVT migration and invasion through effects on IGF availability and/or other actions of the IGFBPs. We employed a well-established model of first trimester EVT to examine the effects of exogenous IGFBP-4 and −5 on the migration-stimulating effects of IGF-I and IGF-II. We focused primarily on the inhibition of the migration, but also tested for potentiation and IGF-independent effects. The IGFBPs are known to be expressed in the maternal decidua [
23] and therefore are well positioned to regulate EVT invasion. However, to examine whether IGFBP-4 and −5 might also regulate processes within the villi, we examined the expression of these binding proteins in first-trimester villi using immunohistochemistry.
Discussion
To investigate the potential mechanism underlying the associations between levels of PAPP-A and PAPP-A2 and pregnancy complications such as PE and IUGR, we studied the effects of pappalysin substrates IGFBP-4 and −5 on IGF-I and -II in a model of EVT migration. We also examined the location and timing of IGFBP-4 and −5 expression to determine whether the pappalysins might also influence processes occurring within the villi early in pregnancy.
Consistent with previous findings in various models of first trimester human EVT [
30‐
36], both IGF-I and IGF-II at 2 nM significantly stimulated migration of HTR-8/SVneo cells in a cell-wounding assay. Furthermore, IGF-I had greater stimulatory effects than IGF-II at this concentration. The difference in effects of IGF-I and –II may be due to the presence of both type 1 and type 2 IGF receptors (IGF1R and IGF2R) in HTR-8/SVneo cells [
26,
36], and the higher binding affinity of IGF1R for IGF-I than for IGF-II [
37].
IGFBP-4 and −5 showed different levels of IGF inhibition. IGFBP-4 was able to block the migration-stimulating effects of both IGF-I and IGF-II to control levels. In contrast, IGFBP-5 was able to inhibit the migration-stimulating effects of IGF-II to control levels, but only partially inhibited IGF-I. While previous work has shown IGFBP-5 blocks IGF-II stimulation of migration in a cell line model of EVT [
26], this is to our knowledge the first data regarding the effects of exogenous IGFBP-4 on trophoblast migration. IGFBP-4 has however been found to influence migration and invasion in cancer studies, with inhibitory or stimulatory effects on migration depending on the model examined [
38‐
42]. Similarly, the effects of IGFBP-5 on cellular proliferation and invasion in breast cancer studies appear to be cell line dependent [
43]. The inhibitory effect of IGFBP-4 on trophoblast migration may, at least in part, underlie the association between elevated circulating levels of IGFBP-4 in early pregnancy and the subsequent development of fetal growth restriction [
44].
There was no evidence of potentiation of the effects of IGF-I by low doses of IGFBP-5, either because no such effect exists
in vivo, or because IGF potentiation requires interaction between IGFBP-5 and the extracellular matrix [
45]. Similarly, the lack of IGF-independent effects of IGFBP-4 or −5 in our experiments may be due to a real absence of effects
in vivo, to the absence of as-yet-uncharacterized cell surface IGFBP-4 and −5 receptors in HTR-8/SVneo cells [
29], or to a requirement for interaction with the extracellular matrix [
45] or proteolysis of the IGFBPs [
12,
14].
IGF-I and -II have different primary sources in the placenta. IGF-II is strongly expressed by EVT and syncytiotrophoblast in the human placenta [
3,
4,
23], whereas the predominant source of IGF-I in the placenta is the maternal circulation, as it is only weakly expressed by the placenta [
4,
23]. Low levels of IGF-I mRNA relative to IGF-II mRNA have been detected in the cytotrophoblast, mesodermal core and endothelium of human placental villi (in first, second and third trimesters), but not in EVT [
30,
46,
47]. IGF-I has been detected less consistently at the protein level. In one study, IGF-I protein was undetectable in human placental lysates [
48], and in another IGF-I protein was detected in first trimester villous cytotrophoblasts, but at much lower levels than IGF-II [
47].
A number of observations from previous work and this study suggest that PAPP-A and PAPP-A2 play very different roles in normal placental development and disease, which may explain why PAPP-A and PAPP-A2 show contrasting levels in relation to adverse pregnancy outcomes (i.e., PAPP-A being down-regulated and PAPP-A2 being upregulated in association with pregnancy complications [
16]). PAPP-A is a protease of both IGFBP-4 and IGFBP-5, whereas PAPP-A2 only proteolyzes IGFBP-5. Therefore our data suggest that PAPP-A2 has less of a stimulatory effect on EVT migration than PAPP-A, as IGFBP-5 shows less inhibition of IGF-I, which is more potent than IGF-II. Furthermore, PAPP-A may have a stronger effect modulating the availability of IGF-I from the maternal circulation than PAPP-A2.
PAPP-A and PAPP-A2 are expressed by the syncytiotrophoblast as well as invasive EVT [
49,
50]. It was previously thought that the only location of expression of IGFBP-4 and −5 in the first trimester was the maternal decidua, since IGFBP-5 had only been observed in villi in the second and third trimester [
23]. More recently, IGFBP-4 immunoreactivity has been observed in the chorionic mesoderm of placental villi at 10–13 weeks [
44]. Here, we show that IGFBP-4 and −5 are present in the syncytiotrophoblast of placental villi as early as 5 weeks of gestation. PAPP-A and PAPP-A2 may therefore influence aspects of early placental development in addition to the EVT invasion of the decidua and remodeling of the spiral arteries, e.g., cytotrophoblast proliferation or fusion with the syncytiotrophoblast. While we do not know whether the placentae we sampled were from pregnancies that would have gone on to develop pregnancy complications, we observed consistent results in 6 different placentae, 3 of which were at a gestational age of 7 weeks or less. It is very unlikely that none of these early placentae were from a healthy, uncomplicated pregnancy.
Acknowledgements
This work was supported by a Canadian Institutes of Health Research Master’s Award (Frederick Banting and Charles Best Canada Graduate Scholarships), Simon Fraser University (SFU) graduate fellowships and a Phyllis Carter Burr Scholarship to EJC, and SFU Vice-President, Research, Bridging and NSERC Discovery Grants to JKC. We thank Dr. Gordon Rintoul for the use of his cell culture facilities at Simon Fraser University, Pavel Ogay for laboratory assistance, and Bryce Pasqualotto, Lubna Nadeem, Bo Peng and Tim Beischlag for advice and guidance.
This paper is dedicated to the memory of Dr. Andrée Gruslin, who provided guidance and mentorship in this and so much of our other work.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
EJC performed all cell culture work and statistical analyses and drafted the manuscript. CED and AGB provided guidance with methodology, prepared sections for immunohistochemistry, and provided comments on the manuscript. JKC conceived of the study, participated in its design, performed the immunohistochemistry, and helped to draft the manuscript. All authors read and approved the final manuscript.