Localization of Angiogenic Growth Factors and Their Receptors in the Human Placental Bed Throughout Normal Human Pregnancy
Introduction
Invasion by extravillous trophoblast (EVT) cells into uterine decidua, the inner third of the myometrium (interstitial trophoblast) and uterine spiral arteries (endovascular trophoblast) is an essential feature of normal human placentation. This process results in reversible remodelling of the normal musculo-elastic structure of the media of the spiral arteries which is replaced by fibrinoid material containing trophoblast, converting the spiral arteries into large dilated vessels of low resistance [1], [2], [3]. This so-called ‘physiological transformation’ affects decidual segments of spiral arteries in early pregnancy followed by myometrial segments and is complete by 20–22 weeks of gestation [4]. Failure of trophoblast invasion and spiral artery transformation has been implicated in the pathogenesis of late miscarriage, pre-eclampsia, fetal growth restriction, placental abruption, preterm delivery and preterm premature rupture of membranes [2], [5], [6], [7], [8], [9].
Despite the importance of spiral artery remodelling the controlling mechanisms are poorly understood. Angiogenic growth factors and cytokines, particularly the vascular endothelial growth factor (VEGF) family and the angiopoietin (Ang) family, produced by extravillous trophoblast and various decidual cell populations, including uterine natural killer (NK) cells, have been implicated in this process [10], [11], [12].
The Tie-2 receptor and its ligands Ang-1 and Ang-2 play fundamental roles in angiogenesis and remodelling of vessel structure. Ang-1 is a secreted protein of 498 amino acids that contains an NH2-terminal coiled domain and a COOH-terminal fibrinogen domain [13]. Ang-1 does not induce endothelial cell proliferation in vitro, but is chemotactic for human endothelial cells and promotes angiogenesis in vivo [13]. Ang-2 contains 496 amino acids and is 60% homologous to Ang-1. As an antagonist ligand for Tie-2, Ang-2 plays a role in dilatation of vessels and disruption of vessel integrity [13], [14], [15]. Since Ang-2 antagonizes the stabilizing effect on vessels of Ang-1, the ratio of these two factors appears to be of importance for vessel formation [13].
In humans Ang-2 has been reported to be expressed in villous syncytiotrophoblast and Tie-2 in both fetal and maternal endothelial cells within decidua [13]. Zhang et al. [13] used in situ hybridisation to investigate placental Ang-2 expression and reported lower mRNA levels in villous syncytiotrophoblast at term compared to the first trimester, the time of branching angiogenesis [13]. In contrast, Leach et al. [16] reported predominance of VEGF-A in the human placenta in early pregnancy, with Ang-1 present in large vessels and Ang-2 in terminal villous capillaries only in term placentas [16].
The VEGF-family comprises VEGF-A, -B, -C, -D, -E and placental growth factor (PlGF) which interact with various receptors including VEGF-R1 (Flt-1), -R2 (KDR), -R3 (Flt-4), soluble VEGF-R1 (sFlt-1), neuropilin (NRP) -1, -2 and heparin sulphate proteoglycan [17]. There are five isoforms of VEGF-A derived from alternative mRNA splicing, of which VEGF-A165 is the best studied [17]. VEGF-A has been shown to be a potent angiogenic factor and is critical for placental angiogenesis [18]. In addition, VEGF-A has been reported to inhibit invasiveness of two different trophoblast-like cell lines [19], [20]. VEGF-A is also a permeability factor leading to vascular leakage [17]. The different actions of VEGF-A are mediated by VEGF-R1, VEGF-R2, NRP-1 and NRP-2, whereas sVEGF-R1 acts as a natural inhibitor of VEGF-A [17]. VEGF-C and VEGF-D interact with VEGF-R2 and -R3 and are primarily involved in lymphangiogenesis, although they also possess some angiogenic properties [17].
While both families of angiogenic growth factors have been widely studied in the placenta, their potential role in spiral artery remodelling has not been adequately investigated. Although evidence of spatial and temporal regulation of some of these factors in decidua has been reported [21], [22], experimental numbers were low and examination limited to the superficial decidua attached to the delivered placenta rather than true placental bed. The aim of the current study therefore was to determine the spatial and temporal localization of VEGF-A, -C, -D, -R1, -R2 and -R3 and Ang-1, Ang-2 and their receptor Tie-2 in true placental bed biopsies in normal human pregnancy from 8 weeks to term. We hypothesized that, if these angiogenic growth factors play an important role in spiral artery transformation, their expression may vary with gestational age and differ between different trophoblast subpopulations.
Section snippets
Placental bed biopsies
Placental bed biopsies were obtained after elective termination of apparently normal pregnancies at 8–10 (n = 6), 12–14 (n = 6) and 16–20 weeks (n = 6) of gestation and during elective caesarean section of normal pregnancies at 37–42 weeks (n = 6) at the Royal Victoria Infirmary, Newcastle upon Tyne. Placental bed biopsies were taken as previously described using biopsy forceps (Richard Wolf Endoscopes, Wimbledon, UK) [23]. All women gave informed written consent for the biopsy. The study was approved
Results
Mean immunohistochemical staining scores (±SEM) for Ang-1, Ang-2, Tie-2, VEGF-A, -C, -D, -R1, -R2 and -R3 are summarised in Table 2, Table 3. Representative photomicrographs are shown in Fig. 1A–J. Several different cell types in the placental bed biopsies were positive for Ang-1, Ang-2, Tie-2, VEGF-A, -C, -D, -R1, -R2 and -R3, including extravillous trophoblast, decidual stromal cells, myometrial cells, leucocytes in decidua and myometrium, vascular endothelial cells and vascular medial smooth
Discussion
This is the first report of the temporal and spatial localization of the angiopoietin and vascular endothelial growth factor families in extravillous trophoblast in the true placental bed throughout pregnancy. We report that there is an increase in the level of immunostaining of intramural EVT for Tie-2 and VEGF-C with increasing gestational age. In addition, there was a reduction in Ang-1 and Ang-2 expression by multinucleate interstitial EVT and of VEGF-R1 and VEGF-R2 by endovascular EVT with
Acknowledgements
The authors wish to acknowledge the staff at the Royal Victoria Infirmary, Newcastle upon Tyne for their assistance in sample collection.
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