Published online before print June 30, 2006
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* Schools of Surgical and Reproductive Sciences,
Medical Education Development, and
Clinical and Laboratory Sciences, University of Newcastle upon Tyne, United Kingdom
1 Correspondence: School of Surgical and Reproductive Sciences, 3rd Floor, William Leech Building, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, UK. E-mail: g.e.lash{at}ncl.ac.uk
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Key Words: uNK cells • angiogenesis • VEGF-C • Ang2
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During early pregnancy, extravillous trophoblast cells (EVT), derived from the placenta, invade the decidua and inner third of the myometrium. There are two routes of EVT invasion: through the decidua, giving rise to interstitial EVT, and up the lumen of the spiral arteries, giving rise to endovascular EVT. Intramural EVT are detected within the wall of spiral arteries, but whether these cells are derived from an interstitial or endovascular source remains unclear [1 , 3 , 5 ]. Although it has been suggested that endovascular and interstitial EVT are responsible for spiral artery remodeling, others have observed initiation of this process in the absence of EVT [8 , 9 ] at a time when spiral arteries are surrounded by uterine natural killer (uNK) cells [10 ]. It has recently been postulated that there is a nontrophoblast or decidual component and then a trophoblast-dependent component of spiral artery remodeling [2 ].
uNK cells are a major leukocyte population within the early pregnant uterus, accounting for 
70% of all decidual leukocytes [10
]. These cells are phenotypically characterized as CD56 bright/CD16 dim and are distinct from the majority of peripheral blood NK cells. The role of these cells within the placental bed remains unclear, although they secrete cytokines capable of regulating trophoblast invasion in vitro [11
]. In addition, in the nonpregnant endometrium, in situ hybridization studies have localized mRNA for growth factors that may be involved in spiral artery remodeling [such as angiopoietin 2 (Ang2), placental growth factor (PlGF), and vascular endothelial growth factor-C (VEGF-C)] to uNK cells [12
]. Furthermore, spiral artery remodeling is not observed during pregnancy in mice lacking uNK cells [13
], but on transplantation of bone marrow from mice with NK cells, these animals regained the ability of spiral artery transformation [14
]. Recently, a significant role for uNK cell-derived interferon-
(IFN-) in initiating spiral artery remodeling has been postulated [14
, 15
]. However, in the human, only low levels of uNK cell-derived IFN- have been observed [11
, 16
].
We hypothesized that uNK cells obtained from early pregnancy decidua are a major source of angiogenic growth factors, which are likely to play a key role in spiral artery remodeling in normal pregnancy.
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uNK cell isolation
Total decidual cell suspensions and CD56+ cell-enriched isolates were prepared as described previously [18
]. Briefly, decidual tissue was minced, DNase/collagenase-treated, allowed to adhere overnight, and used as total decidual cell suspensions or subjected to anti-CD56 (Coulter, High Wycombe, UK)-reactive magnetic bead separation (MidiMACS, Miltenyi Biotec, Surrey, UK) to obtain CD56+ cell suspensions of >95% purity. After isolation, a fraction of the total decidual suspension and CD56+ cells was snap-frozen for RNA isolation (t=0). Total decidual cell suspensions or CD56+ uNK cells were plated in a 96-well plate at a concentration of 1 x 105 cells/well in 100 µl RPMI 1640 [containing 1000 U/ml penicillin, 1 mg/ml streptomycin, 2 mM L-glutamine, and 10% fetal bovine serum (all from Sigma Chemical Co., Poole, UK)] and incubated for 24 and 48 h. Conditioned medium was removed and stored at –20°C until required for analysis. Using this methodology, the CD56+ cell-enriched isolate was consistently >95% pure [19
]. Approximately 25% of the total decidual cell suspension is CD56+ cells.
FASTQuant® microspot assays for angiogenesis factor quantification
A FASTQuant human angiogenesis array for angiogenin, Ang2, platelet-derived growth factor (PDGF)-BB, VEGF-A, fibroblast growth factor basic (FGF-b), keratinocyte growth factor (KGF), and intercellular adhesion molecule (ICAM)-1 was run according to the manufacturer’s instructions (Whatman Schleicher and Schuell, Dassel, Germany). Each kit contains four glass slides arranged with 16 nitrocellulose pads on which reference markers and capture antibody for analytes in that array are dotted in triplicate using nanodot technology. Briefly, slides were blocked by incubating in 70 µl blocking buffer for 30 min with shaking at room temperature; the blocking buffer was removed, and 70 µl samples or standards was added to the appropriate well and incubated overnight. The slides were then washed three times in wash buffer, and 70 µl biotinylated detection antibody was added and incubated for 1 h. Following a further three washes, 70 µl streptavidin-Cy5 solution was added, and the slides were incubated for 45 min in the dark, washed three times, and allowed to dry. The slides were imaged using a GenePix scanner (Axon, Molecular Devices, Workingham, Berks, UK), and data analysis was performed using the ArrayVisionTMFAST® software. The dynamic range for angiogenin and VEGF was 2.4–2500 pg/ml and for all other analytes, was 12.2–12,500 pg/ml.
Enzyme-linked immunosorbent assay (ELISA) for VEGF-C, PlGF, transforming-growth factor-ß1 (TGF-ß1), and Ang2
Total decidual cell suspension and CD56+ cell-conditioned medium were tested for levels of secreted VEGF-C (Bender Medsystems, Vienna, Austria), PlGF, TGF-ß1, and Ang1 (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. The dynamic range of the VEGF-C, PlGF, Ang1, and TGF-ß1 ELISAs was 47.5–3000 pg/ml, 15.6–1000 pg/ml, 54.7–3500 pg/ml, and 31.3–2000 pg/ml, respectively.
Immunohistochemistry
For immunohistochemistry, placental bed biopsies were fixed in 10% neutral-buffered formalin for 24–48 h, routinely processed, embedded in paraffin wax, sectioned at 3 µm thickness, and mounted on 3-aminopropyl-triethoxysilane (Sigma Chemical Co.)-coated slides. Serial sections were immunostained for VEGF-A, VEGF-C, VEGF-receptor-(R)2, Ang1, Ang2, TIE2, TGF-ß1, and CD56 using the antibodies detailed in Table 1
, which were detected using an avidin biotin peroxidase technique (mouse or rabbit Vectastain Elite ABC kit, Vector Laboratories, Peterborough, UK), as described previously [20
]. The reaction was developed with 3,3'-diaminobenzidine (Sigma Chemical Co.). The sections were counterstained lightly with Mayer’s haematoxylin (BDH, Poole, UK) and mounted with DPX synthetic resin (Raymond A. Lamb Ltd., London, UK). Immunostaining was not quantified.
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Table 1. Antibodies Used for Immunohistochemistry on Paraffin Sections
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Table 2. Primer and Probe Sequences Used for Real-Time Reverse Transcriptase-PCR
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Figure 1. Levels of secreted protein as determined by FASTQuant angiogenesis array for (A) angiogenin, (B) Ang2, (C) FGF-b, (D) ICAM-1, (E) KGF, (F) PDGF-BB, and (G) VEGF-A in total decidual cell isolates (1x106 cells/ml) and uNK cell-enriched isolates (1x106 cells/ml), which had been cultured for 48 h. n = 5 each group; mean ± SEM.
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Figure 2. Levels of secreted protein as determined by ELISA for (A) VEGF-C, (B) Ang1, (C) PlGF, and (D) TGF-ß1 in total decidual cell isolates (1x106 cells/ml) and uNK cell-enriched isolates (1x106 cells/ml), which had been cultured for 48 h. n = 5 each group; mean ± SEM.
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Unfractionated total decidual cell suspensions versus uNK cells
In 8–10 weeks gestational age samples, protein levels secreted by uNK cells were lower compared with total decidual cell suspensions for five analytes (angiogenin, P=0.05; FGF-b, P=0.009; ICAM-1, P<0.0001; KGF, P=0.009; PDGF-BB, P=0.0002). In contrast, there was significantly more Ang1 secreted by uNK cell samples than the total decidual cell suspensions (P=0.01).
At 12–14 weeks gestation, there was significantly less VEGF-C secreted by the uNK cells compared with total decidual cell suspensions (VEGF-C, P=0.009) and a trend toward lower ICAM-1 secretion (P=0.07). There was no difference in the levels of PlGF secreted by any of the groups tested. However, three of seven total decidual cell suspensions and five of seven uNK cell samples from 8–10 weeks gestation and two of five total decidual cell suspensions and two of five uNK cell samples were below the detection limit of the PlGF assay. TGF-ß1 levels did not differ between total decidual and uNK cell fractions from either gestational age group.
In the total decidual fraction of samples taken at 8–10 weeks gestation, there was 40-fold more Ang2 secreted than Ang1; by 12–14 weeks gestation, this ratio had reduced to 4.3-fold. In the uNK cell fraction of samples taken at 8–10 weeks and 12–14 weeks gestation, there was 5.2- and 6.7-fold more Ang2 secreted than Ang1, respectively.
Immunolocalization of VEGF-C and Ang2 and their receptors, VEGF-R2 and Tie2, within the placental bed
CD56-positive uNK cells were observed in the placental bed in close proximity to spiral arteries undergoing transformation at both gestational ages examined (Fig. 3A
). TGF-ß1 (Fig. 3B)
was immunolocalized to uNK cells as well as interstitial EVT and endothelial cells. VEGF-C (Fig. 3D)
was predominantly immunolocalized to uNK cells, and some staining of interstitial EVT was also observed. VEGF-A (Fig. 3C)
immunostaining of uNK cells was seen in a small proportion of cells. However, VEGF-A was immunolocalized to endothelial cells and EVT. Immunostaining of uNK cells for Ang1 (Fig. 3E)
was observed in a lower proportion of cells than Ang2 (Fig. 3F)
. Ang1 and Ang2 also immunolocalized to EVT and gland epithelium. VEGF-R2 (Fig. 3G)
and Tie2 (Fig. 3H)
were observed on vascular smooth muscle cells in non- and partially remodeled vessels. Immunostaining of Tie2 was much more intense on the vascular endothelial cells than vascular smooth muscle cells.
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Figure 3. Representative photomicrographs of immunostaining for (A) CD56 (original magnification, x250; 8 weeks), (B) TGF-ß1 (12 weeks), (C) VEGF-A (17 weeks), (D) VEGF-C (15 weeks), (E) Ang1 (18 weeks), (F) Ang2 (18 weeks), (G) VEGF-R2 (nontransformed vessel, 15 weeks), (H) TIE2 (nontransformed vessel, 8 weeks). Solid arrows demonstrate CD56-positive cells, and broken arrows demonstrate CD56-negative cells. Original magnification, x400 unless otherwise stated.
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Figure 4. Expression of mRNA as determined by real-time reverse transcriptase-PCR for VEGF-C, Ang2, and TGF-ß1 in total decidual cell isolates and uNK cell-enriched isolates, which had been freshly isolated. n = 5 each group; mean ± SEM.
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There has only been one previous study of uNK cell production of angiogenic growth factors. Using in situ hybridization and CD56 immunohistochemistry, Li et al. [12 ] reported that uNK cells in nonpregnant endometrium expressed mRNA for Ang2, PlGF, and VEGF-C. Hybridization signals for VEGF-C and PlGF were maximal in the midsecretory phase of the menstrual cycle, and levels of Ang2 appeared to be maximal in the late secretory phase. The highest hybridization signals for all three growth factors were concentrated around endometrial vessels and glands. In addition, Li et al. [12 ] demonstrated mRNA for Ang2 and VEGF-C in uNK cells isolated from early pregnant decidua (6–12 weeks gestation). The mRNA results reported in the present study confirm those of Li et al. [12 ] and establish that uNK secretion of Ang2 and VEGF-C protein is temporally regulated. As a result of differences in techniques used in the study of Li et al. [12 ] and the present study, it is not known whether there are differences in levels of uNK cell-derived angiogenic growth factors during the menstrual cycle and early pregnancy. The stimuli for uNK cell production of angiogenic growth factors in the nonpregnant endometrium and pregnant decidua are also not clear and present an interesting area for further research.
Remodeling of the uterine spiral arteries to allow sufficient maternal blood flow into the intervillous space for the nutritional requirements of the fetus is a critical step in the establishment of a successful pregnancy. The histological features of spiral artery remodeling are well-defined (reviewed in ref. [2 ]). In the first instance, there is dilatation of the vessel, swelling of the endothelial cells, followed by deposition of thin strands of periodic acid Schiff reagent (PAS)-positive, "fibrinoid-like" material into the wall of the vessel [2 , 8 , 9 ]. The vascular smooth muscle cell layers then separate, and there is progressive loss of vascular smooth muscle cells until the vessel is completely demuscularized, and the wall of the lumen is stabilized by fibrinoid and intramural EVT [1 , 21 ]. Normal, remuscularized, uterine spiral arteries are observed again soon after the end of pregnancy [2 ]. The composition of the so-called fibrinoid material in the vessel wall remains uncertain [2 ]. In addition, it is not clear whether the thin strands of PAS-positive material seen in the vessel walls in the early stages of remodeling are biochemically similar to the dense fibrinoid deposition that characterizes complete spiral artery remodeling.
It has been postulated that the presence of EVT is critical for these changes to occur [3 , 9 , 22 ]. However, Craven et al. [8 ] demonstrated that approximately 80% of dilated spiral arteries in early pregnancy decidua were not associated with EVT, a finding supported by Kam et al. [9 ]. Although the presence of trophoblast may be required for the latter stages of spiral artery remodeling and to keep vessels in a remodeled state until the end of pregnancy, it appears that other triggers are involved in initiating this process. Recently, Pijnenborg et al. [2 ] has suggested new terminology to separate out the "decidual-associated remodeling" (dilatation, endothelial cell swelling, and deposition of small amounts of PAS-positive, fibrinoid-like material) and the "trophoblast-associated remodeling" (vascular smooth muscle cell separation and ultimate loss, intramural trophoblast, and heavy fibrinoid deposition). It is therefore tempting to speculate that failed remodeling may be a result of a maternal defect in initiating spiral artery remodeling and not a trophoblast defect [2 , 8 ].
The first stages of angiogenesis are similar to the steps of spiral artery remodeling described above. There is initial vessel dilatation and increased vascular permeability leading to deposition of plasma proteins in the vessel wall (reviewed in ref. [23 ]). These processes are thought to be mediated predominantly by nitric oxide (NO) and VEGF-A [23 ]. We have previously demonstrated that extravillous trophoblast-derived NO does not appear to play a role in spiral artery remodeling [24 ]. Although VEGF-A can be detected in the decidual stroma, VEGF-A production by uNK cells is minimal. VEGF-A is the best-characterized, angiogenic growth factor with major effects on endothelial cell proliferation and motility, as well as inducing vascular permeability [25 ]. VEGF-A has also previously been demonstrated to inhibit trophoblast invasion in vitro [26 , 27 ], and the presence of substantial VEGF-A levels in decidua in early pregnancy may reflect a role in the regulation of trophoblast invasion, rather than an angiogenic function. In addition, VEGF-A is a cell survival factor [28 ], and decidual production of this growth factor may be required for survival of the different cell types which make up the pregnant decidua. However, uNK cells are a major source of VEGF-C within the placental bed, and this growth factor is also capable of inducing vascular permeability [29 ]. VEGF-C is better known for its role in lymphangiogenesis [23 ], although it has been shown to have some vascular angiogenic potential [30 ]. We also demonstrate that VEGF-C protein but not mRNA levels secreted by uNK cells decreases with increasing gestational age, an observation consistent with this factor being involved in early stages of vascular remodeling. This may reflect differences in the sensitivity of the different methodologies used or that there is some level of post-translational regulation. However, secreted protein levels are likely to be the key indicator of the level of functional growth factor produced by a cell.
The second step in angiogenesis (and spiral artery remodeling) is separation of the vascular smooth muscle cells and eventual loss of these cells from the vascular wall. Ang2 and TGF-ß1 have been proposed to be key in mediating these events in angiogenesis [23
]. We have shown uNK cell production of Ang1, Ang2, and TGF-ß1 mRNA and protein and confirmed localization of protein to uNK cells around spiral arteries in the placental bed. Ang1 and Ang2 work through the receptor Tie2: Ang1, having proangiogenic properties, and Ang2 inhibits this process via competitive binding to Tie2 [31
]. The ratio of these factors to each other is therefore more critical than their absolute levels, and excess Ang2 leads to vessel destabilization [31
]. uNK cells appear to be a major source of Ang1 and Ang2 within the placental bed, but the high Ang2:Ang1 ratio favors a state of vessel destabilization. In common with VEGF-C, uNK levels of Ang2 decrease with increasing gestational age, again suggesting a role for this growth factor in early spiral artery remodeling. TGF-ß1 has been shown to inhibit vascular smooth muscle cell proliferation and up-regulate levels of 
smooth muscle actin, suggesting a de-differentiated state [23
].
As well as those growth factors secreted predominantly by uNK cells, several other growth factors that have been implicated in angiogenesis were present in the decidua in substantial levels, several of which were temporally regulated. Angiogenin is a member of the RNase A superfamily, which has inherent ribonucleolytic activity required for its angiogenic activity [32 ]. Nuclear translocation of angiogenin induces cellular proliferation and can be induced by a number of angiogenic growth factors including FGF-b and VEGF-A [33 ]. It can be speculated that decreased levels of angiogenin later in gestation may contribute to maintaining vessels in a remodeled state. Although FGF-b has direct effects on endothelial cell proliferation, it has also been demonstrated to up-regulate VEGF-A and urokinase plasminogen activator, potentially contributing to vessel destabilization [34 ]. PDGF-BB has also been shown to play a role in vessel stabilization [35 ]. Hence, decreased decidual levels of these factors with increasing gestational age may help maintain vessels in a remodeled state after the initial destabilization process. Indeed, increased serum levels of PDGF-BB have been observed in women with pregnancy-induced hypertension, in which vascular remodeling is diminished [36 ]. PlGF is a member of the VEGF superfamily, which has been proposed to have a critical role in vessel stabilization [37 ]. Indeed, in the current study, decidual and uNK fractions secreted reasonable levels of this growth factor, suggesting a potential role of this protein in spiral artery remodeling during early pregnancy. The role of ICAM-1 and KGF in the placental bed and spiral artery remodeling remains unclear. In addition, like VEGF-A, all of these proteins may have additional roles in decidual function and the maternal adaptation to early pregnancy, which are not currently evident.
In summary, we have demonstrated that uNK cells are a major source of VEGF-C, Ang1, Ang2, and TGF-ß1. We speculate that secretion of these growth factors by uNK cells initiates decidual-associated remodeling, readying the vessels for trophoblast infiltration and the final stages of spiral artery remodeling.
Received April 5, 2006; accepted May 9, 2006.
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26 mice J. Exp. Med. 187,217-223 contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation during normal murine pregnancy J. Exp. Med. 192,259-270 by uterine CD56++ large granular lymphocytes is amplified by decidual macrophages Hum. Reprod. 12,921-924This article has been cited by other articles:
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