Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0406250 on June 30, 2006

Published online before print June 30, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0406250v1
80/3/572    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lash, G. E.
Right arrow Articles by Bulmer, J. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lash, G. E.
Right arrow Articles by Bulmer, J. N.
(Journal of Leukocyte Biology. 2006;80:572-580.)
© 2006 by Society for Leukocyte Biology

Expression of angiogenic growth factors by uterine natural killer cells during early pregnancy

Gendie E. Lash*,1, Barbara Schiessl*, Maureen Kirkley*, Barbara A. Innes*, Alix Cooper*, Roger F. Searle{dagger}, Stephen C. Robson* and Judith N. Bulmer{ddagger}

* Schools of Surgical and Reproductive Sciences,
{dagger} Medical Education Development, and
{ddagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Remodeling of uterine spiral arteries is critical for the continuation of a successful pregnancy. Uterine natural killer (uNK) cells are the predominant leukocyte population in the early pregnant decidua, and a role for these cells in spiral artery remodeling in pregnancy has been suggested. Angiogenic growth factors were measured in isolated uNK and total (unseparated) decidual cells (8–10 or 12–14 weeks gestation, n=5 each gestational age) after culture for 48 h. Angiopoietin (Ang)1, placental growth factor, transforming growth factor-ß1 (TGF-ß1), and vascular endothelial growth factor (VEGF)-C were measured by enzyme-linked immunosorbent assay. Angiogenin, Ang2, fibroblast growth factor basic, intercellular adhesion molecule (ICAM)-1, keratinocyte growth factor (KGF), platelet-derived growth factor-BB, and VEGF-A were measured using a FASTQuant angiogenic growth factor multiplex protein assay. Levels of Ang2, ICAM-1, and KGF, secreted by the total decidual fraction, decreased with increasing gestational age. uNK levels of Ang2 and VEGF-C also decreased with increasing gestational age. At 8–10 weeks gestation, there was no difference in the level of Ang1, Ang2, TGF-ß1, and VEGF-C secreted by uNK cells and the total decidual fraction. At 12–14 weeks, uNK cells secreted significantly lower levels of VEGF-C than the total decidual fraction. Early pregnancy decidua is a major source of angiogenic growth factors whose levels decrease with increasing gestational age, suggesting that they may play a role in spiral artery remodeling. uNK cells appear to be a prominent source of Ang1, Ang2, TGF-ß1, and VEGF-C within the placental bed.

Key Words: uNK cells • angiogenesis • VEGF-C • Ang2


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One of the major maternal adaptations to pregnancy is remodeling of the spiral arteries, whereby there is a loss of the normal musculoelastic structure with replacement of the vascular media by fibrinoid material, converting the spiral arteries into large, dilated vessels of low resistance (ref. [1 ]; reviewed in ref. [2 ]). Remodeling of the decidual portion of spiral arteries is generally complete by 10–12 weeks gestation, and complete remodeling of myometrial portions of these vessels is complete by 14–16 weeks gestation [3 ]. Failure of spiral artery remodeling has been associated with the development of complications of pregnancy such as pre-eclampsia [4 ], fetal growth restriction [5 ], and second trimester miscarriage [6 , 7 ]. The molecular events underlying spiral artery remodeling in pregnancy remain unclear.

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-{gamma} (IFN-{gamma}) in initiating spiral artery remodeling has been postulated [14 , 15 ]. However, in the human, only low levels of uNK cell-derived IFN-{gamma} 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Samples
Placental bed biopsies were obtained from apparently normal pregnancies at 8–10 weeks (n=6), 12–14 weeks (n=6), and 16–20 weeks (n=6) gestation at elective termination of pregnancy at the Royal Victoria Infirmary (Newcastle upon Tyne, UK). Placental bed biopsies were taken as described previously using biopsy forceps (Richard Wolf Endoscopes, Wimbledon, UK) [17 ]. Only biopsies that contain decidua and myometrium, interstitial extravillous trophoblast, and at least one cross-section of a spiral artery were included in the study. From a separate set of patients, decidua was obtained from apparently normal pregnancies at 8–10 weeks (n=5) and 12–14 weeks (n=5) of gestation as described previously [18 ]. All women gave informed, written consent before termination of pregnancy. The study was approved by the Joint Ethics Committee of Newcastle upon Tyne Health Authority and the University of Newcastle (UK).

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.


View this table:
[in this window]
[in a new window]
  		Table 1. Antibodies Used for Immunohistochemistry on Paraffin Sections

 
Real-time reverse transcriptase-polymerase chain reaction (PCR)
Total RNA was isolated from total decidual cell suspensions and CD56+ cells (t=0) using TRI-reagent, according to the manufacturer’s instructions (TRIZOL, Sigma Chemical Co.). The quantity of RNA was assessed at 260 nm using a 96-well spectrophotometer (Molecular Devices) and ultraviolet-visible 96-well plates (Greiner, Gloucestershire, UK) and 500 ng reverse-transcribed using Superscript III according to the manufacturer’s instructions (InVitrogen, Paisley, UK). Real-time reverse transcriptase-PCR was performed using an ABI7000 (Applied Biosystems, Foster City, CA) PCR machine and probes labeled with the fluorophore 6-Fam and quencher tetramethylrhodamine. Each 20 µl reaction contained 3 µl cDNA, 10 µl ABI Taqman Master Mix (Applied Biosystems,), forward and reverse primers, probe, and water. The concentration of forward and reverse primers and probe was optimized prior to the commencement of the study and is shown in Table 2 along with primer and probe sequences. In each run, a standard curve was produced for each set of primers and probe based on serial dilutions of a standard amount of RNA, which was reverse-transcribed; therefore, a standard curve of threshold cycle number versus amount was created, and for each sample, an amount interpolated from that curve. For each sample, reverse transcriptase-PCR was performed for 18srRNA (housekeeping gene), VEGF-C, TGF-ß1, and Ang2. Data are presented as the ratio of amount of target mRNA (VEGF-C, TGF-ß1, or Ang2) to amount of 18srRNA.


View this table:
[in this window]
[in a new window]
  		Table 2. Primer and Probe Sequences Used for Real-Time Reverse Transcriptase-PCR

 
Statistical analysis
Data are presented as means with standard error. Statistical calculations were performed using the StatView statistical software package (Abacus Concepts Inc., Berkley, CA). Statistical significance was determined by use of ANOVA with Fishers post hoc test unless stated in the text. All statistical tests were two-sided, and differences were considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Secretion of angiogenic growth factors by isolated uNK cells
There was no difference in the levels of any of the analytes tested after culture for 24 or 48 h (data not shown), and therefore, for simplicity, data from cultures incubated for 48 h only are shown (Figs. 1 and 2 ).


Figure 1
View larger version (15K):
[in this window]
[in a new window]
  		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.

 

Figure 2
View larger version (17K):
[in this window]
[in a new window]
  		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.

 
Effect of gestational age on angiogenic growth factor secretion
For three analytes, there was less protein secreted from total decidual cell suspensions at 12–14 weeks gestation compared with 8–10 weeks gestation (Ang2, P=0.009; ICAM-1, P=0.0003; KGF, P=0.01). There was a similar trend for three other analytes tested, which failed to reach statistical significance (angiogenin, P=0.07; FGF-b, P=0.07; PDGF-BB, P=0.09). With respect to uNK cells, there was less Ang2 and VEGF-C protein at 12–14 weeks gestation compared with 8–10 weeks gestation (Ang2, P=0.03; VEGF-C, P=0.0007) and a trend toward lower, secreted Ang1 levels (P=0.07).

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.


Figure 3
View larger version (101K):
[in this window]
[in a new window]
  		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.

 
Expression of TGF-ß1, VEGF-C, and Ang2 mRNA by isolated uNK cells
There was less mRNA for VEGF-C and Ang2 expressed by the purified uNK cells than in the total decidual cell suspension from 8–10 weeks gestation (VEGF-C, P=0.02; Ang2, P=0.02; Fig. 4 ). There was no difference between the two cell types isolated from the 12- to 14-week gestational age group. There were no gestational age differences for VEGF-C or Ang2 in either cell fraction studied. There was no difference in TGF-ß1 mRNA levels between the cellular fractions isolated from either gestational age group.


Figure 4
View larger version (20K):
[in this window]
[in a new window]
  		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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first comprehensive study of the production of angiogenic growth factors by uNK cells isolated from different stages of early pregnancy. We have demonstrated the secretion of several important angiogenic growth factors by "total" decidual cell suspensions and identified uNK cells as a major source of Ang1, Ang2, VEGF-C, and TGF-ß1 within the placental bed. The ELISA results for Ang1, Ang2, VEGF-A, VEGF-C, and TGF-ß1 were confirmed by immunohistochemistry of placental bed biopsies. In general, real-time reverse transcriptase-PCR detection of mRNA for Ang2, TGF-ß1, and VEGF-C was consistent with the protein results. In addition, Tie2 and VEGF-R2 were identified on vascular smooth muscle cells in decidual spiral arteries. Taken together, these findings further support a role for uNK cells in spiral artery remodeling during early pregnancy.

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 {alpha} 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.


   ACKNOWLEDGEMENTS
 
This project was supported by funding from BBSRC (S19967). The authors acknowledge the staff at the Royal Victoria Infirmary, Newcastle upon Tyne, for their assistance in sample collection.

Received April 5, 2006; accepted May 9, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pijnenborg, R., Dixon, G., Robertson, W. B., Brosens, I. (1980) Trophoblastic invasion of human decidua from 8 to 18 weeks of pregnancy Placenta 1,3-19[Medline]
  2. Pijnenborg, R., Vercruysse, L., Hanssens, M. (2006) The uterine spiral arteries in human pregnancy: facts and controversies Placenta Epub ahead of print.
  3. Brosens, I., Robertson, W. B., Dixon, H. G. (1967) The physiological response of the vessels of the placental bed to normal pregnancy J. Pathol. Bacteriol. 93,569-579[CrossRef][Medline]
  4. Pijnenborg, R., Anthony, J., Davey, D. A., Rees, A., Tiltman, A., Vercruysse, L., van Assche, A. (1991) Placental bed spiral arteries in the hypertensive disorders of pregnancy Br. J. Obstet. Gynaecol. 98,648-655[Medline]
  5. Khong, T. Y., Lane, E. B., Robertson, W. B. (1986) An immunocytochemical study of fetal cells at the maternal-placental interface using monoclonal antibodies to keratins, vimentin and desmin Cell Tissue Res. 246,189-195[Medline]
  6. Khong, T. Y., Liddell, H. S., Robertson, W. B. (1987) Defective haemochorial placentation as a cause of miscarriage: a preliminary study Br. J. Obstet. Gynaecol. 94,649-655[Medline]
  7. Ball, E., Bulmer, J., Ayis, S., Lyall, F., Robson, S. (2006) Late sporadic miscarriage is associated with abnormalities in spiral artery transformation and trophoblast invasion J. Pathol. 208,535-542[CrossRef][Medline]
  8. Craven, C. M., Morgan, T., Ward, K. (1998) Decidual spiral artery remodeling begins before cellular interaction with cytotrophoblasts Placenta 19,241-252[CrossRef][Medline]
  9. Kam, E. P., Gardner, L., Loke, Y. W., King, A. (1999) The role of trophoblast in the physiological change in decidual spiral arteries Hum. Reprod. 14,2131-2138[Abstract/Free Full Text]
  10. Bulmer, J. N., Morrison, L., Longfellow, M., Ritson, A., Pace, D. (1991) Granulated lymphocytes in human endometrium: histochemical and immunohistochemical studies Hum. Reprod. 6,791-798[Abstract/Free Full Text]
  11. Jokhi, P. P., King, A., Loke, Y. W. (1997) Cytokine production and cytokine receptor expression by cells of the human first trimester placental-uterine interface Cytokine 9,126-137[CrossRef][Medline]
  12. Li, X. F., Charnock-Jones, D. S., Zhang, E., Hiby, S., Malik, S., Day, K., Licence, D., Bowen, J. M., Gardner, L., King, A., Loke, Y. W., Smith, S. K. (2001) Angiogenic growth factor messenger ribonucleic acids in uterine natural killer cells J. Clin. Endocrinol. Metab. 86,1823-1834[Abstract/Free Full Text]
  13. Croy, B. A., Ashkar, A. A., Foster, R. A., DiSanto, J. P., Magram, J., Carson, D., Gendler, S. J., Grusby, M. J., Wagner, N., Muller, W., Guimond, M. J. (1997) Histological studies of gene-ablated mice support important functional roles for natural killer cells in the uterus during pregnancy J. Reprod. Immunol. 35,111-133[CrossRef][Medline]
  14. Guimond, M. J., Wang, B., Croy, B. A. (1998) Engraftment of bone marrow from severe combined immunodeficient (SCID) mice reverses the reproductive deficits in natural killer cell-deficient tg {epsilon} 26 mice J. Exp. Med. 187,217-223[Abstract/Free Full Text]
  15. Ashkar, A. A., Di Santo, J. P., Croy, B. A. (2000) Interferon {gamma} 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[Abstract/Free Full Text]
  16. Marzusch, K., Buchholz, F., Ruck, P., Handgretinger, R., Geiselhart, A., Engelmann, L., Dietl, J. (1997) Interleukin-12- and interleukin-2-stimulated release of interferon-{gamma} by uterine CD56++ large granular lymphocytes is amplified by decidual macrophages Hum. Reprod. 12,921-924[Abstract/Free Full Text]
  17. Robson, S. C., Simpson, H., Ball, E., Lyall, F., Bulmer, J. N. (2002) Punch biopsy of the human placental bed Am. J. Obstet. Gynecol. 187,1349-1355[CrossRef][Medline]
  18. Vassiliadou, N., Bulmer, J. N. (1998) Functional studies of human decidua in spontaneous early pregnancy loss: effect of soluble factors and purified CD56+ lymphocytes on killing of natural killer- and lymphokine-activated killer-sensitive targets Biol. Reprod. 58,982-987[Abstract/Free Full Text]
  19. Jones, R. K., Bulmer, J. N., Searle, R. F. (1997) Cytotoxic activity of endometrial granulated lymphocytes during the menstrual cycle in humans Biol. Reprod. 57,1217-1222[Abstract]
  20. Pongcharoen, S., Searle, R. F., Bulmer, J. N. (2004) Placental Fas and Fas ligand expression in normal early, term and molar pregnancy Placenta 25,321-330[CrossRef][Medline]
  21. Pijnenborg, R., Bland, J. M., Robertson, W. B., Brosens, I. (1983) Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy Placenta 4,397-413[Medline]
  22. De Wolf, F., De Wolf-Peeters, C., Brosens, I. (1973) Ultrastructure of the spiral arteries in the human placental bed at the end of normal pregnancy Am. J. Obstet. Gynecol. 117,833-848[Medline]
  23. Distler, J. H., Hirth, A., Kurowska-Stolarska, M., Gay, R. E., Gay, S., Distler, O. (2003) Angiogenic and angiostatic factors in the molecular control of angiogenesis Q. J. Nucl. Med. 47,149-161[Medline]
  24. Lyall, F., Bulmer, J. N., Kelly, H., Duffie, E., Robson, S. C. (1999) Human trophoblast invasion and spiral artery transformation: the role of nitric oxide Am. J. Pathol. 154,1105-1114[Abstract/Free Full Text]
  25. Ferrara, N. (2004) Vascular endothelial growth factor: basic science and clinical progress Endocr. Rev. 25,581-611[Abstract/Free Full Text]
  26. Lash, G. E., Cartwright, J. E., Whitley, G., St, J., Trew, A. J., Baker, P. N. (1999) The effects of angiogenic growth factors on extravillous trophoblast invasion and motility Placenta 20,661-667[CrossRef][Medline]
  27. Fitzpatrick, T. E., Lash, G. E., Yanaihara, A., Charnock-Jones, D. S., Macdonald-Goodfellow, S. K., Graham, C. H. (2003) Inhibition of breast carcinoma and trophoblast cell invasiveness by vascular endothelial growth factor Exp. Cell Res. 283,247-255[CrossRef][Medline]
  28. Weis, S., Shintani, S., Weber, A., Kirchmair, R., Wood, M., Cravens, A., McSharry, H., Iwakura, A., Yoon, Y. S., Himes, N., Burstein, D., Doukas, J., Soll, R., Losordo, D., Cheresh, D. (2004) Src blockade stabilizes a Flk/cadherin complex, reducing edema and tissue injury following myocardial infarction J. Clin. Invest. 113,885-894[CrossRef][Medline]
  29. Cao, R., Eriksson, A., Kubo, H., Alitalo, K., Cao, Y., Thyberg, J. (2004) Comparative evaluation of FGF-2-, VEGF-A-, and VEGF-C-induced angiogenesis, lymphangiogenesis, vascular fenestrations, and permeability Circ. Res. 94,664-670[Abstract/Free Full Text]
  30. Bauer, S. M., Bauer, R. J., Liu, Z. J., Chen, H., Goldstein, L., Velazquez, O. C. (2005) Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels J. Vasc. Surg. 41,699-707[CrossRef][Medline]
  31. Maisonpierre, P. C., Suri, C., Jones, P. F., Bartunkova, S., Wiegand, S. J., Radziejewski, C., Compton, D., McClain, J., Aldrich, T. H., Papadopoulos, N., Daly, T. J., Davis, S., Sato, T. N., Yancopoulos, G. D. (1997) Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis Science 277,55-60[Abstract/Free Full Text]
  32. Strydom, D. J. (1998) The angiogenins Cell. Mol. Life Sci. 54,811-824[CrossRef][Medline]
  33. Kishimoto, K., Liu, S., Tsuji, T., Olson, K. A., Hu, G. F. (2005) Endogenous angiogenin in endothelial cells is a general requirement for cell proliferation and angiogenesis Oncogene 24,445-456[CrossRef][Medline]
  34. Presta, M., Dell’Era, P., Mitola, S., Moroni, E., Ronca, R., Rusnati, M. (2005) Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis Cytokine Growth Factor Rev. 16,159-178[CrossRef][Medline]
  35. Cao, R., Brakenhielm, E., Pawliuk, R., Wariaro, D., Post, M. J., Wahlberg, E., Leboulch, P., Cao, Y. (2003) Angiogenic synergism, vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2 Nat. Med. 9,604-613[CrossRef][Medline]
  36. Morita, H., Mizutori, M., Takeuchi, K., Motoyama, S., Maruo, T. (2001) Abundant expression of platelet-derived growth factor in spiral arteries in decidua associated with pregnancy-induced hypertension and its relevance to atherosis Eur. J. Endocrinol. 144,271-276[Abstract]
  37. Tammela, T., Enholm, B., Alitalo, K., Paavonen, K. (2005) The biology of vascular endothelial growth factors Cardiovasc. Res. 65,550-563[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
H. Yadi, S. Burke, Z. Madeja, M. Hemberger, A. Moffett, and F. Colucci
Unique Receptor Repertoire in Mouse Uterine NK cells
J. Immunol., November 1, 2008; 181(9): 6140 - 6147.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
S. Quenby, H. Nik, B. Innes, G. Lash, M. Turner, J. Drury, and J. Bulmer
Uterine natural killer cells and angiogenesis in recurrent reproductive failure
Hum. Reprod., October 3, 2008; (2008) den348v1.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. M. Sharkey, L. Gardner, S. Hiby, L. Farrell, R. Apps, L. Masters, J. Goodridge, L. Lathbury, C. A. Stewart, S. Verma, et al.
Killer Ig-Like Receptor Expression in Uterine NK Cells Is Biased toward Recognition of HLA-C and Alters with Gestational Age
J. Immunol., July 1, 2008; 181(1): 39 - 46.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. Evans, R. D. Catalano, K. Morgan, H. O. D. Critchley, R. P. Millar, and H. N. Jabbour
Prokineticin 1 Signaling and Gene Regulation in Early Human Pregnancy
Endocrinology, June 1, 2008; 149(6): 2877 - 2887.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
S.E. Hiby, L. Regan, W. Lo, L. Farrell, M. Carrington, and A. Moffett
Association of maternal killer-cell immunoglobulin-like receptors and parental HLA-C genotypes with recurrent miscarriage
Hum. Reprod., April 1, 2008; 23(4): 972 - 976.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
M. Plaisier, P. Koolwijk, F. Willems, F. M. Helmerhorst, and V. W.M. van Hinsbergh
Pericellular-acting proteases in human first trimester decidua
Mol. Hum. Reprod., January 1, 2008; 14(1): 41 - 51.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
G. I. Bondarenko, D. W. Burleigh, M. Durning, E. E. Breburda, R. L. Grendell, and T. G. Golos
Passive Immunization against the MHC Class I Molecule Mamu-AG Disrupts Rhesus Placental Development and Endometrial Responses
J. Immunol., December 15, 2007; 179(12): 8042 - 8050.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
C. Tayade, Y. Fang, D. Hilchie, and B. A. Croy
Lymphocyte contributions to altered endometrial angiogenesis during early and midgestation fetal loss
J. Leukoc. Biol., October 1, 2007; 82(4): 877 - 886.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Tayade, D. Hilchie, H. He, Y. Fang, L. Moons, P. Carmeliet, R. A. Foster, and B. A. Croy
Genetic Deletion of Placenta Growth Factor in Mice Alters Uterine NK Cells
J. Immunol., April 1, 2007; 178(7): 4267 - 4275.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0406250v1
80/3/572    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lash, G. E.
Right arrow Articles by Bulmer, J. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lash, G. E.
Right arrow Articles by Bulmer, J. N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS