Oxygen regulates human cytotrophoblast migration by controlling chemokine and receptor expression.

INTRODUCTION: Placental development involves the variation of oxygen supply due to vascular changes and cytotrophoblast invasion. Chemokines and their receptors play an important role during placental formation. Herein, the analysis of the chemokine/receptor pair CXCL12/CXCR4 and further chemokine receptors, such as CCR1, CCR7 and CXCR6 expression in human cytotrophoblasts was conducted. METHODS: Human cytotrophoblasts were examined directly after isolation or after incubation with different oxygen tensions and a chemical HIF-stimulator for 12 h with realtime PCR, immunoblot, immunohistochemistry. Conditioned media of placental villi, decidua, and endothelial cells was used for ELISA analysis of CXL12. Cytotrophoblast migration assays were conducted applying conditioned media of endothelial cells, a CXCL12 gradient, and different oxygen level. Endometrial and decidual tissue was stained for CXCL12 expression. RESULTS: An upregulation of CXCL12, CXCR4, CCR1, CCR7 and CXCR6 was observed after cytotrophoblast differentiation. Low oxygen supply upregulated CXCR4, CCR7 and CXCR6, but downregulated CXCL12 and CCR1. In contrast to the HIF associated upregulation of the aforementioned proteins, downregulation of CXCL12 and CCR1 seemed to be HIF independent. Cytotrophoblast migration was stimulated by low oxygen, the application of a CXCL12 gradient and endothelial cell conditioned media. CXCL12 was detected in endometrial vessels, glands and conditioned media of placental and decidual tissue, but not decidual vessels. DISCUSSION/CONCLUSION: Taken together, oxygen supply and cytotrophoblast differentiation seem to be regulators of chemokine and receptor expression and function in human cytotrophoblasts. Therefore, this system seems to be involved in placental development, directed cytotrophoblast migration in the decidual compartment and a subsequent sufficient supply of the growing fetus.

CXCR7 and syndecan-4 are potential receptors for CXCL12 in human cytotrophoblasts.

The placenta forms the interface between the mother and the fetus. During placental development cytotrophoblasts differentiate to form the syncytium or to invade the decidual wall to breach maternal vessels and establish the blood flow in the intervillous space. This process is still not well understood but it is proposed that chemokines and their receptors are involved in guiding cytotrophoblasts to the decidua and maternal vessels as well as attracting immunocompetent cells to the implantation site. CXCL12 is a chemokine expressed by cytotrophoblasts and is involved in cytotrophoblast invasion, differentiation and survival. One of its receptors, CXCR4, has been detected on cytotrophoblasts. Recent data show that CXCR7 and syndecan-4 might partially mediate CXCL12 function in other cell types. In this study, we examined CXCR7 and syndecan-4 expression at the maternal-fetal interface via immmunolocalization in placental tissue sections and in isolated cytotrophoblasts. We further used immunoblot analyses to confirm the data. We were able to show that cytotrophoblasts express both receptors and that upregulation occurs during the differentiation process of cytotrophoblasts towards the invasive phenotype. On a functional level CXCR7 seems not to be involved in JAR cell chemotaxis, suggesting a different function of this receptor. In conclusion, we propose that CXCL12 binds to CXCR4, but also to CXCR7 and syndecan-4. These three receptors could mediate different functions of CXCL12, such as cell migration, directed invasion, proliferation and survival. The latter molecules might also be involved in the development of placental pathologies, such as preeclampsia or choriocarcinoma growth.

The embryo's cystatin C and F expression functions as a protective mechanism against the maternal proteinase cathepsin S in mice.

A successful implantation of a mammalian embryo into the maternal endometrium depends on a highly synchronized fetal-maternal dialogue involving chemokines, growth factors, and matrix-modifying enzymes. A growing body of evidence suggests an important role for proteinases playing a role in matrix degeneration and enhancing the embryo's invasive capacity and influencing the mother's immunological status in favor of the conceptus. This study focused on the expression of cathepsin S (CTSS) and its inhibitors in the murine fetal-maternal interface as well as the detection of the cellular sources of either proteinase and inhibitors. Nested RT-PCR for detection of embryonic mRNAs, immunohistochemistry of maternal and fetal tissues in B6C3F1 mice, and FACS analysis for determination of immunocompetent cell population were applied. This study shows that the cysteine proteinase CTSS is upregulated in the stroma of the implantation site, and that pregnancy induces an influx of CTSS-positive uterine natural killer cells. Compared to maternal tissues, the CTSS inhibitors cystatin F and C, but not the proteinase itself, are expressed in blastocysts. In conclusion, CTSS underlies a hormonal regulation in the maternal tissue and therewith most likely supports the embryonic implantation. The invading embryo regulates the depth of its own invasion through the expression of the cathepsin inhibitors and furthermore, interleukin-6 to activate CTSS in maternal tissues. Additionally, the observed decrease in CD3(+) cells leads to the hypothesis that cells of the cytotoxic T-cell group are down-regulated in the decidua to support the implantation and ensure the survival of the embryo.

Interleukin-1 system in the human fallopian tube-No spatial but a temporal regulation of mRNA and protein expression.

The human fallopian tube provides the environment for the first 5 days of embryonic development in vivo. The IL-1 system is involved in human embryo implantation. This study aimed to investigate IL-1beta, IL-1ra and IL-1R tI expression within the length of the human fallopian tube on mRNA- and protein-level in samples from proliferative versus secretory phase, postmenopause (PMP) samples and samples from intra- (IUP) and extrauterine pregnancies (EUP) to examine possible spatial and hormonal induced changes (fimbrial, ampullary and isthmic tube segments). On mRNA-level, IL-1beta was expressed in all samples except in PMP. IL-1R tI could be detected in all samples whereas IL-1ra was only expressed in secretory phase and the IUP sample. Immunohistochemically we could detect IL-1beta and IL-1R t1 protein in all proliferative and secretory phase samples with maximum intensity in secretory phase samples whereas IL-1ra was expressed in secretory phase samples only. Overall no spatial but temporal differences possibly due to hormonal changes could be observed suggesting a precise regulation of the IL-1 system, especially for IL-1ra and moreover a stable molecular architecture within the full length of the fallopian tube.

Expression of the vascular endothelial growth factor receptor neuropilin-1 in the human endometrium.

Angiogenesis is a key process in the endometrium which undergoes dramatic changes during the menstrual cycle. Molecules such as vascular endothelial growth factor (VEGF), acting via two tyrosine kinase family receptors (VEGFR1 [Flt-1] and VEGFR2 [KDR/Flk-1]), are potent modulators of angiogenesis and vascular remodelling in the endometrium. Recently, neuropilin-1 (NRP-1) was shown to be expressed in endothelial cells binding VEGF(165) and therewith enhancing the binding of VEGF(165) to VEGFR2. This suggests that NRP-1, in addition to the known VEGF receptors, may play an important role in VEGF-induced angiogenesis. In this study, the expression of NRP-1 in the cycling human endometrium has been investigated by reverse transcription (RT)-polymerase chain reaction (RT-PCR), semi-quantitative competitive RT-PCR (RT-cPCR) and immunohistochemical staining. NRP-1 was expressed in all 32 endometrium samples throughout the menstrual cycle. However, samples from the proliferative phase showed significantly higher expression levels of NRP-1 mRNA compared to samples from the secretory phase (t/c-ratio 2.13 vs. 0.84, p<0.05). Immunohistochemistry confirmed the results showing increased NRP-1 staining in vascular endothelium, glandular epithelium and stromal cells of the proliferative phase endometrium. This study demonstrates mRNA and protein expression of NRP-1 in human endometrium samples throughout the menstrual cycle. The enhanced expression of NRP-1 in the proliferative phase suggests that it may participate in hormonally regulated changes of endometrial angiogenesis, preparing the endometrium for the implantation of an embryo. NRP-1 expression might act as a co-factor for VEGF(165) enhancing the angiogenic stimulus.

Angiopoietin-1 and -2 mRNA and protein expression in mouse preimplantation embryos and uteri suggests a role in angiogenesis during implantation.

After attachment and migration through the endometrial epithelium, the embryo must induce angiogenesis within the endometrial stroma to successfully complete the implantation process. Growth factors have been shown to play an important role in embryo implantation and placentation. The aim of the study was to investigate the expression of angiopoietin-1 and -2 (Ang-1 and -2) mRNA and protein expression during the development of single preimplantation mouse embryos and of possible complementary expression in mouse uteri. Angiopoietin-1 mRNA was expressed throughout development in 78% of zygotes, 66% of 2-cell-embryos, 71% of 4-cell-embryos, 70% of 8-cell-embryos, 60% of morula stages, 48% of early blastocysts and 78% of late blastocysts. The number of Ang-1-expressing embryos in the early-blastocyst group was significantly different in comparison with zygotes, 4-cell-embryos, 8-cell-embryos and late blastocysts. Angiopoietin-2 mRNA and protein expression could not be detected in preimplantation embryos. Examination of the uteri revealed Ang-2 mRNA and protein expression in the oestrogen-dominated cycling phase and the progesterone-dominated mated phase, whereas Ang-1 expression was restricted to the mated phase. Herein, Ang-1 expression in preimplantation mouse embryos as well as Ang-1 and -2 expression in mouse uteri is demonstrated, suggesting a possible role for angiopoietins in the embryo-maternal dialogue of the implantation process via an enhancement of the vascular remodelling in favour of an implanting conceptus.

Habitual smoking causes an abnormality in platelet thromboxane A2 metabolism and results in an altered susceptibility to aspirin effects.

The present study investigates the effects of aspirin (100 mg every second day for 14 days) on platelet function in nine healthy non-smokers and in nine healthy habitual smokers. There was a significantly (P < 0.05) stronger inhibition of collagen (0.6 microgram/ml)- and ADP (2 microM)-induced platelet aggregation by aspirin in smokers as compared to non-smokers. This difference occurred in the presence of an almost complete (> 95%) inhibition of thromboxane A2 (TXA2) synthesis in both groups. The platelet capacity to generate TXA2 in vitro was significantly reduced in smokers, urinary excretion of TXA2, however, was significantly increased. Thus, the better susceptibility of smokers to anti-aggregatory effects of aspirin is very likely to be related to a chronic smoking-induced alteration of platelet TXA2 system. Cessation of smoking should, therefore, be encouraged.

Use of maximum intensity projections in CT angiography: a basic review.

Maximum intensity projection (MIP) is a simple three-dimensional visualization tool that can be used to display computed tomographic angiography data sets. MIP images are not threshold dependent and preserve attenuation information. Thus, they often yield acceptable results even in cases in which shaded surface display images fail because of threshold problems. MIP is particularly useful for depicting small vessels. Because MIP does not allow for differentiation between foreground and background, MIP images are best suited for displaying relatively simple anatomic situations in which superimposition of structures does not occur (eg, the abdominal aorta). If anatomic structures are superimposed over the vessel of interest, the MIP technique can provide images of diagnostic quality as long as the contrast of the vessel of interest is sufficiently high compared with that of surrounding structures. Editing procedures for MIP are usually used to exclude unwanted structures from the volume of interest and include cutting functions and region-growing algorithms. Artifacts from vessel pulsation and respiratory motion may occur and simulate abnormalities, but, with careful attention, they can be distinguished from real disease. MIP images should always be interpreted together with the original transaxial data set. Knowledge of display properties and artifacts is necessary for correct interpretation of MIP images and helps one create images of optimal quality, choose appropriate examination parameters, and distinguish artifacts from disease.