Katsuhiko Naruse

Localization of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitors for MMPs (TIMPs) in uterine natural killer cells in early human pregnancy

BACKGROUND Invasion by extravillous trophoblast into uterine decidua and myometrium with remodeling of spiral arteries is essential for normal human pregnancy and is tightly regulated. Uterine natural killer (uNK) cells appear to be a major maternal regulator of placentation through the secretion of growth factors, cytokines and proteinases. METHOD Matrix metalloproteinase (MMP)-2 and MMP-9 activity in placental bed biopsies was studied using in situ gelatin zymography. Expression by uNK cells of MMP-2, MMP-9 and their tissue inhibitors, TIMP-1, TIMP-2 and TIMP-3, was localized in the placental bed by immunohistochemistry. Levels of MMP-2, MMP-9, TIMP-1, TIMP-2 and TIMP-3 secreted into 24 h cell culture supernatants of isolated uNK and unseparated (total) decidual cells (8-10 and 12-14 weeks gestation, n = 10 each group) were determined by gelatin gel zymography or western blot as appropriate. RESULTS Gelatinase activity in situ appeared greater in decidua than myometrium. uNK cells showed strong immunostaining for MMP-2 and TIMP-2. MMP-9 activity was lower in uNK cells than total decidual supernatants (8-10 weeks: P = 0.0003; 12-14 weeks: P = 0.0012). In contrast, there was no difference in MMP-2 secreted by either uNK cell or total decidual cultures. CONCLUSIONS uNK cells from early human pregnancy decidua possess innate protease activity, especially MMP-2, providing further evidence for a role for these cells in regulation of trophoblast invasion and spiral artery remodeling in early placentation.

Effect of tumour necrosis factor-α in combination with interferon-γ on first trimester extravillous trophoblast invasion

Successful pregnancy is dependent upon invasion of the uterine tissues by extravillous trophoblast cells (EVT). The mechanisms that control trophoblast invasion are unclear, but several cytokines and growth factors appear to be involved. We have previously demonstrated that IFN-γ inhibits EVT invasion via a mechanism partially dependent on an increase in EVT apoptosis and decreased secretion of matrix metalloproteinase (MMP)-2. In the current study we show that TNF-α, both alone and in combination with IFN-γ, inhibits EVT invasion via a mechanism associated with increased trophoblast apoptosis, decreased trophoblast proliferation and/or altered production of active proteases. TNF-α and its receptors, TNF-αRI and TNF-αRII, were immunolocalised in the placental bed. Uterine natural killer (uNK) cells, EVT and villous cytotrophoblast were shown to all produce TNF-α, and TNF-α receptors were primarily immunolocalised to EVT in the placental bed. TNF-α increased EVT apoptosis, decreased villous cytotrophoblast proliferation and increased expression of pro-MMP-9 (but not active MMP-9), urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI)-1 by EVT. The combination of TNF-α and IFN-γ inhibited EVT via a mechanism associated with increased EVT apoptosis, reduced proliferation, reduced pro-MMP-2 secretion and increased secretion of uPA. TNF-α is one of several decidua-derived factors with the capacity to inhibit EVT invasion. The mode of activity of TNF-α was modified by the presence of IFN-γ, suggesting that the local cytokine milieu may be critical in determining spatial and/or temporal changes in EVT invasion.

Hypoxia inhibits invasion of extravillous trophoblast cells through reduction of matrix metalloproteinase (MMP)-2 activation in the early first trimester of human pregnancy.

During early pregnancy, extravillous trophoblast (EVT) cells are exposed to very low pO(2) values. In this study, we investigated the proteolytic functions and invasiveness of human primary EVT cells under hypoxic conditions to show the early placental pathophysiology. Placental samples (from 5 to 10 weeks gestation) were obtained at termination of pregnancy. Cytotrophoblast cells were separated by Percoll(®) gradient method and cultured on Matrigel(®) to obtain an invasive phenotype (similar to EVT). The invasion capacity (Matrigel-coated invasion assay), migration of the cells (wound healing assay), activity and expression of matrix metalloproteinase (MMP)-2 and tissue inhibitor for MMP (TIMP)-2 (gelatin gel zymography, ELISA, and quantitative RT-PCR), and expression of membrane-type (MT)1-MMP (western blot) were investigated. All cultures (except for quantitative RT-PCR) were performed under 20% oxygen, 5% oxygen, and 5% oxygen with 3 repetitions of 0.1% oxygen hypoxic stimulation for 1 h. Invasion and MMP2 activity of the cells were significantly increased in 20% and decreased in 0.1% oxygen. There was no significant difference in cell migration among the oxygen environments. Concentrations of MMP2 in the supernatant and expression of MT1-MMP were increased in both the 0.1% and 20% oxygen environments. The MMP2 mRNA level was increased after 1-h stimulation with 0.1% oxygen. The TIMP2 concentration was increased only in 20% oxygen, but the mRNA level was decreased in 0.1% oxygen. These results suggested that hypoxia might inhibit the invasive capacity and MMP2 activation of EVT cells in the early first trimester of pregnancy. Decrease in TIMP2 production may reduce the MMP2/TIMP2/MT1-MMP complex and lead to this unique behavior of EVT cells under hypoxic conditions.

Secretion of cytokines by villous cytotrophoblast and extravillous trophoblast in the first trimester of human pregnancy

Cytokines are proposed to play roles in regulation of trophoblast invasion, spiral artery remodeling and immunoregulation during early pregnancy. Secretion of 12 cytokines (interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-13, IFNγ, GM-CSF, MCP-1 and RANTES) by first trimester extravillous trophoblast and villous cytotrophoblast cells was examined using multiplex cytokine array technology. Seven (IL-1β, IL-8, IL-12p70, IL-13, GM-CSF, MCP-1 and RANTES) of the 12 cytokines examined were detectable in the samples studied (n=10 each group). Villous cytotrophoblast production of IL-1β and IL-8 increased with gestational age. Extravillous trophoblast production of IL-8, IL-13 and RANTES increased with gestational age. At 12-14 weeks gestation extravillous trophoblast cells secreted higher levels of IL-8, IL-13 and RANTES than villous cytotrophoblast cells.

Fatal Factors of Clinical Manifestations and Laboratory Testing in Patients with Amniotic Fluid Embolism

Aims: To identify factors leading to fatality of patients with amniotic fluid embolism (AFE). Methods: Patients who had fatal or nonfatal AFE were registered at the Hamamatsu University School of Medicine in the Department of Obstetrics and Gynecology from 1992 to 2006. Data collected included information about demographics and clinical characteristics. The fatal factors among these data were identified using χ2 analysis and the Mann-Whitney test. Results: One hundred and thirty-five patients met the criteria, which included fatal (n = 65) and nonfatal AFE (n = 70). Maternal full-term gestational weeks, multiparous and noncesarean sections were the risk factors for death found in this study (p < 0.01). Sialyl Tn levels (mean ± SD) in the serum of patients with fatal AFE (69.7± 126.4 U/ml) were higher compared to those with nonfatal AFE (48.3± 161.8 U/ml; p = 0.003). Each of three items (cardiac arrest, dyspnea or loss of consciousness) was more common in fatal AFE (p < 0.01). Maternal pregnancy and labor complications were not associated with the distinction between fatal and nonfatal AFE. Conclusion: Factors associated with patients with fatal AFE were identified. These included multiparity, noncesarean section at full-term and the three symptoms mentioned above. Sialyl Tn levels could be a possible prognostic fatality factor.

Interaction between uterine natural killer cells and extravillous trophoblast cells: effect on cytokine and angiogenic growth factor production

BACKGROUND Uterine natural killer (uNK) cells are a major source of cytokines and angiogenic growth factors (AGFs), with AGF levels decreasing and cytokine levels increasing with gestational age. The factors that regulate AGF and cytokine secretion are unclear but may involve interactions between uNK cells and extravillous trophoblast (EVT) cells. We hypothesize that uNK cell interaction with EVT cells alters their cytokine and AGF secretion. METHODS Ex vivo co-cultures of uNK cells with either EVT (irradiated or fresh) or villous cytotrophoblast (CTB; control cell type) cells isolated from the same patients at 8-10 or 12-14 weeks gestational age (n = 10 each group) were established. Co-cultures were established with either direct contact between the different cell types or with the cells separated by a 0.4 µm filter. AGFs and cytokines were measured in cell culture supernatants using multiplex analysis (FAST Quant) or ELISA. RESULTS Secretion of angiopoietin-1 (P < 0.006) and vascular endothelial growth factor-C (P < 0.001) by uNK cells was lower when these cells were co-cultured, either directly or indirectly, with both trophoblast cell types at both gestational ages tested compared with when cultured alone. In contrast, interleukin (IL)-6 (P < 0.0001), IL-8 (P < 0.0001) and transforming growth factor-β1 (P < 0.002) were decreased only in direct uNK/EVT and uNK/CTB co-culture conditions at 8-10 and 12-14 weeks gestational age. CONCLUSIONS AGF and cytokine secretion was reduced after co-culture of uNK cells and both EVT and CTB cells. It remains unclear whether uNK cell AGF and cytokine production was reduced after co-culture with trophoblast cells (EVT or CTB) or whether trophoblast cell (EVT or CTB) AGF and cytokine production was reduced after co-culture with uNK cells. Local production of AGFs and cytokines in the placental bed may be lowered when uNK cells come in direct contact with EVT cells.

Evidence for activation of Toll-like receptor and receptor for advanced glycation end products in preterm birth.

Objective. Individuals with inflammation have a myriad of pregnancy aberrations including increasing their preterm birth risk. Toll-like receptors (TLRs) and receptor for advanced glycation end products (RAGE) and their ligands were all found to play a key role in inflammation. In the present study, we reviewed TLR and RAGE expression, their ligands, and signaling in preterm birth. Research Design and Methods. A systematic search was performed in the electronic databases PubMed and ScienceDirect up to July 2010, combining the keywords “preterm birth,” “TLR”, “RAGE”, “danger signal”, “alarmin”, “genomewide,” “microarray,” and “proteomics” with specific expression profiles of genes and proteins. Results. This paper provides data on TLR and RAGE levels and critical downstream signaling events including NF-kappaB-dependent proinflammatory cytokine expression in preterm birth. About half of the genes and proteins specifically present in preterm birth have the properties of endogenous ligands “alarmin” for receptor activation. The interactions between the TLR-mediated acute inflammation and RAGE-mediated chronic inflammation have clear implications for preterm birth via the TLR and RAGE system, which may be acting collectively. Conclusions. TLR and RAGE expression and their ligands, signaling, and functional activation are increased in preterm birth and may contribute to the proinflammatory state.

Peripheral RAGE (Receptor for Advanced Glycation Endproducts)-ligands in normal pregnancy and preeclampsia: novel markers of inflammatory response

Inflammatory response in preeclampsia (PE) is a key feature in its pathophysiology. Advanced Glycation Endproducts (AGEs), receptors for AGEs (RAGE), and RAGE ligands are involved in systemic inflammation in various pathological conditions. In this study, we measured serum RAGE ligands in normal pregnancy controls and PE patients. Levels of Carboxymethyl Lysine (CML), HMGB1 and S100A12/EN-RAGE were measured in thirty-three normal pregnant women 3 times at 10-12 (1st measurement), 28 (2nd measurement), and 36 (3rd measurement) weeks during gestation for paired analysis. We also measured those in serum samples from 17 severe PE patients at admission using ELISA. Early onset (EO, <32 weeks) and late onset (LO, ≥32 weeks) PE patients were compared with the 2nd and 3rd measurements of normal controls, respectively. CML and HMGB1 did not change during normal pregnancy. However, S100A12/EN-RAGE decreased from the 1st to 2nd measurement (P<0.0001). Across all PE patients, serum CML was unaltered, while HMGB1 significantly increased compared to 2nd (P=0.0002) and 3rd (P<0.0001) measurement as well as individually compared to both EO (P=0.018) and LO groups (P=0.0001). S100A12 in all PE patients increased over 2nd (P=0.0015) and 3rd (P=0.0002) measurements, although only LO was significantly increased compared to the 3rd measurement (P=0.0005). Our data suggest that patterns of serum RAGE ligand concentration indicate different inflammatory pathways in normal pregnancy, EO-PE, and LO-PE.

Inflammatory pattern recognition receptors and their ligands: factors contributing to the pathogenesis of preeclampsia

ProblemPreeclampsia, a pregnancy-specific hypertensive syndrome, is one of the leading causes of premature births as well as fetal and maternal death. Preeclampsia lacks effective therapies because of the poor understanding of disease pathogenesis. The aim of this paper is to review molecular signaling pathways that could be responsible for the pathogenesis of preeclampsia.Method of studyThis article reviews the English-language literature for pathogenesis and pathophysiological mechanisms of preeclampsia based on genome-wide gene expression profiling and proteomic studies.ResultsWe show that the expression of the genes and proteins involved in response to stress, host-pathogen interactions, immune system, inflammation, lipid metabolism, carbohydrate metabolism, growth and tissue remodeling was increased in preeclampsia. Several significant common pathways observed in preeclampsia overlap the datasets identified in TLR (Toll-like receptor)- and RAGE (receptor for advanced glycation end products)-dependent signaling pathways. Placental oxidative stress and subsequent chronic inflammation are considered to be major contributors to the development of preeclampsia.ConclusionThis review summarizes recent advances in TLR- and RAGE-mediated signaling and the target molecules, and provides new insights into the pathogenesis of preeclampsia.

Iron-dependent oxidative stress as a pathogenesis for preterm birth.

Problem: Preterm birth (PTB) is an oxidative stress-related disease that lacks effective therapies partly because of the poor understanding of disease pathogenesis. The aim of this manuscript was to review molecular pathways that could be responsible for the pathogenesis of PTB. Genomic and proteomic studies have started to delineate the wide array of mediators involved in this disorder. Understanding the mechanisms of the development of PTB and elucidating pathogenesis and pathophysiology are intrinsic to prevention and effective therapies for this disorder. Method of Study. This article reviews the English language literature for pathogenesis and pathophysiological studies on PTB. Several recent genomic and proteomic studies are discussed in the context of PTB biology. Results: Decidual hemorrhage has been identified histologically in the placentas of patients with PTB, which may result in high levels of free heme and iron. Several important PTB-specific genes and proteins overlap with those known to be regulated by iron. Others were genes involved in oxidative stress and detoxification. Free iron oxidatively modifies lipid and protein, leading to DNA and cell damage. This signaling pathway of PTB will be discussed as it provides new insights into regulation of inflammation, oxidative stress, and detoxification. Conclusion: This review summarizes recent advances in heme/iron-mediated signaling, the target genes thereof, and the potential challenges to the understanding of pathogenesis and pathophysiology of PTB. A novel model is proposed. Collectively, decidual hemorrhage and inflammation are considered to be major contributors to the pathogenesis of PTB. Target Audience: Obstetricians & Gynecologists, Family Physicians Learning Objectives: After completion of this article, the reader should be able to paraphrase the role of oxidative stress in pathogenesis of preterm birth, explain the idea of preterm birth as a “syndrome,” and summarize the potential role of early uterine bleeding in pathophysiology of preterm birth.