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| Project Details |
| Funding Scheme : | General Research Fund | ||||||||||||||||||||||||
| Project Number : | 17122415 | ||||||||||||||||||||||||
| Project Title(English) : | The role of glycodelin-A on the vascular remodeling function of decidual natural killer cells during early pregnancy and its association with preeclampsia | ||||||||||||||||||||||||
| Project Title(Chinese) : | Glycodelin-A對孕早期蛻膜自然殺傷細胞血管重塑功能的作用及其與子癇前期的相關性研究 | ||||||||||||||||||||||||
| Principal Investigator(English) : | Dr Chiu, Chi Ngong | ||||||||||||||||||||||||
| Principal Investigator(Chinese) : | |||||||||||||||||||||||||
| Department : | Department of Obstetrics & Gynaecology | ||||||||||||||||||||||||
| Institution : | The University of Hong Kong | ||||||||||||||||||||||||
| E-mail Address : | pchiucn@hku.hk | ||||||||||||||||||||||||
| Tel : | |||||||||||||||||||||||||
| Co - Investigator(s) : |
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| Panel : | Biology & Medicine | ||||||||||||||||||||||||
| Subject Area : | Medicine, Dentistry & Health | ||||||||||||||||||||||||
| Exercise Year : | 2015 / 16 | ||||||||||||||||||||||||
| Fund Approved : | 1,064,451 | ||||||||||||||||||||||||
| Project Status : | Completed | ||||||||||||||||||||||||
| Completion Date : | 30-9-2018 | ||||||||||||||||||||||||
| Project Objectives : |
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| Abstract as per original application (English/Chinese): |
妊娠期母體螺旋動脈的重塑是形成胎盤並成功妊娠的必要條件，重塑失敗可導致多種妊娠並發症，危及孕婦和胎兒生命。子癇前期是常見的妊娠並發症之一，主要認為與滋養細胞侵入過淺，子宮螺旋動脈重塑障礙有關。子癇前期影響著2-5%的妊娠產婦，是產前發病率和死亡率的首要原因。目前尚無可靠的預測或早期診斷的方法。 蛻膜自然殺傷(dNK)細胞是孕早期蛻膜組織中最豐富的淋巴細胞群，妊娠第三月末期其數量約佔蛻膜淋巴細胞總量的70%。與外周血自然殺傷(pNK)細胞不同，dNK細胞具有極低的細胞殺傷活性，並能分泌各種大量的細胞因子，直接和間接作用於子宮螺旋動脈與滋養細胞，最終促進胎盤血管重塑。 Glycodelin-A（GdA)主要由蛻膜間質細胞分泌，是妊娠早期蛻膜中最豐富的糖蛋白。它對調節蛻膜內不同免疫細胞的細胞活性至關重要。我們未公開的數據表明GdA能特異性地與dNK細胞結合，促進其血管重塑功能。 因此我們提出假設：蛻膜內GdA通過影響孕早期dNK細胞的活性從而調節胎盤血管重塑；GdA表達下調會參與子癇前期的病理生理髮生機制。此假設將通過研究以下五個方面來驗證：(1) GdA對dNK細胞直接調節血管重塑的影響；(2) GdA對dNK細胞通過作用於滋養細胞而間接調節血管重塑的影響；(3) GdA對dNK細胞表面活化受體表達的影響；(4) GdA對dNK細胞的作用機制；(5) 子癇前期GdA及其受體的表達。 此研究有助於更深入理解人類早期胎盤形成的機制。在臨床方面，其結果或預示著可以使用GdA及/或其受體的表達水平作為預測或早期診斷子癇前期的指標之一。 |
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| Realisation of objectives: | Glycodelin-A (GdA) is an abundant glycoprotein in the decidua during early pregnancy. It plays an important role in regulating the cellular activity of different immune cells that reside in the decidua. In this study, we hypothesize that decidual GdA regulates the placental vascular remodeling in human by modulating the activities of dNK cells during early pregnancy and the down-regulation of GdA contributes to the pathophysiology of preeclampsia. There were 4 objectives in this project, and they had all been achieved. Objective 1. To study the effect of glycodelin-A on decidual natural killer cell-mediated vascular remodeling GdA was purified from amniotic fluid by affinity chromatography. Its identity was confirmed by tandem mass spectrometry. Insoluble particles and endotoxin were removed by a 0.22 μm filter and endotoxin removing kit, respectively. Buffy coats were obtained from Hong Kong Red Cross Blood Transfusion Service. Peripheral blood CD16-56bright NK cells, the suggested tissue-specific NK cells precursors, were isolated by fluorescence-activated cell sorting or by CD56 MicroBeads after negative immuno-magnetic NK cell isolation steps. Decidual natural killer (dNK) cells were isolated from first trimester termination of pregnancy tissues. The tissue was minced, digested with DNase I and collagenase and passed through a 70 μm cell filter to obtain single-cell suspension. dNK cells were obtained by Ficoll-Plaque followed by CD56+ positive immuno-magnetic isolation. GdA was purified from amniotic fluid by anti-glycodelin affinity chromatography. In addition, primary trophoblast was isolated from tissues obtained from patients undergoing surgical termination of first trimester pregnancy. NK cells (1x106) were cultured in the Roswell Park Memorial Institute (RPMI-1640) medium supplemented with 10% fetal bovine serum (FBS), 500 IU/mL of recombinant human interleukin (IL)-2, 5 ng/mL of recombinant human IL-15, and 5 μg/mL of GdA for 5 days. NK cells cultured under the same condition without GdA were used as control. GdA treatment significantly enhanced the expression of dNK cell markers CD9 and CD49a and the production of the dNK pro-angiogenic factor VEGF in the peripheral blood CD16-CD56bright NK cells. The spent media of GdA-treated CD16-CD56bright NK cells promoted tube formation of human umbilical vein endothelial cells (HUVEC). Supplementation by anti-VEGF antibody reduced the tube formation induction by GdA-treated peripheral blood CD16-56bright NK cell medium. Objective 2. To study the regulatory role of decidual natural killer cells on the vascular remodeling activity of trophoblast and its association with glycodelin-A The vascular remodeling activity of trophoblast was determined by the ability of the trophoblast to migrate/invade and then integrate into the endothelial cells network. Spent medium derived from GdA-treated peripheral blood CD16-56bright NK cell induced the invasion of primary extravillous trophoblast and JEG-3 cells. In contrast, the medium did not affect the migration of the trophoblast. Anti-IGFBP-1 antibody supplementation nullified the biological activity of GdA-treated peripheral blood CD16-56bright NK cell culture medium, i.e. down-regulated the invasiveness of JEG-3 cells when compared to GdA-treated NK cell medium group. On the other hand, anti-VEGF antibody has no such effect. Besides, spent medium derived from GdA-treated CD16-56bright NK cell has no effect on trophoblast integration to the endothelial cell network. Objective 3. To study the effect of glycodelin-A on the expression of activating receptors in decidual natural killer cell Flow cytometry was used to determine the surface markers of decidual NK cell markers (CD9, CD151 and L-selectin). GdA treatment significantly induced the expression of CD9 in peripheral blood CD16-CD56bright NK and the tissue-specific NK cells precursors subset, but not CD16+CD56dim NK cells. GdA has no effect on the expression of activating receptor, including LILRB1, NKp44 and KIR2DS1 in all NK cell subsets. Objective 4. To investigate the mechanism of glycodelin-A action on decidual natural killer cell GdA had a stronger binding affinity to the CD16-CD56bright NK cells when compared to the CD16+CD56dim NK cells. This GdA-NK cell interaction was reduced by de-sialylation. The involvement of L-selectin on GdA-NK cells interaction was confirmed by (1) immuno-precipitation with the recombinant L-selectin; (2) L-selectin expression on monocyte and macrophage surface; and (3) the ability of anti-L-selectin antibody to block the GdA binding to the monocytes and macrophage. The intracellular signaling pathways induced by GdA treatment was also analyzed. The biological activities of GdA were demonstrated to be mediated by the activation of ERK signaling pathways in NK cells. Objective 5. To determine the expression of glycodelin-A and its receptor in pregnancy complicated by preeclampsia Chorionic villus sampling (CVS) is used to obtained placental tissues in early pregnancy for prenatal diagnosis. In this study, CVS surplus to diagnostic needs (~1 mg) was collected. As there is no reliable marker in predicting PE in early pregnancy, spare CVS samples from all women consented to participate in the study were stored until delivery. The expressions of glycodelin-A and L-selectin in CVS was determined by immunohistochemistry using specific antibodies followed by fluorescent-labelled secondary antibody. Results from samples diagnosed to have PE at delivery was compared with that of the matched controls. PE is defined as maternal hypertension >140/90 mmHg without previous hypertensive history and 300 mg/l proteinuria without any history of renal disease. A total of 163 CVS samples have obtained during the study period and 5 of them were obtained from PE pregnancy. Our results showed that there was no difference on the expressions of glycodelin-A and L-selectin between first trimester CVS samples from PE patients and normal pregnancy control. | ||||||||||||||||||||||||
| Summary of objectives addressed: |
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| Research Outcome | |||||||||||||||||||||||||
| Major findings and research outcome: | - GdA treatment enhanced the expression of dNK cell markers CD9 and CD49a and the production of the functional dNK secretory product VEGF in the peripheral blood CD16-CD56bright NK cells. - The spent media of GdA-treated CD16-CD56bright NK cells promoted tube formation of human umbilical vein endothelial cells and invasiveness of trophoblasts. - The stimulatory effect of GdA on angiogenesis and trophoblast invasion were mediated by the up-regulation of vascular endothelial growth factor (VEGF) and insulin-like growth factor-binding protein 1 (IGFBP-1), respectively (Lee et al., 2019). GdA is involved in angiogenesis during embryogenesis as well as in endometrial cancer (Lee et al., 2016). -The stimulatory effect of GdA on angiogenesis and trophoblast invasion were mediated by the stimulatory activities of GdA on the extracellular signal-regulated protein kinases (ERK) signaling pathways activation (Lee et al., 2019). - Glycosylation is important in the biological activities of GdA. Only native GdA, but not deglycosylated or desialylated GdA, binds to NK cells (Lee et al., 2019). Deglycosylation also abolishes the regulatory activities of GdA on trophoblasts, monocyte and spermatozoa (Lee et al., 2016). - GdA interacted with L-selectin, expressed only in the CD16-CD56bright NK cells, but not in the CD16+CD56dim NK cells (Lee et al., 2019). | ||||||||||||||||||||||||
| Potential for further development of the research and the proposed course of action: |
There are two important areas for further development: 1. Based on the important role of GdA in regulating immune cell and trophoblast functions during early pregnancy, it is reasonable to speculate that deficiency in the normal functions of GdA may contribute to the pathophysiology of various pregnancy-associated complications. We are planning to determine the expression of GdA and its receptors in the placenta complicated by intrauterine growth restriction and gestational trophoblastic disease. 2. We have shown that GdA binding to L-selectina to monocyte and macrophage via carbohydrate recognition. Removal of sialic acid, the outmost sugar residue of the glycan chain, has lost the binding the GdA-mediated biological functions. In a recent study on its molecular structure using x-ray crystallography indicated a unique structure of glycodelin in presenting its glycan chains. Further study of glycodelin with homogeneous glycan structure is required to define the mechanistic aspects of these glycans. | ||||||||||||||||||||||||
| Layman's Summary of Completion Report: | The vascular remodeling of maternal spiral artery is crucial to placentation and successful pregnancy. Decidual natural killer (dNK) cells are the most abundant (~70%) leukocyte population at the maternal-fetal interface in early pregnancy. dNK cells secrete immunomodulatory factors that contributes to vascular remodeling. Dysregulation of vascular remodeling and aberrant levels of dNK cells are associated with various pregnancy complications leading to significant maternal and fetal death. Glycodelin-A (GdA) is a decidua-abundant glycoprotein regulators immune cell function. The project tested the hypothesis that GdA regulates the placental vascular remodeling in human by modulating the activities of dNK cells during early pregnancy. The results demonstrated a role of GdA in driving the NK cell differentiation into a dNK cells, which promotes vascular remodeling and placental development via secretion of cytokine IGFBP-1 and angiogenic factor VEGF. The project also identified the mechanism of GdA by binding to the surface receptors L-selectin and activate ERK signaling pathway. The outcome of this project gives a better understanding on the role of dNK cells in pregnancy. In long term, the present findings enhance our understanding of the cause of pregnancy loses and provide the basis for development of new treatment strategies. | ||||||||||||||||||||||||
| Research Output | |||||||||||||||||||||||||
| Peer-reviewed journal publication(s) arising directly from this research project : (* denotes the corresponding author) |
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| Recognized international conference(s) in which paper(s) related to this research project was/were delivered : |
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| Other impact (e.g. award of patents or prizes, collaboration with other research institutions, technology transfer, etc.): |
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| SCREEN ID: SCRRM00542 |