27 results found
Huang Z, Lai PF, Cocker ATH, et al., 2023, Roles of N-linked glycosylation and glycan-binding proteins in placentation: trophoblast infiltration, immunomodulation, angiogenesis, and pathophysiology, Biochemical Society Transactions, Vol: 51, Pages: 639-653, ISSN: 0300-5127
Protein N-linked glycosylation is a structurally diverse post-translational modification that stores biological information in a larger order of magnitude than other post-translational modifications such as phosphorylation, ubiquitination and acetylation. This gives N-glycosylated proteins a diverse range of properties and allows glyco-codes (glycan-related information) to be deciphered by glycan-binding proteins (GBPs). The intervillous space of the placenta is richly populated with membrane-bound and secreted glycoproteins. Evidence exists to suggest that altering the structural nature of their N-glycans can impact several trophoblast functions, which include those related to interactions with decidual cells. This review summarizes trophoblast-related activities influenced by N-glycan-GBP recognition, exploring how different subtypes of trophoblasts actively adapt to characteristics of the decidualized endometrium through cell-specific expression of N-glycosylated proteins, and how these cells receive decidua-derived signals via N-glycan-GBP interactions. We highlight work on how changes in N-glycosylation relates to the success of trophoblast infiltration, interactions of immunomodulators, and uterine angiogenesis. We also discuss studies that suggest aberrant N-glycosylation of trophoblasts may contribute to the pathogenesis of pregnancy complications (e.g. pre-eclampsia, early spontaneous miscarriages and hydatidiform mole). We propose that a more in-depth understanding of how N-glycosylation shapes trophoblast phenotype during early pregnancy has the potential to improve our approach to predicting, diagnosing and alleviating poor maternal/fetal outcomes associated with placental dysfunction.
Cocker ATH, Whettlock EM, Browne B, et al., 2022, Isolation of single cells from human uterus in the third trimester of pregnancy: myometrium, decidua, amnion and chorion, Oxford Open Immunology, Vol: 3, Pages: 1-12, ISSN: 2633-6960
During pregnancy, interactions between uterine immune cells and cells of the surrounding reproductive tissues are thought to be vital for regulating labour. The mechanism that specifically initiates spontaneous labour has not been determined, but distinct changes in uterine immune cell populations and their activation status have been observed during labour at term gestation. To understand the regulation of human labour by the immune system, the ability to isolate both immune cells and non-immune cells from the uterus is required. Here, we describe protocols developed in our laboratory to isolate single cells from uterine tissues, which preserve both immune and non-immune cell populations for further analysis. We provide detailed methods for isolating immune and non-immune cells from human myometrium, chorion, amnion and decidua, together with representative flow cytometry analysis of isolated cell populations present. The protocols can be completed in tandem and take approximately 4–5 h, resulting in single-cell suspensions that contain viable leucocytes, and non-immune cells in sufficient numbers for single-cell analysis approaches such as flow cytometry and single cell RNA sequencing (scRNAseq).
Li J, 2022, PKA and EPAC: cyclic AMP effectors in the regulation of myometrial inflammatory responses in parturition
The timing of parturition is key to a successful conclusion of pregnancy. For births that occur before 37 weeks, which is defined as preterm birth (PTB), the baby is at increased risk of neonatal morbidity and mortality. PTB remains a significant public health concern, affecting around 1 in 10 births worldwide. The initiation of parturition is caused by an accumulation of factors that promote myometrial contractions, and inflammation is often proposed to be one such factor. At the same time, it is also proposed that a withdrawal of signalling pathways that support myometrial quiescence is sufficient to trigger parturition. Progesterone is most recognised for this role in maintaining myometrial quiescence. There is also ample evidence to suggest that cAMP signalling also plays a role. Recent published studies have suggested that PKA can enhance the effects of progesterone and suppress myometrial inflammatory responses. Additionally, a pro-contractile role for cAMP via EPAC-mediated pathways has also been proposed. The overall aim for this thesis was to investigate the role of cAMP in the regulation of myometrial inflammatory responses in the context of parturition.Using primary myometrial cells cultured from human myometrial biopsies, which were all obtained from term gestation non-labouring study participants at time of elective/planned Caesarean section, my studies demonstrated that cAMP’s anti-inflammatory effects are mediated by PKA. PKA-specific activator N6-benzoyl-cAMP (6-Bnz-cAMP) suppressed myometrial expression of IL-8 in response to IL-1β stimuli. Furthermore, siRNA knockdown of catalytic PKA subunit (PKAc) partially reduced forskolin’s ability to suppress IL-8 expression. My data also showed forskolin’s ability to reduce NF-κB translocation is lost in the event of PKAc knockdown. The role of EPAC was investigated using EPAC activator 8-pCPT-2'-O-Me-cAMP and inhibitor ESI-09. My experiments showed EPAC activation was able to in
Zhang J, Chen J, Richardson JP, et al., 2022, Targeting an Initiator Allergen Provides Durable and Expansive Protection against House Dust Mite Allergy, ACS PHARMACOLOGY & TRANSLATIONAL SCIENCE, Vol: 5, Pages: 735-751
Lai PF, Lei K, Zhan X, et al., 2021, Labour classified by cervical dilatation & fetal membrane rupture demonstrates differential impact on RNA-seq data for human myometrium tissues., PLoS One, Vol: 16, Pages: 1-22, ISSN: 1932-6203
High throughput sequencing has previously identified differentially expressed genes (DEGs) and enriched signalling networks in human myometrium for term (≥37 weeks) gestation labour, when defined as a singular state of activity at comparison to the non-labouring state. However, transcriptome changes that occur during transition from early to established labour (defined as ≤3 and >3 cm cervical dilatation, respectively) and potentially altered by fetal membrane rupture (ROM), when adapting from onset to completion of childbirth, remained to be defined. In the present study, we assessed whether differences for these two clinically observable factors of labour are associated with different myometrial transcriptome profiles. Analysis of our tissue ('bulk') RNA-seq data (NCBI Gene Expression Omnibus: GSE80172) with classification of labour into four groups, each compared to the same non-labour group, identified more DEGs for early than established labour; ROM was the strongest up-regulator of DEGs. We propose that lower DEGs frequency for early labour and/or ROM negative myometrium was attributed to bulk RNA-seq limitations associated with tissue heterogeneity, as well as the possibility that processes other than gene transcription are of more importance at labour onset. Integrative analysis with future data from additional samples, which have at least equivalent refined clinical classification for labour status, and alternative omics approaches will help to explain what truly contributes to transcriptomic changes that are critical for labour onset. Lastly, we identified five DEGs common to all labour groupings; two of which (AREG and PER3) were validated by qPCR and not differentially expressed in placenta and choriodecidua.
Lai PF, Young RC, Tribe RM, et al., 2021, Evaluating aminophylline and progesterone combination treatment to modulate contractility and labor‐related proteins in pregnant human myometrial tissues, Pharmacology Research & Perspectives, Vol: 9, Pages: 1-18, ISSN: 2052-1707
Progesterone (P4) and cyclic adenosine monophosphate (cAMP) are regarded as pro-quiescent factors that suppress uterine contractions during pregnancy. We previously used human primary cells in vitro and mice in vivo to demonstrate that simultaneously enhancing myometrial P4 and cAMP levels may reduce inflammation-associated preterm labor. Here, we assessed whether aminophylline (Ami; phosphodiesterase inhibitor) and P4 can reduce myometrial contractility and contraction-associated proteins (CAPs) better together than individually; both agents are clinically used drugs. Myometrial tissues from pregnant non-laboring women were treated ex vivo with Ami acutely (while spontaneous contracting) or throughout 24-h tissue culture (±P4); isometric tension measurements, PKA assays, and Western blotting were used to assess tissue contractility, cAMP action, and inflammation. Acute (1 h) treatment with 250 and 750 μM Ami reduced contractions by 50% and 84%, respectively, which was not associated with a directly proportional increase in whole tissue PKA activity. Sustained myometrial relaxation was observed during 24-h tissue culture with 750 μM Ami, which did not require P4 nor reduce CAPs. COX-2 protein can be reduced by 300 nM P4 but this did not equate to myometrial relaxation. Ami (250 μM) and P4 (100 and 300 nM) co-treatment did not prevent oxytocin-augmented contractions nor reduce CAPs during interleukin-1β stimulation. Overall, Ami and P4 co-treatment did not suppress myometrial contractions more than either agent alone, which may be attributed to low specificity and efficacy of Ami; cAMP and P4 action at in utero neighboring reproductive tissues during pregnancy should also be considered.
Li JKH, Lai PF, Tribe RM, et al., 2021, Transcription factors regulated by cAMP in smooth muscle of the myometrium at human parturition., Biochemical Society Transactions, Vol: 49, Pages: 997-1011, ISSN: 0300-5127
Cyclic adenosine monophosphate (cAMP) contributes to maintenance of a quiescent (relaxed) state in the myometrium (i.e. uterine smooth muscle) during pregnancy, which most commonly has been attributed to activation of protein kinase A (PKA). PKA-mediated phosphorylation of cytosolic contractile apparatus components in myometrial smooth muscle cells (mSMCs) are known to promote relaxation. Additionally, PKA also regulates nuclear transcription factor (TF) activity to control expression of genes important to the labour process; these are mostly involved in actin-myosin interactions, cell-to-cell connectivity and inflammation, all of which influence mSMC transition from a quiescent to a contractile (pro-labour) phenotype. This review focuses on the evidence that cAMP modulates the activity of TFs linked to pro-labour gene expression, predominantly cAMP response element (CRE) binding TFs, nuclear factor κB (NF-κB), activator protein 1 (AP-1) family and progesterone receptors (PRs). This review also considers the more recently described exchange protein directly activated by cAMP (EPAC) that may oppose the pro-quiescent effects of PKA, as well as explores findings from other cell types that have the potential to be of novel relevance to cAMP action on TF function in the myometrium.
Lai PF, Georgiou EX, Tribe RM, et al., 2020, The impact of progesterone and RU-486 on classic pro-labour proteins & contractility in human myometrial tissues during 24-hour exposure to tension & Interleukin-1β, Molecular and Cellular Endocrinology, Vol: 500, ISSN: 0303-7207
Increased expression of pro-labour genes that encode cyclooxygenase-2 (COX-2), oxytocin receptor (OTR) and connexin-43 (Cx43) at parturition is often attributed to P4 functional withdrawal, based on findings from animal models and human primary myometrial cells. However, the cause of reduced myometrial P4 responsiveness that promotes contractions at labour is not fully determined. Uterine stretch occurs with advancing gestation but most in vitro experimental models do not take this into consideration. We aimed to examine whether tissue-level myometrial stretch influences the ability of P4 to regulate pro-labour protein abundance by using myometrial biopsies from term gestation pregnant women to assess the impact of 24 h exposure to combinations of (i) stretch-mediated tension, (ii) P4 (100 nM) and (iii) an anti-progestin, RU-486 (1 μM). Firstly, we observed baseline COX-2 and Cx43 protein levels increased, whereas P4 content along with calponin-1 and progesterone receptor (PR) protein abundance decreased, in vehicle-treated tissues. P4 supplementation subtly reduced COX-2 levels in un-stretched tissues. Spontaneous and oxytocin-augmented contractility were unchanged by tissue culture exposure to P4 and/or RU-486. Interleukin-1β (IL-1β; 1 ng/ml) enhanced COX-2 protein and PGE2 content in un-stretched tissues. Overall, tissue stretch may, in part, regulate P4-sensitive pro-labour protein levels, but this is likely to be reliant on interaction with other in utero factors that were absent in our tissue cultures. More complex culture conditions should be evaluated in future to aid further development of a physiologically relevant model to improve our understanding of in utero myometrial P4 responsiveness.
Herbert BR, Markovic D, Georgiou E, et al., 2019, Aminophylline and progesterone prevent inflammation-induced preterm parturition in the mouse, Biology of Reproduction, Vol: 101, Pages: 813-822, ISSN: 1529-7268
Although progesterone (P4) supplementation is the most widely used therapy for the prevention of preterm labor (PTL), reports of its clinical efficacy have been conflicting. We have previously shown that the anti-inflammatory effects of P4 can be enhanced by increasing intracellular cAMP levels in primary human myometrial cells. Here we have examined whether adding aminophylline (Am), a non-specific phosphodiesterase (PDE) inhibitor that increases intracellular cyclic adenosine monophosphate (cAMP) levels, to P4 might improve its efficacy using in vivo and in vitro models of PTL. In a mouse model of lipopolysaccharide (LPS)-induced PTL, we found that the combination of P4 and Am delayed the onset of LPS-induced PTL, while the same dose of P4 and Am alone had no effect. Pup survival was not improved by either agent alone or in combination. Myometrial prolabor and inflammatory cytokine gene expression was reduced, but the reduction was similar in P4 and P4/Am treated mice. There was no effect of the combination of P4 and Am on an ex vivo assessment of myometrial contractility. In human myometrial cells and myometrial tissue explants, we found that the combination had marked anti-inflammatory effects, reducing cytokine and COX-2 mRNA and protein levels to a greater extent than either agent alone. These data suggest that the combination of P4 and Am has a more potent anti-inflammatory effect than either agent alone and may be an effective combination in women at high-risk of PTL.
Stanfield Z, Lai PF, Lei K, et al., 2019, Corrigendum: Myometrial transcriptional signatures of human parturition, Frontiers in Genetics, Vol: 10, ISSN: 1664-8021
A Corrigendum onMyometrial Transcriptional Signatures of Human Parturitionby Stanfield, Z., Lai, P. F., Lei, K., Johnson, M. R., Blanks, A. M., Romero, R., et al. (2019). Front. Genet. 10:185. doi: 10.3389/fgene.2019.00185“Pei F. Lai” and “Kaiyu Lei” were not included as authors in the published article. Due to the addition of authors, the list of affiliations had been updated accordingly. The corrected Author Contributions Statement appears below.
Stanfield Z, Johnson MR, Blanks AM, et al., 2019, Myometrial transcriptional signatures of human parturition, Frontiers in Genetics, Vol: 10, ISSN: 1664-8021
The process of parturition involves the transformation of the quiescent myometrium (uterine smooth muscle) to the highly contractile laboring state. This is thought to be driven by changes in gene expression in myometrial cells. Despite the existence of multiple myometrial gene expression studies, the transcriptional programs that initiate labor are not known. Here, we integrated three transcriptome datasets, one novel (NCBI Gene Expression Ominibus: GSE80172) and two existing, to characterize the gene expression changes in myometrium associated with the onset of labor at term. Computational analyses including classification, singular value decomposition, pathway enrichment, and network inference were applied to individual and combined datasets. Outcomes across studies were integrated with multiple protein and pathway databases to build a myometrial parturition signaling network. A high-confidence (significant across all studies) set of 126 labor genes were identified and machine learning models exhibited high reproducibility between studies. Labor signatures included both known (interleukins, cytokines) and unknown (apoptosis, MYC, cell proliferation/differentiation) pathways while cyclic AMP signaling and muscle relaxation were associated with non-labor. These signatures accurately classified and characterized the stages of labor. The data-derived parturition signaling networks provide new genes/signaling interactions to understand phenotype-specific processes and aid in future studies of parturition.
Shah NM, Lai PF, Imami N, et al., 2019, Progesterone-related immune modulation of pregnancy and labor, Frontiers in Endocrinology, Vol: 10, ISSN: 1664-2392
Pregnancy involves a complex interplay between maternal neuroendocrine and immunological systems in order to establish and sustain a growing fetus. It is thought that the uterus at pregnancy transitions from quiescent to laboring state in response to interactions between maternal and fetal systems at least partly via altered neuroendocrine signaling. Progesterone (P4) is a vital hormone in maternal reproductive tissues and immune cells during pregnancy. As such, P4 is widely used in clinical interventions to improve the chance of embryo implantation, as well as reduce the risk of miscarriage and premature labor. Here we review research to date that focus on the pathways through which P4 mediates its actions on both the maternal reproductive and immune system. We will dissect the role of P4 as a modulator of inflammation, both systemic and intrinsic to the uterus, during human pregnancy and labor.
Lai PF, Young RC, Tribe RM, et al., 2018, The Efficacy of Aminophylline and Progesterone Co-Treatment to Suppress Human Myometrial Contractions during Prolonged Exposure., 65th Annual Scientific Meeting of the Society-for-Reproductive-Investigation (SRI), Publisher: SAGE PUBLICATIONS INC, Pages: 104A-104A, ISSN: 1933-7191
Lai PF, Tribe RM, Johnson MR, 2017, Effects of Tension on cAMP-Driven Control of Contractility in Human Myometrial Tissue., 64th Annual Scientific Meeting of the Society-for-Reproductive-Investigation (SRI), Publisher: SAGE PUBLICATIONS INC, Pages: 244A-244A, ISSN: 1933-7191
Georgiou E, Lei K, Lai P, et al., 2016, Progesterone repression of IL-1 beta action is maintained in human myometrium after the onset of labour, Publisher: WILEY-BLACKWELL, Pages: E12-E13, ISSN: 1470-0328
Lai PF, Tribe RM, Johnson MR, 2016, Differential impact of acute and prolonged cAMP agonist exposure on protein kinase A activation and human myometrium contractile activity, Journal of Physiology-London, Vol: 594, Pages: 6369-6393, ISSN: 1469-7793
Acute cAMP elevation inhibits myometrial contractility, but the mechanisms responsible are not fully defined and the long-term effects uncertain. These need to be defined in pregnant human myometrium before the therapeutic potential of cAMP-elevating agents in the prevention of preterm labour can be realised. In the present study, we tested the hypotheses that PKA activity is necessary for cAMP-induced myometrial relaxation and prolonged cAMP elevation can prevent myometrial contractions. Myometrial tissues obtained from term, pre-labour elective Caesarean sections were exposed to receptor-independent cAMP agonists to determine the relationship between myometrial contractility (spontaneous and oxytocin-induced), PKA activity, HSP20 phosphorylation and expression of contraction-associated and cAMP signalling proteins. Acute (1 h) application of cAMP agonists promoted myometrial relaxation but this was weakly related to PKA activation. PKA-specific activator, 6-Bnz-cAMP, increased PKA activity (6.8 ± 2.0 mean fold versus vehicle; P = 0.0313) without inducing myometrial relaxation. Spontaneous myometrial contractility declined after 24 h but was less marked when tissues were constantly exposed to cAMP agonists, especially for 8-bromo-cAMP (4.3 ± 1.2 mean fold versus vehicle; P = 0.0043); this was associated with changes to calponin, cofilin and HSP20 phosphorylated/total protein levels. Oxytocin-induced contractions were unaffected by pre-incubation with cAMP agonists despite treatments being able to enhance PKA activity and HSP20 phosphorylation. These data suggest that cAMP-induced myometrial relaxation is not solely dependent on PKA activity and the ability of cAMP agonists to repress myometrial contractility is lost with prolonged exposure. We conclude that cAMP agonist treatment alone may not prevent preterm labour.
Georgiou EX, Lei K, Lai PF, et al., 2016, The study of progesterone action in human myometrial explants., Molecular Human Reproduction, ISSN: 1460-2407
STUDY HYPOTHESIS: Myometrial explants represent a superior model compared with cell culture models for the study of human myometrial progesterone (P4) signalling in parturition. STUDY FINDING: Gene expression analysis showed myometrial explants closely resemble the in vivo condition and the anti-inflammatory action of P4 is not lost with labour onset. WHAT IS KNOWN ALREADY: Circulating P4 levels decline before the onset of parturition in most animals, but not in humans. This has led to the suggestion that there is a functional withdrawal of P4 action at the myometrial level prior to labour onset. However, to date, no evidence of a loss of P4 function has been provided, with studies hampered by a lack of a physiologically relevant model. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Myometrial biopsies obtained at Caesarean section were dissected into explants after a portion was immediately snap frozen (t = 0). Microarray analysis was used to compare gene expression of t = 0 with paired (i) explants, (ii) passage 4 myometrial cell cultures or (iii) the hTERT myometrial cell line. Western blotting and chemokine/cytokine assays were used to study P4 signalling in myometrial explants. MAIN RESULTS AND THE ROLE OF CHANCE: Gene expression comparison of t = 0 to the three models demonstrated that explants more closely resemble the in vivo status. At the protein level, explants maintain both P4 receptor (PR) and glucocorticoid receptor (GR) levels versus t = 0 whereas cells only maintain GR levels. Additionally, treatment with 1 µM P4 led to a reduction in interleukin-1 (IL-1) β-driven cyclooxygenase-2 in explants but not in cells. P4 signalling in explants was PR-mediated and associated with a repression of p65 and c-Jun phosphorylation. Furthermore, the anti-inflammatory action of P4 was maintained after labour onset. LIMITATIONS/REASONS FOR CAUTION: There is evidence of basal inflammation in the myometrial explant model. WIDER IMPLICATIONS OF THE FINDINGS: Myomet
Lai PF, Tribe RM, Johnson MR, 2015, Sustained Presence of Progesterone Reduces Spontaneous Contractility Enhanced By Prolonged Exposure To 8-bromo-cAMP., Publisher: SAGE PUBLICATIONS INC, Pages: 334A-334A, ISSN: 1933-7191
Georgiou EX, Lai PF, Yulia A, et al., 2015, Progesterone Repression of IL-1 beta-Driven COX-2 Expression in Myometrial Explant Cultures, Publisher: SAGE PUBLICATIONS INC, Pages: 134A-134A, ISSN: 1933-7191
Newton GK, Perrior TR, Jenkins K, et al., 2014, The Discovery of Potent, Selective, and Reversible Inhibitors of the House Dust Mite Peptidase Allergen Der p 1: An Innovative Approach to the Treatment of Allergic Asthma, JOURNAL OF MEDICINAL CHEMISTRY, Vol: 57, Pages: 9447-9462, ISSN: 0022-2623
Lai PF, Tribe RM, Johnson MR, 2014, Prolonged Exposure to cAMP Enhancers Increases Pregnant Human Myometrium Contractility and Alters Contraction-Associated Protein Expression, 61st Annual Scientific Meeting of the Society-for-Gynecologic-Investigation (SGI), Publisher: SAGE PUBLICATIONS INC, Pages: 242A-243A, ISSN: 1933-7191
Lai PF, Tribe RM, Johnson MR, 2013, Prolonged Elevation of cAMP Enhances Spontaneous Contractility in Human Myometrium from Pregnant Women at Term, 60th Annual Scientific Meeting of the Society-for-Gynecologic-Investigation (SGI), Publisher: SAGE PUBLICATIONS INC, Pages: 173A-173A, ISSN: 1933-7191
Lai P, Michelangeli F, 2012, Bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)-methane (bis-phenol) is a potent and selective inhibitor of the secretory pathway Ca2+ ATPase (SPCA1), BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol: 424, Pages: 616-619, ISSN: 0006-291X
Lai P, Yip NC, Michelangeli F, 2011, Regucalcin (RGN/SMP30) alters agonist- and thapsigargin- induced cytosolic [Ca2+] transients in cells by increasing SERCA Ca(2+)ATPase levels, FEBS LETTERS, Vol: 585, Pages: 2291-2294, ISSN: 0014-5793
Lai P, Michelangeli F, 2009, Changes in expression and activity of the secretory pathway Ca2+ ATPase 1 (SPCA1) in A7r5 vascular smooth muscle cells cultured at different glucose concentrations, BIOSCIENCE REPORTS, Vol: 29, Pages: 397-404, ISSN: 0144-8463
Michelangeli F, Ogunbayo OA, Wootton LL, et al., 2008, Endocrine disrupting alkylphenols: Structural requirements for their adverse effects on Ca2+ pumps, Ca2+ homeostasis & Sertoli TM4 cell viability, CHEMICO-BIOLOGICAL INTERACTIONS, Vol: 176, Pages: 220-226, ISSN: 0009-2797
Ogunbayo OA, Lai PF, Connolly TJ, et al., 2008, Tetrabromobisphenol A (TBBPA), induces cell death in TM4 Sertoli cells by modulating Ca2+ transport proteins and causing dysregulation of Ca2+ homeostasis, TOXICOLOGY IN VITRO, Vol: 22, Pages: 943-952, ISSN: 0887-2333
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