1,721,121 research outputs found
Characterisation of placental and platelet exosome-bound cardiotrophin-1 in cardiomyocyte and endothelial cell function
Get PDFMaternal cardiovascular adaptions during pregnancy have been well described and include characteristic changes in cardiac morphology and function. The mechanisms behind these changes remain largely unknown but are tied to the developing feto-placental unit. Extracellular vesicles are membrane-encapsulated particles carrying cargo originating from their parental cells and are critical mediators of intercellular communication. Syncytiotrophoblast extracellular vesicles (STB-EV), comprising of microvesicles and exosomes, are released by the placenta into the maternal circulation throughout pregnancy. We hypothesized that there might be molecules on the STB-EV that had cardiac effects. In this thesis, we characterised the expression of Cardiotrophin 1 (CT-1), a known potent inducer of cardiac hypertrophy and regulator of cardiac physiology, as being present on the human placenta. Importantly, we demonstrated that CT-1 is present on both microvesicles and exosomes, with a greater expression on exosomes. Furthermore, we show that CT-1 is biologically active and can phosphorylate its cognate receptor gp130/LIFR. Moreover, we demonstrate downstream signaling pathways that are mediated by STB-EV, most notably STAT 3 and ERK 1/2. We subsequently conducted a transcriptomic analysis using microarrays to investigate the effects of CT-1 positive STB-EV on cardiomyocytes derived from human induced pluripotent stem cells. This analysis showed significantly upregulated and downregulated genes involved in cardiovascular function. Furthermore, gene networking using Ingenuity Pathway Analysis (IPA) revealed STAT 3 as the most likely mediator of these gene changes. Intriguingly, real-time quantitative PCR validation confirmed the vesicular changes seen in the array but revealed that while some of the gene changes are directly due to CT-1, some are almost certainly due to the cargo within the vesicles. As a corollary to our experimental investigations we also identified, for the first time that CT-1 was present in platelets and platelet extracellular vesicles (PEV). We found in vitro evidence that PEV-bound CT-1 phosphorylated gp130 signaling leading to the activation of STAT 3 and ERK 1/2. Furthermore, we demonstrated that CT-1 positive vesicles protect against cytotoxicity in endothelial cells raising the possibility that this cytokine performs a cellular protective role.
This thesis thus reports the first observation of active CT-1 expressed in STB-EV from normal placentae and points to a novel finding that the placenta may well play a role in the orchestration of cardiovascular adaptation in pregnancy. Moreover, our results suggest that the placenta can communicate with the maternal heart. Additionally, it places CT-1 in vesicles as playing a significant role in both cardiomyocyte and vascular biology. Finally, the finding that platelets contain Cardiotrophin-1 positive vesicles suggests that this cytokine may well have a role in cellular protection outside pregnancy.</p
Developing exocounter platform for the detection of placenta small extracellular vesicle (psEV) in serum/plasma from normal pregnancy and preeclampsia patients and dissecting the potential role of psEV on the renal function
No full textIntroduction: Preeclampsia (PE) is a complication of pregnancy that affects 2-8% of women worldwide. It is one of the leading causes of maternal deaths and premature birth. PE is characterised by high blood pressure, proteinuria, and/or end-organ damage after 20 weeks of gestation. The placenta is believed to play a crucial role in the development of PE, with altered extracellular vesicle (pEV) being released into the maternal circulation. However, the potential diagnostic utility of pEV for early detection of PE remains unclear. Furthermore, the impact of pEV on renal functions, specifically regarding proteinuria and reduced renal function in PE, is not fully understood. This thesis aims to address these questions and shed light on the involvement of pEV in PE pathogenesis.
Aim: My research investigates the potential of utilizing pEV as a diagnostic tool for early detection of preeclampsia, while also examining the impact of pEV on renal functions in individuals with normal pregnancy (NP) and PE.
Methodology: I have investigated the potential diagnostic value of pEV for early detection of PE, using a novel EV characterising device named ExoCounter. Additionally, I conducted comprehensive studies to understand the functional and proteomics effects of pEV derived from NP and PE on podocytes, a type of renal cells.
Results: The serum of women who developed early onset preeclampsia (WWD-EOPE) during the first trimester showed significantly higher counts of CD10-placental alkaline phosphatase (PLAP) and CD63-PLAP placental small extracellular vesicle (psEV) compared to normal pregnancy (NP). While previous studies have demonstrated that the levels of CD10-PLAP and CD63-PLAP placental extracellular vesicles (pEV) are elevated in PE compared to NP in the third trimester, this is the first study to report that this elevation can also be detected as early as the first trimester. These psEV can serve as biomarkers to identify patients at risk of developing EOPE, allowing for early intervention. Additionally, psEV isolated from ex vivo placental dual-lobe perfusion were found to be internalized by podocytes. Flow cytometry analysis revealed no significant difference in the levels of podocyte-internalized EV from NP psEV and PE psEV. NP psEV induced increased migration ability in podocytes, potentially through mechanisms involving enhanced nephrin expression and strengthened cytoskeleton filamentous F-actin. On the other hand, PE psEV impaired podocyte adhesion ability and did not stimulate migration, potentially due to the formation of stressed F-actin. Subsequent analysis using proteomics analysis and functional enrichment revealed that differentially expressed proteins (DEPs) in podocytes treated with NP psEV were found to be associated with cellular component organization or biogenesis, intracellular transport, and mitochondrial functions. Conversely, DEPs in podocytes treated with PE psEV were potentially involved in metabolic processes, nitrogen compound metabolic processes, and gene expression.
Conclusions: The findings presented in this thesis have advanced the knowledge of psEV in the context of preeclampsia, offering insights into their diagnostic potential and proposing effective methodologies for their evaluation. Furthermore, the examination of NP and PE psEV’s impact on podocyte functions has provided valuable insights that could guide the development of future therapeutic strategies to enhance maternal and fetal well-being
Optimisation of ExoCounter technology for use in cerebrospinal fluid and investigation of placenta-derived exosomes within cerebrospinal fluid
Get PDFPregnancy is associated with neurological changes and increased risk of certain neurological pathologies. The mechanisms and causes of these changes are not entirely clear. Placenta-derived exosomes are small vesicles released from the placenta into maternal circulation. Research suggests that these exosomes are bioactive and can affect cells and tissues they encounter. There is evidence that exosomes of non-placental origin can cross the blood-brain barrier and be taken up by central nervous tissues, and that placenta-derived exosomes may increase the permeability of the blood-brain barrier. At the time of writing, no human studies have addressed the possibility that placenta-derived exosomes may cross the blood-brain barrier and affect neurological tissues. This project used a novel technology for exosome quantification, the ExoCounter assay, to analyse placenta-derived exosomes in matched cerebrospinal fluid and plasma samples from normotensive, preeclamptic and eclamptic pregnant women. This project did not find evidence that placenta-derived exosomes are present in cerebrospinal fluid. No difference was seen in plasma or cerebrospinal fluid counts between the three clinical groups, but this was affected by small samples sizes and unmatched gestational age between groups. In addition to work on the samples, this project assessed performance of the ExoCounter assay, and found high linearity, high repeatability, and dependence of the assay on intact exosome membranes. The impact of human/pipetting error and human anti-mouse antibodies was assessed, with no evidence found of interference by either factor
Sex differences in type I interferon production by plasmacytoid dendritic cells
Get PDFThe importance of immune sex differences is widely gaining recognition as critically important in determining differential outcome in health and disease between males and females. The female immune response is generally more pro-inflammatory and shows a greater TH2-skew than the male response which is generally more tolerant and TH1-skewed. Central to this difference the production of type I interferon (IFN-I) by the principal responsible cell type, plasmacytoid dendritic cells (pDCs), is greater in females as the upstream receptor, TLR7, is encoded by the X-chromosome and is biallelically expressed in ∼30% of female immune cells. However, study of pDCs is historically difficult as they constitute only 0.1-0.5% of peripheral blood mononuclear cells and their activity declines rapidly ex-vivo.
The aim of this thesis was therefore to investigate further the sex-differential behaviour of pDCs; for which a new assay to stimulate their IFN-I production within whole blood was developed and revealed that adult male pDCs respond to stimulation with greater upregulation of activation markers despite lower IFN-I production than adult female cells. The pDC response between sexes was also compared within a cohort of adolescents co-vaccinated with Pfizer BioNTech BNT162b2 and live-attenuated influenza vaccine where increased activation in male pDCs corresponded to a delay in reaching the same level of IFN-I production as female pDCs. Additionally, male androgens were found to suppress pDC IFN-I which paradoxically increased antibody titres to BNT162b2 and suggests high immune activation is not beneficial to new mRNA vaccines as it is for traditional protein- subunit vaccines.
Despite these differences, transcriptional profiling of the largest pDC single-cell RNA sequencing dataset generated to-date revealed remarkable similarities between male and female pDCs with no difference for transcription of IFN-I or markers of activation. Differentially-expressed genes revealed subtle differences with a focus on cytokine signalling in female pDCs and antigen presentation in male pDCs; further supporting their delayed activation compared to female pDCs.
Lastly, sex-differential IFN-I has been linked to selection for increased IFN-I - resistance and decreased viral replicative capacity (VRC) during in-utero transmission of HIV-1 to females. Here no such selection for VRC is seen for heterosexual adult-to-adult HIV-1 transmission which suggests the adult-to-adult transmission bottleneck differs from the in-utero mother-to-child bottleneck.
Overall, data in this thesis supports a model wherein monoallelic TLR7 expression increases the activation threshold for male pDCs which delays IFN-I production relative to female pDCs with biallelic TLR7 expression
Sexual dimorphism in Hofbauer cells
Get PDFSexual dimorphism is defined as the existing differences between individuals of different genders in features other than gender itself. In addition to well described differences in physical traits, reproductive organs, or hormones the term also indicates the differences which are present in cognition, immunity, and the biological responses to various medical conditions.
Regarding sexual dimorphism in the placenta; although it is known that male foetuses have more severe cases of preeclampsia and cases of gestational diabetes mellitus, on the other hand, female foetuses suffer more HIV infections transmitted from the mother, however, the detailed mechanisms underlying to explain these intriguing facts are unknown.
Hofbauer cells are immune cells located in the placenta that are of fetal origin and can be identified from an early stage of pregnancy - only after 18 days of gestation. They are known to function as macrophages including phagocytosis, antigen presentation, angiogenesis and vasculogenesis, and also known to be involved in several medical conditions during pregnancy such as villitis of unknown etiology and preeclampsia. In spite of their important roles, research focused on Hofbauer cells has been limited, largely due to difficulties in their isolation.
I successfully established a new methodology to isolate Hofbauer cells from whole term placenta with high yield and purity and subjected Hofbauer cells isolated from 6 normal male placentae and 6 normal female placentae to mass spectrometry analysis. From the result of spectrometry analysis, 86 out of 5426 proteins were found to be significantly differentially expressed between male and female placentae (p<0.05). These 86 proteins were bioinformatically analysed utilising STRING® and found to connectively form four different pathways engaged in immunity, mitochondrial function, extracellular vesicle functions and transcriptional regulation function. Results from mass spectrometry were validated further using western blotting, which corroborated the proteomics differences.
Furthermore, I extracted RNAs from the cells that were subjected to proteomics and next-generation sequencing was performed. Of 6844 included transcripts, 26 transcripts were significantly up-regulated in male HBCs samples and 23 transcripts were significantly up-regulated in female HBCs samples (adjusted p value<0.05). To identify the signalling pathways between differentially expressed genes, results from RNA-sequencing data was applied to KEGG (Kyoto Encyclopedia of Genes and Genomics) and “cytokine- cytokine receptor interaction pathway” was most related to the functions of Hofbauer cells as immune cells and had the smallest p value (0.001) and adjusted p value (0.073). From the results, it was clear that although there are some genes which are related to immune functions and are located on X chromosomes, such as IL2RG, are highly up-regulated in female HBCs, male HBCs manage their immunocompetence by regulating the expressions of chemokines, chemokine receptors and several cytokines, cytokine receptors including IL-11, IL23A, IL-1 (IL1A, IL1B, IL1RN and IL1F5), IL22RA1, IL34 and IL2RB, as well as members from the TNF and TGF-β family. Some of them are heavily involved in the pathogenesis of pregnancy related conditions represented by preeclampsia and GDM.
In summary, HBCs exhibit significant differences in proteomic and genetic signature in male and female placentae. It is likely these differences confer functional alterations in immune cell function which may impact a number of critical functions
Investigating the role of syncytiotrophoblast-derived extracellular vesicles in the pathogenesis of preeclampsia
Get PDFIntroduction: Preeclampsia (PE), a multi-systemic disorder of pregnancy affecting 2-8% of women globally, is one of the leading causes of maternal and neonatal morbidity and mortality. PE is diagnosed by de novo hypertension and end-organ damage. Although precise mechanisms are not fully elucidated, placenta is involved in the pathogenesis. Syncytiotrophoblast-derived extracellular vesicles (STB-EVs), which are membrane-bound biomolecules ranging between 30 – 1000 nm in size are gaining attention due to their diagnostic potential and their role in pathogenesis of PE. This thesis explores the roles of STB-EVs in pregnancy, focusing on the effects of their cargo (proteins and miRNA) on altering maternal physiology in PE and their potential to serve as non-invasive biomarkers. This research aimed to establish reliable protocols for isolating STB-EVs and to investigate their protein and genetic cargo, particularly focusing on kynurenine metabolising enzymes (KYNMEs) and microRNAs (miRNAs) relevant to preeclampsia.
Aims: The study's objectives included isolating and characterizing STB-EVs, analysing their protein (kynurenine metabolising enzymes, KYNME) and genetic cargo (hsa-miR-9-5p), and exploring their functional implications PE.
Methods: STB-EVs were isolated and enriched using ex vivo dual lobe placental perfusion and differential ultracentrifugation. Characterisation involved nanoparticle tracking analysis, western blotting, transmission electron microscopy, and flow cytometry. The protein cargo, particularly KYNMEs and their functionality, were analysed using immunohistochemistry, capillary electrophoresis and western blotting, and gas chromatography-mass spectrometry. Differentially expressed microRNAs were validated using quantitative polymerase chain reaction and their functional impacts were assessed in vitro on cerebral microvascular endothelial cells via assays for cell proliferation, viability and migration.
Results: The isolation protocol successfully enriched STB-EVs. STB-EVs from normal and PE pregnancies contained KYNMEs with no significant differences between the two groups. Serum kynurenine levels were significantly lower in preeclamptic pregnancies. KYNMEs on STB-EVs demonstrated a dose-dependent attenuation of kynurenine levels in vitro. Additionally, differentially expressed miRNAs were validated, with hsa-miR-9-5p identified as a key target over-expressed in medium/large STB-EVs and serum EVs in preeclampsia. miR-9-5p attenuated cell proliferation and migration and altered the expression of angiogenesis-related proteins in cerebral microvascular endothelial cells.
Conclusion: This thesis for the first time highlights the potential role of STB-EVs in kynurenine pathway and describes the anti-angiogenic effect of hsa-miR-9-5p on cerebral microvascular endothelial cells. The findings suggest that STB-EVs could be critical in regulating vascular and angiogenic responses in preeclampsia, providing a foundation for further understanding of the pathogenesis and future diagnostic strategies
tRNA fragments in preeclampsia: expression, function and biomarker potential
No full textBackground: Preeclampsia is a placental disease, resulting in maternal hypertension and multi-organ impairment. The relationship between placental pathology and the widespread inflammation and endothelial dysfunction in preeclampsia is incompletely characterised. Mismatch between placental nutrient supply and fetal demands induces stress in the syncytiotrophoblast, the placental layer in contact with maternal blood. Such stress alters the content and release of extracellular vesicles (STB-EVs) into the maternal circulation. I have previously shown 5’-tRNA fragments (5’-tRFs) constitute the majority of small RNA in STB-EVs in healthy pregnancies. 5’-tRFs are produced in response to stress. In this thesis, I hypothesise STB-EV 5’-tRFs release might change in early-onset preeclampsia. I aim to 1) characterise STB-EV 5’-tRFs in normal pregnancy and preeclampsia; 2) investigate STB-EV tRFs in maternal blood; 3) evaluate tRFs as circulating biomarkers in preeclampsia; 4) explore functional activity of STB-EV tRFs in preeclampsia dysfunctional cells.
Methods: Placentas from eight cases and six controls were perfused, comparing smallRNA sequences from STB-EVs using various bioinformatic approaches. EVs, STB-EVs and total RNA were isolated from different plasma samples and tRFs quantified using qPCR. The actions of the most abundant preeclampsia-dysregulated 5’-tRFs were investigated in monocytes, macrophages and endothelial cells.
Results: 5’-tRFs constitute the majority of small RNA in STB-EVs from both preeclampsia and normal pregnancies. >4000 small RNA fragments are differentially expressed in preeclampsia STB-EVs. Preeclampsia-dysregulated 5’-tRFs are detectable in maternal plasma, where a placentally-derived load circulates. 5’-tRFs do not currently represent blood biomarkers for preeclampsia. The most abundant preeclampsia-upregulated small RNA, 5’-tRF-Glu-CTC, induces inflammation in macrophages but has no direct effect on monocytes or endothelial cells. The conditioned media from 5’-tRF-Glu-CTC-activated macrophages reduces endothelial nitric oxide synthase expression and increases adhesion molecule expression in endothelial cells.
Conclusions: STB-EV tRFs are placental endocrine signals which contribute to the pathogenesis of preeclampsia at a molecular level
Syncytiotrophoblast extracellular vesicles:their role in gestational diabetes mellitus
Get PDFGestational diabetes mellitus (GDM) is the most common metabolic complication of pregnancy. It affects more than 17 million women each year and causes adverse consequences for both mother and baby. Previous research has observed that the placenta plays an important role in the pathogenesis of GDM, since delivery of the placenta results in the immediate reversal of diabetic symptoms. The aim of this thesis is to investigate the nature of the placenta’s role in GDM, building on the knowledge that the organ releases extracellular vesicles (EVs) into the maternal circulation from six weeks gestation until delivery. EVs carry a variety of ligands, including both proteins and RNA species, between the cells. As such, they have an important role in cell-cell communication in both physiological and pathophysiological conditions, and may provide novel biomarkers of disease. We hypothesized that placental EVs might carry factors impacting insulin availability, a hallmark of pregnancy altered during the establishment of GDM. We identified two novel biomarkers (dipeptidyl peptidase 4 (DPPIV) and insulin receptor (IR)) on placental EVs. DPPIV is a glycoprotein that rapidly degrades incretin hormones, which are known to increase insulin secretion, and thus regulate glucose homeostasis. DPPIV inhibitors are established as therapeutic agents for type 2 diabetes mellitus, and we confirmed that they can be used to inhibit placental DPPIV-EV activity. IR is a key regulator of glucose uptake by skeletal muscles and adipose cells. We showed that placental IR-EVs can act as decoy receptors capable of neutralising maternal insulin. We demonstrated that GDM patients exhibited significantly higher levels of circulating placental DPPIV-EVs and IR-EVs. The final aim of this project was to characterise the protein cargo of placental EVs from GDM pregnancy using mass spectrometry. We identified 56 proteins as differentially expressed between GDM and normal pregnancy. In conclusion, this DPhil project shows that STB-EVs carry biologically active molecules that have the potential to regulate maternal insulin levels in GDM and could represent biologically plausible biomarkers for the disease
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