Trophoblast Microvesicles, Exosomes, miRNAs, Danger Molecules and Pre-eclampsia

Lead Research Organisation: University of Oxford
Department Name: Obstetrics and Gynaecology

Abstract

Pre-eclampsia is a complication of the second half of pregnancy which affects 2.5-3.0% of women. It has the potential to kill mother or baby or both, but cannot yet be prevented. It is the commonest reason for induced preterm delivery, which can lead to fetal handicap and has long term health consequences for both mother and child. No other complication of pregnancy is so common and so dangerous for both mother and baby. The disease originates in the placenta, the supply line for the unborn child, and evolves in two stages. The first stage, without any symptoms at 2-4 months of pregnancy, involves poor development of the blood supply to the placenta, leading to placental damage. The second stage, which occurs in the second half of pregnancy, involves activation of an inflammatory response in the mother's blood stream with damage to the blood vessels, leading to the high blood pressure and protein in the urine, which characterise the disorder. The first and second stages are believed to be linked by the release of inflammatory factors (collectively known as "danger" molecules) from the surface of the damaged placenta into the maternal blood, which interact with maternal inflammatory and endothelial cells to cause the disease. These danger molecules are the focus of this programme.

We believe that pre-eclampsia is caused not by one, but by many different danger molecules working together. These may be shed from the placenta either as soluble factors or associated with tiny fragments of cells called microvesicles and exosomes. These sub-cellular vesicles are now known to be an important way that cells communicate with each other. We have previously found that there are significantly more placental vesicles in the circulation of pre-eclamptic compared to normal pregnant women and that they can affect the function of immune and endothelial cells in the laboratory. We have found that these vesicles carry over 2,500 different molecules, many of which may be dangerous to the mother. These vesicles may, in effect, be acting like "cluster bombs", carrying multiple warheads to their target cells to cause the maximum damage. Thus they may play a key role in the disease process.

The purpose of this programme is to learn more about these vesicles, how they differ between normal and pre-eclamptic pregnancy and their role in the development of pre-eclampsia. As they are released into the mother's blood they provide us with easily accessible tiny biopsies of the placenta which give us a unique window on what is happening in the placenta during the development of the disease. The ultimate aim therefore is to isolate them from the maternal circulation and use them as markers to diagnose or predict pre-eclampsia. Understanding the make up of these vesicles may also help us develop potential treatments in the future.

Until recently most of these vesicles have been inaccessible because of their small size - below the limits of microscopy and other standard laboratory equipment. In this research we will use unique technology that we have developed that allows us to see the full range of circulating vesicles and distinguish those that come from the placenta from those that come from the mother's own systems. Our preliminary work, demonstrates that these previously 'invisible' particles form the major part of circulating vesicles; hence they are completely new territory for pre-eclampsia research.

Technical Summary

We will use placental perfusion to examine, (by proteomics and miRNA arrays), which danger molecules and miRNAs, shed from the placenta's syncytiotrophoblast surface, are soluble, and which are part of syncytiotrophoblast vesicles (STBM). As STBM comprise both smaller exosomes (STBex) and larger microvesicles (STBmic), with different possible biological functions, we will fractionate them (e.g. by nanofiltration) to determine their phenotypes and the differences between normal pregnancy and pre-eclampsia. To interpret our findings, the BeWo trophoblast cell line will be used to model the pre-eclamptic damage by exposing syncytialised BeWo to oxidative and inflammatory stress and inducers of apoptosis and necrosis. We will measure changes in soluble molecules released, proportions of STBex and STBmic and the molecules they carry, in response to each treatment to mimic the changes in pre-eclampsia. STBex and STBmic will be phenotyped using fluorescence Nanoparticle Tracking Analysis (fl-NTA - a technique we have pioneered) together with 6 colour flow cytometry. The further development of fl-NTA is a key part of this programme. We will then examine how STBex and STBmic and the miRNAs they carry are taken up by (using imaging flow cytometry, RT-PCR) and affect leucocyte and endothelial functions (cytokine/chemokine production and endothelial cell proliferation/permeability). Based on the danger molecules and miRNAs identified above, we will investigate their effect on syncytialised BeWo and placental explants to determine whether this triggers a vicious cycle of activation which could amplify the underlying problem. Our findings will be used to achieve our final aim, to characterize STBM from peripheral blood of normal pregnant and pre-eclamptic women, in samples from the Oxford Pregnancy Biobank, taken before and during active disease, to determine their use as diagnostic or predictive biomarkers.

Planned Impact

Who will benefit from this research?

The main beneficiaries from this research apart from other researchers (detailed elsewhere) will be 1) the women who suffer from pre-eclampsia, their unborn children and their families 2) the NHS in terms of cost savings through better prediction and management of pre-eclampsia 3) commercial companies through the exploitation of any biomarkers discovered and the development and sales of new instrumentation for the measurement of microvesicles and exosomes.

How will they benefit?

1) Patients: The women, children and families will benefit by the development of predictive tests which enable clinicians to identify who is at risk so that antenatal care is focused on them. This will help to avoid unnecessary admissions to hospital which can be very disruptive to family life. The research will also help us develop new treatments which can ameliorate the maternal problems to buy time for the fetus so that it does not need to be delivered too early, with all the inherent risks of prematurity, especially fetal handicap. There is growing evidence that inflammatory stress in pregnancy can cause epigenetic changes in both mother and child which lead to increased risk of cardiovascular disease in later life. Early treatment of these disorders by drugs or lifestyle changes could prevent the development of these problems and lead to significant improvements in quality of life.

2) NHS: These measures would have a major impact on the NHS in terms of cost savings in that they would 1) provide better prediction and management of pre-eclampsia allowing antenatal care to be focused on those who most need it and reduce the amount of unnecessary precautionary admissions of pregnant women to hospital 2) significantly reduce the incidence of pre-term delivery and fetal handicap with its lifelong associated costs 3) reduce the incidence of cardiovascular disease in mother and child in later life and 4) reduce the enormously expensive litigation costs often associated with these cases.

3) Industry: Commercial companies may benefit through the exploitation of any new biomarkers for pre-eclampsia. The applicants have considerable experience with working with industry. They have established an excellent working relationship with Nanosight Ltd over the last three years for the development of Nanoparticle Tracking Analysis (NTA) technology. This collaboration has put the UK at the forefront of the development of technology to rapidly measure and phenotype microvesicles and exosomes in biological fluids and there is huge potential in terms of instrument sales world wide. The applicants also have links with four companies (Beckman Coulter, Alere, Cyathus and Critical Diagnostics) for the evaluation of biomarkers for pre-eclampsia, one of which has led to a patent application. These companies would be natural partners for future biomarker development.

Publications

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publication icon
Carnell-Morris P (2017) Analysis of Extracellular Vesicles Using Fluorescence Nanoparticle Tracking Analysis. in Methods in molecular biology (Clifton, N.J.)

 
Description Advised on ise of PlGF based therapies for the prediction of preeclampsia - our data was used as part of the submission
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Characterisation of Trophoblast Extracellular Vesicles in Gestational Diabetes Mellitus
Amount £400,000 (GBP)
Organisation Janssen Diagnostics 
Sector Private
Country United States
Start 01/2017 
End 12/2019
 
Description ERASMUS
Amount € 1,320 (EUR)
Funding ID 28972-IC-1-2007-1-NL-ERASMUS-EUCX-1 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 01/2012 
 
Description Placental Biomarkers for Assessment of Preeclampsia
Amount £250,000 (GBP)
Organisation F. Hoffmann-La Roche AG 
Department Roche Diagnostics
Sector Private
Country Global
Start 02/2014 
End 02/2017
 
Title Exocounter 
Description We have begun a collaboration with Sysmex-JVC to us etheir exocounter to quantify extracellular vesicles - we are the first to use this tool in the UK 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact We will attempt to devlop this tool as a diagnotic assessment of circulating placental extracellular vesicles 
 
Description Characterisation of exosomes for use in gene therapy, molecular diagnostics and basic neuroscience research 
Organisation University of Oxford
Department Department of Physiology, Anatomy and Genetics
Country United Kingdom 
Sector Academic/University 
PI Contribution We have developed standard protocols for Nanoparticle Tracking Analysis of exosomes and microvesicles as well as techniques for the characterisation of exosomes expressing green fluorescent proteins.
Collaborator Contribution Provision of unlabelled and green fluorescent protein expressing exosomes for Nanoparticle Tracking Analysis.
Impact Publication of the methodology in Nature Protocols (in press).
Start Year 2011
 
Description EURAMET 
Organisation Academic Medical Center
Country Netherlands 
Sector Academic/University 
PI Contribution The European Association of National Metrology Institutes is supporting a Joint Research Programme entitled "Metrological characterisation of micro-vesicles from body fluids as non-invasive diagnostic biomarkers". The aim of this programme is to develop reliable, comparable and quantitative methods for analysis of microvesicles in biological fluids. Professor Sargent and Dr Gardiner are Stakeholders in this programme and their contribution is to analyse standard vesicle preparations (which are circulated to all members of the programme) using Nanoparticle Tracking Analysis to compare with other technologies used by other groups.
Collaborator Contribution The research of this JRP is divided into 4 Work Packages (WP). In WP1, methods will be developed for the standardized collection and handling of human body fluids for isolation of MV, which will be used as input for the other WP's. Methods, available in metrological institutes, will be explored to measure the size of single MV, and the size distribution of total MV populations (WP2), chemical composition, morphology and concentration (WP3). Synthetic and biological reference materials will be tested as standards for MV measurements (WP4). Selected reference materials will be analysed by all JRP-Partners and data will be compared between them. The results of this JRP will enable JRP-Partners to perform traceable calibrations of MV measurements.
Impact It is too early in the programme for any measureable outcomes.
Start Year 2012
 
Description EURAMET 
Organisation Aix-Marseille University
Country France 
Sector Academic/University 
PI Contribution The European Association of National Metrology Institutes is supporting a Joint Research Programme entitled "Metrological characterisation of micro-vesicles from body fluids as non-invasive diagnostic biomarkers". The aim of this programme is to develop reliable, comparable and quantitative methods for analysis of microvesicles in biological fluids. Professor Sargent and Dr Gardiner are Stakeholders in this programme and their contribution is to analyse standard vesicle preparations (which are circulated to all members of the programme) using Nanoparticle Tracking Analysis to compare with other technologies used by other groups.
Collaborator Contribution The research of this JRP is divided into 4 Work Packages (WP). In WP1, methods will be developed for the standardized collection and handling of human body fluids for isolation of MV, which will be used as input for the other WP's. Methods, available in metrological institutes, will be explored to measure the size of single MV, and the size distribution of total MV populations (WP2), chemical composition, morphology and concentration (WP3). Synthetic and biological reference materials will be tested as standards for MV measurements (WP4). Selected reference materials will be analysed by all JRP-Partners and data will be compared between them. The results of this JRP will enable JRP-Partners to perform traceable calibrations of MV measurements.
Impact It is too early in the programme for any measureable outcomes.
Start Year 2012
 
Description EURAMET 
Organisation Curie Institute Paris (Institut Curie)
Department Immunity and Cancer INSERM (U932)
Country France 
Sector Academic/University 
PI Contribution The European Association of National Metrology Institutes is supporting a Joint Research Programme entitled "Metrological characterisation of micro-vesicles from body fluids as non-invasive diagnostic biomarkers". The aim of this programme is to develop reliable, comparable and quantitative methods for analysis of microvesicles in biological fluids. Professor Sargent and Dr Gardiner are Stakeholders in this programme and their contribution is to analyse standard vesicle preparations (which are circulated to all members of the programme) using Nanoparticle Tracking Analysis to compare with other technologies used by other groups.
Collaborator Contribution The research of this JRP is divided into 4 Work Packages (WP). In WP1, methods will be developed for the standardized collection and handling of human body fluids for isolation of MV, which will be used as input for the other WP's. Methods, available in metrological institutes, will be explored to measure the size of single MV, and the size distribution of total MV populations (WP2), chemical composition, morphology and concentration (WP3). Synthetic and biological reference materials will be tested as standards for MV measurements (WP4). Selected reference materials will be analysed by all JRP-Partners and data will be compared between them. The results of this JRP will enable JRP-Partners to perform traceable calibrations of MV measurements.
Impact It is too early in the programme for any measureable outcomes.
Start Year 2012
 
Description EURAMET 
Organisation Free University of Amsterdam
Department Department of Gynaecologic Oncology
Country Netherlands 
Sector Academic/University 
PI Contribution The European Association of National Metrology Institutes is supporting a Joint Research Programme entitled "Metrological characterisation of micro-vesicles from body fluids as non-invasive diagnostic biomarkers". The aim of this programme is to develop reliable, comparable and quantitative methods for analysis of microvesicles in biological fluids. Professor Sargent and Dr Gardiner are Stakeholders in this programme and their contribution is to analyse standard vesicle preparations (which are circulated to all members of the programme) using Nanoparticle Tracking Analysis to compare with other technologies used by other groups.
Collaborator Contribution The research of this JRP is divided into 4 Work Packages (WP). In WP1, methods will be developed for the standardized collection and handling of human body fluids for isolation of MV, which will be used as input for the other WP's. Methods, available in metrological institutes, will be explored to measure the size of single MV, and the size distribution of total MV populations (WP2), chemical composition, morphology and concentration (WP3). Synthetic and biological reference materials will be tested as standards for MV measurements (WP4). Selected reference materials will be analysed by all JRP-Partners and data will be compared between them. The results of this JRP will enable JRP-Partners to perform traceable calibrations of MV measurements.
Impact It is too early in the programme for any measureable outcomes.
Start Year 2012
 
Description EURAMET 
Organisation University of North Carolina at Chapel Hill
Department UNC School of Medicine
Country United States 
Sector Academic/University 
PI Contribution The European Association of National Metrology Institutes is supporting a Joint Research Programme entitled "Metrological characterisation of micro-vesicles from body fluids as non-invasive diagnostic biomarkers". The aim of this programme is to develop reliable, comparable and quantitative methods for analysis of microvesicles in biological fluids. Professor Sargent and Dr Gardiner are Stakeholders in this programme and their contribution is to analyse standard vesicle preparations (which are circulated to all members of the programme) using Nanoparticle Tracking Analysis to compare with other technologies used by other groups.
Collaborator Contribution The research of this JRP is divided into 4 Work Packages (WP). In WP1, methods will be developed for the standardized collection and handling of human body fluids for isolation of MV, which will be used as input for the other WP's. Methods, available in metrological institutes, will be explored to measure the size of single MV, and the size distribution of total MV populations (WP2), chemical composition, morphology and concentration (WP3). Synthetic and biological reference materials will be tested as standards for MV measurements (WP4). Selected reference materials will be analysed by all JRP-Partners and data will be compared between them. The results of this JRP will enable JRP-Partners to perform traceable calibrations of MV measurements.
Impact It is too early in the programme for any measureable outcomes.
Start Year 2012
 
Description EURAMET 
Organisation University of Twente
Department Faculty of Science and Technology
Country Netherlands 
Sector Academic/University 
PI Contribution The European Association of National Metrology Institutes is supporting a Joint Research Programme entitled "Metrological characterisation of micro-vesicles from body fluids as non-invasive diagnostic biomarkers". The aim of this programme is to develop reliable, comparable and quantitative methods for analysis of microvesicles in biological fluids. Professor Sargent and Dr Gardiner are Stakeholders in this programme and their contribution is to analyse standard vesicle preparations (which are circulated to all members of the programme) using Nanoparticle Tracking Analysis to compare with other technologies used by other groups.
Collaborator Contribution The research of this JRP is divided into 4 Work Packages (WP). In WP1, methods will be developed for the standardized collection and handling of human body fluids for isolation of MV, which will be used as input for the other WP's. Methods, available in metrological institutes, will be explored to measure the size of single MV, and the size distribution of total MV populations (WP2), chemical composition, morphology and concentration (WP3). Synthetic and biological reference materials will be tested as standards for MV measurements (WP4). Selected reference materials will be analysed by all JRP-Partners and data will be compared between them. The results of this JRP will enable JRP-Partners to perform traceable calibrations of MV measurements.
Impact It is too early in the programme for any measureable outcomes.
Start Year 2012
 
Description New Methods of Extracellular Vesicle Detection 
Organisation University of Washington
Country United States 
Sector Academic/University 
PI Contribution Chemistry department and us working to develop methods to measure circulating extracellular vesicles
Collaborator Contribution Our partnetrs have devised a chemical which can reveribly bind to vesicles which is non toxic and permits them to be counted. We have supplied extracellular vesicles for them to optimise the technique. We will attempt to use this in human plasma samples.
Impact Still in inception - multi-disciplinary - obstetrics, chemistry
Start Year 2017
 
Description Standardisation of methodologies for microvesicle and exosome measurement 
Organisation Academic Medical Center
Country Netherlands 
Sector Academic/University 
PI Contribution We have exchanged man made and biological vesicles in order to compare different methodologies for microvesicle and exosome measurement. A manuscript based on this work will be submitted for publication later this year
Collaborator Contribution Exchange of man made and biological vesicles in order to compare different methodologies for microvesicle and exosome measurement.
Impact This collaboration has paved the way for the European Metrology Research Programme which is detailed elsewhere.
Start Year 2010
 
Description The role of microvesicles and exosomes in the systemic response to brain injury 
Organisation University of Oxford
Department Department of Pharmacology
Country United Kingdom 
Sector Academic/University 
PI Contribution We have used Nanoparticle Tracking Analysis to the study the role of microvesicles and exosomes and the pharmacological control of the systemic response to brain injury in a mouse model.
Collaborator Contribution Establishment of the mouse model of brain injury and provision of plasma for Nanoparticle Tracking Analysis of microvesicles and exosomes.
Impact The first publication is in preparation.
Start Year 2011
 
Description The role of placenta vesicles and free fetal haemoglobin in pre-eclampsia 
Organisation Lund University
Department Faculty of Medicine
Country Sweden 
Sector Academic/University 
PI Contribution Measurement of microvesicles and exosomes in perfusates from placentas treated with fetal haemoglobin
Collaborator Contribution Provision of perfusates from placentas treated with fetal haemoglobin
Impact The first publication is under review by Placenta
Start Year 2011
 
Company Name Nanosight Ltd 
Description NanoSight visualizes, measures and characterizes virtually all nanoparticles. Particle size, concentration, zeta potential and aggregation can all be analyzed while a fluorescence mode provides speciation of labeled particles. NanoSight provides real time monitoring of the subtle changes in the characteristics of particle populations with all of these analyses uniquely confirmed by visual validation. http://www.nanosight.com/ 
Impact In 2011 NanoSight won Technology World's 2011 Business Innovation Award and was recognised by Deloitte as the UK's Fastest Growing Biotech Company . In 2012 NanoSight wins Queen's Award for Enterprise for International Trade, having achieved their 450th instrument sale with more than 400 third party papers citing NanoSight technology. Much of this success has come directly from our collaboration with them in which we use their technology to measure microvesicles and exosomes in biological fluids. 2011 NanoSight wins Technology World's 2011 Business Innovation Award NanoSight Recognised by Deloitte as the UK's Fastest Growing Biotech Company in their 2011 Technology Fast 50 Rankings 2013 On the basis of the above success, Nanosight Ltd was sold to Malvern Instruments for £15 million.
Website http://www.nanosight.com/
 
Description Oxford Science Fair 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The Oxford Science Fair is an annual event designed to increase awareness of research activity - there are approximately 50 "stalls" which are competitively awareded.
We presented on extracellular vesicles using animation, posters and origami
Year(s) Of Engagement Activity 2018