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

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.

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.

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.