Aberrant first trimester placental endothelial cell biology in fetal growth restriction

Lead Research Organisation: St George's University of London
Department Name: Molecular & Clinical Sci Research Inst

Abstract

The size we are born should be determined by the genes we inherit from our parents, however many babies are born smaller than they should be. Some of these babies may suffer from a form of starvation during pregnancy, not receiving the vital oxygen and nutrients they need from their mother. It is thought that babies suffering the effects of starvation during pregnancy are not only more likely to have problems at birth but these stay with them throughout their life with long lasting and damaging consequences. Smaller babies are more likely to suffer from health problems in later life, including an increased risk of cardiovascular disease and diabetes. Estimates suggest that this affects up to 3 million individuals in the UK alone and, over their lifetime, this places a huge financial burden on health services. In order for a baby to receive the nourishment it needs from its mother during pregnancy it must have a healthy placenta with a rich network of blood vessels. This network develops early in pregnancy long before the time when an accurate measure of the baby's growth can be made. Failure of the placental blood vessels to develop into this network is a common feature of pregnancies with small babies. With the help of our clinical colleagues we have used a technique called uterine artery Doppler ultrasound to identify pregnancies that are likely to result in a small baby and have found that the placental blood vessels are different to those from pregnancies with a normal sized baby. The aim of this project is to look more closely at why these cells are different. We will look at the ability of the cells that form blood vessels to grow and form vascular networks. We will also see why cells from pregnancies that would result in small babies are more sensitive to factors that result in their death. It is only by understanding, why the cells from some pregnancies are different that we may one day be able to develop ways of managing these differences. Having babies that are born the right size will help them in early life but also help keep them healthy for longer as an adult.

Technical Summary

Poor placental vascular development at term is seen in pregnancies complicated by fetal growth restriction (FGR). FGR affects 5-8% of human pregnancies worldwide and is a major cause of neonatal morbidity and mortality. Although the pathological changes responsible are believed to occur early in pregnancy, studies directed towards the cellular and molecular mechanisms have been hampered by the lack of appropriate cellular models and the ability to determine, at a clinically relevant time, which pregnancies are at risk of developing FGR. Studies have established a correlation between high first trimester uterine artery Doppler resistance indices (hRI) and birth weight due to placental insufficiency. We will use Doppler ultrasound in the first trimester as a proxy measure of placental function. Using this unique approach and methods developed here to isolate first trimester placental endothelial cells (PEC), we have found that PEC isolated from pregnancies with hRI are more sensitive to apoptotic factors than cells from pregnancies with a normal RI (nRI). It is our hypothesis that poor placental capillary network development is the result of changes to PEC migration/motility and/or sensitivity to apoptotic stimulation and we will seek to establish the mechanism of these differences at a cellular and molecular level. We will investigate whether these differences are the result of changes in gene and/or protein expression in PEC. As placental angiogenesis and PEC survival may also be dependent on factors secreted by surrounding placental stromal cells (PSC) we will determine whether they support an anti-angiogenic, pro-apoptotic phenotype in hRI pregnancies by characterizing differences in gene expression and protein secretion profiles. In vitro findings will be supported by immunohistochemical studies performed on first trimester placental tissue sections.

Planned Impact

The major beneficiaries of this research will be as follows:

1) Students at St George's. We actively participate in the teaching of undergraduate medical, biomedical science degree students and post-graduate MRes, PhD and MDRes students. The best way to inspire this group is for them work alongside young enthusiastic scientists. We would anticipate the appointment of two such individuals to the group will provide such an environment. The results obtained and the techniques developed will have a longer term impact on the education of these and future students that may join the group.

2) Clinicians within the SouthWest London Academic Network will benefit from an increased awareness of the research in this area through the Grand Round seminar series and an annual St George's Research Day organised by members of our research institute. Other clinical beneficiaries will be members of the Department of Obstetrics and Gynaecology and, in particular, those within the Fetal Medicine Unit here at St George's. Findings will be disseminated more widely to similar beneficiaries outside St George's through targeted press releases from our media office and through attendance at Clinical meetings such as Society of Reproductive Investigation held internationally each year. In particular, International dissemination will be achieved through the TRUFFLE group of collaborators, of which St Georges is a member. The TRUFFLE group are responsible for the conduct of a multi-centre European study on the diagnosis and management of early-onset FGR (Lancet 2014; in press).

3) The aim of this proposal is to improve our understanding of the fundamental processes controlling placental angiogenesis in both health and disease. We do not anticipate any immediate impact on the commercial sector however increased knowledge in this important area may identify therapeutic targets or provide diagnostic tools that could ultimately change clinical practice.

4) There are patient liaison groups within the hospital that may be interested as might Action on Pre-eclampsia (APEC) a group that aims to raise public and professional awareness of pre-eclampsia, a syndrome often associated with FGR. Charities that directly fund research in to child health such as Action Medical Research, Wellbeing of Women, Tommy's Campaign may also benefit from the increased knowledge obtained as might the the British Heart Foundation because of the direct link with increased cardiovascular disease in later life.

Publications

10 25 50
 
Title First trimester placental stromal cell gene array 
Description First trimester placental stromal cells were isolated by negative selection using CD31 coated magnetic beads. Immediately after isolation mRNA was extracted and subsequently analysed using Illumina technology. Two comparisons were made and each group consisted of 8 gestationally age matched patients (8-10 weeks) from pregnancies with normal and poor placental perfusion. To determine changes with gestation a second comparison was made between samples from normal pregnancies gestational ages 8-10 and 12-14. Statistical analysis identified over 18 genes that were significantly different and or were 1.5 fold or more different. Analysis between early and late gestational ages in normal first trimester pregnancies identified a further /// genes that were either up or down regulated during this critical transition phase. Both arrays have been validated by PCR using different patient samples. 
Type Of Material Biological samples 
Provided To Others? No  
Impact As far as we are aware this is the first attempt to isolate first trimester placental stromal cells and characterise their gene expression profiles. We the only group worldwide that characterise our first trimester samples using Doppler ultrasound we are therefore the only group able to make comparisons between stromal cells isolated from normal and poorly perfused placenta. The importance of these cells is in their ability to regulate the growth and development of the placental vasculature. Characterising the factors that are expressed by these cells will have a fundamental bearing on the way the placenta develops and the therefore how well the fetus grows in utero. 
 
Title Lentiviral vectors 
Description Previously in this project, using a microarray approach, we performed a gene expression profile of placental endothelial cells (PEC) from high resistance (HRI) and normal resistance index (NRI) samples from first-trimester terminations of pregnancy. Applying a moderate t-test, we found 58 genes that were significantly upregulated (ranging from 1.5-2.8 fold) in the HRI samples when compared with NRI samples, and 175 genes that were significantly downregulated (ranging from 1.5-3.5 fold) for the same comparison. Having previously verified the array using quantitative real time PCR, we started this year's work by verifying these findings at the protein level. We embarked on checking this by western blotting in a subset of proteins, namely heme oxygenase 1 (HMOX1), hemoglobin a (HBA1) and superoxide dismutase 2 (SOD2), as they relate to our previous published work, where we showed that PEC from HRI pregnancies are more sensitive to apoptosis (Charolidi et al., 2018). We confirmed that hemoglobin a was significantly upregulated in HRI PEC. To check the mechanism of sensitivity to apoptosis of the HRI PEC, as well as other aspects, such as angiogenic ability, motility and tube formation, that may differ between HRI and NRI PEC, we selected a set of genes that relate to these processes from the array and moved to functional studies. To do that, we cloned and constructed 3 types of lentiviral plasmids for manipulating expression of these genes in endothelial cell lines, and with the scope to use these constructs in freshly isolated PEC. The three types of plasmids we developed are: 1) an sgRNA lentiviral plasmid, for complete deletion of a candidate gene through CRISPER 2) a flag overexpression lentiviral plasmid for overexpression of a candidate gene, and 3) a series of shRNA lentiviral plasmids for variable downregulation of a candidate gene We then created a library each of the above plasmids for the genes AKAP12, CXCL8, HBA, SOD2, HMOX1, POSTN and HLX-1. Briefly, to do that, we amplified these genes from endothelial cell cDNA through gene-specific reverse transcription, purified the product, and cloned it into each of the above plasmids. We then transformed bacteria, selected colonies and grew them further for large scale plasmid DNA isolation. The resulting plasmids were linearised and sequenced before packaged into virus particles by HEK cells. Following, the viral titter was collected and used for stable transfection of puromycin-selected endothelial cell lines. So far, we have manipulated Hemoglobin a in an endothelial cell line and performed a series of time-lapse microscopy experiments for studying sensitivity to apoptosis in complete absence of hemoglobin alpha (stable transfectants with the sgRNA plasmid) and overexpression of haemoglobin alpha (stable transfectants with the flag overexpression plasmid). In parallel, we are checking expression of periostin (POSTN) in our endothelial cell lines and started a series of experiments in which we treat them with recombinant periostin in order to assess changes in motility and apoptosis. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact These tools are the result of recent developments and so the impact has not yet been felt. 
 
Title Placental endohelial cell gene array 
Description First trimester placental endothelial cells were isolated using CD31 coated magnetic beads. Immediately after isolation mRNA was extracted and subsequently analysed using Illumina technology. Two comparisons were made and each group consisted of 8 gestationally age matched patients (8-10 weeks) from pregnancies with normal and poor placental perfusion. To determine changes with gestation a second comparison was made between samples from normal pregnancies gestational ages 8-10 and 12-14. Statistical analysis identified over 200 genes that were significantly different and or were 1.5 fold or more different. Analysis between early and late gestational ages in normal first trimester pregnancies identified a further 270 genes that were either up or down regulated during this critical transition phase. Both arrays have been validated by PCR using different patient samples. 
Type Of Material Biological samples 
Provided To Others? No  
Impact As far as we are aware this is the first attempt to isolate first trimester placental endothelial cells and characterise the gene expression profiles and the first to classify these pregnancies according to spiral artery resistance, an indirect measure of placental perfusion. To our knowledge it is also the first study to examine gene expression before and immediately after the loss of the trophoblast plug in the first trimester. 
 
Description Gene array analysis 
Organisation National Academy of Sciences of Ukraine (NASU)
Department Institute of Molecular Biology and Genetics (IMGB)
Country Ukraine 
Sector Public 
PI Contribution Alina Frolova, Institute for Molecular Biology and Genetics Kyiv Ukraine
Collaborator Contribution Dr Frolova is a bioinfomatician and is helping us analyse the array data from the PEC and stromal cell array. She has visited the group for a week to familiarise herself with the data.
Impact None yet
Start Year 2017
 
Description Placental endothielial cells studies in normal and growth restricted pregnancies 
Organisation Monash University
Department Monash Business School
Country Australia 
Sector Academic/University 
PI Contribution We are collaborating with the group of Dr Padma Murthi who has an interest in homeobox gene expression in placental cells in normal and growth restricted pregnancies. Our initial collaboration has been to provide endothelial cells for existing studies. However this has now broadened since we have been able to analyse the endothelial and stromal cell arrays which have identified a change in the expression of a number of key homeobox genes that are differentially expressed in the first trimester between pregnacies with normal and poor placental perfusion.
Collaborator Contribution Our partners have provided significant insight in to the regulation of homebox genes and in will the furture be instrumental in analysisng gene expression in a larger cohort.
Impact Outputs to date doi: 10.1530/REP-16-0068 This is not a multi-disciplinary collaboration.
Start Year 2015
 
Description A visit to the Houses of Parliament (Research, Impact and the UK Parliament) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Attended this event organised by the UK Parliament's University Programme, which aimed to engage with postgraduate students, postdocs and junior academics that perform research in a variety of fields. Their call of engagement stated 'Whether looking at prison reform, legislating on embryology or scrutinising Government preparedness for unexpected space weather, Parliament needs to hear from the experts. Academics can help to ensure that the decisions that affect people's lives are taken with the best available evidence'
Year(s) Of Engagement Activity 2016
 
Description Fertility Fest 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This was a four day event attended by NC the Post doctoral fellow on the grant.

Fertility Fest is an arts festival dedicated to fertility, infertility, the science of making babies and modern families. Its mission is 1) 'To improve understanding of the emotional journey of people who struggle or go on a complex journey to conceive' 2 'To improve the level of public conversation about infertility and reproductive science' 3 'To improve fertility education'

It was an opportunity for NC to meet and discuss possible future developments with the founders of the event
Year(s) Of Engagement Activity 2018
URL https://www.fertilityfest.com/our-mission
 
Description NC joined the Science Media Centre's database of expert scientists with the potential to offer opinions in reproductive and cardiovascular biology. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Introduction to the News Media (http://www.sciencemediacentre.org/)
Year(s) Of Engagement Activity 2018
 
Description Writing for the Progress Educational Trust 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The Progress Educational Trust (PET) advances public understanding of science, law and ethics in the fields of human genetics, assisted reproduction, embryology and stem cell research. (https://www.progress.org.uk)

NC regularly helps with the organisation of events, such as 'Make do or amend: should we update UK fertility and embryo law?', and writes commentaries, and science news articles
Year(s) Of Engagement Activity 2016,2017,2018,2019
URL https://www.bionews.org.uk/nicolettacharolidi