Mechanical sensing in placental vascular endothelium

Background

Diseases of placental vascular function can result in recurrent miscarriages, intrauterine growth restriction (IUGR) of the developing fetus and stillbirth. Babies who are born with IUGR are at greater risk of becoming obese and developing heart disease and diabetes in later life. Pregnant women are also at risk and may develop the high blood pressure condition, preeclampsia, which complicates 5% of pregnancies (ref). Severe preeclampsia can result in haemorrhage and stroke, and is a cause of maternal mortality (ref).
There are limited management options for placental dysfunction, and early delivery of the baby is often necessary. More information on what regulates the function of blood vessels running within the placenta would be extremely useful and could potentially lead to new treatments.
Endothelial cells line blood vessels and contain ion channels, which have an important role in regulating calcium entry. Modulation of these calcium channels has previously been used as a strategy for the development of medications, such as treatment for high blood pressure. Calcium channels can be activated by mechanical forces, such as blood flow.
A recently discovered mechanically sensitive calcium-permeable channel is the Piezo1 channel. Importantly, our research group has shown that mice engineered to have the Piezo1 gene disrupted die as embryos (Li et al. 2014). These murine embryos showed impaired blood vessel formation and were smaller than their normal comparators.

Pilot data

I have already successfully extracted, grown and characterised endothelial cells from healthy placentas (PlECs) using a technique which I developed; alongside I have isolated human umbilical vein endothelial cells (HUVECs). Characterisation of these cells included measuring responses to vascular endothelial growth factor (VEGF), which were clear and reproducible. I have already shown Piezo1 DNA to be present in normal human placental tissue and endothelial cells from the placenta and umbilical cord. Knockdown of Piezo1 in HUVECs and PlECs strikingly abolished response of the cells to mechanical force (shear stress). This suggests that Piezo1 is present and functionally active within the human placenta.

Aims

1. To determine the role of mechanically sensitive ion channels, such as Piezo1, within the placenta.
2. To compare the functional differences between endothelial cells and sections of blood vessel from the umbilical cord and placenta from healthy pregnancies and those affected by IUGR and preeclampsia - particularly in relation to mechanical sensitivity.
3. To work towards new strategies to control and modulate the regulation of placental blood vessels to improve fetal blood supply.

Objectives

I will compare the function of placental blood vessels from healthy and abnormal placentas by studying whole vessel sections, PlECs and HUVECs. I will determine the relative quantities of Piezo1 and other mechanically sensitive ion channels in these cells. The effect of both activating and depleting Piezo1 on cell function will be explored. Cell signalling pathways downstream of Piezo1 will be investigated to further understand the regulation of blood vessel contraction and relaxation.

Application
The results of this project may shed new light on the control of blood vessel function within the placenta. The ability to modulate vascular tone though ion channels would be a major step forward on the path to developing new treatment strategies for placental vascular disorders, such as recurrent miscarriage, IUGR and preeclampsia.

Background
Piezo1 is a mechanosensitive ion channel, which may have a critical role in shear stress sensing within endothelial cells. Isolated and cultured human placental endothelial cells (PlECs) and human umbilical vein endothelial cells (HUVECs) are positive for CD31, alignment to shear stress and respond reliably to vascular endothelial growth factor (VEGF) and the Piezo1 activator, Yoda1. Piezo1 mRNA has been detected in these cells. When PlECs and HUVECs undergo depletion of Piezo1, responses to shear stress, Yoda1 and, strikingly, VEGF are suppressed, suggesting an important regulatory role for Piezo1 in endothelial function within the placenta.
Aims
The aim of this project is to establish the importance of mechanical sensing and Piezo1 function in normal placental vascular function, and in preeclampsia and intrauterine growth restriction, and to determine the effect of Piezo1 modulation.

Objectives and methodology
The response of PlECs and HUVECs to shear stress will be measured in Ibidi chambers and the relative quantity of Piezo1 DNA and protein expression determined. Tube formation in co-culture, cell migration and apoptosis will be assessed the IncuCyte and complementary capabilities. Pressure and wire myography will be used to observe endothelium-dependent properties of fresh placental vessels with and without luminal flow and stretch. In partnership with Dundee Cell Products, phosphoproteomics will indicate protein differences in PLECs from normal versus IUGR-affected placentas. Piezo1 activity will be assessed by depletion (short interfering RNA), inhibition (GsMTx4 toxin), and Yoda1 activation. The InterPregGen database will be used to explore variations in Piezo1 expression between patients with and without preeclampsia.

Opportunities
This novel project will increase understanding of shear stress sensing within normal placentas and in vascular dysfunction. This could potentially lead to the development of new treatment targets.

Placental vascular disease can affect women at all gestations of pregnancy, resulting in miscarriage, intrauterine growth restriction (IUGR) and stillbirth. Preeclampsia is associated with systemic endothelial dysfunction in the maternal circulation resulting from placental dysfunction. Preeclampsia affects 5% of UK pregnancies and remains a significant cause of maternal morbidity and mortality, particularly in the developing world (Krause et al. 2013). For the fetus, preeclampsia in the mother is associated iatrogenic prematurity.

In later life, IUGR is associated with increased risk of cardiovascular disease, obesity and type 2 diabetes mellitus (Zhodi et al. 2015).
The management of preeclampsia and IUGR is currently limited to maternal anti-hypertensives and early delivery of the baby (RCOG, 2013). As such, novel treatments that can alter placental vascular dysfunction would have a significant impact on fetal, and potentially maternal health.
Neonatal care costs associated with caring for premature and growth-restricted babies could be reduced with the development of new treatments. Similarly, cost-benefit and economic impact could be achieved by prevention of the long-term sequelae associated with IUGR and prematurity.

There remains a paucity of information on the endothelial cell function within the placenta and how this correlates with different clinical phenotypes, such as early versus late onset preeclampsia plus/minus IUGR. Greater understanding of pathways to vascular insult may influence clinicians' antenatal management of these patients. Likewise increased understanding with the potential for novel treatments, could impact on policy-makers and the development of clinical guidelines, such as NICE.
We have developed a novel method of placental endothelial cell isolation and culture, which could be applied to study other disorders, such as diabetes mellitus in pregnancy, broadening the impact of this project.

This fellowship would significantly impact on my personal academic development, as I will learn a vast array of new scientific skills. I will also gain greater experience of writing basic science and presenting within in the scientific community. I will have the opportunity to network, be mentored and further develop my career aims.

Timescales to impact:
6 months: Determine differences in endothelial function between cells isolated from normal versus pathological placentas.
1-2 years: Detailed understanding of the molecular pathways involved in controlling vasomotor tone in placental blood vessels.
2-3 years: Manipulation of placental endothelial cells and whole vessels plus/minus testing novel treatments in the in vitro setting.