Measurement of Extracellular Vesicles as novel biomarkers of inflammation by Nanoparticle Tracking Analysis

Microvesicles(MV), exosomes and apoptotic bodies (collectively known as extracellular vesicles, EV) are becoming recognised as important biomarkers. It is becoming clear that they play important roles in normal cellular physiology and provide novel intercellular communication mechanisms. Their levels in body fluids are probably an accurate reflection of health or disease of specific organs and tissues and the underlying inflammatory and activation status of the vascular endothelium and circulating blood cells. The circulating pool of EV are heterogeneous (derived from platelets, leucocytes, erythrocytes, the vascular endothelium or other tissues), small (exosomes ranging from 50-100 nm, MV from 100-1000 nm and apoptotic vesicles 1-3 microns in size), express antigens from their cell of origin, are procoagulant (expressing phosphatidylserine) and contain nucleic acids (e.g. mRNA and microRNA). There is also a significant and growing literature on the measurement and potential importance of EV in patients with cardiovascular disease. However, there remain significant problems with the standardisation of measurements and it is clear that the conventional methods (e.g.flowcytometry) are very limited for measuring all sizes of EV. The novel methodology of Nanoparticle Tracking Analysis (NTA) suggests that there are vast numbers of EV within plasma (~1010/ml) which is 3 orders of magnitude higher than previous estimates by flow cytometry (Dragovic et al, 2011). We have also demonstrated that the size distribution of the majority of plasma EV is surprisingly small i.e. between 100 and 200 nm. One possible explanation of this discrepancy is that the vast numbers of EV produce significant coincident events or “Swarming” within the cytometer laser beam resulting in overestimation of size and underestimation of concentration (Harrison and Gardiner, 2011). Although phenotyping of EV using fluorescent labels (e.g. quantum dots) is possible within the NS500 NTA system (Dragovic et al, 2011) there remain significant obstacles to this becoming a reliable measurement e.g. photobleaching of conventional fluorophores, high background labelling, aggregation of quantum dots. In this project we aim to develop new high affinity fluorescent probes (in collaboration with the School of Chemistry, University of Birmingham) to enable us to fully characterise the phenotype of EV and their concentration within plasma from normal subjects and patients with inflammatory disease e.g. cardiovascular disease, pre-eclampsia and cancer. We will collaborate with Oxford University and Nanosight to optimise the conditions of analysis to reduce problems with photobleaching and the signal to noise ratio in the NS500 (e.g. by using pulse analysis of samples, by modulating laser power and using alternative lasers and fluorophores). Once optimal labelling of EV is obtained we will then be able to fully characterise EV for the first time and evaluate their potential role as novel biomarkers of inflammation in cardiovascular disease, cancer and pre-eclampsia.

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