Extracellular Vesicles in cell-cell communication for health across the life course

Project Outline

Extracellular vesicles (EV) represent a novel method of cell communication and we are only beginning to understand the mechanisms by which they transfer messages between cells. The importance of these messages however is very clear as these EV can help to control inflammatory responses, promote homeostasis and support healthy ageing. By expanding our detailed understanding of how EV communicate in physiology, we can shed light on approaches for novel therapy in chronic disease and to address the negative consequences of ageing.

Whilst EV are known to be of greatly varying size (<50nm to >1µm) and derived from different compartments of both viable and apoptotic cells, remarkably little is known of the composition of the EV and how this links to function1,2. Over recent years, our BBSRC-funded work has yielded extensive detail of the EV composition revealing the presence of key inflammation-controlling enzymes and their products (eicosanoids: small lipid molecules involved in the control of inflammation), exposed phosphatidylserine3 as well as many membrane receptors and transporters. The functional significance of these components in controlling inflammation, promoting wound healing and underpinning other EV functions is not known. The projects outlined below will help us to address these knowledge gaps directly, paving the way for novel therapeutic approaches to important conditions associated with ageing.

Defining the role of EV communication in wound healing

(with Prof Devitt, Dr Milic, Dr Bellary and Prof Wollfsohn)

Inflammation and wound healing are important physiological responses that, because they get better (i.e. resolve), we take for granted. Whilst EV play an important part in these processes, this project will define the importance of specific EV-components to these processes. As we age, wound healing and inflammatory control can be impaired. Here we will address the role of key EV components (e.g. enzyme activities) on wound healing (using in vitro models of wound healing relevant to the skin and eye) and will address the impact of ageing on these processes using a range of EV from stem cells and immune cells.

Extracellular Vesicle mediated communication with the immune system: It’s as easy as ABC (transporters)

(with Prof Devitt, Dr Milic and Dr Rothnie)

Inflammation and wound healing are important physiological responses that, because they get better (i.e. resolve), we take for granted. Whilst EV play an important part in these processes, this project will define the importance of specific EV-components to these processes. As we age, wound healing and inflammatory control can be impaired. Here we will address the role of key EV components (e.g. enzyme activities) on wound healing (using in vitro models of wound healing relevant to the skin and eye) and will address the impact of ageing on these processes using a range of EV from stem cells and immune cells.

These projects will use a broad range of techniques to answer these questions including cell culture, particle isolation and analysis, flow cytometry, imaging (light and electron microscopy), assays of immune cell function and inflammation and will harness the power of available inhibitors (e.g. enzyme and ABC-transporter inhibitors).

References

¹Grant, L., Milic, I. & Devitt, A. (2019). Apoptotic cell derived extracellular vesicles: structure-function relationships. Biochemical Society Transactions: 47(2): 509-516.

²Van Niel, G., D’Angelo, G., & Raposo (2018). Shedding light on the cell biology of extracellular vesicles. Nature Reviews: Molecular Cell Biology: DOI: 10.1038/nrm.2017.125

³Devitt, A., Griffiths, H.R. & Milic I. (2018). Communicating with the dead: lipids, lipid mediators and extracellular vesicles. Biochemical Society Transactions: DOI: 10.1042/BST2016477

Techniques

• Analysis and interrogation of extensive proteomics data: to identify lead targets for further analysis using systems biology approaches
• Cell culture: Isolation of primary cells from human peripheral blood; cell culture of a range of cell lines and primary cells. Mesenchymal stem cell culture to derive viable cell EV involved in regenerative responses. Culture of a range of primary cells from skin (fibroblasts and keratinocytes) and eye (conjunctival and corneal cells).
• Extracellular Vesicle analyses: isolation and analysis of EV structure and function using Exoid tunable resistive pulse sensing, flow cytometry, electron microscopy, analysis of immune-modulating capability in vitro using chemoattraction assays and flow cytometry, enzyme assays and analysis of wound healing using scratch assays.
• Imaging and analysis: Flow cytometry, Western blotting, light and fluorescence microscopy including real-time microscopy and confocal microscopy, automated cell imaging for functional studies
• Cell assays: Induction and analysis of cell death, EV binding and uptake assays;
• Mass spectrometry: for the analysis of EV proteome and lipidome