Scott, Aaron Jon; (2022) In vivo characterisation of endogenous cardiovascular extracellular vesicles and their dynamics in response to ischaemic cardiac injury in zebrafish. Doctoral thesis (Ph.D), University of Bristol.

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Text Scott, 2022. Richardson Lab. In vivo characterisation of endogenous cardiovascular extracellular vesicles and their dynamics in response to ischaemic cardiac injury in zebrafish.pdf - Published Version Download (9MB) | Preview

Abstract

Cell-cell communication is integral to the intricately synchronized cellular response that drives successful recovery from ischaemic cardiac damage. Extracellular vesicles (EVs) are cellularly produced, small, lipid membrane bound vesicles, encapsulating a variety of cargoes, that are transported through extracellular space, and can be picked up by recipient cells, mediating local and systemic signalling in health and disease. Extensive, mostly in vitro experimentation has revealed functional roles for EVs, but our understanding of endogenous EVs in vivo is still underdeveloped. The zebrafish is both genetically tractable and optically transparent, providing opportunity for unparalleled subcellular observations in a living vertebrate. Stable transgenesis, using cell-type-specific promoters to drive membrane-anchored fluorophore expression, labels both the cells plasma membrane and membrane bound particles produced by those cells. Initially established in larval zebrafish, permitting sophisticated live imaging of EV dynamics, this model was refined and used to identify endothelial- and cardiomyocyte-derived EVs in adult zebrafish. Finally, this model allowed the assessment of EVs from repairing/regenerating cardiac tissue, resulting from an ischaemic cardiac injury model in adult zebrafish. In both the larval and adult zebrafish models, cardiomyocyte and endothelial cell derived EVs were imaged in vivo by high spatiotemporal resolution confocal/light sheet imaging, revealing EV dynamics within the peripheral circulation and pericardial fluid, and likely uptake by recipient cells. Optimised flow cytometry approaches allowed these EVs to be extracted and verified ex vivo. Ultrastructural and protein-based analysis provided reliable identification of EVs, and considerable morphological heterogeneity was discovered by cryo-electron microscopy. Multiple analyses revealed ischaemic cardiac injury to differentially alter EV production during repair and regeneration. The proteome of these post-injury EVs was assessed and suggests potentially diverse functional roles. The establishment of this new model presents exciting opportunities to decipher the complexity of EV function in vivo in a vertebrate model of human disease.

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