Measurement of pathologically relevant adipocyte-derived extracellular vesicles

Extracellular vesicles (EVs) are microscopic membrane-bound vesicles, habitually released by different cell types into the local tissue and circulation to serve as intercellular signal communicators. They play an active role in the normal physiology as well as pathogenesis of diseases. In vitro studies from animal models and human adipose tissue explants have established the release of EVs from adipocytes, also known as adipocyte-derived EVs (ADEVs). These have been identified in vitro studies as possible endocrine mediators in the metabolic function of adipose tissue. Biological fluids, particularly, plasma is one of the channels for movement of EVs. Therefore, this thesis aimed to provide evidence for the presence of ADEVs in circulating plasma. With the field being relatively young and expanding, there is a lack of standardisation in EV isolation and analytical techniques. Ultracentrifugation (UC) and size-exclusion chromatography (SEC) proved efficient in isolating an optimal population of EVs from plasma, whereas nanoparticle tracking analysis, time resolved fluorescence and western blotting were used to phenotype EV populations. EV-rich sample was isolated from platelet-free plasma obtained from healthy individuals. Two techniques of magnetic bead capture and solid-phase immunoassay was used to selectively and sequentially deplete major circulating EV populations derived from platelets, leukocytes, endothelial and erythrocytes. Post-depleted samples retained an EV population positive for adipocyte markers and contained a range of adipokines. Accumulating evidence has implicated ADEVs in obesity-associated cardiovascular diseases, through onset of endothelial dysfunction mediated by inflammation. Leukocyte-adhesion assays performed on HUVECs pre-treated with ADEVs, showed enhanced endothelial activation by ADEVs obtained from obese as compared to healthy subjects. This implies adipocytes release EVs into the circulation and could have a significant role in the development of endothelial dysfunction and atherosclerosis. This thesis suggests the role of plasma-borne ADEVs warrants further investigation as novel biomarkers and for potential therapeutic opportunities in treating cardiovascular diseases.