Das, D; (2016) The Role of exosomes in reperfusion injury and cardio-protection. Doctoral thesis , UCL (University College London).

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Abstract

The mechanism by which remote ischaemic preconditioning (RIPC) exerts its cardioprotective effects is not fully understood. Exosomes are lipid bound membranous vesicles that range from 30-100nm in size. With increasing interest in the scientific community about these vesicles as a mode of intercellular communication, in this project we explore the possible mechanistic role of human plasma exosomes in RIPC. We first developed a consistent and reliable model for the isolation of human plasma exosomes through a process of differential centrifugation and ultracentrifugation. Further scientific models for biomarker analysis and quantification of the isolated human plasma exosomes were developed and validated through western blotting and flow cytometry and nanosight analysis. In addition we successfully developed a protocol for the isolation of microRNA from our human plasma exosomes. Confident with our isolation and biomarker analysis models we investigated the content differences in human plasma exosomes isolated after a RIPC (3 cycles of fore arm cuff inflation and deflation) and compared those to control human plasma exosomes. Our results show that RIPC lead to a rise in plasma exosome concentration with a peak rise in exosome concentrations occurring only after the last 5 minutes of cuff deflation (reperfusion) in the 3 cycles of the RIPC protocol. We probed both RIPC and control human plasma exosomes for known pro-survival protein kinases of the RISK pathway (P13K, MEK 1/2, AKT, ERK1/2) though were not able to detect these in any sample. We then tested the hypothesis that the cardio-protection conferred through RIPC is mediated through circulating plasma exosomes. RIPC and control human plasma exosomes were added to an invitro model of ischaemia reperfusion using rat cardiomyocyte cells. Interestingly our results demonstrate that human plasma exosomes are cardio-protective against ischaemia reperfusion injury irrespective if they are control or RIPC plasma exosomes. The cardio-protection conferred by human plasma exosomes is comparable to Insulin, a known cardio-protective agent, used as our positive control. These results suggest that plasma exosomes in general are cardio-protective and may not have a mechanistic role in the cardio-protection conferred through RIPC. Further experiments must be carried out to ascertain the exact mechanism through which human plasma exosomes confer cardio-protection.

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