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|Title: ||Exosomal proteome as a source of biomarkers for human disease|
|Authors: ||Street, Jonathan Mark|
|Supervisor(s): ||Dear, James|
|Issue Date: ||25-Nov-2011|
|Publisher: ||The University of Edinburgh|
|Abstract: ||Exosomes are small lipid membrane bound vesicles formed as part of the
endosomal pathway and released into the extracellular space following
fusion of late endosomes with the plasma membrane. Exosomes have been
shown to have a variety of biological roles and may represent a novel source
of disease biomarkers.
The objectives of this project were to develop a panel of techniques for
identifying exosomes in human urine then establish an in vitro model to
determine whether exosomes change with cellular activation. We then used
the techniques developed with human urine to determine whether human
cerebrospinal fluid (CSF) contains exosomes and applied a mass
spectrometry based approach to characterise the exosomal proteome.
We used western blot for three exosomal markers (tsg101, CD24 and flotillin-
1), isopycnic centrifugation on a sucrose density gradient and direct
visualisation using transmission electron microscopy (TEM) to verify the
presence of exosomes. Using GeLC-MS/MS, 88 proteins were identified in
the urinary exosomes. Several of these proteins could be linked to diseases
and specific sections of the nephron.
A murine cortical collecting duct cell line was used to model exosome release
into the urine. Firstly, exosome release was verified using the approach
developed in the urine. Stimulation of the cells with desmopressin caused an
increase in the presence of aquaporin 2 in the exosomes. This increase reflected a similar change in the cells and occurred over a similar time
course. This supports the hypothesis that the exosomes reflect the state of
the kidney cells. In contrast, stimulation with cisplatin did not alter the
presence of Fetuin-A, a proposed biomarker of cisplatin-induced acute
kidney injury, in exosomes and this was consistent with no change in Fetuin-
A expression in the cells.
The released exosomes may act as mediators of communication to other cells.
Following incubation of mCCD cells with AQP2 containing exosomes AQP2
in the cell lysate was increased indicating interaction between the cells and
exosomes and potentially internalisation.
Exosomes have been shown to be released by neuronal cells in vitro. We
identified exosomes in the CSF of humans using western blot for known
exosomal markers, density determination and direct visualisation with TEM
and Immuno-TEM using an antibody specific for the exosomal marker
flotillin-1. Label-free quantitative mass spectrometry was used to compare
multiple CSF samples. On a whole protein analysis 86% of the proteins
identified varied by less than 2-fold in comparison to the average across
samples. On a tryptic peptide analysis 75% of the peptides identified varied
by less than 2-fold in comparison with the average across samples.
We have demonstrated exosomes are present in urine, CSF and mCCD cell
conditioned media. In the mCCD cell derived exosomes we have
demonstrated that following stimulation the proteome of the exosomes
changes and that this change reflects the change seen in the cells. For the
urinary and CSF exosomes we have characterised their proteomes using GeLC-MS/MS. These findings are consistent with the hypothesis that
exosomes are a rich source of information, including biomarkers, on their
cells of origin.|
|Sponsor(s): ||British Heart Foundation|
Alzheimer’s Research UK
|Appears in Collections:||School of Clinical Sciences thesis and dissertation collection|
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