Project title: Endothelial cell extracellular vesicles in cerebral small vessel disease

Cerebral small vessel disease (SVD) is the commonest cause of vascular dementia, and also contributes to Alzheimer's disease, with its biggest risk factor being increasing age. However, although very common, there are no therapies that directly target the pathology1. The LACI-2 trial (https://www.ed.ac.uk/usher/edinburgh-clinical-trials/our-studies/ukcrc-studies/laci) has just finished testing two repurposed drugs (Cilostazol and isosorbide mononitrate (ISMN)) for effect in patients with SVD, and is due to report very soon. These drugs have been chosen as they stabilize endothelial cell (EC) function and it is now known that a key mechanism in SVD is EC dysregulation. This leads to changes of crosstalk between ECs and surrounding brain cells, particularly the oligodendrocytes and myelin of the white matter2, as well as altering blood flow and blood brain barrier integrity.
We have generated a model of SVD in a transgenic rat (Atp11bKO) which shows EC dysregulation, white matter abnormalities, Magnetic resonance imaging and behavioural changes consistent with human SVD3. ECs in this model lack ATP11B and a SNP in this gene is associated with sporadic human SVD2. The white matter changes in human are used for diagnosis of SVD, and increased extent correlates with worse cognition, yet we are unclear about how EC dysregulation causes the secondary effect of white matter change. As ATP11B is involved in vesicular release through the endoplasmic reticulum3, we hypothesise that this crosstalk may be through extracellular vesicles (EVs) released from ECs to the surrounding brain, as well as into the blood.
This PhD project will address this hypothesis, taking both a candidate approach (with already identified candidates4) and an unbiased approach (proteomics) in rat EC cultures and in vivo, and then moving this to humans where possible, to determine if there is a correlation between these levels and response in the LACI-2 trial.

Aims
1) Determine the number and content of rat brain EC EVs in the Atp11bKO rat model and controls at different ages in isolated cells and circulating in serum in vivo, using a candidate approach (from current work) and an unbiased approach using proteomics.

2) Repeat this in the Atp11bKO rat model and controls after treatment with cilostazol and ISMN, to reflect the LACI-2 trial protocol, to identify if the cargo changes after treatment and can be correlated to outcome/hallmarks of SVD.

3) Analyse EVs from already collected LACI-2 trial patient serum (using Simoa technology) for key candidates and correlate with trial response, to determine if patients can be stratified on this basis.