Supervisors: Dr P Kasher, Prof Craig Smith, Prof Stuart Allan, Prof E Kontopantelis

Background, hypothesis and aims:

Systemic infections can be associated with neurovascular damage and increased susceptibility to blood vessel rupture and haemorrhagic stroke (PMID:32897811). Mechanisms thought to underpin this increased risk include infection-induced hypertension and endothelial dysfunction leading to reduced neurovascular integrity. A less well understood clinical risk factor for haemorrhagic stroke is hypocholesterolemia, where an inverse correlation with LDL cholesterol and haemorrhage risk exists (PMID:23704101).  Pathogens require cholesterol for replication and cellular entry, therefore, the body’s response to infection is to temporally reduce available cholesterol levels (PMID:26686653). Cholesterol is essential for adherins junction formation and function, therefore a reduction in cholesterol can impair endothelial integrity (PMID:10903311). 

We therefore hypothesise that a reduction in cholesterol levels caused by infection/inflammation leads to neuroendothelial instability and brain haemorrhage. To support this hypothesis, we have used zebrafish disease modelling and in vitro experiments, to show that anti-viral signalling can induce neuroendothelial dysfunction and increased risk of brain haemorrhage, which is dependent on cholesterol (https://doi.org/10.1101/2024.05.10.590792). This work provides a potential mechanistic link between two clinical risk factors for haemorrhagic stroke and the aim of this PhD project is to expand on these observations to address three key objectives: 

1. Assess a range of infections/inflammatory conditions in a zebrafish model of brain haemorrhage risk and determine if dependent on cholesterol. 

2. Utilise electronic health records data to investigate the associations between systemic infections, cholesterol and haemorrhagic stroke, and whether cholesterol is causally related to haemorrhagic stroke. 

3. Assess whether infection increases haemorrhagic stroke risk in a stroke-prone rat model and determine role of cholesterol. 

Methods:

For objective 1, a combination of zebrafish disease modelling and high-resolution live imaging will be used to determine risk/severity of brain haemorrhage following infection, alongside gene expression analysis and genetic/pharmacological interventions to assess role of cholesterol in these models. For objective 2, epidemiological approaches using the Greater Manchester Care Record (GMCR) will develop a cohort study to investigate infections, cholesterol and haemorrhagic stroke. The GMCR includes linked healthcare records for >10 years across primary and secondary care, spanning the patient journey. Analysis will enable integration and appropriate adjustment of key confounding factors such as demographics, medications, prevalent medical history, vaccination status, laboratory measures of inflammation and investigate whether cholesterol is causally related to haemorrhagic stroke. For objective 3, neuroimaging, behavioural assays and histological techniques will be used to assess risk/severity of haemorrhage following infection in stroke-prone rats, together with multi-omics to assess cholesterol in the brain/neuroendothelium following infection/haemorrhage.  

Potential outcomes:

This study will provide new evidence to support a mechanistic link between infection/inflammation, cholesterol metabolism and haemorrhagic stroke risk, which holds the potential to inform future prevention studies. In the final year of the PhD, we will seek funding for formal pre-clinical trials to assess targeting cholesterol synthesis as a protective factor for haemorrhagic stroke. 

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Supervisors: Dr Kieron South, Prof Craig Smith, Prof Stuart Allan, Dr Lucy Roberts

Around 80% of ischaemic stroke patients are ineligible to receive the only licenced thrombolytic drug, recombinant tissue plasminogen activator (rt-PA). rt-PA is not always effective in breaking down clots, achieving reperfusion of occluded arteries in <50% of cases. More effective and safer thrombolytic therapies for AIS are therefore urgently required. rt-PA induces the degradation of fibrin through the generation of plasmin. However, many thrombi contain little fibrin and are instead comprised of other matrix components including von-Willebrand factor (VWF). Using clots collected from thrombectomy patients, we have shown that clot fibrinogen content ranges from 15-60% of total clot area and VWF ranges from 5-50%. The extent of leukocyte infiltration and activation correlates with VWF content of the clots. It remains unclear which biological factors determine the differences in clot composition between patients, but the presence of preceding infection or systemic inflammation may play a role. 

We have developed and patented a novel thrombolytic, a constitutively active(ca) variant of the VWF-cleaving protease ADAMTS13 which, in a rt-PA-resistant mouse model of stroke, rapidly increased cerebral reperfusion and reduced infarct volume (PMID:34780600 and 40171654). Unlike rt-PA, caADAMTS13 can break down both fibrin-rich and platelet-rich clots and shows potentially beneficial anti-inflammatory activity (PMID: 39029401). Before first-in-man clinical trials, it is critical to assess the efficacy of caADAMTS13 against human clots of varying composition ex vivo to ensure translational feasibility in patients. 

Hypothesis/Aims – We hypothesise that thrombolytic susceptibility of ischaemic stroke patient thrombi is determined by their cellular and molecular composition which is, in turn, determined by clinical characteristics, particularly preceding infection or systemic inflammation.  

This project will utilise fresh stroke thrombi collected from patients receiving mechanical thrombectomy (MT) at Salford Royal Hospital (IRAS No. 328538) and historic stroke patient thrombi and matched plasma samples.  

Objective 1 – Determine susceptibility of ex vivo human stroke thrombi to thrombolysis 

Fresh stroke thrombi will be weighed before and after incubation with either rt-PA, caADAMTS13 or vehicle to allow direct comparison of thrombolysis. Remaining material will be immediately fixed in 4% PFA and paraffin embedded for use in Objective 2. We will also extract routinely collected clinical and neuroimaging data including evidence of preceding infection/inflammation from electronic patient records. 

Objective 2 – Detailed analysis of ex vivo human stroke thrombi using Hyperion imaging mass cytometry (IMC) 

We will investigate the relationship between the more complex elements of clot composition and responses to thrombolytic agents (rt-PA, caADAMTS13) using a validated antibody panel to simultaneously label matrix components, platelet surface markers, red blood cells, immune cell markers and NETosis markers. 

Objective 3 – Investigate peripheral blood markers of clot composition and thrombolytic susceptibility 

The Hyperion imaging and analysis pipeline will be applied to the fixed thrombi. Matching plasma samples will be used to quantify thromboinflammatory factors including VWF and ADAMTS13, platelet activation markers, neutrophil activation markers and NETosis markers. We will investigate the relationship between these thromboinflammatory markers, clot composition and each patient’s response to thrombolysis. This will help inform the design of future clinical trials of caADAMTS13 within specific patient groups. 

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