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Title: The role of neutrophil elastase in abdominal aortic aneurysm and aortic dissection
Author: Pearce, Stuart
ISNI:       0000 0004 9355 6648
Awarding Body: Queen Mary University of London
Current Institution: Queen Mary, University of London
Date of Award: 2020
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Background: Abdominal Aortic Aneurysm (AAA) affects 4-5% of men over 65, and Aortic Dissection (AD) is a life-threatening aortic pathology where 75% of patients die within 2-weeks post-onset. Relatively little is known about the underlying mechanisms, which warrants further investigation. Neutrophil Elastase (NE) is an enzyme with roles in priming of the immune system, clearance of large pathogens and remodelling of extracellular-matrix proteins, all influential in AAA and AD. The Angiotensin II (Ang II) and Calcium Chloride (CaCL2) procedures provide methods to model the AAA pathology within small rodents, which enables the study of the disease. Furthermore, the β-aminopropionitrile monofumarate (BAPN) model provides an animal model which mimics the AD pathology and allows in-depth study of both changes in mortality and pathological severity. Within AAA and AD, there is known to be a large immune system implication in the production and development of both pathologies, with enzymatic activity seemingly pivotal in the initiation of the AD and AAA phenotype. With the impact of enzymes such as immune cell-derived matrix metalloproteinases (MMPs)-2 and -9 studied in depth. Neutrophil Elastase (NE) is an, as yet, unstudied enzyme within these processes. Purpose: Current treatment regimen for AAA and AD both have significant flaws and is entirely surgical, with most at-risk groups being elderly and treatment consequentially of increased risk of complications. Surgical intervention has complex post-operative care considerations, with reintervention and morbidity being high within the following weeks after the procedures take place. Understanding of the underlying mechanisms contributing to AAA and AD is poor and therefore designing a pharmacological agent to improve therapy is difficult. Understanding the role of NE and its implications within AAA will allow us to identify whether it has an impact within the pathology and, if so if it is an appropriate target for therapeutic intervention. Therefore, the primary aims of this project was to identify if Neutrophil Elastase is involved within the AAA and AD processes, to detect via what mechanism it may act and if NE is a potential novel drugs target for treatment of AAA and AD. Methods and Results: To study if NE gene expression is regulated by AAA pathologies, in Vitro experiments were conducted with multiple cell lines (human aortic SMCs, Raw264.7 macrophages and HuVECs). Data from RT-qPCR analysis was insignificant and failed to show a relation between NE, MMP2 and MMP9, when cells were treated with Angiotensin II (Ang II) and Calcium Chloride, respectively. Development of an MRI scanning technique to monitor the progression of AAA overtime was undertaken. A semi-automatic methodology was developed with representative and highly reproducible results achieved. This enabled the detailed study of animals whilst undergoing AAA-induction procedures, namely the CaCl2 and Ang II animal models. By utilising two in vivo models, so far we have observed a significant difference that the two Aneurysm models result in lower AAA incidence within NE deficient mice, with infrarenal aortic expansion significantly reduced. Investigations into flow and other dynamic aspects of the vessel differ between animal models and consequentially need to be furthered but at present no significant differences were observable. Underlying mechanisms for AAA and NEs contribution towards the pathology were assessed, with novel associations between NE and MMPs identified as well as the involvement of proteins such as TBL1x, Caveolin-2 and COPS8 following proteomics analysis techniques. Similar results were seen within AD experimentation, with knockout of NE resulting in significantly lower mortality and vessel dissection rate. Similar histological staining patterns were observed within this model when compared to the two AAA animal models. This suggests a potential common pathway between the two that results in aortic medial degradation, and rupture of the vessel. Early Human translational experiments were carried out with a AAA patient audit, there were no significant findings that correlated any basic blood profile characteristic to size or progression of AAA. This work was continued, working with Barts BioBank Resource to obtain tissues from surgical repair patients. Diseased tissues displayed expression of NE with small areas of staining being seen alongside MMPs, such as within the remodelled medial portion of the vessel, similar to that of previous mouse works. Furthermore, blood serum samples from patients had no significant change in level of NE when compared to Healthy Controls. Conclusion: This study identifies NE as having potential therapeutic applications if similar results are seen within pharmacological inhibition of the enzyme as were produced as a result of genetic knockout in mouse studies, especially within AD models. Early Translational experimental results have not been as promising, but more work is needed to fully understand whether NE could play a similar role within the Human condition and therefore warrant further study as a potential drugs target, although current results need to be developed prior to this. Wider Implications: Findings outlined within this thesis suggest NE has a potential role within both AD and AAA pathologies, with implications in the regulation of MMPs as well as potential direct enzymatic action within the modification of extracellular matrix proteins regulated within the AAA and AD conditions. Results so far show areas of promise, and with further investigation, NE inhibition could be established as a therapeutic option for AAA and/ or AD. In order to establish the link further, there is a need to determine if pharmacological knockdown of NE can produce the same results as those seen within in vivo experiments where genetic knockout was established.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available