PhD ThesisBackground:
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
