Abstract
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Abstract
Background
Abdominal aortic aneurysm (AAA) represents a significant health burden,
affecting approximately 4% of men over the age of 65 years in western countries. To
date, no clinically useful circulating AAA biomarkers have been identified. In order
to address this problem, a preliminary proteomics analysis was conducted in our
research lab to compare differences between aneurysmal and non-aneurysmal
abdominal aortic tissues, with a total of ten differentially expressed proteins being
identified. This thesis aimed to investigate whether some of these putative
biomarkers could be validated. Furthermore, the subsequently validated (tissue)
proteins were also examined in a more clinically relevant setting, as circulating
biomarkers for AAA, to better test their potential utility.
Methods
Initially, a preliminary 2D-PAGE/mass-spectrometry analysis was performed
on AAA (n=7) and control (n=3) abdominal aortic adventitial layer homogenates. A
total of ten differential proteins were identified. Based on the literature four of the
proteins, including 14-3-3 θ, 14-3-3 β, heat shock protein-27 (Hsp27) and serum
amyloid protein p component (SAP), were chosen for validation.
In the first study, two methods were chosen for the validation of 14-3-3 θ, 14-
3-3 β, using 14-3-3 γ as a comparative control isoform. Initially, these three 14-3-3
isoforms were examined by western blot in tissues from 82 AAAs and 16 controls.
Subsequently, the three isoforms were also quantified in 36 AAAs and 30 controls
using a commercially available ELISA kits.
In a second study, in order to provide a better understanding of 14-3-3
isoforms and to test their utilities as potential clinically useful biomarkers, plasma
samples were measured using ELISA. In addition, separate layers of aortic walls
(intima/media & adventitia) were also examined. Immunohistochemistry was also
performed to localize the tissue expression of 14-3-3 proteins.
In a third study, the potential functions of 14-3-3 isoforms, specifically on cell
proliferation and apoptosis, were tested in Jurkat T lymphocytes and vascular smooth
muscle cells by inhibiting protein expression using small interference RNA
(siRNA) which targeted specific 14-3-3 isoforms.
Finally, both Hsp27 and SAP were examined in abdominal aortic wall tissues
from both AAAs and controls (20 of each). In addition, 10 samples from each group
also had separate micro-dissected adventitia and intima/media specimens examined.
Plasma Hsp27 and SAP were also tested in an elderly cohort of 280 subjects, which
included otherwise healthy controls, AAA, CAD and PAD patients.
Results
(1) Western blot and ELISA analysis confirmed that 14-3-3 θ had significantly higher
protein expression in AAA wall homogenates compared with controls (P<0.01).
Subsequent ELISA analysis on separate aortic wall (adventitial and
intimal/medial) layers and immunohistochemistry indicated that 14-3-3 θ was
predominantly localized within adventitial T-cell rich lymphoid aggregates.
Plasma analysis failed to detect 14-3-3 θ in either AAA or controls.
(2) Treatment of T-cells with 14-3-3 θ siRNA appeared to lead to slowed cell growth,
however, this effect was not observed in vascular smooth muscle cells.
(3) Tissue Hsp27 appeared elevated in adventitia, media and total walls of AAA
tissues compared to elderly controls (P<0.01). Plasma Hsp27 levels were
significantly lower in all arterial disease patients, including CAD, PAD and AAA,
compared with otherwise healthy elderly controls (P<0.01).
Conclusion
Our 2D-PAGE and subsequent validation studies revealed significantly
increased 14-3-3 θ in AAA tissues. 14-3-3 isoforms are known anti-apoptotic
proteins, whose association with adventitia lymphocytes may prolong T-cell survival
and thereby contribute to the characteristic enhanced adventitial inflammation
observed in AAA. Unfortunately, the inability to detect 14-3-3 θ in the circulation
may suggest that this protein has limited potential as a disease biomarker.
Nevertheless, this study promotes our understanding of the pathobiological
mechanisms underlying the formation of abdominal aortic aneurysm and may offer a
novel potential therapeutic target.