Biomarkers for abdominal aortic aneurysm

Abstract

i 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.