Integrated Plasma and Tissue Proteomics Reveals Attractin Release by Intraluminal Thrombus of Abdominal Aortic Aneurysms and Improves Aneurysm Growth Prediction in Humans

Abstract

Background: Abdominal aortic aneurysms (AAA) are pathological dilatations of the aorta which can result in rupture and mortality. Novel methods of predicting AAA growth is a recognised priority in AAA research. Patient with AAAs have increased risk of cardiovascular morbidity. We have previously observed accelerated systemic endothelial dysfunction (measured by brachial artery FMD) in AAA patients and FMD correlates with future AAA growth. Further, systemic endothelial dysfunction is reversed by AAA repair. AAAs contain intra-luminal thrombus (ILT). Since ILT is either removed or excluded from circulation after successful repair of AAAs, we hypothesise that ILT to be the source of mediators that contribute to AAA growth. Methods: Patients were prospectively recruited to the Study (Ethics Ref SC/13/0250). Plasma samples were collected at baseline and at 1 year from each patient. Plasma samples were also collected before and at 10-12 weeks after surgery from each patient (n=29). Paired aneurysm wall, ILT, omental biopsies were collected intra-operatively during open surgical repair (n=3). In addition to analyses of the tissue, supernatant was obtained from ex vivo culture of these paired tissue samples. Samples were subjected to non-targeted LC-MSMS workflow after trypsin digest, using the Universal method to discover novel proteins. LC-MSMS data was analysed using the Progenesis QI pipeline. Results: The median AAA size at baseline was 48 mm. 59 patients were prospectively followed for 12 months. The median growth rate of AAA was 3.8%/year (IQR 1.9% to 6.8%). Comparison between patients with the fastest vs the slowest (n=10 each) showed 116 proteins to be differentially expressed in their plasma. Among these proteins, 35 also changed significantly before and after AAA repair, suggesting their origin to from the AAA complex. Comparison of the proteomics profile of aneurysm tissue, ILT, and omental artery show 128 proteins to be uniquely present in ILT. Analyses of the tissue culture supernatant further revealed 3 proteins that are: (i) uniquely present in ILT; (ii) released by ILT; (iii) systemic levels reduced after AAA surgery; (iv) differs between fast and slow growth AAAs. One of these proteins is attractin. To validate the LC-MSMS data, attractin level in individual patient was measured by ELISA. Consistent with the LC-MSMS data, plasma attractin level is higher in patients with fast AAA growth. Plasma attractin level correlates significantly with future AAA growth rate (Spearman r=0.35, P<;0.005). Using attractin and AAA diameter as input variables, the AUROC for predicting no growth of AAA at 12 months is 85% (P<0.001). Conclusion: We show that ILT of AAAs releases mediators (such as attractin) during the natural history of AAA growth. These are novel biomarkers for AAA growth prediction in humans.