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Title: Proteomics analysis of intracellular and extracellular proteome in aneurysmal patients with bicuspid and tricuspid aortic valve
Author: Fava, Marika
ISNI:       0000 0004 6057 6597
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2016
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Background. Thoracic aortic aneurysm (TAA) is a degenerative disease of the aortic wall and is associated with an increased risk of aortic rupture. TAA is a more prevalent complication in patients with bicuspid aortic valve (BAV) compared to those with tricuspid aortic valve (TAV). Objectives. We aimed to investigate first, changes in intracellular proteins by comparing BAV and TAV patients with aneurysm, and second, regional changes in extracellular matrix (ECM) proteins by comparing the lesser (concavity) and the greater (convexity) aortic curvatures of BAV patients with and without aneurysm. The findings obtained in human patients were followed up by mechanistic studies in mice. Methods. Human and murine aortic specimens were analysed by mass spectrometry (MS) after extraction of intracellular and ECM proteins. Immunoblotting and gene expression analysis were used to validate the proteomics findings. An ECM protease knockout mouse model was used to better characterise proteolytic processing of ECM large proteoglycan. Similar techniques were used as above. Results. To compare the intracellular proteome of aneurysmal BAV and TAV patients we used two-dimensional fluorescence difference in-gel electrophoresis. This approach revealed regulation of 24 proteins. BAV aneurysms were associated with up-regulation of smooth muscle cell related proteins and down­regulation of glycolytic and oxidative stress related proteins. Next, we focused on alterations in ECM proteins and compared the concave and convex areas in aortas of BAV patients with and without TAA using liquid chromatography and tandem MS. In aortas of aneurysmal BAV patients, versican, a large proteoglycan important for tissue integrity, was up-regulated in the concave area; conversely, versican was down-regulated in the same area of non-aneurysmal BAV samples. Gene expression analysis showed no differences in versican levels between concavity and convexity of aneurysmal patients, suggesting that regulation of versican protein levels may be related to proteolytic processing. ADAMTSs (a disintegrin and metalloproteases with thrombospondin motifs) are the main proteases cleaving versican. Notably, in aneurysmal BAV patients there was an inverse association between the abundance of versican and gene expression levels of members of the ADAMTS family. To study the role of ADAMTS in the remodelling of the ECM in aorta, we used ADAMTS-5 knockout mice. After angiotensin II infusion to induce aortic dilatation, versican levels were higher in aortas of ADAMTS-5 knockout mice activity compared to wild-type controls. Conclusion. Our results suggest that ADAMTS proteases may be important for versican regulation during aortic ECM remodelling, as supported by proteomics changes in the ECM of aneurysmal BAV patients and ADAMTS-5 knockout mice.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available