Valvular aortic stenosis: a proteomic insight

Abstract

Calcified aortic valve disease is a slowly progressive disorder that ranges from mild valve thickening with no obstruction of blood flow, known as aortic sclerosis, to severe calcification with impaired leaflet motion or aortic stenosis. In the present work we describe a rapid, reproducible and effective method to carry out proteomic analysis of stenotic human valves by conventional 2-DE and 2D-DIGE, minimizing the interference due to high calcium concentrations. Furthermore, the protocol permits the aortic stenosis proteome to be analysed, advancing our knowledge in this area.

Summary: Until recently, aortic stenosis (AS) was considered a passive process secondary to calcium deposition in the aortic valves. However, it has recently been highlighted that the risk factors associated with the development of calcified AS in the elderly are similar to those of coronary artery disease. Furthermore, degenerative AS shares histological characteristics with atherosclerotic plaques, leading to the suggestion that calcified aortic valve disease is a chronic inflammatory process similar to atherosclerosis. Nevertheless, certain data does not fit with this theory making it necessary to further study this pathology. The aim of this study is to develop an effective protein extraction protocol for aortic stenosis valves such that proteomic analyses can be performed on these structures. In the present work we have defined a rapid, reproducible and effective method to extract proteins and that is compatible with 2-DE, 2D-DIGE and MS techniques. Defining the protein profile of this tissue is an important and challenging task that will help to understand the mechanisms of physiological/pathological processes in aortic stenosis valves.

Keywords: aortic stenosis; human aortic valves; proteomics.

Figure 1

Diagram illustrating the protein extraction method employed for AS valve proteomic analysis. A) Flowchart representing the analysis of human AS valves. B) Sequential extraction protein protocol scheme.

Figure 2

Two dimensional gel analysis of homogenates from AS valves: A) when focused at pH 4–7 containing the soluble protein extract (E1); B) the extract rich in hydrophobic proteins focused at pH 4–7 (E2); C) the pooled extracts focused at pH 4–7 (E1 + E2); D) the soluble protein extract (E1) focused at pH 3–10; E) the hydrophobic rich protein extract focused at pH 3–10 (E2); and F) the pooled extract when focused at pH3-10 (E1 + E2).

Figure 3

A) 2D-DIGE gels. The area marked in blue shows the spots obtained from the E1 extract and the area marked in red shows the spots obtained from E2. B) Representative image of 2-DE silver stained gel of AS valve extracts (IEF: 4–7 pH range, second dimension, 12% acrylamide) and the protein spots identified. Numbers in red corresponded to two different proteins identified in the same spot.