Felix Gil-Dones¹, Tatiana Martin-Rojas¹, Luis F. Lopez-Almodovar², Fernando de la Cuesta³, Veronica M. Darde⁴, Gloria Alvarez-Llamas³, Rocio Juarez-Tosina⁵, Gemma Barroso⁴, Fernando Vivanco^3,6, Luis R. Padial^1,7 and Maria G. Barderas¹

¹Department of Vascular Physiopathology, Hospital Nacional de Paraplejicos (HNP), SESCAM, Toledo. ²Cardiac Surgery, Hospital Virgen de la Salud, Toledo. ³Department of Immunology, Fundacion Jimenez Diaz, Madrid. ⁴Proteomic Unit, Hospital Nacional de Parapléjicos, SESCAM, Toledo. ⁵Department of Pathology, Hospital Virgen de la Salud, Toledo. ⁶Department of Biochemistry and Molecular Biology I, Universidad Complutense, Madrid. ⁷Department of Cardiology, Hospital Virgen de la Salud, Toledo.

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.