**1. Introduction**

#### **1.1. Aortic stenosis**

Aortic stenosis is characterized by the abnormal narrowing of the aortic valve (AV) opening, producing a blockage of the blood flow from the left ventricle into the aorta. Two different types of aortic stenosis can be distinguished. In congenital aortic stenosis an inherited abnor‐ mal formation of the AV exists. Otherwise, in acquired aortic stenosis external causes such as rheumatic fever or valve degeneration occur. Calcific aortic stenosis (AS) is the most common valvular disease in elder population and remain the main cause of aortic valve replacement in developed countries [1].

AS progresses from a primary stage of aortic sclerosis, with thickening and stiffness of the AV, to severe calcific stenosis. Its most common symptoms are dyspnea, angina and syncope, which predict the rapid deterioration of left ventricular function, the development of heart failure and, if the pathology progresses, the patient's death. Therefore, the appearance of any of these symptoms is considered as an indication for the treatment of this pathology. In these cases, surgery is recommended to replace the AV since there is not treatment to delay the progression of the disease.

Classically, this disease has been considered as a consequence of the aging process of the valve. However, recent studies have provided evidences that inflammation plays a key role in the physiopathology of AS as well as classical cardiovascular risk factors such as hypertension, hypercholesterolemia, diabetes, smoking, age or sex [2]. Degeneration of the valve begins with

© 2013 Mourino-Alvarez et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

an endothelial dysfunction in the aortic side as a result of the abovementioned risk factors. Low density lipoproteins (LDLs) are accumulated in the subendothelial space of the valve, where they are oxidized, resulting in the activation of the endothelial cells. These cells express adhesion and chemotactic molecules, which attract inflammatory cells such as monocytes and T lymphocytes. Monocytes extravasate to the fibrous layer and differentiate into macrophages, which capture oxidized lipoproteins and become foam cells. Proinflammatory cytokines released by both cell types induce phenotypic differentiation of a subset of myofibroblasts to osteoblasts, which leads to the subsequent formation of calcium nodules [3]. These numerous similarities suggest a common relationship between atherosclerosis and AS. 3 58 Figure 1 GENOMICS

Understanding of the Disease

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#### **1.2. Proteomic and metabolomic study of healthy valves**

A complete knowledge of the structures involved in a disease it is important to understand its development. Previously, healthy tissue such as vasculature and myocardial have been succesfully studied to better understand the molecular mechanisms involved in vascular develepment and angiogenesis as well as biochemical changes that occur during physiological ageing [4, 5].

Histologically, AV consists of three layers: fibrosa, spongiosa and ventricularis. The fibrosa is located in the aortic side of the leaflets and it is mainly composed of fibroblasts and collagen fibers. The spongiosa is located below and it is formed by fibroblasts, mesenchymal cells and a polysaccharides-rich matrix. The ventricularis, found in the ventricular side of the leaflet, is made up of elastic fibers radially distributed. The AV is externally covered by several layers of endothelial cells. Collagen is responsible for the mechanical integrity of the valve, the spongiosa serves as a shock absorber and elastic fibers contract cusps during systole [3, 6]. 3 14 aortic valve AV

**2. Proteomics**

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Chapter Title: Proteomics and Metabolomics in Aortic Stenosis: Studying Healthy Valves for a Better

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Author(s) Name(s): L. Mourino-Alvarez, C.M. Laborde and M.G. Barderas

<sup>1</sup>11 Aortic stenosis is characterized by the

valve degeneration occur.

abnormal narrowing of the aortic valve

(AV) opening, producing a blockage of the

blood flow from the left ventricle into

the aorta. Two different types of AS can

inherited abnormal formation of the aortic

external causes such as rheumatic fever or

valve exists. Otherwise, in acquired AS

1 18 aortic valve AV

1 22 aortic valve AV

2 19 aortic valve AV

be distinguished. In congenital AS an

**2.1. Sample preparation**

Proteomics is the large-scale study of the proteins content in a given sample (ie. biofluid or tissue) [9]. Since proteins are the final product of genes expression, proteomics constitutes a powerful tool for the study of biological systems thanks to proteome reflects the current state of the organism and it varies according to its functional situation [10]. These studies can be performed through a wide variety of techniques and methodologies, depending on the sample and the experimental design. In the case of descriptive proteomics, in which the most usually is the study of very complex samples, it is essential to perform certain steps to facilitate the study: 1) sample preparation, 2) protein separation and 3) analysis by mass spectrometry.

**Figure 1.** Dynamic study of the physiophatology of a disease through "-omics" technologies.

Genetic information Metabolic pathways

Proteomics and Metabolomics in Aortic Stenosis: Studying Healthy Valves for a Better Understanding of the Disease

Genotipe Fenotipe

Aortic stenosis is characterized by the

flow from the left ventricle into the

aorta. Two different types of aortic

abnormal formation of the AV exists.

Otherwise, in acquired aortic stenosis

external causes such as rheumatic fever or

PROTEOMICS Proteins

METABOLOMICS Metabolites

http://dx.doi.org/10.5772/55589

153

Dinamic interactions

congenital aortic stenosis an inherited

stenosis can be distinguished. In

valve degeneration occur.

TRANSCRIPTOMICS mRNA

Transcription Traduction

abnormal narrowing of the aortic valve (AV)

opening, producing a blockage of the blood

Preparation of the samples prior to the analysis using proteomic techniques is an essential step for obtaining robust and reproducible data. Between the large number of standardized protocols, the selected one must be carefully chosen to suit the sample to be analyzed, as well as for the proteins of interest. In the case of the AV an effective and suitable protocol has been previously described [11] (Figure 2). Briefly, within 2 hours after surgery, valves were washed in PBS and then ground into a powder in liquid N2 with a mortar. 0.2 g of this powder was resuspended in 400 μl of protein extraction buffer (Tris 10 mM [pH 7.5], 500 mM NaCl, 0.1% Triton x-100, 1% β-mercaptoethanol, 1 mM PMSF) and then centrifuged to precipitate mem‐ branes and tissue debris [12]. Supernatant containing most of the soluble proteins was collected

1

However, it is also essential to perform studies at the molecular level of the tissue, looking for the discovery of tissue- and disease-specific markers. For this purpose, proteomic and metabolomic analyses can be ideally used since they allow the unbias analysis of hundreds or thousands of molecules at a time, detecting and identifying which molecules are present. In contrast to genomics and transcriptomics, proteomics and metabolomics study dynamic protein products, low molecular weight compounds and their interactions, which have a direct effect on the phenotype of the tissue (Figure 1). 4 17 Proteomics proteomics 4 21 aortic valve AV

Descriptive proteomics is a methodology that enables unbiased large-scale study of the set of all proteins in a biologic system at any given time. Thus, the expression, localization, interac‐ tion, structural domains and activity of these proteins, including splice isoforms and posttranslational modifications (PTMs), can be studied [7]. Metabolomics is the study of a complete metabolome or a single group of particular metabolites, which are small molecules that participate in general metabolic reactions and that are required for the maintenance, growth and normal function of a cell [8]. The study of healthy valves through proteomic and metab‐ olomic approaches and the subsequent integration of data, can provide molecular level information of the metabolic pathways that are more active in that tissue and will help to understand the mechanisms of physiological/pathological processes in aortic stenosis valves. This makes it easier the search for potential markers for early diagnosis of the disease, thus being able to predict which people may develop aortic stenosis in the future. 5 6 2DE 2-DE 5 6 2DE regime can enhance these 2-DE regime enhance separation of these

2 26 studies study Proteomics and Metabolomics in Aortic Stenosis: Studying Healthy Valves for a Better Understanding of the Disease http://dx.doi.org/10.5772/55589 153

Aortic stenosis is characterized by the

flow from the left ventricle into the

aorta. Two different types of aortic

abnormal formation of the AV exists.

Otherwise, in acquired aortic stenosis

external causes such as rheumatic fever or

congenital aortic stenosis an inherited

stenosis can be distinguished. In

valve degeneration occur.

abnormal narrowing of the aortic valve (AV)

opening, producing a blockage of the blood

**Figure 1.** Dynamic study of the physiophatology of a disease through "-omics" technologies.
