Elsevier

International Journal of Cardiology

Volume 225, 15 December 2016, Pages 99-106
International Journal of Cardiology

Patients with calcific aortic stenosis exhibit systemic molecular evidence of ischemia, enhanced coagulation, oxidative stress and impaired cholesterol transport

https://doi.org/10.1016/j.ijcard.2016.09.089Get rights and content

Highlights

  • A pro-coagulation and pro-inflammatory state exists in aortic stenosis patients.

  • Stenotic patients show impairment in lipid metabolism and response to ischemia.

  • Four sensitive and specific molecular panels for aortic stenosis have been defined.

Abstract

Background

The most common valve diseases are calcific aortic stenosis (AS) and aortic regurgitation (AR). The former is characterized by thickening of valve leaflets followed by progressive calcification, which produces progressive aortic valve (AV) narrowing, increased pressure afterload on the left ventricle (LV) and subsequent LV hypertrophy. On the other hand, AR is due to malcoaptation of the valve leaflets with resultant diastolic reflux of blood from aorta back to the LV producing volume and pressure overload and progressive LV dilatation. In order to isolate the molecular mechanisms taking place during AS, we have used an integrated “–omic” approach to compare plasma samples from AS and from AR patients used as controls. The final purpose of this work is to find molecular changes in response to the calcification of the AV, diminishing the effects of the AV dysfunction.

Methods and results

Using two-dimensional difference gel electrophoresis (2D-DIGE) and gas chromatography coupled to mass spectrometry (GC-MS) in a cohort of 6 subjects, we have found differences in 24 protein spots and 19 metabolites, respectively. Among them, 7 proteins and 3 metabolites have been verificated by orthogonal techniques (SRM or turbidimetry): fibrinogen beta and gamma chain, vitronectin, apolipoprotein C-II, antithrombin III, haptoglobin, succinic acid, pyroglutamic acid and alanine. Classification according to their main function showed alterations related to coagulation, inflammation, oxidative stress, response to ischemia and lipid metabolism, defining 4 different molecular panels that characterize AS with high specificity and sensitivity.

Conclusion

These results may facilitate management of these patients by making faster diagnostics of the disease and better understand these pathways for regulating its progression.

Graphical abstract

Figure 1 Scheme of the workflow of this study. A combined proteomics and metabolomics approach was performed for the discovery phase. Verification of these results was carried out using SRM and turbidimetry to describe a molecular panel of AS.

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Introduction

The two main valvulopathies affecting the aortic valve (AV) are calcific aortic stenosis (AS) and aortic regurgitation (AR). In AS there is AV calcification which results in narrowing of the valve which in turn causes increased pressure afterload on the left ventricle (LV). To restore wall stress there is a hypertrophic response of the LV although eventually LV dilatation and systolic dysfunction occur during the latter stages of the disease. It was believed that calcification of the AV is a passive process occurring as a natural result of aging. However, more recent studies demonstrated that it is a complex mechanism, involving deposition of lipoproteins, chronic inflammation and calcification [1]. On the contrary, in AR there is malcoaptation of the AV leaflets that results in diastolic reflux of blood from the aorta to the LV. This leads to volume and pressure overload on the LV producing progressive LV dilatation and eventually systolic dysfunction [2], [3]. In both AS and AR, there is a relatively long initial asymptomatic phase that normally lasts several years. However, when the pathophysiological response ultimately becomes maladaptive, the patient becomes symptomatic (angina, dyspnea and syncope in AS, and primarily dyspnea in AR) and there is no turning back. Despite current efforts, the disease management is still an important clinical dilemma for patients with AS partly since the only effective treatment is AV replacement either surgically or by transcatheter AV implantation (TAVI) which has emerged as an alternative to surgery during recent years [4], [5]. Furthermore, valve replacement is only indicated in symptomatic patients with severe AS for which reason patients with AS need regular clinical and echocardiographic follow-up, even at the earlier stages of the disease.

With these premises in mind, the search for new diagnostic and prognostic tools in AS is thus clinically relevant, especially since it may help to identify patients as high risk of rapid disease progression that would be subjected to frequent regular follow-up visits and those classified as low risk that would receive less frequent follow-ups, thus allowing for optimization of health care resources. Previous AS studies developed in our lab were performed by comparison of AS patients with control subjects [6], [7]. Nevertheless, with that experimental design it was not possible to evaluate which alterations are due to the calcification of the AV and which ones only appear as a consequence of the cardiocirculatory alterations caused by the AV dysfunction that exists in both AS and AR. Regarding these and with the purpose to isolate the effects of the calcification, we have decided to use an integrated “–omic” approach to compare plasma samples from AS and from AR patients due to aortic root dilation. This experimental design allows the isolation of systemic indicators due to the AS pathological process from these which appear due to the functional alteration of the valve. The combination of proteomics and metabolomics, two powerful techniques, allows us the identification and quantification of these proteins and metabolites which define the phenotype of the disease. Besides, as plasma is a biological sample easy to obtain, the molecules of interest could be easily measured by SRM and/or by turbidimetry, which facilitates their implementation in clinic in the near future.

Section snippets

Patient selection and blood extraction

Heart valves and peripheral blood samples of patients with severe AS and AR (n = 44) were obtained from subjects who underwent AV replacement, both in Hospital Virgen de la Salud (Toledo, Spain) and Hospital Universitario Gregorio Marañon (Madrid, Spain). Samples from patients with bicuspid AV, concomitant AS and AR or mitral valve disease were excluded. First of all, we analyze the valves using hematoxilin/eosin analysis, in order to ensure that AR valves were histologically healthy. Furthermore

Altered proteins related to AS

We first investigated the most significant protein changes in plasma in response to AS according to groups division detailed in the Material and methods section. We used AR samples as control, considering that it reflects changes in AV function, allowing us to focus on alterations originated by the degenerative process. The differential protein expression analysis using 2D-DIGE was performed using 6 subjects per group matched by age, gender and basal characteristics. Gel images were imported to

Discussion

Our double approach investigates main alterations, both at protein and metabolite levels, with the idea of looking in depth into the processes taking place in AS to find out the strongest variations which could serve as potential indicators of the disease. The observations carried out reinforce the need of searching for novel measurable indicators that would help in the identification of AS subjects. To the best of our knowledge, this is the first time that metabolomics and proteomics are

Conflicts of interest

The authors have no conflicts of interest.

Acknowledgements

This work was supported by grants from the Instituto de Salud Carlos III [FIS PI07-0537, PI11-02239, PI11/01401, PI13/01873, PI14-01917, PI14/01650, CP09/00229], FONDOS FEDER, RD06/0014/1015, RD12/0042/0071, RD012/0021, IF08/3667-1, IDCSalud (3371/002) and Fundación Conchita Rábago de Jiménez Díaz,. These results are lined up with the Spanish initiative on the Human Proteome Project (SpHPP).

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    This author takes responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation.

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