Modification of the Secretion Pattern of Proteases,Inflammatory Mediators,and Extracellular Matrix Proteins by Human Aortic Valve is Key in Severe Aortic Stenosis

One of the major challenges in cardiovascular medicine is to identify candidate biomarker proteins. Secretome analysis is particularly relevant in this search as it focuses on a subset of proteins released by a cell or tissue under certain conditions. The sample can be considered as a plasma subproteome and it provides a more direct approximation to the in vivo situation. Degenerative aortic stenosis is the most common worldwide cause of valve replacement. Using a proteomic analysis of the secretome from aortic stenosis valves we could identify candidate markers related to this pathology, which may facilitate early diagnosis and treatment. For this purpose, we have designed a method to validate the origin of secreted proteins, demonstrating their synthesis and release by the tissue and ruling out blood origin. The nLC-MS/MS analysis showed the labeling of 61 proteins, 82% of which incorporated the label in only one group. Western blot and selective reaction monitoring differential analysis, revealed a notable role of the extracellular matrix. Variation in particular proteins such as PEDF, cystatin and clusterin emphasizes the link between aortic stenosis and atherosclerosis. In particular, certain proteins variation in secretome levels correlates well, not only with label incorporation trend (only labeled in aortic stenosis group) but, more importantly, with alterations found in plasma from an independent cohort of samples, pointing to specific candidate markers to follow up in diagnosis, prognosis, and therapeutic intervention.Degenerative aortic stenosis (AS)¹ is currently the most common cause of valve replacement in Western countries, and a significant increase in its prevalence is expected in the future because of increasing longevity (1). At the same time that our population age increases it can be expected that cardiac valve disease, and AS in particular, will increase in parallel. More and more, clinicians are seeing patients with symptomatic severe AS who are very advanced in age and have severe comorbidities or significant frailty, making operative intervention either impossible or of very high risk in the eyes of the cardiac surgeon. For this reason, the need of new diagnostic and prognostic methods, together with the urgent need of new drugs for therapy, has increased making a correct diagnosis in the early stages of the disease thereby reducing the cost burden to society. Several lines of evidence have demonstrated that degenerative AS is an active process in which inflammation plays a key role. As such, preventative approaches similar to those used in coronary artery disease (CAD) may be also applicable to AS (2, 3, 4, 5, 6). However, recent studies reported no reduction in later states of AS disease using statins, which significantly benefit patients with atherosclerosis (7, 8). Hence, further research is required to elucidate the pathogenic mechanism of this prevalent disease and to identify the similarities and differences in relation to atherosclerosis.Proteomics has emerged as a particularly suitable platform for the nonbiased analysis of proteins involved in the pathogenesis of various diseases, such as AS (9, 10). This type of approach provided the basis for the development of biomarkers to detect patients at risk of developing degenerative AS. Plasma and serum have been the main source used in proteomic studies to identify candidate protein biomarkers, followed by tissue and cell samples. However, the use of plasma and serum is hampered by their complex nature and by the large dynamic range of protein concentrations, which may favor the detection of abundant proteins at the expense of those present at lower concentrations (11, 12). As such, the aortic valve secretome has emerged as an attractive target to further understand the AS pathogenic process. Tissue secretomes provide a more accurate model of the in vivo situation and, by minimizing serum contaminants, they facilitate the detection of low abundance proteins secreted into the blood (13).In the present study, aortic valves from AS patients and nonaffected control subjects were cultured and the secretome analyzed by nLC-MS/MS. The addition of labeled amino acids to the culture media enabled the origin of the secreted proteins to be validated (truly secreted versus serum contaminants) and provided with information related to the dynamics of their synthesis and release from tissue.