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

Abstract

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. Molecular & Cellular Proteomics 12:

A clinical perspective on the utility of alpha 1 antichymotrypsin for the early diagnosis of calcific aortic stenosis

“…Due to the high social costs associated with CAS healthcare, there is a strong unmet clinical need to find markers that aid early diagnosis, estimate the long-term prognosis, monitor treatment response and predict potential adverse effects in CAS patients. By using multi-omics approaches, we hoped to define markers that may potentially serve to diagnose the disease earlier than traditional methods, such as echocardiography [ 11 ]. Novel tools for easier diagnosis will reduce the high morbidity and mortality associated to surgery, and they may provide a solution for those patients who cannot be operated on, as well as improving patient management.…”

“…For secretome analysis, the second valve leaaflet was cultured as described elsewhere [ 11 , 12 ] using medium supplemented with antibiotics and amphotericin B (Fungizone ® ) to avoid contamination. Samples were transferred to a Petri dish (Cell Star ® ), cut into pieces and incubated at 37 °C in lysine-arginine free 1640 RPMI medium (Cell Culture Technologies Invitrus) supplemented with 5 mg/ml fungizone, 250 mg/ml amikacin, 2 mg/ml l -lysine 2HCl (U-13C6, 97–99%) and 10 mg/ml l -Arginine HCl (U-13C6, 97–98%: Cambridge Isotope Laboratories Inc., Andover, MA) in a humidified atmosphere of 5% CO 2 .…”

iTRAQ proteomic analysis of extracellular matrix remodeling in aortic valve disease

“…Despite the growing evidence connecting ECM remodeling with AS pathogenesis, the underlying regulatory mechanisms for such remodeling and stratification are not well defined, even though they seem relevant to interpret aortic valve degeneration. In previous studies in our lab 6 , we conducted an in-depth analysis of the secretome of aortic valves which pointed out an outstanding role of valvular remodeling in the pathophysiological mechanisms of AS. The functional classification of the differentially secreted proteins showed a significant number of ECM proteins.…”

The plasma proteomic signature as a strategic tool for early diagnosis of acute coronary syndrome

“…Additionally, in spite of the large variety of biological processes of the identified proteins, based on our previous experience in secretome studies, the tissue is the origin of many of them [ 33 , 34 ]. As previously explained in the Background section, the wide number of biochemical disorders secondary to hypoxia suffered by the myocardium lead to tissue breakdown and the releasing of a large number of proteins of potential interest into the blood stream.…”