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

Abstract

Background: Calcific aortic stenosis (CAS) is the most common heart valve disease in the elderly, representing an important economic and social burden in developed countries. Currently, there is no way to predict either the onset or progression of CAS, emphasizing the need to identify useful biomarkers for this condition.

Methods: We performed a multi-proteomic analysis on different kinds of samples from CAS patients and healthy donors: tissue, secretome and plasma. The results were validated in an independent cohort of subjects by immunohistochemistry, western blotting and selected reaction monitoring.

Results: Alpha 1 antichymotrypsin (AACT) abundance was altered in the CAS samples, as confirmed in the validation phase. The significant changes observed in the amounts of this protein strongly suggest that it could be involved in the molecular mechanisms underlying CAS. In addition, our results suggest there is enhanced release of AACT into the extracellular fluids when the disease commences.

Conclusions: The significant increase of AACT in CAS patients suggests it fulfils an important role in the physiopathology of this disease. These results permit us to propose that AACT may serve as a potential marker for the diagnosis of CAS, with considerable clinical value.

Keywords: Alpha 1 antichymotrypsin; Biomarker; Calcific aortic stenosis; Multi-proteomic.

Fig. 1

Schematic representation of the workflow. Samples were collected from valve replacement surgeries (AS) and autopsies (controls). During the discovery phase the whole tissue and secretome were analyzed and AACT was identified. Finally, AACT was validated as a potential biomarker in WBs, and by IHC and SRM

Fig. 2

2D-DIGE results. a Representative fluorescence DIGE image, showing the differentially expressed spots corresponding to AACT. On the right, the spot intensity in 3D of one of the three spots corresponding to AACT is shown. The differences between controls and patients were consistent in the 8 gels studied. b Fragmentation spectra of the proteotypic peptides used for AACT identification by MALDI-MS/MS. The fragments of the y ion series are shown in blue, while the b ion series are shown in red

Fig. 3

Validation of the AACT protein using IHC. Stronger expression of AACT is observed in the endothelium on the aortic side and, to a greater extent in the fibrosa of the stenotic valves when compared to the controls. DAB staining is brown and is indicated by the arrows. Statistical analyses showed significant differences (*p = 0.023). Amplified images at ×100 magnification

Fig. 4

Validation of the AACT protein in Western Blots. a Bidimensional immunodetection analysis of tissue samples in which differences in the expression of the isoforms is indicated by arrows. b Immunodetection of the secretome samples. The band was more intense in the patient group than in the controls. c Immunodetection of the plasma samples. Again, the band in the plasma from the patients was more intense than in the controls. Quantification by densitometry is also shown in the figure and the AACT levels are clearly higher in the CAS patients in all cases

Fig. 5

SRM validation of the differences in AACT found using LC-MS/MS. a Chromatograms of secretome samples showing the transitions of the 2 proteotypic peptides: ITLLSALVETR (A.1) and LYGSEAFATDFQDSAAAK (A.2). As it is shown in the figure, the m/z values selected in the first quadrupole were 608.37 and 946.44, respectively. On the right, quantification of the three transitions (area under the curve) that were monitored for each peptide. The different m/z values selected in the third quadrupole are indicated in the figure. b Chromatograms of plasma samples showing the transitions of the proteotypic peptide AVLDVFEEGTEASAATAVK (m/z 954.48) and quantification of each transition (area under the curve) in plasma samples. The different m/z values selected in the third quadrupole are indicated in the figure