Dissecting sexual dimorphism in aortic valve stenosis by proteomics

Abstract Background The treatment of aortic valve stenosis (AVS) remains limited to aortic valve replacement (AVR). No pharmacotherapy has yet proven efficacious, and its development is challenged by sexual dimorphism. Women display extensive valve fibrosis, and men present remarkably higher valve calcification. To accelerate the development of sex-personalised therapies, deeper molecular insights are needed. Hence, we aimed to characterise AVS sexual dimorphism using proteomics. Methods Fifty surgically excised valves (50% women) were homogenised, and the proteins were quantified by LC-MS/MS. The influence of differentially expressed proteins (DEPs) in sexual dimorphism was appraised using bioinformatics. DEPs were validated using immunohistochemistry, qRT-PCR and ELISA, with 30 additional valves. Results We quantified ~ 4,000 proteins and 76 DEPs between sexes. CD163, CD74, and NADPH oxidase-2 (NOX2) were more abundant in men’s valves and central in a protein-protein interaction network. Functional enrichment analysis (FEA) supported increased lipoprotein binding and macrophage activation in men’s valves, confirmed by increased CD74 + cell infiltration (immunohistochemistry). Aminopeptidase N, coagulation factor XIII, and metalloreductase STEAP4 were more abundant in men’s valves at the transcript and protein levels. FEA indicated a women-specific dysregulation of spliceosomal proteins that may dictate a pro-fibrotic phenotype, which was observed histologically. A higher glutathione peroxidase-1/NOX2 ratio (ELISA) was found in women, suggesting increased protection against oxidative stress. Conclusions Proteomics confirms sexual dimorphism in AVS. Women displayed a higher degree of fibrotic remodelling, whereas men displayed greater immune cell infiltration and were less protected from oxidation, favouring calcification. Proteomics identified putative targets for a sex-personalised AVS modulation. Graphical abstract We studied sexual dimorphism in aortic valve stenosis following a proteomic approach. Proteins were quantified by mass spectrometry and sex differences were uncovered by differential expression, network and pathway analyses. Some targets were selected for validation by PCR, immunohistochemistry and ELISA. Collectively, our experiments support a higher propensity for men’s valves to accumulate lipoproteins, red and white blood cells, with concomitantly greater activation of macrophages, particularly CD74+. Men’s valves also show a higher propensity for oxidative stress and calcification. Women’s valves are less prone to oxidative stress (higher GPX1/NOX2) but show a greater extent of fibrosis, which might result from an alternative splicing program that translates into a significant dysregulation of focal adhesion proteins. Some graphical elements were retrieved from Flaticon (https://www.flaticon.com/).