Proteomic and degradomic signatures of extracellular matrix remodeling in calcific aortic valve stenosis

Aortic stenosis is a progressive fibro-inflammatory valvular disorder with major clinical burden and no disease-modifying pharmacological therapy. Defining molecular circuits associated with thrombo-inflammatory activation to extracellular matrix remodeling may enable future therapeutic targeting and biomarker development. Human aortic valves explanted from patients with severe aortic stenosis and non-stenotic surgical controls (aortic regurgitation) were profiled using a multi-layer mass spectrometry strategy. Global protein changes were quantified by label-free proteomics (data-independent acquisition). Extracellular matrix proteolysis was interrogated using an extracellular matrix-focused semi-tryptic peptide workflow. Collagen qualitative remodeling was assessed by mapping hydroxyproline enrichment. Differential abundance was evaluated using multiple-testing correction (false discovery rate). Selected candidates were validated by targeted multiple reaction monitoring, and elastin integrity was assessed histologically. We identified 594 significantly modulated proteins in severe aortic stenosis, with predominant upregulation of complement/coagulation and extracellular matrix-related pathways. Targeted Multiple Reaction Monitoring confirmed key thrombo-inflammatory and matrix-associated candidates. Semi-tryptic profiling revealed a focused extracellular matrix degradomic signature dominated by small leucine-rich proteoglycans (decorin, lumican, PRELP), fibrillin-1, and collagen VI, consistent with preferential proteolytic targeting of structural matrix scaffolds. Histology showed marked elastin fragmentation in stenotic leaflets. Collagen post-translational modification analysis revealed increased hydroxyproline- bearing peptides across selected collagen chains despite minimal changes in total collagen abundance, indicating qualitative remodeling beyond protein accumulation. An integrated proteomic-degradomic-post-translational modification framework reveals concomitant thrombo-inflammatory activation to matrix breakdown and qualitative collagen remodeling in severe aortic stenosis, highlighting molecular circuits that may inform future biomarker development and therapeutic target discovery.