Screening Analysis of Proteolytic Enzymes and Their Inhibitors in the Leaflets of Epoxy-Treated Bioprosthetic Heart Valves Explanted due to Dysfunction

— Bioprosthetic heart valves (BHVs) have lower thrombogenicity rates and excellent hemodynamic parameters similar to native valves. However, the lifespan of these medical devices is limited to 15 years due to the structural valve degeneration (SVD). One of the mechanisms underlying functional impairment and calcification of BHVs includes proteolytic degradation of biomaterials. However, proteases found in xenogeneic tissue of BHVs remain poorly studied. In this study using the dot blot assay, we have performed a screening analysis of proteolytic enzymes and their inhibitors in the leaflets of five BHVs explanted due to dysfunction. Five aortic valves (AVs) explanted due to calcific aortic valve disease were used as a comparison group. The results of the study have demonstrated the presence of at least 17 proteases and 19 of their inhibitors in BHVs. In the AVs 20 proteases and 21 of their inhibitors were identified. Small quantitative differences were found between proteomic profiles of BHVs and AVs. Matrix metalloproteinases (MMPs) were expressed in BHVs and AVs at comparable levels, but the level of tissue inhibitors of metalloproteinases-1/-2 and reversion-inducing-cysteine-rich protein with Kazal motifs in implant tissues was lower than in native valves. This suggests that excessive activity of MMPs cannot be counterbalanced by their specific inhibitors in BHVs and therefore MMPs initiate the process of degeneration. Moreover, the detection of a wide range of proteolytic enzymes and their inhibitors in the degenerated BHVs suggests the existence of several pathophysiological pathways that can lead to SVD.