Glutaraldehyde-fixed bovine pericardium valves are the predominantly used devices in the clinic. Proteomic analysis of explanted degeneration bioprosthetics is, therefore, a cross-species challenge, as the degenerated explants are suspected to contain proteins from both the donor species (Bos taurus) and the host species (Homo sapiens). However, in traditional bottom-up proteomic approaches, shared peptide amino acid sequences across donor versus host species render protein-level specificity a challenge. Previous bioprosthetic research has been limited by the lack of an appropriate processing workflow to consider the background proteome of the xenogenic ECM (glutaraldehyde-fixed bovine pericardium). During this fellowship, for the first time, a proteomic pipeline was developed including non-implanted BP tissues to perform species differentiation and exclude any non-implanted BP background proteins.
Previous bioprosthetic (BP) research has been limited by the disregard for the heterogeneous nature of degeneration. Guided by gross pathological features and a review of the histopathology literature, we defined for the first time subtypes of BP degeneration and then conducted label-free proteomic profiling, in comparison with well-established disease stage-associated regions of native aortic valve disease. In the histopathological analyses, different regions of calcification were identified in BP. The fellow used laser capture microdissection to microscopically isolate these different subtypes of bioprosthetic calcification for independent downstream processing and proteomic analysis. Finally, as extracellular vesicles (EVs) have been implicated in the pathogenesis of aortic stenosis and are widely appreciated as building blocks of cardiovascular calcification, an ultrastructural assessment of explanted degenerated bioprosthetic tissue was conducted, confirming the presence of EVs. Using tissue EV isolation strategies developed at CICS, EVs were isolated from degeneration BP tissue for downstream processing and proteomic analysis.
The pathogenesis of BHV degeneration likely stems from complex interactions between the biomechanical environment, host response, and leaflet matrix. During each cardiac cycle, pressure changes cause the aortic valve to open and close, inducing repeated flexural deformation, radial stresses, and shear forces on the leaflets. Although the solid biomechanics of valve tissue have been widely studied to predict fatigue and tearing, recent clinical and explanted tissue studies have observed that shear stress correlates with the incidence of degeneration and calcification. The fellow observed the acquisition of circulating inflammatory cells at the BHV blood-contacting surface. However, the role of inflammatory cells in BHV degeneration, especially in response to shear stress, remains poorly understood. Experiments were designed to determine whether differential shear patterns experienced by acquired host inflammatory cells at the BHV blood contacting surface drive bioprosthetic degeneration.
