Project Summary Oxidative stress plays a key but unknown role in fibrocalcific aortic valve stenosis (FAVS). FAVS is a disease of aortic valve (AV), where leaflets progressively stiffen and eventually calcify, causing cardiac malfunction. The only treatment is surgical valve replacement. Increased oxidative stress due to redox imbalance is believed to be an initiating factor for end point calcification of the aortic valve (AV) leaflets, but redox status during FAVS remains undefined. Our current data shows that oxidative stress is increased in a very young adult mouse model of aortic valve stiffening, concomitant with extracellular matrix remodeling, implicating that redox mechanisms play a much earlier role in FAVS than previously described. We hypothesize that early redox imbalances are associated with the ECM disorganization that results in valvular stiffening. To delineate redox mechanisms, Aim 1 will define redox status in bulk, whole mount and histological cross sectional measurements of the AV leaflet during early sequential aortic valve stiffening. Physiological measurements of heart function and biomechanical properties of the AV will be used to define levels of AV stiffening parallel with redox status. Aim 2 will utilize proteomic techniques on extracts from decellularized valve leaflets to identify redox sensitive cellular pathways and protein interaction networks (PINs) during sequential valve stiffening. Bioinformatic approaches will be used to assess PINs regulated during valve stiffening, earmarking new candidates of FAVs. Extracellular matrix (ECM) from the decellularized valve structure will be interrogated by proteomics and immunoblotting to report on ECM changes correlating to increased oxidative stress. Bioinformatics tools and immunoblotting techniques will be used to report on oxidative stress induced post translational modifications. Aim 3 will delineate regulation in protein interaction networks coinciding with increased oxidative stress in de-identified normal and FAVS clinical specimens. A combination of immunohistochemistry, immunoblotting, and proteomics will be used to define the redox status of normal AV compared to FAVS at a cellular level and assess overall contribution to valve structure phenotype. Candidates from the murine model of early valve stiffening will be evaluated for contribution to FAVS in clinical specimens. This investigation relies on the unique resources of the Medical University of South Carolina's COBRE in Oxidants, Redox Balance and Stress Signaling and would not be accomplished without use of these resources. This study will allow us to define the role of redox balance and affected protein expression during the early stages of valve stiffening in FAVS, identifying new therapeutic targets that might inhibit disease progression, improve quality of life and decrease mortality.