PROJECT SUMMARY/ABSTRACT The bicuspid aortic valve (BAV) is the most common cardiac congenital defect and contains two, as opposed to the normal three, leaflet tissues. BAVs commonly become diseased at a faster rate than structurally normal aortic valves (AVs) most often due to calcium build up which eventually leads to aortic stenosis (AS). Current clinical treatments for AS in BAV patients consist only of surgical options such as AV repair and replacement, with replacement being the more common. Bioprosthetic valves are routinely used in replacement scenarios despite their limited lifespan of 10-15 years. In the context of BAV patients, who tend to disease at earlier time points in life, bioprosthetic valves are not an indefinite solution and will most likely require follow-up surgical operations. Alternatively, mechanical valves are employed for the younger BAV patient population but require the indefinite need for anticoagulants which substantially hinders patient quality of life. Thus, no optimal nor indefinite surgical intervention currently exists to treat BAV disease. Previous work from our lab and others have elucidated drastic differences in extracellular matrix (ECM) composition and structure as well as differences in the mechanical stress-strain environment between AVs and BAVs. However, it has yet to be elucidated as to how these changes affect BAV interstitial cell (BAVIC) functional remodeling behaviors. In addition, limited work has been done to explore whether BAV disease may be caused by intrinsic differences between the BAVICs and normal AV interstitial cells (AVICs). We hypothesize that the intrinsic differences of BAVICs, the altered microenvironment, and the altered BAV leaflet strains enhance BAV disease progression through cell-mediated ECM remodeling and biosynthesis brought on by phenotypic activation of the BAVIC population. We will address this hypothesis with the following three aims: Identifying the 3D morphological and ECM regional variations within the BAV. We will utilize state-of-the- art methods including 3D small angle light scattering, quantitative histology, and focused-ion beam scanning electron microscopy to assess the differences in ECM between the BAV and AV. Delineating the biophysical state and biosynthetic behaviors of isolated BAVICs and AVICs within peptide-modified poly (ethylene glycol) (PEG) hydrogels of varying stiffness. We will assess the contractile and biosynthetic properties of isolated BAVICs and AVICs within PEG hydrogels to investigate intrinsic differences among the cell groups. Emulating BAV leaflet strains to assess BAVIC remodeling behaviors in vitro. Here we will use a uniaxial stretch bioreactor to emulate BAV strain levels and assess how altered kinematics affect BAVIC responses.