PROJECT SUMMARY: While 1.4% of people have the congenital defect of bicuspid aortic valve (BAV), BAV patients make up 50% of the patients that receive aortic valve replacements. Moreover, patients with BAVs develop aortic stenosis (AS) earlier, and thus require replacements at younger ages than patients with normal tricuspid aortic valves (TAV). Given the limited durability of replacements, BAV patients have a high procedure burden that negatively affects their length and quality of life. Therefore, development of a pharmacological therapy will reduce morbidity and mortality from AS. Our long-term goal is to understand the BAV disease process at the cellular level to develop effective treatments that mitigate AS. Since no pharmacological treatment has been forthcoming, our hypothesis is that abnormal valve interstitial cell (VIC) deformation patterns present in BAVs have a crucial role in the biochemical signaling events in AS. Since AS is associated with VIC activation into myofibroblasts and transforming growth factor-beta (TGFB) signaling, this study addresses the relationship between mechanically-conditioned cellular morphology and TGFB signaling in two specific aims: 1. Determine what parameters of mechanical conditioning experienced in varying aortic valve anatomies affect VIC morphology. The level of myofibroblast activation will first be determined in native human valve leaflet explants from BAV and TAV. The morphologies of cells from native tissues will be compared to that of cells mechanically conditioned in a novel 3D high-throughput biaxial oscillatory stretch screen (3D HT-BOSS) to determine what cyclical biaxial stretch, matrix stiffness, and VIC basal contractility is required to produce morphologies seen in BAVs and TAVs. 2. Ascertain how altering mechanical pattern will modify VIC response to TGFB. Quantitative proteomics will be employed to develop steady-state models of VIC TGFB signaling from VICs freshly isolated from native human valve leaflets. Microscopy of 3D HT-BOSS samples will then be employed to analyze shifts in: 1) EC50 of αSMA protein and 2) nuclear localization of TGFB- mediated transcription factors upon exogenous TGFB stimulation. Through this investigation, underlying drivers of AS and novel target pathways for pharmacological treatment will therefore be uncovered. Furthermore, the training that the fellowship applicant, Dr. Toni West, will receive will enable her to make the leap to becoming an independent investigator. Dr. West will be conducting research in the lab of her sponsor, Dr. Michal Sacks, and in the lab of her collaborator, Dr. Aaron Baker, at the University of Texas. As part of her training, Dr. West will travel to Columbia University, where her co- sponsor Dr. Giovanni Ferrari runs the biobank she will be collecting tissues and cells from.

PROJECT NARRATIVE Patients with the congenital defect of bicuspid aortic valve (BAV) develop aortic stenosis (AS) earlier and more often than patients with normal tricuspid aortic valves (TAV), which adversely affects BAV patients’ length and quality of life. The goal of this study is to understand changes in biochemical signaling that occur in the major cell type of valves, valve interstitial cells, due to abnormal deformation patterns present in BAVs. Two-photon microscopy of excised valves, proteomics-based steady state biochemical modeling, and 3D biaxial oscillatory stretch screens will be employed to determine the relationships between micromechanical environments and biochemical signaling cascades so that novel pharmacological targets for AS can be identified.