Project Summary Calcific Aortic Valve Disease (CAVD) is the gradual stiffening and calcification of the aortic heart valve, which serves as a gateway for unidirectional flow of oxygenated blood from the left ventricle to the aorta during each heartbeat. In severe cases of CAVD, the valve cannot open to allow blood to travel from the heart to the body, which can cause severe damage to the left ventricle resulting in heart failure and death. Despite its high prevalence, there are no effective pharmacological therapeutics to date for CAVD, forcing patients to undergo heart surgery to receive life-saving treatment. We believe that a lack of knowledge about the dynamic stages of early, mid, and late disease combined with a limited understanding of the heterogenous differentiated diseased cell populations and the use of in-vitro drug testing platforms that do not capture the full complexity of the disease contribute to this lack of clinically effective pharmacological therapeutics to date. To address these critical gaps in the CAVD field, I will first combine our lab’s tissue engineered mechanically active co-culture valve model with cutting-edge live-OCT imaging to acquire 3D time-lapse images of the calcification process. Next, I will utilize single-cell sequencing of our tissue-engineered valve model to uncover characteristics of heterogeneous disease-prone and disease-resistant valvular cells. Finally, I will combine the information and technologies produced from the first two stages of the project in a multi-dimensional drug testing platform to investigate when and why a previously discovered pharmacological drug target, NFkB pathway, reduces calcification in-vitro. At the conclusion of this project, we will uncover never-before seen time-lapse stages in ECM, cellular morphology and mineralization changes that are critical for valvular calcification, as well as molecular pathways related to heterogenous cell groups that are inductive or preventative of calcification. We will utilize these advancements together to evaluate a promising therapeutic target not just for whether it has an effect, but rather when and why it has an effect on 3D in-vitro valvular mineralization. The drug-testing platform produced as a result of this project may be utilized to rigorously evaluate other promising therapeutic targets identified by our single-cell sequencing results and by other labs, allowing for more rapid and cost-effective evaluation of CAVD pharmacological therapeutics.