Calcific aortic valve disease (CAVD) is a fatal condition that affects the aortic valve of the heart. Currently, there are no pharmacological treatments for the disease; the only intervention is heart valve replacement at CAVD end-stages. The goal of this thesis is to unravel mechanisms of CAVD emergence for pharmacological development, through first identifying live biomarkers of early pathogenesis, then identifying unique subgroups of valvular cells with differing propensities towards pathogenesis for pharmacological targeting. This was accomplished utilizing a 3D valvular endothelial cell (VEC) and endothelial interstitial cell (VIC) co-culture model that forms spatiotemporally complex lesions involving both cell types when induced with osteogenic media (OGM). To identify live pathogenic biomarkers, a novel combination 3D optical coherence microscopy + confocal fluorescence imaging platform was utilized to image VEC, VIC, and matrix before, during, and after calcific lesion formation. A new mechanism of lesion formation was discovered involving VEC delamination adjacent to VEC aggregations in areas of calcifications. Investigation into molecular pathways involved with these processes revealed that the RhoA GTPase pathway was active in OGM-treated samples, and could be inhibited to prevent lesion onset. Second, we performed single-cell sequencing to identify disease-prone and disease-resistant VIC and VEC subgroups. We were able to demonstrate similarities between our model and human sequencing datasets, identify novel VIC and VEC populations that received OGM treatment but did not differentiate to disease-transformed cells, and identify and evaluate a pharmacological target. We are optimistic that the insights gained from this work will help aid in earlier live detection and identification of pharmacological windows in CAVD, and help enable more rapid pharmacological target discovery with targeted modulation of unique valve cell sub-populations.