PROJECT SUMMARY/ABSTRACT Vascular calcification affects ~60% of adults over 60 years of age and is frequently seen in patients with atherosclerosis. Atherosclerotic calcification is an independent risk factor for cardiovascular morbidity and mortality. Despite decades of research, no medical therapy has been convincingly established to prevent or reverse vascular calcification. The intimal (luminal) endothelium forms the innermost layer of the vasculature. Under disease conditions, the intimal endothelial cells (ECs) are stimulated to transform into osteoprogenitor cells through endothelial-mesenchymal transitions and contribute to vascular calcification. ECs in the adventitia contribute to neoangiogenesis in vascular disease. However, little is known about whether adventitial ECs are associated with vascular calcification. During bone development, a subset of ECs defined by high expression of the EC marker CD31 and the glycoprotein Endomucin (Emcn) are critical for bone formation. Our preliminary study using Matrix Gla Protein (Mgp) knockout (Mgp-/-) mice as a model of vascular calcification revealed two distinct populations of ECs in the calcified aortas, the intimal ECs (i-ECs) defined by CD31+Emcn- and the adventitial ECs (a-ECs) characterized by CD31+Emcn+. These two EC subtypes with distinct Emcn expression levels were also detected in human calcified arteries. Bulk RNA sequencing studies showed i-ECs were enriched in stem cell and osteogenic markers and a-ECs exhibited upregulated Notch expression. Endothelial deletion of the Notch1 gene reduced vascular calcification and increased the survival of the Mgp-/- mice. In this proposal, we hypothesize that a-ECs support the biomineralization in the vascular calcification in animal models and human atherosclerotic lesions, and are closely regulated by Notch signaling. In Aim 1, we will define the molecular signature of the a-EC (CD31+Emcn+) population in vascular calcification using single cell RNA sequencing. We will delineate the developmental trajectories of a-ECs in vascular calcification with correlation to the extent and severity of calcification. In Aim 2, we will determine the contribution of endothelial subtypes (a- ECs and i-ECs) and Notch signaling in atherosclerotic calcification. We will investigate the effect of endothelial- specific deletion of the Notch1 receptor on EC subtypes, calcification and transcriptional profiles in atherosclerotic lesions. The proposed studies will provide novel insight into the fundamental mechanisms of endothelial cell biology in atherosclerotic calcification and may identify potential gene targets for selective therapeutic modulation. Together with the mentored career development plan, the completion of the projects will serve as a foundation to facilitate the candidate to transition into a successful and independent physician scientist in cardiovascular research.