SUMMARY Therapeutic advances in vascular calcification may have far-reaching public benefits. Vascular calcification is a frequent complication of diabetes mellitus and associated with the increase of morbidity and mortality. Although the precise mechanism has not been determined, vascular calcification is known to be an active process involving ectopic bone formation, in which osteogenic differentiation occurs in the cells transdifferentiated from other lineages. Previous studies have shown that vascular endothelial cells (ECs) switch cell fate to differentiate into osteoblastic-like cells to contribute to vascular calcification. However, it is unknown if shifting the transcriptional landscape of EC-derived osteoblast-like cells back to endothelial differentiation ameliorates vascular calcification. In present proposal, we take advantage of single-cell RNA sequencing (scRNA-seq) and connectivity Map (CMap) to identify a novel approach, aiming to shift the transcriptional landscape of EC-derived osteoblast-like cells to endothelial differentiation and investigate its effect on vascular calcification in diabetes mellitus. In preliminary study, we use lineage tracing, scRNA-seq and CMap query to identify cyclin-dependent kinase 1 (CDK1) inhibition to redirect EC-derived osteoblast-like cells to endothelial differentiation and significantly improve vascular calcification. We find a specific induction of CDK1 in EC-derived osteoblast-like cells. We show that CDK1 deletion or its inhibitor AT7519 increases E-twenty-six specific sequence variant 2 (ETV2), which is responsible for shifting the transcriptional landscape of EC-derived osteoblast-like cells to endothelial differentiation. Furthermore, CDK1 deletion in ECs or AT7519 reduces EC-derived osteogenesis and decreases aortic calcification in diabetic Ins2Akita/+ mice without affecting other tissues. Therefore, we hypothesize that CDK1 inhibition induces ETV2 to redirect EC-derived osteoblast-like cells back to endothelial differentiation in turn to ameliorate vascular calcification in diabetes mellitus. In specific Aim 1, we will elucidate the mechanism underlying CDK1 inhibition that shifts EC-derived osteoblast-like cells toward endothelial differentiation. In specific Aim 2, we will determine if CDK1 inhibition ameliorates vascular calcification in a diabetic mouse model. If successful, it will build the redirection of ill-fated cells back to normalization as a new concept, and the CDK1 inhibition may emerge as a new therapeutic approach to treat calcification in acquired vascular diseases.