Abstract Body (Do not enter title and authors here): Background
Medial arterial calcification (MAC) significantly contributes to cardiovascular mortality, yet effective treatments remain limited due to unclear molecular mechanisms. This study aimed to develop a mouse model of MAC using O-ring-induced transverse aortic constriction (OTAC) and to elucidate key genes, pathways, and cellular interactions involved in MAC formation through lineage tracing and single-cell RNA sequencing (scRNA-seq).

Methods
An OTAC mouse model was established to replicate MAC. Adult C57BL/6J male mice underwent OTAC, and subsequent analyses were conducted at various time points. Histological and immunohistochemical assessments were performed to monitor changes in vascular smooth muscle cells (VSMCs). Lineage tracing was utilized to verify the origin of osteochondrogenic VSMCs. Furthermore, scRNA-seq was employed to capture dynamic changes in VSMCs and other cell types contributing to MAC development.

Results
The OTAC model successfully induced osteochondrogenic transdifferentiation of VSMCs, leading to MAC. Immunohistochemical analysis demonstrated time-dependent decreases in α-SMA expression and increases in SOX9 and RUNX2 expression. Lineage tracing using Myh11CreERT2;ROSA26-EGFP mice confirmed that osteochondrogenic VSMCs originated from contractile VSMCs. scRNA-seq identified osteochondrogenic VSMCs and revealed dynamic changes in surrounding cells, including macrophages and fibroblasts, which were implicated in calcification-related and inflammatory processes.

Conclusion:
The OTAC method effectively mimicked human MAC pathology, offering comprehensive insights into the microenvironmental dynamics of MAC progression at the single-cell level. This model serves as a robust platform for future research on therapeutic interventions.