Vascular calcification was induced in mice via subcutaneous injection of high-dose vitamin D3 (vD3). The treatment group received vD3 (5×10^5 IU/kg/day; Sigma-Aldrich, #PHR1237) dissolved in the vehicle, while the control group received the vehicle alone. Injections were administered for three consecutive days. Animals were euthanized, and aortic tissues were collected at two time points: day 4 and day 7 after the first injection. Single-cell suspensions were prepared from pooled aortic arch (AA) or descending thoracic aorta (DTA) segments (n=6 pools per group). Briefly, aortas were dissected from mice at day 0, 4, and 7 post-vitamin D3 treatment, cleaned of adherent tissues in HBSS under a stereomicroscope, and segmented into AA and DTA. Tissues from the same type and time point were pooled, minced, and enzymatically digested at 37°C using a solution of collagenase type I and dispase. The dissociated cell suspension was then triturated, filtered through a 70-μm strainer, and centrifuged. The resulting cell pellet was resuspended, and cell viability was confirmed to be >80% prior to single-cell library preparation.

This study investigates why the aortic arch is more prone to calcification than the descending aorta. Vascular calcification is a serious condition with no effective treatment, where blood vessel cells become similar to bone-forming cells. We used single-cell RNA sequencing on a mouse model of vitamin D-induced calcification to compare these two aortic regions. Our key discovery was a specific subpopulation of Msx2-positive vascular smooth muscle cells that is uniquely found in the aortic arch and appears to drive its increased calcification. This finding provides new insights into the cellular basis of regional calcification differences and could lead to new therapeutic targets.

Raw sequencing reads were processed using the 10x Genomics Cell Ranger pipeline for demultiplexing, alignment to the reference genome (e.g., mm10), cell barcode assignment, and unique molecular identifier (UMI) counting to generate the final gene expression matrix.