Endothelial-to-mesenchymal transition primes vascular endothelial cells toward an osteochondrogenic fate

Endothelial-to-mesenchymal transition (EndMT), in which endothelial cells (ECs) lose their endothelial identity and acquire mesenchymal-like features, contributes to vascular dysfunction and remodeling in atherosclerosis. However, the fate and function of these cells remain unclear. Here, we investigated their differentiation potential and functional properties to define how EndMT contributes to vascular dysfunction. Human umbilical vein ECs (HUVECs) were treated with transforming growth factor-β2 (TGF-β2) and interleukin-1β (IL-1β) for seven days to induce EndMT. Mesenchymal stem/stromal cell (MSC) identity was assessed by flow cytometry for canonical markers (CD44, CD73, CD105, CD90). Differentiation states were evaluated using published single-cell RNA sequencing (scRNA-seq) data of EndMT-treated HUVECs and validated under lineage-specific culture environments. In vivo analysis was performed using scRNA-seq data from EC lineage reporter mice in atherosclerosis models. EndMT-treated HUVECs displayed an intermediate mesenchymal phenotype, expressing CD44, CD73 and CD105 but lacking CD90, failing to meet MSC criteria. Potency analysis showed that 77 % of EndMT-treated HUVECs remained oligopotent, while 19 % acquired osteogenic and chondrogenic potential, accompanied by activation of lineage-associated transcriptional programs (RUNX2, BMPR1A, NOTCH2, WNT5A; CD151, ANXA6, DCN). In vivo, endothelial lineage-traced cells in atherosclerotic mice formed an EndMT cluster enriched for osteogenic and chondrogenic gene programs, including ossification and cartilage development pathways. We define a primed oligopotent state of EndMT-derived cells both in vitro and in vivo, marked by transition toward osteogenic and chondrogenic fates. These findings suggest that EndMT contributes to atherosclerosis by generating osteogenic- and chondrogenic-like cells, linking endothelial dysfunction to vascular calcification in disease.