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bioRxiv · 10.1101/2025.10.28.684711

Osteochondrogenic Transdifferentiation of Vascular Smooth Muscle Cells and Microenvironmental Dynamics in Medial Arterial Calcification

Nakao, Y.·Sakaue, T.·Ito, J.·Hamaguchi, M.·Takahashi, K.·Kukida, M.·Seike, F.·Aono, J.·Omiya, S.·Sakahashi, Y.·Yoshida, M.·Wakabayashi, M.·Shirai, M.·Yasuda, K.·Inoue, K.·Ikeda, S.·Yamaguchi, O.
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Abstract

BackgroundVascular calcification, particularly medial arterial calcification (MAC), considerably affects cardiovascular mortality. Current treatments are limited because of the unclear molecular mechanisms of MAC. This study aimed to establish an MAC mouse model using O-ring-induced transverse aortic constriction (OTAC) and to identify critical genes, pathways, and cellular interactions involved in MAC formation by combining lineage tracing technology and single-cell RNsA sequencing (scRNA-seq). MethodsWe developed an OTAC mouse model to mimic MAC. Adult C57BL/6J male mice underwent OTAC, and subsequent analyses were performed at various intervals. Histological and immunohistochemical evaluations were conducted to observe changes in vascular smooth muscle cells (VSMCs). Lineage tracing was used to confirm the origin of osteochondrogenic cells in the tunica media. Additionally, scRNA-seq was performed to capture the dynamic changes in VSMCs and other cell types involved in MAC development. ResultsOsteochondrogenic cells and the subsequent MAC in OTAC mice were observed using histological evaluations. Immunohistochemistry showed time-dependent decreases in -SMA expression and increases in SOX9 and RUNX2 expression in VSMCs. Lineage tracing using Myh11CreERT2;ROSA26-EGFP mice confirmed that osteochondrogenic cells originated from contractile VSMCs. scRNA-seq confirmed osteochondrogenic transdifferentiation of VSMCs and highlighted dynamic changes in surrounding cells involved in calcification-related and inflammatory processes. ConclusionThe OTAC method enabled a comprehensive understanding of the microenvironmental dynamics during MAC progression at the single-cell level. This powerful model provides a robust platform for future therapeutic intervention research. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/684711v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@c64634org.highwire.dtl.DTLVardef@12b0b9aorg.highwire.dtl.DTLVardef@106c5b4org.highwire.dtl.DTLVardef@19dd4f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Nakao, Y., Sakaue, T., Ito, J., Hamaguchi, M., Takahashi, K., Kukida, M., Seike, F., Aono, J., Omiya, S., Sakahashi, Y., Yoshida, M., Wakabayashi, M., Shirai, M., Yasuda, K., Inoue, K., Ikeda, S., Yamaguchi, O.. 2025-10-28. Osteochondrogenic Transdifferentiation of Vascular Smooth Muscle Cells and Microenvironmental Dynamics in Medial Arterial Calcification. https://doi.org/10.1101/2025.10.28.684711

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