Substrate Stiffness Modulates TGF-β1-Induced Lineage Specification in Multipotent Vascular Stem Cells

Multipotent vascular stem cells (MVSCs) are found in the vascular wall and surrounding tissues and possess the ability to differentiate into mesenchymal lineages. Previous studies have shown that MVSCs can be activated in response to vascular injury and differentiate into vascular smooth muscle cells (SMCs), contributing to vascular remodeling and microvessel formation. However, it remains unclear as to whether and how microenvironmental changes in the extracellular matrix, such as substrate stiffness, modulates MVSC differentiation under pathological conditions. This study demonstrated that MVSCs cultured on stiff substrates exhibited increased cell spreading, stronger cell adhesion, and a higher expression of SMC markers, including myosin heavy chain (MHC), myocardin (MYCD), calponin 1 (CNN1), and smooth muscle α-actin (SMA). In contrast, MVSCs on soft substrates showed an elevated expression of the chondrogenic markers aggrecan 1 (AGC1) and collagen-II (COL2A1). The presence of TGF-β1 further increased the expression of SMC markers on stiff substrates and chondrogenic markers on the soft substrates. Collectively, these results establish substrate stiffness as a key regulator of MVSC lineage commitment through cytoskeletal reorganization, with TGF-β1 acting as a biochemical amplifier. Our findings highlight the substrate-stiffness-dependent differentiation of MVSCs and provide mechanistic insights into the role of MVSCs in vascular remodeling during atherosclerosis development and blood vessel regeneration.