Pressure and stiffness sensing together regulate vascular smooth muscle cell phenotype switching

This study investigated how two pathological mechanical cues implicated in atherosclerosis—hypertensive pulsatile pressure and a compliant extracellular matrix—combine to drive vascular smooth muscle cell (VSMC) phenotypic switching. VSMCs cultured on defined-stiffness substrates (spanning very soft, intima-like compliance to stiff, calcified-like conditions) showed measurable cytoskeletal and morphometric changes in response to either substrate compliance or pressure alone, but a full switch was only observed when hypertensive pressure was applied on compliant substrates. Under these combined conditions, VSMCs robustly formed matrix-degrading podosomes (confirmed by cortactin core with zyxin/vinculin ring organization and gelatin degradation), exhibited reduced actin alignment and altered cell shape distributions, and displayed broad proteomic remodeling (TMT-based quantitative proteomics) that was most pronounced specifically in the compliant-plus-hypertensive condition. Mechanistically, the work identified convergent regulation of actin dynamics through cofilin: stiffness sensing modulated cofilin activity via a RhoA–ROCK2–LIMK2 axis, while pressure (and PKC activation) promoted Ca2+- and slingshot-dependent cofilin dephosphorylation, together maximizing podosome formation and ECM remodeling—linking distinct mechanosensing pathways to early atherosclerotic progression.