Neuro-Mechanical Regulation of Vascular Smooth Muscle Cell Behaviour Under Ageing-Associated Substrate Stiffness

Cardiovascular diseases (CVDs) remain a leading cause of mortality worldwide, and ageing is strongly associated with progressive arterial stiffening. Age-related alterations in extracellular matrix (ECM) mechanics influence vascular smooth muscle cell (VSMC) behaviour, while sympathetic innervation represents an additional regulator of vascular homeostasis. However, how neural signalling interacts with ageing-associated mechanical conditions to regulate VSMC behaviour remains unclear. In this study, an in vitro sympathetic neuron-VSMC co-culture model was established to investigate neuro-mechanical regulation. Primary rat sympathetic neurons and A7r5 VSMCs were cultured on glass or polydimethylsiloxane (PDMS) substrates with defined stiffness (20 and 130 kPa), representing healthy and ageing-associated stiffened arterial environments, respectively. VSMC behaviour was assessed through analysis of cell area, proliferation, migration, cellular Young's modulus (YM), and DNA damage marker γH2AX. Sympathetic neuronal co-culture was associated with reduced VSMC spreading and decreased γH2AX levels. Under the conditions tested, neural signalling exerted limited effects on cell proliferation and migration. In contrast, increased substrate stiffness promoted cell proliferation and elevated YM. Both neuronal input and substrate stiffness were associated with increased cellular YM. Together, these findings indicate that neural and mechanical cues may jointly influence VSMC behaviour within ageing-associated mechanical environments. This co-culture system provides a controllable platform for studying neuro-mechanical interactions in vascular biology.