3D confinement alters smooth muscle cell responses to chemical and mechanical cues

Smooth muscle cell (SMC) phenotypic switching is a hallmark of many vascular diseases. Although prior work has established that chemical and mechanical cues contribute to SMC phenotypic switching, the impact of three-dimensional (3D) confinement on this process remains elusive. Yet, <i>in vivo</i>, arterial SMCs reside within confined environments. In this study, we designed a microfluidic assay to investigate the interplay between 3D confinement and different environmental stimuli in SMC function. Our results show that tightly, but not moderately, confined SMCs acquire a contractile phenotype when exposed to collagen I. Elevated compressive forces induced by hydrostatic pressure abolish this upregulation of the contractile phenotype and compromise SMC survival, particularly in tightly confined spaces. Transforming growth factor beta 1, which promotes the contractile state in moderate confinement, fails to enhance the contractility of tightly confined cells. Fibronectin and engagement of cadherin 2 suppress the contractile phenotype of SMCs regardless of the degree of confinement. In contrast, homophilic engagement of cadherin 11 upregulates SMC-specific genes and enhances contractility in both moderately and tightly confined cells. Overall, our work introduces 3D confinement as a regulator of SMC phenotypic responses to chemical and mechanical signals.