Mechanical stretch regulates inflammatory signaling in human smooth muscle cells

AimsDuring atherosclerosis progression, vascular smooth muscle cells (SMCs) undergo phenotypic modulation from a contractile state to alternative modulated and proliferative states. Similar transitions occur in vitro, likely due to loss of physiological cues such as specific extracellular matrix (ECM) components and mechanical forces. Here, we investigated how defined ECM substrates and stretch conditions influence the phenotype of human aortic SMCs. Methods and resultsSMCs were cultured on collagen I-, fibronectin-, or laminin-coated plates under static conditions, physiological stretch (10% elongation), or pathological stretch (15% elongation), followed by bulk RNA sequencing. Mechanical stretch regulated genes involved in cell cycle regulation, contractile function, and inflammatory signaling. While functional effects on proliferation and contractility were modest, inflammatory pathways were strongly affected by stretch intensity. Physiological stretch suppressed basal and TNF-induced inflammatory gene expression, whereas pathological stretch enhanced it, with consistent effects across all ECM substrates. Physiological stretch downregulated multiple NF-{kappa}B target genes and reduced IKBKB expression. IKBKB knockdown lowered baseline inflammatory gene expression and abolished stretch-induced suppression of CCL2, indicating an NF-{kappa}B-dependent mechanism, likely downstream of p65 translocation. Single-cell RNA sequencing revealed pronounced phenotypic heterogeneity in cultured SMCs. Integration with human atherosclerosis datasets showed that in vitro SMC states partially overlapped with plaque SMC phenotypes but displayed a globally enhanced pro-inflammatory phenotype. Importantly, stretch-induced suppression of inflammatory signaling was observed across the heterogeneous SMC population. ConclusionsPhysiological mechanical stretch induces strong anti-inflammatory effects in human SMCs. Single-cell transcriptomic analysis further revealed marked heterogeneity among cultured SMCs, recapitulating the phenotypic diversity seen in human atherosclerotic plaques. The stretch-induced anti-inflammatory response in SMCs was observed across all cell clusters, highlighting a conserved protective effect of physiological mechanical forces.