ECM stiffness governs endothelial barrier integrity through YAP-mediated stabilization of ZO-1.

The integrity of intercellular junctions is essential for maintaining tissue barriers and preventing pathological fluid leakage. While reduced extracellular matrix (ECM) stiffness has emerged as a biomechanical trigger for barrier dysfunction, the underlying mechanotransduction mechanisms remain poorly understood. In this study, employing tunable polyacrylamide hydrogels to model physiological and pathological ECM stiffness, we demonstrate that matrix softening occurs earlier than morphological changes in corneal endothelial cells (CEnCs) and triggers junctional disassembly by impairing the membrane localization and stability of the tight junction protein ZO-1. Mechanistically, we uncover a novel non-canonical role for YAP in binding and stabilizing ZO-1, thereby shielding it from ubiquitin-proteasome degradation. Furthermore, we elucidate a synergistic interplay between FAK and Hippo signaling pathways, which converges on YAP to modulate junctional integrity in response to ECM stiffness. Computational modeling further elucidates the spatiotemporal dynamics of stress propagation during ECM softening, providing a biomechanical framework for intercellular gap formation and cell detachment. Collectively, these results establish matrix stiffness as a pivotal modulator of cell-cell junctions, yielding fresh perspectives for harnessing engineered biomaterials in the fabrication of robust tissue barriers.