FAK- and TGF-β1-dependent biomechanical alterations in microvascular endothelial cells exposed to fluid shear stress gradients

Abstract Endothelial cells (EC) form a dynamic monolayer barrier that regulates vascular permeability and is highly sensitive to mechanical cues, including shear stress (SS) generated by blood flow. While spatial shear stress gradients (SSG) are strongly linked to vascular pathologies such as atherosclerosis, their direct effects on EC kinematics, mechanics, and signaling, and whether these responses vary across EC subtypes, remain poorly understood. Here, using an impinging flow device compatible with long-term videomicroscopy, we demonstrate that exposure of two distinct EC types to SSG leads to an immediate and sustained two-fold decrease in migration speed, accompanied by enhanced coordination of collective motion and directional migration against the flow. Concurrently, EC traction forces and monolayer stresses increase acutely upon flow onset and continue to rise linearly over time. Mechanistically, FAK-mediated mechanotransduction governs flow-opposed migration, whereas the time-dependent increase in traction depends on TGF-β1 signaling and actomyosin contractility but is not required for setting migration direction. Together, these findings identify SSG as key regulators of EC mechanics, collective dynamics, and signaling, with important implications for vascular disease diagnostics and therapeutic strategies.