Dynamic and functional changes in vascular endothelial cell monolayers under combined hypoxic and inflammatory...

In sepsis, dysregulated inflammatory responses cause microthrombi formation, leading to organ hypoxia. While endothelial cell (EC) responses to inflammation are well-studied, the combined effects of hypoxia and inflammation remain unclear. We investigated EC dynamics and function under simultaneous hypoxia and lipopolysaccharide (LPS) exposure using microfluidic devices with controllability of oxygen concentration. Collective cell migration was measured by particle image velocimetry, while monolayer permeability was assessed by diffusion of fluorescently labeled dextran. Additionally, we examined adhesion and signaling protein expression. Migration speed decreased under approximately 1% O₂, but LPS exposure delayed this decrease, attenuating hypoxic responses. Both hypoxia and LPS increased permeability, though this effect was partially suppressed by LPS preconditioning at a specific concentration. LPS exposure increased ICAM-1 and VCAM-1 expression on endothelial surfaces, except for VCAM-1 under hypoxia, suggesting complex interactions between HIF-1α and NF-κB pathways activated by hypoxia and LPS, respectively. NF-κB inhibition suppressed both collective migration and permeability, confirming its role in these dynamic and functional changes. These findings provide novel insights into EC phenotypic alterations under combined hypoxic and inflammatory conditions in an in vitro model that effectively recapitulates key aspects of sepsis pathophysiology.