Observation of Collective Migration of Vascular Endothelial Cells Exposed to Lipopolysaccharide and Hypoxia

During sepsis, when the immune system overreacts to an infection, large amounts of inflammatory cytokines are secreted, leading to microthrombus formation and consequent hypoxia. Hence, both the inflammatory response to the infection and the in vivo oxygen environment significantly affect the progression of sepsis. However, few studies have simultaneously exposed cells to inflammatory and hypoxic stresses; thus, their combined effects on cellular dynamics remain unclear. Here, we simultaneously exposed monolayers of vascular endothelial cells to inflammatory and hypoxic stresses using a microfluidic device that enables control of the oxygen concentration. A preliminary investigation examined the effects of lipopolysaccharide (LPS) derived from gram-negative bacteria (one of the main causes of sepsis) on vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were cultured in multiwell plates and exposed to different concentrations of LPS (0-100 μg/ml) and oxygen (21% or 5% O2) using a multi-gas incubator. Under each oxygen condition, the cell viability changed minimally with LPS concentration, but the expression of glycocalyx on HUVECs decreased with increasing LPS concentration. Based on these results, the LPS concentration was set to 1 and 10 μg/ml for subsequent experiments using the microfluidic device. A monolayer of HUVECs was formed in the device, and collective cell migration was observed under different LPS and oxygen concentrations. Exposure of HUVECs to 10 μg/ml LPS temporarily increased the cell migration speed, which then decreased over time after 1 h, ultimately reaching a lower value than that under the control condition. Moreover, the addition of LPS suppressed the hypoxia-induced increase in migration speed. Thus, cell migration is altered by simultaneous exposure to inflammation and hypoxia stresses.