P21-activated kinase regulates oxygen-dependent migration of vascular endothelial cells in monolayers.

The collective migration of vascular endothelial cells plays important roles in homeostasis and angiogenesis. Oxygen concentration in vivo, which is lower than in the atmosphere and changes due to diseases, is a key factor affecting the cellular dynamics of vascular endothelial cells. We previously reported that hypoxic conditions promote the internalization of vascular endothelial (VE)-cadherin, a specific cell-cell adhesion molecule, and increase the velocity of the collective migration of vascular endothelial cells. However, the mechanism through which cells regulate collective migration as affected by oxygen tension is not fully understood. Here, we investigated oxygen-dependent collective migration, focusing on intracellular protein p21-activated kinase (PAK) and hypoxia-inducing factor (HIF)-1α. A monolayer of human umbilical vein vascular endothelial cells (HUVECs) was formed in a microfluidic device with controllability of oxygen tension. The HUVECs were then exposed to various oxygen conditions in a range from 0.8% to 21% O2, with or without PAK inhibition or chemical stabilization of HIF-1α. Collective cell migration was measured by particle image velocimetry with time-lapse phase-contrast microscopic images. Localizations of VE-cadherin and HIF-1α were quantified by immunofluorescent staining. The collective migration of HUVECs varied in an oxygen-dependent fashion; the migration speed was increased by hypoxic exposure down to 1% O2, while it decreased under an extremely low oxygen tension of less than 1% O2. PAK inhibition suppressed the hypoxia-induced increase of the migration speed by preventing VE-cadherin internalization into HUVECs. A decrease in the migration speed was also obtained by chemical stabilization of HIF-1α, suggesting that excessive accumulation of HIF-1α diminishes collective cell migration. These results indicate that the oxygen-dependent variation of the migration speed of vascular endothelial cells is mediated by the regulation of VE-cadherin through the PAK pathway, as well as other mechanisms via HIF-1α, especially under extreme hypoxic conditions.

Microfluidic platform for three-dimensional cell culture under spatiotemporal heterogeneity of oxygen tension.

Cells in a tumor microenvironment are exposed to spatial and temporal variations in oxygen tension due to hyperproliferation and immature vascularization. Such spatiotemporal oxygen heterogeneity affects the behavior of cancer cells, leading to cancer growth and metastasis, and thus, it is essential to clarify the cellular responses of cancer cells to oxygen tension. Herein, we describe a new double-layer microfluidic device allowing the control of oxygen tension and the behavior of cancer cells under spatiotemporal oxygen heterogeneity. Two parallel gas channels were located above the media and gel channels to enhance gas exchange, and a gas-impermeable polycarbonate film was embedded in the device to prevent the diffusion of atmospheric oxygen. Variations in oxygen tension in the device with the experimental parameters and design variables were investigated computationally and validated by using oxygen-sensitive nanoparticles. The present device can generate a uniform hypoxic condition at oxygen levels down to 0.3% O2, as well as a linear oxygen gradient from 3% O2 to 17% O2 across the gel channel within 15 min. Moreover, human breast cancer cells suspended in type I collagen gel were introduced in the gel channel to observe their response under controlled oxygen tension. Hypoxic exposure activated the proliferation and motility of the cells, which showed a local maximum increase at 5% O2. Under the oxygen gradient condition, the increase in the cell number was relatively high in the central mild hypoxia region. These findings demonstrate the utility of the present device to study cellular responses in an oxygen-controlled microenvironment.

Migration of vascular endothelial cells in monolayers under hypoxic exposure.

The hypoxic microenvironment existing in vivo is known to significantly affect cell morphology and dynamics, and cell group behaviour. Collective migration of vascular endothelial cells is essential for vasculogenesis and angiogenesis, and for maintenance of monolayer integrity. Although hypoxic stress increases vascular endothelial permeability, the changes in collective migration and intracellular junction morphology of vascular endothelial cells remain poorly understood. This study reveals the migration of confluent vascular endothelial cells and changes in their adherens junction, as reflected by changes in the vascular endothelial (VE)-cadherin distribution, under hypoxic exposure. Vascular endothelial monolayers of human umbilical vein endothelial cells (HUVECs) were formed in microfluidic devices with controllability of oxygen tension. The oxygen tension was set to either normoxia (21% O2) or hypoxia (<3% O2) by supplying gas mixtures into separate gas channels. The migration velocity of HUVECs was measured using particle image velocimetry with a time series of phase-contrast microscopic images of the vascular endothelial monolayers. Hypoxia inducible factor-1α (HIF-1α) and VE-cadherin in HUVECs were observed after exposure to normoxic or hypoxic conditions using immunofluorescence staining and quantitative confocal image analysis. Changes in the migration speed of HUVECs were observed in as little as one hour after exposure to hypoxic condition, showing that the migration speed was increased 1.4-fold under hypoxia compared to that under normoxia. Nuclear translocation of HIF-1α peaked after the hypoxic gas mixture was supplied for 2 h. VE-cadherin expression was also found to be reduced. When ethanol was added to the cell culture medium, cell migration increased. By contrast, by strengthening VE-cadherin junctions with forskolin, cell migration decreased gradually in spite the effect of ethanol to stimulate migration. These results indicate that the increase of cell migration by hypoxic exposure was attributable to loosening of intercellular junction resulting from the decrease of VE-cadherin expression.