Unforeseen decreases in dissolved oxygen levels affect tube formation kinetics in collagen gels

Abstract

The availability of oxygen (O(2)) is a critical parameter affecting vascular tube formation. In this study, we hypothesize that dissolved oxygen (DO) levels in collagen gels change during the three-dimensional (3D) culture of human umbilical vein endothelial cells (HUVECs) in atmospheric conditions and that such changes affect the kinetics of tube formation through the production of reactive oxygen species (ROS). We demonstrate a decrease in O(2) tension during 3D cultures of HUVECs. Noninvasive measurements of DO levels during culture under atmospheric conditions revealed a profound decrease that reached as low as 2% O(2) at the end of 24 h. After media replacement, DO levels rose rapidly and equilibrated at ∼15% O(2), creating a reoxygenated environment. To accurately estimate DO gradients in 3D collagen gels, we developed a 3D mathematical model and determined the Michaelis-Menten parameters, V(max) and K(m), of HUVECs in collagen gels. We detected an increase in ROS levels throughout the culture period. Using diphenyliodonium to inhibit ROS production resulted in the complete inhibition of tube formation. Interference RNA studies further showed that hypoxia-inducible factors (HIFs)-1α and -2α are not involved in the formation of 3D tubes in collagen gels. We conclude that ROS affect the tubulogenesis process through HIFα-independent pathways, where the levels of ROS are influenced by the uncontrolled variations in DO levels. This study is the first demonstration of the critical and unexpected role of O(2) during 3D in vitro culture models of tubulogenesis in atmospheric conditions.

Fig. 1.

Dissolved oxygen (DO) levels and cell viability during three-dimensional (3D) culture of human umbilical vein endothelial cells (HUVECs) in collagen gels. A: measured DO levels during 48 h of HUVEC culture in 3D collagen gels in atmospheric conditions, with and without reactive oxygen species (ROS) inhibitor diphenyleneiodonium (DPI), with media replacement after 24 h (Ai) and no media replacement (Aii). B: percentage of viable HUVECs at multiple time points during the culture period in untreated and DPI-treated 3D cultured HUVECs. **P < 0.01, ***P < 0.001; n = 3.

Fig. 2.

Model prediction of DO levels and gradients. A: steady-state DO at the bottom of collagen gels at different gel thicknesses. We fitted model numerical predictions (dashed black line) and analytical prediction (solid red line) to the experimental values (○) to determine V_max and K_m using the residual sum of squares method. B: model predictions for oxygen gradient after 24 h of culture in the three-layer model (Bi) and across the gel (Bii).

Fig. 3.

Intracellular ROS generation during 3D culture of HUVECs in collagen gels. A: fluorescence images of intracellular ROS in untreated and DPI-treated 3D HUVEC cultures after 48 h (left) and quantification of the ROS fluorescence intensity at multiple points, normalized by cell viability (right). **P < 0.01. B: tube formation of untreated and DPI-treated HUVECs after 48 h using light microscope images (top) and fluorescence microscope images (bottom; phalloidin in green, nuclei in blue). Scale bars, 100 μm.

Fig. 4.

Small interfering RNA (siRNA) studies of hypoxia-inducible factor-α (HIFα). A: light microscope images (top) and fluorescence microscope images (bottom; phalloidin in green, nuclei in blue) of 3D collagen culture of HUVECs transfected with siRNA of luciferase, HIF1α, and HIF2α after 48 h (scale bars, 100 μm). B: tube length, thickness, and area coverage of untreated cells, DPI-treated cells, and HIF1α- and HIF2α-silenced cells. *P < 0.05, **P < 0.01, ***P < 0.001. C: viability of HUVECs at multiple time points as a comparison of luciferase-transfected cells to HIF1α and HIF2α siRNA-treated cells.

Fig. 5.

Possible mechanisms involved in tubulogenesis of HUVECs in 3D collagen gel in atmospheric conditions. Varying experimental conditions, such as cell-seeding density, collagen gel and media thickness, and oxygen uptake rate, lower the available DO levels and determine the severity of hypoxia, which may lead to HIFα stabilization. Subsequent changes of media result in the reoxygenation of the cells and promote intracellular ROS generation, which, in turn, mediates tubulogenesis through both HIFα-dependent and -independent pathways. Moreover, adding DPI to the culture medium precludes the rise of ROS both by inhibiting NOX-derived ROS and by the formation of hypoxic conditions via the blocking of mitochondrial respiration and therefore ROS generation derived by reoxygenation. The gray and black solid lines denote the evidence shown in this study and in others (shown in brackets), respectively, whereas dashed black lines denote results still under debate.