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.