Oxygen tension modulates neurite outgrowth in PC12 cells through a mechanism involving HIF and VEGF

Abstract

Cell-based approaches are a promising therapeutic strategy for treating injuries to the nervous system, but the optimal means to promote neurite extension and direct cellular behavior are unclear. Previous studies have examined the behavior of neural-like cells in ambient air (21% oxygen tension), yet these conditions are not representative of the physiological oxygen microenvironment of neural tissues. We hypothesized that neuronal differentiation of a model neural cell line (PC12) could be controlled by modulating local oxygen tension. Compared to ambient conditions, PC12 cells cultured in reduced oxygen exhibited significant increases in neurite extension and total neurite length, with 4% oxygen yielding the highest levels of both indicators. We confirmed neurite extension was mediated through oxygen-responsive mechanisms using small molecules that promote or inhibit HIF-1alpha stabilization. The hypoxic target gene Vegf was implicated as a neurotrophic factor, as neurite formation at 21% oxygen was mimicked with exogenous VEGF, and a VEGF-neutralizing antibody attenuated neurite formation under reduced oxygen conditions. These findings demonstrate that behavior of neural-like cells is driven by the oxygen microenvironment via VEGF function, and suggest promising approaches for future applications in neural repair.

Figure 1

Reductions in local oxygen tension promote neurite formation in PC12 cells. a PC12 cells display differences in neurite extension when cultured at indicated oxygen tensions. Arrowheads indicate neurites; scale bar represents 50 µm. b Fraction of cells extending neurites under varying oxygen tension. a indicates p < 0.001 by ANOVA compared to 21% oxygen; b indicates p < 0.001 by ANOVA compared to 21%, 12%, or 1% oxygen. n = minimum of four per condition. c Quantification of average total neurite length of 100 randomly selected cells in each condition. a indicates p < 0.001 by ANOVA compared to 21% oxygen; b indicates p < 0.001 by ANOVA compared to 21%, 12%, or 1% oxygen. n = minimum of four per condition

Figure 2

Manipulation of HIF signaling mediates neurite formation. a Western blotting for HIF-1α after 8 h (left) and 72 h (right) for PC12 cells cultured in 21%, 4%, or 1% oxygen tension. b The hypoxia mimetic 100 μM CoCl₂ increases the frequency of PC12 cells elaborating neurites under 21% oxygen culture, whereas HIF-α inhibition with 100 μM YC-1 abrogated hypoxia-driven neurite formation under 4% culture. a indicates p < 0.001 by t test compared to 21% oxygen; b indicates p < 0.001 by t test compared to 4% oxygen. n = minimum of four per condition

Figure 3

VEGF signaling mediates neurite formation. a Levels of Vegf transcript after culture for 8 or 72 h under 21%, 4%, or 1% oxygen tension culture. a indicates p < 0.05 compared to 21% or 1% oxygen at 8 h; b indicates p < 0.05 compared to 21% oxygen at 72 h; c indicates p < 0.01 compared to 21% oxygen at 72 h. b Addition of exogenous VEGF (50 ng/mL) increases the frequency of PC12 cells elaborating neurites under culture at 21% oxygen tension, whereas cells cultured at 4% oxygen in the presence of a VEGF-neutralizing antibody failed to extend neurites. a indicates p < 0.001 by t test compared to 21% oxygen; b indicates p < 0.001 by t test compared to 4% oxygen. n = minimum of four per condition

Figure 4

PC12 metabolic activity is altered by hypoxia and VEGF signaling. a PC12 cells reveal increased metabolic activity when cultured under 4% oxygen tension. b The hypoxia mimetic 100 μM CoCl₂ or 50 ng/mL VEGF increase metabolic activity, whereas the hypoxia antagonist YC-1 or VEGF-inhibiting antibody attenuates hypoxic increases in metabolic activity. n = 3 per condition