Preconditioning protects against oxidative injury involving hypoxia-inducible factor-1 and vascular endothelial growth factor in cultured astrocytes

Tolerance to brain injury involves hypoxia-inducible factor-1 (HIF-1) and its target genes as the key pathway mediating a cascade of events including cell survival, energetics, and angiogenesis. In this study, we established the treatment paradigms for an in vitro model of tolerance to oxidative injury in primary astrocytic cultures and further examined the roles for the HIF-1 signalling cascade. Isolated murine astrocytes were preconditioned with sub-toxic concentrations of HIF-1 inducers and subsequently exposed to a H2O2 insult, where changes in cell viability and protein expression were determined. Preconditioning with non-damaging concentrations of desferrioxamine (DFO) and ethyl-3,4-dihydroxybenzoate (EDHB) significantly improved cellular viability after H2O2 injury treatment. Time course studies revealed that DFO and EDHB treatments alone induced sequential activation of HIF-1 signal transduction where nuclear HIF-1α protein accumulation was detected as early as 2 h, followed by downstream upregulation of intracellular and released VEGF from 4 h and 8 h onwards, respectively. The protective effects of DFO and EDHB preconditioning against H2O2 injury were abolished by co-treatment with cycloheximide, an inhibitor of protein synthesis. Importantly, when the anti-HIF-1 compound, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) was used, the cytoprotection and VEGF accumulation produced by DFO and EDHB preconditioning were diminished. These results indicate the essential role of the HIF-1 pathway in our model of tolerance against oxidative injury in cultured astrocytes, and suggest roles for astrocytic HIF-1 expression and VEGF release which may influence the function of surrounding cells and vasculature during oxidative stress-related brain diseases.