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 H(2)O(2) 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 H(2)O(2) injury treatment. Time course studies revealed that DFO and EDHB treatments alone induced sequential activation of HIF-1 signal transduction where nuclear HIF-1alpha protein accumulation was detected as early as 2h, followed by downstream upregulation of intracellular and released VEGF from 4h and 8h onwards, respectively. The protective effects of DFO and EDHB preconditioning against H(2)O(2) 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.
Molecular Neuropharmacology, Florey Neuroscience Institutes, University of Melbourne, VIC 3010, Australia.
Percy W Y
PW
Chu
Philip M
PM
Beart
Nicole M
NM
Jones
D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D019610:Cytoprotection / Q000187:drug effects; D003676:Deferoxamine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000037:antagonists & inhibitors / Q000633:toxicity; D062385:Hydroxybenzoates / Q000037:antagonists & inhibitors / Q000494:pharmacology; D051793:Hypoxia-Inducible Factor 1 / Q000378:metabolism*; D007191:Indazoles / Q000494:pharmacology; D051379:Mice; D018384:Oxidative Stress / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D004791:Enzyme Inhibitors; D062385:Hydroxybenzoates; D051793:Hypoxia-Inducible Factor 1; D007191:Indazoles; D042461:Vascular Endothelial Growth Factor A; C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; C018063:ethyl protocatechuate; D003513:Cycloheximide; D006861:Hydrogen Peroxide; D003676:Deferoxamine