The role of HIF in cobalt-induced ischemic tolerance.

Understanding the endogenous survival pathways induced by ischemic tolerance may yield targets for neuroprotection from stroke. One well-studied pathway reported to be evoked by preconditioning stimuli is the transcription factor HIF (hypoxia-inducible factor). However, whether HIF induction by ischemic insults is neuroprotective or toxic is still unclear. We examined the ability of three prolyl-hydroxylase inhibitors, which induce HIF, to protect hippocampal cultures from oxygen-glucose deprivation. Hippocampal cultures were exposed to ischemic preconditioning or various concentrations of cobalt chloride, deferoxamine (DFO) or dimethyloxylalyglycine (DMOG), prior to lethal oxygen-glucose deprivation (OGD). Cell survival of neurons and astrocytes was determined with dual-label immunocytochemistry. The induction of HIF targets was assessed in mixed as well as astrocyte-enriched cultures. Ischemic preconditioning, as well as low concentrations of cobalt and DFO, enhanced the survival of neurons following OGD. However, DMOG exacerbates OGD-induced neuronal death. At low concentrations, all three prolyl-hydroxylase (PHD) inhibitors increased the survival of astrocytes. Neuroprotective concentrations of cobalt induced the transcription of the cytokine erythropoietin (EPO) in astrocyte cultures. In addition, pretreatment with recombinant human erythropoietin (rH-EPO) also protected neurons from OGD. Our data suggest that HIF-induced EPO, released from astrocytes, protects neurons from OGD. However, the three PHD inhibitors each exhibited different neuroprotective profiles at low concentrations, suggesting that not all PHD inhibitors are created equal. The protective effects at low doses is reminiscent of HIF involvement in ischemic tolerance, in which sub-lethal insults induce HIF pathways resulting in neuroprotection, whereas the high-dose toxicity suggests that over-activation of HIF is not always protective. Therefore, the choice of inhibitor and dose may determine the clinical utility of these compounds. Deferoxamine exhibited little toxicity even at higher doses, and therefore appears a promising candidate for clinical use.