Abstract
Cells universally adapt to ischemic conditions by turning on a transcription factor hypoxia-inducible factor (HIF), in which its role is known to differ widely across many different types of cells. Given that microglia have been reported as an essential mediator of neuroinflammation in many brain diseases, we examined the role of HIF in microglia in the progression of an acute phase of ischemic stroke by challenging our novel strains of myeloid-specific Hif-1α or Hif-2α knockout (KO) mice created by Cre-loxP system via middle cerebral artery occlusion (MCAO). We observed that Hif-1α but not Hif-2α KO mice exhibited an improved recovery compared to wild-type (WT) mice determined by behavioral tests. Immunostaining analyses revealed that there were increased numbers of both mature and immature neurons while microglia and apoptotic cells were significantly decreased in the dentate gyrus of Hif-1α KO mice following MCAO. By isolating microglia with fluorescence-activated cell sorter, we found that HIF-1α-deficient microglia were impaired in phagocytosis, reactive oxygen species (ROS) production, and tumor necrosis factor-α (TNF-α) secretion. We further observed a significant decrease in the expression of Cd36 and milk fat globule-epidermal growth factor 8 (Mfg-e8) genes, both of which contain hypoxia-responsive element (HRE). Knocking down either of these genes in BV2 microglial cells was sufficient to abrogate HIF-mediated increase in phagocytosis, production of intracellular ROS, or TNF-α secretion. Our results therefore suggest that HIF-1α in microglia is a novel therapeutic target to protect neuronal survival following an acute phase of ischemic stroke.
Highlights
Ischemic stroke occurs by blood clots obstructing the blood flow to the brain leading to permanent neuronal disabilities in the affected individuals [1]
Given that microglia have been reported as an essential mediator of neuroinflammation in many brain diseases, we examined the role of hypoxia-inducible factor (HIF) in microglia in the progression of an acute phase of ischemic stroke by challenging our novel strains of myeloid-specific Hif-1α or Hif-2α knockout (KO) mice created by Cre-loxP system via middle cerebral artery occlusion (MCAO)
We found in Hif-1α KO mice that Hif-1α gene deletion efficiency was approximately 70% in Iba-1-positive microglia isolated by fluorescence-activated cell sorting (FACS) (Supplementary Figure 1A)
Summary
Ischemic stroke occurs by blood clots obstructing the blood flow to the brain leading to permanent neuronal disabilities in the affected individuals [1]. Cells respond to ischemic conditions by activating hypoxia-inducible factor (HIF), a transcription factor regulating numerous genes involved in cellular survival, migration, metabolism, and www.impactjournals.com/oncotarget angiogenesis [2]. Three HIF-α isoforms (HIF-1α, HIF2α, and HIF-3α) have been identified where HIF-α form is being hydroxylated under well-oxygenated conditions by proline hydroxylases, which eventually becomes degraded by proteasome via von Hippel Lindau E3 ubiquitin ligase complex [3]. HIF-α cannot be hydroxylated due to the lack of oxygen inactivating proline hydroxylase activity [4]. HIF-α is stabilized in the cytoplasm where it interacts with HIF-1β, followed by translocation into the nucleus where it binds to hypoxiaresponsive elements (HREs) of HIF target genes [4]
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