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Role of the Transcription Factor HIF‐2α in Promoting the Ischemia‐Tolerant Phenotype in Cerebral Endothelial Cells
Author(s) -
Pan Jason J,
Zhang Yunhong,
Gidday Jeffrey M.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1276-a
Subject(s) - gene knockdown , viability assay , transfection , small interfering rna , rna interference , biology , endothelial stem cell , transcription factor , cell culture , hypoxia inducible factors , microbiology and biotechnology , gene , rna , in vitro , biochemistry , genetics
Brief exposure to hypoxia, known as hypoxic preconditioning (HPC), prompts adaptive cellular responses that induce a state of ischemic tolerance, or an increased resistance to ischemic injury. We hypothesized that the expression of hypoxia‐inducible factor‐2α (HIF‐2α), a transcription factor preferentially expressed in endothelial cells, is increased by HPC and activates endothelial cell survival genes. To provide causal evidence for the contributory role of HIF‐2α in HPC‐induced protection, a specific knockdown model was developed, utilizing HIF‐2α RNA interference (RNAi) in a mouse brain endothelial cell line (bEnd.3). Small interfering RNA (siRNA) oligonucleotides were delivered to the endothelial cells by a liposome transfection approach. Cy3‐labelled scrambled siRNA identified a high transfection efficiency and minimal cytotoxicity. Cells were preconditioned in a hypoxic incubator (1% O2, 5% CO2, and 94% N2) for 6 hours; after a 16‐h period of reoxygenation, cells were then exposed to oxygen‐glucose deprivation (OGD) for 24 h. MTT viability assays 24 h later confirmed that HPC protected endothelial cells from OGD injury, but revealed that endothelial cell viability of the HIF‐2α RNAi transfected group was significantly lower than that of matched preconditioned controls. HPC‐induced increases in HIF‐2α, and HIF‐2α knockdown by RNAi, was confirmed by immunoblotting. These findings indicate that genes transactivated by HIF‐2α, secondary to HPC, contribute to the protection of cerebral endothelium from ischemia. Experiments designed to identify the HIF‐2α target genes underlying endothelial ischemic tolerance are underway.