Brain-specific knock-out of hypoxia-inducible factor-1alpha reduces rather than increases hypoxic-ischemic damage

Abstract

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays an essential role in cellular and systemic O(2) homeostasis by regulating the expression of genes important in glycolysis, erythropoiesis, angiogenesis, and catecholamine metabolism. It is also believed to be a key component of the cellular response to hypoxia and ischemia under pathophysiological conditions, such as stroke. To clarify the function of HIF-1alpha in the brain, we exposed adult mice with late-stage brain deletion of HIF-1alpha to hypoxic injuries. Contrary to expectations, the brains from the HIF-1alpha-deficient mice were protected from hypoxia-induced cell death. These surprising findings suggest that decreasing the level of HIF-1alpha can be neuroprotective. Gene chip expression analysis revealed that, contrary to expectations, the majority of hypoxia-dependent gene-expression changes were unaltered, whereas a specific downregulation of apoptotic genes was observed in the HIF-1alpha-deficient mice. Although the role of HIF-1alpha has been extensively characterized in vitro, in cancer models, and in chronic preconditioning paradigms, this is the first study to evaluate the role of HIF-1alpha in vivo in the brain in response to acute hypoxia/ischemia. Our data suggest, that in acute hypoxia, the neuroprotection found in the HIF-1alpha-deficient mice is mechanistically consistent with a predominant role of HIF-1alpha as proapoptotic and loss of function leads to neuroprotection. Furthermore, our data suggest that functional redundancy develops after excluding HIF-1alpha, leading to the preservation of gene expression regulating the majority of other previously characterized HIF-dependent genes.

Figure 1.

Quantitative results based on cell counting are graphed as the percentage of apoptotic cells in the heterozygous HIF-1α^+/Neo (n = 5) and WT (n = 9) CA1 region of the hippocampus. The abscissa indicates the genotype. The ordinate indicates the percentage of TUNEL-positive cells. Statistical significance was calculated using a Student's t test (^*p < 0.001). Error bars indicate SD.

Figure 2.

Efficiency of CRE recombinase-mediated deletion. A, Immunohistochemistry for CRE shows that neurons in the hippocampus (top right panel, white arrows) and cerebellar Purkinje cells (bottom right panel, white arrows) express CRE in the CAMCRE transgenic mouse (CAMCRE⁺). In contrast, no staining is detected in the control CRE^- mice (CAMCRE^-, top and bottom left panels). Scale bars, 500 μm. B, Southern blot analysis performed on DNA isolated from the cortex (lane A) and hippocampus (lane C) of an HIF-1α^F/F genotype mouse and from the cortex (lane B) and hippocampus (lane D) of an HIF-1α^Δ/Δ genotype mouse. The line labeled Flox indicates the undeleted 2.2 kb HIF-1αflox allele. The line labeled Δ indicates the 1.2 kb HIF-1α-deleted allele. Percentage of deletion is represented below the corresponding lanes. C, PCR fragments from DNA isolated from the cortex (lane A) and hippocampus (lane B) of an HIF-1α^Δ/Δ genotype mouse and from the cortex (lane C) of an HIF-1α^F/F mouse. The line labeled Flox indicates the undeleted 1 kb HIF-1α allele. The line labeled Δ indicates the 300 bp-deleted allele.

Figure 3.

Absence of HIF-1α is protective during global ischemia. A, A representation of acid fusin- and cresyl violet-stained sections from HIF-1α^F/F (wild-type) and HIF-1α^Δ/Δ (mutant) hippocampus shows a substantial decrease in neuronal damage in the HIF-1α^Δ/Δ mice. Arrows indicate regions of neuronal damage. B, Mean neuronal damage in the cortex and hippocampus (Hipp) in HIF-1α^F/F and HIF-1α^Δ/Δ mice after a 75 min BCCAo and 3 d reperfusion is shown. Ischemic neuronal damage was graded on a four-point scale basis. Neuronal damage in the cortex and hippocampus was significantly greater in HIF-1α^F/F (n = 6) versus HIF-1α^Δ/Δ (n = 13) mice. ^*p < 0.05; ^**p < 0.005. Sections were viewed at 2× magnification. The data were compared using a Student's t test.

Figure 4.

HIF-1α is not required for the classic transcriptional response to hypoxia. Venn diagram (A) of the number of differentially expressed genes and the direction of change in response to hypoxia in the hippocampus of HIF-1α^F/F animals, HIF-1α^Δ/Δ animals, or both. The differentially expressed genes were first identified for each of the genotypes; then the gene lists from each group were analyzed for unique and overlapping gene expression (see Materials and Methods). Of the four genes changed uniquely in the HIF-1α^F/F hippocampus after hypoxia, three genes were increased and one was decreased (Table 1). Of the 16 genes unique to the HIF-1α^Δ/Δ hippocampus after hypoxia, five increased and 11 decreased (Table 4). Of the 49 genes in common to both genotypes after hypoxia, 27 increased and 22 decreased (Table 2). One gene was oppositely expressed, showing increased expression in the HIF-1α^Δ/Δ hippocampus and decreased expression in the HIF-1α^F/F hippocampus after hypoxia (Table 5). ↑ or ↓ indicates direction change in response to hypoxia; (^* ↑ ↓) indicates that this gene is expressed in opposite directions in HIF-1α^F/F (↓) and HIF-1α^Δ/Δ (↑). B, Ingenuity pathway analyses are shown for the genes induced by hypoxia in common to both mutant and normal animals. Two networks were identified and are displayed graphically as nodes (genes/gene products) and edges (the biological relationships between the nodes). Of the 49 genes in common to both genotypes, 19 mapped to two specific networks and one connected to HIF-1α (^*p < 1.0 × 10^-8) and CDKN1A (p21) (^*p < 1.0 × 10^-19). C, Quantitative results are graphed as the biological process using the Gene Ontology Tree Machine program. The abscissa indicates the pathways interrogated using the program, and only cell death showed a significance of enrichment (^*p < 0.02). The ordinate indicates the number of genes observed in each category compared with the number of genes expected.