Mitochondria and hypoxia-induced gene expression mediated by hypoxia-inducible factors.

The influence of mitochondrial activity on gene expression programs, particularly those involved in neuroprotection and repair, is likely to play an important role in the pathophysiology of neurodegenerative diseases. One such gene expression program is activated by the cellular pathway that senses a decrease in optimal oxygen levels and leads to activation of a family of transcriptional activators called hypoxia-inducible factors (HIFs). HIFs are members of the bHLH-PAS family of transcription factors and are heterodimers composed of HIF-alpha and HIF-beta (also known as aryl hydrocarbon receptor nuclear translocator) subunits that bind to canonical DNA sequences (hypoxia-regulated elements) in the promoters or enhancers of target genes. HIFs activate the expression of more than a hundred genes encoding proteins that regulate cell metabolism, survival, angiogenesis, vascular tone, hematopoiesis, and other functions. There is considerable evidence showing a bidirectional crosstalk between mitochondrial signals and HIF activity. For instance, mitochondrial reactive oxygen species and metabolic substrates from the tricarboxylic acid cycle are implicated in the regulation of the HIF pathway. Conversely, HIF activity leads to the expression of target genes that influence mitochondrial function. In this chapter we will review the complex interactions between mitochondria and the HIF pathway and we will discuss the relevance of this interaction for metabolic adaptation to hypoxia.

The brain in the age of old: the hippocampal formation is targeted differentially by diseases of late life.

OBJECTIVE: To rely on the anatomical organization of the hippocampal formation in understanding whether and how late-life diseases such as diabetes and stroke contribute to age-related cognitive decline. METHODS: Magnetic resonance imaging (MRI) was used to document brain infarcts and to generate high-resolution functional maps of the hippocampal formation in 240 community-based nondemented elders (mean age, 79.7 years) who received a comprehensive medical evaluation. Sixty participants had type 2 diabetes mellitus, whereas 74 had MRI-documented brain infarcts, and the first analysis was designed to pinpoint hippocampal subregions differentially linked to each disorder. Then, guided by the results, additional functional MRI studies in aging rhesus monkeys and mice were used to test proposed mechanisms of dysfunction. RESULTS: Although both diabetes and brain infarcts were associated with hippocampal dysfunction, each was linked to separate hippocampal subregions, suggesting distinct underlying mechanisms. The hippocampal subregion linked to diabetes implicated blood glucose as a pathogenic mechanism, a hypothesis confirmed by imaging aging rhesus monkeys and a mouse model of diabetes. The hippocampal subregion linked to infarcts suggested transient hypoperfusion as a pathogenic mechanism, a hypothesis provisionally confirmed by comparing anatomical patterns across subjects with infarcts in different vascular territories. INTERPRETATION: Taken together with previous findings, these results clarify how diseases of late life differentially target the hippocampal formation, identify elevations in blood glucose as a contributing cause of age-related memory decline, and suggest specific interventions that can preserve cognitive health.

Hypoxia-inducible factor-1 mediates neuronal expression of the receptor for advanced glycation end products following hypoxia/ischemia.

Activation of the receptor for advanced glycation endproducts (RAGE) by its multiple ligands can trigger diverse signaling pathways with injurious or pro-survival consequences. In this study, we show that Rage mRNA and protein levels were stimulated in the mouse brain after experimental stroke and systemic hypoxia. In both cases, RAGE expression was primarily associated with neurons. Activation of RAGE-dependent pathway(s) post-ischemia appears to have a neuroprotective role because mice genetically deficient for RAGE exhibited increased infarct size 24 h after injury. Up-regulation of RAGE expression was also observed in primary neurons subjected to hypoxia or oxygen-glucose deprivation, an in vitro model of ischemia. Treatment of neurons with low concentrations of S100B decreased neuronal death after oxygen-glucose deprivation, and this effect was abolished by a neutralizing antibody against RAGE. Conversely, high concentrations of exogenous S100B had a cytotoxic effect that seems to be RAGE-independent. As an important novel finding, we demonstrate that hypoxic stimulation of RAGE expression is mediated by the transcription factor hypoxia-inducible factor-1. This conclusion is supported by the finding that HIF-1alpha down-regulation by Cre-mediated excision drastically decreased RAGE induction by hypoxia or desferrioxamine. In addition, we showed that the mouse RAGE promoter region contains at least one functional HIF-1 binding site, located upstream of the proposed transcription start site. A luciferase reporter construct containing this RAGE promoter fragment was activated by hypoxia, and mutation at the potential HIF-1 binding site decreased hypoxia-dependent promoter activation. Specific binding of HIF-1 to this putative HRE in hypoxic cells was detected by chromatin immunoprecipitation assay.

Neuron-specific inactivation of the hypoxia inducible factor 1 alpha increases brain injury in a mouse model of transient focal cerebral ischemia.

In the present study, we show a biphasic activation of hypoxia inducible factor 1alpha (HIF-1) after stroke that lasts for up to 10 d, suggesting the involvement of the HIF pathway in several aspects of the pathophysiology of cerebral ischemia. We provide evidence that HIF-1-mediated responses have an overall beneficial role in the ischemic brain as indicated by increased tissue damage and reduced survival rate of mice with neuron-specific knockdown of HIF-1alpha that were subjected to transient focal cerebral ischemia. In addition, we demonstrated that drugs known to activate HIF-1 in cultured cells as well as in vivo had neuroprotective properties in this model of cerebral ischemia. This protective effect was significantly attenuated but not completely abolished in neuron-specific HIF-1alpha-deficient mice, suggesting that alternative mechanisms of neuroprotection are also implicated. Last, our study showed that hypoxia-induced tolerance to ischemia was preserved in neuron-specific HIF-1alpha-deficient mice, indicating that the neuroprotective effects of hypoxic preconditioning do not depend on neuronal HIF-1 activation.

The transcriptional activator hypoxia inducible factor 2 (HIF-2/EPAS-1) regulates the oxygen-dependent expression of erythropoietin in cortical astrocytes.

In the ischemic or hypoxic brain, astrocytes appear to be one of the main sources of erythropoietin (EPO). In this study, we investigated the differential contribution of hypoxia inducible factor (HIF) isoforms to the regulation of hypoxic EPO expression in cultured astrocytes. In addition, using an in vitro model of oxygen-glucose deprivation (OGD), we studied the role of HIF-1alpha and HIF-2alpha in the generation of paracrine protective signals by astrocytes that modulate the survival of neurons exposed to OGD. Expression of HIF-1alpha or HIF-2alpha was abrogated by infecting astrocytes with lentiviral particles encoding small interference RNA specific for HIF-1alpha or HIF-2alpha (siHIF-1alpha or siHIF-2alpha). Astrocytes infected with siHIF-1alpha showed abrogated hypoxic induction of vascular endothelial growth factor (VEGF) and lactate dehydrogenase (LDH) but normal EPO induction. In contrast, reduction of HIF-2alpha expression by siHIF-2alpha led to a drastic decrease of EPO hypoxic expression, but it did not affect LDH or VEGF upregulation. To further test whether HIF-2 is sufficient to drive EPO upregulation, we expressed oxygen-insensitive mutant forms of HIF-1alpha (mtHIF-1alpha) (P402A/P577A) and HIF-2alpha (mtHIF-2alpha) (P405A/P530A). Expression of mtHIF-2alpha but not mtHIF-1alpha in normoxic astrocytes resulted in a significant upregulation of EPO mRNA and protein. Accordingly, HIF-2alpha but not HIF-1alpha was found to be associated with the EPO hypoxia-response element by a chromatin immunoprecipitation assay. Interestingly, conditioned medium from astrocytes challenged by sublethal OGD improved neuronal survival to OGD; however, this effect was abolished during the downregulation of astrocytic HIF-2alpha using siHIF-2alpha. These results indicate that HIF-2alpha mediates the transcriptional activation of EPO expression in astrocytes, and this pathway may promote astrocytic paracrine-dependent neuronal survival during ischemia.

Structural and functional adaptation to hypoxia in the rat brain.

Chronic exposure to a hypoxic environment leads to structural and functional adaptations in the rat brain. One significant adaptation is a decrease in intercapillary distances through a near doubling of the capillary density, which begins after about 1 week of hypoxic exposure and is completed by 3 weeks. Hypoxic angiogenesis is controlled by activation of downstream genes by Hypoxia Inducible Factor-1 and Angiopoietin-2. The processes that increase capillary density are reversible upon restoration of the ambient oxygen concentration. Capillary regression, which also occurs over a 3-week period, is accomplished through activation of apoptosis. The implication from these observations is that the brain naturally functions in a low, but controlled, oxygen environment. Acute imbalances in oxygen delivery and metabolic demand are addressed through changes in blood flow; persistent imbalances activate mechanisms that adjust capillary density. The mechanisms that control these processes decline with age.

Hypoxic regulation of angiopoietin-2 expression in endothelial cells.

Exposure of endothelial cells to hypoxia-induced angiopoietin-2 (Ang2) expression. The increase in Ang2 mRNA levels occurred by transcriptional regulation and by post-transcriptional increase in mRNA stability. Induction of Ang2 mRNA resulted in an increase of intracellular and secreted Ang2 protein levels. Since the transcriptional regulation of several genes involved in angiogenesis during hypoxia is mediated by hypoxia-inducible factor-1 (HIF-1), it was conceivable that Ang2 expression might be regulated by the same oxygen-dependent mechanism. However, our data showed that pharmacological HIF inducers, CoCl(2) and DFO, did not affect Ang2 expression. Moreover, HIF-1-deficient hepatoma cell (Hepa1 c4) and its wild-type counterpart (Hepa1 c1c4) up-regulates Ang2 during hypoxia. These results indicated that hypoxia-driven Ang2 expression may be independent of the HIF pathway. Using neutralizing VEGF antibody or pharmacological inhibitors of VEGF receptors, we showed that hypoxia-induced VEGF participates but could not account completely for Ang2 expression during hypoxia. In addition, hypoxia elicited an increase of cyclooxygenase-2 (COX-2) expression and a parallel increase in prostanglandin E(2) (PGE(2)) and prostacyclin (PGI(2)) production. COX-2 inhibitors decreased the hypoxic induction of Ang2 and the hypoxic induction of PGE(2) and PGI(2) in a dose-dependent manner. Similarly, COX-2 but not COX-1 antisense treatment decreased hypoxic induction of Ang2 expression, and this effect was reversed by exogenous PGE(2). Finally, exogenous PGE(2) and PGI(2) were able to stimulate Ang2 under normoxic conditions. These findings suggest that COX-2-dependent prostanoids may play an important role in the regulation of hypoxia-induced Ang2 expression.

Angiopoietin-2 and rat brain capillary remodeling during adaptation and deadaptation to prolonged mild hypoxia.

Angiogenesis is a crucial component of rat brain adaptation to prolonged hypoxia, but it is not known whether this structural change is permanent or reversed on return to normoxia. Also, the intrinsic mechanisms controlling brain microvascular plasticity in response to oxygen availability remains unclear. Our results indicate that capillary density in the rat cerebral cortex increased by 60% after 3 wk of hypoxia and that it progressively decreased to prehypoxic values after 3 wk of normoxic recovery (deadaptation). Angiopoietin-2 (Ang2) expression in the capillary endothelium was induced between 6 h and 14 days of hypoxia but fell to control levels at 21 days of hypoxia. During deadaptation, Ang2 levels were elevated at 1-14 days but decreased to baseline at 21 days. In contrast, the constitutive expression of Ang1 and Tie2 was not affected during hypoxia or deadaptation. TUNEL-positive endothelial cells and caspase-3 activation were observed at 7 and 14 days of deadaptation. These data suggest that Ang2 might modulate both angiogenesis and vascular regression in the rat brain and that capillary regression occurring during deadaptation involves activation of apoptosis.

Role of nitric oxide in the regulation of HIF-1alpha expression during hypoxia.

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor consisting of HIF-1alpha and HIF-1beta subunits, controls the expression of a large number of genes involved in the regulation of cellular responses to reduced oxygen availability. The oxygen-regulated subunit, HIF-1alpha, is stabilized in cells exposed to hypoxia. The regulation of hypoxic responses by nitric oxide (NO) is believed to have wide pathophysiological relevance, thus we investigated whether NO affects HIF-1 activation in hypoxic cells. Here we show that NO generated from NO donors prevented HIF-1alpha hypoxic accumulation in Hep 3B and PC-12 cells. Addition of a glutathione analog or peroxynitrite scavengers prevented the NO-induced inhibition of HIF-1alpha accumulation in both cell lines. Exposure to NO was associated with inhibition of mitochondrial electron transport and compensatory glycolysis, which maintained normal cellular ATP content. Succinate, a Krebs cycle intermediate and respiratory chain substrate, restored HIF-1alpha hypoxic induction in the cells, suggesting involvement of mitochondria in regulation of HIF-1alpha accumulation during hypoxia. Regulation of HIF-1alpha by NO is an additional important mechanism by which NO might modulate cellular responses to hypoxia in mammalian cells.

Inhibitors of mitochondrial complex I attenuate the accumulation of hypoxia-inducible factor-1 during hypoxia in Hep3B cells.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that regulates transcriptional activation of several genes that are responsive to oxygen lack, including erythropoietin, vascular endothelial growth factor, various glycolytic enzymes and the GLUT-1 glucose transporter. Because mitochondria have been postulated to be involved in the regulation of HIF-1, we tested the effects of mitochondrial electron transport chain complex I inhibitors, rotenone and 1-methyl-4-phenylpiridinium (MPP(+)), on hypoxic-induced accumulation of HIF-1 alpha, the regulated component of the dimer. We found, consistent with our previous observations in Cath.a and PC12 cells, that rotenone and MPP(+) attenuated the HIF-1 alpha hypoxic response. Thus, it can be concluded that an intact, functional mitochondrial respiratory chain is required for HIF-1 alpha accumulation.