Guoxiang Yuan

Heterozygous HIF-1α deficiency impairs carotid body-mediated systemic responses and reactive oxygen species generation in mice exposed to intermittent hypoxia

Chronic intermittent hypoxia (CIH) occurs in patients with sleep apnoea and has adverse effects on multiple physiological functions. Previous studies have shown that reflexes arising from carotid bodies mediate CIH‐evoked cardio‐respiratory responses, and reactive oxygen species (ROS) play important roles in eliciting systemic responses to CIH. Very little is known about the molecular mechanisms underlying CIH. The transcriptional activator hypoxia‐inducible factor‐1 (HIF‐1) mediates a broad range of cellular and systemic responses to hypoxia, and HIF‐1 is activated in cell cultures exposed to IH. In the present study we examined whether CIH activates HIF‐1 and if so whether it contributes to cardio‐respiratory responses and ROS generation in mice. Experiments were performed on male littermate wild‐type (WT) and heterozygous (HET) mice partially deficient in HIF‐1α, the O2 regulated subunit of the HIF‐1 complex. Both groups of mice were exposed to either 10 days of CIH (15 s of hypoxia followed by 5 min of normoxia, 9 episodes h−1, 8 h day−1) or to 10 days of 21% O2 (controls). Carotid body response to hypoxia was augmented, and acute intermittent hypoxia (AIH) induced sensory long‐term facilitation (sLTF) of the chemoreceptor activity in CIH‐exposed WT mice. In striking contrast, hypoxic sensory response was unaffected and AIH was ineffective in eliciting sLTF in CIH‐exposed HET mice. Analysis of cardio‐respiratory responses in CIH‐exposed WT mice revealed augmented hypoxic ventilatory response, LTF of breathing, elevated blood pressures and increased plasma noradrenaline. In striking contrast these responses were either absent or attenuated in HET mice exposed to CIH. In CIH‐exposed WT mice, ROS were elevated and this response was absent in HET mice. Manganese (III) tetrakis(1‐methyl‐4‐pyridyl) porphyrin pentachloride, a potent scavenger of superoxide, not only prevented CIH‐induced increases in ROS but also CIH‐evoked HIF‐1α up‐regulation in WT mice. These results indicate that: (a) HIF‐1 activation is critical for eliciting CIH‐induced carotid body‐mediated cardio‐respiratory responses; (b) CIH increases ROS; and (c) the effects of CIH involve complex positive interactions between HIF‐1 and ROS.

Induction of HIF-1α expression by intermittent hypoxia: Involvement of NADPH oxidase, Ca2+ signaling, prolyl hydroxylases, and mTOR

Sleep‐disordered breathing with recurrent apnea (periodic cessation of breathing) results in chronic intermittent hypoxia (IH), which leads to cardiovascular and respiratory pathology. Molecular mechanisms underlying IH‐evoked cardio‐respiratory co‐morbidities have not been delineated. Mice with heterozygous deficiency of hypoxia‐inducible factor 1α (HIF‐1α) do not develop cardio‐respiratory responses to chronic IH. HIF‐1α protein expression and HIF‐1 transcriptional activity are induced by IH in PC12 cells. In the present study, we investigated the signaling pathways associated with IH‐evoked HIF‐1α accumulation. PC12 cells were exposed to aerobic conditions (20% O2) or 60 cycles of IH (30 sec at 1.5% O2 followed by 5 min at 20% O2). Our results show that IH‐induced HIF‐1α accumulation is due to increased generation of ROS by NADPH oxidase. We further demonstrate that ROS‐dependent Ca2+ signaling pathways involving phospholipase Cγ (PLCγ) and protein kinase C activation are required for IH‐evoked HIF‐1α accumulation. IH leads to activation of mTOR and S6 kinase (S6K) and rapamycin partially inhibited IH‐induced HIF‐1α accumulation. IH also decreased hydroxylation of HIF‐1α protein and anti‐oxidants as well as inhibitors of Ca+2 signaling prevented this response. Thus, both increased mTOR‐dependent HIF‐1α synthesis and decreased hydroxylase‐dependent HIF‐1α degradation contribute to IH‐evoked HIF‐1α accumulation. Following IH, HIF‐1α, and phosphorylated mTOR levels remained elevated during 90 min of re‐oxygenation despite re‐activation of prolyl hydroxylase. Rapamycin or cycloheximide, blocked increased HIF‐1α levels during re‐oxygenation indicating that mTOR‐dependent protein synthesis is required for the persistent elevation of HIF‐1α levels during re‐oxygenation. J. Cell. Physiol. 217: 674–685, 2008.

NADPH Oxidase Is Required for the Sensory Plasticity of the Carotid Body by Chronic Intermittent Hypoxia

Respiratory motoneuron response to hypoxia is reflex in nature and carotid body sensory receptor constitutes the afferent limb of this reflex. Recent studies showed that repetitive exposures to hypoxia evokes long term facilitation of sensory nerve discharge (sLTF) of the carotid body in rodents exposed to chronic intermittent hypoxia (CIH). Although studies with anti-oxidants suggested the involvement of reactive oxygen species (ROS)-mediated signaling in eliciting sLTF, the source of and the mechanisms associated with ROS generation have not yet been investigated. We tested the hypothesis that ROS generated by NADPH oxidase (NOX) mediate CIH-evoked sLTF. Experiments were performed on ex vivo carotid bodies from rats and mice exposed either to 10 d of CIH or normoxia. Acute repetitive hypoxia evoked a ∼12-fold increase in NOX activity in CIH but not in control carotid bodies, and this effect was associated with upregulation of NOX2 mRNA and protein, which was primarily localized to glomus cells of the carotid body. sLTF was prevented by NOX inhibitors and was absent in mice deficient in NOX2. NOX activation by CIH required 5-HT release and activation of 5-HT2 receptors coupled to PKC signaling. Studies with ROS scavengers revealed that H2O2 generated from O2·− contributes to sLTF. Priming with H2O2 elicited sLTF of carotid bodies from normoxic control rats and mice, similar to that seen in CIH-treated animals. These observations reveal a novel role for NOX-induced ROS signaling in mediating sensory plasticity of the carotid body.

Role of oxidative stress in intermittent hypoxia‐induced immediate early gene activation in rat PC12 cells

Intermittent hypoxia (IH) occurs in many pathophysiological conditions. The molecular mechanisms associated with IH, however, have received little attention. Previous studies have reported that the c‐fos gene via formation of activator protein‐1 (AP‐1) transcription factor contributes to adaptive responses to continuous hypoxia. In the present study, using a cell culture model we examined whether IH activates c‐fos and AP‐1 and if so, by what mechanisms. Experiments were performed on rat phaeochromocytoma cells exposed to 21% O2 (normoxia) or 60 and 120 cycles of IH, each cycle consisting 15 s of hypoxia followed by 4 min of normoxia. IH resulted in a significant elevation of c‐fos mRNA as well as transcriptional activation. IH was more potent and induced a longer lasting activation of c‐fos than comparable cumulative duration of continuous hypoxia. IH increased AP‐1 activity and tyrosine hydroxylase (TH) mRNA, an AP‐1‐regulated downstream gene, and these effects were prevented by antisense c‐fos. Superoxide dismutase mimetic, a potent scavenger of superoxide anions, prevented IH‐induced c‐fos, AP‐1 and TH activations. IH increased superoxide anion levels in mitochondria as evidenced by decreased aconitase enzyme activity and increased levels of hydrogen peroxide, a stable dismutated product of superoxide anions. Complex I of the mitochondrial electron transport chain was markedly inhibited in IH exposed cells. Pharmacological inhibitors of complex I mimicked the effects of IH during normoxia and occluded the effects of IH on c‐fos activation, suggesting the involvement of the mitochondrial electron transport chain in the generation of superoxide anions during IH. These results suggest IH‐induced c‐fos‐mediated transcriptional activation involves oxidative stress.

Hypoxia-Inducible Factor 1 Mediates Increased Expression of NADPH Oxidase-2 in Response to Intermittent Hypoxia

Sleep‐disordered breathing with recurrent apnea is associated with intermittent hypoxia (IH). Cardiovascular morbidities caused by IH are triggered by increased generation of reactive oxygen species (ROS) by pro‐oxidant enzymes, especially NADPH oxidase‐2 (Nox2). Previous studies showed that (i) IH activates hypoxia‐inducible factor 1 (HIF‐1) in a ROS‐dependent manner and (ii) HIF‐1 is required for IH‐induced ROS generation, indicating the existence of a feed‐forward mechanism. In the present study, using multiple pharmacological and genetic approaches, we investigated whether IH‐induced expression of Nox2 is mediated by HIF‐1 in the central and peripheral nervous system of mice as well as in cultured cells. IH increased Nox2 mRNA, protein, and enzyme activity in PC12 pheochromocytoma cells as well as in wild‐type mouse embryonic fibroblasts (MEFs). This effect was abolished or attenuated by blocking HIF‐1 activity through RNA interference or pharmacologic inhibition (digoxin or YC‐1) or by genetic knockout of HIF‐1α in MEFs. Increasing HIF‐1α expression by treating PC 12 cells with the iron chelator deferoxamine for 20 h or by transfecting them with HIF‐1alpha expression vector increased Nox2 expression and enzyme activity. Exposure of wild‐type mice to IH (8 h/day for 10 days) up‐regulated Nox2 mRNA expression in brain cortex, brain stem, and carotid body but not in cerebellum. IH did not induce Nox2 expression in cortex, brainstem, carotid body, or cerebellum of Hif1a+/− mice, which do not manifest increased ROS or cardiovascular morbidities in response to IH. These results establish a pathogenic mechanism linking HIF‐1, ROS generation, and cardiovascular pathology in response to IH. J. Cell. Physiol. 226: 2925–2933, 2011.

Intermittent hypoxia degrades HIF-2α via calpains resulting in oxidative stress: Implications for recurrent apnea-induced morbidities

Intermittent hypoxia (IH) occurs in many pathological conditions including recurrent apneas. Hypoxia-inducible factors (HIFs) 1 and 2 mediate transcriptional responses to low O2. A previous study showed that HIF-1 mediates some of the IH-evoked physiological responses. Because HIF-2α is an orthologue of HIF-1α, we examined the effects of IH on HIF-2α, the O2-regulated subunit expression, in pheochromocytoma 12 cell cultures. In contrast to the up-regulation of HIF-1α, HIF-2α was down-regulated by IH. Similar down-regulation of HIF-2α was also seen in carotid bodies and adrenal medullae from IH-exposed rats. Inhibitors of calpain proteases (ALLM, ALLN) prevented IH-evoked degradation of HIF-2α whereas inhibitors of prolyl hydroxylases or proteosome were ineffective. IH activated calpain proteases and down-regulated the endogenous calpain inhibitor calpastatin. IH-evoked HIF-2α degradation led to inhibition of SOD2 transcription, resulting in oxidative stress. Over-expression of transcriptionally active HIF-2α prevented IH-evoked oxidative stress and restored SOD2 activity. Systemic treatment of IH-exposed rats with ALLM rescued HIF-2α degradation and restored SOD2 activity, thereby preventing oxidative stress and hypertension. These observations demonstrate that, unlike continuous hypoxia, IH leads to down-regulation of HIF-2α via a calpain-dependent signaling pathway and results in oxidative stress as well as autonomic morbidities.

Transcriptional responses to intermittent hypoxia

Recurrent apneas are characterized by transient repetitive cessations of breathing (two breaths duration or longer) resulting in periodic decreases in arterial blood PO2 or chronic intermittent hypoxia (IH). Patients with recurrent apneas and experimental animals exposed to chronic IH exhibit cardio-respiratory morbidities. The purpose of this article is to highlight the current information on the transcriptional mechanisms associated with chronic IH. Studies on rodents and cell cultures have shown that IH activates a variety of transcription factors including the hypoxia-inducible factor-1 (HIF-1), c-fos (immediate early gene), nuclear factor of activated T-cells (NFAT), and nuclear factor kB (NF-kB). The signaling pathways associated with transcriptional activation associated with IH differ from continuous hypoxia (CH). Compared to same duration and intensity of CH, IH is more potent in activating HIF-1 and c-fos and also results in long-lasting accumulation of HIF-1alpha and c-fos mRNA, a phenomenon that was not seen with CH. IH-evoked transcriptional activation by HIF-1, c-fos as well as the resulting activator protein-1 (AP-1) requires reactive oxygen species (ROS)-mediated signaling and involves complex feed forward interactions between HIF-1 and ROS. Chronic IH-evoked cardio-respiratory responses are absent in Hif-1alpha+/- mice, and hypertension elicited by chronic IH is absent in mice lacking NFAT3c. These studies indicate that cardiorespiratory responses to chronic IH depend on complex interactions between various transcription factors resulting in alterations in several down stream genes and their protein products.

Epigenetic regulation of hypoxic sensing disrupts cardiorespiratory homeostasis

Recurrent apnea with intermittent hypoxia is a major clinical problem in preterm infants. Recent studies, although limited, showed that adults who were born preterm exhibit increased incidence of sleep-disordered breathing and hypertension, suggesting that apnea of prematurity predisposes to autonomic dysfunction in adulthood. Here, we demonstrate that adult rats that were exposed to intermittent hypoxia as neonates exhibit exaggerated responses to hypoxia by the carotid body and adrenal chromaffin cells, which regulate cardio-respiratory function, resulting in irregular breathing with apneas and hypertension. The enhanced hypoxic sensitivity was associated with elevated oxidative stress, decreased expression of genes encoding antioxidant enzymes, and increased expression of pro-oxidant enzymes. Decreased expression of the Sod2 gene, which encodes the antioxidant enzyme superoxide dismutase 2, was associated with DNA hypermethylation of a single CpG dinucleotide close to the transcription start site. Treating neonatal rats with decitabine, an inhibitor of DNA methylation, during intermittent hypoxia exposure prevented oxidative stress, enhanced hypoxic sensitivity, and autonomic dysfunction. These findings implicate a hitherto uncharacterized role for DNA methylation in mediating neonatal programming of hypoxic sensitivity and the ensuing autonomic dysfunction in adulthood.

Hypoxia-inducible factor 2α (HIF-2α) heterozygous-null mice exhibit exaggerated carotid body sensitivity to hypoxia, breathing instability, and hypertension

Cardiorespiratory functions in mammals are exquisitely sensitive to changes in arterial O2 levels. Hypoxia-inducible factors (e.g., HIF-1 and HIF-2) mediate transcriptional responses to reduced oxygen availability. We demonstrate that haploinsufficiency for the O2-regulated HIF-2α subunit results in augmented carotid body sensitivity to hypoxia, irregular breathing, apneas, hypertension, and elevated plasma norepinephrine levels in adult Hif-2α+/− mice. These dysregulated autonomic responses were associated with increased oxidative stress and decreased mitochondrial electron transport chain complex I activity in adrenal medullae as a result of decreased expression of major cytosolic and mitochondrial antioxidant enzymes. Systemic administration of a membrane-permeable antioxidant prevented oxidative stress, normalized hypoxic sensitivity of the carotid body, and restored autonomic functions in Hif-2α+/− mice. Thus, HIF-2α–dependent redox regulation is required for maintenance of carotid body function and cardiorespiratory homeostasis.

5‐HT evokes sensory long‐term facilitation of rodent carotid body via activation of NADPH oxidase

5‐Hydroxytryptamine (5‐HT) evokes long‐term activation of neuronal activity in the nervous system. Carotid bodies, the sensory organs for detecting arterial oxygen, express 5‐HT. In the present study we examined whether 5‐HT evokes sensory long‐term facilitation (LTF) of the carotid body, and if so by what mechanism(s). Experiments were performed on anaesthetized adult rats and mice. Sensory activity was recorded from carotid bodies ex vivo. Spaced (3 × 15 s of 100 nm at 5 min intervals) but not mass (300 nm, 45 s) application of 5‐HT elicited LTF, whereas both modes of 5‐HT application evoked initial sensory excitation of the carotid bodies in rats. Ketanserin, a 5‐HT2 receptor antagonist prevented sensory LTF but not the initial sensory excitation. Spaced application of 5‐HT activated protein kinase C (PKC) as evidenced by increased phosphorylations of PKC at Thr514 and myristoylated alanine‐rich C kinase substrate (MARCKS) and these effects were abolished by ketanserin as well as bisindolylmaleimide (Bis‐1), an inhibitor of PKC. Bis‐1 prevented 5‐HT‐evoked sensory LTF. 5‐HT increased NADPH oxidase activity and PKC‐dependent phosphorylation of p47phox subunit of the oxidase complex. NADPH oxidase inhibitors (apocynin and diphenyl iodinium), as well as an anti‐oxidant (N‐acetyl cysteine), prevented 5‐HT‐evoked sensory LTF. Mice deficient in gp91phox, the membrane subunit of the NADPH oxidase complex, showed no sensory LTF, although responding to 5‐HT with initial afferent nerve activation, whereas both LTF and initial excitation by 5‐HT were seen in wild‐type mice. These results demonstrate that spaced but not mass application of 5‐HT elicits sensory LTF of the carotid body via activation of 5‐HT2 receptors, which involves a novel signalling mechanism coupled to PKC‐dependent activation of NADPH oxidase.