Shakil A. Khan

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.

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.

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.

Reactive oxygen species-dependent endothelin signaling is required for augmented hypoxic sensory response of the neonatal carotid body by intermittent hypoxia

We previously reported that intermittent hypoxia (IH) augments hypoxic sensory response (HSR) and increases the number of glomus cells in neonatal carotid bodies. In the present study, we tested the hypothesis that recruitment of endothelin-1 (ET-1) signaling by reactive oxygen species (ROS) plays a critical role in IH-evoked changes in neonatal carotid bodies. Experiments were performed on neonatal rats exposed either to 10 days of IH (P0-P10; 8 h/day) or to normoxia. IH augmented HSR of the carotid bodies ex vivo and resulted in hyperplasia of glomus cells. The effects of IH were associated with enhanced basal release of ET-1 under normoxia, sensitization of carotid body response to exogenous ET-1, and upregulation of ET(A) but not an ET(B) receptor mRNA without altering the ET-1 content. An ET(A) but not ET(B) receptor antagonist prevented augmented HSR by IH. ROS levels were elevated in carotid bodies from IH-treated rat pups as evidenced by increased levels of malondialdehyde. Systemic administration of manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 5 mg/kg ip), a scavenger of O(2)(*-), prevented IH-induced elevation of ROS, basal release of ET-1, upregulation of ET(A) mRNA, and augmented HSR. In striking contrast, MnTMPyP treatment had no significant effect on IH-induced hyperplasia of glomus cells. These results demonstrate that IH-evoked increase in HSR involve a ROS-mediated increase in basal ET-1 release and upregulation of ET(A) receptor mRNA.

NADPH Oxidase 2 Mediates Intermittent Hypoxia-Induced Mitochondrial Complex I Inhibition: Relevance to Blood Pressure Changes in Rats

Previous studies identified NADPH oxidases (Nox) and mitochondrial electron transport chain at complex I as major cellular sources of reactive oxygen species (ROS) mediating systemic and cellular responses to intermittent hypoxia (IH). In the present study, we investigated potential interactions between Nox and the mitochondrial complex I and assessed the contribution of mitochondrial ROS in IH-evoked elevation in blood pressure. IH treatment led to stimulus-dependent activation of Nox and inhibition of complex I activity in rat pheochromocytoma (PC)12 cells. After re-oxygenation, Nox activity returned to baseline values within 3 h, whereas the complex I activity remained downregulated even after 24 h. IH-induced complex I inhibition was prevented by Nox inhibitors, Nox2 but not Nox 4 siRNA, in cell cultures and was absent in gp91(phox-/Y) (Nox2 knock-out; KO) mice. Using pharmacological inhibitors, we show that ROS generated by Nox activation mobilizes Ca(2+) flux from the cytosol to mitochondria, leading to S-glutathionylation of 75- and 50-kDa proteins of the complex I and inhibition of complex I activity, which results in elevated mitochondrial ROS. Systemic administration of mito-tempol prevented the sustained but not the acute elevations of blood pressure in IH-treated rats, suggesting that mitochondrial-derived ROS contribute to sustained elevation of blood pressure.

Mutual antagonism between hypoxia-inducible factors 1α and 2α regulates oxygen sensing and cardio-respiratory homeostasis

Significance The carotid body (CB) chemosensory reflex and catecholamine secretion by the adrenal medulla (AM) are principal regulators of cardio-respiratory function during hypoxia, but the molecular mechanisms by which the CB and AM respond to hypoxia with changes in breathing and blood pressure are unknown. Hypoxia-inducible factor-1 (HIF-1) and HIF-2 mediate adaptive transcriptional responses to hypoxia. Herein, we demonstrate that a mutual functional antagonism between HIF-1 and HIF-2 plays a critical role in O2 sensing by establishing a dynamic balance that determines the proper redox set point in the CB and AM, which is essential for maintenance of cardio-respiratory homeostasis. Breathing and blood pressure are under constant homeostatic regulation to maintain optimal oxygen delivery to the tissues. Chemosensory reflexes initiated by the carotid body and catecholamine secretion from the adrenal medulla are the principal mechanisms for maintaining respiratory and cardiovascular homeostasis; however, the underlying molecular mechanisms are not known. Here, we report that balanced activity of hypoxia-inducible factor-1 (HIF-1) and HIF-2 is critical for oxygen sensing by the carotid body and adrenal medulla, and for their control of cardio-respiratory function. In Hif2α+/− mice, partial HIF-2α deficiency increased levels of HIF-1α and NADPH oxidase 2, leading to an oxidized intracellular redox state, exaggerated hypoxic sensitivity, and cardio-respiratory abnormalities, which were reversed by treatment with a HIF-1α inhibitor or a superoxide anion scavenger. Conversely, in Hif1α+/− mice, partial HIF-1α deficiency increased levels of HIF-2α and superoxide dismutase 2, leading to a reduced intracellular redox state, blunted oxygen sensing, and impaired carotid body and ventilatory responses to chronic hypoxia, which were corrected by treatment with a HIF-2α inhibitor. None of the abnormalities observed in Hif1α+/− mice or Hif2α+/− mice were observed in Hif1α+/−;Hif2α+/− mice. These observations demonstrate that redox balance, which is determined by mutual antagonism between HIF-α isoforms, establishes the set point for hypoxic sensing by the carotid body and adrenal medulla, and is required for maintenance of cardio-respiratory homeostasis.

Regulation of hypoxia-inducible factor-α isoforms and redox state by carotid body neural activity in rats

Rats exposed to chronic intermittent hypoxia (CIH) exhibited imbalanced expression of hypoxia‐inducible factor (HIF)‐α isoforms and oxidative stress in brainstem regions associated with the carotid body (CB) chemoreflex, and in the adrenal medulla, an end organ of the sympathetic nervous system. Selective ablation of the CB abolished the effects of CIH on HIF‐α isoform expression and oxidative stress. In the adrenal medulla, chemoreflex‐mediated sympathetic activation regulates HIF‐α isoform expression via muscarinic acetylcholine receptor‐mediated Ca2+ influx and the resultant activation of the mammalian target of rapamycin pathway and calpain proteases. Thus, CB neural activity regulates HIF‐α isoform expressions and redox state in the central and peripheral nervous system associated with the chemoreflex pathway under the setting of CIH.

Xanthine Oxidase Mediates Hypoxia-Inducible Factor-2α Degradation by Intermittent Hypoxia

Sleep-disordered breathing with recurrent apnea produces chronic intermittent hypoxia (IH). We previously reported that IH leads to down-regulation of HIF-2α protein via a calpain-dependent signaling pathway resulting in oxidative stress. In the present study, we delineated the signaling pathways associated with calpain-dependent HIF-2α degradation in cell cultures and rats subjected to chronic IH. Reactive oxygen species (ROS) scavengers prevented HIF-2α degradation by IH and ROS mimetic decreased HIF-2α protein levels in rat pheochromocytoma PC12 cell cultures, suggesting that ROS mediate IH-induced HIF-2α degradation. IH activated xanthine oxidase (XO) by increased proteolytic conversion of xanthine dehydrogenase to XO. ROS generated by XO activated calpains, which contributed to HIF-2α degradation by IH. Calpain-induced HIF-2α degradation involves C-terminus but not the N-terminus of the HIF-2α protein. Pharmacological blockade as well as genetic knock down of XO prevented IH induced calpain activation and HIF-2α degradation in PC12 cells. Systemic administration of allopurinol to rats prevented IH-induced hypertension, oxidative stress and XO activation in adrenal medulla. These results demonstrate that ROS generated by XO activation mediates IH-induced HIF-2α degradation via activation of calpains.