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.blood pressure | developmental programming | norepinephrine I n preterm infants, respiratory disorders with recurrent apnea and the associated intermittent hypoxemia (IH) are major clinical problems (1). Infants with recurrent apnea exhibit an enhanced hypoxic ventilatory response (2), an effect that was attributed to a heightened chemo-reflex arising from the carotid body, which is a sensory organ that detects changes in arterial blood O 2 levels (3). Carotid body sensitivity to hypoxia is reset after birth and this effect is modulated by chronic hypoxia (4-6). Neonatal rats exposed to IH showed exaggerated carotid body and ventilatory responses to hypoxia (7,8). Catecholamine secretion from the adrenal medulla is another important homeostatic mechanism that preserves cardiovascular function under hypoxia (9, 10). In neonates, hypoxia facilitates catecholamine secretion by directly affecting the excitability of adrenal chromaffin cells (11), a response that is markedly augmented in neonatal rats subjected to IH (12,13). Exaggerated hypoxic sensing of carotid body and adrenal chromaffin cells in neonates by IH is attributed to increased oxidative stress (12,14). IH also augments hypoxic responses of the carotid body and adrenal medulla in adult rats (15, 16), which is completely reversed following reoxygenation (15). In striking contrast, in neonates the augmented hypoxic sensitivity that is induced by IH persists into adulthood (8,12,14). The molecular mechanisms underlying the long-lasting effects of neonatal IH on hypoxic sensing and its physiological consequences in adult life are not known.It is being increasingly recognized that environm...

Key pointsr 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. r Selective ablation of the CB abolished the effects of CIH on HIF-α isoform expression and oxidative stress.r In the adrenal medulla, chemoreflex-mediated sympathetic activation regulates HIF-α isoform expression via muscarinic acetylcholine receptor-mediated Ca 2+ influx and the resultant activation of the mammalian target of rapamycin pathway and calpain proteases.r 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.Abstract Previous studies reported that chronic intermittent hypoxia (CIH) results in an imbalanced expression of hypoxia-inducible factor-α (HIF-α) isoforms and oxidative stress in rodents, which may be due either to the direct effect of CIH or indirectly via hitherto uncharacterized mechanism(s). As neural activity is a potent regulator of gene transcription, we hypothesized that carotid body (CB) neural activity contributes to CIH-induced HIF-α isoform expression and oxidative stress in the chemoreflex pathway. Experiments were performed on adult rats exposed to CIH for 10 days. Rats exposed to CIH exhibited: increased HIF-1α and decreased HIF-2α expression; increased NADPH oxidase 2 and decreased superoxide dismutase 2 expression; and oxidative stress in the nucleus tractus solitarius and rostral ventrolateral medulla as well as in the adrenal medulla (AM), a major end organ of the sympathetic nervous system. Selective ablation of the CB abolished these effects. In the AM, sympathetic activation by the CB chemoreflex mediates CIH-induced HIF-α isoform imbalance via muscarinic acetylcholine receptor-mediated Ca 2+ influx, and the resultant activation of mammalian target of rapamycin pathway and calpain proteases. Rats exposed to CIH presented with hypertension, elevated sympathetic activity and increased circulating catecholamines. Selective ablation of either the CB (afferent pathway) or sympathetic innervation to the AM (efferent pathway) abolished these effects. These observations uncover CB neural activity-dependent regulation of HIF-α isoforms Abbreviations Ac-LLM-CHO, N-acetyl-leucine-leucine-methionine-aldehyde; AChR, acetylcholine receptor; AIH, acute intermittent hypoxia; AM, adrenal medulla; ASA, adrenal sympathetic ablation; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid; BP, blood pressure; CA, catecholamines; CB, carotid body; CBA, carotid body ablation; CIH, chronic intermittent hypoxia; HIF-α, hypoxia-inducible factor alpha; HR, heart rate; mAChR, muscarinic ACh receptor; MDA, malondialdehyde; mTOR, mammalian target of rapamycin; nAChR, nicotinic ACh receptor; Nox2, NADPH oxidase 2; nTS, nucleus tractus solitarious; PC12, phe...

Cardiorespiratory functions in mammals are exquisitely sensitive to changes in arterial O 2 levels. Hypoxia-inducible factors (e.g., HIF-1 and HIF-2) mediate transcriptional responses to reduced oxygen availability. We demonstrate that haploinsufficiency for the O 2 -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.blood pressure | control of ventilation | catecholamines H ypoxia-inducible factors (HIFs) mediate transcriptional responses to reduced O 2 availability (1). HIF-1, the first identified member of the HIF family, is comprised of an O 2 -regulated α subunit and a constitutively expressed β subunit (2). Complete deficiency of HIF-1α in Hif-1α −/− mice results in embryonic lethality at midgestation with major malformations of the central nervous system, heart, and vasculature (3). Hif-1α +/− mice, which are partially deficient in HIF-1α expression, develop normally and are indistinguishable from their WT littermates in the sedentary state with respect to autonomic functions, including blood pressure (BP), ventilatory responses to hypoxia or hypercapnia, and plasma catecholamine levels (4). However, Hif-1α +/− mice exhibit impaired O 2 sensing by the carotid body (4, 5), the primary sensory organ for detecting hypoxemia (6, 7), and altered physiological adaptations to chronic hypoxia (5, 8).The HIF-2α subunit, also known as endothelial PAS domain protein-1 (EPAS-1), shares 48% amino acid sequence identity with HIF-1α (9). As in the case of HIF-1α, continuous hypoxia leads to HIF-2α accumulation and subsequent dimerization with HIF-1β. Transcriptional activation by HIF-2 regulates some target genes in common with HIF-1 as well as other genes that are uniquely regulated by HIF-2 (10-12). Homozygous deficiency of HIF-2α is often lethal with the surviving Hif-2α -/− mice exhibiting multiple organ pathology and increased oxidative stress as a result of impaired induction of genes encoding major antioxidant enzymes (AOEs) (12).Hif-2α +/− mice, like Hif-1α +/− mice, are phenotypically indistinguishable from WT littermates under sedentary conditions, but exhibit impaired responses to chronic hypoxia such as impaired pulmonary vascular remodeling, erythropoiesis, and retinal neovascularization (13-15). Although the carotid body is a prominent site of HIF-2α expression (16), the effects of Hi...

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 Hif1αmice. 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 cardiorespiratory homeostasis.blood pressure regulation | ventilatory adaptation | reactive oxygen species | Nox2 | Sod2

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.

Chronic alcohol consumption causes numerous biochemical and biophysical changes in the central nervous system, in which mitochondria is the primary organelle affected. In the present study, we hypothesized that alcohol alters the mitochondrial membrane properties and leads to mitochondrial dysfunction via mitochondrial reactive oxygen species (mROS) and reactive nitrogen species (RNS). Alcohol-induced hypoxia further enhances these effects. Administration of alcohol to rats significantly increased the mitochondrial lipid peroxidation and protein oxidation with decreased SOD2 mRNA and protein expression was decreased, while nitric oxide (NO) levels and expression of iNOS and nNOS in brain cortex were increased. In addition, alcohol augmented HIF-1α mRNA and protein expression in the brain cortex. Results from this study showed that alcohol administration to rats decreased mitochondrial complex I, III, IV activities, Na(+)/K(+)-ATPase activity and cardiolipin content with increased anisotropic value. Cardiolipin regulates numerous enzyme activities, especially those related to oxidative phosphorylation and coupled respiration. In the present study, decreased cardiolipin could be ascribed to ROS/RNS-induced damage. In conclusion, alcohol-induced ROS/RNS is responsible for the altered mitochondrial membrane properties, and alcohol-induced hypoxia further enhance these alterations, which ultimately leads to mitochondrial dysfunction.

Hypoxia-inducible factor 1 (HIF-1) mediates many of the systemic and cellular responses to intermittent hypoxia (IH), which is an experimental model that simulates O2 saturation profiles occurring with recurrent apnea. IH-evoked HIF-1α synthesis and stability are due to increased reactive oxygen species (ROS) generated by NADPH oxidases, especially Nox2. However, the mechanisms by which IH activates Nox2 are not known. We recently reported that IH activates xanthine oxidase (XO) and the resulting increase in ROS elevates intracellular calcium levels. Since Nox2 activation requires increased intracellular calcium levels, we hypothesized XO-mediated calcium signaling contributes to Nox activation by IH. We tested this possibility in rat pheochromocytoma PC12 cells subjected to IH consisting alternating cycles of hypoxia (1.5% O2 for 30 sec) and normoxia (21% O2 for 5 min). Kinetic analysis revealed that IH-induced XO preceded Nox activation. Inhibition of XO activity either by allopurinol or by siRNA prevented IH-induced Nox activation, translocation of the cytosolic subunits p47phox and p67phox to the plasma membrane and their interaction with gp91phox. ROS generated by XO also contribute to IH-evoked Nox activation via calcium-dependent protein kinase C stimulation. More importantly, silencing XO blocked IH-induced upregulation of HIF-1α demonstrating that HIF-1α activation by IH requires Nox2 activation by XO.

Aim: Cigarette smoking is a recognized risk factor for cardiovascular diseases and has been implicated in the pathogenesis of atherosclerosis. Platelet adhesiveness and aggregation increases as a result of smoking. Cigarette smoking modifies haemostatic parameters via thrombosis with a consequently higher rate of cardiovascular events, but smoking-induced alterations of platelet membrane fluidity and other changes have not been studied. Methods: Thirty experimental and control subjects (mean age 35 8) were selected for the study. Experimental subjects had smoked 10 2 cigarettes per day for 7 − 10 years. The plasma lipid profile, platelet carbonyls, sulfhydryl groups, Na /K -ATPase activity, fluidity using a fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), total cholesterol and phospholipids as well individual phospholipids were determined. Results: Increases in the platelet membrane cholesterol phospholipid (C/P) ratio, phosphotidylethanolamine, phosphotidylserine with decreased phosphotidylcholine, Na /K -ATPase activity, fluidity and no significant change in phosphotidylinositol and sphingomylein, as well as increases in plasma total cholesterol, LDL-cholesterol, protein carbonyls with decreased HDL-cholesterol and sulfhydryl groups were observed in cigarette smokers. Platelet membrane total phospholipids were positively correlated with plasma LDL-cholesterol (r 0.568) and VLDL-cholesterol (r 0.614) in cigarette smokers. Conclusions: Increased plasma LDL-cholesterol, VLDL-cholesterol and total cholesterol might have resulted in the increased C/P ratio and decreased platelet membrane fluidity of cigarette smokers. J Atheroscler Thromb, 2010; 17:619-627.