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1.  DNA Methylation in Inflammatory Genes among Children with Obstructive Sleep Apnea 
Background: Pediatric obstructive sleep apnea (OSA) leads to multiple end-organ morbidities that are mediated by the cumulative burden of oxidative stress and inflammation. Because not all children with OSA exhibit increased systemic inflammation, genetic and environmental factors may be affecting patterns of DNA methylation in genes subserving inflammatory functions.
Methods: DNA from matched children with OSA with and without high levels of high-sensitivity C-reactive protein (hsCRP) were assessed for DNA methylation levels of 24 inflammatory-related genes. Primer-based polymerase chain reaction assays in a case-control setting involving 47 OSA cases and 31 control subjects were conducted to confirm the findings; hsCRP and myeloid-related protein (MRP) 8/14 levels were also assayed.
Measurements and Main Results: Forkhead box P3 (FOXP3) and interferon regulatory factor 1 (IRF1) showed higher methylation in six children with OSA and high hsCRP levels compared with matched children with OSA and low hsCRP levels (P < 0.05). In the case-control cohort, children with OSA and high CRP levels had higher log FOXP3 DNA methylation levels compared with children with OSA and low CRP levels and control subjects. IRF1 did not exhibit significant differences. FOXP3 DNA methylation levels correlated with hsCRP and MRP 8/14 levels and with apnea-hypopnea index (AHI), BMI z score, and apolipoprotein B levels. A stepwise multiple regression model showed that AHI was independently associated with FOXP3 DNA methylation levels (P < 0.03).
Conclusions: The FOXP3 gene, which regulates expression of T regulatory lymphocytes, is more likely to display increased methylation among children with OSA who exhibit increased systemic inflammatory responses. Thus, epigenetic modifications may constitute an important determinant of inflammatory phenotype in OSA, and FOXP3 DNA methylation levels may provide a potential biomarker for end-organ vulnerability.
PMCID: PMC3297110  PMID: 22077067
obstructive sleep apnea; epigenetics; DNA methylation; T regulatory lymphocytes; inflammation
2.  A Mediation Model Linking Body Weight, Cognition, and Sleep-Disordered Breathing 
Rationale: Academic success involves the ability to use cognitive skills in a school environment. Poor academic performance has been linked to disrupted sleep associated with sleep-disordered breathing (SDB). In parallel, poor sleep is associated with increased risk for obesity, and weight management problems have been linked to executive dysfunction, suggesting that interactions may be operational between SDB and obesity to adversely affect neurocognitive outcomes.
Objectives: To test whether mediator relationships exist between body weight, SDB, and cognition.
Methods: Structural equation modeling was conducted on data from 351 children in a community-based cohort assessed with the core subtests of the Differential Abilities Scales after an overnight polysomnogram. Body mass index, apnea–hypopnea index, and cognitive abilities were modeled as latent constructs.
Measurements and Main Results: In a sample of predominantly white children 6 to 10 years of age, SDB amplified the adverse cognitive and weight outcomes by 0.55- to 0.46-fold, respectively. Weight amplified the risk by 0.39- to 0.40-fold for SDB and cognitive outcomes, respectively. Poor ability to perform complex mental processing functions increased the risk of adverse weight and SDB outcomes by 2.9- and 7.9-fold, respectively.
Conclusions: Cognitive functioning in children is adversely affected by frequent health-related problems, such as obesity and SDB. Furthermore, poorer integrative mental processing may place a child at a bigger risk for adverse health outcomes.
PMCID: PMC3297090  PMID: 22071385
sleep-disordered breathing; weight; BMI; cognition; verbal abilities
3.  Sleep Fragmentation Induces Cognitive Deficits Via Nicotinamide Adenine Dinucleotide Phosphate Oxidase–dependent Pathways in Mouse 
Rationale: Sleep fragmentation (SF) is one of the major characteristics of sleep apnea, and has been implicated in its morbid consequences, which encompass excessive daytime sleepiness and neurocognitive impairments. We hypothesized that absence of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity is neuroprotective in SF-induced cognitive impairments.
Objectives: To examine whether increased NADPH oxidase activity may play a role in SF-induced central nervous system dysfunction.
Methods: The effect of chronic SF during the sleep-predominant period on sleep architecture, sleep latency, spatial memory, and oxidative stress parameters was assessed in mice lacking NADPH oxidase activity (gp91phox-/Y) and wild-type littermates.
Measurements and Main Results: SF for 15 days was not associated with differences in sleep duration, sleep state distribution, or sleep latency in both gp91phox-/Y and control mice. However, on a standard place training task, gp91phox-/Y mice displayed normal learning and were protected from the spatial learning deficits observed in wild-type littermates exposed to SF. Moreover, anxiety levels were increased in wild-type mice exposed to SF, whereas no changes emerged in gp91phox-/Y mice. Additionally, wild-type mice, but not gp91phox-/Y mice, had significantly elevated NADPH oxidase gene expression and activity, and in malondialdehyde and 8-oxo-2′-deoxyguanosine levels in cortical and hippocampal lysates after SF exposures.
Conclusions: This work substantiates an important role for NADPH oxidase in hippocampal memory impairments induced by SF, modeling sleep apnea. Targeting NADPH oxidase, therefore, is expected to minimize hippocampal impairments from both intermittent hypoxia and SF associated with the disease.
PMCID: PMC3262045  PMID: 21868506
NADPH oxidase; sleep fragmentation; neurocognitive impairments
4.  Leukotriene B4 Receptor-1 Mediates Intermittent Hypoxia-induced Atherogenesis 
Rationale: Obstructive sleep apnea, which is characterized by intermittent hypoxia (IH) during sleep, has emerged as an independent risk factor for cardiovascular disease, including atherosclerosis. Leukotriene B4 (LTB4) production is increased in patients with obstructive sleep apnea and negatively correlates to hypoxic levels during sleep, with continuous positive airway pressure therapy decreasing LTB4 production.
Objectives: Determine the potential role of LTB4 in IH-induced atherosclerosis in a monocyte cellular model and a murine model.
Methods: THP-1 cells were exposed to IH for 3, 6, 24, and 48 hours. Macrophage transformation and foam cell formation were assessed after IH exposures. Apolipopotein E (ApoE)−/− or BLT1−/−/ApoE−/− mice were fed an atherogenic diet and exposed to IH (alternating 21% and 5.7% O2 from 7 am to 7 PM each day) for 10 weeks. Atherosclerotic lesion formation in en face aorta was examined by oil red O staining.
Measurements and Main Results: IH increased production of LTB4 and the expression of 5-lipoxygenase and leukotriene A4 hydrolase, the key enzymes for producing LTB4. IH was associated with transformation of monocytes to activated macrophages, as evidenced by increased expression of CD14 and CD68. In addition, IH exposures promoted increased cellular cholesterol accumulation and foam cell formation. The LTB4 receptor 1 (BLT1) antagonist U-75302 markedly attenuated IH-induced changes. Furthermore, IH promoted atherosclerotic lesion formation in ApoE−/− mice. IH-induced lesion formation was markedly attenuated in BLT1−/−/ApoE−/− mice.
Conclusions: BLT1-dependent pathways underlie IH-induced atherogenesis, and may become a potential novel therapeutic target for obstructive sleep apnea–associated cardiovascular disease.
PMCID: PMC3172891  PMID: 21493735
obstructive sleep apnea; inflammation; monocyte; atherosclerosis
5.  Physical Activity Attenuates Intermittent Hypoxia-induced Spatial Learning Deficits and Oxidative Stress 
Rationale: Exposure to intermittent hypoxia (IH), such as occurs in sleep-disordered breathing, is associated with substantial cognitive impairments, oxidative stress and inflammation, and increased neuronal cell losses in brain regions underlying learning and memory in rats. Physical activity (PA) is now recognized as neuroprotective in models of neuronal injury and degeneration.
Objectives: To examine whether PA will ameliorate IH-induced deficits.
Methods: Young adult Sprague-Dawley rats were randomly assigned to one of four treatment groups including normal activity (NA) or PA for 3 months and then subjected to either normoxia (RA) or exposure to IH during the light phase during the last 14 days.
Measurements and Main Results: Significant impairments in IH-exposed rats emerged on both latency and pathlength to locate the hidden platform in a water maze and decreased spatial bias during the probe trials. These impairments were not observed in PA-IH rats. In addition, the PA-IH group, relative to NA-IH, conferred greater resistance to both lipid peroxidation and 8-hydroxy-2′-deoxyguanosine (DNA damage) in both the cortex and hippocampus. In support of a neuroprotective effect from PA, PA-IH versus NA-IH rats showed greater AKT activation and neuronal insulin growth factor-1 in these regions.
Conclusions: Behavioral modifications such as increased physical activity are associated with decreased susceptibility to IH-induced spatial task deficits and lead to reduced oxidative stress, possibly through improved preservation of insulin growth factor-1–Akt neuronal signaling. Considering the many advantages of PA, interventional strategies targeting behavioral modifications leading to increased PA should be pursued in patients with sleep-disordered breathing.
PMCID: PMC2902754  PMID: 20224062
sleep apnea; oxidative stress; neurotrophic factors
6.  Endothelial Progenitor Cells and Vascular Dysfunction in Children with Obstructive Sleep Apnea 
Rationale: Endothelial dysfunction is a potential complication of obstructive sleep apnea syndrome (OSAS) in children ascribed to systemic inflammatory changes. However, not all children with OSAS will manifest endothelial dysfunction.
Objectives: The variability in endothelial function in pediatric OSAS may be related to the ability to recruit repair mechanisms such as endothelial progenitor cells (EPCs).
Methods: Prepubertal nonhypertensive children with or without polysomonographically confirmed OSAS had endothelial function assessed in a morning fasted state using a modified hyperemic test involving cuff-induced occlusion of the radial and ulnar arteries. Blood was drawn and EPCs were assessed by flow cytometry and triple staining using antibodies against CD133, CD34, and vascular endothelial growth factor receptor-2 after isolation of peripheral blood mononuclear cells. SDF-1 levels were measured by ELISA.
Measurements and Main Results: Eighty children with OSAS (mean age 8.2 ± 1.4 yr, mean body mass index [BMI] z-score, 1.43 ± 0.3) and 20 controls (CO) matched for BMI, age, sex, and ethnicity were studied. Significant delays to peak capillary reperfusion after occlusion release (Tmax) occurred in OSAS children, but substantial variability was present. Despite similar OSAS severity, EPC counts, and stromal cell–derived factor-1 (SDF-1) levels were significantly lower among the 20 OSAS children with the longest Tmax, when compared with either the 20 children with normal Tmax values or to CO ( P < 0.01). Furthermore, Tmax was significantly and inversely correlated with EPCs (r2, 0.51; P < 0.01), but neither EPCs nor Tmax were associated with apnea-hyponea index (AHI).
Conclusions: Endothelial dysfunction is frequently present in OSAS. Variance in endothelial functional phenotype may not only reside in the individual susceptibility but also in the ability to recruit endothelial repair mechanisms.
PMCID: PMC2902761  PMID: 20203242
sleep apnea; endothelial progenitor cells; endothelial function; serum lipids; atherosclerosis
7.  Transcriptomic Analysis Identifies Phosphatases as Novel Targets for Adenotonsillar Hypertrophy of Pediatric Obstructive Sleep Apnea 
Rationale: Obstructive sleep apnea (OSA) is a highly prevalent disorder in children, in which enlarged adenotonsillar tissues (AT) play a major pathophysiologic role. Mechanisms leading to the proliferation and hypertrophy of AT in children who subsequently develop OSA remain unknown, and surgical extirpation of AT is associated with potential morbidity and mortality.
Objectives: We hypothesized that a computationally based analysis of gene expression in tonsils from children with OSA and children with recurrent tonsillitis without OSA can identify putative mechanistic pathways associated with tonsillar proliferation and hypertrophy in OSA.
Methods: Palatine tonsils from children with either polysomnographically documented OSA or recurrent infectious tonsillitis were subjected to whole-genome microarray and functional enrichment analyses followed by significance score ranking based on gene interaction networks. The latter enabled identification and confirmation of a candidate list of tonsil-proliferative genes in OSA.
Measurements and Main Results: In vitro studies using a mixed tonsil cell culture system targeting one of these candidates, phosphoserine phosphatase, revealed that it was more abundantly expressed in tonsils of children with OSA, and that pharmacological inhibition of phosphoserine phosphatase led to marked reductions in T- and B-lymphocyte cell proliferation and increased apoptosis.
Conclusions: A systems biology approach revealed a restricted set of candidate genes potentially underlying the heightened proliferative properties of AT in children with OSA. Furthermore, functional studies confirm a novel role for protein phosphatases in AT hypertrophy, and may provide a promising strategy for discovery of novel, nonsurgical therapeutic targets in pediatric OSA.
PMCID: PMC2874453  PMID: 20093640
sleep apnea; phosphatase; adenotonsillar; genetic network; microarray
8.  Two-Dimensional Differential In-Gel Electrophoresis Proteomic Approaches Reveal Urine Candidate Biomarkers in Pediatric Obstructive Sleep Apnea 
Rationale: Sleep studies are laborious, expensive, inaccessible, and inconvenient for diagnosing obstructive sleep apnea (OSA) in children.
Objectives: To examine whether the urinary proteome uncovers specific clusters that are differentially expressed in the urine of children with OSA.
Methods: Two-dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry proteomics followed by validation with western blot of ELISA.
Measurements and Main Results: Morning urine proteins from 60 children with polysomnographically confirmed OSA and from matched children with primary snoring (n = 30) and control subjects (n = 30) were assessed. A total of 16 proteins that are differentially expressed in OSA were identified, and 7 were confirmed by either immunoblots or ELISA. Among the latter, receiver–operator curve analyses of urinary concentrations of uromodulin, urocortin-3, orosomucoid-1, and kallikrein assigned favorable predictive properties to these proteins. Furthermore, combinatorial approaches indicated that the presence of values beyond the calculated cutoff concentrations for three or more of the proteins yielded a sensitivity of 95% and a specificity of 100%.
Conclusions: Proteomic approaches reveal that pediatric OSA is associated with specific and consistent alterations in urinary concentrations of specific protein clusters. Future studies aiming to validate this approach as a screening method of habitually snoring children appears warranted.
PMCID: PMC2796735  PMID: 19797158
sleep apnea; biomarkers; children; urine
9.  Erectile Dysfunction in a Murine Model of Sleep Apnea 
Rationale: Erectile dysfunction (ED) is frequent in obstructive sleep apnea syndrome (OSAS). Chronic intermittent hypoxia (CIH), one of the hallmarks of OSAS, could mediate ED.
Objectives: To determine whether intermittent hypoxia during sleep affects erectile dysfunction in mice.
Methods: Three groups of C57BL/6 mice were exposed to CIH for 5 or 24 weeks. Sexual function was evaluated by in vivo telemetry of corpus spongiosum pressure. Spontaneous erections, sexual activity during mating, and noncontact tests were assessed after 5 weeks of CIH and after treatment with tadalafil. Plasma testosterone was measured after 8 and 24 weeks of CIH, and the expression of nitric oxide synthase (NOS) isoforms was examined in penile tissue.
Measurements and Main Results: Noncontact, spontaneous, and contact sexual activity in the mice was suppressed after CIH. Spontaneous erection counts decreased after the first week of CIH by 55% (P < 0.001) and remained unchanged thereafter. Recovery of erectile activity during normoxia for 6 weeks was incomplete. Compared with control mice, latencies for mounts and intromissions increased by 60- and 40-fold, respectively (P < 0.001), and the sexual activity index decreased sixfold. Tadalafil treatment significantly attenuated these effects. Immunoblot analyses of NOS proteins in the erectile tissue showed decreased expression of endothelial NOS after CIH (P < 0.01), with no changes in plasma testosterone levels after 8 and 24 weeks of CIH.
Conclusions: CIH during sleep is associated with decreased libido in mice. The decreased expression of endothelial NOS protein in erectile tissue and the favorable response to tadalafil suggest that altered nitric oxide mechanisms underlie CIH-mediated ED. No changes in testosterone emerge after intermittent hypoxia.
PMCID: PMC2542437  PMID: 18535258
nitric oxide synthase; erectile dysfunction; sleep apnea; intermittent hypoxia
10.  Metabolic Alterations and Systemic Inflammation in Obstructive Sleep Apnea among Nonobese and Obese Prepubertal Children 
Rationale: Obstructive sleep apnea (OSA) has been associated with a higher prevalence and severity of the metabolic syndrome in adult patients, even after controlling for obesity. In contrast, OSA in prepubertal children does not appear to correlate with the magnitude of such metabolic derangements.
Objectives: To further establish the potential mechanistic role of OSA in metabolic regulation in prepubertal children.
Methods: Fasting glucose, insulin, C-reactive protein, apolipoprotein B, and serum lipid concentrations were determined during the initial polysomnographic diagnosis of OSA and 6–12 months after adenotonsillectomy in both obese and nonobese children.
Measurements and Main Results: Sixty-two children with OSA (37 obese and 25 nonobese), age 7.40 ± 2.6 years (mean ± SD) completed the study. After adenotonsillectomy, significant improvements in apnea–hypopnea index and sleep fragmentation occurred, particularly among nonobese children. In nonobese children, adenotonsillectomy was associated with mild increases in body mass index z scores, no changes in either fasting glucose or insulin, significant increases in high-density lipoprotein and reciprocal decreases in low-density lipoprotein, and reductions in plasma C-reactive protein and apolipoprotein B levels. In obese children, adenotonsillectomy did not result in body mass index or glucose changes, but was associated with marked improvements in all other measures.
Conclusions: OSA does not appear to induce insulin resistance in nonobese pediatric patients but seems to play a significant role in obese patients. The significant improvements in lipid profiles, C-reactive protein, and apolipoprotein B after adenotonsillectomy in the two groups suggest a pathogenic role for OSA in lipid homeostasis and systemic inflammation independent of the degree of adiposity.
PMCID: PMC2383995  PMID: 18276939
obstructive sleep apnea; inflammation; obesity; serum lipids; diabetes
11.  Green Tea Catechin Polyphenols Attenuate Behavioral and Oxidative Responses to Intermittent Hypoxia 
Rationale: The intermittent hypoxia (IH) that characterizes sleep-disordered breathing impairs spatial learning and increases NADPH oxidase activity and oxidative stress in rodents. We hypothesized that green tea catechin polyphenols (GTPs) may attenuate IH-induced neurobehavioral deficits by reducing IH-induced NADPH oxidase expression, lipid peroxidation, and inflammation.
Objectives: To assess the effects of GTP administered in drinking water on the cognitive, inflammatory, and oxidative responses to long-term (>14 d) IH during sleep in male Sprague-Dawley rats.
Methods: Cognitive assessments were conducted in the Morris water maze. We measured levels and expression of malondialdehyde (MDA), prostaglandin E2, p47phox subunit of NADPH oxidase, receptor for advanced glycation end products (RAGE), and glial fibrillary acidic protein expression in rodent brain tissue.
Measurements and Main Results: GTP treatment prevented IH-induced decreases in spatial bias for the hidden platform during the Morris water maze probe trails as well as IH-induced increases in p47phox expression within the hippocampal CA1 region. In untreated animals, IH exposure was associated with doubling of cortical MDA levels in comparison to room air control animals, and GTP-treated animals exposed to IH showed a 40% reduction in MDA levels. Increases in brain RAGE and glial fibrillary acidic protein expression were observed in IH-exposed animals, and these increases were attenuated in animals treated with GTP.
Conclusions: Oral GTP attenuates IH-induced spatial learning deficits and mitigates IH-induced oxidative stress through multiple beneficial effects on oxidant pathways. Because oxidative processes underlie neurocognitive deficits associated with IH, the potential therapeutic role of GTP in sleep-disordered breathing deserves further exploration.
PMCID: PMC2383994  PMID: 18276944
sleep apnea; cognition; inflammation; oxidative stress; hypoxia
12.  Cardiovascular Morbidity in Obstructive Sleep Apnea 
Sleep-disordered breathing and obstructive sleep apnea (OSA) are highly prevalent disorders throughout the lifespan, which may affect up to 2–10% of the population, and have now been firmly associated with an increased risk for cardiovascular and neurobehavioral complications. Nevertheless, the overall pathophysiologic mechanisms mediating end-organ injury in OSA remain undefined, particularly due to the very frequent coexistence of other disease states, such as obesity, that clearly complicate the potential cause–effect relationships. Two major, and to some extent overlapping, mechanisms have been proposed to explain the morbid consequences of OSA, namely increased generation and propagation of reactive oxygen species and initiation and amplification of inflammatory processes. The evidence supporting the validity of these concepts as well as that detracting from such mechanisms will be critically reviewed in the context of clinical and laboratory-based approaches. In addition, some of the contradictory issues raised by such evaluation of the literature will be interpreted in the context of putative modifications of the individual responses to OSA, as determined by genetic variants among susceptibility-related genes, and also by potential environmental modulators of the phenotypic expression of any particular end-organ morbidity associated with OSA.
PMCID: PMC2258438  PMID: 17975198
inflammation; oxidative stress; reactive oxygen species; intermittent hypoxia; sleep fragmentation
13.  C-reactive Protein, Obstructive Sleep Apnea, and Cognitive Dysfunction in School-aged Children 
Rationale: Obstructive sleep apnea (OSA) in children is associated with substantial neurobehavioral and cognitive dysfunction. However, not all children with OSA exhibit altered cognitive performance.
Objectives: To assess the magnitude of the systemic inflammatory response, as measured by high-sensitivity C-reactive protein (hsCRP) serum levels which may identify children with OSA at higher susceptibility for cognitive morbidity.
Methods: Habitually snoring children and nonsnoring children (total, 278; age range, 5–7 yr) were recruited from the community, and underwent overnight polysomnography and neurocognitive testing and a blood draw the next morning. Snoring children were divided into OSA and no-OSA groups, and children with OSA were further subdivided into those with two or more abnormal cognitive subtests and into those with normal cognitive scores. Serum levels of hsCRP were also measured.
Measurements and Main Results: Among snoring children without OSA, mean hsCRP was 0.19 ± 0.07 mg/dl compared with 0.36 ± 0.11 mg/dl in those with OSA (p < 0.01). Furthermore, hsCRP was 0.48 ± 0.12 mg/dl in children with OSA and cognitive deficits, compared with 0.21 ± 0.08 mg/dl in children with OSA and normal cognitive scores (p < 0.002).
Conclusions: hsCRP levels are higher in children with OSA, and particularly in those who develop neurocognitive deficits, suggesting that the magnitude of the inflammatory responses elicited by OSA is a major determinant of increased risk for neurocognitive dysfunction.
PMCID: PMC1994211  PMID: 17400731
sleep-disordered breathing; systemic inflammation; hypoxia; sleep fragmentation
14.  Leukotriene Modifier Therapy for Mild Sleep-disordered Breathing in Children 
Background: Children with mild sleep-disordered breathing (SDB), who may not be recommended for adenotonsillectomy, frequently exhibit neurocognitive and behavioral morbidity, and may benefit from alternative therapeutic interventions, such as leukotriene modifier therapy. Methods: Twenty-four children with SDB completed an open-label intervention study for 16 weeks with daily montelukast therapy. Sleep studies and adenoid size estimates from lateral X-ray films of the neck were obtained before and after treatment. In a parallel study, adenoid and tonsillar tissues from children with obstructive sleep apnea or recurrent throat infections were subjected to quantitative polymerase chain reaction, immunohistochemistry, and Western blotting for gene and protein expression of leukotriene receptors LT1-R and LT2-R, and for concentrations of LTB4 and LTC4/D4/E4. Results: Montelukast treatment induced significant reductions in adenoid size and respiratory-related sleep disturbances, which were absent in 16 children with SDB who did not receive treatment. LT1-R and LT2-R mRNA was similarly abundant in adenoid tissues, but increased LT1-R and LT2-R protein expression and higher levels of LTB4 and LTC4/D4/E4 emerged in children with obstructive sleep apnea. Conclusions: Oral therapy with a leukotriene modifier appears to be associated with improved breathing during sleep. Double-blind, placebo-controlled trials will be needed to corroborate current findings and solidly establish antiinflammatory strategies, such as leukotriene modifiers, as therapeutic alternatives in children with SDB too mild to justify referral for adenotonsillectomy.
PMCID: PMC2718474  PMID: 15879419
leukotriene receptors; lymphoid hyperplasia; sleep apnea; adenotonsillectomy; tonsils

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