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.

Adenotonsillar hypertrophy is the major pathophysiological mechanism underlying obstructive sleep apnea (OSA) and recurrent tonsillitis (RI) in children. The increased expression of various mediators of the inflammatory response in tonsils of patients with OSA prompted our hypothesis that the enhanced local and systemic inflammation in children with OSA would promote tonsillar proliferation. Mixed cell cultures from tonsils recovered during adenotonsillectomy in children with OSA and RI were established, and proliferative rates were assessed. Cells were also cultured to determine the levels of proinflammatory cytokines and antioxidant protein levels and mRNA expression. Global cell proliferative rates from OSA tonsils were significantly higher than RI (p < 0.01), with CD3⁺, CD4⁺, and CD8⁺ cell proliferation being higher in OSA (p < 0.05). Moreover, proinflammatory cytokines, such as TNF-α, IL-6, and IL-1α, were highly expressed in OSA-derived tonsils. Furthermore, thioredoxin (TRX), an antioxidant protein, was also highly expressed in OSA tonsils at the mRNA and protein levels (p < 0.01). Thus, T cells are in a highly proliferative state in the tonsils of children with OSA and are associated with increased production of proinflammatory cytokines and TRX, when compared with children with RI.

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.

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.