Jinkwan Kim

Obesity and Obstructive Sleep Apnea Syndrome in Children: A Tale of Inflammatory Cascades

Obesity has emerged as one of the most prevalent diseases in the western hemisphere, and its prevalence continues to increase. Obese children are at increased risk for several disorders, particularly affecting the cardiovascular and metabolic systems. The mechanisms leading to obesity‐related morbidities are likely multifactorial, and include activation of inflammatory pathways ultimately leading to end‐organ injury. Furthermore, the concurrent presence of obesity and other diseases facilitated by increased fat deposition poses a theoretical risk of accentuating obesity‐related complications. One of the conditions whose prevalence is increased by obesity in childhood is the obstructive sleep apnea syndrome (OSAS). OSAS in non‐obese children may lead to co‐morbidities that are not only remarkably similar to those associated with obesity but recruit similar inflammatory mechanisms as those activated by obesity, suggesting that the two disorders may amplify each other and synergistically augment the magnitude of their respective adverse consequences. The objective of this review is to critically review the effects of both obesity and OSAS in inducing systemic inflammation in children and will examine the latest evidence pertaining to the up‐regulation of specific inflammatory mediators. Pediatr Pulmonol. 2011; 46:313–323.

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 (r(2), 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.

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.

Endothelial Dysfunction in Children Without Hypertension: Potential Contributions of Obesity and Obstructive Sleep Apnea

BACKGROUND Endothelial dysfunction can develop in the context of both obesity and obstructive sleep apnea (OSA) in children. However, the potential interactions between OSA and obesity have not been defined. METHODS Children who were prepubertal and nonhypertensive were recruited. Endothelial function was assessed in a morning fasted state, using a modified hyperemic test involving cuff-induced occlusion of the radial and ulnar arteries, and blood was drawn for assessment of myeloid-related protein 8/14 (MRP8/14) levels using a commercial enzyme-linked immunosorbent assay. Overnight polysomnography defined the presence of OSA or absence of OSA (NOSA) in subjects investigated for sleep-disordered breathing. Anthropometric measurements were performed to assign subjects to obese (OB) and nonobese (NOB) categories. RESULTS Fifty-four children with OSA who were obese and nonobese (mean age, 7.90 ± 0.26 years; mean BMI z-score, 1.70 ± 0.3; obstructive apnea-hypopnea index [OAHI], 7.36 ± 1.09) were compared with 54 children without OSA who were obese and nonobese (mean age, 8.26 ± 0.24 years; mean BMI z-score, 1.41 ± 0.18; OAHI, 0.86 ± 0.07). Of those subjects, 62.5% of the OB-OSA category, 38.7% of the OB-NOSA category, and 20.0% of the NOB-OSA category had evidence of endothelial dysfunction, compared with 0.0% of the NOB-NOSA category (P < .01). The degree of endothelial dysfunction in all groups was associated with circulating MRP8/14 levels (r = 0.343, P < .001). CONCLUSIONS Both obesity and OSA can independently increase the risk for endothelial dysfunction, and the concurrent presence of both markedly increases such risk. Although the mechanisms underlying endothelial dysfunction remain unclear, a potential role for MRP8/14 as an inflammatory biomarker of endothelial dysfunction is suggested.

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.

Original ResearchSleep DisordersEndothelial Dysfunction in Children Without Hypertension: Potential Contributions of Obesity and Obstructive Sleep Apnea

BACKGROUND Endothelial dysfunction can develop in the context of both obesity and obstructive sleep apnea (OSA) in children. However, the potential interactions between OSA and obesity have not been defined. METHODS Children who were prepubertal and nonhypertensive were recruited. Endothelial function was assessed in a morning fasted state, using a modified hyperemic test involving cuff-induced occlusion of the radial and ulnar arteries, and blood was drawn for assessment of myeloid-related protein 8/14 (MRP8/14) levels using a commercial enzyme-linked immunosorbent assay. Overnight polysomnography defined the presence of OSA or absence of OSA (NOSA) in subjects investigated for sleep-disordered breathing. Anthropometric measurements were performed to assign subjects to obese (OB) and nonobese (NOB) categories. RESULTS Fifty-four children with OSA who were obese and nonobese (mean age, 7.90 ± 0.26 years; mean BMI z-score, 1.70 ± 0.3; obstructive apnea-hypopnea index [OAHI], 7.36 ± 1.09) were compared with 54 children without OSA who were obese and nonobese (mean age, 8.26 ± 0.24 years; mean BMI z-score, 1.41 ± 0.18; OAHI, 0.86 ± 0.07). Of those subjects, 62.5% of the OB-OSA category, 38.7% of the OB-NOSA category, and 20.0% of the NOB-OSA category had evidence of endothelial dysfunction, compared with 0.0% of the NOB-NOSA category (P < .01). The degree of endothelial dysfunction in all groups was associated with circulating MRP8/14 levels (r = 0.343, P < .001). CONCLUSIONS Both obesity and OSA can independently increase the risk for endothelial dysfunction, and the concurrent presence of both markedly increases such risk. Although the mechanisms underlying endothelial dysfunction remain unclear, a potential role for MRP8/14 as an inflammatory biomarker of endothelial dysfunction is suggested.

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% O(2) 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.

Increased Cellular Proliferation and Inflammatory Cytokines in Tonsils Derived From Children With Obstructive Sleep Apnea

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.

Inflammatory pathways in children with insufficient or disordered sleep

Sleep is not only an essential physiological function, but also serves important roles in promoting growth, maturation, and overall health of children and adolescents. There is increasing interest regarding the impact of sleep and its disorders on the regulation of inflammatory processes and end-organ morbidities, particularly in the context of metabolic and cardiovascular diseases (CVD) and their complications. Obstructive sleep apnea syndrome (OSAS) is an increasingly common health problem in children, and in the last decade, the emergence of increasing obesity rates has further led to remarkable increases in the prevalence of OSAS, along with more prominent neurocognitive, behavioral, cardiovascular and metabolic morbidities. Although the underlying mechanisms leading to OSAS-induced morbidities are likely multi-factorial, and remain to be fully elucidated, activation of inflammatory pathways by OSAS has emerged as an important pathophysiological component of the end-organ injury associated with this disorder. To this effect, it would appear that OSAS could be viewed as a chronic, low-grade inflammatory disorder. Furthermore, the concurrent presence of obesity and OSAS poses a theoretically increased risk of OSAS-related complications. In this review, we will critically review the current state of research regarding the impact of insufficient and disrupted sleep and OSAS on the immune processes and inflammatory pathways that underlie childhood OSAS as a distinctive systemic inflammatory condition in children, and will explore potential interactions between OSAS and obesity.

Fatty-acid binding protein 4 gene polymorphisms and plasma levels in children with obstructive sleep apnea

INTRODUCTION Obstructive sleep apnea (OSA) is associated with increased risk for metabolic syndrome in both adults and children. In adults with OSA, serum levels of fatty acid binding protein 4 (FABP4) are elevated and associated with the degree of metabolic insulin resistance, independent of obesity. Therefore, we assessed plasma FABP4 levels and FABP4 allelic variants in obese and non-obese children with and without OSA. METHODS A total of 309 consecutive children ages 5-8years were recruited. Children were divided into those with OSA and without OSA (NOSA) based on the apnea-hypopnea index (AHI). Subjects were also subdivided into obese (OB) and non-obese (NOB) based on BMI z score. Morning fasting plasma FABP4 levels were assayed using ELISA, and 11 single-nucleotide polymorphisms (SNPs) within the FABP4 region were genotyped. RESULTS Morning plasma FABP4 levels were increased in all children with OSA, even in NOB children. However, plasma FABP4 levels were strongly associated with BMI z score. Of the 11 SNPs tested, the frequency of rs1054135 (A/G) minor allele (A) was significantly increased in OSA. This SNP was also associated with increased plasma FABP4 levels in both OSA and obese subjects. The minor allele frequency of all other SNPs was similar in OSA and NOSA groups. CONCLUSIONS Childhood obesity and OSA are associated with higher plasma FABP4 levels and thus promote cardiometabolic risk. The presence of selective SNP (e.g., rs1054135) in the FABP4 gene may account for increased plasma FABP4 levels in the context of obesity and OSA in children.