Atherosclerosis 240 (2015) 222e227
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Endothelial dysfunction and oxidative stress in children with sleep disordered breathing: Role of NADPH oxidase Lorenzo Loffredo a, *, Anna Maria Zicari b, Francesca Occasi b, Ludovica Perri a, Roberto Carnevale a, Francesco Angelico c, Maria Del Ben a, Francesco Martino b, Cristina Nocella a, Vincenzo Savastano b, Azzurra Cesoni Marcelli b, Marzia Duse b, Francesco Violi a a
Department of Internal Medicine and Medical Specialties, Sapienza University of Rome, Italy Department of Pediatrics, Sapienza University of Rome, Italy c Department of Public Health and Infectious Diseases, Sapienza University of Rome, Italy b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 January 2015 Received in revised form 9 March 2015 Accepted 17 March 2015 Available online 18 March 2015
Objective: Oxidative stress plays a crucial role in impairing endothelial function in sleep disordered breathing (SDB) but the underlying mechanism is still undefined. The objective of this study was to evaluate the interplay between oxidative stress, assessed by serum isoprostanes (8-iso-PGF2a) and soluble NOX2-dp (sNOX2-dp), and endothelial function, assessed by flow-mediated dilation (FMD), in children with SDB and healthy controls (HC). Methods: One-hundred forty-four children including 45 with primary snoring (PS), 22 with obstructive sleep apnea (OSA) and 67 HC were recruited in this study; in 15 out of 22 OSA children FMD, serum 8-isoPGF2a and sNOX2-dp were assessed before and after one month post adeno-tonsillectomy (AT). Results: Compared with HC, OSA and PS children had significantly higher sNOX2-dp and serum 8-isoPGF2a levels and lower FMD; compared with PS, FMD was significantly lower in OSA children. No significant difference for sNOX2-dp and serum 8-iso-PGF2a was observed between OSA and PS children. FMD was inversely correlated with sNOX2-dp levels (p < 0.001) and with serum 8-iso-PGF2a (p < 0.001). In multiple linear regression analysis, sNOX2-dp (p < 0.001) and serum 8-iso-PGF2a (p < 0.001) were the only independent predictive variables associated with FMD. AT significantly decreased sNOX2-dp and serum 8-iso-PGF2a levels (from 38.2 ± 8.8 to 22.4 ± 11.1 pg/ml, p < 0.001, and from 281.4 ± 69.7 to 226.0 ± 66.4 pg/ml, p < 0.001, respectively); conversely, FMD significantly increased after AT in OSA children (from 3.0 ± 1.5 to 8.0 ± 2.8%, p < 0.001). Conclusion: This study suggests that NOX2-derived oxidative stress is involved in artery dysfunction in SDB children. Such hypothesis is reinforced by FMD improvement after AT coincidentally with oxidative stress lowering. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02247167. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Sleep disorder breathing Endothelial function Atherosclerosis Oxidative stress
1. Introduction Endothelial dysfunction is widely considered as a hallmark of systemic atherosclerosis and an effective marker to evaluate the
Abbreviations: SDB, sleep disordered breathing; PS, primary snoring; OSA, obstructive sleep apnea; NADPH oxidase, nicotinamide-adenine dinucleotide phosphate oxidase; AT, adenotonsillectomy; IMT, intima media thickness. * Corresponding author. Divisione I Clinica Medica, Viale del Policlinico 155, Rome 00161, Italy. E-mail address: [email protected] (L. Loffredo). http://dx.doi.org/10.1016/j.atherosclerosis.2015.03.024 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
cardiovascular risk [1]. Flow-mediated dilatation (FMD) is considered a useful tool to assess endothelial dysfunction in humans [1,2]. Sleep disordered breathing (SDB) is very common in children [3e5] and includes diseases ranging from Primary Snoring (PS) to obstructive sleep apnea (OSA) [6]. SDB is characterized by precocious cardiovascular abnormalities (e.g. hypertension, vagal and sympathetic over-discharge, ventricular hypertrophy and endothelial dysfunction). In particular, children with PS have increased blood pressure and reduced arterial distensibility [7]. Children affected by OSA have intermittent episodes of complete or partial obstruction leading to intermittent desaturations and/or arousals
L. Loffredo et al. / Atherosclerosis 240 (2015) 222e227
223
[4]. This condition of intermittent hypoxemia and reoxygenation provokes damages to endothelium [8], determining significant changes in the cardiovascular system [9] in OSA. The mechanism accounting for endothelial dysfunction in children with SDB has not been fully elucidated. Oxidative stress plays a pivotal role to elicit atherosclerosis [10]. In particular, oxidative stress leads to an imbalance between reactive oxygen species (ROS) and nitric oxide production that could be accountable for endothelial dysfunction [10e13]. Several ROS-generating enzymes, as xanthine oxidase, myeloperoxidase and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase may be responsible for arterial dysfunction [14]. NADPH oxidase represents the most important source of cellular superoxide anion production in humans [12e15]. Studies performed in animal and humans suggest that NADPH oxidase modulates arterial tone [14]. Previously, we provided evidence of a major role for NADPH oxidase in modulating arterial tone in humans, since in patients with chronic granulomatous disease with hereditary deficiency of NOX2, the catalytic core of NADPH oxidase, FMD had improved compared to healthy controls (HC) [12,13]. Oxidative stress and endothelial dysfunction characterize SDB in children [16e18]. Previous studies showed high levels of urinary isoprostanes [17,19] and reduced FMD [5] in children with SDB. We postulated that NOX2-derived oxidative stress may be involved in artery dysfunction in children with SDB. To explore this issue we performed a cross-sectional study to assess oxidative stress and endothelial function in SDB children and controls. Because adeno-tonsillectomy (AT) is known to improve or resolve SDB [20e22], we performed an interventional study in OSA children with enlarged tonsils and adenoids to evaluate FMD and oxidative stress before and after surgical removal.
endothelial function were performed before and 30 ± 5 days after the surgical procedure. Exclusion criteria were as follows: presence of epilepsy, acute or chronic cardio-respiratory diseases, neuromuscular diseases, chronic inflammatory diseases, liver disease, serious kidney disorders (e.g. serum creatinine >2.8 mg/dl), smoking or vitamin assumption. All procedures performed in this study were in accordance to the ethical guidelines of the 1975 Declaration of Helsinki; the study was approved by the Ethical Committee of “Sapienza” University of Rome and was registered on ClinicalTrials.gov (Identifier: NCT02247167). Written parental informed consent was obtained. In all subjects we performed a complete physical examination and a full medical history.
2. Materials and methods
Standard overnight polysomnography recordings were performed by a Grass Heritage polygraph as previously described [27]. The following variables were recorded: body position and oxygen saturation, thoracic and abdominal respiratory effort, six electroencephalogram channels, right and left electrooculogram, electromyogram of left and right tibialis anterior muscles, chin electromyogram, electrocardiogram nasal flow [27]. Obstructive apnea was defined as absence of airflow with constant respiratory effort for more than two baseline breaths, independently of arterial oxygen saturation changes [27]. Hypopnea was defined as a reduction of at least 50% but not more than 90% in the amplitude of the airflow signal and it was only quantified if longer than two baseline breaths and associated with arousals and/or an oxygen desaturations >3% [28]. The obstructive apnea-hypopnea index (OAHI) was defined as the total number of apneic and hypopneic episodes per hour of sleep [28]. PS was diagnosed when OAHI was
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Endothelial dysfunction and oxidative stress in children with sleep disordered breathing: Role of NADPH oxidase Lorenzo Loffredo a, *, Anna Maria Zicari b, Francesca Occasi b, Ludovica Perri a, Roberto Carnevale a, Francesco Angelico c, Maria Del Ben a, Francesco Martino b, Cristina Nocella a, Vincenzo Savastano b, Azzurra Cesoni Marcelli b, Marzia Duse b, Francesco Violi a a
Department of Internal Medicine and Medical Specialties, Sapienza University of Rome, Italy Department of Pediatrics, Sapienza University of Rome, Italy c Department of Public Health and Infectious Diseases, Sapienza University of Rome, Italy b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 January 2015 Received in revised form 9 March 2015 Accepted 17 March 2015 Available online 18 March 2015
Objective: Oxidative stress plays a crucial role in impairing endothelial function in sleep disordered breathing (SDB) but the underlying mechanism is still undefined. The objective of this study was to evaluate the interplay between oxidative stress, assessed by serum isoprostanes (8-iso-PGF2a) and soluble NOX2-dp (sNOX2-dp), and endothelial function, assessed by flow-mediated dilation (FMD), in children with SDB and healthy controls (HC). Methods: One-hundred forty-four children including 45 with primary snoring (PS), 22 with obstructive sleep apnea (OSA) and 67 HC were recruited in this study; in 15 out of 22 OSA children FMD, serum 8-isoPGF2a and sNOX2-dp were assessed before and after one month post adeno-tonsillectomy (AT). Results: Compared with HC, OSA and PS children had significantly higher sNOX2-dp and serum 8-isoPGF2a levels and lower FMD; compared with PS, FMD was significantly lower in OSA children. No significant difference for sNOX2-dp and serum 8-iso-PGF2a was observed between OSA and PS children. FMD was inversely correlated with sNOX2-dp levels (p < 0.001) and with serum 8-iso-PGF2a (p < 0.001). In multiple linear regression analysis, sNOX2-dp (p < 0.001) and serum 8-iso-PGF2a (p < 0.001) were the only independent predictive variables associated with FMD. AT significantly decreased sNOX2-dp and serum 8-iso-PGF2a levels (from 38.2 ± 8.8 to 22.4 ± 11.1 pg/ml, p < 0.001, and from 281.4 ± 69.7 to 226.0 ± 66.4 pg/ml, p < 0.001, respectively); conversely, FMD significantly increased after AT in OSA children (from 3.0 ± 1.5 to 8.0 ± 2.8%, p < 0.001). Conclusion: This study suggests that NOX2-derived oxidative stress is involved in artery dysfunction in SDB children. Such hypothesis is reinforced by FMD improvement after AT coincidentally with oxidative stress lowering. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02247167. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Sleep disorder breathing Endothelial function Atherosclerosis Oxidative stress
1. Introduction Endothelial dysfunction is widely considered as a hallmark of systemic atherosclerosis and an effective marker to evaluate the
Abbreviations: SDB, sleep disordered breathing; PS, primary snoring; OSA, obstructive sleep apnea; NADPH oxidase, nicotinamide-adenine dinucleotide phosphate oxidase; AT, adenotonsillectomy; IMT, intima media thickness. * Corresponding author. Divisione I Clinica Medica, Viale del Policlinico 155, Rome 00161, Italy. E-mail address: [email protected] (L. Loffredo). http://dx.doi.org/10.1016/j.atherosclerosis.2015.03.024 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
cardiovascular risk [1]. Flow-mediated dilatation (FMD) is considered a useful tool to assess endothelial dysfunction in humans [1,2]. Sleep disordered breathing (SDB) is very common in children [3e5] and includes diseases ranging from Primary Snoring (PS) to obstructive sleep apnea (OSA) [6]. SDB is characterized by precocious cardiovascular abnormalities (e.g. hypertension, vagal and sympathetic over-discharge, ventricular hypertrophy and endothelial dysfunction). In particular, children with PS have increased blood pressure and reduced arterial distensibility [7]. Children affected by OSA have intermittent episodes of complete or partial obstruction leading to intermittent desaturations and/or arousals
L. Loffredo et al. / Atherosclerosis 240 (2015) 222e227
223
[4]. This condition of intermittent hypoxemia and reoxygenation provokes damages to endothelium [8], determining significant changes in the cardiovascular system [9] in OSA. The mechanism accounting for endothelial dysfunction in children with SDB has not been fully elucidated. Oxidative stress plays a pivotal role to elicit atherosclerosis [10]. In particular, oxidative stress leads to an imbalance between reactive oxygen species (ROS) and nitric oxide production that could be accountable for endothelial dysfunction [10e13]. Several ROS-generating enzymes, as xanthine oxidase, myeloperoxidase and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase may be responsible for arterial dysfunction [14]. NADPH oxidase represents the most important source of cellular superoxide anion production in humans [12e15]. Studies performed in animal and humans suggest that NADPH oxidase modulates arterial tone [14]. Previously, we provided evidence of a major role for NADPH oxidase in modulating arterial tone in humans, since in patients with chronic granulomatous disease with hereditary deficiency of NOX2, the catalytic core of NADPH oxidase, FMD had improved compared to healthy controls (HC) [12,13]. Oxidative stress and endothelial dysfunction characterize SDB in children [16e18]. Previous studies showed high levels of urinary isoprostanes [17,19] and reduced FMD [5] in children with SDB. We postulated that NOX2-derived oxidative stress may be involved in artery dysfunction in children with SDB. To explore this issue we performed a cross-sectional study to assess oxidative stress and endothelial function in SDB children and controls. Because adeno-tonsillectomy (AT) is known to improve or resolve SDB [20e22], we performed an interventional study in OSA children with enlarged tonsils and adenoids to evaluate FMD and oxidative stress before and after surgical removal.
endothelial function were performed before and 30 ± 5 days after the surgical procedure. Exclusion criteria were as follows: presence of epilepsy, acute or chronic cardio-respiratory diseases, neuromuscular diseases, chronic inflammatory diseases, liver disease, serious kidney disorders (e.g. serum creatinine >2.8 mg/dl), smoking or vitamin assumption. All procedures performed in this study were in accordance to the ethical guidelines of the 1975 Declaration of Helsinki; the study was approved by the Ethical Committee of “Sapienza” University of Rome and was registered on ClinicalTrials.gov (Identifier: NCT02247167). Written parental informed consent was obtained. In all subjects we performed a complete physical examination and a full medical history.
2. Materials and methods
Standard overnight polysomnography recordings were performed by a Grass Heritage polygraph as previously described [27]. The following variables were recorded: body position and oxygen saturation, thoracic and abdominal respiratory effort, six electroencephalogram channels, right and left electrooculogram, electromyogram of left and right tibialis anterior muscles, chin electromyogram, electrocardiogram nasal flow [27]. Obstructive apnea was defined as absence of airflow with constant respiratory effort for more than two baseline breaths, independently of arterial oxygen saturation changes [27]. Hypopnea was defined as a reduction of at least 50% but not more than 90% in the amplitude of the airflow signal and it was only quantified if longer than two baseline breaths and associated with arousals and/or an oxygen desaturations >3% [28]. The obstructive apnea-hypopnea index (OAHI) was defined as the total number of apneic and hypopneic episodes per hour of sleep [28]. PS was diagnosed when OAHI was
