Clinical Science (2014) 127, 323–330 (Printed in Great Britain) doi: 10.1042/CS20130679
Nitric oxide production by monocytes in children with OSA and endothelial dysfunction
Clinical Science
www.clinsci.org
Leila KHEIRANDISH-GOZAL∗1 , Yang WANG∗1 , Ryan C. DUGGAN†, Sindhuja HARSHAN VARDHAN∗ , Hui-Leng TAN∗ , Helena MOLERO RAMIREZ∗ , Abdelnaby KHALYFA∗ , Rakesh BHATTACHARJEE∗ , Hari P.R. BANDLA∗ and David GOZAL∗ ∗ Section of Sleep Medicine, Department of Pediatrics, University of Chicago, Chicago, IL 60637, U.S.A. †Flow Cytometry Core Facility, Pritzker School of Medicine, Biological Sciences Division, University of Chicago, Chicago, IL 60637, U.S.A.
Abstract OSA (obstructive sleep apnoea) is associated with a higher risk for alterations in post-occlusive hyperaemia, an eNOS (endothelial NO synthase)-dependent endothelial response. However, since not all children manifest endothelial dysfunction, we hypothesized that differences in circulating monocyte subsets and NO production may underlie the vascular phenotype in paediatric OSA. Matched pre-pubertal children with OSA with abnormal endothelial function (OSAab) and with normal endothelial function (OSAn), and controls (CO) were recruited. Peripheral blood mononuclear cells were subtyped into CD14 + and CD16 + cells, and NO production was assessed using flow cytometry. Endothelial dysfunction was defined as T max (time to reach maximal reperfusion) >45 s by laser Doppler flowmetry. A total of 11 OSAab, 12 OSAn and 12 CO-matched children completed the study. The OSAab group had increased CD16 + and decreased CD14 + cell numbers. They also had increased CX3CR1 (CX3C chemokine receptor 1) expression in CD16 + monocytes (P < 0.01). Furthermore, monocytes from the OSAab group exhibited overall reduced NO production (787 + − 71 compared with 1226 + − 229 and 1089 + − 116 median fluorescence intensity in the OSAn group and CO children respectively; P < 0.01). Significant bivariate associations emerged between NO production, monocyte subsets, CX3CR1 in CD16 + monocytes, the CD14 + /CD16 + ratio and T max . Thus OSA in children is associated with increased numbers of pro-inflammatory monocytes and reduced NO production in circulating monocytes that are closely associated with endothelial function. Key words: children, endothelial function, inflammation, monocyte, sleep apnoea
INTRODUCTION OSA (obstructive sleep apnoea) is a highly prevalent paediatric disorder that is typically associated with increased upper airway resistance during sleep, leading to major alterations in intrathoracic pressure swings during breathing, abnormal gas exchange and disrupted sleep patterns [1]. Autonomic imbalance, particularly manifesting as increased sympathetic activation [2,3], along with evidence of systemic oxidative stress and inflammation, are therefore probable players in the putative adverse cardiovascular consequences of OSA, including pro-atherogenic lipid alterations [4–6]. In children, cross-sectional studies have now repeatedly shown that moderate-to-severe OSA is associated with a higher
risk of cardiovascular morbidity, primarily presenting as increased systemic and pulmonary vascular blood pressures that can lead to ventricular remodelling [7–12]. However, more subtle phenotypic changes, such as disturbances in blood pressure regulation (e.g. loss of sleep-associated blood pressure dipping) or altered endothelial function, an early precursor of atherosclerosis, can be frequently detected as well [13–16]. In the context of endothelial dysfunction, it is important to note that not every child, even with severe OSA, displays abnormal endothelial function as measured by post-occlusive hyperaemic responses [16–19]. Despite a large degree of variance, circulating biomarkers of inflammation and vascular injury, such as adhesion molecules, circulating microparticles and myeloid-related protein 8/14, and other inflammatory mediators correlate with OSA severity in
Abbreviations: AHI, apnoea/hypopnoea index; BMI, body mass index; CO, control; CX3CR1, CX3C chemokine receptor 1; DAF-2DA, 4,5-diaminofluorescein diacetate; eNOS, endothelial NOS; hrTST, hour of total sleep time; L-NAME, NG -nitro-L-arginine methyl ester; MFI, median fluorescence intensity; NOS, NO synthase; OSA, obstructive sleep apnoea; OSAab, children with OSA with abnormal endothelial function; OSAn, children with OSA with normal endothelial function; PBMC, peripheral blood mononuclear cell; PE, phycoerythrin; SpO2 , peripheral oxygen saturation; T max , time to reach maximal reperfusion. 1 These authors contributed equally to the study. Correspondence: Professor Leila Kheirandish-Gozal (email [email protected]).
C The Authors Journal compilation C 2014 Biochemical Society
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children, as well as with the presence of endothelial dysfunction [20–23]. On the basis of such observations, we sought to explore the potential factors underlying the large spectrum of individual susceptibility of the vascular system in paediatric OSA [24]. In this context, we have identified genetic and immunological processes that appear to significantly contribute to vascular dysfunction [18,19,25]. A critical step in atherogenesis is the infiltration of monocytes into the sub-endothelial space of large arteries where they differentiate into macrophages and become functionally active. Macrophage accumulation within atheromatous plaques is a hallmark finding of all stages of atherosclerosis [26–28]. Fractalkine [CX3CL1 (CX3C chemokine ligand 1)] is a proinflammatory chemokine that induces chemotaxis of circulating monocytes through interaction with its ligand, CX3CR1 (CX3C chemokine receptor 1), that is highly expressed on activated pro-inflammatory monocytes and plays an important mechanistic role in atherosclerosis [29]. Since post-occlusive hyperaemic responses are NO-dependent [30] and monocytes express functional eNOS [endothelial NOS (NO synthase)] [31], we hypothesized that children with OSA and abnormal endothelial function (OSAab) would exhibit changes in monocyte subsets that would be associated with reductions in NO production, whereas children with OSA but with preserved endothelial function (OSAn) would have preserved monocyte populations with intact NO release. In other words, it was postulated that monocytes that express eNOS [32,33] would be less pro-inflammatory than those with reduced NO production, the latter expressing higher levels of CX3CR1, and therefore reflect improved preservation of endothelial functional integrity.
MATERIALS AND METHODS Subjects The study was approved by the University of Chicago Human Research Committee and informed consent was obtained from the legal caregiver of each participant. Consecutive healthy prepubertal children (age, 4–12 years) from the community and consecutive patients being evaluated for habitual snoring who were polysomnographically diagnosed with OSA and agreed to participate were recruited to investigate endothelial function. All participants underwent baseline overnight polysomnography and measurement of endothelial function, followed by a fasting blood draw in the morning.
Anthropometry Height was measured with a stadiometer and recorded to the nearest 0.1 cm. Weight was recorded to the nearest 0.1 kg. Height and weight centiles were calculated using the Centre for Disease Control 2000 growth charts for the U.S.A. BMI (body mass index) Z-scores were calculated using The Children’s Hospital of Philadelphia BMI and Z-score calculation in children online software (http://stokes.chop.edu/web/zscore).
Sphygmomanometry All children had arterial blood pressure measured non-invasively using an automated mercury sphygmomanometer (Welch Allyn) at the brachial artery using a guidelines-defined appropriate cuff size on the non-dominant arm [34]. Two consecutive blood pressure measurements were made while children lay supine with the head of the bed elevated to 45◦ . A non-standard posture of blood pressure measurements was chosen in order to mimic the posture utilized during endothelial function testing, which was both comfortable for children and best minimized arm motion artifact during measurement. Blood pressure measurements were made in the evening before the sleep study.
Overnight polysomnography Polysomnography was conducted and scored using standard approaches [35,36], and an obstructive AHI (apnoea/hypopnoea index) >2/hrTST (hour of total sleep time) along with a nadir SpO2 (peripheral oxygen saturation) 2/hrTST served as OSA criteria.
Endothelial function Endothelial function was assessed upon awakening from the sleep study in the morning, using the hyperaemic test after cuff-induced occlusion of wrist arteries [16]. In brief, a laser Doppler sensor (Periflux 5000 System; Perimed) was placed over the volar aspect of the hand at the first finger distal metacarpal surface and the hand was secured and immobilized. Once cutaneous blood flow readings became stable, a cuff placed at the forearm and connected to a computer was inflated to supra-systolic pressures and the blood flow signal declined to undetectable levels. The cuff was rapidly deflated and the laser Doppler measured hyperaemic responses. The time to maximal regional blood flow after occlusion release [T max (time to reach maximal reperfusion)] is representative of the post-occlusion hyperaemic response, an index of endothelial function. A T max >45 s was considered as indicative of abnormal endothelial function [37].
Blood tests Exclusion criteria All children found hypertensive or using anti-hypertensive therapies were excluded (n = 4). Furthermore, children with diabetes (fasting serum glucose 120 mg/dl; n = 2), with a craniofacial, neuromuscular or defined genetic syndrome, and children on chronic anti-inflammatory therapy (n = 7), or with any known acute or chronic illness were excluded. In addition, children placed on sympathomimetic agents such as psychostimulants were not tested (n = 13).
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C The Authors Journal compilation C 2014 Biochemical Society
Fasting blood samples were drawn by venipuncture in the morning immediately after endothelial function testing, and serum lipid concentrations were examined using standard laboratory techniques.
Processing of peripheral blood mononuclear cells PBMCs (peripheral blood mononuclear cells) were immediately isolated with Ficoll–Paque PREMIUM (GE Healthcare Life Sciences). PBMCs were counted, tested for viability (>90 % viable) and frozen using 20 % DMSO in FBS at a concentration of
Monocytes and endothelial function in OSA
(3–5)×106 cells/ml of freezing medium. Cryovials were immediately transferred to a controlled cooling container (Mr. Frosty Freezing Container; Thermo Scientific), kept at − 80 ◦ C overnight, and transferred to a liquid nitrogen tank for storage. All samples were processed in batches of 6–12 samples within
Nitric oxide production by monocytes in children with OSA and endothelial dysfunction
Clinical Science
www.clinsci.org
Leila KHEIRANDISH-GOZAL∗1 , Yang WANG∗1 , Ryan C. DUGGAN†, Sindhuja HARSHAN VARDHAN∗ , Hui-Leng TAN∗ , Helena MOLERO RAMIREZ∗ , Abdelnaby KHALYFA∗ , Rakesh BHATTACHARJEE∗ , Hari P.R. BANDLA∗ and David GOZAL∗ ∗ Section of Sleep Medicine, Department of Pediatrics, University of Chicago, Chicago, IL 60637, U.S.A. †Flow Cytometry Core Facility, Pritzker School of Medicine, Biological Sciences Division, University of Chicago, Chicago, IL 60637, U.S.A.
Abstract OSA (obstructive sleep apnoea) is associated with a higher risk for alterations in post-occlusive hyperaemia, an eNOS (endothelial NO synthase)-dependent endothelial response. However, since not all children manifest endothelial dysfunction, we hypothesized that differences in circulating monocyte subsets and NO production may underlie the vascular phenotype in paediatric OSA. Matched pre-pubertal children with OSA with abnormal endothelial function (OSAab) and with normal endothelial function (OSAn), and controls (CO) were recruited. Peripheral blood mononuclear cells were subtyped into CD14 + and CD16 + cells, and NO production was assessed using flow cytometry. Endothelial dysfunction was defined as T max (time to reach maximal reperfusion) >45 s by laser Doppler flowmetry. A total of 11 OSAab, 12 OSAn and 12 CO-matched children completed the study. The OSAab group had increased CD16 + and decreased CD14 + cell numbers. They also had increased CX3CR1 (CX3C chemokine receptor 1) expression in CD16 + monocytes (P < 0.01). Furthermore, monocytes from the OSAab group exhibited overall reduced NO production (787 + − 71 compared with 1226 + − 229 and 1089 + − 116 median fluorescence intensity in the OSAn group and CO children respectively; P < 0.01). Significant bivariate associations emerged between NO production, monocyte subsets, CX3CR1 in CD16 + monocytes, the CD14 + /CD16 + ratio and T max . Thus OSA in children is associated with increased numbers of pro-inflammatory monocytes and reduced NO production in circulating monocytes that are closely associated with endothelial function. Key words: children, endothelial function, inflammation, monocyte, sleep apnoea
INTRODUCTION OSA (obstructive sleep apnoea) is a highly prevalent paediatric disorder that is typically associated with increased upper airway resistance during sleep, leading to major alterations in intrathoracic pressure swings during breathing, abnormal gas exchange and disrupted sleep patterns [1]. Autonomic imbalance, particularly manifesting as increased sympathetic activation [2,3], along with evidence of systemic oxidative stress and inflammation, are therefore probable players in the putative adverse cardiovascular consequences of OSA, including pro-atherogenic lipid alterations [4–6]. In children, cross-sectional studies have now repeatedly shown that moderate-to-severe OSA is associated with a higher
risk of cardiovascular morbidity, primarily presenting as increased systemic and pulmonary vascular blood pressures that can lead to ventricular remodelling [7–12]. However, more subtle phenotypic changes, such as disturbances in blood pressure regulation (e.g. loss of sleep-associated blood pressure dipping) or altered endothelial function, an early precursor of atherosclerosis, can be frequently detected as well [13–16]. In the context of endothelial dysfunction, it is important to note that not every child, even with severe OSA, displays abnormal endothelial function as measured by post-occlusive hyperaemic responses [16–19]. Despite a large degree of variance, circulating biomarkers of inflammation and vascular injury, such as adhesion molecules, circulating microparticles and myeloid-related protein 8/14, and other inflammatory mediators correlate with OSA severity in
Abbreviations: AHI, apnoea/hypopnoea index; BMI, body mass index; CO, control; CX3CR1, CX3C chemokine receptor 1; DAF-2DA, 4,5-diaminofluorescein diacetate; eNOS, endothelial NOS; hrTST, hour of total sleep time; L-NAME, NG -nitro-L-arginine methyl ester; MFI, median fluorescence intensity; NOS, NO synthase; OSA, obstructive sleep apnoea; OSAab, children with OSA with abnormal endothelial function; OSAn, children with OSA with normal endothelial function; PBMC, peripheral blood mononuclear cell; PE, phycoerythrin; SpO2 , peripheral oxygen saturation; T max , time to reach maximal reperfusion. 1 These authors contributed equally to the study. Correspondence: Professor Leila Kheirandish-Gozal (email [email protected]).
C The Authors Journal compilation C 2014 Biochemical Society
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L. Kheirandish-Gozal and others
children, as well as with the presence of endothelial dysfunction [20–23]. On the basis of such observations, we sought to explore the potential factors underlying the large spectrum of individual susceptibility of the vascular system in paediatric OSA [24]. In this context, we have identified genetic and immunological processes that appear to significantly contribute to vascular dysfunction [18,19,25]. A critical step in atherogenesis is the infiltration of monocytes into the sub-endothelial space of large arteries where they differentiate into macrophages and become functionally active. Macrophage accumulation within atheromatous plaques is a hallmark finding of all stages of atherosclerosis [26–28]. Fractalkine [CX3CL1 (CX3C chemokine ligand 1)] is a proinflammatory chemokine that induces chemotaxis of circulating monocytes through interaction with its ligand, CX3CR1 (CX3C chemokine receptor 1), that is highly expressed on activated pro-inflammatory monocytes and plays an important mechanistic role in atherosclerosis [29]. Since post-occlusive hyperaemic responses are NO-dependent [30] and monocytes express functional eNOS [endothelial NOS (NO synthase)] [31], we hypothesized that children with OSA and abnormal endothelial function (OSAab) would exhibit changes in monocyte subsets that would be associated with reductions in NO production, whereas children with OSA but with preserved endothelial function (OSAn) would have preserved monocyte populations with intact NO release. In other words, it was postulated that monocytes that express eNOS [32,33] would be less pro-inflammatory than those with reduced NO production, the latter expressing higher levels of CX3CR1, and therefore reflect improved preservation of endothelial functional integrity.
MATERIALS AND METHODS Subjects The study was approved by the University of Chicago Human Research Committee and informed consent was obtained from the legal caregiver of each participant. Consecutive healthy prepubertal children (age, 4–12 years) from the community and consecutive patients being evaluated for habitual snoring who were polysomnographically diagnosed with OSA and agreed to participate were recruited to investigate endothelial function. All participants underwent baseline overnight polysomnography and measurement of endothelial function, followed by a fasting blood draw in the morning.
Anthropometry Height was measured with a stadiometer and recorded to the nearest 0.1 cm. Weight was recorded to the nearest 0.1 kg. Height and weight centiles were calculated using the Centre for Disease Control 2000 growth charts for the U.S.A. BMI (body mass index) Z-scores were calculated using The Children’s Hospital of Philadelphia BMI and Z-score calculation in children online software (http://stokes.chop.edu/web/zscore).
Sphygmomanometry All children had arterial blood pressure measured non-invasively using an automated mercury sphygmomanometer (Welch Allyn) at the brachial artery using a guidelines-defined appropriate cuff size on the non-dominant arm [34]. Two consecutive blood pressure measurements were made while children lay supine with the head of the bed elevated to 45◦ . A non-standard posture of blood pressure measurements was chosen in order to mimic the posture utilized during endothelial function testing, which was both comfortable for children and best minimized arm motion artifact during measurement. Blood pressure measurements were made in the evening before the sleep study.
Overnight polysomnography Polysomnography was conducted and scored using standard approaches [35,36], and an obstructive AHI (apnoea/hypopnoea index) >2/hrTST (hour of total sleep time) along with a nadir SpO2 (peripheral oxygen saturation) 2/hrTST served as OSA criteria.
Endothelial function Endothelial function was assessed upon awakening from the sleep study in the morning, using the hyperaemic test after cuff-induced occlusion of wrist arteries [16]. In brief, a laser Doppler sensor (Periflux 5000 System; Perimed) was placed over the volar aspect of the hand at the first finger distal metacarpal surface and the hand was secured and immobilized. Once cutaneous blood flow readings became stable, a cuff placed at the forearm and connected to a computer was inflated to supra-systolic pressures and the blood flow signal declined to undetectable levels. The cuff was rapidly deflated and the laser Doppler measured hyperaemic responses. The time to maximal regional blood flow after occlusion release [T max (time to reach maximal reperfusion)] is representative of the post-occlusion hyperaemic response, an index of endothelial function. A T max >45 s was considered as indicative of abnormal endothelial function [37].
Blood tests Exclusion criteria All children found hypertensive or using anti-hypertensive therapies were excluded (n = 4). Furthermore, children with diabetes (fasting serum glucose 120 mg/dl; n = 2), with a craniofacial, neuromuscular or defined genetic syndrome, and children on chronic anti-inflammatory therapy (n = 7), or with any known acute or chronic illness were excluded. In addition, children placed on sympathomimetic agents such as psychostimulants were not tested (n = 13).
324
C The Authors Journal compilation C 2014 Biochemical Society
Fasting blood samples were drawn by venipuncture in the morning immediately after endothelial function testing, and serum lipid concentrations were examined using standard laboratory techniques.
Processing of peripheral blood mononuclear cells PBMCs (peripheral blood mononuclear cells) were immediately isolated with Ficoll–Paque PREMIUM (GE Healthcare Life Sciences). PBMCs were counted, tested for viability (>90 % viable) and frozen using 20 % DMSO in FBS at a concentration of
Monocytes and endothelial function in OSA
(3–5)×106 cells/ml of freezing medium. Cryovials were immediately transferred to a controlled cooling container (Mr. Frosty Freezing Container; Thermo Scientific), kept at − 80 ◦ C overnight, and transferred to a liquid nitrogen tank for storage. All samples were processed in batches of 6–12 samples within
