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Study of endothelial dysfunction and vascular inflammation in sleep apnea, obesity and aged humans
Egyptian Journal of Chest Diseases and Tuberculosis (2012) 61, 469–476
The Egyptian Society of Chest Diseases and Tuberculosis
Egyptian Journal of Chest Diseases and Tuberculosis www.elsevier.com/locate/ejcdt www.sciencedirect.com
ORIGINAL ARTICLE
Study of endothelial dysfunction and vascular inflammation in sleep apnea, obesity and aged humans Essam El-Shamy a, Samir Eskaros a, Abeer E. Dief a, Seham Z. Nassar a, Anwar Algenady b,*, Nermeen Hossam Eldin c a b c
Physiology Department, Egypt Chest Diseases Department, Egypt Clinical and Chemical Pathology Department, Egypt
Received 22 August 2012; accepted 3 September 2012 Available online 1 February 2013
KEYWORDS Obstructive sleep apnea; Nuclear factor jB; Cardiovascular diseases; Endothelial dysfunction; Inflammation
Abstract Background: Obstructive sleep apnea (OSA) has been associated with cardiovascular complications. The overnight repetitive hypoxia represents a form of oxidative stress in the vasculature which may activate the oxidant-sensitive, proinflammatory transcription factor nuclear factor jB (NF-jB), affecting endothelial function and atherosclerosis. Aim: We investigated whether the endothelial alterations attributed to OSA rather than to other confounding factors. Also, the production of inflammatory cytokine nuclear factor-kappa b (NFKb) was investigated as the molecular mechanism involved in vascular endothelial dysfunction with OSA. Material and methods: Sixty subjects underwent attended nocturnal polysomnography were grouped by apnea hypopnea index: control (AHI<5/h) and OSA cases (AHI>5/h) the cases were further classified according to age and BMI into subgroup IIA: OSA, non-obese, middle age (35–52 y), subgroup IIB: OSA, non-obese, older age group (55–68 y), subgroup IIIA: OSA, obese, middle age group (35–52 y) and subgroup IIIB: OSA, obese, older age group (55–68 y).
Abbreviations: OSA, Obstructive sleep apnea; AHI, Apnea hypopnea index; BMI, body mass index; NF-jB, Nuclear factor jB; sVCAM-1, soluble Vascular cell adhesion molecule; FMD, Flow mediated dilatation * Corresponding author. Tel.: +20 1223926049. E-mail address: [email protected] (A. Algenady). Peer review under responsibility of The Egyptian Society of Chest Diseases and Tuberculosis.
Production and hosting by Elsevier 0422-7638 ª 2012 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejcdt.2012.09.013
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E. El-Shamy et al. A morning venous blood sample was obtained. Neutrophils were isolated, and NF-jB activity was determined. Plasma sVCAM-1 was assayed by enzyme-linked immunosorbent assay and flow-mediated dilation (FMD) was performed. Results: NF-jB activation and plasma level of sVCAM-1 were significantly increased in OSA patients as compared to the control group and there was no significant difference between the obese and non-obese cases also no significant difference between the middle and old age cases. The degree of NF-jB activation was positively correlated with indices of apnea severity(r = 0.938; p < 0.001). FMD was significantly decreased in OSA patients as compared to the control group. Conclusion: These findings suggested that OSA is an independent risk factor for cardiovascular morbidity also that OSA leads to NF-jB activation, which may constitute an important pathway linking OSA with systemic inflammation and cardiovascular disease. ª 2012 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V. All rights reserved.
Introduction The endothelium is a thin mono cellular layer that covers all the inner surface of the blood vessels, separating the circulating blood from the tissues. In the past, the endothelium was considered to be inert and inactive, However, this view has been completely changed with founding that endothelial cells are dynamic and exert significant autocrine, paracrine and endocrine actions that influence smooth muscle cells, platelets and peripheral leucocytes. It releases agents that regulate vasomotor function, trigger inflammatory processes, and affect hemostasis [1]. It was postulated that impaired endothelial function exerts an important role in the development of cardiovascular diseases associated with obstructive sleep apnea [2]. Obstructive sleep apnea (OSA) is characterized by repeated episodes of apnea during sleep. Each cycle of apnea and resumption of ventilation is accompanied by arterial oxyhaemoglobin desaturation and resaturation. This overnight repetitive hypoxia and reoxygenation may represent a form of oxidative stress, which generates reactive oxygen species [3]. Many investigations, had established that OSA is an independent risk factor for many cardiovascular diseases. OSA is often associated with risk factors as aging, obesity, arterial hypertension, diabetes mellitus and ischemic heart disease. This increase in cardiovascular morbidity in patients with OSA is attributed to accelerated atherosclerosis. An early event of pathophysiologic importance in the atherosclerotic process is endothelial dysfunction [4]. Endothelial dysfunction is a broad term that was initially identified as impaired vasodilation due to diminished production or availability of nitric oxide (NO) and/or an imbalance in the relative contribution of endothelium-derived relaxing and contracting factors, such as endothelin-1 (ET-1), angiotensin, and oxidants. A broader understanding of the term would include not only reduced vasodilation but also a proinflammatory and prothrombic state associated with dysfunction of the endothelium [5]. Endothelial cells regulate leucocyte movement into tissues via carefully regulated processes involving adhesion molecules. Adhesion molecules mediate the adhesion of leucocytes to the endothelium by binding to specific ligands on the leucocytes. Adhesion molecules allow movement of activated inflammatory cells out of the circulation to the site of infection or injury whilst at the same time, maintaining the integrity of the endothelium itself [6]. Endothelial cells express E-selectin, P-selectin, ICAM-1 and VCAM-1. VCAM-1 binds monocytes and T lymphocytes, the
first step of invasion of the vessel wall by inflammatory cells. Once adherent, the monocytes transmigrate into the tunica intima, the innermost layer of the arterial wall [7]. Once within the arterial intima, the monocytes develop into macrophages and begin to express scavenger receptors, that internalize modified lipoproteins. Internalization of these lipoprotein particles gives rise to lipid-laden macrophages or foam cells, which characterize early atherosclerotic lesions [8]. Oxidative stress may activate endothelial cells and leukocytes, resulting in increased expression of several glycoproteins, including the adhesion molecules as vascular cell adhesion molecule-1 (VCAM-1) [9]. These molecules facilitate the attachment of macrophages and leukocytes and their migration to the subendothelial space, where their products facilitate the atherogenic process. Adhesion molecules on these cells are a well-defined early marker of premature atherosclerosis [8]. This reactive oxygen species may also activate the oxidantsensitive, proinflammatory transcription factor nuclear factor kappa b (NF-Kb), increasing systemic inflammation [10]. NF-jB is one of the most important redox responsive transcription factors, which has a role in the activation of the promoter activity of over 200 genes of many cytokines, growth factors, adhesion molecules and enzymes involved in immune and inflammatory responses, many of which play an important role in the pathophysiology of athrescleosis and other cardiovascular diseases [11]. Thus, it is possible that OSA could promote systemic inflammation and cardiovascular pathology by inducing NF-jB-mediated expression of proinflammatory and proatherogenic genes. Several tools has been done to assess the endothelium dysfunction as measurement of circulating markers of endothelial cell damage as ICAM-1, VCAM-1, E-selectin and plasma endothelin-1. A widely reported noninvasive method to assess endothelial dysfunction, relies on high-resolution ultrasound measurements of flow-mediated dilation of the brachial artery to reactive hyperemia after a short period of forearm ischemia [12]. Aim of the work This work was designed to investigate whether OSA is associated with endothelium dysfunction compared with age and body mass index (BMI)–matched control subjects as indicated by VCAM-1 and FMD. Also, to investigate the production of
Study of endothelial dysfunction and vascular inflammation in sleep apnea, obesity and aged humans inflammatory cytokine nuclear factor-kappa b (NF-Kb) as the molecular mechanism involved in vascular endothelial dysfunction with OSA. Subjects and methods The present study was carried out on 60 persons referred to sleep laboratory in the chest department in the main University hospital. The inclusion criteria for the studied subjects were; patients with newly diagnosed obstructive sleep apnea (OSA) [defined as an apnea-hypopnea index (AHI) of 5 obstructive events per hour of sleep], no any other clinical diseases as assessed by medical history, physical examination, blood chemistry, and resting ECGs. However, the exclusion criteria were smoking, diabetes mellitus, and subjects taking any medications or herbal supplements. The studied subjects were subdivided according to their age, body mass index and polysomnography data into 3 groups: Group I: (control group) consisted of 20 nonsmokers healthy volunteers (BMI < 25) (AHI < 5); Group II (OSA, non-obese group) consisted of 20 non-obese OSA patients (BMI < 25) (AHI P 5) and Group III (OSA, obese group) consisted of 20 obese OSA patients (BMI > 25) (AHI P 5). All the 3 groups were subdivided into two subgroups 10 persons each, subgroup A (Middle age group 35–52 y) and subgroup B (old age group 55–68 y). All the participants were subjected to; history taking, clinical examination, routine laboratory investigations, resting ECG, assessment of anthropometric data using fat absorbetiometer in the physiology department. Nocturnal polysomnography at sleep lab chest department, Alexandria university hospital were done and flow-mediated dilation of the brachial artery (FMD) was done using high-resolution ultrasonography in the Radiology department [13]. Moreover, fasting blood samples were collected from all participants between 9 and 11 AM within 48 h of polysomnography for estimation of: NF-jB by ELISA Neutrophils (95–98% purity) were isolated using Ficoll–Paque centrifugation and were subsequently purified by dextran sedimentation and hypotonic lysis of residual erythrocytes. Purification of cellular nuclear extracts using Cayman’s nuclear extraction kit to isolate nuclear protein. NF-jB was quantified using NF-jB (human P50/P65) combo transcription factor assay kit (Caymann) [14]. o Soluble vascular cell adhesion molecule-1 (sVCAM-1) using ELIZA Kit [15]. Statistics The values of the measured parameters were expressed as mean ± SD or as median (min–max). The difference between the studied groups was determined using ANOVA test (F-test) and The Kruskal-Wallis test (H-test). Pearson’s correlation coefficient was performed for evaluating the biochemical and radiological variables. P < 0.05 values were considered significant. All statistical analyses were processed using SPSS for windows Version 18.
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Results Clinical characteristics and laboratory values of the patients upon initial admission are summarized in Table 1. All the 3 groups were subdivided into two subgroups, subgroup A (Middle age group 35–52 y) and subgroup B (old age group 55–68 y).It was observed that BMI is significantly increased in group III as compared to the other two groups. There were no significant differences between the 3 groups in the fasting blood sugar and serum cholesterol levels. Sleep studies data AHI, ODI, Mini PaO2, and average pulse rate are shown in Table 2. Apnea-hypopnea index (AHI) and oxygen desaturation index (ODI) increased significantly as expected between the control group I with AHI (2.25 ± 1.16) and ODI (1.53 ± 0.32) as compared to OSA patient with AHI (24.07 ± 15.14) and ODI (23.49 ± 22.07). Mini PaO2 was significantly decreased in OSA patients (76.20 ± 6.76) as compared to the control group I (92.45 ± 1.66) and average pulse rate was significantly increased in OSA patients as compared to the control group. Endothelial dysfunction and vascular inflammation investigations are shown in Table 3. It is observed that NF-jB activation was significantly increased in OSA patients as compared to the control group. On the other hand there was no significant difference between the obese and non-obese cases also no significant difference between the middle and old age cases. Serum sVCAM-1 was significantly increased in OSA patients as compared to the control group. On the other hand there was no significant difference between the obese and non-obese cases also no significant difference between the middle and old age cases. However, there was a significant difference in the control group between the middle age as compared to the old age subgroup. The reactive dilatation of the brachial artery was significantly decreased in OSA patients as compared to the control group. On the other hand there was no significant difference between the obese and non-obese cases also no significant difference between the middle and old age cases. However, there was a significant difference in the control group between the middle age as compared to the old age subgroup.
Correlation study between NF-jB activity, sVCAM-1 and the severity of OSA
n Neutrophil NF-jB activity correlates with indices of OSA severity as assessed by AHI. Neutrophil NF-jB activity was positively correlated with AHI (r = 0.938; p < 0.001), ODI (r = 0.927; p < 0.001), and negative with Mini PaO2 (r = 0.888; p < 0.001). n A significant strong positive correlation was found between sVCAM-1 level and AHI (r = 0.914, p < 0.001) and also with the degree of hypoxaemia as assessed by ODI (r = .928, p < 0.001) and strong negatively correlation with Mini PaO2 (r = .877, p < 0.001) (see Table 4).
E. El-Shamy et al.
Values are mean ± SD. Except serum cholesterol is median (min–max). * p < 0.05 vs group IA.
B
F = 715 (.615) H = 11.54 (.042)
40 ± 3.68 46.77 ± 16.13* 7/3 83.20 ± 11.37 189.00(175 198)
A B A
38.7 ± 2.83 23.99 ± 0.60 7/3 84.00 ± 12.84 181.50(157 193)
B
59.8 ± 1.75* 23.92 ± 0.60 6/4 87.40 ± 7.72 188.00(157 197) Age (years) BMI (kg/m2) Sex M/F Fasting blood glucose (mg/dl) Serum Cholesterol (mg/dl)
39 ± 2.94 23.55 ± 0.63 7/3 87.20 ± 12.69 172.50(152 197)
A
59.3 ± 1.69 * 24.36 ± 0.74 6/4 90.40 ± 7.59 175.00(145 197)
III II I
Clinical characteristics of the 3 study groups. Table 1
Discussion
58.2 ± 2.04* 39.29 ± 4.30* 7/3 89.10 ± 9.79 189.50(179 198)
F = 172.81 (<.001) F = 21.98 (<.001)
Test statistic (p-value)
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The vascular endothelium participates in control of various vascular functions through regulation of vasoactive mediators in response to physical or biochemical stimuli in the body [1]. Endothelial injury, at cellular level or tissue level, is an important initial event in atherogenesis, preceding thickening of intima and formation of atherosclerotic plaques [8]. Endothelial dysfunction was shown to have a predictive value for cardiovascular events in patients with chest pain and/or coronary artery disease [16]. It was shown also to be the early event in accelerated atherosclerosis attributed to OSA [4]. Obstructive sleep apnea (OSA) is currently considered to be an inflammatory disorder. Evidence suggests that the chronic intermittent hypoxia and, possibly, sleep loss and fragmentation associated with OSA increase the levels of various markers of inflammation. In particular, increased levels of inflammatory cytokines, adhesion molecules, and activation of circulating neutrophils [8]. The present work was designed to assess the development of endothelial dysfunction and inflammation in patients with OSA compared with age and body mass index (BMI)– matched control subjects. Regarding the NF-jB activity in circulating neutrophils of OSA patients and the controls the current data demonstrated significant increase NF-jB activity in circulating neutrophils of OSA patients. Moreover, the degree of NF-jB activity is positively correlated with OSA severity. Neutrophil NF-jB activity was positively correlated with AHI, ODI, and mini Pao2. It has been well documented that NF-jB activation is an integral part of the pathophysiology of numerous cardiovascular disorders [11]. our findings suggest that NF-jB could be an important molecule linking the patho-physiological consequences of OSA with cardiovascular disease. There is ample evidence supporting the essential role of NF-jB activation in the pathogenesis of various cardiovascular diseases. Many genes, whose expression is regulated by NF-jB, have been implicated in atherogenesis and cerebrovascular disease [11,17]. The relevance of NF-jB activation in OSA is demonstrated by the presence of elevated levels of the products of several NF-jB controlled genes in OSA patients. Data from previous studies have shown that OSA patients have increased levels of the adhesion molecules sEselectin, sVCAM-1, ICAM-1, and L-selectin as well as the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-a, all of which are produced by NF-jB regulated genes [18]. Because these inflammatory proteins have been shown to play a crucial role in the pathogenesis of atherosclerosis [17], these observations collectively suggested that OSA may contribute to atherogenesis by stimulating NF-jB, with subsequent activation of cell adhesion and other proinflammatory pathways. It has been postulated that intermittent hypoxia associated with recurrent obstructive apneas is a crucial pathophysiological feature of OSA which generates increased ROS [19]. These free radicals may then activate oxidant-sensitive transcription factors such as NF-jB. As a result, production of proinflammatory mediators and adhesion molecules is increased, in addition to activation of neutrophils and endothelial cells. Increased adhesion of activated inflammatory cells
Study of endothelial dysfunction and vascular inflammation in sleep apnea, obesity and aged humans Table 2
473
sleep studies data AHI, ODI, Mini PaO2, and average pulse rate. Control
Cases
Mean AHI (events/h) ODI (events/h) Mini PaO2 (mmHg) Average pulse (beats/min)
SD
2.25 1.53 92.45 66.90
t-test
Mean *
1.16 .32 1.66 3.67
24.07 23.49* 76.20* 84.13*
SD
t
p-value
15.14 22.07 6.76 10.39
9.06 6.29 14.35 9.38
<.001 <.001 <.001 <.001
Values are mean ± SD. * p < 0.05 vs control group I.
Table 3
The NF-kB activity, serum s-VCAM-1 and FMD in the studied groups. Group I
II
A NF-KB(lg/well) sVCAM (ng/ml) FMD% a,b,c
B a
5.50 (3.0–8.0) 538.00a (470–708) 9.10a (8.10–9.60)
III
A a
5.00 (2.00–8.0) 762.00c (724–816) 7.90c (7.10–8.20)
B a
21.00 (15.0–65.0) 1617.0b (1224–2304) 4.30b (3.10–5.60)
A a
21.00 (15.0–65.0) 1686.00b (1492–3002) 4.20b (2.90–4.60)
B a
19.50 (15.0–58.0) 1375.00b (1260–2574) 4.85b (3.20–5.40)
25.00a (14.0–65.0) 1960.00b (1242–4378) 3.65b (2.50–4.50)
Values are median (min–max). The groups with the same small letter have no significant difference.
Table 4 Correlation between NF-jB activity, sVCAM-1 and severity of OSA. NFKB
r(p-value)
sVCAM1
R (p-value)
AHI
ODI
Mini PaO2
.938** (<.001) .955** (<.001)
.927** (<.001) .928** (<.001)
.888** (<.001) .877** (<.001)
to the endothelium is an important factor contributing to endothelial dysfunction and atherogenesis [8,9]. In Animal studies, Shuo et al, 2011 had studied the systemic production of inflammatory factors and activation of NF-jB in response to different levels of intermittent hypoxia in 160 male Wistar rats, which has a typical breathing pattern of OSA. They have found that the correlation of NFjB activation under intermittent hypoxia implies an important role of this transcription factor in inflammation-induced cardiovascular damage occurring during OSA [20]. Furthermore, other in vitro studies done by Ryan and colleagues, 2005 reported that, in an in vitro cell culture model, alternating cycles of hypoxia and re-oxygenation resulted in selective and dose-dependent activation of inflammatory NF-jB-dependent pathways [21]. These data go in line with other studies by Aung et al, 2006 who reported that neutrophils demonstrated significantly greater NF-jB activity in the mild to moderate and severe OSA groups than in the control subjects and this increased neutrophil NF-jB activity is reversed by CPAP treatment [10], Yamauchi et al, 2006 have measured activation of the NF-jB, in nine age-matched, nonsmoking, and non-hypertensive men with OSA symptoms and seven similar healthy subjects were measured at the same time and after the next
night of continuous positive airway pressure (CPAP). NF-kB activity in the OSA group were significantly higher than in the control group. One night of CPAP resulted in a reduction in NF-kappaB. They concluded that NF-kappaB activation occurs with sleep-disordered breathing. Such activation of NF-kappaB may contribute to the pathogenesis of atherosclerosis in OSA patients [22]. The present study has focused on activation of NF-jB in circulating neutrophils. While both monocytes and neutrophils have been implicated in the inflammatory processes leading to atherosclerosis, we chose to investigate circulating neutrophils since Richard Schulz et al, 2000 had demonstrated the release of superoxide from circulating neutrophils of patients with OSA in response to ex vivo stimulation, and is reversible with nasal CPAP therapy [23]. Furthermore, the neutrophil is known to play an important role in cardiovascular disease since infiltration of neutrophils into atherosclerotic plaque has been associated with plaque rupture and the occurrence of acute coronary events [24]. However, it is likely that OSA may also activate NF-jB in other cell types such as endothelial cells. This study also demonstrated that there was no significant difference between the obese and non-obese cases also no significant difference between the middle and old age cases which means that the co-morbid factors are not influencing our findings. This evidence indicates that the observed increase in NFjB activity is causally related to OSA, rather than to comorbid factors or to differences in BMI between groups. Thus, while obesity has been characterized as a proinflammatory state [25], and we cannot completely rule out a contribution of obesity itself to NF-jB activity, our findings support an independent effect of OSA on NF-jB activation as our study use a lean OSA group to definitely separate effects of obesity from those of OSA.
474 To confirm the biologic relevance of the observed change in NF-jB, we measured serum levels sVCAM-1, an important NF-jB controlled gene product, in OSA patients and in control subjects. There was a significant increase in the biochemical marker (sVCAM-1) in OSA patients as compared to age matched control groups. Furthermore our findings have shown that the concentration of circulating adhesion molecules correlated with the severity of sleep apnea. The current study also showed a significant relation between cellular adhesion molecules and the severity of hypoxemia as indicated by the ODI and mini PaO2 suggesting that the risk of cardiovascular events is perhaps related to the intermittent hypoxia observed during sleep and the level of hypoxaemia. It is plausible that intermittent hypoxia promotes oxygen radical formation [26] that leads to activation of transcriptional factors that upregulate the genetic expression of adhesion molecules [27]. Although the pathologic role of these cellular adhesion molecules in OSA is uncertain, studies have shown elevated circulating levels of adhesion molecules in subjects with OSA compared to those without sleep-disordered breathing [18]. Various circulating markers of endothelial dysfunction, including nitric oxide, soluble cell adhesion molecules, fibrinogen, and plasminogen activator inhibitor, have been reported to be altered in OSA [19,28]. This coincides with recent studies of Bernabe Jurado-Gamez et al,2012 who found that patients with OSA and greater intermittent hypoxia had worse endothelial function, and higher levels of ICAM-1 and P-selectin [29]. Hypoxia has been implicated in the induction of interleukin-1 and cellular adhesion molecules gene expression [30]. The present data confirm previous results that OSA syndrome is associated with a rise in circulating levels of adhesion molecules. Aung. et al, 2006 have found that the circulating levels of VCAM-1 are significantly increased in patients with OSA compared with those of a matched control group.[10] Ohga and coworkers, 1999 reported increased levels of ICAM-1, VCAM-1, and L-selectin levels in seven patients with OSA compared to a control group of ‘‘normal subjects’’. The authors suggested that the repetitive hypoxic stress during sleep might induce activation and provoke sustained release of these inflammatory mediators. Since cellular adhesion molecules mediate cellular interactions and transmigration of leukocytes across the vascular endothelial wall [31]. our findings suggest that OSA could contribute potentially to the inflammatory process implicated in atherogenesis. On the other hand, other studies have found no significant change. As Klein et al, 1995 who studied the in vitro influences of hypoxia on endothelial cell proliferation and expression of cell adhesion molecules. They found that the presentation of ICAM-1, VCAM-1, and E-Selectin was not affected by hypoxia or even reduced and they are not appear to be the major aspect in hypoxic injury [32]. The reactive dilatation of the brachial artery was assessed as a physical marker of endothelial dysfunction. FMD was significantly decreased in OSA patients as compared to the control group. On the other hand there was no significant difference between the obese and non-obese cases also no significant difference between the middle and old age cases. This indicates impaired reactive hyperaemia in OSA cases which indicates endothelial dysfunction. The mechanisms of vascular response to vessel occlusion followed by reactive hyperemia are clearly complex, involving
E. El-Shamy et al. myogenic, neurogenic, and vasculogenic components, mediated by a variety of metabolic alterations and vasoactive factors. The exact mechanisms apparently differ with vascular beds and evoking stimuli [33]. Flow-mediated dilation refers to nitric oxide-mediated vasodilatation resulting from shearmediated activation of endothelial nitric oxide synthase in response to an acute increase in blood flow [13]. The test has been shown to be accurate and reproducible [34]. Previous studies on vascular endothelial function in OSA have shown conflicting data. These studies have used different methods for evaluation of vascular endothelial function. For instance, Blunted vasodilation in response to infusion of acetylcholine, a vasodilator that stimulates endothelial release of nitric oxide, was demonstrated in forearm resistance vessels using occlusion plethysmography [35], but not confirmed in another study [36]. Impaired relaxation response to bradykinin in the forearm venous vasculature has been reported, suggesting endothelial dysfunction in the venous vasculature [37]. Using ultrasound Doppler method similar to ours, Mathias Grebe et al, 2006 had measured flow-mediated dilation (FMD) of the brachial artery by ultrasound in 10 otherwise healthy, untreated patients with OSA and 10 age-and sex-matched control subjects without sleep-disordered breathing before and after intravenous injection of the antioxidant vitamin C. They found that the reduced endothelial-dependent vasodilation in untreated patients with OSA acutely improves by the free radical scavenger vitamin C. These results are in favor of oxidative stress being responsible for the endothelial dysfunction in OSA [38]. In contrast to our findings, another study found no significant difference in conduit–vessel dilation between sleep apnea and control subjects. In that study, subjects underwent conduit–vessel studies at least 1 hour after resistance–vessel studies, which involved intra-arterial infusion of acetylcholine, nitroprusside and verapamil, and residual effect of drugs on the vasculature, which affected their response to reactive hyperemia, cannot be completely excluded [39]. To exclude the effect of obesity, we compare the FMD in lean and obese OSA patients, and there was no significant difference between both groups. In another study, OSA and subjects without OSA were matched for obesity, and the body mass index was not a significant independent determinant of FMD on multiple regression analysis. The change in FMD with treatment of OSA without any concomitant change in body weight and the use of a control group who did not receive nCPAP treatment provided further evidence that the improvement in endothelial function in this study sample, most of whom were obese, was attributed to the use of nCPAP, whereas a placebo effect could not be definitively excluded. OSA, therefore, may be associated with functional impairment (‘‘a premature aging effect’’) on the endothelium and on arterial stiffness [40]. Conclusions The findings of endothelial dysfunction in otherwise healthy subjects with OSA lend strong circumstantial evidence to an independent contribution of OSA to atherosclerosis. The present data also provide evidence for activation of the proinflammatory transcription factor NF-jB in OSA. This finding lends further support to an emerging hypothesis which postulates
Study of endothelial dysfunction and vascular inflammation in sleep apnea, obesity and aged humans that OSA contributes to cardiovascular disease by increasing systemic inflammation.
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[20] Li Shuo, Q. Xue-han, Zh Wei, Zh Yan, F. Jing, W. Nan-sheng, Zh Zhen, G. Run, C. Bao-yuan, Time-dependent inflammatory factor production and NFjB activation in a rodent model of intermittent hypoxia, Swiss Med. Wkly. 141 (2011) w13309. [21] S. Ryan, C.T. Taylor, W.T. McNicholas, Selective activation of inflammatory pathways by intermittent hypoxia in obstructive sleep apnea syndrome, Circulation 112 (2005) 2660–2667. [22] M. Yamauchi, S. Tamaki, K. Tomoda, M. Yoshikawa, A. Fukuoka, K. Makinodan, N. Koyama, T. Suzuki, Kimura H Evidence for activation of nuclear factor kappaB in obstructive sleep apnea, Sleep Breath 10 (4) (2006) 189–193. [23] R. Schulz, S. Mahmoudi, K. Hattar, U. Sibelius, H. Olschewski, K. Mayer, W. Seeger, F. Grimminger, Enhanced release of superoxide from polymorphonuclear neutrophils in obstructive sleep apnea. Impact of continuous positive airway pressure therapy., Am. J. Respir. Crit. Care Med. 162 (2 Pt 1) (2000) 566– 570. [24] T. Naruko, M. Ueda, K. Haze, A.C. van der Wal, C.M. van der Loos, A. Itoh, R. Komatsu, Y. Ikura, M. Ogami, Y. Shimada, S. Ehara, M. Yoshiyama, K. Takeuchi, J. Yoshikawa, A.E. Becker, Neutrophil infiltration of culprit lesions in acute coronary syndromes, Circulation 106 (23) (2002) 2894–2900. [25] H. Ghanim, A. Aljada, D. Hofmeyer, T. Syed, P. Mohanty, P. Dandona, Circulating mononuclear cells in the obese are in a proinflammatory state, Circulation 110 (12) (2004) 1564–1571. [26] R. Schulz, S. Mahmoudi, K. Hattar, Enhanced release of free oxygen radicals from polymorphonuclear neutrophils in obstructive sleep apnea, Am. J. Respir. Crit. Care Med. 159 (1999) A527. [27] C.D. Kim, Y.K. Kim, S.H. Lee, Rebamipide inhibits neutrophil adhesion to hypoxia/reoxygenation-stimulated endothelial cells via nuclear factor-B dependent pathway, J. Pharmacol. Exp. Ther. 294 (2000) 864–869. [28] T.J. Anderson, Assessment and treatment of endothelial dysfunction in human, J. Am. Coll. Cardiol. 34 (1999) 631–638. [29] J.-G. Bernabe, B.C. Carlos, C.B. Laura, M.H. Carmen, M.C. Luis, P.-J. Francisco, L.M. Jose, Association of cellular adhesion molecules and oxidative stress with endothelial function in obstructive sleep apnea, Intern. Med. 51 (2012) 363–368. [30] C. Combe, C.J. Burton, P. Dufourco, Hypoxia induces intercellular adhesion molecule-1 on cultured human tubular cells, Kidney Int. 51 (1997) 1703–1709. [31] E. Ohga, T. Nagase, T. Tomita, S. Teramoto, T. Matsuse, H. Katayama, Y. Ouchi, Increased levels of circulating ICAM-1, VCAM-1, and L-selectin in obstructive sleep apnea syndrome, J. Appl. Physiol. 87 (1) (1999) 10–14. [32] C.L. Klein, H. Kohler, F. Bittinger, Comparative studies on vascular endothelium in vitro; 2. Hypoxia: its influences on endothelial cell proliferation and expression of cell adhesion molecules, Pathology 63 (1995) 1–8. [33] M.J. Joyner, N.M. Dietz, Nitric oxide and vasodilation in human limbs, J. Appl. Physiol. 83 (1997) 1785–1796. [34] K.E. Sorensen, D.S. Celermajer, D.J. Spiegelhalter, D. Georgakopoulos, J. Robinson, O. Thomas, J.E. Deanfield, Noninvasive measurement of human endothelium-dependent arterial responses: accuracy and reproducibility, Br. Heart. J. 74 (1995) 247–253. [35] Y. Ohike, K. Kozaki, K. Iijima, M. Eto, T. Kojima, E. Ohga, Amelioration of vascular endothelial dysfunction in obstructive sleep apnea syndrome by nasal continuous positive airway pressure. Possible involvement of nitric oxide and asymmetric NG, NG Dimethylarginine, Circ. J. 69 (2005) 221–226. [36] H. Kraiczi, J. Hedner, Y. Peker, J. Carlson, Increased vasoconstrictor sensitivity in obstructive sleep apnea, J. Appl. Physiol. 89 (2000) 493–498. [37] H.W. Duchna, C. Guilleminault, R.A. Stoohs, J.L. Faul, H. Moreno, B.B. Hoffman, T.F. Blaschke, Vascular reactivity in
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The Egyptian Society of Chest Diseases and Tuberculosis
Egyptian Journal of Chest Diseases and Tuberculosis www.elsevier.com/locate/ejcdt www.sciencedirect.com
ORIGINAL ARTICLE
Study of endothelial dysfunction and vascular inflammation in sleep apnea, obesity and aged humans Essam El-Shamy a, Samir Eskaros a, Abeer E. Dief a, Seham Z. Nassar a, Anwar Algenady b,*, Nermeen Hossam Eldin c a b c
Physiology Department, Egypt Chest Diseases Department, Egypt Clinical and Chemical Pathology Department, Egypt
Received 22 August 2012; accepted 3 September 2012 Available online 1 February 2013
KEYWORDS Obstructive sleep apnea; Nuclear factor jB; Cardiovascular diseases; Endothelial dysfunction; Inflammation
Abstract Background: Obstructive sleep apnea (OSA) has been associated with cardiovascular complications. The overnight repetitive hypoxia represents a form of oxidative stress in the vasculature which may activate the oxidant-sensitive, proinflammatory transcription factor nuclear factor jB (NF-jB), affecting endothelial function and atherosclerosis. Aim: We investigated whether the endothelial alterations attributed to OSA rather than to other confounding factors. Also, the production of inflammatory cytokine nuclear factor-kappa b (NFKb) was investigated as the molecular mechanism involved in vascular endothelial dysfunction with OSA. Material and methods: Sixty subjects underwent attended nocturnal polysomnography were grouped by apnea hypopnea index: control (AHI<5/h) and OSA cases (AHI>5/h) the cases were further classified according to age and BMI into subgroup IIA: OSA, non-obese, middle age (35–52 y), subgroup IIB: OSA, non-obese, older age group (55–68 y), subgroup IIIA: OSA, obese, middle age group (35–52 y) and subgroup IIIB: OSA, obese, older age group (55–68 y).
Abbreviations: OSA, Obstructive sleep apnea; AHI, Apnea hypopnea index; BMI, body mass index; NF-jB, Nuclear factor jB; sVCAM-1, soluble Vascular cell adhesion molecule; FMD, Flow mediated dilatation * Corresponding author. Tel.: +20 1223926049. E-mail address: [email protected] (A. Algenady). Peer review under responsibility of The Egyptian Society of Chest Diseases and Tuberculosis.
Production and hosting by Elsevier 0422-7638 ª 2012 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejcdt.2012.09.013
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E. El-Shamy et al. A morning venous blood sample was obtained. Neutrophils were isolated, and NF-jB activity was determined. Plasma sVCAM-1 was assayed by enzyme-linked immunosorbent assay and flow-mediated dilation (FMD) was performed. Results: NF-jB activation and plasma level of sVCAM-1 were significantly increased in OSA patients as compared to the control group and there was no significant difference between the obese and non-obese cases also no significant difference between the middle and old age cases. The degree of NF-jB activation was positively correlated with indices of apnea severity(r = 0.938; p < 0.001). FMD was significantly decreased in OSA patients as compared to the control group. Conclusion: These findings suggested that OSA is an independent risk factor for cardiovascular morbidity also that OSA leads to NF-jB activation, which may constitute an important pathway linking OSA with systemic inflammation and cardiovascular disease. ª 2012 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V. All rights reserved.
Introduction The endothelium is a thin mono cellular layer that covers all the inner surface of the blood vessels, separating the circulating blood from the tissues. In the past, the endothelium was considered to be inert and inactive, However, this view has been completely changed with founding that endothelial cells are dynamic and exert significant autocrine, paracrine and endocrine actions that influence smooth muscle cells, platelets and peripheral leucocytes. It releases agents that regulate vasomotor function, trigger inflammatory processes, and affect hemostasis [1]. It was postulated that impaired endothelial function exerts an important role in the development of cardiovascular diseases associated with obstructive sleep apnea [2]. Obstructive sleep apnea (OSA) is characterized by repeated episodes of apnea during sleep. Each cycle of apnea and resumption of ventilation is accompanied by arterial oxyhaemoglobin desaturation and resaturation. This overnight repetitive hypoxia and reoxygenation may represent a form of oxidative stress, which generates reactive oxygen species [3]. Many investigations, had established that OSA is an independent risk factor for many cardiovascular diseases. OSA is often associated with risk factors as aging, obesity, arterial hypertension, diabetes mellitus and ischemic heart disease. This increase in cardiovascular morbidity in patients with OSA is attributed to accelerated atherosclerosis. An early event of pathophysiologic importance in the atherosclerotic process is endothelial dysfunction [4]. Endothelial dysfunction is a broad term that was initially identified as impaired vasodilation due to diminished production or availability of nitric oxide (NO) and/or an imbalance in the relative contribution of endothelium-derived relaxing and contracting factors, such as endothelin-1 (ET-1), angiotensin, and oxidants. A broader understanding of the term would include not only reduced vasodilation but also a proinflammatory and prothrombic state associated with dysfunction of the endothelium [5]. Endothelial cells regulate leucocyte movement into tissues via carefully regulated processes involving adhesion molecules. Adhesion molecules mediate the adhesion of leucocytes to the endothelium by binding to specific ligands on the leucocytes. Adhesion molecules allow movement of activated inflammatory cells out of the circulation to the site of infection or injury whilst at the same time, maintaining the integrity of the endothelium itself [6]. Endothelial cells express E-selectin, P-selectin, ICAM-1 and VCAM-1. VCAM-1 binds monocytes and T lymphocytes, the
first step of invasion of the vessel wall by inflammatory cells. Once adherent, the monocytes transmigrate into the tunica intima, the innermost layer of the arterial wall [7]. Once within the arterial intima, the monocytes develop into macrophages and begin to express scavenger receptors, that internalize modified lipoproteins. Internalization of these lipoprotein particles gives rise to lipid-laden macrophages or foam cells, which characterize early atherosclerotic lesions [8]. Oxidative stress may activate endothelial cells and leukocytes, resulting in increased expression of several glycoproteins, including the adhesion molecules as vascular cell adhesion molecule-1 (VCAM-1) [9]. These molecules facilitate the attachment of macrophages and leukocytes and their migration to the subendothelial space, where their products facilitate the atherogenic process. Adhesion molecules on these cells are a well-defined early marker of premature atherosclerosis [8]. This reactive oxygen species may also activate the oxidantsensitive, proinflammatory transcription factor nuclear factor kappa b (NF-Kb), increasing systemic inflammation [10]. NF-jB is one of the most important redox responsive transcription factors, which has a role in the activation of the promoter activity of over 200 genes of many cytokines, growth factors, adhesion molecules and enzymes involved in immune and inflammatory responses, many of which play an important role in the pathophysiology of athrescleosis and other cardiovascular diseases [11]. Thus, it is possible that OSA could promote systemic inflammation and cardiovascular pathology by inducing NF-jB-mediated expression of proinflammatory and proatherogenic genes. Several tools has been done to assess the endothelium dysfunction as measurement of circulating markers of endothelial cell damage as ICAM-1, VCAM-1, E-selectin and plasma endothelin-1. A widely reported noninvasive method to assess endothelial dysfunction, relies on high-resolution ultrasound measurements of flow-mediated dilation of the brachial artery to reactive hyperemia after a short period of forearm ischemia [12]. Aim of the work This work was designed to investigate whether OSA is associated with endothelium dysfunction compared with age and body mass index (BMI)–matched control subjects as indicated by VCAM-1 and FMD. Also, to investigate the production of
Study of endothelial dysfunction and vascular inflammation in sleep apnea, obesity and aged humans inflammatory cytokine nuclear factor-kappa b (NF-Kb) as the molecular mechanism involved in vascular endothelial dysfunction with OSA. Subjects and methods The present study was carried out on 60 persons referred to sleep laboratory in the chest department in the main University hospital. The inclusion criteria for the studied subjects were; patients with newly diagnosed obstructive sleep apnea (OSA) [defined as an apnea-hypopnea index (AHI) of 5 obstructive events per hour of sleep], no any other clinical diseases as assessed by medical history, physical examination, blood chemistry, and resting ECGs. However, the exclusion criteria were smoking, diabetes mellitus, and subjects taking any medications or herbal supplements. The studied subjects were subdivided according to their age, body mass index and polysomnography data into 3 groups: Group I: (control group) consisted of 20 nonsmokers healthy volunteers (BMI < 25) (AHI < 5); Group II (OSA, non-obese group) consisted of 20 non-obese OSA patients (BMI < 25) (AHI P 5) and Group III (OSA, obese group) consisted of 20 obese OSA patients (BMI > 25) (AHI P 5). All the 3 groups were subdivided into two subgroups 10 persons each, subgroup A (Middle age group 35–52 y) and subgroup B (old age group 55–68 y). All the participants were subjected to; history taking, clinical examination, routine laboratory investigations, resting ECG, assessment of anthropometric data using fat absorbetiometer in the physiology department. Nocturnal polysomnography at sleep lab chest department, Alexandria university hospital were done and flow-mediated dilation of the brachial artery (FMD) was done using high-resolution ultrasonography in the Radiology department [13]. Moreover, fasting blood samples were collected from all participants between 9 and 11 AM within 48 h of polysomnography for estimation of: NF-jB by ELISA Neutrophils (95–98% purity) were isolated using Ficoll–Paque centrifugation and were subsequently purified by dextran sedimentation and hypotonic lysis of residual erythrocytes. Purification of cellular nuclear extracts using Cayman’s nuclear extraction kit to isolate nuclear protein. NF-jB was quantified using NF-jB (human P50/P65) combo transcription factor assay kit (Caymann) [14]. o Soluble vascular cell adhesion molecule-1 (sVCAM-1) using ELIZA Kit [15]. Statistics The values of the measured parameters were expressed as mean ± SD or as median (min–max). The difference between the studied groups was determined using ANOVA test (F-test) and The Kruskal-Wallis test (H-test). Pearson’s correlation coefficient was performed for evaluating the biochemical and radiological variables. P < 0.05 values were considered significant. All statistical analyses were processed using SPSS for windows Version 18.
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Results Clinical characteristics and laboratory values of the patients upon initial admission are summarized in Table 1. All the 3 groups were subdivided into two subgroups, subgroup A (Middle age group 35–52 y) and subgroup B (old age group 55–68 y).It was observed that BMI is significantly increased in group III as compared to the other two groups. There were no significant differences between the 3 groups in the fasting blood sugar and serum cholesterol levels. Sleep studies data AHI, ODI, Mini PaO2, and average pulse rate are shown in Table 2. Apnea-hypopnea index (AHI) and oxygen desaturation index (ODI) increased significantly as expected between the control group I with AHI (2.25 ± 1.16) and ODI (1.53 ± 0.32) as compared to OSA patient with AHI (24.07 ± 15.14) and ODI (23.49 ± 22.07). Mini PaO2 was significantly decreased in OSA patients (76.20 ± 6.76) as compared to the control group I (92.45 ± 1.66) and average pulse rate was significantly increased in OSA patients as compared to the control group. Endothelial dysfunction and vascular inflammation investigations are shown in Table 3. It is observed that NF-jB activation was significantly increased in OSA patients as compared to the control group. On the other hand there was no significant difference between the obese and non-obese cases also no significant difference between the middle and old age cases. Serum sVCAM-1 was significantly increased in OSA patients as compared to the control group. On the other hand there was no significant difference between the obese and non-obese cases also no significant difference between the middle and old age cases. However, there was a significant difference in the control group between the middle age as compared to the old age subgroup. The reactive dilatation of the brachial artery was significantly decreased in OSA patients as compared to the control group. On the other hand there was no significant difference between the obese and non-obese cases also no significant difference between the middle and old age cases. However, there was a significant difference in the control group between the middle age as compared to the old age subgroup.
Correlation study between NF-jB activity, sVCAM-1 and the severity of OSA
n Neutrophil NF-jB activity correlates with indices of OSA severity as assessed by AHI. Neutrophil NF-jB activity was positively correlated with AHI (r = 0.938; p < 0.001), ODI (r = 0.927; p < 0.001), and negative with Mini PaO2 (r = 0.888; p < 0.001). n A significant strong positive correlation was found between sVCAM-1 level and AHI (r = 0.914, p < 0.001) and also with the degree of hypoxaemia as assessed by ODI (r = .928, p < 0.001) and strong negatively correlation with Mini PaO2 (r = .877, p < 0.001) (see Table 4).
E. El-Shamy et al.
Values are mean ± SD. Except serum cholesterol is median (min–max). * p < 0.05 vs group IA.
B
F = 715 (.615) H = 11.54 (.042)
40 ± 3.68 46.77 ± 16.13* 7/3 83.20 ± 11.37 189.00(175 198)
A B A
38.7 ± 2.83 23.99 ± 0.60 7/3 84.00 ± 12.84 181.50(157 193)
B
59.8 ± 1.75* 23.92 ± 0.60 6/4 87.40 ± 7.72 188.00(157 197) Age (years) BMI (kg/m2) Sex M/F Fasting blood glucose (mg/dl) Serum Cholesterol (mg/dl)
39 ± 2.94 23.55 ± 0.63 7/3 87.20 ± 12.69 172.50(152 197)
A
59.3 ± 1.69 * 24.36 ± 0.74 6/4 90.40 ± 7.59 175.00(145 197)
III II I
Clinical characteristics of the 3 study groups. Table 1
Discussion
58.2 ± 2.04* 39.29 ± 4.30* 7/3 89.10 ± 9.79 189.50(179 198)
F = 172.81 (<.001) F = 21.98 (<.001)
Test statistic (p-value)
472
The vascular endothelium participates in control of various vascular functions through regulation of vasoactive mediators in response to physical or biochemical stimuli in the body [1]. Endothelial injury, at cellular level or tissue level, is an important initial event in atherogenesis, preceding thickening of intima and formation of atherosclerotic plaques [8]. Endothelial dysfunction was shown to have a predictive value for cardiovascular events in patients with chest pain and/or coronary artery disease [16]. It was shown also to be the early event in accelerated atherosclerosis attributed to OSA [4]. Obstructive sleep apnea (OSA) is currently considered to be an inflammatory disorder. Evidence suggests that the chronic intermittent hypoxia and, possibly, sleep loss and fragmentation associated with OSA increase the levels of various markers of inflammation. In particular, increased levels of inflammatory cytokines, adhesion molecules, and activation of circulating neutrophils [8]. The present work was designed to assess the development of endothelial dysfunction and inflammation in patients with OSA compared with age and body mass index (BMI)– matched control subjects. Regarding the NF-jB activity in circulating neutrophils of OSA patients and the controls the current data demonstrated significant increase NF-jB activity in circulating neutrophils of OSA patients. Moreover, the degree of NF-jB activity is positively correlated with OSA severity. Neutrophil NF-jB activity was positively correlated with AHI, ODI, and mini Pao2. It has been well documented that NF-jB activation is an integral part of the pathophysiology of numerous cardiovascular disorders [11]. our findings suggest that NF-jB could be an important molecule linking the patho-physiological consequences of OSA with cardiovascular disease. There is ample evidence supporting the essential role of NF-jB activation in the pathogenesis of various cardiovascular diseases. Many genes, whose expression is regulated by NF-jB, have been implicated in atherogenesis and cerebrovascular disease [11,17]. The relevance of NF-jB activation in OSA is demonstrated by the presence of elevated levels of the products of several NF-jB controlled genes in OSA patients. Data from previous studies have shown that OSA patients have increased levels of the adhesion molecules sEselectin, sVCAM-1, ICAM-1, and L-selectin as well as the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-a, all of which are produced by NF-jB regulated genes [18]. Because these inflammatory proteins have been shown to play a crucial role in the pathogenesis of atherosclerosis [17], these observations collectively suggested that OSA may contribute to atherogenesis by stimulating NF-jB, with subsequent activation of cell adhesion and other proinflammatory pathways. It has been postulated that intermittent hypoxia associated with recurrent obstructive apneas is a crucial pathophysiological feature of OSA which generates increased ROS [19]. These free radicals may then activate oxidant-sensitive transcription factors such as NF-jB. As a result, production of proinflammatory mediators and adhesion molecules is increased, in addition to activation of neutrophils and endothelial cells. Increased adhesion of activated inflammatory cells
Study of endothelial dysfunction and vascular inflammation in sleep apnea, obesity and aged humans Table 2
473
sleep studies data AHI, ODI, Mini PaO2, and average pulse rate. Control
Cases
Mean AHI (events/h) ODI (events/h) Mini PaO2 (mmHg) Average pulse (beats/min)
SD
2.25 1.53 92.45 66.90
t-test
Mean *
1.16 .32 1.66 3.67
24.07 23.49* 76.20* 84.13*
SD
t
p-value
15.14 22.07 6.76 10.39
9.06 6.29 14.35 9.38
<.001 <.001 <.001 <.001
Values are mean ± SD. * p < 0.05 vs control group I.
Table 3
The NF-kB activity, serum s-VCAM-1 and FMD in the studied groups. Group I
II
A NF-KB(lg/well) sVCAM (ng/ml) FMD% a,b,c
B a
5.50 (3.0–8.0) 538.00a (470–708) 9.10a (8.10–9.60)
III
A a
5.00 (2.00–8.0) 762.00c (724–816) 7.90c (7.10–8.20)
B a
21.00 (15.0–65.0) 1617.0b (1224–2304) 4.30b (3.10–5.60)
A a
21.00 (15.0–65.0) 1686.00b (1492–3002) 4.20b (2.90–4.60)
B a
19.50 (15.0–58.0) 1375.00b (1260–2574) 4.85b (3.20–5.40)
25.00a (14.0–65.0) 1960.00b (1242–4378) 3.65b (2.50–4.50)
Values are median (min–max). The groups with the same small letter have no significant difference.
Table 4 Correlation between NF-jB activity, sVCAM-1 and severity of OSA. NFKB
r(p-value)
sVCAM1
R (p-value)
AHI
ODI
Mini PaO2
.938** (<.001) .955** (<.001)
.927** (<.001) .928** (<.001)
.888** (<.001) .877** (<.001)
to the endothelium is an important factor contributing to endothelial dysfunction and atherogenesis [8,9]. In Animal studies, Shuo et al, 2011 had studied the systemic production of inflammatory factors and activation of NF-jB in response to different levels of intermittent hypoxia in 160 male Wistar rats, which has a typical breathing pattern of OSA. They have found that the correlation of NFjB activation under intermittent hypoxia implies an important role of this transcription factor in inflammation-induced cardiovascular damage occurring during OSA [20]. Furthermore, other in vitro studies done by Ryan and colleagues, 2005 reported that, in an in vitro cell culture model, alternating cycles of hypoxia and re-oxygenation resulted in selective and dose-dependent activation of inflammatory NF-jB-dependent pathways [21]. These data go in line with other studies by Aung et al, 2006 who reported that neutrophils demonstrated significantly greater NF-jB activity in the mild to moderate and severe OSA groups than in the control subjects and this increased neutrophil NF-jB activity is reversed by CPAP treatment [10], Yamauchi et al, 2006 have measured activation of the NF-jB, in nine age-matched, nonsmoking, and non-hypertensive men with OSA symptoms and seven similar healthy subjects were measured at the same time and after the next
night of continuous positive airway pressure (CPAP). NF-kB activity in the OSA group were significantly higher than in the control group. One night of CPAP resulted in a reduction in NF-kappaB. They concluded that NF-kappaB activation occurs with sleep-disordered breathing. Such activation of NF-kappaB may contribute to the pathogenesis of atherosclerosis in OSA patients [22]. The present study has focused on activation of NF-jB in circulating neutrophils. While both monocytes and neutrophils have been implicated in the inflammatory processes leading to atherosclerosis, we chose to investigate circulating neutrophils since Richard Schulz et al, 2000 had demonstrated the release of superoxide from circulating neutrophils of patients with OSA in response to ex vivo stimulation, and is reversible with nasal CPAP therapy [23]. Furthermore, the neutrophil is known to play an important role in cardiovascular disease since infiltration of neutrophils into atherosclerotic plaque has been associated with plaque rupture and the occurrence of acute coronary events [24]. However, it is likely that OSA may also activate NF-jB in other cell types such as endothelial cells. This study also demonstrated that there was no significant difference between the obese and non-obese cases also no significant difference between the middle and old age cases which means that the co-morbid factors are not influencing our findings. This evidence indicates that the observed increase in NFjB activity is causally related to OSA, rather than to comorbid factors or to differences in BMI between groups. Thus, while obesity has been characterized as a proinflammatory state [25], and we cannot completely rule out a contribution of obesity itself to NF-jB activity, our findings support an independent effect of OSA on NF-jB activation as our study use a lean OSA group to definitely separate effects of obesity from those of OSA.
474 To confirm the biologic relevance of the observed change in NF-jB, we measured serum levels sVCAM-1, an important NF-jB controlled gene product, in OSA patients and in control subjects. There was a significant increase in the biochemical marker (sVCAM-1) in OSA patients as compared to age matched control groups. Furthermore our findings have shown that the concentration of circulating adhesion molecules correlated with the severity of sleep apnea. The current study also showed a significant relation between cellular adhesion molecules and the severity of hypoxemia as indicated by the ODI and mini PaO2 suggesting that the risk of cardiovascular events is perhaps related to the intermittent hypoxia observed during sleep and the level of hypoxaemia. It is plausible that intermittent hypoxia promotes oxygen radical formation [26] that leads to activation of transcriptional factors that upregulate the genetic expression of adhesion molecules [27]. Although the pathologic role of these cellular adhesion molecules in OSA is uncertain, studies have shown elevated circulating levels of adhesion molecules in subjects with OSA compared to those without sleep-disordered breathing [18]. Various circulating markers of endothelial dysfunction, including nitric oxide, soluble cell adhesion molecules, fibrinogen, and plasminogen activator inhibitor, have been reported to be altered in OSA [19,28]. This coincides with recent studies of Bernabe Jurado-Gamez et al,2012 who found that patients with OSA and greater intermittent hypoxia had worse endothelial function, and higher levels of ICAM-1 and P-selectin [29]. Hypoxia has been implicated in the induction of interleukin-1 and cellular adhesion molecules gene expression [30]. The present data confirm previous results that OSA syndrome is associated with a rise in circulating levels of adhesion molecules. Aung. et al, 2006 have found that the circulating levels of VCAM-1 are significantly increased in patients with OSA compared with those of a matched control group.[10] Ohga and coworkers, 1999 reported increased levels of ICAM-1, VCAM-1, and L-selectin levels in seven patients with OSA compared to a control group of ‘‘normal subjects’’. The authors suggested that the repetitive hypoxic stress during sleep might induce activation and provoke sustained release of these inflammatory mediators. Since cellular adhesion molecules mediate cellular interactions and transmigration of leukocytes across the vascular endothelial wall [31]. our findings suggest that OSA could contribute potentially to the inflammatory process implicated in atherogenesis. On the other hand, other studies have found no significant change. As Klein et al, 1995 who studied the in vitro influences of hypoxia on endothelial cell proliferation and expression of cell adhesion molecules. They found that the presentation of ICAM-1, VCAM-1, and E-Selectin was not affected by hypoxia or even reduced and they are not appear to be the major aspect in hypoxic injury [32]. The reactive dilatation of the brachial artery was assessed as a physical marker of endothelial dysfunction. FMD was significantly decreased in OSA patients as compared to the control group. On the other hand there was no significant difference between the obese and non-obese cases also no significant difference between the middle and old age cases. This indicates impaired reactive hyperaemia in OSA cases which indicates endothelial dysfunction. The mechanisms of vascular response to vessel occlusion followed by reactive hyperemia are clearly complex, involving
E. El-Shamy et al. myogenic, neurogenic, and vasculogenic components, mediated by a variety of metabolic alterations and vasoactive factors. The exact mechanisms apparently differ with vascular beds and evoking stimuli [33]. Flow-mediated dilation refers to nitric oxide-mediated vasodilatation resulting from shearmediated activation of endothelial nitric oxide synthase in response to an acute increase in blood flow [13]. The test has been shown to be accurate and reproducible [34]. Previous studies on vascular endothelial function in OSA have shown conflicting data. These studies have used different methods for evaluation of vascular endothelial function. For instance, Blunted vasodilation in response to infusion of acetylcholine, a vasodilator that stimulates endothelial release of nitric oxide, was demonstrated in forearm resistance vessels using occlusion plethysmography [35], but not confirmed in another study [36]. Impaired relaxation response to bradykinin in the forearm venous vasculature has been reported, suggesting endothelial dysfunction in the venous vasculature [37]. Using ultrasound Doppler method similar to ours, Mathias Grebe et al, 2006 had measured flow-mediated dilation (FMD) of the brachial artery by ultrasound in 10 otherwise healthy, untreated patients with OSA and 10 age-and sex-matched control subjects without sleep-disordered breathing before and after intravenous injection of the antioxidant vitamin C. They found that the reduced endothelial-dependent vasodilation in untreated patients with OSA acutely improves by the free radical scavenger vitamin C. These results are in favor of oxidative stress being responsible for the endothelial dysfunction in OSA [38]. In contrast to our findings, another study found no significant difference in conduit–vessel dilation between sleep apnea and control subjects. In that study, subjects underwent conduit–vessel studies at least 1 hour after resistance–vessel studies, which involved intra-arterial infusion of acetylcholine, nitroprusside and verapamil, and residual effect of drugs on the vasculature, which affected their response to reactive hyperemia, cannot be completely excluded [39]. To exclude the effect of obesity, we compare the FMD in lean and obese OSA patients, and there was no significant difference between both groups. In another study, OSA and subjects without OSA were matched for obesity, and the body mass index was not a significant independent determinant of FMD on multiple regression analysis. The change in FMD with treatment of OSA without any concomitant change in body weight and the use of a control group who did not receive nCPAP treatment provided further evidence that the improvement in endothelial function in this study sample, most of whom were obese, was attributed to the use of nCPAP, whereas a placebo effect could not be definitively excluded. OSA, therefore, may be associated with functional impairment (‘‘a premature aging effect’’) on the endothelium and on arterial stiffness [40]. Conclusions The findings of endothelial dysfunction in otherwise healthy subjects with OSA lend strong circumstantial evidence to an independent contribution of OSA to atherosclerosis. The present data also provide evidence for activation of the proinflammatory transcription factor NF-jB in OSA. This finding lends further support to an emerging hypothesis which postulates
Study of endothelial dysfunction and vascular inflammation in sleep apnea, obesity and aged humans that OSA contributes to cardiovascular disease by increasing systemic inflammation.
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