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* From the Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Chuo-ku, Kobe, Japan.
Correspondence to: Yoshihiro Nishimura, MD, Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7–5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan; e-mail: nishiy{at}med.kobe-u.ac.jp
Abstract
Background: Obstructive sleep apnea syndrome (OSAS) is an independent risk factor for atherosclerosis. CD40-CD40 ligand interaction promotes several proinflammatory mediators and plays a pivotal role in the various stages of atherosclerotic diseases. The present study examines whether CD40 ligation contributes to outcomes in patients with OSAS.
Methods: The study population comprised OSAS patients with an apnea hypopnea index (AHI) 
30 (n = 35) and control subjects (AHI < 5; n = 16). We measured serum levels of soluble CD40 ligand (sCD40L), tumor necrosis factor (TNF)-
, and hypersensitive C-reactive protein (hsCRP) before and after nasal continuous positive airway pressure (nCPAP) therapy for 3 months.
Results: Baseline levels of sCD40L were significantly higher in patients with OSAS (6.93 ± 4.64 ng/mL) [mean ± SD] than in control subjects (3.43 ± 2.11 ng/mL, p < 0.01). Baseline levels of sCD40L positively correlated with TNF- but not with hsCRP. The elevation of sCD40L was improved for 1 night after nCPAP therapy (3.83 ± 2.78 ng/mL, p < 0.001). Even though patients with severe OSAS did not receive any other medication to control atherosclerotic risk factors for 3 months, nCPAP was continued to reduce the levels of sCD40L.
Conclusion: The present study suggested that sCD40L is a key factor that links OSAS and atherosclerotic progression.
Key Words: atherosclerosis • hypoxia • obstructive sleep apnea • soluble CD40 ligand
Obstructive sleep apnea syndrome (OSAS) is the most widespread form of sleep-related breathing disorders that include snoring, upper airway resistance syndrome, and central sleep apnea. Since a relationship between snoring and cardiovascular diseases was discovered,1 subsequent investigations23456 have demonstrated that OSAS is associated with not only an increased prevalence of cardiovascular disease but also with cerebrovascular morbidity and mortality. Hypoxic stress elicited by OSAS increases superoxide production by monocytes and neutrophils,7 and it might be involved in the development of such vascular disorders. However, the exact mechanisms remain unclear. Levels of circulating proinflammatory cytokines and chemokines, such as tumor necrosis factor (TNF)-, interleukin (IL)-6, and IL-8, are increased in patients with OSAS.89 In addition, OSAS induces the activation of adhesion molecules, including intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and L-selectin.10 Furthermore, platelet aggregation and activation increase during sleep in patients with OSAS.11 These risk factors of atherosclerosis and vascular diseases are improved by therapy using nasal continuous positive airway pressure (nCPAP).
CD40 ligand (CD40L, CD154) was originally identified in T-lymphocytes, and it plays an important role in the immune response by binding to its receptor CD40 on B-cells. Both CD40 and CD40L have been identified on several other types of cells, including endothelial cells, smooth-muscle cells, monocytes, macrophages, and platelets. CD40 signaling (ligation with CD40) in these cells triggers the expression of various proinflammatory mediators; the cytokines, IL-1, IL-6, IL-12 and TNF-; the chemokines, monocyte chemoattractant protein-1 and IL-8; the adhesion molecules, ICAM-1 and VCAM-1; matrix metalloproteinases; growth factors; and procoagulant tissue factor.12 These observations support the notion that CD40L plays a pivotal role in the various stages of atherosclerosis.
CD40 ligand can be membrane associated, and it assumes a soluble form in plasma (soluble CD40 ligand [sCD40L]). Patients with unstable angina have higher plasma levels of sCD40L than healthy individuals or those with stable angina,13 and elevated sCD40L in this setting indicates a high risk for recurrent events.14 High plasma concentrations of sCD40L are associated with an increased vascular risk in healthy women.15 Furthermore, the CD40 system is up-regulated in patients with acute cerebral ischemia.16 These studies demonstrated that sCD40L has an independent prognostic value in vascular diseases.
We thus supposed that sCD40L is the key factor involved in the aggravation of atherosclerosis and vascular diseases induced by OSAS. The present study evaluated the relationship between sCD40L levels and outcomes in patients with OSAS and whether treatment with nCPAP decreases plasma levels of sCD40L.
Materials and Methods
Study Population and Polysomnography
Table 1
shows the study population and their clinical profiles. We recruited outpatients with characteristic symptoms of sleep apnea, snoring, daytime fatigue, or drowsiness, and examined them using an apnomonitor (Apnomonitor 3; CHEST; Tokyo, Japan) or by polysomnography (Alice 3 Diagnostics System; CHEST). The apnomonitor does not have an EEG sleep staging monitor. Apnea was defined as the continuous cessation of airflow for > 10 s, and hypopnea was defined as a reduction in airflow for > 10 s with > 4% oxygen desaturation. The apnea hypopnea index (AHI) was calculated as the total number of episodes of apnea and hypopnea per hour of total sleep. Patients with AHI < 5 were regarded as control patients (without OSAS), and those with AHI 30 were assumed to have OSAS. Ten of 16 control patients were examined by polysomnography after apnomonitor screening. All OSAS patients were examined by polysomnography and treated with nCPAP.
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Sample Collection and Cytokine Analysis
All OSAS patients retired at 9 PM, and peripheral venous blood samples were collected at the moment of waking. In addition, blood samples from patients with severe OSAS were collected every morning, as well as 1 night, 1 month, and 3 months after nCPAP therapy. All blood samples were centrifuged at 3,000g for 10 min and stored at – 80°C. Levels of sCD40L, TNF-, and hypersensitive C-reactive protein (hsCRP) were determined by enzyme-linked immunosorbent assays (TECHNE; Minneapolis, MN).
Statistical Analysis
The results for each experiment are presented as mean ± SD and were considered statistically significant at p < 0.05. The data were analyzed by nonparametric methods to avoid assumptions about the distribution of the measured variables. Differences between pretreatment and posttreatment values were analyzed using the Wilcoxon signed-rank test. The Spearman correlation coefficient with a rank test was used to examine correlations among cytokine levels.
Results
Patient Background
Table 1 shows that the background features of OSAS patients and control patients did not significantly differ. Table 2
shows the results of polysomnography, which all patients underwent after screening with the apnomonitor.
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were also significantly higher in OSAS patients than control patients (1.11 ± 0.46 pg/mL vs 0.62 ± 0.44 pg/mL, p < 0.01), whereas hsCRP levels did not significantly differ between the two groups (Fig 1, center, B, and bottom, C).
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(p = 0.02) but not with hsCRP before nCPAP therapy (Fig 2
, top, A, and center, B). Serum sCD40L levels in OSAS patients also positively correlated with the amount of time spent at < 90% oxygen saturation by pulse oximetry (SpO2) [p = 0.017; Fig 2, bottom, C].
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levels for 1 night but not those of hsCRP (Fig 3, center, B, and bottom, C).
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The present study demonstrated higher serum levels of sCD40L and TNF- in OSAS patients than in control patients. In addition, treatment with nCPAP improved these levels in the OSAS patients.
The association of OSAS with vascular morbidity, including hypertension, coronary artery diseases, and stroke, is a major concern. Since OSAS might independently contribute to these vascular diseases, the pathogenetic mechanisms of atherosclerosis caused by OSAS should be understood. Atherosclerosis has recently been viewed as a chronic inflammatory disease of the large arteries.17 CD40-CD40 ligand interactions play an important role in a wide range of chronic inflammatory diseases, including atherosclerosis.12 Studies181920 in vitro have shown that CD40 ligation results in the activation of proatherogenic pathways. Ligation of CD40 on endothelial cells stimulates the expression of leukocyte adhesion molecules such as ICAM-1 and VCAM-1,18 as well as the proatherogenic cytokines IL-1ß and IL-6, but not TNF-. CD40 ligation in macrophages induces the production of the cytokines, IL-1ß, IL-6, and IL-12, as well as TNF-.19 Furthermore, CD40 ligand on platelets can induce human umbilical vein endothelial cells to express both monocyte chemoattractant protein-1 and IL-8.20
Although others8910 have found that the serum levels of these proinflammatory mediators are elevated in patients with OSAS and that nCPAP therapy decreases these levels, the mechanism through which OSAS induces these mediators remains obscure. The present data revealed positive correlations between sCD40L and the amount of time spent at < 90% SpO2, and the fact that nCPAP therapy decreased the elevation of serum sCD40L in OSAS patients were in parallel with the improved clinical symptoms and AHI. Precise evidence has yet to confirm that hypoxic stress directly or indirectly induces the elevation of sCD40L mediated via other inflammatory factors. Therefore, the mechanism of sCD40L elevation in relation to atherosclerotic progression in patients with OSAS should be studied in more detail.
Platelets play a pivotal role in the pathogenesis of acute cardiovascular syndrome. Patients with OSAS have increased aggregation of activated platelets21 that release vascular endothelial growth factor (VEGF) triggered by CD40 ligation. VEGF, a key mediator in angiogenesis, is up-regulated under hypoxic conditions in cardiac myocytes, vascular smooth-muscle cells, and endothelial cells.222324 Schulz et al25 reported that the elevated serum levels of VEGF in OSAS patients are related to atherosclerotic progression. The present data are compatible with those of Schulz et al25 and should help to elucidate how several vascular disorders develop in OSAS in relation to VEGF expression.
Monocytes spontaneously produce TNF-, serum levels of which are elevated in OSAS patients.26 Monocytes are activated by hypoxia in OSAS and release TNF-. The present study showed that levels of sCD40L and TNF- changed in parallel, implying that sCD40L is expressed in apnea-related hypoxia. The expression of adhesion molecules, CD15 and CD11c, on monocytes was also increased in OSAS and improved by nCPAP therapy.7 CD40 ligation also caused expression of these adhesion molecules. Therefore, CD40 ligation on monocytes plays an important role in atherosclerotic progression.
Levels of C-reactive protein (CRP) and IL-6 are reportedly elevated in OSAS patients and are decreased by CPAP.9 However, levels of CRP and IL-6 did not significantly differ in the present study between OSAS and control patients (data not shown). Both CRP and IL-6 are so sensitive to various types of inflammation that they could easily fluctuate over a wide range. Therefore, these indexes are unsuitable for the practical assessment of inflammatory status in OSAS. Meanwhile, our results showed that sCD40L levels did not vary quite so obviously. Moreover, since the level of sCD40L was improved early during therapy, we propose that sCD40L is a clinical predictive factor of atherosclerotic progression in OSAS. Future large-cohort studies will be helpful to understand the clinical course of OSAS.
Footnotes
Abbreviations: AHI = apnea-hypopnea index; CRP = C-reactive protein; hsCRP = hypersensitive C-reactive protein; ICAM = intercellular adhesion molecule; IL = interleukin; nCPAP = nasal continuous positive airway pressure; OSAS = obstructive sleep apnea syndrome; sCD40L = soluble CD40 ligand; SpO2 = oxygen saturation by pulse oximetry; TNF = tumor necrosis factor; VCAM = vascular cell adhesion molecule; VEGF = vascular endothelial growth factor
Received for publication June 28, 2005. Accepted for publication September 24, 2005.
References
by monocytes in patients with obstructive sleep apnea syndrome. Chest 2004;126,1473-1479
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