Increased Carotid Intima-Media Thickness and Serum Inflammatory Markers in Obstructive Sleep Apnea
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美国呼吸和危急护理医学 2005年第9期
First Department of Internal Medicine, Showa University, Tokyo, Japan
ABSTRACT
Increased carotid intima-media thickness (IMT) and increased serum levels of inflammatory markers, such as C-reactive protein (CRP), interleukin (IL)-6, and IL-18, are associated with an increased risk of cardiovascular and cerebrovascular diseases. The aim of this study was to evaluate whether carotid IMT, a useful marker for early atherosclerosis, is associated with these inflammatory markers in patients with obstructive sleep apnea (OSA). Carotid IMT was investigated with ultrasonography in 36 patients with OSA and 16 obese control subjects. Serum levels of CRP, IL-6, and IL-18 were measured at 5:00 A.M. Carotid IMT (p < 0.001) and serum levels of CRP (p < 0.003), IL-6 (p < 0.005), and IL-18 (p < 0.03) of patients with OSA were significantly higher than those of obese control subjects. Carotid IMT was significantly correlated with serum levels of CRP (r = 0.61, p = 0.0001), IL-6 (r = 0.41, p = 0.01), and IL-18 (r = 0.45, p = 0.005), duration of OSA-related hypoxia (r = 0.60, p = 0.0001), and severity of OSA (r = 0.50, p = 0.002). In addition, the primary factor influencing carotid IMT was duration of hypoxia during total sleep time (p = 0.036). These results suggest that OSA-related hypoxia and systemic inflammation might be associated with the progression of atherosclerosis and thus might increase the risks of cardiovascular and cerebrovascular morbidity in patients with OSA.
Key Words: atherosclerosis; cytokine; hypoxia; inflammation; sleep apnea
Obstructive sleep apnea (OSA) is associated with increased cardiovascular and cerebrovascular morbidity (1eC5). Ongoing inflammatory responses play important roles in atherosclerosis (6, 7). Recent studies have demonstrated that increased serum levels of C-reactive protein (CRP), interleukin (IL)-6, and tumor necrosis factor (TNF)- are important risk factors for atherosclerosis, stroke, and cardiovascular diseases (8eC10). We have demonstrated that serum levels of CRP, IL-6, TNF-, and matrix metalloproteinase-9 are elevated in patients with OSA (11eC13). Intracellular content of TNF- in T cells was also increased in patients with sleep apnea (14). In addition, levels of circulating soluble adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and chemokines, such as monocyte chemoattractant protein-1, are elevated in patients with OSA (15, 16). Moreover, plasma levels of vascular endothelial growth factor, a potent angiogenic cytokine that also contributes to the progression of atherosclerosis, are increased in patients with OSA (17, 18). Therefore, these findings suggest that OSA may accelerate the progression of atherosclerosis.
Although IL-18 is a potent proinflammatory cytokine that stimulates IFN- production by murine splenocytes, it also promotes atherosclerosis and increases the vulnerability of atherosclerotic plaques (19). One recent study demonstrated that IL-18 is a strong predictor of cardiovascular death in patients with stable or unstable angina (20). Furthermore, serum levels of IL-18 are increased in patients with acute coronary syndrome (21). These findings suggest a close relationship exists between elevated levels of IL-18 and cardiovascular events. However, levels of IL-18 in patients with OSA have not been investigated.
Carotid intima-media thickness (IMT) is a useful marker for investigating the degree of early atherosclerosis (22). Several clinical studies have demonstrated that an increase in ultrasonographically measured carotid IMT is associated with elevated risks of cardiovascular diseases and stroke (23eC26). In addition, carotid IMT is associated with inflammatory markers for atherosclerosis such as CRP, IL-6, and IL-18 (27eC29). Moreover, carotid IMT is higher in patients with OSA than in obese control subjects (30eC33). Therefore, OSA is independently associated with the progression of atherosclerosis. However, the relationship between carotid IMT and levels of inflammatory markers for atherosclerosis have not been previously studied in patients with OSA.
The purpose of this study in patients with OSA was to evaluate whether carotid IMT and serum levels of inflammatory markers, such as CRP, IL-6, and IL-18, are elevated; to investigate the correlation between carotid IMT and serum levels of these inflammatory markers; and to identify factors that are independent predictors of carotid IMT.
METHODS
Patients
Thirty-six men with newly diagnosed OSA (age, 23eC60 years) and 16 obese male control subjects (age, 25eC66 years) were enrolled in this study (Table 1) and underwent polysomnography. All subjects who were free from other diseases and were taking no medications were enrolled into the present study. Subjects who had smoked and were exposed to second-hand smoke or who had systemic infections at the time of the study or within 2 weeks before the study were also excluded. The study was approved by the Ethics Committee of Showa University, and all patients gave written, informed consent.
Polysomnography
Full polysomnographic monitoring was performed with the Compumedics P-series Sleep System (Compumedics Sleep, Abbotsford, Australia). An apneaeChypopnea index (AHI) greater than 5/hour in conjunction with sleep-related symptoms was considered as diagnostic of OSA. An AHI of 5 or more and less than 20 indicated mild OSA, an AHI of 20 or more and less than 30 indicated moderate OSA, and an AHI of 30 or more indicated severe OSA. The Epworth Sleepiness Scale was used to investigate changes in subjective daytime sleepiness (34).
Measurement of Carotid IMT and Plaque
Carotid atherosclerosis was evaluated with high-resolution B-mode ultrasonography with a 7.5-MHz linear-array transducer (PEL-705S; Toshiba, Tokyo, Japan). The far wall of both sides of the right and left common carotid arteries and the carotid bifurcation were analyzed. The mean IMT was calculated as the average of eight measurements (excluding sites of plaque) in the right and left sides during end diastole. Plaques were defined as the presence of focal, severe wall thickening (IMT > 1.2 mm), wall irregularity, and calcification. Plaque formation was graded as absent (0), mild (1: < 30% of the vessel diameter), moderate (2: 30eC50% of the vessel diameter), or severe (3: > 50% of the vessel diameter) according to methods described previously (35). All subjects were examined by the same investigator who was blinded to clinical characteristics.
Measurement of IL-6, IL-18, and CRP
All subjects went to bed at 9:00 P.M. and were awakened at 5:00 A.M. Samples of peripheral venous blood were collected at 5:00 A.M. Samples were stored at eC80°C until the time of assay. Serum levels of IL-6 and IL-18 were measured with an ELISA, which could detect concentrations as low as 0.104 and 2.0 pg/ml, respectively. ELISA kits for IL-6 and IL-18 were obtained from Biosource International (Camarillo, CA). High-sensitivity CRP was measured with a latex particle-enhanced immunoturbidimetric assay as described previously (36).
Statistical Analysis
The significance of the differences between two groups was analyzed with Student's t test. To compare three groups, the data were analyzed by analysis of variance with Bonferroni correction. The correlation was analyzed with Spearman's rank correlation. To assess the relative strength of association of carotid IMT with possible contributing factors, we used a stepwise multiple regression analysis to the patients with OSA and obese control subjects as a single group. Data are expressed as mean ± SEM, and a probability of less than 0.05 was considered to indicate significance.
RESULTS
Subject Characteristics
Characteristics of the patients with OSA and obese control subjects, including age, sex, body mass index, neck, waist circumference, waist/hip ratio, metabolic variables, polysomnography variables, Epworth Sleepiness Scale, and serum levels of CRP, IL-6, and IL-18 are shown in Table 1. AHI, percentage of time with SaO2 less than 90%, arousal index, and Epworth Sleepiness Scale were significantly higher in patients with moderate to severe OSA than in those with mild OSA or in obese control subjects. Lowest SaO2 and total sleep time were significantly lower in patients with moderate to severe OSA than those in mild OSA or in obese control subjects.
Carotid IMT in Patients with OSA and Obese Control Subjects
Carotid IMT was significantly elevated in patients with OSA compared with obese control subjects (1.07 ± 0.05 vs. 0.71 ± 0.03 mm, p < 0.001). In addition, carotid IMT in patients with moderate to severe OSA (1.16 ± 0.05 mm) was significantly elevated compared with patients with mild OSA (0.92 ± 0.07 mm, p < 0.003) or compared with obese control subjects (0.71 ± 0.03 mm, p < 0.0001; Figure 1). Plaques were detected in 30.4% (26.1% in Grade 1 and 4.3% in Grade 2) in patients with moderate to severe OSA, 23.1% (23.1% in Grade 1) in patients with mild OSA, and 12.5% (12.5% in Grade 1) in obese control subjects. We identified calcified plaques in four patients with OSA (11.1%) but calcified plaques were not observed in any obese control subjects by ultrasonograpy.
Serum Levels of CRP, IL-6, and IL-18
Serum levels of CRP, IL-6, and IL-18 are shown in Table 1 and Figure 2. Serum levels of CRP, IL-6, and IL-18 in patients with OSA (0.23 ± 0.03 mg/dl, 1.88 ± 0.20 pg/ml, and 250.5 ± 15.2 pg/ml, respectively) were significantly higher than those in obese control subjects (0.09 ± 0.02 mg/dl, p < 0.003; 0.91 ± 0.15 pg/ml, p < 0.005; and 181.9 ± 20.3 pg/ml, p < 0.003, respectively). Moreover, serum levels of CRP, IL-6, and IL-18 in patients with moderate to severe OSA (0.28 ± 0.04 mg/dl, 2.25 ± 0.28 pg/ml, and 273.5 ± 16.8 pg/ml, respectively) were significantly elevated compared with patients with mild OSA (0.15 ± 0.03 mg/dl, p < 0.01; 1.23 ± 0.14 pg/ml, p < 0.01; and 209.7 ± 27.0 pg/ml, p < 0.05, respectively) or compared with obese control subjects (0.09 ± 0.02 mg/dl, p < 0.0001; 0.91 ± 0.15 pg/ml, p < 0.001; and 181.9 ± 20.3 pg/ml, p < 0.003, respectively).
Correlation between Carotid IMT and Age, Polysomnography Variables, Metabolic Variables, Epworth Sleepiness Scale, Body Mass Index, and Levels of CRP, IL-6, and IL-18 in Patients with OSA
The correlations between carotid IMT and age, polysomnography variables, metabolic variables, and serum levels of CRP, IL-6, or IL-18 in patients with OSA are shown in Table 2. Carotid IMT was positively correlated with age (r = 0.41, p = 0.004), AHI (r = 0.50, p = 0.002), and percentage of time with SaO2 less than 90% (r = 0.60, p = 0.0001). Carotid IMT was also positively correlated with serum levels of CRP (r = 0.61, p = 0.0001; Figure 3A), IL-6 (r = 0.41, p = 0.01; Figure 3B), and IL-18 (r = 0.45, p = 0.005; Figure 3C). There was a significant correlation between AHI and serum levels of CRP (r = 0.60, p < 0.001), and IL-6 (r = 0.41, p = 0.048), but no significant correlation was found between AHI and serum levels of IL-18 (r = 0.27, p = 0.09) in patients with OSA. In addition, serum levels of IL-18 were weakly but significantly correlated with serum levels of CRP (r = 0.35, p = 0.04) and IL-6 (r = 0.39, p = 0.02). Percentage of time with SaO2 less than 90% was positively correlated with serum levels of CRP (r = 0.61, p < 0.0001) and IL-6 (r = 0.39, p = 0.049). Thus, in men with OSA, elevated carotid IMT was observed more often in those who had higher levels of inflammatory markers for atherosclerosis, and had more severe OSA with hypoxia during sleep period.
Stepwise Multiple Regression Analysis in Patients with OSA and Obese Control Subjects
This study used carotid IMT as a dependent variable and evaluated the order of inclusion in the model of the following independent variables: age, total cholesterol, triglyceride, body mass index, waist/hip ratio, Epworth Sleepiness Scale, AHI, percentage of time with SaO2 less than 90%, and serum levels of CRP, IL-6, and IL-18. Of the independent variables, percentage of time with SaO2 less than 90% was the strongest predictor of carotid IMT in patients with OSA and obese control subjects (p = 0.036).
DISCUSSION
The present study found that carotid IMT and serum levels of CRP, IL-6, and IL-18 were significantly higher in patients with OSA than in obese control subjects. In addition, carotid IMT was significantly correlated with levels of CRP, IL-6, and IL-18; duration of OSA-related hypoxia; and severity of OSA. Moreover, the duration of hypoxia during total sleep time was the strongest independent predictor of carotid IMT in patients with OSA and obese control subjects.
Our study confirmed results of previous studies that found that patients with OSA have an increased predisposition to atherosclerotic changes, as determined by carotid IMT and plaque formation (30eC33). However, our study differed from previous studies in that patients with hypertension, diabetes, or hyperlipidemia were excluded because these conditions can increase carotid IMT (37eC39). In addition, age and body mass index, which also affect carotid IMT, were matched between patients with OSA and obese control subjects. Therefore, our results extend the previous findings by showing that a significant relationship exists between OSA and increased carotid IMT, even in the absence of significant comorbidities.
Many studies suggest that carotid IMT is associated with an increased risk of cardiovascular and cerebrovascular disease, and therefore the severity of generalized atherosclerosis (23eC26). Here we show that OSA is also associated with increased carotid IMT. Moreover, several clinical studies have reported high prevalence of stroke and myocardial infarction in patients with OSA (1, 2, 40). Taken together, the data suggest that increased carotid IMT may be also a risk factor for cardiovascular and cerebrovascular disease in patients with OSA.
The ratio of plaque formation we report in the current study was not as profound as a previous report in patients with OSA (86%) and control subjects (64%) by Schultz and coworkers (33). The higher prevalence of plaque formation in the Schultz study may be related to multiple confounding factors, including age, hypertension, diabetes mellitus, hypercholesterolemia, and smoking, that were present in subjects with OSA and control subjects. Similarly, the percentage of patients with calcified plaques in the present study (11.1%) was less than in a previous study by Friedlander and colleagues (41), which showed that 23.3% of patients with OSA have calcified atheromas as assessed by cephalography. Thus, the present data demonstrated that, even in the absence of multiple risk factors, OSA is associated with significant degree of plaque formation and calcification.
Ongoing inflammatory responses play an important role in atherosclerosis (3, 4). Recent studies suggest that CRP is both a marker of inflammation and a factor in the pathogenesis of atherosclerosis, in part by activating endothelial cells and coronary artery smooth muscle cells. CRP is a prooxidant that induces production of monocyte chemoattractant protein-1 and expression of adhesion molecules, such as ICAM-1 and VCAM-1 (42, 43). Although several studies have shown that plasma levels of CRP are significantly associated with carotid IMT, the relation between carotid IMT and CRP levels has not been studied previously in patients with OSA. The present study demonstrated that serum levels of CRP were significantly higher in patients with OSA than in obese control subjects and were significantly correlated with carotid IMT. Therefore, our results suggest that increased levels of CRP may play an important role in the progression of atherosclerosis in patients with OSA.
IL-6 is an important proinflammatory cytokine that may also be involved in the pathogenesis of atherosclerosis. Plasma levels of IL-6 are reportedly correlated with mortality rate in patients with unstable coronary artery disease and with the risk of future myocardial infarction in apparently healthy men and well-functioning subjects (9, 44). In addition, carotid IMT is correlated with IL-6 levels in patients with pituitary deficiency (28). The present study confirmed previous reports that levels of IL-6 are significantly higher in patients with OSA than in obese control subjects. Moreover, we found that carotid IMT was significantly correlated with serum levels of IL-6 in patients with OSA. Therefore, increased serum levels of IL-6 may be a risk factor for accelerated progression of atherosclerosis, as demonstrated by carotid IMT, in patients with OSA.
One recent study suggests that IL-18 is a strong predictor of cardiovascular death in patients with stable or unstable angina (20). In fact, serum levels of IL-18 are increased in patients with acute coronary syndrome (21). The present study found that serum levels of IL-18 were significantly higher in patients with OSA than in obese control subjects and were significantly correlated with serum levels of CRP and IL-6. In patients with type 2 diabetes, carotid IMT is greater in those with high plasma levels of IL-18 than in those with normal levels of IL-18 (29). Similarly, our results show that serum levels of IL-18 are significantly correlated with carotid IMT in patients with OSA. These results suggest that IL-18 may contribute to the progression of atherosclerosis in patients with OSA.
Several factors may explain the elevated levels of IL-18 in patients with OSA. First, proinflammatory cytokines, including IL-1, TNF-, and IL-6, induce IL-18 expression (45). Because the present study and previous studies have shown that, in patients with OSA, serum levels of TNF- and IL-6 are increased and levels of IL-6 are positively correlated with levels of IL-18, these cytokines may be associated with the increased levels of IL-18 in these patients. Second, although production of IL-18 in adipose tissue has not been clearly demonstrated, body mass index and plasma levels of IL-18 are positively correlated in obese subjects (46). Third, sleep disturbance with repeated nocturnal hypoxia may affect serum levels of IL-18. For example, a recent study suggests that IL-18 contributes to the development of hypoxic-ischemic brain injury in rats (47). Although direct evidence that hypoxia increases IL-18 levels is lacking in humans, hypoxia might contribute to elevated levels of IL-18 in patients with OSA. Therefore, proinflammatory cytokines, obesity, and hypoxia may be involved in the elevated levels of IL-18 in patients with OSA.
The intermittent hypoxia observed in patients with sleep apnea has been proposed to represent a form of oxidative stress that results in increased generation of oxygen species. Oxidative damage is involved in the pathogenesis of atherosclerosis and cardiovascular diseases (48, 49). In fact, production of oxygen species from neutrophils and monocytes obtained from patients with OSA was increased (50, 51). These results suggest that oxidative stress is increased in the peripheral circulation in patients with OSA. Recent studies also suggest an association exists between the duration of OSA-related hypoxia and carotid IMT (31, 33). Similarly, our study has found that the duration of hypoxia during total sleep time is significantly associated with carotid IMT, and is the strongest predictor of carotid IMT in patients with OSA and obese control subjects. In addition, we have found that the duration of OSA-related hypoxia is significantly correlated with serum levels of CRP and IL-6 in patients with OSA. Because hypoxia increases IL-6 production through activation of nuclear factoreCB and may thus increase the CRP production by the liver, hypoxia caused by repeated apnea and hypopnea during sleep may be indirectly involved in the pathogenesis of atherosclerosis in patients with OSA (52). Therefore, OSA-related hypoxia may be an important factor for the progression of atherosclerosis in patients with OSA.
Limitations of the present study are the inclusion of only male subjects and the relatively small numbers of patients with OSA and obese control subjects. In addition, although a recent study suggests that snoring affects the progression of atherosclerosis (53), we could not compare the present data with those of obese nonsnoring subjects or normative nonsnoring subjects to investigate the effect of snoring on carotid IMT because sleep studies were not performed in these subjects. Finally, we did not include a continuous positive air pressure intervention in our study, which may have provided further evidence of a link between OSA, inflammation, and atherosclerosis by demonstrating that treatment of apnea-related hypoxia reduces inflammatory markers and progression of atherosclerosis in patients with OSA.
In conclusion, we have shown that patients with OSA have increased carotid IMT and increased serum levels of CRP, IL-6, and IL-18. The carotid IMT is significantly correlated with serum levels of CRP, IL-6, and IL-18; the severity of OSA; and duration of OSA-related hypoxia. In addition, the duration hypoxia during total sleep time is an independent predictor of carotid IMT in patients with OSA and obese control subjects. Therefore, OSA-related hypoxia and systemic inflammation may be associated with the progression of atherosclerosis and potentially increase the risks of cardiovascular and cerebrovascular morbidity.
Acknowledgments
The authors thank Mrs. Hiroko Takeuchi for her skillful technical assistance and Dr. Masao Okazaki for careful review of this manuscript.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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ABSTRACT
Increased carotid intima-media thickness (IMT) and increased serum levels of inflammatory markers, such as C-reactive protein (CRP), interleukin (IL)-6, and IL-18, are associated with an increased risk of cardiovascular and cerebrovascular diseases. The aim of this study was to evaluate whether carotid IMT, a useful marker for early atherosclerosis, is associated with these inflammatory markers in patients with obstructive sleep apnea (OSA). Carotid IMT was investigated with ultrasonography in 36 patients with OSA and 16 obese control subjects. Serum levels of CRP, IL-6, and IL-18 were measured at 5:00 A.M. Carotid IMT (p < 0.001) and serum levels of CRP (p < 0.003), IL-6 (p < 0.005), and IL-18 (p < 0.03) of patients with OSA were significantly higher than those of obese control subjects. Carotid IMT was significantly correlated with serum levels of CRP (r = 0.61, p = 0.0001), IL-6 (r = 0.41, p = 0.01), and IL-18 (r = 0.45, p = 0.005), duration of OSA-related hypoxia (r = 0.60, p = 0.0001), and severity of OSA (r = 0.50, p = 0.002). In addition, the primary factor influencing carotid IMT was duration of hypoxia during total sleep time (p = 0.036). These results suggest that OSA-related hypoxia and systemic inflammation might be associated with the progression of atherosclerosis and thus might increase the risks of cardiovascular and cerebrovascular morbidity in patients with OSA.
Key Words: atherosclerosis; cytokine; hypoxia; inflammation; sleep apnea
Obstructive sleep apnea (OSA) is associated with increased cardiovascular and cerebrovascular morbidity (1eC5). Ongoing inflammatory responses play important roles in atherosclerosis (6, 7). Recent studies have demonstrated that increased serum levels of C-reactive protein (CRP), interleukin (IL)-6, and tumor necrosis factor (TNF)- are important risk factors for atherosclerosis, stroke, and cardiovascular diseases (8eC10). We have demonstrated that serum levels of CRP, IL-6, TNF-, and matrix metalloproteinase-9 are elevated in patients with OSA (11eC13). Intracellular content of TNF- in T cells was also increased in patients with sleep apnea (14). In addition, levels of circulating soluble adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and chemokines, such as monocyte chemoattractant protein-1, are elevated in patients with OSA (15, 16). Moreover, plasma levels of vascular endothelial growth factor, a potent angiogenic cytokine that also contributes to the progression of atherosclerosis, are increased in patients with OSA (17, 18). Therefore, these findings suggest that OSA may accelerate the progression of atherosclerosis.
Although IL-18 is a potent proinflammatory cytokine that stimulates IFN- production by murine splenocytes, it also promotes atherosclerosis and increases the vulnerability of atherosclerotic plaques (19). One recent study demonstrated that IL-18 is a strong predictor of cardiovascular death in patients with stable or unstable angina (20). Furthermore, serum levels of IL-18 are increased in patients with acute coronary syndrome (21). These findings suggest a close relationship exists between elevated levels of IL-18 and cardiovascular events. However, levels of IL-18 in patients with OSA have not been investigated.
Carotid intima-media thickness (IMT) is a useful marker for investigating the degree of early atherosclerosis (22). Several clinical studies have demonstrated that an increase in ultrasonographically measured carotid IMT is associated with elevated risks of cardiovascular diseases and stroke (23eC26). In addition, carotid IMT is associated with inflammatory markers for atherosclerosis such as CRP, IL-6, and IL-18 (27eC29). Moreover, carotid IMT is higher in patients with OSA than in obese control subjects (30eC33). Therefore, OSA is independently associated with the progression of atherosclerosis. However, the relationship between carotid IMT and levels of inflammatory markers for atherosclerosis have not been previously studied in patients with OSA.
The purpose of this study in patients with OSA was to evaluate whether carotid IMT and serum levels of inflammatory markers, such as CRP, IL-6, and IL-18, are elevated; to investigate the correlation between carotid IMT and serum levels of these inflammatory markers; and to identify factors that are independent predictors of carotid IMT.
METHODS
Patients
Thirty-six men with newly diagnosed OSA (age, 23eC60 years) and 16 obese male control subjects (age, 25eC66 years) were enrolled in this study (Table 1) and underwent polysomnography. All subjects who were free from other diseases and were taking no medications were enrolled into the present study. Subjects who had smoked and were exposed to second-hand smoke or who had systemic infections at the time of the study or within 2 weeks before the study were also excluded. The study was approved by the Ethics Committee of Showa University, and all patients gave written, informed consent.
Polysomnography
Full polysomnographic monitoring was performed with the Compumedics P-series Sleep System (Compumedics Sleep, Abbotsford, Australia). An apneaeChypopnea index (AHI) greater than 5/hour in conjunction with sleep-related symptoms was considered as diagnostic of OSA. An AHI of 5 or more and less than 20 indicated mild OSA, an AHI of 20 or more and less than 30 indicated moderate OSA, and an AHI of 30 or more indicated severe OSA. The Epworth Sleepiness Scale was used to investigate changes in subjective daytime sleepiness (34).
Measurement of Carotid IMT and Plaque
Carotid atherosclerosis was evaluated with high-resolution B-mode ultrasonography with a 7.5-MHz linear-array transducer (PEL-705S; Toshiba, Tokyo, Japan). The far wall of both sides of the right and left common carotid arteries and the carotid bifurcation were analyzed. The mean IMT was calculated as the average of eight measurements (excluding sites of plaque) in the right and left sides during end diastole. Plaques were defined as the presence of focal, severe wall thickening (IMT > 1.2 mm), wall irregularity, and calcification. Plaque formation was graded as absent (0), mild (1: < 30% of the vessel diameter), moderate (2: 30eC50% of the vessel diameter), or severe (3: > 50% of the vessel diameter) according to methods described previously (35). All subjects were examined by the same investigator who was blinded to clinical characteristics.
Measurement of IL-6, IL-18, and CRP
All subjects went to bed at 9:00 P.M. and were awakened at 5:00 A.M. Samples of peripheral venous blood were collected at 5:00 A.M. Samples were stored at eC80°C until the time of assay. Serum levels of IL-6 and IL-18 were measured with an ELISA, which could detect concentrations as low as 0.104 and 2.0 pg/ml, respectively. ELISA kits for IL-6 and IL-18 were obtained from Biosource International (Camarillo, CA). High-sensitivity CRP was measured with a latex particle-enhanced immunoturbidimetric assay as described previously (36).
Statistical Analysis
The significance of the differences between two groups was analyzed with Student's t test. To compare three groups, the data were analyzed by analysis of variance with Bonferroni correction. The correlation was analyzed with Spearman's rank correlation. To assess the relative strength of association of carotid IMT with possible contributing factors, we used a stepwise multiple regression analysis to the patients with OSA and obese control subjects as a single group. Data are expressed as mean ± SEM, and a probability of less than 0.05 was considered to indicate significance.
RESULTS
Subject Characteristics
Characteristics of the patients with OSA and obese control subjects, including age, sex, body mass index, neck, waist circumference, waist/hip ratio, metabolic variables, polysomnography variables, Epworth Sleepiness Scale, and serum levels of CRP, IL-6, and IL-18 are shown in Table 1. AHI, percentage of time with SaO2 less than 90%, arousal index, and Epworth Sleepiness Scale were significantly higher in patients with moderate to severe OSA than in those with mild OSA or in obese control subjects. Lowest SaO2 and total sleep time were significantly lower in patients with moderate to severe OSA than those in mild OSA or in obese control subjects.
Carotid IMT in Patients with OSA and Obese Control Subjects
Carotid IMT was significantly elevated in patients with OSA compared with obese control subjects (1.07 ± 0.05 vs. 0.71 ± 0.03 mm, p < 0.001). In addition, carotid IMT in patients with moderate to severe OSA (1.16 ± 0.05 mm) was significantly elevated compared with patients with mild OSA (0.92 ± 0.07 mm, p < 0.003) or compared with obese control subjects (0.71 ± 0.03 mm, p < 0.0001; Figure 1). Plaques were detected in 30.4% (26.1% in Grade 1 and 4.3% in Grade 2) in patients with moderate to severe OSA, 23.1% (23.1% in Grade 1) in patients with mild OSA, and 12.5% (12.5% in Grade 1) in obese control subjects. We identified calcified plaques in four patients with OSA (11.1%) but calcified plaques were not observed in any obese control subjects by ultrasonograpy.
Serum Levels of CRP, IL-6, and IL-18
Serum levels of CRP, IL-6, and IL-18 are shown in Table 1 and Figure 2. Serum levels of CRP, IL-6, and IL-18 in patients with OSA (0.23 ± 0.03 mg/dl, 1.88 ± 0.20 pg/ml, and 250.5 ± 15.2 pg/ml, respectively) were significantly higher than those in obese control subjects (0.09 ± 0.02 mg/dl, p < 0.003; 0.91 ± 0.15 pg/ml, p < 0.005; and 181.9 ± 20.3 pg/ml, p < 0.003, respectively). Moreover, serum levels of CRP, IL-6, and IL-18 in patients with moderate to severe OSA (0.28 ± 0.04 mg/dl, 2.25 ± 0.28 pg/ml, and 273.5 ± 16.8 pg/ml, respectively) were significantly elevated compared with patients with mild OSA (0.15 ± 0.03 mg/dl, p < 0.01; 1.23 ± 0.14 pg/ml, p < 0.01; and 209.7 ± 27.0 pg/ml, p < 0.05, respectively) or compared with obese control subjects (0.09 ± 0.02 mg/dl, p < 0.0001; 0.91 ± 0.15 pg/ml, p < 0.001; and 181.9 ± 20.3 pg/ml, p < 0.003, respectively).
Correlation between Carotid IMT and Age, Polysomnography Variables, Metabolic Variables, Epworth Sleepiness Scale, Body Mass Index, and Levels of CRP, IL-6, and IL-18 in Patients with OSA
The correlations between carotid IMT and age, polysomnography variables, metabolic variables, and serum levels of CRP, IL-6, or IL-18 in patients with OSA are shown in Table 2. Carotid IMT was positively correlated with age (r = 0.41, p = 0.004), AHI (r = 0.50, p = 0.002), and percentage of time with SaO2 less than 90% (r = 0.60, p = 0.0001). Carotid IMT was also positively correlated with serum levels of CRP (r = 0.61, p = 0.0001; Figure 3A), IL-6 (r = 0.41, p = 0.01; Figure 3B), and IL-18 (r = 0.45, p = 0.005; Figure 3C). There was a significant correlation between AHI and serum levels of CRP (r = 0.60, p < 0.001), and IL-6 (r = 0.41, p = 0.048), but no significant correlation was found between AHI and serum levels of IL-18 (r = 0.27, p = 0.09) in patients with OSA. In addition, serum levels of IL-18 were weakly but significantly correlated with serum levels of CRP (r = 0.35, p = 0.04) and IL-6 (r = 0.39, p = 0.02). Percentage of time with SaO2 less than 90% was positively correlated with serum levels of CRP (r = 0.61, p < 0.0001) and IL-6 (r = 0.39, p = 0.049). Thus, in men with OSA, elevated carotid IMT was observed more often in those who had higher levels of inflammatory markers for atherosclerosis, and had more severe OSA with hypoxia during sleep period.
Stepwise Multiple Regression Analysis in Patients with OSA and Obese Control Subjects
This study used carotid IMT as a dependent variable and evaluated the order of inclusion in the model of the following independent variables: age, total cholesterol, triglyceride, body mass index, waist/hip ratio, Epworth Sleepiness Scale, AHI, percentage of time with SaO2 less than 90%, and serum levels of CRP, IL-6, and IL-18. Of the independent variables, percentage of time with SaO2 less than 90% was the strongest predictor of carotid IMT in patients with OSA and obese control subjects (p = 0.036).
DISCUSSION
The present study found that carotid IMT and serum levels of CRP, IL-6, and IL-18 were significantly higher in patients with OSA than in obese control subjects. In addition, carotid IMT was significantly correlated with levels of CRP, IL-6, and IL-18; duration of OSA-related hypoxia; and severity of OSA. Moreover, the duration of hypoxia during total sleep time was the strongest independent predictor of carotid IMT in patients with OSA and obese control subjects.
Our study confirmed results of previous studies that found that patients with OSA have an increased predisposition to atherosclerotic changes, as determined by carotid IMT and plaque formation (30eC33). However, our study differed from previous studies in that patients with hypertension, diabetes, or hyperlipidemia were excluded because these conditions can increase carotid IMT (37eC39). In addition, age and body mass index, which also affect carotid IMT, were matched between patients with OSA and obese control subjects. Therefore, our results extend the previous findings by showing that a significant relationship exists between OSA and increased carotid IMT, even in the absence of significant comorbidities.
Many studies suggest that carotid IMT is associated with an increased risk of cardiovascular and cerebrovascular disease, and therefore the severity of generalized atherosclerosis (23eC26). Here we show that OSA is also associated with increased carotid IMT. Moreover, several clinical studies have reported high prevalence of stroke and myocardial infarction in patients with OSA (1, 2, 40). Taken together, the data suggest that increased carotid IMT may be also a risk factor for cardiovascular and cerebrovascular disease in patients with OSA.
The ratio of plaque formation we report in the current study was not as profound as a previous report in patients with OSA (86%) and control subjects (64%) by Schultz and coworkers (33). The higher prevalence of plaque formation in the Schultz study may be related to multiple confounding factors, including age, hypertension, diabetes mellitus, hypercholesterolemia, and smoking, that were present in subjects with OSA and control subjects. Similarly, the percentage of patients with calcified plaques in the present study (11.1%) was less than in a previous study by Friedlander and colleagues (41), which showed that 23.3% of patients with OSA have calcified atheromas as assessed by cephalography. Thus, the present data demonstrated that, even in the absence of multiple risk factors, OSA is associated with significant degree of plaque formation and calcification.
Ongoing inflammatory responses play an important role in atherosclerosis (3, 4). Recent studies suggest that CRP is both a marker of inflammation and a factor in the pathogenesis of atherosclerosis, in part by activating endothelial cells and coronary artery smooth muscle cells. CRP is a prooxidant that induces production of monocyte chemoattractant protein-1 and expression of adhesion molecules, such as ICAM-1 and VCAM-1 (42, 43). Although several studies have shown that plasma levels of CRP are significantly associated with carotid IMT, the relation between carotid IMT and CRP levels has not been studied previously in patients with OSA. The present study demonstrated that serum levels of CRP were significantly higher in patients with OSA than in obese control subjects and were significantly correlated with carotid IMT. Therefore, our results suggest that increased levels of CRP may play an important role in the progression of atherosclerosis in patients with OSA.
IL-6 is an important proinflammatory cytokine that may also be involved in the pathogenesis of atherosclerosis. Plasma levels of IL-6 are reportedly correlated with mortality rate in patients with unstable coronary artery disease and with the risk of future myocardial infarction in apparently healthy men and well-functioning subjects (9, 44). In addition, carotid IMT is correlated with IL-6 levels in patients with pituitary deficiency (28). The present study confirmed previous reports that levels of IL-6 are significantly higher in patients with OSA than in obese control subjects. Moreover, we found that carotid IMT was significantly correlated with serum levels of IL-6 in patients with OSA. Therefore, increased serum levels of IL-6 may be a risk factor for accelerated progression of atherosclerosis, as demonstrated by carotid IMT, in patients with OSA.
One recent study suggests that IL-18 is a strong predictor of cardiovascular death in patients with stable or unstable angina (20). In fact, serum levels of IL-18 are increased in patients with acute coronary syndrome (21). The present study found that serum levels of IL-18 were significantly higher in patients with OSA than in obese control subjects and were significantly correlated with serum levels of CRP and IL-6. In patients with type 2 diabetes, carotid IMT is greater in those with high plasma levels of IL-18 than in those with normal levels of IL-18 (29). Similarly, our results show that serum levels of IL-18 are significantly correlated with carotid IMT in patients with OSA. These results suggest that IL-18 may contribute to the progression of atherosclerosis in patients with OSA.
Several factors may explain the elevated levels of IL-18 in patients with OSA. First, proinflammatory cytokines, including IL-1, TNF-, and IL-6, induce IL-18 expression (45). Because the present study and previous studies have shown that, in patients with OSA, serum levels of TNF- and IL-6 are increased and levels of IL-6 are positively correlated with levels of IL-18, these cytokines may be associated with the increased levels of IL-18 in these patients. Second, although production of IL-18 in adipose tissue has not been clearly demonstrated, body mass index and plasma levels of IL-18 are positively correlated in obese subjects (46). Third, sleep disturbance with repeated nocturnal hypoxia may affect serum levels of IL-18. For example, a recent study suggests that IL-18 contributes to the development of hypoxic-ischemic brain injury in rats (47). Although direct evidence that hypoxia increases IL-18 levels is lacking in humans, hypoxia might contribute to elevated levels of IL-18 in patients with OSA. Therefore, proinflammatory cytokines, obesity, and hypoxia may be involved in the elevated levels of IL-18 in patients with OSA.
The intermittent hypoxia observed in patients with sleep apnea has been proposed to represent a form of oxidative stress that results in increased generation of oxygen species. Oxidative damage is involved in the pathogenesis of atherosclerosis and cardiovascular diseases (48, 49). In fact, production of oxygen species from neutrophils and monocytes obtained from patients with OSA was increased (50, 51). These results suggest that oxidative stress is increased in the peripheral circulation in patients with OSA. Recent studies also suggest an association exists between the duration of OSA-related hypoxia and carotid IMT (31, 33). Similarly, our study has found that the duration of hypoxia during total sleep time is significantly associated with carotid IMT, and is the strongest predictor of carotid IMT in patients with OSA and obese control subjects. In addition, we have found that the duration of OSA-related hypoxia is significantly correlated with serum levels of CRP and IL-6 in patients with OSA. Because hypoxia increases IL-6 production through activation of nuclear factoreCB and may thus increase the CRP production by the liver, hypoxia caused by repeated apnea and hypopnea during sleep may be indirectly involved in the pathogenesis of atherosclerosis in patients with OSA (52). Therefore, OSA-related hypoxia may be an important factor for the progression of atherosclerosis in patients with OSA.
Limitations of the present study are the inclusion of only male subjects and the relatively small numbers of patients with OSA and obese control subjects. In addition, although a recent study suggests that snoring affects the progression of atherosclerosis (53), we could not compare the present data with those of obese nonsnoring subjects or normative nonsnoring subjects to investigate the effect of snoring on carotid IMT because sleep studies were not performed in these subjects. Finally, we did not include a continuous positive air pressure intervention in our study, which may have provided further evidence of a link between OSA, inflammation, and atherosclerosis by demonstrating that treatment of apnea-related hypoxia reduces inflammatory markers and progression of atherosclerosis in patients with OSA.
In conclusion, we have shown that patients with OSA have increased carotid IMT and increased serum levels of CRP, IL-6, and IL-18. The carotid IMT is significantly correlated with serum levels of CRP, IL-6, and IL-18; the severity of OSA; and duration of OSA-related hypoxia. In addition, the duration hypoxia during total sleep time is an independent predictor of carotid IMT in patients with OSA and obese control subjects. Therefore, OSA-related hypoxia and systemic inflammation may be associated with the progression of atherosclerosis and potentially increase the risks of cardiovascular and cerebrovascular morbidity.
Acknowledgments
The authors thank Mrs. Hiroko Takeuchi for her skillful technical assistance and Dr. Masao Okazaki for careful review of this manuscript.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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