Journal of Human Hypertension (2015), 1–6 © 2015 Macmillan Publishers Limited All rights reserved 0950-9240/15 www.nature.com/jhh
ORIGINAL ARTICLE
Relationship between serum levels of endogenous secretory RAGE and blood pressure in male nondiabetic patients with obstructive sleep apnea W Cai1,2, J-F Sun1, Y Liu1, J-X Xu1, J-R Xiao1, X-M Duan1, J-Y Liu1 and W Zhang3 The interaction of advanced glycation end products (AGE) and their specific cell-surface receptor (RAGE) has an important role in the pathogenesis of cardiovascular disease and diabetic complications. Two isoforms of C-truncated RAGE, soluble RAGE (sRAGE) and endogenous secretory RAGE (esRAGE), may prevent activation of RAGE signaling by acting as decoys. This study investigated whether serum esRAGE and sRAGE levels are associated with blood pressure in nondiabetic patients with obstructive sleep apnea (OSA). Male nondiabetic patients (n = 139) with OSA were enrolled. Serum esRAGE and sRAGE levels were examined using enzyme-linked immunosorbent assay. Three consecutive seated systolic blood pressure (SBP) and diastolic blood pressure (DBP) measurements were obtained at 5-min intervals in the morning. In univariate analysis, there was a significant correlation between serum esRAGE and SBP or DBP, but not between serum sRAGE and SBP or DBP. Multiple regression analysis showed that SBP was independently associated with waist circumference, HbA1c, minimum SaO2 and serum esRAGE, and that DBP was independently associated with low-density lipoprotein cholesterol, apnea–hypopnea index, serum AGE and body mass index, but not with serum esRAGE. These results indicated that serum esRAGE levels were inversely associated with blood pressure, especially SBP, in male nondiabetic patients with OSA. esRAGE may have a protective role against hypertension in patients with OSA, and it may be a novel biomarker for OSA patients at high risk of developing cardiovascular diseases. Journal of Human Hypertension advance online publication, 21 May 2015; doi:10.1038/jhh.2015.46
INTRODUCTION Epidemiological data from community populations have provided evidence suggestive of an independent association between obstructive sleep apnea (OSA) and hypertension.1,2 OSA has also been identified as a secondary cause of hypertension.3,4 Although studies have reported that the intermittent hypoxia and sleep deprivation that OSA patients experience can result in increased nervous system activity,5,6 which can eventually lead to elevated blood pressure, the mechanisms behind the pathogenesis of hypertension in patients with OSA are complicated and require clarification. Advanced glycation end products (AGE) are protein products generated by the Maillard reaction. Classically, AGE formation has been described by a nonenzymatic reaction between proteins and glucose under hyperglycemic conditions.7 The intermittent hypoxemia that occurs in patients with OSA may induce the formation of AGE.8 Several studies have reported that serum AGE levels increase in nondiabetic people with OSA,9,10 and continuous positive airway pressure therapy was found to reduce serum AGE levels.10–12 In our previous study, we found that serum AGE levels in nondiabetic patients with OSA were associated with insulin resistance,13 which is an important risk factor for hypertension. There is growing evidence to suggest that interaction between AGE and their specific cell-surface receptor, the receptor for AGE (RAGE), stimulates oxidative stress generation and has an
important role in cardiovascular disease and diabetic complications.14–16 Recently, two circulating isoforms of RAGE have been identified: soluble RAGE (sRAGE) and endogenous secretory RAGE (esRAGE).17 Administration of recombinant sRAGE inhibited the development and progression of atherosclerosis in animal models,18,19 and esRAGE was found to be capable of neutralizing the effects of AGE on endothelial cells in culture.20 These findings suggest that sRAGE and esRAGE may capture circulating AGE by acting as decoy receptors. Geroldi et al.21 reported that decreased levels of plasma sRAGE were related to pulse pressure in patients with essential hypertension. However, Momma et al.22 reported that circulating esRAGE levels, but not sRAGE, inversely correlated with several components of metabolic syndrome, including blood pressure. The relationship between serum esRAGE, sRAGE and blood pressure in patients with OSA remains unclear. In this study, we examined whether serum esRAGE and sRAGE levels were associated with blood pressure in a population of male nondiabetic patients with OSA. MATERIALS AND METHODS Study participants Participants with OSA were recruited from the sleep laboratory at the First Affiliated Hospital of Nanchang University between 2013 and 2014.
1 Department of Endocrinology and Metabolism, First Affiliated Hospital of Nanchang University, Nanchang, People’s Republic of China; 2Department of Medical Biology, Medical College of Nanchang University, Nanchang, People’s Republic of China and 3Department of Respiratory Medicine, First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China. Correspondence: Professor J-X Xu, Department of Endocrinology and Metabolism, First Affiliated Hospital of Nanchang University, 17 Yongwaizheng Street, Nanchang, Jiangxi 330006, People’s Republic of China. E-mail: [email protected] Received 29 January 2015; revised 9 April 2015; accepted 16 April 2015
Serum levels of endogenous secretory RAGE and blood pressure W Cai et al
2 An overnight sleep study was conducted using a computerized polysomnogram system (Alice LE, Philips Respironics California, Inc., Carlsbad, CA, USA). Apneas were defined as complete cessation of air flow for ⩾ 10 s. Hypopnea was defined as a ⩾ 50% reduction of air flow or a o50% reduction of air flow accompanied by a ⩾ 3% decrease in oxygen saturation, or followed by an arousal. Measurement of the apnea– hypopnea index (AHI; the total number of apnea and hypopnea episodes divided by hours of sleep) during the night were obtained. Finger pulse oximetry was used to determine arterial blood oxygen saturation (SaO2). Time of SaO2 o90% and the lowest SaO2 (%) were calculated. Time spent in rapid eye movement sleep and nonrapid eye movement sleep (stages 1–4) were calculated as a percentage of the total sleep time. Stages 3 and 4 of nonrapid eye movement sleep are also known as slow-wave sleep (SWS). Procedures were performed by experienced technicians. Computerized sleep data were manually analyzed by permanent professional staff. Participants had their blood pressure measured when they were referred to the sleep laboratory in the morning. Three consecutive seated blood pressure measurements were obtained at 5-min intervals after the patient had rested for 15 min. Measurements used an appropriately sized cuff and were performed in a quiet room. The average of the last two blood pressure readings was used for the analysis. The following morning, participants with an AHI415 underwent an oral glucose tolerance test for which they received 75 g of glucose orally, and blood samples were obtained before and after 2 h (at 0 and 120 min) for measurement of glucose and insulin. A total of 139 male participants with an AHI415, defined as nondiabetic by 1999 World Health Organization diagnostic criteria, were enrolled in the study. Participants with neoplastic, hepatic, renal, lung, and heart diseases or inflammatory diseases were excluded.
Table 1.
Furthermore, people who received glucocorticoids, or antidiabetic, antihypertensive, antiobesity or lipid-lowering drugs, were excluded. All participants gave written informed consent before sample collection. This study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University.
Biochemical analyses Fasting blood samples were taken for measurement of fasting plasma glucose, fasting plasma insulin, hemoglobin A1c (HbA1c), high-sensitivity C-reactive protein, total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol, creatinine, serum AGE, serum esRAGE and serum sRAGE. All samples were stored at − 80 °C before analysis. Serum AGE was measured using a competitive inhibition enzyme-linked immunosorbent assay (ELISA) with an intra-assay coefficient of variation (CV) of o 10% and an inter-assay CV of o 12% according to the supplier’s recommendation (Uscn Life Science Inc., Wuhan, China). Serum sRAGE was measured using the R&D Systems Quantikine Immunoassay (Minneapolis, MN, USA) specific for the extracellular domain of human RAGE, with an intra-assay CV of o 6.2% and an inter-assay CV of o8.2%. Serum esRAGE was measured using B-Bridge ELISA (B-Bridge International, Cupertino, CA, USA), with an intra-assay CV of o4.4% and an inter-assay CV of o5.3%. Insulin resistance was determined using the homeostasis model assessment index (HOMA-IR): fasting plasma glucose (mmol l − 1) × fasting plasma insulin (mIU l − 1)/22.5. The creatinine clearance rate (Ccr) was calculated using the Cockcroft– Gault equation.
Clinical and metabolic characteristics of subjects categorized by AHI tertiles Total
First tertile (AHIo42)
Second tertile (42 ⩽ AHIo66)
Third tertile (AHI ⩾ 66)
n Age (years) Smoking (n(%)) BMI (kg m − 2) WC (cm) SBP (mm Hg) DBP (mm Hg) HbA1c (%) FPG(mmol l − 1) PPG (mmol l − 1) FPI (mIU l − 1) HOMA-IR TC (mmol l − 1) TG (mmol l − 1) LDL-C (mmol l − 1) HDL-C (mmol l − 1) Creatinine(μmol l − 1) Ccr (ml min − 1) hsCRP (mg l − 1) AHI (events h − 1)
139 41.8 ± 11.1 53 (38.1) 28.10 ± 3.19 96.46 ± 9.72 133.0 ± 13.6 90.9 ± 12.4 5.85 ± 0.79 5.64 ± 0.82 7.19 ± 2.04 13.6 (9.7 − 19.1) 3.15 (2.05 − 4.29) 5.27 ± 1.09 1.86 (1.17 − 2.73) 3.26 ± 0.98 1.28 (1.10 − 1.62) 76.6 ± 16.5 85.8 ± 19.9 2.35 (1.41 − 4.13) 53.1 (33.6 − 73.7)
46 41.8 ± 10.5 17 (37.0) 26.49 ± 3.20 94.12 ± 9.95 132.3 ± 14.8 89.4 ± 12.5 5.70 ± 0.56 5.39 ± 0.87 7.10 ± 2.17 10.0 (8.1 − 21.3) 2.69 (1.71 − 3.43) 5.08 ± 1.04 1.48 (1.01 − 2.22) 3.17 ± 0.89 1.32 (1.15 − 1.50) 76.8 ± 16.9 86.3 ± 20.2 2.04 (1.34 − 2.78) 28.3 ± 5.6
46 41.9 ± 11.3 20 (39.1) 28.38 ± 3.35† 97.31 ± 9.15† 133.7 ± 15.7 91.5 ± 13.4 5.75 ± 0.61 5.63 ± 0.83† 7.18 ± 2.22 13.4 (8.8 − 20.6)† 2.99 (1.73 − 4.45)† 5.15 ± 1.12 1.97 (1.33 − 2.72)† 3.25 ± 1.09 1.30 (1.11 − 1.87) 75.9 ± 14.3 86.0 ± 22.4 2.24 (1.34 − 3.71)† 53.6 ± 6.1†
47 42.0 ± 10.4 18 (38.3) 28.63 ± 3.41† 97.82 ± 8.84† 134.9 ± 12.4 91.6 ± 11.8 6.08 ± 1.06† 5.88 ± 0.75† 7.23 ± 1.79† 15.5 (10.7 − 25.6)†‡ 3.55 (2.73 − 4.66)†‡ 5.51 ± 1.10† 2.31 (1.64 − 3.88)†‡ 3.35 ± 1.00 1.23 (0.97 − 1.58) 78.1 ± 15.5 84.7 ± 25.6 2.62 (1.46 − 4.41)†‡ 79.3 ± 9.1†‡
SaO2 Durationo90%(min) Minimum (%) TST (min)
31.5 (8.3 − 104.6) 69.7 ± 12.6 317.3 ± 12.5
27.1 (8.3 − 52.4) 77.8 ± 10.9 326.9 ± 11.2
31.6 (11.1 − 83.2)† 75.3 ± 11.3 318.1 ± 10.8
34.2 (14.8 − 101.8)†‡ 67.1 ± 10.3†‡ 314.2 ± 11.8
10.9 ± 2.3 52.7 ± 14.6 16.5 ± 3.7 17.8 ± 2.6 2.63 ± 0.61 1032.6 ± 233.9 225.3 ± 43.7
7.7 ± 1.8 52.2 ± 13.1 23.9 ± 4.7 17.2 ± 2.4 2.27 ± 0.29 1143.2 ± 223.6 241.0 ± 69.4
10.6 ± 2.5† 52.5 ± 12.8 19.6 ± 4.3† 17.9 ± 2.8 2.51 ± 0.40† 1021.5 ± 208.1 217.3 ± 60.7†
14.2 ± 2.7†‡ 52.1 ± 12.4 15.8 ± 4.1†‡ 18.0 ± 2.6 2.98 ± 0.46†‡ 985.7 ± 187.3† 170.1 ± 65.3†‡
NREM sleep S1 (%) S2 (%) S3+S4 (%) REM sleep(%) AGE (μg ml − 1 ) sRAGE (pg ml − 1) esRAGE (pg ml − 1)
Abbreviations: AGE, advanced glycation end products; AHI, apnea–hypopnea index; BMI, body mass index; Ccr, creatinine clearance rate; DBP, diastolic blood pressure; esRAGE, endogenous secretory; FPG, fasting plasma glucose; FPI, fasting plasma insulin; HbA1c, glycated hemoglobin A1c; HDL-C, high-density lipoprotein cholesterol; hsCRP, high-sensitivity C-reactive protein; IQR, interquartile ranges; LDL-C, low-density lipoprotein cholesterol; NREM sleep, nonrapid eye movement sleep; PPG, postprandial plasma glucose; REM sleep, rapid eye movement sleep; SBP, systolic blood pressure; sRAGE, soluble RAGE; SaO2, oxygen saturation; S1,stage 1; S2, stage 2; S3+S4, stage 3 and stage 4; TC, total cholesterol; TG, triglycerides; TST, total sleep time; WC, waist circumference. Data are presented as mean ± s.d., median (IQR) or n (%). †Po0.05 versus first tertile. ‡Po 0.05 versus second tertile.
Journal of Human Hypertension (2015), 1 – 6
© 2015 Macmillan Publishers Limited
Serum levels of endogenous secretory RAGE and blood pressure W Cai et al
r=−0.273, p=0.006
r=−0.213, p=0.029
Figure 1. Correlation between serum levels of esRAGE and SBP (a) and DBP (b).
Statistical analysis Results are expressed as mean ± s.d. or as median and interquartile ranges if the data were found to be non-normally distributed. Natural logarithmic transformations were made for non-normally distributed variables before analyses, and differences among groups were analyzed by one-way analysis of variance. Pearson’s correlation analyses were used to determine the relationship between two variables. Partial correlation analyses were used to test the relationship between two variables, while adjusting for potential confounders. Multiple stepwise regression analyses were performed to determine the independent parameters of systolic blood pressure (SBP) and diastolic blood pressure (DBP). Age-, waist circumference- and body mass index (BMI)-adjusted SBP and DBP levels stratified by serum esRAGE tertiles were compared using analyses of covariance. All statistical tests were two-sided, with a P-value of o0.05 taken to indicate statistical significance. Statistical analyses were carried out using SPSS Version 16 (SPSS, Chicago, IL, USA). © 2015 Macmillan Publishers Limited
RESULTS Clinical characteristics and serum esRAGE and sRAGE levels of participants Clinical and metabolic characteristics of subjects categorized by AHI tertiles are shown in Table 1. Subjects were categorized by AHI tertiles into three groups as follows: first tertile of AHI, AHI o 42, n = 46; second tertile of AHI, 42 ⩽ AHIo 66, n = 46; and third tertile of AHI, AHI ⩾ 66, n = 47. BMI, waist circumference, duration of SaO2 o 90%, SBP, HbA1c, fasting plasma glucose, postprandial plasma glucose, fasting plasma insulin, HOMA-IR, TC, TG, highsensitivity C-reactive protein and AGEs levels showed a significant increase with increasing AHI (P o0.05); minimum SaO2, duration of SWS, serum sRAGE and serum esRAGE showed a significant decrease with increasing AHI (P o 0.05); and total sleep time and the duration of REM sleep did not differ significantly among the three groups by AHI tertiles, which is consistent with sleep fragmentation and poor sleep quality associated with the severity of OSA. In addition, serum esRAGE significantly correlated with SBP (r = − 0.273, P = 0.006), DBP (r = − 0.213, P = 0.029) and hs-CRP (r = − 0.225, P = 0.022). There were no significant correlations between serum sRAGE and SBP (P40.05) or DBP (P40.05). Serum sRAGE significantly correlated with serum AGE (r = 0.199, P = 0.038). There was no significant correlation between serum esRAGE and serum AGE (P40.05). Relationship between SBP and serum esRAGE levels Pearson’s correlation analyses showed that SBP significantly correlated with BMI (r = 0.345, P = 0.002), waist circumference (r = 0.364, P o0.001), smoking status (r = 0.211, P = 0.041), fasting blood glucose (r = 0.217, P = 0.034), HOMA-IR (r = 0.214, P = 0.033), HbA1c (r = 0.261, P = 0.014), minimum SaO2 (r = − 0.359, P = 0.001), duration of SWS (r = − 0.229, P = 0.040), serum AGE (r = 0.245, P = 0.022) and serum esRAGE (r = − 0.273, P = 0.006) (Figure 1a). No significant correlations were found with age, smoking status, TG, TC, high-density lipoprotein cholesterol, LDL-C, AHI, hs-CRP and serum sRAGE (P40.05). To determine whether the association between SBP and serum esRAGE was independent of other confounders, multiple linear regression analysis was performed. Waist circumference (β = 0.380, P o 0.0001), HbA1c (β = 0.213, P = 0.043), minimum SaO2 (β = − 0.264, P = 0.013) and serum esRAGE (β = − 0.270, P = 0.011) were found to be independently associated with SBP (R2 = 0.347, F = 8.22, P o 0.0001). Participants were categorized by SBP tertiles into three groups. Serum esRAGE level in participants in the third tertile of SBP (170.8 ± 42.6 pg ml − 1) was significantly lower than that in participants in the second tertile of SBP (230.4 ± 65.3 pg ml − 1) (P = 0.024) and in the first tertile of SBP (261.4 ± 71.4 pg ml − 1) (P = 0.001), and serum esRAGE level in participants in the second tertile of SBP was also lower than that in participants in the first tertile of SBP, but the difference was not statistically significant (P40.05). After adjustment for age, BMI and waist circumference, analysis of covariance showed a significant decreasing trend in serum esRAGE levels among the three groups (P = 0.002 for trend) (Figure 2a), meaning that serum esRAGE levels significantly decreased with SBP levels increasing. Relationship between DBP and serum esRAGE levels Pearson’s correlation analyses showed that DBP significantly correlated with BMI (r = 0.314, P = 0.001), waist circumference (r = 0.265, P = 0.009), smoking status (r = 0.231, P = 0.021), fasting blood glucose (r = 0.208, P = 0.036), LDL-C (r = 0.330, P = 0.002), AHI (r = 0.289, P = 0.004), minimum SaO2 (r = − 0.216, P = 0.031), serum AGE (r = 0.266, P = 0.010) and serum esRAGE (r = − 0.213, P = 0.029) (Figure 1b). No significant correlations were observed for age, Journal of Human Hypertension (2015), 1 – 6
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Serum levels of endogenous secretory RAGE and blood pressure W Cai et al
4 p =0.002 for trend
†‡
Tertiles of SBP levels (mmHg)
p =0.056 for trend
S
†
Tertiles of DBP levels (mmHg)
Figure 2. Age, BMI and waist circumference adjusted for serum esRAGE levels (mean ± s.d.) stratified by SBP (a) and DBP (b) level tertiles. †P o0.05 versus first tertile of SBP. ‡P o0.05 versus second tertile of SBP.
HOMA-IR, TG, TC, high-density lipoprotein cholesterol, duration of SWS, hs-CRP and serum sRAGE (P40.05). Although the correlation between esRAGE and DBP remained significant after adjustment for potential confounders such as age, waist circumference (or BMI), Ccr and HbA1c (P = 0.031), multiple linear regression analysis showed that serum esRAGE was not independently associated with DBP (P40.05). LDL-C (β = 0.243, P = 0.028), AHI (β = 0.235, P = 0.036), serum AGE (β = 0.260, P = 0.013) and BMI (β = 0.256, P = 0.018) were independently associated with DBP (R2 = 0.331, F = 7.37, P o 0.0001). Participants were categorized by DBP tertiles into three groups. Serum esRAGE level in participants in the third tertile of DBP Journal of Human Hypertension (2015), 1 – 6
(189.2 ± 46.9 pg ml − 1) was significantly lower than that in participants in the first tertile of SBP (247.9 ± 73.5 pg ml − 1) (P = 0.021), and it was lower than that in participants in the second tertile of DBP (227.3 ± 56.2 pg ml − 1), but the difference was not statistically significant (P40.05). After adjustment for age, BMI and waist circumference, analysis of covariance showed a decreasing trend in serum esRAGE levels among the three groups, but this trend was not statistically significant (P = 0.056 for trend) (Figure 2b). DISCUSSION This is the first comparative study of serum esRAGE and sRAGE as markers of disease in nondiabetic participants with OSA. This study found that serum esRAGE, but not serum sRAGE, significantly correlated with blood pressure. Serum esRAGE levels were inversely associated with SBP level, independent of other potential confounding factors. Obesity is a common risk factor for OSA, type 2 diabetes and high blood pressure. In this study, BMI, and particularly waist circumference, which reflects abdominal obesity, correlated with SBP and DBP, and with the severity of OSA, as evaluated by AHI and minimum SaO2. OSA patients experience intermittent hypoxia and sleep deprivation, especially deep SWS deprivation during the night, which elicit sustained sympathetic activation driving an increase in blood pressure.23,24 Data from this study suggest that minimum SaO2 was independently associated with elevated SBP, and AHI was independently associated with elevated DBP, and duration of SWS also significantly correlated with SBP. The relationship between serum esRAGE and SBP was independent of waist circumference, BMI, minimum SaO2, AHI, duration of SWS and other conventional risk factors such as age, smoking, HbA1c, HOMA-IR, TG, TC, high-density lipoprotein cholesterol, LDL-C and Ccr in this study. Although serum esRAGE was not a main determinant of DBP, the correlation between esRAGE and DBP remained significant after adjustment for classical confounders such as age, waist circumference (or BMI), Ccr and HbA1c. Serum AGE also significantly correlated with SBP and DBP, and it was the main determinant of DBP. These results suggest a close relationship between the AGE–RAGE axis and hypertension in patients with OSA. There is growing evidence to suggest that the AGE–RAGE axis has an important role in the development and progression of atherosclerosis,14,16 one of the pathogenic factors for hypertension. It has been suggested that esRAGE, acting as an antagonistic decoy receptor, may exhibit a feedback mechanism by which esRAGE prevents the activation of RAGE signaling. esRAGE was found to be capable of neutralizing the effects of AGE on endothelial cells in culture.25 Overexpression of esRAGE in vivo in mice reverses diabetic impairment of vascular dysfunction.20 Although there remains insufficient evidence of the relationship between serum esRAGE and atherosclerosis in patients with OSA, studies have found an inverse correlation between serum esRAGE and carotid atherosclerosis in type 1 and type 2 diabetic patients.26,27 In an observational cohort of patients with endstage renal disease, the cumulative incidence of cardiovascular death was significantly higher in patients with lower plasma esRAGE levels.28 These results suggest that esRAGE may act as a protective factor against cardiovascular diseases. In the present study, a significant association between serum esRAGE and blood pressure in male nondiabetic participants with OSA was observed. No significant correlation between serum sRAGE and blood pressure was found. Such findings are similar to previous studies examining other diseases, which reported that circulating esRAGE levels, but not sRAGE levels, inversely correlated with several components of metabolic syndrome, including blood pressure.22,29 However, Geroldi et al.21 reported that decreased levels of plasma sRAGE correlated with SBP in © 2015 Macmillan Publishers Limited
Serum levels of endogenous secretory RAGE and blood pressure W Cai et al
patients with essential hypertension. In addition, Hudson et al.30 reported that serum sRAGE levels inversely correlated with several components of metabolic syndrome, including hypertension. Circulating esRAGE levels were not measured in these studies. From these results, it is possible that serum sRAGE and esRAGE levels are distinct biomarkers, and may have different roles in various disease states. sRAGE and esRAGE are two isoforms of C-truncated RAGE.17 sRAGE is formed by the cleavage of native membrane receptor mediated by disintegrin and matrix metalloproteinase. esRAGE is formed from alternative splicing of native membrane receptor, and it is secreted from cells.31 The regulatory mechanisms for alternative splicing to generate esRAGE and for proteolytic shedding of cell surface RAGE to generate sRAGE remain unclear.32 A weak correlation between serum sRAGE and serum AGE, but no correlation between serum esRAGE and serum AGE, was observed in the current study. Similarly, Yamagishi et al.33 reported that serum sRAGE was positively associated with serum AGE in nondiabetic participants. These results suggest that circulating sRAGE and esRAGE levels may be under the control of different mechanisms. It is unclear whether the pathophysiological significance of circulating esRAGE and sRAGE levels is distinct in different clinical settings, and further examination is required. This study had some limitations. First, this was a cross-sectional study using correlation analysis. Therefore, the results can only suggest possible associations and not causal relationships between serum esRAGE and blood pressure in OSA patients. Although evidence suggestive of mechanistic links between RAGE and cardiovascular diseases has been reported in in vitro and in vivo experiments,14–16 further studies using a large prospective cohort are needed to elucidate the relationship between serum esRAGE and blood pressure in patients with OSA. Katakami et al. reported that esRAGE, but not sRAGE, was associated with carotid atherosclerosis in type 1 diabetes patients in a cross-sectional study.34 However, in a 4-year follow-up study, the researchers found that circulating esRAGE and sRAGE levels were associated with the progression of carotid atherosclerosis in type 1 diabetes patients.26 Second, analyses in the current study were based on office blood pressure measurements, which are subject to methodological limitations. Although we took care to optimize the procedure (sufficient resting period, appropriate cuff size and use of mean from two measurements), we acknowledge that the use of 24-h ambulatory blood pressure measurements would strengthen our analyses. Third, all participants were male, because most patients with OSA undergoing polysomnography at our hospital were male. Although studies of patients without OSA have reported that serum esRAGE levels were associated with hypertension in female, as well as in male, patients,22,29 further research that includes both male and female patients is needed to confirm the results. In addition, there were no mild cases of OSA or participants without OSA in the current study, because most patients with OSA undergoing polysomnography at our hospital had moderate-to-severe OSA. Although previous studies had suggested that circulating esRAGE levels inversely correlated with blood pressure in other diseases such as metabolic syndrome,22,29 and the strength of the correlation between serum esRAGE levels and blood pressure in patients with different severities of OSA was similar when participants in this study were categorized by AHI tertiles for analysis, this study would have benefited from a control group, which would have allowed us to establish the relationship between serum esRAGE levels and blood pressure in individuals without OSA. In conclusion, we found that serum esRAGE levels were inversely associated with blood pressure, especially SBP, in male nondiabetic patients with OSA. These findings suggest that esRAGE may have a protective role against hypertension in © 2015 Macmillan Publishers Limited
5 patients with OSA, and may be a novel biomarker for OSA patients at high risk of developing cardiovascular diseases. What is known about this topic? ● Interaction of advanced glycation end products (AGE) and their specific cell-surface receptor (RAGE) has an important role in the pathogenesis of cardiovascular disease, and two isoforms of C-truncated RAGE, soluble RAGE (sRAGE) and endogenous secretory RAGE (esRAGE), may prevent activation of RAGE signaling by acting as decoys. ● It has been reported that circulating sRAGE levels correlated with blood pressure in patients with essential hypertension and metabolic syndrome. ● Less is known about the relationship between serum esRAGE, sRAGE and blood pressure in patients with obstructive sleep apnea (OSA). What this study adds? ● Serum esRAGE levels, but not serum sRAGE levels, were inversely associated with blood pressure, especially SBP, in male nondiabetic patients with OSA. ● esRAGE may have a protective role against hypertension in patients with OSA, and may be a novel biomarker for OSA patients at high risk of developing cardiovascular diseases.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS The authors thank all patients who participated in this study. The study was supported by grants from the National Natural Science Funds of China (No. 81160105, 81360017, 81460018) and the Jiangxi Provincial Natural Science Funds of China (No. 2010GZY0325, 20114BAB205006).
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6 12 Kotani K, Kimura S, Komada I, Sakane N, Gugliucci A. Continuous positive air pressure treatment reduces serum advanced glycation end products in patients with obstructive sleep apnoea syndrome: a pilot study. Prim Care Respir J 2011; 20 (3): 336–337. 13 Xu JX, Cai W, Sun JF, Liao WJ, Liu Y, Xiao JR et al. Serum advanced glycation end products are associated with insulin resistance in male nondiabetic patients with obstructive sleep apnea. Sleep Breath (e-pub ahead of print 2 January 2015; doi: 10.1007/s11325-014-1100-z). 14 Basta G, Lazzerini G, Massaro M, Simoncini T, Tanganelli P, Fu C et al. Advanced glycation end products activate endothelium through signal transduction receptor RAGE: a mechanism for amplification of inflammatory responses. Circulation 2002; 105: 816–822. 15 Elmasry A, Lindberg E, Hedner J, Janson C, Boman G. RAGE-induced cytosolic ROS promote mitochondrial superoxide generation in diabetes. J Am Soc Nephrol 2009; 20: 742–752. 16 Yan SF, Ramasamy R, Schmidt AM. The RAGE Axis: a fundamental mechanism signaling danger to the vulnerable vasculature. Circ Res 2010; 106: 842–853. 17 Hudson BI, Carter AM, Harja E, Kalea AZ, Arriero M, Yang H et al. Identification, classification, an expression of RAGE gene splice variants. FASEB J 2008; 22: 1572–1580. 18 Bucciarelli LG, Wendt T, Qu W, Lu Y, Lalla E, Rong LL et al. RAGE blockade stabilizes established atherosclerosis in diabetic apolipoprotein E-null mice. Circulation 2002; 106: 2827–2835. 19 YanS F, D’Agati V, Schmidt AM, Ramasamy R. Receptor for Advanced Glycation End products (RAGE): a formidable force in the pathogenesis of the cardiovascular complications of diabetes & aging. Curr Mol Med 2007; 7: 699–710. 20 Shoji T, Koyama H, Morioka T, Tanaka S, Kizu A, Motoyama K et al. Receptor for advanced glycation end products is involved in impaired angiogenic response in diabetes. Diabetes 2006; 55: 2245–2255. 21 Geroldi D, Falcone C, Emanuele E, D'Angelo A, Calcagnino M, Buzzi MP et al. Decreased plasma levels of soluble receptor for advanced glycation end-products in patients with essential hypertension. J Hypertens 2005; 23: 1725–1729. 22 Momma H, Niu K, Kobayashi Y, Huang C, Chujo M, Otomo A et al. Lower serum endogenous secretory receptor for advanced glycation end product level as a risk factor of metabolic syndrome among Japanese adult men: a 2-year longitudinal study. J Clin Endocrinol Metab 2014; 99: 587–593. 23 Narkiewicz K, van de Borne PJ, Cooley RL, Dyken ME, Somers VK. Sympathetic activity in obese subjects with and without obstructive sleep apnea. Circulation 1998; 98: 772–776.
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24 Narkiewicz K, Somers VK. Sympathetic nerve activity in obstructive sleep apnoea. Acta Physiol Scand 2003; 177: 385–390. 25 Yonekura H, Yamamoto Y, Sakurai S, Petrova RG, Abedin MJ, Li H et al. Novel splice variants of the receptor for advanced glycation end-products expressed in human vascular endothelial cells and pericytes, and their putative roles in diabetes-induced vascular injury. Biochem J 2003; 370: 1097–1109. 26 Katakami N, Matsuhisa M, Kaneto H, Matsuoka TA, Sakamoto K, Yasuda T et al. Serum endogenous secretory RAGE level is an independent risk factor for the progression of carotid atherosclerosis in type 1 diabetes. Atherosclerosis 2009; 204: 288–292. 27 Katakami N, Matsuhisa M, Kaneto H, Yamasaki Y. Serum endogenous secretory RAGE levels are inversely associated with carotid IMT in type 2 diabetic patients. Atherosclerosis 2007; 190: 22–23. 28 Koyama H, Yamamoto H, Nishizawa Y. Endogenous secretory RAGE as a novel biomarker for metabolic syndrome and cardiovascular diseases. Biomark Insights 2007; 2: 331–339. 29 Koyama H, Shoji T, Yokoyama H, Motoyama K, Mori K, Fukumoto S et al. Plasma level of endogenous secretory RAGE is associated with components of the metabolic syndrome and atherosclerosis. Arterioscler Thromb Vasc Biol 2005; 25: 2587–2593. 30 Hudson BI, Dong C, Gardener H, Elkind MS, Wright CB, Goldberg R et al. Serum levels of soluble receptor for advanced glycation end-products and metabolic syndrome: the Northern Manhattan Study. Metabolism 2014; 63: 1125–1130. 31 Raucci A, Cugusi S, Antonelli A, Barabino SM, Monti L, Bierhaus A et al. A soluble form of the receptor for advanced glycation end products(RAGE) is produced by proteolytic cleavage of the membrane-bound form by the sheddase a disintegrin and metalloprotease 10 (ADAM10). FASEB J 2008; 22: 3716–3727. 32 Ohe K, Watanabe T, Harada SI, Munesue S, Yamamoto Y, Yonekura H et al. Regulation of alternative splicing of the receptor for advanced glycation endproducts (RAGE) through G-rich cis-elements and heterogenous nuclear ribonucleoprotein H. J. Biochem 2010; 147: 651–659. 33 Yamagishi S, Adachi H, Nakamura K, Matsui T, Jinnouchi Y, Takenaka K et al. Positive association between serum levels of advanced glycation end products and the soluble form of receptor for advanced glycation end products in nondiabetic subjects. Metabolism 2006; 55: 1227–1231. 34 Katakami N, Matsuhisa M, Kaneto H, Matsuoka TA, Sakamoto K, Yasuda T et al. Endogenous secretory RAGE but not soluble RAGE is associated with carotid atherosclerosis in type 1 diabetes patients. Diabetes Vasc Dis Res 2008; 5: 190–197.
© 2015 Macmillan Publishers Limited
ORIGINAL ARTICLE
Relationship between serum levels of endogenous secretory RAGE and blood pressure in male nondiabetic patients with obstructive sleep apnea W Cai1,2, J-F Sun1, Y Liu1, J-X Xu1, J-R Xiao1, X-M Duan1, J-Y Liu1 and W Zhang3 The interaction of advanced glycation end products (AGE) and their specific cell-surface receptor (RAGE) has an important role in the pathogenesis of cardiovascular disease and diabetic complications. Two isoforms of C-truncated RAGE, soluble RAGE (sRAGE) and endogenous secretory RAGE (esRAGE), may prevent activation of RAGE signaling by acting as decoys. This study investigated whether serum esRAGE and sRAGE levels are associated with blood pressure in nondiabetic patients with obstructive sleep apnea (OSA). Male nondiabetic patients (n = 139) with OSA were enrolled. Serum esRAGE and sRAGE levels were examined using enzyme-linked immunosorbent assay. Three consecutive seated systolic blood pressure (SBP) and diastolic blood pressure (DBP) measurements were obtained at 5-min intervals in the morning. In univariate analysis, there was a significant correlation between serum esRAGE and SBP or DBP, but not between serum sRAGE and SBP or DBP. Multiple regression analysis showed that SBP was independently associated with waist circumference, HbA1c, minimum SaO2 and serum esRAGE, and that DBP was independently associated with low-density lipoprotein cholesterol, apnea–hypopnea index, serum AGE and body mass index, but not with serum esRAGE. These results indicated that serum esRAGE levels were inversely associated with blood pressure, especially SBP, in male nondiabetic patients with OSA. esRAGE may have a protective role against hypertension in patients with OSA, and it may be a novel biomarker for OSA patients at high risk of developing cardiovascular diseases. Journal of Human Hypertension advance online publication, 21 May 2015; doi:10.1038/jhh.2015.46
INTRODUCTION Epidemiological data from community populations have provided evidence suggestive of an independent association between obstructive sleep apnea (OSA) and hypertension.1,2 OSA has also been identified as a secondary cause of hypertension.3,4 Although studies have reported that the intermittent hypoxia and sleep deprivation that OSA patients experience can result in increased nervous system activity,5,6 which can eventually lead to elevated blood pressure, the mechanisms behind the pathogenesis of hypertension in patients with OSA are complicated and require clarification. Advanced glycation end products (AGE) are protein products generated by the Maillard reaction. Classically, AGE formation has been described by a nonenzymatic reaction between proteins and glucose under hyperglycemic conditions.7 The intermittent hypoxemia that occurs in patients with OSA may induce the formation of AGE.8 Several studies have reported that serum AGE levels increase in nondiabetic people with OSA,9,10 and continuous positive airway pressure therapy was found to reduce serum AGE levels.10–12 In our previous study, we found that serum AGE levels in nondiabetic patients with OSA were associated with insulin resistance,13 which is an important risk factor for hypertension. There is growing evidence to suggest that interaction between AGE and their specific cell-surface receptor, the receptor for AGE (RAGE), stimulates oxidative stress generation and has an
important role in cardiovascular disease and diabetic complications.14–16 Recently, two circulating isoforms of RAGE have been identified: soluble RAGE (sRAGE) and endogenous secretory RAGE (esRAGE).17 Administration of recombinant sRAGE inhibited the development and progression of atherosclerosis in animal models,18,19 and esRAGE was found to be capable of neutralizing the effects of AGE on endothelial cells in culture.20 These findings suggest that sRAGE and esRAGE may capture circulating AGE by acting as decoy receptors. Geroldi et al.21 reported that decreased levels of plasma sRAGE were related to pulse pressure in patients with essential hypertension. However, Momma et al.22 reported that circulating esRAGE levels, but not sRAGE, inversely correlated with several components of metabolic syndrome, including blood pressure. The relationship between serum esRAGE, sRAGE and blood pressure in patients with OSA remains unclear. In this study, we examined whether serum esRAGE and sRAGE levels were associated with blood pressure in a population of male nondiabetic patients with OSA. MATERIALS AND METHODS Study participants Participants with OSA were recruited from the sleep laboratory at the First Affiliated Hospital of Nanchang University between 2013 and 2014.
1 Department of Endocrinology and Metabolism, First Affiliated Hospital of Nanchang University, Nanchang, People’s Republic of China; 2Department of Medical Biology, Medical College of Nanchang University, Nanchang, People’s Republic of China and 3Department of Respiratory Medicine, First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China. Correspondence: Professor J-X Xu, Department of Endocrinology and Metabolism, First Affiliated Hospital of Nanchang University, 17 Yongwaizheng Street, Nanchang, Jiangxi 330006, People’s Republic of China. E-mail: [email protected] Received 29 January 2015; revised 9 April 2015; accepted 16 April 2015
Serum levels of endogenous secretory RAGE and blood pressure W Cai et al
2 An overnight sleep study was conducted using a computerized polysomnogram system (Alice LE, Philips Respironics California, Inc., Carlsbad, CA, USA). Apneas were defined as complete cessation of air flow for ⩾ 10 s. Hypopnea was defined as a ⩾ 50% reduction of air flow or a o50% reduction of air flow accompanied by a ⩾ 3% decrease in oxygen saturation, or followed by an arousal. Measurement of the apnea– hypopnea index (AHI; the total number of apnea and hypopnea episodes divided by hours of sleep) during the night were obtained. Finger pulse oximetry was used to determine arterial blood oxygen saturation (SaO2). Time of SaO2 o90% and the lowest SaO2 (%) were calculated. Time spent in rapid eye movement sleep and nonrapid eye movement sleep (stages 1–4) were calculated as a percentage of the total sleep time. Stages 3 and 4 of nonrapid eye movement sleep are also known as slow-wave sleep (SWS). Procedures were performed by experienced technicians. Computerized sleep data were manually analyzed by permanent professional staff. Participants had their blood pressure measured when they were referred to the sleep laboratory in the morning. Three consecutive seated blood pressure measurements were obtained at 5-min intervals after the patient had rested for 15 min. Measurements used an appropriately sized cuff and were performed in a quiet room. The average of the last two blood pressure readings was used for the analysis. The following morning, participants with an AHI415 underwent an oral glucose tolerance test for which they received 75 g of glucose orally, and blood samples were obtained before and after 2 h (at 0 and 120 min) for measurement of glucose and insulin. A total of 139 male participants with an AHI415, defined as nondiabetic by 1999 World Health Organization diagnostic criteria, were enrolled in the study. Participants with neoplastic, hepatic, renal, lung, and heart diseases or inflammatory diseases were excluded.
Table 1.
Furthermore, people who received glucocorticoids, or antidiabetic, antihypertensive, antiobesity or lipid-lowering drugs, were excluded. All participants gave written informed consent before sample collection. This study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University.
Biochemical analyses Fasting blood samples were taken for measurement of fasting plasma glucose, fasting plasma insulin, hemoglobin A1c (HbA1c), high-sensitivity C-reactive protein, total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol, creatinine, serum AGE, serum esRAGE and serum sRAGE. All samples were stored at − 80 °C before analysis. Serum AGE was measured using a competitive inhibition enzyme-linked immunosorbent assay (ELISA) with an intra-assay coefficient of variation (CV) of o 10% and an inter-assay CV of o 12% according to the supplier’s recommendation (Uscn Life Science Inc., Wuhan, China). Serum sRAGE was measured using the R&D Systems Quantikine Immunoassay (Minneapolis, MN, USA) specific for the extracellular domain of human RAGE, with an intra-assay CV of o 6.2% and an inter-assay CV of o8.2%. Serum esRAGE was measured using B-Bridge ELISA (B-Bridge International, Cupertino, CA, USA), with an intra-assay CV of o4.4% and an inter-assay CV of o5.3%. Insulin resistance was determined using the homeostasis model assessment index (HOMA-IR): fasting plasma glucose (mmol l − 1) × fasting plasma insulin (mIU l − 1)/22.5. The creatinine clearance rate (Ccr) was calculated using the Cockcroft– Gault equation.
Clinical and metabolic characteristics of subjects categorized by AHI tertiles Total
First tertile (AHIo42)
Second tertile (42 ⩽ AHIo66)
Third tertile (AHI ⩾ 66)
n Age (years) Smoking (n(%)) BMI (kg m − 2) WC (cm) SBP (mm Hg) DBP (mm Hg) HbA1c (%) FPG(mmol l − 1) PPG (mmol l − 1) FPI (mIU l − 1) HOMA-IR TC (mmol l − 1) TG (mmol l − 1) LDL-C (mmol l − 1) HDL-C (mmol l − 1) Creatinine(μmol l − 1) Ccr (ml min − 1) hsCRP (mg l − 1) AHI (events h − 1)
139 41.8 ± 11.1 53 (38.1) 28.10 ± 3.19 96.46 ± 9.72 133.0 ± 13.6 90.9 ± 12.4 5.85 ± 0.79 5.64 ± 0.82 7.19 ± 2.04 13.6 (9.7 − 19.1) 3.15 (2.05 − 4.29) 5.27 ± 1.09 1.86 (1.17 − 2.73) 3.26 ± 0.98 1.28 (1.10 − 1.62) 76.6 ± 16.5 85.8 ± 19.9 2.35 (1.41 − 4.13) 53.1 (33.6 − 73.7)
46 41.8 ± 10.5 17 (37.0) 26.49 ± 3.20 94.12 ± 9.95 132.3 ± 14.8 89.4 ± 12.5 5.70 ± 0.56 5.39 ± 0.87 7.10 ± 2.17 10.0 (8.1 − 21.3) 2.69 (1.71 − 3.43) 5.08 ± 1.04 1.48 (1.01 − 2.22) 3.17 ± 0.89 1.32 (1.15 − 1.50) 76.8 ± 16.9 86.3 ± 20.2 2.04 (1.34 − 2.78) 28.3 ± 5.6
46 41.9 ± 11.3 20 (39.1) 28.38 ± 3.35† 97.31 ± 9.15† 133.7 ± 15.7 91.5 ± 13.4 5.75 ± 0.61 5.63 ± 0.83† 7.18 ± 2.22 13.4 (8.8 − 20.6)† 2.99 (1.73 − 4.45)† 5.15 ± 1.12 1.97 (1.33 − 2.72)† 3.25 ± 1.09 1.30 (1.11 − 1.87) 75.9 ± 14.3 86.0 ± 22.4 2.24 (1.34 − 3.71)† 53.6 ± 6.1†
47 42.0 ± 10.4 18 (38.3) 28.63 ± 3.41† 97.82 ± 8.84† 134.9 ± 12.4 91.6 ± 11.8 6.08 ± 1.06† 5.88 ± 0.75† 7.23 ± 1.79† 15.5 (10.7 − 25.6)†‡ 3.55 (2.73 − 4.66)†‡ 5.51 ± 1.10† 2.31 (1.64 − 3.88)†‡ 3.35 ± 1.00 1.23 (0.97 − 1.58) 78.1 ± 15.5 84.7 ± 25.6 2.62 (1.46 − 4.41)†‡ 79.3 ± 9.1†‡
SaO2 Durationo90%(min) Minimum (%) TST (min)
31.5 (8.3 − 104.6) 69.7 ± 12.6 317.3 ± 12.5
27.1 (8.3 − 52.4) 77.8 ± 10.9 326.9 ± 11.2
31.6 (11.1 − 83.2)† 75.3 ± 11.3 318.1 ± 10.8
34.2 (14.8 − 101.8)†‡ 67.1 ± 10.3†‡ 314.2 ± 11.8
10.9 ± 2.3 52.7 ± 14.6 16.5 ± 3.7 17.8 ± 2.6 2.63 ± 0.61 1032.6 ± 233.9 225.3 ± 43.7
7.7 ± 1.8 52.2 ± 13.1 23.9 ± 4.7 17.2 ± 2.4 2.27 ± 0.29 1143.2 ± 223.6 241.0 ± 69.4
10.6 ± 2.5† 52.5 ± 12.8 19.6 ± 4.3† 17.9 ± 2.8 2.51 ± 0.40† 1021.5 ± 208.1 217.3 ± 60.7†
14.2 ± 2.7†‡ 52.1 ± 12.4 15.8 ± 4.1†‡ 18.0 ± 2.6 2.98 ± 0.46†‡ 985.7 ± 187.3† 170.1 ± 65.3†‡
NREM sleep S1 (%) S2 (%) S3+S4 (%) REM sleep(%) AGE (μg ml − 1 ) sRAGE (pg ml − 1) esRAGE (pg ml − 1)
Abbreviations: AGE, advanced glycation end products; AHI, apnea–hypopnea index; BMI, body mass index; Ccr, creatinine clearance rate; DBP, diastolic blood pressure; esRAGE, endogenous secretory; FPG, fasting plasma glucose; FPI, fasting plasma insulin; HbA1c, glycated hemoglobin A1c; HDL-C, high-density lipoprotein cholesterol; hsCRP, high-sensitivity C-reactive protein; IQR, interquartile ranges; LDL-C, low-density lipoprotein cholesterol; NREM sleep, nonrapid eye movement sleep; PPG, postprandial plasma glucose; REM sleep, rapid eye movement sleep; SBP, systolic blood pressure; sRAGE, soluble RAGE; SaO2, oxygen saturation; S1,stage 1; S2, stage 2; S3+S4, stage 3 and stage 4; TC, total cholesterol; TG, triglycerides; TST, total sleep time; WC, waist circumference. Data are presented as mean ± s.d., median (IQR) or n (%). †Po0.05 versus first tertile. ‡Po 0.05 versus second tertile.
Journal of Human Hypertension (2015), 1 – 6
© 2015 Macmillan Publishers Limited
Serum levels of endogenous secretory RAGE and blood pressure W Cai et al
r=−0.273, p=0.006
r=−0.213, p=0.029
Figure 1. Correlation between serum levels of esRAGE and SBP (a) and DBP (b).
Statistical analysis Results are expressed as mean ± s.d. or as median and interquartile ranges if the data were found to be non-normally distributed. Natural logarithmic transformations were made for non-normally distributed variables before analyses, and differences among groups were analyzed by one-way analysis of variance. Pearson’s correlation analyses were used to determine the relationship between two variables. Partial correlation analyses were used to test the relationship between two variables, while adjusting for potential confounders. Multiple stepwise regression analyses were performed to determine the independent parameters of systolic blood pressure (SBP) and diastolic blood pressure (DBP). Age-, waist circumference- and body mass index (BMI)-adjusted SBP and DBP levels stratified by serum esRAGE tertiles were compared using analyses of covariance. All statistical tests were two-sided, with a P-value of o0.05 taken to indicate statistical significance. Statistical analyses were carried out using SPSS Version 16 (SPSS, Chicago, IL, USA). © 2015 Macmillan Publishers Limited
RESULTS Clinical characteristics and serum esRAGE and sRAGE levels of participants Clinical and metabolic characteristics of subjects categorized by AHI tertiles are shown in Table 1. Subjects were categorized by AHI tertiles into three groups as follows: first tertile of AHI, AHI o 42, n = 46; second tertile of AHI, 42 ⩽ AHIo 66, n = 46; and third tertile of AHI, AHI ⩾ 66, n = 47. BMI, waist circumference, duration of SaO2 o 90%, SBP, HbA1c, fasting plasma glucose, postprandial plasma glucose, fasting plasma insulin, HOMA-IR, TC, TG, highsensitivity C-reactive protein and AGEs levels showed a significant increase with increasing AHI (P o0.05); minimum SaO2, duration of SWS, serum sRAGE and serum esRAGE showed a significant decrease with increasing AHI (P o 0.05); and total sleep time and the duration of REM sleep did not differ significantly among the three groups by AHI tertiles, which is consistent with sleep fragmentation and poor sleep quality associated with the severity of OSA. In addition, serum esRAGE significantly correlated with SBP (r = − 0.273, P = 0.006), DBP (r = − 0.213, P = 0.029) and hs-CRP (r = − 0.225, P = 0.022). There were no significant correlations between serum sRAGE and SBP (P40.05) or DBP (P40.05). Serum sRAGE significantly correlated with serum AGE (r = 0.199, P = 0.038). There was no significant correlation between serum esRAGE and serum AGE (P40.05). Relationship between SBP and serum esRAGE levels Pearson’s correlation analyses showed that SBP significantly correlated with BMI (r = 0.345, P = 0.002), waist circumference (r = 0.364, P o0.001), smoking status (r = 0.211, P = 0.041), fasting blood glucose (r = 0.217, P = 0.034), HOMA-IR (r = 0.214, P = 0.033), HbA1c (r = 0.261, P = 0.014), minimum SaO2 (r = − 0.359, P = 0.001), duration of SWS (r = − 0.229, P = 0.040), serum AGE (r = 0.245, P = 0.022) and serum esRAGE (r = − 0.273, P = 0.006) (Figure 1a). No significant correlations were found with age, smoking status, TG, TC, high-density lipoprotein cholesterol, LDL-C, AHI, hs-CRP and serum sRAGE (P40.05). To determine whether the association between SBP and serum esRAGE was independent of other confounders, multiple linear regression analysis was performed. Waist circumference (β = 0.380, P o 0.0001), HbA1c (β = 0.213, P = 0.043), minimum SaO2 (β = − 0.264, P = 0.013) and serum esRAGE (β = − 0.270, P = 0.011) were found to be independently associated with SBP (R2 = 0.347, F = 8.22, P o 0.0001). Participants were categorized by SBP tertiles into three groups. Serum esRAGE level in participants in the third tertile of SBP (170.8 ± 42.6 pg ml − 1) was significantly lower than that in participants in the second tertile of SBP (230.4 ± 65.3 pg ml − 1) (P = 0.024) and in the first tertile of SBP (261.4 ± 71.4 pg ml − 1) (P = 0.001), and serum esRAGE level in participants in the second tertile of SBP was also lower than that in participants in the first tertile of SBP, but the difference was not statistically significant (P40.05). After adjustment for age, BMI and waist circumference, analysis of covariance showed a significant decreasing trend in serum esRAGE levels among the three groups (P = 0.002 for trend) (Figure 2a), meaning that serum esRAGE levels significantly decreased with SBP levels increasing. Relationship between DBP and serum esRAGE levels Pearson’s correlation analyses showed that DBP significantly correlated with BMI (r = 0.314, P = 0.001), waist circumference (r = 0.265, P = 0.009), smoking status (r = 0.231, P = 0.021), fasting blood glucose (r = 0.208, P = 0.036), LDL-C (r = 0.330, P = 0.002), AHI (r = 0.289, P = 0.004), minimum SaO2 (r = − 0.216, P = 0.031), serum AGE (r = 0.266, P = 0.010) and serum esRAGE (r = − 0.213, P = 0.029) (Figure 1b). No significant correlations were observed for age, Journal of Human Hypertension (2015), 1 – 6
3
Serum levels of endogenous secretory RAGE and blood pressure W Cai et al
4 p =0.002 for trend
†‡
Tertiles of SBP levels (mmHg)
p =0.056 for trend
S
†
Tertiles of DBP levels (mmHg)
Figure 2. Age, BMI and waist circumference adjusted for serum esRAGE levels (mean ± s.d.) stratified by SBP (a) and DBP (b) level tertiles. †P o0.05 versus first tertile of SBP. ‡P o0.05 versus second tertile of SBP.
HOMA-IR, TG, TC, high-density lipoprotein cholesterol, duration of SWS, hs-CRP and serum sRAGE (P40.05). Although the correlation between esRAGE and DBP remained significant after adjustment for potential confounders such as age, waist circumference (or BMI), Ccr and HbA1c (P = 0.031), multiple linear regression analysis showed that serum esRAGE was not independently associated with DBP (P40.05). LDL-C (β = 0.243, P = 0.028), AHI (β = 0.235, P = 0.036), serum AGE (β = 0.260, P = 0.013) and BMI (β = 0.256, P = 0.018) were independently associated with DBP (R2 = 0.331, F = 7.37, P o 0.0001). Participants were categorized by DBP tertiles into three groups. Serum esRAGE level in participants in the third tertile of DBP Journal of Human Hypertension (2015), 1 – 6
(189.2 ± 46.9 pg ml − 1) was significantly lower than that in participants in the first tertile of SBP (247.9 ± 73.5 pg ml − 1) (P = 0.021), and it was lower than that in participants in the second tertile of DBP (227.3 ± 56.2 pg ml − 1), but the difference was not statistically significant (P40.05). After adjustment for age, BMI and waist circumference, analysis of covariance showed a decreasing trend in serum esRAGE levels among the three groups, but this trend was not statistically significant (P = 0.056 for trend) (Figure 2b). DISCUSSION This is the first comparative study of serum esRAGE and sRAGE as markers of disease in nondiabetic participants with OSA. This study found that serum esRAGE, but not serum sRAGE, significantly correlated with blood pressure. Serum esRAGE levels were inversely associated with SBP level, independent of other potential confounding factors. Obesity is a common risk factor for OSA, type 2 diabetes and high blood pressure. In this study, BMI, and particularly waist circumference, which reflects abdominal obesity, correlated with SBP and DBP, and with the severity of OSA, as evaluated by AHI and minimum SaO2. OSA patients experience intermittent hypoxia and sleep deprivation, especially deep SWS deprivation during the night, which elicit sustained sympathetic activation driving an increase in blood pressure.23,24 Data from this study suggest that minimum SaO2 was independently associated with elevated SBP, and AHI was independently associated with elevated DBP, and duration of SWS also significantly correlated with SBP. The relationship between serum esRAGE and SBP was independent of waist circumference, BMI, minimum SaO2, AHI, duration of SWS and other conventional risk factors such as age, smoking, HbA1c, HOMA-IR, TG, TC, high-density lipoprotein cholesterol, LDL-C and Ccr in this study. Although serum esRAGE was not a main determinant of DBP, the correlation between esRAGE and DBP remained significant after adjustment for classical confounders such as age, waist circumference (or BMI), Ccr and HbA1c. Serum AGE also significantly correlated with SBP and DBP, and it was the main determinant of DBP. These results suggest a close relationship between the AGE–RAGE axis and hypertension in patients with OSA. There is growing evidence to suggest that the AGE–RAGE axis has an important role in the development and progression of atherosclerosis,14,16 one of the pathogenic factors for hypertension. It has been suggested that esRAGE, acting as an antagonistic decoy receptor, may exhibit a feedback mechanism by which esRAGE prevents the activation of RAGE signaling. esRAGE was found to be capable of neutralizing the effects of AGE on endothelial cells in culture.25 Overexpression of esRAGE in vivo in mice reverses diabetic impairment of vascular dysfunction.20 Although there remains insufficient evidence of the relationship between serum esRAGE and atherosclerosis in patients with OSA, studies have found an inverse correlation between serum esRAGE and carotid atherosclerosis in type 1 and type 2 diabetic patients.26,27 In an observational cohort of patients with endstage renal disease, the cumulative incidence of cardiovascular death was significantly higher in patients with lower plasma esRAGE levels.28 These results suggest that esRAGE may act as a protective factor against cardiovascular diseases. In the present study, a significant association between serum esRAGE and blood pressure in male nondiabetic participants with OSA was observed. No significant correlation between serum sRAGE and blood pressure was found. Such findings are similar to previous studies examining other diseases, which reported that circulating esRAGE levels, but not sRAGE levels, inversely correlated with several components of metabolic syndrome, including blood pressure.22,29 However, Geroldi et al.21 reported that decreased levels of plasma sRAGE correlated with SBP in © 2015 Macmillan Publishers Limited
Serum levels of endogenous secretory RAGE and blood pressure W Cai et al
patients with essential hypertension. In addition, Hudson et al.30 reported that serum sRAGE levels inversely correlated with several components of metabolic syndrome, including hypertension. Circulating esRAGE levels were not measured in these studies. From these results, it is possible that serum sRAGE and esRAGE levels are distinct biomarkers, and may have different roles in various disease states. sRAGE and esRAGE are two isoforms of C-truncated RAGE.17 sRAGE is formed by the cleavage of native membrane receptor mediated by disintegrin and matrix metalloproteinase. esRAGE is formed from alternative splicing of native membrane receptor, and it is secreted from cells.31 The regulatory mechanisms for alternative splicing to generate esRAGE and for proteolytic shedding of cell surface RAGE to generate sRAGE remain unclear.32 A weak correlation between serum sRAGE and serum AGE, but no correlation between serum esRAGE and serum AGE, was observed in the current study. Similarly, Yamagishi et al.33 reported that serum sRAGE was positively associated with serum AGE in nondiabetic participants. These results suggest that circulating sRAGE and esRAGE levels may be under the control of different mechanisms. It is unclear whether the pathophysiological significance of circulating esRAGE and sRAGE levels is distinct in different clinical settings, and further examination is required. This study had some limitations. First, this was a cross-sectional study using correlation analysis. Therefore, the results can only suggest possible associations and not causal relationships between serum esRAGE and blood pressure in OSA patients. Although evidence suggestive of mechanistic links between RAGE and cardiovascular diseases has been reported in in vitro and in vivo experiments,14–16 further studies using a large prospective cohort are needed to elucidate the relationship between serum esRAGE and blood pressure in patients with OSA. Katakami et al. reported that esRAGE, but not sRAGE, was associated with carotid atherosclerosis in type 1 diabetes patients in a cross-sectional study.34 However, in a 4-year follow-up study, the researchers found that circulating esRAGE and sRAGE levels were associated with the progression of carotid atherosclerosis in type 1 diabetes patients.26 Second, analyses in the current study were based on office blood pressure measurements, which are subject to methodological limitations. Although we took care to optimize the procedure (sufficient resting period, appropriate cuff size and use of mean from two measurements), we acknowledge that the use of 24-h ambulatory blood pressure measurements would strengthen our analyses. Third, all participants were male, because most patients with OSA undergoing polysomnography at our hospital were male. Although studies of patients without OSA have reported that serum esRAGE levels were associated with hypertension in female, as well as in male, patients,22,29 further research that includes both male and female patients is needed to confirm the results. In addition, there were no mild cases of OSA or participants without OSA in the current study, because most patients with OSA undergoing polysomnography at our hospital had moderate-to-severe OSA. Although previous studies had suggested that circulating esRAGE levels inversely correlated with blood pressure in other diseases such as metabolic syndrome,22,29 and the strength of the correlation between serum esRAGE levels and blood pressure in patients with different severities of OSA was similar when participants in this study were categorized by AHI tertiles for analysis, this study would have benefited from a control group, which would have allowed us to establish the relationship between serum esRAGE levels and blood pressure in individuals without OSA. In conclusion, we found that serum esRAGE levels were inversely associated with blood pressure, especially SBP, in male nondiabetic patients with OSA. These findings suggest that esRAGE may have a protective role against hypertension in © 2015 Macmillan Publishers Limited
5 patients with OSA, and may be a novel biomarker for OSA patients at high risk of developing cardiovascular diseases. What is known about this topic? ● Interaction of advanced glycation end products (AGE) and their specific cell-surface receptor (RAGE) has an important role in the pathogenesis of cardiovascular disease, and two isoforms of C-truncated RAGE, soluble RAGE (sRAGE) and endogenous secretory RAGE (esRAGE), may prevent activation of RAGE signaling by acting as decoys. ● It has been reported that circulating sRAGE levels correlated with blood pressure in patients with essential hypertension and metabolic syndrome. ● Less is known about the relationship between serum esRAGE, sRAGE and blood pressure in patients with obstructive sleep apnea (OSA). What this study adds? ● Serum esRAGE levels, but not serum sRAGE levels, were inversely associated with blood pressure, especially SBP, in male nondiabetic patients with OSA. ● esRAGE may have a protective role against hypertension in patients with OSA, and may be a novel biomarker for OSA patients at high risk of developing cardiovascular diseases.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS The authors thank all patients who participated in this study. The study was supported by grants from the National Natural Science Funds of China (No. 81160105, 81360017, 81460018) and the Jiangxi Provincial Natural Science Funds of China (No. 2010GZY0325, 20114BAB205006).
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