Clinica Chimica Acta 448 (2015) 1–7

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Increased circulating endothelial progenitor cells and anti-oxidant capacity in obstructive sleep apnea after surgical treatment Cheng-Hsien Lu a,b,c,⁎, Hsin-Ching Lin d, Chih-Cheng Huang a, Wei-Che Lin e, Hsiu-Ling Chen e, Hsueh-Wen Chang b, Michael Friedman f,g, Chao Tung Chen h, Nai-Wen Tsai a, Hung-Chen Wang i, Chia-Te Kung j, Yu-Jih Su b,k, Ben-Chung Cheng b,k a

Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan Department of Biological Science, National Sun Yat-Sen University, Kaohsiung, Taiwan c Department of Neurology, Xiamen Chang Gung Memorial Hospital, Xiamen, Fujian, China d Department of Otolaryngology and Sleep Center, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan e Department of Radiology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan f Department of Otolaryngology — Head and Neck Surgery, Rush University Medical Center, Chicago, IL, United States g Department of Otolaryngology, Advanced Center for Specialty Care, Advocate Illinois Masonic Medical Center, Chicago, IL, United States h Department of Family Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan i Department of Neurosurgery, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan j Department of Emergency Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan k Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan b

a r t i c l e

i n f o

Article history: Received 27 January 2015 Received in revised form 23 May 2015 Accepted 26 May 2015 Available online 18 June 2015 Keywords: Endothelial progenitor cells Oxidative stress Obstructive sleep apnea Polysomnography

a b s t r a c t Background: Obstructive sleep apnea (OSA) has increased risk of cardiovascular diseases. Profiles of endothelial progenitor cells (EPCs) reflect the degree of endothelial impairment. This study tested the hypothesis that surgical treatment not only improves clinical outcomes but also increases the number of circulating EPCs and antioxidant capacity. Methods: The number of circulating EPCs (CD133+/CD34+ [%], KDR+/CD34+ [%]), biomarkers for oxidative stress (thiols and TBARS), and polysomnography (PSG) study was prospectively evaluated in 62 OSA patients at two time points (pre-operative and at least 3-month post-operative). The biomarkers and PSG were compared with those of 31 age- and body mass index (BMI)-matched healthy controls. Results: Levels of HbA1c and LDL-C were significantly higher while CD133+/CD34+ (%) and HDL were significantly lower in OSA patients than in healthy controls. The levels of CD133+/CD34+ (%) and thiols significantly increased in both mild/moderate and severe OSA. The TBAR levels also significantly decreased in severe OSA patients after N3 months of follow-up. The number of CD133+/CD34+ (%) negatively correlated with both age and mO2 of b90% but positively correlated with thiols. Clinical efficiency after OSA surgery assessed by PSG showed improvement and mean systolic blood pressure (SBP) (night and morning) reduction and improved lipid profile in the severe OSA group while only the snoring index improved in the mild/moderate OSA group. Conclusions: OSA surgery not only improves clinical outcomes, SBP reduction and improved lipid profile but also increases the number of circulating EPCs and antioxidant capacity, especially in patients with severe OSA. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Obstructive sleep apnea (OSA) is associated with loud snoring, recurrent upper airway obstruction, recurrent apneas/hypopnea, oxygen desaturation, and arousals during sleep [1]. Nocturnal hypoxemia has been proposed to increase the generation of free radicals and other reactive species, leading to increased oxidative stress, produce cell adhesion molecules, and add to endothelial dysfunction in OSA [2]. ⁎ Corresponding author at: Department of Neurology, Chang Gung Memorial Hospital, 123, Ta Pei Road, Niao Sung District, Kaohsiung, Taiwan. Tel.: +886 7 7317123x2283. E-mail addresses: [email protected], [email protected] (C.-H. Lu).

http://dx.doi.org/10.1016/j.cca.2015.05.023 0009-8981/© 2015 Elsevier B.V. All rights reserved.

Endothelial progenitor cells (EPCs) contribute to vascular homeostasis and may serve as a circulating pool of cells to replace dysfunctional endothelia [3,4]. EPCs are exposed to oxidative stress during vascular injury as residents of blood vessel walls or as circulating cells homing on to sites of neovascularization [5]. Decreased numbers of circulating EPCs are reported to be predictive of future cardiovascular events in patients with cardiovascular conditions like coronary artery disease and stroke [6,7]. Epidemiologic studies indicate that patients with OSA have an increased risk of cardiovascular disease [8,9]. Furthermore, recent findings suggest that vascular function is impaired in OSA patients and that endothelial dysfunction may precede and predispose these patients to cardiovascular events [10,11].

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Continuous positive airway pressure (CPAP) is the primary treatment of OSA, but CPAP compliance varies from 28% to 80% [12, 13]. Surgery for OSA is not a substitute for CPAP but is a salvage procedure for those who fail CPAP and other conservative therapies and have no other options. The principles behind surgical treatment for OSA are based on the reduction of the volume of redundant tissues, stiffening of the flaccid soft palate, and suspension of the collapsed tongue base to maintain airway patency for improving symptoms and reducing the sequelae of OSA. The efficacy and safety of multi-level surgery are demonstrated in literature [14–16]. To date, there is paucity of information on the effects of surgical treatment for OSA patients in terms of clinical efficiency and biomarkers of endothelial dysfunction. This study tested the hypothesis that OSA surgery not only improves the clinical outcomes but also increases the number of circulating EPCs and anti-oxidant capacity. The successful clinical translation of these approaches has the potential of reducing cardiovascular risks and improving the quality of life for patients with OSA. 2. Patients and methods

electromyography, and electro-oculography were recorded with surface electrodes by standard techniques. Nasal and oral airflow were recorded by thermistors. Oxygen saturation was measured by pulse oximetry. Sleep stage scoring was done at 30 s intervals by experienced technicians according to the standard criteria [20]. 2.5. Surgical procedures All of the procedures were performed by one of the co-authors (H-C Lin) under general anesthesia and with oro-tracheal intubation. The techniques used were determined upon the discretion of the treating sleep surgeon based on the severity of OSA with PSG and conditions of upper airway abnormality, as examined by a flexible fiberscope. The modified uvulo-palato-pharyngoplasty (UPPP), or Zpalatopharyngoplasty (ZPPP), was used for the retro-palatal obstruction. ZPPP was designed to create the necessary supero-anterolateral tension and further widen the pharynx. It has been demonstrated to increase the surgical success rate and ameliorate some possible complications of traditional UPPP. Trans-oral endoscopic tongue base tissue volume reduction with radiofrequency/coblator was performed for the retro-lingual obstruction [14–16].

2.1. Study design 2.6. Biochemical analysis This is a single-center, prospective case–control study conducted at Chang Gung Memorial Hospital-Kaohsiung. 2.2. Diagnostic criteria of obstructive sleep apnea The severity of sleep-disordered breathing was classified according to the number of apnea and hypopnea during sleep. By definition, obstructive apnea was a cessation of airflow for at least 10 s with effort to breath during apnea. Obstructive hypopnea was defined as an abnormal respiratory event with at least a 30% reduction in thoracoabdominal movement or airflow compared with baseline, lasting at least 10 s, and with ≥ 4% oxygen desaturation. The apnea/hypopnea index (AHI) was defined as the total number of apnea and hypopnea per hour of electroencephalographic sleep. Central respiratory events were excluded for severity classification. Moreover, OSA was defined as a severe type of sleep-disordered breathing with AHI of more than 5 per hour. An AHI of 5–15 was classified as mild OSA, 15–30 as moderate OSA, and N30 as severe OSA. 2.3. Inclusion and exclusion criteria Sixty-two patients with OSA who had undergone surgical treatment were enrolled. For comparison, 31 age-, sex-, and body mass index (BMI)-matched subjects without any known cardiovascular risk factor or diseases and who did not take any medications were included as healthy controls. The hospital's Institutional Review Committee on Human Research approved the study protocol and all enrolled patients provided full informed written consent. Patients with any of the following were excluded from this study: 1) atherosclerotic narrowing on intracranial and extracranial vessels (N 50% stenosis) with or without evidence of old cerebral infarctions, coronary artery diseases status post percutaneous trans-luminal coronary angioplasty or bypass surgery, and renal failure requiring hemodialysis or peritoneal dialysis; 2) moderate-to-severe heart failure (NYHA class III and IV); 3) metabolic syndrome based on the World Health Organization Clinical Criteria [17]; or 4) intake of medications that might influence the number of circulating EPCs [18,19]. 2.4. Sleep study — polysomnography All-night attended comprehensive diagnostic sleep studies were performed at the hospital's Sleep Center, in a temperature-controlled and sound-attenuated room. Electroencephalography, submental

Blood samples were obtained by antecubital vein puncture in a fasting, non-sedative state between 09:00 and 10:00 AM in the control and study groups to exclude the possible influence of circadian variations, and were analyzed by the hospital's central laboratory. Serum levels of triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), blood sugar, HBA1c, and high sensitive C-reactive protein (hs-CRP) were determined. 2.7. Biomarkers for oxidative stress Blood samples were centrifuged at 3000 rpm for 10 min. Each serum sample was collected and frozen at − 80 °C prior to biochemical measurement. Measurement of thiobarbituric acid-reactive substances (TBARS) was a well-established method for detecting lipid peroxidation [21]. A TBAR Assay Kit was used according to the manufacturer's instructions (cat. 10009055; Cayman Chemical, USA). Briefly, serum (100 ml) was added in duplicate to sodium dodecyl sulfate (SDS) (100 ml) and color reagent (4 ml). Reaction mixtures were then incubated for 1 h in boiling water and centrifuged at 1600 × g for 10 min at 4 °C. After warming for 5 min at 25 °C, the samples were read on a micro-plate spectrophotometer (Beckman Coulter). Values for the samples were calculated from a linear calibration curve prepared using pure MDA-containing samples (range, 0–50 μmol/l). The ability of anti-oxidative defense in response to increased oxidative damage was evaluated by measuring the serum levels of total reduced thiols, since serum thiols were physiologic free radical scavengers. Serum total protein thiols were estimated by directly reacting thiols with 5,5-dithiobis 2-nitrobenzoic acid (DTNB) (#D8130, Sigma-Aldrich) to form 5-thio-2- nitrobenzoic acid (TNB). The amount of thiols in the sample was calculated from the absorbance determined by the extinction coefficient of TNB (A412 = 13,600 l/mol·cm). 2.8. Assessment of circulating EPC level To assess the circulating EPC numbers, blood samples were collected at baseline (before surgery) and at 6-month follow-up after surgery. Peripheral blood EPC level was measured by flow cytometry. To determine the EPC surface markers of CD45/CD34/CD133 and CD45/CD34/KDR, we incubated 106 PBMNCs with fluorescein isothiocyanate-conjugated anti-CD45 antibodies (#IM1870, Beckman

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Coulter), phycoerythrin-Cy5-conjugated anti-CD45 antibodies (#A07785, Beckman Coulter), phycoerythrin-conjugated anti-CD133 (#130-090-853, Miltenyi Biotec) and phycoerythrin-conjugated antiKDR (#130-093-598, Miltenyi Biotec) antibodies for 30 min at 4 °C according to the manufacturers' instructions or with appropriate isotype controls. After staining, the mononuclear cells were fixed in 1% para-formaldehyde. An Epics XL flow cytometer (Beckman Coulter Inc.) was used for quantitative three-color flow cytometry analysis (1,000,000 cells per sample). Intra-individual variability and mean intra-assay CVs of circulating EPC numbers were all b 4.0%. 2.9. Clinical assessment All of the patients underwent complete medical and neurological examinations and PSG study. The clinical records of OSA patients were reviewed, BMI was calculated, and the age of onset and follow-up period after surgery were registered. Daytime sleepiness was assessed using the Epworth Sleepiness Scale (ESS). Snoring was estimated with a standard visual analogue scale (1–10, with 1 as “no snoring” and 10 as “very intensive snoring”) by the bed-partner. 2.10. Statistical analysis Three separate statistical analyses were performed. Categorical variables were compared using the chi-square or Fisher's exact tests. Continuous variables within the two groups were compared using independent t-test for parametric data and the Mann–Whitney U test for non-parametric data. First, demographic data between OSA patients and healthy controls were compared. Second, correlation analysis was used to explore the relationship between EPCs and peripheral blood testing, and parameters of the PSG study. Third, changes between preoperative and 6-months post-operative values of biomarkers and PSG parameters were compared by way of paired t-test. Further, repeated measures of ANOVA were used to compare biomarkers and leukocyte apoptosis at two different time points (preoperative and at 6 months post-operative). Analysis of covariance (ANCOVA) was used to compare subgroups (mild–moderate OSA and severe OSA) after controlling for potential confounding variables. Levene's test of equality of error variance was used to ensure equal variance existing in both groups. For comparison of the biomarkers between the subgroups, ANCOVA was used, with BMI and age as potential confounding variables. All statistical analyse were conducted using the SAS software package, ver. 9.1 (SAS Statistical Institute). 3. Results 3.1. Baseline characteristics of the study patients The baseline characteristics and laboratory data of the 62 adult OSA cases and 31 healthy controls revealed that the two groups were similar in terms of age (p = 0.09), sex (p = 1.0), and BMI (p = 0.08) (Table 1). The levels of mean systolic blood pressure (SBP) and mean diastolic blood pressure (DSP) (both at night and in the morning) were significantly higher in OSA patients than in control subjects (p = 0.001, p = 0.001, p b 0.001, and p b 0.001, respectively). The levels of LDL-C (p = 0.004) and HbA1c (p = 0.003) was significantly higher in OSA patients (p b 0.05). The HDL-C (p = 0.004) and CD133+/CD34+ (%) (p = 0.004) levels were significantly lower in OSA patients than in the controls (p = 0.015), but the levels of KDR+/CD34+ (%), TBARS, and thiols were similar between the 2 groups. The characteristics of PSG studies between the two groups were similar as regards the mean ESS (p = 0.68) and sleep efficiency (%) (p = 0.51). The apnea/hypopnea index-total sleep time (AHI-TST), mO2 of b 90% (%/night), average O2, de-saturation index (events/h/ night), arousal-TST (events/h/night), and snoring index (events/h/

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Table 1 Baseline characteristics and laboratory data of the patients and controls. OSA patients (n = 62)

Controls (n = 31)

p value

42.4 ± 10.0 8; 54 25.5 ± 2.7

40.2 ± 7.7 4; 27 24.8 ± 2.7

NS NS NS

137.3 ± 16.2

127.0 ±

0.001

84.0 ± 12.9 135.5 ± 14.9

15.3 75.7 ± 9.1 125.6 ±

b0.001 0.001

85.0 ± 15.8

15.3 75.8 ± 10.0

b0.001

192.0 ± 31.5

188.5 ±

NS

HDL-C, mg/dl LDL-C, mg/dl

52.9 ± 12.6 122.0 ± 31.8

38.4 60.6 ± 16.0 108.0 ±

0.004 0.004

Triglyceride, mg/dl

139.9 ± 48.8

37.8 105.7 ±

NS

5.7 ± 0.3 92.9 ± 9.1 2.2 ± 1.7

39.2 5.5 ± 0.3 91.2 ± 7.7 2.0 ± 1.5

0.003 NS NS

25.9 ± 14.5 1.8 ± 1.0

36.3 ± 15.1 2.0 ± 0.7

0.004 NS

14.8 ± 6.3 1.2 ± 0.3

14.1 ± 4.1 1.3 ± 0.2

NS NS

9.5 ± 4.8 84.6 ± 13.1 41.0 ± 25.6 10.7 ± 4.7 94.8 ± 2.7 31.5 ± 24.9 39.0 ± 25.5 380.3 ±

9.0 ± 5.2 86.2 ± 8.1 2.4 ± 1.5 0.3 ± 0.1 96.8 ± 1.0 0.7 ± 0.5 12.5 ± 7.1 170.6 ±

NS NS b0.0001 b0.0001 b0.0001 b0.0001 b0.0001 b0.0001

179.1

97.8

Age, years Sex (female; male) Body mass index, kg/m2 Mean blood pressure Mean systolic blood pressure (night) Mean diastolic blood pressure (night) Mean systolic blood pressure (morning) Mean diastolic blood pressure (morning) Peripheral blood studies Total cholesterol, mg/dl

HbA1c Glucose, mg/dl hs-CRP, mg/l Numbers of EPC CD133+/CD34+ (%) KDR+/CD34 + (%) Biomarkers of oxidative stress TBARS, μmol/l Thiols, μmol/l Polysomnography studies Epworth Sleepiness Scale (ESS) Sleep efficiency (%) AHI-TST mO2 of b90% (%/night) Average O2 De-saturation index (events/h/night) Arousal-TST (time/h/night) Snoring index (events/h/night)

Values are expressed in mean ± SD unless otherwise indicated. Abbreviations: HBA1c, glycosylated hemoglobin; EPC, endothelial progenitor cells; OSA, obstructive sleep apnea/hypopnea syndrome; AHI, apnea/hypopnea index; REM, rapid eye movement; mO2, mean oxygen saturation; ESS, Epworth sleepiness scale; and TST, total sleep time.

night) were statistically significantly different between patients with severe OSA and the controls (all p b 0.0001). 3.2. Effect of EPCs on peripheral blood testing, biomarkers of oxidative stress, and PSG parameters Correlation analysis was used to test the influence of EPC level on peripheral blood testing, biomarkers of oxidative stress, and PSG parameters (Table 2). The statistically significant results (correlation coefficient, p value) of CD133+/CD34+ EPCs (%) were age (r = − 0.19, p = 0.038), thiols (r = 0.34, p b 0.0001) and mO2 of b90% (%/night) (r = − 0.17, p = 0.043). The mean levels of thiols negatively correlated with age (average) (r = − 0.264, p = 0.14), while the mean levels of CD133+/CD34+ EPCs (%) positively correlated with the mean levels of thiols (r = 0.267, p = 0.013) after controlling for age (Fig. 1). 3.3. Comparison of the baseline characteristics and biomarkers in OSA subgroups The baseline biomarkers in mild/moderate and severe OSA (Table 3) revealed that the 2 groups were similar in terms of sex, mean SBP (nighttime and daytime) and mean DSP (nighttime and

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Table 2 Correlation among EPCs, peripheral blood testing, biomarkers of oxidative stress, and parameters of polysomnography study. CD133+/CD34+ EPCs (%)

KDR+/CD34+ EPC (%)

Spearman's correlation

r

p

r

p

Age, years Body mass index, kg/m2 Epworth sleepiness scale (ESS) Sleep efficiency (%) AHI-TST mO2 of b90% (%/night) Average O2 De-saturation index (time/h/night) Arousal-TST (time/h/night) Snoring index (events/h/night) TBARS, μmol/l Thiols, μmol/l Total cholesterol hs-CRP, mg/l HDL-C LDL-C Triglyceride, mg/dl HbA1c

−0.19 −0.067 0.005 −0.133 −0.088 −0.17 0.151 −1.15 −0.019 0.04 −0.087 0.34 −0.049 0.086 −0.068 −0.024 −0.058 −0.002

0.038⁎ NS NS NS NS 0.043⁎

0.12 −0.073 0.022 0.03 −0.12 −0.123 0.114 −0.102 −0.09 0.033 0.102 −0.006 −0.067 −0.07 −0.037 −0.046 −0.071 −0.018

NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS

NS NS NS NS NS b0.0001⁎ NS NS NS NS NS NS

Values are expressed in mean ± SD unless otherwise indicated. Abbreviations: IQR, inter-quartile range; HbA1c, hemoglobin A1c; EPC, endothelial progenitor cells; VEGF, vascular endothelial growth factor; SDF-1, stromal cell-derived factor-1; HBA1c, glycosylated hemoglobin; OSA, obstructive sleep apnea/hypopnea syndrome; AHI, apnea/hypopnea index; TST, total sleep time mO2, mean oxygen saturation; and ESS, Epworth sleepiness scale. ⁎ Significant correlation at p b 0.05.

daytime), and levels of cholesterol, triglyceride, hs-CRP, LDL-C, glucose, CD133+ /CD34 + (%), KDR+/CD34 + (%), TBARS, and thiols. Moreover, BMI (p = 0.004) was higher but HDL level (p = 0.028) was lower in patients with severe OSA. 3.4. Comparison of the baseline PSG study and clinical score in OSA subgroups The characteristics of PSG studies between the 2 groups were similar in terms of mean ESS, sleep efficiency (%), and snoring index (events/h/ night). The mean AHI-TST (p b 0.0001), mO2 of b 90% (%/night) (p b 0.001), average O2 (p b 0.001), desaturation index (events/h/night) (p b 0.0001), and arousal-TST (time/h/night) (p b 0.0001) were significantly different between patients with severe OSA and those with mild/moderate OSA. 3.5. Changes of biomarkers pre- and post-operatively in OSA subgroups Changes of biomarkers of biochemical data, oxidative stress, and level of EPC at baseline and at least 3 months after surgery (Table 3) showed significantly increased changes at least 3 months of follow-up in levels of CD133+/CD34+ (%) and thiols in the mild/moderate OSA group. The mean levels of total cholesterol, HDL-C, LDL-C, triglyceride, HbA1c, glucose, and hs-CRP did not show significant changes at least 3 months of follow-up. In the severe OSA group, the level of HDL and number of CD133+/CD34+ (%) and thiols showed significantly increased changes while the levels of LDL and TBARS show significantly

Fig. 1. The figure shows the levels of CD133+/CD34+ (%) and KDR+/CD34+ (%) in one patient with obstructive sleep apnea before and after surgical treatment and one normal control. Blood samples from one normal control is shown in a and d; one patient with pre-operative state is shown in b and e; and post-operative state is shown in c and f. Multi-color flow cytometry analyses of human mononuclear cells in whole blood samples. Staining for CD45, CD34 and CD133 of mononuclear cells, and events are gating from CD45dim events on the CD45-side scatter plot (a–c). Staining for CD45, CD34 and KDR of mononuclear cells, and events are also gating from CD45dim events on the CD45-side scatter plot (d–f).

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Table 3 Serial changes of polysomnography and biomarker study in patients with obstructive sleep apnea in terms of severity. Mild-moderate OSA (n = 22)

Mean systolic blood pressure (night) Mean diastolic blood pressure (night) Mean systolic blood pressure (morning) Mean diastolic blood pressure (morning) Biomarkers of EPC CD133+/CD34+ (%) KDR+/CD34+ (%) Biomarkers of oxidative stress TBARS, μmol/l Thiols, μmol/l Peripheral blood studies Total cholesterol HDL-C LDL-C Triglyceride, mg/dl HbA1c hs-CRP, mg/l Polysomnography studies Epworth sleepiness scale (ESS) Sleep efficiency (%) AHI-TST mO2 of b90% (%/night) Average O2 De-saturation index (events/h/night) Arousal-TST (time/h/night) Snoring index (events/h/night)

Severe OSA (n = 40)

Pre-op

Post-op

Pre-op

Post-op

135.8 ± 20.5 84.5 ± 15.2 133.3 ± 16.3 85.5 ± 12.6

134.8 ± 15.4 83.2 ± 14.7 134.7 ± 15.3 87.0 ± 13.9

138.0 ± 14.9 83.5 ± 10.9 138.1 ± 12.1 86.7 ± 9.3

132.3 ± 14.7⁎ 80.4 ± 11.4 133.2 ± 11.1⁎ 85.8 ± 10.8

27.4 ± 14.3 2.1 ± 1.7

36.2 ± 15.0⁎⁎ 2.8 ± 2.0

28.7 ± 15.8 1.7 ± 1.5

34.9 ± 15.9⁎⁎⁎ 2.1 ± 1.9

17.5 ± 5.3 1.0 ± 0.4

13.4 ± 10.7 1.3 ± 0.3⁎⁎

14.5 ± 5.5 1.1 ± 0.2

10.7 ± 2.5⁎⁎ 1.3 ± 0.3⁎⁎

190.0 ± 26.7 54.6 ± 14.1 118.6 ± 27.9 153.2 ± 133.0 5.6 ± 0.3 1.5 ± 0.9

191.3 ± 29.3 56.9 ± 13.4 116.6 ± 30.6 142.7 ± 132.7 5.6 ± 0.3 1.5 ± 0.9

189.6 ± 27.4 52.3 ± 10.2 123.8 ± 27.0 138.6 ± 48.7 5.8 ± 0.3 2.1 ± 1.1

188.5 ± 29.2 55.9 ± 12.1⁎ 115.5 ± 34.2⁎

9.5 ± 4.6 81.6 ± 14.1 17.4 ± 7.1 2.3 ± 2.1 96.3 ± 1.2 9.5 ± 5.6 24.8 ± 14.8 350.5 ± 214.3

7.5 ± 4.1 85.0 ± 15.9 12.3 ± 10.1 3.1 ± 2.0 96.0 ± 1.5 7.3 ± 6.0 22.5 ± 12.5 191.6 ± 151.1⁎⁎⁎

9.3 ± 5.2 85.9 ± 12.2 59.8 ± 17.2 16.8 ± 14.5 93.6 ± 3.3 46.8 ± 20.7 49.5 ± 26.1 446.7 ± 190.4

8.2 ± 3.8 88.5 ± 9.1 32.9 ± 23.1⁎⁎⁎⁎⁎ 7.2 ± 4.6⁎⁎⁎⁎⁎ 95.3 ± 1.89⁎⁎⁎⁎⁎ 20.2 ± 19.4⁎⁎⁎⁎ 38.1 ± 26.5⁎ 340.8 ± 227.3⁎⁎⁎

120.1 ± 46.6 5.8 ± 0.4 1.8 ± 1.6

Values are expressed in mean ± SD unless otherwise indicated. Abbreviations: Pre-op: pre-operatively; Post-op: post-operatively; IQR, inter-quartile range; HBA1c, glycosylated hemoglobin; VEGF, vascular endothelial growth factor; SDF-1, stromal cell-derived factor-1; OSA, obstructive sleep apnea/hypopnea syndrome; AHI, apnea/hypopnea index; REM, rapid eye movement; mO2, mean oxygen saturation; ESS, Epworth sleepiness scale; and TST, total sleep time. The changes (pre-operative evaluation and at 6 months post-operatively) of biomarkers and parameters of polysomnography study in different OSA subgroup patients (mild–moderate and severe) were compared using paired t-test. ⁎ p b 0.05. ⁎⁎ p b 0.01. ⁎⁎⁎ p b 0.005. ⁎⁎⁎⁎ p b 0.001. ⁎⁎⁎⁎⁎ p b 0.0001.

decreased changes at least 3 months of follow-up after surgery. The mean levels of total cholesterol, triglyceride, HbA1c, and hs-CRP showed non-significant changes at least 3 months of follow-up. To exclude the possible effects of BMI and age on biomarkers of EPC and oxidative stress, the hypothesis that the levels of EPC and oxidative stress were equal between BMI and age was tested using ANCOVA. Univariate analysis of covariance between the two treated groups at the two different time points (baseline and at least 3 months of follow-up after surgery) showed that TBARS (p = 0.038) were statistically different while CD133+/CD34+ (%), thiols, HDL-C, and LDL-C were not.

3.6. Changes in PSG study and clinical score at baseline and follow-up after surgery among OSA subgroups Changes in PSG study and clinical score at baseline and at least 3 months after surgery (Table 3) showed significant changes in snoring index (events/h/night) (p = 0.001) in the mild/moderate OSA group, but AHI-TST, ESS, sleep efficiency (%), mO2 of b90% (%/night), average O2, arousal-TST (time/h/night), and mean SBP (nighttime and daytime) and mean DBP (nighttime and daytime) did not. In severe OSA group, the mean AHI-TST (p = 0.001), mO2 of b90% (%/night) (p b 0.0001), average O2 (p b 0.0001), desaturation index (p b 0.0001) (events/h/night), arousal-TST (time/h/night) (p = 0.034), snoring index (events/h/night) (p = 0.004), and mean SBP (nighttime and daytime) (p = 0.025 and 0.013, respectively) showed significant changes, whereas the mean ESS, sleep efficiency (%), and mean DBP (nighttime and daytime) did not.

To exclude the possible effects of BMI and age on parameters of PSG and mean SBP (nighttime and daytime), the hypothesis that the levels of parameters of PSG and mean DBP (nighttime and daytime) were equal between BMI and age was tested using ANCOVA. Univariate analysis of covariance between the two treated groups at two different time points (baseline and follow-up after surgery) showed that desaturation index (p = 0.008) and mO2 of b 90% (p = 0.007) were statistically different while average O2 and arousal-TST, mean AHI-TST, snoring index, and mean SBP (nighttime and daytime) were not. 4. Discussion This study confirmed the hypothesis that OSA surgery not only improves clinical outcomes but also increases the number of circulating EPCs and anti-oxidant capacity (increased thiol level and decreased TBAR level). The present study examines changes of biomarkers on EPC and oxidative stress and clinical efficiency in OSA patients after upper airway surgery for OSA and on follow-up for at least 3 months. There are 5 major findings. First, the biomarkers of metabolic risk factors (HbA1c and LDL-C) and both mean SBP and DBP (nighttime and daytime) are significantly increased in OSA patients compared with healthy controls. The levels of CD133+/CD34+ (%) and HDL are significantly lower in OSA patients than in healthy control. Second, the levels of CD133+/CD34+ (%) and thiols are significantly increased in OSA patients regardless of severity, but the levels of TBARS are significantly decreased in patients with severe OSA at least 3 months of follow-up. Third, the number of CD133+/CD34+ (%) is negatively correlated with both age and mO2 of b 90%, but it is positively correlated with thiols.

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The mean number of CD133+/CD34+ EPCs (%) controlling for age remains positively correlated with the mean levels of thiols. Fourth, the mean desaturation index, mO2 of b90%, and TBAR levels between the mild/moderate and severe OSA groups at two different time points (pre-operatively and post-operatively) are statistically different after controlling for BMI and age. Five, the clinical efficiency after OSA surgery as assessed by PSG reveals that AHI-TST, mO2 of b 90% (%/night), average O2, desaturation index (events/h/night), arousal-TST (time/h/night), and snoring index (events/h/night) are statistically improved in the severe OSA group, while only snoring index (events/h/night) is improved in the mild/moderate OSA group. The importance of these findings is the implication that surgical treatment is more efficient in both alleviating symptoms of snoring and in reducing signs of sleep apnea in the severe OSA group. Circulating endothelial progenitor cells (EPCs) were identified in adult human peripheral blood. Since their original identification, EPCs have been extensively studied as biomarkers to assess the risk of cardiovascular diseases [6–9] and as a potential cell therapeutic for vascular regeneration. At present, there is no specific marker for EPCs and the term EPC is routinely used to encompass a group of cells ranging from circulating endothelial cells (ECs) to hemangioblasts. These cells migrate from the bone marrow into the peripheral circulation and are able to differentiate into mature endothelial cells in vitro and in vivo. Flow cytometry quantification of EPCs is based on the analysis of cell surface expression within the mononuclear cell gate. These EPCs should be identified by the surface expression of at least one immaturity/stem cell antigen (e.g. CD34 or CD133) plus at least one endothelial antigen (usually KDR or CD31), but negative for leukocyte antigens (e.g., CD45). Thus, CD34+KDR+, CD133+KDR+, CD34+CD133+KDR+, CD34+CD31+, CD133+CD31+, and CD34+CD133+CD31+ are all theoretically possible EPC phenotypes. Several studies using variations of basic EPC culture methods and flow cytometry techniques demonstrate that changes in circulatory EPC concentration can now be used to correlate a wide variety of human diseases, including cardiovascular disorders, diabetic vasculopathy, and stroke [3,4,22,23]. Several clinical studies have investigated the association between OSA and circulatory EPC. The findings have been inconclusive, with both negative [10,24] and positive results [2,25,26]. Nonetheless, most studies have focused on the number of circulatory EPCs in different severities of OSA [24], between OSA patients and healthy controls [10, 26], and in serial changes of circulatory EPCs following short-term (4– 6 weeks) CPAP treatment [2,24,25]. The present study demonstrates that surgery for OSA increases the number of circulating EPCs even after six months. Although supporting data are sparse, OSA-related repetitive episodes of hypoxia/re-oxygenation have generally been associated with increased vascular production of reactive oxygen species (ROS), leading to increase oxidative stress, while reduced anti-oxidant defense may exacerbate the detrimental effects of increased oxidative stress on the vascular endothelium of patients with OSA [27]. Numerous disease states enhance oxidant stress in vivo prior to clinically significant vascular disease [28–30]. More importantly, the intracellular redox environment has a critical role in controlling apoptosis, proliferation, self-renewal, senescence, and differentiation [31]. Dysregulation of any one of the phenotypes in EPCs will alter endothelial cell function, predisposing to the development of vascular pathology. Clinical studies show that levels of oxidative stress normalize with 4 months of effective CPAP therapy, whereas anti-oxidant defense remains partially impaired after one year of therapy in OSA patients [27, 32]. The present study demonstrates increased anti-oxidant capacity (increased thiol levels and decreased TBAR levels), especially in the severe OSA (AHI N 30) group at least 3 months after surgery. The effects of CPAP treatment on blood pressure (BP) control have been extensively investigated and both short- and long-term CPAP treatments have been found to lower BP in OSA patients [33,34]. The results of meta-analyses in OSA patients who underwent CPAP

treatment revealed that the effects of CPAP were variable and dependent on the severity of disease and the level of BP [35,36]. Our study showed that SBP reduction was more pronounced in normotensive severe OSA patients after OSA surgery. Based on the results here, OSA surgery can improve the clinical outcomes and significant SBP reduction and improved lipid profile, and increased the number of circulating EPCs and anti-oxidant capacity, in normotensive severe OSA patients. Therefore, more prospective multi-center investigations with long-term follow-up are warranted to confirm the predictive value of these biomarkers on the outcome of patients with OSA. Acknowledgments This study was supported by grants from the National Science Council Research Project (NSC 101-2314-B-182A-084-MY3). The authors wish to thank Dr. Gene Alzona Nisperos for editing and reviewing the manuscript for English language considerations. References [1] T. Young, M. Palta, J. Dempsey, J. Skatrud, S. Weber, S. Badr, The occurrence of sleepdisordered breathing among middle aged adults, N Engl J Med 328 (1993) 1230–1235. [2] S. Jelic, M. Padeletti, S.M. Kawut, et al., Inflammation, oxidative stress, and repair capacity of the vascular endothelium in obstructive sleep apnea, Circulation 117 (2008) 2270–2278. [3] A.Y. Khakoo, T. Finkel, Endothelial progenitor cells, Annu Rev Med 56 (2005) 79–101. [4] M. Hristov, W. Erl, P.C. Weber, Endothelial progenitor cells: mobilization, differentiation, and homing, Arterioscler Thromb Vasc Biol 23 (2003) 1185–1189. [5] J. Case, D.A. Ingram, L.S. Haneline, Oxidative stress impairs endothelial progenitor cell function, Antioxid Redox Signal 10 (2008) 1895–1907. [6] C. Schmidt-Lucke, L. Rössig, S. Fichtlscherer, et al., Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events: proof of concept for the clinical importance of endogenous vascular repair, Circulation 111 (2005) 2981–2987. [7] N.W. Tsai, S.H. Hung, C.R. Huang, et al., The association between circulating endothelial progenitor cells and outcome in different subtypes of acute ischemic stroke, Clin Chim Acta 427 (2014) 6–10. [8] F.J. Nieto, T.B. Young, B.K. Lind, et al., Association of sleep-disordered breathing, sleep apnea, and hypertension in a large community-based study. Sleep Heart Health Study, JAMA 283 (2000) 1829–1836. [9] P. Peppard, T. Young, M. Palta, J. Skatrud, Prospective study of the association between sleep-disordered breathing and hypertension, N Engl J Med 342 (2000) 1378–1384. [10] K. Martin, M. Stanchina, N. Kouttab, E.O. Harrington, S. Rounds, Circulating endothelial cells and endothelial progenitor cells in obstructive sleep apnea, Lung 186 (2008) 145–150. [11] M.M. Lui, H.F. Tse, J.C. Mak, et al., Altered profile of circulating endothelial progenitor cells in obstructive sleep apnea, Sleep Breath 17 (2013) 937–942. [12] M. Haniffa, T.J. Lasserson, I. Smith, Interventions to improve compliance with continuous positive airway pressure for obstructive sleep apnoea, Cochrane Database Syst Rev (2004) CD003531. [13] H.S. Lin, G. Zuliani, E.H. Amjad, et al., Treatment compliance in patients lost to follow-up after polysomnography, Otolaryngol Head Neck Surg 136 (2007) 236–240. [14] H.C. Lin, M. Friedman, H.W. Chang, B. Gurpinar, The efficacy of multilevel surgery of the upper airway in adults with obstructive sleep apnea/hypopnea syndrome, Laryngoscope 118 (2008) 902–908. [15] H.C. Lin, M. Friedman, H.W. Chang, M.C. Su, M. Wilson, Z-palato-pharyngoplasty plus radiofrequency tongue base reduction for moderate/severe obstructive sleep apnea/ hypopnea syndrome, Acta Otolaryngol 130 (2010) 1070–1076. [16] H.C. Lin, M. Friedman, H.W. Chang, S. Yalamanchali, Z-palatopharyngoplasty combined with endoscopic coblator open tongue base resection for severe obstructive sleep apnea/hypopnea syndrome, Otolaryngol Head Neck Surg 150 (2014) 1078–1085. [17] S.M. Grundy, H.B. Brewer Jr., J.I. Cleeman, et al., Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition, Circulation 109 (2004) 433–438. [18] J. Llevadot, S. Murasawa, Y. Kureishi, et al., HMG-CoA reductase inhibitor mobilizes bone marrow-derived endothelial progenitor cells, J Clin Invest 108 (2001) 399–405. [19] F.H. Bahlmann, K. de Groot, O. Mueller, B. Hertel, H. Haller, D. Fliser, Stimulation of endothelial progenitor cells: a new putative therapeutic effect of angiotensin II receptor antagonists, Hypertension 45 (2005) 526–529. [20] A. Rechtschaffen, A. Kales (Eds.), A manual of standardized terminology techniques and scoring system for sleep stages of human subjects, UCLA Brain Information Service, Brain Research Institute, 1968.

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