Association between Upper-airway Surgery and Ameliorative Risk Markers of Endothelial Function in Obstructive Sleep Apnea

The objective of our study was to evaluate the effects of upper-airway surgery on improvement of endothelial function-related markers in patients with obstructive sleep apnea (OSA). Subjects with moderate to severe OSA who underwent upper-airway surgery, with a follow-up duration of at least 6 months, were included. Pre- and postoperative polysomnographic variables and endothelial function-related markers were compared. Subgroup and correlation analyses were conducted to find possible indicators for better endothelial function-related markers after upper-airway surgery. In total, 44 patients with OSA were included. The mean follow-up duration was 1.72 ± 0.92 years. Serum VEGFA [−20.29 (CI: −35.27, −5.31), p < 0.05], Ang2 [−0.06 (CI: −0.16, 0.03), p < 0.05], E-selectin [−7.21 (CI: −11.01, −3.41), p < 0.001], VWF [−58.83 (CI: −103.93, −13.73), p < 0.05], VWFCP [−33.52 (CI: −66.34, −0.70), p < 0.05], and TM [−0.06 (CI: −0.09, −0.03), p < 0.05] were significantly lower after upper-airway surgery. However, other risk markers of endothelial function, such as Ang1, ICAM1, VEGFR1, and VCAM, did not change significantly. Correlations between improved endothelial function-related markers and ameliorated oxyhemoglobin saturation and glucolipid metabolism were established. Upper-airway surgery might be associated with an improvement in endothelial function in patients with OSA. These changes may be associated with improved oxygen saturation after upper-airway surgery.


Sleep evaluation.
Every participant underwent all-night standardized PSG monitoring (Alice 4 or 5; Respironics, Pittsburgh, PA, USA) at the sleep center of our clinical institute before and after surgery to objectively evaluate sleep condition. Nocturnal PSG included electroencephalography (EEG) (F3, F4, C3, C4, O3, O4 leads), electrooculography, chin and leg electromyography, nasal and oral airflow measures, thoracic abdominal movement measurement, oxygen saturation measurement, electrocardiography, snoring measurement, and body position. The PSG data were manually assessed by professional technicians in strict accordance with the 2007 criteria of the American Academy of Sleep Medicine (AASM) 19 . Obstructive apnea was defined as the absence of airflow for at least 10 seconds, or ≥90% decrease of airflow for at least 10 seconds. Hypopnea was defined by a greater than 50% reduction of airflow lasting ≥10 seconds accompanied by reduction of oxygen saturation ≥4% or occurrence of an arousal during sleep. The AHI was the number of apnea and hypopnea events per hour of sleep. OSA was diagnosed if the AHI was ≥5 [mild (5 ≤ AHI < 15), moderate (15 ≤ AHI < 30), or severe (AHI ≥ 30)]. Arousal was defined as abrupt switches in frequency of EEG ≥3 seconds. The micro-arousal index (MAI) was defined as the average number of microarousals per hour of total sleep time. The oxygen desaturation index (ODI) was the number of desaturations ≥4% per hour of total sleep time. The percentage of time with oxyhemoglobin saturation below 90% (CT90), mean saturation level (mean SaO 2 ), and lowest saturation level (LSaO 2 ) were also calculated. The Chinese version of the Epworth Sleepiness Scale (ESS) was applied to determine excessive daytime sleepiness or hypersomnia. Total score >10 was considered to indicate clinically significant hypersomnia.
Surgical procedure. The operative technique was based on the preoperative evaluation of obstruction levels and patient preferences. Surgeries performed in those with palatal obstructions included UPPP, Han-UPPP, and Z-palatopharyngoplasty (ZPPP); in patients with multilevel obstructions, these procedures were combined with GAHM. All operations were finished by the same set of experienced doctors. Surgical success was defined as ≥50% reduction in the preoperative AHI and a postoperative AHI <20/h 20 . clinical and biochemical measurement. Physical examinations, questionnaire surveys, and blood tests were performed twice, on the mornings after the preoperative and postoperative PSG monitoring; this included anthropometric indices covering height, weight, and waist circumference (WC). The means of measurements were calculated. Body mass index (BMI) was calculated as weight/height squared (kg/m 2 ). Conforming to the recommendations of the Working Group on Obesity in China, obesity was defined as BMI ≥28 kg/m 2 , and abdominal obesity was defined as waist circumference ≥90 cm in males and ≥85 cm in females. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured to the nearest 2 mmHg by mercury sphygmomanometry, with all patients sitting after 15 minutes rest. High blood pressure was defined when SBP ≥140 mmHg or DBP ≥90 mmHg 21 . Age, sex, and medical history, including nature of the disease, timing of first diagnosis, and treatments, were also recorded. We applied and translated the Epworth sleepiness scale (ESS) questionnaire 22 surveys into Chinese version to collect self-administered information referring to eight situations when OSA patients are known to be soporific based on their usual way of life.
After peripheral blood was drawn from each subject on the morning after PSG monitoring, blood samples were centrifuged at 3000 rpm for 10 min following standing at normal temperature for 30 min. The serum profile including glucose, insulin, triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), apolipoprotein A (Apo A), and apolipoprotein B (Apo B), was examined at the hospital's clinical laboratory. Following the World Health Organization criteria 23 , hyperglycemia was diagnosed with fasting plasma glucose ≥7 mmol/L. The homeostatic model of insulin resistance (HOMA-IR) was calculated as glucose (mmol/L) × insulin (μU/mL)/22.5. Insulin resistance (IR) was defined as a HOMA-IR value >2.50 24 . Referring to the National Cholesterol Education Program Adult Treatment 25 , normal fasting triglyceride level was <1.7 mmol/L. High triglyceride (TG) was ≥2.3 mmol/L. Borderline-high triglyceride was ≥1.7 mmol/L and <2.3 mmol/L. Normal TC was diagnosed when the fasting total cholesterol level was <5.2 mmol/L. High TC was ≥6.2 mmol/L. Borderline high TC was ≥5.2 mmol/L and <6.2 mmol/L. Normal HDL-C was diagnosed when the fasting HDL-C was ≥1.03 mmol/L. High HDL-C was <1.03 mmol/L. Normal LDL-C was diagnosed when the fasting LDL-C level was <3.4 mmol/L. High LDL-C was ≥4.1 mmol/L. Borderline high LDL-C was ≥3.4 mmol/L and <4.1 mmol/L. Normal Apo A was diagnosed when the fasting level was ≥1.2 g/L and ≤16 g/L. Low Apo A was <1.2 g/L. Normal Apo B was ≥0.8 g/L and ≤1.1 g/L. High Apo B was >1.1 g/L.
The resting serum was stored at -80 °C for further endothelial injury biomarker analysis. Concentrations of serum VEGFA, VEGFR1, Ang1, Ang2, ICAM1, VCAM, E-selectin, VWF, VWFCP, and TM were measured using commercially available ELISA kits (Cloud-Clone Corp, Katy, TX, USA) in duplicate according to the manufacturer's instructions. Each serum sample was loaded twice to guarantee the accuracy of detection. Statistical analysis. Study data were presented with descriptive statistics such as mean, median, standard deviation, 95% confidence interval (CI), and percentage. The normality of distribution of continuous variables was analyzed using the Kolmogorov-Smirnov test. The change between preoperative and postoperative variables with time was analyzed by paired sample t-test for normally distributed variables. Variables with skewed distributions were analyzed with the Wilcoxon signed rank test. We used Mann-Whitney or Kruskal-Wallis among-subgroup tests to evaluate the possible predictive utility of marker levels (VEGFA, VEGFR1, Ang1, Ang2, ICAM1, VCAM, E-selectin, VWF, VWFCP, and TM) by sex (male or female), preoperative OSA severity (AHI <30 or >30), preoperative obesity (BMI ≥28 kg/m 2 or <28 kg/m 2 ), preoperative abdominal obesity (yes or no), follow-up duration (<1 year or ≥1 year), successful operation or not, preoperative blood pressure levels, and preoperative metabolic status of blood glucose and lipids. Spearman's rank correlation coefficients were calculated to determine the influence of PSG changes on the levels of variables that differed significantly pre-and postoperatively. Multivariable linear regression analyses with robust variances were performed to assess specific improved PSG parameters as a dependent variable to influence levels of EC markers. Statistical analyses were performed using SPSS software (Statistical Package for Social Sciences, version 22.0, SPSS Inc., Chicago, IL, USA). Two-sided P values < 0.05 were considered significant. www.nature.com/scientificreports www.nature.com/scientificreports/ ethical approval. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments. informed consent. Informed consent was obtained from all individual participants included in the study.

Results
Subject characteristics. In total, 44 participants were included in this study. The male:female ratio was  Table 2 lists the demographic characteristics and sleep data of subjects in the preoperative and postoperative groups.
Neither BMI nor WC of the patients changed significantly after surgery. Table 2 lists the changes in blood pressure indices, glycometabolism indices, and lipid metabolism indices. Reductions were evident in a number of cardiovascular-related biomarkers, including fasting glucose (p < 0.05), HOMA-IR (p < 0.05), fasting TC (p < 0.001), fasting LDL-C (p < 0.05), and fasting Apo B (p < 0.001). SBP, DBP, TG, HDL-C, and Apo A did not change significantly after surgery. changes in ten endothelial function markers. Table 3 lists the preoperative and postoperative serum levels of endothelial function-related markers. Reductions of serum levels of VEGFA, Ang2, E-selectin, VWF, VWFCP, and TM after surgery were significant (P < 0.05). Levels of Ang1 and ICAM1 increased without significance, and levels of VEGFR1 and VCAM decreased without significance. We also searched for normal values of 10 endothelium function-related markers in previous published studies. Preoperative and postoperative serum levels of VEGFA. VEGFR1, VCAM, E-selectin and TM were lower than healthy values. Preoperative and postoperative serum levels of Ang1, Ang2, ICAM1, VWF and VWFCP were higher than healthy values. Preoperative level of   www.nature.com/scientificreports www.nature.com/scientificreports/ VEGFR1 in male subgroup was higher than female subgroup. Compared with male subgroup, the preoperative serum level of E-selectin in female subgroup was higher. There was no significant difference in preoperative level of other endothelial cytokines between different sex. We also found no evident difference of postoperative serum values of 10 EC-function related markers between male and female subgroups. Table 4 presents subgroup comparisons. VEGFA was associated with preoperative Apo A level (p < 0.05 between normal and abnormal subgroups); VEGFR1 was associated with preoperative hypertension (p < 0.05); VWF was associated with surgery success (p < 0.05); and TM was associated with preoperative WC (p < 0.05 for abdominal obesity), follow-up duration (p < 0.05 between <1 year and >1 year subgroups), and preoperative hypertension (p < 0.05). Table 5 shows that among the biomarkers that improved significantly after surgery, Spearman's correlation test revealed positive associations between ΔAng2 and ΔApo B (r = 0.372, p = 0.013), ΔVEGFA and ΔCT90% (r = 0.298, p = 0.049), ΔVWFCP and Δglucose (r = 0.317, p = 0.036), and ΔTM and ΔCT90% (r = 0.343, p = 0.023).

Discussion
Our pilot study might be the first to explore changes of 10 risk markers of endothelial function in patients with OSA after upper-airway surgery. In comparison with previous research, we found similar declining tendency in ICAM1 7 , E-selectin 8 and VWF 10 and increasing tendency in VCAM 6 . We found that upper-airway surgery can lead to significant improvements in the levels of endothelial function-related markers, including VEGFA, Ang2, E-selectin, VWF, VWFCP, and TM. We also established correlations between VEGFA, TM, VWFCP, and Ang2 and ameliorated oxyhemoglobin saturation and glucolipid metabolism.
Several prior studies have focused on the pernicious impact of OSA-related pathological features on vascular endothelial dysfunction, such as increases in EC instability molecules 14,15 , circulating cell adhesion cytokines 6,26 , and hyper-coagulability 27,28 . Recurrent intermittent hypoxia during sleep and the concomitant high level of endothelial nitric oxide 29,30 aggravate oxidative stress 31 and systemic inflammation 32 . Endothelial dysfunction is considered to be one of the crucial mechanisms leading to adverse cardiovascular consequences in OSA patients 33 .    www.nature.com/scientificreports www.nature.com/scientificreports/ In our study, we were pleased to find that the reductions in levels of VEGFA and Ang2 were significant. Therefore, the synergistic effects of VEGFA [34][35][36] and Ang2 37-40 on EC instability and hyperpermeability were significantly reduced by upper-airway surgery. Higher expressions of VEGFR1 and Ang1, as representational markers of protection and stabilization, were increased after upper-airway surgery, although the changes were not statistically significant. Together with the increased Ang1/Ang2 40 ratio, this finding implies a reduced risk of CVD by reducing EC permeability and promoting healthy status of vessels after surgery. Hypoxia, even hypoxia-induced oxidative stress, coagulation, inflammation, and/or a hyperlipidemic environment, can all induce release of CAMs 8,41-43 . Postoperative results indicated that surgical treatment decreased expression of VCAM and E-selectin 44 , (regardless of whether upper-airway surgery outcome was successful), along with evident improvement of sleep parameters. However, the serum level of ICAM1 was increased. This finding suggests that initial CAM-induced risk of atherosclerosis formation still existed, and other therapies are necessary to eliminate the possibility of atherosclerosis progression. In our study, we observed decreases in VWF and TM      www.nature.com/scientificreports www.nature.com/scientificreports/ accompanied with reduction of VWFCP (all significant). Reduction in VWFCP 44,45 may be due to synchronization with low VWF, so this finding implies that long-term risk of CVD, particularly the possibility of artery atherosclerosis and thrombus, might be significantly reduced after surgical therapy. Almost all of the ten endothelial function markers were improved to some extent postoperatively, with the exception of ICAM1. We found that most of the values in post-operative subjects were higher than healthy individuals (normal values of previous published studies as reference). Upper-airway surgery might not reverse values of endothelial function in a relative short follow-up time. Gender seemed not to be associated with these endothelial cytokines we focused on.
We also found that different preoperative conditions (including sex, AHI, BMI, fasting glucose levels, fasting insulin levels, fasting TC levels, fasting TG levels, fasting HDL-C levels, fasting LDL-C levels, and fasting Apo B levels) might not be associated with the mean change of the ten endothelial function markers. Only respective ΔAHI ΔMean  Table 6. Multivariable linear regression analyses with changes in serum levels of 10 markers, PSG parameters and biomarker levels. Abbreviations: VEGFA = vascular endothelium growth factor A, Ang2 = angiopoietin-2, E-selectin = endothelial-leukocyte adhesion molecule, vWF = von willebrand factor, vWFCP = von willebrand factor cleavage protease, TM = thrombomodulin, AHI = apnea-hypopnea index, SaO 2 = oxygen saturation, LSpO 2 = lowest pulse oxygen saturation, CT90 = percentage of time during which oxyhemoglobin saturation was < 90%, ODI = oxygen desaturation index, MAI = micro-arousal index, ESS = Epworth Sleepiness Scale, HOMA-IR = homeostasis model of assessment for insulin resistance index, TC = total cholesterol, LDL-C = low-density lipoprotein cholesterol, Apo B = apolipoprotein B. *p < 0.05. www.nature.com/scientificreports www.nature.com/scientificreports/ markers in respective conditions, i.e., VEGFA (with or without preoperative hypertension), VEGFR1 (with or without normal level of fasting Apo A), VWF (successful or unsuccessful surgery), TM (with or without preoperative abdominal obesity, follow-up time >1 year or not, and with or without preoperative hypertension), differed significantly, with similar variation tendencies. This finding implies that the beneficial incomes of improved endothelial function after surgery were relatively steady.
After comprehensively comparing the changes of sleep parameters, glucolipid metabolism indices, and endothelial function-related markers, we found improvements of the potent markers VEGFA and TM might be associated with down-regulation of CT90%. Improvement of Ang2 was associated with down-regulation of fasting Apo B levels. Improvement of VWFCP might be associated with down-regulation of fasting glucose levels. Improvement of VEGFA could be associated with melioration of CT90% and fasting glucose levels, especially with down-regulation of blood glucose level. Improvement of Ang2 was associated with down-regulation of CT90%, ODI, fasting glucose levels and fasting Apo B levels, in especial with melioration of Apo B levels. These findings suggest that improved endothelial function-related markers are associated with ameliorated oxyhemoglobin saturation and glucolipid metabolism indices after upper-airway surgery.
Our findings are consistent with results of experimental studies reporting vascular hyperpermeability, hypercoagulability, and unbalance of the steady endovascular environment in response to chronic intermittent hypoxia 46,47 and impaired sleep architecture 48 . Potential physiological pathways may include oxidative stress, inflammation, and sympathetic activation [49][50][51] .
Several limitations to this study should be acknowledged. First, the sample size in our pilot study was small, so selection bias might exist. Second, this study was not a randomized controlled trial (RCT); although a RCT could upgrade the level of evidence and identify the causal relationships, it is difficult to perform one in this context. Third, confounding factors, such as diet and exercise, could also affect endothelial function markers. Fourth, we did not measure endothelial dysfunction assessed by flow-mediated dilatation (FMD). Fourth, though we search for normal values of 10 endothelium function-related markers in previous published studies, we did not set a control group simultaneously. Finally, this was a preliminary hypothesis-generating study; we plan to confirm the results in an independent sample of patients in the near future. Further more, given that the majority of endothelium function-related markers influence the process of endothelial colony-forming cells (ECFC) 52,53 transforming into mature EC, we consider that further research about isolated ECFC from circulation is essential to explore the potential pathological procedure of endothelial injury induced by unbalanced EC markers. We suspect that intermittent hypoxia and sleep fragmentation is likely the key factors.
In conclusion, the results of this study suggest that upper-airway surgery might be associated with an improvement in endothelial dysfunction-related markers in patients with OSA, probably through downregulation of inflammatory adhesion molecules, endothelial cell (EC) propermeability, inflammatory factor overflow, and hypercoagulability and upregulation of endothelium-stabilizing markers. The improvements in endothelial function biomarkers might be related to increased oxygen saturation after surgery. More prospective research on this issue will be needed to verify our findings and demonstrate the potential benefit of airway surgery for OSA patients Fig. 1.

Data availability
The corresponding authors will provide the accessibility of clinical data applied to support conclusions after receiving request.