The role of hyaluronic acid and hyaluronidase-1 in obstructive sleep apnoea

Biological functions of hyaluronic acid (HA) depend on its molecular size. High-molecular weight HA (HMW-HA) is an important component of the endothelial wall and has anti-inflammatory and antioxidant properties. Under inflammation or hypoxia, HMW-HA is degraded by hyaluronidases, such as HYAL-1 resulting in pro-inflammatory low-molecular weight fragments. Obstructive sleep apnoea (OSA) is characterised by intermittent hypoxia and systemic inflammation. Our aim was to evaluate circulating HMW-HA and HYAL-1 in OSA. We recruited 68 patients with OSA and 40 control volunteers. After full-night sleep study blood samples were taken for HMW-HA and HYAL-1 measurements. HYAL-1 levels were significantly higher in patients with OSA compared to controls (0.59/0.31–0.88/ng/mL vs. 0.31/0.31–0.58/ng/mL; p = 0.005) after adjustment for gender, age, BMI and smoking. There was a trend for reduced HMW-HA concentrations in OSA (31.63/18.11–59.25/ng/mL vs. 46.83/25.41–89.95/ng/mL; p = 0.068). Significant correlation was detected between circulating HMW-HA and apnoea-hypopnoea-index (r = − 0.195, p = 0.043), HYAL-1 and apnoea-hypopnoea-index (r = 0.30, p < 0.01) as well as oxygen desaturation index (r = 0.26, p < 0.01). Our results suggest that chronic hypoxia is associated with increased plasma HYAL-1 concentration and accelerated HMW-HA degradation. Altered hyaluronan metabolism may be involved in the inflammatory cascade potentially leading to endothelial dysfunction in OSA.

www.nature.com/scientificreports/ pathophysiology during inflammation. Altered HA production has been detected in airway diseases characterised by inflammation and oxidative stress, such as asthma and COPD 13,14 . Fragments derived from HMW-HA stimulate several inflammatory cells in the airways, such as alveolar macrophages to produce mediators and these mechanisms enhance inflammation 7 . The expression of HYAL enzymes is highly induced in the airway epithelial cells by pro-inflammatory cytokines suggesting that the epithelium could be the source for LMW-HA in airway inflammation 13 . Preliminary reports detected elevated HYAL-1 activity in the airways of asthma and COPD patients 15 . Moreover, increased enzymatic degradation of HMW-HA in bronchoalveolar lavage fluid and serum of COPD patients were associated with airflow limitation 14,16 . The same mechanisms can contribute to accelerated metabolism of HMW-HA and enhanced airway and systemic inflammation in OSA. However, HA levels have not been analysed in this disease. Therefore, our aim was to investigate the levels of HMW-HA and HYAL-1 in patients with OSA and understand the role of hyaluronan metabolism in the pathogenesis of OSA.

Results
Patient characteristics. Sixty-eight patients were diagnosed with OSA (median age: 53 years/47-64/, 49 men). Twenty-five of them had mild (AHI 5-14.9 events/h), 16 moderate (AHI 15-29.9 events/h) and 27 severe (AHI > 30 events/h) disorder. Subjects' characteristics and comparisons between the OSA and control groups are shown in Table 1. Patients with OSA were older (p = 0.08) and had significantly higher BMI, systolic (SBP) and diastolic blood pressure (DBP) and ESS values. They had higher levels of CRP, glucose, total triglyceride and lipoprotein (a) and lower levels of HDL-C (all p < 0.01). There was no difference in total cholesterol and LDL-C levels (p = 0.43; p = 0.73). No differences were detected in the prevalence of comorbidities except for hyperten-  39; p = 0.005). After adjustment for hypertension and cardiovascular diseases HYAL-1 concentrations remained significantly higher (p = 0.005), HMW-HA levels were lower (p = 0.068) in OSA group. The difference between the two groups remained significant for HMW-HA/HYAL-1 ratio (p = 0.005).
We noticed 10 outliers (2 controls and 4 patients for HMW-HA as well as 1 control and 3 patients for HYAL-1). These subjects were not different in their general characteristics or comorbidities from the others. After excluding them, the HYAL-1 levels and HMW-HA/HYAL-1 ratio were still significantly different (p = 0.013; p = 0.002) and the difference in HMW-HA concentrations between the two groups became significant (p = 0.027).
When excluding patients with chronic airway disease and chronic heart failure (33 controls vs. 50 patients) we significantly lower HMW-HA (28.   www.nature.com/scientificreports/ Eighteen participants were treated with statins and three patients were treated with systemic steroids (low dose methylprednisolone). After adjustment for the statin and steroid usage, HYAL-1 levels (p = 0.005) and the HMW-HA/HYAL-1 ratio (p = 0.005) were still significantly different and the difference in HMW-HA concentrations between the two groups did not change (p = 0.068).

Discussion
We reported higher plasma HYAL-1 and lower HMW-HA levels in OSA. Chronic intermittent hypoxia, enhanced oxidative stress and inflammation characterising OSA may explain our results. Chronic intermittent hypoxia influences the expression of several hypoxia-related transcriptional factors, such as hypoxia-inducible factor-1 and nuclear factor kappa-B (NF-kB) which play role in inflammatory responses 17,18 . Gao et al. have demonstrated that hyaluronan metabolism is regulated by hypoxia in vitro as it upregulates the expression of HYAL 19 . In line with this, plasma HYAL-1 levels were associated with the markers of overnight hypoxia in our current study. Although CIH can upregulate the anti-inflammatory HMW-HA, it is quickly degraded by increased concentrations of HYAL enzymes resulting higher LMW-HA levels 19 . Hypoxia can also increase the expression of CD44 which is the most important receptor of HA fragments and it is expressed on a wide range of inflammatory cells 20 . This may result in a consequentially higher HA uptake from the circulation.
Increased production of ROS and RNS is well described in OSA 21 . Oxidative stress can degrade HA through direct and indirect mechanisms. HA polymers are susceptible to direct oxidation by ROS and RNS 11 . Oxidative stress can also induce the expression of HYAL 22 . In OSA patients CIH decreases the plasma concentration of superoxide dismutase (SOD) 23 . In SOD3 deficient mice elevated lung HYAL expression was detected due to hypoxic injury. Moreover, the loss of SOD3 increased the hypoxia-induced HYAL expression and activity in human pulmonary artery smooth muscle cells in vitro 24 .
Pro-inflammatory cytokines, such as interleukin 1-beta (IL-1β) and tumour necrosis factor-alpha (TNF-α) increase HA levels by upregulating HA-synthase enzymes 25 . However, these cytokines elevate HYAL levels in plasma as well as in the endothelium leading to an accelerated HA clearance from the circulation. Previous studies have shown that LMW-HA fragments stimulate the production of IL-1β, IL-8 via NF-kB signalling 26 and potently enhance the activation and recruitment of neutrophils 27 . The activation of NF-kB and consequential elevated levels of these cytokines are well described in OSA 17 .
In contrast to the pro-inflammatory molecules, the role of anti-inflammatory processes in OSA has not been fully elucidated. Only a few anti-inflammatory biomarkers have been examined with altered levels in OSA 3,4 . HMW-HA is a potent anti-inflammatory and immunosuppressive molecule. It is well known that HMW-HA was able to down-regulate the genes of NF-kB and several interleukins 6 . Moreover, HMW-HA is capable of neutralizing intra-and extracellular ROS 28 . Protective activation of HYAL enzymes has been also demonstrated by decreasing the expression of TNF-α and IL-6 29 . A recent study has shown that HYAL suppressed neutrophil recruitment and cytokine production in a murine model of acute inflammation 30 . However, the immunoregulatory functions of HYAL-1 are contradictory. High activity of HYAL enzymes elevate pro-inflammatory LMW-HA levels and itself can damage endothelial glycocalyx resulting in endothelial dysfunction and vascular inflammation 31 .
OSA is associated with increased cardiovascular morbidity and mortality 32 . Alterations of hyaluronan metabolism have been demonstrated in association with endothelial dysfunction through glycocalyx destabilization with attenuated endothelial nitric oxide availability contributing to atherosclerosis 31,33 . The degradation of HMW-HA under inflammatory conditions reduces extracellular HA viscosity and increase tissue permeability 34 resulting extravasation of macrophages to the arterial wall. The damaged endothelium facilitates the adhesion of leukocytes and platelets via adhesion molecules, and the levels were found to be elevated in OSA 2, 35 . Dogné et al. demonstrated that the lack of HYAL-1 is associated with lower levels of P-selectin suggesting that HYAL-1 deficiency may be protective in endothelial dysfunction 33 . Elevated HYAL levels are implicated in intima aging 36 and increased HYAL activity was detected in patients with coronary artery disease 37 indicating its potential role in atherosclerosis. In addition, HYAL enzymes impair the endothelial mechanosensitive response by glycocalyx shedding and potentiate the dysregulation of vascular tone 38,39 . Supporting this, arterial stiffness was significantly associated with increased plasma HYAL concentrations suggesting that altered hyaluronan metabolism may be involved in the pathophysiology of hypertension 40 . www.nature.com/scientificreports/ Obstructive sleep apnoea is often associated with obesity. Fat tissue is a major source for inflammatory molecules and obesity itself can be associated with elevated levels of inflammatory molecules 41 . The hyaluronic acid has a well-described role in adipogenesis 42 . We found a significant relationship between HYAL-1 levels and BMI, and HYAL-1 concentrations were higher in the obese compared to the non-obese controls. This suggests that obesity could partially contribute to our findings. Interestingly, plasma HMW-HA levels were not related to obesity. Most importantly, our primary analyses were adjusted to age, gender, smoking and BMI and the investigated molecules were not different in obese vs. non-obese OSA. This suggests that OSA-related changes in HMW-HA and HYAL-1 levels were not exclusively due to differences in BMI between the two groups.
Hyaluronic acid has been implicated in the pathophysiology on chronic airway diseases [13][14][15] and chronic heart failure 43 . Therefore, we analysed our data also following exclusion of patients with these diseases. Exclusion did not affect the HYAL-1 or HMW-HA/HYAL-1 results, while the difference in HMW-HA concentrations between patients with OSA and controls became significant. This suggests that further studies analysing hyaluronic acid metabolism should take into account these diseases as confounders to their results.
This study has limitations. We did not measure LMW-HA as it less stable, has significantly shorter half-life compared to HMW-HA 44 and is also quickly eliminated by the kidney 45 . However, to have a deeper understanding of hyaluronic acid metabolism in OSA, further studies may consider measuring LMW-HA as well. The levels of HYAL-1 and HMW-HA were below the detection limit in 39% and 18% of all cases. There are multiple techniques how to handle results with values below the lower limit of detection, including exclusion of these results, assigning the detection limit, detection limit value/√2 or zero. However, all these methods have limitations and replacing the values with the detection limit has the least bias 46,47 . Of note, this led to skewing our data. Therefore, our results need to be interpreted with caution. Nevertheless, significantly more patients had detectable levels of HYAL-1 than controls strengthening our conclusion that this enzyme is elevated in OSA. The study was powered to find differences in either HMW-HA or HYAL-1 between OSA and controls. The comparison has been adjusted to age, gender and BMI. As confirmed in this study, these parameters are often different in OSA compared to controls. Apart from the correlations with the BMI, we observed significant relationships between HMW-HA and age as well as HYAL-1 and gender. Although the primary analyses were adjusted on these factors, further studies should aim for better balanced case-control groups. The relationships between HYAL-1 and glucose, triglyceride as well as HDL-C levels also need to be interpreted carefully, as they may have been affected by OSA itself and does not necessarily represent and independent association. Circulating markers of hyaluronan metabolism were examined in diabetic patients and they were correlated with serum glucose and lipid parameters 48 . One may argue if these were independent associations as the potential presence of OSA was not considered. Nevertheless, the relationships between hyaluronan metabolism and metabolic profile should be investigated in non-OSA populations. Most notably, the correlation analyses in our study are exploratory and need to be interpreted with caution. Only 60 subjects had polysomnography, therefore data on TST, SPT and AI has to be interpreted carefully. Potentially, sleep fragmentation could also have contributed to our results, however this cannot be concluded as PSG has not been performed in everybody. In addition, being a more sensitive test, higher AHI values may have been obtained with PSG compared to PG as hypopnoeas resulting arousals, but no desaturation can be scored only with polysomnography. Therefore, correlations between biomarkers and AHI may have been stronger if only one sleep test has been used. None of the volunteers has been treated for their OSA before and a follow-up measurement after CPAP therapy in moderate-to-severe patients would have given additional information on the role of this molecular pathway. We believe that our study would provide an essential basis for sample size estimation when designing a prospective study evaluating the effect of CPAP.
In conclusion, our study indicates that plasma HYAL-1 levels are elevated in OSA and may decrease the circulating HMW-HA concentrations. These alterations potentially contribute to the enhanced systemic inflammation in OSA and could represent a potential link between OSA and its comorbidities.

Methods
Study design and subjects. We recruited 108 participants who were referred to the Sleep Unit of the Department of Pulmonology, Semmelweis University due to suspected OSA (i.e. symptoms of snoring, witnessed apnoea, daytime sleepiness, obesity or cardiometabolic comorbidities). None of the patients had previously been diagnosed with OSA and they had not been treated with continuous positive airway pressure (CPAP) or mandibular advancement devices (MAD). Patients with malignancy within 10 years, infection within 2 months, autoimmune disorders or uncontrolled chronic disease (for example acute heart failure, uncontrolled diabetes), were excluded.
In the evening, detailed medical history was taken, and patients filled out the Epworth Sleepiness Scale (ESS). Full-night, inpatient diagnostic cardiorespiratory polygraphy (PG, n = 48) or polysomnography (PSG, n = 60) were performed. The decision which diagnostic test to use was made by an expert somnologist who triaged the patients into low and high-likelihood groups. In the following morning between 6:00 and 8:00 a.m, systolic and diastolic blood pressure was measured and venous blood was taken to evaluate plasma HMW-HA and HYAL-1, as well as fasting serum glucose, total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride, lipoprotein(a) and C-reactive protein (CRP) levels. Comorbidities were defined according to the patients' report, medical history, and current medications. In particular, cardiovascular diseases included current or previous stable and unstable angina, stroke, transient ischaemic attack and significant atherosclerosis. Smoking status was defined as self-reported current smoking or previous history of smoking.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study protocol has been approved by the Semmelweis Sleep studies. Full-night cardiorespiratory polygraphy and polysomnography were performed using the Somnoscreen Plus Tele PSG (Somnomedics GmbH Germany) as described previously 49 . Sleep stages, movements and cardiopulmonary events were scored manually according to the American Academy of Sleep Medicine (AASM) guideline 50 . Apnoea was defined as a 90% decrease in airflow lasting for at least 10 s. Hypopnoea was defined as at least 30% decrease in airflow which lasted for more than 10 s with a ≥ 3% oxygen desaturation or an arousal. Total sleep time (TST), sleep period time (SPT) and minimal oxygen saturation (MinSatO 2 ) were recorded, apnoea-hypopnoea index (AHI), oxygen desaturation index (ODI), total sleep time with saturation below 90% (TST90%) and arousal index (AI) were calculated to evaluate the severity of OSA. OSA was defined with an AHI > 5/h.

Biomarker measurements.
Blood samples were taken into EDTA tubes. They were processed within 30 min after collection and centrifuged at 4 °C for 10 min at 1500 rpm. Immediately after centrifugation plasma samples were separated and stored at − 80 °C until further analysis. Plasma HMW-HA and HYAL-1 levels were measured using commercially available ELISA kits (Human Hyaluronic Acid (HA) ELISA kit from Corgenix Inc (Catalogue number: 029-001), Colorado, USA; Human Hyaluronidase-1 (HYAL-1) ELISA Kit from Cusabio Technology Llc (Catalogue number: CSB-EL010918HU), Houston, USA). Plasma HMW-HA and HYAL-1 measurements were performed in duplicates according to the manufacturers' instructions and the mean concentrations were used as inputs for analysis. The detection limits (DL) were 11 ng/mL for HMW-HA and 0.31 ng/ mL for HYAL-1. If the measured concentration was below the detection limit, the DL value was assigned to the sample. The intra-assay coefficients of variation were 4.0 ± 2.9% for HA and 8.6 ± 7.9% for HYAL-1, respectively.

Statistical analysis.
Statistical analyses were performed with JASP 0.11.1 (Amsterdam, Netherlands) and Graph Pad Prism 5.0 (GraphPad Software, San Diego, CA, USA). The normality of the data was assessed with the Shapiro-Wilk test, which showed non-parametric distribution for HMW-HA and HYAL-1 levels. Clinical data, categorical variables and biomarkers were compared between OSA and control groups with t-test, Mann-Whitney U-test and Chi-square test. We applied non-parametric ANCOVA after adjustment on age, gender, BMI and smoking to evaluate the differences in HMW-HA and HYAL-1 levels as well as HMW-HA/HYAL-1 ratio between OSA and control groups as well as among controls, mild, moderate and severe patients with OSA. Comparisons between the OSA and control groups were performed also after excluding outlier data (defined as 75th percentile + 1.5 × interquartile range) as well as patients with chronic airway disease (asthma and COPD) and chronic heart failure. Further sensitivity analyses were performed when the comparisons were adjusted for statin and steroid use. Plasma HA and HYAL-1 levels were compared with clinical parameters and markers of sleep architecture with the Spearman test. The HMW-HA, HYAL-1 and HMW-HA/HYAL-1 results are presented as median with interquartile range. A p-value < 0.05 was considered significant. The sample size was calculated to find difference in HMW-HA or HYAL-1 between the OSA and control groups with an effect size of 0.60, α error probability of 0.05 and power of 0.80 taking into the asymptotic relative efficiency of non-parametric tests. Post-hoc sensitivity analyses revealed that using this sample size we were able to find correlations with greater than − 0.19 or 0.19 critical r values 51 .

Data availability
The data are available from the corresponding author on request. www.nature.com/scientificreports/