Correlation between Endothelial Dysfunction,Rho-associated Protein Kinase Activity,C-reactive Protein and Obstructive Sleep Apnoea Syndrome in Male Patients

Abstract

Objective:

This study investigated the association between obstructive sleep apnoea syndrome (OSAS) and flow-mediated dilatation (FMD), Rho-associated protein kinase (ROCK) activity, and C-reactive protein (CRP) concentrations in male patients.

Methods:

Consecutive patients with symptoms suggestive of OSAS were recruited and divided into non-OSAS (n = 18) and OSAS (n = 32) groups. FMD was measured in the brachial artery; blood samples were taken to measure ROCK activity and CRP concentrations.

Results:

ROCK activity and CRP concentrations were significantly higher, and FMD was significantly lower, in the OSAS group than in the non-OSAS group. There was a correlation between ROCK activity and FMD. In stepwise multiple regression analyses, the proportion of sleep time spent with an oxygen saturation < 90% was a significant determinant of ROCK activity, while body mass index was the only significant determinant of CRP concentration. The oxygen desaturation index was a significant determinant of FMD.

Conclusions:

OSAS increased ROCK activity and was a major determinant of endothelial dysfunction.

Keywords

Obstructive sleep apnoea syndrome Males Flow-mediated dilatation Brachial artery Rho-associated protein kinase C-reactive protein

Introduction

Obstructive sleep apnoea syndrome (OSAS) is characterized by repetitive episodes of upper airway obstruction during sleep with associated intermittent hypoxaemia, which affects ≥ 4% of adult males and ≥ 2% of adult females.¹ It is widely accepted that repetitive nocturnal hypoxaemia in OSAS patients is associated with increased risk of stroke and cardiovascular disease.^2,3 The underlying pathophysiology linking these disorders with OSAS has not been fully elucidated. It is likely to be a multifactorial process involving a diverse range of mechanisms, including inflammation and vascular endothelial dysfunction. Atherosclerosis is considered to be the main cause of the cardiovascular complications observed in OSAS patients.⁴ Research has demonstrated that endothelial dysfunction, as an early marker of atherosclerosis, was significantly correlated with OSAS.⁵ Endothelial function can be measured noninvasively using flow-mediated dilatation (FMD) in the brachial artery,⁶ which is a nitric oxide (NO)-dependent vasodilatation.⁷ NO, constitutively produced by endothelial NO synthase (eNOS), regulates cerebral blood flow and vascular tone, and protects against ischaemic stroke by increasing collateral flow to the ischaemic area.⁸ Indirect evidence of reduced NO availability is suggestive of vascular endothelial dysfunction in patients with OSAS.⁹ It has been reported that the neuroprotective effect of Rho-associated protein kinase (ROCK) inhibition is mediated by endothelium-derived NO.⁸ C-reactive protein (CRP) is an important marker of inflammation and has been shown to be a powerful mediator of inflammation in atherosclerosis.¹⁰

As FMD, ROCK activity and CRP are involved in the development of atherosclerosis, the present study investigated the association between FMD, ROCK activity and CRP and the severity of OSAS, in order to identify variables that might explain change in FMD, ROCK activity and CRP in OSAS.

Patients and methods

Study Population and Design

This prospective study enrolled consecutive male patients who were referred to the Sleep Laboratory of Changzheng Hospital, Second Military Medical University, Shanghai, China, for evaluation of suspected sleep apnoea, between June 2009 and May 2010. Patients with a history of diabetes mellitus or cardiac, renal, liver and chronic inflammatory diseases were excluded. Women were also excluded, to avoid potential sex-associated differences.^11,12 Sleep habits, symptoms, demographic characteristics (age, weight, height, and body mass index [BMI]), blood pressure and medication use were reviewed before the night of the polysomnography (PSG) assessment. Patients also completed the Epworth Sleepiness Scale, which scores the likelihood of dozing in eight situations (0, no likelihood; 1, slight likelihood; 2, moderate likelihood; 3, high likelihood), giving a maximum score of 24.¹³

The study was approved by the Medical Ethical Committee of Changzheng Hospital. All participants signed an informed consent form prior to participation in the study.

Polysomnography Assessment

All participants underwent an overnight attended PSG assessment using an Embla TMN 7000 PSG Amplifier (Embla, Reykjavik, Iceland), and standard electrodes and sensors. The PSG assessment included the following: left and right electro-oculography; electroencephalography (C4-A2, C3-A1, O2-A1, O1-A2); electromyogram of submental muscles; electromyogram of the anterior tibialis muscle of the legs; electro -cardiography; a sensor on the thoracic belt to evaluate the body posture; airflow (with an oronasal thermistor); left and right anterior tibialis movement sensors; respiratory effort by thoracoabdominal piezoelectric belts; finger pulse oximetry; a neck microphone for recording snoring. Based on standard criteria,¹⁴ PSG recordings were analysed at 30-s intervals. Apnoea was defined as a complete cessation of airflow for ≥ 10 s. Hypopnoea was defined as a significant reduction in airflow (> 50%) for ≥ 10 s or a moderate reduction in airflow for ≥ 10 s together with an arousal or oxygen desaturation (≥ 4%).¹⁵ The apnoea– hypopnoea index (AHI) was defined as the total number of apnoea and hypopnoea episodes per h of sleep; the oxygen desaturation index (ODI) was defined as the number of oxygen desaturations (≥ 4%) per h of sleep. OSAS was defined as an AHI ≥ 5 obstructive events/h of sleep.⁵ Study participants were allocated to either the OSAS group or the non-OSAS group, based on this definition.

Assessment of CRP Concentration and Rock Activity

All venous blood samples were drawn in the morning, between 06:00 h and 08:00 h after an overnight fast of ∼ 10 h, in commercial Vacutainer™ tubes containing thrombin as an anticoagulant. Blood was collected at the same time as the PSG assessment and at 4 weeks after continuous positive airway pressure treatment. Blood samples for lipid profile analysis were sent immediately to the laboratory for testing. Total cholesterol was measured by an autoanalyser using standard laboratory protocols. Blood samples for the measurement of CRP concentrations and ROCK activity were immediately frozen at –80 °C until analysis. High-sensitivity CRP (hs-CRP) was measured using a commercial chemiluminescence immunoassay (Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA). ROCK activity was measured as described previously, as the ratio of phosphorylated threonine 696 myosin phosphatase target (p-Thr696 MYPT) to total MYPT, using an enhanced chemiluminescence immunoassay.¹⁶

Assessment of FMD

Vascular reactivity of the brachial artery was assessed in the arm, from the anterior intima to the posterior intima, by FMD according to the guidelines of the International Brachial Artery Reactivity Task Force.¹⁷ Digitized images were captured and analysed using Quantity One^® software, (Bio-Rad, Hercules, CA, USA).

Statistical Analyses

All statistical analyses were performed using the SPSS^® statistical package, version 12.0 (SPSS Inc., Chicago, IL, USA) for Windows^®. Continuous variables were presented as mean ± SE. Pearson's correlation coefficients were calculated to identify associations between clinical variables. Stepwise multiple regression analysis was performed to identify variables that contributed statistically to the hs-CRP concentration, ROCK activity and FMD. Comparison of the continuous variables between the two groups was made using analysis of variance with Student's t-test. Categorical variables were compared using the χ²-test. A P-value < 0.05 was considered statistically significant.

Results

A total of 50 male participants were enrolled in the study: 32 satisfied the diagnostic criteria for OSAS; 18 were diagnosed as not having OSAS. Clinical characteristics of the participants with and without OSAS are presented in Table 1. There were no significant differences in age, BMI, blood pressure and total cholesterol level between the two groups. There were also no significant differences in prevalence of hypertension or smoking habits between the two groups (data not shown), which are both factors that affect endothelial function. Compared with subjects in the non-OSAS group, patients in the OSAS group had significantly higher AHI and ODI scores, a significantly higher proportion of sleep time spent with an oxygen saturation < 90% and significantly more daytime sleepiness, as measured by the Epworth Sleepiness Scale¹³ (P < 0.05 for all comparisons).

Table 1:

Baseline demographic and clinical characteristics of patients with obstructive sleep apnoea syndrome⁵ (OSAS group) and subjects without OSAS (non-OSAS group)

Characteristic	OSAS group n = 32	Non-OSAS group n = 18
Age, years	47.2 ± 12.5	51.2 ± 13.6
Body mass index, kg/m²	28.7 ± 4.6	30.2 ± 4.2
Systolic blood pressure, mmHg	125.3 ± 16.8	118.2 ± 17.1
Diastolic blood pressure, mmHg	74.9 ± 12.3	79.1 ± 13.1
Apnoea–hypopnoea index	27.9 ± 10.2*	3.1 ± 1.2
Oxygen desaturation index	36.8 ± 14.4*	2.8 ± 1.4
Proportion of sleep spent with an oxygen saturation < 90%, %	22.3 ± 3.1*	0.6 ± 0.9
Epworth sleepiness scale score¹³	12.1 ± 3.8*	7.2 ± 4.2
Total cholesterol, mg/dl	181.6 ± 35.4	175.3 ± 37.1

Data presented as mean ± SE.

P < 0.05 compared with the non-OSAS group (analysis of variance with Student's t-test).

Brachial artery FMD is an indirect marker of endothelial NO-mediated reactivity. No differences in the resting brachial diameter and percentage reactive hyperaemia after cuff deflation were observed between the OSAS and non-OSAS groups (data not shown). Baseline mean ± SE FMD was significantly lower in the OSAS group compared with the non-OSAS group (4.0 ± 3.0% versus 9.5 ± 2.8%, respectively; P < 0.05). Patients in the OSAS group had a significantly higher mean ± SE ROCK activity (ratio of p-Thr696 MYPT to total MYPT) (0.7 ± 0.2 versus 0.3 ± 0.1; P < 0.01) and mean ± SE hs-CRP concentration (21.6 ± 19.7 versus 9.4 ± 6.5 IU/ml; P < 0.05) compared with those in the non-OSAS group at baseline.

Pearson's correlation coefficients for the clinical variables are shown in Table 2. After controlling for age and BMI, FMD was found to be significantly correlated with AHI (P < 0.05), ODI (P < 0.05), systolic blood pressure (P < 0.05), proportion of sleep time spent with an oxygen saturation < 90% (P < 0.01), and ROCK activity (P < 0.05). ROCK activity was significantly correlated with AHI (P < 0.05), ODI (P < 0.05), systolic blood pressure (P < 0.05) and the proportion of sleep time spent with an oxygen saturation < 90% (P < 0.05). Hs-CRP concentration was significantly correlated with ODI (P < 0.05) and proportion of sleep time spent with an oxygen saturation < 90% (P < 0.05).

Table 2:

Pearson's correlation coefficients between clinical variables and the flow-mediated dilatation (FMD), Rho-associated protein kinase (ROCK) activity, and high-sensitivity C-reactive protein (hs-CRP) concentration calculated for 32 patients with obstructive sleep apnoea syndrome (OSAS)⁵ and 18 subjects without OSAS

Clinical variable	FMD	ROCK activity	hs-CRP concentration
Apnoea–hypopnoea index	−0.32*	0.26*	0.07
Oxygen desaturation index	−0.31*	0.21*	0.33*
Proportion of sleep spent with an oxygen saturation < 90%	−0.41**	0.22*	0.27*
Systolic blood pressure	−0.23*	0.25*	0.18
Diastolic blood pressure	−0.10	0.16	0.19
Total cholesterol	−0.11	0.19	0.13
Hs-CRP concentration	0.17	0.15	1.00
ROCK activity	−0.28*	1.00
FMD	1.00

P < 0.05,

P < 0.01.

Stepwise multiple regression analyses were performed to identify the factors that contributed to FMD, ROCK activity, and hs-CRP concentration (Table 3). ODI was a significant determinant of FMD (adjusted R² = 15%, β = −0.33, P < 0.05). The proportion of sleep time spent with an oxygen saturation < 90% was a significant determinant of ROCK activity (adjusted R² = 18%, β = 0.26, P < 0.05). BMI was a significant determinant of hs-CRP concentration (adjusted R² = 13%, β = 0.25, P < 0.05).

Table 3:

Stepwise multiple regression analyses of the effect of different clinical variables on the flow-mediated dilatation (FMD), Rho-associated protein kinase (ROCK) activity, and high-sensitivity C-reactive protein (hs-CRP) concentration calculated for 32 patients with obstructive sleep apnoea syndrome (OSAS)⁵ and 18 subjects without OSAS

Clinical variable	FMD	ROCK activity	hs-CRP concentration
Age	−0.05	−0.07	−0.13
Body mass index	−0.12	0.14	0.25*
Apnoea–hypopnoea index	0.15	−0.03	0.08
Oxygen desaturation index	−0.33*	0.21	0.09
Proportion of sleep spent with an oxygen saturation < 90%	−0.04	0.26*	−0.07

P < 0.05.

Discussion

The present study provided direct evidence of vascular endothelial dysfunction and inflammation in patients with OSAS, compared with healthy control subjects. FMD was significantly lower in patients with OSAS compared with healthy control subjects. On multiple regression analysis and after adjustment for important covariates, ODI was the only variable to be significantly correlated with FMD. Endothelial dysfunction plays a role in the occurrence of various diseases and is commonly measured (using a noninvasive flow-mediated technique) as the relative increase in brachial artery diameter in response to peak reactive hyperaemia.¹⁷ The vascular endothelium participates in the control of various vascular functions and regulates vasoactive mediators in response to physical or biochemical stimuli in the body.¹⁸ Endothelial dysfunction has been detected in atherosclerosis, and in cigarette smoking and hypercholesterolaemia that predispose to atherosclerosis, indicating that it is a marker of early atherogenesis.^19,20 Endothelial dysfunction was shown to have a predictive value for cardiovascular events.²¹ In previous studies, endothelial dysfunction (as measured by FMD) was associated with AHI and a high hypoxaemia index in patients with OSAS.²² Endothelium-dependent vasodilatation is mediated by NO, which is an intracellular signalling molecule and is the most potent vascular relaxant.¹⁸ Oxygen tension plays an important role in regulating NO synthases,²³ and hypoxia may alter NO biosynthesis.²⁴ OSAS reduces endothelial NO availability,²⁵ which results in endothelial dysfunction, thereby increasing the risk for atherosclerosis and vascular diseases in OSAS patients.²⁶ Thus, the repetitive episodes of hypoxia/ reoxygenation that characterize OSAS may be responsible for triggering endothelial dysfunction.

Research suggests that ROCK plays an important role in pathological conditions including arteriosclerosis,²⁷ vascular inflammation and remodelling,²⁸ cerebral and coronary vasospasm^29,30 and hypertension.³¹ ROCK inhibition reversed the downregulation of eNOS under hypoxic conditions.¹⁶ A strong association was previously observed between FMD and ROCK activity;³² thus, the present study measured ROCK activity in OSAS patients and in healthy control subjects. It is likely that the effect of sleep apnoea would be greatest in the morning after a night of repetitive apnoeic events. Thus, a fasting blood sample was taken in the morning for measurement of ROCK activity and was found to be significantly higher in OSAS patients compared with healthy control subjects. There was a significant correlation between ROCK activity and sleep apnoeic activity as indicated by AHI, ODI, and the proportion of sleep time spent with an oxygen saturation < 90%. The significant correlation between ROCK activity and FMD suggested that ROCK may be involved in the regulation of NO synthesis in OSAS. Stepwise multiple regression analysis indicated that the proportion of sleep time spent with an oxygen saturation < 90% was the only significant determinant of ROCK activity. These results suggested that ROCK activity was related to OSAS.

Patients with OSAS in the present study had significantly higher CRP concentrations than healthy control subjects, and a correlation was found between CRP concentration and hypoxaemia in OSAS patients, as indicated by ODI and proportion of sleep time spent with an oxygen saturation < 90%. CRP reflects the inflammatory response and is also an important risk factor for the development of atherosclerosis.¹⁰ In OSAS patients, an elevated CRP concentration was found to be significantly and positively correlated with the severity of sleep-disordered breathing.⁵ It has also been suggested that CRP is the important pathophysiological link between OSAS and coronary artery disease.³³ Research has reported that BMI was the only significant variable associated with log-CRP by multiple regression analysis,^34,35 which was also demonstrated in the present study. Obesity is associated with both the severity of sleep apnoea and an elevated serum CRP level; thus, it is probably a major confounding factor in this association.³⁶ The effect of OSAS on serum CRP could be differentiated from the effect of obesity, because Asian patients with OSAS are less obese than Caucasian patients.³⁴

There were a number of limitations to the present study. The sample size was small and although the correlations were significant, they were often quite low, which could have been a chance finding.

In conclusion, FMD was found to be significantly correlated with the severity of OSAS, and ODI was a significant determinant of FMD. The present study also demonstrated that ROCK activity was one of the mediators involved in OSAS, and that sleep time spent with an oxygen saturation < 90% was associated with ROCK activity. Moreover, CRP was not found to be correlated with OSAS severity. Although the sample size was small, this study showed that the presence of OSAS increased ROCK activity and was a major determinant of endothelial dysfunction.

Footnotes

Acknowledgements

This study was supported by Key Medical Funds of Science and Technology Commission of Shanghai Municipality (Project No. 11411950203), Shanghai, China.

Conflicts of interest: The authors had no conflicts of interest to declare in relation to this article.

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