Sleep Breath DOI 10.1007/s11325-013-0923-3


Endothelial injury markers before and after nasal continuous positive airway pressure treatment for obstructive sleep apnoea hypopnoea syndrome Maria Wilczynska & Samuel Rice & Gareth Davies & Keir E Lewis

Received: 11 June 2013 / Revised: 23 November 2013 / Accepted: 27 November 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Purpose The purpose of this study was to evaluate whether serum amyloid A (SAA), C-reactive protein (CRP), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) levels are elevated in obstructive sleep apnoea hypopnoea syndrome (OSAHS), and whether they change following acute- and medium-term CPAP treatment. Methods Consecutive subjects (n =40) referred to the Sleep Disordered Breathing Unit were included in the research. Sera were sampled in the afternoon prior to an in-hospital limitedchannel sleep study and on the next morning. Those diagnosed with OSAHS were commenced on CPAP and had further blood samples collected in the morning after the first night and then after a month of treatment. Results We had 20 subjects with moderate/severe OSAHS (mean ± SD), 4 % desaturation rate (4 % DR) 44.3±31.4 events/h, and 20 comparator subjects with symptoms but negative sleep studies, 4 % DR 5.6±2.9 events/h. There was no difference in the morning and afternoon vascular injury marker levels between the OSAHS and comparator groups. However, CRP (6.52±9.53 vs. 5.58±8.47, p =0.04) and VCAM-1 (366.30±90.11 vs. 339.60±95.87, p =0.02) levels showed significant diurnal variation within the OSAHS group

M. Wilczynska (*) : S. Rice : K. E. Lewis Department of Medicine, Prince Philip Hospital, Hywel Dda Health Board, Llanelli SA14 8QF, UK e-mail: [email protected] M. Wilczynska : S. Rice : K. E. Lewis College of Medicine, Institute of Life Sciences, Swansea University, Swansea SA2 8PP, UK G. Davies Public Health Wales, St David’s Park, Job’s Well Rd, Carmarthen SA31 3WY, UK

with higher afternoon levels compared to morning measurements. There were no changes in any of the vascular injury marker levels following CPAP. Conclusions This study demonstrates that OSAHS leads to endothelial dysfunction as reflected by higher afternoon than morning CRP and VCAM-1 levels. However, despite a good CPAP compliance, a month of treatment does not decrease vascular injury marker levels. Keywords Adhesion molecules . Inflammation . Serum amyloid A . C-reactive protein . Vascular cell adhesion molecule-1 . Intercellular adhesion molecule-1

Introduction Obstructive sleep apnoea hypopnoea syndrome (OSAHS) affects 2–4 % of adults and is characterized by the coexistence of excessive daytime sleepiness and obstructive sleepdisordered breathing (SDB) [1, 2]. OSAHS is an important contributor to cardiovascular disease (CVD), being an independent risk factor for the prevalence and incidence of hypertension, coronary artery disease, arrhythmias, heart failure, stroke and overall cardiovascular death [3-8]. Vascular inflammation is believed to have a role in the pathogenesis of cardiovascular events in OSAHS, with established CVD seen as the end of a long process of inflammation-mediated atherosclerosis [9]. The inflammatory response depends on the presence of both cytokines and adhesion molecules that mediate neutrophil–endothelial cell adhesive interactions [10]. Once in the tissue, activated leukocytes can subsequently enhance the localized inflammatory response by releasing toxic metabolites such as proteases and reactive oxygen species, resulting in damage to the surrounding tissue [11]. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion

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molecule-1 (VCAM-1) [12], C-reactive protein (CRP) [13-15] and serum amyloid A (SAA) [16] are associated with the vascular inflammatory process through various mechanisms. SAA has also been linked to high-density lipoprotein (HDL) metabolism and cholesterol transport and might overrule HDL's protective effect against atherosclerosis [17, 18]. ICAM-1, VCAM-1, CRP and SAA levels were all found to be increased in subjects with OSAHS compared to controls [19-21]. Such vascular injury markers act as surrogates of CVD risk, and therefore, any decrease with CPAP therapy could translate into a reduction in risk. The reported effects of CPAP on vascular marker levels in OSAHS are conflicting. In those with moderate to severe OSAHS, 6 months of CPAP treatment was associated with a significant decrease in CRP levels in those with initially high CRP levels and who had good compliance (CPAP use ≥4 h/night and >5 days/week) [22, 23]. A meta-analysis on the influence of CPAP therapy on CRP levels in OSAHS concluded that at least 3 months of treatment is required to significantly decrease levels [24], but a few more studies found no changes in CRP regardless of the CPAP duration (between 6 weeks and 9 months) [25-29]. Others report significant reduction in ICAM-1 levels after 8 and 12 months of CPAP treatment [30, 31]. In contrast, SAA levels remained unchanged after the first night of CPAP but decreased after 3 months of CPAP treatment only in subjects with severe OSAHS [21, 32]. We could not find reports on CPAP and VCAM-1 levels in OSAHS nor studies looking at diurnal variation in these vascular markers before and after treatment.

Aims and objectives The purpose of this study was to see if morning levels of the vascular injury markers (SAA, CRP, VCAM-1 and ICAM-1) are different in moderate/severe OSAHS compared to those of controls. Our secondary aims were to see if there was any diurnal variation in the baseline measurements of these vascular markers and to determine whether CPAP treatment affects morning levels of the markers in those with OSAHS only.

Methods Subjects We performed a prospective study on 40 consecutive subjects attending a SDB unit for the first time. All subjects were referred with symptoms suggestive of OSAHS, including excessive daytime sleepiness, snoring, choking during sleep and witnessed apnoeas.

This study was approved by the local research ethics committee, and each participant provided written informed consent. Following enrolment, subjects underwent a detailed evaluation that included a clinical history concentrating on sleep-related symptoms, co-morbidities and a physical examination. Their subjective daytime somnolence was partially determined using the Epworth sleepiness scale. Those with known cardiovascular illnesses (e.g. hypertension) were included in the research to limit the number of ineligible subjects. We included only those patients who did not have changes in their medications/illnesses during the study period. None of the subjects was on a long-term oxygen therapy or suffered from a severe respiratory disease. Exclusion criteria included refusal to provide written informed consent, personal or family history of prothrombotic or bleeding disorders, severe liver disease (clotting problems) and those prescribed warfarin or heparin, and age 80 years old. Limited-channel sleep study All subjects underwent overnight, inpatient, unattended limited-channel sleep study (Visilab, Oakwood Scientific, Oxford, UK), including video recording, measuring of thoraco-abdominal movements, finger pulse oximetry, sound monitoring of snoring level and a single modified type II electrocardiography lead for cardiac monitoring. An airflow measurement was not included, but all video recordings and tracings were manually reviewed by a sleep technician to confirm that episodes were predominantly obstructive rather than central. We have measured a mean nocturnal SaO2, percentage of total sleep time (%TST) spent with SaO2 10 events/h were classified as OSAHS. Those with symptoms but with a 4 % DR ≤10 events/h were classified as the comparator group. CPAP treatment Patients with OSAHS underwent a single night auto-CPAP titration at home to determine the appropriate mask pressure settings. Subsequently, subjects were issued with a Sullivan C8 CPAP machine (Resmed, Sydney, New South Wales, Australia) with a fixed predetermined pressure and asked to use a mask for at least 4 h/night for at least 5 days a week. Further equipment (humidifiers, masks and new headgear) was issued at the discretion of sleep technicians. OSAHS patients re-attended after 1 month of starting CPAP to check for symptom resolution, confirm resolution of DR on oximetry (while on CPAP) and calculate average nightly machine on time.

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Blood collection and analysis Blood samples were collected for CRP, SAA, ICAM-1 and VCAM-1 serum levels in the afternoon (4–5 p.m.) prior to and on the following morning (7–8 a.m.) immediately after the sleep study. Further samples were drawn again between 8 and 10 a.m. after the first night and then after 1 month of CPAP treatment in those with OSAHS. Venous citrated blood samples were centrifuged at 3,500 rpm for 10 min during which the platelet-poor plasma component was separated, put into aliquots, and then stored at –80C. Samples were analyzed by an experienced senior biotechnologist at the internationally accredited Central Biotechnology Services, Cardiff University School of Medicine in Cardiff. Vascular marker concentrations were run in duplicate and determined with the Meso Scale Discovery (MSD) Vascular Injury Panel-II assay (Meso Scale Discovery, Gaithersburg, MD, USA) using an electro-chemiluminescence detection system with multi-array technology. Multi-array plates are supplied by the manufacturer fitted with multi-electrodes per well with each electrode being coated with a different catching antibody. The plates were pre-incubated with 5 % Blocker A solution and left at room temperature for an hour. In the next step, they were washed three times with a mixture of buffered phosphate saline and 0.05 % Tween-20 (PBS-T). Following the pre-incubation, 10 μl of calibrator or diluted sample (1:200) was added in duplicate to the appropriate wells. The array was then incubated at room temperature for 2 h. After incubation, the plates were washed three times with PBS-T, and analyte-specific ruthenium-conjugated antibody was added. Following a further 1-h room-temperature incubation, the array was washed three times with PBS-T, and the Read Buffer T containing Triton X-100 was added to induce electrochemical reaction. The assay results were read immediately using MSD Sector Imager 6000. Vascular injury marker concentrations were determined with Discovery Workbench software as supplied by MSD. Intra-assay coefficients of variation were 3.7 % for VCAM-1, 3.2 % for ICAM-1, 2.4 % for CRP and 3.3 % for SAA. Calculated concentration mean was used to average out the results. Statistical analysis Statistical analysis was performed using SPSS version 20.0 software (SPSS Inc. Chicago, IL, USA). Continuous variables were checked for normality. Spearman's correlation coefficients were used to test measures of association, betweengroup differences were assessed using the independent t test or Mann–Whitney test, and within-group differences were measured using paired t test or Wilcoxon tests. Categorical data were analyzed using the chi-square test. All p values are reported as two-tailed with statistical significance set at

Endothelial injury markers before and after nasal continuous positive airway pressure treatment for obstructive sleep apnoea hypopnoea syndrome.

The purpose of this study was to evaluate whether serum amyloid A (SAA), C-reactive protein (CRP), vascular cell adhesion molecule-1 (VCAM-1) and inte...
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