ORIGINAL ARTICLE

MECHANISMS OF LUNG DISEASE

Intermittent hypoxia-activated
cyclooxygenase pathway: role in
atherosclerosis
Elodie Gautier-Veyret1,2, Claire Arnaud1, Magnus Bäck3, Jean-Louis Pépin1,4,5,
Marcelo H. Petri3, Jean-Philippe Baguet4,6, Renaud Tamisier1,4,5, Patrick Lévy1,4,5
and Françoise Stanke-Labesque1,2,4

Affiliations: 1INSERM, U1042, Grenoble, 2CHU, Hôpital A. Michallon, Laboratoire de Pharmacologie, BP217,
Grenoble, 4Université Grenoble 1, Faculté de Médecine, UFR1, Grenoble, 5CHU, Hôpital A. Michallon, Pôle
rééducation et Physiologie, BP217, Grenoble, 6CHU, Hôpital A. Michallon, Clinique de Cardiologie, BP217,
Grenoble, France, 3Dept of Medicine, Karolinska Institute and University Hospital, CMM L8, 03, Stockholm,
Sweden.

Correspondence: F. Stanke-Labesque, Laboratory of Pharmacology, Grenoble University Hospital, BP 217,

F-38043 Grenoble Cedex 9, France. E-mail: [email protected]

ABSTRACT Intermittent hypoxia, the main stimulus of obstructive sleep apnoea (OSA), induces
inflammation, leading to early atherosclerosis. Whether the cyclooxygenase (COX) pathway contributes to
intermittent hypoxia-induced atherosclerosis remains to be determined.
We studied the effects of 8-weeks of intermittent hypoxia exposure on COX-pathway gene expression and
atherosclerosis, and the influence of COX-1 inhibition by SC-560 on atherosclerosis progression in aortas of
apolipoprotein E-/- mice. Urinary 11-dehydrothromboxane B2 (11-dTXB2) was assessed in 50 OSA subjects
free of cardiovascular risk factor matched for age and body mass index with 25 controls, and 56 OSA with
cardiovascular risk factor.
Intermittent hypoxia significantly increased atherosclerotic lesion sizes, mRNA levels of COX-1 and
thromboxane synthase (TXBS). Lesion sizes correlated to COX-1 (r50.654, p50.0003) and TXBS (r50.693,
p,0.0001) mRNA levels. COX-1 inhibition reduced lesion progression in intermittent hypoxia mice only
(p50.04). Urinary 11-dTXB2 was similar in OSA subjects free of cardiovascular risk factor and controls, but
was increased by 13% (p50.007) in OSA subjects with cardiovascular risk factor compared with those
without.
Although OSA itself was not associated with increased urinary 11-dTXB2 concentration, the COX-1
pathway was activated in intermittent hypoxia-exposed mice and in OSA subjects presenting with
cardiovascular risk factor, and may contribute to intermittent hypoxia-induced atherogenesis. COX-1
inhibition could be of clinical interest in the prevention of cardiovascular morbidity in OSA.

@ERSpublications
COX-1 inhibition could be of clinical interest in the prevention of cardiovascular morbidity in
obstructive sleep apnoea http://ow.ly/lUIb5

This article has supplementary material available from www.erj.ersjournals.com

Received: June 18 2012 | Accepted after revision: Sept 26 2012 | First published online: Oct 11 2012

Clinical trial: The study is registered at www.clinicaltrials.gov with identifier number NCT01089257.
Support statement: This study was supported by a grant from ‘‘le vivier de la recherche médicale’’ from Grenoble
University of Medicine, PHRC 2010, ResMed Foundation, Mairie de Paris (‘‘Research in Paris’’), The French-Swedish
Foundation and the Swedish Heart and Lung Foundation.
Conflict of interest: None declared.
Copyright ßERS 2013

404 Eur Respir J 2013; 42: 404–413 | DOI: 10.1183/09031936.00096512

 MECHANISMS OF LUNG DISEASE | E. GAUTIER-VEYRET ET AL.

Introduction
Obstructive sleep apnoea (OSA) is characterised by recurrent episodes of partial or complete upper airway
obstruction occurring during sleep, leading to chronic intermittent hypoxia (CIH). This hallmark is the
main factor involved in cardiovascular remodelling in OSA [1]. In OSA patients, early signs of
atherosclerosis correlated to hypoxia severity [2], even after adjustment for confounding factors. OSA is
associated with increased cardiovascular morbidity and mortality, and is identified as an independent
cardiovascular risk factor (CVRF) [3]. Moreover, exposure of apolipoprotein E-deficient (ApoE-/-) mice [4, 5]
and C57BL/6J mice [6] to CIH accelerated atherogenesis progression.
Atherosclerosis is a chronic inflammatory disease. Among the many inflammatory mediators involved in
atherogenesis, we previously demonstrated the contribution of arachidonic acid-derived metabolites in OSA
patients. Notably, OSA patients exhibited elevated levels of leukotriene B4 [7, 8] and cysteinyl-leukotrienes
[9] that were associated with vascular remodelling. However, the cyclooxygenase (COX)-dependant
pathway of arachidonic acid metabolism was poorly studied in OSA patients, and its implication in CIH-
induced atherogenesis is unknown.
Thromboxane A2 (TXA2) and prostacyclin (PGI2) are two COX-derived metabolites of arachidonic acid
metabolism. TXA2 is predominantly generated by platelets through COX type 1 isoform (COX-1) and
thromboxane synthase (TXBS). TXA2 is quickly metabolised to thromboxane B2 (TXB2) and 11-
dehydrothromboxane B2 (11-dTXB2), two metabolites respectively quantifiable in plasma and urine. TXA2
binding on its TP receptors induces platelet activation, vasoconstriction, vascular smooth muscle cell
proliferation and increases expression of adhesion molecules [10]. The production of PGI2 mainly depends on
endothelial COX-2 and prostacyclin synthase (PGIS). PGI2 is rapidly metabolised to 6-keto-prostaglandin F1a
(6-keto-PGF1a in plasma and 2,3-dinor-6-ketoprostaglandin F1a (PGI-M) excreted in urine. In contrast to
TXA2, PGI2 binding on its IP receptors inhibits platelet aggregation and vasoconstriction, and reduces
chemotaxis and expression of adhesion molecules [10]. Thus, TXA2 and PGI2 have antagonist properties.
A recent growing body of evidence suggests a major role of the COX pathway in the pathogenesis and
progression of atherosclerosis. In fact, pharmacological inhibition of COX pathway [11, 12] or genetic
deletion [13, 14] delayed atherosclerosis in different mouse models of atherosclerosis. Furthermore, the
urinary 11-dTXB2/PGI2-M ratio is enhanced in elderly subjects with past history of serious cardiovascular
event (stroke and myocardial infarction) [15]. As selective COX-2 inhibitors may have nonfavourable
effects on this ratio and increase cardiovascular risk [16], the role of COX-1 inhibition has received less
attention. In addition, data in OSA are very limited and conflicting, showing either an increased [17] or a
decreased [18] urinary ratio 11-dTXB2/PGI2-M. Thus, the aim of the present study was to characterise the
COX pathway in ApoE-/- mice exposed to CIH and in OSA patients, and to investigate its link with CIH-
induced atherogenesis and OSA-associated early vascular remodelling.

Materials and methods

Experimental animal study
Male ApoE-/- mice (14 weeks old) were purchased from the Charles River Laboratories (L’Arbresle, France).
All animal procedures were conducted in accordance with the European Convention for the Protection of
Vertebrate Animals used for Experimental and Other Scientific Purposes (Council of Europe, European
Treaties ETS 123, Strasbourg, March 18, 1986) and to the Guide for the Care and Use of Laboratory
Animals (NIH Publication no. 85–23, revised 1996).
In a first series of experiments, mice were randomised to 8 weeks of CIH (cyclic 21–25% inspiratory oxygen
fraction (FIO2), 60-s cycle for 8 h?day-1) or normoxic air as previously described (n515 in each group) [1].
In a second series of experiments, mice were exposed to 8 weeks of CIH or normoxic air (n520 in each
group), and randomised to receive either placebo or the selective COX-1 inhibitor SC-560 (Interchim,
Montluçon, France) (15 mg?kg-1 daily [12]) in their food, for the last 4 weeks of exposure. All mice were fed
with a normal diet, ad libitum, during all experiments.
At the end of exposure, blood was collected under anaesthesia (ketamine/xylazine 100 mg?kg-1/10 mg?kg-1
by intraperitoneal injection) for haematocrit and lipid measurements. Entire aortas were harvested.
Abdominal aortas were placed in RNAlater (Life Technologies, Villebon-sur-Yvette, France), frozen in
liquid nitrogen and stored at -80uC until analysis. Immediately after their sampling, thoracic aortas were
placed in Tyrode solution (137 mM NaCl, 2.7 mM KCl, 0.41 mM NaHPO4, 2 mM CaCl2, 5 mM MgCl2,
11.9 mM NaHCO3 and 5.5 mM glucose) for prostanoid secretion measurements.

Atherosclerotic lesion size quantification

Atherosclerotic lesions of aortic roots were studied by Oil-Red-O (Sigma Aldrich, Saint Quentin-Fallavier,
France) staining, as previously described [5]. For each aorta, lipid deposition was quantified from five

DOI: 10.1183/09031936.00096512 405

MECHANISMS OF LUNG DISEASE | E. GAUTIER-VEYRET ET AL.

sections (8-mm thickness), separated by 80 mm from each other, using computer image analysis
(NisElement; Nikon Instruments Inc., Melville, NY, USA).

Aortic secretion of prostanoids

Thoracic aortas were incubated for 15 min in Tyrode solution maintained at 37uC, aerated with 95% O2
and 5% CO2, in presence of calcium ionophore A23187 (Sigma Aldrich, Saint Quentin Fallavier, France) at
10-6 M. Supernatants were immediately frozen at -80uC for prostanoid measurement, while aortas were
dried for measurement of dry tissue weight.

COX-pathway gene expression

Total mRNA was isolated from aorta using the RNeasy kit (Qiagen, Hilden, Germany) as previously
described [19] and reverse-transcribed using Superscript II (Invitrogen, Carlsbad, CA, USA) with random
hexamers according to the manufacturer’s instructions. Quantitative TaqMan PCR was performed on a
7900 HT using primer/probe pairs designed with Assay-On-Demand (both Applied Biosystems; Life
Technologies SAS, Saint Aubin, France) (online supplementary table S1). Data were normalised to 18S
ribosomal protein mRNA and expressed as 2-DCT.

Clinical study
The local ethics committee approved this study according to the Declaration of Helsinki. All participants
gave written informed consent.
113 newly diagnosed OSA patients were consecutively entered into the study between January 2007 and
April 2011, as well as 25 controls. Patients were referred to the sleep laboratory of Grenoble University
Hospital for symptoms suggesting OSA. Exclusion criteria were past history of stroke or myocardial
infarction, known hypertension, and treatment with nonsteroidal anti-inflammatories, aspirin, steroids,
antidiabetic, antihypertensive and lipid-lowering drugs.

Design
The specific contribution of CIH on 11-dTXB2 urinary concentrations was assessed in 25 controls
carefully matched for age and body mass index (BMI) with 50 nonobese OSA patients (i.e. one healthy
subject for two OSA subjects). All subjects were free of any known CVRFs. A sample size of 50 OSA
patients and 25 controls was calculated as adequate to show an increase of 30% in urinary 11-dTXB2
excretion with at least 90% power and at the 5% significance level. This sample size calculation was based
on preliminary data obtained on healthy subjects showing a mean concentration of 11-dTXB2 of
647 pg?mL-1 with a standard deviation of 287 pg?mL-1. As the dispersion of 11-dTXB2 was higher in
patients with cardiovascular disease than in controls, we chose a mixed model including one healthy for
every two OSA subjects.
The influence of CVRFs (which comprise obesity (BMI .30 kg?m-2); hypertension (clinical diastolic blood
pressure (BP) .90 mmHg and systolic BP .140 mmHg); dyslipidemia (low-density lipoprotein (LDL)
cholesterol .4.13 mmol?l-1 or association of total cholesterol .5.16 mmol?l-1 and high-density lipoprotein
(HDL) cholesterol ,1.03 mmol?l-1); smoking; and metabolic syndrome (defined by criteria of International
Diabetes Federation [20])) on urinary excretion of 11-dTXB2 was evaluated in the whole cohort of 113 OSA
subjects. Patients were stratified on the presence or not of one of these CVRFs.
The influence of continuous positive airway pressure (CPAP) on urinary 11-dTXB2 concentration was
studied in 14 OSA subjects free of any CVRF and 21 OSA patients with CVRF(s) who were adherent to
CPAP. CPAP adherence was defined as CPAP daily use for .4 h [9].
All subjects underwent an overnight polysomnography as described [9]. Sleep apnoea was defined as an
apnoea/hypopnoea index (AHI) o5 events?h-1 of sleep and symptoms or respiratory disturbance index
(RDI), including flow limitation episodes .15 events?h-1 [21].
Urine sample for 11-dTXB2 quantification and venous blood for biochemical measurements were collected
at 07:00 h at the end of the nocturnal polysomnographic recordings and were stored at -80uC until later
analysis.
Carotid ultrasonography was performed in 97 OSA patients and 24 controls as previously described [2].

Biological measurements
Plasma cholesterol, triglycerides, glucose and high-sensitivity C reactive protein (hsCRP) concentrations
were determined using enzymatic colorimetric methods on a Dimension Vista analyser (Siemens AG,
Erlangen, Germany). LDL cholesterol was calculated using the Friedewald formula (total cholesterol-HDL

406 DOI: 10.1183/09031936.00096512

 MECHANISMS OF LUNG DISEASE | E. GAUTIER-VEYRET ET AL.

cholesterol-(triglycerides/5)). Urinary 11-dTXB2 of patients, TXB2 and 6-keto-PGF-1a in supernatants of

mice were measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS) as previously
described [9, 22]. The limits of quantitation of TXB2 and 6-keto-PGF1a were 14 pg?mL-1 and 7.25 pg?mL-1,
respectively, and their inter- and intra-assay coefficients of variation were ,12% for both analytes.

Statistical analysis
Statistical analyses were performed using NCSS97 (Kaysville, Utah) or SAS (SAS 9.1, Cary, NC, USA) for the
mixed model. Data were expressed as median and 10th and 90th percentiles. Normal distribution was tested
using Kolmogorov–Smirnov nonparametric test. Comparisons between normoxic air and CIH mice were
made using the t-test. Comparisons between OSA patients and controls matched for age and BMI (one
control for two OSA subjects) were made using a mixed model. Noncontinuous variables were compared
using Fisher’s test. Comparisons between more than three groups were made using the Kruskal–Wallis test
and subsequent pairwise comparisons were made with the Bonferroni multiple-comparison test. For
patients treated by CPAP, differences between baseline and post-CPAP values were analysed using a paired
t-test or the Wilcoxon signed-rank test. Relationships between two variables were studied with single
regression. A multiple linear regression analysis was performed, taking into account the variables that
correlated with dependant variable urinary 11-dTXB2 in humans. A p-value of ,0.05 was considered
significant.

Results
Experimental animal study
Weight, lipid levels and haematocrit
After 8-weeks of exposure, bodyweight and plasma cholesterol levels were similar in CIH and normoxic air
mice (table 1). Haematocrit was significantly higher in CIH mice compared with normoxic air mice
(table 1).

Atherosclerotic lesion sizes

Atherosclerotic lesions on aortic roots were higher (p50.008) in CIH mice compared with normoxic mice
(CIH 66 532 (32 741–163 224) versus normoxic air 37 675 (6798–75 596) mm2; p50.03). Lesion sizes
correlated with plasma total cholesterol in normoxic air mice (r50.501, p50.04) but not in CIH mice.

COX-pathway gene expression

COX-1 and TXBS aortic mRNA levels were significantly increased in CIH group compared with normoxic
air, whereas COX-2, PGIS, TP and IP receptors aortic mRNA levels were not significantly different between
both groups (fig. 1a). mRNA levels of COX-1 and TXBS significantly correlated with lesion sizes (fig. 1b).

Aortic prostanoid secretion

A23187-stimulated 6-keto-PGF1a and TXB2 secretions and 6-keto-PGF1a/TXB2 ratio were similar in aorta
from CIH and normoxic air mice (table 2). 6-keto-PGF1a and TXB2 concentrations were highly correlated
(r50.842, p,0.0001).

Effects of COX-1 inhibition

In both normoxic air and CIH groups, treatment with SC-560 significantly decreased TXB2 and 6-keto-
PGF1a aortic secretion versus placebo (table 2). There was no difference between groups for total
cholesterol and bodyweight (table 2). Treatment with SC-560 significantly reduced lesion size by 35% in
CIH mice whereas it had no effect in normoxic air mice (fig. 2).

TABLE 1 Bodyweight, plasma cholesterol level and haematocrit of apolipoprotein E-deficient

(ApoE-/-) mice exposed for 8 weeks to chronic intermittent hypoxia (CIH) or normoxia

Normoxia CIH p-value

Subjects n 15 15
Bodyweight after exposure g 31.7 (28.5–33.9) 30.3 (27.8–32.6) 0.08
Total cholesterol mmol?L-1 12.2 (5.24–18.7) 14.6 (7.10–19.1) 0.46
Haematocrit % 45.0 (41.4–47.0) 47.0 (44.2–55.0) 0.006

Data are presented as median (10th–90th percentiles).

DOI: 10.1183/09031936.00096512 407

MECHANISMS OF LUNG DISEASE | E. GAUTIER-VEYRET ET AL.

a) 2.5 b) 250000 r=0.512

●
p=0.01
225000
2.0 * 200000
*
Fold change compared

Lesion size µm2

175000
●
with control

1.5 150000
125000
● ●
1.0 100000
75000 ● ● ●● ● ●
● ● ●
●
50000 ● ● ●
0.5 ● ●
●
●
25000 ●● ●
●
●●
0 0
Control COX-1 COX-2 TXBS PGIS TP IP 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
receptor receptor mRNA COX-1

c) 250000 r=0.602
●
p=0.003
225000
200000
Lesion size µm2

175000
●
150000
125000
● ●
100000
75000 ●● ●●
● ●
●●
50000 ● ●
● ● FIGURE 1 a) mRNA levels of COX-pathway genes in mice exposed to
● ●
● chronic intermittent hypoxia in the abdominal aorta. Regressions are shown
●
25000 ● ● ● between atherosclerotic lesion size and aortic mRNA levels of b) COX-1 and
● c) thromboxane synthase (TXBS). Data are expressed as fold-changes
● ●
0 compared with normoxic mice (control). *: p,0.05 versus normoxic group.
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 COX-1: cyclooxygenase type 1 isoform; COX-2: COX type 2 isoform; TXBS:
mRNA TXBS thromboxane synthase; PGIS: prostacyclin synthase.

TABLE 2 Bodyweight, plasma cholesterol level, haematocrit and aortic prostanoid secretion of apolipoprotein E-deficient
(ApoE-/-) mice exposed for 8 weeks to chronic intermittent hypoxia (CIH) or normoxia, with treatment by SC-560 or placebo
during the last 4 weeks of exposure

Normoxia CIH

Placebo SC-560 Placebo SC-560

Subjects n 10 9 10 10
Bodyweight after exposure g 28.8 (25.8–31.0) 28.9 (27.1–32.6) 28.9 (25.9–31.5) 31.2 (28.4–32.0)
Total cholesterol mmol?L-1 14.2 (9.13–20.2) 11.1 (8.44–17.6) 15.3 (12.8–21.0) 12.3 (8.46–16.1)
Haematocrit % 44.5 (42.5–45.0) 45.0 (41.8–47.0) 46.0 (45.0–47.6)# 45.5 (43.5–48.5)
Aortic secretion of TXB2 55.2 (35.3–94.7) 3.13 (1.80–22.6)" 56.7 (44.0–80.8) 7.86 (2.65–11.86)+
ng?L-1?mg-1
Aortic secretion of 6-keto-PGF1a 778.3 (559.7–1059.1) 91.8 (45.9–285.7)" 802.1 (638.3–1152.8) 107.9 (68.1–198.6)+
ng?L-1?mg-1
Aortic 6-keto-PGF1a/TXB2 ratio 13.4 (9.9–21.6) 24.1 (9.3–40.8) 14.0 (11.6–18.1) 17.0 (10.9–26.7)
#
Data are presented as median (10th–90th percentiles). TXB2: thromboxane; 6-keto-PGF1a: 6-keto-prostaglandin F1a. : p,0.05 normoxic mice
treated by placebo versus hypoxic mice treated by placebo; ": p,0.05 normoxic mice treated by placebo versus normoxic mice treated by SC-560;
+
: p,0.05 hypoxic mice treated by placebo versus hypoxic mice treated by SC-560.

408 DOI: 10.1183/09031936.00096512

 MECHANISMS OF LUNG DISEASE | E. GAUTIER-VEYRET ET AL.

b) c)
NS

p=0.004 p=0.04
a) 350000

300000
Lesion size µm2

NS
250000

200000
d) e)
150000

100000

50000

0
N placebo N SC-560 CIH placebo CIH SC-560
(n=10) (n=9) (n=10) (n=10)

FIGURE 2 Effects of cyclooxygenase type 1 isoform (COX-1) inhibition on atherosclerosis in mice exposed to chronic intermittent hypoxia (CIH) or normoxia
(N). a) Lesion sizes. Data are presented as interquartile range (boxes), data range (whiskers) and median (horizontal line). NS: nonsignificant. Representative
photographs of Oil-Red-O staining for b) normoxia placebo, c) normoxia SC-560, d) CIH placebo and e) CIH SC-560.

TABLE 3 Baseline characteristics of controls and obstructive sleep apnoea (OSA) patients free
of any known cardiovascular risk factor, matched for age and body mass index (BMI)

Controls OSA patients p-value

Subjects n 25 50
Males % 76.0 88.0 0.20
BMI kg?m-2 25.0 (21.6–27.3) 24.9 (21.8–27.9) 0.83
Age years 51.2 (42.7–62.6) 51.1 (43.8–60.9) 0.99
Clinical systolic BP mmHg 127 (112–148) 125 (110–146) 0.72
Clinical diastolic BP mmHg 85 (76–97) 80 (69–93) 0.29
AHI events?h-1 5.0 (0.0–9.2) 35.4 (14.9–59.4) ,0.0001
RDI events?h-1 6.6 (1.0–15.7) 35.9 (24.1–71.6) ,0.0001
Minimal SaO2 % 90.0 (85.4–92.5) 84.5 (75.3–90.0) ,0.0001
SaO2,90% % TST 0.00 (0.00–1.00) 1.00 (0.00–13.2) ,0.0001
RAI events?h-1 4.9 (1.9–11.0) 28.0 (12.0–49.1) ,0.0001
Plasma glucose mmol?L-1 4.7 (4.3–5.3) 5.1 (4.5–6.1) 0.03
Plasma insulin mUI?mL-1 4.2 (2.2–7.11) 5.48 (2.85–8.57) 0.11
HOMA-IR index 0.86 (0.50–1.68) 1.21 (0.63–2.22) 0.13
Total cholesterol mmol?L-1 4.77 (3.41–6.17) 5.68 (4.41–6.62) 0.004
LDL cholesterol mmol?L-1 2.76 (1.57–3.92) 3.55 (2.13–4.36) 0.003
HDL cholesterol mmol?L-1 1.52 (0.98–2.40) 1.52 (1.10–2.13) 0.94
Triglycerides mmol?L-1 0.89 (0.52–1.90) 1.11 (0.68–1.84) 0.07
hsCRP mg?L-1 1.00 (0.38–1.74) 0.95 (0.30–3.30) 0.61
Right carotid IMT mm 593 (515–653) 604 (508–805) 0.44
Left carotid IMT mm 622 (527–780) 647 (515–872) 0.32
Mean carotid IMT mm 606 (523–710) 620 (539–838) 0.52
Overweight/normal BMI % 48/52 46/56 0.87
Smoking % 0 0 1
Hypertension % 4 6 1
Metabolic syndrome % 0 0 1
Dyslipidemia % 0 20 0.03
Urinary 11-dTXB2 609.0 (265.8–945.3) 629.3 (266.4–933.1) 0.56
pg?mg-1 creatinine

Data are presented as median (10th–90th percentiles), unless otherwise stated. BP: blood pressure; AHI:
apnoea/hypopnoea index; RDI: respiratory disturbance index; SaO2: arterial oxygen saturation; TST: total sleep
time; RAI: respiratory arousal index; HOMA-IR: homeostasis model assessment of insulin resistance; LDL:
low-density lipoprotein; HDL: high-density lipoprotein; hsCRP: high-sensitivity C reactive protein; IMT: intima-
media thickness; 11-dTXB2: 11-dehydrothromboxane. Bold indicates statistical significance.

DOI: 10.1183/09031936.00096512 409

MECHANISMS OF LUNG DISEASE | E. GAUTIER-VEYRET ET AL.

TABLE 4 Baseline characteristics of obstructive sleep apnoea (OSA) patients without (CVRF-)
or with (CVRF+) associated cardiovascular risk factors

OSA

CVRF- CVRF+ p-value

Subjects n 57 56
Males % 84.5 84.0 1
BMI kg?m-2 25.6 (21.5–28.4) 28.2 (23.0–34.6) ,0.0001
Age years 51.3 (44.5–65.3) 53.1 (39.9–66.6) 0.72
Clinical systolic BP mmHg 124 (110–138) 132 (118–160) 0.0002
Clinical diastolic BP mmHg 80 (65–90) 88 (72–108) ,0.0001
AHI events?h-1 30.0 (17.0–50.0) 40.2 (19.4–67.0) 0.0004
RDI events?h-1 35.8 (24.3–64.3) 51.0 (27.7–80.3) 0.0002
Minimal SaO2 % 85.0 (76.0–90.0) 82.0 (74.9–88.2) 0.04
Mean SaO2 % 94.0 (92.0–96.0) 93.0 (90.0–95.0) 0.0009
SaO2,90% % TST 1.0 (0.0–11.5) 4.0 (0.0–35.0) 0.002
RAI events?h-1 27.9 (12.4–44.2) 35.0 (12.6–59.2) 0.003
Plasma glucose mmol?L-1 4.9 (4.5–5.9) 5.2 (4.7–6.4) 0.02
Plasma insulin mUI?mL-1 4.8 (2.7–9.0) 6.4 (3.7–12.2) 0.0002
HOMA-IR index 1.0 (0.6–2.1) 1.5 (0.9–3.1) 0.0001
Total cholesterol mmol?L-1 5.47 (4.02–5.91) 5.81 (4.45–6.73) 0.006
LDL cholesterol mmol?L-1 3.35 (2.01–3.95) 3.66 (2.59–4.55) 0.008
HDL cholesterol mmol?L-1 1.55 (1.14–2.19) 1.42 (0.98–1.91) 0.13
Triglycerides mmol?L-1 1.01 (0.62–1.59) 1.27 (0.79–2.11) 0.002
hsCRP mg?L-1 1.30 (0.20–3.42) 1.30 (0.60–3.74) 0.50
Right carotid IMT mm 596 (498–821) 602 (457–815) 0.66
Left carotid IMT mm 613 (512–876) 663 (527–897) 0.20
Mean carotid IMT mm 608 (520–848) 628 (531–848) 0.21
Obesity/overweight/normal 0/46/54 39/34/27 ,0.0001
BMI %
Smoking % 0 2 0.001
Hypertension % 0 25 ,0.0001
Metabolic syndrome % 0 30 ,0.0001
Dyslipidemia % 0 50 ,0.0001
Urinary 11-dTXB2 612.6 (289.0–866.3) 677.9 (389.5–1247.6) 0.008
pg?mg-1 creatinine

Data are presented as median (10th–90th percentiles), unless otherwise stated. BMI: body mass index; BP:
blood pressure; AHI: apnoea/hypopnoea index; RDI: respiratory disturbance index; SaO2: arterial oxygen
saturation; TST: total sleep time; RAI: respiratory arousal index; HOMA-IR: homeostasis model assessment of
insulin resistance; LDL: low-density lipoprotein; HDL: high-density lipoprotein; hsCRP: high-sensitivity C
reactive protein; IMT: intima-media thickness; 11-dTXB2: 11-dehydrothromboxane. Bold indicates statistical
significance.

Clinical study
11-dTXB2 and OSA
Baseline characteristics of the 25 controls and the 50 OSA patients matched for age and BMI are described
in table 3. There was no significant difference for plasma insulin, hsCRP, HDL cholesterol, homeostatic
model assessment insulin resistance index (HOMA-IR), BP, carotid intima-media thickness (IMT) and sex
ratio. As expected, polysomnographic parameters were different between OSA and control groups. Plasma
glucose, total and LDL cholesterol levels were significantly increased in OSA patients versus controls.
Urinary 11-dTXB2 was not significantly different between OSA patients and controls (table 3).

11-dTXB2, OSA and cardiovascular risk factor

Clinical, biological and polysomnographic parameters of OSA patients with or without CVRFs are shown in
table 4. The OSA group with CVRFs had polysomnographic parameters, BP, BMI, plasma triglycerides,
total and LDL cholesterol, glucose, insulin and HOMA-IR higher than the OSA group free of CVRFs
(table 4). Conversely, these two groups were similar with regard to age, sex ratio, carotid IMT, HDL
cholesterol and hsCRP (table 4). Urinary 11-dTXB2 was significantly increased in OSA patients with CVRFs
compared with OSA patients free of CVRFs (fig. 3a). In OSA patients with or without CVRFs, 11-TXB2

410 DOI: 10.1183/09031936.00096512

 MECHANISMS OF LUNG DISEASE | E. GAUTIER-VEYRET ET AL.

p=0.009 p=0.02
a) 1400 b) 1400

1200 1200

1000 1000

pg.mg-1 creatinine
pg.mg-1 creatinine

Urinary 11-dTXB2
Urinary 11-dTXB2

800 800

600 600

400 400

200 200

0 0
CVRF- CVRF+ Normal carotid Carotid wall
(n=57) (n=56) wall (n=48) hypertrophy (n=8)

FIGURE 3 a) Urinary 11-dehydrothromboxane B2 (11-dTXB2) concentrations in obstructive sleep apnoea (OSA) patients with (CVRF+) and without (CVRF-)
known cardiovascular risk factor. b) Urinary 11-dTXB2 concentrations in OSA patients with or without carotid wall hypertrophy. Data are presented as
interquartile range (boxes), data range (whiskers) and median (horizontal line).

levels were similar in patients with mild-to-moderate (AHI ,30 events?h-1) and severe OSA (AHI
o30 events?h-1) (data not shown).
To determine the CVRF involved in the increase of urinary 11-dTXB2, simple regressions were performed.
Hypertension (r50.190, p50.05) and obesity (r50.242, p50.01) were weakly associated with increased
urinary excretion of 11-dTXB2. In multiple regression model, obesity remained the sole independent
predictive factor of urinary 11-dTXB2 (r50.257, p50.04).

Effect of CPAP treatment on urinary 11-dTXB2 concentration

CPAP treatment for at least 8 weeks significantly decreased AHI, RDI and respiratory arousal index,
increased minimal nocturnal arterial oxygen saturation (SaO2) and mean nocturnal Sa,O2, and decreased the
percentage of time spent with a mean SaO2 ,90%. CPAP treatment induced no change on 11-dTXB2
urinary concentration in OSA without or with CVRF (online supplementary tables S2 and S3).

11-dTXB2, OSA and vascular remodelling

In OSA patients free of CVRF, vascular hypertrophy (defined by carotid IMT .0.8 mm) was associated
with increased urinary 11-dTXB2 concentrations compared with subjects without vascular hypertrophy
(p50.02) (fig. 3b).

Discussion
Our study demonstrated for the first time an activation of the COX pathway in ApoE-/- mice exposed to
CIH and also in OSA patients with other CVRFs; this activation is associated with increased atherosclerotic
lesions in mice and with early markers of atherosclerosis in OSA patients.
We showed that CIH mice had higher atherosclerotic lesions than normoxic air mice, suggesting that, in our
model, CIH might have accelerated atherosclerosis development. This result is in accordance with previous
works in ApoE-/- mice exposed to CIH from 2 to 4 weeks [4, 5] or in C57BL/6J mice exposed to CIH for
12 weeks [6].
In the present study, plasma cholesterol levels were not significantly different between normoxic air and
CIH groups and atherosclerotic lesion sizes correlated with plasma cholesterol in normoxic air mice, but not
in CIH mice. These data suggested that, in our model, CIH-induced atherosclerosis might be independent
of lipid disorders, and contrast with previous works showing that concomitant exposure to a high-fat high-
cholesterol diet and CIH aggravate both atherosclerosis and dyslipidemia in ApoE-/- mice [4] and in C57BL/
6J mice [6, 23]. However, we recently showed that CIH also exerts pro-atherogenic effects through other
contributing factors, notably the inflammatory process [5]. In agreement with this later hypothesis, our data
showed an activation of the thromboxane pathway in CIH mice, as mRNA levels of COX-1 and TXBS were
increased in aortic tissue of CIH mice. Furthermore, the correlation between these mRNA levels and
atherosclerotic lesion size was consistent with direct effects of TXA2 on macrophages [24]. Unexpectedly,
the aortic secretion of TXB2 and 6-keto-PGF1a, as well as the TXB2/6-keto-PGF1a ratio were similar in CIH
and normoxic air mice. These results could be explained by the fact that we measured prostanoid secretion

DOI: 10.1183/09031936.00096512 411

MECHANISMS OF LUNG DISEASE | E. GAUTIER-VEYRET ET AL.

after aorta stimulation with A23187, and not the basal aortic production. A previous study performed in
LDL r-knockout (r-KO) mice demonstrated that the acceleration of atherogenesis in response to high fat
diet was associated with increased basal levels of TXB2 and 6-keto-PGF1a in the aortic arch [25], but
measurements of prostanoids were performed by ELISA, which is not a specific method.
We chose to measure TXB2 and 6-keto-PGF1a upon aorta stimulation with A23187 to obtain detectable
prostanoid levels by our analytical technique (LC-MS/MS), which is highly specific but probably less sensitive.
However, in our model, A23187 appears to stimulate TXB2 and 6-keto-PGF1a release to the same extent, as
both levels were highly correlated, which could explain the similar TXB2/6-keto-PGF1a ratio in aorta from CIH
and normoxic air mice. We acknowledge that comparisons of basal TXB2 and 6-keto-PGF1a production and
Western blot analysis of COX-1 and TXBS would have been of interest with regard to the increased mRNA
levels of COX-1 and TXBS induced by CIH, and that their absence may represent a limitation of our study.
Moreover, treatment with the selective COX-1 inhibitor SC-560 during the last 4 weeks of CIH exposure
reduced atherosclerosis progression in CIH mice, providing further evidence for a CIH-dependent
activation of the COX pathway. We already demonstrated that 4 weeks of CIH exposure were sufficient to
induce atherosclerosis in ApoE-/- mice [5], thus it was of interest to explore the effects of COX-1 inhibition
on established atherosclerotic lesions. As previously described [12], treatment with SC-560 had no effect on
established atherosclerotic lesions in normoxic air mice, although it was effective in inhibiting COX-1
pathway as assessed by the measurement of aortic TXB2 and 6-keto-PGF1a secretion. Collectively, these
data demonstrated that in ApoE-/- mice, the atherogenic process was accelerated by CIH exposure, at least in
part through COX-1 pathway activation. CIH activates leukocytes [26] and OSA patients display leukocyte
activation [7, 8, 27]. By regulating the interaction between leukocytes, smooth muscle cells and endothelial
cells, TXA2 promotes and PGI2 prevents the initiation and progression of atherogenesis [14].
To extend the thromboxane-pathway activation observed in CIH mice to OSA patients, we measured the
urinary excretion of 11-dTXB2, a validated biomarker of systemic TXA2 production [10]. Urinary 11-dTXB2
concentrations of OSA patients free of CVRF were not different to those of controls carefully matched for
age and BMI, two major confounding factors often present in studies focused on the underlying
inflammation associated to OSA. These data suggested that OSA itself was not associated with an increased
urinary 11-dTXB2 excretion, an hypothesis confirmed by the observation that CPAP treatment had no
influence on urinary 11-dTXB2 level. However, increased urinary 11-dTXB2 concentrations have previously
been described in patients with cardiovascular diseases including obesity [28] and hypertension [29].
Consistent with these data, we showed that OSA patients with an associated CVRF had higher urinary
concentrations of 11-dTXB2 than OSA patients free of a known CVRF. Furthermore, among the studied
CVRFs, obesity was the sole independent predictor of urinary 11-dTXB2 excretion, which is in agreement
with a previous study demonstrating an increased urinary 11-dTXB2 excretion in obese females [28]. This
result provided further evidence for the major role of obesity in arachidonic acid metabolism activation in
OSA patients, as we previously described for the 5-lipooxygenase pathway [9]. Finally, urinary 11-dTXB2
excretion was associated with carotid wall hypertrophy in OSA patients. Again, these findings were
consistent with the proliferative effects of TXA2 on vascular smooth muscle cells [10], although it must be
kept in mind that vascular remodelling is a complex process implying many mediators and that COX
pathway activation might not be the sole mechanism of vascular remodelling in OSA patients with CVRFs.
In conclusion, our study showed an activation of the COX-1 pathway in ApoE-/- mice exposed to CIH,
which contributed to the acceleration of the atherogenic process induced by this stimulus. Such an
activation of the COX-1 pathway was also found in OSA patients with associated CVRFs. This study extends
the activation of the arachidonic acid metabolism previously described for the leukotriene pathway [7, 9] to
the COX-1 pathway in OSA in relation to vascular remodelling. These findings open the field to the interest
of new pharmacological approaches (dual COX and 5-lipoxygenase inhibitor [30]) in the prevention of
cardiovascular morbidity in OSA patients.

Acknowledgements
The authors thank Nathalie Arnold (CHU, Hôpital A. Michallon, Pôle rééducation et Physiologie, BP217, Grenoble,
France) for statistical analyses, Jean-François Jourdil, Karine Scalabrino, Cécile Girard (CHU, Hôpital A. Michallon,
Laboratoire de Pharmacologie, BP217, Grenoble) and Sandrine Cachot (INSERM, U1042, Grenoble) for expert technical
assistance.

References
1 Dematteis M, Godin-Ribuot D, Arnaud C, et al. Cardiovascular consequences of sleep-disordered breathing:
contribution of animal models to understanding the human disease. ILAR J 2009; 50: 262–281.
2 Baguet JP, Hammer L, Lévy P, et al. The severity of oxygen desaturation is predictive of carotid wall thickening and
plaque occurrence. Chest 2005; 128: 3407–3412.

412 DOI: 10.1183/09031936.00096512

 MECHANISMS OF LUNG DISEASE | E. GAUTIER-VEYRET ET AL.

3 Somers VK, White DP, Amin R, et al. Sleep apnea and cardiovascular disease: an American Heart Association/
American College of Cardiology Foundation Scientific Statement from the American Heart Association Council for
High Blood Pressure Research Professional Education Committee, Council on Clinical Cardiology, Stroke Council,
and Council on Cardiovascular Nursing. J Am Coll Cardiol 2008; 52: 686–717.
4 Jun J, Reinke C, Bedja D, et al. Effect of intermittent hypoxia on atherosclerosis in apolipoprotein E-deficient mice.
Atherosclerosis 2010; 209: 381–386.
5 Arnaud C, Poulain L, Lévy P, et al. Inflammation contributes to the atherogenic role of intermittent hypoxia in
apolipoprotein-E knock out mice. Atherosclerosis 2011; 219: 425–431.
6 Savransky V, Nanayakkara A, Li J, et al. Chronic intermittent hypoxia induces atherosclerosis. Am J Respir Crit Care
Med 2007; 175: 1290–1297.
7 Lefebvre B, Pépin JL, Baguet JP, et al. Leukotriene B4: early mediator of atherosclerosis in obstructive sleep apnoea?
Eur Respir J 2008; 32: 113–120.
8 Stanke-Labesque F, Pepin JL, de Jouvencel T, et al. Leukotriene B4 pathway activation and atherosclerosis in
obstructive sleep apnea. J Lipid Res 2012; 53: 1944–1951.
9 Stanke-Labesque F, Bäck M, Lefebvre B, et al. Increased urinary leukotriene E4 excretion in obstructive sleep apnea:
effects of obesity and hypoxia. J Allergy Clin Immunol 2009; 124: 364–370.
10 Capra V, Bäck M, Barbieri SS, et al. Eicosanoids and their drugs in cardiovascular diseases: focus on atherosclerosis
and stroke. Med Res Rev 2013; 33: 364–438.
11 Cyrus T, Sung S, Zhao L, et al. Effect of low-dose aspirin on vascular inflammation, plaque stability, and
atherogenesis in low-density lipoprotein receptor-deficient mice. Circulation 2002; 106: 1282–1287.
12 Belton OA, Duffy A, Toomey S, et al. Cyclooxygenase isoforms and platelet vessel wall interactions in the
apolipoprotein E knockout mouse model of atherosclerosis. Circulation 2003; 108: 3017–3023.
13 McClelland S, Gawaz M, Kennerknecht E, et al. Contribution of cyclooxygenase-1 to thromboxane formation,
platelet-vessel wall interactions and atherosclerosis in the ApoE null mouse. Atherosclerosis 2009; 202: 84–91.
14 Kobayashi T, Tahara Y, Matsumoto M, et al. Roles of thromboxane A(2) and prostacyclin in the development of
atherosclerosis in apoE-deficient mice. J Clin Invest 2004; 114: 784–794.
15 Montine TJ, Sonnen JA, Milne G, et al. Elevated ratio of urinary metabolites of thromboxane and prostacyclin is
associated with adverse cardiovascular events in ADAPT. PLoS One 2010; 5: e9340.
16 Bäck M, Yin L, Ingelsson E. Cyclooxygenase-2 inhibitors and cardiovascular risk in a nation-wide cohort study after
the withdrawal of rofecoxib. Eur Heart J 2012; 33: 1928–1933.
17 Krieger J, Benzoni D, Sforza E, et al. Urinary excretion of prostanoids during sleep in obstructive sleep apnoea
patients. Clin Exp Pharmacol Physiol 1991; 18: 551–555.
18 Kimura H, Niijima M, Abe Y, et al. Compensatory excretion of prostacyclin and thromboxane metabolites in
obstructive sleep apnea syndrome. Intern Med 1998; 37: 127–133.
19 Bäck M, Sultan A, Ovchinnikova O, et al. 5-Lipoxygenase-activating protein: a potential link between innate and
adaptive immunity in atherosclerosis and adipose tissue inflammation. Circ Res 2007; 100: 946–949.
20 Holt RI. International Diabetes Federation re-defines the metabolic syndrome. Diabetes Obes Metab 2005; 7: 618–620.
21 Hosselet J, Ayappa I, Norman RG, et al. Classification of sleep-disordered breathing. Am J Respir Crit Care Med
2001; 163: 398–405.
22 Casetta B, Vecchione G, Tomaiuolo M, et al. Setting up a 2D-LC/MS/MS method for the rapid quantitation of the
prostanoid metabolites 6-oxo-PGF(1a) and TXB2 as markers for hemostasis assessment. J Mass Spectrom 2009; 44:
346–352.
23 Li J, Thorne LN, Punjabi NM, et al. Intermittent hypoxia induces hyperlipidemia in lean mice. Circ Res 2005; 97:
698–706.
24 Caughey GE, Pouliot M, Cleland LG, et al. Regulation of tumor necrosis factor-a and IL-1b synthesis by
thromboxane A2 in nonadherent human monocytes. J Immunol 1997; 158: 351–358.
25 Cyrus T, Ding T, Praticò D. Expression of thromboxane synthase, prostacyclin synthase and thromboxane receptor
in atherosclerotic lesions: correlation with plaque composition. Atherosclerosis 2010; 208: 376–381.
26 Arnaud C, Beguin PC, Lantuejoul S, et al. The inflammatory preatherosclerotic remodeling induced by intermittent
hypoxia is attenuated by RANTES/CCL5 inhibition. Am J Respir Crit Care Med 2011; 184: 724–731.
27 Lavie L. Oxidative stress inflammation and endothelial dysfunction in obstructive sleep apnea. Front Biosci (Elite
Ed) 2012; 4: 1391–1403.
28 Davì G, Guagnano MT, Ciabattoni G, et al. Platelet activation in obese women: role of inflammation and oxidant
stress. JAMA 2002; 288: 2008–2014.
29 Minuz P, Patrignani P, Gaino S, et al. Determinants of platelet activation in human essential hypertension.
Hypertension 2004; 43: 64–70.
30 Choi JH, Jeon HJ, Park JG, et al. Anti-atherogenic effect of BHB-TZD having inhibitory activities on
cyclooxygenase and 5-lipoxygenase in hyperlipidemic mice. Atherosclerosis 2010; 212: 146–152.

DOI: 10.1183/09031936.00096512 413