leukotriene b4 pathway activation and atherosclerosis in
TRANSCRIPT
Leukotriene B4 pathway activation and atherosclerosis
in obstructive sleep apnea
Françoise Stanke-Labesque,1,2,3, Jean-Louis Pépin 1,2,4, Tiphaine de Jouvencel 5, Claire
Arnaud1, Jean-Philippe Baguet2,6, Marcelo H. Petri7, Renaud Tamisier 1,2,4, Jean François
Jourdil3, Patrick Lévy 1,2,4, Magnus Bäck7,8
1INSERM, U1042, Grenoble, F-38042 France, 2Université Grenoble 1, Faculté de Médecine,
IFR1, Grenoble, F-38042 France, 3CHU, Hôpital A. Michallon, Laboratoire de
pharmacologie, BP217, Grenoble, F-38043, 4CHU, Hôpital A. Michallon, Pôle réeducation et
Physiologie, BP217, Grenoble, F-38043 France , 5INSERM U698, Paris 7 Denis Diderot
University, Bichat University Hospital, 75018 Paris, France, 6CHU, Hôpital A. Michallon,
Clinique de cardiologie, BP217, Grenoble, F-38043 France , 7Department of Medicine,
Karolinska Institutet, 17176 Stockholm, Sweden and 8Department of Cardiology, Karolinska
University Hospital, CMM L8:03, 17176 Stockholm, Sweden
Corresponding authors. Françoise Stanke-Labesque
Laboratory of Pharmacology, Grenoble University Hospital
BP 217, F-38043 Grenoble Cedex 9, France,
Tel: +33-4-76765492. Fax: +33-4-76768938.
E-mail: [email protected]
Magnus Bäck
Karolinska University Hospital, CMM L8:03
17176 Stockholm, Sweden
Tel: +46-8-51770000. Fax: +33-8-313147.
E-mail: [email protected]
Running title: LTB4 and atherosclerosis in sleep apnea
Clinical Trial registration: NCT01089257
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Abstract
Leukotriene B4 (LTB4) production increases in obstructive sleep apnea syndrome (OSA) and
is linked to early vascular remodeling, the mechanism of which is unknown. The objective of
this study was to to determine the molecular mechanisms of LTB4 pathway activation in
polymorphonuclear cells (PMNs) and early vascular remodeling in OSA, and the specific
contribution of intermittent hypoxia (IH). PMNs were isolated from 120 OSA patients and 33
healthy subjects and used for measurements of LTB4 production, determination of mRNA and
protein expression levels, or exposed for 4 cycles of in vitro IH. PMNs derived from OSA
patients exhibited increased LTB4 production, for which apnea-hypopnea index was an
independent predictor (P=0.042). Five lipoxygenase activating protein (FLAP) mRNA and
protein increased significantly in PMNs from OSA patients versus controls and were
associated with carotid luminal diameter and intima-media thickness. LTB4 (10 ng/mL)
increased IL-6 (P=0.006) and MCP-1 (P=0.002) production in OSA patient monocytes. In
vitro exposure of PMNs from controls to IH enhanced FLAP mRNA levels (P= 0.027) and
induced a 2.7-fold increase (P=0.028) in LTB4 secretion compared to PMNs exposed to
normoxia. In conclusion, upregulation of FLAP in PMNs in response to IH may participate in
early vascular remodeling in OSA patients, suggesting FLAP as a potential therapeutic target
for the cardiovascular morbidity associated with OSA.
Abstract word count: 215
Supplementary keywords: Arachidonic acid, Eicosanoids, Granulocytes, Intermittent
hypoxia, Lipoxygenase, Vascular remodeling
This article has online supplementary data.
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Abbreviations
AHI, apnea-hypopnea index
5-LO, 5-lipoxygenase
BMI, body mass index
CysLTs, cysteinyl leukotrienes
CPAP, continuous positive air pressure
DBP, diastolic blood pressure
FLAP, 5-lipoxygenase-activating protein
LC-MS/MS, liquid chromatography/tandem mas spectrometry
LTs, leukotrienes
OSA, obstructive sleep apnea
RAI, respiratory-related arousal index
RDI, respiratory disturbance index
SaO2, oxygen saturation
SBP, systolic blood pressure
TST, total sleep time
hsCRP, high-sensitivity C-reactive protein
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Introduction
Obstructive sleep apnea (OSA) patients experience recurring episodes of partial or complete
upper airway obstruction during sleep. Upper airway collapse is usually associated with
desaturation-reoxygenation, harming the cardiovascular system. Moderate to severe OSA
patients show signs of early atherosclerosis (1-3).
Recent studies have associated the inflammatory mediators leukotrienes (LTs), with both
OSA (4, 5, 6 , 7) and atherosclerosis (8, 9). However, the mechanisms involved in the role of
LTs as a link between OSA and atherosclerosis have remained largely unexplored.
LTs are synthesized on activation of 5-lipoxygenase (5-LO) (10), which interacts with nuclear
membrane-bound 5-LO-activating protein (FLAP) (11), generating leukotriene A4 (LTA4) in
inflammatory cells (12). In polymorphonuclear neutrophils (PMNs), LTA4 is converted by
LTA4 hydrolase (LTA4H) into LTB4 which modulates transcription or is secreted to mediate
autocrine or paracrine effects through the BLT1 and BLT2 receptors. LTB4 is a potent
chemoattractant, facilitating leukocyte adhesion to endothelial cell and recruitment - critical
steps in atherosclerosis. Thus, LTB4 function in the pathogenesis of atherosclerosis is well
established (8, 13).
In addition to monocytes and macrophages, PMNs mediate the onset and progression of
atherosclerosis (14). As the principal source of LTB4, the capacity of PMNs to produce LTB4
is measured by ex vivo production of LTB4 in response to calcium ionophore; LTB4
production by stimulated PMNs rises in patients with a history of myocardial infarction (15)
and in nonobese cardiovascular disease-free OSA patients (4).
Recent findings suggest that intermittent hypoxia (IH) induces LTB4 activation in monocytes
in vitro and that the LTB4 pathway contributes in the development of atherosclerosis in
chronic IH-exposed ApoE-/- mice (16). However, the effects of OSA on LTB4 pathway
transcription and atherosclerosis have not been examined in humans.
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The aims of the present study were: 1) to examine PMN production of LTB4 in a large cohort
of OSA patients reflecting routine clinical practice; 2) to determine the molecular mechanisms
of LTB4 pathway activation and early vascular remodeling in OSA; 3/ to study the
consequences of LTB4 pathway activation in OSA in terms of paracrine effect on monocytes,
another cellular type greatly involved in atherogenesis and 4) to explore the contribution of
the hypoxic component of OSA on LTB4 pathway activation in isolated PMNs exposed to in
vitro IH.
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Methods
Patients
This study was approved by the local ethics committee per the Declaration of Helsinki. All
participants gave written informed consent. Patients were referred to the Grenoble University
Hospital sleep laboratory for suspicion of OSA. Controls were healthy volunteers who were
free of inflammatory and sleep disorders. All subjects underwent a full polysomnography,
which was scored blinded to biological parameters, and biochemical measures were analyzed
blinded to these results. A flow chart detailing patient inclusion in the different experiments is
shown in Figure 1.
The exclusion criteria were: cancer, infectious or autoimmune disease, diabetes mellitus,
disease that potentially affected blood pressure, atrial fibrillation or frequent premature beats
(>10/min), shift work, asthma, chronic obstructive pulmonary disease, atopy, rhinitis,
arthritis, oral appliances, maxillofacial surgery, and pharmacological treatments that could
affect LT concentration, including non steroidal anti-inflammatory drugs, corticosteroids.
Nocturnal polysomnography was performed in all subjects as described (5). Sleep apnea was
defined as apnea-hypopnea index (AHI) >5 per hour of sleep and respiratory disturbance
index (RDI), including flow limitation episodes >15 and symptoms (17). OSA was considered
mild (5>AHI<15), moderate (15>AHI<30), or severe (>30).
Subjects were enrolled regardless of previous cardiovascular medical history, hence reflecting
routine clinical activity. They were non-overlapping with cardiovascular free subjects
included in our previous study (4).
After the nocturnal polysomnography, peripheral blood was sampled. Plasma glucose and
serum triglycerides levels were measured on an automat (Modular 700, Roche, Meylan,
France). Serum insulin was measured by radioimmunometric sandwich assay (CIS bio
international, Gif-Sur-Yvette, France). Serum hs-CRP was measured by automated
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immunonephelometry (Behring Nephelometer II Analyzer, Dade Behring, Germany). Carotid
ultrasonography was performed in 29 OSA and 4 controls as described (1).
PMN preparation
Human PMNs were isolated as previously described (4) and used for one of the following
protocols: 1) PMNs were resuspended in PBS, pH 7.4 containing 0.133 g/l CaCl2 and 0.1 g/l
Mg2+ for A23187 stimulation (68 OSA patients and 19 controls); 2) resuspended in RNAlater
(Ambion) for RT-PCR (25 OSA patients and 9 controls); or 3) resuspended in PBS with
protease inhibitors (Sigma) for Western blot (12 OSA patients and 5 controls).
LTB4 production in A23187-treated PMNs
LTB4 production by PMNs was measured on stimulation with A23187 (4). Viability exceeded
98% by Trypan blue exclusion method. PMNs (2x106 cells/ml) were incubated for 15 min at
37°C with 10 µmol/l A23187 or vehicle (PBS). In some experiments, PMNs were incubated
with the 5-LO inhibitor AA861 (10 µM) or the FLAP inhibitor MK886 (10 µM) for 30 min
before A23187 stimulation. Incubations were stopped by centrifugation (5000 rpm) for 5 min
at 4°C, and supernatants were stored at -80°C until use.
LTB4 was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as
previously described (4). This method enabled the adequate chromatographic separation of
isomeric non-enzymatic products 6-trans-LTB4 and 6-trans-12-epi LTB4 from enzymatically-
generated LTB4 as shown in Figure 2.
FLAP, 5-LO, LTA4H, and BLT1 and BLT2 receptor mRNA levels in PMNs
Total mRNA was isolated from PMNs using the RNeasy kit (Qiagen) as described (18) and
reverse-transcribed using Superscript II (Invitrogen, Carlsbad, CA) with random hexamers per
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the manufacturer`s instructions. Quantitative TaqMan PCR was performed on a 7900HT
using primer/probe pairs designed with Assay-On-Demand™ (both Applied Biosystems), as
indicated in Supplementary Table 1. Data were normalized to cyclophilin A and GAPDH
mRNA and expressed as 2-CT.
FLAP and LTA4H proteins expression in PMNs from OSA
PMNs were subjected to 3 freeze/thaw cycles in liquid nitrogen and ultracentrifuged for 30
min at 100,000 g. The supernatant (cytosolic extract) was collected, and the pellet was
resuspended in 500 µL PBS. After ultracentrifugation at 100,000 g for 30 min, the pellet was
resuspended in RIPA containing protease inhibitors (Sigma). Protein concentration was
measured by Bradford assay.
Ten micrograms of proteins (cytosolic and membrane extract) was resolved by 12% SDS-
PAGE; transferred onto nitrocellulose membranes; blocked with 5% milk powder in TBS, pH
7.4, containing 0.1% Tween 20; probed with polyclonal anti-FLAP for membrane extract or
goat anti-LTA4H for cytosolic extract (200 ng/mL, Santa Cruz Biotechnology) and
peroxidase-conjugated secondary anti-rabbit or -goat (1:25,000 Jackson ImmunoResearch
Laboratories); and detected by enhanced chemiluminescence (ECL), as previously described
(19). In separate experiments, cytosolic fractions from PMNs derived from healthy subjects
were analyzed by Western blot and membranes probed with rabbit GAPDH antibody
(AbCam) to confirm that loading control did not differ between samples.
In vitro exposure of PMNs from healthy subjects to IH
Purified PMNs from 8 healthy subjects underwent 4 cycles of IH using a modified protocol
(20). In a hypoxia chamber with atmospheric pressure maintained, a 35-minute hypoxic
period (95% N2 and 5% CO2) was followed by 25 minutes of reoxygenation (95% O2 and 5%
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CO2), after which the cells were resuspended in RNAlater (Ambion) for LTB4 pathway RT-
PCR analysis (n =4) or in PBS for A23187 1 µM stimulation (n =6). Control-PMNs from the
same donors were maintained in normoxic conditions for the same durations.
Isolation of monocytes and cytokine production in response to LTB4
Monocytes were isolated using standard methods (21). Briefly, after separation by dextran
sedimentation and centrifugation through a discontinuous ficoll, mononuclear cells were
placed on plastic tissue culture dishes (Falcon 3003) precoated with pooled human serum
(PHS) for 15 min at 37° C. After 2 h culture at 37°C in RPMI 1640 with 10% PHS,
nonadherent cells were removed. Plastic adherent cells (monocytes) were collected by
scraping with a rubber policeman, washed twice in RPMI and suspended in 2 ml RPMI to be
counted.
Enriched adherent cells (monocytes 2x105 cells) were resuspended in RPMI 1640, 10% heat-
inactivated FBS (Invitrogen) and 2 mM glutamine (Invitrogen), and incubated in a 96-well
plate overnight at 37°C in a humidified 5% CO2 incubator with 10 ng/ml LTB4 or vehicle.
The supernatants were collected and stored at -80°C until use.
CCL5/RANTES, CCL2/MCP-1 (monocyte chemoattractant protein 1), IL-6, and TNFα were
measured by multiplex bead immunoassay (Fluorokine MAP Multiplex Human Cytokine
Panel, R&D Systems, Minneapolis, USA) on a Bioplex 200 (Bio-Rad Laboratories, Hercules,
CA, USA) using Luminex xMAP™ Technology (Luminex Corporation, Austin, TX, USA) as
described (22).
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Statistical analysis
Statistical analyses were performed using NCSS97 (Kaysville, Utah). Continuous data were
expressed as median and 10th and 90th percentiles. Noncontinuous data were expressed as
numbers and percentages and compared by chi-square test. When necessary, LTB4 was log-
transformed to normalize data, or appropriate nonparametric tests were used (Spearman
correlation coefficient, Kruskall-Wallis method, and Mann-Whitney U test). LTB4 production
between OSA subgroups, stratified by AHI, and controls was compared by Kruskall-Wallis
method, and subsequent pairwise comparisons were made by nonparametric Bonferroni
multiple comparison test. The impact of pharmacological treatments and polysomonographic
parameters on log-LTB4 concentrations was analyzed by multiple regression. Differences
between chemokine and cytokine concentrations at baseline and after challenge with LTB4
were analyzed by Wilcoxon signed-rank test. P<0.05 was considered significant.
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Results
LTB4 production in OSA patients and controls
Table 1 shows the baseline characteristics of subjects in whom A23187-induced stimulation
of LTB4 was measured, stratified by AHI. A23187-induced LTB4 production increased with
OSA severity, rising significantly in severe OSA patients versus controls (Table 1).
Pretreatment with AA861 or MK886 inhibited A23187-mediated LTB4 synthesis (data not
shown).
Confounders of LTB4 production in OSA patients
LTB4 production correlated significantly with AHI and percentage of time spent with SaO2<
90% (Table 2). LTB4 concentrations were unrelated to age, BMI, or metabolic variables.
Gender, smoking status, and different drug treatments (lipid-lowering, antihypertensive or
anti-platelet treatments) did not influence LTB4 concentration. A multiple-linear regression
including pharmacological treatments, AHI, glycemia and LDL-cholesterol (variables with P
value<0.2 in the univariated analysis) indicated that AHI (P=0.042) was an independent
predictor of log LTB4 concentrations but this model merely explained 14 % of the variance.
Increased FLAP mRNA expression in OSA patient PMNs
Supplementary Table E2 shows the baseline characteristics of the 9 controls and 25 OSA
patients included in the RT-PCR experiments. Among the patients included in the mRNA
study, 13 were severe, 8 were moderated and 4 were mild OSA patients. FLAP mRNA levels
were significantly higher in PMNs derived from OSA patients versus controls (Figure 3A).
Conversely, 5-LO, LTA4H (Figure 3A), and BLT1 and BLT2 receptor mRNA (data not
shown) did not differ significantly between the groups. Although FLAP mRNA and OSA
severity did not correlate significantly, there was a trend correlation of FLAP mRNA with
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percentage of time spent with SaO2<90% (r=0.358, P=0.0613). FLAP mRNA was unrelated
to BMI (P=0.276).
Increased FLAP protein expression in OSA patient PMNs
Since FLAP and LTA4H mRNA levels rose in OSA patients, protein expression of these
LTB4 pathway components was examined by Western blot. Online supplementary Table E3
shows the baseline characteristics of the 5 controls and 12 OSA patients included in the
Western Blot experiments. In line with the PCR results, FLAP increased in the membrane
fraction of PMNs derived from OSA patients versus controls, whereas cytosolic LTA4H did
not significantly differ (Figure 3B). FLAP expression correlated with AHI (r= 0.536, P=0.03),
but not with RDI, min SaO2, and mean SO2. FLAP (r=0.664, P=0.01) and LTA4H (r= 0.677,
P=0.0098) levels were associated with BMI.
Specific effect of IH on the LTB4 pathway
In vitro exposure of PMNs from healthy subject to IH consisting of 4 cycles of 35 min
hypoxia followed by 25 min reoxygenation, increased FLAP and LTA4H mRNA levels
(Figure 4A) versus PMNs under normoxic conditions. In contrast, IH did not alter 5-LO
(Figure 4A) or BLT1 or BLT2 receptor mRNA (data not shown). As shown in Figure 4B,
LTB4 secretion in response to A23181 challenge was significantly 2.7-fold enhanced in
PMNs exposed to IH (ng/ml/2.106cells): 1.6±0.4 (normoxia conditions) vs 3.3±0.4 (hypoxic
conditions).
Associations of the LTB4 pathway with atherosclerosis
The correlations between PMN mRNA and protein levels for FLAP and LTA4H with
measures collected at carotid artery sonography are shown in Table 3. FLAP mRNA and
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protein in circulating PMNs correlated significantly with mean luminal diameter and mean
intima-media thickness (IMT) of common carotid arteries (Table 3). Similarly, PMN 5-LO
mRNA was associated with carotid luminal diameter and IMT (Table 3).
5-LO mRNA levels were greater (P=0.049) in subjects with atherosclerotic plaques (n=17)
versus those without plaque (n=18) and higher (P=0.024) in patients with carotid wall
hypertrophy, defined as IMT >0.8 mm (n=13) than in subjects without hypertrophy (n=19).
Although LTA4H mRNA levels in PMNs did not correlate with these markers of early
vascular remodeling, LTA4H protein in PMNs was associated with right carotid luminal
diameter and left IMT (Table 3). BLT1 and BLT2 mRNA in PMNs and monocytes was
unrelated to these markers (see online supplemental Table EIV).
Paracrine effects of LTB4 pathway activation in OSA patients
LTB4 (10 ng/mL) increased IL-6 and MCP-1 production in OSA patient monocytes (Figure
5). Conversely, LTB4 did not alter TNF-alpha or RANTES secretion from monocytes.
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Discussion
Our results point to an important role of the LTB4 pathway in PMNs for atherosclerosis
associated with OSA. Transcriptional alterations of the LTB4 pathway in PMNs, with a major
contributing role of IH, may represent a potential molecular mechanism of leukotriene-
induced atherogenesis in OSA. In particular, this study is the first to correlate subclinical
atherosclerosis with expression levels of the LTB4 pathway in PMNs, and to show that IH
increased LTB4 production in PMNs associated with greater FLAP mRNA and protein
expression in OSA patients. Collectively, these data suggest an important role of the LTB4
pathway in sleep apnea-related atherosclerosis.
Increased LTB4 production in A23187-stimulated PMNs has previously been
demonstrated in patients with a history of myocardial infarction (15) and in cardiovascular
disease-free OSA patients (4). The present study for the first time showed increased LTB4
production in A23187-stimulated PMNs from severe OSA patients presenting cardiovascular
comorbitities as seen in clinical practice, and its correlation with AHI and percentage of total
sleep time with SaO2<90%.
Although the present and previous (4, 15) studies demonstrate LTB4 pathway
activation in PMNs and suggested an association with cardiovascular disease and OSA, the
molecular mechanisms have remained largely unexplored. In the present study, the increase in
LTB4 production in OSA was associated with an increased mRNA expression of FLAP,
suggesting transcriptional activation of LTB4 pathway components in OSA patients, which
was also translated into higher protein levels. Our in vitro exposure of PMNs to IH, which
increased LTB4 production and induced FLAP upregulation, suggests that IH directly
participates to LTB4 pathway activation in OSA. The latter results are consistent with recent
findings in another cell type, showing that in vitro exposure of monocyte THP-1 cells to IH
increases expression levels of LTB4 synthesizing enzymes (23). Although IH in vitro might
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not reflect the repetitive desaturation-reoxygenation sequences in OSA, hypoxic conditions in
vitro are linked to NF-kB and HIF1 activation, delaying PMN apoptosis (20) and
upregulating FLAP (24).
LTB4 pathway activation in OSA may contribute to explain the association between
OSA and atherosclerosis. In the present study, the expression levels of LTB4 synthesizing
enzymes correlated with findings on carotid ultrasound, linking for the first time transcription
of the LTB4 pathway in peripheral leukocytes with atherosclerosis and early vascular
remodeling. For example, 5-LO transcript levels were higher in PMNs derived from subjects
with carotid atherosclerotic plaques compared with those derived from subjects without
atherosclerosis. The latter finding is in line with the proinflammatory and proatherogenic
effects of LTB4, which has been previously established (8, 9). In addition, the correlation
between FLAP and 5-LO expression in PMNs with carotid wall hypertrophy, measured as
IMT, is consistent with the direct chemotactic and proliferative effects of LTB4 on vascular
smooth muscle cells (25).
The association of transcriptional levels of the LTB4 pathway in PMNs with
atherosclerosis and vascular remodelling is in line with PMNs being a major source of LTB4
production. However, since monocytes/macrophages are major effectors in atherosclerosis, it
is also important that our results indicate that LTB4 production in PMNs may act in a
paracrine way to induce proinflammatory IL-6 and MCP-1 in monocytes. These findings are
supported by the upregulation of leukotriene B4 receptors by IH in THP-1 cells (16).
Similarly, proatherosclerotic MCP-1 levels rise in monocytes after sleep in severe OSA
patients (26) and IH-increased MCP-1 expression is markedly attenuated by BLT1 receptor
antagonist (16). Thus, LTB4-induced proinflammatory monocyte signaling might be a link
between PMN-derived LTB4 and atherosclerosis.
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Although our results link IH in OSA patients and LTB4 pathway activation in PMNs,
leading to LTB4-induced proinflammatory effects in monocytes and atherosclerosis, it must
be acknowledged that atherosclerosis is a multifactorial disease. Due to confounders and
cardiovascular risk factors in our patients, we cannot exclude that LTB4 pathway activation by
IH may not be the sole mechanism of vascular remodeling. Notably, LTA4H expression
correlated with BMI but not oxygen desaturation, whereas FLAP expression was influenced
by oxygen desaturation and obesity, as reported (5, 18). Similarly, FLAP mRNA and protein
levels were significantly higher in PMNs derived from OSA patients versus controls, which
supports the suggested mechanism but our data demonstrated only trend correlation of FLAP
mRNA with percentage of time spent with SaO2<90%. In addition, since the protein analysis
did not include an internal control for each sample, it cannot be fully excluded that subtle
differences in protein loading may have influenced the evaluation of protein levels. Finally,
the presence of cardiovascular risk factors in our population may contribute to blunt the
correlation between hypoxia severity and FLAP mRNA levels. In particular, our observations
support that obesity is a major confounding factor of the inflammatory state in OSA patients.
However, considering BMI and other potential cardiovascular comorbidities, AHI remained
an independent predictor of LTB4 production in OSA patients by multivariate analysis.
In summary, we have demonstrated activation of the LTB4 pathway in PMNs from
OSA patients, through transcriptional upregulation, which correlated with carotid
atherosclerosis and IMT. LTB4-induced proinflammatory monocyte signaling might be a link
between PMN-derived LTB4 and atherosclerosis. Together with the up- regulation of FLAP
and LTA4H in PMNs exposed in vitro to IH our data provide evidence that IH is a major
feature of OSA involved in LTB4 pathway activation underlying vascular remodeling and
atherosclerosis. Our results implicate LTB4 pathway, notably FLAP, as a therapeutic target in
OSA-associated metabolic and cardiovascular morbidity.
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Acknowledgments
We thank Chantal Nahum, Karine Scalabrino and Sophie Machuca for expert technical
assistance; Nathalie Arnol for statistical analysis; Pascale Roux-Lombard for multiplex
analysis; and Professor Göran K. Hansson for helpful advice.
This study was supported by grants from ResMed Foundation, “AgirAdom” scientific
council, PHRC 2006, Mairie de Paris (“Research in Paris”), The French-Swedish Foundation,
and the Swedish Heart and Lung Foundation.
Conflicts of interest: none declared
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References 1. Baguet, J. P., L. Hammer, P. Levy, H. Pierre, S. Launois, J. M. Mallion, and J. L. Pepin. 2005. The severity of oxygen desaturation is predictive of carotid wall thickening and plaque occurrence. Chest 128: 3407-3412. 2. Minoguchi, K., T. Yokoe, T. Tazaki, H. Minoguchi, A. Tanaka, N. Oda, S. Okada, S. Ohta, H. Naito, and M. Adachi. 2005. Increased carotid intima-media thickness and serum inflammatory markers in obstructive sleep apnea. Am J Respir Crit Care Med 172: 625-630. 3. Drager, L. F., L. A. Bortolotto, M. C. Lorenzi, A. C. Figueiredo, E. M. Krieger, and G. Lorenzi-Filho. 2005. Early signs of atherosclerosis in obstructive sleep apnea. Am J Respir Crit Care Med 172: 613-618. 4. Lefebvre, B., J. L. Pepin, J. P. Baguet, R. Tamisier, M. Roustit, K. Riedweg, G. Bessard, P. Levy, and F. Stanke-Labesque. 2008. Leukotriene B4: early mediator of atherosclerosis in obstructive sleep apnoea? Eur Respir J 32: 113-120. 5. Stanke-Labesque, F., M. Bäck, B. Lefebvre, R. Tamisier, J. P. Baguet, N. Arnol, P. Levy, and J. L. Pepin. 2009. Increased urinary leukotriene E4 excretion in obstructive sleep apnea: effects of obesity and hypoxia. J Allergy Clin Immunol 124: 364-370, 370 e361-362. 6. Goldbart, A. D., J. Krishna, R. C. Li, L. D. Serpero, and D. Gozal. 2006. Inflammatory Mediators in Exhaled Breath Condensate of Children With Obstructive Sleep Apnea Syndrome. Chest 130: 143-148. 7. Kaditis, A. G., E. Alexopoulos, K. Chaidas, G. Ntamagka, A. Karathanasi, I. Tsilioni, T. S. Kiropoulos, E. Zintzaras, and K. Gourgoulianis. 2009. Urine concentrations of cysteinyl leukotrienes in children with obstructive sleep-disordered breathing. Chest 135: 1496-1501. 8. Back, M., and G. K. Hansson. 2006. Leukotriene receptors in atherosclerosis. Annals of medicine 38: 493-502. 9. Back, M. 2009. Leukotriene signaling in atherosclerosis and ischemia. Cardiovascular drugs and therapy / sponsored by the International Society of Cardiovascular Pharmacotherapy 23: 41-48. 10. Rouzer, C. A., and S. Kargman. 1988. Translocation of 5-lipoxygenase to the membrane in human leukocytes challenged with ionophore A23187. J Biol Chem 263: 10980-10988. 11. Dixon, R. A., R. E. Diehl, E. Opas, E. Rands, P. J. Vickers, J. F. Evans, J. W. Gillard, and D. K. Miller. 1990. Requirement of a 5-lipoxygenase-activating protein for leukotriene synthesis. Nature 343: 282-284. 12. Samuelsson, B., S. E. Dahlen, J. A. Lindgren, C. A. Rouzer, and C. N. Serhan. 1987. Leukotrienes and lipoxins: structures, biosynthesis, and biological effects. Science (New York, N.Y 237: 1171-1176. 13. Hlawaty, H., M. P. Jacob, L. Louedec, D. Letourneur, C. Brink, J. B. Michel, L. Feldman, and M. Back. 2009. Leukotriene receptor antagonism and the prevention of extracellular matrix degradation during atherosclerosis and in-stent stenosis. Arteriosclerosis, thrombosis, and vascular biology 29: 518-524. 14. Leclercq, A., X. Houard, M. Philippe, V. Ollivier, U. Sebbag, O. Meilhac, and J. B. Michel. 2007. Involvement of intraplaque hemorrhage in atherothrombosis evolution via neutrophil protease enrichment. Journal of leukocyte biology 82: 1420-1429. 15. Helgadottir, A., A. Manolescu, G. Thorleifsson, S. Gretarsdottir, H. Jonsdottir, U. Thorsteinsdottir, N. J. Samani, G. Gudmundsson, S. F. Grant, G. Thorgeirsson, S. Sveinbjornsdottir, E. M. Valdimarsson, S. E. Matthiasson, H. Johannsson, O. Gudmundsdottir, M. E. Gurney, J. Sainz, M. Thorhallsdottir, M. Andresdottir, M. L. Frigge, E. J. Topol, A. Kong, V. Gudnason, H. Hakonarson, J. R. Gulcher, and K. Stefansson. 2004.
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Table 1: Baseline characteristics of controls and OSA patients enrolled in the LTB4
production study
Controls OSA
mild moderate Severe
N 19 16 25 27 Male, % 68.4 81.3 84,0 81,4
smokers % 36.8 62.5 52.0 59.2
Age, years 47 (36-64) 51 (41-61) 57(45-64) 56 (35-66)
BMI, kg/m2 24.0 (20.3-30.7) 25.9 (22.9-31.9) 27.0 (24.8-34.6) 29.6 (25.3-34.8)*
SBP, mm Hg 130 (108-149) 130 (117-158) 134 (120-157) 138 (118-159)
DBP, mm Hg 85 (67-103) 82 (69-105) 87 (73-100) 87 (72-99)
AHI, events/hour 2.8 (0.9-8.0) 9.1 (5.9-13.6) 20.7 (15.7-27.1)* 41.5 (32.0-62.6)*$†
RDI, events/hour 9.6 (3.1-17.1) 22.1 (9.9-40.7) 35.0 (24.0-44.7)* 55.0 (40.0-70.3)*$†
Mean nocturnal SaO2, % 94.0 (92.0-99.4) 94.7 (91.8-99.4) 93.9 (91.0-95.0) 93.0 (88.9-96.0)*
Minimal nocturnal SaO2, % 88.0 (85.4-93.0) 89.5 (81.6-92.6) 85.0 (77.7-89.0)*$ 81.0 (68.7-88.6)*$
SaO2< 90%, %TST 0 .0 (0.0-1.0) 0.0 (0.0-6.6) 1.0 (0.0-8.5)* 4.5 (0.0-50.0)*$
RAI, events/hour 6.0 (1.0-11.8) 17.0 (8.6-21.5) 23.5 (14.0.-39.1)* 38.6 (27.8.-56.6)*$†
Fasting plasma glucose, mM 4.7 (4.4-5.2) 4.9 (4.4-6.4) 5.3 (4.5-9.6)* 5.2 (4.5-6.4)
Fasting plasma insulin, µUI/mL 4.8 (3.1-10.2) 5.8 (2.9-12.4) 5.8 (3.5-10.6) 8.6 (5.2-14.2)*†
HOMA-R index 1.1 (0.6-2.1) 1.2 (0.7-2.6) 1.5 (0.8-3.1) 2.0 (0.8-3.5)*
Total cholesterol, g/L 1.9 (1.3-2.4) 2.1 (1.4-3.2) 1.9 (1.3-2.3) 2.0 (1.6-2.5)
HDL-cholesterol , g/L 0.6 (0.4-0.9) 0.6 (0.4-0.7) 0.6 (0.4-0.9) 0.5 (0.4-0.7)
LDL-cholesterol , g/L 1.2 (0.7-1.5) 1.3 (0.7-1.9) 1.1 (0.5-1.6) 1.2 (0.8-1.6)
Triglycerides, g/L 0.8 (0.4-2.2) 1.4 (0.7-2.5) 0.9 (0.7-1.7) 1.2 (0.7-2.6)*
hsCRP, mg/L 1.0 (0.5-10.4) 1.5 (0.3-4.8) 1.0 (0.3-8.0) 1.6 (0.4-6.3)
LTB4, ng/ml for 2.106 PMNs 11.7 (6.7-16.6) 12.6 (4.3-26.6) 13.8 (4.8-26.6) 16.5 (9.3-29.9)*
Treatment, % Lipid-lowering drugs 10.5 18.8 33.3 10.3 Antihypertensives 42.1 25.0 29.2 31.0 Aspirine-AAP 21.1 25.0 25.0 13.8
Values are medians (10th-90th percentiles). BMI, body mass index; SBP, systolic blood pressure; DBP,
diastolic blood pressure; AHI, apnea-hypopnea index; RDI, respiratory disturbance index; SaO2, O2
saturation; TST, total sleep time; RAI, respiratory arousal index.
*: P<0.05 vs controls, $: P<0.05 vs mild OSA, †: P<0.05 vs moderate OSA. Lipid lowering drugs
were statines and fibrates, antihypertensive drugs were ACE inhibitors, and angiotensine receptor
antagonists.
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Table 2: Correlation coefficients between LTB4 production and age, BMI, and
polysomnographic and metabolic variables (n=87).
R P
Age 0.145 0.189
BMI 0.072 0.505
AHI 0.280 0.0095
RDI 0.182 0.092
Mean nocturnal SaO2 -0.186 0.084
Minimal nocturnal SaO2 -0.169 0.111
SaO2<90%, %TST 0.347 0.0013
RAI 0.208 0.066
Fasting plasma glucose 0.160 0.139
Fasting plasma insulin -0.038 0.728
HOMA-R index 0.031 0.773
Total cholesterol -0.035 0.749
HDL-cholesterol 0.089 0.412
LDL-cholesterol -0.158 0.148
Triglycerides 0.086 0.427
BMI, body mass index; AHI, apnea-hypopnea index; RDI, respiratory disturbance index; SaO2, O2
saturation; TST, total sleep time; RAI, respiratory-related arousal index
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Table 3: Correlation between FLAP, 5-LO, and LTA4H mRNA and protein expression with early vascular remodelling. IMT=intima-media
thickness.
mRNA Protein
FLAP 5-LO LTA4 H FLAP LTA4H
Right carotid luminal diameter r=0.550, P = 0.001 r=0.663, P=0.011 r=0.176, P=0.18 r=0.555, P=0.07 r=0.609, P=0.05
Left carotid luminal diameter r=0.457, P=0.04 r=0.426, P=0.048 r=0.079, P=0.644 r=0.685, P= 0.04 r=0.527, P=0.11
Right common carotid IMT r=0.576, P=0.01 r=0.645, P=0.002 r=-0.072, P=0.682 r=0.15, P=0.671 r=0.150, P=0.671
Left common carotid IMT r=0.584, P=0.02 r=0.512, P=0.017 r=0.182, P=0.302 r=0.933, P=0.008 r=0.700, P=0.047
Mean common carotid IMT r=0.617, P=0.007 r=0.637, P=0.002 r=0.276, P=0.11 r=0.783, P=0.026 r=0.617, P=0.081
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Figure legends
Figure 1: Flow chart detailing patient inclusion in the different experiments
Figure 2: Representative typical LC-MS/MS chromatogram of LTB4, 6 trans-LTB4 and 6-
trans-12-epi LTB4 obtained from PMNs stimulated with A23187 10 µM.
Figure 3: A/ Messenger RNA levels of FLAP, 5-LO, and LTA4H in PMNs from OSA
patients. B/ membrane FLAP and cytosolic LTA4H protein expression in PMNs derived from
controls and OSA patients.
Figure 4: A/ Messenger RNA levels of 5-LO, FLAP, and LTA4H in PMNs from healthy
subjects exposed in vitro to intermittent hypoxia (IH) or normoxia (NX).B/A23187-mediated
LTB4 production by PMNs exposed to in vitro IH or NX.
Figure 5: Cytokine production in monocytes from OSA patients in response to LTB4
challenge 10 ng/ml (+) or vehicle (+).
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LTB4 analysis by LC‐MS/MS
120 patients with newly diagnosed OSA and 33 controls
Carotid ultrasound29 OSA patients
15 OSA patients for monocyte isolation
68 OSA patients and 19 controls
25 OSA patients and 9 controls
12 OSA patients and 5 controls
qPCRWestern Blot
A23187 –stimulation
Protein extraction mRNA extraction
Figure 1
LTB4 –stimulation
Cytokine production
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6-trans LTB4m/z 335.0/195.1
6-trans-12-epi LTB4m/z 335.0/195.1
LTB4m/z 335.0/195.1
LTB4-D4m/z 339.1/196.9
Figure 2
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5-LOFLAP LTA4H
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