Leukotriene B4 pathway activation and atherosclerosis in ...

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

by guest, on March 27, 2018

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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 upregulation 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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The gene encoding 5-lipoxygenase activating protein confers risk of myocardial infarction and stroke. Nat Genet 36: 233-239. 16. Li, R. C., B. Haribabu, S. P. Mathis, J. Kim, and D. Gozal. Leukotriene B4 receptor-1 mediates intermittent hypoxia-induced atherogenesis. Am J Respir Crit Care Med 184: 124-131. 17. Hosselet, J., I. Ayappa, R. G. Norman, A. C. Krieger, and D. M. Rapoport. 2001. Classification of sleep-disordered breathing. Am J Respir Crit Care Med 163: 398-405. 18. Bäck, M., A. Sultan, O. Ovchinnikova, and G. K. Hansson. 2007. 5-Lipoxygenase-activating protein: a potential link between innate and adaptive immunity in atherosclerosis and adipose tissue inflammation. Circ Res 100: 946-949. 19. Dejouvencel, T., D. Feron, P. Rossignol, M. Sapoval, C. Kauffmann, J. M. Piot, J. B. Michel, I. Fruitier-Arnaudin, and O. Meilhac. 2010. Hemorphin 7 reflects hemoglobin proteolysis in abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol 30: 269-275. 20. Dyugovskaya, L., A. Polyakov, P. Lavie, and L. Lavie. 2008. Delayed neutrophil apoptosis in patients with sleep apnea. Am J Respir Crit Care Med 177: 544-554. 21. Havlir, D. V., J. J. Ellner, K. A. Chervenak, and W. H. Boom. 1991. Selective expansion of human gamma delta T cells by monocytes infected with live Mycobacterium tuberculosis. The Journal of clinical investigation 87: 729-733. 22. Borel, J. C., P. Roux-Lombard, R. Tamisier, C. Arnaud, D. Monneret, N. Arnol, J. P. Baguet, P. Levy, and J. L. Pepin. 2009. Endothelial dysfunction and specific inflammation in obesity hypoventilation syndrome. PloS one 4: e6733. 23. . 1999. Sleep-related breathing disorders in adults: recommendations for syndrome definition and measurement techniques in clinical research. The Report of an American Academy of Sleep Medicine Task Force. Sleep 22: 667-689. 24. Gonsalves, C. S., and V. K. Kalra. Hypoxia-mediated expression of 5-lipoxygenase-activating protein involves HIF-1alpha and NF-kappaB and microRNAs 135a and 199a-5p. J Immunol 184: 3878-3888. 25. Back, M., D. X. Bu, R. Branstrom, Y. Sheikine, Z. Q. Yan, and G. K. Hansson. 2005. Leukotriene B4 signaling through NF-kappaB-dependent BLT1 receptors on vascular smooth muscle cells in atherosclerosis and intimal hyperplasia. Proceedings of the National Academy of Sciences of the United States of America 102: 17501-17506. 26. Tamaki, S., M. Yamauchi, A. Fukuoka, K. Makinodan, N. Koyama, K. Tomoda, M. Yoshikawa, and H. Kimura. 2009. Production of inflammatory mediators by monocytes in patients with obstructive sleep apnea syndrome. Internal medicine (Tokyo, Japan) 48: 1255-1262.


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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



qPCRWestern Blot

A23187 –stimulation

Protein extraction mRNA extraction

Figure 1

LTB4 –stimulation

Cytokine production


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