Keywords: (6 max) Obstructive sleep apnea --Cardiovascular disease --Intermittent hypoxia --Oxidative stress--Hypoxia inducible factor--1 --Endothelin--1 AHI, apnea--hypopnea index, ARBs, angiotensin II receptor blockers, BP, blood pressure, CAD, coronary artery disease, CamK, calcium--calmodulin kinase, CPAP, continuous positive airway pressure, ET--1, endothelin--1, ET--A, type A endothelin receptor, FIH, factor inhibiting HIF--1, HF, heart failure, HIF--1, hypoxia inducible factor--1, IH, intermittent hypoxia, LV, left ventricle, mTOR, mammalian target of rapamycin, miRNA, micro--ribonucleic acid, NADPH, nicotinamide adenine dinucleotide phosphate, NO, nitric oxide, NOX, NADPH oxidase, OSA, obstructive sleep apnea syndrome, PHDs, HIF--1 prolyl--hydroxylases, PKC, protein kinase C, PLC, phospholipase C, ROS, reactive oxygen species

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Targeting the ROS-HIF-1-endothelin axis as a therapeutic approach for the treatment of obstructive sleep apnea-related cardiovascular complications

Elise Belaidi, Jessica Morand, Emmanuelle Gras, Jean-Louis Pépin, Diane

Godin-Ribuot

To cite this version:

Elise Belaidi, Jessica Morand, Emmanuelle Gras, Jean-Louis Pépin, Diane Godin-Ribuot. Targeting the ROS-HIF-1-endothelin axis as a therapeutic approach for the treatment of obstructive sleep apnearelated cardiovascular complications.

Introduction

Obstructive sleep apnea (OSA) is characterized by repetitive upper airway occlusions during sleep leading to intermittent hypoxia. Factors promoting OSA include functional or anatomical anomalies of the upper airways, obesity, age over 60 years, smoking and alcohol consumption [START_REF] Young | Epidemiology of obstructive sleep apnea: a population health perspective[END_REF]. According to recent estimates, the prevalence of the disease has more than doubled in the last 20 years and is now estimated to be between 9 and 17% in women and men, respectively, aged 50 to 70 years [START_REF] Peppard | Increased prevalence of sleep--disordered breathing in adults[END_REF]. It is a major public health concern worldwide and in Europe since it is associated with high cardiovascular morbidity and mortality [START_REF] Baguet | Mechanisms of cardiac dysfunction in obstructive sleep apnea[END_REF][START_REF] Lévy | Obstructive sleep apnoea syndrome[END_REF]. Intermittent hypoxia (IH), the landmark of OSA, induces oxidative stress and consequently promotes low-grade inflammation, endothelial dysfunction and cardiometabolic co--morbidities. Thus, effective treatment of OSA is expected to represent an important target for improving cardiometabolic risk.

Continuous positive airway pressure (CPAP) is the first line therapy for OSA. While CPAP therapy clearly improves vigilance and cognitive function, it is still debated whether CPAP alone may substantially reduce the rate of new cardiovascular events [START_REF] Barbe | Effect of continuous positive airway pressure on the incidence of hypertension and cardiovascular events in nonsleepy patients with obstructive sleep apnea: a randomized controlled trial[END_REF].

One of the major barriers to CPAP efficacy is patient adherence. Indeed, 15% of OSA patients initially refuse the treatment and 20% of those treated discontinue or use it irregularly or suboptimally. Moreover, the response to CPAP therapy, in terms of cardiovascular and metabolic outcomes, is obtained only in CPAP compliers [START_REF] Bratton | CPAP vs Mandibular Advancement Devices and Blood Pressure in Patients With Obstructive Sleep Apnea: A Systematic Review and Meta--analysis[END_REF] and differs in non--obese and obese OSA patients. Thus, in obese OSA patients, CPAP alone has a limited effect, compared to weight loss, on blood pressure, insulin sensitivity, lipid profile and inflammation [START_REF] Chirinos | CPAP, weight loss, or both for obstructive sleep apnea[END_REF]. The failure of CPAP to improve cardiometabolic and inflammatory markers in obese OSA patients [START_REF] Jullian--Desayes | Impact of obstructive sleep apnea treatment by continuous positive airway pressure on cardiometabolic biomarkers: a systematic review from sham CPAP randomized controlled trials[END_REF] emphasizes the need for combined therapeutic strategies [START_REF] Pepin | Obstructive sleep apnoea and metabolic syndrome: put CPAP efficacy in a more realistic perspective[END_REF]. Hence, angiotensin II receptor blockers (ARBs) are more effective than CPAP to lower blood pressure (BP), but there is an added benefit of combining both treatments especially on nighttime BP (Pepin et al., 2010;[START_REF] Thunstrom | Blood Pressure Response to Losartan and Continuous Positive Airway Pressure in Hypertension and Obstructive Sleep Apnea[END_REF]. Therefore, it is important to identify the best pharmacological approach to counteract the key intermediary mechanisms responsible for OSA--related cardiovascular alterations, namely oxidative stress, sympathetic activation and inflammation.

As previously described [START_REF] Dematteis | Cardiovascular consequences of sleep--disordered breathing: contribution of animal models to understanding the human disease[END_REF], IH is a major consequence of OSA in terms of impact on the cardiovascular system, in particular through the generation of reactive oxygen species (ROS) [START_REF] Lévy | Obstructive sleep apnoea syndrome[END_REF]. Indeed, ROS enhance the stabilization and activity of the hypoxia inducible factor--1 (HIF--1) transcription factor well known to promote adaptive and maladaptive responses to hypoxia [START_REF] Semenza | Regulation of oxygen homeostasis by hypoxia--inducible factor 1[END_REF]. Amongst the various HIF--1 target gene products, some have beneficial effects on the cardiovascular system, such as nitric oxide and carbon monoxide [START_REF] Andreadou | The role of gasotransmitters NO, H2S and CO in myocardial ischaemia/reperfusion injury and cardioprotection by preconditioning, postconditioning and remote conditioning[END_REF], while others, like endothelin--1 (ET--1), are involved in several pathophysiological pathways associated with cardiovascular disease [START_REF] Kaoukis | The role of endothelin system in cardiovascular disease and the potential therapeutic perspectives of its inhibition[END_REF]. Differential modulation of these gene pathways could account for the dual aspect of IH, which has been shown to be protective or deleterious according to the duration and severity of the hypoxia--reoxygenation cycles [START_REF] Verges | Hypoxic Conditioning as a New Therapeutic Modality[END_REF].

In the present review, we will focus on the involvement of the ROS--HIF--1--endothelin signaling pathway in the deleterious cardiovascular consequences induced by the rapid IH cycles and low desaturation levels encountered in moderate to severe OSA patients (Fig. 1).

We will propose new therapeutic avenues targeting this pathway to treat or prevent the OSA--related cardiovascular complications. We will discuss the rationale for innovative clinical trials based on the mechanistic insights presented in this review.

Cardiovascular impact of OSA and intermittent hypoxia

Systemic hypertension

It is now well established that OSA and intermittent hypoxia causes arterial hypertension [START_REF] Pepin | Hypertension and sleep: Overview of a tight relationship[END_REF][START_REF] Torres | Relationship Between OSA and Hypertension[END_REF]. There is indeed a linear relationship between the severity of sleep--disordered breathing, determined by the apnea hypopnea index (AHI), and blood pressure elevation, independently of confounding factors [START_REF] Young | Population-based study of sleep--disordered breathing as a risk factor for hypertension[END_REF].

Fletcher et al. were the first to demonstrate that intermittent hypoxia was one of the landmark features of OSA, showing its role in the development of hypertension [START_REF] Fletcher | Sympathetic denervation blocks blood pressure elevation in episodic hypoxia[END_REF]. This has been confirmed in several rodent models of IH [START_REF] Dematteis | Cardiovascular consequences of sleep--disordered breathing: contribution of animal models to understanding the human disease[END_REF] as well in healthy human volunteers exposed exclusively to intermittent hypoxia [START_REF] Tamisier | 14 nights of intermittent hypoxia elevate daytime blood pressure and sympathetic activity in healthy humans[END_REF].

Vascular dysfunction and remodeling, atherosclerosis

OSA promotes endothelial dysfunction (Hoyos et al., 2015), vascular remodeling [START_REF] Baguet | The severity of oxygen desaturation is predictive of carotid wall thickening and plaque occurrence[END_REF] and atherosclerosis [START_REF] Drager | Obstructive sleep apnea: an emerging risk factor for atherosclerosis[END_REF][START_REF] Stanke--Labesque | Leukotrienes as a molecular link between obstructive sleep apnoea and atherosclerosis[END_REF][START_REF] Trzepizur | Microparticles and vascular dysfunction in obstructive sleep apnoea[END_REF]. Similarly, IH exposure results in endothelial dysfunction in various vascular territories [START_REF] Krause | Arginase--endothelial nitric oxide synthase imbalance contributes to endothelial dysfunction during chronic intermittent hypoxia[END_REF][START_REF] Phillips | Chronic intermittent hypoxia impairs endothelium--dependent dilation in rat cerebral and skeletal muscle resistance arteries[END_REF][START_REF] Totoson | Atorvastatin protects against deleterious cardiovascular consequences induced by chronic intermittent hypoxia[END_REF], in inflammatory and fibrotic vascular remodeling (Arnaud et al., 2011a;[START_REF] Castro--Grattoni | Intermittent Hypoxia--Induced Cardiovascular Remodeling is Reversed by Normoxia in A Mouse Model of Sleep Apnea[END_REF][START_REF] Gras | Endothelin--1 mediates intermittent hypoxia--induced inflammatory vascular remodeling through HIF--1 activation[END_REF][START_REF] Yang | Restoring leptin signaling reduces hyperlipidemia and improves vascular stiffness induced by chronic intermittent hypoxia[END_REF] and in accelerated atherosclerosis (Arnaud et al., 2011b;[START_REF] Drager | Chronic intermittent hypoxia induces atherosclerosis via activation of adipose angiopoietin--like 4[END_REF][START_REF] Savransky | Dyslipidemia and atherosclerosis induced by chronic intermittent hypoxia are attenuated by deficiency of stearoyl coenzyme A desaturase[END_REF].

Myocardial infarction and stroke

Moderate to severe OSA is associated by an enhanced risk of coronary heart disease (CAD) [START_REF] Arzt | Sleep--Disordered Breathing and Coronary Artery Disease[END_REF] and stroke [START_REF] Arzt | Association of sleep-disordered breathing and the occurrence of stroke[END_REF][START_REF] Dong | Obstructive sleep apnea and cardiovascular risk: meta--analysis of prospective cohort studies[END_REF]. OSA patients with acute myocardial infarction have more severe CAD, prolonged myocardial ischemia and less salvaged myocardium than non--apneic patients [START_REF] Arzt | Sleep--Disordered Breathing and Coronary Artery Disease[END_REF]. Thus, a high AHI is independently associated with a larger infarct size 3 months after acute myocardial infarction (S. [START_REF] Buchner | Impact of sleep--disordered breathing on myocardial salvage and infarct size in patients with acute myocardial infarction[END_REF]. IH appears to be a major contributor since several experimental studies have demonstrated an increase in myocardial infarct size following IH exposure [START_REF] Belaidi | Major role for hypoxia inducible factor--1 and the endothelin system in promoting myocardial infarction and hypertension in an animal model of obstructive sleep apnea[END_REF]Belaidi et al., 2016;Bourdier et al., 2016;[START_REF] Joyeux--Faure | Chronic intermittent hypoxia increases infarction in the isolated rat heart[END_REF][START_REF] Ramond | Oxidative stress mediates cardiac infarction aggravation induced by intermittent hypoxia[END_REF][START_REF] Yeung | Cardioprotective effects of melatonin against myocardial injuries induced by chronic intermittent hypoxia in rats[END_REF]. Cerebral infarct size and neuronal excitotoxicity (an index of stroke vulnerability) are also increased by IH [START_REF] Jackman | Dichotomous effects of chronic intermittent hypoxia on focal cerebral ischemic injury[END_REF]) [START_REF] Jagadapillai | Ceftriaxone preserves glutamate transporters and prevents intermittent hypoxia--induced vulnerability to brain excitotoxic injury[END_REF].

Cardiac dysfunction and heart failure

Left ventricular dysfunction and remodeling have been reported in OSA patients prior to the development of hypertension or other cardiovascular diseases [START_REF] Aslan | Early left ventricular functional alterations in patients with obstructive sleep apnea syndrome[END_REF][START_REF] Baguet | Mechanisms of cardiac dysfunction in obstructive sleep apnea[END_REF]. Following acute myocardial infarction, cardiac dysfunction and remodeling is worsened in OSA compared to non--apneic patients [START_REF] Arzt | Sleep--Disordered Breathing and Coronary Artery Disease[END_REF]. Thus, not only is OSA a significant risk factor for the development of incident heart failure [START_REF] Gottlieb | Prospective study of obstructive sleep apnea and incident coronary heart disease and heart failure: the sleep heart health study[END_REF], but it also enhances mortality in heart failure patients [START_REF] Khayat | Sleep disordered breathing and post--discharge mortality in patients with acute heart failure[END_REF][START_REF] Wang | Influence of obstructive sleep apnea on mortality in patients with heart failure[END_REF].

In rodent models, prolonged IH exposure results in left ventricular dysfunction [START_REF] Chen | Left ventricular dysfunction and associated cellular injury in rats exposed to chronic intermittent hypoxia[END_REF], fibrotic ventricular remodeling [START_REF] Hayashi | Role of gp91phox--containing NADPH oxidase in left ventricular remodeling induced by intermittent hypoxic stress[END_REF] [START_REF] Ding | Adiponectin protects rat heart from left ventricular remodeling induced by chronic intermittent hypoxia via inhibition of TGF--beta/smad2/3 pathway[END_REF] and cardiac hypertrophy [START_REF] Chen | The Na+/Ca2+ exchanger--1 mediates left ventricular dysfunction in mice with chronic intermittent hypoxia[END_REF].

Regarding heart failure (HF), a recent study in rats has shown that a 4--week IH exposure exacerbated myocardial remodeling and cardiac dysfunction in a pressure overload--induced HF model (S. [START_REF] Li | Delayed neutrophil apoptosis mediates intermittent hypoxia--induced progressive heart failure in pressure-overloaded rats[END_REF].

3. The ROS--HIF--1--endothelin pathway in OSA and intermittent hypoxia and its role in the cardiovascular alterations

Why the ROS--HIF--1--ET--1 pathway?

OSA is widely recognized as an oxidative stress disorder [START_REF] Badran | Cardiovascular Complications of Sleep Apnea: Role of Oxidative Stress[END_REF][START_REF] Eisele | Obstructive Sleep Apnea, Oxidative Stress, and Cardiovascular Disease: Evidence from Human Studies[END_REF][START_REF] Lavie | Oxidative stress in obstructive sleep apnea and intermittent hypoxia---revisited----the bad ugly and good: implications to the heart and brain[END_REF][START_REF] Lavie | Molecular mechanisms of cardiovascular disease in OSAHS: the oxidative stress link[END_REF]. As recently reviewed by Eisele et al., OSA patients present with increased superoxide anion release by circulating leucocytes associated with an upregulation of NADPH oxidase (NOX), reduced nitric oxide bioavailability reflected by a decrease in total nitrate and nitrite, increased lipid peroxidation and reduced antioxidant status characterized by lower plasma levels of antioxidant substances, such as vitamin A and E, or antioxidant enzymes, such as superoxide dismutase and catalase [START_REF] Eisele | Obstructive Sleep Apnea, Oxidative Stress, and Cardiovascular Disease: Evidence from Human Studies[END_REF].

Accordingly, lipid peroxidation products, such as thiobarbituric reactive substances [START_REF] Barcelo | Abnormal lipid peroxidation in patients with sleep apnoea[END_REF][START_REF] Lavie | Evidence for lipid peroxidation in obstructive sleep apnea[END_REF] or isoprostanes [START_REF] Biltagi | Correlation of 8--isoprostane, interleukin--6 and cardiac functions with clinical score in childhood obstructive sleep apnoea[END_REF][START_REF] Carpagnano | 8--Isoprostane, a marker of oxidative stress, is increased in exhaled breath condensate of patients with obstructive sleep apnea after night and is reduced by continuous positive airway pressure therapy[END_REF][START_REF] Del Ben | Oxidative stress mediated arterial dysfunction in patients with obstructive sleep apnoea and the effect of continuous positive airway pressure treatment[END_REF][START_REF] Monneret | Association of urinary 15--F2t--isoprostane level with oxygen desaturation and carotid intima--media thickness in nonobese sleep apnea patients[END_REF] are increased in OSA patients and their levels are correlated with sleep apnea severity [START_REF] Biltagi | Correlation of 8--isoprostane, interleukin--6 and cardiac functions with clinical score in childhood obstructive sleep apnoea[END_REF][START_REF] Lavie | Evidence for lipid peroxidation in obstructive sleep apnea[END_REF][START_REF] Monneret | Association of urinary 15--F2t--isoprostane level with oxygen desaturation and carotid intima--media thickness in nonobese sleep apnea patients[END_REF]. Few studies have investigated the correlation of OSA--related oxidative stress with cardiovascular disease markers but we have demonstrated a direct relationship between urinary 15--F2t--isoprostane levels and carotid intima--media thickness in non--obese OSA patients, in support for a role of oxidative stress in the early atherosclerotic process [START_REF] Monneret | Association of urinary 15--F2t--isoprostane level with oxygen desaturation and carotid intima--media thickness in nonobese sleep apnea patients[END_REF]. Indeed, there is a linear relationship between oxidative stress and sleep apnea severity [START_REF] Franco | Obstructive sleep apnea severity correlates with cellular and plasma oxidative stress parameters and affective symptoms[END_REF]. Thus the lowest antioxidant status and the highest ROS production by circulating leucocytes are observed in the most severe OSA patients [START_REF] Mancuso | Oxidative stress biomarkers in patients with untreated obstructive sleep apnea syndrome[END_REF][START_REF] Pilkauskaite | Reactive oxygen species production in peripheral blood neutrophils of obstructive sleep apnea patients[END_REF]. In accordance, the severity of the cardiometabolic alterations is correlated to that of hypoxemia, with alterations most significant in patients with severe nocturnal hypoxia [START_REF] Cakmak | Association Between the Severity of Nocturnal Hypoxia in Obstructive Sleep Apnea and Non--Alcoholic Fatty Liver Damage[END_REF][START_REF] Gami | Obstructive sleep apnea and the risk of sudden cardiac death: a longitudinal study of 10,701 adults[END_REF][START_REF] He | Relationship of daytime blood pressure and severity of obstructive sleep apnea among Chinese: a multi--center investigation in China[END_REF]. In a recent retrospective follow--up study on 1,068 men originally referred to night polysomnography due to suspected OSA, Muraja--Murro et al. conclude that adjusting AHI with severity of obstruction events enhances the detection of patients with the highest risk of cardiovascular morbidity or mortality [START_REF] Muraja--Murro | Adjustment of apnea--hypopnea index with severity of obstruction events enhances detection of sleep apnea patients with the highest risk of severe health consequences[END_REF].

Animal models of chronic short--cycling IH exposure closely reproduce the desaturation pattern and cardiovascular alterations seen in moderate to severe OSA patients [START_REF] Dematteis | Cardiovascular consequences of sleep--disordered breathing: contribution of animal models to understanding the human disease[END_REF]. Chronic cyclic IH exposure also induces an oxidative stress in the brain, lung and liver, in proportion to hypoxia intensity [START_REF] Quintero | The effects of intermittent hypoxia on redox status, NF--kappaB activation, and plasma lipid levels are dependent on the lowest oxygen saturation[END_REF] while a 3--day exposure is sufficient to produce oxidative--stress related kidney damage [START_REF] Wu | Metallothionein deletion exacerbates intermittent hypoxia--induced renal injury in mice[END_REF]. Likewise, others and we have shown that IH induces an important oxidative stress in cardiovascular tissues [START_REF] Chen | Oxidative stress and left ventricular function with chronic intermittent hypoxia in rats[END_REF][START_REF] Friedman | Intermittent hypoxia--induced increases in reactive oxygen species activate NFATc3 increasing endothelin--1 vasoconstrictor reactivity[END_REF][START_REF] Ramond | Oxidative stress mediates cardiac infarction aggravation induced by intermittent hypoxia[END_REF][START_REF] Troncoso Brindeiro | Reactive oxygen species contribute to sleep apnea--induced hypertension in rats[END_REF]. Moreover, animals exposed to IH experience the same alterations in oxidant status than OSA patients, namely increased NOX expression and decreased catalase activity [START_REF] Totoson | Atorvastatin protects against deleterious cardiovascular consequences induced by chronic intermittent hypoxia[END_REF].

The hypoxia inducible factor--1 (HIF--1) is a fundamental transcription factor in the molecular physiology of O 2 homeostasis. HIF--1 is a complex protein composed of 2 subunits, the cytosolic HIF--1α (O 2 --sensitive) subunit and the nuclear HIF--1β subunit, that dimerize in the nucleus to form the functional HIF--1 transcription factor and activate gene transcription.

The N--terminal portion of HIF--1α and HIF--1β contains bHLH (basic helix--loop--helix) and PAS (Per--ARNT--Sim homology) domains necessary for heterodimerization and DNA binding [START_REF] Xia | Recent advances in hypoxia--inducible factor (HIF)--1 inhibitors[END_REF]. The C--terminal portion contains two transactivation domains (TAD), called TAD--N and TAD--C separated by an inhibitory domain. Activation of transcription by HIF--1 requires various co activators such as the CREB Binding Protein (CBP)/p300 co--activator. The O 2 --sensitive region of HIF--1α is called the oxygen degradation domain and is pivotal for the stabilization of this subunit under hypoxic conditions.

In well--oxygenated cells, proline hydroxylation of the HIF--1α subunit by prolyl--hydroxylase enzymes (PHDs) leads to binding of the von Hippel--Lindau (VHL) protein, recruitment of an E3--ubiquitin protein ligase complex with subsequent ubiquitination and proteasomal degradation. Moreover, the factor inhibiting HIF--1 (FIH) negatively regulates HIF--1 transactivation by asparagine hydroxylation, which blocks HIF--1 binding on its co--activator CBP/p300 (Fig. 2).

In sustained hypoxia, there is a general consensus that oxygen deprivation is the main mechanism behind HIF--1 activation with an on--going debate on a potential role of mitochondrial ROS [START_REF] Hagen | Oxygen versus Reactive Oxygen in the Regulation of HIF--1alpha: The Balance Tips[END_REF][START_REF] Movafagh | Regulation of hypoxia--inducible factor--1a by reactive oxygen species: new developments in an old debate[END_REF]. In contrast, ROS production by the repetitive desaturation--reoxygenation sequences induced by IH appears to play a major role in up--regulating HIF--1 activity through complex mechanisms involving prolyl--hydroxylase inactivation and HIF--1 transcription and transactivation (Fig. 3). Briefly, repetitive apneas and intermittent hypoxia trigger nicotinamide adenine dinucleotide phosphate (NADPH) oxidase--dependent production of ROS, mostly superoxide anions, which in turn activate phospholipase C (PLC) leading to calcium--calmodulin kinase (CamK) and protein kinase C (PKC) activation. PKC stimulates mammalian target of rapamycin (mTOR)--dependent synthesis of HIF--1α and inhibits PHD--dependent HIF--1α degradation [START_REF] Prabhakar | Adaptive and maladaptive cardiorespiratory responses to continuous and intermittent hypoxia mediated by hypoxia--inducible factors 1 and 2[END_REF]. CamK also promotes the interaction between HIF--1α and its coactivator p300, thereby leading to increased transcriptional activation upon dimerization with HIF--1β. While HIF--1α is rapidly degraded (t1/2 < 5 min) upon reoxygenation following sustained hypoxia, HIF--1α levels remain elevated following intermittent hypoxia due to the persistent activation of mTOR [START_REF] Semenza | Regulation of oxygen homeostasis by hypoxia--inducible factor 1[END_REF].

Although in vitro studies established that the pro--inflammatory transcription factor nuclear factor--κB (NF--κB) plays a more important role than HIF--1 in the response to IH [START_REF] Taylor | Human adipocytes are highly sensitive to intermittent hypoxia induced NF--kappaB activity and subsequent inflammatory gene expression[END_REF]) [START_REF] Polotsky | Intermittent and sustained hypoxia induce a similar gene expression profile in human aortic endothelial cells[END_REF][START_REF] Ryan | Selective activation of inflammatory pathways by intermittent hypoxia in obstructive sleep apnea syndrome[END_REF], we and others have observed that chronic in vivo exposure to IH leads to a strong and sustained activation of HIF--1 in the heart [START_REF] Belaidi | Prevention of HIF--1 activation and iNOS gene targeting by low--dose cadmium results in loss of myocardial hypoxic preconditioning in the rat[END_REF][START_REF] Belaidi | Major role for hypoxia inducible factor--1 and the endothelin system in promoting myocardial infarction and hypertension in an animal model of obstructive sleep apnea[END_REF][START_REF] Belaidi | Endoplasmic reticulum stress as a novel inducer of hypoxia inducible factor--1 activity: its role in the susceptibility to myocardial ischemia--reperfusion induced by chronic intermittent hypoxia[END_REF], aorta [START_REF] Gras | Endothelin--1 mediates intermittent hypoxia--induced inflammatory vascular remodeling through HIF--1 activation[END_REF], brain [START_REF] Chou | C/EBP homologous binding protein (CHOP) underlies neural injury in sleep apnea model[END_REF]; Z. F. Wang et al., 2009) and liver [START_REF] Da Rosa | Simulating sleep apnea by exposure to intermittent hypoxia induces inflammation in the lung and liver[END_REF]. Nevertheless, the important role of pro--inflammatory pathways is in accordance with these observations since there is a strong crosstalk and synergistic activity between NF--κB and HIF--1 [START_REF] Bruning | NFkappaB and HIF display synergistic behaviour during hypoxic inflammation[END_REF][START_REF] Gorlach | The cross--talk between NF--kappaB and HIF--1: further evidence for a significant liaison[END_REF]) (Fig. 1). An interesting aspect of HIF--1 activation by IH is that it persists when the animals are back in normoxic conditions. This is in agreement with in vitro observations showing that HIF--1 activity remains lastingly elevated after termination of IH [START_REF] Yuan | Induction of HIF--1alpha expression by intermittent hypoxia: involvement of NADPH oxidase, Ca2+ signaling, prolyl hydroxylases, and mTOR[END_REF] and can explain the long--lasting HIF--1 levels and activity observed hours after termination of IH in experimental animals. Although data in OSA patients are scarce, Kazmareck et al. have confirmed that HIF--1α and target genes mRNA are increased in skin biopsies from severe OSA patients and concluded that HIF--1 expression profile may correlate with disease severity and could possibly be used to predict cardiovascular risk [START_REF] Kaczmarek | Molecular biomarkers of vascular dysfunction in obstructive sleep apnea[END_REF].

Among the various HIF--1 target gene products, some encode for proteins that have an impact on the cardiovascular system, such as erythropoietin, inducible/endothelial nitric oxide synthases, vascular endothelial growth factor and ET--1. We have identified ET--1 as a major gene product of interest based on the fact that:

-Through its vasoconstrictive, growth promoting and proinflammatory properties, ET--1 has been implicated in several pathophysiological pathways associated with cardiovascular disease [START_REF] Kaoukis | The role of endothelin system in cardiovascular disease and the potential therapeutic perspectives of its inhibition[END_REF][START_REF] Ohkita | Pathophysiological roles of endothelin receptors in cardiovascular diseases[END_REF].

-Plasma big ET--1 and ET--1 are increased in OSA patients [START_REF] Gjorup | Abnormally increased endothelin--1 in plasma during the night in obstructive sleep apnea: relation to blood pressure and severity of disease[END_REF][START_REF] Karkoulias | The role of Endothelin--1 in obstructive sleep apnea syndrome and pulmonary arterial hypertension: pathogenesis and Endothelin--1 antagonists[END_REF][START_REF] Phillips | Effects of obstructive sleep apnea on endothelin--1 and blood pressure[END_REF][START_REF] Saarelainen | Circulating endothelin--1 in obstructive sleep apnea[END_REF][START_REF] Zamarron | Circulating levels of vascular endothelial markers in obstructive sleep apnoea syndrome. Effects of nasal continuous positive airway pressure[END_REF], in correlation with BP values [START_REF] Anunciato | Big endothelin--1 and nitric oxide in hypertensive elderly patients with and without obstructive sleep apnea--hypopnea syndrome[END_REF][START_REF] Phillips | Effects of obstructive sleep apnea on endothelin--1 and blood pressure[END_REF], and in plasma and tissues of animals exposed to IH [START_REF] Belaidi | Major role for hypoxia inducible factor--1 and the endothelin system in promoting myocardial infarction and hypertension in an animal model of obstructive sleep apnea[END_REF][START_REF] Capone | Endothelin 1--dependent neurovascular dysfunction in chronic intermittent hypoxia[END_REF][START_REF] Kanagy | Role of endothelin in intermittent hypoxia-induced hypertension[END_REF][START_REF] Peng | Endothelin--1 mediates attenuated carotid baroreceptor activity by intermittent hypoxia[END_REF].

-Endothelin receptors, in particular ET--A receptors, are upregulated by IH and the vascular response to ET--1 is increased [START_REF] Allahdadi | Augmented endothelin vasoconstriction in intermittent hypoxia--induced hypertension[END_REF][START_REF] Belaidi | Major role for hypoxia inducible factor--1 and the endothelin system in promoting myocardial infarction and hypertension in an animal model of obstructive sleep apnea[END_REF][START_REF] Friedman | Intermittent hypoxia--induced increases in reactive oxygen species activate NFATc3 increasing endothelin--1 vasoconstrictor reactivity[END_REF][START_REF] Lefebvre | Functional assessment of vascular reactivity after chronic intermittent hypoxia in the rat[END_REF][START_REF] Pawar | Reactive oxygen species--dependent endothelin signaling is required for augmented hypoxic sensory response of the neonatal carotid body by intermittent hypoxia[END_REF][START_REF] Rey | Expression and immunolocalization of endothelin peptides and its receptors, ETA and ETB, in the carotid body exposed to chronic intermittent hypoxia[END_REF][START_REF] Snow | Role for PKCbeta in enhanced endothelin--1--induced pulmonary vasoconstrictor reactivity following intermittent hypoxia[END_REF][START_REF] Wang | Effects of cyclic intermittent hypoxia on ET--1 responsiveness and endothelial dysfunction of pulmonary arteries in rats[END_REF].

-ET--1 has the ability to increase NADPH oxidase activity and HIF--1 expression in part through its ET--A receptors (L. [START_REF] Li | Endothelin--1 increases vascular superoxide via endothelin(A)--NADPH oxidase pathway in low--renin hypertension[END_REF][START_REF] Pisarcik | Activation of hypoxia--inducible factor--1 in pulmonary arterial smooth muscle cells by endothelin--1[END_REF], thus further promoting superoxide production and HIF--1 activity in a feed--forward mechanism (Fig. 3).

In the following sections, we will present the experimental pharmacological evidence in favor of an inhibition of the ROS--HIF--1--endothelin axis in the prevention of the cardiovascular complications induced by IH.

Role in systemic hypertension

Numerous experimental studies have confirmed that reduction in oxidative stress level, either by NOX--2 deficiency [START_REF] Schulz | Arterial hypertension in a murine model of sleep apnea: role of NADPH oxidase 2[END_REF] or antioxidant treatments [START_REF] Hung | Melatonin ameliorates endothelial dysfunction, vascular inflammation, and systemic hypertension in rats with chronic intermittent hypoxia[END_REF][START_REF] Ramond | Oxidative stress mediates cardiac infarction aggravation induced by intermittent hypoxia[END_REF] prevents the development of hypertension in response to intermittent hypoxia exposure. Endothelin receptor antagonists have also been shown to be very effective in preventing or reversing hypertension in rodents exposed to IH [START_REF] Allahdadi | Endothelin type A receptor antagonist normalizes blood pressure in rats exposed to eucapnic intermittent hypoxia[END_REF][START_REF] Belaidi | Major role for hypoxia inducible factor--1 and the endothelin system in promoting myocardial infarction and hypertension in an animal model of obstructive sleep apnea[END_REF][START_REF] De Frutos | NFATc3 contributes to intermittent hypoxia--induced arterial remodeling in mice[END_REF][START_REF] Kanagy | Role of endothelin in intermittent hypoxia-induced hypertension[END_REF].

Role in vascular dysfunction and remodeling

In rats exposed to intermittent hypoxia, allopurinol treatment (through its xanthine oxidase-inhibiting property) attenuated the impairment in acetylcholine--mediated vasodilation [START_REF] Dopp | Xanthine oxidase inhibition attenuates endothelial dysfunction caused by chronic intermittent hypoxia in rats[END_REF]. In a comprehensive study in mice, Capone et al. confirmed the role of both oxidative stress and ET--1 on the impairment in cerebrovascular endothelial--dependent relaxation induced by IH. Hence, cerebrovascular dysfunction was abolished in mice lacking the NOX2 subunit of NADPH oxidase as well as by local perfusion with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (a free radical scavenger) or with BQ--123 (a selective ET--A receptor antagonist) [START_REF] Capone | Endothelin 1--dependent neurovascular dysfunction in chronic intermittent hypoxia[END_REF]. We have recently demonstrated that oxidative stress was responsible for the IH--induced vascular dysfunction in the rat ophthalmic artery, in particular regarding the increased contractile response to ET--1 [START_REF] Mentek | Effects of intermittent hypoxia on rat ophthalmic artery reactivity: role of endothelin 1, oxidative stress and endothelium derived hyperpolarizing factors[END_REF].

In the case of vascular remodeling, de Frutos et al. demonstrated in mice that IH--induced remodeling of mesenteric arteries could be prevented by the mixed endothelin receptor antagonist bosentan [START_REF] De Frutos | NFATc3 contributes to intermittent hypoxia--induced arterial remodeling in mice[END_REF]. Finally, we have recently established that ET--1 was involved in the inflammatory vascular remodeling induced by IH, through a feed-forward mechanism implicating HIF--1 and its crosstalk with NF--κB [START_REF] Gras | Endothelin--1 mediates intermittent hypoxia--induced inflammatory vascular remodeling through HIF--1 activation[END_REF].

Role in myocardial infarction and stroke

There is experimental data in favor of a major role for oxidative stress and endothelin system activation in the increased sensibility to myocardial infarction induced by chronic IH exposure in rodents. Thus, in mice, the increase in infarct size is associated with enhanced protein carbonylation and lipid peroxidation and decreased thioredoxin content [START_REF] Park | Effects of intermittent hypoxia on oxidative stress--induced myocardial damage in mice[END_REF]. In rats, tempol, melatonin as well as atorvastatin treatment during IH exposure reduce myocardial free radical production and prevent the increase in infarct size [START_REF] Ramond | Oxidative stress mediates cardiac infarction aggravation induced by intermittent hypoxia[END_REF][START_REF] Totoson | Atorvastatin protects against deleterious cardiovascular consequences induced by chronic intermittent hypoxia[END_REF].

The cardiac endothelin system is activated by HIF--1 in rats exposed to IH, resulting in increased tissue ET--1 levels, upregulation of coronary ET--A receptors and enhanced vasoconstriction. The deleterious impact of these effects on the myocardium is evidenced by the ability of bosentan to completely prevent the related increase in infarct size [START_REF] Belaidi | Major role for hypoxia inducible factor--1 and the endothelin system in promoting myocardial infarction and hypertension in an animal model of obstructive sleep apnea[END_REF]. Similar effects on the brain's susceptibility to ischemia remain to be demonstrated. However, Capone et al. have observed that alterations of the cerebral circulation induced by chronic IH in mice were prevented by the selective ET--A receptor antagonist BQ123 [START_REF] Capone | Endothelin 1--dependent neurovascular dysfunction in chronic intermittent hypoxia[END_REF]. In a recent study, we have shown that endoplasmic reticulum (ER) stress and HIF--1 were responsible for the infarct size expansion induced by IH [START_REF] Belaidi | Endoplasmic reticulum stress as a novel inducer of hypoxia inducible factor--1 activity: its role in the susceptibility to myocardial ischemia--reperfusion induced by chronic intermittent hypoxia[END_REF].

Role in myocardial remodeling and dysfunction

In rats, treatment with allopurinol throughout IH exposure significantly improved LV dysfunction [START_REF] Williams | Effects of allopurinol on cardiac function and oxidant stress in chronic intermittent hypoxia[END_REF]. Mice deficient for gp91phox (the membrane--bound catalytic subunit of NOX) did not develop LV remodeling in response to IH [START_REF] Hayashi | Role of gp91phox--containing NADPH oxidase in left ventricular remodeling induced by intermittent hypoxic stress[END_REF]. Furthermore, pitavastatin treatment during IH exposure significantly reduced NADPH oxidase--related superoxide production as well as LV remodeling in mice [START_REF] Inamoto | Pitavastatin reduces oxidative stress and attenuates intermittent hypoxia--induced left ventricular remodeling in lean mice[END_REF].

There is ample evidence that activation of the endothelin system promotes cardiac hypertrophy and HF. Hence, plasma and myocardial ET--1 levels [START_REF] Cavero | Endothelin in experimental congestive heart failure in the anesthetized dog[END_REF][START_REF] Margulies | Increased endothelin in experimental heart failure[END_REF][START_REF] Yorikane | Increased production of endothelin--1 in the hypertrophied rat heart due to pressure overload[END_REF] and ET--1 receptors [START_REF] Sakai | Endogenous endothelin--1 participates in the maintenance of cardiac function in rats with congestive heart failure. Marked increase in endothelin--1 production in the failing heart[END_REF] are increased in animal models of congestive HF and in HF patients [START_REF] Mcmurray | Plasma endothelin in chronic heart failure[END_REF][START_REF] Zolk | Expression of endothelin--1, endothelin--converting enzyme, and endothelin receptors in chronic heart failure[END_REF]. Moreover, ET--1 receptor antagonists [START_REF] Fraccarollo | Chronic endothelin receptor blockade attenuates progressive ventricular dilation and improves cardiac function in rats with myocardial infarction: possible involvement of myocardial endothelin system in ventricular remodeling[END_REF][START_REF] Mishima | Effects of long--term therapy with bosentan on the progression of left ventricular dysfunction and remodeling in dogs with heart failure[END_REF][START_REF] Mulder | Role of endogenous endothelin in chronic heart failure: effect of long--term treatment with an endothelin antagonist on survival, hemodynamics, and cardiac remodeling[END_REF] have been shown to improve LV dysfunction and remodeling and to enhance survival [START_REF] Mulder | Role of endogenous endothelin in chronic heart failure: effect of long--term treatment with an endothelin antagonist on survival, hemodynamics, and cardiac remodeling[END_REF]Sakai et al., 1996a) in HF models. Nevertheless, the role of ET--1 in the development of cardiac remodeling and HF in response to IH still remains to be investigated.

Table 1 summarizes the various experimental studies associating chronic IH to cardiovascular diseases described in this section as well the beneficial effects of targeting ROS or the endothelin system in this context.

Targeting the ROS--HIF--1--ET--1 axis as a novel therapeutic approach in OSA

The experimental evidence presented above clearly underscores the therapeutic potential of interventions targeting this axis in the prevention and/or reduction of cardiovascular alterations in moderate to severe OSA patients. While large--scale clinical investigations specifically designed to address this issue are still pending, we have identified several potentially interesting pharmacological agents that have already been tested clinically or are in pre--clinical development.

Antioxidant treatment

As mentioned earlier, there is an emerging consensus that OSA is an oxidative stress disorder. In accordance, various studies have shown that reversal of apneas by CPAP therapy can reduce OSA--related oxidative stress markers [START_REF] Eisele | Obstructive Sleep Apnea, Oxidative Stress, and Cardiovascular Disease: Evidence from Human Studies[END_REF][START_REF] Jullian--Desayes | Impact of obstructive sleep apnea treatment by continuous positive airway pressure on cardiometabolic biomarkers: a systematic review from sham CPAP randomized controlled trials[END_REF]. However, other studies report limited effects [START_REF] Barceló | Antioxidant status in patients with sleep apnoea and impact of continuous positive airway pressure treatment[END_REF][START_REF] Del Ben | Oxidative stress mediated arterial dysfunction in patients with obstructive sleep apnoea and the effect of continuous positive airway pressure treatment[END_REF][START_REF] Jullian--Desayes | Impact of obstructive sleep apnea treatment by continuous positive airway pressure on cardiometabolic biomarkers: a systematic review from sham CPAP randomized controlled trials[END_REF] and a recent sham--controlled randomized study specifically designed to test the effect of CPAP treatment on primary markers of lipid peroxidation failed to observe significant effects [START_REF] Sivam | Effects of 8 weeks of CPAP on lipid--based oxidative markers in obstructive sleep apnea: a randomized trial[END_REF]. These data, plus the fact that a significant proportion of OSA patients are unable or unwilling to use CPAP therapy, represent a strong argument in favor of studies investigating the effects of antioxidants in OSA patients. Still, apart from some reports of beneficial effects of acute intravenous vitamin C injection (N. J. [START_REF] Buchner | Microvascular endothelial dysfunction in obstructive sleep apnea is caused by oxidative stress and improved by continuous positive airway pressure therapy[END_REF][START_REF] Grebe | Antioxidant vitamin C improves endothelial function in obstructive sleep apnea[END_REF] or of oral allopurinol treatment [START_REF] El Solh | Allopurinol improves endothelial function in sleep apnoea: a randomised controlled study[END_REF], no large--scale clinical studies have been performed. The reason behind this discrepancy could be that while antioxidants have proven effective in preventing or reverting oxidative stress--related cardiovascular alterations in experimental models, they have generally yielded disappointing results when translated to human clinical practice, as recently reviewed in this journal [START_REF] Farías | Pharmacological models and approaches for pathophysiological conditions associated with hypoxia and oxidative stress[END_REF].

Nevertheless, the fact that part of the effectiveness of medications such as statins, angiotensin--converting enzyme (ACE) inhibitors or ARBs is due to their pleiotropic antioxidant and anti--inflammatory properties [START_REF] Siti | The role of oxidative stress, antioxidants and vascular inflammation in cardiovascular disease (a review)[END_REF] supports the use of these agents in the prevention or treatment of the cardiovascular complications associated with OSA (Pepin et al., 2010;[START_REF] Thunstrom | Blood Pressure Response to Losartan and Continuous Positive Airway Pressure in Hypertension and Obstructive Sleep Apnea[END_REF]. This is strengthened by the fact that these drugs interfere with NADPH oxidase activation and expression, a therapeutic strategy potentially more efficient than non--selective ROS scavenging [START_REF] Schramm | Targeting NADPH oxidases in vascular pharmacology[END_REF], particularly in the context of IH and OSA.

Hence, we have shown that the IH--induced vascular remodeling and increase in blood pressure and infarct size were prevented by atorvastatin along with a decrease in NADPH oxidase upregulation and a preservation of superoxide dismutase activity [START_REF] Totoson | Atorvastatin protects against deleterious cardiovascular consequences induced by chronic intermittent hypoxia[END_REF]. In OSA patients, 3 months of atorvastatin treatment lowered blood pressure and improved lipid profile [START_REF] Joyeux--Faure | Response to statin therapy in obstructive sleep apnea syndrome: a multicenter randomized controlled trial[END_REF]. Also, Increased internalization of endothelial CD59 in IH appears to be cholesterol--dependent and is reversed by statins in a CD59--dependent manner [START_REF] Emin | Increased internalization of complement inhibitor CD59 may contribute to endothelial inflammation in obstructive sleep apnea[END_REF].

In accordance, we have shown that valsartan induced a fourfold greater decrease in mean 24--hour BP compared to CPAP in a small cohort of untreated hypertensive OSA patients [START_REF] Pepin | Comparison of continuous positive airway pressure and valsartan in hypertensive patients with sleep apnea[END_REF]. In view of the antioxidant, anti--inflammatory and anti--hypertensive actions of statins, ACE inhibitors and ARBs, their beneficial effects in OSA patients should definitely be confirmed in large--scale clinical trials.

Another interesting therapeutic candidate appears to be melatonin, a circadian rhythm regulating hormone with potent pleiotropic antioxidant properties [START_REF] Tan | Melatonin as a Potent and Inducible Endogenous Antioxidant: Synthesis and Metabolism[END_REF] and whose secretion profile is altered in OSA patients [START_REF] Zirlik | Melatonin and omentin: influence factors in the obstructive sleep apnoea syndrome?[END_REF]. We were among the first to report the potent cardioprotective effects of melatonin against ischemia--reperfusion injuries (ventricular arrhythmias and infarct size) [START_REF] Lagneux | Protective effects of melatonin against ischemia--reperfusion injury in the isolated rat heart[END_REF]. More recently, others and we have shown that melatonin administration throughout IH exposure was very effective in preventing the development of myocardial susceptibility to infarction, hypertension and endothelial dysfunction [START_REF] Hung | Melatonin ameliorates endothelial dysfunction, vascular inflammation, and systemic hypertension in rats with chronic intermittent hypoxia[END_REF][START_REF] Ramond | Oxidative stress mediates cardiac infarction aggravation induced by intermittent hypoxia[END_REF][START_REF] Yeung | Cardioprotective effects of melatonin against myocardial injuries induced by chronic intermittent hypoxia in rats[END_REF]. These effects were also associated with a reduction in IH--induced NADPH oxidase expression and with an increase in antioxidant enzyme activity [START_REF] Hung | Melatonin ameliorates endothelial dysfunction, vascular inflammation, and systemic hypertension in rats with chronic intermittent hypoxia[END_REF]. Of particular relevance to the topic of present review, melatonin also has the ability to inhibit HIF--1 activity and target gene expression, possibly through its antioxidant actions [START_REF] Vriend | Melatonin and the von Hippel--Lindau/HIF--1 oxygen sensing mechanism: A review[END_REF].

Finally, the novel therapeutic concept of replacing antioxidant supplementation by activation of endogenous antioxidants was recently addressed in this journal (J. Y. Chan & Chan, 2015). This approach is particularly interesting in the context of OSA where antioxidant defenses are lowered. Moreover, this could be achieved by non--pharmacological means such as exercise. In a recent study, moderate exercise was shown to increase endogenous antioxidant levels in healthy as well as in diabetic patients and to reduce oxidative DNA damage [START_REF] Pittaluga | Physical exercise and redox balance in type 2 diabetics: effects of moderate training on biomarkers of oxidative stress and DNA damage evaluated through comet assay[END_REF]. In that respect, we have recently shown that high--intensity training during IH exposure reduces pro--apoptotic ER stress and prevents the development of hypertension and the increase in infarct size [START_REF] Bourdier | High--intensity training reduces intermittent hypoxia--induced ER stress and myocardial infarct size[END_REF].

Endothelin system antagonism

Although the major role of endothelin and its receptors in the development of systemic and pulmonary hypertension, coronary atherosclerosis, myocardial infarction and heart failure is well documented [START_REF] Ohkita | Pathophysiological roles of endothelin receptors in cardiovascular diseases[END_REF], endothelin receptor antagonists are currently mainly used for the treatment of pulmonary hypertension where they have proven effective in decreasing pulmonary vascular resistance and increasing cardiac index [START_REF] Channick | Effects of the dual endothelin--receptor antagonist bosentan in patients with pulmonary hypertension: a randomised placebo--controlled study[END_REF]). Yet, mixed receptor antagonists like bosentan [START_REF] Krum | The effect of an endothelin--receptor antagonist, bosentan, on blood pressure in patients with essential hypertension. Bosentan Hypertension Investigators[END_REF], or selective ET--A receptor antagonists like darusentan [START_REF] Black | Efficacy and safety of darusentan in patients with resistant hypertension: results from a randomized, double--blind, placebo--controlled dose--ranging study[END_REF][START_REF] Nakov | Darusentan: an effective endothelinA receptor antagonist for treatment of hypertension[END_REF], have been shown to significantly decrease blood pressure in patients with essential or resistant arterial hypertension. Bosentan improved peripheral [START_REF] Rafnsson | The endothelin receptor antagonist bosentan improves peripheral endothelial function in patients with type 2 diabetes mellitus and microalbuminuria: a randomised trial[END_REF] and the ET--A receptor antagonist atrasentan improved coronary [START_REF] Reriani | Long--term administration of endothelin receptor antagonist improves coronary endothelial function in patients with early atherosclerosis[END_REF] endothelial function in patients with type--2 diabetes and early atherosclerosis, respectively. Finally, in a study in patients with heart failure resistant to standard therapy, bosentan treatment increased cardiac output and decreased systemic and pulmonary vascular resistance [START_REF] Sutsch | Short-term oral endothelin--receptor antagonist therapy in conventionally treated patients with symptomatic severe chronic heart failure[END_REF] although subsequent clinical trials with mixed and ET--A antagonists have failed to show clear clinical benefits [START_REF] Kaoukis | The role of endothelin system in cardiovascular disease and the potential therapeutic perspectives of its inhibition[END_REF]. Moreover, the safety profile of these drugs has been disappointing due to a high incidence of hepatotoxicity, headache and edema.

Nevertheless, a new generation of endothelin system antagonists is emerging such as ET--converting enzyme inhibitors, mixed ECE and neutral endopeptidase inhibitors, ET--B receptor agonists or new potent non--toxic mixed antagonists, such as macitentan [START_REF] Maguire | Endothelin@25 --new agonists, antagonists, inhibitors and emerging research frontiers: IUPHAR Review 12[END_REF]. These promising agents should definitely be tested in experimental IH models in order to compare their efficacy to that of bosentan or ET--A receptor antagonists.

Some authors argue that selective ET--A receptor antagonists are better suited than mixed antagonists for the prevention or treatment of cardiovascular diseases [START_REF] Nasser | Endothelin ETA receptor antagonism in cardiovascular disease[END_REF] but this remains to be confirmed by specifically--designed clinical studies, particularly in the context of OSA since the deleterious effects of IH are mainly due to ET--A receptor upregulation [START_REF] Allahdadi | Endothelin type A receptor antagonist normalizes blood pressure in rats exposed to eucapnic intermittent hypoxia[END_REF][START_REF] Belaidi | Major role for hypoxia inducible factor--1 and the endothelin system in promoting myocardial infarction and hypertension in an animal model of obstructive sleep apnea[END_REF][START_REF] Briancon--Marjollet | Endothelin regulates intermittent hypoxia--induced lipolytic remodelling of adipose tissue and phosphorylation of hormone--sensitive lipase[END_REF][START_REF] Capone | Endothelin 1--dependent neurovascular dysfunction in chronic intermittent hypoxia[END_REF][START_REF] De Frutos | NFATc3 contributes to intermittent hypoxia--induced arterial remodeling in mice[END_REF][START_REF] Mentek | Effects of intermittent hypoxia on rat ophthalmic artery reactivity: role of endothelin 1, oxidative stress and endothelium derived hyperpolarizing factors[END_REF].

In a small randomized controlled pilot study in OSA patients (n=16) with mild untreated hypertension (clinical BD of 142/85 mmHg), we recently showed that a 4--week bosentan treatment resulted in a 3 mmHg greater decrease in 24--h DBP than CPAP, but this effect was minimal and not significant compared to baseline (Joyeux--Faure et al., 2015). This could be due to the fact that these patients were not severe enough in terms of hypertension and sleep apnea level. Hence, in view of the clear involvement of the ET--1 system in the severe IH--induced cardiovascular alterations and of the dramatic effects of ET--1 receptor antagonists in this context [START_REF] Belaidi | Major role for hypoxia inducible factor--1 and the endothelin system in promoting myocardial infarction and hypertension in an animal model of obstructive sleep apnea[END_REF][START_REF] Briancon--Marjollet | Endothelin regulates intermittent hypoxia--induced lipolytic remodelling of adipose tissue and phosphorylation of hormone--sensitive lipase[END_REF][START_REF] Gras | Endothelin--1 mediates intermittent hypoxia--induced inflammatory vascular remodeling through HIF--1 activation[END_REF], large--scale clinical investigations of ET--1 system antagonism in moderate to severe OSA patients are undoubtedly warranted.

HIF--1 inhibition

Since both ROS and ET--1 have the ability to promote HIF--1 activity in a feed--forward manner (Fig. 3), HIF--1 appears to be a pivotal target to reverse the deleterious cardiovascular consequences of chronic IH and OSA. Various novel molecular and pharmacological interventions to reduce HIF--1 expression or activity have reached the phase of clinical translation, mostly in the field of cancer research (Fig. 4). These could potentially be investigated in OSA patients after prior pre--clinical studies evaluating the most appropriate therapeutic approaches to prevent excessive HIF--1 activation without suppressing its essential physiological actions.

Genetic strategies to decrease HIF--1α protein levels

MicroRNAs (miRNAs) are a class of single--stranded non--coding RNAs that negatively regulate eukaryotic gene expression at the post--transcriptional and translational levels. Several miRNAs, named hypoxamiRs, are upregulated by hypoxia through various transcription factors including HIF--1 and NF--κB [START_REF] Cottrill | Hypoxamirs and mitochondrial metabolism[END_REF][START_REF] Nallamshetty | Hypoxia: a master regulator of microRNA biogenesis and activity[END_REF]. Amongst them, miR--210 is a ubiquitous HIF--1 target gene that has been identified has a major prognostic and therapeutic target in a number of hypoxia--related diseases such as tumor progression, myocardial infarction and pulmonary hypertension [START_REF] Cottrill | Hypoxamirs and mitochondrial metabolism[END_REF].

Reciprocally, miR--210 and other hypoxamiRs engage in regulatory feedback loops with HIF--1.

Hence, miR--210 and miR--424 have the ability to promote HIF--1 stabilization through suppression of glycerol--3--phosphate dehydrogenase 1--like enzyme and cullin 2, respectively [START_REF] Nallamshetty | Hypoxia: a master regulator of microRNA biogenesis and activity[END_REF]. Moreover, miR--210 appears to be involved in the ROS--mediated HIF--1 stabilization [START_REF] Movafagh | Regulation of hypoxia--inducible factor--1a by reactive oxygen species: new developments in an old debate[END_REF][START_REF] Nallamshetty | Hypoxia: a master regulator of microRNA biogenesis and activity[END_REF].

On the other hand, targeting of the 3' untranslated region (3' UTR) of Hif1a by miR--155 [START_REF] Bruning | MicroRNA--155 promotes resolution of hypoxia--inducible factor 1alpha activity during prolonged hypoxia[END_REF], miR--519 [START_REF] Cha | MicroRNA--519c suppresses hypoxia--inducible factor--1alpha expression and tumor angiogenesis[END_REF], miR--20b [START_REF] Lei | Regulation of HIF--1alpha and VEGF by miR--20b tunes tumor cells to adapt to the alteration of oxygen concentration[END_REF] or the miR--17/92 cluster [START_REF] Taguchi | Identification of hypoxia--inducible factor--1 alpha as a novel target for miR--17--92 microRNA cluster[END_REF] decreases HIF--1α protein levels.

Thus delivery of miRNA antagonists (miR--210, miR--424) or mimics (miR--155, miR--519, miR--20b, miR17/92) represents a promising new approach to reduce HIF--1 activity that should be tested in pre--clinical IH models. Translation to clinical studies is possible in view of the rapid development of miRNA--based therapy and of its apparent lack of adverse effects [START_REF] Bader | Developing therapeutic microRNAs for cancer[END_REF].

Chemical inhibitors

Several anti--cancer agents acting at different levels of HIF--1 signaling have been tested in clinical trials, or are being investigated in pre--clinical studies. These molecules inhibit HIF--1 activity by interfering with HIF--1α mRNA expression, translation, dimerisation with HIF--1β

and DNA binding or by promoting HIF--1α degradation [START_REF] Xia | Recent advances in hypoxia--inducible factor (HIF)--1 inhibitors[END_REF]. Below is presented a selection of small molecular HIF--1 inhibitors along with some experimental data in favor of their potential use in the prevention or treatment of IH--and OSA--induced cardiovascular alterations.

Camptothecin, a natural inhibitor of topoisomerase I, prevents HIF--1α transcription and has proven beneficial in phase I and II clinical studies [START_REF] Rapisarda | Topoisomerase I--mediated inhibition of hypoxia--inducible factor 1: mechanism and therapeutic implications[END_REF]. Interestingly, camptothecin also has the ability to decrease HIF--1 expression and activity by up--regulating miR--155 expression [START_REF] Bertozzi | The natural inhibitor of DNA topoisomerase I, camptothecin, modulates HIF--1alpha activity by changing miR expression patterns in human cancer cells[END_REF].

Antisense oligodeoxynucleotide HIF--1α mRNA inhibitors, such as EZN--2968, have been shown to reduce the expression of its target genes in cell and animal models [START_REF] Greenberger | A RNA antagonist of hypoxia--inducible factor--1alpha, EZN--2968, inhibits tumor cell growth[END_REF] as well as in cancer patients [START_REF] Jeong | Pilot trial of EZN--2968, an antisense oligonucleotide inhibitor of hypoxia--inducible factor--1 alpha (HIF--1alpha), in patients with refractory solid tumors[END_REF].

YC--1, 3--(5--hydroxymethyl--2--furyl)--1--benzyl indazole, first described as a platelet guanylate cyclase activator [START_REF] Ko | YC--1, a novel activator of platelet guanylate cyclase[END_REF], is an approved cancer drug that prevents HIF--1α from interacting with the transcriptional co--activators p300/CBP by activating FIH (S. H. Li et al., 2008). In cardiomyocytes, HIF--1 inhibition by YC--1 effectively represses VEGF induction [START_REF] Hui | M3--mAChR stimulation exerts anti--apoptotic effect via activating the HIF--1alpha/HO--1/VEGF signaling pathway in H9c2 rat ventricular cells[END_REF].

Acriflavine, a cutaneous antibacterial and antiviral agent, prevents HIF--1 dimerisation by binding to the PAS subdomain of HIF--1α, thereby inhibiting HIF--1 DNA--binding and transcriptional activity [START_REF] Lee | Acriflavine inhibits HIF--1 dimerization, tumor growth, and vascularization[END_REF]. Moreover, acriflavine prevents HIF--1 activity in C2C12 mouse myocytes exposed to IH [START_REF] Cai | Hypoxia--inducible factor 1 is required for remote ischemic preconditioning of the heart[END_REF].

Echinomycin is an antibiotic that can bind DNA in a sequence--specific fashion and that interferes with HIF--1--induced gene transcription by binding to its hypoxia response elements (D. [START_REF] Kong | Echinomycin, a small--molecule inhibitor of hypoxia--inducible factor--1 DNA--binding activity[END_REF].

Finally, the antifungal antibiotic trichostatin, a class I and II histone deacetylase inhibitor, promotes the proteasomal degradation of HIF--1α [START_REF] Geng | HIF1alpha protein stability is increased by acetylation at lysine 709[END_REF][START_REF] Kong | Histone deacetylase inhibitors induce VHL and ubiquitin--independent proteasomal degradation of hypoxia--inducible factor 1alpha[END_REF]. It was recently shown to repress HIF--1--VEGF signaling and to inhibit choroidal angiogenesis in vivo (N. Chan et al., 2015).

Natural inhibitors

Various natural substances possess HIF--1 inhibiting activities. Moracin O, from Morus Alba also known as white mulberry, inhibits HIF--1 translocation and activity due to its particular structure containing a 2--arylbenzofuran ring [START_REF] Xia | HIF--1alpha inhibitors: synthesis and biological evaluation of novel moracin O and P analogues[END_REF]. Curcumin, a yellow spice from Curcuma longa, is known to repress HIF--1 activity via its action on HIF--1β [START_REF] Choi | Curcumin inhibits hypoxia-inducible factor--1 by degrading aryl hydrocarbon receptor nuclear translocator: a mechanism of tumor growth inhibition[END_REF]. And finally, Manassantin A, from the water plant Saururuschinensis, prevents HIF--1α nuclear translocation [START_REF] Hossain | Saururus cernuus lignans----potent small molecule inhibitors of hypoxia-inducible factor--1[END_REF].

Conclusion

Although continuous positive airway pressure treatment, the first line therapy for OSA, clearly improves quality of life and sleepiness, positive response to CPAP in terms of cardiovascular and metabolic outcome is hampered by poor patient adherence, suboptimal use or limited efficacy. Novel therapeutic strategies are thus required to help counteract the key mechanisms responsible for OSA--related cardiovascular consequences. These therapeutic agents could be used in OSA patients unable or unwilling to use CPAP or in combination with CPAP in order to increase its efficacy.

This review supports a role for the ROS--HIF--1--endothelin axis in the deleterious cardiovascular consequences of the chronic intermittent hypoxia associated with moderate to severe OSA. It also stresses the importance to extend fundamental and clinical research around the pivotal role of the HIF--1 transcription factor in this context.

Table 1. Summary of experimental studies demonstrating the beneficial effects of targeting reactive oxygen species (ROS), HIF--1 and the endothelin--1 (ET--1) system in animal models of cardiovascular complications associated with intermittent hypoxia (IH). Gp91phox: MnTBAP: Mn(III)tetrakis(4--benzoic acid)porphyrin chloride; NOX2: gp91phox subunit of NADPH oxidase. FIH' YC21'

a

  Pharmacology and Therapeutics, 2016, [Epub ahead of print]. Université Grenoble Alpes --Laboratoire HP2 --Grenoble --F--38042 France b INSERM --U1042 --Grenoble --F--38042 France c CHU de Grenoble --Grenoble --F--38042 France

Fig. 3 .

 3 Fig. 3.Signal transduction pathways by which obstructive sleep apnea (OSA), intermittent hypoxia (IH) and reactive oxygen species (ROS) induce HIF--1α protein stability, synthesis and transactivation and the promoting effect of endothelin--1 (ET--1) (adapted with permission from(Semenza)). See text for detailed description and additional abbreviations.

Conflicts of interest

None. ET--1 BQ--123 (10 μM perfusion) [START_REF] Capone | Endothelin 1--dependent neurovascular dysfunction in chronic intermittent hypoxia[END_REF]

Cardiovascular


Samir Vartabi Kashanian

Aurélien Eloy

William Guerin

Michel Lintz

Mathilde Fouché
email: mathilde.fouche@inln.cnrs.fr

Robin Kaiser

Noise spectroscopy with large clouds of cold atoms

Noise measurement is a powerful tool to investigate many phenomena from laser characterization to quantum behavior of light. In this paper, we report on intensity noise measurements obtained when a laser beam is transmitted through a large cloud of cold atoms. While this measurement could possibly investigate complex processes such as the influence of atomic motion, one is first limited by the conversion of the intrinsic laser frequency noise to intensity noise via the atomic resonance. This conversion is studied here in details. We show that, while experimental intensity noise spectra collapse onto the same curve at low Fourier frequencies, some differences appear at higher frequencies when the probe beam is detuned from the center of the resonance line. A simple model, based on a mean-field approach, which corresponds to describing the atomic cloud by a dielectric susceptibility, is sufficient to understand the main features. Using this model, the noise spectra allow extracting some quantitative informations on the laser noise as well as on the atomic sample.

I. INTRODUCTION

Progress in the study of light-matter interaction has opened the way to important developments in quantum optics. In particular, laser-cooled atoms are used to develop quantum memories [1], novel laser designs [2,3], or to prepare non classical states for potential use in metrology [4,5]. To investigate the nonclassical behavior of light, the study of average intensities is not sufficient and one needs to measure coincidence rates [6,7], correlation and anticorrelation functions [7][8][9][10][11] or noise and fluctuations [12][13][14][15]. Measuring the light fluctuations and correlations after their interaction with cold atomic samples also provides information on the atomic motion [16][17][18] and could be used to characterize more subtle effects due to interference effects in multiple scattering [19] or obtain direct evidence of the random laser operation in cold atoms [20].

In this work, we address a particular configuration in which intensity noise measurements are performed on a laser beam transmitted through a sample of laser-cooled atoms. This transmission geometry is relevant to investigate different properties, such as the reduction of the noise below the shot-noise level (squeezing) [21,22], the extra noise due to the atomic internal structure via Raman scattering [23], the cooperative fluorescence from a strongly driven dilute cloud of atoms [24], or two-photon optical nonlinearity [11]. However in this kind of geometry, and contrary to the fluorescence configuration, the contribution of the intrinsic noise of the involved lasers can be especially important, with in particular the conversion of laser frequency or phase noise to intensity noise through the atomic resonance. This technical noise may be hard to distinguish from the signal under study and a good understanding of this process is thus essential.

The frequency to intensity conversion, in which the atomic resonance acts as a frequency discriminator, was first reported in [37]. It was then studied theoretically [38][39][40] as well as experimentally, using atomic or molecular resonance, either to measure the laser properties [41,42], to extract atomic characteristics [43,44] or to study the light-matter interaction [45][46][47][48]. This phenomenon has been thus extensively studied. However, all previous experimental studies used room-temperature or hot vapors. In this case, the Doppler effect needs to be taken into account and limits the atomic spectral linewidth. On the other hand, cold atomic samples correspond to a system where the Doppler effect can generally be ignored. The atomic spectral linewidth is reduced, improving the frequency to intensity noise conversion. This results in a quantitative change of the noise spectra, but also in a qualitative change as we will see in this paper, with in particular the appearance of structures for Fourier frequencies higher than the atomic linewidth.

In this paper, we study the frequency to intensity noise conversion of a laser going through a cold atomic cloud. The experimental setup and the results are presented in Sec. II. Whereas all the noise spectra collapse onto the same curve at low Fourier frequencies, some differences appear at higher Fourier frequencies when the probe beam is detuned by various amonts from the center of the resonance line. We show in Sec. III that the low Fourier frequencies components are well understood using the frequency discriminator approach, corresponding to what has been already obtained with room temperature or hot vapors. The apparent discrepancies at high Fourier frequencies are addressed in Sec. IV. We will show that a 'mean-field approach' [25], in which the atomic cloud is described by a complex index of refraction, is sufficient to explain, qualitatively and quantitatively, the measured spectra of the intensity noise, even when the on-resonance optical thickness is large. Finally, Appendix A presents the characterization of our probe laser with standard techniques, which serves as a bench-mark for our measurements with cold atoms.

II. NOISE SPECTROSCOPY WITH COLD ATOMS

A. Apparatus

The experimental setup, based on measuring the intensity noise of a weak probe beam transmitted through a cloud of cold atoms, is depicted in Fig. 1a. The atomic cloud is obtained by loading a magneto-optical trap (MOT) with 85 Rb atoms. A compression is applied to increase the atomic density [49]. The maximum number of atoms is N ≃ 10 10 with a temperature of about 100 µK and a cloud rms radius of R ≃ 1 mm. The first pulse provides the transmission through the atomic cloud, and the second one allows us to measure the incident intensity without atoms in order to calculate the normalized transmission for each cycle. The atoms are removed by applying the MOT beams at resonance during t push = 6 ms between the two probe pulses. For the PSD measurements the time window of the oscilloscope is set tpause = 200 µs after the beginning of the first probe within tosc = 100 µs.

The probe beam is delivered by a distributed-feedback (DFB) laser. This laser, also used for the MOT beams, is amplified by a tapered amplifier. The laser frequency is locked using a master/slave configuration with an offset locking scheme [26] and set close to the F = 3 → F ′ = 4 hyperfine transition of the D2 resonance line of 85 Rb. A double-pass acousto-optical modulator (AOM) is used to change the laser detuning δ = (ω L -ω 0 )/Γ from this transition. The parameter ω L /2π = ν L is the laser frequency, ω 0 /2π the atomic transition frequency and Γ/2π = 6.07 MHz the natural transition linewidth. The laser beam inside the atomic cloud is linearly polarized and its waist is about 300 µm. The intensity is adjusted to have a saturation parameter lower than 0.1. The measurements are realized after a fixed t TOF = 4 ms time of flight (TOF) and the laser beam path has been aligned to correspond to the centre of the atomic cloud after this TOF.

The laser beam after propagation through the atomic cloud is collected by a homemade transimpedance photodiode which has two outputs ports. The first port, corresponding to the DC output, is used to measure the probe transmission. The intensity noise is measured thanks to the AC output of the photodiode amplified by a low noise AC amplifier. The frequency response of the photodiode has been measured by illuminating it with a shot noise limited thermal light bulb. Its bandwidth ranges from about 10 kHz to 10 MHz. The power spectral density (PSD) of the detected signal is computed by an oscilloscope and the measured intensity noise PSD is finally normalized by the frequency response of the detection system.

The probe beam is applied during t p = 1.2 ms but we fix the oscilloscope time window to t osc = 100 µs, t pause = 200 µs after the beginning of the probe pulse. To increase the signal to noise ratio, data are integrated over 100 cycles. We also record for each cycle the power of the probe beam without atoms I 0 , which is needed for intensity to frequency noise conversion. This measurement is done by applying a second probe pulse after having removed all the atoms by shining the MOT beams at resonance during t push = 6 ms. The time sequence is sketched in Fig. 1b. The duration t i includes the loading and compression stages. The optical thickness is varied by changing the total number of atoms through the MOT loading time.

B. Transmission curve and frequency discriminator

Before noise measurements, we acquire the transmission curve by scanning the laser through the atomic transition. A typical transmission curve is plotted in Fig. 2. The on-resonance optical thickness b 0 is extracted by fitting the data by the expected transmission,

T ≡ I a I 0 = e -b(δ) , (1) with b
(δ) = b 0 1 + 4δ 2 .
(

) 2
The parameter I a corresponds to the power measured with atoms while I 0 is measured without atoms.

The dependance of the transmission with the laser frequency allows us to use it as a frequency discriminator. The relation between the PSD S T in [Hz -1 ] of the transmitted intensity normalized by the intensity without atoms, T = I a /I 0 , and the laser frequency noise PSD (FNPSD) S νL in [Hz 2 /Hz] is given by the following equation,

S T = dT dν L 2 S νL (3) = D 2 S νL , (4)
valid when the transmission curve can be locally approximated by a line whose slope is equal to D. As we will see in Sec. IV B, this is a good approximation for low Fourier frequencies. Using Eqs. ( 1) and ( 2), one finds:

D = dT dν L = 16πδb 0 Γ (1 + 4δ 2 ) 2 e - b 0 1+4δ 2 . (5)
The discriminator slope D depends on the optical thickness b 0 as well as the laser detuning δ. A typical curve is plotted in Fig. 2 for an optical thickness of 19. The best conversion is obtained when |D| is maximum, corresponding to |δ| ≃ 1.8 for b 0 = 19.

C. Experimental results

We have measured the transmitted intensity noise PSD for three different on-resonance optical thicknesses: b 0 = 6.5, 19 and 51.5. For each b 0 , the laser detuning is adjusted, thanks to the double-pass AOM, to be at the maximum of the discriminator slope on the blue side of the atomic transition. We have first checked that the detection background and the intrinsic laser intensity noise, measured without atoms, are well below the intensity noise PSD measured with atoms. The PSD S T is then converted to FNPSD using Eq. ( 3).

The results are plotted in Fig. 3 for three optical thicknesses. Whereas all the PSDs are consistent at low frequencies, typically below 1 MHz, some differences appear at higher frequencies. 3) is valid and the curves thus correspond to the laser frequency noise PSD (FNPSD). The FNPSD measured with a Fabry-Perot cavity as a frequency discriminator is plotted in grey (see appendix A for more details). The dashed line corresponds to the β-separation line used to estimate the laser linewidth.

III. NOISE SPECTROSCOPY AT LOW FREQUENCIES: LASER SPECTRAL PROPERTIES

For low Fourier frequencies, typically for f n below 1 MHz, Eq. ( 3) is valid and the curves S T /D 2 plotted in Fig. 3 thus correspond to the laser FNPSD S νL . This is also confirmed by comparing these curves to the one obtained with a Fabry-Perot (FP) cavity used as frequency discriminator (see appendix A for more details).

The measurements of the laser FNPSD can be used to recover the laser linewidth. The first method is to use the formula which links the FNPSD and the optical spectrum [28]:

S E (ν) = 2 ∞ -∞ e -2iπντ E 2 0 e 2iπνLτ exp -2 ∞ 0 S νL (f n ) sin 2 (πf n τ ) f 2 n df n dτ.(6)
However, most of the time, there is no analytical solution for this equation and one needs to perform a tedious numerical integration. As an alternative method, the laser linewidth can be estimated using a simple geometrical approach, based on the so-called β-separation line. Since the first theoretical study [29], this approach has been applied in many experimental setups [30][31][32][33] and further refined [34,35]. The β-separation line is defined as [29]:

S β (f n ) = 8ln(2) π 2 f n . (7)
This line, plotted in Fig. 3 and in Fig. 10, separates the noise spectrum in two regions. For S β (f n ) < S νL (f n ), frequency noise contributes to the central Gaussian part of the laser line shape and thus to the linewidth. For S β (f n ) > S νL (f n ), frequency noise contributes to the Lorentzian wings of the line shape and does not significantly affect the linewidth. This method approximates the laser linewidth ∆ν L , corresponding to the FWHM of the central part of the line shape, by ∆ν L = 8ln(2)A, (8) with A the area below S νL in the frequency range where S νL is above the β-separation line, i.e.:

A = +∞ 1/T obs H S νL (f n ) -S β (f n ) S νL (f n )df n , (9)
where T obs is the observation time, and H is the Heaviside step function.

The values obtained with this approach are listed in Table I together with the values obtained with the Fabry-Perot cavity. The uncertainties obtained with the cold atomic cloud take into account the statistical uncertainty (standard deviation of the linewidth measurements obtained in similar conditions) as well as the estimation of the maximum error due to the β-separation line approach [34]. The laser linewidth has also been measured using the beat-note technique (see appendix A). All these results are compatible, validating the fact that the noise measured with the cold atomic sample at low Fourier frequencies corresponds to the intrinsic laser frequency noise.

IV. NOISE SPECTROSCOPY AT HIGH FREQUENCIES: ATOMIC CLOUD PROPERTIES A. Experimental results

We can see in Fig. 3 that the PSDs differ at high frequencies, with in particular the appearance of a small "bump". However, these curves becomes limited by the noise floor of the photodiode for frequencies higher than 1 MHz. To overcome this problem, the photodiode and the amplifier have been replaced by a new photodiode, with a high cutoff frequency of 240 MHz and with a lower noise floor.

Typical FNPSDs, obtained with this low noise photodiode, are zoomed at high frequencies in Fig. 4. The three curves have been measured with the same optical thickness b 0 = 19 but for three different laser detunings. We clearly see the appearance of bumps whose frequency positions depend on the laser detuning. These positions also depend on the optical thickness as shown in Fig. 6, where the frequency position of the first and second bump is plotted as a function of the laser detuning and for the three previous optical thicknesses.

B. Modeling

To understand what happens at high frequencies, we model the laser frequency noise as a carrier at frequency ν L with two weak sidebands at ν L ±f n as done in Ref. [47]. It corresponds to a phase modulation where the laser field can be written as follows:

E = E 0 e i[2πνLt+B φ sin(2πfnt)] , (10)
≃ E 0 e i2πνLt + B φ 2 e i2π(νL+fn)t - B φ 2 e i2π(νL-fn)t . (11)
The parameter B φ corresponds to the phase modulation depth. The corresponding amplitude of the frequency noise at frequency f n is B φ f n . When the laser goes through the atoms, the carrier and the two sidebands experience different transmissions √ T and phase shifts φ:

T (δ) = e - b 0 2(1+4δ 2 ) , (12)
φ(δ) = - b 0 δ 1 + 4δ 2 . ( 13
)
The laser intensity transmission as a function of time t becomes

I a I 0 = T 0 1 + B φ C φ √ T 0 cos (2πf n t + ψ) , (14)
where ψ is a phase shift and

C φ = T 1 + T 2 -2 T 1 T 2 cos(2φ 0 -φ 1 -φ 2 ), ( 15
)
where T 0 and φ 0 are the intensity transmission and phase shift induced by the atoms on the carrier and T 1 , T 2 , φ 1 , φ 2 the intensity transmission and phase shifts induced on the two sidebands. The theoretical frequency to intensity noise conversion is thus given by:

S T,th = δI a I 0 2 = T 0 C 2 φ f 2 n S νL . (16)
We can show that we recover the conversion given by Eqs.

(3) and ( 5) for f n ≪ Γ and f n ≪ δΓ respectively. The same approach can be used if one deals with laser amplitude noise instead of frequency noise. In this case, the laser field can be written as follows:

E = E 0 1 + B E sin(2πf n t)e i2πνLt , (17)
= E 0 e i2πνLt -i B E 2 e i2π(νL+fn)t + i B E 2 e i2π(νL-fn)t , (18)
with B E the amplitude modulation depth. The laser intensity transmission is calculated as previously and one gets:

I a I 0 = T 0 1 + B E C E √ T 0 cos (2πf n t + ψ) , (19)
with

C E = T 1 + T 2 + 2 T 1 T 2 cos(2φ 0 -φ 1 -φ 2 ). ( 20
)
The normalized intensity PSD of the transmitted beam is finally given by:

S T,th = δI a I 0 2 = T 0 C 2 E S E , (21)
where S E corresponds to the normalized laser amplitude noise PSD.

In either case, the shape of S T depends on the Fourier frequency through the phase shifts φ 1 and φ 2 induced by the atoms. We can also see that the two types of noises give the same typical equations with only a change of sign in Eqs. ( 15) and ( 20). This change of sign is then responsible for the shape dependance of the PSD S T on the type of laser noise.

C. Comparison between experimental and modeling results

The previous model is used to calculate the expected noise of the transmitted intensity. We assume a white frequency noise for frequencies higher than 1 MHz. Its value, extracted from the measured PSD at f n = 1 MHz, is set to (B φ f n ) 2 ≃ S νL ≃ 10 5 Hz 2 /Hz. This is injected in Eq. ( 16) to calculate the expected transmission noise PSD S T,th . We then divide it by the discriminator slope given by Eq. ( 5) in order to compare it to the measurements.

Typical calculated and measured PSD are compared in Fig. 5. The optical thickness is b 0 = 19 and the laser detuning is δ = 3. We see a good overlap between the measured and the calculated PSD, without any free parameter. In particular, the model predicts the existence of two bumps whose frequency positions correspond to the ones experimentally observed. These bumps are intrinsically related to the fact that we deal with frequency noise. On the contrary, if we do the calculations assuming an incident laser with amplitude noise, one sees the appearance of dips instead of bumps as shown in Fig. 5. The analysis of the noise spectra at high Fourier frequencies, with the presence of bumps or dips, thus allows to extract the nature of the laser noise. 15) and ( 16) assuming a white frequency noise. Dashed line: S T,th /D 2 calculated using Eq. ( 20) assuming a white amplitude noise.

Finally, we have compared the measured and the calculated bump positions, corresponding to the frequency position of the local maxima, as a function of the laser detuning and for the three different optical thicknesses. The results are plotted in Fig. 6, assuming laser frequency noise for the calculations. We obtain a very good agreement between measurements and calculations, validating the model used to understand the frequency to intensity noise conversion. We can also notice that the frequency difference between both bump positions remains constant, at least for sufficiently high laser detuning, and that this difference roughly corresponds to the frequency range where the transmission curve is close to zero. Both bumps can thus be interpreted as the signature of the beat-note between the carrier and the phase shifted and attenuated sideband that goes from one side of the transmission curve to the other.

The frequency positions of the bumps in the FNPSD as well as the difference between both bump positions depend on the laser detuning from the atomic transition and on the optical thickness. Their measurements thus allow extracting information on the discriminator medium itself. One could imagine to extract the two last quantities by directly measuring one FNPSD instead of measuring the entire transmission curve by scanning the laser frequency around the atomic transition.

V. CONCLUSION

In this paper, we have studied the intensity noise on a laser beam transmitted through a cold atomic cloud. In this forward configuration, we have observed the conversion of the intrinsic laser frequency noise to intensity noise, the atomic transition playing the role of a frequency discriminator whose slope is adjustable through the optical thickness. While we recover the same FNPSD at low Fourier frequencies using a Fabry-Perot cavity, some differences appear at higher Fourier frequencies and one needs to go beyond the linear response approximation of the discriminator given in Eq. (3). We have shown that a simple model in which the frequency noise is modeled as a carrier with two sidebands and the atomic cloud as a medium with an index of refraction is sufficient to describe the observations. The measurement of one single spectrum of the intensity noise allows us to extract much information. The conversion of the laser frequency noise to intensity noise can be used to characterize the laser noise. However one can also extract the nature of the laser noise or some important characteristics of the atomic sample such as its optical thickness. Usually perceived as a drawback, frequency to intensity noise conversion can clearly be seen as an important source of information.

Finally, in this forward direction, the conversion of the intrinsic laser frequency noise to intensity noise is usually an important source of noise. An accurate understand-ing of this effect is thus of crucial importance. With this conversion now well characterized, intensity noise measurements could possibly be used to extract some signatures of more involved phenomena, such as the observation of the influence of atomic motion and quantum optical properties [11,[21][22][23].

Line shape and linewidth

The optical spectrum corresponds to the power spectral density (PSD) of the laser electric field S E (ν). It is measured using a beat-note technique sketched in Fig. 7. The beat-note signal is obtained using two different lasers. The first one is the DFB laser we want to characterize. For the second laser we have used a DL commercial external-cavity diode laser (ECDL) from TOPTICA, whose linewidth is specified to be lower than 500 kHz [50]. The lasers are independently frequency locked and the frequency difference between them is typically 1 GHz. The two lasers are injected into a 50 : 50 fiber coupler. The beat-note signal is then detected by a 9.5 GHz bandwidth photodiode and its power spectral density is measured by a spectrum analyzer.

The beat-note PSD is plotted in Fig. 8. It corresponds to the convolution of the optical spectrum of the two lasers, but since the linewidth of the TOPTICA laser is much smaller than the DFB laser one, it is mainly dominated by the DFB optical spectrum. It contains a central part at the frequency difference of the two lasers, which can be fitted by a Gaussian, superposed on large wings, which can be fitted by a Lorentzian. The linewidth, given

Frequency noise PSD

Measurements of the laser optical spectrum, and the corresponding linewidth, and the laser FNPSD are complementary. But, while the first one is convenient to quickly compare different types of lasers, the second one gives a much more complete knowledge of the laser spectral properties.

One of the most common frequency discriminator, used to measure laser FNPSD S νL (f n ), is the Fabry-Perot (FP) cavity. Its transmission depends on the light frequency which allows to convert frequency noise into intensity noise. For a laser linewidth smaller than the cavity linewidth ∆ν c , the conversion between the FNPSD and the normalized intensity noise PSD (INPSD) of the transmitted beam is The experimental setup is shown in Fig. 9. We use a free-running confocal FP cavity with a length L c ≃ 10 cm. The corresponding free spectral range is ∆ν FSR = c/4L c ≃ 750 MHz and the cavity linewidth is ∆ν c ≃ 20 MHz. A piezoelectric transducer is placed on one of the mirrors, allowing us to adjust the cavity-to-laser detuning to the half of the cavity resonance. The laser transmitted by the cavity is detected by the same homemade transimpedance photodiode used for the first measurements with the cold atomic cloud. The PSD of the detected signal is recorded by an oscilloscope computing its power spectrum and normalized by the frequency response of the detection system. It is finally converted to FNPSD using Eq. (A2).

The FNPSD for the DFB laser is depicted in Fig. 10. We have checked that the PSD is not limited by the detection background or by the intrinsic laser intensity noise. The FNPSD essentially decreases as 1/f n up to 1 MHz, corresponding to a flicker noise.

As done in section III, we can extract the laser linewidth using the β-separation line approach. We obtain a linewidth of 3.4 ± 0.4 MHz for the DFB laser. The 10 % relative uncertainty takes into account the maximum typical error introduced by the β-line approach [34], which is an approximate method to estimate the laser linewidth. The laser linewidth is compatible with the one obtained from the beat-note measurement.

FIG. 1 :

 1 FIG. 1: (a) Schematic of our noise detection setup. PD: photodiode, Amp.: low noise amplifier. (b) Experimental time sequence. Typically 10 10 atoms are loaded in the MOT and compressed during ti. Then the trapping system switches off and atoms are released. Two probe pulses are applied during tp = 1.2 ms, after a time of flight of tTOF = 4 ms.The first pulse provides the transmission through the atomic cloud, and the second one allows us to measure the incident intensity without atoms in order to calculate the normalized transmission for each cycle. The atoms are removed by applying the MOT beams at resonance during t push = 6 ms between the two probe pulses. For the PSD measurements the time window of the oscilloscope is set tpause = 200 µs after the beginning of the first probe within tosc = 100 µs.

FIG. 2 :

 2 FIG.2: Grey curve: experimental probe transmission as a function of the laser detuning. Red (dashed) curve: fit of the transmission curve, giving an on-resonant optical thickness of 19. Black curve: fit derivative used for the intensity to frequency noise conversion (right axis).

5 FIG. 3 :

 53 FIG.3: Laser transmission noise PSD ST divided by the square of the discriminator slope D 2 , measured using a cold atomic cloud as a frequency discriminator (from the black curve to the top: b0 = 19, 6.5 and 51.5). For low Fourier frequencies, Eq. (3) is valid and the curves thus correspond to the laser frequency noise PSD (FNPSD). The FNPSD measured with a Fabry-Perot cavity as a frequency discriminator is plotted in grey (see appendix A for more details). The dashed line corresponds to the β-separation line used to estimate the laser linewidth.

5 FIG. 4 :

 54 FIG. 4: Zoom at high Fourier frequencies of the laser transmission noise PSD ST divided by the square of the discriminator slope D 2 using a cold atomic cloud with an optical thickness of b0 = 19 and for three different laser detunings (lower curve: δ = 3; curve in the middle: δ = 4; upper curve: δ = 5).

10

 10

FIG. 5 :

 5 FIG.5: Solid green line: laser transmission noise PSD ST divided by the square of the discriminator slope D 2 using a cold atomic cloud with an optical thickness of b0 = 19 and a laser detuning of δ = 3. Solid black (thin) line: S T,th /D 2 calculated using Eqs. (15) and (16) assuming a white frequency noise. Dashed line: S T,th /D 2 calculated using Eq. (20) assuming a white amplitude noise.

5 FIG. 6 :

 56 FIG.6: Points: experimental frequency position of the bumps (empty icon: first bump, filled icon: second bump) observed in the FNPSD, obtained with the cold atomic cloud, as a function of the laser detuning. Solid line: calculated frequency position of the first bump. Dashed line: calculated frequency position of the second bump assuming frequency noise.

FIG. 7 :

 7 FIG.7: Setup for the beat-note measurement. Two laser beams are injected in a 50 : 50 coupler. Both lasers are independently frequency locked, and the frequency difference between them is typically 1 GHz. The interference signal is collected by a fast photodiode (PD) and then analyzed by a spectrum analyzer.

FIG. 8 :

 8 FIG. 8: Beat-note signal PSD between the DFB laser and the TOPTICA laser. The PSD has been averaged 100 times with a sweep time of 0.5 s. The center of the spectrum has been shifted to the origin. Since the TOPTICA laser has a much smaller linewidth than the DFB laser one, it can be treated as a reference laser and the PSD mainly corresponds to the optical spectrum of the DFB laser. The central part can be fitted by a Gaussian (dashed line) with a FWHM ∆νBN ≃ 3 MHz, and the wings are well fitted by a Lorentzian (solid line). The horizontal grey dashed line corresponds to the typical noise floor. Inset: zoom on the Gaussian part of the optical spectrum. Red curve: beat-note signal PSD. Dashed curve: Gaussian fit.Due to the convolution of the two laser Gaussian optical spectra, the square of the beat-note FWHM is the quadratic sum of the two laser linewidths ∆ν L :∆ν 2 BN = ∆ν 2 L,1 + ∆ν 2 L,2 . (A1)To deduce each laser linewidth, three different beat-note signals from three different lasers are needed. The third one is a homemade, etalon-based ECDL with an intermediate linewidth[27]. The results are summarized in Tab. II. The uncertainties are given at 1σ and have been obtained with a statistical analysis of different beat-note signals recorded in the same conditions.

FIG. 9 :

 9 FIG.9: Schematic of frequency noise PSD measurement using a Fabry-Perot (FP) cavity as a frequency discriminator. PD: photodiode, Amp.: low noise amplifier.

FIG. 10 :

 10 FIG. 10: Frequency noise PSD for the DFB laser. The dashed line corresponds to the β-separation line used to estimate the laser linewidth. Grey area: contribution to the DFB laser linewidth. See section III for further details.

TABLE I :

 I DFB laser linewidth obtained with different techniques.

TABLE II :

 II Laser linewidth measured using the beat-note setup, with lasers independently frequency locked. The uncertainties are given at 1σ.

Acknowledgments

We thank Gabriel Hétet for the very first measurements on this setup. A. Eloy acknowledges the support of the DGA.

Appendix A: Intrinsic laser noise characterization

The aim of this appendix is to present the characterization of our laser intrinsic noise using standard techniques. The corresponding results serve as a benchmark for FNPSD measured with cold atoms.

The laser is the one used as the probe beam in our cold atoms experiment. It is delivered by a DFB laser amplified by a tapered amplifier, and frequency locked close to the F = 3 → F ′ = 4 hyperfine transition of the D 2 line of 85 Rb. The laser spectral properties can be described through the optical spectrum and the corresponding linewidth or through the frequency noise power spectral density (FNPSD). These two complementary approaches are presented in this appendix.