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Chapter 21

Oxidative stress and

inflammation in OSAL. Lavie

Summary

Enormous progress has been made in the last decade inunderstanding the impact obstructive sleep apnoea syndrome

(OSAS) has on the cardiovascular system and the associatedcomorbidities. However, the mechanisms governing theseassociations are poorly understood. The accumulated evidenceimplicates oxidative stress and inflammation as two basicmechanisms associated with OSAS and with various metabolicdysregulations. The recent development of various experimen-tal models of intermittent hypoxia, based on animals andcultured cells, has helped to unveil some of these mechanismsand pathways affected by the intermittent hypoxia. It is likelythat an improved understanding of these mechanisms may leadto the development of new and more effective treatmentmodalities to abort the cardiovascular risk, resulting fromoxidative stress and inflammation.

Keywords: Adhesion molecules, cardiovascular morbidity,cytokines, inflammation, obstructive sleep apnoea, oxidativestress

Correspondence: L. Lavie, Unit ofAnatomy and Cell Biology. The Ruthand Bruce Rappaport Faculty ofMedicine, Technion, POB 9649,

31096, Haifa, Israel,Email [email protected]

Eur Respir Mon 2010. 50, 360380.Printed in UK all rights reserved.Copyright ERS 2010.European Respiratory Monograph;ISSN: 1025-448x.DOI: 10.1183/1025448x.00025509

The increased awareness of the impact obstructive sleep apnoea syndrome (OSAS) has on thecardiovascular system, quality of life, and its association with risk factors such as obesity andhypertension has lead to intensive research on these associations and the mechanisms involved [1].OSAS, a sleep-breathing disorder, is characterised by intermittent and recurrent pauses inrespiration. Based on this trait that implicates the apnoea-related multiple cycles of hypoxia/reoxygenation with an increased production of reactive oxygen species (ROS), similar toischaemia/reperfusion, it was proposed that oxidative stress and inflammation play a key role [2].However, oxidative stress and inflammation are also fundamental to the development ofatherosclerosis, cardiovascular and other morbidities associated with the metabolic syndrome.This oxidative/inflammatory cycle, which leads to the development of atherosclerosis andcardiovascular morbidity in OSAS due to the intermittent hypoxia (IH), is illustrated in figure 1.

This chapter covers the main findings in this area of oxidative stress/inflammation and itsconsequences in OSAS. These may lead to the development of endothelial dysfunction and earlyclinical signs of atherosclerosis and eventually, cardiovascular morbidity and mortality. The potentialrole of various inflammatory blood cells in initiating and propagating oxidative stress/inflammation

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is also described and a discussionis devoted to the potential role ofassociated comorbidities in exacer-bating these effects.

Obstructive sleep

apnoea andcardiovascularmorbidity

In recent years OSAS has emerged asa major public health problem, dueto its profound impact on patientshealth and quality of life [3, 4].OSAS is primarily characterised byrecurrent pauses in respiration,

which result in cyclic decreases inblood oxygen saturation that isdenoted as IH, and brief arousalsfrom sleep due to obstruction of theupper airway. The severity of OSASis defined by the apnoea/hypopnoeaindex (AHI), which denotes thenumber of apnoeas plus hypop-noeas divided by hours of sleep.The prevalence is particularly high

in males, but is also high due tocentral obesity, smoking and inpost-menopausal females. It is esti-mated that 4% of males and 2% offemales have at least five disordered-breathing events in each hour ofsleep in the form of apnoeas or hypopnoeas, accompanied mostly by day time somnolence [3, 5]. Butthe prevalence may be higher in non-symptomatic subjects, increasing to 24% and 9% of adult malesand females, respectively [3, 5]. In the obese and the elderly these values may increase to 60% [36].

Notably, OSAS has profound effects on the cardio-cerebrovascular system. This is based on a greatnumber of small and large scale cross-sectional, prospective, population-based and interventionstudies, which identified OSAS as an independent risk factor for cardiovascular morbidity andmortality [1]. Generally, OSAS is closely associated with an increased risk for hypertension,ischaemic heart disease, strokes, arrhythmias, chronic heart failure, as well as cardiovascularmortality. However, the impact of OSAS on the cardiovascular system is mainly evident inrelatively young populations, i.e. aged 50-yrs-old or younger, and less so with the elderlypopulation [7]. Moreover, evidence from recent years has also shown that OSAS is associated withadditional risk factors of the metabolic syndrome, such as hyperlipidemia, insulin resistance,hypertension and obesity, which also promote cardiovascular morbidity. These additional riskfactors, when clustered with OSAS, may further augment the risk for cardiovascular morbidity byacting synergistically with the apnoeic events [810].

Since the development of cardiovascular morbidity is of a multifactorial nature it could be largelyaffected by the genetic makeup, associated comorbidities and lifestyle related variables, such as physicalactivity, smoking and diet [11]. However, based on the ramifications of the IH, which is the hallmark ofOSAS, cardiovascular morbidity could be the outcome of such hypoxic events, although additional

OSAS/intermittent hypoxia

ROS

Endothelial dysfunction

Atherosclerosis

Activation

Adhesion

Injury

Cardiovascular morbidity

Cytokines, adhesion molecules

Transcription factor activation: NF-BAP-1

Leukocytes,platelets

Endothelialcells

Inflamm

ation

Figure 1. Oxidative stress and inflammation in obstructive sleepapnoea syndrome (OSAS). The intermittent hypoxia inducesreactive oxygen species (ROS) formation, which in turn activate an

inflammatory cascade via activation of transcription factors and

downstream genes as inflammatory cytokines and adhesionmolecules. These in turn can further activate transcription factors

and various blood cells. Activated leukocytes and platelets producehigher amounts of ROS, adhesion molecules and pro-inflammatory

cytokines, exacerbating this oxidative-inflammatory cycle andfacilitating endothelial dysfunction, which is the prelude to athero-

sclerosis and cardiovascular morbidity [2]. NF-kB: nuclear factor-kB;AP-1: activator protein 1.

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factors could also contribute. Early studies have established that OSAS is accompanied by activation ofthe sympathetic nervous system, swings in intrathoracic pressure and altered blood coagulability [12].Indeed these measures were all shown to increase with the severity of OSAS and could largely contributeto cardiovascular morbidity. Owing to the fact that treatment with nasal continuous positive airwaypressure (nCPAP) attenuated some of these measures, it clearly attests to their significance [13, 14].Although these mechanisms contribute to the development of endothelial dysfunction, atherosclerosisand cardiovascular morbidity in OSAS, a large body of evidence supports a role for oxidative stress and

inflammation as the initiating factors. This is mainly attributed to the fact that oxidative stress, which ispromoted in conditions such as ischaemia/reperfusion, is also manifested in OSAS due to the IH. Onceoxidative stress ensues it elicits inflammation and vice versa, inflammation promotes oxidative stress,thereby creating a vicious cycle by exacerbating each other. Additionally, both oxidative stress andinflammation are critical components in eliciting endothelial dysfunction and promotingatherosclerosis and consequently cardiovascular morbidity, as described in figure 1.

ROS and oxidative stress in physiology/pathphysiology

ROS molecules and oxidative stress have a central role in normal cellular function, as well as in various

pathological conditions. In the last two decades their key role as signalling molecules, which activateand control various transduction pathways in physiological as well as pathophysiological conditions,has been increasingly established. However, it should be acknowledged that this is still a controversialarea and while severe oxidative stress can certainly activate these pathways it is not entirely clear if ROSplay a role in the physiological signalling processes, most likely due to the large intracellular pool ofantioxidants. Yet in pathological conditions the excessive formation of ROS is prone to damagingsurrounding tissues by promoting signalling and activation of inflammatory pathways [15].

Sources and activity of ROS

Free radicals or ROS are atoms or molecules with one or more unpaired electron in their outerorbit. This trait enables them to chemically react with other molecules [15]. When two freeradicals react with each other a nonradical molecule is formed, but when a radical reacts with anonradical molecule the product is a new radical that can react with either another radical or anonradical, thereby propagating free radical chain reactions.

The most abundant ROS molecule is the superoxide (O2N-) radical. It is formed by a univalent

reduction of molecular oxygen, mainly during normal aerobic metabolism. Thus, mitochondrialrespiration is its major source. It is estimated that,5% of cellular ROS molecules are by-products ofnormal mitochondrial respiration. However, additional enzymatic systems also produce superoxide.

NADPH oxidase from primed leukocytes and endothelial cells is another major source ofsuperoxide [2, 16]. Other enzymes include xanthine oxidase and possibly uncoupled endothelialnitric oxide synthase (eNOS) [2, 16]. Although the superoxide is relatively a weak radical, itsreaction with other molecules yields additional, more potent ROS molecules and oxidants, such ashydrogen peroxide (H2O2), hydroxyl radical (OH

?) and lipid peroxides, when reacting with lipids.Apart from ROS, also nitrogen reactive species (RNS) are considered free radicals and they toocontribute to oxidative-nitrosative stress. One such toxic molecule with relevance to endothelialfunction is peroxynitrite (OONO-), which is formed by the reaction of the potent vasodilator nitricoxide (NO) with superoxide. Consequently, the availability of NO is diminished and properendothelial function is hampered [17]. A schematic and simplified illustration describing the

possible sources for ROS/RNS production and their fate in OSAS is presented in figure 2.

Oxidative stress

As stated previously, the formation of ROS is as a by-product of normal cellular respiration andaerobic metabolism. Thus, various antioxidant defense mechanisms have evolved to counteract

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excessive formation of ROS andsustain the oxidation-reduction(redox) status of a cell under strictregulation.

Antioxidants can be divided intoenzymes, such as superoxide dismu-tase (SOD), catalase and glutathione

peroxidase, and small molecules,e.g.vitamins C and E, glutathione anduric acid. Together these help pre-serve the tightly regulated balancebetween oxidants and antioxidants.Thus, oxidative stress is viewed as adisruption of this balance throughexcessive production of ROS. Suchoverproduction of ROS/RNS mayhinder cell activity and function

by damaging various biomolecules,such as DNA, lipids, proteins andcarbohydrates [15].

The signallingfunctions of ROS

ROS act like a double-edged sword,apart from injury to macromole-

cules and cellular components, ROSare vital regulators of a plethora ofsignal-transduction pathways andfunction as second messengers inmany physiological, as well as path-ological, conditions [15, 18]. More-over, their importance in maintaining stringent cellular redox homeostasis is well established[15, 19]. ROS were implicated in the activation signalling pathways involved in the initiation ofinflammatory and adaptive pathways, through activation of various nuclear transcription factors,such as hypoxia inducible factor (HIF)-1a, nuclear factor (NF)-kB, activator protein (AP)-1, sterol

regulator element binding proteins (SREBPs), GATA-4 [15, 2022] and NF-erythroid derived 2-related factor (Nrf2)-antioxidant responsive element (ARE), that regulates antioxidant genes [23].These pathways are critical to the development of atherosclerosis in many pathological conditions,including those of the metabolic syndrome [24]. Additional information on transcription factorsinvolved with ROS molecules, with potential importance to OSAS pathology, have been describedpreviously [25]. However, it should be stressed that activation of these transcription factors is notsolely dependent on ROS and in some instances there is a controversy regarding the function of ROSas activators, as is specified later. Nonetheless, the interactions of transcription factors with ROS areintricate and intermingled [26].

Effects of hypoxia/reoxygenation and intermittent hypoxiaIschaemia/reperfusion or hypoxia/reoxygenation is one of the most fundamental and relevantdiscoveries to OSAS. It established the involvement of superoxide radicals in injury of variousmacromolecules and cellular components in conditions that involve ischaemia or hypoxia followedby reperfusion/reoxygenation due to over production of ROS and subsequently oxidative stress [27].

++ +

SOD

O2-

Lipids

ONOO-

+

Cat/GPx

OSAS/intermittent hypoxia

Mitochondrialdysfunction

Xanthineoxidase

NADPHoxidase

Lipidperoxidation

NO

H2O2

H2O+O2

Figure 2. A schematic illustration of reactive oxygen species(ROS) and nitrogen reactive species production in obstructive sleep

apnoea syndrome (OSAS), during intermittent hypoxia, describingthe main sources of ROS. The superoxide (O2

N-) formed can react

with lipids to induce lipid peroxidation and a reaction with nitric

oxide (NO) promotes the formation of peroxynitrite (ONOO-).

Superoxide dismutase (SOD) sequestrates the superoxide yieldinghydrogen peroxide (H2O2), while catalase (Cat) and glutathione

peroxidase (GPx) may convert the hydrogen peroxide to H2O and

O2. Note the equations are not balanced. NADPH: reducednicotinamide adenine dinucleotide phosphate.

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Primarily, the relevance of ischaemia/reperfusion injury was shown in conditions such asischaemic heart disease, stroke, surgery, and organ transplantation. In the past 20 yrs at least 80pathologies were associated with excess ROS and oxidative stress. ROS were shown to increase inall inflammatory diseases studied, in ischaemic heart diseases, stroke, cancers, diabetes,hypertension, neurodegenerative disorders and atherosclerosis [28]. In the last decade, OSAS hasalso been shown to contain an oxidative stress component [22]. It was suggested that theseintermittent breathing arrests during sleep, which are accompanied by drastic changes in arterial

oxygen tension, bear similarities to ischaemia/reperfusion processes and may therefore promotethe formation of ROS, thereby inducing oxidative stress [2]. Consequently, the critical role ofROS molecules in inducing inflammatory pathways through increased activation of variousinflammatory blood cells and endothelial cells in OSAS was investigated. Such sequelae weresuggested to lead to the development of endothelial dysfunction and thereby enhance cerebro/cardiovascular morbidities [29].

Oxidative stress in OSAS

Sources and targets of ROS in IH and OSAS

There is ample evidence that ROS molecules are increased in response to IH and in OSAS. Studiesin cells in culture, organs, animal models that mimic OSAS and in humans all demonstrated thatOSAS and IH can induce mitochondrial dysfunction and thereby increase oxidative stress [3034].In many animal models NADPH oxidase was also reported to increase in tissues like the brain andcarotid body [3537]. In humans there is indirect evidence for NADPH oxidase activation invarious inflammatory cells [3840]. There is also indirect evidence implicating activation ofxanthine oxidase in OSAS by using allopurinol, which is a specific xanthine oxidase inhibitor [41],and identifing some of its metabolic by products in the plasma [2, 42].

The primary targets of oxidative stress in OSAS are likely to be endothelial cells and the

vasculature in general. However, since IH also induces inflammatory cell activation, activatedleukocytes could in turn contribute to increased oxidative stress in the vasculature. Yet, studies inanimal models utilising IH have shown that in tissues, such as the heart, brain and the liver,oxidative stress is also increased [37, 4347]. But since mitochondrial dysfunction is a likely sourceof ROS in IH/OSAS, it is reasonable to assume that each cell type would eventually be affected,depending on the severity of OSAS and the individual sensitivity of each cell type or organ, to thedecreased oxygen levels and ROS generation [45].

Evidence for ROS and oxidative stress

As aforementioned, the production of ROS and oxidative stress in OSAS are attributed to the cyclicnature of the multiple intermittent hypoxia episodes that resemble ischaemia/repefusion. Indeed a greatnumber of studies in the last decade clearly point to increased ROS formation and oxidative stress as aresult of this trait of OSAS. However, only a limited number of studies have shown a direct increase inROS formation by OSAS patients by utilising inflammatory cells, which primarily function in hostdefense and release large quantities of ROS [3840]. A study by SCHULZ et al. [40] demonstratedincreased ROS production in OSAS stimulated neutrophils that was attenuated after nCPAP treatment.In the studies conducted by DYUGOVSKAYA and co-workers [38, 39], stimulated monocytes andneutrophils and nonstimulated monocytes of patients with OSAS released higher amounts of ROS ascompared with controls. However, most of the data regarding increased ROS and oxidative stress inpatients with OSAS is provided by indirect evidence; mainly from circulating markers of oxidative stress.

Lipid peroxidation is a sensitive marker due to the high liklihood of lipids to undergo oxidation,and therefore is a highly used oxidative stress marker. Thus, increased oxidative stress in OSAS wasprimarily shown by using various markers of lipid peroxidation in plasma and serum [4850].Also, the levels of circulating oxidised low-density lipoprotein (oxLDL) were shown to increase in

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patients with OSAS [51, 52]. Oxidative stress was also demonstrated in DNA by an increasedurinary excretion of 8-hydroxy-29deoxyguanosine [53] and in exhaled breath [54]. Treatment withnCPAP or a dental device attenuated the oxidative stress markers [48, 49, 54, 55]. Increasedoxidative stress and lipid peroxidation in OSAS was also associated with an increase in carotidintima media thickness [56]. Moreover, in young healthy adults exposed to 4 days of IH,overproduction of ROS modulated increased acute-hypoxic ventilatory responses [57]. Alsogenomic microarray studies indicate that oxidative stress was increased in OSAS [58].

Diminished antioxidant capacities, which disrupt the tightly regulated balance and promoteoxidative stress, were also described in OSAS. Total antioxidant capacity in OSAS was reported todecrease in a number of studies and lowered levels of vitamins A and E were also described [50, 59].Additionally, the ability of serum albumin to act as an antioxidant was impaired in OSAS and wasimproved after nCPAP treatment [60]. Also, the activity of the antioxidant enzyme paraoxonse(PON)-1 was attenuated in OSAS [48]. This finding is in line with the increases observed in oxLDLlevels and the appearance of dysfunctional high-density lipoprotein (HDL) in OSAS [51]. SincePON-1 is physically bound to HDL and protects both LDL and HDL from oxidative modification.Importantly, PON-1 activity was also significantly correlated negatively with the severity of thesyndrome but not with age or body mass index (data not shown). Although in a small number of

studies negative results were reported, the overwhelming number of studies presenting positivefindings clearly attest to altered ROS balance and increased oxidative stress in OSAS.

Oxidative stress and endothelial dysfunction

NO is a potent vasodilator that regulates and maintains vasodilatation of blood vessels andattenuates expression of adhesion molecules. These properties prevent vasoconstriction andinhibit leukocytes/endothelial cells interactions. Thus, NO maintains a healthy and functionalendothelium. Attenuated NO levels in OSAS were reported in several instances [6163]. Thesestudies indicate that NO bioavailability is compromised, most likely through deactivation by ROS,

which result in endothelial dysfunction [2, 64]. This assumption was reinforced by a more recentstudy demonstrating that the activity of eNOS and its phosphorylated active form (p-eNOS) wereattenuated in freshly harvested venous endothelial cells from patients with OSAS. Moreover, theoxidative stress marker nitrotyrosine, which is indicative of NO inactivation by superoxide wasalso elevated [65]. More recently these findings were expanded to show that untreated OSAS,rather than obesity, was a major determinant of vascular endothelial dysfunction and increasedoxidative stress [66]. It is noteworthy that in both studies treatment with nCPAP improvedendothelial function, as determined by flow mediated dilation of the brachial artery [65, 66].Accordingly, treatment of OSAS patients with antioxidants, such as the xanthine oxidase inhibitorallopurinol, or by injecting a single bolus of vitamin C, improved endothelial function [41, 67].

Also, treatment with nCPAP was shown to reverse the diminished serum nitrite and nitrate inobese patients with OSAS [68]. These findings are in line with the involvement of oxidative stressin inducing endothelial dysfunction in OSAS.

Furthermore, both endothelial dysfunction, which is the subclinical condition that precedesatherosclerosis, and atherosclerosis were reported in patients with OSAS [6972]. For instance,measures, such as increased intima-media thickness, arterial plaque formation and calcified arteryatheromas, were documented in OSAS patients free of comorbidities or cardiovascular disease [7376].Treatments with nCPAP or a dental device improved some of these subclinical signs [55, 77, 78].

Inflammatory and adaptive pathways in OSASNF-kB

NF-kB is a pleiotropic transcription factor and a major regulator of a great number of vital cellularfunctions such as proliferation, death and immune responses. It has a key role in initiating

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inflammation by inducing expression of adhesion molecules, inflammatory cytokines andadipokines, which promote atherosclerotic sequelae [79]. NF-kB is activated by a diverse array ofstimuli including inflammatory cytokines and tumour necrosis factor (TNF)-a in particular aswell as various adhesion molecules and ROS. However, in many instances the participation of ROSremains a controversial issue, in particular when studying various cell types, e.g.cancer cells [80].Yet, ROS-dependent NF-kB activation was recently described in human endothelial cells derivedfrom obese individuals [81]. Thus, it appears that ROS dependent NF-kB activation is cell type-

specific and cell state-specific.In patients with OSAS the upregulated expression of NF-kB was demonstrated in neutrophils andmonocytes [8284]. More recently increased expression of NF-kB was detected in venousendothelial cells obtained from obese patients with OSAS that were compared with obese non-OSAS subjects, demonstrating the contribution of OSAS, rather than obesity, to its upregulation[66]. Additionally these finding were verified in neutrophils of healthy subjects that were treatedwith IH in vitro, specifically implicating IH in activating NF-kB, rather than other causes orproposed mechanisms such as sleep fragmentation or sympathetic activity [85]. In addition,increased expression of adhesion molecules and inflammatory cytokines and adipocytokines, thegene products of NF-kB, was also noted in OSAS [38, 86, 87]. Such increases in the gene productsof NF-kB clearly indicates that activation of NF-kB is associated with OSAS [38, 39, 88, 89]. It isalso noteworthy that a study by RYANet al.[90], who used HeLa cells that were treated with IH invitro, demonstrated a selective activation of NF-kB over HIF-1a, directly implicating IH withupregulated NF-kB expression [90].

HIF-1a

The transcription factor HIF-1a is the master regulator for cellular oxygen homeostasis andadaptive responses to hypoxia. Its activity is upregulated and stabilised under hypoxic conditions,

while regulating hundreds of genes that facilitate oxygen supply to ischaemic/hypoxic tissues [91].Under IH conditions its activation was mainly shown in cells in tissue culture and in experimentalmodels of rodents shown in reviews by SEMENZA and PRABHAKAR[92] and PRABHAKARet al. [93].However, these findings were inconsistent [90, 9496]. Obviously, using various cell types andapplying different patterns and intervals with more or less extreme exposure to IH for varyingdurations of hypoxia and reoxygenation may lead to such inconsistencies [90].

Unlike in tissue culture, studies conducted on animal models treated with chronic IH reveal acritical role for HIF-1a. In the myocardium, HIF-1aactivity and endothelin-1, a gene product ofHIF-1a, were increased in spontaneously hypertensive rats exposed to chronic IH [97]. Of note,HIF-1a was implicated in hypertension [98] and in components of the metabolic syndrome inrodents treated with chronic IH [99]. However, studies demonstrating activation of HIF-1a inpatients with OSAS were not described thus far. Yet, upregulated expression of some of its geneproducts attests to a possible involvement of HIF-1a. However, again regarding some of the HIF-1agene products investigated in OSAS, such as erythropoietin and vascular endothelial growth factor(VEGF), data are not uniform in all studies [2, 100102].

Nuclear factor erythroid derived 2-related factor

Another transcription factor with potential implications to OSAS is Nrf2. This is a true redox

regulated transcription factor that is ubiquitously expressed at low levels in various cells andtissues. It regulates a great number of antioxidant genes and drug-detoxifying enzymes, and bythat serves a cellular protective mechanism. Importantly, it is also activated in response toenvironmental and endogenous stresses and thus helps to maintain health by preventing varioushuman diseases, such as cardiovascular, neurodegenerative, cancer as well as inflammatory andischaemic conditions [103].

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Two recent studies in tissue culture implicated upregulated expression of Nrf2 under IHconditions [96, 104]. Although the intervals of hypoxia and reoxygenation employed did notmimic IH as in OSAS, nonetheless the findings described are interesting. In the first study usinghuman aortic endothelial cells, gene expression profiles were determined. By using IH cyclesalternating between 10 min hypoxia and 10 min reoxygenation Nrf2 gene expression wasupregulated by more than three-fold over normoxia but classical genes regulated by HIF-1 werenot [96]. In the second study, which was conducted on human lung adenocarcinoma A549 cells by

using 2-h periods and alternating between hypoxia and reoxygenation, Nrf2 as well as HIF-1 werefound to be upregulated. Moreover, NADPH oxidase derived ROS were implicated in theupregulation of Nrf2 [104]. These studies clearly emphasise the relevance of the different patterns,durations and the severity of the IH employed as determinants for activation of distinct pathways,such that may lead to contradictory findings in OSAS.

It is noteworthy that there is a crosstalk between the various transcription factors mentionedabove. For instance, while hypoxia upregulates the transcription of HIF-1a by an NF-kBdependent mechanism, also the NF-kB pathway is affected by the HIF pathway [105, 106]. Suchfindings may also implicate the participation of HIF-1a in inflammatory pathways [25]. Also,there appears to be a crosstalk between Nrf2 and NF-kB, since the Nrf2 and NF-kB signalling

pathways interface at several points to control the transcription or function of the downstreamtarget proteins [103]. Also, thioredoxin-1, the downstream gene of Nrf2, has been shown to inducethe amount of HIF-1a protein and thereby may indicate a possible crosstalk between Nrf2 andHIF-1a[107]. These complexities in the interactions between the various transcription factors canillustrate the difficulties in teasing out the function of each pathway under IH conditions. Findingsdescribing some of the possible intricate interactions between these transcription factors have beendescribed elsewhere [103, 108].

Activation of cellular inflammatory pathways in OSAS

As stated above, increased expression of ROS and augmented oxidative stress activate a plethora ofinflammatory pathways. Notably, NF-kB activation in OSAS is a prominent feature that promotesover-expression of adhesion molecules. These facilitate recruitment and accumulation of bloodcells on the endothelium lining the vasculature. Activation of NF-kB also promotes increasedexpression of pro-inflammatory cytokines that further exacerbate endothelial cells/blood cellsinteractions leading to endothelial cell injury and dysfunction [2].

Typically, circulating blood cells express low levels of adhesion molecules and intracellularinflammatory cytokines enabling blood cells to flow freely in the circulation, while withstandinginteractions with endothelial cells. However, expression of adhesion molecules and cytokines is

upregulated by a great number of stimuli. Infections, cytokines, hypoxia/reoxygenation, as well assleep apnoea were shown to increase the expression of various adhesion molecules and otherinflammatory molecules in blood cells and endothelial cells.

Adhesion molecules and blood leukocytes/endothelial cellinteractions

The expression of adhesion molecules is a highly regulated and sequential process. It facilitatesinteractions between activated blood cells and endothelial cells, promoting adhesion and injury to

the vascular endothelium. The most notable groups of adhesion molecules include the selectins.There are three major groups these are: 1) the L-selectins that are expressed on leukocytes, 2) the E-selectins in endothelial cells, and 3) P-selectins that are expressed in platelets as well as endothelialcells. This group of adhesion molecules promotes the initial interactions allowing for weak bindingbetween leukocytes and endothelial cells. However, the firm binding to endothelial cells is mediatedby the integrins, which also enables transmigration into the interstitial layer through the endothelial

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fundamental injury-limiting mechanism to control inflammation, its suppression together withincreased expression of selectins may heighten PMNs/endothelial cells interactions in OSAS, andaugment their destructive potential towards the endothelium [39].

Monocytes

Monocytes also participate in host defense mechanisms by acting as professional phagocytes. However,

they also participate in various stages of atherosclerosis and contribute to its development throughactivation and foam cell formation [24, 113]. Similar to PMNs, also monocytes of patients with OSASdisplay activation and increased expression of selectins (CD15), integrins (CD11c), ROS, cytokines andincreased adhesion to endothelial cellsin vitro[38, 89, 114], while nCPAP treatment effectively lowersadhesion molecules expression (CD15, CD11c) and adhesion to endothelial cells (table 1) [38].

Cytotoxic T-lymphocytes

Several subpopulations of T-lymphocytes were shown to be prevalent in atherosclerotic lesionsand to modulate atherosclerotic responses [115, 116]. These were mainly implicated in variousatherogenic processes via cytokine secretion and antibody production. Table 1 presents asummary of the findings on CD8+, CD4+, andcdT-cells of patients with OSAS. In fact, all T-cellsinvestigated expressed an activated and a cytotoxic phenotype in OSAS [88, 110, 111]. However,OSAS cd T-cells expressed the highest cytotoxic potential against endothelial cells as comparedwith OSAS CD8+, and the cytotoxicity of CD4+ T-cells was the lowest.

Platelets

Similarly to white blood cells, platelets were shown to be activated in OSAS. In physiologicalconditions platelets are circulating in a quiescent state protected from activation by inhibitorymediators released from intact endothelum. This helps to maintain vascular homeostasis.

However, platelets rapidly undergo activation due to oxidative stress, endothelial dysfunction orvascular damage. Subsequently, increasing the interactions with monocytes and PMNs is followedby increased adhesion and aggregation at the vessel wall [117]. In OSAS patients, plateletsexpressed increased activation, adhesion molecules and aggregability in vitro. The most notableadhesion molecule, which is upregulated in activated platelets of OSAS, is the P-selectin (CD62P)that was attenuated by nCPAP treatment [118121]. Additional measures of atherosclerosis, suchas increased blood viscosity, fibrinogen and hematocrit, were also heightened. These may furthercontribute to the increased incidence of cardiovascular events in OSAS [122124].

Endothelial cells

The functional endothelium consists of a cell layer that protects the vasculature by providing apermeability barrier. It regulates vessel tone and maintains an anti-inflammatory and antithromboticphenotype. While in their nonactivated state endothelial cells resist adhesion to various blood cells.However, activation or injury by various factors including hypoxia/reoxygenation, triggers theexpression of adhesion molecules [125, 126]. A number of adhesion molecules and inflammatorymarkers derived from endothelial cells were identified in the circulation of patients with OSAS. Theseincluded E-and P-selectin [127, 128], intracellular adhesion molecule-1 (ICAM-1), and vascular celladhesion molecule-1 (VCAM-1) [127, 129131]. Such circulating adhesion molecules were identifiedas markers of active atherosclerotic diseases, and as predictors of future cardiovascular events [64]. Also

in activated endothelial cells that were directly harvested form the vasculature of OSAS patients,increased oxidative stress and inflammation were noted. Moreover, nitric oxide bioavailability andtheir repair capacity were compromised [65]. More recently several studies addressed the presence ofendothelial progenitor cells (EPCs) and their repair capacity in the circulation of OSAS patients.However, these studies did not yield conclusive findings, most likely due to the very low numbers inthe circulation [132134]. Interestingly, in a recent study conducted on children with OSAS two

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Inflammatory cytokines and adipokines in OSAS

Inflammatory cytokines

Expression of pro-inflammatory cytokines, like adhesion molecules, is also affected by oxidativestress resulting in activation of NF-kB in various blood cells and endothelial cells. While adhesionmolecules facilitate the recruitment and accumulation of platelets and leukocytes to injure the

endothelium, cytokines may further augment this damage to endothelial cells.

The cytokines are multipurpose molecules. They are synthesised and released by many cell typesand regulate both the innate and adaptive immune system by complex interactions with varioustranscription factors and cytokines. Many of their functions including macrophage activation,smooth muscle cell proliferation, diminished nitric oxide activity and endothelial cell activationare involved in the progression of atherosclerosis.

TNF-a is one of the most important cytokines and has a key role in a plethora of cellularfunctions. It is synthesised by a variety of cells including inflammatory leukocytes and adipocytesand is involved with the initiation and progression of cardiovascular pathology [137, 138].

Additionally TNF-ais capable of inducing oxidative stress, expression of adhesion molecules andstimulating cytokine production viaNF-kB dependent pathways.

In OSAS, circulating TNF-a levels in serum or plasma were increased [86, 139, 140]. Also, thelevels of other inflammatory cytokines, such as interleukin (IL)-6 and IL-8 were increased.Conversely, the levels of the anti-inflammatory cytokine IL-10 were attenuated in OSAS [73, 87,140142]. Since IH and OSAS initiate the inflammatory response, these pro-inflammatorycytokines can in turn activate NF-kB thereby further augmenting inflammation (fig. 1).

Apart from circulating levels of cytokines, elevated cytokines levels were described in monocytes andin various cytotoxic T-lymphocytes [110, 111, 114]. Increased TNF-aand IL-8 and decreased IL-10

levels were detected incd+ T-lymphocytes of patients with OSAS [111]. TNF-awas also increased inCD8+T-cells but did not change in CD4+T-cells [88, 143]. Thus, when using specific cell sourcesundoubtedly TNF-a and other pro-inflammatory cytokines increase while anti-inflammatorycytokines decrease. Still each cell type has a unique profile of cytokine expression [87].

Inflammatory cytokines/adipokines and obesity

Cytokines released from adipose tissue are termed adipocytokines or adipokines. The mainadipokines include TNF-a, IL-6, CRP, adiponectin, leptin, resistin, and angiotensinogen [144].However not all adipokines are synthesised exclusively by adipose tissue. Most circulating

adipokines levels were shown to increase in obese patients and in patients with OSAS, whileanti-inflammatory adipokines such as adiponectin decreased resulting in low-grade inflammationin both instances [87, 145]. As, apart from leukocytes, the adipose tissue is a major source forcytokines/adipokines, determination of circulating levels in OSAS does not allow the identificationof their source. Moreover, OSAS patients are mostly obese individuals. Therefore, it is not clearwhether adipokines are synthesised due to obesityper seand/or because of OSAS. Given that inmost studies cytokines/adipokines levels were determined in the circulation of OSAS patients, thevalues obtained reflect the overall pool of cytokines/adipokines released from variousinflammatory cells, adipocytes, the liver, and other tissues. Obviously such findings do notprovide information on specific inflammatory/anti-inflammatory responses, or on an ongoing

process in a specific inflammatory cell [87].

Inflammatory adipokines in OSAS

One of the most studied adipokines in OSAS is adiponectin. It is an anti-inflammatory andantidiabetogenic cytokine with cardiovascular protective effects. It is the most abundant of all

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circulating adipokines, and is exclusively produced by the adipose tissue. Adiponectin wasshown to decrease in obese individuals and in patients with type 2 diabetes [146, 147]. Due toits anti-inflammarory properties, adiponectin also attenuates the levels of adhesion moleculesand pro-inflammatory cytokines as TNF-a, IL-8 and IL-6. In parallel it increases the levels ofthe anti-inflammatory cytokine IL-10.

In OSAS, adipokines levels were mainly unaffected by the syndrome. This is particularly evident instudies selecting nonobese, comorbidity free patients [39, 148, 149]. Notably, decreased

adiponectin levels in OSAS were attributed to obesity [149]. Thus, long-term treatment withnCPAP did not affect adiponectin levels [150]. However in obese patients with OSAS treatmentwith nCPAP reversed the diminished plasma adiponectin levels [68]. Collectively these findingssuggest that obesity, rather then OSAS, is the determining factor for adiponectin levels.

C-reactive protein (CRP) is another inflammatory molecule that is affected by oxidative stress andis associated with inflammation and atherosclerosis. It is an acute phase protein that is induced bythe pro-inflammatory cytokine IL-6 and primarily secreted by the liver but also by other cell types.In endothelial cells it induces the expression of adhesion molecules [151] and inhibits endotheliumdependent NO-mediated dilatation [152]. Moreover, in many epidemiological studies it was

identified as a strong predictor of coronary heart disease and of future cardiovascular events [153,154]. These effects on endothelial cells can induce endothelial dysfunction and atherotrombosis,indicating that it is not simply an inflammatory marker but rather an active participant ininflammatory/atherosclerotic processes [151].

As with adiponectin it is not conclusive whether CRP levels are elevated in OSAS. Thus far CRP wasreported to increase in a severity dependent manner [155] and to decrease with nCPAP [141].However, additional studies implicated obesity, rather than OSAS, in elevating CRP levels [156160].Thus, as with circulating levels of inflammatory cytokines, it is difficult to tease out the independentcontribution of obesity from OSAS in increasing CRP levels.

Oxidative stress/inflammation in OSAS and aggregatedcomorbidities

In recent years it has become increasingly evident that a variety of metabolic risk factors andmorbidities, such as obesity, hypertension, hyperlipidemia and diabetes, are associated withoxidative stress and inflammation. In fact, these metabolic disorders are the very same risk factorsthat cluster with OSAS. Thus, in OSAS there could be a confluence of different potentiallyindependent sources of ROS, which could greatly amplify its effects and contribute to thedevelopment of cardiovascular morbidity as described previously [22]. The central role of ROS

and the associations with comorbidities are depicted in figure 4. Therefore, in conditions likeobesity and morbidities,e.g.hypertension, hyperlipidemia, or insulin resistance, as in OSAS; thereis a common denominator in the form of oxidative stress/inflammation that promotes endothelialdysfunction and atherosclerosis. In some of these morbidities it is not always clear which factor isthe initiator. Such morbidities could develop independently of OSAS due to the geneticbackground, hormonal, dietary or lifestyle-related variables that greatly affect metabolicdysregulations and obesity. However, it is also likely that such morbidities could develop inmany instances due to OSAS and their consequences as described in figure 4. These mayaccelerate the development of hypertension, hyperlipidemia, insulin resistance, and diabetes [22].It is also very likely that obesity could act as an initiating factor. However, regardless of which

precedes which, i.e. whether it is OSAS or metabolic dysregulation, once OSAS develops andclusters with components of metabolic dyregulation the nightly initiated oxidative stress turns outto be a key factor in inducing the chain of events that promotes atherosclerosis and eventuallycardiovascular morbidities. It is noteworthy that since most OSAS are obese, apart from the IHwhich is characteristic of OSAS, the subjects are also exposed to continuous sustained hypoxia.This, since sustained hypoxia, is a major feature of the adipose tissue in the obese [161]. Therefore,

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sustained hypoxia and intermit-tent hypoxia may act synergisti-cally to exacerbate the oxidativestress but may also activate differ-ent ROS-dependent signallingpathways [25]. Additional inter-actions of obesity and oxidative

stress have been described pre-viously [162].

Inter-individualdifferences in hypoxicstimulation: a targetfor intervention?

Although OSAS has been re-peatedly demonstrated to be anindependent risk factor for car-diovascular morbidity, not allpatients develop cardiovascularcomplications. These individualdifferences merit a special atten-tion. One highly likely possibilityresides in the existence of large,individual differences in the re-

sponse of an individual to a givenhypoxia stimulus. These differ-ences may be of such magnitudeand importance as to whether anindividual will withstand a suddenmyocardial infarction, developatherosclerosis or remain free of cardiovascular complications. Although the existing informationregarding individual differences in OSAS is very limited it warrants special attention. Several linesof evidence point to this possibility. First, as previously discussed, some discrepancies wereobserved at the level of the HIF transcription factor and its gene products in models of IH and

OSAS [90, 9496, 99102]. Such discrepancies could stem from inter-individual differences in theresponse to a given hypoxia stimulus. A study in support of this concept was conducted onpatients with coronary artery disease [163]. In that study, the levels of VEGF produced by monocytesof these patients were measured before and after an identical hypoxic stimulus. There was a largeindividual variability in the VEGF-fold induction among patients. Yet, this increase in VEGF levelswas highly correlated with angiographically documented collateral formation. Such findings indicatethat there are individuals who are high responders to hypoxia and therefore, more prone toproduce VEGF and other vascular growth factors and to develop collateral circulation around theheart, as opposed to low responders with low collateralisation. Additional evidence supporting thisassumption is based on the response of peripheral blood lymphocytes of healthy individuals exposed

to hypoxia [164]. In this study, the expression of four HIF regulated genes was followed after exposureto various oxygen concentrations. A significant variation was found within the cellular response tohypoxia between normal humans. It was suggested that the common nature of the variability across allfour HIF-regulated genes resides within the HIF system itself. Evidence that variations in HIF-1agenotype influence the development of coronary artery collaterals in patients with significantcoronary artery diseases, indeed suggests a genetic basis for these individual difference [165].

Dyslipidaemia

Independent

HTNDMDyslipidaemia

Obesity

Oxidative stress

SA

DM

Inflammation

Cardiovascularmorbidity

HTN

OSAS

Figure 4. A schematic illustration suggestive of the central roleplayed by oxidative stress and inflammation in obstructive sleepapnoea and the development of associated conditions and

comorbidities. Associated conditions and comorbidities can be

induced by oxidative stress or develop independently. Once theseconditions and comorbidities develop, regardless of the initiating

factors, they elicit a series of intricate interactions with various

transduction pathways, promoting oxidative stress and inflammation.

The enhanced oxidative stress exacerbates inflammation, which inturn further exacerbates oxidative stress, generating a vicious cycle

of oxidative stress and inflammation, eventually leading to cardio-

vascular morbidity. OSAS: obstructive sleep apnoea syndrome; SA:sympathetic activation; HTN: hypertension; DM: type 2 diabetes

mellitus. Reproduced from [25] with the permission from the

publisher.

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Collectively, these findings could explain the inconsistencies in measuring various HIF-1a geneproducts in OSAS.

The first evidence that OSAS could be associated with an active adaptive mechanism wasprovided by a recent study conducted on patients undergoing elective coronary angiography whowere also screened for OSAS [166]. Interestingly, coronary collateralisation was more developedin the patients with OSAS who had at least one blocked artery, than in their matchedcounterparts without OSAS [166, 167]. Collaterals may protect the myocardium from infarction

and ischaemia by providing alternative routes of blood supply to regions of the heart supplied byoccluded coronary arteries. Yet, the ability of an individual to develop coronary collateralcirculation is highly variable between patients. It is, therefore, likely that genetic variations in theHIF system of patients with OSAS will result in genetically determined variations in thesensitivity to hypoxia. Thus, patients with a higher sensitivity to the lack of oxygen are morelikely to develop coronary collaterals in response to the nocturnal IH, as opposed to those withlower sensitivity. Several studies conducted on animal models treated with IH are in line with thehuman collaterals findings reported by STEINER et al. [166]. For instance, isolated rat heartstreated with IH were protected against post-ischaemic injury. Moreover, this cardiac protectionwas critically dependent on the dosage of HIF-1a gene [168]. However, several factors, such as

the depth, the duration and the intermittency of the hypoxia, are crucial and should be explored[169]. In accordance with this line, treatment of mice with IH also resulted in an increase in leftventricular contractility [170]. Thus, the data of STEINERet al. [166] indicate the presence of anadaptive mechanism in the elderly OSAS patient. These findings may also explain some of theclinical observations regarding the age decline trends in mortality of OSAS patients [7, 171].Undoubtedly, the potentially protective qualities of IH in OSAS should be further investigated asthey may have important clinical implications for treatment. Moreover, such findings, ifcombined with individual gene analysis, and personalised medicine, may provide new treatmentstrategies for cardiovascular protection.

Conclusion

In conclusion the key important factors to remember when understanding oxidative stress andinflammation in obstructive sleep apnoea patients are as follows. 1) OSAS is an independent riskfactor for cardiovascular morbidity and mortality. 2) It is characterised by intermittent recurrentpauses in respiration termed IH and therefore it is considered as an in vivomodel of hypoxia/reoxygenation. 3) In response to the IH a vicious cycle of oxidative stress/inflammation ensues inOSAS, resulting in endothelial dysfunction and atherosclerosis. 4) Conditions and morbidities thatcluster with OSAS are characterised with an oxidative stress/inflammation component andendothelial dysfunction. These may exacerbate atherosclerotic sequelae. 5) Oxidative stress inOSAS, which results from increased production of ROS and/or decreased antioxidant capacity,alters the redox balance and is likely to activate redox sensitive transcription factors. 6) Thesetranscription factors activate inflammatory and adaptive pathways. 7) Activation of theinflammatory pathways is characterised by increased expression of adhesion molecules and pro-inflammatory cytokines, which promote adhesion and cytotoxicity of various leukocytesubpopulations to endothelial cells. 8) The chain of events characteristic of IH, which promotesatherosclerotic sequelae, can result in cardiovascular morbidity and mortality. However it could beexacerbated by the presence of obesity and metabolic dysregulation. 9) Treatment of OSAS withnCPAP aborts some of these atherosclerotic sequelae. 10) IH may confer beneficial effects in someindividuals in the form of increased collateral circulation.

Support Statement

This study was supported in part by a grant from the Binational US-Israel foundation grant#1006695.

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Statement of Interest

None declared.

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