Cardiovascular Consequences of Sleep-Disordered Breathing ...

Cardiovascular Consequences of Sleep-Disordered Breathing: Contribution of Animal Models to Understanding of the Human Disease

Maurice Dematteis, Diane Godin-Ribuot, Claire Arnaud, Christophe Ribuot, Françoise Stanke-Labesque, Jean-Louis Pépin, and Patrick Lévy

Abstract Sleep-disordered breathing, and particularly the highly prevalent obstructive sleep apnea syndrome, is a multicomponent disorder combining intermittent hypoxia (IH), sleep fragmentation, and obstructed respiratory efforts. It is frequently associated with comorbidities and leads to numerous complications, including cardiovascular consequences that are conditioned by genetic predisposition and environment. The complexity of the disease and the reduced possibilities for patient investigations, especially at the tissue level, have limited progress in the understanding of sleep apnea pathophysiology and in the development of specific treatments. Animal models make it possible to study the causative mechanisms (essentially upper airway dysfunction) and the consequences (cardiovascular, metabolic, and neurological alterations) of nocturnal respiratory events without the confounding factors that occur in humans. Such studies have revealed some of the pathophysiological mechanisms and enabled the recognition of IH as the most important sleep apnea component underlying cardiovascular complications. We review different animal models used to assess detrimental sleep apnea–related cardiovascular consequences: blood pressure elevation, impaired vasoreactivity, structural arterial remodeling leading to atherosclerosis, cardiac remodeling, and myocardial infarction. We also review experimental evidence of beneficial effects of IH. By combining clinical and experimental research, these models will contribute to the understanding of differential patient susceptibility and to the elaboration of prevention strategies and tailored treatments for sleep apnea patients.

All the authors are in the HP2 Laboratory, INSERM ERI17, at the Université Joseph Fourier in Grenoble, France. Maurice Dematteis, MD, PhD, is an associate professor and Diane Godin-Ribuot, PhD, Jean-Louis Pépin, MD, PhD, and Patrick Lévy, MD, PhD, are professors of physiology; Christophe Ribuot, PharmD, PhD, is a professor and Françoise StankeLabesque, PharmD, PhD, an associate professor of pharmacology; and Claire Arnaud, PharmD, PhD, is an INSERM researcher. Drs. Dematteis, Lévy, Pépin, and Stanke-Labesque are also at the Centre Hospitalier Universitaire (CHU) Hôpital A. Michallon in Grenoble, in the Laboratoires du Sommeil et d’Explorations Fonctionnelles CardioRespiratoires, and in the Laboratoire de Pharmacologie, respectively. Address correspondence and reprint requests to Drs. Maurice Dematteis and Patrick Lévy, Laboratoire HP2, Institut Jean Roget, Faculté de Médecine de Grenoble, 38042 Grenoble CEDEX 9, France or email Maurice. [email protected] and PLé[email protected].

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Key Words: atherosclerosis; cardiovascular remodeling; hypertension; inflammation; intermittent hypoxia; myocardial infarction; pre-/postconditioning; sleep apnea

Introduction

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leep-disordered breathing (SDB1) encompasses different respiratory disorders. Obstructive SDB, the most frequent, includes different nocturnal respiratory events, from snoring to upper airway resistance episodes, hypopnea and apnea (Figure 1A). Apnea and hypopnea are classified as central or obstructive according to the reduction or increase in thoracoabdominal respiratory movements, respectively. Obstructive sleep apnea (OSA1) and hypopnea are the most frequent and correspond to recurrent episodes of complete or partial pharyngeal collapse during sleep resulting in intermittent hypoxia (IH1), sleep fragmentation, and obstructed respiratory efforts (Figure 1B,C; Deegan and McNicholas 1995; Malhotra and White 2002). Thus SDB, and more precisely OSA, is a multicomponent disease and a heterogeneous clinical entity in terms of severity (i.e., the number and type of respiratory events, and hypoxia length and depth), with different consequences according to individual susceptibility. OSA of at least mild severity (five or more episodes per hour of sleep) affects 5-20% of the general population (Young et al. 1993, 2002). It represents a growing and serious health hazard, and is recognized as an independent risk factor for hypertension, arrhythmias, stroke, and coronary artery disease (Arzt et al. 2005; Gami et al. 2005; Marin et al. 2005; Mehra et al. 2006; for review, Bradley and Floras 2009; Somers et al. 2008). OSA patients have an increased rate of cardiovascular morbidity and mortality, with an augmented risk of sudden death during sleep (Gami et al. 2005; Marin et al. 2005). Sleep apnea is also associated with several cardiovascular subclinical or clinical conditions, including diastolic hypertension (Baguet et al. 2005b, 2008), early atherosclerosis (Baguet et al. 2005a; Drager et al. 2005), diastolic ventricular 1BP,

blood pressure; CPAP, continuous positive airway pressure; ET-1, endothelin-1; FiO2, fraction of inspired oxygen; HIF-1, hypoxia-inducible factor 1; IH, intermittent hypoxia; I/R, ischemia/reperfusion; NF-kB, nuclear factor-kappa B; NTS, nucleus of the solitary tract; OSA, obstructive sleep apnea; PECAM-1, platelet-endothelial cell adhesion molecule-1; SDB, sleep-disordered breathing; SHR, spontaneous hypertensive rat

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Figure 1 Sleep-disordered breathing: A multicomponent disease. (A) Obstructive sleep-disordered breathing (SDB) is the most frequent SDB and encompasses different severity of respiratory events, from snoring to apnea, leading to different consequences. (B) The different severity of respiratory events is represented according to their pharyngeal collapsibility, oronasal airflow, esophageal pressure reflecting respiratory efforts, and nocturnal oxygen desaturation. The obstructive apnea episode is characterized by a cessation of oronasal airflow due to complete upper airway collapse, leading to greater respiratory efforts and oxygen desaturation compared to other respiratory events. (C) Polysomnogram tracing showing sleep fragmentation, fluctuating oxygen saturation, crescendo in thoracoabdominal respiratory efforts, and blood pressure surges at each apnea episode. EEG, electroencephalogram; EMG, electromyogram; SaO2, arterial oxygen saturation.

dysfunction (Alchanatis et al. 2002), and cardiac diseases requiring long-term pacing (Garrigue et al. 2007). Treatment of OSA significantly improves blood pressure (Haentjens et al. 2007; Pepperell et al. 2002) as well as daytime functioning (e.g., wakefulness, cognitive performance, mood, and general productivity; Engleman et al. 1994; Jenkinson et al. 1999; Montserrat et al. 2001; for review, Patel et al. 2003). However, the impact of OSA treatment on overall cardiovascular morbidity is not clear because only observational data (Marin et al. 2005) are available; there are no published prospective data from large randomized controlled trials (RCTs), although several RCTs are under way in specific subgroups. Moreover, the effectiveness of continuous positive airway pressure (CPAP1) on metabolic changes appears to be limited, at least in obese subjects (Coughlin et al. 2007; West et al. 2007); comorbidities and confounding factors (e.g., visceral obesity) may explain these discrepancies. Low long-term compliance also reduces the therapeutic benefit of CPAP, as reports indicate that 46-83% of OSA patients are nonadherent to the treatment (Gay et al. 2006; Weaver and Grunstein 2008). Limited knowledge of OSA pathophysiology has dramatically constrained the development of treatments. Therefore, animal models have been developed during the last 15 Volume 50, Number 3

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years to enable researchers to evaluate controlled conditions in the absence of confounders (e.g., disease duration, chronology of events, contribution of the different OSA components, and genetic and environmental factors; Baekey et al. 2009), which typically affect the outcome of clinical investigations (Figure 2). In addition, animal models have permitted the study of parameters that were difficult or impossible to assess in humans, particularly when organ harvesting was necessary to analyze mechanisms underlying IH consequences at the molecular level. Yet none of the current sleep apnea models is ideal. There have been attempts to reproduce repeated upper airway obstructions mainly in large animals such as dogs and pigs (Brooks et al. 1997; Kimoff et al. 1994; Pinto et al. 1993), but most OSA research has used rodents to study the effects of IH on the cardiovascular system (Fletcher 2001; Morgan 2007; Neubauer 2001). The focus on IH is based on the hypothesis that among the different stimuli related to OSA, IH is probably the most important with respect to the cardiovascular system. In this article we review the various models that have been used in the past two decades and focus on the acute and chronic effects of IH on blood pressure as well as on vascular and cardiac functioning and structures. 263

Models of Sleep-Disordered Breathing A model is an experimental preparation that allows the assessment of the pathophysiology and treatment of a human disease in a reproducible and standardized manner. Based on this general definition, SDB studies include animal as well as cell culture models (Figure 2). Model validation requires the fulfillment of at least one of three criteria: homology, predictiveness, and isomorphism. Homologous models share the cause or pathophysiology of the human disease. Predictive models respond to treatment similarly to the human disease. Isomorphic models display symptoms similar to those of the human disease although their cause and pathophysiology may differ; many SDB models are only partially isomorphic, as they focus on limited aspects of the human disease. In addition to these criteria, animal models used to mimic and study SDB are either spontaneous or induced (Figure 2).

Spontaneous Models The historic natural model of SDB is the English bulldog (Hendricks et al. 1987), which has an abnormal upper airway anatomy with soft palate enlargement and narrowing of the oropharynx. This model reproduces all the clinical features of the human disease including disordered respiration with episodes of oxygen desaturation during sleep as well as sleepiness during wakefulness. There are other spontaneous models such as obese miniature pigs for studies of sleep apnea in obesity (Lonergan et al. 1998), and Sprague-Dawley as well as spontaneous-hypertensive rats (SHRs1; Carley et al.

1998, 2000; Mendelson et al. 1988), which exhibit sleeprelated central apneas. Several of these models have proven useful for testing drugs to prevent obstructive and central apneas (Carley and Radulovacki 1999; Carley et al. 1998; Veasey et al. 1996, 1999, 2001).

Induced Models SDB can be experimentally induced in numerous animal species and strains to create models that mimic at least one important aspect of the human disease. These models, also called experimental models, enable researchers to study SDB consequences and the conditions that may modulate their occurrence and severity; such conditions include age, gender, weight, strain (with differing susceptibility to complications), and experimental procedure (acute or chronic exposure in awake, asleep, or anesthetized animals). The existence of numerous experimental models of SDB reflects the absence of a single ideal model, and may account for some of the discrepancies between studies. Induced models of SDB are either surgical or nonsurgical. Surgical Models Surgical models reproduce OSA in tracheostomized animals equipped with an intermittently occluded endotracheal tube. The models have mainly used large animals—dogs (Brooks et al. 1997; Kimoff et al. 1997; Scharf et al. 1992), lambs (Fewell et al. 1988), baboons (White et al. 1995), and piglets (Launois et al. 2001; Pinto et al. 1993)—but more recently

Figure 2 Sleep-disordered breathing: From patients to experimental models. Experimental models of sleep-disordered breathing (SDB) mimic several or only one aspect of the human disease. They allow investigation of the different components of the human disease without the confounding factors met in humans. The experimental model is appropriately selected according to the issue under study. FiO2, fraction of inspired oxygen. 264

ILAR Journal

similar approaches have also been effective in rats (Farré et al. 2003; Nácher et al. 2007). Alternatively, the tracheostomy can be used for intermittent administration of hypoxic gas (Katayama et al. 2007). There are other models that mimic the human upper airway dysfunction by inducing snoring-like vibrations in the upper airway of rats (Almendros et al. 2007) or injecting liquid collagen in the tongue and pharynx of monkeys (Philip et al. 2005). Although these isomorphic models reproduce some or even all of the characteristics of the human respiratory disorder (intermittent hypoxia-hypercapnia, obstructed respiratory efforts, and sleep fragmentation), they are limited by the animal’s size, the number of animals that can be processed, and side effects (including stress related to the procedure).

Nonsurgical Models To avoid the limitations of invasive obstructive apnea models, a noninvasive method has recently been described in rats (Farré et al. 2007); however this method is potentially stressful, as rats are individually placed in a cylindric chamber that restrains animal movements, with the head of the animal through a latex neck collar, which requires an adaptation period. Other noninvasive models, such as the IH model, are easier to use as they reproduce a single aspect of the human disease, such as the repetitive hypoxia-reoxygenation sequence (i.e., the consequence due to either obstructive or central apneas). Since the first description of these partial models in the early 1990s (Fletcher et al. 1992a,c), they have been widely used to assess various SDB consequences. Apparatus for the IH model. Depending on the species, animals are either ventilated with a mask (piglets) or placed in a specific cage or chamber (rats, mice) (Figure 3A,B,C,D,E); in both cases, they breathe intermittently a nitrogen-enriched air to create hypoxia, alternating with oxygen or air for the reoxygenation phase. Pure oxygen, used particularly in large hypoxic chambers, requires a perfect control of the gas administration to avoid exceeding 20.8% FiO21 (fraction of inspired oxygen). The duration of the hypoxic and normoxic phases of the IH cycle, as well as the slopes of FiO2 decrease and increase, are conditioned by the cage/chamber size and the gas flows and mixtures that result in different IH paradigms (Figure 3G). Control animals are usually placed in the same type of cage or chamber and exposed to air stimulus at similar flows to ensure similar levels of noise and turbulence related to gas circulation. The IH stimulus. The IH model permits evaluation of the effects of varying oxygen desaturation severity, IH cycle patterns, and exposure duration (short- vs. long-term), as well as comparison of the contribution to SDB consequences of IH as distinct from CO2 (Fletcher et al. 1995), continuous (sustained) hypoxia (Hui et al. 2003), or sleep fragmentation (Polotsky et al. 2006; Rubin et al. 2003).2 Although there is no upper airway 2It

is possible that sleep impairment itself contributes to some IH effects. In mice, IH impairs sleep architecture through sleep fragmentation, resulting

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occlusion in this model, some respiratory efforts (intermittent tachypnea) occur, corresponding to a fluctuating hyperventilation that follows the IH cycles. Indeed, IH models usually reproduce severe forms of the human disease, as FiO2 of about 5% and less produces maximal changes in blood pressure and heart rate (Bakehe et al. 1995; Fletcher et al. 1992a,b,c), but a higher FiO2 nadir has been reported in both mice (9-10% FiO2; Veasey et al. 2004; Zhan et al. 2005a,b) and rats (8-10% FiO2; Gozal et al. 2001; Soukhova-O’Hare et al. 2006). The IH stimulus is usually applied automatically during the daytime, as rodents preferentially sleep during this period, but with substantial differences between studies regarding durations of daytime exposure—from 4 hours/day (Kalaria et al. 2004) to 12 or 24 hours/day (Klein et al. 2005; Lin et al. 2007)—and frequencies of desaturating events during these periods. The choice of IH-inducing device contributes to this heterogeneity in the literature as it produces different stimulus frequencies and patterns: the higher the frequency the shorter the IH cycles, ranging from 120 cycles/hour (30-second cycle; Figure 3C; Dematteis et al. 2008; Fletcher et al. 1992c; Julien et al. 2003) to 60 cycles/hour (1-min cycle; Figure 3B,G; Campen et al. 2005; Polotsky et al. 2006; Figure 3D,G; Arnaud et al. 2008a,b). Lower IH frequencies with longer cycles are also used, particularly with large hypoxic chambers (Figure 3E), ranging from 6 to 10 cycles/hour, with cycles of 6 to 7 minutes (Klein et al. 2005; Figure 3E,G) or 9 minutes (Braga et al. 2006; Zoccal et al. 2008), and up to 1 cycle/hour (60-min cycle; Gozal et al. 2001; Figure 3E). The choice of IH stimulus thus results in different oxyhemoglobin saturations, ranging from 60% to 80% in mice and rats exposed to 30-second cycles with rapidly decreasing FiO2 up to 2-5% (Dematteis et al. 2008; Fletcher et al. 1992c; Julien et al. 2003; Lesske et al. 1997), and from 83% to 86% in mice exposed to more progressive and longer IH cycles with a 6-10% FiO2 nadir (Lee et al. 2008; Zhan et al. 2005b). These oxygen desaturations mimic moderate to severe forms of the human disease. However, besides daytime sleepiness, severity criteria of sleep apnea in patients are based on both the arterial oxygen desaturation and the frequency of respiratory events (apnea-hypopnea index, AHI) for classification as mild (AHI: 5-15/h), moderate (AHI: 15-30/h), or severe (AHI >30/h) (AASM Task Force 1999). Thus modeling the different levels of sleep apnea severity requires the appropriate selection of both the FiO2 and number of IH cycles per hour to be comparable to the apneahypopnea and/or oxygen desaturation index in patients. The difference in the IH stimulus, ranging from short and frequent to long and infrequent cycles per hour (Figure 3G), could contribute to discrepancies in the literature and may explain some similarities with continuous hypoxia in the case of longer IH desaturating cycles. Whether long periods of hypoxia followed by reoxygenation using pure in decreased delta power and rapid eye movement (REM) sleep (Polotsky et al. 2006). Additionally, mice chronically exposed to IH exhibit longlasting sleep alterations and hypersomnolence that may be due to oxidative injury to sleep-wake brain regions (Veasey et al. 2004; Zhan et al. 2005a,b).

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Figure 3 Noninvasive models of intermittent hypoxia. Different apparatus for achieving intermittent hypoxia (IH) in animals. In large animals such as piglets, the apparatus can be a mask (A). Hypoxic tents (F) are an alternative for large animals and humans. For rodents, different cage shapes are available to house one (B) or several mice (C); the cylinder-shaped IH cage (C) is the first developed for rats. IH using commercially available ventilated cages (D) has been recently developed, avoiding stress related to housing and investigator manipulation. Animal cages can be placed in hypoxic chambers that hold one or several cages at a time (E). With each apparatus, hypoxia results from nitrogen enrichment of the air, followed by reoxygenation using either air (A,B,C,D) or oxygen (B,E,F); reoxygenation using air instead of oxygen avoids any risk of hyperoxia. As shown in (G), IH cycles correspond to the different hypoxic cages, where the fraction of inspired oxygen (FiO2) oscillates from 20.8% to 5-6%. The IH cycle duration is conditioned by the gas flows and mixtures, and mainly by the size of the experimental apparatus: pyramidal (B) and cylinder-shaped cages (C) as well as ventilated rectangular cages (D) allow short IH cycles (1 min) compared to longer IH cycles (6-7 min) with large hypoxic chambers (E). On the right, the magnification shows different IH patterns according to the experimental hypoxic cages B, C, D, and E. In humans, the hypoxic tent (F) permits short IH cycles, as the tent is continuously maintained under hypoxia and subjects intermittently receive oxygen through a nasal cannula.

oxygen, possibly resulting in mild hyperoxia, could differently trigger oxidative and inflammatory responses as well as other consequences remains to be determined. In keeping with the high breathing frequency and metabolic rate of rodents as well as the characteristics of the human disease, a short IH cycle should be logical and also limits the hypoxiainduced hyperventilation and subsequent hypocapnia. Other factors to consider. Rodent sleep patterns are another relevant parameter to consider, as rodents do not sleep for 7 or 8 consecutive hours as humans do. They instead have polyphasic sleep-wakefulness, with sleep episodes throughout the 24-hour day-night cycle but primarily during daytime. Automatic systems have therefore been developed 266

to trigger the IH stimulus during sleep only (Hamrahi et al. 2001; Tagaito et al. 2001). Researchers using a sleep-induced procedure measured the number and duration of respiratory events in mice (Rubin et al. 2003) and, based on their results, it appears that the automatic application of the IH stimulus every 30 to 60 seconds (60-120 desaturating events/hour, 8h/ day) corresponds to a “physiological reality” in mice. Compared to other SDB models, IH models also permit very long exposures (lasting several months; Savransky et al. 2007; Zhu et al. 2007), allowing the investigation of chronic consequences as occur in the human disease. Thus, since the first studies (Fletcher et al. 1992a,c), the relevance of the IH model has been repeatedly confirmed, and evidence has ILAR Journal

accumulated suggesting that IH could be the most detrimental stimulus responsible for cardiovascular complications.

Alternative Models As in all fields of research, adherence to the 3Rs (refinement, reduction, replacement) is strongly encouraged to reduce the use of animals. To that end, cell cultures represent a complementary SDB model rather than an alternative technique to replace animals (Figure 2). In vitro models of SDB are more recent and have allowed the investigation of IH-induced alterations through different IH-exposed cell lines (Dyugovskaya et al. 2008; Gozal et al. 2005a; Kumar et al. 2003; Lattimore et al. 2005; Ryan et al. 2005; we discuss below the contributions of these studies, particularly those investigating signaling). However, as standard cell cultures do not permit rapid oxygen changes in the culture media, specific devices are necessary to perform oxygen fluctuations at frequencies relevant to sleep apnea–related IH (Baumgardner and Otto 2003; Kumar et al. 2003; Ryan et al. 2005). The use of in vitro models will likely be more extensive in the near future, but they will not replace animal models, which have provided most of the understanding of SDB pathophysiology and are therefore indispensable for SDB studies.

Intermittent Hypoxia and Blood Pressure Characteristics of Blood Pressure Alterations Animal models have enabled confirmation of the causal relationship between the hypoxic component of sleep apnea and blood pressure (BP1) elevation. IH in rodents induces a moderate BP elevation (Campen et al. 2005; Fletcher et al. 1992c; Joyeux-Faure et al. 2005; Lai et al. 2006; Lin et al. 2007; Tahawi et al. 2001), starting 5 to 8 days after the onset of IH (Lai et al. 2006; Tahawi et al. 2001), then plateauing, with no further elevation even after 90 days of IH exposure (Lin et al. 2007). Indeed, two different studies found that C57BL6 mice exhibited a similar increase after 14 days (21.4 millimeters of mercury [mmHg]; Dematteis et al. 2008) and 90 days (19.8 mmHg; Lin et al. 2007). In the canine model, experimentally induced OSA for 1 to 3 months produces a sustained daytime BP elevation of a maximum of 15.7 ± 4.3 mmHg (Brooks et al. 1997). In summary, in animals that are not prone to hypertension, IH induces a rapid but moderate elevation in BP, even after several months of exposure. In contrast, in animals prone to develop hypertension (e.g., SHRs), IH accelerates its occurrence, with high BP values after 14 days of IH (175 ± 3 vs. 147 ± 4 mmHg, IH vs. normoxic SHRs) (Belaidi et al. 2009). However, after 70 days of IH, BP does not further increase, and both hypoxic and normoxic SHRs have similar BP elevation (171.3 ± 21 vs. 178.3 ± 16.5 mmHg, means ± SE; Kraiczi et al. 1999). Overall, these results suggest an accelerating role of IH that decompensates a hemodynamic state prone to hypertension but is insufficient alone to induce hypertension. (IH Volume 50, Number 3

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appears similarly to be an aggravating factor that requires additional factors to induce atherosclerosis; see below.) Such moderate BP elevation in response to IH has been repeatedly confirmed in both rats (Fletcher et al. 1992c; Joyeux-Faure et al. 2005; Tahawi et al. 2001) and mice (Campen et al. 2005; Dematteis et al. 2008; Lin et al. 2007), using different techniques of BP measurements such as tail cuff (Fletcher et al. 1992), telemetry (Lai et al. 2006; Tahawi et al. 2001), and artery catheter (Campen et al. 2005; Dematteis et al. 2008; Joyeux-Faure et al. 2005; Lin et al. 2007) in awake as well as anesthetized animals. As in the human disease (Leung and Bradley 2001; Peppard et al. 2000; Weiss et al. 1996), BP elevation can be predominantly diastolic (Dematteis et al. 2008; Fletcher et al. 1992c). In addition to the sustained BP elevation observed between periods of IH exposure, mice exhibit BP surges at each IH cycle. These surges occur from the first day of IH, but after 14 days they occur in response to lower oxygen desaturation (12.2% vs. 8.5% FiO2), suggesting enhanced chemoreflex activity (Dematteis et al. 2008). The experimental model thus reproduces both the acute and chronic BP alterations that occur in humans, who also exhibit a rapid rise in BP at the end of an apnea, decreasing when respiration resumes. Paradoxically, we also found in mice a more pronounced BP decline during the daily 8-hour exposure to IH, compared to normoxic animals (Dematteis et al. 2008), and a similar finding has been reported in mice acutely exposed to hypoxia (4 min at 10% FiO2; Campen et al. 2005). These observations may seem to contradict those of the human disease, as sleep apnea patients may lose the sleep-related dip in blood pressure, and these “nondipper” patients are more susceptible to cardiovascular complications even without daytime hypertension (Leung and Bradley 2001; Suzuki et al. 1996). However, a similar result has recently been reported in healthy human subjects exposed to IH for 14 nights using the hypoxic tent (Figure 3F; Tamisier et al. 2009): they developed sustained BP elevation during the day and exhibited a steeper BP decrease at night compared with preexposure BP values (Tamisier et al. 2007).3 The animal model of IH has thus been predictive of the experimental results in humans, confirming the clinical relevance of this experimental model.

IH, Carbon Dioxide, and Blood Pressure As described above, depending on the depth and length of the IH cycle, the hypoxic stimulus can elicit a fluctuating hyperventilation that may result in hypocapnia. This outcome differs from that of clinical situations, as sleep apnea patients exhibit eucapnia (normal arterial CO2 pressure) or even intermittent hypercapnia (elevated arterial CO2 pressure). Investigators have therefore evaluated the contribution of CO2 to sleep 3The

concomitant BP decline with IH exposure could be related to the hypoxic diuretic response (Claustre and Peyrin 1982) and/or to the hypoxic vasodilation involving the adenosine-prostaglandin-nitrous oxide cascade, as adenosine resulting from adenosine triphosphate (ATP) degradation accumulates during hypoxia (Ray et al. 2002).

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apnea–related BP changes. Fletcher’s group found that eucapnic IH in Sprague-Dawley rats is a more potent stimulus to induce acute BP elevation and bradycardia than hypocapnic IH, and this stronger effect is associated with enhanced sympathetic nerve activity (Bao et al. 1997b). However, after chronic exposure for 35 days, the additive effect of CO2 in BP elevation disappears (Fletcher et al. 1995), while some differential catecholamine alterations in the hippocampus and hypothalamus persist (Li et al. 1996). These results reinforce the likelihood that among the different blood gas alterations induced by sleep apnea (hypoxia vs. CO2), IH is the more detrimental factor for cardiovascular complications.

IH, Sleep Fragmentation, and Blood Pressure The contribution of sleep fragmentation to the numerous complications associated with sleep apnea is likely underestimated.4 However, the comparison of dogs exposed to either recurrent arousals or intermittent upper airway occlusions during sleep for up to 3 months showed that only dogs in the second group developed sustained hypertension (Brooks et al. 1997). Similarly, sleep fragmentation in rats using acoustic stimuli for 35 days did not elicit BP elevation, possibly due to some habituation (Bao et al. 1999). However, like CO2, sleep fragmentation seems to have an additive effect on respiratory consequences, as respiratory arousals in pigs induced stronger vasopressor responses and BP increase than nonrespiratory arousals (Launois et al. 2001).

Chemoreflex, Baroreflex, and Sympathoadrenergic Activity Numerous hormonal changes may contribute to IH-induced BP elevation, including alterations of the renin-angiotensin (Fletcher et al. 1999, 2002) and endothelin systems (Allahdadi et al. 2005; Belaidi et al. 2009; Kanagy et al. 2001). As these alterations are detailed elsewhere in this issue (Kanagy 2009), we focus on sympathoadrenergic regulations, including chemo- and baroreflexes. In sleep apnea patients, all three components of obstructive respiratory events—IH, sleep fragmentation, and respiratory efforts leading to abrupt intrathoracic pressure changes on venous return and cardiac output—can contribute to the acute and chronic BP alterations described above (Figure 1B,C). Regarding IH, it has been well established that BP elevation is mediated by an increased sympathoadrenergic activity. Animal and cell culture models have enabled the study of central and peripheral regulation implicated in this increased activity and of the associated cellular signaling alterations.

Baro- and chemoreflexes are both involved in sympathoadrenergic and BP regulation (for review, Guyenet 2006). Baroreceptors, principally located in the carotid sinuses and aortic arch, detect BP elevations. Through glossopharyngeal and vagus nerves, baroreceptor information reaches the nucleus of the solitary tract (NTS1) in the brainstem. NTS stimulation of the caudal ventrolateral medulla leads to inhibition of the rostral ventrolateral medulla and sympathetic preganglionic neurons in the spinal cord. In addition, the NTS stimulates the nucleus ambiguus, which regulates parasympathetic activity. In brief, BP-induced baroreflex activation decreases sympathetic and increases parasympathetic activities, thereby reducing heart rate and BP. Similarly, peripheral chemoreceptors located in the aortic and carotid bodies are sensitive to decreased arterial oxygen and, to a lesser degree, arterial CO2 and pH, whereas central chemoreceptors are more sensitive to pH. Chemoreceptor activation by hypoxia leads to enhanced breathing and sympathoadrenergic activity. Like baroreceptors, glossopharyngeal and vagus ascending fibers reach the NTS, which is thus an important structure for integrating baro- and chemoinformation and processing adapted cardiovascular regulations.

Baroreflex Activity As described above, activation of the arterial baroreflex reduces sympathetic activity and inhibits the chemoreflex (Somers et al. 1991); in particular, with chronic hypertension the baroreflex is impaired and chronically reset (Brown et al. 1976). Similarly, studies have reported a reduction in baroreflex activity in OSA patients (Baguet et al. 2008; Bonsignore et al. 2002, 2006; Carlson et al. 1996a; Cooper et al. 2004) and its improvement in response to CPAP therapy (Belozeroff et al. 2002; Bonsignore et al. 2002; Logan et al. 2003). Baroreflex impairment may be due to decreased sensitivity and/or increased set point (Carlson et al. 1996a; Leung and Bradley 2001). Indeed, a canine model with repeated obstructions of the upper airway showed that baroreceptors reset to a higher pressure without a change in baroreflex sensitivity (Brooks et al. 1999). In contrast, baroreflex sensitivity decreases in IH-exposed rodents (Lai et al. 2006) and is associated with both cell loss in the nucleus ambiguus (Yan et al. 2008) and remodeling of cardiac ganglia and vagal projections to these ganglia (Lin et al. 2008; Soukhova-O’Hare et al. 2006). Paradoxically, a recent study (Lin et al. 2007) found enhanced cardiac chronotropic responsiveness to vagal stimulation in IH mice with blunted baroreflex, suggesting a possible compensatory upregulation, whereas other alterations at a peripheral and/or central level could contribute to the baroreflex impairment.

4In

particular, studies of the role of sleep fragmentation in cardiovascular alterations are few compared to those for IH. They have mainly used dogs and pigs, with acoustic and tactile arousals (Brooks et al. 1997; Launois et al. 1998, 2001). Studies of sleep fragmentation in rodents are recent and have used movement-induced arousals (Guzman-Marin et al. 2007; McCoy et al. 2007) or auditory/tactile stimulus with high-flow blasts of air (10-50 liters/min; Polotsky et al. 2006; Rubin et al. 2003).

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Chemoreflex and Sympathoadrenergic Activity Chemoreflex and sympathetic activity. Animal models have revealed that chronic IH stimulates chemoreceptors and thus ILAR Journal

causes sympathetically mediated vasoconstriction (Fletcher 2001; Greenberg et al. 1999a; Lesske et al. 1997). Indeed, chemodenervation by carotid sinus nerve section (Fletcher et al. 1992a) or chemically induced peripheral sympathetic denervation with the neurotoxin 6-OH dopamine (Fletcher et al. 1992b), as well as adrenal medullectomy (Bao et al. 1997a), prevents the BP-elevating effect of chronic IH. The hypertensive effect of IH also occurs through the renal sympathetic nerve that stimulates the renin-angiotensin system with subsequent activation of angiotensin II type 1 receptors (Fletcher et al. 1999). An increased carotid body response to IH has also been reported in mice (Dematteis et al. 2008; Peng et al. 2006) and cats (Rey et al. 2004). In our study (Dematteis et al. 2008), mice exposed to IH for 14 days exhibited BP surges and increased heart rate fluctuations with lower oxygen desaturation compared to the first day of exposure. These alterations could contribute to the arrhythmic complications associated with sleep apnea (Weiss et al. 1996). In chronic hypoxic conditions such as those at high altitude or associated with chronic cardiopulmonary diseases, carotid bodies enlarge and change their hypoxic sensitivity; with IH, the increased chemosensitivity appears related to functional plasticity supported by long-term facilitation (Peng et al. 2003). These functional changes are reactive oxygen species (ROS)–dependent and may contribute to persistent reflex activation of sympathetic nerve activity in sleep apnea. Recent studies also report that endothelin-1 (Rey et al. 2008) and increased expression of N-methyl-D-aspartate (NMDA) receptors (Liu et al. 2008) could contribute to this increased sensitivity. Catecholamine release. Several studies have provided evidence of an increased sympathetic nerve activity in response to IH in both rats (Braga et al. 2006; Dick et al. 2007; Fletcher 2001; Lesske et al. 1997; Prabhakar et al. 2005; Zoccal et al. 2008) and mice (Julien et al. 2003; Dematteis et al. 2008) as well as increased levels of circulating catecholamines in the two species (Dematteis et al. 2008; Lesske et al. 1997; Zoccal et al. 2007). Interestingly, one report described hypoxia-induced catecholamine effluxes in ex vivo adrenal medullae from IH-exposed rats, whereas this response was not present in either hypercapnic animals or those exposed to continuous hypoxia (Kumar et al. 2006). Similarly, chronic IH in mice increased the release-ready pool of secretory granules in adrenal chromaffin cells via ROS-mediated activation of protein kinase C (PKC), whereas continuous hypoxia did not (Kuri et al. 2007). Collectively, these results suggest that IH can directly affect the secretory capacity of chromaffin cells and may thus contribute to elevated catecholamine levels. The differential response between IH and continuous hypoxia has also been reported in cell cultures using pheochromocytoma (PC12) cells as a sympathetic neural model. In particular, IH appears to be more potent than continuous hypoxia in triggering activator protein-1 and hypoxia-inducible factor 1 (HIF-11) transcription factors. Intracellular signaling pathways also seem distinct between IH and continuous hypoxia, partly due to ROS generation during the reoxygenation phase of IH (Gozal et al. 2005a; Prabhakar 2001; Prabhakar et al. 2007). Volume 50, Number 3

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Catecholamine synthesis. Tyrosine hydroxylation is the rate-limiting step of catecholamine biosynthesis. In PC12 cells, IH activates tyrosine hydroxylase via phosphorylation of serine residues (including Ser40), in part by Ca2+/ calmodulin-dependent protein kinase (CaMK) and protein kinase A (PKA). This phosphorylation may facilitate the removal of endogenous inhibition of tyrosine hydroxylase, leading to increased synthesis of dopamine (Kumar et al. 2003). Ser40 tyrosine hydroxylase phosphorylation also occurs in rat cortex (Gozal et al. 2005b) and carotid bodies (Hui et al. 2003), with different time courses for IH and continuous hypoxia. Brain alterations and sympathoadrenergic regulation. Tyrosine hydroxylase, which is useful for mapping the catecholaminergic brainstem structures in rats, showed higher levels in the NTS after 3 weeks of IH (Pépin et al. 1996). The use of c-Fos labelling as a marker of neuronal activation in rats revealed that IH activated several structures that could contribute to alterations of sympathetic and cardiac activities (Sica et al. 2000a,b), including the NTS and other brainstem structures (Greenberg et al. 1999b). In developing piglets, short exposure to intermittent hypercapnic hypoxia (6 min 8% O2, 7% CO2 alternating with 6 min air over 48 min, for 2 or 4 days) increased neuronal death in several brainstem nuclei involved in cardiorespiratory, sleep, and arousal control (Machaalani and Waters 2003). A recent study that focused on mouse catecholaminergic wake-active neurons showed that 6 months of IH resulted in a 40% loss of these neurons through nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activation (Zhu et al. 2007). Overall, there is accumulating evidence that IH induces numerous structural and functional brain alterations, with different brain region susceptibility that could contribute to cardiovascular dysregulations associated with sleep apnea.

Intermittent Hypoxia and Vascular Consequences Animal models of sleep apnea, and especially of IH, make it possible to test the specific role of hypoxia in arterial alterations and to assess both plasma and tissue alterations that may contribute to the development and progression of atherosclerosis. Atherosclerotic plaque is the final stage of artery remodeling, leading to complications such as chronic tissue hypoxia and acute ischemic events (i.e., myocardial infarction and strokes). With currently available medical treatments, the reversibility of this late structural remodeling is challenging. The development of atherosclerotic plaque follows localized and systemic biological events that may be initiated or aggravated by sleep apnea (Figure 4). Assessment of these early events is therefore critical to identify hypoxia-related atherogenesis and thus prevent or treat it, particularly in apneic patients who are at risk of cardiovascular disease. Animal models have enabled the dissection of functional and structural preatherosclerotic remodeling and of their respective time course and relationships. 269

Vasoconstriction

Figure 4 Atherosclerosis and intermittent hypoxia (IH). In the context of genetic susceptibility and/or life habits, IH can induce multiple proatherogenic consequences (hemodynamic, hormonometabolic, and oxidative-inflammatory factors) that may interact and lead to atherosclerosis.

Functional Vascular Remodeling The numerous differences between studies—IH paradigms and durations, time points of exposure, artery territories, animal species, experimental preparations (in vivo vs. ex vivo), and measurement techniques—have led to some discrepancies in published reports of vasoconstriction and vasodilation responses. Nonetheless, studies have collectively demonstrated impairment of either vasodilation and/or vasoconstriction that may result in increased arterial stiffness and reduced vessel compliance. Vasodilation As is true in sleep apnea patients (Carlson et al. 1996b; Imadojemu et al. 2002; Kato et al. 2000), IH-exposed rats showed impaired relaxation to acetylcholine. The impairment involved gracilis muscle and middle cerebral arteries after 14 days (Phillips et al. 2004) and cremaster arterioles after 35 days of IH (Tahawi et al. 2001). The reduced vasodilation may be related to decreased nitrous oxide (NO) release or production (Tahawi et al. 2001). These findings were not confirmed in the rat aorta and mouse hindquarters as there was no change in the endothelium-dependent and -independent vasodilation after up to 35 days of IH (Dematteis et al. 2008; Julien et al. 2003; Lefebvre et al. 2006). Similarly, relaxation to acetylcholine was not affected in mesenteric vessels and carotid arteries (Lefebvre et al. 2006), resembling rat aorta vasoresponses after continuous hypoxia applied intermittently (10% FiO2, 8 h/day; Thomas and Wanstall 2003). 270

Exaggerated vasoconstriction occurs in both sleep apnea patients (Kraiczi et al. 2000) and animals exposed to IH. We found an increased vasoconstrictive response to norepinephrine in mouse hindquarters (Dematteis et al. 2008; Julien et al. 2003) that seems related to sympathetic hyperactivity, without obvious alterations in pre- and postsynaptic alphaadrenoceptor vasoresponses (Dematteis et al. 2008). In contrast, contractile responses to KCl, norepinephrine, angiotensin II, endothelin-1 (ET-11), serotonin, and thromboxane A2-mimetic U46619 were not affected in the rat aorta after 35 days of IH (Lefebvre et al. 2006). This last result is again in agreement with findings obtained after continuous hypoxia applied intermittently (Thomas and Wanstall 2003). Carotid arteries, on the other hand, exhibited an increased contractile response to ET-1 (Lefebvre et al. 2006), possibly due to their higher vascular ET-1 peptide expression compared to that of the aorta (Goettsch et al. 2001). In line with this hypothesis, two studies reported an augmented response to ET-1 in rat mesenteric (Allahdadi et al. 2005) and coronary (Belaidi et al. 2009) arteries associated with an increased endothelin-A receptor density. The increased vasoconstriction to ET-1 may involve the release of cyclooxygenase-derived products (Lefebvre et al. 2006; Troncoso Brindeiro et al. 2007) and PKC pathway upregulation (Allahdadi et al. 2008b). Regarding the relationship between vasoreactivity alterations and BP changes, although vascular alterations may contribute to IH-induced BP elevation, concomitant assessment of BP and vasoresponses in mice showed that BP changes occurred first (Dematteis et al. 2008).

Structural Vascular Remodeling Preatherosclerotic Lesions Alterations of the constitutive components of the arterial wall. Increased carotid intima-media thickness (IMT) is an early sign of atherosclerosis in sleep apnea patients (Drager et al. 2005) and correlates positively with serum inflammatory markers (Minoguchi et al. 2005) as well as oxygen desaturation severity (Baguet et al. 2005a). We recently assessed this structural remodeling in the rodent thoracic aorta and found that 14 days of IH induced an enlarged IMT in adult male C57BL6 mice (Arnaud et al. 2008a; Dematteis et al. 2008) and in Wistar rats (unpublished data from our group). The enlarged aortic IMT affected only the tunica media, with a magnitude similar to changes observed in the human disease, suggesting an expansive remodeling as no aortic dilation was associated. The enlarged IMT included thicker and disorganized elastic fibers, with an increased distance between fibers, likely due to smooth-muscle cell hypertrophy. Whereas some mucoid material depositions were present, the enlarged aortic wall was free of any lipidic depositions and fibrosis (Arnaud et al. 2008a). Collectively, these results are highly suggestive of mechanical adaptations of ILAR Journal

the arterial wall, as IH-exposed mice develop sustained arterial BP elevation with additional surges during IH episodes (Dematteis et al. 2008). We also found decreased intimal platelet-endothelial cell adhesion molecule-1 (PECAM-11) expression in the descending thoracic aorta, especially at the dorsal wall. Similar decreased PECAM-1 expression was observed in the left ventricular myocardium, with almost no staining in the papillary muscles, whereas the right ventricle was less or not affected. Since this alteration occurred without endothelial denudation in the aorta or decreased capillary density in the myocardium, the topographic distribution pattern of PECAM-1 changes suggest functional alterations that may result from IH-induced hemodynamic strains (Dematteis et al. 2008). Indeed, PECAM-1 is a cell-cell adhesion molecule involved in cell adhesion and leukocyte transmigration through the endothelium (van Buul and Hordijk 2004), but recent evidence also implicates it as a mechanoreceptive molecule. Thus alteration of PECAM-1 expression and distribution may represent a mechanism that modulates endothelial cell sensitivity to mechanical stimuli (Tzima et al. 2005). In addition to hemodynamic strains, hypoxia itself and many factors generated by IH (e.g., sympathoadrenergic hyperactivity; hormones such as leptin, insulin, angiotensin, endothelin, and vasopressin; oxidative, inflammatory, and growth factors) can contribute to these adaptive and degenerative structural alterations (Figure 4). Collectively, these histologic changes are in line with vasoreactivity studies (see above) implicating endothelial and smooth-muscle cells in both impaired vasoresponses and structural remodeling due to IH. Inflammation. There is growing evidence that sleep apnea is associated with low-grade inflammation, a key factor in atherogenesis (Foster et al. 2007; Hansson and Libby 2006). Recent in vivo and in vitro studies have shown that IH activates the proinflammatory transcription factor nuclear factor-kappa B (NF-kB1) in mouse cardiovascular tissue (Greenberg et al. 2006) as well as in cell culture (Ryan et al. 2005). This IH-induced NF-kB activation could underlie the increased proinflammatory NF-kB-dependent gene expression reported in sleep apnea patients, such as the intercellular and vascular adhesion molecule 1 (ICAM-1 and VCAM-1, respectively; Ohga et al. 1999, 2003). Indeed, only 3 hours of experimentally induced obstructive apneas in rats increases leukocyte rolling and P-selectin expression in colonic venules (Nácher et al. 2007). Leukocyte recruitment into inflamed tissues involves successive steps: leukocyte rolling, adhesion, and transmigration through the endothelium; leukocyte rolling corresponds to repeated contacts of leukocytes with the endothelial cells, whereas leukocyte arrest and adhesion to the endothelium surface are due to the interaction between the activated leukocytes and endothelium ligands such as ICAM-1. We recently found that IH induced systemic inflammation in mice, including splenocyte (mainly lymphocyte) activation characterized by increases in proliferation, migration toward chemotactic chemokines, and chemokine expression (Arnaud Volume 50, Number 3

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et al. 2008a). This systemic inflammation seems to be an early event (starting at the fifth day of IH exposure) and was further associated with inflammation of small and large arteries. Vascular inflammation consisted of enhanced leukocyte rolling and ICAM-1 expression in mesenteric resistance vessels as well as overexpression of the chemotactic chemokine RANTES/CCL5 and ICAM-1, NF-kB activation, and T lymphocyte infiltration in the aortic wall. Collectively, these results provide evidence of a proatherogenic role of IH through systemic and tissue inflammatory alterations that create favorable conditions for atherogenesis. Although additional factors seem to be necessary for the development of atherosclerotic plaques (see below and Figure 4), these experimental results open the possibility of new therapeutic strategies, as decreasing T cell activity or blocking chemotactic chemokines are both effective to reduce the development and progression of atherosclerosis in mice (Braunersreuther et al. 2008; Steffens et al. 2006; Veillard et al. 2004).

Atherosclerosis Two recent studies have demonstrated the link between experimental IH and the development of atherosclerotic plaques in C57BL6 mice (Savransky et al. 2007, 2008). However, long-term IH exposure (10-12 weeks) was required together with a high-cholesterol diet, suggesting that IH alone is not sufficient for atherogenesis. Indeed, beyond the atherogenic role of sleep apnea and its hypoxic component, genetic susceptibility to atherosclerosis, as well as comorbidities such as obesity, and metabolic and hemodynamic factors, are likely determinant (Figure 4). Metabolic proatherogenic factors, including dyslipidemia and glucose homeostasis dysregulation, are detailed elsewhere in this issue (Jun and Polotsky 2009). We found in IH-exposed mice hemodynamic alterations (sustained BP elevation with surges at each IH episode; Dematteis et al. 2008) that can alter shear stress, a critical determinant of cardiovascular homeostasis, which regulates vascular remodeling and atherogenesis (Chatzizisis et al. 2007). Severe sleep apnea patients may exhibit elevated hematocrit (Choi et al. 2006; Hoffstein et al. 1994), which contributes to blood rheology alterations and atherogenesis (Lee et al. 1998). IH-exposed rats can exhibit a clearly increased hematocrit (Joyeux-Faure et al. 2005; McGuire and Bradford 1999, 2001; Pépin et al. 1996), whereas mice show moderate elevations (Dematteis et al. 2008) even after long-term IH exposure (Campen et al. 2005). Because of this species difference, the contribution of this biological parameter to IHrelated atherogenesis remains unclear. Impaired coagulation is another biological parameter associated with sleep apnea (Robinson et al. 2004; Steiner et al. 2005) and is known to contribute to atherosclerosis, but to our knowledge it has not been studied in rodents. And the contribution of IH-associated sleep impairment (Polotsky et al. 2006; Veasey et al. 2004), particularly through the proatherogenic factors triggered by IH (Figure 4), also remains to be determined. 271

As IH requires additional factors for the development of atherosclerosis, two groups (Arnaud et al. 2008b; Jun et al. 2008) recently used more susceptible animals such as apoE knockout mice that spontaneously develop atherosclerotic plaques (this strain is characterized by atherogenic dyslipidemia and systemic inflammation; Hofker et al. 1998; Zhang et al. 1992). Both groups demonstrated an accelerating role of IH on atherosclerosis progression. In the study by Jonathan Jun and colleagues (2008), this detrimental effect was associated with elevations in plasma LDL cholesterol, diastolic BP, and oxidative stress. In our study (Arnaud et al. 2008b), the IH exposure was shorter (2 vs. 12 weeks), and the proatherogenic role of IH was associated with greater systemic and vascular inflammation as shown by macrophage infiltration that contributed to plaque instability. Both of these studies confirmed that IH needs additional factors such as dyslipidemia or inflammation to cause or accelerate atherosclerosis (Figure 4).

Intermittent Hypoxia and Cardiac Consequences Chronic IH is detrimental to the heart, with acute and chronic consequences (Figure 5). The acute consequences (e.g., myocardial infarction) have been assessed through an experimental model of acute myocardial ischemia/reperfusion (I/R1), involving coronary artery occlusion-reperfusion in hearts isolated from animals chronically exposed to IH. This model mimics the biological changes that occur during myocardial infarction, including the oxidative-inflammatory cascade in response to the ischemic and reperfusion injury (Ytrehus 2000). Chronic cardiac consequences correspond to functional and structural alterations (e.g., cardiac remodeling) that progressively develop under chronic IH.

Myocardial Infarction In addition to causing vessel obstruction through atherosclerotic remodeling that increases the risk of acute ischemic events, IH aggravates the detrimental consequences of artery occlusion. It is therefore a susceptibility factor for more frequent and severe lesions due to ischemia. Wistar rats exposed to 35 days of IH (1-min cycles, 21-5% FiO2, 8h/day) developed enhanced myocardial sensitivity to infarction after global I/R (Joyeux-Faure et al. 2005), and in SHRs 14 days of IH were sufficient to enlarge infarct size (Belaidi et al. 2009). However, this IH-induced susceptibility to I/R may be transient, due to protective adaptive mechanisms. Indeed, in mice, the IH sensitization that enhanced I/R injury and oxidative stress after 2 weeks of IH ceased after 4 weeks of exposure, with increased expression of the H2O2 scavenger thioredoxin (Park and Suzuki 2007). Beyond a direct effect on the myocardial tissue, additional IH-related consequences may aggravate myocardial ischemia; such consequences include increased chemoresponsiveness and adrenergic tone (Prabhakar et al. 2007; 272

Schömig and Richardt 1990) as well as activation of the renin-angiotensin system (Fletcher 2001), with angiotensin II inducing both coronary vasoconstriction and noradrenaline release (Wang et al. 2002). Furthermore, hypoxia is a powerful stimulus for ET-1 production under the transcriptional control of HIF-1 by both endothelial cells and cardiomyocytes (Yamashita et al. 2001). Chronic IH activates the ET-1 system (Allahdadi et al. 2005; Kanagy et al. 2001; Lefebvre et al. 2006), and endogenous ET-1 release in the ischemic myocardium contributes to the development of I/R injury (Pernow and Wang 1997). In line with this hypothesis, we found that SHRs exposed to 14 days of IH had increases in myocardial ET-1 and big ET-1 concentrations, coronary vascular response to ET-1 (as measured by perfusion pressure), and infarct size (Belaidi et al. 2009). Therapeutic interventions—including free-radical scavenging (Troncoso Brindeiro et al. 2007), sympathetic denervation, and blockade of angiotensin II (Fletcher 2001) and ET-1 receptors (Allahdadi et al. 2008a; Kanagy et al. 2001)—prevent IH-induced BP elevation. Whether these treatments are effective against the enhanced myocardial susceptibility to ischemia, as recently shown with the endothelin-A and -B receptor antagonist bosentan (Belaidi et al. 2009), requires further investigation.

Cardiac Remodeling It is well known that chronic hypoxia induces pulmonary hypertension with muscularization of pulmonary arteries, resulting in right heart hypertrophy and failure. IH can also lead to pulmonary hypertension and ventricular hypertrophy; Eugene Fletcher and colleagues (1992a,b,c, 1995) repeatedly reported left ventricular hypertrophy, and other groups have found that IH induces only right hypertrophy (McGuire and Bradford 1999) or hypertrophy of both ventricles (Campen et al. 2005). Although IH shares some similarities with continuous hypoxia regarding ventricular hypertrophy, the comparison of the two hypoxic stimuli using cDNA microarrays of mouse heart tissue has shown a differential gene response pattern (Fan et al. 2005). Interestingly, left ventricular hypertrophy still occurs when sympathetic denervation prevents IH-related BP augmentation (Fletcher et al. 1992b), suggesting a direct effect of IH on the myocardium. Indeed, IH may affect both the myocardial vascularization and cardiomyocytes. In mice exposed to 14 days of IH, we found a decreased expression of PECAM-1, with almost no expression in the deepest layers of the left myocardium (Dematteis et al. 2008); this reduced expression could reflect possible endothelial dysfunction as it was not associated with capillary rarefaction. In rats exposed to 35 days of IH, we even found an increased capillary density and vascular endothelial growth factor (VEGF) expression in the myocardium (Ramond et al. 2008). IH induces further functional and structural myocardium alterations. In rats, 5 weeks of IH led to left ventricular hypertrophy and dilation and decreased cardiac output, with myocardial oxidative stress evident in increased lipid ILAR Journal

Figure 5 Dual effects of intermittent hypoxia. (A) According to the severity and duration of exposure, intermittent hypoxia (IH) may have either beneficial effects, involving pre- and postconditioning, or detrimental effects as in sleep apnea. It is not clear whether pre-/ postconditioning-like phenomena occur during chronic exposure and contribute to the differential susceptibility between patients for IHrelated consequences and/or to the age-related decline in mortality observed in sleep apnea patients (blue arrow). The left inset illustrates rat myocardial slices after ischemia/reperfusion, with and without IH preconditioning (PC); the infarcted area is white/light pink. (B) According to individual susceptibility (critical threshold) and IH severity, IH induces either adaptive protection or detrimental disorders. Three subjects with three different susceptibilities to IH are represented; the IH-induced injuries of subject 1 will occur with less severe IH compared with subjects 2 or 3. But with persistent sleep apnea, subject 3, with a low susceptibility to IH, may become more susceptible (and thus more like subject 2 or 1), due to aging, life habits (e.g., smoking, high-fat diet, lack of exercise), and comorbidities (e.g., weight gain, diabetes, dyslipidemia), and vice versa. Volume 50, Number 3

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peroxidation and decreased superoxide dismutase levels (Chen et al. 2005). In mice, 10 days of IH induced cardiomyocyte enlargement and interstitial fibrosis with oxidative stress and inflammation that may involve gp91(phox)containing NADPH oxidase (Hayashi et al. 2008). These morphologic alterations were confirmed in rats, with evidence of inflammation and apoptosis as well as involvement of remodeling gene markers, trophic factors (insulin-like growth factor II), and the interleukin 6–related MEK5-ERK5 and STAT-3 pathways (Chen et al. 2007, 2008).

The Duality of Intermittent Hypoxia: When a Detrimental Stimulus Becomes Protective Depending on the duration, severity, and pattern of the hypoxic stimulus, IH can induce both beneficial and detrimental effects (Figure 5). The detrimental effects correspond to the cardiovascular complications secondary to long-lasting exposure to IH, as described above for the human disease. The beneficial effects result from short exposures to IH and resemble to some extent the homeopathic concept, that what is detrimental at a high dose may be beneficial at a very low dose. The resemblance is limited, however, as the beneficial effect of IH is not achieved with a markedly attenuated stimulus (see below). The biphasic and dual effect of IH is rather more in keeping with the adage “what doesn’t kill you makes you stronger”: in response to an aggressive stimulus (e.g., ischemia, hypoxia, hyperthermia), organs respond through a variety of mechanisms that induce greater tolerance to subsequent exposure, corresponding to a preconditioning phenomenon (Yellon and Dana 2000). Thus, for example, despite greater comorbidities in older subjects, researchers have reported a paradoxical age-related decline in mortality in sleep apneic patients (Lavie 2007; Lavie et al. 2005). It is not clear, however, whether this observation corresponds to a selection bias, differences in apnea severity or in treatment compliance between young and old patients, or to the development of protective mechanisms such as preconditioning (Brzecka 2005; Lavie and Lavie 2006). Similarly, negative animal models— those that fail to develop consequences or diseases induced in other animal models—are of particular interest in efforts to understand the mechanisms conferring this resistance. The possible dual effects of IH—beneficial versus detrimental—and the selection of the appropriate animal model could help to explain the differential susceptibility among sleep apnea patients to IH-induced deleterious consequences (Figure 5). Animal models of OSA and IH represent a useful tool for identifying the underlying pathophysiological mechanisms and hence new targets for future therapeutic strategies.

IH Preconditioning and the Heart The lower incidence of myocardial infarction in people who live at high altitude has prompted experimental studies using animal models of simulated high-altitude hypoxia (Poupa 274

et al. 1966), revealing the cardioprotective effects of hypoxia (for review, Kolář and Ošťádal 2004). Rats exposed to simulated high altitude (5000 m, 8 h/day, 5 days/week, 24-32 days) have reduced I/R-induced myocardial necrosis (Neckář et al. 2002), ventricular arrhythmia (Asemu et al. 1999; Meerson and Malyshev 1989; Meerson et al. 1987), and apoptosis (Dong et al. 2003). The IH pattern of sleep apnea also provides cardioprotection when applied for a brief period, reducing myocardial infarction in mice (Cai et al. 2003). However, the duration, depth, and type of hypoxia need to be considered. Preconditioning with 4 hours of IH (1-min cycle, 10% FiO2) reduced myocardial infarction size in rats, whereas 30 minutes of IH, or 4 hours of continuous hypoxia at 10% FiO2, did not (Béguin et al. 2005). However, additional factors require attention, such as the I/R model and animal species; for example, in a permanent left anterior descending coronary artery occlusion, preconditioning with continuous hypoxia (10% FiO2, 4 h) also provided delayed cardioprotection in rats (Sasaki et al. 2002) and mice (Xi et al. 2002). In contrast to ischemic preconditioning that gives both early and delayed protection, acute IH seems to activate only the delayed cardioprotection (Cai et al. 2003). Like ischemic preconditioning (Yellon and Downey 2003), IH preconditioning is mediated by p38 mitogen–activated protein kinase (MAPK), extracellular signal-regulated kinase, KATP channels (Béguin et al. 2005, 2007), and NO (due to the inducible NO synthase, iNOS; Belaidi et al. 2008; Xi et al. 2002). Like VEGF, erythropoietin, and heme oxygenase genes, the iNOS gene is involved in the preconditioning cardioprotection and is upregulated by HIF-1 (Semenza 1999). HIF-1 has a major role in the preconditioning-induced protection, as this beneficial effect is completely lost in mice with partial deficiency in HIF-1α (Cai et al. 2008). Similarly, after acute IH, using chromatin immunoprecipitation of rat myocardium tissue, we showed that HIF-1 directly interacted with the iNOS gene promoter and that prevention of this interaction abolished the delayed protection (Belaidi et al. 2008). In addition to HIF-1, the GATA-4 transcription factor also contributes to the cardioprotective effect of IH by increasing the cardiac expression of the antiapoptotic bcl-2 and bcl-xL proteins (Park et al. 2007). Ischemic preconditioning usually requires invasive in situ artery occlusion but can be induced distantly, for example by the occlusion of a femoral instead of coronary artery. This remote ischemic preconditioning is a more manageable cardioprotective strategy, essentially reflecting interorgan protection against I/R injury (Hausenloy and Yellon 2008a). Through systemic distribution, hypoxia may induce both in situ and remote preconditioning. Although there are some specificities according to the initial stimulus (e.g., hypoxia vs. ischemia), the type of preconditioning (in situ vs. remote), the window of protection (early vs. delayed), and the target organ, there is a final common pathway conferring interorgan and interstimuli (e.g., hypoxia or hyperthermia vs. ischemia or epileptic discharge) protection. We do not describe these powerful, innate mechanisms of defense in this ILAR Journal

review, but they involve numerous pathways (e.g., stress proteins such as heat shock proteins, oxidative and immunoinflammatory responses; for review, Hausenloy and Yellon 2008a,b; Yellon and Downey 2003). The brain is also prone to IH-induced injury (degeneration and strokes) and may therefore benefit from ischemic and hypoxic preconditioning protection (Jung et al. 2008; Ran et al. 2005; Steiger and Hänggi 2007). However, the IH stimulus used to precondition rodents is different from that used to mimic sleep apnea, with fewer and/or longer hypoxic episodes. The IH-induced neuroprotective effect may involve multiple signaling cascades, including HIF-1 (Shao et al. 2005), cAMP response element-binding protein (CREB), NF-kB (Gao et al. 2006; Rybnikova et al. 2008), MAPK cascade (Huang et al. 2007; Long et al. 2006), PKC (Li et al. 2005; Niu et al. 2005), and modulation of NO (Lu and Liu 2001), antioxidant enzymes (Stroev et al. 2005), and the pro-/antiapoptotic balance (Rybnikova et al. 2006).

IH Postconditioning and the Heart In contrast to preconditioning, which is of little practical use because the onset of myocardial infarction is unpredictable, cardioprotection is also possible after an ischemic event. Ischemic postconditioning with brief intermittent episodes of ischemia upon reperfusion reduces infarct size to a similar extent as preconditioning (Zhao et al. 2003). Hypoxic postconditioning also confers cardioprotection, which has been demonstrated in vitro and ex vivo. Exposure of cultured rat cardiomyocytes to hypoxia-reoxygenation results in the generation of ROS, intracellular Ca2+ overload, and cardiomyocyte apoptosis, all of which are reduced with the application of short cycles of hypoxia-reoxygenation just before abrupt reoxygenation (Sun et al. 2005; Wang et al. 2006). Ex vivo, a brief hypoxic perfusion at the onset of reperfusion reduces myocardial injury in the isolated rat heart following global ischemia (Serviddio et al. 2005). A recent study reported promising results with pharmacological postconditioning that inhibits the mitochondrial permeability transition pore in patients with myocardial infarction (Piot et al. 2008). However, whether IH shares similar beneficial effects in vivo requires investigation.

IH in the Course of Sleep Apnea: Pathophysiological Continuum between Beneficial and Detrimental Effects of IH Beyond an acute event (e.g., myocardial infarction), the question of whether similar protection (pre- and/or postconditioning) triggered by IH could exist over the long term in sleep apnea patients is of interest (Figure 5). Exacerbation in the course of a chronic disease such as chronic obstructive pulmonary disease is classically harmful. However, the application of IH in these respiratory diseases as well as in nonrespiratory diseases could be beneficial (Burtscher et al. Volume 50, Number 3

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2009; Serebrovskaya 2002; Serebrovskaya et al. 2003, 2008). Indeed, the IH stress could stimulate an adaptive response that confers resistance. Oxidative stress has been recognized as a main component in the pathophysiology of sleep apnea (Lavie 2009). Physiological ROS generation is necessary for signaling, but can become detrimental in the event of overproduction and/or decreased antioxidant defenses. Indeed, ROS signaling is involved not only in cardiac injury but also in pre- and postconditioning cardioprotection (Hausenloy and Yellon 2008b). Because of this pivotal role of ROS, IH training could be beneficial, at least in part, by switching the ROS signaling to positive effects, through enhancement of the antioxidant defenses (Serebrovskaya et al. 2008). However, both the severity and pattern of the IH stimulus, as well as the individual response, are likely determinants for this possible switch. Moreover, the pathophysiology is certainly more complex, and the critical switch modulated by IH could apply to numerous adaptive and detrimental systems, with critical thresholds varying by individual and also by organ. On a pathophysiological continuum, the beneficial effects of mild or moderate IH become detrimental with excessive application, overwhelming the body’s capacity for an adaptive protective response (Figure 5B). The heterogeneity of sleep apnea, in terms of respiratory events and oxygen desaturation (Figure 1), may therefore partly underlie the complex and proteiform picture of sleep apnea–induced consequences, both among patients and in the same patient over the course of the disease. Similarly, the heterogeneous IH paradigm used in the different experimental models of IH could therefore contribute to discrepancies between studies.

Conclusion Sleep-disordered breathing, and in particular the highly prevalent obstructive sleep apnea, is a multicomponent and heterogeneous disease leading to numerous complications, including cardiovascular and metabolic consequences that differ among individuals according to their genetic susceptibility, life habits, and environment. Animal models of SDB have enabled researchers to identify the contribution of each component as well as some of the pathophysiological mechanisms involved in the detrimental and beneficial effects associated with these components, particularly IH. Together with clinical research, these investigations will help to determine future prevention strategies and treatments adapted to the pathophysiology of each individual; indeed, novel findings from experimental research using the models described in this review are currently being tested in sleep apnea patients. The reciprocal relationship between clinical and experimental research is necessary to achieve these goals.

Acknowledgments This work was supported by a grant from AGIR@dom to MD, DGR, and CA. CA is also the recipient of a fellowship from the Fondation pour la Recherche Médicale (France). 275

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