Obstructive Sleep Apnea and Hallmarks of Aging

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Opinion

Obstructive Sleep Apnea and Hallmarks of Aging Laetitia S. Gaspar,1,y Ana Rita Álvaro,1,y Joaquim Moita,2 and Cláudia Cavadas1,3,* Obstructive sleep apnea (OSA) is one of the most common sleep disorders. Since aging is a risk factor for OSA development, it is expected that its prevalence will increase with the current increase in life span. In recent years, several studies have shown that OSA potentially contributes to functional decline, mainly prompted by chronic intermittent hypoxia and sleep fragmentation. Here, we propose that OSA might anticipate/aggravate aging by inducing cellular and molecular impairments that characterize the aging process, such as stem cell exhaustion, telomere attrition and epigenetic changes. We suggest that further knowledge on the impact of OSA on aging mechanisms might contribute to a better understanding of how OSA might putatively accelerate aging and aging-related diseases. Obstructive Sleep Apnea According to the International Classification of Sleep Disorders, OSA is a sleep-related breathing disorder characterized by recurrent episodes of complete (apnea) or partial (hypoapnea) obstruction of the upper airway during sleep while respiratory efforts continue, frequently inducing arousals (Box 1) [1]. Successive airflow interruptions or reductions during sleep culminate in intermittent hypoxia (IH) (see Glossary) and repetitive arousals result in sleep fragmentation (SF), inducing sympathetic neural activity (SNA), with increases and swings in both systemic blood and arterial pulmonary pressures [2,3]. OSA has been recognized as one of the most common sleep disorders. Indeed, according to World Health Organization, 100 million individuals worldwide exhibit some degree of OSA, a prevalence that has substantially increased over the past two decades, and which is expected to increase in the near future ([4,5], reviewed in [6]). The expected increase in OSA has been related to an increased prevalence of obesity and to aging (Box 2), two important risk factors for OSA [1]. Despite the high prevalence of OSA, it is estimated that 80–90% of total OSA cases remain undiagnosed [4]. The gold standard of OSA diagnosis is polysomnography (PSG), performed overnight in a sleep laboratory (Box 3). Unfortunately, PSG is labor intensive and expensive, which results in unnecessary delays in OSA diagnosis and treatment [7]. In addition, OSA has been recognized as a multifactorial and heterogeneous disease, showing variability in its symptoms, etiology, comorbid conditions, and outcomes, which are only poorly predicted based on the severity of disease as determined from PSG [2,3,8]. In this context, efforts should be made to determine the specific pathophysiology of each patient, paving the way for personalized OSA medicine. Application of continuous positive airway pressure (CPAP) via a mask, which alleviates upper airway obstruction, constitutes the first line in OSA treatment. However, the CPAP mask is not effective in some patients, while others are unable to tolerate its use (Box 4). In this context, OSA may have a high economic impact on healthcare systems, mainly due to

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Trends Obstructive sleep apnea (OSA) prevalence has substantially increased over the past two decades and, in industrialized countries, it is estimated to become one of the most common chronic diseases. OSA has recently been associated with earlier cognitive decline; thus, OSA treatment might delay agingassociated functional decline. OSA-associated epigenetic changes may underlie disease-associated heterogeneity in terms of symptoms, outcomes, and even treatment response. Telomere length is reduced in leukocytes from patients with OSA, suggesting accelerated cellular aging and inducing a higher risk for agerelated diseases. Blood-circulating endothelial progenitor cells may be reduced in patients with OSA, compromising the endothelial repair capacity and contributing to endothelial apoptosis.

1 CNC – Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal 2 Sleep Medicine Unit, Coimbra Hospital and University Center (CHUC), Coimbra, Portugal 3 Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal y These authors contributed equally.

*Correspondence: [email protected] (C. Cavadas).

http://dx.doi.org/10.1016/j.molmed.2017.06.006 ©

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undiagnosed and untreated cases that might culminate in further OSA-associated morbidities and healthcare expenditure [9]. Over the years, untreated OSA has been linked to increased predisposition for a variety of conditions and diseases, including hypertension, stroke, type 2 diabetes mellitus, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, as well as cancer and depression ([10–19], reviewed in [2,3,20]). In addition, patients with moderate-to-severe OSA [Apnea and Hypopnea Index (AHI) 15] also show significant impairment in attention and/or vigilance, executive function, and verbal memory tests compared with healthy subjects [21–23]. Although these cognitive impairments are also observed in older people, patients with OSA exhibit such impairments at a younger age (45–59 years of age) compared with age-matched controls [24]. Of relevance, treatment with a CPAP mask only slightly delays cognitive impairment by improving the attention of the user [25]. In this context, recent findings have associated OSA with an increased vulnerability for cognitive decline, early stages of dementia, and neurodegenerative diseases, such as Alzheimer’s or Parkinson’s diseases ([26–28], reviewed in [29]). Structural and functional imaging data, obtained by magnetic resonance imaging techniques, have revealed neurodegenerative-related changes in patients with OSA, namely: abnormal myelin levels; aberrant axonal and glial integrity; loss of white and gray matter in cortical areas and hippocampus; and global and regional neural network dysfunction [30– 36]. Indeed, impairments (DNA damage, inflammation, and oxidative stress damage) in brain regions associated with early-stage neurodegenerative disorders (i.e., substantia nigra, entorhinal cortex, and hippocampus) were observed in a rat model of chronic IH [6-min cycles of low oxygen (10%) followed by reoxygenation (21%) for 8 h/day during a light period, for 7 days] compared with normoxia rats [37]. These observations suggest that patients with OSA are particularly vulnerable to developing cerebral and cognitive impairments at an earlier age than would ordinarily be expected in healthy individuals. Moreover, several studies suggest a higher risk of osteoporosis in adult patients with OSA compared with healthy subjects [reviewed in 38]. Thus, some changes induced by OSA resemble those induced by aging [39]. In addition, aging also increases vulnerability to major human pathologies, including OSA [40]. Here, rather than discussing how aging might contribute to OSA development, we focus on how OSA might potentiate and/or accelerate age-related functional decline. Indeed, a similar hypothesis was previously proposed by others, but focused only on O2 deregulation induced by OSA [41]. In fact, chronic IH has been proposed to decrease the ability to respond to stressors, increasing susceptibility to disease, and mimicking an aging context [41]. However, since OSA involves more than IH, the disruption of sleep architecture by SF is a relevant OSA feature that might also contribute to aging. For instance, sleep disruption has been associated with physiological human aging [42]. Nonetheless, the cellular and molecular mechanisms by which OSA might induce or accelerate aging are not completely known. Recently, nine hallmarks of aging were proposed, that is, specific biological events that might contribute to aging and to defining the aging process: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication [43]. These hallmarks of aging may contribute to a better understanding of the potential contribution of OSA to the aging process. In recent years, several studies have focused on the cellular and molecular outcomes of OSA to better understand the development of OSAassociated comorbidities and impairments. To support our hypothesis that OSA might lead to age-related diseases, we discuss the hallmarks of aging in an OSA-dependent context. Indeed, recent evidence indicated that OSA might contribute to telomere attrition, might affect the behavior and functionality of stem cells, and might induce epigenetic changes that may underlie different OSA phenotypes. Given these connections, the early diagnosis, treatment,

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Glossary Adenotonsillectomy: a surgical operation to remove both the adenoids and tonsils. Autophagy: the endogenously regulated cellular ‘housekeeping’ mechanism responsible for the degradation of damaged and dysfunctional cellular organelles and protein aggregates. Berlin questionnaire: assesses three categories related to the risk of suffering OSA: (i) presence and frequency of snoring behavior; (ii) wake-time sleepiness or fatigue; and (iii) history of obesity and/or hypertension. Patients can be classified as high risk or low risk based on their responses to individual items and their overall scores in symptom categories. Micronuclei (MN): small, extranuclear bodies that arise in dividing cells from fragmented chromosomes (without centromere) or whole chromosomes and/or chromatids lagging behind in anaphase. Cellular senescence: the stable arrest of the cell cycle attributable to stereotyped phenotypic changes. Continuous positive airway pressure (CPAP): a device connected to a nasal or facial mask that supports breathing during sleep. CPAP blows compressed air into the airways and keeps the upper airway free. Endothelial progenitor cells (EPCs): circulating cells that express a variety of cell surface markers similar to those expressed by vascular endothelial cells, adhere to endothelium at sites of hypoxia and/ or ischemia, and participate in new vessel formation. Epworth Sleepiness Scale: measures daytime sleepiness. It comprises a questionnaire that assesses retrospective reports of the likelihood of dozing off or falling asleep in different situations. The total score estimates whether the individual experiences daytime sleepiness that requires medical attention. Exosomes: cell-derived vesicles, present in biological fluids, involved in intercellular communication (via transfer of proteins, lipids and nucleic acids) and regulation of normal physiological processes. Glucose effectiveness: the ability of glucose to facilitate its own

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and prevention of OSA become relevant, anticipating the potential to delay aging-specific processes. In addition, as human life expectancy increases, delaying aging and the onset of age-related diseases becomes critical to society. Indeed, this discussion is timely, given the currently unmet need to fully diagnose and treat OSA. In addition, there is a need to increase public awareness of the importance of healthy sleep.

OSA and the Hallmarks of Aging Genomic Instability Throughout life, continuous exposure to exogenous and endogenous threats, such as oxidative stress, challenges DNA integrity and stability [43]. Genetic damage, accumulated over time, leads to genomic instability [43]. In the OSA context, little attention has been paid to possible DNA damage. Nonetheless, relative to patients with nonsevere OSA (AHI <30), patients with severe OSA (AHI 30) showed increased DNA oxidation, as evidenced by increased excretion of urinary 8-hydroxy-2-deoxyguanosine (8-OHdG), a parameter of genomic instability [44]. In agreement with this finding, lymphocytes from patients with severe OSA (relative to healthy controls) showed increased basal nuclear DNA damage (DNA breaks, as evidenced by the comet assay), were more sensitive to DNA damage produced by exogenous agents (hydrogen peroxide, ethanol, and g-irradiation), and had a less-efficient DNA repair system [45]. Recently, patients with severe OSA were reported to exhibit an increase in the proportion of binucleated cells with micronuclei (MN), nucleoplasmic bridges (NB), and nuclear buds (NBUD) in peripheral blood mononuclear cells (PBMCs) compared with healthy controls [46]. Indeed, the formation of MN, NB, and NBUD has been closely linked to the incidence of chromosomal aberrations, aneuploidy, and DNA damage, suggesting that OSA might contribute to cytogenetic damage and chromosome instability, in some yet-to-be-determined mechanism [47]. In line with this observation, 4 weeks after treatment with nasal CPAP, a decreased frequency of MN, NB, and NBUD in the PBMCs of patients with OSA was reported [46]. These studies suggest that OSA participates in nuclear genomic instability and cellular damage in some capacity, which might contribute to carcinogenesis and aging. However, these correlations need to be tested further, and it remains to be elucidated whether OSA also induces mitochondrial DNA (mtDNA) damage. Telomere Attrition Telomeres, the nucleoprotein structures that protect and stabilize chromosome ends, are particularly susceptible to age-related deterioration. These regions are maintained by telomerase, an enzyme that is repressed in most mammalian somatic cells. Consequently, during normal aging, these chromosomal regions tend to shorten [48]. Both telomere dysfunction and telomerase deficiency have been associated with several age-related diseases and premature aging in mice and humans [43,48]. An emergent topic of research is the link between telomere length and sleep, with cross-sectional and case–control studies showing associations between sleep disturbances and shorter telomere lengths (reviewed in [49]). In 2010, the first work focusing on telomere length in patients with OSA was published, showing that telomeres in leukocytes of patients with severe OSA were shorter than those of healthy control subjects [50]. Subsequently, several authors have observed similar results in different OSA cohorts with older subjects, middle-aged individuals, and even young adults [51–55]. These observations provide strong evidence that OSA might anticipate and/or aggravate the aging process. However, the correlation between telomere length and disease severity remains controversial and further studies are necessary [50,54,55]. For example, snoring was recently associated with shorter leukocyte telomere lengths, independently of the presence of OSA, indicating that snoring could be a primary driver of telomere attrition in patients with OSA [56]. By contrast, children with different OSA severity have been confirmed to exhibit increased telomere lengths [57]. However, the expression of telomerase has been found to be variable during development, which may underlie this paradoxical result [58]. Consequently, longitudinal basic and clinical

disposal independent of an insulin response. Heat shock protein (HSP)-70 protein: a central component of the cellular network of molecular chaperones and folding catalysts. Intermittent hypoxia (IH): repeated episodes of hypoxia (any oxygen levels lower than normoxia) interspersed with normoxia (earth atmosphere oxygen level 21% O2) or less hypoxic conditions. Mesenchymal stem cells (MSCs): adult, nonhematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineages, such as osteocytes, adipocytes, and chondrocytes, as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). Nucleoplasmic bridges (NB): originate from dicentric chromosomes, which may occur due to misrepair of DNA breaks, telomere end fusions, or the defective separation of sister chromatids at anaphase. Nuclear buds (NBUD): nuclear anomalies associated with chromosomal instability events, commonly seen in cancer. Polysomnography (PSG): an overnight sleep test, performed inlaboratory, able to monitor sleep time and stages (electroencephalogram; EEG), eye movements (electrooculogram), muscle tone (chin electromyogram), heart rhythm (electrocardiogram), airflow, thoracic and abdominal respiratory effort, arterial oxygen saturation (oximetry) and carbon dioxide levels (capnography), body orientation, limb movements (leg electromyogram), and snoring. Postocclusive reactive hyperemia: used to assess endothelial function, based on blood perfusion measurements before, during, and after occlusion. Proteosomal activity: the ability of the proteasome to degrade unneeded or damaged proteins by proteolysis. Proteostasis: involves mechanisms for the stabilization of correctly folded proteins, most prominently, the heatshock family of proteins, and mechanisms for the degradation of proteins (i.e., proteasome, lysosome, or autophagy-mediated mechanisms). Proteotoxic stress: results from protein misfolding or aggregation, which may impair cellular function

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studies are needed to further understand both the relationship between OSA and telomere length and the biological mechanisms underlying this association. Epigenetic Alterations Epigenetic patterns change during aging in response to exogenous and endogenous factors. Epigenetic mechanisms include DNA methylation, post-translational modifications of histone proteins, chromatin remodeling, and transcriptional regulation by noncoding RNAs (as miRNAs). Although epigenetic changes do not alter the DNA primary sequence, modifications in transcription patterns can occur, impacting cellular homeostasis [43]. Recently, OSA was linked to changes in epigenetic patterns. A large-scale DNA methylation analysis identified 636 differentially methylated loci (DML) in PBMCs of patients with severe OSA compared with healthy subjects [59]. Although some of these changes were revealed to be false positives, aberrant DNA methylation in the promoter regions of specific genes [hypomethylation in the genes encoding interleukin 1 receptor 2 (IL1R2), and natriuretic peptide receptor 2 (NPR2); and hypermethylation in the genes encoding androgen receptor (AR) and speckled protein 140 (SP140)] were supported by protein expression changes, providing evidence for a potential role of some of these DML in regulating gene expression [59]. These epigenetic alterations in PBMCs were shown to correlate with both AHI and the patients’ susceptibility to excessive daytime sleepiness, suggesting different epigenetic-dependent phenotypes [59]. Accordingly, the DNA methylation profile of blood monocytes from patients with OSA and an obesity hypoventilation syndrome (OHS) phenotype revealed several differentially methylated regions, before and after positive airway pressure (PAP) treatment. These differences occurred mainly on peroxisome proliferation-activated receptor (PPAR)-responsive elements, which showed increased methylation after PAP treatment [60]. Based on these findings, different patterns of epigenetic modifications might underlie OSA heterogeneity and contribute to different OSA outcomes, and perhaps even treatment responses. For example, increased methylation levels in the gene encoding endothelial nitric oxide synthase (eNOS) in blood were shown to underlie abnormal eNOS-dependent vascular responses in children with OSA and abnormal endothelial function compared with children with OSA and normal endothelial function and with healthy children [61]. Similarly, plasma-derived exosomes have shown different miRNA signatures, namely, decreased hsa-miR-630 expression, in the normal and abnormal endothelial function of children with mild to moderate OSA [62]. Furthermore, treatment of children with OSA with surgical adenotonsillectomy increased the levels of hsa-miR-630 and reduced endothelial dysfunction (as evidenced by normalization of postocclusive reactive hyperemia responses) [62]. Another study reported increased DNA methylation levels in FOXP3 (a gene involved in modulating the expression and function of T regulatory lymphocytes) in blood cells of children with OSA also presenting high levels of high-sensitivity C-reactive protein (hsCRP) compared with either children with OSA and low hsCRP levels or healthy children [63]. In this context, although the exact significance of these alterations remains to be dissected, epigenetic modifications may constitute important determinants of endothelial dysfunction and inflammatory phenotypes in OSA [61,63,64]. Does OSA promote epigenetic changes or do epigenetic differences contribute to OSA etiology? Studies in rats have suggested that chronic IH, either during neonatal or adulthood stages (pups exposed during the neonatal stage, along with their mothers, to 15 s of hypoxia and 5 min of normoxia per cycle, between P1 and P10, during daylight, or exposed during adulthood to 5% O2 for 5 s and 21% for 5m, 9 episodes/h, 8 h/day for 10 days), induces the hypermethylation of antioxidant enzyme-encoding genes, such as that encoding superoxide dismutase 2, in the carotid body and adrenal medulla, resulting in oxidative stress relative to controls (i.e., rats under normoxia) [65,66]. The authors suggested that these epigenetic modifications induce autonomic dysfunction and exaggerated hypoxic sensing [65,66]. These impairments reduce the ability to respond to stressors and may increase susceptibility for

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and may affect the fate of nonrenewable cells of long-lived organisms. Senescent associate secretory phenotype (SASP): a senescent cellular state characterized by an increased release of inflammatory cytokines and chemokines. Sleep fragmentation (SF): brief awakenings or microarousals during the sleep period that induce short increases in EEG frequency. Such episodes may occur spontaneously or in response to respiratory events or tones. Stem cell exhaustion: decrease in the number and functional competence of adult stem cells, compromising their role in tissue renewal. STOP-Bang questionnaire: one of the most validated screening and predictive tools developed to estimate the probability of OSA in various clinical populations. It assesses snoring, fatigue, observed events, blood pressure, BMI, age, neck circumference, and gender. Telomere attrition: the progressive and cumulative loss of telomereprotective sequences from chromosome ends that explains the limited proliferative capacity of some types of cell. Toll-like receptors (TLRs): recognize specific patterns of microbial components, especially those from pathogens, and regulate the activation of both innate and adaptive immunity. Unfolded protein response (UPR): an adaptive response to endoplasmic reticulum (ER) stress, restoring ER homeostasis. Very small embryonic-like cells (VSELs): a population of epiblastderived cells that express several markers of pluripotent stem cells (PSC) that are characteristic for epiblast/germ line-derived stem cells. VSELs are deposited during early gastrulation in developing tissues and/or organs and have an important role in the turnover of tissue-specific and/or committed stem cells.

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Box 1. Defining OSA Obstructive sleep apnea (OSA) is a sleep-related breathing disorder in which repeated episodes of complete (apnea) or partial (hypoapnea) obstruction of the upper airway occur during sleep while breathing efforts continue [1]. Each hypopnea or apnea episode is often accompanied by reduced oxygen saturation and hypercapnia, which stimulate chemoreceptors, induce increases in blood and intrathoracic pressures, and stimulate sympathetic neural activity (SNA). Upper airway obstruction often ends with an arousal to restore upper airway patency, normalize blood oxygen levels, and suppress SNA [2,3]. Thus, repetitive obstruction episodes while sleeping culminate in cyclical deoxygenation–reoxygenation, over activation of SNA, bursts in systemic blood and arterial pulmonary pressures, and several arousals and microarousals that result in sleep fragmentation (SF) and, subsequently, nonrestorative sleep [2,3]. In this context, patients with OSA may complain of poor sleep quality, daytime fatigue, morning headaches, concentration problems, forgetfulness, and excessive daytime sleepiness [1]. Consequently, OSA is associated with considerable increased risk of motor vehicle accidents, work-related injuries, and disability [132–134]. Other long-term consequences of OSA include molecular and cellular impairments that contribute to the development or accelerated progression and severity of several diseases, including cardiovascular and metabolic diseases, depression, cancer, and neurodegenerative diseases [12,15,18,19,29] (Figure I). Various factors contribute to a higher propensity of developing OSA, in particular, excessive weight, old age, male gender, postmenopausal status in women, facial and upper airway abnormalities, family history, smoking, use of alcohol or sedatives, nasal congestion, and ethnicity [1–3]. Although familial clustering studies have shown that a positive family history is an important OSA risk factor, a gene or genes responsible for OSA heritability have not yet been identified, apart from a putative association with the gene encoding the apolipoprotein E allele (e4), although this has been inconsistently replicated in studies of patients with OSA [136]. Thus, the manifestation of OSA is likely to be determined by multiple genetic and environmental factors and their interactions, and the apparent heritability of OSA may arise from heritable unfavorable craniofacial morphology and familiar obesity [1–3].

Figure I. Pathophysiological Mechanisms and Outcomes of Obstructive Sleep Apnea (OSA). Representative scheme of the pathophysiological mechanisms of OSA and associated consequences: (A) OSA is characterized by recurrent episodes of complete (apnea, represented in this figure) or partial (hypoapnea) obstruction of the upper airway during sleep. (B) Apnea episodes (between red broken lines) result in cessation of the airflow, often accompanied by reduced oxygen (O2) saturation and increased systemic blood and arterial pulmonary pressure. As a result, sympathetic neural activity (SNA) increases and apnea episodes end with an arousal of the central nervous system to restore upper airway patency, marked by an increased electroencephalogram (EEG) wave frequency. Thus, repetitive obstruction episodes while sleeping culminate in cyclical deoxygenation–reoxygenation (intermittent hypoxia; IH), overactivation of SNA, bursts in systemic blood and arterial pulmonary pressures, and several arousals and microarousals that result in sleep fragmentation (SF). (C) As a result, OSA is associated with oxidative stress, inflammation, endothelial dysfunction, and changes in circulating factors. (D) Untreated OSA has been associated with an increased predisposition for several impairments and diseases, including hypertension, cardiovascular diseases, metabolic disorders, stroke, depression, cancer, and neurodegenerative diseases.

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Box 2. Impact of Aging on OS Obstructive sleep apnea (OSA) has been considered an age-related sleep disorder, since its prevalence increases two to three times in older adults compared with middle-aged adults (reviewed in [6]). Facing such numbers, researchers have attempted to explain the increased OSA prevalence among older people. The obesity prevalence among older people is known to contribute to higher rates of OSA. However, independent of gender and body mass index (BMI), physiological aging has been reported to induce structural and functional changes in the upper airways, causing increased pharyngeal airway collapsibility during sleep. With aging, there is an increased tendency for pharyngeal fat deposition, the soft palate lengthens, and muscle tone and pharyngeal sensory discrimination become impaired, resulting in older people being more vulnerable to the occurrence and persistence of obstruction episodes during sleep [40]. In this context, two distinct OSA phenotypes have been defined, based on aging and OSA crosstalk: (i) an agerelated phenotype: the classic obese 50+-year-old male, with evidence of clinical outcomes (e.g., hypertension and cardiovascular diseases); and (ii) an age-dependent phenotype, less dependent on sex and obesity and more dependent on aging, with unclear clinical consequences [136,137]. Indeed, despite being associated with an increased predisposition for OSA, aging has been shown to confer protection against the deleterious effects of chronic IH. Namely, aged OSA rat models [submitted to 50 apneas/h, lasting 15 s each, for 50 min or 4 h, through a nasal mask, or submitted to cycles of 40 s, 5% O2/80 s, air, equivalent to an Apnea-Hypopnea Index (AHI) of 30, 8 h per day, for 14 days] have shown lower cerebral oxidative stress (e.g., decreased lipid peroxidation and higher expression of antioxidant enzymes) and decreased sympathetic tone (decreased norepinephrine) compared with young rats [138,139]. Such a protective mechanism could be due to different brain oxygen partial pressures among age groups and/or increased antioxidant expression in aged rats, which may translate into less susceptibility to comorbidity [138]. However, cellular and molecular mechanisms underlying aging protection against OSA effects must be investigated further.

disease, putatively mimicking an aging context. The ‘Epigenetics Modifications in OSA’ (EPIOSA) study is a 5-year non-interventional longitudinal prospective study that is currently exploring epigenetic changes in the regulation of systemic inflammation and metabolic dysfunction that might contribute to accelerated cardiovascular morbidity, as well as atherosclerosis prevalence and progression, in patients with OSA compared with treated and control subjects [67]. Although all these studies present results correlating OSA with epigenetic alterations in cells from humans and from different experimental models of OSA, further studies Box 3. Diagnosing OSA Obstructive sleep apnea (OSA) symptoms are difficult to quantify and are poorly correlated with the frequency of apneas and hypopneas during sleep. At first glance, sleepiness may be assessed by subjective tools, such as the Epworth Sleepiness Scale [1]. More recent screening tools, including Berlin and STOP-Bang questionnaires, may also be used to identify other relevant symptoms (i.e., snoring, apneas, and hypertension) [140,141]. Head and neck morphometric analysis are also relevant OSA indicators [142]. The gold-standard method for OSA diagnosis is polysomnography (PSG), a test performed in the laboratory that allows the monitoring of sleep and recording of, among other relevant parameters, apneas, hypopneas and respiratory effortrelated arousals (RERAs), per hour of sleep. Other in-laboratory methods include assessment of sleep nasal airflow, respiratory effort, and/or events of oxygen desaturation in blood. However, these tests are labor intensive, expensive, and associated with long waiting periods, delaying OSA diagnosis and treatment. Home-based tests with portable sleep monitors constitute an alternative strategy but present limitations and may lead to erroneous interpretations that require confirmation by PSG [10]. Key factors commonly used to classify OSA severity include the Apnea-Hypopnea Index (AHI), the number of apneas and hypopneas per hour, and Respiratory Disturbance Index (RDI), a summary of apneas, hypopneas, and RERAs per hour. Accordingly, an adult patient with OSA can be classified as mild (5 AHI/RDI < 15), moderate (15  AHI/ RDI < 30), or severe (AHI/RDI  30) [1]. However, depending on the recording method and index, different types of diagnosis may be made, leading to inaccurate diagnoses and inappropriate treatments. Moreover, OSA is now recognized as a multifactorial and heterogeneous disease, showing diverse physiological etiologies, symptom variability, and different comorbid conditions. Thus, factors other than upper airway collapsibility may contribute to OSA, and must be taken into consideration, particularly nonanatomical parameters (i.e., ventilator control system, respiratory arousal threshold, or pharyngeal muscle responsiveness) and patient history [2,3,8]. Efforts should be made to differentiate the specific pathophysiology of each patient from a personalized medicine perspective. For this approach, new complementary diagnostic strategies are needed. Extensive research has been conducted to identify OSA biomarkers, through omics technology. Although several candidates have been proposed, no ideal biomarker has yet to be widely implemented (reviewed in [144]). Interlaboratory variations regarding assessing methods may also challenge the validation of emerging diagnostic strategies [7].

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Box 4. Treating OSA At first, all patients are encouraged to control their BMI and, if applicable, lose body weight to ameliorate their pharyngeal structure and function, neuromuscular control and functional lung residual capacity. Avoiding certain substances (e.g., sedatives, tranquilizers, antihistamines, and alcohol) and avoiding sleeping in a supine position, through wearable devices or alarms, may also attenuate upper airway collapsibility [7]. For mild obstructive sleep apnea (OSA) cases, these lifestyle changes may be sufficient. However, additional intervention is recommended for patients with mild OSA and OSA-associated comorbidities or intrusive sleepiness and for moderate and severe cases [1]. The first line of OSA therapy is the use of continuous positive airway pressure (CPAP) via a mask interface that supports breathing during sleep. However, despite its general high efficacy on decreasing upper airway collapsibility, many patients do not tolerate the discomfort of sleeping with a mask. More personalized devices have been developed to increase adherence rates, but with limited success [7]. Alternatively, there are nasal and mandibular advancement devices that can be used during sleep. Such appliances may also be recommended to patients whose OSA severity does not justify the use of the CPAP mask [7]. Surgical intervention may also be considered, especially when CPAP or oral appliances are not recommended or have failed. Surgical procedures may correct anatomical deformities, or even remove tissue to widen the airway. However, such procedures are invasive and may not be efficacious in all patients [7]. Consequently, surgical procedures remain controversial and require further investigation. By contrast, pharmacological treatments have also been developed to improve OSA symptoms. Clinical trials were performed with different therapeutic strategies for OSA: to increase respiratory control and the upper airway tone, or to reduce the cross-sectional area or the surface tension of the upper airway, to decrease REM sleep, or to increase the arousal threshold [20]. However, until now, none of these drugs have been approved for OSA treatment. Drugs with high efficacy, few adverse effects, and long-term viability to alleviate OSA are not yet available [144]. Moreover, variability in treatment response strongly encourages the adoption of personalized medicine approaches. For specific patients, combining two or more strategies may lead to greater OSA alleviation [7]. A follow-up of sleep might be recommended to monitor treatment efficacy, independent of the recommended treatment. In the future, treatment-responsive biomarkers may overcome such a need [3,10].

are needed to clarify the cause–effect relationship between OSA and epigenetic regulation, as well as the significance of observed epigenetic alterations to the aging process and to agerelated diseases. Loss of Proteostasis Protein homeostasis, or proteostasis, is ensured through the coordination of several cellular systems that are able to control protein synthesis, folding, aggregation, and degradation. Such systems include translational machinery, molecular chaperones, the ubiquitin-proteasome system, lysosomes, and the autophagy machinery [68]. Aging and aging-related diseases, including neurodegenerative disorders, have been associated with proteostasis impairments and the subsequent presence of unfolded, misfolded, and aggregated proteins [43,68]. Proteostasis networks are vulnerable to several stressors, including oxidative stress, often associated with aging [43,68]. In this context, it would appear likely that, due to increased oxidative stress in OSA, alterations in proteostasis networks could also occur, contributing to aging and age-related diseases. Unfortunately, only a few studies have explored this topic. Such studies have mainly focused on molecular heat shock proteins (HSP), which are molecular chaperones expressed under stress conditions (heat, hypoxia) that restore aberrantly misfolded proteins and prevent their aggregation, protecting cells against stress. These studies showed that protein levels of the molecular chaperone HSP70 progressively decreased in PBMCs of patients with severe OSA during sleep, contrasting with the constant levels that are observed during the sleep of control subjects [69]. Upon CPAP treatment, the HSP70 decrease during sleep was no longer observed in PBMCs from patients with OSA, suggesting that repetitive stress during sleep in untreated patients could lead to HSP70 exhaustion [69]. HSP exhaustion may challenge cell proteostasis, because protein modifications and structural changes may easily occur and aberrant proteins might then accumulate [69,70]. By contrast, HSP70 levels in monocytes of patients with moderate-severe OSA were reported to be higher during the wake daytime cycle compared with healthy subjects; this could indicate a protective mechanism against the oxidative stress derived from sleep IH events [69,70]. However, the same study showed that, following a night of sleep, when monocytes from patients with OSA were submitted to a second challenge (heat stress), HSP70 protein levels decreased, suggesting an impairment in OSA adaptive responses to stress [70]. On the one hand, HSP failure to

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bind and deliver proteins to degradation systems might contribute to protein aggregation and accumulation, although this hypothesis has not yet been explored. On the other hand, in animal studies, both young and aging rats under IH (10% O2 every 90 s, 14 days) have shown decreases in proteosomal activity in cells from the hippocampus, as evidenced by a marked inability to adequately clear degraded proteins compared with rats exposed to room air [71]. Impairments in proteasomal activity have been related to reduced peptide and protein hydrolysis, the accumulation and aggregation of ubiquitinated proteins, and neuronal cytotoxicity, and thus, have been associated with neurodegenerative disorders [68]. In mice, moderatesevere SF (4 weeks of SF induced by a motorized intermittent tactile stimulation) was shown to induce endoplasmic reticulum (ER) stress and to activate the unfolded protein response (UPR) in the hypothalamus compared with sleep control mice [72]. Similarly, rats exposed to chronic IH (9% O2 for 30 s and 21% over the next 12 s, repeated every 90 s over 7.5 h during sleep, for 2 or 4 weeks) induced ER and UPR in the hippocampus and prefrontal cortex compared with normoxia rats [73]. The UPR is reported to occur under ER stress conditions and contributes to the restoration of ER function (through chaperone upregulation and increased protein folding), pausing translation and promoting the degradation of misfolded proteins. Thus, SF may also constitute an important player contributing to OSA-mediated proteostasis impairments. Chronic SF [21 days of SF through bar rotation for 30 s every 2 min followed by 90 s rest which disturbs sleep in a similar manner to gentle handling] was also reported to blunt the circadian rhythm of autophagy-related proteins [microtubule-associated protein 1 light chain 3 (LC3) and beclin-1] in the hippocampus of mice [74]. Thus, although there is evidence of the effects of OSA on proteostasis systems, it remains scarce, and further studies are needed to elucidate the precise impact of OSA on proteostasis and its relationship to OSA clinical outcomes. Deregulated Nutrient Sensing The cellular ability to sense and respond to nutrient level fluctuations is essential for cellular homeostasis. Insulin and insulin-like growth factor 1 (IGF-1) signaling constitute one of the most evolutionarily conserved nutrient-controlling pathways, affecting multiple targets [43,75]. Of relevance, aging can lead to nutrient-sensing pathway deregulation in mouse models and humans [43]. Similarly, over the past decade, several studies have suggested a bidirectional link between OSA and metabolic dysfunction, evidencing possible deregulated nutrient-sensing pathways (reviewed in [76]). For example, independently of any incidence of obesity, patients with mild-to-severe OSA showed insulin resistance (increased fasting insulin) and glucose intolerance (increased glucose levels and decreased glucose effectiveness in peripheral blood) compared with healthy control subjects [14,77–80]. After CPAP treatment, insulin sensitivity was reported to rapidly improve in patients with moderate-to-severe OSA relative to nontreated patients [81,82]. Accordingly, lower blood and serum IGF-1 protein levels were reported in patients with OSA compared with healthy subjects [83,84]. By contrast, following CPAP treatment, IGF-1 levels increased, approaching the levels observed in control subjects and suggesting that OSA treatment impacts specific components of the IGF-1 axis [84]. In addition, patients with moderate-to-severe OSA have also demonstrated impairments in pancreatic b cell function compared with healthy control subjects or those with mild OSA, as shown through an integrated measure of pancreatic b cell function [the disposition index (DI)/ homeostasis model assessment estimates of steady-state pancreatic b cell function (HOMAB)] [79,85]. It is possible that such an impairment is caused by an excessive functional demand of OSA-associated insulin resistance on pancreatic b cells, leading over time to b cell exhaustion and abnormal insulin secretory capacity; however, such a hypothesis remains to be tested [79,85]. Nevertheless, which OSA-associated mechanisms could trigger such impairments? Mice exposed to IH (cyclical pattern every 180 s of alternating 5.7% and 21% O2, for 24 h/day for 4 days or 5–6% O2 for 60 cycles/h for 9 h during 1 day) exhibited increased b cell proliferation and cell death compared with room air-treated mice [86,87]. A hypothesis

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underlying the impact of OSA on nutrient sensing involves the reduced oxygen delivery to tissues, which may directly impair their capacity to oxidatively metabolize glucose, contributing to b cell dysfunction and insulin resistance [86]. Further experiments are necessary to confirm and elucidate the underlying mechanisms of deregulated nutrient sensing in OSA; nevertheless, these observations emphasize a possible contribution of OSA to metabolic disorders, in particular, glucose intolerance, type 2 diabetes mellitus and, putatively, to the acceleration of aging. Mitochondrial Dysfunction As cells age, mitochondrial integrity can become compromised, resulting in the reduced efficacy of the respiratory chain, decreased ATP generation, increased leakage of electrons, as well as the formation of reactive oxygen species (ROS), and enhanced oxidative stress [88]. In turn, ROS and oxidative stress may cause further mitochondrial dysfunction and lead, in a vicious cycle, to cellular damage [43,88]. As a result, nuclear and mitochondrial communication may become impaired, which may contribute further to cellular aging and pathology [43,89]. Several studies have suggested a significant relationship between OSA and oxidative stress. Different oxidative stress markers (ROS production, increased levels of 8-OHdG, 8-isoprostane and peroxide levels, as well as decreased nitrate and nitrite levels) have been reported to be increased in PBMCs, polymorphonuclear neutrophils, plasma, exhaled breath condensates, and urine from patients with moderate-severe OSA relative to patients with mild OSA or healthy control subjects [44,90–92]. Moreover, the antioxidant capacity of patients with severe OSA was also found to be impaired, as evidenced by decreased total antioxidant status (TAS) and levels of antioxidant vitamins (A and E), and increased values of the antioxidant enzyme g-glutamyl transferase (GGT) in plasma, relative to healthy subjects [93]. Antioxidant enzymes and vitamins counterbalance ROS and contribute to reduce ROS-associated cellular damage, indicating that impairments in antioxidant capacity may further exacerbate oxidative stress in patients with OSA. However, CPAP treatment was shown to ameliorate these alterations, improving antioxidant capacity (increased TAS and decreased GGT) in plasma, reducing 8isoprostane levels in exhaled breath condensate and plasma, decreasing superoxide release from polymorphonuclear neutrophils and reducing basal ROS production in monocytes [90– 93]. These results strengthen the link between OSA and oxidative stress and suggest the occurrence of mitochondrial dysfunction in cells from patients with OSA. In accordance, reduced mtDNA copy numbers from whole-blood DNA have been noted in patients with mild-severe OSA relative to healthy controls, correlating with OSA severity [94]. Indeed, lower mtDNA copy numbers are associated with reduced mitochondrial biogenesis and energy metabolism, as commonly seen during aging [95]. In addition, in contrast to rats under normoxia, rats exposed to long-term IH (12% O2, 8 h/day, for at least 4 weeks) exhibited decreased mitochondrial biogenesis and function (e.g., by inhibiting cytochrome C oxidase activity and activating the mitochondrial- and Fas death receptor-dependent apoptotic pathways) in cardiac tissue extracts obtained from the left ventricle and genioglossi [96,97]. Such mitochondrial impairments may lead to increased levels of superoxide formation, aggravation of cellular oxidative stress, and further mitochondrial damage and dysfunction [96,97]. IH has also been shown to stimulate the expression of glycolytic enzymes and the lactate monocarboxylate transporter MCT4 in cultured mouse cortical astrocytes (via a hypoxia-inducible factor-1amediated transcriptional regulation), This reflects the astrocyte–neuron–lactate shuttle hypothesis, which posits that interfering with mitochondrial metabolism and neurometabolic coupling leads to impairments associated with learning deficits [98]. Thus, OSA may contribute to mitochondrial dysfunction, a hypothesis that must be validated further. The underlying mechanisms responsible for mitochondrial dysfunction induced by OSA remain unknown and it is not clear how such impairments could contribute to aging or age-related disorders. Chronic IH may have a role in mitochondrial dysfunction (and result from OSA), but SF and inflammation should also be evaluated in further studies.

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Cellular Senescence Cellular senescence refers to a state in which cells stop dividing, triggered by stereotyped phenotypic changes that culminate in cell cycle arrest. Although cellular senescence was initially associated with telomere shortening, several other aging-associated changes contribute to it, particularly DNA damage and oxidative and proteotoxic stress [43,99]. Human aged adult senescent cells are characterized by the reduced expression of telomerase reverse transcriptase (TERT), increased expression of cyclin-dependent kinase inhibitor 2A (p16INK4a), and a particular secretory pattern, the senescent associate secretory phenotype (SASP), which involves the increased release of inflammatory cytokines and chemokines ([43], reviewed in [99]). Evidence for OSA-associated DNA damage and oxidative and proteotoxic stress has been discussed in previous sections. In addition, SF has been shown to induce SASP and increase cellular senescence; specifically, relative to control sleep mice, mice under chronic and severe SF (20 weeks of SF induced by a motorized intermittent tactile stimulation) demonstrated reduced TERT and cyclin A protein levels and increased p16INK4a protein expression in the ascending aorta, while higher levels of the inflammatory marker interleukin 6 (IL-6) were measured in plasma [100]. In agreement, using a chronic sleep disruption protocol in mice (employing an orbital rotor working on repeated cycles of 10 s on and 50 s off, continuously for 14 weeks) increased levels of lipofuscin (a marker of cellular senescence) were noted in murine locus coeruleus and orexinergic wake-activated neurons, relative to mice under control conditions [101]. Although these observations suggest that OSA contributes to inducing cellular senescence, little evidence exists on the nature of the senescence-related pathways and on the putative causative and/or mechanistic roles of OSA. Further studies should help to elucidate the exact impact of OSA on cellular senescence. Stem Cell Exhaustion When faced with certain stimuli, such as inflammation or oxidative stress, stem cells can mobilize to injured sites to regenerate by differentiating into organ and/or tissue-specialized cells [43]. However, with aging, the regenerative capacity of tissues declines, in part as a consequence of stem cell exhaustion, which implies the loss of proliferative and/or self-renewal potential and the ability to effectively differentiate [43,102]. This decreased regenerative capacity may in turn contribute to aging states, as observed in myeloid malignancies [102]. Recently, interest in OSA-associated oxidative stress, inflammation, and endothelial dysfunction has prompted research in the field of stem cell exhaustion. However, available data are limited and, in some cases, involve contradictory results. Briefly, three stem cell niches have been evaluated within the OSA context: endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs) and very small embryonic-like cells (VSELs) (reviewed in [103]). So far, studies focusing on EPCs have been exclusively conducted in patients with OSA; however, the data obtained so far are contradictory, suggesting either a reduced, unchanged, or increased number of EPCs in the blood of patients with OSA compared with healthy subjects (reviewed in [103]). Contradictory results may derive from different EPC isolation and quantification methods; sample heterogeneity (males or females, children or adults, exclusion of co-morbidities or not); and/or sample scarcity (i.e., a low number of EPCs in the circulating blood). Still, despite contradictory results, most studies point to a reduced number of circulating EPCs in the blood of patients with mild-severe OSA compared with healthy control subjects ([104], reviewed in [103]). Indeed, increased levels of an apoptosis marker in endothelial cells were reported in patients with mild-severe OSA compared with healthy subjects [105]. This observation suggests that OSA promotes endothelial cell apoptosis and exhausts the repair capacity of the vascular endothelium, resulting in continuous endothelial damage [105]. Interestingly, CPAP treatment was shown to re-establish the number of EPCs, emphasizing a potential impact on EPC niches [105]. From another angle, MSC and VSEL data have been solely derived from OSA animal models and revealed different outcomes (reviewed in [103]). Regarding MSCs, it was observed that serum from rats subjected to recurrent obstructive apneas (using an

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electronically controlled nasal mask causing 60 apneas/h, lasting 15 s each, for 5 h), activated MSCs, by promoting their early release into circulation and increasing their motility (measured through transwell assays), and increased MSC adherence and endothelial repair capacity on monolayers of cultured endothelial cells from rat aorta, compared with serum from control rats under normoxia [106]. However, could such an effect be maintained after continuous submission to recurrent obstructive apneas? Could a long-term effect lead to MSC exhaustion? Further studies are needed to understand the potential impact of OSA on MSCs. Finally, in the case of VSELs, mice under IH (alternating 21.0% and 5.7% O2 every 180 s for 12-h light period over 48 h) showed increased migration of VSELs and significant changes in their transcriptional signature, marked by activation of multiorgan developmental programs, angiogenesis, central nervous system (CNS) development, and tube/lung development compared with mice under normoxia conditions [107]. However, would VSELs increased migration upon short-term IH be maintained under a chronic IH condition? Of note, studies with MSCs and VSELs were performed after the short exposure of the animals to IH and may not mimic the impact of OSA on these stem cell niches. Rodent models of long-term IH and also those exhibiting SF may result in different outcomes and will better mimic an OSA condition. Thus, more studies are needed to better understand the impact of chronic OSA on different stem cell niches and whether such changes in stem cell functions contribute to age-related impairments. In addition, it will be interesting to analyze whether OSA can have effects on cognitive function that are potentially related to alterations in neural progenitor cells and impairments of neurogenesis. Rats exposed to IH (90 s of 10% O2 alternated with 90 s of room air, for 12 h, up to 30 days), show an initial decrease in basal neurogenesis, as evidenced by positive staining of cells colabeled for bromodeoxyuridine (BrdU) and neurofilaments in the dentate gyrus of the hippocampus [108]. However, upon continued IH exposure (after 3 days), increased expression of neuronal progenitors and mature neurons (shown by nestin and BrdU-neurofilament positively labeled cells, respectively) were observed [108]. Thus, it is possible that IH can alter neurogenesis. However, could neuronal progenitor cells become exhausted upon continuous stress? Would IH and SF induce a different response? Could such an impairment underlie part of the neurodegeneration observed in the brains of patients with OSA [30–34]? Altered Intra- and Intercellular Communication: Neuroendocrine Function, Inflammation, and Immunity Aging is accompanied by neuroendocrine dysfunction, increased inflammation, immune surveillance decline, as well as changes in peri- and extracellular environments that may challenge intercellular communication [43]. Neuroendocrine dysfunction has been associated with OSA; however, data are scarce and contradictory, likely due to the presence of comorbidities, such as obesity, which may mask whether OSA can cause endocrine alterations (reviewed in [109,110]). Most neuroendocrine studies in patients with OSA point to decreased levels of spontaneous and stimulated growth hormone (GH) in serum; reduced GH peripheral sensitivity [observed following short treatment with the minimal recombinant human (rh)GH effective dose] [111]; reduced serum IGF-I levels [111,112]; and increased serum prolactin levels and impaired secretory circadian rhythm [113–115] compared with matched obese controls. Studies focused on other pituitary, adrenal, thyroid, and gonadal axis hormones (e.g., adrenocorticotropic hormone, cortisol, and TSH) show contradictory results regarding hormone levels in blood, serum, plasma, or saliva, with studies showing increases, decreases, or no alterations (reviewed in [109,110]). Of relevance, several studies have also reported increased urine and plasma levels of catecholamines, namely, norepinephrine (NE), in patients with OSA compared with control subjects ([117], reviewed in [118]). Such an increase is believed to have a role in the pathophysiology of OSA-associated cardiovascular morbidity [116,117]. Thus, although the cause–effect relationship between

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OSA and neuroendocrine dysfunction needs to be robustly dissected, evidence of altered hormonal levels and profiles exists that must be further explored to understand their potential contribution to an aging phenotype. In addition, impairments in hormones involved in hunger and satiety (leptin, ghrelin, and orexin secretion) and peptide control (NPY) have also been reported in patients with OSA compared with control subjects, with potential impact on energy balance parameters (reviewed in 118). However, again, significant variability has also been observed among these studies, either in study design, or in obtained data and CPAP effects. Most studies in the field have focused on leptin, a satiety hormone, with the majority pointing towards increased leptin levels in the serum and plasma of patients with OSA compared with body mass index (BMI)-matched controls (119,120, reviewed in [118]). Based on these results, it was suggested that OSA may induce a leptin-resistance state that might underlie a reciprocal relationship between OSA and obesity [118]. Of relevance, other studies have also showed increased plasma levels of ghrelin and decreased levels of plasma orexin, both hunger signals, in patients with OSA compared with control subjects ([121,122], reviewed in [118]). Such abnormal satiety and hunger control might predispose patients to several metabolic diseases and premature aging. Data regarding NPY are scarce and contradictory, with studies reporting either increases or no alterations in OSA (reviewed in [118]). From another angle, several reports have shown increased inflammation in an OSA context: in particular, when compared with control subjects, circulating neutrophils in patients with mildsevere OSA had elevated expression of nuclear factor-kB (NF-kB), a transcription factor with key roles in inflammatory cascades [123]. Furthermore, increased plasma and serum levels of several systemic inflammatory markers (C-reactive protein, TNF-a, IL-6, IL-8, ICAM, E-selectins, and VCAM-1) were observed in patients with mild-severe OSA compared with control subjects [123–125]. Moreover, unlike mice under normoxia, chronic IH (21% FiO2 alternated with 5% FiO2, 30 s each, 60-s cycle, 8 h/day for 14 days) in mice, has been reported to induce increased lymphocyte proliferation and chemokine expression in mouse spleens, subsequently resulting in systemic and vascular inflammation, as measured by inflammatory chemokine and cytokine expression and based on microglia histological analyses [126]. In addition, the levels and activity of sirtuin 1 (SIRT1), a protein that downregulates inflammation-related genes, were also shown to be reduced in PBMCs from patients with moderate-severe OSA, compared with control subjects [43,127]. By contrast, successful treatment with nasal CPAP restored SIRT1 protein and activity levels [127]. SIRT1 has also been shown to be involved in other cellular processes, namely, cell cycle regulation, DNA damage repair, telomere attrition, and, upon deregulation, the aging process [43,127]. Thus, lower SIRT1 levels may also contribute to the potential effect of OSA on aging. Finally, it has been posited that increased inflammation, as observed in patients with OSA, could interfere with lymphocyte traffic, circulation, proliferation, cytokine production, and functional activities [128]. Indeed, this was shown in patients with moderate-to-severe OSA, who, compared with healthy subjects, presented functional changes in blood-derived CD8+ T lymphocytes, namely, an increased cytotoxic potential, as evidenced by cytotoxicity assays performed against K562 target cells and human umbilical vein endothelial cells [129]. Furthermore, these cells presented increased CD56 and CD16 receptor expression, which are perforin and natural killer receptors, respectively, key components of the innate immune system [129]. Similarly, there is also evidence of increased expression of Toll-like receptor (TLR) 2 and 6 (key players of innate immunity) on the cell surface of peripheral blood neutrophils and mononuclear cells from patients with moderate-severe OSA compared with control subjects [130]. In agreement with this finding, exposure of these blood cell types from healthy subjects to IH with reoxygenation also resulted in TLR2/6 upregulation in vitro [130]. Immune cell alterations in patients with OSA may lead to a decline of the innate and adaptive

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immune systems, and possibly immunosenescence, which may all contribute to an aging phenotype [43]. Indeed, when considering the effects of OSA on cancer pathogenesis, OSA mouse models presented reduced immunosurveillance upon injection with TC1 cells derived from a lung epithelial tumor (mice exposed to IH in alternated cycles of 90 s 6% FiO2 followed by 21% FiO2, 20 cycles/h for 12 h/day and mice submitted to SF through a near-silent motorized mechanical sweeper that provoked automated intermittent mechanically induced arousal) [131]. In a tumor environment context, OSA-mimicking mice exhibited more adverse outcomes, as evidenced by a decreased number and frequency of cytotoxic CD8+ T cells, as well as increased cancer stemness markers in TC1 tumor cells (Oct4+ and CD44+CD133+) compared with controls [131]. Thus, both IH and SF resulted in reduced effector T lymphocyte functions against malignant cells, which could hasten malignant transformations [131]. Although the direct effects of OSA on malignant responses and impaired immune function need to be robustly tested, the prior observations are consistent with the presumed increased susceptibility to carcinogenesis triggered by OSA [18]. In addition, full supportive evidence of the potential effect of OSA on immunosenescence remains to be validated and warrants further studies. In summary, several OSA studies have pointed to alterations in processes that are essential to both intra- and intercellular communication, challenging peri- and extracellular environments, and contributing to a functional decline that might potentiate and/or accelerate the aging process. It is clear that a better understand of the impact of OSA on these processes will be crucial to defining the putative contribution of OSA to aging.

Concluding Remarks Over recent decades, several studies have improved our understanding of the physiological mechanisms and outcomes of OSA, providing relevant insights into its potential contribution to the development of various diseases and impairments. These findings involve nine cellular and molecular impairments generally associated with the aging process: the hallmarks of aging (Figure 1, Key Figure). In fact, several studies have suggested that OSA induces oxidative stress, a link to mitochondrial dysfunction, compromise intra- and intercellular communication, and impair nutrient sensing; recent reports have also shown that OSA potentially induces epigenetic changes, modifies stem cells, and decreases telomere length; only a few studies suggest that OSA impacts other hallmarks of aging, such as on DNA damage and repair, proteostatic mechanisms, and cellular senescence (as summarized in Table S1 in the supplemental information online). Collectively, these observations have led us to propose that OSA not only constitutes an age-related disorder, but also accelerates and/or potentiates aging mechanisms and, possibly, age-related disorders. However, studies that elucidate the causative and/or mechanistic roles of OSA in aging are missing. Further studies are needed to clearly distinguish between correlative and causal observations in the proposed links between OSA, aging, and age-related diseases. Much remains to be explored, regarding both the nature of OSA crosstalk with aging and the induction of age-related phenotypes that might be associated with OSA heterogeneity (see Outstanding Questions and Box 5). OSA is a major health problem, impacting quality of life, health, and socioeconomic sectors. Moreover, the high rates of undiagnosed cases and the low level of public awareness of this disease constitute a barrier that has been difficult to overcome. Thus, it is crucial to develop early, personalized, and robust diagnosis strategies, and improve its treatment efficacy and adherence. OSA patient stratification with respect to disease manifestations may contribute considerably to better diagnoses and treatments under an umbrella aimed at personalized medicine. Effective early OSA diagnosis and therapy might help halt the progression of cellular and molecular changes that may become irreversible, hopefully contributing to delaying aging progression and certain age-related disorders. The hypothesis that OSA might contribute to aging and disease might also help emphasize the importance of healthy sleep, a current societal issue.

Outstanding Questions Could OSA impacts on aging and agerelated impairments be blocked and/or reversed with OSA treatment? To what extent? Could OSA heterogeneity derive from or induce different patterns of epigenetic modifications? Could those alterations contribute to different OSA outcomes and even treatment responses? OSA has been associated with an increased risk for the development of age-related neurodegenerative disorders. Could OSA promote protein misfolding, aggregation, and accumulation, key molecular pathways implicated in diverse neurodegenerative disorders, through impairments of proteostasis mechanisms? Neurogenesis is an essential component of cognition. Are OSA-associated cognitive impairments related to the exhaustion of neural progenitor stem cells and impaired neurogenesis? OSA may induce genomic instability and cellular damage. Are DNA repair mechanisms impaired in OSA? Could those impairments be the basis of the increased tumorigenesis seen in patients with OSA and, potentially, aging? Does OSA also induce mtDNA damage? Mitochondria–nucleus communication is essential for appropriate mitochondrial activity, and its disruption often accompanies aging and age-related diseases. Supporting evidence suggests that OSA induces mitochondrial dysfunction, one of the hallmarks of aging. Is mitochondria–nucleus communication impaired in OSA? What is the impact of OSA on agingrelated dysfunctions, namely, in fertility, locomotor activity, eye pathologies, or skin aging? Are age-related impairments correlated with OSA severity? Aging leads to functional deterioration of the circadian clock. The circadian clock is involved in the control of brain metabolism, ROS homeostasis, hormone secretion, autophagy, and stem cell proliferation. Could OSA contributions to aging and age-related hallmarks derive from disruptions to the circadian clock? Is the central circadian clock impaired in OSA?

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Key Figure

Obstructive Sleep Apnea (OSA) and the Hallmarks of Aging

Figure 1. The figure summarizes the hypothesis of the contribution of OSA to aging and to age-related diseases. OSA-associated outcomes may accelerate and/or potentiate aging, prompting the nine hallmarks of aging: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. Adapted from [43].

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Box 5. Clinician’s Corner  Obstructive sleep apnea (OSA) is a sleep-related breathing disorder characterized by recurrent episodes of complete









(apnea) or partial (hypopnea) obstruction of the upper airway during sleep, while respiratory efforts continue. With aging, there is an increased tendency for pharyngeal fat deposition, the soft palate lengthens and muscle tone and pharyngeal sensory discrimination become impaired, resulting in older people being more vulnerable to the occurrence and persistence of obstruction episodes during sleep. OSA is a multifactorial and heterogeneous disease, showing symptom variability, diverse physiological etiologies, and different comorbid conditions and outcomes. Thus, the AHI or Respiratory Disturbance Index should not be used as single OSA indicators, but should rather be complemented by other important nonanatomical parameters (i. e., ventilator control system, respiratory arousal threshold, or pharyngeal muscle responsiveness) and patient history. New diagnostic strategies are urgently required to counteract the high number of underdiagnosed OSA cases. Identification of OSA biomarkers, if and when available, might provide valuable tools, allowing easier, earlier, and personalized diagnoses that might increase treatment efficacy. Untreated OSA has been associated with the development of several age-related diseases and impairments, namely, cardiovascular diseases, type 2 diabetes mellitus, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, cancer, and depression ([10–19], reviewed in [2,3]). Recent findings have also associated OSA with both cognitive decline and early stages of dementia and neurodegenerative diseases, such as Alzheimer’s or Parkinson’s diseases, impairments commonly associated with aging ([26], reviewed in [29]). Middle-aged individuals with OSA present with alterations in brain performance and function earlier than would ordinarily be expected compared with age-matched controls [24]. Furthermore, supportive evidence shows that OSA may accelerate cognitive decline onset in older patients. Not only might aging contribute to OSA development, but OSA might also potentiate and/or accelerate aging and age-related declines. However, further testing is required to validate this hypothesis. Several studies suggest that OSA contributes to all nine hallmarks of cellular aging, namely, genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. Further studies are needed to confirm and validate such a hypothesis. However, it is possible that future effective OSA diagnosis and treatment strategies might help to counteract the progression of age-related diseases and aid a healthier lifespan.

Acknowledgments This work was funded by FEDER (QREN) through Programa Mais Centro, under projects CENTRO-07-ST24-FEDER002006; the Operational Programme Factors Competitiveness-COMPETE 2020; and National Funds through FCTFoundation for Science and Technology under the Strategic Project (UID/NEU/04539/2013) and HEALTHYAGING 2020 (CENTRO-01-0145-FEDER-000012).

Supplemental Information Supplemental information associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. molmed.2017.06.006.

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