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Nocturnal hypoxia in obese-related obstructive sleep apnea as a putative trigger of oxidative stress in pediatric NAFLD progression

  • Author Footnotes
    † These authors contributed equally to the manuscript preparation.
    Maurizio Parola
    Correspondence
    Corresponding author. Address: Dept. Clinical and Biological Sciences, Unit of Experimental Medicine and Clinical Pathology, University of Torino – School of Medicine Corso Raffaello 30, 10125 Torino, Italy. Tel.: +39 011 6707772; fax: +39 011 6707753.
    Footnotes
    † These authors contributed equally to the manuscript preparation.
    Affiliations
    Dept. Clinical and Biological Sciences, Unit of Experimental Medicine and Clinical Pathology, University of Torino, Italy
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  • Author Footnotes
    † These authors contributed equally to the manuscript preparation.
    Pietro Vajro
    Footnotes
    † These authors contributed equally to the manuscript preparation.
    Affiliations
    Dept. Medicine and Surgery, Unit of Pediatrics, University of Salerno, Italy
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  • Author Footnotes
    † These authors contributed equally to the manuscript preparation.
Published:August 05, 2016DOI:https://doi.org/10.1016/j.jhep.2016.05.042

      Abbreviations:

      ALT (alanine aminotransferase), BS (bariatric surgery), BMI (body mass index), CPAP (continuous positive airway pressure), FFA (free fatty acids), GGT (γ-glutamyl transferase), HCC (hepatocellular carcinoma), Hct (blood hematocrit), HSCs (hepatic stellate cells), HIFs (hypoxia-inducible factors), NAFLD (non-alcoholic fatty liver disease), NASH (non-alcoholic steato-hepatitis), NH (nocturnal hypoxia), OSA (obstructive sleep apnea), OSAS (obstructive sleep apnea syndrome), ROS (reactive oxygen species), WC (waist circumference)
      Non-Alcoholic Fatty Liver Disease (NAFLD) is now considered as the hepatic component of metabolic syndrome. Its prevalence, paralleling the growing diffusion of obesity and type II diabetes, is increasing worldwide both in adults and children. NAFLD is estimated to affect up to 30% of the general population in Western countries, reaching an even higher prevalence among diabetic and obese individuals [
      • Chalasani N.
      • Younossi Z.
      • Lavine J.E.
      • et al.
      The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology.
      ,
      EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease.
      ]. From 20 to 30% of NAFLD patients are expected to develop non-alcoholic steatohepatitis (NASH), characterized by steatosis, parenchymal damage, lobular/portal inflammation with or without perisinusoidal and/or portal fibrosis. NASH can eventually progress to more severe fibrosis and cirrhosis [
      • Yeh M.M.
      • Brunt E.M.
      Pathological features of fatty liver disease.
      ], with development of hepatocellular carcinoma (HCC) detected in both cirrhotic and non-cirrhotic adults [
      • Torres D.M.
      • Harrison S.A.
      Nonalcoholic steatohepatitis and noncirrhotic hepatocellular carcinoma: fertile soil.
      ] and sometimes also in children/young adults [
      • Goyal N.P.
      • Schwimmer J.B.
      The progression and natural history of pediatric nonalcoholic fatty liver disease.
      ].
      Notwithstanding the relevant clinical and social impact of NAFLD/NASH, the exact mechanisms responsible for NAFLD progression to more severe liver injury in pediatric and adult individuals, remain elusive and we lack effective therapies and diagnostic tools to identify NAFLD patients at risk of progression. According to the hypothesis of multiple parallel hits, current literature indicates oxidative stress, inflammasome activation, lipotoxicity and changes in gut microbiota as most relevant mechanisms [
      • Tilg H.
      • Moschen A.R.
      Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis.
      ].
      The involvement of oxidative stress, in particular, has been unequivocally documented in both pediatric and adult conditions of NAFLD, reproduced by experimental studies in rodents and suggested to favor NAFLD progression [
      • Koek G.H.
      • Liedorp P.R.
      • Bast A.
      The role of oxidative stress in non-alcoholic steatohepatitis.
      ,
      • Novo E.
      • Cannito S.
      • Paternostro C.
      • et al.
      Cellular and molecular mechanisms in liver fibrogenesis.
      ]. In experimental NAFLD oxidative stress can result from increased generation of reactive oxygen species (ROS) depending on several critical events, including impairment of mitochondria due to overload of free fatty acids (FFA), increased FFA metabolism in endoplasmic reticulum by cytochrome P450 isoforms 2E1 and 4A or in peroxisomes, lipotoxicity and endoplasmic reticulum stress, the presence of hypoxic conditions, as well as ROS generation through NADPH-oxidase isoforms associated to ligand-receptor interactions (growth factors, cytokines, chemokines, adipokines) or by activated inflammatory cells. Whatever the source, ROS and oxidative stress are widely accepted to contribute to NAFLD progression by eliciting parenchymal death and by sustaining both inflammatory and fibrogenic responses [
      • Koek G.H.
      • Liedorp P.R.
      • Bast A.
      The role of oxidative stress in non-alcoholic steatohepatitis.
      ,
      • Novo E.
      • Cannito S.
      • Paternostro C.
      • et al.
      Cellular and molecular mechanisms in liver fibrogenesis.
      ].
      On this background, we find the results reported by Sundaram et al. in this issue of Journal of Hepatology [
      • Sundaram S.
      • Halbower A.
      • Pan Z.
      • et al.
      Nocturnal hypoxia induced oxidative stress promotes progression of pediatric non-alcoholic fatty liver disease.
      ] intriguing. They provide clinically sound data suggesting that nocturnal hypoxia (NH), associated with obesity-related obstructive sleep apnea (OSA), may promote the progression of pediatric NAFLD by significantly contributing to hepatic oxidative stress. According to current guidelines OSA is defined as a “disorder of breathing during sleep characterized by prolonged partial upper airway obstruction and/or intermittent complete obstruction (obstructive apnea) that disrupts normal ventilation during sleep and normal sleep patterns” [
      • Marcus C.L.
      • Brooks L.J.
      • Draper K.A.
      • et al.
      Diagnosis and management of childhood obstructive sleep apnea syndrome.
      ]. Patients with OSA syndrome (OSAS), in particular, can experience repeated episodes of NH alternating with normoxia (i.e., chronic intermittent hypoxia). This is accompanied by symptoms or signs that include habitual snoring (often with intermittent pauses, snorts, or gasps), disturbed sleep, and daytime neurobehavioral problems. Daytime sleepiness may occur, but is uncommon in young children. Pediatric OSA instead is also associated with neurocognitive impairment, behavioral problems, hypertension, cardiac dysfunction, and systemic inflammation [
      • Marcus C.L.
      • Brooks L.J.
      • Draper K.A.
      • et al.
      Diagnosis and management of childhood obstructive sleep apnea syndrome.
      ]. Interestingly, in the present obesity epidemics, obese children represent a category of patients where those with OSA may reach a proportion of up to 36% [
      • Kassim R.
      • Harris M.A.
      • Leong G.M.
      • et al.
      Obstructive sleep apnea in children with obesity.
      ].
      Recent studies have reported a link not only between OSAS and cardiometabolic alterations, but also between OSAS and either direct (hepatic histology) or indirect (ALT or GGT levels) markers of liver damage in morbidly obese NAFLD adult and pediatric patients [reviewed in [
      • Paschetta E.
      • Belci P.
      • Alisi A.
      • et al.
      OSAS-related inflammatory mechanisms of liver injury in nonalcoholic fatty liver disease.
      ,
      • Petta S.
      • Marrone O.
      • Torres D.
      • et al.
      Obstructive sleep apnea is associated with liver damage and atherosclerosis in patients with non-alcoholic fatty liver disease.
      ]]. Two years ago the same group of Sundaram reported an alarming 60% OSA incidence in pediatric NAFLD. The severity/duration of hypoxiemia was associated with biochemical and histologic measures of NAFLD severity [
      • Sundaram S.S.
      • Sokol R.J.
      • Capocelli K.E.
      • et al.
      Obstructive sleep apnea and hypoxemia are associated with advanced liver histology in pediatric nonalcoholic fatty liver disease.
      ]. The same year Nobili et al. confirmed similar incidence and the presence and severity of OSAS associated with the presence of NASH significant fibrosis, and NAFLD activity score independently of body mass index, abdominal adiposity, metabolic syndrome, and insulin resistance. This relationship held also in non-obese children with NAFLD and the duration of hemoglobin desaturation correlated with increased intrahepatic leukocytes and activated macrophages/Kupffer cells and with circulating markers of hepatocyte apoptosis and fibrogenesis [
      • Nobili V.
      • Cutrera R.
      • Liccardo D.
      • et al.
      Obstructive sleep apnea syndrome affects liver histology and inflammatory cell activation in pediatric nonalcoholic fatty liver disease, regardless of obesity/insulin resistance.
      ]. The association OSA-NASH severity finally was found to correlate with increased endotoxemia coupled with impaired gut barrier function, leading to increased TLR-4-mediated hepatic susceptibility to endotoxemia [
      • Nobili V.
      • Alisi A.
      • Cutrera R.
      • et al.
      Altered gut-liver axis and hepatic adiponectin expression in OSAS: novel mediators of liver injury in paediatric non-alcoholic fatty liver.
      ], in keeping with the concept that gut microbiota plays a role in the development of hepatic steatosis and its progression to NASH [
      • Vajro P.
      • Paolella G.
      • Fasano A.
      Microbiota and gut-liver axis: their influences on obesity and obesity-related liver disease.
      ].
      The study by Sundaram and colleagues has addressed now NH as a potential source of oxidative stress in NAFLD [
      • Sundaram S.
      • Halbower A.
      • Pan Z.
      • et al.
      Nocturnal hypoxia induced oxidative stress promotes progression of pediatric non-alcoholic fatty liver disease.
      ]. Authors have studied 14 lean control adolescents and 36 adolescents (25 with OSA/NH) with biopsy proven NAFLD, polysomnographic characterization, liver histology scoring, and laboratory testing. Oxidative stress has been convincingly documented in this study by evaluating systemic (urine F2-isoprostanes) and hepatic (immunohistochemistry for 4-hydroxynonenal) reliable markers of in vivo lipid peroxidation [
      • Koek G.H.
      • Liedorp P.R.
      • Bast A.
      The role of oxidative stress in non-alcoholic steatohepatitis.
      ,
      • Novo E.
      • Cannito S.
      • Paternostro C.
      • et al.
      Cellular and molecular mechanisms in liver fibrogenesis.
      ].
      This study is particularly welcome because of conflicting results reported in literature, correctly quoted in the Sundaram et al. paper [
      • Sundaram S.
      • Halbower A.
      • Pan Z.
      • et al.
      Nocturnal hypoxia induced oxidative stress promotes progression of pediatric non-alcoholic fatty liver disease.
      ], some reporting increased oxidative stress in adults with OSA as compared to healthy controls but others suggesting that oxidative stress in OSA patients was related to obesity rather than intermittent hypoxia. In their study, in addition to confirm the frequent association of OSA in pediatric NAFLD with deficiency of serum anti-oxidants, authors call attention to a number of relevant points: i) NAFLD subjects with definite NASH (NAS histologic score >5) had more severe sleep disordered breathing and significantly higher apnea/hypopnea index scores compared to those without definite NASH; ii) a correlation between the more severe fibrosis detected in patients with OSA/hypoxia vs. those without OSA/hypoxia; iii) a clear correlation between severity of the indexes of oxidative stress and the severity of the indexes used to evaluate OSA/NH; iv) the two populations of obese NAFLD adolescents did not differ for other relevant serum liver indexes and liver histology score (steatosis, ballooning degeneration, inflammation, NAS score). The suggestion is that obese NAFLD adolescents experiencing OSA/NH will then experience a surplus of hepatic oxidative stress that correlates with a more severe fibrosis than in non-OSA/NH NAFLD patients. This is reasonable since the repeated episodes of NH and normoxia (chronic intermittent hypoxia) in OSA/NH can easily lead to enhanced mitochondrial generation of ROS. Moreover, increased ROS generation in a hypoxic environment can sustain liver fibrogenesis through either ROS themselves or through responses operating through hypoxia-inducible factors (HIFs; i.e., known to be stabilized and then amplified by intracellular ROS) [
      • Koek G.H.
      • Liedorp P.R.
      • Bast A.
      The role of oxidative stress in non-alcoholic steatohepatitis.
      ,
      • Novo E.
      • Cannito S.
      • Paternostro C.
      • et al.
      Cellular and molecular mechanisms in liver fibrogenesis.
      ]. Although further studies designed to investigate specifically the role of HIFs are needed, both events have been suggested to modulate either the release of pro-inflammatory and pro-fibrogenic mediators by macrophages or activated hepatic stellate cells (HSCs) or by directly targeting HSCs. It should be noted, in particular, that the most relevant pro-fibrogenic phenotypical responses by activated HSCs are sustained by increased intracellular levels of ROS not able to induce cytotoxicity [reviewed in ref. [
      • Novo E.
      • Cannito S.
      • Paternostro C.
      • et al.
      Cellular and molecular mechanisms in liver fibrogenesis.
      ]]. Accordingly, this scenario closely resembles the described increase in liver injury and fibrosis reported in a rodent dietary model (high fat diet) of NAFLD/NASH when associated with chronic intermittent hypoxia [
      • Feng S.Z.
      • Tian J.L.
      • Zhang Q.
      • et al.
      An experimental research on chronic intermittent hypoxia leading to liver injury.
      ].
      However, like most investigations on this topic and as the authors themselves acknowledge, this well-designed study is not exempt from some inevitable and evitable limitations. The major inevitable limitations are the poor generalizability (89% Hispanic subjects), the small sample size, the cross-sectional nature of the study not proving a causal link between OSA and liver damage, the possible bias towards a focus on more severe liver disease due to enrollment of subjects who underwent liver biopsy for chronic elevation of aminotransferases, and the intrinsic ethics issues for the invasiveness of liver biopsy itself. Evitable limitations are the absence of a “healthy” obese control group and the lack of information about patients’ adiposity criteria other than BMI (e.g., the waist-to-height ratio, or WC cm >95 percentile for age and gender [
      • Motamed N.
      • Sohrabi M.
      • Ajdarkosh H.
      • et al.
      Fatty liver index vs. waist circumference for predicting non-alcoholic fatty liver disease.
      ]. For lean controls, a lesser disproportion of Hispanic subjects, polysomnography and liver ultrasounds data, and/or a specific validated OSA clinical score might have added weight to the authors’ conclusions and possible insights into the bi-directionality of the events studied.
      In any case, the study by Sundaram et al. [
      • Sundaram S.
      • Halbower A.
      • Pan Z.
      • et al.
      Nocturnal hypoxia induced oxidative stress promotes progression of pediatric non-alcoholic fatty liver disease.
      ] has merit and outlines a number of further relevant issues and perspectives. The reported significant relationships between i) blood hematocrit (Hct) and NAFLD fibrosis stage and ii) anti-oxidant blood values, and NAFLD and lipid peroxidation parameters, may suggest that their combined evaluation should help in deciding whether histological ad polysomnographic evaluation are needed in order to early recognize subjects with more severe NAFLD and/or more severe OSA and hypoxia. Finally, since nocturnal continuous positive airway pressure (CPAP) is a treatment modality also for pediatric NAFLD patients with OSA/hypoxia, we definitively need trials designed to investigate whether CPAP treatment may significantly affect NAFLD progression in this age range. In fact, at present the only randomized controlled trial on the possible efficacy of CPAP treatment was of relatively short duration, performed on adult patients with mild OSA/hypoxia and normal baseline transaminases, and apparently did not demonstrate any impact on steatosis, NASH or liver fibrosis, as evaluated by FibroMax score [
      • Jullian-Desayes I.
      • Tamisier R.
      • Zarski J.P.
      • et al.
      Impact of effective versus sham continuous positive airway pressure on liver injury in obstructive sleep apnoea: Data from randomized trials.
      ].

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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      Linked Article

      • Nocturnal hypoxia-induced oxidative stress promotes progression of pediatric non-alcoholic fatty liver disease
        Journal of HepatologyVol. 65Issue 3
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          Non-alcoholic fatty liver disease (NAFLD), characterized by abnormal lipid deposition in hepatocytes in the absence of excess alcohol intake, is a disease of epidemic proportions in both children and adults, paralleling the obesity epidemic [1]. NAFLD affects up to 9.6% of all children and 38% of obese children across a spectrum of disease, including isolated hepatic steatosis, non-alcoholic steatohepatitis (NASH, defined as steatosis, hepatocyte ballooning and inflammation), and cirrhosis [1,2].
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