Senolytic-facilitated Reversal of End-Organ Dysfunction in a Murine Model of Obstructive Sleep Apnea.

Rationale: Obstructive sleep apnea (OSA) is a highly prevalent condition that is associated with accelerated biological aging and multiple end-organ morbidities. Current treatments, such as continuous positive airway pressure (CPAP), have shown limited cognitive, metabolic, and cardiovascular beneficial outcomes despite adherence. Thus, adjunct therapies aiming to reduce OSA burden, such as senolytics, could improve OSA outcomes.Objectives: To assess if targeting senescence in addition to partial normoxia mimicking "good" CPAP adherence can improve physiological outcomes in mice exposed to chronic intermittent hypoxia.Methods: We compared the effects of 6 weeks of therapy with either partial normoxic recovery alone or combined with the senolytic navitoclax after 16 weeks of intermittent hypoxia exposures, a hallmark of OSA, on multiphenotypic cardiometabolic and neurocognitive parameters.Measurements and Main Results: Our findings indicate that only when combined with navitoclax, partial normoxic recovery significantly improved sleepiness (sleep in the dark phase: 34% ± 4% vs. 26% ± 3%; P < 0.01), cognition (preference score: 51% ± 19% vs. 70% ± 11%; P = 0.048), coronary artery function (response to acetylcholine [vasodilation]: 56% ± 13% vs. 72% ± 10%; P < 0.001), glucose, and lipid metabolism and reduced intestinal permeability and senescence in multiple organs.Conclusions: These findings indicate that the reversibility of end-organ morbidities induced by OSA is not only contingent on restoration of normal oxygenation patterns but can be further enhanced by targeting other OSA-mediated detrimental cellular processes, such as accelerated senescence.

Gut microbiota mediate vascular dysfunction in a murine model of sleep apnoea: effect of probiotics.

BACKGROUND: Obstructive sleep apnoea (OSA) is a chronic prevalent condition characterised by intermittent hypoxia (IH), and is associated with endothelial dysfunction and coronary artery disease (CAD). OSA can induce major changes in gut microbiome diversity and composition, which in turn may induce the emergence of OSA-associated morbidities. However, the causal effects of IH-induced gut microbiome changes on the vasculature remain unexplored. Our objective was to assess if vascular dysfunction induced by IH is mediated through gut microbiome changes. METHODS: Faecal microbiota transplantation (FMT) was conducted on C57BL/6J naïve mice for 6 weeks to receive either IH or room air (RA) faecal slurry with or without probiotics (VSL#3). In addition to 16S rRNA amplicon sequencing of their gut microbiome, FMT recipients underwent arterial blood pressure and coronary artery and aorta function testing, and their trimethylamine N-oxide (TMAO) and plasma acetate levels were determined. Finally, C57BL/6J mice were exposed to IH, IH treated with VSL#3 or RA for 6 weeks, and arterial blood pressure and coronary artery function assessed. RESULTS: Gut microbiome taxonomic profiles correctly segregated IH from RA in FMT mice and the normalising effect of probiotics emerged. Furthermore, IH-FMT mice exhibited increased arterial blood pressure and TMAO levels, and impairments in aortic and coronary artery function (p<0.05) that were abrogated by probiotic administration. Lastly, treatment with VSL#3 under IH conditions did not attenuate elevations in arterial blood pressure or CAD. CONCLUSIONS: Gut microbiome alterations induced by chronic IH underlie, at least partially, the typical cardiovascular disturbances of sleep apnoea and can be mitigated by concurrent administration of probiotics.

Multi-Omics Analysis of Circulating Exosomes in Adherent Long-Term Treated OSA Patients.

Obstructive sleep apnea (OSA) is a highly prevalent chronic disease affecting nearly a billion people globally and increasing the risk of multi-organ morbidity and overall mortality. However, the mechanisms underlying such adverse outcomes remain incompletely delineated. Extracellular vesicles (exosomes) are secreted by most cells, are involved in both proximal and long-distance intercellular communication, and contribute toward homeostasis under physiological conditions. A multi-omics integrative assessment of plasma-derived exosomes from adult OSA patients prior to and after 1-year adherent CPAP treatment is lacking. We conducted multi-omic integrative assessments of plasma-derived exosomes from adult OSA patients prior to and following 1-year adherent CPAP treatment to identify potential specific disease candidates. Fasting morning plasma exosomes isolated from 12 adult patients with polysomnographically-diagnosed OSA were analyzed before and after 12 months of adherent CPAP therapy (mean ≥ 6 h/night) (OSAT). Exosomes were characterized by flow cytometry, transmission electron microscopy, and nanoparticle tracking analysis. Endothelial cell barrier integrity, wound healing, and tube formation were also performed. Multi-omics analysis for exosome cargos was integrated. Exosomes derived from OSAT improved endothelial permeability and dysfunction as well as significant improvement in tube formation compared with OSA. Multi-omic approaches for OSA circulating exosomes included lipidomic, proteomic, and small RNA (miRNAs) assessments. We found 30 differentially expressed proteins (DEPs), 72 lipids (DELs), and 13 miRNAs (DEMs). We found that the cholesterol metabolism (has04979) pathway is associated with lipid classes in OSA patients. Among the 12 subjects of OSA and OSAT, seven subjects had complete comprehensive exosome cargo information including lipids, proteins, and miRNAs. Multi-omic approaches identify potential signature biomarkers in plasma exosomes that are responsive to adherent OSA treatment. These differentially expressed molecules may also play a mechanistic role in OSA-induced morbidities and their reversibility. Our data suggest that a multi-omic integrative approach might be useful in understanding how exosomes function, their origin, and their potential clinical relevance, all of which merit future exploration in the context of relevant phenotypic variance. Developing an integrated molecular classification should lead to improved diagnostic classification, risk stratification, and patient management of OSA by assigning molecular disease-specific therapies.

The Mystery of Red Blood Cells Extracellular Vesicles in Sleep Apnea with Metabolic Dysfunction.

Sleep is very important for overall health and quality of life, while sleep disorder has been associated with several human diseases, namely cardiovascular, metabolic, cognitive, and cancer-related alterations. Obstructive sleep apnea (OSA) is the most common respiratory sleep-disordered breathing, which is caused by the recurrent collapse of the upper airway during sleep. OSA has emerged as a major public health problem and increasing evidence suggests that untreated OSA can lead to the development of various diseases including neurodegenerative diseases. In addition, OSA may lead to decreased blood oxygenation and fragmentation of the sleep cycle. The formation of free radicals or reactive oxygen species (ROS) can emerge and react with nitric oxide (NO) to produce peroxynitrite, thereby diminishing the bioavailability of NO. Hypoxia, the hallmark of OSA, refers to a decline of tissue oxygen saturation and affects several types of cells, playing cell-to-cell communication a vital role in the outcome of this interplay. Red blood cells (RBCs) are considered transporters of oxygen and nutrients to the tissues, and these RBCs are important interorgan communication systems with additional functions, including participation in the control of systemic NO metabolism, redox regulation, blood rheology, and viscosity. RBCs have been shown to induce endothelial dysfunction and increase cardiac injury. The mechanistic links between changes of RBC functional properties and cardiovascular are largely unknown. Extracellular vesicles (EVs) are secreted by most cell types and released in biological fluids both under physiological and pathological conditions. EVs are involved in intercellular communication by transferring complex cargoes including proteins, lipids, and nucleic acids from donor cells to recipient cells. Advancing our knowledge about mechanisms of RBC-EVs formation and their pathophysiological relevance may help to shed light on circulating EVs and to translate their application to clinical practice. We will focus on the potential use of RBC-EVs as valuable diagnostic and prognostic biomarkers and state-specific cargoes, and possibilities as therapeutic vehicles for drug and gene delivery. The use of RBC-EVs as a precision medicine for the diagnosis and treatment of the patient with sleep disorder will improve the prognosis and the quality of life in patients with cardiovascular disease (CVD).

Monocarboxylate Transporter-2 Expression Restricts Tumor Growth in a Murine Model of Lung Cancer: A Multi-Omic Analysis.

Monocarboxylate transporter 2 (MCT2) is a major high-affinity pyruvate transporter encoded by the SLC16A7 gene, and is associated with glucose metabolism and cancer. Changes in the gut microbiota and host immune system are associated with many diseases, including cancer. Using conditionally expressed MCT2 in mice and the TC1 lung carcinoma model, we examined the effects of MCT2 on lung cancer tumor growth and local invasion, while also evaluating potential effects on fecal microbiome, plasma metabolome, and bulk RNA-sequencing of tumor macrophages. Conditional MCT2 mice were generated in our laboratory using MCT2loxP mouse intercrossed with mCre-Tg mouse to generate MCT2loxP/loxP; Cre+ mouse (MCT2 KO). Male MCT2 KO mice (8 weeks old) were treated with tamoxifen (0.18 mg/g BW) KO or vehicle (CO), and then injected with mouse lung carcinoma TC1 cells (10 × 105/mouse) in the left flank. Body weight, tumor size and weight, and local tumor invasion were assessed. Fecal DNA samples were extracted using PowerFecal kits and bacterial 16S rRNA amplicons were also performed. Fecal and plasma samples were used for GC-MS Polar, as well as non-targeted UHPLC-MS/MS, and tumor-associated macrophages (TAMs) were subjected to bulk RNAseq. Tamoxifen-treated MCT2 KO mice showed significantly higher tumor weight and size, as well as evidence of local invasion beyond the capsule compared with the controls. PCoA and hierarchical clustering analyses of the fecal and plasma metabolomics, as well as microbiota, revealed a distinct separation between the two groups. KO TAMs showed distinct metabolic pathways including the Acetyl-coA metabolic process, activation of immune response, b-cell activation and differentiation, cAMP-mediated signaling, glucose and glutamate processes, and T-cell differentiation and response to oxidative stress. Multi-Omic approaches reveal a substantial role for MCT2 in the host response to TC1 lung carcinoma that may involve alterations in the gut and systemic metabolome, along with TAM-related metabolic pathway. These findings provide initial opportunities for potential delineation of oncometabolic immunomodulatory therapeutic approaches.

Cell-Selective Altered Cargo Properties of Extracellular Vesicles Following In Vitro Exposures to Intermittent Hypoxia.

Intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), is associated with cardiovascular and metabolic dysfunction. However, the mechanisms underlying these morbidities remain poorly delineated. Extracellular vesicles (EVs) mediate intercellular communications, play pivotal roles in a multitude of physiological and pathological processes, and could mediate IH-induced cellular effects. Here, the effects of IH on human primary cells and the release of EVs were examined. Microvascular endothelial cells (HMVEC-d), THP1 monocytes, THP1 macrophages M0, THP1 macrophages M1, THP1 macrophages M2, pre-adipocytes, and differentiated adipocytes (HAd) were exposed to either room air (RA) or IH for 24 h. Secreted EVs were isolated and characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. The effects of each of the cell-derived EVs on endothelial cell (EC) monolayer barrier integrity, on naïve THP1 macrophage polarity, and on adipocyte insulin sensitivity were also evaluated. IH did not alter EVs cell quantal release, but IH-EVs derived from HMVEC-d (p < 0.01), THP1 M0 (p < 0.01) and HAd (p < 0.05) significantly disrupted HMVEC-d monolayer integrity, particularly after H2O2 pre-conditioning. IH-EVs from HMVEC-d and THP1 M0 elicited M2-polarity changes did not alter insulin sensitivity responses. IH induces cell-selective changes in EVs cargo, which primarily seem to target the emergence of endothelial dysfunction. Thus, changes in EVs cargo from selected cell sources in vivo may play causal roles in some of the adverse outcomes associated with OSA.

Hypoxia differently modulates the release of mitochondrial and nuclear DNA.

BACKGROUND: We investigated the influence of hypoxia on the concentration of mitochondrial and nuclear cell-free DNA (McfDNA and NcfDNA, respectively). METHOD: By an ultra-sensitive quantitative PCR-based assay, McfDNA and NcfDNA were measured in the supernatants of different colorectal cell lines, and in the plasma of C57/Bl6 mice engrafted with TC1 tumour cells, in normoxic or hypoxic conditions. RESULTS: Our data when setting cell culture conditions highlighted the higher stability of McfDNA as compared to NcfDNA and revealed that cancer cells released amounts of nuclear DNA equivalent to the mass of a chromosome over a 6-h duration of incubation. In cell model, hypoxia induced a great increase in NcfDNA and McfDNA concentrations within the first 24 h. After this period, cfDNA total concentrations remained stable in hypoxia consecutive to a decrease of nuclear DNA release, and noteworthy, to a complete inhibition of daily mitochondrial DNA release. In TC1-engrafted mice submitted to intermittent hypoxia, plasma NcfDNA levels are much higher than in mice bred in normoxia, unlike plasma McfDNA concentration that is not impacted by hypoxia. CONCLUSION: This study suggests that hypoxia negatively modulates nuclear and, particularly, mitochondrial DNA releases in long-term hypoxia, and revealed that the underlying mechanisms are differently regulated.

Circulating plasma exosomes in obstructive sleep apnoea and reverse dipping blood pressure.

BACKGROUND: Obstructive sleep apnoea (OSA) increases the risk of an abnormal nondipping 24 h blood pressure profile, an independent risk factor for cardiovascular disease (CVD). We examined differential exosomal microRNA (miRNA) expression in untreated OSA patients with normal dipping blood pressure (NDBP) and reverse dipping blood pressure (RDBP), an extreme form of nondipping, to understand the mechanisms underlying nondipping blood pressure in OSA. METHODS: 46 patients (15 RDBP versus 31 NDBP) matched for OSA severity (respiratory event index 32.6±22.5 versus 32.2±18.1 events·h-1; p=0.9), age (54.8±12.9 versus 49±9.9 years; p=0.09) and body mass index (36.2±6.6 versus 34.4±6.8 kg·m-2; p=0.4) were included. Plasma exosomes were characterised by flow cytometry and functional in vitro reporter assays were conducted on cultured endothelial cells. Exosome miRNA cargo was profiled with microarrays followed by bioinformatics analyses. RESULTS: Exosomes from RDBP patients increased the permeability of endothelial cell tight junctions and adhesion molecule expression. Principal component analyses of miRNA array data showed strict separation and identification of the two groups. A restricted and validated signature of exosomal miRNAs was identified in the RDBP versus NDBP group. Their predicted target genes involved phosphatidylinositol 3-kinase-Akt (p=0.004), Ras (p=3.42E-05), Wnt (p=0.003) and hypoxia inducible factor-1 signalling (p=0.04), inflammatory mediator regulation of transient receptor potential channels (p=0.01), and several cancer-related pathways. CONCLUSIONS: Patients with RDBP have altered miRNA cargoes in circulating exosomes that invoke in vitro endothelial dysfunction. A selected number of circulating exosomal miRNAs play an important role in abnormal circadian regulation of blood pressure and may provide prognostic biomarkers of CVD risk in OSA.

Placental growth factor negatively regulates retinal endothelial cell barrier function through suppression of glucose-6-phosphate dehydrogenase and antioxidant defense systems.

We report that placental growth factor (PlGF) negatively affects the endothelial cell (EC) barrier function through a novel regulatory mechanism. The PlGF mAb promotes (but recombinant protein disrupts) EC barrier function, thus affecting the barrier-forming protein levels, membrane distribution, and EC monolayer impedance by the electrical cell-impedance sensing system, Western blot, and immunofluorescence staining. RNA sequencing-based transcriptome analysis identified the up-regulation of the pentose phosphate pathway (PPP) and the antioxidant defense protein by PlGF blockade. The PlGF and PlGF/VEGF dimers (but not VEGF-A) down-regulated the protein expression of glucose-6-phosphate dehydrogenase (G6PD) and peroxiredoxin (PRDX). G6PD inhibition and gene silencing (small interfering RNA) abolished the beneficial effects of PlGF inhibition on EC barrier function and PRDX3/6 protein expression. VEGF receptor (VEGFR)1 or VEGFR2 blockade prevented the inhibitory effect of PlGF on G6PD protein expression and EC barrier function. The PRDX6 played dual roles in EC barrier function through glutathione peroxidase and phospholipase A2 activity. In sum, PlGF negatively regulates EC barrier function through the activation of VEGFR1 and VEGFR2 and the suppression of the G6PD/PPP and the antioxidant pathways.-Huang, H., Lennikov, A., Saddala, M. S., Gozal, D., Grab, D. J., Khalyfa, A., Fan, L. Placental growth factor negatively regulates endothelial cell barrier function through suppression of glucose-6-phosphate dehydrogenase and antioxidant defense systems.

Transcriptomic Changes of Murine Visceral Fat Exposed to Intermittent Hypoxia at Single Cell Resolution.

Intermittent hypoxia (IH) is a hallmark of obstructive sleep apnea (OSA) and induces metabolic dysfunction manifesting as inflammation, increased lipolysis and insulin resistance in visceral white adipose tissues (vWAT). However, the cell types and their corresponding transcriptional pathways underlying these functional perturbations are unknown. Here, we applied single nucleus RNA sequencing (snRNA-seq) coupled with aggregate RNA-seq methods to evaluate the cellular heterogeneity in vWAT following IH exposures mimicking OSA. C57BL/6 male mice were exposed to IH and room air (RA) for 6 weeks, and nuclei from vWAT were isolated and processed for snRNA-seq followed by differential expressed gene (DEGs) analyses by cell type, along with gene ontology and canonical pathways enrichment tests of significance. IH induced significant transcriptional changes compared to RA across 14 different cell types identified in vWAT. We identified cell-specific signature markers, transcriptional networks, metabolic signaling pathways, and cellular subpopulation enrichment in vWAT. Globally, we also identify 298 common regulated genes across multiple cellular types that are associated with metabolic pathways. Deconvolution of cell types in vWAT using global RNA-seq revealed that distinct adipocytes appear to be differentially implicated in key aspects of metabolic dysfunction. Thus, the heterogeneity of vWAT and its response to IH at the cellular level provides important insights into the metabolic morbidity of OSA and may possibly translate into therapeutic targets.

Circulating Exosomal miRNAs Signal Circadian Misalignment to Peripheral Metabolic Tissues.

Night shift work increases risk of metabolic disorders, particularly obesity and insulin resistance. While the underlying mechanisms are unknown, evidence points to misalignment of peripheral oscillators causing metabolic disturbances. A pathway conveying such misalignment may involve exosome-based intercellular communication. Fourteen volunteers were assigned to a simulated day shift (DS) or night shift (NS) condition. After 3 days on the simulated shift schedule, blood samples were collected during a 24-h constant routine protocol. Exosomes were isolated from the plasma samples from each of the blood draws. Exosomes were added to naïve differentiated adipocytes, and insulin-induced pAkt/Akt expression changes were assessed. ChIP-Seq analyses for BMAL1 protein, mRNA microarrays and exosomal miRNA arrays combined with bioinformatics and functional effects of agomirs and antagomirs targeting miRNAs in NS and DS exosomal cargo were examined. Human adipocytes treated with exosomes from the NS condition showed altered Akt phosphorylation responses to insulin in comparison to those treated with exosomes from the DS condition. BMAL1 ChIP-Seq of exosome-treated adipocytes showed 42,037 binding sites in the DS condition and 5538 sites in the NS condition, with a large proportion of BMAL1 targets including genes encoding for metabolic regulators. A significant and restricted miRNA exosomal signature emerged after exposure to the NS condition. Among the exosomal miRNAs regulated differentially after 3 days of simulated NS versus DS, proof-of-concept validation of circadian misalignment signaling was demonstrated with hsa-mir-3614-5p. Exosomes from the NS condition markedly altered expression of key genes related to circadian rhythm in several cultured cell types, including adipocytes, myocytes, and hepatocytes, along with significant changes in 29 genes and downstream gene network interactions. Our results indicate that a simulated NS schedule leads to changes in exosomal cargo in the circulation. These changes promote reduction of insulin sensitivity of adipocytes in vitro and alter the expression of core clock genes in peripheral tissues. Circulating exosomal miRNAs may play an important role in metabolic dysfunction in NS workers by serving as messengers of circadian misalignment to peripheral tissues.

Genetics and Extracellular Vesicles of Pediatrics Sleep Disordered Breathing and Epilepsy.

Sleep remains one of the least understood phenomena in biology, and sleep disturbances are one of the most common behavioral problems in childhood. The etiology of sleep disorders is complex and involves both genetic and environmental factors. Epilepsy is the most popular childhood neurological condition and is characterized by an enduring predisposition to generate epileptic seizures, and the neurobiological, cognitive, psychological, and social consequences of this condition. Sleep and epilepsy are interrelated, and the importance of sleep in epilepsy is less known. The state of sleep also influences whether a seizure will occur at a given time, and this differs considerably for various epilepsy syndromes. The development of epilepsy has been associated with single or multiple gene variants. The genetics of epilepsy is complex and disorders exhibit significant genetic heterogeneity and variability in the expressivity of seizures. Phenobarbital (PhB) is the most widely used antiepileptic drug. With its principal mechanism of action to prolong the opening time of the γ-aminobutyric acid (GABA)-A receptor-associated chloride channel, it enhances chloride anion influx into neurons, with subsequent hyperpolarization, thereby reducing excitability. Enzymes that metabolize pharmaceuticals including PhB are well known for having genetic polymorphisms that contribute to adverse drug-drug interactions. PhB metabolism is highly dependent upon the cytochrome P450 (CYP450) and genetic polymorphisms can lead to variability in active drug levels. The highly polymorphic CYP2C19 isozymes are responsible for metabolizing a large portion of routinely prescribed drugs and variants contribute significantly to adverse drug reactions and therapeutic failures. A limited number of CYP2C19 single nucleotide polymorphisms (SNPs) are involved in drug metabolism. Extracellular vesicles (EVs) are circular membrane fragments released from the endosomal compartment as exosomes are shed from the surfaces of the membranes of most cell types. Increasing evidence indicated that EVs play a pivotal role in cell-to-cell communication. Theses EVs may play an important role between sleep, epilepsy, and treatments. The discovery of exosomes provides potential strategies for the diagnosis and treatment of many diseases including neurocognitive deficit. The aim of this study is to better understand and provide further knowledge about the metabolism and interactions between phenobarbital and CYP2C19 polymorphisms in children with epilepsy, interplay between sleep, and EVs. Understanding this interplay between epilepsy and sleep is helpful in the optimal treatment of all patients with epileptic seizures. The use of genetics and extracellular vesicles as precision medicine for the diagnosis and treatment of children with sleep disorder will improve the prognosis and the quality of life in patients with epilepsy.

Plasma Extracellular Vesicles in Children with OSA Disrupt Blood-Brain Barrier Integrity and Endothelial Cell Wound Healing in Vitro.

Pediatric obstructive sleep apnea (P-OSA) is associated with neurocognitive deficits and endothelial dysfunction, suggesting the possibility that disruption of the blood-brain barrier (BBB) may underlie these morbidities. Extracellular vesicles (EVs), which include exosomes, are small particles involved in cell-cell communications via different mechanisms and could play a role in OSA-associated end-organ injury. To examine the roles of EVs in BBB dysfunction, we recruited three groups of children: (a) absence of OSA or cognitive deficits (CL, n = 6), (b) OSA but no evidence of cognitive deficits (OSA-NC(-), n = 12), and (c) OSA with evidence of neurocognitive deficits (OSA-NC(+), n = 12). All children were age-, gender-, ethnicity-, and BMI-z-score-matched, and those with OSA were also apnea-hypopnea index (AHI)-matched. Plasma EVs were characterized, quantified, and applied on multiple endothelial cell types (HCAEC, HIAEC, human HMVEC-D, HMVEC-C, HMVEC-L, and hCMEC/D3) while measuring monolayer barrier integrity and wound-healing responses. EVs from OSA children induced significant declines in hCMEC/D3 transendothelial impedance compared to CL (p < 0.001), and such changes were greater in NC(+) compared to NC(-) (p < 0.01). The effects of EVs from each group on wound healing for HCAEC, HIAEC, HMVED-d, and hCMEC/D3 cells were similar, but exhibited significant differences across the three groups, with evidence of disrupted wound healing in P-OSA. However, wound healing in HMVEC-C was only affected by NC(+) (p < 0.01 vs. NC(-) or controls (CO). Furthermore, no significant differences emerged in HMVEC-L cell wound healing across all three groups. We conclude that circulating plasma EVs in P-OSA disrupt the integrity of the BBB and exert adverse effects on endothelial wound healing, particularly among OSA-NC(+) children, while also exhibiting endothelial cell type selectivity. Thus, circulating EVs cargo may play important roles in the emergence of end-organ morbidity in pediatric OSA.

Sleep-disordered breathing, circulating exosomes, and insulin sensitivity in adipocytes.

BACKGROUND: Sleep-disordered-breathing (SDB), which is characterized by chronic intermittent hypoxia (IH) and sleep fragmentation (SF), is a prevalent condition that promotes metabolic dysfunction, particularly among patients suffering from obstructive hypoventilation syndrome (OHS). Exosomes are generated ubiquitously, are readily present in the circulation, and their cargo may exert substantial functional cellular alterations in both physiological and pathological conditions. However, the effects of plasma exosomes on adipocyte metabolism in patients with OHS or in mice subjected to IH or SF mimicking SDB are unclear. METHODS: Exosomes from fasting morning plasma samples from obese adults with polysomnographically-confirmed OSA before and after 3 months of adherent CPAP therapy were assayed. In addition, C57BL/6 mice were randomly assigned to (1) sleep control (SC), (2) sleep fragmentation (SF), and (3) intermittent hypoxia (HI) for 6 weeks, and plasma exosomes were isolated. Equivalent exosome amounts were added to differentiated adipocytes in culture, after which insulin sensitivity was assessed using 0 nM and 5 nM insulin-induced pAKT/AKT expression changes by western blotting. RESULTS: When plasma exosomes were co-cultured and internalized by human naive adipocytes, significant reductions emerged in Akt phosphorylation responses to insulin when compared to exosomes obtained after 24 months of adherent CPAP treatment (n = 24; p < 0.001), while no such changes occur in untreated patients (n = 8). In addition, OHS exosomes induced significant increases in adipocyte lipolysis that were attenuated after CPAP, but did not alter pre-adipocyte differentiation. Similarly, exosomes from SF- and IH-exposed mice induced attenuated p-AKT/total AKT responses to exogenous insulin and increased glycerol content in naive murine adipocytes, without altering pre-adipocyte differentiation. CONCLUSIONS: Using in vitro adipocyte-based functional reporter assays, alterations in plasma exosomal cargo occur in SDB, and appear to contribute to adipocyte metabolic dysfunction. Further exploration of exosomal miRNA signatures in either human subjects or animal models and their putative organ and cell targets appears warranted.

Exosome and Macrophage Crosstalk in Sleep-Disordered Breathing-Induced Metabolic Dysfunction.

Obstructive sleep apnea (OSA) is a highly prevalent worldwide public health problem that is characterized by repetitive upper airway collapse leading to intermittent hypoxia, pronounced negative intrathoracic pressures, and recurrent arousals resulting in sleep fragmentation. Obesity is a major risk factor of OSA and both of these two closely intertwined conditions result in increased sympathetic activity, oxidative stress, and chronic low-grade inflammation, which ultimately contribute, among other morbidities, to metabolic dysfunction, as reflected by visceral white adipose tissue (VWAT) insulin resistance (IR). Circulating extracellular vesicles (EVs), including exosomes, are released by most cell types and their cargos vary greatly and reflect underlying changes in cellular homeostasis. Thus, exosomes can provide insights into how cells and systems cope with physiological perturbations by virtue of the identity and abundance of miRNAs, mRNAs, proteins, and lipids that are packaged in the EVs cargo, and are secreted from the cells into bodily fluids under normal as well as diseased states. Accordingly, exosomes represent a novel pathway via which a cohort of biomolecules can travel long distances and result in the modulation of gene expression in selected and targeted recipient cells. For example, exosomes secreted from macrophages play a critical role in innate immunity and also initiate the adaptive immune response within specific metabolic tissues such as VWAT. Under normal conditions, phagocyte-derived exosomes represent a large portion of circulating EVs in blood, and carry a protective signature against IR that is altered when secreting cells are exposed to altered physiological conditions such as those elicited by OSA, leading to emergence of IR within VWAT compartment. Consequently, increased understanding of exosome biogenesis and biology should lead to development of new diagnostic biomarker assays and personalized therapeutic approaches. Here, the evidence on the major biological functions of macrophages and exosomes as pathophysiological effectors of OSA-induced metabolic dysfunction is discussed.

Visceral White Adipose Tissue after Chronic Intermittent and Sustained Hypoxia in Mice.

Angiogenesis, a process induced by hypoxia in visceral white adipose tissues (vWAT) in the context of obesity, mediates obesity-induced metabolic dysfunction and insulin resistance. Chronic intermittent hypoxia (IH) and sustained hypoxia (SH) induce body weight reductions and insulin resistance of different magnitudes, suggesting different hypoxia inducible factor (HIF)-1α-related activity. Eight-week-old male C57BL/6J mice (n = 10-12/group) were exposed to either IH, SH, or room air (RA). vWAT were analyzed for insulin sensitivity (phosphorylated (pAKT)/AKT), HIF-1α transcription using chromatin immunoprecipitation (ChIP)-sequencing, angiogenesis using immunohistochemistry, and gene expression of different fat cell markers and HIF-1α gene targets using quantitative polymerase chain reaction or microarrays. Body and vWAT weights were reduced in hypoxia (SH > IH > RA; P < 0.001), with vWAT in IH manifesting vascular rarefaction and increased proinflammatory macrophages. HIF-1α ChIP-sequencing showed markedly increased binding sites in SH-exposed vWAT both at 6 hours and at 6 weeks compared with IH, the latter also showing decreased vascular endothelial growth factor, endothelial nitric oxide synthase, P2RX5, and PAT2 expression, and insulin resistance (IH > > > SH = RA; P < 0.001). IH induces preferential whitening of vWAT, as opposed to prominent browning in SH. Unlike SH, IH elicits early HIF-1α activity that is unsustained over time and is accompanied by concurrent vascular rarefaction, inflammation, and insulin resistance. Thus, the dichotomous changes in HIF-1α transcriptional activity and brown/beige/white fat balance in IH and SH should enable exploration of mechanisms by which altered sympathetic outflow, such as that which occurs in apneic patients, results in whitening, rather than the anticipated browning of adipose tissues that occurs in SH.

Activation of the Integrated Stress Response and Metabolic Dysfunction in a Murine Model of Sleep Apnea.

Intermittent hypoxia (IH) induces activation of the integrated stress response (ISR), but its role in IH-induced visceral white adipose tissue (vWAT) insulin resistance is unknown. CHOP is activated by chronic ISR, whereas GADD34 dephosphorylates the subunit of translation initiation factor 2 (eIF2α), leading to termination of the ISR. We hypothesized that CHOP/Gadd34 null mice would not manifest evidence of insulin resistance after IH exposures. Eight-week-old CHOP/GADD34-/- (double mutant [DM]) and wild-type (WT) littermates were randomly assigned to IH or room air (RA) exposures for 6 weeks. Glucose and insulin tolerance tests were performed, and regulatory T cells (Tregs) and macrophages in vWAT were assessed. Phosphorylated eIF2α:total eIF2α, ATF4, XBP1 expression, and insulin-induced pAKT/AKT expression changes were examined in vWATs. Single GADD34-/- and PERK+/- mice were also evaluated. Body weight and vWAT mass were reduced in DM and WT mice after IH. M1/M2 macrophages and inflammatory macrophages (Ly-6chigh) were significantly increased in WT vWAT but remained unchanged in DM mice. Tregs were significantly decreased in WT vWAT but not in DM mice. Systemic insulin and glucose tolerance tests revealed insulin resistance in IH-WT but not in IH-DM mice. Similarly, decreased pAKT/AKT responses to exogenous insulin emerged in IH-WT compared with RA-WT mice, whereas no significant differences emerged in IH-DM compared with DM-RA. Chronic ISR activation appears to contribute to the insulin resistance and vWAT inflammation that characteristically emerge after long-term IH exposures in a murine model of obstructive sleep apnea.

Late gestational intermittent hypoxia induces metabolic and epigenetic changes in male adult offspring mice.

KEY POINTS: Late gestation during pregnancy has been associated with a relatively high prevalence of obstructive sleep apnoea (OSA). Intermittent hypoxia, a hallmark of OSA, could impose significant long-term effects on somatic growth, energy homeostasis and metabolic function in offspring. Here we show that late gestation intermittent hypoxia induces metabolic dysfunction as reflected by increased body weight and adiposity index in adult male offspring that is paralleled by epigenomic alterations and inflammation in visceral white adipose tissue. Fetal perturbations by OSA during pregnancy impose long-term detrimental effects manifesting as metabolic dysfunction in adult male offspring. ABSTRACT: Pregnancy, particularly late gestation (LG), has been associated with a relatively high prevalence of obstructive sleep apnoea (OSA). Intermittent hypoxia (IH), a hallmark of OSA, could impose significant long-term effects on somatic growth, energy homeostasis, and metabolic function in offspring. We hypothesized that IH during late pregnancy (LG-IH) may increase the propensity for metabolic dysregulation and obesity in adult offspring via epigenetic modifications. Time-pregnant female C57BL/6 mice were exposed to LG-IH or room air (LG-RA) during days 13-18 of gestation. At 24 weeks, blood samples were collected from offspring mice for lipid profiles and insulin resistance, indirect calorimetry was performed and visceral white adipose tissues (VWAT) were assessed for inflammatory cells as well as for differentially methylated gene regions (DMRs) using a methylated DNA immunoprecipitation on chip (MeDIP-chip). Body weight, food intake, adiposity index, fasting insulin, triglycerides and cholesterol levels were all significantly higher in LG-IH male but not female offspring. LG-IH also altered metabolic expenditure and locomotor activities in male offspring, and increased number of pro-inflammatory macrophages emerged in VWAT along with 1520 DMRs (P < 0.0001), associated with 693 genes. Pathway analyses showed that genes affected by LG-IH were mainly associated with molecular processes related to metabolic regulation and inflammation. LG-IH induces metabolic dysfunction as reflected by increased body weight and adiposity index in adult male offspring that is paralleled by epigenomic alterations and inflammation in VWAT. Thus, perturbations to fetal environment by OSA during pregnancy can have long-term detrimental effects on the fetus, and lead to persistent metabolic dysfunction in adulthood.

Intermittent hypoxia causes NOX2-dependent remodeling of atrial connexins.

BACKGROUND: Obstructive sleep apnea has been linked to the development of heart disease and arrhythmias, including atrial fibrillation. Since altered conduction through gap junction channels can contribute to the pathogenesis of such arrhythmias, we examined the abundance and distributions of the major cardiac gap junction proteins, connexin40 (Cx40) and connexin43 (Cx43) in mice treated with sleep fragmentation or intermittent hypoxia (IH) as animal models of the components of obstructive sleep apnea. RESULTS: Wild type C57BL/6 mice or mice lacking NADPH 2 (NOX2) oxidase activity (gp91phox(-/Y)) were exposed to room air or to SF or IH for 6 weeks. Then, the mice were sacrificed, and atria and ventricles were immediately dissected. The abundances of Cx40 or Cx43 in atria and ventricles were unaffected by SF. In contrast, immunoblots showed that the abundance of atrial Cx40 and Cx43 and ventricular Cx43 were reduced in mice exposed to IH. qRT-PCR demonstrated significant reductions of atrial Cx40 and Cx43 mRNAs. Immunofluorescence microscopy revealed that the abundance and size of gap junctions containing Cx40 or Cx43 were reduced in atria by IH treatment of mice. However, no changes of connexin abundance or gap junction size/abundance were observed in IH-treated NOX2-null mice. CONCLUSIONS: These results demonstrate that intermittent hypoxia (but not sleep fragmentation) causes reductions and remodeling of atrial Cx40 and Cx43. These alterations may contribute to the substrate for atrial fibrillation that develops in response to obstructive sleep apnea. Moreover, these connexin changes are likely generated in response to reactive oxygen species generated by NOX2.

Temporal trajectories of novel object recognition performance in mice exposed to intermittent hypoxia.

Intermittent hypoxia is one of the major perturbations of sleep-disordered breathing and has been causally implicated in neurocognitive deficits. However, the reversibility of such deficits is unclear.Male C57BL/6J mice were exposed to either intermittent hypoxia or room air for 3-240 days, and then half were randomly selected and allowed to recover in normoxic conditions for the same duration of the previous exposure. A novel object recognition (NOR) test was performed.NOR performance was stable over time in room air. Intermittent hypoxia induced significant reductions in recognition index that progressed over the first 45 days and stabilised thereafter. Normoxic recovery of recognition index was essentially complete and indistinguishable from room air in mice exposed to shorter intermittent hypoxia times (<90 days). However, significant residual deficits emerged after normoxic recovery following prolonged intermittent hypoxia exposures (p<0.01). In addition, gradual attenuation of the magnitude of recovery in recognition index occurred with increasingly longer intermittent hypoxia exposures (MANOVA p<0.0001).Intermittent hypoxia during the resting period reduces NOR performance in a time-dependent fashion. Reversal of NOR performance deficits is unlikely after prolonged intermittent hypoxia duration. These findings suggest that early recognition of sleep apnoea and effective treatment are critical for restoration of the adverse cognitive effects of the disease.