Alex Gileles-Hillel

Chronic sleep fragmentation induces endothelial dysfunction and structural vascular changes in mice.

STUDY OBJECTIVES Sleep fragmentation (SF) is a common occurrence and constitutes a major characteristic of obstructive sleep apnea (OSA). SF has been implicated in multiple OSA-related morbidities, but it is unclear whether SF underlies any of the cardiovascular morbidities of OSA. We hypothesized that long-term SF exposures may lead to endothelial dysfunction and altered vessel wall structure. METHODS AND RESULTS Adult male C57BL/6J mice were fed normal chow and exposed to daylight SF or control sleep (CTL) for 20 weeks. Telemetric blood pressure and endothelial function were assessed weekly using a modified laser-Doppler hyperemic test. Atherosclerotic plaques, elastic fiber disruption, lumen area, wall thickness, foam cells, and macrophage recruitment, as well as expression of senescence-associated markers were examined in excised aortas. Increased latencies to reach baseline perfusion levels during the post-occlusive period emerged in SF mice with increased systemic BP values starting at 8 weeks of SF and persisting thereafter. No obvious atherosclerotic plaques emerged, but marked elastic fiber disruption and fiber disorganization were apparent in SF-exposed mice, along with increases in the number of foam cells and macrophages in the aorta wall. Senescence markers showed reduced TERT and cyclin A and increased p16INK4a expression, with higher IL-6 plasma levels in SF-exposed mice. CONCLUSIONS Long-term sleep fragmentation induces vascular endothelial dysfunction and mild blood pressure increases. Sleep fragmentation also leads to morphologic vessel changes characterized by elastic fiber disruption and disorganization, increased recruitment of inflammatory cells, and altered expression of senescence markers, thereby supporting a role for sleep fragmentation in the cardiovascular morbidity of OSA.

Chronic sleep disruption alters gut microbiota, induces systemic and adipose tissue inflammation and insulin resistance in mice.

Chronic sleep fragmentation (SF) commonly occurs in human populations, and although it does not involve circadian shifts or sleep deprivation, it markedly alters feeding behaviors ultimately promoting obesity and insulin resistance. These symptoms are known to be related to the host gut microbiota. Mice were exposed to SF for 4 weeks and then allowed to recover for 2 weeks. Taxonomic profiles of fecal microbiota were obtained prospectively, and conventionalization experiments were performed in germ-free mice. Adipose tissue insulin sensitivity and inflammation, as well as circulating measures of inflammation, were assayed. Effect of fecal water on colonic epithelial permeability was also examined. Chronic SF-induced increased food intake and reversible gut microbiota changes characterized by the preferential growth of highly fermentative members of Lachnospiraceae and Ruminococcaceae and a decrease of Lactobacillaceae families. These lead to systemic and visceral white adipose tissue inflammation in addition to altered insulin sensitivity in mice, most likely via enhanced colonic epithelium barrier disruption. Conventionalization of germ-free mice with SF-derived microbiota confirmed these findings. Thus, SF-induced metabolic alterations may be mediated, in part, by concurrent changes in gut microbiota, thereby opening the way for gut microbiome-targeted therapeutics aimed at reducing the major end-organ morbidities of chronic SF.

Inflammatory Markers and Obstructive Sleep Apnea in Obese Children: The NANOS Study

Introduction. Obesity and obstructive sleep apnea syndrome (OSA) are common coexisting conditions associated with a chronic low-grade inflammatory state underlying some of the cognitive, metabolic, and cardiovascular morbidities. Aim. To examine the levels of inflammatory markers in obese community-dwelling children with OSA, as compared to no-OSA, and their association with clinical and polysomnographic (PSG) variables. Methods. In this cross-sectional, prospective multicenter study, healthy obese Spanish children (ages 4–15 years) were randomly selected and underwent nocturnal PSG followed by a morning fasting blood draw. Plasma samples were assayed for multiple inflammatory markers. Results. 204 children were enrolled in the study; 75 had OSA, defined by an obstructive respiratory disturbance index (RDI) of 3 events/hour total sleep time (TST). BMI, gender, and age were similar in OSA and no-OSA children. Monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in OSA children, with interleukin-6 concentrations being higher in moderate-severe OSA (i.e., AHI > 5/hrTST; P < 0.01), while MCP-1 levels were associated with more prolonged nocturnal hypercapnia (P < 0.001). Conclusion. IL-6, MCP-1, and PAI-1 are altered in the context of OSA among community-based obese children further reinforcing the proinflammatory effects of sleep disorders such as OSA. This trial is registered with ClinicalTrials.gov NCT01322763.

Adipose tissue macrophage polarization by intermittent hypoxia in a mouse model of OSA: effect of tumor microenvironment.

Intermittent hypoxia (IH)-induces alterations in tumor-associated macrophages (TAMs) that are associated with adverse cancer outcomes, as reported in patients suffering from sleep apnea. Adipose tissues (AT) and bone-marrow (BM)-derived cells are the inferred sources of macrophages infiltrating malignant tumors. Here, the sources of TAMs and the phenotypic changes induced by IH in the ipsilateral and contralateral AT were investigated by using a syngeneic murine solid tumor model (TC1). C57/B6 male mice were exposed to either IH or room air (RA) for 6 weeks, with TC1 cells being inoculated in the 2nd week. Macrophage content, phenotype and tissue origin were assessed in tumors, and ipsilateral and contralateral AT. IH induced a ~2.2-fold increase in TAM tumor infiltration. However, differential responses in the tumor ipsilateral and contralateral AT emerged: IH increased infiltration of preferentially M1 macrophages in contralateral AT, while reductions in macrophages emerged in ipsilateral AT and primarily consisted of the M2 phenotype. These changes were accompanied by reciprocal increases in resident and BM-derived TAMs in the tumor. IH-induced phenotypic alterations in AT macrophages surrounding the tumor and their increased infiltration within the tumor may contribute to the accelerated tumor progression associated with IH.

Biological plausibility linking sleep apnoea and metabolic dysfunction

Obstructive sleep apnoea (OSA) is a very common disorder that affects 10–25% of the general population. In the past two decades, OSA has emerged as a cardiometabolic risk factor in both paediatric and adult populations. OSA-induced metabolic perturbations include dyslipidaemia, atherogenesis, liver dysfunction and abnormal glucose metabolism. The mainstay of treatment for OSA is adenotonsillectomy in children and continuous positive airway pressure therapy in adults. Although these therapies are effective at resolving the sleep-disordered breathing component of OSA, they do not always produce beneficial effects on metabolic function. Thus, a deeper understanding of the underlying mechanisms by which OSA influences metabolic dysfunction might yield improved therapeutic approaches and outcomes. In this Review, we summarize the evidence obtained from animal models and studies of patients with OSA of potential mechanistic pathways linking the hallmarks of OSA (intermittent hypoxia and sleep fragmentation) with metabolic dysfunction. Special emphasis is given to adipose tissue dysfunction induced by sleep apnoea, which bears a striking resemblance to adipose dysfunction resulting from obesity. In addition, important gaps in current knowledge and promising lines of future investigation are identified.

Effects of adenotonsillectomy on plasma inflammatory biomarkers in obese children with obstructive sleep apnea: A community-based study

Background:Obesity and obstructive sleep apnea syndrome (OSA) are highly prevalent and frequently overlapping conditions in children that lead to systemic inflammation, the latter being implicated in the various end-organ morbidities associated with these conditions.Aim:To examine the effects of adenotonsillectomy (T&A) on plasma levels of inflammatory markers in obese children with polysomnographically diagnosed OSA who were prospectively recruited from the community.Methods:Obese children prospectively diagnosed with OSA, underwent T&A and a second overnight polysomnogram (PSG) after surgery. Plasma fasting morning samples obtained after each of the two PSGs were assayed for multiple inflammatory and metabolic markers including interleukin (IL)-6, IL-18, plasminogen activator inhibitor-1 (PAI-1), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9), adiponectin, apelin C, leptin and osteocrin.Results:Out of 122 potential candidates, 100 obese children with OSA completed the study with only one-third exhibiting normalization of their PSG after T&A (that is, apnea-hypopnea index (AHI) ≤1/hour total sleep time). However, overall significant decreases in MCP-1, PAI-1, MMP-9, IL-18 and IL-6, and increases in adropin and osteocrin plasma concentrations occurred after T&A. Several of the T&A-responsive biomarkers exhibited excellent sensitivity and moderate specificity to predict residual OSA (that is, AHI⩾5/hTST).Conclusions:A defined subset of systemic inflammatory and metabolic biomarkers is reversibly altered in the context of OSA among community-based obese children, further reinforcing the concept on the interactive pro-inflammatory effects of sleep disorders such as OSA and obesity contributing to downstream end-organ morbidities.

Early Intermittent Hypoxia Induces Proatherogenic Changes in Aortic Wall Macrophages in a Murine Model of Obstructive Sleep Apnea

To the Editor: Obstructive sleep apnea (OSA) is a highly prevalent condition throughout the lifespan, affecting 2–10% of the general population at any given age. It is associated with an extensive array of cognitive, behavioral, metabolic, and cardiovascular morbidities (1). In recent years, OSA has emerged as an independent risk factor for cardiovascular disease (2) and has been causally associated with a high prevalence of hypertension, atrial fibrillation, congestive heart failure, stroke, and more specifically, coronary heart disease (3). OSA is associated with activation of multiple inflammatory pathways, disruption of lipid metabolism, and endothelial dysfunction through oxidative stress mechanisms (4), all of which predispose to atherogenesis. Long-term chronic intermittent hypoxia during sleep (IH), a prototypic constitutive element of OSA, induces atherosclerosis in murine models and plays a critical role in OSA-associated cardiovascular morbidities (5). However, the molecular mechanisms underlying OSA-induced atherosclerosis are not well understood. Atherosclerosis is currently viewed as a chronic inflammatory process, in which macrophages play a key pathophysiologic role (6). Monocyte recruitment to the circulation, their entrapment in the vascular wall, and their subsequent differentiation into lipid-laden macrophages are fundamental processes involved in atheromatous plaque formation (7, 8). Shifts across the spectrum of heterogeneous macrophage populations encompassing a continuum between major macrophage phenotypes have been described as a fundamental process in atherogenesis. Generally speaking, proinflammatory macrophages are implicated in foam cell generation, lipid loading, plaque formation, and plaque rupture, whereas anti-inflammatory macrophages play an atheroprotective role (9). In addition to changes in macrophage polarity within the vascular wall, macrophage proliferation has also emerged as a critical determining factor in atherogenesis, although the origin of the expanding macrophage population (i.e., tissue-resident vs. bone-marrow derived) is still controversial (10, 11). We hypothesized that IH during the sleep period will induce shifts toward a proatherogenic state in the spectrum of macrophages within the vascular wall. To examine our hypothesis, we exposed 8-week-old male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) to IH with alternating 90-second cycles (21% FiO2 followed by 6% FiO2, 20 cycles/h) for 12 hours/day or to room air for 6 weeks. All mice were kept on a regular low-fat chow diet (n = 12–20/group). After exposures, mice were asphyxiated using CO2 and were exsanguinated via cardiac puncture. The vasculature was perfused with PBS containing 20 U/ml heparin. Full-length aortas were dissected, cleaned, and enzymatically digested. Single-cell suspensions were prepared from the digested tissues by cycles of washing and resuspending. Cells were incubated with Fc blocker for 30 minutes to reduce autofluorescence and were subsequently fixed with 1% paraformaldehyde. After two cycles of washing, pelleted cells were incubated with antibodies for macrophage markers previously implicated in atherogenesis and metabolic dysregulation: CD11b-PB, F4/80-PE/Cy7, Ly6c-APC-Cy7, CD36-FITC, and CD64-PE (Biolegend, San Jose, CA). Subsequently, cells were washed and analyzed using BD FACS CANTO II (BD Biosciences, San Jose, CA). Results were analyzed using FlowJo software. Data are expressed as mean ± standard error of the mean. Room air and IH conditions were compared with Student’s t tests or nonparametric testing as appropriate. Statistical significance was assumed at P < 0.05. Macrophages were defined as CD11b and F4/80 double-positive cells (Figure 1). Significant increases in the percentage of macrophages out of total cell number emerged in IH-exposed mice (6.4% ± 0.3% vs. 8.1% ± 0.3%; P = 0.003). In addition, a trend toward increase in the absolute macrophage number was observed (cells/mg tissue: 247 ± 28 vs. 414 ± 93; P = 0.18). Furthermore, IH also induced shifts in the macrophage population toward a proinflammatory phenotype. Specifically, the IH group showed significantly higher expression (measured by mean fluorescence intensity) of Ly6c (522 ± 23 vs. 699 ± 43; P = 0.004) and increased the percentage of Ly6c(hi) cells (9.2% ± 0.5% vs. 11.7% ± 0.9%; P = 0.03). In contrast, tissue-resident marker CD64 expression in the aortic macrophages was downregulated after IH exposures (mean fluorescence intensity: 3,207 ± 453 vs. 2,967 ± 483; P = 0.03), as well as the percentage of CD64-positive cells (46.4% ± 2.4% vs. 36.9% ± 2.1%; P = 0.006). CD64 expression was threefold higher in Ly6c(lo) compared with Ly6c(hi) cells, thus reinforcing the notion of two distinct macrophage populations. Finally, scavenger receptor CD36 expression was increased in the IH group (mean fluorescence intensity: 1,694 ± 56 vs. 2,129 ± 209; P = 0.005), as was the percentage of CD36+ macrophages (46.7% ± 1.7% vs. 57.3% ± 3.3%; P = 0.03). Of note, despite the decrease in the number of CD64+ cells, the proportion of CD36+ cells out of CD64+ cells was increased in the IH group (14.1% ± 0.9% vs. 20.8% ± 2.2%; P = 0.01). Figure 1. Changes in macrophage populations in the aortic wall of mice exposed to intermittent hypoxia (IH) or room air (RA) during sleep for 6 weeks. (A) Representative example of fluorescence-activated cell sorter analysis of macrophages (CD11b and F4/80 positively ... Current findings show, for the first time, an increase in the number of macrophages in the aortic wall after IH exposures. Macrophages have been extensively studied in the context of atherosclerosis, and the presence of CD11b cells is crucial to the initial steps of atherogenesis (6, 8). The expansion of this population at this early stage in the course of IH exposures is suggestive of this being a key step in IH-induced atherosclerotic plaque formation. Furthermore, IH induced several interesting changes in the phenotype of aortic wall macrophages. First, the Ly6c(hi) population was increased, as was the total expression of Ly6c on the aortic wall macrophages. Ly6c is a well-established marker of proinflammatory macrophages recruited from circulating bone marrow-derived monocytes into the arterial wall in the process of atherogenesis (6). Second, increases in Ly6c were accompanied by reciprocal decreases in CD64, a marker of tissue-resident macrophages (12). These findings add to the current debate regarding the source of the expanding macrophage population in the vascular wall during atherogenesis. Indeed, Robbins and colleagues (10) showed that resident macrophages predominate in apolipoprotein E −/− and low-density lipoprotein receptor −/− mice on a high-fat diet. Although our experiments were not designed to resolve this debate (which would require more complex techniques, such as bone marrow depletion and parabiosis), our findings point to the bone marrow as the source of the expanding macrophage population in IH, rather than to resident macrophages, as seen in high-fat diets. Aligned with this interpretation, OSA has been previously shown by our group and others to target and activate specifically the myeloid population in other disease models, such as obesity and cancer (13). IH also induced increased expression of CD36 surface marker within the macrophage population. CD36 is a membrane glycoprotein participating in oxidized low-density lipoprotein uptake and foam cell formation, the initial critical stage of atherosclerosis (14). Blood monocytes and their lesional progeny also have the potential to emigrate from the plaque (6), and CD36 pathway activation has been shown to inhibit this migration process, which might in turn cause macrophage entrapment in atherosclerotic lesions. Because of its critical role in atherosclerosis, CD36 has also been suggested as a treatment target for atherosclerosis (14). In addition, in line with the proinflammatory interpretation of the changes in our study, CD36 expression has been recently described as identifying a novel metabolically activated macrophage phenotype, which expresses proinflammatory cytokines, but not the classic proinflammatory macrophage surface markers (15). In summary, IH during the sleep period for 6 weeks leads to expansion of the macrophage population in the aortic wall. In addition to modestly increased cellularity, IH induces changes in macrophage population composition with a shift toward a proinflammatory, metabolically activated phenotype and recruitment of bone marrow-derived macrophages. These changes occur in a relatively resistant animal model for development of atherosclerosis; that is, in the absence of a high-fat diet or of high atherogenic risk predisposing gene knockout mice, thus adding to their potential biological significance. Plans for future studies include the effect of longer IH exposures on macrophage populations within the aortic wall and elucidation of the functional properties associated with the phenotypic switch induced by IH.

Circulating exosomes potentiate tumor malignant properties in a mouse model of chronic sleep fragmentation.

Background Chronic sleep fragmentation (SF) increases cancer aggressiveness in mice. Exosomes exhibit pleiotropic biological functions, including immune regulatory functions, antigen presentation, intracellular communication and inter-cellular transfer of RNA and proteins. We hypothesized that SF-induced alterations in biosynthesis and cargo of plasma exosomes may affect tumor cell properties. Results SF-derived exosomes increased tumor cell proliferation (~13%), migration (~2.3-fold) and extravasation (~10%) when compared to exosomes from SC-exposed mice. Similarly, Pre exosomes from OSA patients significantly enhanced proliferation and migration of human adenocarcinoma cells compared to Post. SF-exosomal cargo revealed 3 discrete differentially expressed miRNAs, and exploration of potential mRNA targets in TC1 tumor cells uncovered 132 differentially expressed genes that encode for multiple cancer-related pathways. Methods Plasma-derived exosomes from C57/B6 mice exposed to 6 wks of SF or sleep control (SC), and from adult human patients with obstructive sleep apnea (OSA) before (Pre) and after adherent treatment for 6 wks (Post) were co-cultured with mouse lung TC1 or human adenocarcinoma tumor cell lines, respectively. Proliferation, migration, invasion, endothelial barrier integrity and extravasation assays of tumor cells were performed. Plasma mouse exosomal miRNAs were profiled with arrays, and transcriptomic assessments of TC1 cells exposed to SF or SC exosomes were conducted to identify gene targets. Conclusions Chronic SF induces alterations in exosomal miRNA cargo that alter the biological properties of TC1 lung tumor cells to enhance their proliferative, migratory and extravasation properties, and similar findings occur in OSA patients, in whom SF is a constitutive component of their sleep disorder. Thus, exosomes could participate, at least in part, in the adverse cancer outcomes observed in OSA.

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

&NA; 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)‐1&agr;‐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‐1&agr; transcription using chromatin immunoprecipitation (ChIP)‐sequencing, angiogenesis using immunohistochemistry, and gene expression of different fat cell markers and HIF‐1&agr; 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‐1&agr; 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‐1&agr; activity that is unsustained over time and is accompanied by concurrent vascular rarefaction, inflammation, and insulin resistance. Thus, the dichotomous changes in HIF‐1&agr; 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.

Association of chronic Candida albicans respiratory infection with a more severe lung disease in patients with cystic fibrosis

Despite the increase in fungal isolates, the significance of chronic Candida albicans airway colonization in CF is unclear.