Obstructive sleep apnea (OSA) is a common disorder characterized by episodic obstruction to breathing due to upper airway collapse during sleep. Because of the episodic airway obstruction, intermittently low O₂ (hypoxia) and high CO₂ (hypercapnia) ensue. OSA has been associated with adverse cardiovascular and metabolic outcomes, although data regarding potential causal pathways are still evolving. As changes in inspired O₂ and CO₂ can affect the ecology of the gut microbiota and the microbiota has been shown to contribute to various cardiometabolic disorders, we hypothesized that OSA alters the gut ecosystem, which, in turn, exacerbates the downstream physiological consequences. Here, we model human OSA and its cardiovascular consequence using Ldlr^−/− mice fed a high-fat diet and exposed to intermittent hypoxia and hypercapnia (IHH). The gut microbiome and metabolome were characterized longitudinally (using 16S rRNA amplicon sequencing and untargeted liquid chromatography-tandem mass spectrometry [LC-MS/MS]) and seen to covary during IHH. Joint analysis of microbiome and metabolome data revealed marked compositional changes in both microbial (>10%, most remarkably in Clostridia) and molecular (>22%) species in the gut. Moreover, molecules that altered in abundance included microbe-dependent bile acids, enterolignans, and fatty acids, highlighting the impact of IHH on host-commensal organism cometabolism in the gut. Thus, we present the first evidence that IHH perturbs the gut microbiome functionally, setting the stage for understanding its involvement in cardiometabolic disorders. IMPORTANCE Intestinal dysbiosis mediates various cardiovascular diseases comorbid with OSA. To understand the role of dysbiosis in cardiovascular and metabolic disease caused by OSA, we systematically study the effect of intermittent hypoxic/hypercapnic stress (IHH, mimicking OSA) on gut microbes in an animal model. We take advantage of a longitudinal study design and paired omics to investigate the microbial and molecular dynamics in the gut to ascertain the contribution of microbes on intestinal metabolism in IHH. We observe microbe-dependent changes in the gut metabolome that will guide future research on unrecognized mechanistic links between gut microbes and comorbidities of OSA. Additionally, we highlight novel and noninvasive biomarkers for OSA-linked cardiovascular and metabolic disorders.

Hypoxia-induced cell injury has been related to multiple pathological conditions. In order to render hypoxia-sensitive cells and tissues resistant to low O₂ environment, in this current study, we used Drosophila melanogaster as a model to dissect the mechanisms underlying hypoxia-tolerance. A D. melanogaster strain that lives perpetually in an extremely low-oxygen environment (4% O₂, an oxygen level that is equivalent to that over about 4,000 m above Mt. Everest) was generated through laboratory selection pressure using a continuing reduction of O₂ over many generations. This phenotype is genetically stable since selected flies, after several generations in room air, survive at this low O₂ level. Gene expression profiling showed striking differences between tolerant and naïve flies, in larvae and adults, both quantitatively and qualitatively. Up-regulated genes in the tolerant flies included signal transduction pathways (e.g., Notch and Toll/Imd pathways), but metabolic genes were remarkably down-regulated in the larvae. Furthermore, a different allelic frequency and enzymatic activity of the triose phosphate isomerase (TPI) was present in the tolerant versus naïve flies. The transcriptional suppressor, hairy, was up-regulated in the microarrays and its binding elements were present in the regulatory region of the specifically down-regulated metabolic genes but not others, and mutations in hairy significantly reduced hypoxia tolerance. We conclude that, the hypoxia-selected flies: (a) altered their gene expression and genetic code, and (b) coordinated their metabolic suppression, especially during development, with hairy acting as a metabolic switch, thus playing a crucial role in hypoxia-tolerance. Hypoxia-induced injury has been related to multiple pathological conditions. In order to render mammalian cells and tissues resistant to low O₂ environment, we wished to first understand the mechanisms underlying hypoxia-tolerance in resistant animals. Therefore, we generated a D. melanogaster strain that is tolerant to severe hypoxic conditions through long-term experimental selection. Several adaptive changes were identified in the hypoxia-selected flies that included up-regulation of multiple signal transduction pathways (such as Notch pathway, Insulin pathway, EGF receptor pathway, and Toll/Imd pathway), modulation of cellular respiration enzymes, and polymorphic differences in metabolic enzymes (such as TPI). While we believe that multiple pathways contribute to the hypoxia-tolerant trait in this Drosophila strain, we demonstrate that hairy-mediated metabolic suppression is a critical mechanism for reducing the mismatch between supply and demand of O₂.

Chronic hypoxia, whether continuous (CCH) or intermittent (CIH), occurs in many neonatal pathological conditions, such as bronchopulmonary dysplasia and obstructive sleep apnea. In this study, we explored the effect of CCH and CIH on cerebral capillary density and myelination. We subjected CD-1 mice starting at postnatal day 2 to either CCH 11% oxygen (O₂), or CIH 11% O₂(4-min cycles), for periods of 2 and 4 wk followed by reoxygenation for 4 wk. Mice were deeply anesthetized and perfused. Brains were removed to fixative for 24 h, then paraffin-embedded. Coronal brain sections were taken for analysis. Immunocytochemistry for glucose transporter 1 was used to assess angiogenesis, and Luxol fast blue and fluoromyelin stains were used to assess myelination. Capillary density increased after 2-wk exposure to CIH and CCH. By 4 wk, capillary density increased in both CIH and CCH by 25% and 47%, respectively, in cortex and by 29% and 44%, respectively, in hippocampus ( P < 0.05). There was a decrease in myelination in the corpus callosum of mice exposed to CIH (75% of control) and CCH (50% of control) ( P < 0.05). Reoxygenation reversed the increased capillary density seen in CCH to normoxic values. However, dysmyelination that occurred in CCH-exposed mice did not show any improvement upon reoxygenation. We conclude that neonatal chronic hypoxia 1) induces brain angiogenesis, which is reversible with reoxygenation, and 2) irreversibly reduces the extent of myelination in the corpus callosum. This potential irreversible effect on myelination in early life can, therefore, have long-term and devastating effects.

Through long-term laboratory selection (over 200 generations), we have generated Drosophila melanogaster populations that tolerate severe, normally lethal, levels of hypoxia. Because of initial experiments suspecting genetic mechanisms underlying this adaptation, we compared the genomes of the hypoxia-selected flies with those of controls using deep resequencing. By applying unique computing and analytical methods we identified a number of DNA regions under selection, mostly on the X chromosome. Several of the hypoxia-selected regions contained genes encoding or regulating the Notch pathway. In addition, previous expression profiling revealed an activation of the Notch pathway in the hypoxia-selected flies. We confirmed the contribution of Notch activation to hypoxia tolerance using a specific γ-secretase inhibitor, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), which significantly reduced adult survival and life span in the hypoxia-selected flies. We also demonstrated that flies with loss-of-function Notch mutations or RNAi-mediated Notch knockdown had a significant reduction in hypoxia tolerance, but those with a gain-of-function had a dramatic opposite effect. Using the UAS-Gal4 system, we also showed that specific overexpression of the Notch intracellular domain in glial cells was critical for conferring hypoxia tolerance. Unique analytical tools and genetic and bioinformatic strategies allowed us to discover that Notch activation plays a major role in this hypoxia tolerance in Drosophila melanogaster .