Real-time visualization of dextran extravasation in intermittent hypoxia mice using noninvasive SWIR imaging.

Imaging tools are crucial for studying the vascular network and its barrier function in various physiopathological conditions. Shortwave infrared (SWIR) window optical imaging allows noninvasive, in-depth exploration. We applied SWIR imaging, combined with vessel segmentation and deep learning analyses, to study real-time dextran probe extravasation in mice experiencing intermittent hypoxia (IH)-a characteristic of obstructive sleep apnea associated with potential cardiovascular alterations due to early vascular permeability. Evidence for permeability in this context is limited, making our investigation significant. C57Bl/6 mice were exposed to normoxia or intermittent hypoxia for 14 days. Then SWIR imaging between 1,250 and 1,700 nm was performed on the saphenous artery and vein and on the surrounding tissue after intravenous injection of labeled dextrans of two different sizes (10 or 70 kDa). Postprocessing and segmentation of the SWIR images were conducted using deep learning treatment. We monitored high-resolution signals, distinguishing arteries, veins, and surrounding tissues. In the saphenous artery and vein, after 70-kD dextran injection, tissue/vessel ratio was higher after intermittent hypoxia (IH) than normoxia (N) over 500 seconds (P < 0.05). However, the ratio was similar in N and IH after 10-kD dextran injection. The SWIR imaging technique allows noninvasive, real-time monitoring of dextran extravasation in vivo. Dextran 70 extravasation is increased after exposure to IH, suggesting an increased vessel permeability in this mice model of obstructive sleep apnea.NEW & NOTEWORTHY We demonstrate that SWIR imaging technique is a useful tool to monitor real-time dextran extravasation from vessels in vivo, with a high resolution. We report for the first time an increased real-time dextran (70 kD) extravasation in mice exposed to intermittent hypoxia for 14 days compared with normoxic controls.

Intermittent hypoxia increases lipid insulin resistance in healthy humans: A randomized crossover trial.

Sympathetic overactivity caused by chronic intermittent hypoxia is a hallmark of obstructive sleep apnea. A high sympathetic tone elicits increases in plasma free fatty acid and insulin. Our objective was to assess the impact of 14 nights of chronic intermittent hypoxia exposure on sympathetic activity, glucose control, lipid profile and subcutaneous fat tissue remodelling in non-obese healthy humans. In this prospective, double-blinded crossover study, 12 healthy subjects were randomized, among them only nine underwent the two phases of exposures of 14 nights chronic intermittent hypoxia versus air. Sympathetic activity was measured by peroneal microneurography (muscle sympathetic nerve activity) before and after each exposure. Fasting glucose, insulin, C-peptide and free fatty acid were assessed at rest and during a multisampling oral glucose tolerance test. We assessed histological remodelling, adrenergic receptors, lipolysis and lipogenesis genes expression and functional changes of the adipose tissue. Two weeks of exposure of chronic intermittent hypoxia versus ambient air significantly increased sympathetic activity (p = 0.04). Muscle sympathetic nerve activity increased from 24.5 [18.9; 26.8] before to 21.7 [13.8; 25.7] after ambient air exposure, and from 20.6 [17.4; 23.9] before to 28.0 [24.4; 31.5] bursts per min after exposure to chronic intermittent hypoxia. After chronic intermittent hypoxia, post-oral glucose tolerance test circulating free fatty acid area under the curve increased (p = 0.05) and free fatty acid sensitivity to insulin decreased (p = 0.028). In adipocyte tissue, intermittent hypoxia increased expression of lipolysis genes (adipocyte triglyceride lipase and hormone-sensitive lipase) and lipogenesis genes (fatty acid synthase; p < 0.05). In this unique experimental setting in healthy humans, chronic intermittent hypoxia induced high sympathetic tone, lipolysis and decreased free fatty acid sensitivity to insulin. This might participate in the trajectory to systemic insulin resistance and diabetes for patients with obstructive sleep apnea.

Early Increase in Blood-Brain Barrier Permeability in a Murine Model Exposed to Fifteen Days of Intermittent Hypoxia.

Obstructive sleep apnea (OSA) is characterized by intermittent repeated episodes of hypoxia-reoxygenation. OSA is associated with cerebrovascular consequences. An enhanced blood-brain barrier (BBB) permeability has been proposed as a marker of those disorders. We studied in mice the effects of 1 day and 15 days intermittent hypoxia (IH) exposure on BBB function. We focused on the dorsal part of the hippocampus and attempted to identify the molecular mechanisms by combining in vivo BBB permeability (Evans blue tests) and mRNA expression of several junction proteins (zona occludens (ZO-1,2,3), VE-cadherin, claudins (1,5,12), cingulin) and of aquaporins (1,4,9) on hippocampal brain tissues. After 15 days of IH exposure we observed an increase in BBB permeability, associated with increased mRNA expressions of claudins 1 and 12, aquaporins 1 and 9. IH seemed to increase early for claudin-1 mRNA expression as it doubled with 1 day of exposure and returned near to its base level after 15 days. Claudin-1 overexpression may represent an immediate response to IH exposure. Then, after 15 days of exposure, an increase in functional BBB permeability was associated with enhanced expression of aquaporin. These BBB alterations are possibly associated with a vasogenic oedema that may affect brain functions and accelerate neurodegenerative processes.

Loss of Blood-Brain Barrier Integrity in an In Vitro Model Subjected to Intermittent Hypoxia: Is Reversion Possible with a HIF-1α Pathway Inhibitor?

Several sleep-related breathing disorders provoke repeated hypoxia stresses, which potentially lead to neurological diseases, such as cognitive impairment. Nevertheless, consequences of repeated intermittent hypoxia on the blood-brain barrier (BBB) are less recognized. This study compared two methods of intermittent hypoxia induction on the cerebral endothelium of the BBB: one using hydralazine and the other using a hypoxia chamber. These cycles were performed on an endothelial cell and astrocyte coculture model. Na-Fl permeability, tight junction protein, and ABC transporters (P-gp and MRP-1) content were evaluated with or without HIF-1 inhibitors YC-1. Our results demonstrated that hydralazine as well as intermittent physical hypoxia progressively altered BBB integrity, as shown by an increase in Na-Fl permeability. This alteration was accompanied by a decrease in concentration of tight junction proteins ZO-1 and claudin-5. In turn, microvascular endothelial cells up-regulated the expression of P-gp and MRP-1. An alteration was also found under hydralazine after the third cycle. On the other hand, the third intermittent hypoxia exposure showed a preservation of BBB characteristics. Furthermore, inhibition of HIF-1α with YC-1 prevented BBB dysfunction after hydralazine treatment. In the case of physical intermittent hypoxia, we observed an incomplete reversion suggesting that other biological mechanisms may be involved in BBB dysfunction. In conclusion, intermittent hypoxia led to an alteration of the BBB model with an adaptation observed after the third cycle.

Differential Impact of Intermittent vs. Sustained Hypoxia on HIF-1, VEGF and Proliferation of HepG2 Cells.

Obstructive sleep apnea (OSA) is an emerging risk factor for cancer occurrence and progression, mainly mediated by intermittent hypoxia (IH). Systemic IH, a main landmark of OSA, and local sustained hypoxia (SH), a classical feature at the core of tumors, may act separately or synergistically on tumor cells. Our aim was to compare the respective consequences of intermittent and sustained hypoxia on HIF-1, endothelin-1 and VEGF expression and on cell proliferation and migration in HepG2 liver tumor cells. Wound healing, spheroid expansion, proliferation and migration were evaluated in HepG2 cells following IH or SH exposure. The HIF-1α, endothelin-1 and VEGF protein levels and/or mRNA expression were assessed, as were the effects of HIF-1 (acriflavine), endothelin-1 (macitentan) and VEGF (pazopanib) inhibition. Both SH and IH stimulated wound healing, spheroid expansion and proliferation of HepG2 cells. HIF-1 and VEGF, but not endothelin-1, expression increased with IH exposure but not with SH exposure. Acriflavine prevented the effects of both IH and SH, and pazopanib blocked those of IH but not those of SH. Macitentan had no impact. Thus, IH and SH stimulate hepatic cancer cell proliferation via distinct signaling pathways that may act synergistically in OSA patients with cancer, leading to enhanced tumor progression.

Intermittent hypoxia-related alterations in vascular structure and function: a systematic review and meta-analysis of rodent data.

BACKGROUND: Obstructive sleep apnoea and the related intermittent hypoxia (IH) are widely recognised as risk factors for incident cardiovascular diseases. Numerous studies support the deleterious vascular impact of IH in rodents but an overall interpretation is challenging owing to heterogeneity in rodent species investigated and the severity and duration of IH exposure. To clarify this major issue, we conducted a systematic review and meta-analysis to quantify the impact of IH on systemic artery structure and function depending on the different IH exposure designs. METHODS: We searched PubMed, Embase and Web of Science, and included 125 articles in a meta-analysis, among them 112 using wild-type rodents and 13 using apolipoprotein E knockout (ApoE-/-) mice. We used the standardised mean difference (SMD) to compare results between studies. RESULTS: IH significantly increased mean arterial pressure (+13.90 (95% CI 11.88-15.92) mmHg), and systolic and diastolic blood pressure. Meta-regressions showed that mean arterial pressure change was associated with strain and year of publication. IH altered vasodilation in males but not in females and increased endothelin-1-induced but not phenylephrine-induced vasoconstriction. Intima-media thickness significantly increased upon IH exposure (SMD 1.10 (95% CI 0.58-1.62); absolute values +5.23 (2.81-7.84) µm). This increase was observed in mice but not in rats and was negatively associated with age. Finally, IH increased atherosclerotic plaque size in ApoE-/- mice (SMD 1.08 (95% CI 0.80-1.37)). CONCLUSIONS: Our meta-analysis established that IH, independently of other confounders, has a strong effect on vascular structure and physiology. Our findings support the interest of identifying and treating sleep apnoea in routine cardiology practice.

Inhibition of Vascular Endothelial Cadherin Cleavage Prevents Elastic Fiber Alterations and Atherosclerosis Induced by Intermittent Hypoxia in the Mouse Aorta.

Intermittent hypoxia (IH), the major feature of obstructive sleep apnea syndrome (OSAS), induces atherosclerosis and elastic fiber alterations. VE-cadherin cleavage is increased in OSAS patients and in an IH-cellular model. It is mediated by HIF-1 and Src-tyr-kinases pathways and results in endothelial hyperpermeability. Our aim was to determine whether blocking VE-cadherin cleavage in vivo could be an efficient strategy to inhibit deleterious IH-induced vascular remodeling, elastic fiber defects and atherogenesis. VE-cadherin regulation, aortic remodeling and atherosclerosis were studied in IH-exposed C57Bl/6J or ApoE-/-mice treated or not with Src-tyr-kinases inhibitors (Saracatinib/Pazopanib) or a HIF-1 inhibitor (Acriflavine). Human aortic endothelial cells were exposed to IH and treated with the same inhibitors. LDL and the monocytes transendothelium passage were measured. In vitro, IH increased transendothelium LDL and monocytes passage, and the tested inhibitors prevented these effects. In mice, IH decreased VE-cadherin expression and increased plasmatic sVE level, intima-media thickness, elastic fiber alterations and atherosclerosis, while the inhibitors prevented these in vivo effects. In vivo inhibition of HIF-1 and Src tyr kinase pathways were associated with the prevention of IH-induced elastic fiber/lamella degradation and atherogenesis, which suggests that VE-cadherin could be an important target to limit atherogenesis and progression of arterial stiffness in OSAS.

Physiological Impact of a Synthetic Elastic Protein in Arterial Diseases Related to Alterations of Elastic Fibers: Effect on the Aorta of Elastin-Haploinsufficient Male and Female Mice.

Elastic fibers, made of elastin (90%) and fibrillin-rich microfibrils (10%), are the key extracellular components, which endow the arteries with elasticity. The alteration of elastic fibers leads to cardiovascular dysfunctions, as observed in elastin haploinsufficiency in mice (Eln+/-) or humans (supravalvular aortic stenosis or Williams-Beuren syndrome). In Eln+/+ and Eln+/- mice, we evaluated (arteriography, histology, qPCR, Western blots and cell cultures) the beneficial impact of treatment with a synthetic elastic protein (SEP), mimicking several domains of tropoelastin, the precursor of elastin, including hydrophobic elasticity-related domains and binding sites for elastin receptors. In the aorta or cultured aortic smooth muscle cells from these animals, SEP treatment induced a synthesis of elastin and fibrillin-1, a thickening of the aortic elastic lamellae, a decrease in wall stiffness and/or a strong trend toward a reduction in the elastic lamella disruptions in Eln+/- mice. SEP also modified collagen conformation and transcript expressions, enhanced the aorta constrictive response to phenylephrine in several animal groups, and, in female Eln+/- mice, it restored the normal vasodilatory response to acetylcholine. SEP should now be considered as a biomimetic molecule with an interesting potential for future treatments of elastin-deficient patients with altered arterial structure/function.

VE-cadherin cleavage in sleep apnoea: new insights into intermittent hypoxia-related endothelial permeability.

BACKGROUND: Obstructive sleep apnoea (OSA) causes intermittent hypoxia that in turn induces endothelial dysfunction and atherosclerosis progression. We hypothesised that VE-cadherin cleavage, detected by its released extracellular fragment solubilised in the blood (sVE), may be an early indicator of emergent abnormal endothelial permeability. Our aim was to assess VE-cadherin cleavage in OSA patients and in in vivo and in vitro intermittent hypoxia models to decipher the cellular mechanisms and consequences. METHODS: Sera from seven healthy volunteers exposed to 14 nights of intermittent hypoxia, 43 OSA patients and 31 healthy control subjects were analysed for their sVE content. Human aortic endothelial cells (HAECs) were exposed to 6 h of intermittent hypoxia in vitro, with or without an antioxidant or inhibitors of hypoxia-inducible factor (HIF)-1, tyrosine kinases or vascular endothelial growth factor (VEGF) pathways. VE-cadherin cleavage and phosphorylation were evaluated, and endothelial permeability was assessed by measuring transendothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran flux. RESULTS: sVE was significantly elevated in sera from healthy volunteers submitted to intermittent hypoxia and OSA patients before treatment, but conversely decreased in OSA patients after 6 months of continuous positive airway pressure treatment. OSA was the main factor accounting for sVE variations in a multivariate analysis. In in vitro experiments, cleavage and expression of VE-cadherin increased upon HAEC exposure to intermittent hypoxia. TEER decreased and FITC-dextran flux increased. These effects were reversed by all of the pharmacological inhibitors tested. CONCLUSIONS: We suggest that in OSA, intermittent hypoxia increases endothelial permeability in OSA by inducing VE-cadherin cleavage through reactive oxygen species production, and activation of HIF-1, VEGF and tyrosine kinase pathways.

Zinc deficiency promotes endothelin secretion and endothelial cell migration through nuclear hypoxia-inducible factor-1 translocation.

Zinc is involved in the expression and function of various transcription factors, including the hypoxia-inducible factor-1 (HIF-1). HIF-1 and its target gene endothelin-1 (ET-1) are activated by intermittent hypoxia (IH), one of the main consequences of obstructive sleep apnea (OSA), and both play a key role in the cardiovascular consequences of IH. Because OSA and IH are associated with zinc deficiency, we investigated the effect of zinc deficiency caused by chelation on the HIF-1/ET-1 pathway and its functional consequences in endothelial cells. Primary human microvascular endothelial cells (HMVEC) were incubated with submicromolar doses of the zinc-specific membrane-permeable chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylene diamine (TPEN, 0.5 µM) or ET-1 (0.01 µM) with or without bosentan, a dual ET-1-receptor antagonist. HIF-1α expression was silenced by transfection with specific siRNA. Nuclear HIF-1 content was assessed by immunofluorescence microscopy and Western blot. Migratory capacity of HMVEC was evaluated with a wound-healing scratch assay. Zinc chelation by TPEN exposure induced the translocation of the cytosolic HIF-1α subunit of HIF-1 to the nucleus as well as an HIF-1-mediated ET-1 secretion by HMVEC. Incubation with either TPEN or ET-1 increased endothelial wound-healing capacity. Both HIF-1α silencing or bosentan abolished this effect. Altogether, these results suggest that zinc deficiency upregulates ET-1 signaling through HIF-1 activation and stimulates endothelial cell migration, suggesting an important role of zinc in the vascular consequences of IH and OSA mediated by HIF-1-ET- signaling.

A midline switch of receptor processing regulates commissural axon guidance in vertebrates.

Commissural axon guidance requires complex modulations of growth cone sensitivity to midline-derived cues, but underlying mechanisms in vertebrates remain largely unknown. By using combinations of ex vivo and in vivo approaches, we uncovered a molecular pathway controlling the gain of response to a midline repellent, Semaphorin3B (Sema3B). First, we provide evidence that Semaphorin3B/Plexin-A1 signaling participates in the guidance of commissural projections at the vertebrate ventral midline. Second, we show that, at the precrossing stage, commissural neurons synthesize the Neuropilin-2 and Plexin-A1 Semaphorin3B receptor subunits, but Plexin-A1 expression is prevented by a calpain1-mediated processing, resulting in silencing commissural responsiveness. Third, we report that, during floor plate (FP) in-growth, calpain1 activity is suppressed by local signals, allowing Plexin-A1 accumulation in the growth cone and sensitization to Sema3B. Finally, we show that the FP cue NrCAM mediates the switch of Plexin-A1 processing underlying growth cone sensitization to Sema3B. This reveals pathway-dependent modulation of guidance receptor processing as a novel mechanism for regulating guidance decisions at intermediate targets.

An innovative intermittent hypoxia model for cell cultures allowing fast Po2 oscillations with minimal gas consumption.

Performing hypoxia-reoxygenation cycles in cell culture with a cycle duration accurately reflecting what occurs in obstructive sleep apnea (OSA) patients is a difficult but crucial technical challenge. Our goal was to develop a novel device to expose multiple cell culture dishes to intermittent hypoxia (IH) cycles relevant to OSA with limited gas consumption. With gas flows as low as 200 ml/min, our combination of plate holders with gas-permeable cultureware generates rapid normoxia-hypoxia cycles. Cycles alternating 1 min at 20% O2 followed by 1 min at 2% O2 resulted in Po2 values ranging from 124 to 44 mmHg. Extending hypoxic and normoxic phases to 10 min allowed Po2 variations from 120 to 25 mmHg. The volume of culture medium or the presence of cells only modestly affected the Po2 variations. In contrast, the nadir of the hypoxia phase increased when measured at different heights above the membrane. We validated the physiological relevance of this model by showing that hypoxia inducible factor-1α expression was significantly increased by IH exposure in human aortic endothelial cells, murine breast carcinoma (4T1) cells as well as in a blood-brain barrier model (2.5-, 1.5-, and 6-fold increases, respectively). In conclusion, we have established a new device to perform rapid intermittent hypoxia cycles in cell cultures, with minimal gas consumption and the possibility to expose several culture dishes simultaneously. This device will allow functional studies of the consequences of IH and deciphering of the molecular biology of IH at the cellular level using oxygen cycles that are clinically relevant to OSA.

Endothelin regulates intermittent hypoxia-induced lipolytic remodelling of adipose tissue and phosphorylation of hormone-sensitive lipase.

Obstructive sleep apnoea syndrome is characterized by repetitive episodes of upper airway collapse during sleep resulting in chronic intermittent hypoxia (IH). Obstructive sleep apnoea syndrome, through IH, promotes cardiovascular and metabolic disorders. Endothelin-1 (ET-1) secretion is upregulated by IH, and is able to modulate adipocyte metabolism. Therefore, the present study aimed to characterize the role of ET-1 in the metabolic consequences of IH on adipose tissue in vivo and in vitro. Wistar rats were submitted to 14 days of IH-cycles (30 s of 21% FiO2 and 30 s of 5% FiO2 ; 8 h day(-1) ) or normoxia (air-air cycles) and were treated or not with bosentan, a dual type A and B endothelin receptor (ETA-R and ETB-R) antagonist. Bosentan treatment decreased plasma free fatty acid and triglyceride levels, and inhibited IH-induced lipolysis in adipose tissue. Moreover, IH induced a 2-fold increase in ET-1 transcription and ETA-R expression in adipose tissue that was reversed by bosentan. In 3T3-L1 adipocytes, ET-1 upregulated its own and its ETA-R transcription and this effect was abolished by bosentan. Moreover, ET-1 induced glycerol release and inhibited insulin-induced glucose uptake. Bosentan and BQ123 inhibited these effects. Bosentan also reversed the ET-1-induced phosphorylation of hormone-sensitive lipase (HSL) on Ser(660) . Finally, ET-1-induced lipolysis and HSL phosphorylation were also observed under hypoxia. Altogether, these data suggest that ET-1 is involved in IH-induced lipolysis in Wistar rats, and that upregulation of ET-1 production and ETA-R expression by ET-1 itself under IH could amplify its effects. Moreover, ET-1-induced lipolysis could be mediated through ETA-R and activation of HSL by Ser(660) phosphorylation.

Intermittent hypoxia in obese Zucker rats: cardiometabolic and inflammatory effects.

NEW FINDINGS: What is the central question of this study? This study addresses the relative impact of obesity and intermittent hypoxia in the pathophysiological process of obstructive sleep apnoea by investigating the metabolic, inflammatory and cardiovascular consequences of intermittent hypoxia in lean and obese Zucker rats. What is the main finding and its importance? We found that obesity and intermittent hypoxia have mainly distinct consequences on the investigated inflammatory and cardiometabolic parameters in Zucker rats. This suggests that, for a given severity of sleep apnea, the association of obesity and obstructive sleep apnoea may not necessarily be deleterious. Obstructive sleep apnoea is associated with obesity with a high prevalence, and both co-morbidities are independent cardiovascular risk factors. Intermittent hypoxia (IH) is thought to be the main factor responsible for the obstructive sleep apnoea-related cardiometabolic alterations. The aim of this study was to assess the respective impact of obesity and IH on the inflammatory and cardiometabolic state in rats. Lean and obese Zucker rats were exposed to normoxia or chronic IH, and we assessed metabolic and inflammatory parameters, such as plasma lipids and glucose, serum leptin and adiponectin, liver cytokines, nuclear factor-κB activity and cardiac endothelin-1 levels. Myocardial infarct size was also evaluated following in vitro ischaemia-reperfusion. Circulating lipids, insulin, homeostasis model assessment of insulin resistance (HOMA-IR), leptin and adiponectin levels were higher in obese versus lean rats. Chronic IH did not have a significant impact on metabolic parameters in lean rats. In obese rats, IH increased glycaemia and HOMA-IR. Liver interleukin-6 and tumour necrosis factor-α levels were elevated in lean rats exposed to IH; obesity prevented the increase in interleukin-6 but not in tumour necrosis factor-α. Finally, IH exposure enhanced myocardial sensitivity to infarction in both lean and obese rats and increased cardiac endothelin-1 in lean but not obese rats. In conclusion, this study shows that the dyslipidaemia and insulin resistance induced by obesity of genetic origin does not enhance the deleterious cardiovascular response to IH and may even partly protect against IH-induced inflammation.

Portrait of intense communications within microfluidic neural networks.

In vitro model networks could provide cellular models of physiological relevance to reproduce and investigate the basic function of neural circuits on a chip in the laboratory. Several tools and methods have been developed since the past decade to build neural networks on a chip; among them, microfluidic circuits appear to be a highly promising approach. One of the numerous advantages of this approach is that it preserves stable somatic and axonal compartments over time due to physical barriers that prevent the soma from exploring undesired areas and guide neurites along defined pathways. As a result, neuron compartments can be identified and isolated, and their interconnectivity can be modulated to build a topological neural network (NN). Here, we have assessed the extent to which the confinement imposed by the microfluidic environment can impact cell development and shape NN activity. Toward that aim, microelectrode arrays have enabled the monitoring of the short- and mid-term evolution of neuron activation over the culture period at specific locations in organized (microfluidic) and random (control) networks. In particular, we have assessed the spike and burst rate, as well as the correlations between the extracted spike trains over the first stages of maturation. This study enabled us to observe intense neurite communications that would have been weaker and more delayed within random networks; the spiking rate, burst and correlations being reinforced over time in terms of number and amplitude, exceeding the electrophysiological features of standard cultures. Beyond the enhanced detection efficiency that was expected from the microfluidic channels, the confinement of cells seems to reinforce neural communications and cell development throughout the network.

Intermittent Hypoxia Mediates Cancer Development and Progression Through HIF-1 and miRNA Regulation.

INTRODUCTION: There is still a debate for the link between obstructive sleep apnoea (OSA) and cancer. The mechanisms underlying this causality are poorly understood. Several miRNAs are involved in cancer development and progression with expression being influenced by hypoxia. The aims of this work were (i) to compare miRNAs expression in controls versus patients affected by OSA without or with cancer (ONCO-OSA) and (ii) in colorectal cancer cells exposed to intermittent hypoxia (IH), to evaluate miRNAs impact on tumor progression in vitro. METHODS: We detected miRNAs by qRT-PCR in patients' sera and in CaCo2 cells exposed to 2-32h of IH with or without acriflavine (ACF), a HIF-1 inhibitor. Viability and transwell invasion test were applied to investigate the proliferation and migration of CaCo2 exposed to IH and treated with miRNA inhibitors or acriflavine. HIF-1α activity was evaluated in CaCo2 cells after IH. RESULTS: The levels of miR-21, miR-26a and miR-210 increased in OSA and ONCO-OSA patients compared to controls. MiR-23b increased in ONCO-OSA patients, and miR-27b and miR-145 increased in OSA but not ONCO-OSA patients. MiR-21, miR-26a, miR-23b and miR-210 increased in cells after IH. IH stimulated cell proliferation and migration. This effect was reduced after either miRNA inhibition or acriflavine treatment. MiRNA inhibition reduces HIF-1α gene expression. Conversely, acriflavine reduced the expression of these miRNAs. CONCLUSIONS: We identified a signature of miRNAs, induced by the IH environment. They could be implicated in cancer development and progression through a regulatory loop involving HIF-1.

Cardiac consequences of intermittent hypoxia: a matter of dose? A systematic review and meta-analysis in rodents.

AIM: Intermittent hypoxia (IH) is considered to be a major contributor to obstructive sleep apnoea-related cardiovascular consequences. The present meta-analysis aimed to assess the effects of IH on cardiac remodelling, function and infarct size after myocardial ischaemia across different rodent species and IH severities. METHODS AND RESULTS: Relevant articles from PubMed, Embase and Web of Science were screened. We performed a random effect meta-analysis to assess the effect of IH on myocardium in rodents by using standardised mean difference (SMD). Studies using rodents exposed to IH and outcomes related to cardiac remodelling, contractile function and response to myocardial ischaemia-reperfusion were included. 5217 articles were screened and 92 were included, demonstrating that IH exposure induced cardiac remodelling, characterised by cardiomyocyte hypertrophy (cross-sectional area: SMD=2.90, CI (0.82-4.98), I2=94.2%), left ventricular (LV) dilation (LV diameter: SMD=0.64, CI (0.18-1.10), I2=88.04%), interstitial fibrosis (SMD=5.37, CI (3.22-7.53), I2=94.8) and apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelling: SMD=6.70, CI (2.96-10.44), I2=95.9). These structural changes were accompanied by a decrease in LV ejection fraction (SMD=-1.82, CI (-2.52--1.12), I2=94.22%). Importantly, most of the utilised IH protocols mimicked extremely severe hypoxic disease. Concerning infarct size, meta-regression analyses highlighted an ambivalent role of IH, depending on its severity. Indeed, IH exposure with inspiratory oxygen fraction (F IO2 ) <7% was associated with an increase in infarct size, whereas a reduced infarct size was reported for F IO2 levels above 10%. Heterogeneity between studies, small study effect and poor reporting of methods in included articles limited the robustness of the meta-analysis findings. CONCLUSION: This meta-analysis demonstrated that severe IH systematically induces cardiac remodelling and contractile dysfunction in rodents, which might trigger or aggravate chronic heart failure. Interestingly, this meta-analysis showed that, depending on stimulus severity, IH exhibits both protective and aggravating effects on infarct size after experimental ischaemia-reperfusion procedures.

Chronic intermittent hypoxia, a hallmark of obstructive sleep apnea, promotes 4T1 breast cancer development through endothelin-1 receptors.

The association between obstructive sleep apnea (OSA) and cancer is still debated and data are scarce regarding the link between OSA and breast cancer progression. Since conclusive epidemiological studies require large sample sizes and sufficient duration of exposure before incident cancer occurrence, basic science studies represent the most promising approach to appropriately address the topic. Here we assessed the impact of intermittent hypoxia (IH), the major hallmark of OSA, on the development of breast cancer and explored the specific involvement of the endothelin signaling pathway. Original in vitro and in vivo models were used where 3D-spheroids or cultures of murine 4T1 breast cancer cells were submitted to IH cycles, and nude NMRI mice, orthotopically implanted with 4T1 cells, were submitted to chronic IH exposure before and after implantation. The role of the endothelin-1 in promoting cancer cell development was investigated using the dual endothelin receptor antagonist, macitentan. In vitro exposure to IH significantly increased 4T1 cell proliferation and migration. Meta-analysis of 4 independent in vivo experiments showed that chronic IH exposure promoted tumor growth, assessed by caliper measurement (overall standardized mean difference: 1.00 [0.45-1.55], p < 0.001), bioluminescence imaging (1.65 [0.59-2.71]; p < 0.01) and tumor weight (0.86 [0.31-1.41], p < 0.01), and enhanced metastatic pulmonary expansion (0.77 [0.12-1.42]; p = 0.01). Both in vitro and in vivo tumor-promoting effects of IH were reversed by macitentan. Overall, these findings demonstrate that chronic intermittent hypoxia exposure promotes breast cancer growth and malignancy and that dual endothelin receptor blockade prevents intermittent hypoxia-induced tumor development.

NG2-expressing glial precursor cells are a new potential oligodendroglioma cell initiating population in N-ethyl-N-nitrosourea-induced gliomagenesis.

Gliomas are the most common primary brain tumor affecting human adults and remain a therapeutic challenge because cells of origin are still unknown. Here, we investigated the cellular origin of low-grade gliomas in a rat model based on transplacental exposure to N-ethyl-N-nitrosourea (ENU). Longitudinal magnetic resonance imaging coupled to immunohistological and immunocytochemical analyses were used to further characterize low-grade rat gliomas at different stages of evolution. We showed that early low-grade gliomas have characteristics of oligodendroglioma-like tumors and exclusively contain NG2-expressing slow dividing precursor cells, which express early markers of oligodendroglial lineage. These tumor-derived precursors failed to fully differentiate into oligodendrocytes and exhibited multipotential abilities in vitro. Moreover, a few glioma NG2+ cells are resistant to radiotherapy and may be responsible for tumor recurrence, frequently observed in humans. Overall, these findings suggest that transformed multipotent NG2 glial precursor cell may be a potential cell of origin in the genesis of rat ENU-induced oligodendroglioma-like tumors. This work may open up new perspectives for understanding biology of human gliomas.