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BackgroundObstructive 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.MethodsWe 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.ResultsIH 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)).ConclusionsOur 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.

BackgroundObstructive 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.MethodsSera 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.ResultssVE 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.ConclusionsWe 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.

International audience; 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.

Electronic micro and nano-devices are suitable tools to monitor the activity of many individual neurons over mesoscale networks. However the inorganic materials currently used in microelectronics are barely accepted by neural cells and tissues, thus limiting both the sensor lifetime and efficiency. In particular, penetrating intracortical probes face high failure rate because of a wide immune response of cells and tissues. This adverse reaction called gliosis leads to the rejection of the implanted probe after few weeks and prevent long-lasting recordings of cortical neurons. Such acceptance issue impedes the realization of many neuro-rehabilitation projects. To overcome this, graphene and related carbon-based materials have attracted a lot of interest regarding their positive impact on the adhesion and regeneration of neurons, and their ability to provide high-sensitive electronic devices, such as graphene field effect transistor (G-FET). Such devices can also be implemented on numerous suitable substrates including soft substrates to match the mechanical compliance of cells and tissues, improving further the biocompatibility of the implants. Thus, using graphene as a coating and sensing device material could significantly enhance the acceptance of intracortical probes. However, such a thin monolayer of carbon atoms could be teared off during manipulation and insertion within the brain, and could also display degradation over time. In this work, we have investigated the ability to protect graphene with a natural, biocompatible and degradable polymeric film derivated from hyaluronic acid (HA). We demonstrate that HA-based coatings can be deposited over a wide range of substrates, including intracortical probes and graphene FET arrays without altering the underlying device material, its biocompatibility and sensitivity. Moreover, we show that this coating can be monitored in situ by quantifying the number of deposited charges with the G-FET arrays. The reported graphene functionalization offers promising alternatives for improving the acceptance of various neural interfaces.

Graphene, the atomically-thin honeycomb carbon lattice, is a highly conducting 2D material whose exposed electronic structure offers an ideal platform for chemical and biological sensing. Its biocompatible, flexible and chemically inert nature associated with the lack of dangling bonds, offers novel perspectives for direct interfacing with biological molecules. Combined with its exceptional electronic and optical properties, this promotes graphene as a unique platform for bioelectronics. Among the successful bio-integrations of graphene, the detection of action potentials in numerous electrogenic cells including neurons has paved the road for the high spatio-temporal and wide-field mapping of neuronal activity. Ultimate resolution of sensing ion channel activity can be achieved with neural interfaces, and it was shown that macroscale electrodes can record extracellular current of individual ion channels in model systems, by charging the quantum capacitance of large graphene monolayer (mm2). Here, we show the field effect detection of ion channel activity within neuron networks, cultured during several weeks above graphene transistor arrays. Dependences upon drugs, reference potential gating and device geometry confirm the field effect detection of individual ion channel and suggest a significant contribution of grain boundaries, which provide highly sensitive nanoscale-sized sensing sites. Our theoretical analysis and simulations demonstrate that the ion gating of a single grain boundary in liquid affects the electronic transmission of the whole transistor channel, resulting in significant conductance variations. Monitoring the ion channels activity is of great interest as most of neurodegenerative diseases relied on channelopathies, which rely on ion channel abnormal activity. Thus, such highly sensitive and biocompatible neuro-electronics which open the way to FET detection at the sub-cell precision should be useful for a wide range of fundamental and applied research areas, including brain-on-chip, pharmacology, and in vivo monitoring or diagnosis., The authors gratefully acknowledge financial support from the University Joseph Fourrier (SMINGUE project), from la Région Rhône-Alpes (COOPERA project), and from the french Agence Nationale de la Recherche under the projects ANR-10-LABX-51-01 (Labex LANEF du Programme d'Investissements d'Avenir) the Lab Alliances on Nanosciences—Energies for the Future. ICN2 is supported by the CERCA programme/Generalitat de Catalunya. And the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (Project No. FIS2015-67767-P MINECO/FEDER), MINECO (Grant No. SEV-2013-0295) and funded by the CERCA Programme/Generalitat de Catalunya. The authors acknowledge the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (Project No. FIS2015-67767-P MINECO/FEDER).

We thank S.Q. Li and colleagues for their interest in our work [1] and comments allowing us to extend the knowledge gain from our data. Our experiments in cultured human endothelial cells have shown that intermittent hypoxia (IH) directly lead to increased endothelial VE-cadherin cleavage, increased sVE release and endothelial permeability, without involvement of systemic inflammatory processes. Endothelial permeability is a key event in early vascular alterations leading to remodeling and atherosclerosis and it is of crucial importance to decipher this initial process. Although endothelial adhesion markers were already studied in OSA patients [2, 3], this is the first time to our knowledge that VE-cadherin regulation and its consequences in terms of endothelial permeability was addressed in OSA. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Olfa Harki has nothing to disclose. Conflict of interest: Gilles Faury has nothing to disclose. Conflict of interest: Isabelle Vilgrain has nothing to disclose. Conflict of interest: Jean-Louis Pepin has nothing to disclose. Conflict of interest: Anne Briancon-Marjollet has nothing to disclose.

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.

In the arteries of vertebrates, evolution has given rise to resilient macromolecular structures, elastin and elastic fibers, capable of sustaining an elevated blood pressure and smoothening the discontinuous blood flow and pressure generated by the heart. Elastic fibers are produced only during development and childhood, before being progressively degraded by mechanical stress and enzymatic activities during adulthood and aging. During this period, arterial elastic fiber calcification and loading of lipids also occur, all of these events conducting to arteriosclerosis. This leads to a progressive dysfunction of the large elastic arteries inducing elevated blood pressure as well as altered hemodynamics and organ perfusion, which induce more global malfunctions of the body during normal aging. Additionally, some arterial conditions occur more frequently with advancing age, such as atherosclerosis or aneurysms, which are called age-related diseases or pathological aging. The physiological or pathological degradation of elastic fibers and function of elastic arteries seemed to be rather inevitable over time. However, during the recent years, different molecules - including several ATP-dependent potassium channel openers, such as minoxidil - have been shown to re-induce elastin production and elastic fiber assembly, leading to improvements in the arterial structure and function or in organ perfusion. This review summarizes the changes in the arterial elastic fibers and structure from development until aging, and presents some of the potential pharmacotherapies leading to elastic fiber neosynthesis and arterial function improvement.

Electronic micro and nano-devices are suitable tools to monitor the activity of many individual neurons over mesoscale networks. However the inorganic materials currently used in microelectronics are barely accepted by neural cells and tissues, thus limiting both the sensor lifetime and efficiency. In particular, penetrating intracortical probes face high failure rate because of a wide immune response of cells and tissues. This adverse reaction called gliosis leads to the rejection of the implanted probe after few weeks and prevent long-lasting recordings of cortical neurons. Such acceptance issue impedes the realization of many neuro-rehabilitation projects. To overcome this, graphene and related carbon-based materials have attracted a lot of interest regarding their positive impact on the adhesion and regeneration of neurons, and their ability to provide high-sensitive electronic devices, such as graphene field effect transistor (G-FET). Such devices can also be implemented on numerous suitable substrates including soft substrates to match the mechanical compliance of cells and tissues, improving further the biocompatibility of the implants. Thus, using graphene as a coating and sensing device material could significantly enhance the acceptance of intracortical probes. However, such a thin monolayer of carbon atoms could be teared off during manipulation and insertion within the brain, and could also display degradation over time. In this work, we have investigated the ability to protect graphene with a natural, biocompatible and degradable polymeric film derivated from hyaluronic acid (HA). We demonstrate that HA-based coatings can be deposited over a wide range of substrates, including intracortical probes and graphene FET arrays without altering the underlying device material, its biocompatibility and sensitivity. Moreover, we show that this coating can be monitored

session 2Db; International audience

Obstructive Sleep Apnea (OSA) 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 OSA-related cardiometabolic alterations. This study aimed at assessing 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 metabolic and inflammatory parameters were assessed such as plasma lipids and glucose, serum leptin and adiponectin, liver cytokines, NF-κB activity and cardiac endothelin-1 levels. Myocardial infarct size was also evaluated following in vitro ischemia-reperfusion. Circulating lipids, insulin, 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 IL-6 and TNF-α levels were elevated in lean rats exposed to IH; obesity prevented the increase in IL-6 but not in TNF- 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 dyslipidemia and insulin resistance induced by obesity of genetic origin does not enhance the deleterious cardiovascular response to IH and may even partially protect against IH-induced inflammation. This article is protected by copyright. All rights reserved

Obstructive sleep apnea (OSA) is a major risk factor for cardiovascular mortality, and apnea-induced intermittent hypoxia (IH) is known to promote various cardiovascular alterations such as vascular remodeling. However, the mechanisms that underlie IH remain incompletely investigated. We previously demonstrated that the hypoxia-inducible factor-1 (HIF-1) and endothelin-1 (ET-1) are involved in arterial hypertension and myocardial susceptibility to infarction induced by IH. Thus the objective of the present study was to investigate whether both ET-1 and HIF-1 were also involved in the vascular inflammatory remodeling induced by IH. Mice partially deficient for the Hif1α gene (HIF-1α+/−) and their wild-type equivalents, as well as C57BL/6J mice, treated or not with bosentan, a dual endothelin receptor antagonist, were exposed to IH or normoxia for 2 wk, 8 h/day. Splenocyte proliferative and secretory capacities, aortic nuclear factor-κB (NF-κB) and HIF-1 activities, and expression of cytokines and intima-media thickness (IMT) were measured. IH induced a systemic and aortic inflammation characterized by an increase in splenocyte proliferative and secretory capacities, aortic NF-κB activity, and cytokine expression in the aortic wall. This was accompanied by an increase in IMT. These modifications were prevented in HIF-1α+/− and bosentan-treated mice. The results of this study suggest that ET-1 is a major contributor to the vascular inflammatory remodeling induced by OSA-related IH, probably through HIF-1-dependent activation of NF-κB.

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.

Background Obstructive sleep apnea is characterized by repetitive pharyngeal collapses during sleep, leading to intermittent hypoxia ( IH ), the main contributor of obstructive sleep apnea–related cardiovascular morbidity. In patients and rodents with obstructive sleep apnea exposed to IH, vascular inflammation and remodeling, endothelial dysfunction, and circulating inflammatory markers are linked with IH severity. The nonmuscle myosin light chain kinase (nm MLCK ) isoform contributes to vascular inflammation and oxidative stress in different cardiovascular and inflammatory diseases. Thus, in the present study, we hypothesized that nm MLCK plays a key role in the IH ‐induced vascular dysfunctions and inflammatory remodeling. Methods and Results Twelve‐week‐old nm MLCK +/+ or nm MLCK −/− mice were exposed to 14‐day IH or normoxia. IH was associated with functional alterations characterized by an elevation of arterial blood pressure and stiffness and perturbations of NO signaling. IH caused endothelial barrier dysfunction (ie, reduced transendothelial resistance in vitro) and induced vascular oxidative stress associated with an inflammatory remodeling, characterized by an increased intima‐media thickness and an increased expression and activity of inflammatory markers, such as interferon‐γ and nuclear factor‐κB, in the vascular wall. Interestingly, nm MLCK deletion prevented all IH ‐induced functional and structural alterations, including the restoration of NO signaling, correction of endothelial barrier integrity, and reduction of both oxidative stress and associated inflammatory response. Conclusions nm MLCK is a key mechanism in IH ‐induced vascular oxidative stress and inflammation and both functional and structural remodeling.

Background Obstructive Sleep Apnea syndrome (OSAS), characterized by intermittent hypoxia (IH) cycles during sleep, induces endothelial dysfunction and atherogenesis lesions, which could be mediated by an increase in endothelial permeability. VE-Cadherin (VECad) cleavage, detected by the soluble extracellular fragment released (sVE), may be a key step in this regulation of endothelial permeability. Objective Our aim was to search for sVE in sera from healthy volunteers submitted to IH, and from OSAS patients before and after treatment by continuous positive airway pressure (CPAP) and to characterize mechanisms regulating VECad cleavage in endothelial cells submitted to IH. Methods sVE was searched in sera from 7 healthy volunteers exposed to IH, 44 OSAS patients and 31 control subjects. Human Aortic Endothelial Cells (HAEC) were exposed to 6 hours of IH in vitro, then endothelial permeability was assessed by measuring trans-endothelial electrical resistance (TEER) and VECad cleavage was evaluated by detecting sVE in cells supernatants after treatment by IH and tyrosine kinase inhibitor (30 μM Genistein or 10 μM PP2), HIF-1 inhibitor (1 μM 2-Methoxyestradiol), an anti-oxidant (100 μM Tempol) or anti-hVEGF blocking antibody (0.5 μg/mL). Results sVE was significantly elevated in healthy volunteers submitted to IH and in OSAS patient sera before treatment, but decreased in OSAS patients after 6 months of CPAP therapy. We found a significant positive correlation between sVE and OSAS severity and between sVE and serum VEGF. In HAEC supernatants, TEER decreased by 37.5% and sVE increased by 39% after cell exposure to IH. These effects were reversed by all the pharmacological inhibitors tested. Conclusion we suggest that, in OSAS, IH increases endothelial permeability by inducing VECad cleavage via the ROS, HIF1, VEGF and tyrosine kinase pathways. Future studies will determine whether sVE could be a potential biomarker to evaluate early endothelial alterations in OSAS.

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% O

Introduction Associated cardiovascular morbidity in Obstructive Sleep Apnea Syndrome is mainly caused by chronic intermittent hypoxia (CIH) stimulus originating sympathetic hyperactivity. In rodent exposed to CIH, the adipose tissue is the host of local inflammation and metabolism alterations. Our objective was to assess the impact of increased sympathetic activity induced by 14 nights of CIH exposure on Glucose control and lipid profile and subcutaneous fat tissue in non-obese healthy humans. Methods In this prospective, single-blinded cross-over study, 12 healthy subjects underwent 2 phases of exposure of 14 nights randomized to CIH vs. air. Sympathetic activity was measured by peroneal microneurography. Fasting glucose, insulin, c-peptide and FFA were assessed at rest and during a multisampling oral glucose tolerance test. Subcutaneous adipose tissue biopsies were obtained after each phase of exposure. We assessed morphological and functional changes of the adipose tissue using immuno-histology and RT-PCR. Results After two weeks exposures of CIH versus room air, sympathetic activity significantly increased (p 3 μm 2 ; p=0.03), increased lipolysis (Hormon Sensitive Lipase expression x 2.95; p=0.02) and increased lipogenesis (Fatty Acid Synthase expression x 3.62; p=0.04). No difference was found in markers of inflammation in the subcutaneous adipose tissue. Conclusion In this experimental setting of high sympathetic tone, CIH induced an increase in lipolysis that may be partly responsible for associated cardio metabolic consequences.

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.

International audience; Due to its outstanding mechanical and electrical properties as well as chemical inertness, graphene has attracted a growing interest in the field of bioelectric interfacing. Herein, we investigate the suitability of pristine, i.e. without a cell adhesive coating, chemical vapor deposition (CVD) grown monolayer graphene to act as a platform for neuronal growth. We study the development of primary hippocampal neurons grown on bare graphene (transferred on glass coverslip) for up to 5 days and show that pristine graphene significantly improves the neurons adhesion and outgrowth at the early stage of culture (1-2 days in vitro). At the later development stage, neurons grown on coating free graphene (untreated with poly-L-lysine) show remarkably well developed neuritic architecture similar to those cultured on conventional poly-L-lysine coated glass coverslips. This exceptional possibility to bypass the adhesive coating allows a direct electrical contact of graphene to the cells and reveals its great potential for chronic medical implants and tissue engineering. Moreover, regarding the controversial results obtained on the neuronal affinity of pristine graphene and its ability to support neuronal growth without the need of polymer or protein coating, we found that the crystallinity of CVD grown graphene play an important role in neuronal attachment, outgrowth and axonal growth. In particular, we show that the decreasing crystalline quality of graphene tunes the neuronal affinity from highly adhesive to fully repellent.

Obstructive sleep apnea syndrome (OSA) is characterized by repetitive upper airway collapses during sleep inducing chronic intermittent hypoxia (IH). IH promotes adipose tissue remodelling and dyslipidaemia. Endothelin-1 (ET-1) production is upregulated by IH and may participate to changes in adipose tissue biology. Wistar rats were submitted to 14 days of IH (60-sec cycles alternating 21 and 5% FiO 2 , 8h/day) or control normoxia and treated or not with bosentan, a dual type A (ETA-R) and B (ETB-R) endothelin receptor antagonist. IH induced a lipolytic remodelling and a 2-fold increase in ET-1 transcription and ETA-R expression in adipose tissue that were reversed by bosentan. In 3T3-L1 adipocytes, ET-1 induced an upregulation of its own and of ETA-R transcription. Moreover, ET-1 promoted a glycerol release inhibited by bosentan and BQ123 (ETA-R antagonist) but not by BQ788 (ETB-R antagonist). Finally, bosentan reversed the ET-1-induced phosphorylation of Hormone-Sensitive Lipase (HSL) on Serine 660. ET-1 is thus involved in IH-induced lipolysis in Wistar rats, and upregulation of ET-1 production and ETA-R expression by ET-1 itself under IH could amplify its effects. ET-1-induced lipolysis appears to be mediated through ETA receptors and the activation of HSL by Serine 660 phosphorylation.

International audience; Nous décrivons et analysons dans cette communication le fonctionnement d’un groupe d’enseignants et d’enseignants-chercheurs qui contribue au développement professionnel de chacun de ses membres d’une part, et qui devient une source durable d’innovations pédagogiques dans notre établissement d’autre part.

Modern lifestyle has profoundly modified human sleep habits. Sleep duration has shortened over recent decades from 8 to 6.5 hours resulting in chronic sleep deprivation. Additionally, irregular sleep, shift work and travelling across time zones lead to disruption of circadian rhythms and asynchrony between the master hypothalamic clock and pacemakers in peripheral tissues. Furthermore, obstructive sleep apnea syndrome (OSA), which affects 4 - 15% of the population, is not only characterized by impaired sleep architecture but also by repetitive hemoglobin desaturations during sleep. Epidemiological studies have identified impaired sleep as an independent risk factor for all cause of-, as well as for cardiovascular, mortality/morbidity. More recently, sleep abnormalities were causally linked to impairments in glucose homeostasis, metabolic syndrome and Type 2 Diabetes Mellitus (T2DM). This review summarized current knowledge on the metabolic alterations associated with the most prevalent sleep disturbances, i.e. short sleep duration, shift work and OSA. We have focused on various endocrine and molecular mechanisms underlying the associations between inadequate sleep quality, quantity and timing with impaired glucose tolerance, insulin resistance and pancreatic β-cell dysfunction. Of these mechanisms, the role of the hypothalamic-pituitary-adrenal axis, circadian pacemakers in peripheral tissues, adipose tissue metabolism, sympathetic nervous system activation, oxidative stress and whole-body inflammation are discussed. Additionally, the impact of intermittent hypoxia and sleep fragmentation (key components of OSA) on intracellular signaling and metabolism in muscle, liver, fat and pancreas are also examined. In summary, this review provides endocrine and molecular explanations for the associations between common sleep disturbances and the pathogenesis of T2DM.

Study objectives Although obstructive sleep apnea (OSA) causes cardiovascular morbidities through atherosclerosis induced by inflammation and endothelial dysfunction, OSA patients exhibit elevated plasma vascular endothelial growth factor (VEGF), which may represent an adaptive response to intermittent hypoxia. The aims of this study were to investigate whether in vitro endothelial wound healing and monocyte migration are affected by patient serum, and to determine the implication of circulating factors (VEGF and C-reactive protein). Patients Serum was collected from healthy controls (HC), "healthy" OSA, and metabolic syndrome (MS) patients with or without OSA. Measurements and results Along with the presence of OSA and/or MS, both VEGF and hsCRP were significantly elevated in patient serum. Their specific role was tested with blocking antibodies on primary endothelial cells for wound healing assay and on human monocytes for migration assay. Endothelial wound healing was reduced with OSA compared to HC serum, and even more significantly using MS+OSA patient serum. Altered wound healing with OSA serum was unmasked when blocking VEGF and restored when blocking CRP. Monocyte migration was activated with OSA serum, and further enhanced by MS+OSA patient serum. Blocking CRP in serum inhibited this migration. Conclusions Serum from OSA patient alters in vitro endothelial cell repair function and activates monocyte migration; this is further aggravated with the presence of metabolic syndrome. These effects are partly driven by VEGF and CRP, suggesting an unfavorable balance between the pro healing (VEGF) and pro injury (CRP) factors that may promote vascular injury in OSA with and without metabolic syndrome.

International audience; The transcription factor Elk-1 is a nuclear target of mitogen-activated protein kinases and regulates immediate early gene activation by extracellular signals. We show that Elk-1 is also conjugated to SUMO on either lysines 230, 249, or 254. Mutation of all three sites is necessary to fully block SUMOylation in vitro and in vivo. This Elk-1 mutant, Elk-1(3R), shuttles more rapidly to nuclei of Balb/C cells fused to transfected HeLa cells. Coexpression of SUMO-1 or -2 strongly reduces shuttling by Elk-1 without affecting that of Elk-1(3R), indicating that SUMOylation regulates nuclear retention of Elk-1. Accordingly, overexpression of Elk-1(3R) in PC12 cells, where cytoplasmic relocalization of Elk-1 has been linked to differentiation, enhances neurite extension relative to Elk-1. The effect of Elk-1, but not of the 3R mutant, was blocked upon cotransfection with SUMO-1 or -2 and enhanced by coexpression with mutant Ubc-9. Thus, SUMO conjugation is a novel regulator of Elk-1 function through the control of its nuclear-cytoplasmic shuttling.

Guanine nucleotide exchange factors for Rho-GTPases (Rho-GEFs) invariably share a catalytic Dbl-Homology (DH) domain associated with a Pleckstrin Homology (PH) domain, whose function in Rho-GEF activation is not well understood. Trio is the first member of an emerging family of Dbl proteins containing two Rho-GEF domains (GEFD1 and GEFD2). TrioGEFD1 activates the GTPases RhoG and Rac1, while TrioGEFD2 acts on RhoA. In this study, we have investigated the roles of the two PH domains of Trio in Rho-GEF activity. We show that TrioPH1 is required for GEFD1-mediated induction of actin cytoskeleton remodeling and JNK activation. TrioPH1 is involved both in the catalytic activity and in the subcellular localization of its associated DH domain, by acting as a cytoskeletal targeting signal. Moreover, TrioPH1 in association with DH2 activates the JNK pathway, by an unknown mechanism independent of DH2 catalytic activity. TrioPH2 does not behave as a targeting module in intact cells. TrioPH2 inhibits DH2-dependent stress fiber formation, which correlates with the TrioPH2-mediated inhibition of DH2 GEF activity. In addition, expression in the neuron-like PC12 cell line of the intact Trio protein deleted of each PH domain shows that only TrioPH1 is required for Trio-induced neurite outgrowth. Taken together, these data demonstrate that the two PH domains play a different role in the control of Trio Rho-GEF function.

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.

International audience; 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.

The chemotropic guidance cue netrin-1 promotes neurite outgrowth through its receptor Deleted in Colorectal Cancer (DCC) via activation of Rac1. The guanine nucleotide exchange factor (GEF) linking netrin-1/DCC to Rac1 activation has not yet been identified. Here, we show that the RhoGEF Trio mediates Rac1 activation in netrin-1 signaling. We found that Trio interacts with the netrin-1 receptor DCC in mouse embryonic brains and that netrin-1-induced Rac1 activation in brain is impaired in the absence of Trio. Trio(-/-) cortical neurons fail to extend neurites in response to netrin-1, while they are able to respond to glutamate. Accordingly, netrin-1-induced commissural axon outgrowth is reduced in Trio(-/-) spinal cord explants, and the guidance of commissural axons toward the floor plate is affected by the absence of Trio. The anterior commissure is absent in Trio-null embryos, and netrin-1/DCC-dependent axonal projections that form the internal capsule and the corpus callosum are defective in the mutants. Taken together, these findings establish Trio as a GEF that mediates netrin-1 signaling in axon outgrowth and guidance through its ability to activate Rac1.

Thèse proposée pour un prix de thèse.; Multiple sclerosis (MS) is a chronic demyelinating and inflammatory disease of the central nervous system. MS affects about 80 000 people in France and represents the first cause of non-traumatic disability among young adults (20-40 years old). The major disease’s characteristic is the progressive loss of myelin sheath, which is destroyed by the immune system itself, provoking a diminution of the nerve impulse conduction. The current medical therapies are only effective to target inflammatory components. A spontaneous reparation of the damaged myelin sheath in MS is well known, but unfortunately still partial. The main source of remyelination is oligodendrocyte precursor cells (OPCs). Stem cell transplantation is studied to achieve an exogenous remyelination of the lesions without OPCs. Furthermore, antibodies (anti-Lingo and anti-semaphorin 4D) are currently in clinical trials in order to promote an endogen remyelination of lesions with persisting OPCs. Neuronal activity can modulate myelination and cause calcium increases in OPCs. The intracellular calcium of OPCs is known as a secondary messenger capable of regulating the transcription of genes, especially those involved in proliferation, differentiation of OPCs into OLs and in myelination. The main goal of the experimental work carried out in this thesis is to understand whether calcium signals depend on neuronal activity during remyelination with a murin model. The development of remyelination knowledge will result in the discovery of new therapeutic targets and might promote remyelinating and neuroprotective therapeutic strategies in MS.; La sclérose en plaques (SEP) est une maladie démyélinisante inflammatoire chronique du système nerveux central. En France, la SEP touche environ 80 000 personnes et représente la première cause de handicap sévère non traumatique chez les trentenaires. La maladie se caractérise par une perte progressive de la gaine de myéline détruite par le système immunitaire qui provoque la diminution de l’influx nerveux. Les thérapies actuelles ciblent uniquement des composantes inflammatoires. Au niveau de certaines lésions démyélinisées, une réparation endogène de la gaine de myéline est possible mais reste partielle. Les cellules précurseurs d’oligodendrocytes (OPCs) sont la principale source de remyélinisation. La transplantation de cellules souches est à l’étude pour réaliser une remyélinisation exogène des lésions absentes d’OPCs. Des anticorps anti-Lingo et anti-sémaphorine 4D sont notamment en cours d’essais cliniques pour favoriser la remyélinisation endogène des lésions où les OPCs persisteraient. L’activité neuronale module la myélinisation et provoque des fluctuations calciques au niveau des OPCs. Le calcium régule la transcription de gènes impliqués dans la prolifération, différenciation des OPCs en oligodendrocytes et dans la myélinisation. La partie expérimentale de cette thèse étudie les mécanismes responsables des signaux calciques des OPCs au cours de la remyélinisation et tente de comprendre s'ils dépendent de l’activité neuronale chez un modèle murin. Un progrès des connaissances sur la remyélinisation permettra peut-être la découverte de nouvelles cibles thérapeutiques pour réparer la gaine de myéline endommagée et éviter la neurodégénérescence.

Les molécules tératogènes nécessitant la présentation d’un accord de soins et de contraception lors de leur dispensation en officine sont les rétinoïdes, l’acide valproïque et ses sels, le mycophénolate ainsi que le vismodegib. Après un rappel de quelques généralités sur le risque tératogène, cette thèse présente les données pré-cliniques et les malformations recensées chez les enfants exposés pour chacune de ces molécules. Il est également proposé une conduite à tenir en cas d'exposition pendant la grossesse et les alternatives thérapeutiques possibles en cas de découverte ou de désir de grossesse pour éviter un arrêt ou un changement brutal de traitement qui pourrait avoir de graves conséquences pour la mère et l'enfant à naître. Enfin, l'ensemble des mesures de minimisation du risque tératogène mises en place par les autorités sanitaires et notamment les conditions de prescription et de délivrance sont abordées et leur impact évalué par le nombre de grossesses encore exposées. En pratique, ces mesures ne sont pas toujours bien connues et respectées comme le montrent les résultats du questionnaire distribué en officine. Cette thèse s’achève donc par l’élaboration et la diffusion d’un dépliant destiné aux équipes officinales reprenant des notions de base sur le risque tératogène mais surtout toutes les informations nécessaires pour sécuriser la dispensation de ces molécules au comptoir. Ce dépliant a été réalisé dans le but d'aider les officinaux à participer activement à la lutte contre l’exposition d’une grossesse à une molécule tératogène.