IH-induced Increase Research Articles

L-Citrulline Supplementation Reduces Blood Pressure and Myocardial Infarct Size under Chronic Intermittent Hypoxia, a Major Feature of Sleep Apnea Syndrome.

Intermittent hypoxia (IH) is a landmark of obstructive sleep apnea (OSA) at the core of the cardiovascular consequences of OSA. IH triggers oxidative stress, a major underlying mechanism for elevated blood pressure (BP) and increased infarct size. L-citrulline is an amino acid that has been demonstrated to be protective of the cardiovascular system and exert pleiotropic effects. Therefore, we tested the impact of citrulline supplementation on IH-induced increase in BP and infarct size. Four groups of rats exposed to normoxia (N) or IH [14 days (d), 8 h/day, 30 s-O2 21%/30 s-O2 5%] and were supplemented or not with citrulline (1 g·kg-1·d-1). After 14 d, BP was measured, and hearts were submitted to global ischemia-reperfusion to measure infarct size. Histological and biochemical analyses were conducted on hearts and aorta to assess oxidative stress. Citrulline significantly reduced BP (-9.92%) and infarct size (-18.22%) under IH only. In the aorta, citrulline supplementation significantly decreased superoxide anion and nitrotyrosine levels under IH and abolished the IH-induced decrease in nitrite. Citrulline supplementation significantly decreased myocardial superoxide anion levels and xanthine oxidase enzyme activity under IH. Citrulline shows a cardioprotective capacity by limiting IH-induced pro-oxidant activity. Our results suggest that citrulline might represent a new pharmacological strategy in OSA patients with high cardiovascular risk.

L-citrulline supplementation reduces systolic arterial pressure and myocardial infarct size in a rat model of sleep apnea syndrome

Sleep apnea syndrome is characterized by intermittent hypoxia (IH), leading to an increase in blood pressure (BP) and infarct size. Independently, IH has been demonstrated to alter arginine (Arg) metabolism. As Arg availability directly impacts nitric oxide production, we speculate that plasmatic Arg deficiency during IH may contribute to cardiovascular alterations induced by IH. Knowing that citrulline (Cit) is a better precursor of Arg than Arg itself, we propose to assess the impact of chronic Cit supplementation on IH-induced an increase in BP and infarct size. Four groups of rats ( n = 11–14), were submitted to normoxia (N) or IH [14 days (d), 8 h per d, 30 s-O2 21%/30 s-O2 5%] and supplemented or not with Cit (1 g.kg −1 .d −1 ). After 14 d, BP was measured by carotid catheterization; hearts were perfused by the Langendorff method and submitted to global and total ischemia (30 min)-reperfusion (120 min) before measuring infarct size related to left ventricle area (I/V) by colorimetry and planimetry. Another set of rats was added ( n = 5–6) in order to collect the heart and aorta for histological and biochemical analyses. IH increases systolic BP (144.5 ± 3.5 vs. 165.1 ± 4.2 mmHg in N and IH respectively, n = 11–14, P < 0.01) and I/V (33.7 ± 4.4 vs. 46 ± 2.5% in N and IH, respectively, n = 10–12, P < 0.05). Cit abolishes IH-induced increase in both systolic BP (139.8 ± 4. vs. 155.6 ± 6.6 mmHg NCit and IHCit, respectively) and I/V (35.7 ± 3.3 vs. 38.2 ± 2.1% in NCit and IHCit). Preliminary western blot results show that IH decreases T495P-eNOS in heart whereas it did not affect S1177P-eNOS. Interestingly, Cit supplementation seems to decrease even more the T495P-eNOS in the IHCit group compared to IH and NCit groups. Cit supplementation abolishes the deleterious effects of IH in terms of BP and infarct size. This suggests that citrulline could be a complementary or alternative treatment to the current sleep apnea standard treatment that is not fully efficient. However, biochemical and histological studies (NOx, oxidative stress, S-nitrosylation…) are ongoing to enlighten the specific mechanisms involved in cardiovascular protection induced by citrulline chronic supplementation during IH.

Intermittent Hypoxia-Induced Activation of Endothelial Cells Is Mediated via Sympathetic Activation-Dependent Catecholamine Release.

Obstructive sleep apnea (OSA) is a common breathing disorder affecting a significant percentage of the adult population. OSA is an independent risk factor for cardiovascular disease (CVD); however, the underlying mechanisms are not completely understood. Since the severity of hypoxia correlates with some of the cardiovascular effects, intermittent hypoxia (IH) is thought to be one of the mechanisms by which OSA may cause CVD. Here, we investigated the effect of IH on endothelial cell (EC) activation, characterized by the expression of inflammatory genes, that is known to play an important role in the pathogenesis of CVD. Exposure of C57BL/6 mice to IH led to aortic EC activation, while in vitro exposure of ECs to IH failed to do so, suggesting that IH does not induce EC activation directly, but indirectly. One of the consequences of IH is activation of the sympathetic nervous system and catecholamine release. We found that exposure of mice to IH caused elevation of circulating levels of catecholamines. Inhibition of the IH-induced increase in catecholamines by pharmacologic inhibition or by adrenalectomy or carotid body ablation prevented the IH-induced EC activation in mice. Supporting a key role for catecholamines, epinephrine alone was sufficient to cause EC activation in vivo and in vitro. Together, these results suggested that IH does not directly induce EC activation, but does so indirectly via release of catecholamines. These results suggest that targeting IH-induced sympathetic nerve activity and catecholamine release may be a potential therapeutic target to attenuate the CV effects of OSA.

L-Citrulline supplementation reduces myocardial infarct size in a rat model of sleep apnea syndrome

Sleep apnea syndrome is characterized by intermittent hypoxia (IH), leading to an increase in blood pressure (BP), infarct size (IS), endothelial dysfunction, insulin resistance, and dyslipidemia. Insulin resistance and the metabolic syndrome associated with IH are also accompanied by an alteration in the metabolism of Arginine (Arg) [and its precursor Citrulline (Cit)]. As Arg availability directly impacts nitric oxide (NO) production, we speculate that myocardial Arg deficiency during IH may contribute to cardiovascular alterations induced by IH. Knowing that Cit is a much better precursor of Arg than Arg itself, we propose to use Cit during IH to protect against IH-induced increase in BP and IS. Four groups of rats ( n = 8), were submitted to normoxia (N) or IH [14 days (d), 8 h per d, 30 s-O 2 21%/30 s-O 2 5%] and supplemented or not with Cit (1 g.kg −1 .d −1 ). One normoxic per-feed (NPf) supplementary group was added to assess the impact of anorexia associated with IH. BW and food intake were daily measured. After 14 d, BP was measured by carotid catheterization. Hearts were perfused by the Langendorff method and submitted to global and total ischemia (30 min)-reperfusion (120 min) before measuring IS related to left ventricle area (I/V) by colorimetry and planimetry. Irrespective of diet, IH increases both systolic BP (141.3 ± 1.1 vs. 170.6 ± 5.5 mmHg in N and IH respectively, n = 4–8, P = 0.05) and I/V (27.2 ± 4.7% vs. 43.6 ± 4.5% in N and IH, respectively, n = 5–8, P < 0.05). Even if Cit increases BP under IH (170.6 ± 5.5 vs. 176.4 ± 4.4 mmHg IH and IHCit, respectively), it reduces I/V in IH groups only (43.6 ± 4.5 vs. 38.7 ± 2.9 in IH and IHCit). Under IH, Cit seems to increase the BP but decrease I/V. After completing groups, further biochemical and histological analyses will be performed to enlighten the mechanisms (NOx, NO synthase, oxidative stress…) potentially involved in the impact of Cit chronic supplementation.

Metformin improves infarct size in mice exposed to chronic intermittent hypoxia, a major feature of obstructive sleep apnea

Chronic intermittent hypoxia (IH), the major feature of obstructive sleep apnea (OSA) increases myocardial infarct size. Recent data demonstrate that IH-induced cardiomyocyte susceptibility to infarction could be explained by mitochondrial function and/or dynamics alterations. Moreover, AMP-activated protein kinase (AMPK), a major protein involved in cardiomyocyte metabolism is activated by IH in striated skeletal muscle. However, we recently found that AMPK failed to be activated in myocardium from mice exposed to IH. We aimed at demonstrating whether AMPK activation can be beneficial for ischemia-reperfusion injury following chronic IH exposure. Mice were exposed to 21 days of IH (21-5% FiO 2 , 60 s-cycles, 8 h/day), or normoxia (N) and treated or not with metformin (300 mg.kg −1 .day −1 ). Infarct size was measured after in vivo ischemia (45 min) and reperfusion (90 min). Mitochondrial function was assessed using oxygraphy and spectrofluorimetry. Mitochondrial dynamics and AMPK activation were assessed by Western blot. An insulin tolerance test was also performed. Metformin abolished the IH-induced increase in infarct size whereas it did not improve systemic insulin sensitivity. Metformin activated AMPK and did not impact the decrease in mitochondrial respiration (O 2 maximal consumption in complex I and II and respiratory control ratio). However, AMPK activation by Metformin seems to reestablish the distinctive IH impact on mitochondrial dynamics (fission and mitophagy). Metformin treatment is protective against ischemia-reperfusion injury induced by IH only. This suggests that metformin could be an alternative treatment to the currently restrictive one for apneic patients with a high CV risk. Ongoing studies will aim to better characterize the mechanisms by which metformin specifically decreases infarct size under IH.

Mechanisms of intermittent hypoxia-mediated macrophage activation - potential therapeutic targets for obstructive sleep apnoea.

Intermittent hypoxia (IH) plays a key role in the pathogenesis of insulin resistance (IR) in obstructive sleep apnoea (OSA). IH induces a pro-inflammatory phenotype of the adipose tissue with M1 macrophage polarisation, subsequently impeding adipocyte insulin signalling, and these changes are in striking similarity to those seen in obesity. However, the detailed molecular mechanisms of IH-induced macrophage polarisation are unknown and identification of same should lead to the identification of novel therapeutic targets. In the present study, we tested the hypothesis that IH acts through similar mechanisms as obesity, activating Toll-like-receptor (TLR)4/nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) signalling pathways leading to the upregulation and secretion of the key cytokines interleukin (IL)-1β and IL-6. Bone-marrow derived macrophages (BMDMs) from lean and obese C57BL/6 male mice were exposed to a state-of-the-art in vitro model of IH. Independent of obesity, IH led to a pro-inflammatory M1 phenotype characterised by increased inducible nitric oxide synthase and IL-6 mRNA expression, robust increase in NF-κB DNA-binding activity and IL-6 secretion. Furthermore, IH significantly increased pro-IL-1β mRNA and protein expression and mature IL-1β secretion compared to control treatment. Providing mechanistic insight, pre-treatment with the TLR4 specific inhibitor, TAK-242, prevented IH-induced M1 polarisation and upregulation of IL-1β mRNA and pro-IL-1β protein expression. Moreover, IH-induced increase in IL-1β secretion was prevented in BMDMs isolated from NLRP3 knockout mice. Thus, targeting TLR4/NF-κB and NLRP3 signalling pathways may provide novel therapeutic options for metabolic complications in OSA.

Obesity attenuates the effect of sleep apnea on active TGF-\xdf1 levels and tumor aggressiveness in patients with melanoma

Active transforming growth factor-β1 (TGF-β1), a cytokine partially regulated by hypoxia and obesity, has been related with poor prognosis in several tumors. We determine whether obstructive sleep apnea (OSA) increases serum levels of active TGF-β1 in patients with cutaneous melanoma (CM), assess their relationship with melanoma aggressiveness and analyze the factors related to TGF-β1 levels in obese and non-obese OSA patients. In a multicenter observational study, 290 patients with CM were underwent sleep studies. TGF-β1 was increased in moderate-severe OSA patients vs. non-OSA or mild OSA patients with CM. In OSA patients, TGF-β1 levels correlated with mitotic index, Breslow index and melanoma growth rate, and were increased in presence of ulceration or higher Clark levels. In CM patients, OSA was associated with higher TGF-β1 levels and greater melanoma aggressiveness only in non-obese subjects. An in vitro model showed that IH-induced increases of TGF-β1 expression in melanoma cells is attenuated in the presence of high leptin levels. In conclusion, TGF-β1 levels are associated with melanoma aggressiveness in CM patients and increased in moderate-severe OSA. Moreover, in non-obese patients with OSA, TGF-β1 levels correlate with OSA severity and leptin levels, whereas only associate with leptin levels in obese OSA patients.

Intermittent Hypoxia Augments Pulmonary Vasoconstrictor Reactivity through PKCβ/Mitochondrial Oxidant Signaling.

Pulmonary vasoconstriction resulting from intermittent hypoxia (IH) contributes to pulmonary hypertension (pHTN) in patients with sleep apnea (SA), although the mechanisms involved remain poorly understood. Based on prior studies in patients with SA and animal models of SA, the objective of this study was to evaluate the role of PKCβ and mitochondrial reactive oxygen species (mitoROS) in mediating enhanced pulmonary vasoconstrictor reactivity after IH. We hypothesized that PKCβ mediates vasoconstriction through interaction with the scaffolding protein PICK1 (protein interacting with C kinase 1), activation of mitochondrial ATP-sensitive potassium channels (mitoKATP), and stimulated production of mitoROS. We further hypothesized that this signaling axis mediates enhanced vasoconstriction and pHTN after IH. Rats were exposed to IH or sham conditions (7 h/d, 4 wk). Chronic oral administration of the antioxidant Tempol or the PKCβ inhibitor LY-333531 abolished IH-induced increases in right ventricular systolic pressure and right ventricular hypertrophy. Furthermore, scavengers of O2- or mitoROS prevented enhanced PKCβ-dependent vasoconstrictor reactivity to endothelin-1 in pulmonary arteries from IH rats. In addition, this PKCβ/mitoROS signaling pathway could be stimulated by the PKC activator PMA in pulmonary arteries from control rats, and in both rat and human pulmonary arterial smooth muscle cells. These responses to PMA were attenuated by inhibition of mitoKATP or PICK1. Subcellular fractionation and proximity ligation assays further demonstrated that PKCβ acutely translocates to mitochondria upon stimulation and associates with PICK1. We conclude that a PKCβ/mitoROS signaling axis contributes to enhanced vasoconstriction and pHTN after IH. Furthermore, PKCβ mediates pulmonary vasoconstriction through interaction with PICK1, activation of mitoKATP, and subsequent mitoROS generation.

Acute Fluoxetine Administration Improves Intermittent Hypoxia‐induced Inspiratory Burst Amplitude Deficits During Peak LPS‐induced Neuroinflammatory Phase in Anesthetized Spontaneously Breathing Adult Male Rats

Studies examining the impact of neuroinflammation on various aspects of neural control of breathing typically use systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) and evaluate alterations in ventilatory control during either the induction (~2–4 hr) or resolution (~24 hr) phases of the acute neuroinflammatory response. Changes in LPS‐regulated pro‐inflammatory gene/protein expression, however, have been shown to peak at ~4–6 hrs following LPS exposure, but little is known about the impact of LPS‐induced neuroinflammation on ventilatory control at this time point. Systemic LPS administration has also been shown to increase serotonin (5‐HT) transporter activity and reduce 5‐HT levels. Since numerous inspiratory motor behaviors are 5‐HT‐dependent, including some hypoxic ventilatory behaviors, LPS‐induced changes in 5‐HT could contribute to ventilatory deficits noted in LPS‐induced neuroinflammation studies. To begin to address these issues, we examined the impact of peak phase LPS‐induced neuroinflammation on inspiratory motor behaviors elicited by intermittent hypoxia (IH) in urethane‐anesthetized vagal intact spontaneously breathing adult male Sprague‐Dawley rats in both the absence and presence of acute administration of the SSRI fluoxetine hydrochloride (FLX). Beginning at ~4 hr after systemic LPS (3 mg/kg, ip) or saline (CON) treatment, basal (BL) diaphragm EMG activity was recorded for ~30 min (40% O2), FLX (10 mg/kg) or vehicle (VEH, 0.9% saline) was injected (iv) and allowed 30 min to exert steady‐state effects, and then IH exposure composed of 5 alternating 3‐min episodes of hypoxia (11% O2; 2%CO2) and hyperoxia (40% O2) was delivered, after which the rat was allowed to recover. We found that during the first 30–60s following FLX injection in both CON‐ and LPS‐treated rats, burst amplitude (by ~12%) and frequency (by ~40%) were increased, after which amplitude returned to BL levels and frequency attained a new steady‐state ~10% above BL levels; VEH injection was ineffective in altering either burst amplitude or frequency. As expected during IH exposure in CON‐treated VEH‐injected rats, a robust increase in burst frequency and amplitude were observed with the subsequent hypoxic exposures eliciting progressive augmentation of burst amplitude (PA; IH1, ~45%; IH5, ~65%). In contrast in CON‐treated FLX‐injected rats, IH‐induced amplitude effects were markedly attenuated (IH1‐IH5, ~11–21%), and similar to those seen during IH exposure in LPS‐treated VEH‐injected rats (~7–11%). In LPS‐treated FLX‐injected rats, however, an increase in both burst amplitude and PA (IH1, ~19%; IH5, ~35%) were noted. Similar IH‐induced increases in burst frequency and post hypoxic frequency decline were also observed in both LPS‐ and CON‐treated FLX‐ and VEH‐injected rats. These preliminary observations suggest that acute FLX administration may partially correct the blunted increase in burst amplitude and induce PA in response to IH exposure during peak phase in LPS‐treated rats albeit similar FLX treatment appears to impair IH‐induced amplitude effects in CON‐treated rats.Support or Funding InformationNIH NS101737; SBU Thomas Hartman Center for Parkinson’s Disease Research

Differential vasomotor responses to isocapnic hyperoxia: cerebral versus peripheral circulation.

Isocapnic hyperoxia (IH) evokes cerebral and peripheral hypoperfusion via both disturbance of redox homeostasis and reduction in nitric oxide (NO) bioavailability. However, it is not clear whether the magnitude of the vasomotor responses depends on the vessel network exposed to IH. To test the hypothesis that the magnitude of IH-induced reduction in peripheral blood flow (BF) may differ from the hypoperfusion response observed in the cerebral vascular network under oxygen-enriched conditions, nine healthy men (25 ± 3 yr, mean ± SD) underwent 10 min of IH during either saline or vitamin C (3 g) infusion, separately. Femoral artery (FA), internal carotid artery (ICA), and vertebral artery (VA) BF (Doppler ultrasound), as well as arterial oxidant (8-isoprostane), antioxidant [ascorbic acid (AA)], and NO bioavailability (nitrite) markers were simultaneously measured. IH increased 8-isoprostane levels and reduced nitrite levels; these responses were followed by a reduction in both FA BF and ICA BF, whereas VA BF did not change. Absolute and relative reductions in FA BF were greater than IH-induced changes in ICA and VA perfusion. Vitamin C infusion increased arterial AA levels and abolished the IH-induced increase in 8-isoprostane levels and reduction in nitrite levels. Whereas ICA and VA BF did not change during the vitamin C-IH trial, FA perfusion increased and reached similar levels to those observed during normoxia with saline infusion. Therefore, the magnitude of IH-induced reduction in femoral blood flow is greater than that observed in the vessel network of the brain, which might involve the determinant contribution that NO has in the regulation of peripheral vascular perfusion.

1959-P: Upregulation of PYY, GLP-1, and NTS by Intermittent Hypoxia in Enteroendocrine Cells

The patients with sleep apnea syndrome (SAS) are exposed to intermittent hypoxia (IH) during sleep. Although accumulating evidence indicates a strong relationship between SAS and obesity, we previously demonstrated that IH up-regulates the mRNA levels of anorexigenic peptides POMC and CART via GATA transcription factors in human neuronal cells (Int. J. Biochem. Cell Biol. 95, 100, 2018). Appetite is regulated not only by the central nervous system but also by gastrointestinal peptides. Here, we investigated the effect of IH on the appetite-inhibiting gut hormone expression using in vitro IH system. Human and mouse enteroendocrine cells, Caco-2 and STC-1, were exposed to IH [64 cycles of 5 min hypoxia (1% O2) and 10 min normoxia (21% O2)] or normoxia for 24 hours. Real-time RT-PCR revealed that IH significantly increased the mRNA levels of PYY, GLP-1, and neurotensin (NTS) in both Caco-2 and STC-1. ELISA showed that the concentrations of PYY, GLP-1, and NTS in the culture medium were significantly increased by IH. To determine whether the IH-induced increases in PYY, GLP-1, and NTS in enteroendocrine cells were caused by transcriptional activation, reporter plasmids for PYY, GCG, which encodes a preprotein, part of which is cleaved into GLP-1, and NTS were transfected into Caco-2, and the luciferase activity was measured after the IH treatment. The promoter activities of PYY, GCG and NTS were not increased by IH. We then searched targeted miRNA and found that PYY, GLP-1 and NTS mRNAs have potential target sequences for miR-96, -527, and -2116. We measured the levels of the miRNAs and found that the levels of the miRNAs were not decreased by IH. Furthermore, the mRNA levels of PYY, GLP-1, and NTS were significantly up-regulated even in normoxia by trichostatin A and were significantly decreased even in IH by 5-azacytidine, suggesting that IH increases PYY, GLP-1, and NTS mRNAs via alterations in the chromatin structure in enteroendocrine cells. IH might have an anorexigenic influence on the enteric nervous system. Disclosure R. Shobatake: None. H. Ota: None. A. Itaya-Hironaka: None. A. Yamauchi: None. M. Makino: None. S. Sakuramoto-Tsuchida: None. T. Uchiyama: None. N. Takahashi: None. S. Ueno: None. K. Sugie: None. S. Takasawa: None.

Intermittent Hypoxia Mimicking Sleep Apnea Increases Passive Stiffness of Myocardial Extracellular Matrix. A Multiscale Study.

Background: Tissue hypoxia-reoxygenation characterizes obstructive sleep apnea (OSA), a very prevalent respiratory disease associated with increased cardiovascular morbidity and mortality. Experimental studies indicate that intermittent hypoxia (IH) mimicking OSA induces oxidative stress and inflammation in heart tissue at the cell and molecular levels. However, it remains unclear whether IH modifies the passive stiffness of the cardiac tissue extracellular matrix (ECM).Aim: To investigate multiscale changes of stiffness induced by chronic IH in the ECM of left ventricular (LV) myocardium in a murine model of OSA.Methods: Two-month and 18-month old mice (N = 10 each) were subjected to IH (20% O2 40 s–6% O2 20 s) for 6 weeks (6 h/day). Corresponding control groups for each age were kept under normoxia. Fresh LV myocardial strips (∼7 mm × 1 mm × 1 mm) were prepared, and their ECM was obtained by decellularization. Myocardium ECM macroscale mechanics were measured by performing uniaxial stress–strain tensile tests. Strip macroscale stiffness was assessed as the stress value (σ) measured at 0.2 strain and Young’s modulus (EM) computed at 0.2 strain by fitting Fung’s constitutive model to the stress–strain relationship. ECM stiffness was characterized at the microscale as the Young’s modulus (Em) measured in decellularized tissue slices (∼12 μm tick) by atomic force microscopy.Results: Intermittent hypoxia induced a ∼1.5-fold increase in σ (p < 0.001) and a ∼2.5-fold increase in EM (p < 0.001) of young mice as compared with normoxic controls. In contrast, no significant differences emerged in Em among IH-exposed and normoxic mice. Moreover, the mechanical effects of IH on myocardial ECM were similar in young and aged mice.Conclusion: The marked IH-induced increases in macroscale stiffness of LV myocardium ECM suggests that the ECM plays a role in the cardiac dysfunction induced by OSA. Furthermore, absence of any significant effects of IH on the microscale ECM stiffness suggests that the significant increases in macroscale stiffening are primarily mediated by 3D structural ECM remodeling.

Intermittent Hypoxia Induces Expression of Epiregulin mRNA via Upregulation of Interleukin-6 in Human Coronary Artery Smooth Muscle Cells

Sleep apnea syndrome (SAS) is characterized by repetitive episodes of oxygen desaturation and re-saturation, such as intermittent hypoxia (IH), and well known as an independent risk factor for diabetes and atherosclerosis. We previously showed that IH directly induced the proliferation of cultured rat aorta smooth muscle cells through upregulations of epidermal growth factor family, especially epiregulin (EREG), and their erbB2 receptor. However, its underlying mechanism remains unclear. On the other hand, we also reported that IH increased interleukin (IL)-6 mRNA in pancreatic beta cells. In this study, we investigated the influence of IH on IL-6 production, and the impact of IL-6 on the expression of EREG, using human coronary artery smooth muscle cells (hCASMC) and in vitro IH system. Quantitative RT-PCR revealed that EREG mRNA was significantly increased by IH, but not by sustained hypoxia. IH also increased expression of IL-6 mRNA in IH cycle dependent manner (3∼72 cycles in 1∼24 hours). Mature IL-6 increased in IH-exposed cell conditioned medium as similar time-dependent manner as seen in mRNA. We next investigated whether IL-6 increases the expression of EREG mRNA in hCASMCs. The addition of IL-6 (100 ng/mL) induced a significant upregulation of EREG mRNA expression. Taking into account the IH-induced increase of IL-6 and IL-6-induced upregulation of EREG mRNA in hCASMCs, we hypothesized that elevated IL-6 production due to IH was responsible for the IH-induced increase in EREG mRNA. To determine the direct role of IL-6 in IH-induced increases in EREG mRNA expression, we applied siRNAs for IL-6 and the IL-6 receptor and obtained the result that both siRNAs for IL-6 and IL-6 receptor significantly suppressed the IH-induced increase of EREG mRNA. These results indicated that IL-6 plays a pivotal role in EREG upregulation by IH and consequently OSA-related atherosclerosis. Disclosure Y. Kyotani: None. J. Zhao: None. A. Itaya-Hironaka: None. A. Yamauchi: None. S. Sakuramoto-Tsuchida: None. M. Makino: None. S. Takasawa: None. M. Yoshizumi: None.

Reduced arterial vasodilatation in response to hypoxia impairs cerebral and peripheral oxygen delivery in hypertensive men

Hypoxaemia evokes a repertoire of homeostatic adjustments that maintain oxygen supply to organs and tissues including the brain and skeletal muscles. Because hypertensive patients have impaired endothelial-dependent vasodilatation and an increased sympathetic response to arterial oxygen desaturation, we investigated whether hypertension impairs isocapnic hypoxia-induced cerebral and skeletal muscle hyperaemia to an extent that limits oxygen supply. In middle-aged hypertensive men, vertebral and femoral artery blood flow do not increase in response to isocapnic hypoxia, limiting brain and peripheral hyperaemia and oxygen supply. Increased chemoreflex-induced sympathetic activation impairs skeletal muscle perfusion and oxygen supply, whereas an attenuation of local vasodilatory signalling in the posterior cerebrovasculature reduced brain hyperperfusion of hypertensive middle-aged men in response to isocapnic hypoxia. The present study investigated whether hypertension impairs isocapnic hypoxia (IH)-induced cerebral and skeletal muscle hyperaemia to an extent that limits oxygen supply. Oxygen saturation (oxymetry), mean arterial pressure (photoplethysmography) and muscle sympathetic nerve activity (MSNA; microneugraphy), as well as femoral artery (FA), internal carotid artery and vertebral artery (VA) blood flow (BF; Doppler ultrasound), were quantified in nine normotensive (NT) (aged 40±11years, systolic pressure 119±7mmHg and diastolic pressure 73±6mmHg) and nine hypertensive men (HT) (aged 44±12years, systolic pressure 152±11mmHg and diastolic pressure 90±9mmHg) during 5min of normoxia (21% O2 ) and IH (10% O2 ). Total cerebral blood flow (tCBF), brain (CDO2 ) and leg (LDO2 ) oxygen delivery were estimated. IH provoked similar oxygen desaturation without changing mean arterial pressure. Internal carotid artery perfusion increased in both groups during IH. However, VA and FA BF only increased in NT. Thus, IH-induced increase in tCBF was smaller in HT. CDO2 only increased in NT and LDO2 decreased in HT. Furthermore, IH evoked a greater increase in HT MSNA. Changes in MSNA were inversely related to FA BF, LDO2 and end-tidal oxygen tension. In conclusion, hypertension disturbs regional and total cerebrovascular and peripheral responses to IH and consequently limits oxygen supply to the brain and skeletal muscle. Although increased chemoreflex-induced sympathetic activation may explain impaired peripheral perfusion, attenuated vasodilatory signalling in the posterior cerebrovasculature appears to be responsible for the small increase in tCBF when HT were exposed to IH.

Endurance training attenuates the increase in peripheral chemoreflex sensitivity with intermittent hypoxia.

Patients with heart failure and sleep apnea have greater chemoreflex sensitivity, presumably due to intermittent hypoxia (IH), and this is predictive of mortality. We hypothesized that endurance training would attenuate the effect of IH on peripheral chemoreflex sensitivity in healthy humans. Fifteen young healthy subjects (9 female, 26 ± 1 yr) participated. Between visits, 11 subjects underwent 8 wk of endurance training that included running four times/wk at 80% predicted maximum heart rate and interval training, and four control subjects did not change activity. Chemoreflex sensitivity (the slope of ventilation responses to serial oxygen desaturations), blood pressure, heart rate, and muscle sympathetic nerve activity (MSNA) were assessed before and after 30 min of IH. Endurance training decreased resting systolic blood pressure (119 ± 3 to 113 ± 3 mmHg; P = 0.027) and heart rate (67 ± 3 to 61 ± 2 beats/min; P = 0.004) but did not alter respiratory parameters at rest (P > 0.2). Endurance training attenuated the IH-induced increase in chemoreflex sensitivity (pretraining: Δ 0.045 ± 0.026 vs. posttraining: Δ -0.028 ± 0.040 l·min-1·% O2 desaturation-1; P = 0.045). Furthermore, IH increased mean blood pressure and MSNA burst rate before training (P < 0.05), but IH did not alter these measures after training (P > 0.2). All measurements were similar in the control subjects at both visits (P > 0.05). Endurance training attenuates chemoreflex sensitization to IH, which may partially explain the beneficial effects of exercise training in patients with cardiovascular disease.

Inflammatory Role of ROS-Sensitive AMP-Activated Protein Kinase in the Hypersensitivity of Lung Vagal C Fibers Induced by Intermittent Hypoxia in Rats.

Obstructive sleep apnea (OSA), manifested by airway exposure to intermittent hypoxia (IH), is associated with excess reactive oxygen species (ROS) production in airways, airway inflammation, and hyperreactive airway diseases. The cause-effect relationship for these events remains unclear. We investigated the inflammatory role of ROS-sensitive AMP-activated protein kinase (AMPK) in IH-induced airway hypersensitivity mediated by lung vagal C fibers (LVCFs) in rats. Conscious rats were exposed to room air (RA) or IH with or without treatment with N-acetyl-L-cysteine (NAC, an antioxidant), Compound C (an AMPK inhibitor), ibuprofen (a cyclooxygenase inhibitor), or their vehicles. Immediately after exposure (24 h), we found that intravenous capsaicin, phenylbiguanide, or α,β-methylene-ATP evoked augmented LVCF-mediated apneic responses and LVCF afferent responses in rats subjected to IH exposure in comparison with those in RA rats. The potentiating effect of IH on LVCF responses decreased at 6 h after and vanished at 12 h after the termination of IH exposure. The potentiating effect of IH on LVCF-mediated apneic and LVCF afferent responses was significantly attenuated by treatment with NAC, compound C, or ibuprofen, but not by their vehicles. Further biochemical analysis revealed that rats exposed to IH displayed increased lung levels of lipid peroxidation (an index of oxidative stress), AMPK phosphorylation (an index of AMPK activation), and prostaglandin E2 (a cyclooxygenase metabolite), compared with those exposed to RA. IH-induced increase in lipid peroxidation was considerably suppressed by treatment with NAC but not by compound C or ibuprofen. IH-induced increase in AMPK phosphorylation was totally abolished by NAC or compound C but not by ibuprofen. IH-induced increase in prostaglandin E2 was considerably prevented by any of these three inhibitor treatments. The vehicles of these inhibitors exerted no significant effect on the three IH-induced responses. These results suggest that 24-h IH exposure sensitizes LVCFs, leading to an exaggerated reflex and afferent responses to chemical stimulants in rats. Moreover, this IH-induced LVCF sensitization is mediated through a cascade of inflammatory responses in the airways involving increases in ROS, AMPK activation, and cyclooxygenase metabolite release.

Protective effect of resveratrol against chronic intermittent hypoxia-induced spatial memory deficits, hippocampal oxidative DNA damage and increased p47Phox NADPH oxidase expression in young rats

Long-term intermittent hypoxia (IH) is a characteristic hallmark of obstructive sleep apnea (OSA) and causes most of the neurological aspects of OSA, such as spatial memory and learning deficits. These deficits are accompanied by an increase in oxidative stress and inflammation in brain areas involved in cognition, such as the hippocampus, particularly in children. Resveratrol is a natural polyphenolic compound with potent antioxidant, anti-inflammatory and neuroprotective properties. AimThe aim of this work is to study the possible protective effect of resveratrol against IH-induced neurobehavioral deficits and to investigate the possible mechanism of this protective effect in the young rat model of OSA. MethodsThe effect of resveratrol (5 and 10mg/kg, orally) on anxiety, spatial memory and learning deficits in young rats exposed to IH for 6 weeks and the corresponding biochemical changes were studied. ResultsResveratrol attenuated IH-induced anxiety and spatial memory deficits, as indicated by the elevated plus maze and Morris water maze tests, respectively, in a dose-dependent manner. In addition, resveratrol antagonized IH-induced increases in hippocampal glutamate, TBARS and 8-OHdG levels and p47Phox expression and decreases in GSH levels and GSH-Px activity in the hippocampus of IH-exposed young rats. ConclusionResveratrol ameliorates IH-induced anxiety and spatial learning deficits through multiple beneficial effects on hippocampal oxidative pathways that involve decreased expression of the p47Phox subunit of NADPH oxidase. Hence, the potential therapeutic role of resveratrol in OSA may be utilized in the near future and deserves further exploration.

Endoplasmic reticulum stress as a novel inducer of hypoxia inducible factor-1 activity: its role in the susceptibility to myocardial ischemia‐reperfusion induced by chronic intermittent hypoxia

BackgroundObstructive sleep apnea (OSA) is a highly prevalent disease and a risk factor for myocardial infarction expansion in humans. Intermittent hypoxia (IH) is known to be the most important OSA feature in terms of cardiovascular morbi-mortality.Since ER stress and HIF-1 are known to be involved in cardiomyocyte life or death, this study investigates the role of ER stress on HIF-1 activation in myocardial susceptibility to ischemia‐reperfusion (I/R) induced by IH. MethodsC57Bl6J, HIF-1α+/‐ and their respective control mice were exposed to 14days of IH (21–5% FiO2, 60scycle, 8h/day). Myocardial inter-organelle calcium exchanges, ER stress and HIF-1 activity were investigated and in vivo I/R was performed to measure infarct size. In additional groups, tauroursodeoxycholic acid (TUDCA, 75mg·kg‐1), an ER stress inhibitor, was administered daily during exposure. ResultsIn C57Bl6J mice, chronic IH induced an increase in ER-Ca2+ content, ER stress markers and HIF-1 activity, associated with an enhanced infarct size (33.7±9.4 vs. 61.0±5.6% in N and IH, respectively, p<0.05). IH failed to increase infarct size in HIF-1α deficient mice (42.4±2.7 and 24.7±3.4% N and IH, respectively). Finally, TUDCA totally abolished the IH-induced increase in HIF-1 activity (1.3±0.04 vs. 0.14±0.02 fold increase in IH vs. IH-TUDCA respectively, p<0.0001) and in infarct size (55.5±7.6 vs. 49.9±3.0 in N-TUDCA and IH-TUDCA, respectively). ConclusionThis novel regulatory mechanism of HIF-1 activity by ER stress should be considered as a potential diagnostic tool for cardiovascular complications in OSA patients as well as a therapeutic target to limit myocardial ischemic damage.

Intermittent hypoxia-induced increases in reactive oxygen species activate NFATc3 increasing endothelin-1 vasoconstrictor reactivity

Sleep apnea (SA), defined as intermittent respiratory arrest during sleep, is associated with increased incidence of hypertension, peripheral vascular disease, stroke, and sudden cardiac death. We have shown that intermittent hypoxia with CO2 supplementation (IH), a model for SA, increases blood pressure and circulating ET-1 levels, upregulates lung pre-pro ET-1 mRNA, increases vasoconstrictor reactivity to ET-1 in rat small mesenteric arteries (MA) and increases vascular reactive oxygen species (ROS). NFAT activity is increased in the aorta (AO) and MA of mice exposed to IH in an ET-1-dependent manner, and the genetic ablation of the isoform NFATc3 prevents IH-induced hypertension. We hypothesized that IH causes an increase in arterial ROS generation, which activates NFATc3 to increase vasoconstrictor reactivity to ET-1. In support of our hypothesis, we found that IH increases ROS in AO and MA. In vivo administration of the SOD mimetic tempol during IH exposure prevents IH-induced increases in NFAT activity in mouse MA and AO. We found that IH causes an NFATc3-dependent increase in vasoconstrictor reactivity to ET-1, accompanied by an increase in vessel wall [Ca2+]. Our results indicate that IH exposure causes an increase in arterial ROS to activate NFATc3, which then increases vasoconstrictor reactivity and Ca2+ response to ET-1. These studies highlight a novel regulatory pathway, and demonstrate the potential clinical relevance of NFAT inhibition to prevent hypertension in SA patients.

Neuropeptide Y Signaling in Altered Catecholamine Synthesis during Intermittent Hypoxia

Intermittent hypoxia (IH) increases catecholamine (CA) levels in the adrenal medulla. In this study we examined whether the IH‐induced increase in CA is due to alterations in tyrosine hydroxylase (TH), the rate‐limiting enzyme in CA biosynthesis. Rats and mice were exposed to IH (15s of 5% O2; 5 min of 21% O2; 8 h per day for 10 days) or normoxia (21% O2) and adrenal medullae were harvested for analyses. IH increased TH activity and TH phosphorylation at ser‐40 (~ 3 fold), without altering TH protein levels. In addition to CA, adrenal medulla also expresses neuropeptide Y (NPY) which is known to modulate TH activity via activation of G‐protein coupled Y1 receptors (Y1R). IH markedly up regulated the expression of NPY levels, Y1R expression, cAMP levels and protein kinase A (PKA) activity. The contribution of NPY in IH‐induced TH activation was examined using 4‐phenyl‐3‐butenoic acid (PBA), an inhibitor of bioactive NPY synthesis. PBA treatment prevented IH‐induced increases in NPY levels, TH activity, TH phosphorylation (ser‐40) and CA levels. Furthermore IH‐induced increases in TH phosphorylation and activity and up regulation of PKA activity were absent in Y1R−/−but not in Y2R−/− mice. Taken together, these results suggest that NPY‐Y1R‐PKA signaling mediates IH‐induced increase in CA synthesis in the adrenal medulla (supported by grants from NIH HL‐89616 and HL‐90554).