Giant phagocytes (Gφ) and neutrophil-macrophage hybrids in human carotid atherosclerotic plaques - An activated phenotype.

Introduction: A small subpopulation of CD66b+ neutrophils with extended lifespan and immensely large size was identified in vitro. They internalized dead neutrophil remnants and cellular debris, transforming them into giant phagocytes (Gφ) resembling macrophage-foam cells with a massive lipid content and CD68+ scavenger receptor expression. Thus, we sought to investigate if similar CD66b+ neutrophils with altered morphology and functions exist in inflammatory/atherosclerotic conditions in vivo, by using human carotid atherosclerotic plaques. Methods: Thirty-three plaques were obtained from 31 patients undergoing endarterectomy. Carotid plaques were analyzed for CD markers by immunohistochemistry and immunofluorescence and quantitatively analyzed by confocal microscopy. Intra-plaque lipids were stained with Oil Red O. Results: Plaque CD66b+ neutrophils co-expressed myeloperoxidase (MPO)+ and neutrophil elastase (NE)+. Also, co-expression of CD66b+/CD68+, CD66b+/CD36+, CD66b+/vascular-endothelial-growth- factor (VEGF)+ and 3-nitrotyrosine (3-NT)+/NE+ was noted. Similarly, macrophages co-expressed CD163+/CD68+, CD163+/VEGF+ and CD163+/3-NT+. Both cell types were predominantly localized in lipid-rich areas and stained for lipids. CD66b+ and CD163+ expressions were highly positively correlated with each other and each with CD68+, and 3-NT+. Morphologically, CD66+ cells were big, had a rounded nucleus, and resembled macrophage-foam cell morphology as well as that of Gφ in vitro. To clarify whether CD66b+ and CD163+ cells represent two distinct plaque-populations, plaques were double-stained for CD66b/CD163 co-localization. A third of the plaques was negative for CD66b/CD163 co-localization. Other plaques had a low co-localization, but in few plaques, co-localization was high, collectively, indicating that in some of plaques there were two distinct cell populations, those resembling Gφ, and those co-expressing CD66b+/CD163+, demonstrating a hybrid neutrophil-macrophage phenotype. Interestingly, CD66b+/CD163+ co-localization was highly positively correlated with the oxidant 3-NT, hence, supporting trans-differentiation of CD66b+ cells to CD163+ Cells. Conversely, phagocytosis of dead neutrophils by macrophages might have also occurred. Discussion: Thus, we conclude that in some of the plaques CD66b+ cells might represent cells resembling Gφ that developed in prolonged culture conditions. Yet, CD66b+/CD163+ co-expressing cells represent a new neutrophil-macrophage hybrid population of unknown transitioning point, possibly by adopting macrophage markers or contrariwise. Nonetheless, the significance and functions of these cells in plaque biology or other inflammatory/atherosclerotic conditions should be unveiled.

Markers of Carotid Plaque Destabilization in Patients With Sleep-Disordered Breathing.

Sleep-disordered breathing (SDB) is a nightly respiratory condition characterized by intermittent hypoxia, leading to oxidative stress, inflammation, and atherosclerosis. However, most cellular markers of human carotid plaques in SDB have not yet been assessed. We aimed at characterizing the cellular, inflammatory, and nitro-oxidative stress markers in carotid plaques obtained from 25 patients undergoing endarterectomy and screened for SDB. Sleep studies were performed during their preoperative hospitalization night using the Watch-PAT 100 device. Oxygen desaturation index (ODI) was used for dividing patients into two groups. Fourteen patients with ODI >5 were designated as SDB and 11 patients with ODI ≤ 5 as non-SDB. Demographics, comorbidities, cardiovascular risk factors, and medications were recorded. Cellular markers in plaques were analyzed by immunofluorescence using confocal microscopy. The expression of neutrophils was identified by CD66b+ and neutrophil elastase, macrophage-foam cells were identified by CD163+, and scavenger receptors by CD68+ and CD36+ expression. Additional markers included 3-nitrotyrosine, endothelial CD31, and smooth muscle cell-actin (SMC-actin). Plaques' lipids were determined by immunohistochemistry with Oil Red O staining. Notably, significantly higher values were found for SDB as compared to patients with non-SDB for 3-nitrotyrosine (p <0.004) and intracellular lipids' content (p <0.02), whereas SMC-actin was lower (p <0.006). There were no significant differences between patients with carotid-associated symptoms (symptomatic) and patients without carotid-associated symptoms (asymptomatic). However, a sub-group of symptomatic patients with co-existent SDB expressed the highest 3-nitrotyrosin, and intracellular lipids levels, and the lowest SMC-actin levels, whereas non-SDB/asymptomatic patients expressed the lowest 3-nitrotyrosin and lipids levels and the highest SMS-actin levels among all patients. Accordingly, ODI was lowest in non-SDB/asymptomatic patients and highest in SDB/symptomatic. In conclusion, plaques of patients with SDB were characterized by markedly increased levels of 3-nitrotyrosine and intracellular lipids content. Conversely, SMC-actin levels were significantly lower. These three markers, such as increased 3-nitrotyrosine and intracellular lipids and decreased SMC-actin are associated with plaque vulnerability and instability. These findings are in line with earlier reports demonstrating increased intima-media thickness in large cohorts of sleep apnea and patients with SDB, and thus, may indicate a higher susceptibility to plaque vulnerability and rapture in patients with SDB.

Oxidative stress in obese children and adolescents with and without type 2 diabetes mellitus is not associated with obstructive sleep apnea.

PURPOSE: Obesity, obstructive sleep apnea (OSA), and type 2 diabetes mellitus (T2DM) are associated with chronic low-grade inflammation and oxidative stress. In adults, increased lipid peroxidation, a marker of oxidative stress, was found in both metabolic syndrome and OSA. Studies on oxidative stress in children with T2DM and OSA are scarce. METHODS: Plasma oxidized low-density lipoprotein (Ox-LDL) levels were evaluated in obese children and adolescents with/without T2DM, and the contribution of OSA to oxidative stress was investigated. RESULTS: Ten patients with T2DM, 8 with impaired glucose tolerance (IGT), and 20 body mass index-standard deviation score (BMI-SDS)-matched non-diabetic children (controls) were studied. They all underwent overnight polysomnography. Fasting plasma concentrations of Ox-LDL were measured and compared to the glycemic status and to the presence of OSA. Fourteen patients (36%) were diagnosed with OSA and 21 (55%) with hypertension. There were no significant group differences in plasma Ox-LDL levels or between patients with/without OSA. Plasma Ox-LDL levels were significantly higher among patients with hypertension compared to controls (P = 0.01), while they correlated with homeostasis model assessment (P = 0.02), BMI-SDS (P = 0.049), and systolic blood pressure (P = 0.002). CONCLUSIONS: The findings of this pilot study suggest that increased lipid peroxidation is associated with insulin resistance and hypertension in obese children and adolescents, while OSA has most likely minor influence.

Intermittent Hypoxia Induced Formation of "Endothelial Cell-Colony Forming Units (EC-CFUs)" Is Affected by ROS and Oxidative Stress.

Intermittent hypoxia (IH)-the hallmark of obstructive sleep apnea (OSA)-increases leukocyte activation, production of NADPH-oxidase dependent reactive oxygen species (ROS) and oxidative stress, affecting endothelial function. However, IH and oxidative stress can also stimulate adaptive-protective mechanisms by inducing the development of Endothelial Cell-Colony Forming Units (EC-CFUs), which are considered as a good surrogate marker for endothelial progenitor cells (EPCs), and likely reflect a reparatory response to vascular damage or tissue ischemia by leukocytes. Blood samples were obtained from 15 healthy consenting volunteers to evaluate the effects of IH and sustained hypoxia (SH) in vitro on EC-CFUs development and functions. The variables measured included: their numbers, the area, the proliferative capacity and ROS production. Additionally, NADPH-oxidase, VEGF and nuclear factor-erythroid 2 related factor 2 (Nrf2) expression, as well as their paracrine effects on endothelial tube formation were determined. The involvement of ROS was probed using the anti-oxidant N-acetylcysteine (NAC) and NADPH-oxidase inhibitors apocynin and diphenyl-iodide. Compared to normoxia, IH-dependent increases in EC-CFUs numbers were observed, showing an individual donor-dependent trait. Also, the expression of VEGF and gp91phox, a subunit of NADPH-oxidase, were significantly increased. ROS production and oxidative stress markers were also significantly increased, but Nrf2 expression and colony size were unaffected by IH. Additionally, conditioned media harvested from IH- and SH-treated mature EC-CFUs, significantly increased endothelial tube formation. These effects were markedly attenuated or diminished by the ROS and NADPH-oxidase inhibitors employed. In conclusion, we show here for the first time that IH-associated oxidative stress promotes EC-CFUs' vascular and paracrine capacities through ROS. However, the large inter-individual variability expressed in EC-CFUs numbers and functions to a given IH stimulus, may represent an individual trait with a potential clinical significance.

Intermittent Hypoxia and Unsaturated Aldehydes: Effects on Oral Epithelial Wound Healing.

Obstructive sleep apnea (OSA) is a highly prevalent sleep breathing disorder characterized by intermittent hypoxia (IH), leading to blood hypoxemia, hypercapnia, and sleep fragmentation. Studies on the effects of OSA on oral epithelial tissue healing are limited. Smoking is considered a risk factor for OSA through the exposure to chemically active toxins, present in the smoke. Acrolein is the most chemically active unsaturated aldehyde, impairing a variety of biological processes. The aim of this study was to determine the effect of IH on oral epithelial tissue healing, with and without acrolein. HaCaT cells were wounded by a cross-scratch made in the cell cultures, considered as time zero. Then, cells were exposed to 28 IH cycles (5-20% oxygen) during 12 h using the BioSpherix OxyCycler-C42 system. Control cells were maintained in normoxic conditions or in sustained hypoxia (SH) (5% oxygen) for the same durations, after which all cells were maintained for additional 12 h in normoxia. The migrating abilities of cells were measured after 24 h by calculating the percent of the residual cross-scratch area. In parallel experiments, 25 µM acrolein were added to each treatment. We found that the scratch closure was the slowest under IH. After 24 h, the residual scratch area in the IH treated cells was 29.5 ± 13.4% of the initial area, while in normoxia and SH it was 9.2 ± 5.8% and 10.3 ± 11.3%, respectively (p < 0.01 for both vs. IH). Adding acrolein further attenuated the migratory ability in IH as compared to normoxia and SH. We conclude that IH delays the healing process of oral epithelial tissue by slowing the cells' migratory abilities. The healing might be further attenuated by chemically active unsaturated aldehydes such as acrolein.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro.

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (Gϕ). Unlike neutrophils, Gϕ are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. Gϕ also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing Gϕ in order to further investigate their significance and functions.

Intermittent Hypoxia Affects the Spontaneous Differentiation In Vitro of Human Neutrophils into Long-Lived Giant Phagocytes.

Previously we identified, for the first time, a new small-size subset of neutrophil-derived giant phagocytes (Gϕ) which spontaneously develop in vitro without additional growth factors or cytokines. Gϕ are CD66b(+)/CD63(+)/MPO(+)/LC3B(+) and are characterized by extended lifespan, large phagolysosomes, active phagocytosis, and reactive oxygen species (ROS) production, and autophagy largely controls their formation. Hypoxia, and particularly hypoxia/reoxygenation, is a prominent feature of many pathological processes. Herein we investigated Gϕ formation by applying various hypoxic conditions. Chronic intermittent hypoxia (IH) (29 cycles/day for 5 days) completely abolished Gϕ formation, while acute IH had dose-dependent effects. Exposure to 24 h (56 IH cycles) decreased their size, yield, phagocytic ability, autophagy, mitophagy, and gp91-phox/p22-phox expression, whereas under 24 h sustained hypoxia (SH) the size and expression of LC3B and gp91-phox/p22-phox resembled Gϕ formed in normoxia. Diphenyl iodide (DPI), a NADPH oxidase inhibitor, as well as the PI3K/Akt and autophagy inhibitor LY294002 abolished Gϕ formation at all oxygen conditions. However, the potent antioxidant, N-acetylcysteine (NAC) abrogated the effects of IH by inducing large CD66b(+)/LC3B(+) Gϕ and increased both NADPH oxidase expression and phagocytosis. These findings suggest that NADPH oxidase, autophagy, and the PI3K/Akt pathway are involved in Gϕ development.

Oxidative stress in obstructive sleep apnea and intermittent hypoxia--revisited--the bad ugly and good: implications to the heart and brain.

Obstructive sleep apnea (OSA), characterized by intermittent hypoxia (IH), is linked with increased reactive oxygen species/reactive nitrogen species (ROS/RNS) and oxidative stress, which adversely affect the associated cardio-/cerebro-vascular disease in OSA. Yet, animal and a small number of human studies support activation of cardio-/cerebro-protective mechanisms as well. ROS/RNS are intricate and multifaceted molecules with multiple functions. At low-moderate concentrations ROS/RNS are considered "good", by regulating vital cellular functions. At higher levels, they are considered "bad" by promoting oxidative stress and damaging vital macromolecules through ischemia and reperfusion (I/R) injury. Subsequently, ROS/RNS can get "ugly" by eliciting sterile inflammation and a multitude of deadly pathologies. What makes ROS/RNS good, bad, or ugly? A dynamic interplay between a large number of factors determines the outcomes. These include the types of ROS/RNS produced, their quantity, duration, frequency, intracellular localization, micro-environmental antioxidants, as well as the genetic make-up and life style related variables. This review presents the currently available data on redox biology in physiological/pathophysiological conditions and in OSA/IH, in order to better understand the apparently contradictory findings on damage vs. repair. These findings are discussed within the context of the prevailing views on I/R associated ROS/RNS, and their potential implications to OSA.

Obstructive sleep apnoea syndrome.

Obstructive sleep apnoea syndrome (OSAS) is a common clinical condition in which the throat narrows or collapses repeatedly during sleep, causing obstructive sleep apnoea events. The syndrome is particularly prevalent in middle-aged and older adults. The mechanism by which the upper airway collapses is not fully understood but is multifactorial and includes obesity, craniofacial changes, alteration in upper airway muscle function, pharyngeal neuropathy and fluid shift towards the neck. The direct consequences of the collapse are intermittent hypoxia and hypercapnia, recurrent arousals and increase in respiratory efforts, leading to secondary sympathetic activation, oxidative stress and systemic inflammation. Excessive daytime sleepiness is a burden for the majority of patients. OSAS is also associated with cardiovascular co-morbidities, including hypertension, arrhythmias, stroke, coronary heart disease, atherosclerosis and overall increased cardiovascular mortality, as well as metabolic dysfunction. Whether treating sleep apnoea can fully reverse its chronic consequences remains to be established in adequately designed studies. Continuous positive airway pressure (CPAP) is the primary treatment modality in patients with severe OSAS, whereas oral appliances are also widely used in mild to moderate forms. Finally, combining different treatment modalities such as CPAP and weight control is beneficial, but need to be evaluated in randomized controlled trials. For an illustrated summary of this Primer, visit: http://go.nature.com/Lwc6te.

Oxidative stress in children with obstructive sleep apnea syndrome.

STUDY OBJECTIVES: Pediatric obstructive sleep apnea (OSA) is associated with cardiovascular consequences, including accelerated atherosclerosis and endothelial dysfunction. Increased lipid peroxidation, a marker of oxidative stress, has been identified in adults with OSA in a severity-dependent manner, with attenuation following treatment with continuous positive airway pressure therapy. Studies on oxidative stress in children with OSA are sparse and results are inconclusive. The objective of this study was to compare lipid peroxidation in children with OSA to non-OSA children. METHODS: A prospective cross-sectional study of 26 children with polysomnography-confirmed OSA (oAHI ≥ 5/h TST) was conducted. Thirty age- and body mass index z-score-matched children with primary snoring (PS) served as a comparison group (oAHI ≤ 1/h TST). Fasting blood samples were obtained on the morning following the sleep study. Plasma oxidized low-density lipoprotein (oxLDL) concentrations were measured by enzyme-linked immunosorbent assay. RESULTS: There were no group differences in patient characteristics and their lipid profiles. The mean oxLDL levels of the OSA group were significantly higher than those of the comparison group (53.1 ± 13.0 vs. 45.7 ± 10.0 U/L, respectively, p = 0.02). There was a significant positive correlation between plasma oxLDL and the apnea hypopnea index (r = 0.29, p = 0.03) and between oxLDL and the oxygen desaturation index (r = 0.51, p = 0.003), and a significant negative correlation between SpO2 nadir and oxLDL (r = -0.29, p = 0.03). CONCLUSIONS: OSA in children is associated with increased lipid peroxidation in a severity-dependent manner. Lipid peroxidation levels correlate with the degree of intermittent hypoxia.

The development of giant phagocytes in long-term neutrophil cultures.

We tested the hypothesis that in long-term culture conditions, some neutrophils remain viable and participate in debris clearance, and autophagy is involved in their prolonged survival. Neutrophils, classified as professional phagocytes, have the shortest half-life among leukocytes and are constitutively committed to apoptosis. Apoptotic neutrophils are actively removed by Mφ/DCs. However, early and acute inflammatory infiltrates primarily consist of neutrophils. Recently, neutrophils were suggested to facilitate debris clearance at inflammatory sites when the Mφ/DC system is insufficient. Here, purified CD15(+)/CD66b(+)/CD63(+) neutrophils were followed up to 7 days in culture using light, time-lapse, and confocal microscopy. After 3 days in culture, Annexin-V(-)/LC3B(+) large vacuolated cells, engulfing cellular residues, were noted among apoptotic neutrophils and cell debris. Thereafter, these cells were vastly enlarged and exhibited a neutrophilic phenotype (CD15(+)/CD63(+)/MPO(+)/CD66b(+)), phagocytosis, and oxidative burst activity. They also expressed CD68 scavenger receptors and internalized oxLDL. But, unlike in fresh neutrophils or cultured monocytes, oxLDL treatment increased their ROS production. Additionally, these phagocytes contained LC3B-coated vacuoles and LC3B aggregates, indicating the activation of autophagy. An intensive LC3B accumulation was also noted during oxLDL internalization. Importantly, the inhibition of autophagy by 3-MA or BafA1 prevented their development. In conclusion, the internalization of neutrophil remnants may induce activation of autophagic mechanisms in some neutrophil subsets or precursors. This may lead to cell adaptation and survival, resulting in their transformation into long-lived Gφ and potentially suggesting their involvement in inflammatory/anti-inflammatory processes.

Clinical implications of sleep disordered breathing in acute myocardial infarction.

BACKGROUND: Sleep disordered breathing (SDB), characterized by nightly intermittent hypoxia, is associated with multiple pathophysiologic alterations that may adversely affect patients with acute myocardial infarction (AMI). This prospective study investigated whether the metabolic perturbations associated with SDB are present when these patients develop AMI and if they affect clinical outcomes. METHODS: We prospectively enrolled 180 AMI patients. SDB was defined as oxygen desaturation index (ODI) >5 events/hour based on a Watch Pat-100 sleep study. Blood samples were obtained for high-sensitivity C-reactive protein (hs-CRP) and markers of oxidative stress (lipid peroxides [PD] and serum paraoxonase-1 [PON-1] (arylesterase activity). Echocardiography was performed to evaluate cardiac dimensions and pulmonary artery systolic pressure. RESULTS: SDB was present in 116 (64%) patients. Hs-CRP levels, PD and PON-1 were similar in patients with and without SDB. Echocardiography revealed higher left atrial dimension (4.1 ± 0.5 vs 3.8 ± 0.5 cm; P = 0.003) and a significant positive correlation between ODI and pulmonary artery systolic pressure (r = 0.41, P<0.0001). After a median follow up of 68 months, no significant differences were observed between the study groups with regard to clinical outcomes, including death, heart failure, myocardial infarction and unstable angina. CONCLUSION: There is a high prevalence of previously undiagnosed SDB among patients with AMI. SDB in the setting of AMI is associated with higher pulmonary artery systolic pressure. SDB was not associated with adverse clinical outcomes.

Endothelial progenitor cells in acute myocardial infarction and sleep-disordered breathing.

RATIONALE: Mobilization and functions of endothelial progenitor cells (EPCs) are increased in patients with acute myocardial infarction (AMI). Yet, sleep-disordered breathing (SDB) is highly prevalent in patients with AMI. OBJECTIVES: To compare EPC numbers and functions in patients with AMI with SDB (AMI-SDB) and without SDB (AMI-only) and to determine the effects of intermittent hypoxia (IH) in vitro on EPC proliferative and angiogenic properties. METHODS: Forty male patients with AMI underwent a whole-night sleep study using ambulatory monitoring. Nineteen had SDB (oxygen desaturation index > 5 events/h). AMI-SDB and AMI-only patients were matched by age, body mass index, blood chemistry, and comorbidities. Blood samples were analyzed by flow cytometry, endothelial cell colony-forming units (EC-CFU), paracrine measures, blood chemistry, and oxidative stress, inflammatory, and angiogenic markers. Effects of IH in vitro were studied in 12 healthy subjects. MEASUREMENTS AND MAIN RESULTS: Circulating EPCs (CD34(+)/KDR(+)), angiogenic T cells (CD3(+)/CD31(+)/CXCR4(+)), and vascular endothelial growth factor in monocytes were significantly higher in AMI-SDB patients, whereas plasma stromal cell-derived factor (SDF)-1α levels were significantly lower. Also, EC-CFU numbers and EC-CFU paracrine effects on endothelial tube formation were significantly higher in AMI-SDB compared with AMI-only patients. Similarly, in cell cultures from healthy subjects, EC-CFU numbers and their paracrine effects on endothelial tube formation were increased after exposure to IH in vitro compared with normoxia. CONCLUSIONS: Coexistent mild to moderate SDB in patients with AMI increased the mobilization, proliferative and angiogenic capacities of EPCs, angiogenic T-cell numbers, and vascular endothelial growth factor expression in monocytes compared with patients with AMI without SDB. IH in vitro had similar effects on healthy EPC functions.

Bax/Mcl-1 balance affects neutrophil survival in intermittent hypoxia and obstructive sleep apnea: effects of p38MAPK and ERK1/2 signaling.

BACKGROUND: Prolonged neutrophil survival is evident in various cardiovascular and respiratory morbidities, in hypoxic conditions in-vitro and in patients with obstructive sleep apnea (OSA) characterized by nightly intermittent hypoxia (IH). This may lead to persistent inflammation, tissue injury and dysfunction. We therefore investigated by a translational approach the potential contribution of the intrinsic stress-induced mitochondrial pathway in extending neutrophil survival under IH conditions. Thus, neutrophils of healthy individuals treated with IH in-vitro and neutrophils of OSA patients undergoing nightly IH episodes in-vivo were investigated. Specifically, the balance between pro-apoptotic Bax and anti-apoptotic Mcl-1 protein expression, and the potential involvement of p38MAPK and ERK1/2 signaling pathways in the control of Mcl-1 expression were investigated. METHODS: Purified neutrophils were exposed to IH and compared to normoxia and to sustained hypoxia (SH) using a BioSpherix-OxyCycler C42 system. Bax and Mcl-1 levels, and p38MAPK and ERK1/2 phosphorylation were determined by western blotting. Also, Bax/Mcl-1 expression and Bax translocation to the mitochondria were assessed by confocal microscopy in pre-apoptotic neutrophils, before the appearance of apoptotic morphology. Co-localization of Bax and mitochondria was quantified by LSM 510 CarlZeiss MicroImaging using Manders Overlap Coefficient. A paired two-tailed t test, with Bonferroni correction for multiple comparisons, was used for statistical analysis. RESULTS: Compared to normoxia, IH and SH up-regulated the anti-apoptotic Mcl-1 by about 2-fold, down-regulated the pro-apoptotic Bax by 41% and 27%, respectively, and inhibited Bax co-localization with mitochondria before visible morphological signs of apoptosis were noted. IH induced ERK1/2 and p38MAPKs phosphorylation, whereas SH induced only p38MAPK phosphorylation. Accordingly, both ERK and p38MAPK inhibitors attenuated the IH-induced Mcl-1 increase. In SH, only p38MAPK inhibition decreased Mcl-1 expression. Similar to neutrophils of healthy subjects exposed to IH (0.97± 0.2), in OSA neutrophils, Bax/Mcl-1 ratio was significantly lower compared to normoxic controls (1.0±0.5 vs.1.99±0.3, p=0.015), and Bax did not co-localize with mitochondria. CONCLUSIONS: These findings suggest that decreased Bax/Mcl-1 balance promotes neutrophil survival in IH in-vitro as well as in OSA patients. Moreover, Bax/Mcl-1 protein function in IH and SH might be regulated by different signal transduction pathways, highlighting a novel regulatory function through ERK1/2 signaling in IH.

Sleep-disordered breathing in acute ischemic stroke and transient ischemic attack: effects on short- and long-term outcome and efficacy of treatment with continuous positive airways pressure--rationale and design of the SAS CARE study.

OBJECTIVES: Sleep-disordered breathing represents a risk factor for cardiovascular morbidity and mortality and negatively affects short-term and long-term outcome after an ischemic stroke or transient ischemic attack. The effect of continuous positive airways pressure in patients with sleep-disordered breathing and acute cerebrovascular event is poorly known. The SAS CARE 1 study assesses the effects of sleep-disordered breathing on clinical evolution, vascular functions, and markers within the first three-months after an acute cerebrovascular event. The SAS CARE 2 assesses the effect of continuous positive airways pressure on clinical evolution, cardiovascular events, and mortality as well as vascular functions and markers at 12 and 24 months after acute cerebrovascular event. METHODS: SAS CARE 1 is an open, observational multicenter study in patients with acute cerebrovascular event acutely admitted in a stroke unit: a sample of 200 acute cerebrovascular event patients will be included. Vascular functions and markers (blood pressure, heart rate variability, endothelial function by peripheral arterial tonometry and specific humoral factors) will be assessed in the acute phase and at three-months follow-up. SAS CARE 2 will include a sample of patients with acute cerebrovascular event in the previous 60-90 days. After baseline assessments, the patients will be classified according to their apnea hypopnea index in four arms: non-sleep-disordered breathing patients (apnea hypopnea index <10), patients with central sleep-disordered breathing, sleepy patients with obstructive apnea hypopnea index ≥20, which will receive continuous positive airways pressure treatment, nonsleepy patients with obstructive sleep-disordered breathing (apnea hypopnea index ≥20), which will be randomized to receive continuous positive airways pressure treatment or not. CONCLUSIONS: The SAS CARE study will improve our understanding of the clinical sleep-disordered breathing in patients with acute cerebrovascular event and the feasibility/efficacy of continuous positive airways pressure treatment in selected patients with acute cerebrovascular event and sleep-disordered breathing.