Molecular mechanisms of cardiovascular disease in OSAHS: the oxidative stress link.

Obstructive sleep apnoea/hypopnoea syndrome (OSAHS) is a highly prevalent breathing disorder in sleep that is an independent risk factor for cardiovascular morbidity and mortality. A large body of evidence, including clinical studies and cell culture and animal models utilising intermittent hypoxia, delineates the central role of oxidative stress in OSAHS as well as in conditions and comorbidities that aggregate with it. Intermittent hypoxia, the hallmark of OSAHS, is implicated in promoting the formation of reactive oxygen species (ROS) and inducing oxidative stress. The ramifications of increased ROS formation are pivotal. ROS can damage biomolecules, alter cellular functions and function as signalling molecules in physiological as well as in pathophysiological conditions. Consequently, they promote inflammation, endothelial dysfunction and cardiovascular morbidity. Oxidative stress is also a crucial component in obesity, sympathetic activation and metabolic disorders such as hypertension, dyslipidaemia and type 2 diabetes/insulin resistance, which aggregate with OSAHS. These conditions and comorbidities could result directly from the oxidative stress that is characteristic of OSAHS or could develop independently. Hence, oxidative stress represents the common underlying link in OSAHS and the conditions and comorbidities that aggregate with it.

Virioplankton 'pegylation': use of PEG (polyethylene glycol) to concentrate and purify viruses in pelagic ecosystems.

We have described the use of Polyethylene glycol (PEG) for the precipitation of natural communities of aquatic viruses, and its comparison with the usual concentration method based on ultracentrifugation. Experimental samples were obtained from different freshwater ecosystems whose trophic status varied. Based on transmission electron microscope observations and counting of phage-shaped particles, our results showed that the greatest recovery efficiency for all ecosystems was obtained when we used the PEG protocol. On average, this protocol allowed the recovery of >2-fold more viruses, compared to ultracentrifugation. In addition, the diversity of virioplankton, based on genomic size profiling using pulsed field gel electrophoresis, was higher and better discriminated when we used the PEG method. We conclude that pegylation offers a valid, simple and cheaper alternative method to ultracentrifugation, for the concentration and the purification of pelagic viruses.

Interindividual heterogeneity in the hypoxic regulation of VEGF: significance for the development of the coronary artery collateral circulation.

BACKGROUND: The coronary artery collateral circulation may be beneficial in protecting against myocardial ischemia and necrosis. However, there is a tremendous interindividual variability in the degree of new collateral formation in patients with coronary artery disease. The basis for this interindividual heterogeneity is not understood. In this study we test the hypothesis that failure to generate collateral vessels is associated with a failure to appropriately induce with hypoxia or ischemia the angiogenic factor, vascular endothelial growth factor (VEGF). METHODS AND RESULTS: We correlated the VEGF response to hypoxia in the monocytes harvested from patients with coronary artery disease with the presence of collaterals visualized during routine angiography. We found that there was a highly significant difference in the hypoxic induction of VEGF in patients with no collaterals compared with patients with some collaterals (mean fold induction 1.9+/-0.2 versus 3.2+/-0.3, P<0.0001). After subjecting the data to ANCOVA, using as covariates a number of factors that might influence the amount of collateral formation (ie, age, sex, diabetes, smoking, hypercholesterolemia), patients with no collaterals still have a significantly lower hypoxic induction of VEGF than patients with collaterals. CONCLUSIONS: This study provides evidence in support of the hypothesis that the ability to respond to progressive coronary artery stenosis is strongly associated with the ability to induce VEGF in response to hypoxia. The observed interindividual heterogeneity in this response may be due to environmental, epigenetic, or genetic causes. This interindividual heterogeneity may also help to explain the variable angiogenic responses seen in other conditions such as diabetic retinopathy and solid tumors.

The presence of NADPH-glyceraldehyde 3-phosphate oxidoreductase in macrophages.

The existence of an NADPH-oxidoreductase which utilizes D-glyceraldehyde 3-phosphate as substrate has been demonstrated in mouse peritoneal macrophages. D-Glyceraldehyde could also serve as substrate, albeit with a 10-fold lower efficiency. No NADH oxidation could be demonstrated with either substrate. Addition of D-glyceraldehyde to cultured macrophages increased the rate of activity of the hexose monophosphate shunt to about 65% of the level observed in zymosan A-stimulated macrophages. The possible involvement of the oxidoreductase in this phenomenon and in the inhibitory effect of D-glyceraldehyde on the production of oxygen free radicals by zymosan-stimulated cells is discussed.

Oxygen free radical production by mouse peritoneal macrophages as a function of age.

The ability of thioglycollate-elicited peritoneal macrophages (PM) from young and senescent C57BL/6J mice to produce oxygen free radicals was assessed by luminol-dependent chemiluminescence (CL) after introduction of phagocytic stimuli. A significant age-dependent variation in the CL response was detected. A 2-fold increase in the oxygen reactive species was produced by senescent PM in response to latex and zymosan stimulation; but, the capacity to ingest latex and zymosan A particles did not vary significantly between PM from young and senescent mice. Peritoneal macrophages from both age groups responded much more vigorously to opsonized zymosan. The response of the PM from young mice was, however, 2.8-fold higher than that of old ones. There was no age-related difference in oxygen free radical production after stimulation with phorbol myristate acetate (PMA). Also, no age-dependent differences were found in the relative contribution of the various oxygen reactive species O2.-, OH., 1O2 and H2O2) to the overall oxidative burst, with latex zymosan A or PMA.

Age- and strain-related changes in tissue transglutaminase activity in murine macrophages: the effects of inflammation and induction by retinol.

We describe differences in the activity of tissue transglutaminase (TGase) between resident and inflammatory mouse peritoneal macrophages as a function of age. Our results established the following observations: (a) resident macrophages from senescent mice expressed higher basal TGase activity than those from young mice; (b) Maximal TGase activity on day 3 of thioglycollate injection was lower by 24% in inflammatory macrophages from senescent as compared to young animals; (c) in contrast, as the inflammatory response abated (days 4-6), the incremental decrease in TGase activity in old was lower than in young animals; (d) in vitro activation of resident macrophages by retinol and mouse serum was more effective in inducing TGase activity from outbred CD-1 young mice than from inbred C57BL/6J young mice (age differences were also more prominent in the CD-1 mouse strain); and (e) Retinol and mouse serum effectively inhibited the production of superoxide in young mice, thereby demonstrating an inverse correlation between TGase activity and superoxide production. In old animals, however, the production of superoxide was not decreased, nor was TGase increased. Although, paradoxically, resident macrophages from senescent mice were a priori more activated than those from young ones, it is concluded that macrophages from young mice respond better than those from old ones to stimuli they encounter, either during inflammation or under physiological stimulation.

Transglutaminase in Plasmodium parasites: activity and putative role in oocysts and blood stages.

Transglutaminase was identified in malaria parasites by immunofluorescence microscopy using alpha-transglutaminase antiserum. Functional enzyme was demonstrated in vivo and in vitro using labeled polyamines that become incorporated into protein substrates through TGase activity. In Plasmodium falciparum intraerythrocytic parasites, transglutaminase activity was stage-dependent: it was weak in ring-forms but much stronger in trophozoites and schizonts. High levels of activity were detected in P. gallinaceum zygotes and ookinetes and in capsules of oocysts developing on mosquito midguts. Unlike most known transglutaminases, the enzymatic activity in Plasmodium was Ca(2+)-independent. Furthermore, levels of activity were similar at 37 and 26 degrees C. Parasite transglutaminase may be responsible for the modification of erythrocytic cytoskeleton in infected cells and it may facilitate the construction of oocyst capsules by cross-linking mosquito-derived basement membrane components with Plasmodium-derived proteins.

Plasma homocysteine levels in obstructive sleep apnea: association with cardiovascular morbidity.

OBJECTIVES: Obstructive sleep apnea (OSA) is associated with cardiovascular morbidity and mortality. Plasma levels of homocysteine are also associated with cardiovascular morbidity and mortality. We therefore investigated homocysteine and conventional cardiovascular risk factors in OSA patients with and without cardiovascular morbidity in comparison with normal control subjects and ischemic heart disease (IHD) patients without OSA. SETTING: Technion Sleep Medicine Center, Haifa, Israel. METHODS AND PARTICIPANTS: Levels of homocysteine, cholesterol, low-density lipoprotein, high-density lipoprotein, triglycerides, creatinine, vitamins B(12) and B(6), and folic acid were determined in 345 participants after overnight fasting. These included OSA patients with IHD (n = 49), with hypertension (n = 61), or without any cardiovascular disease (n = 127). Two control groups were employed: IHD patients without or with low likelihood for sleep apnea (n = 35), and healthy control subjects (n = 73). RESULTS: After adjustment for age, body mass index, creatinine, and existence of diabetes mellitus, OSA patients with IHD had significantly higher homocysteine levels (14.6 +/- 6.77 micromol/L) than all other groups including the IHD-only patients. Hypertensive OSA patients had comparable homocysteine levels to IHD patients (11.80 +/- 5.28 micromol/L and 11.92 +/- 5.7 micromol/L, respectively), while patients with OSA only had comparable levels to normal control subjects (9.85 +/- 2.99 micromol/L and 9.78 +/- 3.49 micromol/L, respectively). No differences in conventional cardiovascular risk factors or in vitamin levels were found between groups. CONCLUSIONS: Patients with the combination of IHD and OSA have elevated homocysteine levels. We hypothesize that these results may be explained by endothelial dysfunction combined with excess free-radical formation in OSA patients.

Selective MAO-A and B inhibitors, radical scavengers and nitric oxide synthase inhibitors in Parkinson's disease.

In the absence of identification of either an endogenously or an exogenously derived dopaminergic neurotoxin, the most valid hypothesis currently envisaged for etiopathology of Parkinson's disease (PD) is selective oxidative stress (OS) in substantia nigra (SN). Although OS is not proven, a significant body of evidence from studies on animal and Parkinsonian brain neurochemistry supports it. This hypothesis is based on excessive formation of reactive oxygen species (O2 and OH.) and demise of systems involved with scavenging or preventing the formation of such radicals from H2O2, generated as a consequence of dopamine oxidation (autoxidation and deamination). Since MAO (monoamine oxidase A and B are the major H2O2 generating enzymes in the SN much attention has been paid to their selective inhibitors as symptomatic and neuroprotective agents in PD. Attention should also be given to radical scavengers (e.g. iron chelators, lipid peroxidative inhibitors and Vitamin E derivatives) as therapeutic neuroprotective agents in PD. This is considered valid since a significant elevation of iron is known to occur selectively in SN zone compacta and within the remaining melanized dopamine neurons of Parkinsonian brains. Although all the mechanism of iron induced oxygen free radical formation is not fully known there is no doubt that it participates with H2O2 (Fenton chemistry) to generate cytotoxic hydroxyl radical (OH.) and induce tissue OS and neurodegeneration in 6-hydroxydopamine model of PD. The dramatic proliferation of reactive amoeboid macrophages and microglia seen in SN of PD brains together with OS is highly compatible with an inflammatory process, similar to what has been observed in Alzheimer's disease and multiple sclerosis brains. This has led us to examine the ability of reactive macrophages to produce oxygen free radicals in response to nitric oxide (NO) production. The latter radical has been implicated in the excitotoxicity of glutaminergic neurons innervating the striatum and SN. Indeed we have now observed that in reactive macrophages NO acts as a signal transducer of O2 production which can synergize with dopamine oxidation.

The macrophage in cell biology of aging.

Peritoneal macrophages (PM) offer a convenient system to study biochemical and physiological aspects of cellular aging, as well as gene expression, signal transduction and cytoplasmic functions. They are harvested directly from animals whose ages are well defined, and in good yields. PM are easily maintained in culture, and their unique physiological or biochemical characteristics can be manipulated either in-vivo or in-vitro. During inflammation, or upon interaction with soluble or particulate stimuli and consequent phagocytosis, macrophages undergo respiratory burst activation, producing large quantities of superoxide anions by NADPH oxidase. Increased superoxide production in response to zymosan was found in resident PM from senescent mice. These were non-specifically activated as compared to young, as shown also by increased tissue transglutaminase (TGase) activity, and increased cell yield. Moreover, PM from senescent mice were not affected by the retinoid induced up-regulation of TGase activity or down-regulation of superoxide release, as were PM from young mice. Also, altered receptor function and enzymic activities were demonstrated. Thus, apart from its function in immunity, the macrophage offers the possibility to study cell biology of aging as well.

Age-related alterations in respiratory burst activation induced by various stimuli in mouse peritoneal macrophages treated with thermal stress.

The respiratory burst stimuli zymosan, opsonized zymosan (OZ), or phorbol myristate acetate (PMA) were used in order to assess the ability of thioglycolate elicited peritoneal macrophages (PM) from young and senescent mice to generate superoxide anions. The amount of superoxide generated by PM from senescent mice in response to zymosan was higher by 20% than in PM from young mice. However, superoxide release in response to stimulation by OZ or PMA was 30% and 60% higher in PM from young mice. Thermal stress reduced the amounts of superoxide generated in response to zymosan in both age groups. However, while at 42.5 degrees C PM from senescent mice demonstrated irreversible loss of activity. PM from young mice recovered up to 70% of their superoxide generating activity. Thermal stress also affected protein synthesis in a temperature and age-dependent manner. Macrophage activation level increased in the old but remained unaffected in the young as attested by transglutaminase activity. This suggests that the mechanisms of up- or down-regulation of superoxide release in the old differ under heat stress conditions.

Management of obstructive sleep apnea in Europe.

OBJECTIVES: In Europe, the services provided for the investigation and management of obstructive sleep apnoea (OSA) varies from country to country. The aim of this questionnaire-based study was to investigate the current status of diagnostic pathways and therapeutic approaches applied in the treatment of OSA in Europe, qualification requirements of physicians involved in diagnosis and treatment of OSA, and reimbursement of these services. METHODS: Two questionnaires were sent to 39 physicians in 22 countries in Europe. In order to standardize the responses, the questionnaire was accompanied by an example. RESULTS: Sleep centers from 21 countries (38 physicians) participated. A broad consistency among countries with respect to the following was found: pathways included referral to sleep physicians/sleep laboratories, necessity for objective diagnosis (primarily by polysomnography), use of polygraphic methods, analysis of polysomnography (PSG), indications for positive airway pressure (PAP) therapy, application of standard continuous PAP (CPAP) therapy (100% with an CPAP/APAP ratio of 2.24:1), and the need (90.5%) and management of follow-up. Differences were apparent in reimbursement of the diagnostic procedures and follow-up, in the procedures for PAP titration from home APAP titration with portable sleep apnea monitoring (38.1%) up to hospital monitoring with PSG and APAP (85.7%), and in the qualification requirements of sleep physicians. CONCLUSIONS: Management of OSA in different European countries is similar except for reimbursement rules, qualification of sleep specialists and procedures for titration of the CPAP treatment. A European network (such as the one accomplished by the European Cooperation in Science and Technology [COST] B26 Action) could be helpful for implementing these findings into health-service research in order to standardize management in a cost effective perspective.

All-cause mortality in males with sleep apnoea syndrome: declining mortality rates with age.

The objective of this study was to assess whether an increasing severity of sleep apnoea is associated with increased all-cause mortality hazards and to assess whether the syndrome is associated with excess mortality, in comparison with the general population. Participants included 14,589 adult males, aged 20-93 yrs, referred to the sleep clinics with suspected sleep apnoea or diagnosed with sleep apnoea. Altogether, 372 deaths were recorded after a median follow-up of 4.6 yrs. The crude all-cause mortality rate was 5.55/1,000 patient yrs, increasing with apnoea severity. Cox proportional analysis revealed that both respiratory disturbance index (RDI) and body mass index significantly influenced all-cause mortality hazard but there was no interaction between them. Males with respiratory disturbance index >30 had a significantly higher mortality hazard rate than the reference group of males with RDI < or =10. Comparing mortality rates of males with moderate/severe sleep apnoea to the general population revealed that only males aged <50 yrs showed an excess mortality rate. The hazard of mortality in sleep apnoea increases with apnoea severity as indexed by respiratory disturbance index. Moderate and severe levels of sleep apnoea are moderately associated with an increased risk of all-cause mortality, in comparison with the general population, particularly in males aged <50 yrs. The lack of information about possible confounders and treatment effects should be taken into consideration in the interpretation of these results.

Activated CD8+ T-lymphocytes in obstructive sleep apnoea.

T-lymphocytes are implicated in the development of atherosclerosis. The aim of this study was to assess whether the CD8+ T-lymphocytes of obstructive sleep apnoea (OSA) patients undergo phenotypic and functional changes that may exaggerate atherogenic sequelae in OSA. A total of 36 OSA patients, 17 controls and 15 single-night-treated OSA patients were studied. Phenotype and cytotoxicity against K562 target cells were analysed by flow cytometry. Cytotoxicity against human umbilical vein endothelial cells (HUVECs) was assessed by 51Cr release assay. The cytotoxicity of the CD8+ T-lymphocytes of OSA patients against K562 and HUVECs was significantly greater than controls. This increased cytotoxicity directly depended on the presence of perforin and natural killer receptors (CD56, CD16), which were significantly increased in OSA CD8+ T-lymphocytes. Also the percentage of the CD56bright subset, which mediates initial interactions with vascular endothelium, significantly increased in OSA. Nasal continuous positive airway pressure treatment significantly decreased CD8+ T-cell cytotoxicity and CD56 expression, and was positively correlated with natural killer inhibitory NKB1 receptor expression either after a single-night treatment or after a prolonged treatment. In conclusion, the CD8+ T-lymphocytes of obstructive sleep apnoea patients undergo phenotypic and functional changes, rendering them cytotoxic to target cells via increased CD56+/perforin+ expression, which can be ameliorated by nasal continuous positive airway pressure treatment. These results are compatible with the current authors' hypothesis of atherogenic sequelae in obstructive sleep apnoea.

Age-related alterations in superoxide anion generation in mouse peritoneal macrophages studied by repeated stimulations and heat shock treatment.

The ability of thioglycollate-elicited peritoneal macrophages (PM) from young and senescent mice to generate superoxide anions (O2-) under repeated stimulation or thermal stress was studied using either zymosan, opsonized zymosan (OZ), or phorbol myristate acetate (PMA). A diminished capacity to recover from repeated stimulation was found with aging. When stimulated for a second time 24 hours after the primary stimulation, PM from young animals generated 80% of the initial O2- responses to either zymosan, or OZ. Under the same conditions, PM from senescent mice generated 62% of the initial O2- produced in response to zymosan, and 45% in response to OZ. In both age groups the response to a second PMA stimulation comprised only 10% of the primary response. A considerably diminished capacity to generate O2- was also demonstrated in PM from senescent mice after recovery from exposure to thermal stress. Exposure to 42.5 degrees C for 20 minutes was found to be the threshold temperature for irreversible loss of activity in senescent PM, whereas at this temperature, PM from young animals recovered up to 70% of their O2- generating activity. Since NADPH oxidase and superoxide dismutase activities were only mildly affected by the hyperthermia in all age groups, they could not account for the age-related decline in the recovery from stress. Age-related alterations in signal transduction or receptor alterations could possibly play a primary role in this decline.

Homocysteine and cardiovascular risk in patients with diabetes mellitus.

BACKGROUND AND AIM: Diabetes mellitus is the most common metabolic disorder in the Western world with coronary artery disease as its leading cause of mortality. Conventional coronary risk factors do not explain the high morbidity rate and there is strong evidence linking hyperhomocystinemia and premature atherosclerosis. Therefore, we reviewed the relationship between homocysteine and diabetes mellitus. DATA SYNTHESIS: The literature dealing with diabetes mellitus, B vitamins and metformin was reviewed. Some studies show higher than normal fasting and postmethionine load levels of plasma homocysteine in diabetes patients, particularly in those with nephropathy and microalbuminutia. Metformin use and low plasma B vitamins are other potential mechanisms resulting in hyperhomocysteinemia in these patients. CONCLUSIONS: Abnormal homocysteine concentration is prevalent in subsets of diabetic patients; its relationship with excess cardiovascular morbidity is not yet clear. Consequently, large prospective studies are essential in order to follow the effects of homocysteine and its normalization on accelerated artherosclerosis in diabetes.

Non-histone chromosomal proteins. Evidence for their role in mediating the binding of histones to deoxyribonucleic acid during the cell cycle.

By selective dissociation of histones with the ionic detergent sodium deoxycholate, we have demonstrated that these basic chromosomal polypeptides, which are effective inhibitors of transcription, are more tenaciously bound to DNA in mitotic than in S-phase chromatin. Evidence is presented which suggests that cell-cycle-stage-specific non-histone chromosomal proteins can account for such variations in the association of histones with DNA. When chromatin is reconstituted with DNA and histones are pooled from S-phase and mitotic cells and either S-phase or mitotic non-histone chromosomal proteins, a preferential extraction of histones with sodium deoxycholate from chromatin reconstituted with S-phase rather than mitotic non-histone chromosomal proteins is observed. In contrast, the extractability of histones with sodium deoxycholate from nucleohistone complexes reconstituted with DNA pooled from S-phase and mitotic cells and either S-phase or mitotic histones is identical. Since non-histone chromosomal proteins rather than histones are responsible for the differences in chromatin template activity during S-phase and mitosis, we propose that non-histone chromosomal proteins may modify gene expression during the cell cycle by mediating the binding of histones to DNA.