Estrogen-dependent dynamic profile of eNOS-DNA associations in prostate cancer

In previous work we have documented the nuclear translocation of endothelial NOS (eNOS) and its participation in combinatorial complexes with Estrogen Receptor Beta (ERβ) and Hypoxia Inducible Factors (HIFs) that determine localized chromatin remodeling in response to estrogen (E2) and hypoxia stimuli, resulting in transcriptional regulation of genes associated with adverse prognosis in prostate cancer (PCa). To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells. We found that: 1. the eNOS-bound regions (peaks) are widely distributed across the genome encompassing multiple transcription factors binding sites, including Estrogen Response Elements. 2. E2 increased the number of peaks, indicating hormone-dependent eNOS re-localization. 3. Peak distribution was similar with/without E2 with ≈ 55% of them in extragenic DNA regions and an intriguing involvement of the 5′ domain of several miRs deregulated in PCa. Numerous potentially novel eNOS-targeted genes have been identified suggesting that eNOS participates in the regulation of large gene sets. The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa

Tonic and phasic nitric oxide signals in hippocampal long-term potentiation

Nitric oxide ( NO) participates in long-term potentiation (LTP) and other forms of synaptic plasticity in many different brain areas but where it comes from and how it acts remain controversial. Using rat and mouse hippocampal slices, we tested the hypothesis that tonic and phasic NO signals are needed and that they derive from different NO synthase isoforms. NMDA increased NO production in a manner that was potently inhibited by three different neuronal NO synthase ( nNOS) inhibitors. Tonic NO could be monitored after sensitizing guanylyl cyclase-coupled NO receptors, allowing the very low ambient NO concentrations to be detected by cGMP measurement. The levels were unaffected by inhibition of NMDA receptors, nNOS, or the inducible NO synthase ( iNOS). iNOS was also undetectable in protein or activity assays. Tonic NO was susceptible to agents inhibiting endothelial NO synthase ( eNOS) and was missing in eNOS knock-out mice. The eNOS knock-out sexhibited a deficiency in LTP resembling that seen in wild-types treated with a NO synthase inhibitor. LTP in the knock-outs could be fully restored by supplying a low level of NO exogenously. Inhibition of nNOS also caused a major loss of LTP, particularly of late-LTP. Again, exogenous NO could compensate, but higher concentrations were needed compared with those restoring LTP in the eNOS knock-outs. It is concluded that tonic and phasic NO signals are both required for hippocampal LTP and the two are generated, respectively, by eNOS and nNOS, the former in blood vessels and the latter in neurons

Enabling onshore CO2 storage in Europe: fostering international cooperation around pilot and test sites

To meet the ambitious EC target of an 80% reduction in greenhouse gas emissions by 2050, CO2 Capture and Storage (CCS) needs to move rapidly towards full scale implementation with geological storage solutions both on and offshore. Onshore storage offers increased flexibility and reduced infrastructure and monitoring costs. Enabling onshore storage will support management of decarbonisation strategies at territory level while enhancing security of energy supply and local economic activities, and securing jobs across Europe. However, successful onshore storage also requires overcoming some unique technical and societal challenges. ENOS will provide crucial advances to help foster onshore CO2 storage across Europe through: 1. Developing, testing and demonstrating in the field, under "real-life conditions", key technologies specifically adapted to onshore storage. 2. Contributing to the creation of a favourable environment for onshore storage across Europe. The ENOS site portfolio will provide a great opportunity for demonstration of technologies for safe and environmentally sound storage at relevant scale. Best practices will be developed using experience gained from the field experiments with the participation of local stakeholders and the lay public. This will produce improved integrated research outcomes and increase stakeholder understanding and confidence in CO2 storage. In this improved framework, ENOS will catalyse new onshore pilot and demonstration projects in new locations and geological settings across Europe, taking into account the site-specific and local socio-economic context. By developing technologies from TRL4/5 to TRL6 across the storage lifecycle, feeding the resultant knowledge and experience into training and education and cooperating at the pan-European and global level, ENOS will have a decisive impact on innovation and build the confidence needed for enabling onshore CO2 storage in Europe. ENOS is initiating strong international collaboration between European researchers and their counterparts from the USA, Canada, South Korea, Australia and South Africa for sharing experience worldwide based on real-life onshore pilots and field experiments. Fostering experience-sharing and research alignment between existing sites is key to maximise the investment made at individual sites and to support the efficient large scale deployment of CCS. ENOS is striving to promote collaboration between sites in the world through a programme of site twinning, focus groups centered around operative issues and the creation of a leakage simulation alliance

Endothelial nitric oxide synthase gene T-786C and 27-bp repeat gene polymorphisms in retinopathy of prematurity

PURPOSE: Retinopathy of prematurity (ROP), which is associated with abnormal retinal vessel development, is the leading cause of visual loss in preterm infants. Endothelial nitric oxide synthase (eNOS) is believed to play a central role in both retinal angiogenesis and vasculogenesis. The aim of this study was to investigate functional genetic polymorphisms of eNOS in the pathogenesis of ROP. METHODS: eNOS T(−786)C and 27-bp repeat (eNOS, b: wild-type, a: mutant) genotypes were determined using allele-specific polymerase chain reaction in 105 low birth weight (LBW) preterm infants treated for ROP (treated group). A control group was set up and composed of 127 LBW infants with stage 1 or 2 ROP that did not not require treatment (untreated group). RESULTS: The genotype distribution of eNOS 27-bp repeat polymorphism was found to significantly differ (p=0.015) between the two groups, whereas the genotype distribution of eNOS T(−786)C did not differ (p=0.984) between the groups. There was no difference in the distribution of either the “a” allele (p=0.153) nor of the C allele (p=0.867) in a groups comparison. Multiple logistic regression analysis revealed that male gender (p=0.046) and eNOS aa genotype (p=0.047 versus ab genotype and p=0.022 versus bb genotype) were significantly associated severe ROP that required treatment. The haplotype estimations based on the detected genotype distributions showed that the prevalence of aT and bT haplotypes was significantly increased in the group treated for ROP. CONCLUSIONS: Functional eNOS 27-bp repeat polymorphism might be associated with the risk of severe ROP, however we found no association between the eNOS T(−786)C and the pathogenesis of ROP

Experiments to Establish Current-carrying Capacity of Thermionic-emitting Cathodes Final Report, 11 Jan. 1966 - 30 Jan. 1967

Current carrying capabilities of thermionic cathodes in nitrogen at pressure levels above one atmospher

Ассоциация аллельных полиморфизмов гена эндотелиальной NO-синтазы с развитием ишемической болезни сердца (литературный обзор)

Проведений аналіз вітчизняних та закордонних досліджень стосовно вивчення впливу Т-786С, G894T, 4a/b поліморфізмів гену eNOS на ризик розвитку ІХС у представників різних популяцій. Доведена роль Т-786 С поліморфізма гену eNOS у розвитку ІХС у представників японської, української, італійської популяції, причому в останніх він пов’язаний із багатосудинним ураженням. G894T поліморфізм гену eNOS пов’язаний із підвищеним ризиком розвитку ІХС, ішемічних інсультів в італійській, турецькій, азіатській популяціях, а в російській ─ із рестенозами стентів. Доведений зв’язок 4а/4b поліморфізму гену eNOS із виникненням ІХС у турецькій, японській, корейській, афро-американській, іранській, російській популяціях, а в японській популяції ─ гендерна специфіка даної асоціації. В окремих дослідженнях отримані суперечливі дані щодо впливу Т-786 С поліморфізму гену eNOS в турецькій популяції. Не виявлено асоціації 4а/4b поліморфізму гену eNOS у чоловіків Словенії, Фінляндії, G894T поліморфізму гену eNOS у корейській популяції, а у представників білої австралійської популяцій не виявлено асоціації генотипів 4а/4b, G894T, Т-786С поліморфізму гену eNOS із ризиком розвитку ІХС.В статье проведен анализ отечественных и зарубежных исследований, посвященных изучению влияния Т-786С, G894T, 4a/b полиморфизмов гена eNOS на риск развития ИБС у представителей различных популяций. Доказана роль Т-786 С полиморфизма гена eNOS в развитии ИБС у представителей японской, украинськой, итальянской популяции, причем у последних он связан с многососудистым поражением. G894T полиморфизм гена eNOS связан с повышеным риском развития ИХС, ишемических инсультов в итальянской, турецкой, азиатской популяциях, а в российской ─ с рестенозами стентов. Доказана связь 4а/4b полиморфизма гена eNOS с возникновением ИБС в турецкой, японской, корейской, афро-американской, иранськой, российской популяциях, а в японской популяции ─ гендерная специфика данной ассоциации. В отдельных исследованиях получены противоречивые данные о влиянии Т-786 С полиморфизма гена eNOS в турецкой популяции. Не выявлено ассоциации 4а/4b полиморфизма гена eNOS у мужчин Словении, Финляндии, G894T полиморфизма гена eNOS в корейской популяции, а у представителей белой австралийской популяции не выявлено ассоциации генотипов 4а/4b, G894T, Т-786С полиморфизму гену eNOS с риском развития ИБС.The article analyzed Ukrainian and foreign research on the impact study T-786С, G894T, 4a /b polymorphisms of the eNOS gene on the risk of coronary artery disease (CAD) among representatives of different populations. The role of T-786C polimorphism of the eNOS gene was proven in the development of CAD among Japanese, Ukrainian, Italian population, and in the past it is associated with multivessel disease. G894T polymorphism of the eNOS gene is associated with high risk of CAD, ischemic stroke in Italian, Turkish, Asian populations. In the Russian population this polymorphism assotiated with restenosis of stents. The 4a/4b polymorphism of the eNOS gene has significant influence on risk of CAD in Turkish, Japanese, Korean, AfricanAmerican, Iranian and Russian populations. Japanese population has gender specificity of the association. Conflicting data obtained in separate studies of the influence of T-786C polymorphism of the eNOS gene in the Turkish population. There was no association 4a /4b polymorphism of the eNOS gene in men Slovenia’s men and in Finland. Wasn’t identify association of G894T polymorphism of the eNOS gene in Korean population. Wasn’t detected association of genotypes 4a/4b, G894T, T-786S of the eNOS gene polymorphisms with risk of CAD in white Australians. Due to the existence of common pathogenetic mechanisms, involving NO, polymorphism eNOS gene presence may increases the risk of developing COPD. So perspective is study of polymorphisms eNOS gene in patients with COPD and CAD of Ukrainian population. Investigate their role as candidate genes can help to predict and prevent the appearance of comorbid disorders

Postconditioning: a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-akt pathway

Brief intermittent episodes of ischemia and reperfusion, at the onset of reperfusion after a prolonged period of ischemia, confer cardioprotection, a phenomenon termed "ischemic postconditioning" (Postcond). We hypothesized that this phenomenon may just represent a modified form of reperfusion that activates the reperfusion injury salvage kinase (RISK) pathway. Isolated perfused rat hearts were subjected to: (a) 35 minutes of ischemia and 120 minutes of reperfusion, and infarct size was determined by tetrazolium staining; or (b) 35 minutes of ischemia and 7 minutes of reperfusion, and the phosphorylation states of Akt, endothelial NO synthase (eNOS), and p70S6K were determined. Postcond reduced infarct size from 51.2±3.4% to 31.5±4.1% (P<0.01), an effect comparable with ischemic preconditioning (IPC; 27.5±2.3%; P<0.01). Of interest, the combined protective effects of IPC and Postcond were not additive (30.1±4.8% with IPC+Postcond; P=NS). Inhibiting phosphatidylinositol 3-kinase (PI3K) at reperfusion using LY or Wortmannin (Wort) during the first 15 minutes of reperfusion completely abolished Postcond-induced protection (31.5±4.1% with Postcond versus 51.7±4.5% with Postcond+LY, P<0.01; 56.2±10.1% with Postcond+ Wort; P<0.01), suggesting that Postcond protects the heart by activating PI3K-Akt. Western blot analysis demonstrated that Postcond induced a significant increase in phosphorylation of Akt, eNOS, and p70S6K in an LY- and Wort-sensitive manner. In conclusion, we show for the first time that ischemic Postcond protects the myocardium by activating the prosurvival kinases PI3K-Akt, eNOS, and p70S6K in accordance with the RISK pathway

Plasma from Volunteers Breathing Helium Reduces Hypoxia-Induced Cell Damage in Human Endothelial Cells-Mechanisms of Remote Protection Against Hypoxia by Helium.

PurposeRemote ischemic preconditioning protects peripheral organs against prolonged ischemia/reperfusion injury via circulating protective factors. Preconditioning with helium protected healthy volunteers against postischemic endothelial dysfunction. We investigated whether plasma from helium-treated volunteers can protect human umbilical vein endothelial cells (HUVECs) against hypoxia in vitro through release of circulating of factors.MethodsHealthy male volunteers inhaled heliox (79% helium, 21% oxygen) or air for 30 min. Plasma was collected at baseline, directly after inhalation, 6 h and 24 h after start of the experiment. HUVECs were incubated with either 5% or 10% of the plasma for 1 or 2 h and subjected to enzymatically induced hypoxia. Cell damage was measured by LDH content. Furthermore, caveolin 1 (Cav-1), hypoxia-inducible factor (HIF1α), extracellular signal-regulated kinase (ERK)1/2, signal transducer and activator of transcription (STAT3) and endothelial nitric oxide synthase (eNOS) were determined.ResultsPrehypoxic exposure to 10% plasma obtained 6 h after helium inhalation decreased hypoxia-induced cell damage in HUVEC. Cav-1 knockdown in HUVEC abolished this effect.ConclusionsPlasma of healthy volunteers breathing helium protects HUVEC against hypoxic cell damage, possibly involving circulating Cav-1

A computational model for analysis of uncoupled NO synthase on nitric oxide and superoxide interaction in microcirculation

Nitric oxide (NO) produced by endothelial cells is a key component for blood-vessel dilation. Dilation is achieved through smooth muscle relaxation as a response to NO transport. Inhibition of this process occurs through the inactivation of NO by reactive oxygen species, especially superoxide (O2 -). NO and superoxide react quickly, forming peroxynitrite (ONOO-). Both superoxide and peroxynitrite apply oxidative stress on vascular tissue. Experimental studies investigating NO interactions are difficult since these reactions occur rapidly and over small distances. This study presents a computational model to describe the interactions of NO, superoxide, and peroxynitrite across an arteriole/venule pair. Based on principles of mass transport, and using knowledge of chemical concentrations and reaction rates, a mathematical model was developed to generate the concentration profiles for NO, O2 -, and ONOO-. We simulated excessive oxidative stress by uncoupled eNOS and determined its effect on NO concentration profiles throughout the region. Based on our understanding of the interactions involved, we predicted 1) increased oxidative stress in the venule decreases NO levels in regions of both the venule and neighboring arteriole, and 2) the amount of NO reduction will vary depending on the location of O2 - increase. The model demonstrates that different sources of O2 - have varied effects on NO concentration profiles, and excessive oxidative stress in the venule can impact NO levels in the venule as well as the arteriole. The results provide a more complete description of nitric oxide transfer, which is an important step toward understanding vascular complications in many pathological conditions

Hydrogen Sulfide Donor GYY4137 Acts Through Endothelial Nitric Oxide to Protect Intestine in Murine Models of Necrotizing Enterocolitis and Intestinal Ischemia

BACKGROUND: Necrotizing enterocolitis (NEC) in premature infants is often a devastating surgical condition with poor outcomes. GYY4137 is a long-acting donor of hydrogen sulfide, a gasotransmitter that is protective against intestinal injury in experimental NEC, likely through protection against injury secondary to ischemia. We hypothesized that administration of GYY4137 would improve mesenteric perfusion, reduce intestinal injury, and reduce inflammatory responses in experimental NEC and ischemia-reperfusion injury, and that these benefits would be mediated through endothelial nitric oxide synthase-dependent pathways. METHODS: NEC was induced in C57BL/6 wild-type (WT) and endothelial nitric oxide synthase (eNOS) knockout (eNOSKO) pups via maternal separation, formula feeding, enteral lipopolysaccharide, and intermittent hypoxic and hypothermic stress. Pups received daily intraperitoneal injections of 50 mg/kg GYY4137 or phosphate buffered saline vehicle. In separate groups, adult male WT and eNOSKO mice underwent superior mesenteric artery occlusion for 60 min. Before abdominal closure, 50 mg/kg GYY4137 or phosphate buffered saline vehicle was administered into the peritoneal cavity. Laser doppler imaging was used to assess mesenteric perfusion of pups at baseline and on postnatal day 9, and the adult mice at baseline and 24 h after ischemic insult. After euthanasia, the terminal ileum of each animal was fixed, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Sections were blindly graded using published injury scores. Intestinal tissue was homogenized and cytokines measured by ELISA. Data were compared using Mann-Whitney U test, and P-values <0.05 were significant. RESULTS: After NEC and ischemia reperfusion (I/R) injury, GYY4137 improved perfusion in WT mice compared to vehicle, but this effect was lost in the eNOSKO animals. Histologic injury followed a similar pattern with reduced intestinal injury in WT mice treated with GYY4137, and no significant improvement in the eNOSKO group. Cytokine expression after GYY4137 administration was altered by the ablation of eNOS in both NEC and I/R injury groups, with significant differences noted in Interleukin 6 and vascular endothelial growth factor. CONCLUSIONS: GYY4137, a long-acting donor of hydrogen sulfide, has potential as a therapeutic compound for NEC. It improves mesenteric perfusion and intestinal injury in experimental NEC and intestinal I/R injury, and these benefits appear to be mediated through eNOS-dependent pathways