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

PURPOSE: Remote 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. METHODS: Healthy 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. RESULTS: Prehypoxic 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. CONCLUSIONS: Plasma of healthy volunteers breathing helium protects HUVEC against hypoxic cell damage, possibly involving circulating Cav-1.

Helium alters the cytoskeleton and decreases permeability in endothelial cells cultured in vitro through a pathway involving Caveolin-1.

Caveolins are involved in anaesthetic-induced cardioprotection. Actin filaments are located in close connection to Caveolins in the plasma membrane. We hypothesised that helium might affect the cytoskeleton and induce secretion of Caveolin. HCAEC, HUVEC and Cav-1 siRNA transfected HUVEC were exposed for 20 minutes to either helium (5% CO2, 25% O2, 70% He) or control gas (5% CO2, 25% O2, 70% N2). Cells and supernatants were collected for infrared Western blot analysis, immunofluorescence staining, nanoparticle tracking analysis and permeability measurements. Helium treatment increased the cortical localisation of F-actin fibers in HUVEC. After 6 hours, helium decreased cellular Caveolin-1 (Cav-1) levels and increased Cav-1 levels in the supernatant. Cell permeability was decreased 6 and 12 hours after helium treatment, and increased levels of Vascular Endothelial - Cadherin (VE-Cadherin) and Connexin 43 (Cx43) were observed. Transfection with Cav-1 siRNA abolished the effects of helium treatment on VE-Cadherin, Cx43 levels and permeability. Supernatant obtained after helium treatment reduced cellular permeability in remote HUVEC, indicating that increased levels of Cav-1 are responsible for the observed alterations. These findings suggest that Cav-1 is secreted after helium exposure in vitro, altering the cytoskeleton and increasing VE-Cadherin and Cx43 expression resulting in decreased permeability in HUVEC.

Effect of helium pre- or postconditioning on signal transduction kinases in patients undergoing coronary artery bypass graft surgery.

BACKGROUND: The noble gas helium induces pre- and postconditioning in animals and humans. Volatile anesthetics induce cardioprotection in humans undergoing coronary artery bypass graft (CABG) surgery. We hypothesized that helium induces pre- and postconditioning in CABG-patients, affecting signaling molecules protein kinase C-epsilon (PKC-ε), p38 mitogen activated protein kinase (p38 MAPK), extracellular signal-regulated kinase 1/2 (ERK-1/2) and heat shock protein 27 (HSP-27) within cardiac tissue, and reducing postoperative troponin levels. METHODS: After ethical approval and informed consent, 125 elective patients undergoing CABG surgery were randomised into this prospective, placebo controlled, investigator blinded, parallel arm single-centre study. Helium preconditioning (3 × 5 min of 70 % helium and 30 % oxygen) was applied before aortic cross clamping; postconditioning (15 min of helium) was applied before release of the aortic cross clamp. Signaling molecules were measured in right atrial appendix specimens. Troponin-T was measured at 4, 12, 24 and 48 h postoperatively. RESULTS: Baseline characteristics of all groups were similar. Helium preconditioning did not significantly alter the primary outcome (molecular levels of kinases PKC-ε and HSP-27, ratio of activated p38 MAPK or ERK ½). Postoperative troponin T was 11 arbitrary units [5, 31; area-under-the-curve (interquartile range)] for controls, and no statistically significant changes were observed after helium preconditioning [He-pre: 11 (6, 18)], helium postconditioning [He-post: 11 (8, 15)], helium pre- and postconditioning [He-PP: 14 (6, 20)] and after sevoflurane preconditioning [APC: 12 (8, 24), p = 0.13]. No adverse effects related to study treatment were observed in this study. CONCLUSIONS: No effect was observed of helium preconditioning, postconditioning or the combination thereof on activation of p38 MAPK, ERK 1/2 or levels of HSP27 and PKC-ε in the human heart. Helium pre- and postconditioning did not affect postoperative troponin release in patients undergoing CABG surgery. Clinical trial number Dutch trial register ( http://www.trialregister.nl/ ) number NTR1226.

Effects of helium on inflammatory and oxidative stress-induced endothelial cell damage.

Helium induces preconditioning in human endothelium protecting against postischemic endothelial dysfunction. Circulating endothelial microparticles are markers of endothelial dysfunction derived in response to injury. Another noble gas, xenon, protected human umbilical vein endothelial cells (HUVEC) against inflammatory stress in vitro. We hypothesised that helium protects the endothelium in vitro against inflammatory and oxidative stress. HUVEC were isolated from fresh umbilical cords and grown upon confluence. Cells were subjected to starving medium for 12h before the experiment and treated for either 3 × 5 min or 1 × 30 min with helium (5% CO2, 25% O2, 70% He) or control gas (5% CO2, 25% O2, 70% N2) in a specialised gas chamber. Subsequently, cells were stimulated with TNF-α (40 ng/ml for 24h or 10 ng/ml for 2h) or H2O2 (500 µM for 2h) or left untreated. Adhesion molecule expression was analysed using real-time quantitative polymerase chain reaction. Caspase-3 expression and viability of the cells was measured by flowcytometry. Microparticles were investigated by nanoparticle tracking analysis. Helium had no effect on adhesion molecule expression after TNF-α stimulation but in combination with oxidative stress decreased cell viability (68.9 ± 1.3% and 58 ± 1.9%) compared to control. Helium further increased TNF-α induced release of caspase-3 containing particles compared to TNF-α alone (6.4 × 10(6) ± 1.1 × 10(6) and 2.9 × 10(6) ± 0.7 × 10(6), respectively). Prolonged exposure of helium increased microparticle formation (2.4 × 10(9) ± 0.5 × 10(9)) compared to control (1.7 × 10(9) ± 0.2 × 10(9)). Summarized, helium increases inflammatory and oxidative stress-induced endothelial damage and is thus not biologically inert. A possible noxious effects on the cellular level causing alterations in microparticle formation both in number and content should be acknowledged.

Plasma from human volunteers subjected to remote ischemic preconditioning protects human endothelial cells from hypoxia-induced cell damage.

Short repeated cycles of peripheral ischemia/reperfusion (I/R) can protect distant organs from subsequent prolonged I/R injury; a phenomenon known as remote ischemic preconditioning (RIPC). A RIPC-mediated release of humoral factors might play a key role in this protection and vascular endothelial cells are potential targets for these secreted factors. In the present study, RIPC-plasma obtained from healthy male volunteers was tested for its ability to protect human umbilical endothelial cells (HUVEC) from hypoxia-induced cell damage. 10 healthy male volunteers were subjected to a RIPC-protocol consisting of 4 × 5 min inflation/deflation of a blood pressure cuff located at the upper arm. Plasma was collected before (T0; control), directly after (T1) and 1 h after (T2) the RIPC procedure. HUVEC were subjected to 24 h hypoxia damage and simultaneously incubated with 5% of the respective RIPC-plasma. Cell damage was evaluated by lactate dehydrogenase (LDH)-measurements. Western blot experiments of hypoxia inducible factor 1 alpha (HIF1alpha), phosphorylated signal transducer and activator of transcription 5 (STAT5), protein kinase B (AKT) and extracellular signal-related kinase 1/2 (ERK-1/2) were performed. Furthermore, the concentrations of hVEGF were evaluated in the RIPC-plasma by sandwich ELISA. Hypoxia-induced cell damage was significantly reduced by plasma T1 (p = 0.02 vs T0). The protective effect of plasma T1 was accompanied by an augmentation of the intracellular HIF1alpha (p = 0.01 vs T0) and increased phosphorylation of ERK-1/2 (p = 0.03 vs T0). Phosphorylation of AKT and STAT5 remained unchanged. Analysis of the protective RIPC-plasma T1 showed significantly reduced levels of hVEGF (p = 0.01 vs T0). RIPC plasma protects endothelial cells from hypoxia-induced cell damage and humoral mediators as well as intracellular HIF1alpha may be involved.

Targets Involved in Cardioprotection by the Non-Anesthetic Noble Gas Helium.

Research data from the past decade indicate that noble gases like xenon and helium exert profound cardioprotection when applied before, during or after organ ischemia. Of all noble gases, especially helium, has gained interest in the past years because it does not have an anesthetic "side effect" like xenon, allowing application of this specific gas in numerous clinical ischemia/reperfusion situations. Because helium has several unique characteristics and no hemodynamic side effects, helium could be administered in severely ill patients. Investigations in animals as well as in humans have proven that this noble gas is not completely inert and can induce several biological effects. Though the underlying molecular mechanisms of helium-induced cardiac protection are still not yet fully understood, recently different signaling pathways have been elucidated.

Noble gases as cardioprotectants - translatability and mechanism.

Several noble gases, although classified as inert substances, exert a tissue-protective effect in different experimental models when applied before organ ischaemia as an early or late preconditioning stimulus, after ischaemia as a post-conditioning stimulus or when given in combination before, during and/or after ischaemia. A wide range of organs can be protected by these inert substances, in particular cardiac and neuronal tissue. In this review we summarize the data on noble gas-induced cardioprotection, focusing on the underlying protective mechanisms. We will also look at translatability of experimental data to the clinical situation.

Helium induces preconditioning in human endothelium in vivo.

AIMS: Helium protects myocardium by inducing preconditioning in animals. We investigated whether human endothelium is preconditioned by helium inhalation in vivo. METHODS AND RESULTS: Forearm ischemia-reperfusion (I/R) in healthy volunteers (each group n = 10) was performed by inflating a blood pressure cuff for 20 min. Endothelium-dependent and endothelium-independent responses were measured after cumulative dose-response infusion of acetylcholine and sodium nitroprusside, respectively, at baseline and after 15 min of reperfusion using strain-gauge, venous occlusion plethysmography. Helium preconditioning was applied by inhalation of helium (79% helium, 21% oxygen) either 15 min (helium early preconditioning [He-EPC]) or 24 h before I/R (helium late preconditioning). Additional measurements of He-EPC were done after blockade of endothelial nitric oxide synthase. Plasma levels of cytokines, adhesion molecules, and cell-derived microparticles were determined. Forearm I/R attenuated endothelium-dependent vasodilation (acetylcholine) with unaltered endothelium-independent response (sodium nitroprusside). Both He-EPC and helium late preconditioning attenuated I/R-induced endothelial dysfunction (max increase in forearm blood flow in response to acetylcholine after I/R was 180 ± 24% [mean ± SEM] without preconditioning, 573 ± 140% after He-EPC, and 290 ± 32% after helium late preconditioning). Protection of helium was comparable to ischemic preconditioning (max forearm blood flow 436 ± 38%) and was not abolished after endothelial nitric oxide synthase blockade. He-EPC did not affect plasma levels of cytokines, adhesion molecules, or microparticles. CONCLUSION: Helium is a nonanesthetic, nontoxic gas without hemodynamic side effects, which induces early and late preconditioning of human endothelium in vivo. Further studies have to investigate whether helium may be an instrument to induce endothelial preconditioning in patients with cardiovascular risk factors.

Effects of helium and air inhalation on the innate and early adaptive immune system in healthy volunteers ex vivo.

BACKGROUND: Helium inhalation protects myocardium, brain and endothelium against ischemia/reperfusion injury in animals and humans, when applied according to specific "conditioning" protocols. Before widespread use of this "conditioning" agent in clinical practice, negative side effects have to be ruled out. We investigated the effect of prolonged helium inhalation on the responsiveness of the human immune response in whole blood ex vivo. METHODS: Male healthy volunteers inhaled 30 minutes heliox (79%He/21%O(2)) or air in a cross over design, with two weeks between measurements. Blood was withdrawn at T0 (baseline), T1 (25 min inhalation) and T2-T5 (1, 2, 6, 24 h after inhalation) and incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), T-cell stimuli anti-CD3/ anti-CD28 (TCS) or RPMI (as control) for 2, 4 and 24 hours or not incubated (0 h). An additional group of six volunteers inhaled 60 minutes of heliox or air, followed by blood incubation with LPS and RPMI. Tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-γ (IFN-γ) and interleukin-2 (IL-2) was analyzed by cytometric bead array. Statistical analysis was performed by the Wilcoxon test for matched samples. RESULTS: Incubation with LPS, LTA or TCS significantly increased TNF-α, IL-1ß, IL-6, IL-8, IFN-γ and IL-2 in comparison to incubation with RPMI alone. Thirty min of helium inhalation did not influence the amounts of TNF-α, IL-1ß, IL-6, IL-8, IFN-γ and IL-2 in comparison to air. Sixty min of helium inhalation did not affect cytokine production after LPS stimulation. CONCLUSIONS: We conclude that 79% helium inhalation does not affect the responsiveness of the human immune system in healthy volunteers. TRIAL REGISTRATION: Dutch Trial Register: http://www.trialregister.nl/ NTR2152.