Untersuchungen zur Bedeutung von CAVEOLIN-1, VE-CADHERIN und CONNEXIN-43 an der Helium-vermittelten Regulation der endothelialen Permeabilität

Background: Recent studies suggested that helium is able to protect the heart against ischemia/reperfusion injury. We have recently shown that helium attenuates permeability of monolayers of HUVEC and that helium leads to decreased intra-cellular protein levels of caveolin-1 (Cav-1). Nevertheless, the underlying cellular and molecular mechanisms and the precise role of Cav-1 in helium-mediated regulation of endothelial barrier integrity are still unclear. We therefore investigated whether Cav-1 is involved in helium-mediated stabilization of the endothelial barrier and whether the expression of the key junctional molecules vascular endothelial-cadherin (VE-cadherin) and connexin-43 (Cx43) is regulated by helium and might be involved in the effects of helium on the endothelial barrier function. Methods: HUVEC were either exposed to 20 min of helium or control gas. Permeability was measured using confluent monolayers of HUVEC on microporous membranes and FITC-BSA flow through. To investigate if the effects of helium on the endothelial barrier function are related to Cav-1, HUVEC were transfected with Cav-1 siRNA prior to helium application. Expression of VE-cadherin and Cx43 was analyzed by western blotting. Results: While helium attenuated permeability of HUVEC monolayers in non-transfected HUVEC, effects of helium on cell permeability were abrogated in Cav-1 siRNA transfected HUVEC, suggesting a key role of Cav-1 in endothelial permeability. In the helium group, protein analyses in cell lysates showed a significantly higher expression of VE-cadherin and Cx43 at 6 and 12 hours. Transfecting HUVEC with Cav-1 siRNA abolished the effect of helium on VE-cadherin and Cx43 expression, pointing towards a role of Cav-1 in the helium-induced alteration of junctional molecule expression. Conclusion: Our findings suggest that Cav-1 is involved in the helium-mediated attenuation of HUVEC permeability and in the helium-mediated increase in the expression of VE-cadherin and Cx43.Hintergrund: Wir zeigten kürzlich, dass Helium die Permeabilität von HUVEC-Monolayers und die intrazellulären Proteinspiegel von Caveolin-1 (Cav-1) verringert. Dennoch sind die zugrundeliegenden zellulären und molekularen Mechanismen und die genaue Rolle von Cav-1 bei der Helium-vermittelten Regulation der Endothelbarriere noch unklar. Wir untersuchten daher, ob Cav-1 an der Helium-vermittelten Stabilisierung der endothelialen Barrierefunktion beteiligt ist und ob die Expression von VE-Cadherin und Connexin-43 (Cx43) durch Helium reguliert wird und möglicherweise an der Wirkung von Helium auf die endotheliale Barrierefunktion beteiligt ist. Methoden: HUVEC wurden entweder mit 20 min Helium oder Kontrollgas behandelt. Die Permeabilität wurde mittels konfluenter HUVEC-Monolayers auf mikroporösen Membranen und FITC-BSA Durchflusses gemessen. Um zu untersuchen, ob die Auswirkungen von Helium auf die endotheliale Barrierefunktion von Cav-1 abhängig sind, wurden HUVEC vor der Heliumapplikation mit Cav-1-siRNA transfiziert. Die Expression von VE-Cadherin und Cx43 wurde durch Western Blot gemessen. Ergebnisse: Während Helium die Permeabilität von HUVEC-Monolayers in nicht transfizierten HUVEC verminderte, wurde die Wirkung von Helium auf die Permeabilität in Cav-1-siRNA-transfizierten HUVEC aufgehoben, was auf eine Schlüsselrolle von Cav-1 für die Endothelpermeabilität hindeutet. In der Heliumgruppe zeigten Proteinanalysen von Zelllysaten eine signifikant höhere Expression von VE-Cadherin und Cx43 nach 6 und 12 Stunden. Die Transfektion von HUVEC mit Cav-1-siRNA hob die Wirkung von Helium auf die Expression von VE-Cadherin und Cx43 auf und wies auf eine Rolle von Cav-1 bei der Helium-induzierten Regulation der Expression von VE-Cadherin und Cx43 hin. Schlussfolgerung: Unsere Ergebnisse legen nahe, dass Cav-1 an der Helium-vermittelten Verminderung der endothelialen Permeabilität und erhöhten Expression von VE-Cadherin und Cx43 beteiligt ist

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

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

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

Reducing mitochondrial bound hexokinase II mediates transition from non-injurious into injurious ischemia/reperfusion of the intact heart

textabstractIschemia/reperfusion (I/R) of the heart becomes injurious when duration of the ischemic insult exceeds a certain threshold (approximately ≥20 min). Mitochondrial bound hexokinase II (mtHKII) protects against I/R injury, with the amount of mtHKII correlating with injury. Here, we examine whether mtHKII can induce the transition from non-injurious to injurious I/R, by detaching HKII from mitochondria during a non-injurious I/R interval. Additionally, we examine possible underlying mechanisms (increased reactive oxygen species (ROS), increased oxygen consumption (MVO2) and decreased cardiac energetics) associated with this transition. Langendorff perfused rat hearts were treated for 20 min with saline, TAT-only or 200 nM TAT-HKII, a peptide that translocates HKII from mitochondria. Then, hearts were exposed to non-injurious 15-min ischemia, followed by 30-min reperfusion. I/R injury was determined by necrosis (LDH release) and cardiac mechanical recovery. ROS were measured by DHE fluorescence. Changes in cardiac respiratory activity (cardiac MVO2 and efficiency and mitochondrial oxygen tension (mitoPO2) using protoporphyrin IX) and cardiac energetics (ATP, PCr, ∆GATP) were determined following peptide treatment. When exposed to 15-min ischemia, control hearts had no necrosis and 85% recovery of function. Conversely, TAT-HKII treatment resulted in significant LDH release and reduced cardiac recovery (25%), indicating injurious I/R. This was associated with increased ROS during ischemia and reperfusion. TAT-HKII treatment reduced MVO2 and improved energetics (increased PCr) before ischemia, without affecting MVO2/RPP ratio or mitoPO2. In conclusion, a reduction in mtHKII turns non-injurious I/R into injurious I/R. Loss of mtHKII was associated with increased ROS during ischemia and reperfusion, but not with increased MVO2 or decreased cardiac energetics before damage occurs

Antimicrobial effect of diallyl sulphide on Campylobacter jejuni biofilms

OBJECTIVES: Bacterial biofilms pose significant food safety risks because of their attachment to fomites and food surfaces, including fresh produce surfaces. The purpose of this study was to systematically investigate the activity of selected antimicrobials on Campylobacter jejuni biofilms. METHODS: C. jejuni biofilms and planktonic cells were treated with ciprofloxacin, erythromycin and diallyl sulphide and examined using infrared and Raman spectroscopies coupled with imaging analysis. RESULTS: Diallyl sulphide eliminated planktonic cells and sessile cells in biofilms at a concentration that was at least 100-fold less than used for either ciprofloxacin or erythromycin on the basis of molarity. Distinct cell lysis was observed in diallyl sulphide-treated planktonic cells using immunoblot analysis and was confirmed by a rapid decrease in cellular ATP. Two phases of C. jejuni biofilm recalcitrance modes against ciprofloxacin and erythromycin were validated using vibrational spectroscopies: (i) an initial hindered adsorption into biofilm extracellular polymeric substance (EPS) and delivery of antibiotics to sessile cells within biofilms; and (ii) a different interaction between sessile cells in a biofilm compared with their planktonic counterparts. Diallyl sulphide destroyed the EPS structure of the C. jejuni biofilm, after which the sessile cells were killed in a similar manner as planktonic cells. Spectroscopic models can predict the survival of sessile cells within biofilms. CONCLUSIONS: Diallyl sulphide elicits strong antimicrobial activity against planktonic and sessile C. jejuni and may have applications for reducing the prevalence of this microbe in foods, biofilm reduction and, potentially, as an alternative chemotherapeutic agent for multidrug-resistant bacterial strains

Infrared and Raman Spectroscopic Studies of the Antimicrobial Effects of Garlic Concentrates and Diallyl Constituents on Foodborne Pathogens

The antimicrobial effects of garlic (Allium sativum) extract (25, 50, 75, 100, and 200 μl/ml) and diallyl sulfide (5, 10 and 20 μM) on Listeria monocytogenes and Escherichia coli O157:H7 cultivated in tryptic soy broth at 4, 22 and 35°C for up to 7 days were investigated. L. monocytogenes was more resistant to garlic extract and diallyl compounds treatment than E. coli O157:H7. Fourier transform Infrared (FT-IR) spectroscopy indicated that diallyl constituents contributed more to the antimicrobial effect than phenolic compounds. This effect was verified by Raman spectroscopy and Raman mapping on single bacteria. Scanning electron microscope (SEM) and transmission electron microscope (TEM) showed cell membrane damage consistent with spectroscopic observation. The degree of bacterial cell injury could be quantified using chemometric methods