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

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Effects of helium on inflammatory and oxidative stress-induced endothelial cell damage Kirsten F. Smit a, Raphaela P Kerindongo a, Anita Böing b, Rienk Nieuwland b, Markus W. Hollmann a, Benedikt Preckel a, Nina C. Weber a,n a Academic Medical Centre (AMC), Department of Anesthesiology, Laboratory of Experimental Intensive Care and Anesthesiology (L.E.I.C.A.), Academic Medical Centre (AMC), University of Amsterdam, Meibergdreef 9, 1100 DD Amsterdam, The Netherlands b Department of Experimental Clinical Chemistry, Academic Medical Centre (AMC), Meibergdreef 9, 1100 DD Amsterdam, The Netherlands

art ic l e i nf o

a b s t r a c t

Article history: Received 30 April 2015 Received in revised form 8 June 2015 Accepted 10 June 2015

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 12 h 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 24 h or 10 ng/ml for 2 h) or H2O2 (500 μM for 2 h) 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 71.3% and 58 71.9%) compared to control. Helium further increased TNF-α induced release of caspase-3 containing particles compared to TNF-α alone (6.4
106 7 1.1
106 and 2.9
106 7 0.7
106, respectively). Prolonged exposure of helium increased microparticle formation (2.4
109 70.5
109) compared to control (1.7
109 7 0.2
109). 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. & 2015 Elsevier Inc. All rights reserved.

Keywords: Noble gases Preconditioning Inflammatory stress Microparticles Human umbilical vein endothelial cells

1. Introduction Ischemic preconditioning results in protection of organs against ischemia/reperfusion (I/R) injury by short, non-lethal periods of ischemia [1]. Besides ischemia, inhalation of volatile anesthetics [2] and noble gases [3] can induce preconditioning. The noble gas helium, which is already routinely and safely used in hospitals worldwide for asthma treatment, has no relevant hemodynamic and neurocognitive side effects, and could be the perfect preconditioning agent for future clinical applications. We recently demonstrated that helium induces both early and late preconditioning in human endothelium in vivo and attenuates postischemic endothelial dysfunction following 20 min of forearm I/R n

Corresponding author. Fax: þ 31 20 6979441. E-mail address: [email protected] (N.C. Weber).

in healthy volunteers [3]. Decreased expression of the pro-inflammatory marker CD11b and intracellular adhesion molecule 1 (ICAM-1) on leucocytes[4] after helium treatment in human volunteers has been reported. In a former study we could show that intermitted treatment with the noble gas xenon decreased ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) expression after stimulation with TNF-α in human umbilical vein endothelial cells (HUVEC), thereby protecting endothelial cells against TNF-α induced damage [5]. These data seem to be of special importance since the endothelium plays a major role during I/R and serves as a first line defence mechanism against organ and tissue injury. The protective functions of the endothelium include anti-coagulation, anti-inflammation, prevention of platelet function and regulation of permeability and vascular tone. During I/R, interactions between endothelial cells and blood constituents result in recruitment of

http://dx.doi.org/10.1016/j.yexcr.2015.06.004 0014-4827/& 2015 Elsevier Inc. All rights reserved.

Please cite this article as: K.F. Smit, et al., Effects of helium on inflammatory and oxidative stress-induced endothelial cell damage, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.06.004i

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circulating leucocytes at inflammation sites. These processes are regulated by cytoskeleton alterations and expression of cell adhesion molecules such as ICAM-1, VCAM-1, and E-selectin [6]. The release of pro-inflammatory cytokines such as TNF-α accelerate this process. In myocardial injury, reperfusion after ischemia leads to formation of reactive oxygen species that contribute to the damage inflicted.[7] Increased levels of reactive oxygen species may lead to apoptosis. Apoptosis of endothelial cells precede that of myocardial cells following I/R of the heart, and is assumed to be mediated by caspase-3 release.[8] Circulating microparticles in plasma are a marker of endothelial damage. These particles are derived from endothelial cells after injury and are used as a quantitative marker of endothelial cell dysfunction in patients [9, 10]. Interestingly, exposure to high pressure noble gases, including helium, caused oxidative stress-induced microparticles production in neutrophils [11]. We investigated two different protocols, the first consisting of short, repetitive stimuli (3
5 min) helium administration and the second consisting of one prolonged stimulus of 30 min helium administration. We here hypothesized that pre-treatment with helium protects HUVEC against inflammatory and oxidative stress-induced damage and decreases adhesion molecules, caspase-3 expression, and endothelial cell-derived microparticles, and increases cell viability after I/R.

2. Material and methods All experiments were performed in a specialised temperature controlled gas chamber (Fig. 1). Gas mixtures were administered via standard procedure as described before [5], and outlet gas concentrations were monitored by a gas analyzer (Capnomatic Ultima, Datex, Helsinki, Finland). We used a mixture of helium (5% CO2, 25% O2, 70% helium) and a mixture of control gas (5% CO2, 25% O2, 70% N2) both provided by Linde Gas Benelux, Schiedam, the Netherlands). 2.1. Materials If not mentioned otherwise, all materials used were from Sigma (Zwijndrecht, the Netherlands). Endothelial cell growth medium was obtained from Promocell (Heidelberg, Germany), medium M199 from PAN biotech (Aidenbach, Germany), Fetal Bovine Serum (FBS) from PAA (Colbe, Germany), Penicillin-Streptomycin, Amphotericin B, Trypsine-EDTA from Gibco (Paisley, UK), L-glutamine 200 mM from Gibco (Paisley, UK), and collagenase A from Roche (Mannheim, Germany). AnnexinV-Fluorescein isothiocyanate (FITC) and IgGpoly-FITC

were obtained from Immuno Quality Products (Groningen, The Netherlands), anti-human Caspase 3 monoclonal antibody from BD Pharmingen, Franklin Lakes, NJ) and sheep anti human von Willebrand factor-FITC from Serotec (Wiesbaden, Germany). 2.2. Isolation of human umbilical vein endothelial cells (HUVEC) HUVEC were collected from human umbilical veins as described previously [5] (Waiver: W12-167#12.17.096, Ethical Committee AMC, Amsterdam). Cells were cultured in gelatine (0.75%) coated 25- cm2 flasks (passage 0). Experiments were performed with cells of passage 3 and 4. HUVEC were identified using antibodies against von Willebrand factor. Fluorescence-activated cell sorting analysis (FACS) of von Willebrand factor revealed that the cell preparation was 99.6% pure (data not shown). All experiments were performed 3 times. 2.3. Experimental protocol The experimental protocol is outlined in Fig. 1. Upon confluence, cells were put in a resting medium (M199, containing 100 mM L-glutamine) supplemented with penicillin-streptomycin, amphotericin B, and extra L-glutamine (200 mM) for 10 h. After each cycle the medium was refreshed to assure complete washout of the treatment. The short pre-treatment protocol consisted of 3
5 min of gas (3 L/min) subsequently followed by 3
5 min of rest medium. The long pre-treatment consisted of 30 min of gas treatment (3 L/min) without interruptions. After the respective pre-treatment protocol, HUVEC were either stimulated with H2O2 (500 μM for 2 h), TNF-α (10ng/ml for 2 h for analysis of adhesion molecules, and 40 ng/ml for 24 h for viability) or left untreated. 2.4. Flowcytometry analysis Attached cells were removed using trypsine and subsequently neutralized with M199 supplemented with FBS10%. Detached cells were isolated from culture supernatants by centrifugation (218g, 4 °C for 10 min). Both cell suspensions were separately centrifuged (218g, 4 °C for 10 min) and resuspended with PBS supplemented with 1% FBS. Adherent cells were resuspended with 600 μl PBS/ FCS1%, and detached cells were resuspended in 300 μl PBS/FCS1%. Cell suspensions of both, attached and detached cells, and supernatant were prepared for analysis of caspase 3 and annexin V as previously described [12]. For analysis of caspase 3 we used the FITC active caspase 3 apoptosis kit from BD biosciences (San Diego, CA, USA). Both, attached and detached cell suspensions were prepared for analysis as previously described [12]. Samples were analysed in a fluorescence automated cell sorter (FACS Calibur)

Fig. 1. Protocol Outline. The short protocol consisted of 3 times 5 min helium (70%), after each cycle, media were exchanged to ensure washout. Long protocol consisted of one cycle of 30 min helium after which medium was exchanged.

Please cite this article as: K.F. Smit, et al., Effects of helium on inflammatory and oxidative stress-induced endothelial cell damage, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.06.004i

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Target

Primer Sequence (5ʹ to 3ʹ)

ICAM-1

Forward: TATGGCAACGACTCCTTCT Reverse: CAT-TCA-GCG-TCA-CCT-TGG

VCAM-1

Forward: CTA GCG TGT ACC CCC TTG AC Reverse: AGT GTT TGC GTA CTC TGC CT

E-Selectine

Forward: CTC-TGA-CAG-AAG-AAG-CCA-A Reverse: ACT-TGA-GTC-CAG-TGA-AGC-CA

28S

Forward: AAC GAG ATT CCC ACT GTC CC Reverse: CTT CAC CGT GCC AGA CTA GAG

3

significantly increased expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin compared to controls (Fig. 2). Both helium protocols did not affect adhesion molecule expression after TNF-α stimulation, nor did helium alone. Stimulation with 500 μM H2O2 did not increase the expression of adhesion molecules ICAM-1 and E-selectin compared to controls. Oxidative stress did significantly decrease the amount of VCAM-1 expression with and without 30 min of helium. No effects were seen in VCAM-1 expression in the 3
5 min protocol (see supplementary data figure S1). Concluding, helium had no effect on inflammatory stress induced adhesion molecule expression, and oxidative stress did not increase adhesion molecule expression in HUVEC. Since we did not investigate the effect of helium on protein levels, we cannot exclude a translational effect of helium in inflammatory and oxidative stress.

with CellQuest software (Becton Dickinson, San Jose, CA, USA). 3.2. Effect of helium on cell viability and necrosis 2.5. Real-time quantitative PCR and Data Analysis For the measurement of mRNA for ICAM-1, VCAM-1, E-Selectin and 28S 1 ml cDNA was used in a total volume of 10 ml PCR mix per reaction. Each mixture contained 10 mM of primer pairs (see tas ble 1) and 2x LightCycler 480 SYBR Green I Master (Roche, The Netherlands). Real-time qPCR amplification was carried out using s the LightCycler 480 instrument (Roche, The Netherlands) under the following conditions: pre-incubation at 95 ˚C for 10 min, followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C for 15 s. The raw data analysis was conducted using the programs LC480Converter and LinRegPCR. For each sample PCR efficiency was determined. Subsequently the means of the PCR efficiencies per target were used to calculate the estimated starting concentration per sample. Afterwards, each target gene was normalized to the gene 28S [13]. 2.6. Nanoparticle tracking analysis Particle concentration and size distribution in collected supernatant was measured with NTA (NS500; Nanosight, Amesbury, UK) as described previously [14]. Calibration and configuration were done with silica beads (100 nm diameter; Microspheres– Nanospheres, Cold Spring, NY). Fractions were diluted x-fold in PBS and of each fraction, 10 videos of 30-s duration were captured. All fractions were analysed using the same threshold, which was calculated by custom-made software (MATLAB v.7.9.0.529). Analysis was performed by the instrument software (NTA 2.3.0.15). 2.7. Statistics Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA). Except nanoparticles tracking analysis data, all data were normally distributed. NTA data were normalised using log transformation. All data were analysed using a one way analysis of variance (ANOVA) with a Bonferroni correction for multiple testing. Values of P o 0.05 were considered statistically significant.

3. Results 3.1. Effect of helium on cellular activation after inflammatory and oxidative stress

To examine the effect of helium and inflammatory and oxidative stress on cell viability we assessed cytometric staining of Annexin V and PI. TNF-α stimulation (40ng/ml TNF-α for 24 h) did not decrease cell viability (see supplementary data figure S2). Oxidative stress in combination with helium administered 3
5 min or 1
30 min significantly decreased cell viability compared to controls (Fig. 3A þB, dark blue bar, left panel, respectively). This reduction of cell viability appears to be due to an increased percentage of necrotic cells, which stained positive for PI and negative for Annexin V (Fig. 3 A and B, dark blue bar, right panel respectively). Exposure of 30 min of helium alone did increase the percentage of necrotic cells whereas exposure of 3
5 min of helium did not (Fig. 3B). No increase in early apoptotic cells was observed in cells after H2O2-stimulation and 3
5 min helium plus H2O2 (9.072) or 1
30 min helium (15.4 71) compared to controls (14.1 70.1 and 15.1 70.2), respectively. Summarizing, inflammatory stress did not affect cell viability. Oxidative stress in combination with 30 min of helium decreased cell viability and increased necrosis. 3.3. Effect of helium on caspase-3 production Caspase-3 production of cells is a marker of cellular apoptosis, and we investigated the effect of helium and inflammatory and oxidative stress on apoptosis by measuring caspase-3 production. Following inflammatory stress, both detached and adherent cells were collected and stained for caspase-3. However, no significant effect on detached and adherent cells was observed in caspase-3 positive cells in any of the groups. Further analysis demonstrated that inflammatory stress significantly increased the amount of caspase-3 positive microparticles. Exposure of cells to helium 3
5 min further increased TNF-α induced increment of caspase-3 containing particles compared to TNF-α alone (6.4
105 71.1
105 and 2.9
105 70.7
105, respectively see also Fig. 4A). Exposure to 1
30 min of helium also showed a trend towards further increased caspase-3 containing microparticles (Fig. 4B). Oxidative stress did not increase the amount of caspase-3 in cells (Fig. 4C þD). Exposure of 30 minutes of helium alone and in combination with oxidative stress reduced caspase-3 positive cells. In conclusion helium increases apoptosis after inflammatory stress, and oxidative stress does not induce apoptosis in this model. 3.4. Helium induces microparticle release in HUVEC

We investigated the effect of helium on adhesion molecule expression after inflammatory stress and oxidative stress. TNF-α stimulation (10ng/ml) causes activation of HUVEC resulting in

Helium further increased caspase-3 containing microparticles, and we investigated the effect of helium and inflammatory and

Please cite this article as: K.F. Smit, et al., Effects of helium on inflammatory and oxidative stress-induced endothelial cell damage, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.06.004i

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Fig. 2. Effect of helium on cellular activation after inflammatory stress. Results of real time quantitative PCR of ICAM-1, VCAM-1 and E-Selectin normalized to 28S (panel A: 3
5 min helium, panel B: 1
30 min helium). Data are mean 7SEM. N ¼5. *(Po 0.05) represents significantly different value compared to controls. One way ANOVA for multiple comparisons and bonferroni correction. Con ¼controls, TNF¼ TNF-α, He ¼ Helium, HeTNF ¼ Heliumþ TNF-α.

oxidative stress on general microparticle production. Regardless of the damage model used (inflammatory or oxidative stress), 3
5 min helium induced no differences in the amount of particles

released (Fig. 5 A and C). However, prolonged exposure of 1
30 min of helium, caused an increase in the amount of particles released after both inflammatory and oxidative stress.

1x30 protocol H2O2

3x5 protocol: H2O2 % necrotic cells

100

*

#

5

20 0

10

*

Con H2O2 He HeH 2O2

0

80

% cells

15

% cells

% cells

40

#

% necrotic cells

100

20

80 60

% viable cells

60 40

20

#

15

% cells

% viable cells

5

20 Con H2O2 He HeH 2O2

0

10

Con H2O2 He HeH 2O2

0

#

*

Con H2O2 He HeH 2O2

Fig. 3. Effect of helium on cell viability after oxidative stress. Results of flowcytometry of harvested cells stained with annexin-V and propidium iodide. Cells that are negative for both annexin-V and PI are considered viable cells. Annexin-V and PI positive cells are considered necrotic cells. Panel A: 3
5 min helium, panel B: 1
30 min helium. Data are mean7 SEM. N ¼ 3. * (Po 0.05) represents values significantly different compared to controls, # (Po 0.05) represents value significantly different compared to all groups. Data were analysed with one way ANOVA for multiple comparisons and Bonferroni correction. Con ¼ controls, He ¼ Helium, HeH2O2 ¼Helium þH2O2.

Please cite this article as: K.F. Smit, et al., Effects of helium on inflammatory and oxidative stress-induced endothelial cell damage, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.06.004i

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Fig. 4. Effect of helium on apoptosis after inflammatory and oxidative stress. Results of cytometry of caspase 3 on cells (both adherent and detached) and in microparticles (mipa). The amount of microparticles are values *10 [5]. Panel A and C: 3
5 min helium, panels B and D: 1
30 min helium. Data are represented as mean7 SEM. N¼ 3. * (Po 0.05) values significantly different compared to controls, # (Po 0.05) represents value significantly different compared to all groups. Data were analysed with ANOVA for multiple comparisons and Bonferroni correction. Con ¼ controls, TNF ¼ TNF-α, He ¼Helium, HeTNF ¼Helium þTNF-α, HeH2O2 ¼ Helium þH2O2.

TNF-α stimulation (4.3
109 70.5
109), helium alone (3.1
109 70.5109), and the combination of helium and TNF-α 3.6
109 70.3
109) significantly increased the amount of particles released compared to controls (1.9
109 70.3
109, Fig. 5B). Additionally, exposure to 1
30 min of helium alone and in combination with H2O2-stimulation significantly increased the amount of particles compared to controls (3.4
109 70.5
109 and 3.6
109 70.6
109 respectively; Fig. 5D). Stimulation with H2O2 without helium did not increase the amount of particles released (P4 0.05 with ANOVA and Bonferroni correction after log transformation).

4. Discussion The major findings of the present study are that pre-treating HUVEC with different protocols of helium does not protect endothelial cells against inflammatory or oxidative stress, but surprisingly enough increased damage when given in combination with TNF-α or H2O2. Inflammatory stress increased adhesion molecules ICAM-1, VCAM-1 and E-selectin, but pre-treatment of helium had no effect on expression of those adhesion molecules. This is in contrast to pre-treatment of HUVEC with other noble gases, for example xenon, which decreased inflammatory stress induced expression of ICAM-1 and VCAM-1 [3]. Unlike helium, xenon has anesthetic properties and shares some mechanisms in preconditioning with other volatile anesthetics [5]. Stimulation with 100 and 500 μM H2O2 did not increase

adhesion molecules in a consistent manner. This is consistent with earlier findings, showing that 100 μM H2O2 did not increase E-selectin and VCAM-1 expression in HUVEC [15], and 800 mM H2O2 did not affect E-selectin expression and even decreased vascular endothelial cadherin and platelet endothelial cell adhesion molecule-1 expression in aortic endothelial cells measured by flowcytometric analysis [16]. Caspase-3 is produced after cell activation and is a crucial step in regulated cell death. Employing HUVEC, adherent cells did not show any signs of apoptosis or accumulation of caspase-3 following stimulation with interleukin (Il)-1α, but interestingly caspase-3 containing microparticles were found in the supernatant [12]. Apparently, these caspase-3 containing microparticles originated from viable cells, since the use of blockers to inhibit microparticle formation resulted in accumulation of caspase-3 in adherent cells and ultimately led to increased cell death [17]. It was postulated that active caspase-3 is sorted in microparticles as a cellular survival mechanism against apoptosis. Our data seem to share these findings since no increase in caspase-3 positive adherent or detached cells was observed, but a significant increase in production of caspase-3 containing microparticles was found [18]. This suggests that HUVEC successfully survived TNF-α stimulation by producing caspase-3 containing microparticles. In the present study, helium interestingly affects this mechanism by increasing the amount of caspase-3 containing microparticles after TNF-α stimulation without affecting cell viability. The exact mechanism of protein sorting of caspase-3 is yet not fully understood, however data suggest that active caspase-3 is colocalized with Caveolin-1 in cardiac endothelial cells [19].

Please cite this article as: K.F. Smit, et al., Effects of helium on inflammatory and oxidative stress-induced endothelial cell damage, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.06.004i

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Fig. 5. Effect of helium of microparticles release. Data of nanoparticle tracking analysis. Data are represented as means 7 SEM. N ¼3. * P o0.05 represents values significantly different compared to controls. Data were analysed with one-way ANOVA for multiple comparisons and Bonferroni correction after log transformation. Panels A and C: 3
5 min helium, panels B and D: 1
30 min helium pretreatment.

Caveolins are structural proteins that are essential in formation of caveolae or “little caves”, which are cholesterol- and sphingolipidenriched invaginations of the plasma membrane and are considered a subset of lipid rafts [20]. Caveolins are known to activate the protective cell survival pathway Phosphatidylinositol 3 Kinase/ Akt, that ultimately preconditions the heart [21]. With respect to helium, Caveolin-1 and 3 are secreted into the blood after helium inhalation in mice which supports the hypothesis that circulating factors in the blood stream may be involved in inducing organ protection by helium [22]. Nanoparticle tracking analysis of the cell culture supernatant showed that 30 min of helium indeed caused a significant increase in microparticle production compared to exposure to control gas. This is in contrast to exposure to 3
5 min of helium, which did not increase the amount of released particles. Thus, these data show for the first time that prolonged exposure to 70% helium under atmospheric conditions increases microparticle formation in endothelial cells. As helium is used in clinical practice in similar concentration and at a similar pressure, these effects may be present in endothelium of patients as well. Helium-induced increase in microparticle production was previously described in neutrophils exposed to partial pressures of helium up to 690 kPa [11]. We did not find an increased number of endothelial cell derived microparticles after helium inhalation [3]. It might be that the stimulus of 3
5 min helium application was not strong enough to induce microparticle release; however, measurement was also complicated by the low amount of endothelial cell derived microparticles found in the plasma of healthy volunteers [3]. A limitation of our study was the failure of inducing apoptosis in HUVEC after exposing them to 40 ng/ml TNF-α for 24 h. This is in contrast to other findings [23,24] showing not only induced apoptosis after this dosage of TNF-α but also a dose dependent protection by propofol preconditioning. However, these previous

experiments were all performed in the pre-established cell line ECV304, which is not of HUVEC origin and is claimed to be an inappropriate cell line to investigate endothelial cell biology [25]. Earlier research demonstrated that in HUVEC without alterations, TNF-α does not induce apoptosis and does not decrease cell viability under physiological conditions, even in extremely high dosages [26] (4 10 fold the concentration we used). Moreover, aging cells were shown to be more susceptible to TNF-α, and only in aged HUVEC significant differences in viability or caspase-3 were observed [27]. This could explain the failure to induce apoptosis in our cultivated cells, because we only processed young cells and kept physiological conditions. For stimulation with H2O2 we encountered similar problems, as 100 mM and 500 mM H2O2 for 2 h did not decrease cell viability. Other studies, using the pre-established cell line CRL-1730, were able to reduce viability and induce apoptosis with 100 μM H2O2 [28,29]. Another study demonstrated that young endothelial cells are able to protect themselves against the detrimental effects of oxidative stress and stimulation with 500 μM H2O2 for 18 h was needed to induce apoptosis, indicating that our stimulus may have been too short to induce cell death [30]. With respect to cell viability, we found no decrease after inflammatory or oxidative stress. Surprisingly, oxidative stress in combination with helium pretreatment for 3
5 min or 1
30 min did in fact decrease cell viability. This may indicate that the combination of helium and H2O2 is strong enough to cause cell death in HUVEC. H2O2 stimulation in endothelial cells leads to superoxide production via (uncoupled) nitric oxide synthase and NAPDH oxidase [31] which can induce cellular injury [32]. Treating neutrophils with hyperbaric noble gases increased formation of reactive oxygen species, mediated by collision-induced superoxide formation dependent of the properties and size of the gas used (helium oargon) [11]. Also, helium (and argon) increased activity of inducible nitric oxide synthase resulting in increased NO2 and peroxynitrite production, which ultimately increases microparticle formation [11]. Hypothetically, helium may aggravate H2O2 induced oxidative stress in HUVEC. The exact role of helium in oxidative stress needs to be investigated further, as well as the role of nitric oxide synthase in this scenario. Previously, we blocked eNOS during helium preconditioning in human volunteers, but this did not block post-ischemic helium induced endothelial protection [3]. Normobaric application of helium to our cells did not result in a decreased cell viability, but we did observe an increase in necrotic cells (PI positive, Annexin-V negative) after treatment with helium for 30 min but not for 3
5 min. This may suggest that 30 min of helium is harmful, and intermitted treatment by 3
5 min of helium is not. Recent research shows that oxidative stress may result in programmed cell death without involvement of caspases, so-called “necroptosis” [33]. We were able to show an increase of PI-positive cells after oxidative stress, yet no increase of annexin-V positive cells, indicating necrosis instead of apoptosis. When looking at the data of caspase-3 positive cells, exposure of helium seems to reduce the amount of caspase-3 positive cells. This would suggest that exposure to 30 min of helium lowers the normal caspase-3 metabolism by stimulating oxidative stress induced necrosis. There is compelling evidence that helium administered in vivo induces preconditioning in humans [3] and animals [34], although the mechanisms underlying this protection remains unclear [35]. Recent data shows helium postconditioning (15 min of helium administered after I/R) involved upregulation of genes involved in autophagy and inhibition of apoptosis. Helium upregulated 27 of 30 genes involved in autophagy, and 12 of 14 antiapoptotic genes [36]. In conclusion, helium administered in vitro for 3
5 min or 1
30 min does not reduce inflammatory and oxidative stress-

Please cite this article as: K.F. Smit, et al., Effects of helium on inflammatory and oxidative stress-induced endothelial cell damage, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.06.004i

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induced damage in HUVEC. In fact, helium treatment for 1
30 min aggravates oxidative stress induced damage. Regardless of the damage model employed, helium is not biologically inert but induces cellular activation leading to increased microparticle formation and altered content. Furthermore, helium increased the amount of caspase-3 containing microparticles after TNF-α stimulation. Further research is necessary to identify the mechanism behind these effects of helium.

[15.]

[16.]

[17.]

Summary statement in table of contents The major finding of the present study is that helium treatment increases inflammatory and oxidative stress-induced endothelial damage.

[18.]

[19.] [20.]

Acnowledgments None.

[21.]

[22.]

Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2015.06.004.

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Please cite this article as: K.F. Smit, et al., Effects of helium on inflammatory and oxidative stress-induced endothelial cell damage, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.06.004i