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Organ protection by the noble gas helium
Smit, K.F.
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Smit, K. F. (2017). Organ protection by the noble gas helium
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Download date: 17 Jun 2017
Chapter 7
ơ‡…–•‘ˆŠ‡Ž‹—‘‹ƪƒƒ–‘”›ƒ†‘š‹†ƒ–‹˜‡
stress-induced endothelial cell damage
Kirsten F. Smit
Raphaela P Kerindongo
Anita Böing
Rienk Nieuwland
Markus W. Hollmann
Benedikt Preckel
Nina C. Weber
Experimental Cell Research 2015;337(1):37-43.
ABSTRACT
Background: 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
Š—ƒ—„‹Ž‹…ƒŽ˜‡‹‡†‘–Š‡Ž‹ƒŽ…‡ŽŽ•ȋȌƒ‰ƒ‹•–‹ƪƒƒ–‘”›•–”‡••‹˜‹–”‘Ǥ‡Š›’‘–Š‡•‹•‡†–Šƒ–Š‡Ž‹—’”‘–‡…–•–Š‡‡†‘–Š‡Ž‹—‹˜‹–”‘ƒ‰ƒ‹•–‹ƪƒƒ–‘”›ƒ†‘š‹†ƒ–‹˜‡
stress.
Methods:™‡”‡‹•‘Žƒ–‡†ˆ”‘ˆ”‡•Š—„‹Ž‹…ƒŽ…‘”†•ƒ†‰”‘™—’‘…‘ƪ—‡…‡Ǥ‡ŽŽ•
were subjected to starving medium for 12 h before the experiment and treated for either 3x5
min or 1x30 min with helium (5%CO2, 25%O2, 70%helium) or control gas (5%CO2, 25%O2,
70%N2Ȍ‹ƒ•’‡…‹ƒŽ‹•‡†‰ƒ•…Šƒ„‡”Ǥ—„•‡“—‡–Ž›ǡ…‡ŽŽ•™‡”‡•–‹—Žƒ–‡†™‹–ŠǦȽȋ͘͜‰Ȁ
ml for 24 hours or 10ng/ml for 2 hours) or H2O2ȋ͘͘͝Ɋˆ‘”͚Š‘—”•Ȍ‘”Ž‡ˆ–—–”‡ƒ–‡†Ǥ†Š‡•‹‘
molecule expression was analysed using real-time quantitative polymerase chain reaction.
ƒ•’ƒ•‡Ǧ͛‡š’”‡••‹‘ƒ†˜‹ƒ„‹Ž‹–›‘ˆ–Š‡…‡ŽŽ•™ƒ•‡ƒ•—”‡†„›ƪ‘™…›–‘‡–”›Ǥ‹…”‘’ƒ”–‹…Ž‡•
were investigated by nanoparticle tracking analysis.
Results:‡Ž‹—Šƒ†‘‡ơ‡…–‘ƒ†Š‡•‹‘‘Ž‡…—Ž‡‡š’”‡••‹‘ƒˆ–‡”ǦȽ•–‹—Žƒ–‹‘„—–‹
combination with oxidative stress decreased cell viability (68.9±1.3% and 58±1.9%) compared
–‘…‘–”‘ŽǤ‡Ž‹—ˆ—”–Š‡”‹…”‡ƒ•‡†ǦȽ‹†—…‡†”‡Ž‡ƒ•‡‘ˆ…ƒ•’ƒ•‡Ǧ͛…‘–ƒ‹‹‰’ƒ”–‹…Ž‡•
…‘’ƒ”‡† –‘ ǦȽ ƒŽ‘‡ ȋ͞Ǥ͜ȗ͙͘6±1.1*106 and 2.9*106±0.7*106, respectively). Prolonged
exposure of helium increased microparticle formation (2.4*109 ±0.5*109) compared to control
(1.7*109 ±0.2*109).
Conclusion: ‡Ž‹— ‹…”‡ƒ•‡• ‹ƪƒƒ–‘”› ƒ† ‘š‹†ƒ–‹˜‡ •–”‡••Ǧ‹†—…‡† ‡†‘–Š‡Ž‹ƒŽ †ƒƒ‰‡ƒ†‹•–Š—•‘–„‹‘Ž‘‰‹…ƒŽŽ›‹‡”–Ǥ’‘••‹„Ž‡‘š‹‘—•‡ơ‡…–•‘–Š‡…‡ŽŽ—Žƒ”Ž‡˜‡Ž…ƒ—•‹‰
alterations in microparticle formation both in number and content should be acknowledged.
118
Chapter 7 : Effect of helium on inflammatory and oxidative stress
INTRODUCTION
•…Š‡‹… ’”‡…‘†‹–‹‘‹‰ ”‡•—Ž–• ‹ ’”‘–‡…–‹‘ ‘ˆ ‘”‰ƒ• ƒ‰ƒ‹•– ‹•…Š‡‹ƒȀ”‡’‡”ˆ—•‹‘ ȋȀȌ
injury by short, non-lethal periods of ischemia.1 Besides ischemia, inhalation of volatile anesthetics2 and noble gases3 can induce preconditioning. The noble gas helium, which is already
routinely and safely used in hospitals worldwide for asthma treatment, has no relevant he‘†›ƒ‹…ƒ†‡—”‘…‘‰‹–‹˜‡•‹†‡‡ơ‡…–•ǡƒ†…‘—Ž†„‡–Š‡’‡”ˆ‡…–’”‡…‘†‹–‹‘‹‰ƒ‰‡–
for future clinical applications. We recently demonstrated that helium induces both early and
late preconditioning in human endothelium in vivo and attenuates post-ischemic endothelial
†›•ˆ—…–‹‘ˆ‘ŽŽ‘™‹‰͚͘‹—–‡•‘ˆˆ‘”‡ƒ”Ȁ‹Š‡ƒŽ–Š›˜‘Ž—–‡‡”•Ǥ3 Decreased expression
‘ˆ –Š‡ ’”‘Ǧ‹ƪƒƒ–‘”› ƒ”‡” ͙͙„ ƒ† ‹–”ƒ…‡ŽŽ—Žƒ” ƒ†Š‡•‹‘ ‘Ž‡…—Ž‡ ͙ ȋǦ͙Ȍ ‘
leucocytes4 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
™‹–ŠǦȽ‹Š—ƒ—„‹Ž‹…ƒŽ˜‡‹‡†‘–Š‡Ž‹ƒŽ…‡ŽŽ•ȋȌǡ–Š‡”‡„›’”‘–‡…–‹‰‡†‘–Š‡Ž‹ƒŽ
…‡ŽŽ•ƒ‰ƒ‹•–ǦȽ‹†—…‡††ƒƒ‰‡Ǥ5
These data seem to be of special importance since the endothelium plays a major role dur‹‰Ȁƒ†•‡”˜‡•ƒ•ƒƤ”•–Ž‹‡†‡ˆ‡…‡‡…Šƒ‹•ƒ‰ƒ‹•–‘”‰ƒƒ†–‹••—‡‹Œ—”›ǤŠ‡’”‘–‡…–‹˜‡ˆ—…–‹‘•‘ˆ–Š‡‡†‘–Š‡Ž‹—‹…Ž—†‡ƒ–‹Ǧ…‘ƒ‰—Žƒ–‹‘ǡƒ–‹Ǧ‹ƪƒƒ–‹‘ǡ’”‡˜‡–‹‘
‘ˆ’Žƒ–‡Ž‡–ˆ—…–‹‘ƒ†”‡‰—Žƒ–‹‘‘ˆ’‡”‡ƒ„‹Ž‹–›ƒ†˜ƒ•…—Žƒ”–‘‡Ǥ—”‹‰Ȁǡ‹–‡”ƒ…–‹‘•
between endothelial cells and blood constituents result in recruitment of circulating leuco…›–‡• ƒ– ‹ƪƒƒ–‹‘ •‹–‡•ǤŠ‡•‡ ’”‘…‡••‡• ƒ”‡ ”‡‰—Žƒ–‡† „› …›–‘•‡Ž‡–‘ ƒŽ–‡”ƒ–‹‘• ƒ†
expression of cell adhesion molecules such as ICAM-1, VCAM-1, and E-selectin.6 The release
‘ˆ ’”‘Ǧ‹ƪƒƒ–‘”› …›–‘‹‡• •—…Š ƒ•ǦȽ ƒ……‡Ž‡”ƒ–‡ –Š‹• ’”‘…‡••Ǥ  ›‘…ƒ”†‹ƒŽ ‹Œ—”›ǡ
reperfusion after ischemia leads to formation of reactive oxygen species that contribute to the
†ƒƒ‰‡‹ƪ‹…–‡†Ǥ7 Increased levels of reactive oxygen species may lead to apoptosis. Apop–‘•‹•‘ˆ‡†‘–Š‡Ž‹ƒŽ…‡ŽŽ•’”‡…‡†‡–Šƒ–‘ˆ›‘…ƒ”†‹ƒŽ…‡ŽŽ•ˆ‘ŽŽ‘™‹‰Ȁ‘ˆ–Š‡Š‡ƒ”–ǡƒ†‹•ƒ•sumed 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
‡ ‹˜‡•–‹‰ƒ–‡† –™‘ †‹ơ‡”‡– ’”‘–‘…‘Ž•ǡ –Š‡ Ƥ”•– …‘•‹•–‹‰ ‘ˆ •Š‘”–ǡ ”‡’‡–‹–‹˜‡ •–‹—Ž‹
(3x5 minutes) helium administration and the second consisting of one prolonged stimulus
of 30 minutes helium administration. We here hypothesized that pre-treatment with helium
’”‘–‡…–• ƒ‰ƒ‹•– ‹ƪƒƒ–‘”› ƒ† ‘š‹†ƒ–‹˜‡ •–”‡••Ǧ‹†—…‡† †ƒƒ‰‡ ƒ† †‡…”‡ƒ•‡•
adhesion molecules, caspase-3 expression, and endothelial cell-derived microparticles, and
‹…”‡ƒ•‡•…‡ŽŽ˜‹ƒ„‹Ž‹–›ƒˆ–‡”ȀǤ
119
MATERIAL AND METHODS
All experiments were performed in a specialised temperature controlled gas chamber (Ƥ‰—”‡
1). Gas mixtures were administered via standard procedure as described before5, and outlet
gas concentrations were monitored by a gas analyzer (Capnomatic Ultima, Datex, Helsinki,
‹Žƒ†ȌǤ‡—•‡†ƒ‹š–—”‡‘ˆŠ‡Ž‹—ȋ͝ά2, 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).
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Figure 1: Protocol Outline
The short protocol consisted of 3 times 5 minutes helium (70%), after each cycle, media were exchanged
to ensure washout. Long protocol consisted of one cycle of 30 minutes helium after which medium was
exchanged.
Materials
If not mentioned otherwise, all materials used were from Sigma (Zwijndrecht, the Netherlands). Endothelial cell growth medium was obtained from Promocell (Heidelberg, Germany),
‡†‹— ͙͡͡ ˆ”‘ „‹‘–‡…Š ȋ‹†‡„ƒ…Šǡ ‡”ƒ›Ȍǡ ‡–ƒŽ ‘˜‹‡ ‡”— ȋȌ ˆ”‘
PAA (Colbe, Germany), Penicillin-Streptomycin, Amphotericin B, Trypsine-EDTA from Gibco
ȋƒ‹•Ž‡›ǡȌǡ Ǧ‰Ž—–ƒ‹‡ ͚͘͘ ˆ”‘
‹„…‘ ȋƒ‹•Ž‡›ǡȌǡ ƒ† …‘ŽŽƒ‰‡ƒ•‡ ˆ”‘ ‘…Š‡
ȋƒŠ‡‹ǡ
‡”ƒ›ȌǤ‡š‹ǦŽ—‘”‡•…‡‹ ‹•‘–Š‹‘…›ƒƒ–‡ ȋȌ ƒ† ‰
’‘Ž›Ǧ ™‡”‡
obtained from Immuno Quality Products (Groningen, The Netherlands), anti-human Caspase
͛‘‘…Ž‘ƒŽƒ–‹„‘†›ˆ”‘Šƒ”‹‰‡ǡ”ƒŽ‹ƒ‡•ǡȌƒ†•Š‡‡’ƒ–‹Š—ƒ˜‘
‹ŽŽ‡„”ƒ†ˆƒ…–‘”Ǧˆ”‘‡”‘–‡…ȋ‹‡•„ƒ†‡ǡ
‡”ƒ›ȌǤ
120
Chapter 7 : Effect of helium on inflammatory and oxidative stress
Isolation of human umbilical vein endothelial cells (HUVEC)
HUVEC were collected from human umbilical veins as described previously5 (Waiver: W12167#12.17.096, Ethical Committee AMC, Amsterdam). Cells were cultured in gelatine (0.75%)
coated 25-cm2ƪƒ••ȋ’ƒ••ƒ‰‡͘ȌǤš’‡”‹‡–•™‡”‡’‡”ˆ‘”‡†™‹–Š…‡ŽŽ•‘ˆ’ƒ••ƒ‰‡͛ƒ†͜Ǥ
™‡”‡‹†‡–‹Ƥ‡†—•‹‰ƒ–‹„‘†‹‡•ƒ‰ƒ‹•–˜‘‹ŽŽ‡„”ƒ†ˆƒ…–‘”ǤŽ—‘”‡•…‡…‡Ǧƒ…–‹˜ƒ–‡†
…‡ŽŽ •‘”–‹‰ ƒƒŽ›•‹• ȋȌ ‘ˆ ˜‘‹ŽŽ‡„”ƒ† ˆƒ…–‘” ”‡˜‡ƒŽ‡† –Šƒ– –Š‡ …‡ŽŽ ’”‡’ƒ”ƒ–‹‘ ™ƒ•
99.6% pure (data not shown). All experiments were performed 3 times.
Experimental Protocol
The experimental protocol is outlined in Ƥ‰—”‡͙Ǥ’‘…‘ƪ—‡…‡ǡ…‡ŽŽ•™‡”‡’—–‹ƒ”‡•–‹‰
medium (M199, containing 100mM L-glutamine) supplemented with penicillin-streptomycin,
amphotericin B, and extra L-glutamine (200mM) for 10 hours. After each cycle the medium
was refreshed to assure complete washout of the treatment. The short pre-treatment protocol consisted of 3x5 minutes of gas (3L/min) subsequently followed by 3x5 minutes of rest
medium. The long pre-treatment consisted of 30 minutes of gas treatment (3L/min) without
interruptions. After the respective pre-treatment protocol, HUVEC were either stimulated
with H2O2ȋ͘͘͝Ɋˆ‘”͚Š‘—”•ȌǡǦȽȋ͙͘‰ȀŽˆ‘”͚Š‘—”•ˆ‘”ƒƒŽ›•‹•‘ˆƒ†Š‡•‹‘‘Ž‡…—Ž‡•ǡ
and 40ng/ml for 24 hours for viability) or left untreated.
Flowcytometry analysis
Attached cells were removed using trypsine and subsequently neutralized with M199 supple‡–‡†™‹–Š͙͘άǤ‡–ƒ…Š‡†…‡ŽŽ•™‡”‡‹•‘Žƒ–‡†ˆ”‘…—Ž–—”‡•—’‡”ƒ–ƒ–•„›…‡–”‹ˆ—‰ƒtion (218g, 4°C for 10 minutes). Both cell suspensions were separately centrifuged (218g, 4°C
ˆ‘”͙͘‹—–‡•Ȍƒ†”‡•—•’‡†‡†™‹–Š•—’’Ž‡‡–‡†™‹–Š͙άǤ†Š‡”‡–…‡ŽŽ•™‡”‡
”‡•—•’‡†‡† ™‹–Š ͘͘͞ɊŽ Ȁ͙άǡ ƒ† †‡–ƒ…Š‡† …‡ŽŽ• ™‡”‡ ”‡•—•’‡†‡† ‹ ͛͘͘ɊŽ Ȁ
͙άǤ‡ŽŽ•—•’‡•‹‘•‘ˆ„‘–Šǡƒ––ƒ…Š‡†ƒ††‡–ƒ…Š‡†…‡ŽŽ•ǡƒ†•—’‡”ƒ–ƒ–™‡”‡’”‡’ƒ”‡†
for analysis of caspase 3 and annexin V as previously described.12‘”ƒƒŽ›•‹•‘ˆ…ƒ•’ƒ•‡͛™‡
—•‡†–Š‡ƒ…–‹˜‡…ƒ•’ƒ•‡͛ƒ’‘’–‘•‹•‹–ˆ”‘„‹‘•…‹‡…‡•ȋƒ‹‡‰‘ǡǡȌǤ‘–Šǡ
attached and detached cell suspensions were prepared for analysis as previously described12.
ƒ’Ž‡•™‡”‡ƒƒŽ›•‡†‹ƒƪ—‘”‡•…‡…‡ƒ—–‘ƒ–‡†…‡ŽŽ•‘”–‡”ȋƒŽ‹„—”Ȍ™‹–Š‡ŽŽ—‡•–
software (Becton Dickinson, San Jose, CA, USA).
Real-time quantitative PCR and Data Analysis
‘”–Š‡‡ƒ•—”‡‡–‘ˆˆ‘”Ǧ͙ǡǦ͙ǡǦ‡Ž‡…–‹ƒ†͚͙͠σŽ…™ƒ•—•‡†
‹ƒ–‘–ƒŽ˜‘Ž—‡‘ˆ͙͘σŽ‹š’‡””‡ƒ…–‹‘Ǥƒ…Š‹š–—”‡…‘–ƒ‹‡†͙͘σ‘ˆ’”‹‡”’ƒ‹”•
(see table 1Ȍƒ†͚š‹‰Š–›…Ž‡”̾͘͜͠
”‡‡ƒ•–‡”ȋ‘…Š‡ǡŠ‡‡–Š‡”Žƒ†•ȌǤ‡ƒŽǦ
–‹‡“ƒ’Ž‹Ƥ…ƒ–‹‘™ƒ•…ƒ””‹‡†‘—–—•‹‰–Š‡‹‰Š–›…Ž‡”̾͘͜͠‹•–”—‡–ȋ‘…Š‡ǡŠ‡
‡–Š‡”Žƒ†•Ȍ—†‡”–Š‡ˆ‘ŽŽ‘™‹‰…‘†‹–‹‘•ǣ’”‡Ǧ‹…—„ƒ–‹‘ƒ–͡͝Ǐˆ‘”͙͘‹ǡˆ‘ŽŽ‘™‡†„›
͜͝…›…Ž‡•‘ˆ͡͝Ǐˆ‘”͙͘•‡…ǡ͘͞Ǐˆ‘”͙͘•‡…ƒ†͚͟Ǐˆ‘”͙͝•‡…Ǥ
121
Š‡”ƒ™†ƒ–ƒƒƒŽ›•‹•™ƒ•…‘†—…–‡†—•‹‰–Š‡’”‘‰”ƒ•͘͜͠‘˜‡”–‡”ƒ†‹‡‰Ǥ
‘” ‡ƒ…Š •ƒ’Ž‡ ‡ƥ…‹‡…› ™ƒ• †‡–‡”‹‡†Ǥ —„•‡“—‡–Ž› –Š‡ ‡ƒ• ‘ˆ –Š‡ ‡ƥciencies per target were used to calculate the estimated starting concentration per sample.
Afterwards, each target gene was normalized to the gene 28S.13
Nanoparticle Tracking Analysis
Particle concentration and size distribution in collected supernatant was measured with NTA
ȋ͘͘͝Ǣ ƒ‘•‹‰Š–ǡ‡•„—”›ǡȌ ƒ• †‡•…”‹„‡† ’”‡˜‹‘—•Ž›Ǥ14ƒŽ‹„”ƒ–‹‘ ƒ† …‘Ƥ‰—”ƒ–‹‘
™‡”‡†‘‡™‹–Š•‹Ž‹…ƒ„‡ƒ†•ȋ͙͘͘†‹ƒ‡–‡”Ǣ‹…”‘•’Š‡”‡•Ǧƒ‘•’Š‡”‡•ǡ‘Ž†’”‹‰ǡȌǤ
”ƒ…–‹‘•™‡”‡†‹Ž—–‡†šǦˆ‘Ž†‹ƒ†‘ˆ‡ƒ…Šˆ”ƒ…–‹‘ǡ͙͘˜‹†‡‘•‘ˆ͛͘Ǧ•‡…‘†•†—”ƒ–‹‘
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).
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
‘ˆ˜ƒ”‹ƒ…‡ȋȌ™‹–Šƒ‘ˆ‡””‘‹…‘””‡…–‹‘ˆ‘”—Ž–‹’Ž‡–‡•–‹‰ǤƒŽ—‡•‘ˆζ͘Ǥ͘͝™‡”‡
…‘•‹†‡”‡†•–ƒ–‹•–‹…ƒŽŽ›•‹‰‹Ƥ…ƒ–Ǥ
RESULTS
ơ‡…–‘ˆŠ‡Ž‹—‘…‡ŽŽ—Žƒ”ƒ…–‹˜ƒ–‹‘ƒˆ–‡”‹ƪƒƒ–‘”›ƒ†‘š‹†ƒ–‹˜‡•–”‡••
‡ ‹˜‡•–‹‰ƒ–‡† –Š‡ ‡ơ‡…– ‘ˆ Š‡Ž‹— ‘ ƒ†Š‡•‹‘ ‘Ž‡…—Ž‡ ‡š’”‡••‹‘ ƒˆ–‡” ‹ƪƒƒ–‘”›
•–”‡••ƒ†‘š‹†ƒ–‹˜‡•–”‡••ǤǦȽ•–‹—Žƒ–‹‘ȋ͙͘‰ȀŽȌ…ƒ—•‡•ƒ…–‹˜ƒ–‹‘‘ˆ”‡•—Ž–‹‰
‹ •‹‰‹Ƥ…ƒ–Ž› ‹…”‡ƒ•‡† ‡š’”‡••‹‘ ‘ˆ ƒ†Š‡•‹‘ ‘Ž‡…—Ž‡• Ǧ͙ǡǦ͙ ƒ† Ǧ•‡Ž‡…–‹
compared to controls (‹‰—”‡ ͚ȌǤ ‘–Š Š‡Ž‹— ’”‘–‘…‘Ž• †‹† ‘– ƒơ‡…– ƒ†Š‡•‹‘ ‘Ž‡…—Ž‡
‡š’”‡••‹‘ƒˆ–‡”ǦȽ•–‹—Žƒ–‹‘ǡ‘”†‹†Š‡Ž‹—ƒŽ‘‡Ǥ
122
Chapter 7 : Effect of helium on inflammatory and oxidative stress
3x5 protocol: TNF-Į
ICAM-1
VCAM-1
2.0
5
ICAM -1/28S
1.0
n.s.
0.5
VCAM -1/28S
4
1.5
*
E-selectin
2.0
n.s.
E-Selectine/28S
A
*
3
2
1
0.0
0
Con TNF He HeTNF
1.5
n.s.
1.0
*
0.5
0.0
Con TNF He HeTNF
Con TNF He HeTNF
1x30 protocol: TNF-Į
B
ICAM-1
VCAM-1
2.0
1.0
n.s.
*
0.0
n.s.
3
*
2
1
0
Con TNF He HeTNF
E-Selectine/28S
4
VCAM -1/28S
ICAM -1/28S
2.0
5
1.5
0.5
E-selectin
1.5
1.0
0.5
n.s.
*
0.0
Con TNF He HeTNF
Con TNF He HeTNF
‹‰—”‡͚ǣ ơ‡…–‘ˆŠ‡Ž‹—‘…‡ŽŽ—Žƒ”ƒ…–‹˜ƒ–‹‘ƒˆ–‡”‹ƪƒƒ–‘”›•–”‡••
‡•—Ž–•‘ˆ”‡ƒŽ–‹‡“—ƒ–‹–ƒ–‹˜‡‘ˆǦ͙ǡǦ͙ƒ†Ǧ‡Ž‡…–‹‘”ƒŽ‹œ‡†–‘͚͠ȋ’ƒ‡Žǣ͛š͝‹—–‡•Š‡Ž‹—ǡ’ƒ‡Žǣ͙š͛͘‹—–‡•Š‡Ž‹—ȌǤƒ–ƒƒ”‡‡ƒΰǤγ͝Ǥȗȋζ͘Ǥ͘͝Ȍ”‡’”‡•‡–••‹‰‹Ƥ…ƒ–Ž›
†‹ơ‡”‡–˜ƒŽ—‡…‘’ƒ”‡†–‘…‘–”‘Ž•Ǥ‡™ƒ›ˆ‘”—Ž–‹’Ž‡…‘’ƒ”‹•‘•ƒ†„‘ˆ‡””‘‹…‘””‡…–‹‘Ǥ
‘γ…‘–”‘Ž•ǡγǦȽǡ‡γ‡Ž‹—ǡ‡γ‡Ž‹—ήǦȽǤ
–‹—Žƒ–‹‘™‹–Š͘͘͝Ɋ2O2 did not increase the expression of adhesion molecules ICAM-1
ƒ†Ǧ•‡Ž‡…–‹…‘’ƒ”‡†–‘…‘–”‘Ž•Ǥš‹†ƒ–‹˜‡•–”‡••†‹†•‹‰‹Ƥ…ƒ–Ž›†‡…”‡ƒ•‡–Š‡ƒ‘—–‘ˆ
Ǧ͙‡š’”‡••‹‘™‹–Šƒ†™‹–Š‘—–͛͘‹—–‡•‘ˆŠ‡Ž‹—Ǥ‘‡ơ‡…–•™‡”‡•‡‡‹Ǧ͙
‡š’”‡••‹‘‹–Š‡͛š͝‹—–‡•’”‘–‘…‘Žȋ•‡‡•—’’Ž‡‡–ƒ”›†ƒ–ƒƤ‰—”‡͙ȌǤ‘…Ž—†‹‰ǡŠ‡Ž‹—
Šƒ† ‘ ‡ơ‡…– ‘ ‹ƪƒƒ–‘”› •–”‡•• ‹†—…‡† ƒ†Š‡•‹‘ ‘Ž‡…—Ž‡ ‡š’”‡••‹‘ǡ ƒ† ‘š‹†ƒ–‹˜‡
stress did not increase adhesion molecule expression in HUVEC. Since we did not investigate
123
–Š‡‡ơ‡…–‘ˆŠ‡Ž‹—‘’”‘–‡‹Ž‡˜‡Ž•ǡ™‡…ƒ‘–‡š…Ž—†‡ƒ–”ƒ•Žƒ–‹‘ƒŽ‡ơ‡…–‘ˆŠ‡Ž‹—‹
‹ƪƒƒ–‘”›ƒ†‘š‹†ƒ–‹˜‡•–”‡••Ǥ
ơ‡…–‘ˆŠ‡Ž‹—‘…‡ŽŽ˜‹ƒ„‹Ž‹–›ƒ†‡…”‘•‹•
‘ ‡šƒ‹‡ –Š‡ ‡ơ‡…– ‘ˆ Š‡Ž‹— ƒ† ‹ƪƒƒ–‘”› ƒ† ‘š‹†ƒ–‹˜‡ •–”‡•• ‘ …‡ŽŽ ˜‹ƒ„‹Ž‹–› ™‡
assessed cytometric staining of Annexin V and PI.
ǦȽ•–‹—Žƒ–‹‘ȋ͘͜‰ȀŽǦȽˆ‘”͚͜Š‘—”•Ȍ†‹†‘–†‡…”‡ƒ•‡…‡ŽŽ˜‹ƒ„‹Ž‹–›ȋ•‡‡•—’’Ž‡‡–ƒ”›†ƒ–ƒƤ‰—”‡͚ȌǤš‹†ƒ–‹˜‡•–”‡••‹…‘„‹ƒ–‹‘™‹–ŠŠ‡Ž‹—ƒ†‹‹•–‡”‡†͛š͝‹—–‡•‘”͙š͛͘‹—–‡••‹‰‹Ƥ…ƒ–Ž›†‡…”‡ƒ•‡†…‡ŽŽ˜‹ƒ„‹Ž‹–›…‘’ƒ”‡†–‘…‘–”‘Ž•ȋ‹‰—”‡͛A+B,
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 (‹‰—”‡͛ A and B, dark blue bar, right panel respectively). Exposure of 30 minutes of helium
alone did increase the percentage of necrotic cells whereas exposure of 3x5 minutes of helium
did not (‹‰—”‡͛B). No increase in early apoptotic cells was observed in cells after H2O2 -stimulation and 3x5 minutes helium plus H2O2 (9.0±2) or 1x30 minutes helium (15.4±1) compared
–‘ …‘–”‘Ž• ȋ͙͜Ǥ͙ΰ͘Ǥ͙ ƒ† ͙͝Ǥ͙ΰ͘Ǥ͚Ȍǡ ”‡•’‡…–‹˜‡Ž›Ǥ—ƒ”‹œ‹‰ǡ ‹ƪƒƒ–‘”› •–”‡•• †‹† ‘–
ƒơ‡…–…‡ŽŽ˜‹ƒ„‹Ž‹–›Ǥš‹†ƒ–‹˜‡•–”‡••‹…‘„‹ƒ–‹‘™‹–Š͛͘‹—–‡•‘ˆŠ‡Ž‹—†‡…”‡ƒ•‡†…‡ŽŽ
viability and increased necrosis.
3x5 protocol: H2O2
% necrotic cells
100
40
% cells
% cells
60
#
15
#
*
*
10
5
20
0
% necrotic cells
80
60
40
20
#
15
0
Con H2O2 He HeH 2O2
10
5
20
0
Con H2O2 He HeH 2O2
% viable cells
100
20
80
1x30 protocol H2O2
% cells
% viable cells
B
% cells
A
#
*
0
Con H2O2 He HeH 2O2
Con H2O2 He HeH2O2
Figure 3: ơ‡…–‘ˆŠ‡Ž‹—‘…‡ŽŽ˜‹ƒ„‹Ž‹–›ƒˆ–‡”‘š‹†ƒ–‹˜‡•–”‡••
‡•—Ž–• ‘ˆ ƪ‘™…›–‘‡–”› ‘ˆ Šƒ”˜‡•–‡† …‡ŽŽ• •–ƒ‹‡† ™‹–Š ƒ‡š‹Ǧ ƒ† ’”‘’‹†‹— ‹‘†‹†‡Ǥ‡ŽŽ• –Šƒ– ƒ”‡
negative for both annexin-V and PI are considered viable cells. Annexin-V and PI positive cells are considered
necrotic cells. Panel A: 3x5 minutes helium, panel B: 1x30 minutes helium. Data are mean ± SEM. N=3. *
ȋζ͘Ǥ͘͝Ȍ”‡’”‡•‡–•˜ƒŽ—‡••‹‰‹Ƥ…ƒ–Ž›†‹ơ‡”‡–…‘’ƒ”‡†–‘…‘–”‘Ž•ǡ #ȋζ͘Ǥ͘͝Ȍ”‡’”‡•‡–•˜ƒŽ—‡•‹‰‹Ƥ…ƒ–Ž›†‹ơ‡”‡–…‘’ƒ”‡†–‘ƒŽŽ‰”‘—’•Ǥƒ–ƒ™‡”‡ƒƒŽ›•‡†™‹–Š‘‡™ƒ›ˆ‘”—Ž–‹’Ž‡…‘’ƒ”‹•‘•
and Bonferroni correction. Con=controls, He=Helium, HeH2O2=Helium + H2O2.
124
Chapter 7 : Effect of helium on inflammatory and oxidative stress
ơ‡…–‘ˆŠ‡Ž‹—‘…ƒ•’ƒ•‡Ǧ͛’”‘†—…–‹‘
Caspase-3 production of cells is a marker of cellular apoptosis, and we investigated the efˆ‡…– ‘ˆ Š‡Ž‹— ƒ† ‹ƪƒƒ–‘”› ƒ† ‘š‹†ƒ–‹˜‡ •–”‡•• ‘ ƒ’‘’–‘•‹• „› ‡ƒ•—”‹‰ …ƒ•’ƒ•‡Ǧ͛
’”‘†—…–‹‘Ǥ‘ŽŽ‘™‹‰‹ƪƒƒ–‘”›•–”‡••ǡ„‘–Š†‡–ƒ…Š‡†ƒ†ƒ†Š‡”‡–…‡ŽŽ•™‡”‡…‘ŽŽ‡…–‡†
ƒ†•–ƒ‹‡†ˆ‘”…ƒ•’ƒ•‡Ǧ͛Ǥ‘™‡˜‡”ǡ‘•‹‰‹Ƥ…ƒ–‡ơ‡…–‘†‡–ƒ…Š‡†ƒ†ƒ†Š‡”‡–…‡ŽŽ•™ƒ•
‘„•‡”˜‡†‹…ƒ•’ƒ•‡Ǧ͛’‘•‹–‹˜‡…‡ŽŽ•‹ƒ›‘ˆ–Š‡‰”‘—’•Ǥ—”–Š‡”ƒƒŽ›•‹•†‡‘•–”ƒ–‡†–Šƒ–
‹ƪƒƒ–‘”›•–”‡•••‹‰‹Ƥ…ƒ–Ž›‹…”‡ƒ•‡†–Š‡ƒ‘—–‘ˆ…ƒ•’ƒ•‡Ǧ͛’‘•‹–‹˜‡‹…”‘’ƒ”–‹…Ž‡•Ǥ
š’‘•—”‡ ‘ˆ …‡ŽŽ• –‘ Š‡Ž‹— ͛š͝ ‹—–‡• ˆ—”–Š‡” ‹…”‡ƒ•‡†ǦȽ ‹†—…‡† ‹…”‡‡– ‘ˆ …ƒ•’ƒ•‡Ǧ͛…‘–ƒ‹‹‰’ƒ”–‹…Ž‡•…‘’ƒ”‡†–‘ǦȽƒŽ‘‡ȋ͞Ǥ͜ȗ͙͘5±1.1*105 and 2.9*105±0.7*105,
respectively see also ‹‰—”‡͜A). Exposure to 1x30 minutes of helium also showed a trend towards further increased caspase-3 containing microparticles (‹‰—”‡͜B). Oxidative stress did
not increase the amount of caspase-3 in cells (‹‰—”‡͜C+D). Exposure of 30 minutes of helium
alone and in combination with oxidative stress reduced caspase-3 positive cells. In conclusion
Š‡Ž‹— ‹…”‡ƒ•‡• ƒ’‘’–‘•‹• ƒˆ–‡” ‹ƪƒƒ–‘”› •–”‡••ǡ ƒ† ‘š‹†ƒ–‹˜‡ •–”‡•• †‘‡• ‘– ‹†—…‡
apoptosis in this model.
1500
1000
500
0
6
4
500
0
Con H2O2 He HeH 2O2
500
0
D
2
0
1500
1000
500
*
*
0
Con H2O2 He HeH 2O2
n.s.
6
*
4
2
0
1x30 protocol: H2O2
2000
4
8
Con TNF He HeTNF
Caspase 3 cells
8
6
Caspase 3 mipa
Con TNF He HeTNF
Amount of cells
1000
1000
Caspase 3 mipa
Amount of microparticles
1500
1500
Con TNF He HeTNF
3x5 protocol: H2O2
2000
2000
0
Caspase 3 cells
Amount of cells
*
2
Con TNF He HeTNF
C
Caspase 3 cells
#
Amount of cells
amount of microparticles
Amount of cells
Caspase 3 mipa
8
Amount of microparticles
Caspase 3 cells
2000
1x30 protocol: TNF-Į
B
Con H2O2 He HeH 2O2
Caspase 3 mipa
Amount of microparticles
3x5 protocol: TNF-Į
A
8
6
4
2
0
Con H2O2 He HeH 2O2
Figure 4: ơ‡…–‘ˆŠ‡Ž‹—‘ƒ’‘’–‘•‹•ƒˆ–‡”‹ƪƒƒ–‘”›ƒ†‘š‹†ƒ–‹˜‡•–”‡••
‡•—Ž–•‘ˆ…›–‘‡–”›‘ˆ…ƒ•’ƒ•‡͛‘…‡ŽŽ•ȋ„‘–Šƒ†Š‡”‡–ƒ††‡–ƒ…Š‡†Ȍƒ†‹‹…”‘’ƒ”–‹…Ž‡•ȋ‹’ƒȌǤŠ‡
amount of microparticles are values *105. Panel A and C: 3x5 minutes helium, panel B and D: 1x30 minutes
helium. Data are represented as mean ± SEM. N=3. *ȋζ͘Ǥ͘͝Ȍ˜ƒŽ—‡••‹‰‹Ƥ…ƒ–Ž›†‹ơ‡”‡–…‘’ƒ”‡†–‘
controls, # ȋζ͘Ǥ͘͝Ȍ ”‡’”‡•‡–• ˜ƒŽ—‡ •‹‰‹Ƥ…ƒ–Ž› †‹ơ‡”‡– …‘’ƒ”‡† –‘ ƒŽŽ ‰”‘—’•Ǥ ƒ–ƒ ™‡”‡ ƒƒŽ›•‡†
™‹–Šˆ‘”—Ž–‹’Ž‡…‘’ƒ”‹•‘•ƒ†‘ˆ‡””‘‹…‘””‡…–‹‘Ǥ‘γ…‘–”‘Ž•ǡγǦȽǡ‡γ‡Ž‹—ǡ
‡γ‡Ž‹—ήǦȽǡ‡2O2=Helium + H2O2
125
Helium induces microparticle release in HUVEC
‡Ž‹—ˆ—”–Š‡”‹…”‡ƒ•‡†…ƒ•’ƒ•‡Ǧ͛…‘–ƒ‹‹‰‹…”‘’ƒ”–‹…Ž‡•ǡƒ†™‡‹˜‡•–‹‰ƒ–‡†–Š‡‡ơ‡…–
‘ˆŠ‡Ž‹—ƒ†‹ƪƒƒ–‘”›ƒ†‘š‹†ƒ–‹˜‡•–”‡••‘‰‡‡”ƒŽ‹…”‘’ƒ”–‹…Ž‡’”‘†—…–‹‘Ǥ‡‰ƒ”†Ž‡••‘ˆ–Š‡†ƒƒ‰‡‘†‡Ž—•‡†ȋ‹ƪƒƒ–‘”›‘”‘š‹†ƒ–‹˜‡•–”‡••Ȍǡ͛š͝‹—–‡•Š‡Ž‹—‹†—…‡†
‘ †‹ơ‡”‡…‡• ‹ –Š‡ ƒ‘—– ‘ˆ ’ƒ”–‹…Ž‡• ”‡Ž‡ƒ•‡† ȋ‹‰—”‡ ͝ A and C). However, prolonged
exposure of 1x30 minutes of helium, caused an increase in the amount of particles released
ƒˆ–‡”„‘–Š‹ƪƒƒ–‘”›ƒ†‘š‹†ƒ–‹˜‡•–”‡••Ǥ
3x5 protocol: TNF-Į
B
6.0×10 9
1x30 protocol: TNF-Į
amount of microparticles
amount of microparticles
A
4.0×10 9
2.0×10 9
0
6.0×10 9
*
2.0×10 9
0
Con TNF He HeTNF
D
6.0×10 9
1x30 protocol: H2O2
amount of microparticles
amount of microparticles
3x5 protocol: H2O2
*
4.0×10 9
Con TNF He HeTNF
C
*
4.0×10 9
2.0×10 9
0
Con H2O2 He HeH 2O2
6.0×10 9
*
*
4.0×10 9
2.0×10 9
0
Con H2O2 He HeH 2O2
Figure 5: ơ‡…–‘ˆŠ‡Ž‹—‘ˆ‹…”‘’ƒ”–‹…Ž‡•”‡Ž‡ƒ•‡
ƒ–ƒ ‘ˆ ƒ‘’ƒ”–‹…Ž‡ –”ƒ…‹‰ ƒƒŽ›•‹•Ǥ ƒ–ƒ ƒ”‡ ”‡’”‡•‡–‡† ƒ• ‡ƒ•ΰ Ǥ γ͛Ǥ ȗ ζ͘Ǥ͘͝ ”‡’”‡•‡–•
˜ƒŽ—‡••‹‰‹Ƥ…ƒ–Ž›†‹ơ‡”‡–…‘’ƒ”‡†–‘…‘–”‘Ž•Ǥƒ–ƒ™‡”‡ƒƒŽ›•‡†™‹–Š‘‡Ǧ™ƒ›ˆ‘”—Ž–‹’Ž‡
comparisons and Bonferroni correction after log transformation. Panel A and C: 3x5 minutes helium, panel B
and D: 1x30 minutes helium pretreatment.
ǦȽ•–‹—Žƒ–‹‘ȋ͜Ǥ͛ȗ͙͘9±0.5*109), helium alone (3.1*109±0.5109), and the combination of
Š‡Ž‹—ƒ†ǦȽ͛Ǥ͞ȗ͙͘9±0.3*109Ȍ•‹‰‹Ƥ…ƒ–Ž›‹…”‡ƒ•‡†–Š‡ƒ‘—–‘ˆ’ƒ”–‹…Ž‡•”‡Ž‡ƒ•‡†
compared to controls (1.9*109±0.3*109, ‹‰—”‡͝B).
Additionally, exposure to 1x30 minutes of helium alone and in combination with H2O2-stim—Žƒ–‹‘•‹‰‹Ƥ…ƒ–Ž›‹…”‡ƒ•‡†–Š‡ƒ‘—–‘ˆ’ƒ”–‹…Ž‡•…‘’ƒ”‡†–‘…‘–”‘Ž•ȋ͛Ǥ͜ȗ͙͘9±0.5*109
and 3.6*109±0.6*109”‡•’‡…–‹˜‡Ž›Ǣ‹‰—”‡͝D). Stimulation with H2O2 without helium did not
126
Chapter 7 : Effect of helium on inflammatory and oxidative stress
‹…”‡ƒ•‡ –Š‡ ƒ‘—– ‘ˆ ’ƒ”–‹…Ž‡• ”‡Ž‡ƒ•‡† ȋη͘Ǥ͘͝ ™‹–Š ƒ† ‘ˆ‡””‘‹ …‘””‡…–‹‘
after log transformation).
DISCUSSION
Š‡ƒŒ‘”Ƥ†‹‰•‘ˆ–Š‡’”‡•‡–•–—†›ƒ”‡–Šƒ–’”‡Ǧ–”‡ƒ–‹‰™‹–Š†‹ơ‡”‡–’”‘–‘…‘Ž•
‘ˆ Š‡Ž‹— †‘‡• ‘– ’”‘–‡…– ‡†‘–Š‡Ž‹ƒŽ …‡ŽŽ• ƒ‰ƒ‹•– ‹ƪƒƒ–‘”› ‘” ‘š‹†ƒ–‹˜‡ •–”‡••ǡ „—–
•—”’”‹•‹‰Ž›‡‘—‰Š‹…”‡ƒ•‡††ƒƒ‰‡™Š‡‰‹˜‡‹…‘„‹ƒ–‹‘™‹–ŠǦȽ‘”2O2.
ƪƒƒ–‘”› •–”‡•• ‹…”‡ƒ•‡† ƒ†Š‡•‹‘ ‘Ž‡…—Ž‡• Ǧ͙ǡ Ǧ͙ ƒ† Ǧ•‡Ž‡…–‹ǡ „—–
’”‡Ǧ–”‡ƒ–‡– ‘ˆ Š‡Ž‹— Šƒ† ‘ ‡ơ‡…– ‘ ‡š’”‡••‹‘ ‘ˆ –Š‘•‡ ƒ†Š‡•‹‘ ‘Ž‡…—Ž‡•ǤŠ‹• ‹•
in contrast to pre-treatment of HUVEC with other noble gases, for example xenon, which
†‡…”‡ƒ•‡† ‹ƪƒƒ–‘”› •–”‡•• ‹†—…‡† ‡š’”‡••‹‘ ‘ˆ Ǧ͙ ƒ†Ǧ͙Ǥ3 Unlike helium,
xenon has anesthetic properties and shares some mechanisms in preconditioning with other
volatile anesthetics.5
–‹—Žƒ–‹‘™‹–Š͙͘͘ƒ†͘͘͝Ɋ2O2 did not increase adhesion molecules in a consistent
ƒ‡”ǤŠ‹•‹•…‘•‹•–‡–™‹–Š‡ƒ”Ž‹‡”Ƥ†‹‰•ǡ•Š‘™‹‰–Šƒ–͙͘͘Ɋ2O2 did not increase Eselectin and VCAM-1 expression in HUVEC15ǡƒ†͘͘͠σ2O2†‹†‘–ƒơ‡…–Ǧ•‡Ž‡…–‹‡š’”‡•sion and even decreased vascular endothelial cadherin and platelet endothelial cell adhesion
‘Ž‡…—Ž‡Ǧ͙‡š’”‡••‹‘‹ƒ‘”–‹…‡†‘–Š‡Ž‹ƒŽ…‡ŽŽ•‡ƒ•—”‡†„›ƪ‘™…›–‘‡–”‹…ƒƒŽ›•‹•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
…ƒ•’ƒ•‡Ǧ͛ˆ‘ŽŽ‘™‹‰•–‹—Žƒ–‹‘™‹–Š‹–‡”Ž‡—‹ȋŽȌǦ͙Ƚǡ„—–‹–‡”‡•–‹‰Ž›…ƒ•’ƒ•‡Ǧ͛…‘–ƒ‹ing 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
…‡ŽŽ—Žƒ” •—”˜‹˜ƒŽ ‡…Šƒ‹• ƒ‰ƒ‹•– ƒ’‘’–‘•‹•Ǥ—” †ƒ–ƒ •‡‡ –‘ •Šƒ”‡ –Š‡•‡ Ƥ†‹‰• •‹…‡
‘‹…”‡ƒ•‡‹…ƒ•’ƒ•‡Ǧ͛’‘•‹–‹˜‡ƒ†Š‡”‡–‘”†‡–ƒ…Š‡†…‡ŽŽ•™ƒ•‘„•‡”˜‡†ǡ„—–ƒ•‹‰‹Ƥ…ƒ–
increase in production of caspase-3 containing microparticles was found.18 This suggests that
•—……‡••ˆ—ŽŽ›•—”˜‹˜‡†ǦȽ•–‹—Žƒ–‹‘„›’”‘†—…‹‰…ƒ•’ƒ•‡Ǧ͛…‘–ƒ‹‹‰‹…”‘’ƒ”–‹…Ž‡•Ǥ  –Š‡ ’”‡•‡– •–—†›ǡ Š‡Ž‹— ‹–‡”‡•–‹‰Ž› ƒơ‡…–• –Š‹• ‡…Šƒ‹• „› increasing the
ƒ‘—–‘ˆ…ƒ•’ƒ•‡Ǧ͛…‘–ƒ‹‹‰‹…”‘’ƒ”–‹…Ž‡•ƒˆ–‡”ǦȽ•–‹—Žƒ–‹‘™‹–Š‘—–ƒơ‡…–‹‰…‡ŽŽ
viability.
The exact mechanism of protein sorting of caspase-3 is yet not fully understood, however
data suggest that active caspase-3 is co-localized with Caveolin-1 in cardiac endothelial cells.19
ƒ˜‡‘Ž‹•ƒ”‡•–”—…–—”ƒŽ’”‘–‡‹•–Šƒ–ƒ”‡‡••‡–‹ƒŽ‹ˆ‘”ƒ–‹‘‘ˆ…ƒ˜‡‘Žƒ‡‘”DzŽ‹––Ž‡…ƒ˜‡•dzǡ
which are cholesterol- and sphingolipid-enriched invaginations of the plasma membrane and
are considered a subset of lipid rafts.20 Caveolins are known to activate the protective cell
127
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 minutes of heŽ‹—‹†‡‡†…ƒ—•‡†ƒ•‹‰‹Ƥ…ƒ–‹…”‡ƒ•‡‹‹…”‘’ƒ”–‹…Ž‡’”‘†—…–‹‘…‘’ƒ”‡†–‘‡š’‘•—”‡–‘
control gas. This is in contrast to exposure to 3x5 minutes of helium, which did not increase the
ƒ‘—–‘ˆ”‡Ž‡ƒ•‡†’ƒ”–‹…Ž‡•ǤŠ—•ǡ–Š‡•‡†ƒ–ƒ•Š‘™ˆ‘”–Š‡Ƥ”•––‹‡–Šƒ–’”‘Ž‘‰‡†‡š’‘•—”‡
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,
–Š‡•‡‡ơ‡…–•ƒ›„‡’”‡•‡–‹‡†‘–Š‡Ž‹—‘ˆ’ƒ–‹‡–•ƒ•™‡ŽŽǤ‡Ž‹—Ǧ‹†—…‡†‹…”‡ƒ•‡‹
microparticle production was previously described in neutrophils exposed to partial pressures
of helium up to 690kPa.11‡ †‹† ‘– Ƥ† ƒ ‹…”‡ƒ•‡† —„‡” ‘ˆ ‡†‘–Š‡Ž‹ƒŽ …‡ŽŽ †‡”‹˜‡†
microparticles after helium inhalation.3 It might be that the stimulus of 3x5 minutes helium
ƒ’’Ž‹…ƒ–‹‘ ™ƒ• ‘– •–”‘‰ ‡‘—‰Š –‘ ‹†—…‡ ‹…”‘’ƒ”–‹…Ž‡ ”‡Ž‡ƒ•‡Ǣ Š‘™‡˜‡”ǡ ‡ƒ•—”‡‡–
was also complicated by the low amount of endothelial cell derived microparticles found in
the plasma of healthy volunteers3.
A limitation of our study was the failure of inducing apoptosis in HUVEC after exposing
–Š‡ –‘ ͘͜‰ȀŽǦȽ ˆ‘” ͚͜ Š‘—”•ǤŠ‹• ‹• ‹ …‘–”ƒ•– –‘ ‘–Š‡” Ƥ†‹‰•23, 24 showing not
‘Ž› ‹†—…‡† ƒ’‘’–‘•‹• ƒˆ–‡” –Š‹• †‘•ƒ‰‡ ‘ˆ ǦȽ „—– ƒŽ•‘ ƒ †‘•‡ †‡’‡†‡– ’”‘–‡…–‹‘
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
–Šƒ–‹™‹–Š‘—–ƒŽ–‡”ƒ–‹‘•ǡǦȽ†‘‡•‘–‹†—…‡ƒ’‘’–‘•‹•ƒ††‘‡•‘–†‡…”‡ƒ•‡
cell viability under physiological conditions, even in extremely high dosages 26ȋη͙͘ˆ‘Ž†–Š‡
…‘…‡–”ƒ–‹‘™‡—•‡†ȌǤ‘”‡‘˜‡”ǡƒ‰‹‰…‡ŽŽ•™‡”‡•Š‘™–‘„‡‘”‡•—•…‡’–‹„Ž‡–‘ǦȽǡ
ƒ†‘Ž›‹ƒ‰‡†•‹‰‹Ƥ…ƒ–†‹ơ‡”‡…‡•‹˜‹ƒ„‹Ž‹–›‘”…ƒ•’ƒ•‡Ǧ͛™‡”‡‘„•‡”˜‡†Ǥ27 This
could explain the failure to induce apoptosis in our cultivated cells, because we only processed
›‘—‰ …‡ŽŽ• ƒ† ‡’– ’Š›•‹‘Ž‘‰‹…ƒŽ …‘†‹–‹‘•Ǥ ‘” •–‹—Žƒ–‹‘ ™‹–Š 2O2 we encountered
similar problems, as 100mM and 500mM H2O2 for 2 hours did not decrease cell viability. Other
•–—†‹‡•ǡ—•‹‰–Š‡’”‡Ǧ‡•–ƒ„Ž‹•Š‡†…‡ŽŽŽ‹‡Ǧ͙͛͘͟ǡ™‡”‡ƒ„Ž‡–‘”‡†—…‡˜‹ƒ„‹Ž‹–›ƒ†‹†—…‡
ƒ’‘’–‘•‹•™‹–Š͙͘͘Ɋ2O2.28, 29 Another study demonstrated that young endothelial cells are
ƒ„Ž‡–‘’”‘–‡…––Š‡•‡Ž˜‡•ƒ‰ƒ‹•––Š‡†‡–”‹‡–ƒŽ‡ơ‡…–•‘ˆ‘š‹†ƒ–‹˜‡•–”‡••ƒ†•–‹—Žƒ–‹‘
™‹–Š͘͘͝Ɋ2O2 for 18 hours was needed to induce apoptosis, indicating that our stimulus
may have been too short to induce cell death.30
‹–Š”‡•’‡…––‘…‡ŽŽ˜‹ƒ„‹Ž‹–›ǡ™‡ˆ‘—†‘†‡…”‡ƒ•‡ƒˆ–‡”‹ƪƒƒ–‘”›‘”‘š‹†ƒ–‹˜‡•–”‡••Ǥ
Surprisingly, oxidative stress in combination with helium pretreatment for 3x5 minutes or 1x30
minutes 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
128
Chapter 7 : Effect of helium on inflammatory and oxidative stress
leads to superoxide production via (uncoupled) nitric oxide synthase and NAPDH oxidase31
which can induce cellular injury.32 Treating neutrophils with hyperbaric noble gases increased
formation of reactive oxygen species, mediated by collision-induced superoxide formation
†‡’‡†‡– ‘ˆ –Š‡ ’”‘’‡”–‹‡• ƒ† •‹œ‡ ‘ˆ –Š‡ ‰ƒ• —•‡† ȋŠ‡Ž‹—ζƒ”‰‘ȌǤ11 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
minutes but not for 3x5 minutes. This may suggest that 30 minutes of helium is harmful, and
intermitted treatment by 3x5 minutes of helium is not.
‡…‡–”‡•‡ƒ”…Š•Š‘™•–Šƒ–‘š‹†ƒ–‹˜‡•–”‡••ƒ›”‡•—Ž–‹’”‘‰”ƒ‡†…‡ŽŽ†‡ƒ–Š™‹–Š‘—–
‹˜‘Ž˜‡‡– ‘ˆ …ƒ•’ƒ•‡•ǡ •‘Ǧ…ƒŽŽ‡† Dz‡…”‘’–‘•‹•dzǤ33 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 minutes 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
humans3 and animals,34 although the mechanisms underlying this protection remains unclear.35‡…‡–†ƒ–ƒ•Š‘™•Š‡Ž‹—’‘•–…‘†‹–‹‘‹‰ȋ͙͝‹—–‡•‘ˆŠ‡Ž‹—ƒ†‹‹•–‡”‡†ƒˆ–‡”
ȀȌ‹˜‘Ž˜‡†—’”‡‰—Žƒ–‹‘‘ˆ‰‡‡•‹˜‘Ž˜‡†‹ƒ—–‘’Šƒ‰›ƒ†‹Š‹„‹–‹‘‘ˆƒ’‘’–‘•‹•Ǥ‡Ž‹—
upregulated 27 of 30 genes involved in autophagy, and 12 of 14 antiapoptotic genes.36
In conclusion, helium administered in vitro for 3x5 minutes or 1x30 minutes does not reduce
‹ƪƒƒ–‘”›ƒ†‘š‹†ƒ–‹˜‡•–”‡••Ǧ‹†—…‡††ƒƒ‰‡‹. In fact, helium treatment for
͙š͛͘‹—–‡•ƒ‰‰”ƒ˜ƒ–‡•‘š‹†ƒ–‹˜‡•–”‡••‹†—…‡††ƒƒ‰‡Ǥ‡‰ƒ”†Ž‡••‘ˆ–Š‡†ƒƒ‰‡‘†‡Ž
employed, helium is not biologically inert but induces cellular activation leading to increased
‹…”‘’ƒ”–‹…Ž‡ˆ‘”ƒ–‹‘ƒ†ƒŽ–‡”‡†…‘–‡–Ǥ—”–Š‡”‘”‡ǡŠ‡Ž‹—‹…”‡ƒ•‡†–Š‡ƒ‘—–‘ˆ
…ƒ•’ƒ•‡Ǧ͛…‘–ƒ‹‹‰‹…”‘’ƒ”–‹…Ž‡•ƒˆ–‡”ǦȽ•–‹—Žƒ–‹‘Ǥ—”–Š‡””‡•‡ƒ”…Š‹•‡…‡••ƒ”›–‘
‹†‡–‹ˆ›–Š‡‡…Šƒ‹•„‡Š‹†–Š‡•‡‡ơ‡…–•‘ˆŠ‡Ž‹—Ǥ
129
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Chapter 7 : Effect of helium on inflammatory and oxidative stress
A
3x5 protocol: H2O2
VCAM-1
2.0
4
1.5
1.0
E-selectin
2.0
E-Se le ctine /28S
5
VCAM -1/28S
ICAM -1/28S
ICAM-1
2.5
3
2
1
0.5
0
0.0
1.0
0.5
0.0
Con H2O2 He HeH2O2
Con H2O2 He He+H 2O2
B
1.5
Con H2O2 He HeH 2O2
1x30 protocol: H2O2
VCAM-1
5
2.0
4
1.5
1.0
0.5
0.0
2.0
3
n.s.
2
1
*
0
Con H2O2 He HeH 2O2
E-selectin
E-Se le ctine /28S
2.5
VCAM -1/28S
ICAM -1/28S
ICAM-1
1.5
1.0
0.5
0.0
Con H2O2 He HeH 2O2
Con H2O2 He HeH 2O2
‹‰—”‡͙ǣ ơ‡…–‘ˆŠ‡Ž‹—‘…‡ŽŽ—Žƒ”ƒ…–‹˜ƒ–‹‘ƒˆ–‡”‘š‹†ƒ–‹˜‡•–”‡••
‡•—Ž–•‘ˆ”‡ƒŽ–‹‡“—ƒ–‹–ƒ–‹˜‡‘ˆǦ͙ǡǦ͙ƒ†Ǧ‡Ž‡…–‹‘”ƒŽ‹œ‡†–‘͚͠ȋ’ƒ‡Žǣ͛š͝‹—–‡•Š‡Ž‹—ǡ’ƒ‡Žǣ͙š͛͘‹—–‡•Š‡Ž‹—ȌǤƒ–ƒƒ”‡‡ƒΰǤγ͝Ǥȗȋζ͘Ǥ͘͝Ȍ”‡’”‡•‡–••‹‰‹Ƥ…ƒ–Ž›
†‹ơ‡”‡–˜ƒŽ—‡…‘’ƒ”‡†–‘…‘–”‘Ž•Ǥ‡™ƒ›ˆ‘”—Ž–‹’Ž‡…‘’ƒ”‹•‘•ƒ†„‘ˆ‡””‘‹…‘””‡…–‹‘Ǥ
‘γ…‘–”‘Ž•ǡγǦȽǡ‡γ‡Ž‹—ǡ‡γ‡Ž‹—ήǦȽǤ
133
Cell viability
3x5 protocol: TNF-Į
80
100
% viable cells
% viable cells
100
60
40
20
1x30 protocol: TNF-Į
80
60
40
20
0
0
Con TNF He HeTNF
Con TNF He HeTNF
Figure S2: ơ‡…–‘ˆŠ‡Ž‹—‘…‡ŽŽ˜‹ƒ„‹Ž‹–›ƒˆ–‡”‹ƪƒƒ–‘”›•–”‡••
‡•—Ž–• ‘ˆ ƪ‘™…›–‘‡–”› ‘ˆ Šƒ”˜‡•–‡† …‡ŽŽ• •–ƒ‹‡† ™‹–Š ƒ‡š‹Ǧ ƒ† ’”‘’‹†‹— ‹‘†‹†‡Ǥ‡ŽŽ• –Šƒ– ƒ”‡
negative for both annexin-V and PI are considered viable cells. Annexin-V and PI positive cells are considered
necrotic cells. Panel A: 3x5 minutes helium, panel B: 1x30 minutes helium. Data are mean ± SEM. N=3. *
ȋζ͘Ǥ͘͝Ȍ”‡’”‡•‡–•˜ƒŽ—‡••‹‰‹Ƥ…ƒ–Ž›†‹ơ‡”‡–…‘’ƒ”‡†–‘…‘–”‘Ž•Ǥƒ–ƒ™‡”‡ƒƒŽ›•‡†™‹–Š‘‡™ƒ›
ANOVA for multiple comparisons and Bonferroni correction. Con=controls, He=Helium, HeH2O2=Helium +
H2O2.
134