Research Article Nitric Oxide and Superoxide Anion Balance in Rats Exposed to Chronic and Long Term Intermittent Hypoxia

Research ArticleNitric Oxide and Superoxide Anion Balance in Rats Exposed toChronic and Long Term Intermittent Hypoxia

Patricia Siques1 Aacutengel Luis Loacutepez de Pablo23 Julio Brito1 Silvia M Arribas23

Karen Flores1 Karem Arriaza1 Nelson Naveas1 M Carmen Gonzaacutelez23

Alexander Hoorntje2 Fabiola Leoacuten-Velarde4 and M Rosario Loacutepez5

1 Instituto de Estudios de la Salud Universidad Arturo Prat Avenida Arturo Prat 2120 11100939 Iquique Chile2 Departamento de Fisiologıa Facultad de Medicina Universidad Autonoma de Madrid 28029 Madrid Spain3 Instituto de Investigacion Hospital Universitario Gregorio Maranon 28007 Madrid Spain4Departamento de Ciencias Biologicas y Fisiologicas Facultad de Ciencias y FilosofıaIIA Universidad Peruana Cayetano HerediaLima 31 Lima Peru

5 Departamento de Medicina Preventiva Salud Publica y Microbiologıa Facultad de Medicina Universidad Autonoma de Madrid28029 Madrid Spain

Correspondence should be addressed to Patricia Siques psiquestiecl

Received 5 December 2013 Accepted 15 January 2014 Published 26 February 2014

Academic Editor Iveta Bernatova

Copyright copy 2014 Patricia Siques et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Work at high altitude in shifts exposes humans to a new form of chronic intermittent hypoxia with still unknown healthconsequences We have established a rat model resembling this situation which develops a milder form of right ventricularhypertrophy and pulmonary artery remodelling compared to continuous chronic exposure We aimed to compare the alterationsin pulmonary artery nitric oxide (NO) availability induced by these forms of hypoxia and the mechanisms implicated Rats wereexposed for 46 days to normoxia or hypobaric hypoxia either continuous (CH) or intermittent (2 day shifts CIH2x2) andassessed NO and superoxide anion availability (fluorescent indicators and confocal microscopy) expression of phosphorylatedendothelial NO synthase (eNOS) NADPH-oxidase (p22phox) and 3-nitrotyrosine (western blotting) and NADPH-oxidaselocation (immunohistochemistry) Compared to normoxia (1) NO availability was reduced and superoxide anion was increased inboth hypoxic groups with a larger effect in CH (2) eNOS expression was only reduced in CH (3) NADPH-oxidase was similarlyincreased in both hypoxic groups and (4) 3-nitrotyrosinewas increased to a larger extent inCH In conclusion intermittent hypoxiareduces NO availability through superoxide anion destruction without reducing its synthesis while continuous hypoxia affectsboth producing larger nitrosative damage which could be related to the more severe cardiovascular alterations

1 Introduction

Exposure to hypoxia in either chronic or intermittentconditions is associated with cardiovascular alterationsHypoxia-related diseases are well characterized in peopleliving at high altitude (chronic hypoxia CH) [1] and inobstructive sleep apnea (OSA) where hypoxic conditionsare maintained intermittently for brief periods [2] Anothermode of intermittent hypoxia has arisen as a result of therecent settlements of mines and other activities at highaltitude (chronic intermittent hypoxia CIH) where subjectsrepeatedly ascend from sea level to 3800ndash4200m and work

in shifts being exposed to hypoxia for longer periods thanin OSA conditions [3] The large number of workers underthese circumstances together with the known deleteriouscardiovascular effects reported in OSA patients [4] makesit of utmost importance to study the mechanisms implicatedin CIH alterations Since this condition is a relatively recentphenomenon studies are still limited and animal models arevaluableWe have used an experimental model of CIH whererats are exposed to hypobaric hypoxia in shifts resembling thehuman situation [5] We and others have found that underthese experimental conditions the rats develop pulmonaryhypertension pulmonary vascular remodeling and right

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 610474 10 pageshttpdxdoiorg1011552014610474

2 BioMed Research International

ventricular hypertrophy [6 7] which are milder comparedto rats chronically exposed to hypoxia [8]

Hypoxia-induced pulmonary hypertension and rightventricular hypertrophy are related to pulmonary arteryvasoconstriction and vascular remodeling [9 10] endothelialnitric oxide (NO) being an important modulator of theseresponses [11 12] Human and animal studies demonstratethat OSA is associated with reduced NO availability dueto a decreased production or destruction by an excess ofreactive oxygen species (ROS) [2] and pulmonary arteryvasoconstriction has been suggested to result from imbalancebetween endothelial vasodilator factors and ROS [13] NO-ROS misbalance can also contribute to remodeling pro-cess in the pulmonary vasculature through modification ofcell migration proliferation dedifferentiation and apoptosis[14] We have previously demonstrated that rats exposedto long term CIH exhibit pulmonary artery remodelingwith characteristic features which differ from those foundunder chronic exposure conditions [15 16] While thereare several studies in OSA conditions to the best of ourknowledge there are no data regarding the effect of hypoxiamaintained intermittently during days on NOROS balancein the pulmonary vasculatureTherefore we aimed to analyzethe possible alterations under this condition and to comparethem with those induced by continuous hypoxic exposureWe have used a rat model mimicking the conditions ofsubjects working in shifts or permanently living at highaltitude

2 Materials and Methods

21 Animal Model Three-month-old male Wistar rats wereused The rats were exposed to 22 plusmn 2∘C 12 h lightdarkcycle and were maintained in separate cages with foodmade available to them (20 g pelletsday per rat) and waterad libitum Standard veterinary care was used during allof the experiments following institutional protocols for thestudy of animals and the procedures used were approved bythe Institutional Research Ethics Committee of UniversidadArturo Prat (Chile) The rats were randomly assigned to oneof the following groups

normoxia control group (NX 119899 = 10)chronic intermittent hypoxia 2 days under hypobarichypoxia and 2 days under normobaric normoxia for46 days (CIH2x2 119899 = 10)chronic hypoxia continuous hypobaric hypoxia for46 days (CH 119899 = 10)

Hypobaric hypoxia was simulated at Universidad ArturoPrat facilities in a hypobaric chamber at 428 Torr which isequivalent to an altitude of 4600m (PO

2= 89mmHg PCO

2

= 015mmHg temperature 22 plusmn 2∘C and humidity 35 plusmn 5)Control (sea level) conditions were PO

2= 159mmHg PCO

2

= 029mmHg temperature 22 plusmn 2∘C and humidity 35 plusmn 5The animal model for chronic intermittent hypoxia exposure(CIH2x2) has been described previously [5 8 17]Thismodelinvolves 2 days of hypoxia and 2 days of normoxia over aperiod of 46 days We maintained the rats for 46 days since

previous data indicate that the hematological and cardiovas-cular effects in the rat are maximal between 30 and 45 daysof exposure to hypoxia [5] The 2x2 regimen was chosen inorder to mimic long term exposure to intermittent hypoxicconditions as experienced by human subjects working inshifts at high altitude which is in the order of days (usually7ndash14) [18] and including at least one full circadian cycle Thecontrol groupwas placed in the same room at sea level duringthe 46-day period

All the rats were weighted at day 1 and at day 46 using anAcculab V-1200 electronic balance At day 46 a blood samplewas taken from the tail and hematocrit was measured usinga microcentrifuge (Eppendorf AG Hamburg Germany)Thereafter the animals were euthanized with an overdose ofanesthesia (Ketamine 8mg ip) and the heart and the lungswere removed in a block for further dissection

The heart was cut down and the right ventricle wasdetached from the heart leaving in situ the septum portiontogether with the left ventricle Both ventricles were weighedin an analytic balance (AcculabV-1200 Illinois USA) and theratio between the right ventricleleft ventricle plus the septumweight was used to measure the grade of right ventricularhypertrophy We did not use total heart weight or rightventricular weightbody weight since we have previouslyreported that exposure to hypoxia produces alterations inbody weight gain [5] The removed lung was placed ina Petri dish and pulmonary artery branches (4th order)were dissected and stored for further experiments in salinesolution (09 NaCl)

22 Confocal Microscopy

221 Determination of NO Availability Basal NO avail-ability was determined by the fluorescent NO indicator45-diaminofluorescein diacetate (DAF-2 DA Sigma) asdescribed previously [19] Briefly 3mm length pulmonaryartery segments were stabilized in physiological salt solu-tion (PSS 115mmolL NaCl 46mmolL KCl 25mmolLNaHCO3 12mmolL KH2PO4 12mmolL MgSO4 and25mmolL CaCl2) for 30min at 37∘C and oxygenatedwith carbogen (95 O

2and 5 CO

2) Thereafter they were

stained with oxygenated DAF-2 DA solution (10 120583molL) for30min in the darkness at 37∘C in a shaking water bath Thisexperimental procedure in saturated oxygen ensures thatNOrather than O

2 is the limiting factor in the reaction and the

fluorescence is directly proportional to NO [19] Negativecontrols for DAF-2 DA were incubated in 01mmolL L-NAME throughout the experimental period The segmentswere then washed 3 times for 1min each in PSS and fixedin 4 (wv) paraformaldehyde The segments were cut inrings with a blade and mounted on a slide equipped witha small well made of spacers filled with mounting medium(Citifluor Aname Spain) and covered with a cover glassThe arterial rings were visualized with a Leica TCS SP2confocal system (Leica Microsystems Wetzlar Germany) atUniversidad Autonoma de Madrid of Spain facilities usingthe 488 nm515 nm line 1 120583m thick serial images (25120583m intotal) were captured with a 63x objective at zoom 2 in 3

BioMed Research International 3

randomly chosen areas of the ring at identical conditions ofbrightness contrast and laser power for all of the experimen-tal groups MetaMorph image analysis software (UniversalImaging Co UK) was used for quantification of fluorescenceintensity Briefly the serial images were first reconstructedin a confocal projection and fluorescence intensity wasquantified in several regions of the smooth muscle cellswhere the dye is trapped avoiding the elastic lamella which isalso fluorescent in the same wavelength

222 Determination of O2

∙minus Availability Dihydroethidium(DHE Sigma) was used to determine basal O

2

∙minus as described[19] Briefly 3mm long pulmonary arteries were stabilized inPSS (30min at 37∘C) Thereafter they were incubated with3 120583molL DHE washed 3 times for 1min each in PSS andfixed in 4 (wv) paraformaldehyde Negative controls forDHE were incubated in 15 unitsmL superoxide dismutase(SOD) throughout the incubation periodThe segments werethen washed 3 times for 1min each in PSS fixed in 4 (wv)paraformaldehyde cut in rings and mounted as describedabove 1 120583m thick serial images (25 120583m in total) were capturedwith a 63x objective at zoom 2 in 3 randomly chosen areasof the ring at identical conditions of brightness contrastand laser power for all of the experimental groups withthe 488 nm590ndash620 nm line of the microscope MetaMorphimage analysis software (Universal Imaging Co UK) wasused for quantification of fluorescence intensity which waslocated in the nuclei The serial images were first recon-structed in a confocal projection and fluorescence intensitywas quantified in several regions along the ring

223 NADPH-Oxidase Detection by ImmunohistochemistryTo detect the presence of NADPH oxidase in the adventitiallayer pulmonary arteries were first incubated with the pri-mary antibody of the p22phox subunit of the enzyme (rabbitpolyclonal Santa Cruz Biotechnology USA) (60min 1 200in saline solution) at room temperature (RT) and thenwashedwith saline solution (30min RT) Thereafter the segmentswere incubated with the secondary antibody-Alexa Fluor 647goat anti-rabbit IgG (H + L) (Invitrogen Madrid Spain)(60min 1 200 RT in the darkness) followed by washingfor 30min RT Finally they were incubated with the nucleardye 410158406-diamidino-2-phenylindole (DAPI 1 500 from a5mgmL stock 30min RT in the darkness) andwashed twice(30min RT) The pulmonary arteries were longitudinallysectioned and mounted with the adventitial side facing upas described above The arteries were visualized with aLeica TCS confocal system using the 405 nm excitation410ndash475 nm emission wavelength (DAPI) to locate the cellsand the 633 nm excitation640ndash650 nm emission wavelength(secondary antibody-Alexa 647) to detect the protein 1 120583mthick serial images of the adventitial layer (12 120583m in total)were captured at both wavelengths with a 40x objective atzoom 4 from 3 different regions MetaMorph software wasused to count the total number of cells (DAPI positive) andthose stained with p22phox Cell number (DAPI or p22phoxpositive) was counted always in the same volume which wascalculated from the layer thickness (12120583m) and the image

area at times40 zoom 4 We calculated the total number ofp22phox positive cells and the relative number of positivecells (positive cellstotal cells) in the mentioned volume

23 Western Blot Western blot was performed accordingto the standard methods Briefly total pulmonary arterytissues were frozen in liquid nitrogen and then homoge-nized into homogenization buffer containing 50mM Tris-HCl 150mM NaCl 100mM NaF 1 Triton X-100 1mMdithiothreitol 01mM phenyl methylsulfonyl fluoride 1mMleupeptin 002M Hepes 10minus3M EDTA 10minus3M EGTA and20 glycerol followed by centrifugation to 10000timesg by10min Supernatant was removed and protein quantifica-tion was carried out by Bradford assay Equal amount ofprotein (25 120583g) was resolved on 75ndash12 SDS-PAGE andproteins were transferred to a PVDF membrane The non-specific binding sites on the membrane were blocked using5 nonfat dry milk in TBS-T buffer (10mM Tris-HCl150mM NaCl 005 tween 20 and pH 74) by 1 hourMembranes were incubated with primary antibody rabbitpolyclonal to PNK (3-nitrotyrosine) and p22phox and goatpolyclonal pNOS-3 (phosphorylated form of e-NOS SantaCruz Biotechnology USA) by overnight and washed threetimes for 15 10 5min with TBS-T followed by incubationwith horseradish peroxidase-conjugated secondary antibody(1 2000 Santa Cruz Biotechnology USA) for 1 hour andwashed 3 times with TBS-T and once with TBS Blots werevisualized using a West Pico Chemiluminescence System(Pierce USA) and then analyzed using Image J Expressionlevels of p22phox PNK and pNOS3 were normalized to 120573-actin expression

24 Statistical Analysis The experimental data were enteredinto a database and were analyzed using SPSS 170 statisticalpackage (SPSS Inc Chicago Ill USA) Mean standarddeviation and standard error were calculated for eachparameter Normality was established using the Kolmogorov-Smirnov test Statistical analysis of the differences across alltesting conditions was established using analysis of variance(ANOVA) of one factor and less significant difference posthoc tests All of the variables were normally distributedStatistical significance was established at a 119875 value lt 005

3 Results

31 Body Weight Gain Hematocrit and Right VentricularHypertrophy Body weight at day 1 was not statisticallydifferent between experimental groups (NX 2478 plusmn 166 grCIH2x2 2469plusmn 112 gr and CH 2515plusmn 172 gr) While NXrats gained weight over the 46-day period (final body weightNX = 330 plusmn 135 g) there was a gradual weight loss in bothhypoxic groups (final bodyweight CIH2x2 = 206plusmn 803 g CH= 169 plusmn 36 g) being significantly smaller compared to NX(119875 lt 0001) Hematocrit at the end of experimental periodwas significantly higher in CH (66 plusmn 11) and in CIH2x2(58 plusmn 18) compared to NX rats (51 plusmn 10) (119875 lt 001)CIH2x2 hematocrit was significantly smaller compared toCH (119875 lt 001) Right ventricular weighttotal heart weight

4 BioMed Research International

NX CIH2x2 CH

50120583m

(a)

dagger

Inte

nsity

(rel

ativ

e uni

ts)

00

200

400

600

800

1000

1200

1400

1600

1800

2000

NXCIH2x2CH

lowast

lowast

(b)

Figure 1 DAF-2 DA intensity levels in pulmonary arteries from rats exposed to normoxia (NX 119899 = 10) intermittent hypoxia (CIH2x2119899 = 10) or chronic hypoxia (CH 119899 = 10) (a) Representative examples of projections obtained from confocal microscopy images (times40 zoom2) (b) Quantitative analysis 119899 represents the number of animals lowast119875 lt 005 compared to NX dagger119875 lt 005 compared to CIH2x2

was smaller in NX (NX = 023 plusmn 002 119875 lt 001) comparedto CIH2x2 rats (034 plusmn 002) and CH group (040 plusmn 002)which was also significantly larger compared to intermittentexposure group (119875 lt 005)

32 NO Availability The fluorescence emitted by DAF-2DA was located in the cytoplasm of smooth muscle cellsPulmonary arteries from NX group exhibited a significantlyhigher DAF-2 DA emitted fluorescence compared to bothhypoxic groups suggesting a larger basal NO availability [19]Fluorescence was significantly lower in CH rats compared toCIH2x2 (Figure 1)

33 O2

∙minus Availability DHE fluorescence was located in thecell nuclei NX rats showed a smaller level of fluorescenceintensity compared to both hypoxic groups being higher inCH compared to CIH2x2 (Figure 2)

34 Phosphorylated eNOS p22phox and 3-NitrotyrosineExpression Expression of the phosphorylated form of eNOSwas significantly reduced in CH compared to NX Theexpression levels in CIH2x2 were not statistically differentfrom NX (Figure 3(a))

p22phox expressionwas scarcely detectable in pulmonaryarteries from NX but was largely increased in both hypoxicrats without differences between CH and CIH2x2 groups(Figure 3(b))

3-Nitrotyrosine expression a marker of nitrosative dam-age was significantly elevated in pulmonary arteries fromhypoxic rats compared toNX being significantly larger inCHcompared to CIH2x2 (Figure 3(c))

35 p22phox Location in the Adventitia The total number ofadventitial cells quantified by the nuclear dye DAPI in a fixedvolume was significantly higher in pulmonary arteries from

BioMed Research International 5

NX CIH2x2 CH

50120583m

(a)

dagger

00

100

200

300

400

500

600

700

800

900

1000

NXCIH2x2CH

Inte

nsity

(rel

ativ

e uni

ts)

lowast

lowast

(b)

Figure 2 DHE intensity levels in pulmonary arteries from rats exposed to normoxia (NX 119899 = 10) intermittent hypoxia (CIH2x2 119899 = 10)or chronic hypoxia (CH 119899 = 10) (a) Representative examples of projections obtained from confocal microscopy images (times40 zoom 2) (b)Quantitative analysis 119899 represents the number of animals lowast119875 lt 005 compared to NX dagger119875 lt 005 compared to CIH2x2

both hypoxic rats (CIH2x2 = 1447 plusmn 132 CH = 1571 plusmn 97)compared to control (NX = 749 plusmn 26 119875 lt 0001)

P22phox staining was observed in the three experimentalgroups and was located around some of the adventitial cellsPulmonary arteries from hypoxic rats exhibited a larger levelof staining compared to NX (Figure 4(a)) Both total andrelative numbers of p22phox positive cells were significantlylarger in the adventitial layer of hypoxic rats compared toNXwith no statistical difference between CIH2x2 and CH rats(Figure 4(b))

4 Discussion

The main findings of the current study are that exposure tochronic intermittent hypoxia reduces NO availability in thepulmonary vasculature This decrease is likely due to NOdestruction by O

2

∙minus generated by NADPH-oxidase whileeNOS is not altered On the other hand in continuous

exposure to hypoxia NO availability is further reducedthrough the combination of diminished NO synthesis andincreased destruction In consequence chronic hypoxia pro-duces larger nitrosative damage compared to intermittentexposure which likely contributes to the higher impact onpulmonary artery remodeling and right ventricular hypertro-phy (Figure 5 summarizes these results)

The present data confirmed that hypoxia induced weightloss right ventricular hypertrophy and hematocrit increaseas previously described by us [8] and others [6 7] Wehave also described remodeling of the pulmonary vasculature[15 16] these alterations being less severe under intermittentexposure We aimed to assess if the above mentioned cardio-vascular alterations are linked toNOROSmisbalance To thebest of our knowledge there is virtually no information inlong term intermittent hypoxia conditions the majority ofevidence coming from OSA studies [20] where low oxygenlevels are maintained for very brief periods

6 BioMed Research International

NXCIH2x2CH

000

020

040

060

080

100

120

Relat

ive p

rote

in co

nten

t (O

D)

lowast

(a) eNOS

NXCIH2x2CH

Relat

ive p

rote

in co

nten

t (O

D)

000

010

020

030

040

050

060

lowast

lowast

(b) p22phox

000

010

020

030

040

050

060dagger

Relat

ive p

rote

in co

nten

t (O

D)

lowast

lowast

NXCIH2x2CH

(c) 3-Nitrotyrosine

NX CIH CH

p-NOS3

-Actin

p22phox

3-NT

(d) Representative examples

Figure 3 Western blot analysis of (a) phosphorylated eNOS (b) p22phox and (c) 3-nitrotyrosine from pulmonary arteries of rats exposedto normoxia (NX 119899 = 9) intermittent hypoxia (CIH2x2 119899 = 10) or chronic hypoxia (CH 119899 = 10) (d) Representative examples 119899 representsthe number of animals lowast119875 lt 005 compared to NX dagger119875 lt 005 compared to CIH2x2

We focused on NO a key factor for pulmonary arteryresistance [12] since diminished NO availability likely affectsboth pulmonary artery structure and function Furthermorestudies on rodents have revealed that intermittent exposureto hypoxia is associated with NO reduction in the systemicand cerebral vasculature [21 22] Decreased NO availabilityin the pulmonary vasculature can be the result of a reducedproduction by eNOS andor an increased destruction byROS particularly O

2

∙minus which has been implicated in hypoxicpulmonary vasoconstriction [13 23ndash25] To determine NOavailability we used DAF-2 DA a fluorescent indicatordirectly proportional to the amount of NO [26 27] Usingconfocal microscopy and image analysis software we have

previously demonstrated that this method is sufficientlysensitive for the quantification of basal NO in resistance andconduit arteries [19 28 29] Continuous hypoxia induced alarger reduction of NO availability compared to intermittentexposureThis can be explained by a reducedNOproductionas suggested by the decreased expression of phosphorylatedeNOSmdashthe active form of the enzymemdashfound in chronichypoxia onlyThe lack of effect of intermittent exposure couldbe explained by the functional ldquoon-offrdquo (hypoxia-normoxia)switch [30] which is not able to induce downregulation of theNO biosynthetic machinery

Reduced NO availability in hypoxic conditions seemsalso to be related to NO destruction by O

2

∙minus This is

BioMed Research International 7

NX CIH2x2 CH

(a)

0

2

4

6

8

10

12

14

Cel

l num

ber

0

5

10

15

20

25

NXCIH2x2CH

NXCIH2x2CH

Posit

ive c

ells

tota

l cel

ls

lowast

lowast

lowast

lowast

(b) p22phox positive cells

Figure 4 Immunohistochemical detection of p22phox positive cells in the adventitia of pulmonary arteries from rats exposed to normoxia(NX 119899 = 10) intermittent hypoxia (CIH2x2 119899 = 10) or chronic hypoxia (CH 119899 = 10) (a) Representative examples of projections obtainedfrom confocal microscopy images (times40 zoom 4) (b) Quantitative analysis 119899 represents the number of animals lowast119875 lt 005 compared to NX

suggested by the increased superoxide anion productionfound in CIH2x2 and CH pulmonary arteries similarlyto data described in an OSA rat model [31] Superoxideanion levels were even higher in continuous compared tointermittent hypoxia suggesting that continuous exposurefurther stimulates the enzymatic systems responsible for ROSsynthesis In the vascular wall several enzymes produceO

2

∙minusNADPH oxidase being the main system in the pulmonaryvasculature [13 32] We found a remarkable expressionof this enzymemdashconfirmed by immunohistochemistrymdashinboth CH and CIH2x2 while it was very low in normoxic ratsThe important role of this enzyme in intermittent hypoxia hasbeen previously demonstrated in NADPH-oxidase knock-out mice exposed to OSA [33] Our data show that O

2

∙minus

levels were larger in continuous compared to intermittent

hypoxia despite the similar p22phox expression in bothgroups This could be explained by O

2

∙minus production byxanthine oxidase as suggested in OSA patients and animalmodels [34 35] Since it has been reported that underhypoxic conditions O

2

∙minus generated by xanthine oxidase isvery small [36] alternatively O

2

∙minus can be produced byldquouncoupledrdquo dysfunctional eNOS which can be inducedby peroxynitrite [37] We did not measure peroxynitritedirectly but quantified 3-nitrotyrosine which is currentlyaccepted as evidence of peroxynitrite generation The largercontent of 3-nitrotyrosine found in CH suggests that eNOSuncoupling can contribute to larger O

2

∙minus production foundin continuous hypoxic conditions compared to intermittentconditions despite the similar NADPH oxidase expression inboth groups

8 BioMed Research International

NO availability

NO production(eNOS)

eNOS

UncoupledeNOS

NO

NONONONO

NO

NONO

NONO

NO

NONO

NO

INormoxia

ONOOminus

Oxidative damage(3-NT)

availability

Long-termintermittent hypoxia Chronic hypoxia

+

minus

O2

∙minusproduction(NADPH oxidase)O2

∙minus

O2

∙minus

O2

∙minus

O2

∙minus

O2

∙minus

O2

∙minusO2

∙minus

O2

∙minus

O2

∙minus O2

∙minus

O2

∙minus

O2

∙minus

Figure 5 Schematic diagram showing the main results and the proposed mechanism implicated in the NOO2

∙minus misbalance induced bychronic and intermittent hypoxia

Oxidative-nitrosative stress is associated with vascularremodeling in hypoxia-induced pulmonary hypertension[38]We have previously found several signs of remodeling inpulmonary arteries fromCH and CIH2x2 rats including wallhypertrophy due to increased smooth muscle and adventitialcells [15 16] Adventitial NADPH oxidase has been describedto be involved in pulmonary artery adventitial fibroblastsproliferation [39 40] and seems to be a primary site ofsuperoxide anion production in the vessel wall [41ndash43]We found that chronic or intermittent hypoxia substantiallyincreased adventitial cell number together with a largerpercentage of NADPH-positive cells Moreover we [44] andothers [43] have previously demonstrated that adventitia is akey layer regarding NO inactivation by ROS Since NO is anantiproliferative agent we suggest that an imbalance betweenNOO

2

∙minus can be linked to the vascular remodeling processunder continuous or intermittent hypoxic exposure

It was beyond the scope of this study to investigatethe mechanisms implicated in hypoxia-induced NOROSdisbalance However we can speculate on the possible roleof inflammation Alveolar hypoxia produces widespreadsystemic inflammation [45] and it also promotes thedevelopment of a pulmonary artery chronic inflammatorymicroenvironment [46] We also have evidence of infiltratedmacrophages in pulmonary arteries from CH and CIH2x2(unpublished results) suggesting that local inflammation inthe vascularwallmight contribute to theNOROSdisbalanceIn support of this hypothesis there is evidence that tumornecrosis factor-120572 can reduce eNOS expression and activity inpulmonary arteries [47] Moreover inflammation associatedwith macrophage infiltration can also contribute to ROS gen-eration through stimulation of NADPH oxidase expressionas previously found in the carotid body under intermittenthypoxic conditions [48]

In conclusion the present study suggests that hypobarichypoxia under intermittent conditions reduces NO availabil-ity due to destruction by superoxide anion without affectingNO synthesis while continuous exposure is associated withboth increased degradation and reducedNOproductionTheoxidative-nitrosative stress induced by long term intermittenthypoxia might participate in the observed cardiovascularstructural alterations but represents a milder form of damagecompared to continuous exposure These data suggest thatthe alterations in oxidative status of humans chronically orintermittently exposed to high altitude need to be evaluatedin order to improve the associated cardiovascular alterations

Conflict of Interests

There is no conflict of interests

Funding

Thisworkwas funded byGORE-TARAPACA (BIP 30125349-0) and ALTMEDFIS (CYTED 213RT0478) Grants

References

[1] F Leon-Velarde M Maggiorini J T Reeves et al ldquoConsensusstatement on chronic and subacute high altitude diseasesrdquoHighAltitude Medicine and Biology vol 6 no 2 pp 147ndash157 2005

[2] R Dumitrascu J Heitmann W Seeger N Weissmann andR Schulz ldquoObstructive sleep apnea oxidative stress and car-diovascular disease lessons from animal studiesrdquo OxidativeMedicine and Cellular Longevity vol 2013 Article ID 2346317 pages 2013

BioMed Research International 9

[3] J-P Richalet M V Donoso D Jimenez et al ldquoChilean minerscommuting from sea level to 4500 m a prospective studyrdquoHighAltitude Medicine and Biology vol 3 no 2 pp 159ndash166 2002

[4] G E Foster J V Brugniaux V Pialoux et al ldquoCardiovascu-lar and cerebrovascular responses to acute hypoxia followingexposure to intermittent hypoxia in healthy humansrdquo Journalof Physiology vol 587 no 13 pp 3287ndash3299 2009

[5] P Siques Lee J Brito F Leon-Velarde et al ldquoTime course ofcardiovascular and hematological responses in rats exposedto chronic intermittent hypobaric hypoxia (4600 m)rdquo HighAltitude Medicine and Biology vol 7 no 1 pp 72ndash80 2006

[6] A F Corno G Milano S Morel et al ldquoHypoxia uniquemyocardial morphologyrdquo Journal of Thoracic and Cardiovas-cular Surgery vol 127 no 5 pp 1301ndash1308 2004

[7] M McGuire and A Bradford ldquoChronic intermittent hypoxiaincreases haematocrit and causes right ventricular hypertrophyin the ratrdquo Respiration Physiology vol 117 no 1 pp 53ndash58 1999

[8] J Brito P Siques F Leon-Velarde et al ldquoVarying exposureregimes to long term chronic intermittent hypoxia exert differ-ent outcomes and morphological effects on Wistar rats at 4600mrdquo Toxicological and Environmental Chemistry vol 90 no 1pp 169ndash179 2008

[9] A G Durmowicz and K R Stenmark ldquoMechanisms ofstructural remodeling in chronic pulmonary hypertensionrdquoPediatrics in Review vol 20 no 11 pp e91ndashe102 1999

[10] N Sommer A Dietrich R T Schermuly et al ldquoRegulationof hypoxic pulmonary vasoconstriction basic mechanismsrdquoEuropean Respiratory Journal vol 32 no 6 pp 1639ndash1651 2008

[11] M S Wolin S A Gupte C J Mingone B H Neo Q Gaoand M Ahmad ldquoRedox regulation of responses to hypoxia andNO-cGMP signaling in pulmonary vascular pathophysiologyrdquoAnnals of the New York Academy of Sciences vol 1203 pp 126ndash132 2010

[12] W Steudel M Scherrer-Crosbie K D Bloch et al ldquoSustainedpulmonary hypertension and right ventricular hypertrophyafter chronic hypoxia in mice with congenital deficiency ofnitric oxide synthaserdquo Journal of Clinical Investigation vol 101no 11 pp 2468ndash2477 1998

[13] G Frazziano H C Champion and P J Pagano ldquoNADPHoxidase-derived ROS and the regulation of pulmonary vesseltonerdquo The American Journal of Physiology vol 302 no 11 ppH2166ndashH2177 2012

[14] K K Griendling andM Ushio-Fukai ldquoReactive oxygen speciesas mediators of angiotensin II signalingrdquo Regulatory Peptidesvol 91 no 1ndash3 pp 21ndash27 2000

[15] M C Gonzalez ldquoConfocal Microscopy as New Tool for theStudy of Pulmonary Artery Remodelling at a Cellular Level inRats Exposed to Chronic Hypobaric Hypoxiardquo vol 11 pp 2622010

[16] P Siques ldquoStructural changes in pulmonary artery of exposedrats to chronic intermittent hypobaric hypoxiardquo High AltitudeMedicine and Biology vol 11 article 290 2010

[17] R Germack F Leon-Velarde R Valdes De La Barra J Farias GSoto and J P Richalet ldquoEffect of intermittent hypoxia on car-diovascular function adrenoceptors and muscarinic receptorsin Wistar ratsrdquo Experimental Physiology vol 87 no 4 pp 453ndash460 2002

[18] J Brito P Siques F Leon-Velarde J J De La Cruz V Lopezand R Herruzo ldquoChronic intermittent hypoxia at high altitude

exposure for over 12 years assessment of hematological cardio-vascular and renal effectsrdquoHigh Altitude Medicine and Biologyvol 8 no 3 pp 236ndash244 2007

[19] J M Gonzalez B Somoza M V Conde M S Fernandez-Alfonso M C Gonzalez and S M Arribas ldquoHypertensionincreases middle cerebral artery resting tone in spontaneouslyhypertensive rats role of tonic vasoactive factor availabilityrdquoClinical Science vol 114 no 9-10 pp 651ndash659 2008

[20] ZWang A Y Li Q H Guo et al ldquoEffects of cyclic intermittenthypoxia on ET-1 responsiveness and endothelial dysfunction ofpulmonary arteries in ratsrdquo PLoS ONE vol 8 no 3 Article IDe58078 2013

[21] Z Tahawi N Orolinova I G Joshua M Bader and E CFletcher ldquoAltered vascular reactivity in arterioles of chronicintermittent hypoxic ratsrdquo Journal of Applied Physiology vol 90no 5 pp 2007ndash2000 2001

[22] S A Phillips E B Olson B J Morgan and J H Lom-bard ldquoChronic intermittent hypoxia impairs endothelium-dependent dilation in rat cerebral and skeletal muscle resistancearteriesrdquoThe American Journal of Physiology vol 286 no 1 ppH388ndashH393 2004

[23] D C Irwin J M McCord E Nozik-Grayck et al ldquoA potentialrole for reactive oxygen species and the HIF-1120572-VEGF pathwayin hypoxia-induced pulmonary vascular leakrdquo Free RadicalBiology and Medicine vol 47 no 1 pp 55ndash61 2009

[24] NWeissmann R T Schermuly H A Ghofrani et al ldquoHypoxicpulmonary vasoconstriction triggered by an increase in reac-tive oxygen speciesrdquoNovartis Foundation Symposium vol 272pp 196ndash208 2006

[25] N Weissmann S Zeller R U Schafer et al ldquoImpact ofmitochondria and NADPH oxidases on acute and sustainedhypoxic pulmonary vasoconstrictionrdquoThe American Journal ofRespiratory Cell and Molecular Biology vol 34 no 4 pp 505ndash513 2006

[26] H Kojima N Nakatsubo K Kikuchi et al ldquoDirect evidenceof NO production in rat hippocampus and cortex using a newfluorescent indicator DAF-2 DArdquo NeuroReport vol 9 no 15pp 3345ndash3348 1998

[27] F-X Yi A Y Zhang W B Campbell A-P Zou C VanBreemen and P-L Li ldquoSimultaneous in situ monitoring ofintracellular Ca2+ and NO in endothelium of coronary arter-iesrdquo The American Journal of Physiology vol 283 no 6 ppH2725ndashH2732 2002

[28] S M Arribas C J Daly M C Gonzalez and J C McgrathldquoImaging the vascular wall using confocal microscopyrdquo Journalof Physiology vol 584 no 1 pp 5ndash9 2007

[29] B Somoza F Abderrahim J M Gonzalez et al ldquoShort-term treatment of spontaneously hypertensive rats with livergrowth factor reduces carotid artery fibrosis improves vascularfunction and lowers blood pressurerdquo Cardiovascular Researchvol 69 no 3 pp 764ndash771 2006

[30] F L Powell and N Garcia ldquoPhysiological effects of intermittenthypoxiardquo High Altitude Medicine and Biology vol 1 no 2 pp125ndash136 2000

[31] C E Norton N L Jernigan N L Kanagy B R Walker and TC Resta ldquoIntermittent hypoxia augments pulmonary vascularsmooth muscle reactivity to NO regulation by reactive oxygenspeciesrdquo Journal of Applied Physiology vol 111 no 4 pp 980ndash988 2011

10 BioMed Research International

[32] B Fuchs N Sommer A Dietrich et al ldquoRedox signaling andreactive oxygen species in hypoxic pulmonary vasoconstric-tionrdquo Respiratory Physiology and Neurobiology vol 174 no 3pp 282ndash291 2010

[33] R E Nisbet A S Graves D J Kleinhenz et al ldquoThe roleof NADPH oxidase in chronic intermittent hypoxia-inducedpulmonary hypertension in micerdquo The American Journal ofRespiratory Cell and Molecular Biology vol 40 no 5 pp 601ndash609 2009

[34] J M Dopp N R Philippi N J Marcus et al ldquoXanthine oxidaseinhibition attenuates endothelial dysfunction caused by chronicintermittent hypoxia in ratsrdquo Respiration vol 82 no 5 pp 458ndash467 2011

[35] A A El Solh R Saliba T Bosinski B J B Grant E Berbaryand N Miller ldquoAllopurinol improves endothelial functionin sleep apnoea a randomised controlled studyrdquo EuropeanRespiratory Journal vol 27 no 5 pp 997ndash1002 2006

[36] I Al Ghouleh N K H Khoo U G Knaus et al ldquoOxidases andperoxidases in cardiovascular and lung disease new conceptsin reactive oxygen species signalingrdquo Free Radical Biology andMedicine vol 51 no 7 pp 1271ndash1288 2011

[37] U Forstermann ldquoNitric oxide and oxidative stress in vasculardiseaserdquo Pflugers Archiv vol 459 no 6 pp 923ndash939 2010

[38] S Aggarwal C M Gross S Sharma J R Fineman and SM Black ldquoReactive oxygen species in pulmonary vascularremodelingrdquo Comprehensive Physiology vol 3 no 3 pp 1011ndash1034

[39] E Panzhinskiy W M Zawada K R Stenmark and M DasldquoHypoxia induces unique proliferative response in adventitialfibroblasts by activating PDGFbeta receptor-JNK1 signallingrdquoCardiovascular Research vol 95 no 3 pp 356ndash365 2012

[40] S Li S S Tabar V Malec et al ldquoNOX4 regulates ROS levelsunder normoxic and hypoxic conditions triggers proliferationand inhibits apoptosis in pulmonary artery adventitial fibrob-lastsrdquoAntioxidants and Redox Signaling vol 10 no 10 pp 1687ndash1697 2008

[41] C Berry C AHamiltonM J Brosnan et al ldquoInvestigation intothe sources of superoxide in humanblood vessels angiotensin IIincreases superoxide production in human internal mammaryarteriesrdquo Circulation vol 101 no 18 pp 2206ndash2212 2000

[42] P J Pagano Y Ito K Tornheim P M Gallop A I Tauberand R A Cohen ldquoAn NADPH oxidase superoxide-generatingsystem in the rabbit aortardquoThe American Journal of Physiologyvol 268 no 6 pp H2274ndashH2280 1995

[43] H D Wang P J Pagano Y Du et al ldquoSuperoxide anion fromthe adventitia of the rat thoracic aorta inactivates nitric oxiderdquoCirculation Research vol 82 no 7 pp 810ndash818 1998

[44] B Somoza M C Gonzalez J M Gonzalez F AbderrahimS M Arribas and M S Fernandez-Alfonso ldquoModulatoryrole of the adventitia on noradrenaline and angiotensin IIresponses role of endothelium and AT2 receptorsrdquo Cardiovas-cular Research vol 65 no 2 pp 478ndash486 2005

[45] J Chao J G Wood V G Blanco and N C GonzalezldquoThe systemic inflammation of alveolar hypoxia is initiated byalveolar macrophage-bornemediator(s)rdquoTheAmerican Journalof Respiratory Cell andMolecular Biology vol 41 no 5 pp 573ndash582 2009

[46] D L Burke M G Frid C L Kunrath et al ldquoSustainedhypoxia promotes the development of a pulmonary artery-specific chronic inflammatory microenvironmentrdquo The Amer-ican Journal of Physiology vol 297 no 2 pp L238ndashL250 2009

[47] J Zhang J M Patel Y D Li and E R Block ldquoProinflam-matory cytokines downregulate gene expression and activityof constitutive nitric oxide synthase in porcine pulmonaryartery endothelial cellsrdquo Research Communications inMolecularPathology and Pharmacology vol 96 no 1 pp 71ndash88 1997

[48] S-Y Lam Y Liu K-M Ng et al ldquoChronic intermittent hypoxiainduces local inflammation of the rat carotid body via func-tional upregulation of proinflammatory cytokine pathwaysrdquoHistochemistry and Cell Biology vol 137 no 3 pp 303ndash317 2012

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Evidence-Based Complementary and Alternative Medicine

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