research article adenosine 2b receptor activation reduces...

Research ArticleAdenosine 2B Receptor Activation Reduces MyocardialReperfusion Injury by Promoting Anti-InflammatoryMacrophages Differentiation via PI3KAkt Pathway

Yikui Tian12 Bryan A Piras3 Irving L Kron1 Brent A French3 and Zequan Yang13

1Department of Surgery University of Virginia PO Box 800709 Charlottesville VA 22908 USA2Department of Cardiovascular Surgery Tianjin Medical University General Hospital Tianjin 300052 China3Department of Biomedical Engineering University of Virginia Charlottesville VA 22908 USA

Correspondence should be addressed to Zequan Yang zy6bvirginiaedu

Received 18 September 2014 Revised 9 December 2014 Accepted 11 December 2014

Academic Editor Zhengyuan Xia

Copyright copy 2015 Yikui Tian 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

Background Activation of the adenosineA2B receptor (A2BR) can reducemyocardial ischemiareperfusion (IR) injury However themechanism underlying the A2BR-mediated cardioprotection is less clearThe present study was designed to investigate the potentialmechanisms of cardioprotection mediated by A2BR Methods and Results C57BL6 mice underwent 40-minute ischemia and 60-minute reperfusion ATL-801 a potent selective A2BR antagonist could not block ischemic preconditioning induced protectionBAY 60-6583 a highly selective A2BR agonist significantly reducedmyocardial infarct size and its protective effect could be blockedby either ATL-801 or wortmannin BAY 60-6583 increased phosphorylated Akt (p-Akt) levels in the heart at 10min of reperfusionand this phosphorylation could also be blocked by ATL-801 or wortmannin Furthermore BAY 60-6583 significantly increasedM2 macrophages and decreased M1 macrophage and neutrophils infiltration in reperfused hearts which also could be blocked bywortmannin Meanwhile confocal imaging studies showed that the majority of Akt phosphorylation in the heart was colocalizedto CD206+ cells in both control and BAY 60-6583 pretreated hearts Conclusion Our results indicated that pretreatment with BAY60-6583 protects the heart against myocardial IR injury by its anti-inflammatory effects probably by modulating macrophagesphenotype switching via a PI3KAkt pathway

1 Introduction

The adenosine receptor (AR) family comprises four sub-types A

1 A2A A2B and A

3 They are widely distributed

in mammalian species The A2BR is the fourth AR subtype

identified and to date there ismuch less information availableon the precise role of this receptor compared to the otherAR subtypes This notwithstanding an increasing body ofevidence demonstrates that activation of A

2BRs by the selec-tive A

2BR agonist BAY 60-6583 either before ischemia [1 2]or before reperfusion [3 4] reduces myocardial infarct sizeThus pharmaceutical preconditioning with A

2BR activationhas been shown to protect against myocardial IR injury butthe role of A

2BRs in IPC remains controversialUsing an in vivo mouse model of IPC and genetic

knockouts of ARs Eckle et al challenged the mechanism of

A1R-mediated IPC [5ndash7] by proposing that A

2BRs not A1Rsare essential in mediating IPC via an adenosine signalingpathway involving ecto-51015840-nucleotidase [1] HoweverMaas etal reported that A

2BR activation is not required for IPC ineither rat or mouse models Nevertheless they did find thatpretreatment prior to index ischemia with an A

2BR agonistdid reduce IR injury but to a lesser degree compared to thatof IPC [2] Interestingly another study reported by Ecklersquosgroup [8] recently demonstrated that BAY 60-6583 protectedthe heart against IR injury not by acting on cardiomyocytesbut by activating the A

2BR on bone marrow derived cellsand reducing inflammatory cell infiltration in reperfusedheart The discrepancies between these published results callfor more studies to clarify the role of A

2BRs in IPC andto explore the mechanisms underlying the A

2BR-mediatedcardioprotection against IR injury

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 585297 8 pageshttpdxdoiorg1011552015585297

2 Oxidative Medicine and Cellular Longevity

By using a well-established intact mouse model withmyocardial IR injury the current study was undertaken tofurther define the roles of the A

2BR in IPC and its anti-inflammatory effects during myocardial IR injury

2 Materials and Methods

This study conformed to the Guide for the Care and Useof Laboratory Animals published by the National Institutesof Health (Eighth Edition revised 2011) and was conductedunder protocols approved by the University of VirginiarsquosInstitutional Animal Care and Use Committee

21 Agents and Chemicals 235-Triphenyltetrazolium chlo-ride (TTC) and wortmannin were purchased from Sigma-Aldrich (St Louis MO) Phthalo blue was purchased fromHeucotech Ltd (Fairless Hills PA) BAY 60-6583 was pur-chased from Tocris Bioscience (Bristol UK) ATL-801 waskindly provided by Lewis and Clark Pharmaceuticals Inc(Charlottesville VA) Antibodies against phospho-Akt andtotal Akt were purchased from Cell Signaling Technology(Beverly MA) Ly-6B2 and CD206 antibodies for neu-trophils staining were fromABDSerotec (Oxford UK) CD45antibody was from BD Biosciences (San Jose CA) Allfluorochrome-conjugated secondary antibodies and ProlongGold antifade reagentwithDAPIwere fromLife Technologies(Grand Island NY)

22 Animals and Experimental Protocol C57BL6 mice (9-13 weeks old purchased from Jackson Laboratories) wereassigned to 6 different groups as shown in Figure 1 Thesemice underwent 40min of LADocclusion followed by 60minof reperfusion with or without IPC IPC was applied tomice with two cycles of 5- minute ischemia and 5-minutereperfusion In the treated groups BAY 60-6583 (100 120583gkgiv) was administered 15 minutes before index LAD occlusionATL-801 (100 120583gkg iv) was administered 5 minutes beforeeither BAY 60-6583 injection or IPCWortmannin (25 120583gkgiv) was administered 5minutes before BAY60-6583 injectionThe doses of the BAY 60-6583 ATL-801 and wortmanninused in this study were equal to or less than those usedin the literature which reported no significant effect onhemodynamics Additional 3 mice from each group wereundergoing sham surgery We monitored heart rate andfound no significant difference when compared to controlgroups (Table 1) The hearts were harvested at the end of theexperiments for infarct size measurement or immunostain-ing Effects of BAY 60-6583 on phosphorylated-Akt (p-Akt)levels in hearts which underwent 40 minutes of ischemiafollowed by 10 minutes of reperfusion were tested by westernblot The hearts from BAY 60-6583 + ATL-801 or BAY 60-6583 + wortmannin groups were also harvested for p-Aktanalysis

23 Myocardial IschemiaReperfusion Injury and Measure-ment of Infarct Size Mice were subjected to 40 minutesof coronary occlusion followed by 60 minutes of reper-fusion as detailed previously [5 9ndash11] Briefly mice were

Occlusion Reperfusion






Group 1Control

Group 2IPC

Group 4BAY 60-6583

Group 3ATL-801 + IPC

ATL-801 100120583gkg IV

minus50min 0min 40min 100min






BAY 60-6583

Group 5ATL-801 +

BAY 60-6583

Group 6Wortmannin +

BAY 60-6583 100120583gkg IV

BAY 60-6583100120583gkg IV

BAY 60-6583100120583gkg IV

ATL-801100120583gkg IV

Wortmannin25120583gkg IV

Figure 1 Experimental protocol All mice underwent 40minischemia followed by 60min reperfusion At the end of experimentshearts were harvested for TTC staining to determine themyocardialinfarct size IPC ischemic preconditioning

anesthetized with sodium pentobarbital (100mgkg ip) andorally intubated Artificial respiration was maintained witha FiO

2of 080 100 strokes per minute and a 02 to 05

mL stroke volume The heart was exposed through a leftthoracotomy A 7-0 silk suture was placed around LAD at alevel 1mm inferior to the left auricle and a miniature balloonoccluder fashioned from Microbore Tygon tubing (SmallParts Inc Seattle WA) was affixed over the LAD Ischemiaand reperfusion were induced by inflating or deflating theballoon respectively ECG was monitored perioperativelyusing PowerLab instrumentation (ADInstruments ColoradoSprings CO) The mice were euthanized 60 minutes afterreperfusion and the hearts were cannulated through theascending aorta for perfusion with 3 to 4mL of 10 TTCThe LAD was then reoccluded with the same suture used forcoronary occlusion prior to 10 Phthalo blue perfusion todetermine risk region (RR) The left ventricle was then cutinto 5 to 7 transverse slices that were weighed and digitallyphotographed to determine infarct size as a percent of RR

24 Western Blot Analysis The total protein was extractedfrom the indicated experimental groups using RIPA bufferand protein concentration was determined by BCA proteinassay (Thermo Scientific Rockford IL) All western blotswere performed according to standard procedures Twentymicrograms of protein was separated by 10 SDS-PAGEAfter transfer nitrocellulose membranes (BioRad Hercules

Oxidative Medicine and Cellular Longevity 3

Table 1 Perioperative heart rates

Groups Baseline During ischemia ReperfusionControl 437 plusmn 20 479 plusmn 18

lowast480 plusmn 20

lowast

IPC 438 plusmn 9 492 plusmn 8lowast

494 plusmn 6lowast

ATL + IPC 440 plusmn 8 486 plusmn 6lowast

496 plusmn 5lowast

BAY 431 plusmn 13 485 plusmn 11lowast

481 plusmn 13lowast

ATL + BAY 411 plusmn 10 503 plusmn 15lowast

501 plusmn 11lowast

Wort + BAY 433 plusmn 6 502 plusmn 9lowast

501 plusmn 8lowast

IPC ischemic preconditioning ATL ATL-801 BAY BAY 60-6583 Wortwortmanninlowast119875 lt 005 versus baseline

CA) were probed with primary antibodies against total Akt(t-Akt) or S473 (p-Akt) at a 1 2000 dilution and secondaryantibodies (Promega Madison WI) at a 1 5000 dilution inblocking solution (05 BSA in TBS-T) Proteins were visu-alized with enhanced chemiluminescent substrate (ThermoScientific Rockford IL) followed by densitometry analysisusing Fluorchem 8900 imaging system (Alpha InnotechSanta Clara CA)

25 Immunohistochemistry for Neutrophils The hearts wereharvested and immediately fixed in 4 paraformaldehydein PBS (pH 74) for paraffin embedding Paraffin-embeddedsections (5 120583m) were rehydrated and incubated with 1hydrogen peroxide After being rinsed in PBS the sectionswere incubated with 10 blocking serum Immunostain-ing was performed with rat anti-mouse Ly-6B2 antibodyBiotinylated secondary antibody was then applied for 1 hourat room temperature After incubation with avidin-biotincomplex immunoreactivity was visualized by incubating thesections with 33-diaminobenzidine tetrahydrochloride toproduce a brown precipitate

26 Immunofluorescence Staining In order to determine thelocalization of p-Akt expression immunofluorescence stain-ingwas performed on hearts after 40minutes of ischemia and10 minutes of reperfusion in control or BAY 60-6583 treatedgroups Cardiac macrophage polarization was detected after60 minutes of reperfusion by defining M1 and M2 subsetsof macrophages Hearts were fixed in 4 paraformaldehydein PBS (pH 74) for 1 hour at room temperature and thenincubated in 30 sucrose overnight at 4∘C before freezingin OCT Frozen sections were cut and permeabilized by03 TritonX-100 in PBS After blocking with 10 normalserum for 1 hour specimens were colabeled with anti-p-Akt and anti-CD45 or anti-CD206 antibody overnight at4∘C for colocalization detection For macrophage stainingspecimens were incubated with CD86 or CD163 antibodiesto identify the M1 and M2 macrophages respectively Afterwashing sections were incubated with a mixture of AlexaFluor 488- and Alexa Fluor 594-conjugated secondary anti-bodies for 1 hour Prolong Gold antifade reagent with DAPIwas used to mount the specimens All images were acquiredunder the same parameters for each fluorochrome using anOlympusBX-41Microscope (OlympusAmerica Inc CenterValley PA) with a Retiga-2000R camera (QImaging Surrey

BC) Further image processing was performed using Image Jsoftware (NIH)

27 Statistical Analysis All data are presented as the mean plusmnSEM (standard error of the mean) Peri-ischemic heart ratechanges were analyzed using a repeated measures ANOVAfollowed by Bonferroni pairwise comparisons All other datawere compared using one-way ANOVA followed by 119905-test forunpaired data with Bonferroni correction

3 Results

31 Perioperative Heart Rate Changes Table 1 shows changesin heart rate before during and after LAD occlusionConsistent with previous reports heart rate was increasedsignificantly after LADocclusion and remained elevated untilearly reperfusion compared with baseline in all groupsTherewas no significant difference in heart rates between controland treated mice

32 Role of A2119861R in IPC-Induced Infarct-Sparing Effect Three

groups of mice which underwent 40 minutes of LAD occlu-sion followed by 60 minutes of reperfusion were designed toinvestigate the role of A

2BR in IPC phenomenon There wasno significant difference of risk region (RR) among the threegroups Infarct size in the IPC-treated group (192 plusmn 27of RR) was significantly reduced compared with the controlgroup (496 plusmn 14of RR119875 lt 005) Administration of A

2BRselective antagonist ATL-801 5 minutes before IPC couldnot block the cardioprotective effects (187 plusmn 14 versus192 plusmn 27 of RR 119875 gt 005) (Figure 2)

33 Activation of A2119861R ReducedMyocardial IR Injury Is Medi-

ated by PI3KAkt Pathway BAY 60-6583 had no effects onheart rate during the peri-ischemic phase (Table 1) Admin-istration of BAY 60-6583 15 minutes before LAD occlusionhad significant infarct-sparing effects against myocardialreperfusion injury (210 plusmn 13 versus 496 plusmn 14 of RR119875 lt 005) Pretreating the mice with ATL-801 before BAY 60-6583 administration completely blocked the cardioprotectiveeffect (486 plusmn 24 of RR 119875 lt 005 compared withBAY group) (Figure 3) As shown in Figure 3 the infarct-sparing effect of BAY 60-6583 was completely abrogatedby wortmannin a selective PI3K inhibitor administered 5minutes before BAY administration (450 plusmn 32 of RR 119875 lt005 compared with BAY group) Furthermore western blotresults showed that BAY 60-6583 significantly increased p-Akt levels in heart tissue undergoing 40 minutes of ischemiaand 10 minutes of reperfusion This effect was completelyabolished by pretreating the mice with either ATL-801 orwortmannin 5 minutes before BAY 60-6583 administration(Figure 4)

34 Administration of A2119861R Agonist Presents Local Anti-

Inflammatory Effects after Myocardial IR Injury It has beenreported that activation of A

2BR has anti-inflammatoryeffects by promotingmacrophages phenotype switching Alsoactivated PI3KAkt pathway has been shown to modulate


0

10

20

30

40

50

60

70

IF ( RR) RR ( LV)

()

Control (n = 8)IPC (n = 8)ATL + IPC (n = 10)

lowastlowast

Figure 2 Role of A2BR antagonist in ischemic preconditioning

PretreatingmicewithATL-801 anA2BR selective antagonist did not

block the cardioprotective effect of IPC as comparedwith the controlgroup (lowast119875 lt 005) IF infarct size RR risk region LV left ventricleIPC ischemic preconditioning ATL ATL-801

010203040506070

IF ( RR) RR ( LV)

()

Control (n = 8)BAY (n = 9)

ATL + BAY (n = 8)Wort + BAY (n = 7)

lowast

Figure 3 Role of A2BR in myocardial IR injury Pretreating mice

with BAY 60-6583 a potent A2BR agonist reduced myocardial

infarct size an effect that was abolished by either ATL-801 orthe PI3K inhibitor wortmannin IF infarct size RR risk regionBAY BAY 60-6583 ATL ATL-801 Wort wortmannin lowast119875 lt 005compared with control group

macrophages to M2 anti-inflammatory subset Thus wehypothesized that BAY 60-6583 may protect the heart by reg-ulating cardiac macrophages phenotype via PI3KAkt path-way and presenting anti-inflammatory effects Immunofluo-rescence staining was used to identify macrophage subsetsIn sham mouse heart there were few M1 macrophages(CD86+) but more M2 macrophages (CD163+) IR signifi-cantly increased the M1 and decreased the M2 macrophagesnumber Compared with the IR group BAY 60-6583 restoredthe macrophage polarization to a M2 phenotype (Figure 5)which could also be abolished by wortmannin In additionconfocal staining results showed that remarkable p-Akt

p-Akt

t-Akt

0

02

04

06

08

1

12

Control BAY

p-A

kt S

473

t-Akt

amp amp

ATL + BAY Wort + BAY

lowast

Figure 4 Phospho-Akt to total Akt ratio in heart tissue The ratioof phospho-Akt S473 to total Akt (the bar graph) was measuredby densitometry where the total Akt inputs were normalized to 1BAY 60-6583 pretreatment increased p-Akt level after 10 minutes ofreperfusion which was blocked by either ATL-801 or wortmanninBAY BAY 60-6583 ATL ATL-801 Wort wortmannin lowast119875 lt 005compared with control group amp119875 lt 005 compared with BAY group

expressions were colocalized with CD45+ cells (Figure 6(a))In order to further identify these CD45+ cells we per-formed additional confocal microscopy studies on tissuesections immunostained with p-Akt and CD206 antibodiesanother specific marker of M2 macrophages As shown inFigure 6(b) although a few of the p-Akt+ cells were CD206negative the majority of the p-Akt expression colocalizedwith M2 macrophages These results suggest that BAY 60-6583 may protect the heart by promoting macrophagephenotype switching to anti-inflammatory subsets via thePI3KAkt pathway Immunohistochemistry was used to stainneutrophils in hearts undergoing 40 minutes of ischemiaand 60 minutes of reperfusion When pretreated with BAY60-6583 before IR neutrophil infiltration was significantlyreduced Consistent with the infarct size results this anti-inflammatory effect of BAY 60-6583 could be blocked by bothATL-801 and wortmannin respectively (Figure 7)

4 Discussion

For over a decade abundant evidence has accumulated todemonstrate that brief periods of myocardial ischemia (IPC)activate the A1R to protect the heart against the subsequentprolonged ischemia [5 12ndash15] Activation of A1Rs triggers thesurvival signal transduction pathway through the enhancedphosphorylation of Akt [15ndash17] This event mostly likelyoccurs inside cardiomyocytes thus rendering them moreresistant to the subsequent ischemic insult [5 12] Eckle andcolleagues [1] recently proposed that cardioprotective effectof IPC was mediated via CD73-dependent generation ofextracellular adenosine and signaling through the A

2BR thelowest-affinity receptor among the 4 subtypes of adenosinereceptor A

2BRKO mice demonstrate increased susceptibilityto acute IR injury and are not protected by IPC HoweverMaas and colleagues evaluated the role of A

2BR activation


0

20

40

60

80

100

Sham Control BAY

CD163

CD163 DAPI Merged CD86 DAPI Merged

0

5

10

15

20

25

30

Sham Control BAY

CD86

amp

amp

BAY + Wort

Sham

Control

BAY

BAY + Wort

Cell

coun

thig

h fie

ld (20

x)

Cell

coun

thig

h fie

ld (20

x)

BAY + Wort

lowast

lowast

Figure 5 Cardiac macrophages polarization after IR injury There are mainly M2 subsets macrophages in normal heart IR reduced thenumber of M2 subset (CD163 green staining) macrophages and increased M1 subset (CD86 red staining) BAY 60-6583 pretreatmentsignificantly restored the macrophage polarization during reperfusion Wortmannin a PI3K inhibitor blocked the effects of BAY 60-6583DAPI was used to stain the nuclei Scale bar 50 120583m IR ischemia and reperfusion injury BAY BAY 60-6583 Wort wortmannin lowast119875 lt 005compared with sham or BAY groups amp119875 lt 005 compared with BAY groups

p-Akt CD45 DAPI Merged

Sham

Control

BAY

(a)


Sham

Control

BAY

(b)

Figure 6 Confocal staining of p-Akt and immune cells in the heart Hearts were harvested from mice with 40min10min IR injury forstaining to determine the localization of p-Akt expression (a) In the ischemic area or lower anterior wall of the left ventricle higher p-Aktexpression (red staining) was colocalized with CD45 signal (green staining) in all groups Scale bar 20 120583m (b) The majority of strong p-Aktexpression (red staining) was colocated with CD206 expression (green staining) which is a specific marker of M2 macrophages Scale bar20 120583m Arrows indicate coexpression of p-Akt and CD45 or CD206 410158406-Diamidino-2-phenylindole (DAPI blue staining) was used to stainthe nuclei IR ischemia and reperfusion injury BAY BAY 60-6583


02468

101214161820

Control BAY

Cell

coun

thig

h fie

ld (20

x)

Wort + BAYATL + BAY

Figure 7 Neutrophils infiltration after IR injury BAY 60-6583significantly reduced neutrophil infiltration (brown staining) afterIR injury which could be blocked by either ATL-801 orwortmanninScale bar 50 120583m Arrows indicate neutrophils BAY BAY 60-6583ATL ATL-801 Wort wortmannin lowast119875 lt 005 compared with othergroups

or inhibition in both ex vivo and in vivo mouserat modelswith myocardial IR injury and found that IPC-inducedcardioprotection was not dependent upon A

2BR activationand also existed in a different strain of A

2BRKO mice [2] Itshould be noted that the lowest affinity for adenosine couldmake A

2BR be the last receptor activated by endogenousadenosine which is the coremechanism of IPC [18] althoughA2BR might be sensitized by PKC at reperfusion phase [19]

Also A2BR expression at the mRNA level is very low in the

heart when compared to other organs [20] or other adenosinereceptors [21] Moreover there has been no physical evidenceso far to demonstrate that A

2BRs can be detected on thesarcolemma of cardiomyocytes but increased A

2BR mRNAexpression has been reported after IR injury [22] which maybe derived from infiltrating inflammatory cells By employingthe same in vivo mouse model we used previously [9 1117 23] the current study demonstrated activation of A

2BRsbefore ischemia reduced myocardial infarct size probably byinhibiting inflammatory responses during reperfusion not bypreconditioning cardiomyocytes (Figures 2 and 3)

Accumulating evidences have shown that inflammatoryresponses play important roles during myocardial reperfu-sion injury [10 11 24 25] A

2BR has been shown to haveanti-inflammatory effects in several in vitro and in vivoanimal models Yang and colleagues [26] found that A

2BRKOmice present proinflammatory phenotype They found thatmore inflammatory cytokines such as TNF-alpha and IL-6increased in serum of A

2BRKO mice compared with wild-type mice Konrad and colleagues [27] reported that BAY60-6583 a specific A

2BR agonist acts on hematopoietic cellsto inhibit PMN migration into lung interstitium BAY 60-6583 also has been shown to inhibit TNF-alpha secretionfrom macrophages after vascular injury [28] Consistentwith these studies we found that BAY 60-6583 significantlypromoted macrophages phenotype switching to a M2 (anti-inflammatory) subset and reduced neutrophils infiltrationaftermyocardial IR injury (Figures 5 and 7) Indeed A

2BR hasbeen shown to be able tomodulate macrophage phenotype to

an anti-inflammatory M2 subset and increase IL-10 expres-sion in both cultured macrophage and dendritic cell [29 30]which are consistent with our findings that A

2BR agonistpromoted the cardiac macrophage transforming to an anti-inflammatory M2 phenotype Indeed previous studies haveshown that BAY 60-6583 provided potent cardioprotectionin a Krebs-perfused isolated heart model However it isworthwhile to note that there actually are several residentialmacrophages and dendritic cells in the heart which maymediate the cardioprotection of the A

2BR agonist Furtherstudy is warranted to investigate the mechanism of how theseresidential immune cells regulate myocardial IR injury

Activation of PI3KAkt pathway in immune cells has beenreported providing anti-inflammatory effect by upregulatingIL-10 expression [31 32]The relationship between A

2BR acti-vation and PI3KAkt pathway is not well established Kunoand colleagues [3] reported that activation of A

2BR increasedp-Akt levels in a rabbit model although a nonselectiveadenosine receptor agonist was used It is also reported thatactivation of A

2BR contributes to PI3KAkt activation andsubsequent eNOS phosphorylation in penile endothelia [33]In present study we found that after 40 minutes of ischemiaand 10 minutes of reperfusion phosphorylation of Akt in theheart was significantly increased in mice treated with A

2BRagonist which could be blocked by either ATL-801 a selectiveA2BR antagonist or wortmannin a PI3K inhibitor (Figure 4)

Since activation of PI3KAkt pathway in nonimmune cellssuch as cardiomyocytes [34] or endothelial cells [35] couldenhance cellular survival or inhibit oxidative stress wefurther performed confocal imaging analysis to determinethe location of p-Akt expression The results showed thatthe majority of p-Akt expression was localized to CD206+cells a specific marker for M2 macrophages in both controland BAY 60-6583 pretreated mice In addition the PI3Kinhibitor attenuated the anti-inflammatory effects of BAY 60-6583 (Figures 5ndash7) These findings strongly suggest that BAY60-6583 exerts its infarct-limiting effect not by acting oncardiomyocytes but possibly by acting on macrophages via aPI3KAkt pathway

It should be noted that a regulatory inflammation envi-ronment could promote macrophages phenotype switch toM2 subset [36] Although our results strongly indicatedBAY 60-6583 modulates macrophages phenotype switchingafter myocardial IR injury there are several cell types otherthan macrophages which may mediate BAY 60-6583 inducedcardioprotection which warrants further studies Koeppenand his colleagues [8] demonstrated that BAY 60-6583 pro-tects heart from myocardial ischemia and reperfusion injuryby acting on bone marrow derived cells They found thatBAY 60-6583 inhibited tumor necrosis factor 120572 release ofPMNs which limited myocardial injury van der Hoeven andhis colleagues [37] showed that A

2BR activation suppressedoxidase activity in neutrophils Moreover A

2BR was alsofound in dendritic cells and lymphocytes Studies are neededto define the roles of these cells during myocardial IR injuryand the cardioprotection effects of A

2BRIn present study we did not analyze the cardiac function

aftermyocardial reperfusion injuryHowever it is well knownthat histological determination of myocardial infarction has


been widely used to estimate the cardiac injury in animalmodels [38] and utilized as the golden standard to val-idate novel imaging methods of infarction quantificationincluding cardiac MRI [39] and contrast echocardiography[40] Moreover using a cardiac MRI imaging technique wehave defined in our previous publication that left ventricularfunction is depressed proportionally to the size of infarctionwhich was determined by the same TTC staining techniquein the present study [41] Hence it is reasonable to speculatethat BAY 60-6583 could improve the cardiac function afterreperfusion injury However further study is warranted todetermine the role of A

2BR activation during long-termventricular remodeling after myocardial infarction

In summary our work demonstrates that the A2BR

agonists reducemyocardial IR injury by inhibiting inflamma-tory responses in reperfused heart probably by promotingmacrophage phenotype switching to an anti-inflammatoryM2 subset

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was funded in part by NIH R01 HL 092305 toIrving L Kron and Brent A French andTheNational NaturalScience Foundation of China (NSFC 81400213) to Yikui Tian

References

[1] T Eckle T Krahn A Grenz et al ldquoCardioprotection by ecto-51015840-nucleotidase (CD73) and A2B adenosine receptorsrdquo Circulationvol 115 no 12 pp 1581ndash1590 2007

[2] J E Maas T C Wan R A Figler G J Gross and J AAuchampach ldquoEvidence that the acute phase of ischemic pre-conditioning does not require signaling by the A2B adenosinereceptorrdquo Journal of Molecular and Cellular Cardiology vol 49no 5 pp 886ndash893 2010

[3] A Kuno S D Critz L Cui et al ldquoProtein kinase C protectspreconditioned rabbit hearts by increasing sensitivity of adeno-sine A2b-dependent signaling during early reperfusionrdquo Journalof Molecular and Cellular Cardiology vol 43 no 3 pp 262ndash2712007

[4] C Methner K Schmidt M V Cohen J M Downey and TKrieg ldquoBoth A2a and A2b adenosine receptors at reperfusionare necessary to reduce infarct size in mouse heartsrdquo AmericanJournal of PhysiologymdashHeart and Circulatory Physiology vol299 no 4 pp H1262ndashH1264 2010

[5] A R Lankford J-N Yang R RosersquoMeyer et al ldquoEffect ofmodulating cardiac A1 adenosine receptor expression on pro-tection with ischemic preconditioningrdquo The American Journalof PhysiologymdashHeart and Circulatory Physiology vol 290 no 4pp H1469ndashH1473 2006

[6] J A Auchampach and G J Gross ldquoAdenosine A1 receptorsK(ATP) channels and ischemic preconditioning in dogsrdquo TheAmerican Journal of PhysiologymdashHeart and Circulatory Physiol-ogy vol 264 no 5 pp H1327ndashH1336 1993

[7] M E Reichelt A Shanu LWillems et al ldquoEndogenous adeno-sine selectively modulates oxidant stress via the A1 receptor inischemic heartsrdquo Antioxidants and Redox Signaling vol 11 no11 pp 2641ndash2650 2009

[8] M Koeppen P N Harter S Bonney et al ldquoAdora2b signalingon bone marrow derived cells dampens myocardial ischemia-reperfusion injuryrdquoAnesthesiology vol 116 no 6 pp 1245ndash12572012

[9] Z Yang V E Laubach B A French and I L KronldquoAcute hyperglycemia enhances oxidative stress and exacerbatesmyocardial infarction by activating nicotinamide adenine din-ucleotide phosphate oxidase during reperfusionrdquoThe Journal ofThoracic and Cardiovascular Surgery vol 137 no 3 pp 723ndash7292009

[10] Z Yang Y-J Day M-C Toufektsian et al ldquoInfarct-sparingeffect of A2A-adenosine receptor activation is due primarily toits action on lymphocytesrdquo Circulation vol 111 no 17 pp 2190ndash2197 2005

[11] Z Yang Y-J Day M-C Toufektsian et al ldquoMyocardial infarct-sparing effect of adenosine A2A receptor activation is due to itsaction on CD4+ T lymphocytesrdquo Circulation vol 114 no 19 pp2056ndash2064 2006

[12] C Dougherty J Barucha P R Schofield K A Jacobson andB T Liang ldquoCardiac myocytes rendered ischemia resistant byexpressing the human adenosine A1 or A3 receptorrdquoThe FASEBJournal vol 12 no 15 pp 1785ndash1792 1998

[13] Z Yang R L J Cerniway A M Byford S S Berr B AFrench and G Paul Matherne ldquoCardiac overexpression ofA1-adenosine receptor protects intact mice against myocardial

infarctionrdquo American Journal of PhysiologymdashHeart and Circu-latory Physiology vol 282 no 3 pp H949ndashH955 2002

[14] Z-Q Zhao K Nakanishi D Scott McGee P Tan and JVinten-Johansen ldquoA1 receptor mediatedmyocardial infarct sizereduction by endogenous adenosine is exerted primarily duringischaemiardquo Cardiovascular Research vol 28 no 2 pp 270ndash2791994

[15] J D Joo M Kim V D DrsquoAgati and H T Lee ldquoIschemicpreconditioning provides both acute and delayed protectionagainst renal ischemia and reperfusion injury in micerdquo Journalof the American Society of Nephrology vol 17 no 11 pp 3115ndash3123 2006

[16] NV Solenkova V SolodushkoMV Cohen and JMDowneyldquoEndogenous adenosine protects preconditioned heart duringearly minutes of reperfusion by activating Aktrdquo AmericanJournal of PhysiologymdashHeart and Circulatory Physiology vol290 no 1 pp H441ndashH449 2006

[17] Z Yang Y Tian Y Liu SHennessy I L Kron andBA FrenchldquoAcute hyperglycemia abolishes ischemic preconditioning byinhibiting Akt phosphorylation normalizing blood glucosebefore ischemia restores ischemic preconditioningrdquo OxidativeMedicine and Cellular Longevity vol 2013 Article ID 329183 8pages 2013

[18] K Mubagwa andW Flameng ldquoAdenosine adenosine receptorsandmyocardial protection an updated overviewrdquo Cardiovascu-lar Research vol 52 no 1 pp 25ndash39 2001

[19] X YangMVCohen and JMDowney ldquoMechanismof cardio-protection by early ischemic preconditioningrdquo CardiovascularDrugs andTherapy vol 24 no 3 pp 225ndash234 2010

[20] A K Dixon A K Gubitz D J S Sirinathsinghji P J Richard-son and T C Freeman ldquoTissue distribution of adenosinereceptor mRNAs in the ratrdquo British Journal of Pharmacologyvol 118 no 6 pp 1461ndash1468 1996


[21] P C Chandrasekera V J McIntosh F X Cao and R DLasley ldquoDifferential effects of adenosine A2a and A2b receptorson cardiac contractilityrdquoAmerican Journal of PhysiologymdashHeartand Circulatory Physiology vol 299 no 6 pp H2082ndashH20892010

[22] R R Morrison B Teng P J Oldenburg L C Katwa J BSchnermann and S J Mustafa ldquoEffects of targeted deletion ofA1adenosine receptors on postischemic cardiac function and

expression of adenosine receptor subtypesrdquo American Journalof PhysiologymdashHeart and Circulatory Physiology vol 291 no 4pp H1875ndashH1882 2006


[24] J Han D Wang B Yu et al ldquoCardioprotection againstischemiareperfusion by licochalcone B in isolated rat heartsrdquoOxidative Medicine and Cellular Longevity vol 2014 Article ID134862 11 pages 2014

[25] J Li C Xie J Zhuang et al ldquoResveratrol attenuates inflam-mation in the rat heart subjected to ischemia-reperfusion roleof the TLR4NF-120581B signaling pathwayrdquo Molecular MedicineReports 2014

[26] D Yang Y Zhang H G Nguyen et al ldquoThe A2B adenosinereceptor protects against inflammation and excessive vascularadhesionrdquo Journal of Clinical Investigation vol 116 no 7 pp1913ndash1923 2006

[27] F M Konrad E Witte I Vollmer S Stark and J Reuter-shan ldquoAdenosine receptor A2b on hematopoietic cells mediatesLPS-induced migration of PMNs into the lung interstitiumrdquoAmerican Journal of PhysiologymdashLung Cellular and MolecularPhysiology vol 303 no 5 pp L425ndashL438 2012

[28] H Chen D Yang S H Carroll H K Eltzschig and KRavid ldquoActivation of the macrophage A2b adenosine receptorregulates tumor necrosis factor-120572 levels following vascularinjuryrdquo Experimental Hematology vol 37 no 5 pp 533ndash5382009

[29] J Ham and D A Rees ldquoThe adenosine A2b receptor its rolein inflammationrdquo Endocrine Metabolic and Immune DisordersDrug Targets vol 8 no 4 pp 244ndash254 2008

[30] E Sciaraffia A Riccomi R Lindstedt et al ldquoHumanmonocytesrespond to extracellular cAMP through A2A and A2B adeno-sine receptorsrdquo Journal of Leukocyte Biology vol 96 no 1 pp113ndash122 2014

[31] W Zhang W Xu and S Xiong ldquoMacrophage differentiationand polarization via phosphatidylinositol 3-kinaseAkt-ERKsignaling pathway conferred by serum amyloid P componentrdquoJournal of Immunology vol 187 no 4 pp 1764ndash1777 2011

[32] L Tarassishin H-S Suh and S C Lee ldquoInterferon regulatoryfactor 3 plays an anti-inflammatory role in microglia by activat-ing the PI3KAkt pathwayrdquo Journal of Neuroinflammation vol8 article 187 2011

[33] J Wen A Grenz Y Zhang et al ldquoA2B adenosine receptorcontributes to penile erection via PI3KAKT signaling cascade-mediated eNOS activationrdquo The FASEB Journal vol 25 no 8pp 2823ndash2830 2011

[34] Y Cao Y Ruan T Shen et al ldquoAstragalus polysaccharidesuppresses doxorubicin-induced cardiotoxicity by regulatingthe PI3kAkt and p38MAPKpathwaysrdquoOxidativeMedicine andCellular Longevity vol 2014 Article ID 674219 12 pages 2014

[35] S Xing X Yang W Li et al ldquoSalidroside stimulates mitochon-drial biogenesis and protects against H

2O2-induced endothelial

dysfunctionrdquo Oxidative Medicine and Cellular Longevity vol2014 Article ID 904834 13 pages 2014

[36] M Nahrendorf and F K Swirski ldquoMonocyte and macrophageheterogeneity in the heartrdquo Circulation Research vol 112 no 12pp 1624ndash1633 2013

[37] D vanderHoeven TCWan E TGizewski et al ldquoA role for thelow-affinity A2B adenosine receptor in regulating superoxidegeneration by murine neutrophilsrdquo Journal of Pharmacologyand Experimental Therapeutics vol 338 no 3 pp 1004ndash10122011

[38] J P Borges K S Verdoorn A Daliry et al ldquoDelta opioidreceptors the link between exercise and cardioprotectionrdquoPLoSONE vol 9 no 11 Article ID e113541 2014

[39] S M Grieve J Mazhar F Callaghan et al ldquoAutomated quan-tification of myocardial salvage in a rat model of ischemia-reperfusion injury using 3D high-resolution magnetic reso-nance imaging (MRI)rdquo Journal of the American Heart Associ-ation vol 3 no 4 2014

[40] A Filusch S Buss S Hardt H A Katus H F Kuecherer andA Hansen ldquoEvaluation cardioprotective effects of atorvastatinin rats by real time myocardial contrast echocardiographyrdquoEchocardiography vol 25 no 9 pp 974ndash981 2008

[41] Z Yang J Linden S S Berr I L Kron G A Beller andB A French ldquoTiming of adenosine 2A receptor stimulationrelative to reperfusion has differential effects on infarct size andcardiac function as assessed in mice byMRIrdquo American Journalof PhysiologymdashHeart and Circulatory Physiology vol 295 no 6pp H2328ndashH2335 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


By using a well-established intact mouse model withmyocardial IR injury the current study was undertaken tofurther define the roles of the A

2BR in IPC and its anti-inflammatory effects during myocardial IR injury

2 Materials and Methods

This study conformed to the Guide for the Care and Useof Laboratory Animals published by the National Institutesof Health (Eighth Edition revised 2011) and was conductedunder protocols approved by the University of VirginiarsquosInstitutional Animal Care and Use Committee

21 Agents and Chemicals 235-Triphenyltetrazolium chlo-ride (TTC) and wortmannin were purchased from Sigma-Aldrich (St Louis MO) Phthalo blue was purchased fromHeucotech Ltd (Fairless Hills PA) BAY 60-6583 was pur-chased from Tocris Bioscience (Bristol UK) ATL-801 waskindly provided by Lewis and Clark Pharmaceuticals Inc(Charlottesville VA) Antibodies against phospho-Akt andtotal Akt were purchased from Cell Signaling Technology(Beverly MA) Ly-6B2 and CD206 antibodies for neu-trophils staining were fromABDSerotec (Oxford UK) CD45antibody was from BD Biosciences (San Jose CA) Allfluorochrome-conjugated secondary antibodies and ProlongGold antifade reagentwithDAPIwere fromLife Technologies(Grand Island NY)

22 Animals and Experimental Protocol C57BL6 mice (9-13 weeks old purchased from Jackson Laboratories) wereassigned to 6 different groups as shown in Figure 1 Thesemice underwent 40min of LADocclusion followed by 60minof reperfusion with or without IPC IPC was applied tomice with two cycles of 5- minute ischemia and 5-minutereperfusion In the treated groups BAY 60-6583 (100 120583gkgiv) was administered 15 minutes before index LAD occlusionATL-801 (100 120583gkg iv) was administered 5 minutes beforeeither BAY 60-6583 injection or IPCWortmannin (25 120583gkgiv) was administered 5minutes before BAY60-6583 injectionThe doses of the BAY 60-6583 ATL-801 and wortmanninused in this study were equal to or less than those usedin the literature which reported no significant effect onhemodynamics Additional 3 mice from each group wereundergoing sham surgery We monitored heart rate andfound no significant difference when compared to controlgroups (Table 1) The hearts were harvested at the end of theexperiments for infarct size measurement or immunostain-ing Effects of BAY 60-6583 on phosphorylated-Akt (p-Akt)levels in hearts which underwent 40 minutes of ischemiafollowed by 10 minutes of reperfusion were tested by westernblot The hearts from BAY 60-6583 + ATL-801 or BAY 60-6583 + wortmannin groups were also harvested for p-Aktanalysis

23 Myocardial IschemiaReperfusion Injury and Measure-ment of Infarct Size Mice were subjected to 40 minutesof coronary occlusion followed by 60 minutes of reper-fusion as detailed previously [5 9ndash11] Briefly mice were







Group 1Control

Group 2IPC

Group 4BAY 60-6583

Group 3ATL-801 + IPC

ATL-801 100120583gkg IV







BAY 60-6583

Group 5ATL-801 +

BAY 60-6583

Group 6Wortmannin +

BAY 60-6583 100120583gkg IV

BAY 60-6583100120583gkg IV

BAY 60-6583100120583gkg IV

ATL-801100120583gkg IV

Wortmannin25120583gkg IV

Figure 1 Experimental protocol All mice underwent 40minischemia followed by 60min reperfusion At the end of experimentshearts were harvested for TTC staining to determine themyocardialinfarct size IPC ischemic preconditioning

anesthetized with sodium pentobarbital (100mgkg ip) andorally intubated Artificial respiration was maintained witha FiO

2of 080 100 strokes per minute and a 02 to 05

mL stroke volume The heart was exposed through a leftthoracotomy A 7-0 silk suture was placed around LAD at alevel 1mm inferior to the left auricle and a miniature balloonoccluder fashioned from Microbore Tygon tubing (SmallParts Inc Seattle WA) was affixed over the LAD Ischemiaand reperfusion were induced by inflating or deflating theballoon respectively ECG was monitored perioperativelyusing PowerLab instrumentation (ADInstruments ColoradoSprings CO) The mice were euthanized 60 minutes afterreperfusion and the hearts were cannulated through theascending aorta for perfusion with 3 to 4mL of 10 TTCThe LAD was then reoccluded with the same suture used forcoronary occlusion prior to 10 Phthalo blue perfusion todetermine risk region (RR) The left ventricle was then cutinto 5 to 7 transverse slices that were weighed and digitallyphotographed to determine infarct size as a percent of RR

24 Western Blot Analysis The total protein was extractedfrom the indicated experimental groups using RIPA bufferand protein concentration was determined by BCA proteinassay (Thermo Scientific Rockford IL) All western blotswere performed according to standard procedures Twentymicrograms of protein was separated by 10 SDS-PAGEAfter transfer nitrocellulose membranes (BioRad Hercules




lowast480 plusmn 20

lowast


494 plusmn 6lowast


496 plusmn 5lowast


481 plusmn 13lowast


501 plusmn 11lowast


501 plusmn 8lowast







3 Results












0

10

20

30

40

50

60

70

IF ( RR) RR ( LV)

()


lowastlowast




010203040506070

IF ( RR) RR ( LV)

()



lowast





p-Akt

t-Akt

0

02

04

06

08

1

12

Control BAY

p-A

kt S

473

t-Akt

amp amp


lowast



4 Discussion




2BR activation


0

20

40

60

80

100

Sham Control BAY

CD163


0

5

10

15

20

25

30

Sham Control BAY

CD86

amp

amp

BAY + Wort

Sham

Control

BAY

BAY + Wort

Cell

coun

thig

h fie

ld (20

x)

Cell

coun

thig

h fie

ld (20

x)

BAY + Wort

lowast

lowast



Sham

Control

BAY

(a)


Sham

Control

BAY

(b)



02468

101214161820

Control BAY

Cell

coun

thig

h fie

ld (20

x)

Wort + BAYATL + BAY






































Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















lowast480 plusmn 20

lowast


494 plusmn 6lowast


496 plusmn 5lowast


481 plusmn 13lowast


501 plusmn 11lowast


501 plusmn 8lowast







3 Results












0

10

20

30

40

50

60

70

IF ( RR) RR ( LV)

()


lowastlowast




010203040506070

IF ( RR) RR ( LV)

()



lowast





p-Akt

t-Akt

0

02

04

06

08

1

12

Control BAY

p-A

kt S

473

t-Akt

amp amp


lowast



4 Discussion




2BR activation


0

20

40

60

80

100

Sham Control BAY

CD163


0

5

10

15

20

25

30

Sham Control BAY

CD86

amp

amp

BAY + Wort

Sham

Control

BAY

BAY + Wort

Cell

coun

thig

h fie

ld (20

x)

Cell

coun

thig

h fie

ld (20

x)

BAY + Wort

lowast

lowast



Sham

Control

BAY

(a)


Sham

Control

BAY

(b)



02468

101214161820

Control BAY

Cell

coun

thig

h fie

ld (20

x)

Wort + BAYATL + BAY






































Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

50

60

70

IF ( RR) RR ( LV)

()


lowastlowast




010203040506070

IF ( RR) RR ( LV)

()



lowast





p-Akt

t-Akt

0

02

04

06

08

1

12

Control BAY

p-A

kt S

473

t-Akt

amp amp


lowast



4 Discussion




2BR activation


0

20

40

60

80

100

Sham Control BAY

CD163


0

5

10

15

20

25

30

Sham Control BAY

CD86

amp

amp

BAY + Wort

Sham

Control

BAY

BAY + Wort

Cell

coun

thig

h fie

ld (20

x)

Cell

coun

thig

h fie

ld (20

x)

BAY + Wort

lowast

lowast



Sham

Control

BAY

(a)


Sham

Control

BAY

(b)



02468

101214161820

Control BAY

Cell

coun

thig

h fie

ld (20

x)

Wort + BAYATL + BAY






































Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

Sham Control BAY

CD163


0

5

10

15

20

25

30

Sham Control BAY

CD86

amp

amp

BAY + Wort

Sham

Control

BAY

BAY + Wort

Cell

coun

thig

h fie

ld (20

x)

Cell

coun

thig

h fie

ld (20

x)

BAY + Wort

lowast

lowast



Sham

Control

BAY

(a)


Sham

Control

BAY

(b)



02468

101214161820

Control BAY

Cell

coun

thig

h fie

ld (20

x)

Wort + BAYATL + BAY






































Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















02468

101214161820

Control BAY

Cell

coun

thig

h fie

ld (20

x)

Wort + BAYATL + BAY






































Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article adenosine 2b receptor activation reduces...

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